黄嘌呤氧化酶多酚抑制剂的筛选及其作用机制

黄嘌呤氧化酶(xanthine oxidase,XOD)是人体内次黄嘌呤和黄嘌呤代谢产生尿酸的关键酶,而多酚单体可抑制黄嘌呤氧化酶从而减少尿酸合成进而缓解痛风症状。为探究来源于天然产物的多酚单体对XOD的抑制作用机制,首先研究几种常见活性较好的多酚单体对黄嘌呤氧化酶的抑制率,及其对XOD的抑制动力学,并且通过荧光猝灭实验与分子对接模拟多酚单体与XOD之间的相互作用形式。在6种多酚单体中,白藜芦醇、表没食子儿茶素没食子酸酯(epigallocatechin gallate,EGCG)和橙皮苷对黄嘌呤氧化酶活性的抑制效果较好,室温下IC50分别为261.22、150.51、97.08μmol/L,是黄嘌呤氧化酶的竞争性抑制剂。荧光猝灭实验表明,单体与XOD结合使酶的内源荧光发生了猝灭现象,猝灭机制为静态猝灭。分子对接模拟表明,多酚单体能与XOD的氨基酸残基结合,结合位点位于钼蝶呤(molybdopterin,Mo-Pt)辅因子或黄素腺嘌呤二核苷酸(flavine-adenine dinucleotide,FAD)结构域的异恶嗪环附近。多酚单体是有效的XOD竞争性抑制剂,可以通过抑制黄嘌呤氧化酶的活性有效降低尿酸水平,在天然产物的开发和功能性食品的应用中具有很好的前景。

The Inhibitory Kinetics and Mechanism of Xanthine Oxidase by Screened Polyphenols

Xanthine oxidase(XOD)is used as a key enzyme in the metabolism of hypoxanthine and xanthine to produce uric acid in human body,but polyphenols can inhibit Xanthine Oxidase,thereby reducing uric acid synthesis and alleviating gout symptoms. To investigate the inhibition mechanism of polyphenols derived from natural products on XOD,the inhibition rate of XOD by several polyphenol monomers with good activity and inhibition kinetics of XOD were conducted,and the interaction between polyphenol monomers and XOD was simulated by fluorescence quenching experiment and molecular docking. Among the six polyphenol monomers,resveratrol,epigallocatechin gallate(EGCG)and hesperidin had better inhibitory effect on xanthine oxidase activity,and the IC50 of resveratrol,EGCG and hesperidin were 261.22,150.51,97.08 μmol/L,respectively,which were competitive inhibitors of XOD. Fluorescence quenching experiments showed that the binding of monomer to XOD caused the endogenous fluorescence of the enzyme to quench,and the quenching mechanism was static quenching. Molecular docking results showed that polyphenols could bind to amino acid residues of XOD and enter the molybdopterin(Mo-Pt)center or flavine-adenine dinucleotide(FAD)domain near the isoxazine ring to form a complex. Polyphenol monomers are effective competitive inhibitors,and can reduce uric acid levels by inhibiting the activity of XOD,which had a good prospect in the development of natural products and the application of functional food.