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Original Research
Identification of Structure-Linked Activity on Bioactive Peptides from
Sea Cucumber (Stichopus japonicus): A Compressive In Silico/In Vitro
Study
Hyo-Geun Lee1,†, D.P. Nagahawatta1,†, Jun-Geon Je1, Jae-Young Oh2,
H.H.A.C.K. Jayawardhana1, N.M. Liyanage1, M.J.M.S. Kurera1,3, Si-Hyeong Park4,
You-Jin Jeon1, Won-Kyo Jung5, Yu Ri Choe4,* , Hyun-Soo Kim4,*
1Department of Marine Life Science, Jeju National University, 63243 Jeju, Republic of Korea
2Food Safety and Processing Research Division, National Institute of Fisheries Science, 46083 Busan, Republic of Korea
3Department of Biotechnology, Faculty of Agriculture and Plantation Management, Wayamba University of Sri Lanka, Makandura, 60170 Gonawila
(NWP), Sri Lanka
4Department of Seafood Science and Technology, The Institute of Marine Industry, Gyeongsang National University, 53064 Tongyeongsi,
Gyeongsangnamdo, Republic of Korea
5Major of Biomedical Engineering, Division of Smart Healthcare, College of Information Technology and Convergence and New-Senior Healthcare
Innovation Center (BK21 Plus), Pukyong National University, Namgu, 48513 Busan, Republic of Korea
*Correspondence: dbfl3319@gmail.com (Yu Ri Choe); gustn783@gnu.ac.kr (Hyun-Soo Kim)
†These authors contributed equally.
Academic Editors: Marcello Iriti and Graham Pawelec
Submitted: 15 July 2024 Revised: 28 August 2024 Accepted: 14 September 2024 Published: 23 October 2024
Abstract
Background: A sea cucumber (Stichopus japonicus) is an invertebrate rich in high-quality protein peptides that inhabits the coastal
seas around East Asian countries. Such bioactive peptides can be utilized in targeted disease therapies and practical applications in
the nutraceutical industry. Methods: Bioactive peptides were isolated from Stichopus japonicus through ultrafiltration and Sephadex
G-10 size exclusion chromatography. The low-molecular-weight fraction (ACSH-III) showed the highest hydroxyl radical scavenging
and angiotensin-converting enzyme (ACE) inhibitory activities. Subsequent purification of ACSH-III resulted in four fractions, of which
ACSH-III-F3 and ACSH-III-F4 exhibited significant bioactivity. Results: Peptides identified in these fractions, including Phenylalanine-
Proline-Threonine-Tyrosine (FPTY) and Tyrosine-Proline-Serine-Tyrosine-Proline-Serine (YPSYPS), were characterized using high-
performance liquid chromatography (HPLC) and quadrupole time-of-flight mass spectrometry (QTOF-MS). FPTY demonstrated the
most potent antioxidant and antihypertensive activities among these peptides, with IC50 values of 0.11 ±0.01 mg/mL for hydroxyl
radicals and 0.03 ±0.01 mg/mL for ACE inhibition. Docking simulations revealed strong binding affinities of these peptides to the
active site of the ACE, with FPTY displaying interactions similar to those of the synthetic inhibitor lisinopril. Conclusions: These
findings suggest that the identified peptides, particularly FPTY, have potential applications as natural antioxidants and functional foods.
Keywords: Stichopus japonicus; bioactive peptide; antioxidant activity; antihypertensive activity; nutraceuticals
1. Introduction
Cardiovascular disease is widely regarded in indus-
trialized nations as one of the most pressing health con-
cerns. Indeed, cardiovascular diseases are the leading cause
of death globally, with hypertension emerging as the pri-
mary 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]. Hy-
pertension is commonly managed with medication, in par-
ticular ACE inhibitors, which are widely accessible, cost-
effective, and also recommended as an initial treatment
for other prevalent chronic conditions such as heart fail-
ure with reduced ejection fraction and chronic kidney dis-
ease [3]. Lisinopril, trandolapril, ramipril, moexipril, and
quinapril hydrochlorides are chemically synthesized anti-
hypertensive 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 di-
etary sources can lower high blood pressure without caus-
ing these adverse effects [5,6]. There is also increasing evi-
dence 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 pri-
mary 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 stabi-
lize the configuration of highly reactive free radicals and
prevent cellular damage. The primary antioxidant defense
mechanism is enzymatic, involving enzymes such as su-
peroxide dismutase, glutathione peroxidase, catalase, per-
oxidase, and glutathione reductase [9]. However, external
factors such as environmental pollution, ultraviolet (UV) ir-
radiation, and exposure to chemical reagents can disrupt an-
tioxidant systems, resulting in severe oxidative stress [10].
