- 學位論文
香豆素及其衍生物在黃嘌呤氧化酶抑制和自由基清除之結構-活性相關性分析
Structure-Activity Relationship of Coumarin and Derivatives on Xanthine Oxidase-Inhibiting and Free Radical Scavenging Activities
摘要
我們利用DNA鬆懈實驗、DPPH清除能力及電子自旋共振法的技術去研究八種香豆素及其衍生物在抗氧化活性、自由基捕捉能力的表現。此外，我們去評估這些香豆素及其衍生物對於黃嘌呤氧化酶活性的抑制能力，且進一步利用分子模擬技術去解釋這些衍生物對於黃嘌呤氧化酶活性的抑制結構-活性關係。我們發現esculetin 和 4-methylesculetin具有最佳的自由基清除能力，推測其結構中的取代的官能基種類和氫氧基團數目與自由基清除能力有關。結果發現所有的香豆素衍生物都具有抑制黃嘌呤氧化酶活性的能力，且其抑制型態屬於競爭型抑制劑。由此可確認在以分子模擬時可與黃嘌呤氧化酶活性中心對位。由分子模擬的結果，可以發現香豆素衍生物的α-pyrone環結構可與黃嘌呤氧化酶受質結合位入口的苯丙胺酸（Phe）914具π-π重疊力，而O-1和C-2羰基上的氧原子可與黃嘌呤氧化酶的精胺酸（Arg）880或透過水分子與精胺酸（Arg）880的NH基團、酥胺酸（Thr）1010的OH基團形成氫鍵。在8種香豆素衍生物中發現esculetin與黃嘌呤氧化酶的親和力最高，因為在esculetin的結構上，C-6的OH基團可與麩胺酸（Glu）802的COOH基團形成氫鍵，此鍵結對於抑制黃嘌伶氧化酶活性是很重要的，因而增加了兩者結合的穩定性。最後以電子自旋共振法，利用 Hypoxanthine/ Xanthine Oxidase反應體系，產生之•O2-自由基，去評估香豆素及其衍生物對於黃嘌呤氧化酶活性抑制和自由基清除的能力的整體表現，結果顯示esculetin 和 4-methylesculetin具有最佳的活性。由於Aβ肽造成神經細胞傷害涉及自由基的過度生成，因此我們將上述結構-活性關係結論進一步應用在活體細胞實驗的表現，結果顯示esculetin可以透過抑制自由基生成或自由基清除作用而降低Aβ所引發的細胞毒性。
並列摘要
We employed the techniques of DNA relaxation, DPPH, and DMPO-electron spin resonance (ESR), to study the effects of the reactive oxygen species (ROS) suppression by 8 selected coumarin and derivatives under oxidative conditions. We also investigated the effect of these derivatives on the inhibition of xanthine oxidase (XO) activity, and the structure-activity relationships (SARs) of these derivatives against XO activity were further examined using computer and molecular modeling. Esculetin and 4-methylesculetin express the more potent radical scavenger among the 8 test compounds. Our results suggest that the chemical structure and number of hydroxyl groups on the benzene ring of coumarin were correlated with the effects of ROS suppression. All test derivatives were competitive inhibitors of XO. The results of the structure-based molecular modeling exhibited interactions between coumarin derivatives and the molybdopterin region of XO. The α-pyrone ring of coumarin derivatives stacked with the phenyl at Phe 914. Two hydrogen bonding interactions were observed involving the O-1 of the coumarin derivatives with the guanidinium group of Arg 880, carbonyl group of coumarin derivatives with Thr 1010. Esculetin had the highest affinity toward the binding site of XO, and this was due to hydrogen bonding interactions. The hydroxyl group at C-6 formed hydrogen bonds with the carboxyl group Glu802 that played critical role on XO binding. The hypoxanthine/XO reaction in the DMPO-ESR technique was used to correlate the effect on enzyme inhibition and ROS suppression by these coumarin derivatives, and the results showed that esculetin and 4-methylesculetin were the two most potent agents among the tested compounds. Aβ（β-amyloid peptide） induce neuronal cell damage by increasing the generation of ROS. Therefore, we further assessed the effects of test compounds on living cells. Esculetin was the most potent agent at protecting cells against Aβ induced cell cytotoxisity via inhibiting the generation of ROS or ROS scavenging among the tested coumarin derivatives.
參考文獻
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