Glycative Stress Research
Online edition : ISSN 2188-3610
Print edition : ISSN 2188-3602
Received : April 20, 2022
Accepted : May 20, 2022
Published online : June 30, 2022
doi:10.24659/gsr.9.2_97

Original article

Effects of rooster comb degradation product containing low-molecular
hyaluronic acid (INJUV) in individuals with eye dryness;
open-label trial with no control group.
Masahiko Ayaki 1), Sachio Wakayama 2), Yoshikazu Yonei 3)1) Department of Ophthalmology, Keio University School of Medicine, Tokyo, Japan
2) Laimu Corporation. Yokohama, Kanagawa, Japan3) Anti-Aging Medical Research Center and Glycative Stress Research Center,
Faculty of Life and Medical Sciences, Doshisha University, Kyoto, Japan

Abstract

Purpose: An open-level study with no control was conducted to confirm the changes in subjective and objective symptoms andsafety of a food test product (INJUV) containing a rooster comb enzymatic degradation product that contains low molecularweight hyaluronic acid (HA) as a major component.
Methods: From 25 healthy subjects who were aware of dry or eye fatigue, 12 subjects with severe symptoms (male: 6, female: 6,
age: 37.8 ± 10.1 years) were selected as subjects. The subjects orally ingested INJUV for 4 weeks, and eye examinations andquestionnaires were conducted before and after ingestion, and the results were compared.
Results: Subjective symptoms that improved significantly were “eye dryness,” “eye pain,” “eye fatigue,” and “eyelid heaviness.”
The nine items that improved significantly in the Anti-Aging QOL Common Questionnaire were “eye fatigue,” “ blurredvision,” “eye pain,” “stiff shoulders,” “muscle pain/stiffness,” “lethargy,” “skin problems,” “headache,” and “dizziness.” Tearlayer breakdown time improved from 3.6 seconds to 5. 5 seconds (p < 0.05), intraocular pressure (IOP) decreased from 13.0mmHg to 11.7 mmHg (p < 0.01), and diastolic blood pressure decreased from 73.3 mmHg to 68.9 mmHg (p < 0.05). No
adverse events were observed during the study period.
Conclusion: Oral intake of INJUV was shown to be safe and improve ocular symptoms, quality of life and ocular surface,
as well as lower IOP and diastolic blood pressure.

KEY WORDS: low molecular weight hyaluronic acid, dry eye, Dry Eye-related Quality of Life Score (DEQS),		

tear layer breakup time, intraocular pressure, blood pressure

Introduction

The test ingredient INJUV (hereafter referred to as“ test food” ) used in this study is a powdered substancemade by lyophilization of rooster comb enzyme degradation
products, the main component of which is ultra-low
molecular weight hyaluronic acid (HA), produced by room
temperature enzymatic degradation of whole rooster comb
containing high molecular weight HA. In the past, an open
pilot study on arthralgia and lumbago showed improvementin subjective symptoms of arthralgia and knee joint pain,
and an increase in the range of motion of the knee joint 1).
Analysis of the Anti-Aging Quality of Life Common

Questionnaire (AAQol) for changes in subjective symptoms
showed significant improvement in scores for ocularsymptoms “eye fatigue” and “blurred vision” in addition to“arthralgia” and “lumbago.” These symptoms are associatedwith the ophthalmic disease “dry eye,” which led us to
conduct this study. Dry eye is usually treated with eye
drops, and oral pilocarpine hydrochloride and supplements
containing lactobacillus and lactoferrin are also used, butthey are not established management. Here, an open study
without a control group was conducted on “those who areaware of eye dryness and eye fatigue” for the purpose of
examining the effect of the test food on dry eye symptoms.

Contact Address: Professor Yoshikazu Yonei, MD, PhD
Anti-Aging Medical Research Center,
Graduate School of Life and Medical Sciences, Doshisha University
1- 3, Tatara Miyakodani, Kyotanabe, Kyoto, 610- 0394 Japan
TEL & FAX: +81-774 -65-6394 e-mail: yyonei@mail.doshisha.ac. jp
Co-authors; Ayaki M, ayaki@keio.jp; Wakayama S, s.wakayama@laimu.jp

Glycative Stress Research 2022; 9 (2) : 97-105(c) Society for Glycative Stress Research_ 97 _

Low-Molecular Hyaluronic Acid ( INJUV) and Eye Dryness

MethodSubjects

The subjects were 25 healthy men and women between
the ages of 20 and 60 who were aware of eye dryness andeye fatigue in their daily lives. In the preliminary survey, 12
subjects were selected for the study in order of their QOL
score in the DEQS (Dry Eye Quality of Life Questionnairescore) 2), who met the selection criteria and did not violate
the exclusion criteria. The main study was initiated with 12
subjects as described above, completed with all 12 subjects
without any discontinuations, and all subjects were includedin the analysis. The age of the 12 subjects (6 males and 6
females) in the test food (INJUV- containing food ) groupwas 37. 8 ± 10.1 years (males: 37.8 ± 8.9 years, females:
37.7 ± 12.0 years), and the DEQS QOL score at allocation
was 52.4 ± 13.6 (males: 56. 5 ± 15.7, females: 48.3 ± 10.9).
Selection and exclusion criteria are presented next.

