Elucidating the Inhibitory Mechanism of Raisin Polyphenols Against α-Amylase: Insights from Multispectral Analysis and Computational Simulations

Natural α-amylase inhibitors from plant sources remain underexplored, whereas raisin polyphenols exhibit considerable inhibitory potential. In this study, raisin polyphenols were purified with D101 macroporous resin, and the adsorption process followed the pseudo-first-order kinetic model and the Langmuir isotherm model, indicating monolayer, exothermic adsorption. UHPLC-MS/MS analysis of the purified raisin polyphenols (PRP) identified 10 phenolic compounds, with ferulic acid, quercetin, and isoquercetin as the major constituents. The inhibitory mechanism of PRP against porcine pancreatic α-amylase (PPA) was systematically investigated using inhibition kinetics, fluorescence spectroscopy, circular dichroism (CD) spectroscopy, molecular docking, and molecular dynamics (MD) simulations. PRP exhibited strong inhibitory activity (IC50=3.15±0.16 mg/mL) and acted as a mixed-type inhibitor, whereas the major monomers showed distinct inhibition behaviors. Fluorescence and CD analyses indicated that PRP and its monomers altered the microenvironment around tryptophan and tyrosine residues, induced secondary-structure rearrangement in PPA, and reduced enzymatic activity. MD simulations confirmed the stability of the polyphenol-PPA complexes. Binding free-energy calculations showed that quercetin had the most favorable overall binding affinity and complex stability among the tested monomers, driven mainly by hydrogen bonding and hydrophobic interactions. These findings clarify the α-amylase inhibitory mechanisms of PRP and its key constituents and provide a theoretical basis for developing natural hypoglycemic functional ingredients and plant-derived α-amylase inhibitors.