This document summarizes research purifying and characterizing a novel antioxidant peptide from the hard-shelled mussel Mytilus coruscus. Enzymatic hydrolysis was used to generate hydrolysates from M. coruscus, which were screened for antioxidant activity. The papain hydrolysate showed the highest free radical scavenging activity. Further purification using chromatography yielded a novel 10 amino acid peptide. In vitro and in vivo assays found the peptide to have potent antioxidant effects, inhibiting oxidative stress markers and enhancing antioxidant enzyme activity in mice. This is the first report of an antioxidant peptide from M. coruscus with potential anti-inflammatory properties.

1. Purification of a novel peptide derived from Mytilus coruscus and in vitro/in vivo
evaluation of its bioactive properties
Eun-Kyung Kim a,f
, Hyun-Jung Oh b
, Yon-Suk Kim b
, Jin-Woo Hwang b
, Chang-Bum Ahn c
, Jung Suck Lee d
,
You-Jin Jeon e
, Sang-Ho Moon f
, Si Heung Sung f
, Byong-Tae Jeon f
, Pyo-Jam Park b,f,*
a
Department of Natural Science, Konkuk University, Chungju 380-701, Republic of Korea
b
Department of Biotechnology, Konkuk University, Chungju 380-701, Republic of Korea
c
Department of Food Science and Nutrition, Chonam National University, Yosu 550-749, Republic of Korea
d
Industry-Academic Cooperation Foundation, Jeju National University, Jeju 690-756, Republic of Korea
e
Faculty of Applied Marine Science, Jeju National University, Jeju 690-756, Republic of Korea
f
Korea Nokyong Research Center, Konkuk University, Chungju 380-701, Republic of Korea
a r t i c l e i n f o
Article history:
Received 7 November 2012
Received in revised form
9 January 2013
Accepted 20 January 2013
Available online 9 February 2013
Keywords:
Mytilus coruscus
Enzymatic hydrolysis
Inflammatory response
Antioxidant peptide
Anti-inflammatory
a b s t r a c t
Excess oxidant can promote inflammatory responses. Moreover, chronic inflammation accompanied by
oxidative stress is connected various steps involved in many diseases. From the aspect, we investigated
an antioxidant peptide to prevent inflammatory response against oxidant overexpression. To prepare the
peptide, eight proteases were employed for enzymatic hydrolysis, and the antioxidant properties of the
hydrolysates were investigated using free radical scavenging activity by electron spin resonance (ESR)
spectrometry. Papain hydrolysates, which showed clearly superior free radical scavenging activity, were
further purified using consecutive chromatographic methods. Finally, a novel antioxidant peptide was
obtained, and the sequence was identified as Ser-Leu-Pro-Ile-Gly-Leu-Met-Ile-Ala-Met at N-terminal.
Oral administration of the peptide to mice effectively inhibited malondialdehyde (MDA) levels in a thi-
obarbituric acid reactive substances (TBARS) assay, and we also confirmed the antioxidative enzyme
activities in superoxide dismutase (SOD) and glutathione-s-transferase (GST) assays. This is the first
report of an antioxidant peptide derived from the hydrolysate of Mytilus coruscus, and also these results
suggest that the peptide possesses potent antioxidant activity, and potential to enhance anti-
inflammatory response.
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
Although reactive oxygen species (ROS) play an important role
in host defense against microbial infection, their overexpression
and residual ROS can cause cellular damage [1,2], and are impli-
cated in many inflammatory conditions [3]. Mitochondria are the
major organelles that produce ROS and the main target of ROS-
induced damage, as observed in various pathological states
including aging, malaria, acquired immunodeficiency syndrome,
heart disease, stroke, arteriosclerosis, diabetes, cancer, and gastric
ulcer [4,5]. Major cellular defenses against ROS include superoxide
dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and
glutathione-s-transferase (GST) [6]. Therefore, the development
and utilization of effective antioxidants that enhance the activity of
the antioxidative enzymes are desired.
Meanwhile, acute inflammation is part of the defense response,
but chronicinflammationhasbeen found to mediate awide varietyof
disease, including cardiovascular disease, cancers, diabetes, arthritis,
Alzheimer’s disease, pulmonary disease, and autoimmune disease.
Moreover, ROS production by H2O2 activates the inflammasome
which can promote inflammatory responses [7]. ROS may either
directly trigger inflammasome assembly or be indirectly sensed
through cytoplasmic proteins that modulate inflammasome activity
[7]. From the view point of cellular biology, accordingly, chronic
inflammation accompanied by oxidative stress is linked to various
steps involved in many diseases mentioned above [8]. Therefore
many antioxidants also have a natural anti-inflammatory action [3].
Peptides generated by digesting various proteins, including
animal and plant sources, possess biofunctional activity. These
peptides are inactive within the sequences of their parent proteins
but are released during gastrointestinal digestion or food
* Corresponding author. Department of Biotechnology, Konkuk University, #518,
Sang-Heo Research Building, Chungju 380-701, Republic of Korea. Tel.: þ82 43 840
3588; fax: þ82 43 852 3616.
E-mail address: parkpj@kku.ac.kr (P.-J. Park).
Contents lists available at SciVerse ScienceDirect
Fish & Shellfish Immunology
journal homepage: www.elsevier.com/locate/fsi
1050-4648/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.fsi.2013.01.013
Fish & Shellfish Immunology 34 (2013) 1078e1084

2. processing [9]. Once such bioactive peptides are liberated,
depending on their structural, compositional, and sequential
properties, they may exhibit various biofunctional activities. The
functional properties of a protein can be also improved by enzy-
matic hydrolysis under controlled conditions [10]. Several studies
have reported antioxidant peptides generated from seafood sources
and their potential for use as alternative antioxidants [11e15].
However, few studies have evaluated antioxidant properties of
peptides derived from enzymatic hydrolysates of Mytilus coruscus.
The hard-shelled mussel, M. coruscus, belongs to the family
Mytiloidae, is one of the most important marine shellfish species,
and is widely cultured throughout coastal areas of the Bohai Sea,
Yellow Sea, and East Sea in Korea and China [16]. Although it is an
important commercial shellfish species, little is known about its
antioxidant activity. Therefore, the aim of this study was to identify
new antioxidant peptide from M. coruscus, and characterize its
antioxidant properties in vitro and in vivo as an anti-inflammatory.
2. Materials and methods
2.1. Materials
Fresh M. coruscus was obtained from a local market (Jeonnam,
Korea). 5,5-Dimethyl-1-pyrroline N-oxide (DMPO), 2,2-azobis(2-
amidinopropane) hydrochloride (AAPH), 1,1-diphenyl-2-picrylhy-
drazyl (DPPH), (4-pyridyl-1- oxide)-N-tert-butylnitrone (4-POBN),
trichloroacetic acid (TCA), thiobarbituric acid (TBA), butylatedhy-
droxytoluene (BHT) and four enzymes including papain, pepsin, a-
chymotrypsin, and trypsin were obtained from Sigma Chemical Co.
(St. Louis, MO, USA), and the other four enzymes including Fla-
vourzyme, Neutrase, Protamex, and Alcalase were donated from
Novozyme Co. (Bagsvaerd, Denmark). All other reagents were of the
highest grade commercially available.
2.2. Preparation of enzymatic hydrolysates from M. coruscus
Prior to enzymatic hydrolysis, M. coruscus were pulverized into
a powder using a grinder (FM-909T, Hanil Co., Korea), and the enzy-
matic hydrolysates were obtained according to the method described
by Park etal. [17]. The optimum pH, temperature andcharacterization
of various enzymes are summarized in Table 1. Briefly, one hundred
milliliter of buffer solution was added to 2 g of the dried sample, and
then 40 mL (or mg) of each enzymewas addedafter pre-incubation for
30 min. The enzymatic hydrolysis reactions were performed for 8 h to
achieve an optimum hydrolytic level, and followed by immediate
heating at 100 C for 10 min to inactivate the enzyme. Finally, the
enzymatic hydrolysates were rapidly cooled to 20e25 C in an ice
bath, filtered, lyophilized, and stored at À20 C until use.
2.3. Measurement of free radical scavenging activity
2.3.1. Hydroxyl radical scavenging activity
Hydroxyl radicals were generated by the iron-catalyzed Habere
Weiss reaction (Fenton-driven HabereWeiss reaction), and the
hydroxyl radicals generated reacted rapidly with nitrone spin-trap
DMPO. The resultant DMPO-OH adduct was detectable with an ESR
spectrometer. Briefly, 0.2 ml of each enzymatic hydrolysate with
various concentrations was mixed with 0.2 ml DMPO (0.3 M),
0.2 ml FeSO4 (10 mM), and 0.2 ml H2O2 (10 mM) in a phosphate
buffer solution (pH 7.2), and then transferred to a 100 mL Teflon
capillary tube. After 2.5 min, the ESR spectrum was recorded using
a JES-FA ESR spectrometer (JEOL Ltd., Tokyo, Japan). The hydroxyl
radical scavenging activity was expressed as IC50 value, which
means concentration for scavenging 50% of hydroxyl radicals.
Experimental conditions were as follows: central field, 3475 G;
modulation frequency, 100 kHz; modulation amplitude, 2 G; mi-
crowave power, 1 mW; gain, 6.3 Â 105
and temperature, 298 K.
2.3.2. DPPH radical scavenging activity
DPPH radical scavenging activity was measured using the
method described by Nanjo et al. [18]. Briefly, 60 mL of various
concentrations of each enzymatic hydrolysate was added to 60 mL
of DPPH (60 mM) in methanol solution. After mixing vigorously for
10 s, the solution was transferred to a 100 mL Teflon capillary tube,
and the scavenging activity of each enzymatic hydrolysate on the
DPPH radical was measured using an ESR spectrometer. The spin
adduct was measured on an ESR spectrometer exactly 2 min later.
The DPPH radical scavenging activity was expressed as IC50 value,
which means concentration for scavenging 50% of DPPH radicals.
Experimental conditions were as follows: central field, 3475 G;
modulation frequency, 100 kHz; modulation amplitude, 2 G; mi-
crowave power, 5 mW; gain, 6.3 Â 105
and temperature, 298 K.
2.3.3. Superoxide radical scavenging activity
Superoxide radicals were generated by UV irradiation of a ribo-
flavin/ethylenediaminetetra-acetic acid solution. The reaction
mixtures, containing 0.1 ml of 0.8 mM riboflavin, 0.1 ml of 1.6 mM
EDTA, 0.1 ml of 800 mM DMPO and 0.1 ml of various concentrations
of peptide, were irradiated for 1 min under a UV lamp at 365 nm.
The mixtures were transferred to a 100 mL quartz capillary tube of
the ESR spectrometer for measurement. The superoxide radical
scavenging activity was expressed as IC50 value, which means
concentration for scavenging 50% of superoxide radicals. Exper-
imental conditions were as follows: central field, 3475 G; modu-
lation frequency, 100 kHz; modulation amplitude, 2 G; microwave
power, 10 mW; gain, 6.3 Â 105
and temperature, 298 K.
2.3.4. Peroxyl radical scavenging activity
Peroxyl radicals were generated by AAPH. The PBS (pH 7.4) re-
action mixtures containing 0.1 ml of 10 mM AAPH, 0.1 ml of 10 mM
4-POBN, and 0.1 ml of the indicated concentrations of the tested
samples were incubated at 37 C in a water bath for 30 min and
then transferred to a 100 mL Teflon capillary tube. The spin adduct
was recorded on an ESR spectrometer. The peroxyl radical scav-
enging activity was expressed as IC50 value, which means concen-
tration for scavenging 50% of peroxyl radicals. Measurement
conditions were as follows: central field, 3475 G; modulation fre-
quency, 100 kHz; modulation amplitude, 2 G; microwave power,
1 mW; gain, 6.3 Â 105
and temperature, 298 K.
2.4. Purification of antioxidant peptides from M. coruscus
2.4.1. Fractions from a tangential flow filtration (TFF) system
The enzymatic hydrolysates of M. coruscus were fractionated
through ultrafiltration (UF) membranes with a range of MWCO of
Table 1
Optimum hydrolysis conditions and apoptosis rate of various enzymatic extracts
from M. coruscus.
Enzyme Optimum conditions Buffer Description
pH Temperature
Flavourzyme 7.0 50
C Phosphate Endo- and exo-peptidase
activities
Neutrase 7.0 50
C Phosphate An endoprotease
Protamex 7.0 50
C Phosphate Hydrolysis of food proteins
Alcalase 7.0 50
C Phosphate A endoprotease
Papain 6.0 37
C Phosphate Endolytic cysteine protease
Pepsin 2.0 37
C Glycin-HCl From porcin gastric mucosa
a-Chymotrypsin 7.0 37
C Phosphate From bovine pancrease
Trypsin 7.0 37
C Phosphate A serine protease
E.-K. Kim et al. / Fish Shellfish Immunology 34 (2013) 1078e1084 1079

3. 30, 10, and 5 kDa using a TFF system, respectively. The fractions
were designated as follows: MWCO I referred to the filtrates that
were not passed through a 30-K MWCO membrane. MWCO II
referred to the filtrates that were passed through a 30-K MWCO
membrane but not passed through a 10-K MWCO membrane.
