2.6. In Vitro Antidiabetic Activity (α-Amylase Inhibition Assay)

The α-amylase inhibitory potential was evaluated through employing the starch-iodine assay method with minor modification, proposed by Xiao, et al. [66]. Prior to testing, α-amylase solution (0.04 mg/mL) was prepared by dissolving 4 mg of α-amylase in 100 mL of 0.02 M sodium phosphate buffer (containing 0.006 M of NaCl, pH 6.9). In brief, 500 mL of test extract/standard (acarbose) at a concentration of 125–1000 µg/mL was added to 500 μL of α-amylase solution (0.04 mg/mL) and incubated at 37 °C for 10 min. Subsequently, 500 μL of 1% (w/v) soluble starch was added to each test solution and re-incubated at unchanged temperature for 15 min. After re-incubation, the enzymatic reaction was terminated by adding 20 μL of 1M HCl. At last, 100 μL of iodine reagent (0.005 M I₂ and 0.005 M KI) was added and a change in solution color was noted. The absorbance was read at OD₆₂₀ nm (optical density) on a UV-visible spectrometer. In control, the plant sample was replaced with buffer which represents 100% α-amylase activity. Blank contained only buffer solution instead of the enzyme. The percentage (%) of α-amylase inhibitory activity of test samples and acarbose were calculated by applying the following Equation (3):

where, A_c and A_s denote absorbance of control reaction and absorbance of test sample or standard, respectively.