Research ArticleInfluence of the Melissa officinalis Leaf Extract onLong-Term Memory in Scopolamine Animal Model withAssessment of Mechanism of Action

Marcin Ozarowski12 Przemyslaw L Mikolajczak23 Anna Piasecka4 Piotr Kachlicki4

Radoslaw Kujawski2 Anna Bogacz56 Joanna Bartkowiak-Wieczorek5 Michal Szulc3

Ewa Kaminska3 Malgorzata Kujawska7 Jadwiga Jodynis-Liebert7

Agnieszka Gryszczynska2 Bogna Opala2 Zdzislaw Lowicki2

Agnieszka Seremak-Mrozikiewicz289 and Boguslaw Czerny610

1 Department of Pharmaceutical Botany and Plant Biotechnology Poznan University of Medical Sciences Sw Marii Magdaleny 1461-861 Poznan Poland

2 Department of Pharmacology and Phytochemistry Institute of Natural Fibres and Medicinal Plants Wojska Polskiego 71b60-630 Poznan Poland

3 Department of Pharmacology University of Medical Sciences Rokietnicka 5a 60-806 Poznan Poland4 Department of Pathogen Genetics and Plant Resistance Metabolomics Team Institute of Plant Genetics ofthe Polish Academy of Science Strzeszynska 34 60-479 Poznan Poland

5 Laboratory of Experimental Pharmacogenetics Department of Clinical Pharmacy and Biopharmacy University of Medical Sciences14 Sw Marii Magdaleny 61-861 Poznan Poland

6 Department of Stem Cells and Regenerative Medicine Institute of Natural Fibres and Medicinal Plants Wojska Polskiego 71b60-630 Poznan Poland

7 Department of Toxicology Poznan University of Medical Sciences Dojazd 30 60-631 Poznan Poland8 Division of Perinatology and Womenrsquos Diseases Poznan University of Medical Sciences Polna 3360-535 Poznan Poland

9 Laboratory of Molecular Biology Poznan University of Medical Sciences Polna 33 60-535 Poznan Poland10Department of General Pharmacology and Pharmacoeconomics Pomeranian Medical University Zolnierska 4870-204 Szczecin Poland

Correspondence should be addressed to Marcin Ozarowski mozarowumpedupl

Received 30 September 2015 Accepted 3 December 2015

Academic Editor Helmut Hugel

Copyright copy 2016 Marcin Ozarowski et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Melissa officinalis (MO English lemon balm Lamiaceae) one of the oldest and still most popular aromatic medicinal plants isused in phytomedicine for the prevention and treatment of nervous disturbances The aim of our study was to assess the effect ofsubchronic (28-fold) administration of a 50 ethanol extract ofMO leaves (200mgkg po) compared with rosmarinic acid (RA10mgkg po) and huperzine A (HU 05mgkg po) on behavioral and cognitive responses in scopolamine-induced rats Theresults were linked with acetylcholinesterase (AChE) butyrylcholinesterase (BuChE) and beta-secretase (BACE-1) mRNA levelsand AChE and BuChE activities in the hippocampus and frontal cortex of rats In our studyMO and HU but not RA showed animprovement in long-term memory The results were in line with mRNA levels since MO produced a decrease of AChE mRNAlevel by 52 in the cortex and caused a strong significant inhibition of BACE1 mRNA transcription (64 in the frontal cortex 50in the hippocampus) However the extract produced only an insignificant inhibition of AChE activity in the frontal cortex Themechanisms ofMO action are probably more complicated since its role as a modulator of beta-secretase activity should be takeninto consideration

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2016 Article ID 9729818 17 pageshttpdxdoiorg10115520169729818

2 Evidence-Based Complementary and Alternative Medicine

1 Introduction

Neurodegenerative disorders including Alzheimerrsquos diseasecharacterized by loss of memory and learning ability arethe increasing public health problem worldwide [1 2] Plantswith neurobiological activity may be potential targets fordrug discovery [3] Searching for new drugs and explainingtheir mechanisms of action are one of the most intensivelydeveloping areas of scientific platformMoreover plant originsubstances can be a valuable alternative way in the preventionand treatment of dementias as component of healthy diet

Melissa officinalis (MO English lemon balm Lamiaceae)one of the oldest and still most popular aromatic medicinalplants is used in phytotherapy for the prevention and treat-ment of nervous disturbances of sleep and gastrointestinaldisorders as sedative and antispasmodic medicine [4] Newneuropharmacological investigations showed that ethanolextracts of MO exerted also neuroprotective [5 6] antiox-idant cyclooxygenase-2 inhibitory [7] and antinociceptiveactivities [6 8] Moreover it is known that MO is used formemory-enhancing effects in European folkmedicine [9ndash12]Indeed Akhondzadeh et al [13] carried out the clinical trialin whichMO extract produced a significantly better outcomeon cognitive function than placebo in patients with mildto moderate Alzheimerrsquos disease In other clinical studiesKennedy et al [14ndash16] observed that a treatment combiningboth calming effects and beneficial cholinergic modulationmay well prove to be a novel treatment for Alzheimerrsquosdisease Studies of molecular mechanisms showed that MOextracts exhibited cholinergic (nicotinic and muscarinic)receptor-binding properties in human cerebral cortex tissue[15] Moreover it was observed that both fractions andcrude ethanol extract of MO inhibited acetylcholinesterase(AChE) of rats brain [17 18] and also in vitro [9 1920] but only two studies analyzed behavioral mechanismof action of MO extracts on scopolamine-induced mem-ory impairment in rats [12 18] One study showed thatMO extract (after intraperitoneal injection) significantlyenhanced learning and memory of rats and significantlyameliorated scopolamine-induced learning deficit [18] how-ever in another study it was observed that MO extract wascompletely inactive [12] Moreover in these studies attentionhas not been paid to the influence of the MO extract onthe expression of genes participating in the conditioningof the synaptic cholinergic equilibrium AChE and bytyryl-cholinesterase (BuChE) or even the beta-secretase (BACE1)in rats brain being responsible for beta-amyloid depositionin Alzheimerrsquos disease [1]

On the other hand it is well known that essential oilin leaf of MO is considered to be the therapeutic principlemainly responsible formost of the abovementioned activitiesbut also plant phenolics are considered as an importantfactor in MO therapeutic effects [6 21] It was shown thatethanol extract contains rosmarinic acid (RA) as the majorcompound [7] however there is no detailed informationabout full phenolic profile of extract from MO leaves beingprobably responsible for pharmacological effects as wellThisbecomes especially important given the results of previous

studies showing that RA did not affect short- and long-termmemory [22] or marginally improved long-term memoryin rats model [23] although it was observed that RA hadan ability to inhibit AChE in the frontal cortex and in thehippocampus of rats [23] On the other hand polyphenolsstill constitute a promising source of new drugs and thereis a high interest in understanding their mechanisms in pre-vention and treatment of Alzheimerrsquos disease [16 23 24]

2 Objectives

The aim of this study was to evaluate the influence of sub-chronic (28-fold) intragastrical administration of ethanolextract of MO leaves and rosmarinic acid on scopolamine(SC) impaired memory in animal model Furthermore ace-tylcholinesterase (AChE) and butyrylcholinesterase (BuChE)activities assessment in hippocampus and frontal cortex werestudied Moreover gene expression levels for AChE BuChEand BACE-1 in the hippocampus and frontal cortex wereinvestigated The MO ethanolic extract was phytochemicallyinvestigated (HPLC-ESI-MSn UPLC-PDA) in order to iden-tify phytochemicals present in plant extract

3 Materials and Methods

31 Plant Material The leaves ofMelissa officinalis L (Lami-aceae) were obtained from an herbal company ldquoKawon-Hurtrdquo(Gostyn Wlkp Poland) The plant material was identifiedin the Department of Pharmaceutical Botany and PlantBiotechnology Faculty of Pharmacy Poznan University ofMedical Sciences The voucher specimen has been depositedin the Herbarium of the Institute of Natural Fibres andMedicinal Plants in Poznan (Plewiska) Poland

32 Chemical and Drugs All reagents for HPLC analysisscopolamine hydrobromide trihydrate (SC) and rosmarinicacid (RA) and reagents for biochemical analyses were pur-chased from Sigma-Aldrich (Poland) Huperzine A (HU)was obtained from Enzo Life Sciences AG (Alexis Corpo-ration Biomibo Distribution Poland) Chemicals for geneexpression analysis were obtained from Roche DiagnosticandALAB (Poland) All chemicals and drugs were ex temporeprepared on the day of the experiment

33 Preparation of the Extract 1000 g of raw plant materialwas extracted with 50 ethanol by percolation (24 h) atroom temperature (22 plusmn 1∘C) After filtration the extract wasconcentrated under vacuum to eliminate the ethanol contentThe concentrated extract was frozen and freeze-dried Thefinal product yielded 24821 g of solid extract

34 Metabolites Identification with LC-MS Metabolomicanalyses were performed using two complementary LC-MSsystems The first one HPLC-DAD-MSn consisted of anAgilent 1100 HPLC instrument with a diode-array detector(DAD) (Agilent Palo Alto CA USA) and an Esquire 3000ion trap mass spectrometer (Bruker Daltonics Bremen Ger-many) Chromatographic separations by HPLC were carried

Evidence-Based Complementary and Alternative Medicine 3

out on an XBridge C18 column (150 times 21mm 35 120583mparticlesize) using water acidified with 01 formic acid (solvent A)and acetonitrile (solvent B) with the mobile phase flow of02mLmin in the following gradient 0ndash25min from 10to 30 B 25ndash46min to 98 B and being maintained atthese conditions until 51min Up to 52min system returnedto the starting conditions and was reequilibrated for 5minThe most important MS parameters were as follows the ionsource ESI voltage minus4 kV or 4 kV nebulization of nitrogenat a pressure of 30 psi at a gas flow rate 9 Lmin ion sourcetemperature at 310∘C and skimmer 1 minus10VThe spectra werescanned in the range of 50ndash3000119898119911 The second systemconsisted of UPLC (the Acquity system Waters MilfordUSA) hyphenated to QExactive hybrid MSMS quadrupole-Orbitrap mass spectrometer Chromatographic separationsin this system were carried out using water acidified with01 formic acid (solvent A) and acetonitrile (solvent B)with the mobile phase flow of 04mLmin in the followinggradient 0ndash5min from 10 to 25 B 5ndash13min to 98 Band being maintained at these conditions until 145min Upto 15min system returned to the starting conditions andwas reequilibrated for 3min QExactive MS operated uponthe following settings the HESI ion source voltage minus3 kVor 3 kV The sheath gas flow was 48 Lmin auxiliary gasflow 13 Lmin ion source capillary temperature 250∘C andauxiliary gas heater temperature 380∘C The CID MSMSexperiments were performed using collision energy of 15 eVThe MSn (up to the MS5) and MSMS spectra were recordedin the negative and positive ion modes using the previouslypublished approach [23 25 26] The individual compoundswere identified via comparison of the exact molecular masses(measured in most cases with Δ below 1 ppm) mass spectraand retention times to those of the standard compoundsas well as the databases available online (PubChem ChEBIMetlin and KNApSAck) and literature data

35 Quantitative HPLC Analysis Sample was extracted by70 ethanol After sonification solution was cooled downand filtered through membrane filter HPLC method wasused to determine RA in a dry ethanolic extract The Lichro-spher 100 RP-18e (125mm 40mm 5 umMerck) was appliedfor identification of this active compound Temperature ofcolumn was 35∘C detection of RA was at 205 nm andflow rate was 15mLmin Mobile phase A was H

3PO4 H2O

(1 999)mobile phase Bwas acetonitrile Timewas as follows0min 10 B 13min 22 B 14min 40 B 25min 40 B

Moreover for identification of other chemical com-pounds a Zorbax Poroshell 120 SB-C18 column 27mm30mm times 100mm (Agilent) was used The lithospermic acidand salvianolic acid B were detected at 250 nm salvianolicacid A was detected at 280 nm The gradient mixtures ofphase Amdashwater H

3PO4(100 002 VV)mdashand of phase Bmdash

acetonitrile tetrahydrofurane (100 2 VV)mdashwere used aseluents Peaks were identified by addition of standard solu-tions and by UV-Vis spectra The quantification of thesecompounds was achieved using calibration curves preparedwith pure compoundsThe flow rate was 07mLmin columntemperature 27∘C and sample injection 5mL The gradient

mixtures program was as follows 0min 5 B 3min 10B 5min 12 B 11min 217 B 15min 39 B 39min 39B 70min 70 B and detection of compounds took place at250 nm 280 nm and 330 nm [27]

36 Determination of Total Phenolic Compounds in theExtract The calculation of polyphenols to gallic acid wasdone using the Folin-Ciocalteu reagent with the spectropho-tometric modified method described by Slinkart and Single-ton [28]

37 Determination of Total Hydroxycinnamic AcidDerivativesDetermination of total hydroxycinnamic acid derivativescalculated on rosmarinic acidwas performed according to theprocedure described in EurPh 50

38 Distillation of Essential Oil The essential oil contentswere determined by way of stream distillation in Deryngrsquosapparatus according to EurPh 50 1000 g of the dry hy-droethanolic MO leaf extract (separate sample) was placedin a round-bottom flask Then 5000mL distilled water and03mL xylenwere added and boiled inDeryngrsquos apparatus for3 h

39 Gas Chromatography Analysis Gas chromatography(GC) analyses were carried out using a Perkin-Elmer Clarus500 gas chromatograph with a data processing system andan FID (GC-FID) Separation was achieved by using an EliteFFAP fused-silica capillary column (30m long 032mm ininternal diameter and 025 120583mof film thickness)The injectorand detector temperatures were 220∘C Helium was used as acarrier gas with a flow of 15mLminminus1 A sample of 10 120583Lwasinjected using slit mode (split ratio 1 100) The results werereported as the relative percentage of the total peak area

310 Animals Experiments with rats were performed inaccordance with Polish governmental regulations (Dz U0533289) The study was conducted in accordance withethical guidelines for investigations in conscious animals andthe study protocol was approved by the Local Ethics Com-mittee of the Use of Laboratory Animals in Poznan Poland(642008) The experiments were performed on male six-week-oldWistar rats housed in controlled room temperature(20 plusmn 02 C) and humidity (65ndash75) under a 12 h 12 h light-dark cycle (lights on at 7 am) Animals were kept in groupsin amounts of 8ndash10 in light plastic cages (60times 40times 40 cm) andhad a free access to standard laboratory diet (pellets-LabofeedB) and to tap water in their cages

311 Treatments The rats were treated with hydroethanolicextract ofMelissa officinalis leaf (MO) in a dose of 200mgkgbw intragastrically (po) (groups MO + H

2O and MO +

SC) for 28 (28x) consecutive days For comparative purposeshuperzine A (HU) was administered chronically (28x) ina dose 05mgkg bw (po) (groups HU + H

2O and HU

+ SC) as a known acetylcholinesterase inhibitor Moreoverrosmarinic acid (Sigma-Aldrich) (RA) was applied (28x) in adose of 10mgkg bw (po) (groups RA + H

2O and RA + SC)

4 Evidence-Based Complementary and Alternative Medicine

as a comparative chemical compound On the last day 30minafter the last dose of MO or HU SC was given intraperi-toneally (ip) in a dose of 05mgkg bw Control groups weretreated with 05 methylcellulose (MC) whereas water forinjection (H

2O) was used as a vehicle for SC (groups MC +

H2O and MC + SC) MO was prepared ex tempore before

administration and suspended in MC in concentrations of20mgmLOnday 28 of the experiment 1 h after the last dosethe animals were killed by decapitation and hippocampus andpart of frontal cortex were collected from brain of rats Thetissue samples were then stored at minus80∘C until measurementof acetylcholinesterase (AChE) and butyrylcholinesterase(BuChE) activities or mRNA level changes

312 Cognitive and Behavioral Tests Cognitive and behav-ioral tests were used in the present study similarly as in ourprevious report [23] (1) sedative activity was assessed usinga locomotor activity test (2) motor coordination assessmentwas done using a ldquochimneyrdquo test (3) the passive avoidance testwas performed as an animalmodel for the assessment of long-termmemory and (4) the object recognition test was used asan animal model for the assessment of short-term memory

3121 Measurement of Locomotor Activity Locomotor activ-ity assessment was performed with licensed activity meter(Activity Cage Ugo Basile Italy) by placing the animals in thecentre of the apparatus and recording their horizontal activity[23] The data obtained were expressed as signals corre-sponding to animalmovements for 5minutesThe locomotoractivity was measured 30 minutes after the administration ofa single dose of SCH

2OAny distracting factors were reduced

to the minimum (noise presence of people and presence ofother rats)

3122Measurement ofMotorCoordination Motor coordina-tion was evaluated using ldquochimneyrdquo test described originallyfor mice [29] Thirty minutes after SC or vehicle injectionrat was allowed to enter a glass laboratory cylinder that is500mm long and 80mm in diameter laid on its side Uponreaching its bottom by the animal position of the cylinderwas rapidly changed from horizontal to vertical and a timerstarted The animal immediately began to move backwardsThe timer was stopped after the rat left the cylinder andassumed a sitting posture on the top of the vessel The timeof exit from the cylinder was accepted as a measure of motorcoordinationMotor impairment was assessed as the inabilityof rats to climb backwards up the tube within 60 s

3123 Passive Avoidance Test Passive avoidance test wasused as an animal model for the assessment of long-termmemory (effects on retrieval and memory consolidation)[30] The test relies on the natural preference of rats for dark-ness After 2 minutes of habituation to a dark compartmenta rat was placed on the illuminated platform and allowedto enter the dark compartment using licensed apparatus(Passive Avoidance System step through Ugo Basile Italy)Two more approach trials were allowed on the following daywith a two-minute interval between them At the end of

the second trial an unavoidable scrambled electric footshock(500120583A AC 3 s) was delivered through the grid floor of thedark compartment (learning trial) Retention of the passiveavoidance response (latency) was tested 24 h later by placingthe animal on the platformandmeasuring the latency in reen-tering the dark compartment against the arbitrary maximumtime of 180 s The test was performed after 30 minutes afterthe administration of a single dose of SC or the vehicle

3124 Object Recognition Test Object recognition test wasused as an animal model for the assessment of short-termmemory [31]The object recognition task took place in a 40 times60 cm open box surrounded by 40 cm high walls made ofplywood with a frontal glass wall All animals were submittedto a habituation session where they were allowed to freelyexplore the open field for 5min No objects were placed inthe box during the habituation trial On the day of testing theanimals were given an additional 3min rehabituation periodprior to commencing the test The test was divided into threephases with two trials the acquisition trial the retention trialand an intertrial interval of varying times

(i) Acquisition trial in this first trial the animals ex-plored two identical objects (1198601 and 1198602) for a periodof 3min positioned in two adjacent corners 10 cmfrom the walls

(ii) Intertrial interval (ITI) the animals were returned tothe home cage for 30min

(iii) Retention trial in this second trial the animalsexplored a familiar object (119860lowast) that is a duplicate ofthose objects from the acquisition trial (to minimizeolfactory cues) and a novel object (119861) for a further3min

They were made of a biologically inert substance (plastic)and were chosen to enable ease of cleaning (10 alcohol)between subjects in an attempt to remove olfactory cuesObject exploration is defined by animals licking sniffing ortouching the object whilst sniffing but not leaning againstturning round or standing or sitting on the object Objectswere of sufficient weight and were secured to the floor of thearena to ensure that they could not be knocked over ormovedaround by the animal The exploration time (s) of all objectswas recorded via stopwatch for subsequent statistical analysisThe time measured as an exploration behavior was used tocalculate amemory discrimination index (OR) as reported byBlalock et al [31]OR = (119861minus119860lowast)(119861+119860) where119861was the timespent exploring the new object and 119860lowast was the time spentexploring the familiar object Higher OR was considered toreflect greater memory ability [31] The test was performedafter 30 minutes after the administration of a single dose ofSC or the vehicle

313 Acetylcholinesterase and Butyrylcholinesterase ActivitiesAssay in Brain of Rats Acetylcholinesterase (AChE) andbutyrylcholinesterase (BuChE) activities were performed bymodifying spectrophotometric Ellmanrsquos method accordingto Isomae et al [32] The activities of AChE and BuChE

Evidence-Based Complementary and Alternative Medicine 5

028

021

014

007

000

200100 300 400 500 600 700 800 900(Minutes)

(AU

)

1 23

4

56 7 8 9 10

1112

1314

1617

19

15 1820

2122

23

2425

2627

28

2930

31

37

3234

3536 38 39

40

33

Figure 1 Chromatogram UV ofMelissa officinalis leaf extract obtained at 270 nm with peaks identified by HPLC-UV-MS

were determined by measuring the formation of the yellowanion obtained from the reaction between Ellmanrsquos reagentand the thiocholine generated by the enzymatic hydrolysisof acetylthiocholine iodide (ATCh) and butyrylthiocholine(BTCh) respectively (sample 01mL PBS 08mL DTNB01mL ATCh 020mL and BTCh 020mL)The biochemicalassay of AChE and BuChE in homogenate of brain sampleswas expressed as 120583molminmg protein by using spectropho-tometric method (120582 = 412 nm)

314 RNA Isolation and Reverse Transcription Reaction TotalRNA isolation from the rats brain tissues homogenates(frontal cortex hippocampus) was carried out using TriPureIsolation Reagent (Roche) according to manufacturerrsquos pro-tocol The integrity of RNA was visually assessed by a con-ventional agarose gel electrophoresis and the concentrationwill be evaluated by measuring the absorbance at 260 and280 nm in a spectrophotometer (BioPhotometer Eppendorf)RNA samples were stored at minus80∘C until use The 1 120583gof total RNA from all samples was used for the reversetranscription into cDNA using Transcriptor First StrandSynthesis Kit (Roche) according to manufacturerrsquos protocolObtained cDNA samples was stored at minus20∘C or used directlyfor the quantitative real-time PCR (qRT-PCR)

315 Real-Time PCR mRNA Quantification The acetylcho-linesterase (AChE) butyrylcholinesterase (BChE) and beta-secretase (BACE1) genes expression level was analyzed bytwo-step quantitative real-time PCR (qRT-PCR) in a vol-ume of 10 120583L reaction mixture using relative quantificationmethodology with a LightCycler TM Instrument (RocheGermany) and a LightCycler Fast Start DNA Master SYBRGreen I kit (Roche Applied Science) according to theinstructions of the manufacturer All primers sequences weredesigned and custom-designed using the Oligo 60 software(National Biosciences) and were verified by assessment ofa single PCR product on agarose gel and by a single tem-perature dissociation peak (melting curve analysis) of eachcDNA amplification product An GAPDH gene was usedas a housekeeping gene (endogenous internal standard) for

normalization of qPCR For each quantified gene standardcurves were prepared from dilution of cDNA and gener-ated from a minimum of four data points All quantitativePCR were repeated twice The data were evaluated usingLightCycler Run 45 software (Roche Applied Science) EachPCR run included a nontemplate control to detect potentialcontamination of reagents

316 Statistical Analysis All values were expressed as meansplusmn SEM The statistical comparison of results was carried outusing one-way analysis of variance (ANOVA) followed byDuncanrsquos post hoc test for detailed data analysisThe values of119901 lt 005were considered as a statistical significant difference

4 Results

41 Phytochemical Profile of Extract

411 Identification ofMetabolites Forty phenolicmetaboliteswere identified in hydroethanolic Melissa officinalis leafextract (Table 1 Figure 1) The predominant identified com-pounds were bioactive caffeic acid esters and glycosides offlavones The caffeic acid dimer rosmarinic acid (metabolite23) and caffeate trimer (lithospermic acid 31) were the prin-cipal caffeic acid derivatives in the analyzed samples Hydrox-yjasmonic acid and its derivatives teucrol as well as sage-coumarin were identified for the first time in the genusMelissa while luteolin and apigenin glycoconjugates arewell known phytochemicals in this species [33] Multistepfragmentation with accurate mass measurement enabledconfirming that the losses of fragment 799573 amu from the[M-H]minus ions of compounds 4 9 27 30 and 32 referredto sulphate groups The first-order fragmentation of 30revealed the loss of 799573 and yielded the product ion at7191622119898119911 The following fragmentation of this ion cor-responded to that of the sagerinic acid (dimer of rosmarinicacid) described by Barros et al [34] The exact placementof the sulphation position would require in-depth chemicalanalysis Thus 30 was tentatively assigned as sulphated sag-erinic acid

6 Evidence-Based Complementary and Alternative MedicineTa

ble1Metabolitesd

etectedin

Melissa

officin

alisleafextractb

yUPL

C-MS

No

RT [min]

Metabolite

identifi

catio

nCh

emical

form

ula

Exactm

asso

f[M-H

]minusΔpp

mFragmentatio

nin

120582max[nm]

CID

aIdentifi

catio

nlevelb

Reference

Measured

Calculated

Negativeion

mod

e(ESIminus)

Positiveion

mod

e(ESI+)

114

1

2-Hydroxy-3-(34-

dihydroxyphenyl)-

prop

anoic

acid

C 9H9

O5

1970

451970

455minus41775

197179135

199163

283312

8143997

2[34]

217

3Dihydroxybenzoica

cid

hexosid

eC 13

H15

O9

315072

3150722

08346

315153109

317155

282

54726828

3[35]

319

8Ca

ftaric

acid

C 13

H11

O9

3110

413110

409

06141

311221179149

318

6440397

2[35]

4224

2-Hydroxy-3-(34-

dihydroxyphenyl)-

prop

anoica

cid

sulphated

C 9H9

O8

S2770

032770

024

03998

277197179135

312

3[35]

5256

Hydroxyjasm

onicacid

hexosid

eC 18

H27

O9

387166

3871661

09214

387207163

323

44237366

2[17]

6276

Caffeicacid

C 9H7

O4

1790

341790

451minus49075

179135

181163

275

689043

1std

7289

Salviano

licacid

EC 36

H29

O16

7171450

717146

72313

717519339321

295277

275325

49770697

2[35]

8317

Salviano

licacid

HI

(isom

er)

C 27

H21

O12

537104

5371038

08562

537493359295

281325

2[35]

9345

Hydroxyjasm

onicacid

sulphated

C 12

H17

O7

S30507

30507

1117

305225194147

275330

3[17]

10362

NepetoidinB

C 17

H13

O6

313072

3130718

11232

nd282316

5316819

2[73]

1137

Yunn

aneica

cidF

C 26

H25

O14

5971255

5971244

18743

597509311197

Masked

2[34]

12383

Decarbo

xyrosm

arinicacid

(teucrol)

C 17

H15

O6

315088

3150874

09396

315179135

275330

637829

2[74]

1339

Caffeoylcaft

aricacid

C 22

H17

O12

473073

4730725

07555

173311149

Masked

65018

3[35]

14397

Apigenin

glucosylrham

nosid

eC 27

H29

O14

577157

5771563

11136

577269

579433271

275340

92741003

3[33]

15404

Luteolin

7-O-glucosid

e31015840-O

-glucuronide

C 27

H27

O17

623126

6231254

09696

62346144

7285

255

625463287

273343

2[33]

16421

Rosm

arinicacid

hexosid

eC 24

H25

O13

52113

5211301

03551

521359161

523361325163

329

25245848

2[34]

1743

Luteolin

O-diglucosid

eC 27

H29

O16

609147

6091461

07183

609285

611287

269349

3[33]

18441

Luteolin

glucosylrham

nosid

eC 27

H29

O15

593152

5931512

08077

59344

7285

59544

9287

271343

3[33]

19449

Luteolin

41015840-O

-glucosid

eC 21

H19

O11

4470

9444

70933

09728

447285

449287

271343

5319116

3[33]

2045

Sagerin

icacid

2-hydroxy-3-

(34-dihydroxyph

enyl)-

prop

anoide

C 45

H39

O20

8992

058992

0410

448

899719591475

295

Masked

3[35]

2149

Salviano

licacid

B(lithosperm

icacid

B)C 36

H29

O16

717146

7171461

01846

717519359161

327

6441188

2[34]

22504

Sagerin

icacid

C 36

H31

O16

7191

637191

618

09979

719519359161

287330

2[34]

23514

Rosm

arinicacid

C 18

H15

O8

3590

773590

772

0002

359161

361163

329

5281792

1std

Evidence-Based Complementary and Alternative Medicine 7

Table1Con

tinued

No

RT [min]

Metabolite

identifi

catio

nCh

emical

form

ula

Exactm

asso

f[M-H

]minusΔpp

mFragmentatio

nin

120582max[nm]

CID

aIdentifi

catio

nlevelb

Reference

Measured

Calculated

Negativeion

mod

e(ESIminus)

Positiveion

mod

e(ESI+)

24555

Salviano

licacid

B2-hydroxy-3-(34-

dihydroxyphenyl)-

prop

anoide

C 45

H37

O20

89719

8971884

16873

897717519359

161

330

3[35]

2556

Sagerin

icacid

di-2-hydroxy-3-(34-

dihydroxyphenyl)-

prop

anoide

C 54

H47

O24

1079244

910792457minus07911

1079897719539

359295

288330

3[35]

26592

Sagecoum

arin

di-2-hydroxy-3-(34-

dihydroxyphenyl)-

prop

anoide

caffeide

C 54

H43

O24

10752156

10752150

0559

1077897717537

409359339277

322

3[35]

27598

Rosm

arinicacid

sulphated

Iisomer

C 18

H15

O11

S4390

354390

341

20214

439359341163

Masked

2[34]

28613

Luteolin

31015840-O

-glucuronide

C 21

H17

O12

4610

734610

7216

34461285

463287

269340

170474237

2[33]

29632

Salviano

licacid

AC 26

H21

O10

493115

493114

11011

493359295179

298327

5281793

[34]

30656

Sagerin

icacid

sulphated

C 36

H31

O19

S7991

196

7991

186

0954

799719619519

359161

325

3[35]

31671

Lithosperm

icacid

C 27

H21

O12

537104

537104

809698

537493359161

292329

6441498

2[35]

3268

Rosm

arinicacid

sulphated

IIiso

mer

C 18

H15

O11

S4390

344390

341

02837

439359341163

Masked

2[34]

33689

Sagecoum

arin

C 27

H19

O12

535088

5350882

01204

535311267177

Masked

2[36]

3470

3Salviano

licacid

LIisomer

C 36

H29

O16

717146

7171461

02697

717519359

284329

2[35]

3571

Salviano

licacid

Lhydroxycaffeide

C 45

H35

O20

895173

8951727

06282

895519359161

Masked

3[35]

3674

Sagecoum

arin

cafta

ride

C 40

H29

O20

8291

268291

258

06953

829667535355

311

Masked

3[36]

37751

Sagecoum

arin

2-hydroxy-

3-(34-dihydroxyph

enyl)-

prop

anoide

C 36

H27

O16

715131

7151305

08176

715535311267

319

3[34]

3877

1Unk

nown

C 36

H57

O14

S745348

7453475

044

02281326

4[35]

3978

5Methylrosmarinate

C 19

H17

O8

373093

3730929

01765

373359161

284323

6479915

2[75]

40869

Salviano

licacid

Ccaffeoylhydroxycaffeide

C 44

H33

O18

8491

688491

672

08615

849687491359

327255

286318

3[35]

a CID

identifier

fora

chem

icalstr

ucture

intheP

ubCh

emCom

poun

ddatabase

b Metabolite

identifi

catio

nlevelaccording

toMetabolom

icsS

tand

ards

Initiativer

ecom

mendatio

n[76]

stdidentificatio

non

theb

asisof

stand

ardcompo

undfragmentatio

nnd

not

detected

8 Evidence-Based Complementary and Alternative Medicine

100 200 300 400 500 600 700 800 900 1000 1100 1200

mz

70

75

80

85

90

95

100

minus198 (2-hydroxy-3-(34-dihydroxyphenyl)

-porpanoide)

minus180 (caffeide)

minus180 (2-hydroxy-3-(34-dihydroxyphenyl)

-propanoide)

minus180 (2-hydroxy-3-(34-dihydroxyphenyl)

-propanoide)

minus180 (2-hydroxy-3-(34-dihydroxyphenyl)

-propanoide)

1350438

2191749

HO

HO

HO HO

OH

OH

OH

OH OH OH

OHOHOHOHHOOC

COOH

COOH COOH COOH

24612ppmC36H55O37 = 10792422

minus07911ppmC54H47O24 = 10792457

10792449

10448ppmC45H39O20 = 8992040

899205018453ppm

C36H31O16 = 7191612

7191625

20501ppmC27H23O12 = 5391190

5391201

14852ppmC23H33O10 = 4692074

469208119316ppm

C17H11O5 = 2950606

2950612

0

5

10

15

20

25

30

35

40

45

50

55

60

65

Rela

tive a

bund

ance

22903ppmC18H15O8 = 3590767

3590775

minus37218 ppmC9H5O3 = 1610239

1610233

(a)

100 200 300 400 500 600 700 800 900 1000

mz

100 200 300 400 500 600 700 800 900 1000

mz

100 200 300 400 500 600 700 800 900 1000

mz

100 200 300 400 500 600 700 800 900 1000

mz

0

250

500

750

1000

00

04

08

times104

times104

01234

0

1

2

times105

Ints

Ints

Ints

Ints

MS1

MS2

MS3

MS4

[M-H]minus10749

minus224 (acidic part

minus224 (acidic part

of sagecoumarin)

of sagecoumarin)

minus360 (rosmarinic acid)

4908

7148

5348 minus180 minus162 (caffeide)8769

31074909

5348

minus180 (2-hydroxy-3-(34-dihydroxyphenyl)-propanoide)

3108

35474909

1

(b)

Figure 2 Continued

Evidence-Based Complementary and Alternative Medicine 9

150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900

mz

0

5

10

15

20

25

30

35

40

45

50

55

60

65

Rela

tive a

bund

ance

O

O

O O

O

O

OH OH

OH

OH

OH

OHOH

OH

HO

HO

HO

COOH

OC

1529946

1970447

C9H9O5 = 1970455

minus41775ppm1610232

2552328

C16H31O2 = 2552330

minus04091ppm

3110563

C17H11O6 = 3110561

05243ppm

minus224 (fragment of sagecoumarin)

3912257

4092361

4832730

C29H39O6 = 4832752

minus44931ppm

5350885

C27H19O12 = 5350882

05640ppm

minus132 (tartaric acid)

5770990

C29H21O13 = 5770988

06486ppm

6670947

C31H23O17 = 6670941

09808ppm

minus162 (caffeic acid)

8291267

C40H29O20 = 8291258

11370ppm

(c)

Figure 2 (a) Mass spectra in negative ionization mode and simplified fragmentation scheme of compound 25 (pentameric ester ofcaffeic acid) (b) Mass spectra in negative ionization mode and simplified fragmentation scheme of compound 26 (pentameric structureof sagecoumarin di-2-hydroxy-3-(34-dihydroxyphenyl)-propanoide caffeide) (c) Mass spectra in negative ionization mode and simplifiedfragmentation scheme of compound 36 (pentameric structure of sagecoumarin caftaride)

Tetrameric structures of hydroxycinnamic acids wereidentified in lemon balm recently [34 35] The measure-ment of accurate masses allowed the identification of com-pound 25 which was tentatively identified as pentamericester of caffeic acid (Figure 2(a)) In 25 the double loss of1800421 amu corresponded to fragments with the molecularformula of C

9H8O4 adequate to dehydroxylated 2-hydroxy-

3-(34-dihydroxyphenyl)-propanoic acid The product ionat 7191623119898119911 and its further fragmentation are sim-ilar to those of metabolite 22 as described previously [34 35]Therefore metabolite 25was tentatively assigned as sagerinicacid di-2-hydroxy-3-(34-dihydroxyphenyl)-propanoideNev-ertheless comprehensive studies by nuclear magnetic reso-nance are required to complete elucidation of substitutionpattern for particular components of those pentamers

Sagecoumarins were previously identified in Salvia offic-inalis as caffeic acid trimers [36] Our MS analysis indicatedthe presence of such compounds and their derivatives alsoin M officinalis The pseudomolecular ion of compound33 observed in the negative ionization mode had theaccurate mass of 5350880119898119911 which corresponded to thechemical formula C

27H19O12

adequate for sagecoumarin(according to the Metlin and KNApSAck databases) Note-worthy 26 33 36 and 37 had the same fragmentationpattern of the product ion obtained in the MSMS andMSn in the negative ionization mode The [M-H-1800422]minusion corresponded to the detachment of 2-hydroxy-3-(34-dihydroxyphenyl)-propanoic acid thus 37 was tentatively

assigned as tetrameric sagecoumarin 2-hydroxy-3-(34-dihy-droxyphenyl)-propanoide The [M-H-3600846]minus ion in 26was indicated on rosmarinic acid substitution to 37 Massspectra of MSn in negative ionization of the compoundprovided complementary information to HR-MSMS massspectra indicated on caffeic acid as internal componentof the dimer In addition simultaneous loss of fragments180 amu and 224 amu in MS3 and MS4 indicated that thetwo components cannot be linked (Figure 2(b)) How-ever detailed analysis of substitution pattern of 26 shouldbe done Therefore 26 was tentatively assigned as penta-meric structure of sagecoumarin di-2-hydroxy-3-(34-dihy-droxyphenyl)-propanoide caffeide Rupture of caffeic andtartaric acid moieties from the product ion at 5350885119898119911was observed for compound36 (Figure 2(c))Detection of theaccurate masses of these two detached fragments with Δ lessthan 1 ppm eliminated the possibility of hexose and pentosesubstitution which have the same nominal masses as caffeicand tartaric acid respectively Therefore 36 was tentativelyidentified as another pentameric structure of sagecoumarincaftaride

Metabolite 28 with the accurate masses of 461073119898119911was tentatively identified as luteolin O-glucuronide The [M-H-1760324]minus ion is indicated on loss of structure C

6H8O6

corresponding to glucuronide moiety The product ion at2850405119898119911 was indicated on flavone luteolin The placeof substitution of the carboxylic acid on flavone skeleton isproblematic due to different isomers reported in lemon balm

10 Evidence-Based Complementary and Alternative Medicine

Table 2 Effect ofMelissa officinalis leaf extract (200mgkg po) treatment on sedative activity motor coordination and memory in rats

Group 119899Locomotor activity

[number of impulses5min]Motor coordination

exit time [s]Short-term memorye

ORLong-term memory

latency [s]MC + H

2

O 18 390 plusmn 24 17 plusmn 3 040 plusmn 006 47 plusmn 14MC + SC 18 526 plusmn 48lowast 32 plusmn 5lowast 032 plusmn 005 12 plusmn 3lowast

MO + H2

O 10 231 plusmn 48lowast 32 plusmn 7lowast 043 plusmn 007 169 plusmn 11lowast

MO + SC 9 436 plusmn 60 43 plusmn 7 009 plusmn 011lowast 23 plusmn 6HU + H

2

O 9 515 plusmn 32 15 plusmn 2 037 plusmn 009 158 plusmn 14lowast

HU + SC 8 639 plusmn 71lowast 56 plusmn 3 022 plusmn 006 49 plusmn 18

RA + H2

O 8 406 plusmn 59 21 plusmn 5 045 plusmn 005 58 plusmn 28RA + SC 8 605 plusmn 55 28 plusmn 7 045 plusmn 005 20 plusmn 7Means plusmn SEM119899Number of animalsMC + H

2O control rats

SC scopolamine (05mgkg bw ip)HU huperzine A (05mgkg bw po)RA rosmarinic acid (10mgkg bw po)eExpressed as ratio OR = (119861 minus 119860lowast)(119861 + 119860) for details see Section 3lowastVersus MC + H

2O 119901 lt 005

Versus MC + SC 119901 lt 005

luteolin 31015840-O- and 7-O-glucuronide [33] It is impossible todistinguish both structures by mass spectrometry Only onechromatographic peak corresponding to the [M-H]minus ion at461073119898119911 was observed in our study Since luteolin 31015840-O-glucuronide was assigned as the most abundant flavonoid inlemon balm [33] we assumed that 28 corresponded to thisstructure

412 Flavonoids and Polyphenolic Acids The major com-pound from the 40 chemical compounds identified in hy-droethanolic MO leaf extract established by HPLC was RA(885 g100 g) (Table 1 Figure 1)

Other chemical compoundswith neuromodulatory activ-ities such as lithospermic acid (0042 g100 g) salvianolicacid A (0040 g100 g) salvianolic acid B (0023 g100 g) andcaffeic acid (0087 g100 g) were also documented in litera-ture Moreover the total polyphenolic compounds content ofMO determined with the use of FolinndashCiocalteu assay was3397 calculated as gallic acid The total hydroxycinnamicderivatives content expressed spectrophotometrically as ros-marinic acid was 2115 g100 g

413 Essential Oil Composition Hydroethanolic MO leafextract contained 008 of total essential oil The GCFIDanalysis showed that the extract comprised camphene(004) alfa-pinene (007) beta-pinene (1647) andmyrcene (1951) Moreover according to retention time16 compounds were identified as follows alfa-bisabololborneol carvone chamazulene cineole eugenol gamma-terpineol guaiazulene isopulegol linalool limonene men-thol menthyl acetate pulegone terpine and thymol Thesecompounds are present in the essential oil in trace amountswhich do not allow the quantitative interpretation

42 Cognitive and Behavioral Experiments

421 Locomotor Activity A one-way ANOVA analysisrevealed significant differences in the locomotor activity of

rats expressed as their horizontal spontaneous activity afterMO administration (ANOVA 119865(7 80) = 646 119901 lt 005)(Table 2) Detailed post hoc analysis showed thatMO + H

2O

decreased the locomotor activity of rats by 4031 but HU +H2O did not affect this activity when compared with control

group (MC + H2O) We observed also that RA + H

2O did

not change the locomotor activity of rats Stimulating effectsin the locomotor activity of rats were observed after an acuteSC injection (MC + SC versus MC + H

2O 119901 lt 005) and

this effect was observed in all SC-treated rats when comparedwith the proper non-SC-treated animals On the contrarythese SC-treated animals did not differ in comparison toanimals that received SC only (MO + SC versus MC + SC119901 gt 005 HU + SC versus MC + SC 119901 gt 005 RA + SCversus MC + SC 119901 gt 005)

422 Motor Coordination A one-way ANOVA analysisrevealed significant differences in motor coordination of ratsexpressed as their exit time from the cylinder (119865(5 73) =284 119901 lt 005) (Table 2) Detailed analysis showed that themultiple administration of RA + H

2O and HU + H

2O did

not affect significantly this paradigm when compared withcontrol rats (119901 gt 005) whereas MO treatment led to pro-longed exit time (MO + H

2O versus MC + H

2O 119901 lt 005)

Moreover generally SC-treated animals showed producedprolongation of exit time and the effects were statisticallysignificant not only in control groups (MC + SC versus MC +H2O 119901 lt 005) but also in RA-treated rats (RA + SC versus

MC + SC 119901 lt 005) However the rest of the SC-treatedanimals did not differ in comparison to animals receiving SConly (MO + SC versus MC + SC 119901 gt 005 HU + SC versusMC + SC 119901 gt 005)

423 Long-Term Memory A one-way ANOVA analysisrevealed significant differences in long-term memory afterusing a passive avoidance test (119865(7 77) = 201 119901 lt 005Table 2) It was shown that the strongest effect leading to

Evidence-Based Complementary and Alternative Medicine 11

Table 3 The effect ofMelissa officinalis leaf extract on acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) activities and AChEBuAChE or beta-secretase (BACE1) mRNA expression levels in frontal cortex (FC) or hippocampus (Hipp) of rats

Group119899Enzyme activity

[nmolminmg protein]mRNA expression

[]AChE BuChE ACHE BuChE BACE1

FC Hipp FC Hipp FC Hipp FC Hipp FC HippMC + H

2

O 363 plusmn 49 439 plusmn 73 65 plusmn 11 53 plusmn 8 100 plusmn 12 100 plusmn 11 100 plusmn 18 100 plusmn 11 100 plusmn 16 100 plusmn 8MO + H

2

O 276 plusmn 34 409 plusmn 28 69 plusmn 6 62 plusmn 4 48 plusmn 4lowast 31 plusmn 7lowast 16 plusmn 2lowast 64 plusmn 21amp 36 plusmn 3lowast 50 plusmn 8lowast

HU + H2

O 189 plusmn 15lowast 239 plusmn 15lowast 58 plusmn 6 51 plusmn 4 53 plusmn 13lowast 85 plusmn 5 42 plusmn 9lowast 102 plusmn 11 62 plusmn 6lowast 98 plusmn 4RA + H

2

O 224 plusmn 16lowast 251 plusmn 12lowast 77 plusmn 7 99 plusmn 7lowast 101 plusmn 12 103 plusmn 5 184 plusmn 31lowast 56 plusmn 7lowast 126 plusmn 13amp 98 plusmn 3Means plusmn SEM119899Number of animals 7ndash10Values expressed as a ratio the geneGAPDHMC + H

2O control group

HU huperzine A (05mgkg bw po)RA rosmarinic acid (10mgkg bw po)lowastampVersus MC + H

2O 119901 lt 005 or 119901 lt 007 respectively

an improvement of this paradigm was produced by extractof MO and HU but not RA when compared with controlanimals (MO + H

2O versus MC + H

2O 119901 lt 005 HU +

H2O versus MC + H

2O 119901 lt 005 RA + H

2O versus MC +

H2O 119901 gt 005) However the administration of SC to rats

significantly decreased the latency time of passive avoidancetask (MC + SC versus MC + H

2O 119901 lt 005) After MO or

RA combined treatment with SC no improvement of long-term memory was observed but HU given with SC showedenhancement of this paradigm in rats (HU + SC versusMC + SC 119901 lt 005) Therefore it can be concluded thatadministration of HU overcomes the effect shown by SC only(Table 1)

424 Short-Term Memory The results of the object recogni-tion test showed that an administration of the compoundsor extract did affect the ratsrsquo short-term memory (ANOVA119865(7 78) = 273 119901 lt 005) (Table 2)

Detailed post hoc analysis showed that only SC signifi-cantly decreased the short-term memory in MO-treated rats(MO + SC versusMO + H

2O 119901 lt 005MO + SC versus MC

+ SC 119901 lt 005) whereas the differences between the rest ofthe animals did not reach statistical significance (119901 gt 005)

43 AChE and BuChE Activities in Rat Brain A one-wayANOVA revealed significant differences between groups inthe activity of AChE in both the cortex and the hippocampus(frontal cortex 119865(3 27) = 565 119901 lt 005 hippocampusANOVA 119865(3 29) = 796 119901 lt 005) It was found out thatMOshowed an insignificant inhibition of AChE activity in thefrontal cortex by 24 when compared with control rats (MC+ H2O 119901 lt 006) and in the hippocampus by 7 (Table 3)

whereas HU produced a distinct significant inhibition ofAChE activity in comparison to control group by 48 (119901 lt005) and 47 (119901 lt 005) in the cortex and the hippocampusrespectively Also RA lowered significantly AChE activityboth in the cortex (38) and in the hippocampus (43)Moreover there were not significant differences between thevalues of BuChE activities for MO and HU when compared

with control group in the frontal cortex (ANOVA 119865(3 30) =101 119901 gt 005) whereas in the hippocampus the differencesreached statistical significance (ANOVA 119865(3 27) = 141119901 lt 005) Detailed analysis showed that only RA effect wassignificant and increased BuChE activity in the hippocampuswhen compared with the control rats (119901 lt 005)

44 AChE BuChE and BACE1 mRNA Level Changes in RatBrain A one-way ANOVA analysis revealed significant dif-ferences of AChE mRNA transcription profile in the cortex(ANOVA 119865(3 27) = 857 119901 lt 005) As shown in Table 3the multiple treatment of MO produced in the cortex astatistically significant decrease of AChEmRNA level by 52the administration of HU caused decrease of its level by 44(119901 lt 005) whereas RA did not affect this parameter whencompared to the control

There were significant differences between the relativevalues of BuChE mRNA levels in this region of brain in rats(frontal cortex ANOVA 119865(3 25) = 162 119901 lt 005) TheMOtreatment led to a decrease in the BuChEmRNA level by 84(versus MC + H

2O 119901 lt 005) the prolonged HU admin-

istration resulted in a decrease of the transcript level in thecortex by 58 (versus MC + H

2O 119901 lt 005) but RA

increased this parameter in the cortex by 84 (versus MC +H2O 119901 lt 005)The significant differences ofmRNA transcription level of

AChE mRNA level in the hippocampus (ANOVA 119865(3 31) =212 119901 lt 005) have been observed The detailed analysisshown that in the case of AChE afterMO treatment mRNAlevel significantly decreased by 69 (versus MC + H

2O

119901 lt 005) while the administration of HU resulted in astatistically insignificant decrease of AChE mRNA level by18 when compared with control group Also RA did notchange the level of transcript

In the case of BuChEmRNA expression in the hippocam-pus there were statistically significant differences betweengroups (ANOVA 119865(3 27) = 310 119901 lt 005) Detailed analysisshowed that in theMO + H

2O treated group the expression

lowered by 36 but the difference did not reach strong

12 Evidence-Based Complementary and Alternative Medicine

significance when compared with the control values (119901 lt007) and no change in the expression level of this enzymewas shown after the administration of HU On the contraryRA produced an increase of BuChE mRNA expression in thehippocampus by 44 (versus MC + H

2O 119901 lt 005)

Further analysis showed the significant differences inBACE1 mRNA expression in both brain regions of rats (inthe cortex and the hippocampus) (frontal cortex ANOVA119865(3 27) = 163 119901 lt 005 hippocampus ANOVA 119865(3 27) =133 119901 gt 005) It was observed that MO produced astatistically significant decrease of the BACE1 expression levelby 64 in the cortex (versus MC + H

2O 119901 lt 005) and by

50 in the hippocampus (versus MC + H2O 119901 lt 005) For

comparison HU treatment led to a decrease in the mRNAexpression level by 38 in the cortex (versus MC + H

2O

119901 lt 005) but not in the hippocampus On the contrary RAproduced an increase of this transcript in the cortex by 26but the effect did not reach a strong statistical significance(versus MC + H

2O 119901 lt 007) whereas in the hippocampus

there was no difference between RA and control group

5 Discussion

Cognitive and Behavioral Experiments The present studyinvestigated the influence of subchronic (28-fold) adminis-tration of standardized 50 EtOH extract ofMelisa officinalisleaf extract (MO) (200mgkg po containing 177mgkg ofRA) on SC-induced impairment of short-term and long-term memory in rats The results were compared with theactivity of cholinesterases (AChE and BuChE) as well aswith AChE BuChE and BACE1 gene expression levels in thecortex and hippocampus of the rat brain So far little evidenceis yet available as regards mechanisms of MO leaf extractaction that are potentially relevant to cognitive function ofrats after per os administration MO is traditionally used intreating neurological disorders through its anti-AChE [18]and antiagitation properties [37] Moreover Wake et al [38]and Kennedy et al [15] showed thatMO extract has nicotinicreceptor activity and that it can displace [3H]-(N)-nicotinefrom nicotinic receptors in homogenates of human cerebralcortex tissue and they suggested that these mechanisms canexplain activity of MO extract in amnesia model RecentlySoodi et al [18] observed that intraperitoneal injections ofMO extract (200mgkg) in rats could significantly enhancelearning and memory processes in animals since the extractsignificantly ameliorates SC-induced learning deficit in Mor-ris water maze test On the contrary in higher dose itcan be observed that MO extract (400mgkg) could notreverse SC-induced memory impairment [18] In our studyadministration of MO extract at a dose of 200mgkg (po)showed the effect leading to improvement of long-termmemory in a passive avoidance test in rats However afterMO combined treatment with SCMO did not overcome theimpairment shown by SC (Table 2) It should be emphasizedthat it is not clear whether the effect shown in our study byMO in non-SC-treated rats is specific since theMO treatmentproduced significant lowering of locomotor activity of ratstherefore the sedative profile ofMO cannot be excluded

On the other hand Ryu et al [39] observed that agitationand aggression are highly prevalent in patientswith dementiaAccording to Gitlin et al [40] nonpharmacological inter-ventions are recommended as first-line therapy Althoughantipsychotics have shown benefit for Alzheimerrsquos disease-related psychosis their use is associated with several seriousadverse effects [40]Thus it seems that the use ofMO extractcan provide dual benefits both in aspect of inhibition ofagitation and in improving the memory of patients withAlzheimerrsquos disease

Furthermore in our study RA in the dose of 10mgkgbw (po) did not affect either short- or long-term memoryalthough RA lowered significantly AChE activity both inthe cortex and in the hippocampus Moreover we observedthat the repeated administration of RA in non-scopolamine-treated rats did not produce any changes of locomotoractivity similarly to our previous study [23]

It is possible that other chemical compounds can influ-ence the memory in rats by synergic interactions in plantextract According to our calculations caffeic acid (0174mgin a single dose of extract administered to animals per kgbw) lithospermic acid (0084mgkg) salvianolic acid A(008mgkg) and salvianolic acid B (0046mgkg) may beresponsible for observed pharmacological effects On theother hand results from few studies showed that salvianolicacids do not cross the blood brain barrier (BBB) [41 42]and also lithospermic acid does not efficiently cross the BBB[43] For this reason the interpretation of our results is morecomplicated

Firstly Xu et al [44] demonstrated that Sal A is ametabol-ically unstable compound that would undergo rapid methy-lationmetabolism catalyzed by catechol O-methyltransferasein vivo into four major methylated metabolites of Sal A(3-O-methyl 31015840-O-methyl 3310158401015840-O-dimethyl and 31015840310158401015840-O-dimethyl salvianolic acid A) These generated O-methylatedmetabolites may be largely responsible for its in vivo phar-macological effects Although there are no available recentstudies on the ability of these compounds to pass the BBBsuch possibility should not be excluded

Secondly several studies showed central pharmacolog-ical effects in animals after per os administration of Sal B[45 46] Sal A [47] and caffeic acid [48] It was provedfor example that salvianolic acid B (10mgkg po) sig-nificantly rescued the A12057325ndash35 peptide-induced decreaseof choline acetyltransferase and brain-derived neurotrophicfactor protein levels in an amyloid 120573 (A120573) peptide-inducedAlzheimerrsquos disease mouse model [49] It also significantlyreversed (10mgkg po) the cognitive impairments inducedby scopolamine (1mgkg ip) or A120573 (25ndash35) (10 nmol5 120583Licv) injection inmice [45] Previous studies [46 47] showedalso that both Sal A and Sal B are able to improve theimpaired memory function induced by cerebral ischemia-reperfusion in miceThe stimulation of neurogenesis processin both subgranular zone (SGZ) and subventricular zone(SVZ) after brain ischemia and also alleviation neural cellsloss and improved motor function recovery after brainischemia in rats after the Sal B administration were alsoobserved by Zhong et al [50] Moreover the exposure to SalB can maintain the proliferation of neural stemprogenitor

Evidence-Based Complementary and Alternative Medicine 13

cells (NSPCs) after cerebral ischemia and improve cognitivepostischemic impairment after stroke in rats using Morriswater maze test therefore authors concluded that Sal B mayact as a potential drug in treatment of brain injury or neu-rodegenerative disease [51] Additionally the improvement ofmotor function after cerebral ischemia in rats after salvianolicacid B administrationwas also demonstrated [52]The clue topass through the BBB Sal B provided also results of Li et alindicating its protective effect on BBB in rats after cerebralischemia-reperfusion by inhibiting the MAPK pathway [53]In addition to this it was shown that lithospermic acid andsalvianolic acids exerted neuroprotective activity in variousexperimental models Lithospermic acid significantly attenu-ates neurotoxicity in vitro and in vivo induced by 1-methyl-4-phenylpyridin (MPP(+)) by blocking neuronal apoptotic andneuroinflammatory pathways [54] Salvianolic acid B inhib-ited amyloid beta-protein aggregation and fibril formation aswell as directly inhibiting the cellular toxicity of amyloid beta-protein in PC12 cells [55] and significantly reduced its cyto-toxic effects on human neuroblastoma SH-SY5Y cells [56]

For an explanation of our results observations of Pin-heiro Fernandes et al [57] may be very helpful whichshowed that caffeic acid nonflavanoid catecholic compoundwhose derivatives are occurring in MO extract improvedthe working spatial and long-term aversive memory deficitsinduced by focal cerebral ischemia in mice Anwar et al [48]showed also that caffeic acid (100mgkg) improved the step-down latencies in the inhibitory avoidance in rats Tsai et al[58] showed that caffeic acid is a potent neuroprotective agentin brain of 1-methyl-4-phenyl-1236-tetrahydropyridine(MPTP) treatedmiceMoreover caffeic acid improves A12057325ndash35-induced memory deficits and cognitive impairment inmice [59] In another study it was also shown that this com-pound has a significant protective effect on global cerebralischemia-reperfusion injury in rats [60] For instance 124 plusmn18mg100 g of caffeic acid was detected in the brain of micewith a diet containing 2 caffeic acid for 4 weeks [58] Thusthere is progressive evidence that this compound passesthrough the BBB and has a central pharmacological activity

Also results by Yoo et al are also worth noting [61] whichshowed also in a ratmodel of scopolamine-induced amnesiathat administration of luteolin (a common flavonoid frommany plants including M officinalis) at dose of 10mgkgcaused the increase in the brain-derived neurotrophic factor(BDNF) acetylcholine and the decrease in lipid peroxida-tion Liu et al [62] observed that chronic treatment withluteolin (50 and 100mgkg) improved neuronal injury andcognitive performance by attenuating oxidative stress andcholinesterase activity in streptozotocin-induced diabetes inrats In our study ethanolic extract of MO administered torats significantly improved long-term memory after using apassive avoidance test but afterMO combined treatmentwithSC no improvement of this paradigmwas observed (Table 2)

Thirdly available pharmacokinetic studies were carriedout for the pure compounds (Sal A and B) and the extractof the roots of Salvia miltiorrhiza [42 63] but there are notavailable studies of bioavailability of these compounds afteradministration of Melissa officinalis leaf extract containingother compounds as compared with the extract of Salvia

miltiorrhiza Moreover there is a lack of data about howscopolamine may influence the penetration of the salvianolicacids (and other compounds) across the BBB Hence thereis a need to study the pharmacokinetic parameters of thesecompounds in the group of animals treatedwith scopolaminein comparison with control group

AChEandBuChEActivities in Rat Brain To date several stud-ies have focused on explaining themechanismof action of theMS extract and its active compounds Soodi et al [18] showedthat treatment of animals withMO extract (400mgkg) priorto scopolamine injection could ameliorate scopolamine-induced enhancement in AChE activity This dose of MOextract inhibited the AChE activity in the hippocampus ofrats (519 versus 1004 in normal saline group 119901 lt 005914 in group treated with scopolamine +MO versus 1281in scopolamine group 119901 lt 005) In another study Anwaret al [48] showed that 50 and 100mgkg of caffeic aciddecreased in vivo the AChE activity in the cerebral cortexand striatum and increased the activity of this enzyme inthe cerebellum hippocampus hypothalamus pons lympho-cytes and muscles when compared to the control group(119901 lt 005) Our study showed that ethanolic extract of MOproduced an insignificant and slight inhibition of AChE andBuChE activity in the frontal cortex and in the hippocampus(especially) However it was observed previously [23] thatRA inhibited the AChE activity in the rats frontal cortex andhippocampusMoreover we showed that RA possess a strongstimulatory effect on BuChE in the hippocampus

AChE BuChE and BACE1mRNA Level Changes in Rat BrainSo far no results have been published of studies concerningthe in vivo assessment of changes in AChE BuChE andBACE1 gene expression profile in different brain regionsunder the influence of MO extracts or their key bioactivemetabolites Such studies focused overwhelmingly on theanalysis of their in vitro activities to a lesser extent in animalexperiments

In our study in the frontal cortex we have observedthe strongest inhibition of both AChE and BuCHE mRNAtranscription under the influence ofM officinalis extract andhuperzine A (Table 3) However a more significant differencein the level of transcript was seen in the frontal cortex inparticular in the case of BuCHE mRNA of experimentalrats (a decrease by 516 and 44 for MO and HU versuscontrol resp)The strong inhibition of transcription ofAChEmRNAwas also observed in the hippocampus ofMO-treatedrats (a decrease by 69) The BuChE mRNA transcriptionin animals receiving MO was lowered in a moderate way(decrease by 36)HuperzineA alone has not caused changesthat were observed in the group ofMO (Table 2) Our findingsmostly correlate with the observed changes in activities ofAChE and BuChE in different groups of animals In the caseof BuChE slightly different results between its activity andexpression profile were especially seen in the frontal cortexand hippocampus of animals receivingMO (Table 3)

To date there is a lack of published results of studiesmaking an attempt to clarify the potential differences in thetranscriptional profile and activity of AChE and BuChE

14 Evidence-Based Complementary and Alternative Medicine

under the influence of MO and its bioactive metabolites Itcannot be excluded that the key for the observed differencesin the level of AChE activities and mRNA level can becaused by changes in the activity of AChE in other regionsof the brain not analyzed in this study such as substantianigra cerebellum globus pallidus and hypothalamus whereit exerts nonenzymatic neuromodulatory functions affect-ing neurite outgrowth and synaptogenesis modulating theactivity of other proteins regional cerebral blood flow andother functions [64] But it is difficult to clearly explain whydiminishing of AChE and BuChEmRNAs did not always cor-relate with the lowering of activity of these enzymes althoughit has been recently noted that AChE activity was notparalleled by an increase in mRNA levels [65] The authorsexplained this fact by stating that AChE levels are regulatedat transcriptional posttranscriptional and posttranslationallevels leading to complex expression patterns which canbe modulated by physiological and pathological conditionsHowever these mechanisms are not fully understood andfurther studies are needed in this field

The cause of observed different degree of inhibition ofactivity and transcription status of studied genes especiallyof BuChE (and AChE) may lie in a so-called ldquonegative feed-backrdquo consisting of a complicated transcriptiontranslationregulation protein-protein interactionsmodifications and ametabolic network together forming a system that allows thecell to respond sensibly to the multiple signal molecules thatexist in its environment [66]

Because of that we propose that the reason for differencesbetween AChE and BuChE activities in the cortex and hip-pocampus under the MO may be due to the fact of insuf-ficiency of applied dose and the duration of the experi-ment affecting the transcriptional tissue-specific cellularmachinery regulating BuChE transcription without affectingits activity Moreover the administration of higher doses ofMO and extended period of time could lead to sufficientinhibition of its activity

There is a need to conduct further studies to determinethe molecular degree of dependence between changes inAChE and BuChE activities and activities of potential keyfactors regulating expression of these genes in the frontalcortex and hippocampus under the influence of extracts ofMO and active metabolites Another study determining theeffectiveness of their actions on the cholinergic system inexperimental animal models of memory impairment withparticular emphasis on scopolamine including the determi-nation of changes at the molecular level should be thereforecarried out

Alterations in BACE1 protein level have been proved inpostmortem brain tissue from individuals with AD withincreases decreases and also no change reported [1 67 68]An example of confirmation of these findings at the mRNAlevel is results of study by Coulson et al [69] Based onconducted studies some authors suggested that increasedBACE1mRNA transcription in remaining neuronal cells maycontribute to the increased BACE1 protein levels and activityfound in brain regions affected by AD [70]

Results of many studies concerning the elevated level ofBACE1 mRNA and protein in Alzheimerrsquos disease provide

direct and compelling reasons to develop therapies directed atBACE1 inhibition thus reducing120573-amyloid and its associatedtoxicities [67 68]

In our study we have observed that BACEmRNA expres-sion statistically significantly decreased after MO adminis-tration in frontal cortex and hippocampus These resultssuggest that the MO extract may act to inhibit BACE1mRNA level given the fact that the percentage of inhibitionof the expression (64 in the frontal cortex 50 in thehippocampus) is higher than that in the case of HU (38in the cortex and the lack of changes in the hippocampus)A careful analysis of the literature data shows that there areno studies which analyzed the impact of MO extract on theexpression level of BACE1 in Alzheimerrsquos disease

Furthermore a literature analysis does not indicatealready published results conducted by other teams attempt-ing to assess the impact of RA on the transcriptional activityof AChE BuChE and BACE1 Although several studies(already mentioned and others [70 71]) highlighted theRA and other caffeic acid derivatives capability of acetyl-cholinesterases inhibition none of these studies does nottouch the question of the molecular basis of their impacton the transcriptional machinery that regulates in vivo theexpression of studied genes Hence in our opinion obtainedby our team results they are one of the first of this typeand in general correlate with the results of our previousstudy [23] In this case there is no clear evidence explainingdifferent responses at the transcriptional level under theinfluence of RA especially in the case of BuChE encodinggene in the hippocampus (Table 2) It is possible that theobserved differentiation of BuChE transcriptional activitybetween the frontal cortex and the hippocampus may bedue to the differences of butyrylcholinesterase localizationand substrate affinity [72] Since in our experiment we havecarried out a quantitative analysis of AChE BuChE andBACE1 transcripts in brain homogenates of tested animalsrather than in individual isolated cell fractions therefore theobtained results constitute an overall ldquopicturerdquo of both studiedgenes transcriptional changes occurring in the brain areas ofstudied animals under the influence of the RA and the wholeplant extract as well

6 Conclusion

The subchronic administration ofMO led to an improvementof long-term memory of rats however the mechanisms ofMO action are probably more complicated since its role asa modulator of beta-secretase activity (due to inhibition ofBACE1 mRNA expression in frontal cortex) should be takeninto consideration

It should be noted that we have studied a crude extractfrom leaf of Melissa officinalis not a single pure chemicalcompound This plant extract is a complex mixture and itsaction may be a result of the summation of activities ofseveral components (synergismadditive action of caffeic acidwith salvianolic acids rosmarinic acid and others) In thecase of extract from leaves ofMelissa officinalis it is possiblethat interactions occur between the 40 chemical compoundsidentified by HPLC system

Evidence-Based Complementary and Alternative Medicine 15

Taken together it seems that the MO activity representsa possible option as complementary interventions to relievethe symptoms of mild dementia

Competing Interests

The authors declare that they have no competing interests

Acknowledgments

This work was supported by the Ministry of Science andHigher EducationWarsaw Poland from educational sources(2008ndash2011 Grant no N 405417836)

References

[1] S L Cole and R Vassar ldquoThe Alzheimerrsquos disease 120573-secretaseenzyme BACE1rdquo Molecular Neurodegeneration vol 2 no 1article 22 2007

[2] LACraigN SHong andR JMcDonald ldquoRevisiting the cho-linergic hypothesis in the development of Alzheimerrsquos diseaserdquoNeuroscience amp Biobehavioral Reviews vol 35 no 6 pp 1397ndash1409 2011

[3] N G M Gomes M G Campos J M C Orfao and C A FRibeiro ldquoPlants with neurobiological activity as potential tar-gets for drug discoveryrdquo Progress in Neuro-Psychopharmacologyamp Biological Psychiatry vol 33 no 8 pp 1372ndash1389 2009

[4] WHO WHO Monographs on Selected Medicinal PlantsmdashVolume 2 WHO Geneva Switzerland 2004 httpappswhointmedicinedocsendJs4927e18htmlJs4927e18

[5] M Bayat A A Tameh M H Ghahremani et al ldquoNeuropro-tective properties of Melissa officinalis after hypoxic-ischemicinjury both in vitro and in vivordquo DARU Journal of Pharmaceu-tical Sciences vol 20 article 42 2012

[6] J P Kamdem A Adeniran A A Boligon et al ldquoAntioxidantactivity genotoxicity and cytotoxicity evaluation of lemon balm(Melissa officinalis L) ethanolic extract Its potential role inneuroprotectionrdquo Industrial Crops and Products vol 51 pp 26ndash34 2013

[7] J-T Lin Y-C Chen Y-C Lee C-W R Hou F-L Chen andD-J Yang ldquoAntioxidant anti-proliferative and cyclooxygenase-2 inhibitory activities of ethanolic extracts from lemon balm(Melissa officinalis L) leavesrdquo LWTmdashFood Science and Technol-ogy vol 49 no 1 pp 1ndash7 2012

[8] G Guginski A P Luiz M D Silva et al ldquoMechanisms involvedin the antinociception caused by ethanolic extract obtainedfrom the leaves of Melissa officinalis (lemon balm) in micerdquoPharmacology Biochemistry amp Behavior vol 93 no 1 pp 10ndash162009

[9] N Perry G Court N Bidet J Court and E Perry ldquoEuropeanherbs with cholinergic activities potential in dementia therapyrdquoInternational Journal of Geriatric Psychiatry vol 11 no 12 pp1063ndash1069 1996

[10] E K Perry A T Pickering W W Wang P J Houghtonand N S L Perry ldquoMedicinal plants and Alzheimerrsquos diseasefrom ethnobotany to phytotherapyrdquo Journal of Pharmacy andPharmacology vol 51 no 5 pp 527ndash534 1999

[11] M-J R Howes N S L Perry and P J Houghton ldquoPlants withtraditional uses and activities relevant to the management ofAlzheimerrsquos disease and other cognitive disordersrdquo Phytother-apy Research vol 17 no 1 pp 1ndash18 2003

[12] I Orhan and M Aslan ldquoAppraisal of scopolamine-inducedantiamnesic effect in mice and in vitro antiacetylcholinesteraseand antioxidant activities of some traditionally used Lamiaceaeplantsrdquo Journal of Ethnopharmacology vol 122 no 2 pp 327ndash332 2009

[13] S Akhondzadeh M Noroozian M Mohammadi S OhadiniaA H Jamshidi and M Khani ldquoMelissa officinalis extract inthe treatment of patients with mild to moderate Alzheimerrsquosdisease a double blind randomised placebo controlled trialrdquoJournal of Neurology Neurosurgery and Psychiatry vol 74 no 7pp 863ndash866 2003

[14] D O Kennedy A B Scholey N T J Tildesley E K Perry andK AWesnes ldquoModulation ofmood and cognitive performancefollowing acute administration of Melissa officinalis (lemonbalm)rdquo Pharmacology Biochemistry amp Behavior vol 72 no 4pp 953ndash964 2002

[15] D O Kennedy G Wake S Savelev et al ldquoModulation of moodand cognitive performance following acute administration ofsingle doses of Melissa officinalis (Lemon balm) with humanCNS nicotinic and muscarinic receptor-binding propertiesrdquoNeuropsychopharmacology vol 28 no 10 pp 1871ndash1881 2003

[16] D O Kennedy and E L Wightman ldquoHerbal extracts and phy-tochemicals plant secondarymetabolites and the enhancementof human brain functionrdquoAdvances inNutrition vol 2 no 1 pp32ndash50 2011

[17] R P Pereira A A Boligon A S Appel et al ldquoChemical com-position antioxidant and anticholinesterase activity of Melissaofficinalisrdquo Industrial Crops and Products vol 53 pp 34ndash452014

[18] M Soodi N Naghdi H Hajimehdipoor S Choopani andE Sahraei ldquoMemory-improving activity of Melissa officinalisextract in naıve and scopolamine-treated ratsrdquo Research inPharmaceutical Sciences vol 9 no 2 pp 107ndash114 2014

[19] K Dastmalchi V Ollilainen P Lackman et al ldquoAcetyl-cholinesterase inhibitory guided fractionation of Melissa offic-inalis Lrdquo Bioorganic amp Medicinal Chemistry vol 17 no 2 pp867ndash871 2009

[20] W Chaiyana and S Okonogi ldquoInhibition of cholinesterase byessential oil from food plantrdquo Phytomedicine vol 19 no 8-9 pp836ndash839 2012

[21] K Dastmalchi H J D Dorman P P Oinonen Y Darwis ILaakso and R Hiltunen ldquoChemical composition and in vitroantioxidative activity of a lemon balm (Melissa officinalis L)extractrdquo LWTmdashFood Science and Technology vol 41 no 3 pp391ndash400 2008

[22] P Pereira D Tysca P Oliveira L F D S Brum J N Picadaand P Ardenghi ldquoNeurobehavioral and genotoxic aspects ofrosmarinic acidrdquo Pharmacological Research vol 52 no 3 pp199ndash203 2005

[23] M Ozarowski P L Mikolajczak A Bogacz et al ldquoRosmarinusofficinalis L leaf extract improves memory impairment andaffects acetylcholinesterase and butyrylcholinesterase activitiesin rat brainrdquo Fitoterapia vol 91 pp 261ndash271 2013

[24] G P Kumar and F Khanum ldquoNeuroprotective potential of phy-tochemicalsrdquo Pharmacognosy Reviews vol 6 no 12 pp 81ndash902012

[25] M Stobiecki A Staszkow A Piasecka P M Garcia-LopezF Zamora-Natera and P Kachlicki ldquoLC-MSMS profiling offlavonoid conjugates in wild mexican lupine Lupinus reflexusrdquoJournal of Natural Products vol 73 no 7 pp 1254ndash1260 2010

[26] A Wojakowska A Piasecka P M Garcıa-Lopez et al ldquoStruc-tural analysis and profiling of phenolic secondarymetabolites of

16 Evidence-Based Complementary and Alternative Medicine

Mexican lupine species using LC-MS techniquesrdquo Phytochem-istry vol 92 pp 71ndash86 2013

[27] A Gryszczynska B Opala Z Lowicki et al ldquoBioactive com-pounds determination in the callus and hydroalcoholic extractsfrom Salvia miltiorrhiza and Salvia przewalskiimdashpreliminarystudy on their anti-alcoholic activity effectsrdquo PhytochemistryLetters vol 11 pp 399ndash403 2015

[28] K Slinkart and V L Singleton ldquoTotal phenol analysis automa-tion and comparison with manual methodrdquo American Journalof Enology and Viticulture vol 28 no 1 pp 49ndash55 1977

[29] P J R Boissier J Tardy and J C Diverres ldquoUne nouvellemethode simple pour explorer lrsquoaction lsquotranquillisantersquo le testde la chemineerdquo Medicina Experimentalis vol 3 no 1 pp 81ndash84 1960

[30] R Ader J A Weijnen and P Moleman ldquoRetention of a passiveavoidance response as a function of the intensity and durationof electric shockrdquo Psychonomic Science vol 26 no 3 pp 125ndash128 1972

[31] E M Blalock K-C Chen K Sharrow et al ldquoGene microar-rays in hippocampal aging statistical profiling identifies novelprocesses correlated with cognitive impairmentrdquo Journal ofNeuroscience vol 23 no 9 pp 3807ndash3819 2003

[32] K Isomae S Morimoto H Hasegawa K Morita and JKamei ldquoEffects of T-82 a novel acetylcholinesterase inhibitoron impaired learning and memory in passive avoidance task inratsrdquo European Journal of Pharmacology vol 465 no 1-2 pp97ndash103 2003

[33] J Patora and B Klimek ldquoFlavonoids from lemon balm (Melissaofficinalis L Lamiaceae)rdquo Acta Poloniae Pharmaceutica-DrugResearch vol 59 no 2 pp 139ndash143 2002

[34] L Barros M Duenas M I Dias M J Sousa C Santos-Buelgaand I C F R Ferreira ldquoPhenolic profiles of cultivated invitro cultured and commercial samples of Melissa officinalis Linfusionsrdquo Food Chemistry vol 136 no 1 pp 1ndash8 2013

[35] T L Miron M Herrero and E Ibanez ldquoEnrichment of antiox-idant compounds from lemon balm (Melissa officinalis) bypressurized liquid extraction and enzyme-assisted extractionrdquoJournal of Chromatography A vol 1288 pp 1ndash9 2013

[36] Y Lu L Y Foo and H Wong ldquoSagecoumarin a novel caffeicacid trimer from Salvia officinalisrdquo Phytochemistry vol 52 no6 pp 1149ndash1152 1999

[37] S Abuhamdah LHuangM S J Elliott et al ldquoPharmacologicalprofile of an essential oil derived from Melissa officinalis withanti-agitation properties focus on ligand-gated channelsrdquo Jour-nal of Pharmacy and Pharmacology vol 60 no 3 pp 377ndash3842008

[38] G Wake J Court A Pickering R Lewis R Wilkins andE Perry ldquoCNS acetylcholine receptor activity in Europeanmedicinal plants traditionally used to improve failing memoryrdquoJournal of Ethnopharmacology vol 69 no 2 pp 105ndash114 2000

[39] S-H Ryu C Katona B Rive and G Livingston ldquoPersistenceof and changes in neuropsychiatric symptoms in Alzheimerdisease over 6months the LASER-AD studyrdquoAmerican Journalof Geriatric Psychiatry vol 13 no 11 pp 976ndash983 2005

[40] L N Gitlin H C Kales and C G Lyketsos ldquoNonpharma-cologic management of behavioral symptoms in dementiardquoJournal of the AmericanMedical Association vol 308 no 19 pp2020ndash2029 2012

[41] A Konczol J Muller E Foldes et al ldquoApplicability of a blood-brain barrier specific artificial membrane permeability assay atthe early stage of natural product-based CNS drug discoveryrdquoJournal of Natural Products vol 76 no 4 pp 655ndash663 2013

[42] Y-J Zhang L Wu Q-L Zhang J Li F-X Yin and Y YuanldquoPharmacokinetics of phenolic compounds of Danshen extractin rat blood and brain by microdialysis samplingrdquo Journal ofEthnopharmacology vol 136 no 1 pp 129ndash136 2011

[43] X Li C Yu Y Lu et al ldquoPharmacokinetics tissue distributionmetabolism and excretion of depside salts from Salvia miltior-rhiza in ratsrdquo Drug Metabolism and Disposition vol 35 no 2pp 234ndash239 2007

[44] H Xu Y Li X Che H Tian H Fan and K Liu ldquoMetabolismof salvianolic acid a and antioxidant activities of its methylatedmetabolitesrdquo Drug Metabolism and Disposition vol 42 no 2pp 274ndash281 2014

[45] D H Kim S J Park J M Kim et al ldquoCognitive dysfunctionsinduced by a cholinergic blockade and A120573 25-35 peptide areattenuated by salvianolic acid Brdquo Neuropharmacology vol 61no 8 pp 1432ndash1440 2011

[46] G-H Du Y Qiu and J-T Zhang ldquoSalvianolic acid B protectsthe memory functions against transient cerebral ischemia inmicerdquo Journal of Asian Natural Products Research vol 2 no 2pp 145ndash152 2000

[47] G Du and J Zhang ldquoProtective effects of salvianolic acid Aagainst impairment of memory induced by cerebral ischemia-reperfusion in micerdquo Chinese Medical Journal vol 110 no 1 pp65ndash68 1997

[48] J Anwar R M Spanevello G Thome et al ldquoEffects of caffeicacid on behavioral parameters and on the activity of acetyl-cholinesterase in different tissues from adult ratsrdquo Pharmacol-ogy Biochemistry and Behavior vol 103 no 2 pp 386ndash394 2012

[49] YW Lee D H Kim S J Jeon et al ldquoNeuroprotective effects ofsalvianolic acid B on anA12057325-35 peptide-inducedmousemodelof Alzheimerrsquos diseaserdquo European Journal of Pharmacology vol704 no 1ndash3 pp 70ndash77 2013

[50] J Zhong M-K Tang Y Zhang Q-P Xu and J-T ZhangldquoEffect of salvianolic acid B on neural cells damage and neu-rogenesis after brain ischemia-reperfusion in ratsrdquo Yao Xue XueBao vol 42 no 7 pp 716ndash721 2007

[51] P Zhuang Y Zhang G Cui et al ldquoDirect stimulation of adultneural stemprogenitor cells in vitro and neurogenesis in vivo bysalvianolic acid Brdquo PLoS ONE vol 7 no 4 Article ID e356362012

[52] M TangW Feng Y Zhang J Zhong and J Zhang ldquoSalvianolicacid B improves motor function after cerebral ischemia in ratsrdquoBehavioural Pharmacology vol 17 no 5-6 pp 493ndash498 2006

[53] Q Li L-P Han Z-H Li J-T Zhang and M-K Tang ldquoSal-vianolic acid B alleviate the disruption of blood-brain barrierin rats after cerebral ischemia-reperfusion by inhibiting MAPKpathwayrdquo Yao Xue Xue Bao vol 45 no 12 pp 1485ndash1490 2010

[54] Y-L LinH-J Tsay T-H Lai T-T Tzeng andY-J Shiao ldquoLith-ospermic acid attenuates 1-methyl-4-phenylpyridine-inducedneurotoxicity by blocking neuronal apoptotic and neuroinflam-matory pathwaysrdquo Journal of Biomedical Science vol 22 article37 2015

[55] M-K Tang and J-T Zhang ldquoSalvianolic acid B inhibits fibrilformation and neurotoxicity of amyloid beta-protein in vitrordquoActa Pharmacologica Sinica vol 22 no 4 pp 380ndash384 2001

[56] S S K Durairajan Q Yuan L Xie et al ldquoSalvianolic acid Binhibits A120573 fibril formation and disaggregates preformed fibrilsand protects against A120573-induced cytotoxictyrdquo NeurochemistryInternational vol 52 no 4-5 pp 741ndash750 2008

[57] FD Pinheiro Fernandes A P FonteneleMenezes J C de SousaNeves et al ldquoCaffeic acid protects mice from memory deficits

Evidence-Based Complementary and Alternative Medicine 17

induced by focal cerebral ischemiardquo Behavioural Pharmacologyvol 25 no 7 pp 637ndash647 2014

[58] S-J Tsai C-Y Chao andM-C Yin ldquoPreventive and therapeu-tic effects of caffeic acid against inflammatory injury in striatumof MPTP-treated micerdquo European Journal of Pharmacology vol670 no 2-3 pp 441ndash447 2011

[59] J H Kim QWang J M Choi S Lee and E J Cho ldquoProtectiverole of caffeic acid in an 119860120573

25minus35

-induced Alzheimerrsquos diseasemodelrdquo Nutrition Research and Practice vol 9 no 5 pp 480ndash488 2015

[60] G Liang B Shi W Luo and J Yang ldquoThe protective effect ofcaffeic acid on global cerebral ischemia-reperfusion injury inratsrdquo Behavioral and Brain Functions vol 11 no 1 article 182015

[61] D Y Yoo J H ChoiW Kim et al ldquoEffects of luteolin on spatialmemory cell proliferation and neuroblast differentiation in thehippocampal dentate gyrus in a scopolamine-induced amnesiamodelrdquo Neurological Research vol 35 no 8 pp 813ndash820 2013

[62] Y Liu X Tian L Gou L Sun X Ling and X Yin ldquoLuteolinattenuates diabetes-associated cognitive decline in ratsrdquo BrainResearch Bulletin vol 94 pp 23ndash29 2013

[63] S-M Liu Z-H Yang andX-B Sun ldquoSimultaneous determina-tion of six Salvia miltiorrhiza gradients in rat plasma and brainby LC-MSMSrdquo Zhongguo Zhongyao Zazhi vol 39 no 9 pp1704ndash1708 2014

[64] M A Papandreou A Dimakopoulou Z I Linardaki et alldquoEffect of a polyphenol-rich wild blueberry extract on cognitiveperformance of mice brain antioxidant markers and acetyl-cholinesterase activityrdquo Behavioural Brain Research vol 198 no2 pp 352ndash358 2009

[65] M-S Garcıa-Ayllon O Cauli M-X Silveyra et al ldquoBraincholinergic impairment in liver failurerdquo Brain vol 131 no 11pp 2946ndash2956 2008

[66] G K Ferreira M Carvalho-Silva C L Goncalves et al ldquoL-Tyrosine administration increases acetylcholinesterase activityin ratsrdquo Neurochemistry International vol 61 no 8 pp 1370ndash1374 2012

[67] R Vassar P-H Kuhn C Haass et al ldquoFunction therapeuticpotential and cell biology of BACE proteases current status andfuture prospectsrdquo Journal of Neurochemistry vol 130 no 1 pp4ndash28 2014

[68] R Vassar ldquoThe 120573-secretase BACE a prime drug target forAlzheimerrsquos diseaserdquo Journal of Molecular Neuroscience vol 17no 2 pp 157ndash170 2001

[69] D T R Coulson N Beyer J G Quinn et al ldquoBACE1 mRNAexpression in Alzheimerrsquos disease postmortem brain tissuerdquoJournal of Alzheimerrsquos Disease vol 22 no 4 pp 1111ndash1122 2010

[70] S Vladimir-Knezevic B Blazekovic M Kindl J Vladic A DLower-Nedza and A H Brantner ldquoAcetylcholinesterase inhib-itory antioxidant and phytochemical properties of selectedmedicinal plants of the lamiaceae familyrdquoMolecules vol 19 no1 pp 767ndash782 2014

[71] F Marcelo C Dias A Martins et al ldquoMolecular recognitionof rosmarinic acid from Salvia sclareoides extracts by ace-tylcholinesterase a new binding site detected by NMR spec-troscopyrdquo ChemistrymdashA European Journal vol 19 no 21 pp6641ndash6649 2013

[72] G Johnson and S W Moore ldquoWhy has butyrylcholinesterasebeen retained Structural and functional diversification in aduplicated generdquoNeurochemistry International vol 61 no 5 pp783ndash797 2012

[73] R J Grayer M R Eckert N C Veitch et al ldquoThe chemo-taxonomic significance of two bioactive caffeic acid estersnepetoidins A and B in the Lamiaceaerdquo Phytochemistry vol 64no 2 pp 519ndash528 2003

[74] A M D El-Mousallamy U W Hawas and S A M HusseinldquoTeucrol a decarboxyrosmarinic acid and its 41015840-O-triglycosideteucroside from Teucrium pilosumrdquo Phytochemistry vol 55 no8 pp 927ndash931 2000

[75] I Fecka and S Turek ldquoDetermination of water-soluble polyphe-nolic compounds in commercial herbal teas from Lamiaceaepeppermint melissa and sagerdquo Journal of Agricultural and FoodChemistry vol 55 no 26 pp 10908ndash10917 2007

[76] L W Sumner A Amberg D Barrett et al ldquoProposed min-imum reporting standards for chemical analysis WorkingGroup (CAWG) Metabolomics Standards Initiative (MSI)rdquoMetabolomics vol 3 pp 211ndash221 2007

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Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

2 Evidence-Based Complementary and Alternative Medicine

1 Introduction

Neurodegenerative disorders including Alzheimerrsquos diseasecharacterized by loss of memory and learning ability arethe increasing public health problem worldwide [1 2] Plantswith neurobiological activity may be potential targets fordrug discovery [3] Searching for new drugs and explainingtheir mechanisms of action are one of the most intensivelydeveloping areas of scientific platformMoreover plant originsubstances can be a valuable alternative way in the preventionand treatment of dementias as component of healthy diet

Melissa officinalis (MO English lemon balm Lamiaceae)one of the oldest and still most popular aromatic medicinalplants is used in phytotherapy for the prevention and treat-ment of nervous disturbances of sleep and gastrointestinaldisorders as sedative and antispasmodic medicine [4] Newneuropharmacological investigations showed that ethanolextracts of MO exerted also neuroprotective [5 6] antiox-idant cyclooxygenase-2 inhibitory [7] and antinociceptiveactivities [6 8] Moreover it is known that MO is used formemory-enhancing effects in European folkmedicine [9ndash12]Indeed Akhondzadeh et al [13] carried out the clinical trialin whichMO extract produced a significantly better outcomeon cognitive function than placebo in patients with mildto moderate Alzheimerrsquos disease In other clinical studiesKennedy et al [14ndash16] observed that a treatment combiningboth calming effects and beneficial cholinergic modulationmay well prove to be a novel treatment for Alzheimerrsquosdisease Studies of molecular mechanisms showed that MOextracts exhibited cholinergic (nicotinic and muscarinic)receptor-binding properties in human cerebral cortex tissue[15] Moreover it was observed that both fractions andcrude ethanol extract of MO inhibited acetylcholinesterase(AChE) of rats brain [17 18] and also in vitro [9 1920] but only two studies analyzed behavioral mechanismof action of MO extracts on scopolamine-induced mem-ory impairment in rats [12 18] One study showed thatMO extract (after intraperitoneal injection) significantlyenhanced learning and memory of rats and significantlyameliorated scopolamine-induced learning deficit [18] how-ever in another study it was observed that MO extract wascompletely inactive [12] Moreover in these studies attentionhas not been paid to the influence of the MO extract onthe expression of genes participating in the conditioningof the synaptic cholinergic equilibrium AChE and bytyryl-cholinesterase (BuChE) or even the beta-secretase (BACE1)in rats brain being responsible for beta-amyloid depositionin Alzheimerrsquos disease [1]

On the other hand it is well known that essential oilin leaf of MO is considered to be the therapeutic principlemainly responsible formost of the abovementioned activitiesbut also plant phenolics are considered as an importantfactor in MO therapeutic effects [6 21] It was shown thatethanol extract contains rosmarinic acid (RA) as the majorcompound [7] however there is no detailed informationabout full phenolic profile of extract from MO leaves beingprobably responsible for pharmacological effects as wellThisbecomes especially important given the results of previous

studies showing that RA did not affect short- and long-termmemory [22] or marginally improved long-term memoryin rats model [23] although it was observed that RA hadan ability to inhibit AChE in the frontal cortex and in thehippocampus of rats [23] On the other hand polyphenolsstill constitute a promising source of new drugs and thereis a high interest in understanding their mechanisms in pre-vention and treatment of Alzheimerrsquos disease [16 23 24]

2 Objectives

The aim of this study was to evaluate the influence of sub-chronic (28-fold) intragastrical administration of ethanolextract of MO leaves and rosmarinic acid on scopolamine(SC) impaired memory in animal model Furthermore ace-tylcholinesterase (AChE) and butyrylcholinesterase (BuChE)activities assessment in hippocampus and frontal cortex werestudied Moreover gene expression levels for AChE BuChEand BACE-1 in the hippocampus and frontal cortex wereinvestigated The MO ethanolic extract was phytochemicallyinvestigated (HPLC-ESI-MSn UPLC-PDA) in order to iden-tify phytochemicals present in plant extract

3 Materials and Methods

31 Plant Material The leaves ofMelissa officinalis L (Lami-aceae) were obtained from an herbal company ldquoKawon-Hurtrdquo(Gostyn Wlkp Poland) The plant material was identifiedin the Department of Pharmaceutical Botany and PlantBiotechnology Faculty of Pharmacy Poznan University ofMedical Sciences The voucher specimen has been depositedin the Herbarium of the Institute of Natural Fibres andMedicinal Plants in Poznan (Plewiska) Poland

32 Chemical and Drugs All reagents for HPLC analysisscopolamine hydrobromide trihydrate (SC) and rosmarinicacid (RA) and reagents for biochemical analyses were pur-chased from Sigma-Aldrich (Poland) Huperzine A (HU)was obtained from Enzo Life Sciences AG (Alexis Corpo-ration Biomibo Distribution Poland) Chemicals for geneexpression analysis were obtained from Roche DiagnosticandALAB (Poland) All chemicals and drugs were ex temporeprepared on the day of the experiment

33 Preparation of the Extract 1000 g of raw plant materialwas extracted with 50 ethanol by percolation (24 h) atroom temperature (22 plusmn 1∘C) After filtration the extract wasconcentrated under vacuum to eliminate the ethanol contentThe concentrated extract was frozen and freeze-dried Thefinal product yielded 24821 g of solid extract

34 Metabolites Identification with LC-MS Metabolomicanalyses were performed using two complementary LC-MSsystems The first one HPLC-DAD-MSn consisted of anAgilent 1100 HPLC instrument with a diode-array detector(DAD) (Agilent Palo Alto CA USA) and an Esquire 3000ion trap mass spectrometer (Bruker Daltonics Bremen Ger-many) Chromatographic separations by HPLC were carried

Evidence-Based Complementary and Alternative Medicine 3

out on an XBridge C18 column (150 times 21mm 35 120583mparticlesize) using water acidified with 01 formic acid (solvent A)and acetonitrile (solvent B) with the mobile phase flow of02mLmin in the following gradient 0ndash25min from 10to 30 B 25ndash46min to 98 B and being maintained atthese conditions until 51min Up to 52min system returnedto the starting conditions and was reequilibrated for 5minThe most important MS parameters were as follows the ionsource ESI voltage minus4 kV or 4 kV nebulization of nitrogenat a pressure of 30 psi at a gas flow rate 9 Lmin ion sourcetemperature at 310∘C and skimmer 1 minus10VThe spectra werescanned in the range of 50ndash3000119898119911 The second systemconsisted of UPLC (the Acquity system Waters MilfordUSA) hyphenated to QExactive hybrid MSMS quadrupole-Orbitrap mass spectrometer Chromatographic separationsin this system were carried out using water acidified with01 formic acid (solvent A) and acetonitrile (solvent B)with the mobile phase flow of 04mLmin in the followinggradient 0ndash5min from 10 to 25 B 5ndash13min to 98 Band being maintained at these conditions until 145min Upto 15min system returned to the starting conditions andwas reequilibrated for 3min QExactive MS operated uponthe following settings the HESI ion source voltage minus3 kVor 3 kV The sheath gas flow was 48 Lmin auxiliary gasflow 13 Lmin ion source capillary temperature 250∘C andauxiliary gas heater temperature 380∘C The CID MSMSexperiments were performed using collision energy of 15 eVThe MSn (up to the MS5) and MSMS spectra were recordedin the negative and positive ion modes using the previouslypublished approach [23 25 26] The individual compoundswere identified via comparison of the exact molecular masses(measured in most cases with Δ below 1 ppm) mass spectraand retention times to those of the standard compoundsas well as the databases available online (PubChem ChEBIMetlin and KNApSAck) and literature data

35 Quantitative HPLC Analysis Sample was extracted by70 ethanol After sonification solution was cooled downand filtered through membrane filter HPLC method wasused to determine RA in a dry ethanolic extract The Lichro-spher 100 RP-18e (125mm 40mm 5 umMerck) was appliedfor identification of this active compound Temperature ofcolumn was 35∘C detection of RA was at 205 nm andflow rate was 15mLmin Mobile phase A was H

3PO4 H2O

(1 999)mobile phase Bwas acetonitrile Timewas as follows0min 10 B 13min 22 B 14min 40 B 25min 40 B

Moreover for identification of other chemical com-pounds a Zorbax Poroshell 120 SB-C18 column 27mm30mm times 100mm (Agilent) was used The lithospermic acidand salvianolic acid B were detected at 250 nm salvianolicacid A was detected at 280 nm The gradient mixtures ofphase Amdashwater H

3PO4(100 002 VV)mdashand of phase Bmdash

acetonitrile tetrahydrofurane (100 2 VV)mdashwere used aseluents Peaks were identified by addition of standard solu-tions and by UV-Vis spectra The quantification of thesecompounds was achieved using calibration curves preparedwith pure compoundsThe flow rate was 07mLmin columntemperature 27∘C and sample injection 5mL The gradient

mixtures program was as follows 0min 5 B 3min 10B 5min 12 B 11min 217 B 15min 39 B 39min 39B 70min 70 B and detection of compounds took place at250 nm 280 nm and 330 nm [27]

36 Determination of Total Phenolic Compounds in theExtract The calculation of polyphenols to gallic acid wasdone using the Folin-Ciocalteu reagent with the spectropho-tometric modified method described by Slinkart and Single-ton [28]

37 Determination of Total Hydroxycinnamic AcidDerivativesDetermination of total hydroxycinnamic acid derivativescalculated on rosmarinic acidwas performed according to theprocedure described in EurPh 50

38 Distillation of Essential Oil The essential oil contentswere determined by way of stream distillation in Deryngrsquosapparatus according to EurPh 50 1000 g of the dry hy-droethanolic MO leaf extract (separate sample) was placedin a round-bottom flask Then 5000mL distilled water and03mL xylenwere added and boiled inDeryngrsquos apparatus for3 h

39 Gas Chromatography Analysis Gas chromatography(GC) analyses were carried out using a Perkin-Elmer Clarus500 gas chromatograph with a data processing system andan FID (GC-FID) Separation was achieved by using an EliteFFAP fused-silica capillary column (30m long 032mm ininternal diameter and 025 120583mof film thickness)The injectorand detector temperatures were 220∘C Helium was used as acarrier gas with a flow of 15mLminminus1 A sample of 10 120583Lwasinjected using slit mode (split ratio 1 100) The results werereported as the relative percentage of the total peak area

310 Animals Experiments with rats were performed inaccordance with Polish governmental regulations (Dz U0533289) The study was conducted in accordance withethical guidelines for investigations in conscious animals andthe study protocol was approved by the Local Ethics Com-mittee of the Use of Laboratory Animals in Poznan Poland(642008) The experiments were performed on male six-week-oldWistar rats housed in controlled room temperature(20 plusmn 02 C) and humidity (65ndash75) under a 12 h 12 h light-dark cycle (lights on at 7 am) Animals were kept in groupsin amounts of 8ndash10 in light plastic cages (60times 40times 40 cm) andhad a free access to standard laboratory diet (pellets-LabofeedB) and to tap water in their cages

311 Treatments The rats were treated with hydroethanolicextract ofMelissa officinalis leaf (MO) in a dose of 200mgkgbw intragastrically (po) (groups MO + H

2O and MO +

SC) for 28 (28x) consecutive days For comparative purposeshuperzine A (HU) was administered chronically (28x) ina dose 05mgkg bw (po) (groups HU + H

2O and HU

+ SC) as a known acetylcholinesterase inhibitor Moreoverrosmarinic acid (Sigma-Aldrich) (RA) was applied (28x) in adose of 10mgkg bw (po) (groups RA + H

2O and RA + SC)

4 Evidence-Based Complementary and Alternative Medicine

as a comparative chemical compound On the last day 30minafter the last dose of MO or HU SC was given intraperi-toneally (ip) in a dose of 05mgkg bw Control groups weretreated with 05 methylcellulose (MC) whereas water forinjection (H

2O) was used as a vehicle for SC (groups MC +

H2O and MC + SC) MO was prepared ex tempore before

administration and suspended in MC in concentrations of20mgmLOnday 28 of the experiment 1 h after the last dosethe animals were killed by decapitation and hippocampus andpart of frontal cortex were collected from brain of rats Thetissue samples were then stored at minus80∘C until measurementof acetylcholinesterase (AChE) and butyrylcholinesterase(BuChE) activities or mRNA level changes

312 Cognitive and Behavioral Tests Cognitive and behav-ioral tests were used in the present study similarly as in ourprevious report [23] (1) sedative activity was assessed usinga locomotor activity test (2) motor coordination assessmentwas done using a ldquochimneyrdquo test (3) the passive avoidance testwas performed as an animalmodel for the assessment of long-termmemory and (4) the object recognition test was used asan animal model for the assessment of short-term memory

3121 Measurement of Locomotor Activity Locomotor activ-ity assessment was performed with licensed activity meter(Activity Cage Ugo Basile Italy) by placing the animals in thecentre of the apparatus and recording their horizontal activity[23] The data obtained were expressed as signals corre-sponding to animalmovements for 5minutesThe locomotoractivity was measured 30 minutes after the administration ofa single dose of SCH

2OAny distracting factors were reduced

to the minimum (noise presence of people and presence ofother rats)

3122Measurement ofMotorCoordination Motor coordina-tion was evaluated using ldquochimneyrdquo test described originallyfor mice [29] Thirty minutes after SC or vehicle injectionrat was allowed to enter a glass laboratory cylinder that is500mm long and 80mm in diameter laid on its side Uponreaching its bottom by the animal position of the cylinderwas rapidly changed from horizontal to vertical and a timerstarted The animal immediately began to move backwardsThe timer was stopped after the rat left the cylinder andassumed a sitting posture on the top of the vessel The timeof exit from the cylinder was accepted as a measure of motorcoordinationMotor impairment was assessed as the inabilityof rats to climb backwards up the tube within 60 s

3123 Passive Avoidance Test Passive avoidance test wasused as an animal model for the assessment of long-termmemory (effects on retrieval and memory consolidation)[30] The test relies on the natural preference of rats for dark-ness After 2 minutes of habituation to a dark compartmenta rat was placed on the illuminated platform and allowedto enter the dark compartment using licensed apparatus(Passive Avoidance System step through Ugo Basile Italy)Two more approach trials were allowed on the following daywith a two-minute interval between them At the end of

the second trial an unavoidable scrambled electric footshock(500120583A AC 3 s) was delivered through the grid floor of thedark compartment (learning trial) Retention of the passiveavoidance response (latency) was tested 24 h later by placingthe animal on the platformandmeasuring the latency in reen-tering the dark compartment against the arbitrary maximumtime of 180 s The test was performed after 30 minutes afterthe administration of a single dose of SC or the vehicle

3124 Object Recognition Test Object recognition test wasused as an animal model for the assessment of short-termmemory [31]The object recognition task took place in a 40 times60 cm open box surrounded by 40 cm high walls made ofplywood with a frontal glass wall All animals were submittedto a habituation session where they were allowed to freelyexplore the open field for 5min No objects were placed inthe box during the habituation trial On the day of testing theanimals were given an additional 3min rehabituation periodprior to commencing the test The test was divided into threephases with two trials the acquisition trial the retention trialand an intertrial interval of varying times

(i) Acquisition trial in this first trial the animals ex-plored two identical objects (1198601 and 1198602) for a periodof 3min positioned in two adjacent corners 10 cmfrom the walls

(ii) Intertrial interval (ITI) the animals were returned tothe home cage for 30min

(iii) Retention trial in this second trial the animalsexplored a familiar object (119860lowast) that is a duplicate ofthose objects from the acquisition trial (to minimizeolfactory cues) and a novel object (119861) for a further3min

They were made of a biologically inert substance (plastic)and were chosen to enable ease of cleaning (10 alcohol)between subjects in an attempt to remove olfactory cuesObject exploration is defined by animals licking sniffing ortouching the object whilst sniffing but not leaning againstturning round or standing or sitting on the object Objectswere of sufficient weight and were secured to the floor of thearena to ensure that they could not be knocked over ormovedaround by the animal The exploration time (s) of all objectswas recorded via stopwatch for subsequent statistical analysisThe time measured as an exploration behavior was used tocalculate amemory discrimination index (OR) as reported byBlalock et al [31]OR = (119861minus119860lowast)(119861+119860) where119861was the timespent exploring the new object and 119860lowast was the time spentexploring the familiar object Higher OR was considered toreflect greater memory ability [31] The test was performedafter 30 minutes after the administration of a single dose ofSC or the vehicle

313 Acetylcholinesterase and Butyrylcholinesterase ActivitiesAssay in Brain of Rats Acetylcholinesterase (AChE) andbutyrylcholinesterase (BuChE) activities were performed bymodifying spectrophotometric Ellmanrsquos method accordingto Isomae et al [32] The activities of AChE and BuChE

Evidence-Based Complementary and Alternative Medicine 5

028

021

014

007

000

200100 300 400 500 600 700 800 900(Minutes)

(AU

)

1 23

4

56 7 8 9 10

1112

1314

1617

19

15 1820

2122

23

2425

2627

28

2930

31

37

3234

3536 38 39

40

33

Figure 1 Chromatogram UV ofMelissa officinalis leaf extract obtained at 270 nm with peaks identified by HPLC-UV-MS

were determined by measuring the formation of the yellowanion obtained from the reaction between Ellmanrsquos reagentand the thiocholine generated by the enzymatic hydrolysisof acetylthiocholine iodide (ATCh) and butyrylthiocholine(BTCh) respectively (sample 01mL PBS 08mL DTNB01mL ATCh 020mL and BTCh 020mL)The biochemicalassay of AChE and BuChE in homogenate of brain sampleswas expressed as 120583molminmg protein by using spectropho-tometric method (120582 = 412 nm)

314 RNA Isolation and Reverse Transcription Reaction TotalRNA isolation from the rats brain tissues homogenates(frontal cortex hippocampus) was carried out using TriPureIsolation Reagent (Roche) according to manufacturerrsquos pro-tocol The integrity of RNA was visually assessed by a con-ventional agarose gel electrophoresis and the concentrationwill be evaluated by measuring the absorbance at 260 and280 nm in a spectrophotometer (BioPhotometer Eppendorf)RNA samples were stored at minus80∘C until use The 1 120583gof total RNA from all samples was used for the reversetranscription into cDNA using Transcriptor First StrandSynthesis Kit (Roche) according to manufacturerrsquos protocolObtained cDNA samples was stored at minus20∘C or used directlyfor the quantitative real-time PCR (qRT-PCR)

