全 文 ： 辽宁大学学报
自然科学版
第 38卷 第 1期 2011年
JOURNALOFLIAONINGUNIVERSITY
NaturalSciencesEdition
Vol.38 No.1 2011
Isolation, Purificationandantioxidantactivitiesofpolysaccharides
fromtheleavesofCastaneamolissimaBl.
WANGXin1 , KONGYu-mei1 , LIUBin1 , WANGJun2
(1.SchoolofPharmaceuticalSciences, LiaoningUniversity, Shenyang110036, China;
2.CollegeofChemistry, LiaoningUniversity, Shenyang110036, China)
Abstract: ThepolysaccharideswereisolatedandpurifiedfromtheleavesofCastaneamolissimaBl.andtheir
antioxidantactivitieswerestudiedinvitro.Crudepolysaccharides(CLP)wasisolatedfromtheleavesofCastanea
molissimaBl.CLPwaspurifiedbyapplyingacelluloseDE-52column, andthepolysaccharidesobtainedfrom
theeluentwithwaterweretermedasCLP1.TheantioxidantactivitiesofCLPandCLP1, suchasscavengingabili-
tytohydroxylradicals, superoxideradicalsandhydrogenperoxide, andreducingpowerwereinvestigatedinvitro.
TheresultshowedthatCLPandCLP1 possessedmoderatescavengingactivitiestohydroxylradicals, superoxide
anionradicalsandhydrogenperoxide, andmoderatereducingpowerinaconcentration-dependentmanner.In
addition, thescavengingcapacityofCLPonhydrogenperoxidecorrespondedwiththatofascorbicacid.Thestudy
suggestedthatCLPandCLP1 couldpotentialybeexploredasnaturalantioxidants.
Keywords: theleavesofCastaneamolissimaBl.;polysaccharide;purification;antioxidantactivities
中图分类号:R284.2 文献标识码:A 文章编号:1000-5846(2011)01-0054-06
＊作者简介:王新(1976-),男 ,辽宁东港人 ,硕士 ,辽宁大学讲师 ,从事天然产物活性成分研究.
基金项目:辽宁省教育厅科学研究计划资助(2008565, 05L162)
收稿日期:2010-11-09
Reactiveoxygenspecies(ROS)intheformsof
superoxideanionradical(· O-2 ), hydroxylradical
(· OH)andhydrogenperoxide(H2O2)aregener-
atedbythenormalmetabolicprocessorfromexoge-
nousagents, asmediatorsofcarcinogenesis, ische-
mia/reperfusionandinflammationinjury[ 1] .Al-
thoughalmostalorganismspossessantioxidantde-
fenseandrepairsystemsthathaveevolvedtoprotect
themagainstoxidativedamage, thesesystemsarein-
suficienttopreventthedamageentirely[ 2] .Soitis
essentialtodevelopandutilizeefectiveantioxidants
sothattheycanscavengeROSinthehumanbody.
Inordertoreducedamagetothehumanbody, syn-
theticantioxidantsarecurentlyusedforindustrial
procesing.However, themostcommonantioxidants
suchasbutylatedhydroxyanisoleand butylated
hydroxytoluenehavebeensuspectedofbeingrespon-
sibleforliverdamageandcarcinogenesis[ 3] .Inre-
centyears, moreandmoreatentionhasbeenpaid
tofindingnaturalnon-toxicantioxidantsandanin-
creasingpileofevidencesindicatesthatplantpoly-
saccharidesingeneralhavestrongantioxidantactivi-
tiesandcanbeexploredasnovelpotentialantioxida-
nts[ 4] .
TheleavesofCastaneamolissimaBl.were
usedastraditionalChinesemedicineinsingleand
compoundingprescriptionsfortreatingsorethroat
andulcer.However, fewstudiesonthecharacter-
izationofbioactiveconstituentsintheleavesofCas-
taneamolissimaBl.havebeenreported.Inthis
work, onepolysaccharidefractionwasisolatedfrom
theleavesofCastaneamolissimaBl.andpurifiedin
anion-exchangecolumnchromatography.Further-
more, theirinvitroantioxidantactivities, suchas
scavengingabilityon· OH, O-2 , H2O2 , andre-
ducingpower, wereinvestigated.
1 Materialsandmethods
1.1 Materialsandreagents
ThefreshleavesofCastaneamolissimaBl.
wereharvestedfromthesuburbofDandong, Liaon-
ingProvince, China.CeluloseDE-52 waspur-
chasedfromWhatmanKent.England.Otherchemi-
calsusedinthestudywereofanalyticalgrade.
