Food Chemistry 364 (2021) 130432

Contents lists available at ScienceDirect

Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Study on the structure–activity relationship of watermelon seed antioxidant

peptides by using molecular simulations
Chaoting Wen a, Jixian Zhang b, Haihui Zhang a, Yuqing Duan a, *, Haile Ma a
a
School of Food and Biological Engineering, Jiangsu University, Zhenjiang 212013, China
b
College of Food Science and Engineering, Yangzhou University, Yangzhou 225127, China

A R T I C L E I N F O A B S T R A C T

Keywords: Our previous studies had shown that watermelon seed antioxidant peptides (WSAPs: P1-P5) possessed good
Watermelon seed antioxidant peptides activities. However, the structure–activity relationship of P1 is still unclear. Quantum chemistry and molecular
Structure-activity relationship docking were used to investigate the antioxidant mechanism of P1. The active site of P1 is located at C6H14 on
Quantum chemistry
Arg. P1 can bind to DPPH/ABTS through hydrogen bond and hydrophobic interaction. Compared with P2-P4, P1
Molecular docking
Keap1-Nrf2
has the strongest antioxidant capacity. The molecular simulation showed that P1 could enhance the stability of
Keap1 by interacting with Asn382, Arg380 and Tyr 334 in the active sites. Compared with the model group, the
expression of Keap1 was down-regulated (p < 0.05), while the expression of Nrf2 and HO-1 was up-regulated (p
< 0.05) after P1 treatment. P1 has the potential ability to activate the signaling pathway Keap1-Nrf2 and
improve the antioxidant defense system. This study provides a new perspective for the rational design and
mechanism of antioxidant peptides.

1. Introduction
Endogenous or exogenous stimulation can induce the body to pro­
duce excessive free radicals, which will lead to oxidative stress, and then
reduce the activity of antioxidant enzymes, damage biological macro­
molecules (DNA, protein, lipid, etc.), and finally cause cell dysfunction
and diseases (Waris & Ahsan, 2006). The antioxidant defense system
(non-enzyme antioxidant system and antioxidant enzyme system) of the
body can maintain the dynamic balance of free radicals (Wen, Zhang,
Zhang, Duan, & Ma, 2020). It is worth noting that antioxidant peptides
can activate the antioxidant defense system of the body, maintain the
balance between oxidative damage and antioxidant defense, and reduce
oxidative stress damage (Wen et al., 2020, 2018). In recent years,
antioxidant peptides from by-products of agricultural processing have
attracted wide attention due to their sustainable reuse, easy absorption,
diverse activity, high safety and bioavailability (Lemes et al., 2016).
Watermelon seeds are high-quality protein resources, with a large yield,
high protein content (30.63–43.60%) and balanced distribution of
amino acids, especially rich in arginine, glutamine, asparagine and
leucine (Arise, Yekeen, & Ekun, 2016), which is a potential resource for
the preparation of antioxidant peptides. Although our previous studies
had shown that watermelon seed antioxidant peptides (WSAPs: P1-P5)
possessed good antioxidant activities in vitro (Wen et al., 2020), the
specific antioxidant mechanism of P1-P5 remains unclear.
As we all know, the molecular structure is the material basis of
peptide activity expression, and the study on the structure–activity
relationship of antioxidant peptides is contribute to clarify the mecha­
nism of active peptides. A large number of studies have shown that the
activity of antioxidant peptides is closely related to their molecular
weight (Ahmed, Bassiony, Elmalt, & Ibrahim, 2015), hydrophobicity
(Barbosa et al., 2018), amino acid species (Chi et al., 2014) and spatial
conformation (Delgado et al., 2016). Quantum chemistry (Delgado
et al., 2016) and molecular docking (Chen, Zhao, He, & Yang, 2021; Fan
et al., 2020) technology can be used to investigate the structure–activity
relationship, interaction site and mechanism of peptides. Wang et al.
(2019) determined that the active sites of EAAY, FSAP, PVETVR and
QEPLLR for scavenging free radicals by using quantum chemistry were
the phenolic hydroxyl structure of tyrosine, the hydroxyl structure of
tryptophan, the guanine structure of arginine, and the guanine structure
of arginine, respectively. Wu et al. (2021) used quantum chemistry to
calculate the active site of PMRGGGGYHY, the results showed that the
active site was tyrosine. Besides, molecular docking can also be used to

