Food Chemistry 118 (2010) 333–340

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

Marine collagen peptide isolated from Chum Salmon (Oncorhynchus keta) skin
facilitates learning and memory in aged C57BL/6J mice
Xinrong Pei a, Ruiyue Yang a, Zhaofeng Zhang a, Lifang Gao b, Junbo Wang a, Yajun Xu a, Ming Zhao a,
Xiaolong Han a, Zhigang Liu a, Yong Li a,*
a
Department of Nutrition and Food Hygiene, School of Public Health, Peking University, Beijing 100191, China
b
Center for Food and Drug Safety Evaluation, Capital Medical University, Beijing, China

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

Article history: To observe the neuroprotective effects of marine collagen peptide (MCP) isolated from Chum Salmon
Received 3 November 2008 (Oncorhynchus keta) skin by enzymatic hydrolysis, 20-month-old female C57BL/6J mice were fed with
Received in revised form 21 April 2009 chow diet, 0.22%, 0.44% or 1.32% (wt/wt) MCP diet for 3 months. Comparing with aged control group,
Accepted 29 April 2009
the abilities of passive avoidance and spatial memory and learning were significantly enhanced evaluated
by step-down test and Morris water maze respectively. Furthermore, the abilities of learning and mem-
ory had no significant difference between 0.44% and 1.32% MCP treated groups and young control group.
Keywords:
The alleviated oxidative stress, reduced apoptotic neurons, up-regulated expression of brain-derived neu-
Bioactive peptides
Learning and memory
rotrophic factor (BDNF) and postsynaptic density protein 95 (PSD95) were observed in MCP treated
Aging groups compared with aged control group. Our research revealed that there were no significant differ-
Brain-derived neurotrophic factor ence between 0.44% and 1.32% MCP treated groups and young control group. These findings suggest that
Postsynaptic density protein 95 MCP could be a candidate for functional food to relieve memory deficits associated with aging.
Ó 2009 Elsevier Ltd. All rights reserved.

1. Introduction interest and attention (Korhonen & Pihlanto, 2003; Mellander,

Cognitive deficits including learning impairment and delayed
amnesia may be caused by age-related deterioration in brain func-
tion (Erickson & Barnes, 2003). While the world’s population is
aging in the 21st century (Kalache, Aboderin, & Hoskins, 2002), a
fast growing number of aged people suffer from neurodegenerative
diseases, such as Alzheimer’s disease (AD) and Parkinson’s disease
(PD), two of the most common diseases among the elderly that im-
pairs the cognitive ability (Morrison & Hof, 1997).
Mounting evidence suggests that lifestyle factors, especially the
diet, may account for the development of chronic diseases, such as
cardiovascular disease, diabetes and neurodegenerative disease
(Ferrari, 2007). Nowadays, while major dietary intervention has
been applied to prevent such diseases as diabetes and heart dis-
ease, the potential impact of diet on aging process-associated neu-
rodegeneration has been neglected. Apparently, the potential effect
of nutrition on slowing down the neuropathies deserves increasing
public concern (Paliyath, 2006). And it is well known that in pro-
tein, it is bioactive peptides that act out the biological activity.
Since the first bioactive peptide was revealed by Mellander
(1950), the potential physiological functions of bioactive peptides
derived from dietary protein have aroused a lot of scientific
* Corresponding author. Tel./fax: +86 10 82801177.
E-mail addresses: liyongbmu@163.com, liyong@bjmu.edu.cn (Y. Li).
1950). As specific protein fragments, a wide range of activities of
bioactive peptides have been described, including antimicrobial,
blood pressure-lowering, cholesterol-lowering, antioxidative and
immunomodulatory effects, etc. (Hartmann & Meisel, 2007; Mira-
liakbari & Shahidi, 2008). Moreover, some peptides can influence
higher brain functions, such as learning and memory, in humans
and animals (McLay, Pan, & Kastin, 2001).
Theoretically, bioactive peptides can be released from dietary
proteins through digestion in the gut; the amount of peptides,
however, is too small to induce any significant effects. In contrast,
enzymatic hydrolysis of dietary proteins offers a rapid and
reproducible method for the production of considerable bioactive
peptide fractions, and they are likely to become potential
health-beneficial-food ingredients or nutraceutical preparations
(Hartmann & Meisel, 2007).
During the past five decades, fish proteins were widely studied
and their various multifunctional properties were well described
(Aneiros & Garateix, 2004; Chavan, McKenzie, & Shahidi, 2001).
We still do not know, however, whether bioactive peptides from
fish proteins have preventive effect against age-related cognitive
decline. It is certain now that oxidative stress can lead to cell and
tissue damage, thus resulting in age-related cognitive decline and
our previous research demonstrated that bioactive peptides de-
rived from fish skin has antioxidant function in D-galactose induced
Sprague-Dawley rats. Hence, it is reasonable to hypothesise that