Excessive ROS-mediated oxidative stress is strongly asso-
ciated with cell death, deoxyribonucleic acid (DNA) frag-
mentation, 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 reg-
ulating excessive ROS generation would reduce its inci-
dence, improve patient outcomes across a broad spectrum
of disorders, and be clinically beneficial [17].
Notably, there is growing recognition of the bioactiv-
ities of naturally derived peptides; bioactive peptides are
potent antioxidant candidates with biotechnological appli-
cations 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 com-
prise 2–20 amino acids and are abundant in bioresources,
especially in animals with high peptide content. More-
over, these peptides have been shown to possess antioxi-
dant [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]. There-
fore, they have received significant attention in the food
and functional food industries. Marine-derived functional
ingredients and bioactive peptides obtained from enzy-
matically hydrolyzed fish exhibit various functionalities
highlighting their potential applications in food technol-
ogy for developing functional foods and nutraceuticals [28].
Marine-derived peptides were found to promote antihyper-
tensive 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 vas-
cular endothelial cells [29]. Hence, several marine bioac-
tive peptides have been commercialized in food and func-
tional 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 nutri-
tional and biological activities, such as lipid metabolism
and antioxidant, antihypertensive, anticancer, antifatigue,
and regenerative capacities [33]. In our previous study, α-
chymotrypsin-assisted hydrolysis highly increased the an-
tioxidant ability and provided protective effects against hy-
drogen peroxide-induced oxidative damages in vitro and in
vivo [34]. The biological activity of peptides highly af-
fects the peptide structure, amino acid sequence, and com-
position of their position in the structure. Therefore, this
present study investigated the antioxidant and antihyper-
tensive 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. japoni-
cus could contribute to developing the food and functional
food industries.
2. Materials and Methods
2.1 Materials
Sephadex G-10 gel filtration resin (catalog: 07-
0010-01), used for peptide separation, was purchased
from GE Healthcare (Uppsala, Sweden). Bovine pancre-
atic α-chymotrypsin enzyme (catalog: A4531) was pur-
chased from PanReac AppliChem (Barcelona, Spain). Liq-
uid chromatography (LC)-grade acetonitrile (ACN) (cat-
alog 34998) was purchased from Honeywell B&J (MI,
USA). Analytical high-performance liquid chromatography
(HPLC)-grade formic acid (FA; catalog: 063-05895) was
purchased from Wako Pure Chemical Corp. (Osaka, Japan).
The Millipore Direct Q3 water purification system was pur-
chased from Millipore (Billerica, MA, USA). HPLC sepa-
ration was performed using a 3.0 ×150 mm C18 Atlantis
T3 column (Waters Corporation, MA, USA).
2.2 Purification of Bioactive Peptides from S. japonicus
S. japonicus samples were collected from Jeju Is-
land, South Korea. The captured adult S. japonicus was
then dried and homogenized. After homogenization, 1 g
of powdered S. japonicus and 10 mg of α-chymotrypsin
were suspended in 1 L of deionized water (pH adjusted
to 8.00 and 37 °C) and incubated in a shaking incubator
for 24 h. Dried S. japonicus was hydrolyzed with food
grade α-chymotrypsin, and the hydrolysis process followed
the previously established method by Lee et al. (2021)
[34]. The resulting α-chymotrypsin-assisted hydrolysate
from Stichopus japonicus (ACSH) was fractionated using a
tangential flow ultrafiltration (UF) system (Lab scale TFF
system; Millipore) equipped with molecular weight (MW)
cut-off membranes. Peptide fractions were separated by
2
MW using a decreasing molecular mass order from 10–5
kDa. UF separation was performed under low-temperature
conditions (4 °C) and followed a modified method from Lee
et al. (2021) [34]. Three ultrafiltration fractions (above 10
kDa, ACSH-I; 5–10 kDa, ACSH-II; below 5 kDa, ACSH-
III) were obtained from ACSH. ACSH-III was further fil-
tered using a Sephadex G-10 gel column (2.5 ×100 cm)
to isolate biologically active peptides. The loaded ACSH-
III was eluted using distilled water as the mobile phase at
a 1.5 mL/min flow rate. The eluents were collected using
an automatic fraction collector (Young In, Anyang, Korea).
