全 文 ：天然产物研究与开发 NatProdResDev2009, 21:822-825, 839
文章编号:1001-6880(2009)05-0822-05
ReceivedMay15, 2008;AcceptedOctober14, 2008
＊CorespondingauthorTel:86-21-62479808-463;E-mail:yangpeiming
@yahoo.cn
高速逆流色谱法分离凤尾草中的芹菜素和木犀草素
欧阳丹薇 ,孔德云 ,许海燕 ,倪 翔 ,陈 佳 ,杨培明＊
上海医药工业研究院中药室 ,上海 200040
摘 要:建立了高速逆流色谱分离纯化芹菜素和木犀草素的方法。两相溶剂系统为氯仿-甲醇-水(4∶3∶2), 上相
为固定相 , 下相为流动相进行洗脱。从 100mg粗提物中一步分离得到 17.0 mg芹菜素和 22.5 mg木犀草素 ,经
高效液相色谱分析 ,纯度分别为 92%和 96%。其化学结构由 1H和 13CNMR鉴定。
关键词:高速逆流色谱;凤尾草;黄酮;芹菜素;木犀草素
中图分类号:R284.2;Q946.91 文献标识码:A
IsolationandPurificationofApigeninandLuteolinfromPteris
multifidaPoir.byHigh-speedCounter-currentChromatography
OUYANGDan-wei, KONGDe-yun, XUHai-yan, NIXiang, CHENJia, YANGPei-ming＊
DepartmentofChineseTraditionalMedicine, ShanghaiInstituteofPharmaceuticalIndustry, Shanghai200040 , China
Abstract:High-speedcounter-currentchromatography(HSCCC)wasappliedtoisolateandpurifybioactivecompounds
fromPterismultifidaPoir.Twoflavonoidsapigeninandluteolinweresuccessfullyisolatedandpurifiedbyusingtwo-
phasesolventsystem, whichwascomposedofchloroform-methanol-water(4∶3∶2, v/v/v)inheadtotailelutionmode.
Consequently, 17.0 mgofapigeninand22.5 mgofluteolinwereobtainedfrom 100 mgcrudesamplewithpuritiesof
92% and96%, respectively, asdeterminedbyHPLC.Thechemicalstructureswereidentifiedby1Hand13CNMR.
Keywords:high-speedcounter-currentchromatography;PterismultifidaPoir.;flavonoids;apigenin;luteolin
Introduction
Fengweicao, thedriedherbofPterismultifidaPoir., is
atraditionalChinesemedicine.Itcouldbeeasilyfound
andwidelyusedinChina.Ithastheeficacyofenteri-
tis, hepatitis, bacterialdysentery, haematemesis, haema-
turia, tonsilitis, parotitisandeczema[ 1, 2] .Theefective
componentsofthisherbareflavonoids, sesquiterpe-
noidsandditerpenoids[ 3, 4] .Pharmacologicaltestre-
vealedthattheflavonoidsapigeninandluteolinhave
efectsofanti-bacterialandanti-virus[ 5] .
High-speedcounter-currentchromatography(HSCCC)
isanal-liquidchromatographictechniquebasedon
partitioningofcompoundsbetweentwoimmiscibleliq-
uidphases.HSCCCdoesnotemployasolidphasesup-
portandthusthereisnoirreversibleadsorptionassoci-
atedwiththesolidsupports.Inaddition, itoperatesun-
derverygentleconditionsandalowsnon-destructive
separationofcompounds[ 6] .Asaresult, thistechnology
hasrecentlygainedincreasinglyimportanceinthesep-
arationofnaturalyoccuringproducts, andhasbeen
succesfulyappliedtotheseparationofanumberof
naturalproducts[ 7-10] .Althoughapigeninandluteolin
havebeenpurifiedfromP.multifidaPoir., noreport
hasbeenrevealedconcerningusageofHSCCCforthe
isolationandpurificationofapigeninandluteolinfrom
P.multifidaPoir..Therefore, thisstudyaimstodevelop
aneficientmethodfortheisolationandpurificationof
apigeninandluteolinwithhighpuritiesfromP.multifi-
daPoir.byHSCCC.
