4.7. α-glucosidase Inhibition Assay

The inhibitory activity of α-glucosidase was determined by slight modifications of the procedure previously reported [62]. Shortly, 15 μL of the enzyme (0.5 U/mL in 0.1 M phosphate buffer (pH-6.8)) and 15 μL of the samples were successively added to 96-well plates and were incubated together at 37 °C for 10 min. Then, 15 μL of the substrate p-nitrophenyl α-d-glucopyranoside (p-PNP-G) (0.5 mM solution in 0.1 M phosphate buffer (pH-6.8)) was added and the enzymatic reaction was performed at 37 °C for 30 min, after that 100 μL of sodium carbonate (0.2 M) was added as a stop buffer. Finally, the solvent was monitored spectrophotometrically by measuring the absorbance at 405 nm. Acarbose was used as a positive control and the uninhibited enzyme was taken as a negative control. The assay was performed in triplicate. The extracts and fractions were dissolved in DMSO and diluted to desired concentrations with 0.1 M phosphate buffer. The final DMSO concentration was maintained below 2% (v/v), which was found not to have any effect on the enzyme activities. Different concentrations were tested of each crude extract (0.1, 0.2, 0.3, 0.4, 0.5, 1 and 2 mg/mL) and compounds (0.001, 0.005, 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5 mg/mL). Appropriate blank was used for all the extracts.

The α-glucosidase inhibition percentage was calculated as follows: