抗氧化活性测定方法

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：10μL sample (不同浓度) + 50 μL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 μL of sample solution （样品液）+ 50 μL of a 1.0 mmol/L CuSO4 solution （CuSO4 溶液）+ 50 μL of a 1 mmol/L 1,10-phenanthroline solution（邻二氮菲溶液）+ 700 μL of a 0.05mol/L borate buffer (pH 9.0) （硼酸缓冲液）+ 100 μL of a 1 mmol/L ascorbate solution + 50 μL 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 system
2、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 g/mL DNA was incubated with a phen-Cu solution.
步骤：800 L of phen-Cu/DNA solution + 100 L of 4.2 *10-3 mol/L ascorbate + 200 L of 6% H2O2 + were added without interval to a 100 L 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 mixture without the sample solution, and the background was detected without H2O2 addition.)
4、1.0 mmol/L CuSO4 solution （CuSO4 溶液）+ 50 L 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EPR
3.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 μL) was manually pipetted into the wells containing the extract or standards (20 μL) 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 μL 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 230nm
5.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: 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235 nm下测定，摩尔消光系数为100 M-1cm-1
样品和阿魏酸均溶于50mM ph7.4PBS
100ul氨基乙磺酸（150mM）+100ul 样+100ul HOCL+700ul PBS------10min室温，10ul碘化钾（2M）加入混合物
空：样+pbs 350nm。