On the mechanism of small molecule peptides purified from Antheraea pernyi pupae in preventing cellular oxidative damage

Abstract Antheraea pernyi pupae, recognized as a novel food resource by the National Health Commission of China, are rich in easily digestible proteins and amino acids. In this study defatted Antheraea pernyi pupae powder was used as the raw material for the enzymatic preparation of crude polypeptides. The free radical scavenging activities of each component were evaluated using 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assays. The component exhibiting the strongest antioxidant activity was selected for structural identification. This component was then used to establish an oxidative damage model in Human hepatocellular carcinoma (HepG2) cells, induced by hydrogen peroxide (H 2 O 2 ), to investigate its mechanism of action in preventing oxidative stress. The results show that the antioxidant peptides PH1, PH2, and PH3, after isolation and purification, exhibited DPPH scavenging rates of 83.33%, 84.37% and 70.6%, respectively, and ABTS scavenging rates of 88.84%, 86.57% and 78.97%, respectively. Structural identification using liquid chromatography-tandem mass spectrometry (LC-MS/MS) revealed their amino-acid sequences as GSPDRPWAKPDDPQLM, HADPDDLGLGGHE, and LGNDVDDIKERVCWN. The molecular weights of the peptides were determined to be 1808.85, 1331.57, and 1272.63 Da, respectively. The results of cell experiments demonstrated that the antioxidant peptide components inhibited oxidative stress by modulating antioxidant enzyme levels. Immunoblotting analysis revealed that all three peptides activated the Kelch-like ECH-associated protein 1-Nuclear factor erythroid 2-related factor 2 (Keap1-Nrf2) signaling pathway, thereby suppressing oxidative stress and preventing cellular damage. Among these, alkaline protease digests exhibited superior effectiveness. The research results provide valuable insights into the antioxidant molecular mechanisms of Antheraea pernyi pupae peptides.