全 文 ：第 28卷第 1期
2008年 2月
林 产 化 学 与 工 业
ChemistryandIndustryofForestProducts
Vol.28 No.1
Feb.2008
CharacterizationandAntioxidantActivityof
FlavonoidExtractfromLeavesof
AdinandranitidaMerr.exLi
收稿日期:2007-02-05
基金项目:广东省科技计划支持项目(2005A20303002);广州市科技局项目(2006J1-C0251)
作者简介:刘本国(1978-), 男 , 湖北武汉人 ,讲师 ,博士 ,主要从事食品化学与天然产物的研究工作
＊通讯作者:宁正祥 ,博士生导师 ,研究领域为食品化学与天然产物;E-mail:fezhning@scut.edu.cn。
LIUBen-guo
LIUBen-guo1, 2 , ZHANYu3 , NINGZheng-xiang1＊ , GAOJian-hua1 , XUKe-yong1
(1.ColegeofLightIndustryandFoodScience, SouthChinaUniversityofTechnology, Guangzhou510640,
China;2.SchoolofFoodScience, HenanInstituteofScienceandTechnology, Xinxiang453003, China;
3.SchoolofChemistryandChemicalEngineering, GuangzhouUniversity, Guangzhou510091, China)
Abstract:InordertodevelopAdinandranitidaMerr.exLi(akindofparticularplantinChina),
flavonoidextractfromleavesofA.nitida(FE)wasobtainedbytraditionalsolventextraction.Byhigh
performanceliquidchromatograph(HPLC)andtandemelectrosprayionizationmassspectrometry(ESI-MSn), themajorfla-
vonoidinleaveswasidentifiedascamelianinA, whosecontentinFEwasashighas51.19%±1.13%.Theflavonoidextract
showedhighantioxidantactivitybymeasuringabilitiesofscavengingDPPHradicalsandhydroxylradicals.Italsoexhibitedstrong
reducingpower.TheseresultsrevealedthatFEcouldbeservedasanewnaturalantioxidant.
Keywords:AdinandranitidaMerr.exLi;camellianinA;flavonoids;freeradicalscavenging
CLCnumbers:TQ91;S792 Documentcode:A ArticleID:0253-2417(2008)01-0006-05
亮叶杨桐叶黄酮类提取物的鉴定及其抗氧化活性研究
刘本国 1, 2 , 战 宇 3 , 宁正祥1 , 高建华 1 , 许克勇 1
(1.华南理工大学 轻工与食品学院 , 广东 广州 510640;2.河南科技学院 食品学院 , 河南 新乡 453003;
3.广州大学 化学化工学院 , 广东 广州 510091)
摘 要:为了开发亮叶杨桐———这种中国特有的植物 , 采用传统的有机溶剂提取法获得了亮叶杨桐叶的黄酮类提取物
(FE)。高效液相色谱(HPLC)和多级串联电喷雾质谱 (ESI-MSn)的分析表明 ,亮叶杨桐叶中的主要黄酮类化合物为山
茶苷 A,其在 FE中的质量分数高达 51.19% ±1.13%。在 DPPH自由基清除力 、还原力 、羟自由基清除力等抗氧化实验
中 , 亮叶杨桐叶的黄酮类提取物展现了较强的抗氧化能力。
关键词:亮叶杨桐;山茶苷 A;黄酮类化合物;自由基清除
Flavonoids, abundantinfruits, teas, vegetablesandmedicinalplants, havereceivedgreatatentionand
havebeeninvestigatedextensively, sincetheyarehighlyefectivefreeradicalscavengersandareassumedto
belesstoxicthansyntheticantioxidantssuchasBHAandBHT, whicharesuspectedofbeingcarcinogenicand
causingliverdamage[ 1] .Tothisday, morethan4 000 kindsofflavonoidshavebeenidentifiedorsynthe-
sized, butfewofthemcanbewidelyusedinfieldsoffood, medicine.Why? Thoughflavonoidsubiquitously
existinplants, fewkindsofplantscontainenoughflavonoidstoachievelarge-scaleproduction.Inthisstudy,
leavesofAdinandranitidaMerr.exLi, whichwestudied, areakindofflavonoid-richplantsource.A.nitida
isakindofparticularwildplantinSouthChina.Itsleaveshavebeenconsumedashealthtea(Shiyacha)and
第 1期 刘本国 ,等:亮叶杨桐叶黄酮类提取物的鉴定及其抗氧化活性研究 7
herbalmedicineforhundredsyears, whichwasreportedtohavemanycurativeefects, suchasreducingblood
pressure, antibiosis, antitumor, analgesic, etc.[ 2-3] .Today, A.nitidahasbeensuccessfulycultivatedin
Guangxiprovince, China.However, thecontentsofthemajorflavonoid, camelianinA, initsleavesand
extractshaven′tbeenstudied, andtheantioxidantactivityofitsextractshasn′tbeenevaluatedbyusingcom-
monlyacceptedassays.So, theobjectiveofthisstudywastoexploretheantioxidantpotentialofitsextractby
DPPHradicalscavengingassay, hydroxylradicalsscavengingasayandreducingpowerassay, onthebasisof
characterizationofHPLCandESI-MSn.
