全 文 ：淡竹叶中总黄酮和三种黄酮苷的同步 HPLC检测(英文)
刘体云 , 卢艳花＊ , 魏东芝 , 周文瑜
(生物反应器工程国家重点实验室 ,鲁华生物技术研究所 ,华东理工大学 ,上海 200237)
收稿日期:2008-02-12
作者简介:刘体云(1983-),女 ,硕士研究生 ,研究方向:中药活性成分分离及分析。 E-mail:liutiyun@sohu.com
＊通讯作者:卢艳花 , Tel:(021)-64253715 E-mail:luyanhua@ecust.edu.cn
关键词:淡竹叶;黄酮苷;质量控制;HPLC/DAD
摘要:目的:建立检测中药材淡竹叶中的总黄酮(以芦丁为外标)和 3种黄酮苷(异荭草素 、牡荆苷 、苜蓿素 7-O葡萄糖
苷)的有效 、精确而可靠的 HPLC/DAD方法 。方法:色谱柱为 AgilentSB-C18柱 (250 mm×4.6 mm, 5.0 μm),用乙腈和
0.5%的冰醋酸为流动相梯度洗脱 ,波长为 350 nm。结果:其加样回收率为 97.1% ～ 102.3%,芦丁和 3种黄酮苷均得到
满意的分辨率和线性范围 , 同时为了确保方法的有效性 , 还分析了不同产地淡竹叶中的总黄酮和 3种黄酮苷的含量。结
论:HPLC检测方法可用于淡竹叶及其相关产品的质量控制。
中图分类号:R284.1 文献标识码:A 文章编号:1001-1528(2009)01-0096-05
Simultaneousdeterminationoftotalflavonoidsandthreeflavonoidglycosidesin
LophatherumgracileBrongn.byHPLC
LIUTi-yun, LUYan-hua＊ , WEIDong-zhi, ZHOUWen-yu
(StateKeyLaboratoryofBioreactorEngineering, NewWorldInstituteofBiotechnology, EastChinaUniversityofScienceandTechnology, Shanghai,
200237 , China)
KEYWORDS:LophatherumgracileBrongn.;flavonoidglycosides;qualitycontrol;HPLC/DAD
ABSTRACT:AIM:Todevelopanefective, accurateandreliableHPLC/DADmethodfordeterminationoftotal
flavonoids(rutinservedasexternalstandard)andthreeflavonoidglycosides(isoorientin, vitexinandtricin7-O-
glucoside)intraditionalChineseherbLophatherumgracileBrongn.METHODS:Thechromatographicseparation
wasperformedonanAgilentSB-C18 column(250 mm×4.6mm, 5.0μm)withagradientelutionprogrammeu-
singthemixtureofacetonitrileand0.5%aqueousaceticacid(v/v)asmobilephaseat350 nm.RESULTS:The
recoveryofthemethodwasintherangeof97.1%-102.3%, andthefourcompoundsshowedgoodlinearrelation
(R2 >0.999)inarelativelywideconcentrationrange.Inaddition, thecontentoftotalflavonoidsandthreefla-
vonoidglycosidesinL.gracilegrowingindiferentlocationsofChinawasdeterminedtoestablishtheefectiveness
ofthemethod.CONCLUSION:ThedevelopedHPLCassaycouldbereadilyutilizedasaqualitycontrolmethod
forL.gracileanditsrelatedmedicinalpreparations.
1 Introduction
ThestemandleafofLophatherumgracileBrongn.
(Gramineae)havebeenoficialylistedintheChinese
Pharmacopoeia, andhavebeenusedasadrugoftrea-
tingfeveranddiuresisintraditionalChineseherbal
medicinesforalongtime[ 1] .Phytochemicalstudieson
L.gracilehaverevealedthepresenceofphenolic
acids, flavonoids, etc.Flavonoidsincludingisoorien-
tin, vitexinandtricin7-O-glucosidearethemajorac-
tivecomponentsinL.gracile.Theyhaveprovedto
posessvariousactivities, suchasantimicrobialactivi-
ty, hepatoprotectiveefect, anti-oxidationandotherbi-
ologicalactivities[ 2, 3] .Hence, thetotalflavonoidsand
threeflavonoidglycosideswereselectedforanalyzing
andevaluatingL.gracile.
