3.3.1. α-Amylase Inhibition Assay

The assay was performed as described before [43]: 20 µL of sample extract (serial dilutions) and 40 μL of 2 g L starch solution were mixed with 20 μL of α-amylase (0.1 mg mL⁻¹). All solutions were prepared in 0.1 M phosphate buffer (pH 6.9). After incubation (20 min; 37 °C), the reaction was stopped by the addition of 80 μL of 0.4 M HCl followed by 100 μL of 5 mmol L⁻¹ I2 (in 5 mmol L⁻¹ KI) and the absorbance was read at 620 nm (Victor3 microtiter reader, PerkinElmer, Waltham, MA, USA). Acarbose was used as positive control. Inhibition (% I) of α-amylase activity was calculated using the following formula:

where A_C, A_CB, A_S, and A_SB are the absorbance of control, control blank, sample and sample blank, respectively. Control of the experiment contains all the reagents except extracts, whereas sample blanks were without the enzyme. The IC₅₀ values (mg mL⁻¹ of dry extract or reference compound) were determined from the least-squares regression line of the logarithmic concentrations plotted against percentage inhibition. This value corresponds to the concentration of the extracts able to reduce the enzyme activity by 50% with reference to the control.