α-Amylase Inhibition Assay of A. pavonina Leaf Extract

The α-amylase inhibition assay was performed using the 3,5-dinitrosalicylic acid (DNSA) method [8 (link)]. The leaf extract of A. pavonina was dissolved in minimum amount of 10% DMSO and was further dissolved in buffer ((Na₂HPO₄/NaH₂PO₄ (0.02 M), NaCl (0.006 M) at pH 6.9) to give concentrations ranging from 10 to 1000 μg/ml. A volume of 200 μl of α-amylase solution (2 units/ml) was mixed with 200 μl of the extract and was incubated for 10 min at 30 °C. Thereafter 200 μl of the starch solution (1% in water (w/v)) was added to each tube and incubated for 3 min. The reaction was terminated by the addition of 200 μl DNSA reagent (12 g of sodium potassium tartrate tetrahydrate in 8.0 mL of 2 M NaOH and 20 mL of 96 mM of 3,5-dinitrosalicylic acid solution) and was boiled for 10 min in a water bath at 85–90 °C. The mixture was cooled to ambient temperature and was diluted with 5 ml of distilled water, and the absorbance was measured at 540 nm using a UV-Visible spectrophotometer. The blank with 100% enzyme activity was prepared by replacing the plant extract with 200 μl of buffer. A blank reaction was similarly prepared using the plant extract at each concentration in the absence of the enzyme solution. A positive control sample was prepared using Acarbose (100 μg/ml–2 μg/ml) and the reaction was performed similarly to the reaction with plant extract as mentioned above. The α-amylase inhibitory activity was expressed as percent inhibition and was calculated using the equation given below: The % α-amylase inhibition was plotted against the extract concentration and the IC₅₀values were obtained from the graph. $\%\alpha \mathrm{amylase}\mathrm{inhibition}=100\times \frac{\mathrm{Ab}{\mathrm{s}}_{100\%\mathrm{control}}-\mathrm{Ab}{\mathrm{s}}_{\mathrm{Sample}}}{\mathrm{Ab}{\mathrm{s}}_{100\%\mathrm{Control}}}$