Food Chemistry 124 (2011) 188194

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Food Chemistry
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In vitro antioxidant activity and in vivo anti-fatigue effect of loach

(Misgurnus anguillicaudatus) peptides prepared by papain digestion
Lijun You a, Mouming Zhao a,*, Joe M. Regenstein b, Jiaoyan Ren a
a
College of Light Industry and Food Sciences, South China University of Technology, Guangzhou 510640, China
b
Department of Food Science, Cornell University, Ithaca, NY 14853-7201, USA

a r t i c l e i n f o a b s t r a c t

Article history: The in vitro antioxidant activity and in vivo anti-fatigue activity of loach peptide (LP) were determined.
Received 5 November 2009 Results showed that LP contained the amino acids, which were expected to contribute to its antioxidant
Received in revised form 19 April 2010 and anti-fatigue activities. LP could scavenge 1,1-diphenyl-2-picrylhydrazyl (DPPH) (IC50 17.0 0.54 mg/
Accepted 3 June 2010
ml) and hydroxyl radicals (IC50 2.64 0.29 mg/ml). It could chelate cupric ion and inhibit the lipid perox-
idation in a linoleic acid emulsion system. It also prolonged the swimming time to exhaustion of mice by
2028% compared to the control. It increased the levels of blood glucose (2842% increase) and liver gly-
Keywords:
cogen (2.33.0-fold increase). It decreased the levels of lactic acid and blood urea nitrogen by 10.927.5%
Loach peptide
Antioxidant activity
and 8.617.5%, respectively. It also improved the endogenous cellular antioxidant enzymes in mice by
Anti-fatigue effect increasing the activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase
Free radicals (GSH-Px). Therefore, LP can increase an endurance capacity and facilitate recovery from fatigue.
Blood glucose 2010 Elsevier Ltd. All rights reserved.

1. Introduction
Fatigue is best dened as the difculty in initiating or sustaining
voluntary activities, and can be classied into mental and physical
fatigue. Physical fatigue is thought to be accompanied by deterio-
ration in performance (Tanaka et al., 2008). There are several the-
ories about the mechanisms of exercise-induced fatigue:
exhaustion theory, clogging theory, radical theory, homeo-
stasis disturbance theory, protective inhibition theory, and
mutation theory (Wang et al., 2008). The exhaustion theory
suggests that during exercise, many energy sources, such as glu-
cose (GLU) and liver glycogen, will be exhausted, thus leading to
physical fatigue. Several reports showed that post-exercise nutri-
tion through the administration of proteins, peptides or amino
acids can facilitate recovery from fatigue (Wang et al., 2008). Com-
pared with proteins, peptides, as nutrition supplements, not only
are absorbed quickly and easily without competition from amino
acids, but also promote the use of amino acids, proteins and glu-
cose (van Loon, Saris, Kruijshoop, & Wagenmakers, 2000). There-
fore, peptides may be useful in assisting in counteracting and
ameliorating physical fatigue. According to the clogging theory,
the over accumulation of serum lactic acid (LA) and blood urea
nitrogen (BUN) will also result in metabolic disorders leading to
fatigue.
* Corresponding author. Tel./fax: +86 20 87113914.
E-mail address: femmzhao@scut.edu.cn (M. Zhao).
Among the fatigue mechanisms, the radical theory has been
attracting more interest. Free radicals are intermediate metabolites
of many vital biochemical events in the body, and they are in a
dynamic balance between their production and clearance. Har-
mans classical radical theory suggests that intense exercise
can produce an imbalance between the bodys oxidation system
and its anti-oxidation system. The accumulation of reactive free
radicals will put the body in a state of oxidative stress and bring
injury to the body by attacking large molecules and cell organs
(Wang et al., 2008). Muscle cells contain complex endogenous cel-
lular defense mechanisms to eliminate reactive oxygen species,
such as superoxide dismutase (SOD), glutathione peroxidase
(GSH-Px) and catalase (CAT), and to protect among other things
against exercise-induced oxidative injury. Moreover, some reports
(Yu et al., 2006) showed that exogenous dietary antioxidants can
also decrease the contribution of exercise-induced oxidative stress
and improve the animals physiological condition. The reason may
be that exogenous antioxidants can promote or interact with
endogenous antioxidants to form a cooperative network of cellular
antioxidants (Mizuno et al., 2008). However, the mechanisms have
not been fully elucidated. Nevertheless, consumers are seeking
more natural antioxidant components in their diet to reduce oxida-
tive damage and ght against fatigue, without the side effects of
other anti-fatigue drugs. Such antioxidant components have been
found in plants, such as red mold rice (Wang, Shieh, Kuo, Lee, &
Pan, 2006) and tissue culture extracts of Saussurea (Jia & Wu,
2008), and in herbs, such as Trichopus zeylanicus (Tharakan, Dhan-
asekaran, & Manyam, 2005). However, there are few reports

0308-8146/$ - see front matter 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodchem.2010.06.007
 L. You et al. / Food Chemistry 124 (2011) 188194
regarding antioxidant peptides from animal sources having anti-
fatigue activity.
