Evaluation of the cytotoxic and cytostatic activities of alkaloid extracts from different parts of Peganum harmala L.(Zygophyllaceae)

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Research

paper

Evaluation

of

the

cytotoxic

and

cytostatic

activities

of

alkaloid

extracts

from

different

parts

of

Peganum

harmala

L. (Zygophyllaceae)

Lamine

Bournine

a,b,

*

,

Sihem

Bensalem

b

,

So

ﬁane

Fatmi

c,d

,

Fatiha

Bedjou

b

,

Véronique

Mathieu

e

,

Mokrane

Iguer-Ouada

c

,

Robert

Kiss

e

,

Pierre

Duez

f

a

DépartementdeBiologie,FacultédesSciencesdelaNatureetdelaVieetdesSciencesdelaTerre,UniversitédeBouira,10000Bouira,Algeria

b

LaboratoiredeBiotechnologieVégétalesetEthnobotanique,FacultédesSciencesdelaNatureetdelaVie,UniversitédeBejaia,06000Bejaia,Algeria

c

LaboratoireAssociéenEcosystèmesMarinsetAquacoles,FacultédesSciencesdelaNatureetdelaVie,UniversitédeBejaia,06000Bejaia,Algeria

d_Laboratoire_de_Technologie_{Pharmaceutique,}_Département_de_Génie_des_procédés,_Université_de_Bejaia,₀₆₀₀₀_Bejaia,_Algeria e

LaboratoiredeCancérologieetdeToxicologieExpérimentale,FacultédePharmacie,UniversitéLibredeBruxelles(ULB),1050Bruxelles,Belgium

f

ServicedeChimieThérapeutiqueetdePharmacognosie,FacultédeMédecineetdePharmacie,UniversitédeMons(UMONS),7000Mons,Belgium

ARTICLE INFO Articlehistory: Received3August2016

Receivedinrevisedform19October2016 Accepted19October2016 Availableonlinexxx Keywords: PeganumharmalaL. Zygophyllaceae Alkaloids Cytotoxicactivity MTTassay Videomicroscopyanalysis ABSTRACT

Introduction:PeganumharmalaL.(P.harmala)isbroadlyusedinfolkmedicineforthetreatmentofvarious

diseasesincluding cancer. Theseeds are particularlyemployed fortherapeutic.The presentpaper

investigatesthecytotoxiceffectsofrawalkaloidextractsfromdifferentpartsofP.harmalaincluding

fruits(TAFr),seeds(TASe),roots(TAR)andaerialparts(TAAp);inordertoassesstraditionalclaimsabout

thetherapeuticpotentialofthisplant.

Methods:Thecytotoxiceffectswereevaluatedonsixmalignantcancercells(A549,U373,Hs683,MCF7,

B16F10andSKMEL-28;A549,U373,MCF7andSKMEL-28areresistanttoproapoptoticstimuli)byMTT

assayandquantitativevideomicroscopyanalysis.ThemainalkaloidswerequantiﬁedbyHPLCinthe

differentpartsofplant.

Results:TotalalkaloidsofthedifferentpartswerecytotoxictowardspracticallyallcancercelllineswithIC

50ranging1–52mg/mLafter72hoftreatment.VideomicroscopyanalysisindicatedthattheTAFr,TASe

andTARalkaloidsaffectA549lungcarcinomacellsbehaviourandinduceacytostaticeffectwhereas

TAApextractwascytotoxicratherthancytostatic.TAAp,TAFr,TASeandTARtreatmentinducedglobal

growthratioindexes(GGR)of0.19,0.26,0.3and0.62,respectively,after72hoftreatment.Dependingon

theorgane,harmine,harmaline, harmolandvasicinehave,rangebetween2and90%w/woftotal

alkaloids.

Conclusion:ThesedataindicatethatP.harmalaalkaloidsextractmaysupportthetraditionalclaims

regardingitsanticanceruseswhichcouldbehelpfulinprovidingofnewcytotoxicagentsagainst

chemo-resistantcancercells.

