沒食子酸之微生物轉換及抗氧化活性探討

Microbial transformation of gallic acid and antioxidant evaluation

詹雅棋(Ya-Chi Jan)

指導教授 ： 徐鳳麟 ； 林淑娟

臺北醫學大學/藥學院/生藥學研究所/碩士(2005年)

摘要

沒食子酸 (Gallic acid, 3,4,5-trihydroxy-benzoic acid)為水解型單寧之基本單元，可從Hamamelis Leaf (Hamamelidaceae)、Japanese galls及Chinese galls等萃取而得，通常都會與一些蛋白質和多醣形成複合體，進而引起生物活性。根據文獻報導，gallic acid的生理活性包括：抗細胞增生、抗氧化、cytotoxicity、genotoxicity、血管收縮、抗真菌、抗過敏、抗細菌及抑制DNA breaking activity等。經由篩選26種菌種後，選出Beauveria sulfurescens ATCC 7159進行微生物轉換gallic acid。將gallic acid經過微生物轉換可得4,5-dihydroxy-3-methoxy-benzoic acid (2)，3,5-dihydroxy-4-methoxy-benzoic acid (3)，4-(5¢-hydroxymethylene-4¢-methoxy-tetrahydro-pyran-2¢,3¢-diol)-3-methoxy-5-hydroxy-benzoic acid (4)，4-hydroxy-3,5-dimethoxy-benzoic acid (5)，5-hydroxy-3,4-dimethoxy-benzoic acid (6)，1-(5¢-hydroxy-methylene-4¢-methoxy-tetrahydro-pyran-2¢,3¢-diol)-3,4-dimethoxy-5-hydroxy-benzoate (7)共六個代謝物，其中4及7為新的代謝物。此外，將其它水解型單寧化合物如：geraniin及tannic acid經由Beauveria sulfurescens轉換，並以薄層色層分析法與gallic acid轉換之結果比較，同樣可得上述之代謝物。另外，以化學合成的方式進行甲基化反應，可得methyl 5-hydroxy-3,4-dimethoxy-benzoate (8)，methyl 3,5-dihydroxy-4-methoxy-benzoate (9)，methyl 3,4,5-trihydroxy-benzoate (10)及methyl 3,4,5-trimethoxy-benzoate (11)，這些化合物的結構皆經由一維、二維核磁共振光譜及高解析質譜來決定。將上述六個代謝物2-7及四個合成之化合物8-11進行DPPH自由基清除試驗、超氧自由基清除試驗以及電子自旋共振清除氫氧自由基試驗，發現在DPPH自由基清除試驗及超氧自由基清除試驗中，代謝物及合成化合物的抗氧化活性都比受質差，但是在以電子自旋共振清除氫氧自由基試驗中，化合物2、3、7及9-11則有較受質佳之清除氫氧自由基的活性。分離之代謝物2、3及6與gallic acid於哺乳類之代謝物是相同的，說明了微生物具有與哺乳類相似之代謝酵素。這些分離之代謝物將繼續進行其它生物活性試驗，及作為日後研究水解型單寧於哺乳類代謝之標準品。

關鍵字

微生物轉換 ； 沒食子酸 ； 抗氧化

並列摘要

Gallic acid ( 3,4,5-trihydroxy-benzoic acid), a basic unit of hydrolysable tannin, is extracted from Hamamelis Leaf (Hamamelidaceae), Japanese galls, and Chinese galls. It could bind with protein and polysaccharide and cause many biological activities. According to the literature, the biological activities of gallic acid include antiproliferative, antioxidant, cytotoxicity, genotoxicity, vasoconstrictive, antifungal, antiallergic, antibacterial and inhibition of DNA breaking activities. By screening twenty-six microorganisms, Beauveria sulfurescens ATCC 7159 was selected for the preparative-scale microbial transformation of gallic acid. Microbial transformation of gallic acid yielded six metabolites including 4,5-dihydroxy-3-methoxy-benzoic acid (2), 3,5-dihydroxy-4-methoxy-benzoic acid (3), 4-(5¢-hydroxymethylene- 4¢-methoxy-tetrahydro-pyran-2¢,3¢-diol)-3-methoxy-5-hydroxy-benzoic acid (4), 4-hydroxy-3,5-dimethoxy-benzoic acid (5), 5-hydroxy-3,4-dimethoxy-benzoic acid (6) and 1-(5¢-hydroxymethylene-4¢-methoxy-tetrahydro-pyran-2¢,3¢-diol)-3,4-dimethoxy-5-hydroxy-benzoate (7). Among them, 4 and 7 are new compounds. In addition, microbial transformation of geraniin and tannic acid with Beauveria sulfurescens also yielded the metabolites 2-7 which were detected by thin layer chromatography. Furthemore, chemical methylation of gallic acid yielded methyl 5-hydroxy-3,4-dimethoxy-benzoate (8), methyl 3,5-dihydroxy-4-methoxy-benzoate (9), methyl 3,4,5-trihydroxy-benzoate (10) and methyl 3,4,5-trimethoxy-benzoate (11). The structures of compounds were established on the basis of 1D-, 2D-NMR, and HRFABMS. Six metabolites 2-7, four synthetic compounds 8-11 were assayed for the antioxidant activity by DPPH radical scavenging, superoxide anion radical scavenging, and hydroxyl radical scavenging. The results showed that the metabolites and synthetic compounds were less scavenging activity than the parent compound on DPPH radical and superoxide anion radical, whereas the metabolites 2, 3, 7, and synthetic compounds 9-11 were more scavenging activity than the parent compound on hydroxyl radical. Furthermore, the metabolites 2, 3 and 6 are in accord with the mammalian metabolites of gallic acid. These isolated metabolites will be tested for other biological activities and as the standards for monitoring our continuing studies on the mammalian metabolism of hydrolysable tannin.

並列關鍵字

microbial transformation ； gallic acid ； antioxidant

參考文獻

Davis P. J., Glade J. C., Clark A. M., Smith R.V., Appl. Environ. Microbiol., 1979, 38, 891-893..

Parmar V. S., Kumar A., Bisht K. S., Mukherjee S., Prasad A. K.,Tetrahedron, 1997, 53, 2163-2176..

Cai Y. J., Ma L. P., Hou L. F., Zhou B. Y., Yang L., Liu Z. L., Chem. Phys. Lipids, 2002, 120, 109-117..

Lin J. K., Chen P. C., Ho C. T., Lin S., Shoei Y., J. Agric. Food Chem., 2000, 48, 2736-2743..

Moody J. D., Zhang D., Heinze T. M., Cerniglia C. E., Appl. Environ Microbiol, 2000, 66, 3646-3649..