Masaki H, Okamoto N, Sakaki S, Sakurai H
Shiga Central Laboratories, Noevir Co., Ltd., Japan.
Biol Pharm Bull. 1997 Apr;20(4):304-8. doi: 10.1248/bpb.20.304.
The purpose of this study was to evaluate the hydroxyl radical scavenging activities of hydroxybenzoic acids and their esters from both chemical and biological aspects. These activities of hydroxybenzoic acids and their related compounds were estimated by ESR-spin trapping method, in which 3,4,5-trihydroxybenzoic acid and its ethyl and propyl esters showed the highest activities as estimated by IC50 value (50% inhibition concentration of hydroxyl radicals generated in the system): 78.04 +/- 11.23, 95.95 +/- 2.64, and 86.46 +/- 2.31 microM, respectively. In addition, 3,4,5-trihydroxybenzoic acid (gallic acid) at a concentration of 25 microM, protected against dermal fibroblast cell damage induced by H2O2, and enhanced the survival to 83.8 +/- 3.1%, in which the survival of control was 44.2 +/- 1.0%. Based on these results, the pretreatment effects of 3,4,5-trihydroxybenzoic acid n-alkyl esters on cell damage induced by H2O2 were examined. The survival of fibroblasts pretreated with the esters increased depending on the alkyl chain-length. Both C12 and C16 alkyl esters gave almost complete cell survival of 89.5 +/- 2.0% and 91.3 +/- 1.0%, respectively. The order of the protective effects of the compounds was in good agreement with that of their partition coefficients, suggesting that 3,4,5-trihydroxybenzoic acid alkyl esters are incorporated into fibroblasts, and thus prevent the cells from the toxicity caused by H2O2. In addition, an increase of intracellular peroxide formation in fibroblasts induced by UVA-irradiation, was suppressed to 2.27 +/- 0.41 nmol/10(4) cells by pretreatment with C16 alkyl ester at a concentration of 25 microM. Since 3,4,5-trihydroxybenzoic group has been demonstrated to possess a potent scavenging activity of hydroxyl radicals, this moiety was indicated to be important in preventing cell damage induced by UVA or H2O2: in turn, these produce hydroxyl radicals in the presence of trace metal ions such as iron and copper in cells.
本研究的目的是从化学和生物学两个方面评估羟基苯甲酸及其酯类的羟自由基清除活性。通过电子自旋共振（ESR）自旋捕集法估算了羟基苯甲酸及其相关化合物的这些活性，其中3,4,5 - 三羟基苯甲酸及其乙酯和丙酯的活性最高，以IC50值（系统中产生的羟自由基50%抑制浓度）估算：分别为78.04±11.23、95.95±2.64和86.46±2.31微摩尔。此外，浓度为25微摩尔的3,4,5 - 三羟基苯甲酸（没食子酸）可保护皮肤成纤维细胞免受H2O2诱导的损伤，并将细胞存活率提高到83.8±3.1%，其中对照组的存活率为44.2±1.0%。基于这些结果，研究了3,4,5 - 三羟基苯甲酸正烷基酯对H2O2诱导的细胞损伤的预处理效果。用这些酯预处理的成纤维细胞的存活率随着烷基链长度的增加而提高。C12和C16烷基酯分别使细胞存活率几乎达到100%，分别为89.5±2.0%和91.3±1.0%。这些化合物的保护作用顺序与其分配系数顺序高度一致，表明3,4,5 - 三羟基苯甲酸烷基酯可进入成纤维细胞，从而防止细胞受到H2O2引起的毒性作用。此外，用浓度为25微摩尔的C16烷基酯预处理可将UVA照射诱导的成纤维细胞内过氧化物形成量抑制至2.27±0.41纳摩尔/10(4)个细胞。由于已证明3,4,5 - 三羟基苯甲酸基团具有强大的羟自由基清除活性，该部分在预防UVA或H2O2诱导的细胞损伤中起重要作用：反过来，这些物质在细胞中存在铁和铜等痕量金属离子时会产生羟自由基。