1 Experiments
1.1 Materialsandchemicals
LeavesofA.nitida(2006 springproduction, moisturecontent9.3%)forthisstudywerepurchasedin
Laibin, China.1, 1-diphenyl-2-picrylhydrazyl(DPPH)waspurchasedfromSigmaChemicalsCo.(St.
Louis, MO).HPLCgrademethanolwassuppliedbyDIKMA.Otherchemicalswereofanalyticalgrade.
1.2 PurificationandESI-MSnanalysisofcamelianinAinleavesofA.nitida
LeavesofA.nitida(about300g)wasextractedwith6Lboilingwaterfor1handthenfiltered.Thefil-
tratewasleftfor24hatambienttemperatureandthenfilteredthroughaporousfilter.Theresultantprecipitate
wascolectedandpurifiedtogetthecrystalofthemajorflavonoidbyrecrystalizingfor10 timesfromwater.
Afterfreeze-drying(α1-4, Christ, Germany), 1.84goflightyelowpowderwasobtained.ESI-MSnanalysis
ofcamelianinAwastakenonanEsquireHCTPLUSelectrosprayionizationmassspectrometer(Bruker,
Germany)inthenegativeionmodewithaflowrateof0.1mL/h.Thedatawererecordedandprocessedby
BrukerDaltonicsDataAnalysis3.3 software.
1.3 PreparationofFE
PowderedleavesofA.nitida(13.52g)wasextractedwith120mLof63% ethanolinawaterbathat
70℃ for10minandthenfiltered.Thefiltratewasconcentratedundervacuumat45℃ andfreeze-dried(α
1-4, Christ, Germany), 6.61goftheextractwasobtained.
1.4 QuantitativeanalysisofcamelianinAintheleavesandFE
CamelianinA 30.8mg(producedin1.2)wasdissolvedinmethanoltoproducestocksolutionof
0.308g/L.Forthecalibrationcurve, thestocksolutionwasdilutedwithmethanolintheconcentrationrange
from0.015 4to0.308g/L.FE20.8mgwasdissolvedinmethanoltoproducestocksolutionof0.208g/Lfor
HPLCanalysis.Driedpowderedleaves1.079gwasplacedinaSoxhletextractorandrefluxedat80℃ for
10hwith150mLmethonal, andthentheextractwascolectedanddilutedto250mLwithmethanolforHPLC
analysis.
Thesampleswereseparatedonareversedphasecolumn, Diamonsil® C18column(4.6mm×250mm,
5μmparticlesize)manufacturedbyDIKMA.Themobilephasewasconsistedofmethanolandwater(1∶1)
withaflowrateof0.8mL/min.TheHPLCsystemwasconsistedofaWaters1525 BinaryHPLCpumpanda
Waters2487dualλabsorbancedetector.Theinjectionvolumewas10μLandwavelengthfordetectionwasset
at265nm.BeforeHPLCanalysis, alsamplesmustbepassedthrougha0.45μmmiliporefilter.Thequanti-
tativeanalysisofcamelianinAinthesampleswasbasedonanexternalstandard.Thechromatographicdata
wererecordedandprocessedbyBreezeSystem3.30 software.
1.5 DPPHradicalscavengingassay
DPPHradicalscavengingassaywasdoneaccordingtoapublishedmethod[ 4] .Briefly, DPPHsolution
2mL(0.2mmol/L, inethanol) wasincubatedwith diferentconcentrationsofFE and butylated
8 林 产 化 学 与 工 业 第 28卷
hydroxytoluene(BHT).Thereactionmixturewasshakenandincubatedinthedarkfor30min, atroom
temperature, andtheabsorbancewasreadat517nmagainstethanol.Controlscontainingethanolinsteadof
theantioxidantsolution, andblankscontainingethanolinsteadofDPPHsolutionwerealsomade.Theinhibi-
tionofDPPHradicalbythesamplewascalculatedaccordingtothefolowingformula:
S=[ 1-(M1 -M2)/M] ×100%
Where:S—DPPHscavengingactivity;M—abs.ofcontrol;M1— abs.ofsample;M2— abs.ofblank.