Selection criteria
1) Men and women between 20 and 60 years of age at thetime of obtaining consent to participate in the study.
2) Healthy subjects with no chronic physical diseases,
including ocular or skin diseases.
3) Subjects who are aware of dry eye-like symptoms in daily
life (e.g., eye fatigue, eye irritation, eye dryness, eyediscomfort, eye pain, red eyes, difficulty opening eyes in
the morning, burning or itchy eyes).
4) Subjects who perform VDT (visual display terminals)
work in their daily lives for 20 hours or more per week,
5 days per week (including the time spent playing video
games and operating computers and cell phones).
5) Subjects must have corrected visual acuity of 1.0 or
better in both eyes and not wear contact lenses or be able
to change to glasses during the examination period.
6 ) Subjects who have been fully informed of the purpose
and content of this examination, have the ability toconsent, understand it well, volunteer to participate, andagree to participate in this examination in writing.
7 ) Subjects who are able to come to the study site on thedesignated examination date and undergo the examination.
8) Subjects who are deemed appropriate by the investigatorto participate in the study.

Exclusion criteria
1) Subjects currently receiving drug treatment for any disease.
2) Subjects who have taken or applied drugs in the pastmonth for the purpose of treatment of disease (e.g.,
excluding abrupt application of drugs for headache,menstrual cramps, common cold).
3) Subjects with a history or current history of mental illness,sleep disorders, hypertension, diabetes, dyslipidemia, orother serious illnesses.
4) Subjects with a history or current medical history ofserious disorders of the liver, kidney, heart, lungs, blood,etc.
5) Subjects with co-morbidities or a serious history ofgastrointestinal diseases.
6 ) Subjects who use artificial tears (eye drops) more than 6times a day on a daily basis.

7 ) Subjects who have been diagnosed with presbyopia or areaware of presbyopia.
8 ) Subjects with ocular surface disease, entropion, or
trichiasis.
9) Subjects with diagnosed dry eye.
10) Subjects who are using eye drops for the treatment ofocular diseases.
11) Subjects with refractive errors that have not been properly
corrected.
12) Subjects who have undergone corneal surgery such as
LASIK (laser in situ keratomileusis).
13) Subjects with severe astigmatism ( > 2.0 D).
14) Subjects whose eye fatigue is caused by neurological orother regulatory dysfunctions.
15) Subjects with a body mass index (BMI) of 30.0 kg/m 2 orhigher.
16) Subjects with drug or food allergies.
17) Subjects currently, or within the past 3 months, whohave or will take functional foods, health foods, or
supplements claiming to improve eye-related functions
on a regular basis during the study period.
18) Subjects currently, and within the past 3 months, whohave or will take health foods containing rooster comb
enzyme degradation products, hyaluronic acid (HA),
collagen, proteoglycans, elastin, or their precursors on aregular basis during the study period.
19) Subjects whose daily alcohol consumption exceeds anaverage of 60 g/day of pure alcohol equivalent.
20) Subjects who may change their lifestyle during theexamination period.
21) Subjects who work at night.
22) Subjects pregnant, lactating, or possibly pregnant.
23) Subjects who are currently participating in anotherhuman clinical trial or who have not yet completed 3
months of participation in another human clinical trial.
24) Subjects whose family members are engaged in thedevelopment, manufacture, or sale of health/functional
foods and cosmetics.
25) Other subjects who are judged by the investigator to beunsuitable for this study.

Test food
The test food was “ INJUV capsulesˮ provided Laimu
Corporation ( Yokohama. Kanagawa, Japan). INJUV is
a powdered substance made by lyophilization of rooster
comb enzyme degradation products. This test food contains
HA with ultra-low molecular weight (molecular weight 380~ 5,000) and other collagen peptides as characteristic main
ingredients. HA is distributed throughout the body, but injoints, it is produced by synovial membranes and is a major
component of joint fluid and also a component of articularcartilage aggrecan 3). HA plays important roles in water
retention 4, 5), joint lubrication 6), intercellular adhesion 7), andimmunomodulation 8). HA- containing eye drops are often
used for dry eyes 9).
In this study, four capsules containing 150 mg of INJUV
were taken twice daily with water or lukewarm water for fourweeks (600 mg of INJUV twice daily = 1,200 mg/day).

