Optimisation of a fluorimetric assay for the screening of potential alpha-glucosidase inhibitors in coloured plant extracts and foods

The frequently used method for the in vitro screening of inhibitors of yeast α-glucosidase is based on the hydrolysis of p-nitrophenol-α-D-glucopyranoside to yield p-nitrophenol and glucose. After adjusting to pH 8 to form the yellow nitrophenolate anion, the absorbance at 405 nm is measured [1,2]. During our investigations into potential α-glucosidase inhibitors from sugarcane products, we found a decrease in sensitivity when highly coloured extracts were assayed. Therefore, an optimised method of the α-glucosidase assay was developed, using the fluorogenic substrate 4-methylumbelliferyl-α-D-glucopyranoside (4-MUG), to ensure a more reproducible and reliable screening tool for highly coloured plant extracts and foods. The optimised conditions for the assay were determined: α-glucosidase (2mU/mL), 4-MUG (84µM), incubation time (20 minutes), incubation temperature (37°C) and assay buffer pH (5.5). A molasses extract was screened for α-glucosidase inhibition using the optimised conditions with 80% inhibition seen at 150µg/mL and no negative quenching effects at concentrations within the linear range of the instrument. The optimised assay was also used to determine IC50 values for acarbose and fucoidan. The results suggest that acarbose does inhibit yeast α-glucosidase, which contradicts earlier work [3]. Overall, this optimised assay will be a valuable tool for the screening of highly coloured plant extracts and foods for α-glucosidase activity