EXPERIMENTAL AND THERAPEUTIC MEDICINE 4: 577-580, 2012
Abstract. In a previous study, we revealed that a commercially
available product of dietary supplement containing a chicken
comb extract (CCE), which is rich in hyaluronan, not only
relieves joint pain and other symptoms, but also potentially
improves the balance of type II collagen degradation/synthesis
in patients with knee osteoarthritis. Since soccer is one of the
sports most likely to cause knee osteoarthritis (OA), we evalu-
ated the effect of a CCE-containing supplement on cartilage
and bone metabolism in athletes. Fourteen and 15 subjects
(all midfielders) were randomly assigned to receive the test
product (test group) and the dummy placebo containing
only vehicle (placebo group), respectively, for 12 weeks. The
daily oral intake of the CCE-containing test product clearly
decreased the urinary levels of both C-terminal crosslinked
telopeptides of cartilage-specific type II collagen (CTX-II) as
a type II collagen degradation marker and the N-terminal telo-
peptides of bone-specific type I collagen (NTx) as a marker of
bone resorption at 12 weeks after the initiation of the interven-
tion. By contrast, no significant reduction was detected in the
placebo group at any timepoint during the intervention. These
observations indicate that the test product is effective in inhibi-
ting, not only cartilage degradation, but also bone remodeling.
Thus, the CCE-containing supplement may be useful for the
management of joint health in athletes.
The failure of any component of joints participating in load
transmission, particularly bone and articular cartilage, may
lead to sport-induced joint injuries. The skeletal challenges
appear to vary considerably among different sports. In soccer,
the incidence of knee- and ankle-joint injuries is conceivably
higher than in any other sports, possibly since soccer athletes
expose the lower extremity joints to excessive and repetitive
axial loading. Such skeletal injuries are predicted, not only to
prevent players from participating in practice and competition,
but also to increase the risk of osteoarthritis (OA). Results of
various clinical studies have indicated that soccer is one of
the sports most likely to cause knee OA (1-4). Gelber et al (5)
reported that young adults with knee injuries have a consider-
ably increased risk of developing OA.
It is possible that soccer- and certain other sports-related
skeletal injuries, as well as acute or chronic physical loadings,
are expected to affect the turnover rate of the afflicted parts
of the skeleton, particularly the joint tissues, and are detected
using systemic biomarker assays. Various molecular markers
have been reported as indicators of bone turnover and carti-
lage metabolism in patients with bone and joint disorders (6,7).
Among the biomarkers that have been extensively used are
the C-terminal crosslinked telopeptides of type II collagen
(CTX-II) and C-terminal propeptides of type II collagen (CPII)
as the cartilage-specific type II collagen (CII) degradation
and synthesis markers, respectively. N-terminal telopeptides
of bone-specific type I collagen (NTx) have been used as
the marker of bone resorption (8). Previously, we revealed
that CTX-II, CPII and NTx are significantly or substantially
elevated in the urine or sera of young athletes compared with
non-athlete controls (9).
A commercially available product of dietary supplement
containing a chicken comb extract (CCE), which is rich
in hyaluronan, (Kojun®) has been shown relieve joint pain
and other symptoms, as well as to potentially improve the
Evaluation of the effect of a chicken comb extract-containing
supplement on cartilage and bone metabolism in athletes
MASAFUMI YOSHIMURA1, YUKIHIRO AOBA1, TAIJI WATARI2, REI MOMOMURA2, KEITA WATANABE3,
AKIHITO TOMONAGA4, MICHITAKA MATSUNAGA5, YOSHIMASA SUDA6, WOO YOUNG LEE6,
KATSUHITO ASAI7, KAORI YOSHIMURA8, TAKASHI NAKAGAWA8, TETSURO YAMAMOTO8,
HIDEYO YAMAGUCHI8 and ISAO NAGAOKA9
1Graduate School of Health and Sports Science, Juntendo University, Chiba; 2Department of Medicine for Motor Organ,
Graduate School of Medicine, Juntendo University, Tokyo; 3Kitashinyokohama Orthopedic Surgery;
4Tana Orthopedic Surgery; 5Kanagawa University; 6Institute of Physical Education, Keio University, Kanagawa;
7Everlife Co., Ltd., Fukuoka; 8Total Technological Consultant Co., Ltd.; 9Department of Host Defense
and Biochemical Research, Graduate School of Medicine, Juntendo University, Tokyo, Japan
Received April 8, 2012; Accepted June 25, 2012
Correspondence to: Professor Isao Nagaoka, Department of Host
Defense and Biochemical Research, Graduate School of Medicine,
Juntendo University, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan
Key words: hyaluronan, cartilage and bone metabolism, biomarker,
supplementary diet, athlete
YOSHIMURA et al: CHICKEN COMB EXTRACT EFFECT ON CARTILAGE AND BONE METABOLISM IN ATHLETES
balance of CII degradation/synthesis in patients with knee
OA (10,11). Based on these observations, we hypothesized that
the CCE-containing supplement product (test product) has an
effect on the three biomarkers in athletes. Therefore, in this
study, we evaluated the effect of a CCE-containing supplement
on cartilage and bone metabolism in athletes.