MWCO III referred to the filtrates that were passed through a 10-K
MWCO membrane but not passed through a 5-K MWCO mem-
brane. MWCO IV referred to filtrates that were passed through a 5-
K MWCO membrane. All fractions recovered were lyophilized in
a freeze drier for 3 days.
2.4.2. Ion exchange chromatography using a diethylaminoethyl
(DEAE)-Sephacel
Among the four MWCO fractions, MWCO I fraction, showing the
highest hydroxyl radical scavenging activity, were selected as pu-
rification material. Four milliliter of MWCO I (250 mg/ml) was
loaded onto a DEAE-Sephacel ion exchange column (74 Â 280 mm)
equilibrated with 1.0 M TriseHCl buffer (pH. 8.0) and eluted with
a linear gradient of NaCl (0e1.0 M) in the same buffer at a flow rate
of 2.0 ml/min. Each fraction was monitored at 280 nm, collected at
a volume of 3.0 ml, and desalted using dialysis membrane, then
lyophilized, and antioxidant activity was investigated. Five frac-
tions were pooled, dialyzed overnight, and lyophilized for 3 days.
The strongest antioxidant fraction was lyophilized, and chroma-
tography was conducted as the next step.
2.4.3. High-performance liquid chromatography (HPLC) using a C18
column
The fraction exhibiting the highest antioxidant activity was
further purified using reversed-phase HPLC (RP-HPLC) on a C18
column (20 Â 250 mm) with a linear gradient of acetonitrile (0e
70%) at a flow rate of 2.0 ml/min. The elution peaks were detec-
ted at 215 nm, concentrated using a rotary evaporator, and
lyophilized for 3 days.
For further purification, the fraction with the highest hydroxyl
radical scavenging activity from RP-HPLC was loaded onto a C18
column (4.0 Â 250 mm) with a linear gradient of acetonitrile (0e
70%) at a flow rate 0.5 ml/min. Potent peaks were collected, eval-
uated for antioxidant activity and then lyophilized. The fraction
that showed the highest hydroxyl radical scavenging activity on
hydroxyl radical was further applied to a gel permeation chroma-
tography (GPC) column (4.0 Â 250 mm) at a flow rate 0.5 ml/min.
The final purified peptides were analyzed for amino acid sequence.
2.5. Identification of the amino acid sequence of the purified
peptides by Edman degradation
The molecular mass and amino acid sequence of the purified
peptides were determined by automated Edman degradation using
a Milligen 6600 protein sequencer (Milligen, Watford, UK). The
purified peptide was searched against the NCBI non-redundant
peptide database (http://www.ncbi.nlm.nih.gov/blast).
2.6. Animals
Adult male mice were obtained from Samtaco Bio Korea (Osan,
Korea). The animals were kept in the animal house of the Depart-
ment of Biotechnology, Konkuk University, Chungju, under a 12 h
light and 12 h dark cycle. The mice were allowed free access to
laboratory diet and tap water. All animal experiments were carried
out as per the guidelines for animal experiments at the Bio-Food
Drug Research Center, Konkuk University and the Institutional Re-
view Board of Korean government. After a weeklong adaptation
period, mice were randomized into four groups of 10 mice each.
Control group mice received water only, the oxidative stress group
mice were administered AAPH (50 mg/kg/day), the M. coruscus
group mice were administered AAPH and the novel peptide from
M. coruscus, (5 mg/kg/day) by oral gavage. Body weight and food
intake were measured at the beginning and the end of the
experiment, respectively.
2.7. Antioxidant defenses and oxidative stress biomarkers in liver
homogenate
2.7.1. The level of thiobarbituric acid reactive substances (TBARS)
TBARS level was estimated by the method of Buege and Aust
[19]. Liver homogenate supernatants were mixed with TCA (28% w/
v in 0.25 N HCl), TBA (1% in 0.25 M acetic acid) and BHT (125 mM in
ethanol), heated for 15 min at 95 C and then placed in an ice bath.
