2.4. Amylase inhibition assay

The amylase inhibition assay was performed using the preincubation chromogenic method adapted from Geethalakshmi, Sarada, Marimuthu, and Ramasamy (²⁰¹⁰). The plant extract (40 μl, 20 mg/ml in dimethylsulfoxide, DMSO), 160 μl of distilled water, was preincubated with the addition of 200 μl of the enzyme solution for 5 min at 37°C before reacting with the starch solution (400 μl) for 15 min. The mixture (200 μL) was removed and added into a separate tube containing 100 μL 3,5‐dinitrosalicylic acid (DNS) color reagent solution and placed in a 85°C water bath. After 15 min, this mixture was diluted with 900 μl distilled water and removed from the water bath. α‐Amylase activity was determined by measuring the absorbance of the mixture at 540 nm. Acarbose was used as the control/standard inhibitor.

Percentage maltose generated was calculated from the equation obtained from the maltose standard calibration curve (0%–0.2%, w/v maltose)

% reaction = (mean maltose in test/mean maltose in control) × 100.

% Amylase inhibition activity = (100−% reaction)