超临界流体萃取_高效液相色谱法测定百合中秋水仙碱_英文_何纯莲

1、2003Vo1.15No.1天然产物研究与开发NATURALPRODUCTRESEARCHANDDEVELOPMENT5SUPERCRITICALFLUIDEXTRACTION-HIGHPERFORMANCELIQUIDCHROMATOGRAPHYDETERMINATIONOFCOLCHICINEINLILYHEChun-lian1LIGu-caiRENFeng-lianLIXiang-qun222(1.Medicalcollege,HunanNormalUniversity,Changsha410006,China;2.CollegeofChemistryandChemicalEnginee

2、ring,CentralSouthUniversity,Changsha410083,China)AbstractColchicinewasextractedfromthebulbsoflilybyusingsupercriticalcarbondioxidefluidextraction(SFE)andtraditionalorganicsolventextraction(OSE)respectively.Itscontentsinthetwoextractsweredeter-mineddirectlybyhighperformanceliquidchromatographyandthec

3、olchicinecontentinthecormsoflilywasobtained.TheSFEconditionswereasfollows.Pre-soaker:ethanol,extractionpressure:18MPa,extractiontemperature:40.TheHPLCconditionswere:column:HypersilODS5m,mobilephase:CH3OH:KH2PO4(0.05mol/L)=5050,detectionwavelength:220nm.Themethodissimple,rapid,accurateandcanbeusedtod

4、eterminethecontentsofcolchicineincolchicinecrudedrugs,pharmaceuticalpreparationsandotherplants.Atthesametime,theresultalsosuggestedthatitispossibletoextractcolchicinefromthebulbsoflilyinin-dustryanditmaybringaboutagreatereconomicefficiency.KeywordsSupercriticalfluidextraction;highperformanceliquidch

5、romatography;colchicine;lilyIntroductionInChina,lilyhasbeenusedformorethantwothou-sandyearsasafolkmedicine.Modernmedicalstudiesshowedthatlilycontainscolchicinewhichisacompo-nentwithbiologicalactivity.Asaprincipaltropholonealkaloid,itcanrestraincell'smitosisandDNA'ssyn-thesis.Italsocanbeusedt

6、oholdbackcancercell'sgrowth.Inclinics,it'susuallyservedasananticanceragentandithasavarietyofmedicalpurposeinthetreatmentsofbreastcancer,skincancer,leukemia.Hodgkin'sdisease,FamilialMediterraneanFeverandotherdiseases.Especially,itisanimportantandeffec-tivepainreliefforacutegoutyarthritisa

7、nditmayworkin1224hours.Inagriculture,colchicineiswidelyusedinpolyploidbreedingandit'salsoanec-essaryagentincytobiologicaltechniques.Atpresent,themajormethodsforthedeterminationofcolchicineareultravioletspectrometry1,fluorime-try,polarography,voltammetry,etc.Amongthem,theultravioletspectrometryis

8、anofficialmethodforthedeterminationofcolchicineinthePharmacopoeiaofthePeople'sRepublicofChina(1990,thesecondpart).However,becausethechemi-calconstituentsoflilyaretoocomplicated,thecontentofcolchicineinthebulbsoflilyisnotdeterminedre-portedly.Therefore,itisnecessaryandmeaningfultodeterminethecolc

9、hicinecontentinlily.Thepurposeofthisstudyistoestablishanewwaywhichissim-ple,rapidandaccurateforthedeterminationofcolchicineinthebulbsoflily.2341Experimental1.1InstrumentsandreagentsHA-120-40-0.5supercriticalfluidextractionappara-tus(madebyGuizhouProvince,WujiangMachine,h收稿日期:2002-09-25修回日期:2002-11-2

10、2HeChun-lian(1969-),woman,xinshaoCounty,HunanProvince,lecturer.Majorstudydirections:instrumentalanalysisandnaturalprod-ucts.6天然产物研究与开发2003Vo1.15No.1(HP1100),RE-52Arotaryevaporator(madebyShanghaiYayongBiochemistryInstrumentsPlant),CSF-1Aultra-sonicgenerator(ShanghaiUltra-sonicInstrumentsFactory),bulb

11、soflily(cultivatedinLongshan,HunanProvince),standardcolchicinesam-ple(99%purity,gotfromShanghaiChem.ReagentsCo.),HPLCgrademethanol(SuppliedbyWujingChemicalsWork),theotherchemicalsandagentswereanalyticallypure.1.2ExtractionAfterthefreshbulbsoflilywerewashedcleananddriedat50forabout12hours,thentheywer

12、egroundto20meshsizeforuse.Inconsiderationofcolchicine'spolarityandalkales-cence,itmaycombinewithplant'sacidiccomponentsandexistintheformofsaltsinplant.sothelilypow-dermustbealkalifiedbyammonialiquororotheral-kalinereagentsbeforeextractioninordertomakecolchicineexistasfreealkalianditwillmakee

13、xtracteasierandmorefully.Moreover,duetoitsbiggermolecularweight,itsdissolvabilityincarbondioxidefluidissmall.Thus,properpre-soakermustbeem-ployedinSFEprocesssoastoincreasethefluid'sdis-solvingcapacityandselectivity.Apolarsolventsuchasmethanol,ethanolmaybesuitable.Aftercondition-alexperimentswere

14、made,ethanolwaschosenasabetterentrainer.1.2.1SupercriticalCO2colchicinefluidextractionof1.2.2OrganicsolventextractionofcolchicineAfterhavingbeenalkalified,lilypowder(50g)wasextractedwith95%ethanolatabout85for5hours.Nextthesolutionwasfilteredandevaporatedunderreducedpressure.Thentheresiduewasdis-solv

15、edinwater(adjustitspHvalue)followedbyex-tractionwithchloroform(3×50mL)andfinallyOSEextractingliquorwasobtained.1.3HPLCdeterminationofcolchicinecontentTheSFEandtheOSEextractingliquorswereevapo-ratedunderreducedpressurerespectivelyandtworesidueswereproduced.Afterweighedaccurately,theresidueswered

16、issolvedina50mLcontainerwithmethanol,andthenfilteredthrougha0.45mHPLCfilter.Thefilterliquorsweredegassedbyanultra-sonicgeneratorbeforedetermination.TheHPLCde-tectionconditionswereasfollows:Column:HypersilODS5mMobilephase:CH3OHKH2PO4(0.05mol/L)=5050Temperature:25Pressure:173barFlowrate:1mL/minInjecti

17、onvolume:10LDetectionwavelength:220nmThetwoextracts'weightsandcolchicinecontentsweregivenintable2.Table2Theresidues'weightsandcolchicinecontentsExtractweight(mg)SFEOSE380652Colchicinecontentinextract(%)6.383.66Colchicinemassfractioninlily(%)0.04850.0477TheSFEprocesswasasfollows:theweighedlil

18、ypowder(50g)wassoakedinammonialiquorfullyfor30minutesbeforebeingloadedintoa500mlhighpressurestainlesssteelextractingvessel.Thenthesamplewaspre-soakedbyethanolforanother30min-utesbeforeextractedbysupercriticalCO2fluidfor1.5hours.Thespecificextractionconditionswerelistedintable1.Table1SupercriticalCO2

19、fluidextractionconditionsExtractionpressure(MPa)186.57.535Extractiontemperature()404040CO2flowrate(m3/h)2.12.12.1Alltheresultswereanaveragesof3parallelexperiments'.2ResultsanddiscussionTheHPLCanalysesofdifferentcolchicinesamplesweremadeaccordingtotheabovechromatographyconditionsandtheirchromatog

20、rammapswereshowninFig.14.Thecolchicinewasidentifiedbyitsretentiontimeinchromatogrammapincomparisonwithpurecolch-icinesample's,andthequantitativeanalysesweredoneinaccordancewithchromatogramcalculations.ExtractionkettleLiberationkettleLiberationkettle2003Vo1.15No.1何纯莲等:超临界流体萃取高效液相色谱法测定百合中秋水仙碱7Fig.

21、1HPLCanalysisofthestandardcolchicinesampleFig.2HPLCanalysisofSFEsample(pre-soakedwithethanol)Fig.3HPLCanalysisofSFEsamplewithaddingstandardcolchicinesampleFig.4HPLCanalysisofOSEsampleTable2showedthatthetotalcolchicineintheSFEextractwasalittlemorethanthatintheOSEex-tract.Itcouldbeattributedtothehighe

22、rextraction,posedthatmoreconstituentswerecontainedinOSEextractthaninSFEextract.ThedifferencebetweenFig.2andFig.4suggestedthatSFEhashigherselec-8天然产物研究与开发2003Vo1.15No.1easier.Moreimportant,theprocesswascarriedoutatlowertemperature(soitdidnotdestroyconstituents'exist-ingform)andthesamplelossinSFEp

23、rocesscanbereducedgreatlyandtheHPLCdeterminationisadi-rectassay.Sothecontentofcolchicineinthebulbsoflilycanbedeterminedaccuratelybythecombinedmethod.it.Italsocanbepredictedthatthiscombinedmethodcanbeusedtoextract-determineothercomponents'contentinplants.Reference1PharmacopoeiaEditoralBoardofMini

24、stryofPublicHealthofthePeople'sRepublicofChina.PharmacopoeiaofthePeople'srepublicofChina,1990,Part2(inChinese)Bei-jing:ChemicalIndustryPress,1990.3592LiuYong-ming,LiGui-Zhi.DeterminationofcolchicineinTabletbyFluorimetry.ChineseJournalofAnalyticalChem-istry(inChinese).2000,28(3):3303323HeShi-hong,RenFeng-lian,SongGe.DeterminationofColchicinebySecondaryDerivativePolarography.NaturalScienceJournalofXiangtanUniversity(inChinese).2001,23(4):78804YuSu-hua,HuXiao-ya,LengZong-zhou.Me