0308-8146/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodchem.2009.04.120
334 X. Pei et al. / Food Chemistry 118 (2010) 333–340
fish protein may be a valuable source of neuroprotective peptides.
The purpose of the present study was to investigate the promnestic
effects of Marine Collagen Peptide (MCP), compounds of low molec-
ular weight peptides derived from Chum Salmon (Oncorhynchus
keta) skin by enzymatic hydrolysis, on the cognition of aged
C57BL/6J mice. Despite broadly comparable patterns of cognitive
decline with normal aging for males and females, some groups of
females may be more vulnerable than males to cognitive dysfunc-
tion as they age. Most remarkable is the observation that Alzhei-
mer’s disease is two to threefold higher in women than in men
after the age of 65 years (Andersen, Launer, & Dewey, 1999; Heb-
da-Bauer, Luo, Watson, & Akil, 2007). This gender difference per-
sists even after correcting for differences in life expectancy. So,
we used female mice in this study. In addition, we intended to ex-
plore cellular and molecular mechanisms and how MCP regulates
the plasticity of the brain to repair itself in response to the effect
of aging. Furthermore, we also wanted to determine whether MCP
might become a candidate for functional food or pharmaceutical
applications to relieve memory deficits associated with aging.
2. Materials and methods
2.1. Chemicals
Commercial kits used for determination of T-SOD, GSH-Px and
TBARS were purchased from Jiancheng Institute of Biotechnology
(Nanjing, China). Mouse monoclonal anti-BDNF was purchased
from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Rabbit
monoclonal anti-PSD95 was purchased from Chimicon (Temecula,
CA). ECL plus western blotting detection system was obtained from
Amerscham Pharmacia Biotech (Buckinghamshire, England). All
other chemicals were reagent grade or the highest quality available
from Sigma–Aldrich Co. (St. Louis, MO, USA). MCP was purified by
reversed phase chromatography using a 250  10 mm C18 column
(Phenomenex Inc., Torrace, CA, USA).
2.2. Preparation and identification of MCP
Marine Collagen Peptide (MCP) was derived from the skin of
wild-caught Chum Salmon ( O. keta) (average body weight,
1.47 kg) and donated by CF Haishi Biotechnology Ltd. Co. (Beijing,
China). In brief, skin of Chum Salmon was cleaned and the adher-
ent tissue was scraped manually by a scalpel. Then the skin was
minced and defatted by vigorous stirring in chill water. The mate-
rials were homogenised and emulsified in distilled water, and
added into complex protease (3000 U/g protein), which including
7% of trypsin, 65% of papain and 28% of alkaline proteinase, with
the constant temperature at 40 °C and pH value of 8, for 3 h. The
resultant hydrolysate was centrifuged at 10,000g by decanter cen-
trifuge at first, and then it was centrifuged at 15,000g by tube cen-
trifuge to get rid of the impurity. After centrifuge, the liquid was
subsequently separated through ceramic membrane (200 lm) to
purify it. The mineral salt was removed from the liquid through a
procedure of nanofiltration at pH 6.5–7.5 with a concentration of
solid material between 2.2–2.6 g/100 ml. Then the purified liquid
was condensed by cryoconcentration under vacuum at 70 °C with
an evaporation rate of 500 kg/h. When the concentration of the
condensed liquid was almost 30 Baume degrees, it was decoloured
with 12% of active carbon at 75 °C for 1 h, and then got rid of the
carbon by filtration. When all the process above was finished, re-
moved most of the water by spray drying with the pressure of
20 MPa at an evaporation rate of 200 kg/h, MCP powder used in
the following investigation was obtained.
High-performance liquid chromatography (HPLC, Waters Corp.,
Milford, MA, USA) of peptide sample was performed using a Phe-
nomenex C18 column (10  250 mm), which used acetonitrile-
0.05 mol/L phosphate buffer (PH 3.2) (10:90) as the mobile phase
with a flow rate of 2.0 mL/min and monitored at 208 nm using a
Water 486 tunable UV detector. Then the molecular weight distribu-
tion of the sample was ascertained by LDI-1700 matrix-assisted la-
ser desorption ionisation time-of-flight mass spectrometry (MALDI-
TOF-MS) (Liner Scientific Inc., Reno, NV, USA). In addition, the amino
acid composition was further analysed by an H835–50 automatic
amino acid analyzer (Hitachi, Tokyo, Japan). The result indicated
that the molecular weight distribution of MCP was 100–860 Da. Fur-
thermore, 85.86% of the molecular weight distributed between 300
and 860 Da. So, the main composition of MCP was oligopeptides. The
amino acid composition of MCP was shown in Table 1.
2.3. Animals and diet
C57BL/6 J female mice were obtained from the Animal Service
of Health Science Center, Peking University. Control animals were
fed AIN93M Diet from Vital River Limited Company (Beijing, Chi-
na). The mice in the experimental groups were fed with 0.22%,
0.44% or 1.32% (wt/wt) MCP in AIN93M diet, respectively. The com-
position of the AIN93M diet include 20.26% of protein, 4.24% of fat,
25.19% of carbohydrate, 3.60 mg/100 g vitminA, 0.25 mg/100 g vit-
minD, 44.00 mg/100 g vitminE, 5.00 mg/100 g vitminK, 1.10 g/kg
choline chloride and 25.90 g/kg mineral mix. The weight and food
consumption were measured every week. No significant differ-
ences in diet or weight gain had been found between the un-sup-
plemented aged mice and the old supplemented mice (data were
not shown). The young mice were 3 month-old, while aged mice
20 monthly at the start of the experiment. All animals were accli-
matised at animal colony facilities in laboratory of animal service
of Peking University for at least 1 week before treatment. Mice
were housed four to five per cage and provided with MCP-supple-
mented diet or standard diet for 3 months. All animals were main-
tained at a constant temperature (23 ± 1 °C) and humidity
(60 ± 10%) environment under a 12-h light/dark cycle (light on
07:30–19:30 h) with free access to food and water. Animal treat-
ment and maintenance were carried out in accordance with the
Principle of Laboratory Animal Care (NIH publication No. 85–23,
revised 1985) and the guidelines of the Peking University Animal
Research Committee.
2.4. Behavioral test
2.4.1. Passive avoidance task (step-down test)
A step-down passive avoidance was examined by using appara-
tus consisted of a box (25  25  40 cm), a floor with stainless-
Table 1
Amino acid composition of MCP from salmon skin.
Amino acid No. residues/100 residues
Glycine 23.77