Fig.1 Chemicalstructuresofapigeninandluteolin
DOI:10.16333/j.1001-6880.2009.05.030
Experimental
Apparatus
TheHSCCCinstrumentemployedinthepresentstudy
isTBE-300Ahigh-speedcounter-currentchromatogra-
phy(ShanghaiTautoBiotech, Shanghai, China), with
threepreparativemultilayercoilsseparationcolumn
connectedinseries(i.d.ofthetubing=1.6 mm, total
volume=260 mL)anda20mLsampleloop.Theβ val-
uesofthispreparativecolumnrangefrom0.5to0.8(β
=r/R, R=5 cm, whereristhedistancefromthecoil
totheholdershaft, andR, therevolutionradiusorthe
distancebetweentheholderaxisandcentralaxisofthe
centrifuge).Therevolutionspeedoftheapparatuscan
beregulatedwithaspeedcontrolerintherangebe-
tween0 and1000 r/min.AModelHX-1050 constant-
temperaturecirculatingimplement(BeijingBoyikang
LabInstrumentCompany, Beijing, China)wasusedto
controltheseparationtemperature.Thetwo-phasesol-
ventwaspumpedintothecolumnwithaModelTBP-
50Aconstant-flowpump(ShanghaiTautoBiotech,
Shanghai, China).Continuousmonitoringoftheeflu-
entwasachievedwithaModel8823BUVdetector
(BeijingBingdayingTechnology, Beijing, China).A
N2000workstation(ZhejiangUniversityZhidaInforma-
tionTech, Hangzhou, China)wasemployedtorecord
thechromatogram.
Thehigh-performanceliquidchromatography(HPLC)
usedwasaWaters510 pump, aWaters2487 dualλ
absorbancedetector, a7725iinjectionvavlewitha20
μLsampleloop, andMilennium32 HPLCworkstation.
Reagentsandmaterials
AlorganicsolventsusedforHSCCCwereofanalytical
gradeandpurchasedfromShanghaiZhenxinChemical
Factory(Shanghai, China).MethanolusedforHPLCa-
nalysiswasofchromatographicgrade, andwaterused
wasdistiledwater.
Preparationofcrudeextract
P.multifidaPoir.waspurchasedfromShanghaiHuayu
PharmaceuticalCompanyLtd.andidentifiedbyDr.
TongWu(ShanghaiInstituteofPharmaceuticalIndus-
try, Shanghai, China).
TheaerialpartofP.multifidaPoir.(24.0kg)wasex-
tractedtwicewith70% aqueousethanolunderreflu-
xing(2 hforeachtime).Thentheextractwasfiltered
andevaporatedto55 Lwatersolution.Thewatersolu-
tionwaschromatographedonD101 macroporousresin
column, elutedwithdeionedwater, 25% ethanol, 35%
ethanol, 50% ethanoland95% ethanol.Theefluentof
50% ethanolwascolectedandevaporatedundervac-
cum.Crudesample(28.5g)wasobtainedandstoredin
arefrigeratorforthesubsequentHSCCCseperation.
Selectionoftwo-phasesolventsystem
SelectingthesolventsystemforHSCCCmeanssimulta-
neouslychoosingthestationaryphaseandthemobile
phase.Thesolventsystemwasselectedaccordingtothe
partitioncoeficients(K)ofthetargetcompounds.The
K-valuesweredeterminedbyHPLCasfolowing:suit-
ableamountofcrudeextractpowderwasdissolvedin
theupperphaseofthesolventsystemandanalyzedby
HPLC.Thepeakareasofthepeakswererecordedas
A1.Thenequalvolumeofthelowerphasewasaddedto
thesolutionandmixedthoroughlytoreachpartitione-
quilibrium.Theupperphasewasthenanalyzedby
HPLCagain.Thelaterpeakareaswererecordedas
A2.TheK-valueswerecalculatedaccordingtothefol-
lowingequation:K=A2 /(A1 -A2).
