Synergistic Inhibition of α-Glucosidase by Hyperin, Isoquercitrin, and Quercetin: Mechanistic Insights from Multi-spectroscopic, Calorimetric, Computational, and Caco-2 Cell Assays

Abstract Naturally abundant flavonoids inhibit α-glucosidase (α-GLU), but the mechanisms underlying the synergistic effects among key active compounds remain poorly understood. In this study, the synergistic inhibition of α-GLU by hyperin, isoquercitrin, and quercetin was systematically investigated using fluorescence quenching, isothermal titration calorimetry, circular dichroism, differential scanning calorimetry, computational models, and Caco-2 cell assays. The ternary combination synergistically inhibited α-GLU (IC50 = 0.022 mg/mL, combination index <1) and showed the greatest α-helix reduction and denaturation temperature decrease of α-GLU. Molecular docking and molecular dynamics simulations suggested that quercetin preferentially occupied the hydrophobic catalytic pocket, whereas hyperin and isoquercitrin primarily interacted with peripheral regions. In differentiated Caco-2 cells, hyperin and isoquercitrin exhibited stronger inhibition toward sucrase than maltase, whereas quercetin showed the opposite trend. Collectively, the results demonstrate that the synergistic inhibition of α-GLU arises from complementary binding modes, cooperative conformational perturbation, and differential targeting of intestinal disaccharidase activities.