Glycative Stress Research
Online edition : ISSN 2188-3610
Print edition : ISSN 2188-3602
Received : October 7, 2017
Accepted : December 27, 2017
Published online : March 31, 2018

Original article

Study for investigation of symptomatic improvement and safety of the ingestionof rooster comb degradation product containing low-molecular hyaluronic acid
(INJUV) in individuals with knee and lower back pain; open-label trial with nocontrol group
Mari Ogura 1, 2), Wakako Takabe 1), Masayuki Yagi 1), Sachio Wakayama 3), Yoshikazu Yonei 1)
1) Anti-Aging Medical Research Center and Glycative Stress Research Center, Faculty of Life and Medical Sciences,
Doshisha University, Kyoto, Japan2) Kyoto Bunkyo Junior College, Kyoto, Japan
3) Laimu Corporation. Yokohama, Kanagawa, Japan

Abstract

Objectives: The objectives of this study were to verify the efficacy in subjective and objective symptoms and safety of theingestion of food sample, rooster comb degradation product containing low-molecular hyaluronic acid (INJUV) under an
open-label trial with no control group.
Methods: The subjects were 12 healthy adults (6 males and 6 females, age: 57.5 ± 3.5 years old) selected from 30
individuals who suffered with gonalgia and lumbago. INJUV was administered to the subjects by oral ingestion for 8
weeks.
Results: Improvements of gonalgia and lumbago were observed as subjective symptoms. The range of motion in the knee
joint was extended as physical findings (left and right average; before 137.4 ± 1.8, 2.9%, p < 0.05). Furthermore, diastolicblood pressure was lowered (before 75.7 ± 3.2 mmHg, –8.2%, p < 0.05) and HbA1c was decreased (before 5.47 ± 0.08%,– 3.3%, p < 0.01). No adverse effect was observed during the trial.
Conclusion: The test results showed the improvement in gonalgia and lumbago, and the extension of the range of motion in
the knee joint, as well as the safety of INJUV. It was assumed that this low-molecular samples, characterized by a high rateof absorption, exerted beneficial effects on articular cartilage tissues, synovial cells and immunocompetent cells. Further
examinations are needed to clarify the efficacy and the mechanism of action.

KEY WORDS: low-molicular hyaluronic acid, arthralgia, lumbago, osteoarthritis, range of motion

Introduction

Locomotive syndrome means the state of lowered mobile
or locomotive function due to disorders of locomotoriums
such as muscle, bones and joint. In progression of locomotive
syndrome (herein after referred as “locomo” ), people areforced to walk with a cane and move using a wheelchair,
which lowers quality of life (QOL). This could lead to aperson being bedridden and requiring long-term care level
5. Japan has been facing a situation where the population
is rapidly aging. Countermeasures for the prevention andtreatment for locomo could not be achieved without theextension of health expectancy in Japanese society.
This time we focus on osteoarthritis (OA), which isone of the factors inducing locomo. We examined whether
or not symptoms of gonalgia and lumbago were mitigatedby the ingestion of functional food. It has been reported
that glycative stress involves pathogenic mechanisms ofrheumatoid arthritis (RA), and OA 1-5). INJUV, which is arooster comb degradation product, whose main component ishyaluronic acid was chosen as a test sample in this study 6).
This product is popular among users in post marketingsurveillance and is known to have repeat customers.The
subjects were healthy males and females who suffered frompain in the knee and lower backin their daily lives. INJUV
efficacies on OA and lumbago were examined for 8 weeks;
this study was an open trial with no control group.

Contact Address: Professor Yoshikazu Yonei, MD, PhD
Glycative Stress Research Center,
Faculty of Life and Medical Sciences, Doshisha University
1- 3, Tatara Miyakodani, Kyotanabe, Kyoto, 610 - 0394 Japan
Tel & FAX: + 81-774 - 65- 6394 eMail: yyonei@mail.doshisha.ac.jp
Co-authors: Ogura M, m-ogura@po.kbu.ac.jp ; Takabe W, wtakabe@mail.doshisha.ac.jp ;Yagi M, yagi@yonei-labo.com ; Wakayama S, s.wakayama@laimu.jp_ 55 _

Glycative Stress Research 2018; 5 (1): 055 - 067(c) Society for Glycative Stress ResearchAn Open-label Trial: Rooster Comb Degradation Product containing Low-molecular Hyaluronic Acid

Methods
Subjects

With a recruitment notification, males and females
aged 55-69 years, with knee and/or low back pain in daily
life were asked to participate. The selected subjects wereas follows; among the 32 potential individuals who met theeligibility requirements, the inclusion criteria, and did notmeet the exclusion criteria, the 12 subjects with the greatest
representation in the questionnaire regarding the following
index 1) and 2) were selected (male; 6, female; 6, age; 57.5 ±3.5 years).
1) Japanese Knee Osteoarthritis Measure 7-11): JKOMⅠ.
Score in “Degree of keen pain”
2) Japan Lower back pain Evaluation. Questionnaire 12-15):
JLEQ Ⅰ. Score in “ Degree of lower back pain”
Inclusion criteria and exclusion criteria were as follows;

Inclusion criteriag

1) Males and females aged 55-69 years.
2) Healthy individuals not receiving any medical disease
treatment
3) Individuals with knee and/or lower back pain in daily
life (in walking, in sitting with one's legs bent beneathoneself and in ascending and descending stairs)
4) Individuals who are able to sign and provide writtenconsent for participation, after receiving enoughexplanation of the purpose and details of the study,
understanding the study well, and deciding to participate
in the study with their own will5) Individuals who are willing and able to comply withscheduled visits and examinations
6) Individuals judged appropriate for the study by theprincipal investigator