Precipitated material was removed by centrifugation, and the
absorbance of the sample at 535 nm was determined. TBARS level
was calculated using the molar absorption coefficient of MDA
(154,000 MÀ1
cmÀ1
).
2.7.2. Superoxide dismutase (SOD)
SOD was estimated by the method described by Misra and Fri-
dovich [20]. An aliquot of 0.25 ml ice-cold chloroform was added to
0.1 ml of supernatant followed by adding 0.15 ml ice-cold ethanol.
The mixture was centrifuged at 3000 rpm for 10 min at 4 C. A
0.2 ml aliquot of the supernatant was taken, and 1.3 ml buffer,
0.5 ml EDTA, and 0.8 ml water were added. The reaction was started
by adding 0.2 ml epinephrine. Change in absorbance (DOD/min) at
480 nm was read for 3 min. The result was expressed in terms of
nmol/ml/min.
2.7.3. Catalase (CAT)
CAT activity was evaluated by the rate of H2O2 decomposition
[21]. The method was based on H2O2 degradation by the action of
CAT contained in the examined samples. In this procedure, 50 mM
phosphate buffer (pH 7.0) was used with 30 mM H2O2 as substrate.
CAT activity was expressed as mM H2O2/ml/min.
2.7.4. Glutathione peroxidase (GPx)
GPx activity was determined following the oxidation of nicotin
amide adenine dinucleotide phosphate (NADPH) with t-butyl hy-
droperoxide as a substrate [22]. This reaction was proceeded by the
action of GPx contained in the samples examined with t-butyl
hydroperoxide (3 mM) as substrate in 0.5 M phosphate buffer, pH
7.0, at 37 C. GPx activity was expressed as nmol NADPH/ml/min.
2.7.5. Glutathione-s-transferase (GST)
The activity of GST toward 1-chloro-2,4-dinitrobenzene (CDNB)
was determined by a previous method [23]. The method was based
on the reaction of CDNB with the eSH group of GSH catalyzed by
GST contained in the samples. The reaction proceeded in the
presence of 1 mM GSH in phosphate buffer (pH 6.5) at 37 C. GST
activity was expressed as nmol GSH/ml/min. A GST Assay Kit was
utilized with 1-Chloro-2,4-dinitrobenzene (CDNB) which is suit-
able for the broadest GST isozyme range. The increase in absorb-
ance at 340 nm was determined upon conjugation of the thiol
group of glutathione to the CDNB substrate.
2.8. Statistical methods
Statistical analysis of data was carried out with the GraphPad
PRISM program (GraphPad Software, Inc., San Diego, CA, USA).
Multiple group data were analyzed using a one-way analysis of
variance followed by Bonferroni post-hoc tests. All results are
expressed as mean Æ standard deviation of comparative fold dif-
ferences. Data are representative of three independent experiments.
E.-K. Kim et al. / Fish Shellfish Immunology 34 (2013) 1078e10841080

4. 3. Results
3.1. Preparation of M. coruscus protein hydrolysates and their
antioxidant properties
The free radical scavenging activities of the hydrolysates were
evaluated using four free radical including hydroxyl, DPPH, alkyl
and superoxide by employing an ESR spectrometer. As shown in
Table 2, the highest antioxidant activity was observed for papain
hydrolysates on hydroxyl, DPPH and alkyl radical, and also high
activity with superoxide radical. Therefore, papain hydrolysates
were selected for further study.
3.2. Purification of an antioxidant peptide from M. coruscus
The 50% inhibitory concentrations of the A: MWCO I, B: MWCO II
and C: MWCO III were 0.368, 0.825, and 0.745 mg/ml, respectively
(Table 3).
The MWCO I fraction exhibited the highest hydroxyl radical,
which is the strongest free radical, scavenging activity and was
selected for further study. The lyophilized active fraction MWCO I
was a further separated by ion exchange chromatography, and the
fraction was divided into five subfractions (Fig.1). Each fraction was
pooled, lyophilized, and measured for antioxidant activity in the
hydroxyl radical scavenging activity. As a result, A, B, C, and E had
scavenging activities, and the IC50 values were 0.261, 0.373,
0.318,and 0.274 mg/ml on the hydroxyl radical, respectively (Fig. 1).