Four Sephadex G-10 fractions (ACSH-III-F1, ACSH-III-
F2, ACSH-III-F3, and ACSH-III-F4) were obtained and an-
alyzed using a HPLC separation module (Waters Corpora-
tion) equipped with a 2998 photodiode array detector (Wa-
ters, Milford, MA, USA) and C18 Atlantis T3 column (3.0
×150 mm; Waters Corporation). Gradient elution was per-
formed using ACN (A) and water (B) as the mobile and
stationary phases, respectively. The optimal gradient con-
ditions were as follows: 0 min, 0% A, 100% B; 20 min,
10% A, 90% B; 30 min, 20% A, 80% B; 40 min, 50% A,
50% B; 45 min, 100% A, 0% B; 50 min, 100% A, 0% B.
The flow rate was maintained at 0.3 mL/min, and the ab-
sorption peaks were analyzed at 220 nm using Waters 2998
photodiode array detector (Milford, USA).
2.3 Sequencing and Synthesis of Bioactive Peptides from
S. Japonicus
The peptides in two of the four Sephadex G-10 frac-
tions (ACSH-III-F3 and ACSH-III-F4) were sequenced and
synthesized according to the protocol described by Kim et
al. (2019) [35]. The sequences and masses of the pep-
tides in ACSH-III-F3 and ACSH-III-F4 were analyzed us-
ing the LC UltiMate 3000 LC system (Dionex, Sunnyvale,
CA, USA) equipped with Poroshell 120 EC C18 separa-
tion columns (2.1 ×100 mm, 2.7 µm; Agilent, Santa Clara,
CA, USA). For liquid chromatographic detection, solvent
A (H2O/FA = 100/0.2 (v/v)) and solvent B (ACN/FA =
100/0.2 (v/v)) were used and gradient elution performed at
a flow rate of 200 µL/min. The gradient elution was ap-
plied as follows: 0 min 95% A, 5% B; 5 min 95% A, 5%
B; 28 min 70% A, 30% B; 33 min 5% A, 95% B; 40 min
5% A, 95% B; 41 min 95% A, 5% B; 46 min 95% A, 5% B.
Absorption spectra were recorded at 220 nm using the PDA
detector. Molecular masses and sequences were analyzed in
the positive ion mode using quadrupole time-of-flight mass
spectrometry (QTOF-MS) (Micro Q-TOF III mass spec-
trometer; Bruker Daltonics, Bremen, Germany). The in-
strument settings for QTOF-MS detection were as follows:
flow rate, 200 µL/min; scan range, 50–2000 m/z; rolling
average spectra rate, 2 ×2.0 Hz; source temperature, 180
°C.
2.4 Hydrogen Peroxide Scavenging Activity
A hydrogen peroxide scavenging assay was performed
on the purified peptide fractions to assess their free radical
scavenging activities. Hydroxyl radical scavenging activ-
ity was determined using the method described by Lee et
al. (2023) [36]. Briefly, 50 µL 0.1 M phosphate buffer
(Welgene Inc., Daegu, Korea) and 50 µL peptide sam-
ples were added to 96-well plates. Then, 10 µL 10 mM
hydrogen peroxide (Junsei, Chemical, Tokyo, Japan) was
added to each well, and the plates were incubated at 37 °C
for 5 min. After incubation, 15 µL 1 U/mL peroxidase
(Fluka, Buchs, Switzerland) and 1.25 mM 2,2′-azino-bis
(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt
(Sigma, St Louis, MO, USA) were added to the wells. Af-
ter that, the mixtures were incubated in a shaking incubator
at 37 °C for 10 min. Following an additional incubation
period, the absorbance was measured at 405 nm using a
microplate reader (Synergy HT Multi Detection microplate
reader; BioTek, Winooski, VT, USA).