Preparationoftwo-phasesolventsystemandsam-
plesolution
Inthecurentstudy, chloroform-methanol-water(4∶3∶2,
v/v)wasusedasthetwo-phasesolventsystemfor
HSCCCseparation.Itwaspreparedbyaddingthesol-
ventstoaseparationfunnelaccordingtothevolumera-
tiosandthoroughlyequilibratedbyshakingrepeatedly.
Then, theupperphaseandthelowerphaseweresepa-
ratedanddegassedbysonicationfor30 minbefore
use.
ThesamplesolutionforHSCCCseparationwaspre-
paredbydissolving100 mgofthesampleinthemix-
tureof2mLoftheupperphaseand2mLofthelower
phaseofchloroform-methanol-water(4∶3∶2, v/v).
HSCCCseparationprocedure
HSCCCseparationwasperformedasfolows:theupper
phase(stationaryphase)andthelowerphase(mobile
phase)ofchloroform-methanol-water(4∶3∶2, v/v)were
pumpedintotheseparationcolumnsimultaneouslyu-
823Vol.21 OUYANGDan-wei, etal:IsolationandPurificationofApigeninandLuteolinfromPterismultifidaPoir.byHigh-speedCounter-currentChromatography
singTBP-50Asystem, accordingtothevolumeratioof
50∶50.Afterthecolumnwastotalyfiledwiththetwo
phases, theHSCCCapparatuswasrunattherevolution
speedof850r/min.Atthesametime, thelowerphase
waspumpedintothecolumnataflowrateof2.0mL/
min.Afterhydrodynamicequilibriumwasreached(a-
bouthalfanhourlater), thesamplesolutionwasinjec-
tedintotheseparationcolumnthroughtheinjection
valve.Theseparationtemperaturewascontroledat30
℃ althroughtheexperiment.Theefluentfromthetail
endoftheseparationcolumnwascontinuouslymoni-
toredat280 nm.Thedatawerecolectedimmediately
afterthesampleinjection.Eachfractionwascolected
manualyaccordingtotheobtainedchromatogramand
thenevaporatedunderreducedpressure.Theresiduals
weredissolvedinmethanolforsubsequentpurityanaly-
sisbyHPLC.
HPLCanalysisandidentificationofHSCCCpeak
fractions
TheHPLCanalysiswasperformedwithaDikmaDia-
mondC18 column(250 mm×4.6 mm, 5 μm)atroom
temperature.Themobilephasewasmethanol-water(65
∶35, v/v).Theefluentwasmonitoredat280 nmand
theflowratewaskeptat1.0 mL/minconstantly.The
structureidentificationofHSCCCpeakfractionswas
carriedoutby1Hand13CNMRofBrukerUltraShield
600NMR.
ResultsandDiscussion
OptimizationofHPLCmethod
Thepartitioncoeficientofeachcomponentincrude
samplewasdeterminedbyHPLCanalysis.Sointhe
firstplace, agoodHPLCmethodshouldbedeveloped
foranalysisofcrudesample.Diferentmobilephases
(methanol-water, acetonitrile-water)were used in
HPLCtoseparateapigeninandluteolinfromthecrude
sampleofP.multifidaPoir.Theresultsindicatedthat
whenmethanol-water(65∶35, v/v)wasusedasthemo-
bilephase, apigeninandluteolincouldobtainbaseline
separation.BeinganalyzedbyWatersworkstation, the
purityfactorofeachpeakwaswithinthecalculated
thresholdlimit.TheHPLCchromatogramofthecrude
samplefromP.multifidaPoirwasilustratedinFig.2.
