Antioxidant activities of three enzymatic hydrolysates from Stichopus japonicus on HaCaT cells in vitro and zebrafish in vivo

To explore the antioxidant activity of enzymatic hydrolysates of S. japonicus from Dalian and preliminarily elucidate their mechanisms of action both in vitro and in vivo. Samples were hydrolysed using alcalase, protamex, and neutrase. 2,2-diphenyl-1-(2,4,6-trinitrophenyl)hydrazyl (DPPH) and 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) radical scavenging assays showed that the alcalase hydrolysate had the highest antioxidant activity, with IC₅₀ values of 4.233 ± 0.067 mg/mL (DPPH) and 1.188 ± 0.066 mg/mL (ABTS). Further cell experiments indicated that the alcalase hydrolysate effectively inhibited intracellular reactive oxygen species (ROS), with a fluorescence rate of 4.29% ± 1.98%, and modulated antioxidant-related enzymes (superoxide dismutase: SOD, glutathione peroxidase: GSH-PX, malondialdehyde: MDA). The obtained optimal enzymatic hydrolysate was analysed by LC-MS/MS. The fluorescence rate of zebrafish (AAPH: 100%) decreased to 29.24% ± 1.10% (acridine orange: AO) and 56.71% ± 0.02% (ROS), with no observed embryonic toxicity. This study provides a foundation for the development of natural antioxidants.