1.6 Reducingpowerassay
ReducingpowerofFEandBHTwasdeterminedaccordingtothemethodofYen[ 5] .Extractinethanol
(1mL)wasmixedwithphosphatebufer(2.5mL, 0.2mol/L, pHvalue6.6)andpotassiumferricyanide
(2.5mL, 1%).Themixturewasincubatedat50℃ for20min.Aportion(2.5mL)oftrichloroaceticacid
(10%)wasaddedtothemixture, whichwasthencentrifugedat3 000r/minfor10min.Theupperlayerof
solution(2.5mL)wasmixedwithdistiledwater(2.5mL)andFeCl3 (0.5mL, 0.1%), andtheabsor-
bancewasmeasuredat700nm.Increasedabsorbanceofreactionmixtureindicatedincreasedreducingpower.
1.7 Hydroxylradicalsscavengingasay
ThehydroxylradicalscavengingactivityoftheextractwasdeterminedaccordingtothemethodofJia[ 6] .
1, 10-Phenanthroline0.6mL(5mmol/L)wasmixedwithphosphatebufer(0.6mL, 0.1mol/L, pHvalue
7.4), FeSO4(0.6mL, 5mmol/L), EDTA(15mmol/L, 0.6mL).Distiledwater(Adamage, Acontrol)or
sample(Asample)0.6mL, H2O2 0.8mL(0.1%)(Adamage, Asample)ordistiledwater(Acontrol)was
addedintothemixture.Themixturewasincubatedat37℃ for1h, andtheabsorbancewasreadat536nm.
Thehydroxylradicalsscavengingactivity(T)wascalculatedusingthefolowingequation:
T=[ (Asample-Adamage)/(Acontrol-Adamage)] ×100%
2 Resultsanddiscussion
2.1 AnalysisofflavonoidsinFE
Fig.1 ChemicalstructureofcamellianinA
Accordingtoourpreviousstudy[ 7] , themainflavonoidin
leavesofA.nitidawasidentifiedascamelianinA(Fig.1), and
Fig.2 showsthenegativeionsESI-MSandESI-MS2 spectraof
camelianinAobtainedinthisstudy.The[ M-H]-ofcamelianin
Awas619.2.TheMS/MSproductionsobtainedfrom[ M-H] -
wereasfolowing:577.2 ([ M-Acetyl-H]-), 473.1([ M-Rham-
H]-), 268.9 ([ M-Acetyl-Rham-Glu-H] -).AlthoughZhanget
al[ 8] hadreportedtheidentificationofflavonoidsinleavesofA.
nitidabyHPLC-MS, thenamesofcamelianinAandcamelianin
Bintheirreportwereconfused, andthechemicalstructureofthe
flavonewithm/z619, namedcamelianinBintheirreport, wasexpressedasApigenin-Rham-Glc-OCCH3
(Glc:glucose;Rham:rhamnose), whichdidn′tcoincidewiththeresultsofESI-MS2 inthisstudy(Fig.2),
andtheoriginalreportaboutcamelianinAandB[ 9] .ForabsenceofcommercialstandardofcamelianinA,
therewerenopublishedreportsondeterminationofcamelianinAcontentsinleavesanditsextracts.Inthis
study, thecontentofcamelianinAwasmeasuredbyHPLCanalysis, withcamelianinAobtainedbyrecrystal-
lizationfor10timesfromwaterasstandard, thecontentsofcamelianinAintherawmaterialsandextractwere
27.57%±0.92% and51.19%±1.13% (mean±SDofn=3samples), respectively.Tothebestofour
knowledge, therearefewplantsourcescontainingsomuchflavonoidlikeleavesofA.nitida.