Fig.2 HPLCchromatogramofthecrudesampleof
PterismultifidaPoir.
Note:Column:DikmaDiamondC18 column(250mm×4.6mm, 5
μm);mobilephase:methanol-water(65∶35, v/v);flowrate:1.0
mL/min;UVwavelength:280nm;1:apigenin;2:luteolin.
OptimizationofHSCCCconditions
AccordingtotheHPLCanalysisresults(Fig.2), there
aretwomajorcompoundsappearedinthecrudesam-
ple.Inordertoobtaingoodresolutionoftargetcom-
poundsinHSCCCseparation, theK-valuesofthesetwo
compoundsindiferentsolventsystemsweredeter-
minedbyHPLCastheprocedureshowninSection2.
4.TheresultsarestatedinTable1.
Table1 K-valuesofapigeninandluteolin
Solventsystem Apigenin Luteolin
chloroform-methanol-water(4∶2∶2, v/v) 3.37 8.90
chloroform-methanol-water(4∶3∶2, v/v) 1.98 4.03
chloroform-methanol-water(4∶4∶2v/v) 1.15 1.73
AccordingtotheK-values, chloroform-methanol-water
solventsystemswithdiferentvolumeratiosweretested
inHSCCCseparation.TheresultsofHSCCCtestsindi-
catedthatwhenchloroform-methanol-water(4∶2∶2, v/
v)wasusedasthesolventsystem, theseparationtime
wastoolong.Whenchloroform-methanol-water(4∶4∶2,
v/v)wasused, thetwocompoundscouldnotgetsatis-
factoryseparation.Whenchloroform-methanol-water(4
∶3∶2, v/v)wasusedasthesolventsystem, goodsepa-
rationresultsandacceptableseparationtimecouldbe
obtained.
Theflowrateofthemobilephase, theseparationtem-
perature, theretentionpercentageofthestationary
phaseandtherevolutionspeedoftheseparationcoil
werealsooptimized.
Fig.3 showstheresultobtainedfrom 100 mgcrude
sampleofPterismultifidaPoir.bypreparativeHSCCC.
824 NatProdResDev Vol.21
Fig.3 HSCCCchromatogramofthecrudesample
ofPterismultifidaPoir.
Solventsystem:chloroform-methanol-water(4∶3∶2, v/v);sta-
tionaryphase:upperphase;mobilephase:lowerphase;flowrate:
2.0mL/min;revolutionspeed:850rpm;separationtemperature:
30℃;samplesize:100mg;retentionofstationaryphase:60%;
detectionwavelength:280nm.
Afterthisseparation, 17.0mgofapigenin(peak1, col-
lectedduring225-245 min)and22.5 mgofluteolin
(peak2 , colectedduring300-320 min)couldbeob-
tained, respectively.
HPLCanalysisandidentificationofHSCCCpeak
fractions
TheanalysisofthesefractionsindicatedthatthePeak1
fractioncontainedapigeninwithpurityof92%, andthe
Peak2fractioncontainedluteolinwithpurityof96%,
asasayedbyHPLC(Fig.4AandB).Identificationof
eachHSCCCpeakfractionwasperformedby1HNMR
and13 CNMRanalysisasfolows:HSCCCPeak1:1 H
Fig.4 HPLCchromatogramofHSCCCfractions
Note:Column:DikmaDiamondC18 column(250mm×4.6mm, 5μm);mobilephase:methanol-water(65∶35, v/v);flowrate:1.0mL/min;UV
wavelength:280 nm;(A):fraction1;(B):fraction2.