Alanine 6.59
Serine 4.23
Proline 9.79
Valine 2.94
Threonine 2.53
Leucine 4.64
Isoleucine 2.57
Aspartic acid 7.29
Lysine 5.66
Arginine 6.08
Glutamic acid 12.22
Methionine 0.03
Histidine 1.61
Phenylalanine 2.51
Tyrosine 0.03
Hydroxy proline 7.51
 X. Pei et al. / Food Chemistry 118 (2010) 333–340
steel grids 2 mm in diameter at 8 mm intervals, and a rubber plat-
form (4 cm diameter, 4 cm height) set on the grid in one corner.
Electric stimulation was given through the grid connected with a
scrambled shock generator. After 3 months of treatment, an acqui-
sition trial was performed. In this trial, each mouse was placed
gently on the platform and allowed to habituate freely for 3 min,
and then electric shocks (0.4 mA) were delivered to the grid. If
the mouse stepped down from the platform, the electric shock
was delivered to the mouse on the grid floor. The cutoff time
was 5 min. A retention trial was performed 24 h after the acquisi-
tion trial. Each mouse was again placed on the platform. The time
(step-down latency) that elapsed until the mouse stepped down
from the platform was recorded. If the mouse did not step down
from the platform within 300 s, the retention trial was terminated
and the maximal step down latency of 300 s was recorded. Each er-
ror was counted whenever the mouse stepped down from the plat-
form and the number of errors made within 5 min was recorded.
2.4.2. Morris water maze
The experimental apparatus which used in the Morris water
maze test consisted of a circular water tank (120 cm in diameter,
35 cm in height), containing water (24 ± 1 °C) to a depth of
20 cm, which was rendered opaque by adding milk. A platform
(4.5 cm in diameter, 19 cm in height) was submerged 1 cm below
the water surface and placed at the midpoint of one quadrant.
The pool was located in a test room, which contained various
prominent visual cues. Each mouse received four training periods
per day for seven consecutive days. Before the first trail, each
mouse was put on the platform for 10 s, and then it was given
30 s free swim and then was assisted to the platform where it
was remained for another 10 s rest. For each trial, mouse was
placed in the water facing the wall at one of four starting positions,
and the time required for the released mouse to find the hidden
platform was recorded. Mouse that found the platform was al-
lowed to remain on the platform for 10 s and then returned to its
cage for the inter-trial interval. A mouse failed to find the platform
within 90 s was placed on the platform for 10 s at the end of the
trial. Latency to escape from the water maze (finding the sub-
merged escape platform), swimming distance and average speed
to reach the platform were recorded for each trial.
To measure the strength of the spatial memory, on day 7, a
‘‘probe test” was performed, during which, mice were swimming
freely for 60 s in the pool without platform. In the ‘‘probe test” cal-
culated were: (1) the time (in seconds) spent by mice in the target
quadrant in which the platform was hidden during acquisition
trails, and (2) the number of times exactly crossing over the previ-
ous position of the platform.
Swimming behavior during the test was video-recorded using a
commercial VCR and analysed by a PC computer.
2.5. Detection of SOD, GSH-Px and TBARS in serum
Superoxide dismutase (SOD), glutathione peroxidase (GSH-Px)
and thiobarbituric acid-reactive substances (TBARS) in serum were
examined by reagent kit (Jiancheng Institute of Biotechnology,
Nanjing, China). The principles of these kits are as follows. The
method which was used to measure SOD employs xanthine and
xanthine oxidase to generate superoxide radicals. Activity of
GSH-Px was reflected by the speed of enzymatic reaction, in which
GSH-Px promote glutathione hormone (GSH) to generate oxidised
form glutathione (GSSG). Lipid peroxidation is known to result in
formation of malonaldehyde (MDA) and other structurally similar
compounds, which react with triobarbitrric acid (TBA) to form
TBARS that absorbs at 532 nm. The TBARS levels in serum were ex-
pressed as malondialdehyde (MDA) equivalents (Shahidi & Ho,
2007).
335
2.6. Morphological analysis
After behavioral analyses, five mice in each group were deeply
anesthetised with pentobarbital and perfused intracardially with
normal saline followed by 4% paraformaldehyde in phosphatebuf-
fered saline (PBS). The brains were removed, post-fixed with the
same fixative and cryoprotected with 30% sucrose containing
PBS. Sections (10 lm) containing hippocampus were obtained
using a rotary microtome (HM505E; MICROM International GmbH,
Walldorf, Germany), mounted on slides, and stored at 20 °C until
use.
Morphology of the hippocampal was monitored using nissl
staining. The slide-mounted brain sections were soaked in cresyl
violet solution for 10 min, dehydrated through a graded series of
ethanol–water solutions, coverslipped, and analysed under a bright
field microscope. Nissl-positive neuronal cell numbers were man-
ually and rigidly counted within the hippocampal pyramidal cell
layer (CA1 and CA3 regions and the dentate gyrus). The total cell
counts were averaged from at least three sections per animal.
2.7. Western blot analysis
Mice were deeply anesthetised and their hippocampuses were
rapidly removed and stored at 80 °C until use. The tissue samples
were pooled and lysed in a buffer containing 50 mM Tris–HCl, pH
6.8, 10% glycerol, 2% sodium dodecyl sulfate, and 5% b-mercap-
toethanol. 50 lg protein lysate were separated by 8% SDS–PAGE
and transferred onto polyvinylidene difluoride (PVDF) membrane.
After blocking in a 5% non-fat dry milk solution in washing buffer
containing 10 mM Tris (pH7.5), 150 mM NaCl, and 0.05% Tween-
20, membranes were incubated overnight at 4 °C with primary
antibody (monoclonal anti-BDNF, 1:1000, Santa Cruz Biotechnol-
ogy, USA; monoclonal anti-PSD95, 1:250, Chemicon, Temecula,
CA). After washing three times with TBS, membranes were incu-
bated for 1.5 h with horseradish peroxidase-coupled secondary
antibody at room temperature. Following the post-secondary
washes, the resulting antigen–antibody-peroxidase complex was
detected using the ECL kit (Amersham Pharmacia Biotech) and vis-
ualised by exposures of various lengths to Kodak film.
2.8. Statistical analyses
All statistical analyses were performed with the Statistical Pack-
age for social sciences for Windows version 11.0 (SPSS Inc. Chicago,
IL. USA). Data were expressed as mean ± SEM. Effects on escape