* Corresponding author.
E-mail addresses: hopewen177@163.com (C. Wen), zjx@yzu.edu.cn (J. Zhang), zhanghh@ujs.edu.cn (H. Zhang), dyq101@ujs.edu.cn (Y. Duan), mhl@ujs.edu.cn
(H. Ma).

https://doi.org/10.1016/j.foodchem.2021.130432
Received 27 April 2021; Received in revised form 16 June 2021; Accepted 18 June 2021
Available online 21 June 2021
0308-8146/© 2021 Elsevier Ltd. All rights reserved.
C. Wen et al.
elucidate the structure–activity relationship and antioxidant mechanism
of peptides. Tonolo et al. (2020) used molecular docking to analyze the
interaction between Keap1 and antioxidant peptides from milk. The
results showed that the peptide could activate the endogenous Nrf2-
Keap1 antioxidant pathway, which inhibited the binding of Nrf2 and
Keap1 by occupying the Nrf2 site in the Keap1 pocket, and then pro­
moted the transfer of Nrf2 from the cytoplasm into the nucleus. The
results of Wang et al. (2020) showed that the glutamate of the soft-
shelled turtle antioxidant peptide (EDYGA) could bind to the arginine
415 residue of Keap1 by using molecular docking to inhibit the degra­
dation of Nrf2. In our previous study, WSAPs (P1-P5) exhibited a good
ability to scavenge DPPH and ABTS radicals (Wen et al., 2020). How­
ever, the structure–activity relationship of WSAPs has not been studied
by using quantum chemistry and molecular docking technology.
In view of our previous research, the antioxidant peptides P1
(RDPEER), P2(KELEEK), P3(DAAGRLQE), P4 (LDDDGRL) and P5
(GFAGDDAPRA) were identified from the watermelon seed protein hy­
drolysates (Wen et al., 2020). As a continued study, the purpose of the
current research was to (1) obtain the energy, structural parameters and
active site of P1-P5 by using quantum chemistry calculation; (2) analyze
the docking dominant conformation and interaction between P1-P5 and
free radicals (DPPH, ABTS) by using molecular docking; (4) predict the
docking dominant conformation and interaction between P1 and Keap1-
Nrf2 by using molecular docking; (5) verify the interaction between P1
and Keap1-Nrf2 by western-blot analysis. The results were expected to
provide new ideas for the engineering design of antioxidant peptides and
provide the theoretical basis of structure for the study of the antioxidant
mechanism of the peptides.
2. Materials and methods
2.1. Materials
The watermelon seed antioxidant peptides (WSAPs), including
RDPEER (P1), KELEEK (P2), DAAGRLQE (P3), LDDDGRL (P4), and
GFAGDDAPRA (P5) with a purity of 98% were synthesized by GL Bio­
chem Ltd. (Shanghai, China). Glutathione was obtained from the Nanj­
ing Jiancheng Bioengineering Research Institute (Jiangsu, China).
2.2. Bioinformatics determination of physicochemical properties of P1-P5
Potential toxicity and crucial physicochemical properties of P1-P5
were predicted through the tool ToxinPred (https://webs.iiitd.edu.
in/raghava/toxinpred/multi_submit.php). The resistance of P1-P5 to
digestion was analyzed using the program ExPASy peptide cutter
(https://web.expasy.org/peptide_cutter/). These peptides were evalu­
ated against pepsin (pH = 1.3, and > 2.0), trypsin and chymotrypsin.
The lowest cleavage probability was 20%.
2.3. Optimization of peptide configuration based on quantum chemistry
The two-dimensional structure of P1-P5 were drawn by using ACD
labs software. The initial geometry of the P1-P5 was constructed and
optimized by using the molecular force field and semi-empirical algo­
rithm method of HyperChem 8.0 software. The full molecular geometry
optimization of the P1-P5 was calculated by using the Gaussian 09
software. HF method at sto-3 g/6-31G level was employed to stabilize
the configuration to the virtual frequency.
2.4. Calculation on system energy and structure index of the peptides
The energy index (total energy, the energy level of highest electron
occupied orbit (EHOMO), the lowest electron unoccupied orbit (ELOMO))
and structure index (highest electron occupied orbit, atomic net charge
distribution, bond length) of the peptide molecule were calculated.
2
Food Chemistry 364 (2021) 130432
2.5. Molecular docking of P1-P5 with DPPH and ABTS
The three-dimensional structures of DPPH (CID: 2735032) and ABTS
(CID: 5360881) were obtained from PubChem database
(https://pubchem.ncbi.nlm.nih.gov/). The 3D structure of P1-P5 was
constructed using the method as same section 2.3.1. Peptide and ligand
(DPPH and ABTS) were docked by using Autodock software and its
conformation was optimized by using a genetic algorithm based on the
principle of minimizing docking energy. The stereoscopic and planar
interactions between the receptor peptide and ligand were analyzed by
PyMOL and Liglot software.
2.6. Molecular docking of P1 and Keap1
The 3D structure of ligand (P1) was constructed by using the method
as same section 2.3.1. The water molecules and 16-mer Nrf2 in Nrf2-
keap1 protein were removed for later docking calculation. The dock­
ing procedure was conducted by using Autodock software and its
conformation was optimized by using a genetic algorithm based on the
principle of minimizing docking energy.
2.7. Western blot analysis of interaction between P1 and Keap1-Nrf2
Protein expression of Nrf2, HO-1, Keap1 was analyzed by Western
blot. HepG2 cells (5.0 × 105 cells/well) were seeded into the 6-well
plates for 12 h. After P1 and H2O2 treatment, the cells of each group
were digested and lysed (ice bath) for 30 min. Then, after the lysed cell
suspension was centrifuged (13,000 rpm/min, 4 ℃) for 5 min, the su­
pernatant was collected and its total protein content was determined.
The supernatant was mixed with 5 × loading buffer (containing 5%
β-mercaptoethanol) at the ratio of 1:5 and boiled for 5 min. The protein
solution of each group was separated by loading on the SDS-PAGE (10%)
electrophoretic. The proteins were transferred onto a PVDF membrane
and blocked with 5% skim milk for 2 h at room temperature. The
membranes were incubated with diluted primary antibodies (Nrf2, HO-
1, Keap1) overnight at 4 ◦ C. After washing five times with TBST, the
membranes were incubated with secondary antibodies goat anti-mouse
IgG-HRP (1:5000) for 2 h and then washed three times in PBST. Sub­
sequently, the proteins were detected by using an enhanced chem­
iluminescence (ECL) kit. The related expression of proteins was analyzed
using ImageJ software, and β-actin was used as the internal reference.
2.8. Statistical analysis
All data were analyzed by SPSS 18.0 (SPSS Inc. USA). One way
ANOVA was used to analyze the differences among the groups. p < 0.05
means significant difference, p < 0.01 means extremely significant
difference.
3. Results and discussion
3.1. Analysis of physicochemical properties of P1-P5 by using
bioinformatics
It is necessary to evaluate the toxicity and anti-digestion properties
of antioxidant peptides before they can be successfully developed into
functional foods. As shown in Table 1S, the five antioxidant peptides
from watermelon seeds protein have no toxicity. Besides, gastrointes­
tinal digestion can degrade peptides and change their antioxidant ca­
pacity. The results showed that P1 could completely resist
gastrointestinal digestion, while other peptides were broken up in
varying degrees. Thus, P1 might possess good antioxidant capacity after
