Structure, stability, and antioxidative mechanism of novel antioxidant peptides from Agaricus bisporus prepared by three-step purification.

Three novel antioxidant peptides (FPEF, LGFD, MGFGL, molecular weight <1000 Da) were isolated from Agaricus bisporus via sequential DA201-C macroporous resin, Sephadex G-100 gel filtration, and RP-HPLC. Molecular docking revealed strong Keap1 binding (energies: -8.2 to -8.7 kcal/mol) via hydrogen bonds and π-alkyl interactions. Cellular assays confirmed that all peptides alleviated H₂O₂-induced oxidative damage in HepG2 cells by reducing ROS/MDA levels, enhancing SOD/CAT/GSH-Px activities, and upregulating Nrf2-target genes (HO-1, NQO-1). Structural analysis indicated that FPEF's stability arose from balanced secondary structure and flexibility, rather than high ordered-structure content. FPEF exhibited superior stability under heat (100 °C), pH (3-11), and simulated gastrointestinal digestion (>90% retention), while MGFGL showed the highest ABTS radical scavenging activity (98.62% at 1.5 mg/mL). Collectively, FPEF shows promise as a natural antioxidant for functional food and nutricosmetic applications.