ã2016ElsevierGmbH.Allrightsreserved.

1.Introduction

Cancer remains the second leading cause of death behind cardiovascular diseases; cancer-related deaths are predicted to increase to over 24 million in 2035 [1,2]. The use of cancer chemotherapybeganatthestartofthe20thcenturyand,forthe time being, small molecules remain the principal mode of treatmentforvariouscancers[3].Atotalof174newanticancer

drugswereapprovedduringthetimeperiod1981–2014bytheFDA (food and drug administration) [4]. Chemotherapy drugs can potentiallyeliminatecancercellsbutcanalsodamageperfectly healthycellsandtissues;theseinducesideeffectsthroughoutthe body, dramatically reducing the qualityof life for patients[5]. Hence,thereisarequirementtodiscovernewpotentanticancer drugs with fewer side effects. Based on past major successes, medicinalplants are considereda promising sourceto develop newchemicalcompoundswithpotentialanticanceractivities[6]. P. harmala belongs to the family of Zygophyllaceae. Widely distributedinNorthAfrica,itgrowsnaturallyinsemi-aridand pre-desertarea.Ithaswideusesinfolkmedicineforthetreatmentof various diseases; it has anti-inﬂammatory [7], anticancer [8], analgesic, antiseptic [9], and stimulant emmenagogue [10] * Correspondingauthorat:DépartementdeBiologie,FacultédesSciencesdela

NatureetdelaVieetdesSciencesdelaTerre,UniversitédeBouira,10000,Bouira, Algeria.

E-mailaddresses:lbournine@yahoo.com,lbournine06@gmail.com(L.Bournine) .

http://dx.doi.org/10.1016/j.eujim.2016.10.002

1876-3820/ã2016ElsevierGmbH.Allrightsreserved.

xxx–xxx

ContentslistsavailableatScienceDirect

European

Journal

of

Integrative

Medicine

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propertiesandistraditionallyusedtotreatdiabetes,hypertension [11],lumbago,asthma,colicandjaundice[10].Severalstudieshave alsodescribedtherapeuticeffectsofP.harmala,suchas antibacte-rial[12],antiplasmodial,vasorelaxant[13,14],hypoglycemic[15] andphytotoxiceffects [16]. Theantitumor effectsof P.harmala seedsoncancerouscelllineshavebeendemonstratedinprevious studiesbutmechanismsofactionarestillunknown[2,8,17–19]. Some studies also reported the cytotoxic effects of harmine derivatives[20,21].

Tothebestofourknowledge,therearenopublicationsfocusing on the cytotoxic activity of alkaloid extracts obtained from differentparts(seeds,fruits,rootsandaerialparts)ofP.harmala. Inthisstudy,weevaluatefortheﬁrsttimethecytotoxiceffectsof totalalkaloidextractsonsixcancercelllinesusingMTTassay.In addition, the changes in cell proliferation, cell death and cell morphology induced by tested extracts were investigated by meansofcomputer-assistedphase-contrastmicroscopy (quanti-tativevideomicroscopyanalysis).

2.Materialandmethods 2.1.Plantextraction

ThedifferentpartsofP.harmala(aerialparts,fruits,seedsand roots)werecollectedin thehighlands ofSétif(Algeria) in June 2011.AvoucherspecimenwasdepositedattheHerbariumofthe Botanical Garden of Meise, Belgium (BR0000013496968). The extractionwascarriedoutaccordingtothemethoddescribedby Kartal et al. (2003) with some modifications [22]. Dried plant materials(20g) were crushedand then extracted with200mL ethanol(80%,v/v)inasoxhletapparatus.Theethanolextractswere filteredandconcentratedundervacuum,acidifiedwithHCl(2%,v/ v)andextractedwithpetroleumethertoremovefattymaterials.