_ 98 _Glycative Stress Research

Study design

Eye examination

The study format was an uncontrolled open study.
In this study, the DEQS, VAS test for subjective
symptoms, BUT (tear film breakup time), visual acuity test,
and intraocular pressure (IOP) test were conducted before
and 4 weeks after intake of the test food as indicators related
to eye moisture. Schirmer test, visual acuity measurement,
and IOP measurement were conducted. In addition, a survey
was conducted on the effects of the intake of the test foodson QOL using AAQol.
The safety of the test food after 4 weeks of continuous
intake was confirmed by fundus examination, slit-lamp
microscopy, blood pressure/pulse rate, physician interview,
occurrence of adverse events/side effects, and subject
logbooks. In addition, background investigation, predominant
eye determination, and refraction examination wereconducted in the preliminary examination.
The study period was from September 2021 to November
2021.

The following eye examinations were performed todetermine the dominant eye, and all items were tabulated
as dominant eye values and nondominant eye values, andevaluated by comparing before and after 4 weeks of intakeof the test food. All eye examinations were performed at
a medical institution and the results were evaluated by anophthalmologist.

Assessment Items

BUT
Subjects were asked to hold back blinking, and the BUT,
or the time it takes for the BU T (a phenomenon in which thearea not covered by stained tears on the cornea appears andgradually expands), was measured.

Schirmer's test
A defined filter paper was placed between the lower
eyelids of the subjects and the length of wetting in 5 minutes
was determined (measurement/mm).

Visual acuity test

Subjective symptom
DEQS

In the DEQS, subjects first responded to a total of
15 items, six related to “ Eye symptoms” and nine related
to “ Impact on daily life,” with questions asking about thefrequency of the symptoms (Column A). Consequently, forsubjects who answered that the symptoms were at least alittle bothersome, they were asked to indicate whether thesymptoms bothered them and to what extent (Column B), andthe “ QOL score” was calculated by summing the Column
B scores/number of valid responses*25. The severity of dryeye symptoms and their “ Impact on daily life” and mental
health were evaluated by comparing before and 4 weeks after
consumption.

Experiential VAS questionnaire on subjective
symptoms (Visual Analogue Scale).

Uncorrected and corrected visual acuity were measured
in the right and left eyes. The dominant eye was determined
and analyzed. Results of visual acuity were converted to
LogMAR (logarithm of minimum angular resolution) fromthe decimal acuity values obtained in the measurements,
followed by statistical analysis. The conversion formula is
LogMAR = −Log10 (decimal acuity value). This is because
decimal acuity is the reciprocal of the minimum visual angle
at which two points can be distinguished, and statisticalprocessing is not possible with the number as it is. For
example, a decimal acuity of 1.2 is converted to − 0.08 in
LogMAR, 1.0 to 0.00, and 0.8 to 0.10, so a smaller LogMAR
acuity value means better acuity. Results are shown for both
LogMAR and the decimal acuity values obtained by reconverting LogMAR to decimal acuity values.

Refraction test

In the VAS test, participants were asked to respond to
the degree of “ eye dryness,” “ eye irritation.” “ eye pain,”“ blurred vision,” “ eye fatigue”, “ visual clarity,” “ stiffshoulders,” “headache,” and “sleep depth” for the past week
using the VAS on a 100 -mm line. The left end was definedas the best condition (no symptoms) and the right end as theworst condition (greatest ever symptoms). The results were
evaluated by comparing before and one week after, 2 weeks
after, 3 weeks after, and 4 weeks after consumption.

AAQol.
The AAQol 10, 11) was used to evaluate subjective symptoms.
The AAQol was divided into “ physical symptoms” and“ mental symptoms.” Then, the scores were evaluated on a
5-point scale from 1 to 5, with a before-and-after comparison
between before and 4 weeks after consumption of the test
foods.

Examination was performed in a dark or dim room(light-shielding curtains on windows).

IOP testThree measurements were taken for both the right andleft eyes using a pneumatic tonometer. If an outlier occurred
due to blinking, eyelash, etc., the measurement was repeated,
and the average IOP of the three measurements excluding theoutlier was calculated.

SafetyFundus examination, slit-lamp microscopy, bloodpressure/pulse measurements, and physician interview/
adverse drug reaction/adverse event assessment wereconducted.