Loach (Misgurnus anguillicaudatus) is a common freshwater sh
in East Asia. It has been familiar to the Chinese since ancient times,
for its desirable taste and avour. It has also traditionally been con-
sidered as a folk remedy for physical and mental fatigue in the
southern part of China. However, the mechanisms of its anti-fati-
gue activity have not been explored.
In this study, we investigated the in vitro antioxidant activities
and the in vivo anti-fatigue effect of loach peptide (LP) previously
prepared by papain digestion. The in vitro antioxidant activities
were studied by determining the 1,1-diphenyl-2-picrylhydrazyl
(DPPH) and the hydroxyl radicals scavenging activities, the chelat-
ing ability with Cu2+ ions, and the lipid peroxidation inhibitory
activity in a linoleic acid emulsion system, using glutathione
(GSH) as a control. In the subsequent in vivo study, mice were
fed with high or low doses of LP for 4 weeks. Then, an exhaustive
swimming time was recorded and several biochemical parameters
related to fatigue [GLU, liver glycogen, LA, BUN, lactate dehydroge-
nase (LDH), creatine kinase (CK), SOD, CAT, GSH-Px] were also
determined to explore the anti-fatigue activity of loach peptides.
2. Materials and methods
2.1. Loach peptide preparation
Live loach (Misgurnus anguilliacaudatus, 8.4 1.1 g body weight
and 9.8 3 cm body length) were purchased from a local market in
Guangzhou, China, and transported to the lab within 10 min. After
killing, the meat (without head, tail, skin, bone and blood) was col-
lected and ground twice through a meat grinder with a plate with
4 mm holes (MM12, Shaoguan Food Machine Co., Shaoguan, Chi-
na). The ground meat was stored in a polyethylene bag at 18 C
until use, for a maximum of 4 months. Fifty grams of loach meat
were mixed with 100 ml of distilled water and homogenised at a
speed of 10,000 rpm for 1 min using a basic homogeniser (T25,
Ika, Staufen, Germany). The homogenate was hydrolyzed with pa-
pain (6  105 U/g) (Baiao Biochemistry Co., Jiangmen, China) at
55 C for 4.5 h. The enzyme to substrate ratio was 3:1000 (w/w).
The hydrolysis was conducted at pH 7.0 (SL1-PHS-3B pH-metre,
Wuhan Midwest Instrument Co. Ltd., Wuhan, China) in a water
bath shaker (New Brunswick Scientics C24, Jintan, China). After
hydrolysis, the enzyme was inactivated by placing the samples in
boiling water for 15 min. The hydrolysates were centrifuged in a
GL-21M refrigerated centrifuge (Xiangyi Instrument Co. Ltd.,
Changsha, China) at 5000g for 20 min, and the supernatants were
fractionated through ultraltration membranes using a bioreactor
system (Vivaow 200, Vivascience, Sartorius, Goettingen, Ger-
many) with a molecular weight cutoff (MWCO) of 5 kDa. The frac-
tion with molecular weight less than 5 kDa was lyophilised (R2L-
100KPS, Kyowa Vacuum Engineering, Tokyo, Japan) and stored in
a desiccator for further use. The estimated protein content of the
LP powder was 85%, determined by the Kjeldahl method (AOAC,
1995) using a conversion factor of 6.25. The fractions in the LP with
molecular weight <1 kDa, 13 kDa, 35 kDa and >5 kDa were 32%,
48%, 18% and 2%, respectively, determined by gel permeation chro-
matography, as described in our previous report (You, Zhao, Cui,
Zhao, & Yang, 2008).
2.2. Animals
All the in vivo tests were carried by the School of Pharmaceuti-
cal Sciences of Southern Medical University (Guangzhou, China),
which obtained the permission for performing the research proto-
cols and all animal experiments conducted during the present
189
study from the ethics committee of Southern Medical University.
