抗氧化活性测定方法

抗氧化方法一、DeterminationofSuperoxideAnionScavengingAbility（超氧阴离子清除能力）1、determinedbyaCLmethodinthepyrogallol-luminolsystemonaBPCLUltra-weakluminescenceanalyzer。（焦性没食子酸-发光胺，荧光检测）2、步骤：10Lsample(不同浓度)+50L焦棓酸(6.25*10-4mol/L)+0.94mLofamixturecontainingluminol(0.05mol/L)andcarbonatebuffer(pH10.2)（发光胺用pH10.2碳酸盐缓冲液配成0.05mol/L）3、Hi-V,800;Kv,-1;thespectralrangeofCL（波长范围）180-800nm;温度：30°C.总时间：300S，每2S读数一次。4、对照：不加样品（样品用水代替）。空白不加焦棓酸（用来调零）。二、DeterminationofScavengingAbilityonHydroxideRadicals（羟基自由基清除能力）1、CuSO4-Phen-Vc-H2O2CLsystem（1mL体系）2、50Lofsamplesolution（样品液）+50Lofa1.0mmol/LCuSO4solution（CuSO4溶液）+50Lofa1mmol/L1,10-phenanthrolinesolution（邻二氮菲溶液）+700Lofa0.05mol/Lboratebuffer(pH9.0)（硼酸缓冲液）+100Lofa1mmol/Lascorbatesolution+50Lofa0.15%H2O2solution3、总时间400S，间隔3S。Hi-V,800;Kv,-1;thespectralrangeofCL（波长范围）180-800nm;温度：30°C.4、阳性对照：不加样品（样品用水代替）。空白不加H2O2（用来调零）。三、DeterminationofScavengingEffectonHydrogenPeroxide（过氧化氢清除能力）1、luminol-H2O2system2、Theluminescentreactionwasinitiatedbymanuallyadding1mLofasolutioncontaining0.15mol/Lhydrogenperoxideand0.1mol/Lluminolperliterofcarbonatebuffer(0.05mol/L,pH9.4).Lightemissionvstimewasrecordedfor3minat2sintervals.步骤：0.15mol/L的过氧化氢+0.1mol/L发光胺（用pH9.4的碳酸缓冲液配），总体系1ml.3、BPCLUltra-weakluminescenceanalyzer，Hi-V,800;Kv,-1;thespectralrangeofCL（波长范围）180-800nm;温度：30°C.4、Thecontrolwasperformedinthesamemannerinthemixturewithoutthesamplesolution,andthebackgroundwasdetectedwithoutH2O2addition。四、DeterminationofPreventingDNADamageEffect（DNA损伤）1、CuSO4-Phen-Vc-H2O2-DNACLsystem.2、步骤：Copperand1,10-phenanthrolinewerepremixedin0.1mol/LNaOAc/HOAc(pH5.5)buffer。-Cusolution.步骤：800Lofphen-Cu/DNAsolution+100Lof4.2*10-3mol/Lascorbate+200Lof6%H2O2+wereaddedwithoutintervaltoa100Lsamplesolutiontogiveafinalvolumeof1.2mL.（总体系1.2mL）3、ThekineticcurveofCLproducedinthephen/Cu/H2O2/ascorbatesystemwasimmediatelyrecorded.(Thecontrolwasperformedinthesamemannerinthemixturewithoutthesamplesolution,andthebackgroundwasdetectedwithoutH2O2addition.)4、1.0mmol/LCuSO4solution（CuSO4溶液）+50Lofa1mmol/L1,10-phenanthrolinesolution（邻二氮菲溶液）（文献来源J.Agric.FoodChem.EvaluationofAntioxidantActivityandPreventingDNADamageEffectofPomegranateExtractsbyChemiluminescenceMethod）1.DPPH:0.5ml样品+2mlDPPH--------漩涡，室温暗处样品梯度：0.062-2.5mg/ml5个梯度，甲醇溶DPPH：0.19m/M甲醇溶电子顺磁共振（EPR）侧吸光值2.羟基自由基清除活力：可溶和结合酚类都溶于去离子水，稀释100ul提取液+100ulH2O2（10mM）+200ulDMPO（17.6mM）+100ulFeSO4(0.1mM)----1min后EPR3.氧自由基吸收力测定：测定样品对AAPH产生的过氧化氢的抗氧化活性样品和其他试剂均用75mM(pH7.0)的磷酸缓冲液配置295ul体系：200ul（0.11uM）荧光素+20ul样品孵化15min37摄氏度+75ulAAPH（63.4mM）Fluorescein(200μL)wasmanuallypipettedintothewellscontainingtheextractorstandards(20μL)followedbyincubationfor15minat37_C.TheinjectorpumpwasprogrammedtoinjectAPPHattheendofincubationduringthefirstcycle.Theplatewasautomaticallyshakenfor4saftereachaddition,andthemicroplatereaderwasprogrammedtoperformadditionalshakingofthecontentsinwellsbeforeeachreadingwastaken.Againadjustmentwasperformedbeforethebeginningofthemeasurementstooptimizethemaximumsensitivity,bymanuallypipetting200μLoffluoresceinintowells.Fluorescencewasrecordedeveryminutefor25cycles,andeachcyclewas210s.4.H2O2清除能力：pbs45mMpH7.40.4ml样品（蒸馏水溶）+0.6mlH2O2(40mM)+1mlPBS30摄氏度40min230nm5.单线态氧抑制：pbs45mMpH7.40.4ml样品+0.5mlDPN(200um)+0.2ml组氨酸（100mM）+0.2ml次氯酸钠（100mM）+0.2mlH2O2(100mM)+0.5mlPBS---------40min30摄氏度440nm空白：样+PBScontrol:PBS取代样品6：HOCl清除能力HOcl：1%（v/v）Naocl调至ph6.2(用1%（v/v）硫酸)HOcl含量在235nm下测定，摩尔消光系数为100M-1cm-1样品和阿魏酸均溶于50mMph7.4PBS100ul氨基乙磺酸（150mM）+100ul样+100ulHOCL+700ulPBS------10min室温，10ul碘化钾（2M）加入混合物空：样+pbs350nm