Novel cottonseed-derived antioxidant peptides with dual mechanisms: direct free radical scavenging and Nrf2/Keap1 activation for alleviating oxidative stress

Cottonseed protein, a major agricultural by-product, represents a promising and sustainable resource for bioactive peptides discovery. In this study, four novel peptides (P1: EGPGCPMMER, P2: ETEDACR, P3: LLLNCKADK and P4: SSRFCTLPQQ) were isolated from cottonseed protein hydrolysate using multi-steps separation. All four peptides demonstrated dual antioxidant activities: directly scavenging Reactive Oxygen Species (ROS) and activating the Kelch-like ECH-associated protein 1/Nuclear factor erythroid 2-related factor 2 (Keap1/Nrf2) signaling pathway, albeit with varying efficacy. Radical scavenging assays confirmed their potent activity, with 1,1-diphenyl-2-picrylhydrazyl (DPPH) EC 50 values of 0.18 ± 0.02, 0.45 ± 0.05, 0.89 ± 0.07, and 0.43 ± 0.03 mg/mL for P1 to P4, respectively. EGPGCPMMER (P1) exhibited the most potent direct scavenging ability, with efficacy comparable to glutathione. In cell assays, pretreatment with all four peptides activated the Keap1/Nrf2 pathway, as evidenced by promoted Nrf2 nuclear translocation in Hepatocellular carcinoma (HepG2) cells, leading to an enhanced cellular antioxidant defense system. While P1 excelled in direct scavenging, the P3 group demonstrated the most significant reduction in malondialdehyde (MDA) levels, indicating a superior capacity to mitigate lipid peroxidation. Molecular docking analysis revealed stable interactions between all four peptides and the Kelch domain of Keap1 for key residues such as Arg380, Arg415, and Tyr334. Molecular dynamics simulations further demonstrated that all four peptides formed stable complexes with Keap1. Overall, these newly identified cottonseed-derived peptides exhibit complementary antioxidant functions, underscoring their potential as natural agents against oxidative stress in various applications. • Four novel antioxidant peptides (EGPGCPMMER, ETEDACR, LLLNCKADK, SSRFCTLPQQ) identified from cottonseed protein. • EGPGCPMMER showed glutathione-comparable DPPH scavenging with EC 50 of 0.18 mg/mL. • Peptides bind Keap1 Kelch domain, disrupt Keap1-Nrf2 interaction, and drive Nrf2 nuclear translocation. • Peptides protect HepG 2 cells from H 2 O 2 -induced oxidative stress damage.