Fifty-four male NIH mice (1822 g, specic pathogen-free grade,
SPF, Approval No. 2007A058) were purchased from the Academy
of Experimental Animal Center of Southern Medical University
(Guangzhou, China). They were housed in a SPF level laboratory
in the School of Pharmaceutical Sciences of Southern Medical Uni-
versity. All experimental procedures were conducted under the
oversight and approval of the Academy of Experimental Animal
Center of Southern Medical University and in strict accordance
with the NIH Guide for the Care and Use of Laboratory Animals
(NIH, 2002). Animals were allowed to adapt to their surroundings
for 1 week before starting the experiments. Mice were housed 56
per cage at room temperature (22 2 C) and moderate humidity
(50 10%), with a 12/12-h lightdark cycle; noise was <60 dB. They
were fed a balanced murine diet provided by the Academy of
Experimental Animal Center of Southern Medical University, and
had drinking water available ad libitum. After adaptation, the 54
mice were randomly divided into six groups: two control groups,
two LP treatment groups at a high dose (LP-H), and two groups
at a low dose (LP-L), with 9 mice each. One group from each set
was used for the exhaustive swimming test. The other group was
used for collecting the blood to determine biochemical parameters
related to fatigue, after swimming for 30 min. For 4 week, the LP-H
mice were given 5 mg/(g d) of LP, and the LP-L mice were given
1 mg/(g d) by intraperitoneal injection every day at 1:00
3:00 pm. The control groups received distilled water. After each
treatment, all groups of the mice were allowed to rest 30 min
and were forced to swim for 20 min to become accustomed to
swimming (see below).
2.3. Amino acid analysis
The amino acid prole of LP was determined according to the
method of You et al. (2008). The amino acid composition was
determined by high-performance liquid chromatography (Waters,
Milford, MA, USA) equipped with a PICO.TAG column. The total
amino acids (except for tryptophan) were determined after hydro-
lysis at 110 C for 24 h with 6 M HCl, prior to the derivatisation
with phenyl isothiocyanate. Alkaline hydrolysis was also done for
the determination of tryptophan level. The amino acid standards
(SigmaAldrich, St. Louis, MO, USA) were analysed using the same
conditions as for the samples.
2.4. Determination of in vitro antioxidant activities of LP
2.4.1. Hydroxyl radical scavenging activity assay
The hydroxyl radical scavenging activity was assayed according
to the method of Li, Jiang, Zhang, Mu, and Liu (2008), with some
modications. A mix of 600 ll of 1,10-phenanthroline (5.0 mM),
600 ll of FeSO4 (5.0 mM) and 600 ll of ethylenediaminetetraacetic
acid (EDTA) (15 mM) were mixed with 400 ll of sodium phosphate
buffer (0.2 M, pH 7.4). Then, 600 ll of LP (025 mg/ml) and 800 ll
of H2O2 (0.01%) were added. The mixture was incubated at 37 C
for 60 min, and the absorbance was measured at 536 nm (UV754,
Xianjian Scientic Instrument Co., Shanghai, China). GSH (Sigma
Aldrich, St. Louis, MO, USA) at 010 mg/ml was used as a control.
The following equation was used:
Hydroxyl radical scavenging activity %
As  A0  100=Ac  A0
where As is the absorbance of the sample; A0 is the absorbance of
the blank solution using distilled water instead of sample; and Ac
is the absorbance of a control solution in the absence of H2O2. The
plot of scavenging activity against the concentration of the hydroly-
190 L. You et al. / Food Chemistry 124 (2011) 188194
sate was prepared, and the IC50 (concentration of samples to de-
crease the scavenging activity by 50%) obtained.
2.4.2. DPPH radical scavenging activity assay
The DPPH radical scavenging activity was determined by the
method of Wu, Chen, and Shiau (2003), with a slight modication.
Two millilitres of LP (05 mg/ml) were mixed with 2.0 ml of
0.15 mM DPPH that was dissolved in 95% ethanol. The mixture
was then shaken vigorously using a mixer (QT-1 Mixer, Tianchen
Technological Co. Ltd., Shanghai, China), and was kept for 30 min
in the dark. The absorbance of the resulting solution was recorded
at 517 nm. GSH at 05 mg/ml was used as a control. The scaveng-
ing activity was calculated using the following equation:
Scavenging activity % ADPPH sample  Asample control
 100=ADPPH blank
where ADPPH sample is the value for the 2 ml of sample solution mixed
with the DPPH solution; Asample control is the value for the 2 ml of
sample solution mixed with the 2 ml of 95% ethanol; and ADPPH blank
is the value for the 2 ml of 95% ethanol mixed with the DPPH solu-
tion. The plot of the scavenging activity against the concentration of
sample was prepared, and the IC50 obtained.