The aqueous layer was brought to pH 9 with ammonia and extractedthreetimeswithchloroform(20mL).Theorganiclayer wasevaporatedundervacuumtoobtaintotalalkaloidsfromaerial parts(TAAp),fruits(TAFr),seeds(ATSe)androots(ATR). 2.2.Quantiﬁcationofalkaloids

Inthedifferentextracts,themajoralkaloidswerequantiﬁedby HPLCmethod.Thechromatographicseparationwasperformedon aHPLCKontronSystem.ThecompoundswereelutedonaLuna 5

m

m phenyl hexyl column (2504.60mm) (Phenomenex, Utrecht, Netherlands) using a gradient of 0.1% formic acid in water(phaseA)andCH3CN CH3OH(1:1,v/v;phaseB)ataﬂow

rate of 1.5mL/min at 40C. The gradient was as follows: equilibration time, 15min at 0% B; 0min, 0% B; 5min,10% B; 10min,25%B;15min,50%B;17min,100%B.TheUV-detectorwas setat254nmand20

m

Lsampleswereinjected[7].

2.3.Cytotoxicactivity

2.3.1.Preparationoftestsolutions

The stock solutions of P. harmala alkaloid extracts were preparedinDMSO(10mg/mL)anddilutedwiththeculturemedia to achieve the indicated ﬁnal concentrations (0.5–100

m

g/mL; maximum1%DMSOincontactwithcells).Theetoposideusedas positivecontrolwaspurchasedfromSigma(SaintLouis,Missouri, USA). The stock solution of etoposide was prepared in DMSO (10 2M)anddilutedwiththeculturemediaatdesired concentra-tion.

2.3.2.Cellculture

HumancancercelllineswereobtainedfromtheAmericanType Culture Collection (ATCC, Manassas, USA) and the Deutsche

Fig.1.P.harmalaalkaloidextractsinhibitedtheviabilityofsixmalignantcancercells(MCF7,Hs683,SKMEL-28,U373,A549andB16F10)inadose-dependentmanner.Cells weretreatedwithDMSOvehicleortheindicatedconcentrationsofTAFr(A),TASe(B),TAR(C),andTAApextracts(D)for72h.CellviabilitywasdeterminedusingMTTassay andexpressedasmeansSEMinthreeindependentexperiments.TAFr:totalalkaloidoffruits;TASe:totalalkaloidofseeds;TAR:totalalkaloidofroots;TAAp:totalalkaloidof aerialparts.

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Sammlung von Mikroorganismen und Zellkulturen (DSMZ, Braunschweig, Germany). Six cell lines were used to evaluate the cytotoxic activity: human A549 non-small-cell-lung cancer (NSCLC)(DSMZcodeACC107),humanU373glioblastoma(ATCC codeHTB-17),humanHs683anaplasticoligodendroglioma(ATCC codeHTB-138),humanMCF7breastcancer(ATCCcodeHTB-22), human SKMEL-28 melanoma (ATCC code HTB-72) and mouse B16F10melanoma(ATCCcodeCRL-6475).Thecellswerecultured in Dulbecco’s ModiﬁedEagle’sMedium (DMEM) supplemented

with10%fetal bovineserum(FBS), 4mMglutamine,100

m

g/mL gentamicin, and penicillin-streptomycin (200U/mL and 200

m

g/ mL)24hbeforethestartoftheexperiment.Cellsweregrownat 37_C_in_a_humidi_ﬁed_5%_CO

2incubator.

