Glycative Stress Research
Online edition : ISSN 2188-3610
Print edition : ISSN 2188-3602
Received : August 31, 2023
Accepted : September 20, 2023
Published online : September 30, 2023
doi:10.24659/gsr.10.3_110

Original article

Ocular moisturizing effects of rooster comb degradation product containing
low-molecular hyaluronic acid and collagen peptides (CRISTA®) in individuals
with dry eye-related symptoms: A randomized, double-blind, placebo-controlled,parallel-group study
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: A double-blind randomized controlled clinical trial with parallel-group comparison was conducted to confirm
the alternations in subjective and objective symptoms, and the safety of ingestion of a test food product (CRISTA®), whichcontains a high concentration of rooster comb enzymatic degradation product. The primary components are low molecularweight hyaluronic acid (HA) and collagen peptide. Effectiveness on ocular moisturizing effects and safety were assessed.
Methods: Thirty healthy subjects who were experienced dry or eye fatigue on a daily basis due to viewing an electronic
display for work, were randomly classified into two groups, the test food product group and the control (placebo) group.
Among them, excluding one participant (the test food product group), who met exclusion criteria, twenty-nine subjects were
subject of analysis for validity verification assessment items: fourteen food product group participants (male: seven, female:seven, age: 38.2 ± 3.2 years) and control food group participants (male: eight, female: seven, age: 38.4 ± 2.7 years).
Results: After four-week ingestion of test product, significant increase of lacrimation was confirmed with Schirmer's test.
Significant differences were shown in both the test product group and the control group. The effectiveness of the test productwas verified to keep the eyes moisturized. There were no differences between two groups regarding Dry Eye Quality of Life
(QOL) Questionnaire, Visual Analog Scale for subjective symptoms, Anti-aging QOL Common Questionnaire, tear breakup time, visual acuity, and intra-ocular pressure. The safety of the continuous oral ingestion for as long as four weeks wasverified.
Conclusion: This test food product is promising as an effective and safe oral ingestion functional food to prevent eye
dryness.

KEY WORDS: randomized controlled clinical comparative trial, low molecular weight hyaluronic acid, dry eye,		Schirmer's test, tear break-up time

The intercellular matrix is a network of diverse molecular
structures existing in the outer space of parenchymal cells.
Intercellular matrix in humans contains glycoproteins suchas collagen, proteoglycan, fibronectin, and laminin, andalso mucopolysaccharides such as hyaluronic acid andchondroitin sulfa. Quantitative and qualitative deteriorationsof intercellular matrix are intensely related to the onset andprogression of symptoms of skin aging and osteoarthritis 1).
The Society for Glycative Stress Research have beencommitted to studies for people who suffer from knee joint

and lower back pains. An uncontrolled trial was conducted
for the ingestion of the test food product (INJUV®), whichcontained a rooster comb enzymatic degradation product andthe main components were low molecular weight hyaluronic
acid (HA) and collagen peptide, to examine alternations ofsubjective and objective symptoms. The trial confirmed theimprovements in the knee joint and lower back pains and theeffectiveness on enlargements of articulatio genus. Moreover,
improvements of asthenopia were observed 2). Thus, an
uncontrolled trial was conducted for individuals who were
aware of eye dryness or eye strain, because tears contain
HA, which has protective function against ocular dryness

Contact Address: Professor Yoshikazu Yonei, MD, PhD
Anti-Aging Medical Research Center, Graduate School of Life andMedical 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 2023; 10 (3): 110-123(c) Society for Glycative Stress Research

Introduction_ 110 _Glycative Stress Researchand dry eye. This trial confirmed significant improvement intear break-up time and significant decrease in intra-ocularpressure, suggesting a possibility of effectiveness on oculardryness 3).
People have been increasingly exposed to risk factors
for eye health conditions in modern society due to long work
hours in front of personal computers, dryness due to the useof air conditioners, and wearing contact lenses. An increasing
number of patients complain of dry eye syndrome, feelingsof dryness, eye pains, hazy and cloudy vision, and blurredvision. According to Osaka Study in 2013, 79.5 % of females
and 60.2% of males are diagnosed with dry eye syndrome 4).
Since the Covid-19 pandemic, patients with dry eye syndrome
have increased since 2020 5). These exacerbated medical
conditions could remarkably reduce quality of life. Leaving
damage without clinical treatments would lead to theaggravation of diseases. Consequently, cornea and conjunctiva
are injured, and disordered physical conditions could occur
such as headache and dizziness or vertigo. Therefore, wheneye conditions are deteriorated, immediate treatments are
essentially required to improve symptoms.
It is common for the improvement of eye conditions toapply eyedrops directly to eyes such as artificial tears, HA
drug formulation, medical agent promoting the formationand secretion of mucin, and eyedrops containing antiinflammatory drug, such as an adrenocortical steroid (Patent
Literature: unexamined patent application 201938783
patent publication 1). The applications of these eye drops,
ophthalmic solutions, could be burdensome. Instructions on

