Study On α-Glucosidase Inhibitory Activity And Mechanism Of Indirubin Derivatives

α-Glycosidase,a hydrolytic enzyme,plays a role in the α-glucoside of disaccharide,oligosaccharides,aromatic glucoside.α-Glycosidase is one of the important members of glucose metabolic pathway and directly involved in starch and glycogen metabolism in organism.α-Glycosidase inhibitors can delay the decomposition of polysaccharides in the digestive tract and effectively prevent diabetics from raising postprandial blood glucose.Moreover,the burden of pancreatic β-cells will not be increased by α-glycosidase inhibitors.Therefore,it is an effective method for the prevention and treatment of diabetes.Isatin and indirubin,oxoindole derivatives,are biologically active and naturally occurring molecules found in both human organs and plants.Recently,oxoindole derivatives were reported as potent α-glycosidase inhibitors.In this study,the α-glycosidase inhibitory activity and mechanism of our synthetic isatin and indirubin derivatives were investigated.The assay system of α-glycosidase inhibitory activity in our lab was established and optimized.The reaction system contains 140μL of 50 mM potassium phosphate buffer(pH 6.8),20μL of 0.04 U/mL α-glycosidase solution and 10μL of samples.The reaction mixture was incubated for 5 min at 37℃,and was mixed with 30μL of 0.5 mM 4-nitrophenyl-a-D-glucoside substrate solution.After incubation for 30 min at 37℃,the absorbance value at 405 nm was tested.Based on the system,activity screening of isatin and indirubin,which were the potential inhibitors for a-glycosidase,was carried out.The results suggest that the 3-carbonyl on isatin and indirubin was the key site for a-glycosidase inhibitory activity.Bulky electron-withdrawing groups,such as the hydroxy phenyl and methoxy benzyl,were introduced at N-1 and C-5 of indirubin could increase the activity.Moreover,the indirubin75 was identified the most inhibitory activity with IC50＝1.88μM and is 1/27 of that of standard acarbose.It is proved that compound 75 was reversible competitively combined with α-glycosidase by kinetic determination.Fluorescence analysis showed that compound 75 directly combined with the hydrophobic domains ofα-glycosidase might reduce hydrophobic surfaces and induce the conformational changes of the free tryptophan in α-glycosidase,thereby preventing the formation of active center and/or the combination to substrate.The most trusted combination mode to α-glycosidase was simulated by the molecular docking test.The 3T3-L1 preadipocyte experimental results showed that compound 75 has significant inhibitiory effects on blood sugar comsuption.In this study,the inhibitory effects on α-glycosidase of compound 75 were screened,and the inhibitory mechanism was studied.The compound 75 is a potential small molecule hypoglycemic drug,its activity determination lays a foundation for the development of new drugs regarding such compounds.