2.4.3. Cupric ion chelating activity
The ability of LP to chelate pro-oxidative Cu2+ was investigated
according to Zhu, Chen, Tang, and Xiong (2008). In the chelation
test, 1 ml of 2 mM CuSO4 was mixed with 1 ml of pyridine (pH
7.0) and 20 ll of 0.1% pyrocatechol violet. After the addition of
1 ml of LP (05 mg/ml), the disappearance of the blue color, due
to dissociation of Cu2+, was recorded by measuring the absorbance
at 632 nm after 5 min of reaction. An equivalent volume of distilled
water instead of the sample was used for the blank. GSH at 0
5 mg/ml was used as a control. The Cu2+ chelating activity of the
GI digests were calculated as:
Cu chelating activity A0  As =A0   100%
where As is the absorbance of the sample and A0 is the absorbance of
the blank solution using distilled water instead of sample. The plot
of the scavenging activity against the concentration of sample was
prepared, and the IC50 obtained.
2.4.4. Measurement of the lipid peroxidation inhibition activity in a
linoleic acid emulsion system
The lipid peroxidation inhibition activity of LP was measured in
a linoleic acid emulsion system according to the methods of Qian,
Jung, Byun, and Kim (2008). Briey, 2.0 ml of LP (020 mg/ml) was
mixed with 2 ml of 2.5% linoleic acid dissolved in 95% ethanol.
Then, 4 ml of 50 mM sodium phosphate buffer (pH 7.0) and 2 ml
of distilled water were added. The mixture was incubated in a
50 ml conical ask with a screw cap at 40 1 C in a dark room,
and the degree of oxidation was evaluated by measuring the FeSCN
values described below. The reaction solution (100 ll), incubated
in the linoleic acid model system, was mixed at different intervals
during the incubation period with 9.7 ml of 75% ethanol, 0.1 ml of
30% NH4SCN, and 0.1 ml of 20 mM FeCl2 solution in 3.5% HCl. After
3 min, the SCN value was measured by reading the absorbance at
500 nm. An equivalent volume of distilled water was used as blank.
GSH at 020 mg/ml was used as a control.
The lipid peroxidation inhibition activity %
1  As;t144 h  As;t0 h   100=A0;t144 h  A0;t0 h
where As,t=144 h and As,t =0 h are the absorbances for the sample at
144 h and 0 h, respectively & A0,t=144 h and A0,t=0 h are the absor-
bances for the blank at 144 h and 0 h, respectively. The plot of the
scavenging activity against the concentration of sample was pre-
pared, and the IC50 obtained.
2.5. In vivo anti-fatigue effect of LP
2.5.1. Exhaustive swimming test
After the nal treatment with LP or distilled water, the mice
were allowed to rest for 30 min. Then, they were placed in the
swimming tank (50 cm  50 cm  40 cm) with 30 cm deep water
at 25 1 C. The current in the pool was generated by circulating
water with a pump, and the strength of the current was adjusted
to 8 l/min with a water ow metre (type F45500, Blue White Co.,
Westminster, CA, USA). The water was agitated to make the mice
limbs keep moving. The mice were determined to be exhausted
when they failed to rise to the surface to breathe after 7 s (Jung,
Han, Kwon, Lee, & Kim, 2007).
2.5.2. Measuring biochemical parameters related to fatigue
After the nal treatment with LP or distilled water, the mice
were allowed to rest for 30 min. Then they were placed in the
swimming tank (50 cm  50 cm  40 cm) with 30 cm deep of
water (temperature: 25 1 C). After swimming for 30 min, they
were taken out. Blood was collected from the orbital sinus to deter-
mine GLU, LA and BUN content and CK, SOD, GSH-Px, LDH and CAT
activity (Jung et al., 2007). The livers of the mice were also taken to
determine the content of liver glycogen (see below). All of the bio-
chemical parameters were determined by using an automated Bio-
chemistry Analyzer (7600-010, Hitachi, Japan).