latencies and swimming distance in Morris water maze between
groups were evaluated using Repeated-measures two-way ANOVA
followed by a least significant difference test as a post hoc test. To
evaluate other effects of MCP, difference between all groups were
compared using one-way ANOVA followed by a least significant
difference test as a post hoc test or Dunnett’s test. For all statistical
tests p < 0.05 was considered significant.
3. Results
3.1. Effect of MCP on latency and number of errors in step-down test
One-way ANOVA revealed that the latency and number of er-
rors in step-down test had significant difference among all five
groups (p < 0.01, p < 0.05 respectively). As shown in Fig. 1, aged
control showed significantly memory loss with respect to young
mice. The error-number increased while latency reduced signifi-
cantly in aged control with respect to young mice (p < 0.01).
Treatment with 0.44% and 1.32% MCP prolonged the latency in
the testing day compared with aged control (p < 0.05). Further-
336 X. Pei et al. / Food Chemistry 118 (2010) 333–340
more, there was no significant latency difference between young
mice and 0.44% or 1.32% MCP treated mice. Regarding to mice
treated with 0.22% MCP, a reduction in the latency was showed
with respect to young control (p < 0.05) (Fig. 1A). As shown in
Fig. 1B, treatment with 0.44% MCP showed a significant difference
in the error-number compared to aged control (p < 0.05). No dif-
ference was observed between 0.22% or 1.32% MCP supplementa-
tion groups and aged control group. Compared with young mice,
the error-number in all MCP treated mice had no significant
difference.
3.2. MCP improves spatial learning and memory in Morris water maze
Morris water maze is a sensitive method for revealing the
impairment of spatial learning and memory. Once animals learn
where the hidden platform is located, they can remember the loca-
tion and swim rapidly to it from any starting point. Both the time
taken to reach the platform and the swimming distance traveled
were measured. As shown in Fig. 2A and B, the mean latency to find
the platform and swimming distance declined progressively during
Fig. 1. Effects of MCP on latency (A) and number of errors (B) in step-down passive
avoidance test. After daily oral treatment with MCP supplemented diet or standard
diet for 3 months, animals were tested in step-down avoidance task. Values were
presented as means ± SEMs of 15–16 mice. *p < 0.05 compared to aged control
mice; **p < 0.01 compared to aged control mice;  p < 0.05 compared to young
control mice;   p < 0.01 compared to young control mice (By Xinrong Pei, Ruiyue
Yang, Zhaofeng Zhang, Lifang Gao, Junbo Wang, Yajun Xu, Ming Zhao, Xiaolong Han,
Zhigang Liu, Yong Li*).
the training days in all animals. However, the aged control demon-
strated markedly longer escape latencies and swim-traveling dis-
tance than young control group. MCP treatment to aged mice
resulted in a significant decrease in escape latency and swim-trav-
eling distance. The main effects for day on escape latency and
swim-traveling distance were statistically significantly
[F(5, 370) = 210.32, p < 0.01; F(5, 370) = 292.22, p < 0.01 respec-
tively]. The main effects for group on escape latency and swim-
traveling distance were also statistically significant
[F(4, 74) = 9.71, p < 0.01; F(4, 74) = 21.38, p < 0.01 respectively].
The day  group interaction was not significant [F(20, 370) = 0.73,
p > 0.05; F(20, 370) = 1.20, p > 0.05 respectively]. Post hoc test re-
vealed a significant increase in escape latency and swim-traveling
distance in aged control mice compared with young control mice
and MCP-treated mice (p < 0.05). There were no differences be-
tween MCP treated and control mice in the swimming speed (data
not shown).
During the probe test, mice were swimming in the vicinity of
the place that contained escape platform during the acquisition
trial. There were significant differences on the time spent at tar-
get quadrant and number of times crossing over the platform
among five groups (p < 0.01, p < 0.05 respectively). Young mice
showed higher precision of the search for the platform as re-
vealed by more time spent at the former platform position and
higher number of crossing than aged control mice did (Fig. 2C
and D). MCP seemed to restore the lost procedural subcompo-
nent of spatial memory in aged mice, but only the 0.44% MCP
had significantly effect on the time spent at target quadrant.
On the other hand, 1.32% MCP could increase the number of
times crossing over the platform site compared with aged
control.
3.3. Effects of MCP on SOD, GSH-Px activities and TBARS level in serum
As regard to activity of SOD, all five groups of animals had sig-
nificant difference (p < 0.01). Compared with young mice, the
activity of SOD in serum significantly declined by 33.6% in aged
control mice and MCP (0.22%, 0.44% or 1.32%) increased the activ-
ities of SOD nearly to 70.9%, 85.7% and 77.3% of the young mice,
respectively (Table 2). The dose group of 0.44% MCP reached signif-
icant levels (p < 0.05) versus aged control.
Analysis on activity of GSH-Px also revealed a significant differ-
ence in five groups (p < 0.01). The activity of GSH-Px in serum of
aged control group mice was significantly lower compared with
young control group (Table 2, p < 0.01). MCP treatment resulted
in an elevation in the activity of this enzyme. The dose group of
1.32% MCP (p < 0.05 versus aged control group) had more signifi-
cant effect than other groups.
Aged control mice showed significant increase in TBARS level
compared with the young mice (p < 0.01). This increase in TBARS
was also attenuated in MCP treated mice.
3.4. Effect of MCP on morphological change in aged mice hippocampus
In order to discuss the neuroprotective effect of MCP, morpho-
logical change in hippocampus was observed using nissl staining
method. In the young control group, multiple layers of pyramidal
neurons with a dense and orderly arrangement were defined eas-
ily. However, in the aged control group, pyramidal neurons were
less compact than those of young control group. Table 3 numeri-
cally expresses nissl positive cells in the CA1, CA3 regions and den-
tate gyrus (DG) of hippocampus. Measurement of total number in
hippocampal CA1, CA3 and DG did not reveal any significant age-
related change, although a slight age-related decline was observed.
Similarly, no difference was seen in hippocampus between aged
control and MCP treated groups.
 X. Pei et al. / Food Chemistry 118 (2010) 333–340 337

Fig. 2. Effects of MCP on spatial learning and memory of mice in Morris water maze test. The number for aged control group was 15, and 16 for other groups. (A) Escape
latency to find the hidden platform. Two-way repeated measures ANOVA revealed statistically significant difference [group, F(4, 74) = 9.71, p < 0.01, day, F(5, 370) = 210.32,
p < 0.01]. This difference was attributed to the longer latencies of aged control mice (post hoc LSD test p < 0.01 vs. all other groups) and shorter latencies of young control mice
(post hoc LSD test p < 0.01vs. aged control group, p < 0.05 vs. 0.22% and 1.32% MCP treated groups). (B) Swimming distance travel to the goal platform. Two-way repeated
measures ANOVA revealed statistically significant difference [group, F(4, 74) = 21.38, p < 0.01, day, F(5, 370) = 292.22, p < 0.01]. This difference was attributed to the longer
distance of aged control (post hoc LSD test p < 0.01 vs. all other groups) and shorter distance of young control mice (post hoc LSD test p < 0.01vs. all other groups). (C)
Presented were means ± SEMs time spent in target quadrant [one way ANOVA using LSD test, *p < 0.05 compare with aged control mice, **p < 0.01 compare with aged control;
 
p < 0.05 comparing with young control mice,   p < 0.01 comparing with young control mice]. (D) Presented were means ± SEMs numbers of crossing during the ‘‘probe test”
[one way ANOVA using LSD test, *p < 0.05 when compared with aged control mice;  p < 0.05 as compare with young control mice] (By Xinrong Pei, Ruiyue Yang, Zhaofeng
Zhang, Lifang Gao, Junbo Wang, Yajun Xu, Ming Zhao, Xiaolong Han, Zhigang Liu, Yong Li*).