Thedevelopmentofqualitycontrolmethodisan
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essentialissuefortheefectiveclinicaluseoftheme-
dicinalherb.Butsofar, thereisnospecificquantita-
tivedeterminationtocontrolthequalityofL.gracilein
theChinesePharmacopoeia, andonlyafewmethods
havebeendocumentedforthedeterminationoftotal
flavonoidspresentinL.gracilebyUV[ 4, 5] .Toour
knowledge, thereisnoreportaboutanalyticalmethods
fordeterminationoftotalflavonoidsandthreeflavonoid
glycosidessimultaneouslyinL.gracile.
Thus, withtheincreasingapplicationsofL.grac-
ileinfood, healthcareproductsandthemedicinal
herbindustry, itisessentialtoestablishananalytical
methodforqualitycontrol.Thestrategyweappliedwas
todeterminetotalflavonoidsandthreeactivecom-
poundsfortheassessmentofthequalityofL.gracile.
Thisisthefirstreporttodeterminetotalflavonoids,
isoorientin, vitexinandtricin7-O-glucosideinL.
gracilebyHPLC/DAD.Inaddition, thecontentofto-
talflavonoidsandthosethreecompoundsintenL.
gracilesamplesgrowingindiferentpartsofChinawas
determinedtoestablishtheefectivenessofthemethod.
2 Materialsandmethods
2.1 Plantmaterialsandchemicals
TheaerialofL.gracilewasusedinourexperi-
ment.Commercialherbalsampleswerecolectedfrom
localdrugstoresindiferentprovincesinChina.The
samplesweretaxonomicalyidentifiedonthebasisof
morphologicalcharacteristics by ProfessorWANG
Zheng-tao.Theair-dried leavesofsampleswere
smashedintopowderanddepositedatNewWorldInsti-
tuteofBiotechnology, EastChinaUniversityofScience
andTechnology, Shanghai, China.
HPLC-grademethanolandacetonitrilewerepur-
chasedfromCALEDONLABORATORIESLtd., Cana-
da.Redistiledwaterwaspreparedinourownlab.
GlacialaceticacidofanalyticalgradewasfromShang-
haiChemicalCo., Ltd.Althesolutionswerefiltered
through0.45 μmmembranes(Schleicher＆ Schuel,
Germany)anddegassedbyanultrasonicbathbefore
use.
RutinwasgiftedbyprofessorLUYan-hua.The
threecompoundsofflavonoidglycosideswereextrac-
ted, isolatedandpurifiedfromL.gracileinourlab.
Thestandardcompounds(isoorientin, vitexinandtri-
cin 7-O-glucoside) wereconfirmed by ESI-MS,
1HNMRand13 CNMRspectrometrictechniquesand
werecomparedwithliteratures[ 6, 7, 8] .Thepurities
werehigherthan98% basedonHPLCanalysis, and
showedverystableinmethanolsolution.Thestructures
andUVspectraofflavonoidglycosidesweregivenbe-
low(Fig.1).
Fig1 Thechemicalstructuresofisoorientin,
vitexinandtricin7-O-glycoside
2.2 Analyticalmethod
ExperimentswereperformedonanAgilent1100
seriesHPLC-DADsystem, equippedwithaphotodiode
arraydetectorworkingintherangeof190-400 nm, a
quaternarysolventdeliverysystem, acolumntempera-
turecontrolerandanautosampler.Thechromato-
graphicdatawererecordedandprocessedwithAgilent
ChromatographicWorkStationsoftware.Analysiswas
cariedoutat30°ConanAgilentEclipseSB-C18col-
umn(250mm×4.6mm, 5μm), whichwasprotec-
tedbyaguardcolumn(12.5 mm×4.6mm, 5μm).
