抗氧化剂抗氧化活性的测定方法-二-王会

()＊王　会1　郭　立2　谢文磊11(,,450052)　2(,,450003)　　综述了抗氧化剂抑制脂质氧化、清除自由基、抑制自由基对底物的氧化降解以及还原能力4类测定抗氧化活性的方法,并讨论了评价或表征测定结果的方法和参数以及对测定方法的要求。　抗氧化剂,抗氧化活性,测定方法　:()。＊2006,32(3):92～96　:2005-12-053.3　3.1.3.2,,,,,。3.3.1　ORAC法ORAC(oxygenradicalabsorbancecapacity,)Cao[31]Glaz-er[32],:β-(β-phycoerythrin,β-PE)540nm565nm。,β-PE;,β-PE。AAPH()、H2O2-Cu2+(HO·)Cu2+。AAPH,,、α-、β-。Cu2+-H2O2,、、,、α-[33]。Trolox()。、。[32,34]2:(1)(AUC),。。(2),,,PE。ORAC,AAPH(2000),,,,。,、,。,(、、)。3.3.2　TRAP法WaynerTRAP(totalradicaltrappingan-tioxidantparameter,)[35]10。AAPH,AAPH,。,。Trolox。GhiselliTRAP[34]Glazer[32]。Glazer1990TRAPAAPHCu2+-HO·,B-R-PE()。GlazerHO·,PE。PEHO·Trolox。,PEHO·,[36]。GhiselliTRAP[34]ORAC,。TRAP[34]PE(),80%;ORAC[37]()。PE。TRAPFOODANDFERMENTATIONINDUSTRIES98　　　2006Vol.32No.4(Total220)、;ORAC,。Trolox。3.3.3　DCFH-DA法ValkonenKuusiDCFH-DA(dichoroflu-orescin-diacetane,)[38]。AAPHDCFH-DA,DCF(dichlorofluorescein,)。DCF(Ex480nm,Fm526nm),504nm,。,DCF4:(thefirstlagphase),(thefirstpropagationphase);Trolox,,Trolox。。3.3.4　化学发光法(chemiluminescence,CL)(luminol()、lucigenin(),,,。,CL,,(tIND)。Trolox。CL(tIND)TroloxCL。CLMetsa-ketela,White-head[39],。H2O2luminol,(HRP)。,,。-(),。,。,CL(2)。CL、,,。CL,。3.3.5　β-胡萝卜素漂白法[45]β-(β-carotenebleachingtest),β-,β-(470nm);,β-。2　CLAAPHluminol40ABAPluminol41EDTA/Co(+2)/H2O2·OH42-hypoxantine-O2·luminol43/O2-·lucigenin44　　,(),,。(),。β-,,。,β-,。,。3.3.6　藏花素漂白法(crocinbleachingtest)Bors[46]、Tubaro[47],。(),(443nm)[46,47]。(AAPHABAP),();,。。A,(),1min,(V0)(V),10min,V0/V=1+(ka/kc)·([A]/[C])。,[A][C],kcka[46,47],ka/kc[A]/[C]V0/V,ka/kc,V0/V。Troloxka/kcka/kc,“Trolox”2006324(220)99　　　。,。Tubaro,[47]。-,(10μmol/L),。Lyssignoli———(microplate-basedcolori-metricassay),[48]。%=(Ac-Aa)/Ac×100,,AcAa。3.3.7　KI法[49],。PV,AAPH,KI。,。。,。3.3.8　TOSC法TOSC(totaloxyradicalscavengingcapacity,)RegoliWinston[50,51]。、·OHKMBA(α-ketoγ-methiolbutyricacid),(ethylene),。,KMBA,。TOSCTOSC=100-(∫SA/∫CA×100),∫SA∫CA,TOSC0～100。。KMBA。,TOSC。TOSC,、,,(ABAP)。TOSC,(μmol/L)[51];,;,,。,,,,。,。3.4　3.4.1　FRAP法FRAP(ferricreducingantioxidantpower,):pH,()Fe3+-TPTZ(tripyridyltriazine,)Fe2+-TPTZ,Fe2+-TPTZ,593nm[52],585nm[53]600nm[54]。Fe2+。Fe3+Fe2+。()(·OH),Fe3+,Fe3+,Fe3+。,Fe3+,GSH。FRAP,,,,、、、。SH—。3.4.2　循环伏安法,(cyclicvoltammetry,CV)。Kohen[55]CV。CV()、(Ag/AgCl)()。(100mV/s),,。,,,S。,,,,。(x-),[Ep(a)](,,E1/2)(2),Ep(a)FOODANDFERMENTATIONINDUSTRIES100　　2006Vol.32No.4(Total220)E1/2。2:[Ep(a)](anodiccurrent,AC)。E1/2:E1/2,。AC(y)(,),[56],[55]。2()。2　(0.5mmol/Lβ-NADPH(0.5mmol/L)],CV。,CV。,[57][58]。CV,。,。,()[57]。,。,()[55],(Au/Hg)。CV,(1～10μmol/L)[55],,,。CV、HPLC。4　各种测试方法的比较,。(1),TEACORAC,FRAP。