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Low-Molecular Hyaluronic Acid ( INJUV) and Eye Dryness

Statistical analysis

AAQol (Table 2)

Statistical analysis was performed using SAS (SAS 9.4;
SAS Institute Japan, Minato-ku, Tokyo) or SPSS (Statistics
26; IBM Japan, Chuo-ku, Tokyo) statistical analysis software,and a paired-t test was performed for comparison with before
intake. Scores obtained from the DEQS and the AAQol andvalues of visual acuity tests were treated as nonparametric,and Wilcoxon signed rank tests were performed forcomparison to preintake. The significance level for all tests
was set at 5 % for two-tailed tests.

The AAQol was used to evaluate physical symptoms
4 weeks after compared to before intake of the test food.
The results showed significant improvement ( p < 0.05) in“eye fatigue,” “blurred vision,” “eye pain,” “stiff shoulders,”“ muscle pain/stiffness,” “ tiredness,” “ skin problems,”“headache,” and “dizziness” 4 weeks after compared tobefore intake.

Ethical standard
This study was conducted under the approval of the
Human Study Ethics Committee at the “Glycation StressResearch Society” (GSE #2021010) in Nakano, Tokyo, Japan.
Clinical trial pre-registration was conducted for this study(UMIN # 000045484).

Results

Evaluation of subjective symptoms
After 4 weeks of consumption of the test food,subjective symptoms improved as follows.

Eye examination (Table 1)
BUT test

BUT is a test to determine how long it takes for thetears covering the surface of the eye to begin to dry out.
When the patient stops blinking and looks at the front of theeye, the surface of the eye gradually dries out and the shapeof the surface becomes uneven. This time is measured as
BUT. Normal BUT is 10 seconds or more; if it is less than
5 seconds, the patient is diagnosed with probable dry eye.
BUT (binocular mean) was less than 5 seconds in 9 of the
12 subjects. Compared to the pre-food intake, the dominant
eye improved significantly from 4.1 ± 2.6 before to 5.6 ± 2.6
after 4 weeks ( p = 0.194), the non-dominant eye improved
from 3. 2 ± 1. 3 before to 5. 5 ± 2.3 after 4 weeks ( p < 0.01),
and the binocular mean improved from 3.63 ± 1.72 before to
5.54 ± 2.36 after 4 weeks (p < 0.05).

Schirmer' s test

DEQS
In the DEQS “ Eye symptoms” (6 items), all 6 items
in Column A, “eye irritation,” “eye dryness,” “eye pain,”“eye fatigue”, “eyelid heaviness,” and “eye redness” weresignificantly improved ( p < 0.05) at 4 weeks after intake
compared to before intake of the test food. Column B also
showed significant improvement (p < 0.05) for four items: “eyedryness,” “eye pain,” “eyes fatigue,” and “eyelid heaviness.”
In the “ Impact on daily life” (9 items), the results
after 4 weeks of intake compared to the results before theintake of the test foods were significantly improved (p <0.05), “eye symptoms worsen when watching TV or using
a computer or cell phone,” “concentration is impaired due
to eye symptoms,” and “eye symptoms interfere with work,
housework, or study.” Column B also showed significant
improvement (p < 0.05) for the two items “eye symptoms getworse when watching TV or using a computer/mobile phone”
and “concentration is impaired due to eye symptoms.”
The QOL score, calculated using the Column B (degree)score, improved significantly from 52.4 ± 13.6 before
consumption of the test food to 23.6 ± 10.6 four weeks after
consumption (p < 0.01, Table 1 ).

VAS test ( Table 1)
In the VAS test (9 items), “eye dryness,” “blurredvision,” “eye fatigue,” “visual clarity,” and “stiff shoulders”
improved significantly (p < 0.05) after 1 week compared tobefore intake of the test food. After 2 and 3 weeks, “sleep
depth” was also significantly improved (p < 0.05), in addition
to the 5 items that were significantly improved after 1 week.
After 4 weeks, 8 items including “eye irritation” and “eyepain” were significantly improved (p < 0.05).

The Schirmer's test measures the amount of tears
produced. The normal level is 10 mm or more, and if it is lessthan 5 mm, the volume is judged to be low. The subjects' prevalues (binocular mean) were in the normal range (> 10 mm)
in 5 of the 12 subjects, with the lowest value being 2.0 mm.
No significant change in tear fluid volume was observed
after 4 weeks of intake compared to the pre-value.

Visual acuity test

The binocular mean corrected visual acuity was 1. 2or better in 12 of the 12 subjects, with a minimum value of
1.2. Binocular means of noncorrected and corrected visualacuity were not significantly different after 4 weeks of intake
compared to before. Table 1 shows both decimal acuity and
LogMAR acuity.