how to apply eye drops are as follows: Everytime wash yourhands before handling your eye drops or touching your eyes.
Tilt your head back and pull your lower eyelid down. Don’t
let the bottle touch to your eye, eyelid, and eyelashes. Squeeze
the bottle gently and let one drip of eye drop fall into the
conjunctival sac. Close your eyes lightly. Wipe out excessive
eyedrops from edge of eyes and skin using facial tissues. The
directions for usage are bothersome. In addition, you have to
be cautious of storage methods, expiration date, and allergic
reactions due to preservative. Therefore, the development ofa desirable oral ingestion is expected. It would be simple andeasy to ingest and store to effectively improve eye conditions.
However, there are few proven functional components via
oral ingestion to effectively improve ocular problems.
Considering these backgrounds, we conducted the
double-blind randomized placebo-controlled clinical trial
with comparison of parallel-group design, to verify the test
food product (CRISTA®), which is condensed in a highconcentration from INJUV®.

Subjects and method

1. Implementation structure of the trial

GlcCer and Cer (Fig. 1) were purified according to ourprevi The present study was conducted, for the human rights
and safety of research participants and the data trust of thetrial, after obtaining the examination and approval of theAssessed for eligibility(SCR &Visit -1)(n = 58)

Excluded (n = 28)
Not meeting inclusion criteria (n = 22)
Other reasons (n = 6 )Randomized (n = 30)

Test food group (n = 15)

Placebo food group (n = 15)

Inspection 4 weeks after intake(Visit -2) (n = 15)

Inspection 4 weeks after intake(Visit -2) (n = 15)

Lost to the follow (n = 0)

Lost to the follow (n = 0)

Analyzed (PPS) (n = 14)

Analyzed (PPS) (n = 15)

Excluded Analyzed (PPS) (n = 1)
• Not meeting inclusion criteria(n = 1)

Fig. 1. Flow of analysis from subject enrollment.
PPS, Per Protocol Set.

_ 111 _

Effects of Low-Molecular Hyaluronic Acid and Collagen Peptides on Ocular Dryness

Ethical Committee of the third party, general incorporated
association Society for Glycative Stress Research, regarding“ Research involving human subjects” (approval number:
2022-008, December 21st, 2022). The trial was performed
by Ueno Asagao Clinic (principal investigator of the trial:
the president of the hospital, Atsushi Nakajima), and Hongo
san-chome eye clinic (Sub investigator: the president of thehospital, Kayako Shibuya) based on the clinical trial protocol(UMIN trial ID: UMIN000049882). The present trial was
conducted in compliance with ethical principles based on the
Declaration of Helsinki (Amendment, 2013 WMA Fortaleza“Brazil”) and Ethical Guideline for Medical and BiologicalResearch principles involving human subjects (Implemented
in 2021, Ministry of Education, Culture, Sports, Scienceand Technology, Ministry of Health, Labour and Welfare,and Ministry of Economy, Trade and Industry. Notification:
No. 1).

2. Research Participants

In the recruitment of research participants, target
participants were provided with explanation of the trial, and58 candidates who satisfied the inclusion criteria underwent
prior inspections. Thirty participants were selected for the
final examination, among candidates who investigator andsubinvestigators decided as appropriate participants basedon the selection standard, satisfying the selection criteria anddid not match the exclusion criteria. The number of cases
was decided and set up as follows: In a trial with comparison
between groups, thirteen participates per group were required
such as comparisons in the primary endpoint between groups
and comparisons before- and after-ingestion to performstatistical tests and estimations. With the consideration ofrisks for social circumstances and discontinuation/dropout,the necessary number of cases was decided as fifteen cases
per group.
Key inclusion criteria for subjects as follows:
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 indaily life (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 best corrected visual acuity of 1.0
or better in both eyes and not wear contact lenses or beable to change them to eyeglasses during the examination
period.
6) Subjects who have been fully informed of the purpose andcontent of this examination, have the ability to consent,
understand it well, volunteer to participate, and agree toparticipate 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.

