Upload
others
View
3
Download
0
Embed Size (px)
Citation preview
Hindawi Publishing CorporationBioMed Research InternationalVolume 2013 Article ID 510736 9 pageshttpdxdoiorg1011552013510736
Research ArticleAntibacterial Antioxidant and Anticholinesterase Activities ofPlant Seed Extracts from Brazilian Semiarid Region
Davi Felipe Farias1 Terezinha Maria Souza2 Martocircnio Ponte Viana1
Bruno Marques Soares3 Arcelina Pacheco Cunha4 Ilka Maria Vasconcelos1
Naacutegila Maria Pontes Silva Ricardo4 Paulo Michel Pinheiro Ferreira56
Vacircnia Maria Maciel Melo7 and Ana Fontenele Urano Carvalho7
1 Departamento de Bioquımica e Biologia Molecular Universidade Federal do Ceara 60440-970 Fortaleza CE Brazil2 Netherlands Toxicogenomics Centre Maastricht University Universiteitssingel 50 6229ER Maastricht The Netherlands3 Departamento de Fisiologia e Farmacologia Faculdade de Medicina Universidade Federal do Ceara60430-270 Fortaleza CE Brazil
4Departamento de Quımica Organica e Inorganica Universidade Federal do Ceara 60440-970 Fortaleza CE Brazil5 Departamento de Ciencias Biologicas Campus Senador Helvıdio Nunes de Barros Universidade Federal do Piauı64600-000 Picos PI Brazil
6 Programa de Pos-Graduacao em Ciencias Farmaceuticas Universidade Federal do Piauı Avenida Universitariasn 64049-550 Teresina PI Brazil
7 Departamento de Biologia Universidade Federal do Ceara Campus do Pici 60451-970 Fortaleza CE Brazil
Correspondence should be addressed to Ana Fontenele Urano Carvalho auranoufcbr
Received 9 July 2013 Accepted 28 October 2013
Academic Editor Kazim Husain
Copyright copy 2013 Davi Felipe Farias 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
The antimicrobial antioxidant and anticholinesterase activities of ethanolic seed extracts of twenty-one plant species fromBraziliansemiarid region were investigated The extracts were tested for antimicrobial activity against six bacteria strains and three yeastsSix extracts presented activity against the Gram (minus) organism Salmonella choleraesuis and the Gram (+) organisms Staphylococcusaureus and Bacillus subtilis The MIC values ranged from 496 to 3732mgmL The Triplaris gardneriana extract presented activityagainst the three species withMIC values 188 1376 and 1115mgmL respectively Five extracts presented antioxidant activity withEC50 values ranging from 6973 120583gmL (T gardneriana) to 48751 120583gmL (Licania rigida) For the anticholinesterase activity elevenextracts were capable of inhibiting the enzyme activity From those T gardneriana Parkia platycephala and Connarus detersuspresented the best activities with inhibition values of 767 715 and 919 respectively The extracts that presented antimicrobialactivity were tested for hemolytic assay against humanA B andOblood types and rabbit blood From those only theMyracrodruonurundeuva extract presented activity (about 20 of hemolysis at the lowest tested concentration 19120583gmL) Infrared spectroscopyof six representative extracts attested the presence of tannins polyphenols and flavonoids which was confirmed by a qualitativephytochemical assay
1 Introduction
Plants are considered natural sources of new compounds ofmedical and biotechnological interest since they synthesizea large variety of bioactive compounds An analysis intothe sources of new drugs from 1981 to 2007 reveals thatalmost half of the drugs approved since 1994 were based on
natural products Among these there are various examplesof development of new drugs from plant sources [1] Manyresearches have dedicated great effort to detect secondarymetabolites with biological activities against cancer microor-ganisms tropical diseases and others [2 3] Well-knownexamples of these compounds include flavonoids phe-nols and phenolic glycosides unsaturated lactones sulphur
2 BioMed Research International
compounds saponins cyanogenic glycosides and glucosino-lates [4ndash6]
In this context the Brazilian semiarid region is a richand underexploited source of bioactive molecules This areais covered mainly by xeric shrublands known as ldquoCaatingardquoThis dry land vegetation grows over an area of 800000 km2in Northeastern Brazil and out of 1000 vascular plant speciesdescribed many are endemic [7 8] Many of these plants areused in folk medicine by local communities to treat severaldiseases pointing out the potential to discover new sourcesof useful compounds for several applications [9 10] Recentlyour team has reported for seed ethanolic extracts of BrazilianNortheastern plants promising results of insecticidal activityagainst Aedes aegypti [11] and antiproliferative effects totumor cell lines [12] In order to extend our knowledgeon the Brazilian semiarid vegetation this work aimed toevaluate the presence of antimicrobial anticholinesteraseand antioxidant activities in ethanolic seed extracts of twenty-one plant species The antimicrobial activity was performedby the agar diffusion assay using pathogenic bacteria andyeast antioxidant activity by the DPPH radical scavengingand anticholinesterase activity were measured In addition ahemolytic activity assay was done for antibacterial samples toassess the effects on nontarget cells
2 Materials and Methods
21 Plant Collection Mature wild seeds of twenty-one plantspecies were harvested (at least 500 g of each species) duringthe dry period (from January 2005 to March 2008) in thesemiarid region of the Ceara State Northeastern BrazilPlants were identified by taxonomist Dr Edson de PaulaNunes and voucher specimens were deposited at HerbariumPrisco Bezerra (EAC) at the Federal University of Ceara(Fortaleza Brazil)The studied species are depicted in Table 1which shows harvest information voucher numbers com-mon names and the traditional medicinal use by localpopulation
22 Preparation of Ethanolic Seed Extracts Seeds of freshlycollected plant material were separated from other plantmaterials air-dried and ground in a laboratory mill to amoderately fine powder (particle size le 05mm) Powderedmaterial (500 g) was submitted to extraction with 99ethanol (15 L) at room temperature (25ndash27∘C) for 3 daysThe supernatant was removed and filtered throughWhatmannumber 1 paper After that the initial powder sample wasstill submitted to two extractions which resulted in a finalvolume of extract near 45 L The ethanolic extracts resultingfrom three consecutive extractions were mixed together thenconcentrated under reduced pressure in rotary evaporatoruntil complete elimination of solvent and stored in a freezerat minus20∘C
A stock solution containing 50000 120583gsdotmLminus1 of eachcrude extract was prepared by suspending 50000mg ofextract in 5mL of pure dimethylsulphoxide (DMSO AldrichMilwaukee WI USA) and mixed by sonication (model RK-100 Bandelin Berlin Germany) for 20min The volume was
adjusted to 1000mL with distilled water to provide assaysolutions as required
23 Microorganisms Six bacterial strains were used Staphy-lococcus aureus (ATCC 25923) Bacillus subtilis (ATCC 6633)Enterobacter aerogenes (ATCC 13048) Salmonella cholerae-suis (ATCC 10708) Klebsiella pneumoniae (ATCC 10031)and Pseudomonas aeruginosa (ATCC 25619)These standardswere obtained from the American Type Culture Collection(ATCC) (Manassas VA USA) The stock culture was main-tained on Mueller Hinton agar (HiMedia Laboratories PvtLtdMumbai India)medium at 4∘CThree pathogenic yeastswere usedCandida albicansC krusei andC tropicalisThesefungal strains were obtained from the Microbial Ecologyand Biotechnology Laboratory Biology Department of theFederal University of Ceara Brazil The stock cultures weremaintained on Sabouraud Dextrose agar (HiMedia Labora-tories Pvt Ltd Mumbai India) medium at 4∘C
24 Antibacterial and Antifungal Activity The disc diffu-sion method was followed for antibacterial and antifungalsusceptibility test as described previously [16] with somemodifications Petri dishes were prepared by pouring 20mLof Mueller-Hinton agar for bacteria and Sabouraud Dextroseagar for fungi and allowed to solidify Dishes were dried and01mL of standardized inoculum suspension (106 CFUmLfor bacteria and absorbance of 0600 at 450 nm for fungi)was poured and uniformly spread The excess inoculum wasdrained and the inoculum was allowed to dry for 5min Thepaper filter discs (7mm of diameter) containing 20120583L ofeach ethanolic extract were then applied and the dishes wereincubated at 37∘C for 18 h for bacterial growth and at thesame temperature for 48 h for fungal growth Tetracycline(30mgdisc) was used as positive control for the antibacterialassay Nystatin solution (100000 IUmL EMS SP Brazil)(20120583Ldisc) was used as positive control for the antifungalassayThe inhibition zone was measured from the edge of thedisc to the inner margin of the surrounding pathogens Alldeterminations were run in triplicate
25 Minimum Inhibitory Concentration (MIC) Minimuminhibitory concentrations of the seed ethanolic extracts weretested as described previously by the twofold serial dilutionmethodThe test extract was dissolved in 5DMSO to obtain50mgsdotmLminus1 stock solution Stock solution was diluted withdistilled water to achieve 45 40 35 30 25 20 15 10 and5mgsdotmLminus1 or as required The wet paper discs with dilutionswere placed over the dish and the cultures were incubated inBOD incubators at 37∘C for 18 h (bacteria) and 37∘C for 48 h(yeasts) The lowest concentration which inhibits the visiblegrowth of tested organism after macroscopic evaluation wasdetermined asMICAll determinationswere run in triplicate
26 Antioxidant Activity The capacity to scavenge the 22-diphenyl-1-picrylhydrazyl (DPPH) free radical was moni-tored [17] The diluted crude extracts (01mL) in differ-ent concentrations (5 to 1000 120583gsdotmLminus1) were mixed with39mL of methanolic solution containing DPPH radicals
BioMed Research International 3
Table1Antibacteria
l(MIC)antio
xidantand
hemolyticactiv
ities
ofethano
licseed
extractsof
plantsfro
mBrazilian
semiarid
region
a
Family
Botanicaln
ame
Com
mon
name
(localitydateof
collection)V
oucher
number
Tradition
almedicinaluse
Yield
()b
Antioxidant
activ
ity(EC 5
0120583gmL)
cAnticho
linesterase
activ
ity(
)d
Antibacteria
lactivity
MIC
(mgmL)
e
SCh
oleraesuis
Bsubtilis
Saureus
Anacardiaceae
Myracrodruonurun
deuvaFrA
llAroeira-do-sertao
(Ararip
eNationalForest0107)
EAC34865
Usedto
treatcutaneou
sand
gynecologicalaffections
and
kidn
eyandrespira
tory
prob
lemsantiinfl
ammatory
antiu
lcerandhealing[13]
antih
istam
inicand
antim
icrobial[14
]analgesic
andantid
iarrheal[15]
252
ND
fND
ND
678
ND
Schinopsisbrasiliensis
Engler
Brauna
(Ararip
eNationalForest0107)
EAC35643
Usedto
treatnervou
snessa
ndhyste
riaanalgesic[15]
288
ND
ND
3732
ND
ND
Caryocaraceae
Caryocar
coria
ceum
Wittm
Pequ
i(Ararip
eNationalForest0905)
EAC35251
Not
describ
ed231
ND
634
ND
ND
ND
Chrysobalana
ceae
Licaniatomentosa
Benth
Oiti(Coastzone010
8)EA
C40215
Not
describ
ed44
21672
139
ND
ND
649
Licaniarig
idaBe
nth
Oiticica
(Coastzone1207)
EAC40216
Usedto
treatdiabetes
anti-inflammatory[15]
281
48751
524
ND
ND
863
Conn
araceae
Conn
arus
detersus
Planch
Cabelode
negro
(Ararip
eNationalForest0105)
EAC34733
Not
describ
ed285
ND
919
ND
ND
ND
4 BioMed Research International
Table1Con
tinued
Family
Botanicaln
ame
Com
mon
name
(localitydateof
collection)V
oucher
number
Tradition
almedicinaluse
Yield
()b
Antioxidant
activ
ity(EC 5
0120583gmL)
cAnticho
linesterase
activ
ity(
)d
Antibacteria
lactivity
MIC
(mgmL)
e
SCh
oleraesuis
Bsubtilis
Saureus
Fabaceae
Adenantherapavonina
LFalso
-sandalo
(Coastzone0807)
EAC38697
Not
describ
ed612
ND
ND
ND
ND
ND
Amburana
cearensis
(Allemao)A
C
Smith
Cumaruandam
burana
(Ca
atinga
vegetatio
n1107)
EAC39618
Usedto
treatrheumatism
coldand
sinusitis
antispasm
odichealin
gand
anti-inflammatory[15]
analgesic
[13]
643
ND
ND
ND
ND
ND
Anadenantheramacrocarpa
(Benth)Brenan
Ang
icoverm
elho
(Ca
atinga
vegetatio
n0705)
EAC38389
Usedto
treatrespira
tory
prob
lemsgono
rrheaand
diarrheaastrin
gent
antirheum
atic
anti-inflammatorysedativ
eandhealing[15]
935
ND
541
ND
ND
ND
Diocle
amegacarpa
Rolfe
Mucun
aolho
-de-bo
i(Ararip
eNationalForest0105)
EAC38110
Not
describ
ed240
ND
ND
ND
ND
ND
Enterolobium
contortisiliquum
(Vell)
Moron
gOrelha-de-negro
(Ararip
eNationalForest0905)
EAC38115
Not
describ
ed280
ND
ND
ND
ND
ND
Hym
enaeacourbaril
LJatoba(Ararip
eNationalForest0105)
EAC38108
Not
describ
ed95
02479
5ND
ND
ND
ND
Lonchocarpus
seric
eus(Po
iret)Ku
nth
Inga(Coastzone1006)
EAC39615
Not
describ
ed1060
ND
ND
ND
ND
ND
Luetzelburgiaauric
ulata(A
llemao)
Ducke
Pau-moco
(Ca
atinga
vegetatio
n1106)
EAC40369
Not
describ
ed840
ND
ND
ND
ND
ND
BioMed Research International 5Ta
ble1Con
tinued
Family
Botanicaln
ame
Com
mon
name
(localitydateof
collection)V
oucher
number
Tradition
almedicinaluse
Yield
()b
Antioxidant
activ
ity(EC 5
0120583gmL)
cAnticho
linesterase
activ
ity(
)d
Antibacteria
lactivity
MIC
(mgmL)
e
SCh
oleraesuis
Bsubtilis
Saureus
ParkiaplatycephalaBe
nth
Visgueiro
(Ararip
eNationalForest0106)
EAC3
8109
Not
describ
ed1750
ND
715
ND
ND
ND
Piptadeniamoniliform
isBe
nth
Catand
uva
(Ca
atinga
vegetatio
n0105)
EAC3
5974
Not
describ
ed618
ND
502
ND
ND
ND
Senn
aobtusifolia(L)HSIrw
inamp
Barneby
Matap
asto
(Ca
atinga
vegetatio
n0705)
EAC3
9320
Not
describ
ed570
ND
ND
ND
496
ND
Polyg
onaceae
Triplaris
gardneria
naWedd
Pajeu
(Ca
atinga
vegetatio
n1106)
EAC3
9600
Not
describ
ed260
6973
798
188
1376
1115
Rham
naceae
Zizip
husjoazeiro
Mart
Juaa
ndjuazeiro
(Coastzone0308)
EAC40366
Expectorantand
antip
yretic
used
totre
atskinblood
sto
machandliver
diseases
ulcerantim
icrobial[15]
722
ND
494
ND
ND
ND
Sapind
aceae
Talisiaesculen
ta(A
St-Hil)
Radlk
Pitomba
(Coastzone0308)
EAC40
685
Not
describ
ed15
0ND
6124
ND
ND
ND
Sapind
ussaponaria
LSabo
nete
(Ca
atinga
vegetatio
n1106)
EAC3
9601
Not
describ
ed107
ND
5860
ND
ND
ND
Controlsg
Quercetin
mdashmdash
5552
mdashmdash
mdashmdash
Vitamin
Cmdash
mdash26027
mdashmdash
mdashmdash
a Thev
aluesa
remeans
ofatrip
licateTh
estand
arddeviationwas
lessthan
5of
them
ean
b (Amou
nt(g)o
fsolid
recoveredfro
mthee
thanolicextracta
mou
nt(g)o
fseedflo
urused)times
100
c Efficientcon
centratio
n(EC)
as120583
gof
sampler
equiredto
decrease
oneg
oftheinitia
l22-diphenyl-1-picrylhydrazyl(DPP
Hsdot)con
centratio
nby
50
d Totalinhibitio
nof
cholinesterase
enzymea
fterc
omparis
onto
theb
lank
Con
centratio
nteste
d500120583
gmL
e Minim
uminhibitory
concentration
thelow
estcon
centratio
nwhich
didno
tsho
wanygrow
thof
teste
dorganism
after
macroscop
icevaluatio
nf N
Dnot
detected
g Sub
stancesthatare
frequ
ently
used
assta
ndardfora
ntioxidant
studies
6 BioMed Research International
(65 times 10minus1molsdotLminus1) The reduction of the DPPH radicalwas measured by monitoring continuously the decrease ofabsorption at 515 nm The percentage scavenging of theDPPH radical was calculated according to the followingformula scavenging effect = 100 times [(119860DPPH minus As)119860DPPH]where As is the absorbance of the solution when the samplehas been added at a particular level and 119860DPPH is theabsorbance of the DPPH solution The percentage of theremaining DPPH was plotted against the samplestandardconcentration to obtain the amount of antioxidant necessaryto decrease the initial concentration of DPPH by 50 (EC
50)
Based on the parameter EC50 the result was expressed in
terms of 120583g of seed extract per mL of DPPH in the reactionmedium All determinations were run in triplicate
27 Anticholinesterase Activity The anticholinesterase activ-ity of the ethanolic extracts was measured as described by[18] with slight modifications Acetylcholinesterase activitywas measured using a 96-well microplate reader basedon Ellmanrsquos method [19] In this method the enzymehydrolyzes the substrate acetylthiocholine resulting in theproduction of thiocholine which reacts with 551015840-dithiobis(2-nitrobenzoic acid) (DTNB) to produce 2-nitrobenzoate-5-mercaptothiocholine and 5-thio-2-nitrobenzoate which canbe detected at 405 nm In the 96-well plates 25120583L of 15mMacetylthiocholine iodide (ATCI) in water 125 120583L of 3mMDTNB in 50mM Tris-HCl buffer containing 01M NaCland 002M MgCl
2sdot6H2O 50 120583L of 50mM Tris-HCl buffer
containing 01 BSA and 25 120583L of plant extract sample(5mgsdotmLminus1 in MeOH diluted ten times with 50mM Tris-HCl buffer to give a concentration of 05mgsdotmLminus1) wereadded and the absorbance was measured at 405 nm every30 s for three times Then 25120583L of 022UsdotmLminus1 of acetyl-cholinesterase fromelectric eel was added and the absorbancewas again read every 45 s for eight times Any increase inabsorbance due to the spontaneous hydrolysis of the substratewas corrected by subtracting the rate of the reaction beforethe addition of the enzyme from the rate of the enzymereaction The percentage of inhibition was calculated bycomparison of sample rates to a blank
28 Hemolytic Activity Assay The test was performed in15mL microtubes following the method described by [20]with some modifications Firstly a twofold serial dilution ofeach seed ethanolic extract was prepared with 09 NaClranging from 1000 to 19120583gsdotmLminus1 and reservedThen 100120583Lof a 1 red blood cells (A B and O human blood types orrabbit blood) suspension was added to a newmicrotube con-taining 900 120583L of each seed extract dilution which was thentaken to incubator at 37∘C for 1 h After that the tubes werecentrifuged at 3000timesg for 5min The supernatant (200120583L)was placed in a 96-well plate and led to a microplate reader(Epoch BioTek Vermont USA) to measure the absorbanceat 540 nm The cell suspensions of each human blood typeor those of rabbits (100120583L) were mixed with distilled wateror 09 NaCl (900120583L) to obtain the absorbances for 100and 0 of cell lysis respectivelyThe percentage of hemolysiswas calculated as follows hemolysis = AbstestAbs119901119888times 100
where Abstest = Abs540 of the 1 cell suspension treated withsample test and Abs
119901119888= Abs
540of the 1 cell suspension
treated with distilled water To calculate the relation betweenpercentage of hemolysis and seed extract concentration thehemolytic activity was expressed as the lowest seed extractconcentration (120583gsdotmLminus1) capable of causing hemolysis ge20All determinations were run in triplicate
29 Infrared Spectroscopy The infrared spectrum wasrecorded from KBr discs on a FTIR Perkin Elmer model16 PC spectrophotometer (PerkinElmer Waltham USA)Absorptionmaxima (Egraveđmax) were reported in wavenumbers(cmminus1) The spectra were used to determine organic groupsindicative of the main classes of secondary metabolites
210 Phytochemical Study The ethanolic seed extracts werescreened for phytochemical compounds such as phenols andtannins (reaction with ferric chloride) leucoanthocyanidins(heat treatment followed by alkalinization and acidification ofsample) flavonoids and xanthones (reaction of magnesiumgranules with hydrochloric acid) steroids and triterpenes(extraction with chloroform acetic anhydride and sulfuricacid) saponins (foam production after water solubilizationand stirring) and alkaloids (precipitation with Hager Mayerand Dragendorff reagents) [21] These analyses were basedon visual observation of color modification or precipitateformation after addition of specific reagents
3 Results and Discussion
The ethanolic seed extracts did not show activity againstthe Gram (minus) organisms E aerogenes K pneumonia and Paeruginosa nor against the yeastsC albicansC krusei andCtropicalis MIC values obtained against the Gram (+) organ-isms S aureus and B subtilis and the Gram (minus) organism Scholeraesuis are shown in Table 1 Among all samples testedand microorganisms used the seed extract of S obtusifoliapresented the lowest MIC value (496mgsdotmLminus1) against thepathogenic bacteria S aureus However the speciesT gardne-riana showed the broadest spectrum of activity being activeagainst B subtilis S choleraesuis and S aureuswithMIC val-ues of 188 1376 and 1115mgsdotmLminus1 respectively Althoughthere are a great number of studies reporting antimicrobialactivity of plant parts like leaves roots and stems antimi-crobial screenings using specifically seed extracts are veryrare The reason for that is not easy to explain since seedsare admittedly a rich source of compounds involved inplant defense [22] A good example is the seed extract ofBixa orellana that has been described to have antimicrobialactivity against six strains of bacteria among these are Bsubtilis S aureus and P aeruginosa and against the yeastC albicans [23] Likewise guarana (Paullinia cupana) seedextracts presented strong antimicrobial activity against a hugenumber of damaging fungi and bacteria [24] As to thepotency of the antibacterial activities observed these weremuch lower than those described for Syzygium jambolanumseed extracts against the same bacterial strains used in thiswork (MIC values ranging from 006 to 025mgsdotmLminus1) even
BioMed Research International 7
when compared to the best result observed as describedabove for S obtusifolia (MIC 496mgsdotmLminus1) In additionthe S jambolanum seed extracts were also effective againstdamaging-fungi strains The low potency of the ethanolicseed extracts could be due to the chemical composition of thesamples with low concentration of antimicrobial substancesIn fact the results of phytochemical study revealed that allseed extracts with activity against bacteria showed in theircomposition classes of secondary metabolites widely knownas efficient antimicrobial compounds (alkaloids flavonesflavonoids phenols saponins and tannins) [25] A fraction-ation of these samples in a polarity gradient could improvetheir activities Therefore the absence or low activity of theseed extracts could be a problem of concentration
The hemolytic activity assay was performed with theseed extracts that were active against the microorganismsused Among six species tested only M urundeuva causedhemolysis in all blood types (1 erythrocytes suspension ofA B and O human blood types and rabbit blood) employedbeing the lowest concentration tested (19120583gsdotmLminus1) capableof causing more than 20 hemolysis These results show thatalthough the antimicrobial extracts are not very potent theyare more specific to bacterial cells except for M urundeuvaseed extract It is well reported that some classes of plantsecondarymetabolites are potent hemolytic compounds [26]such as phenols that were also detected forMurundeuva seedextract in the phytochemical study