2.3.3.MTTassay

The overall growth level of human cancer cell lines was determinedusingthecolorimetricMTT (3-[4,5-dimethylthiazol-2-yl]diphenyltetrazoliumbromide,Sigma)assay[23].Brieﬂy,thecell Table1

IC50values(mg/mL)oftotalalkaloidsfromdifferentpartsofP.harmalaagainstvariouscancercelllinesafter72hincubation.IC50valuesweredeterminedbasedonthe

dose-responsecurvesshowninFig.1(meansSEMofthreeseparateexperiments). Totalalkaloidextract IC50(mg/mL)

MCF7 Hs683 SKMEL-28 U373 A549 B16F10

TAApa 15.54.1 52.77.0 80.23.7 46.65.5 55.57.5 20.00.8 TAFr 8.11.4 12.12.6 7.91.7 15.61.6 18.11.2 3.40.4 TASe 6.60.5 4.40.8 11.71.4 5.40.3 8.71.5 4.10.2 TAR 3.70.4 4.60.5 6.20.5 2.60.3 3.80.4 1.00.1 Etoposideb _3.5_0.6 _25.5_0.9 _4.2_0.3 _48.0_1.1 _0.9_0.03 _n.d.c a

TAAp:totalalkaloidofaerialparts;TAFr:totalalkaloidoffruits;TASe:totalalkaloidofseeds;TAR:totalalkaloidofroots.

b _the_growth_inhibitory_effects_of_etoposide_are_expressed_as_m_M. c_not_determined.

Fig.2.MorphologicalillustrationsofA549humanlungcarcinomacellpopulationsuntreated(control)ortreatedfor72hwithrespectiveinvitrogrowthinhibitoryIC50value

ofTAFr,TASe,TARandTAAp,asdeterminedusingtheMTTassayinA549lungcarcinoma(seeTable1).TAFr:totalalkaloidoffruits;TASe:totalalkaloidofseeds;TAR:total alkaloidofroots;TAAp:totalalkaloidofaerialparts.

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lineswereincubatedfor24hin96-microwellplates(atadensityof 5000–8000cells/well).Theassessmentofcellpopulationgrowth bymeansoftheMTTcolorimetricassayisbasedonthecapability of living cells to reduce the yellow product MTT to a purple formazanbyareductionreactionoccurringinthemitochondria.As incubationover1cellcycle(24h)doesnotallowtorevealall possibleeventsaffectingcellviability,cytotoxicitystudiesduration wasﬁxedat72h.Thenumberofmetabolicallyactive(“living”)cells after72hofcultureinthepresence(orabsence:control)ofthe alkaloid extractsis directly proportionaltotheintensity ofthe purpleformazanproductmeasuredquantitativelyby spectropho-tometry (Biorad model 680XR, Biorad, Nazareth, Belgium) at 570nmwavelength(withareferenceof630nm).Themeasured absorbancedirectlycorrelatestothenumberofviablecells.The percentage of cell survival was calculated as follows: % cell survival=(absorbance of treated cells/absorbance of cells with vehiclesolvent)100.Thehalfinhibitoryconcentration(IC50)was

calculatedfromthedose-responsecurveobtainedbyplottingthe percentageofcellsurvivalversustheconcentrationofplantextract used. Each set of experimental conditions was carried out in triplicates[24].

2.3.4.Computer-assistedphase-contrastmicroscopy(quantitative videomicroscopy)

ThedirectvisualizationofA549lungcarcinomabehaviourafter treatmentwiththealkaloidextractsunderstudy,i.e.inhibitionof cellproliferation(acytostaticeffect)versusdirectinductionofcell death(acytotoxiceffect),wascarriedoutbymeansof computer-assistedphasecontrastmicroscopy,i.e.quantitative videomicro-scopy,aspreviouslydetailed[25].Brieﬂy,animageoftheseeded ﬂaskbottomwasacquiredevery4minoverthecourseofa72h observationperiod.Thus,1080digitizedimageswereavailablefor each experimental conditionandan approximately40smovies weregeneratedfromtheseimages.Basedonthephase-contrast picturesofeachanalyzedextract,aglobalgrowthratioindex(GGR index)wascalculated[25]andcomparedtotheMTTIC50value.