Key exclusion criteria for the subjects as follows:
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 (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 who have currently or within the past threemonths 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 who have currently or within the past threemonths, 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 have perennial allergy or have a risk ofseasonal allergy such as pollen allergy during the study
period.
21) Subjects who may change their lifestyle during the test
period.
22) Subjects who work at night.
23) Subjects pregnant, lactating, or possibly pregnant.
24) Subjects who are currently participating in anotherhuman clinical trial or who have not yet completed 3
months of participation in another human clinical trial.
25) Subjects whose family members are engaged in thedevelopment, manufacture, or sale of health/functional
foods and cosmetics.
26) Other subjects who are judged by the investigator andsubinvestigators to be unsuitable for this study.

_ 112 _Glycative Stress Research

3. Selection of research participants, randomization
and blind test

A mong candidates who the investigator andsubinvestigators judged as appropriate, satisfying the inclusion
criteria and not matching the exclusion criteria, thirty subjects
were selected for the trial of the present study in order ofthe highest scores in QOL score for Questionnaire of Dry
Eye related Quality of life Score (DEQS). Furthermore, forparticipants’ selection, their backgrounds as well as otherindexes were considered ( Fig. 1 ).
Gender, age at the prior test, and QOL score in DEQS
were stratified factors. After randomly allocating participants
into two groups by Stratified Block Randomization, it was
finally confirmed that there were no significant differences
between the two allocated groups.
Two food products were employed for the trial: the test
product was CRISTA®, which contained a rooster comb
enzymatic degradation product in a high concentration,and the control food product was the product which did
not contain a rooster comb enzymatic degradation productin a high concentration (Free of HA and collagen). The
test sponsor made sure that products were unable to be
distinguished by their packages, printing an identification
symbol on products and sealing up them. They sent productswith identification unseen to the tester, who confirmedthat each product sent from the producer was unable to berecognized even if they smelled products or observed theappearance of packages. The tester then newly marked withanother type of symbols for food usage management, onboth test food and control food. This replacement enabled
the blind clinical trial. Both products were handed over toa person responsible for management of the food products.
The tester kept both the correspondence table sheet and thesymbols for management. They were sealed and kept in alocked storage until the storage was unlocked and opened.

4. Test food product
Test food product was a capsule containing a highconcentration of rooster comb enzymatic degradation
product. Daily ingestion quantity was four 100 mg capsules.
The method of ingestion as follows: Two capsules were taken
by subjects twice a day with water or lukewarm water for
four weeks. The main components were shown in Table 1.
Analysis of the laboratory center of additive free foodcooperative marketing association confirmed one 100 mg
capsule contains 2.375 mg of HA in total and 43.6 mg ofcollagen in total (Dosage quantity for one day: HA 9. 5 mg,
174.4 mg).
Table 1. Total composition and content of test food(250 mg/1 capsule).
Ingredient name

Test foodPlacebo food

INJUV® (mg)

10 0

−

Vegetable oil (mg)