The glucose, BUN, CK and LDH activities were determined using
commercial diagnostic kits (Product Nos. 000060110, 000000280,
000000090 and 000000131, respectively, Biosino Bio-technology
and Science Inc., Beijing, China). LA was determined using a com-
mercial diagnostic kit (Product No. LC6351, Beijing Leadman Bio-
chemistry Technology Co. Ltd., Beijing, China). The SOD activity
was determined using SOD Assay Kit A001 (Institute of Biological
Engineering of Nanjing Jianchen, Nanjing, China).
The GSH-Px activity was determined with a GSH-Px Assay Kit
A005 (Institute of Biological Engineering of Nanjing Jianchen, Nan-
jing, China). The GSH-Px has the ability to decompose hydrogen
peroxide (H2O2) and other organic hydroperoxides (ROOH). The
reaction uses GSH to complete the reaction using H2O2, as the sub-
strate. The consumption of nicotinamide adenine dinucleotide
phosphate (NADPH) is used to determine the GSH-Px activity.
The catalase activity was determined colorimetrically with a
CAT Assay Kit A007 (Institute of Biological Engineering of Nanjing
Jianchen, Nanjing, China). The test is based on the decomposition
of the H2O2 optical density at 415 nm by CAT.
The livers of the mice were dissected immediately after taking
them out, washed with 0.9% saline, and blotted dry with lter pa-
pers. Liver samples (100 mg) were accurately weighed, and
homogenised in 8 ml of homogenisation buffer from a Liver Glyco-
gen Assay Kit A043 (Institute of Biological Engineering of Nanjing
Jianchen, Nanjing, China). The liver glycogen was determined
according to the recommended procedures.
2.6. Statistical analysis
All the tests were conducted in triplicate. The experimental data
were expressed as mean standard error. The results were sub-
jected to one-way analysis of variance (ANOVA). LSD and Dunnetts
T3 tests were performed to determine the signicant difference be-
tween samples within the 95% condence interval, using SPSS 13.0
software (SPSS Inc., Chicago, IL, USA).
 L. You et al. / Food Chemistry 124 (2011) 188194 191

3. Results and discussion no acids, proteins and DNA. This can lead to physiological disorders
(Cacciuttolo, Trinh, Lumpkin, & Rao, 1993). DPPH has been used
3.1. Amino acid composition of LP extensively as the free radical to evaluate reducing substances.
As shown in Table 2, LP had a hydroxyl IC50 value of
The amino acid compositions of the protein hydrolysates are di- 17.0 0.54 mg/ml and a DPPH IC50 value of 2.64 0.29 mg/ml,
rectly related to their bioactive activities. Histidine or histidine- which was 2.6-fold and 2.4-fold higher than that of GSH, respec-
containing peptides have chelating and lipid radical trapping abil- tively. The IC50 value for the chelation activity of Cu2+ ion was
ity due to the imidazole ring (Uchida & Kawakishi, 1992). Aromatic 2.89 0.33 mg/ml, which was 1.8-fold higher than that of GSH.
amino acids, such as tryptophan exhibit antioxidant activity since The lipid peroxidation is thought to proceed via a radical-mediated
they can donate protons easily to electron-decient radicals. Sev- abstraction of hydrogen atoms from methylene carbons in polyun-
eral amino acids, such as tyrosine, methionine, histidine, lysine, saturated fatty acids (Rajapakse, Mendis, Jung, Je, & Kim, 2005). The
and tryptophan, have generally been considered as antioxidants lipid peroxidation inhibitory activity of LP of the linoleic acid sys-
(Chen, Muramoto, Yamauchi, & Nokihara, 1996). As shown in Table tem gave an IC50 value of 12.3 0.98 mg/ml, which was not statis-
1, the loach peptide contains 24.8% of the above ve amino acids, tical different from that of GSH (P > 0.05). Nevertheless, the
suggesting that it might have potential antioxidant activity. average molecular weight of LP was four times more than that of
Amino acids also play an important role in the regulatory metab- GSH (data not shown). That is to say that LP had stronger antioxi-
olism involved in muscular activity. Some amino acid, especially dant activities than GSH at the same molecular concentration.
the branched chain amino acids, can improve the exercise capabil- Therefore, LP has in vitro antioxidant activities, which indicates
ity and markedly retard the catabolism of protein in the muscle that it may have the potential to reduce the oxidative stress
during exercise (Blomstrand & Newsholme, 1992). Bazzarre, Mur- in vivo and to ght fatigue.
