Inhibitory mechanisms of baicalein, genistein, and isoliquiritigenin on α-amylase: Multispectral characterization and molecular simulations reveal binding interactions and conformational changes

To mitigate postprandial hyperglycemia, acarbose's excessive α-amylase (AMY) inhibition causes gastrointestinal side effects. Baicalein (Bai), genistein (Gen), and isoliquiritigenin (Isl), featuring 5,6-dihydroxyflavone structures, mildly inhibit AMY. In this study, the inhibitory mechanism of Bai, Gen, and Isl on AMY was determined by integrating multiple analytical techniques and molecular simulation technologies. The results showed that Bai/Gen/Isl exhibited strong inhibitory activity against AMY with IC 50 values of 22.44 ± 0.92 μmoL·L −1 , 24.78 ± 1.91 μmoL·L −1 , and 26.78 ± 1.79 μmoL·L −1 , respectively, which belonged to the mixed inhibition type dominated by competitive inhibition. The three form a ground state complex with AMY through a static quenching mechanism, and the order of binding constants is Bai > Gen > Isl. Their binding significantly changed the microenvironment polarity of Trp residues in AMY and induced secondary structure rearrangement. Differential scanning calorimetry (DSC), X-ray diffraction (XRD), and scanning electron microscopy (SEM) showed that the relative crystallinity of AMY increased, forming dense aggregates and increasing thermal stability. Molecular simulation showed that the interactions between AMY and Bai/Gen were dominated by van der Waals forces or hydrogen bonds, while the interactions with Isl were dominated by hydrophobic interactions. By employing a novel integrative approach combining fluorescence/UV–Vis/FTIR spectroscopy, DSC-XRD-SEM characterization, and 120 ns molecular dynamics simulation, this study overcomes the single-technique limitations of previous research and provides a theoretical basis for the development of blood glucose control agents based on natural products.