2.8. α-Amylase Inhibition

The α-amylase inhibition assay was done according to procedure conducted by N. Nirmali et al. [11] with some modifications. The assay was performed using the 3,5-dinitrosalicylic acid (DNSA) method. M fruticosa serpyllifolia VOs stock solution (S.S) of 1 mg/mL was prepared in a minimum amount of DMSO 10% and was further dissolved in buffer (Na₂HPO₄/NaH₂PO₄ (0.02 M), NaCl (0.006 M) at pH 6.9). Working solution of concentrations 10, 50, 100, 500, and 1000 μg/mL was prepared using buffer (Na₂HPO₄/NaH₂PO₄ (0.02 M)), NaCl (0.006 M) at pH 6.9). Acarbose was used as a reference. The stock and working solutions of Acarbose were prepared using the same procedure of M fruticosa serpyllifolia VOs. α-Amylase solution (2 unit/mL) was prepared by dissolving 12.5 mg of amylase enzyme in buffer [(Na₂HPO₄/NaH₂PO₄ (0.02 M)), NaCl (0.006 M) at pH 6.9]. A volume of 200 μL of α-amylase solution (2 unit/mL) was mixed with 200 μL of each VOs working solution and incubated for 10 min at 37°C. Then 200 μL of the starch solution was added and incubated for 3 min. The reaction was stopped by the addition of 200 μL DNSA reagent and boiled for 10 min in a water bath at 85–90°C. The mixture was cooled to ambient temperature and diluted with 5 mL of distilled water, and the absorbance was measured at 540 nm using a UV-Vis. spectrophotometer. The blank with 100% enzyme activity was prepared by replacing the plant extract with 200 μL of buffer. Acarbose was used as a positive control sample. The α-amylase inhibitory activity was expressed as percent inhibition and was calculated using (2). The %  α-amylase inhibition was plotted against the extract concentration and the IC₅₀ values were obtained from the graph [11].

where %  α-amylase inhibition is the percentage inhibition of amylase.