α-葡萄糖苷酶论文：电泳中介微分析法用于酶抑制剂筛选的研究.docVIP

α-葡萄糖苷酶论文：电泳中介微分析法用于酶抑制剂筛选的研究 【中文摘要】威胁人类健康的几类重大疾病都被发现与相关酶的失调密切相关,所以酶是新药研制的重要靶标之一。因此,发展和建立快速、可靠、低成本及高通量的酶抑制剂类药物筛选技术对于研发创新药物具有重要的现实意义。本论文的是通过对电泳中介微分析法用于酶抑制剂筛选的研究,将毛细管电泳发展成为简便可靠的高通量筛选平台用于创新药物的研究开发。第一部分运用电泳中介微分析技术,建立了一种新的α-葡萄糖苷酶抑制剂的快速筛选方法。该方法简便可靠,只需将底物溶液(含或不含待筛选化合物)和酶溶液依次导入毛细管,加上一小电压使其混合,接着进行电泳分离,通过紫外直接测定酶催化水解产物峰面积的变化实现酶抑制剂的筛选。通过对各实验参数的考察和优化,用已知的酶抑制剂对所建立的筛选方法进行验证,并将其用于中药粗提物中α-葡萄糖苷酶的快速筛选。研究结果表明,五倍子的抑制率高于对照品阿卡波糖,达到100%；丁香、大黄、桑叶、葛根的抑制率在45%-70%。第二部分介绍了电泳中介微分析法筛选蘑菇酪氨酸酶抑制剂的模型,实验中优化了蘑菇酪氨酸酶与底物L-酪氨酸的反应条件,测定了酪氨酸酶的米氏常数为0.0532 mM,抑制剂曲酸的抑制机制并计算得到曲酸的抑制常数,与文献报道相符,证实了该筛选方法的可靠性。并用该方法对中草药粗提物、金属配合物、有机合成物进行了筛选。研究结果表明,20种中草药中山楂的抑制活性较高,达到58.3%；61种金属配合物中,除一种抑制率达到40%,其他均较低。 【英文摘要】The most prevalent diseases which threat people health are found to linked to related enzyme disorders. So the enzyme is one of the most important targets for new drug development. So it is important to develop one fast and lower cost screening enzyme inhibitors method. In this paper, Electrophoretically Mediated Microanalysis(EMMA) method in the research of kinetics of enzyme reactions of a-glucosidase and Tyrosinase were introduced, including the measurement of kinetic parameters of enzyme reactions and screening the inhibitors of a-glucosidase and Tyrosinase and the research of kinetics of inhibitory activities.In the first part of the study of screening a-glucosidase inhibitors, we optimized the reaction condition.40 mM sodium phosphate buffers for the reaction and the incuration time 0 were selected for the experiment. The Michaelis-Menton constant of a-glucosidase and the inhibitive mechanism of acarbose were studied, which were in the same range as previous literature data. Furthermore, the inhibitory ratios of enzymatic activity (IRE) of 20 traditional Chinese drugs were determined. The IRE of Galla Chinensis attained 100%, the inhibitory active are too strong, and may lead to hypoglycemia. The IRE of Flos Caryophylli, Radix et Rhizoma Rhei,Folium Mori, Radix Puerariae, were between 45% and 70%, similar with a