1.2 Apparatus
EQUINOX55 FT-IRSpectrometer(Bruker
Co., Germany);UV - 1201 Spectrophotometer
(BeijingRayleighAnalyticalInstrumentCo., Ltd,
China);DBS-100 Ful-AutomatedFractionCol-
lectorsandBT-200BPeristalticPumps(Shanghai
Qingpu-HuxiInstrumentsFactory, China).
1.3 Methods
1.3.1 Isolationandpurificationofpolysaccharides
TheleavesofCastaneamolissimaBl.(50 g)
wereextractedwith95% ethanol(500 ml)at50℃
for6 h, dried, andthenextractedtwicewithdis-
tiledwater(1000 mL)at95 ℃ for1.5 h.After
eachextraction, thesolublepolymerswereseparated
fromresiduesbyfiltration, andextractswerecom-
bined, concentratedanddeproteinized3 timeswith
equalvolumeof4∶1CHCl3 -n-BuOH.Theabove
extractwassubmitedtogradedprecipitationwith4
-foldvolumesofethanolandthemixturewaskept
overnightat4℃ toprecipitatethepolysaccharides.
Theprecipitatewascolectedbycentrifugation,
washedsuccessivelywithanhydrousethanolande-
ther, givingCLPasacrudepolysaccharide.
Theanion-exchangechromatographywasused
topreparethefractionation.ThesampleCLP(300
mg)wasdisolvedin25 mldistiledwater, centri-
fuged, andthenthesupernatantwasinjectedtoa
column(3.0×60 cm)ofCeluloseDE-52.After
loadingwithsample, thecolumnwaselutedwithin
stepwiseelutionwithincreasedconcentrationofNaCl
(0.0, 0.1, 0.5 mol/L)solutioninturnataflow
rateof60ml/h.Theelutionwasdetectedbyphenol
sulfuricacidmethod.Themajorpolysaccharidefrac-
tionswerecolectedwithafractioncolector, dia-
lyzedagainsttapwateranddistiledwaterfor48 h,
respectively.
Thecarbohydratecontentsofthecrudeandpu-
rifiedpolysaccharidesweredeterminedbythephenol
sulfuricacidmethod, usingglucoseasstandard.
Theabsorptionspectraofsolutionsofthecrudeand
purifiedpolysaccharideswererecordedfrom 200 to
800nmwiththeslitwidthsetat1nm.Thepurified
polysaccharidewasgroundwithKBrpowderand
thenpressedintopeletsforFT-IRmeasurementin
thefrequencyrangeof4000 to400 cm-1.
1.3.2 ·OHscavengingassay[ 5]
Samplesof2.5ml(atdiferentconcentrations)
weremixedwith4.0 mlsodiumphosphatebufer
(0.75 mol/L, pH7.4), 1.5 mlferrosindehydra-
tedalcoholsolution(5mmol/L), 1.0mlFeSO4(7.
5 mmol/L), and1%H2O2 solution(v/v).Theso-
lutionswereincubatedat37 ℃ for1 h, anddetec-
tedbymonitoringtheabsorbanceat536 nm.The
capabilityofscavengingto· O-2 wascalculatedby
thefolowingequation:
Scavengingefect(%)=Asample-AblankAcontrol-Ablank ×100
whereAcontrolistheabsorbanceofthecontrol
(distiledwater, insteadofH2O2), Asampletheab-
sorbanceofthetestgroupandAblank istheabsor-
banceoftheblank(distiledwater, insteadofthe
samples).
1.3.3 O-2 scavengingassay[ 6]
Samplesof1.0ml(atdiferentconcentrations)
weremixedwith7.0 mlTris– HClbufer(50
mmol/L, pH8.2)containing1 mmol/LEDTAand
incubatedat25℃ inawaterbathfor20 min.Then
1.0mlpyrogalol(3 mmol/L)wasadded, andthe
55 第 1期 WANGXin:Isolation, PurificationandantioxidantactivitiesofpolysaccharidesfromtheleavesofCastaneamolissimaBl.
mixturewasshakenrapidlyatroom temperature.
Theabsorbanceofthemixturewasmeasuredat325
nmper30sfor5minagainstablank.Thecapabili-
tyofscavengingto· O-2 wascalculatedbythefol-
lowingequation:
Scavengingefect(%)=1 -BA×100
whereAisthechangespeedofabsorbanceof
thecontrolgroupinthesurperoxideradicalgenera-
tionsystemandBisthechangespeedofabsorbance
ofthetestsample.