315 Real-Time PCR mRNA Quantification The acetylcho-linesterase (AChE) butyrylcholinesterase (BChE) and beta-secretase (BACE1) genes expression level was analyzed bytwo-step quantitative real-time PCR (qRT-PCR) in a vol-ume of 10 120583L reaction mixture using relative quantificationmethodology with a LightCycler TM Instrument (RocheGermany) and a LightCycler Fast Start DNA Master SYBRGreen I kit (Roche Applied Science) according to theinstructions of the manufacturer All primers sequences weredesigned and custom-designed using the Oligo 60 software(National Biosciences) and were verified by assessment ofa single PCR product on agarose gel and by a single tem-perature dissociation peak (melting curve analysis) of eachcDNA amplification product An GAPDH gene was usedas a housekeeping gene (endogenous internal standard) for

normalization of qPCR For each quantified gene standardcurves were prepared from dilution of cDNA and gener-ated from a minimum of four data points All quantitativePCR were repeated twice The data were evaluated usingLightCycler Run 45 software (Roche Applied Science) EachPCR run included a nontemplate control to detect potentialcontamination of reagents

316 Statistical Analysis All values were expressed as meansplusmn SEM The statistical comparison of results was carried outusing one-way analysis of variance (ANOVA) followed byDuncanrsquos post hoc test for detailed data analysisThe values of119901 lt 005were considered as a statistical significant difference

4 Results

41 Phytochemical Profile of Extract

411 Identification ofMetabolites Forty phenolicmetaboliteswere identified in hydroethanolic Melissa officinalis leafextract (Table 1 Figure 1) The predominant identified com-pounds were bioactive caffeic acid esters and glycosides offlavones The caffeic acid dimer rosmarinic acid (metabolite23) and caffeate trimer (lithospermic acid 31) were the prin-cipal caffeic acid derivatives in the analyzed samples Hydrox-yjasmonic acid and its derivatives teucrol as well as sage-coumarin were identified for the first time in the genusMelissa while luteolin and apigenin glycoconjugates arewell known phytochemicals in this species [33] Multistepfragmentation with accurate mass measurement enabledconfirming that the losses of fragment 799573 amu from the[M-H]minus ions of compounds 4 9 27 30 and 32 referredto sulphate groups The first-order fragmentation of 30revealed the loss of 799573 and yielded the product ion at7191622119898119911 The following fragmentation of this ion cor-responded to that of the sagerinic acid (dimer of rosmarinicacid) described by Barros et al [34] The exact placementof the sulphation position would require in-depth chemicalanalysis Thus 30 was tentatively assigned as sulphated sag-erinic acid

6 Evidence-Based Complementary and Alternative MedicineTa

ble1Metabolitesd

etectedin

Melissa

officin

alisleafextractb

yUPL

C-MS

No

RT [min]

Metabolite

identifi

catio

nCh

emical

form

ula

Exactm

asso

f[M-H

]minusΔpp

mFragmentatio

nin

120582max[nm]

CID

aIdentifi

catio

nlevelb

Reference

Measured

Calculated

Negativeion

mod

e(ESIminus)

Positiveion

mod

e(ESI+)

114

1

2-Hydroxy-3-(34-

dihydroxyphenyl)-

prop

anoic

acid

C 9H9

O5

1970

451970

455minus41775

197179135

199163

283312

8143997

2[34]

217

3Dihydroxybenzoica

cid

hexosid

eC 13

H15

O9

315072

3150722

08346

315153109

317155

282

54726828

3[35]

319

8Ca

ftaric

acid

C 13

H11

O9

3110

413110

409

06141

311221179149

318

6440397

2[35]

4224

2-Hydroxy-3-(34-

dihydroxyphenyl)-

prop

anoica

cid

sulphated

C 9H9

O8

S2770

032770

024

03998

277197179135

312

3[35]

5256

Hydroxyjasm

onicacid

hexosid

eC 18

H27

O9

387166

3871661

09214

387207163

323

44237366

2[17]

6276

Caffeicacid

C 9H7

O4

1790

341790

451minus49075

179135

181163

275

689043

1std

7289

Salviano

licacid

EC 36

H29

O16

7171450

717146

72313

717519339321

295277

275325

49770697

2[35]

8317

Salviano

licacid

HI

(isom

er)

C 27

H21

O12

537104

5371038

08562

537493359295

281325

2[35]

9345

Hydroxyjasm

onicacid

sulphated

C 12

H17

O7

S30507

30507

1117

305225194147

275330

3[17]

10362

NepetoidinB

C 17

H13

O6

313072

3130718

11232

nd282316

5316819

2[73]

1137

Yunn

aneica

cidF

C 26

H25

O14

5971255

5971244

18743

597509311197

Masked

2[34]

12383

Decarbo

xyrosm

arinicacid

(teucrol)

C 17

H15

O6

315088

3150874

09396

315179135

275330

637829

2[74]

1339

Caffeoylcaft

aricacid

C 22

H17

O12

473073

4730725

07555

173311149

Masked

65018

3[35]

14397

Apigenin

glucosylrham

nosid

eC 27

H29

O14

577157

5771563

11136

577269

579433271

275340

92741003

3[33]

15404

Luteolin

7-O-glucosid

e31015840-O

-glucuronide

C 27

H27

O17

623126

6231254

09696

62346144

7285

255

625463287

273343

2[33]

16421

Rosm

arinicacid

hexosid

eC 24

H25

O13

52113

5211301

03551

521359161

523361325163

329

25245848

2[34]

1743

Luteolin

O-diglucosid

eC 27

H29

O16

609147

6091461

07183

609285

611287

269349

3[33]

18441

Luteolin

glucosylrham

nosid

eC 27

H29

O15

593152

5931512

08077

59344

7285

59544

9287

271343

3[33]

19449

Luteolin

41015840-O

-glucosid

eC 21

H19

O11

4470

9444

70933

09728

447285

449287

271343

5319116

3[33]

2045

Sagerin

icacid

2-hydroxy-3-

(34-dihydroxyph

enyl)-

prop

anoide

C 45

H39

O20

8992

058992

0410

448

899719591475

295

Masked

3[35]

2149

Salviano

licacid

B(lithosperm

icacid

B)C 36

H29

O16

717146

7171461

01846

717519359161

327

6441188

2[34]

22504

Sagerin

icacid

C 36

H31

O16

7191

637191

618

09979

719519359161

287330

2[34]

23514

Rosm

arinicacid

C 18

H15

O8

3590

773590

772

0002

359161

361163

329

5281792

1std

Evidence-Based Complementary and Alternative Medicine 7

Table1Con

tinued

No

RT [min]

Metabolite

identifi

catio

nCh

emical

form

ula

Exactm

asso

f[M-H

]minusΔpp

mFragmentatio

nin

120582max[nm]

CID

aIdentifi

catio

nlevelb

Reference

Measured

Calculated

Negativeion

mod

e(ESIminus)

Positiveion

mod

e(ESI+)

24555

Salviano

licacid

B2-hydroxy-3-(34-

dihydroxyphenyl)-

prop

anoide

C 45

H37

O20

89719

8971884

16873

897717519359

161

330

3[35]

2556

Sagerin

icacid

di-2-hydroxy-3-(34-

dihydroxyphenyl)-

prop

anoide

C 54

H47

O24

1079244

910792457minus07911

1079897719539

359295

288330

3[35]

26592

Sagecoum

arin

di-2-hydroxy-3-(34-

dihydroxyphenyl)-

prop

anoide

caffeide

C 54

H43

O24

10752156

10752150

0559

1077897717537

409359339277

322

3[35]

27598

Rosm

arinicacid

sulphated

Iisomer

C 18

H15

O11

S4390

354390

341

20214

439359341163

Masked

2[34]

28613

Luteolin

31015840-O

-glucuronide

C 21

H17

O12

4610

734610

7216

34461285

463287

269340

170474237

2[33]

29632

Salviano

licacid

AC 26

H21

O10

493115

493114

11011

493359295179

298327

5281793

[34]

30656

Sagerin

icacid

sulphated

C 36

H31

O19

S7991

196

7991

186

0954

799719619519

359161

325

3[35]

31671

Lithosperm

icacid

C 27

H21

O12

537104

537104

809698

537493359161

292329

6441498

2[35]

3268

Rosm

arinicacid

sulphated

IIiso

mer

C 18

H15

O11

S4390

344390

341

02837

439359341163

Masked

2[34]

33689

Sagecoum

arin

C 27

H19

O12

535088

5350882

01204

535311267177

Masked

2[36]

3470

3Salviano

licacid

LIisomer

C 36

H29

O16

717146

7171461

02697

717519359

284329

2[35]

3571

Salviano

licacid

Lhydroxycaffeide

C 45

H35

O20

895173

8951727

06282

895519359161

Masked

3[35]

3674

Sagecoum

arin

cafta

ride

C 40

H29

O20

8291

268291

258

06953

829667535355

311

Masked

3[36]

37751

Sagecoum

arin

2-hydroxy-

3-(34-dihydroxyph

enyl)-

prop

anoide

C 36

H27

O16

715131

7151305

08176

715535311267

319

3[34]

3877

1Unk

nown

C 36

H57

O14

S745348

7453475

044

02281326

4[35]

3978

5Methylrosmarinate

C 19

H17

O8

373093

3730929

01765

373359161

284323

6479915

2[75]

40869

Salviano

licacid

Ccaffeoylhydroxycaffeide

C 44

H33

O18

8491

688491

672

08615

849687491359

327255

286318

3[35]

a CID

identifier

fora

chem

icalstr

ucture

intheP

ubCh

emCom

poun

ddatabase

b Metabolite

identifi

catio

nlevelaccording

toMetabolom

icsS

tand

ards

Initiativer

ecom

mendatio

n[76]

stdidentificatio

non

theb

asisof

stand

ardcompo

undfragmentatio

nnd

not

detected

8 Evidence-Based Complementary and Alternative Medicine

100 200 300 400 500 600 700 800 900 1000 1100 1200

mz

70

75

80

85

90

95

100

minus198 (2-hydroxy-3-(34-dihydroxyphenyl)

-porpanoide)

minus180 (caffeide)

minus180 (2-hydroxy-3-(34-dihydroxyphenyl)

-propanoide)

minus180 (2-hydroxy-3-(34-dihydroxyphenyl)

-propanoide)

minus180 (2-hydroxy-3-(34-dihydroxyphenyl)

-propanoide)

1350438

2191749

HO

HO

HO HO

OH

OH

OH

OH OH OH

OHOHOHOHHOOC

COOH

COOH COOH COOH

24612ppmC36H55O37 = 10792422

minus07911ppmC54H47O24 = 10792457

10792449

10448ppmC45H39O20 = 8992040

899205018453ppm

C36H31O16 = 7191612

7191625

20501ppmC27H23O12 = 5391190

5391201

14852ppmC23H33O10 = 4692074

469208119316ppm

C17H11O5 = 2950606

2950612

0

5

10

15

20

25

30

35

40

45

50

55

60

65

Rela

tive a

bund

ance

22903ppmC18H15O8 = 3590767

3590775

minus37218 ppmC9H5O3 = 1610239

1610233

(a)

100 200 300 400 500 600 700 800 900 1000

mz

100 200 300 400 500 600 700 800 900 1000

mz

100 200 300 400 500 600 700 800 900 1000

mz

100 200 300 400 500 600 700 800 900 1000

mz

0

250

500

750

1000

00

04

08

times104

times104

01234

0

1

2

times105

Ints

Ints

Ints

Ints

MS1

MS2

MS3

MS4

[M-H]minus10749

minus224 (acidic part

minus224 (acidic part

of sagecoumarin)

of sagecoumarin)

minus360 (rosmarinic acid)

4908

7148

5348 minus180 minus162 (caffeide)8769

31074909

5348

minus180 (2-hydroxy-3-(34-dihydroxyphenyl)-propanoide)

3108

35474909

1

(b)

Figure 2 Continued

Evidence-Based Complementary and Alternative Medicine 9

150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900

mz

0

5

10

15

20

25

30

35

40

45

50

55

60

65

Rela

tive a

bund

ance

O

O

O O

O

O

OH OH

OH

OH

OH

OHOH

OH

HO

HO

HO

COOH

OC

1529946

1970447

C9H9O5 = 1970455

minus41775ppm1610232

2552328

C16H31O2 = 2552330

minus04091ppm

3110563

C17H11O6 = 3110561

05243ppm

minus224 (fragment of sagecoumarin)

3912257

4092361

4832730

C29H39O6 = 4832752

minus44931ppm

5350885

C27H19O12 = 5350882

05640ppm

minus132 (tartaric acid)

5770990

C29H21O13 = 5770988

06486ppm

6670947

C31H23O17 = 6670941

09808ppm

minus162 (caffeic acid)

8291267

C40H29O20 = 8291258

11370ppm

(c)

Figure 2 (a) Mass spectra in negative ionization mode and simplified fragmentation scheme of compound 25 (pentameric ester ofcaffeic acid) (b) Mass spectra in negative ionization mode and simplified fragmentation scheme of compound 26 (pentameric structureof sagecoumarin di-2-hydroxy-3-(34-dihydroxyphenyl)-propanoide caffeide) (c) Mass spectra in negative ionization mode and simplifiedfragmentation scheme of compound 36 (pentameric structure of sagecoumarin caftaride)

Tetrameric structures of hydroxycinnamic acids wereidentified in lemon balm recently [34 35] The measure-ment of accurate masses allowed the identification of com-pound 25 which was tentatively identified as pentamericester of caffeic acid (Figure 2(a)) In 25 the double loss of1800421 amu corresponded to fragments with the molecularformula of C

9H8O4 adequate to dehydroxylated 2-hydroxy-

3-(34-dihydroxyphenyl)-propanoic acid The product ionat 7191623119898119911 and its further fragmentation are sim-ilar to those of metabolite 22 as described previously [34 35]Therefore metabolite 25was tentatively assigned as sagerinicacid di-2-hydroxy-3-(34-dihydroxyphenyl)-propanoideNev-ertheless comprehensive studies by nuclear magnetic reso-nance are required to complete elucidation of substitutionpattern for particular components of those pentamers

Sagecoumarins were previously identified in Salvia offic-inalis as caffeic acid trimers [36] Our MS analysis indicatedthe presence of such compounds and their derivatives alsoin M officinalis The pseudomolecular ion of compound33 observed in the negative ionization mode had theaccurate mass of 5350880119898119911 which corresponded to thechemical formula C

27H19O12

adequate for sagecoumarin(according to the Metlin and KNApSAck databases) Note-worthy 26 33 36 and 37 had the same fragmentationpattern of the product ion obtained in the MSMS andMSn in the negative ionization mode The [M-H-1800422]minusion corresponded to the detachment of 2-hydroxy-3-(34-dihydroxyphenyl)-propanoic acid thus 37 was tentatively

assigned as tetrameric sagecoumarin 2-hydroxy-3-(34-dihy-droxyphenyl)-propanoide The [M-H-3600846]minus ion in 26was indicated on rosmarinic acid substitution to 37 Massspectra of MSn in negative ionization of the compoundprovided complementary information to HR-MSMS massspectra indicated on caffeic acid as internal componentof the dimer In addition simultaneous loss of fragments180 amu and 224 amu in MS3 and MS4 indicated that thetwo components cannot be linked (Figure 2(b)) How-ever detailed analysis of substitution pattern of 26 shouldbe done Therefore 26 was tentatively assigned as penta-meric structure of sagecoumarin di-2-hydroxy-3-(34-dihy-droxyphenyl)-propanoide caffeide Rupture of caffeic andtartaric acid moieties from the product ion at 5350885119898119911was observed for compound36 (Figure 2(c))Detection of theaccurate masses of these two detached fragments with Δ lessthan 1 ppm eliminated the possibility of hexose and pentosesubstitution which have the same nominal masses as caffeicand tartaric acid respectively Therefore 36 was tentativelyidentified as another pentameric structure of sagecoumarincaftaride

Metabolite 28 with the accurate masses of 461073119898119911was tentatively identified as luteolin O-glucuronide The [M-H-1760324]minus ion is indicated on loss of structure C

6H8O6

corresponding to glucuronide moiety The product ion at2850405119898119911 was indicated on flavone luteolin The placeof substitution of the carboxylic acid on flavone skeleton isproblematic due to different isomers reported in lemon balm

10 Evidence-Based Complementary and Alternative Medicine

Table 2 Effect ofMelissa officinalis leaf extract (200mgkg po) treatment on sedative activity motor coordination and memory in rats

Group 119899Locomotor activity

[number of impulses5min]Motor coordination

exit time [s]Short-term memorye

ORLong-term memory

latency [s]MC + H

2

O 18 390 plusmn 24 17 plusmn 3 040 plusmn 006 47 plusmn 14MC + SC 18 526 plusmn 48lowast 32 plusmn 5lowast 032 plusmn 005 12 plusmn 3lowast

MO + H2

O 10 231 plusmn 48lowast 32 plusmn 7lowast 043 plusmn 007 169 plusmn 11lowast

MO + SC 9 436 plusmn 60 43 plusmn 7 009 plusmn 011lowast 23 plusmn 6HU + H

2

O 9 515 plusmn 32 15 plusmn 2 037 plusmn 009 158 plusmn 14lowast

HU + SC 8 639 plusmn 71lowast 56 plusmn 3 022 plusmn 006 49 plusmn 18

RA + H2

O 8 406 plusmn 59 21 plusmn 5 045 plusmn 005 58 plusmn 28RA + SC 8 605 plusmn 55 28 plusmn 7 045 plusmn 005 20 plusmn 7Means plusmn SEM119899Number of animalsMC + H

2O control rats

SC scopolamine (05mgkg bw ip)HU huperzine A (05mgkg bw po)RA rosmarinic acid (10mgkg bw po)eExpressed as ratio OR = (119861 minus 119860lowast)(119861 + 119860) for details see Section 3lowastVersus MC + H

2O 119901 lt 005

Versus MC + SC 119901 lt 005

luteolin 31015840-O- and 7-O-glucuronide [33] It is impossible todistinguish both structures by mass spectrometry Only onechromatographic peak corresponding to the [M-H]minus ion at461073119898119911 was observed in our study Since luteolin 31015840-O-glucuronide was assigned as the most abundant flavonoid inlemon balm [33] we assumed that 28 corresponded to thisstructure

412 Flavonoids and Polyphenolic Acids The major com-pound from the 40 chemical compounds identified in hy-droethanolic MO leaf extract established by HPLC was RA(885 g100 g) (Table 1 Figure 1)

Other chemical compoundswith neuromodulatory activ-ities such as lithospermic acid (0042 g100 g) salvianolicacid A (0040 g100 g) salvianolic acid B (0023 g100 g) andcaffeic acid (0087 g100 g) were also documented in litera-ture Moreover the total polyphenolic compounds content ofMO determined with the use of FolinndashCiocalteu assay was3397 calculated as gallic acid The total hydroxycinnamicderivatives content expressed spectrophotometrically as ros-marinic acid was 2115 g100 g

413 Essential Oil Composition Hydroethanolic MO leafextract contained 008 of total essential oil The GCFIDanalysis showed that the extract comprised camphene(004) alfa-pinene (007) beta-pinene (1647) andmyrcene (1951) Moreover according to retention time16 compounds were identified as follows alfa-bisabololborneol carvone chamazulene cineole eugenol gamma-terpineol guaiazulene isopulegol linalool limonene men-thol menthyl acetate pulegone terpine and thymol Thesecompounds are present in the essential oil in trace amountswhich do not allow the quantitative interpretation

42 Cognitive and Behavioral Experiments

421 Locomotor Activity A one-way ANOVA analysisrevealed significant differences in the locomotor activity of

rats expressed as their horizontal spontaneous activity afterMO administration (ANOVA 119865(7 80) = 646 119901 lt 005)(Table 2) Detailed post hoc analysis showed thatMO + H

2O

decreased the locomotor activity of rats by 4031 but HU +H2O did not affect this activity when compared with control

group (MC + H2O) We observed also that RA + H

2O did

not change the locomotor activity of rats Stimulating effectsin the locomotor activity of rats were observed after an acuteSC injection (MC + SC versus MC + H

2O 119901 lt 005) and

this effect was observed in all SC-treated rats when comparedwith the proper non-SC-treated animals On the contrarythese SC-treated animals did not differ in comparison toanimals that received SC only (MO + SC versus MC + SC119901 gt 005 HU + SC versus MC + SC 119901 gt 005 RA + SCversus MC + SC 119901 gt 005)

422 Motor Coordination A one-way ANOVA analysisrevealed significant differences in motor coordination of ratsexpressed as their exit time from the cylinder (119865(5 73) =284 119901 lt 005) (Table 2) Detailed analysis showed that themultiple administration of RA + H

2O and HU + H

2O did

not affect significantly this paradigm when compared withcontrol rats (119901 gt 005) whereas MO treatment led to pro-longed exit time (MO + H

2O versus MC + H

2O 119901 lt 005)

Moreover generally SC-treated animals showed producedprolongation of exit time and the effects were statisticallysignificant not only in control groups (MC + SC versus MC +H2O 119901 lt 005) but also in RA-treated rats (RA + SC versus

MC + SC 119901 lt 005) However the rest of the SC-treatedanimals did not differ in comparison to animals receiving SConly (MO + SC versus MC + SC 119901 gt 005 HU + SC versusMC + SC 119901 gt 005)

423 Long-Term Memory A one-way ANOVA analysisrevealed significant differences in long-term memory afterusing a passive avoidance test (119865(7 77) = 201 119901 lt 005Table 2) It was shown that the strongest effect leading to

Evidence-Based Complementary and Alternative Medicine 11

Table 3 The effect ofMelissa officinalis leaf extract on acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) activities and AChEBuAChE or beta-secretase (BACE1) mRNA expression levels in frontal cortex (FC) or hippocampus (Hipp) of rats

Group119899Enzyme activity

[nmolminmg protein]mRNA expression

[]AChE BuChE ACHE BuChE BACE1

FC Hipp FC Hipp FC Hipp FC Hipp FC HippMC + H

2

O 363 plusmn 49 439 plusmn 73 65 plusmn 11 53 plusmn 8 100 plusmn 12 100 plusmn 11 100 plusmn 18 100 plusmn 11 100 plusmn 16 100 plusmn 8MO + H

2

O 276 plusmn 34 409 plusmn 28 69 plusmn 6 62 plusmn 4 48 plusmn 4lowast 31 plusmn 7lowast 16 plusmn 2lowast 64 plusmn 21amp 36 plusmn 3lowast 50 plusmn 8lowast

HU + H2

O 189 plusmn 15lowast 239 plusmn 15lowast 58 plusmn 6 51 plusmn 4 53 plusmn 13lowast 85 plusmn 5 42 plusmn 9lowast 102 plusmn 11 62 plusmn 6lowast 98 plusmn 4RA + H

2

O 224 plusmn 16lowast 251 plusmn 12lowast 77 plusmn 7 99 plusmn 7lowast 101 plusmn 12 103 plusmn 5 184 plusmn 31lowast 56 plusmn 7lowast 126 plusmn 13amp 98 plusmn 3Means plusmn SEM119899Number of animals 7ndash10Values expressed as a ratio the geneGAPDHMC + H

2O control group

HU huperzine A (05mgkg bw po)RA rosmarinic acid (10mgkg bw po)lowastampVersus MC + H

2O 119901 lt 005 or 119901 lt 007 respectively

an improvement of this paradigm was produced by extractof MO and HU but not RA when compared with controlanimals (MO + H

2O versus MC + H

2O 119901 lt 005 HU +

H2O versus MC + H

2O 119901 lt 005 RA + H

2O versus MC +

H2O 119901 gt 005) However the administration of SC to rats

significantly decreased the latency time of passive avoidancetask (MC + SC versus MC + H

2O 119901 lt 005) After MO or

RA combined treatment with SC no improvement of long-term memory was observed but HU given with SC showedenhancement of this paradigm in rats (HU + SC versusMC + SC 119901 lt 005) Therefore it can be concluded thatadministration of HU overcomes the effect shown by SC only(Table 1)

424 Short-Term Memory The results of the object recogni-tion test showed that an administration of the compoundsor extract did affect the ratsrsquo short-term memory (ANOVA119865(7 78) = 273 119901 lt 005) (Table 2)

Detailed post hoc analysis showed that only SC signifi-cantly decreased the short-term memory in MO-treated rats(MO + SC versusMO + H

2O 119901 lt 005MO + SC versus MC

+ SC 119901 lt 005) whereas the differences between the rest ofthe animals did not reach statistical significance (119901 gt 005)

43 AChE and BuChE Activities in Rat Brain A one-wayANOVA revealed significant differences between groups inthe activity of AChE in both the cortex and the hippocampus(frontal cortex 119865(3 27) = 565 119901 lt 005 hippocampusANOVA 119865(3 29) = 796 119901 lt 005) It was found out thatMOshowed an insignificant inhibition of AChE activity in thefrontal cortex by 24 when compared with control rats (MC+ H2O 119901 lt 006) and in the hippocampus by 7 (Table 3)

whereas HU produced a distinct significant inhibition ofAChE activity in comparison to control group by 48 (119901 lt005) and 47 (119901 lt 005) in the cortex and the hippocampusrespectively Also RA lowered significantly AChE activityboth in the cortex (38) and in the hippocampus (43)Moreover there were not significant differences between thevalues of BuChE activities for MO and HU when compared

with control group in the frontal cortex (ANOVA 119865(3 30) =101 119901 gt 005) whereas in the hippocampus the differencesreached statistical significance (ANOVA 119865(3 27) = 141119901 lt 005) Detailed analysis showed that only RA effect wassignificant and increased BuChE activity in the hippocampuswhen compared with the control rats (119901 lt 005)

44 AChE BuChE and BACE1 mRNA Level Changes in RatBrain A one-way ANOVA analysis revealed significant dif-ferences of AChE mRNA transcription profile in the cortex(ANOVA 119865(3 27) = 857 119901 lt 005) As shown in Table 3the multiple treatment of MO produced in the cortex astatistically significant decrease of AChEmRNA level by 52the administration of HU caused decrease of its level by 44(119901 lt 005) whereas RA did not affect this parameter whencompared to the control

There were significant differences between the relativevalues of BuChE mRNA levels in this region of brain in rats(frontal cortex ANOVA 119865(3 25) = 162 119901 lt 005) TheMOtreatment led to a decrease in the BuChEmRNA level by 84(versus MC + H

2O 119901 lt 005) the prolonged HU admin-

istration resulted in a decrease of the transcript level in thecortex by 58 (versus MC + H

2O 119901 lt 005) but RA

increased this parameter in the cortex by 84 (versus MC +H2O 119901 lt 005)The significant differences ofmRNA transcription level of

AChE mRNA level in the hippocampus (ANOVA 119865(3 31) =212 119901 lt 005) have been observed The detailed analysisshown that in the case of AChE afterMO treatment mRNAlevel significantly decreased by 69 (versus MC + H

2O

119901 lt 005) while the administration of HU resulted in astatistically insignificant decrease of AChE mRNA level by18 when compared with control group Also RA did notchange the level of transcript

In the case of BuChEmRNA expression in the hippocam-pus there were statistically significant differences betweengroups (ANOVA 119865(3 27) = 310 119901 lt 005) Detailed analysisshowed that in theMO + H

2O treated group the expression

lowered by 36 but the difference did not reach strong

12 Evidence-Based Complementary and Alternative Medicine

significance when compared with the control values (119901 lt007) and no change in the expression level of this enzymewas shown after the administration of HU On the contraryRA produced an increase of BuChE mRNA expression in thehippocampus by 44 (versus MC + H

2O 119901 lt 005)

Further analysis showed the significant differences inBACE1 mRNA expression in both brain regions of rats (inthe cortex and the hippocampus) (frontal cortex ANOVA119865(3 27) = 163 119901 lt 005 hippocampus ANOVA 119865(3 27) =133 119901 gt 005) It was observed that MO produced astatistically significant decrease of the BACE1 expression levelby 64 in the cortex (versus MC + H

2O 119901 lt 005) and by

50 in the hippocampus (versus MC + H2O 119901 lt 005) For

comparison HU treatment led to a decrease in the mRNAexpression level by 38 in the cortex (versus MC + H

2O

119901 lt 005) but not in the hippocampus On the contrary RAproduced an increase of this transcript in the cortex by 26but the effect did not reach a strong statistical significance(versus MC + H

2O 119901 lt 007) whereas in the hippocampus

there was no difference between RA and control group

5 Discussion

Cognitive and Behavioral Experiments The present studyinvestigated the influence of subchronic (28-fold) adminis-tration of standardized 50 EtOH extract ofMelisa officinalisleaf extract (MO) (200mgkg po containing 177mgkg ofRA) on SC-induced impairment of short-term and long-term memory in rats The results were compared with theactivity of cholinesterases (AChE and BuChE) as well aswith AChE BuChE and BACE1 gene expression levels in thecortex and hippocampus of the rat brain So far little evidenceis yet available as regards mechanisms of MO leaf extractaction that are potentially relevant to cognitive function ofrats after per os administration MO is traditionally used intreating neurological disorders through its anti-AChE [18]and antiagitation properties [37] Moreover Wake et al [38]and Kennedy et al [15] showed thatMO extract has nicotinicreceptor activity and that it can displace [3H]-(N)-nicotinefrom nicotinic receptors in homogenates of human cerebralcortex tissue and they suggested that these mechanisms canexplain activity of MO extract in amnesia model RecentlySoodi et al [18] observed that intraperitoneal injections ofMO extract (200mgkg) in rats could significantly enhancelearning and memory processes in animals since the extractsignificantly ameliorates SC-induced learning deficit in Mor-ris water maze test On the contrary in higher dose itcan be observed that MO extract (400mgkg) could notreverse SC-induced memory impairment [18] In our studyadministration of MO extract at a dose of 200mgkg (po)showed the effect leading to improvement of long-termmemory in a passive avoidance test in rats However afterMO combined treatment with SCMO did not overcome theimpairment shown by SC (Table 2) It should be emphasizedthat it is not clear whether the effect shown in our study byMO in non-SC-treated rats is specific since theMO treatmentproduced significant lowering of locomotor activity of ratstherefore the sedative profile ofMO cannot be excluded

On the other hand Ryu et al [39] observed that agitationand aggression are highly prevalent in patientswith dementiaAccording to Gitlin et al [40] nonpharmacological inter-ventions are recommended as first-line therapy Althoughantipsychotics have shown benefit for Alzheimerrsquos disease-related psychosis their use is associated with several seriousadverse effects [40]Thus it seems that the use ofMO extractcan provide dual benefits both in aspect of inhibition ofagitation and in improving the memory of patients withAlzheimerrsquos disease

Furthermore in our study RA in the dose of 10mgkgbw (po) did not affect either short- or long-term memoryalthough RA lowered significantly AChE activity both inthe cortex and in the hippocampus Moreover we observedthat the repeated administration of RA in non-scopolamine-treated rats did not produce any changes of locomotoractivity similarly to our previous study [23]

It is possible that other chemical compounds can influ-ence the memory in rats by synergic interactions in plantextract According to our calculations caffeic acid (0174mgin a single dose of extract administered to animals per kgbw) lithospermic acid (0084mgkg) salvianolic acid A(008mgkg) and salvianolic acid B (0046mgkg) may beresponsible for observed pharmacological effects On theother hand results from few studies showed that salvianolicacids do not cross the blood brain barrier (BBB) [41 42]and also lithospermic acid does not efficiently cross the BBB[43] For this reason the interpretation of our results is morecomplicated

Firstly Xu et al [44] demonstrated that Sal A is ametabol-ically unstable compound that would undergo rapid methy-lationmetabolism catalyzed by catechol O-methyltransferasein vivo into four major methylated metabolites of Sal A(3-O-methyl 31015840-O-methyl 3310158401015840-O-dimethyl and 31015840310158401015840-O-dimethyl salvianolic acid A) These generated O-methylatedmetabolites may be largely responsible for its in vivo phar-macological effects Although there are no available recentstudies on the ability of these compounds to pass the BBBsuch possibility should not be excluded

Secondly several studies showed central pharmacolog-ical effects in animals after per os administration of Sal B[45 46] Sal A [47] and caffeic acid [48] It was provedfor example that salvianolic acid B (10mgkg po) sig-nificantly rescued the A12057325ndash35 peptide-induced decreaseof choline acetyltransferase and brain-derived neurotrophicfactor protein levels in an amyloid 120573 (A120573) peptide-inducedAlzheimerrsquos disease mouse model [49] It also significantlyreversed (10mgkg po) the cognitive impairments inducedby scopolamine (1mgkg ip) or A120573 (25ndash35) (10 nmol5 120583Licv) injection inmice [45] Previous studies [46 47] showedalso that both Sal A and Sal B are able to improve theimpaired memory function induced by cerebral ischemia-reperfusion in miceThe stimulation of neurogenesis processin both subgranular zone (SGZ) and subventricular zone(SVZ) after brain ischemia and also alleviation neural cellsloss and improved motor function recovery after brainischemia in rats after the Sal B administration were alsoobserved by Zhong et al [50] Moreover the exposure to SalB can maintain the proliferation of neural stemprogenitor

Evidence-Based Complementary and Alternative Medicine 13

cells (NSPCs) after cerebral ischemia and improve cognitivepostischemic impairment after stroke in rats using Morriswater maze test therefore authors concluded that Sal B mayact as a potential drug in treatment of brain injury or neu-rodegenerative disease [51] Additionally the improvement ofmotor function after cerebral ischemia in rats after salvianolicacid B administrationwas also demonstrated [52]The clue topass through the BBB Sal B provided also results of Li et alindicating its protective effect on BBB in rats after cerebralischemia-reperfusion by inhibiting the MAPK pathway [53]In addition to this it was shown that lithospermic acid andsalvianolic acids exerted neuroprotective activity in variousexperimental models Lithospermic acid significantly attenu-ates neurotoxicity in vitro and in vivo induced by 1-methyl-4-phenylpyridin (MPP(+)) by blocking neuronal apoptotic andneuroinflammatory pathways [54] Salvianolic acid B inhib-ited amyloid beta-protein aggregation and fibril formation aswell as directly inhibiting the cellular toxicity of amyloid beta-protein in PC12 cells [55] and significantly reduced its cyto-toxic effects on human neuroblastoma SH-SY5Y cells [56]

For an explanation of our results observations of Pin-heiro Fernandes et al [57] may be very helpful whichshowed that caffeic acid nonflavanoid catecholic compoundwhose derivatives are occurring in MO extract improvedthe working spatial and long-term aversive memory deficitsinduced by focal cerebral ischemia in mice Anwar et al [48]showed also that caffeic acid (100mgkg) improved the step-down latencies in the inhibitory avoidance in rats Tsai et al[58] showed that caffeic acid is a potent neuroprotective agentin brain of 1-methyl-4-phenyl-1236-tetrahydropyridine(MPTP) treatedmiceMoreover caffeic acid improves A12057325ndash35-induced memory deficits and cognitive impairment inmice [59] In another study it was also shown that this com-pound has a significant protective effect on global cerebralischemia-reperfusion injury in rats [60] For instance 124 plusmn18mg100 g of caffeic acid was detected in the brain of micewith a diet containing 2 caffeic acid for 4 weeks [58] Thusthere is progressive evidence that this compound passesthrough the BBB and has a central pharmacological activity

Also results by Yoo et al are also worth noting [61] whichshowed also in a ratmodel of scopolamine-induced amnesiathat administration of luteolin (a common flavonoid frommany plants including M officinalis) at dose of 10mgkgcaused the increase in the brain-derived neurotrophic factor(BDNF) acetylcholine and the decrease in lipid peroxida-tion Liu et al [62] observed that chronic treatment withluteolin (50 and 100mgkg) improved neuronal injury andcognitive performance by attenuating oxidative stress andcholinesterase activity in streptozotocin-induced diabetes inrats In our study ethanolic extract of MO administered torats significantly improved long-term memory after using apassive avoidance test but afterMO combined treatmentwithSC no improvement of this paradigmwas observed (Table 2)

Thirdly available pharmacokinetic studies were carriedout for the pure compounds (Sal A and B) and the extractof the roots of Salvia miltiorrhiza [42 63] but there are notavailable studies of bioavailability of these compounds afteradministration of Melissa officinalis leaf extract containingother compounds as compared with the extract of Salvia

miltiorrhiza Moreover there is a lack of data about howscopolamine may influence the penetration of the salvianolicacids (and other compounds) across the BBB Hence thereis a need to study the pharmacokinetic parameters of thesecompounds in the group of animals treatedwith scopolaminein comparison with control group

AChEandBuChEActivities in Rat Brain To date several stud-ies have focused on explaining themechanismof action of theMS extract and its active compounds Soodi et al [18] showedthat treatment of animals withMO extract (400mgkg) priorto scopolamine injection could ameliorate scopolamine-induced enhancement in AChE activity This dose of MOextract inhibited the AChE activity in the hippocampus ofrats (519 versus 1004 in normal saline group 119901 lt 005914 in group treated with scopolamine +MO versus 1281in scopolamine group 119901 lt 005) In another study Anwaret al [48] showed that 50 and 100mgkg of caffeic aciddecreased in vivo the AChE activity in the cerebral cortexand striatum and increased the activity of this enzyme inthe cerebellum hippocampus hypothalamus pons lympho-cytes and muscles when compared to the control group(119901 lt 005) Our study showed that ethanolic extract of MOproduced an insignificant and slight inhibition of AChE andBuChE activity in the frontal cortex and in the hippocampus(especially) However it was observed previously [23] thatRA inhibited the AChE activity in the rats frontal cortex andhippocampusMoreover we showed that RA possess a strongstimulatory effect on BuChE in the hippocampus

AChE BuChE and BACE1mRNA Level Changes in Rat BrainSo far no results have been published of studies concerningthe in vivo assessment of changes in AChE BuChE andBACE1 gene expression profile in different brain regionsunder the influence of MO extracts or their key bioactivemetabolites Such studies focused overwhelmingly on theanalysis of their in vitro activities to a lesser extent in animalexperiments

In our study in the frontal cortex we have observedthe strongest inhibition of both AChE and BuCHE mRNAtranscription under the influence ofM officinalis extract andhuperzine A (Table 3) However a more significant differencein the level of transcript was seen in the frontal cortex inparticular in the case of BuCHE mRNA of experimentalrats (a decrease by 516 and 44 for MO and HU versuscontrol resp)The strong inhibition of transcription ofAChEmRNAwas also observed in the hippocampus ofMO-treatedrats (a decrease by 69) The BuChE mRNA transcriptionin animals receiving MO was lowered in a moderate way(decrease by 36)HuperzineA alone has not caused changesthat were observed in the group ofMO (Table 2) Our findingsmostly correlate with the observed changes in activities ofAChE and BuChE in different groups of animals In the caseof BuChE slightly different results between its activity andexpression profile were especially seen in the frontal cortexand hippocampus of animals receivingMO (Table 3)

To date there is a lack of published results of studiesmaking an attempt to clarify the potential differences in thetranscriptional profile and activity of AChE and BuChE

14 Evidence-Based Complementary and Alternative Medicine

under the influence of MO and its bioactive metabolites Itcannot be excluded that the key for the observed differencesin the level of AChE activities and mRNA level can becaused by changes in the activity of AChE in other regionsof the brain not analyzed in this study such as substantianigra cerebellum globus pallidus and hypothalamus whereit exerts nonenzymatic neuromodulatory functions affect-ing neurite outgrowth and synaptogenesis modulating theactivity of other proteins regional cerebral blood flow andother functions [64] But it is difficult to clearly explain whydiminishing of AChE and BuChEmRNAs did not always cor-relate with the lowering of activity of these enzymes althoughit has been recently noted that AChE activity was notparalleled by an increase in mRNA levels [65] The authorsexplained this fact by stating that AChE levels are regulatedat transcriptional posttranscriptional and posttranslationallevels leading to complex expression patterns which canbe modulated by physiological and pathological conditionsHowever these mechanisms are not fully understood andfurther studies are needed in this field

The cause of observed different degree of inhibition ofactivity and transcription status of studied genes especiallyof BuChE (and AChE) may lie in a so-called ldquonegative feed-backrdquo consisting of a complicated transcriptiontranslationregulation protein-protein interactionsmodifications and ametabolic network together forming a system that allows thecell to respond sensibly to the multiple signal molecules thatexist in its environment [66]

Because of that we propose that the reason for differencesbetween AChE and BuChE activities in the cortex and hip-pocampus under the MO may be due to the fact of insuf-ficiency of applied dose and the duration of the experi-ment affecting the transcriptional tissue-specific cellularmachinery regulating BuChE transcription without affectingits activity Moreover the administration of higher doses ofMO and extended period of time could lead to sufficientinhibition of its activity

There is a need to conduct further studies to determinethe molecular degree of dependence between changes inAChE and BuChE activities and activities of potential keyfactors regulating expression of these genes in the frontalcortex and hippocampus under the influence of extracts ofMO and active metabolites Another study determining theeffectiveness of their actions on the cholinergic system inexperimental animal models of memory impairment withparticular emphasis on scopolamine including the determi-nation of changes at the molecular level should be thereforecarried out

Alterations in BACE1 protein level have been proved inpostmortem brain tissue from individuals with AD withincreases decreases and also no change reported [1 67 68]An example of confirmation of these findings at the mRNAlevel is results of study by Coulson et al [69] Based onconducted studies some authors suggested that increasedBACE1mRNA transcription in remaining neuronal cells maycontribute to the increased BACE1 protein levels and activityfound in brain regions affected by AD [70]

Results of many studies concerning the elevated level ofBACE1 mRNA and protein in Alzheimerrsquos disease provide

direct and compelling reasons to develop therapies directed atBACE1 inhibition thus reducing120573-amyloid and its associatedtoxicities [67 68]

In our study we have observed that BACEmRNA expres-sion statistically significantly decreased after MO adminis-tration in frontal cortex and hippocampus These resultssuggest that the MO extract may act to inhibit BACE1mRNA level given the fact that the percentage of inhibitionof the expression (64 in the frontal cortex 50 in thehippocampus) is higher than that in the case of HU (38in the cortex and the lack of changes in the hippocampus)A careful analysis of the literature data shows that there areno studies which analyzed the impact of MO extract on theexpression level of BACE1 in Alzheimerrsquos disease

Furthermore a literature analysis does not indicatealready published results conducted by other teams attempt-ing to assess the impact of RA on the transcriptional activityof AChE BuChE and BACE1 Although several studies(already mentioned and others [70 71]) highlighted theRA and other caffeic acid derivatives capability of acetyl-cholinesterases inhibition none of these studies does nottouch the question of the molecular basis of their impacton the transcriptional machinery that regulates in vivo theexpression of studied genes Hence in our opinion obtainedby our team results they are one of the first of this typeand in general correlate with the results of our previousstudy [23] In this case there is no clear evidence explainingdifferent responses at the transcriptional level under theinfluence of RA especially in the case of BuChE encodinggene in the hippocampus (Table 2) It is possible that theobserved differentiation of BuChE transcriptional activitybetween the frontal cortex and the hippocampus may bedue to the differences of butyrylcholinesterase localizationand substrate affinity [72] Since in our experiment we havecarried out a quantitative analysis of AChE BuChE andBACE1 transcripts in brain homogenates of tested animalsrather than in individual isolated cell fractions therefore theobtained results constitute an overall ldquopicturerdquo of both studiedgenes transcriptional changes occurring in the brain areas ofstudied animals under the influence of the RA and the wholeplant extract as well

6 Conclusion

The subchronic administration ofMO led to an improvementof long-term memory of rats however the mechanisms ofMO action are probably more complicated since its role asa modulator of beta-secretase activity (due to inhibition ofBACE1 mRNA expression in frontal cortex) should be takeninto consideration

It should be noted that we have studied a crude extractfrom leaf of Melissa officinalis not a single pure chemicalcompound This plant extract is a complex mixture and itsaction may be a result of the summation of activities ofseveral components (synergismadditive action of caffeic acidwith salvianolic acids rosmarinic acid and others) In thecase of extract from leaves ofMelissa officinalis it is possiblethat interactions occur between the 40 chemical compoundsidentified by HPLC system

Evidence-Based Complementary and Alternative Medicine 15

Taken together it seems that the MO activity representsa possible option as complementary interventions to relievethe symptoms of mild dementia

Competing Interests

The authors declare that they have no competing interests

Acknowledgments

This work was supported by the Ministry of Science andHigher EducationWarsaw Poland from educational sources(2008ndash2011 Grant no N 405417836)

References

[1] S L Cole and R Vassar ldquoThe Alzheimerrsquos disease 120573-secretaseenzyme BACE1rdquo Molecular Neurodegeneration vol 2 no 1article 22 2007

[2] LACraigN SHong andR JMcDonald ldquoRevisiting the cho-linergic hypothesis in the development of Alzheimerrsquos diseaserdquoNeuroscience amp Biobehavioral Reviews vol 35 no 6 pp 1397ndash1409 2011

[3] N G M Gomes M G Campos J M C Orfao and C A FRibeiro ldquoPlants with neurobiological activity as potential tar-gets for drug discoveryrdquo Progress in Neuro-Psychopharmacologyamp Biological Psychiatry vol 33 no 8 pp 1372ndash1389 2009

[4] WHO WHO Monographs on Selected Medicinal PlantsmdashVolume 2 WHO Geneva Switzerland 2004 httpappswhointmedicinedocsendJs4927e18htmlJs4927e18

[5] M Bayat A A Tameh M H Ghahremani et al ldquoNeuropro-tective properties of Melissa officinalis after hypoxic-ischemicinjury both in vitro and in vivordquo DARU Journal of Pharmaceu-tical Sciences vol 20 article 42 2012

[6] J P Kamdem A Adeniran A A Boligon et al ldquoAntioxidantactivity genotoxicity and cytotoxicity evaluation of lemon balm(Melissa officinalis L) ethanolic extract Its potential role inneuroprotectionrdquo Industrial Crops and Products vol 51 pp 26ndash34 2013

[7] J-T Lin Y-C Chen Y-C Lee C-W R Hou F-L Chen andD-J Yang ldquoAntioxidant anti-proliferative and cyclooxygenase-2 inhibitory activities of ethanolic extracts from lemon balm(Melissa officinalis L) leavesrdquo LWTmdashFood Science and Technol-ogy vol 49 no 1 pp 1ndash7 2012

[8] G Guginski A P Luiz M D Silva et al ldquoMechanisms involvedin the antinociception caused by ethanolic extract obtainedfrom the leaves of Melissa officinalis (lemon balm) in micerdquoPharmacology Biochemistry amp Behavior vol 93 no 1 pp 10ndash162009

[9] N Perry G Court N Bidet J Court and E Perry ldquoEuropeanherbs with cholinergic activities potential in dementia therapyrdquoInternational Journal of Geriatric Psychiatry vol 11 no 12 pp1063ndash1069 1996

[10] E K Perry A T Pickering W W Wang P J Houghtonand N S L Perry ldquoMedicinal plants and Alzheimerrsquos diseasefrom ethnobotany to phytotherapyrdquo Journal of Pharmacy andPharmacology vol 51 no 5 pp 527ndash534 1999

[11] M-J R Howes N S L Perry and P J Houghton ldquoPlants withtraditional uses and activities relevant to the management ofAlzheimerrsquos disease and other cognitive disordersrdquo Phytother-apy Research vol 17 no 1 pp 1ndash18 2003

[12] I Orhan and M Aslan ldquoAppraisal of scopolamine-inducedantiamnesic effect in mice and in vitro antiacetylcholinesteraseand antioxidant activities of some traditionally used Lamiaceaeplantsrdquo Journal of Ethnopharmacology vol 122 no 2 pp 327ndash332 2009

[13] S Akhondzadeh M Noroozian M Mohammadi S OhadiniaA H Jamshidi and M Khani ldquoMelissa officinalis extract inthe treatment of patients with mild to moderate Alzheimerrsquosdisease a double blind randomised placebo controlled trialrdquoJournal of Neurology Neurosurgery and Psychiatry vol 74 no 7pp 863ndash866 2003

[14] D O Kennedy A B Scholey N T J Tildesley E K Perry andK AWesnes ldquoModulation ofmood and cognitive performancefollowing acute administration of Melissa officinalis (lemonbalm)rdquo Pharmacology Biochemistry amp Behavior vol 72 no 4pp 953ndash964 2002

[15] D O Kennedy G Wake S Savelev et al ldquoModulation of moodand cognitive performance following acute administration ofsingle doses of Melissa officinalis (Lemon balm) with humanCNS nicotinic and muscarinic receptor-binding propertiesrdquoNeuropsychopharmacology vol 28 no 10 pp 1871ndash1881 2003

[16] D O Kennedy and E L Wightman ldquoHerbal extracts and phy-tochemicals plant secondarymetabolites and the enhancementof human brain functionrdquoAdvances inNutrition vol 2 no 1 pp32ndash50 2011

[17] R P Pereira A A Boligon A S Appel et al ldquoChemical com-position antioxidant and anticholinesterase activity of Melissaofficinalisrdquo Industrial Crops and Products vol 53 pp 34ndash452014

[18] M Soodi N Naghdi H Hajimehdipoor S Choopani andE Sahraei ldquoMemory-improving activity of Melissa officinalisextract in naıve and scopolamine-treated ratsrdquo Research inPharmaceutical Sciences vol 9 no 2 pp 107ndash114 2014

[19] K Dastmalchi V Ollilainen P Lackman et al ldquoAcetyl-cholinesterase inhibitory guided fractionation of Melissa offic-inalis Lrdquo Bioorganic amp Medicinal Chemistry vol 17 no 2 pp867ndash871 2009

[20] W Chaiyana and S Okonogi ldquoInhibition of cholinesterase byessential oil from food plantrdquo Phytomedicine vol 19 no 8-9 pp836ndash839 2012

[21] K Dastmalchi H J D Dorman P P Oinonen Y Darwis ILaakso and R Hiltunen ldquoChemical composition and in vitroantioxidative activity of a lemon balm (Melissa officinalis L)extractrdquo LWTmdashFood Science and Technology vol 41 no 3 pp391ndash400 2008

[22] P Pereira D Tysca P Oliveira L F D S Brum J N Picadaand P Ardenghi ldquoNeurobehavioral and genotoxic aspects ofrosmarinic acidrdquo Pharmacological Research vol 52 no 3 pp199ndash203 2005

[23] M Ozarowski P L Mikolajczak A Bogacz et al ldquoRosmarinusofficinalis L leaf extract improves memory impairment andaffects acetylcholinesterase and butyrylcholinesterase activitiesin rat brainrdquo Fitoterapia vol 91 pp 261ndash271 2013

[24] G P Kumar and F Khanum ldquoNeuroprotective potential of phy-tochemicalsrdquo Pharmacognosy Reviews vol 6 no 12 pp 81ndash902012

[25] M Stobiecki A Staszkow A Piasecka P M Garcia-LopezF Zamora-Natera and P Kachlicki ldquoLC-MSMS profiling offlavonoid conjugates in wild mexican lupine Lupinus reflexusrdquoJournal of Natural Products vol 73 no 7 pp 1254ndash1260 2010

[26] A Wojakowska A Piasecka P M Garcıa-Lopez et al ldquoStruc-tural analysis and profiling of phenolic secondarymetabolites of

16 Evidence-Based Complementary and Alternative Medicine

Mexican lupine species using LC-MS techniquesrdquo Phytochem-istry vol 92 pp 71ndash86 2013

[27] A Gryszczynska B Opala Z Lowicki et al ldquoBioactive com-pounds determination in the callus and hydroalcoholic extractsfrom Salvia miltiorrhiza and Salvia przewalskiimdashpreliminarystudy on their anti-alcoholic activity effectsrdquo PhytochemistryLetters vol 11 pp 399ndash403 2015

[28] K Slinkart and V L Singleton ldquoTotal phenol analysis automa-tion and comparison with manual methodrdquo American Journalof Enology and Viticulture vol 28 no 1 pp 49ndash55 1977

[29] P J R Boissier J Tardy and J C Diverres ldquoUne nouvellemethode simple pour explorer lrsquoaction lsquotranquillisantersquo le testde la chemineerdquo Medicina Experimentalis vol 3 no 1 pp 81ndash84 1960

[30] R Ader J A Weijnen and P Moleman ldquoRetention of a passiveavoidance response as a function of the intensity and durationof electric shockrdquo Psychonomic Science vol 26 no 3 pp 125ndash128 1972

[31] E M Blalock K-C Chen K Sharrow et al ldquoGene microar-rays in hippocampal aging statistical profiling identifies novelprocesses correlated with cognitive impairmentrdquo Journal ofNeuroscience vol 23 no 9 pp 3807ndash3819 2003

[32] K Isomae S Morimoto H Hasegawa K Morita and JKamei ldquoEffects of T-82 a novel acetylcholinesterase inhibitoron impaired learning and memory in passive avoidance task inratsrdquo European Journal of Pharmacology vol 465 no 1-2 pp97ndash103 2003

[33] J Patora and B Klimek ldquoFlavonoids from lemon balm (Melissaofficinalis L Lamiaceae)rdquo Acta Poloniae Pharmaceutica-DrugResearch vol 59 no 2 pp 139ndash143 2002

[34] L Barros M Duenas M I Dias M J Sousa C Santos-Buelgaand I C F R Ferreira ldquoPhenolic profiles of cultivated invitro cultured and commercial samples of Melissa officinalis Linfusionsrdquo Food Chemistry vol 136 no 1 pp 1ndash8 2013

[35] T L Miron M Herrero and E Ibanez ldquoEnrichment of antiox-idant compounds from lemon balm (Melissa officinalis) bypressurized liquid extraction and enzyme-assisted extractionrdquoJournal of Chromatography A vol 1288 pp 1ndash9 2013

[36] Y Lu L Y Foo and H Wong ldquoSagecoumarin a novel caffeicacid trimer from Salvia officinalisrdquo Phytochemistry vol 52 no6 pp 1149ndash1152 1999

[37] S Abuhamdah LHuangM S J Elliott et al ldquoPharmacologicalprofile of an essential oil derived from Melissa officinalis withanti-agitation properties focus on ligand-gated channelsrdquo Jour-nal of Pharmacy and Pharmacology vol 60 no 3 pp 377ndash3842008

[38] G Wake J Court A Pickering R Lewis R Wilkins andE Perry ldquoCNS acetylcholine receptor activity in Europeanmedicinal plants traditionally used to improve failing memoryrdquoJournal of Ethnopharmacology vol 69 no 2 pp 105ndash114 2000

[39] S-H Ryu C Katona B Rive and G Livingston ldquoPersistenceof and changes in neuropsychiatric symptoms in Alzheimerdisease over 6months the LASER-AD studyrdquoAmerican Journalof Geriatric Psychiatry vol 13 no 11 pp 976ndash983 2005

[40] L N Gitlin H C Kales and C G Lyketsos ldquoNonpharma-cologic management of behavioral symptoms in dementiardquoJournal of the AmericanMedical Association vol 308 no 19 pp2020ndash2029 2012

[41] A Konczol J Muller E Foldes et al ldquoApplicability of a blood-brain barrier specific artificial membrane permeability assay atthe early stage of natural product-based CNS drug discoveryrdquoJournal of Natural Products vol 76 no 4 pp 655ndash663 2013

[42] Y-J Zhang L Wu Q-L Zhang J Li F-X Yin and Y YuanldquoPharmacokinetics of phenolic compounds of Danshen extractin rat blood and brain by microdialysis samplingrdquo Journal ofEthnopharmacology vol 136 no 1 pp 129ndash136 2011

[43] X Li C Yu Y Lu et al ldquoPharmacokinetics tissue distributionmetabolism and excretion of depside salts from Salvia miltior-rhiza in ratsrdquo Drug Metabolism and Disposition vol 35 no 2pp 234ndash239 2007

[44] H Xu Y Li X Che H Tian H Fan and K Liu ldquoMetabolismof salvianolic acid a and antioxidant activities of its methylatedmetabolitesrdquo Drug Metabolism and Disposition vol 42 no 2pp 274ndash281 2014

[45] D H Kim S J Park J M Kim et al ldquoCognitive dysfunctionsinduced by a cholinergic blockade and A120573 25-35 peptide areattenuated by salvianolic acid Brdquo Neuropharmacology vol 61no 8 pp 1432ndash1440 2011

[46] G-H Du Y Qiu and J-T Zhang ldquoSalvianolic acid B protectsthe memory functions against transient cerebral ischemia inmicerdquo Journal of Asian Natural Products Research vol 2 no 2pp 145ndash152 2000

[47] G Du and J Zhang ldquoProtective effects of salvianolic acid Aagainst impairment of memory induced by cerebral ischemia-reperfusion in micerdquo Chinese Medical Journal vol 110 no 1 pp65ndash68 1997

[48] J Anwar R M Spanevello G Thome et al ldquoEffects of caffeicacid on behavioral parameters and on the activity of acetyl-cholinesterase in different tissues from adult ratsrdquo Pharmacol-ogy Biochemistry and Behavior vol 103 no 2 pp 386ndash394 2012

[49] YW Lee D H Kim S J Jeon et al ldquoNeuroprotective effects ofsalvianolic acid B on anA12057325-35 peptide-inducedmousemodelof Alzheimerrsquos diseaserdquo European Journal of Pharmacology vol704 no 1ndash3 pp 70ndash77 2013

[50] J Zhong M-K Tang Y Zhang Q-P Xu and J-T ZhangldquoEffect of salvianolic acid B on neural cells damage and neu-rogenesis after brain ischemia-reperfusion in ratsrdquo Yao Xue XueBao vol 42 no 7 pp 716ndash721 2007

[51] P Zhuang Y Zhang G Cui et al ldquoDirect stimulation of adultneural stemprogenitor cells in vitro and neurogenesis in vivo bysalvianolic acid Brdquo PLoS ONE vol 7 no 4 Article ID e356362012

[52] M TangW Feng Y Zhang J Zhong and J Zhang ldquoSalvianolicacid B improves motor function after cerebral ischemia in ratsrdquoBehavioural Pharmacology vol 17 no 5-6 pp 493ndash498 2006

[53] Q Li L-P Han Z-H Li J-T Zhang and M-K Tang ldquoSal-vianolic acid B alleviate the disruption of blood-brain barrierin rats after cerebral ischemia-reperfusion by inhibiting MAPKpathwayrdquo Yao Xue Xue Bao vol 45 no 12 pp 1485ndash1490 2010

[54] Y-L LinH-J Tsay T-H Lai T-T Tzeng andY-J Shiao ldquoLith-ospermic acid attenuates 1-methyl-4-phenylpyridine-inducedneurotoxicity by blocking neuronal apoptotic and neuroinflam-matory pathwaysrdquo Journal of Biomedical Science vol 22 article37 2015

[55] M-K Tang and J-T Zhang ldquoSalvianolic acid B inhibits fibrilformation and neurotoxicity of amyloid beta-protein in vitrordquoActa Pharmacologica Sinica vol 22 no 4 pp 380ndash384 2001

[56] S S K Durairajan Q Yuan L Xie et al ldquoSalvianolic acid Binhibits A120573 fibril formation and disaggregates preformed fibrilsand protects against A120573-induced cytotoxictyrdquo NeurochemistryInternational vol 52 no 4-5 pp 741ndash750 2008

[57] FD Pinheiro Fernandes A P FonteneleMenezes J C de SousaNeves et al ldquoCaffeic acid protects mice from memory deficits

Evidence-Based Complementary and Alternative Medicine 17

induced by focal cerebral ischemiardquo Behavioural Pharmacologyvol 25 no 7 pp 637ndash647 2014

[58] S-J Tsai C-Y Chao andM-C Yin ldquoPreventive and therapeu-tic effects of caffeic acid against inflammatory injury in striatumof MPTP-treated micerdquo European Journal of Pharmacology vol670 no 2-3 pp 441ndash447 2011

[59] J H Kim QWang J M Choi S Lee and E J Cho ldquoProtectiverole of caffeic acid in an 119860120573

25minus35

-induced Alzheimerrsquos diseasemodelrdquo Nutrition Research and Practice vol 9 no 5 pp 480ndash488 2015

[60] G Liang B Shi W Luo and J Yang ldquoThe protective effect ofcaffeic acid on global cerebral ischemia-reperfusion injury inratsrdquo Behavioral and Brain Functions vol 11 no 1 article 182015

[61] D Y Yoo J H ChoiW Kim et al ldquoEffects of luteolin on spatialmemory cell proliferation and neuroblast differentiation in thehippocampal dentate gyrus in a scopolamine-induced amnesiamodelrdquo Neurological Research vol 35 no 8 pp 813ndash820 2013

[62] Y Liu X Tian L Gou L Sun X Ling and X Yin ldquoLuteolinattenuates diabetes-associated cognitive decline in ratsrdquo BrainResearch Bulletin vol 94 pp 23ndash29 2013

[63] S-M Liu Z-H Yang andX-B Sun ldquoSimultaneous determina-tion of six Salvia miltiorrhiza gradients in rat plasma and brainby LC-MSMSrdquo Zhongguo Zhongyao Zazhi vol 39 no 9 pp1704ndash1708 2014

[64] M A Papandreou A Dimakopoulou Z I Linardaki et alldquoEffect of a polyphenol-rich wild blueberry extract on cognitiveperformance of mice brain antioxidant markers and acetyl-cholinesterase activityrdquo Behavioural Brain Research vol 198 no2 pp 352ndash358 2009

[65] M-S Garcıa-Ayllon O Cauli M-X Silveyra et al ldquoBraincholinergic impairment in liver failurerdquo Brain vol 131 no 11pp 2946ndash2956 2008

[66] G K Ferreira M Carvalho-Silva C L Goncalves et al ldquoL-Tyrosine administration increases acetylcholinesterase activityin ratsrdquo Neurochemistry International vol 61 no 8 pp 1370ndash1374 2012

[67] R Vassar P-H Kuhn C Haass et al ldquoFunction therapeuticpotential and cell biology of BACE proteases current status andfuture prospectsrdquo Journal of Neurochemistry vol 130 no 1 pp4ndash28 2014

[68] R Vassar ldquoThe 120573-secretase BACE a prime drug target forAlzheimerrsquos diseaserdquo Journal of Molecular Neuroscience vol 17no 2 pp 157ndash170 2001

[69] D T R Coulson N Beyer J G Quinn et al ldquoBACE1 mRNAexpression in Alzheimerrsquos disease postmortem brain tissuerdquoJournal of Alzheimerrsquos Disease vol 22 no 4 pp 1111ndash1122 2010

[70] S Vladimir-Knezevic B Blazekovic M Kindl J Vladic A DLower-Nedza and A H Brantner ldquoAcetylcholinesterase inhib-itory antioxidant and phytochemical properties of selectedmedicinal plants of the lamiaceae familyrdquoMolecules vol 19 no1 pp 767ndash782 2014

[71] F Marcelo C Dias A Martins et al ldquoMolecular recognitionof rosmarinic acid from Salvia sclareoides extracts by ace-tylcholinesterase a new binding site detected by NMR spec-troscopyrdquo ChemistrymdashA European Journal vol 19 no 21 pp6641ndash6649 2013

[72] G Johnson and S W Moore ldquoWhy has butyrylcholinesterasebeen retained Structural and functional diversification in aduplicated generdquoNeurochemistry International vol 61 no 5 pp783ndash797 2012

[73] R J Grayer M R Eckert N C Veitch et al ldquoThe chemo-taxonomic significance of two bioactive caffeic acid estersnepetoidins A and B in the Lamiaceaerdquo Phytochemistry vol 64no 2 pp 519ndash528 2003

[74] A M D El-Mousallamy U W Hawas and S A M HusseinldquoTeucrol a decarboxyrosmarinic acid and its 41015840-O-triglycosideteucroside from Teucrium pilosumrdquo Phytochemistry vol 55 no8 pp 927ndash931 2000

[75] I Fecka and S Turek ldquoDetermination of water-soluble polyphe-nolic compounds in commercial herbal teas from Lamiaceaepeppermint melissa and sagerdquo Journal of Agricultural and FoodChemistry vol 55 no 26 pp 10908ndash10917 2007

[76] L W Sumner A Amberg D Barrett et al ldquoProposed min-imum reporting standards for chemical analysis WorkingGroup (CAWG) Metabolomics Standards Initiative (MSI)rdquoMetabolomics vol 3 pp 211ndash221 2007

Submit your manuscripts athttpwwwhindawicom

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Evidence-Based Complementary and Alternative Medicine

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Evidence-Based Complementary and Alternative Medicine 3

out on an XBridge C18 column (150 times 21mm 35 120583mparticlesize) using water acidified with 01 formic acid (solvent A)and acetonitrile (solvent B) with the mobile phase flow of02mLmin in the following gradient 0ndash25min from 10to 30 B 25ndash46min to 98 B and being maintained atthese conditions until 51min Up to 52min system returnedto the starting conditions and was reequilibrated for 5minThe most important MS parameters were as follows the ionsource ESI voltage minus4 kV or 4 kV nebulization of nitrogenat a pressure of 30 psi at a gas flow rate 9 Lmin ion sourcetemperature at 310∘C and skimmer 1 minus10VThe spectra werescanned in the range of 50ndash3000119898119911 The second systemconsisted of UPLC (the Acquity system Waters MilfordUSA) hyphenated to QExactive hybrid MSMS quadrupole-Orbitrap mass spectrometer Chromatographic separationsin this system were carried out using water acidified with01 formic acid (solvent A) and acetonitrile (solvent B)with the mobile phase flow of 04mLmin in the followinggradient 0ndash5min from 10 to 25 B 5ndash13min to 98 Band being maintained at these conditions until 145min Upto 15min system returned to the starting conditions andwas reequilibrated for 3min QExactive MS operated uponthe following settings the HESI ion source voltage minus3 kVor 3 kV The sheath gas flow was 48 Lmin auxiliary gasflow 13 Lmin ion source capillary temperature 250∘C andauxiliary gas heater temperature 380∘C The CID MSMSexperiments were performed using collision energy of 15 eVThe MSn (up to the MS5) and MSMS spectra were recordedin the negative and positive ion modes using the previouslypublished approach [23 25 26] The individual compoundswere identified via comparison of the exact molecular masses(measured in most cases with Δ below 1 ppm) mass spectraand retention times to those of the standard compoundsas well as the databases available online (PubChem ChEBIMetlin and KNApSAck) and literature data

35 Quantitative HPLC Analysis Sample was extracted by70 ethanol After sonification solution was cooled downand filtered through membrane filter HPLC method wasused to determine RA in a dry ethanolic extract The Lichro-spher 100 RP-18e (125mm 40mm 5 umMerck) was appliedfor identification of this active compound Temperature ofcolumn was 35∘C detection of RA was at 205 nm andflow rate was 15mLmin Mobile phase A was H

3PO4 H2O

(1 999)mobile phase Bwas acetonitrile Timewas as follows0min 10 B 13min 22 B 14min 40 B 25min 40 B

Moreover for identification of other chemical com-pounds a Zorbax Poroshell 120 SB-C18 column 27mm30mm times 100mm (Agilent) was used The lithospermic acidand salvianolic acid B were detected at 250 nm salvianolicacid A was detected at 280 nm The gradient mixtures ofphase Amdashwater H

3PO4(100 002 VV)mdashand of phase Bmdash

acetonitrile tetrahydrofurane (100 2 VV)mdashwere used aseluents Peaks were identified by addition of standard solu-tions and by UV-Vis spectra The quantification of thesecompounds was achieved using calibration curves preparedwith pure compoundsThe flow rate was 07mLmin columntemperature 27∘C and sample injection 5mL The gradient

mixtures program was as follows 0min 5 B 3min 10B 5min 12 B 11min 217 B 15min 39 B 39min 39B 70min 70 B and detection of compounds took place at250 nm 280 nm and 330 nm [27]

36 Determination of Total Phenolic Compounds in theExtract The calculation of polyphenols to gallic acid wasdone using the Folin-Ciocalteu reagent with the spectropho-tometric modified method described by Slinkart and Single-ton [28]

37 Determination of Total Hydroxycinnamic AcidDerivativesDetermination of total hydroxycinnamic acid derivativescalculated on rosmarinic acidwas performed according to theprocedure described in EurPh 50

38 Distillation of Essential Oil The essential oil contentswere determined by way of stream distillation in Deryngrsquosapparatus according to EurPh 50 1000 g of the dry hy-droethanolic MO leaf extract (separate sample) was placedin a round-bottom flask Then 5000mL distilled water and03mL xylenwere added and boiled inDeryngrsquos apparatus for3 h

39 Gas Chromatography Analysis Gas chromatography(GC) analyses were carried out using a Perkin-Elmer Clarus500 gas chromatograph with a data processing system andan FID (GC-FID) Separation was achieved by using an EliteFFAP fused-silica capillary column (30m long 032mm ininternal diameter and 025 120583mof film thickness)The injectorand detector temperatures were 220∘C Helium was used as acarrier gas with a flow of 15mLminminus1 A sample of 10 120583Lwasinjected using slit mode (split ratio 1 100) The results werereported as the relative percentage of the total peak area

310 Animals Experiments with rats were performed inaccordance with Polish governmental regulations (Dz U0533289) The study was conducted in accordance withethical guidelines for investigations in conscious animals andthe study protocol was approved by the Local Ethics Com-mittee of the Use of Laboratory Animals in Poznan Poland(642008) The experiments were performed on male six-week-oldWistar rats housed in controlled room temperature(20 plusmn 02 C) and humidity (65ndash75) under a 12 h 12 h light-dark cycle (lights on at 7 am) Animals were kept in groupsin amounts of 8ndash10 in light plastic cages (60times 40times 40 cm) andhad a free access to standard laboratory diet (pellets-LabofeedB) and to tap water in their cages

311 Treatments The rats were treated with hydroethanolicextract ofMelissa officinalis leaf (MO) in a dose of 200mgkgbw intragastrically (po) (groups MO + H

2O and MO +

SC) for 28 (28x) consecutive days For comparative purposeshuperzine A (HU) was administered chronically (28x) ina dose 05mgkg bw (po) (groups HU + H

2O and HU

+ SC) as a known acetylcholinesterase inhibitor Moreoverrosmarinic acid (Sigma-Aldrich) (RA) was applied (28x) in adose of 10mgkg bw (po) (groups RA + H

2O and RA + SC)

4 Evidence-Based Complementary and Alternative Medicine

as a comparative chemical compound On the last day 30minafter the last dose of MO or HU SC was given intraperi-toneally (ip) in a dose of 05mgkg bw Control groups weretreated with 05 methylcellulose (MC) whereas water forinjection (H

2O) was used as a vehicle for SC (groups MC +

H2O and MC + SC) MO was prepared ex tempore before

administration and suspended in MC in concentrations of20mgmLOnday 28 of the experiment 1 h after the last dosethe animals were killed by decapitation and hippocampus andpart of frontal cortex were collected from brain of rats Thetissue samples were then stored at minus80∘C until measurementof acetylcholinesterase (AChE) and butyrylcholinesterase(BuChE) activities or mRNA level changes

312 Cognitive and Behavioral Tests Cognitive and behav-ioral tests were used in the present study similarly as in ourprevious report [23] (1) sedative activity was assessed usinga locomotor activity test (2) motor coordination assessmentwas done using a ldquochimneyrdquo test (3) the passive avoidance testwas performed as an animalmodel for the assessment of long-termmemory and (4) the object recognition test was used asan animal model for the assessment of short-term memory

3121 Measurement of Locomotor Activity Locomotor activ-ity assessment was performed with licensed activity meter(Activity Cage Ugo Basile Italy) by placing the animals in thecentre of the apparatus and recording their horizontal activity[23] The data obtained were expressed as signals corre-sponding to animalmovements for 5minutesThe locomotoractivity was measured 30 minutes after the administration ofa single dose of SCH

2OAny distracting factors were reduced

to the minimum (noise presence of people and presence ofother rats)

3122Measurement ofMotorCoordination Motor coordina-tion was evaluated using ldquochimneyrdquo test described originallyfor mice [29] Thirty minutes after SC or vehicle injectionrat was allowed to enter a glass laboratory cylinder that is500mm long and 80mm in diameter laid on its side Uponreaching its bottom by the animal position of the cylinderwas rapidly changed from horizontal to vertical and a timerstarted The animal immediately began to move backwardsThe timer was stopped after the rat left the cylinder andassumed a sitting posture on the top of the vessel The timeof exit from the cylinder was accepted as a measure of motorcoordinationMotor impairment was assessed as the inabilityof rats to climb backwards up the tube within 60 s

3123 Passive Avoidance Test Passive avoidance test wasused as an animal model for the assessment of long-termmemory (effects on retrieval and memory consolidation)[30] The test relies on the natural preference of rats for dark-ness After 2 minutes of habituation to a dark compartmenta rat was placed on the illuminated platform and allowedto enter the dark compartment using licensed apparatus(Passive Avoidance System step through Ugo Basile Italy)Two more approach trials were allowed on the following daywith a two-minute interval between them At the end of

the second trial an unavoidable scrambled electric footshock(500120583A AC 3 s) was delivered through the grid floor of thedark compartment (learning trial) Retention of the passiveavoidance response (latency) was tested 24 h later by placingthe animal on the platformandmeasuring the latency in reen-tering the dark compartment against the arbitrary maximumtime of 180 s The test was performed after 30 minutes afterthe administration of a single dose of SC or the vehicle

3124 Object Recognition Test Object recognition test wasused as an animal model for the assessment of short-termmemory [31]The object recognition task took place in a 40 times60 cm open box surrounded by 40 cm high walls made ofplywood with a frontal glass wall All animals were submittedto a habituation session where they were allowed to freelyexplore the open field for 5min No objects were placed inthe box during the habituation trial On the day of testing theanimals were given an additional 3min rehabituation periodprior to commencing the test The test was divided into threephases with two trials the acquisition trial the retention trialand an intertrial interval of varying times

(i) Acquisition trial in this first trial the animals ex-plored two identical objects (1198601 and 1198602) for a periodof 3min positioned in two adjacent corners 10 cmfrom the walls

(ii) Intertrial interval (ITI) the animals were returned tothe home cage for 30min

(iii) Retention trial in this second trial the animalsexplored a familiar object (119860lowast) that is a duplicate ofthose objects from the acquisition trial (to minimizeolfactory cues) and a novel object (119861) for a further3min

They were made of a biologically inert substance (plastic)and were chosen to enable ease of cleaning (10 alcohol)between subjects in an attempt to remove olfactory cuesObject exploration is defined by animals licking sniffing ortouching the object whilst sniffing but not leaning againstturning round or standing or sitting on the object Objectswere of sufficient weight and were secured to the floor of thearena to ensure that they could not be knocked over ormovedaround by the animal The exploration time (s) of all objectswas recorded via stopwatch for subsequent statistical analysisThe time measured as an exploration behavior was used tocalculate amemory discrimination index (OR) as reported byBlalock et al [31]OR = (119861minus119860lowast)(119861+119860) where119861was the timespent exploring the new object and 119860lowast was the time spentexploring the familiar object Higher OR was considered toreflect greater memory ability [31] The test was performedafter 30 minutes after the administration of a single dose ofSC or the vehicle

313 Acetylcholinesterase and Butyrylcholinesterase ActivitiesAssay in Brain of Rats Acetylcholinesterase (AChE) andbutyrylcholinesterase (BuChE) activities were performed bymodifying spectrophotometric Ellmanrsquos method accordingto Isomae et al [32] The activities of AChE and BuChE

Evidence-Based Complementary and Alternative Medicine 5

028

021

014

007

000

200100 300 400 500 600 700 800 900(Minutes)

(AU

)

1 23

4

56 7 8 9 10

1112

1314

1617

19

15 1820

2122

23

2425

2627

28

2930

31

37

3234

3536 38 39

40

33

Figure 1 Chromatogram UV ofMelissa officinalis leaf extract obtained at 270 nm with peaks identified by HPLC-UV-MS

were determined by measuring the formation of the yellowanion obtained from the reaction between Ellmanrsquos reagentand the thiocholine generated by the enzymatic hydrolysisof acetylthiocholine iodide (ATCh) and butyrylthiocholine(BTCh) respectively (sample 01mL PBS 08mL DTNB01mL ATCh 020mL and BTCh 020mL)The biochemicalassay of AChE and BuChE in homogenate of brain sampleswas expressed as 120583molminmg protein by using spectropho-tometric method (120582 = 412 nm)

314 RNA Isolation and Reverse Transcription Reaction TotalRNA isolation from the rats brain tissues homogenates(frontal cortex hippocampus) was carried out using TriPureIsolation Reagent (Roche) according to manufacturerrsquos pro-tocol The integrity of RNA was visually assessed by a con-ventional agarose gel electrophoresis and the concentrationwill be evaluated by measuring the absorbance at 260 and280 nm in a spectrophotometer (BioPhotometer Eppendorf)RNA samples were stored at minus80∘C until use The 1 120583gof total RNA from all samples was used for the reversetranscription into cDNA using Transcriptor First StrandSynthesis Kit (Roche) according to manufacturerrsquos protocolObtained cDNA samples was stored at minus20∘C or used directlyfor the quantitative real-time PCR (qRT-PCR)

315 Real-Time PCR mRNA Quantification The acetylcho-linesterase (AChE) butyrylcholinesterase (BChE) and beta-secretase (BACE1) genes expression level was analyzed bytwo-step quantitative real-time PCR (qRT-PCR) in a vol-ume of 10 120583L reaction mixture using relative quantificationmethodology with a LightCycler TM Instrument (RocheGermany) and a LightCycler Fast Start DNA Master SYBRGreen I kit (Roche Applied Science) according to theinstructions of the manufacturer All primers sequences weredesigned and custom-designed using the Oligo 60 software(National Biosciences) and were verified by assessment ofa single PCR product on agarose gel and by a single tem-perature dissociation peak (melting curve analysis) of eachcDNA amplification product An GAPDH gene was usedas a housekeeping gene (endogenous internal standard) for

normalization of qPCR For each quantified gene standardcurves were prepared from dilution of cDNA and gener-ated from a minimum of four data points All quantitativePCR were repeated twice The data were evaluated usingLightCycler Run 45 software (Roche Applied Science) EachPCR run included a nontemplate control to detect potentialcontamination of reagents

316 Statistical Analysis All values were expressed as meansplusmn SEM The statistical comparison of results was carried outusing one-way analysis of variance (ANOVA) followed byDuncanrsquos post hoc test for detailed data analysisThe values of119901 lt 005were considered as a statistical significant difference

4 Results

41 Phytochemical Profile of Extract

411 Identification ofMetabolites Forty phenolicmetaboliteswere identified in hydroethanolic Melissa officinalis leafextract (Table 1 Figure 1) The predominant identified com-pounds were bioactive caffeic acid esters and glycosides offlavones The caffeic acid dimer rosmarinic acid (metabolite23) and caffeate trimer (lithospermic acid 31) were the prin-cipal caffeic acid derivatives in the analyzed samples Hydrox-yjasmonic acid and its derivatives teucrol as well as sage-coumarin were identified for the first time in the genusMelissa while luteolin and apigenin glycoconjugates arewell known phytochemicals in this species [33] Multistepfragmentation with accurate mass measurement enabledconfirming that the losses of fragment 799573 amu from the[M-H]minus ions of compounds 4 9 27 30 and 32 referredto sulphate groups The first-order fragmentation of 30revealed the loss of 799573 and yielded the product ion at7191622119898119911 The following fragmentation of this ion cor-responded to that of the sagerinic acid (dimer of rosmarinicacid) described by Barros et al [34] The exact placementof the sulphation position would require in-depth chemicalanalysis Thus 30 was tentatively assigned as sulphated sag-erinic acid

6 Evidence-Based Complementary and Alternative MedicineTa

ble1Metabolitesd

etectedin

Melissa

officin

alisleafextractb

yUPL

C-MS

No

RT [min]

Metabolite

identifi

catio

nCh

emical

form

ula

Exactm

asso

f[M-H

]minusΔpp

mFragmentatio

nin

120582max[nm]

CID

aIdentifi

catio

nlevelb

Reference

Measured

Calculated

Negativeion

mod

e(ESIminus)

Positiveion

mod

e(ESI+)

114

1

2-Hydroxy-3-(34-

dihydroxyphenyl)-

prop

anoic

acid

C 9H9

O5

1970

451970

455minus41775

197179135

199163

283312

8143997

2[34]

217

3Dihydroxybenzoica

cid

hexosid

eC 13

H15

O9

315072

3150722

08346

315153109

317155

282

54726828

3[35]

319

8Ca

ftaric

acid

C 13

H11

O9

3110

413110

409

06141

311221179149

318

6440397

2[35]

4224

2-Hydroxy-3-(34-

dihydroxyphenyl)-

prop

anoica

cid

sulphated

C 9H9

O8

S2770

032770

024

03998

277197179135

312

3[35]

5256

Hydroxyjasm

onicacid

hexosid

eC 18

H27

O9

387166

3871661

09214

387207163

323

44237366

2[17]

6276

Caffeicacid

C 9H7

O4

1790

341790

451minus49075

179135

181163

275

689043

1std

7289

Salviano

licacid

EC 36

H29

O16

7171450

717146

72313

717519339321

295277

275325

49770697

2[35]

8317

Salviano

licacid

HI

(isom

er)

C 27

H21

O12

537104

5371038

08562

537493359295

281325

2[35]

9345

Hydroxyjasm

onicacid

sulphated

C 12

H17

O7

S30507

30507

1117

305225194147

275330

3[17]

10362

NepetoidinB

C 17

H13

O6

313072

3130718

11232

nd282316

5316819

2[73]

1137

Yunn

aneica

cidF

C 26

H25

O14

5971255

5971244

18743

597509311197

Masked

2[34]

12383

Decarbo

xyrosm

arinicacid

(teucrol)

C 17

H15

O6

315088

3150874

09396

315179135

275330

637829

2[74]

1339

Caffeoylcaft

aricacid

C 22

H17

O12

473073

4730725

07555

173311149

Masked

65018

3[35]

14397

Apigenin

glucosylrham

nosid

eC 27

H29

O14

577157

5771563

11136

577269

579433271

275340

92741003

3[33]

15404

Luteolin

7-O-glucosid

e31015840-O

-glucuronide

C 27

H27

O17

623126

6231254

09696

62346144

7285

255

625463287

273343

2[33]

16421

Rosm

arinicacid

hexosid

eC 24

H25

O13

52113

5211301

03551

521359161

523361325163

329

25245848

2[34]

1743

Luteolin

O-diglucosid

eC 27

H29

O16

609147

6091461

07183

609285

611287

269349

3[33]

18441

Luteolin

glucosylrham

nosid

eC 27

H29

O15

593152

5931512

08077

59344

7285

59544

9287

271343

3[33]

19449

Luteolin

41015840-O

-glucosid

eC 21

H19

O11

4470

9444

70933

09728

447285

449287

271343

5319116

3[33]

2045

Sagerin

icacid

2-hydroxy-3-

(34-dihydroxyph

enyl)-

prop

anoide

C 45

H39

O20

8992

058992

0410

448

899719591475

295

Masked

3[35]

2149

Salviano

licacid

B(lithosperm

icacid

B)C 36

H29

O16

717146

7171461

01846

717519359161

327

6441188

2[34]

22504

Sagerin

icacid

C 36

H31

O16

7191

637191

618

09979

719519359161

287330

2[34]

23514

Rosm

arinicacid

C 18

H15

O8

3590

773590

772

0002

359161

361163

329

5281792

1std

Evidence-Based Complementary and Alternative Medicine 7

Table1Con

tinued

No

RT [min]

Metabolite

identifi

catio

nCh

emical

form

ula

Exactm

asso

f[M-H

]minusΔpp

mFragmentatio

nin

120582max[nm]

CID

aIdentifi

catio

nlevelb

Reference

Measured

Calculated

Negativeion

mod

e(ESIminus)

Positiveion

mod

e(ESI+)

24555

Salviano

licacid

B2-hydroxy-3-(34-

dihydroxyphenyl)-

prop

anoide

C 45

H37

O20

89719

8971884

16873

897717519359

161

330

3[35]

2556

Sagerin

icacid

di-2-hydroxy-3-(34-

dihydroxyphenyl)-

prop

anoide

C 54

H47

O24

1079244

910792457minus07911

1079897719539

359295

288330

3[35]

26592

Sagecoum

arin

di-2-hydroxy-3-(34-

dihydroxyphenyl)-

prop

anoide

caffeide

C 54

H43

O24

10752156

10752150

0559

1077897717537

409359339277

322

3[35]

27598

Rosm

arinicacid

sulphated

Iisomer

C 18

H15

O11

S4390

354390

341

20214

439359341163

Masked

2[34]

28613

Luteolin

31015840-O

-glucuronide

C 21

H17

O12

4610

734610

7216

34461285

463287

269340

170474237

2[33]

29632

Salviano

licacid

AC 26

H21

O10

493115

493114

11011

493359295179

298327

5281793

[34]

30656

Sagerin

icacid

sulphated

C 36

H31

O19

S7991

196

7991

186

0954

799719619519

359161

325

3[35]

31671

Lithosperm

icacid

C 27

H21

O12

537104

537104

809698

537493359161

292329

6441498

2[35]

3268

Rosm

arinicacid

sulphated

IIiso

mer

C 18

H15

O11

S4390

344390

341

02837

439359341163

Masked

2[34]

33689

Sagecoum

arin

C 27

H19

O12

535088

5350882

01204

535311267177

Masked

2[36]

3470

3Salviano

licacid

LIisomer

C 36

H29

O16

717146

7171461

02697

717519359

284329

2[35]

3571

Salviano

licacid

Lhydroxycaffeide

C 45

H35

O20

895173

8951727

06282

895519359161

Masked

3[35]

3674

Sagecoum

arin

cafta

ride

C 40

H29

O20

8291

268291

258

06953

829667535355

311

Masked

3[36]

37751

Sagecoum

arin

2-hydroxy-

3-(34-dihydroxyph

enyl)-

prop

anoide

C 36

H27

O16

715131

7151305

08176

715535311267

319

3[34]

3877

1Unk

nown

C 36

H57

O14

S745348

7453475

044

02281326

4[35]

3978

5Methylrosmarinate

C 19

H17

O8

373093

3730929

01765

373359161

284323

6479915

2[75]

40869

Salviano

licacid

Ccaffeoylhydroxycaffeide

C 44

H33

O18

8491

688491

672

08615

849687491359

327255

286318

3[35]

a CID

identifier

fora

chem

icalstr

ucture

intheP

ubCh

emCom

poun

ddatabase

b Metabolite

identifi

catio

nlevelaccording

toMetabolom

icsS

tand

ards

Initiativer

ecom

mendatio

n[76]

stdidentificatio

non

theb

asisof

stand

ardcompo

undfragmentatio

nnd

not

detected

8 Evidence-Based Complementary and Alternative Medicine

100 200 300 400 500 600 700 800 900 1000 1100 1200

mz

70

75

80

85

90

95

100

minus198 (2-hydroxy-3-(34-dihydroxyphenyl)

-porpanoide)

minus180 (caffeide)

minus180 (2-hydroxy-3-(34-dihydroxyphenyl)

-propanoide)

minus180 (2-hydroxy-3-(34-dihydroxyphenyl)

-propanoide)

minus180 (2-hydroxy-3-(34-dihydroxyphenyl)

-propanoide)

1350438

2191749

HO

HO

HO HO

OH

OH

OH

OH OH OH

OHOHOHOHHOOC

COOH

COOH COOH COOH

24612ppmC36H55O37 = 10792422

minus07911ppmC54H47O24 = 10792457

10792449

10448ppmC45H39O20 = 8992040

899205018453ppm

C36H31O16 = 7191612

7191625

20501ppmC27H23O12 = 5391190

5391201

14852ppmC23H33O10 = 4692074

469208119316ppm

C17H11O5 = 2950606

2950612

0

5

10

15

20

25

30

35

40

45

50

55

60

65

Rela

tive a

bund

ance

22903ppmC18H15O8 = 3590767

3590775

minus37218 ppmC9H5O3 = 1610239

1610233

(a)

100 200 300 400 500 600 700 800 900 1000

mz

100 200 300 400 500 600 700 800 900 1000

mz

100 200 300 400 500 600 700 800 900 1000

mz

100 200 300 400 500 600 700 800 900 1000

mz

0

250

500

750

1000

00

04

08

times104

times104

01234

0

1

2

times105

Ints

Ints

Ints

Ints

MS1

MS2

MS3

MS4

[M-H]minus10749

minus224 (acidic part

minus224 (acidic part

of sagecoumarin)

of sagecoumarin)

minus360 (rosmarinic acid)

4908

7148

5348 minus180 minus162 (caffeide)8769

31074909

5348

minus180 (2-hydroxy-3-(34-dihydroxyphenyl)-propanoide)

3108

35474909

1

(b)

Figure 2 Continued

Evidence-Based Complementary and Alternative Medicine 9

150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900

mz

0

5

10

15

20

25

30

35

40

45

50

55

60

65

Rela

tive a

bund

ance

O

O

O O

O

O

OH OH

OH

OH

OH

OHOH

OH

HO

HO

HO

COOH

OC

1529946

1970447

C9H9O5 = 1970455

minus41775ppm1610232

2552328

C16H31O2 = 2552330

minus04091ppm

3110563

C17H11O6 = 3110561

05243ppm

minus224 (fragment of sagecoumarin)

3912257

4092361

4832730

C29H39O6 = 4832752

minus44931ppm

5350885

C27H19O12 = 5350882

05640ppm

minus132 (tartaric acid)

5770990

C29H21O13 = 5770988

06486ppm

6670947

C31H23O17 = 6670941

09808ppm

minus162 (caffeic acid)

8291267

C40H29O20 = 8291258

11370ppm

(c)

Figure 2 (a) Mass spectra in negative ionization mode and simplified fragmentation scheme of compound 25 (pentameric ester ofcaffeic acid) (b) Mass spectra in negative ionization mode and simplified fragmentation scheme of compound 26 (pentameric structureof sagecoumarin di-2-hydroxy-3-(34-dihydroxyphenyl)-propanoide caffeide) (c) Mass spectra in negative ionization mode and simplifiedfragmentation scheme of compound 36 (pentameric structure of sagecoumarin caftaride)

Tetrameric structures of hydroxycinnamic acids wereidentified in lemon balm recently [34 35] The measure-ment of accurate masses allowed the identification of com-pound 25 which was tentatively identified as pentamericester of caffeic acid (Figure 2(a)) In 25 the double loss of1800421 amu corresponded to fragments with the molecularformula of C

9H8O4 adequate to dehydroxylated 2-hydroxy-

3-(34-dihydroxyphenyl)-propanoic acid The product ionat 7191623119898119911 and its further fragmentation are sim-ilar to those of metabolite 22 as described previously [34 35]Therefore metabolite 25was tentatively assigned as sagerinicacid di-2-hydroxy-3-(34-dihydroxyphenyl)-propanoideNev-ertheless comprehensive studies by nuclear magnetic reso-nance are required to complete elucidation of substitutionpattern for particular components of those pentamers

Sagecoumarins were previously identified in Salvia offic-inalis as caffeic acid trimers [36] Our MS analysis indicatedthe presence of such compounds and their derivatives alsoin M officinalis The pseudomolecular ion of compound33 observed in the negative ionization mode had theaccurate mass of 5350880119898119911 which corresponded to thechemical formula C

27H19O12

adequate for sagecoumarin(according to the Metlin and KNApSAck databases) Note-worthy 26 33 36 and 37 had the same fragmentationpattern of the product ion obtained in the MSMS andMSn in the negative ionization mode The [M-H-1800422]minusion corresponded to the detachment of 2-hydroxy-3-(34-dihydroxyphenyl)-propanoic acid thus 37 was tentatively

assigned as tetrameric sagecoumarin 2-hydroxy-3-(34-dihy-droxyphenyl)-propanoide The [M-H-3600846]minus ion in 26was indicated on rosmarinic acid substitution to 37 Massspectra of MSn in negative ionization of the compoundprovided complementary information to HR-MSMS massspectra indicated on caffeic acid as internal componentof the dimer In addition simultaneous loss of fragments180 amu and 224 amu in MS3 and MS4 indicated that thetwo components cannot be linked (Figure 2(b)) How-ever detailed analysis of substitution pattern of 26 shouldbe done Therefore 26 was tentatively assigned as penta-meric structure of sagecoumarin di-2-hydroxy-3-(34-dihy-droxyphenyl)-propanoide caffeide Rupture of caffeic andtartaric acid moieties from the product ion at 5350885119898119911was observed for compound36 (Figure 2(c))Detection of theaccurate masses of these two detached fragments with Δ lessthan 1 ppm eliminated the possibility of hexose and pentosesubstitution which have the same nominal masses as caffeicand tartaric acid respectively Therefore 36 was tentativelyidentified as another pentameric structure of sagecoumarincaftaride

Metabolite 28 with the accurate masses of 461073119898119911was tentatively identified as luteolin O-glucuronide The [M-H-1760324]minus ion is indicated on loss of structure C

6H8O6

corresponding to glucuronide moiety The product ion at2850405119898119911 was indicated on flavone luteolin The placeof substitution of the carboxylic acid on flavone skeleton isproblematic due to different isomers reported in lemon balm

10 Evidence-Based Complementary and Alternative Medicine

Table 2 Effect ofMelissa officinalis leaf extract (200mgkg po) treatment on sedative activity motor coordination and memory in rats

Group 119899Locomotor activity

[number of impulses5min]Motor coordination

exit time [s]Short-term memorye

ORLong-term memory

latency [s]MC + H

2

O 18 390 plusmn 24 17 plusmn 3 040 plusmn 006 47 plusmn 14MC + SC 18 526 plusmn 48lowast 32 plusmn 5lowast 032 plusmn 005 12 plusmn 3lowast

MO + H2

O 10 231 plusmn 48lowast 32 plusmn 7lowast 043 plusmn 007 169 plusmn 11lowast

MO + SC 9 436 plusmn 60 43 plusmn 7 009 plusmn 011lowast 23 plusmn 6HU + H

2

O 9 515 plusmn 32 15 plusmn 2 037 plusmn 009 158 plusmn 14lowast

HU + SC 8 639 plusmn 71lowast 56 plusmn 3 022 plusmn 006 49 plusmn 18

RA + H2

O 8 406 plusmn 59 21 plusmn 5 045 plusmn 005 58 plusmn 28RA + SC 8 605 plusmn 55 28 plusmn 7 045 plusmn 005 20 plusmn 7Means plusmn SEM119899Number of animalsMC + H

2O control rats

SC scopolamine (05mgkg bw ip)HU huperzine A (05mgkg bw po)RA rosmarinic acid (10mgkg bw po)eExpressed as ratio OR = (119861 minus 119860lowast)(119861 + 119860) for details see Section 3lowastVersus MC + H

2O 119901 lt 005

Versus MC + SC 119901 lt 005

luteolin 31015840-O- and 7-O-glucuronide [33] It is impossible todistinguish both structures by mass spectrometry Only onechromatographic peak corresponding to the [M-H]minus ion at461073119898119911 was observed in our study Since luteolin 31015840-O-glucuronide was assigned as the most abundant flavonoid inlemon balm [33] we assumed that 28 corresponded to thisstructure

412 Flavonoids and Polyphenolic Acids The major com-pound from the 40 chemical compounds identified in hy-droethanolic MO leaf extract established by HPLC was RA(885 g100 g) (Table 1 Figure 1)

Other chemical compoundswith neuromodulatory activ-ities such as lithospermic acid (0042 g100 g) salvianolicacid A (0040 g100 g) salvianolic acid B (0023 g100 g) andcaffeic acid (0087 g100 g) were also documented in litera-ture Moreover the total polyphenolic compounds content ofMO determined with the use of FolinndashCiocalteu assay was3397 calculated as gallic acid The total hydroxycinnamicderivatives content expressed spectrophotometrically as ros-marinic acid was 2115 g100 g

413 Essential Oil Composition Hydroethanolic MO leafextract contained 008 of total essential oil The GCFIDanalysis showed that the extract comprised camphene(004) alfa-pinene (007) beta-pinene (1647) andmyrcene (1951) Moreover according to retention time16 compounds were identified as follows alfa-bisabololborneol carvone chamazulene cineole eugenol gamma-terpineol guaiazulene isopulegol linalool limonene men-thol menthyl acetate pulegone terpine and thymol Thesecompounds are present in the essential oil in trace amountswhich do not allow the quantitative interpretation

42 Cognitive and Behavioral Experiments

421 Locomotor Activity A one-way ANOVA analysisrevealed significant differences in the locomotor activity of

rats expressed as their horizontal spontaneous activity afterMO administration (ANOVA 119865(7 80) = 646 119901 lt 005)(Table 2) Detailed post hoc analysis showed thatMO + H

2O

decreased the locomotor activity of rats by 4031 but HU +H2O did not affect this activity when compared with control

group (MC + H2O) We observed also that RA + H

2O did

not change the locomotor activity of rats Stimulating effectsin the locomotor activity of rats were observed after an acuteSC injection (MC + SC versus MC + H

2O 119901 lt 005) and

this effect was observed in all SC-treated rats when comparedwith the proper non-SC-treated animals On the contrarythese SC-treated animals did not differ in comparison toanimals that received SC only (MO + SC versus MC + SC119901 gt 005 HU + SC versus MC + SC 119901 gt 005 RA + SCversus MC + SC 119901 gt 005)

422 Motor Coordination A one-way ANOVA analysisrevealed significant differences in motor coordination of ratsexpressed as their exit time from the cylinder (119865(5 73) =284 119901 lt 005) (Table 2) Detailed analysis showed that themultiple administration of RA + H

2O and HU + H

2O did

not affect significantly this paradigm when compared withcontrol rats (119901 gt 005) whereas MO treatment led to pro-longed exit time (MO + H

2O versus MC + H

2O 119901 lt 005)

Moreover generally SC-treated animals showed producedprolongation of exit time and the effects were statisticallysignificant not only in control groups (MC + SC versus MC +H2O 119901 lt 005) but also in RA-treated rats (RA + SC versus

MC + SC 119901 lt 005) However the rest of the SC-treatedanimals did not differ in comparison to animals receiving SConly (MO + SC versus MC + SC 119901 gt 005 HU + SC versusMC + SC 119901 gt 005)

423 Long-Term Memory A one-way ANOVA analysisrevealed significant differences in long-term memory afterusing a passive avoidance test (119865(7 77) = 201 119901 lt 005Table 2) It was shown that the strongest effect leading to

Evidence-Based Complementary and Alternative Medicine 11

Table 3 The effect ofMelissa officinalis leaf extract on acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) activities and AChEBuAChE or beta-secretase (BACE1) mRNA expression levels in frontal cortex (FC) or hippocampus (Hipp) of rats

Group119899Enzyme activity

[nmolminmg protein]mRNA expression

[]AChE BuChE ACHE BuChE BACE1

FC Hipp FC Hipp FC Hipp FC Hipp FC HippMC + H

2

O 363 plusmn 49 439 plusmn 73 65 plusmn 11 53 plusmn 8 100 plusmn 12 100 plusmn 11 100 plusmn 18 100 plusmn 11 100 plusmn 16 100 plusmn 8MO + H

2

O 276 plusmn 34 409 plusmn 28 69 plusmn 6 62 plusmn 4 48 plusmn 4lowast 31 plusmn 7lowast 16 plusmn 2lowast 64 plusmn 21amp 36 plusmn 3lowast 50 plusmn 8lowast

HU + H2

O 189 plusmn 15lowast 239 plusmn 15lowast 58 plusmn 6 51 plusmn 4 53 plusmn 13lowast 85 plusmn 5 42 plusmn 9lowast 102 plusmn 11 62 plusmn 6lowast 98 plusmn 4RA + H

2

O 224 plusmn 16lowast 251 plusmn 12lowast 77 plusmn 7 99 plusmn 7lowast 101 plusmn 12 103 plusmn 5 184 plusmn 31lowast 56 plusmn 7lowast 126 plusmn 13amp 98 plusmn 3Means plusmn SEM119899Number of animals 7ndash10Values expressed as a ratio the geneGAPDHMC + H

2O control group

HU huperzine A (05mgkg bw po)RA rosmarinic acid (10mgkg bw po)lowastampVersus MC + H

2O 119901 lt 005 or 119901 lt 007 respectively

an improvement of this paradigm was produced by extractof MO and HU but not RA when compared with controlanimals (MO + H

2O versus MC + H

2O 119901 lt 005 HU +

H2O versus MC + H

2O 119901 lt 005 RA + H

2O versus MC +

H2O 119901 gt 005) However the administration of SC to rats

significantly decreased the latency time of passive avoidancetask (MC + SC versus MC + H

2O 119901 lt 005) After MO or

RA combined treatment with SC no improvement of long-term memory was observed but HU given with SC showedenhancement of this paradigm in rats (HU + SC versusMC + SC 119901 lt 005) Therefore it can be concluded thatadministration of HU overcomes the effect shown by SC only(Table 1)

424 Short-Term Memory The results of the object recogni-tion test showed that an administration of the compoundsor extract did affect the ratsrsquo short-term memory (ANOVA119865(7 78) = 273 119901 lt 005) (Table 2)

Detailed post hoc analysis showed that only SC signifi-cantly decreased the short-term memory in MO-treated rats(MO + SC versusMO + H

2O 119901 lt 005MO + SC versus MC

+ SC 119901 lt 005) whereas the differences between the rest ofthe animals did not reach statistical significance (119901 gt 005)

43 AChE and BuChE Activities in Rat Brain A one-wayANOVA revealed significant differences between groups inthe activity of AChE in both the cortex and the hippocampus(frontal cortex 119865(3 27) = 565 119901 lt 005 hippocampusANOVA 119865(3 29) = 796 119901 lt 005) It was found out thatMOshowed an insignificant inhibition of AChE activity in thefrontal cortex by 24 when compared with control rats (MC+ H2O 119901 lt 006) and in the hippocampus by 7 (Table 3)

whereas HU produced a distinct significant inhibition ofAChE activity in comparison to control group by 48 (119901 lt005) and 47 (119901 lt 005) in the cortex and the hippocampusrespectively Also RA lowered significantly AChE activityboth in the cortex (38) and in the hippocampus (43)Moreover there were not significant differences between thevalues of BuChE activities for MO and HU when compared

with control group in the frontal cortex (ANOVA 119865(3 30) =101 119901 gt 005) whereas in the hippocampus the differencesreached statistical significance (ANOVA 119865(3 27) = 141119901 lt 005) Detailed analysis showed that only RA effect wassignificant and increased BuChE activity in the hippocampuswhen compared with the control rats (119901 lt 005)

44 AChE BuChE and BACE1 mRNA Level Changes in RatBrain A one-way ANOVA analysis revealed significant dif-ferences of AChE mRNA transcription profile in the cortex(ANOVA 119865(3 27) = 857 119901 lt 005) As shown in Table 3the multiple treatment of MO produced in the cortex astatistically significant decrease of AChEmRNA level by 52the administration of HU caused decrease of its level by 44(119901 lt 005) whereas RA did not affect this parameter whencompared to the control

There were significant differences between the relativevalues of BuChE mRNA levels in this region of brain in rats(frontal cortex ANOVA 119865(3 25) = 162 119901 lt 005) TheMOtreatment led to a decrease in the BuChEmRNA level by 84(versus MC + H

2O 119901 lt 005) the prolonged HU admin-

istration resulted in a decrease of the transcript level in thecortex by 58 (versus MC + H

2O 119901 lt 005) but RA

increased this parameter in the cortex by 84 (versus MC +H2O 119901 lt 005)The significant differences ofmRNA transcription level of

AChE mRNA level in the hippocampus (ANOVA 119865(3 31) =212 119901 lt 005) have been observed The detailed analysisshown that in the case of AChE afterMO treatment mRNAlevel significantly decreased by 69 (versus MC + H

2O

119901 lt 005) while the administration of HU resulted in astatistically insignificant decrease of AChE mRNA level by18 when compared with control group Also RA did notchange the level of transcript