Fraction A exhibited the highest hydroxyl radical scavenging ac-
tivity. Therefore, fraction A was selected for the next step. The
lyophilized active fraction A was further separated by RP-HPLC on
a C18 column (20 Â 250 mm) using a linear gradient of acetonitrile
(0e70%), and the fraction was divided into six subfractions (A-I, A-
II, A-III, A-IV, A-V and A-VI) (Fig. 2). The lyophilized active fraction
A-II had the highest hydroxyl radical scavenging activity with the
IC50 value of 0.221 mg/ml and was further subjected to a second
separation by RP-HPLC on a C18 column (4.0 Â 250 mm) using
a linear gradient of acetonitrile (0e70%), and fractionated into two
subfractions (Fig. 3). The fractions were pooled and lyophilized.
Among all fractions collected, fraction A-II-i exhibited the strongest
hydroxyl radical scavenging activity (IC50, 0.160 mg/ml). To obtain
a purified peptide, A-II-i was rechromatographed by a third sepa-
ration on HPLC using a GPC column (8.0 Â 300 mm). Finally, we
obtained the purified antioxidant peptide (Fig. 4). The amino acid
sequence was Ser-Leu-Pro-Ile-Gly-Leu-Met-Ile-Ala-Met. The puri-
fied peptide was searched against the NCBI database, and the range
of the homology identification of the peptide is 41%e83%. Most of
them are hypothetical things from microorganism such as fungi
and bacteria. Interestingly, some of them were nitric-oxide reduc-
tase subunit B (homology 73%); therefore the amino acid is able to
be expected to possess anti-inflammatory property. In addition, the
free radical scavenging activity of the peptide was investigated, and
the IC50 values of the hydroxyl, DPPH, superoxide, and peroxyl
radical scavenging activities were 0.118, 0.154, 0.316, and 0.243 mg/
ml, respectively (Fig. 4).
3.3. Effect of M. coruscus on antioxidant defense and oxidative
stress biomarkers in liver
Body weight gain and food intake did not significantly differ
among the groups (data not shown). To explore the effects of
antioxidant defenses on oxidative stress, the TBARS and anti-
oxidative enzymes levels (SOD, CAT, GPx, and GST) were evaluated
in liver tissues. Compared to control animals, the TBARS level in the
oxidative stress mice group increased significantly, whereas the
level in the M. coruscus mice group level was similar to that
observed in the control (Fig. 5A). SOD is an actual indicator of the
antioxidant capacity of the body and is involved in the protection
against damage caused by oxidative stress. The oxidative stress and
M. coruscus mice groups showed increased SOD activity of 1.35 and
1.5-fold compared to that in the control group (Fig. 5B). The results
presented in Fig. 5C show that the oxidative stress group had sig-
nificantly increased GST activity, and that the M. coruscus group had
a decreased GST level compared to that in the oxidative stress
group (Fig. 5C). Meanwhile, M. coruscus had no effect on CAT and
GPx activities (data not shown).
4. Discussion
The peptides are produced by enzymatic hydrolysis of food
proteins to release the peptide sequences, followed by post-hy-
drolysis processing to isolate bioactive peptides from a complex
mixture of other inactive molecules [24]. Dietary consumption of
antioxidants appears to provide further benefits to the endogenous
antioxidant defense in the fight against oxidative stress [25]. Many
of these properties are attributed to physiologically active peptides
in protein molecules [24].These peptides are present in the raw
material or are generated during food processing and protein hy-
drolysis by digestive enzymes. Many antioxidant peptides have
Table 2
IC50 values of the radical scavenging activities of various enzymatic hydrolysates from M. coruscus.
Enzyme Radical
Hydroxyl DPPH Alkyl Superoxide
Flavourzyme 1.191 Æ 0.092 0.912 Æ 0.039 0.520 Æ 0.053* 3.030 Æ 0.165
Neutrase 1.305 Æ 0.103 1.180 Æ 0.143 0.610 Æ 0.072 2.430 Æ 0.134
Protamex 1.030 Æ 0.035 1.459 Æ 0.156 0.770 Æ 0.035 1.660 Æ 0.542*
Alcalase 0.874 Æ 0.021 1.773 Æ 0.109 0.673 Æ 0.098 10.02 Æ 1.470
Papain 0.532 Æ 0.023* 0.299 Æ 0.035** 0.531 Æ 0.074* 2.330 Æ 0.938
Pepsin 3.632 Æ 0.523 0.681 Æ 0.017 0.622 Æ 0.091 1.434 Æ 0.053*
a-chymotrypsin 3.384 Æ 0.721 0.896 Æ 0.020 0.713 Æ 0.054 2.077 Æ 0.641
Trypsin 0.735 Æ 0.074 0.613 Æ 0.045 0.851 Æ 0.087 2.155 Æ 0.852
* and ** are significantly different at p 0.05 and p 0.01 as analyzed by Bonferroni post-hoc test, respectively.