2.5 ACE Inhibition Assay
An ACE inhibition assay was performed to deter-
mine the potential antihypertensive activity of the iso-
lated peptides. The peptides were dissolved in deion-
ized water, and different concentrations were mixed with
an enzyme-working solution (3-hyppurylbutyryl-Gly-Gly-
Gly, aminoacylase). The principle of this assay is as fol-
lows: 3-hyppurylbutyryl-Gly-Gly-Gly (3HB-GGG) is con-
verted by the ACE to 3-hyppurylbutyryl-Gly (3HB-G).
Then, 3HB-G is converted into 3-hyppurylbutyryl (3HB)
by an aminoacylase. Finally, 3HB reacts with a water-
soluble tetrazolium salt (WST) colorimetric indicator to
form a WST formazan. These WST formazan concentra-
tions were measured at 450 nm using a microplate reader.
The ACE inhibition assay and activity calculation were per-
formed according to the manufacturer’s instructions (ACE-
WST ELISA kit; Rockville, MD, USA).
2.6 Molecular Docking
In silico evaluation was performed using Discovery
Studio V3.0 (Accelrys Inc., San Diego, CA, USA) in accor-
dance with a previously published method [37]. Briefly, the
X-ray crystallographic protein structure of the ACE was ob-
tained from the Protein Data Bank (PDB) (PDB ID: 1O86).
The obtained structure was corrected using the “clean pro-
tein” tool by removing the water and heteroatoms and in-
serting any missing atoms. Then, the structure was prepared
using the “prepare protein” tool by correcting the missing
loops and atoms. The regenerated structure was then super-
imposed on the raw structure, and the root mean square de-
viation (RMSD) value was calculated. After confirming the
accuracy of the prepared structure, the active site was pre-
pared. The ACE structure is available in the PDB as a com-
plex with lisinopril (https://www.rcsb.org/structure/1O86).
Therefore, the amino acid residues in the ACE responsible
for binding were considered as the geometrical center of
the active site, and an active site sphere was generated. The
automated workflow “flexible docking” was used to per-
form in silico simulations. After obtaining the most likely
3
ligand–receptor confirmation, the binding energy was cal-
culated using the “calculate binding energy” tool. The bind-
ing energy was calculated using the equation presented be-
low [37].
Energy binding =
Energy Complex −(Energy Ligand +Energy Receptor)
2.7 Statistical Analysis
All experiments were conducted in triplicate, and the
results are presented as the mean ±standard error. Statis-
tical analyses were performed using GraphPad Prism ver-
sion 6.01 (GraphPad Software, Inc., San Diego, CA, USA).
Mean values were compared using a one-way analysis of
variance (ANOVA), followed by Dunnett’s multiple com-
parison test for mean separation. Statistical significance
was set at p<0.05.
3. Results
3.1 Isolation of Peptides from S. Japonicus via Sephadex
G-10 Purification
To purify the active peptides from S. japonicus, ACSH
was fractionated using a series of UF membranes with
reduced MW cut-offs (10 and 5 kDa), resulting in three
fractions (above 10 kDa, ACSH-I; 5–10 kDa, ACSH-II;
below 5 kDa, ACSH-III). A schematic representation of
the UF fractionation method is shown in Fig. 1A. Among
the UF fractions, low-molecular-mass ACSH-III exhibited
the greatest hydroxyl radical scavenging activity, while
the ACE inhibitory activity in ACSH-III also gradually
increased; thus, ACSH-III was selected for peptide pu-
rification. Four fractions were identified from ACSH-
III using Sephadex G-10 size exclusion chromatography;
the resulting chromatograms are shown in Fig. 1B. HPLC
was performed to identify peptides in the Sephadex G-10
fractions. Analytical HPLC was performed with silica-
based Atlantis T3 columns using an increasing gradi-
ent of ACN eluent. The HPLC chromatograms of the
Sephadex G-10 fractions are presented in Fig. 1C and show
two major HPLC peaks for ACSH-III-F3 and ACSH-III-
F4 at a wavelength of 280 nm. Ultimately, these re-
sults indicate that the Sephadex G-10 fractions, ACSH-
III-F3 and ACSH-III-F4, contained peptides. Fig. 1D,E
show the ACSH-III-F3 peptide sequences identified using
quadrupole time-of-flight mass spectrometry (QTOF-MS).
