Antioxidant peptides of sheep placental extract digestion product: In vitro and in silico study

This study systematically investigated the gastrointestinal digestion-driven release of antioxidant peptides from sheep placental extract (SPE) through in vitro simulated digestion, coupled with structural characterization and exploration of molecular mechanisms. The hydrolysis process significantly reduced the molecular weight (Mw) of SPE, as evidenced by SDS-PAGE, while inducing protein conformational unfolding and enhancing structural disorder. Ultrafiltration of the gastrointestinal digest (SPGI) yielded three fractions (<3 kDa, 3-10 kDa, and >10 kDa), with the <3 kDa fraction exhibiting the most potent ABTS radical scavenging capacity. Nano-HPLC-MS/MS analysis identified a novel antioxidant peptide, HFFNPVPL, within the <3 kDa fraction. Molecular docking revealed its strong binding affinity to Keap1, targeting critical residues (Tyr334, Arg415, Gln530) through hydrogen bonding and hydrophobic interactions. Molecular dynamics simulations further elucidated the stabilizing roles of Arg380, Asn414, and Tyr572 in the Keap1-peptide complex. Suggest that the peptide has the potential to competitively inhibit Keap1-Nrf2 binding, which may in turn activate antioxidant-responsive pathways. These findings demonstrate that gastrointestinal digestion liberates low-Mw peptides (<3 kDa) as the primary bioactive components in SPE, with HFFNPVPL serving as a Keap1-targeted antioxidant agent. This work provides mechanistic insights into the antioxidant potential of sheep placenta-derived peptides, offering promising candidates for the development of natural therapeutics against oxidative stress-related pathologies.