oral administration due to it had low sensitivity to gastrointestinal
protease, but the results still need to be verified in vivo.
C. Wen et al.
3.2. Quantum chemistry calculation of conformation and molecular
energy of P1-P5
As shown in Fig. 1S, the dominant conformations of P1-P5 were
obtained. Compared with the conformational structures of P2, P3, P4
and P5, P1 had a more compact spatial structure, which was mainly
since the arginine side chain of P1 can form multiple hydrogen bonds
with the oxygen atoms of the carboxyl group of the adjacent glutamic
acid, which in turn making the structure of P1 more compact (Shah &
Shaikh, 2016). In addition, the spatial structure of P2 is relatively
stretched, which is mainly due to its relatively small number of amino
acids, resulting in the smaller intramolecular interaction. LaPointe,
Farrag, Bohórquez, & Boyd (2009) reported that the α-helical structure
of peptides can not only significantly affect the steric hindrance,
conformational entropy, electrostatic and hydrophobic properties of
amino acids, but also the structure is closely related to the formation of
hydrogen bonds. Compared with the peptide chain lengths of P1 and P2,
the peptide chains of P3, P4 and P5 are relatively long, which is
conducive to the formation of secondary structure with α-helix and in­
crease the possibility of forming hydrogen bonds in the molecule.
Moreover, the spatial conformation of P5 has a significant turn at its
Fig. 1. HOMO distribution of P1-P5. Note: red ball represents the oxygen atom; blue ball represents the nitrogen atom; light gray ball represents the nitrogen atom;
dark gray ball represents the carbon atom. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
3
Food Chemistry 364 (2021) 130432
proline residues, which results in the peptide molecule being able to
exhibit an irregular coiled state. Furthermore, according to quantum
chemistry theory, a molecule has smaller total energy, its structure and
activity can be more stable and higher (Anouar et al., 2009). As shown in
Table 2S, the molecular total energy order of WSAPs was P2 > P3 > P5
> P4 > P1, which indicated that the order of the antioxidant activity of
the peptides was P1 > P4 > P5 > P3 > P2.
3.3. Highest occupied molecular orbital of P1-P5
The highest occupied molecular orbital (HOMO) and the lowest
unoccupied molecular orbital (LOMO) not only significantly affect the
chemical reactions of various saturated and unsaturated compounds but
also are closely related to the antioxidant capacity of compounds
(Benayahoum, Amira-Guebailia, & Houache, 2013). HOMO distribution
of the five peptides was shown in Fig. 1. HOMO of P1 is more evenly
distributed on the guanidine group of Arg-6, which is mainly due to the
conjugation system between the double bond of guanidine and isolated
electron pair of nitrogen, which makes the peptide easy to lose electrons
(Zheng, Zhao, Dong, Su, & Zhao, 2016). HOMO of P2 was mainly
distributed near the Lys-1 and Glu-2. HOMO of P3 was mainly
C. Wen et al.
distributed in Gly-3. In addition, HOMO of P4 was mainly distributed in
Arg-6 and HOMO of P5 was mainly distributed in Asp-5.
3.4. Frontier molecular orbital energy of P1-P5
The highest occupied orbital energy level (EHOMO) of a molecule can
reflect the supplied ability of electrons (Hossen, Ali, & Reza, 2021).
Molecule possesses the larger EHOMO, which means it has a more un­
stable electron. As shown in Table 3S, the order of EHOMO is P1 > P4 >
P5 > P3 > P2. Peptides with the higher EHOMO are more likely to react
with other molecules, which can be used as hydrogen donors to chelate
metal ions or scavenge free radicals to play an antioxidant role
