不同酶源乙酰胆碱酯酶的抑制剂筛选方法比较  

Comparative Study on Screening Acetylcholinesterase Inhibitors from Different Enzyme Sources

作　　者：刘佳莉[1,2] 苑玉和[1] 陈乃宏[1] 

机构地区：[1]天然药物活性物质与功能国家重点实验室中国医学科学院北京协和医学院药物研究所 [2]天津中医药大学

出　　处：《神经药理学报》2011年第3期27-30,共4页Acta Neuropharmacologica

基　　金：国家自然科学基金项目(No.U832008;30973887;81073078;81102831;90713045;81072541);科技部项目(No.2010DFB32900);国家科技重大专项(No.2012ZX09301002-004)

摘　　要：目的:建立一种可靠、快速、便捷、经济的乙酰胆碱酯酶(acetylcholinesterase,AChE)抑制剂的筛选方法,为新药发现阶段AChE抑制剂的筛选提供经济可靠的微量筛选方法,然后对植物提取物样品进行筛选。方法:分别采用电鳗AChE、人源红细胞AChE、大鼠海马区、纹状体匀浆液中的AChE为酶源。以96孔板为载体,利用优化的Ellman方法,在生理pH值和温度的条件下,以他克林和石杉碱甲作为阳性对照,检测对不同酶源AChE的抑制作用,确定最佳酶反应条件(最佳底物浓度、反应时间、显色剂浓度),并对植物提取物样品的AChE抑制作用进行检测。结果:采用上述四种酶源的AChE进行AChE抑制剂微量筛选方法操作简便快速、可靠经济。其中应用电鳗AChE进行筛选显示灵敏度较高,他克林和石杉碱甲对电鳗AChE的抑制作用相当,而对人源红细胞AChE、大鼠海马区、纹状体匀浆酶源的抑制作用,石杉碱甲明显优于他克林,所筛选植物提取物对AChE均没有抑制作用。结论:本实验中所建立的筛选方法操作简便、快速、可靠经济,可用于纯化合物的筛选。Objective:To establish a reliable,fast,convenient and economical method of screening acetylcholinesterase inhibitors,and use it to screen acetylcholinesterase inhibitors from extracts of plants.Methods:Using the optimized Ellman method,we examined the inhibition effects of tacrine and huperzine on acetylcholinesterase enzyme from electric eel,human erythrocyte,rat hippocampus,striatum homogenate in 96-well plates under physiological pH value and temperature.The concentration of AChE,substrate and DTNB,and reaction time were optimized.We also detected the inhibition effect of plant extracts on AChE.Results:Practical microassays for screening AChE inhibitors were successfully established by using AChEs mentioned above.The data analysis of screening results revealed that electric eel AChE possessed a high sensitivity to inhibitors,whereas human erythrocyte AChE shared high similarity with rat brain homogenate,on which the inhibitory effect of huperzine was superior to tacrine.However,the plant extracts showed no inhibitory effect on AChE.Conclusion:The screening method established in this experiment is simple,fast,reliable and economical,and it can be used for pure compound screening.

关 键 词：乙酰胆碱酯酶 石杉碱甲 他克林 阿尔茨海默病 

分 类 号：R96[医药卫生—药理学]