In the case of BuChEmRNA expression in the hippocam-pus there were statistically significant differences betweengroups (ANOVA 119865(3 27) = 310 119901 lt 005) Detailed analysisshowed that in theMO + H

2O treated group the expression

lowered by 36 but the difference did not reach strong

12 Evidence-Based Complementary and Alternative Medicine

significance when compared with the control values (119901 lt007) and no change in the expression level of this enzymewas shown after the administration of HU On the contraryRA produced an increase of BuChE mRNA expression in thehippocampus by 44 (versus MC + H

2O 119901 lt 005)

Further analysis showed the significant differences inBACE1 mRNA expression in both brain regions of rats (inthe cortex and the hippocampus) (frontal cortex ANOVA119865(3 27) = 163 119901 lt 005 hippocampus ANOVA 119865(3 27) =133 119901 gt 005) It was observed that MO produced astatistically significant decrease of the BACE1 expression levelby 64 in the cortex (versus MC + H

2O 119901 lt 005) and by

50 in the hippocampus (versus MC + H2O 119901 lt 005) For

comparison HU treatment led to a decrease in the mRNAexpression level by 38 in the cortex (versus MC + H

2O

119901 lt 005) but not in the hippocampus On the contrary RAproduced an increase of this transcript in the cortex by 26but the effect did not reach a strong statistical significance(versus MC + H

2O 119901 lt 007) whereas in the hippocampus

there was no difference between RA and control group

5 Discussion

Cognitive and Behavioral Experiments The present studyinvestigated the influence of subchronic (28-fold) adminis-tration of standardized 50 EtOH extract ofMelisa officinalisleaf extract (MO) (200mgkg po containing 177mgkg ofRA) on SC-induced impairment of short-term and long-term memory in rats The results were compared with theactivity of cholinesterases (AChE and BuChE) as well aswith AChE BuChE and BACE1 gene expression levels in thecortex and hippocampus of the rat brain So far little evidenceis yet available as regards mechanisms of MO leaf extractaction that are potentially relevant to cognitive function ofrats after per os administration MO is traditionally used intreating neurological disorders through its anti-AChE [18]and antiagitation properties [37] Moreover Wake et al [38]and Kennedy et al [15] showed thatMO extract has nicotinicreceptor activity and that it can displace [3H]-(N)-nicotinefrom nicotinic receptors in homogenates of human cerebralcortex tissue and they suggested that these mechanisms canexplain activity of MO extract in amnesia model RecentlySoodi et al [18] observed that intraperitoneal injections ofMO extract (200mgkg) in rats could significantly enhancelearning and memory processes in animals since the extractsignificantly ameliorates SC-induced learning deficit in Mor-ris water maze test On the contrary in higher dose itcan be observed that MO extract (400mgkg) could notreverse SC-induced memory impairment [18] In our studyadministration of MO extract at a dose of 200mgkg (po)showed the effect leading to improvement of long-termmemory in a passive avoidance test in rats However afterMO combined treatment with SCMO did not overcome theimpairment shown by SC (Table 2) It should be emphasizedthat it is not clear whether the effect shown in our study byMO in non-SC-treated rats is specific since theMO treatmentproduced significant lowering of locomotor activity of ratstherefore the sedative profile ofMO cannot be excluded

On the other hand Ryu et al [39] observed that agitationand aggression are highly prevalent in patientswith dementiaAccording to Gitlin et al [40] nonpharmacological inter-ventions are recommended as first-line therapy Althoughantipsychotics have shown benefit for Alzheimerrsquos disease-related psychosis their use is associated with several seriousadverse effects [40]Thus it seems that the use ofMO extractcan provide dual benefits both in aspect of inhibition ofagitation and in improving the memory of patients withAlzheimerrsquos disease

Furthermore in our study RA in the dose of 10mgkgbw (po) did not affect either short- or long-term memoryalthough RA lowered significantly AChE activity both inthe cortex and in the hippocampus Moreover we observedthat the repeated administration of RA in non-scopolamine-treated rats did not produce any changes of locomotoractivity similarly to our previous study [23]

It is possible that other chemical compounds can influ-ence the memory in rats by synergic interactions in plantextract According to our calculations caffeic acid (0174mgin a single dose of extract administered to animals per kgbw) lithospermic acid (0084mgkg) salvianolic acid A(008mgkg) and salvianolic acid B (0046mgkg) may beresponsible for observed pharmacological effects On theother hand results from few studies showed that salvianolicacids do not cross the blood brain barrier (BBB) [41 42]and also lithospermic acid does not efficiently cross the BBB[43] For this reason the interpretation of our results is morecomplicated

Firstly Xu et al [44] demonstrated that Sal A is ametabol-ically unstable compound that would undergo rapid methy-lationmetabolism catalyzed by catechol O-methyltransferasein vivo into four major methylated metabolites of Sal A(3-O-methyl 31015840-O-methyl 3310158401015840-O-dimethyl and 31015840310158401015840-O-dimethyl salvianolic acid A) These generated O-methylatedmetabolites may be largely responsible for its in vivo phar-macological effects Although there are no available recentstudies on the ability of these compounds to pass the BBBsuch possibility should not be excluded

Secondly several studies showed central pharmacolog-ical effects in animals after per os administration of Sal B[45 46] Sal A [47] and caffeic acid [48] It was provedfor example that salvianolic acid B (10mgkg po) sig-nificantly rescued the A12057325ndash35 peptide-induced decreaseof choline acetyltransferase and brain-derived neurotrophicfactor protein levels in an amyloid 120573 (A120573) peptide-inducedAlzheimerrsquos disease mouse model [49] It also significantlyreversed (10mgkg po) the cognitive impairments inducedby scopolamine (1mgkg ip) or A120573 (25ndash35) (10 nmol5 120583Licv) injection inmice [45] Previous studies [46 47] showedalso that both Sal A and Sal B are able to improve theimpaired memory function induced by cerebral ischemia-reperfusion in miceThe stimulation of neurogenesis processin both subgranular zone (SGZ) and subventricular zone(SVZ) after brain ischemia and also alleviation neural cellsloss and improved motor function recovery after brainischemia in rats after the Sal B administration were alsoobserved by Zhong et al [50] Moreover the exposure to SalB can maintain the proliferation of neural stemprogenitor

Evidence-Based Complementary and Alternative Medicine 13

cells (NSPCs) after cerebral ischemia and improve cognitivepostischemic impairment after stroke in rats using Morriswater maze test therefore authors concluded that Sal B mayact as a potential drug in treatment of brain injury or neu-rodegenerative disease [51] Additionally the improvement ofmotor function after cerebral ischemia in rats after salvianolicacid B administrationwas also demonstrated [52]The clue topass through the BBB Sal B provided also results of Li et alindicating its protective effect on BBB in rats after cerebralischemia-reperfusion by inhibiting the MAPK pathway [53]In addition to this it was shown that lithospermic acid andsalvianolic acids exerted neuroprotective activity in variousexperimental models Lithospermic acid significantly attenu-ates neurotoxicity in vitro and in vivo induced by 1-methyl-4-phenylpyridin (MPP(+)) by blocking neuronal apoptotic andneuroinflammatory pathways [54] Salvianolic acid B inhib-ited amyloid beta-protein aggregation and fibril formation aswell as directly inhibiting the cellular toxicity of amyloid beta-protein in PC12 cells [55] and significantly reduced its cyto-toxic effects on human neuroblastoma SH-SY5Y cells [56]

For an explanation of our results observations of Pin-heiro Fernandes et al [57] may be very helpful whichshowed that caffeic acid nonflavanoid catecholic compoundwhose derivatives are occurring in MO extract improvedthe working spatial and long-term aversive memory deficitsinduced by focal cerebral ischemia in mice Anwar et al [48]showed also that caffeic acid (100mgkg) improved the step-down latencies in the inhibitory avoidance in rats Tsai et al[58] showed that caffeic acid is a potent neuroprotective agentin brain of 1-methyl-4-phenyl-1236-tetrahydropyridine(MPTP) treatedmiceMoreover caffeic acid improves A12057325ndash35-induced memory deficits and cognitive impairment inmice [59] In another study it was also shown that this com-pound has a significant protective effect on global cerebralischemia-reperfusion injury in rats [60] For instance 124 plusmn18mg100 g of caffeic acid was detected in the brain of micewith a diet containing 2 caffeic acid for 4 weeks [58] Thusthere is progressive evidence that this compound passesthrough the BBB and has a central pharmacological activity

Also results by Yoo et al are also worth noting [61] whichshowed also in a ratmodel of scopolamine-induced amnesiathat administration of luteolin (a common flavonoid frommany plants including M officinalis) at dose of 10mgkgcaused the increase in the brain-derived neurotrophic factor(BDNF) acetylcholine and the decrease in lipid peroxida-tion Liu et al [62] observed that chronic treatment withluteolin (50 and 100mgkg) improved neuronal injury andcognitive performance by attenuating oxidative stress andcholinesterase activity in streptozotocin-induced diabetes inrats In our study ethanolic extract of MO administered torats significantly improved long-term memory after using apassive avoidance test but afterMO combined treatmentwithSC no improvement of this paradigmwas observed (Table 2)

Thirdly available pharmacokinetic studies were carriedout for the pure compounds (Sal A and B) and the extractof the roots of Salvia miltiorrhiza [42 63] but there are notavailable studies of bioavailability of these compounds afteradministration of Melissa officinalis leaf extract containingother compounds as compared with the extract of Salvia

miltiorrhiza Moreover there is a lack of data about howscopolamine may influence the penetration of the salvianolicacids (and other compounds) across the BBB Hence thereis a need to study the pharmacokinetic parameters of thesecompounds in the group of animals treatedwith scopolaminein comparison with control group

AChEandBuChEActivities in Rat Brain To date several stud-ies have focused on explaining themechanismof action of theMS extract and its active compounds Soodi et al [18] showedthat treatment of animals withMO extract (400mgkg) priorto scopolamine injection could ameliorate scopolamine-induced enhancement in AChE activity This dose of MOextract inhibited the AChE activity in the hippocampus ofrats (519 versus 1004 in normal saline group 119901 lt 005914 in group treated with scopolamine +MO versus 1281in scopolamine group 119901 lt 005) In another study Anwaret al [48] showed that 50 and 100mgkg of caffeic aciddecreased in vivo the AChE activity in the cerebral cortexand striatum and increased the activity of this enzyme inthe cerebellum hippocampus hypothalamus pons lympho-cytes and muscles when compared to the control group(119901 lt 005) Our study showed that ethanolic extract of MOproduced an insignificant and slight inhibition of AChE andBuChE activity in the frontal cortex and in the hippocampus(especially) However it was observed previously [23] thatRA inhibited the AChE activity in the rats frontal cortex andhippocampusMoreover we showed that RA possess a strongstimulatory effect on BuChE in the hippocampus

AChE BuChE and BACE1mRNA Level Changes in Rat BrainSo far no results have been published of studies concerningthe in vivo assessment of changes in AChE BuChE andBACE1 gene expression profile in different brain regionsunder the influence of MO extracts or their key bioactivemetabolites Such studies focused overwhelmingly on theanalysis of their in vitro activities to a lesser extent in animalexperiments

In our study in the frontal cortex we have observedthe strongest inhibition of both AChE and BuCHE mRNAtranscription under the influence ofM officinalis extract andhuperzine A (Table 3) However a more significant differencein the level of transcript was seen in the frontal cortex inparticular in the case of BuCHE mRNA of experimentalrats (a decrease by 516 and 44 for MO and HU versuscontrol resp)The strong inhibition of transcription ofAChEmRNAwas also observed in the hippocampus ofMO-treatedrats (a decrease by 69) The BuChE mRNA transcriptionin animals receiving MO was lowered in a moderate way(decrease by 36)HuperzineA alone has not caused changesthat were observed in the group ofMO (Table 2) Our findingsmostly correlate with the observed changes in activities ofAChE and BuChE in different groups of animals In the caseof BuChE slightly different results between its activity andexpression profile were especially seen in the frontal cortexand hippocampus of animals receivingMO (Table 3)

To date there is a lack of published results of studiesmaking an attempt to clarify the potential differences in thetranscriptional profile and activity of AChE and BuChE

14 Evidence-Based Complementary and Alternative Medicine

under the influence of MO and its bioactive metabolites Itcannot be excluded that the key for the observed differencesin the level of AChE activities and mRNA level can becaused by changes in the activity of AChE in other regionsof the brain not analyzed in this study such as substantianigra cerebellum globus pallidus and hypothalamus whereit exerts nonenzymatic neuromodulatory functions affect-ing neurite outgrowth and synaptogenesis modulating theactivity of other proteins regional cerebral blood flow andother functions [64] But it is difficult to clearly explain whydiminishing of AChE and BuChEmRNAs did not always cor-relate with the lowering of activity of these enzymes althoughit has been recently noted that AChE activity was notparalleled by an increase in mRNA levels [65] The authorsexplained this fact by stating that AChE levels are regulatedat transcriptional posttranscriptional and posttranslationallevels leading to complex expression patterns which canbe modulated by physiological and pathological conditionsHowever these mechanisms are not fully understood andfurther studies are needed in this field

The cause of observed different degree of inhibition ofactivity and transcription status of studied genes especiallyof BuChE (and AChE) may lie in a so-called ldquonegative feed-backrdquo consisting of a complicated transcriptiontranslationregulation protein-protein interactionsmodifications and ametabolic network together forming a system that allows thecell to respond sensibly to the multiple signal molecules thatexist in its environment [66]

Because of that we propose that the reason for differencesbetween AChE and BuChE activities in the cortex and hip-pocampus under the MO may be due to the fact of insuf-ficiency of applied dose and the duration of the experi-ment affecting the transcriptional tissue-specific cellularmachinery regulating BuChE transcription without affectingits activity Moreover the administration of higher doses ofMO and extended period of time could lead to sufficientinhibition of its activity

There is a need to conduct further studies to determinethe molecular degree of dependence between changes inAChE and BuChE activities and activities of potential keyfactors regulating expression of these genes in the frontalcortex and hippocampus under the influence of extracts ofMO and active metabolites Another study determining theeffectiveness of their actions on the cholinergic system inexperimental animal models of memory impairment withparticular emphasis on scopolamine including the determi-nation of changes at the molecular level should be thereforecarried out

Alterations in BACE1 protein level have been proved inpostmortem brain tissue from individuals with AD withincreases decreases and also no change reported [1 67 68]An example of confirmation of these findings at the mRNAlevel is results of study by Coulson et al [69] Based onconducted studies some authors suggested that increasedBACE1mRNA transcription in remaining neuronal cells maycontribute to the increased BACE1 protein levels and activityfound in brain regions affected by AD [70]

Results of many studies concerning the elevated level ofBACE1 mRNA and protein in Alzheimerrsquos disease provide

direct and compelling reasons to develop therapies directed atBACE1 inhibition thus reducing120573-amyloid and its associatedtoxicities [67 68]

In our study we have observed that BACEmRNA expres-sion statistically significantly decreased after MO adminis-tration in frontal cortex and hippocampus These resultssuggest that the MO extract may act to inhibit BACE1mRNA level given the fact that the percentage of inhibitionof the expression (64 in the frontal cortex 50 in thehippocampus) is higher than that in the case of HU (38in the cortex and the lack of changes in the hippocampus)A careful analysis of the literature data shows that there areno studies which analyzed the impact of MO extract on theexpression level of BACE1 in Alzheimerrsquos disease

Furthermore a literature analysis does not indicatealready published results conducted by other teams attempt-ing to assess the impact of RA on the transcriptional activityof AChE BuChE and BACE1 Although several studies(already mentioned and others [70 71]) highlighted theRA and other caffeic acid derivatives capability of acetyl-cholinesterases inhibition none of these studies does nottouch the question of the molecular basis of their impacton the transcriptional machinery that regulates in vivo theexpression of studied genes Hence in our opinion obtainedby our team results they are one of the first of this typeand in general correlate with the results of our previousstudy [23] In this case there is no clear evidence explainingdifferent responses at the transcriptional level under theinfluence of RA especially in the case of BuChE encodinggene in the hippocampus (Table 2) It is possible that theobserved differentiation of BuChE transcriptional activitybetween the frontal cortex and the hippocampus may bedue to the differences of butyrylcholinesterase localizationand substrate affinity [72] Since in our experiment we havecarried out a quantitative analysis of AChE BuChE andBACE1 transcripts in brain homogenates of tested animalsrather than in individual isolated cell fractions therefore theobtained results constitute an overall ldquopicturerdquo of both studiedgenes transcriptional changes occurring in the brain areas ofstudied animals under the influence of the RA and the wholeplant extract as well

6 Conclusion

The subchronic administration ofMO led to an improvementof long-term memory of rats however the mechanisms ofMO action are probably more complicated since its role asa modulator of beta-secretase activity (due to inhibition ofBACE1 mRNA expression in frontal cortex) should be takeninto consideration

It should be noted that we have studied a crude extractfrom leaf of Melissa officinalis not a single pure chemicalcompound This plant extract is a complex mixture and itsaction may be a result of the summation of activities ofseveral components (synergismadditive action of caffeic acidwith salvianolic acids rosmarinic acid and others) In thecase of extract from leaves ofMelissa officinalis it is possiblethat interactions occur between the 40 chemical compoundsidentified by HPLC system

Evidence-Based Complementary and Alternative Medicine 15

Taken together it seems that the MO activity representsa possible option as complementary interventions to relievethe symptoms of mild dementia

Competing Interests

The authors declare that they have no competing interests

Acknowledgments

This work was supported by the Ministry of Science andHigher EducationWarsaw Poland from educational sources(2008ndash2011 Grant no N 405417836)

References

[1] S L Cole and R Vassar ldquoThe Alzheimerrsquos disease 120573-secretaseenzyme BACE1rdquo Molecular Neurodegeneration vol 2 no 1article 22 2007

[2] LACraigN SHong andR JMcDonald ldquoRevisiting the cho-linergic hypothesis in the development of Alzheimerrsquos diseaserdquoNeuroscience amp Biobehavioral Reviews vol 35 no 6 pp 1397ndash1409 2011

[3] N G M Gomes M G Campos J M C Orfao and C A FRibeiro ldquoPlants with neurobiological activity as potential tar-gets for drug discoveryrdquo Progress in Neuro-Psychopharmacologyamp Biological Psychiatry vol 33 no 8 pp 1372ndash1389 2009

[4] WHO WHO Monographs on Selected Medicinal PlantsmdashVolume 2 WHO Geneva Switzerland 2004 httpappswhointmedicinedocsendJs4927e18htmlJs4927e18

[5] M Bayat A A Tameh M H Ghahremani et al ldquoNeuropro-tective properties of Melissa officinalis after hypoxic-ischemicinjury both in vitro and in vivordquo DARU Journal of Pharmaceu-tical Sciences vol 20 article 42 2012

[6] J P Kamdem A Adeniran A A Boligon et al ldquoAntioxidantactivity genotoxicity and cytotoxicity evaluation of lemon balm(Melissa officinalis L) ethanolic extract Its potential role inneuroprotectionrdquo Industrial Crops and Products vol 51 pp 26ndash34 2013

[7] J-T Lin Y-C Chen Y-C Lee C-W R Hou F-L Chen andD-J Yang ldquoAntioxidant anti-proliferative and cyclooxygenase-2 inhibitory activities of ethanolic extracts from lemon balm(Melissa officinalis L) leavesrdquo LWTmdashFood Science and Technol-ogy vol 49 no 1 pp 1ndash7 2012

[8] G Guginski A P Luiz M D Silva et al ldquoMechanisms involvedin the antinociception caused by ethanolic extract obtainedfrom the leaves of Melissa officinalis (lemon balm) in micerdquoPharmacology Biochemistry amp Behavior vol 93 no 1 pp 10ndash162009

[9] N Perry G Court N Bidet J Court and E Perry ldquoEuropeanherbs with cholinergic activities potential in dementia therapyrdquoInternational Journal of Geriatric Psychiatry vol 11 no 12 pp1063ndash1069 1996

[10] E K Perry A T Pickering W W Wang P J Houghtonand N S L Perry ldquoMedicinal plants and Alzheimerrsquos diseasefrom ethnobotany to phytotherapyrdquo Journal of Pharmacy andPharmacology vol 51 no 5 pp 527ndash534 1999

[11] M-J R Howes N S L Perry and P J Houghton ldquoPlants withtraditional uses and activities relevant to the management ofAlzheimerrsquos disease and other cognitive disordersrdquo Phytother-apy Research vol 17 no 1 pp 1ndash18 2003

[12] I Orhan and M Aslan ldquoAppraisal of scopolamine-inducedantiamnesic effect in mice and in vitro antiacetylcholinesteraseand antioxidant activities of some traditionally used Lamiaceaeplantsrdquo Journal of Ethnopharmacology vol 122 no 2 pp 327ndash332 2009

[13] S Akhondzadeh M Noroozian M Mohammadi S OhadiniaA H Jamshidi and M Khani ldquoMelissa officinalis extract inthe treatment of patients with mild to moderate Alzheimerrsquosdisease a double blind randomised placebo controlled trialrdquoJournal of Neurology Neurosurgery and Psychiatry vol 74 no 7pp 863ndash866 2003

[14] D O Kennedy A B Scholey N T J Tildesley E K Perry andK AWesnes ldquoModulation ofmood and cognitive performancefollowing acute administration of Melissa officinalis (lemonbalm)rdquo Pharmacology Biochemistry amp Behavior vol 72 no 4pp 953ndash964 2002

[15] D O Kennedy G Wake S Savelev et al ldquoModulation of moodand cognitive performance following acute administration ofsingle doses of Melissa officinalis (Lemon balm) with humanCNS nicotinic and muscarinic receptor-binding propertiesrdquoNeuropsychopharmacology vol 28 no 10 pp 1871ndash1881 2003

[16] D O Kennedy and E L Wightman ldquoHerbal extracts and phy-tochemicals plant secondarymetabolites and the enhancementof human brain functionrdquoAdvances inNutrition vol 2 no 1 pp32ndash50 2011

[17] R P Pereira A A Boligon A S Appel et al ldquoChemical com-position antioxidant and anticholinesterase activity of Melissaofficinalisrdquo Industrial Crops and Products vol 53 pp 34ndash452014

[18] M Soodi N Naghdi H Hajimehdipoor S Choopani andE Sahraei ldquoMemory-improving activity of Melissa officinalisextract in naıve and scopolamine-treated ratsrdquo Research inPharmaceutical Sciences vol 9 no 2 pp 107ndash114 2014

[19] K Dastmalchi V Ollilainen P Lackman et al ldquoAcetyl-cholinesterase inhibitory guided fractionation of Melissa offic-inalis Lrdquo Bioorganic amp Medicinal Chemistry vol 17 no 2 pp867ndash871 2009

[20] W Chaiyana and S Okonogi ldquoInhibition of cholinesterase byessential oil from food plantrdquo Phytomedicine vol 19 no 8-9 pp836ndash839 2012

[21] K Dastmalchi H J D Dorman P P Oinonen Y Darwis ILaakso and R Hiltunen ldquoChemical composition and in vitroantioxidative activity of a lemon balm (Melissa officinalis L)extractrdquo LWTmdashFood Science and Technology vol 41 no 3 pp391ndash400 2008

[22] P Pereira D Tysca P Oliveira L F D S Brum J N Picadaand P Ardenghi ldquoNeurobehavioral and genotoxic aspects ofrosmarinic acidrdquo Pharmacological Research vol 52 no 3 pp199ndash203 2005

[23] M Ozarowski P L Mikolajczak A Bogacz et al ldquoRosmarinusofficinalis L leaf extract improves memory impairment andaffects acetylcholinesterase and butyrylcholinesterase activitiesin rat brainrdquo Fitoterapia vol 91 pp 261ndash271 2013

[24] G P Kumar and F Khanum ldquoNeuroprotective potential of phy-tochemicalsrdquo Pharmacognosy Reviews vol 6 no 12 pp 81ndash902012

[25] M Stobiecki A Staszkow A Piasecka P M Garcia-LopezF Zamora-Natera and P Kachlicki ldquoLC-MSMS profiling offlavonoid conjugates in wild mexican lupine Lupinus reflexusrdquoJournal of Natural Products vol 73 no 7 pp 1254ndash1260 2010

[26] A Wojakowska A Piasecka P M Garcıa-Lopez et al ldquoStruc-tural analysis and profiling of phenolic secondarymetabolites of

16 Evidence-Based Complementary and Alternative Medicine

Mexican lupine species using LC-MS techniquesrdquo Phytochem-istry vol 92 pp 71ndash86 2013

[27] A Gryszczynska B Opala Z Lowicki et al ldquoBioactive com-pounds determination in the callus and hydroalcoholic extractsfrom Salvia miltiorrhiza and Salvia przewalskiimdashpreliminarystudy on their anti-alcoholic activity effectsrdquo PhytochemistryLetters vol 11 pp 399ndash403 2015

[28] K Slinkart and V L Singleton ldquoTotal phenol analysis automa-tion and comparison with manual methodrdquo American Journalof Enology and Viticulture vol 28 no 1 pp 49ndash55 1977

[29] P J R Boissier J Tardy and J C Diverres ldquoUne nouvellemethode simple pour explorer lrsquoaction lsquotranquillisantersquo le testde la chemineerdquo Medicina Experimentalis vol 3 no 1 pp 81ndash84 1960

[30] R Ader J A Weijnen and P Moleman ldquoRetention of a passiveavoidance response as a function of the intensity and durationof electric shockrdquo Psychonomic Science vol 26 no 3 pp 125ndash128 1972

[31] E M Blalock K-C Chen K Sharrow et al ldquoGene microar-rays in hippocampal aging statistical profiling identifies novelprocesses correlated with cognitive impairmentrdquo Journal ofNeuroscience vol 23 no 9 pp 3807ndash3819 2003

[32] K Isomae S Morimoto H Hasegawa K Morita and JKamei ldquoEffects of T-82 a novel acetylcholinesterase inhibitoron impaired learning and memory in passive avoidance task inratsrdquo European Journal of Pharmacology vol 465 no 1-2 pp97ndash103 2003

[33] J Patora and B Klimek ldquoFlavonoids from lemon balm (Melissaofficinalis L Lamiaceae)rdquo Acta Poloniae Pharmaceutica-DrugResearch vol 59 no 2 pp 139ndash143 2002

[34] L Barros M Duenas M I Dias M J Sousa C Santos-Buelgaand I C F R Ferreira ldquoPhenolic profiles of cultivated invitro cultured and commercial samples of Melissa officinalis Linfusionsrdquo Food Chemistry vol 136 no 1 pp 1ndash8 2013

[35] T L Miron M Herrero and E Ibanez ldquoEnrichment of antiox-idant compounds from lemon balm (Melissa officinalis) bypressurized liquid extraction and enzyme-assisted extractionrdquoJournal of Chromatography A vol 1288 pp 1ndash9 2013

[36] Y Lu L Y Foo and H Wong ldquoSagecoumarin a novel caffeicacid trimer from Salvia officinalisrdquo Phytochemistry vol 52 no6 pp 1149ndash1152 1999

[37] S Abuhamdah LHuangM S J Elliott et al ldquoPharmacologicalprofile of an essential oil derived from Melissa officinalis withanti-agitation properties focus on ligand-gated channelsrdquo Jour-nal of Pharmacy and Pharmacology vol 60 no 3 pp 377ndash3842008

[38] G Wake J Court A Pickering R Lewis R Wilkins andE Perry ldquoCNS acetylcholine receptor activity in Europeanmedicinal plants traditionally used to improve failing memoryrdquoJournal of Ethnopharmacology vol 69 no 2 pp 105ndash114 2000

[39] S-H Ryu C Katona B Rive and G Livingston ldquoPersistenceof and changes in neuropsychiatric symptoms in Alzheimerdisease over 6months the LASER-AD studyrdquoAmerican Journalof Geriatric Psychiatry vol 13 no 11 pp 976ndash983 2005

[40] L N Gitlin H C Kales and C G Lyketsos ldquoNonpharma-cologic management of behavioral symptoms in dementiardquoJournal of the AmericanMedical Association vol 308 no 19 pp2020ndash2029 2012

[41] A Konczol J Muller E Foldes et al ldquoApplicability of a blood-brain barrier specific artificial membrane permeability assay atthe early stage of natural product-based CNS drug discoveryrdquoJournal of Natural Products vol 76 no 4 pp 655ndash663 2013

[42] Y-J Zhang L Wu Q-L Zhang J Li F-X Yin and Y YuanldquoPharmacokinetics of phenolic compounds of Danshen extractin rat blood and brain by microdialysis samplingrdquo Journal ofEthnopharmacology vol 136 no 1 pp 129ndash136 2011

[43] X Li C Yu Y Lu et al ldquoPharmacokinetics tissue distributionmetabolism and excretion of depside salts from Salvia miltior-rhiza in ratsrdquo Drug Metabolism and Disposition vol 35 no 2pp 234ndash239 2007

[44] H Xu Y Li X Che H Tian H Fan and K Liu ldquoMetabolismof salvianolic acid a and antioxidant activities of its methylatedmetabolitesrdquo Drug Metabolism and Disposition vol 42 no 2pp 274ndash281 2014

[45] D H Kim S J Park J M Kim et al ldquoCognitive dysfunctionsinduced by a cholinergic blockade and A120573 25-35 peptide areattenuated by salvianolic acid Brdquo Neuropharmacology vol 61no 8 pp 1432ndash1440 2011

[46] G-H Du Y Qiu and J-T Zhang ldquoSalvianolic acid B protectsthe memory functions against transient cerebral ischemia inmicerdquo Journal of Asian Natural Products Research vol 2 no 2pp 145ndash152 2000

[47] G Du and J Zhang ldquoProtective effects of salvianolic acid Aagainst impairment of memory induced by cerebral ischemia-reperfusion in micerdquo Chinese Medical Journal vol 110 no 1 pp65ndash68 1997

[48] J Anwar R M Spanevello G Thome et al ldquoEffects of caffeicacid on behavioral parameters and on the activity of acetyl-cholinesterase in different tissues from adult ratsrdquo Pharmacol-ogy Biochemistry and Behavior vol 103 no 2 pp 386ndash394 2012

[49] YW Lee D H Kim S J Jeon et al ldquoNeuroprotective effects ofsalvianolic acid B on anA12057325-35 peptide-inducedmousemodelof Alzheimerrsquos diseaserdquo European Journal of Pharmacology vol704 no 1ndash3 pp 70ndash77 2013

[50] J Zhong M-K Tang Y Zhang Q-P Xu and J-T ZhangldquoEffect of salvianolic acid B on neural cells damage and neu-rogenesis after brain ischemia-reperfusion in ratsrdquo Yao Xue XueBao vol 42 no 7 pp 716ndash721 2007

[51] P Zhuang Y Zhang G Cui et al ldquoDirect stimulation of adultneural stemprogenitor cells in vitro and neurogenesis in vivo bysalvianolic acid Brdquo PLoS ONE vol 7 no 4 Article ID e356362012

[52] M TangW Feng Y Zhang J Zhong and J Zhang ldquoSalvianolicacid B improves motor function after cerebral ischemia in ratsrdquoBehavioural Pharmacology vol 17 no 5-6 pp 493ndash498 2006

[53] Q Li L-P Han Z-H Li J-T Zhang and M-K Tang ldquoSal-vianolic acid B alleviate the disruption of blood-brain barrierin rats after cerebral ischemia-reperfusion by inhibiting MAPKpathwayrdquo Yao Xue Xue Bao vol 45 no 12 pp 1485ndash1490 2010

[54] Y-L LinH-J Tsay T-H Lai T-T Tzeng andY-J Shiao ldquoLith-ospermic acid attenuates 1-methyl-4-phenylpyridine-inducedneurotoxicity by blocking neuronal apoptotic and neuroinflam-matory pathwaysrdquo Journal of Biomedical Science vol 22 article37 2015

[55] M-K Tang and J-T Zhang ldquoSalvianolic acid B inhibits fibrilformation and neurotoxicity of amyloid beta-protein in vitrordquoActa Pharmacologica Sinica vol 22 no 4 pp 380ndash384 2001

[56] S S K Durairajan Q Yuan L Xie et al ldquoSalvianolic acid Binhibits A120573 fibril formation and disaggregates preformed fibrilsand protects against A120573-induced cytotoxictyrdquo NeurochemistryInternational vol 52 no 4-5 pp 741ndash750 2008

[57] FD Pinheiro Fernandes A P FonteneleMenezes J C de SousaNeves et al ldquoCaffeic acid protects mice from memory deficits

Evidence-Based Complementary and Alternative Medicine 17

induced by focal cerebral ischemiardquo Behavioural Pharmacologyvol 25 no 7 pp 637ndash647 2014

[58] S-J Tsai C-Y Chao andM-C Yin ldquoPreventive and therapeu-tic effects of caffeic acid against inflammatory injury in striatumof MPTP-treated micerdquo European Journal of Pharmacology vol670 no 2-3 pp 441ndash447 2011

[59] J H Kim QWang J M Choi S Lee and E J Cho ldquoProtectiverole of caffeic acid in an 119860120573

25minus35

-induced Alzheimerrsquos diseasemodelrdquo Nutrition Research and Practice vol 9 no 5 pp 480ndash488 2015

[60] G Liang B Shi W Luo and J Yang ldquoThe protective effect ofcaffeic acid on global cerebral ischemia-reperfusion injury inratsrdquo Behavioral and Brain Functions vol 11 no 1 article 182015

[61] D Y Yoo J H ChoiW Kim et al ldquoEffects of luteolin on spatialmemory cell proliferation and neuroblast differentiation in thehippocampal dentate gyrus in a scopolamine-induced amnesiamodelrdquo Neurological Research vol 35 no 8 pp 813ndash820 2013

[62] Y Liu X Tian L Gou L Sun X Ling and X Yin ldquoLuteolinattenuates diabetes-associated cognitive decline in ratsrdquo BrainResearch Bulletin vol 94 pp 23ndash29 2013

[63] S-M Liu Z-H Yang andX-B Sun ldquoSimultaneous determina-tion of six Salvia miltiorrhiza gradients in rat plasma and brainby LC-MSMSrdquo Zhongguo Zhongyao Zazhi vol 39 no 9 pp1704ndash1708 2014

[64] M A Papandreou A Dimakopoulou Z I Linardaki et alldquoEffect of a polyphenol-rich wild blueberry extract on cognitiveperformance of mice brain antioxidant markers and acetyl-cholinesterase activityrdquo Behavioural Brain Research vol 198 no2 pp 352ndash358 2009

[65] M-S Garcıa-Ayllon O Cauli M-X Silveyra et al ldquoBraincholinergic impairment in liver failurerdquo Brain vol 131 no 11pp 2946ndash2956 2008

[66] G K Ferreira M Carvalho-Silva C L Goncalves et al ldquoL-Tyrosine administration increases acetylcholinesterase activityin ratsrdquo Neurochemistry International vol 61 no 8 pp 1370ndash1374 2012

[67] R Vassar P-H Kuhn C Haass et al ldquoFunction therapeuticpotential and cell biology of BACE proteases current status andfuture prospectsrdquo Journal of Neurochemistry vol 130 no 1 pp4ndash28 2014

[68] R Vassar ldquoThe 120573-secretase BACE a prime drug target forAlzheimerrsquos diseaserdquo Journal of Molecular Neuroscience vol 17no 2 pp 157ndash170 2001

[69] D T R Coulson N Beyer J G Quinn et al ldquoBACE1 mRNAexpression in Alzheimerrsquos disease postmortem brain tissuerdquoJournal of Alzheimerrsquos Disease vol 22 no 4 pp 1111ndash1122 2010

[70] S Vladimir-Knezevic B Blazekovic M Kindl J Vladic A DLower-Nedza and A H Brantner ldquoAcetylcholinesterase inhib-itory antioxidant and phytochemical properties of selectedmedicinal plants of the lamiaceae familyrdquoMolecules vol 19 no1 pp 767ndash782 2014

[71] F Marcelo C Dias A Martins et al ldquoMolecular recognitionof rosmarinic acid from Salvia sclareoides extracts by ace-tylcholinesterase a new binding site detected by NMR spec-troscopyrdquo ChemistrymdashA European Journal vol 19 no 21 pp6641ndash6649 2013

[72] G Johnson and S W Moore ldquoWhy has butyrylcholinesterasebeen retained Structural and functional diversification in aduplicated generdquoNeurochemistry International vol 61 no 5 pp783ndash797 2012

[73] R J Grayer M R Eckert N C Veitch et al ldquoThe chemo-taxonomic significance of two bioactive caffeic acid estersnepetoidins A and B in the Lamiaceaerdquo Phytochemistry vol 64no 2 pp 519ndash528 2003

[74] A M D El-Mousallamy U W Hawas and S A M HusseinldquoTeucrol a decarboxyrosmarinic acid and its 41015840-O-triglycosideteucroside from Teucrium pilosumrdquo Phytochemistry vol 55 no8 pp 927ndash931 2000

[75] I Fecka and S Turek ldquoDetermination of water-soluble polyphe-nolic compounds in commercial herbal teas from Lamiaceaepeppermint melissa and sagerdquo Journal of Agricultural and FoodChemistry vol 55 no 26 pp 10908ndash10917 2007

[76] L W Sumner A Amberg D Barrett et al ldquoProposed min-imum reporting standards for chemical analysis WorkingGroup (CAWG) Metabolomics Standards Initiative (MSI)rdquoMetabolomics vol 3 pp 211ndash221 2007

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

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PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

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Diabetes ResearchJournal of

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Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

4 Evidence-Based Complementary and Alternative Medicine

as a comparative chemical compound On the last day 30minafter the last dose of MO or HU SC was given intraperi-toneally (ip) in a dose of 05mgkg bw Control groups weretreated with 05 methylcellulose (MC) whereas water forinjection (H

2O) was used as a vehicle for SC (groups MC +

H2O and MC + SC) MO was prepared ex tempore before

administration and suspended in MC in concentrations of20mgmLOnday 28 of the experiment 1 h after the last dosethe animals were killed by decapitation and hippocampus andpart of frontal cortex were collected from brain of rats Thetissue samples were then stored at minus80∘C until measurementof acetylcholinesterase (AChE) and butyrylcholinesterase(BuChE) activities or mRNA level changes

312 Cognitive and Behavioral Tests Cognitive and behav-ioral tests were used in the present study similarly as in ourprevious report [23] (1) sedative activity was assessed usinga locomotor activity test (2) motor coordination assessmentwas done using a ldquochimneyrdquo test (3) the passive avoidance testwas performed as an animalmodel for the assessment of long-termmemory and (4) the object recognition test was used asan animal model for the assessment of short-term memory

3121 Measurement of Locomotor Activity Locomotor activ-ity assessment was performed with licensed activity meter(Activity Cage Ugo Basile Italy) by placing the animals in thecentre of the apparatus and recording their horizontal activity[23] The data obtained were expressed as signals corre-sponding to animalmovements for 5minutesThe locomotoractivity was measured 30 minutes after the administration ofa single dose of SCH

2OAny distracting factors were reduced

to the minimum (noise presence of people and presence ofother rats)

3122Measurement ofMotorCoordination Motor coordina-tion was evaluated using ldquochimneyrdquo test described originallyfor mice [29] Thirty minutes after SC or vehicle injectionrat was allowed to enter a glass laboratory cylinder that is500mm long and 80mm in diameter laid on its side Uponreaching its bottom by the animal position of the cylinderwas rapidly changed from horizontal to vertical and a timerstarted The animal immediately began to move backwardsThe timer was stopped after the rat left the cylinder andassumed a sitting posture on the top of the vessel The timeof exit from the cylinder was accepted as a measure of motorcoordinationMotor impairment was assessed as the inabilityof rats to climb backwards up the tube within 60 s

3123 Passive Avoidance Test Passive avoidance test wasused as an animal model for the assessment of long-termmemory (effects on retrieval and memory consolidation)[30] The test relies on the natural preference of rats for dark-ness After 2 minutes of habituation to a dark compartmenta rat was placed on the illuminated platform and allowedto enter the dark compartment using licensed apparatus(Passive Avoidance System step through Ugo Basile Italy)Two more approach trials were allowed on the following daywith a two-minute interval between them At the end of

the second trial an unavoidable scrambled electric footshock(500120583A AC 3 s) was delivered through the grid floor of thedark compartment (learning trial) Retention of the passiveavoidance response (latency) was tested 24 h later by placingthe animal on the platformandmeasuring the latency in reen-tering the dark compartment against the arbitrary maximumtime of 180 s The test was performed after 30 minutes afterthe administration of a single dose of SC or the vehicle

3124 Object Recognition Test Object recognition test wasused as an animal model for the assessment of short-termmemory [31]The object recognition task took place in a 40 times60 cm open box surrounded by 40 cm high walls made ofplywood with a frontal glass wall All animals were submittedto a habituation session where they were allowed to freelyexplore the open field for 5min No objects were placed inthe box during the habituation trial On the day of testing theanimals were given an additional 3min rehabituation periodprior to commencing the test The test was divided into threephases with two trials the acquisition trial the retention trialand an intertrial interval of varying times

(i) Acquisition trial in this first trial the animals ex-plored two identical objects (1198601 and 1198602) for a periodof 3min positioned in two adjacent corners 10 cmfrom the walls

(ii) Intertrial interval (ITI) the animals were returned tothe home cage for 30min

(iii) Retention trial in this second trial the animalsexplored a familiar object (119860lowast) that is a duplicate ofthose objects from the acquisition trial (to minimizeolfactory cues) and a novel object (119861) for a further3min

They were made of a biologically inert substance (plastic)and were chosen to enable ease of cleaning (10 alcohol)between subjects in an attempt to remove olfactory cuesObject exploration is defined by animals licking sniffing ortouching the object whilst sniffing but not leaning againstturning round or standing or sitting on the object Objectswere of sufficient weight and were secured to the floor of thearena to ensure that they could not be knocked over ormovedaround by the animal The exploration time (s) of all objectswas recorded via stopwatch for subsequent statistical analysisThe time measured as an exploration behavior was used tocalculate amemory discrimination index (OR) as reported byBlalock et al [31]OR = (119861minus119860lowast)(119861+119860) where119861was the timespent exploring the new object and 119860lowast was the time spentexploring the familiar object Higher OR was considered toreflect greater memory ability [31] The test was performedafter 30 minutes after the administration of a single dose ofSC or the vehicle

313 Acetylcholinesterase and Butyrylcholinesterase ActivitiesAssay in Brain of Rats Acetylcholinesterase (AChE) andbutyrylcholinesterase (BuChE) activities were performed bymodifying spectrophotometric Ellmanrsquos method accordingto Isomae et al [32] The activities of AChE and BuChE

Evidence-Based Complementary and Alternative Medicine 5

028

021

014

007

000

200100 300 400 500 600 700 800 900(Minutes)

(AU

)

1 23

4

56 7 8 9 10

1112

1314

1617

19

15 1820

2122

23

2425

2627

28

2930

31

37

3234

3536 38 39

40

33

Figure 1 Chromatogram UV ofMelissa officinalis leaf extract obtained at 270 nm with peaks identified by HPLC-UV-MS

were determined by measuring the formation of the yellowanion obtained from the reaction between Ellmanrsquos reagentand the thiocholine generated by the enzymatic hydrolysisof acetylthiocholine iodide (ATCh) and butyrylthiocholine(BTCh) respectively (sample 01mL PBS 08mL DTNB01mL ATCh 020mL and BTCh 020mL)The biochemicalassay of AChE and BuChE in homogenate of brain sampleswas expressed as 120583molminmg protein by using spectropho-tometric method (120582 = 412 nm)

314 RNA Isolation and Reverse Transcription Reaction TotalRNA isolation from the rats brain tissues homogenates(frontal cortex hippocampus) was carried out using TriPureIsolation Reagent (Roche) according to manufacturerrsquos pro-tocol The integrity of RNA was visually assessed by a con-ventional agarose gel electrophoresis and the concentrationwill be evaluated by measuring the absorbance at 260 and280 nm in a spectrophotometer (BioPhotometer Eppendorf)RNA samples were stored at minus80∘C until use The 1 120583gof total RNA from all samples was used for the reversetranscription into cDNA using Transcriptor First StrandSynthesis Kit (Roche) according to manufacturerrsquos protocolObtained cDNA samples was stored at minus20∘C or used directlyfor the quantitative real-time PCR (qRT-PCR)

315 Real-Time PCR mRNA Quantification The acetylcho-linesterase (AChE) butyrylcholinesterase (BChE) and beta-secretase (BACE1) genes expression level was analyzed bytwo-step quantitative real-time PCR (qRT-PCR) in a vol-ume of 10 120583L reaction mixture using relative quantificationmethodology with a LightCycler TM Instrument (RocheGermany) and a LightCycler Fast Start DNA Master SYBRGreen I kit (Roche Applied Science) according to theinstructions of the manufacturer All primers sequences weredesigned and custom-designed using the Oligo 60 software(National Biosciences) and were verified by assessment ofa single PCR product on agarose gel and by a single tem-perature dissociation peak (melting curve analysis) of eachcDNA amplification product An GAPDH gene was usedas a housekeeping gene (endogenous internal standard) for

normalization of qPCR For each quantified gene standardcurves were prepared from dilution of cDNA and gener-ated from a minimum of four data points All quantitativePCR were repeated twice The data were evaluated usingLightCycler Run 45 software (Roche Applied Science) EachPCR run included a nontemplate control to detect potentialcontamination of reagents

316 Statistical Analysis All values were expressed as meansplusmn SEM The statistical comparison of results was carried outusing one-way analysis of variance (ANOVA) followed byDuncanrsquos post hoc test for detailed data analysisThe values of119901 lt 005were considered as a statistical significant difference

4 Results

41 Phytochemical Profile of Extract

411 Identification ofMetabolites Forty phenolicmetaboliteswere identified in hydroethanolic Melissa officinalis leafextract (Table 1 Figure 1) The predominant identified com-pounds were bioactive caffeic acid esters and glycosides offlavones The caffeic acid dimer rosmarinic acid (metabolite23) and caffeate trimer (lithospermic acid 31) were the prin-cipal caffeic acid derivatives in the analyzed samples Hydrox-yjasmonic acid and its derivatives teucrol as well as sage-coumarin were identified for the first time in the genusMelissa while luteolin and apigenin glycoconjugates arewell known phytochemicals in this species [33] Multistepfragmentation with accurate mass measurement enabledconfirming that the losses of fragment 799573 amu from the[M-H]minus ions of compounds 4 9 27 30 and 32 referredto sulphate groups The first-order fragmentation of 30revealed the loss of 799573 and yielded the product ion at7191622119898119911 The following fragmentation of this ion cor-responded to that of the sagerinic acid (dimer of rosmarinicacid) described by Barros et al [34] The exact placementof the sulphation position would require in-depth chemicalanalysis Thus 30 was tentatively assigned as sulphated sag-erinic acid

6 Evidence-Based Complementary and Alternative MedicineTa

ble1Metabolitesd

etectedin

Melissa

officin

alisleafextractb

yUPL

C-MS

No

RT [min]

Metabolite

identifi

catio

nCh

emical

form

ula

Exactm

asso

f[M-H

]minusΔpp

mFragmentatio

nin

120582max[nm]

CID

aIdentifi

catio

nlevelb

Reference

Measured

Calculated

Negativeion

mod

e(ESIminus)

Positiveion

mod

e(ESI+)

114

1

2-Hydroxy-3-(34-

dihydroxyphenyl)-

prop

anoic

acid

C 9H9

O5

1970

451970

455minus41775

197179135

199163

283312

8143997

2[34]

217

3Dihydroxybenzoica

cid

hexosid

eC 13

H15

O9

315072

3150722

08346

315153109

317155

282

54726828

3[35]

319

8Ca

ftaric

acid

C 13

H11

O9

3110

413110

409

06141

311221179149

318

6440397

2[35]

4224

2-Hydroxy-3-(34-

dihydroxyphenyl)-

prop

anoica

cid

sulphated

C 9H9

O8

S2770

032770

024

03998

277197179135

312

3[35]

5256

Hydroxyjasm

onicacid

hexosid

eC 18

H27

O9

387166

3871661

09214

387207163

323

44237366

2[17]

6276

Caffeicacid

C 9H7

O4

1790

341790

451minus49075

179135

181163

275

689043

1std

7289

Salviano

licacid

EC 36

H29

O16

7171450

717146

72313

717519339321

295277

275325

49770697

2[35]

8317

Salviano

licacid

HI

(isom

er)

C 27

H21

O12

537104

5371038

08562

537493359295

281325

2[35]

9345

Hydroxyjasm

onicacid

sulphated

C 12

H17

O7

S30507

30507

1117

305225194147

275330

3[17]

10362

NepetoidinB

C 17

H13

O6

313072

3130718

11232

nd282316

5316819

2[73]

1137

Yunn

aneica

cidF

C 26

H25

O14

5971255

5971244

18743

597509311197

Masked

2[34]

12383

Decarbo

xyrosm

arinicacid

(teucrol)

C 17

H15

O6

315088

3150874

09396

315179135

275330

637829

2[74]

1339

Caffeoylcaft

aricacid

C 22

H17

O12

473073

4730725

07555

173311149

Masked

65018

3[35]

14397

Apigenin

glucosylrham

nosid

eC 27

H29

O14

577157

5771563

11136

577269

579433271

275340

92741003

3[33]

15404

Luteolin

7-O-glucosid

e31015840-O

-glucuronide

C 27

H27

O17

623126

6231254

09696

62346144

7285

255

625463287

273343

2[33]

16421

Rosm

arinicacid

hexosid

eC 24

H25

O13

52113

5211301

03551

521359161

523361325163

329

25245848

2[34]

1743

Luteolin

O-diglucosid

eC 27

H29

O16

609147

6091461

07183

609285

611287

269349

3[33]

18441

Luteolin

glucosylrham

nosid

eC 27

H29

O15

593152

5931512

08077

59344

7285

59544

9287

271343

3[33]

19449

Luteolin

41015840-O

-glucosid

eC 21

H19

O11

4470

9444

70933

09728

447285

449287

271343

5319116

3[33]

2045

Sagerin

icacid

2-hydroxy-3-

(34-dihydroxyph

enyl)-

prop

anoide

C 45

H39

O20

8992

058992

0410

448

899719591475

295

Masked

3[35]

2149

Salviano

licacid

B(lithosperm

icacid

B)C 36

H29

O16

717146

7171461

01846

717519359161

327

6441188

2[34]

22504

Sagerin

icacid

C 36

H31

O16

7191

637191

618

09979

719519359161

287330

2[34]

23514

Rosm

arinicacid

C 18

H15

O8

3590

773590

772

0002

359161

361163

329

5281792

1std

Evidence-Based Complementary and Alternative Medicine 7

Table1Con

tinued

No

RT [min]

Metabolite

identifi

catio

nCh

emical

form

ula

Exactm

asso

f[M-H

]minusΔpp

mFragmentatio

nin

120582max[nm]

CID

aIdentifi

catio

nlevelb

Reference

Measured

Calculated

Negativeion

mod

e(ESIminus)

Positiveion

mod

e(ESI+)

24555

Salviano

licacid

B2-hydroxy-3-(34-

dihydroxyphenyl)-

prop

anoide

C 45

H37

O20

89719

8971884

16873

897717519359

161

330

3[35]

2556

Sagerin

icacid

di-2-hydroxy-3-(34-

dihydroxyphenyl)-

prop

anoide

C 54

H47

O24

1079244

910792457minus07911

1079897719539

359295

288330

3[35]

26592

Sagecoum

arin

di-2-hydroxy-3-(34-

dihydroxyphenyl)-

prop

anoide

caffeide

C 54

H43

O24

10752156

10752150

0559

1077897717537

409359339277

322

3[35]

27598

Rosm

arinicacid

sulphated

Iisomer

C 18

H15

O11

S4390

354390

341

20214

439359341163

Masked

2[34]

28613

Luteolin

31015840-O

-glucuronide

C 21

H17

O12

4610

734610

7216

34461285

463287

269340

170474237

2[33]

29632

Salviano

licacid

AC 26

H21

O10

493115

493114

11011

493359295179

298327

5281793

[34]

30656

Sagerin

icacid

sulphated

C 36

H31

O19

S7991

196

7991

186

0954

799719619519

359161

325

3[35]

31671

Lithosperm

icacid

C 27

H21

O12

537104

537104

809698

537493359161

292329

6441498

2[35]

3268

Rosm

arinicacid

sulphated

IIiso

mer

C 18

H15

O11

S4390

344390

341

02837

439359341163

Masked

2[34]

33689

Sagecoum

arin

C 27

H19

O12

535088

5350882

01204

535311267177

Masked

2[36]

3470

3Salviano

licacid

LIisomer

C 36

H29

O16

717146

7171461

02697

717519359

284329

2[35]

3571

Salviano

licacid

Lhydroxycaffeide

C 45

H35

O20

895173

8951727

06282

895519359161

Masked

3[35]

3674

Sagecoum

arin

cafta

ride

C 40

H29

O20

8291

268291

258

06953

829667535355

311

Masked

3[36]

37751

Sagecoum

arin

2-hydroxy-

3-(34-dihydroxyph

enyl)-

prop

anoide

C 36

H27

O16

715131

7151305

08176

715535311267

319

3[34]

3877

1Unk

nown

C 36

H57

O14

S745348

7453475

044

02281326

4[35]

3978

5Methylrosmarinate

C 19

H17

O8

373093

3730929

01765

373359161

284323

6479915

2[75]

40869

Salviano

licacid

Ccaffeoylhydroxycaffeide

C 44

H33

O18

8491

688491

672

08615

849687491359

327255

286318

3[35]

a CID

identifier

fora

chem

icalstr

ucture

intheP

ubCh

emCom

poun

ddatabase

b Metabolite

identifi

catio

nlevelaccording

toMetabolom

icsS

tand

ards

Initiativer

ecom

mendatio

n[76]

stdidentificatio

non

theb

asisof

stand

ardcompo

undfragmentatio

nnd

not

detected

8 Evidence-Based Complementary and Alternative Medicine

100 200 300 400 500 600 700 800 900 1000 1100 1200

mz

70

75

80

85

90

95

100

minus198 (2-hydroxy-3-(34-dihydroxyphenyl)

-porpanoide)

minus180 (caffeide)

minus180 (2-hydroxy-3-(34-dihydroxyphenyl)

-propanoide)

minus180 (2-hydroxy-3-(34-dihydroxyphenyl)

-propanoide)

minus180 (2-hydroxy-3-(34-dihydroxyphenyl)

-propanoide)

1350438

2191749

HO

HO

HO HO

OH

OH

OH

OH OH OH

OHOHOHOHHOOC

COOH

COOH COOH COOH

24612ppmC36H55O37 = 10792422

minus07911ppmC54H47O24 = 10792457

10792449

10448ppmC45H39O20 = 8992040

899205018453ppm

C36H31O16 = 7191612

7191625

20501ppmC27H23O12 = 5391190

5391201

14852ppmC23H33O10 = 4692074

469208119316ppm

C17H11O5 = 2950606

2950612

0

5

10

15

20

25

30

35

40

45

50

55

60

65

Rela

tive a

bund

ance

22903ppmC18H15O8 = 3590767

3590775

minus37218 ppmC9H5O3 = 1610239

1610233

(a)

100 200 300 400 500 600 700 800 900 1000

mz

100 200 300 400 500 600 700 800 900 1000

mz

100 200 300 400 500 600 700 800 900 1000

mz

100 200 300 400 500 600 700 800 900 1000

mz

0

250

500

750

1000

00

04

08

times104

times104

01234

0

1

2

times105

Ints

Ints

Ints

Ints

MS1

MS2

MS3

MS4

[M-H]minus10749

minus224 (acidic part

minus224 (acidic part

of sagecoumarin)

of sagecoumarin)

minus360 (rosmarinic acid)

4908

7148

5348 minus180 minus162 (caffeide)8769

31074909

5348

minus180 (2-hydroxy-3-(34-dihydroxyphenyl)-propanoide)

3108

35474909

1

(b)

Figure 2 Continued

Evidence-Based Complementary and Alternative Medicine 9

150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900

mz

0

5

10

15

20

25

30

35

40

45

50

55

60

65

Rela

tive a

bund

ance

O

O

O O

O

O

OH OH

OH

OH

OH

OHOH

OH

HO

HO

HO

COOH

OC

1529946

1970447

C9H9O5 = 1970455

minus41775ppm1610232

2552328

C16H31O2 = 2552330

minus04091ppm

3110563

C17H11O6 = 3110561

05243ppm

minus224 (fragment of sagecoumarin)

3912257

4092361

4832730

C29H39O6 = 4832752

minus44931ppm

5350885

C27H19O12 = 5350882

05640ppm

minus132 (tartaric acid)

5770990

C29H21O13 = 5770988

06486ppm

6670947

C31H23O17 = 6670941

09808ppm

minus162 (caffeic acid)

8291267

C40H29O20 = 8291258

11370ppm

(c)

Figure 2 (a) Mass spectra in negative ionization mode and simplified fragmentation scheme of compound 25 (pentameric ester ofcaffeic acid) (b) Mass spectra in negative ionization mode and simplified fragmentation scheme of compound 26 (pentameric structureof sagecoumarin di-2-hydroxy-3-(34-dihydroxyphenyl)-propanoide caffeide) (c) Mass spectra in negative ionization mode and simplifiedfragmentation scheme of compound 36 (pentameric structure of sagecoumarin caftaride)

Tetrameric structures of hydroxycinnamic acids wereidentified in lemon balm recently [34 35] The measure-ment of accurate masses allowed the identification of com-pound 25 which was tentatively identified as pentamericester of caffeic acid (Figure 2(a)) In 25 the double loss of1800421 amu corresponded to fragments with the molecularformula of C

9H8O4 adequate to dehydroxylated 2-hydroxy-

3-(34-dihydroxyphenyl)-propanoic acid The product ionat 7191623119898119911 and its further fragmentation are sim-ilar to those of metabolite 22 as described previously [34 35]Therefore metabolite 25was tentatively assigned as sagerinicacid di-2-hydroxy-3-(34-dihydroxyphenyl)-propanoideNev-ertheless comprehensive studies by nuclear magnetic reso-nance are required to complete elucidation of substitutionpattern for particular components of those pentamers

Sagecoumarins were previously identified in Salvia offic-inalis as caffeic acid trimers [36] Our MS analysis indicatedthe presence of such compounds and their derivatives alsoin M officinalis The pseudomolecular ion of compound33 observed in the negative ionization mode had theaccurate mass of 5350880119898119911 which corresponded to thechemical formula C

27H19O12

adequate for sagecoumarin(according to the Metlin and KNApSAck databases) Note-worthy 26 33 36 and 37 had the same fragmentationpattern of the product ion obtained in the MSMS andMSn in the negative ionization mode The [M-H-1800422]minusion corresponded to the detachment of 2-hydroxy-3-(34-dihydroxyphenyl)-propanoic acid thus 37 was tentatively

assigned as tetrameric sagecoumarin 2-hydroxy-3-(34-dihy-droxyphenyl)-propanoide The [M-H-3600846]minus ion in 26was indicated on rosmarinic acid substitution to 37 Massspectra of MSn in negative ionization of the compoundprovided complementary information to HR-MSMS massspectra indicated on caffeic acid as internal componentof the dimer In addition simultaneous loss of fragments180 amu and 224 amu in MS3 and MS4 indicated that thetwo components cannot be linked (Figure 2(b)) How-ever detailed analysis of substitution pattern of 26 shouldbe done Therefore 26 was tentatively assigned as penta-meric structure of sagecoumarin di-2-hydroxy-3-(34-dihy-droxyphenyl)-propanoide caffeide Rupture of caffeic andtartaric acid moieties from the product ion at 5350885119898119911was observed for compound36 (Figure 2(c))Detection of theaccurate masses of these two detached fragments with Δ lessthan 1 ppm eliminated the possibility of hexose and pentosesubstitution which have the same nominal masses as caffeicand tartaric acid respectively Therefore 36 was tentativelyidentified as another pentameric structure of sagecoumarincaftaride

Metabolite 28 with the accurate masses of 461073119898119911was tentatively identified as luteolin O-glucuronide The [M-H-1760324]minus ion is indicated on loss of structure C

6H8O6

corresponding to glucuronide moiety The product ion at2850405119898119911 was indicated on flavone luteolin The placeof substitution of the carboxylic acid on flavone skeleton isproblematic due to different isomers reported in lemon balm

10 Evidence-Based Complementary and Alternative Medicine

Table 2 Effect ofMelissa officinalis leaf extract (200mgkg po) treatment on sedative activity motor coordination and memory in rats

Group 119899Locomotor activity

[number of impulses5min]Motor coordination

exit time [s]Short-term memorye

ORLong-term memory

latency [s]MC + H

2

O 18 390 plusmn 24 17 plusmn 3 040 plusmn 006 47 plusmn 14MC + SC 18 526 plusmn 48lowast 32 plusmn 5lowast 032 plusmn 005 12 plusmn 3lowast

MO + H2

O 10 231 plusmn 48lowast 32 plusmn 7lowast 043 plusmn 007 169 plusmn 11lowast

MO + SC 9 436 plusmn 60 43 plusmn 7 009 plusmn 011lowast 23 plusmn 6HU + H

2

O 9 515 plusmn 32 15 plusmn 2 037 plusmn 009 158 plusmn 14lowast

HU + SC 8 639 plusmn 71lowast 56 plusmn 3 022 plusmn 006 49 plusmn 18

RA + H2

O 8 406 plusmn 59 21 plusmn 5 045 plusmn 005 58 plusmn 28RA + SC 8 605 plusmn 55 28 plusmn 7 045 plusmn 005 20 plusmn 7Means plusmn SEM119899Number of animalsMC + H

2O control rats

SC scopolamine (05mgkg bw ip)HU huperzine A (05mgkg bw po)RA rosmarinic acid (10mgkg bw po)eExpressed as ratio OR = (119861 minus 119860lowast)(119861 + 119860) for details see Section 3lowastVersus MC + H

2O 119901 lt 005

Versus MC + SC 119901 lt 005

luteolin 31015840-O- and 7-O-glucuronide [33] It is impossible todistinguish both structures by mass spectrometry Only onechromatographic peak corresponding to the [M-H]minus ion at461073119898119911 was observed in our study Since luteolin 31015840-O-glucuronide was assigned as the most abundant flavonoid inlemon balm [33] we assumed that 28 corresponded to thisstructure

412 Flavonoids and Polyphenolic Acids The major com-pound from the 40 chemical compounds identified in hy-droethanolic MO leaf extract established by HPLC was RA(885 g100 g) (Table 1 Figure 1)

Other chemical compoundswith neuromodulatory activ-ities such as lithospermic acid (0042 g100 g) salvianolicacid A (0040 g100 g) salvianolic acid B (0023 g100 g) andcaffeic acid (0087 g100 g) were also documented in litera-ture Moreover the total polyphenolic compounds content ofMO determined with the use of FolinndashCiocalteu assay was3397 calculated as gallic acid The total hydroxycinnamicderivatives content expressed spectrophotometrically as ros-marinic acid was 2115 g100 g

413 Essential Oil Composition Hydroethanolic MO leafextract contained 008 of total essential oil The GCFIDanalysis showed that the extract comprised camphene(004) alfa-pinene (007) beta-pinene (1647) andmyrcene (1951) Moreover according to retention time16 compounds were identified as follows alfa-bisabololborneol carvone chamazulene cineole eugenol gamma-terpineol guaiazulene isopulegol linalool limonene men-thol menthyl acetate pulegone terpine and thymol Thesecompounds are present in the essential oil in trace amountswhich do not allow the quantitative interpretation

42 Cognitive and Behavioral Experiments

421 Locomotor Activity A one-way ANOVA analysisrevealed significant differences in the locomotor activity of

rats expressed as their horizontal spontaneous activity afterMO administration (ANOVA 119865(7 80) = 646 119901 lt 005)(Table 2) Detailed post hoc analysis showed thatMO + H

2O

decreased the locomotor activity of rats by 4031 but HU +H2O did not affect this activity when compared with control

group (MC + H2O) We observed also that RA + H

2O did

not change the locomotor activity of rats Stimulating effectsin the locomotor activity of rats were observed after an acuteSC injection (MC + SC versus MC + H

2O 119901 lt 005) and

this effect was observed in all SC-treated rats when comparedwith the proper non-SC-treated animals On the contrarythese SC-treated animals did not differ in comparison toanimals that received SC only (MO + SC versus MC + SC119901 gt 005 HU + SC versus MC + SC 119901 gt 005 RA + SCversus MC + SC 119901 gt 005)

422 Motor Coordination A one-way ANOVA analysisrevealed significant differences in motor coordination of ratsexpressed as their exit time from the cylinder (119865(5 73) =284 119901 lt 005) (Table 2) Detailed analysis showed that themultiple administration of RA + H

2O and HU + H

2O did

not affect significantly this paradigm when compared withcontrol rats (119901 gt 005) whereas MO treatment led to pro-longed exit time (MO + H

2O versus MC + H

2O 119901 lt 005)

Moreover generally SC-treated animals showed producedprolongation of exit time and the effects were statisticallysignificant not only in control groups (MC + SC versus MC +H2O 119901 lt 005) but also in RA-treated rats (RA + SC versus

MC + SC 119901 lt 005) However the rest of the SC-treatedanimals did not differ in comparison to animals receiving SConly (MO + SC versus MC + SC 119901 gt 005 HU + SC versusMC + SC 119901 gt 005)

423 Long-Term Memory A one-way ANOVA analysisrevealed significant differences in long-term memory afterusing a passive avoidance test (119865(7 77) = 201 119901 lt 005Table 2) It was shown that the strongest effect leading to

Evidence-Based Complementary and Alternative Medicine 11

Table 3 The effect ofMelissa officinalis leaf extract on acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) activities and AChEBuAChE or beta-secretase (BACE1) mRNA expression levels in frontal cortex (FC) or hippocampus (Hipp) of rats

Group119899Enzyme activity

[nmolminmg protein]mRNA expression

[]AChE BuChE ACHE BuChE BACE1

FC Hipp FC Hipp FC Hipp FC Hipp FC HippMC + H

2

O 363 plusmn 49 439 plusmn 73 65 plusmn 11 53 plusmn 8 100 plusmn 12 100 plusmn 11 100 plusmn 18 100 plusmn 11 100 plusmn 16 100 plusmn 8MO + H

2

O 276 plusmn 34 409 plusmn 28 69 plusmn 6 62 plusmn 4 48 plusmn 4lowast 31 plusmn 7lowast 16 plusmn 2lowast 64 plusmn 21amp 36 plusmn 3lowast 50 plusmn 8lowast

HU + H2

O 189 plusmn 15lowast 239 plusmn 15lowast 58 plusmn 6 51 plusmn 4 53 plusmn 13lowast 85 plusmn 5 42 plusmn 9lowast 102 plusmn 11 62 plusmn 6lowast 98 plusmn 4RA + H

2

O 224 plusmn 16lowast 251 plusmn 12lowast 77 plusmn 7 99 plusmn 7lowast 101 plusmn 12 103 plusmn 5 184 plusmn 31lowast 56 plusmn 7lowast 126 plusmn 13amp 98 plusmn 3Means plusmn SEM119899Number of animals 7ndash10Values expressed as a ratio the geneGAPDHMC + H

2O control group

HU huperzine A (05mgkg bw po)RA rosmarinic acid (10mgkg bw po)lowastampVersus MC + H

2O 119901 lt 005 or 119901 lt 007 respectively

an improvement of this paradigm was produced by extractof MO and HU but not RA when compared with controlanimals (MO + H

2O versus MC + H

2O 119901 lt 005 HU +

H2O versus MC + H

2O 119901 lt 005 RA + H

2O versus MC +

H2O 119901 gt 005) However the administration of SC to rats

significantly decreased the latency time of passive avoidancetask (MC + SC versus MC + H

2O 119901 lt 005) After MO or

RA combined treatment with SC no improvement of long-term memory was observed but HU given with SC showedenhancement of this paradigm in rats (HU + SC versusMC + SC 119901 lt 005) Therefore it can be concluded thatadministration of HU overcomes the effect shown by SC only(Table 1)

424 Short-Term Memory The results of the object recogni-tion test showed that an administration of the compoundsor extract did affect the ratsrsquo short-term memory (ANOVA119865(7 78) = 273 119901 lt 005) (Table 2)

Detailed post hoc analysis showed that only SC signifi-cantly decreased the short-term memory in MO-treated rats(MO + SC versusMO + H

2O 119901 lt 005MO + SC versus MC

+ SC 119901 lt 005) whereas the differences between the rest ofthe animals did not reach statistical significance (119901 gt 005)

43 AChE and BuChE Activities in Rat Brain A one-wayANOVA revealed significant differences between groups inthe activity of AChE in both the cortex and the hippocampus(frontal cortex 119865(3 27) = 565 119901 lt 005 hippocampusANOVA 119865(3 29) = 796 119901 lt 005) It was found out thatMOshowed an insignificant inhibition of AChE activity in thefrontal cortex by 24 when compared with control rats (MC+ H2O 119901 lt 006) and in the hippocampus by 7 (Table 3)

whereas HU produced a distinct significant inhibition ofAChE activity in comparison to control group by 48 (119901 lt005) and 47 (119901 lt 005) in the cortex and the hippocampusrespectively Also RA lowered significantly AChE activityboth in the cortex (38) and in the hippocampus (43)Moreover there were not significant differences between thevalues of BuChE activities for MO and HU when compared

with control group in the frontal cortex (ANOVA 119865(3 30) =101 119901 gt 005) whereas in the hippocampus the differencesreached statistical significance (ANOVA 119865(3 27) = 141119901 lt 005) Detailed analysis showed that only RA effect wassignificant and increased BuChE activity in the hippocampuswhen compared with the control rats (119901 lt 005)

44 AChE BuChE and BACE1 mRNA Level Changes in RatBrain A one-way ANOVA analysis revealed significant dif-ferences of AChE mRNA transcription profile in the cortex(ANOVA 119865(3 27) = 857 119901 lt 005) As shown in Table 3the multiple treatment of MO produced in the cortex astatistically significant decrease of AChEmRNA level by 52the administration of HU caused decrease of its level by 44(119901 lt 005) whereas RA did not affect this parameter whencompared to the control

There were significant differences between the relativevalues of BuChE mRNA levels in this region of brain in rats(frontal cortex ANOVA 119865(3 25) = 162 119901 lt 005) TheMOtreatment led to a decrease in the BuChEmRNA level by 84(versus MC + H

2O 119901 lt 005) the prolonged HU admin-

istration resulted in a decrease of the transcript level in thecortex by 58 (versus MC + H

2O 119901 lt 005) but RA

increased this parameter in the cortex by 84 (versus MC +H2O 119901 lt 005)The significant differences ofmRNA transcription level of

AChE mRNA level in the hippocampus (ANOVA 119865(3 31) =212 119901 lt 005) have been observed The detailed analysisshown that in the case of AChE afterMO treatment mRNAlevel significantly decreased by 69 (versus MC + H

2O

119901 lt 005) while the administration of HU resulted in astatistically insignificant decrease of AChE mRNA level by18 when compared with control group Also RA did notchange the level of transcript

In the case of BuChEmRNA expression in the hippocam-pus there were statistically significant differences betweengroups (ANOVA 119865(3 27) = 310 119901 lt 005) Detailed analysisshowed that in theMO + H

2O treated group the expression

lowered by 36 but the difference did not reach strong

12 Evidence-Based Complementary and Alternative Medicine

significance when compared with the control values (119901 lt007) and no change in the expression level of this enzymewas shown after the administration of HU On the contraryRA produced an increase of BuChE mRNA expression in thehippocampus by 44 (versus MC + H

2O 119901 lt 005)

Further analysis showed the significant differences inBACE1 mRNA expression in both brain regions of rats (inthe cortex and the hippocampus) (frontal cortex ANOVA119865(3 27) = 163 119901 lt 005 hippocampus ANOVA 119865(3 27) =133 119901 gt 005) It was observed that MO produced astatistically significant decrease of the BACE1 expression levelby 64 in the cortex (versus MC + H

2O 119901 lt 005) and by

50 in the hippocampus (versus MC + H2O 119901 lt 005) For

comparison HU treatment led to a decrease in the mRNAexpression level by 38 in the cortex (versus MC + H

2O

119901 lt 005) but not in the hippocampus On the contrary RAproduced an increase of this transcript in the cortex by 26but the effect did not reach a strong statistical significance(versus MC + H

2O 119901 lt 007) whereas in the hippocampus

there was no difference between RA and control group

5 Discussion

Cognitive and Behavioral Experiments The present studyinvestigated the influence of subchronic (28-fold) adminis-tration of standardized 50 EtOH extract ofMelisa officinalisleaf extract (MO) (200mgkg po containing 177mgkg ofRA) on SC-induced impairment of short-term and long-term memory in rats The results were compared with theactivity of cholinesterases (AChE and BuChE) as well aswith AChE BuChE and BACE1 gene expression levels in thecortex and hippocampus of the rat brain So far little evidenceis yet available as regards mechanisms of MO leaf extractaction that are potentially relevant to cognitive function ofrats after per os administration MO is traditionally used intreating neurological disorders through its anti-AChE [18]and antiagitation properties [37] Moreover Wake et al [38]and Kennedy et al [15] showed thatMO extract has nicotinicreceptor activity and that it can displace [3H]-(N)-nicotinefrom nicotinic receptors in homogenates of human cerebralcortex tissue and they suggested that these mechanisms canexplain activity of MO extract in amnesia model RecentlySoodi et al [18] observed that intraperitoneal injections ofMO extract (200mgkg) in rats could significantly enhancelearning and memory processes in animals since the extractsignificantly ameliorates SC-induced learning deficit in Mor-ris water maze test On the contrary in higher dose itcan be observed that MO extract (400mgkg) could notreverse SC-induced memory impairment [18] In our studyadministration of MO extract at a dose of 200mgkg (po)showed the effect leading to improvement of long-termmemory in a passive avoidance test in rats However afterMO combined treatment with SCMO did not overcome theimpairment shown by SC (Table 2) It should be emphasizedthat it is not clear whether the effect shown in our study byMO in non-SC-treated rats is specific since theMO treatmentproduced significant lowering of locomotor activity of ratstherefore the sedative profile ofMO cannot be excluded

On the other hand Ryu et al [39] observed that agitationand aggression are highly prevalent in patientswith dementiaAccording to Gitlin et al [40] nonpharmacological inter-ventions are recommended as first-line therapy Althoughantipsychotics have shown benefit for Alzheimerrsquos disease-related psychosis their use is associated with several seriousadverse effects [40]Thus it seems that the use ofMO extractcan provide dual benefits both in aspect of inhibition ofagitation and in improving the memory of patients withAlzheimerrsquos disease

Furthermore in our study RA in the dose of 10mgkgbw (po) did not affect either short- or long-term memoryalthough RA lowered significantly AChE activity both inthe cortex and in the hippocampus Moreover we observedthat the repeated administration of RA in non-scopolamine-treated rats did not produce any changes of locomotoractivity similarly to our previous study [23]

It is possible that other chemical compounds can influ-ence the memory in rats by synergic interactions in plantextract According to our calculations caffeic acid (0174mgin a single dose of extract administered to animals per kgbw) lithospermic acid (0084mgkg) salvianolic acid A(008mgkg) and salvianolic acid B (0046mgkg) may beresponsible for observed pharmacological effects On theother hand results from few studies showed that salvianolicacids do not cross the blood brain barrier (BBB) [41 42]and also lithospermic acid does not efficiently cross the BBB[43] For this reason the interpretation of our results is morecomplicated

Firstly Xu et al [44] demonstrated that Sal A is ametabol-ically unstable compound that would undergo rapid methy-lationmetabolism catalyzed by catechol O-methyltransferasein vivo into four major methylated metabolites of Sal A(3-O-methyl 31015840-O-methyl 3310158401015840-O-dimethyl and 31015840310158401015840-O-dimethyl salvianolic acid A) These generated O-methylatedmetabolites may be largely responsible for its in vivo phar-macological effects Although there are no available recentstudies on the ability of these compounds to pass the BBBsuch possibility should not be excluded

Secondly several studies showed central pharmacolog-ical effects in animals after per os administration of Sal B[45 46] Sal A [47] and caffeic acid [48] It was provedfor example that salvianolic acid B (10mgkg po) sig-nificantly rescued the A12057325ndash35 peptide-induced decreaseof choline acetyltransferase and brain-derived neurotrophicfactor protein levels in an amyloid 120573 (A120573) peptide-inducedAlzheimerrsquos disease mouse model [49] It also significantlyreversed (10mgkg po) the cognitive impairments inducedby scopolamine (1mgkg ip) or A120573 (25ndash35) (10 nmol5 120583Licv) injection inmice [45] Previous studies [46 47] showedalso that both Sal A and Sal B are able to improve theimpaired memory function induced by cerebral ischemia-reperfusion in miceThe stimulation of neurogenesis processin both subgranular zone (SGZ) and subventricular zone(SVZ) after brain ischemia and also alleviation neural cellsloss and improved motor function recovery after brainischemia in rats after the Sal B administration were alsoobserved by Zhong et al [50] Moreover the exposure to SalB can maintain the proliferation of neural stemprogenitor

Evidence-Based Complementary and Alternative Medicine 13

cells (NSPCs) after cerebral ischemia and improve cognitivepostischemic impairment after stroke in rats using Morriswater maze test therefore authors concluded that Sal B mayact as a potential drug in treatment of brain injury or neu-rodegenerative disease [51] Additionally the improvement ofmotor function after cerebral ischemia in rats after salvianolicacid B administrationwas also demonstrated [52]The clue topass through the BBB Sal B provided also results of Li et alindicating its protective effect on BBB in rats after cerebralischemia-reperfusion by inhibiting the MAPK pathway [53]In addition to this it was shown that lithospermic acid andsalvianolic acids exerted neuroprotective activity in variousexperimental models Lithospermic acid significantly attenu-ates neurotoxicity in vitro and in vivo induced by 1-methyl-4-phenylpyridin (MPP(+)) by blocking neuronal apoptotic andneuroinflammatory pathways [54] Salvianolic acid B inhib-ited amyloid beta-protein aggregation and fibril formation aswell as directly inhibiting the cellular toxicity of amyloid beta-protein in PC12 cells [55] and significantly reduced its cyto-toxic effects on human neuroblastoma SH-SY5Y cells [56]

For an explanation of our results observations of Pin-heiro Fernandes et al [57] may be very helpful whichshowed that caffeic acid nonflavanoid catecholic compoundwhose derivatives are occurring in MO extract improvedthe working spatial and long-term aversive memory deficitsinduced by focal cerebral ischemia in mice Anwar et al [48]showed also that caffeic acid (100mgkg) improved the step-down latencies in the inhibitory avoidance in rats Tsai et al[58] showed that caffeic acid is a potent neuroprotective agentin brain of 1-methyl-4-phenyl-1236-tetrahydropyridine(MPTP) treatedmiceMoreover caffeic acid improves A12057325ndash35-induced memory deficits and cognitive impairment inmice [59] In another study it was also shown that this com-pound has a significant protective effect on global cerebralischemia-reperfusion injury in rats [60] For instance 124 plusmn18mg100 g of caffeic acid was detected in the brain of micewith a diet containing 2 caffeic acid for 4 weeks [58] Thusthere is progressive evidence that this compound passesthrough the BBB and has a central pharmacological activity

Also results by Yoo et al are also worth noting [61] whichshowed also in a ratmodel of scopolamine-induced amnesiathat administration of luteolin (a common flavonoid frommany plants including M officinalis) at dose of 10mgkgcaused the increase in the brain-derived neurotrophic factor(BDNF) acetylcholine and the decrease in lipid peroxida-tion Liu et al [62] observed that chronic treatment withluteolin (50 and 100mgkg) improved neuronal injury andcognitive performance by attenuating oxidative stress andcholinesterase activity in streptozotocin-induced diabetes inrats In our study ethanolic extract of MO administered torats significantly improved long-term memory after using apassive avoidance test but afterMO combined treatmentwithSC no improvement of this paradigmwas observed (Table 2)

Thirdly available pharmacokinetic studies were carriedout for the pure compounds (Sal A and B) and the extractof the roots of Salvia miltiorrhiza [42 63] but there are notavailable studies of bioavailability of these compounds afteradministration of Melissa officinalis leaf extract containingother compounds as compared with the extract of Salvia

miltiorrhiza Moreover there is a lack of data about howscopolamine may influence the penetration of the salvianolicacids (and other compounds) across the BBB Hence thereis a need to study the pharmacokinetic parameters of thesecompounds in the group of animals treatedwith scopolaminein comparison with control group

AChEandBuChEActivities in Rat Brain To date several stud-ies have focused on explaining themechanismof action of theMS extract and its active compounds Soodi et al [18] showedthat treatment of animals withMO extract (400mgkg) priorto scopolamine injection could ameliorate scopolamine-induced enhancement in AChE activity This dose of MOextract inhibited the AChE activity in the hippocampus ofrats (519 versus 1004 in normal saline group 119901 lt 005914 in group treated with scopolamine +MO versus 1281in scopolamine group 119901 lt 005) In another study Anwaret al [48] showed that 50 and 100mgkg of caffeic aciddecreased in vivo the AChE activity in the cerebral cortexand striatum and increased the activity of this enzyme inthe cerebellum hippocampus hypothalamus pons lympho-cytes and muscles when compared to the control group(119901 lt 005) Our study showed that ethanolic extract of MOproduced an insignificant and slight inhibition of AChE andBuChE activity in the frontal cortex and in the hippocampus(especially) However it was observed previously [23] thatRA inhibited the AChE activity in the rats frontal cortex andhippocampusMoreover we showed that RA possess a strongstimulatory effect on BuChE in the hippocampus

AChE BuChE and BACE1mRNA Level Changes in Rat BrainSo far no results have been published of studies concerningthe in vivo assessment of changes in AChE BuChE andBACE1 gene expression profile in different brain regionsunder the influence of MO extracts or their key bioactivemetabolites Such studies focused overwhelmingly on theanalysis of their in vitro activities to a lesser extent in animalexperiments

In our study in the frontal cortex we have observedthe strongest inhibition of both AChE and BuCHE mRNAtranscription under the influence ofM officinalis extract andhuperzine A (Table 3) However a more significant differencein the level of transcript was seen in the frontal cortex inparticular in the case of BuCHE mRNA of experimentalrats (a decrease by 516 and 44 for MO and HU versuscontrol resp)The strong inhibition of transcription ofAChEmRNAwas also observed in the hippocampus ofMO-treatedrats (a decrease by 69) The BuChE mRNA transcriptionin animals receiving MO was lowered in a moderate way(decrease by 36)HuperzineA alone has not caused changesthat were observed in the group ofMO (Table 2) Our findingsmostly correlate with the observed changes in activities ofAChE and BuChE in different groups of animals In the caseof BuChE slightly different results between its activity andexpression profile were especially seen in the frontal cortexand hippocampus of animals receivingMO (Table 3)

To date there is a lack of published results of studiesmaking an attempt to clarify the potential differences in thetranscriptional profile and activity of AChE and BuChE

14 Evidence-Based Complementary and Alternative Medicine

under the influence of MO and its bioactive metabolites Itcannot be excluded that the key for the observed differencesin the level of AChE activities and mRNA level can becaused by changes in the activity of AChE in other regionsof the brain not analyzed in this study such as substantianigra cerebellum globus pallidus and hypothalamus whereit exerts nonenzymatic neuromodulatory functions affect-ing neurite outgrowth and synaptogenesis modulating theactivity of other proteins regional cerebral blood flow andother functions [64] But it is difficult to clearly explain whydiminishing of AChE and BuChEmRNAs did not always cor-relate with the lowering of activity of these enzymes althoughit has been recently noted that AChE activity was notparalleled by an increase in mRNA levels [65] The authorsexplained this fact by stating that AChE levels are regulatedat transcriptional posttranscriptional and posttranslationallevels leading to complex expression patterns which canbe modulated by physiological and pathological conditionsHowever these mechanisms are not fully understood andfurther studies are needed in this field

The cause of observed different degree of inhibition ofactivity and transcription status of studied genes especiallyof BuChE (and AChE) may lie in a so-called ldquonegative feed-backrdquo consisting of a complicated transcriptiontranslationregulation protein-protein interactionsmodifications and ametabolic network together forming a system that allows thecell to respond sensibly to the multiple signal molecules thatexist in its environment [66]

Because of that we propose that the reason for differencesbetween AChE and BuChE activities in the cortex and hip-pocampus under the MO may be due to the fact of insuf-ficiency of applied dose and the duration of the experi-ment affecting the transcriptional tissue-specific cellularmachinery regulating BuChE transcription without affectingits activity Moreover the administration of higher doses ofMO and extended period of time could lead to sufficientinhibition of its activity

There is a need to conduct further studies to determinethe molecular degree of dependence between changes inAChE and BuChE activities and activities of potential keyfactors regulating expression of these genes in the frontalcortex and hippocampus under the influence of extracts ofMO and active metabolites Another study determining theeffectiveness of their actions on the cholinergic system inexperimental animal models of memory impairment withparticular emphasis on scopolamine including the determi-nation of changes at the molecular level should be thereforecarried out

Alterations in BACE1 protein level have been proved inpostmortem brain tissue from individuals with AD withincreases decreases and also no change reported [1 67 68]An example of confirmation of these findings at the mRNAlevel is results of study by Coulson et al [69] Based onconducted studies some authors suggested that increasedBACE1mRNA transcription in remaining neuronal cells maycontribute to the increased BACE1 protein levels and activityfound in brain regions affected by AD [70]

Results of many studies concerning the elevated level ofBACE1 mRNA and protein in Alzheimerrsquos disease provide

direct and compelling reasons to develop therapies directed atBACE1 inhibition thus reducing120573-amyloid and its associatedtoxicities [67 68]

In our study we have observed that BACEmRNA expres-sion statistically significantly decreased after MO adminis-tration in frontal cortex and hippocampus These resultssuggest that the MO extract may act to inhibit BACE1mRNA level given the fact that the percentage of inhibitionof the expression (64 in the frontal cortex 50 in thehippocampus) is higher than that in the case of HU (38in the cortex and the lack of changes in the hippocampus)A careful analysis of the literature data shows that there areno studies which analyzed the impact of MO extract on theexpression level of BACE1 in Alzheimerrsquos disease

Furthermore a literature analysis does not indicatealready published results conducted by other teams attempt-ing to assess the impact of RA on the transcriptional activityof AChE BuChE and BACE1 Although several studies(already mentioned and others [70 71]) highlighted theRA and other caffeic acid derivatives capability of acetyl-cholinesterases inhibition none of these studies does nottouch the question of the molecular basis of their impacton the transcriptional machinery that regulates in vivo theexpression of studied genes Hence in our opinion obtainedby our team results they are one of the first of this typeand in general correlate with the results of our previousstudy [23] In this case there is no clear evidence explainingdifferent responses at the transcriptional level under theinfluence of RA especially in the case of BuChE encodinggene in the hippocampus (Table 2) It is possible that theobserved differentiation of BuChE transcriptional activitybetween the frontal cortex and the hippocampus may bedue to the differences of butyrylcholinesterase localizationand substrate affinity [72] Since in our experiment we havecarried out a quantitative analysis of AChE BuChE andBACE1 transcripts in brain homogenates of tested animalsrather than in individual isolated cell fractions therefore theobtained results constitute an overall ldquopicturerdquo of both studiedgenes transcriptional changes occurring in the brain areas ofstudied animals under the influence of the RA and the wholeplant extract as well

6 Conclusion

The subchronic administration ofMO led to an improvementof long-term memory of rats however the mechanisms ofMO action are probably more complicated since its role asa modulator of beta-secretase activity (due to inhibition ofBACE1 mRNA expression in frontal cortex) should be takeninto consideration

It should be noted that we have studied a crude extractfrom leaf of Melissa officinalis not a single pure chemicalcompound This plant extract is a complex mixture and itsaction may be a result of the summation of activities ofseveral components (synergismadditive action of caffeic acidwith salvianolic acids rosmarinic acid and others) In thecase of extract from leaves ofMelissa officinalis it is possiblethat interactions occur between the 40 chemical compoundsidentified by HPLC system

Evidence-Based Complementary and Alternative Medicine 15

Taken together it seems that the MO activity representsa possible option as complementary interventions to relievethe symptoms of mild dementia

Competing Interests

The authors declare that they have no competing interests

Acknowledgments

This work was supported by the Ministry of Science andHigher EducationWarsaw Poland from educational sources(2008ndash2011 Grant no N 405417836)

References

[1] S L Cole and R Vassar ldquoThe Alzheimerrsquos disease 120573-secretaseenzyme BACE1rdquo Molecular Neurodegeneration vol 2 no 1article 22 2007

[2] LACraigN SHong andR JMcDonald ldquoRevisiting the cho-linergic hypothesis in the development of Alzheimerrsquos diseaserdquoNeuroscience amp Biobehavioral Reviews vol 35 no 6 pp 1397ndash1409 2011

[3] N G M Gomes M G Campos J M C Orfao and C A FRibeiro ldquoPlants with neurobiological activity as potential tar-gets for drug discoveryrdquo Progress in Neuro-Psychopharmacologyamp Biological Psychiatry vol 33 no 8 pp 1372ndash1389 2009

[4] WHO WHO Monographs on Selected Medicinal PlantsmdashVolume 2 WHO Geneva Switzerland 2004 httpappswhointmedicinedocsendJs4927e18htmlJs4927e18

[5] M Bayat A A Tameh M H Ghahremani et al ldquoNeuropro-tective properties of Melissa officinalis after hypoxic-ischemicinjury both in vitro and in vivordquo DARU Journal of Pharmaceu-tical Sciences vol 20 article 42 2012

[6] J P Kamdem A Adeniran A A Boligon et al ldquoAntioxidantactivity genotoxicity and cytotoxicity evaluation of lemon balm(Melissa officinalis L) ethanolic extract Its potential role inneuroprotectionrdquo Industrial Crops and Products vol 51 pp 26ndash34 2013

[7] J-T Lin Y-C Chen Y-C Lee C-W R Hou F-L Chen andD-J Yang ldquoAntioxidant anti-proliferative and cyclooxygenase-2 inhibitory activities of ethanolic extracts from lemon balm(Melissa officinalis L) leavesrdquo LWTmdashFood Science and Technol-ogy vol 49 no 1 pp 1ndash7 2012

[8] G Guginski A P Luiz M D Silva et al ldquoMechanisms involvedin the antinociception caused by ethanolic extract obtainedfrom the leaves of Melissa officinalis (lemon balm) in micerdquoPharmacology Biochemistry amp Behavior vol 93 no 1 pp 10ndash162009

[9] N Perry G Court N Bidet J Court and E Perry ldquoEuropeanherbs with cholinergic activities potential in dementia therapyrdquoInternational Journal of Geriatric Psychiatry vol 11 no 12 pp1063ndash1069 1996

[10] E K Perry A T Pickering W W Wang P J Houghtonand N S L Perry ldquoMedicinal plants and Alzheimerrsquos diseasefrom ethnobotany to phytotherapyrdquo Journal of Pharmacy andPharmacology vol 51 no 5 pp 527ndash534 1999

[11] M-J R Howes N S L Perry and P J Houghton ldquoPlants withtraditional uses and activities relevant to the management ofAlzheimerrsquos disease and other cognitive disordersrdquo Phytother-apy Research vol 17 no 1 pp 1ndash18 2003

[12] I Orhan and M Aslan ldquoAppraisal of scopolamine-inducedantiamnesic effect in mice and in vitro antiacetylcholinesteraseand antioxidant activities of some traditionally used Lamiaceaeplantsrdquo Journal of Ethnopharmacology vol 122 no 2 pp 327ndash332 2009

[13] S Akhondzadeh M Noroozian M Mohammadi S OhadiniaA H Jamshidi and M Khani ldquoMelissa officinalis extract inthe treatment of patients with mild to moderate Alzheimerrsquosdisease a double blind randomised placebo controlled trialrdquoJournal of Neurology Neurosurgery and Psychiatry vol 74 no 7pp 863ndash866 2003

[14] D O Kennedy A B Scholey N T J Tildesley E K Perry andK AWesnes ldquoModulation ofmood and cognitive performancefollowing acute administration of Melissa officinalis (lemonbalm)rdquo Pharmacology Biochemistry amp Behavior vol 72 no 4pp 953ndash964 2002

[15] D O Kennedy G Wake S Savelev et al ldquoModulation of moodand cognitive performance following acute administration ofsingle doses of Melissa officinalis (Lemon balm) with humanCNS nicotinic and muscarinic receptor-binding propertiesrdquoNeuropsychopharmacology vol 28 no 10 pp 1871ndash1881 2003

[16] D O Kennedy and E L Wightman ldquoHerbal extracts and phy-tochemicals plant secondarymetabolites and the enhancementof human brain functionrdquoAdvances inNutrition vol 2 no 1 pp32ndash50 2011

[17] R P Pereira A A Boligon A S Appel et al ldquoChemical com-position antioxidant and anticholinesterase activity of Melissaofficinalisrdquo Industrial Crops and Products vol 53 pp 34ndash452014

[18] M Soodi N Naghdi H Hajimehdipoor S Choopani andE Sahraei ldquoMemory-improving activity of Melissa officinalisextract in naıve and scopolamine-treated ratsrdquo Research inPharmaceutical Sciences vol 9 no 2 pp 107ndash114 2014

[19] K Dastmalchi V Ollilainen P Lackman et al ldquoAcetyl-cholinesterase inhibitory guided fractionation of Melissa offic-inalis Lrdquo Bioorganic amp Medicinal Chemistry vol 17 no 2 pp867ndash871 2009

[20] W Chaiyana and S Okonogi ldquoInhibition of cholinesterase byessential oil from food plantrdquo Phytomedicine vol 19 no 8-9 pp836ndash839 2012

[21] K Dastmalchi H J D Dorman P P Oinonen Y Darwis ILaakso and R Hiltunen ldquoChemical composition and in vitroantioxidative activity of a lemon balm (Melissa officinalis L)extractrdquo LWTmdashFood Science and Technology vol 41 no 3 pp391ndash400 2008

[22] P Pereira D Tysca P Oliveira L F D S Brum J N Picadaand P Ardenghi ldquoNeurobehavioral and genotoxic aspects ofrosmarinic acidrdquo Pharmacological Research vol 52 no 3 pp199ndash203 2005

[23] M Ozarowski P L Mikolajczak A Bogacz et al ldquoRosmarinusofficinalis L leaf extract improves memory impairment andaffects acetylcholinesterase and butyrylcholinesterase activitiesin rat brainrdquo Fitoterapia vol 91 pp 261ndash271 2013

[24] G P Kumar and F Khanum ldquoNeuroprotective potential of phy-tochemicalsrdquo Pharmacognosy Reviews vol 6 no 12 pp 81ndash902012

[25] M Stobiecki A Staszkow A Piasecka P M Garcia-LopezF Zamora-Natera and P Kachlicki ldquoLC-MSMS profiling offlavonoid conjugates in wild mexican lupine Lupinus reflexusrdquoJournal of Natural Products vol 73 no 7 pp 1254ndash1260 2010

[26] A Wojakowska A Piasecka P M Garcıa-Lopez et al ldquoStruc-tural analysis and profiling of phenolic secondarymetabolites of

16 Evidence-Based Complementary and Alternative Medicine

Mexican lupine species using LC-MS techniquesrdquo Phytochem-istry vol 92 pp 71ndash86 2013

[27] A Gryszczynska B Opala Z Lowicki et al ldquoBioactive com-pounds determination in the callus and hydroalcoholic extractsfrom Salvia miltiorrhiza and Salvia przewalskiimdashpreliminarystudy on their anti-alcoholic activity effectsrdquo PhytochemistryLetters vol 11 pp 399ndash403 2015

[28] K Slinkart and V L Singleton ldquoTotal phenol analysis automa-tion and comparison with manual methodrdquo American Journalof Enology and Viticulture vol 28 no 1 pp 49ndash55 1977

[29] P J R Boissier J Tardy and J C Diverres ldquoUne nouvellemethode simple pour explorer lrsquoaction lsquotranquillisantersquo le testde la chemineerdquo Medicina Experimentalis vol 3 no 1 pp 81ndash84 1960

[30] R Ader J A Weijnen and P Moleman ldquoRetention of a passiveavoidance response as a function of the intensity and durationof electric shockrdquo Psychonomic Science vol 26 no 3 pp 125ndash128 1972

[31] E M Blalock K-C Chen K Sharrow et al ldquoGene microar-rays in hippocampal aging statistical profiling identifies novelprocesses correlated with cognitive impairmentrdquo Journal ofNeuroscience vol 23 no 9 pp 3807ndash3819 2003

[32] K Isomae S Morimoto H Hasegawa K Morita and JKamei ldquoEffects of T-82 a novel acetylcholinesterase inhibitoron impaired learning and memory in passive avoidance task inratsrdquo European Journal of Pharmacology vol 465 no 1-2 pp97ndash103 2003

[33] J Patora and B Klimek ldquoFlavonoids from lemon balm (Melissaofficinalis L Lamiaceae)rdquo Acta Poloniae Pharmaceutica-DrugResearch vol 59 no 2 pp 139ndash143 2002

[34] L Barros M Duenas M I Dias M J Sousa C Santos-Buelgaand I C F R Ferreira ldquoPhenolic profiles of cultivated invitro cultured and commercial samples of Melissa officinalis Linfusionsrdquo Food Chemistry vol 136 no 1 pp 1ndash8 2013

[35] T L Miron M Herrero and E Ibanez ldquoEnrichment of antiox-idant compounds from lemon balm (Melissa officinalis) bypressurized liquid extraction and enzyme-assisted extractionrdquoJournal of Chromatography A vol 1288 pp 1ndash9 2013

[36] Y Lu L Y Foo and H Wong ldquoSagecoumarin a novel caffeicacid trimer from Salvia officinalisrdquo Phytochemistry vol 52 no6 pp 1149ndash1152 1999

[37] S Abuhamdah LHuangM S J Elliott et al ldquoPharmacologicalprofile of an essential oil derived from Melissa officinalis withanti-agitation properties focus on ligand-gated channelsrdquo Jour-nal of Pharmacy and Pharmacology vol 60 no 3 pp 377ndash3842008

[38] G Wake J Court A Pickering R Lewis R Wilkins andE Perry ldquoCNS acetylcholine receptor activity in Europeanmedicinal plants traditionally used to improve failing memoryrdquoJournal of Ethnopharmacology vol 69 no 2 pp 105ndash114 2000

[39] S-H Ryu C Katona B Rive and G Livingston ldquoPersistenceof and changes in neuropsychiatric symptoms in Alzheimerdisease over 6months the LASER-AD studyrdquoAmerican Journalof Geriatric Psychiatry vol 13 no 11 pp 976ndash983 2005

[40] L N Gitlin H C Kales and C G Lyketsos ldquoNonpharma-cologic management of behavioral symptoms in dementiardquoJournal of the AmericanMedical Association vol 308 no 19 pp2020ndash2029 2012

[41] A Konczol J Muller E Foldes et al ldquoApplicability of a blood-brain barrier specific artificial membrane permeability assay atthe early stage of natural product-based CNS drug discoveryrdquoJournal of Natural Products vol 76 no 4 pp 655ndash663 2013

[42] Y-J Zhang L Wu Q-L Zhang J Li F-X Yin and Y YuanldquoPharmacokinetics of phenolic compounds of Danshen extractin rat blood and brain by microdialysis samplingrdquo Journal ofEthnopharmacology vol 136 no 1 pp 129ndash136 2011

[43] X Li C Yu Y Lu et al ldquoPharmacokinetics tissue distributionmetabolism and excretion of depside salts from Salvia miltior-rhiza in ratsrdquo Drug Metabolism and Disposition vol 35 no 2pp 234ndash239 2007

[44] H Xu Y Li X Che H Tian H Fan and K Liu ldquoMetabolismof salvianolic acid a and antioxidant activities of its methylatedmetabolitesrdquo Drug Metabolism and Disposition vol 42 no 2pp 274ndash281 2014

[45] D H Kim S J Park J M Kim et al ldquoCognitive dysfunctionsinduced by a cholinergic blockade and A120573 25-35 peptide areattenuated by salvianolic acid Brdquo Neuropharmacology vol 61no 8 pp 1432ndash1440 2011

[46] G-H Du Y Qiu and J-T Zhang ldquoSalvianolic acid B protectsthe memory functions against transient cerebral ischemia inmicerdquo Journal of Asian Natural Products Research vol 2 no 2pp 145ndash152 2000

[47] G Du and J Zhang ldquoProtective effects of salvianolic acid Aagainst impairment of memory induced by cerebral ischemia-reperfusion in micerdquo Chinese Medical Journal vol 110 no 1 pp65ndash68 1997

[48] J Anwar R M Spanevello G Thome et al ldquoEffects of caffeicacid on behavioral parameters and on the activity of acetyl-cholinesterase in different tissues from adult ratsrdquo Pharmacol-ogy Biochemistry and Behavior vol 103 no 2 pp 386ndash394 2012

[49] YW Lee D H Kim S J Jeon et al ldquoNeuroprotective effects ofsalvianolic acid B on anA12057325-35 peptide-inducedmousemodelof Alzheimerrsquos diseaserdquo European Journal of Pharmacology vol704 no 1ndash3 pp 70ndash77 2013

[50] J Zhong M-K Tang Y Zhang Q-P Xu and J-T ZhangldquoEffect of salvianolic acid B on neural cells damage and neu-rogenesis after brain ischemia-reperfusion in ratsrdquo Yao Xue XueBao vol 42 no 7 pp 716ndash721 2007

[51] P Zhuang Y Zhang G Cui et al ldquoDirect stimulation of adultneural stemprogenitor cells in vitro and neurogenesis in vivo bysalvianolic acid Brdquo PLoS ONE vol 7 no 4 Article ID e356362012

[52] M TangW Feng Y Zhang J Zhong and J Zhang ldquoSalvianolicacid B improves motor function after cerebral ischemia in ratsrdquoBehavioural Pharmacology vol 17 no 5-6 pp 493ndash498 2006

[53] Q Li L-P Han Z-H Li J-T Zhang and M-K Tang ldquoSal-vianolic acid B alleviate the disruption of blood-brain barrierin rats after cerebral ischemia-reperfusion by inhibiting MAPKpathwayrdquo Yao Xue Xue Bao vol 45 no 12 pp 1485ndash1490 2010

[54] Y-L LinH-J Tsay T-H Lai T-T Tzeng andY-J Shiao ldquoLith-ospermic acid attenuates 1-methyl-4-phenylpyridine-inducedneurotoxicity by blocking neuronal apoptotic and neuroinflam-matory pathwaysrdquo Journal of Biomedical Science vol 22 article37 2015

[55] M-K Tang and J-T Zhang ldquoSalvianolic acid B inhibits fibrilformation and neurotoxicity of amyloid beta-protein in vitrordquoActa Pharmacologica Sinica vol 22 no 4 pp 380ndash384 2001

[56] S S K Durairajan Q Yuan L Xie et al ldquoSalvianolic acid Binhibits A120573 fibril formation and disaggregates preformed fibrilsand protects against A120573-induced cytotoxictyrdquo NeurochemistryInternational vol 52 no 4-5 pp 741ndash750 2008

[57] FD Pinheiro Fernandes A P FonteneleMenezes J C de SousaNeves et al ldquoCaffeic acid protects mice from memory deficits

Evidence-Based Complementary and Alternative Medicine 17

induced by focal cerebral ischemiardquo Behavioural Pharmacologyvol 25 no 7 pp 637ndash647 2014

[58] S-J Tsai C-Y Chao andM-C Yin ldquoPreventive and therapeu-tic effects of caffeic acid against inflammatory injury in striatumof MPTP-treated micerdquo European Journal of Pharmacology vol670 no 2-3 pp 441ndash447 2011

[59] J H Kim QWang J M Choi S Lee and E J Cho ldquoProtectiverole of caffeic acid in an 119860120573

25minus35

-induced Alzheimerrsquos diseasemodelrdquo Nutrition Research and Practice vol 9 no 5 pp 480ndash488 2015