(Benayahoum, Amira-Guebailia, & Houache, 2013). Besides, the lowest
unoccupied orbital energy level (ELOMO) is related to the nucleophilic
potential energy of the molecule. The order of ELOMO was P1 < P4 < P5
< P3 < P2. Molecule possesses the lower ELOMO, which indicated that
the molecular possessed the better ability to accept electrons (Cheng
et al., 2015). Moreover, the difference between ELOMO and EHOMO rep­
resents the energy required for the electron transition from the basic
state to the excited state. The molecular has the smaller Δ EL-H, which
indicated the molecular possessed the stronger activity. The order of
ΔEL-H is P1 < P4 < P5 < P3 < P2, which showed that the order of the
antioxidant activity of the peptides is P1 > P4 > P5 > P3 > P2.
3.5. Active sites of P1-P5
Generally, the activity of antioxidants is closely related to the EHOMO
of molecular, HOMO, charge distribution and bond length of atoms in
the frontier molecular orbital (Frei & Higdon, 2003). The molecular has
the higher EHOMO, which indicated that this molecular may be contrib­
uted to the electrophilic reagents obtain hydrogen atoms from nucleo­
philic reagents. In addition, the active sites of molecular are located on
HOMO (Frei & Higdon, 2003). A previous study reported that the free
radical scavenging sites of antioxidants mainly occurred on the rela­
tively negative charge atoms, which can enhance antioxidants to pro­
vide hydrogen atoms (Nagaoka, Kuranaka, Tsuboi, Nagashima, &
Mukai, 1992). Moreover, Mladenović et al. (2011) showed the net
charge of two atoms with a smaller difference can conductive to the
molecular lose hydrogen atoms.
Mulliken atomic charges distribution of P1-P5 was shown in Table 4-
8S. The five peptides possessed a similar distribution pattern, the
negative charge of the atom was mainly distributed on the nitrogen and
oxygen atoms, while the positive charge of the atom was mainly
distributed on the carbon atom connected with the oxygen and nitrogen
atoms. Furthermore, the molecular bond theory indicated that the weak
attraction force between the molecule connected hydrogen atom of the
longer bond length and the negatively charged atom, which makes the
molecule easily lose the hydrogen atom and has a stronger antioxidant
capacity (Pingaew et al., 2013).
HOMO, charge distribution and maximum bond length of peptide
molecules were used to evaluate the active site (Mladenović et al.,
2011). HOMO of P1 was mainly distributed on Arg (84–108 atoms). C89
(0.0475e) and H98 (0.0860e) atoms had the smallest net charge differ­
ence. The bond length of C89H98 was 1.0989. Thus, the active site of P1
was located in C89H98 on Arg, which could provide H14 as a hydrogen
donor to realize its own antioxidant activity (Table 9S).
HOMO of P2 was mainly distributed on Lys and Glu (1–57 atoms).
C29 (0.0383e) and H36 (0.0793e) atoms had the smallest net charge
difference. The bond length of C29H36 was 1.0907. Thus, the active site
of P2 was located in C29H36 on Glu, which could provide H36 as a
hydrogen donor to realize its own antioxidant activity (Table 9S).
HOMO of P3 was mainly distributed on Gly (42–64 atoms). C47
(0.0410e) and H55 (0.0813e) atoms had the smallest net charge differ­
ence. The bond length of C47H55 was 1.0911. Thus, the active site of P3
was located in C47H55 on Gly, which could provide H55 as a hydrogen
donor to realize its own antioxidant activity (Table 9S).
4
Food Chemistry 364 (2021) 130432
HOMO of P4 was mainly distributed on Arg (67–89 atoms). C75
(-0.0122e) and H85 (0.0558e) atoms had the smallest net charge dif­
ference. The bond length of C75H85 was 1.0947. Thus, the active site of