The peptide sequences were identified as TRP-VAL-ASP-
GLN (WVDQ; MW: 547.26), GLU-ALA-GLU-GLY-ARG
(DADGR; MW: 533.24), TYR-PRO-SER-TYR-PRO-SER
(YPSYPS; MW: 713.31), TYR-PRO-SER-TYR-PRO (YP-
SYP; MW: 626.28), PHE-PRO-THR-TYR (FPTY; MW:
527.25), VAL-PRO-PRO-TYR-PHE-GLU-TRP-GLY (VP-
PYPEWG; MW: 944.45), and VAL-PRO-PRO-TYR-PHE-
GLU-TRP (VPPYPEW; MW: 887.43). The ACSH-III-
F4 peptide sequences were identified as TYR-PRO-SER-
TYR-PRO-SER (YPSYPS; MW: 713.31), TYR-PRO-
GLN-TRP (YPQW; MW: 593.27), and TYR-PRO-PRO-
TRP (YPPW; MW: 562.26).
3.2 Determination of Potential Antioxidant and
Antihypertensive Activities
The hydrogen peroxide scavenging and ACE in-
hibitory activities of the Sephadex G10-derived fractions
were determined. The 50% inhibitory concentration (IC50)
of hydrogen peroxide and the ACE are shown in Table 1.
The IC50 of hydrogen peroxide and the ACE were recorded
as 1.97 ±0.01 and 1.82 ±0.02 mg/mL, respectively. In ad-
dition, these activities increased significantly following UF
purification. Among the UF-derived fractions, ACSH-III-
F3 showed the highest hydroxyl radical scavenging (IC50
= 0.45 ±0.07 mg/mL) and ACE inhibitory (IC50 = 0.65 ±
0.02 mg/mL) activities. Among the Sephadex G-10 frac-
tions, ACSH-III-F3 and ACSH-III-F4 showed relatively
strong inhibitory effects against hydroxyl radicals and the
ACE. These results strongly indicate that potential antiox-
idant and antihypertensive peptides are present in ACSH-
III-F3 and ACSH-III-F4. Therefore, these two Sephadex
G-10 fractions were selected as candidates for peptide
sequencing and synthesis. Notably, the Phenylalanine-
Proline-Threonine-Tyrosine (FPTY) peptide from ACSH-
III-F3 showed the highest hydroxyl radical scavenging and
ACE inhibitory activities. The IC50 value of FPTY was
recorded as 0.11 ±0.01 and 0.03 ±0.01 mg/mL for hy-
droxyl radicals and the ACE, respectively.
3.3 Docking Simulation of Antihypertensive Peptides on
Angiotensin-Converting Enzyme Inhibition
The ACE crystal structure was obtained from the PDB
(PDB ID: 1O86) and processed using Discovery Studio
V3.0. The resulting active sites of the ACE comprised
VAL318, HIS353, ALA354, GLU411, LYS511, HIS513,
TYR520, and TYR523 amino acid residues (Fig. 2).
Lisinopril was bound to the ACE at ASN277, GLN 281,
THR282, and TYR520 via four conventional hydrogen
bonds, one van der Waals bond at GLU384, and ten carbon–
hydrogen bonds formed at TRP279, CYS352, CYS370,
GLN369, ASP377, VAL379, ASP453, LYS454, PHE457,
and PHE527. Furthermore, lisinopril formed one salt
bridge and one attractive charge alongside two cation
pi bonds and one anion–pi bond at ASP415, TYR523,
GLU162, GLU376, and HIS383, respectively. Aside from
these bonds, lisinopril also formed weaker bonds, such as
one π–πT-shaped bond with HIS353, one alkyl bond with
VAL380, and one alkyl–pi bond with ALA354 (Fig. 2A–C).