IOP test

The reference value for IOP testing is 10 ~ 21 mmHg,
with diurnal variation. IOP tests showed that 11 out of 12
patients were within the reference range. IOP significantly
decreased from a previous value of 13.0 ± 2.4 in thedominant eye to 11.7 ± 2.2 at 4 weeks (p < 0.05), from 13.0
± 2.3 in the non-dominant eye to 11.7 ± 1.8 ( p < 0.05), andfrom 13.0 ± 2.2 to the binocular mean of 11.7 ± 1.9 ( p <0.01).

Safety endpoints
No abnormal findings were observed in fundus
examination or slit-lamp microscopy after 4 weeks of intake
compared to pre-food intake. Diastolic blood pressure
significantly decreased from 73. 3 ± 10.7 before consumption
to 68.9 ± 9.7 after 4 weeks (p < 0.05).

_ 10 0 _Glycative Stress Research

Table 1. Results

Item
Age (years)

Blood pressure

Systolic (mmHg)

diastolic (mmHg)

Pulse (/min)

DEQS

Visual Analogue Scale
Eye dryness

Before4 weeks

p value

117.7 ± 13.0

113.4 ± 13.0

0.219

76. 5 ± 11. 3

81.1 ± 14.8

0.241

37.8 ± 10.1

68.9 ± 9.7

73.3 ± 10.7

52.4 ± 13.6

23.6 ± 10.6

67.7 ± 18.9

34.7 ± 17.2

0.001*

27.7 ± 20.0

0.017*

Eye irritation

46.5 ± 28.8

25.1 ± 19.4

Blurred vision

68.6 ± 25.1

32.0 ± 24.4

Eye painEye fatigue

51.9 ± 28.3

84.8 ± 10.1

Visual clarity

60.5 ± 22.6

Headache46.9 ± 40.5

Stiff shoulders
Sleep depth

Eye examination
BUT (second)

Dominant eye

0.013*

0.002*< 0.001*37.6 ± 21.3

< 0.001*31.9 ± 18.6

0.013*

22.9 ± 21.4

65.3 ± 25.8

0.0 21*44.2 ± 19.8
28.8 ± 21.6

75.3 ± 23.1

0.002*

4.1 ± 2.6

0.007*
0.058

5.6 ± 2.6

0.194

5. 5 ± 2.4

0.023*

3.2 ± 1. 3

5. 5 ± 2.3

10.7 ± 9.1

12. 5 ± 7.8

0.280

Binocular mean

11.0 ± 9. 4

11.9 ± 7. 0

0.632

Dominant eye

0.43 ± 0.30 (0.36 ± 0.53)

0.46 ± 0.27 (0.34 ± 0.57)

0.43 ± 0.30 (0.36 ± 0.53)

Binocular mean

0.54 ± 0. 52 (0.26 ± 0.29)

0.35 ± 0.22 (0.46 ± 0.65)

0.54 ± 0.52 (0.26 ± 0.29)

Dominant eye

1.22 ± 0.94 (− 0.09 ± 0.03)

Nondominant eye
Binocular mean

Schirmer's test (mm)Dominant eye

Nondominant eye
Visual acuity (uncorrected) A
Nondominant eye
Visual acuity (corrected) A

3.6 ± 1.7

11.3 ± 6.9

11.4 ± 10. 5

0.42 ± 0.31 (0.38 ± 0.51)

0.43 ± 0.30 (0.37 ± 0.52)

0. 001*

0.972

0.42 ± 0.31 (0.38 ± 0.51)

1.29 ± 0.90 (− 0.11 ± 0.04)

0. 2501.22 ± 0.93 (− 0.09 ± 0.04)

0. 375

Binocular mean

1.22 ± 0.94 (− 0.09 ± 0.03)

1.28 ± 0.94 (− 0.11 ± 0.03)

1.27 ± 0.90 (− 0.10 ± 0.04)

Dominant eye− 1.52 ± 2.85

−1.56 ± 2.78

0.6 58− 1.46 ± 2.79

−1.54 ± 2.76

0. 2 07

Dominant eye

0.56 ± 0.66

0. 58 ± 0.55

0.845

Binocular mean

0.58 ± 0. 57

0. 55 ± 0.43

0.7 7113.02 ± 2.35

11.74 ± 2.18

0.014*

11.70 ± 1.86

0.004*

Nondominant eye
Spherical refraction (D)Nondominant eye
Binocular mean

Cylindrrical refraction (D)Nondominant eye
IOP (mmHg)

Dominant eye

Nondominant eye
Binocular mean

− 1.40 ± 2.75

0.60 ± 0. 52

12.96 ± 2.30

12.99 ± 2.20

−1.52 ± 2.77

0. 52 ± 0.36

11.66 ± 1.75

0. 5000. 0 82

0.4 740.015*

Results are expressed as mean ± SD, n = 12, *p < 0.05, paired-t test or Wilcoxon test. LogMAR in parenthesis; Decimal visual acuity was converted to
LogMAR (logarithm of minimum angular resolution; DEQS, Dry Eye-related Quality of life Score; BUT, tear film breakup time; IOP, intraocular pressure;SD, standard deviation.
A_ 101 _

Low-Molecular Hyaluronic Acid ( INJUV) and Eye Dryness

Table 2. AAQol scores.