135

250

Other excipients (mg)15−

5. Study design
The present study employed a double-blind randomized
controlled clinical trial with parallel-group comparison.
Primary endpoints were Dry Eye related Quality oflife Score (DEQS), Visual Analogue Scale (VAS) regarding
subjective symptoms, Tear break-up time and Schirmer's test.
Secondary endpoints were visual acuity test, intra-ocularpressure, and Anti-aging QOL Common Questionnaire(AAQOL). To verify safety of the trial, examinations were
performed such as ophthalmoscopy, slit-lamp microscopy,
refractometry, blood pressure/pulse, consultation with adoctor, and assessments which were performed regarding
absence/presence of adverse medicine reactions and adverse
events.
Alcohol consumption and excessive exercise were
not allowed for trial participants on the day before the test.
Moreover, they were required to have sufficient sleep. On the
day of the test, usage of artificial tears (eye drops) was notallowed until the completion of the test.
During the test period, usage of contact lenses was notallowed. The quantity of working length on a daily basiswith visual display terminals (VDT) was maintained during
the test period. The usage of eye drops as a measure for theprevention of dryness which was used until the test was not
limited during the test period except for the day of the testday. Changing to another product was not allowed. In an
unavoidable change, participants were required to record anychanges in a participant journal. Eye drops containing HA
were not allowed.
Trial participants were required to avoid irregular
lifestyles such as insufficient sleep and excessive eating anddrinking. In addition, they were supposed to maintain theirhabits in eating, doing exercise and sleeping quantitatively
and qualitatively to be the same as before the commencementof the test.
Medicine, including external agents, quasi-pharmaceuticalproducts, and herbal medicine were prohibited in principle.
For unavoidable usage, they were required to report it to theconsulting counter, recording the name of products used,
the manufacturer, and the reason for the usage in participant
journal. It was prohibited to start using or ingesting a new
product of foods with functional claim, health food products,
and supplements during the trial. When participants had
regularly used health food products for the health maintenancebefore the participation in the trial, they were required tocontinue using them, not changing the method and usage
quantity of products. In an unavoidable usage, they should
record it in the participant journal stating the name of
product used, its manufacturer, and the reason for the usage.
It was prohibited for participants of the present trial to
newly participate in another human clinical trial within thetimeframe of trial initiation to the completion of the test.

6. Examination Items

Questionnaire of Dry Eye related Quality of life Score(DEQS)In the same method as the previous report 3), research
participants answered DEQS questions for frequency of
symptoms first; fifteen symptoms consisting of six “eyesymptoms” and nine “influences to their daily life” (Column_ 113 _

Effects of Low-Molecular Hyaluronic Acid and Collagen Peptides on Ocular Dryness

A). After participants answered column A, the participants,
who answered that they had any symptoms, recorded answers
to questions (Column B) regarding whether or not they wereconcerned about them, and the severity of the problems.
“ QOL Score” was calculated with the formular: total
score of column B / the number of valid responses × 25. We
investigated severity in dry eye symptoms, impacts on daily
life and psychological impacts.

Visual Analogue Scale questionnaire on subjective
symptoms (VAS)

In the same method as the previous report 3), VAS
investigations were conducted. Participants reported degreesof their conditions during past one week such as “eyedryness,” “eye irritation,” “eye pain,” “blurred vision,” “eyefatigue,” “visual clarity,” “stiff shoulders,” “headache,” and“sleep quality,” employing the VAS scale of 100 mm linewith the left side as the best condition (no symptoms) and theright side as the worst condition (the most severe symptoms
they had experienced).

Anti-aging QOL Common Questionnaire (AAQOL)

Assessment in subjective symptoms employed AAQOL. Participants answered 33 questions regarding physicalconditions and 21 questions regarding mental conditions,choosing one answer among five levels: 1) never, 2) rarely,3) sometimes, 4) middle-level frequency, and 5) high-level
frequency. In addition, they answered six questions regarding
lifestyle habits, recoding numerical values.
6, 7)

The following tests of eye conditions were performed
evaluating a dominant eye for assessments. All the test items
were calculated with values of dominant eyes and nondominant eyes, the mean values of left and right eyes, and themean values of both eyes. All eye tests were performed ina medical institution and a board-certified ophthalmologist
accessed the data.

Tear break-up Time Test (BUT)
In the same method of the previous report 3), participants
were asked not to blink. The time of tear break-up was
measured (measure time in seconds); the time taken for the
first dry spot to appear on the cornea after a complete blink.
The dry spot gradually expanded.

Schirmer’ test
In the same method of the previous report 3), a prescribed
strip of filter paper was placed inside the lower eyelidof participants. Their eyes produced tears and the strips
absorbed them. After five minutes, the amount of moisture,
length of wetness on the strip, was measured in millimeters.

Visal acuity test
Using a NIDEK space saving chart, SSC-370 Type D,
unaided and best corrected visual acuity were measured on
the right and the left eyes.