Theglobalgrowth(GG)iscalculatedineachcontrolandineach treatedconditionbydividingthenumberofcellsonthelastimage bythenumberofcellsontheﬁrstimage.TheGGRindexisobtained foreachextractbydividingtheGGvaluescalculatedfortreated A549cellsbytheGGvaluescalculatedforthecontrol.

2.4.Statisticalanalysis

Results are expressed as meanS.E.M. of at least three independentexperiments.Student’st-testwasusedtocompare the differencebetween each treated group and thecontrol. P-valuesoflessthan0.05wereconsideredsigniﬁcant.

3.Results

3.1.DeterminationoftheIC50ofP.harmalaalkaloidextractsoncancer

celllines

TheMTTassayisamethodgenerallyusedtostudytheactionof natural compounds and plant extracts on cell proliferation, viabilityand cytotoxicity,asdescribed by Mosmann,1983 [26]. In this experiment, we determined the IC50 in vitro growth

inhibitoryactivitiesoffourextractsofP.harmalaunderstudy,at concentrations of up to 100

m

g/mL. These extracts exhibit a markedgrowthinhibitoryactivitytowardallthecancercelllines tested,regardlessofwhetherthecancercellsareresistant(A549, U373, MCF7 and SKMEL-28 models) or sensitive (Hs683 and B16F10models)toproapoptoticstimuli(Fig.1)withIC50sranging

1–52

m

g/mL(Table1).However, theviabilityof SKMEL-28cells

24 48 72 0 2 4 6 8 10 Control TAFr TASe TAR TAAp * *** ** ** * * ** *** ** * Time (h) Global growth (GG)

Fig.3.EffectofIC50treatmentontheglobalgrowthofA549humanlungcancer

cells.ThedataareexpressedasmeansS.E.Mofthreeseparateexperiments(n=3). *p<0.05;**p<0.01;***p<0.001comparedwithcontrolgroup.TAFr:totalalkaloid offruits;TASe:totalalkaloidofseeds;TAR:totalalkaloidofroots;TAAp:total alkaloidofaerialparts.

Table2

Quantitativevideomicroscopydata. Alkaloid

extracts

Globalgrowthratio (GGRindex)a

Globalproﬁleofthe

compoundintermsofgrowthinhibition

%Celldeath (SEM) TAFrb 24h 0.65 Cytostatic 0.70.7 48h 0.43 1.60.3 72h 0.26 1.30.2 TASe 24h 0.73 Cytostatic 1.40.7 48h 0.44 2.80.9 72h 0.30 2.10.6 TAR 24h 0.96 Cytostatic 1.01.0 48h 0.72 2.01.2 72h 0.62 1.80.7 TAAp 24h 0.64 Cytotoxic 4.73.8 48h 0.31 19.512.7 72h 0.19 27.919.6 a

globalgrowthratioindex(GGRindex)determinedbyquantitativevideomicroscopyonA549lungcancercelllineattheMTTcolorimetricassayrelatedIC50concentration

ofeachalkaloidextract.

b

TAFr:totalalkaloidoffruits;TASe:totalalkaloidofseeds;TAR:totalalkaloidofroots;TAAp:totalalkaloidofaerialparts. xxx–xxx

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wasonlyslightlydecreasedaftertreatmentwithTAApat80

m

g/ mL;besides,upto38%ofSKMEL-28cellsurvivedaftertreatment with100

m

g/mL(thehighesttesteddose).Alltotalalkaloidextracts exhibitedapotentinhibitionontheviabilityofmammaliancancer cells,MCF7(IC50<15

m

g/mL)(Table1).TheTAFr, TASeand TAR

exhibitedasigniﬁcantreductioninthecellviabilitywithalowIC50

incomparisonwithcontrol(etoposide).Especially,TARdisplayeda markedly decreased on cancer cells growth (IC50< 6

m

g/mL)

(Table1).

