硅胶柱色谱结合高速逆流色谱法分离纯化荷花中黄酮类化合物徐双双

201112Vol．29No．12December2011ChineseJournalofChromatography1244～1248DOI10．3724/SP．J．1123．2011．01244*[e-mail]．2010GSF10287201004010．2011-09-03121313*1．2710182．2503553．2500143。---4∶1∶5∶0.025v/v/v/v800r/min、2.0mL/min、254nm150mg6.1mg-3-O-β-D-I14.8mg-3-O-β-D-II20.2mgIII97.0%、95.4%、96.3%、。、、。-3-O-β-D--3-O-β-D-O658A1000-8713201112-1244-05SeparationandpurificationofflavonesfromNelumbonuciferaGaertn．bysilicagelchromatographyandhigh-speedcounter-currentchromatographyXUShuangshuang1SUNYu2JINGFeng1DUANWenjuan3DUJinhua1WANGXiao3*1．CollegeofFoodScienceandEngineeringShandongAgriculturalUniversityTaian271018China2．CollegeofPharmacyShandongUniversityofTraditionalChineseMedicineJinan250355China3．AnalysisandTestCenterShandongAcademyofSciencesJinan250014ChinaAbstractThreeflavoneswereisolatedandpurifiedfromNelumbonuciferaGaertn．bythecombinationofsilicagelchromatographyandhigh-speedcounter-currentchromatographyHSCCC．ThecrudeextractofN．nuciferawasseparatedbysilicagelchromatographyandthefractioncontainingflavoneswasobtained．ThenthefractionwasseparatedbyHSCCCwithtwophasesolventsystemscomposedofethylacetate-ethanol-water-aceticacid4∶1∶5∶0.025v/v/v/v．Theupperphasewasasthestationaryphaseandthelowerphaseasthemobilephase．Undertheconditionsofaflowrateof2.0mL/minwhiletheapparatusrotatedat800r/minandthedetectionwavelengthwasat254nm6.1mgofquercetin-3-O-β-D-glucuronide14.8mgofmyricetin-3-O-β-D-glucopyranosideand20.2mgofastragalinwereobtainedfrom150mgofthecrudesampleinonestep．ThepuritiesdeterminedbyhighperformanceliquidchromatographyHPLCwere97.0%95.4%and96.3%respectively．ThestructuresofthetargetcompoundswereidentifiedbyelectrosprayionisationmassspectrometryESI-MS1H-nuclearmagneticresonance1H-NMRand13C-nuclearmagneticresonance13C-NMR．Thismethodthathaspracticalvaluenotonlysavessolventbutalsoisconvenient．ItiseffectiveintheseparationofflavonesfromN．nuciferaandprovidestheoreticalfoundationforthefurtherdevelopmentanduseofN．nuciferaresources．Keywordssilicagelchromatographyhigh-speedcounter-currentchromatographyHSCCC12quercetin-3-O-β-D-glucuronidemyricetin-3-O-β-D-glucopyranosideastragalinNelumbonucif-eraGaertn．NelumbonuciferaGaertn．、、Nymphaeaceae、、1。。。2。Yang3-/HPLC-DAD/MS15。454。。high-speedcounter-currentchromatographyHSCCC-、67。HSCCC、、。HSCCC、3-3-O-β-D-I、-3-O-β-D-IIIII1。13Fig．1Chemicalstructuresofthe3flavonecompounds11．1GS10A、2.3mm230mLβ0.5～0.8、TBP、8823B-、3057-11Waters600-996PDAWa-tersVarianINOVA-600VarianAgilent1100Series6320ion-trapAgilent。、、200～300。。1．2。5kg4095%8L2h3。