Isolation, Identification And Characterization Of The α-amylase Inhibitor From Cicer Arietinum L.

α-Amylase inhibitor （α-AI） is of glycosidase inhibitors, which widely exists in theendosperm of plant seeds and microorganisms metabolites. As a new diabetes drug, itfunctions in hypoglycemic and lipid-lowering by reducing the body’s digestion and absorptionof dietary carbohydrate and fat production through inbihiting the biological activity ofamylase, a key enzyme in the body glucose metabolism. At present, α-AI has been appliedpopularly in the field of medicine and agriculture, exhibits unique traits in clinically treatmentof diabetes, hyperglycemia, hyperlipemia, lipomatosis. The efficacy and safety of the amylaseinhibitor depend on the processing and extraction techniques used.Biochemical methods including ammonium sulfate fractional precipitation, ion-exchangechromatography and gel filtration column chromatography were used to isolate this novelα-amylase inhibitor, and together with its partial properties were characterized in this paper.The crude extract was obtained by decorticating, grinding, as well as defatting of Cicerarietinum seeds with petroleum ether. We employed the orthogonal design method to test theeffects of four factors, including temperature of extraction solution （A）, pH value ofextraction solution （B）, time of extraction （C） and volume of extraction solution （D）, on theextraction of α-amylase inhibitor, then after practised the optimized procedure. The optimizedcriteria of α-amylase inhibitor extraction procedures were pH4.0,35times of solvent volume,50℃and4.0h for extraction per time. The α-amylase inhibitor was purified further byprecipitating with35%⁶5%ammonium sulfate, ion-exchange chromatography on CMcellulose （CM-52） and gel filtration on Sephadex G-75. The approximate molecular weight ofα-AI was estimated to be40.4kDa by SDS-PAGE. The kinetic analysis showed that α-AIinhibited α-amylase following the competitive model with an inhibition constant Kiof3.14×10^-6mol·L^-1, and the half maximum inhibitory concentration is1.03×10^-6mol·L^-1. Itsoptimal incubation time and concentration were10min and1.0×102μg· mL^-1, respectively.α-AI was stable in pH3.0¹1.0, was also heat-resistant and maintained85%of the activityafter heated in80℃for30min.