P4 was located in C75H85 on Arg, which could provide H85 as a hydrogen
donor to realize its own antioxidant activity (Table 9S).
HOMO of P5 was mainly distributed on Asp (59–71 atoms). C63
(0.0554e) and H69 (0.0821e) atoms had the smallest net charge differ­
ence. The bond length of C63H69 was 1.0924. Thus, the active site of P5
was located in C63H69 on Asp, which could provide H69 as a hydrogen
donor to realize its own antioxidant activity (Table 9S).
In general, compared with the other four peptides (P2, P3, P4, P5),
P1 had the highest EHOMO, the lowest E(L-H) and the longest L (X-H), which
indicated that P1 had the highest antioxidant activity, which was
confirmed in our previous research (Wen et al., 2020).
3.6. Docking and interaction model between P1-P5 and DPPH
Molecular docking technology has potential application prospects,
which is widely used to study the interaction between antioxidant
peptides and free radicals, the research, design and synthesis of anti­
oxidant drugs (Zhang et al., 2021). As shown in Fig. 2, the optimal
binding configuration of P1-P5 and DPPH was obtained based on the
minimum binding energy after docking. The minimum binding energies
of P1-P5 were − 3.44, − 1.99, − 2.11, − 3.32 and − 2.79 kcal / mol,
respectively. Compared with the binding energy of other peptides, P1
possessed the lowest binding energy.
As shown in Fig. 2S, the Arg1 and Arg6 residues of P1 has formed two
hydrogen bonds with DPPH and Glu 5 residue of P1 has formed one
hydrophobic interaction with DPPH. One hydrogen bond was formed
between Lys-1 residue of P2 and DPPH with a length of 2.52. DPPH also
possessed hydrophobic interactions with Leu-3, Glu-2, Glu-4, Glu-5 and
Lys-6 residue of P2. In addition, two hydrogen bonds were formed be­
tween the Arg-5 residue of P3 and DPPH. The residues of P3 (Ala-3, Gly-
4, Glu-8 and Leu-6) and DPPH interacted with hydrophobic interaction,
respectively. A hydrogen bond was formed between the Arg-6 residue of
P4 and DPPH, while no hydrogen bond was formed between the P5 and
DPPH. Moreover, the residues of P4 (Asp-2 and Leu-7) and DPPH
interacted with hydrophobic interaction, respectively. The Ala residues
(Ala-3, Ala-10), Asp-5, Arg-9 and Gly-4 of P5 were also interacting with
DPPH based on the hydrophobic interaction, respectively. Based on the
above results, the WSAPs have a strong hydrogen bond and hydrophobic
interaction with the DPPH molecule. The amino acid residues in the
peptide can effectively interact with DPPH free radical molecules, which
makes the peptide have a strong DPPH radical scavenging activity.
These results were confirmed in our previous research (Wen et al.,
2020).
3.7. Docking and interaction model between P1-P5 and ABTS
As shown in Fig. 3, the optimal binding configuration of P1-P5 and
ABTS was obtained based on the minimum binding energy after dock­
ing. The minimum binding energies of P1-P5 were − 3.87, − 2.28, − 2.73,
− 3.4 and − 3.05 kcal/mol, respectively. Compared with the binding
energy of other peptides, P1 possessed the lowest binding energy. As
shown in Fig. 3S, the Arg-1 and Glu-5 residues of P1 formed two
hydrogen bonding with ABTS. Asp-2, Arg-6, Glu-4 residues of P1 formed
three hydrophobic interactions with ABTS. Two hydrogen bonds were
formed between amino residues (Lys-6, Glu-4) of P2 and ABTS. ABTS
also possessed hydrophobic interactions with Leu-3, Glu-2, Glu-5 and
Lys-1 residues of P2. Besides, Ala-3 and Gln-7 residues of P3 formed two
hydrogen bonds with ABTS. ABTS also possessed hydrophobic in­
teractions with Ala-2, Arg-5 and Asp-1 residue of P3. The Asp-4 and Arg-