600mL、390.5g4℃。1．340g-100∶160∶140∶120∶110∶15∶·5421·2913∶11∶1HPLCF1～F88TLCHPLCF7。1．4HSCCC1．4．1---4∶1∶5∶0.025v/v/v/v1000mL30min。1.3F7150mg5mL。1．4．2KDKD82mgF710mL2mL1min5μLHPLCA1A2KD=A1/A2。1．4．3HSCCC20mL/minHSCCC800r/min2mL/min254nm。10mLF7HSCCC。1．5HPLCInertsilODS-SP250mm×4.6mm5μm。25℃。AB0.1%v/v0～25min30%A～90%A1.0mL/min10μL254nm。1．6HSCCCESI-MS、1H-NMR13C-NMR。0.2mL/min350℃9.0L/min24.1Pa4kVm/z50～1200。DMSOTMS。22．1-、-TLC2---。2Fig．2TLCchromatogramsofthecrudeextractofN．nuciferaDevelopingsolventsa．chloroform-methanolb．petroleumether-ethylacetate．2．2HSCCCHSCCCKD。HSCCCKD0.5～2.09。1.4---、--、--3KD。--4∶1∶5v/v/vKDKD。3KD---4∶1∶5∶0.01v/v/v/vKDKD---4∶1∶5∶0.025v/v/v/vKD1。。III。2.0mL/min。·6421·1213Table1PartitioncoefficientsKDofthe3flavonecompoundsinseveralsolventsystemsSolventsystemIIIIIIPetroleumether-ethylacetate-methanol-water1∶1∶1∶1v/v/v/v8．38．711．2Ethylacetate-methanol-water4∶1∶5v/v/v5．67．17．4Ethylacetate-ethanol-water4∶1∶5v/v/v3．95．16．7Ethylacetate-ethanol-water-aceticacid4∶1∶5∶0．01v/v/v1．13．05．4Ethylacetate-ethanol-water-aceticacid4∶1∶5∶0．025v/v/v/v0．81．92．9ForcompoundsI－IIIseeFig．1．4aF7b-3-O-β-D-I、c-3-O-β-D-IIdIIIHPLCFig．4HPLCchromatogramsofathecomponentF7bquercetin-3-O-β-D-glucuronideIcmyricetin-3-O-β-D-glucopyranosideIIanddastragalinIII2．3HSCCC---4∶1∶5∶0.025v/v1.41.3HSCCCHSCCC3。HPLCI～III6.1、14.8、20.2mg97.0%、95.4%、96.3%。3Fig．3HSCCCchromatogramofflavonecompoundsfromNelumbonuciferaGaertn．I．quercetin-3-O-β-D-glucuronideII．myricetin-3-O-β-D-glu-copyranosideIII．astragalin．2．4HPLC-、--0.1%。AB0.1%0～25minA30%90%1.0mL/minF7-3-O-β-D-、-3-O-β-D-、HPLC4。2．5IESI-MSm/z479M+H+1H-NMRDMSO-d6600MHzδ7.601HsH-2'7.591HdJ=7.2HzH-6'6.841HdJ=7.2HzH-5'6.421HsH-86.221HsH-65.491HdJ=5.4·7421·29HzH-1″13C-NMRDMSO-d6150MHzδ156.3C-2133.2C-3177.2C-4161.2C-598.8C-6164.3C-793.6C-8156.4C-9103.9C-10120.8C-1'115.3C-2'144.9C-3'148.6C-4'116.2C-5'121.6C-6'101.2C-1″75.8C-2″75.9C-3″71.4C-4″73.8C-5″170.0C-6″。10-3-O-β-D-1。IIESI-MSm/z481M+H+1H-NMRDMSO-d6600MHzδ6.191HdJ=1.2HzH-66.371HdJ=1.2HzH-87.192HsH-2'H-6'5.461HdJ=7.8HzH-1″13C-NMRDMSO-d6150MHzδ156.7C-2133.8C-3177.8C-4161.7C-599.0C-6164.5C-793.7C-8156.6C-9104.3C-10120.4C-1'108.9C-2'145.8C-3'137.0C-4'145.8C-5'108.9C-6'101.2C-1″71.3C-2″76.9C-3″70.3C-4″78.0C-5″60.9C-6″。11-3-O-β-D-1。IIIESI-MSm/z449M+H+1H-NMRDMSO-d6600MHzδ6.441HdJ=1.2HzH-86.221HdJ=1.2HzH-68.042HdJ=9.0HzH-2'H-6'6.892HdJ=9.0HzH-3'H-5'12.621Hs5-OH10.88brs7-OH10.20brs4'-OH5.481HdJ=7.8HzH-1″3.08～3.586HmH-2″～6″13C-NMRDMSO-d6150MHzδ156.7C-2133.6C-3177.9C-4161.6C-599.1C-6164.5C-794.0C-8156.7C-9104.4C-10121.3C-1'13