α -amylase inhibitory assay

The enzyme inhibition studies were performed using a modified procedure of McCue and Shetty, 2004.¹³ The set of tubes containing 500 μl of extract (1.25–10 mg/ml) was kept along with 500 μl of 0.02 M sodium phosphate buffer (pH 6.9) containing α-amylase solution (0.5 mg/ml) in water bath for 10 min at 25°C . 500 μl of 1% starch solution made in 0.02 M sodium phosphate buffer of pH 6.9 was added at regular interval and further incubated at 25°C for 10 min. The reaction was stopped by adding 500 μl of dinitrosalicylic acid (DNS) reagent. The tubes were kept in boiling water bath for 5 min and cooled to room temperature. 5 ml distilled water was added and the absorbance was measured at 540 nm using a spectrophotometer. Control was made using the same protocol by only replacing the extract with distilled water. The α-amylase inhibitory activity was evaluated. Concentrations of extracts resulting in 50% inhibition of enzyme activity (IC₅₀) were determined. The IC₅₀ value was defined as the concentration of the extract, containing the α-amylase inhibitor that inhibited 50% of the PPA activity and calculated by given formula below: