Hindawi Publishing CorporationJournal of ChemistryVolume 2013 Article ID 717232 6 pageshttpdxdoiorg1011552013717232

Research ArticleAcetylcholinesterase and Butyrylcholinesterase InhibitoryActivities of 120573-Carboline and Quinoline Alkaloids Derivativesfrom the Plants of Genus Peganum

Ting Zhao1 Ke-min Ding1 Lei Zhang1 Xue-mei Cheng123

Chang-hong Wang123 and Zheng-tao Wang123

1 Institute of Chinese Materia Medica Shanghai University of Traditional Chinese Medicine1200 Cailun Road Zhangjiang Hi-Tech Park Shanghai 201210 China

2The MOE Key Laboratory for Standardization of Chinese Medicines and The SATCM Key Laboratory forNew Resources and Quality Evaluation of Chinese Medicines Shanghai 201210 China

3 Shanghai RampD Center for Standardization of Chinese Medicines Shanghai 201210 China

Correspondence should be addressed toChang-hong Wang wchcxmhotmailcom and Zheng-tao Wang wangzhthotmailcom

Received 29 June 2012 Accepted 29 January 2013

Academic Editor A Hamid A Hadi

Copyright copy 2013 Ting Zhao et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

It was reported that the main chemical constituents in plants of genus Peganum were a serial of 120573-carboline and quinolinealkaloids These alkaloids were quantitatively assessed for selective inhibitory activities on acetylcholinesterase (AChE) andbutyrylcholinesterase (BChE) by in vitro Ellman method The results indicated that harmane was the most potent selective AChEinhibitor with an IC

50of 711 plusmn 200 120583M and AChE selectivity index (SI IC

50of BChEIC

50of AChE) of 1082 Vasicine was the

most potent BChE inhibitor with feature of dual AChEBChE inhibitory activity with an IC50

versus AChEBChE of 1368 plusmn125260 plusmn 147120583M and AChE SI of 019 By analyzing and comparing the IC

50and SI of those chemicals it was indicated that the

120573-carboline alkaloids displayed more potent AChE inhibition but less BChE inhibition than quinoline alkaloidsThe substituent atthe C7 position of the 120573-carboline alkaloids and C3 and C9 positions of quinoline alkaloids played a critical role in AChE or BChEinhibitionThe potent inhibition suggested that those alkaloids may be used as candidates for treatment of Alzheimerrsquos diseaseTheanalysis of the quantitative structure-activity relationship of those compounds investigated might provide guidance for the designand synthesis of AChE and BChE inhibitors

1 Introduction

Alzheimerrsquos disease (AD) was characterized by dementia thattypically begins with subtle recognition failure and poormemory It slowly becomes more severe and eventuallyincapacitating The cholinergic system seemed particularlysusceptible to synapse loss especially in cortical regionsassociated with memory and executive function [1] Recentstudies showed that the main cause of the loss of cognitivefunctions in AD patients was a continuous decline of thecholinergic neurotransmission in cortical and other regionsof the human brain [2] Acetylcholinesterase (AChE) andbutyrylcholinesterase (BChE) are hydrolytic enzymes thatact on acetylcholine (ACh) to terminate its actions in the

synaptic cleft by cleaving the neurotransmitter to choline andacetate Both enzymes are present in the brain and detected inneurofibrillary tangles and neuritic plaques It was suggestedthat AChE predominates in the healthy brain with BChEconsidered to play aminor role in regulating brainACh levelsHowever BChE activity progressively increases in patientswithADwhileAChE activity remains unchanged or declinesBoth enzymes therefore represent legitimate therapeutictargets for ameliorating the cholinergic deficit consideredto be responsible for the declines in cognitive behavioraland global functioning characteristics of AD [3] Despite theunknown etiology of AD those findings supported that it wasneeded to control the activity of the cholinesterase (ChE) atdifferent stages of AD progression One of the most effective

2 Journal of Chemistry

treatment strategies was suggested to restrain cholinergicfunction and elevate ACh level through inhibiting AChE andBChETherefore AChE and BChE inhibitors were developedfor the treatment of AD [4 5]

The history of drug discovery showed that plants wererich sources of new active compounds and many syntheticdrugs owed their origin to plant-based medicine The genusPeganum consists of six species and one subspecies and threeof them that is P harmala Linn P nigellastrum Bunge and Pmultisectum (Maxim) Bobr aremainly distributed in the aridand semiarid areas in the northwest of China [6] P harmalais a well-known and effective herbal medicine in TurkeyIran and China [6ndash8] The seeds and whole plants appear topossess medicinal properties and can be used to treat variousailments including rheumatism hypertension diabetes andasthma jaundice and as antiparasitic agents [6 9ndash11] It wasreported that themain chemical constituents in the seeds andwhole plant of genus Peganum were a serial of 120573-carbolineand quinoline alkaloids [12 13]

As part of the same project we first reported thescreening of AChE inhibition activity of seeds extracts fromgenus Peganum with rapid bioautographic assay on TLCplates [14 15] It was found that the alkaloids fraction(mainly 120573-carboline and quinoline alkaloids) showed poten-tial inhibitory effects on the AChE activity A number ofalkaloids including two new compounds nigellastrine I andnigellastrine II along with eight known alkaloids vasici-none vasicine harmine deoxyvasicinone deoxyvasicineharmaline harmol and harmane were isolated with theirstructures identified from P nigellastrum Bunge and theirAChE inhibition activity was semiquantitatively evaluatedby TLC-bioautographic assay [14] Subsequently intriguedby the interesting biological activities Boerth and Rasapallideveloped a synthetic route for nigellastrine vasicinone andluotonins [16] In addition it was also proved that the 120573-carboline alkaloids from P harmala exhibited improvementeffects on learning and memory of model dementia miceinduced by aluminum [17]

The present study was undertaken to quantificationallyevaluate the inhibitory activity of those alkaloids againstAChE and BChE by in vitro Ellman method [18 19] Fur-thermore by comparing the IC

50values and AChE selectivity

index (SI IC50

of BChEIC50

of AChE) and analyzing thestructure-activity relationship of those compounds investi-gated on selective AChE or BChE inhibition it could providea direction for the design and synthesis of new AChE andBChE inhibitors

2 Materials and Methods

21 Chemicals and Instruments The following chemicalswere obtained from Sigma-Aldrich (USA) AChE from Elec-trophorus electricus BChE from equine serum acetylth-iocholine iodide butyrylthiocholine iodide 551015840-dithiobis-bis-nitrobenzoic acid (DTNB) L-glutathione (L-GSH) har-maline harmine harmane 1234-tetrahydroharmane-3-carboxylic acid and galanthamine harmalol hydrochlo-ride dehydrate and harmol were purchased from Wako

Pure Chemical Industries Ltd (Japan) vasicine vasicinonedeoxyvasicinone and deoxyvasicine were separated andpurified in our laboratory from seeds of P harmala andnigellastrine I and nigellastrine II were separated and purifiedin our laboratory from seeds of P nigellastrum (ge98 purity)and their physical and spectral data were in good agreementwith the literature data [14] The structures of those com-pounds investigated were showed in Figure 1

Microplate reader (Power wave XS Bio-Tek InstrumentsWinooski VT USA) precise pH instrument (PB-10 sar-torius Germany) and Eyela bath SB-9 (NTT-2200 TokyoRikakikal Co Ltd) were used

22 Preparation of Solutions The enzymes AChE and BChEwere dissolved in 20mM sodium phosphate buffer (pH 76)to make the 347 unitmL stock solution and the solutionswere stored at minus80∘C before use The stock solutions ofthe test compounds (harmaline harmine harmalol harmolharmane vasicine 1234-tetrahydroharmane-3-carboxylicacid vasicinone deoxyvasicinone deoxyvasicine nigellas-trine I nigellastrine II and galanthamine) were preparedrespectively by dissolving an adequate quantity of eachcompound in 02 DMSO to obtain 20mM solutions Allthe stock solutions were diluted to a series of concentrationswith 20mM sodium phosphate buffer solution (pH 76)before each experiment DTNB-phosphate-ethanol reagentwas prepared as follows to 124mg of DTNB dissolved in120mL of 96 ethanol 80mL of distilled water and 50mLof 01mM phosphate buffer (pH 76) were added [20]

23 In Vitro Anticholinesterase Assays The AChE and BChEactivity assay was carried out using acetylthiocholine iodideand butyrylthiocholine iodide as substrates respectivelybased on a colorimetric method as described previously[18ndash20] 10 120583L of the test compounds solution in 02DMSO 79120583L of 20mM sodium phosphate buffer (pH 76)and 1 120583L enzyme preparation (with final concentrations0087 unitmL for AChE or 0035 unitmL for BChE andfinal concentrations 1 to 5001000120583Mfor compounds tested)were mixed and preincubated for 15min To the mixture10 120583L substrate solution was added (final concentrations15mM for acetylthiocholine iodide or 4mM for butyrylth-iocholine iodide) and incubated for 30min The reactionwas stopped by adding 900 120583L DTNB-phosphate-ethanolreagent The absorption was read immediately at 412 nm ona microplate reader The concentration of the test compoundrequired to inhibit AChE or BChE activity by 50 (IC

50)

was calculated using an enzyme inhibition dose responsecurve with galanthamine as a positive control and L-GSH asstandard to draw the standard curves AChE SIwas calculatedby using the following formula SI = IC

50of BChEIC

50of

AChE

24 Data Analysis The assays were conducted in triplicateand all tabulated results were expressed as means plusmn SD andthe IC

50values were calculated by Boltzmannrsquos dose response

analysis using Origin 80 software

Journal of Chemistry 3

HO

HO

N

N

N

NH

N NH

HH

H3COH3CON

NH 1

1

2345

6

78

2

345

87

6

9

9 10

11

Harmine

Harmol

Harmaline

Harmane

Harmalol

Vasicine

Deoxyvasicine DeoxyvasicinoneVasicinone

Nigellastrine IINigellastrine I

Galanthamine

O

O

O

OO

O

OH

OH

OH

NHN

N

NN

NN

N

N

NN

N N

NNN

N

N

N

N

H

HH

OH

1 2 3 4-tetrahydroharmane-3-carboxylic acid

middotN CH3

Figure 1 Structures of the compounds investigated

3 Results

The inhibitory activities of the 120573-carboline and quinolinealkaloids onAChE and BChEwere evaluated in vitro using aninhibition assay with AChE (electric eel) and BChE (equineserum) and were summarized in Table 1

First of all it was observed that the inhibition data ofreference compounds galanthamine within the limits of thedifferent enzyme source and assay experimental conditionsare in good agreement with the literature data [21]

All tested compounds exhibited some degree of activityon both AChE and BChE and their IC

50values were in

the low micromolar range for at least one enzyme with

the exception of compounds deoxyvasicine and 1234-tetrahydroharmane-3-carboxylic acid Harmane showed themost potent inhibitory activity with IC

50value of 711 plusmn

200 120583M against AChE However harmane had a lowinhibitory activity against BChE (IC

50of 7691 plusmn 157 120583M)

Harmine and harmaline significantly inhibited AChE activitywith IC

50values of 905 plusmn 108 120583M and 1058 plusmn 201 120583M

but had lower inhibitory activity against BChE with IC50

values of 7507 plusmn 129 120583M and 10139 plusmn 139 120583M respectivelyVasicine exhibited strong inhibition both on AChE andBChE activities with IC

50values of 1368 plusmn 125 and 260 plusmn

147 120583M respectively As the main metabolites of harmalineand harmine in vivo [22 23] harmalol and harmol exhibited

4 Journal of Chemistry

Table 1 Inhibitory activity (IC50) and selectivity index (SI) of thecompounds investigated against AChE and BChE

Compounds IC50 (120583M plusmn SD) SIAChE BChE

Galanthamine 118 plusmn 105 08 [21] 73 [21] 913

Harmane 711 plusmn 200 7691 plusmn 157 1082Harmine 905 plusmn 108 7507 plusmn 129 830Harmaline 1058 plusmn 201 10139 plusmn 139 958Harmol 2158 plusmn 139 821 plusmn 365 038Harmalol 2788 plusmn 113 948 plusmn 203 034Vasicine 1368 plusmn 125 260 plusmn 147 019Nigellastrine II 4046 plusmn 109 926 plusmn 147 023Nigellastrine I 7050 plusmn 114 1824 plusmn 323 026Vasicinone 37078 plusmn 107 mdashDeoxyvasicinone 29444 plusmn 147 mdashDeoxyvasicine mdash mdash1234-tetrahydroharmane-3-carboxylicacid

mdash mdash

IC50 values were determined by regression analyses and expressed as themeansplusmn SDof three replicate determinations SI is theAChE selectivity indexdefined as IC50 BChEIC50 AChE affinity ratiomdash no inhibitory activity

moderate inhibitory activity against AChE with IC50

valuesof 2788 plusmn 113 and 2158 plusmn 139 120583M and stronger inhibitoryactivity against BChE with IC

50values of 948 plusmn 203 and

821 plusmn 365 120583M Nigellastrine I and nigellastrine II hadstronger inhibitory activity against BChE with IC

50values of

1824 plusmn 323 and 926 plusmn 147 120583M but with weaker inhibitoryactivity against AChE with IC

50values of 7050 plusmn 114 and

4046 plusmn 109 120583M

4 Discussion

Results from previous studies showed that the alkaloids frac-tion containing mainly 120573-carboline and quinoline alkaloidsfrom the seeds ofP nigellastrum exhibited potential inhibitiveactivity on AchE [14] In our ongoing studies the inhibitoryactivities of those individual 120573-carboline and quinoline alka-loids separated from genus Peganum on AChE and BChEwere determined using the in vitro Ellman method Based onthe inhibitory potency and selectivity of those alkaloids onAChE and BChE the structure-activity relationship of thosealkaloids could be speculated as follows

Amongst the series of 120573-carboline alkaloids (Figure 1)varying the substituent at C7 modulated the ChE inhibitoryprofile The presence of an O-methyl substituent at C7(harmine) led to the inhibition and selection on AChEslightly reduce with IC

50of 905 plusmn 108 120583M and AChE SI

of 830 compared to harmane with IC50

of 711 plusmn 200 120583Mand AChE SI of 913 No obvious difference was observedbetween the inhibitory activity of harmane and harmine

against BChE When oxidation at C7 occurs such as harmolthe inhibition against BChE was enhanced (IC

50= 821 plusmn

365 120583M) but with reduced activity on AChE (IC50= 2158 plusmn

139 120583M)Therefore the hydroxyl substituent at C7 obviouslyreduced the selectivity for AChE inhibition According tothe above analysis C7 could be regarded as a possible activesite for 120573-carboline alkaloids and varying the substituentat C7 could significantly affect the inhibition on AChE andBChE The double bond at C3-C4 seemed to have a certaindegree of influence on the inhibitory activity against BChE asdemonstrated by the IC

50values of harmaline and harmine

As the main O-demethylation metabolites of harmaline andharmine harmalol and harmol showed moderate inhibitorypotency on AChE with IC

50values of 2788 plusmn 113 and

2158 plusmn 139 120583M and stronger inhibitory potency on BChEwith IC

50values of 948 plusmn 203 and 821 plusmn 365 120583M Namely

the selective inhibition of harmalol and harmol versus AChEdeduced compared with their parent compound harmalineand harmine On the contrary their selective inhibitionagainst BChE enhanced

Amongst the quinoline alkaloids (Figure 1) vasicine wasidentified as the potent inhibitor with IC

50values of 1368 plusmn

125 120583Mon AChE and 260 plusmn 147 120583Mon BChE respectivelySelective BChE inhibitors have already been reported toincrease the ACh levels in the brain and to also reduce theformation of abnormal amyloid Therefore the discovery ofpotent and highly selective BChE inhibitors andor of dualAChEBChE inhibitors is an actively pursued goal in ADtreatment drug discovery [21] Very interestingly althoughwith poor selectivity vasicine may be a very potential leadcompound with the feature of dual AChEBChE inhibitoryactivity

Vasicinone and deoxyvasicinone possessing a carbony-lation at C9 or dehydroxylation at C3 exhibited no muchinhibitory activity on either AChE or BChE with IC

50values

of 37078plusmn107 and 29444plusmn147 120583MonAChE and with IC50

values of above 1000120583M on BChE Those data indicated thatthe inhibitive potency of quinoline alkaloids on both AChEand BChE was affected by the substitution at C3 and C9Deoxyvasicine possessing a carbonylation reaction at C9 anddehydroxylation at C3 simultaneously showed no inhibitorypotency on both AChE and BChE with larger IC

50values

more than 1000 120583MAs seen from Table 1 nigellastrine I and nigellastrine II

(Figure 1) two dimers of quinoline (deoxyvasicinone) and 120573-carboline alkaloids (harmane or dehydroharmane) displayeda stronger activity on BChE inhibition and weaker activityon AChE inhibition When compared with deoxyvasicinonethose dimers enhanced the inhibitor potency on both AChEand BChE but more on BChE On the other hand bycomparison with harmane the two dimmers seemed to havecertain inhibitory selectivity for BChE

From the above analysis harmane harmine and har-maline the main active constituent in genus Peganum hadgood selective inhibitory activities against AChE and theoxidation of the substituent at C7 could significantly modifythe inhibitory potency and selectivity for both AChE andBChE C3 and C9 positions of quinoline alkaloids wereinferred to be the active site for both AChE and BChE

Journal of Chemistry 5

inhibitionMoreover it was found that by the polymerizationof quinoline and carboline alkaloids the inhibitory activitydeclined on AChE but increased on BChE In the lightof those findings it could be concluded that the alkaloidsfrom the plants of genus Peganum showed inhibitory activityagainst both AChE and BChE In addition those findingsmay provide some guidance for the design and synthesis orsemisynthesis of potential inhibitors on AChE or BChE

Conflict of Interests

Theauthors have declared that there is no conflict of interests

Acknowledgments

The authors gratefully acknowledge the award from the KeyProgram of Joint Funds of the National Natural ScienceFoundation of China and Xinjiang Uygur AutonomousRegion of China (no U1130303) the National Nature ScienceFoundation of China (no 81173119) the Key Project of Min-istry of Science and Technology of China (2012ZX09103201-051) and the Program for Shanghai Innovative ResearchTeam in University (2009) to Professor C-H Wang for thefinancial support of this study The authors would also liketo thank Professor Shouming Zhong for providing languagehelp during the writing of this paper

References

[1] J A Bailey and D K Lahiri ldquoA novel effect of rivastigmineon pre-synaptic proteins and neuronal viability in a neurode-generation model of fetal rat primary cortical cultures and itsimplication in Alzheimerrsquos diseaserdquo Journal of Neurochemistryvol 112 no 4 pp 843ndash853 2010

[2] D Schuster M Spetea M Music et al ldquoMorphinans andisoquinolines acetylcholinesterase inhibition pharmacophoremodeling and interaction with opioid receptorsrdquo Bioorganic ampMedicinal Chemistry vol 18 no 14 pp 5071ndash5080 2010

[3] N H Greig D K Lahiri and K Sambamurti ldquoButyryl-cholinesterase an important new target in Alzheimerrsquos diseasetherapyrdquo International Psychogeriatrics vol 14 supplement 1pp 77ndash91 2002

[4] I Orhan B Sener M I Choudhary and A Khalid ldquoAcetyl-cholinesterase and butyrylcholinesterase inhibitory activity ofsome Turkish medicinal plantsrdquo Journal of Ethnopharmacologyvol 91 no 1 pp 57ndash60 2004

[5] T Mohamed and P P N Rao ldquoDesign synthesis and evaluationof 24-disubstituted pyrimidines as cholinesterase inhibitorsrdquoBioorganic and Medicinal Chemistry Letters vol 20 no 12 pp3606ndash3609 2010

[6] X M Cheng T Zhao T Yang C H Wang S W A Blighand Z T Wang ldquoHPLC fingerprints combined with principalcomponent analysis hierarchical cluster analysis and lineardiscriminant analysis for the classification and differentiationof Peganum sp indigenous to Chinardquo Phytochemical Analysisvol 21 no 3 pp 279ndash289 2010

[7] M Kartal M L Altun and S Kurucu ldquoHPLC method for theanalysis of harmol harmalol harmine and harmaline in theseeds of Peganum harmala Lrdquo Journal of Pharmaceutical andBiomedical Analysis vol 31 no 2 pp 263ndash269 2003

[8] B Hemmateenejad A Abbaspour H Maghami R Miri andM R Panjehshahin ldquoPartial least squares-based multivariatespectral calibration method for simultaneous determination ofbeta-carboline derivatives in Peganum harmala seed extractsrdquoAnalytica Chimica Acta vol 575 no 2 pp 290ndash299 2006

[9] Chinese Pharmacopoeia Committee Drug Standards of theMinistry of Public Health of the Peoplersquos Republic of China UygurPharmaceutical Section 1998

