Cardiovascular disease is widely regarded in industrialized nations as one of the most pressing health concerns. Indeed, cardiovascular diseases are the leading cause of death globally, with hypertension emerging as the primary risk factor in their development [1]. The angiotensin-converting enzyme (ACE) breaks down active bradykinin, which is essential for blood pressure control, and converts inactive angiotensin I into active angiotensin II, resulting in arterial contractions and elevated blood pressure [2]. Hypertension is commonly managed with medication, in particular ACE inhibitors, which are widely accessible, cost-effective, and also recommended as an initial treatment for other prevalent chronic conditions such as heart failure with reduced ejection fraction and chronic kidney disease [3]. Lisinopril, trandolapril, ramipril, moexipril, and quinapril hydrochlorides are chemically synthesized antihypertensive drugs [4]. ACE inhibitors are associated with a modest risk of bradykinin-mediated angioedema, acute kidney injury (AKI), hyperkalemia, and chronic cough [3]. However, studies have demonstrated that, unlike synthetic ACE inhibitors, ACE inhibitory peptides derived from dietary sources can lower high blood pressure without causing these adverse effects [5, 6]. There is also increasing evidence suggesting a connection between oxidative stress and the onset of numerous diseases, including hypertension [7]. Utilizing antioxidant compounds may present a promising strategy for mitigating oxidative stress and the associated cardiovascular diseases.

Excessive reactive oxygen species (ROS) levels can cause irreversible oxidative stress or damage; one of the primary drivers of oxidative damage is the imbalance between the oxidant and antioxidant systems in the human body [8]. Under normal conditions, the antioxidant defense system plays an essential role in scavenging excess ROS to stabilize the configuration of highly reactive free radicals and prevent cellular damage. The primary antioxidant defense mechanism is enzymatic, involving enzymes such as superoxide dismutase, glutathione peroxidase, catalase, peroxidase, and glutathione reductase [9]. However, external factors such as environmental pollution, ultraviolet (UV) irradiation, and exposure to chemical reagents can disrupt antioxidant systems, resulting in severe oxidative stress [10]. Excessive ROS-mediated oxidative stress is strongly associated with cell death, deoxyribonucleic acid (DNA) fragmentation, and tissue oxidation [11], which contribute to various human diseases, including neurodegenerative [12], cardiovascular [13], chronic kidney diseases [14], and lung cancer [15]. Recently, some publications have reported that excessive oxidative stress can induce metabolic syndromes such as diabetes, obesity, and cardiovascular disease [16]. Therefore, developing therapeutic agents capable of regulating excessive ROS generation would reduce its incidence, improve patient outcomes across a broad spectrum of disorders, and be clinically beneficial [17].

Notably, there is growing recognition of the bioactivities of naturally derived peptides; bioactive peptides are potent antioxidant candidates with biotechnological applications in the nutraceutical industry [18]. Therefore, many researchers are focusing on the therapeutic development of novel peptides isolated from land or marine animals and plants. Therapeutically active peptides generally comprise 2–20 amino acids and are abundant in bioresources, especially in animals with high peptide content. Moreover, these peptides have been shown to possess antioxidant [19], antimicrobial [20], antiwrinkle [21], anticancer [22], and antihypertensive [23] activities; likewise, a study has also demonstrated these properties in low-molecular-weight peptides [24]. Study has reported that the bioactivity of peptides originates from their specific compositional and structural characteristics [25]. Over the past few decades, biotechnological study has characterized many bioactive peptides isolated from marine animals [26].

A study has reported the importance of marine-derived peptides since these can also provide anti-inflammatory, antibacterial, and antihypertensive effects [27]. Therefore, they have received significant attention in the food and functional food industries. Marine-derived functional ingredients and bioactive peptides obtained from enzymatically hydrolyzed fish exhibit various functionalities highlighting their potential applications in food technology for developing functional foods and nutraceuticals [28]. Marine-derived peptides were found to promote antihypertensive properties by inhibiting the ACE, which reduces the production of angiotensin II (Ang II), a vasoconstrictor, and increases nitric oxide and endothelin in HUVECs and vascular endothelial cells [29]. Hence, several marine bioactive peptides have been commercialized in food and functional food industries [30].

