α-Amylase and α-Glucosidase Inhibition Assay

The effect of the crude extract and its fractions on α-amylase and α-glucosidase activity was determined according to the method described by Kazeem et al²⁹ with modifications.

To 250 μL of each extract concentration in a test tube (0-360 μg/mL), the following was added sequentially: buffered α-amylase (250 μL, 0.05 mg/mL) and starch (250 μL, 1% w/v). The reaction mixture was incubated for 10 minutes at 25°C. DNSA (500 μL) was added subsequently and then boiled for 5 minutes. It was then cooled and diluted with 5 mL of dH₂O. The control was prepared in the same manner as the test samples with distilled water replacing the extract. The absorbance of each test tube content was taken at 540 nm and the percentage inhibition calculated as follows:

where A_c and A_t are the absorbance of the control and test, respectively.

To 50 μL of each extract concentration in a test tube (0-40 μg/mL) the following were added sequentially: buffered α-glucosidase (100 μL, 1.0 U/mL) and incubated at 37°C for 10 minutes, then pNPG (50 μL, 3.0 mM) and incubated at 37°C for 20 minutes, and then Na₂CO₃ (5% w/v), cooled to 25°C, and lastly 5 mL H₂O was added and vortexed. The absorbance of the resulting yellow p-nitrophenol from the different test tubes was taken at 405 nm and the percentage inhibition calculated as follows:

where A_c and A_t are the absorbance of control and test, respectively

The concentration of the extracts resulting in 50% inhibition of the enzyme activity (IC₅₀) was determined graphically.