The results for antioxidant capacity are also shown inTable 1 Only five seed extracts were able to capture free rad-icals in the concentrations used according to DPPHmethodthese were Hymenaea courbaril (EC
5024795 120583gsdotmLminus1) L
rigida (48751120583gsdotmLminus1) L tomentosa (21672 120583gsdotmLminus1) andT gardneriana (6973120583gsdotmLminus1) The L tomentosa and Tgardneriana seed extracts were more potent than the positivecontrol vitamin C (EC
5026027120583gsdotmLminus1) and the second one
was just a bit less potent than quercetin (EC505552120583gsdotmLminus1)
one of the most potent antioxidant molecules A previousstudy [27] showed that out of 25 plant extracts (hexanedichloromethane and methanol) only 12 presented someradical scavenging activity and the most potent activityshowed an IC
50value of 11199 120583gsdotmLminus1This promising result
for T gardneriana could be attributed to components such astannins flavones flavonoids and especially phenols [28 29]Further steps of fractionation and purification may show themolecule(s) responsible for this excellent activity
Concerning the anticholinesterase activity (Table 1)among the 21 ethanolic extracts tested 11 presented inhibitionof the cholinesterase enzyme T gardneriana P platycephalaand C detersus extracts presented the strongest activitywith inhibition values of respectively 767 715 and 919at the final concentration of 500120583gmL These values aresignificant when compared to those obtained in previousstudies [18] which have shown inhibition values of 17 plantextracts ranging from 9 to 81 using a single concentrationof 1mgmL Several plant-derived drugs such as galantamineand rivastigmine are used to inhibit AChE and thus increaseendogenous levels of acetylcholine to improve cholinergictransmission [30] Many studies described alkaloids as the
So
Pp
Lr
Tg
Hc
Lt
Tran
smitt
ance
()
4000 3500 3000 2500 2000 1500 1000 500
Wavenumber (cmminus1)
Pp
Lr
Tg
Hc
Lt
Figure 1 Infrared absorption spectra of some ethanolic extractstested in this study So Senna obtusifolia Pp Parkia platycephala LrLicania rigida Tg Triplaris gardneriana Hc Hymenaea courbarilLt Licania tomentosa
main compounds capable of inhibiting AChE enzyme [31]but recent studies have pointed out several new classes ofsecondarymetabolites as potent inhibitors such as flavonoids[32] flavones [33] and xanthones [34] as well as steroids ter-penoids oils and other phenolic compounds [35] Thus thephytochemical analysis data in the present study corroboratepreviousmolecular findings since polyphenols were detectedin all extracts Additionally the extract of T gardnerianashowed the presence of chalcones and aurones Thereforefurther studies should be conducted to assess the potential ofthese extracts and their respective molecules to inhibit AChEenzyme and thus offer a potential to fight neurodegenerativediseases such as Alzheimerrsquos
After running all the assays described above infraredspectroscopywith themost promising ethanolic seed extractswas also performed (S obtusifolia P platycephala L rigida Ltomentosa and T gardneriana) to confirm the results of phy-tochemical qualitative studyThe phytochemical studies withthese ethanolic extracts showed the presence of tannins andflavonoids which were confirmed by infrared spectroscopy(Figure 1)The spectra obtained between 1800 and 800 cmminus1are present at the fingerprint zone of aromatic compoundswhich can be found in tannins polyphenols and flavonoids[36] Further steps of fractionation andpurificationmay showthe molecule(s) responsible for this excellent activity
4 Conclusions
This study revealed antibacterial antioxidant and anti-cholinesterase activities in some seed ethanolic extracts ofBrazilian semiarid region plants emphasizing the antioxidantactivity presented by T gardneriana seeds This showeda potent capacity to catch free radicals by DPPH assayThis promising result could be attributed to its composition
8 BioMed Research International
featuring phenols tannins flavones and flavonoidsNewphy-tochemical and molecular studies are underway to identifythe bioactive compound(s) responsible for this activity
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgment
The authors thank theMinisterio da Saude Departamento deCiencias e Tecnologia (DECIT) Secretaria de Ciencias Tec-nologia e Insumos estrategicos (SCTIE) Conselho Nacionalde Desenvolvimento Cientıfico e Tecnologico (CNPq) andFundacao Cearense deApoioCientıfico e Tecnologico (FUN-CAP) for financial support of this research
References
[1] C Katiyar A Gupta S Kanjilal and S Katiyar ldquoDrug discoveryfromplant sources an integrated approachrdquoAyu vol 33 pp 10ndash19 2012
[2] P S Luize T S Tiuman L G Morello et al ldquoEffects of medic-inal plant extracts on growth of Leishmania (L) amazonensisand Trypanosoma cruzirdquo Brazilian Journal of PharmaceuticalSciences vol 41 no 1 pp 85ndash94 2005
[3] A E Edris ldquoAnti-cancer properties of Nigella spp essential oilsand their major constituents thymoquinone and 120573-ElemenerdquoCurrent Clinical Pharmacology vol 4 no 1 pp 43ndash46 2009
[4] R N Bennett and R M Wallsgrove ldquoSecondary metabolites inplant defence mechanismsrdquo New Phytologist vol 127 no 4 pp617ndash633 1994
[5] R J Grayer and J B Harborne ldquoA survey of antifungal com-pounds from higher plants 1982-1993rdquo Phytochemistry vol 37no 1 pp 19ndash42 1994
[6] A E Osbourn ldquoPreformed antimicrobial compounds and plantdefense against fungal attackrdquo Plant Cell vol 8 no 10 pp 1821ndash1831 1996
[7] E V S B Sampaio ldquoVegetacao e flora da Caatingardquo APNECNIP Recife 2000
[8] C F C B R De Almeida T C De Lima E Silva E L CDe Amorim M B D S Maia and U P De AlbuquerqueldquoLife strategy and chemical composition as predictors of theselection of medicinal plants from the caatinga (NortheastBrazil)rdquo Journal of Arid Environments vol 62 no 1 pp 127ndash1422005
[9] U P de Albuquerque P M de Medeiros A L S de Almeida etal ldquoMedicinal plants of the caatinga (semi-arid) vegetation ofNE Brazil a quantitative approachrdquo Journal of Ethnopharmacol-ogy vol 114 no 3 pp 325ndash354 2007
[10] U P de Albuquerque V A da Silva M D C Cabral NLeal Alencar and L D H C Andrade ldquoComparisons betweenthe use of medicinal plants in indigenous and rural caatinga(dryland) communities in NE Brazilrdquo Boletin Latinoamericanoy del Caribe de Plantas Medicinales y Aromaticas vol 7 no 3pp 156ndash170 2008
[11] T M Souza D F Farias B M Soares et al ldquoToxicity ofBrazilian plant seed extracts to two strains of Aedes aegypti(Diptera Culicidae) andNontargetAnimalsrdquo Journal ofMedicalEntomology vol 48 no 4 pp 846ndash851 2011
[12] P M P Ferreira D F Farias M P Viana et al ldquoStudy of theantiproliferative potential of seed extracts from NortheasternBrazilian plantsrdquo Anais da Academia Brasileira de Ciencias vol83 no 3 pp 1045ndash1058 2011
[13] F J AMatosM EOMatosM P SousaM I LMachado andA A Craveiro Constituintes Quımicos Ativos e PropriedadesBiologicas de Plantas Medicinais Brasileiras UFC FortalezaBrazil 1st edition 2004
[14] F J A Matos Plantas Medicinais Guia de Selecao e Empregode Plantas Usadas em Fitoterapia no Nordeste do Brasil IUFortaleza Brazil 2nd edition 2000
[15] G N Maia Caatinga Arvores e Arbustos e Suas Utilidades D ampZ Computacao Grafica e Editora Sao Paulo Brazil 1st edition2004
[16] M Chandrasekaran and V Venkatesalu ldquoAntibacterial andantifungal activity of Syzygium jambolanum seedsrdquo Journal ofEthnopharmacology vol 91 no 1 pp 105ndash108 2004
[17] B Yepez M Espinosa S Lopez and G Bolaos ldquoProducingantioxidant fractions from herbaceousmatrices by supercriticalfluid extractionrdquo Fluid Phase Equilibria vol 194ndash197 pp 879ndash884 2002
[18] E M Cardoso-Lopes J A Maier M R Da Silva et alldquoAlkaloids from stems of esenbeckia leiocarpa Engl (Rutaceae)as potential treatment for alzheimer diseaserdquoMolecules vol 15no 12 pp 9205ndash9213 2010
[19] G L Ellman K D Courtney V Andres Jr and R MFeatherstone ldquoA new and rapid colorimetric determination ofacetylcholinesterase activityrdquo Biochemical Pharmacology vol 7no 2 pp 88ndash95 1961
[20] A W Bernheimer ldquoAssay of hemolytic toxinsrdquo Methods inEnzymology vol 165 pp 213ndash217 1988
[21] F J A Matos Introducao A Fitoquımica Experimental EditoraUFC Ceara Brazil 1st edition 2009
[22] J Xavier-Filho ldquoSementes e suas defesas contra insetos ProjetoMultinacional de Biotecnologia e Alimentosrdquo Organizacao dosEstados Americanos (OEA) pp1ndash31 1993
[23] T C Fleischer E P K Ameade M L K Mensah and I KSawer ldquoAntimicrobial activity of the leaves and seeds of Bixaorellanardquo Fitoterapia vol 74 no 1-2 pp 136ndash138 2003
[24] L Majhenic M Skerget and Z Knez ldquoAntioxidant and antimi-crobial activity of guarana seed extractsrdquo Food Chemistry vol104 no 3 pp 1258ndash1268 2007
[25] MM Cowan ldquoPlant products as antimicrobial agentsrdquoClinicalMicrobiology Reviews vol 12 no 4 pp 564ndash582 1999
[26] A Rafat K Philip and SMuniandy ldquoAntioxidant potential andcontent of phenolic compounds in ethanolic extracts of selectedparts of Andrographis paniculatardquo Journal of Medicinal PlantResearch vol 4 no 3 pp 197ndash202 2010
[27] O M Mosquera Y M Correa D C Buitrago and J NinoldquoAntioxidant activity of twenty five plants from ColombianbiodiversityrdquoMemorias do Instituto Oswaldo Cruz vol 102 no5 pp 631ndash634 2007
[28] N Cotelle J-L Bernier J-P Catteau J Pommery J-C Wal-let and E M Gaydou ldquoAntioxidant properties of hydroxy-flavonesrdquo Free Radical Biology and Medicine vol 20 no 1 pp35ndash43 1996
[29] H Oh D-H Kim J-H Cho and Y-C Kim ldquoHepatoprotectiveand free radical scavenging activities of phenolic petrosinsand flavonoids isolated from Equisetum arvenserdquo Journal ofEthnopharmacology vol 95 no 2-3 pp 421ndash424 2004
BioMed Research International 9
[30] S Lopez J Bastida F Viladomat and C Codina ldquoAcetyl-cholinesterase inhibitory activity of some Amaryllidaceae alka-loids and Narcissus extractsrdquo Life Sciences vol 71 no 21 pp2521ndash2529 2002
[31] M G Ortega A M Agnese and J L Cabrera ldquoAnti-cholinesterase activity in an alkaloid extract ofHuperzia sauru-rusrdquo Phytomedicine vol 11 no 6 pp 539ndash543 2004
[32] B J Hillhouse D S Ming C J French and G H NTowers ldquoAcetylcholine esterase inhibitors in Rhodiola roseardquoPharmaceutical Biology vol 42 no 1 pp 68ndash72 2004
[33] P Sawasdee C Sabphon D Sitthiwongwanit and U KokpolldquoAnticholinesterase activity of 7-methoxyflavones isolated fromKaempferia parviflorardquo Phytotherapy Research vol 23 no 12pp 1792ndash1794 2009
[34] 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
[35] H-F Ji andH-Y Zhang ldquoMultipotent natural agents to combatAlzheimerrsquos disease Functional spectrum and structural fea-turesrdquo Acta Pharmacologica Sinica vol 29 no 2 pp 143ndash1512008
[36] K Fernandez and E Agosin ldquoQuantitative analysis of red winetannins using Fourier-transform mid-infrared spectrometryrdquoJournal of Agricultural and Food Chemistry vol 55 no 18 pp7294ndash7300 2007
Submit your manuscripts athttpwwwhindawicom
PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom
Volume 2014
ToxinsJournal of
VaccinesJournal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
AntibioticsInternational Journal of
ToxicologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Drug DeliveryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in Pharmacological Sciences
Tropical MedicineJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Medicinal ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
AddictionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Autoimmune Diseases
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anesthesiology Research and Practice
ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Pharmaceutics
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
2 BioMed Research International
compounds saponins cyanogenic glycosides and glucosino-lates [4ndash6]
In this context the Brazilian semiarid region is a richand underexploited source of bioactive molecules This areais covered mainly by xeric shrublands known as ldquoCaatingardquoThis dry land vegetation grows over an area of 800000 km2in Northeastern Brazil and out of 1000 vascular plant speciesdescribed many are endemic [7 8] Many of these plants areused in folk medicine by local communities to treat severaldiseases pointing out the potential to discover new sourcesof useful compounds for several applications [9 10] Recentlyour team has reported for seed ethanolic extracts of BrazilianNortheastern plants promising results of insecticidal activityagainst Aedes aegypti [11] and antiproliferative effects totumor cell lines [12] In order to extend our knowledgeon the Brazilian semiarid vegetation this work aimed toevaluate the presence of antimicrobial anticholinesteraseand antioxidant activities in ethanolic seed extracts of twenty-one plant species The antimicrobial activity was performedby the agar diffusion assay using pathogenic bacteria andyeast antioxidant activity by the DPPH radical scavengingand anticholinesterase activity were measured In addition ahemolytic activity assay was done for antibacterial samples toassess the effects on nontarget cells
2 Materials and Methods
21 Plant Collection Mature wild seeds of twenty-one plantspecies were harvested (at least 500 g of each species) duringthe dry period (from January 2005 to March 2008) in thesemiarid region of the Ceara State Northeastern BrazilPlants were identified by taxonomist Dr Edson de PaulaNunes and voucher specimens were deposited at HerbariumPrisco Bezerra (EAC) at the Federal University of Ceara(Fortaleza Brazil)The studied species are depicted in Table 1which shows harvest information voucher numbers com-mon names and the traditional medicinal use by localpopulation
22 Preparation of Ethanolic Seed Extracts Seeds of freshlycollected plant material were separated from other plantmaterials air-dried and ground in a laboratory mill to amoderately fine powder (particle size le 05mm) Powderedmaterial (500 g) was submitted to extraction with 99ethanol (15 L) at room temperature (25ndash27∘C) for 3 daysThe supernatant was removed and filtered throughWhatmannumber 1 paper After that the initial powder sample wasstill submitted to two extractions which resulted in a finalvolume of extract near 45 L The ethanolic extracts resultingfrom three consecutive extractions were mixed together thenconcentrated under reduced pressure in rotary evaporatoruntil complete elimination of solvent and stored in a freezerat minus20∘C
A stock solution containing 50000 120583gsdotmLminus1 of eachcrude extract was prepared by suspending 50000mg ofextract in 5mL of pure dimethylsulphoxide (DMSO AldrichMilwaukee WI USA) and mixed by sonication (model RK-100 Bandelin Berlin Germany) for 20min The volume was
adjusted to 1000mL with distilled water to provide assaysolutions as required
23 Microorganisms Six bacterial strains were used Staphy-lococcus aureus (ATCC 25923) Bacillus subtilis (ATCC 6633)Enterobacter aerogenes (ATCC 13048) Salmonella cholerae-suis (ATCC 10708) Klebsiella pneumoniae (ATCC 10031)and Pseudomonas aeruginosa (ATCC 25619)These standardswere obtained from the American Type Culture Collection(ATCC) (Manassas VA USA) The stock culture was main-tained on Mueller Hinton agar (HiMedia Laboratories PvtLtdMumbai India)medium at 4∘CThree pathogenic yeastswere usedCandida albicansC krusei andC tropicalisThesefungal strains were obtained from the Microbial Ecologyand Biotechnology Laboratory Biology Department of theFederal University of Ceara Brazil The stock cultures weremaintained on Sabouraud Dextrose agar (HiMedia Labora-tories Pvt Ltd Mumbai India) medium at 4∘C
24 Antibacterial and Antifungal Activity The disc diffu-sion method was followed for antibacterial and antifungalsusceptibility test as described previously [16] with somemodifications Petri dishes were prepared by pouring 20mLof Mueller-Hinton agar for bacteria and Sabouraud Dextroseagar for fungi and allowed to solidify Dishes were dried and01mL of standardized inoculum suspension (106 CFUmLfor bacteria and absorbance of 0600 at 450 nm for fungi)was poured and uniformly spread The excess inoculum wasdrained and the inoculum was allowed to dry for 5min Thepaper filter discs (7mm of diameter) containing 20120583L ofeach ethanolic extract were then applied and the dishes wereincubated at 37∘C for 18 h for bacterial growth and at thesame temperature for 48 h for fungal growth Tetracycline(30mgdisc) was used as positive control for the antibacterialassay Nystatin solution (100000 IUmL EMS SP Brazil)(20120583Ldisc) was used as positive control for the antifungalassayThe inhibition zone was measured from the edge of thedisc to the inner margin of the surrounding pathogens Alldeterminations were run in triplicate
25 Minimum Inhibitory Concentration (MIC) Minimuminhibitory concentrations of the seed ethanolic extracts weretested as described previously by the twofold serial dilutionmethodThe test extract was dissolved in 5DMSO to obtain50mgsdotmLminus1 stock solution Stock solution was diluted withdistilled water to achieve 45 40 35 30 25 20 15 10 and5mgsdotmLminus1 or as required The wet paper discs with dilutionswere placed over the dish and the cultures were incubated inBOD incubators at 37∘C for 18 h (bacteria) and 37∘C for 48 h(yeasts) The lowest concentration which inhibits the visiblegrowth of tested organism after macroscopic evaluation wasdetermined asMICAll determinationswere run in triplicate
26 Antioxidant Activity The capacity to scavenge the 22-diphenyl-1-picrylhydrazyl (DPPH) free radical was moni-tored [17] The diluted crude extracts (01mL) in differ-ent concentrations (5 to 1000 120583gsdotmLminus1) were mixed with39mL of methanolic solution containing DPPH radicals
BioMed Research International 3
Table1Antibacteria
l(MIC)antio
xidantand
hemolyticactiv
ities
ofethano
licseed
extractsof
plantsfro
mBrazilian
semiarid
region
a
Family
Botanicaln
ame
Com
mon
name
(localitydateof
collection)V
oucher
number
Tradition
almedicinaluse
Yield
()b
Antioxidant
activ
ity(EC 5
0120583gmL)
cAnticho
linesterase
activ
ity(
)d
Antibacteria
lactivity
MIC
(mgmL)
e
SCh
oleraesuis
Bsubtilis
Saureus
Anacardiaceae
Myracrodruonurun
deuvaFrA
llAroeira-do-sertao
(Ararip
eNationalForest0107)
EAC34865
Usedto
treatcutaneou
sand
gynecologicalaffections
and
kidn
eyandrespira
tory
prob
lemsantiinfl
ammatory
antiu
lcerandhealing[13]
antih
istam
inicand
antim
icrobial[14
]analgesic
andantid
iarrheal[15]
252
ND
fND
ND
678
ND
Schinopsisbrasiliensis
Engler
Brauna
(Ararip
eNationalForest0107)
EAC35643
Usedto
treatnervou
snessa
ndhyste
riaanalgesic[15]
288
ND
ND
3732
ND
ND
Caryocaraceae
Caryocar
coria
ceum
Wittm
Pequ
i(Ararip
eNationalForest0905)
EAC35251
Not
describ
ed231
ND
634
ND
ND
ND
Chrysobalana
ceae
Licaniatomentosa
Benth
Oiti(Coastzone010
8)EA
C40215
Not
describ
ed44
21672
139
ND
ND
649
Licaniarig
idaBe
nth
Oiticica
(Coastzone1207)
EAC40216
Usedto
treatdiabetes
anti-inflammatory[15]
281
48751
524
ND
ND
863
Conn
araceae
Conn
arus
detersus
Planch
Cabelode
negro
(Ararip
eNationalForest0105)
EAC34733
Not
describ
ed285
ND
919
ND
ND
ND
4 BioMed Research International
Table1Con
tinued
Family
Botanicaln
ame
Com
mon
name
(localitydateof
collection)V
oucher
number
Tradition
almedicinaluse
Yield
()b
Antioxidant
activ
ity(EC 5
0120583gmL)
cAnticho
linesterase
activ
ity(
)d
Antibacteria
lactivity
MIC
(mgmL)
e
SCh
oleraesuis
Bsubtilis
Saureus
Fabaceae
Adenantherapavonina
LFalso
-sandalo
(Coastzone0807)
EAC38697
Not
describ
ed612
ND
ND
ND
ND
ND
Amburana
cearensis
(Allemao)A
C
Smith
Cumaruandam
burana
(Ca
atinga
vegetatio
n1107)
EAC39618
Usedto
treatrheumatism
coldand
sinusitis
antispasm
odichealin
gand
anti-inflammatory[15]
analgesic
[13]
643
ND
ND
ND
ND
ND
Anadenantheramacrocarpa
(Benth)Brenan
Ang
icoverm
elho
(Ca
atinga
vegetatio
n0705)
EAC38389
Usedto
treatrespira
tory
prob
lemsgono
rrheaand
diarrheaastrin
gent
antirheum
atic
anti-inflammatorysedativ
eandhealing[15]
935
ND
541
ND
ND
ND
Diocle
amegacarpa
Rolfe
Mucun
aolho
-de-bo
i(Ararip
eNationalForest0105)
EAC38110
Not
describ
ed240
ND
ND
ND
ND
ND
Enterolobium
contortisiliquum
(Vell)
Moron
gOrelha-de-negro
(Ararip
eNationalForest0905)
EAC38115
Not
describ
ed280
ND
ND
ND
ND
ND
Hym
enaeacourbaril
LJatoba(Ararip
eNationalForest0105)
EAC38108
Not
describ
ed95
02479
5ND
ND
ND
ND
Lonchocarpus
seric
eus(Po
iret)Ku
nth
Inga(Coastzone1006)
EAC39615
Not
describ
ed1060
ND
ND
ND
ND
ND
Luetzelburgiaauric
ulata(A
llemao)
Ducke
Pau-moco
(Ca
atinga
vegetatio
n1106)
EAC40369
Not
describ
ed840
ND
ND
ND
ND
ND
BioMed Research International 5Ta
ble1Con
tinued
Family
Botanicaln
ame
Com
mon
name
(localitydateof
collection)V
oucher
number
Tradition
almedicinaluse
Yield
()b
Antioxidant
activ
ity(EC 5
0120583gmL)
cAnticho
linesterase
activ
ity(
)d
Antibacteria
lactivity
MIC
(mgmL)
e
SCh
oleraesuis
Bsubtilis
Saureus
ParkiaplatycephalaBe
nth
Visgueiro
(Ararip
eNationalForest0106)
EAC3
8109
Not
describ
ed1750
ND
715
ND
ND
ND
Piptadeniamoniliform
isBe
nth
Catand
uva
(Ca
atinga
vegetatio
n0105)
EAC3
5974
Not
describ
ed618
ND
502
ND
ND
ND
Senn
aobtusifolia(L)HSIrw
inamp
Barneby
Matap
asto
(Ca
atinga
vegetatio
n0705)
EAC3
9320
Not
describ
ed570
ND
ND
ND
496
ND
Polyg
onaceae
Triplaris
gardneria
naWedd
Pajeu
(Ca
atinga
vegetatio
n1106)
EAC3
9600
Not
describ
ed260
6973
798
188
1376
1115
Rham
naceae
Zizip
husjoazeiro
Mart
Juaa
ndjuazeiro
(Coastzone0308)
EAC40366
Expectorantand
antip
yretic
used
totre
atskinblood
sto
machandliver
diseases
ulcerantim
icrobial[15]
722
ND
494
ND
ND
ND
Sapind
aceae
Talisiaesculen
ta(A
St-Hil)
Radlk
Pitomba
(Coastzone0308)
EAC40
685
Not
describ
ed15
0ND
6124
ND
ND
ND
Sapind
ussaponaria
LSabo
nete
(Ca
atinga
vegetatio
n1106)
EAC3
9601
Not
describ
ed107
ND
5860
ND
ND
ND
Controlsg
Quercetin
mdashmdash
5552
mdashmdash
mdashmdash
Vitamin
Cmdash
mdash26027
mdashmdash
mdashmdash
a Thev
aluesa
remeans
ofatrip
licateTh
estand
arddeviationwas
lessthan
5of
them
ean
b (Amou
nt(g)o
fsolid
recoveredfro
mthee
thanolicextracta
mou
nt(g)o
fseedflo
urused)times
100
c Efficientcon
centratio
n(EC)