Physical symptoms
Item

Eye fatigue
Blurred vision
Eye pain
Stiff shoulders
Mascular pain / stiffness
Palpitations
Shortness of breath
Tendency to gain weight
Weight loss/; thin

Before4.3 ± 0.6

4 weeks

2.9 ± 0.5

3.4 ± 1.2

2.2 ± 0.7

4.1 ± 1.2

3.1 ± 0.8

3.1 ± 1.2

3.4 ± 1.3

1.5 ± 0.7

1.8 ± 0.7

2.6 ± 1.2
1.4 ± 0.7

1.6 ± 0.7

1. 5 ± 0.7

1.6 ± 0.9

1.3 ± 0.5

2.0 ± 1.0

2.1 ± 1.2

Lethargy

3.2 ± 1.4

2.3 ± 0.9

Thirst2.1 ± 0.8

1. 8 ± 0.8

No feeling of good health 2.5 ± 1.0
Skin problems
Anorexia
Early satiety
Epigastralgia
Liable to catch cold
Coughing and sputum
Diarrhea
Constipation
Gray hair
Hair loss
HeadacheDizziness
Tinnitus

2.5 ± 0.7

1.9 ± 0.8
1.9 ± 0.9

1.7 ± 0.7

1. 3 ± 0.5

1.8 ± 1.0

1.3 ± 0.6

1.9 ± 1.1

1.4 ± 0.5

1.4 ± 0.7

1.6 ± 0.9

1.7 ± 1.0 1.58 ± 0.8
1.7 ± 0.8

1.6 ± 0.8

1.9 ± 1.2

1.5 ± 0.7

1.8 ± 1.0

2.0 ± 1.2

1.6 ± 0.7

1.8 ± 1.2

2.8 ± 0.8

2.1 ± 0.9

1.8 ± 0.8

1.8 ± 0.7

2.3 ± 1.0

1.3 ± 0.5

2.8 ± 1.1

2.7 ± 0.9

Edematous

2.2 ± 1.1

1.9 ± 0.7

Easily breaking intoa sweat

2.6 ± 1.2

LumbagoArthralgia

Frequent urination
Hot flush
Cold skin

1.7 ± 0.9

1.8 ± 1.0

1.5 ± 0.7

2.5 ± 1.4

1.8 ± 0.8

p valueItem

0.003*

Irritability

0.006*
0.006*
0.025*

0.039*
0. 564
0. 5640.739
0.48

0.026*
0.143
0.083

0.020*
0.059
0.109
0.131

0.706

0.578
0.739

0.257
0.103

0.083

Loss of motivation
No feeling of happiness
Nothing to look forward to in life
Daily life is not enjoyable
Loss of confidence
Reluctance to talk with others
Depressed
Feeling of usefulness
Sallow sleep
Difficulty in falling asleep
Pessimism
Lapse of memory
Inability to concentrate
Inability to solve problems
Inability to make judgments readily
Inability to sleep because of worries

2.3 ± 1.1
2.3 ± 1.1

2.0 ± 1.0

1.8 ± 0.9
1.8 ± 1.1

2.1 ± 1.2

2.1 ± 2.1
2.1 ± 1.0

1.9 ± 0.9
1.7 ± 1.0

1.6 ± 0.8

1.8 ± 0.9

1.8 ± 0.9

1.9 ± 1.2

1.8 ± 1.0

2.0 ± 1.2
1.6 ± 0.7

1.8 ± 1.0

1.7 ± 0.8

2.8 ± 1.1

2.3 ± 0.8

1.8 ± 0.9

1.8 ± 1.1

2.3 ± 1.1

2.3 ± 0.8

2.1 ± 0.9

1.8 ± 0.8

2.3 ± 1.1

2.1 ± 1.0

2.0 ± 1.0

2.0 ± 1.0

1.8 ± 0.9
1.9 ± 0.9

2.1 ± 1.0
1.8 ± 1.0

2.0 ± 1.0

Vague feeling of fear

1.3 ± 0.5

1.5 ± 0.8

Feeling of anxiety for no special reason 1.7 ± 0.7

0.763

0.706

0.257

1

2.6 ± 1.2

4 weeks

2.1 ± 1.0

11.8 ± 0.8

BeforeA sense of tension

0.016*

0.581

2. ± 1.4

Easily angered

0.014*

2.4 ± 0.9

1.5 ± 0.8

Mental symptoms

1

0.748

Results are expressed as mean ± SD, n = 12, *p < 0.05, Wilcoxon test.
AAQol, Anti-Aging QOL Common Questionaries; SD, standard
deviation.