[10] J Riba M Valle G Urbano M Yritia A Morte and M JBarbanoj ldquoHuman pharmacology of ayahuasca subjective andcardiovascular effects monoamine metabolite excretion andpharmacokineticsrdquo Journal of Pharmacology and ExperimentalTherapeutics vol 306 no 1 pp 73ndash83 2003

[11] A Tahraoui J El-Hilaly Z H Israili and B LyoussildquoEthnopharmacological survey of plants used in the tradi-tional treatment of hypertension and diabetes in south-easternMorocco (Errachidia province)rdquo Journal of Ethnopharmacologyvol 110 no 1 pp 105ndash117 2007

[12] Z L Fan and X S Yao ldquoThe research of constituents andpharmacology of the genus Peganumrdquo Journal of ShenyangCollege of Pharmacy vol 9 pp 144ndash151 1992

[13] T Zhao C H Wang and Z T Wang ldquoChemical constituentsand pharmacologic actions of genus Peganum researchadvancesrdquo Journal of International Pharmaceutical Researchvol 37 no 5 pp 333ndash345 2010

[14] X Y Zheng Z J Zhang G X Chou et al ldquoAcetylcholinesteraseinhibitive activity-guided isolation of two new alkaloids fromseeds of Peganum nigellastrum Bunge by an in vitro TLC-bioautographic assayrdquo Archives of Pharmacal Research vol 32no 9 pp 1245ndash1251 2009

[15] X Y Zheng L Zhang X M Cheng Z J Zhang C HWang and Z T Wang ldquoIdentification of acetylcholinesteraseinhibitors from seeds of plants of genus Peganum by thin-layer chromatography-bioautographyrdquo JPC-Journal of PlanarChromatography-Modern TLC vol 24 no 6 pp 470ndash474 2011

[16] J A Boerth and S Rasapalli ldquoStudies toward the total synthesisof quinazoline alkaloids vasicinone luotonin and nigellas-trinerdquo in Proceedings of the 244th ACS National Meeting ampExposition Philadelphia Pa USA August 2012 CHED-304httpabstractsacsorgchem244nmprogramviewphp

[17] J R Fu L Teng X Y Dai and F S Yu ldquoOptimization ofextraction and purification of harmala alkaloids and the effectson learning and memory of model dementia mice induced byaluminumrdquo Chinese Traditional Patent Medicine vol 33 pp975ndash979 2011

[18] G L Ellman K D Courtney V Andres and R M Feather-stone ldquoA new and rapid colorimetric determination of acetyl-cholinesterase activityrdquo Biochemical Pharmacology vol 7 no 2pp 88ndash95 1961

[19] M A Gordon D E Carpenter H W Barrett and I B WilsonldquoDetermination of the normality of cholinesterase solutionsrdquoAnalytical Biochemistry vol 85 no 2 pp 519ndash527 1978

[20] V Gorun I Proinov V Baltescu G Balaban and O BarzuldquoModified Ellman procedure for assay of cholinesterases incrude enzymatic preparationsrdquoAnalytical Biochemistry vol 86no 1 pp 324ndash326 1978

[21] B TassoMCattoONicolotti et al ldquoQuinolizidinyl derivativesof bi- and tricyclic systems as potent inhibitors of acetyl- andbutyrylcholinesterase with potential in Alzheimerrsquos diseaserdquoEuropean Journal of Medicinal Chemistry vol 46 no 6 pp2170ndash2184 2011

6 Journal of Chemistry

[22] T Zhao S S Zheng B F Zhang et al ldquoMetabolic pathways ofthe psychotropic-carboline alkaloids harmaline and harmineby liquid chromatographymass spectrometry and NMR spec-troscopyrdquo Food Chemistry vol 134 no 2 pp 1096ndash1105 2012

[23] A M Yu J R Idle K W Krausz A Kupfer and F J GonzalezldquoContribution of individual cytochrome P450 isozymes tothe O-demethylation of the psychotropic 120573-carboline alkaloidsharmaline and harminerdquo Journal of Pharmacology and Experi-mental Therapeutics vol 305 no 1 pp 315ndash322 2003

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CatalystsJournal of

2 Journal of Chemistry

treatment strategies was suggested to restrain cholinergicfunction and elevate ACh level through inhibiting AChE andBChETherefore AChE and BChE inhibitors were developedfor the treatment of AD [4 5]

The history of drug discovery showed that plants wererich sources of new active compounds and many syntheticdrugs owed their origin to plant-based medicine The genusPeganum consists of six species and one subspecies and threeof them that is P harmala Linn P nigellastrum Bunge and Pmultisectum (Maxim) Bobr aremainly distributed in the aridand semiarid areas in the northwest of China [6] P harmalais a well-known and effective herbal medicine in TurkeyIran and China [6ndash8] The seeds and whole plants appear topossess medicinal properties and can be used to treat variousailments including rheumatism hypertension diabetes andasthma jaundice and as antiparasitic agents [6 9ndash11] It wasreported that themain chemical constituents in the seeds andwhole plant of genus Peganum were a serial of 120573-carbolineand quinoline alkaloids [12 13]

As part of the same project we first reported thescreening of AChE inhibition activity of seeds extracts fromgenus Peganum with rapid bioautographic assay on TLCplates [14 15] It was found that the alkaloids fraction(mainly 120573-carboline and quinoline alkaloids) showed poten-tial inhibitory effects on the AChE activity A number ofalkaloids including two new compounds nigellastrine I andnigellastrine II along with eight known alkaloids vasici-none vasicine harmine deoxyvasicinone deoxyvasicineharmaline harmol and harmane were isolated with theirstructures identified from P nigellastrum Bunge and theirAChE inhibition activity was semiquantitatively evaluatedby TLC-bioautographic assay [14] Subsequently intriguedby the interesting biological activities Boerth and Rasapallideveloped a synthetic route for nigellastrine vasicinone andluotonins [16] In addition it was also proved that the 120573-carboline alkaloids from P harmala exhibited improvementeffects on learning and memory of model dementia miceinduced by aluminum [17]

The present study was undertaken to quantificationallyevaluate the inhibitory activity of those alkaloids againstAChE and BChE by in vitro Ellman method [18 19] Fur-thermore by comparing the IC

50values and AChE selectivity

index (SI IC50

of BChEIC50

of AChE) and analyzing thestructure-activity relationship of those compounds investi-gated on selective AChE or BChE inhibition it could providea direction for the design and synthesis of new AChE andBChE inhibitors

2 Materials and Methods

21 Chemicals and Instruments The following chemicalswere obtained from Sigma-Aldrich (USA) AChE from Elec-trophorus electricus BChE from equine serum acetylth-iocholine iodide butyrylthiocholine iodide 551015840-dithiobis-bis-nitrobenzoic acid (DTNB) L-glutathione (L-GSH) har-maline harmine harmane 1234-tetrahydroharmane-3-carboxylic acid and galanthamine harmalol hydrochlo-ride dehydrate and harmol were purchased from Wako

Pure Chemical Industries Ltd (Japan) vasicine vasicinonedeoxyvasicinone and deoxyvasicine were separated andpurified in our laboratory from seeds of P harmala andnigellastrine I and nigellastrine II were separated and purifiedin our laboratory from seeds of P nigellastrum (ge98 purity)and their physical and spectral data were in good agreementwith the literature data [14] The structures of those com-pounds investigated were showed in Figure 1

Microplate reader (Power wave XS Bio-Tek InstrumentsWinooski VT USA) precise pH instrument (PB-10 sar-torius Germany) and Eyela bath SB-9 (NTT-2200 TokyoRikakikal Co Ltd) were used

22 Preparation of Solutions The enzymes AChE and BChEwere dissolved in 20mM sodium phosphate buffer (pH 76)to make the 347 unitmL stock solution and the solutionswere stored at minus80∘C before use The stock solutions ofthe test compounds (harmaline harmine harmalol harmolharmane vasicine 1234-tetrahydroharmane-3-carboxylicacid vasicinone deoxyvasicinone deoxyvasicine nigellas-trine I nigellastrine II and galanthamine) were preparedrespectively by dissolving an adequate quantity of eachcompound in 02 DMSO to obtain 20mM solutions Allthe stock solutions were diluted to a series of concentrationswith 20mM sodium phosphate buffer solution (pH 76)before each experiment DTNB-phosphate-ethanol reagentwas prepared as follows to 124mg of DTNB dissolved in120mL of 96 ethanol 80mL of distilled water and 50mLof 01mM phosphate buffer (pH 76) were added [20]

23 In Vitro Anticholinesterase Assays The AChE and BChEactivity assay was carried out using acetylthiocholine iodideand butyrylthiocholine iodide as substrates respectivelybased on a colorimetric method as described previously[18ndash20] 10 120583L of the test compounds solution in 02DMSO 79120583L of 20mM sodium phosphate buffer (pH 76)and 1 120583L enzyme preparation (with final concentrations0087 unitmL for AChE or 0035 unitmL for BChE andfinal concentrations 1 to 5001000120583Mfor compounds tested)were mixed and preincubated for 15min To the mixture10 120583L substrate solution was added (final concentrations15mM for acetylthiocholine iodide or 4mM for butyrylth-iocholine iodide) and incubated for 30min The reactionwas stopped by adding 900 120583L DTNB-phosphate-ethanolreagent The absorption was read immediately at 412 nm ona microplate reader The concentration of the test compoundrequired to inhibit AChE or BChE activity by 50 (IC

50)

was calculated using an enzyme inhibition dose responsecurve with galanthamine as a positive control and L-GSH asstandard to draw the standard curves AChE SIwas calculatedby using the following formula SI = IC

50of BChEIC

50of

AChE

24 Data Analysis The assays were conducted in triplicateand all tabulated results were expressed as means plusmn SD andthe IC

50values were calculated by Boltzmannrsquos dose response

analysis using Origin 80 software

Journal of Chemistry 3

HO

HO

N

N

N

NH

N NH

HH

H3COH3CON

NH 1

1

2345

6

78

2

345

87

6

9

9 10

11

Harmine

Harmol

Harmaline

Harmane

Harmalol

Vasicine

Deoxyvasicine DeoxyvasicinoneVasicinone

Nigellastrine IINigellastrine I

Galanthamine

O

O

O

OO

O

OH

OH

OH

NHN

N

NN

NN

N

N

NN

N N

NNN

N

N

N

N

H

HH

OH

1 2 3 4-tetrahydroharmane-3-carboxylic acid

middotN CH3

Figure 1 Structures of the compounds investigated

3 Results

The inhibitory activities of the 120573-carboline and quinolinealkaloids onAChE and BChEwere evaluated in vitro using aninhibition assay with AChE (electric eel) and BChE (equineserum) and were summarized in Table 1