Exclusion Criteria

1) Individuals recieving medical products for treatment ofchronic diseases
2) Individuals receiving outpatient care (except forbonesetters, chiropractors, massagers) for knee and/or
lower back pain3) Individuals with a history of palliative therapy(hyaluronic acid, steroid injection, etc.) or surgical therapy(arthroscopic osteotomy, artificial joint, bolt fixation
surgery, etc.) on knees and/or waist4) Individuals who have a present or past medical history ofpsychiatric disease, sleep disorder, high blood pressure,diabetes, dyslipidemia and or other serious diseases
5) Individuals who have or had administration of a drug
to treat a disease in the past month (except temporary
usage for headache, menstrual pain, common cold andpollenosis)
6) Individuals who have a present or past medical historyof serious hepatopathy, renal dysfunction, heart disease,respilatory disease or hyper blood disease7) Individuals who have a comorbidity and a medical history
with gastrointestinal disease (except a medical history of
caecum)8) Individuals with inflammatory disorders such as chronicsinusitis, periodontal disease, bronchitis and othere> 30.0 kg/m 29) Individuals with body mass index: BMI =10) Individuals who donated blood over 200 mL in the pastmonth or over 400 mL in the past three months
11) Individuals with serious anemia
12) Individuals who could develop an allergy to the foodcomponents contained in the test substance or havea serious allergic reaction to other foods or medical
products
13) Individuals with continuous ingestion of food that could
affect study results such as health food, food professing
improvement of knee and/or low back pain and component
expecting effects that may interfere with the study results(especially glucosamine, chondroitin and others) in thepast three months, at present or during the examination
period, except for general health food for maintaining
health14) Individuals with intake of alcohol 40 mg/day or more, or
weekly 200 mg/week or more in average15) Individuals who are smokers or ex-smokers within one
year after quitting16) Individuals with possible changes of exercise habits orlife style during the examination period17) Individuals who are currently participating at or haveparticipated in other clinical studies in the past 3 months18) Individuals who are pregnant, are possibly or hope tobecome pregnant, or are lactating19) Individuals who are or whose family are engaged inhealth and functional foods, and/or cosmetic companies
for development, manufacture and marketing20) Individuals judged inappropriate for the study by theprincipal investigator

Test sample

The test sample was “INJUVcapsule”. Useful component
injuv (herein after referred as “INJUV”) was a powdery
material, which was obtained through a freeze-drying
process of rooster comb, and is manufactured and sold by
Laimu Co., Ltd. (Kohoku-ku, Yokohama, Kanagawa, Japan).
INJUV was developed through a unique enzyme process at
normal temperature, using rooster comb as a raw material.
Products have been sold not only in Japan but also in 11
countries all over the world since 2001, and especiallyin the United States sales performance is over forty-two
million capsules a year. The test samples for this study weresponsored by this company.
This sample is a powder material produced from rooster
comb. The raw material, rooster comb was degraded andprocessed by the food-derived enzyme and this rooster
comb enzyme decomposition product was freeze-dried
and processed into powder with three times the amount of
dextrin (binding food additive) as rooster comb powder 6) .
This sample contained 150 mg of INJUV per one capsule,the same as the commercial product does. INJUV is anaturally-derived degradation product with components of
a whole rooster comb, which contains HA, collagen peptide,proteoglycan (aggrecan), these precursors, amino acid andvitamins. Generally, oral administration of HA with a highmolecular weight of more than a million or a low molecular
wight of between one hundred thousand and two hundred
thousand has a limited effectiveness. However, this producthas been developed by enzyme degradation to be a molecularweight of between 380 and 5,000, which is regarded as anultra-low molecular weight.
Table 1 shows “Nutrition component of the test product.”
Table 2 shows “ Free amino acid component of the test
product ” 6). The weight of the smallest unit of HA is a

_ 56 _Glycative Stress Research

Table 1. Nutrition component of the test product.

Component
Water
NitrogenProteinFree amino acid
N-Acetylglucosamine
Dextrin (additives)

Content (%)

Method

2.2 ~ 2.6
3.84

Semimicro-Kjeldahl method

4.08

Ninhydrin assay

3.04

Lowry protein assay

0

Elson-Morgan method

75.0

Table 2. Free amino acid component of the test product.

Amino acidρ-Serine
TaurineAsparatic acidThreonine
SerineGlutamic acid
Glutamine
Sarcosine
Glycine
AlanineCitrulline
α-Amino butyric acid
Cysteine
Methionine

Content (%)
1.71

3.30

2.94

Amino acid
Cystine2.78

Isoleucine

6.27

2.26

Leucin1.30

T yrosine

2.18

β-amino isobutyric acid

2.20
0.48
1.81

2.26
3.52

0.92
2.18

1.03
1.97

Content (%)

Phenylalanine
Ornithine
Lysinel -Methylhistidine
Anserine
Arginine

Total amino acid identified
Unknown amino acidmolecular weight of 411, which is a molecular bond of
one molecule of glucuronic acid and one molecule ofN-acetylglucosamine. Listing the assumed molecular weightof HA contained in the sample, in order of component weightratio, showed 1,520 (47%), 5,000 (33%), 1,140 (10%), 760(6%) and 380 (4%), and revealed that the main components
are low molecular HA, which have high absorption
efficiency. The N-acetylglucosamine content is 0%, as HA
degradation product is not contained.
Macromolecule HA has a strong water retention capacity
and is utilized for instillation and as a medical agent for
intra-articular injection and skin moisturizing lotion.
However, there has been only one report about the effectsof oral administration of low molecular HA, which was arandomized double-blind clinical trial conducted at Beijing
University of Chinese Medicine Dongzhimen Hospital (thePeople's Republic of China). It was shown that this sample
showed an enhanced water retention property in keratin 6) .
The safety of the sample, INJUV, was confirmed> 5,050 mg/kg body weight) by a 14 -day INJUV(LD 50 =administration test consigned to Still Meadow Inc. in U.S.A.,
which corresponded to GLP test.

