Abstract: This study utilized peptidomics and bioinformatics to investigate the structure-hypocholesterolemic activity relationships of oyster peptides (OPs). Ion-exchange chromatography separated OP into three fractions (OP-1, OP-2, and OP-3). OP-2 showed the strongest inhibitory effect on pancreatic lipase (PL) (76.16%) and cholesterol esterase (CE) (66.43%), reducing total cholesterol (TC) in a concentration-dependent manner. Analysis by liquid chromatography-mass spectrometry (LC-MS) indicated that active peptides (< 3 kDa) were rich in hydrophobic (Leu, Ala) and acidic (Asp, Glu) residues. Bioinformatics and molecular docking identified key peptides (LPFQ, LNFP) with dual-enzyme inhibitory activity. Furthermore, LNFP demonstrated a significant cholesterol-lowering effect in a HepG2 cell model (seeded at 5.0 × 105 cells/well in six-well plates) by reducing intracellular cholesterol accumulation and enhancing low-density lipoprotein (LDL) uptake. At 50 μmol/L, its cholesterol lowering effect was similar to that of simvastatin at 10 μmol/L. These findings highlight the structural motifs crucial for dual-enzyme inhibition and hepatic cholesterol regulation, emphasizing the potential of OPs as a natural cholesterol-lowering agent and promising candidate for future therapeutic development. Further studies on their gastrointestinal stability and in vivo efficacy are warranted to fully assess their potential for dietary intervention.

Key words: cholesterol-lowering peptides; molecular mechanisms; cardiovascular health; HepG2; bioinformatics