Unraveling Inhibition Mechanism of Structurally Distinct Flavonoids Against α-Amylase and α-Glucosidase: Insights from Multi-Spectroscopic and In Silico Analyses

Type II diabetes mellitus is a growing global health burden, and postprandial hyperglycemia is an independent risk factor for diabetic complications. α-amylase (αA) and α-glucosidase (αG) are key digestive enzymes involved in carbohydrate hydrolysis and represent important targets for investigating the regulation of postprandial carbohydrate digestion. In this study, baicalein (BA), liquiritigenin (LI), and puerarin (PU) were systematically investigated against αA and αG. Results showed that the three flavonoids inhibited αA and αG in a dose-dependent manner, with the inhibitory potency following the order of PU > BA > LI. Kinetic analyses revealed that three flavonoids exhibited mixed-type inhibition toward αA but non-competitive inhibition toward αG. Fluorescence results indicated that flavonoids quenched intrinsic fluorescence of αA and αG through a static mechanism with approximately one binding site, and the binding was spontaneous and driven predominantly by hydrogen bonding and van der Waals forces. Multi-spectroscopic analyses further demonstrated that flavonoid binding induced conformational rearrangements of enzymes, manifested by the burial of aromatic residues into more hydrophobic microenvironments and the conversion of secondary structures from random coil to ordered α-helix and β-sheet. Moreover, in silico analysis revealed that hydrogen bonding and van der Waals forces were the key driving forces between αA/αG and BA/LI/PU. Furthermore, abundant hydroxyl groups of PU provided additional interaction sites for hydrogen-bond formation, contributing to its superior inhibitory efficiency. These findings provide molecular-level evidence for the structure-dependent inhibition of αA/αG by flavonoids under the present in vitro conditions and provide a basis for further investigation of their physiological relevance.