Differential Inhibitory Mechanisms of Myricetin and Dihydromyricetin on α-Glucosidase: A Combined Molecular Docking, Isothermal Titration Calorimetry and Surface Plasmon Resonance Study

α-Glucosidase inhibitors (AGIs) significantly regulate blood glucose by delaying carbohydrate digestion and slowing glucose absorption, thus playing a critical role in glycemic control. Structurally, dihydromyricetin (Unless otherwise stated, the term dihydromyricetin used throughout this manuscript refers to trans-(2R,3R)-(+)-dihydromyricetin.) differs from myricetin in that the C2=C3 double bond in the C-ring is saturated, resulting in a dihydroflavonol instead of a flavonol. This study investigated the inhibition mechanism of α-glucosidase by the C2=C3 double bond structure using a set of integrated and multi-perspective approaches combining enzyme kinetics, multi-spectroscopic methods, molecular docking, isothermal titration calorimetry (ITC), and surface plasmon resonance (SPR). Myricetin (IC50 = 13.648 ± 0.157 μM) was found to be a more potent α-glucosidase inhibitor than dihydromyricetin (IC50 = 453.922 ± 1.643 μM). Enzyme kinetics indicated that myricetin acted as a competitive inhibitor, whereas dihydromyricetin functioned as a non-competitive inhibitor. To further examine these interactions, multi-spectroscopic analysis demonstrated that binding of myricetin caused significant changes in the microenvironment around fluorescent amino acids (such as tyrosine and tryptophan) in α-glucosidase, resulting in slight unfolding of the enzyme structure. Additionally, molecular docking provided a detailed molecular perspective, identifying hydrogen bonding and hydrophobic interactions as the primary forces driving the binding of two flavonoids to α-glucosidase. Delving deeper into the binding mechanism, ITC analysis provided thermodynamic evidence that myricetin (KD = 6.215 ± 0.022 μM) exhibited a stronger binding affinity to α-glucosidase than dihydromyricetin (KD = 232.648 ± 1.236 μM), with both interactions being enthalpy-driven and primarily mediated by hydrogen bonds. Building on this, SPR analysis offered additional insights into the binding process, showing that myricetin not only had a higher binding affinity (KD = 3.416 ± 0.015 μM) but also a faster association rate (ka = 1668 ± 23 M−1 s−1) compared to dihydromyricetin (KD = 11.539 ± 0.056 μM, ka = 339.7 ± 17.1 M−1 s−1). In conclusion, this study demonstrated that the C2=C3 double bond plays a key role in enhancing α-glucosidase/inhibitor interactions, providing a theoretical basis for the design of novel AGIs and proposing a new set of multi-perspective methods for elucidating these inhibition mechanisms.