_ 10 2 _1.8 ± 1.0

p value
0.059
0.18

0.578

0.564
0.157
0.157

0.564
0.18

0.564
0.564
0.196

0.366
1

0.16

0.18

0.408
1

0.564
0.706
0.414

0.527

Glycative Stress Research

Discussion

Role of hyaluronic acid (HA)

Result summary
Characteristics of the 12 men and women who
experienced ocular dryness included low BUT (5 mm), normaltear fluid volume, generally normal corrected visual acuity,and IOP in the normal reference range. All patients met
the new 2016 diagnostic criteria of “dry eye syndrome.”
BUT less than 5 seconds and subjective symptoms (ocular
discomfort or abnormal visual function). After a 4-weektest food feeding, the patients showed improvement in dry
eye-related symptoms, prolonged BUT (binocular mean
+ 34.5 %), and decreased IOP (binocular mean − 9.9 %). No
adverse events were observed.

Lacrimal function and dynamics of tear fluid
Tears protect the cells on the surface of the eye fromdrying out, supply nutrients and oxygen to the tissues, andprevent the entry of foreign substances and bacteria fromthe outside world. They also provide a smooth curve tothe surface of the cornea and deliver clear images to thebrain. Therefore, a decrease in tear fluid or a declined tearfluid function may result in a decrease in functional visualacuity (practical acuity value expressed as a continuous
measurement over a period of one minute).
Lacrimal fluid consists of three layers from the depth(surface of the corneal epithelium): mucin layer, water layer,
and oil layer.
Mucin layer: mucus secreted by the goblet cells of the
conjunctiva. Lack of the mucin layer causes a loss of tearfluid from corneoconjunctival epithelium.
Water (Aqueous) Layer: The main component of tears
secreted by the lacrimal gland. The aqueous layer accounts
for 95 % of tears and contains various components such asproteins and HA. It plays an important role in the nutritional
supply to the cornea, prevention of infection, wound healing,
and other important functions of tears.
Oil layer: Secreted by the meibomian glands and richin oil components. It covers the surface of the tear fluid andserves to prevent evaporation of tears and to protect against
infection. The lipids present in the oil layer are chain-likemolecules called meibum, the main components of which arecholesterol esters and wax esters 12) .
The three layers of lacrimal fluid are driven by theblinking eye and, after moistening the ocular surface in aconstant direction, flow into the upper and lower lacrimalducts on the inner side of the eyelid margin and thenthrough the upper and lower lacrimal ducts, lacrimal sac,nasolacrimal duct, and nasal cavity.
According to dry eye practice guidelines 13), the following
mechanisms are postulated. When the stability of the tearfluid layer is reduced by various upstream risk factors,
epithelial damage to the corneal conjunctiva occurs due todrying stress. Consequently, mucins on the epithelial surface
are impaired and water wettability is reduced. A vicious cycle(core mechanism) occurs in which the stability of the tearfluid layer is further reduced 14 -18). This vicious cycle further
results in inflammation and contributes to epithelial damage.