Materials and methods
Subjects. The study was approved by the Human
Experimentation Ethics Committee of Juntendo University
(Japan) and informed consent was obtained from the partici-
pants. A total of 66 collegiate athletes belonging to three
different intercollegiate soccer teams were recruited to
participate in the study. It was expected that the rate of skeletal
injuries and the levels of soccer-related activities or physical
loadings are influenced by the position of players within the
team and/or on the pitch. These levels were predicted to be
higher in the midfielder position compared to any other player
positions, including the forward, defender and goal keeper,
since midfielders cover a longer distance than the players of
other positions (12). For this reason, only midfielders were
selectively analyzed in this study.
Intervention and subject group assignment. The test product
was a 300-mg capsule preparation consisting of 157.5 mg of
CCE, of which approximately 4.5 mg was hyaluronan, 20 mg
calcium lactate, 10 mg propolis extract, 4.9 mg chitosan
oligosaccharide, 5.0 mg each of vitamins B1 and B6, 2.5 mg
vitamin E, 2.0 mg ferric pyrophosphate, 0.1 mg vitamin B12 and
192.5 mg of vehicle (a mixture of crystalline cellulose, dextrin
and fatty acid sugar esters) (11). Fourteen and 15 subjects
were randomly assigned to receive 16 capsules/day of the test
product (test group) and those of dummy placebo containing
only vehicle (placebo group), respectively. The subjects in the
two groups were instructed to take allocated capsules at a dose
of 8 capsules twice daily for 12 weeks. The study was executed
in accordance with the principles of the amended Declaration
of Helsinki and the Ethical Guidelines for Epidemiological
Research (established by the Japanese Government in 2004)
during the 2010 summer-fall soccer competition season.
Procedures. The urine and blood/serum samples were
collected at baseline (before the intervention) and at 4, 8 and
12 weeks after the intervention, and stored at less than -40˚C
before assay. Urinal CTX-II and NTx were measured using
CartiLaps EIA® (Immunodiagnostic Systems, Inc., Tyne &
Wear, UK) and Osteomark® (Inverness Medical Japan Co.,
Ltd., Tokyo, Japan), respectively, and the values were normal-
ized with creatinine (Cr). Serum CPII was measured using
Procollagen II C Propeptide ELISA® (Ibex Pharmaceuticals,
Inc., Mont-Royal, QC, Canada).
Statistical analysis. Unpaired and paired Student's t-tests
were used for the between-group comparison and within-
group comparison, respectively. Values were presented as the
means ± SD. P<0.05 was considered to indicate a statistically
Data from 29 subjects (14 and 15 in the test and placebo
groups, respectively) who completed the study were evalu-
ated. Demographic characteristics [age, height, weight, body
mass index (BMI), blood pressures and pulse rate] as well as
biomarker profiles (CTX-II, CPII, CTX-II/CPII ratio and NTx)
were not significantly different between the test and placebo
groups (Table I).
Table II shows the changes in the biomarker profiles
during the intervention. CTX-II levels were clearly reduced
from the baseline in the test group with a statistical signifi-
cance at week 12 (P<0.01), while no significant reduction
was observed in the placebo group at any timepoint during
the intervention. Unlike CTX-II, CPII levels were elevated
significantly from the baseline at all timepoints (weeks 4, 8
and 12; P<0.01 or <0.05). As a result, the ratios of CTX-II to
Table I. Baseline data of subjects in the test and placebo groups who completed the study.
Test group (n=14)
(mean ± SD)
Placebo group (n=15)
(mean ± SD) P-value
Body mass index (kg/m2)
Systolic blood pressure (mmHg)
Diastolic blood pressure (mmHg)
Pulse rate (beats/min)
Urinary CTX-II (ng/mmol Cr)
Serum CPII (ng/ml)
Urinary CTX-II/serum CPII ratio
Urinary NTx (nmol BCE/mmol Cr)
BCE, bone collagen equivalent; CTX-II, C-terminal crosslinked telopeptides of cartilage-specific type II collagen; CPII, cartilage-specific
type II collagen; NTx, N-terminal telopeptides of bone-specific type I collagen; Cr, creatinine.
EXPERIMENTAL AND THERAPEUTIC MEDICINE 4: 577-580, 2012
CPII were decreased to a significant level (P<0.01 or <0.05)
at weeks 4, 8 and 12 in the test group and at weeks 8 and
12 in the placebo group. Notably, NTx levels were reduced
only in the test group, reaching a significant level at week 12
Fig. 1 shows the changed values from the baseline in all
biomarkers used in this study during the intervention. The
changes in the CPII levels were not different between the two
groups at any timepoints. By contrast, the changes from the
baseline levels of CTX-II, the CTX-II/CPII ratio and NTx
were greater in the test group than in the placebo group, and
the between-group difference in the NTx was statistically
significant at week 12 (P<0.05).
Joint injury and OA are similarly characterized by the remode-
ling and degradation of cartilage, bone and other joint tissues.
Lohmander et al (13) reported the release of CTX-II into
synovial fluid in human OA and joint injury, which provides
the evidence that the integrity of the CII network of cartilage
is compromised shortly after joint injury and in arthritis. With
regard to OA, accumulating evidence indicates that serum or
urinary levels of CTX-II and/or certain other CII degradation
markers, including C1, 2C and C2C, increase with the progres-
sion of OA (10,11). The presence of NTx in the urine has been
evaluated by Bettica et al (8) as a risk marker in progressive
knee OA and was found to be elevated in patients with the
radiographic progression of knee OA. This finding suggests
that the progression of OA reflects the changes of both carti-
lage metabolism and bone turnover, and that the subchondral
bone alterations play a role in the pathogenesis of OA. Based
on these findings with regard to OA and the similarity of the
change in the skeletal marker profiles between OA and joint
injury, we have hypothesized that, similar to OA, joint injury
is also characterized by cartilage degradation and subchondral
bone abnormalities in athletes.
In the present study, we have shown that the daily oral
intake of the CCE-containing test product clearly decreases
the urinary levels of both CTX-II and NTx at 12 weeks after
the initiation of the intervention. The results suggest that the
Table II. Changes in the levels of urinary CTX-II, serum CPII, the CTX-II/CPII ratio and urinary NTx during the 12-week
intervention period in the test (n=14) and placebo groups (n=15).
Groups Baselinea Week 4a Week 8a Week 12a
CTX-II (ng/mmol Cr)
NTx (nmol BCE/mmol Cr)
aValues are expressed as the mean ± SD and the percentage changes from the baseline are indicated in parentheses. Values are compared between
the baseline and the timepoints after the intervention. bP<0.01; cP<0.05. BCE, bone collagen equivalent; CTX-II, C-terminal crosslinked
telopeptides of cartilage-specific type II collagen; CPII, cartilage-specific type II collagen; NTx, N-terminal telopeptides of bone-specific type I
collagen; Cr, creatinine.
Figure 1. Changes in the levels of (A) urinary CTX-II, (B) serum CPII,
(C) CTX-II/CPII ratio and (D) urinary NTx from the baseline in the test
group (n=14; black bar) and the placebo group (n=15; whites bar) during the
12-week intervention. Changes in the levels from the baseline are expressed
as delta, and are compared between the test and placebo groups. *P<0.05.
YOSHIMURA et al: CHICKEN COMB EXTRACT EFFECT ON CARTILAGE AND BONE METABOLISM IN ATHLETES Download full-text
test product is effective in inhibiting cartilage degradation and
bone remodeling. In this context, hyaluronan, a putative active
component of the CCE-containing supplement, has been
reported to stimulate bovine chondrocytes, being effective in
maintaining the cell phenotype with increased matrix deposi-
tion of glycosaminoglycan and CII (14,15). Although the actual
mechanism of this beneficial action on joint injury remains
unclear, the CCE-containing supplement may be useful for the
management of joint health in athletes.
In conclusion, the oral administration of the CCE-
containing supplement decreases the levels of CTX-II and
NTx in the urine of young athletes, suggesting a potential use
for relieving the joint injury-associated cartilage degradation
and bone remodeling.
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