3.2.CytotoxicandcytostaticeffectsofdifferentalkaloidextractsofP. harmalaonA549lungcancercells

In order to determine whether the plant extracts induced cytotoxicorcytostaticeffects,wesubmittedthehumanA549cell linetoquantitativevideomicroscopyanalysis(computer-assisted phase-contrastmicroscopy).Themorphologicaldata(Figs.2and3) indicatethattheTAFr,TASeandTARdisplayedcytostaticandnot cytotoxiceffectsontheA549cells,whichareresistanttovarious proapoptotic stimuli (percentage of cell death was very low <2.8%); by contrast, the TAAp displayed cytotoxic rather than cytostaticeffects (Table 2).Consequently, theGGR valueof the alkaloidextractsfromdifferentpartsofP.harmaladecreasedina time-dependentmanner(Table2).

TheTAAp,TASeandTAFrdisplayedaGGRvalueof0.19(means that 19% of cells growth) and 0.26 (26% of cells growth), respectively after 72h of treatment. However, a GGR value of 0.62(62%ofcellsgrowth)wasobtainedfortheTAR(Table2).The percentagesof cell death, calculatedfor each alkaloid extracts, indicatethat,whenassayedattheirMTTIC50s,TAFr,TASeandTAR

inhibitcancercellproliferationwithoutinducingcelldeath(Fig.4) intheA549NSCLCcellline.Bycontrast,theTAApextractledto29% celldeathinthesamecells(Table2).

3.3.Alkaloidsquantiﬁcation

InthedifferentpartsofP.harmala,themajorquinazolineand

b

-carbolinealkaloidswerequantiﬁedbyHPLC.AR2_value_typically

higher than 0.98 was observed for linear ﬁtting of the curves obtainedusingstandardspreparedin0.1%HCOOH:harmineand harmol(10–80mg/mL),harmaline(25–250mg/mL)andvasicine (10–70

m

g/mL). TAAp contained vasicine (81.8%) and harmine (2.9%)asmajoralkaloids,TARcontainedmainlyharmine(90.9%) and harmol (2.85%). The TASe of P. harmala mainly contained harmine andharmalinethat reachedthevery highamountsof 37.9%and48.75%,respectively(Table3).

4.Discussion

Chemotherapyplaysanimportantroleinthepreventionand treatmentofcancer;butitsef_ﬁciencyisrestrictedbycellresistance tothedrugs,requiringconstantimprovementoftreatments.New classes of chemotherapeutic agents have been continuously developed, among which naturally occurring compounds have increasingly attracted interest as lead compounds for drug development [5,27]. The ﬁrst group of agents to advance into clinicaluseweretheVincaalkaloids,vinblastineand vincristine isolatedfromCatharanthusroseus[28].

P.harmalaiswidelyreported,intraditionalmedicine,forthe treatment of many diseases, including cancer [17]. Cytotoxic effectsoftherawextractofseedsofthisplant,suchasaqueous, hydro-alcoholic and methanolic extracts, were previously reported,indicating aconsiderablecytotoxicpotential [8,20,29– 33].

ThehighalkaloidcontentofP.harmalaextracts[7]promptedus toinvestigatetheirpotentialcytotoxicity;toourbestknowledge,

theactivitiesofaerialparts,fruitsandrootsofP.harmalaagainst severalcancercelllinesi.e.A549,U373,Hs683,MCF7,B16F10and SKMEL-28 have not been investigated so far. All total alkaloid extracts of fruits, seeds, roots and aerial parts decreased the viability of all analyzed cancer cell lines in a dose-dependent manner.Particularly,TARinducedasigniﬁcantdecreaseoncancer cellviability.Theresultssuggestthatalkaloidsareamajorclassof compounds in different parts of P. harmala and probably responsible for the observed cytotoxic effects. The differences betweentheactivitiesofthedifferentpartsoftheplantstrongly dependontheiralkaloidscomposition;also,asynergiceffectofthe differentalkaloidscannotbeexcluded.