Themobilephaseconsistedofacetonitrile(A)and
0.5% aqueousglacialaceticacid(v/v, B), usinga
gradientprogramof13% Ain0-5 min, 13%-25% A
in5– 20minandheldfor5min, 25%– 60% Ain
25– 30 minandlineargradientto13% Ain1 min.
Itwasfolowedbya5 minequilibriumperiodpriorto
theinjectionofnextsample.Theflowratewas1.0
ml/min.Thedetectionwavelengthwassetat350 nm
foracquiringchromatograms.
2.3 Samplepreparation
ThedriedpowderofL.gracilesamples(1.0 g)
wasaccuratelyweighedandextractedbyUltrasonic
with15ml50%methanolfor1h, then, forcooling15
min.Aftercooling, theextractwasfilteredthrough
glasswoolforsamplecleanupanddilutedto25 ml
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with50% methanol.Thesamplesolutionwasfiltered
througha0.45 μmmembranedirectlyintoaHPLC
samplevial, justpriortoHPLCanalysis.Theinjection
volumewas20μL.
2.4 Standardpreparation
Thereferencestandardsofthethreecompounds
(isoorientin, vitexinandtricin7-O-glucoside)andru-
tinwereaccuratelyweightedanddissolvedinmetha-
nol, thendilutedtoappropriateconcentrationranges
fortheestablishmentofcalibrationcurves.Alstock
andworkingstandardsolutionswerestoredat4°Cuntil
usedforanalysis.
3 Resultsanddiscussion
3.1 Separationofthecompounds
Severalmobilephasesweretested.Eventualy, it
wasfoundthatanacetonitrile-watersystemcontaining
0.5% glacialaceticacid(v/v)gavethebestsepara-
tionofthreeflavonoidcompounds.Fig.2demonstrates
HPLCchromatogramoffourcompoundsandthefour
insetswereDADUVscanofisoorientin(A), vitexin
(B), tricin7-O-glucoside(C)andrutin(D)peak
(190-400 nm).Itcouldbeseenthatagoodseparation
wasachievedwithin30 minusingtheconditionsde-
scribed.Theremainderofthegradientconditionsen-
suredeficientcolumnwashing.
Fig2 HPLCchromatogramoffourcompoundsat350nmwavelength.ThefourinsetsareDADUVscanofisoorientin(A),
vitexin(B), tricin7-O-glucoside(C)andrutin(D)peak(190-400 nm).
3.2 Optimizationofextractionprocedure
Inordertoobtainquantitativeextraction, thein-
volvedvariablesintheproceduresuchastheextraction
solvents, extractionmethodsandextractiontimewere
optimized.Aqueousmethanolwasthepreferedchoice
ofextractionsolventbecauseavarietyofcompounds
withdiferentpolaritycouldbeco-extractedavaila-
bly[ 9, 10] .Inthisstudy, itwasfoundthatUltrasonic
extractionwith50% methanolfor1 hgavethehighest
extractionyieldforthetotalflavonoidsinL.gracile.
Ultrasonicandrefluxextractionwerealsocompared,
andtheultrasonicmethodwasfoundtobemoresuit-
able.
3.3 Validationofthemethod
3.3.1 Linearcalibration
TypicalchromatogramwasshowedinFig.2.The
retentiontimeofisoorientin, vitexin, tricin7-O-gluco-
sideandrutinwere13.75 min, 17.47 min, 25.89
minand17.41 min, respectively.Thelinearityfor
eachcompoundwasestablishedbyplotingthepeakar-
ea(y)versusconcentration(x).Linearregresiona-
nalysiswasperformedbytheexternalstandardmethod.
ThecontentsofthetotalflavonoidsinL.gracilewere
determinedbyreferencetostandardrutin.Theresults
wereshowninTable1.Alcompoundsdisplayedgood
linearity(R2 >0.999)inthegivenconcentration
range.
3.3.2 Precisiontest
Aprecisiontestwasdonewithsixcontinuousin-
jectionsandtheRSDs(relativestandarddeviation)
were1.12%, 1.31%, 0.93% and1.52% forrutin,
isoorientin, vitexinandtricin7-O-glucoside, respec-
tively.