Stichopus japonicus (S. japonicus) is a teleost of the genus Stichopus and is mainly found in East Asian countries [31]. S. japonicus is a commercially valuable species that functions as a seafood and critical raw material in traditional medicine [32]. The body of S. japonicus consists mainly of collagen and mucopolysaccharides that possess nutritional and biological activities, such as lipid metabolism and antioxidant, antihypertensive, anticancer, antifatigue, and regenerative capacities [33]. In our previous study, $\alpha$-chymotrypsin-assisted hydrolysis highly increased the antioxidant ability and provided protective effects against hydrogen peroxide-induced oxidative damages in vitro and in vivo [34]. The biological activity of peptides highly affects the peptide structure, amino acid sequence, and composition of their position in the structure. Therefore, this present study investigated the antioxidant and antihypertensive activities of peptides from S. japonicus in relation to their structure, constituent amino acids, and amino acid positions. Furthermore, we suggest that the identification and biological evaluation of these peptides from S. japonicus could contribute to developing the food and functional food industries.

Marine animal-derived secondary metabolites have been proven to possess highly effective biological activities. Fish have relatively high protein content ratios compared to other nutritional components, and several bioactive peptides from these sources have been proven beneficial in human diseases. Fish-derived low-molecular-weight bioactive peptides have been recognized as functional ingredients that exhibit various biological properties, such as antioxidant, antifatigue, and immunoregulatory activities [38, 39]. Thus, extensive research has been conducted to develop efficient methods for isolating and identifying novel lead compounds with versatile uses in functional food and pharmaceutical industries. In this study, S. japonicus samples were enzymatically hydrolyzed with $\alpha$-chymotrypsin, resulting in the isolation of nine bioactive peptides. These peptides were characterized using liquid chromatography-mass spectrometry (LC–MS) sequencing, and their potential antioxidant and antihypertensive activities were evaluated using free-radical scavenging and ACE inhibitory assays. Peptides from S. japonicus significantly stabilize free radicals and inhibit the activity of the ACE. Previous study has investigated the correlation between potential antioxidant activity, peptide composition, and structural properties. Zou et al. (2016) [40] established a relationship between the quantitative structure of peptides and their antioxidant activity, whereby the amino acid composition and sequence play crucial roles in determining the potency of antioxidant activity. Previous study has reported that specific hydrophobic amino acids (valine, histidine, phenylalanine, proline, glycine, lysine, and isoleucine) facilitate the entry of antioxidant peptides into target organs through increased hydrophobicity, allowing antioxidant peptides to scavenge free radicals in cells [41]. A previous study demonstrated that exogenous proline effectively increases antioxidant activity by inhibiting H₂O₂ diffusion in cells [42]. In addition, tyrosine and tryptophan residues increase the lipid density of the cellular membranes, conferring protection against oxidative damage [43]. Previous research has also indicated that specific amino acids such as cysteine, glycine, lysine, tryptophan, and phenylalanine exhibit strong antioxidant activities by donating electrons to free radicals [44]. In negatively charged peptides, aromatic (histidine, phenylalanine, tryptophan, and tyrosine) and hydrophobic amino acids, including alanine, valine, leucine, isoleucine, proline, and tryptophan, contribute to strong antioxidant activity by regulating the catalytic activity of antioxidant enzymes [45]. FPTY exhibited the strongest hydroxyl radical scavenging activity in chemical assays among the purified peptide sequences. We propose that this antioxidant activity is primarily due to aromatic amino acids such as phenylalanine and tyrosine, as well as the hydrophobic amino acid proline. Regarding antihypertensive ACE inhibition, the proline and tyrosine C-terminal effectively inhibit and increase hydrophobic interactions at the ACE catalytic site [46]. Ding et al. (2023) [47] also reported that the frequency of the N- and C-terminal hydrophobic amino acids strongly affects ACE inhibition. Among the ACE inhibitory peptides, leucine, valine, isoleucine, alanine, glycine, tyrosine, and phenylalanine showed a high-frequency rate in the N-terminal amino acid residue, while proline, tyrosine, phenylalanine, isoleucine, and leucine showed a high-frequency rate in the C-terminal amino acid residue [48]. Earlier publications reported that $\alpha$-chymotrypsin cleaves the peptide bonds adjacent to aromatic ring amino acids, such as phenylalanine, tryptophan, and tyrosine [49]. This processing likely increases the concentration of peptides with aromatic rings at both the N- and C-terminal, thereby enhancing ACE inhibitory activity. Our findings indicate that the selective cleavage of these peptide bonds by $\alpha$-chymotrypsin significantly contributes to the increased ACE inhibition by elevating the levels of aromatic ring peptides. Notably, the purified FPTY peptide, which exhibits the highest ACE inhibitory activity in our chemical assays, contains phenylalanine at the N-terminus and tyrosine at the C-terminus. This specific arrangement of aromatic amino acids is crucial, as it enhances the peptide’s interaction with the ACE enzyme, leading to improved inhibitory efficacy. Collectively, we propose that FPTY is a promising candidate for an ACE inhibitory peptide derived from S. japonicus.