2.65
3.30
5.45

1.05
1.17

0.78

1.92
1.93

57.36

42.64

Trial design

The trial design was as follows; type of control: none,
trial blinding schema: open, intervention: oral ingestion of
the test product, 8 capsules a day (as INJUV, 1,200 mg/day),which were ingested as 4 capsules twice a day with water or
tepid water.
Blood examinations were performed before and 8 weeks
after the administration. Medical questionnaire sheet survey,
physical examination, specialized blood examination (hsCRP) and specialized urine examination (pentosidine andcreatinine) were performed before, 2 weeks after, 4 weeks
after, 6 week after and 8 weeks after the administration. The
test period was from March of 2017 to August of 2017.

Assessment items• Subjective symptoms
Japanese Knee Osteoarthritis Measure (JKOM)The Japanese Knee Osteoarthritis Measure (JKOM) 7-11)
used a Visual Analogue Scaleas the method of measurement
for a patient to indicate a position along a continuous line
between two end-points.One edge of a line with the length_ 57 _An Open-label Trial: Rooster Comb Degradation Product containing Low-molecular Hyaluronic Acidof 100 mm was a mark of “no pain” and the other edge was“the most severe pain that the patient have ever experienced.”
Patients marked a point on the line to express the degree of
their pain and the distance from the point of “no pain” to“the marked point” was measured to assess their pain.
In a multiple choice section of “Pain and stiffness inknees,” “Condition in daily life” and “General activities,”
the lightest state was scored as “0” and the most severe state
was scored as “4.”Other multiple choice states were assessedas “1”, “2” and “3” in accordance with the intensity of thesymptoms. Total score was obtained by calculation.
Western Ontario and McMaster Universities
Osteoarthritis Index (WOMAC) (Japanese Version)Western Ontario and McMaster Universities Osteoarthritis
Index (Japanese Version) 16-20) assessed pain in the knee joint
and physical function. Scale of the degree of pain; No pain
= 5, Slight pain = 4, Medium pain = 3, Severe pain = 2 andExtreme pain = 1. Scale of difficulty in physical function;
Not difficut = 5, Slightly difficult = 4, Moderately difficult =3, Very difficult = 2 and Extremely difficult =1.
Japan Lower Back Pain Evaluation Questionnaire (JLEQ)
Japan Lower Back Pain Evaluation Questionnaire(JLEQ) 12-15) assessed the intensity of lower back pain using
a Visual Analogue Scale. One edge of a line with the lengthof 100 mm was a mark of “no pain” and the other edge was“the most severe pain that the patient have ever experienced”.
Patients marked a point on the line to express the degree of
their pain and the distance from the point of “no pain” to“the marked point” was measured to assess their pain.
In a multiply choice section of “Problems in daily life”
and “Conditions in the last one month,” the lightest state wasscored as “0” and the most severe state was scored as “4.”Other multiple choice states were assessed as “1,” “2” and“ 3” in the accordance with the intensity of symptoms. Total
score was obtained by calculation.

Anti-Aging QOL Common questionnaire

An Anti-Aging QOL Common Questionnaire (AAQOL) 21)
was employed to assess subjective symptoms, which wereclassified into “physical symptoms” and “mental symptoms.”
Scores are assessed in five steps, from point 1 to point 5.
• Physical examination
Measurements of the range of motion in the knee joint
were performed by orthopedic medicine specialists, using agoniometer as a measurement device ( Todai 30 cm; Z813153A).
As for physical measurements, height, weight, body fat
percentage, BMI, blood pressure systolic and diastolic andpulse were measured. Body composition was examined bya biological component measuring device (DC-320; Tanita
Corporation, Itabashi-ku, Tokyo, Japan).
Blood pressure and pulse were measured by an electronic
sphygmomanometer (H55 Eremano sphygmomanometer,
Terumo Corporation, Shibuya-ku, Tokyo, Japan).
• Blood and urine examination
A blood examination was conducted in the Health
Sciences Research Institute, Inc. (Chofu, Tokyo, Japan) for
the following; leukocyte count (WBC), red blood count (RBC),
hemoglobin content (Hb), hematocrit value (Ht), mean cellvolume (MCV), mean corpuscular hemoglobin (MCH), mean

corpuscular hemoglobin concentration (MCHC), blood
platelet count (PLT), total protein (TP), albumin (ALB), urea
nitrogen (UN), creatinine (CRE), uric acid (UA), aspartate
aminotransferase (AST), alanine aminotransferase (ALT),
γ-glutamyl- transpeptidase ( γ- GT), alkaline phosphatase(ALP), lactic acid dehydrogenase (LDH), creatine kinase (CK),
total cholesterol (TC), triglyceride (TG), LDL cholesterol(LDL-C), HDL-cholesterol (HDL-C), natrium (Na), kalium(K), chlorine (Cl), calcium (Ca), fasting blood sugar (FPG),
HbA1c, CRP and rheumatoid factor (RF).
A urine examination was conducted in Health Sciences
Research Institute, Inc. (Chofu, Tokyo, Japan) forthe following;urobilinogen, occult blood reaction, bilirubin, ketone bodies
qualitative, urine glucose, protein, specific weight and pH.
Examination of urinary pentosidine and urinary creatinine
were conducted in LSI Medience Corporation (Chiyoda-ku,
Tokyo, Japan).

