抗氧化活性测定方法.doc

抗氧化方法一、 Determination of Superoxide Anion Scavenging Ability（超氧阴离子清除能力）1、 determined by a CL method in the pyrogallol-luminol system on a BPCL Ultra-weak luminescence analyzer。（焦性没食子酸-发光胺，荧光检测）2、 步骤：10mL sample (不同浓度) + 50 mL焦棓酸(6.25*10-4 mol/L) + 0.94 mL of a mixture containing luminol (0.05 mol/L) and carbonate buffer (pH 10.2) （发光胺用pH 10.2碳酸盐缓冲液配成0.05 mol/L）3、 Hi-V, 800; Kv, -1; the spectral range of CL（波长范围）180-800 nm; 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、Determination of Scavenging Ability on Hydroxide Radicals（羟基自由基清除能力）1、CuSO4-Phen-Vc-H2O2 CL system （1 mL体系）2、50 mL of sample solution （样品液） + 50 mL of a 1.0 mmol/L CuSO4 solution （CuSO4 溶液）+ 50 mL of a 1 mmol/L 1,10-phenanthroline solution（邻二氮菲溶液）+ 700 mL of a 0.05mol/L borate buffer (pH 9.0) （硼酸缓冲液）+ 100 mL of a 1 mmol/L ascorbate solution + 50 mL of a 0.15% H2O2 solution 3、总时间400S，间隔3S。Hi-V, 800; Kv, -1; the spectral range of CL（波长范围）180-800 nm; 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、 Determination of Scavenging Effect on Hydrogen Peroxide（过氧化氢清除能力）1、luminol-H2O2 system2、The luminescent reaction was initiated by manually adding 1 mL of a solution containing 0.15 mol/L hydrogen peroxide and 0.1mol/L luminol per liter of carbonate buffer (0.05 mol/L, pH 9.4). Light emission vs time was recorded for 3 min at 2 s intervals. 步骤：0.15 mol/L的过氧化氢 + 0.1mol/L发光胺（用pH 9.4的碳酸缓冲液配），总体系1ml. 3、BPCL Ultra-weak luminescence analyzer，Hi-V, 800; Kv, -1; the spectral range of CL（波长范围）180-800 nm; 温度：30 C.4、The control was performed in the same manner in the mixture without the sample solution, and the background was detected without H2O2 addition。四、 Determination of Preventing DNA Damage Effect（DNA损伤）1、CuSO4-Phen-Vc-H2O2-DNA CL system.2、步骤：Copper and 1,10-phenanthroline were premixed in 0.1 mol/L NaOAc/HOAc (pH 5.5) buffer。 3 mg/mL DNA was incubated with a phen-Cu solution. 步骤：800 mL of phen-Cu/DNA solution + 100 mL of 4.2 *10-3 mol/L ascorbate + 200 mL of 6% H2O2 + were added without interval to a 100 mL sample solution to give a final volume of 1.2 mL. （总体系1.2 mL）3、The kinetic curve of CL produced in the phen/Cu/H2O2/ascorbate system was immediately recorded. (The control was performed in the same manner in the mixturewithout the sample solution, and the background was detected without H2O2 addition.)4、1.0 mmol/L CuSO4 solution （CuSO4 溶液）+ 50 mL of a 1 mmol/L 1,10-phenanthroline solution（邻二氮菲溶液）（文献来源J. Agric. Food Chem. Evaluation of Antioxidant Activity and Preventing DNA Damage Effect of Pomegranate Extracts by Chemiluminescence Method）1.DPPH: 0.5 ml 样品+ 2 ml DPPH-漩涡，室温暗处 样品梯度：0.062-2.5 mg/ml 5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 100 ul 提取液+ 100ul H2O2（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(pH 7.0)的磷酸缓冲液配置 295ul体系：200ul （0.11uM）荧光素 + 20ul 样品 孵化 15min 37摄氏度+75 ul AAPH（63.4 mM）Fluorescein (200 L) was manually pipetted into the wells containing the extract or standards (20 L) followed by incubation for 15 min at 37 _C. The injector pump was programmed to injectAPPHat the end of incubation during the first cycle.The plate was automatically shaken for 4 s after each addition, and the microplate reader was programmed to perform additional shaking of the contents in wells before each reading was taken. A gain adjustment was performed before the beginning of the measurements to optimize the maximum sensitivity, by manually pipetting 200 L of fluorescein into wells. Fluorescence was recorded everyminute for 25 cycles, and each cycle was 210 s.4.H2O2清除能力：pbs 45 mM pH 7.4 0.4 ml 样品（蒸馏水溶）+ 0.6 ml H2O2 (40mM)+1ml PBS 30摄氏度40min 230nm5.单线态氧抑制：pbs 45 mM pH 7.4 0.4ml 样品 + 0.5 ml DPN (200um)+0.2ml组氨酸（100mM）+0.2ml 次氯酸钠（100mM）+0.2mlH2O2(100mM)+0.5 ml PBS-40min 30摄氏度 440nm 空白：样+PBS control