## 3 Results
### Identification of Angiotensin-Converting Enzyme (ACE) Inhibitory Activity
As they possesses four residues in their structures, the three peptides WLQL (IC\({}_{50}\): 16.87 \(\upmu\)M), LPLF (IC\({}_{50}\): 300.08 \(\upmu\)M) and LPSW (IC\({}_{50}\): 20.80 \(\upmu\)M) from the current study seem to exhibit high ACE inhibitory activity, as compared to ELPF (IC\({}_{50}\): 4.24 mM) derived from chicken egg white [10], HLHT (IC\({}_{50}\): 458 \(\upmu\)M) obtained from pearl oyster (_Pinctada tucata martensii_) protein hydrolyses (HLHT and GWA with the IC\({}_{50}\) values of 458.06 \(\pm\) 3.24 \(\upmu\)M and 109.25 \(\pm\) 1.45 \(\upmu\)M, respectively) [27], and KYKA (5.63 \(\upmu\)M) from spent hen muscle proteins [28]. Furthermore, other ACEI peptides were also derived from various natural sources, such as marine macroalga _Ulta intestinalis_ hydrolyses (HLHT and GWA with the IC\({}_{50}\) values of 458.06 \(\pm\) 3.24 \(\upmu\)M and 109.25 \(\pm\) 1.45 \(\upmu\)M, respectively), MELVLR (IC\({}_{50}\): 236.85 \(\upmu\)M) from marine hydrolyses [29], and naked oat globulin hydrolyses (SSYYPFK, IC\({}_{50}\): 91.82 \(\upmu\)M) [3]. Furthermore, the appearance of a branched-chain aliphatic amino acid (isoleucine, leucine, or valine) at the N-terminal end of peptides can improve ACE inhibitory activity [20,30]. Hydrophobic amino acids can ameliorate the efficiency of ACEI peptides [20,22]. These amino acid characteristics can be identified in almost all of these peptides (WLQL, LPSW, LPLF, VPGALAL, and LVGLPL), and all of them showed prominent ACE inhibitory effects, except LVGLPL. In the literature, it was reported that a Leu (L) residue at the N-terminal of ACEI peptides was determined in egg white hydrolyses (LKYKA, IC\({}_{50}\): 0.09 \(\upmu\)M [11] and LPR, IC\({}_{50}\): 1.30 mM [10]) and spent hen muscle (LKYKA, IC\({}_{50}\): 0.054 \(\upmu\)M and LKY, IC\({}_{50}\): 1.91 \(\upmu\)M) [28], in addition to our current study (LPSW, IC\({}_{50}\): 20.80 \(\upmu\)M; LPLF, IC\({}_{50}\): 300.08 \(\upmu\)M; and LVGLPL, IC\({}_{50}\): \(>\)2000 \(\upmu\)M). Interestingly, LPSW has tryptophan at its C-terminal, leucine at its N-terminal, and proline in its sequence. It matches the hypothesis related to potential ACEI peptides. Moreover, the results of in vitro experiments have also proven its effects on ACE inhibitory activity (Figure A1 and Table 1). In our previous study, other peptides that contained LPSW in their sequence, such as AKLPSW, also played a role in ACEI influence with an IC\({}_{50}\) value of 15.3 \(\upmu\)M [17], which indicates higher ACEI activity than LPSW (IC\({}_{50}\) value of 20.80 \(\upmu\)M). With an aromatic residue also at the C terminal, GVGSPY derived from pearl oyster hydrolyses that contain Tyr (\(\upgamma\)) displayed great ACEI activity with an IC\({}_{50}\) value of 10 \(\upmu\)M [21]. In a similar manner, VRY and VRY from spent hen muscle hydrolyses showed excellent ACEI activity with an IC\({}_{50}\) value of 13.19 \(\upmu\)M and 1.91 \(\upmu\)M, respectively [28]. Currently, various publications have reported dual or multiple bioactivities in the same individual peptide, leading to the growing trend of discovering potentially novel peptides. This result can create opportunities for treating several diseases with one inhibitor. This will motivate developments to decrease the cost of care, as well as the anxiety of patients surrounding the consumption of too many kinds over a long treatment period. Recently, dipeptidyl peptidase-IV (DPP-IV) inhibitors were reported to possess the ability to inhibit ACE because they share a common metabolic pathway and features [19,31]. For instance, ten peptides (CF, KM, ELPF, AM, ADHPF, LPR, PR, FR, PRM, and GR) derived from chicken egg white ovalbumin have shown their ability to inhibit ACE and DPP-IV enzymes simultaneously; however, their efficacy is still moderate according to their IC\({}_{50}\) values, which range from 1.82 to 5.47 mM and 1.43 to 9.92 mM [10].