Antioxidative Function of Plant Polyphenols in Mammalian Cells

(1)以中国仓鼠V79细胞为实验对象，分别用集落形成实验和DNA沉淀实验检测H_2O_2对V79细胞的细胞毒作用和DNA单链断裂作用。在这两种检测中，咖啡酸乙酯都显示出保护作用。构效关系表明咖啡酸乙酯的欧米克-二羟基结构是其保护作用的关键。(2)采用集落形成实验检测姜黄素及其化学修饰同系物对H_2O_2诱导的中国仓鼠长成纤维细胞V79细胞毒的抑制作用。在4种姜黄素中，二羟基姜黄素和二羟基四氢姜黄素可抑制H_2O_2诱导的细胞毒作用。相反，姜黄素和四氢姜黄素均未表现出抑制作用。(3)建立了儿茶素溶液在有氧加热过程中产生的过氧化氢(H_2O_2)的简单定量方法，发现儿茶素非酶促H_2O_2生成依赖于pH、温度、孵育时间和溶液中O_2的存在。(+)-儿茶素氧化产物的形成也取决于这些因素，通过测量溶液在430 nm处的吸光度来估计氧化产物的形成。不同种类的超氧化物歧化酶(SOD)对H_2O_2生成的抑制作用几乎具有相同的剂量依赖关系。过氧化氢酶和H_2O_2对(+)-儿茶素的氧化均无影响，但超氧阴离子(O_2-)促进了儿茶素的氧化。这些结果表明，O_2-而不是H_2O_2参与了(+)-儿茶素的自氧化反应，并与H_2O_2的生成相耦合。

(1) Cytotoxicity and DNA single-strand breaks caused by H_2O_2 were assessed by a colony formation assay and a DNA precipitation assay, respectively, with Chinese hamster V79 cells. In both assays, caffeic acid ethyl ester showed protective effects. The structure-activity relationship showed that the omicron-dihydroxy structure of caffeic acid ethyl ester was essential for the protective effects.(2) We assessed inhibitory effects of curcumin and its chemically modified homologues against H_2O_2-induced cytotoxicity toward Chinese hamster long fibroblasts V79 cells with a colony formation assay. Among 4 curcuminoids, dihydroxycurcumin and dihydroxytetrahydrocurcumin suppressed H_2O_2-induced cytotoxicity. On the contrary, neither curcumin nor tetrahydrocurcumin showed any suppressive effects. These results support out empirical rule that omicron-dihydroxy (catechol) moiety is essential for the inhibitory effects.(3) We established a simple method to quantify hydrogen peroxide (H_2O_2) generated during the aerobic heating process of (+) -catechin solution Using this method, we found that nonenzymatic H_2O_2 formation from catechin depended on pH,temperature, incubation time, and the presence of O_2 in the solution. The formation of oxidezed products of (+) -catechin, which was estimatied by measuring the absorbance of the solution at 430 nm, also depended on these factors. The H_2O_2 formation was inhibited by various kinds of superoxide dismutase (SOD) with almost the same dose dependency. Although the oxidation of (+) -catechin was enhanced by superoxide (O_2-), neither catalase nor H_2O_2 had any effects on the oxidation. These results suggest that O_2-, rather than H_2O_2, participated in the autoxidation of (+) -catechin, which was coupled with the H_2O_2 formation.