[60] G Liang B Shi W Luo and J Yang ldquoThe protective effect ofcaffeic acid on global cerebral ischemia-reperfusion injury inratsrdquo Behavioral and Brain Functions vol 11 no 1 article 182015

[61] D Y Yoo J H ChoiW Kim et al ldquoEffects of luteolin on spatialmemory cell proliferation and neuroblast differentiation in thehippocampal dentate gyrus in a scopolamine-induced amnesiamodelrdquo Neurological Research vol 35 no 8 pp 813ndash820 2013

[62] Y Liu X Tian L Gou L Sun X Ling and X Yin ldquoLuteolinattenuates diabetes-associated cognitive decline in ratsrdquo BrainResearch Bulletin vol 94 pp 23ndash29 2013

[63] S-M Liu Z-H Yang andX-B Sun ldquoSimultaneous determina-tion of six Salvia miltiorrhiza gradients in rat plasma and brainby LC-MSMSrdquo Zhongguo Zhongyao Zazhi vol 39 no 9 pp1704ndash1708 2014

[64] M A Papandreou A Dimakopoulou Z I Linardaki et alldquoEffect of a polyphenol-rich wild blueberry extract on cognitiveperformance of mice brain antioxidant markers and acetyl-cholinesterase activityrdquo Behavioural Brain Research vol 198 no2 pp 352ndash358 2009

[65] M-S Garcıa-Ayllon O Cauli M-X Silveyra et al ldquoBraincholinergic impairment in liver failurerdquo Brain vol 131 no 11pp 2946ndash2956 2008

[66] G K Ferreira M Carvalho-Silva C L Goncalves et al ldquoL-Tyrosine administration increases acetylcholinesterase activityin ratsrdquo Neurochemistry International vol 61 no 8 pp 1370ndash1374 2012

[67] R Vassar P-H Kuhn C Haass et al ldquoFunction therapeuticpotential and cell biology of BACE proteases current status andfuture prospectsrdquo Journal of Neurochemistry vol 130 no 1 pp4ndash28 2014

[68] R Vassar ldquoThe 120573-secretase BACE a prime drug target forAlzheimerrsquos diseaserdquo Journal of Molecular Neuroscience vol 17no 2 pp 157ndash170 2001

[69] D T R Coulson N Beyer J G Quinn et al ldquoBACE1 mRNAexpression in Alzheimerrsquos disease postmortem brain tissuerdquoJournal of Alzheimerrsquos Disease vol 22 no 4 pp 1111ndash1122 2010

[70] S Vladimir-Knezevic B Blazekovic M Kindl J Vladic A DLower-Nedza and A H Brantner ldquoAcetylcholinesterase inhib-itory antioxidant and phytochemical properties of selectedmedicinal plants of the lamiaceae familyrdquoMolecules vol 19 no1 pp 767ndash782 2014

[71] F Marcelo C Dias A Martins et al ldquoMolecular recognitionof rosmarinic acid from Salvia sclareoides extracts by ace-tylcholinesterase a new binding site detected by NMR spec-troscopyrdquo ChemistrymdashA European Journal vol 19 no 21 pp6641ndash6649 2013

[72] G Johnson and S W Moore ldquoWhy has butyrylcholinesterasebeen retained Structural and functional diversification in aduplicated generdquoNeurochemistry International vol 61 no 5 pp783ndash797 2012

[73] R J Grayer M R Eckert N C Veitch et al ldquoThe chemo-taxonomic significance of two bioactive caffeic acid estersnepetoidins A and B in the Lamiaceaerdquo Phytochemistry vol 64no 2 pp 519ndash528 2003

[74] A M D El-Mousallamy U W Hawas and S A M HusseinldquoTeucrol a decarboxyrosmarinic acid and its 41015840-O-triglycosideteucroside from Teucrium pilosumrdquo Phytochemistry vol 55 no8 pp 927ndash931 2000

[75] I Fecka and S Turek ldquoDetermination of water-soluble polyphe-nolic compounds in commercial herbal teas from Lamiaceaepeppermint melissa and sagerdquo Journal of Agricultural and FoodChemistry vol 55 no 26 pp 10908ndash10917 2007

[76] L W Sumner A Amberg D Barrett et al ldquoProposed min-imum reporting standards for chemical analysis WorkingGroup (CAWG) Metabolomics Standards Initiative (MSI)rdquoMetabolomics vol 3 pp 211ndash221 2007

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

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Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

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PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Computational and Mathematical Methods in Medicine

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Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Evidence-Based Complementary and Alternative Medicine 5

028

021

014

007

000

200100 300 400 500 600 700 800 900(Minutes)

(AU

)

1 23

4

56 7 8 9 10

1112

1314

1617

19

15 1820

2122

23

2425

2627

28

2930

31

37

3234

3536 38 39

40

33

Figure 1 Chromatogram UV ofMelissa officinalis leaf extract obtained at 270 nm with peaks identified by HPLC-UV-MS

were determined by measuring the formation of the yellowanion obtained from the reaction between Ellmanrsquos reagentand the thiocholine generated by the enzymatic hydrolysisof acetylthiocholine iodide (ATCh) and butyrylthiocholine(BTCh) respectively (sample 01mL PBS 08mL DTNB01mL ATCh 020mL and BTCh 020mL)The biochemicalassay of AChE and BuChE in homogenate of brain sampleswas expressed as 120583molminmg protein by using spectropho-tometric method (120582 = 412 nm)

314 RNA Isolation and Reverse Transcription Reaction TotalRNA isolation from the rats brain tissues homogenates(frontal cortex hippocampus) was carried out using TriPureIsolation Reagent (Roche) according to manufacturerrsquos pro-tocol The integrity of RNA was visually assessed by a con-ventional agarose gel electrophoresis and the concentrationwill be evaluated by measuring the absorbance at 260 and280 nm in a spectrophotometer (BioPhotometer Eppendorf)RNA samples were stored at minus80∘C until use The 1 120583gof total RNA from all samples was used for the reversetranscription into cDNA using Transcriptor First StrandSynthesis Kit (Roche) according to manufacturerrsquos protocolObtained cDNA samples was stored at minus20∘C or used directlyfor the quantitative real-time PCR (qRT-PCR)

315 Real-Time PCR mRNA Quantification The acetylcho-linesterase (AChE) butyrylcholinesterase (BChE) and beta-secretase (BACE1) genes expression level was analyzed bytwo-step quantitative real-time PCR (qRT-PCR) in a vol-ume of 10 120583L reaction mixture using relative quantificationmethodology with a LightCycler TM Instrument (RocheGermany) and a LightCycler Fast Start DNA Master SYBRGreen I kit (Roche Applied Science) according to theinstructions of the manufacturer All primers sequences weredesigned and custom-designed using the Oligo 60 software(National Biosciences) and were verified by assessment ofa single PCR product on agarose gel and by a single tem-perature dissociation peak (melting curve analysis) of eachcDNA amplification product An GAPDH gene was usedas a housekeeping gene (endogenous internal standard) for

normalization of qPCR For each quantified gene standardcurves were prepared from dilution of cDNA and gener-ated from a minimum of four data points All quantitativePCR were repeated twice The data were evaluated usingLightCycler Run 45 software (Roche Applied Science) EachPCR run included a nontemplate control to detect potentialcontamination of reagents

316 Statistical Analysis All values were expressed as meansplusmn SEM The statistical comparison of results was carried outusing one-way analysis of variance (ANOVA) followed byDuncanrsquos post hoc test for detailed data analysisThe values of119901 lt 005were considered as a statistical significant difference

4 Results

41 Phytochemical Profile of Extract

411 Identification ofMetabolites Forty phenolicmetaboliteswere identified in hydroethanolic Melissa officinalis leafextract (Table 1 Figure 1) The predominant identified com-pounds were bioactive caffeic acid esters and glycosides offlavones The caffeic acid dimer rosmarinic acid (metabolite23) and caffeate trimer (lithospermic acid 31) were the prin-cipal caffeic acid derivatives in the analyzed samples Hydrox-yjasmonic acid and its derivatives teucrol as well as sage-coumarin were identified for the first time in the genusMelissa while luteolin and apigenin glycoconjugates arewell known phytochemicals in this species [33] Multistepfragmentation with accurate mass measurement enabledconfirming that the losses of fragment 799573 amu from the[M-H]minus ions of compounds 4 9 27 30 and 32 referredto sulphate groups The first-order fragmentation of 30revealed the loss of 799573 and yielded the product ion at7191622119898119911 The following fragmentation of this ion cor-responded to that of the sagerinic acid (dimer of rosmarinicacid) described by Barros et al [34] The exact placementof the sulphation position would require in-depth chemicalanalysis Thus 30 was tentatively assigned as sulphated sag-erinic acid

6 Evidence-Based Complementary and Alternative MedicineTa

ble1Metabolitesd

etectedin

Melissa

officin

alisleafextractb

yUPL

C-MS

No

RT [min]

Metabolite

identifi

catio

nCh

emical

form

ula

Exactm

asso

f[M-H

]minusΔpp

mFragmentatio

nin

120582max[nm]

CID

aIdentifi

catio

nlevelb

Reference

Measured

Calculated

Negativeion

mod

e(ESIminus)

Positiveion

mod

e(ESI+)

114

1

2-Hydroxy-3-(34-

dihydroxyphenyl)-

prop

anoic

acid

C 9H9

O5

1970

451970

455minus41775

197179135

199163

283312

8143997

2[34]

217

3Dihydroxybenzoica

cid

hexosid

eC 13

H15

O9

315072

3150722

08346

315153109

317155

282

54726828

3[35]

319

8Ca

ftaric

acid

C 13

H11

O9

3110

413110

409

06141

311221179149

318

6440397

2[35]

4224

2-Hydroxy-3-(34-

dihydroxyphenyl)-

prop

anoica

cid

sulphated

C 9H9

O8

S2770

032770

024

03998

277197179135

312

3[35]

5256

Hydroxyjasm

onicacid

hexosid

eC 18

H27

O9

387166

3871661

09214

387207163

323

44237366

2[17]

6276

Caffeicacid

C 9H7

O4

1790

341790

451minus49075

179135

181163

275

689043

1std

7289

Salviano

licacid

EC 36

H29

O16

7171450

717146

72313

717519339321

295277

275325

49770697

2[35]

8317

Salviano

licacid

HI

(isom

er)

C 27

H21

O12

537104

5371038

08562

537493359295

281325

2[35]

9345

Hydroxyjasm

onicacid

sulphated

C 12

H17

O7

S30507

30507

1117

305225194147

275330

3[17]

10362

NepetoidinB

C 17

H13

O6

313072

3130718

11232

nd282316

5316819

2[73]

1137

Yunn

aneica

cidF

C 26

H25

O14

5971255

5971244

18743

597509311197

Masked

2[34]

12383

Decarbo

xyrosm

arinicacid

(teucrol)

C 17

H15

O6

315088

3150874

09396

315179135

275330

637829

2[74]

1339

Caffeoylcaft

aricacid

C 22

H17

O12

473073

4730725

07555

173311149

Masked

65018

3[35]

14397

Apigenin

glucosylrham

nosid

eC 27

H29

O14

577157

5771563

11136

577269

579433271

275340

92741003

3[33]

15404

Luteolin

7-O-glucosid

e31015840-O

-glucuronide

C 27

H27

O17

623126

6231254

09696

62346144

7285

255

625463287

273343

2[33]

16421

Rosm

arinicacid

hexosid

eC 24

H25

O13

52113

5211301

03551

521359161

523361325163

329

25245848

2[34]

1743

Luteolin

O-diglucosid

eC 27

H29

O16

609147

6091461

07183

609285

611287

269349

3[33]

18441

Luteolin

glucosylrham

nosid

eC 27

H29

O15

593152

5931512

08077

59344

7285

59544

9287

271343

3[33]

19449

Luteolin

41015840-O

-glucosid

eC 21

H19

O11

4470

9444

70933

09728

447285

449287

271343

5319116

3[33]

2045

Sagerin

icacid

2-hydroxy-3-

(34-dihydroxyph

enyl)-

prop

anoide

C 45

H39

O20

8992

058992

0410

448

899719591475

295

Masked

3[35]

2149

Salviano

licacid

B(lithosperm

icacid

B)C 36

H29

O16

717146

7171461

01846

717519359161

327

6441188

2[34]

22504

Sagerin

icacid

C 36

H31

O16

7191

637191

618

09979

719519359161

287330

2[34]

23514

Rosm

arinicacid

C 18

H15

O8

3590

773590

772

0002

359161

361163

329

5281792

1std

Evidence-Based Complementary and Alternative Medicine 7

Table1Con

tinued

No

RT [min]

Metabolite

identifi

catio

nCh

emical

form

ula

Exactm

asso

f[M-H

]minusΔpp

mFragmentatio

nin

120582max[nm]

CID

aIdentifi

catio

nlevelb

Reference

Measured

Calculated

Negativeion

mod

e(ESIminus)

Positiveion

mod

e(ESI+)

24555

Salviano

licacid

B2-hydroxy-3-(34-

dihydroxyphenyl)-

prop

anoide

C 45

H37

O20

89719

8971884

16873

897717519359

161

330

3[35]

2556

Sagerin

icacid

di-2-hydroxy-3-(34-

dihydroxyphenyl)-

prop

anoide

C 54

H47

O24

1079244

910792457minus07911

1079897719539

359295

288330

3[35]

26592

Sagecoum

arin

di-2-hydroxy-3-(34-

dihydroxyphenyl)-

prop

anoide

caffeide

C 54

H43

O24

10752156

10752150

0559

1077897717537

409359339277

322

3[35]

27598

Rosm

arinicacid

sulphated

Iisomer

C 18

H15

O11

S4390

354390

341

20214

439359341163

Masked

2[34]

28613

Luteolin

31015840-O

-glucuronide

C 21

H17

O12

4610

734610

7216

34461285

463287

269340

170474237

2[33]

29632

Salviano

licacid

AC 26

H21

O10

493115

493114

11011

493359295179

298327

5281793

[34]

30656

Sagerin

icacid

sulphated

C 36

H31

O19

S7991

196

7991

186

0954

799719619519

359161

325

3[35]

31671

Lithosperm

icacid

C 27

H21

O12

537104

537104

809698

537493359161

292329

6441498

2[35]

3268

Rosm

arinicacid

sulphated

IIiso

mer

C 18

H15

O11

S4390

344390

341

02837

439359341163

Masked

2[34]

33689

Sagecoum

arin

C 27

H19

O12

535088

5350882

01204

535311267177

Masked

2[36]

3470

3Salviano

licacid

LIisomer

C 36

H29

O16

717146

7171461

02697

717519359

284329

2[35]

3571

Salviano

licacid

Lhydroxycaffeide

C 45

H35

O20

895173

8951727

06282

895519359161

Masked

3[35]

3674

Sagecoum

arin

cafta

ride

C 40

H29

O20

8291

268291

258

06953

829667535355

311

Masked

3[36]

37751

Sagecoum

arin

2-hydroxy-

3-(34-dihydroxyph

enyl)-

prop

anoide

C 36

H27

O16

715131

7151305

08176

715535311267

319

3[34]

3877

1Unk

nown

C 36

H57

O14

S745348

7453475

044

02281326

4[35]

3978

5Methylrosmarinate

C 19

H17

O8

373093

3730929

01765

373359161

284323

6479915

2[75]

40869

Salviano

licacid

Ccaffeoylhydroxycaffeide

C 44

H33

O18

8491

688491

672

08615

849687491359

327255

286318

3[35]

a CID

identifier

fora

chem

icalstr

ucture

intheP

ubCh

emCom

poun

ddatabase

b Metabolite

identifi

catio

nlevelaccording

toMetabolom

icsS

tand

ards

Initiativer

ecom

mendatio

n[76]

stdidentificatio

non

theb

asisof

stand

ardcompo

undfragmentatio

nnd

not

detected

8 Evidence-Based Complementary and Alternative Medicine

100 200 300 400 500 600 700 800 900 1000 1100 1200

mz

70

75

80

85

90

95

100

minus198 (2-hydroxy-3-(34-dihydroxyphenyl)

-porpanoide)

minus180 (caffeide)

minus180 (2-hydroxy-3-(34-dihydroxyphenyl)

-propanoide)

minus180 (2-hydroxy-3-(34-dihydroxyphenyl)

-propanoide)

minus180 (2-hydroxy-3-(34-dihydroxyphenyl)

-propanoide)

1350438

2191749

HO

HO

HO HO

OH

OH

OH

OH OH OH

OHOHOHOHHOOC

COOH

COOH COOH COOH

24612ppmC36H55O37 = 10792422

minus07911ppmC54H47O24 = 10792457

10792449

10448ppmC45H39O20 = 8992040

899205018453ppm

C36H31O16 = 7191612

7191625

20501ppmC27H23O12 = 5391190

5391201

14852ppmC23H33O10 = 4692074

469208119316ppm

C17H11O5 = 2950606

2950612

0

5

10

15

20

25

30

35

40

45

50

55

60

65

Rela

tive a

bund

ance

22903ppmC18H15O8 = 3590767

3590775

minus37218 ppmC9H5O3 = 1610239

1610233

(a)

100 200 300 400 500 600 700 800 900 1000

mz

100 200 300 400 500 600 700 800 900 1000

mz

100 200 300 400 500 600 700 800 900 1000

mz

100 200 300 400 500 600 700 800 900 1000

mz

0

250

500

750

1000

00

04

08

times104

times104

01234

0

1

2

times105

Ints

Ints

Ints

Ints

MS1

MS2

MS3

MS4

[M-H]minus10749

minus224 (acidic part

minus224 (acidic part

of sagecoumarin)

of sagecoumarin)

minus360 (rosmarinic acid)

4908

7148

5348 minus180 minus162 (caffeide)8769

31074909

5348

minus180 (2-hydroxy-3-(34-dihydroxyphenyl)-propanoide)

3108

35474909

1

(b)

Figure 2 Continued

Evidence-Based Complementary and Alternative Medicine 9

150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900

mz

0

5

10

15

20

25

30

35

40

45

50

55

60

65

Rela

tive a

bund

ance

O

O

O O

O

O

OH OH

OH

OH

OH

OHOH

OH

HO

HO

HO

COOH

OC

1529946

1970447

C9H9O5 = 1970455

minus41775ppm1610232

2552328

C16H31O2 = 2552330

minus04091ppm

3110563

C17H11O6 = 3110561

05243ppm

minus224 (fragment of sagecoumarin)

3912257

4092361

4832730

C29H39O6 = 4832752

minus44931ppm

5350885

C27H19O12 = 5350882

05640ppm

minus132 (tartaric acid)

5770990

C29H21O13 = 5770988

06486ppm

6670947

C31H23O17 = 6670941

09808ppm

minus162 (caffeic acid)

8291267

C40H29O20 = 8291258

11370ppm

(c)

Figure 2 (a) Mass spectra in negative ionization mode and simplified fragmentation scheme of compound 25 (pentameric ester ofcaffeic acid) (b) Mass spectra in negative ionization mode and simplified fragmentation scheme of compound 26 (pentameric structureof sagecoumarin di-2-hydroxy-3-(34-dihydroxyphenyl)-propanoide caffeide) (c) Mass spectra in negative ionization mode and simplifiedfragmentation scheme of compound 36 (pentameric structure of sagecoumarin caftaride)

Tetrameric structures of hydroxycinnamic acids wereidentified in lemon balm recently [34 35] The measure-ment of accurate masses allowed the identification of com-pound 25 which was tentatively identified as pentamericester of caffeic acid (Figure 2(a)) In 25 the double loss of1800421 amu corresponded to fragments with the molecularformula of C

9H8O4 adequate to dehydroxylated 2-hydroxy-

3-(34-dihydroxyphenyl)-propanoic acid The product ionat 7191623119898119911 and its further fragmentation are sim-ilar to those of metabolite 22 as described previously [34 35]Therefore metabolite 25was tentatively assigned as sagerinicacid di-2-hydroxy-3-(34-dihydroxyphenyl)-propanoideNev-ertheless comprehensive studies by nuclear magnetic reso-nance are required to complete elucidation of substitutionpattern for particular components of those pentamers

Sagecoumarins were previously identified in Salvia offic-inalis as caffeic acid trimers [36] Our MS analysis indicatedthe presence of such compounds and their derivatives alsoin M officinalis The pseudomolecular ion of compound33 observed in the negative ionization mode had theaccurate mass of 5350880119898119911 which corresponded to thechemical formula C

27H19O12

adequate for sagecoumarin(according to the Metlin and KNApSAck databases) Note-worthy 26 33 36 and 37 had the same fragmentationpattern of the product ion obtained in the MSMS andMSn in the negative ionization mode The [M-H-1800422]minusion corresponded to the detachment of 2-hydroxy-3-(34-dihydroxyphenyl)-propanoic acid thus 37 was tentatively

assigned as tetrameric sagecoumarin 2-hydroxy-3-(34-dihy-droxyphenyl)-propanoide The [M-H-3600846]minus ion in 26was indicated on rosmarinic acid substitution to 37 Massspectra of MSn in negative ionization of the compoundprovided complementary information to HR-MSMS massspectra indicated on caffeic acid as internal componentof the dimer In addition simultaneous loss of fragments180 amu and 224 amu in MS3 and MS4 indicated that thetwo components cannot be linked (Figure 2(b)) How-ever detailed analysis of substitution pattern of 26 shouldbe done Therefore 26 was tentatively assigned as penta-meric structure of sagecoumarin di-2-hydroxy-3-(34-dihy-droxyphenyl)-propanoide caffeide Rupture of caffeic andtartaric acid moieties from the product ion at 5350885119898119911was observed for compound36 (Figure 2(c))Detection of theaccurate masses of these two detached fragments with Δ lessthan 1 ppm eliminated the possibility of hexose and pentosesubstitution which have the same nominal masses as caffeicand tartaric acid respectively Therefore 36 was tentativelyidentified as another pentameric structure of sagecoumarincaftaride

Metabolite 28 with the accurate masses of 461073119898119911was tentatively identified as luteolin O-glucuronide The [M-H-1760324]minus ion is indicated on loss of structure C

6H8O6

corresponding to glucuronide moiety The product ion at2850405119898119911 was indicated on flavone luteolin The placeof substitution of the carboxylic acid on flavone skeleton isproblematic due to different isomers reported in lemon balm

10 Evidence-Based Complementary and Alternative Medicine

Table 2 Effect ofMelissa officinalis leaf extract (200mgkg po) treatment on sedative activity motor coordination and memory in rats

Group 119899Locomotor activity

[number of impulses5min]Motor coordination

exit time [s]Short-term memorye

ORLong-term memory

latency [s]MC + H

2

O 18 390 plusmn 24 17 plusmn 3 040 plusmn 006 47 plusmn 14MC + SC 18 526 plusmn 48lowast 32 plusmn 5lowast 032 plusmn 005 12 plusmn 3lowast

MO + H2

O 10 231 plusmn 48lowast 32 plusmn 7lowast 043 plusmn 007 169 plusmn 11lowast

MO + SC 9 436 plusmn 60 43 plusmn 7 009 plusmn 011lowast 23 plusmn 6HU + H

2

O 9 515 plusmn 32 15 plusmn 2 037 plusmn 009 158 plusmn 14lowast

HU + SC 8 639 plusmn 71lowast 56 plusmn 3 022 plusmn 006 49 plusmn 18

RA + H2

O 8 406 plusmn 59 21 plusmn 5 045 plusmn 005 58 plusmn 28RA + SC 8 605 plusmn 55 28 plusmn 7 045 plusmn 005 20 plusmn 7Means plusmn SEM119899Number of animalsMC + H

2O control rats

SC scopolamine (05mgkg bw ip)HU huperzine A (05mgkg bw po)RA rosmarinic acid (10mgkg bw po)eExpressed as ratio OR = (119861 minus 119860lowast)(119861 + 119860) for details see Section 3lowastVersus MC + H

2O 119901 lt 005

Versus MC + SC 119901 lt 005

luteolin 31015840-O- and 7-O-glucuronide [33] It is impossible todistinguish both structures by mass spectrometry Only onechromatographic peak corresponding to the [M-H]minus ion at461073119898119911 was observed in our study Since luteolin 31015840-O-glucuronide was assigned as the most abundant flavonoid inlemon balm [33] we assumed that 28 corresponded to thisstructure

412 Flavonoids and Polyphenolic Acids The major com-pound from the 40 chemical compounds identified in hy-droethanolic MO leaf extract established by HPLC was RA(885 g100 g) (Table 1 Figure 1)

Other chemical compoundswith neuromodulatory activ-ities such as lithospermic acid (0042 g100 g) salvianolicacid A (0040 g100 g) salvianolic acid B (0023 g100 g) andcaffeic acid (0087 g100 g) were also documented in litera-ture Moreover the total polyphenolic compounds content ofMO determined with the use of FolinndashCiocalteu assay was3397 calculated as gallic acid The total hydroxycinnamicderivatives content expressed spectrophotometrically as ros-marinic acid was 2115 g100 g

413 Essential Oil Composition Hydroethanolic MO leafextract contained 008 of total essential oil The GCFIDanalysis showed that the extract comprised camphene(004) alfa-pinene (007) beta-pinene (1647) andmyrcene (1951) Moreover according to retention time16 compounds were identified as follows alfa-bisabololborneol carvone chamazulene cineole eugenol gamma-terpineol guaiazulene isopulegol linalool limonene men-thol menthyl acetate pulegone terpine and thymol Thesecompounds are present in the essential oil in trace amountswhich do not allow the quantitative interpretation

42 Cognitive and Behavioral Experiments

421 Locomotor Activity A one-way ANOVA analysisrevealed significant differences in the locomotor activity of

rats expressed as their horizontal spontaneous activity afterMO administration (ANOVA 119865(7 80) = 646 119901 lt 005)(Table 2) Detailed post hoc analysis showed thatMO + H

2O

decreased the locomotor activity of rats by 4031 but HU +H2O did not affect this activity when compared with control

group (MC + H2O) We observed also that RA + H

2O did

not change the locomotor activity of rats Stimulating effectsin the locomotor activity of rats were observed after an acuteSC injection (MC + SC versus MC + H

2O 119901 lt 005) and

this effect was observed in all SC-treated rats when comparedwith the proper non-SC-treated animals On the contrarythese SC-treated animals did not differ in comparison toanimals that received SC only (MO + SC versus MC + SC119901 gt 005 HU + SC versus MC + SC 119901 gt 005 RA + SCversus MC + SC 119901 gt 005)

422 Motor Coordination A one-way ANOVA analysisrevealed significant differences in motor coordination of ratsexpressed as their exit time from the cylinder (119865(5 73) =284 119901 lt 005) (Table 2) Detailed analysis showed that themultiple administration of RA + H

2O and HU + H

2O did

not affect significantly this paradigm when compared withcontrol rats (119901 gt 005) whereas MO treatment led to pro-longed exit time (MO + H

2O versus MC + H

2O 119901 lt 005)

Moreover generally SC-treated animals showed producedprolongation of exit time and the effects were statisticallysignificant not only in control groups (MC + SC versus MC +H2O 119901 lt 005) but also in RA-treated rats (RA + SC versus

MC + SC 119901 lt 005) However the rest of the SC-treatedanimals did not differ in comparison to animals receiving SConly (MO + SC versus MC + SC 119901 gt 005 HU + SC versusMC + SC 119901 gt 005)

423 Long-Term Memory A one-way ANOVA analysisrevealed significant differences in long-term memory afterusing a passive avoidance test (119865(7 77) = 201 119901 lt 005Table 2) It was shown that the strongest effect leading to

Evidence-Based Complementary and Alternative Medicine 11

Table 3 The effect ofMelissa officinalis leaf extract on acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) activities and AChEBuAChE or beta-secretase (BACE1) mRNA expression levels in frontal cortex (FC) or hippocampus (Hipp) of rats

Group119899Enzyme activity

[nmolminmg protein]mRNA expression

[]AChE BuChE ACHE BuChE BACE1

FC Hipp FC Hipp FC Hipp FC Hipp FC HippMC + H

2

O 363 plusmn 49 439 plusmn 73 65 plusmn 11 53 plusmn 8 100 plusmn 12 100 plusmn 11 100 plusmn 18 100 plusmn 11 100 plusmn 16 100 plusmn 8MO + H

2

O 276 plusmn 34 409 plusmn 28 69 plusmn 6 62 plusmn 4 48 plusmn 4lowast 31 plusmn 7lowast 16 plusmn 2lowast 64 plusmn 21amp 36 plusmn 3lowast 50 plusmn 8lowast

HU + H2

O 189 plusmn 15lowast 239 plusmn 15lowast 58 plusmn 6 51 plusmn 4 53 plusmn 13lowast 85 plusmn 5 42 plusmn 9lowast 102 plusmn 11 62 plusmn 6lowast 98 plusmn 4RA + H

2

O 224 plusmn 16lowast 251 plusmn 12lowast 77 plusmn 7 99 plusmn 7lowast 101 plusmn 12 103 plusmn 5 184 plusmn 31lowast 56 plusmn 7lowast 126 plusmn 13amp 98 plusmn 3Means plusmn SEM119899Number of animals 7ndash10Values expressed as a ratio the geneGAPDHMC + H

2O control group

HU huperzine A (05mgkg bw po)RA rosmarinic acid (10mgkg bw po)lowastampVersus MC + H

2O 119901 lt 005 or 119901 lt 007 respectively

an improvement of this paradigm was produced by extractof MO and HU but not RA when compared with controlanimals (MO + H

2O versus MC + H

2O 119901 lt 005 HU +

H2O versus MC + H

2O 119901 lt 005 RA + H

2O versus MC +

H2O 119901 gt 005) However the administration of SC to rats

significantly decreased the latency time of passive avoidancetask (MC + SC versus MC + H

2O 119901 lt 005) After MO or

RA combined treatment with SC no improvement of long-term memory was observed but HU given with SC showedenhancement of this paradigm in rats (HU + SC versusMC + SC 119901 lt 005) Therefore it can be concluded thatadministration of HU overcomes the effect shown by SC only(Table 1)

424 Short-Term Memory The results of the object recogni-tion test showed that an administration of the compoundsor extract did affect the ratsrsquo short-term memory (ANOVA119865(7 78) = 273 119901 lt 005) (Table 2)

Detailed post hoc analysis showed that only SC signifi-cantly decreased the short-term memory in MO-treated rats(MO + SC versusMO + H

2O 119901 lt 005MO + SC versus MC

+ SC 119901 lt 005) whereas the differences between the rest ofthe animals did not reach statistical significance (119901 gt 005)

43 AChE and BuChE Activities in Rat Brain A one-wayANOVA revealed significant differences between groups inthe activity of AChE in both the cortex and the hippocampus(frontal cortex 119865(3 27) = 565 119901 lt 005 hippocampusANOVA 119865(3 29) = 796 119901 lt 005) It was found out thatMOshowed an insignificant inhibition of AChE activity in thefrontal cortex by 24 when compared with control rats (MC+ H2O 119901 lt 006) and in the hippocampus by 7 (Table 3)

whereas HU produced a distinct significant inhibition ofAChE activity in comparison to control group by 48 (119901 lt005) and 47 (119901 lt 005) in the cortex and the hippocampusrespectively Also RA lowered significantly AChE activityboth in the cortex (38) and in the hippocampus (43)Moreover there were not significant differences between thevalues of BuChE activities for MO and HU when compared

with control group in the frontal cortex (ANOVA 119865(3 30) =101 119901 gt 005) whereas in the hippocampus the differencesreached statistical significance (ANOVA 119865(3 27) = 141119901 lt 005) Detailed analysis showed that only RA effect wassignificant and increased BuChE activity in the hippocampuswhen compared with the control rats (119901 lt 005)

44 AChE BuChE and BACE1 mRNA Level Changes in RatBrain A one-way ANOVA analysis revealed significant dif-ferences of AChE mRNA transcription profile in the cortex(ANOVA 119865(3 27) = 857 119901 lt 005) As shown in Table 3the multiple treatment of MO produced in the cortex astatistically significant decrease of AChEmRNA level by 52the administration of HU caused decrease of its level by 44(119901 lt 005) whereas RA did not affect this parameter whencompared to the control

There were significant differences between the relativevalues of BuChE mRNA levels in this region of brain in rats(frontal cortex ANOVA 119865(3 25) = 162 119901 lt 005) TheMOtreatment led to a decrease in the BuChEmRNA level by 84(versus MC + H

2O 119901 lt 005) the prolonged HU admin-

istration resulted in a decrease of the transcript level in thecortex by 58 (versus MC + H

2O 119901 lt 005) but RA

increased this parameter in the cortex by 84 (versus MC +H2O 119901 lt 005)The significant differences ofmRNA transcription level of

AChE mRNA level in the hippocampus (ANOVA 119865(3 31) =212 119901 lt 005) have been observed The detailed analysisshown that in the case of AChE afterMO treatment mRNAlevel significantly decreased by 69 (versus MC + H

2O

119901 lt 005) while the administration of HU resulted in astatistically insignificant decrease of AChE mRNA level by18 when compared with control group Also RA did notchange the level of transcript

In the case of BuChEmRNA expression in the hippocam-pus there were statistically significant differences betweengroups (ANOVA 119865(3 27) = 310 119901 lt 005) Detailed analysisshowed that in theMO + H

2O treated group the expression

lowered by 36 but the difference did not reach strong

12 Evidence-Based Complementary and Alternative Medicine

significance when compared with the control values (119901 lt007) and no change in the expression level of this enzymewas shown after the administration of HU On the contraryRA produced an increase of BuChE mRNA expression in thehippocampus by 44 (versus MC + H

2O 119901 lt 005)

Further analysis showed the significant differences inBACE1 mRNA expression in both brain regions of rats (inthe cortex and the hippocampus) (frontal cortex ANOVA119865(3 27) = 163 119901 lt 005 hippocampus ANOVA 119865(3 27) =133 119901 gt 005) It was observed that MO produced astatistically significant decrease of the BACE1 expression levelby 64 in the cortex (versus MC + H

2O 119901 lt 005) and by

50 in the hippocampus (versus MC + H2O 119901 lt 005) For

comparison HU treatment led to a decrease in the mRNAexpression level by 38 in the cortex (versus MC + H

2O

119901 lt 005) but not in the hippocampus On the contrary RAproduced an increase of this transcript in the cortex by 26but the effect did not reach a strong statistical significance(versus MC + H

2O 119901 lt 007) whereas in the hippocampus

there was no difference between RA and control group

5 Discussion

Cognitive and Behavioral Experiments The present studyinvestigated the influence of subchronic (28-fold) adminis-tration of standardized 50 EtOH extract ofMelisa officinalisleaf extract (MO) (200mgkg po containing 177mgkg ofRA) on SC-induced impairment of short-term and long-term memory in rats The results were compared with theactivity of cholinesterases (AChE and BuChE) as well aswith AChE BuChE and BACE1 gene expression levels in thecortex and hippocampus of the rat brain So far little evidenceis yet available as regards mechanisms of MO leaf extractaction that are potentially relevant to cognitive function ofrats after per os administration MO is traditionally used intreating neurological disorders through its anti-AChE [18]and antiagitation properties [37] Moreover Wake et al [38]and Kennedy et al [15] showed thatMO extract has nicotinicreceptor activity and that it can displace [3H]-(N)-nicotinefrom nicotinic receptors in homogenates of human cerebralcortex tissue and they suggested that these mechanisms canexplain activity of MO extract in amnesia model RecentlySoodi et al [18] observed that intraperitoneal injections ofMO extract (200mgkg) in rats could significantly enhancelearning and memory processes in animals since the extractsignificantly ameliorates SC-induced learning deficit in Mor-ris water maze test On the contrary in higher dose itcan be observed that MO extract (400mgkg) could notreverse SC-induced memory impairment [18] In our studyadministration of MO extract at a dose of 200mgkg (po)showed the effect leading to improvement of long-termmemory in a passive avoidance test in rats However afterMO combined treatment with SCMO did not overcome theimpairment shown by SC (Table 2) It should be emphasizedthat it is not clear whether the effect shown in our study byMO in non-SC-treated rats is specific since theMO treatmentproduced significant lowering of locomotor activity of ratstherefore the sedative profile ofMO cannot be excluded

On the other hand Ryu et al [39] observed that agitationand aggression are highly prevalent in patientswith dementiaAccording to Gitlin et al [40] nonpharmacological inter-ventions are recommended as first-line therapy Althoughantipsychotics have shown benefit for Alzheimerrsquos disease-related psychosis their use is associated with several seriousadverse effects [40]Thus it seems that the use ofMO extractcan provide dual benefits both in aspect of inhibition ofagitation and in improving the memory of patients withAlzheimerrsquos disease

Furthermore in our study RA in the dose of 10mgkgbw (po) did not affect either short- or long-term memoryalthough RA lowered significantly AChE activity both inthe cortex and in the hippocampus Moreover we observedthat the repeated administration of RA in non-scopolamine-treated rats did not produce any changes of locomotoractivity similarly to our previous study [23]

It is possible that other chemical compounds can influ-ence the memory in rats by synergic interactions in plantextract According to our calculations caffeic acid (0174mgin a single dose of extract administered to animals per kgbw) lithospermic acid (0084mgkg) salvianolic acid A(008mgkg) and salvianolic acid B (0046mgkg) may beresponsible for observed pharmacological effects On theother hand results from few studies showed that salvianolicacids do not cross the blood brain barrier (BBB) [41 42]and also lithospermic acid does not efficiently cross the BBB[43] For this reason the interpretation of our results is morecomplicated

Firstly Xu et al [44] demonstrated that Sal A is ametabol-ically unstable compound that would undergo rapid methy-lationmetabolism catalyzed by catechol O-methyltransferasein vivo into four major methylated metabolites of Sal A(3-O-methyl 31015840-O-methyl 3310158401015840-O-dimethyl and 31015840310158401015840-O-dimethyl salvianolic acid A) These generated O-methylatedmetabolites may be largely responsible for its in vivo phar-macological effects Although there are no available recentstudies on the ability of these compounds to pass the BBBsuch possibility should not be excluded

Secondly several studies showed central pharmacolog-ical effects in animals after per os administration of Sal B[45 46] Sal A [47] and caffeic acid [48] It was provedfor example that salvianolic acid B (10mgkg po) sig-nificantly rescued the A12057325ndash35 peptide-induced decreaseof choline acetyltransferase and brain-derived neurotrophicfactor protein levels in an amyloid 120573 (A120573) peptide-inducedAlzheimerrsquos disease mouse model [49] It also significantlyreversed (10mgkg po) the cognitive impairments inducedby scopolamine (1mgkg ip) or A120573 (25ndash35) (10 nmol5 120583Licv) injection inmice [45] Previous studies [46 47] showedalso that both Sal A and Sal B are able to improve theimpaired memory function induced by cerebral ischemia-reperfusion in miceThe stimulation of neurogenesis processin both subgranular zone (SGZ) and subventricular zone(SVZ) after brain ischemia and also alleviation neural cellsloss and improved motor function recovery after brainischemia in rats after the Sal B administration were alsoobserved by Zhong et al [50] Moreover the exposure to SalB can maintain the proliferation of neural stemprogenitor

Evidence-Based Complementary and Alternative Medicine 13

cells (NSPCs) after cerebral ischemia and improve cognitivepostischemic impairment after stroke in rats using Morriswater maze test therefore authors concluded that Sal B mayact as a potential drug in treatment of brain injury or neu-rodegenerative disease [51] Additionally the improvement ofmotor function after cerebral ischemia in rats after salvianolicacid B administrationwas also demonstrated [52]The clue topass through the BBB Sal B provided also results of Li et alindicating its protective effect on BBB in rats after cerebralischemia-reperfusion by inhibiting the MAPK pathway [53]In addition to this it was shown that lithospermic acid andsalvianolic acids exerted neuroprotective activity in variousexperimental models Lithospermic acid significantly attenu-ates neurotoxicity in vitro and in vivo induced by 1-methyl-4-phenylpyridin (MPP(+)) by blocking neuronal apoptotic andneuroinflammatory pathways [54] Salvianolic acid B inhib-ited amyloid beta-protein aggregation and fibril formation aswell as directly inhibiting the cellular toxicity of amyloid beta-protein in PC12 cells [55] and significantly reduced its cyto-toxic effects on human neuroblastoma SH-SY5Y cells [56]

For an explanation of our results observations of Pin-heiro Fernandes et al [57] may be very helpful whichshowed that caffeic acid nonflavanoid catecholic compoundwhose derivatives are occurring in MO extract improvedthe working spatial and long-term aversive memory deficitsinduced by focal cerebral ischemia in mice Anwar et al [48]showed also that caffeic acid (100mgkg) improved the step-down latencies in the inhibitory avoidance in rats Tsai et al[58] showed that caffeic acid is a potent neuroprotective agentin brain of 1-methyl-4-phenyl-1236-tetrahydropyridine(MPTP) treatedmiceMoreover caffeic acid improves A12057325ndash35-induced memory deficits and cognitive impairment inmice [59] In another study it was also shown that this com-pound has a significant protective effect on global cerebralischemia-reperfusion injury in rats [60] For instance 124 plusmn18mg100 g of caffeic acid was detected in the brain of micewith a diet containing 2 caffeic acid for 4 weeks [58] Thusthere is progressive evidence that this compound passesthrough the BBB and has a central pharmacological activity

Also results by Yoo et al are also worth noting [61] whichshowed also in a ratmodel of scopolamine-induced amnesiathat administration of luteolin (a common flavonoid frommany plants including M officinalis) at dose of 10mgkgcaused the increase in the brain-derived neurotrophic factor(BDNF) acetylcholine and the decrease in lipid peroxida-tion Liu et al [62] observed that chronic treatment withluteolin (50 and 100mgkg) improved neuronal injury andcognitive performance by attenuating oxidative stress andcholinesterase activity in streptozotocin-induced diabetes inrats In our study ethanolic extract of MO administered torats significantly improved long-term memory after using apassive avoidance test but afterMO combined treatmentwithSC no improvement of this paradigmwas observed (Table 2)

Thirdly available pharmacokinetic studies were carriedout for the pure compounds (Sal A and B) and the extractof the roots of Salvia miltiorrhiza [42 63] but there are notavailable studies of bioavailability of these compounds afteradministration of Melissa officinalis leaf extract containingother compounds as compared with the extract of Salvia

miltiorrhiza Moreover there is a lack of data about howscopolamine may influence the penetration of the salvianolicacids (and other compounds) across the BBB Hence thereis a need to study the pharmacokinetic parameters of thesecompounds in the group of animals treatedwith scopolaminein comparison with control group

AChEandBuChEActivities in Rat Brain To date several stud-ies have focused on explaining themechanismof action of theMS extract and its active compounds Soodi et al [18] showedthat treatment of animals withMO extract (400mgkg) priorto scopolamine injection could ameliorate scopolamine-induced enhancement in AChE activity This dose of MOextract inhibited the AChE activity in the hippocampus ofrats (519 versus 1004 in normal saline group 119901 lt 005914 in group treated with scopolamine +MO versus 1281in scopolamine group 119901 lt 005) In another study Anwaret al [48] showed that 50 and 100mgkg of caffeic aciddecreased in vivo the AChE activity in the cerebral cortexand striatum and increased the activity of this enzyme inthe cerebellum hippocampus hypothalamus pons lympho-cytes and muscles when compared to the control group(119901 lt 005) Our study showed that ethanolic extract of MOproduced an insignificant and slight inhibition of AChE andBuChE activity in the frontal cortex and in the hippocampus(especially) However it was observed previously [23] thatRA inhibited the AChE activity in the rats frontal cortex andhippocampusMoreover we showed that RA possess a strongstimulatory effect on BuChE in the hippocampus

AChE BuChE and BACE1mRNA Level Changes in Rat BrainSo far no results have been published of studies concerningthe in vivo assessment of changes in AChE BuChE andBACE1 gene expression profile in different brain regionsunder the influence of MO extracts or their key bioactivemetabolites Such studies focused overwhelmingly on theanalysis of their in vitro activities to a lesser extent in animalexperiments

In our study in the frontal cortex we have observedthe strongest inhibition of both AChE and BuCHE mRNAtranscription under the influence ofM officinalis extract andhuperzine A (Table 3) However a more significant differencein the level of transcript was seen in the frontal cortex inparticular in the case of BuCHE mRNA of experimentalrats (a decrease by 516 and 44 for MO and HU versuscontrol resp)The strong inhibition of transcription ofAChEmRNAwas also observed in the hippocampus ofMO-treatedrats (a decrease by 69) The BuChE mRNA transcriptionin animals receiving MO was lowered in a moderate way(decrease by 36)HuperzineA alone has not caused changesthat were observed in the group ofMO (Table 2) Our findingsmostly correlate with the observed changes in activities ofAChE and BuChE in different groups of animals In the caseof BuChE slightly different results between its activity andexpression profile were especially seen in the frontal cortexand hippocampus of animals receivingMO (Table 3)

To date there is a lack of published results of studiesmaking an attempt to clarify the potential differences in thetranscriptional profile and activity of AChE and BuChE

14 Evidence-Based Complementary and Alternative Medicine

under the influence of MO and its bioactive metabolites Itcannot be excluded that the key for the observed differencesin the level of AChE activities and mRNA level can becaused by changes in the activity of AChE in other regionsof the brain not analyzed in this study such as substantianigra cerebellum globus pallidus and hypothalamus whereit exerts nonenzymatic neuromodulatory functions affect-ing neurite outgrowth and synaptogenesis modulating theactivity of other proteins regional cerebral blood flow andother functions [64] But it is difficult to clearly explain whydiminishing of AChE and BuChEmRNAs did not always cor-relate with the lowering of activity of these enzymes althoughit has been recently noted that AChE activity was notparalleled by an increase in mRNA levels [65] The authorsexplained this fact by stating that AChE levels are regulatedat transcriptional posttranscriptional and posttranslationallevels leading to complex expression patterns which canbe modulated by physiological and pathological conditionsHowever these mechanisms are not fully understood andfurther studies are needed in this field

The cause of observed different degree of inhibition ofactivity and transcription status of studied genes especiallyof BuChE (and AChE) may lie in a so-called ldquonegative feed-backrdquo consisting of a complicated transcriptiontranslationregulation protein-protein interactionsmodifications and ametabolic network together forming a system that allows thecell to respond sensibly to the multiple signal molecules thatexist in its environment [66]

Because of that we propose that the reason for differencesbetween AChE and BuChE activities in the cortex and hip-pocampus under the MO may be due to the fact of insuf-ficiency of applied dose and the duration of the experi-ment affecting the transcriptional tissue-specific cellularmachinery regulating BuChE transcription without affectingits activity Moreover the administration of higher doses ofMO and extended period of time could lead to sufficientinhibition of its activity

There is a need to conduct further studies to determinethe molecular degree of dependence between changes inAChE and BuChE activities and activities of potential keyfactors regulating expression of these genes in the frontalcortex and hippocampus under the influence of extracts ofMO and active metabolites Another study determining theeffectiveness of their actions on the cholinergic system inexperimental animal models of memory impairment withparticular emphasis on scopolamine including the determi-nation of changes at the molecular level should be thereforecarried out

Alterations in BACE1 protein level have been proved inpostmortem brain tissue from individuals with AD withincreases decreases and also no change reported [1 67 68]An example of confirmation of these findings at the mRNAlevel is results of study by Coulson et al [69] Based onconducted studies some authors suggested that increasedBACE1mRNA transcription in remaining neuronal cells maycontribute to the increased BACE1 protein levels and activityfound in brain regions affected by AD [70]

Results of many studies concerning the elevated level ofBACE1 mRNA and protein in Alzheimerrsquos disease provide

direct and compelling reasons to develop therapies directed atBACE1 inhibition thus reducing120573-amyloid and its associatedtoxicities [67 68]

In our study we have observed that BACEmRNA expres-sion statistically significantly decreased after MO adminis-tration in frontal cortex and hippocampus These resultssuggest that the MO extract may act to inhibit BACE1mRNA level given the fact that the percentage of inhibitionof the expression (64 in the frontal cortex 50 in thehippocampus) is higher than that in the case of HU (38in the cortex and the lack of changes in the hippocampus)A careful analysis of the literature data shows that there areno studies which analyzed the impact of MO extract on theexpression level of BACE1 in Alzheimerrsquos disease

Furthermore a literature analysis does not indicatealready published results conducted by other teams attempt-ing to assess the impact of RA on the transcriptional activityof AChE BuChE and BACE1 Although several studies(already mentioned and others [70 71]) highlighted theRA and other caffeic acid derivatives capability of acetyl-cholinesterases inhibition none of these studies does nottouch the question of the molecular basis of their impacton the transcriptional machinery that regulates in vivo theexpression of studied genes Hence in our opinion obtainedby our team results they are one of the first of this typeand in general correlate with the results of our previousstudy [23] In this case there is no clear evidence explainingdifferent responses at the transcriptional level under theinfluence of RA especially in the case of BuChE encodinggene in the hippocampus (Table 2) It is possible that theobserved differentiation of BuChE transcriptional activitybetween the frontal cortex and the hippocampus may bedue to the differences of butyrylcholinesterase localizationand substrate affinity [72] Since in our experiment we havecarried out a quantitative analysis of AChE BuChE andBACE1 transcripts in brain homogenates of tested animalsrather than in individual isolated cell fractions therefore theobtained results constitute an overall ldquopicturerdquo of both studiedgenes transcriptional changes occurring in the brain areas ofstudied animals under the influence of the RA and the wholeplant extract as well

6 Conclusion

The subchronic administration ofMO led to an improvementof long-term memory of rats however the mechanisms ofMO action are probably more complicated since its role asa modulator of beta-secretase activity (due to inhibition ofBACE1 mRNA expression in frontal cortex) should be takeninto consideration

It should be noted that we have studied a crude extractfrom leaf of Melissa officinalis not a single pure chemicalcompound This plant extract is a complex mixture and itsaction may be a result of the summation of activities ofseveral components (synergismadditive action of caffeic acidwith salvianolic acids rosmarinic acid and others) In thecase of extract from leaves ofMelissa officinalis it is possiblethat interactions occur between the 40 chemical compoundsidentified by HPLC system

Evidence-Based Complementary and Alternative Medicine 15

Taken together it seems that the MO activity representsa possible option as complementary interventions to relievethe symptoms of mild dementia

Competing Interests

The authors declare that they have no competing interests

Acknowledgments

This work was supported by the Ministry of Science andHigher EducationWarsaw Poland from educational sources(2008ndash2011 Grant no N 405417836)

References

[1] S L Cole and R Vassar ldquoThe Alzheimerrsquos disease 120573-secretaseenzyme BACE1rdquo Molecular Neurodegeneration vol 2 no 1article 22 2007

[2] LACraigN SHong andR JMcDonald ldquoRevisiting the cho-linergic hypothesis in the development of Alzheimerrsquos diseaserdquoNeuroscience amp Biobehavioral Reviews vol 35 no 6 pp 1397ndash1409 2011

[3] N G M Gomes M G Campos J M C Orfao and C A FRibeiro ldquoPlants with neurobiological activity as potential tar-gets for drug discoveryrdquo Progress in Neuro-Psychopharmacologyamp Biological Psychiatry vol 33 no 8 pp 1372ndash1389 2009

[4] WHO WHO Monographs on Selected Medicinal PlantsmdashVolume 2 WHO Geneva Switzerland 2004 httpappswhointmedicinedocsendJs4927e18htmlJs4927e18

[5] M Bayat A A Tameh M H Ghahremani et al ldquoNeuropro-tective properties of Melissa officinalis after hypoxic-ischemicinjury both in vitro and in vivordquo DARU Journal of Pharmaceu-tical Sciences vol 20 article 42 2012

[6] J P Kamdem A Adeniran A A Boligon et al ldquoAntioxidantactivity genotoxicity and cytotoxicity evaluation of lemon balm(Melissa officinalis L) ethanolic extract Its potential role inneuroprotectionrdquo Industrial Crops and Products vol 51 pp 26ndash34 2013

[7] J-T Lin Y-C Chen Y-C Lee C-W R Hou F-L Chen andD-J Yang ldquoAntioxidant anti-proliferative and cyclooxygenase-2 inhibitory activities of ethanolic extracts from lemon balm(Melissa officinalis L) leavesrdquo LWTmdashFood Science and Technol-ogy vol 49 no 1 pp 1ndash7 2012

[8] G Guginski A P Luiz M D Silva et al ldquoMechanisms involvedin the antinociception caused by ethanolic extract obtainedfrom the leaves of Melissa officinalis (lemon balm) in micerdquoPharmacology Biochemistry amp Behavior vol 93 no 1 pp 10ndash162009

[9] N Perry G Court N Bidet J Court and E Perry ldquoEuropeanherbs with cholinergic activities potential in dementia therapyrdquoInternational Journal of Geriatric Psychiatry vol 11 no 12 pp1063ndash1069 1996

[10] E K Perry A T Pickering W W Wang P J Houghtonand N S L Perry ldquoMedicinal plants and Alzheimerrsquos diseasefrom ethnobotany to phytotherapyrdquo Journal of Pharmacy andPharmacology vol 51 no 5 pp 527ndash534 1999

[11] M-J R Howes N S L Perry and P J Houghton ldquoPlants withtraditional uses and activities relevant to the management ofAlzheimerrsquos disease and other cognitive disordersrdquo Phytother-apy Research vol 17 no 1 pp 1ndash18 2003

[12] I Orhan and M Aslan ldquoAppraisal of scopolamine-inducedantiamnesic effect in mice and in vitro antiacetylcholinesteraseand antioxidant activities of some traditionally used Lamiaceaeplantsrdquo Journal of Ethnopharmacology vol 122 no 2 pp 327ndash332 2009

[13] S Akhondzadeh M Noroozian M Mohammadi S OhadiniaA H Jamshidi and M Khani ldquoMelissa officinalis extract inthe treatment of patients with mild to moderate Alzheimerrsquosdisease a double blind randomised placebo controlled trialrdquoJournal of Neurology Neurosurgery and Psychiatry vol 74 no 7pp 863ndash866 2003

[14] D O Kennedy A B Scholey N T J Tildesley E K Perry andK AWesnes ldquoModulation ofmood and cognitive performancefollowing acute administration of Melissa officinalis (lemonbalm)rdquo Pharmacology Biochemistry amp Behavior vol 72 no 4pp 953ndash964 2002

[15] D O Kennedy G Wake S Savelev et al ldquoModulation of moodand cognitive performance following acute administration ofsingle doses of Melissa officinalis (Lemon balm) with humanCNS nicotinic and muscarinic receptor-binding propertiesrdquoNeuropsychopharmacology vol 28 no 10 pp 1871ndash1881 2003

[16] D O Kennedy and E L Wightman ldquoHerbal extracts and phy-tochemicals plant secondarymetabolites and the enhancementof human brain functionrdquoAdvances inNutrition vol 2 no 1 pp32ndash50 2011

[17] R P Pereira A A Boligon A S Appel et al ldquoChemical com-position antioxidant and anticholinesterase activity of Melissaofficinalisrdquo Industrial Crops and Products vol 53 pp 34ndash452014

[18] M Soodi N Naghdi H Hajimehdipoor S Choopani andE Sahraei ldquoMemory-improving activity of Melissa officinalisextract in naıve and scopolamine-treated ratsrdquo Research inPharmaceutical Sciences vol 9 no 2 pp 107ndash114 2014

[19] K Dastmalchi V Ollilainen P Lackman et al ldquoAcetyl-cholinesterase inhibitory guided fractionation of Melissa offic-inalis Lrdquo Bioorganic amp Medicinal Chemistry vol 17 no 2 pp867ndash871 2009

[20] W Chaiyana and S Okonogi ldquoInhibition of cholinesterase byessential oil from food plantrdquo Phytomedicine vol 19 no 8-9 pp836ndash839 2012

[21] K Dastmalchi H J D Dorman P P Oinonen Y Darwis ILaakso and R Hiltunen ldquoChemical composition and in vitroantioxidative activity of a lemon balm (Melissa officinalis L)extractrdquo LWTmdashFood Science and Technology vol 41 no 3 pp391ndash400 2008

[22] P Pereira D Tysca P Oliveira L F D S Brum J N Picadaand P Ardenghi ldquoNeurobehavioral and genotoxic aspects ofrosmarinic acidrdquo Pharmacological Research vol 52 no 3 pp199ndash203 2005

[23] M Ozarowski P L Mikolajczak A Bogacz et al ldquoRosmarinusofficinalis L leaf extract improves memory impairment andaffects acetylcholinesterase and butyrylcholinesterase activitiesin rat brainrdquo Fitoterapia vol 91 pp 261ndash271 2013

[24] G P Kumar and F Khanum ldquoNeuroprotective potential of phy-tochemicalsrdquo Pharmacognosy Reviews vol 6 no 12 pp 81ndash902012

[25] M Stobiecki A Staszkow A Piasecka P M Garcia-LopezF Zamora-Natera and P Kachlicki ldquoLC-MSMS profiling offlavonoid conjugates in wild mexican lupine Lupinus reflexusrdquoJournal of Natural Products vol 73 no 7 pp 1254ndash1260 2010

[26] A Wojakowska A Piasecka P M Garcıa-Lopez et al ldquoStruc-tural analysis and profiling of phenolic secondarymetabolites of

16 Evidence-Based Complementary and Alternative Medicine

Mexican lupine species using LC-MS techniquesrdquo Phytochem-istry vol 92 pp 71ndash86 2013

[27] A Gryszczynska B Opala Z Lowicki et al ldquoBioactive com-pounds determination in the callus and hydroalcoholic extractsfrom Salvia miltiorrhiza and Salvia przewalskiimdashpreliminarystudy on their anti-alcoholic activity effectsrdquo PhytochemistryLetters vol 11 pp 399ndash403 2015

[28] K Slinkart and V L Singleton ldquoTotal phenol analysis automa-tion and comparison with manual methodrdquo American Journalof Enology and Viticulture vol 28 no 1 pp 49ndash55 1977

[29] P J R Boissier J Tardy and J C Diverres ldquoUne nouvellemethode simple pour explorer lrsquoaction lsquotranquillisantersquo le testde la chemineerdquo Medicina Experimentalis vol 3 no 1 pp 81ndash84 1960

[30] R Ader J A Weijnen and P Moleman ldquoRetention of a passiveavoidance response as a function of the intensity and durationof electric shockrdquo Psychonomic Science vol 26 no 3 pp 125ndash128 1972

[31] E M Blalock K-C Chen K Sharrow et al ldquoGene microar-rays in hippocampal aging statistical profiling identifies novelprocesses correlated with cognitive impairmentrdquo Journal ofNeuroscience vol 23 no 9 pp 3807ndash3819 2003

[32] K Isomae S Morimoto H Hasegawa K Morita and JKamei ldquoEffects of T-82 a novel acetylcholinesterase inhibitoron impaired learning and memory in passive avoidance task inratsrdquo European Journal of Pharmacology vol 465 no 1-2 pp97ndash103 2003

[33] J Patora and B Klimek ldquoFlavonoids from lemon balm (Melissaofficinalis L Lamiaceae)rdquo Acta Poloniae Pharmaceutica-DrugResearch vol 59 no 2 pp 139ndash143 2002

[34] L Barros M Duenas M I Dias M J Sousa C Santos-Buelgaand I C F R Ferreira ldquoPhenolic profiles of cultivated invitro cultured and commercial samples of Melissa officinalis Linfusionsrdquo Food Chemistry vol 136 no 1 pp 1ndash8 2013

[35] T L Miron M Herrero and E Ibanez ldquoEnrichment of antiox-idant compounds from lemon balm (Melissa officinalis) bypressurized liquid extraction and enzyme-assisted extractionrdquoJournal of Chromatography A vol 1288 pp 1ndash9 2013

[36] Y Lu L Y Foo and H Wong ldquoSagecoumarin a novel caffeicacid trimer from Salvia officinalisrdquo Phytochemistry vol 52 no6 pp 1149ndash1152 1999

[37] S Abuhamdah LHuangM S J Elliott et al ldquoPharmacologicalprofile of an essential oil derived from Melissa officinalis withanti-agitation properties focus on ligand-gated channelsrdquo Jour-nal of Pharmacy and Pharmacology vol 60 no 3 pp 377ndash3842008

[38] G Wake J Court A Pickering R Lewis R Wilkins andE Perry ldquoCNS acetylcholine receptor activity in Europeanmedicinal plants traditionally used to improve failing memoryrdquoJournal of Ethnopharmacology vol 69 no 2 pp 105ndash114 2000

[39] S-H Ryu C Katona B Rive and G Livingston ldquoPersistenceof and changes in neuropsychiatric symptoms in Alzheimerdisease over 6months the LASER-AD studyrdquoAmerican Journalof Geriatric Psychiatry vol 13 no 11 pp 976ndash983 2005

[40] L N Gitlin H C Kales and C G Lyketsos ldquoNonpharma-cologic management of behavioral symptoms in dementiardquoJournal of the AmericanMedical Association vol 308 no 19 pp2020ndash2029 2012

[41] A Konczol J Muller E Foldes et al ldquoApplicability of a blood-brain barrier specific artificial membrane permeability assay atthe early stage of natural product-based CNS drug discoveryrdquoJournal of Natural Products vol 76 no 4 pp 655ndash663 2013

[42] Y-J Zhang L Wu Q-L Zhang J Li F-X Yin and Y YuanldquoPharmacokinetics of phenolic compounds of Danshen extractin rat blood and brain by microdialysis samplingrdquo Journal ofEthnopharmacology vol 136 no 1 pp 129ndash136 2011

[43] X Li C Yu Y Lu et al ldquoPharmacokinetics tissue distributionmetabolism and excretion of depside salts from Salvia miltior-rhiza in ratsrdquo Drug Metabolism and Disposition vol 35 no 2pp 234ndash239 2007

[44] H Xu Y Li X Che H Tian H Fan and K Liu ldquoMetabolismof salvianolic acid a and antioxidant activities of its methylatedmetabolitesrdquo Drug Metabolism and Disposition vol 42 no 2pp 274ndash281 2014

[45] D H Kim S J Park J M Kim et al ldquoCognitive dysfunctionsinduced by a cholinergic blockade and A120573 25-35 peptide areattenuated by salvianolic acid Brdquo Neuropharmacology vol 61no 8 pp 1432ndash1440 2011

[46] G-H Du Y Qiu and J-T Zhang ldquoSalvianolic acid B protectsthe memory functions against transient cerebral ischemia inmicerdquo Journal of Asian Natural Products Research vol 2 no 2pp 145ndash152 2000

[47] G Du and J Zhang ldquoProtective effects of salvianolic acid Aagainst impairment of memory induced by cerebral ischemia-reperfusion in micerdquo Chinese Medical Journal vol 110 no 1 pp65ndash68 1997

[48] J Anwar R M Spanevello G Thome et al ldquoEffects of caffeicacid on behavioral parameters and on the activity of acetyl-cholinesterase in different tissues from adult ratsrdquo Pharmacol-ogy Biochemistry and Behavior vol 103 no 2 pp 386ndash394 2012

[49] YW Lee D H Kim S J Jeon et al ldquoNeuroprotective effects ofsalvianolic acid B on anA12057325-35 peptide-inducedmousemodelof Alzheimerrsquos diseaserdquo European Journal of Pharmacology vol704 no 1ndash3 pp 70ndash77 2013

[50] J Zhong M-K Tang Y Zhang Q-P Xu and J-T ZhangldquoEffect of salvianolic acid B on neural cells damage and neu-rogenesis after brain ischemia-reperfusion in ratsrdquo Yao Xue XueBao vol 42 no 7 pp 716ndash721 2007

[51] P Zhuang Y Zhang G Cui et al ldquoDirect stimulation of adultneural stemprogenitor cells in vitro and neurogenesis in vivo bysalvianolic acid Brdquo PLoS ONE vol 7 no 4 Article ID e356362012

[52] M TangW Feng Y Zhang J Zhong and J Zhang ldquoSalvianolicacid B improves motor function after cerebral ischemia in ratsrdquoBehavioural Pharmacology vol 17 no 5-6 pp 493ndash498 2006

[53] Q Li L-P Han Z-H Li J-T Zhang and M-K Tang ldquoSal-vianolic acid B alleviate the disruption of blood-brain barrierin rats after cerebral ischemia-reperfusion by inhibiting MAPKpathwayrdquo Yao Xue Xue Bao vol 45 no 12 pp 1485ndash1490 2010

[54] Y-L LinH-J Tsay T-H Lai T-T Tzeng andY-J Shiao ldquoLith-ospermic acid attenuates 1-methyl-4-phenylpyridine-inducedneurotoxicity by blocking neuronal apoptotic and neuroinflam-matory pathwaysrdquo Journal of Biomedical Science vol 22 article37 2015

[55] M-K Tang and J-T Zhang ldquoSalvianolic acid B inhibits fibrilformation and neurotoxicity of amyloid beta-protein in vitrordquoActa Pharmacologica Sinica vol 22 no 4 pp 380ndash384 2001

[56] S S K Durairajan Q Yuan L Xie et al ldquoSalvianolic acid Binhibits A120573 fibril formation and disaggregates preformed fibrilsand protects against A120573-induced cytotoxictyrdquo NeurochemistryInternational vol 52 no 4-5 pp 741ndash750 2008

[57] FD Pinheiro Fernandes A P FonteneleMenezes J C de SousaNeves et al ldquoCaffeic acid protects mice from memory deficits

Evidence-Based Complementary and Alternative Medicine 17

induced by focal cerebral ischemiardquo Behavioural Pharmacologyvol 25 no 7 pp 637ndash647 2014

[58] S-J Tsai C-Y Chao andM-C Yin ldquoPreventive and therapeu-tic effects of caffeic acid against inflammatory injury in striatumof MPTP-treated micerdquo European Journal of Pharmacology vol670 no 2-3 pp 441ndash447 2011

[59] J H Kim QWang J M Choi S Lee and E J Cho ldquoProtectiverole of caffeic acid in an 119860120573

25minus35

-induced Alzheimerrsquos diseasemodelrdquo Nutrition Research and Practice vol 9 no 5 pp 480ndash488 2015

[60] G Liang B Shi W Luo and J Yang ldquoThe protective effect ofcaffeic acid on global cerebral ischemia-reperfusion injury inratsrdquo Behavioral and Brain Functions vol 11 no 1 article 182015

[61] D Y Yoo J H ChoiW Kim et al ldquoEffects of luteolin on spatialmemory cell proliferation and neuroblast differentiation in thehippocampal dentate gyrus in a scopolamine-induced amnesiamodelrdquo Neurological Research vol 35 no 8 pp 813ndash820 2013

[62] Y Liu X Tian L Gou L Sun X Ling and X Yin ldquoLuteolinattenuates diabetes-associated cognitive decline in ratsrdquo BrainResearch Bulletin vol 94 pp 23ndash29 2013

[63] S-M Liu Z-H Yang andX-B Sun ldquoSimultaneous determina-tion of six Salvia miltiorrhiza gradients in rat plasma and brainby LC-MSMSrdquo Zhongguo Zhongyao Zazhi vol 39 no 9 pp1704ndash1708 2014

[64] M A Papandreou A Dimakopoulou Z I Linardaki et alldquoEffect of a polyphenol-rich wild blueberry extract on cognitiveperformance of mice brain antioxidant markers and acetyl-cholinesterase activityrdquo Behavioural Brain Research vol 198 no2 pp 352ndash358 2009

[65] M-S Garcıa-Ayllon O Cauli M-X Silveyra et al ldquoBraincholinergic impairment in liver failurerdquo Brain vol 131 no 11pp 2946ndash2956 2008

[66] G K Ferreira M Carvalho-Silva C L Goncalves et al ldquoL-Tyrosine administration increases acetylcholinesterase activityin ratsrdquo Neurochemistry International vol 61 no 8 pp 1370ndash1374 2012

[67] R Vassar P-H Kuhn C Haass et al ldquoFunction therapeuticpotential and cell biology of BACE proteases current status andfuture prospectsrdquo Journal of Neurochemistry vol 130 no 1 pp4ndash28 2014

[68] R Vassar ldquoThe 120573-secretase BACE a prime drug target forAlzheimerrsquos diseaserdquo Journal of Molecular Neuroscience vol 17no 2 pp 157ndash170 2001

[69] D T R Coulson N Beyer J G Quinn et al ldquoBACE1 mRNAexpression in Alzheimerrsquos disease postmortem brain tissuerdquoJournal of Alzheimerrsquos Disease vol 22 no 4 pp 1111ndash1122 2010

[70] S Vladimir-Knezevic B Blazekovic M Kindl J Vladic A DLower-Nedza and A H Brantner ldquoAcetylcholinesterase inhib-itory antioxidant and phytochemical properties of selectedmedicinal plants of the lamiaceae familyrdquoMolecules vol 19 no1 pp 767ndash782 2014

[71] F Marcelo C Dias A Martins et al ldquoMolecular recognitionof rosmarinic acid from Salvia sclareoides extracts by ace-tylcholinesterase a new binding site detected by NMR spec-troscopyrdquo ChemistrymdashA European Journal vol 19 no 21 pp6641ndash6649 2013

[72] G Johnson and S W Moore ldquoWhy has butyrylcholinesterasebeen retained Structural and functional diversification in aduplicated generdquoNeurochemistry International vol 61 no 5 pp783ndash797 2012

[73] R J Grayer M R Eckert N C Veitch et al ldquoThe chemo-taxonomic significance of two bioactive caffeic acid estersnepetoidins A and B in the Lamiaceaerdquo Phytochemistry vol 64no 2 pp 519ndash528 2003

[74] A M D El-Mousallamy U W Hawas and S A M HusseinldquoTeucrol a decarboxyrosmarinic acid and its 41015840-O-triglycosideteucroside from Teucrium pilosumrdquo Phytochemistry vol 55 no8 pp 927ndash931 2000

[75] I Fecka and S Turek ldquoDetermination of water-soluble polyphe-nolic compounds in commercial herbal teas from Lamiaceaepeppermint melissa and sagerdquo Journal of Agricultural and FoodChemistry vol 55 no 26 pp 10908ndash10917 2007

[76] L W Sumner A Amberg D Barrett et al ldquoProposed min-imum reporting standards for chemical analysis WorkingGroup (CAWG) Metabolomics Standards Initiative (MSI)rdquoMetabolomics vol 3 pp 211ndash221 2007

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

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Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

6 Evidence-Based Complementary and Alternative MedicineTa

ble1Metabolitesd

etectedin

Melissa

officin

alisleafextractb

yUPL

C-MS

No

RT [min]

Metabolite

identifi

catio

nCh

emical

form

ula

Exactm

asso

f[M-H

]minusΔpp

mFragmentatio

nin

120582max[nm]

CID

aIdentifi

catio

nlevelb

Reference

Measured

Calculated

Negativeion

mod

e(ESIminus)

Positiveion

mod

e(ESI+)

114

1

2-Hydroxy-3-(34-

dihydroxyphenyl)-

prop

anoic

acid

C 9H9

O5

1970

451970

455minus41775

197179135

199163

283312

8143997

2[34]

217

3Dihydroxybenzoica

cid

hexosid

eC 13

H15

O9

315072

3150722

08346

315153109

317155

282

54726828

3[35]

319

8Ca

ftaric

acid

C 13

H11

O9

3110

413110

409

06141

311221179149

318

6440397

2[35]

4224

2-Hydroxy-3-(34-

dihydroxyphenyl)-

prop

anoica

cid

sulphated

C 9H9

O8

S2770

032770

024

03998

277197179135

312

3[35]

5256

Hydroxyjasm

onicacid

hexosid

eC 18

H27

O9

387166

3871661

09214

387207163

323

44237366

2[17]

6276

Caffeicacid

C 9H7

O4

1790

341790

451minus49075

179135

181163

275

689043

1std

7289

Salviano

licacid

EC 36

H29

O16

7171450

717146

72313

717519339321

295277

275325

49770697

2[35]

8317

Salviano

licacid

HI

(isom

er)

C 27

H21

O12

537104

5371038

08562

537493359295

281325

2[35]

9345

Hydroxyjasm

onicacid

sulphated

C 12

H17

O7

S30507

30507

1117

305225194147

275330

3[17]

10362

NepetoidinB

C 17

H13

O6

313072

3130718

11232

nd282316

5316819

2[73]

1137

Yunn

aneica

cidF

C 26

H25

O14

5971255

5971244

18743

597509311197

Masked

2[34]

12383

Decarbo

xyrosm

arinicacid

(teucrol)

C 17

H15

O6

315088

3150874

09396

315179135

275330

637829

2[74]

1339

Caffeoylcaft

aricacid

C 22

H17

O12

473073

4730725

07555

173311149

Masked

65018

3[35]

14397

Apigenin

glucosylrham

nosid

eC 27

H29

O14

577157

5771563

11136

577269

579433271

275340

92741003

3[33]

15404

Luteolin

7-O-glucosid

e31015840-O

-glucuronide

C 27

H27

O17

623126

6231254

09696

62346144

7285

255

625463287

273343

2[33]

16421

Rosm

arinicacid

hexosid

eC 24

H25

O13

52113

5211301

03551

521359161

523361325163

329

25245848

2[34]

1743

Luteolin

O-diglucosid

eC 27

H29

O16

609147

6091461

07183

609285

611287

269349

3[33]

18441

Luteolin

glucosylrham

nosid

eC 27

H29

O15

593152

5931512

08077

59344

7285

59544

9287

271343

3[33]

19449

Luteolin

41015840-O

-glucosid

eC 21

H19

O11

4470

9444

70933

09728

447285

449287

271343

5319116

3[33]

2045

Sagerin

icacid

2-hydroxy-3-

(34-dihydroxyph

enyl)-

prop

anoide

C 45

H39

O20

8992

058992

0410

448

899719591475

295

Masked

3[35]

2149

Salviano

licacid

B(lithosperm

icacid

B)C 36

H29

O16

717146

7171461

01846

717519359161

327

6441188

2[34]

22504

Sagerin

icacid

C 36

H31

O16

7191

637191

618

09979

719519359161

287330

2[34]

23514

Rosm

arinicacid

C 18

H15

O8

3590

773590

772

0002

359161

361163

329

5281792

1std

Evidence-Based Complementary and Alternative Medicine 7

Table1Con

tinued

No

RT [min]

Metabolite

identifi

catio

nCh

emical

form

ula

Exactm

asso

f[M-H

]minusΔpp

mFragmentatio

nin

120582max[nm]

CID

aIdentifi

catio

nlevelb

Reference

Measured

Calculated

Negativeion

mod

e(ESIminus)

Positiveion

mod

e(ESI+)

24555

Salviano

licacid

B2-hydroxy-3-(34-

dihydroxyphenyl)-

prop

anoide

C 45

H37

O20

89719

8971884

16873

897717519359

161

330

3[35]

2556

Sagerin

icacid

di-2-hydroxy-3-(34-

dihydroxyphenyl)-

prop

anoide

C 54

H47

O24

1079244

910792457minus07911

1079897719539

359295

288330

3[35]

26592

Sagecoum

arin

di-2-hydroxy-3-(34-

dihydroxyphenyl)-

prop

anoide

caffeide

C 54

H43

O24

10752156

10752150

0559

1077897717537

409359339277

322

3[35]

27598

Rosm

arinicacid

sulphated

Iisomer

C 18

H15

O11

S4390

354390

341

20214

439359341163

Masked

2[34]

28613

Luteolin

31015840-O

-glucuronide

C 21

H17

O12

4610

734610

7216

34461285

463287

269340

170474237

2[33]

29632

Salviano

licacid

AC 26

H21

O10

493115

493114

11011

493359295179

298327

5281793

[34]

30656

Sagerin

icacid

sulphated

C 36

H31

O19

S7991

196

7991

186

0954

799719619519

359161

325

3[35]

31671

Lithosperm

icacid

C 27

H21

O12

537104

537104

809698

537493359161

292329

6441498

2[35]

3268

Rosm

arinicacid

sulphated

IIiso

mer

C 18

H15

O11

S4390

344390

341

02837

439359341163

Masked

2[34]

33689

Sagecoum

arin

C 27

H19

O12

535088

5350882

01204

535311267177

Masked

2[36]

3470

3Salviano

licacid

LIisomer

C 36

H29

O16

717146

7171461

02697

717519359

284329

2[35]

3571

Salviano

licacid

Lhydroxycaffeide

C 45

H35

O20

895173

8951727

06282

895519359161

Masked

3[35]

3674

Sagecoum

arin

cafta

ride

C 40

H29

O20

8291

268291

258

06953

829667535355

311

Masked

3[36]

37751

Sagecoum

arin

2-hydroxy-

3-(34-dihydroxyph

enyl)-

prop

anoide

C 36

H27

O16

715131

7151305

08176

715535311267

319

3[34]

3877

1Unk

nown

C 36

H57

O14

S745348

7453475

044

02281326

4[35]

3978

5Methylrosmarinate

C 19

H17

O8

373093

3730929

01765

373359161

284323

6479915

2[75]

40869

Salviano

licacid

Ccaffeoylhydroxycaffeide

C 44

H33

O18

8491

688491

672

08615

849687491359

327255

286318

3[35]

a CID

identifier

fora

chem

icalstr

ucture

intheP

ubCh

emCom

poun

ddatabase

b Metabolite

identifi

catio

nlevelaccording

toMetabolom

icsS

tand

ards

Initiativer

ecom

mendatio

n[76]

stdidentificatio

non

theb

asisof

stand

ardcompo

undfragmentatio

nnd

not

detected

8 Evidence-Based Complementary and Alternative Medicine

100 200 300 400 500 600 700 800 900 1000 1100 1200

mz

70

75

80

85

90

95

100

minus198 (2-hydroxy-3-(34-dihydroxyphenyl)

-porpanoide)

minus180 (caffeide)

minus180 (2-hydroxy-3-(34-dihydroxyphenyl)

-propanoide)

minus180 (2-hydroxy-3-(34-dihydroxyphenyl)

-propanoide)

minus180 (2-hydroxy-3-(34-dihydroxyphenyl)

-propanoide)

1350438

2191749

HO

HO

HO HO

OH

OH

OH

OH OH OH

OHOHOHOHHOOC

COOH

COOH COOH COOH

24612ppmC36H55O37 = 10792422

minus07911ppmC54H47O24 = 10792457

10792449

10448ppmC45H39O20 = 8992040

899205018453ppm

C36H31O16 = 7191612

7191625

20501ppmC27H23O12 = 5391190

5391201

14852ppmC23H33O10 = 4692074

469208119316ppm

C17H11O5 = 2950606

2950612

0

5

10

15

20

25

30

35

40

45

50

55

60

65

Rela

tive a

bund

ance

22903ppmC18H15O8 = 3590767

3590775

minus37218 ppmC9H5O3 = 1610239

1610233

(a)

100 200 300 400 500 600 700 800 900 1000

mz

100 200 300 400 500 600 700 800 900 1000

mz

100 200 300 400 500 600 700 800 900 1000

mz

100 200 300 400 500 600 700 800 900 1000

mz

0

250

500

750

1000

00

04

08

times104

times104

01234

0

1

2

times105

Ints

Ints

Ints

Ints

MS1

MS2

MS3

MS4

[M-H]minus10749

minus224 (acidic part

minus224 (acidic part

of sagecoumarin)

of sagecoumarin)

minus360 (rosmarinic acid)

4908

7148

5348 minus180 minus162 (caffeide)8769

31074909

5348

minus180 (2-hydroxy-3-(34-dihydroxyphenyl)-propanoide)

3108

35474909

1

(b)

Figure 2 Continued

Evidence-Based Complementary and Alternative Medicine 9

150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900

mz

0

5

10

15

20

25

30

35

40

45

50

55

60

65

Rela

tive a

bund

ance

O

O

O O

O

O

OH OH

OH

OH

OH

OHOH

OH

HO

HO

HO

COOH

OC

1529946

1970447

C9H9O5 = 1970455

minus41775ppm1610232

2552328

C16H31O2 = 2552330

minus04091ppm

3110563

C17H11O6 = 3110561

05243ppm

minus224 (fragment of sagecoumarin)

3912257

4092361

4832730

C29H39O6 = 4832752

minus44931ppm

5350885

C27H19O12 = 5350882

05640ppm

minus132 (tartaric acid)

5770990

C29H21O13 = 5770988

06486ppm

6670947

C31H23O17 = 6670941

09808ppm

minus162 (caffeic acid)

8291267

C40H29O20 = 8291258

11370ppm

(c)

Figure 2 (a) Mass spectra in negative ionization mode and simplified fragmentation scheme of compound 25 (pentameric ester ofcaffeic acid) (b) Mass spectra in negative ionization mode and simplified fragmentation scheme of compound 26 (pentameric structureof sagecoumarin di-2-hydroxy-3-(34-dihydroxyphenyl)-propanoide caffeide) (c) Mass spectra in negative ionization mode and simplifiedfragmentation scheme of compound 36 (pentameric structure of sagecoumarin caftaride)

Tetrameric structures of hydroxycinnamic acids wereidentified in lemon balm recently [34 35] The measure-ment of accurate masses allowed the identification of com-pound 25 which was tentatively identified as pentamericester of caffeic acid (Figure 2(a)) In 25 the double loss of1800421 amu corresponded to fragments with the molecularformula of C

9H8O4 adequate to dehydroxylated 2-hydroxy-

3-(34-dihydroxyphenyl)-propanoic acid The product ionat 7191623119898119911 and its further fragmentation are sim-ilar to those of metabolite 22 as described previously [34 35]Therefore metabolite 25was tentatively assigned as sagerinicacid di-2-hydroxy-3-(34-dihydroxyphenyl)-propanoideNev-ertheless comprehensive studies by nuclear magnetic reso-nance are required to complete elucidation of substitutionpattern for particular components of those pentamers

Sagecoumarins were previously identified in Salvia offic-inalis as caffeic acid trimers [36] Our MS analysis indicatedthe presence of such compounds and their derivatives alsoin M officinalis The pseudomolecular ion of compound33 observed in the negative ionization mode had theaccurate mass of 5350880119898119911 which corresponded to thechemical formula C

27H19O12

adequate for sagecoumarin(according to the Metlin and KNApSAck databases) Note-worthy 26 33 36 and 37 had the same fragmentationpattern of the product ion obtained in the MSMS andMSn in the negative ionization mode The [M-H-1800422]minusion corresponded to the detachment of 2-hydroxy-3-(34-dihydroxyphenyl)-propanoic acid thus 37 was tentatively

assigned as tetrameric sagecoumarin 2-hydroxy-3-(34-dihy-droxyphenyl)-propanoide The [M-H-3600846]minus ion in 26was indicated on rosmarinic acid substitution to 37 Massspectra of MSn in negative ionization of the compoundprovided complementary information to HR-MSMS massspectra indicated on caffeic acid as internal componentof the dimer In addition simultaneous loss of fragments180 amu and 224 amu in MS3 and MS4 indicated that thetwo components cannot be linked (Figure 2(b)) How-ever detailed analysis of substitution pattern of 26 shouldbe done Therefore 26 was tentatively assigned as penta-meric structure of sagecoumarin di-2-hydroxy-3-(34-dihy-droxyphenyl)-propanoide caffeide Rupture of caffeic andtartaric acid moieties from the product ion at 5350885119898119911was observed for compound36 (Figure 2(c))Detection of theaccurate masses of these two detached fragments with Δ lessthan 1 ppm eliminated the possibility of hexose and pentosesubstitution which have the same nominal masses as caffeicand tartaric acid respectively Therefore 36 was tentativelyidentified as another pentameric structure of sagecoumarincaftaride

Metabolite 28 with the accurate masses of 461073119898119911was tentatively identified as luteolin O-glucuronide The [M-H-1760324]minus ion is indicated on loss of structure C

6H8O6

corresponding to glucuronide moiety The product ion at2850405119898119911 was indicated on flavone luteolin The placeof substitution of the carboxylic acid on flavone skeleton isproblematic due to different isomers reported in lemon balm

10 Evidence-Based Complementary and Alternative Medicine

Table 2 Effect ofMelissa officinalis leaf extract (200mgkg po) treatment on sedative activity motor coordination and memory in rats

Group 119899Locomotor activity

[number of impulses5min]Motor coordination

exit time [s]Short-term memorye

ORLong-term memory

latency [s]MC + H

2

O 18 390 plusmn 24 17 plusmn 3 040 plusmn 006 47 plusmn 14MC + SC 18 526 plusmn 48lowast 32 plusmn 5lowast 032 plusmn 005 12 plusmn 3lowast

MO + H2

O 10 231 plusmn 48lowast 32 plusmn 7lowast 043 plusmn 007 169 plusmn 11lowast

MO + SC 9 436 plusmn 60 43 plusmn 7 009 plusmn 011lowast 23 plusmn 6HU + H

2

O 9 515 plusmn 32 15 plusmn 2 037 plusmn 009 158 plusmn 14lowast

HU + SC 8 639 plusmn 71lowast 56 plusmn 3 022 plusmn 006 49 plusmn 18

RA + H2

O 8 406 plusmn 59 21 plusmn 5 045 plusmn 005 58 plusmn 28RA + SC 8 605 plusmn 55 28 plusmn 7 045 plusmn 005 20 plusmn 7Means plusmn SEM119899Number of animalsMC + H

2O control rats

SC scopolamine (05mgkg bw ip)HU huperzine A (05mgkg bw po)RA rosmarinic acid (10mgkg bw po)eExpressed as ratio OR = (119861 minus 119860lowast)(119861 + 119860) for details see Section 3lowastVersus MC + H

2O 119901 lt 005

Versus MC + SC 119901 lt 005

luteolin 31015840-O- and 7-O-glucuronide [33] It is impossible todistinguish both structures by mass spectrometry Only onechromatographic peak corresponding to the [M-H]minus ion at461073119898119911 was observed in our study Since luteolin 31015840-O-glucuronide was assigned as the most abundant flavonoid inlemon balm [33] we assumed that 28 corresponded to thisstructure

412 Flavonoids and Polyphenolic Acids The major com-pound from the 40 chemical compounds identified in hy-droethanolic MO leaf extract established by HPLC was RA(885 g100 g) (Table 1 Figure 1)

Other chemical compoundswith neuromodulatory activ-ities such as lithospermic acid (0042 g100 g) salvianolicacid A (0040 g100 g) salvianolic acid B (0023 g100 g) andcaffeic acid (0087 g100 g) were also documented in litera-ture Moreover the total polyphenolic compounds content ofMO determined with the use of FolinndashCiocalteu assay was3397 calculated as gallic acid The total hydroxycinnamicderivatives content expressed spectrophotometrically as ros-marinic acid was 2115 g100 g

413 Essential Oil Composition Hydroethanolic MO leafextract contained 008 of total essential oil The GCFIDanalysis showed that the extract comprised camphene(004) alfa-pinene (007) beta-pinene (1647) andmyrcene (1951) Moreover according to retention time16 compounds were identified as follows alfa-bisabololborneol carvone chamazulene cineole eugenol gamma-terpineol guaiazulene isopulegol linalool limonene men-thol menthyl acetate pulegone terpine and thymol Thesecompounds are present in the essential oil in trace amountswhich do not allow the quantitative interpretation

42 Cognitive and Behavioral Experiments

421 Locomotor Activity A one-way ANOVA analysisrevealed significant differences in the locomotor activity of

rats expressed as their horizontal spontaneous activity afterMO administration (ANOVA 119865(7 80) = 646 119901 lt 005)(Table 2) Detailed post hoc analysis showed thatMO + H

2O

decreased the locomotor activity of rats by 4031 but HU +H2O did not affect this activity when compared with control

group (MC + H2O) We observed also that RA + H

2O did

not change the locomotor activity of rats Stimulating effectsin the locomotor activity of rats were observed after an acuteSC injection (MC + SC versus MC + H

2O 119901 lt 005) and

this effect was observed in all SC-treated rats when comparedwith the proper non-SC-treated animals On the contrarythese SC-treated animals did not differ in comparison toanimals that received SC only (MO + SC versus MC + SC119901 gt 005 HU + SC versus MC + SC 119901 gt 005 RA + SCversus MC + SC 119901 gt 005)

422 Motor Coordination A one-way ANOVA analysisrevealed significant differences in motor coordination of ratsexpressed as their exit time from the cylinder (119865(5 73) =284 119901 lt 005) (Table 2) Detailed analysis showed that themultiple administration of RA + H

2O and HU + H

2O did

not affect significantly this paradigm when compared withcontrol rats (119901 gt 005) whereas MO treatment led to pro-longed exit time (MO + H

2O versus MC + H

2O 119901 lt 005)

Moreover generally SC-treated animals showed producedprolongation of exit time and the effects were statisticallysignificant not only in control groups (MC + SC versus MC +H2O 119901 lt 005) but also in RA-treated rats (RA + SC versus

MC + SC 119901 lt 005) However the rest of the SC-treatedanimals did not differ in comparison to animals receiving SConly (MO + SC versus MC + SC 119901 gt 005 HU + SC versusMC + SC 119901 gt 005)

423 Long-Term Memory A one-way ANOVA analysisrevealed significant differences in long-term memory afterusing a passive avoidance test (119865(7 77) = 201 119901 lt 005Table 2) It was shown that the strongest effect leading to

Evidence-Based Complementary and Alternative Medicine 11

Table 3 The effect ofMelissa officinalis leaf extract on acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) activities and AChEBuAChE or beta-secretase (BACE1) mRNA expression levels in frontal cortex (FC) or hippocampus (Hipp) of rats

Group119899Enzyme activity

[nmolminmg protein]mRNA expression

[]AChE BuChE ACHE BuChE BACE1

FC Hipp FC Hipp FC Hipp FC Hipp FC HippMC + H

2

O 363 plusmn 49 439 plusmn 73 65 plusmn 11 53 plusmn 8 100 plusmn 12 100 plusmn 11 100 plusmn 18 100 plusmn 11 100 plusmn 16 100 plusmn 8MO + H

2

O 276 plusmn 34 409 plusmn 28 69 plusmn 6 62 plusmn 4 48 plusmn 4lowast 31 plusmn 7lowast 16 plusmn 2lowast 64 plusmn 21amp 36 plusmn 3lowast 50 plusmn 8lowast

HU + H2

O 189 plusmn 15lowast 239 plusmn 15lowast 58 plusmn 6 51 plusmn 4 53 plusmn 13lowast 85 plusmn 5 42 plusmn 9lowast 102 plusmn 11 62 plusmn 6lowast 98 plusmn 4RA + H

2

O 224 plusmn 16lowast 251 plusmn 12lowast 77 plusmn 7 99 plusmn 7lowast 101 plusmn 12 103 plusmn 5 184 plusmn 31lowast 56 plusmn 7lowast 126 plusmn 13amp 98 plusmn 3Means plusmn SEM119899Number of animals 7ndash10Values expressed as a ratio the geneGAPDHMC + H

2O control group

HU huperzine A (05mgkg bw po)RA rosmarinic acid (10mgkg bw po)lowastampVersus MC + H

2O 119901 lt 005 or 119901 lt 007 respectively

an improvement of this paradigm was produced by extractof MO and HU but not RA when compared with controlanimals (MO + H

2O versus MC + H

2O 119901 lt 005 HU +

H2O versus MC + H

2O 119901 lt 005 RA + H

2O versus MC +

H2O 119901 gt 005) However the administration of SC to rats

significantly decreased the latency time of passive avoidancetask (MC + SC versus MC + H

2O 119901 lt 005) After MO or

RA combined treatment with SC no improvement of long-term memory was observed but HU given with SC showedenhancement of this paradigm in rats (HU + SC versusMC + SC 119901 lt 005) Therefore it can be concluded thatadministration of HU overcomes the effect shown by SC only(Table 1)

424 Short-Term Memory The results of the object recogni-tion test showed that an administration of the compoundsor extract did affect the ratsrsquo short-term memory (ANOVA119865(7 78) = 273 119901 lt 005) (Table 2)

Detailed post hoc analysis showed that only SC signifi-cantly decreased the short-term memory in MO-treated rats(MO + SC versusMO + H

2O 119901 lt 005MO + SC versus MC

+ SC 119901 lt 005) whereas the differences between the rest ofthe animals did not reach statistical significance (119901 gt 005)

43 AChE and BuChE Activities in Rat Brain A one-wayANOVA revealed significant differences between groups inthe activity of AChE in both the cortex and the hippocampus(frontal cortex 119865(3 27) = 565 119901 lt 005 hippocampusANOVA 119865(3 29) = 796 119901 lt 005) It was found out thatMOshowed an insignificant inhibition of AChE activity in thefrontal cortex by 24 when compared with control rats (MC+ H2O 119901 lt 006) and in the hippocampus by 7 (Table 3)

whereas HU produced a distinct significant inhibition ofAChE activity in comparison to control group by 48 (119901 lt005) and 47 (119901 lt 005) in the cortex and the hippocampusrespectively Also RA lowered significantly AChE activityboth in the cortex (38) and in the hippocampus (43)Moreover there were not significant differences between thevalues of BuChE activities for MO and HU when compared

with control group in the frontal cortex (ANOVA 119865(3 30) =101 119901 gt 005) whereas in the hippocampus the differencesreached statistical significance (ANOVA 119865(3 27) = 141119901 lt 005) Detailed analysis showed that only RA effect wassignificant and increased BuChE activity in the hippocampuswhen compared with the control rats (119901 lt 005)

44 AChE BuChE and BACE1 mRNA Level Changes in RatBrain A one-way ANOVA analysis revealed significant dif-ferences of AChE mRNA transcription profile in the cortex(ANOVA 119865(3 27) = 857 119901 lt 005) As shown in Table 3the multiple treatment of MO produced in the cortex astatistically significant decrease of AChEmRNA level by 52the administration of HU caused decrease of its level by 44(119901 lt 005) whereas RA did not affect this parameter whencompared to the control

There were significant differences between the relativevalues of BuChE mRNA levels in this region of brain in rats(frontal cortex ANOVA 119865(3 25) = 162 119901 lt 005) TheMOtreatment led to a decrease in the BuChEmRNA level by 84(versus MC + H

2O 119901 lt 005) the prolonged HU admin-

istration resulted in a decrease of the transcript level in thecortex by 58 (versus MC + H

2O 119901 lt 005) but RA

increased this parameter in the cortex by 84 (versus MC +H2O 119901 lt 005)The significant differences ofmRNA transcription level of

AChE mRNA level in the hippocampus (ANOVA 119865(3 31) =212 119901 lt 005) have been observed The detailed analysisshown that in the case of AChE afterMO treatment mRNAlevel significantly decreased by 69 (versus MC + H

2O

119901 lt 005) while the administration of HU resulted in astatistically insignificant decrease of AChE mRNA level by18 when compared with control group Also RA did notchange the level of transcript

In the case of BuChEmRNA expression in the hippocam-pus there were statistically significant differences betweengroups (ANOVA 119865(3 27) = 310 119901 lt 005) Detailed analysisshowed that in theMO + H

2O treated group the expression

lowered by 36 but the difference did not reach strong

12 Evidence-Based Complementary and Alternative Medicine

significance when compared with the control values (119901 lt007) and no change in the expression level of this enzymewas shown after the administration of HU On the contraryRA produced an increase of BuChE mRNA expression in thehippocampus by 44 (versus MC + H

2O 119901 lt 005)

Further analysis showed the significant differences inBACE1 mRNA expression in both brain regions of rats (inthe cortex and the hippocampus) (frontal cortex ANOVA119865(3 27) = 163 119901 lt 005 hippocampus ANOVA 119865(3 27) =133 119901 gt 005) It was observed that MO produced astatistically significant decrease of the BACE1 expression levelby 64 in the cortex (versus MC + H

2O 119901 lt 005) and by

50 in the hippocampus (versus MC + H2O 119901 lt 005) For

comparison HU treatment led to a decrease in the mRNAexpression level by 38 in the cortex (versus MC + H

2O

119901 lt 005) but not in the hippocampus On the contrary RAproduced an increase of this transcript in the cortex by 26but the effect did not reach a strong statistical significance(versus MC + H

2O 119901 lt 007) whereas in the hippocampus

there was no difference between RA and control group

5 Discussion

Cognitive and Behavioral Experiments The present studyinvestigated the influence of subchronic (28-fold) adminis-tration of standardized 50 EtOH extract ofMelisa officinalisleaf extract (MO) (200mgkg po containing 177mgkg ofRA) on SC-induced impairment of short-term and long-term memory in rats The results were compared with theactivity of cholinesterases (AChE and BuChE) as well aswith AChE BuChE and BACE1 gene expression levels in thecortex and hippocampus of the rat brain So far little evidenceis yet available as regards mechanisms of MO leaf extractaction that are potentially relevant to cognitive function ofrats after per os administration MO is traditionally used intreating neurological disorders through its anti-AChE [18]and antiagitation properties [37] Moreover Wake et al [38]and Kennedy et al [15] showed thatMO extract has nicotinicreceptor activity and that it can displace [3H]-(N)-nicotinefrom nicotinic receptors in homogenates of human cerebralcortex tissue and they suggested that these mechanisms canexplain activity of MO extract in amnesia model RecentlySoodi et al [18] observed that intraperitoneal injections ofMO extract (200mgkg) in rats could significantly enhancelearning and memory processes in animals since the extractsignificantly ameliorates SC-induced learning deficit in Mor-ris water maze test On the contrary in higher dose itcan be observed that MO extract (400mgkg) could notreverse SC-induced memory impairment [18] In our studyadministration of MO extract at a dose of 200mgkg (po)showed the effect leading to improvement of long-termmemory in a passive avoidance test in rats However afterMO combined treatment with SCMO did not overcome theimpairment shown by SC (Table 2) It should be emphasizedthat it is not clear whether the effect shown in our study byMO in non-SC-treated rats is specific since theMO treatmentproduced significant lowering of locomotor activity of ratstherefore the sedative profile ofMO cannot be excluded

On the other hand Ryu et al [39] observed that agitationand aggression are highly prevalent in patientswith dementiaAccording to Gitlin et al [40] nonpharmacological inter-ventions are recommended as first-line therapy Althoughantipsychotics have shown benefit for Alzheimerrsquos disease-related psychosis their use is associated with several seriousadverse effects [40]Thus it seems that the use ofMO extractcan provide dual benefits both in aspect of inhibition ofagitation and in improving the memory of patients withAlzheimerrsquos disease

Furthermore in our study RA in the dose of 10mgkgbw (po) did not affect either short- or long-term memoryalthough RA lowered significantly AChE activity both inthe cortex and in the hippocampus Moreover we observedthat the repeated administration of RA in non-scopolamine-treated rats did not produce any changes of locomotoractivity similarly to our previous study [23]

It is possible that other chemical compounds can influ-ence the memory in rats by synergic interactions in plantextract According to our calculations caffeic acid (0174mgin a single dose of extract administered to animals per kgbw) lithospermic acid (0084mgkg) salvianolic acid A(008mgkg) and salvianolic acid B (0046mgkg) may beresponsible for observed pharmacological effects On theother hand results from few studies showed that salvianolicacids do not cross the blood brain barrier (BBB) [41 42]and also lithospermic acid does not efficiently cross the BBB[43] For this reason the interpretation of our results is morecomplicated

Firstly Xu et al [44] demonstrated that Sal A is ametabol-ically unstable compound that would undergo rapid methy-lationmetabolism catalyzed by catechol O-methyltransferasein vivo into four major methylated metabolites of Sal A(3-O-methyl 31015840-O-methyl 3310158401015840-O-dimethyl and 31015840310158401015840-O-dimethyl salvianolic acid A) These generated O-methylatedmetabolites may be largely responsible for its in vivo phar-macological effects Although there are no available recentstudies on the ability of these compounds to pass the BBBsuch possibility should not be excluded

Secondly several studies showed central pharmacolog-ical effects in animals after per os administration of Sal B[45 46] Sal A [47] and caffeic acid [48] It was provedfor example that salvianolic acid B (10mgkg po) sig-nificantly rescued the A12057325ndash35 peptide-induced decreaseof choline acetyltransferase and brain-derived neurotrophicfactor protein levels in an amyloid 120573 (A120573) peptide-inducedAlzheimerrsquos disease mouse model [49] It also significantlyreversed (10mgkg po) the cognitive impairments inducedby scopolamine (1mgkg ip) or A120573 (25ndash35) (10 nmol5 120583Licv) injection inmice [45] Previous studies [46 47] showedalso that both Sal A and Sal B are able to improve theimpaired memory function induced by cerebral ischemia-reperfusion in miceThe stimulation of neurogenesis processin both subgranular zone (SGZ) and subventricular zone(SVZ) after brain ischemia and also alleviation neural cellsloss and improved motor function recovery after brainischemia in rats after the Sal B administration were alsoobserved by Zhong et al [50] Moreover the exposure to SalB can maintain the proliferation of neural stemprogenitor

Evidence-Based Complementary and Alternative Medicine 13

cells (NSPCs) after cerebral ischemia and improve cognitivepostischemic impairment after stroke in rats using Morriswater maze test therefore authors concluded that Sal B mayact as a potential drug in treatment of brain injury or neu-rodegenerative disease [51] Additionally the improvement ofmotor function after cerebral ischemia in rats after salvianolicacid B administrationwas also demonstrated [52]The clue topass through the BBB Sal B provided also results of Li et alindicating its protective effect on BBB in rats after cerebralischemia-reperfusion by inhibiting the MAPK pathway [53]In addition to this it was shown that lithospermic acid andsalvianolic acids exerted neuroprotective activity in variousexperimental models Lithospermic acid significantly attenu-ates neurotoxicity in vitro and in vivo induced by 1-methyl-4-phenylpyridin (MPP(+)) by blocking neuronal apoptotic andneuroinflammatory pathways [54] Salvianolic acid B inhib-ited amyloid beta-protein aggregation and fibril formation aswell as directly inhibiting the cellular toxicity of amyloid beta-protein in PC12 cells [55] and significantly reduced its cyto-toxic effects on human neuroblastoma SH-SY5Y cells [56]

For an explanation of our results observations of Pin-heiro Fernandes et al [57] may be very helpful whichshowed that caffeic acid nonflavanoid catecholic compoundwhose derivatives are occurring in MO extract improvedthe working spatial and long-term aversive memory deficitsinduced by focal cerebral ischemia in mice Anwar et al [48]showed also that caffeic acid (100mgkg) improved the step-down latencies in the inhibitory avoidance in rats Tsai et al[58] showed that caffeic acid is a potent neuroprotective agentin brain of 1-methyl-4-phenyl-1236-tetrahydropyridine(MPTP) treatedmiceMoreover caffeic acid improves A12057325ndash35-induced memory deficits and cognitive impairment inmice [59] In another study it was also shown that this com-pound has a significant protective effect on global cerebralischemia-reperfusion injury in rats [60] For instance 124 plusmn18mg100 g of caffeic acid was detected in the brain of micewith a diet containing 2 caffeic acid for 4 weeks [58] Thusthere is progressive evidence that this compound passesthrough the BBB and has a central pharmacological activity

Also results by Yoo et al are also worth noting [61] whichshowed also in a ratmodel of scopolamine-induced amnesiathat administration of luteolin (a common flavonoid frommany plants including M officinalis) at dose of 10mgkgcaused the increase in the brain-derived neurotrophic factor(BDNF) acetylcholine and the decrease in lipid peroxida-tion Liu et al [62] observed that chronic treatment withluteolin (50 and 100mgkg) improved neuronal injury andcognitive performance by attenuating oxidative stress andcholinesterase activity in streptozotocin-induced diabetes inrats In our study ethanolic extract of MO administered torats significantly improved long-term memory after using apassive avoidance test but afterMO combined treatmentwithSC no improvement of this paradigmwas observed (Table 2)

Thirdly available pharmacokinetic studies were carriedout for the pure compounds (Sal A and B) and the extractof the roots of Salvia miltiorrhiza [42 63] but there are notavailable studies of bioavailability of these compounds afteradministration of Melissa officinalis leaf extract containingother compounds as compared with the extract of Salvia

miltiorrhiza Moreover there is a lack of data about howscopolamine may influence the penetration of the salvianolicacids (and other compounds) across the BBB Hence thereis a need to study the pharmacokinetic parameters of thesecompounds in the group of animals treatedwith scopolaminein comparison with control group