Table 2
Effects of MCP on superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and thiobarbituric acid-reactive substances (TBARS) in serum.

Group n SOD (U/mg protein) GSH-Px (U/mg protein) TBARS (MDA nmol/mg protein)
     
Aged control 8 81.56 ± 23.18 818.63 ± 116.73 2.67 ± 0.27  
0.22% MCP 8 87.13 ± 25.02  874.02 ± 99.31  2.18 ± 0.24*, 
0.44% MCP 8 105.37 ± 18.22* 886.54 ± 101.01  1.78 ± 0.33*
1.32% MCP 8 95.00 ± 21.39  945.14 ± 109.74* 2.31 ± 0.63 
Young control 8 122.87 ± 13.08** 1028.86 ± 161.80** 1.67 ± 0.52**
*
p < 0.05 compared to aged control.
**
p < 0.01 compared to aged control.
 
p < 0.05 compared to young control.
  
p < 0.01 compared to young control.

3.5. Effect of MCP on expression of BDNF and PSD95 in hippocampus
As shown in Fig. 3, compared the aged control mice with the
young mice, hippocampal BDNF and PSD95 decreased by 30%
(p < 0.01) and 34% (p < 0.01) respectively.
The expression of hippocampal BDNF was significant up-reg-
ulated (29%, 26%) in aged mice treated with 0.44% and 1.32%
MCP for 3 months (p < 0.01, p < 0.05 respectively) (Fig. 3). How-
ever the difference of hippocampal BDNF expression between
aged control mice and 0.22% treated mice had no significant
(p > 0.05).
The results also indicated that the level of PSD95 was signifi-
cantly higher (38%) in 0.44% MCP treated mice than that of the
aged control mice (p < 0.01) (Fig. 3). There was no significant differ-
ence of hippocampal PSD95 expression between aged control mice
and 0.22%, 1.32% MCP treated mice (p > 0.05).
338 X. Pei et al. / Food Chemistry 118 (2010) 333–340

