In vitro and in silico methods to assess modulation of digestive proteases by food-derived phenolics

The interaction of food components with digestive enzymes can modify the digestion process and affect human health. Although polyphenols (PPs) have a beneficial impact, they are often referred to as anti-nutritional factors, having been reported to inhibit proteolytic digestive enzymes. However, studies on this topic are often contradictory, highlighting the necessity of using standardized methods. In this study, the effects of a selected food-derived PPS were assessed "in vitro" on pepsin, trypsin, and chymotrypsin using albumin, gluten, and hemoglobin as substrates. Results show that PPs may affect proteolytic activity in opposite ways, depending on the substrate/enzyme combination. Therefore, a selection of PPs was further investigated via “in silico” approaches to assess a possible structure/activity relationship on the ovalbumin/chymotrypsin system. After searching for putative binding pockets in both the protein substrate and the enzyme, molecular docking and dynamics allowed to point out the possible capability of PPs to interact in stable fashion with either ovalbumin or chymotrypsin. Interestingly, the most potent proteolytic digestion inhibitors according to the “in vitro” results was found to correspond to those best interacting PP within the enzyme binding site. Conversely, digestion-promoting PPs were best interacting with the substrate’s binding pockets. These studies indicate that structural features have a role in eliciting specific effects of PPs binding affinity, orientation, and geometry. The evidence gathered here suggests the possibility of considering some PPs as “digestion-promoting agents” in the formulation of functional foods.