AChEandBuChEActivities in Rat Brain To date several stud-ies have focused on explaining themechanismof action of theMS extract and its active compounds Soodi et al [18] showedthat treatment of animals withMO extract (400mgkg) priorto scopolamine injection could ameliorate scopolamine-induced enhancement in AChE activity This dose of MOextract inhibited the AChE activity in the hippocampus ofrats (519 versus 1004 in normal saline group 119901 lt 005914 in group treated with scopolamine +MO versus 1281in scopolamine group 119901 lt 005) In another study Anwaret al [48] showed that 50 and 100mgkg of caffeic aciddecreased in vivo the AChE activity in the cerebral cortexand striatum and increased the activity of this enzyme inthe cerebellum hippocampus hypothalamus pons lympho-cytes and muscles when compared to the control group(119901 lt 005) Our study showed that ethanolic extract of MOproduced an insignificant and slight inhibition of AChE andBuChE activity in the frontal cortex and in the hippocampus(especially) However it was observed previously [23] thatRA inhibited the AChE activity in the rats frontal cortex andhippocampusMoreover we showed that RA possess a strongstimulatory effect on BuChE in the hippocampus

AChE BuChE and BACE1mRNA Level Changes in Rat BrainSo far no results have been published of studies concerningthe in vivo assessment of changes in AChE BuChE andBACE1 gene expression profile in different brain regionsunder the influence of MO extracts or their key bioactivemetabolites Such studies focused overwhelmingly on theanalysis of their in vitro activities to a lesser extent in animalexperiments

In our study in the frontal cortex we have observedthe strongest inhibition of both AChE and BuCHE mRNAtranscription under the influence ofM officinalis extract andhuperzine A (Table 3) However a more significant differencein the level of transcript was seen in the frontal cortex inparticular in the case of BuCHE mRNA of experimentalrats (a decrease by 516 and 44 for MO and HU versuscontrol resp)The strong inhibition of transcription ofAChEmRNAwas also observed in the hippocampus ofMO-treatedrats (a decrease by 69) The BuChE mRNA transcriptionin animals receiving MO was lowered in a moderate way(decrease by 36)HuperzineA alone has not caused changesthat were observed in the group ofMO (Table 2) Our findingsmostly correlate with the observed changes in activities ofAChE and BuChE in different groups of animals In the caseof BuChE slightly different results between its activity andexpression profile were especially seen in the frontal cortexand hippocampus of animals receivingMO (Table 3)

To date there is a lack of published results of studiesmaking an attempt to clarify the potential differences in thetranscriptional profile and activity of AChE and BuChE

14 Evidence-Based Complementary and Alternative Medicine

under the influence of MO and its bioactive metabolites Itcannot be excluded that the key for the observed differencesin the level of AChE activities and mRNA level can becaused by changes in the activity of AChE in other regionsof the brain not analyzed in this study such as substantianigra cerebellum globus pallidus and hypothalamus whereit exerts nonenzymatic neuromodulatory functions affect-ing neurite outgrowth and synaptogenesis modulating theactivity of other proteins regional cerebral blood flow andother functions [64] But it is difficult to clearly explain whydiminishing of AChE and BuChEmRNAs did not always cor-relate with the lowering of activity of these enzymes althoughit has been recently noted that AChE activity was notparalleled by an increase in mRNA levels [65] The authorsexplained this fact by stating that AChE levels are regulatedat transcriptional posttranscriptional and posttranslationallevels leading to complex expression patterns which canbe modulated by physiological and pathological conditionsHowever these mechanisms are not fully understood andfurther studies are needed in this field

The cause of observed different degree of inhibition ofactivity and transcription status of studied genes especiallyof BuChE (and AChE) may lie in a so-called ldquonegative feed-backrdquo consisting of a complicated transcriptiontranslationregulation protein-protein interactionsmodifications and ametabolic network together forming a system that allows thecell to respond sensibly to the multiple signal molecules thatexist in its environment [66]

Because of that we propose that the reason for differencesbetween AChE and BuChE activities in the cortex and hip-pocampus under the MO may be due to the fact of insuf-ficiency of applied dose and the duration of the experi-ment affecting the transcriptional tissue-specific cellularmachinery regulating BuChE transcription without affectingits activity Moreover the administration of higher doses ofMO and extended period of time could lead to sufficientinhibition of its activity

There is a need to conduct further studies to determinethe molecular degree of dependence between changes inAChE and BuChE activities and activities of potential keyfactors regulating expression of these genes in the frontalcortex and hippocampus under the influence of extracts ofMO and active metabolites Another study determining theeffectiveness of their actions on the cholinergic system inexperimental animal models of memory impairment withparticular emphasis on scopolamine including the determi-nation of changes at the molecular level should be thereforecarried out

Alterations in BACE1 protein level have been proved inpostmortem brain tissue from individuals with AD withincreases decreases and also no change reported [1 67 68]An example of confirmation of these findings at the mRNAlevel is results of study by Coulson et al [69] Based onconducted studies some authors suggested that increasedBACE1mRNA transcription in remaining neuronal cells maycontribute to the increased BACE1 protein levels and activityfound in brain regions affected by AD [70]

Results of many studies concerning the elevated level ofBACE1 mRNA and protein in Alzheimerrsquos disease provide

direct and compelling reasons to develop therapies directed atBACE1 inhibition thus reducing120573-amyloid and its associatedtoxicities [67 68]

In our study we have observed that BACEmRNA expres-sion statistically significantly decreased after MO adminis-tration in frontal cortex and hippocampus These resultssuggest that the MO extract may act to inhibit BACE1mRNA level given the fact that the percentage of inhibitionof the expression (64 in the frontal cortex 50 in thehippocampus) is higher than that in the case of HU (38in the cortex and the lack of changes in the hippocampus)A careful analysis of the literature data shows that there areno studies which analyzed the impact of MO extract on theexpression level of BACE1 in Alzheimerrsquos disease

Furthermore a literature analysis does not indicatealready published results conducted by other teams attempt-ing to assess the impact of RA on the transcriptional activityof AChE BuChE and BACE1 Although several studies(already mentioned and others [70 71]) highlighted theRA and other caffeic acid derivatives capability of acetyl-cholinesterases inhibition none of these studies does nottouch the question of the molecular basis of their impacton the transcriptional machinery that regulates in vivo theexpression of studied genes Hence in our opinion obtainedby our team results they are one of the first of this typeand in general correlate with the results of our previousstudy [23] In this case there is no clear evidence explainingdifferent responses at the transcriptional level under theinfluence of RA especially in the case of BuChE encodinggene in the hippocampus (Table 2) It is possible that theobserved differentiation of BuChE transcriptional activitybetween the frontal cortex and the hippocampus may bedue to the differences of butyrylcholinesterase localizationand substrate affinity [72] Since in our experiment we havecarried out a quantitative analysis of AChE BuChE andBACE1 transcripts in brain homogenates of tested animalsrather than in individual isolated cell fractions therefore theobtained results constitute an overall ldquopicturerdquo of both studiedgenes transcriptional changes occurring in the brain areas ofstudied animals under the influence of the RA and the wholeplant extract as well

6 Conclusion

The subchronic administration ofMO led to an improvementof long-term memory of rats however the mechanisms ofMO action are probably more complicated since its role asa modulator of beta-secretase activity (due to inhibition ofBACE1 mRNA expression in frontal cortex) should be takeninto consideration

It should be noted that we have studied a crude extractfrom leaf of Melissa officinalis not a single pure chemicalcompound This plant extract is a complex mixture and itsaction may be a result of the summation of activities ofseveral components (synergismadditive action of caffeic acidwith salvianolic acids rosmarinic acid and others) In thecase of extract from leaves ofMelissa officinalis it is possiblethat interactions occur between the 40 chemical compoundsidentified by HPLC system

Evidence-Based Complementary and Alternative Medicine 15

Taken together it seems that the MO activity representsa possible option as complementary interventions to relievethe symptoms of mild dementia

Competing Interests

The authors declare that they have no competing interests

Acknowledgments

This work was supported by the Ministry of Science andHigher EducationWarsaw Poland from educational sources(2008ndash2011 Grant no N 405417836)

References

[1] S L Cole and R Vassar ldquoThe Alzheimerrsquos disease 120573-secretaseenzyme BACE1rdquo Molecular Neurodegeneration vol 2 no 1article 22 2007

[2] LACraigN SHong andR JMcDonald ldquoRevisiting the cho-linergic hypothesis in the development of Alzheimerrsquos diseaserdquoNeuroscience amp Biobehavioral Reviews vol 35 no 6 pp 1397ndash1409 2011

[3] N G M Gomes M G Campos J M C Orfao and C A FRibeiro ldquoPlants with neurobiological activity as potential tar-gets for drug discoveryrdquo Progress in Neuro-Psychopharmacologyamp Biological Psychiatry vol 33 no 8 pp 1372ndash1389 2009

[4] WHO WHO Monographs on Selected Medicinal PlantsmdashVolume 2 WHO Geneva Switzerland 2004 httpappswhointmedicinedocsendJs4927e18htmlJs4927e18

[5] M Bayat A A Tameh M H Ghahremani et al ldquoNeuropro-tective properties of Melissa officinalis after hypoxic-ischemicinjury both in vitro and in vivordquo DARU Journal of Pharmaceu-tical Sciences vol 20 article 42 2012

[6] J P Kamdem A Adeniran A A Boligon et al ldquoAntioxidantactivity genotoxicity and cytotoxicity evaluation of lemon balm(Melissa officinalis L) ethanolic extract Its potential role inneuroprotectionrdquo Industrial Crops and Products vol 51 pp 26ndash34 2013

[7] J-T Lin Y-C Chen Y-C Lee C-W R Hou F-L Chen andD-J Yang ldquoAntioxidant anti-proliferative and cyclooxygenase-2 inhibitory activities of ethanolic extracts from lemon balm(Melissa officinalis L) leavesrdquo LWTmdashFood Science and Technol-ogy vol 49 no 1 pp 1ndash7 2012

[8] G Guginski A P Luiz M D Silva et al ldquoMechanisms involvedin the antinociception caused by ethanolic extract obtainedfrom the leaves of Melissa officinalis (lemon balm) in micerdquoPharmacology Biochemistry amp Behavior vol 93 no 1 pp 10ndash162009

[9] N Perry G Court N Bidet J Court and E Perry ldquoEuropeanherbs with cholinergic activities potential in dementia therapyrdquoInternational Journal of Geriatric Psychiatry vol 11 no 12 pp1063ndash1069 1996

[10] E K Perry A T Pickering W W Wang P J Houghtonand N S L Perry ldquoMedicinal plants and Alzheimerrsquos diseasefrom ethnobotany to phytotherapyrdquo Journal of Pharmacy andPharmacology vol 51 no 5 pp 527ndash534 1999

[11] M-J R Howes N S L Perry and P J Houghton ldquoPlants withtraditional uses and activities relevant to the management ofAlzheimerrsquos disease and other cognitive disordersrdquo Phytother-apy Research vol 17 no 1 pp 1ndash18 2003

[12] I Orhan and M Aslan ldquoAppraisal of scopolamine-inducedantiamnesic effect in mice and in vitro antiacetylcholinesteraseand antioxidant activities of some traditionally used Lamiaceaeplantsrdquo Journal of Ethnopharmacology vol 122 no 2 pp 327ndash332 2009

[13] S Akhondzadeh M Noroozian M Mohammadi S OhadiniaA H Jamshidi and M Khani ldquoMelissa officinalis extract inthe treatment of patients with mild to moderate Alzheimerrsquosdisease a double blind randomised placebo controlled trialrdquoJournal of Neurology Neurosurgery and Psychiatry vol 74 no 7pp 863ndash866 2003

[14] D O Kennedy A B Scholey N T J Tildesley E K Perry andK AWesnes ldquoModulation ofmood and cognitive performancefollowing acute administration of Melissa officinalis (lemonbalm)rdquo Pharmacology Biochemistry amp Behavior vol 72 no 4pp 953ndash964 2002

[15] D O Kennedy G Wake S Savelev et al ldquoModulation of moodand cognitive performance following acute administration ofsingle doses of Melissa officinalis (Lemon balm) with humanCNS nicotinic and muscarinic receptor-binding propertiesrdquoNeuropsychopharmacology vol 28 no 10 pp 1871ndash1881 2003

[16] D O Kennedy and E L Wightman ldquoHerbal extracts and phy-tochemicals plant secondarymetabolites and the enhancementof human brain functionrdquoAdvances inNutrition vol 2 no 1 pp32ndash50 2011

[17] R P Pereira A A Boligon A S Appel et al ldquoChemical com-position antioxidant and anticholinesterase activity of Melissaofficinalisrdquo Industrial Crops and Products vol 53 pp 34ndash452014

[18] M Soodi N Naghdi H Hajimehdipoor S Choopani andE Sahraei ldquoMemory-improving activity of Melissa officinalisextract in naıve and scopolamine-treated ratsrdquo Research inPharmaceutical Sciences vol 9 no 2 pp 107ndash114 2014

[19] K Dastmalchi V Ollilainen P Lackman et al ldquoAcetyl-cholinesterase inhibitory guided fractionation of Melissa offic-inalis Lrdquo Bioorganic amp Medicinal Chemistry vol 17 no 2 pp867ndash871 2009

[20] W Chaiyana and S Okonogi ldquoInhibition of cholinesterase byessential oil from food plantrdquo Phytomedicine vol 19 no 8-9 pp836ndash839 2012

[21] K Dastmalchi H J D Dorman P P Oinonen Y Darwis ILaakso and R Hiltunen ldquoChemical composition and in vitroantioxidative activity of a lemon balm (Melissa officinalis L)extractrdquo LWTmdashFood Science and Technology vol 41 no 3 pp391ndash400 2008

[22] P Pereira D Tysca P Oliveira L F D S Brum J N Picadaand P Ardenghi ldquoNeurobehavioral and genotoxic aspects ofrosmarinic acidrdquo Pharmacological Research vol 52 no 3 pp199ndash203 2005

[23] M Ozarowski P L Mikolajczak A Bogacz et al ldquoRosmarinusofficinalis L leaf extract improves memory impairment andaffects acetylcholinesterase and butyrylcholinesterase activitiesin rat brainrdquo Fitoterapia vol 91 pp 261ndash271 2013

[24] G P Kumar and F Khanum ldquoNeuroprotective potential of phy-tochemicalsrdquo Pharmacognosy Reviews vol 6 no 12 pp 81ndash902012

[25] M Stobiecki A Staszkow A Piasecka P M Garcia-LopezF Zamora-Natera and P Kachlicki ldquoLC-MSMS profiling offlavonoid conjugates in wild mexican lupine Lupinus reflexusrdquoJournal of Natural Products vol 73 no 7 pp 1254ndash1260 2010

[26] A Wojakowska A Piasecka P M Garcıa-Lopez et al ldquoStruc-tural analysis and profiling of phenolic secondarymetabolites of

16 Evidence-Based Complementary and Alternative Medicine

Mexican lupine species using LC-MS techniquesrdquo Phytochem-istry vol 92 pp 71ndash86 2013

[27] A Gryszczynska B Opala Z Lowicki et al ldquoBioactive com-pounds determination in the callus and hydroalcoholic extractsfrom Salvia miltiorrhiza and Salvia przewalskiimdashpreliminarystudy on their anti-alcoholic activity effectsrdquo PhytochemistryLetters vol 11 pp 399ndash403 2015

[28] K Slinkart and V L Singleton ldquoTotal phenol analysis automa-tion and comparison with manual methodrdquo American Journalof Enology and Viticulture vol 28 no 1 pp 49ndash55 1977

[29] P J R Boissier J Tardy and J C Diverres ldquoUne nouvellemethode simple pour explorer lrsquoaction lsquotranquillisantersquo le testde la chemineerdquo Medicina Experimentalis vol 3 no 1 pp 81ndash84 1960

[30] R Ader J A Weijnen and P Moleman ldquoRetention of a passiveavoidance response as a function of the intensity and durationof electric shockrdquo Psychonomic Science vol 26 no 3 pp 125ndash128 1972

[31] E M Blalock K-C Chen K Sharrow et al ldquoGene microar-rays in hippocampal aging statistical profiling identifies novelprocesses correlated with cognitive impairmentrdquo Journal ofNeuroscience vol 23 no 9 pp 3807ndash3819 2003

[32] K Isomae S Morimoto H Hasegawa K Morita and JKamei ldquoEffects of T-82 a novel acetylcholinesterase inhibitoron impaired learning and memory in passive avoidance task inratsrdquo European Journal of Pharmacology vol 465 no 1-2 pp97ndash103 2003

[33] J Patora and B Klimek ldquoFlavonoids from lemon balm (Melissaofficinalis L Lamiaceae)rdquo Acta Poloniae Pharmaceutica-DrugResearch vol 59 no 2 pp 139ndash143 2002

[34] L Barros M Duenas M I Dias M J Sousa C Santos-Buelgaand I C F R Ferreira ldquoPhenolic profiles of cultivated invitro cultured and commercial samples of Melissa officinalis Linfusionsrdquo Food Chemistry vol 136 no 1 pp 1ndash8 2013

[35] T L Miron M Herrero and E Ibanez ldquoEnrichment of antiox-idant compounds from lemon balm (Melissa officinalis) bypressurized liquid extraction and enzyme-assisted extractionrdquoJournal of Chromatography A vol 1288 pp 1ndash9 2013

[36] Y Lu L Y Foo and H Wong ldquoSagecoumarin a novel caffeicacid trimer from Salvia officinalisrdquo Phytochemistry vol 52 no6 pp 1149ndash1152 1999

[37] S Abuhamdah LHuangM S J Elliott et al ldquoPharmacologicalprofile of an essential oil derived from Melissa officinalis withanti-agitation properties focus on ligand-gated channelsrdquo Jour-nal of Pharmacy and Pharmacology vol 60 no 3 pp 377ndash3842008

[38] G Wake J Court A Pickering R Lewis R Wilkins andE Perry ldquoCNS acetylcholine receptor activity in Europeanmedicinal plants traditionally used to improve failing memoryrdquoJournal of Ethnopharmacology vol 69 no 2 pp 105ndash114 2000

[39] S-H Ryu C Katona B Rive and G Livingston ldquoPersistenceof and changes in neuropsychiatric symptoms in Alzheimerdisease over 6months the LASER-AD studyrdquoAmerican Journalof Geriatric Psychiatry vol 13 no 11 pp 976ndash983 2005

[40] L N Gitlin H C Kales and C G Lyketsos ldquoNonpharma-cologic management of behavioral symptoms in dementiardquoJournal of the AmericanMedical Association vol 308 no 19 pp2020ndash2029 2012

[41] A Konczol J Muller E Foldes et al ldquoApplicability of a blood-brain barrier specific artificial membrane permeability assay atthe early stage of natural product-based CNS drug discoveryrdquoJournal of Natural Products vol 76 no 4 pp 655ndash663 2013

[42] Y-J Zhang L Wu Q-L Zhang J Li F-X Yin and Y YuanldquoPharmacokinetics of phenolic compounds of Danshen extractin rat blood and brain by microdialysis samplingrdquo Journal ofEthnopharmacology vol 136 no 1 pp 129ndash136 2011

[43] X Li C Yu Y Lu et al ldquoPharmacokinetics tissue distributionmetabolism and excretion of depside salts from Salvia miltior-rhiza in ratsrdquo Drug Metabolism and Disposition vol 35 no 2pp 234ndash239 2007

[44] H Xu Y Li X Che H Tian H Fan and K Liu ldquoMetabolismof salvianolic acid a and antioxidant activities of its methylatedmetabolitesrdquo Drug Metabolism and Disposition vol 42 no 2pp 274ndash281 2014

[45] D H Kim S J Park J M Kim et al ldquoCognitive dysfunctionsinduced by a cholinergic blockade and A120573 25-35 peptide areattenuated by salvianolic acid Brdquo Neuropharmacology vol 61no 8 pp 1432ndash1440 2011

[46] G-H Du Y Qiu and J-T Zhang ldquoSalvianolic acid B protectsthe memory functions against transient cerebral ischemia inmicerdquo Journal of Asian Natural Products Research vol 2 no 2pp 145ndash152 2000

[47] G Du and J Zhang ldquoProtective effects of salvianolic acid Aagainst impairment of memory induced by cerebral ischemia-reperfusion in micerdquo Chinese Medical Journal vol 110 no 1 pp65ndash68 1997

[48] J Anwar R M Spanevello G Thome et al ldquoEffects of caffeicacid on behavioral parameters and on the activity of acetyl-cholinesterase in different tissues from adult ratsrdquo Pharmacol-ogy Biochemistry and Behavior vol 103 no 2 pp 386ndash394 2012

[49] YW Lee D H Kim S J Jeon et al ldquoNeuroprotective effects ofsalvianolic acid B on anA12057325-35 peptide-inducedmousemodelof Alzheimerrsquos diseaserdquo European Journal of Pharmacology vol704 no 1ndash3 pp 70ndash77 2013

[50] J Zhong M-K Tang Y Zhang Q-P Xu and J-T ZhangldquoEffect of salvianolic acid B on neural cells damage and neu-rogenesis after brain ischemia-reperfusion in ratsrdquo Yao Xue XueBao vol 42 no 7 pp 716ndash721 2007

[51] P Zhuang Y Zhang G Cui et al ldquoDirect stimulation of adultneural stemprogenitor cells in vitro and neurogenesis in vivo bysalvianolic acid Brdquo PLoS ONE vol 7 no 4 Article ID e356362012

[52] M TangW Feng Y Zhang J Zhong and J Zhang ldquoSalvianolicacid B improves motor function after cerebral ischemia in ratsrdquoBehavioural Pharmacology vol 17 no 5-6 pp 493ndash498 2006

[53] Q Li L-P Han Z-H Li J-T Zhang and M-K Tang ldquoSal-vianolic acid B alleviate the disruption of blood-brain barrierin rats after cerebral ischemia-reperfusion by inhibiting MAPKpathwayrdquo Yao Xue Xue Bao vol 45 no 12 pp 1485ndash1490 2010

[54] Y-L LinH-J Tsay T-H Lai T-T Tzeng andY-J Shiao ldquoLith-ospermic acid attenuates 1-methyl-4-phenylpyridine-inducedneurotoxicity by blocking neuronal apoptotic and neuroinflam-matory pathwaysrdquo Journal of Biomedical Science vol 22 article37 2015

[55] M-K Tang and J-T Zhang ldquoSalvianolic acid B inhibits fibrilformation and neurotoxicity of amyloid beta-protein in vitrordquoActa Pharmacologica Sinica vol 22 no 4 pp 380ndash384 2001

[56] S S K Durairajan Q Yuan L Xie et al ldquoSalvianolic acid Binhibits A120573 fibril formation and disaggregates preformed fibrilsand protects against A120573-induced cytotoxictyrdquo NeurochemistryInternational vol 52 no 4-5 pp 741ndash750 2008

[57] FD Pinheiro Fernandes A P FonteneleMenezes J C de SousaNeves et al ldquoCaffeic acid protects mice from memory deficits

Evidence-Based Complementary and Alternative Medicine 17

induced by focal cerebral ischemiardquo Behavioural Pharmacologyvol 25 no 7 pp 637ndash647 2014

[58] S-J Tsai C-Y Chao andM-C Yin ldquoPreventive and therapeu-tic effects of caffeic acid against inflammatory injury in striatumof MPTP-treated micerdquo European Journal of Pharmacology vol670 no 2-3 pp 441ndash447 2011

[59] J H Kim QWang J M Choi S Lee and E J Cho ldquoProtectiverole of caffeic acid in an 119860120573

25minus35

-induced Alzheimerrsquos diseasemodelrdquo Nutrition Research and Practice vol 9 no 5 pp 480ndash488 2015

[60] G Liang B Shi W Luo and J Yang ldquoThe protective effect ofcaffeic acid on global cerebral ischemia-reperfusion injury inratsrdquo Behavioral and Brain Functions vol 11 no 1 article 182015

[61] D Y Yoo J H ChoiW Kim et al ldquoEffects of luteolin on spatialmemory cell proliferation and neuroblast differentiation in thehippocampal dentate gyrus in a scopolamine-induced amnesiamodelrdquo Neurological Research vol 35 no 8 pp 813ndash820 2013

[62] Y Liu X Tian L Gou L Sun X Ling and X Yin ldquoLuteolinattenuates diabetes-associated cognitive decline in ratsrdquo BrainResearch Bulletin vol 94 pp 23ndash29 2013

[63] S-M Liu Z-H Yang andX-B Sun ldquoSimultaneous determina-tion of six Salvia miltiorrhiza gradients in rat plasma and brainby LC-MSMSrdquo Zhongguo Zhongyao Zazhi vol 39 no 9 pp1704ndash1708 2014

[64] M A Papandreou A Dimakopoulou Z I Linardaki et alldquoEffect of a polyphenol-rich wild blueberry extract on cognitiveperformance of mice brain antioxidant markers and acetyl-cholinesterase activityrdquo Behavioural Brain Research vol 198 no2 pp 352ndash358 2009

[65] M-S Garcıa-Ayllon O Cauli M-X Silveyra et al ldquoBraincholinergic impairment in liver failurerdquo Brain vol 131 no 11pp 2946ndash2956 2008

[66] G K Ferreira M Carvalho-Silva C L Goncalves et al ldquoL-Tyrosine administration increases acetylcholinesterase activityin ratsrdquo Neurochemistry International vol 61 no 8 pp 1370ndash1374 2012

[67] R Vassar P-H Kuhn C Haass et al ldquoFunction therapeuticpotential and cell biology of BACE proteases current status andfuture prospectsrdquo Journal of Neurochemistry vol 130 no 1 pp4ndash28 2014

[68] R Vassar ldquoThe 120573-secretase BACE a prime drug target forAlzheimerrsquos diseaserdquo Journal of Molecular Neuroscience vol 17no 2 pp 157ndash170 2001

[69] D T R Coulson N Beyer J G Quinn et al ldquoBACE1 mRNAexpression in Alzheimerrsquos disease postmortem brain tissuerdquoJournal of Alzheimerrsquos Disease vol 22 no 4 pp 1111ndash1122 2010

[70] S Vladimir-Knezevic B Blazekovic M Kindl J Vladic A DLower-Nedza and A H Brantner ldquoAcetylcholinesterase inhib-itory antioxidant and phytochemical properties of selectedmedicinal plants of the lamiaceae familyrdquoMolecules vol 19 no1 pp 767ndash782 2014

[71] F Marcelo C Dias A Martins et al ldquoMolecular recognitionof rosmarinic acid from Salvia sclareoides extracts by ace-tylcholinesterase a new binding site detected by NMR spec-troscopyrdquo ChemistrymdashA European Journal vol 19 no 21 pp6641ndash6649 2013

[72] G Johnson and S W Moore ldquoWhy has butyrylcholinesterasebeen retained Structural and functional diversification in aduplicated generdquoNeurochemistry International vol 61 no 5 pp783ndash797 2012

[73] R J Grayer M R Eckert N C Veitch et al ldquoThe chemo-taxonomic significance of two bioactive caffeic acid estersnepetoidins A and B in the Lamiaceaerdquo Phytochemistry vol 64no 2 pp 519ndash528 2003

[74] A M D El-Mousallamy U W Hawas and S A M HusseinldquoTeucrol a decarboxyrosmarinic acid and its 41015840-O-triglycosideteucroside from Teucrium pilosumrdquo Phytochemistry vol 55 no8 pp 927ndash931 2000

[75] I Fecka and S Turek ldquoDetermination of water-soluble polyphe-nolic compounds in commercial herbal teas from Lamiaceaepeppermint melissa and sagerdquo Journal of Agricultural and FoodChemistry vol 55 no 26 pp 10908ndash10917 2007

[76] L W Sumner A Amberg D Barrett et al ldquoProposed min-imum reporting standards for chemical analysis WorkingGroup (CAWG) Metabolomics Standards Initiative (MSI)rdquoMetabolomics vol 3 pp 211ndash221 2007

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

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PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

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Computational and Mathematical Methods in Medicine

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Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Evidence-Based Complementary and Alternative Medicine 7

Table1Con

tinued

No

RT [min]

Metabolite

identifi

catio

nCh

emical

form

ula

Exactm

asso

f[M-H

]minusΔpp

mFragmentatio

nin

120582max[nm]

CID

aIdentifi

catio

nlevelb

Reference

Measured

Calculated

Negativeion

mod

e(ESIminus)

Positiveion

mod

e(ESI+)

24555

Salviano

licacid

B2-hydroxy-3-(34-

dihydroxyphenyl)-

prop

anoide

C 45

H37

O20

89719

8971884

16873

897717519359

161

330

3[35]

2556

Sagerin

icacid

di-2-hydroxy-3-(34-

dihydroxyphenyl)-

prop

anoide

C 54

H47

O24

1079244

910792457minus07911

1079897719539

359295

288330

3[35]

26592

Sagecoum

arin

di-2-hydroxy-3-(34-

dihydroxyphenyl)-

prop

anoide

caffeide

C 54

H43

O24

10752156

10752150

0559

1077897717537

409359339277

322

3[35]

27598

Rosm

arinicacid

sulphated

Iisomer

C 18

H15

O11

S4390

354390

341

20214

439359341163

Masked

2[34]

28613

Luteolin

31015840-O

-glucuronide

C 21

H17

O12

4610

734610

7216

34461285

463287

269340

170474237

2[33]

29632

Salviano

licacid

AC 26

H21

O10

493115

493114

11011

493359295179

298327

5281793

[34]

30656

Sagerin

icacid

sulphated

C 36

H31

O19

S7991

196

7991

186

0954

799719619519

359161

325

3[35]

31671

Lithosperm

icacid

C 27

H21

O12

537104

537104

809698

537493359161

292329

6441498

2[35]

3268

Rosm

arinicacid

sulphated

IIiso

mer

C 18

H15

O11

S4390

344390

341

02837

439359341163

Masked

2[34]

33689

Sagecoum

arin

C 27

H19

O12

535088

5350882

01204

535311267177

Masked

2[36]

3470

3Salviano

licacid

LIisomer

C 36

H29

O16

717146

7171461

02697

717519359

284329

2[35]

3571

Salviano

licacid

Lhydroxycaffeide

C 45

H35

O20

895173

8951727

06282

895519359161

Masked

3[35]

3674

Sagecoum

arin

cafta

ride

C 40

H29

O20

8291

268291

258

06953

829667535355

311

Masked

3[36]

37751

Sagecoum

arin

2-hydroxy-

3-(34-dihydroxyph

enyl)-

prop

anoide

C 36

H27

O16

715131

7151305

08176

715535311267

319

3[34]

3877

1Unk

nown

C 36

H57

O14

S745348

7453475

044

02281326

4[35]

3978

5Methylrosmarinate

C 19

H17

O8

373093

3730929

01765

373359161

284323

6479915

2[75]

40869

Salviano

licacid

Ccaffeoylhydroxycaffeide

C 44

H33

O18

8491

688491

672

08615

849687491359

327255

286318

3[35]

a CID

identifier

fora

chem

icalstr

ucture

intheP

ubCh

emCom

poun

ddatabase

b Metabolite

identifi

catio

nlevelaccording

toMetabolom

icsS

tand

ards

Initiativer

ecom

mendatio

n[76]

stdidentificatio

non

theb

asisof

stand

ardcompo

undfragmentatio

nnd

not

detected

8 Evidence-Based Complementary and Alternative Medicine

100 200 300 400 500 600 700 800 900 1000 1100 1200

mz

70

75

80

85

90

95

100

minus198 (2-hydroxy-3-(34-dihydroxyphenyl)

-porpanoide)

minus180 (caffeide)

minus180 (2-hydroxy-3-(34-dihydroxyphenyl)

-propanoide)

minus180 (2-hydroxy-3-(34-dihydroxyphenyl)

-propanoide)

minus180 (2-hydroxy-3-(34-dihydroxyphenyl)

-propanoide)

1350438

2191749

HO

HO

HO HO

OH

OH

OH

OH OH OH

OHOHOHOHHOOC

COOH

COOH COOH COOH

24612ppmC36H55O37 = 10792422

minus07911ppmC54H47O24 = 10792457

10792449

10448ppmC45H39O20 = 8992040

899205018453ppm

C36H31O16 = 7191612

7191625

20501ppmC27H23O12 = 5391190

5391201

14852ppmC23H33O10 = 4692074

469208119316ppm

C17H11O5 = 2950606

2950612

0

5

10

15

20

25

30

35

40

45

50

55

60

65

Rela

tive a

bund

ance

22903ppmC18H15O8 = 3590767

3590775

minus37218 ppmC9H5O3 = 1610239

1610233

(a)

100 200 300 400 500 600 700 800 900 1000

mz

100 200 300 400 500 600 700 800 900 1000

mz

100 200 300 400 500 600 700 800 900 1000

mz

100 200 300 400 500 600 700 800 900 1000

mz

0

250

500

750

1000

00

04

08

times104

times104

01234

0

1

2

times105

Ints

Ints

Ints

Ints

MS1

MS2

MS3

MS4

[M-H]minus10749

minus224 (acidic part

minus224 (acidic part

of sagecoumarin)

of sagecoumarin)

minus360 (rosmarinic acid)

4908

7148

5348 minus180 minus162 (caffeide)8769

31074909

5348

minus180 (2-hydroxy-3-(34-dihydroxyphenyl)-propanoide)

3108

35474909

1

(b)

Figure 2 Continued

Evidence-Based Complementary and Alternative Medicine 9

150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900

mz

0

5

10

15

20

25

30

35

40

45

50

55

60

65

Rela

tive a

bund

ance

O

O

O O

O

O

OH OH

OH

OH

OH

OHOH

OH

HO

HO

HO

COOH

OC

1529946

1970447

C9H9O5 = 1970455

minus41775ppm1610232

2552328

C16H31O2 = 2552330

minus04091ppm

3110563

C17H11O6 = 3110561

05243ppm

minus224 (fragment of sagecoumarin)

3912257

4092361

4832730

C29H39O6 = 4832752

minus44931ppm

5350885

C27H19O12 = 5350882

05640ppm

minus132 (tartaric acid)

5770990

C29H21O13 = 5770988

06486ppm

6670947

C31H23O17 = 6670941

09808ppm

minus162 (caffeic acid)

8291267

C40H29O20 = 8291258

11370ppm

(c)

Figure 2 (a) Mass spectra in negative ionization mode and simplified fragmentation scheme of compound 25 (pentameric ester ofcaffeic acid) (b) Mass spectra in negative ionization mode and simplified fragmentation scheme of compound 26 (pentameric structureof sagecoumarin di-2-hydroxy-3-(34-dihydroxyphenyl)-propanoide caffeide) (c) Mass spectra in negative ionization mode and simplifiedfragmentation scheme of compound 36 (pentameric structure of sagecoumarin caftaride)

Tetrameric structures of hydroxycinnamic acids wereidentified in lemon balm recently [34 35] The measure-ment of accurate masses allowed the identification of com-pound 25 which was tentatively identified as pentamericester of caffeic acid (Figure 2(a)) In 25 the double loss of1800421 amu corresponded to fragments with the molecularformula of C

9H8O4 adequate to dehydroxylated 2-hydroxy-

3-(34-dihydroxyphenyl)-propanoic acid The product ionat 7191623119898119911 and its further fragmentation are sim-ilar to those of metabolite 22 as described previously [34 35]Therefore metabolite 25was tentatively assigned as sagerinicacid di-2-hydroxy-3-(34-dihydroxyphenyl)-propanoideNev-ertheless comprehensive studies by nuclear magnetic reso-nance are required to complete elucidation of substitutionpattern for particular components of those pentamers

Sagecoumarins were previously identified in Salvia offic-inalis as caffeic acid trimers [36] Our MS analysis indicatedthe presence of such compounds and their derivatives alsoin M officinalis The pseudomolecular ion of compound33 observed in the negative ionization mode had theaccurate mass of 5350880119898119911 which corresponded to thechemical formula C

27H19O12

adequate for sagecoumarin(according to the Metlin and KNApSAck databases) Note-worthy 26 33 36 and 37 had the same fragmentationpattern of the product ion obtained in the MSMS andMSn in the negative ionization mode The [M-H-1800422]minusion corresponded to the detachment of 2-hydroxy-3-(34-dihydroxyphenyl)-propanoic acid thus 37 was tentatively

assigned as tetrameric sagecoumarin 2-hydroxy-3-(34-dihy-droxyphenyl)-propanoide The [M-H-3600846]minus ion in 26was indicated on rosmarinic acid substitution to 37 Massspectra of MSn in negative ionization of the compoundprovided complementary information to HR-MSMS massspectra indicated on caffeic acid as internal componentof the dimer In addition simultaneous loss of fragments180 amu and 224 amu in MS3 and MS4 indicated that thetwo components cannot be linked (Figure 2(b)) How-ever detailed analysis of substitution pattern of 26 shouldbe done Therefore 26 was tentatively assigned as penta-meric structure of sagecoumarin di-2-hydroxy-3-(34-dihy-droxyphenyl)-propanoide caffeide Rupture of caffeic andtartaric acid moieties from the product ion at 5350885119898119911was observed for compound36 (Figure 2(c))Detection of theaccurate masses of these two detached fragments with Δ lessthan 1 ppm eliminated the possibility of hexose and pentosesubstitution which have the same nominal masses as caffeicand tartaric acid respectively Therefore 36 was tentativelyidentified as another pentameric structure of sagecoumarincaftaride

Metabolite 28 with the accurate masses of 461073119898119911was tentatively identified as luteolin O-glucuronide The [M-H-1760324]minus ion is indicated on loss of structure C

6H8O6

corresponding to glucuronide moiety The product ion at2850405119898119911 was indicated on flavone luteolin The placeof substitution of the carboxylic acid on flavone skeleton isproblematic due to different isomers reported in lemon balm

10 Evidence-Based Complementary and Alternative Medicine

Table 2 Effect ofMelissa officinalis leaf extract (200mgkg po) treatment on sedative activity motor coordination and memory in rats

Group 119899Locomotor activity

[number of impulses5min]Motor coordination

exit time [s]Short-term memorye

ORLong-term memory

latency [s]MC + H

2

O 18 390 plusmn 24 17 plusmn 3 040 plusmn 006 47 plusmn 14MC + SC 18 526 plusmn 48lowast 32 plusmn 5lowast 032 plusmn 005 12 plusmn 3lowast

MO + H2

O 10 231 plusmn 48lowast 32 plusmn 7lowast 043 plusmn 007 169 plusmn 11lowast

MO + SC 9 436 plusmn 60 43 plusmn 7 009 plusmn 011lowast 23 plusmn 6HU + H

2

O 9 515 plusmn 32 15 plusmn 2 037 plusmn 009 158 plusmn 14lowast

HU + SC 8 639 plusmn 71lowast 56 plusmn 3 022 plusmn 006 49 plusmn 18

RA + H2

O 8 406 plusmn 59 21 plusmn 5 045 plusmn 005 58 plusmn 28RA + SC 8 605 plusmn 55 28 plusmn 7 045 plusmn 005 20 plusmn 7Means plusmn SEM119899Number of animalsMC + H

2O control rats

SC scopolamine (05mgkg bw ip)HU huperzine A (05mgkg bw po)RA rosmarinic acid (10mgkg bw po)eExpressed as ratio OR = (119861 minus 119860lowast)(119861 + 119860) for details see Section 3lowastVersus MC + H

2O 119901 lt 005

Versus MC + SC 119901 lt 005

luteolin 31015840-O- and 7-O-glucuronide [33] It is impossible todistinguish both structures by mass spectrometry Only onechromatographic peak corresponding to the [M-H]minus ion at461073119898119911 was observed in our study Since luteolin 31015840-O-glucuronide was assigned as the most abundant flavonoid inlemon balm [33] we assumed that 28 corresponded to thisstructure

412 Flavonoids and Polyphenolic Acids The major com-pound from the 40 chemical compounds identified in hy-droethanolic MO leaf extract established by HPLC was RA(885 g100 g) (Table 1 Figure 1)

Other chemical compoundswith neuromodulatory activ-ities such as lithospermic acid (0042 g100 g) salvianolicacid A (0040 g100 g) salvianolic acid B (0023 g100 g) andcaffeic acid (0087 g100 g) were also documented in litera-ture Moreover the total polyphenolic compounds content ofMO determined with the use of FolinndashCiocalteu assay was3397 calculated as gallic acid The total hydroxycinnamicderivatives content expressed spectrophotometrically as ros-marinic acid was 2115 g100 g

413 Essential Oil Composition Hydroethanolic MO leafextract contained 008 of total essential oil The GCFIDanalysis showed that the extract comprised camphene(004) alfa-pinene (007) beta-pinene (1647) andmyrcene (1951) Moreover according to retention time16 compounds were identified as follows alfa-bisabololborneol carvone chamazulene cineole eugenol gamma-terpineol guaiazulene isopulegol linalool limonene men-thol menthyl acetate pulegone terpine and thymol Thesecompounds are present in the essential oil in trace amountswhich do not allow the quantitative interpretation

42 Cognitive and Behavioral Experiments

421 Locomotor Activity A one-way ANOVA analysisrevealed significant differences in the locomotor activity of

rats expressed as their horizontal spontaneous activity afterMO administration (ANOVA 119865(7 80) = 646 119901 lt 005)(Table 2) Detailed post hoc analysis showed thatMO + H

2O

decreased the locomotor activity of rats by 4031 but HU +H2O did not affect this activity when compared with control

group (MC + H2O) We observed also that RA + H

2O did

not change the locomotor activity of rats Stimulating effectsin the locomotor activity of rats were observed after an acuteSC injection (MC + SC versus MC + H

2O 119901 lt 005) and

this effect was observed in all SC-treated rats when comparedwith the proper non-SC-treated animals On the contrarythese SC-treated animals did not differ in comparison toanimals that received SC only (MO + SC versus MC + SC119901 gt 005 HU + SC versus MC + SC 119901 gt 005 RA + SCversus MC + SC 119901 gt 005)

422 Motor Coordination A one-way ANOVA analysisrevealed significant differences in motor coordination of ratsexpressed as their exit time from the cylinder (119865(5 73) =284 119901 lt 005) (Table 2) Detailed analysis showed that themultiple administration of RA + H

2O and HU + H

2O did

not affect significantly this paradigm when compared withcontrol rats (119901 gt 005) whereas MO treatment led to pro-longed exit time (MO + H

2O versus MC + H

2O 119901 lt 005)

Moreover generally SC-treated animals showed producedprolongation of exit time and the effects were statisticallysignificant not only in control groups (MC + SC versus MC +H2O 119901 lt 005) but also in RA-treated rats (RA + SC versus

MC + SC 119901 lt 005) However the rest of the SC-treatedanimals did not differ in comparison to animals receiving SConly (MO + SC versus MC + SC 119901 gt 005 HU + SC versusMC + SC 119901 gt 005)

423 Long-Term Memory A one-way ANOVA analysisrevealed significant differences in long-term memory afterusing a passive avoidance test (119865(7 77) = 201 119901 lt 005Table 2) It was shown that the strongest effect leading to

Evidence-Based Complementary and Alternative Medicine 11

Table 3 The effect ofMelissa officinalis leaf extract on acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) activities and AChEBuAChE or beta-secretase (BACE1) mRNA expression levels in frontal cortex (FC) or hippocampus (Hipp) of rats

Group119899Enzyme activity

[nmolminmg protein]mRNA expression

[]AChE BuChE ACHE BuChE BACE1

FC Hipp FC Hipp FC Hipp FC Hipp FC HippMC + H

2

O 363 plusmn 49 439 plusmn 73 65 plusmn 11 53 plusmn 8 100 plusmn 12 100 plusmn 11 100 plusmn 18 100 plusmn 11 100 plusmn 16 100 plusmn 8MO + H

2

O 276 plusmn 34 409 plusmn 28 69 plusmn 6 62 plusmn 4 48 plusmn 4lowast 31 plusmn 7lowast 16 plusmn 2lowast 64 plusmn 21amp 36 plusmn 3lowast 50 plusmn 8lowast

HU + H2

O 189 plusmn 15lowast 239 plusmn 15lowast 58 plusmn 6 51 plusmn 4 53 plusmn 13lowast 85 plusmn 5 42 plusmn 9lowast 102 plusmn 11 62 plusmn 6lowast 98 plusmn 4RA + H

2

O 224 plusmn 16lowast 251 plusmn 12lowast 77 plusmn 7 99 plusmn 7lowast 101 plusmn 12 103 plusmn 5 184 plusmn 31lowast 56 plusmn 7lowast 126 plusmn 13amp 98 plusmn 3Means plusmn SEM119899Number of animals 7ndash10Values expressed as a ratio the geneGAPDHMC + H

2O control group

HU huperzine A (05mgkg bw po)RA rosmarinic acid (10mgkg bw po)lowastampVersus MC + H

2O 119901 lt 005 or 119901 lt 007 respectively

an improvement of this paradigm was produced by extractof MO and HU but not RA when compared with controlanimals (MO + H

2O versus MC + H

2O 119901 lt 005 HU +

H2O versus MC + H

2O 119901 lt 005 RA + H

2O versus MC +

H2O 119901 gt 005) However the administration of SC to rats

significantly decreased the latency time of passive avoidancetask (MC + SC versus MC + H

2O 119901 lt 005) After MO or

RA combined treatment with SC no improvement of long-term memory was observed but HU given with SC showedenhancement of this paradigm in rats (HU + SC versusMC + SC 119901 lt 005) Therefore it can be concluded thatadministration of HU overcomes the effect shown by SC only(Table 1)

424 Short-Term Memory The results of the object recogni-tion test showed that an administration of the compoundsor extract did affect the ratsrsquo short-term memory (ANOVA119865(7 78) = 273 119901 lt 005) (Table 2)

Detailed post hoc analysis showed that only SC signifi-cantly decreased the short-term memory in MO-treated rats(MO + SC versusMO + H

2O 119901 lt 005MO + SC versus MC

+ SC 119901 lt 005) whereas the differences between the rest ofthe animals did not reach statistical significance (119901 gt 005)

43 AChE and BuChE Activities in Rat Brain A one-wayANOVA revealed significant differences between groups inthe activity of AChE in both the cortex and the hippocampus(frontal cortex 119865(3 27) = 565 119901 lt 005 hippocampusANOVA 119865(3 29) = 796 119901 lt 005) It was found out thatMOshowed an insignificant inhibition of AChE activity in thefrontal cortex by 24 when compared with control rats (MC+ H2O 119901 lt 006) and in the hippocampus by 7 (Table 3)

whereas HU produced a distinct significant inhibition ofAChE activity in comparison to control group by 48 (119901 lt005) and 47 (119901 lt 005) in the cortex and the hippocampusrespectively Also RA lowered significantly AChE activityboth in the cortex (38) and in the hippocampus (43)Moreover there were not significant differences between thevalues of BuChE activities for MO and HU when compared

with control group in the frontal cortex (ANOVA 119865(3 30) =101 119901 gt 005) whereas in the hippocampus the differencesreached statistical significance (ANOVA 119865(3 27) = 141119901 lt 005) Detailed analysis showed that only RA effect wassignificant and increased BuChE activity in the hippocampuswhen compared with the control rats (119901 lt 005)

44 AChE BuChE and BACE1 mRNA Level Changes in RatBrain A one-way ANOVA analysis revealed significant dif-ferences of AChE mRNA transcription profile in the cortex(ANOVA 119865(3 27) = 857 119901 lt 005) As shown in Table 3the multiple treatment of MO produced in the cortex astatistically significant decrease of AChEmRNA level by 52the administration of HU caused decrease of its level by 44(119901 lt 005) whereas RA did not affect this parameter whencompared to the control

There were significant differences between the relativevalues of BuChE mRNA levels in this region of brain in rats(frontal cortex ANOVA 119865(3 25) = 162 119901 lt 005) TheMOtreatment led to a decrease in the BuChEmRNA level by 84(versus MC + H

2O 119901 lt 005) the prolonged HU admin-

istration resulted in a decrease of the transcript level in thecortex by 58 (versus MC + H

2O 119901 lt 005) but RA

increased this parameter in the cortex by 84 (versus MC +H2O 119901 lt 005)The significant differences ofmRNA transcription level of

AChE mRNA level in the hippocampus (ANOVA 119865(3 31) =212 119901 lt 005) have been observed The detailed analysisshown that in the case of AChE afterMO treatment mRNAlevel significantly decreased by 69 (versus MC + H

2O

119901 lt 005) while the administration of HU resulted in astatistically insignificant decrease of AChE mRNA level by18 when compared with control group Also RA did notchange the level of transcript

In the case of BuChEmRNA expression in the hippocam-pus there were statistically significant differences betweengroups (ANOVA 119865(3 27) = 310 119901 lt 005) Detailed analysisshowed that in theMO + H

2O treated group the expression

lowered by 36 but the difference did not reach strong

12 Evidence-Based Complementary and Alternative Medicine

significance when compared with the control values (119901 lt007) and no change in the expression level of this enzymewas shown after the administration of HU On the contraryRA produced an increase of BuChE mRNA expression in thehippocampus by 44 (versus MC + H

2O 119901 lt 005)

Further analysis showed the significant differences inBACE1 mRNA expression in both brain regions of rats (inthe cortex and the hippocampus) (frontal cortex ANOVA119865(3 27) = 163 119901 lt 005 hippocampus ANOVA 119865(3 27) =133 119901 gt 005) It was observed that MO produced astatistically significant decrease of the BACE1 expression levelby 64 in the cortex (versus MC + H

2O 119901 lt 005) and by

50 in the hippocampus (versus MC + H2O 119901 lt 005) For

comparison HU treatment led to a decrease in the mRNAexpression level by 38 in the cortex (versus MC + H

2O

119901 lt 005) but not in the hippocampus On the contrary RAproduced an increase of this transcript in the cortex by 26but the effect did not reach a strong statistical significance(versus MC + H

2O 119901 lt 007) whereas in the hippocampus

there was no difference between RA and control group

5 Discussion

Cognitive and Behavioral Experiments The present studyinvestigated the influence of subchronic (28-fold) adminis-tration of standardized 50 EtOH extract ofMelisa officinalisleaf extract (MO) (200mgkg po containing 177mgkg ofRA) on SC-induced impairment of short-term and long-term memory in rats The results were compared with theactivity of cholinesterases (AChE and BuChE) as well aswith AChE BuChE and BACE1 gene expression levels in thecortex and hippocampus of the rat brain So far little evidenceis yet available as regards mechanisms of MO leaf extractaction that are potentially relevant to cognitive function ofrats after per os administration MO is traditionally used intreating neurological disorders through its anti-AChE [18]and antiagitation properties [37] Moreover Wake et al [38]and Kennedy et al [15] showed thatMO extract has nicotinicreceptor activity and that it can displace [3H]-(N)-nicotinefrom nicotinic receptors in homogenates of human cerebralcortex tissue and they suggested that these mechanisms canexplain activity of MO extract in amnesia model RecentlySoodi et al [18] observed that intraperitoneal injections ofMO extract (200mgkg) in rats could significantly enhancelearning and memory processes in animals since the extractsignificantly ameliorates SC-induced learning deficit in Mor-ris water maze test On the contrary in higher dose itcan be observed that MO extract (400mgkg) could notreverse SC-induced memory impairment [18] In our studyadministration of MO extract at a dose of 200mgkg (po)showed the effect leading to improvement of long-termmemory in a passive avoidance test in rats However afterMO combined treatment with SCMO did not overcome theimpairment shown by SC (Table 2) It should be emphasizedthat it is not clear whether the effect shown in our study byMO in non-SC-treated rats is specific since theMO treatmentproduced significant lowering of locomotor activity of ratstherefore the sedative profile ofMO cannot be excluded

On the other hand Ryu et al [39] observed that agitationand aggression are highly prevalent in patientswith dementiaAccording to Gitlin et al [40] nonpharmacological inter-ventions are recommended as first-line therapy Althoughantipsychotics have shown benefit for Alzheimerrsquos disease-related psychosis their use is associated with several seriousadverse effects [40]Thus it seems that the use ofMO extractcan provide dual benefits both in aspect of inhibition ofagitation and in improving the memory of patients withAlzheimerrsquos disease

Furthermore in our study RA in the dose of 10mgkgbw (po) did not affect either short- or long-term memoryalthough RA lowered significantly AChE activity both inthe cortex and in the hippocampus Moreover we observedthat the repeated administration of RA in non-scopolamine-treated rats did not produce any changes of locomotoractivity similarly to our previous study [23]

It is possible that other chemical compounds can influ-ence the memory in rats by synergic interactions in plantextract According to our calculations caffeic acid (0174mgin a single dose of extract administered to animals per kgbw) lithospermic acid (0084mgkg) salvianolic acid A(008mgkg) and salvianolic acid B (0046mgkg) may beresponsible for observed pharmacological effects On theother hand results from few studies showed that salvianolicacids do not cross the blood brain barrier (BBB) [41 42]and also lithospermic acid does not efficiently cross the BBB[43] For this reason the interpretation of our results is morecomplicated

Firstly Xu et al [44] demonstrated that Sal A is ametabol-ically unstable compound that would undergo rapid methy-lationmetabolism catalyzed by catechol O-methyltransferasein vivo into four major methylated metabolites of Sal A(3-O-methyl 31015840-O-methyl 3310158401015840-O-dimethyl and 31015840310158401015840-O-dimethyl salvianolic acid A) These generated O-methylatedmetabolites may be largely responsible for its in vivo phar-macological effects Although there are no available recentstudies on the ability of these compounds to pass the BBBsuch possibility should not be excluded

Secondly several studies showed central pharmacolog-ical effects in animals after per os administration of Sal B[45 46] Sal A [47] and caffeic acid [48] It was provedfor example that salvianolic acid B (10mgkg po) sig-nificantly rescued the A12057325ndash35 peptide-induced decreaseof choline acetyltransferase and brain-derived neurotrophicfactor protein levels in an amyloid 120573 (A120573) peptide-inducedAlzheimerrsquos disease mouse model [49] It also significantlyreversed (10mgkg po) the cognitive impairments inducedby scopolamine (1mgkg ip) or A120573 (25ndash35) (10 nmol5 120583Licv) injection inmice [45] Previous studies [46 47] showedalso that both Sal A and Sal B are able to improve theimpaired memory function induced by cerebral ischemia-reperfusion in miceThe stimulation of neurogenesis processin both subgranular zone (SGZ) and subventricular zone(SVZ) after brain ischemia and also alleviation neural cellsloss and improved motor function recovery after brainischemia in rats after the Sal B administration were alsoobserved by Zhong et al [50] Moreover the exposure to SalB can maintain the proliferation of neural stemprogenitor

Evidence-Based Complementary and Alternative Medicine 13

cells (NSPCs) after cerebral ischemia and improve cognitivepostischemic impairment after stroke in rats using Morriswater maze test therefore authors concluded that Sal B mayact as a potential drug in treatment of brain injury or neu-rodegenerative disease [51] Additionally the improvement ofmotor function after cerebral ischemia in rats after salvianolicacid B administrationwas also demonstrated [52]The clue topass through the BBB Sal B provided also results of Li et alindicating its protective effect on BBB in rats after cerebralischemia-reperfusion by inhibiting the MAPK pathway [53]In addition to this it was shown that lithospermic acid andsalvianolic acids exerted neuroprotective activity in variousexperimental models Lithospermic acid significantly attenu-ates neurotoxicity in vitro and in vivo induced by 1-methyl-4-phenylpyridin (MPP(+)) by blocking neuronal apoptotic andneuroinflammatory pathways [54] Salvianolic acid B inhib-ited amyloid beta-protein aggregation and fibril formation aswell as directly inhibiting the cellular toxicity of amyloid beta-protein in PC12 cells [55] and significantly reduced its cyto-toxic effects on human neuroblastoma SH-SY5Y cells [56]

For an explanation of our results observations of Pin-heiro Fernandes et al [57] may be very helpful whichshowed that caffeic acid nonflavanoid catecholic compoundwhose derivatives are occurring in MO extract improvedthe working spatial and long-term aversive memory deficitsinduced by focal cerebral ischemia in mice Anwar et al [48]showed also that caffeic acid (100mgkg) improved the step-down latencies in the inhibitory avoidance in rats Tsai et al[58] showed that caffeic acid is a potent neuroprotective agentin brain of 1-methyl-4-phenyl-1236-tetrahydropyridine(MPTP) treatedmiceMoreover caffeic acid improves A12057325ndash35-induced memory deficits and cognitive impairment inmice [59] In another study it was also shown that this com-pound has a significant protective effect on global cerebralischemia-reperfusion injury in rats [60] For instance 124 plusmn18mg100 g of caffeic acid was detected in the brain of micewith a diet containing 2 caffeic acid for 4 weeks [58] Thusthere is progressive evidence that this compound passesthrough the BBB and has a central pharmacological activity

Also results by Yoo et al are also worth noting [61] whichshowed also in a ratmodel of scopolamine-induced amnesiathat administration of luteolin (a common flavonoid frommany plants including M officinalis) at dose of 10mgkgcaused the increase in the brain-derived neurotrophic factor(BDNF) acetylcholine and the decrease in lipid peroxida-tion Liu et al [62] observed that chronic treatment withluteolin (50 and 100mgkg) improved neuronal injury andcognitive performance by attenuating oxidative stress andcholinesterase activity in streptozotocin-induced diabetes inrats In our study ethanolic extract of MO administered torats significantly improved long-term memory after using apassive avoidance test but afterMO combined treatmentwithSC no improvement of this paradigmwas observed (Table 2)

Thirdly available pharmacokinetic studies were carriedout for the pure compounds (Sal A and B) and the extractof the roots of Salvia miltiorrhiza [42 63] but there are notavailable studies of bioavailability of these compounds afteradministration of Melissa officinalis leaf extract containingother compounds as compared with the extract of Salvia

miltiorrhiza Moreover there is a lack of data about howscopolamine may influence the penetration of the salvianolicacids (and other compounds) across the BBB Hence thereis a need to study the pharmacokinetic parameters of thesecompounds in the group of animals treatedwith scopolaminein comparison with control group

AChEandBuChEActivities in Rat Brain To date several stud-ies have focused on explaining themechanismof action of theMS extract and its active compounds Soodi et al [18] showedthat treatment of animals withMO extract (400mgkg) priorto scopolamine injection could ameliorate scopolamine-induced enhancement in AChE activity This dose of MOextract inhibited the AChE activity in the hippocampus ofrats (519 versus 1004 in normal saline group 119901 lt 005914 in group treated with scopolamine +MO versus 1281in scopolamine group 119901 lt 005) In another study Anwaret al [48] showed that 50 and 100mgkg of caffeic aciddecreased in vivo the AChE activity in the cerebral cortexand striatum and increased the activity of this enzyme inthe cerebellum hippocampus hypothalamus pons lympho-cytes and muscles when compared to the control group(119901 lt 005) Our study showed that ethanolic extract of MOproduced an insignificant and slight inhibition of AChE andBuChE activity in the frontal cortex and in the hippocampus(especially) However it was observed previously [23] thatRA inhibited the AChE activity in the rats frontal cortex andhippocampusMoreover we showed that RA possess a strongstimulatory effect on BuChE in the hippocampus

AChE BuChE and BACE1mRNA Level Changes in Rat BrainSo far no results have been published of studies concerningthe in vivo assessment of changes in AChE BuChE andBACE1 gene expression profile in different brain regionsunder the influence of MO extracts or their key bioactivemetabolites Such studies focused overwhelmingly on theanalysis of their in vitro activities to a lesser extent in animalexperiments

In our study in the frontal cortex we have observedthe strongest inhibition of both AChE and BuCHE mRNAtranscription under the influence ofM officinalis extract andhuperzine A (Table 3) However a more significant differencein the level of transcript was seen in the frontal cortex inparticular in the case of BuCHE mRNA of experimentalrats (a decrease by 516 and 44 for MO and HU versuscontrol resp)The strong inhibition of transcription ofAChEmRNAwas also observed in the hippocampus ofMO-treatedrats (a decrease by 69) The BuChE mRNA transcriptionin animals receiving MO was lowered in a moderate way(decrease by 36)HuperzineA alone has not caused changesthat were observed in the group ofMO (Table 2) Our findingsmostly correlate with the observed changes in activities ofAChE and BuChE in different groups of animals In the caseof BuChE slightly different results between its activity andexpression profile were especially seen in the frontal cortexand hippocampus of animals receivingMO (Table 3)

To date there is a lack of published results of studiesmaking an attempt to clarify the potential differences in thetranscriptional profile and activity of AChE and BuChE

14 Evidence-Based Complementary and Alternative Medicine

under the influence of MO and its bioactive metabolites Itcannot be excluded that the key for the observed differencesin the level of AChE activities and mRNA level can becaused by changes in the activity of AChE in other regionsof the brain not analyzed in this study such as substantianigra cerebellum globus pallidus and hypothalamus whereit exerts nonenzymatic neuromodulatory functions affect-ing neurite outgrowth and synaptogenesis modulating theactivity of other proteins regional cerebral blood flow andother functions [64] But it is difficult to clearly explain whydiminishing of AChE and BuChEmRNAs did not always cor-relate with the lowering of activity of these enzymes althoughit has been recently noted that AChE activity was notparalleled by an increase in mRNA levels [65] The authorsexplained this fact by stating that AChE levels are regulatedat transcriptional posttranscriptional and posttranslationallevels leading to complex expression patterns which canbe modulated by physiological and pathological conditionsHowever these mechanisms are not fully understood andfurther studies are needed in this field

The cause of observed different degree of inhibition ofactivity and transcription status of studied genes especiallyof BuChE (and AChE) may lie in a so-called ldquonegative feed-backrdquo consisting of a complicated transcriptiontranslationregulation protein-protein interactionsmodifications and ametabolic network together forming a system that allows thecell to respond sensibly to the multiple signal molecules thatexist in its environment [66]

Because of that we propose that the reason for differencesbetween AChE and BuChE activities in the cortex and hip-pocampus under the MO may be due to the fact of insuf-ficiency of applied dose and the duration of the experi-ment affecting the transcriptional tissue-specific cellularmachinery regulating BuChE transcription without affectingits activity Moreover the administration of higher doses ofMO and extended period of time could lead to sufficientinhibition of its activity

There is a need to conduct further studies to determinethe molecular degree of dependence between changes inAChE and BuChE activities and activities of potential keyfactors regulating expression of these genes in the frontalcortex and hippocampus under the influence of extracts ofMO and active metabolites Another study determining theeffectiveness of their actions on the cholinergic system inexperimental animal models of memory impairment withparticular emphasis on scopolamine including the determi-nation of changes at the molecular level should be thereforecarried out

Alterations in BACE1 protein level have been proved inpostmortem brain tissue from individuals with AD withincreases decreases and also no change reported [1 67 68]An example of confirmation of these findings at the mRNAlevel is results of study by Coulson et al [69] Based onconducted studies some authors suggested that increasedBACE1mRNA transcription in remaining neuronal cells maycontribute to the increased BACE1 protein levels and activityfound in brain regions affected by AD [70]

Results of many studies concerning the elevated level ofBACE1 mRNA and protein in Alzheimerrsquos disease provide

direct and compelling reasons to develop therapies directed atBACE1 inhibition thus reducing120573-amyloid and its associatedtoxicities [67 68]

In our study we have observed that BACEmRNA expres-sion statistically significantly decreased after MO adminis-tration in frontal cortex and hippocampus These resultssuggest that the MO extract may act to inhibit BACE1mRNA level given the fact that the percentage of inhibitionof the expression (64 in the frontal cortex 50 in thehippocampus) is higher than that in the case of HU (38in the cortex and the lack of changes in the hippocampus)A careful analysis of the literature data shows that there areno studies which analyzed the impact of MO extract on theexpression level of BACE1 in Alzheimerrsquos disease

Furthermore a literature analysis does not indicatealready published results conducted by other teams attempt-ing to assess the impact of RA on the transcriptional activityof AChE BuChE and BACE1 Although several studies(already mentioned and others [70 71]) highlighted theRA and other caffeic acid derivatives capability of acetyl-cholinesterases inhibition none of these studies does nottouch the question of the molecular basis of their impacton the transcriptional machinery that regulates in vivo theexpression of studied genes Hence in our opinion obtainedby our team results they are one of the first of this typeand in general correlate with the results of our previousstudy [23] In this case there is no clear evidence explainingdifferent responses at the transcriptional level under theinfluence of RA especially in the case of BuChE encodinggene in the hippocampus (Table 2) It is possible that theobserved differentiation of BuChE transcriptional activitybetween the frontal cortex and the hippocampus may bedue to the differences of butyrylcholinesterase localizationand substrate affinity [72] Since in our experiment we havecarried out a quantitative analysis of AChE BuChE andBACE1 transcripts in brain homogenates of tested animalsrather than in individual isolated cell fractions therefore theobtained results constitute an overall ldquopicturerdquo of both studiedgenes transcriptional changes occurring in the brain areas ofstudied animals under the influence of the RA and the wholeplant extract as well

6 Conclusion

The subchronic administration ofMO led to an improvementof long-term memory of rats however the mechanisms ofMO action are probably more complicated since its role asa modulator of beta-secretase activity (due to inhibition ofBACE1 mRNA expression in frontal cortex) should be takeninto consideration

It should be noted that we have studied a crude extractfrom leaf of Melissa officinalis not a single pure chemicalcompound This plant extract is a complex mixture and itsaction may be a result of the summation of activities ofseveral components (synergismadditive action of caffeic acidwith salvianolic acids rosmarinic acid and others) In thecase of extract from leaves ofMelissa officinalis it is possiblethat interactions occur between the 40 chemical compoundsidentified by HPLC system

Evidence-Based Complementary and Alternative Medicine 15

Taken together it seems that the MO activity representsa possible option as complementary interventions to relievethe symptoms of mild dementia

Competing Interests

The authors declare that they have no competing interests

Acknowledgments

This work was supported by the Ministry of Science andHigher EducationWarsaw Poland from educational sources(2008ndash2011 Grant no N 405417836)

References

[1] S L Cole and R Vassar ldquoThe Alzheimerrsquos disease 120573-secretaseenzyme BACE1rdquo Molecular Neurodegeneration vol 2 no 1article 22 2007

[2] LACraigN SHong andR JMcDonald ldquoRevisiting the cho-linergic hypothesis in the development of Alzheimerrsquos diseaserdquoNeuroscience amp Biobehavioral Reviews vol 35 no 6 pp 1397ndash1409 2011

[3] N G M Gomes M G Campos J M C Orfao and C A FRibeiro ldquoPlants with neurobiological activity as potential tar-gets for drug discoveryrdquo Progress in Neuro-Psychopharmacologyamp Biological Psychiatry vol 33 no 8 pp 1372ndash1389 2009

[4] WHO WHO Monographs on Selected Medicinal PlantsmdashVolume 2 WHO Geneva Switzerland 2004 httpappswhointmedicinedocsendJs4927e18htmlJs4927e18

[5] M Bayat A A Tameh M H Ghahremani et al ldquoNeuropro-tective properties of Melissa officinalis after hypoxic-ischemicinjury both in vitro and in vivordquo DARU Journal of Pharmaceu-tical Sciences vol 20 article 42 2012

[6] J P Kamdem A Adeniran A A Boligon et al ldquoAntioxidantactivity genotoxicity and cytotoxicity evaluation of lemon balm(Melissa officinalis L) ethanolic extract Its potential role inneuroprotectionrdquo Industrial Crops and Products vol 51 pp 26ndash34 2013

[7] J-T Lin Y-C Chen Y-C Lee C-W R Hou F-L Chen andD-J Yang ldquoAntioxidant anti-proliferative and cyclooxygenase-2 inhibitory activities of ethanolic extracts from lemon balm(Melissa officinalis L) leavesrdquo LWTmdashFood Science and Technol-ogy vol 49 no 1 pp 1ndash7 2012

[8] G Guginski A P Luiz M D Silva et al ldquoMechanisms involvedin the antinociception caused by ethanolic extract obtainedfrom the leaves of Melissa officinalis (lemon balm) in micerdquoPharmacology Biochemistry amp Behavior vol 93 no 1 pp 10ndash162009

[9] N Perry G Court N Bidet J Court and E Perry ldquoEuropeanherbs with cholinergic activities potential in dementia therapyrdquoInternational Journal of Geriatric Psychiatry vol 11 no 12 pp1063ndash1069 1996

[10] E K Perry A T Pickering W W Wang P J Houghtonand N S L Perry ldquoMedicinal plants and Alzheimerrsquos diseasefrom ethnobotany to phytotherapyrdquo Journal of Pharmacy andPharmacology vol 51 no 5 pp 527ndash534 1999

[11] M-J R Howes N S L Perry and P J Houghton ldquoPlants withtraditional uses and activities relevant to the management ofAlzheimerrsquos disease and other cognitive disordersrdquo Phytother-apy Research vol 17 no 1 pp 1ndash18 2003

[12] I Orhan and M Aslan ldquoAppraisal of scopolamine-inducedantiamnesic effect in mice and in vitro antiacetylcholinesteraseand antioxidant activities of some traditionally used Lamiaceaeplantsrdquo Journal of Ethnopharmacology vol 122 no 2 pp 327ndash332 2009

[13] S Akhondzadeh M Noroozian M Mohammadi S OhadiniaA H Jamshidi and M Khani ldquoMelissa officinalis extract inthe treatment of patients with mild to moderate Alzheimerrsquosdisease a double blind randomised placebo controlled trialrdquoJournal of Neurology Neurosurgery and Psychiatry vol 74 no 7pp 863ndash866 2003

[14] D O Kennedy A B Scholey N T J Tildesley E K Perry andK AWesnes ldquoModulation ofmood and cognitive performancefollowing acute administration of Melissa officinalis (lemonbalm)rdquo Pharmacology Biochemistry amp Behavior vol 72 no 4pp 953ndash964 2002

[15] D O Kennedy G Wake S Savelev et al ldquoModulation of moodand cognitive performance following acute administration ofsingle doses of Melissa officinalis (Lemon balm) with humanCNS nicotinic and muscarinic receptor-binding propertiesrdquoNeuropsychopharmacology vol 28 no 10 pp 1871ndash1881 2003

[16] D O Kennedy and E L Wightman ldquoHerbal extracts and phy-tochemicals plant secondarymetabolites and the enhancementof human brain functionrdquoAdvances inNutrition vol 2 no 1 pp32ndash50 2011

[17] R P Pereira A A Boligon A S Appel et al ldquoChemical com-position antioxidant and anticholinesterase activity of Melissaofficinalisrdquo Industrial Crops and Products vol 53 pp 34ndash452014

[18] M Soodi N Naghdi H Hajimehdipoor S Choopani andE Sahraei ldquoMemory-improving activity of Melissa officinalisextract in naıve and scopolamine-treated ratsrdquo Research inPharmaceutical Sciences vol 9 no 2 pp 107ndash114 2014

[19] K Dastmalchi V Ollilainen P Lackman et al ldquoAcetyl-cholinesterase inhibitory guided fractionation of Melissa offic-inalis Lrdquo Bioorganic amp Medicinal Chemistry vol 17 no 2 pp867ndash871 2009

[20] W Chaiyana and S Okonogi ldquoInhibition of cholinesterase byessential oil from food plantrdquo Phytomedicine vol 19 no 8-9 pp836ndash839 2012

[21] K Dastmalchi H J D Dorman P P Oinonen Y Darwis ILaakso and R Hiltunen ldquoChemical composition and in vitroantioxidative activity of a lemon balm (Melissa officinalis L)extractrdquo LWTmdashFood Science and Technology vol 41 no 3 pp391ndash400 2008

[22] P Pereira D Tysca P Oliveira L F D S Brum J N Picadaand P Ardenghi ldquoNeurobehavioral and genotoxic aspects ofrosmarinic acidrdquo Pharmacological Research vol 52 no 3 pp199ndash203 2005

[23] M Ozarowski P L Mikolajczak A Bogacz et al ldquoRosmarinusofficinalis L leaf extract improves memory impairment andaffects acetylcholinesterase and butyrylcholinesterase activitiesin rat brainrdquo Fitoterapia vol 91 pp 261ndash271 2013

[24] G P Kumar and F Khanum ldquoNeuroprotective potential of phy-tochemicalsrdquo Pharmacognosy Reviews vol 6 no 12 pp 81ndash902012

[25] M Stobiecki A Staszkow A Piasecka P M Garcia-LopezF Zamora-Natera and P Kachlicki ldquoLC-MSMS profiling offlavonoid conjugates in wild mexican lupine Lupinus reflexusrdquoJournal of Natural Products vol 73 no 7 pp 1254ndash1260 2010

[26] A Wojakowska A Piasecka P M Garcıa-Lopez et al ldquoStruc-tural analysis and profiling of phenolic secondarymetabolites of

16 Evidence-Based Complementary and Alternative Medicine

Mexican lupine species using LC-MS techniquesrdquo Phytochem-istry vol 92 pp 71ndash86 2013

[27] A Gryszczynska B Opala Z Lowicki et al ldquoBioactive com-pounds determination in the callus and hydroalcoholic extractsfrom Salvia miltiorrhiza and Salvia przewalskiimdashpreliminarystudy on their anti-alcoholic activity effectsrdquo PhytochemistryLetters vol 11 pp 399ndash403 2015

[28] K Slinkart and V L Singleton ldquoTotal phenol analysis automa-tion and comparison with manual methodrdquo American Journalof Enology and Viticulture vol 28 no 1 pp 49ndash55 1977

[29] P J R Boissier J Tardy and J C Diverres ldquoUne nouvellemethode simple pour explorer lrsquoaction lsquotranquillisantersquo le testde la chemineerdquo Medicina Experimentalis vol 3 no 1 pp 81ndash84 1960

[30] R Ader J A Weijnen and P Moleman ldquoRetention of a passiveavoidance response as a function of the intensity and durationof electric shockrdquo Psychonomic Science vol 26 no 3 pp 125ndash128 1972

[31] E M Blalock K-C Chen K Sharrow et al ldquoGene microar-rays in hippocampal aging statistical profiling identifies novelprocesses correlated with cognitive impairmentrdquo Journal ofNeuroscience vol 23 no 9 pp 3807ndash3819 2003

[32] K Isomae S Morimoto H Hasegawa K Morita and JKamei ldquoEffects of T-82 a novel acetylcholinesterase inhibitoron impaired learning and memory in passive avoidance task inratsrdquo European Journal of Pharmacology vol 465 no 1-2 pp97ndash103 2003

[33] J Patora and B Klimek ldquoFlavonoids from lemon balm (Melissaofficinalis L Lamiaceae)rdquo Acta Poloniae Pharmaceutica-DrugResearch vol 59 no 2 pp 139ndash143 2002

[34] L Barros M Duenas M I Dias M J Sousa C Santos-Buelgaand I C F R Ferreira ldquoPhenolic profiles of cultivated invitro cultured and commercial samples of Melissa officinalis Linfusionsrdquo Food Chemistry vol 136 no 1 pp 1ndash8 2013

[35] T L Miron M Herrero and E Ibanez ldquoEnrichment of antiox-idant compounds from lemon balm (Melissa officinalis) bypressurized liquid extraction and enzyme-assisted extractionrdquoJournal of Chromatography A vol 1288 pp 1ndash9 2013

[36] Y Lu L Y Foo and H Wong ldquoSagecoumarin a novel caffeicacid trimer from Salvia officinalisrdquo Phytochemistry vol 52 no6 pp 1149ndash1152 1999

[37] S Abuhamdah LHuangM S J Elliott et al ldquoPharmacologicalprofile of an essential oil derived from Melissa officinalis withanti-agitation properties focus on ligand-gated channelsrdquo Jour-nal of Pharmacy and Pharmacology vol 60 no 3 pp 377ndash3842008

[38] G Wake J Court A Pickering R Lewis R Wilkins andE Perry ldquoCNS acetylcholine receptor activity in Europeanmedicinal plants traditionally used to improve failing memoryrdquoJournal of Ethnopharmacology vol 69 no 2 pp 105ndash114 2000

[39] S-H Ryu C Katona B Rive and G Livingston ldquoPersistenceof and changes in neuropsychiatric symptoms in Alzheimerdisease over 6months the LASER-AD studyrdquoAmerican Journalof Geriatric Psychiatry vol 13 no 11 pp 976ndash983 2005

[40] L N Gitlin H C Kales and C G Lyketsos ldquoNonpharma-cologic management of behavioral symptoms in dementiardquoJournal of the AmericanMedical Association vol 308 no 19 pp2020ndash2029 2012

[41] A Konczol J Muller E Foldes et al ldquoApplicability of a blood-brain barrier specific artificial membrane permeability assay atthe early stage of natural product-based CNS drug discoveryrdquoJournal of Natural Products vol 76 no 4 pp 655ndash663 2013

[42] Y-J Zhang L Wu Q-L Zhang J Li F-X Yin and Y YuanldquoPharmacokinetics of phenolic compounds of Danshen extractin rat blood and brain by microdialysis samplingrdquo Journal ofEthnopharmacology vol 136 no 1 pp 129ndash136 2011

[43] X Li C Yu Y Lu et al ldquoPharmacokinetics tissue distributionmetabolism and excretion of depside salts from Salvia miltior-rhiza in ratsrdquo Drug Metabolism and Disposition vol 35 no 2pp 234ndash239 2007

[44] H Xu Y Li X Che H Tian H Fan and K Liu ldquoMetabolismof salvianolic acid a and antioxidant activities of its methylatedmetabolitesrdquo Drug Metabolism and Disposition vol 42 no 2pp 274ndash281 2014

[45] D H Kim S J Park J M Kim et al ldquoCognitive dysfunctionsinduced by a cholinergic blockade and A120573 25-35 peptide areattenuated by salvianolic acid Brdquo Neuropharmacology vol 61no 8 pp 1432ndash1440 2011

[46] G-H Du Y Qiu and J-T Zhang ldquoSalvianolic acid B protectsthe memory functions against transient cerebral ischemia inmicerdquo Journal of Asian Natural Products Research vol 2 no 2pp 145ndash152 2000

[47] G Du and J Zhang ldquoProtective effects of salvianolic acid Aagainst impairment of memory induced by cerebral ischemia-reperfusion in micerdquo Chinese Medical Journal vol 110 no 1 pp65ndash68 1997

[48] J Anwar R M Spanevello G Thome et al ldquoEffects of caffeicacid on behavioral parameters and on the activity of acetyl-cholinesterase in different tissues from adult ratsrdquo Pharmacol-ogy Biochemistry and Behavior vol 103 no 2 pp 386ndash394 2012

[49] YW Lee D H Kim S J Jeon et al ldquoNeuroprotective effects ofsalvianolic acid B on anA12057325-35 peptide-inducedmousemodelof Alzheimerrsquos diseaserdquo European Journal of Pharmacology vol704 no 1ndash3 pp 70ndash77 2013

[50] J Zhong M-K Tang Y Zhang Q-P Xu and J-T ZhangldquoEffect of salvianolic acid B on neural cells damage and neu-rogenesis after brain ischemia-reperfusion in ratsrdquo Yao Xue XueBao vol 42 no 7 pp 716ndash721 2007

[51] P Zhuang Y Zhang G Cui et al ldquoDirect stimulation of adultneural stemprogenitor cells in vitro and neurogenesis in vivo bysalvianolic acid Brdquo PLoS ONE vol 7 no 4 Article ID e356362012

[52] M TangW Feng Y Zhang J Zhong and J Zhang ldquoSalvianolicacid B improves motor function after cerebral ischemia in ratsrdquoBehavioural Pharmacology vol 17 no 5-6 pp 493ndash498 2006

[53] Q Li L-P Han Z-H Li J-T Zhang and M-K Tang ldquoSal-vianolic acid B alleviate the disruption of blood-brain barrierin rats after cerebral ischemia-reperfusion by inhibiting MAPKpathwayrdquo Yao Xue Xue Bao vol 45 no 12 pp 1485ndash1490 2010

[54] Y-L LinH-J Tsay T-H Lai T-T Tzeng andY-J Shiao ldquoLith-ospermic acid attenuates 1-methyl-4-phenylpyridine-inducedneurotoxicity by blocking neuronal apoptotic and neuroinflam-matory pathwaysrdquo Journal of Biomedical Science vol 22 article37 2015

[55] M-K Tang and J-T Zhang ldquoSalvianolic acid B inhibits fibrilformation and neurotoxicity of amyloid beta-protein in vitrordquoActa Pharmacologica Sinica vol 22 no 4 pp 380ndash384 2001

[56] S S K Durairajan Q Yuan L Xie et al ldquoSalvianolic acid Binhibits A120573 fibril formation and disaggregates preformed fibrilsand protects against A120573-induced cytotoxictyrdquo NeurochemistryInternational vol 52 no 4-5 pp 741ndash750 2008

[57] FD Pinheiro Fernandes A P FonteneleMenezes J C de SousaNeves et al ldquoCaffeic acid protects mice from memory deficits

Evidence-Based Complementary and Alternative Medicine 17

induced by focal cerebral ischemiardquo Behavioural Pharmacologyvol 25 no 7 pp 637ndash647 2014

[58] S-J Tsai C-Y Chao andM-C Yin ldquoPreventive and therapeu-tic effects of caffeic acid against inflammatory injury in striatumof MPTP-treated micerdquo European Journal of Pharmacology vol670 no 2-3 pp 441ndash447 2011

[59] J H Kim QWang J M Choi S Lee and E J Cho ldquoProtectiverole of caffeic acid in an 119860120573

25minus35

-induced Alzheimerrsquos diseasemodelrdquo Nutrition Research and Practice vol 9 no 5 pp 480ndash488 2015

[60] G Liang B Shi W Luo and J Yang ldquoThe protective effect ofcaffeic acid on global cerebral ischemia-reperfusion injury inratsrdquo Behavioral and Brain Functions vol 11 no 1 article 182015

[61] D Y Yoo J H ChoiW Kim et al ldquoEffects of luteolin on spatialmemory cell proliferation and neuroblast differentiation in thehippocampal dentate gyrus in a scopolamine-induced amnesiamodelrdquo Neurological Research vol 35 no 8 pp 813ndash820 2013

[62] Y Liu X Tian L Gou L Sun X Ling and X Yin ldquoLuteolinattenuates diabetes-associated cognitive decline in ratsrdquo BrainResearch Bulletin vol 94 pp 23ndash29 2013

[63] S-M Liu Z-H Yang andX-B Sun ldquoSimultaneous determina-tion of six Salvia miltiorrhiza gradients in rat plasma and brainby LC-MSMSrdquo Zhongguo Zhongyao Zazhi vol 39 no 9 pp1704ndash1708 2014

[64] M A Papandreou A Dimakopoulou Z I Linardaki et alldquoEffect of a polyphenol-rich wild blueberry extract on cognitiveperformance of mice brain antioxidant markers and acetyl-cholinesterase activityrdquo Behavioural Brain Research vol 198 no2 pp 352ndash358 2009

[65] M-S Garcıa-Ayllon O Cauli M-X Silveyra et al ldquoBraincholinergic impairment in liver failurerdquo Brain vol 131 no 11pp 2946ndash2956 2008

[66] G K Ferreira M Carvalho-Silva C L Goncalves et al ldquoL-Tyrosine administration increases acetylcholinesterase activityin ratsrdquo Neurochemistry International vol 61 no 8 pp 1370ndash1374 2012

[67] R Vassar P-H Kuhn C Haass et al ldquoFunction therapeuticpotential and cell biology of BACE proteases current status andfuture prospectsrdquo Journal of Neurochemistry vol 130 no 1 pp4ndash28 2014

[68] R Vassar ldquoThe 120573-secretase BACE a prime drug target forAlzheimerrsquos diseaserdquo Journal of Molecular Neuroscience vol 17no 2 pp 157ndash170 2001

[69] D T R Coulson N Beyer J G Quinn et al ldquoBACE1 mRNAexpression in Alzheimerrsquos disease postmortem brain tissuerdquoJournal of Alzheimerrsquos Disease vol 22 no 4 pp 1111ndash1122 2010

[70] S Vladimir-Knezevic B Blazekovic M Kindl J Vladic A DLower-Nedza and A H Brantner ldquoAcetylcholinesterase inhib-itory antioxidant and phytochemical properties of selectedmedicinal plants of the lamiaceae familyrdquoMolecules vol 19 no1 pp 767ndash782 2014

[71] F Marcelo C Dias A Martins et al ldquoMolecular recognitionof rosmarinic acid from Salvia sclareoides extracts by ace-tylcholinesterase a new binding site detected by NMR spec-troscopyrdquo ChemistrymdashA European Journal vol 19 no 21 pp6641ndash6649 2013

[72] G Johnson and S W Moore ldquoWhy has butyrylcholinesterasebeen retained Structural and functional diversification in aduplicated generdquoNeurochemistry International vol 61 no 5 pp783ndash797 2012

[73] R J Grayer M R Eckert N C Veitch et al ldquoThe chemo-taxonomic significance of two bioactive caffeic acid estersnepetoidins A and B in the Lamiaceaerdquo Phytochemistry vol 64no 2 pp 519ndash528 2003

[74] A M D El-Mousallamy U W Hawas and S A M HusseinldquoTeucrol a decarboxyrosmarinic acid and its 41015840-O-triglycosideteucroside from Teucrium pilosumrdquo Phytochemistry vol 55 no8 pp 927ndash931 2000

[75] I Fecka and S Turek ldquoDetermination of water-soluble polyphe-nolic compounds in commercial herbal teas from Lamiaceaepeppermint melissa and sagerdquo Journal of Agricultural and FoodChemistry vol 55 no 26 pp 10908ndash10917 2007

[76] L W Sumner A Amberg D Barrett et al ldquoProposed min-imum reporting standards for chemical analysis WorkingGroup (CAWG) Metabolomics Standards Initiative (MSI)rdquoMetabolomics vol 3 pp 211ndash221 2007

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

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PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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ObesityJournal of

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Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

8 Evidence-Based Complementary and Alternative Medicine

100 200 300 400 500 600 700 800 900 1000 1100 1200

mz

70

75

80

85

90

95

100

minus198 (2-hydroxy-3-(34-dihydroxyphenyl)

-porpanoide)

minus180 (caffeide)

minus180 (2-hydroxy-3-(34-dihydroxyphenyl)

-propanoide)

minus180 (2-hydroxy-3-(34-dihydroxyphenyl)

-propanoide)

minus180 (2-hydroxy-3-(34-dihydroxyphenyl)

-propanoide)

1350438

2191749

HO

HO

HO HO

OH

OH

OH

OH OH OH

OHOHOHOHHOOC

COOH

COOH COOH COOH

24612ppmC36H55O37 = 10792422

minus07911ppmC54H47O24 = 10792457

10792449

10448ppmC45H39O20 = 8992040

899205018453ppm

C36H31O16 = 7191612

7191625

20501ppmC27H23O12 = 5391190

5391201

14852ppmC23H33O10 = 4692074

469208119316ppm

C17H11O5 = 2950606

2950612

0

5

10

15

20

25

30

35

40

45

50

55

60

65

Rela

tive a

bund

ance

22903ppmC18H15O8 = 3590767

3590775

minus37218 ppmC9H5O3 = 1610239

1610233

(a)

100 200 300 400 500 600 700 800 900 1000

mz

100 200 300 400 500 600 700 800 900 1000

mz

100 200 300 400 500 600 700 800 900 1000

mz

100 200 300 400 500 600 700 800 900 1000

mz

0

250

500

750

1000

00

04

08

times104

times104

01234

0

1

2

times105

Ints

Ints

Ints

Ints

MS1

MS2

MS3

MS4

[M-H]minus10749

minus224 (acidic part

minus224 (acidic part

of sagecoumarin)

of sagecoumarin)

minus360 (rosmarinic acid)

4908

7148

5348 minus180 minus162 (caffeide)8769

31074909

5348

minus180 (2-hydroxy-3-(34-dihydroxyphenyl)-propanoide)

3108

35474909

1

(b)

Figure 2 Continued

Evidence-Based Complementary and Alternative Medicine 9

150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900

mz

0

5

10

15

20

25

30

35

40

45

50

55

60

65

Rela

tive a

bund

ance

O

O

O O

O

O

OH OH

OH

OH

OH

OHOH

OH

HO

HO

HO

COOH

OC

1529946

1970447

C9H9O5 = 1970455

minus41775ppm1610232

2552328

C16H31O2 = 2552330

minus04091ppm

3110563

C17H11O6 = 3110561

05243ppm

minus224 (fragment of sagecoumarin)

3912257

4092361

4832730

C29H39O6 = 4832752

minus44931ppm

5350885

C27H19O12 = 5350882

05640ppm

minus132 (tartaric acid)

5770990

C29H21O13 = 5770988

06486ppm

6670947

C31H23O17 = 6670941

09808ppm

minus162 (caffeic acid)

8291267

C40H29O20 = 8291258

11370ppm

(c)

Figure 2 (a) Mass spectra in negative ionization mode and simplified fragmentation scheme of compound 25 (pentameric ester ofcaffeic acid) (b) Mass spectra in negative ionization mode and simplified fragmentation scheme of compound 26 (pentameric structureof sagecoumarin di-2-hydroxy-3-(34-dihydroxyphenyl)-propanoide caffeide) (c) Mass spectra in negative ionization mode and simplifiedfragmentation scheme of compound 36 (pentameric structure of sagecoumarin caftaride)

Tetrameric structures of hydroxycinnamic acids wereidentified in lemon balm recently [34 35] The measure-ment of accurate masses allowed the identification of com-pound 25 which was tentatively identified as pentamericester of caffeic acid (Figure 2(a)) In 25 the double loss of1800421 amu corresponded to fragments with the molecularformula of C

9H8O4 adequate to dehydroxylated 2-hydroxy-

3-(34-dihydroxyphenyl)-propanoic acid The product ionat 7191623119898119911 and its further fragmentation are sim-ilar to those of metabolite 22 as described previously [34 35]Therefore metabolite 25was tentatively assigned as sagerinicacid di-2-hydroxy-3-(34-dihydroxyphenyl)-propanoideNev-ertheless comprehensive studies by nuclear magnetic reso-nance are required to complete elucidation of substitutionpattern for particular components of those pentamers

Sagecoumarins were previously identified in Salvia offic-inalis as caffeic acid trimers [36] Our MS analysis indicatedthe presence of such compounds and their derivatives alsoin M officinalis The pseudomolecular ion of compound33 observed in the negative ionization mode had theaccurate mass of 5350880119898119911 which corresponded to thechemical formula C

27H19O12

adequate for sagecoumarin(according to the Metlin and KNApSAck databases) Note-worthy 26 33 36 and 37 had the same fragmentationpattern of the product ion obtained in the MSMS andMSn in the negative ionization mode The [M-H-1800422]minusion corresponded to the detachment of 2-hydroxy-3-(34-dihydroxyphenyl)-propanoic acid thus 37 was tentatively

assigned as tetrameric sagecoumarin 2-hydroxy-3-(34-dihy-droxyphenyl)-propanoide The [M-H-3600846]minus ion in 26was indicated on rosmarinic acid substitution to 37 Massspectra of MSn in negative ionization of the compoundprovided complementary information to HR-MSMS massspectra indicated on caffeic acid as internal componentof the dimer In addition simultaneous loss of fragments180 amu and 224 amu in MS3 and MS4 indicated that thetwo components cannot be linked (Figure 2(b)) How-ever detailed analysis of substitution pattern of 26 shouldbe done Therefore 26 was tentatively assigned as penta-meric structure of sagecoumarin di-2-hydroxy-3-(34-dihy-droxyphenyl)-propanoide caffeide Rupture of caffeic andtartaric acid moieties from the product ion at 5350885119898119911was observed for compound36 (Figure 2(c))Detection of theaccurate masses of these two detached fragments with Δ lessthan 1 ppm eliminated the possibility of hexose and pentosesubstitution which have the same nominal masses as caffeicand tartaric acid respectively Therefore 36 was tentativelyidentified as another pentameric structure of sagecoumarincaftaride

Metabolite 28 with the accurate masses of 461073119898119911was tentatively identified as luteolin O-glucuronide The [M-H-1760324]minus ion is indicated on loss of structure C

6H8O6

corresponding to glucuronide moiety The product ion at2850405119898119911 was indicated on flavone luteolin The placeof substitution of the carboxylic acid on flavone skeleton isproblematic due to different isomers reported in lemon balm

10 Evidence-Based Complementary and Alternative Medicine

Table 2 Effect ofMelissa officinalis leaf extract (200mgkg po) treatment on sedative activity motor coordination and memory in rats

Group 119899Locomotor activity

[number of impulses5min]Motor coordination

exit time [s]Short-term memorye

ORLong-term memory

latency [s]MC + H

2

O 18 390 plusmn 24 17 plusmn 3 040 plusmn 006 47 plusmn 14MC + SC 18 526 plusmn 48lowast 32 plusmn 5lowast 032 plusmn 005 12 plusmn 3lowast

MO + H2

O 10 231 plusmn 48lowast 32 plusmn 7lowast 043 plusmn 007 169 plusmn 11lowast

MO + SC 9 436 plusmn 60 43 plusmn 7 009 plusmn 011lowast 23 plusmn 6HU + H

2

O 9 515 plusmn 32 15 plusmn 2 037 plusmn 009 158 plusmn 14lowast

HU + SC 8 639 plusmn 71lowast 56 plusmn 3 022 plusmn 006 49 plusmn 18

RA + H2

O 8 406 plusmn 59 21 plusmn 5 045 plusmn 005 58 plusmn 28RA + SC 8 605 plusmn 55 28 plusmn 7 045 plusmn 005 20 plusmn 7Means plusmn SEM119899Number of animalsMC + H

2O control rats

SC scopolamine (05mgkg bw ip)HU huperzine A (05mgkg bw po)RA rosmarinic acid (10mgkg bw po)eExpressed as ratio OR = (119861 minus 119860lowast)(119861 + 119860) for details see Section 3lowastVersus MC + H

2O 119901 lt 005

Versus MC + SC 119901 lt 005

luteolin 31015840-O- and 7-O-glucuronide [33] It is impossible todistinguish both structures by mass spectrometry Only onechromatographic peak corresponding to the [M-H]minus ion at461073119898119911 was observed in our study Since luteolin 31015840-O-glucuronide was assigned as the most abundant flavonoid inlemon balm [33] we assumed that 28 corresponded to thisstructure

412 Flavonoids and Polyphenolic Acids The major com-pound from the 40 chemical compounds identified in hy-droethanolic MO leaf extract established by HPLC was RA(885 g100 g) (Table 1 Figure 1)

Other chemical compoundswith neuromodulatory activ-ities such as lithospermic acid (0042 g100 g) salvianolicacid A (0040 g100 g) salvianolic acid B (0023 g100 g) andcaffeic acid (0087 g100 g) were also documented in litera-ture Moreover the total polyphenolic compounds content ofMO determined with the use of FolinndashCiocalteu assay was3397 calculated as gallic acid The total hydroxycinnamicderivatives content expressed spectrophotometrically as ros-marinic acid was 2115 g100 g

413 Essential Oil Composition Hydroethanolic MO leafextract contained 008 of total essential oil The GCFIDanalysis showed that the extract comprised camphene(004) alfa-pinene (007) beta-pinene (1647) andmyrcene (1951) Moreover according to retention time16 compounds were identified as follows alfa-bisabololborneol carvone chamazulene cineole eugenol gamma-terpineol guaiazulene isopulegol linalool limonene men-thol menthyl acetate pulegone terpine and thymol Thesecompounds are present in the essential oil in trace amountswhich do not allow the quantitative interpretation

42 Cognitive and Behavioral Experiments

421 Locomotor Activity A one-way ANOVA analysisrevealed significant differences in the locomotor activity of

rats expressed as their horizontal spontaneous activity afterMO administration (ANOVA 119865(7 80) = 646 119901 lt 005)(Table 2) Detailed post hoc analysis showed thatMO + H

2O

decreased the locomotor activity of rats by 4031 but HU +H2O did not affect this activity when compared with control

group (MC + H2O) We observed also that RA + H

2O did

not change the locomotor activity of rats Stimulating effectsin the locomotor activity of rats were observed after an acuteSC injection (MC + SC versus MC + H

2O 119901 lt 005) and

this effect was observed in all SC-treated rats when comparedwith the proper non-SC-treated animals On the contrarythese SC-treated animals did not differ in comparison toanimals that received SC only (MO + SC versus MC + SC119901 gt 005 HU + SC versus MC + SC 119901 gt 005 RA + SCversus MC + SC 119901 gt 005)

422 Motor Coordination A one-way ANOVA analysisrevealed significant differences in motor coordination of ratsexpressed as their exit time from the cylinder (119865(5 73) =284 119901 lt 005) (Table 2) Detailed analysis showed that themultiple administration of RA + H

2O and HU + H

2O did

not affect significantly this paradigm when compared withcontrol rats (119901 gt 005) whereas MO treatment led to pro-longed exit time (MO + H

2O versus MC + H

2O 119901 lt 005)

Moreover generally SC-treated animals showed producedprolongation of exit time and the effects were statisticallysignificant not only in control groups (MC + SC versus MC +H2O 119901 lt 005) but also in RA-treated rats (RA + SC versus

MC + SC 119901 lt 005) However the rest of the SC-treatedanimals did not differ in comparison to animals receiving SConly (MO + SC versus MC + SC 119901 gt 005 HU + SC versusMC + SC 119901 gt 005)

423 Long-Term Memory A one-way ANOVA analysisrevealed significant differences in long-term memory afterusing a passive avoidance test (119865(7 77) = 201 119901 lt 005Table 2) It was shown that the strongest effect leading to

Evidence-Based Complementary and Alternative Medicine 11

Table 3 The effect ofMelissa officinalis leaf extract on acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) activities and AChEBuAChE or beta-secretase (BACE1) mRNA expression levels in frontal cortex (FC) or hippocampus (Hipp) of rats

Group119899Enzyme activity

[nmolminmg protein]mRNA expression

[]AChE BuChE ACHE BuChE BACE1

FC Hipp FC Hipp FC Hipp FC Hipp FC HippMC + H

2

O 363 plusmn 49 439 plusmn 73 65 plusmn 11 53 plusmn 8 100 plusmn 12 100 plusmn 11 100 plusmn 18 100 plusmn 11 100 plusmn 16 100 plusmn 8MO + H

2

O 276 plusmn 34 409 plusmn 28 69 plusmn 6 62 plusmn 4 48 plusmn 4lowast 31 plusmn 7lowast 16 plusmn 2lowast 64 plusmn 21amp 36 plusmn 3lowast 50 plusmn 8lowast

HU + H2

O 189 plusmn 15lowast 239 plusmn 15lowast 58 plusmn 6 51 plusmn 4 53 plusmn 13lowast 85 plusmn 5 42 plusmn 9lowast 102 plusmn 11 62 plusmn 6lowast 98 plusmn 4RA + H

2

O 224 plusmn 16lowast 251 plusmn 12lowast 77 plusmn 7 99 plusmn 7lowast 101 plusmn 12 103 plusmn 5 184 plusmn 31lowast 56 plusmn 7lowast 126 plusmn 13amp 98 plusmn 3Means plusmn SEM119899Number of animals 7ndash10Values expressed as a ratio the geneGAPDHMC + H

2O control group

HU huperzine A (05mgkg bw po)RA rosmarinic acid (10mgkg bw po)lowastampVersus MC + H

2O 119901 lt 005 or 119901 lt 007 respectively

an improvement of this paradigm was produced by extractof MO and HU but not RA when compared with controlanimals (MO + H

2O versus MC + H

2O 119901 lt 005 HU +

H2O versus MC + H

2O 119901 lt 005 RA + H

2O versus MC +

H2O 119901 gt 005) However the administration of SC to rats

significantly decreased the latency time of passive avoidancetask (MC + SC versus MC + H

2O 119901 lt 005) After MO or

RA combined treatment with SC no improvement of long-term memory was observed but HU given with SC showedenhancement of this paradigm in rats (HU + SC versusMC + SC 119901 lt 005) Therefore it can be concluded thatadministration of HU overcomes the effect shown by SC only(Table 1)

424 Short-Term Memory The results of the object recogni-tion test showed that an administration of the compoundsor extract did affect the ratsrsquo short-term memory (ANOVA119865(7 78) = 273 119901 lt 005) (Table 2)

Detailed post hoc analysis showed that only SC signifi-cantly decreased the short-term memory in MO-treated rats(MO + SC versusMO + H

2O 119901 lt 005MO + SC versus MC

+ SC 119901 lt 005) whereas the differences between the rest ofthe animals did not reach statistical significance (119901 gt 005)

43 AChE and BuChE Activities in Rat Brain A one-wayANOVA revealed significant differences between groups inthe activity of AChE in both the cortex and the hippocampus(frontal cortex 119865(3 27) = 565 119901 lt 005 hippocampusANOVA 119865(3 29) = 796 119901 lt 005) It was found out thatMOshowed an insignificant inhibition of AChE activity in thefrontal cortex by 24 when compared with control rats (MC+ H2O 119901 lt 006) and in the hippocampus by 7 (Table 3)

whereas HU produced a distinct significant inhibition ofAChE activity in comparison to control group by 48 (119901 lt005) and 47 (119901 lt 005) in the cortex and the hippocampusrespectively Also RA lowered significantly AChE activityboth in the cortex (38) and in the hippocampus (43)Moreover there were not significant differences between thevalues of BuChE activities for MO and HU when compared

with control group in the frontal cortex (ANOVA 119865(3 30) =101 119901 gt 005) whereas in the hippocampus the differencesreached statistical significance (ANOVA 119865(3 27) = 141119901 lt 005) Detailed analysis showed that only RA effect wassignificant and increased BuChE activity in the hippocampuswhen compared with the control rats (119901 lt 005)

44 AChE BuChE and BACE1 mRNA Level Changes in RatBrain A one-way ANOVA analysis revealed significant dif-ferences of AChE mRNA transcription profile in the cortex(ANOVA 119865(3 27) = 857 119901 lt 005) As shown in Table 3the multiple treatment of MO produced in the cortex astatistically significant decrease of AChEmRNA level by 52the administration of HU caused decrease of its level by 44(119901 lt 005) whereas RA did not affect this parameter whencompared to the control

There were significant differences between the relativevalues of BuChE mRNA levels in this region of brain in rats(frontal cortex ANOVA 119865(3 25) = 162 119901 lt 005) TheMOtreatment led to a decrease in the BuChEmRNA level by 84(versus MC + H

2O 119901 lt 005) the prolonged HU admin-

istration resulted in a decrease of the transcript level in thecortex by 58 (versus MC + H

2O 119901 lt 005) but RA

increased this parameter in the cortex by 84 (versus MC +H2O 119901 lt 005)The significant differences ofmRNA transcription level of

AChE mRNA level in the hippocampus (ANOVA 119865(3 31) =212 119901 lt 005) have been observed The detailed analysisshown that in the case of AChE afterMO treatment mRNAlevel significantly decreased by 69 (versus MC + H

2O

119901 lt 005) while the administration of HU resulted in astatistically insignificant decrease of AChE mRNA level by18 when compared with control group Also RA did notchange the level of transcript

In the case of BuChEmRNA expression in the hippocam-pus there were statistically significant differences betweengroups (ANOVA 119865(3 27) = 310 119901 lt 005) Detailed analysisshowed that in theMO + H

2O treated group the expression

lowered by 36 but the difference did not reach strong

12 Evidence-Based Complementary and Alternative Medicine

significance when compared with the control values (119901 lt007) and no change in the expression level of this enzymewas shown after the administration of HU On the contraryRA produced an increase of BuChE mRNA expression in thehippocampus by 44 (versus MC + H

2O 119901 lt 005)

Further analysis showed the significant differences inBACE1 mRNA expression in both brain regions of rats (inthe cortex and the hippocampus) (frontal cortex ANOVA119865(3 27) = 163 119901 lt 005 hippocampus ANOVA 119865(3 27) =133 119901 gt 005) It was observed that MO produced astatistically significant decrease of the BACE1 expression levelby 64 in the cortex (versus MC + H

