香豆素及其衍生物在黃嘌呤氧化?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
中文摘要
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。
英文摘要
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.
