抗氧化研究方法.pptVIP

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 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 氧自由基的反应 超氧阴离子歧化 铁催化的羟自由基产生－Fenton Reaction and Harber-Weiss Reaction 脂质过氧化 －CH2－ + .OH 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) 直接测定DPPH自由基 以4－POBN为捕获剂，测定碳中心自由基 化学法 (Hydroxyl radical) Drug effects were tested on deoxyribose oxidation induced by 60 min incubation at 37?C with nonchelated Fe2+, namely 20 ?M FeSO4, with or without 1.42 mM H2O2 , in PPB, 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 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 4?C; after a brief centrifugation to favor organic phase separation, the upper n-butanol layer was removed and read spectrophotometrically at 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 aliquot of 10?l 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 concentration. ABTS radical cat