Inhibition assay for in vitro α-glucosidase activity

The α-glucosidase inhibition assay was conducted by the chromogenic method described by Watanabe et al. (12) using a readily available yeast enzyme. In brief, yeast α-glucosidase (0.7 units, Sigma, St. Louis, MO, USA) was dissolved in 100 mM phosphate buffer (pH 7.0) containing 2 g/L bovine serum albumin and 0.2 g/L NaN₃ to form the enzyme solution. p-Nitrophenyl-α-D-glucopyranoside (5 μM) was dissolved in the same buffer (pH 7.0) to form the substrate solution. Next, 50 μL of enzyme solution and 10 μL of sample dissolved in dimethylsulfoxide (5 mg/mL) were mixed in a well of a microtiter plate, and the absorbance was measured at 405 nm with a microplate reader (zero time point). After incubation for 5 min, the substrate solution (50 μL) was added, and the mixture was incubated for another 5 min at room temperature. Then, the increase in absorbance from the zero time point was measured. The inhibitory activity at varying concentrations of SSE was expressed as 100 minus the absorbance change of test compounds relative to the absorbance change of the control (%), where the test solution was replaced by the carrier solvent. The measurements were performed in triplicate, and the IC₅₀ value (the concentration of SSE that results in 50% inhibition of maximal activity) was determined.