All peptides showed high binding affinities. In particular,
similar to lisinopril, FPTY formed bonds with the active
site, providing insights into its activity. The WVDQ pep-
tide exhibited the lowest binding affinity (Figs. 3,4,5). The
peptides VPPYPEWG and VPPYPEW did not bind to the
ACE in the molecular docking system; therefore, these re-
sults were not included. The binding affinities and interac-
4
Fig. 1. Purification and identification of bioactive peptides from Stichopus japonicus.(A) Scheme of ultrafiltration methods used
to obtain the three ultrafiltration fractions based on molecular size: above 10 kDa (ACSH-I), 5–10 kDa (ACSH-II), and below 5 kDa
(ACSH-III) (B) Chromatogram of Sephadex G-10 fractions (ACSH-III-F1, ACSH-III-F2, ACSH-III-F3, and ACSH-III-F4). (C) High-
performance liquid chromatograms of Sephadex G-10 fractions (ACSH-III-F1, ACSH-III-F2, ACSH-III-F3, and ACSH-III-F4). Liquid
Chromatography-Mass Spectrometry (LC–MS) analysis and the (D) ACSH-III-F3 and (E) ACSH-III-F4 peptide sequences. ACSH, α-
chymotrypsin assisted hydrolysate from Stichopus japonicus; WVDQ, Tryptophan-Valine-Aspartic acid-Glutamine; DADGR, Aspartic
acid-Alanine-Aspartic acid-Glycine-Arginine; YPSYPS, Tyrosine-Proline-Serine-Tyrosine-Proline-Serine; YPSYP, Tyrosine-Proline-
Serine-Tyrosine-Proline; FPTY, Phenylalanine-Proline-Threonine-Tyrosine; YPQW, Tyrosine-Proline-Glutamine-Tryptophan; YPPW,
Tyrosine-Proline-Proline-Tryptophan.
5
Table 1. Potential hydrogen peroxide scavenging activity and angiotensin converting enzyme (ACE) inhibitory activity of
ACSH, ultrafiltration (UF) fractions, Sephadex G-10 fractions, and peptides from S. japonicus.
Type Sample Hydrogen peroxide scavenging
activity, IC50 value (mg/mL)
ACE inhibitory activity,
IC50 value (mg/mL)
Enzyme-assisted hy-
drolysate
ACSH 1.97 ±0.01 1.82 ±0.02
Ultrafiltration fractions
ACSH-I 1.14 ±0.03 1.78 ±0.08
ACSH-II 0.72 ±0.02 1.51 ±0.05
ACSH-III 0.26 ±0.22 1.68 ±0.01
Sephadex G-10 fractions
ACSH-III-F1 1.51 ±0.06 1.23 ±0.02
ACSH-III-F2 1.60 ±0.07 1.16 ±0.01
ACSH-III-F3 0.45 ±0.07 0.65 ±0.02
ACSH-III-F4 0.35 ±0.10 0.90 ±0.03
Synthesized peptides
Tryptophan-Valine-Aspartic acid-Glutamine
(WVDQ)
>4 1.97 ±0.03
Aspartic acid-Alanine-Aspartic
acid-Glycine-Arginine (DADGR)
>4 2.36 ±0.08
Tyrosine-Proline-Serine-Tyrosine-Proline-Serine
(YPSYPS)
0.17 ±0.02 0.21 ±0.01
Tyrosine-Proline-Serine-Tyrosine-Proline
(YPSYP)
0.16 ±0.01 0.21 ±0.02
Phenylalanine-Proline-Threonine-Tyrosine
(FPTY)
0.11 ±0.01 0.03 ±0.01
Valine-Proline-Proline-Tyrosine-Proline-
Glutamic acid-Glycine (VPPYPEWG)
0.44 ±0.01 0.37 ±0.01
Valine-Proline-Proline-Tyrosine-Proline-
Glutamic acid (VPPYPEW)
0.20 ±0.02 0.08 ±0.00
Tyrosine-Proline-Glutamine-Tryptophan (YPQW) 0.23 ±0.02 0.13 ±0.01
Tyrosine-Proline-Proline-Tryptophan (YPPW) 0.28 ±0.08 0.27 ±0.01
tion energies of lisinopril with each peptide are summarized
in Supplementary Table 1.