6 residues of P4 interacted with ABTS through two hydrogen bonds. Asp-
5 and Asp-9 residue of P5 interacted with ABTS through two hydrogen
bonds. Moreover, the Gly-5 and Leu-7 residues of P4 interacted hydro­
phobically with ABTS, respectively. Ala-3, Pro-8 and Gly-4 residues of
C. Wen et al.
Fig. 2. Optimal docking model of P1-P5 and DPPH.
Fig. 3. Optimal docking model of P1-P5 and ABTS.
P5 interacted hydrophobically with ABTS, respectively. The results
indicated that P1 could effectively interact with ABTS to scavenge free
radical, which were confirmed in our previous study (Wen et al., 2020).
A previous study also reported that serine and threonine residues in
millet antioxidant peptides (TSSSLNMAVRGGLTR, STTVGLGISMR­
SASVR) could effectively form hydrogen bonds with DPPH⋅,
ABTS+⋅(Agrawal, Joshi, & Gupta, 2019).
The above results confirmed that molecular docking could be used to
study the structure–activity relationship between peptides and small
molecules (e.g., DPPH and ABTS). The principle of molecular docking is
a method to predict the optimal location and affinity of ligands at re­
ceptor binding sites based on the principle of space matching and energy
matching between ligands and receptors (Tao, et al., 2020). In addition,
molecular docking can also be applied for the study of the interaction
between peptide and protein (Agrawal, et al., 2019). The study of the
structure–activity relationship of peptides will help to expand the
application of antioxidant peptides. It is worth noting that whether the
molecular docking is suitable for elucidating the use of biologically
active peptides or proteins in aquatic products to prevent myofibrillar
protein degradation is worthy of further study (Feng, Bansal, & Yang,
2016).
3.8. Effect of P1 on the expression of Keap1-Nrf2 pathway related
proteins in HepG2 cells
Keap1-Nrf2 signaling pathway is the most important endogenous
5
Food Chemistry 364 (2021) 130432
antioxidant signaling pathway in cells. The activation of the Keap1-Nrf2
signaling pathway can reduce the oxidative damage caused by exoge­
nous adverse stimulation (Kensler, Wakabayashi, & Biswal, 2007). To
further explore the potential antioxidant mechanism of P1, molecular
docking technology was used to evaluate the interaction between pep­
tide and Keap1-Nrf2.
3.8.1. Docking and interaction model force between P1 and Keap1
The three-dimensional configuration of the Keap1-Kelch region and
Nrf2 were shown in Fig. 4a, Nrf2 can bind to key amino acids in the
central cavity of the Keap1-Kelch domain. Interestingly, as shown in
Fig. 4b, the binding region and conformation of the P1 and Keap1-Kelch
region were similar to those of the Keap1-Nrf2 model (Fig. 4a). Tonolo
et al. (2020) reported that the amino acid residues in Keap1-Kelch re­
gion, including Arg-380, Arg-415, Ser-363, Ser-555, Asn-382, Ser-602,
Tyr-334, Arg-483, Ser-508 and Gln-530, can form hydrogen bonds with
Nrf2. The binding interaction between P1 and Keap1-Kelch region was
shown in Fig. 4C, Glu-4 of P1 formed a hydrogen bond with Asn-382 of
Keap1 and their bond distance was 2.71 Å. Glu 4 of P1 also formed a
hydrogen bond with Arg-380 of Keap1 and their bond distance was 3.15
Å. In addition, Glu-4 of P1 formed hydrophobic interaction with Tyr-334
of Keap1. The amino acid residues (Asn-382, Arg-380, Tyr-334) of P1
could interact with the Keap1-Kelch domain, and these binding sites
were contained in the binding sites of Nrf2 and Keap1 Kelch domains,
which indicated that P1 occupied the binding sites of Nrf2 in the Keap1-
kelch domain, and then prevented the interaction between Keap1 and
C. Wen et al. Food Chemistry 364 (2021) 130432

