Flavonoids and chalcones as selective α-glucosidase inhibitors: from enzymatic screening to validation using the human enzyme.

α-Glucosidase is a key therapeutic target for postprandial glucose control, but most clinically available inhibitors also inhibit α-amylase, leading to gastrointestinal side effects. Natural polyphenols have been widely reported as potent and selective α-glucosidase inhibitors, primarily based on screening assays using yeast enzyme and synthetic substrates. In this study, thirteen flavonoids and five chalcones were initially evaluated using this conventional approach to elucidate the structural determinants of selective α-glucosidase inhibition. Among them, genistein and myricetin emerged as the most potent inhibitors of yeast α-glucosidase, with IC50 values significantly lower than that of acarbose, the most widely used α-glucosidase inhibitor. Both flavonoids showed high selectivity, with minimal or no inhibition of α-amylase. To assess the translational relevance of these findings, these compounds were subsequently tested against human α-glucosidase from C2BBe1 cell extracts using the physiological substrate maltose. Under these conditions, only myricetin retained measurable inhibitory activity at low concentrations, which was further validated through chromatographic analysis. These findings demonstrate that current screening strategies relying solely on yeast α-glucosidase can substantially overestimate the inhibitory activity of natural polyphenols. This work highlights the importance of incorporating physiologically relevant enzyme sources, natural substrates, and controls for spectrophotometric assay interference into the discovery of novel α-glucosidase inhibitors, providing a more reliable workflow for the assessment of natural products.