In Vitro Evaluation of Hesperidin’s α-Glucosidase Inhibitory Kinetics and Its Potential Role in Type 2 Diabetes Management

Background: Type 2 diabetes mellitus is driven in part by postprandial hyperglycemia (PPHG), for which inhibition of intestinal α-glucosidase is a validated strategy. Hesperidin, a citrus-derived flavonoid, has been reported to modulate carbohydrate metabolism, but its kinetic interaction with α-glucosidase has not been fully clarified. Objective: To characterize the inhibitory kinetics of hesperidin against Saccharomyces cerevisiae α-glucosidase and evaluate its potential as a candidate for managing PPHG. Methods: α-Glucosidase activity was assayed using p-nitrophenyl-α-D-glucopyranoside (pNPGP) in 0.1 M phosphate buffer (pH 6.8). Enzyme was pre-incubated with hesperidin (100 μM) at 37 °C for 10 min; reactions were initiated with substrate (0.125–4.000 mM), incubated for 20 min, and quenched with 100 mM Na₂CO₃. Product formation (p-nitrophenol) was monitored at 405 nm. Controls lacked inhibitor; acarbose served as a positive control. Initial velocities were fitted by Lineweaver–Burk plots to estimate kinetic parameters; the inhibition constant (K_i) was derived from secondary plots. Experiments were performed in triplicate and analyzed by linear regression. Results: Hesperidin produced concentration-dependent inhibition consistent with a competitive mechanism: double-reciprocal plots intersected on the y-axis, V_max was effectively unchanged, and K_m increased from 0.046 mM (control) to 2.511 mM (+hesperidin). The calculated K_i = 0.0019 mM indicates high affinity for the free enzyme. Acarbose exhibited the expected inhibitory profile, corroborating assay validity. Conclusions: Hesperidin is a potent competitive inhibitor of α-glucosidase in vitro, markedly reducing apparent substrate affinity without impacting maximal catalytic rate. These kinetics support hesperidin’s promise as a natural adjunct for PPHG control.