complex of Nrf2 and Keap1 start dissociates in the case of oxidative

stress, and then Nrf2 enters the nucleus and binds to the antioxidant
response element (ARE), thus promoting the transcriptional activation
of metabolizing enzyme/antioxidant protease in phase II (Bellezza,
Giambanco, Minelli, & Donato, 2018). Besides, ROS from redox reaction
can activate Nrf2 by promoting the formation of a disulfide bond with
Keap1 (Fourquet, Guerois, Biard, & Toledano, 2010). Therefore, the
Nrf2-ARE signaling pathway activated by antioxidants is the main
antioxidant mechanism, which can protect cells from oxidative stress by
activating phase II metabolizing enzyme / antioxidant protease (Wang,
Ding, Zhang, Ma, & Liu, 2016)).

4. Conclusion

In the present study, quantum chemistry and molecular docking

were used to investigate the structure–activity relationship of water­
melon seed antioxidant peptides (P1-P5) for the first time. The two
methods are contributed to fill the knowledge gap about the molecular
simulation of bioactive peptides. The active site of P1 is located at C6H14
on Arg. P1 can bind to DPPH/ABTS through hydrogen bond and hy­
drophobic interaction. Compared with P2-P4, P1 has the strongest
antioxidant capacity. Moreover, molecular docking was used to predict
whether the novel P1 could react on Keap1-Nrf2 pathway. The results
showed that P1 could firmly enhance the stability of Keap1 by inter­
Fig. 4. Dominant conformation and interaction model of P1 and Keap1 pocket.
acting with Asn382、Arg380 and Tyr 334 in the active sites. The results
Note: A: the three-dimensional conformation of the Kelch domain of Keap1 and
the Nrf2 complex; B: the dominant conformation of P1 and Keap1 pockets; C:
of western-blot analysis confirmed that P1 might has the potential
the interaction force model of P1 and Keap1 pockets. ability to activate the signaling pathway Keap1-Nrf2 and improve the
antioxidant defense system. In general, our results could definite the key
active sites and their structure–activity relationship of P1-P5, provide
Nrf2. These results were similar to the report of Li et al., (2017), who
new ideas for the engineering design and mechanism research of anti­
showed that egg-derived antioxidant peptides (DKK and DDW) could
oxidant peptides. The future research will focus on exploring the key
occupy the binding site of Nrf2 in the Keap1-Kelch domain to inhibit the
target genes of watermelon seed antioxidant peptide based on Keap1-
interaction between Keap1-Nrf2.
Nrf2 signaling pathway.
3.8.2. Western-Blot analysis of Keap1-Nrf2 pathway related proteins
CRediT authorship contribution statement
As shown in Fig. 5, compared with the control group, the expression
of Keap1 protein was significantly up-regulated (p < 0.05) in HepG2
Chaoting Wen: Writing - original draft, Conceptualization, Meth­
cells of the model group, while the expression levels of Nrf2 and HO-1
odology. Jixian Zhang: Writing - review & editing, Software, Investi­
protein remained basically unchanged. Compared with the model
gation. Haihui Zhang: Project administration, Funding acquisition.
group, the expression of Keap1 pretreated by P1 was significantly down-
Yuqing Duan: Funding acquisition, Resources. Haile Ma: Methodology,
regulated (p < 0.05), while the expression of Nrf2 and HO-1 pretreated
Validation.
by P1 was significantly up-regulated (P < 0.05) in a dose-dependent
manner.
Under the cytoplasm homeostatic conditions, Nrf2 as an important
regulator of the antioxidant signal can bind to the Keap1. However, the

Fig. 5. Effects of P1 on oxidative stress related proteins in HepG2 cells; (A) control group; (B) model group; (C) Low dose of P1 prevention group (12.5 μmol/L); (D)
Medium dose of P1 prevention group (50 μmol/L); (E) High dose of P1 prevention group (100 μmol/L); *p < 0.05 and **p < 0.01 compared with the control group;
#p < 0.05 and ##p < 0.01 compared with the model.

6
C. Wen et al.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgements
This work was supported by National Key R & D Program, China
(2016YFD0400303); Priority Academic Program Development of
Jiangsu Higher Education Institutions (PAPD).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.foodchem.2021.130432.
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