葡萄糖醛酸和半乳糖醛酸hydroxamate衍生物之生理活性探討

Studies on the biological activity of hydroxamate derivatives of glucuronic acid and galacturonic acid

李季倞(Chi-Ching Lee)

指導教授 ： 徐鳳麟、侯文琪

臺北醫學大學/藥學院/生藥學研究所/碩士(2005年)

摘要

果膠廣泛存在於水果和蔬菜中，普遍應用於食品、醫藥、化妝品等行業，已經使用多年。本研究以果膠的單體半乳糖醛酸，與立體異構物的葡萄糖醛酸做為材料，進行化學修飾反應。將半乳糖醛酸和葡萄糖醛酸一號碳和六號碳上羧基均接上甲氧基，之後分個別在鹼性溶液中室溫條件下，以及在DMF溶液高溫下，把六號碳上甲氧基，置換成羥?胺酸，再進行一連串抗氧化活性的測試，包括DPPH自由基清除、保護DNA免受氫氧自由基攻擊、氫氧自由基清除、超氧自由基清除、抗脂質過氧化、抑制過氧亞硝酸氧化，實驗結果顯示半乳糖醛酸和葡萄糖醛酸均無效果，但是兩者的羥?胺酸衍生物GluA-NHOH 與 GalA-NHOH皆具有明顯抗氧化效果。 兩個起始反應物與其羥?胺酸衍生物進行抑制一氧化氮生成測定，以LPS誘導細胞株RAW264.7所產生的NO，發現只有半乳糖醛酸衍生物GalA-NHOH具有抑制NO的效果，而在以MTT進行細胞毒性測試，發現GalA-NHOH在測試濃度下，對細胞的毒性相當低。 另一方面，兩起始物和兩羥?胺酸衍生物進行抑制 semicarbazide-sensitive amine oxidase (SSAO)和雙胺氧化?測定中，發現不論酵素來源是小牛血漿或是豬血漿，在SSAO抑制曲線測定和SSAO活性染色，均很清楚顯示兩衍生物具抑制效果，而兩起始反應物半乳糖醛酸和葡萄糖醛酸均無效果，而對於抑制雙胺氧化?曲線測定，兩羥?胺酸衍生物的抑制能力也優於兩起始物。探討SSAO與兩羥?胺酸衍生物的酵素動力學抑制型態，以Lineweaver-Burk分析結果發現，兩條線也是交會於X軸，表示GluA-NHOH 與 GalA-NHOH與基質(benzylamine)對於小牛血漿內SSAO進行非競爭型抑制，GluA-NHOH 與 GalA-NHOH的抑制常數(Ki) 個別為0.039mM 和0.027 mM。

關鍵字

葡萄糖醛酸、半乳糖醛酸、羥?胺酸

並列摘要

The pectin is generally found in fruits and vegetables and has been widely used in food industry, medical, and cosmetics. The galacturonic acid (GalA) is the monomer of pectins, and the glucuronic acid (GluA) is its isomer. In the first, GalA was reacted with equal volumes of acidic methanol solution at 4 ℃ for five days, and GluA was also reacted with equal volumes of methanol solution at 90℃ for 12 hours. The methoxyl group was added at the C-1 and C-6 of them. Then, they were reacted with alkaline hydroxylamine at room temperature and N, N-Dimethylformamide at 80℃ respectively to repare GalA and GluA hydroxamic acid derivatives (GalA-NHOH and GluA-NHOH). They were used to test the antioxidant and antiradical activities in comparison with the GalA and GluA. The scavenging activities of GluA-NHOH and GalA-NHOH for DPPH radicals were very apparent as compared with GluA and GalA. Similarly, using electron spin resonance (ESR) for scavenging hydroxyl radicals, it was also found that the hydroxamic acid derivatives can present quite obvious effects of antiradical activities. Futhermore, GluA-NHOH and GalA-NHOH not only exhibited protection from hydroxyl radical-mediated DNA damage and scavenging superoxide radicals induced by xanthine oxidase. but also showed anti-lipid peroxidation and anti-peroxynitrite through dihydrorhodamine 123. On the other hand, it was found that only GalA-NHOH is proven to inhibit the LPS-induced NO production, and Gal-NHOH is exhibited low cytotoxicity under the measurement condition by MTT assay. While, in the semicarbazide-sensitive amine oxidase (SSAO) inhibitory activity from bovine or pig plasma, both GluA-NHOH and GalA-NHOH can inhibit SSAO effectively, but neither GluA nor GlaA. It was also found that GluA-NHOH and GalA-NHOH could inhibit diamine oxidase (DAO) from porcine kidney as dose-dependent patterns. By using SSAO activity staining on 7.5% polyacrylamide gels, it was comfirmed the inhibitory activities of GluA-NHOH and GalA-NHOH against SSAO. While GluA-NHOH (0.04 mM) or GalA-NHOH (0.03 mM) were added, GluA-NHOH and GalA-NHOH showed noncompetitive inhibition compared to benzylamine. The apperant inhibition constant (Ki) of GluA-NHOH and GalA-NHOH were 0.039 mM and 0.027 mM, respectively.

並列關鍵字

glucuronic acid, galacturonic acid, hydroxamic acid

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