as120583
gof
sampler
equiredto
decrease
oneg
oftheinitia
l22-diphenyl-1-picrylhydrazyl(DPP
Hsdot)con
centratio
nby
50
d Totalinhibitio
nof
cholinesterase
enzymea
fterc
omparis
onto
theb
lank
Con
centratio
nteste
d500120583
gmL
e Minim
uminhibitory
concentration
thelow
estcon
centratio
nwhich
didno
tsho
wanygrow
thof
teste
dorganism
after
macroscop
icevaluatio
nf N
Dnot
detected
g Sub
stancesthatare
frequ
ently
used
assta
ndardfora
ntioxidant
studies
6 BioMed Research International
(65 times 10minus1molsdotLminus1) The reduction of the DPPH radicalwas measured by monitoring continuously the decrease ofabsorption at 515 nm The percentage scavenging of theDPPH radical was calculated according to the followingformula scavenging effect = 100 times [(119860DPPH minus As)119860DPPH]where As is the absorbance of the solution when the samplehas been added at a particular level and 119860DPPH is theabsorbance of the DPPH solution The percentage of theremaining DPPH was plotted against the samplestandardconcentration to obtain the amount of antioxidant necessaryto decrease the initial concentration of DPPH by 50 (EC
50)
Based on the parameter EC50 the result was expressed in
terms of 120583g of seed extract per mL of DPPH in the reactionmedium All determinations were run in triplicate
27 Anticholinesterase Activity The anticholinesterase activ-ity of the ethanolic extracts was measured as described by[18] with slight modifications Acetylcholinesterase activitywas measured using a 96-well microplate reader basedon Ellmanrsquos method [19] In this method the enzymehydrolyzes the substrate acetylthiocholine resulting in theproduction of thiocholine which reacts with 551015840-dithiobis(2-nitrobenzoic acid) (DTNB) to produce 2-nitrobenzoate-5-mercaptothiocholine and 5-thio-2-nitrobenzoate which canbe detected at 405 nm In the 96-well plates 25120583L of 15mMacetylthiocholine iodide (ATCI) in water 125 120583L of 3mMDTNB in 50mM Tris-HCl buffer containing 01M NaCland 002M MgCl
2sdot6H2O 50 120583L of 50mM Tris-HCl buffer
containing 01 BSA and 25 120583L of plant extract sample(5mgsdotmLminus1 in MeOH diluted ten times with 50mM Tris-HCl buffer to give a concentration of 05mgsdotmLminus1) wereadded and the absorbance was measured at 405 nm every30 s for three times Then 25120583L of 022UsdotmLminus1 of acetyl-cholinesterase fromelectric eel was added and the absorbancewas again read every 45 s for eight times Any increase inabsorbance due to the spontaneous hydrolysis of the substratewas corrected by subtracting the rate of the reaction beforethe addition of the enzyme from the rate of the enzymereaction The percentage of inhibition was calculated bycomparison of sample rates to a blank
28 Hemolytic Activity Assay The test was performed in15mL microtubes following the method described by [20]with some modifications Firstly a twofold serial dilution ofeach seed ethanolic extract was prepared with 09 NaClranging from 1000 to 19120583gsdotmLminus1 and reservedThen 100120583Lof a 1 red blood cells (A B and O human blood types orrabbit blood) suspension was added to a newmicrotube con-taining 900 120583L of each seed extract dilution which was thentaken to incubator at 37∘C for 1 h After that the tubes werecentrifuged at 3000timesg for 5min The supernatant (200120583L)was placed in a 96-well plate and led to a microplate reader(Epoch BioTek Vermont USA) to measure the absorbanceat 540 nm The cell suspensions of each human blood typeor those of rabbits (100120583L) were mixed with distilled wateror 09 NaCl (900120583L) to obtain the absorbances for 100and 0 of cell lysis respectivelyThe percentage of hemolysiswas calculated as follows hemolysis = AbstestAbs119901119888times 100
where Abstest = Abs540 of the 1 cell suspension treated withsample test and Abs
119901119888= Abs
540of the 1 cell suspension
treated with distilled water To calculate the relation betweenpercentage of hemolysis and seed extract concentration thehemolytic activity was expressed as the lowest seed extractconcentration (120583gsdotmLminus1) capable of causing hemolysis ge20All determinations were run in triplicate
29 Infrared Spectroscopy The infrared spectrum wasrecorded from KBr discs on a FTIR Perkin Elmer model16 PC spectrophotometer (PerkinElmer Waltham USA)Absorptionmaxima (Egraveđmax) were reported in wavenumbers(cmminus1) The spectra were used to determine organic groupsindicative of the main classes of secondary metabolites
210 Phytochemical Study The ethanolic seed extracts werescreened for phytochemical compounds such as phenols andtannins (reaction with ferric chloride) leucoanthocyanidins(heat treatment followed by alkalinization and acidification ofsample) flavonoids and xanthones (reaction of magnesiumgranules with hydrochloric acid) steroids and triterpenes(extraction with chloroform acetic anhydride and sulfuricacid) saponins (foam production after water solubilizationand stirring) and alkaloids (precipitation with Hager Mayerand Dragendorff reagents) [21] These analyses were basedon visual observation of color modification or precipitateformation after addition of specific reagents
3 Results and Discussion
The ethanolic seed extracts did not show activity againstthe Gram (minus) organisms E aerogenes K pneumonia and Paeruginosa nor against the yeastsC albicansC krusei andCtropicalis MIC values obtained against the Gram (+) organ-isms S aureus and B subtilis and the Gram (minus) organism Scholeraesuis are shown in Table 1 Among all samples testedand microorganisms used the seed extract of S obtusifoliapresented the lowest MIC value (496mgsdotmLminus1) against thepathogenic bacteria S aureus However the speciesT gardne-riana showed the broadest spectrum of activity being activeagainst B subtilis S choleraesuis and S aureuswithMIC val-ues of 188 1376 and 1115mgsdotmLminus1 respectively Althoughthere are a great number of studies reporting antimicrobialactivity of plant parts like leaves roots and stems antimi-crobial screenings using specifically seed extracts are veryrare The reason for that is not easy to explain since seedsare admittedly a rich source of compounds involved inplant defense [22] A good example is the seed extract ofBixa orellana that has been described to have antimicrobialactivity against six strains of bacteria among these are Bsubtilis S aureus and P aeruginosa and against the yeastC albicans [23] Likewise guarana (Paullinia cupana) seedextracts presented strong antimicrobial activity against a hugenumber of damaging fungi and bacteria [24] As to thepotency of the antibacterial activities observed these weremuch lower than those described for Syzygium jambolanumseed extracts against the same bacterial strains used in thiswork (MIC values ranging from 006 to 025mgsdotmLminus1) even
BioMed Research International 7
when compared to the best result observed as describedabove for S obtusifolia (MIC 496mgsdotmLminus1) In additionthe S jambolanum seed extracts were also effective againstdamaging-fungi strains The low potency of the ethanolicseed extracts could be due to the chemical composition of thesamples with low concentration of antimicrobial substancesIn fact the results of phytochemical study revealed that allseed extracts with activity against bacteria showed in theircomposition classes of secondary metabolites widely knownas efficient antimicrobial compounds (alkaloids flavonesflavonoids phenols saponins and tannins) [25] A fraction-ation of these samples in a polarity gradient could improvetheir activities Therefore the absence or low activity of theseed extracts could be a problem of concentration
The hemolytic activity assay was performed with theseed extracts that were active against the microorganismsused Among six species tested only M urundeuva causedhemolysis in all blood types (1 erythrocytes suspension ofA B and O human blood types and rabbit blood) employedbeing the lowest concentration tested (19120583gsdotmLminus1) capableof causing more than 20 hemolysis These results show thatalthough the antimicrobial extracts are not very potent theyare more specific to bacterial cells except for M urundeuvaseed extract It is well reported that some classes of plantsecondarymetabolites are potent hemolytic compounds [26]such as phenols that were also detected forMurundeuva seedextract in the phytochemical study
The results for antioxidant capacity are also shown inTable 1 Only five seed extracts were able to capture free rad-icals in the concentrations used according to DPPHmethodthese were Hymenaea courbaril (EC
5024795 120583gsdotmLminus1) L
rigida (48751120583gsdotmLminus1) L tomentosa (21672 120583gsdotmLminus1) andT gardneriana (6973120583gsdotmLminus1) The L tomentosa and Tgardneriana seed extracts were more potent than the positivecontrol vitamin C (EC
5026027120583gsdotmLminus1) and the second one
was just a bit less potent than quercetin (EC505552120583gsdotmLminus1)
one of the most potent antioxidant molecules A previousstudy [27] showed that out of 25 plant extracts (hexanedichloromethane and methanol) only 12 presented someradical scavenging activity and the most potent activityshowed an IC
50value of 11199 120583gsdotmLminus1This promising result
for T gardneriana could be attributed to components such astannins flavones flavonoids and especially phenols [28 29]Further steps of fractionation and purification may show themolecule(s) responsible for this excellent activity
Concerning the anticholinesterase activity (Table 1)among the 21 ethanolic extracts tested 11 presented inhibitionof the cholinesterase enzyme T gardneriana P platycephalaand C detersus extracts presented the strongest activitywith inhibition values of respectively 767 715 and 919at the final concentration of 500120583gmL These values aresignificant when compared to those obtained in previousstudies [18] which have shown inhibition values of 17 plantextracts ranging from 9 to 81 using a single concentrationof 1mgmL Several plant-derived drugs such as galantamineand rivastigmine are used to inhibit AChE and thus increaseendogenous levels of acetylcholine to improve cholinergictransmission [30] Many studies described alkaloids as the
So
Pp
Lr
Tg
Hc
Lt
Tran
smitt
ance
()
4000 3500 3000 2500 2000 1500 1000 500
Wavenumber (cmminus1)
Pp
Lr
Tg
Hc
Lt
Figure 1 Infrared absorption spectra of some ethanolic extractstested in this study So Senna obtusifolia Pp Parkia platycephala LrLicania rigida Tg Triplaris gardneriana Hc Hymenaea courbarilLt Licania tomentosa
main compounds capable of inhibiting AChE enzyme [31]but recent studies have pointed out several new classes ofsecondarymetabolites as potent inhibitors such as flavonoids[32] flavones [33] and xanthones [34] as well as steroids ter-penoids oils and other phenolic compounds [35] Thus thephytochemical analysis data in the present study corroboratepreviousmolecular findings since polyphenols were detectedin all extracts Additionally the extract of T gardnerianashowed the presence of chalcones and aurones Thereforefurther studies should be conducted to assess the potential ofthese extracts and their respective molecules to inhibit AChEenzyme and thus offer a potential to fight neurodegenerativediseases such as Alzheimerrsquos
After running all the assays described above infraredspectroscopywith themost promising ethanolic seed extractswas also performed (S obtusifolia P platycephala L rigida Ltomentosa and T gardneriana) to confirm the results of phy-tochemical qualitative studyThe phytochemical studies withthese ethanolic extracts showed the presence of tannins andflavonoids which were confirmed by infrared spectroscopy(Figure 1)The spectra obtained between 1800 and 800 cmminus1are present at the fingerprint zone of aromatic compoundswhich can be found in tannins polyphenols and flavonoids[36] Further steps of fractionation andpurificationmay showthe molecule(s) responsible for this excellent activity
4 Conclusions
This study revealed antibacterial antioxidant and anti-cholinesterase activities in some seed ethanolic extracts ofBrazilian semiarid region plants emphasizing the antioxidantactivity presented by T gardneriana seeds This showeda potent capacity to catch free radicals by DPPH assayThis promising result could be attributed to its composition
8 BioMed Research International
featuring phenols tannins flavones and flavonoidsNewphy-tochemical and molecular studies are underway to identifythe bioactive compound(s) responsible for this activity
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgment
The authors thank theMinisterio da Saude Departamento deCiencias e Tecnologia (DECIT) Secretaria de Ciencias Tec-nologia e Insumos estrategicos (SCTIE) Conselho Nacionalde Desenvolvimento Cientıfico e Tecnologico (CNPq) andFundacao Cearense deApoioCientıfico e Tecnologico (FUN-CAP) for financial support of this research
References
[1] C Katiyar A Gupta S Kanjilal and S Katiyar ldquoDrug discoveryfromplant sources an integrated approachrdquoAyu vol 33 pp 10ndash19 2012
[2] P S Luize T S Tiuman L G Morello et al ldquoEffects of medic-inal plant extracts on growth of Leishmania (L) amazonensisand Trypanosoma cruzirdquo Brazilian Journal of PharmaceuticalSciences vol 41 no 1 pp 85ndash94 2005
[3] A E Edris ldquoAnti-cancer properties of Nigella spp essential oilsand their major constituents thymoquinone and 120573-ElemenerdquoCurrent Clinical Pharmacology vol 4 no 1 pp 43ndash46 2009
[4] R N Bennett and R M Wallsgrove ldquoSecondary metabolites inplant defence mechanismsrdquo New Phytologist vol 127 no 4 pp617ndash633 1994
[5] R J Grayer and J B Harborne ldquoA survey of antifungal com-pounds from higher plants 1982-1993rdquo Phytochemistry vol 37no 1 pp 19ndash42 1994
[6] A E Osbourn ldquoPreformed antimicrobial compounds and plantdefense against fungal attackrdquo Plant Cell vol 8 no 10 pp 1821ndash1831 1996
[7] E V S B Sampaio ldquoVegetacao e flora da Caatingardquo APNECNIP Recife 2000
[8] C F C B R De Almeida T C De Lima E Silva E L CDe Amorim M B D S Maia and U P De AlbuquerqueldquoLife strategy and chemical composition as predictors of theselection of medicinal plants from the caatinga (NortheastBrazil)rdquo Journal of Arid Environments vol 62 no 1 pp 127ndash1422005
[9] U P de Albuquerque P M de Medeiros A L S de Almeida etal ldquoMedicinal plants of the caatinga (semi-arid) vegetation ofNE Brazil a quantitative approachrdquo Journal of Ethnopharmacol-ogy vol 114 no 3 pp 325ndash354 2007
[10] U P de Albuquerque V A da Silva M D C Cabral NLeal Alencar and L D H C Andrade ldquoComparisons betweenthe use of medicinal plants in indigenous and rural caatinga(dryland) communities in NE Brazilrdquo Boletin Latinoamericanoy del Caribe de Plantas Medicinales y Aromaticas vol 7 no 3pp 156ndash170 2008
[11] T M Souza D F Farias B M Soares et al ldquoToxicity ofBrazilian plant seed extracts to two strains of Aedes aegypti(Diptera Culicidae) andNontargetAnimalsrdquo Journal ofMedicalEntomology vol 48 no 4 pp 846ndash851 2011
[12] P M P Ferreira D F Farias M P Viana et al ldquoStudy of theantiproliferative potential of seed extracts from NortheasternBrazilian plantsrdquo Anais da Academia Brasileira de Ciencias vol83 no 3 pp 1045ndash1058 2011
[13] F J AMatosM EOMatosM P SousaM I LMachado andA A Craveiro Constituintes Quımicos Ativos e PropriedadesBiologicas de Plantas Medicinais Brasileiras UFC FortalezaBrazil 1st edition 2004
[14] F J A Matos Plantas Medicinais Guia de Selecao e Empregode Plantas Usadas em Fitoterapia no Nordeste do Brasil IUFortaleza Brazil 2nd edition 2000
[15] G N Maia Caatinga Arvores e Arbustos e Suas Utilidades D ampZ Computacao Grafica e Editora Sao Paulo Brazil 1st edition2004
[16] M Chandrasekaran and V Venkatesalu ldquoAntibacterial andantifungal activity of Syzygium jambolanum seedsrdquo Journal ofEthnopharmacology vol 91 no 1 pp 105ndash108 2004
[17] B Yepez M Espinosa S Lopez and G Bolaos ldquoProducingantioxidant fractions from herbaceousmatrices by supercriticalfluid extractionrdquo Fluid Phase Equilibria vol 194ndash197 pp 879ndash884 2002
[18] E M Cardoso-Lopes J A Maier M R Da Silva et alldquoAlkaloids from stems of esenbeckia leiocarpa Engl (Rutaceae)as potential treatment for alzheimer diseaserdquoMolecules vol 15no 12 pp 9205ndash9213 2010
[19] G L Ellman K D Courtney V Andres Jr and R MFeatherstone ldquoA new and rapid colorimetric determination ofacetylcholinesterase activityrdquo Biochemical Pharmacology vol 7no 2 pp 88ndash95 1961
[20] A W Bernheimer ldquoAssay of hemolytic toxinsrdquo Methods inEnzymology vol 165 pp 213ndash217 1988
[21] F J A Matos Introducao A Fitoquımica Experimental EditoraUFC Ceara Brazil 1st edition 2009
[22] J Xavier-Filho ldquoSementes e suas defesas contra insetos ProjetoMultinacional de Biotecnologia e Alimentosrdquo Organizacao dosEstados Americanos (OEA) pp1ndash31 1993
[23] T C Fleischer E P K Ameade M L K Mensah and I KSawer ldquoAntimicrobial activity of the leaves and seeds of Bixaorellanardquo Fitoterapia vol 74 no 1-2 pp 136ndash138 2003
[24] L Majhenic M Skerget and Z Knez ldquoAntioxidant and antimi-crobial activity of guarana seed extractsrdquo Food Chemistry vol104 no 3 pp 1258ndash1268 2007
[25] MM Cowan ldquoPlant products as antimicrobial agentsrdquoClinicalMicrobiology Reviews vol 12 no 4 pp 564ndash582 1999
[26] A Rafat K Philip and SMuniandy ldquoAntioxidant potential andcontent of phenolic compounds in ethanolic extracts of selectedparts of Andrographis paniculatardquo Journal of Medicinal PlantResearch vol 4 no 3 pp 197ndash202 2010
[27] O M Mosquera Y M Correa D C Buitrago and J NinoldquoAntioxidant activity of twenty five plants from ColombianbiodiversityrdquoMemorias do Instituto Oswaldo Cruz vol 102 no5 pp 631ndash634 2007
[28] N Cotelle J-L Bernier J-P Catteau J Pommery J-C Wal-let and E M Gaydou ldquoAntioxidant properties of hydroxy-flavonesrdquo Free Radical Biology and Medicine vol 20 no 1 pp35ndash43 1996
[29] H Oh D-H Kim J-H Cho and Y-C Kim ldquoHepatoprotectiveand free radical scavenging activities of phenolic petrosinsand flavonoids isolated from Equisetum arvenserdquo Journal ofEthnopharmacology vol 95 no 2-3 pp 421ndash424 2004
BioMed Research International 9
[30] S Lopez J Bastida F Viladomat and C Codina ldquoAcetyl-cholinesterase inhibitory activity of some Amaryllidaceae alka-loids and Narcissus extractsrdquo Life Sciences vol 71 no 21 pp2521ndash2529 2002
[31] M G Ortega A M Agnese and J L Cabrera ldquoAnti-cholinesterase activity in an alkaloid extract ofHuperzia sauru-rusrdquo Phytomedicine vol 11 no 6 pp 539ndash543 2004
[32] B J Hillhouse D S Ming C J French and G H NTowers ldquoAcetylcholine esterase inhibitors in Rhodiola roseardquoPharmaceutical Biology vol 42 no 1 pp 68ndash72 2004
[33] P Sawasdee C Sabphon D Sitthiwongwanit and U KokpolldquoAnticholinesterase activity of 7-methoxyflavones isolated fromKaempferia parviflorardquo Phytotherapy Research vol 23 no 12pp 1792ndash1794 2009
[34] 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
[35] H-F Ji andH-Y Zhang ldquoMultipotent natural agents to combatAlzheimerrsquos disease Functional spectrum and structural fea-turesrdquo Acta Pharmacologica Sinica vol 29 no 2 pp 143ndash1512008
[36] K Fernandez and E Agosin ldquoQuantitative analysis of red winetannins using Fourier-transform mid-infrared spectrometryrdquoJournal of Agricultural and Food Chemistry vol 55 no 18 pp7294ndash7300 2007
Submit your manuscripts athttpwwwhindawicom
PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom
Volume 2014
ToxinsJournal of
VaccinesJournal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
AntibioticsInternational Journal of
ToxicologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Drug DeliveryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in Pharmacological Sciences
Tropical MedicineJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Medicinal ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
AddictionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Autoimmune Diseases
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anesthesiology Research and Practice
ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Pharmaceutics
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
BioMed Research International 3
Table1Antibacteria
l(MIC)antio
xidantand
hemolyticactiv
ities
ofethano
licseed
extractsof
plantsfro
mBrazilian
semiarid
region
a
Family
Botanicaln
ame
Com
mon
name
(localitydateof
collection)V
oucher
number
Tradition
almedicinaluse
Yield
()b
Antioxidant
activ
ity(EC 5
0120583gmL)
cAnticho
linesterase
activ
ity(
)d
Antibacteria
lactivity
MIC
(mgmL)
e
SCh
oleraesuis
Bsubtilis
Saureus
Anacardiaceae
Myracrodruonurun
deuvaFrA
llAroeira-do-sertao
(Ararip
eNationalForest0107)
EAC34865
Usedto
treatcutaneou
sand
gynecologicalaffections
and
kidn
eyandrespira
tory
prob
lemsantiinfl
ammatory
antiu
lcerandhealing[13]
antih
istam
inicand
antim
icrobial[14
]analgesic
andantid
iarrheal[15]
252
ND
fND
ND
678
ND
Schinopsisbrasiliensis
Engler
Brauna
(Ararip
eNationalForest0107)
EAC35643
Usedto
treatnervou
snessa
ndhyste
riaanalgesic[15]
288
ND
ND
3732
ND
ND
Caryocaraceae
Caryocar
coria
ceum
Wittm
Pequ
i(Ararip
eNationalForest0905)
EAC35251
Not
describ
ed231
ND
634
ND
ND
ND
Chrysobalana
ceae
Licaniatomentosa
Benth
Oiti(Coastzone010
8)EA
C40215
Not
describ
ed44
21672
139
ND
ND
649
Licaniarig
idaBe
nth
Oiticica
(Coastzone1207)
EAC40216
Usedto
treatdiabetes
anti-inflammatory[15]
281
48751
524
ND
ND
863
Conn
araceae
Conn
arus
detersus
Planch
Cabelode
negro
(Ararip
eNationalForest0105)
EAC34733
Not
describ
ed285
ND
919
ND
ND
ND
4 BioMed Research International
Table1Con
tinued
Family
Botanicaln
ame
Com
mon
name
(localitydateof
collection)V
oucher
number
Tradition
almedicinaluse
Yield
()b
Antioxidant
activ
ity(EC 5
0120583gmL)
cAnticho
linesterase
activ
ity(
)d
Antibacteria
lactivity
MIC
(mgmL)
e
SCh
oleraesuis
Bsubtilis
Saureus
Fabaceae
Adenantherapavonina
LFalso
-sandalo
(Coastzone0807)
EAC38697
Not
describ
ed612
ND
ND
ND
ND
ND
Amburana
cearensis
(Allemao)A
C
Smith
Cumaruandam
burana
(Ca
atinga
vegetatio
n1107)
EAC39618
Usedto
treatrheumatism
coldand
sinusitis
antispasm
odichealin
gand
anti-inflammatory[15]
analgesic
[13]
643
ND
ND
ND
ND
ND
Anadenantheramacrocarpa
(Benth)Brenan
Ang
icoverm
elho
(Ca
atinga
vegetatio
