α-Glucosidase Inhibition Assay for Anti-Diabetic Potential

The anti-diabetic potential of extracts was further determined by α-glucosidase inhibition bioassay using a previously reported protocol with minute modifications [42 (link), 43 (link)]. In the experiment, 50 mL of phosphate buffer (pH 6.8) supplemented with 100-mg BSA (bovine serum albumin) was used to dissolve α-glucosidase (Saccharomyces cerevisiae, Sigma-Aldrich). Reaction mixtures constituting 10 μL of tested sample, phosphate buffer (490 μL; pH 6.8) and p-nitrophenyl α-D-glucopyranoside (5 mM; 250 μL) were kept sepatately for incubation at 37 °C for 5 min. Two hundred fifty microlitre α-glucosidase (0.15 unit/mL) was then added to each mixture followed by incubation for 15 min at 37 °C. After terminating reaction, by adding 2 mL Na₂CO₃ (200 mM) solution, absorption was recorded using a UV-Vis spectrophotometer at 400 nm. The assay is based on the quantification of p-nitrophenol release from p-nitrophenyl α-D-glucopyranoside. In the experiment, acarbose was employed as a positive control and assay was repeated three times.
$$\%\text{Enzyme inhibition}=\left(\frac{\mathit{Abs}\mathit{\text{Sample}}-\mathit{Abs}\mathit{\text{negative control}}}{\mathit{Abs}\mathit{\text{blank}}-\mathit{Abs}\mathit{\text{negative control}}}\right)\times 100$$