doch, Wu, Herr, and Snider (1992) reported that the amount of ami-
no acids, especially alanine, glycine, valine, isoleucine, threonine,
3.3. LP prolonged the exhaustive swimming time
serine and tyrosine in the plasma will decrease rapidly during an
endurance test. As shown in Table 1, the loach peptide contains
A direct measure of an anti-fatigue effect is the increase in exer-
32.2% of the above amino acids, suggesting that it might enhance
cise tolerance. Swimming to exhaustion is an experimental exer-
exercise capability. Glutamic acid was found to have a very positive
cise model to evaluate anti-fatigue; it works well for evaluating
effect on the nervous system and would also be helpful during exer-
the endurance capacity of mice, and gives a high reproducibility
cise (Guezennec et al., 1998). Aspartic acid was considered to be
(Zhang, Yao, Bao, & Zhang, 2006). Reduced susceptibility to fatigue
helpful in the oxidative deamination and could lower the blood
is correlated with longer swimming times. Both the high dose
ammonia concentration, therefore delaying the occurrence of fati-
(5 mg/(g d)) and low dose (1 mg/(g d)) of LP treatments prolonged
gue (Marquezi, Roschel, Costa, Sawada, & Lancha, 2003). The loach
the swimming time of the mice. As shown in Fig. 1, the median
peptide contains 16.7% glutamic acid and 8.74% aspartic acid, sug-
exhaustion time of the LP-H mice was 241 min (28% greater than
gesting that it might have a potential anti-fatigue effect.
that of the water-injected control group); the median exhaustion
time of the LP-L mice was 226 min (20% greater than that of the
3.2. In vitro antioxidant activities of LP control) (P < 0.05), indicating that LP possesses an anti-fatigue ef-
fect. To explore the mechanism(s), some biochemical parameters,
As one of reactive oxygen species generated in the human body, such as GLU, liver glycogen, LA, BUN contents and LDH, CK, SOD,
hydroxyl radicals can react easily with biomolecules, such as ami- CAT, GSH-Px activities were determined in the mice after they
swam for 30 min.
Table 1
The amino acid composition of LP. 3.4. LP increased blood GLU and liver glycogen
Amino acid Concentrationa (mg amino Compositionb (%)
acid residues/g loach peptide powder) The homeostasis of blood glucose plays an important role in
Aspartic acid 69.7 8.74 prolonging endurance exercise (Wagenmakers et al., 1991). Hypo-
Glutamic acid 133 16.7 glycemia can suppress the active functioning of the brain during
Serine 28.1 3.52 exercise, and this often leads to the inability to continue exercise
Glycine 46.9 5.88
(Wang et al., 2006). Thus, the amount of blood glycogen can illus-
Histidine 31.1 3.90
Arginine 35.4 4.43
trate the speed and degree of fatigue development (Wang et al.,
Threonine 37.7 4.72 2008). As shown in Table 3, the blood GLU levels of LP-H and LP-
Alanine 55.9 7.01 L mice were both signicantly higher than that of the control group
Proline 43.7 5.48 (P < 0.05); 28% and 42% higher, respectively. However, there is no
Tyrosine 27.9 3.5
obvious dose-dependence between the high and low doses groups
Valine 29.4 3.69
Methionine 19.6 2.45
Cystine 0.2 0.02
Isoleucine 31.0 3.88 Table 2
Leucine 55.9 7.01 In vitro antioxidant activity of LP.
Tryptophan 65.7 8.23 Sample Antioxidant activity (IC50) (mg/ml)
Phenylalanine 33.0 4.14
Lysine 53.7 6.73 Hydroxyla DPPHb Cupric Lipid peroxidation
ionc inhibitiond
Total 798 100
LP 17.0 0.54 2.64 0.29 2.89 0.33 12.3 0.98
a
One gram of loach protein from 0 to 5 kDa fraction contained 850 mg of total GSH 4.7 0.63 0.77 0.31 1.03 0.21 10.1 1.47
estimated protein. Thus, the approximate yield of the identied amino acid residues
a
(amino acids minus H2O) accounted for 798/850, or approximately 94% of the The scavenging activity for the hydroxyl radical.
b
nominal protein. The scavenging activity for the DPPH radical.
c
b
Normalised so that the observed amino acid residues add up to 100% of the total The chelating activity of Cu2+ ion.
d
amino acid residues. The lipid peroxidation inhibition activity in a linoleic acid emulsion system.
