Novel antioxidant peptides targeting the Keap1-Nrf2 pathway discovered from Ginkgo biloba seeds protein hydrolysate: Combining virtual screening, quantum chemical calculation, and zebrafish models in vivo validation.

Abstract

The development of safe, edible, and highly stable food-derived natural antioxidant peptides holds significant practical value for functional foods and dietary health management. Ginkgo biloba seeds (GBS), as a food resource rich in protein, are an ideal raw material for the preparation of food-grade antioxidant peptides. This study uses papain to hydrolyze Ginkgo biloba seeds protein (GBSP). Two novel peptides (LTGMAFR and IGINGFGR, LR-7 and IR-8) were first identified via peptidomics combined with computational approaches. Molecular docking revealed that the selected peptides all exhibit strong binding affinity to Keap1 (-7.8 to -8.2 kcal/mol), surpassing the positive ligand (9-mer Nrf2, -7.2 kcal/mol). Molecular dynamics simulations validated the complex's dynamic stability. Molecular orbital calculations and Fukui function analysis confirm that these two peptides exhibit excellent electron-donating properties. Furthermore, IR-8 exhibits stronger DPPH and ABTS scavenging activities than LR-7. In an H2O2-induced oxidative stress model in zebrafish, the target peptides reduced ROS and MDA levels in zebrafish larvae, upregulated antioxidant enzyme levels and the mRNA levels of genes downstream of the Nrf2 pathway, and effectively alleviated H2O2-induced oxidative damage. Western blot results further confirmed that H2O2-induced oxidative stress reduces nuclear Nrf2 protein levels and the Nrf2 nuclear-to-cytoplasmic ratio, while peptide intervention effectively restores Nrf2 nuclear accumulation. The Nrf2-specific inhibitor ML385 experiments further confirmed that the antioxidant activity of the target peptides depends on the activation of the Nrf2 pathway. This work elucidates the antioxidant mechanism of GBSP targeting the Keap1-Nrf2 pathway and provides an efficient multi-method strategy for natural antioxidant screening.