n0705)
EAC38389
Usedto
treatrespira
tory
prob
lemsgono
rrheaand
diarrheaastrin
gent
antirheum
atic
anti-inflammatorysedativ
eandhealing[15]
935
ND
541
ND
ND
ND
Diocle
amegacarpa
Rolfe
Mucun
aolho
-de-bo
i(Ararip
eNationalForest0105)
EAC38110
Not
describ
ed240
ND
ND
ND
ND
ND
Enterolobium
contortisiliquum
(Vell)
Moron
gOrelha-de-negro
(Ararip
eNationalForest0905)
EAC38115
Not
describ
ed280
ND
ND
ND
ND
ND
Hym
enaeacourbaril
LJatoba(Ararip
eNationalForest0105)
EAC38108
Not
describ
ed95
02479
5ND
ND
ND
ND
Lonchocarpus
seric
eus(Po
iret)Ku
nth
Inga(Coastzone1006)
EAC39615
Not
describ
ed1060
ND
ND
ND
ND
ND
Luetzelburgiaauric
ulata(A
llemao)
Ducke
Pau-moco
(Ca
atinga
vegetatio
n1106)
EAC40369
Not
describ
ed840
ND
ND
ND
ND
ND
BioMed Research International 5Ta
ble1Con
tinued
Family
Botanicaln
ame
Com
mon
name
(localitydateof
collection)V
oucher
number
Tradition
almedicinaluse
Yield
()b
Antioxidant
activ
ity(EC 5
0120583gmL)
cAnticho
linesterase
activ
ity(
)d
Antibacteria
lactivity
MIC
(mgmL)
e
SCh
oleraesuis
Bsubtilis
Saureus
ParkiaplatycephalaBe
nth
Visgueiro
(Ararip
eNationalForest0106)
EAC3
8109
Not
describ
ed1750
ND
715
ND
ND
ND
Piptadeniamoniliform
isBe
nth
Catand
uva
(Ca
atinga
vegetatio
n0105)
EAC3
5974
Not
describ
ed618
ND
502
ND
ND
ND
Senn
aobtusifolia(L)HSIrw
inamp
Barneby
Matap
asto
(Ca
atinga
vegetatio
n0705)
EAC3
9320
Not
describ
ed570
ND
ND
ND
496
ND
Polyg
onaceae
Triplaris
gardneria
naWedd
Pajeu
(Ca
atinga
vegetatio
n1106)
EAC3
9600
Not
describ
ed260
6973
798
188
1376
1115
Rham
naceae
Zizip
husjoazeiro
Mart
Juaa
ndjuazeiro
(Coastzone0308)
EAC40366
Expectorantand
antip
yretic
used
totre
atskinblood
sto
machandliver
diseases
ulcerantim
icrobial[15]
722
ND
494
ND
ND
ND
Sapind
aceae
Talisiaesculen
ta(A
St-Hil)
Radlk
Pitomba
(Coastzone0308)
EAC40
685
Not
describ
ed15
0ND
6124
ND
ND
ND
Sapind
ussaponaria
LSabo
nete
(Ca
atinga
vegetatio
n1106)
EAC3
9601
Not
describ
ed107
ND
5860
ND
ND
ND
Controlsg
Quercetin
mdashmdash
5552
mdashmdash
mdashmdash
Vitamin
Cmdash
mdash26027
mdashmdash
mdashmdash
a Thev
aluesa
remeans
ofatrip
licateTh
estand
arddeviationwas
lessthan
5of
them
ean
b (Amou
nt(g)o
fsolid
recoveredfro
mthee
thanolicextracta
mou
nt(g)o
fseedflo
urused)times
100
c Efficientcon
centratio
n(EC)
as120583
gof
sampler
equiredto
decrease
oneg
oftheinitia
l22-diphenyl-1-picrylhydrazyl(DPP
Hsdot)con
centratio
nby
50
d Totalinhibitio
nof
cholinesterase
enzymea
fterc
omparis
onto
theb
lank
Con
centratio
nteste
d500120583
gmL
e Minim
uminhibitory
concentration
thelow
estcon
centratio
nwhich
didno
tsho
wanygrow
thof
teste
dorganism
after
macroscop
icevaluatio
nf N
Dnot
detected
g Sub
stancesthatare
frequ
ently
used
assta
ndardfora
ntioxidant
studies
6 BioMed Research International
(65 times 10minus1molsdotLminus1) The reduction of the DPPH radicalwas measured by monitoring continuously the decrease ofabsorption at 515 nm The percentage scavenging of theDPPH radical was calculated according to the followingformula scavenging effect = 100 times [(119860DPPH minus As)119860DPPH]where As is the absorbance of the solution when the samplehas been added at a particular level and 119860DPPH is theabsorbance of the DPPH solution The percentage of theremaining DPPH was plotted against the samplestandardconcentration to obtain the amount of antioxidant necessaryto decrease the initial concentration of DPPH by 50 (EC
50)
Based on the parameter EC50 the result was expressed in
terms of 120583g of seed extract per mL of DPPH in the reactionmedium All determinations were run in triplicate
27 Anticholinesterase Activity The anticholinesterase activ-ity of the ethanolic extracts was measured as described by[18] with slight modifications Acetylcholinesterase activitywas measured using a 96-well microplate reader basedon Ellmanrsquos method [19] In this method the enzymehydrolyzes the substrate acetylthiocholine resulting in theproduction of thiocholine which reacts with 551015840-dithiobis(2-nitrobenzoic acid) (DTNB) to produce 2-nitrobenzoate-5-mercaptothiocholine and 5-thio-2-nitrobenzoate which canbe detected at 405 nm In the 96-well plates 25120583L of 15mMacetylthiocholine iodide (ATCI) in water 125 120583L of 3mMDTNB in 50mM Tris-HCl buffer containing 01M NaCland 002M MgCl
2sdot6H2O 50 120583L of 50mM Tris-HCl buffer
containing 01 BSA and 25 120583L of plant extract sample(5mgsdotmLminus1 in MeOH diluted ten times with 50mM Tris-HCl buffer to give a concentration of 05mgsdotmLminus1) wereadded and the absorbance was measured at 405 nm every30 s for three times Then 25120583L of 022UsdotmLminus1 of acetyl-cholinesterase fromelectric eel was added and the absorbancewas again read every 45 s for eight times Any increase inabsorbance due to the spontaneous hydrolysis of the substratewas corrected by subtracting the rate of the reaction beforethe addition of the enzyme from the rate of the enzymereaction The percentage of inhibition was calculated bycomparison of sample rates to a blank
28 Hemolytic Activity Assay The test was performed in15mL microtubes following the method described by [20]with some modifications Firstly a twofold serial dilution ofeach seed ethanolic extract was prepared with 09 NaClranging from 1000 to 19120583gsdotmLminus1 and reservedThen 100120583Lof a 1 red blood cells (A B and O human blood types orrabbit blood) suspension was added to a newmicrotube con-taining 900 120583L of each seed extract dilution which was thentaken to incubator at 37∘C for 1 h After that the tubes werecentrifuged at 3000timesg for 5min The supernatant (200120583L)was placed in a 96-well plate and led to a microplate reader(Epoch BioTek Vermont USA) to measure the absorbanceat 540 nm The cell suspensions of each human blood typeor those of rabbits (100120583L) were mixed with distilled wateror 09 NaCl (900120583L) to obtain the absorbances for 100and 0 of cell lysis respectivelyThe percentage of hemolysiswas calculated as follows hemolysis = AbstestAbs119901119888times 100
where Abstest = Abs540 of the 1 cell suspension treated withsample test and Abs
119901119888= Abs
540of the 1 cell suspension
treated with distilled water To calculate the relation betweenpercentage of hemolysis and seed extract concentration thehemolytic activity was expressed as the lowest seed extractconcentration (120583gsdotmLminus1) capable of causing hemolysis ge20All determinations were run in triplicate
29 Infrared Spectroscopy The infrared spectrum wasrecorded from KBr discs on a FTIR Perkin Elmer model16 PC spectrophotometer (PerkinElmer Waltham USA)Absorptionmaxima (Egraveđmax) were reported in wavenumbers(cmminus1) The spectra were used to determine organic groupsindicative of the main classes of secondary metabolites
210 Phytochemical Study The ethanolic seed extracts werescreened for phytochemical compounds such as phenols andtannins (reaction with ferric chloride) leucoanthocyanidins(heat treatment followed by alkalinization and acidification ofsample) flavonoids and xanthones (reaction of magnesiumgranules with hydrochloric acid) steroids and triterpenes(extraction with chloroform acetic anhydride and sulfuricacid) saponins (foam production after water solubilizationand stirring) and alkaloids (precipitation with Hager Mayerand Dragendorff reagents) [21] These analyses were basedon visual observation of color modification or precipitateformation after addition of specific reagents
3 Results and Discussion
The ethanolic seed extracts did not show activity againstthe Gram (minus) organisms E aerogenes K pneumonia and Paeruginosa nor against the yeastsC albicansC krusei andCtropicalis MIC values obtained against the Gram (+) organ-isms S aureus and B subtilis and the Gram (minus) organism Scholeraesuis are shown in Table 1 Among all samples testedand microorganisms used the seed extract of S obtusifoliapresented the lowest MIC value (496mgsdotmLminus1) against thepathogenic bacteria S aureus However the speciesT gardne-riana showed the broadest spectrum of activity being activeagainst B subtilis S choleraesuis and S aureuswithMIC val-ues of 188 1376 and 1115mgsdotmLminus1 respectively Althoughthere are a great number of studies reporting antimicrobialactivity of plant parts like leaves roots and stems antimi-crobial screenings using specifically seed extracts are veryrare The reason for that is not easy to explain since seedsare admittedly a rich source of compounds involved inplant defense [22] A good example is the seed extract ofBixa orellana that has been described to have antimicrobialactivity against six strains of bacteria among these are Bsubtilis S aureus and P aeruginosa and against the yeastC albicans [23] Likewise guarana (Paullinia cupana) seedextracts presented strong antimicrobial activity against a hugenumber of damaging fungi and bacteria [24] As to thepotency of the antibacterial activities observed these weremuch lower than those described for Syzygium jambolanumseed extracts against the same bacterial strains used in thiswork (MIC values ranging from 006 to 025mgsdotmLminus1) even
BioMed Research International 7
when compared to the best result observed as describedabove for S obtusifolia (MIC 496mgsdotmLminus1) In additionthe S jambolanum seed extracts were also effective againstdamaging-fungi strains The low potency of the ethanolicseed extracts could be due to the chemical composition of thesamples with low concentration of antimicrobial substancesIn fact the results of phytochemical study revealed that allseed extracts with activity against bacteria showed in theircomposition classes of secondary metabolites widely knownas efficient antimicrobial compounds (alkaloids flavonesflavonoids phenols saponins and tannins) [25] A fraction-ation of these samples in a polarity gradient could improvetheir activities Therefore the absence or low activity of theseed extracts could be a problem of concentration
The hemolytic activity assay was performed with theseed extracts that were active against the microorganismsused Among six species tested only M urundeuva causedhemolysis in all blood types (1 erythrocytes suspension ofA B and O human blood types and rabbit blood) employedbeing the lowest concentration tested (19120583gsdotmLminus1) capableof causing more than 20 hemolysis These results show thatalthough the antimicrobial extracts are not very potent theyare more specific to bacterial cells except for M urundeuvaseed extract It is well reported that some classes of plantsecondarymetabolites are potent hemolytic compounds [26]such as phenols that were also detected forMurundeuva seedextract in the phytochemical study
The results for antioxidant capacity are also shown inTable 1 Only five seed extracts were able to capture free rad-icals in the concentrations used according to DPPHmethodthese were Hymenaea courbaril (EC
5024795 120583gsdotmLminus1) L
rigida (48751120583gsdotmLminus1) L tomentosa (21672 120583gsdotmLminus1) andT gardneriana (6973120583gsdotmLminus1) The L tomentosa and Tgardneriana seed extracts were more potent than the positivecontrol vitamin C (EC
5026027120583gsdotmLminus1) and the second one
was just a bit less potent than quercetin (EC505552120583gsdotmLminus1)
one of the most potent antioxidant molecules A previousstudy [27] showed that out of 25 plant extracts (hexanedichloromethane and methanol) only 12 presented someradical scavenging activity and the most potent activityshowed an IC
50value of 11199 120583gsdotmLminus1This promising result
for T gardneriana could be attributed to components such astannins flavones flavonoids and especially phenols [28 29]Further steps of fractionation and purification may show themolecule(s) responsible for this excellent activity
Concerning the anticholinesterase activity (Table 1)among the 21 ethanolic extracts tested 11 presented inhibitionof the cholinesterase enzyme T gardneriana P platycephalaand C detersus extracts presented the strongest activitywith inhibition values of respectively 767 715 and 919at the final concentration of 500120583gmL These values aresignificant when compared to those obtained in previousstudies [18] which have shown inhibition values of 17 plantextracts ranging from 9 to 81 using a single concentrationof 1mgmL Several plant-derived drugs such as galantamineand rivastigmine are used to inhibit AChE and thus increaseendogenous levels of acetylcholine to improve cholinergictransmission [30] Many studies described alkaloids as the
So
Pp
Lr
Tg
Hc
Lt
Tran
smitt
ance
()
4000 3500 3000 2500 2000 1500 1000 500
Wavenumber (cmminus1)
Pp
Lr
Tg
Hc
Lt
Figure 1 Infrared absorption spectra of some ethanolic extractstested in this study So Senna obtusifolia Pp Parkia platycephala LrLicania rigida Tg Triplaris gardneriana Hc Hymenaea courbarilLt Licania tomentosa
main compounds capable of inhibiting AChE enzyme [31]but recent studies have pointed out several new classes ofsecondarymetabolites as potent inhibitors such as flavonoids[32] flavones [33] and xanthones [34] as well as steroids ter-penoids oils and other phenolic compounds [35] Thus thephytochemical analysis data in the present study corroboratepreviousmolecular findings since polyphenols were detectedin all extracts Additionally the extract of T gardnerianashowed the presence of chalcones and aurones Thereforefurther studies should be conducted to assess the potential ofthese extracts and their respective molecules to inhibit AChEenzyme and thus offer a potential to fight neurodegenerativediseases such as Alzheimerrsquos
After running all the assays described above infraredspectroscopywith themost promising ethanolic seed extractswas also performed (S obtusifolia P platycephala L rigida Ltomentosa and T gardneriana) to confirm the results of phy-tochemical qualitative studyThe phytochemical studies withthese ethanolic extracts showed the presence of tannins andflavonoids which were confirmed by infrared spectroscopy(Figure 1)The spectra obtained between 1800 and 800 cmminus1are present at the fingerprint zone of aromatic compoundswhich can be found in tannins polyphenols and flavonoids[36] Further steps of fractionation andpurificationmay showthe molecule(s) responsible for this excellent activity
4 Conclusions
This study revealed antibacterial antioxidant and anti-cholinesterase activities in some seed ethanolic extracts ofBrazilian semiarid region plants emphasizing the antioxidantactivity presented by T gardneriana seeds This showeda potent capacity to catch free radicals by DPPH assayThis promising result could be attributed to its composition
8 BioMed Research International
featuring phenols tannins flavones and flavonoidsNewphy-tochemical and molecular studies are underway to identifythe bioactive compound(s) responsible for this activity
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgment
The authors thank theMinisterio da Saude Departamento deCiencias e Tecnologia (DECIT) Secretaria de Ciencias Tec-nologia e Insumos estrategicos (SCTIE) Conselho Nacionalde Desenvolvimento Cientıfico e Tecnologico (CNPq) andFundacao Cearense deApoioCientıfico e Tecnologico (FUN-CAP) for financial support of this research
References
[1] C Katiyar A Gupta S Kanjilal and S Katiyar ldquoDrug discoveryfromplant sources an integrated approachrdquoAyu vol 33 pp 10ndash19 2012
[2] P S Luize T S Tiuman L G Morello et al ldquoEffects of medic-inal plant extracts on growth of Leishmania (L) amazonensisand Trypanosoma cruzirdquo Brazilian Journal of PharmaceuticalSciences vol 41 no 1 pp 85ndash94 2005
[3] A E Edris ldquoAnti-cancer properties of Nigella spp essential oilsand their major constituents thymoquinone and 120573-ElemenerdquoCurrent Clinical Pharmacology vol 4 no 1 pp 43ndash46 2009
[4] R N Bennett and R M Wallsgrove ldquoSecondary metabolites inplant defence mechanismsrdquo New Phytologist vol 127 no 4 pp617ndash633 1994
[5] R J Grayer and J B Harborne ldquoA survey of antifungal com-pounds from higher plants 1982-1993rdquo Phytochemistry vol 37no 1 pp 19ndash42 1994
[6] A E Osbourn ldquoPreformed antimicrobial compounds and plantdefense against fungal attackrdquo Plant Cell vol 8 no 10 pp 1821ndash1831 1996
[7] E V S B Sampaio ldquoVegetacao e flora da Caatingardquo APNECNIP Recife 2000
[8] C F C B R De Almeida T C De Lima E Silva E L CDe Amorim M B D S Maia and U P De AlbuquerqueldquoLife strategy and chemical composition as predictors of theselection of medicinal plants from the caatinga (NortheastBrazil)rdquo Journal of Arid Environments vol 62 no 1 pp 127ndash1422005
[9] U P de Albuquerque P M de Medeiros A L S de Almeida etal ldquoMedicinal plants of the caatinga (semi-arid) vegetation ofNE Brazil a quantitative approachrdquo Journal of Ethnopharmacol-ogy vol 114 no 3 pp 325ndash354 2007
[10] U P de Albuquerque V A da Silva M D C Cabral NLeal Alencar and L D H C Andrade ldquoComparisons betweenthe use of medicinal plants in indigenous and rural caatinga(dryland) communities in NE Brazilrdquo Boletin Latinoamericanoy del Caribe de Plantas Medicinales y Aromaticas vol 7 no 3pp 156ndash170 2008
[11] T M Souza D F Farias B M Soares et al ldquoToxicity ofBrazilian plant seed extracts to two strains of Aedes aegypti(Diptera Culicidae) andNontargetAnimalsrdquo Journal ofMedicalEntomology vol 48 no 4 pp 846ndash851 2011
[12] P M P Ferreira D F Farias M P Viana et al ldquoStudy of theantiproliferative potential of seed extracts from NortheasternBrazilian plantsrdquo Anais da Academia Brasileira de Ciencias vol83 no 3 pp 1045ndash1058 2011
[13] F J AMatosM EOMatosM P SousaM I LMachado andA A Craveiro Constituintes Quımicos Ativos e PropriedadesBiologicas de Plantas Medicinais Brasileiras UFC FortalezaBrazil 1st edition 2004
[14] F J A Matos Plantas Medicinais Guia de Selecao e Empregode Plantas Usadas em Fitoterapia no Nordeste do Brasil IUFortaleza Brazil 2nd edition 2000
[15] G N Maia Caatinga Arvores e Arbustos e Suas Utilidades D ampZ Computacao Grafica e Editora Sao Paulo Brazil 1st edition2004
[16] M Chandrasekaran and V Venkatesalu ldquoAntibacterial andantifungal activity of Syzygium jambolanum seedsrdquo Journal ofEthnopharmacology vol 91 no 1 pp 105ndash108 2004
[17] B Yepez M Espinosa S Lopez and G Bolaos ldquoProducingantioxidant fractions from herbaceousmatrices by supercriticalfluid extractionrdquo Fluid Phase Equilibria vol 194ndash197 pp 879ndash884 2002
[18] E M Cardoso-Lopes J A Maier M R Da Silva et alldquoAlkaloids from stems of esenbeckia leiocarpa Engl (Rutaceae)as potential treatment for alzheimer diseaserdquoMolecules vol 15no 12 pp 9205ndash9213 2010
[19] G L Ellman K D Courtney V Andres Jr and R MFeatherstone ldquoA new and rapid colorimetric determination ofacetylcholinesterase activityrdquo Biochemical Pharmacology vol 7no 2 pp 88ndash95 1961
[20] A W Bernheimer ldquoAssay of hemolytic toxinsrdquo Methods inEnzymology vol 165 pp 213ndash217 1988
[21] F J A Matos Introducao A Fitoquımica Experimental EditoraUFC Ceara Brazil 1st edition 2009
[22] J Xavier-Filho ldquoSementes e suas defesas contra insetos ProjetoMultinacional de Biotecnologia e Alimentosrdquo Organizacao dosEstados Americanos (OEA) pp1ndash31 1993
[23] T C Fleischer E P K Ameade M L K Mensah and I KSawer ldquoAntimicrobial activity of the leaves and seeds of Bixaorellanardquo Fitoterapia vol 74 no 1-2 pp 136ndash138 2003
[24] L Majhenic M Skerget and Z Knez ldquoAntioxidant and antimi-crobial activity of guarana seed extractsrdquo Food Chemistry vol104 no 3 pp 1258ndash1268 2007
[25] MM Cowan ldquoPlant products as antimicrobial agentsrdquoClinicalMicrobiology Reviews vol 12 no 4 pp 564ndash582 1999
[26] A Rafat K Philip and SMuniandy ldquoAntioxidant potential andcontent of phenolic compounds in ethanolic extracts of selectedparts of Andrographis paniculatardquo Journal of Medicinal PlantResearch vol 4 no 3 pp 197ndash202 2010
[27] O M Mosquera Y M Correa D C Buitrago and J NinoldquoAntioxidant activity of twenty five plants from ColombianbiodiversityrdquoMemorias do Instituto Oswaldo Cruz vol 102 no5 pp 631ndash634 2007
[28] N Cotelle J-L Bernier J-P Catteau J Pommery J-C Wal-let and E M Gaydou ldquoAntioxidant properties of hydroxy-flavonesrdquo Free Radical Biology and Medicine vol 20 no 1 pp35ndash43 1996
[29] H Oh D-H Kim J-H Cho and Y-C Kim ldquoHepatoprotectiveand free radical scavenging activities of phenolic petrosinsand flavonoids isolated from Equisetum arvenserdquo Journal ofEthnopharmacology vol 95 no 2-3 pp 421ndash424 2004
BioMed Research International 9
[30] S Lopez J Bastida F Viladomat and C Codina ldquoAcetyl-cholinesterase inhibitory activity of some Amaryllidaceae alka-loids and Narcissus extractsrdquo Life Sciences vol 71 no 21 pp2521ndash2529 2002
[31] M G Ortega A M Agnese and J L Cabrera ldquoAnti-cholinesterase activity in an alkaloid extract ofHuperzia sauru-rusrdquo Phytomedicine vol 11 no 6 pp 539ndash543 2004
[32] B J Hillhouse D S Ming C J French and G H NTowers ldquoAcetylcholine esterase inhibitors in Rhodiola roseardquoPharmaceutical Biology vol 42 no 1 pp 68ndash72 2004
[33] P Sawasdee C Sabphon D Sitthiwongwanit and U KokpolldquoAnticholinesterase activity of 7-methoxyflavones isolated fromKaempferia parviflorardquo Phytotherapy Research vol 23 no 12pp 1792ndash1794 2009
[34] 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
[35] H-F Ji andH-Y Zhang ldquoMultipotent natural agents to combatAlzheimerrsquos disease Functional spectrum and structural fea-turesrdquo Acta Pharmacologica Sinica vol 29 no 2 pp 143ndash1512008
[36] K Fernandez and E Agosin ldquoQuantitative analysis of red winetannins using Fourier-transform mid-infrared spectrometryrdquoJournal of Agricultural and Food Chemistry vol 55 no 18 pp7294ndash7300 2007
Submit your manuscripts athttpwwwhindawicom
PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom
Volume 2014
ToxinsJournal of
VaccinesJournal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
AntibioticsInternational Journal of
ToxicologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Drug DeliveryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in Pharmacological Sciences
Tropical MedicineJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Medicinal ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
AddictionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Autoimmune Diseases
