臺灣博碩士論文加值系統

本篇論文描述了Hui-1、gentisic acid、scopoletin的抗氧化特質。 我們從Fujisawa Co.提供的3562株菌的萃取液中, 篩選得到一株具有高抗氧化活性的真菌: Penicillium citrinum (F5), 在大量培養後, 分離得到兩個具抗氧化活性的物質, Hui-1和Hui-2, 經結構鑑定後確定Hui-2為2,5-dihydroxybenzoic acid, 即gentisic acid。 scopoletin的抗氧化性, 也同時一起作探討比較。 scopoletin是實驗室過去從中藥青楓藤中純化到的物質。
從初步活性分析的結果看來, Hui-1和gentisic acid都有著很強的還原力, 還原自由基DPPH的能力和ascorbate及trolox相當。 在LDL氧化實驗中, 100 mM濃度下, 兩者都可以完全保護LDL免於AAPH引起的氧化傷害。 scopoletin則特別具有捕捉superoxide anion的能力。 因為ROS造成的傷害和許多疾病都有關係, 進一步深入的研究自然有其價值。

This paper describes the antioxidant properties of Hui-1, gentisic acid, scopoletin. After screening 3562 strains of microbial extracts from Fujisawa Co., one strain of Penicillium citrinum (F5) with high antioxidant activity was found. Two potent antioxidative compounds, Hui-1 and Hui-2, were isolated from the culture broth of this strain. Hui-2 was identified as 2,5-dihydroxybenzoic acid (gentisic acid). Besides, scopoletin, isolated from Sinomonium acutum (a traditional Chinese herb) in our lab before, was investigated at the same time.
Hui-1 and gentisic acid were as effective as ascorbic acid and trolox in reducing the stable free radical DPPH. They also inhibited oxidation of LDL induced by peroxyl radical (AAPH) completely at 100 mM. Scopoletin scavenged superoxide anion specifically without inhibiting xanthine oxidase but was inactive in other assays. As reactive oxygen species caused damages are related to various diseases, the further research of above novel antioxidants are quite justified.

中文摘要 ---------------------------------------------------- 3
英文摘要 ---------------------------------------------------- 4
縮寫表 ------------------------------------------------------- 5
第一章 緒論 ---------------------------------------------- 6
第一節 抗氧化劑的定義 ------------------------------------------------- 6
第二節 抗氧化劑的評估 ------------------------------------------------- 6
第三節 ROS (reactive oxygen species) 的成員 ----------------------- 9
第四節 抗氧化活性的評估方式 --------------------------------------- 12
第五節 ROS造成的疾病 ------------------------------------------------ 13
第六節 研究動機與目的 ------------------------------------------------ 18
第二章 材料與方法 ------------------------------------ 19
第一節 材料 --------------------------------------------------------------- 19
第二節 方法 --------------------------------------------------------------- 21
第三章 結果 --------------------------------------------- 29
第一節 抗氧化活性菌株的篩選 --------------------------------------- 29
第二節 Penicillium citrinum (F5) 的活性物質純化 ---------------- 36
第三節 Hui-2的結構鑑定結果 ---------------------------------------- 42
第四節 Hui-1及gentisic acid的抗氧化活性分析 ------------------- 42
第五節 scopoletin的抗氧化活性分析 -------------------------------- 47
第四章 討論 --------------------------------------------- 52
第一節 篩選實驗的設計 ------------------------------------------------ 52
第二節 Hui-1和gentisic acid的相關研究 ---------------------------- 52
第三節 scopoletin的相關研究 ----------------------------------------- 53
第四節 實驗總結 --------------------------------------------------------- 53
第五章 參考文獻 --------------------------------------- 56
附錄一、F5菌種鑑定報告 ------------------------------ 60
附錄二、Hui-2的結構鑑定資料 ----------------------- 63

