Objective: To investigate the mechanism of synergistic inhibition of α-amylase (AMY) by three flavonoids [baicalein (Bai)， genistein (Gen)， isoliquiritigenin (Isl)] combined with acarbose (Aca). Through methods such as inhibition rate experiments， combination index (CI) analysis， fluorescence spectroscopy， ultraviolet spectroscopy， infrared spectroscopy， and molecular dynamics simulation， the inhibitory effects and action mechanisms of the combined inhibition on AMY were systematically studied. The results show that the inhibitory effects of Bai/Gen/Isl combined with Aca on AMY were significantly enhanced at molar ratios of 2∶1， 3∶1， and 4∶1. Among them， Bai-Aca and Isl-Aca exhibited a strong synergistic effect at a molar ratio of 3∶1(CI < 1)， while Gen-Aca showed synergism only at a molar ratio of 3∶1 and under the specific condition of 0.3 < Fa < 0.9(fraction affected). Fluorescence spectroscopy analysis showed that the combined inhibitors bind to AMY via a static quenching mechanism. Among them， Isl-Aca exhibits the highest binding constant (Ka) with AMY [K = (4.826±0.206)×105 L/mol at 298 K； Ka = (35.172±0.255)×105 L/mol at 300 K]， and its affinity is significantly enhanced with increasing temperature (P < 0.05)， indicating that the complex formed by Isl-Aca and AMY has the optimal stability. Thermodynamic parameters indicated that hydrophobic interactions were the main driving force for Bai-Aca and Isl-Aca， while Gen-Aca relied on hydrogen-bond binding. Synchronous fluorescence and ultraviolet spectroscopy showed that the combined inhibitors regulated the activity of AMY by changing the microenvironment of tryptophan (Trp) and tyrosine (Tyr) residues， with the inhibitory effect of Trp residues being dominant. Infrared spectroscopy and molecular dynamics simulation further revealed that Bai-Aca and Isl-Aca promoted the transformation of the secondary structure of AMY into a more stable β-turn， while Gen-Aca induced the formation of α-helix. However， the free-energy landscape of the latter complex presented a dispersed energy cluster， indicating weaker stability. In conclusion， the Bai-Aca and Isl-Aca combined inhibition systems significantly reduce the dosage of inhibitors through multi-target synergistic effects， while maintaining the high stability of the AMY complex. This provides a theoretical basis for the development of novel diabetes treatment strategies and functional foods based on flavonoids.

combined inhibition  /  baicalein  /  genistein  /  isoliquiritigenin  /  acarbose  /  molecular dynamics