(2),。,FRAP60%,GSH,pH(pH=3.6)。(3),ORACPE,TEAC(36min)。,,ORACFRAP。,,;,。,。,,,。,。,DPPHABTS。,,(DPPH·ABTS·)。。,。,,,,,。31　CaoG,AlessioHM,CutlerRG.Oxygen-radicalabsorbancecapacityassayforantioxidants[J].FreeRadicBiolMed,1993,14:303～31132　GlazerAN.Phycoerythrinfluorescence-basedassayforreac-tiveoxygenspecies[J].Meth.Enzymol,1990,186:161～16833　CaoG,SoficE,PriorRL.Antioxidantandprooxidantbe-haviorofflavonoids:Structure-activityrelationships[J].FreeRadicBiolMed,1997,22:749～76034　GhiselliA,SerafiniM,MaianiG,etal.Afluorescence-basedmethodformeasuringtotalplasmaantioxidantcapability[J].FreeRadicBiolMed,1995,18:29～3635　WaynerDD,BurtonGW,IngoldKU,etal.Quantitativemeasurementofthetotal,peroxylradical-trappingantioxi-dantcapacityofhumanbloodplasmabycontrolledperoxida-tion[J].FEBSLett,1985,187:33～3736　PriorRL,CaoG.Invivototalantioxidantcapacity:compar-isonofdifferentanalyticalmethods[J].FreeRadicBiol2006324(220)101　　Med,1999,27,1173～118137　CaoG,PriorRL.Measurementofoxygen-radicalabsorbancecapacityinbiologicalsamples[J].MethEnzymol,1999,299:50～6238　ValkonenM.KunsiT.Spectrophotometricassayfortotalperoxylradicaltrappingantioxidantpotentialinhumanserum[J].JLipidRes,1997,38:823～83339　WhiteheadTP,ThorpeGHG,MaxwellSRJ.Enhancedchemiluminescentassayforantioxidantcapacityinbiologicalfluids[J].AnalChimActa,1992,266:265～27740　VisioliF,GalliC.Evaluationofantioxidantcapacitybychemiluminescence[J].Anal.Biochem,1997,249:244～24641　LissiE,PascualC,Del-CastilloMD.Luminolluminescenceinducedby2,2'-azo-bis-2-amidinopropanethermolysis[J].FreeRadicResComm,1992,17:299～31142　ArnousA,MakrisDP,KefalasP.Effectofprincipalpolyphenoliccomponentsinrelationtoantioxidantcharacter-isticsofagedredwines[J].JAgricFoodChem,2001,49:5736～574243　OgawaA,AraiH,TanizawaH,etal.On-linescreeningmethodforantioxidantsbyliquidchromatographywithchemiluminescencedetection[J].Anal.Chin.Acta,1999,383:221～23044　GabbianelliR,FalcioniG,SantroniAM,etal.Effectofaro-maticnitroxidesonhemolysisofhumanerythrocytesen-trappedwithisolatedhemoglobinchains[J].FreeradicBiolMed,1997,23:278～28445　KulisicT,RadonicA,KatalinicV,etal.Useofdifferentmethodsfortestingantioxidativeactivityoforeganoessentialoil[J].FoodChem,2004,85:633～64046　BorsW,MichelC,SaranM.Inhibitionofthebleachingofthecarotenoidcrocin.Arapidtestforquantifyingantioxi-dantactivity[J].Biochim.Biophys.Acta,1984,796:312～31947　TubaroF,GhiselliA,RapuzziP,etal.Analysisofplasmaan-tioxidantcapacitybycompetitionkinetics[J].FreeRadicBi-olMed,1998,24:1228～123448　LussignoliS,FraccaroliM,AndrioliG,etal.Amicro