Enzymatic assays

The inhibitory effect of the synthesized compounds on α-glucosidase (yeast origin), α-fucosidase (bovine kidney origin) and α-mannosidase (jack bean origin) inhibitory activity was determined in 96-well plates employing the substrate PNPG and 4-nitrophenyl α-D-glucopyranoside according to the procedure previously reported by Ferreres et al.⁴². Prior to use, all test compounds were solubilised in solvent, dimethylsulfoxide (DMSO), and then further diluted in DMSO to acquire the desired final maximum test concentration. Briefly, each well in 96-well plates contained 100 µL of 2 mM 4-nitrophenyl α-D-glucopyranoside (PNP-G) in 10 mM potassium phosphate buffer (pH 7.2) and different test concentrations (2–10 µM). The reaction was initiated by the addition of 5 µL of the enzyme solution (0.1 IU per well). α-glucosidase (yeast), α-fucosidase (bovine kidney) and α-mannosidase (jack bean) were purchased from Sigma Aldrich, Bangaluru). The plates were incubated at 37 °C for 10 min. The absorbance was measured spectrophotometrically at 430 nm (Spectra Max M5e micro plate reader). The increase in absorbance (ΔA) was compared with that of the control (buffer instead of test compound) to compute the inhibitory concentrations (IC₅₀) which was determined from two independent assays, performed in duplicate. Acarbose, an illustrious inhibitor of α-glucosidase was employed as positive control.Inhibition (%) = (ΔA_control − ΔAs_ample/ΔA_control × 100).The concentration of compound required to obtain 50% inhibition ofα-glucosidase activity under the assay conditions was defined as the IC₅₀ value.