1.3.4 H2O2 scavengingassay[ 7]
Samplesof1.0ml(atdiferentconcentrations)
and6.0 mlsodiumphosphatebufer(0.1 mol/L,
pH7.4)mixedwith1.0 mlofH2O2 solution(50
mmol/L).AbsorbanceofH2O2 at230 nmwasde-
termined10 minlaterinaspectrophotometer.For
eachconcentration, aseparatedblanksamplewas
usedforbackgroundsubtraction.Thecapabilityof
scavengingtoH2O2 wascalculatedbythefolowing
equation:
Scavengingefect(%)=Asample-AsampleblankAcontrol ×100
whereAcontrolistheabsorbanceofthecontrol
(distiledwater, insteadofthesamples), Asampleis
theabsorbanceofthetestgroupandAsampleblankisthe
absorbanceofthesamplesonly.
1.3.5 Reducingpowerasay[ 8]
Samplesof1.0 ml(atdiferentconcentra-
tions), 2.5 mlsodiumphosphatebufer(0.2 mol/
L, pH6.6)and2.5 ml1% K3 Fe(CN)6 (w/v)
wereincubatedat50 ℃ for20 min.Then, 2.5 ml
10% trichloroaceticacid(w/v)wasaddedtothe
mixturefolowedbycentrifugationat4000 r/minfor
10 min.Theupperlayer5.0mlwasmixedwith0.5
mlfresh0.1%FeCl3(w/v).Themixturewasshak-
enanditsabsorbancewasmeasuredat700 nm.
2 Resultsanddiscussion
2.1 Isolationandpurificationofpolysaccha-
rides
Fig.1 ElutioncurveofCLPonCeluloseDE-52column
Onecrudepolysaccharide, namedCLP, was
obtainedbyhotwaterextractionfolowedbyethanol
precipitation, withyieldof3.67%.CLPwaspuri-
fiedbyapplyingaceluloseDE-52 column, and
thepurifiedpolysaccharide(67 mg)obtainedfrom
theeluatewithwaterwasnotedasCLP1 (Fig.1).
ThecarbohydratecontentsofCLPandCLP1 were
32.98% and72.31%, respectively.
TheUV-visspectraofCLPandCLP1 were
showninFig.2.ItwasobviousthatCLPhadabsor-
banceintheregion260-280nm, thepeaksmaybe
atributedtowater-solubleprotein, suggestingthat
theproteinwasnotremovedcompletelybyprecipita-
tionwithCHCl3 -n-BuOH.Afterpurificationby
celuloseDE-52columnthepeaksat260-280 nm
disappeared, suggestingthattheproteinwasre-
movedcompletely.
Fig.2 TheUV-visspectraofCLPandCLP1
56 辽宁大学学报 自然科学版 2011年
TheinfraredspectrumofCLP1 wasshownin
Fig.3.Thebandsintheregionof3455 cm-1 were
duetothehydroxylstretchingvibrationofthepoly-
saccharides.Thebandsintheregionof2973 cm-1
wereduetoC-Hstretchingvibration.Thecharac-
teristicbandsat1680 cm-1belongedtothehydrate
ofpolysaccharides.Thebandsbetween950 and
1160cm-1 atributedtotwotypesofC-Ostretching
vibrations, C-O-HandC-O-Cofpyranose
ring.Inaddition, thecharacteristicbandsat900
cm-1and868 cm-1belongedtotheBconfiguration
andCconfigurationoffuranose, respectively.
Fig.3 TheinfraredspectrumofCLP1
2.2 · OHscavengingactivity
· OHisconsideredtobeahighlypotentoxi-
dant, whichcanreactwithalbiomacromolecules
functioninginlivingcels.Thescavengingactivityof
CLPandCLP1 againstOHwithascorbicacidasa
positivecontrolwasshownasFig.4.Atthetest
concentrations, bothCLPandCLP1 exhibitedmod-
eratescavengingactivityon·OHinaconcentration
-dependentmanner.TheEC50 valuesofCLP,
CLP1 andascorbicacidfor· OH were101.8,
135.1 and38.9 mg/L, respectively, whichindica-
tedthattheordersof·OHscavengingactivitywere
ascorbicacid>CLP>CLP1.Inaddition, thepu-
rifiedpolysaccharideshowedscavengingactivityof
· OH, indicatingthatitwasmaincontentstoexe-
cuteantioxidantfunctionincrudepolysaccharide.
2.3 · O-2 scavengingactivity
Fig.4 ScavengingactivityofCLP, CLP1 andVC against
· OHatdiferentconcentrations
· O-2 wasgeneratedinthesystemofpyrogal-
lol sautoxidation.CLPandCLP1 werefoundto
possesstheabilitytoscavenge· O-2 atconcentra-
tionsbetween20 and140 mg/L(Fig.5).The
scavengingefectsofthetwopolysaccharidesin-
creasedwithincreasingconcentration.· O-2 isa
precursortoactivefreeradicalsthathavepotentialof
reactingwithbiologicalmacromoleculesandthereby
inducingtissuedamage.·O-2 wouldplayimportant
rolesintheformationofotherROSsuchasH2O2 ,
· OH, andsingletoxygen(1O2), whichinduceox-
idativedamageinlipids, proteins, andDNA.Both
CLPandCLP1 couldinhibittheautoxidationofpy-
rogaloltosomedegree, whichindicatedthatCLP
andCLP1playedmoderaterolesinantioxidation.