Afanas'ev, I. B., Dorozhko, A. I., Brodskii, A. V., Kostyuk, V. A., and Potapovitch, A. I. (1989). Chelating and free radical scavenging mechanisms of inhibitory action of rutin and quercetin in lipid peroxidation. Biochem Pharmacol 38, 1763-9.
Armstrong, D. (1998). Free radical and antioxidant protocols: Humana Press.
Aruoma, O. I. (1993). Free radicals: their role in nutrition: Harwood, London.
Aruoma, O. I. (1994). Nutrition and health aspects of free radicals and antioxidants. Food Chem Toxicol 32, 671-83.
Bielski, B. H. J. (1985). Reactivity of HO2/O2- radicals in aqueous solution. Journal of physical and chemical reference data 14, 1041-1100.
Blois, M. S. (1958). Antioxidant determinations by the use of a stable free radical. Nature 181, 1199-1200.
Brune, M., Rossander, L., and Hallberg, L. (1989). Iron absorption and phenolic compounds: importance of different phenolic structures. Eur J Clin Nutr 43, 547-57.
Budavari, S. (1989). Gentisic acid. Merck index 4290, 688.
Buege, J. A. (1978). Microsomal lipid peroxidaion. Methods in enzymology 52, 302-310.
Chisolm, G. M., and Steinberg, D. (2000). The oxidative modification hypothesis of atherogenesis: an overview. Free radical in biology and medicine 28, 1815-1826.
Dean, R. T., Gieseg, S., and Davies, M. J. (1993). Reactive species and their accumulation on radical-damaged proteins. Trends Biochem Sci 18, 437-41.
el-Saadani, M., Esterbauer, H., el-Sayed, M., Goher, M., Nassar, A. Y., and Jurgens, G. (1989). A spectrophotometric assay for lipid peroxides in serum lipoproteins using a commercially available reagent. J Lipid Res 30, 627-30.
Esterbauer, H., Gebicki, J., Puhl, H., and Jurgens, G. (1992). The role of lipid peroxidation and antioxidants in oxidative modification of LDL. Free Radic Biol Med 13, 341-90.
Esterbauer, H., Striegl, G., Puhl, H., and Rotheneder, M. (1989). Continuous monitoring of in vitro oxidation of human low density lipoprotein. Free Radic Res Commun 6, 67-75.
Exner, M., Hermann, M., Hofbauer, R., Kapiotis, S., Speiser, W., Held, I., Seelos, C., and Gmeiner, B. M. (2000). The salicylate metabolite gentisic acid, but not the parent drug, inhibits glucose autoxidation-mediated atherogenic modification of low density lipoprotein. FEBS Lett 470, 47-50.
Halliwell, B. (1995). Antioxidant characterization. Methodology and mechanism. Biochem Pharmacol 49, 1341-8.
Halliwell, B. (1994). Free radicals and antioxidants: a personal view. Nutr Rev 52, 253-65.
Halliwell, B. (1998). Free radicals in biology and medicine: Oxford.
Halliwell, B. (1990). How to characterize a biological antioxidant. Free Radic Res Commun 9, 1-32.
Halliwell, B., Aeschbach, R., Loliger, J., and Aruoma, O. I. (1995). The characterization of antioxidants. Food Chem Toxicol 33, 601-17.
Hermann, M., Kapiotis, S., Hofbauer, R., Seelos, C., Held, I., and Gmeiner, B. (1999). Salicylate promotes myeloperoxidase-initiated LDL oxidation: antagonization by its metabolite gentisic acid. Free Radic Biol Med 26, 1253-60.
Hussein, M., and Samuelsson, G. (1992). Pharmacologically active phenylpropanoids from Senra incana. Planta medica 58, 14-18.
Laurindo, F. R. M. (1991). Evidence for superoxide -dependent coronary artery vasospasm after angioplasty in intact dogs. Circulation 83, 1705.
Lusis, A. J., and Navab, M. (1993). Lipoprotein oxidation and gene expression in the artery wall. New opportunities for pharmacologic intervention in atherosclerosis. Biochem Pharmacol 46, 2119-26.
Melov, S., Ravenscroft, J., Malik, S., Gill, M. S., Walker, D. W., Clayton, P. E., Wallace, D. C., Malfroy, B., Doctrow, S. R., and Lithgow, G. J. (2000). Extension of life-span with superoxide dismutase/catalase mimetics. Science 289, 1567-9.
Nakazono, K., Watanabe, N., Matsuno, K., Sasaki, J., Sato, T., and Inoue, M. (1991). Does superoxide underlie the pathogenesis of hypertension? Proc Natl Acad Sci U S A 88, 10045-8.
Napoli, C., Mancini, F. P., Corso, G., Malorni, A., Crescenzi, E., Postiglione, A., and Palumbo, G. (1997). A simple and rapid purification procedure minimizes spontaneous oxidative modifications of low density lipoprotein and lipoprotein (a). J Biochem (Tokyo) 121, 1096-101.
Palinski, W., Miller, E., and Witztum, J. L. (1995). Immunization of low density lipoprotein (LDL) receptor-deficient rabbits with homologous malondialdehyde-modified LDL reduces atherogenesis. Proc Natl Acad Sci U S A 92, 821-5.
Parker, J. D., and Parker, J. O. (1998). Nitrate therapy for stable angina pectoris. N Engl J Med 338, 520-31.
Roy, D., Floyd, R. A., and Liehr, J. G. (1991). Elevated 8-hydroxydeoxyguanosine levels in DNA of diethylstilbestrol- treated Syrian hamsters: covalent DNA damage by free radicals generated by redox cycling of diethylstilbestrol. Cancer Res 51, 3882-5.
Sanan, S., and Sharma, G. (1986). Effect of desferrioxamine mesylate (Desferal) in anesthetized dogs with clinical hemorrhagic shock. Ind. J. Med. Res. 83, 655.
Schilderman, P. A., ten Vaarwerk, F. J., Lutgerink, J. T., Van der Wurff, A., ten Hoor, F., and Kleinjans, J. C. (1995). Induction of oxidative DNA damage and early lesions in rat gastro- intestinal epithelium in relation to prostaglandin H synthase-mediated metabolism of butylated hydroxyanisole. Food Chem Toxicol 33, 99-109.
Sies, H., Stahl, W., and Sundquist, A. R. (1992). Antioxidant functions of vitamins. Vitamins E and C, beta-carotene, and other carotenoids. Ann N Y Acad Sci 669, 7-20.
Szabo, C. (1996). The pathophysiological role of peroxynitrite in shock, inflammation, and ischemia-reperfusion injury. Shock 6, 79-88.
Tal, B., and Robeson, D. J. (1986). The metabolism of sunflower phytoalexins: ayapin and scopoletin. Plant physiology 82, 167-176.
Valle, T. (1997). Antifungal activity of scopoletin and its differential accumulation in Ulmus pumila and Ulmus campestris cell suspension cultures infected with Ophiostoma ulmi spores. Plant science 125, 97-101.
van Acker, S. A., van den Berg, D. J., Tromp, M. N., Griffioen, D. H., van Bennekom, W. P., van der Vijgh, W. J., and Bast, A. (1996). Structural aspects of antioxidant activity of flavonoids. Free Radic Biol Med 20, 331-42.
Wettasinghe, M., and Shahidi, F. (2000). Scavenging of reactive-oxygen species and DPPH free radicals by extracts of borage and evening primrose meals. Food chemistry 70, 17-26.
Wiseman, H., and Halliwell, B. (1993). Carcinogenic antioxidants. Diethylstilboestrol, hexoestrol and 17 alpha- ethynyloestradiol. FEBS Lett 332, 159-63.