【doc】 高效液相色谱法对中药复方四君子汤中异甘草素、甘草素、异甘草苷和甘草苷的含量测定

【doc】 高效液相色谱法对中药复方四君子汤中异甘草素、甘草素、异甘草苷和甘草苷的含量测定 高效液相色谱法对中药复方四君子汤中异 甘草素、甘草素、异甘草苷和甘草苷的含量 测定 JournalofChinesePharmaceuticalSciences2005,14(4):227.230 Dq~terminationofLiquiritig”,Liquiritin,Is0liquiritigeninandDeterminationLiquiritigeninLiqmritinanO,,lS0IlqUlrlUgenln IsoliquiritininExtractofTraditionalChineseMedicineSijunziDecoction byHigh—PerformanceLiquidChromatography uuYangandYANGJun—shan (InstituteofMedicinalPlantDevelopment,PekingUnionMedicalCollege, ChineseAcademyofMedicalSciences,&昭100094,China) Abstract:AimAnHPLC.UVmethodfortheanalysisofTraditionalChineseMedicine(TCM) preparation,Sijunzidecoction,hasbeendeveloped.Fourflavonoidmarkercompounds,liquiritigenin, liquiritin,isoliquiritigenin,andisoliquiritin,fromRadixGlycyrrhizaewerequantitativelyanalyzedinthis preparation.Methods111eseparationwasperformedonareversed—phaseC l8columnbyusingagradient elutionwithmobilephaseof(A)water—formicacid(100:0.04,v/v)(pH3)an d(B)acetonitrile,111e mobilephasegradientwassetfrom0—5minat10%ofBand45minto90%ofB .Theassaywascal”- fledoutataflowrateof0.2mL?rainatFoomtemperaturewiththeUVdetectionwavelengthsat280nln and368nln+Results111eextract(25mg?mL)ofSijunzidecoctioncontmns1.47腭’mL,liquiri— tigenin,16.40/.tg’mL,liquiritin,0.66ttg’mL, isoliquiritigenin,and2.12ttg.n山,isoliquiritin. Therecoveriesforthesamplepreparationofthemarkemwere102.2%,97.7%,100.3%,and 99.9%.respectively.Conclusion111emethodissimpleandaccurate.111isstudyreportsaroutine quantitativemethodfortheanalysisofmuhiplecomponentsinSijunzidecoctionbyHPI. Keywords:traditionalChinesemedicine;Sijunzidecoction,HHJC a【nlRlll~r:R284.1;R9.2DDo咖rItc0de:AArticleID:1003.1057(加 aI5)4_227.o4 TraditionalChineseMedicine(TCM)preparations havebeenusedmOllthanthousandsofyearsforthetreat- mentofvariousdiseases.Mostofthemarecomposedof severalmedicinalherbs.ThequalitycontrolofTCMisa realchallenge,whichisveryimportantforsafeandeffec- tiveclinicalapplication.Sijunzidecoctionisafamous TCMpreparation,whichwasfirstrecordedinapharma— ceuticalbookofSongdynasty.Itconsistsoffourmedicinal herbs,Panax,(~nseng),AtractyIMesm伽,D (Baizhu),Por/ao0螂(),andura/ens/s (Cancao).Theformulawasreportedtoeffectivelyimprove dalIlagedlearningandmemoryinmice…;tostrengthenthe Receiveddate:2005—06.27. CorrespondingauIhor!Tel86-10-62899707,Fax86-10-62898425, E-mailjunshanyang@hotmal1.eolll decreasedimmunityduetothelowlevelofI2,II厂6, andtumornecrosisfactor(TNF)inspleen—deficiencysyn- drome引.toregulatehormoneleveIexcretedfromstomach andintestine.andtoinhibitthegrowthoftumorcell【4.. Differentmethodshavebeendevelopedforthedeter- minationofconstituentsineachindividualcrudedrugL一; however,therehavebeenfewreportsonthesimultaneous determinationofthemultipleconstituentsinthisprepara— tion.SincethecompositionsofTCMpreparationarein— deedcomplicated.thesamplepll-treatmentisquiteim- portantinordertokeeptheactivecomponentsandtoex- dudethecontaminants.Inthisstudy,theSijunzidecoc— tionwasextractedwithwaterandprecipitatedbyethano1. Fromtheinitialstudyitwasfoundthatthemaincompo- nentsalefromGancaoandGinseng.Hence,fourmajor activeflavonoidcompoundsliquiritigenin,liquiritin, isoliquiritigenin.andisoliquiritin[一]fromGancaowell selectedasmarkersforquantitativeanalysis. Thisstudvreportsaroutinequantitativemethodfor 228JournalofChinesePharmaceuticalSciences2005,14(4):227-230 theanalysisofmultiplecomponentsinSijunzidec(s:tion byHPLC. Experimental Samplesandreagents Thestandardsofliquiritigenin,liquiritin,isoliquiri— tigenin,andisoliquiritinwerekindlyprovidedbyDr.Liu H.X..PekingUnionMedicalCollege,ChineseAcademy ofMedicalSciences,InstituteofMedicinalPlantDevel— opment,Beijing,China.Ginseng,Baizhu,Fuling,and GancaowerepurchasedfromChineseTraditionalMedicine PharmacyTongrentang,HongKong,China.Thesol— vents,acetonitrile,andmethanolwereofHPLCgrade. Otherreagentswereofanalyticalgrade.Ultra—purewater wascollectedfromaMilli-Qsystem(Millipore,Bedfozrl, MA,USA). Chromatographiccondition AnYMCODSALcolumn(100mm×2.0mm,ID 5tun)(YMC,Kyoto,Japan)wasusedfortheHPLC analysiswithasampleinjectionvolumeof10taL.Agilent 1100pumpsystemwithadiode-arraydetector(Agilent Technologies,Inc.,PaloAlto,CA,USA)wasused. Themobilephaseconsistedof(A)water-formicacid (100:0.04,V/V)(pH3)and(B)acetonitrile.Agra- dientprogramof0——5minat10%ofBand45minto 90%ofBwasused.Theanalysiswascarriedoutata flowrateof200taL?min一atrDomtemperaturewiththe UVdetectionat280nlnand368nm,r6spectively. Samplepreparation Ginseng,Baizhu,Fuling,andGancaowerefirstho— mogenized.ThepowderedsamplesofGinseng(0.4g), Fuling(0.4g),Baizhu(0.4g),andGancao(0.2g) wereitrmaersedin140mLdeionizedwaterforlhand thendeeoetedtoboil,keepingboilingfor30min.This procedurewasrepeatedtwice.Thecombinedwaterextract wascondensedandthendilutedwithethanoltoVea 50%ethanolsolution.Theresultingsolutionwasfiltered afterethanolprecipitation.Ethanolwevaporatedand theresiduewasdissolvedinwatertoproducethesample solution(25mgcrudedrug’mL). Standardsolutions Astockstandardsolutionofliquiritigenin(67g’ mL),liquiritin(205?mL),isoliquiritigenin(55 ?mL),andisoliquiritin(102’mL)waspre— paredinwater:methanol(1:2).Bydilutingthestandard solutionwithwater:methanol(4:1),calibrationsolutions atlevelsof0.335—67g.mLofliquiritigenin, 1.025—205g’mL一ofliquiritin,0.275—55g’ mL,ofisoliquiritigenin.and0.5I一102/4. g’mL,of isoliquiritinwereprepared. Recovery Recoverywasdeterminedbythemethodofstandard addition(1iquiritigenin0.42,0.84,1.68’mL,, liquiritin5.13,10.25,20.50g’mL,,isoliquiritige— nin0.34,0.69,1.38’mI,,andisoliquiritin0.64, 1.28,2.55ttg?mL). Allsampleswerefilteredthrough0.2p.mfilters (Millipore)andintroducedtoHHC.Thesamplesolut— ionswerestoredat4.【=andusedatroomtemperature. Table1Linearregressionresults Table2Intra-andinterdayRSDs(n=5) Table3Recoveriesofllquirifigenin,liquiritin,is(diqlli—tige- nin,andisoHquir~inethylacetateextractofSUun~decoction ThechromatogramofSijunzidecoctionisshownin LIUyang,etaJ:DotorminationofLiquiritig~nin.Liquiritin,Isoliquiritigenin andlsoliquiritininExtractofTraditional ChineseMedicine$ijunziDecoctionbyHigh-PefformanceLiquidChromatography229 Figure1(1iquiritin18.2min,liquiritigenin23.1min, isoliquiritin22.2min,andisoliquiritigenin28.2rain). TheregressionequationsalegiveninTable1,which showedgoodlinearrelationshipsbetweenthepeakalias andconcentrations.TotestthestabilityofSijunzidecoc— tionandtheprecisionofthemethod,detectionsofthe4 markersinsampleusingthesan3ereagentsandinstrument wereperformedinfiveconsecutivedays(Table2).We usedS/N>3tocalculatethelowerlimit-ofdetection. whichwas55ng’IIlL,forisoliquiritigenin.102ng’ mL,forisoliquiritin.67ng’IIlL,forliquiritigenin.and 205ng.mL,forliquiritin.Wealsopreparedotherthree Sijunzidecoctionsamplesunderthesan3econditionto evaluatethestabilityandreproducibilityofsampleprepa- ration,whichgavesatisfactoryresults(RSD:7%of isoliquiritin,7%ofisoliquiritigenin,9%ofliquiritin, and6%ofliquiritigenin.n=4).TheSijunzidecoction (25ITlg’mL)contained1.47ttg?IIlL,liquiritigenin, 16.4’IIlL,liquiritin,0.66ttg’mL,isoliquiri— tigenm.and2.12Pg.mL,isoliquiritin.Thestandard additionmethodwasusedfordeterminingtheaccuracyof l2. l.. 8o 暑6. 40 20 o themethod.Recoveriesarecalculatedinrable3.Analy. sisoftherCMpreparationshowedacceptableprecision andaccuracy. GancaoandFulingcontaintriterpenicacidsand Gancaoalsocontainsflavonoids.Triterpenicacidsand flavonoidshaveacidicca/boxylandphenolichydroxyl groups.Therefore,anacidmobilephasewasemployedto enhancetheirretentionandavoidthetailingpeakinthe analysis.Thestandardshadhighsolubilityinmethano1. 111epuremethanolsolutioninjectedproduceddistorted chromatogrampeaksoffourmarkers,especiallyatlower concentrations.Thusweusedamixtureofwaterand methanoltore—dissolvethestandardcompounds Conclusion Thedescribedmethodwasfoundtobesimple,accu? rate,andreproduciblewithagoodlinearrange.Thedata fromthevalidationexperimentswereexcellent.Themeth— odcouldbeusedtoquantify4markercomponentssimul— taneouslyinSijunzidecoction. 10203040l0203040 t/nlin Figure1OIrwatogramof跚lln商 decoctionatdifferentUvwavelmgflts.Peak1:Liquififin;Peak3:Liquiritigenin ;Peak2: Isoliquififin;Peak4:Isoliquiritigenin References [1]GengY,RenJP,ChenGz.111eeffectofJiaweiSijunziTang (JWSJZT)onlearningandmemo~inmice[J].Pharmacol ainChinMaterMed,1997,13(6):112. 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[10]TakahashiT,TakasukaN,IigoM.Isoliquiritigenin.afla— vonoidfromlicoriee,reducesprostag]landinE2andnitricOX— ide,causesapoptosis,andsuppressesaberrantcryptfoeide— velopment[J].CancerSci,2OO4,95(5):448453. 111JZuoFZ,ZhouM,YanMZ.Metabolismofconstituentsin HuangqinTang,aprescriptionintraditionalChinesemedi— cine,byhumanintestinalflora[J].BiolPhannBull,20o2, 25(5):558—563. 112jZuoF,ZhouZM,ZhangQ.Phannacokinetic~tudyonthe multi-eonstituentsofHuangqindecoctioninrats[J].Biol 口册成,砌.26(7):911-919. 高效液相色谱法对中药复方四君子汤中异甘草素,甘草素, 异甘草苷和甘草苷的含量测定 刘扬,杨峻山 (中国协和医科大学中国医学科学院药用植物研究所,北京100094) 摘要:目的为中药复方四君子汤建立简便的液相分析方法.对复方提取物中异甘草素,甘草素,异甘草苷和甘草苷 进行定量分析.方法色谱条件为(A)O.04%甲酸溶液(pH3)和(B)L腈;0—5min10%B,45min达到90%B;在室温下 c.反相柱.流速为O.2mL?min实现分离和含量测定;检测波长为280n/ll和368nm.结果四君子汤提取物中(25nag?mL.) 含有1.47Pg?mL甘草素,l6.40?mL甘草苷,O.66,ug?mL异甘草素,2.12?mL异甘草苷;回收率分别为lo2.2%, 97.7%,100.3%,和99.9%.结论本文首次报道复方四君子汤中多个黄酮类成分的含量测定. 关键词:中药;四君子汤;高效液相色谱法