COX-2 Inhibition by Bioactive Peptides from Peanut Worm (Siphonosoma australe) Collagen Through in vitro Digestion Simulation

Research background. Chronic, unregulated inflammation is a crucial factor in the development of numerous diseases and is closely linked to the increased expression of cyclooxygenase-2 (COX-2). While various bioactive peptides from marine organisms have shown COX-2 inhibitory effects, peptides derived from the collagen of the peanut worm (Siphonosoma australe) have not yet been demonstrated. The aim of this study is to investigate the potential COX-2 inhibitory activity of peanut worm collagen by simulated digestion in vitro with pepsin-pancreatin followed by molecular docking. Experimental approach. During simulated in vitro digestion, commercial pepsin (at pH=3) and pancreatin (at pH=7.5) were applied for 240 min at 37 °C to evaluate the degree of hydrolysis, peptide concentration and COX-2 inhibitory activity. The samples with the most significant COX-2 inhibitory activity were then separated into fractions and identified. Results and conclusions. The 210-minute simulated digestion in vitro showed the highest COX-2 inhibitory activity (64.31 ). This result was confirmed by the increased degree of hydrolysis (DH) and peptide concentrations observed during the simulated in vitro digestion. The peptide fraction of <1 kDa had the highest inhibitory activity (89.05 ) and was subsequently subjected to sequencing analysis. Three novel peptides, ADIAGQAAQVLR, LNNEITTLR and VGTVEK, were identified and confirmed to contain crucial amino acids and therefore verified as COX-2 inhibitors. VGTVEK has the most potent interaction, as shown by the lowest binding energy (−4.41 kcal/mol). The molecular docking revealed that VGTVEK (631.35 Da) binds to the active site of COX-2 and forms hydrogen bonds with Gln178, Leu338, Ser339, Tyr371, Ile503, Phe504, Val509 and Ser516 and hydrophobic interactions with Met99, Val102, Val330, Ile331, Tyr334, Val335, Leu345, Trp373, Leu517 and Leu520. Other biological activities of the produced peptides included ACE inhibitors, dipeptidyl peptidase-IV (DPP-IV) inhibitors and α-glucosidase inhibitors. After toxicity prediction, the peptides were classified as non-toxic. Novelty and scientific contribution. The study found that peptides derived from peanut worm collagen have the potential to be novel, natural agents for anti-inflammatory therapy. Their broader application in functional foods, nutraceuticals and pharmaceuticals could provide new options for people suffering from inflammation and support both treatment and maintenance of overall health. © 2025, University of Zagreb. All rights reserved.