3.5. α-Amylase Inhibition Assay

α-Amylase inhibitory activity was determined following the starch-iodine method [39] with some modifications. A 1% starch solution was prepared by 1 g of starch in 10 mL of distilled water following gentle boiling and cooling into 100 mL. A reaction mixture, 25 μL sample (0–1 mM) and 50 μL α-amylase (5 U/mL) in phosphate buffer, was incubated at 37 °C for 10 min. Afterwards, the starch (100 μL, 1% w/v) solution was added to the mixture and incubated again at 37 °C for 10 min. The enzymatic reaction was suspended by adding HCl (25 μL, 1 N) followed by the incorporation of 50 μL of iodine reagent (2.5 mM I₂ and 2.5 mM KI). After adding the iodine/iodide solution, based on the colour change, the absorbance was monitored at 630 nm for 10 min. Acarbose was used a positive control. The percentage of inhibition was calculated and results were expressed as IC₅₀ (µM).