Table 3 probably due to the antioxidative activity and protective effect

Effect of MCP on nissl-positive cell numbers in hippocampal CA1, CA3 regions and DG. on BDNF and PSD95 expression. To our knowledge, the present
Group Nissl-positive cells (numbers/mm2) study is the first to reveal the neuro-protective effect of MCP on
CA1 CA3 DG age-related deficit in rodents.
While the exact molecular basis of aging remains uncertain, the
Aged control 7591 ± 1392 5164 ± 665 17822 ± 996
0.22% MCP 7811 ± 1217 5314 ± 857 18302 ± 1292
increasing accumulated related knowledge suggests that it may be
0.44% MCP 7915 ± 1050 5639 ± 591 18115 ± 1026 related with the increase of free radical and neuroplasticity loss
1.32% MCP 7050 ± 1149 5505 ± 586 17989 ± 1134 (Burke & Barnes, 2006). According to Free Radical Theory of Aging,
Young control 7972 ± 759 6213 ± 1128 18044 ± 1058 the increase of lipid peroxides and the decrease activities of antiox-
idant enzymes, such as SOD and GSH-Px, are involved in the course
of aging (Ceballos-Picot, Nicole, Clement, Bourre, & Sinet, 1992).
The alterations are considered to play important roles in learning
and memory deficits. In the present study, the activities of SOD
and GSH-Px in serum showed a significant decline in aged control
mice compared with young control; moreover, the activities of
anti-oxidative enzymes such as SOD and GSH-Px were significantly
increased in MCP treated group. As lipid peroxidation may also
contribute to the neurotoxicity in mice, the peroxides could be
indirectly estimated by measuring TBARS, a measure of lipid per-
oxidation and widely used as a biomarker of oxidative stress
(Shahidi & Ho, 2007; Wanasundara & Shahidi, 1998). An obvious
enhancement of the level of TBARS was shown in the aged control
mice in this study, and it could be significantly reduced after MCP
administration. The results indicate that MCP, to some extent,
attenuates the oxidative injury in aging. In other words, the protec-
tion of MCP against oxidative stress to brain may be involved in the
mechanism of its action to ameliorate the impairments of learning
and memory.
Although neuronal cell loss was considered the main cause of
learning and memory impairments during normal aging histori-
cally, more recent studies of rodents and humans demonstrated
that the number of neurons in the hippocampal formation re-
mained stable in aged subjects, irrespective of cognitive status
(Calhoun et al., 1998; Rasmussen, Schliemann, Sorensen, Zimmer,
& West, 1996). These data indicate that cognitive impairment
may occur in aging, independent of neurodegeneration. Our analy-
Fig. 3. Immunoblot analysis of BDNF and PSD95 in the hippocampus of the young, sis of the morphology of the hippocampal using nissl staining re-
aged and MCP-treated mice. (A) Western blot from the aged group showed weak
vealed that there was no evidence of aged-induced alterations in
hippocampal BDNF and PSD95 compared with the young mice. Reinforced BDNF
and PSD95 expression was observed in the MCP-treated groups, especially the aged subjects compared to young control. In line with our observa-
0.44% MCP treated group. Molecular weight markers (kDa) were shown on the left. tions, Nyffeler also reported that there was no difference in mean
(B) Quantitative analysis showed that hippocampal levels of BDNF and PSD95 total volume of the hippocampus between performance groups
protein of the young control mice and 0.44% MCP-treated mice were obviously as well (Nyffeler, Zhang, Feldon, & Knuesel, 2007). Nyffeler’s and
higher than that of the aged mice. *p < 0.05, **p < 0.01 vs. the aged control group (By
Xinrong Pei, Ruiyue Yang, Zhaofeng Zhang, Lifang Gao, Junbo Wang, Yajun Xu, Ming
our findings support the assumption that aged-related cognitive
Zhao, Xiaolong Han, Zhigang Liu, Yong Li*). impairments are not induced by gross morphological changes but
rather associated with subtle connectional and synaptic reorgani-
sation within the hippocampal formation (Nyffeler et al., 2007).
There is now considerable evidence supporting the view that nor-
4. Discussion mal age-related memory deficits can be caused by functional
changes, without the occurrence of major neuronal cell loss (Rapp,
Unlike other dietary proteins, fish protein is likely to contain a Deroche, Mao, & Burwell, 2002).
lot of specific and potent bioactive sub-sequences in nutritional It is well established that brain-derived neurotrophic factor
perspective for their competitive and aggressive living conditions (BDNF), a crucial mediator of neuronal vitality and function, regu-
(Aneiros & Garateix, 2004). Thus if the potent bioactive compounds lates the morphology through acting on both presynaptic and post-
were extracted and the functions were revealed, it would open a synaptic (Suzuki et al., 2004). It is emerging as a major player in
new perspective for pharmaceutical and functional food neuronal events underlying learning and memory (Castren, Bernin-
development. ger, Leingartner, & Lindholm, 1998). The present study reported
MCP, compounds of proline and hydroxyl proline-rich oligopep- that BDNF in hippocampus of aged control group was lower than
tides, was derived from Chum Salmon (O. keta) skin which usually that of the young group, in agreement with previous data on
causes wastage and pollution in fish-processing industry (Kim age-related BDNF expression (Hwang et al., 2006). Furthermore,
et al., 2001). The low molecular weight distribution (100–860 Da) the results showed that when aged control group compared with
and small amounts of free amino acids, fat and carbohydrate, con- 0.44% or 1.32% MCP-treated group, hippocampal BDNF increased
firmed that the main composition of MCP was oligopeptides. The by 29% and 26%, respectively. Although no information about
present study demonstrated that dietary administration of MCP how MCP regulates BDNF expression is provided in the present
could facilitate acquisition of spatial learning and increase the pas- study, the authors think that there are several possible interpreta-
sive avoidance ability, bringing these to the level observed in tions for MCP increasing BDNF expression: (1) It is well known that
young subjects. Furthermore, we found that these results were the generation of free radicals can lead to cell and tissue damage
 X. Pei et al. / Food Chemistry 118 (2010) 333–340
paralleled by alterations in the function of the genetic apparatus,
resulting in aging and untimely cell death (Droge & Schipper,
2007). MCP is confirmed to have antioxidant function, which can
protect cerebral cells and reduce neuron cell apoptosis, therefore
enhance the BDNF secretion indirectly or may probably protect
the secreted BDNF directly from the oxidant damage. (2) The loss
of elasticity and increasing rigidity of the aging vascular are con-
sidered as one of the factors leading progressively to severe arte-
riosclerosis and vascular dementia (Stewart, 2007). Collagen is
well proved to improve vascular elasticity and reduce vascular
rigidity, consequently increase the blood supply of brain (Bailey,
Paul, & Knott, 1998; Silver, Horvath, & Foran, 2001), therefore pro-
tect the neuron cells and promote the expression of BDNF. Such ef-
fect may be another mechanism of how MCP increases BDNF
expression. Despite lacking of direct evidence, the present results
suggest that chronic MCP supplementation may prevent age-re-
lated learning and memory decline through increasing the expres-
sion of BDNF in hippocampus. However, further examinations are
needed to elucidate the detail mechanisms on how MCP affects
BDNF expression in brain.
BDNF does not only promote neuronal survival and differentia-
tion, but also regulates synaptic transmission and plasticity in the
central nervous system (CNS). There is now some considerable evi-
dence indicating that brain has a decreased capacity for plastic re-
sponse in aging process. Particularly, the hippocampus shows a
reduction of synapticplasticity during aging (Teter & Ashford,
2002; Uylings & de Brabander, 2002). Consistent with these data,
this study provided evidence for a concomitant decrease in PSD95
protein expression in the hippocampus of the aged mice. Nakata
and Nakamura previously demonstrated that the expression of
PSD95, a membrane-associated guanylate kinase protein enriched
in the postsynaptic density (PSD), was increased upon BDNF addi-
tion (Nakata & Nakamura, 2007). Therefore, the PSD95 level in hip-
pocampus was tested using immunoblot analysis in our research. A
remarkable decrease in hippocampal PSD95 expression in the aged
mice was found in the study, which supported previous reports of
reduced PSD expression in aging brain (Head et al., 2007). At the
same time, we presented reliable evidence of increasing hippocam-
pal PSD95 of the aged mice receiving MCP administration. Further-
more, our results showed that PSD95 expression changed in an age-
dependent manner, and BDNF revealed a similar expression pattern
as compared with PSD95 in the hippocampus; also in MCP-treated
