抗氧化剂抗氧化活性的测定方法-一-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:()。:2005-12-05　　,;。、。[1]。、、DNA。,:(1)(primaryantioxidants),(chain-breakingantioxi-dants),(2)(secondaryantioxidants),(preventativeantioxidants)。(AH)L·、LOO·LO·[2]。L·+AHLH+A·LOO·+AHLOOH+A·LO·+AHLOH+A·A·、。LOO·+A·LOOALO·+A·LOA,、、[3]。()。,。1　评价或表征抗氧化活性的方法与参数1.1　　,。;()。:(1);(2);(3)();(4)();(5)。1.2　　1.2.1　诱导期(inductionperiod)tIND(,lagperiod)。,。,、。,。1.2.2　抑制率(percentageofinhibition)和IC50IC50(50%,EC50)。,,。,IC50。,IC50。TEC50FOODANDFERMENTATIONINDUSTRIES92　　　2006Vol.32No.3(Total219)50%,。2　对测定方法的要求:(1),;(2);(3);(4);(5);(6);(7);(8),;(9)。,。,2,2。,,:2,,,。,,。3　测定抗氧化活性的方法(DNA)、、()。。:(1),(2),(3)。(1)(2),;(3),,,ABTSDPPH。3.1　,。,,、、。,。、,。3.1.1　氧化的底物或使用的体系。、(、、、)、,。,,、、[4],。,,。,()。,。,。,,。,,。,(VE),。,。。3.1.2　引发或加速氧化的方法,,,。,、、Rancimat、OSI。。,,。2006323(219)93　　　,,AAPH[2,2'-azobis(2-amidinopropane)dihy-drochloride]AMVN[2,2'-azobis(2,4-dimethylvaleronitrile)]。,ABAP(azo-bis-amidinopropanehydrochloride)、ABIP(2,2'-azobis[2-(2-imidazolin-2-yl)propanel]dihy-drochloride)ADVN(azo-bis-2,4-dimethylyaleronitrile)、AIBN(azobisisobutyrylnitrile),DBHN(di-tert-butylhyponitrite)。,。,。,。,,,,[5]。,Fe2+Cu2+、Fe3+/。(),。()。,,,。3.1.3　脂质氧化的检测、。,、、()、TBARS、、。,、、、、、、、。,(C18:2/C16:0)、、。,,,。3.1.3.1　(1)(peroxidevalue,PV)。,。PV,。,PV[6]。ROOHROOR,:ROOH+2H++2I-I2+ROH+H2OROOR+2H++2I-I2+2ROHI2+2S2O3S4O62-+2I2,。,,,。,、、(、)。(2)。1931,Gillam230～235nm[7],2。1960,。,。。。(、、),。。,。,,,。,。(3)(ferricthiocyanatemethod,FTC)。Fe2+Fe3+,Fe3+,500nm,500nm。3.1.3.2　,()、(、、)、,,1。FOODANDFERMENTATIONINDUSTRIES94　　　2006Vol.32No.3(Total219),TBARS。,TBA。TBA,TBA-,532～535nm。。,HPLC。、、,。,,。,。1　　　(Kreistest)(),546nm(anisidinevalue)α-β,350nm(TBARS)(MDA)(TBA),532535nm(2,4-)(、)2,4-,440nm、、　　,、、TBARS。,。3.2　3.2.1　ABTS法ABTSMarklundABTS[2,2'-azinobis-(3-ethylbenzthiazoline-6-sulfonicacid)diammoniumsalt,2,2--(3--6-)]ABTS·+,[8]。Miller,[9],。ABTS(λmax=342nm)K2S2O8、MnO2、ABAPH2O2(2),ABTS·+(1)。ABTS·+,414、645、734805nm[9]。(),ABTS·+,ABTS。ABTS·+414734nm。TEAC(troloxequivalentan-tioxidantcapacity,trolox),ABTS·+()trolox(VE)ABTS·+。trolox(2-carboxy-2,5,7,8-tetramethyl-chroman-2-carboxylicacid)TEAC1。MillerABTS,ABTS·+ABTS(ferrylmyoglobin),(metamyoglobin)H2O2。,H2O2ABTS·+,,TEAC。ABTS·+,ABTS,ABTS·+,(2)。1　ABTSABTS·+2　ABTSABTS·+/nm　myoglobin/H2O273410H2O2/HRP73511HRP/H2O241412microperoxidase-8(MP8)73413K2S2O873414K2S2O841415H2O266016ABAP73417MnO273418414734192006323(219)95　　　　　ABTS。,。,TEAC,,,TEAC3.1[14]6.4[17]。,,TEAC,,,1.51.0μmol/LTEAC5.66.4[17]。TEAC:ABTS·+、、、pH。ABTS5～6min,ABTS·+,TEAC。