Next, we performed an in silico molecular docking study to verify the binding affinity of the purified peptide for the ACE binding site. ACE inhibitors are the first-line therapeutic agents for the initial prevention of hypertension. Recent study has demonstrated that ACE inhibitors have comparable effects on the long-term prognosis and mortality rates in cardiovascular, cerebrovascular, and renal diseases [47]. Lisinopril can be used to treat acute myocardial infarction and high blood pressure and as an adjunct therapy for heart failure by inhibiting the ACE [50]. Thus, the current study attempted to determine the binding affinity and most stable pose of each isolated peptide in the active site of the ACE to evaluate their potential as small molecules and antihypertensive drugs. The C-terminus of angiotensin I contains a dipeptide consisting of histidine and leucine residues, which are cleaved by the ACE to produce angiotensin II. Therefore, peptides with high binding affinities for this active site would inhibit the histidine-leucine (HIS–LEU) motif cleavage. All peptides isolated from the ACSH-III-F3 and ACSH-III-F4 Sephadex G-10 fractions showed high binding affinity for the ACE active site. Our results also indicated that peptides containing hydrophobic amino acids, such as YPSYPS, YPSYP, FPTY, YPQW, and YPPW, showed strong ACE inhibitory activity. Supplementary Table 2 indicates the ratio of hydrophobic to hydrophilic amino acids in the peptide. Among the tested peptides, FPTY had the highest hydrophobic amino acid content. Thus, the in silico docking results indicated that these peptides have high binding affinities and interaction energies for the ACE. Furthermore, we found that FPTY showed specificity for binding to the ACE compared to other amino acids. FPTY shared the same amino acid residues with lisinopril, which mediates hydrogen bonding with THR166, ASN277, MET278, and GLN281. Furthermore, similar to lisinopril, FPTY forms a salt bridge with ASP415 and interacts with TYR523 via an alkyl–pi bond, whereas lisinopril forms a cation–pi bond. In addition to these bonds, FPTY has an attractive charge with LYS511 and an alkyl–pi bond with both VAL380 and PHE457. The in vitro results revealed that FPTY exhibited the highest antihypertensive activity by inhibiting the ACE.

Our findings suggest that Stichopus japonicus contains bioactive peptides with antioxidant and antihypertensive properties. In particular, FPTY showed promising properties and warrants further investigation for its therapeutic potential in treating oxidative stress and hypertension. FPTY also demonstrated strong ROS scavenging activity and significantly inhibited the ACE activity, which was attributed to its peptide structure and the specific amino acids it contained. However, further studies are required to verify these initial results, such as molecular dynamics simulations to explore the protein–ligand dynamics and additional experimental assays to validate the activity of FPTY. Further investigation into bioactive peptides from Stichopus japonicus could provide valuable insights for targeted therapies in these disease areas and lead to practical applications in the nutraceutical and functional food industries, potentially enhancing the development of new and effective dietary supplements.

Data presented in this study are contained within this article and in the supplementary materials, or are available upon request to the corresponding author.

HGL proposed the conception and design. HGL, DPN, JGJ, HHACKJ, NML and WKJ performed a acquisition of data. JGJ, JYO, YRC, HSK and WKJ performed the analysis and interpretation of data. HHACKJ, NML and MJMSK conducted the initial screening. HGL, DPN, MJMSK, HSK, SHP and YJJ performed the statistical analysis. HGL, JGJ, JYO, HHACKJ and NML organized the figures and tables. WKJ participated in the project design. HGL and DPN wrote this primary article. YRC and HSK revised and reviewed the manuscript. All authors contributed to editorial changes in the manuscript. All authors read and approved final version of the manuscript and are fully prepared to take responsibility for all aspects of the work.

Studies using Stichopus japonicus were exempt from review and approval by the Jeju National University.

This work was supported by the research grant of the Gyeongsang National University in 2023. Further, this research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2021R1A6A1A03039211).

The authors declare no conflict of interest.

Supplementary material associated with this article can be found, in the online version, at https://doi.org/10.31083/j.fbl2910368.