groups, BDNF level increased in hippocampus accompanied with
enhanced PSD95. PSD95 is thought to serve various functions at
the synapse, implicate in the regulation of ion-channel function,
synaptic activity, and intracellular signaling (Craven, El-Husseini,
& Bredt, 1999). Therefore, the increased PSD95 expression induced
by BDNF addition may represent a potential molecular substrate by
which MCP increases hippocampal synaptic plasticity.
As mentioned above, MCP produced a biphasic effect, of which
0.44% MCP is an optical dose for memory, on the learning and
memory in aged mice. This finding is not surprising, because
numerous findings indicate that the U-shaped nature of the
dose–response curve for the effects of promnestic agents on learn-
ing and memory is typical in that low doses have opposite effects
from high dose, such as leptin, neuropeptide Y, colostrinin, et al.
(Farr, Banks, & Morley, 2006; Flood, Hernandez, & Morley, 1987;
Popik, Bobula, Janusz, Lisowski, & Vetulani, 1999).
In conclusion, the present study demonstrates that MCP can
facilitate learning and memory in aged mice through reducing oxi-
dative damage in the brain and increasing BDNF and PSD95 expres-
sion level. The increase in life expectancy in the 21st century has
resulted in an increase in the prevalence of age-dependent diseases
such as depression, Alzheimer’s disease (AD) and other dementias.
It is, therefore, crucial to develop appropriate health care means to
deal with age-related neurodegenerative disorders, including cog-
339
nitive deficits. Considering both the lack and the need of functional
food or drugs proven to be effective in improving memory retrie-
val, the neuroprotective effect of MCP reported here is of vital
importance and practical value. Undoubtedly it could be a candi-
date for functional food or drugs to manage/reduce memory defi-
cits associated with aging.
Acknowledgements
The authors of the paper are grateful to the company CF Haishi
Biotechnology Ltd. for providing the samples used in this study.
This research was supported by the foundation (No.
2006BAD27B08) from the Ministry of Science and Technology of
the People’s Republic of China.
References
Andersen, K., Launer, L. J., & Dewey, M. E. (1999). Gender differences in the
incidence of AD and vascular dementia: The EURODEM Studies.EURODEM
Incidence Research Group. Neurology, 53(9), 1992–1997.
Aneiros, A., & Garateix, A. (2004). Bioactive peptides from marine sources:
Pharmacological properties and isolation procedures. Journal of
Chromatography B, 803(1), 41–53.
Bailey, A. J., Paul, R. G., & Knott, L. (1998). Mechanisms of maturation and ageing of
collagen. Mechanisms of Ageing and Development, 106, 1–56.
Burke, S. N., & Barnes, C. A. (2006). Neural plasticity in the ageing brain. Nature
Reviews Neuroscience, 7(1), 30–40.
Calhoun, M. E., Kurth, D., Phinney, A. L., Long, J. M., Hengemihle, J., Mouton, P. R.,
et al. (1998). Hippocampal neuron and synaptophysin-positive bouton number
in aging C57BL/6 mice. Neurobiology of Aging, 19(6), 599–606.
Castren, E., Berninger, B., Leingartner, A., & Lindholm, D. (1998). Regulation of brain-
derived neurotrophic factor mRNA levels in hippocampus by neuronal activity.
Progress in Brain Research, 117, 57–64.
Ceballos-Picot, I., Nicole, A., Clement, M., Bourre, J. M., & Sinet, P. M. (1992). Age-
related changes in antioxidant enzymes and lipid peroxidation in brains of
control and transgenic mice overexpressing copper–zinc superoxide dismutase.
Mutation Research, 275(3–6), 281–293.
Chavan, U. D., McKenzie, D. B., & Shahidi, F. (2001). Functional properties of protein
isolates from beach pea (Lathyrus maritimus L.). Food Chemistry, 74(2), 177–187.
Craven, S. E., El-Husseini, A. E., & Bredt, D. S. (1999). Synaptic targeting of the
postsynaptic density protein PSD-95 mediated by lipid and protein motifs.
Neuron, 22(3), 497–509.
Droge, W., & Schipper, H. M. (2007). Oxidative stress and aberrant signaling in aging
and cognitive decline. Aging Cell, 6(3), 361–370.
Erickson, C. A., & Barnes, C. A. (2003). The neurobiology of memory changes in
normal aging. Experimental Gerontology, 38(1–2), 61–69.
Farr, S. A., Banks, W. A., & Morley, J. E. (2006). Effects of leptin on memory
processing. Peptides, 27(6), 1420–1425.
Ferrari, C. K. B. (2007). Functional foods and physical activities in health promotion
of aging people. Maturitas, 58(4), 327–339.
Flood, J. F., Hernandez, E. N., & Morley, J. E. (1987). Modulation of memory
processing by neuropeptide Y. Brain Research, 421(1–2), 280–290.
Hartmann, R., & Meisel, H. (2007). Food-derived peptides with biological activity:
From research to food applications. Current Opinion in Biotechnology, 18(2),
163–169.
Head, E., Corrada, M. M., Kahle-Wrobleski, K., Kim, R. C., Sarsoza, F., Goodus, M. &
Kawas, C. H. (2007). Synaptic proteins, neuropathology and cognitive status in
the oldest-old. Neurobiology of Aging. 10.1016/j.neurobiolaging.2007.10.001.
Hebda-Bauer, E. K., Luo, J., Watson, S. J., & Akil, H. (2007). Female CREBa ,- deficient
mice show earlier age-related cognitive deficits than males. Neuroscience,
150(2), 260–272.
Hwang, I. K., Yoo, K. Y., Jung, B. K., Cho, J. H., Kim, D. H., Kang, T. C., et al. (2006).
Correlations between neuronal loss, decrease of memory, and decrease
expression of brain-derived neurotrophic factor in the gerbil hippocampus
during normal aging. Experimental Neurology, 201(1), 75–83.
Kalache, A., Aboderin, I., & Hoskins, I. (2002). Compression of morbidity and active
ageing: Key priorities for public health policy in the 21st century. Bulletin of the
World Health Organization, 80(3), 243–244.
Kim, S. K., Kim, Y. T., Byun, H. G., Nam, K. S., Joo, D. S., & Shahidi, F. (2001). Isolation
and characterization of antioxidative peptides from gelatin hydrolysate of
Alaska pollack skin. Journal of agricultural and food chemistry, 49(4), 1984–1989.
Korhonen, H., & Pihlanto, A. (2003). Food-derived bioactive peptides – opportunities
for designing future foods. Current Pharmaceutical Design, 9(16), 1297–1308.
McLay, R. N., Pan, W., & Kastin, A. J. (2001). Effects of peptides on animal and human
behavior: A review of studies published in the first twenty years of the journal
Peptides. Peptides, 22(12), 2181–2255.
Mellander, O. (1950). The physiological importance of the casein phosphopeptide
calcium salts. II. Peroral calcium dosage of infants. Acta Societatis Medicorum
Upsaliensis, 55(5–6), 247–255.
Miraliakbari, H., & Shahidi, F. (2008). Antioxidant activity of minor components of
tree nut oils. Food Chemistry, 111(2), 421–427.
340 X. Pei et al. / Food Chemistry 118 (2010) 333–340
Morrison, J. H., & Hof, P. R. (1997). Life and death of neurons in the aging brain.
Science, 278(5337), 412–419.
Nakata, H., & Nakamura, S. (2007). Brain-derived neurotrophic factor regulates
AMPA receptor trafficking to post-synaptic densities via IP3R and TRPC calcium
signaling. FEBS Letters, 581(10), 2047–2054.
Nyffeler, M., Zhang, W. N., Feldon, J., & Knuesel, I. (2007). Differential expression of
PSD proteins in age-related spatial learning impairments. Neurobiology of Aging,
28(1), 143–155.
Paliyath, G. (2006). Functional foods, ageing and degenerative disease. Trends in
Food Science & Technology, 17(8), 466–467.
Popik, P., Bobula, B., Janusz, M., Lisowski, J., & Vetulani, J. (1999). Colostrinin, a
polypeptide isolated from early milk, facilitates learning and memory in rats.
Pharmacoogyl Biochemistry and Behavior, 64(1), 183–189.
Rapp, P. R., Deroche, P. S., Mao, Y., & Burwell, R. D. (2002). Neuron number in the
parahippocampal region is preserved in aged rats with spatial learning deficits.
Cerebral Cortex, 12(11), 1171–1179.
Rasmussen, T., Schliemann, T., Sorensen, J. C., Zimmer, J., & West, M. J. (1996).
Memory impaired aged rats: No loss of principal hippocampal and subicular
neurons. Neurobiology of Aging, 17(1), 143–147.
Shahidi, F., & Ho, C. -T. (2007). Antioxidant Measurement and Applications. ACS
Symposium Series (p. 956). Washington, DC: American Chemical Society.
Silver, F. H., Horvath, I., & Foran, D. J. (2001). Viscoelasticity of the vessel wall: The
role of collagen and elastic fibers. Critical Reviews in Biomedical Engineering,
29(3), 279–301.
Stewart, J. T. (2007). Psychiatric and behavioral manifestations of vascular
dementia. The American Journal of Geriatric Cardiology, 16(3), 165–170.
Suzuki, S., Numakawa, T., Shimazu, K., Koshimizu, H., Hara, T., Hatanaka, H., et al.
(2004). BDNF-induced recruitment of TrkB receptor into neuronal lipid rafts:
Roles in synaptic modulation. The Journal of Cell Biology, 167(6), 1205–1215.
Teter, B., & Ashford, J. W. (2002). Neuroplasticity in Alzheimer’s disease. Journal of
Neuroscience Research, 70(3), 402–437.
Uylings, H. B., & de Brabander, J. M. (2002). Neuronal changes in normal human
aging and Alzheimer’s disease. Brain and Cognition, 49(3), 268–276.
Wanasundara, U. N., & Shahidi, F. (1998). Antioxidant and pro-oxidant activity of
green tea extracts in marine oils. Food Chemistry, 63(3), 335–342.