Statistical analysis

Statistical analysis was performed, usinga statisticalanalysis software, SAS (SAS 9.4; SAS Institute Japan,
Minato-ku, Tokyo, Japan) and/or SPSS (Statistics19; IBM
Japan, Ltd., Chuo-ku, Tokyo, Japan) in a paired-t test. Hazard
rate < 0.05 was a significant difference and hazard rate < 0.1
was marginally significant. As for outlier and missing value,
outlier was not set. Provided that data was not obtained
due to troubles on examinations or a problem induced lackcredibility of data, the data was treated as a missing value
and an alternative value was not used.

Ethical review

This study was conducted with the approval of Human
Studies Ethics Committee in “general corporate judicial
person, Society for Glycative Stress Research” (GSE #2017002). This study was registered beforehand to Clinical Trial
Registry (UMIN #000026460).

Results

Subjective symptoms assessment

Administration of thetest sample for 8 weeks improved
subjective symptoms as follows;
In AAQOL, 5 of the 33 physical symptom items weresignificantly improved; “ Tired eyes ” (p < 0.05), “Stiffshoulders” (p < 0.05), “Muscular pain/stiffness” (p < 0.05),“Lumbago” (p < 0.01) and “Arthralgia” (p < 0.01). One itemof 21 mental symptom items, “Pessimism” was significantly
improved (p < 0.05, Table 3 ). Other items did not show any
significant changes.
Table 4 shows that in JKOM, significant improvements
were as follows; “Ⅰ. Degree of knee pain” “Where on thisline would your pain have been over the last several days?”(Before: 6.1 ± 0.4, 8 weeks: 2.6 ± 0.6, p < 0.01), “ Ⅱ. Pain andstiffness of the knees” (Before: 10.1 ± 1.4, 8 weeks: 5.1 ± 1.2,
p < 0.01), “ Ⅲ. Condition in daily life” (Before: 9.2 ± 1.5,
8 weeks: 5.2 ± 1.3, p < 0.01) and “ Total score” (Before: 27.8
± 3.8, 8 weeks: 15.8 ± 3.1, p < 0.01).
Table 5 shows that in WOMAC, Japanese version,
significant improvements were as follows; in “ Ⅰ. Pain,” rightknee (Before: 18.8 ± 1.1, 8 weeks: 22.4 ± 0.8, p < 0.01), left
knee (Before: 18.5 ± 0.9, 8 weeks: 22.2 ± 0.7, p < 0.01) and_ 58 _Glycative Stress Research

Table 3. AntiAging QOL Common questionnaire.

Physical symptoms
Tired eyes
Stiff shoulders
Muscular pain/stiffness
LumbagoArthralgia
Mental symptoms
Pessimism

Before8 weeks

p value

3.4 ± 0.4

2.8 ± 0.3 *0.011

2.1 ± 0.4 *0.028

3.5 ± 0.4
3.2 ± 0.4

2.8 ± 0.4 *3.6 ± 0.3

2.5 ± 0.3 **1.9 ± 0.3

1.6 ± 0.2*

3.6 ± 0.2

2.7 ± 0.3 **

0.021
0.010

0.004
0.046

Data are expressed as mean ± SEM, Wilcoxon signed-rank test, n = 12. SEM, standard error mean.

Table 4. Japanese Knee Osteoarthritis Measure.

Ⅰ. Degree of knee pain
Visual analogue scale asking the degree of knee pain.
Ⅱ. Pain and stiffness in knees
Ⅲ. Condition in daily life
Ⅳ. General activities
Ⅴ. Health conditions
Total score

Before8 weeks

p value

6.1 ± 0.4

2. 6 ± 0.6 **

0.000

10.1 ± 1.4

5.1 ± 1.2 **0.004

3.6 ± 0.6 *0.044

9.2 ± 1. 5

5.2 ± 1.3 **3.0 ± 0.7

2.0 ± 0.5

5.6 ± 0.9

27.8 ± 3.8

15.8 ± 3.1 **

0.002
0.058

0.003

Data are expressed as mean ± SEM,Paired t-test, Wilcoxon signed-rank test, n = 12. SEM, standard error mean.

Table 5. WOMAC (western ontario mcmaster universities osteoarthritis index), japanese version.

Pain
Right knee
Left knee
Total score

Before8 weeks

p value

18.8 ± 1.1

22.4 ± 0.8 **0.006

44.6 ± 1. 5 **0.005

22.2 ± 0.7 **3. 5 ± 0.2

4.2 ± 0.2 *0.020

3.8 ± 0.3

4.6 ± 0.2 *

0.015

37.3 ± 1.9

Physical function
1. Descending stairs
2. Ascending stair
3. Rising from sitting
7. Getting in/out of car
11. Taking off socks
Total score

3.4 ± 0.3

4.0 ± 0.2
3.7 ± 0.3

66.2 ± 3.6

Data are expressed as mean ± SEM, Wilcoxon signed-rank test, n = 12. SEM, standard error mean.