2O 119901 lt 005) and by

50 in the hippocampus (versus MC + H2O 119901 lt 005) For

comparison HU treatment led to a decrease in the mRNAexpression level by 38 in the cortex (versus MC + H

2O

119901 lt 005) but not in the hippocampus On the contrary RAproduced an increase of this transcript in the cortex by 26but the effect did not reach a strong statistical significance(versus MC + H

2O 119901 lt 007) whereas in the hippocampus

there was no difference between RA and control group

5 Discussion

Cognitive and Behavioral Experiments The present studyinvestigated the influence of subchronic (28-fold) adminis-tration of standardized 50 EtOH extract ofMelisa officinalisleaf extract (MO) (200mgkg po containing 177mgkg ofRA) on SC-induced impairment of short-term and long-term memory in rats The results were compared with theactivity of cholinesterases (AChE and BuChE) as well aswith AChE BuChE and BACE1 gene expression levels in thecortex and hippocampus of the rat brain So far little evidenceis yet available as regards mechanisms of MO leaf extractaction that are potentially relevant to cognitive function ofrats after per os administration MO is traditionally used intreating neurological disorders through its anti-AChE [18]and antiagitation properties [37] Moreover Wake et al [38]and Kennedy et al [15] showed thatMO extract has nicotinicreceptor activity and that it can displace [3H]-(N)-nicotinefrom nicotinic receptors in homogenates of human cerebralcortex tissue and they suggested that these mechanisms canexplain activity of MO extract in amnesia model RecentlySoodi et al [18] observed that intraperitoneal injections ofMO extract (200mgkg) in rats could significantly enhancelearning and memory processes in animals since the extractsignificantly ameliorates SC-induced learning deficit in Mor-ris water maze test On the contrary in higher dose itcan be observed that MO extract (400mgkg) could notreverse SC-induced memory impairment [18] In our studyadministration of MO extract at a dose of 200mgkg (po)showed the effect leading to improvement of long-termmemory in a passive avoidance test in rats However afterMO combined treatment with SCMO did not overcome theimpairment shown by SC (Table 2) It should be emphasizedthat it is not clear whether the effect shown in our study byMO in non-SC-treated rats is specific since theMO treatmentproduced significant lowering of locomotor activity of ratstherefore the sedative profile ofMO cannot be excluded

On the other hand Ryu et al [39] observed that agitationand aggression are highly prevalent in patientswith dementiaAccording to Gitlin et al [40] nonpharmacological inter-ventions are recommended as first-line therapy Althoughantipsychotics have shown benefit for Alzheimerrsquos disease-related psychosis their use is associated with several seriousadverse effects [40]Thus it seems that the use ofMO extractcan provide dual benefits both in aspect of inhibition ofagitation and in improving the memory of patients withAlzheimerrsquos disease

Furthermore in our study RA in the dose of 10mgkgbw (po) did not affect either short- or long-term memoryalthough RA lowered significantly AChE activity both inthe cortex and in the hippocampus Moreover we observedthat the repeated administration of RA in non-scopolamine-treated rats did not produce any changes of locomotoractivity similarly to our previous study [23]

It is possible that other chemical compounds can influ-ence the memory in rats by synergic interactions in plantextract According to our calculations caffeic acid (0174mgin a single dose of extract administered to animals per kgbw) lithospermic acid (0084mgkg) salvianolic acid A(008mgkg) and salvianolic acid B (0046mgkg) may beresponsible for observed pharmacological effects On theother hand results from few studies showed that salvianolicacids do not cross the blood brain barrier (BBB) [41 42]and also lithospermic acid does not efficiently cross the BBB[43] For this reason the interpretation of our results is morecomplicated

Firstly Xu et al [44] demonstrated that Sal A is ametabol-ically unstable compound that would undergo rapid methy-lationmetabolism catalyzed by catechol O-methyltransferasein vivo into four major methylated metabolites of Sal A(3-O-methyl 31015840-O-methyl 3310158401015840-O-dimethyl and 31015840310158401015840-O-dimethyl salvianolic acid A) These generated O-methylatedmetabolites may be largely responsible for its in vivo phar-macological effects Although there are no available recentstudies on the ability of these compounds to pass the BBBsuch possibility should not be excluded

Secondly several studies showed central pharmacolog-ical effects in animals after per os administration of Sal B[45 46] Sal A [47] and caffeic acid [48] It was provedfor example that salvianolic acid B (10mgkg po) sig-nificantly rescued the A12057325ndash35 peptide-induced decreaseof choline acetyltransferase and brain-derived neurotrophicfactor protein levels in an amyloid 120573 (A120573) peptide-inducedAlzheimerrsquos disease mouse model [49] It also significantlyreversed (10mgkg po) the cognitive impairments inducedby scopolamine (1mgkg ip) or A120573 (25ndash35) (10 nmol5 120583Licv) injection inmice [45] Previous studies [46 47] showedalso that both Sal A and Sal B are able to improve theimpaired memory function induced by cerebral ischemia-reperfusion in miceThe stimulation of neurogenesis processin both subgranular zone (SGZ) and subventricular zone(SVZ) after brain ischemia and also alleviation neural cellsloss and improved motor function recovery after brainischemia in rats after the Sal B administration were alsoobserved by Zhong et al [50] Moreover the exposure to SalB can maintain the proliferation of neural stemprogenitor

Evidence-Based Complementary and Alternative Medicine 13

cells (NSPCs) after cerebral ischemia and improve cognitivepostischemic impairment after stroke in rats using Morriswater maze test therefore authors concluded that Sal B mayact as a potential drug in treatment of brain injury or neu-rodegenerative disease [51] Additionally the improvement ofmotor function after cerebral ischemia in rats after salvianolicacid B administrationwas also demonstrated [52]The clue topass through the BBB Sal B provided also results of Li et alindicating its protective effect on BBB in rats after cerebralischemia-reperfusion by inhibiting the MAPK pathway [53]In addition to this it was shown that lithospermic acid andsalvianolic acids exerted neuroprotective activity in variousexperimental models Lithospermic acid significantly attenu-ates neurotoxicity in vitro and in vivo induced by 1-methyl-4-phenylpyridin (MPP(+)) by blocking neuronal apoptotic andneuroinflammatory pathways [54] Salvianolic acid B inhib-ited amyloid beta-protein aggregation and fibril formation aswell as directly inhibiting the cellular toxicity of amyloid beta-protein in PC12 cells [55] and significantly reduced its cyto-toxic effects on human neuroblastoma SH-SY5Y cells [56]

For an explanation of our results observations of Pin-heiro Fernandes et al [57] may be very helpful whichshowed that caffeic acid nonflavanoid catecholic compoundwhose derivatives are occurring in MO extract improvedthe working spatial and long-term aversive memory deficitsinduced by focal cerebral ischemia in mice Anwar et al [48]showed also that caffeic acid (100mgkg) improved the step-down latencies in the inhibitory avoidance in rats Tsai et al[58] showed that caffeic acid is a potent neuroprotective agentin brain of 1-methyl-4-phenyl-1236-tetrahydropyridine(MPTP) treatedmiceMoreover caffeic acid improves A12057325ndash35-induced memory deficits and cognitive impairment inmice [59] In another study it was also shown that this com-pound has a significant protective effect on global cerebralischemia-reperfusion injury in rats [60] For instance 124 plusmn18mg100 g of caffeic acid was detected in the brain of micewith a diet containing 2 caffeic acid for 4 weeks [58] Thusthere is progressive evidence that this compound passesthrough the BBB and has a central pharmacological activity

Also results by Yoo et al are also worth noting [61] whichshowed also in a ratmodel of scopolamine-induced amnesiathat administration of luteolin (a common flavonoid frommany plants including M officinalis) at dose of 10mgkgcaused the increase in the brain-derived neurotrophic factor(BDNF) acetylcholine and the decrease in lipid peroxida-tion Liu et al [62] observed that chronic treatment withluteolin (50 and 100mgkg) improved neuronal injury andcognitive performance by attenuating oxidative stress andcholinesterase activity in streptozotocin-induced diabetes inrats In our study ethanolic extract of MO administered torats significantly improved long-term memory after using apassive avoidance test but afterMO combined treatmentwithSC no improvement of this paradigmwas observed (Table 2)

Thirdly available pharmacokinetic studies were carriedout for the pure compounds (Sal A and B) and the extractof the roots of Salvia miltiorrhiza [42 63] but there are notavailable studies of bioavailability of these compounds afteradministration of Melissa officinalis leaf extract containingother compounds as compared with the extract of Salvia

miltiorrhiza Moreover there is a lack of data about howscopolamine may influence the penetration of the salvianolicacids (and other compounds) across the BBB Hence thereis a need to study the pharmacokinetic parameters of thesecompounds in the group of animals treatedwith scopolaminein comparison with control group

AChEandBuChEActivities in Rat Brain To date several stud-ies have focused on explaining themechanismof action of theMS extract and its active compounds Soodi et al [18] showedthat treatment of animals withMO extract (400mgkg) priorto scopolamine injection could ameliorate scopolamine-induced enhancement in AChE activity This dose of MOextract inhibited the AChE activity in the hippocampus ofrats (519 versus 1004 in normal saline group 119901 lt 005914 in group treated with scopolamine +MO versus 1281in scopolamine group 119901 lt 005) In another study Anwaret al [48] showed that 50 and 100mgkg of caffeic aciddecreased in vivo the AChE activity in the cerebral cortexand striatum and increased the activity of this enzyme inthe cerebellum hippocampus hypothalamus pons lympho-cytes and muscles when compared to the control group(119901 lt 005) Our study showed that ethanolic extract of MOproduced an insignificant and slight inhibition of AChE andBuChE activity in the frontal cortex and in the hippocampus(especially) However it was observed previously [23] thatRA inhibited the AChE activity in the rats frontal cortex andhippocampusMoreover we showed that RA possess a strongstimulatory effect on BuChE in the hippocampus

AChE BuChE and BACE1mRNA Level Changes in Rat BrainSo far no results have been published of studies concerningthe in vivo assessment of changes in AChE BuChE andBACE1 gene expression profile in different brain regionsunder the influence of MO extracts or their key bioactivemetabolites Such studies focused overwhelmingly on theanalysis of their in vitro activities to a lesser extent in animalexperiments

In our study in the frontal cortex we have observedthe strongest inhibition of both AChE and BuCHE mRNAtranscription under the influence ofM officinalis extract andhuperzine A (Table 3) However a more significant differencein the level of transcript was seen in the frontal cortex inparticular in the case of BuCHE mRNA of experimentalrats (a decrease by 516 and 44 for MO and HU versuscontrol resp)The strong inhibition of transcription ofAChEmRNAwas also observed in the hippocampus ofMO-treatedrats (a decrease by 69) The BuChE mRNA transcriptionin animals receiving MO was lowered in a moderate way(decrease by 36)HuperzineA alone has not caused changesthat were observed in the group ofMO (Table 2) Our findingsmostly correlate with the observed changes in activities ofAChE and BuChE in different groups of animals In the caseof BuChE slightly different results between its activity andexpression profile were especially seen in the frontal cortexand hippocampus of animals receivingMO (Table 3)

To date there is a lack of published results of studiesmaking an attempt to clarify the potential differences in thetranscriptional profile and activity of AChE and BuChE

14 Evidence-Based Complementary and Alternative Medicine

under the influence of MO and its bioactive metabolites Itcannot be excluded that the key for the observed differencesin the level of AChE activities and mRNA level can becaused by changes in the activity of AChE in other regionsof the brain not analyzed in this study such as substantianigra cerebellum globus pallidus and hypothalamus whereit exerts nonenzymatic neuromodulatory functions affect-ing neurite outgrowth and synaptogenesis modulating theactivity of other proteins regional cerebral blood flow andother functions [64] But it is difficult to clearly explain whydiminishing of AChE and BuChEmRNAs did not always cor-relate with the lowering of activity of these enzymes althoughit has been recently noted that AChE activity was notparalleled by an increase in mRNA levels [65] The authorsexplained this fact by stating that AChE levels are regulatedat transcriptional posttranscriptional and posttranslationallevels leading to complex expression patterns which canbe modulated by physiological and pathological conditionsHowever these mechanisms are not fully understood andfurther studies are needed in this field

The cause of observed different degree of inhibition ofactivity and transcription status of studied genes especiallyof BuChE (and AChE) may lie in a so-called ldquonegative feed-backrdquo consisting of a complicated transcriptiontranslationregulation protein-protein interactionsmodifications and ametabolic network together forming a system that allows thecell to respond sensibly to the multiple signal molecules thatexist in its environment [66]

Because of that we propose that the reason for differencesbetween AChE and BuChE activities in the cortex and hip-pocampus under the MO may be due to the fact of insuf-ficiency of applied dose and the duration of the experi-ment affecting the transcriptional tissue-specific cellularmachinery regulating BuChE transcription without affectingits activity Moreover the administration of higher doses ofMO and extended period of time could lead to sufficientinhibition of its activity

There is a need to conduct further studies to determinethe molecular degree of dependence between changes inAChE and BuChE activities and activities of potential keyfactors regulating expression of these genes in the frontalcortex and hippocampus under the influence of extracts ofMO and active metabolites Another study determining theeffectiveness of their actions on the cholinergic system inexperimental animal models of memory impairment withparticular emphasis on scopolamine including the determi-nation of changes at the molecular level should be thereforecarried out

Alterations in BACE1 protein level have been proved inpostmortem brain tissue from individuals with AD withincreases decreases and also no change reported [1 67 68]An example of confirmation of these findings at the mRNAlevel is results of study by Coulson et al [69] Based onconducted studies some authors suggested that increasedBACE1mRNA transcription in remaining neuronal cells maycontribute to the increased BACE1 protein levels and activityfound in brain regions affected by AD [70]

Results of many studies concerning the elevated level ofBACE1 mRNA and protein in Alzheimerrsquos disease provide

direct and compelling reasons to develop therapies directed atBACE1 inhibition thus reducing120573-amyloid and its associatedtoxicities [67 68]

In our study we have observed that BACEmRNA expres-sion statistically significantly decreased after MO adminis-tration in frontal cortex and hippocampus These resultssuggest that the MO extract may act to inhibit BACE1mRNA level given the fact that the percentage of inhibitionof the expression (64 in the frontal cortex 50 in thehippocampus) is higher than that in the case of HU (38in the cortex and the lack of changes in the hippocampus)A careful analysis of the literature data shows that there areno studies which analyzed the impact of MO extract on theexpression level of BACE1 in Alzheimerrsquos disease

Furthermore a literature analysis does not indicatealready published results conducted by other teams attempt-ing to assess the impact of RA on the transcriptional activityof AChE BuChE and BACE1 Although several studies(already mentioned and others [70 71]) highlighted theRA and other caffeic acid derivatives capability of acetyl-cholinesterases inhibition none of these studies does nottouch the question of the molecular basis of their impacton the transcriptional machinery that regulates in vivo theexpression of studied genes Hence in our opinion obtainedby our team results they are one of the first of this typeand in general correlate with the results of our previousstudy [23] In this case there is no clear evidence explainingdifferent responses at the transcriptional level under theinfluence of RA especially in the case of BuChE encodinggene in the hippocampus (Table 2) It is possible that theobserved differentiation of BuChE transcriptional activitybetween the frontal cortex and the hippocampus may bedue to the differences of butyrylcholinesterase localizationand substrate affinity [72] Since in our experiment we havecarried out a quantitative analysis of AChE BuChE andBACE1 transcripts in brain homogenates of tested animalsrather than in individual isolated cell fractions therefore theobtained results constitute an overall ldquopicturerdquo of both studiedgenes transcriptional changes occurring in the brain areas ofstudied animals under the influence of the RA and the wholeplant extract as well

6 Conclusion

The subchronic administration ofMO led to an improvementof long-term memory of rats however the mechanisms ofMO action are probably more complicated since its role asa modulator of beta-secretase activity (due to inhibition ofBACE1 mRNA expression in frontal cortex) should be takeninto consideration

It should be noted that we have studied a crude extractfrom leaf of Melissa officinalis not a single pure chemicalcompound This plant extract is a complex mixture and itsaction may be a result of the summation of activities ofseveral components (synergismadditive action of caffeic acidwith salvianolic acids rosmarinic acid and others) In thecase of extract from leaves ofMelissa officinalis it is possiblethat interactions occur between the 40 chemical compoundsidentified by HPLC system

Evidence-Based Complementary and Alternative Medicine 15

Taken together it seems that the MO activity representsa possible option as complementary interventions to relievethe symptoms of mild dementia

Competing Interests

The authors declare that they have no competing interests

Acknowledgments

This work was supported by the Ministry of Science andHigher EducationWarsaw Poland from educational sources(2008ndash2011 Grant no N 405417836)

References

[1] S L Cole and R Vassar ldquoThe Alzheimerrsquos disease 120573-secretaseenzyme BACE1rdquo Molecular Neurodegeneration vol 2 no 1article 22 2007

[2] LACraigN SHong andR JMcDonald ldquoRevisiting the cho-linergic hypothesis in the development of Alzheimerrsquos diseaserdquoNeuroscience amp Biobehavioral Reviews vol 35 no 6 pp 1397ndash1409 2011

[3] N G M Gomes M G Campos J M C Orfao and C A FRibeiro ldquoPlants with neurobiological activity as potential tar-gets for drug discoveryrdquo Progress in Neuro-Psychopharmacologyamp Biological Psychiatry vol 33 no 8 pp 1372ndash1389 2009

[4] WHO WHO Monographs on Selected Medicinal PlantsmdashVolume 2 WHO Geneva Switzerland 2004 httpappswhointmedicinedocsendJs4927e18htmlJs4927e18

[5] M Bayat A A Tameh M H Ghahremani et al ldquoNeuropro-tective properties of Melissa officinalis after hypoxic-ischemicinjury both in vitro and in vivordquo DARU Journal of Pharmaceu-tical Sciences vol 20 article 42 2012

[6] J P Kamdem A Adeniran A A Boligon et al ldquoAntioxidantactivity genotoxicity and cytotoxicity evaluation of lemon balm(Melissa officinalis L) ethanolic extract Its potential role inneuroprotectionrdquo Industrial Crops and Products vol 51 pp 26ndash34 2013

[7] J-T Lin Y-C Chen Y-C Lee C-W R Hou F-L Chen andD-J Yang ldquoAntioxidant anti-proliferative and cyclooxygenase-2 inhibitory activities of ethanolic extracts from lemon balm(Melissa officinalis L) leavesrdquo LWTmdashFood Science and Technol-ogy vol 49 no 1 pp 1ndash7 2012

[8] G Guginski A P Luiz M D Silva et al ldquoMechanisms involvedin the antinociception caused by ethanolic extract obtainedfrom the leaves of Melissa officinalis (lemon balm) in micerdquoPharmacology Biochemistry amp Behavior vol 93 no 1 pp 10ndash162009

[9] N Perry G Court N Bidet J Court and E Perry ldquoEuropeanherbs with cholinergic activities potential in dementia therapyrdquoInternational Journal of Geriatric Psychiatry vol 11 no 12 pp1063ndash1069 1996

[10] E K Perry A T Pickering W W Wang P J Houghtonand N S L Perry ldquoMedicinal plants and Alzheimerrsquos diseasefrom ethnobotany to phytotherapyrdquo Journal of Pharmacy andPharmacology vol 51 no 5 pp 527ndash534 1999

[11] M-J R Howes N S L Perry and P J Houghton ldquoPlants withtraditional uses and activities relevant to the management ofAlzheimerrsquos disease and other cognitive disordersrdquo Phytother-apy Research vol 17 no 1 pp 1ndash18 2003

[12] I Orhan and M Aslan ldquoAppraisal of scopolamine-inducedantiamnesic effect in mice and in vitro antiacetylcholinesteraseand antioxidant activities of some traditionally used Lamiaceaeplantsrdquo Journal of Ethnopharmacology vol 122 no 2 pp 327ndash332 2009

[13] S Akhondzadeh M Noroozian M Mohammadi S OhadiniaA H Jamshidi and M Khani ldquoMelissa officinalis extract inthe treatment of patients with mild to moderate Alzheimerrsquosdisease a double blind randomised placebo controlled trialrdquoJournal of Neurology Neurosurgery and Psychiatry vol 74 no 7pp 863ndash866 2003

[14] D O Kennedy A B Scholey N T J Tildesley E K Perry andK AWesnes ldquoModulation ofmood and cognitive performancefollowing acute administration of Melissa officinalis (lemonbalm)rdquo Pharmacology Biochemistry amp Behavior vol 72 no 4pp 953ndash964 2002

[15] D O Kennedy G Wake S Savelev et al ldquoModulation of moodand cognitive performance following acute administration ofsingle doses of Melissa officinalis (Lemon balm) with humanCNS nicotinic and muscarinic receptor-binding propertiesrdquoNeuropsychopharmacology vol 28 no 10 pp 1871ndash1881 2003

[16] D O Kennedy and E L Wightman ldquoHerbal extracts and phy-tochemicals plant secondarymetabolites and the enhancementof human brain functionrdquoAdvances inNutrition vol 2 no 1 pp32ndash50 2011

[17] R P Pereira A A Boligon A S Appel et al ldquoChemical com-position antioxidant and anticholinesterase activity of Melissaofficinalisrdquo Industrial Crops and Products vol 53 pp 34ndash452014

[18] M Soodi N Naghdi H Hajimehdipoor S Choopani andE Sahraei ldquoMemory-improving activity of Melissa officinalisextract in naıve and scopolamine-treated ratsrdquo Research inPharmaceutical Sciences vol 9 no 2 pp 107ndash114 2014

[19] K Dastmalchi V Ollilainen P Lackman et al ldquoAcetyl-cholinesterase inhibitory guided fractionation of Melissa offic-inalis Lrdquo Bioorganic amp Medicinal Chemistry vol 17 no 2 pp867ndash871 2009

[20] W Chaiyana and S Okonogi ldquoInhibition of cholinesterase byessential oil from food plantrdquo Phytomedicine vol 19 no 8-9 pp836ndash839 2012

[21] K Dastmalchi H J D Dorman P P Oinonen Y Darwis ILaakso and R Hiltunen ldquoChemical composition and in vitroantioxidative activity of a lemon balm (Melissa officinalis L)extractrdquo LWTmdashFood Science and Technology vol 41 no 3 pp391ndash400 2008

[22] P Pereira D Tysca P Oliveira L F D S Brum J N Picadaand P Ardenghi ldquoNeurobehavioral and genotoxic aspects ofrosmarinic acidrdquo Pharmacological Research vol 52 no 3 pp199ndash203 2005

[23] M Ozarowski P L Mikolajczak A Bogacz et al ldquoRosmarinusofficinalis L leaf extract improves memory impairment andaffects acetylcholinesterase and butyrylcholinesterase activitiesin rat brainrdquo Fitoterapia vol 91 pp 261ndash271 2013

[24] G P Kumar and F Khanum ldquoNeuroprotective potential of phy-tochemicalsrdquo Pharmacognosy Reviews vol 6 no 12 pp 81ndash902012

[25] M Stobiecki A Staszkow A Piasecka P M Garcia-LopezF Zamora-Natera and P Kachlicki ldquoLC-MSMS profiling offlavonoid conjugates in wild mexican lupine Lupinus reflexusrdquoJournal of Natural Products vol 73 no 7 pp 1254ndash1260 2010

[26] A Wojakowska A Piasecka P M Garcıa-Lopez et al ldquoStruc-tural analysis and profiling of phenolic secondarymetabolites of

16 Evidence-Based Complementary and Alternative Medicine

Mexican lupine species using LC-MS techniquesrdquo Phytochem-istry vol 92 pp 71ndash86 2013

[27] A Gryszczynska B Opala Z Lowicki et al ldquoBioactive com-pounds determination in the callus and hydroalcoholic extractsfrom Salvia miltiorrhiza and Salvia przewalskiimdashpreliminarystudy on their anti-alcoholic activity effectsrdquo PhytochemistryLetters vol 11 pp 399ndash403 2015

[28] K Slinkart and V L Singleton ldquoTotal phenol analysis automa-tion and comparison with manual methodrdquo American Journalof Enology and Viticulture vol 28 no 1 pp 49ndash55 1977

[29] P J R Boissier J Tardy and J C Diverres ldquoUne nouvellemethode simple pour explorer lrsquoaction lsquotranquillisantersquo le testde la chemineerdquo Medicina Experimentalis vol 3 no 1 pp 81ndash84 1960

[30] R Ader J A Weijnen and P Moleman ldquoRetention of a passiveavoidance response as a function of the intensity and durationof electric shockrdquo Psychonomic Science vol 26 no 3 pp 125ndash128 1972

[31] E M Blalock K-C Chen K Sharrow et al ldquoGene microar-rays in hippocampal aging statistical profiling identifies novelprocesses correlated with cognitive impairmentrdquo Journal ofNeuroscience vol 23 no 9 pp 3807ndash3819 2003

[32] K Isomae S Morimoto H Hasegawa K Morita and JKamei ldquoEffects of T-82 a novel acetylcholinesterase inhibitoron impaired learning and memory in passive avoidance task inratsrdquo European Journal of Pharmacology vol 465 no 1-2 pp97ndash103 2003

[33] J Patora and B Klimek ldquoFlavonoids from lemon balm (Melissaofficinalis L Lamiaceae)rdquo Acta Poloniae Pharmaceutica-DrugResearch vol 59 no 2 pp 139ndash143 2002

[34] L Barros M Duenas M I Dias M J Sousa C Santos-Buelgaand I C F R Ferreira ldquoPhenolic profiles of cultivated invitro cultured and commercial samples of Melissa officinalis Linfusionsrdquo Food Chemistry vol 136 no 1 pp 1ndash8 2013

[35] T L Miron M Herrero and E Ibanez ldquoEnrichment of antiox-idant compounds from lemon balm (Melissa officinalis) bypressurized liquid extraction and enzyme-assisted extractionrdquoJournal of Chromatography A vol 1288 pp 1ndash9 2013

[36] Y Lu L Y Foo and H Wong ldquoSagecoumarin a novel caffeicacid trimer from Salvia officinalisrdquo Phytochemistry vol 52 no6 pp 1149ndash1152 1999

[37] S Abuhamdah LHuangM S J Elliott et al ldquoPharmacologicalprofile of an essential oil derived from Melissa officinalis withanti-agitation properties focus on ligand-gated channelsrdquo Jour-nal of Pharmacy and Pharmacology vol 60 no 3 pp 377ndash3842008

[38] G Wake J Court A Pickering R Lewis R Wilkins andE Perry ldquoCNS acetylcholine receptor activity in Europeanmedicinal plants traditionally used to improve failing memoryrdquoJournal of Ethnopharmacology vol 69 no 2 pp 105ndash114 2000

[39] S-H Ryu C Katona B Rive and G Livingston ldquoPersistenceof and changes in neuropsychiatric symptoms in Alzheimerdisease over 6months the LASER-AD studyrdquoAmerican Journalof Geriatric Psychiatry vol 13 no 11 pp 976ndash983 2005

[40] L N Gitlin H C Kales and C G Lyketsos ldquoNonpharma-cologic management of behavioral symptoms in dementiardquoJournal of the AmericanMedical Association vol 308 no 19 pp2020ndash2029 2012

[41] A Konczol J Muller E Foldes et al ldquoApplicability of a blood-brain barrier specific artificial membrane permeability assay atthe early stage of natural product-based CNS drug discoveryrdquoJournal of Natural Products vol 76 no 4 pp 655ndash663 2013

[42] Y-J Zhang L Wu Q-L Zhang J Li F-X Yin and Y YuanldquoPharmacokinetics of phenolic compounds of Danshen extractin rat blood and brain by microdialysis samplingrdquo Journal ofEthnopharmacology vol 136 no 1 pp 129ndash136 2011

[43] X Li C Yu Y Lu et al ldquoPharmacokinetics tissue distributionmetabolism and excretion of depside salts from Salvia miltior-rhiza in ratsrdquo Drug Metabolism and Disposition vol 35 no 2pp 234ndash239 2007

[44] H Xu Y Li X Che H Tian H Fan and K Liu ldquoMetabolismof salvianolic acid a and antioxidant activities of its methylatedmetabolitesrdquo Drug Metabolism and Disposition vol 42 no 2pp 274ndash281 2014

[45] D H Kim S J Park J M Kim et al ldquoCognitive dysfunctionsinduced by a cholinergic blockade and A120573 25-35 peptide areattenuated by salvianolic acid Brdquo Neuropharmacology vol 61no 8 pp 1432ndash1440 2011

[46] G-H Du Y Qiu and J-T Zhang ldquoSalvianolic acid B protectsthe memory functions against transient cerebral ischemia inmicerdquo Journal of Asian Natural Products Research vol 2 no 2pp 145ndash152 2000

[47] G Du and J Zhang ldquoProtective effects of salvianolic acid Aagainst impairment of memory induced by cerebral ischemia-reperfusion in micerdquo Chinese Medical Journal vol 110 no 1 pp65ndash68 1997

[48] J Anwar R M Spanevello G Thome et al ldquoEffects of caffeicacid on behavioral parameters and on the activity of acetyl-cholinesterase in different tissues from adult ratsrdquo Pharmacol-ogy Biochemistry and Behavior vol 103 no 2 pp 386ndash394 2012

[49] YW Lee D H Kim S J Jeon et al ldquoNeuroprotective effects ofsalvianolic acid B on anA12057325-35 peptide-inducedmousemodelof Alzheimerrsquos diseaserdquo European Journal of Pharmacology vol704 no 1ndash3 pp 70ndash77 2013

[50] J Zhong M-K Tang Y Zhang Q-P Xu and J-T ZhangldquoEffect of salvianolic acid B on neural cells damage and neu-rogenesis after brain ischemia-reperfusion in ratsrdquo Yao Xue XueBao vol 42 no 7 pp 716ndash721 2007

[51] P Zhuang Y Zhang G Cui et al ldquoDirect stimulation of adultneural stemprogenitor cells in vitro and neurogenesis in vivo bysalvianolic acid Brdquo PLoS ONE vol 7 no 4 Article ID e356362012

[52] M TangW Feng Y Zhang J Zhong and J Zhang ldquoSalvianolicacid B improves motor function after cerebral ischemia in ratsrdquoBehavioural Pharmacology vol 17 no 5-6 pp 493ndash498 2006

[53] Q Li L-P Han Z-H Li J-T Zhang and M-K Tang ldquoSal-vianolic acid B alleviate the disruption of blood-brain barrierin rats after cerebral ischemia-reperfusion by inhibiting MAPKpathwayrdquo Yao Xue Xue Bao vol 45 no 12 pp 1485ndash1490 2010

[54] Y-L LinH-J Tsay T-H Lai T-T Tzeng andY-J Shiao ldquoLith-ospermic acid attenuates 1-methyl-4-phenylpyridine-inducedneurotoxicity by blocking neuronal apoptotic and neuroinflam-matory pathwaysrdquo Journal of Biomedical Science vol 22 article37 2015

[55] M-K Tang and J-T Zhang ldquoSalvianolic acid B inhibits fibrilformation and neurotoxicity of amyloid beta-protein in vitrordquoActa Pharmacologica Sinica vol 22 no 4 pp 380ndash384 2001

[56] S S K Durairajan Q Yuan L Xie et al ldquoSalvianolic acid Binhibits A120573 fibril formation and disaggregates preformed fibrilsand protects against A120573-induced cytotoxictyrdquo NeurochemistryInternational vol 52 no 4-5 pp 741ndash750 2008

[57] FD Pinheiro Fernandes A P FonteneleMenezes J C de SousaNeves et al ldquoCaffeic acid protects mice from memory deficits

Evidence-Based Complementary and Alternative Medicine 17

induced by focal cerebral ischemiardquo Behavioural Pharmacologyvol 25 no 7 pp 637ndash647 2014

[58] S-J Tsai C-Y Chao andM-C Yin ldquoPreventive and therapeu-tic effects of caffeic acid against inflammatory injury in striatumof MPTP-treated micerdquo European Journal of Pharmacology vol670 no 2-3 pp 441ndash447 2011

[59] J H Kim QWang J M Choi S Lee and E J Cho ldquoProtectiverole of caffeic acid in an 119860120573

25minus35

-induced Alzheimerrsquos diseasemodelrdquo Nutrition Research and Practice vol 9 no 5 pp 480ndash488 2015

[60] G Liang B Shi W Luo and J Yang ldquoThe protective effect ofcaffeic acid on global cerebral ischemia-reperfusion injury inratsrdquo Behavioral and Brain Functions vol 11 no 1 article 182015

[61] D Y Yoo J H ChoiW Kim et al ldquoEffects of luteolin on spatialmemory cell proliferation and neuroblast differentiation in thehippocampal dentate gyrus in a scopolamine-induced amnesiamodelrdquo Neurological Research vol 35 no 8 pp 813ndash820 2013

[62] Y Liu X Tian L Gou L Sun X Ling and X Yin ldquoLuteolinattenuates diabetes-associated cognitive decline in ratsrdquo BrainResearch Bulletin vol 94 pp 23ndash29 2013

[63] S-M Liu Z-H Yang andX-B Sun ldquoSimultaneous determina-tion of six Salvia miltiorrhiza gradients in rat plasma and brainby LC-MSMSrdquo Zhongguo Zhongyao Zazhi vol 39 no 9 pp1704ndash1708 2014

[64] M A Papandreou A Dimakopoulou Z I Linardaki et alldquoEffect of a polyphenol-rich wild blueberry extract on cognitiveperformance of mice brain antioxidant markers and acetyl-cholinesterase activityrdquo Behavioural Brain Research vol 198 no2 pp 352ndash358 2009

[65] M-S Garcıa-Ayllon O Cauli M-X Silveyra et al ldquoBraincholinergic impairment in liver failurerdquo Brain vol 131 no 11pp 2946ndash2956 2008

[66] G K Ferreira M Carvalho-Silva C L Goncalves et al ldquoL-Tyrosine administration increases acetylcholinesterase activityin ratsrdquo Neurochemistry International vol 61 no 8 pp 1370ndash1374 2012

[67] R Vassar P-H Kuhn C Haass et al ldquoFunction therapeuticpotential and cell biology of BACE proteases current status andfuture prospectsrdquo Journal of Neurochemistry vol 130 no 1 pp4ndash28 2014

[68] R Vassar ldquoThe 120573-secretase BACE a prime drug target forAlzheimerrsquos diseaserdquo Journal of Molecular Neuroscience vol 17no 2 pp 157ndash170 2001

[69] D T R Coulson N Beyer J G Quinn et al ldquoBACE1 mRNAexpression in Alzheimerrsquos disease postmortem brain tissuerdquoJournal of Alzheimerrsquos Disease vol 22 no 4 pp 1111ndash1122 2010

[70] S Vladimir-Knezevic B Blazekovic M Kindl J Vladic A DLower-Nedza and A H Brantner ldquoAcetylcholinesterase inhib-itory antioxidant and phytochemical properties of selectedmedicinal plants of the lamiaceae familyrdquoMolecules vol 19 no1 pp 767ndash782 2014

[71] F Marcelo C Dias A Martins et al ldquoMolecular recognitionof rosmarinic acid from Salvia sclareoides extracts by ace-tylcholinesterase a new binding site detected by NMR spec-troscopyrdquo ChemistrymdashA European Journal vol 19 no 21 pp6641ndash6649 2013

[72] G Johnson and S W Moore ldquoWhy has butyrylcholinesterasebeen retained Structural and functional diversification in aduplicated generdquoNeurochemistry International vol 61 no 5 pp783ndash797 2012

[73] R J Grayer M R Eckert N C Veitch et al ldquoThe chemo-taxonomic significance of two bioactive caffeic acid estersnepetoidins A and B in the Lamiaceaerdquo Phytochemistry vol 64no 2 pp 519ndash528 2003

[74] A M D El-Mousallamy U W Hawas and S A M HusseinldquoTeucrol a decarboxyrosmarinic acid and its 41015840-O-triglycosideteucroside from Teucrium pilosumrdquo Phytochemistry vol 55 no8 pp 927ndash931 2000

[75] I Fecka and S Turek ldquoDetermination of water-soluble polyphe-nolic compounds in commercial herbal teas from Lamiaceaepeppermint melissa and sagerdquo Journal of Agricultural and FoodChemistry vol 55 no 26 pp 10908ndash10917 2007

[76] L W Sumner A Amberg D Barrett et al ldquoProposed min-imum reporting standards for chemical analysis WorkingGroup (CAWG) Metabolomics Standards Initiative (MSI)rdquoMetabolomics vol 3 pp 211ndash221 2007

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

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Oxidative Medicine and Cellular Longevity

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PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

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Diabetes ResearchJournal of

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Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Evidence-Based Complementary and Alternative Medicine 9

150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900

mz

0

5

10

15

20

25

30

35

40

45

50

55

60

65

Rela

tive a

bund

ance

O

O

O O

O

O

OH OH

OH

OH

OH

OHOH

OH

HO

HO

HO

COOH

OC

1529946

1970447

C9H9O5 = 1970455

minus41775ppm1610232

2552328

C16H31O2 = 2552330

minus04091ppm

3110563

C17H11O6 = 3110561

05243ppm

minus224 (fragment of sagecoumarin)

3912257

4092361

4832730

C29H39O6 = 4832752

minus44931ppm

5350885

C27H19O12 = 5350882

05640ppm

minus132 (tartaric acid)

5770990

C29H21O13 = 5770988

06486ppm

6670947

C31H23O17 = 6670941

09808ppm

minus162 (caffeic acid)

8291267

C40H29O20 = 8291258

11370ppm

(c)

Figure 2 (a) Mass spectra in negative ionization mode and simplified fragmentation scheme of compound 25 (pentameric ester ofcaffeic acid) (b) Mass spectra in negative ionization mode and simplified fragmentation scheme of compound 26 (pentameric structureof sagecoumarin di-2-hydroxy-3-(34-dihydroxyphenyl)-propanoide caffeide) (c) Mass spectra in negative ionization mode and simplifiedfragmentation scheme of compound 36 (pentameric structure of sagecoumarin caftaride)

Tetrameric structures of hydroxycinnamic acids wereidentified in lemon balm recently [34 35] The measure-ment of accurate masses allowed the identification of com-pound 25 which was tentatively identified as pentamericester of caffeic acid (Figure 2(a)) In 25 the double loss of1800421 amu corresponded to fragments with the molecularformula of C

9H8O4 adequate to dehydroxylated 2-hydroxy-

3-(34-dihydroxyphenyl)-propanoic acid The product ionat 7191623119898119911 and its further fragmentation are sim-ilar to those of metabolite 22 as described previously [34 35]Therefore metabolite 25was tentatively assigned as sagerinicacid di-2-hydroxy-3-(34-dihydroxyphenyl)-propanoideNev-ertheless comprehensive studies by nuclear magnetic reso-nance are required to complete elucidation of substitutionpattern for particular components of those pentamers

Sagecoumarins were previously identified in Salvia offic-inalis as caffeic acid trimers [36] Our MS analysis indicatedthe presence of such compounds and their derivatives alsoin M officinalis The pseudomolecular ion of compound33 observed in the negative ionization mode had theaccurate mass of 5350880119898119911 which corresponded to thechemical formula C

27H19O12

adequate for sagecoumarin(according to the Metlin and KNApSAck databases) Note-worthy 26 33 36 and 37 had the same fragmentationpattern of the product ion obtained in the MSMS andMSn in the negative ionization mode The [M-H-1800422]minusion corresponded to the detachment of 2-hydroxy-3-(34-dihydroxyphenyl)-propanoic acid thus 37 was tentatively

assigned as tetrameric sagecoumarin 2-hydroxy-3-(34-dihy-droxyphenyl)-propanoide The [M-H-3600846]minus ion in 26was indicated on rosmarinic acid substitution to 37 Massspectra of MSn in negative ionization of the compoundprovided complementary information to HR-MSMS massspectra indicated on caffeic acid as internal componentof the dimer In addition simultaneous loss of fragments180 amu and 224 amu in MS3 and MS4 indicated that thetwo components cannot be linked (Figure 2(b)) How-ever detailed analysis of substitution pattern of 26 shouldbe done Therefore 26 was tentatively assigned as penta-meric structure of sagecoumarin di-2-hydroxy-3-(34-dihy-droxyphenyl)-propanoide caffeide Rupture of caffeic andtartaric acid moieties from the product ion at 5350885119898119911was observed for compound36 (Figure 2(c))Detection of theaccurate masses of these two detached fragments with Δ lessthan 1 ppm eliminated the possibility of hexose and pentosesubstitution which have the same nominal masses as caffeicand tartaric acid respectively Therefore 36 was tentativelyidentified as another pentameric structure of sagecoumarincaftaride

Metabolite 28 with the accurate masses of 461073119898119911was tentatively identified as luteolin O-glucuronide The [M-H-1760324]minus ion is indicated on loss of structure C

6H8O6

corresponding to glucuronide moiety The product ion at2850405119898119911 was indicated on flavone luteolin The placeof substitution of the carboxylic acid on flavone skeleton isproblematic due to different isomers reported in lemon balm

10 Evidence-Based Complementary and Alternative Medicine

Table 2 Effect ofMelissa officinalis leaf extract (200mgkg po) treatment on sedative activity motor coordination and memory in rats

Group 119899Locomotor activity

[number of impulses5min]Motor coordination

exit time [s]Short-term memorye

ORLong-term memory

latency [s]MC + H

2

O 18 390 plusmn 24 17 plusmn 3 040 plusmn 006 47 plusmn 14MC + SC 18 526 plusmn 48lowast 32 plusmn 5lowast 032 plusmn 005 12 plusmn 3lowast

MO + H2

O 10 231 plusmn 48lowast 32 plusmn 7lowast 043 plusmn 007 169 plusmn 11lowast

MO + SC 9 436 plusmn 60 43 plusmn 7 009 plusmn 011lowast 23 plusmn 6HU + H

2

O 9 515 plusmn 32 15 plusmn 2 037 plusmn 009 158 plusmn 14lowast

HU + SC 8 639 plusmn 71lowast 56 plusmn 3 022 plusmn 006 49 plusmn 18

RA + H2

O 8 406 plusmn 59 21 plusmn 5 045 plusmn 005 58 plusmn 28RA + SC 8 605 plusmn 55 28 plusmn 7 045 plusmn 005 20 plusmn 7Means plusmn SEM119899Number of animalsMC + H

2O control rats

SC scopolamine (05mgkg bw ip)HU huperzine A (05mgkg bw po)RA rosmarinic acid (10mgkg bw po)eExpressed as ratio OR = (119861 minus 119860lowast)(119861 + 119860) for details see Section 3lowastVersus MC + H

2O 119901 lt 005

Versus MC + SC 119901 lt 005

luteolin 31015840-O- and 7-O-glucuronide [33] It is impossible todistinguish both structures by mass spectrometry Only onechromatographic peak corresponding to the [M-H]minus ion at461073119898119911 was observed in our study Since luteolin 31015840-O-glucuronide was assigned as the most abundant flavonoid inlemon balm [33] we assumed that 28 corresponded to thisstructure

412 Flavonoids and Polyphenolic Acids The major com-pound from the 40 chemical compounds identified in hy-droethanolic MO leaf extract established by HPLC was RA(885 g100 g) (Table 1 Figure 1)

Other chemical compoundswith neuromodulatory activ-ities such as lithospermic acid (0042 g100 g) salvianolicacid A (0040 g100 g) salvianolic acid B (0023 g100 g) andcaffeic acid (0087 g100 g) were also documented in litera-ture Moreover the total polyphenolic compounds content ofMO determined with the use of FolinndashCiocalteu assay was3397 calculated as gallic acid The total hydroxycinnamicderivatives content expressed spectrophotometrically as ros-marinic acid was 2115 g100 g

413 Essential Oil Composition Hydroethanolic MO leafextract contained 008 of total essential oil The GCFIDanalysis showed that the extract comprised camphene(004) alfa-pinene (007) beta-pinene (1647) andmyrcene (1951) Moreover according to retention time16 compounds were identified as follows alfa-bisabololborneol carvone chamazulene cineole eugenol gamma-terpineol guaiazulene isopulegol linalool limonene men-thol menthyl acetate pulegone terpine and thymol Thesecompounds are present in the essential oil in trace amountswhich do not allow the quantitative interpretation

42 Cognitive and Behavioral Experiments

421 Locomotor Activity A one-way ANOVA analysisrevealed significant differences in the locomotor activity of

rats expressed as their horizontal spontaneous activity afterMO administration (ANOVA 119865(7 80) = 646 119901 lt 005)(Table 2) Detailed post hoc analysis showed thatMO + H

2O

decreased the locomotor activity of rats by 4031 but HU +H2O did not affect this activity when compared with control

group (MC + H2O) We observed also that RA + H

2O did

not change the locomotor activity of rats Stimulating effectsin the locomotor activity of rats were observed after an acuteSC injection (MC + SC versus MC + H

2O 119901 lt 005) and

this effect was observed in all SC-treated rats when comparedwith the proper non-SC-treated animals On the contrarythese SC-treated animals did not differ in comparison toanimals that received SC only (MO + SC versus MC + SC119901 gt 005 HU + SC versus MC + SC 119901 gt 005 RA + SCversus MC + SC 119901 gt 005)

422 Motor Coordination A one-way ANOVA analysisrevealed significant differences in motor coordination of ratsexpressed as their exit time from the cylinder (119865(5 73) =284 119901 lt 005) (Table 2) Detailed analysis showed that themultiple administration of RA + H

2O and HU + H

2O did

not affect significantly this paradigm when compared withcontrol rats (119901 gt 005) whereas MO treatment led to pro-longed exit time (MO + H

2O versus MC + H

2O 119901 lt 005)

Moreover generally SC-treated animals showed producedprolongation of exit time and the effects were statisticallysignificant not only in control groups (MC + SC versus MC +H2O 119901 lt 005) but also in RA-treated rats (RA + SC versus

MC + SC 119901 lt 005) However the rest of the SC-treatedanimals did not differ in comparison to animals receiving SConly (MO + SC versus MC + SC 119901 gt 005 HU + SC versusMC + SC 119901 gt 005)

423 Long-Term Memory A one-way ANOVA analysisrevealed significant differences in long-term memory afterusing a passive avoidance test (119865(7 77) = 201 119901 lt 005Table 2) It was shown that the strongest effect leading to

Evidence-Based Complementary and Alternative Medicine 11

Table 3 The effect ofMelissa officinalis leaf extract on acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) activities and AChEBuAChE or beta-secretase (BACE1) mRNA expression levels in frontal cortex (FC) or hippocampus (Hipp) of rats

Group119899Enzyme activity

[nmolminmg protein]mRNA expression

[]AChE BuChE ACHE BuChE BACE1

FC Hipp FC Hipp FC Hipp FC Hipp FC HippMC + H

2

O 363 plusmn 49 439 plusmn 73 65 plusmn 11 53 plusmn 8 100 plusmn 12 100 plusmn 11 100 plusmn 18 100 plusmn 11 100 plusmn 16 100 plusmn 8MO + H

2

O 276 plusmn 34 409 plusmn 28 69 plusmn 6 62 plusmn 4 48 plusmn 4lowast 31 plusmn 7lowast 16 plusmn 2lowast 64 plusmn 21amp 36 plusmn 3lowast 50 plusmn 8lowast

HU + H2

O 189 plusmn 15lowast 239 plusmn 15lowast 58 plusmn 6 51 plusmn 4 53 plusmn 13lowast 85 plusmn 5 42 plusmn 9lowast 102 plusmn 11 62 plusmn 6lowast 98 plusmn 4RA + H

2

O 224 plusmn 16lowast 251 plusmn 12lowast 77 plusmn 7 99 plusmn 7lowast 101 plusmn 12 103 plusmn 5 184 plusmn 31lowast 56 plusmn 7lowast 126 plusmn 13amp 98 plusmn 3Means plusmn SEM119899Number of animals 7ndash10Values expressed as a ratio the geneGAPDHMC + H

2O control group

HU huperzine A (05mgkg bw po)RA rosmarinic acid (10mgkg bw po)lowastampVersus MC + H

2O 119901 lt 005 or 119901 lt 007 respectively

an improvement of this paradigm was produced by extractof MO and HU but not RA when compared with controlanimals (MO + H

2O versus MC + H

2O 119901 lt 005 HU +

H2O versus MC + H

2O 119901 lt 005 RA + H

2O versus MC +

H2O 119901 gt 005) However the administration of SC to rats

significantly decreased the latency time of passive avoidancetask (MC + SC versus MC + H

2O 119901 lt 005) After MO or

RA combined treatment with SC no improvement of long-term memory was observed but HU given with SC showedenhancement of this paradigm in rats (HU + SC versusMC + SC 119901 lt 005) Therefore it can be concluded thatadministration of HU overcomes the effect shown by SC only(Table 1)

424 Short-Term Memory The results of the object recogni-tion test showed that an administration of the compoundsor extract did affect the ratsrsquo short-term memory (ANOVA119865(7 78) = 273 119901 lt 005) (Table 2)

Detailed post hoc analysis showed that only SC signifi-cantly decreased the short-term memory in MO-treated rats(MO + SC versusMO + H

2O 119901 lt 005MO + SC versus MC

+ SC 119901 lt 005) whereas the differences between the rest ofthe animals did not reach statistical significance (119901 gt 005)

43 AChE and BuChE Activities in Rat Brain A one-wayANOVA revealed significant differences between groups inthe activity of AChE in both the cortex and the hippocampus(frontal cortex 119865(3 27) = 565 119901 lt 005 hippocampusANOVA 119865(3 29) = 796 119901 lt 005) It was found out thatMOshowed an insignificant inhibition of AChE activity in thefrontal cortex by 24 when compared with control rats (MC+ H2O 119901 lt 006) and in the hippocampus by 7 (Table 3)

whereas HU produced a distinct significant inhibition ofAChE activity in comparison to control group by 48 (119901 lt005) and 47 (119901 lt 005) in the cortex and the hippocampusrespectively Also RA lowered significantly AChE activityboth in the cortex (38) and in the hippocampus (43)Moreover there were not significant differences between thevalues of BuChE activities for MO and HU when compared

with control group in the frontal cortex (ANOVA 119865(3 30) =101 119901 gt 005) whereas in the hippocampus the differencesreached statistical significance (ANOVA 119865(3 27) = 141119901 lt 005) Detailed analysis showed that only RA effect wassignificant and increased BuChE activity in the hippocampuswhen compared with the control rats (119901 lt 005)

44 AChE BuChE and BACE1 mRNA Level Changes in RatBrain A one-way ANOVA analysis revealed significant dif-ferences of AChE mRNA transcription profile in the cortex(ANOVA 119865(3 27) = 857 119901 lt 005) As shown in Table 3the multiple treatment of MO produced in the cortex astatistically significant decrease of AChEmRNA level by 52the administration of HU caused decrease of its level by 44(119901 lt 005) whereas RA did not affect this parameter whencompared to the control

There were significant differences between the relativevalues of BuChE mRNA levels in this region of brain in rats(frontal cortex ANOVA 119865(3 25) = 162 119901 lt 005) TheMOtreatment led to a decrease in the BuChEmRNA level by 84(versus MC + H

2O 119901 lt 005) the prolonged HU admin-

istration resulted in a decrease of the transcript level in thecortex by 58 (versus MC + H

2O 119901 lt 005) but RA

increased this parameter in the cortex by 84 (versus MC +H2O 119901 lt 005)The significant differences ofmRNA transcription level of

AChE mRNA level in the hippocampus (ANOVA 119865(3 31) =212 119901 lt 005) have been observed The detailed analysisshown that in the case of AChE afterMO treatment mRNAlevel significantly decreased by 69 (versus MC + H

2O

119901 lt 005) while the administration of HU resulted in astatistically insignificant decrease of AChE mRNA level by18 when compared with control group Also RA did notchange the level of transcript

In the case of BuChEmRNA expression in the hippocam-pus there were statistically significant differences betweengroups (ANOVA 119865(3 27) = 310 119901 lt 005) Detailed analysisshowed that in theMO + H

2O treated group the expression

lowered by 36 but the difference did not reach strong

12 Evidence-Based Complementary and Alternative Medicine

significance when compared with the control values (119901 lt007) and no change in the expression level of this enzymewas shown after the administration of HU On the contraryRA produced an increase of BuChE mRNA expression in thehippocampus by 44 (versus MC + H

2O 119901 lt 005)

Further analysis showed the significant differences inBACE1 mRNA expression in both brain regions of rats (inthe cortex and the hippocampus) (frontal cortex ANOVA119865(3 27) = 163 119901 lt 005 hippocampus ANOVA 119865(3 27) =133 119901 gt 005) It was observed that MO produced astatistically significant decrease of the BACE1 expression levelby 64 in the cortex (versus MC + H

2O 119901 lt 005) and by

50 in the hippocampus (versus MC + H2O 119901 lt 005) For

comparison HU treatment led to a decrease in the mRNAexpression level by 38 in the cortex (versus MC + H

2O

119901 lt 005) but not in the hippocampus On the contrary RAproduced an increase of this transcript in the cortex by 26but the effect did not reach a strong statistical significance(versus MC + H

2O 119901 lt 007) whereas in the hippocampus

there was no difference between RA and control group

5 Discussion

Cognitive and Behavioral Experiments The present studyinvestigated the influence of subchronic (28-fold) adminis-tration of standardized 50 EtOH extract ofMelisa officinalisleaf extract (MO) (200mgkg po containing 177mgkg ofRA) on SC-induced impairment of short-term and long-term memory in rats The results were compared with theactivity of cholinesterases (AChE and BuChE) as well aswith AChE BuChE and BACE1 gene expression levels in thecortex and hippocampus of the rat brain So far little evidenceis yet available as regards mechanisms of MO leaf extractaction that are potentially relevant to cognitive function ofrats after per os administration MO is traditionally used intreating neurological disorders through its anti-AChE [18]and antiagitation properties [37] Moreover Wake et al [38]and Kennedy et al [15] showed thatMO extract has nicotinicreceptor activity and that it can displace [3H]-(N)-nicotinefrom nicotinic receptors in homogenates of human cerebralcortex tissue and they suggested that these mechanisms canexplain activity of MO extract in amnesia model RecentlySoodi et al [18] observed that intraperitoneal injections ofMO extract (200mgkg) in rats could significantly enhancelearning and memory processes in animals since the extractsignificantly ameliorates SC-induced learning deficit in Mor-ris water maze test On the contrary in higher dose itcan be observed that MO extract (400mgkg) could notreverse SC-induced memory impairment [18] In our studyadministration of MO extract at a dose of 200mgkg (po)showed the effect leading to improvement of long-termmemory in a passive avoidance test in rats However afterMO combined treatment with SCMO did not overcome theimpairment shown by SC (Table 2) It should be emphasizedthat it is not clear whether the effect shown in our study byMO in non-SC-treated rats is specific since theMO treatmentproduced significant lowering of locomotor activity of ratstherefore the sedative profile ofMO cannot be excluded

On the other hand Ryu et al [39] observed that agitationand aggression are highly prevalent in patientswith dementiaAccording to Gitlin et al [40] nonpharmacological inter-ventions are recommended as first-line therapy Althoughantipsychotics have shown benefit for Alzheimerrsquos disease-related psychosis their use is associated with several seriousadverse effects [40]Thus it seems that the use ofMO extractcan provide dual benefits both in aspect of inhibition ofagitation and in improving the memory of patients withAlzheimerrsquos disease

Furthermore in our study RA in the dose of 10mgkgbw (po) did not affect either short- or long-term memoryalthough RA lowered significantly AChE activity both inthe cortex and in the hippocampus Moreover we observedthat the repeated administration of RA in non-scopolamine-treated rats did not produce any changes of locomotoractivity similarly to our previous study [23]

It is possible that other chemical compounds can influ-ence the memory in rats by synergic interactions in plantextract According to our calculations caffeic acid (0174mgin a single dose of extract administered to animals per kgbw) lithospermic acid (0084mgkg) salvianolic acid A(008mgkg) and salvianolic acid B (0046mgkg) may beresponsible for observed pharmacological effects On theother hand results from few studies showed that salvianolicacids do not cross the blood brain barrier (BBB) [41 42]and also lithospermic acid does not efficiently cross the BBB[43] For this reason the interpretation of our results is morecomplicated

Firstly Xu et al [44] demonstrated that Sal A is ametabol-ically unstable compound that would undergo rapid methy-lationmetabolism catalyzed by catechol O-methyltransferasein vivo into four major methylated metabolites of Sal A(3-O-methyl 31015840-O-methyl 3310158401015840-O-dimethyl and 31015840310158401015840-O-dimethyl salvianolic acid A) These generated O-methylatedmetabolites may be largely responsible for its in vivo phar-macological effects Although there are no available recentstudies on the ability of these compounds to pass the BBBsuch possibility should not be excluded

Secondly several studies showed central pharmacolog-ical effects in animals after per os administration of Sal B[45 46] Sal A [47] and caffeic acid [48] It was provedfor example that salvianolic acid B (10mgkg po) sig-nificantly rescued the A12057325ndash35 peptide-induced decreaseof choline acetyltransferase and brain-derived neurotrophicfactor protein levels in an amyloid 120573 (A120573) peptide-inducedAlzheimerrsquos disease mouse model [49] It also significantlyreversed (10mgkg po) the cognitive impairments inducedby scopolamine (1mgkg ip) or A120573 (25ndash35) (10 nmol5 120583Licv) injection inmice [45] Previous studies [46 47] showedalso that both Sal A and Sal B are able to improve theimpaired memory function induced by cerebral ischemia-reperfusion in miceThe stimulation of neurogenesis processin both subgranular zone (SGZ) and subventricular zone(SVZ) after brain ischemia and also alleviation neural cellsloss and improved motor function recovery after brainischemia in rats after the Sal B administration were alsoobserved by Zhong et al [50] Moreover the exposure to SalB can maintain the proliferation of neural stemprogenitor

Evidence-Based Complementary and Alternative Medicine 13

cells (NSPCs) after cerebral ischemia and improve cognitivepostischemic impairment after stroke in rats using Morriswater maze test therefore authors concluded that Sal B mayact as a potential drug in treatment of brain injury or neu-rodegenerative disease [51] Additionally the improvement ofmotor function after cerebral ischemia in rats after salvianolicacid B administrationwas also demonstrated [52]The clue topass through the BBB Sal B provided also results of Li et alindicating its protective effect on BBB in rats after cerebralischemia-reperfusion by inhibiting the MAPK pathway [53]In addition to this it was shown that lithospermic acid andsalvianolic acids exerted neuroprotective activity in variousexperimental models Lithospermic acid significantly attenu-ates neurotoxicity in vitro and in vivo induced by 1-methyl-4-phenylpyridin (MPP(+)) by blocking neuronal apoptotic andneuroinflammatory pathways [54] Salvianolic acid B inhib-ited amyloid beta-protein aggregation and fibril formation aswell as directly inhibiting the cellular toxicity of amyloid beta-protein in PC12 cells [55] and significantly reduced its cyto-toxic effects on human neuroblastoma SH-SY5Y cells [56]

For an explanation of our results observations of Pin-heiro Fernandes et al [57] may be very helpful whichshowed that caffeic acid nonflavanoid catecholic compoundwhose derivatives are occurring in MO extract improvedthe working spatial and long-term aversive memory deficitsinduced by focal cerebral ischemia in mice Anwar et al [48]showed also that caffeic acid (100mgkg) improved the step-down latencies in the inhibitory avoidance in rats Tsai et al[58] showed that caffeic acid is a potent neuroprotective agentin brain of 1-methyl-4-phenyl-1236-tetrahydropyridine(MPTP) treatedmiceMoreover caffeic acid improves A12057325ndash35-induced memory deficits and cognitive impairment inmice [59] In another study it was also shown that this com-pound has a significant protective effect on global cerebralischemia-reperfusion injury in rats [60] For instance 124 plusmn18mg100 g of caffeic acid was detected in the brain of micewith a diet containing 2 caffeic acid for 4 weeks [58] Thusthere is progressive evidence that this compound passesthrough the BBB and has a central pharmacological activity

Also results by Yoo et al are also worth noting [61] whichshowed also in a ratmodel of scopolamine-induced amnesiathat administration of luteolin (a common flavonoid frommany plants including M officinalis) at dose of 10mgkgcaused the increase in the brain-derived neurotrophic factor(BDNF) acetylcholine and the decrease in lipid peroxida-tion Liu et al [62] observed that chronic treatment withluteolin (50 and 100mgkg) improved neuronal injury andcognitive performance by attenuating oxidative stress andcholinesterase activity in streptozotocin-induced diabetes inrats In our study ethanolic extract of MO administered torats significantly improved long-term memory after using apassive avoidance test but afterMO combined treatmentwithSC no improvement of this paradigmwas observed (Table 2)

Thirdly available pharmacokinetic studies were carriedout for the pure compounds (Sal A and B) and the extractof the roots of Salvia miltiorrhiza [42 63] but there are notavailable studies of bioavailability of these compounds afteradministration of Melissa officinalis leaf extract containingother compounds as compared with the extract of Salvia

miltiorrhiza Moreover there is a lack of data about howscopolamine may influence the penetration of the salvianolicacids (and other compounds) across the BBB Hence thereis a need to study the pharmacokinetic parameters of thesecompounds in the group of animals treatedwith scopolaminein comparison with control group

AChEandBuChEActivities in Rat Brain To date several stud-ies have focused on explaining themechanismof action of theMS extract and its active compounds Soodi et al [18] showedthat treatment of animals withMO extract (400mgkg) priorto scopolamine injection could ameliorate scopolamine-induced enhancement in AChE activity This dose of MOextract inhibited the AChE activity in the hippocampus ofrats (519 versus 1004 in normal saline group 119901 lt 005914 in group treated with scopolamine +MO versus 1281in scopolamine group 119901 lt 005) In another study Anwaret al [48] showed that 50 and 100mgkg of caffeic aciddecreased in vivo the AChE activity in the cerebral cortexand striatum and increased the activity of this enzyme inthe cerebellum hippocampus hypothalamus pons lympho-cytes and muscles when compared to the control group(119901 lt 005) Our study showed that ethanolic extract of MOproduced an insignificant and slight inhibition of AChE andBuChE activity in the frontal cortex and in the hippocampus(especially) However it was observed previously [23] thatRA inhibited the AChE activity in the rats frontal cortex andhippocampusMoreover we showed that RA possess a strongstimulatory effect on BuChE in the hippocampus

AChE BuChE and BACE1mRNA Level Changes in Rat BrainSo far no results have been published of studies concerningthe in vivo assessment of changes in AChE BuChE andBACE1 gene expression profile in different brain regionsunder the influence of MO extracts or their key bioactivemetabolites Such studies focused overwhelmingly on theanalysis of their in vitro activities to a lesser extent in animalexperiments

In our study in the frontal cortex we have observedthe strongest inhibition of both AChE and BuCHE mRNAtranscription under the influence ofM officinalis extract andhuperzine A (Table 3) However a more significant differencein the level of transcript was seen in the frontal cortex inparticular in the case of BuCHE mRNA of experimentalrats (a decrease by 516 and 44 for MO and HU versuscontrol resp)The strong inhibition of transcription ofAChEmRNAwas also observed in the hippocampus ofMO-treatedrats (a decrease by 69) The BuChE mRNA transcriptionin animals receiving MO was lowered in a moderate way(decrease by 36)HuperzineA alone has not caused changesthat were observed in the group ofMO (Table 2) Our findingsmostly correlate with the observed changes in activities ofAChE and BuChE in different groups of animals In the caseof BuChE slightly different results between its activity andexpression profile were especially seen in the frontal cortexand hippocampus of animals receivingMO (Table 3)

To date there is a lack of published results of studiesmaking an attempt to clarify the potential differences in thetranscriptional profile and activity of AChE and BuChE

14 Evidence-Based Complementary and Alternative Medicine

under the influence of MO and its bioactive metabolites Itcannot be excluded that the key for the observed differencesin the level of AChE activities and mRNA level can becaused by changes in the activity of AChE in other regionsof the brain not analyzed in this study such as substantianigra cerebellum globus pallidus and hypothalamus whereit exerts nonenzymatic neuromodulatory functions affect-ing neurite outgrowth and synaptogenesis modulating theactivity of other proteins regional cerebral blood flow andother functions [64] But it is difficult to clearly explain whydiminishing of AChE and BuChEmRNAs did not always cor-relate with the lowering of activity of these enzymes althoughit has been recently noted that AChE activity was notparalleled by an increase in mRNA levels [65] The authorsexplained this fact by stating that AChE levels are regulatedat transcriptional posttranscriptional and posttranslationallevels leading to complex expression patterns which canbe modulated by physiological and pathological conditionsHowever these mechanisms are not fully understood andfurther studies are needed in this field

The cause of observed different degree of inhibition ofactivity and transcription status of studied genes especiallyof BuChE (and AChE) may lie in a so-called ldquonegative feed-backrdquo consisting of a complicated transcriptiontranslationregulation protein-protein interactionsmodifications and ametabolic network together forming a system that allows thecell to respond sensibly to the multiple signal molecules thatexist in its environment [66]

Because of that we propose that the reason for differencesbetween AChE and BuChE activities in the cortex and hip-pocampus under the MO may be due to the fact of insuf-ficiency of applied dose and the duration of the experi-ment affecting the transcriptional tissue-specific cellularmachinery regulating BuChE transcription without affectingits activity Moreover the administration of higher doses ofMO and extended period of time could lead to sufficientinhibition of its activity

There is a need to conduct further studies to determinethe molecular degree of dependence between changes inAChE and BuChE activities and activities of potential keyfactors regulating expression of these genes in the frontalcortex and hippocampus under the influence of extracts ofMO and active metabolites Another study determining theeffectiveness of their actions on the cholinergic system inexperimental animal models of memory impairment withparticular emphasis on scopolamine including the determi-nation of changes at the molecular level should be thereforecarried out

Alterations in BACE1 protein level have been proved inpostmortem brain tissue from individuals with AD withincreases decreases and also no change reported [1 67 68]An example of confirmation of these findings at the mRNAlevel is results of study by Coulson et al [69] Based onconducted studies some authors suggested that increasedBACE1mRNA transcription in remaining neuronal cells maycontribute to the increased BACE1 protein levels and activityfound in brain regions affected by AD [70]

Results of many studies concerning the elevated level ofBACE1 mRNA and protein in Alzheimerrsquos disease provide

direct and compelling reasons to develop therapies directed atBACE1 inhibition thus reducing120573-amyloid and its associatedtoxicities [67 68]

In our study we have observed that BACEmRNA expres-sion statistically significantly decreased after MO adminis-tration in frontal cortex and hippocampus These resultssuggest that the MO extract may act to inhibit BACE1mRNA level given the fact that the percentage of inhibitionof the expression (64 in the frontal cortex 50 in thehippocampus) is higher than that in the case of HU (38in the cortex and the lack of changes in the hippocampus)A careful analysis of the literature data shows that there areno studies which analyzed the impact of MO extract on theexpression level of BACE1 in Alzheimerrsquos disease

Furthermore a literature analysis does not indicatealready published results conducted by other teams attempt-ing to assess the impact of RA on the transcriptional activityof AChE BuChE and BACE1 Although several studies(already mentioned and others [70 71]) highlighted theRA and other caffeic acid derivatives capability of acetyl-cholinesterases inhibition none of these studies does nottouch the question of the molecular basis of their impacton the transcriptional machinery that regulates in vivo theexpression of studied genes Hence in our opinion obtainedby our team results they are one of the first of this typeand in general correlate with the results of our previousstudy [23] In this case there is no clear evidence explainingdifferent responses at the transcriptional level under theinfluence of RA especially in the case of BuChE encodinggene in the hippocampus (Table 2) It is possible that theobserved differentiation of BuChE transcriptional activitybetween the frontal cortex and the hippocampus may bedue to the differences of butyrylcholinesterase localizationand substrate affinity [72] Since in our experiment we havecarried out a quantitative analysis of AChE BuChE andBACE1 transcripts in brain homogenates of tested animalsrather than in individual isolated cell fractions therefore theobtained results constitute an overall ldquopicturerdquo of both studiedgenes transcriptional changes occurring in the brain areas ofstudied animals under the influence of the RA and the wholeplant extract as well

6 Conclusion

The subchronic administration ofMO led to an improvementof long-term memory of rats however the mechanisms ofMO action are probably more complicated since its role asa modulator of beta-secretase activity (due to inhibition ofBACE1 mRNA expression in frontal cortex) should be takeninto consideration

It should be noted that we have studied a crude extractfrom leaf of Melissa officinalis not a single pure chemicalcompound This plant extract is a complex mixture and itsaction may be a result of the summation of activities ofseveral components (synergismadditive action of caffeic acidwith salvianolic acids rosmarinic acid and others) In thecase of extract from leaves ofMelissa officinalis it is possiblethat interactions occur between the 40 chemical compoundsidentified by HPLC system

Evidence-Based Complementary and Alternative Medicine 15

Taken together it seems that the MO activity representsa possible option as complementary interventions to relievethe symptoms of mild dementia

Competing Interests

The authors declare that they have no competing interests

Acknowledgments

This work was supported by the Ministry of Science andHigher EducationWarsaw Poland from educational sources(2008ndash2011 Grant no N 405417836)

References

[1] S L Cole and R Vassar ldquoThe Alzheimerrsquos disease 120573-secretaseenzyme BACE1rdquo Molecular Neurodegeneration vol 2 no 1article 22 2007

[2] LACraigN SHong andR JMcDonald ldquoRevisiting the cho-linergic hypothesis in the development of Alzheimerrsquos diseaserdquoNeuroscience amp Biobehavioral Reviews vol 35 no 6 pp 1397ndash1409 2011

[3] N G M Gomes M G Campos J M C Orfao and C A FRibeiro ldquoPlants with neurobiological activity as potential tar-gets for drug discoveryrdquo Progress in Neuro-Psychopharmacologyamp Biological Psychiatry vol 33 no 8 pp 1372ndash1389 2009

[4] WHO WHO Monographs on Selected Medicinal PlantsmdashVolume 2 WHO Geneva Switzerland 2004 httpappswhointmedicinedocsendJs4927e18htmlJs4927e18

[5] M Bayat A A Tameh M H Ghahremani et al ldquoNeuropro-tective properties of Melissa officinalis after hypoxic-ischemicinjury both in vitro and in vivordquo DARU Journal of Pharmaceu-tical Sciences vol 20 article 42 2012

[6] J P Kamdem A Adeniran A A Boligon et al ldquoAntioxidantactivity genotoxicity and cytotoxicity evaluation of lemon balm(Melissa officinalis L) ethanolic extract Its potential role inneuroprotectionrdquo Industrial Crops and Products vol 51 pp 26ndash34 2013

[7] J-T Lin Y-C Chen Y-C Lee C-W R Hou F-L Chen andD-J Yang ldquoAntioxidant anti-proliferative and cyclooxygenase-2 inhibitory activities of ethanolic extracts from lemon balm(Melissa officinalis L) leavesrdquo LWTmdashFood Science and Technol-ogy vol 49 no 1 pp 1ndash7 2012

[8] G Guginski A P Luiz M D Silva et al ldquoMechanisms involvedin the antinociception caused by ethanolic extract obtainedfrom the leaves of Melissa officinalis (lemon balm) in micerdquoPharmacology Biochemistry amp Behavior vol 93 no 1 pp 10ndash162009

[9] N Perry G Court N Bidet J Court and E Perry ldquoEuropeanherbs with cholinergic activities potential in dementia therapyrdquoInternational Journal of Geriatric Psychiatry vol 11 no 12 pp1063ndash1069 1996

[10] E K Perry A T Pickering W W Wang P J Houghtonand N S L Perry ldquoMedicinal plants and Alzheimerrsquos diseasefrom ethnobotany to phytotherapyrdquo Journal of Pharmacy andPharmacology vol 51 no 5 pp 527ndash534 1999

[11] M-J R Howes N S L Perry and P J Houghton ldquoPlants withtraditional uses and activities relevant to the management ofAlzheimerrsquos disease and other cognitive disordersrdquo Phytother-apy Research vol 17 no 1 pp 1ndash18 2003

[12] I Orhan and M Aslan ldquoAppraisal of scopolamine-inducedantiamnesic effect in mice and in vitro antiacetylcholinesteraseand antioxidant activities of some traditionally used Lamiaceaeplantsrdquo Journal of Ethnopharmacology vol 122 no 2 pp 327ndash332 2009

[13] S Akhondzadeh M Noroozian M Mohammadi S OhadiniaA H Jamshidi and M Khani ldquoMelissa officinalis extract inthe treatment of patients with mild to moderate Alzheimerrsquosdisease a double blind randomised placebo controlled trialrdquoJournal of Neurology Neurosurgery and Psychiatry vol 74 no 7pp 863ndash866 2003

[14] D O Kennedy A B Scholey N T J Tildesley E K Perry andK AWesnes ldquoModulation ofmood and cognitive performancefollowing acute administration of Melissa officinalis (lemonbalm)rdquo Pharmacology Biochemistry amp Behavior vol 72 no 4pp 953ndash964 2002

[15] D O Kennedy G Wake S Savelev et al ldquoModulation of moodand cognitive performance following acute administration ofsingle doses of Melissa officinalis (Lemon balm) with humanCNS nicotinic and muscarinic receptor-binding propertiesrdquoNeuropsychopharmacology vol 28 no 10 pp 1871ndash1881 2003

[16] D O Kennedy and E L Wightman ldquoHerbal extracts and phy-tochemicals plant secondarymetabolites and the enhancementof human brain functionrdquoAdvances inNutrition vol 2 no 1 pp32ndash50 2011

[17] R P Pereira A A Boligon A S Appel et al ldquoChemical com-position antioxidant and anticholinesterase activity of Melissaofficinalisrdquo Industrial Crops and Products vol 53 pp 34ndash452014

[18] M Soodi N Naghdi H Hajimehdipoor S Choopani andE Sahraei ldquoMemory-improving activity of Melissa officinalisextract in naıve and scopolamine-treated ratsrdquo Research inPharmaceutical Sciences vol 9 no 2 pp 107ndash114 2014

[19] K Dastmalchi V Ollilainen P Lackman et al ldquoAcetyl-cholinesterase inhibitory guided fractionation of Melissa offic-inalis Lrdquo Bioorganic amp Medicinal Chemistry vol 17 no 2 pp867ndash871 2009

[20] W Chaiyana and S Okonogi ldquoInhibition of cholinesterase byessential oil from food plantrdquo Phytomedicine vol 19 no 8-9 pp836ndash839 2012

[21] K Dastmalchi H J D Dorman P P Oinonen Y Darwis ILaakso and R Hiltunen ldquoChemical composition and in vitroantioxidative activity of a lemon balm (Melissa officinalis L)extractrdquo LWTmdashFood Science and Technology vol 41 no 3 pp391ndash400 2008

[22] P Pereira D Tysca P Oliveira L F D S Brum J N Picadaand P Ardenghi ldquoNeurobehavioral and genotoxic aspects ofrosmarinic acidrdquo Pharmacological Research vol 52 no 3 pp199ndash203 2005

[23] M Ozarowski P L Mikolajczak A Bogacz et al ldquoRosmarinusofficinalis L leaf extract improves memory impairment andaffects acetylcholinesterase and butyrylcholinesterase activitiesin rat brainrdquo Fitoterapia vol 91 pp 261ndash271 2013

[24] G P Kumar and F Khanum ldquoNeuroprotective potential of phy-tochemicalsrdquo Pharmacognosy Reviews vol 6 no 12 pp 81ndash902012

[25] M Stobiecki A Staszkow A Piasecka P M Garcia-LopezF Zamora-Natera and P Kachlicki ldquoLC-MSMS profiling offlavonoid conjugates in wild mexican lupine Lupinus reflexusrdquoJournal of Natural Products vol 73 no 7 pp 1254ndash1260 2010

[26] A Wojakowska A Piasecka P M Garcıa-Lopez et al ldquoStruc-tural analysis and profiling of phenolic secondarymetabolites of

16 Evidence-Based Complementary and Alternative Medicine

Mexican lupine species using LC-MS techniquesrdquo Phytochem-istry vol 92 pp 71ndash86 2013

[27] A Gryszczynska B Opala Z Lowicki et al ldquoBioactive com-pounds determination in the callus and hydroalcoholic extractsfrom Salvia miltiorrhiza and Salvia przewalskiimdashpreliminarystudy on their anti-alcoholic activity effectsrdquo PhytochemistryLetters vol 11 pp 399ndash403 2015

[28] K Slinkart and V L Singleton ldquoTotal phenol analysis automa-tion and comparison with manual methodrdquo American Journalof Enology and Viticulture vol 28 no 1 pp 49ndash55 1977

[29] P J R Boissier J Tardy and J C Diverres ldquoUne nouvellemethode simple pour explorer lrsquoaction lsquotranquillisantersquo le testde la chemineerdquo Medicina Experimentalis vol 3 no 1 pp 81ndash84 1960

[30] R Ader J A Weijnen and P Moleman ldquoRetention of a passiveavoidance response as a function of the intensity and durationof electric shockrdquo Psychonomic Science vol 26 no 3 pp 125ndash128 1972

[31] E M Blalock K-C Chen K Sharrow et al ldquoGene microar-rays in hippocampal aging statistical profiling identifies novelprocesses correlated with cognitive impairmentrdquo Journal ofNeuroscience vol 23 no 9 pp 3807ndash3819 2003

[32] K Isomae S Morimoto H Hasegawa K Morita and JKamei ldquoEffects of T-82 a novel acetylcholinesterase inhibitoron impaired learning and memory in passive avoidance task inratsrdquo European Journal of Pharmacology vol 465 no 1-2 pp97ndash103 2003

[33] J Patora and B Klimek ldquoFlavonoids from lemon balm (Melissaofficinalis L Lamiaceae)rdquo Acta Poloniae Pharmaceutica-DrugResearch vol 59 no 2 pp 139ndash143 2002

[34] L Barros M Duenas M I Dias M J Sousa C Santos-Buelgaand I C F R Ferreira ldquoPhenolic profiles of cultivated invitro cultured and commercial samples of Melissa officinalis Linfusionsrdquo Food Chemistry vol 136 no 1 pp 1ndash8 2013

[35] T L Miron M Herrero and E Ibanez ldquoEnrichment of antiox-idant compounds from lemon balm (Melissa officinalis) bypressurized liquid extraction and enzyme-assisted extractionrdquoJournal of Chromatography A vol 1288 pp 1ndash9 2013

[36] Y Lu L Y Foo and H Wong ldquoSagecoumarin a novel caffeicacid trimer from Salvia officinalisrdquo Phytochemistry vol 52 no6 pp 1149ndash1152 1999

[37] S Abuhamdah LHuangM S J Elliott et al ldquoPharmacologicalprofile of an essential oil derived from Melissa officinalis withanti-agitation properties focus on ligand-gated channelsrdquo Jour-nal of Pharmacy and Pharmacology vol 60 no 3 pp 377ndash3842008

[38] G Wake J Court A Pickering R Lewis R Wilkins andE Perry ldquoCNS acetylcholine receptor activity in Europeanmedicinal plants traditionally used to improve failing memoryrdquoJournal of Ethnopharmacology vol 69 no 2 pp 105ndash114 2000

[39] S-H Ryu C Katona B Rive and G Livingston ldquoPersistenceof and changes in neuropsychiatric symptoms in Alzheimerdisease over 6months the LASER-AD studyrdquoAmerican Journalof Geriatric Psychiatry vol 13 no 11 pp 976ndash983 2005

[40] L N Gitlin H C Kales and C G Lyketsos ldquoNonpharma-cologic management of behavioral symptoms in dementiardquoJournal of the AmericanMedical Association vol 308 no 19 pp2020ndash2029 2012

[41] A Konczol J Muller E Foldes et al ldquoApplicability of a blood-brain barrier specific artificial membrane permeability assay atthe early stage of natural product-based CNS drug discoveryrdquoJournal of Natural Products vol 76 no 4 pp 655ndash663 2013

[42] Y-J Zhang L Wu Q-L Zhang J Li F-X Yin and Y YuanldquoPharmacokinetics of phenolic compounds of Danshen extractin rat blood and brain by microdialysis samplingrdquo Journal ofEthnopharmacology vol 136 no 1 pp 129ndash136 2011

[43] X Li C Yu Y Lu et al ldquoPharmacokinetics tissue distributionmetabolism and excretion of depside salts from Salvia miltior-rhiza in ratsrdquo Drug Metabolism and Disposition vol 35 no 2pp 234ndash239 2007

[44] H Xu Y Li X Che H Tian H Fan and K Liu ldquoMetabolismof salvianolic acid a and antioxidant activities of its methylatedmetabolitesrdquo Drug Metabolism and Disposition vol 42 no 2pp 274ndash281 2014

[45] D H Kim S J Park J M Kim et al ldquoCognitive dysfunctionsinduced by a cholinergic blockade and A120573 25-35 peptide areattenuated by salvianolic acid Brdquo Neuropharmacology vol 61no 8 pp 1432ndash1440 2011

[46] G-H Du Y Qiu and J-T Zhang ldquoSalvianolic acid B protectsthe memory functions against transient cerebral ischemia inmicerdquo Journal of Asian Natural Products Research vol 2 no 2pp 145ndash152 2000

[47] G Du and J Zhang ldquoProtective effects of salvianolic acid Aagainst impairment of memory induced by cerebral ischemia-reperfusion in micerdquo Chinese Medical Journal vol 110 no 1 pp65ndash68 1997

[48] J Anwar R M Spanevello G Thome et al ldquoEffects of caffeicacid on behavioral parameters and on the activity of acetyl-cholinesterase in different tissues from adult ratsrdquo Pharmacol-ogy Biochemistry and Behavior vol 103 no 2 pp 386ndash394 2012

[49] YW Lee D H Kim S J Jeon et al ldquoNeuroprotective effects ofsalvianolic acid B on anA12057325-35 peptide-inducedmousemodelof Alzheimerrsquos diseaserdquo European Journal of Pharmacology vol704 no 1ndash3 pp 70ndash77 2013

[50] J Zhong M-K Tang Y Zhang Q-P Xu and J-T ZhangldquoEffect of salvianolic acid B on neural cells damage and neu-rogenesis after brain ischemia-reperfusion in ratsrdquo Yao Xue XueBao vol 42 no 7 pp 716ndash721 2007

[51] P Zhuang Y Zhang G Cui et al ldquoDirect stimulation of adultneural stemprogenitor cells in vitro and neurogenesis in vivo bysalvianolic acid Brdquo PLoS ONE vol 7 no 4 Article ID e356362012

[52] M TangW Feng Y Zhang J Zhong and J Zhang ldquoSalvianolicacid B improves motor function after cerebral ischemia in ratsrdquoBehavioural Pharmacology vol 17 no 5-6 pp 493ndash498 2006

[53] Q Li L-P Han Z-H Li J-T Zhang and M-K Tang ldquoSal-vianolic acid B alleviate the disruption of blood-brain barrierin rats after cerebral ischemia-reperfusion by inhibiting MAPKpathwayrdquo Yao Xue Xue Bao vol 45 no 12 pp 1485ndash1490 2010

[54] Y-L LinH-J Tsay T-H Lai T-T Tzeng andY-J Shiao ldquoLith-ospermic acid attenuates 1-methyl-4-phenylpyridine-inducedneurotoxicity by blocking neuronal apoptotic and neuroinflam-matory pathwaysrdquo Journal of Biomedical Science vol 22 article37 2015

[55] M-K Tang and J-T Zhang ldquoSalvianolic acid B inhibits fibrilformation and neurotoxicity of amyloid beta-protein in vitrordquoActa Pharmacologica Sinica vol 22 no 4 pp 380ndash384 2001

[56] S S K Durairajan Q Yuan L Xie et al ldquoSalvianolic acid Binhibits A120573 fibril formation and disaggregates preformed fibrilsand protects against A120573-induced cytotoxictyrdquo NeurochemistryInternational vol 52 no 4-5 pp 741ndash750 2008

[57] FD Pinheiro Fernandes A P FonteneleMenezes J C de SousaNeves et al ldquoCaffeic acid protects mice from memory deficits

Evidence-Based Complementary and Alternative Medicine 17

induced by focal cerebral ischemiardquo Behavioural Pharmacologyvol 25 no 7 pp 637ndash647 2014

[58] S-J Tsai C-Y Chao andM-C Yin ldquoPreventive and therapeu-tic effects of caffeic acid against inflammatory injury in striatumof MPTP-treated micerdquo European Journal of Pharmacology vol670 no 2-3 pp 441ndash447 2011

[59] J H Kim QWang J M Choi S Lee and E J Cho ldquoProtectiverole of caffeic acid in an 119860120573

25minus35

-induced Alzheimerrsquos diseasemodelrdquo Nutrition Research and Practice vol 9 no 5 pp 480ndash488 2015

[60] G Liang B Shi W Luo and J Yang ldquoThe protective effect ofcaffeic acid on global cerebral ischemia-reperfusion injury inratsrdquo Behavioral and Brain Functions vol 11 no 1 article 182015

[61] D Y Yoo J H ChoiW Kim et al ldquoEffects of luteolin on spatialmemory cell proliferation and neuroblast differentiation in thehippocampal dentate gyrus in a scopolamine-induced amnesiamodelrdquo Neurological Research vol 35 no 8 pp 813ndash820 2013

[62] Y Liu X Tian L Gou L Sun X Ling and X Yin ldquoLuteolinattenuates diabetes-associated cognitive decline in ratsrdquo BrainResearch Bulletin vol 94 pp 23ndash29 2013

[63] S-M Liu Z-H Yang andX-B Sun ldquoSimultaneous determina-tion of six Salvia miltiorrhiza gradients in rat plasma and brainby LC-MSMSrdquo Zhongguo Zhongyao Zazhi vol 39 no 9 pp1704ndash1708 2014

[64] M A Papandreou A Dimakopoulou Z I Linardaki et alldquoEffect of a polyphenol-rich wild blueberry extract on cognitiveperformance of mice brain antioxidant markers and acetyl-cholinesterase activityrdquo Behavioural Brain Research vol 198 no2 pp 352ndash358 2009

[65] M-S Garcıa-Ayllon O Cauli M-X Silveyra et al ldquoBraincholinergic impairment in liver failurerdquo Brain vol 131 no 11pp 2946ndash2956 2008

[66] G K Ferreira M Carvalho-Silva C L Goncalves et al ldquoL-Tyrosine administration increases acetylcholinesterase activityin ratsrdquo Neurochemistry International vol 61 no 8 pp 1370ndash1374 2012

[67] R Vassar P-H Kuhn C Haass et al ldquoFunction therapeuticpotential and cell biology of BACE proteases current status andfuture prospectsrdquo Journal of Neurochemistry vol 130 no 1 pp4ndash28 2014

[68] R Vassar ldquoThe 120573-secretase BACE a prime drug target forAlzheimerrsquos diseaserdquo Journal of Molecular Neuroscience vol 17no 2 pp 157ndash170 2001

[69] D T R Coulson N Beyer J G Quinn et al ldquoBACE1 mRNAexpression in Alzheimerrsquos disease postmortem brain tissuerdquoJournal of Alzheimerrsquos Disease vol 22 no 4 pp 1111ndash1122 2010

[70] S Vladimir-Knezevic B Blazekovic M Kindl J Vladic A DLower-Nedza and A H Brantner ldquoAcetylcholinesterase inhib-itory antioxidant and phytochemical properties of selectedmedicinal plants of the lamiaceae familyrdquoMolecules vol 19 no1 pp 767ndash782 2014

[71] F Marcelo C Dias A Martins et al ldquoMolecular recognitionof rosmarinic acid from Salvia sclareoides extracts by ace-tylcholinesterase a new binding site detected by NMR spec-troscopyrdquo ChemistrymdashA European Journal vol 19 no 21 pp6641ndash6649 2013

[72] G Johnson and S W Moore ldquoWhy has butyrylcholinesterasebeen retained Structural and functional diversification in aduplicated generdquoNeurochemistry International vol 61 no 5 pp783ndash797 2012

[73] R J Grayer M R Eckert N C Veitch et al ldquoThe chemo-taxonomic significance of two bioactive caffeic acid estersnepetoidins A and B in the Lamiaceaerdquo Phytochemistry vol 64no 2 pp 519ndash528 2003

[74] A M D El-Mousallamy U W Hawas and S A M HusseinldquoTeucrol a decarboxyrosmarinic acid and its 41015840-O-triglycosideteucroside from Teucrium pilosumrdquo Phytochemistry vol 55 no8 pp 927ndash931 2000

[75] I Fecka and S Turek ldquoDetermination of water-soluble polyphe-nolic compounds in commercial herbal teas from Lamiaceaepeppermint melissa and sagerdquo Journal of Agricultural and FoodChemistry vol 55 no 26 pp 10908ndash10917 2007

[76] L W Sumner A Amberg D Barrett et al ldquoProposed min-imum reporting standards for chemical analysis WorkingGroup (CAWG) Metabolomics Standards Initiative (MSI)rdquoMetabolomics vol 3 pp 211ndash221 2007

Submit your manuscripts athttpwwwhindawicom

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Disease Markers

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Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

10 Evidence-Based Complementary and Alternative Medicine

Table 2 Effect ofMelissa officinalis leaf extract (200mgkg po) treatment on sedative activity motor coordination and memory in rats

Group 119899Locomotor activity

[number of impulses5min]Motor coordination

exit time [s]Short-term memorye

ORLong-term memory

latency [s]MC + H

2

O 18 390 plusmn 24 17 plusmn 3 040 plusmn 006 47 plusmn 14MC + SC 18 526 plusmn 48lowast 32 plusmn 5lowast 032 plusmn 005 12 plusmn 3lowast

MO + H2

O 10 231 plusmn 48lowast 32 plusmn 7lowast 043 plusmn 007 169 plusmn 11lowast

MO + SC 9 436 plusmn 60 43 plusmn 7 009 plusmn 011lowast 23 plusmn 6HU + H

2

O 9 515 plusmn 32 15 plusmn 2 037 plusmn 009 158 plusmn 14lowast

HU + SC 8 639 plusmn 71lowast 56 plusmn 3 022 plusmn 006 49 plusmn 18

RA + H2

O 8 406 plusmn 59 21 plusmn 5 045 plusmn 005 58 plusmn 28RA + SC 8 605 plusmn 55 28 plusmn 7 045 plusmn 005 20 plusmn 7Means plusmn SEM119899Number of animalsMC + H

2O control rats

SC scopolamine (05mgkg bw ip)HU huperzine A (05mgkg bw po)RA rosmarinic acid (10mgkg bw po)eExpressed as ratio OR = (119861 minus 119860lowast)(119861 + 119860) for details see Section 3lowastVersus MC + H

2O 119901 lt 005

Versus MC + SC 119901 lt 005

luteolin 31015840-O- and 7-O-glucuronide [33] It is impossible todistinguish both structures by mass spectrometry Only onechromatographic peak corresponding to the [M-H]minus ion at461073119898119911 was observed in our study Since luteolin 31015840-O-glucuronide was assigned as the most abundant flavonoid inlemon balm [33] we assumed that 28 corresponded to thisstructure

412 Flavonoids and Polyphenolic Acids The major com-pound from the 40 chemical compounds identified in hy-droethanolic MO leaf extract established by HPLC was RA(885 g100 g) (Table 1 Figure 1)

Other chemical compoundswith neuromodulatory activ-ities such as lithospermic acid (0042 g100 g) salvianolicacid A (0040 g100 g) salvianolic acid B (0023 g100 g) andcaffeic acid (0087 g100 g) were also documented in litera-ture Moreover the total polyphenolic compounds content ofMO determined with the use of FolinndashCiocalteu assay was3397 calculated as gallic acid The total hydroxycinnamicderivatives content expressed spectrophotometrically as ros-marinic acid was 2115 g100 g

413 Essential Oil Composition Hydroethanolic MO leafextract contained 008 of total essential oil The GCFIDanalysis showed that the extract comprised camphene(004) alfa-pinene (007) beta-pinene (1647) andmyrcene (1951) Moreover according to retention time16 compounds were identified as follows alfa-bisabololborneol carvone chamazulene cineole eugenol gamma-terpineol guaiazulene isopulegol linalool limonene men-thol menthyl acetate pulegone terpine and thymol Thesecompounds are present in the essential oil in trace amountswhich do not allow the quantitative interpretation

42 Cognitive and Behavioral Experiments

421 Locomotor Activity A one-way ANOVA analysisrevealed significant differences in the locomotor activity of

rats expressed as their horizontal spontaneous activity afterMO administration (ANOVA 119865(7 80) = 646 119901 lt 005)(Table 2) Detailed post hoc analysis showed thatMO + H

2O

decreased the locomotor activity of rats by 4031 but HU +H2O did not affect this activity when compared with control

group (MC + H2O) We observed also that RA + H

2O did

not change the locomotor activity of rats Stimulating effectsin the locomotor activity of rats were observed after an acuteSC injection (MC + SC versus MC + H

2O 119901 lt 005) and

this effect was observed in all SC-treated rats when comparedwith the proper non-SC-treated animals On the contrarythese SC-treated animals did not differ in comparison toanimals that received SC only (MO + SC versus MC + SC119901 gt 005 HU + SC versus MC + SC 119901 gt 005 RA + SCversus MC + SC 119901 gt 005)

422 Motor Coordination A one-way ANOVA analysisrevealed significant differences in motor coordination of ratsexpressed as their exit time from the cylinder (119865(5 73) =284 119901 lt 005) (Table 2) Detailed analysis showed that themultiple administration of RA + H

2O and HU + H

2O did

not affect significantly this paradigm when compared withcontrol rats (119901 gt 005) whereas MO treatment led to pro-longed exit time (MO + H

2O versus MC + H

2O 119901 lt 005)

Moreover generally SC-treated animals showed producedprolongation of exit time and the effects were statisticallysignificant not only in control groups (MC + SC versus MC +H2O 119901 lt 005) but also in RA-treated rats (RA + SC versus

MC + SC 119901 lt 005) However the rest of the SC-treatedanimals did not differ in comparison to animals receiving SConly (MO + SC versus MC + SC 119901 gt 005 HU + SC versusMC + SC 119901 gt 005)

423 Long-Term Memory A one-way ANOVA analysisrevealed significant differences in long-term memory afterusing a passive avoidance test (119865(7 77) = 201 119901 lt 005Table 2) It was shown that the strongest effect leading to

Evidence-Based Complementary and Alternative Medicine 11

Table 3 The effect ofMelissa officinalis leaf extract on acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) activities and AChEBuAChE or beta-secretase (BACE1) mRNA expression levels in frontal cortex (FC) or hippocampus (Hipp) of rats

Group119899Enzyme activity

[nmolminmg protein]mRNA expression

[]AChE BuChE ACHE BuChE BACE1

FC Hipp FC Hipp FC Hipp FC Hipp FC HippMC + H

2

O 363 plusmn 49 439 plusmn 73 65 plusmn 11 53 plusmn 8 100 plusmn 12 100 plusmn 11 100 plusmn 18 100 plusmn 11 100 plusmn 16 100 plusmn 8MO + H

2

O 276 plusmn 34 409 plusmn 28 69 plusmn 6 62 plusmn 4 48 plusmn 4lowast 31 plusmn 7lowast 16 plusmn 2lowast 64 plusmn 21amp 36 plusmn 3lowast 50 plusmn 8lowast

HU + H2

O 189 plusmn 15lowast 239 plusmn 15lowast 58 plusmn 6 51 plusmn 4 53 plusmn 13lowast 85 plusmn 5 42 plusmn 9lowast 102 plusmn 11 62 plusmn 6lowast 98 plusmn 4RA + H

2

O 224 plusmn 16lowast 251 plusmn 12lowast 77 plusmn 7 99 plusmn 7lowast 101 plusmn 12 103 plusmn 5 184 plusmn 31lowast 56 plusmn 7lowast 126 plusmn 13amp 98 plusmn 3Means plusmn SEM119899Number of animals 7ndash10Values expressed as a ratio the geneGAPDHMC + H

2O control group

HU huperzine A (05mgkg bw po)RA rosmarinic acid (10mgkg bw po)lowastampVersus MC + H

2O 119901 lt 005 or 119901 lt 007 respectively

an improvement of this paradigm was produced by extractof MO and HU but not RA when compared with controlanimals (MO + H

2O versus MC + H

2O 119901 lt 005 HU +

H2O versus MC + H

2O 119901 lt 005 RA + H

2O versus MC +

H2O 119901 gt 005) However the administration of SC to rats

significantly decreased the latency time of passive avoidancetask (MC + SC versus MC + H

2O 119901 lt 005) After MO or

RA combined treatment with SC no improvement of long-term memory was observed but HU given with SC showedenhancement of this paradigm in rats (HU + SC versusMC + SC 119901 lt 005) Therefore it can be concluded thatadministration of HU overcomes the effect shown by SC only(Table 1)

424 Short-Term Memory The results of the object recogni-tion test showed that an administration of the compoundsor extract did affect the ratsrsquo short-term memory (ANOVA119865(7 78) = 273 119901 lt 005) (Table 2)

Detailed post hoc analysis showed that only SC signifi-cantly decreased the short-term memory in MO-treated rats(MO + SC versusMO + H

2O 119901 lt 005MO + SC versus MC

+ SC 119901 lt 005) whereas the differences between the rest ofthe animals did not reach statistical significance (119901 gt 005)

43 AChE and BuChE Activities in Rat Brain A one-wayANOVA revealed significant differences between groups inthe activity of AChE in both the cortex and the hippocampus(frontal cortex 119865(3 27) = 565 119901 lt 005 hippocampusANOVA 119865(3 29) = 796 119901 lt 005) It was found out thatMOshowed an insignificant inhibition of AChE activity in thefrontal cortex by 24 when compared with control rats (MC+ H2O 119901 lt 006) and in the hippocampus by 7 (Table 3)

whereas HU produced a distinct significant inhibition ofAChE activity in comparison to control group by 48 (119901 lt005) and 47 (119901 lt 005) in the cortex and the hippocampusrespectively Also RA lowered significantly AChE activityboth in the cortex (38) and in the hippocampus (43)Moreover there were not significant differences between thevalues of BuChE activities for MO and HU when compared

with control group in the frontal cortex (ANOVA 119865(3 30) =101 119901 gt 005) whereas in the hippocampus the differencesreached statistical significance (ANOVA 119865(3 27) = 141119901 lt 005) Detailed analysis showed that only RA effect wassignificant and increased BuChE activity in the hippocampuswhen compared with the control rats (119901 lt 005)

44 AChE BuChE and BACE1 mRNA Level Changes in RatBrain A one-way ANOVA analysis revealed significant dif-ferences of AChE mRNA transcription profile in the cortex(ANOVA 119865(3 27) = 857 119901 lt 005) As shown in Table 3the multiple treatment of MO produced in the cortex astatistically significant decrease of AChEmRNA level by 52the administration of HU caused decrease of its level by 44(119901 lt 005) whereas RA did not affect this parameter whencompared to the control

There were significant differences between the relativevalues of BuChE mRNA levels in this region of brain in rats(frontal cortex ANOVA 119865(3 25) = 162 119901 lt 005) TheMOtreatment led to a decrease in the BuChEmRNA level by 84(versus MC + H

2O 119901 lt 005) the prolonged HU admin-

istration resulted in a decrease of the transcript level in thecortex by 58 (versus MC + H

2O 119901 lt 005) but RA

increased this parameter in the cortex by 84 (versus MC +H2O 119901 lt 005)The significant differences ofmRNA transcription level of

AChE mRNA level in the hippocampus (ANOVA 119865(3 31) =212 119901 lt 005) have been observed The detailed analysisshown that in the case of AChE afterMO treatment mRNAlevel significantly decreased by 69 (versus MC + H

2O

119901 lt 005) while the administration of HU resulted in astatistically insignificant decrease of AChE mRNA level by18 when compared with control group Also RA did notchange the level of transcript

In the case of BuChEmRNA expression in the hippocam-pus there were statistically significant differences betweengroups (ANOVA 119865(3 27) = 310 119901 lt 005) Detailed analysisshowed that in theMO + H

2O treated group the expression

lowered by 36 but the difference did not reach strong

12 Evidence-Based Complementary and Alternative Medicine

significance when compared with the control values (119901 lt007) and no change in the expression level of this enzymewas shown after the administration of HU On the contraryRA produced an increase of BuChE mRNA expression in thehippocampus by 44 (versus MC + H

2O 119901 lt 005)

Further analysis showed the significant differences inBACE1 mRNA expression in both brain regions of rats (inthe cortex and the hippocampus) (frontal cortex ANOVA119865(3 27) = 163 119901 lt 005 hippocampus ANOVA 119865(3 27) =133 119901 gt 005) It was observed that MO produced astatistically significant decrease of the BACE1 expression levelby 64 in the cortex (versus MC + H

2O 119901 lt 005) and by

50 in the hippocampus (versus MC + H2O 119901 lt 005) For

comparison HU treatment led to a decrease in the mRNAexpression level by 38 in the cortex (versus MC + H

2O

119901 lt 005) but not in the hippocampus On the contrary RAproduced an increase of this transcript in the cortex by 26but the effect did not reach a strong statistical significance(versus MC + H

2O 119901 lt 007) whereas in the hippocampus

there was no difference between RA and control group

5 Discussion

Cognitive and Behavioral Experiments The present studyinvestigated the influence of subchronic (28-fold) adminis-tration of standardized 50 EtOH extract ofMelisa officinalisleaf extract (MO) (200mgkg po containing 177mgkg ofRA) on SC-induced impairment of short-term and long-term memory in rats The results were compared with theactivity of cholinesterases (AChE and BuChE) as well aswith AChE BuChE and BACE1 gene expression levels in thecortex and hippocampus of the rat brain So far little evidenceis yet available as regards mechanisms of MO leaf extractaction that are potentially relevant to cognitive function ofrats after per os administration MO is traditionally used intreating neurological disorders through its anti-AChE [18]and antiagitation properties [37] Moreover Wake et al [38]and Kennedy et al [15] showed thatMO extract has nicotinicreceptor activity and that it can displace [3H]-(N)-nicotinefrom nicotinic receptors in homogenates of human cerebralcortex tissue and they suggested that these mechanisms canexplain activity of MO extract in amnesia model RecentlySoodi et al [18] observed that intraperitoneal injections ofMO extract (200mgkg) in rats could significantly enhancelearning and memory processes in animals since the extractsignificantly ameliorates SC-induced learning deficit in Mor-ris water maze test On the contrary in higher dose itcan be observed that MO extract (400mgkg) could notreverse SC-induced memory impairment [18] In our studyadministration of MO extract at a dose of 200mgkg (po)showed the effect leading to improvement of long-termmemory in a passive avoidance test in rats However afterMO combined treatment with SCMO did not overcome theimpairment shown by SC (Table 2) It should be emphasizedthat it is not clear whether the effect shown in our study byMO in non-SC-treated rats is specific since theMO treatmentproduced significant lowering of locomotor activity of ratstherefore the sedative profile ofMO cannot be excluded

On the other hand Ryu et al [39] observed that agitationand aggression are highly prevalent in patientswith dementiaAccording to Gitlin et al [40] nonpharmacological inter-ventions are recommended as first-line therapy Althoughantipsychotics have shown benefit for Alzheimerrsquos disease-related psychosis their use is associated with several seriousadverse effects [40]Thus it seems that the use ofMO extractcan provide dual benefits both in aspect of inhibition ofagitation and in improving the memory of patients withAlzheimerrsquos disease

Furthermore in our study RA in the dose of 10mgkgbw (po) did not affect either short- or long-term memoryalthough RA lowered significantly AChE activity both inthe cortex and in the hippocampus Moreover we observedthat the repeated administration of RA in non-scopolamine-treated rats did not produce any changes of locomotoractivity similarly to our previous study [23]

It is possible that other chemical compounds can influ-ence the memory in rats by synergic interactions in plantextract According to our calculations caffeic acid (0174mgin a single dose of extract administered to animals per kgbw) lithospermic acid (0084mgkg) salvianolic acid A(008mgkg) and salvianolic acid B (0046mgkg) may beresponsible for observed pharmacological effects On theother hand results from few studies showed that salvianolicacids do not cross the blood brain barrier (BBB) [41 42]and also lithospermic acid does not efficiently cross the BBB[43] For this reason the interpretation of our results is morecomplicated