192 L. You et al. / Food Chemistry 124 (2011) 188194

300 Urea is formed in the liver as the end product of protein metab-
a
olism. During digestion, protein is broken down into small peptides
a
250 and amino acids. The amino acid nitrogen is removed as NH 4,
while the rest of the molecule is used to produce energy or other
Swimming time (min)

b substances needed by the cell (Koo, Lee, Hong, & Kim, 2004). Serum
200
urea nitrogen, which is a product of energy metabolism, is another
150 sensitive index of fatigue status. There was a positive correlation
between urea nitrogen in vivo and exercise tolerance. The less an
animal is adapted to exercise, the more the urea nitrogen level in-
100
creases (Zhang et al., 2006). As shown in Table 3, the BUN levels of
the mice were signicantly lower by 17.5% in the LP-H group com-
50
pared to the control (P < 0.05), while the decrease of 8.6% in the LP-
L group was not signicantly different to the control (P > 0.05). The
0 reduced protein metabolism of the high dose of LP is indicative of
control LP-L LP-H
enhanced endurance (Jia and Wu, 2008; Wang et al., 2008).
Group

Fig. 1. Effect of loach peptide on the swimming time in mice. The error bars 3.6. LP increased LDH activity but decreased CK activity
represent one standard deviation. Different letters indicate signicant differences
between groups (P < 0.05). Serum CK and LDH are known to be accurate indicators of mus-
cle damage. The normal function of CK in cells is to add a phos-
phate group to creatine, turning it into the high-energy molecule
Table 3
phosphocreatine, which is burned as a quick source of energy by
The contents of glucose, liver glycogen, lactic acid and blood urea nitrogen in the mice
(mmol/l).
the cells (Coombes & McNaughton, 2000). However, the normal
function of CK is not as relevant here as what happens to CK when
Groups
the muscle is damaged. During the process of muscle degeneration,
Control LP-L LP-H the muscle cells lyse and their contents nd their way into the
Glucose 5.06 1.00b,* 6.47 0.50a 7.20 0.98a bloodstream. Because most of the CK in the body normally exists
Liver glycogen 3.44 0.28c 11.4 1.32b 13.8 1.74a in the muscle, an increase in CK in the blood indicates that muscle
Lactic acid 11.4 1.11a 10.1 0.82b 8.23 0.59c damage has occurred or is occurring. LDH catalyses the intercon-
Blood urea nitrogen 9.98 0.88a 9.12 0.54b 8.23 0.36c
version of pyruvate and lactate (Koo et al., 2004).
*
Different letters indicate signicant differences between groups for the same. As shown in Fig. 2, the LDH level of mice was signicantly in-
creased for LP-H mice (P < 0.05), while the serum CK level de-
creased (P < 0.05) compared to the control. There were no
(P > 0.05). This indicated that the blood-glucose-regulating ability signicant changes for the LDH and CK levels of LP-L. However,
of the treatment groups was higher than that of the control. the statistical differences were signicant between the high and
Liver glycogen is another index of fatigue. The role of hepatic low LP (P < 0.05) for these two biochemical parameters.
glycogen is to complement the consumption of blood glucose
and maintain the blood glucose in the physiologic range. Fatigue 3.7. LP enhanced the antioxidative enzymes in mice
will happen when the liver glycogen is mostly consumed (Jia and
Wu, 2008). As shown in Table 3, the liver glycogen levels of LP-H Growing evidence indicates that reactive oxygen species are
and LP-L mice were both signicantly higher than that of the con- responsible for exercise-induced protein oxidation, and contribute
trol group (P < 0.05), 2.3-fold and 3.0-fold higher, respectively. strongly to muscle fatigue (Powers, DeRuisseau, Quindry, & Hamil-
Moreover, there was a positive dose-dependent effect between ton, 2004). Muscle cells contain two major classes (non-enzymic
LP-H and LP-L (P < 0.05). and enzymic) of endogenous cellular defense mechanisms to elim-
The results show that the anti-fatigue activity of LP may be re-
lated to the improvement in the metabolic control of exercise and
the activation of energy metabolism (Wang et al., 2006). 2500 LDH 2500
CK a
b b
3.5. LP decreased LA and BUN in the blood 2000 2000
LDH activity (U/L)

CK activity (U/L)

The response to exercise in mammals begins with an increase in 1500 1500

aerobic muscular activity, which switches over to anaerobic x x
metabolism if the exercise is intense, which leads to the accumula- y
tion of LA (Evans, Subramoniam, Rajasekharan, & Pushpangadan, 1000 1000
2002). So, the accumulation of blood serum LA is an important
cause of fatigue. With intense exercise, O2 and pyruvic acid are re- 500 500
duced by LDH to LA, which decreases the pH, affecting both the
cardio-circulating system and the skeletal muscle system function.