_ 59 _

0.004

18. 5 ± 0.9

4.3 ± 0.2 **4.6 ± 0.1 *4.3 ± 0.2 *74.9 ± 2.3 *

0.005
0.020
0.038
0.013

An Open-label Trial: Rooster Comb Degradation Product containing Low-molecular Hyaluronic Aciddays” (Before: 21.8 ± 4.2, 8 weeks: 10.6 ± 2.4, p < 0.01), “ Ⅳ.
Your condition in the last month” (Before: 6.1 ± 1.3, 8 weeks:
2.4 ± 0.7, p < 0.01) and “ Total score” (Before: 38.3 ± 7.3, 8
weeks: 17.8 ± 4.1, p < 0.01).

total score (Before: 37.3 ± 1.9, 8 weeks: 44.6 ± 1.5, p < 0.01).
In “ Physical function,” “1. Descending stairs” (p < 0.05),“ 2. Ascending stairs” ( p < 0.01), “ 3. Rising from sitting”( p < 0.05), “ 7. Getting in/out of car ” ( p < 0.05) and “11.
Taking off socks” ( p < 0.05) were significantly improved.
Furthermore, “ Total score” was significantly improved(Before: 66.2 ± 3.6, 8 weeks: 74.9 ± 2.3, p < 0.05).
Table 6 showed that in JLEQ, the following was
significantly improved; “ Ⅰ. Degree of lower back pain”“Where on this line would your pain have been over the last
several days? ” (Before: 6.0 ± 0.7, 8 weeks: 2.5 ± 0.5, p < 0.01),“ Ⅱ. Your lower back pain over the last several days” (Before:
10.4 ± 2.0, 8 weeks: 4.8 ± 1.1, p < 0.01), “ Ⅲ. Problems withyour lifestyle due to lower back pain over the last several

Physical examination (Table 7)

Physical examination showed that blood pressure (diastolic)
was significantly lowered 8 weeks after administration(Before: 75.7 ± 3.2 mmHg, 8 weeks: 69.5 ± 3.3 mmHg, rateof change: −8.2%, p < 0.05).
The assessment on the range of motion of the kneeby orthopedic medicine specialists showed that the rangeof active motion (left) was significantly improved after

Table 6. JLEQ：Japan Low Back Pain Evaluation Questionnaire

I.

Degree of low back pain
Where on this line would your pain have been over the last several days?

6.0 ± 0.7

2.5 ± 0. 5 **0.001

II. Low back pain related to activity of daily living
Your low back pain over the last several days

10.4 ± 2.0

4.8 ± 1.1 **0.007

III. Problems due to low back pain
Problems with your lifestyle due to low back pain overthe last several days

21.8 ± 4.2

10.6 ± 2.4 **0.002

IV. Health and psychological condition
Your condition in the last month

6.1 ± 1.3

2.4 ± 0.7 **0.006

Total score38.3 ± 7.3

17.8 ± 4.1 **0.002

Data are expressed as mean ± SEM, Paired t-test, Wilcoxon signed-rank test, n = 12. SEM, standard error mean.

Table 7. Physical examination.

BeforeHeightWeightBody fat
BMI
Blood pressure (systolic)(diastolic)
Pulse

cm
kg
%
mmHg
mmHg
/min

164.1 ± 2.1

Range of motion at knee joint
activeleft
right
left and right average
passiveleft
right
left and right average

°
°
°
°
°
°
°

64.1 ± 3.2
27.4 ± 1. 5
23.7 ± 0.9

8 weeks

p value

63.9 ± 3. 5

0.775

23.6 ± 1.0

0.743

26.7 ± 1. 5

0.060
0.269

116.8 ± 5.2

112.6 ± 4.6

72.7 ± 4.1

68.0 ± 2.4

138.3 ± 1.7

140.8 ± 1.9 *

0.049

137.4 ± 1.8

141.4 ± 1.8 *

0.044

143.6 ± 2.1

142.8 ± 1.9

0.522

142.8 ± 2.1

143.1 ± 1.8

0.852

75.7 ± 3.2

136. 5 ± 2. 5

141.9 ± 2. 5

Data are expressed as mean ± SEM, Paired t test, n = 12. SEM, standard error mean.

_ 60 _

69. 5 ± 3.3 *141.9 ± 1.7

143.4 ± 1.7

0.015

0.106

0.079

0.559

Glycative Stress Research

8 weeks (Before: 138.3 ± 1.7, 8 weeks: 140.8 ± 1.9, rateof change: 1.8%, p < 0.05). Range of active motion (right)
showed marginally significant improvement after 8 weeks(Before: 136.5 ± 2.5, 8 weeks: 141.9 ± 1.7, rate of change:
4.0%, p < 0.1). Range of active motion (left and right average)
was significantly improved after 8 weeks (Before: 137.4 ±
1.8, 8 weeks: 141.4 ± 1.8, rate of change: 2.9%, p < 0.05).
Range of passive motion of the knee showed no significant
improvement.

Blood and urine examination (Table 8)

A peripheral blood examination showed that the following
items had been significantly reduced 8 weeks after theadministration of the test sample; WBC (– 7.1%, p < 0.05),
RBC (– 3.6%, p < 0.05), Hb (– 4.9%, p < 0.01), Hct (– 3.0%,
p < 0.05), MCH (–1.3%, p < 0.05) and MCHC (–1.8%, p <0.01). The changes in RBC-related index were influenced bycollection of blood sample.
A blood biological examination showed that thefollowing items were significantly lowered 8 weeks after theadministration; total protein (– 4.6%, p< 0.01), Alb (– 3.0%,
p < 0.05), AST (– 12.8%, p < 0.01), ALT (–17.8%, p <0.05), Na (–1.1%, p < 0.01). Carbohydrate metabolism index
showed that FPG did not change but HbA1c was significantly
lowered 8 weeks after administration (– 3.3%, p < 0.01).
There was no significant difference in hs-CRP and
urinary pentosidine.

