Antioxidant Peptides from Hizikia fusiformis: A Study of the Preparation, Identification, Molecular Docking, and Cytoprotective Function of H2O2-Damaged A549 Cells by Regulating the Keap1/Nrf2 Pathway

Hijiki (Hizikia fusiformis) is a seaweed native to warm-temperate and subtropical regions that has a high edible value and economic value, with a production of about 2 × 10⁵ tons/year. Current research has clearly shown that the pharmacological activities of active ingredients from hijiki have covered a broad spectrum of areas, including antioxidant, hypoglycemic, antiviral, anticoagulant, anti-inflammatory, intestinal flora modulation, anti-aging, antineoplastic and antibacterial, and anti-Alzheimer's disease areas. However, no studies have reported on the production of antioxidant peptides from hijiki proteins. The objectives of this study were to optimize the preparation process and explore the cytoprotective function and mechanisms of antioxidant peptides from hijiki protein. The results indicated that papain is more suitable for hydrolyzing hijiki protein than pepsin, trypsin, alkaline protease, and neutral protease. Under the optimized parameters of an enzyme dosage of 3%, a material-liquid ratio of 1:30, and an enzyme digestion time of 5 h, hijiki hydrolysate with a high radical scavenging activity was generated. Using ultrafiltration and serial chromatographic methods, ten antioxidant oligopeptides were purified from the papain-prepared hydrolysate and identified as DGPD, TIPEE, TYRPG, YTPAP, MPW, YPSKPT, YGALT, YTLLQ, FGYGP, and FGYPA with molecular weights of 402.35, 587.61, 592.64, 547.60, 532.53, 691.77, 523.57, 636.73, 539.58, and 553.60 Da, respectively. Among them, tripeptide MPW could regulate the Keap1/Nrf2 pathway to significantly ameliorate H₂O₂-induced oxidative damage of A549 cells by increasing cell viability and antioxidant enzyme (SOD, CAT, and GSH-Px) activity, decreasing ROS and MDA levels, and reducing the apoptosis rate. Molecular docking experiments show that HFP5 (MPW) exerts its inhibitory effect mainly through hydrogen bonds and hydrophobic interactions with the Kelch domain of the Keap1 protein, eventually facilitating the translocation of Nrf2 to the nucleus. Therefore, antioxidant peptides from hijiki can be applied to develop algae-derived health foods for treating diseases associated with oxidative stress.