First of all it was observed that the inhibition data ofreference compounds galanthamine within the limits of thedifferent enzyme source and assay experimental conditionsare in good agreement with the literature data [21]

All tested compounds exhibited some degree of activityon both AChE and BChE and their IC

50values were in

the low micromolar range for at least one enzyme with

the exception of compounds deoxyvasicine and 1234-tetrahydroharmane-3-carboxylic acid Harmane showed themost potent inhibitory activity with IC

50value of 711 plusmn

200 120583M against AChE However harmane had a lowinhibitory activity against BChE (IC

50of 7691 plusmn 157 120583M)

Harmine and harmaline significantly inhibited AChE activitywith IC

50values of 905 plusmn 108 120583M and 1058 plusmn 201 120583M

but had lower inhibitory activity against BChE with IC50

values of 7507 plusmn 129 120583M and 10139 plusmn 139 120583M respectivelyVasicine exhibited strong inhibition both on AChE andBChE activities with IC

50values of 1368 plusmn 125 and 260 plusmn

147 120583M respectively As the main metabolites of harmalineand harmine in vivo [22 23] harmalol and harmol exhibited

4 Journal of Chemistry

Table 1 Inhibitory activity (IC50) and selectivity index (SI) of thecompounds investigated against AChE and BChE

Compounds IC50 (120583M plusmn SD) SIAChE BChE

Galanthamine 118 plusmn 105 08 [21] 73 [21] 913

Harmane 711 plusmn 200 7691 plusmn 157 1082Harmine 905 plusmn 108 7507 plusmn 129 830Harmaline 1058 plusmn 201 10139 plusmn 139 958Harmol 2158 plusmn 139 821 plusmn 365 038Harmalol 2788 plusmn 113 948 plusmn 203 034Vasicine 1368 plusmn 125 260 plusmn 147 019Nigellastrine II 4046 plusmn 109 926 plusmn 147 023Nigellastrine I 7050 plusmn 114 1824 plusmn 323 026Vasicinone 37078 plusmn 107 mdashDeoxyvasicinone 29444 plusmn 147 mdashDeoxyvasicine mdash mdash1234-tetrahydroharmane-3-carboxylicacid

mdash mdash

IC50 values were determined by regression analyses and expressed as themeansplusmn SDof three replicate determinations SI is theAChE selectivity indexdefined as IC50 BChEIC50 AChE affinity ratiomdash no inhibitory activity

moderate inhibitory activity against AChE with IC50

valuesof 2788 plusmn 113 and 2158 plusmn 139 120583M and stronger inhibitoryactivity against BChE with IC

50values of 948 plusmn 203 and

821 plusmn 365 120583M Nigellastrine I and nigellastrine II hadstronger inhibitory activity against BChE with IC

50values of

1824 plusmn 323 and 926 plusmn 147 120583M but with weaker inhibitoryactivity against AChE with IC

50values of 7050 plusmn 114 and

4046 plusmn 109 120583M

4 Discussion

Results from previous studies showed that the alkaloids frac-tion containing mainly 120573-carboline and quinoline alkaloidsfrom the seeds ofP nigellastrum exhibited potential inhibitiveactivity on AchE [14] In our ongoing studies the inhibitoryactivities of those individual 120573-carboline and quinoline alka-loids separated from genus Peganum on AChE and BChEwere determined using the in vitro Ellman method Based onthe inhibitory potency and selectivity of those alkaloids onAChE and BChE the structure-activity relationship of thosealkaloids could be speculated as follows

Amongst the series of 120573-carboline alkaloids (Figure 1)varying the substituent at C7 modulated the ChE inhibitoryprofile The presence of an O-methyl substituent at C7(harmine) led to the inhibition and selection on AChEslightly reduce with IC

50of 905 plusmn 108 120583M and AChE SI

of 830 compared to harmane with IC50

of 711 plusmn 200 120583Mand AChE SI of 913 No obvious difference was observedbetween the inhibitory activity of harmane and harmine

against BChE When oxidation at C7 occurs such as harmolthe inhibition against BChE was enhanced (IC

50= 821 plusmn

365 120583M) but with reduced activity on AChE (IC50= 2158 plusmn

139 120583M)Therefore the hydroxyl substituent at C7 obviouslyreduced the selectivity for AChE inhibition According tothe above analysis C7 could be regarded as a possible activesite for 120573-carboline alkaloids and varying the substituentat C7 could significantly affect the inhibition on AChE andBChE The double bond at C3-C4 seemed to have a certaindegree of influence on the inhibitory activity against BChE asdemonstrated by the IC

50values of harmaline and harmine

As the main O-demethylation metabolites of harmaline andharmine harmalol and harmol showed moderate inhibitorypotency on AChE with IC

50values of 2788 plusmn 113 and

2158 plusmn 139 120583M and stronger inhibitory potency on BChEwith IC

50values of 948 plusmn 203 and 821 plusmn 365 120583M Namely

the selective inhibition of harmalol and harmol versus AChEdeduced compared with their parent compound harmalineand harmine On the contrary their selective inhibitionagainst BChE enhanced

Amongst the quinoline alkaloids (Figure 1) vasicine wasidentified as the potent inhibitor with IC

50values of 1368 plusmn

125 120583Mon AChE and 260 plusmn 147 120583Mon BChE respectivelySelective BChE inhibitors have already been reported toincrease the ACh levels in the brain and to also reduce theformation of abnormal amyloid Therefore the discovery ofpotent and highly selective BChE inhibitors andor of dualAChEBChE inhibitors is an actively pursued goal in ADtreatment drug discovery [21] Very interestingly althoughwith poor selectivity vasicine may be a very potential leadcompound with the feature of dual AChEBChE inhibitoryactivity

Vasicinone and deoxyvasicinone possessing a carbony-lation at C9 or dehydroxylation at C3 exhibited no muchinhibitory activity on either AChE or BChE with IC

50values

of 37078plusmn107 and 29444plusmn147 120583MonAChE and with IC50

values of above 1000120583M on BChE Those data indicated thatthe inhibitive potency of quinoline alkaloids on both AChEand BChE was affected by the substitution at C3 and C9Deoxyvasicine possessing a carbonylation reaction at C9 anddehydroxylation at C3 simultaneously showed no inhibitorypotency on both AChE and BChE with larger IC

50values

more than 1000 120583MAs seen from Table 1 nigellastrine I and nigellastrine II

(Figure 1) two dimers of quinoline (deoxyvasicinone) and 120573-carboline alkaloids (harmane or dehydroharmane) displayeda stronger activity on BChE inhibition and weaker activityon AChE inhibition When compared with deoxyvasicinonethose dimers enhanced the inhibitor potency on both AChEand BChE but more on BChE On the other hand bycomparison with harmane the two dimmers seemed to havecertain inhibitory selectivity for BChE

From the above analysis harmane harmine and har-maline the main active constituent in genus Peganum hadgood selective inhibitory activities against AChE and theoxidation of the substituent at C7 could significantly modifythe inhibitory potency and selectivity for both AChE andBChE C3 and C9 positions of quinoline alkaloids wereinferred to be the active site for both AChE and BChE

Journal of Chemistry 5

inhibitionMoreover it was found that by the polymerizationof quinoline and carboline alkaloids the inhibitory activitydeclined on AChE but increased on BChE In the lightof those findings it could be concluded that the alkaloidsfrom the plants of genus Peganum showed inhibitory activityagainst both AChE and BChE In addition those findingsmay provide some guidance for the design and synthesis orsemisynthesis of potential inhibitors on AChE or BChE

Conflict of Interests

Theauthors have declared that there is no conflict of interests

Acknowledgments

The authors gratefully acknowledge the award from the KeyProgram of Joint Funds of the National Natural ScienceFoundation of China and Xinjiang Uygur AutonomousRegion of China (no U1130303) the National Nature ScienceFoundation of China (no 81173119) the Key Project of Min-istry of Science and Technology of China (2012ZX09103201-051) and the Program for Shanghai Innovative ResearchTeam in University (2009) to Professor C-H Wang for thefinancial support of this study The authors would also liketo thank Professor Shouming Zhong for providing languagehelp during the writing of this paper

References

[1] J A Bailey and D K Lahiri ldquoA novel effect of rivastigmineon pre-synaptic proteins and neuronal viability in a neurode-generation model of fetal rat primary cortical cultures and itsimplication in Alzheimerrsquos diseaserdquo Journal of Neurochemistryvol 112 no 4 pp 843ndash853 2010

[2] D Schuster M Spetea M Music et al ldquoMorphinans andisoquinolines acetylcholinesterase inhibition pharmacophoremodeling and interaction with opioid receptorsrdquo Bioorganic ampMedicinal Chemistry vol 18 no 14 pp 5071ndash5080 2010

[3] N H Greig D K Lahiri and K Sambamurti ldquoButyryl-cholinesterase an important new target in Alzheimerrsquos diseasetherapyrdquo International Psychogeriatrics vol 14 supplement 1pp 77ndash91 2002

[4] I Orhan B Sener M I Choudhary and A Khalid ldquoAcetyl-cholinesterase and butyrylcholinesterase inhibitory activity ofsome Turkish medicinal plantsrdquo Journal of Ethnopharmacologyvol 91 no 1 pp 57ndash60 2004

[5] T Mohamed and P P N Rao ldquoDesign synthesis and evaluationof 24-disubstituted pyrimidines as cholinesterase inhibitorsrdquoBioorganic and Medicinal Chemistry Letters vol 20 no 12 pp3606ndash3609 2010

[6] X M Cheng T Zhao T Yang C H Wang S W A Blighand Z T Wang ldquoHPLC fingerprints combined with principalcomponent analysis hierarchical cluster analysis and lineardiscriminant analysis for the classification and differentiationof Peganum sp indigenous to Chinardquo Phytochemical Analysisvol 21 no 3 pp 279ndash289 2010

[7] M Kartal M L Altun and S Kurucu ldquoHPLC method for theanalysis of harmol harmalol harmine and harmaline in theseeds of Peganum harmala Lrdquo Journal of Pharmaceutical andBiomedical Analysis vol 31 no 2 pp 263ndash269 2003

[8] B Hemmateenejad A Abbaspour H Maghami R Miri andM R Panjehshahin ldquoPartial least squares-based multivariatespectral calibration method for simultaneous determination ofbeta-carboline derivatives in Peganum harmala seed extractsrdquoAnalytica Chimica Acta vol 575 no 2 pp 290ndash299 2006

