2.12. α-Glucosidase and α-Amylase Inhibitor Assay

α-glucosidase and α-amylase inhibitor screening kits (colorimetric) were purchased from Biovision (Milpitas, CA, USA). In total, 10 mM of stock solution of all the tested compounds were dissolved in DMSO and serially diluted in the assay buffer of each kit. Experiments were performed according to the manufacturer’s protocol. Briefly, for the α-glucosidase assay, 10 µL of serially diluted compounds at the corresponding concentration (10 nm–1 mM) were added into designated wells of clear 96 well-plates. Subsequently, 10 µL of the α-glycosidase enzyme was added to each well and volume was adjusted to 80 µL and plates were incubated for 15-min at room temperature in dark condition. Then, 20 µL of α-glycosidase substrate mixture was added in all wells and kinetic of reaction was measured at OD: 410 nm for 60 min at 2 min intervals by using a multiplate reader, Biotek instrument, Inc., Canada. Enzyme control (no inhibitor), background control (no enzyme), solvent control (DMSO) and inhibitor control (acarbose) were included in the plates. For the α-amylase assay, 50 µL of serially diluted compounds (3.25 µM to 500 µM) were added into a clear 96-well plate with 50 µL of assay buffer and 50 µL of α-amylase enzymes. The plate was incubated at room temperature in the dark for 10 min. Then 50 µL of the α-amylase substrate was added in all wells. The kinetic of reaction was measured at OD:410 nm for 26 min at intervals of 2 min by using a multiplate reader. Control α-amylase inhibitor was provided by the manufacturer, enzyme control, background control and solvent control were all included. Enzyme inhibition was calculated according to Zhang et al. (2014) [42]. In summary, ODs were plotted according to the time for each sample. Areas under the curve (AUC) were calculated, and enzyme inhibition was measured as 100−(AUC_compound/AUC_enzyme) × 100 for each dilution of each compound.