## 3 Discussion  
The structural characteristics of APs provide guides for the evaluation of food-derived proteins as potential precursors of APs and predict the possible release of APs from various proteins while using appropriate proteases [1].  
Acidic and basic amino acid residues play critical roles in the metal ion chelating activity, which is related to the carboxyl and amino groups in their side chains [56]. The carboxyl residues of acidic amino acids are deprotonated for rendering the complex formation with metal ions, and the amino nitrogen loses its proton nitrogen to allow for unshared pairs of electrons of nitrogen to bind with metal ions [53]. [PERSON] et al. reported similar results, who found that the basic (Arg) and acidic (Asp and Glu) amino acid residues in the sequences of NTDGSTDYGILQINSR and LDEPDPLI were critical to their antioxidant activities [57]. [PERSON] et al. found that Glu residue is an effective cation chelator that forms complexes with calcium and zinc ions and it may contribute to the antioxidant activity [58]. Therefore, the Glu residue in RSHP-B, RSHP-C and RSHP-D, and Arg residue in RSHP-A might be favorable for their antioxidant activities.  
[PERSON] et al. reported that the pyrrolidine ring of Pro residue can interact with the secondary structure of the peptide, thereby increasing the flexibility, and it is also capable of quenching singlet oxygen due to its low ionization potential [59]. [PERSON] and [PERSON] reported that Pro residue played an important role in the activity of AP that was purified from _Saccharomyces cerevisiae_ protein hydrolysate [60]. Therefore, the Pro residue in the amino acid sequences of RSHP-A and RSHP-B should enhance their radical-scavenging activities.  
Aromatic amino acid residues, such as Phe, Tyr, His, and Trp, and hydrophobic amino acid residues, including Ala, Val, and Leu, have been reported as critical to the activities of APs [1]. The results from [PERSON] et al. indicated that hydrophobic amino acid residues (e.g., Val, Ile, and Leu) and aromatic amino acid residues (Phe, His, Tyr, and Trp) can enhance the radical-scavenging abilities of APs from Chinese cherry seeds [61]. Therefore, the presence of aromatic and hydrophobic amino acid residues in RSHP-A (Val), RSHP-B (Ile and His), RSHP-C (Leu), and RSHP-D (Ile) should have a positive impact on their radical-scavenging and lipid peroxidation inhibitory activities.  
In addition, the activities of APs are dependent on their molecular size. Short peptides with 2-10 amino acid residues have stronger radical-scavenging and lipid peroxidation inhibition activities than their parent native proteins or long-chain peptides [1]. RSHP-A-D exhibited good antioxidant activities, which suggested that they could more effectively and easily interact with free radicals and inhibit the propagation cycles of lipid peroxidation [33]. However, RSHP-B showed the strongest radical scavenging activity (DPPPH\(\bullet\), HO\(\bullet\), and ABTS\({}^{+}\bullet\)), reducing power, and lipid peroxidation inhibition activities, RSHP-A showed the strongest O\({}_{2}^{-}\bullet\) scavenging activity and RSHP-C showed the highest Fe\({}^{2+}\)-chelating ability among the four isolated APs. There was no consistent trend in the  
Figure 9: Lipid peroxidation inhibition abilities of the four isolated APs (RSHP-A\(\sim\)D) from protein hydrolysate of red stingray (_D. akajei_) cartilages. All data are presented as mean \(\pm\) SD (\(n\) = 3).  
different tested assays. Therefore, more detailed study should be performed to clarify the relationship between the activity and structure of the four isolated APs (RSHP-A-D).