[9] Chinese Pharmacopoeia Committee Drug Standards of theMinistry of Public Health of the Peoplersquos Republic of China UygurPharmaceutical Section 1998

[10] J Riba M Valle G Urbano M Yritia A Morte and M JBarbanoj ldquoHuman pharmacology of ayahuasca subjective andcardiovascular effects monoamine metabolite excretion andpharmacokineticsrdquo Journal of Pharmacology and ExperimentalTherapeutics vol 306 no 1 pp 73ndash83 2003

[11] A Tahraoui J El-Hilaly Z H Israili and B LyoussildquoEthnopharmacological survey of plants used in the tradi-tional treatment of hypertension and diabetes in south-easternMorocco (Errachidia province)rdquo Journal of Ethnopharmacologyvol 110 no 1 pp 105ndash117 2007

[12] Z L Fan and X S Yao ldquoThe research of constituents andpharmacology of the genus Peganumrdquo Journal of ShenyangCollege of Pharmacy vol 9 pp 144ndash151 1992

[13] T Zhao C H Wang and Z T Wang ldquoChemical constituentsand pharmacologic actions of genus Peganum researchadvancesrdquo Journal of International Pharmaceutical Researchvol 37 no 5 pp 333ndash345 2010

[14] X Y Zheng Z J Zhang G X Chou et al ldquoAcetylcholinesteraseinhibitive activity-guided isolation of two new alkaloids fromseeds of Peganum nigellastrum Bunge by an in vitro TLC-bioautographic assayrdquo Archives of Pharmacal Research vol 32no 9 pp 1245ndash1251 2009

[15] X Y Zheng L Zhang X M Cheng Z J Zhang C HWang and Z T Wang ldquoIdentification of acetylcholinesteraseinhibitors from seeds of plants of genus Peganum by thin-layer chromatography-bioautographyrdquo JPC-Journal of PlanarChromatography-Modern TLC vol 24 no 6 pp 470ndash474 2011

[16] J A Boerth and S Rasapalli ldquoStudies toward the total synthesisof quinazoline alkaloids vasicinone luotonin and nigellas-trinerdquo in Proceedings of the 244th ACS National Meeting ampExposition Philadelphia Pa USA August 2012 CHED-304httpabstractsacsorgchem244nmprogramviewphp

[17] J R Fu L Teng X Y Dai and F S Yu ldquoOptimization ofextraction and purification of harmala alkaloids and the effectson learning and memory of model dementia mice induced byaluminumrdquo Chinese Traditional Patent Medicine vol 33 pp975ndash979 2011

[18] G L Ellman K D Courtney V Andres and R M Feather-stone ldquoA new and rapid colorimetric determination of acetyl-cholinesterase activityrdquo Biochemical Pharmacology vol 7 no 2pp 88ndash95 1961

[19] M A Gordon D E Carpenter H W Barrett and I B WilsonldquoDetermination of the normality of cholinesterase solutionsrdquoAnalytical Biochemistry vol 85 no 2 pp 519ndash527 1978

[20] V Gorun I Proinov V Baltescu G Balaban and O BarzuldquoModified Ellman procedure for assay of cholinesterases incrude enzymatic preparationsrdquoAnalytical Biochemistry vol 86no 1 pp 324ndash326 1978

[21] B TassoMCattoONicolotti et al ldquoQuinolizidinyl derivativesof bi- and tricyclic systems as potent inhibitors of acetyl- andbutyrylcholinesterase with potential in Alzheimerrsquos diseaserdquoEuropean Journal of Medicinal Chemistry vol 46 no 6 pp2170ndash2184 2011

6 Journal of Chemistry

[22] T Zhao S S Zheng B F Zhang et al ldquoMetabolic pathways ofthe psychotropic-carboline alkaloids harmaline and harmineby liquid chromatographymass spectrometry and NMR spec-troscopyrdquo Food Chemistry vol 134 no 2 pp 1096ndash1105 2012

[23] A M Yu J R Idle K W Krausz A Kupfer and F J GonzalezldquoContribution of individual cytochrome P450 isozymes tothe O-demethylation of the psychotropic 120573-carboline alkaloidsharmaline and harminerdquo Journal of Pharmacology and Experi-mental Therapeutics vol 305 no 1 pp 315ndash322 2003

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of

Journal of Chemistry 3

HO

HO

N

N

N

NH

N NH

HH

H3COH3CON

NH 1

1

2345

6

78

2

345

87

6

9

9 10

11

Harmine

Harmol

Harmaline

Harmane

Harmalol

Vasicine

Deoxyvasicine DeoxyvasicinoneVasicinone

Nigellastrine IINigellastrine I

Galanthamine

O

O

O

OO

O

OH

OH

OH

NHN

N

NN

NN

N

N

NN

N N

NNN

N

N

N

N

H

HH

OH

1 2 3 4-tetrahydroharmane-3-carboxylic acid

middotN CH3

Figure 1 Structures of the compounds investigated

3 Results

The inhibitory activities of the 120573-carboline and quinolinealkaloids onAChE and BChEwere evaluated in vitro using aninhibition assay with AChE (electric eel) and BChE (equineserum) and were summarized in Table 1

First of all it was observed that the inhibition data ofreference compounds galanthamine within the limits of thedifferent enzyme source and assay experimental conditionsare in good agreement with the literature data [21]

All tested compounds exhibited some degree of activityon both AChE and BChE and their IC

50values were in

the low micromolar range for at least one enzyme with

the exception of compounds deoxyvasicine and 1234-tetrahydroharmane-3-carboxylic acid Harmane showed themost potent inhibitory activity with IC

50value of 711 plusmn

200 120583M against AChE However harmane had a lowinhibitory activity against BChE (IC

50of 7691 plusmn 157 120583M)

Harmine and harmaline significantly inhibited AChE activitywith IC

50values of 905 plusmn 108 120583M and 1058 plusmn 201 120583M

but had lower inhibitory activity against BChE with IC50

values of 7507 plusmn 129 120583M and 10139 plusmn 139 120583M respectivelyVasicine exhibited strong inhibition both on AChE andBChE activities with IC

50values of 1368 plusmn 125 and 260 plusmn

147 120583M respectively As the main metabolites of harmalineand harmine in vivo [22 23] harmalol and harmol exhibited

4 Journal of Chemistry

Table 1 Inhibitory activity (IC50) and selectivity index (SI) of thecompounds investigated against AChE and BChE

Compounds IC50 (120583M plusmn SD) SIAChE BChE

Galanthamine 118 plusmn 105 08 [21] 73 [21] 913

Harmane 711 plusmn 200 7691 plusmn 157 1082Harmine 905 plusmn 108 7507 plusmn 129 830Harmaline 1058 plusmn 201 10139 plusmn 139 958Harmol 2158 plusmn 139 821 plusmn 365 038Harmalol 2788 plusmn 113 948 plusmn 203 034Vasicine 1368 plusmn 125 260 plusmn 147 019Nigellastrine II 4046 plusmn 109 926 plusmn 147 023Nigellastrine I 7050 plusmn 114 1824 plusmn 323 026Vasicinone 37078 plusmn 107 mdashDeoxyvasicinone 29444 plusmn 147 mdashDeoxyvasicine mdash mdash1234-tetrahydroharmane-3-carboxylicacid

mdash mdash

IC50 values were determined by regression analyses and expressed as themeansplusmn SDof three replicate determinations SI is theAChE selectivity indexdefined as IC50 BChEIC50 AChE affinity ratiomdash no inhibitory activity

moderate inhibitory activity against AChE with IC50

valuesof 2788 plusmn 113 and 2158 plusmn 139 120583M and stronger inhibitoryactivity against BChE with IC

50values of 948 plusmn 203 and

821 plusmn 365 120583M Nigellastrine I and nigellastrine II hadstronger inhibitory activity against BChE with IC

50values of

1824 plusmn 323 and 926 plusmn 147 120583M but with weaker inhibitoryactivity against AChE with IC

50values of 7050 plusmn 114 and

4046 plusmn 109 120583M

4 Discussion

Results from previous studies showed that the alkaloids frac-tion containing mainly 120573-carboline and quinoline alkaloidsfrom the seeds ofP nigellastrum exhibited potential inhibitiveactivity on AchE [14] In our ongoing studies the inhibitoryactivities of those individual 120573-carboline and quinoline alka-loids separated from genus Peganum on AChE and BChEwere determined using the in vitro Ellman method Based onthe inhibitory potency and selectivity of those alkaloids onAChE and BChE the structure-activity relationship of thosealkaloids could be speculated as follows

Amongst the series of 120573-carboline alkaloids (Figure 1)varying the substituent at C7 modulated the ChE inhibitoryprofile The presence of an O-methyl substituent at C7(harmine) led to the inhibition and selection on AChEslightly reduce with IC

50of 905 plusmn 108 120583M and AChE SI

of 830 compared to harmane with IC50

of 711 plusmn 200 120583Mand AChE SI of 913 No obvious difference was observedbetween the inhibitory activity of harmane and harmine

against BChE When oxidation at C7 occurs such as harmolthe inhibition against BChE was enhanced (IC

50= 821 plusmn

365 120583M) but with reduced activity on AChE (IC50= 2158 plusmn

139 120583M)Therefore the hydroxyl substituent at C7 obviouslyreduced the selectivity for AChE inhibition According tothe above analysis C7 could be regarded as a possible activesite for 120573-carboline alkaloids and varying the substituentat C7 could significantly affect the inhibition on AChE andBChE The double bond at C3-C4 seemed to have a certaindegree of influence on the inhibitory activity against BChE asdemonstrated by the IC

50values of harmaline and harmine

As the main O-demethylation metabolites of harmaline andharmine harmalol and harmol showed moderate inhibitorypotency on AChE with IC

50values of 2788 plusmn 113 and

2158 plusmn 139 120583M and stronger inhibitory potency on BChEwith IC

50values of 948 plusmn 203 and 821 plusmn 365 120583M Namely

the selective inhibition of harmalol and harmol versus AChEdeduced compared with their parent compound harmalineand harmine On the contrary their selective inhibitionagainst BChE enhanced

Amongst the quinoline alkaloids (Figure 1) vasicine wasidentified as the potent inhibitor with IC