An interestingﬁnding in thepresent study is that alkaloids extractedfromourP.harmalasamplesinhibittheviabilityofthe mammary cancercellline(MCF7)at concentration(IC50)lower

thantheIC50valuereportedfortheseedstotalalkaloidextracts

obtainedfromanIranianP.harmala(MCF7cellline;IC50,25

m

g/

mL)[18].Therawalkaloidextractsofseedshavealsobeenrecently reportedtoinhibittheviabilityofMDA-MB-231breastcancercell (IC50, 30

m

g/mL) [34]; also, quinazoline alkaloids puriﬁed from

seeds have been shown active on human gastric cancer cells (IC50>84

m

mol/L) [20]. Consequently,thepotenteffect ofTAAp

extractcouldberelatedtoitshighcontentinvasicine. Table3

Content(%)ofmajoralkaloidsintotalalkaloidextractsofdifferentpartsofP. harmala.

Concentrationsofthemajoralkaloids (%w/w;meanSD;n=3)

TAApa _TASeb _TAR

Totalalkaloids 1.86 5 1.75 Harmine 2.920.03 37.90.1 90.90.6 Harmaline n.d.c 48.750.01 n.d. Harmane n.d. n.d. n.d. Harmol n.d. n.d. 2.850.01 Harmalol n.d. n.d. n.d. Vasicine 81.80.9 n.d. n.d. a

TAAp:totalalkaloidofaerialparts;TASe:totalalkaloidofseeds;TAR:total alkaloidofroots.

b

thealkaloidsquantiﬁcationoffruitsalkaloidextractwassimilartoalkaloid extractofseeds. c notdetermined.

0

20

40

60

80

10

0

10

20

30

24h 48h 72h

TAFr

TASe

TAR

24h 48h 72h 24h 48h 72h

TAA

p

24h 48h 72h

% of Growth Inh

ibition

%

o

f Ce

ll

De

a

th

Fig.4.Illustrationofthe%ofcelldeathinducedbyeachalkaloidextractunder study(quantitativevideomicroscopyanalysis)inrelationtothegrowthinhibitory effectsinducedbytheseextracts.Cellcultureshavebeenmonitoredintriplicatefor eachexperimentalconditionfor24h,48hand72h.TAFr:totalalkaloidoffruits; TASe:totalalkaloidofseeds;TAR:totalalkaloidofroots;TAAp:totalalkaloidof aerialparts.

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Quantitative videomicroscopy indicated that the alkaloid extracts exhibit a cytostatic or cytotoxic effect on A549 lung cancercellline, depending onthetested extract; thecytotoxic activity could be ascribed to the high concentration of the quinazolinealkaloid,vasicine,intheaerialpartsextract.

In conclusion,total alkaloidextractsfromaerialparts, fruits, seeds and roots of P. harmala exert potent cytotoxic and/or cytostaticeffectstowardseveralmulti-drugresistantcancercell lines.In alungcarcinomaA549cellline,fruits,seedsandroots extractdemonstratedmostlycytostaticeffectwhereasacytotoxic activitywasobservedwithaerialpartsextract.Thepromisingdata ofP.harmalaalkaloidextractsmaysupportthetraditionalclaims regardingitsanticanceruses;thesecouldbehelpfulinproviding newanticanceragentsagainstchemo-resistantcancersaswellas newleadsfordrugdiscovery;howeverthepsychotropicactivities ofthesealkaloidsmaylimittheirclinicaluseand pharmacomo-dulationmaybenecessarytodissociatethe2typesofactivities.All thealkaloidsextractedfromdifferentpartsoftheplantshouldbe furtherinvestigatedforinvitrocytotoxicactivity,structure-activity relationshipsandinvivoanticancerstudies.

Conﬂictsofinterest

Theauthorsdeclarethattherearenoconﬂictsofinterest. Acknowledgments

ThisstudywassupportedbygrantsfromtheAlgerianMinister ofHigherEducationandScientiﬁcResearchandtheBelgianFund forScientiﬁcResearch(FRS-FNRS).

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