Discussion

This study was conducted to confirm the physicalchanges and the safety of the ingestion of the test product(INJUV) over 8 weeks in an open-label, non-controlled
clinical trial. The subjects were 12 adults (male; 6, female;6) with pain in the knee joint and/or lower back during
daily life (in walking, in sitting with one's legs bent beneathoneself and in ascending and descending stairs). This study
examined the physical changes and the safety in an openlabel, non-controlled clinical trial of the oral administrationof the specimen containing enzyme degradation product ofrooster comb (main component; low-molecular HA) for 8
weeks. The data results indicated that the subjective symptoms
of knee and lower back pain were improved and the physicalreview showed the extension of knee joint motion range.
Secondarily, the decrease of diastolic blood pressure, thedecrease of TC and the decrease of HbAlc were also observed.
The ingestion of the test product was observed to
improve the subjective symptoms of knee and lower backpain. Anti-Aging QOL Common Questionnaire (AAQOL) 21) ,Japanese Knee Osteoarthritis Measure (JKOM) 10, 11), Western
Ontario and McMaster Universities Osteoarthritis Index(WOMAC) (Japanese Version) 18, 20) and Japan Lower Back
Pain Evaluation Questionnaire (JLEQ) 12, 13) were employed
for the assessment of subjective symptoms. Among thequestionnaire items concerning the whole body in AAQOL,
it was assumed that the pain in the knee joint and lower
back had an efficacy spectrum. A physical examination
showed the decrease of diastolic blood pressure (– 8.2%, p< 0.05), the extension of knee joint motion range (average ofleft and right: 2.9 %, p < 0.05) and the decrease of HbAlc.
A biochemical examination of blood showed a decreaseof WBC (– 7.1%, p < 0.05). An examination of glycolipid

metabolism showed no difference in FPG, LDL-C, HDL-C
and TG but a decrease in TC (– 5.4%, p < 0.05) and adecrease in HbA1c (– 3.3%, p < 0.01) were recognized.
Further, the decrease of the RBC-related indices ( RBC,
Hb, Hct, MCH and MCHC), the decrease of total protein
and the decrease of Alb were observed, which seemed tobe affected by collecting blood samples. The reason why
these phenomena are influences of collecting blood samples
is explained in the section on safety assessment, as this isrelated to the safety of this product. We were not able toclarify the reason for the decrease of HbAlc.
This test product contains HA with a low molecular weightand collagen peptide as related-substances and the mainfunctional component is HA. It has been reported in clinical
trials that the oral ingestion of HA and/or glucosamine
reduces pain in the knee joint or other joints 8, 22-24).
Processed food made with rooster comb extracts
containing low molecular HA was examined in a randomized
double blind comparative trial with 40 cases of knee OA
patients. In the JOA assessment standard, two of fivesubscales,“pain and ability of walking” and “pain and abilityof ascending and descending stairs,” and “ Total score”
were significantly improved 8). Furthermore, in a randomized
double blind comparative trial examining rooster comb
extracted processed food containing low molecular HA
with 66 soccer athletes, there was shown to be a marginallysignificant improvement in “pain when pressure is placed on
the foot joint.” Pain of articulation coxae while exercising”
was significantly improved 22). An examination of preparedsoybean milk with a mix of N-acetylglucosamine wasconducted in a randomized double blind comparative trial,
where sixty-seven subjects with mild pain, stiffness and painin the knee joint showed that “pain in the knee joints on
ascending and descending stairs” and “pain in the knee joints
during resting periods” were significantly improved 23). In aclinical study on the ingestion of glucosamine-chondroitinquercetinglucoside with 46 OA patients and 22 RA patients,
OA patients showed a reduction of pain but RA patients did
not show a reduction of pain 24).
The results of these clinical studies have indicated thepossibilities that HA with a relatively small molecular weight
are effective on OA-derived arthralgia. This clinical trialof roster comb extract is not inconsistent with the efficacy
of previous cases, as RA patients were excluded from thesubjects of this trial. This is a case of OA-derived or agingrelated joint pain, and the ingested test product was rooster
comb degradation product by enzymatic treatment containing
low-molecular hyaluronic acid affluently, and secondarily,
this test product contained collagen peptide. However, acertain amount of unsolved questions remain regarding
action mechanisms for how low molecular HA is digested,degraded and absorbed after oral ingestion, and also how to
reduce pain.
It was assumed that changes in blood pressure could be
affected by pain relief. It has been reported that pain andblood pressure are correlated 25-27). Thus, it was supposedthat pain in the joints and lower back were mitigated, which
induced the lowered blood pressure.
The chronic pain of complex regional pain syndrome(CRPS) is strongly affected by autonomic nerves. The
mechanism shows the activation of the catecholamine
receptor 25). For example, in postoperative temporary pain,
a sympathetic-nerve predominant state is shown in an early_ 61 _An Open-label Trial: Rooster Comb Degradation Product containing Low-molecular Hyaluronic Acid
Table 8. Blood, urine examination.

WBC
RBC
Hb
Ht
MCV
MCH
MCHC
PLT

/μL
10 4 /μL
g/dL
%
fL
pg
%
10 4 /μL

TP
ALB
BUN
CRE
UA
AST
ALT
γ - GTP
ALP
LDH
CK
TC
TG
LDL- C
HDL- C
Na
K
Cl
Ca
FPG
HbA1c
CRP
Pentosidine (urine)

g/dL
g/dL
mg/dL
mg/dL
mg/dL
U/L
U/L
U/L
U/L
U/L
U/L
mg/dL
mg/dL
mg/dL
mg/dL
mEq/L
mEq/L
mEq/L
mg/dL
mg/dL
%
mg/dL

