Molecular interactions between polyphenols and porcine α-amylase: An inhibition study on starch granules probed by kinetic, spectroscopic, calorimetric and in silico techniques

Abstract

Inhibition of α-amylase activity to limit the rise in postprandial glucose is one of the most notable effects of dietary plant (poly)phenols. The rate and extent of inhibition depend on many factors, including the structure of (poly)phenols and the substrate used for the inhibition study. While soluble/gelatinised starch is widely used as a substrate for studying inhibition mechanisms, the formation of (poly)phenol-amylose complexes can reduce susceptibility to α-amylase catalysis. Therefore, the extent of inhibition may not truly represent the inhibition of α-amylase by (poly)phenols. To elucidate mechanisms of interactions between a (poly)phenol and the enzyme, this study compared the inhibition of porcine pancreatic α-amylase (PPA) by three differently structured (poly)phenols: p-coumaric acid, quercetin, and cyanidin-3-glucoside, using solid maize starch as a substrate, that limits the formation of starch-(poly)phenol complexes due to the compact structure of starch granules. The inhibition kinetics were investigated and supported by complementary techniques: in silico analysis (molecular docking and molecular dynamic simulation), fluorescence quenching, circular dichroism and differential scanning calorimetry. Quercetin exhibited the strongest inhibition, followed by cyanidin-3-glucoside and p-coumaric acid, with apparent modes of mixed, mixed, and competitive inhibition, respectively. The specific binding of (poly)phenols to PPA involved hydrogen bonds and hydrophobic interactions, as indicated by in-silico studies. Multi-spectroscopic analysis of the PPA structure unveiled changes or peak shifts due to ligand binding, resulting in structural alterations. These findings were consistent with the kinetic data, emphasising the importance of the structural aspects of each individual (poly)phenol in relation to their interaction with PPA. The study articulated the in-depth mechanism of molecular interaction mechanism between (poly)phenols and enzymes and provided evidence that the structural alterations of enzymes are not a strong indicator of inhibition efficacy, but rather inhibition is due to the binding of (poly)phenols to the enzyme structure limiting the catalysis.