Valorization of crocodile head for anti-inflammatory peptides: In silico screening and cellular validation

In this study, two novel anti-inflammatory peptides were identified from the hydrolysates of crocodile heads using computer-assisted techniques and in vitro cellular experiments. Their interaction mechanisms were elucidated through molecular docking and molecular dynamics simulations. A total of 67 peptides were identified via LC-MS/MS, with AKLDLEEVIK and DFLDLPSIER emerging as promising candidates for anti-inflammatory activity. Results of in vitro cellular experiments demonstrated that these peptides significantly inhibited LPS-stimulated macrophage hyperactivation, resulting in reduced release of nitric oxide (NO) and pro-inflammatory cytokines, i.e., TNF-α, IL-1β, and IL-6. Molecular dynamics (MD) simulations and docking analyses revealed that AKLDLEEVIK and DFLDLPSIER displayed high affinity for the TLR4-MD2 complex, with stable and tight interactions. Key residues Arg90, Ser118, Cys133, and Arg264 were identified as critical for the recognition and interaction between the peptides and TLR4-MD2. This study provides a theoretical foundation for the development of anti-inflammatory functional foods derived from crocodile heads.