pmol/mg・C r

Before8 weeks

p value

5375 ± 224

4992 ± 225 *0.020

13.4 ± 0.4 **

0.002

470.7 ± 9.4

453.9 ± 11.3 *43.0 ± 0.9

41.7 ± 1.0 *14.1 ± 0.3
91.4 ± 1.1

30.0 ± 0.4

91.8 ± 1.1

29.6 ± 0.4 *0.0 11
0.018

0.137

0.013

0.003

32.9 ± 0.2

32.2 ± 0.2 **7.1 ± 0.1

6.8 ± 0.1 **

0.000

14.3 ± 0.9

13.9 ± 0.9

0.504

24.3 ± 1.4

4.3 ± 0.4
0.8 ± 0.1

5.2 ± 0.4

20.3 ± 1.1

23.9 ± 1. 5

4.2 ± 0.04 *0.8 ± 0.04
5.0 ± 0.4

17.7 ± 0.9 **18.0 ± 1.3

14.8 ± 1.1 *209.4 ± 13.2

206.1 ± 10.4

88.3 ± 8.3

95.9 ± 12.6

22.9 ± 3.6

172.0 ± 7.8

142.9 ± 0. 5

141.3 ± 0.4 **106.0 ± 0.6

106.8 ± 0.7

92.3 ± 2.8

96.4 ± 5.1

0.038 ± 0.007

0.034 ± 0.010

5. 5 ± 0.1
7.0 ± 0.7

0.006
0.011

0.473

132.7 ± 7.2

9.4 ± 0.1

0.338

168.3 ± 9.0

137.4 ± 7.6

4.4 ± 0.1

0.084

0.098

214.3 ± 7.9 *64.8 ± 5.6

0.025

19.8 ± 2.3

226.6 ± 9.8

125.8 ± 25.6

0.638

96.3 ± 13.6
63.0 ± 3.6
4.2 ± 0.1
9.4 ± 0.1

5.3 ± 0.1 **6.6 ± 0.7

0.671
0.554
0.014
0.219

0.252
0.452

0.005
0.076

0.096
0.146

0.429
0.000
0.626

0.455

Data are expressed as mean ± SEM, Paired t test, n = 12. SEM, standard error mean.

period but the state does not last (approximately during 5
to 30 minutes). A clinical trial with type 2 diabetic patient
subjects to examine the relation between peripheralneuropathy-induced pain and orthostatic blood pressurechange in the Schellong test showed that patients with
peripheral-neuropathy-induced pain tend to lose normalpressure response 27). Blood donors who have vasovagal
reaction (VVR) in the late stage after blood donation thinkthat they are sensitive to pain and tend to have a decrease indiastolic blood pressure after blood donation 26).
Pain relief exerts a beneficial influence on the bloodpressure condition. Actually, it has been reported that
meditation, relaxation 28, 29), massage 30) and hyperthermia
treatment 31) for pain relief induce a depressing effect onblood pressure rise and a reducing effect on diastolic bloodpressure.
This trial revealed a significant extension effect on knee
joint motion range. A previous study of OA rabbit model witharthrodesis has reported influences on the joint motion range
due to HA. The lowered range of joint motion of this model
was alleviated by HA intra-articular administration 32). HA,_ 62 _Glycative Stress Researchwith a molecular weight of two million and twenty thousand
has a larger effect than HA with a molecular weight of nine
hundred and fifty thousand. There has been no report relating
to HA with a molecular weight as a low as this test product.
Diversified mechanisms of HA effects have been reported asfollows; an inhibitory effect against fibrosis, the involvementof a water retention effect, effects to elevate the fluidity ofsynovial fluid and to restore the function of synovial fluid 33),
and effects to inhibit the isolation of glycosaminoglycan, torestore the quantity of glycosaminoglycan in cartilage and to
suppress the cartilage degeneration 34 -36). Articular cartilage
tissues, by the stimuli of the addition of IL-1 and RA synovial
fluid, promote an extracellular separation of aggrecan andinduce the synthesis and secretion of MMP. These reactions
are suppressed by HA 37). These effects improved articularfunction as well as relieve pain. As the result, it was assumed
knee articulation range was improved.

The damaged articular cartilage releases proinflammatory
cytokine, which induces matrix-degrading enzymes, such as
HA degrading enzymes and aggrecan degrading enzymes.
However, exogenous hyaluronic acid (HA) has an inhibitory
function against the induction of these enzymes. As a result,the separation of the matrix from cartilage (loss of HA andaggrecan) is reduced 37, 38). Thus, it was assumed that the
inflammatory reaction was mitigated and the isolation ofpain stimuli substance from articular tissues was reduced,
which resulted in pain relief.
Relations between HA and glycative stress have not been
paid a full attention to so far. However, that glycative stressinvolves the mechanism of pathogenesis of osteoarthritis(OA) has been gaining an increasing attention 52-55). In thearticular cartilage of OA patients, advanced glycation end
products (AGEs) are accumulated 2) and the expression ofreceptors for AGEs (RAGE) are conspicuous 4).

