一种用微孔板筛选alpha-葡萄糖苷酶抑制剂的方法

中国临床药理学与治疗学 ›› 2005, Vol. 10 ›› Issue (10): 1128-1134.

一种用微孔板筛选alpha-葡萄糖苷酶抑制剂的方法

李婷, 张小东, 宋聿文¹, 刘建文

华东理工大学药学院, ¹生物工程学院, 上海 200237

收稿日期:2005-08-17 修回日期:2005-09-26 发布日期:2020-11-23

A microplate-based screening method for alpha-glucosidase inhibitors

Li Ting, ZHANG Xiao-dong, SONG Yu-wen¹, LIU Jian-wen

East China University of Science and Teecolopgy School of Pharmac, ¹College of Bioengineering, Shanghai 200237, China

Received:2005-08-17 Revised:2005-09-26 Published:2020-11-23
Contact: JU Jja-wen, nale, Ph.D. & M.D, professor, advisorofdoxrtor, engaged in the study of effects and mechanisms of nalural prolucts. Tel: 021-64252044 E-mail: liujian@ecust.edu.cn

摘要/Abstract

摘要： 目的: 建立敏感的微孔板方法检测alpha-葡萄糖苷酶抑制活性,以进行抑制剂体外高通量筛选。方法: 调整酶和底物适当配比,通过标准曲线、波长扫描、动力学分析以及最佳反应条件的研究,用阿卡波糖验证所建方法。结果: 确定反应步骤,96孔板,160μL体系含2.5mmol·L^-1 PNPG和0.2U·ml^-1 alpha-葡萄糖苷酶,37℃,pH7.0反应15min,400nm检测,此法正确、可靠。结论: 本文所述用小体积样品筛选得到大量抑制剂的方法,利于从天然产物中开发改善餐后血糖的糖尿病及其并发症治疗药物。

关键词: 阿卡波糖, alpha-葡萄糖苷酶抑制剂, 药物筛选, 微孔板方法, 餐后血糖

Abstract: AIM: To establish a new sensitive micro-late-based method to determine alpha-glucosidase inhibiting activity and provide a reliable high-through put way for screening alpha-glucosidase inhibitors in vitro.METHODS: The fitting combination of enzyme and sub-strate in a certain reaction was tested. Acarbose, the most popular alpha-glucosidase inhibitor in clinical use was used to validate the established method. Calibration curve, wavelength fidelity and kinetic analysis, together with the effect of altered incubation time, temperature and PH were then studied.RESULTS: The details of assayprocedure and evaluation of factors affecting thee measure-ment are described. As little as 160 μl assaythe combination of 2.5 mmol·L^-1 PNPGU and alpha-glucosidase was performed in a 96-well plate. The optimal action was finally achieved by incubat-ed at 37 C, PH 7.0 for 15 min and measured at 400nm.RESULTS: from the validation exercises by Acartosestrongly demonstrated the accuracy and reliability of the pmposed approach.CONCLUSION: This method re-ported in the current paper makes it possible to rapidlyexamine large numbers of samples for the presence of al-pha-glucosidase inhibitors in very small sample volume Such action may help to pace the development of potentialoral medications from natural products protecting patienagainst postprandial hyperglylyceruic toxicity andoretreating (iabetes mellitus and the related complications.

Key words: acarbose, alpha-glucosidase inhibitorsdrug screening, microplate assay: postprandial hyperglycemla

中图分类号:

R965.2

李婷, 张小东, 宋聿文, 刘建文. 一种用微孔板筛选alpha-葡萄糖苷酶抑制剂的方法[J]. 中国临床药理学与治疗学, 2005, 10(10): 1128-1134.

Li Ting, ZHANG Xiao-dong, SONG Yu-wen, LIU Jian-wen. A microplate-based screening method for alpha-glucosidase inhibitors[J]. Chinese Journal of Clinical Pharmacology and Therapeutics, 2005, 10(10): 1128-1134.

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