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anesthesiology Research and Practice
ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Pharmaceutics
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
4 BioMed Research International
Table1Con
tinued
Family
Botanicaln
ame
Com
mon
name
(localitydateof
collection)V
oucher
number
Tradition
almedicinaluse
Yield
()b
Antioxidant
activ
ity(EC 5
0120583gmL)
cAnticho
linesterase
activ
ity(
)d
Antibacteria
lactivity
MIC
(mgmL)
e
SCh
oleraesuis
Bsubtilis
Saureus
Fabaceae
Adenantherapavonina
LFalso
-sandalo
(Coastzone0807)
EAC38697
Not
describ
ed612
ND
ND
ND
ND
ND
Amburana
cearensis
(Allemao)A
C
Smith
Cumaruandam
burana
(Ca
atinga
vegetatio
n1107)
EAC39618
Usedto
treatrheumatism
coldand
sinusitis
antispasm
odichealin
gand
anti-inflammatory[15]
analgesic
[13]
643
ND
ND
ND
ND
ND
Anadenantheramacrocarpa
(Benth)Brenan
Ang
icoverm
elho
(Ca
atinga
vegetatio
n0705)
EAC38389
Usedto
treatrespira
tory
prob
lemsgono
rrheaand
diarrheaastrin
gent
antirheum
atic
anti-inflammatorysedativ
eandhealing[15]
935
ND
541
ND
ND
ND
Diocle
amegacarpa
Rolfe
Mucun
aolho
-de-bo
i(Ararip
eNationalForest0105)
EAC38110
Not
describ
ed240
ND
ND
ND
ND
ND
Enterolobium
contortisiliquum
(Vell)
Moron
gOrelha-de-negro
(Ararip
eNationalForest0905)
EAC38115
Not
describ
ed280
ND
ND
ND
ND
ND
Hym
enaeacourbaril
LJatoba(Ararip
eNationalForest0105)
EAC38108
Not
describ
ed95
02479
5ND
ND
ND
ND
Lonchocarpus
seric
eus(Po
iret)Ku
nth
Inga(Coastzone1006)
EAC39615
Not
describ
ed1060
ND
ND
ND
ND
ND
Luetzelburgiaauric
ulata(A
llemao)
Ducke
Pau-moco
(Ca
atinga
vegetatio
n1106)
EAC40369
Not
describ
ed840
ND
ND
ND
ND
ND
BioMed Research International 5Ta
ble1Con
tinued
Family
Botanicaln
ame
Com
mon
name
(localitydateof
collection)V
oucher
number
Tradition
almedicinaluse
Yield
()b
Antioxidant
activ
ity(EC 5
0120583gmL)
cAnticho
linesterase
activ
ity(
)d
Antibacteria
lactivity
MIC
(mgmL)
e
SCh
oleraesuis
Bsubtilis
Saureus
ParkiaplatycephalaBe
nth
Visgueiro
(Ararip
eNationalForest0106)
EAC3
8109
Not
describ
ed1750
ND
715
ND
ND
ND
Piptadeniamoniliform
isBe
nth
Catand
uva
(Ca
atinga
vegetatio
n0105)
EAC3
5974
Not
describ
ed618
ND
502
ND
ND
ND
Senn
aobtusifolia(L)HSIrw
inamp
Barneby
Matap
asto
(Ca
atinga
vegetatio
n0705)
EAC3
9320
Not
describ
ed570
ND
ND
ND
496
ND
Polyg
onaceae
Triplaris
gardneria
naWedd
Pajeu
(Ca
atinga
vegetatio
n1106)
EAC3
9600
Not
describ
ed260
6973
798
188
1376
1115
Rham
naceae
Zizip
husjoazeiro
Mart
Juaa
ndjuazeiro
(Coastzone0308)
EAC40366
Expectorantand
antip
yretic
used
totre
atskinblood
sto
machandliver
diseases
ulcerantim
icrobial[15]
722
ND
494
ND
ND
ND
Sapind
aceae
Talisiaesculen
ta(A
St-Hil)
Radlk
Pitomba
(Coastzone0308)
EAC40
685
Not
describ
ed15
0ND
6124
ND
ND
ND
Sapind
ussaponaria
LSabo
nete
(Ca
atinga
vegetatio
n1106)
EAC3
9601
Not
describ
ed107
ND
5860
ND
ND
ND
Controlsg
Quercetin
mdashmdash
5552
mdashmdash
mdashmdash
Vitamin
Cmdash
mdash26027
mdashmdash
mdashmdash
a Thev
aluesa
remeans
ofatrip
licateTh
estand
arddeviationwas
lessthan
5of
them
ean
b (Amou
nt(g)o
fsolid
recoveredfro
mthee
thanolicextracta
mou
nt(g)o
fseedflo
urused)times
100
c Efficientcon
centratio
n(EC)
as120583
gof
sampler
equiredto
decrease
oneg
oftheinitia
l22-diphenyl-1-picrylhydrazyl(DPP
Hsdot)con
centratio
nby
50
d Totalinhibitio
nof
cholinesterase
enzymea
fterc
omparis
onto
theb
lank
Con
centratio
nteste
d500120583
gmL
e Minim
uminhibitory
concentration
thelow
estcon
centratio
nwhich
didno
tsho
wanygrow
thof
teste
dorganism
after
macroscop
icevaluatio
nf N
Dnot
detected
g Sub
stancesthatare
frequ
ently
used
assta
ndardfora
ntioxidant
studies
6 BioMed Research International
(65 times 10minus1molsdotLminus1) The reduction of the DPPH radicalwas measured by monitoring continuously the decrease ofabsorption at 515 nm The percentage scavenging of theDPPH radical was calculated according to the followingformula scavenging effect = 100 times [(119860DPPH minus As)119860DPPH]where As is the absorbance of the solution when the samplehas been added at a particular level and 119860DPPH is theabsorbance of the DPPH solution The percentage of theremaining DPPH was plotted against the samplestandardconcentration to obtain the amount of antioxidant necessaryto decrease the initial concentration of DPPH by 50 (EC
50)
Based on the parameter EC50 the result was expressed in
terms of 120583g of seed extract per mL of DPPH in the reactionmedium All determinations were run in triplicate
27 Anticholinesterase Activity The anticholinesterase activ-ity of the ethanolic extracts was measured as described by[18] with slight modifications Acetylcholinesterase activitywas measured using a 96-well microplate reader basedon Ellmanrsquos method [19] In this method the enzymehydrolyzes the substrate acetylthiocholine resulting in theproduction of thiocholine which reacts with 551015840-dithiobis(2-nitrobenzoic acid) (DTNB) to produce 2-nitrobenzoate-5-mercaptothiocholine and 5-thio-2-nitrobenzoate which canbe detected at 405 nm In the 96-well plates 25120583L of 15mMacetylthiocholine iodide (ATCI) in water 125 120583L of 3mMDTNB in 50mM Tris-HCl buffer containing 01M NaCland 002M MgCl
2sdot6H2O 50 120583L of 50mM Tris-HCl buffer
containing 01 BSA and 25 120583L of plant extract sample(5mgsdotmLminus1 in MeOH diluted ten times with 50mM Tris-HCl buffer to give a concentration of 05mgsdotmLminus1) wereadded and the absorbance was measured at 405 nm every30 s for three times Then 25120583L of 022UsdotmLminus1 of acetyl-cholinesterase fromelectric eel was added and the absorbancewas again read every 45 s for eight times Any increase inabsorbance due to the spontaneous hydrolysis of the substratewas corrected by subtracting the rate of the reaction beforethe addition of the enzyme from the rate of the enzymereaction The percentage of inhibition was calculated bycomparison of sample rates to a blank
28 Hemolytic Activity Assay The test was performed in15mL microtubes following the method described by [20]with some modifications Firstly a twofold serial dilution ofeach seed ethanolic extract was prepared with 09 NaClranging from 1000 to 19120583gsdotmLminus1 and reservedThen 100120583Lof a 1 red blood cells (A B and O human blood types orrabbit blood) suspension was added to a newmicrotube con-taining 900 120583L of each seed extract dilution which was thentaken to incubator at 37∘C for 1 h After that the tubes werecentrifuged at 3000timesg for 5min The supernatant (200120583L)was placed in a 96-well plate and led to a microplate reader(Epoch BioTek Vermont USA) to measure the absorbanceat 540 nm The cell suspensions of each human blood typeor those of rabbits (100120583L) were mixed with distilled wateror 09 NaCl (900120583L) to obtain the absorbances for 100and 0 of cell lysis respectivelyThe percentage of hemolysiswas calculated as follows hemolysis = AbstestAbs119901119888times 100
where Abstest = Abs540 of the 1 cell suspension treated withsample test and Abs
119901119888= Abs
540of the 1 cell suspension
treated with distilled water To calculate the relation betweenpercentage of hemolysis and seed extract concentration thehemolytic activity was expressed as the lowest seed extractconcentration (120583gsdotmLminus1) capable of causing hemolysis ge20All determinations were run in triplicate
29 Infrared Spectroscopy The infrared spectrum wasrecorded from KBr discs on a FTIR Perkin Elmer model16 PC spectrophotometer (PerkinElmer Waltham USA)Absorptionmaxima (Egraveđmax) were reported in wavenumbers(cmminus1) The spectra were used to determine organic groupsindicative of the main classes of secondary metabolites
210 Phytochemical Study The ethanolic seed extracts werescreened for phytochemical compounds such as phenols andtannins (reaction with ferric chloride) leucoanthocyanidins(heat treatment followed by alkalinization and acidification ofsample) flavonoids and xanthones (reaction of magnesiumgranules with hydrochloric acid) steroids and triterpenes(extraction with chloroform acetic anhydride and sulfuricacid) saponins (foam production after water solubilizationand stirring) and alkaloids (precipitation with Hager Mayerand Dragendorff reagents) [21] These analyses were basedon visual observation of color modification or precipitateformation after addition of specific reagents
3 Results and Discussion
The ethanolic seed extracts did not show activity againstthe Gram (minus) organisms E aerogenes K pneumonia and Paeruginosa nor against the yeastsC albicansC krusei andCtropicalis MIC values obtained against the Gram (+) organ-isms S aureus and B subtilis and the Gram (minus) organism Scholeraesuis are shown in Table 1 Among all samples testedand microorganisms used the seed extract of S obtusifoliapresented the lowest MIC value (496mgsdotmLminus1) against thepathogenic bacteria S aureus However the speciesT gardne-riana showed the broadest spectrum of activity being activeagainst B subtilis S choleraesuis and S aureuswithMIC val-ues of 188 1376 and 1115mgsdotmLminus1 respectively Althoughthere are a great number of studies reporting antimicrobialactivity of plant parts like leaves roots and stems antimi-crobial screenings using specifically seed extracts are veryrare The reason for that is not easy to explain since seedsare admittedly a rich source of compounds involved inplant defense [22] A good example is the seed extract ofBixa orellana that has been described to have antimicrobialactivity against six strains of bacteria among these are Bsubtilis S aureus and P aeruginosa and against the yeastC albicans [23] Likewise guarana (Paullinia cupana) seedextracts presented strong antimicrobial activity against a hugenumber of damaging fungi and bacteria [24] As to thepotency of the antibacterial activities observed these weremuch lower than those described for Syzygium jambolanumseed extracts against the same bacterial strains used in thiswork (MIC values ranging from 006 to 025mgsdotmLminus1) even
BioMed Research International 7
when compared to the best result observed as describedabove for S obtusifolia (MIC 496mgsdotmLminus1) In additionthe S jambolanum seed extracts were also effective againstdamaging-fungi strains The low potency of the ethanolicseed extracts could be due to the chemical composition of thesamples with low concentration of antimicrobial substancesIn fact the results of phytochemical study revealed that allseed extracts with activity against bacteria showed in theircomposition classes of secondary metabolites widely knownas efficient antimicrobial compounds (alkaloids flavonesflavonoids phenols saponins and tannins) [25] A fraction-ation of these samples in a polarity gradient could improvetheir activities Therefore the absence or low activity of theseed extracts could be a problem of concentration
The hemolytic activity assay was performed with theseed extracts that were active against the microorganismsused Among six species tested only M urundeuva causedhemolysis in all blood types (1 erythrocytes suspension ofA B and O human blood types and rabbit blood) employedbeing the lowest concentration tested (19120583gsdotmLminus1) capableof causing more than 20 hemolysis These results show thatalthough the antimicrobial extracts are not very potent theyare more specific to bacterial cells except for M urundeuvaseed extract It is well reported that some classes of plantsecondarymetabolites are potent hemolytic compounds [26]such as phenols that were also detected forMurundeuva seedextract in the phytochemical study
The results for antioxidant capacity are also shown inTable 1 Only five seed extracts were able to capture free rad-icals in the concentrations used according to DPPHmethodthese were Hymenaea courbaril (EC
5024795 120583gsdotmLminus1) L
rigida (48751120583gsdotmLminus1) L tomentosa (21672 120583gsdotmLminus1) andT gardneriana (6973120583gsdotmLminus1) The L tomentosa and Tgardneriana seed extracts were more potent than the positivecontrol vitamin C (EC
5026027120583gsdotmLminus1) and the second one
was just a bit less potent than quercetin (EC505552120583gsdotmLminus1)
one of the most potent antioxidant molecules A previousstudy [27] showed that out of 25 plant extracts (hexanedichloromethane and methanol) only 12 presented someradical scavenging activity and the most potent activityshowed an IC
50value of 11199 120583gsdotmLminus1This promising result
for T gardneriana could be attributed to components such astannins flavones flavonoids and especially phenols [28 29]Further steps of fractionation and purification may show themolecule(s) responsible for this excellent activity
Concerning the anticholinesterase activity (Table 1)among the 21 ethanolic extracts tested 11 presented inhibitionof the cholinesterase enzyme T gardneriana P platycephalaand C detersus extracts presented the strongest activitywith inhibition values of respectively 767 715 and 919at the final concentration of 500120583gmL These values aresignificant when compared to those obtained in previousstudies [18] which have shown inhibition values of 17 plantextracts ranging from 9 to 81 using a single concentrationof 1mgmL Several plant-derived drugs such as galantamineand rivastigmine are used to inhibit AChE and thus increaseendogenous levels of acetylcholine to improve cholinergictransmission [30] Many studies described alkaloids as the
So
Pp
Lr
Tg
Hc
Lt
Tran
smitt
ance
()
4000 3500 3000 2500 2000 1500 1000 500
Wavenumber (cmminus1)
Pp
Lr
Tg
Hc
Lt
Figure 1 Infrared absorption spectra of some ethanolic extractstested in this study So Senna obtusifolia Pp Parkia platycephala LrLicania rigida Tg Triplaris gardneriana Hc Hymenaea courbarilLt Licania tomentosa
main compounds capable of inhibiting AChE enzyme [31]but recent studies have pointed out several new classes ofsecondarymetabolites as potent inhibitors such as flavonoids[32] flavones [33] and xanthones [34] as well as steroids ter-penoids oils and other phenolic compounds [35] Thus thephytochemical analysis data in the present study corroboratepreviousmolecular findings since polyphenols were detectedin all extracts Additionally the extract of T gardnerianashowed the presence of chalcones and aurones Thereforefurther studies should be conducted to assess the potential ofthese extracts and their respective molecules to inhibit AChEenzyme and thus offer a potential to fight neurodegenerativediseases such as Alzheimerrsquos
After running all the assays described above infraredspectroscopywith themost promising ethanolic seed extractswas also performed (S obtusifolia P platycephala L rigida Ltomentosa and T gardneriana) to confirm the results of phy-tochemical qualitative studyThe phytochemical studies withthese ethanolic extracts showed the presence of tannins andflavonoids which were confirmed by infrared spectroscopy(Figure 1)The spectra obtained between 1800 and 800 cmminus1are present at the fingerprint zone of aromatic compoundswhich can be found in tannins polyphenols and flavonoids[36] Further steps of fractionation andpurificationmay showthe molecule(s) responsible for this excellent activity
4 Conclusions
This study revealed antibacterial antioxidant and anti-cholinesterase activities in some seed ethanolic extracts ofBrazilian semiarid region plants emphasizing the antioxidantactivity presented by T gardneriana seeds This showeda potent capacity to catch free radicals by DPPH assayThis promising result could be attributed to its composition
8 BioMed Research International
featuring phenols tannins flavones and flavonoidsNewphy-tochemical and molecular studies are underway to identifythe bioactive compound(s) responsible for this activity
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgment
The authors thank theMinisterio da Saude Departamento deCiencias e Tecnologia (DECIT) Secretaria de Ciencias Tec-nologia e Insumos estrategicos (SCTIE) Conselho Nacionalde Desenvolvimento Cientıfico e Tecnologico (CNPq) andFundacao Cearense deApoioCientıfico e Tecnologico (FUN-CAP) for financial support of this research
References
[1] C Katiyar A Gupta S Kanjilal and S Katiyar ldquoDrug discoveryfromplant sources an integrated approachrdquoAyu vol 33 pp 10ndash19 2012
[2] P S Luize T S Tiuman L G Morello et al ldquoEffects of medic-inal plant extracts on growth of Leishmania (L) amazonensisand Trypanosoma cruzirdquo Brazilian Journal of PharmaceuticalSciences vol 41 no 1 pp 85ndash94 2005
[3] A E Edris ldquoAnti-cancer properties of Nigella spp essential oilsand their major constituents thymoquinone and 120573-ElemenerdquoCurrent Clinical Pharmacology vol 4 no 1 pp 43ndash46 2009
[4] R N Bennett and R M Wallsgrove ldquoSecondary metabolites inplant defence mechanismsrdquo New Phytologist vol 127 no 4 pp617ndash633 1994
[5] R J Grayer and J B Harborne ldquoA survey of antifungal com-pounds from higher plants 1982-1993rdquo Phytochemistry vol 37no 1 pp 19ndash42 1994
[6] A E Osbourn ldquoPreformed antimicrobial compounds and plantdefense against fungal attackrdquo Plant Cell vol 8 no 10 pp 1821ndash1831 1996
[7] E V S B Sampaio ldquoVegetacao e flora da Caatingardquo APNECNIP Recife 2000
[8] C F C B R De Almeida T C De Lima E Silva E L CDe Amorim M B D S Maia and U P De AlbuquerqueldquoLife strategy and chemical composition as predictors of theselection of medicinal plants from the caatinga (NortheastBrazil)rdquo Journal of Arid Environments vol 62 no 1 pp 127ndash1422005
[9] U P de Albuquerque P M de Medeiros A L S de Almeida etal ldquoMedicinal plants of the caatinga (semi-arid) vegetation ofNE Brazil a quantitative approachrdquo Journal of Ethnopharmacol-ogy vol 114 no 3 pp 325ndash354 2007
[10] U P de Albuquerque V A da Silva M D C Cabral NLeal Alencar and L D H C Andrade ldquoComparisons betweenthe use of medicinal plants in indigenous and rural caatinga(dryland) communities in NE Brazilrdquo Boletin Latinoamericanoy del Caribe de Plantas Medicinales y Aromaticas vol 7 no 3pp 156ndash170 2008
[11] T M Souza D F Farias B M Soares et al ldquoToxicity ofBrazilian plant seed extracts to two strains of Aedes aegypti(Diptera Culicidae) andNontargetAnimalsrdquo Journal ofMedicalEntomology vol 48 no 4 pp 846ndash851 2011
[12] P M P Ferreira D F Farias M P Viana et al ldquoStudy of theantiproliferative potential of seed extracts from NortheasternBrazilian plantsrdquo Anais da Academia Brasileira de Ciencias vol83 no 3 pp 1045ndash1058 2011
[13] F J AMatosM EOMatosM P SousaM I LMachado andA A Craveiro Constituintes Quımicos Ativos e PropriedadesBiologicas de Plantas Medicinais Brasileiras UFC FortalezaBrazil 1st edition 2004
[14] F J A Matos Plantas Medicinais Guia de Selecao e Empregode Plantas Usadas em Fitoterapia no Nordeste do Brasil IUFortaleza Brazil 2nd edition 2000
[15] G N Maia Caatinga Arvores e Arbustos e Suas Utilidades D ampZ Computacao Grafica e Editora Sao Paulo Brazil 1st edition2004
[16] M Chandrasekaran and V Venkatesalu ldquoAntibacterial andantifungal activity of Syzygium jambolanum seedsrdquo Journal ofEthnopharmacology vol 91 no 1 pp 105ndash108 2004
[17] B Yepez M Espinosa S Lopez and G Bolaos ldquoProducingantioxidant fractions from herbaceousmatrices by supercriticalfluid extractionrdquo Fluid Phase Equilibria vol 194ndash197 pp 879ndash884 2002
[18] E M Cardoso-Lopes J A Maier M R Da Silva et alldquoAlkaloids from stems of esenbeckia leiocarpa Engl (Rutaceae)as potential treatment for alzheimer diseaserdquoMolecules vol 15no 12 pp 9205ndash9213 2010
[19] G L Ellman K D Courtney V Andres Jr and R MFeatherstone ldquoA new and rapid colorimetric determination ofacetylcholinesterase activityrdquo Biochemical Pharmacology vol 7no 2 pp 88ndash95 1961
[20] A W Bernheimer ldquoAssay of hemolytic toxinsrdquo Methods inEnzymology vol 165 pp 213ndash217 1988
[21] F J A Matos Introducao A Fitoquımica Experimental EditoraUFC Ceara Brazil 1st edition 2009
[22] J Xavier-Filho ldquoSementes e suas defesas contra insetos ProjetoMultinacional de Biotecnologia e Alimentosrdquo Organizacao dosEstados Americanos (OEA) pp1ndash31 1993
[23] T C Fleischer E P K Ameade M L K Mensah and I KSawer ldquoAntimicrobial activity of the leaves and seeds of Bixaorellanardquo Fitoterapia vol 74 no 1-2 pp 136ndash138 2003
[24] L Majhenic M Skerget and Z Knez ldquoAntioxidant and antimi-crobial activity of guarana seed extractsrdquo Food Chemistry vol104 no 3 pp 1258ndash1268 2007
[25] MM Cowan ldquoPlant products as antimicrobial agentsrdquoClinicalMicrobiology Reviews vol 12 no 4 pp 564ndash582 1999
[26] A Rafat K Philip and SMuniandy ldquoAntioxidant potential andcontent of phenolic compounds in ethanolic extracts of selectedparts of Andrographis paniculatardquo Journal of Medicinal PlantResearch vol 4 no 3 pp 197ndash202 2010
[27] O M Mosquera Y M Correa D C Buitrago and J NinoldquoAntioxidant activity of twenty five plants from ColombianbiodiversityrdquoMemorias do Instituto Oswaldo Cruz vol 102 no5 pp 631ndash634 2007
[28] N Cotelle J-L Bernier J-P Catteau J Pommery J-C Wal-let and E M Gaydou ldquoAntioxidant properties of hydroxy-flavonesrdquo Free Radical Biology and Medicine vol 20 no 1 pp35ndash43 1996
[29] H Oh D-H Kim J-H Cho and Y-C Kim ldquoHepatoprotectiveand free radical scavenging activities of phenolic petrosinsand flavonoids isolated from Equisetum arvenserdquo Journal ofEthnopharmacology vol 95 no 2-3 pp 421ndash424 2004
BioMed Research International 9
[30] S Lopez J Bastida F Viladomat and C Codina ldquoAcetyl-cholinesterase inhibitory activity of some Amaryllidaceae alka-loids and Narcissus extractsrdquo Life Sciences vol 71 no 21 pp2521ndash2529 2002
[31] M G Ortega A M Agnese and J L Cabrera ldquoAnti-cholinesterase activity in an alkaloid extract ofHuperzia sauru-rusrdquo Phytomedicine vol 11 no 6 pp 539ndash543 2004
[32] B J Hillhouse D S Ming C J French and G H NTowers ldquoAcetylcholine esterase inhibitors in Rhodiola roseardquoPharmaceutical Biology vol 42 no 1 pp 68ndash72 2004
[33] P Sawasdee C Sabphon D Sitthiwongwanit and U KokpolldquoAnticholinesterase activity of 7-methoxyflavones isolated fromKaempferia parviflorardquo Phytotherapy Research vol 23 no 12pp 1792ndash1794 2009
[34] 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
[35] H-F Ji andH-Y Zhang ldquoMultipotent natural agents to combatAlzheimerrsquos disease Functional spectrum and structural fea-turesrdquo Acta Pharmacologica Sinica vol 29 no 2 pp 143ndash1512008