Tonometry test
(measurement of intra-ocular pressure)
Using a Tomey MR-6000 multifunction refractometer,
intra-ocular pressure of both the right and the left eyes were
measured three times. When an outlier was recognized dueto blinks, tonometry was measured once again. Adoped mean
were the mean value of three tonometry measurement values
excluding outliers.
In addition, the absence/presence of abnormal findings
were examined by an ophthalmoscopy test (using a NEITZ
rechargeable type brightscope, BSⅢ LED), a slit lamp
microscopy test (using Takagi slit-lamp microscope, 700GL
0814040), and a refraction test (using Tomey MR-6000
multifunction refraction refractometer).

Statistical analysis

As fundamental statistics, mean value, standard
deviation, standard error, maximum value, and minimum
value were calculated. Examination result data werecompiled to a summary sheet using Microsoft Office Excel
2016 (Microsoft Corp.). Values for analysis were measured
values and alternation quantity compared with values prior
to product ingestions. Statistical analysis software such as
SAS 9.4 (SAS Institute Inc.), and SPSS Statistics 26 (IBM)were employed. Significance level of all tests was 5% witha two-sided test. Tendency level was 10 %. Calculated data
were expressed with mean value ± standard error to record intables and graphs.
For comparison between two groups, the test food groupand the placebo group, data from all tests were analyzed
via t-test (unpaired). The unpaired t-test used P-Value asa homogeneity of variance. Scores obtained in DEQS
and AAQOL were regarded as nonparametric. For groupcomparisons, Mann-Whitney U test was performed.
A paired t-test was performed for statistical data,comparing measured values at time of commencement(before ingestion) and all observation points (duringobservation period). Scores obtained in DEQS and AAQOL
were regarded as nonparametric. For group comparisons, aWilcoxon signed-rank test was performed.

Results

Classification and content of participants

The present study started with 30 subjects and wascompleted with 30 subjects, which included no excluded
cases. Subjects mentioned below were chosen as the target of
statistical analysis in the clinical case conference.
Analysis target for safety assessment/Intention to Treat(ITT) included 30 subjects: 15 subjects in the test products
group, who ingested test food products at least once, and 15
subjects in the control product group.
Analysis target of effectiveness assessment/Per Protocol
Set (PPS) included 29 subjects: excluding one subject from
ITT analysis target (a subject of the test product group who
met the exclusion criteria), 14 subjects of the test productgroup (male: seven, female: seven, age: 38.2 ± 3.2 years old)and 15 subjects of the control product group (male: eight,_ 114 _Glycative Stress Researchfemale: seven, age: 38.4 ± 2.7 years old). The analysis flowof subjects is shown in Fig. 1 and background information isshown in Table 2. It is considered that the present experiment
was not affected by six subjects with eye drops (threesubjects of the test product group who used commerciallyavailable eye drops and three subjects of the control group
who used commercially available eye drops), because thequantity of pharmaceutical ingredients were trace amounts
in the eye drops.

Questionnaire of Dry Eye related Qualityof life Score (DEQS)

Condition of DEQS results changes are shown in Table 3.
The following are items which suggested improvements
in “ Items regarding eye symptoms, which consisted of
six items” among the DEQS for the test product group,comparing before ingestion and after two weeks: two items
in column A, “dry eyes” (p < 0.05) and “eyes fatigue” (p <0.01) and in column B, “dry eyes” (p < 0.01). At four weeks,
for the test group, four items in column A: “dry eyes” (p <0.01), “eyes fatigue” (p < 0.01), “heavy eyelids” (p < 0.05),
and “eyes redness” (p < 0.05) and in column B: two items,“dry eyes” (p < 0.05) and “eyes fatigue” (p < 0.05). However,there were no significant differences between the two groups
for either item.
Improved items in DEQS for the test food product groupregarding “Impacts on daily life” (which consisted of nine
items) were, comparing before ingestion and after two weeks,
three items in column A: “worsened eye symptoms when
watching TV or using a computer or cell phone” (p < 0.05),“lowered concentration due to eye symptoms” (p < 0.01), and“hindrance to work, housework, or study due to eye symptoms,”
and one item in column B: “lowered concentration due to eye
symptoms” (p < 0.05). At four weeks, there were three
improved items in column A: “worsened eye symptoms when
watching TV or using a computer or cell phone” (p < 0.05),“lowered concentration due to eye symptoms” (p < 0.01), and“hindrance to work, housework, or study due to eye symptoms”( p < 0.05), and in column B, “lowered concentration due toeye symptoms” (p < 0.05). However, there were no recognizedsignificant differences between the two groups in either item.
QOL scores, calculated with scores in column B, whichwere related to “degree”, showed significant improvementin the test food product group: before ingestion 46.3 ± 3.5,
after two weeks 29.8 ± 5.0, and after four weeks 26.4 ± 4.7(both two weeks and four weeks: p < 0.01). In the control
product group, scores also improved: before ingestion 51.3 ±
4.8, after two weeks 30.5 ± 4.2, and after four weeks 26.5 ±
4.4 (both two weeks and four weeks: p < 0.01). At two weeks
and four weeks, no significant difference was recognized
between the two groups.