Firstly Xu et al [44] demonstrated that Sal A is ametabol-ically unstable compound that would undergo rapid methy-lationmetabolism catalyzed by catechol O-methyltransferasein vivo into four major methylated metabolites of Sal A(3-O-methyl 31015840-O-methyl 3310158401015840-O-dimethyl and 31015840310158401015840-O-dimethyl salvianolic acid A) These generated O-methylatedmetabolites may be largely responsible for its in vivo phar-macological effects Although there are no available recentstudies on the ability of these compounds to pass the BBBsuch possibility should not be excluded

Secondly several studies showed central pharmacolog-ical effects in animals after per os administration of Sal B[45 46] Sal A [47] and caffeic acid [48] It was provedfor example that salvianolic acid B (10mgkg po) sig-nificantly rescued the A12057325ndash35 peptide-induced decreaseof choline acetyltransferase and brain-derived neurotrophicfactor protein levels in an amyloid 120573 (A120573) peptide-inducedAlzheimerrsquos disease mouse model [49] It also significantlyreversed (10mgkg po) the cognitive impairments inducedby scopolamine (1mgkg ip) or A120573 (25ndash35) (10 nmol5 120583Licv) injection inmice [45] Previous studies [46 47] showedalso that both Sal A and Sal B are able to improve theimpaired memory function induced by cerebral ischemia-reperfusion in miceThe stimulation of neurogenesis processin both subgranular zone (SGZ) and subventricular zone(SVZ) after brain ischemia and also alleviation neural cellsloss and improved motor function recovery after brainischemia in rats after the Sal B administration were alsoobserved by Zhong et al [50] Moreover the exposure to SalB can maintain the proliferation of neural stemprogenitor

Evidence-Based Complementary and Alternative Medicine 13

cells (NSPCs) after cerebral ischemia and improve cognitivepostischemic impairment after stroke in rats using Morriswater maze test therefore authors concluded that Sal B mayact as a potential drug in treatment of brain injury or neu-rodegenerative disease [51] Additionally the improvement ofmotor function after cerebral ischemia in rats after salvianolicacid B administrationwas also demonstrated [52]The clue topass through the BBB Sal B provided also results of Li et alindicating its protective effect on BBB in rats after cerebralischemia-reperfusion by inhibiting the MAPK pathway [53]In addition to this it was shown that lithospermic acid andsalvianolic acids exerted neuroprotective activity in variousexperimental models Lithospermic acid significantly attenu-ates neurotoxicity in vitro and in vivo induced by 1-methyl-4-phenylpyridin (MPP(+)) by blocking neuronal apoptotic andneuroinflammatory pathways [54] Salvianolic acid B inhib-ited amyloid beta-protein aggregation and fibril formation aswell as directly inhibiting the cellular toxicity of amyloid beta-protein in PC12 cells [55] and significantly reduced its cyto-toxic effects on human neuroblastoma SH-SY5Y cells [56]

For an explanation of our results observations of Pin-heiro Fernandes et al [57] may be very helpful whichshowed that caffeic acid nonflavanoid catecholic compoundwhose derivatives are occurring in MO extract improvedthe working spatial and long-term aversive memory deficitsinduced by focal cerebral ischemia in mice Anwar et al [48]showed also that caffeic acid (100mgkg) improved the step-down latencies in the inhibitory avoidance in rats Tsai et al[58] showed that caffeic acid is a potent neuroprotective agentin brain of 1-methyl-4-phenyl-1236-tetrahydropyridine(MPTP) treatedmiceMoreover caffeic acid improves A12057325ndash35-induced memory deficits and cognitive impairment inmice [59] In another study it was also shown that this com-pound has a significant protective effect on global cerebralischemia-reperfusion injury in rats [60] For instance 124 plusmn18mg100 g of caffeic acid was detected in the brain of micewith a diet containing 2 caffeic acid for 4 weeks [58] Thusthere is progressive evidence that this compound passesthrough the BBB and has a central pharmacological activity

Also results by Yoo et al are also worth noting [61] whichshowed also in a ratmodel of scopolamine-induced amnesiathat administration of luteolin (a common flavonoid frommany plants including M officinalis) at dose of 10mgkgcaused the increase in the brain-derived neurotrophic factor(BDNF) acetylcholine and the decrease in lipid peroxida-tion Liu et al [62] observed that chronic treatment withluteolin (50 and 100mgkg) improved neuronal injury andcognitive performance by attenuating oxidative stress andcholinesterase activity in streptozotocin-induced diabetes inrats In our study ethanolic extract of MO administered torats significantly improved long-term memory after using apassive avoidance test but afterMO combined treatmentwithSC no improvement of this paradigmwas observed (Table 2)

Thirdly available pharmacokinetic studies were carriedout for the pure compounds (Sal A and B) and the extractof the roots of Salvia miltiorrhiza [42 63] but there are notavailable studies of bioavailability of these compounds afteradministration of Melissa officinalis leaf extract containingother compounds as compared with the extract of Salvia

miltiorrhiza Moreover there is a lack of data about howscopolamine may influence the penetration of the salvianolicacids (and other compounds) across the BBB Hence thereis a need to study the pharmacokinetic parameters of thesecompounds in the group of animals treatedwith scopolaminein comparison with control group

AChEandBuChEActivities in Rat Brain To date several stud-ies have focused on explaining themechanismof action of theMS extract and its active compounds Soodi et al [18] showedthat treatment of animals withMO extract (400mgkg) priorto scopolamine injection could ameliorate scopolamine-induced enhancement in AChE activity This dose of MOextract inhibited the AChE activity in the hippocampus ofrats (519 versus 1004 in normal saline group 119901 lt 005914 in group treated with scopolamine +MO versus 1281in scopolamine group 119901 lt 005) In another study Anwaret al [48] showed that 50 and 100mgkg of caffeic aciddecreased in vivo the AChE activity in the cerebral cortexand striatum and increased the activity of this enzyme inthe cerebellum hippocampus hypothalamus pons lympho-cytes and muscles when compared to the control group(119901 lt 005) Our study showed that ethanolic extract of MOproduced an insignificant and slight inhibition of AChE andBuChE activity in the frontal cortex and in the hippocampus(especially) However it was observed previously [23] thatRA inhibited the AChE activity in the rats frontal cortex andhippocampusMoreover we showed that RA possess a strongstimulatory effect on BuChE in the hippocampus

AChE BuChE and BACE1mRNA Level Changes in Rat BrainSo far no results have been published of studies concerningthe in vivo assessment of changes in AChE BuChE andBACE1 gene expression profile in different brain regionsunder the influence of MO extracts or their key bioactivemetabolites Such studies focused overwhelmingly on theanalysis of their in vitro activities to a lesser extent in animalexperiments

In our study in the frontal cortex we have observedthe strongest inhibition of both AChE and BuCHE mRNAtranscription under the influence ofM officinalis extract andhuperzine A (Table 3) However a more significant differencein the level of transcript was seen in the frontal cortex inparticular in the case of BuCHE mRNA of experimentalrats (a decrease by 516 and 44 for MO and HU versuscontrol resp)The strong inhibition of transcription ofAChEmRNAwas also observed in the hippocampus ofMO-treatedrats (a decrease by 69) The BuChE mRNA transcriptionin animals receiving MO was lowered in a moderate way(decrease by 36)HuperzineA alone has not caused changesthat were observed in the group ofMO (Table 2) Our findingsmostly correlate with the observed changes in activities ofAChE and BuChE in different groups of animals In the caseof BuChE slightly different results between its activity andexpression profile were especially seen in the frontal cortexand hippocampus of animals receivingMO (Table 3)

To date there is a lack of published results of studiesmaking an attempt to clarify the potential differences in thetranscriptional profile and activity of AChE and BuChE

14 Evidence-Based Complementary and Alternative Medicine

under the influence of MO and its bioactive metabolites Itcannot be excluded that the key for the observed differencesin the level of AChE activities and mRNA level can becaused by changes in the activity of AChE in other regionsof the brain not analyzed in this study such as substantianigra cerebellum globus pallidus and hypothalamus whereit exerts nonenzymatic neuromodulatory functions affect-ing neurite outgrowth and synaptogenesis modulating theactivity of other proteins regional cerebral blood flow andother functions [64] But it is difficult to clearly explain whydiminishing of AChE and BuChEmRNAs did not always cor-relate with the lowering of activity of these enzymes althoughit has been recently noted that AChE activity was notparalleled by an increase in mRNA levels [65] The authorsexplained this fact by stating that AChE levels are regulatedat transcriptional posttranscriptional and posttranslationallevels leading to complex expression patterns which canbe modulated by physiological and pathological conditionsHowever these mechanisms are not fully understood andfurther studies are needed in this field

The cause of observed different degree of inhibition ofactivity and transcription status of studied genes especiallyof BuChE (and AChE) may lie in a so-called ldquonegative feed-backrdquo consisting of a complicated transcriptiontranslationregulation protein-protein interactionsmodifications and ametabolic network together forming a system that allows thecell to respond sensibly to the multiple signal molecules thatexist in its environment [66]

Because of that we propose that the reason for differencesbetween AChE and BuChE activities in the cortex and hip-pocampus under the MO may be due to the fact of insuf-ficiency of applied dose and the duration of the experi-ment affecting the transcriptional tissue-specific cellularmachinery regulating BuChE transcription without affectingits activity Moreover the administration of higher doses ofMO and extended period of time could lead to sufficientinhibition of its activity

There is a need to conduct further studies to determinethe molecular degree of dependence between changes inAChE and BuChE activities and activities of potential keyfactors regulating expression of these genes in the frontalcortex and hippocampus under the influence of extracts ofMO and active metabolites Another study determining theeffectiveness of their actions on the cholinergic system inexperimental animal models of memory impairment withparticular emphasis on scopolamine including the determi-nation of changes at the molecular level should be thereforecarried out

Alterations in BACE1 protein level have been proved inpostmortem brain tissue from individuals with AD withincreases decreases and also no change reported [1 67 68]An example of confirmation of these findings at the mRNAlevel is results of study by Coulson et al [69] Based onconducted studies some authors suggested that increasedBACE1mRNA transcription in remaining neuronal cells maycontribute to the increased BACE1 protein levels and activityfound in brain regions affected by AD [70]

Results of many studies concerning the elevated level ofBACE1 mRNA and protein in Alzheimerrsquos disease provide

direct and compelling reasons to develop therapies directed atBACE1 inhibition thus reducing120573-amyloid and its associatedtoxicities [67 68]

In our study we have observed that BACEmRNA expres-sion statistically significantly decreased after MO adminis-tration in frontal cortex and hippocampus These resultssuggest that the MO extract may act to inhibit BACE1mRNA level given the fact that the percentage of inhibitionof the expression (64 in the frontal cortex 50 in thehippocampus) is higher than that in the case of HU (38in the cortex and the lack of changes in the hippocampus)A careful analysis of the literature data shows that there areno studies which analyzed the impact of MO extract on theexpression level of BACE1 in Alzheimerrsquos disease

Furthermore a literature analysis does not indicatealready published results conducted by other teams attempt-ing to assess the impact of RA on the transcriptional activityof AChE BuChE and BACE1 Although several studies(already mentioned and others [70 71]) highlighted theRA and other caffeic acid derivatives capability of acetyl-cholinesterases inhibition none of these studies does nottouch the question of the molecular basis of their impacton the transcriptional machinery that regulates in vivo theexpression of studied genes Hence in our opinion obtainedby our team results they are one of the first of this typeand in general correlate with the results of our previousstudy [23] In this case there is no clear evidence explainingdifferent responses at the transcriptional level under theinfluence of RA especially in the case of BuChE encodinggene in the hippocampus (Table 2) It is possible that theobserved differentiation of BuChE transcriptional activitybetween the frontal cortex and the hippocampus may bedue to the differences of butyrylcholinesterase localizationand substrate affinity [72] Since in our experiment we havecarried out a quantitative analysis of AChE BuChE andBACE1 transcripts in brain homogenates of tested animalsrather than in individual isolated cell fractions therefore theobtained results constitute an overall ldquopicturerdquo of both studiedgenes transcriptional changes occurring in the brain areas ofstudied animals under the influence of the RA and the wholeplant extract as well

6 Conclusion

The subchronic administration ofMO led to an improvementof long-term memory of rats however the mechanisms ofMO action are probably more complicated since its role asa modulator of beta-secretase activity (due to inhibition ofBACE1 mRNA expression in frontal cortex) should be takeninto consideration

It should be noted that we have studied a crude extractfrom leaf of Melissa officinalis not a single pure chemicalcompound This plant extract is a complex mixture and itsaction may be a result of the summation of activities ofseveral components (synergismadditive action of caffeic acidwith salvianolic acids rosmarinic acid and others) In thecase of extract from leaves ofMelissa officinalis it is possiblethat interactions occur between the 40 chemical compoundsidentified by HPLC system

Evidence-Based Complementary and Alternative Medicine 15

Taken together it seems that the MO activity representsa possible option as complementary interventions to relievethe symptoms of mild dementia

Competing Interests

The authors declare that they have no competing interests

Acknowledgments

This work was supported by the Ministry of Science andHigher EducationWarsaw Poland from educational sources(2008ndash2011 Grant no N 405417836)

References

[1] S L Cole and R Vassar ldquoThe Alzheimerrsquos disease 120573-secretaseenzyme BACE1rdquo Molecular Neurodegeneration vol 2 no 1article 22 2007

[2] LACraigN SHong andR JMcDonald ldquoRevisiting the cho-linergic hypothesis in the development of Alzheimerrsquos diseaserdquoNeuroscience amp Biobehavioral Reviews vol 35 no 6 pp 1397ndash1409 2011

[3] N G M Gomes M G Campos J M C Orfao and C A FRibeiro ldquoPlants with neurobiological activity as potential tar-gets for drug discoveryrdquo Progress in Neuro-Psychopharmacologyamp Biological Psychiatry vol 33 no 8 pp 1372ndash1389 2009

[4] WHO WHO Monographs on Selected Medicinal PlantsmdashVolume 2 WHO Geneva Switzerland 2004 httpappswhointmedicinedocsendJs4927e18htmlJs4927e18

[5] M Bayat A A Tameh M H Ghahremani et al ldquoNeuropro-tective properties of Melissa officinalis after hypoxic-ischemicinjury both in vitro and in vivordquo DARU Journal of Pharmaceu-tical Sciences vol 20 article 42 2012

[6] J P Kamdem A Adeniran A A Boligon et al ldquoAntioxidantactivity genotoxicity and cytotoxicity evaluation of lemon balm(Melissa officinalis L) ethanolic extract Its potential role inneuroprotectionrdquo Industrial Crops and Products vol 51 pp 26ndash34 2013

[7] J-T Lin Y-C Chen Y-C Lee C-W R Hou F-L Chen andD-J Yang ldquoAntioxidant anti-proliferative and cyclooxygenase-2 inhibitory activities of ethanolic extracts from lemon balm(Melissa officinalis L) leavesrdquo LWTmdashFood Science and Technol-ogy vol 49 no 1 pp 1ndash7 2012

[8] G Guginski A P Luiz M D Silva et al ldquoMechanisms involvedin the antinociception caused by ethanolic extract obtainedfrom the leaves of Melissa officinalis (lemon balm) in micerdquoPharmacology Biochemistry amp Behavior vol 93 no 1 pp 10ndash162009

[9] N Perry G Court N Bidet J Court and E Perry ldquoEuropeanherbs with cholinergic activities potential in dementia therapyrdquoInternational Journal of Geriatric Psychiatry vol 11 no 12 pp1063ndash1069 1996

[10] E K Perry A T Pickering W W Wang P J Houghtonand N S L Perry ldquoMedicinal plants and Alzheimerrsquos diseasefrom ethnobotany to phytotherapyrdquo Journal of Pharmacy andPharmacology vol 51 no 5 pp 527ndash534 1999

[11] M-J R Howes N S L Perry and P J Houghton ldquoPlants withtraditional uses and activities relevant to the management ofAlzheimerrsquos disease and other cognitive disordersrdquo Phytother-apy Research vol 17 no 1 pp 1ndash18 2003

[12] I Orhan and M Aslan ldquoAppraisal of scopolamine-inducedantiamnesic effect in mice and in vitro antiacetylcholinesteraseand antioxidant activities of some traditionally used Lamiaceaeplantsrdquo Journal of Ethnopharmacology vol 122 no 2 pp 327ndash332 2009

[13] S Akhondzadeh M Noroozian M Mohammadi S OhadiniaA H Jamshidi and M Khani ldquoMelissa officinalis extract inthe treatment of patients with mild to moderate Alzheimerrsquosdisease a double blind randomised placebo controlled trialrdquoJournal of Neurology Neurosurgery and Psychiatry vol 74 no 7pp 863ndash866 2003

[14] D O Kennedy A B Scholey N T J Tildesley E K Perry andK AWesnes ldquoModulation ofmood and cognitive performancefollowing acute administration of Melissa officinalis (lemonbalm)rdquo Pharmacology Biochemistry amp Behavior vol 72 no 4pp 953ndash964 2002

[15] D O Kennedy G Wake S Savelev et al ldquoModulation of moodand cognitive performance following acute administration ofsingle doses of Melissa officinalis (Lemon balm) with humanCNS nicotinic and muscarinic receptor-binding propertiesrdquoNeuropsychopharmacology vol 28 no 10 pp 1871ndash1881 2003

[16] D O Kennedy and E L Wightman ldquoHerbal extracts and phy-tochemicals plant secondarymetabolites and the enhancementof human brain functionrdquoAdvances inNutrition vol 2 no 1 pp32ndash50 2011

[17] R P Pereira A A Boligon A S Appel et al ldquoChemical com-position antioxidant and anticholinesterase activity of Melissaofficinalisrdquo Industrial Crops and Products vol 53 pp 34ndash452014

[18] M Soodi N Naghdi H Hajimehdipoor S Choopani andE Sahraei ldquoMemory-improving activity of Melissa officinalisextract in naıve and scopolamine-treated ratsrdquo Research inPharmaceutical Sciences vol 9 no 2 pp 107ndash114 2014

[19] K Dastmalchi V Ollilainen P Lackman et al ldquoAcetyl-cholinesterase inhibitory guided fractionation of Melissa offic-inalis Lrdquo Bioorganic amp Medicinal Chemistry vol 17 no 2 pp867ndash871 2009

[20] W Chaiyana and S Okonogi ldquoInhibition of cholinesterase byessential oil from food plantrdquo Phytomedicine vol 19 no 8-9 pp836ndash839 2012

[21] K Dastmalchi H J D Dorman P P Oinonen Y Darwis ILaakso and R Hiltunen ldquoChemical composition and in vitroantioxidative activity of a lemon balm (Melissa officinalis L)extractrdquo LWTmdashFood Science and Technology vol 41 no 3 pp391ndash400 2008

[22] P Pereira D Tysca P Oliveira L F D S Brum J N Picadaand P Ardenghi ldquoNeurobehavioral and genotoxic aspects ofrosmarinic acidrdquo Pharmacological Research vol 52 no 3 pp199ndash203 2005

[23] M Ozarowski P L Mikolajczak A Bogacz et al ldquoRosmarinusofficinalis L leaf extract improves memory impairment andaffects acetylcholinesterase and butyrylcholinesterase activitiesin rat brainrdquo Fitoterapia vol 91 pp 261ndash271 2013

[24] G P Kumar and F Khanum ldquoNeuroprotective potential of phy-tochemicalsrdquo Pharmacognosy Reviews vol 6 no 12 pp 81ndash902012

[25] M Stobiecki A Staszkow A Piasecka P M Garcia-LopezF Zamora-Natera and P Kachlicki ldquoLC-MSMS profiling offlavonoid conjugates in wild mexican lupine Lupinus reflexusrdquoJournal of Natural Products vol 73 no 7 pp 1254ndash1260 2010

[26] A Wojakowska A Piasecka P M Garcıa-Lopez et al ldquoStruc-tural analysis and profiling of phenolic secondarymetabolites of

16 Evidence-Based Complementary and Alternative Medicine

Mexican lupine species using LC-MS techniquesrdquo Phytochem-istry vol 92 pp 71ndash86 2013

[27] A Gryszczynska B Opala Z Lowicki et al ldquoBioactive com-pounds determination in the callus and hydroalcoholic extractsfrom Salvia miltiorrhiza and Salvia przewalskiimdashpreliminarystudy on their anti-alcoholic activity effectsrdquo PhytochemistryLetters vol 11 pp 399ndash403 2015

[28] K Slinkart and V L Singleton ldquoTotal phenol analysis automa-tion and comparison with manual methodrdquo American Journalof Enology and Viticulture vol 28 no 1 pp 49ndash55 1977

[29] P J R Boissier J Tardy and J C Diverres ldquoUne nouvellemethode simple pour explorer lrsquoaction lsquotranquillisantersquo le testde la chemineerdquo Medicina Experimentalis vol 3 no 1 pp 81ndash84 1960

[30] R Ader J A Weijnen and P Moleman ldquoRetention of a passiveavoidance response as a function of the intensity and durationof electric shockrdquo Psychonomic Science vol 26 no 3 pp 125ndash128 1972

[31] E M Blalock K-C Chen K Sharrow et al ldquoGene microar-rays in hippocampal aging statistical profiling identifies novelprocesses correlated with cognitive impairmentrdquo Journal ofNeuroscience vol 23 no 9 pp 3807ndash3819 2003

[32] K Isomae S Morimoto H Hasegawa K Morita and JKamei ldquoEffects of T-82 a novel acetylcholinesterase inhibitoron impaired learning and memory in passive avoidance task inratsrdquo European Journal of Pharmacology vol 465 no 1-2 pp97ndash103 2003

[33] J Patora and B Klimek ldquoFlavonoids from lemon balm (Melissaofficinalis L Lamiaceae)rdquo Acta Poloniae Pharmaceutica-DrugResearch vol 59 no 2 pp 139ndash143 2002

[34] L Barros M Duenas M I Dias M J Sousa C Santos-Buelgaand I C F R Ferreira ldquoPhenolic profiles of cultivated invitro cultured and commercial samples of Melissa officinalis Linfusionsrdquo Food Chemistry vol 136 no 1 pp 1ndash8 2013

[35] T L Miron M Herrero and E Ibanez ldquoEnrichment of antiox-idant compounds from lemon balm (Melissa officinalis) bypressurized liquid extraction and enzyme-assisted extractionrdquoJournal of Chromatography A vol 1288 pp 1ndash9 2013

[36] Y Lu L Y Foo and H Wong ldquoSagecoumarin a novel caffeicacid trimer from Salvia officinalisrdquo Phytochemistry vol 52 no6 pp 1149ndash1152 1999

[37] S Abuhamdah LHuangM S J Elliott et al ldquoPharmacologicalprofile of an essential oil derived from Melissa officinalis withanti-agitation properties focus on ligand-gated channelsrdquo Jour-nal of Pharmacy and Pharmacology vol 60 no 3 pp 377ndash3842008

[38] G Wake J Court A Pickering R Lewis R Wilkins andE Perry ldquoCNS acetylcholine receptor activity in Europeanmedicinal plants traditionally used to improve failing memoryrdquoJournal of Ethnopharmacology vol 69 no 2 pp 105ndash114 2000

[39] S-H Ryu C Katona B Rive and G Livingston ldquoPersistenceof and changes in neuropsychiatric symptoms in Alzheimerdisease over 6months the LASER-AD studyrdquoAmerican Journalof Geriatric Psychiatry vol 13 no 11 pp 976ndash983 2005

[40] L N Gitlin H C Kales and C G Lyketsos ldquoNonpharma-cologic management of behavioral symptoms in dementiardquoJournal of the AmericanMedical Association vol 308 no 19 pp2020ndash2029 2012

[41] A Konczol J Muller E Foldes et al ldquoApplicability of a blood-brain barrier specific artificial membrane permeability assay atthe early stage of natural product-based CNS drug discoveryrdquoJournal of Natural Products vol 76 no 4 pp 655ndash663 2013

[42] Y-J Zhang L Wu Q-L Zhang J Li F-X Yin and Y YuanldquoPharmacokinetics of phenolic compounds of Danshen extractin rat blood and brain by microdialysis samplingrdquo Journal ofEthnopharmacology vol 136 no 1 pp 129ndash136 2011

[43] X Li C Yu Y Lu et al ldquoPharmacokinetics tissue distributionmetabolism and excretion of depside salts from Salvia miltior-rhiza in ratsrdquo Drug Metabolism and Disposition vol 35 no 2pp 234ndash239 2007

[44] H Xu Y Li X Che H Tian H Fan and K Liu ldquoMetabolismof salvianolic acid a and antioxidant activities of its methylatedmetabolitesrdquo Drug Metabolism and Disposition vol 42 no 2pp 274ndash281 2014

[45] D H Kim S J Park J M Kim et al ldquoCognitive dysfunctionsinduced by a cholinergic blockade and A120573 25-35 peptide areattenuated by salvianolic acid Brdquo Neuropharmacology vol 61no 8 pp 1432ndash1440 2011

[46] G-H Du Y Qiu and J-T Zhang ldquoSalvianolic acid B protectsthe memory functions against transient cerebral ischemia inmicerdquo Journal of Asian Natural Products Research vol 2 no 2pp 145ndash152 2000

[47] G Du and J Zhang ldquoProtective effects of salvianolic acid Aagainst impairment of memory induced by cerebral ischemia-reperfusion in micerdquo Chinese Medical Journal vol 110 no 1 pp65ndash68 1997

[48] J Anwar R M Spanevello G Thome et al ldquoEffects of caffeicacid on behavioral parameters and on the activity of acetyl-cholinesterase in different tissues from adult ratsrdquo Pharmacol-ogy Biochemistry and Behavior vol 103 no 2 pp 386ndash394 2012

[49] YW Lee D H Kim S J Jeon et al ldquoNeuroprotective effects ofsalvianolic acid B on anA12057325-35 peptide-inducedmousemodelof Alzheimerrsquos diseaserdquo European Journal of Pharmacology vol704 no 1ndash3 pp 70ndash77 2013

[50] J Zhong M-K Tang Y Zhang Q-P Xu and J-T ZhangldquoEffect of salvianolic acid B on neural cells damage and neu-rogenesis after brain ischemia-reperfusion in ratsrdquo Yao Xue XueBao vol 42 no 7 pp 716ndash721 2007

[51] P Zhuang Y Zhang G Cui et al ldquoDirect stimulation of adultneural stemprogenitor cells in vitro and neurogenesis in vivo bysalvianolic acid Brdquo PLoS ONE vol 7 no 4 Article ID e356362012

[52] M TangW Feng Y Zhang J Zhong and J Zhang ldquoSalvianolicacid B improves motor function after cerebral ischemia in ratsrdquoBehavioural Pharmacology vol 17 no 5-6 pp 493ndash498 2006

[53] Q Li L-P Han Z-H Li J-T Zhang and M-K Tang ldquoSal-vianolic acid B alleviate the disruption of blood-brain barrierin rats after cerebral ischemia-reperfusion by inhibiting MAPKpathwayrdquo Yao Xue Xue Bao vol 45 no 12 pp 1485ndash1490 2010

[54] Y-L LinH-J Tsay T-H Lai T-T Tzeng andY-J Shiao ldquoLith-ospermic acid attenuates 1-methyl-4-phenylpyridine-inducedneurotoxicity by blocking neuronal apoptotic and neuroinflam-matory pathwaysrdquo Journal of Biomedical Science vol 22 article37 2015

[55] M-K Tang and J-T Zhang ldquoSalvianolic acid B inhibits fibrilformation and neurotoxicity of amyloid beta-protein in vitrordquoActa Pharmacologica Sinica vol 22 no 4 pp 380ndash384 2001

[56] S S K Durairajan Q Yuan L Xie et al ldquoSalvianolic acid Binhibits A120573 fibril formation and disaggregates preformed fibrilsand protects against A120573-induced cytotoxictyrdquo NeurochemistryInternational vol 52 no 4-5 pp 741ndash750 2008

[57] FD Pinheiro Fernandes A P FonteneleMenezes J C de SousaNeves et al ldquoCaffeic acid protects mice from memory deficits

Evidence-Based Complementary and Alternative Medicine 17

induced by focal cerebral ischemiardquo Behavioural Pharmacologyvol 25 no 7 pp 637ndash647 2014

[58] S-J Tsai C-Y Chao andM-C Yin ldquoPreventive and therapeu-tic effects of caffeic acid against inflammatory injury in striatumof MPTP-treated micerdquo European Journal of Pharmacology vol670 no 2-3 pp 441ndash447 2011

[59] J H Kim QWang J M Choi S Lee and E J Cho ldquoProtectiverole of caffeic acid in an 119860120573

25minus35

-induced Alzheimerrsquos diseasemodelrdquo Nutrition Research and Practice vol 9 no 5 pp 480ndash488 2015

[60] G Liang B Shi W Luo and J Yang ldquoThe protective effect ofcaffeic acid on global cerebral ischemia-reperfusion injury inratsrdquo Behavioral and Brain Functions vol 11 no 1 article 182015

[61] D Y Yoo J H ChoiW Kim et al ldquoEffects of luteolin on spatialmemory cell proliferation and neuroblast differentiation in thehippocampal dentate gyrus in a scopolamine-induced amnesiamodelrdquo Neurological Research vol 35 no 8 pp 813ndash820 2013

[62] Y Liu X Tian L Gou L Sun X Ling and X Yin ldquoLuteolinattenuates diabetes-associated cognitive decline in ratsrdquo BrainResearch Bulletin vol 94 pp 23ndash29 2013

[63] S-M Liu Z-H Yang andX-B Sun ldquoSimultaneous determina-tion of six Salvia miltiorrhiza gradients in rat plasma and brainby LC-MSMSrdquo Zhongguo Zhongyao Zazhi vol 39 no 9 pp1704ndash1708 2014

[64] M A Papandreou A Dimakopoulou Z I Linardaki et alldquoEffect of a polyphenol-rich wild blueberry extract on cognitiveperformance of mice brain antioxidant markers and acetyl-cholinesterase activityrdquo Behavioural Brain Research vol 198 no2 pp 352ndash358 2009

[65] M-S Garcıa-Ayllon O Cauli M-X Silveyra et al ldquoBraincholinergic impairment in liver failurerdquo Brain vol 131 no 11pp 2946ndash2956 2008

[66] G K Ferreira M Carvalho-Silva C L Goncalves et al ldquoL-Tyrosine administration increases acetylcholinesterase activityin ratsrdquo Neurochemistry International vol 61 no 8 pp 1370ndash1374 2012

[67] R Vassar P-H Kuhn C Haass et al ldquoFunction therapeuticpotential and cell biology of BACE proteases current status andfuture prospectsrdquo Journal of Neurochemistry vol 130 no 1 pp4ndash28 2014

[68] R Vassar ldquoThe 120573-secretase BACE a prime drug target forAlzheimerrsquos diseaserdquo Journal of Molecular Neuroscience vol 17no 2 pp 157ndash170 2001

[69] D T R Coulson N Beyer J G Quinn et al ldquoBACE1 mRNAexpression in Alzheimerrsquos disease postmortem brain tissuerdquoJournal of Alzheimerrsquos Disease vol 22 no 4 pp 1111ndash1122 2010

[70] S Vladimir-Knezevic B Blazekovic M Kindl J Vladic A DLower-Nedza and A H Brantner ldquoAcetylcholinesterase inhib-itory antioxidant and phytochemical properties of selectedmedicinal plants of the lamiaceae familyrdquoMolecules vol 19 no1 pp 767ndash782 2014

[71] F Marcelo C Dias A Martins et al ldquoMolecular recognitionof rosmarinic acid from Salvia sclareoides extracts by ace-tylcholinesterase a new binding site detected by NMR spec-troscopyrdquo ChemistrymdashA European Journal vol 19 no 21 pp6641ndash6649 2013

[72] G Johnson and S W Moore ldquoWhy has butyrylcholinesterasebeen retained Structural and functional diversification in aduplicated generdquoNeurochemistry International vol 61 no 5 pp783ndash797 2012

[73] R J Grayer M R Eckert N C Veitch et al ldquoThe chemo-taxonomic significance of two bioactive caffeic acid estersnepetoidins A and B in the Lamiaceaerdquo Phytochemistry vol 64no 2 pp 519ndash528 2003

[74] A M D El-Mousallamy U W Hawas and S A M HusseinldquoTeucrol a decarboxyrosmarinic acid and its 41015840-O-triglycosideteucroside from Teucrium pilosumrdquo Phytochemistry vol 55 no8 pp 927ndash931 2000

[75] I Fecka and S Turek ldquoDetermination of water-soluble polyphe-nolic compounds in commercial herbal teas from Lamiaceaepeppermint melissa and sagerdquo Journal of Agricultural and FoodChemistry vol 55 no 26 pp 10908ndash10917 2007

[76] L W Sumner A Amberg D Barrett et al ldquoProposed min-imum reporting standards for chemical analysis WorkingGroup (CAWG) Metabolomics Standards Initiative (MSI)rdquoMetabolomics vol 3 pp 211ndash221 2007

Submit your manuscripts athttpwwwhindawicom

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Behavioural Neurology

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Disease Markers

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Oxidative Medicine and Cellular Longevity

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Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Evidence-Based Complementary and Alternative Medicine 11

Table 3 The effect ofMelissa officinalis leaf extract on acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) activities and AChEBuAChE or beta-secretase (BACE1) mRNA expression levels in frontal cortex (FC) or hippocampus (Hipp) of rats

Group119899Enzyme activity

[nmolminmg protein]mRNA expression

[]AChE BuChE ACHE BuChE BACE1

FC Hipp FC Hipp FC Hipp FC Hipp FC HippMC + H

2

O 363 plusmn 49 439 plusmn 73 65 plusmn 11 53 plusmn 8 100 plusmn 12 100 plusmn 11 100 plusmn 18 100 plusmn 11 100 plusmn 16 100 plusmn 8MO + H

2

O 276 plusmn 34 409 plusmn 28 69 plusmn 6 62 plusmn 4 48 plusmn 4lowast 31 plusmn 7lowast 16 plusmn 2lowast 64 plusmn 21amp 36 plusmn 3lowast 50 plusmn 8lowast

HU + H2

O 189 plusmn 15lowast 239 plusmn 15lowast 58 plusmn 6 51 plusmn 4 53 plusmn 13lowast 85 plusmn 5 42 plusmn 9lowast 102 plusmn 11 62 plusmn 6lowast 98 plusmn 4RA + H

2

O 224 plusmn 16lowast 251 plusmn 12lowast 77 plusmn 7 99 plusmn 7lowast 101 plusmn 12 103 plusmn 5 184 plusmn 31lowast 56 plusmn 7lowast 126 plusmn 13amp 98 plusmn 3Means plusmn SEM119899Number of animals 7ndash10Values expressed as a ratio the geneGAPDHMC + H

2O control group

HU huperzine A (05mgkg bw po)RA rosmarinic acid (10mgkg bw po)lowastampVersus MC + H

2O 119901 lt 005 or 119901 lt 007 respectively

an improvement of this paradigm was produced by extractof MO and HU but not RA when compared with controlanimals (MO + H

2O versus MC + H

2O 119901 lt 005 HU +

H2O versus MC + H

2O 119901 lt 005 RA + H

2O versus MC +

H2O 119901 gt 005) However the administration of SC to rats

significantly decreased the latency time of passive avoidancetask (MC + SC versus MC + H

2O 119901 lt 005) After MO or

RA combined treatment with SC no improvement of long-term memory was observed but HU given with SC showedenhancement of this paradigm in rats (HU + SC versusMC + SC 119901 lt 005) Therefore it can be concluded thatadministration of HU overcomes the effect shown by SC only(Table 1)

424 Short-Term Memory The results of the object recogni-tion test showed that an administration of the compoundsor extract did affect the ratsrsquo short-term memory (ANOVA119865(7 78) = 273 119901 lt 005) (Table 2)

Detailed post hoc analysis showed that only SC signifi-cantly decreased the short-term memory in MO-treated rats(MO + SC versusMO + H

2O 119901 lt 005MO + SC versus MC

+ SC 119901 lt 005) whereas the differences between the rest ofthe animals did not reach statistical significance (119901 gt 005)

43 AChE and BuChE Activities in Rat Brain A one-wayANOVA revealed significant differences between groups inthe activity of AChE in both the cortex and the hippocampus(frontal cortex 119865(3 27) = 565 119901 lt 005 hippocampusANOVA 119865(3 29) = 796 119901 lt 005) It was found out thatMOshowed an insignificant inhibition of AChE activity in thefrontal cortex by 24 when compared with control rats (MC+ H2O 119901 lt 006) and in the hippocampus by 7 (Table 3)

whereas HU produced a distinct significant inhibition ofAChE activity in comparison to control group by 48 (119901 lt005) and 47 (119901 lt 005) in the cortex and the hippocampusrespectively Also RA lowered significantly AChE activityboth in the cortex (38) and in the hippocampus (43)Moreover there were not significant differences between thevalues of BuChE activities for MO and HU when compared

with control group in the frontal cortex (ANOVA 119865(3 30) =101 119901 gt 005) whereas in the hippocampus the differencesreached statistical significance (ANOVA 119865(3 27) = 141119901 lt 005) Detailed analysis showed that only RA effect wassignificant and increased BuChE activity in the hippocampuswhen compared with the control rats (119901 lt 005)

44 AChE BuChE and BACE1 mRNA Level Changes in RatBrain A one-way ANOVA analysis revealed significant dif-ferences of AChE mRNA transcription profile in the cortex(ANOVA 119865(3 27) = 857 119901 lt 005) As shown in Table 3the multiple treatment of MO produced in the cortex astatistically significant decrease of AChEmRNA level by 52the administration of HU caused decrease of its level by 44(119901 lt 005) whereas RA did not affect this parameter whencompared to the control

There were significant differences between the relativevalues of BuChE mRNA levels in this region of brain in rats(frontal cortex ANOVA 119865(3 25) = 162 119901 lt 005) TheMOtreatment led to a decrease in the BuChEmRNA level by 84(versus MC + H

2O 119901 lt 005) the prolonged HU admin-

istration resulted in a decrease of the transcript level in thecortex by 58 (versus MC + H

2O 119901 lt 005) but RA

increased this parameter in the cortex by 84 (versus MC +H2O 119901 lt 005)The significant differences ofmRNA transcription level of

AChE mRNA level in the hippocampus (ANOVA 119865(3 31) =212 119901 lt 005) have been observed The detailed analysisshown that in the case of AChE afterMO treatment mRNAlevel significantly decreased by 69 (versus MC + H

2O

119901 lt 005) while the administration of HU resulted in astatistically insignificant decrease of AChE mRNA level by18 when compared with control group Also RA did notchange the level of transcript

In the case of BuChEmRNA expression in the hippocam-pus there were statistically significant differences betweengroups (ANOVA 119865(3 27) = 310 119901 lt 005) Detailed analysisshowed that in theMO + H

2O treated group the expression

lowered by 36 but the difference did not reach strong

12 Evidence-Based Complementary and Alternative Medicine

significance when compared with the control values (119901 lt007) and no change in the expression level of this enzymewas shown after the administration of HU On the contraryRA produced an increase of BuChE mRNA expression in thehippocampus by 44 (versus MC + H

2O 119901 lt 005)

Further analysis showed the significant differences inBACE1 mRNA expression in both brain regions of rats (inthe cortex and the hippocampus) (frontal cortex ANOVA119865(3 27) = 163 119901 lt 005 hippocampus ANOVA 119865(3 27) =133 119901 gt 005) It was observed that MO produced astatistically significant decrease of the BACE1 expression levelby 64 in the cortex (versus MC + H

2O 119901 lt 005) and by

50 in the hippocampus (versus MC + H2O 119901 lt 005) For

comparison HU treatment led to a decrease in the mRNAexpression level by 38 in the cortex (versus MC + H

2O

119901 lt 005) but not in the hippocampus On the contrary RAproduced an increase of this transcript in the cortex by 26but the effect did not reach a strong statistical significance(versus MC + H

2O 119901 lt 007) whereas in the hippocampus

there was no difference between RA and control group

5 Discussion

Cognitive and Behavioral Experiments The present studyinvestigated the influence of subchronic (28-fold) adminis-tration of standardized 50 EtOH extract ofMelisa officinalisleaf extract (MO) (200mgkg po containing 177mgkg ofRA) on SC-induced impairment of short-term and long-term memory in rats The results were compared with theactivity of cholinesterases (AChE and BuChE) as well aswith AChE BuChE and BACE1 gene expression levels in thecortex and hippocampus of the rat brain So far little evidenceis yet available as regards mechanisms of MO leaf extractaction that are potentially relevant to cognitive function ofrats after per os administration MO is traditionally used intreating neurological disorders through its anti-AChE [18]and antiagitation properties [37] Moreover Wake et al [38]and Kennedy et al [15] showed thatMO extract has nicotinicreceptor activity and that it can displace [3H]-(N)-nicotinefrom nicotinic receptors in homogenates of human cerebralcortex tissue and they suggested that these mechanisms canexplain activity of MO extract in amnesia model RecentlySoodi et al [18] observed that intraperitoneal injections ofMO extract (200mgkg) in rats could significantly enhancelearning and memory processes in animals since the extractsignificantly ameliorates SC-induced learning deficit in Mor-ris water maze test On the contrary in higher dose itcan be observed that MO extract (400mgkg) could notreverse SC-induced memory impairment [18] In our studyadministration of MO extract at a dose of 200mgkg (po)showed the effect leading to improvement of long-termmemory in a passive avoidance test in rats However afterMO combined treatment with SCMO did not overcome theimpairment shown by SC (Table 2) It should be emphasizedthat it is not clear whether the effect shown in our study byMO in non-SC-treated rats is specific since theMO treatmentproduced significant lowering of locomotor activity of ratstherefore the sedative profile ofMO cannot be excluded

On the other hand Ryu et al [39] observed that agitationand aggression are highly prevalent in patientswith dementiaAccording to Gitlin et al [40] nonpharmacological inter-ventions are recommended as first-line therapy Althoughantipsychotics have shown benefit for Alzheimerrsquos disease-related psychosis their use is associated with several seriousadverse effects [40]Thus it seems that the use ofMO extractcan provide dual benefits both in aspect of inhibition ofagitation and in improving the memory of patients withAlzheimerrsquos disease

Furthermore in our study RA in the dose of 10mgkgbw (po) did not affect either short- or long-term memoryalthough RA lowered significantly AChE activity both inthe cortex and in the hippocampus Moreover we observedthat the repeated administration of RA in non-scopolamine-treated rats did not produce any changes of locomotoractivity similarly to our previous study [23]

It is possible that other chemical compounds can influ-ence the memory in rats by synergic interactions in plantextract According to our calculations caffeic acid (0174mgin a single dose of extract administered to animals per kgbw) lithospermic acid (0084mgkg) salvianolic acid A(008mgkg) and salvianolic acid B (0046mgkg) may beresponsible for observed pharmacological effects On theother hand results from few studies showed that salvianolicacids do not cross the blood brain barrier (BBB) [41 42]and also lithospermic acid does not efficiently cross the BBB[43] For this reason the interpretation of our results is morecomplicated

Firstly Xu et al [44] demonstrated that Sal A is ametabol-ically unstable compound that would undergo rapid methy-lationmetabolism catalyzed by catechol O-methyltransferasein vivo into four major methylated metabolites of Sal A(3-O-methyl 31015840-O-methyl 3310158401015840-O-dimethyl and 31015840310158401015840-O-dimethyl salvianolic acid A) These generated O-methylatedmetabolites may be largely responsible for its in vivo phar-macological effects Although there are no available recentstudies on the ability of these compounds to pass the BBBsuch possibility should not be excluded

Secondly several studies showed central pharmacolog-ical effects in animals after per os administration of Sal B[45 46] Sal A [47] and caffeic acid [48] It was provedfor example that salvianolic acid B (10mgkg po) sig-nificantly rescued the A12057325ndash35 peptide-induced decreaseof choline acetyltransferase and brain-derived neurotrophicfactor protein levels in an amyloid 120573 (A120573) peptide-inducedAlzheimerrsquos disease mouse model [49] It also significantlyreversed (10mgkg po) the cognitive impairments inducedby scopolamine (1mgkg ip) or A120573 (25ndash35) (10 nmol5 120583Licv) injection inmice [45] Previous studies [46 47] showedalso that both Sal A and Sal B are able to improve theimpaired memory function induced by cerebral ischemia-reperfusion in miceThe stimulation of neurogenesis processin both subgranular zone (SGZ) and subventricular zone(SVZ) after brain ischemia and also alleviation neural cellsloss and improved motor function recovery after brainischemia in rats after the Sal B administration were alsoobserved by Zhong et al [50] Moreover the exposure to SalB can maintain the proliferation of neural stemprogenitor

Evidence-Based Complementary and Alternative Medicine 13

cells (NSPCs) after cerebral ischemia and improve cognitivepostischemic impairment after stroke in rats using Morriswater maze test therefore authors concluded that Sal B mayact as a potential drug in treatment of brain injury or neu-rodegenerative disease [51] Additionally the improvement ofmotor function after cerebral ischemia in rats after salvianolicacid B administrationwas also demonstrated [52]The clue topass through the BBB Sal B provided also results of Li et alindicating its protective effect on BBB in rats after cerebralischemia-reperfusion by inhibiting the MAPK pathway [53]In addition to this it was shown that lithospermic acid andsalvianolic acids exerted neuroprotective activity in variousexperimental models Lithospermic acid significantly attenu-ates neurotoxicity in vitro and in vivo induced by 1-methyl-4-phenylpyridin (MPP(+)) by blocking neuronal apoptotic andneuroinflammatory pathways [54] Salvianolic acid B inhib-ited amyloid beta-protein aggregation and fibril formation aswell as directly inhibiting the cellular toxicity of amyloid beta-protein in PC12 cells [55] and significantly reduced its cyto-toxic effects on human neuroblastoma SH-SY5Y cells [56]

For an explanation of our results observations of Pin-heiro Fernandes et al [57] may be very helpful whichshowed that caffeic acid nonflavanoid catecholic compoundwhose derivatives are occurring in MO extract improvedthe working spatial and long-term aversive memory deficitsinduced by focal cerebral ischemia in mice Anwar et al [48]showed also that caffeic acid (100mgkg) improved the step-down latencies in the inhibitory avoidance in rats Tsai et al[58] showed that caffeic acid is a potent neuroprotective agentin brain of 1-methyl-4-phenyl-1236-tetrahydropyridine(MPTP) treatedmiceMoreover caffeic acid improves A12057325ndash35-induced memory deficits and cognitive impairment inmice [59] In another study it was also shown that this com-pound has a significant protective effect on global cerebralischemia-reperfusion injury in rats [60] For instance 124 plusmn18mg100 g of caffeic acid was detected in the brain of micewith a diet containing 2 caffeic acid for 4 weeks [58] Thusthere is progressive evidence that this compound passesthrough the BBB and has a central pharmacological activity

Also results by Yoo et al are also worth noting [61] whichshowed also in a ratmodel of scopolamine-induced amnesiathat administration of luteolin (a common flavonoid frommany plants including M officinalis) at dose of 10mgkgcaused the increase in the brain-derived neurotrophic factor(BDNF) acetylcholine and the decrease in lipid peroxida-tion Liu et al [62] observed that chronic treatment withluteolin (50 and 100mgkg) improved neuronal injury andcognitive performance by attenuating oxidative stress andcholinesterase activity in streptozotocin-induced diabetes inrats In our study ethanolic extract of MO administered torats significantly improved long-term memory after using apassive avoidance test but afterMO combined treatmentwithSC no improvement of this paradigmwas observed (Table 2)

Thirdly available pharmacokinetic studies were carriedout for the pure compounds (Sal A and B) and the extractof the roots of Salvia miltiorrhiza [42 63] but there are notavailable studies of bioavailability of these compounds afteradministration of Melissa officinalis leaf extract containingother compounds as compared with the extract of Salvia

miltiorrhiza Moreover there is a lack of data about howscopolamine may influence the penetration of the salvianolicacids (and other compounds) across the BBB Hence thereis a need to study the pharmacokinetic parameters of thesecompounds in the group of animals treatedwith scopolaminein comparison with control group

AChEandBuChEActivities in Rat Brain To date several stud-ies have focused on explaining themechanismof action of theMS extract and its active compounds Soodi et al [18] showedthat treatment of animals withMO extract (400mgkg) priorto scopolamine injection could ameliorate scopolamine-induced enhancement in AChE activity This dose of MOextract inhibited the AChE activity in the hippocampus ofrats (519 versus 1004 in normal saline group 119901 lt 005914 in group treated with scopolamine +MO versus 1281in scopolamine group 119901 lt 005) In another study Anwaret al [48] showed that 50 and 100mgkg of caffeic aciddecreased in vivo the AChE activity in the cerebral cortexand striatum and increased the activity of this enzyme inthe cerebellum hippocampus hypothalamus pons lympho-cytes and muscles when compared to the control group(119901 lt 005) Our study showed that ethanolic extract of MOproduced an insignificant and slight inhibition of AChE andBuChE activity in the frontal cortex and in the hippocampus(especially) However it was observed previously [23] thatRA inhibited the AChE activity in the rats frontal cortex andhippocampusMoreover we showed that RA possess a strongstimulatory effect on BuChE in the hippocampus

AChE BuChE and BACE1mRNA Level Changes in Rat BrainSo far no results have been published of studies concerningthe in vivo assessment of changes in AChE BuChE andBACE1 gene expression profile in different brain regionsunder the influence of MO extracts or their key bioactivemetabolites Such studies focused overwhelmingly on theanalysis of their in vitro activities to a lesser extent in animalexperiments

In our study in the frontal cortex we have observedthe strongest inhibition of both AChE and BuCHE mRNAtranscription under the influence ofM officinalis extract andhuperzine A (Table 3) However a more significant differencein the level of transcript was seen in the frontal cortex inparticular in the case of BuCHE mRNA of experimentalrats (a decrease by 516 and 44 for MO and HU versuscontrol resp)The strong inhibition of transcription ofAChEmRNAwas also observed in the hippocampus ofMO-treatedrats (a decrease by 69) The BuChE mRNA transcriptionin animals receiving MO was lowered in a moderate way(decrease by 36)HuperzineA alone has not caused changesthat were observed in the group ofMO (Table 2) Our findingsmostly correlate with the observed changes in activities ofAChE and BuChE in different groups of animals In the caseof BuChE slightly different results between its activity andexpression profile were especially seen in the frontal cortexand hippocampus of animals receivingMO (Table 3)

To date there is a lack of published results of studiesmaking an attempt to clarify the potential differences in thetranscriptional profile and activity of AChE and BuChE

14 Evidence-Based Complementary and Alternative Medicine

under the influence of MO and its bioactive metabolites Itcannot be excluded that the key for the observed differencesin the level of AChE activities and mRNA level can becaused by changes in the activity of AChE in other regionsof the brain not analyzed in this study such as substantianigra cerebellum globus pallidus and hypothalamus whereit exerts nonenzymatic neuromodulatory functions affect-ing neurite outgrowth and synaptogenesis modulating theactivity of other proteins regional cerebral blood flow andother functions [64] But it is difficult to clearly explain whydiminishing of AChE and BuChEmRNAs did not always cor-relate with the lowering of activity of these enzymes althoughit has been recently noted that AChE activity was notparalleled by an increase in mRNA levels [65] The authorsexplained this fact by stating that AChE levels are regulatedat transcriptional posttranscriptional and posttranslationallevels leading to complex expression patterns which canbe modulated by physiological and pathological conditionsHowever these mechanisms are not fully understood andfurther studies are needed in this field

The cause of observed different degree of inhibition ofactivity and transcription status of studied genes especiallyof BuChE (and AChE) may lie in a so-called ldquonegative feed-backrdquo consisting of a complicated transcriptiontranslationregulation protein-protein interactionsmodifications and ametabolic network together forming a system that allows thecell to respond sensibly to the multiple signal molecules thatexist in its environment [66]

Because of that we propose that the reason for differencesbetween AChE and BuChE activities in the cortex and hip-pocampus under the MO may be due to the fact of insuf-ficiency of applied dose and the duration of the experi-ment affecting the transcriptional tissue-specific cellularmachinery regulating BuChE transcription without affectingits activity Moreover the administration of higher doses ofMO and extended period of time could lead to sufficientinhibition of its activity

There is a need to conduct further studies to determinethe molecular degree of dependence between changes inAChE and BuChE activities and activities of potential keyfactors regulating expression of these genes in the frontalcortex and hippocampus under the influence of extracts ofMO and active metabolites Another study determining theeffectiveness of their actions on the cholinergic system inexperimental animal models of memory impairment withparticular emphasis on scopolamine including the determi-nation of changes at the molecular level should be thereforecarried out

Alterations in BACE1 protein level have been proved inpostmortem brain tissue from individuals with AD withincreases decreases and also no change reported [1 67 68]An example of confirmation of these findings at the mRNAlevel is results of study by Coulson et al [69] Based onconducted studies some authors suggested that increasedBACE1mRNA transcription in remaining neuronal cells maycontribute to the increased BACE1 protein levels and activityfound in brain regions affected by AD [70]

Results of many studies concerning the elevated level ofBACE1 mRNA and protein in Alzheimerrsquos disease provide

direct and compelling reasons to develop therapies directed atBACE1 inhibition thus reducing120573-amyloid and its associatedtoxicities [67 68]

In our study we have observed that BACEmRNA expres-sion statistically significantly decreased after MO adminis-tration in frontal cortex and hippocampus These resultssuggest that the MO extract may act to inhibit BACE1mRNA level given the fact that the percentage of inhibitionof the expression (64 in the frontal cortex 50 in thehippocampus) is higher than that in the case of HU (38in the cortex and the lack of changes in the hippocampus)A careful analysis of the literature data shows that there areno studies which analyzed the impact of MO extract on theexpression level of BACE1 in Alzheimerrsquos disease

Furthermore a literature analysis does not indicatealready published results conducted by other teams attempt-ing to assess the impact of RA on the transcriptional activityof AChE BuChE and BACE1 Although several studies(already mentioned and others [70 71]) highlighted theRA and other caffeic acid derivatives capability of acetyl-cholinesterases inhibition none of these studies does nottouch the question of the molecular basis of their impacton the transcriptional machinery that regulates in vivo theexpression of studied genes Hence in our opinion obtainedby our team results they are one of the first of this typeand in general correlate with the results of our previousstudy [23] In this case there is no clear evidence explainingdifferent responses at the transcriptional level under theinfluence of RA especially in the case of BuChE encodinggene in the hippocampus (Table 2) It is possible that theobserved differentiation of BuChE transcriptional activitybetween the frontal cortex and the hippocampus may bedue to the differences of butyrylcholinesterase localizationand substrate affinity [72] Since in our experiment we havecarried out a quantitative analysis of AChE BuChE andBACE1 transcripts in brain homogenates of tested animalsrather than in individual isolated cell fractions therefore theobtained results constitute an overall ldquopicturerdquo of both studiedgenes transcriptional changes occurring in the brain areas ofstudied animals under the influence of the RA and the wholeplant extract as well

6 Conclusion

The subchronic administration ofMO led to an improvementof long-term memory of rats however the mechanisms ofMO action are probably more complicated since its role asa modulator of beta-secretase activity (due to inhibition ofBACE1 mRNA expression in frontal cortex) should be takeninto consideration

It should be noted that we have studied a crude extractfrom leaf of Melissa officinalis not a single pure chemicalcompound This plant extract is a complex mixture and itsaction may be a result of the summation of activities ofseveral components (synergismadditive action of caffeic acidwith salvianolic acids rosmarinic acid and others) In thecase of extract from leaves ofMelissa officinalis it is possiblethat interactions occur between the 40 chemical compoundsidentified by HPLC system

Evidence-Based Complementary and Alternative Medicine 15

Taken together it seems that the MO activity representsa possible option as complementary interventions to relievethe symptoms of mild dementia

Competing Interests

The authors declare that they have no competing interests

Acknowledgments

This work was supported by the Ministry of Science andHigher EducationWarsaw Poland from educational sources(2008ndash2011 Grant no N 405417836)

References

[1] S L Cole and R Vassar ldquoThe Alzheimerrsquos disease 120573-secretaseenzyme BACE1rdquo Molecular Neurodegeneration vol 2 no 1article 22 2007

[2] LACraigN SHong andR JMcDonald ldquoRevisiting the cho-linergic hypothesis in the development of Alzheimerrsquos diseaserdquoNeuroscience amp Biobehavioral Reviews vol 35 no 6 pp 1397ndash1409 2011

[3] N G M Gomes M G Campos J M C Orfao and C A FRibeiro ldquoPlants with neurobiological activity as potential tar-gets for drug discoveryrdquo Progress in Neuro-Psychopharmacologyamp Biological Psychiatry vol 33 no 8 pp 1372ndash1389 2009

[4] WHO WHO Monographs on Selected Medicinal PlantsmdashVolume 2 WHO Geneva Switzerland 2004 httpappswhointmedicinedocsendJs4927e18htmlJs4927e18

[5] M Bayat A A Tameh M H Ghahremani et al ldquoNeuropro-tective properties of Melissa officinalis after hypoxic-ischemicinjury both in vitro and in vivordquo DARU Journal of Pharmaceu-tical Sciences vol 20 article 42 2012

[6] J P Kamdem A Adeniran A A Boligon et al ldquoAntioxidantactivity genotoxicity and cytotoxicity evaluation of lemon balm(Melissa officinalis L) ethanolic extract Its potential role inneuroprotectionrdquo Industrial Crops and Products vol 51 pp 26ndash34 2013

[7] J-T Lin Y-C Chen Y-C Lee C-W R Hou F-L Chen andD-J Yang ldquoAntioxidant anti-proliferative and cyclooxygenase-2 inhibitory activities of ethanolic extracts from lemon balm(Melissa officinalis L) leavesrdquo LWTmdashFood Science and Technol-ogy vol 49 no 1 pp 1ndash7 2012

[8] G Guginski A P Luiz M D Silva et al ldquoMechanisms involvedin the antinociception caused by ethanolic extract obtainedfrom the leaves of Melissa officinalis (lemon balm) in micerdquoPharmacology Biochemistry amp Behavior vol 93 no 1 pp 10ndash162009

[9] N Perry G Court N Bidet J Court and E Perry ldquoEuropeanherbs with cholinergic activities potential in dementia therapyrdquoInternational Journal of Geriatric Psychiatry vol 11 no 12 pp1063ndash1069 1996

[10] E K Perry A T Pickering W W Wang P J Houghtonand N S L Perry ldquoMedicinal plants and Alzheimerrsquos diseasefrom ethnobotany to phytotherapyrdquo Journal of Pharmacy andPharmacology vol 51 no 5 pp 527ndash534 1999

[11] M-J R Howes N S L Perry and P J Houghton ldquoPlants withtraditional uses and activities relevant to the management ofAlzheimerrsquos disease and other cognitive disordersrdquo Phytother-apy Research vol 17 no 1 pp 1ndash18 2003

[12] I Orhan and M Aslan ldquoAppraisal of scopolamine-inducedantiamnesic effect in mice and in vitro antiacetylcholinesteraseand antioxidant activities of some traditionally used Lamiaceaeplantsrdquo Journal of Ethnopharmacology vol 122 no 2 pp 327ndash332 2009

[13] S Akhondzadeh M Noroozian M Mohammadi S OhadiniaA H Jamshidi and M Khani ldquoMelissa officinalis extract inthe treatment of patients with mild to moderate Alzheimerrsquosdisease a double blind randomised placebo controlled trialrdquoJournal of Neurology Neurosurgery and Psychiatry vol 74 no 7pp 863ndash866 2003

[14] D O Kennedy A B Scholey N T J Tildesley E K Perry andK AWesnes ldquoModulation ofmood and cognitive performancefollowing acute administration of Melissa officinalis (lemonbalm)rdquo Pharmacology Biochemistry amp Behavior vol 72 no 4pp 953ndash964 2002

[15] D O Kennedy G Wake S Savelev et al ldquoModulation of moodand cognitive performance following acute administration ofsingle doses of Melissa officinalis (Lemon balm) with humanCNS nicotinic and muscarinic receptor-binding propertiesrdquoNeuropsychopharmacology vol 28 no 10 pp 1871ndash1881 2003

[16] D O Kennedy and E L Wightman ldquoHerbal extracts and phy-tochemicals plant secondarymetabolites and the enhancementof human brain functionrdquoAdvances inNutrition vol 2 no 1 pp32ndash50 2011

[17] R P Pereira A A Boligon A S Appel et al ldquoChemical com-position antioxidant and anticholinesterase activity of Melissaofficinalisrdquo Industrial Crops and Products vol 53 pp 34ndash452014

[18] M Soodi N Naghdi H Hajimehdipoor S Choopani andE Sahraei ldquoMemory-improving activity of Melissa officinalisextract in naıve and scopolamine-treated ratsrdquo Research inPharmaceutical Sciences vol 9 no 2 pp 107ndash114 2014

[19] K Dastmalchi V Ollilainen P Lackman et al ldquoAcetyl-cholinesterase inhibitory guided fractionation of Melissa offic-inalis Lrdquo Bioorganic amp Medicinal Chemistry vol 17 no 2 pp867ndash871 2009

[20] W Chaiyana and S Okonogi ldquoInhibition of cholinesterase byessential oil from food plantrdquo Phytomedicine vol 19 no 8-9 pp836ndash839 2012

[21] K Dastmalchi H J D Dorman P P Oinonen Y Darwis ILaakso and R Hiltunen ldquoChemical composition and in vitroantioxidative activity of a lemon balm (Melissa officinalis L)extractrdquo LWTmdashFood Science and Technology vol 41 no 3 pp391ndash400 2008

[22] P Pereira D Tysca P Oliveira L F D S Brum J N Picadaand P Ardenghi ldquoNeurobehavioral and genotoxic aspects ofrosmarinic acidrdquo Pharmacological Research vol 52 no 3 pp199ndash203 2005

[23] M Ozarowski P L Mikolajczak A Bogacz et al ldquoRosmarinusofficinalis L leaf extract improves memory impairment andaffects acetylcholinesterase and butyrylcholinesterase activitiesin rat brainrdquo Fitoterapia vol 91 pp 261ndash271 2013

[24] G P Kumar and F Khanum ldquoNeuroprotective potential of phy-tochemicalsrdquo Pharmacognosy Reviews vol 6 no 12 pp 81ndash902012

[25] M Stobiecki A Staszkow A Piasecka P M Garcia-LopezF Zamora-Natera and P Kachlicki ldquoLC-MSMS profiling offlavonoid conjugates in wild mexican lupine Lupinus reflexusrdquoJournal of Natural Products vol 73 no 7 pp 1254ndash1260 2010

[26] A Wojakowska A Piasecka P M Garcıa-Lopez et al ldquoStruc-tural analysis and profiling of phenolic secondarymetabolites of

16 Evidence-Based Complementary and Alternative Medicine

Mexican lupine species using LC-MS techniquesrdquo Phytochem-istry vol 92 pp 71ndash86 2013

[27] A Gryszczynska B Opala Z Lowicki et al ldquoBioactive com-pounds determination in the callus and hydroalcoholic extractsfrom Salvia miltiorrhiza and Salvia przewalskiimdashpreliminarystudy on their anti-alcoholic activity effectsrdquo PhytochemistryLetters vol 11 pp 399ndash403 2015

[28] K Slinkart and V L Singleton ldquoTotal phenol analysis automa-tion and comparison with manual methodrdquo American Journalof Enology and Viticulture vol 28 no 1 pp 49ndash55 1977

[29] P J R Boissier J Tardy and J C Diverres ldquoUne nouvellemethode simple pour explorer lrsquoaction lsquotranquillisantersquo le testde la chemineerdquo Medicina Experimentalis vol 3 no 1 pp 81ndash84 1960

[30] R Ader J A Weijnen and P Moleman ldquoRetention of a passiveavoidance response as a function of the intensity and durationof electric shockrdquo Psychonomic Science vol 26 no 3 pp 125ndash128 1972

[31] E M Blalock K-C Chen K Sharrow et al ldquoGene microar-rays in hippocampal aging statistical profiling identifies novelprocesses correlated with cognitive impairmentrdquo Journal ofNeuroscience vol 23 no 9 pp 3807ndash3819 2003

[32] K Isomae S Morimoto H Hasegawa K Morita and JKamei ldquoEffects of T-82 a novel acetylcholinesterase inhibitoron impaired learning and memory in passive avoidance task inratsrdquo European Journal of Pharmacology vol 465 no 1-2 pp97ndash103 2003

[33] J Patora and B Klimek ldquoFlavonoids from lemon balm (Melissaofficinalis L Lamiaceae)rdquo Acta Poloniae Pharmaceutica-DrugResearch vol 59 no 2 pp 139ndash143 2002

[34] L Barros M Duenas M I Dias M J Sousa C Santos-Buelgaand I C F R Ferreira ldquoPhenolic profiles of cultivated invitro cultured and commercial samples of Melissa officinalis Linfusionsrdquo Food Chemistry vol 136 no 1 pp 1ndash8 2013

[35] T L Miron M Herrero and E Ibanez ldquoEnrichment of antiox-idant compounds from lemon balm (Melissa officinalis) bypressurized liquid extraction and enzyme-assisted extractionrdquoJournal of Chromatography A vol 1288 pp 1ndash9 2013

[36] Y Lu L Y Foo and H Wong ldquoSagecoumarin a novel caffeicacid trimer from Salvia officinalisrdquo Phytochemistry vol 52 no6 pp 1149ndash1152 1999

[37] S Abuhamdah LHuangM S J Elliott et al ldquoPharmacologicalprofile of an essential oil derived from Melissa officinalis withanti-agitation properties focus on ligand-gated channelsrdquo Jour-nal of Pharmacy and Pharmacology vol 60 no 3 pp 377ndash3842008

[38] G Wake J Court A Pickering R Lewis R Wilkins andE Perry ldquoCNS acetylcholine receptor activity in Europeanmedicinal plants traditionally used to improve failing memoryrdquoJournal of Ethnopharmacology vol 69 no 2 pp 105ndash114 2000

[39] S-H Ryu C Katona B Rive and G Livingston ldquoPersistenceof and changes in neuropsychiatric symptoms in Alzheimerdisease over 6months the LASER-AD studyrdquoAmerican Journalof Geriatric Psychiatry vol 13 no 11 pp 976ndash983 2005

[40] L N Gitlin H C Kales and C G Lyketsos ldquoNonpharma-cologic management of behavioral symptoms in dementiardquoJournal of the AmericanMedical Association vol 308 no 19 pp2020ndash2029 2012

[41] A Konczol J Muller E Foldes et al ldquoApplicability of a blood-brain barrier specific artificial membrane permeability assay atthe early stage of natural product-based CNS drug discoveryrdquoJournal of Natural Products vol 76 no 4 pp 655ndash663 2013

[42] Y-J Zhang L Wu Q-L Zhang J Li F-X Yin and Y YuanldquoPharmacokinetics of phenolic compounds of Danshen extractin rat blood and brain by microdialysis samplingrdquo Journal ofEthnopharmacology vol 136 no 1 pp 129ndash136 2011

[43] X Li C Yu Y Lu et al ldquoPharmacokinetics tissue distributionmetabolism and excretion of depside salts from Salvia miltior-rhiza in ratsrdquo Drug Metabolism and Disposition vol 35 no 2pp 234ndash239 2007

[44] H Xu Y Li X Che H Tian H Fan and K Liu ldquoMetabolismof salvianolic acid a and antioxidant activities of its methylatedmetabolitesrdquo Drug Metabolism and Disposition vol 42 no 2pp 274ndash281 2014

[45] D H Kim S J Park J M Kim et al ldquoCognitive dysfunctionsinduced by a cholinergic blockade and A120573 25-35 peptide areattenuated by salvianolic acid Brdquo Neuropharmacology vol 61no 8 pp 1432ndash1440 2011

[46] G-H Du Y Qiu and J-T Zhang ldquoSalvianolic acid B protectsthe memory functions against transient cerebral ischemia inmicerdquo Journal of Asian Natural Products Research vol 2 no 2pp 145ndash152 2000

[47] G Du and J Zhang ldquoProtective effects of salvianolic acid Aagainst impairment of memory induced by cerebral ischemia-reperfusion in micerdquo Chinese Medical Journal vol 110 no 1 pp65ndash68 1997

[48] J Anwar R M Spanevello G Thome et al ldquoEffects of caffeicacid on behavioral parameters and on the activity of acetyl-cholinesterase in different tissues from adult ratsrdquo Pharmacol-ogy Biochemistry and Behavior vol 103 no 2 pp 386ndash394 2012

[49] YW Lee D H Kim S J Jeon et al ldquoNeuroprotective effects ofsalvianolic acid B on anA12057325-35 peptide-inducedmousemodelof Alzheimerrsquos diseaserdquo European Journal of Pharmacology vol704 no 1ndash3 pp 70ndash77 2013

[50] J Zhong M-K Tang Y Zhang Q-P Xu and J-T ZhangldquoEffect of salvianolic acid B on neural cells damage and neu-rogenesis after brain ischemia-reperfusion in ratsrdquo Yao Xue XueBao vol 42 no 7 pp 716ndash721 2007

[51] P Zhuang Y Zhang G Cui et al ldquoDirect stimulation of adultneural stemprogenitor cells in vitro and neurogenesis in vivo bysalvianolic acid Brdquo PLoS ONE vol 7 no 4 Article ID e356362012

[52] M TangW Feng Y Zhang J Zhong and J Zhang ldquoSalvianolicacid B improves motor function after cerebral ischemia in ratsrdquoBehavioural Pharmacology vol 17 no 5-6 pp 493ndash498 2006

[53] Q Li L-P Han Z-H Li J-T Zhang and M-K Tang ldquoSal-vianolic acid B alleviate the disruption of blood-brain barrierin rats after cerebral ischemia-reperfusion by inhibiting MAPKpathwayrdquo Yao Xue Xue Bao vol 45 no 12 pp 1485ndash1490 2010

[54] Y-L LinH-J Tsay T-H Lai T-T Tzeng andY-J Shiao ldquoLith-ospermic acid attenuates 1-methyl-4-phenylpyridine-inducedneurotoxicity by blocking neuronal apoptotic and neuroinflam-matory pathwaysrdquo Journal of Biomedical Science vol 22 article37 2015

[55] M-K Tang and J-T Zhang ldquoSalvianolic acid B inhibits fibrilformation and neurotoxicity of amyloid beta-protein in vitrordquoActa Pharmacologica Sinica vol 22 no 4 pp 380ndash384 2001

[56] S S K Durairajan Q Yuan L Xie et al ldquoSalvianolic acid Binhibits A120573 fibril formation and disaggregates preformed fibrilsand protects against A120573-induced cytotoxictyrdquo NeurochemistryInternational vol 52 no 4-5 pp 741ndash750 2008

[57] FD Pinheiro Fernandes A P FonteneleMenezes J C de SousaNeves et al ldquoCaffeic acid protects mice from memory deficits

Evidence-Based Complementary and Alternative Medicine 17

induced by focal cerebral ischemiardquo Behavioural Pharmacologyvol 25 no 7 pp 637ndash647 2014

[58] S-J Tsai C-Y Chao andM-C Yin ldquoPreventive and therapeu-tic effects of caffeic acid against inflammatory injury in striatumof MPTP-treated micerdquo European Journal of Pharmacology vol670 no 2-3 pp 441ndash447 2011

[59] J H Kim QWang J M Choi S Lee and E J Cho ldquoProtectiverole of caffeic acid in an 119860120573

25minus35

-induced Alzheimerrsquos diseasemodelrdquo Nutrition Research and Practice vol 9 no 5 pp 480ndash488 2015

[60] G Liang B Shi W Luo and J Yang ldquoThe protective effect ofcaffeic acid on global cerebral ischemia-reperfusion injury inratsrdquo Behavioral and Brain Functions vol 11 no 1 article 182015

[61] D Y Yoo J H ChoiW Kim et al ldquoEffects of luteolin on spatialmemory cell proliferation and neuroblast differentiation in thehippocampal dentate gyrus in a scopolamine-induced amnesiamodelrdquo Neurological Research vol 35 no 8 pp 813ndash820 2013

[62] Y Liu X Tian L Gou L Sun X Ling and X Yin ldquoLuteolinattenuates diabetes-associated cognitive decline in ratsrdquo BrainResearch Bulletin vol 94 pp 23ndash29 2013

[63] S-M Liu Z-H Yang andX-B Sun ldquoSimultaneous determina-tion of six Salvia miltiorrhiza gradients in rat plasma and brainby LC-MSMSrdquo Zhongguo Zhongyao Zazhi vol 39 no 9 pp1704ndash1708 2014

[64] M A Papandreou A Dimakopoulou Z I Linardaki et alldquoEffect of a polyphenol-rich wild blueberry extract on cognitiveperformance of mice brain antioxidant markers and acetyl-cholinesterase activityrdquo Behavioural Brain Research vol 198 no2 pp 352ndash358 2009

[65] M-S Garcıa-Ayllon O Cauli M-X Silveyra et al ldquoBraincholinergic impairment in liver failurerdquo Brain vol 131 no 11pp 2946ndash2956 2008

[66] G K Ferreira M Carvalho-Silva C L Goncalves et al ldquoL-Tyrosine administration increases acetylcholinesterase activityin ratsrdquo Neurochemistry International vol 61 no 8 pp 1370ndash1374 2012

[67] R Vassar P-H Kuhn C Haass et al ldquoFunction therapeuticpotential and cell biology of BACE proteases current status andfuture prospectsrdquo Journal of Neurochemistry vol 130 no 1 pp4ndash28 2014

[68] R Vassar ldquoThe 120573-secretase BACE a prime drug target forAlzheimerrsquos diseaserdquo Journal of Molecular Neuroscience vol 17no 2 pp 157ndash170 2001

[69] D T R Coulson N Beyer J G Quinn et al ldquoBACE1 mRNAexpression in Alzheimerrsquos disease postmortem brain tissuerdquoJournal of Alzheimerrsquos Disease vol 22 no 4 pp 1111ndash1122 2010

[70] S Vladimir-Knezevic B Blazekovic M Kindl J Vladic A DLower-Nedza and A H Brantner ldquoAcetylcholinesterase inhib-itory antioxidant and phytochemical properties of selectedmedicinal plants of the lamiaceae familyrdquoMolecules vol 19 no1 pp 767ndash782 2014

[71] F Marcelo C Dias A Martins et al ldquoMolecular recognitionof rosmarinic acid from Salvia sclareoides extracts by ace-tylcholinesterase a new binding site detected by NMR spec-troscopyrdquo ChemistrymdashA European Journal vol 19 no 21 pp6641ndash6649 2013

[72] G Johnson and S W Moore ldquoWhy has butyrylcholinesterasebeen retained Structural and functional diversification in aduplicated generdquoNeurochemistry International vol 61 no 5 pp783ndash797 2012

[73] R J Grayer M R Eckert N C Veitch et al ldquoThe chemo-taxonomic significance of two bioactive caffeic acid estersnepetoidins A and B in the Lamiaceaerdquo Phytochemistry vol 64no 2 pp 519ndash528 2003

[74] A M D El-Mousallamy U W Hawas and S A M HusseinldquoTeucrol a decarboxyrosmarinic acid and its 41015840-O-triglycosideteucroside from Teucrium pilosumrdquo Phytochemistry vol 55 no8 pp 927ndash931 2000

[75] I Fecka and S Turek ldquoDetermination of water-soluble polyphe-nolic compounds in commercial herbal teas from Lamiaceaepeppermint melissa and sagerdquo Journal of Agricultural and FoodChemistry vol 55 no 26 pp 10908ndash10917 2007

[76] L W Sumner A Amberg D Barrett et al ldquoProposed min-imum reporting standards for chemical analysis WorkingGroup (CAWG) Metabolomics Standards Initiative (MSI)rdquoMetabolomics vol 3 pp 211ndash221 2007

Submit your manuscripts athttpwwwhindawicom

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Behavioural Neurology

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Disease Markers

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Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

12 Evidence-Based Complementary and Alternative Medicine

significance when compared with the control values (119901 lt007) and no change in the expression level of this enzymewas shown after the administration of HU On the contraryRA produced an increase of BuChE mRNA expression in thehippocampus by 44 (versus MC + H

2O 119901 lt 005)

Further analysis showed the significant differences inBACE1 mRNA expression in both brain regions of rats (inthe cortex and the hippocampus) (frontal cortex ANOVA119865(3 27) = 163 119901 lt 005 hippocampus ANOVA 119865(3 27) =133 119901 gt 005) It was observed that MO produced astatistically significant decrease of the BACE1 expression levelby 64 in the cortex (versus MC + H

2O 119901 lt 005) and by

50 in the hippocampus (versus MC + H2O 119901 lt 005) For

comparison HU treatment led to a decrease in the mRNAexpression level by 38 in the cortex (versus MC + H

2O

119901 lt 005) but not in the hippocampus On the contrary RAproduced an increase of this transcript in the cortex by 26but the effect did not reach a strong statistical significance(versus MC + H

2O 119901 lt 007) whereas in the hippocampus

there was no difference between RA and control group

5 Discussion

Cognitive and Behavioral Experiments The present studyinvestigated the influence of subchronic (28-fold) adminis-tration of standardized 50 EtOH extract ofMelisa officinalisleaf extract (MO) (200mgkg po containing 177mgkg ofRA) on SC-induced impairment of short-term and long-term memory in rats The results were compared with theactivity of cholinesterases (AChE and BuChE) as well aswith AChE BuChE and BACE1 gene expression levels in thecortex and hippocampus of the rat brain So far little evidenceis yet available as regards mechanisms of MO leaf extractaction that are potentially relevant to cognitive function ofrats after per os administration MO is traditionally used intreating neurological disorders through its anti-AChE [18]and antiagitation properties [37] Moreover Wake et al [38]and Kennedy et al [15] showed thatMO extract has nicotinicreceptor activity and that it can displace [3H]-(N)-nicotinefrom nicotinic receptors in homogenates of human cerebralcortex tissue and they suggested that these mechanisms canexplain activity of MO extract in amnesia model RecentlySoodi et al [18] observed that intraperitoneal injections ofMO extract (200mgkg) in rats could significantly enhancelearning and memory processes in animals since the extractsignificantly ameliorates SC-induced learning deficit in Mor-ris water maze test On the contrary in higher dose itcan be observed that MO extract (400mgkg) could notreverse SC-induced memory impairment [18] In our studyadministration of MO extract at a dose of 200mgkg (po)showed the effect leading to improvement of long-termmemory in a passive avoidance test in rats However afterMO combined treatment with SCMO did not overcome theimpairment shown by SC (Table 2) It should be emphasizedthat it is not clear whether the effect shown in our study byMO in non-SC-treated rats is specific since theMO treatmentproduced significant lowering of locomotor activity of ratstherefore the sedative profile ofMO cannot be excluded

On the other hand Ryu et al [39] observed that agitationand aggression are highly prevalent in patientswith dementiaAccording to Gitlin et al [40] nonpharmacological inter-ventions are recommended as first-line therapy Althoughantipsychotics have shown benefit for Alzheimerrsquos disease-related psychosis their use is associated with several seriousadverse effects [40]Thus it seems that the use ofMO extractcan provide dual benefits both in aspect of inhibition ofagitation and in improving the memory of patients withAlzheimerrsquos disease

Furthermore in our study RA in the dose of 10mgkgbw (po) did not affect either short- or long-term memoryalthough RA lowered significantly AChE activity both inthe cortex and in the hippocampus Moreover we observedthat the repeated administration of RA in non-scopolamine-treated rats did not produce any changes of locomotoractivity similarly to our previous study [23]

It is possible that other chemical compounds can influ-ence the memory in rats by synergic interactions in plantextract According to our calculations caffeic acid (0174mgin a single dose of extract administered to animals per kgbw) lithospermic acid (0084mgkg) salvianolic acid A(008mgkg) and salvianolic acid B (0046mgkg) may beresponsible for observed pharmacological effects On theother hand results from few studies showed that salvianolicacids do not cross the blood brain barrier (BBB) [41 42]and also lithospermic acid does not efficiently cross the BBB[43] For this reason the interpretation of our results is morecomplicated

Firstly Xu et al [44] demonstrated that Sal A is ametabol-ically unstable compound that would undergo rapid methy-lationmetabolism catalyzed by catechol O-methyltransferasein vivo into four major methylated metabolites of Sal A(3-O-methyl 31015840-O-methyl 3310158401015840-O-dimethyl and 31015840310158401015840-O-dimethyl salvianolic acid A) These generated O-methylatedmetabolites may be largely responsible for its in vivo phar-macological effects Although there are no available recentstudies on the ability of these compounds to pass the BBBsuch possibility should not be excluded

Secondly several studies showed central pharmacolog-ical effects in animals after per os administration of Sal B[45 46] Sal A [47] and caffeic acid [48] It was provedfor example that salvianolic acid B (10mgkg po) sig-nificantly rescued the A12057325ndash35 peptide-induced decreaseof choline acetyltransferase and brain-derived neurotrophicfactor protein levels in an amyloid 120573 (A120573) peptide-inducedAlzheimerrsquos disease mouse model [49] It also significantlyreversed (10mgkg po) the cognitive impairments inducedby scopolamine (1mgkg ip) or A120573 (25ndash35) (10 nmol5 120583Licv) injection inmice [45] Previous studies [46 47] showedalso that both Sal A and Sal B are able to improve theimpaired memory function induced by cerebral ischemia-reperfusion in miceThe stimulation of neurogenesis processin both subgranular zone (SGZ) and subventricular zone(SVZ) after brain ischemia and also alleviation neural cellsloss and improved motor function recovery after brainischemia in rats after the Sal B administration were alsoobserved by Zhong et al [50] Moreover the exposure to SalB can maintain the proliferation of neural stemprogenitor

Evidence-Based Complementary and Alternative Medicine 13

cells (NSPCs) after cerebral ischemia and improve cognitivepostischemic impairment after stroke in rats using Morriswater maze test therefore authors concluded that Sal B mayact as a potential drug in treatment of brain injury or neu-rodegenerative disease [51] Additionally the improvement ofmotor function after cerebral ischemia in rats after salvianolicacid B administrationwas also demonstrated [52]The clue topass through the BBB Sal B provided also results of Li et alindicating its protective effect on BBB in rats after cerebralischemia-reperfusion by inhibiting the MAPK pathway [53]In addition to this it was shown that lithospermic acid andsalvianolic acids exerted neuroprotective activity in variousexperimental models Lithospermic acid significantly attenu-ates neurotoxicity in vitro and in vivo induced by 1-methyl-4-phenylpyridin (MPP(+)) by blocking neuronal apoptotic andneuroinflammatory pathways [54] Salvianolic acid B inhib-ited amyloid beta-protein aggregation and fibril formation aswell as directly inhibiting the cellular toxicity of amyloid beta-protein in PC12 cells [55] and significantly reduced its cyto-toxic effects on human neuroblastoma SH-SY5Y cells [56]

For an explanation of our results observations of Pin-heiro Fernandes et al [57] may be very helpful whichshowed that caffeic acid nonflavanoid catecholic compoundwhose derivatives are occurring in MO extract improvedthe working spatial and long-term aversive memory deficitsinduced by focal cerebral ischemia in mice Anwar et al [48]showed also that caffeic acid (100mgkg) improved the step-down latencies in the inhibitory avoidance in rats Tsai et al[58] showed that caffeic acid is a potent neuroprotective agentin brain of 1-methyl-4-phenyl-1236-tetrahydropyridine(MPTP) treatedmiceMoreover caffeic acid improves A12057325ndash35-induced memory deficits and cognitive impairment inmice [59] In another study it was also shown that this com-pound has a significant protective effect on global cerebralischemia-reperfusion injury in rats [60] For instance 124 plusmn18mg100 g of caffeic acid was detected in the brain of micewith a diet containing 2 caffeic acid for 4 weeks [58] Thusthere is progressive evidence that this compound passesthrough the BBB and has a central pharmacological activity

Also results by Yoo et al are also worth noting [61] whichshowed also in a ratmodel of scopolamine-induced amnesiathat administration of luteolin (a common flavonoid frommany plants including M officinalis) at dose of 10mgkgcaused the increase in the brain-derived neurotrophic factor(BDNF) acetylcholine and the decrease in lipid peroxida-tion Liu et al [62] observed that chronic treatment withluteolin (50 and 100mgkg) improved neuronal injury andcognitive performance by attenuating oxidative stress andcholinesterase activity in streptozotocin-induced diabetes inrats In our study ethanolic extract of MO administered torats significantly improved long-term memory after using apassive avoidance test but afterMO combined treatmentwithSC no improvement of this paradigmwas observed (Table 2)

Thirdly available pharmacokinetic studies were carriedout for the pure compounds (Sal A and B) and the extractof the roots of Salvia miltiorrhiza [42 63] but there are notavailable studies of bioavailability of these compounds afteradministration of Melissa officinalis leaf extract containingother compounds as compared with the extract of Salvia

miltiorrhiza Moreover there is a lack of data about howscopolamine may influence the penetration of the salvianolicacids (and other compounds) across the BBB Hence thereis a need to study the pharmacokinetic parameters of thesecompounds in the group of animals treatedwith scopolaminein comparison with control group

AChEandBuChEActivities in Rat Brain To date several stud-ies have focused on explaining themechanismof action of theMS extract and its active compounds Soodi et al [18] showedthat treatment of animals withMO extract (400mgkg) priorto scopolamine injection could ameliorate scopolamine-induced enhancement in AChE activity This dose of MOextract inhibited the AChE activity in the hippocampus ofrats (519 versus 1004 in normal saline group 119901 lt 005914 in group treated with scopolamine +MO versus 1281in scopolamine group 119901 lt 005) In another study Anwaret al [48] showed that 50 and 100mgkg of caffeic aciddecreased in vivo the AChE activity in the cerebral cortexand striatum and increased the activity of this enzyme inthe cerebellum hippocampus hypothalamus pons lympho-cytes and muscles when compared to the control group(119901 lt 005) Our study showed that ethanolic extract of MOproduced an insignificant and slight inhibition of AChE andBuChE activity in the frontal cortex and in the hippocampus(especially) However it was observed previously [23] thatRA inhibited the AChE activity in the rats frontal cortex andhippocampusMoreover we showed that RA possess a strongstimulatory effect on BuChE in the hippocampus

AChE BuChE and BACE1mRNA Level Changes in Rat BrainSo far no results have been published of studies concerningthe in vivo assessment of changes in AChE BuChE andBACE1 gene expression profile in different brain regionsunder the influence of MO extracts or their key bioactivemetabolites Such studies focused overwhelmingly on theanalysis of their in vitro activities to a lesser extent in animalexperiments

In our study in the frontal cortex we have observedthe strongest inhibition of both AChE and BuCHE mRNAtranscription under the influence ofM officinalis extract andhuperzine A (Table 3) However a more significant differencein the level of transcript was seen in the frontal cortex inparticular in the case of BuCHE mRNA of experimentalrats (a decrease by 516 and 44 for MO and HU versuscontrol resp)The strong inhibition of transcription ofAChEmRNAwas also observed in the hippocampus ofMO-treatedrats (a decrease by 69) The BuChE mRNA transcriptionin animals receiving MO was lowered in a moderate way(decrease by 36)HuperzineA alone has not caused changesthat were observed in the group ofMO (Table 2) Our findingsmostly correlate with the observed changes in activities ofAChE and BuChE in different groups of animals In the caseof BuChE slightly different results between its activity andexpression profile were especially seen in the frontal cortexand hippocampus of animals receivingMO (Table 3)

To date there is a lack of published results of studiesmaking an attempt to clarify the potential differences in thetranscriptional profile and activity of AChE and BuChE

14 Evidence-Based Complementary and Alternative Medicine

under the influence of MO and its bioactive metabolites Itcannot be excluded that the key for the observed differencesin the level of AChE activities and mRNA level can becaused by changes in the activity of AChE in other regionsof the brain not analyzed in this study such as substantianigra cerebellum globus pallidus and hypothalamus whereit exerts nonenzymatic neuromodulatory functions affect-ing neurite outgrowth and synaptogenesis modulating theactivity of other proteins regional cerebral blood flow andother functions [64] But it is difficult to clearly explain whydiminishing of AChE and BuChEmRNAs did not always cor-relate with the lowering of activity of these enzymes althoughit has been recently noted that AChE activity was notparalleled by an increase in mRNA levels [65] The authorsexplained this fact by stating that AChE levels are regulatedat transcriptional posttranscriptional and posttranslationallevels leading to complex expression patterns which canbe modulated by physiological and pathological conditionsHowever these mechanisms are not fully understood andfurther studies are needed in this field

The cause of observed different degree of inhibition ofactivity and transcription status of studied genes especiallyof BuChE (and AChE) may lie in a so-called ldquonegative feed-backrdquo consisting of a complicated transcriptiontranslationregulation protein-protein interactionsmodifications and ametabolic network together forming a system that allows thecell to respond sensibly to the multiple signal molecules thatexist in its environment [66]

Because of that we propose that the reason for differencesbetween AChE and BuChE activities in the cortex and hip-pocampus under the MO may be due to the fact of insuf-ficiency of applied dose and the duration of the experi-ment affecting the transcriptional tissue-specific cellularmachinery regulating BuChE transcription without affectingits activity Moreover the administration of higher doses ofMO and extended period of time could lead to sufficientinhibition of its activity

There is a need to conduct further studies to determinethe molecular degree of dependence between changes inAChE and BuChE activities and activities of potential keyfactors regulating expression of these genes in the frontalcortex and hippocampus under the influence of extracts ofMO and active metabolites Another study determining theeffectiveness of their actions on the cholinergic system inexperimental animal models of memory impairment withparticular emphasis on scopolamine including the determi-nation of changes at the molecular level should be thereforecarried out

Alterations in BACE1 protein level have been proved inpostmortem brain tissue from individuals with AD withincreases decreases and also no change reported [1 67 68]An example of confirmation of these findings at the mRNAlevel is results of study by Coulson et al [69] Based onconducted studies some authors suggested that increasedBACE1mRNA transcription in remaining neuronal cells maycontribute to the increased BACE1 protein levels and activityfound in brain regions affected by AD [70]

Results of many studies concerning the elevated level ofBACE1 mRNA and protein in Alzheimerrsquos disease provide

direct and compelling reasons to develop therapies directed atBACE1 inhibition thus reducing120573-amyloid and its associatedtoxicities [67 68]

In our study we have observed that BACEmRNA expres-sion statistically significantly decreased after MO adminis-tration in frontal cortex and hippocampus These resultssuggest that the MO extract may act to inhibit BACE1mRNA level given the fact that the percentage of inhibitionof the expression (64 in the frontal cortex 50 in thehippocampus) is higher than that in the case of HU (38in the cortex and the lack of changes in the hippocampus)A careful analysis of the literature data shows that there areno studies which analyzed the impact of MO extract on theexpression level of BACE1 in Alzheimerrsquos disease

Furthermore a literature analysis does not indicatealready published results conducted by other teams attempt-ing to assess the impact of RA on the transcriptional activityof AChE BuChE and BACE1 Although several studies(already mentioned and others [70 71]) highlighted theRA and other caffeic acid derivatives capability of acetyl-cholinesterases inhibition none of these studies does nottouch the question of the molecular basis of their impacton the transcriptional machinery that regulates in vivo theexpression of studied genes Hence in our opinion obtainedby our team results they are one of the first of this typeand in general correlate with the results of our previousstudy [23] In this case there is no clear evidence explainingdifferent responses at the transcriptional level under theinfluence of RA especially in the case of BuChE encodinggene in the hippocampus (Table 2) It is possible that theobserved differentiation of BuChE transcriptional activitybetween the frontal cortex and the hippocampus may bedue to the differences of butyrylcholinesterase localizationand substrate affinity [72] Since in our experiment we havecarried out a quantitative analysis of AChE BuChE andBACE1 transcripts in brain homogenates of tested animalsrather than in individual isolated cell fractions therefore theobtained results constitute an overall ldquopicturerdquo of both studiedgenes transcriptional changes occurring in the brain areas ofstudied animals under the influence of the RA and the wholeplant extract as well

6 Conclusion

The subchronic administration ofMO led to an improvementof long-term memory of rats however the mechanisms ofMO action are probably more complicated since its role asa modulator of beta-secretase activity (due to inhibition ofBACE1 mRNA expression in frontal cortex) should be takeninto consideration

It should be noted that we have studied a crude extractfrom leaf of Melissa officinalis not a single pure chemicalcompound This plant extract is a complex mixture and itsaction may be a result of the summation of activities ofseveral components (synergismadditive action of caffeic acidwith salvianolic acids rosmarinic acid and others) In thecase of extract from leaves ofMelissa officinalis it is possiblethat interactions occur between the 40 chemical compoundsidentified by HPLC system

Evidence-Based Complementary and Alternative Medicine 15

Taken together it seems that the MO activity representsa possible option as complementary interventions to relievethe symptoms of mild dementia

Competing Interests

The authors declare that they have no competing interests

Acknowledgments

This work was supported by the Ministry of Science andHigher EducationWarsaw Poland from educational sources(2008ndash2011 Grant no N 405417836)

References

[1] S L Cole and R Vassar ldquoThe Alzheimerrsquos disease 120573-secretaseenzyme BACE1rdquo Molecular Neurodegeneration vol 2 no 1article 22 2007

[2] LACraigN SHong andR JMcDonald ldquoRevisiting the cho-linergic hypothesis in the development of Alzheimerrsquos diseaserdquoNeuroscience amp Biobehavioral Reviews vol 35 no 6 pp 1397ndash1409 2011

[3] N G M Gomes M G Campos J M C Orfao and C A FRibeiro ldquoPlants with neurobiological activity as potential tar-gets for drug discoveryrdquo Progress in Neuro-Psychopharmacologyamp Biological Psychiatry vol 33 no 8 pp 1372ndash1389 2009

[4] WHO WHO Monographs on Selected Medicinal PlantsmdashVolume 2 WHO Geneva Switzerland 2004 httpappswhointmedicinedocsendJs4927e18htmlJs4927e18

[5] M Bayat A A Tameh M H Ghahremani et al ldquoNeuropro-tective properties of Melissa officinalis after hypoxic-ischemicinjury both in vitro and in vivordquo DARU Journal of Pharmaceu-tical Sciences vol 20 article 42 2012

[6] J P Kamdem A Adeniran A A Boligon et al ldquoAntioxidantactivity genotoxicity and cytotoxicity evaluation of lemon balm(Melissa officinalis L) ethanolic extract Its potential role inneuroprotectionrdquo Industrial Crops and Products vol 51 pp 26ndash34 2013

[7] J-T Lin Y-C Chen Y-C Lee C-W R Hou F-L Chen andD-J Yang ldquoAntioxidant anti-proliferative and cyclooxygenase-2 inhibitory activities of ethanolic extracts from lemon balm(Melissa officinalis L) leavesrdquo LWTmdashFood Science and Technol-ogy vol 49 no 1 pp 1ndash7 2012

[8] G Guginski A P Luiz M D Silva et al ldquoMechanisms involvedin the antinociception caused by ethanolic extract obtainedfrom the leaves of Melissa officinalis (lemon balm) in micerdquoPharmacology Biochemistry amp Behavior vol 93 no 1 pp 10ndash162009

[9] N Perry G Court N Bidet J Court and E Perry ldquoEuropeanherbs with cholinergic activities potential in dementia therapyrdquoInternational Journal of Geriatric Psychiatry vol 11 no 12 pp1063ndash1069 1996

[10] E K Perry A T Pickering W W Wang P J Houghtonand N S L Perry ldquoMedicinal plants and Alzheimerrsquos diseasefrom ethnobotany to phytotherapyrdquo Journal of Pharmacy andPharmacology vol 51 no 5 pp 527ndash534 1999

[11] M-J R Howes N S L Perry and P J Houghton ldquoPlants withtraditional uses and activities relevant to the management ofAlzheimerrsquos disease and other cognitive disordersrdquo Phytother-apy Research vol 17 no 1 pp 1ndash18 2003

[12] I Orhan and M Aslan ldquoAppraisal of scopolamine-inducedantiamnesic effect in mice and in vitro antiacetylcholinesteraseand antioxidant activities of some traditionally used Lamiaceaeplantsrdquo Journal of Ethnopharmacology vol 122 no 2 pp 327ndash332 2009

[13] S Akhondzadeh M Noroozian M Mohammadi S OhadiniaA H Jamshidi and M Khani ldquoMelissa officinalis extract inthe treatment of patients with mild to moderate Alzheimerrsquosdisease a double blind randomised placebo controlled trialrdquoJournal of Neurology Neurosurgery and Psychiatry vol 74 no 7pp 863ndash866 2003

[14] D O Kennedy A B Scholey N T J Tildesley E K Perry andK AWesnes ldquoModulation ofmood and cognitive performancefollowing acute administration of Melissa officinalis (lemonbalm)rdquo Pharmacology Biochemistry amp Behavior vol 72 no 4pp 953ndash964 2002

[15] D O Kennedy G Wake S Savelev et al ldquoModulation of moodand cognitive performance following acute administration ofsingle doses of Melissa officinalis (Lemon balm) with humanCNS nicotinic and muscarinic receptor-binding propertiesrdquoNeuropsychopharmacology vol 28 no 10 pp 1871ndash1881 2003

[16] D O Kennedy and E L Wightman ldquoHerbal extracts and phy-tochemicals plant secondarymetabolites and the enhancementof human brain functionrdquoAdvances inNutrition vol 2 no 1 pp32ndash50 2011

[17] R P Pereira A A Boligon A S Appel et al ldquoChemical com-position antioxidant and anticholinesterase activity of Melissaofficinalisrdquo Industrial Crops and Products vol 53 pp 34ndash452014

[18] M Soodi N Naghdi H Hajimehdipoor S Choopani andE Sahraei ldquoMemory-improving activity of Melissa officinalisextract in naıve and scopolamine-treated ratsrdquo Research inPharmaceutical Sciences vol 9 no 2 pp 107ndash114 2014

[19] K Dastmalchi V Ollilainen P Lackman et al ldquoAcetyl-cholinesterase inhibitory guided fractionation of Melissa offic-inalis Lrdquo Bioorganic amp Medicinal Chemistry vol 17 no 2 pp867ndash871 2009

[20] W Chaiyana and S Okonogi ldquoInhibition of cholinesterase byessential oil from food plantrdquo Phytomedicine vol 19 no 8-9 pp836ndash839 2012

[21] K Dastmalchi H J D Dorman P P Oinonen Y Darwis ILaakso and R Hiltunen ldquoChemical composition and in vitroantioxidative activity of a lemon balm (Melissa officinalis L)extractrdquo LWTmdashFood Science and Technology vol 41 no 3 pp391ndash400 2008

[22] P Pereira D Tysca P Oliveira L F D S Brum J N Picadaand P Ardenghi ldquoNeurobehavioral and genotoxic aspects ofrosmarinic acidrdquo Pharmacological Research vol 52 no 3 pp199ndash203 2005

[23] M Ozarowski P L Mikolajczak A Bogacz et al ldquoRosmarinusofficinalis L leaf extract improves memory impairment andaffects acetylcholinesterase and butyrylcholinesterase activitiesin rat brainrdquo Fitoterapia vol 91 pp 261ndash271 2013

[24] G P Kumar and F Khanum ldquoNeuroprotective potential of phy-tochemicalsrdquo Pharmacognosy Reviews vol 6 no 12 pp 81ndash902012

[25] M Stobiecki A Staszkow A Piasecka P M Garcia-LopezF Zamora-Natera and P Kachlicki ldquoLC-MSMS profiling offlavonoid conjugates in wild mexican lupine Lupinus reflexusrdquoJournal of Natural Products vol 73 no 7 pp 1254ndash1260 2010

[26] A Wojakowska A Piasecka P M Garcıa-Lopez et al ldquoStruc-tural analysis and profiling of phenolic secondarymetabolites of

16 Evidence-Based Complementary and Alternative Medicine

Mexican lupine species using LC-MS techniquesrdquo Phytochem-istry vol 92 pp 71ndash86 2013

[27] A Gryszczynska B Opala Z Lowicki et al ldquoBioactive com-pounds determination in the callus and hydroalcoholic extractsfrom Salvia miltiorrhiza and Salvia przewalskiimdashpreliminarystudy on their anti-alcoholic activity effectsrdquo PhytochemistryLetters vol 11 pp 399ndash403 2015

[28] K Slinkart and V L Singleton ldquoTotal phenol analysis automa-tion and comparison with manual methodrdquo American Journalof Enology and Viticulture vol 28 no 1 pp 49ndash55 1977

[29] P J R Boissier J Tardy and J C Diverres ldquoUne nouvellemethode simple pour explorer lrsquoaction lsquotranquillisantersquo le testde la chemineerdquo Medicina Experimentalis vol 3 no 1 pp 81ndash84 1960

[30] R Ader J A Weijnen and P Moleman ldquoRetention of a passiveavoidance response as a function of the intensity and durationof electric shockrdquo Psychonomic Science vol 26 no 3 pp 125ndash128 1972

[31] E M Blalock K-C Chen K Sharrow et al ldquoGene microar-rays in hippocampal aging statistical profiling identifies novelprocesses correlated with cognitive impairmentrdquo Journal ofNeuroscience vol 23 no 9 pp 3807ndash3819 2003

[32] K Isomae S Morimoto H Hasegawa K Morita and JKamei ldquoEffects of T-82 a novel acetylcholinesterase inhibitoron impaired learning and memory in passive avoidance task inratsrdquo European Journal of Pharmacology vol 465 no 1-2 pp97ndash103 2003

[33] J Patora and B Klimek ldquoFlavonoids from lemon balm (Melissaofficinalis L Lamiaceae)rdquo Acta Poloniae Pharmaceutica-DrugResearch vol 59 no 2 pp 139ndash143 2002

[34] L Barros M Duenas M I Dias M J Sousa C Santos-Buelgaand I C F R Ferreira ldquoPhenolic profiles of cultivated invitro cultured and commercial samples of Melissa officinalis Linfusionsrdquo Food Chemistry vol 136 no 1 pp 1ndash8 2013

[35] T L Miron M Herrero and E Ibanez ldquoEnrichment of antiox-idant compounds from lemon balm (Melissa officinalis) bypressurized liquid extraction and enzyme-assisted extractionrdquoJournal of Chromatography A vol 1288 pp 1ndash9 2013

[36] Y Lu L Y Foo and H Wong ldquoSagecoumarin a novel caffeicacid trimer from Salvia officinalisrdquo Phytochemistry vol 52 no6 pp 1149ndash1152 1999

[37] S Abuhamdah LHuangM S J Elliott et al ldquoPharmacologicalprofile of an essential oil derived from Melissa officinalis withanti-agitation properties focus on ligand-gated channelsrdquo Jour-nal of Pharmacy and Pharmacology vol 60 no 3 pp 377ndash3842008

[38] G Wake J Court A Pickering R Lewis R Wilkins andE Perry ldquoCNS acetylcholine receptor activity in Europeanmedicinal plants traditionally used to improve failing memoryrdquoJournal of Ethnopharmacology vol 69 no 2 pp 105ndash114 2000

[39] S-H Ryu C Katona B Rive and G Livingston ldquoPersistenceof and changes in neuropsychiatric symptoms in Alzheimerdisease over 6months the LASER-AD studyrdquoAmerican Journalof Geriatric Psychiatry vol 13 no 11 pp 976ndash983 2005

[40] L N Gitlin H C Kales and C G Lyketsos ldquoNonpharma-cologic management of behavioral symptoms in dementiardquoJournal of the AmericanMedical Association vol 308 no 19 pp2020ndash2029 2012

[41] A Konczol J Muller E Foldes et al ldquoApplicability of a blood-brain barrier specific artificial membrane permeability assay atthe early stage of natural product-based CNS drug discoveryrdquoJournal of Natural Products vol 76 no 4 pp 655ndash663 2013

