Study On A Microplate Bioassay To Determine The Activity Inhibition Of Glucosidase With Douchi-Extract

The alpha-glucosidase inhibitor can reduce blood glucose concentration by inhibiting the hydrolysis reaction.They always used as oral hypoglycemic agents in the treatment of diabetes.DNJ(1-Deoxynojirimycin),which has a strong inhibitory effect on alpha glucosidase,is a typical alpha-glucosidase inhibitor.The study claim a method to detect the half inhibitory concentration(IC50)of alpha glucosidase inhibitors by microplate assay,which use DNJ as a reference standard.The optimum reaction conditions of the experiment were studied.The results are followed.The optimum enzyme concentration range is 2.0-3.0mg/m L.For the detection wavelength,there are 5 condition of detection wavelength: 405 nm,450nm.492 nm,520nm,630 nm,and the optimum is 520 nm or 492 nm.The detection strength decreased with the decreasing enzyme concentration.For sucrose concentration of substrate,the suitable range should be 20-40 mM/L.For the reaction time,the appropriate range should be 20-30 min.At the same time,the method's applicability for the determination of Touchi-extract IC50 was verified.The conclusion is followed.For Touchi-Extract sample: the linear correlation coefficient R2 was greater than 0.98,and it meets the requirements;the repeatability results is fine and and the relative standard deviation is less than 20% between different test person,also meets the requirements;in the specificity test,the IC50 of Touchi-Extract sample can be effectively detected,but it not happened to the extract of Sauteed Green Beans;in the robustness experiments,compared with Amway R & D laboratory in American,Amway microassay laboratory in China's average results meet the range of ±20%.Therefore,this method is suitable for the determination of Touchi-extract IC50.