3.3.3 Repeatabilitytest
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Table1 Calibrationcurveoffourflavonoids
compounds RT/(min) Linearregression
LinearityRange
/(μg/ml) r
Rutin 17.41 y=9389.9x+2.133 7 36-720 0.9999
Isoorientin 13.75 y=19894x-1.352 7 5-100 0.9993
Vitexin 17.47 y=24247x-6.442 5 2.3-46 0.9999
Tricin7-O-glycoside 25.89 y=47.817x-5.885 1 1.8-36 0.9998
RT:Retentiontime;y:peakarea;x:theconcentrationofeachreferencecompound(μg/ml);R2:corelationcoeficientofregressionequations.
SixindependentlypreparedsamplesolutionsofL.
gracilefromFujianprovincewiththesameamountwere
analyzedandthevariationswithinsixmeasurements
werecalculatedforevaluationofrepeatability.The
procesesofmeasurementswereinaccordancewiththe
“samplepreparation” inparalel.Itwasfoundthatthe
RSDswere1.85%, 1.76%, 2.03%, and2.54% for
rutin, isoorientin, vitexinandtricin7-O-glucoside, re-
spectively.
3.3.4 Recoverytest
L.gracilefromFujianprovincewaschosenfora
recoverytest.Itwasfoundthattherecoverieswere
(97.1±2.3)%, (101.2 ±2.1)%, (97.4 ±
1.6)%, and(102.3±2.8)% forrutin, isoorientin,
vitexinandtricin7-O-glucoside, respectively.High
recoverysuggestedthattheestablishedmethodwasac-
curateenoughforthedeterminationoftotalflavonoids
andthreebioactivecomponentsinL.gracile.
3.4 Quantitativeanalysis
Theestablishedanalyticalmethodwassuccessfuly
appliedtothesimultaneousdeterminationoftotalfla-
vonoids, isoorientin, vitexinandtricin7-O-glucoside
in10 samplesofL.gracile, whichwereobtainedfrom
variousprovincesandcitiesinChina.Eachsamplewas
determinedintriplicate.Peaksinthechromatograms
wereidentifiedbycomparingtheretentiontimeandon-
lineUVspectrawiththoseofthestandards.Thereten-
tiontimeofisoorientin, vitexinandtricin7-O-gluco-
sidewere13.75min, 17.47 minand25.89 min, re-
spectively(seeFig.3).
TheHPLC/DADprofileswereilustratedinFig.
3.Thecontentsofthreeflavonoidglycosideswerede-
terminedbythecorespondingregressionequationand
wereshownwiththemeanvaluesofthreereplicatein-
jections.DADUVscanmethodforidentifingcharac-
teristicpeakandcalculatingtotalpeakareaoffla-
Fig3 HPLCchromatogramsofflavonoidglycosidesisoori-
entin(A), vitexin(B)andtricin7-O-glucoside(C)
fromdifferentsourcesforL.gracileSample.
Number1.Zhejiangprovince(070810) 2.Fujian(070826) 3.
Shanxiprovince(070818) 4.Hunanprovince(070814) 5.Anhui
province(070709) 6.Anhuiprovince(070715) 7.Shanxiprovince
(070828) 8.Hebeiprovince(070830) 9.Guangdongprovince
(070823) 10.Anhuiprovince(070803).
vonoids, thecontentsoftotalflavonoidsweredeter-
minedbythecorespondingregressionequationofru-
tin.Table2showedthecontentsoftotalflavonoidsand
threeflavonoidglycosidesintensamplesofL.gracile.
Itwasfoundthatthecontentsofflavonoidsandthree
flavonoidglycosidesvariedgreatlyamongthediferent
samples.Thecontentsoftotalflavonoidsvariedfrom
2.15to7.95mg/g.Inthemajorityofcasesisoorientin
wasthemaincomponent, whosecontentsvariedfrom
0.18to0.95mg/g.Thesimilarvariationcouldalsobe
foundfortheothercomponents.Thereasonsforthe
variationofthecontentscouldbethediferenceofplant
origin, theefectofenvironment, seasonofcolection,
dryingprocessandstorageconditions, etc.Becauseof
thesignificantvariations, itisnecessarytodevelopan
efectivemethodtoevaluatethequalityofL.gracile.