[42] Y-J Zhang L Wu Q-L Zhang J Li F-X Yin and Y YuanldquoPharmacokinetics of phenolic compounds of Danshen extractin rat blood and brain by microdialysis samplingrdquo Journal ofEthnopharmacology vol 136 no 1 pp 129ndash136 2011

[43] X Li C Yu Y Lu et al ldquoPharmacokinetics tissue distributionmetabolism and excretion of depside salts from Salvia miltior-rhiza in ratsrdquo Drug Metabolism and Disposition vol 35 no 2pp 234ndash239 2007

[44] H Xu Y Li X Che H Tian H Fan and K Liu ldquoMetabolismof salvianolic acid a and antioxidant activities of its methylatedmetabolitesrdquo Drug Metabolism and Disposition vol 42 no 2pp 274ndash281 2014

[45] D H Kim S J Park J M Kim et al ldquoCognitive dysfunctionsinduced by a cholinergic blockade and A120573 25-35 peptide areattenuated by salvianolic acid Brdquo Neuropharmacology vol 61no 8 pp 1432ndash1440 2011

[46] G-H Du Y Qiu and J-T Zhang ldquoSalvianolic acid B protectsthe memory functions against transient cerebral ischemia inmicerdquo Journal of Asian Natural Products Research vol 2 no 2pp 145ndash152 2000

[47] G Du and J Zhang ldquoProtective effects of salvianolic acid Aagainst impairment of memory induced by cerebral ischemia-reperfusion in micerdquo Chinese Medical Journal vol 110 no 1 pp65ndash68 1997

[48] J Anwar R M Spanevello G Thome et al ldquoEffects of caffeicacid on behavioral parameters and on the activity of acetyl-cholinesterase in different tissues from adult ratsrdquo Pharmacol-ogy Biochemistry and Behavior vol 103 no 2 pp 386ndash394 2012

[49] YW Lee D H Kim S J Jeon et al ldquoNeuroprotective effects ofsalvianolic acid B on anA12057325-35 peptide-inducedmousemodelof Alzheimerrsquos diseaserdquo European Journal of Pharmacology vol704 no 1ndash3 pp 70ndash77 2013

[50] J Zhong M-K Tang Y Zhang Q-P Xu and J-T ZhangldquoEffect of salvianolic acid B on neural cells damage and neu-rogenesis after brain ischemia-reperfusion in ratsrdquo Yao Xue XueBao vol 42 no 7 pp 716ndash721 2007

[51] P Zhuang Y Zhang G Cui et al ldquoDirect stimulation of adultneural stemprogenitor cells in vitro and neurogenesis in vivo bysalvianolic acid Brdquo PLoS ONE vol 7 no 4 Article ID e356362012

[52] M TangW Feng Y Zhang J Zhong and J Zhang ldquoSalvianolicacid B improves motor function after cerebral ischemia in ratsrdquoBehavioural Pharmacology vol 17 no 5-6 pp 493ndash498 2006

[53] Q Li L-P Han Z-H Li J-T Zhang and M-K Tang ldquoSal-vianolic acid B alleviate the disruption of blood-brain barrierin rats after cerebral ischemia-reperfusion by inhibiting MAPKpathwayrdquo Yao Xue Xue Bao vol 45 no 12 pp 1485ndash1490 2010

[54] Y-L LinH-J Tsay T-H Lai T-T Tzeng andY-J Shiao ldquoLith-ospermic acid attenuates 1-methyl-4-phenylpyridine-inducedneurotoxicity by blocking neuronal apoptotic and neuroinflam-matory pathwaysrdquo Journal of Biomedical Science vol 22 article37 2015

[55] M-K Tang and J-T Zhang ldquoSalvianolic acid B inhibits fibrilformation and neurotoxicity of amyloid beta-protein in vitrordquoActa Pharmacologica Sinica vol 22 no 4 pp 380ndash384 2001

[56] S S K Durairajan Q Yuan L Xie et al ldquoSalvianolic acid Binhibits A120573 fibril formation and disaggregates preformed fibrilsand protects against A120573-induced cytotoxictyrdquo NeurochemistryInternational vol 52 no 4-5 pp 741ndash750 2008

[57] FD Pinheiro Fernandes A P FonteneleMenezes J C de SousaNeves et al ldquoCaffeic acid protects mice from memory deficits

Evidence-Based Complementary and Alternative Medicine 17

induced by focal cerebral ischemiardquo Behavioural Pharmacologyvol 25 no 7 pp 637ndash647 2014

[58] S-J Tsai C-Y Chao andM-C Yin ldquoPreventive and therapeu-tic effects of caffeic acid against inflammatory injury in striatumof MPTP-treated micerdquo European Journal of Pharmacology vol670 no 2-3 pp 441ndash447 2011

[59] J H Kim QWang J M Choi S Lee and E J Cho ldquoProtectiverole of caffeic acid in an 119860120573

25minus35

-induced Alzheimerrsquos diseasemodelrdquo Nutrition Research and Practice vol 9 no 5 pp 480ndash488 2015

[60] G Liang B Shi W Luo and J Yang ldquoThe protective effect ofcaffeic acid on global cerebral ischemia-reperfusion injury inratsrdquo Behavioral and Brain Functions vol 11 no 1 article 182015

[61] D Y Yoo J H ChoiW Kim et al ldquoEffects of luteolin on spatialmemory cell proliferation and neuroblast differentiation in thehippocampal dentate gyrus in a scopolamine-induced amnesiamodelrdquo Neurological Research vol 35 no 8 pp 813ndash820 2013

[62] Y Liu X Tian L Gou L Sun X Ling and X Yin ldquoLuteolinattenuates diabetes-associated cognitive decline in ratsrdquo BrainResearch Bulletin vol 94 pp 23ndash29 2013

[63] S-M Liu Z-H Yang andX-B Sun ldquoSimultaneous determina-tion of six Salvia miltiorrhiza gradients in rat plasma and brainby LC-MSMSrdquo Zhongguo Zhongyao Zazhi vol 39 no 9 pp1704ndash1708 2014

[64] M A Papandreou A Dimakopoulou Z I Linardaki et alldquoEffect of a polyphenol-rich wild blueberry extract on cognitiveperformance of mice brain antioxidant markers and acetyl-cholinesterase activityrdquo Behavioural Brain Research vol 198 no2 pp 352ndash358 2009

[65] M-S Garcıa-Ayllon O Cauli M-X Silveyra et al ldquoBraincholinergic impairment in liver failurerdquo Brain vol 131 no 11pp 2946ndash2956 2008

[66] G K Ferreira M Carvalho-Silva C L Goncalves et al ldquoL-Tyrosine administration increases acetylcholinesterase activityin ratsrdquo Neurochemistry International vol 61 no 8 pp 1370ndash1374 2012

[67] R Vassar P-H Kuhn C Haass et al ldquoFunction therapeuticpotential and cell biology of BACE proteases current status andfuture prospectsrdquo Journal of Neurochemistry vol 130 no 1 pp4ndash28 2014

[68] R Vassar ldquoThe 120573-secretase BACE a prime drug target forAlzheimerrsquos diseaserdquo Journal of Molecular Neuroscience vol 17no 2 pp 157ndash170 2001

[69] D T R Coulson N Beyer J G Quinn et al ldquoBACE1 mRNAexpression in Alzheimerrsquos disease postmortem brain tissuerdquoJournal of Alzheimerrsquos Disease vol 22 no 4 pp 1111ndash1122 2010

[70] S Vladimir-Knezevic B Blazekovic M Kindl J Vladic A DLower-Nedza and A H Brantner ldquoAcetylcholinesterase inhib-itory antioxidant and phytochemical properties of selectedmedicinal plants of the lamiaceae familyrdquoMolecules vol 19 no1 pp 767ndash782 2014

[71] F Marcelo C Dias A Martins et al ldquoMolecular recognitionof rosmarinic acid from Salvia sclareoides extracts by ace-tylcholinesterase a new binding site detected by NMR spec-troscopyrdquo ChemistrymdashA European Journal vol 19 no 21 pp6641ndash6649 2013

[72] G Johnson and S W Moore ldquoWhy has butyrylcholinesterasebeen retained Structural and functional diversification in aduplicated generdquoNeurochemistry International vol 61 no 5 pp783ndash797 2012

[73] R J Grayer M R Eckert N C Veitch et al ldquoThe chemo-taxonomic significance of two bioactive caffeic acid estersnepetoidins A and B in the Lamiaceaerdquo Phytochemistry vol 64no 2 pp 519ndash528 2003

[74] A M D El-Mousallamy U W Hawas and S A M HusseinldquoTeucrol a decarboxyrosmarinic acid and its 41015840-O-triglycosideteucroside from Teucrium pilosumrdquo Phytochemistry vol 55 no8 pp 927ndash931 2000

[75] I Fecka and S Turek ldquoDetermination of water-soluble polyphe-nolic compounds in commercial herbal teas from Lamiaceaepeppermint melissa and sagerdquo Journal of Agricultural and FoodChemistry vol 55 no 26 pp 10908ndash10917 2007

[76] L W Sumner A Amberg D Barrett et al ldquoProposed min-imum reporting standards for chemical analysis WorkingGroup (CAWG) Metabolomics Standards Initiative (MSI)rdquoMetabolomics vol 3 pp 211ndash221 2007

Submit your manuscripts athttpwwwhindawicom

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

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Disease Markers

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OncologyJournal of

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Oxidative Medicine and Cellular Longevity

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The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Evidence-Based Complementary and Alternative Medicine 13

cells (NSPCs) after cerebral ischemia and improve cognitivepostischemic impairment after stroke in rats using Morriswater maze test therefore authors concluded that Sal B mayact as a potential drug in treatment of brain injury or neu-rodegenerative disease [51] Additionally the improvement ofmotor function after cerebral ischemia in rats after salvianolicacid B administrationwas also demonstrated [52]The clue topass through the BBB Sal B provided also results of Li et alindicating its protective effect on BBB in rats after cerebralischemia-reperfusion by inhibiting the MAPK pathway [53]In addition to this it was shown that lithospermic acid andsalvianolic acids exerted neuroprotective activity in variousexperimental models Lithospermic acid significantly attenu-ates neurotoxicity in vitro and in vivo induced by 1-methyl-4-phenylpyridin (MPP(+)) by blocking neuronal apoptotic andneuroinflammatory pathways [54] Salvianolic acid B inhib-ited amyloid beta-protein aggregation and fibril formation aswell as directly inhibiting the cellular toxicity of amyloid beta-protein in PC12 cells [55] and significantly reduced its cyto-toxic effects on human neuroblastoma SH-SY5Y cells [56]

For an explanation of our results observations of Pin-heiro Fernandes et al [57] may be very helpful whichshowed that caffeic acid nonflavanoid catecholic compoundwhose derivatives are occurring in MO extract improvedthe working spatial and long-term aversive memory deficitsinduced by focal cerebral ischemia in mice Anwar et al [48]showed also that caffeic acid (100mgkg) improved the step-down latencies in the inhibitory avoidance in rats Tsai et al[58] showed that caffeic acid is a potent neuroprotective agentin brain of 1-methyl-4-phenyl-1236-tetrahydropyridine(MPTP) treatedmiceMoreover caffeic acid improves A12057325ndash35-induced memory deficits and cognitive impairment inmice [59] In another study it was also shown that this com-pound has a significant protective effect on global cerebralischemia-reperfusion injury in rats [60] For instance 124 plusmn18mg100 g of caffeic acid was detected in the brain of micewith a diet containing 2 caffeic acid for 4 weeks [58] Thusthere is progressive evidence that this compound passesthrough the BBB and has a central pharmacological activity

Also results by Yoo et al are also worth noting [61] whichshowed also in a ratmodel of scopolamine-induced amnesiathat administration of luteolin (a common flavonoid frommany plants including M officinalis) at dose of 10mgkgcaused the increase in the brain-derived neurotrophic factor(BDNF) acetylcholine and the decrease in lipid peroxida-tion Liu et al [62] observed that chronic treatment withluteolin (50 and 100mgkg) improved neuronal injury andcognitive performance by attenuating oxidative stress andcholinesterase activity in streptozotocin-induced diabetes inrats In our study ethanolic extract of MO administered torats significantly improved long-term memory after using apassive avoidance test but afterMO combined treatmentwithSC no improvement of this paradigmwas observed (Table 2)

Thirdly available pharmacokinetic studies were carriedout for the pure compounds (Sal A and B) and the extractof the roots of Salvia miltiorrhiza [42 63] but there are notavailable studies of bioavailability of these compounds afteradministration of Melissa officinalis leaf extract containingother compounds as compared with the extract of Salvia

miltiorrhiza Moreover there is a lack of data about howscopolamine may influence the penetration of the salvianolicacids (and other compounds) across the BBB Hence thereis a need to study the pharmacokinetic parameters of thesecompounds in the group of animals treatedwith scopolaminein comparison with control group

AChEandBuChEActivities in Rat Brain To date several stud-ies have focused on explaining themechanismof action of theMS extract and its active compounds Soodi et al [18] showedthat treatment of animals withMO extract (400mgkg) priorto scopolamine injection could ameliorate scopolamine-induced enhancement in AChE activity This dose of MOextract inhibited the AChE activity in the hippocampus ofrats (519 versus 1004 in normal saline group 119901 lt 005914 in group treated with scopolamine +MO versus 1281in scopolamine group 119901 lt 005) In another study Anwaret al [48] showed that 50 and 100mgkg of caffeic aciddecreased in vivo the AChE activity in the cerebral cortexand striatum and increased the activity of this enzyme inthe cerebellum hippocampus hypothalamus pons lympho-cytes and muscles when compared to the control group(119901 lt 005) Our study showed that ethanolic extract of MOproduced an insignificant and slight inhibition of AChE andBuChE activity in the frontal cortex and in the hippocampus(especially) However it was observed previously [23] thatRA inhibited the AChE activity in the rats frontal cortex andhippocampusMoreover we showed that RA possess a strongstimulatory effect on BuChE in the hippocampus

AChE BuChE and BACE1mRNA Level Changes in Rat BrainSo far no results have been published of studies concerningthe in vivo assessment of changes in AChE BuChE andBACE1 gene expression profile in different brain regionsunder the influence of MO extracts or their key bioactivemetabolites Such studies focused overwhelmingly on theanalysis of their in vitro activities to a lesser extent in animalexperiments

In our study in the frontal cortex we have observedthe strongest inhibition of both AChE and BuCHE mRNAtranscription under the influence ofM officinalis extract andhuperzine A (Table 3) However a more significant differencein the level of transcript was seen in the frontal cortex inparticular in the case of BuCHE mRNA of experimentalrats (a decrease by 516 and 44 for MO and HU versuscontrol resp)The strong inhibition of transcription ofAChEmRNAwas also observed in the hippocampus ofMO-treatedrats (a decrease by 69) The BuChE mRNA transcriptionin animals receiving MO was lowered in a moderate way(decrease by 36)HuperzineA alone has not caused changesthat were observed in the group ofMO (Table 2) Our findingsmostly correlate with the observed changes in activities ofAChE and BuChE in different groups of animals In the caseof BuChE slightly different results between its activity andexpression profile were especially seen in the frontal cortexand hippocampus of animals receivingMO (Table 3)

To date there is a lack of published results of studiesmaking an attempt to clarify the potential differences in thetranscriptional profile and activity of AChE and BuChE

14 Evidence-Based Complementary and Alternative Medicine

under the influence of MO and its bioactive metabolites Itcannot be excluded that the key for the observed differencesin the level of AChE activities and mRNA level can becaused by changes in the activity of AChE in other regionsof the brain not analyzed in this study such as substantianigra cerebellum globus pallidus and hypothalamus whereit exerts nonenzymatic neuromodulatory functions affect-ing neurite outgrowth and synaptogenesis modulating theactivity of other proteins regional cerebral blood flow andother functions [64] But it is difficult to clearly explain whydiminishing of AChE and BuChEmRNAs did not always cor-relate with the lowering of activity of these enzymes althoughit has been recently noted that AChE activity was notparalleled by an increase in mRNA levels [65] The authorsexplained this fact by stating that AChE levels are regulatedat transcriptional posttranscriptional and posttranslationallevels leading to complex expression patterns which canbe modulated by physiological and pathological conditionsHowever these mechanisms are not fully understood andfurther studies are needed in this field

The cause of observed different degree of inhibition ofactivity and transcription status of studied genes especiallyof BuChE (and AChE) may lie in a so-called ldquonegative feed-backrdquo consisting of a complicated transcriptiontranslationregulation protein-protein interactionsmodifications and ametabolic network together forming a system that allows thecell to respond sensibly to the multiple signal molecules thatexist in its environment [66]

Because of that we propose that the reason for differencesbetween AChE and BuChE activities in the cortex and hip-pocampus under the MO may be due to the fact of insuf-ficiency of applied dose and the duration of the experi-ment affecting the transcriptional tissue-specific cellularmachinery regulating BuChE transcription without affectingits activity Moreover the administration of higher doses ofMO and extended period of time could lead to sufficientinhibition of its activity

There is a need to conduct further studies to determinethe molecular degree of dependence between changes inAChE and BuChE activities and activities of potential keyfactors regulating expression of these genes in the frontalcortex and hippocampus under the influence of extracts ofMO and active metabolites Another study determining theeffectiveness of their actions on the cholinergic system inexperimental animal models of memory impairment withparticular emphasis on scopolamine including the determi-nation of changes at the molecular level should be thereforecarried out

Alterations in BACE1 protein level have been proved inpostmortem brain tissue from individuals with AD withincreases decreases and also no change reported [1 67 68]An example of confirmation of these findings at the mRNAlevel is results of study by Coulson et al [69] Based onconducted studies some authors suggested that increasedBACE1mRNA transcription in remaining neuronal cells maycontribute to the increased BACE1 protein levels and activityfound in brain regions affected by AD [70]

Results of many studies concerning the elevated level ofBACE1 mRNA and protein in Alzheimerrsquos disease provide

direct and compelling reasons to develop therapies directed atBACE1 inhibition thus reducing120573-amyloid and its associatedtoxicities [67 68]

In our study we have observed that BACEmRNA expres-sion statistically significantly decreased after MO adminis-tration in frontal cortex and hippocampus These resultssuggest that the MO extract may act to inhibit BACE1mRNA level given the fact that the percentage of inhibitionof the expression (64 in the frontal cortex 50 in thehippocampus) is higher than that in the case of HU (38in the cortex and the lack of changes in the hippocampus)A careful analysis of the literature data shows that there areno studies which analyzed the impact of MO extract on theexpression level of BACE1 in Alzheimerrsquos disease

Furthermore a literature analysis does not indicatealready published results conducted by other teams attempt-ing to assess the impact of RA on the transcriptional activityof AChE BuChE and BACE1 Although several studies(already mentioned and others [70 71]) highlighted theRA and other caffeic acid derivatives capability of acetyl-cholinesterases inhibition none of these studies does nottouch the question of the molecular basis of their impacton the transcriptional machinery that regulates in vivo theexpression of studied genes Hence in our opinion obtainedby our team results they are one of the first of this typeand in general correlate with the results of our previousstudy [23] In this case there is no clear evidence explainingdifferent responses at the transcriptional level under theinfluence of RA especially in the case of BuChE encodinggene in the hippocampus (Table 2) It is possible that theobserved differentiation of BuChE transcriptional activitybetween the frontal cortex and the hippocampus may bedue to the differences of butyrylcholinesterase localizationand substrate affinity [72] Since in our experiment we havecarried out a quantitative analysis of AChE BuChE andBACE1 transcripts in brain homogenates of tested animalsrather than in individual isolated cell fractions therefore theobtained results constitute an overall ldquopicturerdquo of both studiedgenes transcriptional changes occurring in the brain areas ofstudied animals under the influence of the RA and the wholeplant extract as well

6 Conclusion

The subchronic administration ofMO led to an improvementof long-term memory of rats however the mechanisms ofMO action are probably more complicated since its role asa modulator of beta-secretase activity (due to inhibition ofBACE1 mRNA expression in frontal cortex) should be takeninto consideration

It should be noted that we have studied a crude extractfrom leaf of Melissa officinalis not a single pure chemicalcompound This plant extract is a complex mixture and itsaction may be a result of the summation of activities ofseveral components (synergismadditive action of caffeic acidwith salvianolic acids rosmarinic acid and others) In thecase of extract from leaves ofMelissa officinalis it is possiblethat interactions occur between the 40 chemical compoundsidentified by HPLC system

Evidence-Based Complementary and Alternative Medicine 15

Taken together it seems that the MO activity representsa possible option as complementary interventions to relievethe symptoms of mild dementia

Competing Interests

The authors declare that they have no competing interests

Acknowledgments

This work was supported by the Ministry of Science andHigher EducationWarsaw Poland from educational sources(2008ndash2011 Grant no N 405417836)

References

[1] S L Cole and R Vassar ldquoThe Alzheimerrsquos disease 120573-secretaseenzyme BACE1rdquo Molecular Neurodegeneration vol 2 no 1article 22 2007

[2] LACraigN SHong andR JMcDonald ldquoRevisiting the cho-linergic hypothesis in the development of Alzheimerrsquos diseaserdquoNeuroscience amp Biobehavioral Reviews vol 35 no 6 pp 1397ndash1409 2011

[3] N G M Gomes M G Campos J M C Orfao and C A FRibeiro ldquoPlants with neurobiological activity as potential tar-gets for drug discoveryrdquo Progress in Neuro-Psychopharmacologyamp Biological Psychiatry vol 33 no 8 pp 1372ndash1389 2009

[4] WHO WHO Monographs on Selected Medicinal PlantsmdashVolume 2 WHO Geneva Switzerland 2004 httpappswhointmedicinedocsendJs4927e18htmlJs4927e18

[5] M Bayat A A Tameh M H Ghahremani et al ldquoNeuropro-tective properties of Melissa officinalis after hypoxic-ischemicinjury both in vitro and in vivordquo DARU Journal of Pharmaceu-tical Sciences vol 20 article 42 2012

[6] J P Kamdem A Adeniran A A Boligon et al ldquoAntioxidantactivity genotoxicity and cytotoxicity evaluation of lemon balm(Melissa officinalis L) ethanolic extract Its potential role inneuroprotectionrdquo Industrial Crops and Products vol 51 pp 26ndash34 2013

[7] J-T Lin Y-C Chen Y-C Lee C-W R Hou F-L Chen andD-J Yang ldquoAntioxidant anti-proliferative and cyclooxygenase-2 inhibitory activities of ethanolic extracts from lemon balm(Melissa officinalis L) leavesrdquo LWTmdashFood Science and Technol-ogy vol 49 no 1 pp 1ndash7 2012

[8] G Guginski A P Luiz M D Silva et al ldquoMechanisms involvedin the antinociception caused by ethanolic extract obtainedfrom the leaves of Melissa officinalis (lemon balm) in micerdquoPharmacology Biochemistry amp Behavior vol 93 no 1 pp 10ndash162009

[9] N Perry G Court N Bidet J Court and E Perry ldquoEuropeanherbs with cholinergic activities potential in dementia therapyrdquoInternational Journal of Geriatric Psychiatry vol 11 no 12 pp1063ndash1069 1996

[10] E K Perry A T Pickering W W Wang P J Houghtonand N S L Perry ldquoMedicinal plants and Alzheimerrsquos diseasefrom ethnobotany to phytotherapyrdquo Journal of Pharmacy andPharmacology vol 51 no 5 pp 527ndash534 1999

[11] M-J R Howes N S L Perry and P J Houghton ldquoPlants withtraditional uses and activities relevant to the management ofAlzheimerrsquos disease and other cognitive disordersrdquo Phytother-apy Research vol 17 no 1 pp 1ndash18 2003

[12] I Orhan and M Aslan ldquoAppraisal of scopolamine-inducedantiamnesic effect in mice and in vitro antiacetylcholinesteraseand antioxidant activities of some traditionally used Lamiaceaeplantsrdquo Journal of Ethnopharmacology vol 122 no 2 pp 327ndash332 2009

[13] S Akhondzadeh M Noroozian M Mohammadi S OhadiniaA H Jamshidi and M Khani ldquoMelissa officinalis extract inthe treatment of patients with mild to moderate Alzheimerrsquosdisease a double blind randomised placebo controlled trialrdquoJournal of Neurology Neurosurgery and Psychiatry vol 74 no 7pp 863ndash866 2003

[14] D O Kennedy A B Scholey N T J Tildesley E K Perry andK AWesnes ldquoModulation ofmood and cognitive performancefollowing acute administration of Melissa officinalis (lemonbalm)rdquo Pharmacology Biochemistry amp Behavior vol 72 no 4pp 953ndash964 2002

[15] D O Kennedy G Wake S Savelev et al ldquoModulation of moodand cognitive performance following acute administration ofsingle doses of Melissa officinalis (Lemon balm) with humanCNS nicotinic and muscarinic receptor-binding propertiesrdquoNeuropsychopharmacology vol 28 no 10 pp 1871ndash1881 2003

[16] D O Kennedy and E L Wightman ldquoHerbal extracts and phy-tochemicals plant secondarymetabolites and the enhancementof human brain functionrdquoAdvances inNutrition vol 2 no 1 pp32ndash50 2011

[17] R P Pereira A A Boligon A S Appel et al ldquoChemical com-position antioxidant and anticholinesterase activity of Melissaofficinalisrdquo Industrial Crops and Products vol 53 pp 34ndash452014

[18] M Soodi N Naghdi H Hajimehdipoor S Choopani andE Sahraei ldquoMemory-improving activity of Melissa officinalisextract in naıve and scopolamine-treated ratsrdquo Research inPharmaceutical Sciences vol 9 no 2 pp 107ndash114 2014

[19] K Dastmalchi V Ollilainen P Lackman et al ldquoAcetyl-cholinesterase inhibitory guided fractionation of Melissa offic-inalis Lrdquo Bioorganic amp Medicinal Chemistry vol 17 no 2 pp867ndash871 2009

[20] W Chaiyana and S Okonogi ldquoInhibition of cholinesterase byessential oil from food plantrdquo Phytomedicine vol 19 no 8-9 pp836ndash839 2012

[21] K Dastmalchi H J D Dorman P P Oinonen Y Darwis ILaakso and R Hiltunen ldquoChemical composition and in vitroantioxidative activity of a lemon balm (Melissa officinalis L)extractrdquo LWTmdashFood Science and Technology vol 41 no 3 pp391ndash400 2008

[22] P Pereira D Tysca P Oliveira L F D S Brum J N Picadaand P Ardenghi ldquoNeurobehavioral and genotoxic aspects ofrosmarinic acidrdquo Pharmacological Research vol 52 no 3 pp199ndash203 2005

[23] M Ozarowski P L Mikolajczak A Bogacz et al ldquoRosmarinusofficinalis L leaf extract improves memory impairment andaffects acetylcholinesterase and butyrylcholinesterase activitiesin rat brainrdquo Fitoterapia vol 91 pp 261ndash271 2013

[24] G P Kumar and F Khanum ldquoNeuroprotective potential of phy-tochemicalsrdquo Pharmacognosy Reviews vol 6 no 12 pp 81ndash902012

[25] M Stobiecki A Staszkow A Piasecka P M Garcia-LopezF Zamora-Natera and P Kachlicki ldquoLC-MSMS profiling offlavonoid conjugates in wild mexican lupine Lupinus reflexusrdquoJournal of Natural Products vol 73 no 7 pp 1254ndash1260 2010

[26] A Wojakowska A Piasecka P M Garcıa-Lopez et al ldquoStruc-tural analysis and profiling of phenolic secondarymetabolites of

16 Evidence-Based Complementary and Alternative Medicine

Mexican lupine species using LC-MS techniquesrdquo Phytochem-istry vol 92 pp 71ndash86 2013

[27] A Gryszczynska B Opala Z Lowicki et al ldquoBioactive com-pounds determination in the callus and hydroalcoholic extractsfrom Salvia miltiorrhiza and Salvia przewalskiimdashpreliminarystudy on their anti-alcoholic activity effectsrdquo PhytochemistryLetters vol 11 pp 399ndash403 2015

[28] K Slinkart and V L Singleton ldquoTotal phenol analysis automa-tion and comparison with manual methodrdquo American Journalof Enology and Viticulture vol 28 no 1 pp 49ndash55 1977

[29] P J R Boissier J Tardy and J C Diverres ldquoUne nouvellemethode simple pour explorer lrsquoaction lsquotranquillisantersquo le testde la chemineerdquo Medicina Experimentalis vol 3 no 1 pp 81ndash84 1960

[30] R Ader J A Weijnen and P Moleman ldquoRetention of a passiveavoidance response as a function of the intensity and durationof electric shockrdquo Psychonomic Science vol 26 no 3 pp 125ndash128 1972

[31] E M Blalock K-C Chen K Sharrow et al ldquoGene microar-rays in hippocampal aging statistical profiling identifies novelprocesses correlated with cognitive impairmentrdquo Journal ofNeuroscience vol 23 no 9 pp 3807ndash3819 2003

[32] K Isomae S Morimoto H Hasegawa K Morita and JKamei ldquoEffects of T-82 a novel acetylcholinesterase inhibitoron impaired learning and memory in passive avoidance task inratsrdquo European Journal of Pharmacology vol 465 no 1-2 pp97ndash103 2003

[33] J Patora and B Klimek ldquoFlavonoids from lemon balm (Melissaofficinalis L Lamiaceae)rdquo Acta Poloniae Pharmaceutica-DrugResearch vol 59 no 2 pp 139ndash143 2002

[34] L Barros M Duenas M I Dias M J Sousa C Santos-Buelgaand I C F R Ferreira ldquoPhenolic profiles of cultivated invitro cultured and commercial samples of Melissa officinalis Linfusionsrdquo Food Chemistry vol 136 no 1 pp 1ndash8 2013

[35] T L Miron M Herrero and E Ibanez ldquoEnrichment of antiox-idant compounds from lemon balm (Melissa officinalis) bypressurized liquid extraction and enzyme-assisted extractionrdquoJournal of Chromatography A vol 1288 pp 1ndash9 2013

[36] Y Lu L Y Foo and H Wong ldquoSagecoumarin a novel caffeicacid trimer from Salvia officinalisrdquo Phytochemistry vol 52 no6 pp 1149ndash1152 1999

[37] S Abuhamdah LHuangM S J Elliott et al ldquoPharmacologicalprofile of an essential oil derived from Melissa officinalis withanti-agitation properties focus on ligand-gated channelsrdquo Jour-nal of Pharmacy and Pharmacology vol 60 no 3 pp 377ndash3842008

[38] G Wake J Court A Pickering R Lewis R Wilkins andE Perry ldquoCNS acetylcholine receptor activity in Europeanmedicinal plants traditionally used to improve failing memoryrdquoJournal of Ethnopharmacology vol 69 no 2 pp 105ndash114 2000

[39] S-H Ryu C Katona B Rive and G Livingston ldquoPersistenceof and changes in neuropsychiatric symptoms in Alzheimerdisease over 6months the LASER-AD studyrdquoAmerican Journalof Geriatric Psychiatry vol 13 no 11 pp 976ndash983 2005

[40] L N Gitlin H C Kales and C G Lyketsos ldquoNonpharma-cologic management of behavioral symptoms in dementiardquoJournal of the AmericanMedical Association vol 308 no 19 pp2020ndash2029 2012

[41] A Konczol J Muller E Foldes et al ldquoApplicability of a blood-brain barrier specific artificial membrane permeability assay atthe early stage of natural product-based CNS drug discoveryrdquoJournal of Natural Products vol 76 no 4 pp 655ndash663 2013

[42] Y-J Zhang L Wu Q-L Zhang J Li F-X Yin and Y YuanldquoPharmacokinetics of phenolic compounds of Danshen extractin rat blood and brain by microdialysis samplingrdquo Journal ofEthnopharmacology vol 136 no 1 pp 129ndash136 2011

[43] X Li C Yu Y Lu et al ldquoPharmacokinetics tissue distributionmetabolism and excretion of depside salts from Salvia miltior-rhiza in ratsrdquo Drug Metabolism and Disposition vol 35 no 2pp 234ndash239 2007

[44] H Xu Y Li X Che H Tian H Fan and K Liu ldquoMetabolismof salvianolic acid a and antioxidant activities of its methylatedmetabolitesrdquo Drug Metabolism and Disposition vol 42 no 2pp 274ndash281 2014

[45] D H Kim S J Park J M Kim et al ldquoCognitive dysfunctionsinduced by a cholinergic blockade and A120573 25-35 peptide areattenuated by salvianolic acid Brdquo Neuropharmacology vol 61no 8 pp 1432ndash1440 2011

[46] G-H Du Y Qiu and J-T Zhang ldquoSalvianolic acid B protectsthe memory functions against transient cerebral ischemia inmicerdquo Journal of Asian Natural Products Research vol 2 no 2pp 145ndash152 2000

[47] G Du and J Zhang ldquoProtective effects of salvianolic acid Aagainst impairment of memory induced by cerebral ischemia-reperfusion in micerdquo Chinese Medical Journal vol 110 no 1 pp65ndash68 1997

[48] J Anwar R M Spanevello G Thome et al ldquoEffects of caffeicacid on behavioral parameters and on the activity of acetyl-cholinesterase in different tissues from adult ratsrdquo Pharmacol-ogy Biochemistry and Behavior vol 103 no 2 pp 386ndash394 2012

[49] YW Lee D H Kim S J Jeon et al ldquoNeuroprotective effects ofsalvianolic acid B on anA12057325-35 peptide-inducedmousemodelof Alzheimerrsquos diseaserdquo European Journal of Pharmacology vol704 no 1ndash3 pp 70ndash77 2013

[50] J Zhong M-K Tang Y Zhang Q-P Xu and J-T ZhangldquoEffect of salvianolic acid B on neural cells damage and neu-rogenesis after brain ischemia-reperfusion in ratsrdquo Yao Xue XueBao vol 42 no 7 pp 716ndash721 2007

[51] P Zhuang Y Zhang G Cui et al ldquoDirect stimulation of adultneural stemprogenitor cells in vitro and neurogenesis in vivo bysalvianolic acid Brdquo PLoS ONE vol 7 no 4 Article ID e356362012

[52] M TangW Feng Y Zhang J Zhong and J Zhang ldquoSalvianolicacid B improves motor function after cerebral ischemia in ratsrdquoBehavioural Pharmacology vol 17 no 5-6 pp 493ndash498 2006

[53] Q Li L-P Han Z-H Li J-T Zhang and M-K Tang ldquoSal-vianolic acid B alleviate the disruption of blood-brain barrierin rats after cerebral ischemia-reperfusion by inhibiting MAPKpathwayrdquo Yao Xue Xue Bao vol 45 no 12 pp 1485ndash1490 2010

[54] Y-L LinH-J Tsay T-H Lai T-T Tzeng andY-J Shiao ldquoLith-ospermic acid attenuates 1-methyl-4-phenylpyridine-inducedneurotoxicity by blocking neuronal apoptotic and neuroinflam-matory pathwaysrdquo Journal of Biomedical Science vol 22 article37 2015

[55] M-K Tang and J-T Zhang ldquoSalvianolic acid B inhibits fibrilformation and neurotoxicity of amyloid beta-protein in vitrordquoActa Pharmacologica Sinica vol 22 no 4 pp 380ndash384 2001

[56] S S K Durairajan Q Yuan L Xie et al ldquoSalvianolic acid Binhibits A120573 fibril formation and disaggregates preformed fibrilsand protects against A120573-induced cytotoxictyrdquo NeurochemistryInternational vol 52 no 4-5 pp 741ndash750 2008

[57] FD Pinheiro Fernandes A P FonteneleMenezes J C de SousaNeves et al ldquoCaffeic acid protects mice from memory deficits

Evidence-Based Complementary and Alternative Medicine 17

induced by focal cerebral ischemiardquo Behavioural Pharmacologyvol 25 no 7 pp 637ndash647 2014

[58] S-J Tsai C-Y Chao andM-C Yin ldquoPreventive and therapeu-tic effects of caffeic acid against inflammatory injury in striatumof MPTP-treated micerdquo European Journal of Pharmacology vol670 no 2-3 pp 441ndash447 2011

[59] J H Kim QWang J M Choi S Lee and E J Cho ldquoProtectiverole of caffeic acid in an 119860120573

25minus35

-induced Alzheimerrsquos diseasemodelrdquo Nutrition Research and Practice vol 9 no 5 pp 480ndash488 2015

[60] G Liang B Shi W Luo and J Yang ldquoThe protective effect ofcaffeic acid on global cerebral ischemia-reperfusion injury inratsrdquo Behavioral and Brain Functions vol 11 no 1 article 182015

[61] D Y Yoo J H ChoiW Kim et al ldquoEffects of luteolin on spatialmemory cell proliferation and neuroblast differentiation in thehippocampal dentate gyrus in a scopolamine-induced amnesiamodelrdquo Neurological Research vol 35 no 8 pp 813ndash820 2013

[62] Y Liu X Tian L Gou L Sun X Ling and X Yin ldquoLuteolinattenuates diabetes-associated cognitive decline in ratsrdquo BrainResearch Bulletin vol 94 pp 23ndash29 2013

[63] S-M Liu Z-H Yang andX-B Sun ldquoSimultaneous determina-tion of six Salvia miltiorrhiza gradients in rat plasma and brainby LC-MSMSrdquo Zhongguo Zhongyao Zazhi vol 39 no 9 pp1704ndash1708 2014

[64] M A Papandreou A Dimakopoulou Z I Linardaki et alldquoEffect of a polyphenol-rich wild blueberry extract on cognitiveperformance of mice brain antioxidant markers and acetyl-cholinesterase activityrdquo Behavioural Brain Research vol 198 no2 pp 352ndash358 2009

[65] M-S Garcıa-Ayllon O Cauli M-X Silveyra et al ldquoBraincholinergic impairment in liver failurerdquo Brain vol 131 no 11pp 2946ndash2956 2008

[66] G K Ferreira M Carvalho-Silva C L Goncalves et al ldquoL-Tyrosine administration increases acetylcholinesterase activityin ratsrdquo Neurochemistry International vol 61 no 8 pp 1370ndash1374 2012

[67] R Vassar P-H Kuhn C Haass et al ldquoFunction therapeuticpotential and cell biology of BACE proteases current status andfuture prospectsrdquo Journal of Neurochemistry vol 130 no 1 pp4ndash28 2014

[68] R Vassar ldquoThe 120573-secretase BACE a prime drug target forAlzheimerrsquos diseaserdquo Journal of Molecular Neuroscience vol 17no 2 pp 157ndash170 2001

[69] D T R Coulson N Beyer J G Quinn et al ldquoBACE1 mRNAexpression in Alzheimerrsquos disease postmortem brain tissuerdquoJournal of Alzheimerrsquos Disease vol 22 no 4 pp 1111ndash1122 2010

[70] S Vladimir-Knezevic B Blazekovic M Kindl J Vladic A DLower-Nedza and A H Brantner ldquoAcetylcholinesterase inhib-itory antioxidant and phytochemical properties of selectedmedicinal plants of the lamiaceae familyrdquoMolecules vol 19 no1 pp 767ndash782 2014

[71] F Marcelo C Dias A Martins et al ldquoMolecular recognitionof rosmarinic acid from Salvia sclareoides extracts by ace-tylcholinesterase a new binding site detected by NMR spec-troscopyrdquo ChemistrymdashA European Journal vol 19 no 21 pp6641ndash6649 2013

[72] G Johnson and S W Moore ldquoWhy has butyrylcholinesterasebeen retained Structural and functional diversification in aduplicated generdquoNeurochemistry International vol 61 no 5 pp783ndash797 2012

[73] R J Grayer M R Eckert N C Veitch et al ldquoThe chemo-taxonomic significance of two bioactive caffeic acid estersnepetoidins A and B in the Lamiaceaerdquo Phytochemistry vol 64no 2 pp 519ndash528 2003

[74] A M D El-Mousallamy U W Hawas and S A M HusseinldquoTeucrol a decarboxyrosmarinic acid and its 41015840-O-triglycosideteucroside from Teucrium pilosumrdquo Phytochemistry vol 55 no8 pp 927ndash931 2000

[75] I Fecka and S Turek ldquoDetermination of water-soluble polyphe-nolic compounds in commercial herbal teas from Lamiaceaepeppermint melissa and sagerdquo Journal of Agricultural and FoodChemistry vol 55 no 26 pp 10908ndash10917 2007

[76] L W Sumner A Amberg D Barrett et al ldquoProposed min-imum reporting standards for chemical analysis WorkingGroup (CAWG) Metabolomics Standards Initiative (MSI)rdquoMetabolomics vol 3 pp 211ndash221 2007

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

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PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

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Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

14 Evidence-Based Complementary and Alternative Medicine

under the influence of MO and its bioactive metabolites Itcannot be excluded that the key for the observed differencesin the level of AChE activities and mRNA level can becaused by changes in the activity of AChE in other regionsof the brain not analyzed in this study such as substantianigra cerebellum globus pallidus and hypothalamus whereit exerts nonenzymatic neuromodulatory functions affect-ing neurite outgrowth and synaptogenesis modulating theactivity of other proteins regional cerebral blood flow andother functions [64] But it is difficult to clearly explain whydiminishing of AChE and BuChEmRNAs did not always cor-relate with the lowering of activity of these enzymes althoughit has been recently noted that AChE activity was notparalleled by an increase in mRNA levels [65] The authorsexplained this fact by stating that AChE levels are regulatedat transcriptional posttranscriptional and posttranslationallevels leading to complex expression patterns which canbe modulated by physiological and pathological conditionsHowever these mechanisms are not fully understood andfurther studies are needed in this field

The cause of observed different degree of inhibition ofactivity and transcription status of studied genes especiallyof BuChE (and AChE) may lie in a so-called ldquonegative feed-backrdquo consisting of a complicated transcriptiontranslationregulation protein-protein interactionsmodifications and ametabolic network together forming a system that allows thecell to respond sensibly to the multiple signal molecules thatexist in its environment [66]

Because of that we propose that the reason for differencesbetween AChE and BuChE activities in the cortex and hip-pocampus under the MO may be due to the fact of insuf-ficiency of applied dose and the duration of the experi-ment affecting the transcriptional tissue-specific cellularmachinery regulating BuChE transcription without affectingits activity Moreover the administration of higher doses ofMO and extended period of time could lead to sufficientinhibition of its activity

There is a need to conduct further studies to determinethe molecular degree of dependence between changes inAChE and BuChE activities and activities of potential keyfactors regulating expression of these genes in the frontalcortex and hippocampus under the influence of extracts ofMO and active metabolites Another study determining theeffectiveness of their actions on the cholinergic system inexperimental animal models of memory impairment withparticular emphasis on scopolamine including the determi-nation of changes at the molecular level should be thereforecarried out

Alterations in BACE1 protein level have been proved inpostmortem brain tissue from individuals with AD withincreases decreases and also no change reported [1 67 68]An example of confirmation of these findings at the mRNAlevel is results of study by Coulson et al [69] Based onconducted studies some authors suggested that increasedBACE1mRNA transcription in remaining neuronal cells maycontribute to the increased BACE1 protein levels and activityfound in brain regions affected by AD [70]

Results of many studies concerning the elevated level ofBACE1 mRNA and protein in Alzheimerrsquos disease provide

direct and compelling reasons to develop therapies directed atBACE1 inhibition thus reducing120573-amyloid and its associatedtoxicities [67 68]

In our study we have observed that BACEmRNA expres-sion statistically significantly decreased after MO adminis-tration in frontal cortex and hippocampus These resultssuggest that the MO extract may act to inhibit BACE1mRNA level given the fact that the percentage of inhibitionof the expression (64 in the frontal cortex 50 in thehippocampus) is higher than that in the case of HU (38in the cortex and the lack of changes in the hippocampus)A careful analysis of the literature data shows that there areno studies which analyzed the impact of MO extract on theexpression level of BACE1 in Alzheimerrsquos disease

Furthermore a literature analysis does not indicatealready published results conducted by other teams attempt-ing to assess the impact of RA on the transcriptional activityof AChE BuChE and BACE1 Although several studies(already mentioned and others [70 71]) highlighted theRA and other caffeic acid derivatives capability of acetyl-cholinesterases inhibition none of these studies does nottouch the question of the molecular basis of their impacton the transcriptional machinery that regulates in vivo theexpression of studied genes Hence in our opinion obtainedby our team results they are one of the first of this typeand in general correlate with the results of our previousstudy [23] In this case there is no clear evidence explainingdifferent responses at the transcriptional level under theinfluence of RA especially in the case of BuChE encodinggene in the hippocampus (Table 2) It is possible that theobserved differentiation of BuChE transcriptional activitybetween the frontal cortex and the hippocampus may bedue to the differences of butyrylcholinesterase localizationand substrate affinity [72] Since in our experiment we havecarried out a quantitative analysis of AChE BuChE andBACE1 transcripts in brain homogenates of tested animalsrather than in individual isolated cell fractions therefore theobtained results constitute an overall ldquopicturerdquo of both studiedgenes transcriptional changes occurring in the brain areas ofstudied animals under the influence of the RA and the wholeplant extract as well

6 Conclusion

The subchronic administration ofMO led to an improvementof long-term memory of rats however the mechanisms ofMO action are probably more complicated since its role asa modulator of beta-secretase activity (due to inhibition ofBACE1 mRNA expression in frontal cortex) should be takeninto consideration

It should be noted that we have studied a crude extractfrom leaf of Melissa officinalis not a single pure chemicalcompound This plant extract is a complex mixture and itsaction may be a result of the summation of activities ofseveral components (synergismadditive action of caffeic acidwith salvianolic acids rosmarinic acid and others) In thecase of extract from leaves ofMelissa officinalis it is possiblethat interactions occur between the 40 chemical compoundsidentified by HPLC system

Evidence-Based Complementary and Alternative Medicine 15

Taken together it seems that the MO activity representsa possible option as complementary interventions to relievethe symptoms of mild dementia

Competing Interests

The authors declare that they have no competing interests

Acknowledgments

This work was supported by the Ministry of Science andHigher EducationWarsaw Poland from educational sources(2008ndash2011 Grant no N 405417836)

References

[1] S L Cole and R Vassar ldquoThe Alzheimerrsquos disease 120573-secretaseenzyme BACE1rdquo Molecular Neurodegeneration vol 2 no 1article 22 2007

[2] LACraigN SHong andR JMcDonald ldquoRevisiting the cho-linergic hypothesis in the development of Alzheimerrsquos diseaserdquoNeuroscience amp Biobehavioral Reviews vol 35 no 6 pp 1397ndash1409 2011

[3] N G M Gomes M G Campos J M C Orfao and C A FRibeiro ldquoPlants with neurobiological activity as potential tar-gets for drug discoveryrdquo Progress in Neuro-Psychopharmacologyamp Biological Psychiatry vol 33 no 8 pp 1372ndash1389 2009

[4] WHO WHO Monographs on Selected Medicinal PlantsmdashVolume 2 WHO Geneva Switzerland 2004 httpappswhointmedicinedocsendJs4927e18htmlJs4927e18

[5] M Bayat A A Tameh M H Ghahremani et al ldquoNeuropro-tective properties of Melissa officinalis after hypoxic-ischemicinjury both in vitro and in vivordquo DARU Journal of Pharmaceu-tical Sciences vol 20 article 42 2012

[6] J P Kamdem A Adeniran A A Boligon et al ldquoAntioxidantactivity genotoxicity and cytotoxicity evaluation of lemon balm(Melissa officinalis L) ethanolic extract Its potential role inneuroprotectionrdquo Industrial Crops and Products vol 51 pp 26ndash34 2013

[7] J-T Lin Y-C Chen Y-C Lee C-W R Hou F-L Chen andD-J Yang ldquoAntioxidant anti-proliferative and cyclooxygenase-2 inhibitory activities of ethanolic extracts from lemon balm(Melissa officinalis L) leavesrdquo LWTmdashFood Science and Technol-ogy vol 49 no 1 pp 1ndash7 2012

[8] G Guginski A P Luiz M D Silva et al ldquoMechanisms involvedin the antinociception caused by ethanolic extract obtainedfrom the leaves of Melissa officinalis (lemon balm) in micerdquoPharmacology Biochemistry amp Behavior vol 93 no 1 pp 10ndash162009

[9] N Perry G Court N Bidet J Court and E Perry ldquoEuropeanherbs with cholinergic activities potential in dementia therapyrdquoInternational Journal of Geriatric Psychiatry vol 11 no 12 pp1063ndash1069 1996

[10] E K Perry A T Pickering W W Wang P J Houghtonand N S L Perry ldquoMedicinal plants and Alzheimerrsquos diseasefrom ethnobotany to phytotherapyrdquo Journal of Pharmacy andPharmacology vol 51 no 5 pp 527ndash534 1999

[11] M-J R Howes N S L Perry and P J Houghton ldquoPlants withtraditional uses and activities relevant to the management ofAlzheimerrsquos disease and other cognitive disordersrdquo Phytother-apy Research vol 17 no 1 pp 1ndash18 2003

[12] I Orhan and M Aslan ldquoAppraisal of scopolamine-inducedantiamnesic effect in mice and in vitro antiacetylcholinesteraseand antioxidant activities of some traditionally used Lamiaceaeplantsrdquo Journal of Ethnopharmacology vol 122 no 2 pp 327ndash332 2009

[13] S Akhondzadeh M Noroozian M Mohammadi S OhadiniaA H Jamshidi and M Khani ldquoMelissa officinalis extract inthe treatment of patients with mild to moderate Alzheimerrsquosdisease a double blind randomised placebo controlled trialrdquoJournal of Neurology Neurosurgery and Psychiatry vol 74 no 7pp 863ndash866 2003

[14] D O Kennedy A B Scholey N T J Tildesley E K Perry andK AWesnes ldquoModulation ofmood and cognitive performancefollowing acute administration of Melissa officinalis (lemonbalm)rdquo Pharmacology Biochemistry amp Behavior vol 72 no 4pp 953ndash964 2002

[15] D O Kennedy G Wake S Savelev et al ldquoModulation of moodand cognitive performance following acute administration ofsingle doses of Melissa officinalis (Lemon balm) with humanCNS nicotinic and muscarinic receptor-binding propertiesrdquoNeuropsychopharmacology vol 28 no 10 pp 1871ndash1881 2003

[16] D O Kennedy and E L Wightman ldquoHerbal extracts and phy-tochemicals plant secondarymetabolites and the enhancementof human brain functionrdquoAdvances inNutrition vol 2 no 1 pp32ndash50 2011

[17] R P Pereira A A Boligon A S Appel et al ldquoChemical com-position antioxidant and anticholinesterase activity of Melissaofficinalisrdquo Industrial Crops and Products vol 53 pp 34ndash452014

[18] M Soodi N Naghdi H Hajimehdipoor S Choopani andE Sahraei ldquoMemory-improving activity of Melissa officinalisextract in naıve and scopolamine-treated ratsrdquo Research inPharmaceutical Sciences vol 9 no 2 pp 107ndash114 2014

[19] K Dastmalchi V Ollilainen P Lackman et al ldquoAcetyl-cholinesterase inhibitory guided fractionation of Melissa offic-inalis Lrdquo Bioorganic amp Medicinal Chemistry vol 17 no 2 pp867ndash871 2009

[20] W Chaiyana and S Okonogi ldquoInhibition of cholinesterase byessential oil from food plantrdquo Phytomedicine vol 19 no 8-9 pp836ndash839 2012

[21] K Dastmalchi H J D Dorman P P Oinonen Y Darwis ILaakso and R Hiltunen ldquoChemical composition and in vitroantioxidative activity of a lemon balm (Melissa officinalis L)extractrdquo LWTmdashFood Science and Technology vol 41 no 3 pp391ndash400 2008

[22] P Pereira D Tysca P Oliveira L F D S Brum J N Picadaand P Ardenghi ldquoNeurobehavioral and genotoxic aspects ofrosmarinic acidrdquo Pharmacological Research vol 52 no 3 pp199ndash203 2005

[23] M Ozarowski P L Mikolajczak A Bogacz et al ldquoRosmarinusofficinalis L leaf extract improves memory impairment andaffects acetylcholinesterase and butyrylcholinesterase activitiesin rat brainrdquo Fitoterapia vol 91 pp 261ndash271 2013

[24] G P Kumar and F Khanum ldquoNeuroprotective potential of phy-tochemicalsrdquo Pharmacognosy Reviews vol 6 no 12 pp 81ndash902012

[25] M Stobiecki A Staszkow A Piasecka P M Garcia-LopezF Zamora-Natera and P Kachlicki ldquoLC-MSMS profiling offlavonoid conjugates in wild mexican lupine Lupinus reflexusrdquoJournal of Natural Products vol 73 no 7 pp 1254ndash1260 2010

[26] A Wojakowska A Piasecka P M Garcıa-Lopez et al ldquoStruc-tural analysis and profiling of phenolic secondarymetabolites of

16 Evidence-Based Complementary and Alternative Medicine

Mexican lupine species using LC-MS techniquesrdquo Phytochem-istry vol 92 pp 71ndash86 2013

[27] A Gryszczynska B Opala Z Lowicki et al ldquoBioactive com-pounds determination in the callus and hydroalcoholic extractsfrom Salvia miltiorrhiza and Salvia przewalskiimdashpreliminarystudy on their anti-alcoholic activity effectsrdquo PhytochemistryLetters vol 11 pp 399ndash403 2015

[28] K Slinkart and V L Singleton ldquoTotal phenol analysis automa-tion and comparison with manual methodrdquo American Journalof Enology and Viticulture vol 28 no 1 pp 49ndash55 1977

[29] P J R Boissier J Tardy and J C Diverres ldquoUne nouvellemethode simple pour explorer lrsquoaction lsquotranquillisantersquo le testde la chemineerdquo Medicina Experimentalis vol 3 no 1 pp 81ndash84 1960

[30] R Ader J A Weijnen and P Moleman ldquoRetention of a passiveavoidance response as a function of the intensity and durationof electric shockrdquo Psychonomic Science vol 26 no 3 pp 125ndash128 1972

[31] E M Blalock K-C Chen K Sharrow et al ldquoGene microar-rays in hippocampal aging statistical profiling identifies novelprocesses correlated with cognitive impairmentrdquo Journal ofNeuroscience vol 23 no 9 pp 3807ndash3819 2003

[32] K Isomae S Morimoto H Hasegawa K Morita and JKamei ldquoEffects of T-82 a novel acetylcholinesterase inhibitoron impaired learning and memory in passive avoidance task inratsrdquo European Journal of Pharmacology vol 465 no 1-2 pp97ndash103 2003

[33] J Patora and B Klimek ldquoFlavonoids from lemon balm (Melissaofficinalis L Lamiaceae)rdquo Acta Poloniae Pharmaceutica-DrugResearch vol 59 no 2 pp 139ndash143 2002

[34] L Barros M Duenas M I Dias M J Sousa C Santos-Buelgaand I C F R Ferreira ldquoPhenolic profiles of cultivated invitro cultured and commercial samples of Melissa officinalis Linfusionsrdquo Food Chemistry vol 136 no 1 pp 1ndash8 2013

[35] T L Miron M Herrero and E Ibanez ldquoEnrichment of antiox-idant compounds from lemon balm (Melissa officinalis) bypressurized liquid extraction and enzyme-assisted extractionrdquoJournal of Chromatography A vol 1288 pp 1ndash9 2013

[36] Y Lu L Y Foo and H Wong ldquoSagecoumarin a novel caffeicacid trimer from Salvia officinalisrdquo Phytochemistry vol 52 no6 pp 1149ndash1152 1999

[37] S Abuhamdah LHuangM S J Elliott et al ldquoPharmacologicalprofile of an essential oil derived from Melissa officinalis withanti-agitation properties focus on ligand-gated channelsrdquo Jour-nal of Pharmacy and Pharmacology vol 60 no 3 pp 377ndash3842008

[38] G Wake J Court A Pickering R Lewis R Wilkins andE Perry ldquoCNS acetylcholine receptor activity in Europeanmedicinal plants traditionally used to improve failing memoryrdquoJournal of Ethnopharmacology vol 69 no 2 pp 105ndash114 2000

[39] S-H Ryu C Katona B Rive and G Livingston ldquoPersistenceof and changes in neuropsychiatric symptoms in Alzheimerdisease over 6months the LASER-AD studyrdquoAmerican Journalof Geriatric Psychiatry vol 13 no 11 pp 976ndash983 2005

[40] L N Gitlin H C Kales and C G Lyketsos ldquoNonpharma-cologic management of behavioral symptoms in dementiardquoJournal of the AmericanMedical Association vol 308 no 19 pp2020ndash2029 2012

[41] A Konczol J Muller E Foldes et al ldquoApplicability of a blood-brain barrier specific artificial membrane permeability assay atthe early stage of natural product-based CNS drug discoveryrdquoJournal of Natural Products vol 76 no 4 pp 655ndash663 2013

[42] Y-J Zhang L Wu Q-L Zhang J Li F-X Yin and Y YuanldquoPharmacokinetics of phenolic compounds of Danshen extractin rat blood and brain by microdialysis samplingrdquo Journal ofEthnopharmacology vol 136 no 1 pp 129ndash136 2011

[43] X Li C Yu Y Lu et al ldquoPharmacokinetics tissue distributionmetabolism and excretion of depside salts from Salvia miltior-rhiza in ratsrdquo Drug Metabolism and Disposition vol 35 no 2pp 234ndash239 2007

[44] H Xu Y Li X Che H Tian H Fan and K Liu ldquoMetabolismof salvianolic acid a and antioxidant activities of its methylatedmetabolitesrdquo Drug Metabolism and Disposition vol 42 no 2pp 274ndash281 2014

[45] D H Kim S J Park J M Kim et al ldquoCognitive dysfunctionsinduced by a cholinergic blockade and A120573 25-35 peptide areattenuated by salvianolic acid Brdquo Neuropharmacology vol 61no 8 pp 1432ndash1440 2011

[46] G-H Du Y Qiu and J-T Zhang ldquoSalvianolic acid B protectsthe memory functions against transient cerebral ischemia inmicerdquo Journal of Asian Natural Products Research vol 2 no 2pp 145ndash152 2000

[47] G Du and J Zhang ldquoProtective effects of salvianolic acid Aagainst impairment of memory induced by cerebral ischemia-reperfusion in micerdquo Chinese Medical Journal vol 110 no 1 pp65ndash68 1997

[48] J Anwar R M Spanevello G Thome et al ldquoEffects of caffeicacid on behavioral parameters and on the activity of acetyl-cholinesterase in different tissues from adult ratsrdquo Pharmacol-ogy Biochemistry and Behavior vol 103 no 2 pp 386ndash394 2012

[49] YW Lee D H Kim S J Jeon et al ldquoNeuroprotective effects ofsalvianolic acid B on anA12057325-35 peptide-inducedmousemodelof Alzheimerrsquos diseaserdquo European Journal of Pharmacology vol704 no 1ndash3 pp 70ndash77 2013

[50] J Zhong M-K Tang Y Zhang Q-P Xu and J-T ZhangldquoEffect of salvianolic acid B on neural cells damage and neu-rogenesis after brain ischemia-reperfusion in ratsrdquo Yao Xue XueBao vol 42 no 7 pp 716ndash721 2007

[51] P Zhuang Y Zhang G Cui et al ldquoDirect stimulation of adultneural stemprogenitor cells in vitro and neurogenesis in vivo bysalvianolic acid Brdquo PLoS ONE vol 7 no 4 Article ID e356362012

[52] M TangW Feng Y Zhang J Zhong and J Zhang ldquoSalvianolicacid B improves motor function after cerebral ischemia in ratsrdquoBehavioural Pharmacology vol 17 no 5-6 pp 493ndash498 2006

[53] Q Li L-P Han Z-H Li J-T Zhang and M-K Tang ldquoSal-vianolic acid B alleviate the disruption of blood-brain barrierin rats after cerebral ischemia-reperfusion by inhibiting MAPKpathwayrdquo Yao Xue Xue Bao vol 45 no 12 pp 1485ndash1490 2010

[54] Y-L LinH-J Tsay T-H Lai T-T Tzeng andY-J Shiao ldquoLith-ospermic acid attenuates 1-methyl-4-phenylpyridine-inducedneurotoxicity by blocking neuronal apoptotic and neuroinflam-matory pathwaysrdquo Journal of Biomedical Science vol 22 article37 2015

[55] M-K Tang and J-T Zhang ldquoSalvianolic acid B inhibits fibrilformation and neurotoxicity of amyloid beta-protein in vitrordquoActa Pharmacologica Sinica vol 22 no 4 pp 380ndash384 2001

[56] S S K Durairajan Q Yuan L Xie et al ldquoSalvianolic acid Binhibits A120573 fibril formation and disaggregates preformed fibrilsand protects against A120573-induced cytotoxictyrdquo NeurochemistryInternational vol 52 no 4-5 pp 741ndash750 2008

[57] FD Pinheiro Fernandes A P FonteneleMenezes J C de SousaNeves et al ldquoCaffeic acid protects mice from memory deficits

Evidence-Based Complementary and Alternative Medicine 17

induced by focal cerebral ischemiardquo Behavioural Pharmacologyvol 25 no 7 pp 637ndash647 2014

[58] S-J Tsai C-Y Chao andM-C Yin ldquoPreventive and therapeu-tic effects of caffeic acid against inflammatory injury in striatumof MPTP-treated micerdquo European Journal of Pharmacology vol670 no 2-3 pp 441ndash447 2011

[59] J H Kim QWang J M Choi S Lee and E J Cho ldquoProtectiverole of caffeic acid in an 119860120573

25minus35

-induced Alzheimerrsquos diseasemodelrdquo Nutrition Research and Practice vol 9 no 5 pp 480ndash488 2015

[60] G Liang B Shi W Luo and J Yang ldquoThe protective effect ofcaffeic acid on global cerebral ischemia-reperfusion injury inratsrdquo Behavioral and Brain Functions vol 11 no 1 article 182015

[61] D Y Yoo J H ChoiW Kim et al ldquoEffects of luteolin on spatialmemory cell proliferation and neuroblast differentiation in thehippocampal dentate gyrus in a scopolamine-induced amnesiamodelrdquo Neurological Research vol 35 no 8 pp 813ndash820 2013

[62] Y Liu X Tian L Gou L Sun X Ling and X Yin ldquoLuteolinattenuates diabetes-associated cognitive decline in ratsrdquo BrainResearch Bulletin vol 94 pp 23ndash29 2013

[63] S-M Liu Z-H Yang andX-B Sun ldquoSimultaneous determina-tion of six Salvia miltiorrhiza gradients in rat plasma and brainby LC-MSMSrdquo Zhongguo Zhongyao Zazhi vol 39 no 9 pp1704ndash1708 2014

[64] M A Papandreou A Dimakopoulou Z I Linardaki et alldquoEffect of a polyphenol-rich wild blueberry extract on cognitiveperformance of mice brain antioxidant markers and acetyl-cholinesterase activityrdquo Behavioural Brain Research vol 198 no2 pp 352ndash358 2009

[65] M-S Garcıa-Ayllon O Cauli M-X Silveyra et al ldquoBraincholinergic impairment in liver failurerdquo Brain vol 131 no 11pp 2946ndash2956 2008

[66] G K Ferreira M Carvalho-Silva C L Goncalves et al ldquoL-Tyrosine administration increases acetylcholinesterase activityin ratsrdquo Neurochemistry International vol 61 no 8 pp 1370ndash1374 2012

[67] R Vassar P-H Kuhn C Haass et al ldquoFunction therapeuticpotential and cell biology of BACE proteases current status andfuture prospectsrdquo Journal of Neurochemistry vol 130 no 1 pp4ndash28 2014

[68] R Vassar ldquoThe 120573-secretase BACE a prime drug target forAlzheimerrsquos diseaserdquo Journal of Molecular Neuroscience vol 17no 2 pp 157ndash170 2001

[69] D T R Coulson N Beyer J G Quinn et al ldquoBACE1 mRNAexpression in Alzheimerrsquos disease postmortem brain tissuerdquoJournal of Alzheimerrsquos Disease vol 22 no 4 pp 1111ndash1122 2010

[70] S Vladimir-Knezevic B Blazekovic M Kindl J Vladic A DLower-Nedza and A H Brantner ldquoAcetylcholinesterase inhib-itory antioxidant and phytochemical properties of selectedmedicinal plants of the lamiaceae familyrdquoMolecules vol 19 no1 pp 767ndash782 2014

[71] F Marcelo C Dias A Martins et al ldquoMolecular recognitionof rosmarinic acid from Salvia sclareoides extracts by ace-tylcholinesterase a new binding site detected by NMR spec-troscopyrdquo ChemistrymdashA European Journal vol 19 no 21 pp6641ndash6649 2013

[72] G Johnson and S W Moore ldquoWhy has butyrylcholinesterasebeen retained Structural and functional diversification in aduplicated generdquoNeurochemistry International vol 61 no 5 pp783ndash797 2012

[73] R J Grayer M R Eckert N C Veitch et al ldquoThe chemo-taxonomic significance of two bioactive caffeic acid estersnepetoidins A and B in the Lamiaceaerdquo Phytochemistry vol 64no 2 pp 519ndash528 2003

[74] A M D El-Mousallamy U W Hawas and S A M HusseinldquoTeucrol a decarboxyrosmarinic acid and its 41015840-O-triglycosideteucroside from Teucrium pilosumrdquo Phytochemistry vol 55 no8 pp 927ndash931 2000

[75] I Fecka and S Turek ldquoDetermination of water-soluble polyphe-nolic compounds in commercial herbal teas from Lamiaceaepeppermint melissa and sagerdquo Journal of Agricultural and FoodChemistry vol 55 no 26 pp 10908ndash10917 2007

[76] L W Sumner A Amberg D Barrett et al ldquoProposed min-imum reporting standards for chemical analysis WorkingGroup (CAWG) Metabolomics Standards Initiative (MSI)rdquoMetabolomics vol 3 pp 211ndash221 2007

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Evidence-Based Complementary and Alternative Medicine 15

Taken together it seems that the MO activity representsa possible option as complementary interventions to relievethe symptoms of mild dementia

Competing Interests

The authors declare that they have no competing interests

Acknowledgments

This work was supported by the Ministry of Science andHigher EducationWarsaw Poland from educational sources(2008ndash2011 Grant no N 405417836)

References

[1] S L Cole and R Vassar ldquoThe Alzheimerrsquos disease 120573-secretaseenzyme BACE1rdquo Molecular Neurodegeneration vol 2 no 1article 22 2007

[2] LACraigN SHong andR JMcDonald ldquoRevisiting the cho-linergic hypothesis in the development of Alzheimerrsquos diseaserdquoNeuroscience amp Biobehavioral Reviews vol 35 no 6 pp 1397ndash1409 2011

[3] N G M Gomes M G Campos J M C Orfao and C A FRibeiro ldquoPlants with neurobiological activity as potential tar-gets for drug discoveryrdquo Progress in Neuro-Psychopharmacologyamp Biological Psychiatry vol 33 no 8 pp 1372ndash1389 2009

[4] WHO WHO Monographs on Selected Medicinal PlantsmdashVolume 2 WHO Geneva Switzerland 2004 httpappswhointmedicinedocsendJs4927e18htmlJs4927e18

[5] M Bayat A A Tameh M H Ghahremani et al ldquoNeuropro-tective properties of Melissa officinalis after hypoxic-ischemicinjury both in vitro and in vivordquo DARU Journal of Pharmaceu-tical Sciences vol 20 article 42 2012

[6] J P Kamdem A Adeniran A A Boligon et al ldquoAntioxidantactivity genotoxicity and cytotoxicity evaluation of lemon balm(Melissa officinalis L) ethanolic extract Its potential role inneuroprotectionrdquo Industrial Crops and Products vol 51 pp 26ndash34 2013

[7] J-T Lin Y-C Chen Y-C Lee C-W R Hou F-L Chen andD-J Yang ldquoAntioxidant anti-proliferative and cyclooxygenase-2 inhibitory activities of ethanolic extracts from lemon balm(Melissa officinalis L) leavesrdquo LWTmdashFood Science and Technol-ogy vol 49 no 1 pp 1ndash7 2012

[8] G Guginski A P Luiz M D Silva et al ldquoMechanisms involvedin the antinociception caused by ethanolic extract obtainedfrom the leaves of Melissa officinalis (lemon balm) in micerdquoPharmacology Biochemistry amp Behavior vol 93 no 1 pp 10ndash162009

[9] N Perry G Court N Bidet J Court and E Perry ldquoEuropeanherbs with cholinergic activities potential in dementia therapyrdquoInternational Journal of Geriatric Psychiatry vol 11 no 12 pp1063ndash1069 1996

[10] E K Perry A T Pickering W W Wang P J Houghtonand N S L Perry ldquoMedicinal plants and Alzheimerrsquos diseasefrom ethnobotany to phytotherapyrdquo Journal of Pharmacy andPharmacology vol 51 no 5 pp 527ndash534 1999

[11] M-J R Howes N S L Perry and P J Houghton ldquoPlants withtraditional uses and activities relevant to the management ofAlzheimerrsquos disease and other cognitive disordersrdquo Phytother-apy Research vol 17 no 1 pp 1ndash18 2003

[12] I Orhan and M Aslan ldquoAppraisal of scopolamine-inducedantiamnesic effect in mice and in vitro antiacetylcholinesteraseand antioxidant activities of some traditionally used Lamiaceaeplantsrdquo Journal of Ethnopharmacology vol 122 no 2 pp 327ndash332 2009

[13] S Akhondzadeh M Noroozian M Mohammadi S OhadiniaA H Jamshidi and M Khani ldquoMelissa officinalis extract inthe treatment of patients with mild to moderate Alzheimerrsquosdisease a double blind randomised placebo controlled trialrdquoJournal of Neurology Neurosurgery and Psychiatry vol 74 no 7pp 863ndash866 2003

[14] D O Kennedy A B Scholey N T J Tildesley E K Perry andK AWesnes ldquoModulation ofmood and cognitive performancefollowing acute administration of Melissa officinalis (lemonbalm)rdquo Pharmacology Biochemistry amp Behavior vol 72 no 4pp 953ndash964 2002

[15] D O Kennedy G Wake S Savelev et al ldquoModulation of moodand cognitive performance following acute administration ofsingle doses of Melissa officinalis (Lemon balm) with humanCNS nicotinic and muscarinic receptor-binding propertiesrdquoNeuropsychopharmacology vol 28 no 10 pp 1871ndash1881 2003

[16] D O Kennedy and E L Wightman ldquoHerbal extracts and phy-tochemicals plant secondarymetabolites and the enhancementof human brain functionrdquoAdvances inNutrition vol 2 no 1 pp32ndash50 2011

[17] R P Pereira A A Boligon A S Appel et al ldquoChemical com-position antioxidant and anticholinesterase activity of Melissaofficinalisrdquo Industrial Crops and Products vol 53 pp 34ndash452014

[18] M Soodi N Naghdi H Hajimehdipoor S Choopani andE Sahraei ldquoMemory-improving activity of Melissa officinalisextract in naıve and scopolamine-treated ratsrdquo Research inPharmaceutical Sciences vol 9 no 2 pp 107ndash114 2014

[19] K Dastmalchi V Ollilainen P Lackman et al ldquoAcetyl-cholinesterase inhibitory guided fractionation of Melissa offic-inalis Lrdquo Bioorganic amp Medicinal Chemistry vol 17 no 2 pp867ndash871 2009

[20] W Chaiyana and S Okonogi ldquoInhibition of cholinesterase byessential oil from food plantrdquo Phytomedicine vol 19 no 8-9 pp836ndash839 2012

[21] K Dastmalchi H J D Dorman P P Oinonen Y Darwis ILaakso and R Hiltunen ldquoChemical composition and in vitroantioxidative activity of a lemon balm (Melissa officinalis L)extractrdquo LWTmdashFood Science and Technology vol 41 no 3 pp391ndash400 2008

[22] P Pereira D Tysca P Oliveira L F D S Brum J N Picadaand P Ardenghi ldquoNeurobehavioral and genotoxic aspects ofrosmarinic acidrdquo Pharmacological Research vol 52 no 3 pp199ndash203 2005

[23] M Ozarowski P L Mikolajczak A Bogacz et al ldquoRosmarinusofficinalis L leaf extract improves memory impairment andaffects acetylcholinesterase and butyrylcholinesterase activitiesin rat brainrdquo Fitoterapia vol 91 pp 261ndash271 2013

[24] G P Kumar and F Khanum ldquoNeuroprotective potential of phy-tochemicalsrdquo Pharmacognosy Reviews vol 6 no 12 pp 81ndash902012

[25] M Stobiecki A Staszkow A Piasecka P M Garcia-LopezF Zamora-Natera and P Kachlicki ldquoLC-MSMS profiling offlavonoid conjugates in wild mexican lupine Lupinus reflexusrdquoJournal of Natural Products vol 73 no 7 pp 1254ndash1260 2010

[26] A Wojakowska A Piasecka P M Garcıa-Lopez et al ldquoStruc-tural analysis and profiling of phenolic secondarymetabolites of

16 Evidence-Based Complementary and Alternative Medicine

Mexican lupine species using LC-MS techniquesrdquo Phytochem-istry vol 92 pp 71ndash86 2013

[27] A Gryszczynska B Opala Z Lowicki et al ldquoBioactive com-pounds determination in the callus and hydroalcoholic extractsfrom Salvia miltiorrhiza and Salvia przewalskiimdashpreliminarystudy on their anti-alcoholic activity effectsrdquo PhytochemistryLetters vol 11 pp 399ndash403 2015

[28] K Slinkart and V L Singleton ldquoTotal phenol analysis automa-tion and comparison with manual methodrdquo American Journalof Enology and Viticulture vol 28 no 1 pp 49ndash55 1977

[29] P J R Boissier J Tardy and J C Diverres ldquoUne nouvellemethode simple pour explorer lrsquoaction lsquotranquillisantersquo le testde la chemineerdquo Medicina Experimentalis vol 3 no 1 pp 81ndash84 1960

[30] R Ader J A Weijnen and P Moleman ldquoRetention of a passiveavoidance response as a function of the intensity and durationof electric shockrdquo Psychonomic Science vol 26 no 3 pp 125ndash128 1972

[31] E M Blalock K-C Chen K Sharrow et al ldquoGene microar-rays in hippocampal aging statistical profiling identifies novelprocesses correlated with cognitive impairmentrdquo Journal ofNeuroscience vol 23 no 9 pp 3807ndash3819 2003

[32] K Isomae S Morimoto H Hasegawa K Morita and JKamei ldquoEffects of T-82 a novel acetylcholinesterase inhibitoron impaired learning and memory in passive avoidance task inratsrdquo European Journal of Pharmacology vol 465 no 1-2 pp97ndash103 2003

[33] J Patora and B Klimek ldquoFlavonoids from lemon balm (Melissaofficinalis L Lamiaceae)rdquo Acta Poloniae Pharmaceutica-DrugResearch vol 59 no 2 pp 139ndash143 2002

[34] L Barros M Duenas M I Dias M J Sousa C Santos-Buelgaand I C F R Ferreira ldquoPhenolic profiles of cultivated invitro cultured and commercial samples of Melissa officinalis Linfusionsrdquo Food Chemistry vol 136 no 1 pp 1ndash8 2013

[35] T L Miron M Herrero and E Ibanez ldquoEnrichment of antiox-idant compounds from lemon balm (Melissa officinalis) bypressurized liquid extraction and enzyme-assisted extractionrdquoJournal of Chromatography A vol 1288 pp 1ndash9 2013

[36] Y Lu L Y Foo and H Wong ldquoSagecoumarin a novel caffeicacid trimer from Salvia officinalisrdquo Phytochemistry vol 52 no6 pp 1149ndash1152 1999

[37] S Abuhamdah LHuangM S J Elliott et al ldquoPharmacologicalprofile of an essential oil derived from Melissa officinalis withanti-agitation properties focus on ligand-gated channelsrdquo Jour-nal of Pharmacy and Pharmacology vol 60 no 3 pp 377ndash3842008

[38] G Wake J Court A Pickering R Lewis R Wilkins andE Perry ldquoCNS acetylcholine receptor activity in Europeanmedicinal plants traditionally used to improve failing memoryrdquoJournal of Ethnopharmacology vol 69 no 2 pp 105ndash114 2000

[39] S-H Ryu C Katona B Rive and G Livingston ldquoPersistenceof and changes in neuropsychiatric symptoms in Alzheimerdisease over 6months the LASER-AD studyrdquoAmerican Journalof Geriatric Psychiatry vol 13 no 11 pp 976ndash983 2005

[40] L N Gitlin H C Kales and C G Lyketsos ldquoNonpharma-cologic management of behavioral symptoms in dementiardquoJournal of the AmericanMedical Association vol 308 no 19 pp2020ndash2029 2012

[41] A Konczol J Muller E Foldes et al ldquoApplicability of a blood-brain barrier specific artificial membrane permeability assay atthe early stage of natural product-based CNS drug discoveryrdquoJournal of Natural Products vol 76 no 4 pp 655ndash663 2013

[42] Y-J Zhang L Wu Q-L Zhang J Li F-X Yin and Y YuanldquoPharmacokinetics of phenolic compounds of Danshen extractin rat blood and brain by microdialysis samplingrdquo Journal ofEthnopharmacology vol 136 no 1 pp 129ndash136 2011

[43] X Li C Yu Y Lu et al ldquoPharmacokinetics tissue distributionmetabolism and excretion of depside salts from Salvia miltior-rhiza in ratsrdquo Drug Metabolism and Disposition vol 35 no 2pp 234ndash239 2007

[44] H Xu Y Li X Che H Tian H Fan and K Liu ldquoMetabolismof salvianolic acid a and antioxidant activities of its methylatedmetabolitesrdquo Drug Metabolism and Disposition vol 42 no 2pp 274ndash281 2014

[45] D H Kim S J Park J M Kim et al ldquoCognitive dysfunctionsinduced by a cholinergic blockade and A120573 25-35 peptide areattenuated by salvianolic acid Brdquo Neuropharmacology vol 61no 8 pp 1432ndash1440 2011

[46] G-H Du Y Qiu and J-T Zhang ldquoSalvianolic acid B protectsthe memory functions against transient cerebral ischemia inmicerdquo Journal of Asian Natural Products Research vol 2 no 2pp 145ndash152 2000

[47] G Du and J Zhang ldquoProtective effects of salvianolic acid Aagainst impairment of memory induced by cerebral ischemia-reperfusion in micerdquo Chinese Medical Journal vol 110 no 1 pp65ndash68 1997

[48] J Anwar R M Spanevello G Thome et al ldquoEffects of caffeicacid on behavioral parameters and on the activity of acetyl-cholinesterase in different tissues from adult ratsrdquo Pharmacol-ogy Biochemistry and Behavior vol 103 no 2 pp 386ndash394 2012

[49] YW Lee D H Kim S J Jeon et al ldquoNeuroprotective effects ofsalvianolic acid B on anA12057325-35 peptide-inducedmousemodelof Alzheimerrsquos diseaserdquo European Journal of Pharmacology vol704 no 1ndash3 pp 70ndash77 2013

[50] J Zhong M-K Tang Y Zhang Q-P Xu and J-T ZhangldquoEffect of salvianolic acid B on neural cells damage and neu-rogenesis after brain ischemia-reperfusion in ratsrdquo Yao Xue XueBao vol 42 no 7 pp 716ndash721 2007

[51] P Zhuang Y Zhang G Cui et al ldquoDirect stimulation of adultneural stemprogenitor cells in vitro and neurogenesis in vivo bysalvianolic acid Brdquo PLoS ONE vol 7 no 4 Article ID e356362012

[52] M TangW Feng Y Zhang J Zhong and J Zhang ldquoSalvianolicacid B improves motor function after cerebral ischemia in ratsrdquoBehavioural Pharmacology vol 17 no 5-6 pp 493ndash498 2006

[53] Q Li L-P Han Z-H Li J-T Zhang and M-K Tang ldquoSal-vianolic acid B alleviate the disruption of blood-brain barrierin rats after cerebral ischemia-reperfusion by inhibiting MAPKpathwayrdquo Yao Xue Xue Bao vol 45 no 12 pp 1485ndash1490 2010

[54] Y-L LinH-J Tsay T-H Lai T-T Tzeng andY-J Shiao ldquoLith-ospermic acid attenuates 1-methyl-4-phenylpyridine-inducedneurotoxicity by blocking neuronal apoptotic and neuroinflam-matory pathwaysrdquo Journal of Biomedical Science vol 22 article37 2015

[55] M-K Tang and J-T Zhang ldquoSalvianolic acid B inhibits fibrilformation and neurotoxicity of amyloid beta-protein in vitrordquoActa Pharmacologica Sinica vol 22 no 4 pp 380ndash384 2001

[56] S S K Durairajan Q Yuan L Xie et al ldquoSalvianolic acid Binhibits A120573 fibril formation and disaggregates preformed fibrilsand protects against A120573-induced cytotoxictyrdquo NeurochemistryInternational vol 52 no 4-5 pp 741ndash750 2008

[57] FD Pinheiro Fernandes A P FonteneleMenezes J C de SousaNeves et al ldquoCaffeic acid protects mice from memory deficits

Evidence-Based Complementary and Alternative Medicine 17

induced by focal cerebral ischemiardquo Behavioural Pharmacologyvol 25 no 7 pp 637ndash647 2014

[58] S-J Tsai C-Y Chao andM-C Yin ldquoPreventive and therapeu-tic effects of caffeic acid against inflammatory injury in striatumof MPTP-treated micerdquo European Journal of Pharmacology vol670 no 2-3 pp 441ndash447 2011

[59] J H Kim QWang J M Choi S Lee and E J Cho ldquoProtectiverole of caffeic acid in an 119860120573

25minus35

-induced Alzheimerrsquos diseasemodelrdquo Nutrition Research and Practice vol 9 no 5 pp 480ndash488 2015

[60] G Liang B Shi W Luo and J Yang ldquoThe protective effect ofcaffeic acid on global cerebral ischemia-reperfusion injury inratsrdquo Behavioral and Brain Functions vol 11 no 1 article 182015

[61] D Y Yoo J H ChoiW Kim et al ldquoEffects of luteolin on spatialmemory cell proliferation and neuroblast differentiation in thehippocampal dentate gyrus in a scopolamine-induced amnesiamodelrdquo Neurological Research vol 35 no 8 pp 813ndash820 2013

[62] Y Liu X Tian L Gou L Sun X Ling and X Yin ldquoLuteolinattenuates diabetes-associated cognitive decline in ratsrdquo BrainResearch Bulletin vol 94 pp 23ndash29 2013

[63] S-M Liu Z-H Yang andX-B Sun ldquoSimultaneous determina-tion of six Salvia miltiorrhiza gradients in rat plasma and brainby LC-MSMSrdquo Zhongguo Zhongyao Zazhi vol 39 no 9 pp1704ndash1708 2014

[64] M A Papandreou A Dimakopoulou Z I Linardaki et alldquoEffect of a polyphenol-rich wild blueberry extract on cognitiveperformance of mice brain antioxidant markers and acetyl-cholinesterase activityrdquo Behavioural Brain Research vol 198 no2 pp 352ndash358 2009

[65] M-S Garcıa-Ayllon O Cauli M-X Silveyra et al ldquoBraincholinergic impairment in liver failurerdquo Brain vol 131 no 11pp 2946ndash2956 2008

[66] G K Ferreira M Carvalho-Silva C L Goncalves et al ldquoL-Tyrosine administration increases acetylcholinesterase activityin ratsrdquo Neurochemistry International vol 61 no 8 pp 1370ndash1374 2012

[67] R Vassar P-H Kuhn C Haass et al ldquoFunction therapeuticpotential and cell biology of BACE proteases current status andfuture prospectsrdquo Journal of Neurochemistry vol 130 no 1 pp4ndash28 2014

[68] R Vassar ldquoThe 120573-secretase BACE a prime drug target forAlzheimerrsquos diseaserdquo Journal of Molecular Neuroscience vol 17no 2 pp 157ndash170 2001

[69] D T R Coulson N Beyer J G Quinn et al ldquoBACE1 mRNAexpression in Alzheimerrsquos disease postmortem brain tissuerdquoJournal of Alzheimerrsquos Disease vol 22 no 4 pp 1111ndash1122 2010

[70] S Vladimir-Knezevic B Blazekovic M Kindl J Vladic A DLower-Nedza and A H Brantner ldquoAcetylcholinesterase inhib-itory antioxidant and phytochemical properties of selectedmedicinal plants of the lamiaceae familyrdquoMolecules vol 19 no1 pp 767ndash782 2014

[71] F Marcelo C Dias A Martins et al ldquoMolecular recognitionof rosmarinic acid from Salvia sclareoides extracts by ace-tylcholinesterase a new binding site detected by NMR spec-troscopyrdquo ChemistrymdashA European Journal vol 19 no 21 pp6641ndash6649 2013

[72] G Johnson and S W Moore ldquoWhy has butyrylcholinesterasebeen retained Structural and functional diversification in aduplicated generdquoNeurochemistry International vol 61 no 5 pp783ndash797 2012

[73] R J Grayer M R Eckert N C Veitch et al ldquoThe chemo-taxonomic significance of two bioactive caffeic acid estersnepetoidins A and B in the Lamiaceaerdquo Phytochemistry vol 64no 2 pp 519ndash528 2003

[74] A M D El-Mousallamy U W Hawas and S A M HusseinldquoTeucrol a decarboxyrosmarinic acid and its 41015840-O-triglycosideteucroside from Teucrium pilosumrdquo Phytochemistry vol 55 no8 pp 927ndash931 2000

[75] I Fecka and S Turek ldquoDetermination of water-soluble polyphe-nolic compounds in commercial herbal teas from Lamiaceaepeppermint melissa and sagerdquo Journal of Agricultural and FoodChemistry vol 55 no 26 pp 10908ndash10917 2007

[76] L W Sumner A Amberg D Barrett et al ldquoProposed min-imum reporting standards for chemical analysis WorkingGroup (CAWG) Metabolomics Standards Initiative (MSI)rdquoMetabolomics vol 3 pp 211ndash221 2007

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

16 Evidence-Based Complementary and Alternative Medicine

Mexican lupine species using LC-MS techniquesrdquo Phytochem-istry vol 92 pp 71ndash86 2013

[27] A Gryszczynska B Opala Z Lowicki et al ldquoBioactive com-pounds determination in the callus and hydroalcoholic extractsfrom Salvia miltiorrhiza and Salvia przewalskiimdashpreliminarystudy on their anti-alcoholic activity effectsrdquo PhytochemistryLetters vol 11 pp 399ndash403 2015

[28] K Slinkart and V L Singleton ldquoTotal phenol analysis automa-tion and comparison with manual methodrdquo American Journalof Enology and Viticulture vol 28 no 1 pp 49ndash55 1977

[29] P J R Boissier J Tardy and J C Diverres ldquoUne nouvellemethode simple pour explorer lrsquoaction lsquotranquillisantersquo le testde la chemineerdquo Medicina Experimentalis vol 3 no 1 pp 81ndash84 1960

[30] R Ader J A Weijnen and P Moleman ldquoRetention of a passiveavoidance response as a function of the intensity and durationof electric shockrdquo Psychonomic Science vol 26 no 3 pp 125ndash128 1972

[31] E M Blalock K-C Chen K Sharrow et al ldquoGene microar-rays in hippocampal aging statistical profiling identifies novelprocesses correlated with cognitive impairmentrdquo Journal ofNeuroscience vol 23 no 9 pp 3807ndash3819 2003

[32] K Isomae S Morimoto H Hasegawa K Morita and JKamei ldquoEffects of T-82 a novel acetylcholinesterase inhibitoron impaired learning and memory in passive avoidance task inratsrdquo European Journal of Pharmacology vol 465 no 1-2 pp97ndash103 2003

[33] J Patora and B Klimek ldquoFlavonoids from lemon balm (Melissaofficinalis L Lamiaceae)rdquo Acta Poloniae Pharmaceutica-DrugResearch vol 59 no 2 pp 139ndash143 2002

[34] L Barros M Duenas M I Dias M J Sousa C Santos-Buelgaand I C F R Ferreira ldquoPhenolic profiles of cultivated invitro cultured and commercial samples of Melissa officinalis Linfusionsrdquo Food Chemistry vol 136 no 1 pp 1ndash8 2013

[35] T L Miron M Herrero and E Ibanez ldquoEnrichment of antiox-idant compounds from lemon balm (Melissa officinalis) bypressurized liquid extraction and enzyme-assisted extractionrdquoJournal of Chromatography A vol 1288 pp 1ndash9 2013

[36] Y Lu L Y Foo and H Wong ldquoSagecoumarin a novel caffeicacid trimer from Salvia officinalisrdquo Phytochemistry vol 52 no6 pp 1149ndash1152 1999

[37] S Abuhamdah LHuangM S J Elliott et al ldquoPharmacologicalprofile of an essential oil derived from Melissa officinalis withanti-agitation properties focus on ligand-gated channelsrdquo Jour-nal of Pharmacy and Pharmacology vol 60 no 3 pp 377ndash3842008

[38] G Wake J Court A Pickering R Lewis R Wilkins andE Perry ldquoCNS acetylcholine receptor activity in Europeanmedicinal plants traditionally used to improve failing memoryrdquoJournal of Ethnopharmacology vol 69 no 2 pp 105ndash114 2000

[39] S-H Ryu C Katona B Rive and G Livingston ldquoPersistenceof and changes in neuropsychiatric symptoms in Alzheimerdisease over 6months the LASER-AD studyrdquoAmerican Journalof Geriatric Psychiatry vol 13 no 11 pp 976ndash983 2005

[40] L N Gitlin H C Kales and C G Lyketsos ldquoNonpharma-cologic management of behavioral symptoms in dementiardquoJournal of the AmericanMedical Association vol 308 no 19 pp2020ndash2029 2012

[41] A Konczol J Muller E Foldes et al ldquoApplicability of a blood-brain barrier specific artificial membrane permeability assay atthe early stage of natural product-based CNS drug discoveryrdquoJournal of Natural Products vol 76 no 4 pp 655ndash663 2013

[42] Y-J Zhang L Wu Q-L Zhang J Li F-X Yin and Y YuanldquoPharmacokinetics of phenolic compounds of Danshen extractin rat blood and brain by microdialysis samplingrdquo Journal ofEthnopharmacology vol 136 no 1 pp 129ndash136 2011

[43] X Li C Yu Y Lu et al ldquoPharmacokinetics tissue distributionmetabolism and excretion of depside salts from Salvia miltior-rhiza in ratsrdquo Drug Metabolism and Disposition vol 35 no 2pp 234ndash239 2007

[44] H Xu Y Li X Che H Tian H Fan and K Liu ldquoMetabolismof salvianolic acid a and antioxidant activities of its methylatedmetabolitesrdquo Drug Metabolism and Disposition vol 42 no 2pp 274ndash281 2014

[45] D H Kim S J Park J M Kim et al ldquoCognitive dysfunctionsinduced by a cholinergic blockade and A120573 25-35 peptide areattenuated by salvianolic acid Brdquo Neuropharmacology vol 61no 8 pp 1432ndash1440 2011

[46] G-H Du Y Qiu and J-T Zhang ldquoSalvianolic acid B protectsthe memory functions against transient cerebral ischemia inmicerdquo Journal of Asian Natural Products Research vol 2 no 2pp 145ndash152 2000

[47] G Du and J Zhang ldquoProtective effects of salvianolic acid Aagainst impairment of memory induced by cerebral ischemia-reperfusion in micerdquo Chinese Medical Journal vol 110 no 1 pp65ndash68 1997

[48] J Anwar R M Spanevello G Thome et al ldquoEffects of caffeicacid on behavioral parameters and on the activity of acetyl-cholinesterase in different tissues from adult ratsrdquo Pharmacol-ogy Biochemistry and Behavior vol 103 no 2 pp 386ndash394 2012

[49] YW Lee D H Kim S J Jeon et al ldquoNeuroprotective effects ofsalvianolic acid B on anA12057325-35 peptide-inducedmousemodelof Alzheimerrsquos diseaserdquo European Journal of Pharmacology vol704 no 1ndash3 pp 70ndash77 2013

[50] J Zhong M-K Tang Y Zhang Q-P Xu and J-T ZhangldquoEffect of salvianolic acid B on neural cells damage and neu-rogenesis after brain ischemia-reperfusion in ratsrdquo Yao Xue XueBao vol 42 no 7 pp 716ndash721 2007

[51] P Zhuang Y Zhang G Cui et al ldquoDirect stimulation of adultneural stemprogenitor cells in vitro and neurogenesis in vivo bysalvianolic acid Brdquo PLoS ONE vol 7 no 4 Article ID e356362012

[52] M TangW Feng Y Zhang J Zhong and J Zhang ldquoSalvianolicacid B improves motor function after cerebral ischemia in ratsrdquoBehavioural Pharmacology vol 17 no 5-6 pp 493ndash498 2006

[53] Q Li L-P Han Z-H Li J-T Zhang and M-K Tang ldquoSal-vianolic acid B alleviate the disruption of blood-brain barrierin rats after cerebral ischemia-reperfusion by inhibiting MAPKpathwayrdquo Yao Xue Xue Bao vol 45 no 12 pp 1485ndash1490 2010

[54] Y-L LinH-J Tsay T-H Lai T-T Tzeng andY-J Shiao ldquoLith-ospermic acid attenuates 1-methyl-4-phenylpyridine-inducedneurotoxicity by blocking neuronal apoptotic and neuroinflam-matory pathwaysrdquo Journal of Biomedical Science vol 22 article37 2015

[55] M-K Tang and J-T Zhang ldquoSalvianolic acid B inhibits fibrilformation and neurotoxicity of amyloid beta-protein in vitrordquoActa Pharmacologica Sinica vol 22 no 4 pp 380ndash384 2001

[56] S S K Durairajan Q Yuan L Xie et al ldquoSalvianolic acid Binhibits A120573 fibril formation and disaggregates preformed fibrilsand protects against A120573-induced cytotoxictyrdquo NeurochemistryInternational vol 52 no 4-5 pp 741ndash750 2008

[57] FD Pinheiro Fernandes A P FonteneleMenezes J C de SousaNeves et al ldquoCaffeic acid protects mice from memory deficits

Evidence-Based Complementary and Alternative Medicine 17

induced by focal cerebral ischemiardquo Behavioural Pharmacologyvol 25 no 7 pp 637ndash647 2014

[58] S-J Tsai C-Y Chao andM-C Yin ldquoPreventive and therapeu-tic effects of caffeic acid against inflammatory injury in striatumof MPTP-treated micerdquo European Journal of Pharmacology vol670 no 2-3 pp 441ndash447 2011

[59] J H Kim QWang J M Choi S Lee and E J Cho ldquoProtectiverole of caffeic acid in an 119860120573

25minus35

-induced Alzheimerrsquos diseasemodelrdquo Nutrition Research and Practice vol 9 no 5 pp 480ndash488 2015

[60] G Liang B Shi W Luo and J Yang ldquoThe protective effect ofcaffeic acid on global cerebral ischemia-reperfusion injury inratsrdquo Behavioral and Brain Functions vol 11 no 1 article 182015

[61] D Y Yoo J H ChoiW Kim et al ldquoEffects of luteolin on spatialmemory cell proliferation and neuroblast differentiation in thehippocampal dentate gyrus in a scopolamine-induced amnesiamodelrdquo Neurological Research vol 35 no 8 pp 813ndash820 2013

[62] Y Liu X Tian L Gou L Sun X Ling and X Yin ldquoLuteolinattenuates diabetes-associated cognitive decline in ratsrdquo BrainResearch Bulletin vol 94 pp 23ndash29 2013

[63] S-M Liu Z-H Yang andX-B Sun ldquoSimultaneous determina-tion of six Salvia miltiorrhiza gradients in rat plasma and brainby LC-MSMSrdquo Zhongguo Zhongyao Zazhi vol 39 no 9 pp1704ndash1708 2014

[64] M A Papandreou A Dimakopoulou Z I Linardaki et alldquoEffect of a polyphenol-rich wild blueberry extract on cognitiveperformance of mice brain antioxidant markers and acetyl-cholinesterase activityrdquo Behavioural Brain Research vol 198 no2 pp 352ndash358 2009

[65] M-S Garcıa-Ayllon O Cauli M-X Silveyra et al ldquoBraincholinergic impairment in liver failurerdquo Brain vol 131 no 11pp 2946ndash2956 2008

[66] G K Ferreira M Carvalho-Silva C L Goncalves et al ldquoL-Tyrosine administration increases acetylcholinesterase activityin ratsrdquo Neurochemistry International vol 61 no 8 pp 1370ndash1374 2012

[67] R Vassar P-H Kuhn C Haass et al ldquoFunction therapeuticpotential and cell biology of BACE proteases current status andfuture prospectsrdquo Journal of Neurochemistry vol 130 no 1 pp4ndash28 2014

[68] R Vassar ldquoThe 120573-secretase BACE a prime drug target forAlzheimerrsquos diseaserdquo Journal of Molecular Neuroscience vol 17no 2 pp 157ndash170 2001

[69] D T R Coulson N Beyer J G Quinn et al ldquoBACE1 mRNAexpression in Alzheimerrsquos disease postmortem brain tissuerdquoJournal of Alzheimerrsquos Disease vol 22 no 4 pp 1111ndash1122 2010

[70] S Vladimir-Knezevic B Blazekovic M Kindl J Vladic A DLower-Nedza and A H Brantner ldquoAcetylcholinesterase inhib-itory antioxidant and phytochemical properties of selectedmedicinal plants of the lamiaceae familyrdquoMolecules vol 19 no1 pp 767ndash782 2014

[71] F Marcelo C Dias A Martins et al ldquoMolecular recognitionof rosmarinic acid from Salvia sclareoides extracts by ace-tylcholinesterase a new binding site detected by NMR spec-troscopyrdquo ChemistrymdashA European Journal vol 19 no 21 pp6641ndash6649 2013

[72] G Johnson and S W Moore ldquoWhy has butyrylcholinesterasebeen retained Structural and functional diversification in aduplicated generdquoNeurochemistry International vol 61 no 5 pp783ndash797 2012

[73] R J Grayer M R Eckert N C Veitch et al ldquoThe chemo-taxonomic significance of two bioactive caffeic acid estersnepetoidins A and B in the Lamiaceaerdquo Phytochemistry vol 64no 2 pp 519ndash528 2003

[74] A M D El-Mousallamy U W Hawas and S A M HusseinldquoTeucrol a decarboxyrosmarinic acid and its 41015840-O-triglycosideteucroside from Teucrium pilosumrdquo Phytochemistry vol 55 no8 pp 927ndash931 2000

[75] I Fecka and S Turek ldquoDetermination of water-soluble polyphe-nolic compounds in commercial herbal teas from Lamiaceaepeppermint melissa and sagerdquo Journal of Agricultural and FoodChemistry vol 55 no 26 pp 10908ndash10917 2007

[76] L W Sumner A Amberg D Barrett et al ldquoProposed min-imum reporting standards for chemical analysis WorkingGroup (CAWG) Metabolomics Standards Initiative (MSI)rdquoMetabolomics vol 3 pp 211ndash221 2007

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Evidence-Based Complementary and Alternative Medicine 17

induced by focal cerebral ischemiardquo Behavioural Pharmacologyvol 25 no 7 pp 637ndash647 2014

[58] S-J Tsai C-Y Chao andM-C Yin ldquoPreventive and therapeu-tic effects of caffeic acid against inflammatory injury in striatumof MPTP-treated micerdquo European Journal of Pharmacology vol670 no 2-3 pp 441ndash447 2011

[59] J H Kim QWang J M Choi S Lee and E J Cho ldquoProtectiverole of caffeic acid in an 119860120573

25minus35

-induced Alzheimerrsquos diseasemodelrdquo Nutrition Research and Practice vol 9 no 5 pp 480ndash488 2015

[60] G Liang B Shi W Luo and J Yang ldquoThe protective effect ofcaffeic acid on global cerebral ischemia-reperfusion injury inratsrdquo Behavioral and Brain Functions vol 11 no 1 article 182015

[61] D Y Yoo J H ChoiW Kim et al ldquoEffects of luteolin on spatialmemory cell proliferation and neuroblast differentiation in thehippocampal dentate gyrus in a scopolamine-induced amnesiamodelrdquo Neurological Research vol 35 no 8 pp 813ndash820 2013

[62] Y Liu X Tian L Gou L Sun X Ling and X Yin ldquoLuteolinattenuates diabetes-associated cognitive decline in ratsrdquo BrainResearch Bulletin vol 94 pp 23ndash29 2013

[63] S-M Liu Z-H Yang andX-B Sun ldquoSimultaneous determina-tion of six Salvia miltiorrhiza gradients in rat plasma and brainby LC-MSMSrdquo Zhongguo Zhongyao Zazhi vol 39 no 9 pp1704ndash1708 2014

[64] M A Papandreou A Dimakopoulou Z I Linardaki et alldquoEffect of a polyphenol-rich wild blueberry extract on cognitiveperformance of mice brain antioxidant markers and acetyl-cholinesterase activityrdquo Behavioural Brain Research vol 198 no2 pp 352ndash358 2009

[65] M-S Garcıa-Ayllon O Cauli M-X Silveyra et al ldquoBraincholinergic impairment in liver failurerdquo Brain vol 131 no 11pp 2946ndash2956 2008

[66] G K Ferreira M Carvalho-Silva C L Goncalves et al ldquoL-Tyrosine administration increases acetylcholinesterase activityin ratsrdquo Neurochemistry International vol 61 no 8 pp 1370ndash1374 2012

[67] R Vassar P-H Kuhn C Haass et al ldquoFunction therapeuticpotential and cell biology of BACE proteases current status andfuture prospectsrdquo Journal of Neurochemistry vol 130 no 1 pp4ndash28 2014

[68] R Vassar ldquoThe 120573-secretase BACE a prime drug target forAlzheimerrsquos diseaserdquo Journal of Molecular Neuroscience vol 17no 2 pp 157ndash170 2001

[69] D T R Coulson N Beyer J G Quinn et al ldquoBACE1 mRNAexpression in Alzheimerrsquos disease postmortem brain tissuerdquoJournal of Alzheimerrsquos Disease vol 22 no 4 pp 1111ndash1122 2010

[70] S Vladimir-Knezevic B Blazekovic M Kindl J Vladic A DLower-Nedza and A H Brantner ldquoAcetylcholinesterase inhib-itory antioxidant and phytochemical properties of selectedmedicinal plants of the lamiaceae familyrdquoMolecules vol 19 no1 pp 767ndash782 2014

[71] F Marcelo C Dias A Martins et al ldquoMolecular recognitionof rosmarinic acid from Salvia sclareoides extracts by ace-tylcholinesterase a new binding site detected by NMR spec-troscopyrdquo ChemistrymdashA European Journal vol 19 no 21 pp6641ndash6649 2013

[72] G Johnson and S W Moore ldquoWhy has butyrylcholinesterasebeen retained Structural and functional diversification in aduplicated generdquoNeurochemistry International vol 61 no 5 pp783ndash797 2012

[73] R J Grayer M R Eckert N C Veitch et al ldquoThe chemo-taxonomic significance of two bioactive caffeic acid estersnepetoidins A and B in the Lamiaceaerdquo Phytochemistry vol 64no 2 pp 519ndash528 2003

[74] A M D El-Mousallamy U W Hawas and S A M HusseinldquoTeucrol a decarboxyrosmarinic acid and its 41015840-O-triglycosideteucroside from Teucrium pilosumrdquo Phytochemistry vol 55 no8 pp 927ndash931 2000

[75] I Fecka and S Turek ldquoDetermination of water-soluble polyphe-nolic compounds in commercial herbal teas from Lamiaceaepeppermint melissa and sagerdquo Journal of Agricultural and FoodChemistry vol 55 no 26 pp 10908ndash10917 2007

[76] L W Sumner A Amberg D Barrett et al ldquoProposed min-imum reporting standards for chemical analysis WorkingGroup (CAWG) Metabolomics Standards Initiative (MSI)rdquoMetabolomics vol 3 pp 211ndash221 2007

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom