莪术油口服吸收机理及制剂前研究-8

ThesisforMaster’sdegreeatUniversityofMacau23CHAPTER2:ANALYTICALMETHODSOFZEDOARYTURMERICOIL2.1.Materials2.1.1.CHEMICALSZedoaryTurmericOilwaspurchasedfromTianreiPharmacyLimitedCompany,ZhejiangProvince,China.Curdione,germacrone,furanodiene,curcumolwereseparatedandpurifiedfromInstituteofChineseMedicalSciences,UniversityofMacau(Macau,China)withthemethoddescribedbyF.Q.Yang(2005).Thepurityofabovepurecompoundswasmorethan98%andthestructureswereconfirmedbycomparingtheirUV,EI-MSandNMRdatawithliteratures.AcetonitrileandmethanolforHPLCanalysiswerepurchasedfromMerck(Darmstadt,Germany).WaterforHPLCanalysiswaspreparedusingaMilliporeMilli-Q®Plussystem(Millipore,Bedford,MA,USA).Otherchemicalreagentswereusedatleastofanalyticalgrades.2.1.2.INSTRUMENTSAgilentSeries1100(AgilentTechnologies,USA)liquidchromatograph,equippedwithavacuumdegasser,aquaternarypump,anautosamplerandadiodearraydetection(DAD)system.TheHPLCcolumnemployedwasanAgilentZORBAXODScolumn(250mm×4.6mmI.D.,5μm)withaZORBAXODSguardcolumn(20mm×3.9mmI.D.,5μm).ThesisforMaster’sdegreeatUniversityofMacau242.2.Methods2.2.1.ANALYTICALMETHODSOFZRDOARYTURMERICOILANDITSPURECOMPOUNDS2.2.1.1.HPLCANALYSISOFZEDOARYTURMERICOILThedeterminationofZedoaryTurmericOilwasconductedbyHPLC-DAD.ThemobilephaseforHPLCanalysisofZedoaryTurmericOilconsistedoftwosolventcompositions,water(solventA)andacetonitrile(solventB).Theflowrateofthemobilephasewassetat1.0ml/minwithinjectionvolumeof50μlandthecolumntemperatureat25℃.Thedetectivewavelengthwas214nm.TheHPLCconditionusedforZedoaryTurmericOilanalysiswaslistedinTable2.1.Table2.1:ElutionconditionofZedoaryTurmericOilTime(minutes)0152545SolventA(%)4535200SolventB(%)556580100Figure2.1a:HPLCchromatogramofZedoaryTurmericOilmin0510152025303540mAU020406080100120140ThesisforMaster’sdegreeatUniversityofMacau25Figure2.1b:UVspectrumofcurdioneinZedoaryTurmericOilFigure2.1c:UVspectrumofgermacroneinZedoaryTurmericOilFigure2.1d:UVspectrumoffuranodieneinZedoaryTurmericOilnm200225250275300325350375mAU0100200300400nm200225250275300325350375mAU0100200300400500600200225250275300325350375mAU0100200300400500600700800nmThesisforMaster’sdegreeatUniversityofMacau262.2.1.2.HPLCANALYSISOFINDIVIDUALPURECOMPONENTOFZEDOARYTURMERICOILThechromatogramconditionsofindividualpurecomponentinZedoaryTurmericOilweresimilartotheconditionoftheessentialoil.Themobilephaseconsistedofwater(solventA)andacetonitrile(solventB).Flowrateisat1.0ml/minwithinjectionvolumeof50μlandcolumntemperatureat25℃.Thedetectivewavelengthis214nmwithUVdetection.TheHPLCconditionsusedforanalysisofindividualpurecomponentinZedoaryTurmericOilanalysiswerelistedinTable2.2.Table2.2:ElutionconditionsofindividualpurecomponentinZedoaryTurmericOilCompoundsTime(minutes)SolventA(%)SolventB(%)Curdione04555153565Furanodiene02080150100Germacrone04555153565252080Curcumol04555153565252080ThesisforMaster’sdegreeatUniversityofMacau27Figure2.2a:HPLCchromatogramofcurdioneFigure2.2b:HPLCchromatogramoffuranodieneFigure2.2c:HPLCchromatogramofgermacronemin02468101214mAU020406080100120140160min02468101214mAU020406080100120140160min02.557.51012.51517.52022.5mAU050100150200250300350ThesisforMaster’sdegreeatUniversityofMacau28Figure2.2d:HPLCchromatogramofcurcumolmin02.557.51012.51517.52022.5mAU012345ThesisforMaster’sdegreeatUniversityofMacau29Figure2.2e:UVspectrumofcurdioneFigure2.1f:UVspectrumoffuranodieneFigure2.1g:UVspectrumofgermacronenm200225250275300325350375mAU050100150200250300350nm200220240260280300320340360380mAU0255075100125150175nm200225250275300325350375mAU0100200300400500ThesisforMaster’sdegreeatUniversityofMacau30Figure2.1h:UVspectrumofcurcumol2.2.2.PREFORMULATIONSTUDYOFZEDOARYTURMERICOILPreformulationstudieswerecarriedouttochooseappropriateexperimentalconditionsfortransportstudiesofZedoaryTurmericOilanditsindividualpurecomponent.Phosphatebufferedsalineplus(PBS+)solutionwasusedasthetransportbufferinthetransportstudiesusingCaco-2cellmodel.Itwaspreparedbydissolving1ofphosphatebufferedsalinetabletwhichcontaining0.01Mphosphatebuffer,0.0027Mpotassiumchlorideand0.137Msodiumchloridein200mlwater.Thencalciumchlorideandmagnesiumchloridewereaddedtoreachafinalconcentrationof0.9mMand0.4mMrespectively.ThepHvalueofthebufferwasattainedatpH7.4.2.2.2.1.DETERMINATIONOFPROPORTIONOFEACHCONSTITUENTSThemainconstituentsinZedoaryTurmericOilwerecurdione,curcumenol,germacrone,curzerene,furanodieneandβ-elemeneetcaccordingtotheirpeakareaintheHPLCchromagramofZedoaryTurmericOil.AstheactualproportionofabovepurecompoundsinZedoaryTurmericOilwasnotclear,itisnecessarytoevaluatetheirpositivecontentsinZedoaryTurmericOiltodeterminetheirloadingconcentrationsinthetransportexperiments.ZedoaryTurmericOilandtheactivecompoundsincludingcurdione,germacrone,furanodieneandcurcumolweredissolvedinmethanolrespectivelytoreachthesamenm200225250275300325350375mAU2468101214ThesisforMaster’sdegreeatUniversityofMacau31concentrationof100μg/ml.ByHPLC-DADanalysis,areapercentagesofthesamecomponentsinZedoaryTurmericOilagainstinpurecompoundswerecalculatedtoconfirmtheproportionofeachconstituentmentioned