Preparation of Antioxidant Peptides from Hippocampus Abdominalis via High Hydrostatic Pressure-Assisted Enzymolysis: Evaluation of Their Protective Effects Against Male Reproductive Dysfunction in Mice

Hippocampus abdominalis (H. abdominalis) is an important marine teleost fish with diverse functional activities. Accordingly, the present research was designed to isolate and prepare innovative antioxidant peptides derived from H. abdominalis and to assess the protective and restorative impacts of its protein hydrolysates (HAPH) against reproductive injury caused by triptolide (TP) in male mice. Notably, pepsin was ultimately identified as the optimal protease among seven screened proteases. High hydrostatic pressure (HHP) pretreatment significantly increased peptide yield and antioxidant activity of pepsin hydrolysates. Subsequently, the optimal enzymatic hydrolysis conditions were established as E/S of 3% (w/w), hydrolysis time of 3 h, S/L of 1:25 (w/v), HHP pressure of 200 MPa, and 9 min of HHP time using single-factor experiments and response surface methodology. The peptide fraction with a molecular weight below 3 kDa (HAPH-1) exhibited stronger antioxidant activity and excellent stability against pH, temperature, and simulated gastrointestinal digestion. Furthermore, animal experiments revealed that HAPH-1 alleviated triptolide-induced impaired spermatogenesis and testicular tissue damage in mice, and the underlying mechanisms involve the regulation of the Keap1-Nrf2 pathway and the Bcl-2/Bax/caspase-3 apoptotic signaling pathway. Finally, through LC-MS/MS analysis, molecular docking, and in vitro activity validation, three novel peptides with high antioxidant activity (GPAPWGF, FPALLF, and LPLPLF) were successfully screened. Collectively, this study provides new insights into the high-value utilization of H. abdominalis and the development of novel antioxidant functional components.