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1、抗氧化研究方法氧化损伤的产生 线粒体呼吸链中，由于发生电子漏，导致氧分子经 单电子转移反应，生成了超氧阴离子。 某些氧化酶催化氧化底物时，释放出活性氧，如黄 嘌呤/黄嘌呤氧化酶体系放出超氧阴离子、葡萄糖/葡 萄糖氧化酶体系放出过氧化氢。 巨噬细胞受刺激后呼吸爆发产生活性氧。体内自由基的产生O2O2.H2O2.OHee+2H+e+H+H2O主要自由基简介 单线态氧 超氧阴离子 过氧化氢 脂质过氧自由基 碳中心自由基氧的状态1s*1s2s*1s2pp2pp*2p*2pGround state O2singlet O2Superoxide O2-Peroxide ion O22-氧自由基的反应超氧阴
2、离子歧化2O2-. + 2H+H2O2 + O2过氧化氢的反应：Mn+ + H2O2M (n+1)+ + .OH +OH- 铁催化的羟自由基产生Fenton Reaction and Harber-Weiss ReactionFenton Reaction:Fe2+ + H2O2Fe3+ + .OH + OH-Weiss Reaction:Fe3+ + O2-.Fe2+ + O2Fe2+ + H2O2Fe3+ + .OH + OH-(1)(2)NetO2-. +H2O2O2+.OH +OH-(3)脂质过氧化CH2 + .OH . CH2 + H2OInitiation:CH. + O2CHO2
3、.Propagation:CHO2. +CHO2H +CH2 CH. Termination :CH. +CH. CHCH抗氧化研究内容 清除自由基 抑制脂质过氧化 总抗氧化活力测定 抑制氧化导致的细胞损伤 缺血再灌注损伤及炎症1、清除自由基a. 清除羟自由基b. 清除超氧阴离子c. 清除单线态氧d. 清除DPPH自由基e. 清除碳中心自由基电子顺磁共振法(Electronic Paramagnetic Resonance, EPR) 以DMPO 捕获剂，测定羟自由基(Hydroxyl radical)和 超氧阴离子自由基 以TEMP为捕获剂，测定单线态氧(Singlet oxygen) 直接
4、测定DPPH自由基 以4POBN为捕获剂，测定碳中心自由基典型的羟自由基DMPO加合物图谱典型的超氧阴离子自由基DMPO加合物图谱典型的单线态氧TEMP加合物图谱典型的碳中心自由基4-POBN加合物图谱典型的DPPH自由基图谱化学法 (Hydroxyl radical) Drug effects were tested on deoxyribose oxidation induced by 60 min incubation at 37C with nonchelated Fe2+, namely 20 M FeSO4, with or without 1.42 mM H2O2 , in PP
5、B, pH7.4. The TBA- test, which detects aldehydic products, such as malondialdehyde (MDA), resulting from deoxyribose (or lipid) oxidation, was then carried out adding to 0.5 mL of sample, 1.0 mL of 2.8% trichloroacetic acid and 1.0 mL of 0.6% aqueous solution of TBA; tubes were heated at 95 C for30
6、min to develop the color, and successively cooled in ice. The red chromogen, expression of the TBA:MDA adduct formation, was extrtacted with n-butanol kept at 4C; after a brief centrifugation to favor organic phase separation, the upper n-butanol layer was removed and read spectrophotometrically at
7、532 nm against an appropriate blank. Results were caculated as nmol TBARS per mL, using a molar extinction coefficient of 154,000 at 532 nm. Superoxide anion To a mixture of tested compound (50 M), xanthine oxidase (25 munit/ml), and nitro blue tetrazolium (50 M) in Tris buffer (0.1 M, pH 7.4), and
8、aliquot of 10l hypoxanthine (3.5 mM) was added, the total volume of the mixture was 1 ml. The absorbance of the reaction mixture was monitored spectrophotometrically at 560 nm for 10 min using a UV-VS spectrum meter. Total antioxidant status (TAS) assay ABTS was dissolved in water to a 7 mM concentr
9、ation. ABTS radical cation (ABTS+) was produced by first adding MnO2 powder in the solution and keeping in the dark at room temperature for more than 12 h, then filtrated with syringe filter and kept in dark for another 6 hours. The obtained radical was stable in this form for more than two days whe
10、n stored in the dark at room temperature. Stock solution of ABTS+ was diluted with PBS, pH 7.4, to an absorbance of 0.70 (0.02) at 734 nm and equilibrated at 30C. After adding 1.0 ml of diluted ABTS+ to 10 l of serum or tissue homogenate supernate, the absorbance reading was taken at 30C exactly 2 m
11、in after initial mixing. The total antioxidant capacity concentration was compared to equivalent antioxidant capacity of Trolox and is expressed in mols of Trolox/g of tissue.Quenching DPPH radical DPPH dissolved in ethanol to a concentration of 90M, tested compounds dissolved in ethanol or distille
12、d water to 120 M solution, to 2.5 ml DPPH solution, the suitable amount of tested compound solution was added, the total volume was adjusted to 3 ml with ethanol and mixed thoroughly, the absorbency was recorded at 517 nm exactly at 10 min. Xanthine oxidase inhibition assay Hypoxanthine (35 M) was a
13、dded to a mixture of xanthine oxidase (25 munit/ml) and tested compound (10-50 M) in Tris buffer (pH 7.4, 0.1 M). The change in absorbance was monitored at 293 nm at room temperature for 5 min.TBARS Assay Direct assay: Four hundred microliter sample mixed with 400 l 10% trichloroacetic acid (TCA), 1
14、 ml of 0.6% 2-thiobarbituric acid (TBA), and then incubated in a water bath at 95for 60 min. After cooling the sample with water, 2.0 ml of n-butanol were added and the mixture was shaken vigorously. After centrifugation at 5000 rpm for 10 min, the absorbance of the organic layer was measured at 532
15、 nm. Induced by other chemicals For Fe2+-Ascorbate, usually incubated at 37for 30 min For AAPH, usually incubated at 37for 2 hoursCell culture Mostly used oxidant is H2O2 Direct addition Enzymatic methodGlucose/glucose oxidase system (H2O2)Xanthine/xanthine oxidase (O2-.) Assaya. Morphological study
16、b. MTT assayc. TBARS assayd. apoptosis缺血再灌注损伤 从89年到2002年，国内期刊上发表的有 关缺血再灌注损伤的文章4308篇，其中 2002年全年793篇机理 再灌流时，多种氧化酶系以氧气、NADPH、ATP等 为底物，在Ca(II)、Fe(II)的催化介导下，产生大量的 具有强烈生物氧化活性的自由基。氧自由基使不饱 和脂肪酸部分激活，在金属复合物特别是铁的参与 下，进而引起一系列的自由基反应，造成膜不饱和 脂肪酸的脂质过氧化，导致脂膜局部破坏，直至溶 解。 另外，自由基还可使磷脂酶改变活性，引起花生四 烯酸(arachidonic acid, AA)代谢紊乱，导致微血栓形 成ATPADPAMPAdenosineC