,ABTS·+ABTS·+,TEACTEAC[20]。,chrysin(5,7-)ABTS·+ABTS·+chrysin,troloxABTS·+ABTS·+,TEAC。,。ABTS·+,,,TEAC[21]。3.2.2　DMPD·+法FoglianoABTS:,DMPD(N,N-dimethyl-p-phenylenediamine)ABAP、FeCl3、CuCl2、H2O2,DMPD·+,505nm[22]。。FoglianoFeCl3DMPD。DMPD,。3.2.3　DPPH法DPPH1950,,、。DPPH·(2,2'-diphenyl-1-picrylhydrazyl,2,2-),,515nm。(),DPPH·,。。DPPH2。DPPH·,,,DPPH·[23];DPPH·,DPPH·—OH,IC50[24]。ABTS,DPPH·DPPH·,。Sanchez-Moreno2,AE(antioxidanteffi-ciency),AE1/EC50.t50(t5050%)[25]。DeBeer:RSE(radicalscavengingeffi-ciency),RSEDPPH·EC50[26]。AERSE,。BandonieneMukovicDPPHHPLC[27]。HPLC,(),,、、。ABTS·+DPPH·2,:(1)DPPH·,ABTS·+。(2)ABTS·+,,。DPPH·,,,,。(3),DPPH·ABTS·+。,DPPH·—OH[28],B--OH[29]。ABTS·+。3.2.4　Fremy自由基和galvinoxyl还原法[30]Fremy(potassiumni-trosodisulphonate,),galvinoxyl[2,6-di-tert-butyl-a-(3,5-di-tert-butyl-4-oxo-2,5-cyclohexadien-1-ylidene)-p-tolyloxy],,。(ESR),。GalvinoxylFremyFOODANDFERMENTATIONINDUSTRIES96　　　2006Vol.32No.3(Total219),;,。1　HalliwellB.Antioxidantcharacterization:methodologyandmechanism[J].Biochem.Pharmacol,1995,49:1341～13482　McClementsDJ,DeckerEA.Lipidoxidationinoil-in-wateremulsions:impactofmolecularenvironmentonchemicalreac-tionsinheterogeneousfoodsystems[J].JFoodSci,2000,65:1270～12823　GrafE,MahoneyJR,BryantRG,etal.Ironcatalyzedhy-droxylradicalformation:Stringentrequirementforfreeironcoordinationsite[J].JBiolChem,1984,259:3620～36244　NietoS,GarridoA,SanhuezaJ,etal.Flavonoidsasstabiliz-ersoffishoil,analternativetosyntheticantioxidants[J].JAmOilChemSoc,1993,70:773～7785　RoginskyV.Chain-breakingantioxidantactivityofnaturalpolyphenolsasdeterminedduringthechainoxidationofmethyllinoleateinTritonx-100micelles[J].ArchBiochemBiophys,2003,414:261～2706　GB/T5009.37—2003.[S]7　DormandyTL,WickensDG.Theexperimentalandclinicalpathwayofdieneconjugation[J].ChemPhysLipids,1987,45:353～3648　MarklundS.Asimplespecificmethodforthedeterminationofthehemoglobincontentoftissuehomogenates[J].ClinChimActa,1979,92:229～2349　MillerNJ,Rice-EvansC,DaviesMJ,etal.Anovelmethodformeasuringantioxidantcapacityanditsapplicationtomonitoringtheantioxidantstatusinprematureneonates.[J].ClinSci,1993,84:407～41210　Rice-EvansC,MillerNJ.Totalantioxidantstatusinplasmaandbodyfluids[J].MethodsEnzymol,1994,234:279～29311　ObónJM,CastellarMR,CascalesJA,etal.AssessmentoftheTEACmethodfordeterminingtheantioxidantcapaci-tyofsyntheticredfoodcolorants[J].FoodResInt,2005,38:843～84512　LabrineaEP,GeorgiouGA.Stopped-flowmethodforas-sessmentofpHandtimingeffectontheABTStotalantioxi-dantcapacityassay[J].AnalyticaChimicaActa,