Structure-activity relationship of (poly)phenols on human salivary α-amylase: A stepwise in vitro/in silico analysis of interfacial inhibition

Dietary (poly)phenols (PPs) can reduce the risk of developing type 2 diabetes by inhibiting digestive enzymes. As starchy food digestion begins with human salivary α-amylases (HSA) acting on solid food surfaces, a process previously overlooked, understanding this initial inhibition is critical. Our findings revealed that kaempferol (KA) was highly effective in inhibiting HSA, while others like malvidin-3-glucoside (M3G) and ferulic acid (FA) showed varying degrees of efficacy. Their distinct inhibition kinetic modes, either competitive or mixed, could be interpreted by in silico molecular docking, which visualized interactions with both active and non-active site amino acid residues of HSA. Additionally, PP binding induced conformational changes in HSA, evidenced by a reduced enzyme intrinsic fluorescence. Further, saturation transfer difference – nuclear magnetic resonance (STD-NMR) revealed close interactions between specific parts of the PP's structure (e.g., aromatic rings and hydroxyl groups) and the enzyme surface, highlighting the key role of the structure-activity relationship. The binding epitopes aligned well with predicted binding poses, bridging experimental and computational approaches for a comprehensive understanding.