Table 3
Hydroxyl radical scavenging activities of papain hydrolysates from M. coruscus
with various molecular weights.
MWCO* (Da) IC50 (mg/ml)
30,000 MWCO I 0.368 Æ 0.053**
10,000 MWCO II 30,000 0.825 Æ 0.083
5000 MWCO III 10,000 0.745 Æ 0.071
MWCO IV 5000 e
*Molecular weight cut off.
**Is significantly different at p 0.05 and p 0.01 as analyzed by Bonferroni
post-hoc test.
E.-K. Kim et al. / Fish Shellfish Immunology 34 (2013) 1078e1084 1081

5. been extracted by enzymatic hydrolysis using various enzymes.
One of the approaches for the effective release of bioactive peptides
from protein sources is enzymatic hydrolysis, which is widely
applied to improve and upgrade the functional and nutritional
properties of protein [11]. In the present study, we also utilized
eight proteases to extract antioxidant peptides from M. coruscus,
and we evaluated their antioxidant activity using free radical
scavenging capacity. Among the eight proteases, papain hydroly-
sates were more effective free radical scavengers than other hy-
drolysates. Therefore, we separated papain hydrolysates with
different molecular weight distributions using TFF membranes,
because many researchers have reported that short peptides are
Fig. 2. Reversed-phase high-performance liquid chromatography (HPLC) pattern on
a C18 column (20 Â 250 mm) of the A active fraction, and the hydroxyl radical scav-
enging activities (upper panel) of the fractions. HPLC was carried out with a linear
CH3CN gradient (0e70%) at a 2.0 ml/min flow rate using a UV detector at 215 nm.
Lower case letters aed indicate values are significantly different at p 0.05 as analyzed
by Bonferroni multiple comparison test.
Fig. 3. Reversed-phase HPLC pattern from a C18 column (4.0 Â 250 mm) of the A-I
active fraction, and the hydroxyl radical scavenging activities (upper panel) of the
fractions. HPLC was carried out with a linear CH3CN gradient (0e70%) at a 0.5 ml/min
flow rate using a UV detector at 215 nm. Lower case letters aeb indicate values are
significantly different at p 0.05 as analyzed by Bonferroni multiple comparison test.
Fig. 4. HPLC with a GPC column of the A-I-ii active fraction, and the radical scavenging
activities (upper panel) of the fraction. HPLC was carried out with a 0.5 ml/min flow
rate using a UV detector at 215 nm.
Fig. 1. Ion-exchange chromatogram of molecular weight cut off I (MWCO I). The elu-
tion was performed at 2.0 ml/min flow rate with a linear NaCl gradient (0e1.0 M) in
1.0 M TriseHCl buffer, pH 8.0, and monitored at 280 nm. Lower case letters aed indi-
cate values are significantly different at p 0.05 as analyzed by Bonferroni multiple
comparison test.
E.-K. Kim et al. / Fish Shellfish Immunology 34 (2013) 1078e10841082

6. more potent bioactive peptides [26], but our result did not agree,
and showed that large peptides could also be bioactive. Moreover
the activity is stronger than enzymatic hydrolysates from Laminaria
japonica [11]. Although the possibility, there are still some problem
due to the size of the peptide which greatly affects their absorption
across the enterocytes and bioavailability in target tissues. There-
fore, we may consider other steps to reduce the size as depend on
the animal test.
However, in this study, a large peptide showed stronger free
radical scavenging activity, and it was not concert with previous
researches. Although the size of the peptide from M. coruscus is
larger than other bioactive peptides, its antioxidant activity indi-
cate that the antioxidant peptide from M. coruscus possess potent
possibility to be applied for functional food. This approach, how-
ever, requires an understanding of the structural requirements of
the peptides for bioactivity, and exploiting the unique structural
features in concentrating the particular peptides of interest during
processing [27].
Antioxidant activity has been attributed to certain amino acid
sequences [28]. High amounts of hydrophobic amino acids such as
Leu, Met, and Ile are associated with antioxidant potency [29].
