## 4 Discussion
sigilitatus_) protein hydrolysates also demonstrated anti-inflammatory properties [34]. The study detailed the efficacy of SEC isolated peptide fractions to inhibit lipoxygenase and cyclooxygenase-2 activity with IC\({}_{50}\) values of 0.13 \(\mathrm{\SIUnitSymbolMicro g}\)/mL and 0.26 \(\mathrm{\SIUnitSymbolMicro g}\)/mL, respectively. The authors also noted that better anti-inflammatory activity was observed for the peptide fractions, as compared to the hydrolysates [34]. Cationic bioactive peptides, particularly, were shown to be useful therapeutic agents against human diseases, when exhibiting high bioactive potency and no cytotoxic activity against mammalian cells. Structure-function analyses revealed that the amphipathic structure, positive charge, and hydrophobic properties help facilitate their interaction with cell walls and inserting past the membrane to the site of action [35]. The de novo sequencing revealed that the cationic peptides fractioned from CPHD consisted of peptides with these characteristics and were predicted as hypotensive peptides (Supplementary Materials Table S1), which might have played a role in the superior ACE inhibiting activity exhibited by this fraction (Table 3). Additionally, most identified sequences contain tri-peptides with reported activity, including antioxidant, antimicrobial, anti-glycemic, and immunomodulatory properties [23]. This would explain the multifunctional ability of the cationic peptide fraction observed (i.e., ACE, \(\alpha\)-glucosidase, \(\alpha\)-amylase, and NF-\(\kappa\)B inhibition) in this study. Among the peptides identified, the molecular docking results revealed that the pentapeptides YKPRP, PHGAP, and VGPPQ had binding properties similar to those of commercial ACE, inhibiting drug captopril with different potentialities (Table 3). All three peptides had binding energies lower than captopril, but similar to that of Lisinopril (-39.96 kJ/mol) [36]. The ability of peptides to form multiple hydrogen bonds and hydrophobic interactions is a key characteristic of peptide-induced inhibition, through stabilization of the non-catalytic enzyme-peptide complex structure [37]. All three peptides interacted with ACE active sites mainly via hydrogen bonding and van der Waals interactions. However, peptides PHGAP and VGPPQ showed more interaction with S1 and S2 active site residues. In addition to hydrogen and hydrophobic bonds, the interaction between the Zn\({}^{2+}\) ion and peptides plays a significant role in ACE inhibition [37]. Both pentapeptides PHGAP and VGPPQ also exhibited interaction with ZN\({}^{2+}\) atoms, binding to ACE in an analogous fashion to that observed in captopril (Figure 4), indicating that they would be potent ACE inhibitors. Nevertheless, YKPR is a known neuropeptide, an analogue of the natural immunomodulatory tetrapeptide tufsin (TKPR), and might be active towards hormonal and neurological functions. Similar peptides are reported as neuromodulators isolated from various insect species and other crustaceans, such as lobster and crabs [38]. For example, the penta-peptide RXLPT (proctolin) is the first insect neuropeptide sequenced and chemically characterized. However, a study demonstrated that proctolin, although showed a high degree of binding affinity towards ACE, was resistant to hydrolysis in vivo. [39]. Peptides, like proctolin, which have a prolyl residue near the C-terminal are known to be resistant to hydrolysis by mammalian ACE, due to the structural constraints imposed by the presence of imino acids [39]. This is likely why YKPRP, in this study, demonstrated a lower binding affinity than other peptides, yet less interaction with active site residues. Whereas peptides PHGAP and VGPPQ contained tripeptides GAP and GPP, respectively. These tripeptides are present in many ACE-inhibiting, antioxidant, DPP-IV-inhibiting, and antimicrobial food-derived peptide sequences (Supplementary Materials Table S1) [23]. Additionally, potent ACE inhibitors include hydrophobic amino acid residues near the C-terminal.