[36] K Fernandez and E Agosin ldquoQuantitative analysis of red winetannins using Fourier-transform mid-infrared spectrometryrdquoJournal of Agricultural and Food Chemistry vol 55 no 18 pp7294ndash7300 2007
Submit your manuscripts athttpwwwhindawicom
PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom
Volume 2014
ToxinsJournal of
VaccinesJournal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
AntibioticsInternational Journal of
ToxicologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Drug DeliveryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in Pharmacological Sciences
Tropical MedicineJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Medicinal ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
AddictionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Autoimmune Diseases
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anesthesiology Research and Practice
ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Pharmaceutics
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
BioMed Research International 5Ta
ble1Con
tinued
Family
Botanicaln
ame
Com
mon
name
(localitydateof
collection)V
oucher
number
Tradition
almedicinaluse
Yield
()b
Antioxidant
activ
ity(EC 5
0120583gmL)
cAnticho
linesterase
activ
ity(
)d
Antibacteria
lactivity
MIC
(mgmL)
e
SCh
oleraesuis
Bsubtilis
Saureus
ParkiaplatycephalaBe
nth
Visgueiro
(Ararip
eNationalForest0106)
EAC3
8109
Not
describ
ed1750
ND
715
ND
ND
ND
Piptadeniamoniliform
isBe
nth
Catand
uva
(Ca
atinga
vegetatio
n0105)
EAC3
5974
Not
describ
ed618
ND
502
ND
ND
ND
Senn
aobtusifolia(L)HSIrw
inamp
Barneby
Matap
asto
(Ca
atinga
vegetatio
n0705)
EAC3
9320
Not
describ
ed570
ND
ND
ND
496
ND
Polyg
onaceae
Triplaris
gardneria
naWedd
Pajeu
(Ca
atinga
vegetatio
n1106)
EAC3
9600
Not
describ
ed260
6973
798
188
1376
1115
Rham
naceae
Zizip
husjoazeiro
Mart
Juaa
ndjuazeiro
(Coastzone0308)
EAC40366
Expectorantand
antip
yretic
used
totre
atskinblood
sto
machandliver
diseases
ulcerantim
icrobial[15]
722
ND
494
ND
ND
ND
Sapind
aceae
Talisiaesculen
ta(A
St-Hil)
Radlk
Pitomba
(Coastzone0308)
EAC40
685
Not
describ
ed15
0ND
6124
ND
ND
ND
Sapind
ussaponaria
LSabo
nete
(Ca
atinga
vegetatio
n1106)
EAC3
9601
Not
describ
ed107
ND
5860
ND
ND
ND
Controlsg
Quercetin
mdashmdash
5552
mdashmdash
mdashmdash
Vitamin
Cmdash
mdash26027
mdashmdash
mdashmdash
a Thev
aluesa
remeans
ofatrip
licateTh
estand
arddeviationwas
lessthan
5of
them
ean
b (Amou
nt(g)o
fsolid
recoveredfro
mthee
thanolicextracta
mou
nt(g)o
fseedflo
urused)times
100
c Efficientcon
centratio
n(EC)
as120583
gof
sampler
equiredto
decrease
oneg
oftheinitia
l22-diphenyl-1-picrylhydrazyl(DPP
Hsdot)con
centratio
nby
50
d Totalinhibitio
nof
cholinesterase
enzymea
fterc
omparis
onto
theb
lank
Con
centratio
nteste
d500120583
gmL
e Minim
uminhibitory
concentration
thelow
estcon
centratio
nwhich
didno
tsho
wanygrow
thof
teste
dorganism
after
macroscop
icevaluatio
nf N
Dnot
detected
g Sub
stancesthatare
frequ
ently
used
assta
ndardfora
ntioxidant
studies
6 BioMed Research International
(65 times 10minus1molsdotLminus1) The reduction of the DPPH radicalwas measured by monitoring continuously the decrease ofabsorption at 515 nm The percentage scavenging of theDPPH radical was calculated according to the followingformula scavenging effect = 100 times [(119860DPPH minus As)119860DPPH]where As is the absorbance of the solution when the samplehas been added at a particular level and 119860DPPH is theabsorbance of the DPPH solution The percentage of theremaining DPPH was plotted against the samplestandardconcentration to obtain the amount of antioxidant necessaryto decrease the initial concentration of DPPH by 50 (EC
50)
Based on the parameter EC50 the result was expressed in
terms of 120583g of seed extract per mL of DPPH in the reactionmedium All determinations were run in triplicate
27 Anticholinesterase Activity The anticholinesterase activ-ity of the ethanolic extracts was measured as described by[18] with slight modifications Acetylcholinesterase activitywas measured using a 96-well microplate reader basedon Ellmanrsquos method [19] In this method the enzymehydrolyzes the substrate acetylthiocholine resulting in theproduction of thiocholine which reacts with 551015840-dithiobis(2-nitrobenzoic acid) (DTNB) to produce 2-nitrobenzoate-5-mercaptothiocholine and 5-thio-2-nitrobenzoate which canbe detected at 405 nm In the 96-well plates 25120583L of 15mMacetylthiocholine iodide (ATCI) in water 125 120583L of 3mMDTNB in 50mM Tris-HCl buffer containing 01M NaCland 002M MgCl
2sdot6H2O 50 120583L of 50mM Tris-HCl buffer
containing 01 BSA and 25 120583L of plant extract sample(5mgsdotmLminus1 in MeOH diluted ten times with 50mM Tris-HCl buffer to give a concentration of 05mgsdotmLminus1) wereadded and the absorbance was measured at 405 nm every30 s for three times Then 25120583L of 022UsdotmLminus1 of acetyl-cholinesterase fromelectric eel was added and the absorbancewas again read every 45 s for eight times Any increase inabsorbance due to the spontaneous hydrolysis of the substratewas corrected by subtracting the rate of the reaction beforethe addition of the enzyme from the rate of the enzymereaction The percentage of inhibition was calculated bycomparison of sample rates to a blank
28 Hemolytic Activity Assay The test was performed in15mL microtubes following the method described by [20]with some modifications Firstly a twofold serial dilution ofeach seed ethanolic extract was prepared with 09 NaClranging from 1000 to 19120583gsdotmLminus1 and reservedThen 100120583Lof a 1 red blood cells (A B and O human blood types orrabbit blood) suspension was added to a newmicrotube con-taining 900 120583L of each seed extract dilution which was thentaken to incubator at 37∘C for 1 h After that the tubes werecentrifuged at 3000timesg for 5min The supernatant (200120583L)was placed in a 96-well plate and led to a microplate reader(Epoch BioTek Vermont USA) to measure the absorbanceat 540 nm The cell suspensions of each human blood typeor those of rabbits (100120583L) were mixed with distilled wateror 09 NaCl (900120583L) to obtain the absorbances for 100and 0 of cell lysis respectivelyThe percentage of hemolysiswas calculated as follows hemolysis = AbstestAbs119901119888times 100
where Abstest = Abs540 of the 1 cell suspension treated withsample test and Abs
119901119888= Abs
540of the 1 cell suspension
treated with distilled water To calculate the relation betweenpercentage of hemolysis and seed extract concentration thehemolytic activity was expressed as the lowest seed extractconcentration (120583gsdotmLminus1) capable of causing hemolysis ge20All determinations were run in triplicate
29 Infrared Spectroscopy The infrared spectrum wasrecorded from KBr discs on a FTIR Perkin Elmer model16 PC spectrophotometer (PerkinElmer Waltham USA)Absorptionmaxima (Egraveđmax) were reported in wavenumbers(cmminus1) The spectra were used to determine organic groupsindicative of the main classes of secondary metabolites
210 Phytochemical Study The ethanolic seed extracts werescreened for phytochemical compounds such as phenols andtannins (reaction with ferric chloride) leucoanthocyanidins(heat treatment followed by alkalinization and acidification ofsample) flavonoids and xanthones (reaction of magnesiumgranules with hydrochloric acid) steroids and triterpenes(extraction with chloroform acetic anhydride and sulfuricacid) saponins (foam production after water solubilizationand stirring) and alkaloids (precipitation with Hager Mayerand Dragendorff reagents) [21] These analyses were basedon visual observation of color modification or precipitateformation after addition of specific reagents
3 Results and Discussion
The ethanolic seed extracts did not show activity againstthe Gram (minus) organisms E aerogenes K pneumonia and Paeruginosa nor against the yeastsC albicansC krusei andCtropicalis MIC values obtained against the Gram (+) organ-isms S aureus and B subtilis and the Gram (minus) organism Scholeraesuis are shown in Table 1 Among all samples testedand microorganisms used the seed extract of S obtusifoliapresented the lowest MIC value (496mgsdotmLminus1) against thepathogenic bacteria S aureus However the speciesT gardne-riana showed the broadest spectrum of activity being activeagainst B subtilis S choleraesuis and S aureuswithMIC val-ues of 188 1376 and 1115mgsdotmLminus1 respectively Althoughthere are a great number of studies reporting antimicrobialactivity of plant parts like leaves roots and stems antimi-crobial screenings using specifically seed extracts are veryrare The reason for that is not easy to explain since seedsare admittedly a rich source of compounds involved inplant defense [22] A good example is the seed extract ofBixa orellana that has been described to have antimicrobialactivity against six strains of bacteria among these are Bsubtilis S aureus and P aeruginosa and against the yeastC albicans [23] Likewise guarana (Paullinia cupana) seedextracts presented strong antimicrobial activity against a hugenumber of damaging fungi and bacteria [24] As to thepotency of the antibacterial activities observed these weremuch lower than those described for Syzygium jambolanumseed extracts against the same bacterial strains used in thiswork (MIC values ranging from 006 to 025mgsdotmLminus1) even
BioMed Research International 7
when compared to the best result observed as describedabove for S obtusifolia (MIC 496mgsdotmLminus1) In additionthe S jambolanum seed extracts were also effective againstdamaging-fungi strains The low potency of the ethanolicseed extracts could be due to the chemical composition of thesamples with low concentration of antimicrobial substancesIn fact the results of phytochemical study revealed that allseed extracts with activity against bacteria showed in theircomposition classes of secondary metabolites widely knownas efficient antimicrobial compounds (alkaloids flavonesflavonoids phenols saponins and tannins) [25] A fraction-ation of these samples in a polarity gradient could improvetheir activities Therefore the absence or low activity of theseed extracts could be a problem of concentration
The hemolytic activity assay was performed with theseed extracts that were active against the microorganismsused Among six species tested only M urundeuva causedhemolysis in all blood types (1 erythrocytes suspension ofA B and O human blood types and rabbit blood) employedbeing the lowest concentration tested (19120583gsdotmLminus1) capableof causing more than 20 hemolysis These results show thatalthough the antimicrobial extracts are not very potent theyare more specific to bacterial cells except for M urundeuvaseed extract It is well reported that some classes of plantsecondarymetabolites are potent hemolytic compounds [26]such as phenols that were also detected forMurundeuva seedextract in the phytochemical study
The results for antioxidant capacity are also shown inTable 1 Only five seed extracts were able to capture free rad-icals in the concentrations used according to DPPHmethodthese were Hymenaea courbaril (EC
5024795 120583gsdotmLminus1) L
rigida (48751120583gsdotmLminus1) L tomentosa (21672 120583gsdotmLminus1) andT gardneriana (6973120583gsdotmLminus1) The L tomentosa and Tgardneriana seed extracts were more potent than the positivecontrol vitamin C (EC
5026027120583gsdotmLminus1) and the second one
was just a bit less potent than quercetin (EC505552120583gsdotmLminus1)
one of the most potent antioxidant molecules A previousstudy [27] showed that out of 25 plant extracts (hexanedichloromethane and methanol) only 12 presented someradical scavenging activity and the most potent activityshowed an IC
50value of 11199 120583gsdotmLminus1This promising result
for T gardneriana could be attributed to components such astannins flavones flavonoids and especially phenols [28 29]Further steps of fractionation and purification may show themolecule(s) responsible for this excellent activity
Concerning the anticholinesterase activity (Table 1)among the 21 ethanolic extracts tested 11 presented inhibitionof the cholinesterase enzyme T gardneriana P platycephalaand C detersus extracts presented the strongest activitywith inhibition values of respectively 767 715 and 919at the final concentration of 500120583gmL These values aresignificant when compared to those obtained in previousstudies [18] which have shown inhibition values of 17 plantextracts ranging from 9 to 81 using a single concentrationof 1mgmL Several plant-derived drugs such as galantamineand rivastigmine are used to inhibit AChE and thus increaseendogenous levels of acetylcholine to improve cholinergictransmission [30] Many studies described alkaloids as the
So
Pp
Lr
Tg
Hc
Lt
Tran
smitt
ance
()
4000 3500 3000 2500 2000 1500 1000 500
Wavenumber (cmminus1)
Pp
Lr
Tg
Hc
Lt
Figure 1 Infrared absorption spectra of some ethanolic extractstested in this study So Senna obtusifolia Pp Parkia platycephala LrLicania rigida Tg Triplaris gardneriana Hc Hymenaea courbarilLt Licania tomentosa
main compounds capable of inhibiting AChE enzyme [31]but recent studies have pointed out several new classes ofsecondarymetabolites as potent inhibitors such as flavonoids[32] flavones [33] and xanthones [34] as well as steroids ter-penoids oils and other phenolic compounds [35] Thus thephytochemical analysis data in the present study corroboratepreviousmolecular findings since polyphenols were detectedin all extracts Additionally the extract of T gardnerianashowed the presence of chalcones and aurones Thereforefurther studies should be conducted to assess the potential ofthese extracts and their respective molecules to inhibit AChEenzyme and thus offer a potential to fight neurodegenerativediseases such as Alzheimerrsquos
After running all the assays described above infraredspectroscopywith themost promising ethanolic seed extractswas also performed (S obtusifolia P platycephala L rigida Ltomentosa and T gardneriana) to confirm the results of phy-tochemical qualitative studyThe phytochemical studies withthese ethanolic extracts showed the presence of tannins andflavonoids which were confirmed by infrared spectroscopy(Figure 1)The spectra obtained between 1800 and 800 cmminus1are present at the fingerprint zone of aromatic compoundswhich can be found in tannins polyphenols and flavonoids[36] Further steps of fractionation andpurificationmay showthe molecule(s) responsible for this excellent activity
4 Conclusions
This study revealed antibacterial antioxidant and anti-cholinesterase activities in some seed ethanolic extracts ofBrazilian semiarid region plants emphasizing the antioxidantactivity presented by T gardneriana seeds This showeda potent capacity to catch free radicals by DPPH assayThis promising result could be attributed to its composition
8 BioMed Research International
featuring phenols tannins flavones and flavonoidsNewphy-tochemical and molecular studies are underway to identifythe bioactive compound(s) responsible for this activity
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgment
The authors thank theMinisterio da Saude Departamento deCiencias e Tecnologia (DECIT) Secretaria de Ciencias Tec-nologia e Insumos estrategicos (SCTIE) Conselho Nacionalde Desenvolvimento Cientıfico e Tecnologico (CNPq) andFundacao Cearense deApoioCientıfico e Tecnologico (FUN-CAP) for financial support of this research
References
[1] C Katiyar A Gupta S Kanjilal and S Katiyar ldquoDrug discoveryfromplant sources an integrated approachrdquoAyu vol 33 pp 10ndash19 2012
[2] P S Luize T S Tiuman L G Morello et al ldquoEffects of medic-inal plant extracts on growth of Leishmania (L) amazonensisand Trypanosoma cruzirdquo Brazilian Journal of PharmaceuticalSciences vol 41 no 1 pp 85ndash94 2005
[3] A E Edris ldquoAnti-cancer properties of Nigella spp essential oilsand their major constituents thymoquinone and 120573-ElemenerdquoCurrent Clinical Pharmacology vol 4 no 1 pp 43ndash46 2009
[4] R N Bennett and R M Wallsgrove ldquoSecondary metabolites inplant defence mechanismsrdquo New Phytologist vol 127 no 4 pp617ndash633 1994
[5] R J Grayer and J B Harborne ldquoA survey of antifungal com-pounds from higher plants 1982-1993rdquo Phytochemistry vol 37no 1 pp 19ndash42 1994
[6] A E Osbourn ldquoPreformed antimicrobial compounds and plantdefense against fungal attackrdquo Plant Cell vol 8 no 10 pp 1821ndash1831 1996
[7] E V S B Sampaio ldquoVegetacao e flora da Caatingardquo APNECNIP Recife 2000
[8] C F C B R De Almeida T C De Lima E Silva E L CDe Amorim M B D S Maia and U P De AlbuquerqueldquoLife strategy and chemical composition as predictors of theselection of medicinal plants from the caatinga (NortheastBrazil)rdquo Journal of Arid Environments vol 62 no 1 pp 127ndash1422005
[9] U P de Albuquerque P M de Medeiros A L S de Almeida etal ldquoMedicinal plants of the caatinga (semi-arid) vegetation ofNE Brazil a quantitative approachrdquo Journal of Ethnopharmacol-ogy vol 114 no 3 pp 325ndash354 2007
[10] U P de Albuquerque V A da Silva M D C Cabral NLeal Alencar and L D H C Andrade ldquoComparisons betweenthe use of medicinal plants in indigenous and rural caatinga(dryland) communities in NE Brazilrdquo Boletin Latinoamericanoy del Caribe de Plantas Medicinales y Aromaticas vol 7 no 3pp 156ndash170 2008
[11] T M Souza D F Farias B M Soares et al ldquoToxicity ofBrazilian plant seed extracts to two strains of Aedes aegypti(Diptera Culicidae) andNontargetAnimalsrdquo Journal ofMedicalEntomology vol 48 no 4 pp 846ndash851 2011
[12] P M P Ferreira D F Farias M P Viana et al ldquoStudy of theantiproliferative potential of seed extracts from NortheasternBrazilian plantsrdquo Anais da Academia Brasileira de Ciencias vol83 no 3 pp 1045ndash1058 2011
[13] F J AMatosM EOMatosM P SousaM I LMachado andA A Craveiro Constituintes Quımicos Ativos e PropriedadesBiologicas de Plantas Medicinais Brasileiras UFC FortalezaBrazil 1st edition 2004
[14] F J A Matos Plantas Medicinais Guia de Selecao e Empregode Plantas Usadas em Fitoterapia no Nordeste do Brasil IUFortaleza Brazil 2nd edition 2000
[15] G N Maia Caatinga Arvores e Arbustos e Suas Utilidades D ampZ Computacao Grafica e Editora Sao Paulo Brazil 1st edition2004
[16] M Chandrasekaran and V Venkatesalu ldquoAntibacterial andantifungal activity of Syzygium jambolanum seedsrdquo Journal ofEthnopharmacology vol 91 no 1 pp 105ndash108 2004
[17] B Yepez M Espinosa S Lopez and G Bolaos ldquoProducingantioxidant fractions from herbaceousmatrices by supercriticalfluid extractionrdquo Fluid Phase Equilibria vol 194ndash197 pp 879ndash884 2002
[18] E M Cardoso-Lopes J A Maier M R Da Silva et alldquoAlkaloids from stems of esenbeckia leiocarpa Engl (Rutaceae)as potential treatment for alzheimer diseaserdquoMolecules vol 15no 12 pp 9205ndash9213 2010
[19] G L Ellman K D Courtney V Andres Jr and R MFeatherstone ldquoA new and rapid colorimetric determination ofacetylcholinesterase activityrdquo Biochemical Pharmacology vol 7no 2 pp 88ndash95 1961
[20] A W Bernheimer ldquoAssay of hemolytic toxinsrdquo Methods inEnzymology vol 165 pp 213ndash217 1988
[21] F J A Matos Introducao A Fitoquımica Experimental EditoraUFC Ceara Brazil 1st edition 2009
[22] J Xavier-Filho ldquoSementes e suas defesas contra insetos ProjetoMultinacional de Biotecnologia e Alimentosrdquo Organizacao dosEstados Americanos (OEA) pp1ndash31 1993
[23] T C Fleischer E P K Ameade M L K Mensah and I KSawer ldquoAntimicrobial activity of the leaves and seeds of Bixaorellanardquo Fitoterapia vol 74 no 1-2 pp 136ndash138 2003
[24] L Majhenic M Skerget and Z Knez ldquoAntioxidant and antimi-crobial activity of guarana seed extractsrdquo Food Chemistry vol104 no 3 pp 1258ndash1268 2007
[25] MM Cowan ldquoPlant products as antimicrobial agentsrdquoClinicalMicrobiology Reviews vol 12 no 4 pp 564ndash582 1999
[26] A Rafat K Philip and SMuniandy ldquoAntioxidant potential andcontent of phenolic compounds in ethanolic extracts of selectedparts of Andrographis paniculatardquo Journal of Medicinal PlantResearch vol 4 no 3 pp 197ndash202 2010
[27] O M Mosquera Y M Correa D C Buitrago and J NinoldquoAntioxidant activity of twenty five plants from ColombianbiodiversityrdquoMemorias do Instituto Oswaldo Cruz vol 102 no5 pp 631ndash634 2007
[28] N Cotelle J-L Bernier J-P Catteau J Pommery J-C Wal-let and E M Gaydou ldquoAntioxidant properties of hydroxy-flavonesrdquo Free Radical Biology and Medicine vol 20 no 1 pp35ndash43 1996
[29] H Oh D-H Kim J-H Cho and Y-C Kim ldquoHepatoprotectiveand free radical scavenging activities of phenolic petrosinsand flavonoids isolated from Equisetum arvenserdquo Journal ofEthnopharmacology vol 95 no 2-3 pp 421ndash424 2004
BioMed Research International 9
[30] S Lopez J Bastida F Viladomat and C Codina ldquoAcetyl-cholinesterase inhibitory activity of some Amaryllidaceae alka-loids and Narcissus extractsrdquo Life Sciences vol 71 no 21 pp2521ndash2529 2002
[31] M G Ortega A M Agnese and J L Cabrera ldquoAnti-cholinesterase activity in an alkaloid extract ofHuperzia sauru-rusrdquo Phytomedicine vol 11 no 6 pp 539ndash543 2004
[32] B J Hillhouse D S Ming C J French and G H NTowers ldquoAcetylcholine esterase inhibitors in Rhodiola roseardquoPharmaceutical Biology vol 42 no 1 pp 68ndash72 2004
[33] P Sawasdee C Sabphon D Sitthiwongwanit and U KokpolldquoAnticholinesterase activity of 7-methoxyflavones isolated fromKaempferia parviflorardquo Phytotherapy Research vol 23 no 12pp 1792ndash1794 2009
[34] 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
[35] H-F Ji andH-Y Zhang ldquoMultipotent natural agents to combatAlzheimerrsquos disease Functional spectrum and structural fea-turesrdquo Acta Pharmacologica Sinica vol 29 no 2 pp 143ndash1512008
[36] K Fernandez and E Agosin ldquoQuantitative analysis of red winetannins using Fourier-transform mid-infrared spectrometryrdquoJournal of Agricultural and Food Chemistry vol 55 no 18 pp7294ndash7300 2007
Submit your manuscripts athttpwwwhindawicom
PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom
Volume 2014
ToxinsJournal of
VaccinesJournal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
AntibioticsInternational Journal of
ToxicologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Drug DeliveryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in Pharmacological Sciences
Tropical MedicineJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Medicinal ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
AddictionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Autoimmune Diseases
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anesthesiology Research and Practice
ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Pharmaceutics
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
6 BioMed Research International
(65 times 10minus1molsdotLminus1) The reduction of the DPPH radicalwas measured by monitoring continuously the decrease ofabsorption at 515 nm The percentage scavenging of theDPPH radical was calculated according to the followingformula scavenging effect = 100 times [(119860DPPH minus As)119860DPPH]where As is the absorbance of the solution when the samplehas been added at a particular level and 119860DPPH is theabsorbance of the DPPH solution The percentage of theremaining DPPH was plotted against the samplestandardconcentration to obtain the amount of antioxidant necessaryto decrease the initial concentration of DPPH by 50 (EC
50)
Based on the parameter EC50 the result was expressed in
terms of 120583g of seed extract per mL of DPPH in the reactionmedium All determinations were run in triplicate
27 Anticholinesterase Activity The anticholinesterase activ-ity of the ethanolic extracts was measured as described by[18] with slight modifications Acetylcholinesterase activitywas measured using a 96-well microplate reader basedon Ellmanrsquos method [19] In this method the enzymehydrolyzes the substrate acetylthiocholine resulting in theproduction of thiocholine which reacts with 551015840-dithiobis(2-nitrobenzoic acid) (DTNB) to produce 2-nitrobenzoate-5-mercaptothiocholine and 5-thio-2-nitrobenzoate which canbe detected at 405 nm In the 96-well plates 25120583L of 15mMacetylthiocholine iodide (ATCI) in water 125 120583L of 3mMDTNB in 50mM Tris-HCl buffer containing 01M NaCland 002M MgCl
2sdot6H2O 50 120583L of 50mM Tris-HCl buffer
containing 01 BSA and 25 120583L of plant extract sample(5mgsdotmLminus1 in MeOH diluted ten times with 50mM Tris-HCl buffer to give a concentration of 05mgsdotmLminus1) wereadded and the absorbance was measured at 405 nm every30 s for three times Then 25120583L of 022UsdotmLminus1 of acetyl-cholinesterase fromelectric eel was added and the absorbancewas again read every 45 s for eight times Any increase inabsorbance due to the spontaneous hydrolysis of the substratewas corrected by subtracting the rate of the reaction beforethe addition of the enzyme from the rate of the enzymereaction The percentage of inhibition was calculated bycomparison of sample rates to a blank
28 Hemolytic Activity Assay The test was performed in15mL microtubes following the method described by [20]with some modifications Firstly a twofold serial dilution ofeach seed ethanolic extract was prepared with 09 NaClranging from 1000 to 19120583gsdotmLminus1 and reservedThen 100120583Lof a 1 red blood cells (A B and O human blood types orrabbit blood) suspension was added to a newmicrotube con-taining 900 120583L of each seed extract dilution which was thentaken to incubator at 37∘C for 1 h After that the tubes werecentrifuged at 3000timesg for 5min The supernatant (200120583L)was placed in a 96-well plate and led to a microplate reader(Epoch BioTek Vermont USA) to measure the absorbanceat 540 nm The cell suspensions of each human blood typeor those of rabbits (100120583L) were mixed with distilled wateror 09 NaCl (900120583L) to obtain the absorbances for 100and 0 of cell lysis respectivelyThe percentage of hemolysiswas calculated as follows hemolysis = AbstestAbs119901119888times 100
where Abstest = Abs540 of the 1 cell suspension treated withsample test and Abs
119901119888= Abs
540of the 1 cell suspension
treated with distilled water To calculate the relation betweenpercentage of hemolysis and seed extract concentration thehemolytic activity was expressed as the lowest seed extractconcentration (120583gsdotmLminus1) capable of causing hemolysis ge20All determinations were run in triplicate
29 Infrared Spectroscopy The infrared spectrum wasrecorded from KBr discs on a FTIR Perkin Elmer model16 PC spectrophotometer (PerkinElmer Waltham USA)Absorptionmaxima (Egraveđmax) were reported in wavenumbers(cmminus1) The spectra were used to determine organic groupsindicative of the main classes of secondary metabolites
210 Phytochemical Study The ethanolic seed extracts werescreened for phytochemical compounds such as phenols andtannins (reaction with ferric chloride) leucoanthocyanidins(heat treatment followed by alkalinization and acidification ofsample) flavonoids and xanthones (reaction of magnesiumgranules with hydrochloric acid) steroids and triterpenes(extraction with chloroform acetic anhydride and sulfuricacid) saponins (foam production after water solubilizationand stirring) and alkaloids (precipitation with Hager Mayerand Dragendorff reagents) [21] These analyses were basedon visual observation of color modification or precipitateformation after addition of specific reagents
3 Results and Discussion
The ethanolic seed extracts did not show activity againstthe Gram (minus) organisms E aerogenes K pneumonia and Paeruginosa nor against the yeastsC albicansC krusei andCtropicalis MIC values obtained against the Gram (+) organ-isms S aureus and B subtilis and the Gram (minus) organism Scholeraesuis are shown in Table 1 Among all samples testedand microorganisms used the seed extract of S obtusifoliapresented the lowest MIC value (496mgsdotmLminus1) against thepathogenic bacteria S aureus However the speciesT gardne-riana showed the broadest spectrum of activity being activeagainst B subtilis S choleraesuis and S aureuswithMIC val-ues of 188 1376 and 1115mgsdotmLminus1 respectively Althoughthere are a great number of studies reporting antimicrobialactivity of plant parts like leaves roots and stems antimi-crobial screenings using specifically seed extracts are veryrare The reason for that is not easy to explain since seedsare admittedly a rich source of compounds involved inplant defense [22] A good example is the seed extract ofBixa orellana that has been described to have antimicrobialactivity against six strains of bacteria among these are Bsubtilis S aureus and P aeruginosa and against the yeastC albicans [23] Likewise guarana (Paullinia cupana) seedextracts presented strong antimicrobial activity against a hugenumber of damaging fungi and bacteria [24] As to thepotency of the antibacterial activities observed these weremuch lower than those described for Syzygium jambolanumseed extracts against the same bacterial strains used in thiswork (MIC values ranging from 006 to 025mgsdotmLminus1) even
BioMed Research International 7
when compared to the best result observed as describedabove for S obtusifolia (MIC 496mgsdotmLminus1) In additionthe S jambolanum seed extracts were also effective againstdamaging-fungi strains The low potency of the ethanolicseed extracts could be due to the chemical composition of thesamples with low concentration of antimicrobial substancesIn fact the results of phytochemical study revealed that allseed extracts with activity against bacteria showed in theircomposition classes of secondary metabolites widely knownas efficient antimicrobial compounds (alkaloids flavonesflavonoids phenols saponins and tannins) [25] A fraction-ation of these samples in a polarity gradient could improvetheir activities Therefore the absence or low activity of theseed extracts could be a problem of concentration
The hemolytic activity assay was performed with theseed extracts that were active against the microorganismsused Among six species tested only M urundeuva causedhemolysis in all blood types (1 erythrocytes suspension ofA B and O human blood types and rabbit blood) employedbeing the lowest concentration tested (19120583gsdotmLminus1) capableof causing more than 20 hemolysis These results show thatalthough the antimicrobial extracts are not very potent theyare more specific to bacterial cells except for M urundeuvaseed extract It is well reported that some classes of plantsecondarymetabolites are potent hemolytic compounds [26]such as phenols that were also detected forMurundeuva seedextract in the phytochemical study
The results for antioxidant capacity are also shown inTable 1 Only five seed extracts were able to capture free rad-icals in the concentrations used according to DPPHmethodthese were Hymenaea courbaril (EC
5024795 120583gsdotmLminus1) L
rigida (48751120583gsdotmLminus1) L tomentosa (21672 120583gsdotmLminus1) andT gardneriana (6973120583gsdotmLminus1) The L tomentosa and Tgardneriana seed extracts were more potent than the positivecontrol vitamin C (EC
5026027120583gsdotmLminus1) and the second one
was just a bit less potent than quercetin (EC505552120583gsdotmLminus1)
one of the most potent antioxidant molecules A previousstudy [27] showed that out of 25 plant extracts (hexanedichloromethane and methanol) only 12 presented someradical scavenging activity and the most potent activityshowed an IC
50value of 11199 120583gsdotmLminus1This promising result
for T gardneriana could be attributed to components such astannins flavones flavonoids and especially phenols [28 29]Further steps of fractionation and purification may show themolecule(s) responsible for this excellent activity
Concerning the anticholinesterase activity (Table 1)among the 21 ethanolic extracts tested 11 presented inhibitionof the cholinesterase enzyme T gardneriana P platycephalaand C detersus extracts presented the strongest activitywith inhibition values of respectively 767 715 and 919at the final concentration of 500120583gmL These values aresignificant when compared to those obtained in previousstudies [18] which have shown inhibition values of 17 plantextracts ranging from 9 to 81 using a single concentrationof 1mgmL Several plant-derived drugs such as galantamineand rivastigmine are used to inhibit AChE and thus increaseendogenous levels of acetylcholine to improve cholinergictransmission [30] Many studies described alkaloids as the
So
Pp
Lr
Tg
Hc
Lt
Tran
smitt
ance
()
4000 3500 3000 2500 2000 1500 1000 500
Wavenumber (cmminus1)
Pp
Lr
Tg
Hc
Lt
Figure 1 Infrared absorption spectra of some ethanolic extractstested in this study So Senna obtusifolia Pp Parkia platycephala LrLicania rigida Tg Triplaris gardneriana Hc Hymenaea courbarilLt Licania tomentosa
main compounds capable of inhibiting AChE enzyme [31]but recent studies have pointed out several new classes ofsecondarymetabolites as potent inhibitors such as flavonoids[32] flavones [33] and xanthones [34] as well as steroids ter-penoids oils and other phenolic compounds [35] Thus thephytochemical analysis data in the present study corroboratepreviousmolecular findings since polyphenols were detectedin all extracts Additionally the extract of T gardnerianashowed the presence of chalcones and aurones Thereforefurther studies should be conducted to assess the potential ofthese extracts and their respective molecules to inhibit AChEenzyme and thus offer a potential to fight neurodegenerativediseases such as Alzheimerrsquos
After running all the assays described above infraredspectroscopywith themost promising ethanolic seed extractswas also performed (S obtusifolia P platycephala L rigida Ltomentosa and T gardneriana) to confirm the results of phy-tochemical qualitative studyThe phytochemical studies withthese ethanolic extracts showed the presence of tannins andflavonoids which were confirmed by infrared spectroscopy(Figure 1)The spectra obtained between 1800 and 800 cmminus1are present at the fingerprint zone of aromatic compoundswhich can be found in tannins polyphenols and flavonoids[36] Further steps of fractionation andpurificationmay showthe molecule(s) responsible for this excellent activity
4 Conclusions
This study revealed antibacterial antioxidant and anti-cholinesterase activities in some seed ethanolic extracts ofBrazilian semiarid region plants emphasizing the antioxidantactivity presented by T gardneriana seeds This showeda potent capacity to catch free radicals by DPPH assayThis promising result could be attributed to its composition
8 BioMed Research International
featuring phenols tannins flavones and flavonoidsNewphy-tochemical and molecular studies are underway to identifythe bioactive compound(s) responsible for this activity
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgment
The authors thank theMinisterio da Saude Departamento deCiencias e Tecnologia (DECIT) Secretaria de Ciencias Tec-nologia e Insumos estrategicos (SCTIE) Conselho Nacionalde Desenvolvimento Cientıfico e Tecnologico (CNPq) andFundacao Cearense deApoioCientıfico e Tecnologico (FUN-CAP) for financial support of this research
References
[1] C Katiyar A Gupta S Kanjilal and S Katiyar ldquoDrug discoveryfromplant sources an integrated approachrdquoAyu vol 33 pp 10ndash19 2012
[2] P S Luize T S Tiuman L G Morello et al ldquoEffects of medic-inal plant extracts on growth of Leishmania (L) amazonensisand Trypanosoma cruzirdquo Brazilian Journal of PharmaceuticalSciences vol 41 no 1 pp 85ndash94 2005
[3] A E Edris ldquoAnti-cancer properties of Nigella spp essential oilsand their major constituents thymoquinone and 120573-ElemenerdquoCurrent Clinical Pharmacology vol 4 no 1 pp 43ndash46 2009
[4] R N Bennett and R M Wallsgrove ldquoSecondary metabolites inplant defence mechanismsrdquo New Phytologist vol 127 no 4 pp617ndash633 1994
[5] R J Grayer and J B Harborne ldquoA survey of antifungal com-pounds from higher plants 1982-1993rdquo Phytochemistry vol 37no 1 pp 19ndash42 1994
[6] A E Osbourn ldquoPreformed antimicrobial compounds and plantdefense against fungal attackrdquo Plant Cell vol 8 no 10 pp 1821ndash1831 1996
[7] E V S B Sampaio ldquoVegetacao e flora da Caatingardquo APNECNIP Recife 2000
[8] C F C B R De Almeida T C De Lima E Silva E L CDe Amorim M B D S Maia and U P De AlbuquerqueldquoLife strategy and chemical composition as predictors of theselection of medicinal plants from the caatinga (NortheastBrazil)rdquo Journal of Arid Environments vol 62 no 1 pp 127ndash1422005
[9] U P de Albuquerque P M de Medeiros A L S de Almeida etal ldquoMedicinal plants of the caatinga (semi-arid) vegetation ofNE Brazil a quantitative approachrdquo Journal of Ethnopharmacol-ogy vol 114 no 3 pp 325ndash354 2007
[10] U P de Albuquerque V A da Silva M D C Cabral NLeal Alencar and L D H C Andrade ldquoComparisons betweenthe use of medicinal plants in indigenous and rural caatinga(dryland) communities in NE Brazilrdquo Boletin Latinoamericanoy del Caribe de Plantas Medicinales y Aromaticas vol 7 no 3pp 156ndash170 2008
[11] T M Souza D F Farias B M Soares et al ldquoToxicity ofBrazilian plant seed extracts to two strains of Aedes aegypti(Diptera Culicidae) andNontargetAnimalsrdquo Journal ofMedicalEntomology vol 48 no 4 pp 846ndash851 2011
[12] P M P Ferreira D F Farias M P Viana et al ldquoStudy of theantiproliferative potential of seed extracts from NortheasternBrazilian plantsrdquo Anais da Academia Brasileira de Ciencias vol83 no 3 pp 1045ndash1058 2011
[13] F J AMatosM EOMatosM P SousaM I LMachado andA A Craveiro Constituintes Quımicos Ativos e PropriedadesBiologicas de Plantas Medicinais Brasileiras UFC FortalezaBrazil 1st edition 2004
[14] F J A Matos Plantas Medicinais Guia de Selecao e Empregode Plantas Usadas em Fitoterapia no Nordeste do Brasil IUFortaleza Brazil 2nd edition 2000
[15] G N Maia Caatinga Arvores e Arbustos e Suas Utilidades D ampZ Computacao Grafica e Editora Sao Paulo Brazil 1st edition2004
[16] M Chandrasekaran and V Venkatesalu ldquoAntibacterial andantifungal activity of Syzygium jambolanum seedsrdquo Journal ofEthnopharmacology vol 91 no 1 pp 105ndash108 2004
[17] B Yepez M Espinosa S Lopez and G Bolaos ldquoProducingantioxidant fractions from herbaceousmatrices by supercriticalfluid extractionrdquo Fluid Phase Equilibria vol 194ndash197 pp 879ndash884 2002
[18] E M Cardoso-Lopes J A Maier M R Da Silva et alldquoAlkaloids from stems of esenbeckia leiocarpa Engl (Rutaceae)as potential treatment for alzheimer diseaserdquoMolecules vol 15no 12 pp 9205ndash9213 2010
[19] G L Ellman K D Courtney V Andres Jr and R MFeatherstone ldquoA new and rapid colorimetric determination ofacetylcholinesterase activityrdquo Biochemical Pharmacology vol 7no 2 pp 88ndash95 1961
[20] A W Bernheimer ldquoAssay of hemolytic toxinsrdquo Methods inEnzymology vol 165 pp 213ndash217 1988
[21] F J A Matos Introducao A Fitoquımica Experimental EditoraUFC Ceara Brazil 1st edition 2009
[22] J Xavier-Filho ldquoSementes e suas defesas contra insetos ProjetoMultinacional de Biotecnologia e Alimentosrdquo Organizacao dosEstados Americanos (OEA) pp1ndash31 1993
[23] T C Fleischer E P K Ameade M L K Mensah and I KSawer ldquoAntimicrobial activity of the leaves and seeds of Bixaorellanardquo Fitoterapia vol 74 no 1-2 pp 136ndash138 2003
[24] L Majhenic M Skerget and Z Knez ldquoAntioxidant and antimi-crobial activity of guarana seed extractsrdquo Food Chemistry vol104 no 3 pp 1258ndash1268 2007
[25] MM Cowan ldquoPlant products as antimicrobial agentsrdquoClinicalMicrobiology Reviews vol 12 no 4 pp 564ndash582 1999
[26] A Rafat K Philip and SMuniandy ldquoAntioxidant potential andcontent of phenolic compounds in ethanolic extracts of selectedparts of Andrographis paniculatardquo Journal of Medicinal PlantResearch vol 4 no 3 pp 197ndash202 2010
[27] O M Mosquera Y M Correa D C Buitrago and J NinoldquoAntioxidant activity of twenty five plants from ColombianbiodiversityrdquoMemorias do Instituto Oswaldo Cruz vol 102 no5 pp 631ndash634 2007
[28] N Cotelle J-L Bernier J-P Catteau J Pommery J-C Wal-let and E M Gaydou ldquoAntioxidant properties of hydroxy-flavonesrdquo Free Radical Biology and Medicine vol 20 no 1 pp35ndash43 1996
[29] H Oh D-H Kim J-H Cho and Y-C Kim ldquoHepatoprotectiveand free radical scavenging activities of phenolic petrosinsand flavonoids isolated from Equisetum arvenserdquo Journal ofEthnopharmacology vol 95 no 2-3 pp 421ndash424 2004
BioMed Research International 9
[30] S Lopez J Bastida F Viladomat and C Codina ldquoAcetyl-cholinesterase inhibitory activity of some Amaryllidaceae alka-loids and Narcissus extractsrdquo Life Sciences vol 71 no 21 pp2521ndash2529 2002
[31] M G Ortega A M Agnese and J L Cabrera ldquoAnti-cholinesterase activity in an alkaloid extract ofHuperzia sauru-rusrdquo Phytomedicine vol 11 no 6 pp 539ndash543 2004
[32] B J Hillhouse D S Ming C J French and G H NTowers ldquoAcetylcholine esterase inhibitors in Rhodiola roseardquoPharmaceutical Biology vol 42 no 1 pp 68ndash72 2004
[33] P Sawasdee C Sabphon D Sitthiwongwanit and U KokpolldquoAnticholinesterase activity of 7-methoxyflavones isolated fromKaempferia parviflorardquo Phytotherapy Research vol 23 no 12pp 1792ndash1794 2009
[34] 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
[35] H-F Ji andH-Y Zhang ldquoMultipotent natural agents to combatAlzheimerrsquos disease Functional spectrum and structural fea-turesrdquo Acta Pharmacologica Sinica vol 29 no 2 pp 143ndash1512008
[36] K Fernandez and E Agosin ldquoQuantitative analysis of red winetannins using Fourier-transform mid-infrared spectrometryrdquoJournal of Agricultural and Food Chemistry vol 55 no 18 pp7294ndash7300 2007
Submit your manuscripts athttpwwwhindawicom
PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom
Volume 2014
ToxinsJournal of
VaccinesJournal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
AntibioticsInternational Journal of
ToxicologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Drug DeliveryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in Pharmacological Sciences
Tropical MedicineJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Medicinal ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
AddictionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Autoimmune Diseases
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anesthesiology Research and Practice
ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Pharmaceutics
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
BioMed Research International 7
when compared to the best result observed as describedabove for S obtusifolia (MIC 496mgsdotmLminus1) In additionthe S jambolanum seed extracts were also effective againstdamaging-fungi strains The low potency of the ethanolicseed extracts could be due to the chemical composition of thesamples with low concentration of antimicrobial substancesIn fact the results of phytochemical study revealed that allseed extracts with activity against bacteria showed in theircomposition classes of secondary metabolites widely knownas efficient antimicrobial compounds (alkaloids flavonesflavonoids phenols saponins and tannins) [25] A fraction-ation of these samples in a polarity gradient could improvetheir activities Therefore the absence or low activity of theseed extracts could be a problem of concentration
The hemolytic activity assay was performed with theseed extracts that were active against the microorganismsused Among six species tested only M urundeuva causedhemolysis in all blood types (1 erythrocytes suspension ofA B and O human blood types and rabbit blood) employedbeing the lowest concentration tested (19120583gsdotmLminus1) capableof causing more than 20 hemolysis These results show thatalthough the antimicrobial extracts are not very potent theyare more specific to bacterial cells except for M urundeuvaseed extract It is well reported that some classes of plantsecondarymetabolites are potent hemolytic compounds [26]such as phenols that were also detected forMurundeuva seedextract in the phytochemical study
The results for antioxidant capacity are also shown inTable 1 Only five seed extracts were able to capture free rad-icals in the concentrations used according to DPPHmethodthese were Hymenaea courbaril (EC
5024795 120583gsdotmLminus1) L
rigida (48751120583gsdotmLminus1) L tomentosa (21672 120583gsdotmLminus1) andT gardneriana (6973120583gsdotmLminus1) The L tomentosa and Tgardneriana seed extracts were more potent than the positivecontrol vitamin C (EC
