2006-DPPH清除自由基方法

Evaluationofantioxidantpropertyofextractandfractionsobtainedfromaredalga,PolysiphoniaurceolataXiao-JuanDuan,Wei-WeiZhang,Xiao-MingLi,Bin-GuiWang*KeyLaboratoryofExperimentalMarineBiology,InstituteofOceanology,ChineseAcademyofSciences,NanhaiRoad7,Qingdao266071,PRChinaReceived19July2004;receivedinrevisedform13December2004;accepted13December2004AbstractAntioxidantactivity(AA),totalphenoliccontent,andreducingpowerofthecrudeextract,fractions,andsubfractionsderivedfromaredalga,Polysiphoniaurceolata,wereevaluatedanddetermined.Theantioxidativeactivitywasmeasuredusingthea,a-diphenyl-b-picrylhydrazyl(DPPH)radicalscavengingassayandtheb-carotene–linoleateassaysystems,andcomparedwiththatofbutylatedhydroxytoluene(BHT),gallicacid(GA),andascorbicacid(AscA).Theresultsshowedthatthecrudeextractandtheethylacetate-solublefractionexhibitedhigherAAthanBHTintheDPPHassaymodel,atalloffourconcentrationlevelstested(from0.4to50lg/ml),while,intheb-carotene–linoleateassaysystem,thecrudeextractandtheethylacetate-solublefractionexhib-itedsimilaror,inmostcases,higherAAthanGAandAscAatthesameconcentrations(from10to200lg/ml).Theethylacetate-solublefractionwasfurtherfractionatedintosevensubfractionsF1–F7bysilicagelvacuumliquidchromatography.F1wasfoundtobethemosteffectivesubfractioninbothassaysystems.ThetotalphenoliccontentandreducingpowerweredeterminedusingtheFolin–Ciocalteuandthepotassiumferricyanidereductionmethods,respectively.Statisticalanalysisindicatedasignificantassocia-tionbetweentheantioxidantpotencyandtotalphenoliccontentaswellasbetweentheantioxidantpotencyandreducingpower.
2005ElsevierLtd.Allrightsreserved.Keywords:Polysiphoniaurceolata;Antioxidant;DPPH;b-Carotene–linoleate;Totalphenoliccontent;Reducingpower1.IntroductionReactiveoxygenspecies(ROS),suchassuperoxideradicalðO

2Þ,hydroxylradical(OH
),peroxylradical(ROO
),andnitricoxideradical(NO
),attackbiologicalmolecules,suchaslipids,proteins,enzymes,DNAandRNA,leadingtocellortissueinjuryassociatedwithaging,atherosclerosis,andcarcinogenesis(Fisch,Bo¨hm,Wright,&Ko¨nig,2003;Nakamura,Watanabe,Miyake,Kohno,&Osawa,2003;Shon,Kim,&Sung,2003;Va-lenta˜oetal.,2002).Antioxidantsareeffectiveinprotect-ingthebodyagainstdamagebyreactiveoxygenspecies.Thereisanincreasinginterestinnaturalantioxidantsbecauseofthesafetyandtoxicityproblemsofsyntheticantioxidants,suchasbutylatedhydroxyanisol(BHA)andbutylatedhydroxytoluene(BHT),thatarecom-monlyusedinlipid-containingfood(Amarowicz,Naczk,&Shahidi,2000;Howell,1986;Itoetal.,1986).Manynaturalantioxidantshavealreadybeeniso-latedfromdifferentkindsofplant,suchasoilseeds,cer-ealcrop,vegetables,leaves,roots,spicesandherbs(Ramarathnam,Osawa,Ochi,&Kawakishi,1995;Shonetal.,2003;Wettasinghe&Shahidi,1999).Amongnat-uralantioxidants,phenolicantioxidantsareinthefore-frontastheyarewidelydistributedintheplantkingdom.Plantscontainadiversegroupofphenoliccompounds,includingsimplephenolics,phenolicacids,anthocyanins,hydroxycinnamicacidderivatives,andflavonoids.Allthephenolicclasseshavethestructuralrequirementsoffreeradicalscavengersandhave0308-8146/$-seefrontmatter
2005ElsevierLtd.Allrightsreserved.doi:10.1016/j.foodchem.2004.12.015*Correspondingauthor.Tel.:[phone];fax:[phone].E-mailaddress:[e-mail](B.-G.Wang).(2006)37–43FoodChemistrypotentialasfoodantioxidants(Bandoniene&Murko-vic,2002).However,naturalantioxidantsarenotlimitedtoter-restrialsources.Someoftheseaweedsareconsideredtobearichsourceofantioxidants(Lim,Cheung,Ooi,&Ang,2002).Forexample,chlorophylls,carotenoids,tocopherolderivativessuchasvitaminE,andrelatedisoprenoids,thatarestructurallyrelatedtoplant-derivedantioxidants,werefoundinsomemarineorganisms(Takamatsuetal.,2003).Duringthecourseofantioxi-dantactivityscreeningofseaweedscommonlyfoundintheQingdaocoastline,wemeasuredthetotallipo-philiccontentandantioxidantactivityoflowerpolar(diethylether)extractsof16seaweedsamples(Huang&Wang,2004).Ourpresentworkonmorepolar(chlo-roform:methanol)extractsfoundthatthecrudeextractoftheredalga,Polysiphoniaurceolata,revealedhigherantioxidantactivityinthea,a-diphenyl-b-picrylhydrazyl(DPPH)radicalscavengingassayandintheb-carotene–linoleateassaysystems.Itshouldbementionedthattherawextractsderivedfromdifferentsolventextractionsofthisalgahavepreviouslybeenscreenedforantioxidantactivity(Yan,Nagata,&Fan,1998).Incontrast,thecurrentcommunicationfocussedontheinvestigationandevaluationoftheantioxidativecapacityofextract,fractions,andsemi-purifiedsubfractionswithdifferentpolaritythatwerederivedfromP.urceolata.Further-more,thetotalphenoliccontentandthereducingpowerofallextract,fractionsandsubfractionswerealsodeter-mined.Inaddition,therelationshipsbetweentheantiox-idantactivityandthephenoliccontentaswellasbetweentheantioxidantactivityandthereducingpowerarealsoconsidered.2.Materialandmethods2.1.ChemicalsButylatedhydroxytoluene(BHT),a,a-diphenyl-b-picrylhydrazyl(DPPH),b-caroteneandFolin–CiocalteureagentwerepurchasedfromSigmaChemicalCo.(St.Louis,MO,USA).Linoleicacid,Tween-40(polyoxyeth-ylenesorbita