50values of 1368 plusmn

125 120583Mon AChE and 260 plusmn 147 120583Mon BChE respectivelySelective BChE inhibitors have already been reported toincrease the ACh levels in the brain and to also reduce theformation of abnormal amyloid Therefore the discovery ofpotent and highly selective BChE inhibitors andor of dualAChEBChE inhibitors is an actively pursued goal in ADtreatment drug discovery [21] Very interestingly althoughwith poor selectivity vasicine may be a very potential leadcompound with the feature of dual AChEBChE inhibitoryactivity

Vasicinone and deoxyvasicinone possessing a carbony-lation at C9 or dehydroxylation at C3 exhibited no muchinhibitory activity on either AChE or BChE with IC

50values

of 37078plusmn107 and 29444plusmn147 120583MonAChE and with IC50

values of above 1000120583M on BChE Those data indicated thatthe inhibitive potency of quinoline alkaloids on both AChEand BChE was affected by the substitution at C3 and C9Deoxyvasicine possessing a carbonylation reaction at C9 anddehydroxylation at C3 simultaneously showed no inhibitorypotency on both AChE and BChE with larger IC

50values

more than 1000 120583MAs seen from Table 1 nigellastrine I and nigellastrine II

(Figure 1) two dimers of quinoline (deoxyvasicinone) and 120573-carboline alkaloids (harmane or dehydroharmane) displayeda stronger activity on BChE inhibition and weaker activityon AChE inhibition When compared with deoxyvasicinonethose dimers enhanced the inhibitor potency on both AChEand BChE but more on BChE On the other hand bycomparison with harmane the two dimmers seemed to havecertain inhibitory selectivity for BChE

From the above analysis harmane harmine and har-maline the main active constituent in genus Peganum hadgood selective inhibitory activities against AChE and theoxidation of the substituent at C7 could significantly modifythe inhibitory potency and selectivity for both AChE andBChE C3 and C9 positions of quinoline alkaloids wereinferred to be the active site for both AChE and BChE

Journal of Chemistry 5

inhibitionMoreover it was found that by the polymerizationof quinoline and carboline alkaloids the inhibitory activitydeclined on AChE but increased on BChE In the lightof those findings it could be concluded that the alkaloidsfrom the plants of genus Peganum showed inhibitory activityagainst both AChE and BChE In addition those findingsmay provide some guidance for the design and synthesis orsemisynthesis of potential inhibitors on AChE or BChE

Conflict of Interests

Theauthors have declared that there is no conflict of interests

Acknowledgments

The authors gratefully acknowledge the award from the KeyProgram of Joint Funds of the National Natural ScienceFoundation of China and Xinjiang Uygur AutonomousRegion of China (no U1130303) the National Nature ScienceFoundation of China (no 81173119) the Key Project of Min-istry of Science and Technology of China (2012ZX09103201-051) and the Program for Shanghai Innovative ResearchTeam in University (2009) to Professor C-H Wang for thefinancial support of this study The authors would also liketo thank Professor Shouming Zhong for providing languagehelp during the writing of this paper

References

[1] J A Bailey and D K Lahiri ldquoA novel effect of rivastigmineon pre-synaptic proteins and neuronal viability in a neurode-generation model of fetal rat primary cortical cultures and itsimplication in Alzheimerrsquos diseaserdquo Journal of Neurochemistryvol 112 no 4 pp 843ndash853 2010

[2] D Schuster M Spetea M Music et al ldquoMorphinans andisoquinolines acetylcholinesterase inhibition pharmacophoremodeling and interaction with opioid receptorsrdquo Bioorganic ampMedicinal Chemistry vol 18 no 14 pp 5071ndash5080 2010

[3] N H Greig D K Lahiri and K Sambamurti ldquoButyryl-cholinesterase an important new target in Alzheimerrsquos diseasetherapyrdquo International Psychogeriatrics vol 14 supplement 1pp 77ndash91 2002

[4] I Orhan B Sener M I Choudhary and A Khalid ldquoAcetyl-cholinesterase and butyrylcholinesterase inhibitory activity ofsome Turkish medicinal plantsrdquo Journal of Ethnopharmacologyvol 91 no 1 pp 57ndash60 2004

[5] T Mohamed and P P N Rao ldquoDesign synthesis and evaluationof 24-disubstituted pyrimidines as cholinesterase inhibitorsrdquoBioorganic and Medicinal Chemistry Letters vol 20 no 12 pp3606ndash3609 2010

[6] X M Cheng T Zhao T Yang C H Wang S W A Blighand Z T Wang ldquoHPLC fingerprints combined with principalcomponent analysis hierarchical cluster analysis and lineardiscriminant analysis for the classification and differentiationof Peganum sp indigenous to Chinardquo Phytochemical Analysisvol 21 no 3 pp 279ndash289 2010

[7] M Kartal M L Altun and S Kurucu ldquoHPLC method for theanalysis of harmol harmalol harmine and harmaline in theseeds of Peganum harmala Lrdquo Journal of Pharmaceutical andBiomedical Analysis vol 31 no 2 pp 263ndash269 2003

[8] B Hemmateenejad A Abbaspour H Maghami R Miri andM R Panjehshahin ldquoPartial least squares-based multivariatespectral calibration method for simultaneous determination ofbeta-carboline derivatives in Peganum harmala seed extractsrdquoAnalytica Chimica Acta vol 575 no 2 pp 290ndash299 2006

[9] Chinese Pharmacopoeia Committee Drug Standards of theMinistry of Public Health of the Peoplersquos Republic of China UygurPharmaceutical Section 1998

[10] J Riba M Valle G Urbano M Yritia A Morte and M JBarbanoj ldquoHuman pharmacology of ayahuasca subjective andcardiovascular effects monoamine metabolite excretion andpharmacokineticsrdquo Journal of Pharmacology and ExperimentalTherapeutics vol 306 no 1 pp 73ndash83 2003

[11] A Tahraoui J El-Hilaly Z H Israili and B LyoussildquoEthnopharmacological survey of plants used in the tradi-tional treatment of hypertension and diabetes in south-easternMorocco (Errachidia province)rdquo Journal of Ethnopharmacologyvol 110 no 1 pp 105ndash117 2007

[12] Z L Fan and X S Yao ldquoThe research of constituents andpharmacology of the genus Peganumrdquo Journal of ShenyangCollege of Pharmacy vol 9 pp 144ndash151 1992

[13] T Zhao C H Wang and Z T Wang ldquoChemical constituentsand pharmacologic actions of genus Peganum researchadvancesrdquo Journal of International Pharmaceutical Researchvol 37 no 5 pp 333ndash345 2010

[14] X Y Zheng Z J Zhang G X Chou et al ldquoAcetylcholinesteraseinhibitive activity-guided isolation of two new alkaloids fromseeds of Peganum nigellastrum Bunge by an in vitro TLC-bioautographic assayrdquo Archives of Pharmacal Research vol 32no 9 pp 1245ndash1251 2009

[15] X Y Zheng L Zhang X M Cheng Z J Zhang C HWang and Z T Wang ldquoIdentification of acetylcholinesteraseinhibitors from seeds of plants of genus Peganum by thin-layer chromatography-bioautographyrdquo JPC-Journal of PlanarChromatography-Modern TLC vol 24 no 6 pp 470ndash474 2011

[16] J A Boerth and S Rasapalli ldquoStudies toward the total synthesisof quinazoline alkaloids vasicinone luotonin and nigellas-trinerdquo in Proceedings of the 244th ACS National Meeting ampExposition Philadelphia Pa USA August 2012 CHED-304httpabstractsacsorgchem244nmprogramviewphp

[17] J R Fu L Teng X Y Dai and F S Yu ldquoOptimization ofextraction and purification of harmala alkaloids and the effectson learning and memory of model dementia mice induced byaluminumrdquo Chinese Traditional Patent Medicine vol 33 pp975ndash979 2011

[18] G L Ellman K D Courtney V Andres and R M Feather-stone ldquoA new and rapid colorimetric determination of acetyl-cholinesterase activityrdquo Biochemical Pharmacology vol 7 no 2pp 88ndash95 1961

[19] M A Gordon D E Carpenter H W Barrett and I B WilsonldquoDetermination of the normality of cholinesterase solutionsrdquoAnalytical Biochemistry vol 85 no 2 pp 519ndash527 1978

[20] V Gorun I Proinov V Baltescu G Balaban and O BarzuldquoModified Ellman procedure for assay of cholinesterases incrude enzymatic preparationsrdquoAnalytical Biochemistry vol 86no 1 pp 324ndash326 1978

[21] B TassoMCattoONicolotti et al ldquoQuinolizidinyl derivativesof bi- and tricyclic systems as potent inhibitors of acetyl- andbutyrylcholinesterase with potential in Alzheimerrsquos diseaserdquoEuropean Journal of Medicinal Chemistry vol 46 no 6 pp2170ndash2184 2011

6 Journal of Chemistry

[22] T Zhao S S Zheng B F Zhang et al ldquoMetabolic pathways ofthe psychotropic-carboline alkaloids harmaline and harmineby liquid chromatographymass spectrometry and NMR spec-troscopyrdquo Food Chemistry vol 134 no 2 pp 1096ndash1105 2012

[23] A M Yu J R Idle K W Krausz A Kupfer and F J GonzalezldquoContribution of individual cytochrome P450 isozymes tothe O-demethylation of the psychotropic 120573-carboline alkaloidsharmaline and harminerdquo Journal of Pharmacology and Experi-mental Therapeutics vol 305 no 1 pp 315ndash322 2003

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of

4 Journal of Chemistry

Table 1 Inhibitory activity (IC50) and selectivity index (SI) of thecompounds investigated against AChE and BChE

Compounds IC50 (120583M plusmn SD) SIAChE BChE

Galanthamine 118 plusmn 105 08 [21] 73 [21] 913

Harmane 711 plusmn 200 7691 plusmn 157 1082Harmine 905 plusmn 108 7507 plusmn 129 830Harmaline 1058 plusmn 201 10139 plusmn 139 958Harmol 2158 plusmn 139 821 plusmn 365 038Harmalol 2788 plusmn 113 948 plusmn 203 034Vasicine 1368 plusmn 125 260 plusmn 147 019Nigellastrine II 4046 plusmn 109 926 plusmn 147 023Nigellastrine I 7050 plusmn 114 1824 plusmn 323 026Vasicinone 37078 plusmn 107 mdashDeoxyvasicinone 29444 plusmn 147 mdashDeoxyvasicine mdash mdash1234-tetrahydroharmane-3-carboxylicacid

mdash mdash

IC50 values were determined by regression analyses and expressed as themeansplusmn SDof three replicate determinations SI is theAChE selectivity indexdefined as IC50 BChEIC50 AChE affinity ratiomdash no inhibitory activity

moderate inhibitory activity against AChE with IC50

valuesof 2788 plusmn 113 and 2158 plusmn 139 120583M and stronger inhibitoryactivity against BChE with IC

