α-Glucosidase Inhibitory Assay Protocol

The α-glucosidase inhibitory assay was performed according to a slightly modified method previously reported^{21 ,22} . In brief, a series of reaction solutions, including a fixed amount of α-glucosidase (10 unit/mL 3.75 µL), different concentrations of inhibitors (the tested compounds or positive controls, 37.5 µL), and sodium phosphate buffer (PBS, 596.75 µL, 0.1 M, pH 6.8), were incubated at 37 °C for 10 min. To initiate the reaction, 112.5 µL of pNPG (6.0 mM, as a substrate) was added into the pre-incubated mixtures, and the final volume of reaction system was kept at 750 µL. The p-nitrophenol released from pNPG substrate was used as the target substance to quantify the enzymatic activity. The absorbance of p-nitrophenol was monitored at 405 nm after incubation at 37 °C for 30 min. All samples were analyzed in triplicate, acarbose, and genistein were used as positive controls. The negative control was prepared by adding PBS instead of α-glucosidase, the blank was prepared by adding solvent instead of tested compounds, and the inhibition rate was calculated as the following Equation (1).
$\frac{\left(O{D}_{control}-O{D}_{controlblank}\right)-\left(O{D}_{test}-O{D}_{testblank}\right)}{O{D}_{control}-O{D}_{controlblank}}\times 100\%$
Equation (1). Inhibition rate calculation formula.