α glucosidase type 4 enzyme

The method of Lam et al (2008) was used to evaluate α-glucosidase inhibitory activity (19 (link)). α-Glucosidase type IV enzyme (Sigma Co, St. Louis, USA) from Bacillus stearothermophilus was dissolved in phosphate buffer (0.5 M, pH 6.5). The enzyme solution and extracts dissolved in MeOH-H₂O were preincubated in a 96-well microtiter plate for 15 min at 37 °C. After that, the substrate solution [20 mM p-nitrophenyl-α-d-glucopyranoside (NPG), Sigma] was added. The mixture was incubated for 35 min at 37 °C. The increase in the absorption at 405 nm due to the hydrolysis of NPG by α-glucosidase was measured by an ELISA microtiter plate reader. Acarbose (Bayer Group, Turkey) was used as positive control. The inhibition percentage (%) was calculated by the equation:
Inhibition %=[(A_Control-A_Sample)/A_Control]×100

α-Glucosidase activity was performed according to the method of Lam et al (22 (link)). α-Glucosidase type IV enzyme (Sigma Co., St. Louis, USA) from B. stearothermophilus was dissolved in 0.5 M phosphate buffer (pH 6.5) (3 U/ml). The enzyme solution (20 μl) and test extracts (10 μl) dissolved in MeOH-H₂O (1:9, v/v) were preincubated in a 96-well microtiter plate for 15 min at 37°C. After that, the substrate solution [10 μl, 20 mM p-nitrophenyl-α-d-glucopyranoside (NPG), Sigma] in the same buffer was added. The mixture was incubated for 35 min at 37°C. The increase in the absorption at 405 nm due to the hydrolysis of NPG by α-glucosidase was measured by an ELISA microtiter plate reader. Acarbose (Bayer Group, Turkey) was used as a positive control. The inhibition percentage (%) was calculated by the equation:
Inhibition (%) = [1 − (A _sample/A _control)] × 100
IC₅₀ calculations were done by using Sigma Plot 12.0 software. Minimum of eight different concentrations prepared from the stock solutions of extracts were used for calculating the IC₅₀ value. The logarithmic concentrations (10.000–0.1 μg/ml) were chosen.