50values of 948 plusmn 203 and

821 plusmn 365 120583M Nigellastrine I and nigellastrine II hadstronger inhibitory activity against BChE with IC

50values of

1824 plusmn 323 and 926 plusmn 147 120583M but with weaker inhibitoryactivity against AChE with IC

50values of 7050 plusmn 114 and

4046 plusmn 109 120583M

4 Discussion

Results from previous studies showed that the alkaloids frac-tion containing mainly 120573-carboline and quinoline alkaloidsfrom the seeds ofP nigellastrum exhibited potential inhibitiveactivity on AchE [14] In our ongoing studies the inhibitoryactivities of those individual 120573-carboline and quinoline alka-loids separated from genus Peganum on AChE and BChEwere determined using the in vitro Ellman method Based onthe inhibitory potency and selectivity of those alkaloids onAChE and BChE the structure-activity relationship of thosealkaloids could be speculated as follows

Amongst the series of 120573-carboline alkaloids (Figure 1)varying the substituent at C7 modulated the ChE inhibitoryprofile The presence of an O-methyl substituent at C7(harmine) led to the inhibition and selection on AChEslightly reduce with IC

50of 905 plusmn 108 120583M and AChE SI

of 830 compared to harmane with IC50

of 711 plusmn 200 120583Mand AChE SI of 913 No obvious difference was observedbetween the inhibitory activity of harmane and harmine

against BChE When oxidation at C7 occurs such as harmolthe inhibition against BChE was enhanced (IC

50= 821 plusmn

365 120583M) but with reduced activity on AChE (IC50= 2158 plusmn

139 120583M)Therefore the hydroxyl substituent at C7 obviouslyreduced the selectivity for AChE inhibition According tothe above analysis C7 could be regarded as a possible activesite for 120573-carboline alkaloids and varying the substituentat C7 could significantly affect the inhibition on AChE andBChE The double bond at C3-C4 seemed to have a certaindegree of influence on the inhibitory activity against BChE asdemonstrated by the IC

50values of harmaline and harmine

As the main O-demethylation metabolites of harmaline andharmine harmalol and harmol showed moderate inhibitorypotency on AChE with IC

50values of 2788 plusmn 113 and

2158 plusmn 139 120583M and stronger inhibitory potency on BChEwith IC

50values of 948 plusmn 203 and 821 plusmn 365 120583M Namely

the selective inhibition of harmalol and harmol versus AChEdeduced compared with their parent compound harmalineand harmine On the contrary their selective inhibitionagainst BChE enhanced

Amongst the quinoline alkaloids (Figure 1) vasicine wasidentified as the potent inhibitor with IC

50values of 1368 plusmn

125 120583Mon AChE and 260 plusmn 147 120583Mon BChE respectivelySelective BChE inhibitors have already been reported toincrease the ACh levels in the brain and to also reduce theformation of abnormal amyloid Therefore the discovery ofpotent and highly selective BChE inhibitors andor of dualAChEBChE inhibitors is an actively pursued goal in ADtreatment drug discovery [21] Very interestingly althoughwith poor selectivity vasicine may be a very potential leadcompound with the feature of dual AChEBChE inhibitoryactivity

Vasicinone and deoxyvasicinone possessing a carbony-lation at C9 or dehydroxylation at C3 exhibited no muchinhibitory activity on either AChE or BChE with IC

50values

of 37078plusmn107 and 29444plusmn147 120583MonAChE and with IC50

values of above 1000120583M on BChE Those data indicated thatthe inhibitive potency of quinoline alkaloids on both AChEand BChE was affected by the substitution at C3 and C9Deoxyvasicine possessing a carbonylation reaction at C9 anddehydroxylation at C3 simultaneously showed no inhibitorypotency on both AChE and BChE with larger IC

50values

more than 1000 120583MAs seen from Table 1 nigellastrine I and nigellastrine II

(Figure 1) two dimers of quinoline (deoxyvasicinone) and 120573-carboline alkaloids (harmane or dehydroharmane) displayeda stronger activity on BChE inhibition and weaker activityon AChE inhibition When compared with deoxyvasicinonethose dimers enhanced the inhibitor potency on both AChEand BChE but more on BChE On the other hand bycomparison with harmane the two dimmers seemed to havecertain inhibitory selectivity for BChE

From the above analysis harmane harmine and har-maline the main active constituent in genus Peganum hadgood selective inhibitory activities against AChE and theoxidation of the substituent at C7 could significantly modifythe inhibitory potency and selectivity for both AChE andBChE C3 and C9 positions of quinoline alkaloids wereinferred to be the active site for both AChE and BChE

Journal of Chemistry 5

inhibitionMoreover it was found that by the polymerizationof quinoline and carboline alkaloids the inhibitory activitydeclined on AChE but increased on BChE In the lightof those findings it could be concluded that the alkaloidsfrom the plants of genus Peganum showed inhibitory activityagainst both AChE and BChE In addition those findingsmay provide some guidance for the design and synthesis orsemisynthesis of potential inhibitors on AChE or BChE

Conflict of Interests

Theauthors have declared that there is no conflict of interests

Acknowledgments

The authors gratefully acknowledge the award from the KeyProgram of Joint Funds of the National Natural ScienceFoundation of China and Xinjiang Uygur AutonomousRegion of China (no U1130303) the National Nature ScienceFoundation of China (no 81173119) the Key Project of Min-istry of Science and Technology of China (2012ZX09103201-051) and the Program for Shanghai Innovative ResearchTeam in University (2009) to Professor C-H Wang for thefinancial support of this study The authors would also liketo thank Professor Shouming Zhong for providing languagehelp during the writing of this paper

References

[1] J A Bailey and D K Lahiri ldquoA novel effect of rivastigmineon pre-synaptic proteins and neuronal viability in a neurode-generation model of fetal rat primary cortical cultures and itsimplication in Alzheimerrsquos diseaserdquo Journal of Neurochemistryvol 112 no 4 pp 843ndash853 2010

[2] D Schuster M Spetea M Music et al ldquoMorphinans andisoquinolines acetylcholinesterase inhibition pharmacophoremodeling and interaction with opioid receptorsrdquo Bioorganic ampMedicinal Chemistry vol 18 no 14 pp 5071ndash5080 2010

[3] N H Greig D K Lahiri and K Sambamurti ldquoButyryl-cholinesterase an important new target in Alzheimerrsquos diseasetherapyrdquo International Psychogeriatrics vol 14 supplement 1pp 77ndash91 2002

[4] I Orhan B Sener M I Choudhary and A Khalid ldquoAcetyl-cholinesterase and butyrylcholinesterase inhibitory activity ofsome Turkish medicinal plantsrdquo Journal of Ethnopharmacologyvol 91 no 1 pp 57ndash60 2004

[5] T Mohamed and P P N Rao ldquoDesign synthesis and evaluationof 24-disubstituted pyrimidines as cholinesterase inhibitorsrdquoBioorganic and Medicinal Chemistry Letters vol 20 no 12 pp3606ndash3609 2010

[6] X M Cheng T Zhao T Yang C H Wang S W A Blighand Z T Wang ldquoHPLC fingerprints combined with principalcomponent analysis hierarchical cluster analysis and lineardiscriminant analysis for the classification and differentiationof Peganum sp indigenous to Chinardquo Phytochemical Analysisvol 21 no 3 pp 279ndash289 2010

[7] M Kartal M L Altun and S Kurucu ldquoHPLC method for theanalysis of harmol harmalol harmine and harmaline in theseeds of Peganum harmala Lrdquo Journal of Pharmaceutical andBiomedical Analysis vol 31 no 2 pp 263ndash269 2003

[8] B Hemmateenejad A Abbaspour H Maghami R Miri andM R Panjehshahin ldquoPartial least squares-based multivariatespectral calibration method for simultaneous determination ofbeta-carboline derivatives in Peganum harmala seed extractsrdquoAnalytica Chimica Acta vol 575 no 2 pp 290ndash299 2006

[9] Chinese Pharmacopoeia Committee Drug Standards of theMinistry of Public Health of the Peoplersquos Republic of China UygurPharmaceutical Section 1998

[10] J Riba M Valle G Urbano M Yritia A Morte and M JBarbanoj ldquoHuman pharmacology of ayahuasca subjective andcardiovascular effects monoamine metabolite excretion andpharmacokineticsrdquo Journal of Pharmacology and ExperimentalTherapeutics vol 306 no 1 pp 73ndash83 2003

[11] A Tahraoui J El-Hilaly Z H Israili and B LyoussildquoEthnopharmacological survey of plants used in the tradi-tional treatment of hypertension and diabetes in south-easternMorocco (Errachidia province)rdquo Journal of Ethnopharmacologyvol 110 no 1 pp 105ndash117 2007

[12] Z L Fan and X S Yao ldquoThe research of constituents andpharmacology of the genus Peganumrdquo Journal of ShenyangCollege of Pharmacy vol 9 pp 144ndash151 1992

[13] T Zhao C H Wang and Z T Wang ldquoChemical constituentsand pharmacologic actions of genus Peganum researchadvancesrdquo Journal of International Pharmaceutical Researchvol 37 no 5 pp 333ndash345 2010

[14] X Y Zheng Z J Zhang G X Chou et al ldquoAcetylcholinesteraseinhibitive activity-guided isolation of two new alkaloids fromseeds of Peganum nigellastrum Bunge by an in vitro TLC-bioautographic assayrdquo Archives of Pharmacal Research vol 32no 9 pp 1245ndash1251 2009

[15] X Y Zheng L Zhang X M Cheng Z J Zhang C HWang and Z T Wang ldquoIdentification of acetylcholinesteraseinhibitors from seeds of plants of genus Peganum by thin-layer chromatography-bioautographyrdquo JPC-Journal of PlanarChromatography-Modern TLC vol 24 no 6 pp 470ndash474 2011

[16] J A Boerth and S Rasapalli ldquoStudies toward the total synthesisof quinazoline alkaloids vasicinone luotonin and nigellas-trinerdquo in Proceedings of the 244th ACS National Meeting ampExposition Philadelphia Pa USA August 2012 CHED-304httpabstractsacsorgchem244nmprogramviewphp

[17] J R Fu L Teng X Y Dai and F S Yu ldquoOptimization ofextraction and purification of harmala alkaloids and the effectson learning and memory of model dementia mice induced byaluminumrdquo Chinese Traditional Patent Medicine vol 33 pp975ndash979 2011

[18] G L Ellman K D Courtney V Andres and R M Feather-stone ldquoA new and rapid colorimetric determination of acetyl-cholinesterase activityrdquo Biochemical Pharmacology vol 7 no 2pp 88ndash95 1961

[19] M A Gordon D E Carpenter H W Barrett and I B WilsonldquoDetermination of the normality of cholinesterase solutionsrdquoAnalytical Biochemistry vol 85 no 2 pp 519ndash527 1978

[20] V Gorun I Proinov V Baltescu G Balaban and O BarzuldquoModified Ellman procedure for assay of cholinesterases incrude enzymatic preparationsrdquoAnalytical Biochemistry vol 86no 1 pp 324ndash326 1978

[21] B TassoMCattoONicolotti et al ldquoQuinolizidinyl derivativesof bi- and tricyclic systems as potent inhibitors of acetyl- andbutyrylcholinesterase with potential in Alzheimerrsquos diseaserdquoEuropean Journal of Medicinal Chemistry vol 46 no 6 pp2170ndash2184 2011

6 Journal of Chemistry

[22] T Zhao S S Zheng B F Zhang et al ldquoMetabolic pathways ofthe psychotropic-carboline alkaloids harmaline and harmineby liquid chromatographymass spectrometry and NMR spec-troscopyrdquo Food Chemistry vol 134 no 2 pp 1096ndash1105 2012

[23] A M Yu J R Idle K W Krausz A Kupfer and F J GonzalezldquoContribution of individual cytochrome P450 isozymes tothe O-demethylation of the psychotropic 120573-carboline alkaloidsharmaline and harminerdquo Journal of Pharmacology and Experi-mental Therapeutics vol 305 no 1 pp 315ndash322 2003

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of

Journal of Chemistry 5

inhibitionMoreover it was found that by the polymerizationof quinoline and carboline alkaloids the inhibitory activitydeclined on AChE but increased on BChE In the lightof those findings it could be concluded that the alkaloidsfrom the plants of genus Peganum showed inhibitory activityagainst both AChE and BChE In addition those findingsmay provide some guidance for the design and synthesis orsemisynthesis of potential inhibitors on AChE or BChE

Conflict of Interests

Theauthors have declared that there is no conflict of interests

Acknowledgments

The authors gratefully acknowledge the award from the KeyProgram of Joint Funds of the National Natural ScienceFoundation of China and Xinjiang Uygur AutonomousRegion of China (no U1130303) the National Nature ScienceFoundation of China (no 81173119) the Key Project of Min-istry of Science and Technology of China (2012ZX09103201-051) and the Program for Shanghai Innovative ResearchTeam in University (2009) to Professor C-H Wang for thefinancial support of this study The authors would also liketo thank Professor Shouming Zhong for providing languagehelp during the writing of this paper

References

[1] J A Bailey and D K Lahiri ldquoA novel effect of rivastigmineon pre-synaptic proteins and neuronal viability in a neurode-generation model of fetal rat primary cortical cultures and itsimplication in Alzheimerrsquos diseaserdquo Journal of Neurochemistryvol 112 no 4 pp 843ndash853 2010

[2] D Schuster M Spetea M Music et al ldquoMorphinans andisoquinolines acetylcholinesterase inhibition pharmacophoremodeling and interaction with opioid receptorsrdquo Bioorganic ampMedicinal Chemistry vol 18 no 14 pp 5071ndash5080 2010

[3] N H Greig D K Lahiri and K Sambamurti ldquoButyryl-cholinesterase an important new target in Alzheimerrsquos diseasetherapyrdquo International Psychogeriatrics vol 14 supplement 1pp 77ndash91 2002

[4] I Orhan B Sener M I Choudhary and A Khalid ldquoAcetyl-cholinesterase and butyrylcholinesterase inhibitory activity ofsome Turkish medicinal plantsrdquo Journal of Ethnopharmacologyvol 91 no 1 pp 57ndash60 2004

[5] T Mohamed and P P N Rao ldquoDesign synthesis and evaluationof 24-disubstituted pyrimidines as cholinesterase inhibitorsrdquoBioorganic and Medicinal Chemistry Letters vol 20 no 12 pp3606ndash3609 2010

[6] X M Cheng T Zhao T Yang C H Wang S W A Blighand Z T Wang ldquoHPLC fingerprints combined with principalcomponent analysis hierarchical cluster analysis and lineardiscriminant analysis for the classification and differentiationof Peganum sp indigenous to Chinardquo Phytochemical Analysisvol 21 no 3 pp 279ndash289 2010

[7] M Kartal M L Altun and S Kurucu ldquoHPLC method for theanalysis of harmol harmalol harmine and harmaline in theseeds of Peganum harmala Lrdquo Journal of Pharmaceutical andBiomedical Analysis vol 31 no 2 pp 263ndash269 2003

[8] B Hemmateenejad A Abbaspour H Maghami R Miri andM R Panjehshahin ldquoPartial least squares-based multivariatespectral calibration method for simultaneous determination ofbeta-carboline derivatives in Peganum harmala seed extractsrdquoAnalytica Chimica Acta vol 575 no 2 pp 290ndash299 2006

[9] Chinese Pharmacopoeia Committee Drug Standards of theMinistry of Public Health of the Peoplersquos Republic of China UygurPharmaceutical Section 1998

[10] J Riba M Valle G Urbano M Yritia A Morte and M JBarbanoj ldquoHuman pharmacology of ayahuasca subjective andcardiovascular effects monoamine metabolite excretion andpharmacokineticsrdquo Journal of Pharmacology and ExperimentalTherapeutics vol 306 no 1 pp 73ndash83 2003

[11] A Tahraoui J El-Hilaly Z H Israili and B LyoussildquoEthnopharmacological survey of plants used in the tradi-tional treatment of hypertension and diabetes in south-easternMorocco (Errachidia province)rdquo Journal of Ethnopharmacologyvol 110 no 1 pp 105ndash117 2007

[12] Z L Fan and X S Yao ldquoThe research of constituents andpharmacology of the genus Peganumrdquo Journal of ShenyangCollege of Pharmacy vol 9 pp 144ndash151 1992

[13] T Zhao C H Wang and Z T Wang ldquoChemical constituentsand pharmacologic actions of genus Peganum researchadvancesrdquo Journal of International Pharmaceutical Researchvol 37 no 5 pp 333ndash345 2010

[14] X Y Zheng Z J Zhang G X Chou et al ldquoAcetylcholinesteraseinhibitive activity-guided isolation of two new alkaloids fromseeds of Peganum nigellastrum Bunge by an in vitro TLC-bioautographic assayrdquo Archives of Pharmacal Research vol 32no 9 pp 1245ndash1251 2009

[15] X Y Zheng L Zhang X M Cheng Z J Zhang C HWang and Z T Wang ldquoIdentification of acetylcholinesteraseinhibitors from seeds of plants of genus Peganum by thin-layer chromatography-bioautographyrdquo JPC-Journal of PlanarChromatography-Modern TLC vol 24 no 6 pp 470ndash474 2011

[16] J A Boerth and S Rasapalli ldquoStudies toward the total synthesisof quinazoline alkaloids vasicinone luotonin and nigellas-trinerdquo in Proceedings of the 244th ACS National Meeting ampExposition Philadelphia Pa USA August 2012 CHED-304httpabstractsacsorgchem244nmprogramviewphp

[17] J R Fu L Teng X Y Dai and F S Yu ldquoOptimization ofextraction and purification of harmala alkaloids and the effectson learning and memory of model dementia mice induced byaluminumrdquo Chinese Traditional Patent Medicine vol 33 pp975ndash979 2011

[18] G L Ellman K D Courtney V Andres and R M Feather-stone ldquoA new and rapid colorimetric determination of acetyl-cholinesterase activityrdquo Biochemical Pharmacology vol 7 no 2pp 88ndash95 1961

[19] M A Gordon D E Carpenter H W Barrett and I B WilsonldquoDetermination of the normality of cholinesterase solutionsrdquoAnalytical Biochemistry vol 85 no 2 pp 519ndash527 1978

[20] V Gorun I Proinov V Baltescu G Balaban and O BarzuldquoModified Ellman procedure for assay of cholinesterases incrude enzymatic preparationsrdquoAnalytical Biochemistry vol 86no 1 pp 324ndash326 1978

[21] B TassoMCattoONicolotti et al ldquoQuinolizidinyl derivativesof bi- and tricyclic systems as potent inhibitors of acetyl- andbutyrylcholinesterase with potential in Alzheimerrsquos diseaserdquoEuropean Journal of Medicinal Chemistry vol 46 no 6 pp2170ndash2184 2011

6 Journal of Chemistry

[22] T Zhao S S Zheng B F Zhang et al ldquoMetabolic pathways ofthe psychotropic-carboline alkaloids harmaline and harmineby liquid chromatographymass spectrometry and NMR spec-troscopyrdquo Food Chemistry vol 134 no 2 pp 1096ndash1105 2012

[23] A M Yu J R Idle K W Krausz A Kupfer and F J GonzalezldquoContribution of individual cytochrome P450 isozymes tothe O-demethylation of the psychotropic 120573-carboline alkaloidsharmaline and harminerdquo Journal of Pharmacology and Experi-mental Therapeutics vol 305 no 1 pp 315ndash322 2003

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of

6 Journal of Chemistry

[22] T Zhao S S Zheng B F Zhang et al ldquoMetabolic pathways ofthe psychotropic-carboline alkaloids harmaline and harmineby liquid chromatographymass spectrometry and NMR spec-troscopyrdquo Food Chemistry vol 134 no 2 pp 1096ndash1105 2012

[23] A M Yu J R Idle K W Krausz A Kupfer and F J GonzalezldquoContribution of individual cytochrome P450 isozymes tothe O-demethylation of the psychotropic 120573-carboline alkaloidsharmaline and harminerdquo Journal of Pharmacology and Experi-mental Therapeutics vol 305 no 1 pp 315ndash322 2003

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Chromatography Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Applied ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Theoretical ChemistryJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Spectroscopy

Analytical ChemistryInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Quantum Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Organic Chemistry International

ElectrochemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CatalystsJournal of

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Carbohydrate Chemistry

International Journal of

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Chemistry

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Analytical Methods in Chemistry

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Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of

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The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of

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Chromatography Research International

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Applied ChemistryJournal of

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Theoretical ChemistryJournal of

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Analytical ChemistryInternational Journal of

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ElectrochemistryInternational Journal of

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