Davalos et al. reported that the peptide contained-Met, Trp, and Tyr
showed the highest antioxidant activity [30]. The addition of a Leu
or Pro residue to the N-terminus of a His-His dipeptide enhanced
antioxidant activity and facilitated further synergy with non-
peptide antioxidants such as BHT. The novel peptide from the
M. coruscus enzymatic hydrolysates was not short peptide, but it
had plenty of hydrophobic peptides as well as Met. Therefore, we
speculated that the radical scavenging activity of the peptide
obtained from the M. coruscus hydrolysate could be attributed to
the presence of Leu, Met, and Ile.
ROS are continually formed as by-products of aerobic metabo-
lism; these compounds carry out physiological functions as well as
have deleterious effects [31]. Overproduction of ROS results in
oxidative stress, which can cause significant damage to cellular
proteins, lipids, and DNA, and ROS play an important role in
degenerative and inflammatory diseases and cancer [32]. The po-
tential harmful effects of ROS are controlled by cellular antioxidant
defense mechanisms including enzymatic defense systems such as
SOD, CAT, and GPx, as well as nonenzymatic defense systems such
as GSH, vitamin A, vitamin C, and vitamin E. The toxic effects of free
radicals are kept under control by a fragile balance between the rate
of their production and the rate of their elimination by these de-
fense systems [33]. During aerobic respiration to generate ATP in
mitochondria, leakage of electrons frequently produces mito-
chondrial superoxide anions that are rapidly reduced to H2O2 by
manganese SOD. Because CAT, which metabolizes H2O2, is absent in
the mitochondria of most animal cells [34], mitochondrial GPx and
GST play a key role in metabolizing H2O2. In this study, SOD activity
was increased in the peptide treated-group, and no changes of GST
level. We assumed that the oxidative stress in the M. coruscus group
was eliminated oxidative stress earlier, and that may be why their
GST level was unchanged. Meanwhile, the liver is the principal
organ involved in oxidative and detoxification processes. In the
initial stages of many diseases, oxidative stress biomarkers are
elevated in the liver [35]. TBARS level is widely used as a lipid
peroxidation biomarker [36], and he antioxidant peptide from the
M. coruscus hydrolysates inhibited MDA, and regulated anti-
oxidative enzymes activities in vivo.
It is known that inflammation is a natural local reaction of
mammalian tissue to a variety of hostile agents including parasites,
pathogenic micro-organisms, toxic chemical substances, and
physical damage to tissue [37]. The processes associated with
the inflammatory response are complex, but important aspects,
which have been exploited for screening for anti-inflammatory
Fig. 5. Antioxidant activity of the peptide from M. coruscus in vivo. (A) Effect of the
antioxidant peptide from M. coruscus on lipid peroxidation induced by AAPH treatment
in vivo. (B) Effect of the antioxidant peptide from M. coruscus on SOD in vivo. (C) Effect
of the antioxidant peptide from M. coruscus on GST activity in vivo. Values are given as
mean Æ SD. *(p 0.05) and *** (p 0.001) are significantly different as analyzed by
paired t-test.
E.-K. Kim et al. / Fish Shellfish Immunology 34 (2013) 1078e1084 1083

7. compounds are the various functions of neutrophils, the metabolic
products of arachidonic acid and the role played by ROS [3]. Several
lines of evidence suggest that ROS play a main role in cellular
damage and are implicated in many inflammatory conditions [38].
Therefore, the novel antioxidant peptide from M. coruscus hydro-
lysates possesses a potent antioxidant activity against oxidative
stress in mice, and can promote anti-inflammatory response.
5. Conclusions
We obtained the antioxidant peptide, and the sequence was Ser-
Leu-Pro-Ile-Gly-Leu-Met-Ile-Ala-Met at N-terminal. We inves-
tigated the effects of the peptide on free radical scavenging activity
in vitro and antioxidative enzyme activity in vivo. The novel anti-
oxidant peptide efficiently quenched four free radicals, inhibited
MDA level, and regulated antioxidative enzymes activity.
Taken together, the peptide possesses a potent antioxidant ac-
tivity and great potential to enhance anti-inflammatory system.
However, further researches on the structure of the peptides, and
inflammatory studies are needed.
Acknowledgments
This work was supported by Korea Institute of Planning and
Evaluation for Technology in Agriculture, Forestry and Fisheries
(iPET).
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