Fig.5 ScavengingactivityofCLPandCLP1againstO-2 atdif-
ferentconcentrations
57 第 1期 WANGXin:Isolation, PurificationandantioxidantactivitiesofpolysaccharidesfromtheleavesofCastaneamolissimaBl.
2.4 H2O2 scavengingactivity
Fig.6 ScavengingactivityofCLP, CLP1andVCagainstH2O2
atdiferentconcentrations
H2O2 itselfisnotveryreactive, howeveritcan
sometimesbetoxictocelbecauseitmaygiveriseto
· OHinthecels.AdditionofH2O2 tocelsincul-
turecanleadtotransitionmetaliondependent· OH
mediatedoxidativeDNAdamage.Thus, removing
H2O2 aswelas· O-2 isveryimportantforlifebeing
awayfromdamage.ThescavengingactivityofCLP
andCLP1againstH2O2 withascorbicacidasaposi-
tivecontrolwasshownasFig.6.TheH2O2scaven-
gingactivitiesofCLPandCLP1 increasedverysig-
nificantlywithincreasingconcentrations.TheEC50
valuesofCLPandCLP1 forH2O2 were136.0 and
182.5 mg/L, respectively.Further, thescavenging
capacityonH2O2 ofCLPcorespondedwiththatof
ascorbicacid, thelatersEC50 valuebeing124.4
mg/L.
2.5 Reducingpower
Thereducingpowerofacompoundmayserve
asasignificantindicatorofitspotentialantioxidant
activity.Fig.7 depictsthereducingpowerofCLP
andCLP1.Higherabsorbancevaluemeansstronger
reducingpowerofsamples.Thereducingpowerof
CLPandCLP1 corelatedwelwithincreasingcon-
centration.ThereducingpowerofCLPandCLP1 at
75 mg/Lwere0.829 and0.564, respectively.The
dataonthereducingpowerofCLPandCLP1 sug-
gestedthatitwaslikelytocontributetowardtheob-
servedantioxidantefect.
Fig.7 ReductiveactivityofCLPandCLP1atdiferentconcen-
trations
3 Conclusions
Accordingtotheresultsabove, itwasconclu-
dedthatthewaterextractingcrudepolysaccharide
(CLP)oftheleavesofCastaneamolissimaBl.con-
tainedpredominantlyonepolysaccharidefraction
(CLP1)purifiedbyCeluloseDE-52columnchro-
matography.Antioxidationtestinvitroshowedthat
CLPandCLP1possessedmoderatescavengingactiv-
itiesagainst· OH, O-2 , H2O2 , andmoderatere-
ducingpower.Wemayrationalyasumethatthe
leavesofCastaneamolissimaBl.playtheircurative
efectsinfolkmedicinepartlyastheresultsofthe
mechanismofantioxidationofpolysaccharides, and
sothepolysaccharidesshouldbeexploredasnovel
potentialantioxidant.Furtherstructuralanalysisand
evaluationoftheotherbioactivitiesofthepolysac-
charidesfromtheleavesofCastaneamolissimaBl.
wilbeinvestigatedfortheirapplicationinfoodand
medicinalfields.
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栗叶多糖的分离 、纯化及抗氧化活性
王 新1 , 孔玉梅 1 ,刘 彬 1 ,王君2
(1.辽宁大学 药学院 , 辽宁 沈阳 110036;2.辽宁大学 化学院 ,辽宁 沈阳 110036)
摘 要 从栗叶中分离纯化得到多糖并研究其体外抗氧化活性。从栗叶中提取得到栗叶粗多糖 CLP。
CLP经 DE-52纤维素柱分离纯化后 ,于蒸馏水洗脱液中得到一个纯化多糖组分 CLP1。采用体外试验
研究了 CLP与 CLP1对羟自由基 、超氧阴离子自由基 、过氧化氢的清除能力及还原能力 。结果表明 , CLP
与 CLP1均具有一定的清除羟自由基 、超氧阴离子自由基 、过氧化氢的能力及还原能力 ,而且与多糖的
浓度成正相关性 。其中 , CLP对过氧化氢的清除作用效果与 VC相当 。说明 CLP与 CLP1可以开发用作
天然抗氧化剂。
关键词 栗叶;多糖;纯化;抗氧化活性
(责任编辑 李 超)
59 第 1期 WANGXin:Isolation, PurificationandantioxidantactivitiesofpolysaccharidesfromtheleavesofCastaneamolissimaBl.