Hyaluronic acid (HA) and joint

Assessment of safety

This review was compiled by collecting literature related
to the main component HA of this test product (INJUV)
to examine the action mechanism 38). HA is distributed
throughout the tissues and organs of the human body. HAis produced from articular synovial membranes in the joints
and is the main component of synovial fluid. Further, HA is
composed of articular cartilage aggrecan 39). HA performs thevital roles of water retention 6, 40, 41), lubrication of the joint 4244), intercellular adhesion 45, 46) and immunoregulation effects 47).
Aggrecan is a glycoprotein complex and a main
component of cartilage with a molecular weight of more thanseveral hundred thousand daltons. Aggrecan has diversified
functions within the body, which forms a matrix withcollagen and HA and maintains cartilage and other articular
tissues. These components are functional components
related to maintaining and repairing tissues as a substanceof tissue formation and transmission. The polysaccharide
moiety is called glycosaminoglycan, which are composedof chondroitin sulfate, heparan sulfate, keratan sulfate anddermatan sulfate 48 -50). HA are mainly distributed in surface
cell space and are also present in connective tissue of thehollow spaces surrounding blood vessels. Chondroitin sulfate
is distributed in highly fibrotic parts and connective tissues
surrounding blood vessels. Dermatan sulfate is distributed insurface interstitial tissues and blood vessel endothelial cell,and heparan sulfate is distributed in blood vessel endothelial
cells 49).
Degradation of the aggrecan matrix is related to theprocess in articular cartilage destruction of OA. Articular
extracellular matrix constructs a higher order structure
through the interaction with the HA-aggrecan network andtype II collagen fiber 51). HA binds with aggrecan to endow
articular cartilage with load-bearing properties and to exert
a lubrication effect on the surface of cartilage 42). In thearticular cartilage destruction process, the HA-aggrecan
network is degraded and then collagen fiber degradation
proceeds. MMP (matrix metalloproteinase) and ADAMTS( a disintegrin and metalloproteinase with thrombospondin
motifs) gene family molecules play a significant role indegrading extracellular matrices 51). In an early stage ofarticular cartilage destruction, ADAMTS4 and ADAMTS5,
which are called aggrecanase, play a leading part in aggrecan
degradation. HYBID (hyaluronan-binding protein involved
in hyaluronan depolymerization: KIAA1199) plays animportant role of HA degradation.

In previous reports, the safety of HA has been examined.
Safety of HA intra-articular administration has been confirmed
in subacute toxicity tests of beagle dogs and rabbits 56-61).
The safety of HA oral administration has been confirmed
in human clinical trials 62-65). The safety of rooster combderived HA oral administration, whose raw material is thesame as the test product, has also been confirmed in humanclinical trials 66). The safety of collagen peptide contained in
the test product has also already been confirmed in humanclinical trials 67).
This trial showed a decrease of RBC related indices,
RBC (– 3.6%), Hb (– 5.0 %), and Ht (– 3.0%), and TP(– 4.2%) and ALB (– 2.3%). Data 68 - 82) from 15 clinical trials,
which we performed, showed that in 4 cases 68, 72, 75, 82) out
of 7 cases 68, 70, 72, 75, 77, 78, 82) which conducted blood collection
four weeks following ingestion, RBC, Hb and Ht (at least
either one of three) were confirmed to be lowered by 2%
to 4%; one case showed no significant difference of Ht 4
weeks after ingestion but a significant decrease of Ht after
8 weeks 78). Considering gender differences, the subjects inthese cases were all females; one female subject was postmenopausal and 3 female subjects were with both pre- andpost-menopausal 68, 72, 78). In 2 cases 70, 77), a male and a female
subject, no significant difference was shown 4 weeks afteringestion.
In 8 cases 69, 71, 73, 74, 76, 79-81) for the test where blood samples
were not taken at 4 weeks after ingestion, but only at 8 weeks,
one female subject showed an Ht decrease at 8 weeks 76). Seven
other cases showed no significant difference 69, 71, 73, 74, 79-81).
Among the 7 cases, 6 cases 69, 71, 73, 74, 79, 80) were males and one
case was female who was both pre- and post-menopausal.
Even a small change in blood sampling of 10-20 mL, if thereis the same direction for change, it could possibly appear as asignificant difference in a paired t test.
This kind of phenomenon tends to happen for female
subjects, which could relate to factors of age, the quantityof menstruum, or is affected by the absence or presence ofa latent iron deficiency. The decreased MCH and MCHC in
the data results of the blood tests confirmed the presenceof a latent iron deficiency. However, examinations are alsoneeded to verify the presence of latent vitamin B6. In thecondition of the lack of Hb, coenzyme vitamin B6, whichis related to the synthesis of Hb, is in relatively short supply
to compensate for the deficiency of Hb. Vitamin B6 isessential for the synthesis of proteins other than Hb so that_ 63 _An Open-label Trial: Rooster Comb Degradation Product containing Low-molecular Hyaluronic Acidother protein synthesis abilities are also affected. It is assumed
that TP, ALB, AST (– 12.8%) and ALT (–17.8%) in this blood
test were significantly reduced. Previous clinical trials 68-82),
which we conducted, revealed significant decreases in TP
and ALB in 4 cases 76, 77, 79, 80) (one 80) of 4 cases exhibited adecreasing tendency). Furthermore, significant decreases of
AST and ALT were observed in six cases 68-70, 75-77) (two 68, 76)
of 6 cases showed decreasing tendencies).
These changes were observed in other trials, and it isdifficult to completely deny the impacts of INJUV. However,
it is assumed that the possibility is low and a vitamin B
malabsorption was induced in isolation by the ingestion ofthis product (INJUV). Thus, the safety the product of thistrail was verified.

Conclusion

This clinical trial examined the physical effects andsafety of the oral administration of this test product for 8
weeks, which contains a rooster comb enzyme degradation
product (main component; HA with a low molecule) in aan open-label, non-controlled clinical trial with 12 subjects
with pain in the knee joint and lower back (male; 6, female;6, age; 57.5 ± 3.5 years). The test results showed that as
subjective symptoms, gonalgia and lumbago were improved
and as physical views, the extension of the range of motionin the knee joint were observed (average of left and right;

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Acknowledgement

This work was partially supported by the JapaneseCouncil for Science, Technology and Innovation, SIP(Project ID 14533567 ), “ Technologies for creating nextgeneration agriculture, forestry and fisheries ” (funding
agency: Bio-oriented Technology Research Advancement
Institution, NARO.

Conflict of interest statement

This research received support from Laimu Corporation.

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