The decrease in the contractive strength of the muscle eventually 0 0
induces fatigue (Jia and Wu, 2008). As shown in Table 3, the LA lev- control LP-L LP-H
els of LP-H and LP-L mice were both signicantly lower than that of Group
the control group (P < 0.05), 10.9% and 27.5% decreases, respec-
Fig. 2. Effect of loach peptide on LDH and CK activities in mice. LDH refers to lactate
tively. Moreover, there is a dose-dependent effect between LP-H dehydrogenase and CK refers to creatine kinase. The error bars represent one
and LP-L (P < 0.05), which is another conrmation that LP has an standard deviation. Different letters indicate signicant differences between groups
anti-fatigue effect. within the same procedure (P < 0.05).

CAT
GSH-Px
#
Antioxidant enzymes activity (U/L)
180 # SOD
x,y
150
y We are grateful for the nancial support from the National High
120
90
a a
60 b
30
0 AOAC (1995). Ofcial methods of analysis (16th ed.). Washington, DC, USA:
control LP-L
Group
Fig. 3. Effect of loach peptide on the activities of antioxidant enzymes in mice. SOD
refers to superoxide dismutase; GSH-Px refers to glutathione peroxidase; and CAT
refers to catalase. The error bars represent one standard deviation. Different letters
indicate signicant differences between groups within the same procedure
(P < 0.05).
inate reactive oxygen species. The primary antioxidant enzymes
include SOD, GSH-Px and CAT (Tharakan et al., 2005). SOD dismu-
tates superoxide radicals to form H2O2 and O2. GPH-Px is an en-
zyme responsible for reducing H2O2 or organic hydroperoxides to
water and alcohol, respectively. CAT catalyses the breakdown of
H2O2 to form water and O2. These antioxidant defense mechanisms
become weaker during chronic fatigue and other disease condi-
tions (Powers & Lennon, 1999). So, the improvement in the activi-
ties of these defense mechanisms can help to ght against fatigue.
As shown in Fig. 3, the CAT activities of the LP treatment groups
(both high and low doses) were signicantly higher than that of
the control (49% and 36% greater, respectively) (P < 0.05), while
there was no statistical difference between LP-H and LP-L. The
GSH-Px and SOD activities of LP-H increased by 27% and 8.5%,
respectively, compared to the control (P < 0.05), while those for
the low dose of LP treatment increased by 13% and 1.7%, respec-
tively; however, both were without statistical signicance
(P > 0.05), compared to the control. The results show that LP can
promote increases in the activities of these antioxidant enzymes,
again supporting that LP has an anti-fatigue effect.
4. Conclusions
The results show that a loach peptide prepared by papain diges-
tion has not only in vitro antioxidant activities, but also an in vivo
anti-fatigue effect in mice. It contained the amino acids, which
were expected to contribute to its antioxidant and anti-fatigue
activities. It acted as the scavenger for DPPH and hydroxyl radicals.
It had the ability to chelate Cu2+ ion and to inhibit lipid peroxida-
tion in a linoleic acid emulsion system. The in vivo study showed
that LP prolonged the exhaustion swimming time of the mice. It
improved the metabolic control of exercise and activated the en-
ergy metabolism by increasing the levels of blood GLU and liver
glycogen. It helped to eliminate the accumulated products of
metabolism by decreasing the levels of LA and BUN. It also im-
proved the endogenous cellular antioxidant enzymes in mice by
increasing the activities of SOD, CAT and GSH-Px. Therefore, LP
can elevate the endurance capacity and facilitate recovery from fa-
tigue. The results provide an important basis for developing the
loach peptide as a novel antioxidant and anti-fatigue compound.
However, further research needs to be carried out to evaluate its
L. You et al. / Food Chemistry 124 (2011) 188194 193
anti-fatigue activity on humans and its anti-fatigue mechanism(s)
* at the cellular and molecular levels.
x
Acknowledgements
Technology Research and Development Program of China (863 Pro-
gram) (No. 20576083) and the 11th 5-year National Key Technolo-
gies R&D Program of China (Nos. 2006BAD27B03 and
2006BAD27B04).
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