The mechanism of action of HA, the main componentof HA-containing topical products that are frequently usedwith eye drops for the treatment of dry eye, still remains
unclear. In animal studies, HA- containing topical artificial
tear solutions have been reported to improve corneal
re-epithelialization after corneal epithelial peeling 19).
Biochemically, it has been speculated that phospholipid-HA
interactions may be associated with vitreous liquefaction
associated with aging, treatment of dry eye syndrome,skin care products, and synovial joint lubrication 20). The
affinity of HA with other substances is strongest withphosphatidylcholine sphingomyelin and phosphatidic acid,followed by monoglycerides and palmitoyl palmitate, andweakest with cholesterol 21).
In humans, its efficacy in increasing tear film thickness( TF T) has been reported in a randomized, doublemasked, placebo-controlled study 22). However, there issome argument that artificial tears containing HA onlytransiently alleviate dry eye symptoms, and combination
agents with galactoxyloglucan have also been proposed 23).
In recent years, modern life has become very convenientwith the spread of personal computers and air conditioning
control, whereas, the environment surrounding the eyes
has not improved. According to the Osaka Study reported
in 2013, dry eye examinations and questionnaires wereconducted on 561 company employees, and 79.5 % of womenand 60.2 % of men were diagnosed with dry eye, which wasmore pronounced in the group using VDT for 8 hours ormore per day 24). Even under the pandemic of Covid-19 from
2020, dry eye is increasing 25). Under these circumstances,an effective and safe treatment for dry eye that addresses thepathogenesis of dry eye is desperately needed.
Considering dry eye as a lifestyle-related disease,
lifestyle modification is a prerequisite for prevention andtreatment. Lifestyle guidance through dietary modification,
increased physical activity, and encouragement of positive
thinking has been shown to improve subjective symptoms
associated with dry eye 26) .
The test product used in this study, a food productcontaining HA, is characterized by its main ingredient, HA,
being ultra-low molecular weight (molecular weight 380~ 5,000). Despite the fact that this was an open pilot study,
not only subjective symptoms but also BUT and IOP weresignificantly improved. This effect may be due to the fact thatthe low molecular weight HA contained in the test productwas absorbed into the body and had some effect. Although
the mechanism of action of HA is currently unknown andrequires further investigation, the fact that both subjective
and subjective symptoms improved after oral intake isgroundbreaking. In a previous report, symptoms of the knees
and hips were improved by INJUV administration 1), and it isassumed that the effects on the ocular area were produced bya similar pathway.
Hypothesis raised for IOP and diastolic blood pressure
reduction is as follows. Dry eye is closely related to mental
stress 27) and the autonomic nervous system 28), and the effectof HA-containing foods on the ocular surface first improves
BUT, which in turn improves optical function of the ocularsurface and improves vision. It is possible that these_ 10 3 _Low-Molecular Hyaluronic Acid ( INJUV) and Eye Dryness

improvements in dry eye and vision may have reduced stress,
resulting in a decrease in IOP 29, 30) and diastolic blood pressure.
IOP, like blood pressure, is supposed to fluctuate seasonally
via catecholamines 31), and a reduction in IOP due toimprovement of dry eye or other effects may be expected asa potential effect of INJUV.
HA is also detected in tear fluid. The average HA
concentration has been reported to be 18.9 ± 12.6 ng/mg
protein (range 3.2 ~ 45.0 ng/mg protein) in healthy subjects 32).
There are no differences between men and women or between
contact lens wearers and non-wearers 33). It is increased inpatients with Basedow's disease (hyperthyroidism), reaching
35.1 ± 34.7 ng/mg protein 32). Results of an analysis oftear fluid from patients with unilateral acute adenoviral
conjunctivitis (UAAC), normal donors (controls), and allergic
conjunctivitis showed that HA tear fluid volume wassignificantly increased in patients with UAAC compared tocontrols and allergic conjunctivitis patients 34). These findings
suggest that there may be a mechanism by which HA in thetear fluid is increased by some stimulus to maintain ocularhomeostasis.

Research limitations.
Since this is an uncontrolled, open-label study, thelevel of evidence for the efficacy of this study with respect
to dry eye is not high. There are many unknowns regarding
the disposition of the main component of the test food,ultra-low molecular weight HA, in the body. Whether or not
the HA concentration in the tear fluid is increased by theingestion of the test food is a subject for future verification.
The reason for the significant decrease in IOP and diastolicblood pressure was only hypothesized and should be further
confirmed.

Safety(p < 0.05). This variation was within the reference range. No
significant changes were observed in systolic blood pressure orpulse rate. No other adverse event investigations were found tobe causally related to the test food.

Conclusions
To confirm the changes in subjective and objective
symptoms and safety of a food test product (INJUV)
containing low molecular weight HA-based rooster comb
enzymatic degradation products, an uncontrolled, open-levelstudy was conducted in 12 healthy subjects (6 males, 6 females,age: 37.8 ± 10.1 years) who were aware of eye dryness andeye fatigue. As a result, subjective symptoms such as “eyedryness,” “eye pain,” “eye fatigue,” and “eyelid heaviness”
were significantly improved. In the AAQol, 9 items
significantly improved: “eye fatigue,” “ blurred vision,” “eyepain,” “stiff shoulders,” “muscle pain/stiffness,” “lethargy,”“skin problems,” “ headache,” and “dizziness.” Ocularexamination showed an improvement in BUT, a decrease in
IOP, and a decrease in diastolic blood pressure. No adverse
events were observed during the study period, confirming
safety. Since this is an open, uncontrolled study, results may
include a placebo effect. Further detailed investigation of
the efficacy evaluation and mechanism of action of the test
product is needed in a randomized controlled trial (RCT).

Conflict of interest declaration
In carrying out this study, we received support from
Laimu Co.

No abnormal findings were observed in fundus
examination or slit-lamp microscopy after 4 weeks of intake
compared to pre-food intake. Diastolic blood pressure
significantly decreased from 73.3 ± 10.7 before to 68.9 ± 9.7
after 4 weeks of consumption compared to before consumption

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