NMR(600 MHz, DMSO-d6 )δ:6.18(1H, d, J=1.8
Hz, H-6), 6.47(1H, d, J=1.8 Hz, H-8), 6.76(1H,
s, H-3), 6.92(2H, dd, J=6.6, 2.4 Hz, H-3′, 5′),
7.91(2H, dd, J=6.6, 2.4 Hz, H-2′, 6′);13 CNMR
(150MHz, DMSO-d6)δ:164.2(C-2), 102.8(C-3),
181.7(C-4), 161.4(C-5), 98.8(C-6), 163.7(C-7),
93.9(C-8), 157.3(C-9), 103.6(C-10), 121.1(C-
1′), 161.2(C-4′), 115.9(C-3′, 5′), 128.4(C-2′, 6′)
.Comparingthedatawiththeliterature[ 11] , peak1 was
identifiedasapigenin.
HSCCCPeak2:1HNMR(600MHz, DMSO-d6)δ:6.18
(1H, d, J=1.8Hz, H-6), 6.45(1H, d, J=1.8Hz, H-
8), 6.64(1H, s, H-3), 6.88(1H, d, J=9.0 Hz, H-
5′), 7.39(1H, d, J=2.4 Hz, H-2′), 7.40(1H, dd, J
=9.0, 2.4 Hz, H-6′);13 CNMR(150 MHz, DMSO-
d6)δ:164.3(C-2), 102.8(C-3), 181.6(C-4), 161.4
(C-5), 98.8(C-6), 163.9(C-7), 93.8(C-8), 157.2
(C-9), 103.6(C-10), 118.9(C-1′), 113.3(C-2′),
149.8(C-3′), 145.8(C-4′), 116.0(C-5′), 121.4(C-
6′).Comparingthedatawiththeliterature[ 12] , peak2
wasidentifiedasluteolin.
Conclusion
HSCCCwassuccesfulyusedfortheisolationandpur-
ificationofapigeninandluteolinfrom P.multifida
Poir..Apigenin(17.0 mg)andofluteolin(22.5 mg)
withpuritiesof92% and96%, canbeobtainedfrom
100 mgofthecrudesampleinone-stepseparation.
Acknowledgement WethankProfessorGenjinYang
ofColegeofPharmacy, SecondMilitaryMedicalUni-
versityforthespectralmeasurement.
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825Vol.21 OUYANGDan-wei, etal:IsolationandPurificationofApigeninandLuteolinfromPterismultifidaPoir.byHigh-speedCounter-currentChromatography
性最差 ,其盆栽抑制效果也仅为 1.7%。取代基中
甲氧基的位置与活性关系密切 ,活性顺序为:5、6位
>1、6位 >6位 >8位;6位是决定活性的关键 。例
如 6位为甲氧基时 , Xanthorin-5-methylether活性 >
大黄素-1, 6-二甲醚 >大黄素 -6-甲醚 >大黄素-8-甲
醚 >大黄素甲醚 -8-o-β-D-葡萄糖糖甙 ,其 EC50值分
别为 0.605、0.656、1.469、38.459和 65.901 μg/g,
当剂量为 6.25 μg/g时 , 相应的盆栽效果分别为
84.7%、74.6%、83.1%、47.5%、8.5%。若 6位替
换为其他取代基则效果明显下降 ,其活性顺序为:
OCH3 >OCH2CH3 >OH,相应的化合物活性顺序为:
大黄素 -6-甲醚 >大黄素 -6-乙醚 >大黄素 ,其 EC50值
分别为 1.469、3.871和 18.945μg/g,相应的盆栽抑
制效果分别为 83.1%、33.9%和 15.3%。大黄酚和
大黄素 -8-甲醚等蒽醌衍生物采用离体叶段法活性
不明显 ,其 EC50值分别为 12.798和 38.5 μg/g,而
采用盆栽法具有较好的抑制效果 ,其抑制效果分别
为 40.7%和 47.5%,这可能与这二个蒽醌衍生物具
有一定诱导抗病性作用有关 ,具体的作用机制尚待
进一步研究 。
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839Vol.21 喻大昭等:蒽醌衍生物结构及其对小麦白粉病菌生物活性的关系研究