4. Discussion
Marine animal-derived secondary metabolites have
been proven to possess highly effective biological activi-
ties. Fish have relatively high protein content ratios com-
pared to other nutritional components, and several bioac-
tive peptides from these sources have been proven ben-
eficial in human diseases. Fish-derived low-molecular-
weight bioactive peptides have been recognized as func-
tional ingredients that exhibit various biological properties,
such as antioxidant, antifatigue, and immunoregulatory ac-
tivities [38,39]. Thus, extensive research has been con-
ducted to develop efficient methods for isolating and iden-
tifying novel lead compounds with versatile uses in func-
tional food and pharmaceutical industries. In this study,
S. japonicus samples were enzymatically hydrolyzed with
α-chymotrypsin, resulting in the isolation of nine bioac-
tive peptides. These peptides were characterized using liq-
uid chromatography-mass spectrometry (LC–MS) sequenc-
ing, and their potential antioxidant and antihypertensive ac-
tivities were evaluated using free-radical scavenging and
ACE inhibitory assays. Peptides from S. japonicus signif-
icantly stabilize free radicals and inhibit the activity of the
ACE. Previous study has investigated the correlation be-
tween potential antioxidant activity, peptide composition,
and structural properties. Zou et al. (2016) [40] estab-
lished a relationship between the quantitative structure of
peptides and their antioxidant activity, whereby the amino
acid composition and sequence play crucial roles in de-
termining 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, allow-
ing antioxidant peptides to scavenge free radicals in cells
[41]. A previous study demonstrated that exogenous pro-
line effectively increases antioxidant activity by inhibiting
H2O2diffusion in cells [42]. In addition, tyrosine and tryp-
tophan residues increase the lipid density of the cellular
membranes, conferring protection against oxidative dam-
age [43]. Previous research has also indicated that spe-
cific amino acids such as cysteine, glycine, lysine, trypto-
phan, and phenylalanine exhibit strong antioxidant activi-
ties by donating electrons to free radicals [44]. In negatively
charged peptides, aromatic (histidine, phenylalanine, tryp-
tophan, and tyrosine) and hydrophobic amino acids, includ-
6
Fig. 2. The active site preparation of the angiotensin-converting enzyme (ACE). (A) The ACE structure obtained from the Protein
Data Bank (PDB) (1O86), (B) the ACE structure prepared using Discovery Studio V3.0 (Accelrys Inc.), and (C) the active site of the
ACE that is responsible for converting angiotensin I to angiotensin II. All the structures were analyzed using Discovery Studio visualizer
V3.0 (Accelrys Inc.).
ing alanine, valine, leucine, isoleucine, proline, and trypto-
phan, 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. Regard-
ing antihypertensive ACE inhibition, the proline and tyro-
sine C-terminal effectively inhibit and increase hydropho-
bic 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 pheny-
lalanine 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 re-
ported that α-chymotrypsin cleaves the peptide bonds adja-
cent to aromatic ring amino acids, such as phenylalanine,
tryptophan, and tyrosine [49]. This processing likely in-
creases the concentration of peptides with aromatic rings
at both the N- and C-terminal, thereby enhancing ACE in-
hibitory activity. Our findings indicate that the selective
cleavage of these peptide bonds by α-chymotrypsin signif-
icantly contributes to the increased ACE inhibition by el-
evating the levels of aromatic ring peptides. Notably, the
purified FPTY peptide, which exhibits the highest ACE in-
hibitory activity in our chemical assays, contains phenylala-
nine 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
7
Fig. 3. The angiotensin-converting enzyme (ACE) structure bound to FPTY. (A) The ribbon structure of ACE bound to FPTY, (B)
the solid structure of ACE bound to FPTY, (C) the ligand interaction between FPTY and amino acids in the ACE, and (D) the 2D image
of the bonds between FPTY and the ACE. All the structures were analyzed using Discovery Studio visualizer V3.0 (Accelrys Inc.).
8
Fig. 4. The ligands bound to the active site of the angiotensin-converting enzyme (ACE). (A) The ribbon structure of ACE bound
to lisinopril, (B) the solid structure of ACE bound to lisinopril, and (C) a 2D image of the bonds between ACE and lisinopril. (D) The
bound ribbon structure of ACE to WVDQ, (E) the solid structure of ACE bound to WVDQ, and (F) a 2D image of the bonds between
ACE and WVDQ. (G) The bound ribbon structure of ACE to DADGR, (H) the solid structure of ACE bound to DADGR, and (I) a 2D
image of the bonds between ACE and DADGR. (J) The bound ribbon structure of ACE to YPSYPS, (K) the bound solid structure of
ACE to YPSYPS, and (L) a 2D image of the bonds between ACE and YPSYPS. All the structures were analyzed using Discovery Studio
visualizer V3.0 (Accelrys Inc.).
9
Fig. 5. The ligands bound to the active site of the angiotensin-converting enzyme (ACE). (A) The bound ribbon structure of ACE to
YPSYP, (B) the bound solid structure of ACE to the YPSYP, and (C) a 2D image of the bonds between ACE and YPSYP. (D) The bound
ribbon structure of ACE to the YPQW, (E) the bound solid structure of ACE to YPQW, and (F) a 2D image of the bonds between ACE
and YPQW. (G) The bound ribbon structure of ACE to the YPPW, (H) the solid structure of ACE bound to YPPW, and (I) a 2D image
of the bonds between ACE and YPPW. All the structures were analyzed using Discovery Studio visualizer V3.0 (Accelrys, Inc.).
propose that FPTY is a promising candidate for an ACE in-
hibitory 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 mortal-
ity rates in cardiovascular, cerebrovascular, and renal dis-
eases [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 cur-
rent 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 an-
10
giotensin II. Therefore, peptides with high binding affini-
ties 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 hydropho-
bic amino acids, such as YPSYPS, YPSYP, FPTY, YPQW,
and YPPW, showed strong ACE inhibitory activity. Sup-
plementary 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. Further-
more, 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 an-
tihypertensive activity by inhibiting the ACE.
5. Conclusions
Our findings suggest that Stichopus japonicus con-
tains bioactive peptides with antioxidant and antihyperten-
sive properties. In particular, FPTY showed promising
properties and warrants further investigation for its thera-
peutic potential in treating oxidative stress and hyperten-
sion. FPTY also demonstrated strong ROS scavenging ac-
tivity 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 sim-
ulations to explore the protein–ligand dynamics and addi-
tional experimental assays to validate the activity of FPTY.
Further investigation into bioactive peptides from Sticho-
pus japonicus could provide valuable insights for targeted
therapies in these disease areas and lead to practical appli-
cations in the nutraceutical and functional food industries,
potentially enhancing the development of new and effective
dietary supplements.
Availability of Data and Materials
Data presented in this study are contained within this
article and in the supplementary materials, or are available
upon request to the corresponding author.
Author Contributions
HGL proposed the conception and design. HGL,
DPN, JGJ, HHACKJ, NML and WKJ performed a acquisi-
tion 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 anal-
ysis. HGL, JGJ, JYO, HHACKJ and NML organized the
figures and tables. WKJ participated in the project de-
sign. HGL and DPN wrote this primary article. YRC and
HSK revised and reviewed the manuscript. All authors con-
tributed 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.
Ethics Approval and Consent to Participate
Studies using Stichopus japonicus were exempt from
review and approval by the Jeju National University.
Acknowledgment
We would like to express my gratitude to all those who
helped me during the writing of this manuscript.
Funding
This work was supported by the research grant of
the Gyeongsang National University in 2023. Further,
this research was supported by the Basic Science Re-
search Program through the National Research Founda-
tion of Korea (NRF) funded by the Ministry of Education
(2021R1A6A1A03039211).
Conflict of Interest
The authors declare no conflict of interest.
Supplementary Material
Supplementary material associated with this article
can be found, in the online version, at https://doi.org/10.
31083/j.fbl2910368.
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