5026027120583gsdotmLminus1) and the second one
was just a bit less potent than quercetin (EC505552120583gsdotmLminus1)
one of the most potent antioxidant molecules A previousstudy [27] showed that out of 25 plant extracts (hexanedichloromethane and methanol) only 12 presented someradical scavenging activity and the most potent activityshowed an IC
50value of 11199 120583gsdotmLminus1This promising result
for T gardneriana could be attributed to components such astannins flavones flavonoids and especially phenols [28 29]Further steps of fractionation and purification may show themolecule(s) responsible for this excellent activity
Concerning the anticholinesterase activity (Table 1)among the 21 ethanolic extracts tested 11 presented inhibitionof the cholinesterase enzyme T gardneriana P platycephalaand C detersus extracts presented the strongest activitywith inhibition values of respectively 767 715 and 919at the final concentration of 500120583gmL These values aresignificant when compared to those obtained in previousstudies [18] which have shown inhibition values of 17 plantextracts ranging from 9 to 81 using a single concentrationof 1mgmL Several plant-derived drugs such as galantamineand rivastigmine are used to inhibit AChE and thus increaseendogenous levels of acetylcholine to improve cholinergictransmission [30] Many studies described alkaloids as the
So
Pp
Lr
Tg
Hc
Lt
Tran
smitt
ance
()
4000 3500 3000 2500 2000 1500 1000 500
Wavenumber (cmminus1)
Pp
Lr
Tg
Hc
Lt
Figure 1 Infrared absorption spectra of some ethanolic extractstested in this study So Senna obtusifolia Pp Parkia platycephala LrLicania rigida Tg Triplaris gardneriana Hc Hymenaea courbarilLt Licania tomentosa
main compounds capable of inhibiting AChE enzyme [31]but recent studies have pointed out several new classes ofsecondarymetabolites as potent inhibitors such as flavonoids[32] flavones [33] and xanthones [34] as well as steroids ter-penoids oils and other phenolic compounds [35] Thus thephytochemical analysis data in the present study corroboratepreviousmolecular findings since polyphenols were detectedin all extracts Additionally the extract of T gardnerianashowed the presence of chalcones and aurones Thereforefurther studies should be conducted to assess the potential ofthese extracts and their respective molecules to inhibit AChEenzyme and thus offer a potential to fight neurodegenerativediseases such as Alzheimerrsquos
After running all the assays described above infraredspectroscopywith themost promising ethanolic seed extractswas also performed (S obtusifolia P platycephala L rigida Ltomentosa and T gardneriana) to confirm the results of phy-tochemical qualitative studyThe phytochemical studies withthese ethanolic extracts showed the presence of tannins andflavonoids which were confirmed by infrared spectroscopy(Figure 1)The spectra obtained between 1800 and 800 cmminus1are present at the fingerprint zone of aromatic compoundswhich can be found in tannins polyphenols and flavonoids[36] Further steps of fractionation andpurificationmay showthe molecule(s) responsible for this excellent activity
4 Conclusions
This study revealed antibacterial antioxidant and anti-cholinesterase activities in some seed ethanolic extracts ofBrazilian semiarid region plants emphasizing the antioxidantactivity presented by T gardneriana seeds This showeda potent capacity to catch free radicals by DPPH assayThis promising result could be attributed to its composition
8 BioMed Research International
featuring phenols tannins flavones and flavonoidsNewphy-tochemical and molecular studies are underway to identifythe bioactive compound(s) responsible for this activity
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgment
The authors thank theMinisterio da Saude Departamento deCiencias e Tecnologia (DECIT) Secretaria de Ciencias Tec-nologia e Insumos estrategicos (SCTIE) Conselho Nacionalde Desenvolvimento Cientıfico e Tecnologico (CNPq) andFundacao Cearense deApoioCientıfico e Tecnologico (FUN-CAP) for financial support of this research
References
[1] C Katiyar A Gupta S Kanjilal and S Katiyar ldquoDrug discoveryfromplant sources an integrated approachrdquoAyu vol 33 pp 10ndash19 2012
[2] P S Luize T S Tiuman L G Morello et al ldquoEffects of medic-inal plant extracts on growth of Leishmania (L) amazonensisand Trypanosoma cruzirdquo Brazilian Journal of PharmaceuticalSciences vol 41 no 1 pp 85ndash94 2005
[3] A E Edris ldquoAnti-cancer properties of Nigella spp essential oilsand their major constituents thymoquinone and 120573-ElemenerdquoCurrent Clinical Pharmacology vol 4 no 1 pp 43ndash46 2009
[4] R N Bennett and R M Wallsgrove ldquoSecondary metabolites inplant defence mechanismsrdquo New Phytologist vol 127 no 4 pp617ndash633 1994
[5] R J Grayer and J B Harborne ldquoA survey of antifungal com-pounds from higher plants 1982-1993rdquo Phytochemistry vol 37no 1 pp 19ndash42 1994
[6] A E Osbourn ldquoPreformed antimicrobial compounds and plantdefense against fungal attackrdquo Plant Cell vol 8 no 10 pp 1821ndash1831 1996
[7] E V S B Sampaio ldquoVegetacao e flora da Caatingardquo APNECNIP Recife 2000
[8] C F C B R De Almeida T C De Lima E Silva E L CDe Amorim M B D S Maia and U P De AlbuquerqueldquoLife strategy and chemical composition as predictors of theselection of medicinal plants from the caatinga (NortheastBrazil)rdquo Journal of Arid Environments vol 62 no 1 pp 127ndash1422005
[9] U P de Albuquerque P M de Medeiros A L S de Almeida etal ldquoMedicinal plants of the caatinga (semi-arid) vegetation ofNE Brazil a quantitative approachrdquo Journal of Ethnopharmacol-ogy vol 114 no 3 pp 325ndash354 2007
[10] U P de Albuquerque V A da Silva M D C Cabral NLeal Alencar and L D H C Andrade ldquoComparisons betweenthe use of medicinal plants in indigenous and rural caatinga(dryland) communities in NE Brazilrdquo Boletin Latinoamericanoy del Caribe de Plantas Medicinales y Aromaticas vol 7 no 3pp 156ndash170 2008
[11] T M Souza D F Farias B M Soares et al ldquoToxicity ofBrazilian plant seed extracts to two strains of Aedes aegypti(Diptera Culicidae) andNontargetAnimalsrdquo Journal ofMedicalEntomology vol 48 no 4 pp 846ndash851 2011
[12] P M P Ferreira D F Farias M P Viana et al ldquoStudy of theantiproliferative potential of seed extracts from NortheasternBrazilian plantsrdquo Anais da Academia Brasileira de Ciencias vol83 no 3 pp 1045ndash1058 2011
[13] F J AMatosM EOMatosM P SousaM I LMachado andA A Craveiro Constituintes Quımicos Ativos e PropriedadesBiologicas de Plantas Medicinais Brasileiras UFC FortalezaBrazil 1st edition 2004
[14] F J A Matos Plantas Medicinais Guia de Selecao e Empregode Plantas Usadas em Fitoterapia no Nordeste do Brasil IUFortaleza Brazil 2nd edition 2000
[15] G N Maia Caatinga Arvores e Arbustos e Suas Utilidades D ampZ Computacao Grafica e Editora Sao Paulo Brazil 1st edition2004
[16] M Chandrasekaran and V Venkatesalu ldquoAntibacterial andantifungal activity of Syzygium jambolanum seedsrdquo Journal ofEthnopharmacology vol 91 no 1 pp 105ndash108 2004
[17] B Yepez M Espinosa S Lopez and G Bolaos ldquoProducingantioxidant fractions from herbaceousmatrices by supercriticalfluid extractionrdquo Fluid Phase Equilibria vol 194ndash197 pp 879ndash884 2002
[18] E M Cardoso-Lopes J A Maier M R Da Silva et alldquoAlkaloids from stems of esenbeckia leiocarpa Engl (Rutaceae)as potential treatment for alzheimer diseaserdquoMolecules vol 15no 12 pp 9205ndash9213 2010
[19] G L Ellman K D Courtney V Andres Jr and R MFeatherstone ldquoA new and rapid colorimetric determination ofacetylcholinesterase activityrdquo Biochemical Pharmacology vol 7no 2 pp 88ndash95 1961
[20] A W Bernheimer ldquoAssay of hemolytic toxinsrdquo Methods inEnzymology vol 165 pp 213ndash217 1988
[21] F J A Matos Introducao A Fitoquımica Experimental EditoraUFC Ceara Brazil 1st edition 2009
[22] J Xavier-Filho ldquoSementes e suas defesas contra insetos ProjetoMultinacional de Biotecnologia e Alimentosrdquo Organizacao dosEstados Americanos (OEA) pp1ndash31 1993
[23] T C Fleischer E P K Ameade M L K Mensah and I KSawer ldquoAntimicrobial activity of the leaves and seeds of Bixaorellanardquo Fitoterapia vol 74 no 1-2 pp 136ndash138 2003
[24] L Majhenic M Skerget and Z Knez ldquoAntioxidant and antimi-crobial activity of guarana seed extractsrdquo Food Chemistry vol104 no 3 pp 1258ndash1268 2007
[25] MM Cowan ldquoPlant products as antimicrobial agentsrdquoClinicalMicrobiology Reviews vol 12 no 4 pp 564ndash582 1999
[26] A Rafat K Philip and SMuniandy ldquoAntioxidant potential andcontent of phenolic compounds in ethanolic extracts of selectedparts of Andrographis paniculatardquo Journal of Medicinal PlantResearch vol 4 no 3 pp 197ndash202 2010
[27] O M Mosquera Y M Correa D C Buitrago and J NinoldquoAntioxidant activity of twenty five plants from ColombianbiodiversityrdquoMemorias do Instituto Oswaldo Cruz vol 102 no5 pp 631ndash634 2007
[28] N Cotelle J-L Bernier J-P Catteau J Pommery J-C Wal-let and E M Gaydou ldquoAntioxidant properties of hydroxy-flavonesrdquo Free Radical Biology and Medicine vol 20 no 1 pp35ndash43 1996
[29] H Oh D-H Kim J-H Cho and Y-C Kim ldquoHepatoprotectiveand free radical scavenging activities of phenolic petrosinsand flavonoids isolated from Equisetum arvenserdquo Journal ofEthnopharmacology vol 95 no 2-3 pp 421ndash424 2004
BioMed Research International 9
[30] S Lopez J Bastida F Viladomat and C Codina ldquoAcetyl-cholinesterase inhibitory activity of some Amaryllidaceae alka-loids and Narcissus extractsrdquo Life Sciences vol 71 no 21 pp2521ndash2529 2002
[31] M G Ortega A M Agnese and J L Cabrera ldquoAnti-cholinesterase activity in an alkaloid extract ofHuperzia sauru-rusrdquo Phytomedicine vol 11 no 6 pp 539ndash543 2004
[32] B J Hillhouse D S Ming C J French and G H NTowers ldquoAcetylcholine esterase inhibitors in Rhodiola roseardquoPharmaceutical Biology vol 42 no 1 pp 68ndash72 2004
[33] P Sawasdee C Sabphon D Sitthiwongwanit and U KokpolldquoAnticholinesterase activity of 7-methoxyflavones isolated fromKaempferia parviflorardquo Phytotherapy Research vol 23 no 12pp 1792ndash1794 2009
[34] 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
[35] H-F Ji andH-Y Zhang ldquoMultipotent natural agents to combatAlzheimerrsquos disease Functional spectrum and structural fea-turesrdquo Acta Pharmacologica Sinica vol 29 no 2 pp 143ndash1512008
[36] K Fernandez and E Agosin ldquoQuantitative analysis of red winetannins using Fourier-transform mid-infrared spectrometryrdquoJournal of Agricultural and Food Chemistry vol 55 no 18 pp7294ndash7300 2007
Submit your manuscripts athttpwwwhindawicom
PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom
Volume 2014
ToxinsJournal of
VaccinesJournal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
AntibioticsInternational Journal of
ToxicologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Drug DeliveryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in Pharmacological Sciences
Tropical MedicineJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Medicinal ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
AddictionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Autoimmune Diseases
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anesthesiology Research and Practice
ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Pharmaceutics
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
8 BioMed Research International
featuring phenols tannins flavones and flavonoidsNewphy-tochemical and molecular studies are underway to identifythe bioactive compound(s) responsible for this activity
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgment
The authors thank theMinisterio da Saude Departamento deCiencias e Tecnologia (DECIT) Secretaria de Ciencias Tec-nologia e Insumos estrategicos (SCTIE) Conselho Nacionalde Desenvolvimento Cientıfico e Tecnologico (CNPq) andFundacao Cearense deApoioCientıfico e Tecnologico (FUN-CAP) for financial support of this research
References
[1] C Katiyar A Gupta S Kanjilal and S Katiyar ldquoDrug discoveryfromplant sources an integrated approachrdquoAyu vol 33 pp 10ndash19 2012
[2] P S Luize T S Tiuman L G Morello et al ldquoEffects of medic-inal plant extracts on growth of Leishmania (L) amazonensisand Trypanosoma cruzirdquo Brazilian Journal of PharmaceuticalSciences vol 41 no 1 pp 85ndash94 2005
[3] A E Edris ldquoAnti-cancer properties of Nigella spp essential oilsand their major constituents thymoquinone and 120573-ElemenerdquoCurrent Clinical Pharmacology vol 4 no 1 pp 43ndash46 2009
[4] R N Bennett and R M Wallsgrove ldquoSecondary metabolites inplant defence mechanismsrdquo New Phytologist vol 127 no 4 pp617ndash633 1994
[5] R J Grayer and J B Harborne ldquoA survey of antifungal com-pounds from higher plants 1982-1993rdquo Phytochemistry vol 37no 1 pp 19ndash42 1994
[6] A E Osbourn ldquoPreformed antimicrobial compounds and plantdefense against fungal attackrdquo Plant Cell vol 8 no 10 pp 1821ndash1831 1996
[7] E V S B Sampaio ldquoVegetacao e flora da Caatingardquo APNECNIP Recife 2000
[8] C F C B R De Almeida T C De Lima E Silva E L CDe Amorim M B D S Maia and U P De AlbuquerqueldquoLife strategy and chemical composition as predictors of theselection of medicinal plants from the caatinga (NortheastBrazil)rdquo Journal of Arid Environments vol 62 no 1 pp 127ndash1422005
[9] U P de Albuquerque P M de Medeiros A L S de Almeida etal ldquoMedicinal plants of the caatinga (semi-arid) vegetation ofNE Brazil a quantitative approachrdquo Journal of Ethnopharmacol-ogy vol 114 no 3 pp 325ndash354 2007
[10] U P de Albuquerque V A da Silva M D C Cabral NLeal Alencar and L D H C Andrade ldquoComparisons betweenthe use of medicinal plants in indigenous and rural caatinga(dryland) communities in NE Brazilrdquo Boletin Latinoamericanoy del Caribe de Plantas Medicinales y Aromaticas vol 7 no 3pp 156ndash170 2008
[11] T M Souza D F Farias B M Soares et al ldquoToxicity ofBrazilian plant seed extracts to two strains of Aedes aegypti(Diptera Culicidae) andNontargetAnimalsrdquo Journal ofMedicalEntomology vol 48 no 4 pp 846ndash851 2011
[12] P M P Ferreira D F Farias M P Viana et al ldquoStudy of theantiproliferative potential of seed extracts from NortheasternBrazilian plantsrdquo Anais da Academia Brasileira de Ciencias vol83 no 3 pp 1045ndash1058 2011
[13] F J AMatosM EOMatosM P SousaM I LMachado andA A Craveiro Constituintes Quımicos Ativos e PropriedadesBiologicas de Plantas Medicinais Brasileiras UFC FortalezaBrazil 1st edition 2004
[14] F J A Matos Plantas Medicinais Guia de Selecao e Empregode Plantas Usadas em Fitoterapia no Nordeste do Brasil IUFortaleza Brazil 2nd edition 2000
[15] G N Maia Caatinga Arvores e Arbustos e Suas Utilidades D ampZ Computacao Grafica e Editora Sao Paulo Brazil 1st edition2004
[16] M Chandrasekaran and V Venkatesalu ldquoAntibacterial andantifungal activity of Syzygium jambolanum seedsrdquo Journal ofEthnopharmacology vol 91 no 1 pp 105ndash108 2004
[17] B Yepez M Espinosa S Lopez and G Bolaos ldquoProducingantioxidant fractions from herbaceousmatrices by supercriticalfluid extractionrdquo Fluid Phase Equilibria vol 194ndash197 pp 879ndash884 2002
[18] E M Cardoso-Lopes J A Maier M R Da Silva et alldquoAlkaloids from stems of esenbeckia leiocarpa Engl (Rutaceae)as potential treatment for alzheimer diseaserdquoMolecules vol 15no 12 pp 9205ndash9213 2010
[19] G L Ellman K D Courtney V Andres Jr and R MFeatherstone ldquoA new and rapid colorimetric determination ofacetylcholinesterase activityrdquo Biochemical Pharmacology vol 7no 2 pp 88ndash95 1961
[20] A W Bernheimer ldquoAssay of hemolytic toxinsrdquo Methods inEnzymology vol 165 pp 213ndash217 1988
[21] F J A Matos Introducao A Fitoquımica Experimental EditoraUFC Ceara Brazil 1st edition 2009
[22] J Xavier-Filho ldquoSementes e suas defesas contra insetos ProjetoMultinacional de Biotecnologia e Alimentosrdquo Organizacao dosEstados Americanos (OEA) pp1ndash31 1993
[23] T C Fleischer E P K Ameade M L K Mensah and I KSawer ldquoAntimicrobial activity of the leaves and seeds of Bixaorellanardquo Fitoterapia vol 74 no 1-2 pp 136ndash138 2003
[24] L Majhenic M Skerget and Z Knez ldquoAntioxidant and antimi-crobial activity of guarana seed extractsrdquo Food Chemistry vol104 no 3 pp 1258ndash1268 2007
[25] MM Cowan ldquoPlant products as antimicrobial agentsrdquoClinicalMicrobiology Reviews vol 12 no 4 pp 564ndash582 1999
[26] A Rafat K Philip and SMuniandy ldquoAntioxidant potential andcontent of phenolic compounds in ethanolic extracts of selectedparts of Andrographis paniculatardquo Journal of Medicinal PlantResearch vol 4 no 3 pp 197ndash202 2010
[27] O M Mosquera Y M Correa D C Buitrago and J NinoldquoAntioxidant activity of twenty five plants from ColombianbiodiversityrdquoMemorias do Instituto Oswaldo Cruz vol 102 no5 pp 631ndash634 2007
[28] N Cotelle J-L Bernier J-P Catteau J Pommery J-C Wal-let and E M Gaydou ldquoAntioxidant properties of hydroxy-flavonesrdquo Free Radical Biology and Medicine vol 20 no 1 pp35ndash43 1996
[29] H Oh D-H Kim J-H Cho and Y-C Kim ldquoHepatoprotectiveand free radical scavenging activities of phenolic petrosinsand flavonoids isolated from Equisetum arvenserdquo Journal ofEthnopharmacology vol 95 no 2-3 pp 421ndash424 2004
BioMed Research International 9
[30] S Lopez J Bastida F Viladomat and C Codina ldquoAcetyl-cholinesterase inhibitory activity of some Amaryllidaceae alka-loids and Narcissus extractsrdquo Life Sciences vol 71 no 21 pp2521ndash2529 2002
[31] M G Ortega A M Agnese and J L Cabrera ldquoAnti-cholinesterase activity in an alkaloid extract ofHuperzia sauru-rusrdquo Phytomedicine vol 11 no 6 pp 539ndash543 2004
[32] B J Hillhouse D S Ming C J French and G H NTowers ldquoAcetylcholine esterase inhibitors in Rhodiola roseardquoPharmaceutical Biology vol 42 no 1 pp 68ndash72 2004
[33] P Sawasdee C Sabphon D Sitthiwongwanit and U KokpolldquoAnticholinesterase activity of 7-methoxyflavones isolated fromKaempferia parviflorardquo Phytotherapy Research vol 23 no 12pp 1792ndash1794 2009
[34] 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
[35] H-F Ji andH-Y Zhang ldquoMultipotent natural agents to combatAlzheimerrsquos disease Functional spectrum and structural fea-turesrdquo Acta Pharmacologica Sinica vol 29 no 2 pp 143ndash1512008
[36] K Fernandez and E Agosin ldquoQuantitative analysis of red winetannins using Fourier-transform mid-infrared spectrometryrdquoJournal of Agricultural and Food Chemistry vol 55 no 18 pp7294ndash7300 2007
Submit your manuscripts athttpwwwhindawicom
PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom
Volume 2014
ToxinsJournal of
VaccinesJournal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
AntibioticsInternational Journal of
ToxicologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Drug DeliveryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in Pharmacological Sciences
Tropical MedicineJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Medicinal ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
AddictionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Autoimmune Diseases
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anesthesiology Research and Practice
ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Pharmaceutics
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
BioMed Research International 9
[30] S Lopez J Bastida F Viladomat and C Codina ldquoAcetyl-cholinesterase inhibitory activity of some Amaryllidaceae alka-loids and Narcissus extractsrdquo Life Sciences vol 71 no 21 pp2521ndash2529 2002
[31] M G Ortega A M Agnese and J L Cabrera ldquoAnti-cholinesterase activity in an alkaloid extract ofHuperzia sauru-rusrdquo Phytomedicine vol 11 no 6 pp 539ndash543 2004
[32] B J Hillhouse D S Ming C J French and G H NTowers ldquoAcetylcholine esterase inhibitors in Rhodiola roseardquoPharmaceutical Biology vol 42 no 1 pp 68ndash72 2004
[33] P Sawasdee C Sabphon D Sitthiwongwanit and U KokpolldquoAnticholinesterase activity of 7-methoxyflavones isolated fromKaempferia parviflorardquo Phytotherapy Research vol 23 no 12pp 1792ndash1794 2009
[34] 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
[35] H-F Ji andH-Y Zhang ldquoMultipotent natural agents to combatAlzheimerrsquos disease Functional spectrum and structural fea-turesrdquo Acta Pharmacologica Sinica vol 29 no 2 pp 143ndash1512008
[36] K Fernandez and E Agosin ldquoQuantitative analysis of red winetannins using Fourier-transform mid-infrared spectrometryrdquoJournal of Agricultural and Food Chemistry vol 55 no 18 pp7294ndash7300 2007
Submit your manuscripts athttpwwwhindawicom
PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom
Volume 2014
ToxinsJournal of
VaccinesJournal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
AntibioticsInternational Journal of
ToxicologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Drug DeliveryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in Pharmacological Sciences
Tropical MedicineJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Medicinal ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
AddictionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Autoimmune Diseases
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anesthesiology Research and Practice
ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Pharmaceutics
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Submit your manuscripts athttpwwwhindawicom
PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom
Volume 2014
ToxinsJournal of
VaccinesJournal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
AntibioticsInternational Journal of
ToxicologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Drug DeliveryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in Pharmacological Sciences
Tropical MedicineJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Medicinal ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
AddictionJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Autoimmune Diseases
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Anesthesiology Research and Practice
ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Pharmaceutics
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of