3.4. Assay for α-Glucosidase Inhibitory Activity

The α-glucosidase inhibition assay was performed according to previous protocols [31]. The assay was based on the release of p-nitrophenol from p-nitrophenol-α-d-glucopyranoside (substrate). The test samples were prepared by dissolving in 50% DMSO. Two-fold serial dilution was done for IC₅₀ determination of active compounds. The sample solution (10 μL) and 0.1 U/mL α-glucosidase (40 μL) in phosphate buffer (pH 6.8) were added to a 96-well plate. The mixture was preincubated at 37 °C for 10 min before adding 2 mM p-nitrophenol-α-d-glucopyranoside (50 μL). Then, the reaction was incubated again at 37 °C for 20 min. Finally, 1 M Na₂CO₃ solution (100 μL) was added to stop the reaction. The absorbance of the mixture was determined using a microplate reader at 405 nm. In this assay, acarbose was used as the positive control.

An enzyme kinetic study was conducted based on the α-glucosidase assay as mentioned above. The PNPG concentrations were varied from 0.25 to 2 mM in the absence or presence of compound 2 (11 and 22 µM) or acarbose (930 and 465 µM). The inhibition mode was determined by double-reciprocal Lineweaver–Burk plot (1/V vs. 1/[S]). In order to estimate the K_i value, slopes of double-reciprocal lines were used to construct a secondary plot, and the K_i was calculated from the line equation of the plot [32].