AntiproliferativeactivityofdifferentextractsfromDaphnealtaicaPall.onselectedcancercells

Prof.Dr.MuratKizaibekTraditionalKazakhMedicineResearchInstituteofIliKazakhAutonomousPrefecture,P.R.ChinaAlmatyMay3,2012IntroductionCanceristheworld’ssecondbiggestkilleraftercardiovasculardisease.In2005,7.6millionpeoplediedfromcancer,morethanHIV/AIDS,malariaandtuberculosiscombined.Inaddition,thisnumberisexpectedtoriseto9millionin2015andincreasefurtherto11.5millionin2030(WHO,2007).Clearly,thereisagreatneedtoimprovecurrentcancertherapiesandtosearchfornewtherapies.DatafromStatisticsCanadaThroughouthistory,plantshaveaffordedarichsourceofcompoundsthathavefoundmanyapplicationsinthefieldsofmedicine,pharmacyandbiology.Withinthesphereofcancer,plantshaveplayedanimportantroleasasourceofeffectiveanti-canceragents,anditissignificantthatover60%ofcurrentlyusedanti-canceragentsarederivedinonewayoranotherfromnaturalsources,includingplants,marineorganismsandmicro-organisms(Craggetal.,2005).InIliKazakhAutonomousPrefectureofChina,herbalremediesarefrequentlyusedbyTraditionalKazakhMedicine(TKM)doctorstotreatalargevarietyofdiseases,e.g.,hypertension,cancer,rheumatism,andbonefracture.However,thereislittleinformationabouttheirefficacy.DaphnealtaicaPall.,locallyknownasuwsoyq?orqasq?rjiydek,isadeciduousherboftheThymelaeaceaefamily.ItisendemictothenorthofJungarBasinofXinjiang,China(theTachengandHabaheareas),Altai,ManrakandTarbagataiMountainsofKazakhstanandAltairegionofRussiaaswellasnorthwestMongoliaD.altaicahavelongbeenusedinTKMtotreatesophaguscancer,gastriccancer,tracheitis,commoncold,soarthroat,rheumatism,snakebite,andfortheirantitussiveanddiaphoreticproperties(Xuetal.,2009).ItsmedicinalusewasfirstlyrecordedinaKazakhmedicalclassicShipagerlikBayan,whichwaswrittenbyOteyboydakTleukabylulyin15thcentury(Tleukabyluly,1994).OteyboydakTleukabyluly（1388-？）ShipagerlikBayanTranslation：Prescription4573:addthebarkofDaphneintothemeatbrothandboilthem.Assoonasthebrothcomestoafullboil,removetheresidue,drinkthebrothandeatthemeat.Itcancureasthmaandcough.Atpage420ofthisbook,thereisthefollowingprescription：Althoughmanybenefitsofthisplanthavebeenclaimed,noscientificdatumisavailablesofaraboutitsbiologicalproperties,especiallyitsanticanceractivity.Therefore,thispromptedustoinvestigatetheantiproliferativeactivityofthisplantoncancercells.Forthesepurposes,sixextractswerepreparedandtestedfortheirpotentialantiproliferativepropertieswith3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide(MTT)bioassayondifferentcancercelllines.Toourknowledge,thisisthefirsttimethattheanticanceractivityofD.altaicaisevaluated.MATERIALSANDMETHODSPlantmaterialThebarksofD.altaicawerecollectedfromAltaiMountain,Xinjiang,ChinainJuly2008.TheplantwasidentifiedandauthenticatedbyBahargulKongirkhan,aherbalistattheherbariumofAltayInstituteforDrugControl,Xinjiang,China.Avoucherspecimen(No.050036)wasdepositedatthesameplace.TestedmaterialDriedbarksoftheplant(150g)werechoppedandextractedwith95%EtOHbymacerationfor2weeksinadarkplaceataroomtemperatureof20±2℃.Theprocedurewasrepeatedfortwice.Theextractswerecombined,concentratedunderreducedpressureandfreeze-driedtoyieldtheEtOHextract(DA-Et,12.10g).0.5gofthisextractwaskeptforMTTassayandtherestwassubmittedtoasequentialliquid-liquidextractionwithsolventsofincreasedpolaritytoyieldpetroleumether(DA-Pt，1.7064g),chloroform(DA-Ch,0.6915g),ethylacetate(DA-Ea，1.8237g),n-butanol(DA-Bu，2.5242g)andaqueous(DA-Aq，2.5998g)fractions.Cellculturehumanesophagealsquamouscellcarcinoma(Eca-109)cellsgastriccarcinoma(AGS)cellshepatoma(SMMC-7721)cellscervicalcarcinoma(HeLa)cellsAllcelllineswereculturedat37°Cinahumidifiedatmosphereof5%carbondioxide.CellproliferationassayAllthesamplesweretestedat6.25,12.5，25，50，100μg/mlconcentrations.ThesamplesweredissolvedinDMSOandfurtherdilutedwithcellculturemedium.CellsincubatedwiththesameconcentrationofDMSOwereusedasacontrol.TheDMSOfinalconcentrationwasadjustedto1%ofthetotalvolumeofmediuminalltreatments,includingthecontrol.ForMTTassay,1×105cells/wellwereplatedinto96-wellplates(Nunclon,Denmark)andincubatedfor24hbeforetheadditionofdrugs.After48hofincubationforallcells,20μlofMTT(Sigma,USA)reagent(5mg/ml)inphosphatebufferedsaline(PBS)wasaddedtoeachwell.Theplateswereincubatedat37°Cfor4h.Attheendoftheincubationperiod,themediumwasremovedandpureDMSO(150μl)wasaddedtoeachwell.ThemetabolizedMTTproductwasquantifiedbyreadingtheabsorbanceat490nmonaBeckmanCoulter-AD340(BeckmanCoulter,Fullerton,CA,USA).Resultswereexpressedpercentageofcellviability(%).Allassayswereperformedintriplicate.Themediangrowthinhibitoryconcentrationvalues(IC50)wereusedtocomparetheantiproliferativeactivityofextractsoncancercells.StatisticalanalysisTheresultsofpercentageofcellviabilitywerepresentedasmeans±SD.Statisticalcomparisonsbetweentreatmentgroupsandthecontrolgroupwereperformedusingtheone-wayANOVAfollowedbyposthocTukey(incaseofequalvariance)orDunnett’sT3(incaseofunequalvariance)test.ThesetestswereperformedusingSPSS13forWindows.p<0.05wasconsideredstatisticallysignificant.IC50valuesandtheir95%confidenceinterval(IC95)werecalculatedusingsigmoidaldose-responsemodelwithvariableslopeintheGraphpadPrismsoftware(version4.03).RESULTSANDDISCUSSIONFigure1.AntiproliferativeactivityofDA-Aq,DA-Bu,DA-Ea,DA-Ch,DA-PtandDA-Etonesophagealsquamouscellcarcinoma(Eca-109)cells.Figure2.AntiproliferativeactivityofDA-Aq,DA-Bu,DA-Ea,DA-Ch,DA-PtandDA-Etongastriccarcinoma(AGS)cellsFigure3.AntiproliferativeactivityofDA-Aq,DA-Bu,DA-Ea,DA-Ch,DA-PtandDA-Etonhepatoma(SMMC-7721)cells.Figure4.AntiproliferativeactivityofDA-Aq,DA-Bu,DA-Ea,DA-Ch,DA-PtandDA-Etoncervicalcarcinoma(HeLa)cells.Table1.AntiproliferativeeffectofsixextractsfromthebarksofD.altaicaonfourdifferenttumorcelllines.DataarepresentedasIC50(μg/ml)valuesIndeed,somespeciesofgenusDaphnewerereportedtohavesignificantantitumoractivity.Forexample,itwasdescribedthatextractsandcompoundsofD.genkwa(Zhanetal.,2005),D.mucronata

(Mahdavietal.,2007)andD.odoravar.marginata(Zhangetal.,20