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,