全 文 ：第 38卷第 1期 上海师范大学学报(自然科学版) Vol.38, No.1
2 0 0 9年 2月 JournalofShanghaiNormalUniversity(NaturalSciences) 2 00 9 , Feb.
Distributionofanticancerdrug
inOphiorrhizajaponica
WANGWei, LUYang, LILi, WANGJing,
QIANZhong-ying, ZHOUGen-yu, KAIGuo-yin＊
(CollegeofLifeandEnvironmentSciences, ShanghaiNormalUniversity, Shanghai200234, China)
Abstract:TheanticancerdrugCamptothecin(CPT)andHydroxycamptothecin(HCPT)weredetectedin
Ophiorrhizajaponicabyhigh-performanceliquidchromatogram(HPLC).ThediscoveryofCPTandHCPTinsuch
aChinesetraditionalherbmightprovideanewapproachtoproduceCPTandHCPT, whichalsoservedasanalterna-
tivemodelexperimentalsystemforstudyingCPTbiosynthesisandevenimprovingCPTcontentbygeneticengineering
inthefuture.
Keywords:Ophiorrhizajaponica;camptothecin;Hydroxycamptothecin;HPLC
CLCnumber:Q946.91 Documentcode:A ArticleID:1000-5137(2009)01-0059-04
Receiveddate:2008-09-10
Foundationitem:ShanghaiScienceand TechnologyCommiteeProject(06QA14038, 065458022, 073158202,
075405117, 05ZR14093);ShanghaiEducationCommitteeFoundation(06DZ015;09ZZ138);ZhejiangProvincialNaturalSci-
enceFoundation(Y2080621);FoundationfromShanghaiNormalUniversity(SK200830, CH030);LeadingAcademicDisci-
plineProjectofShanghaiMunicipalEducationCommission(J50401).
Biography:WANGWei(1982-), female, graduatestudent, ColegeofLifeandEnviromentSciences, ShanghaiNormalUni-
versity;KAIGuo-yin(1977-), male, associateprofessor, ColegeofLifeandEnviromentSciences, ShanghaiNormalUniversity.
＊Correspondingauthor.
1 Introduction
Camptothecin(CPT)isakindofmodifiedmonoterpenoidindolealkaloids, originalyidentifiedintheex-
tractsofChinesetreeCamptothecaacuminata(Nyssaceae)[ 1] .Camptothecinexhibitsananti-tumoraction
duetoitsinhibitoryactivitytoDNAtopoisomeraseI[ 2] .CPTderivativessuchasirinothecanandtopothecan
havebeenwidelyusedforthetreatmentofcancerovertheworld[ 3] .Themarketsizeofcamptothecinderiva-
tivesin2002wasestimatedatUS＄1000milion, about200kgcamptothecinasrawmaterialwouldbeneeded
forthemanufactureofthecamptothecinderivatives[ 4] .Inspiteoftherapidgrowthofthepharmaceuticalmar-
ket, theywerestilsynthesizedfromnaturalCPT, whichwasmainlyobtainedbyextractionfromoriginalCPT
-producingplantssuchasC.acuminataandNothapodytesfoetida.Beingawoodyplant, C.acuminata
grewveryslowlyandthesupplyofCPTsustainedbyisolationfromtheoriginalplantsourcewasverylimited,
whilethedemandforCPTwasincreasingrapidlyduetotheexpansionofclinicaltrialsandcancertreatments.
Hence, itwasanimportantandurgenttasktoresolvethesourcescarcityofCPTworldwide.
NowCPTderivativescouldbesynthesizedbychemicaltotalsynthesismethod, butthechemicalsynthesis
proceswaslong, expensive, andtoolowyieldingtobecommercialyfeasiblebecauseofthecomplexityofthe
chemicalstructure.PlanttissueandcelcultureofC.acuminataetc, hadbeentriedextensivelybutwerenot
sosuccessfulregardedtoofcostsandCPTproduction[ 5, 6] .Comparatively, screeningthefast-growingweedy
naturalresourcesproducingCPTwasanalternativemethodintheforeseeablefuture.SeveralOphiorhizaspe-
cies(ofthefamilyRubiaceae)suchasO.mungos, O.filistipula, O.pumila, O.liukiuensis, O.prostrata
hadbeenreportedasthenewsourcesofCPTproduction[ 4, 7 ～ 11] , respectively.AsO.japonica, oneindigenous
Chinesemedicinalplantcommonlyusedforulcers, poisonouswoundsandleprosyinChina, belongsto
Ophiorhizaspecies, itwasspeculatedthatCPTanaloguesmightalsoexistinO.japonicasimilarly.Atthe
present, therehavebeennoreportsonCPTandHCPTinO.japonica.
Inthispaper, anti-cancerdrugsCPTandHCPTweredetectedinO.japonicaforthefirsttime, which
provideanewresourceforCPTandHCPTproductionandanalternativemodelexperimentalsystemforstud-
yingCPTbiosynthesisandevenimprovingCPTcontentbygeneticengineeringinthefuture.
2 Materialsandmethods
2.1 Plantmaterials
PlantmaterialofO.japonicawascolectedfromFujianprovinceinChina, oven-driedandgroundto
finepowderbyamortarandpestle.DriedmaterialsofO.japonicapower(450 mg)weredisolvedin15mL
60% methanolandextractionwascarriedoutbyultrasonicmethodatthe50℃ temperaturefor45 min, and
centrifugedtocolectsupernatant.Themixturewasthenevaporatedatroomtemperatureandtheresiduewasre
-suspendedin1 mL60% methanolandfilteredthrougha0.45 μmpolymericfilterpriortoHPLCanaly-
sis[ 12] .StandardsforCamptothecinandHydroxycamptothecinwerepurchasedfromSigma(USA)andwere
preparedinmethanolatafinalconcentrationof1mg· mL-1.
2.2 HPLCanalysis
CamptothecinandHydroxycamptothecinwereassayedbyHPLC.ThesamplewasloadedontoanAlte-
chEconosiC18 column(4.6mm ×250 mm, Japan)atroomtemperature.Themobilephasewasmadeup
from30%HPLCgradeacetonitrileand70% MiniQgradewater.Theflowratewas1.0 mL· min-1 andthe
injectionvolumewas10μL.Theelutionwasmonitoredat254nmandalsoconfirmedat380nmbyUVdetec-
tor[ 12] .Astandardcurve(5 ～ 100 mg/mL)wasobtainedusingauthenticCPTandHCPTsamples.Retention
timesforCPTandHCPTwere8.746 minand7.450 min, respectively(Figure1, 2).
Figure1 HPLCanalysisoftheCPT(254nmand380nm)retentiontime= 8.746min
3 Resultsandconclusions
CamptothecinandHydroxycamptothecinweredetectedincrudeextractsofwild-typeO.japonicaplantsusing
60 上海师范大学学报(自然科学版) 2009年
Figure2 HPLCanalysisoftheHCPT(254nmand380nm)retentiontime= 7.450min
Figure3 HPLCanalysisofthesample(254nmand380nm)CPTretentiontime=8.820minandHCPTretentiontime=7.515min
reverse-phaseHPLCanalysis.RetentiontimeofpeakobtainedwithstandardCamptothecinandHydroxycamptothe-
cinwasusedtoidentifythecorespondingpeaksintheMeOHcrudeextractsofO.japonica.Inaddition, thecrude
extractswerespikedwithstandardCamptothecinandHydroxycamptothecintoconfirmthepeakscorespondingto
CamptothecinandHydroxycamptothecin.Theelutedcompoundhadthesimilarretentiontime(8.820min)andUV
absorptionspectrumasauthenticCPT.TheHPLCresultsalsoshowedthatthesampleofHCPT, aswefoundasimi-
larretentiontime(7.515min)andthesameUVabsorptionmaximaasauthenticHCPT.
Ourexperimentresultsrevealedthat0.0073% dryweightofcamptothecinwasdetectedinthewhole
O.japonicaplants(andHydroxycamptothecinwasdetectedonlyintraceamountsof0.0001% dryweight),
whosecontentwasmuchlowerthanthatinC.acuminata(about0.05%).Beingakindofwoodyplant, the
growthcycleC.acuminatawastoolongwhiletissuecultureofC.acuminatawasverydificult.Becauseof
thelowconcentrationoftryptamineandlowlevelofactivityofsecologaninsynthaseandstrictosidinesynthase,
thecellineofC.acuminatawasevennotabletoaccumulatecamptothecin.Asakindofherb, Ophiorrhiza
japonicagrewrapidlyandwasusedingenetictransformationeasily, whichmaybeanalternativedrugsource
ofrawmaterialforproductionofcamptothecin.
Althoughtotalsynthesisofcamptothecinwasachieved, theclinicalyformulatedanti-tumormedicines, to-
potecanandirinotecanwerecurentlysemi-synthesizedusingnaturalcamptothecinasastartingmaterialextrac-
tedfromC.acuminataandN.foetida[ 13] .Itwasclearthatintheforeseeablefuturethesupplyofcamptothe-
cinanditssyntheticalyusefulprogenitorsmustdependonbiologicalmethodsofproduction.Thefindingsthat
CPTexistsinO.japonicaprovidedanalternativeresourceforCPTdrugproduction.Atsametime, aneficient
plantregenerationsystemfromleaf-derivedcalusofO.japonicawassuccesfulyestablished[ 14] andseveral
61 第 1期 王 伟 ,陆 杨 ,李 礼 ,等:抗癌药物在日本蛇根草中的检测
genescodingenzymeresponsibleforthebiosynthesisofcamptothecininO.japonicawereclonedasinitial
stepstowardsdetailedunderstandingofthepathwayforcamptothecinbiosynthesisinourlab, whichcouldbe
oferanefectiveapproachfortherapidpropagationofO.japonicaandalsoprovidedthefoundationtoimprove
CPTproductionbygenetictransformationofO.japonicainthefuture.
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抗癌药物在日本蛇根草中的检测
王 伟 ,陆 杨 ,李 礼 ,王 敬 ,钱忠英 ,周根余 ,开国银
(上海师范大学 生命与环境科学学院 , 上海 200234)
摘要:利用 HPLC技术在日本蛇根草中检测到了抗癌药物喜树碱(CPT)和羟基喜树碱(HCPT),而这 2种药物是当今世
界应用比较广泛的抗癌药物.蛇根草是中国传统的中草药 , 检测到日本蛇根草中含有 CPT和 HCPT,为获得这 2种抗癌
药物提供了新的生药途径 ,同时也为利用基因工程手段提高蛇根草中 CPT和 HCPT含量奠定了基础.
关键词:日本蛇根草;喜树碱;羟基喜树碱;HPLC
(责任编辑:郁 慧)
62 上海师范大学学报(自然科学版) 2009年