4 Conclusion
Inourpresentstudy, asimple, accurateandreli-
ableHPLCmethodhasbeendevelopedandthiswas
thefirstreportofaHPLCdeterminationoftotalfla-
vonoidsandthreeflavonoidglycosidesofL.gracile.
Theresultsdemonstratethatthedevelopedmethodis
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Table2 ContentsoftotalflavonoidsandthreeflavonoidglycosidesindifferentL.gracilesamples
Samples
Totalflavonoids
Mean±SD(mg/g),
RSD(%)
Isoorientin
Mean±SD(mg/g),
RSD(%)
Vitexin
Mean±SD(mg/g),
RSD(%)
Tricin7-O-glycoside
Mean±SD(mg/g),
RSD(%)
Zhejiangprovince(070810) 4.40±0.10, 2.27 0.67±0.02, 2.98 0.14±0.00, 0.00 0.05±0.00, 0.00
Fujianprovince(070826) 7.95±0.25, 3.14 0.95±0.02, 2.10 0.13±0.00, 0.00 0.05±0.00, 0.00
Shanxiprovince(070818) 3.12±0.07, 2.24 0.29±0.01, 3.44 0.13±0.00, 0.00 0.04±0.00, 0.00
Hunanprovince(070814) 5.64±0.12, 2.13 0.57±0.02, 3.50 0.12±0.00, 0.00 0.06±0.00, 0.00
Anhuiprovince(070709) 3.26±0.09, 2.76 0.32±0.01, 3.12 0.07±0.00, 0.00 0.03±0.00, 0.00
Anhuiprovince(070715) 2.15±0.04, 1.86 0.22±0.01, 4.54 0.08±0.00, 0.00 0.04±0.00, 0.00
Shanxiprovince(070828) 3.72±0.10, 2.68 0.18±0.00, 0.00 0.44±0.01, 2.27 0.03±0.00, 0.00
Hebeiprovince(070830) 2.78±0.05, 1.79 0.26±0.01, 3.84 0.13±0.00, 0.00 0.04±0.00, 0.00
Guangdongprovince(070823) 2.17±0.03, 1.38 0.19±0.00, 0.00 0.07±0.00, 0.00 0.03±0.00, 0.00
Anhuiprovince(070803) 6.98±0.18, 2.57 0.48±0.01, 2.08 0.16±0.00, 0.00 0.04±0.00, 0.00
±SD=standarddeviation(n=3);RSD=relativestandarddeviation
accurateandreproducibleandcouldbereadilyutilized
asasuitablequalitycontrolmethodforthequantifica-
tionoftheaerialofL.gracile, derivedextractsand
phytomedicines.Theresultsoftheanalysisontheten
L.gracilesamplessuggestedthatcontentsoftotalfla-
vonoidsandthreeflavonoidglycosidesvariedsignifi-
cantlyintheaerialofL.gracilefromdiferentlocations
ofChina.Therefore, theevaluationofdatamightbe
usefulinqualityassuranceaswelasfordetermination
ofadulterationofthecrudedrug.
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HPLC测定不同产地威灵仙中齐墩果酸的含量
谢 乐 , 马晓黎 , 刘丽芳＊ , 池玲玲 , 何 斌 , 王琪琳
(中国药科大学中药分析教研室 ,江苏 南京 210038)
收稿日期:2008-04-12
基金项目:国家自然科学基金项目(3077270);江苏省高技术计划基金项目资助(BG2007613)
作者简介:谢 乐(1983-),男 ,硕士生 ,从事中药分析研究。 Tel:(025)85391253
＊通讯作者:刘丽芳(1969-),博士 ,副教授 ,主要从事中药活性成分和动物药研究。 E-mail:liulifang69@hotmail.com
关键词:威灵仙;齐墩果酸;HPLC
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