第 1期 刘本国 ,等:亮叶杨桐叶黄酮类提取物的鉴定及其抗氧化活性研究 9
Fig.2 NegativeionsESI-MS(a)andESI-MS2(b)spectraofcamellianinA
2.2 DPPHradicalscavengingactivity
Proton-radicalscavengingactivityisanimportantatributeofantioxidants, whichismeasuredbyDPPH
radicalscavengingasay.DPPH, aprotonatedradical, hascharacteristicabsorbancemaximaat517nm,
whichdecreaseswiththescavengingoftheprotonradical.Hydrogen-donatingabilityoftheantioxidantmole-
culecontributestoitsfreeradicalscavengingnature.AhighDPPHradicalscavengingactivityofFEwas
observed(Fig.3), andtheextractwasmoreactivethanBHT, whichshouldbeatributedtothehighcontent
offlavonoidsinit.
2.3 Reducingpower
Itisbelievedthatantioxidantactivityandreducingpowerarecorrelated.Inthisassay, althoughthe
extractfromleavesofA.nitidashowedhighactivity, itsactivitywaslowerthanthatofBHT(Fig.4).The
equationofreducingpower(y)andamountofFE(x)was:y=1.712x-0.0132(R2 =0.995 1), indicating
thatreducingabilitycorelatedwelwithamountofFE.
Fig.3 DPPHradicalsscavengingactivityofFEandBHT Fig.4 ReducingpowerofFEandBHT
Fig.5 Hydroxylradicalsscavengingactivity
ofFE
2.4 Hydroxylradicalsscavengingactivity
Thehydroxylradicalisanextremelyreactivefreeradical
formedinbiologicalsystemsandhasbeenimplicatedasahighly
damagingspeciesinfreeradicalpathology, capableofdamaging
biomoleculesofthelivingcels.Hydroxylradicalhasthecapacity
tocauseDNAstrandbreakage, whichcontributestocarcinogene-
sis, mutagenesisandcytotoxicity.Inaddition, thisradicalspe-
ciesisconsideredasoneofthequickinitiatorsoftheLPO
proces, abstractinghydrogenatomsfromunsaturatedfatyacids.
Inthisassay, extractfromleavesofA.nitidashowedhighactivi-
ty, whichwasconcentration-dependent(Fig.5).
10 林 产 化 学 与 工 业 第 28卷
3 Conclusion
3.1 LeavesofAdinandranitidaMerr.exLiisakindofflavonoid-richplantsource.Thecontentsofthe
majorflavonoid, camelianinA, inleavesandFEwere27.57% ±0.92%and51.19%±1.13%, respec-
tively.
3.2 TheresultsofassaysofDPPHradicalsscavengingability, reducingpowerandhydroxylradicalsscaven-
gingactivityrevealedthatflavonoidextractsfromleavesofA.nitidacouldbeservedasanewkindofnatural
antioxidant.
References:
[ 1] BURDAS, OLESZEKW.Antioxidantandantiradicalactivitiesofflavonoids[ J].JournalofAgriculturalandFoodChemistry, 2001, 49(6):
2774-2779.
[ 2]陈粤 ,佘纲哲 ,陈鸿霖.亮叶杨桐提取物抗肿瘤活性的研究 [ J].汕头大学学报:自然科学版 , 1997, 12(2):43-45.
[ 3]余杰 ,陈美珍.亮叶杨桐中类黄酮提取及其抗氧化 、抑菌作用的研究 [ J].汕头大学学报:自然科学版 , 1997, 12(2):52-57.
[ 4] SUNT, HOC.Antioxidantactivitiesofbuckwheatextracts[ J] .FoodChemistry, 2005, 90(4):743-749.
[ 5] YENGC, CHENHY.Antioxidantactivityofvariousteaextractsinrelationtotheirantimutagenicity[ J].JournalofAgriculturalandFood
Chemistry, 1995, 43(1):27-32.
[ 6]贾之慎 ,邬建敏 ,唐孟成.比色法测定 Fenton反应产生的羟自由基 [ J] .生物化学与生物物理进展 , 1996, 23(2):184-186.
[ 7]刘本国 ,战宇 ,许克勇 ,等.液质联用鉴定亮叶杨桐叶中的类黄酮化合物 [ J] .食品研究与开发 , 2007, 28(3):118-120.
[ 8] ZHANGJie, YANGJun, DUANJi-cheng, etal.QuantitativeandqualitativeanalysisofflavonoidsinleavesofAdinandranitidabyhigh
performanceliquidchromatographywithUVandelectrosprayionizationtandemmassspectrometrydetection[J] .AnalChimActa, 2005, 532:
97-104.
[ 9]成桂仁 ,金静兰 ,文永新.白水茶中二种新黄酮苷的结构 [ J] .药学学报 , 1987, 22(3):203-207.
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