Visual Analogue Scale questionnaire on subjective
symptoms (VAS)

The changes of VAS are shown in Table 4. The following
is improved items in VAS (nine items): Comparing with one
week before-ingestion, the four improved items were “dryeyes” (p < 0.01), “eyes fatigue” (p < 0.01), “stiff shoulder”(p < 0.05), and “headache” (p < 0.05). At two weeks, four
improved items were “dry eyes” (p < 0.05), “eyes fatigue”(p < 0.01), “stiff shoulder” (p < 0.01), and “headache” (p <0.01). At three weeks, five items were “dry eyes” (p < 0.01),“hazy and cloudy vision” (p < 0.05), “eyes fatigue” (p < 0.01),“stiff shoulder” (p < 0.01), and “headache” (p < 0.05). At fourweeks, seven items were “dry eyes” (p < 0.01), “eye irritation”(p < 0.05), “hazy and cloudy vision” (p < 0.05), “eyes fatigue”(p < 0.01), “stiff shoulder” (p < 0.01), “headache” (p < 0.01)
and “depth of sleep” (p < 0.05). No significant difference wasrecognized between the two groups.

Tear break-up time Test (BUT)

The changes in BUT are shown in Table 5. Non-dominant
eye tear break-up time was significantly improved in the test
food product group from before ingestion (5.6 ± 0.4 seconds)
to four weeks (7.3 ± 0.7 seconds) (p < 0.05). The test results
for all eyes also significantly improved in the test productgroup from before ingestion (5.4 ± 0.3 seconds) to four weeks(6.8 ± 0.6 seconds) (p < 0.05). There was no significant
difference in the control product group at both observation
times. In the control group, no significant difference wasrecognized between non-dominant eye and both eyes. There
was no significant difference between the two groups.

Schirmer’ test
The changes in secretion volume of aqueous tears, areshown in Table 6. The length of moisture on the filter paper
for a dominant eye in Schirmer’ test for the test food productgroup was 7.8 ± 1.3 mm before the ingestion. After fourweeks, it was 15.1 ± 2.9 mm (p < 0.05). Similarly, in the test
product group, non-dominant eyes test showed from 7.9 ±1.2 mm before ingestion to 13.6 ± 2.8 mm after four weeks(p < 0.05). The test for mean value of the left and right eyes
showed from 7.86 ± 1.07 mm before ingestion to 14.39 ±2.78 mm after four weeks (p < 0.05). The test for all eyes
showed from 7.9 ± 0.8 mm before ingestion to 14.4 ± 2.0
mm after four weeks (p < 0.01). Significant improvements
were observed in all measurement items of lacrimal secretion
volume. The test product group showed a significantly highvalue in comparison with the control product group regarding
both-eyes measurement after four weeks (p = 0.030).

Table2. Profiles of subjects (PPS).
Group
Test foodPlacebo foodnAge
QOL Score (-)
nAge
QOL Score (-)

Total

Male

Female14
38. 2 ± 3.2
46. 3 ± 3. 5

7
4 4. 3 ± 4.4
43. 4 ± 3. 9

732.1 ± 3. 8
49.1 ± 5. 9

15
38. 4 ± 2.7
51. 3 ± 4.8

Data are shown as the mean ± SEM.

_ 115 _

8
4 0.5 ± 4.2
45.0 ± 6.0

736.0 ± 3. 3
58.4 ± 7. 0

_ 116 _9) Sensitivity to bright light9) Sensitivity to bright light( Column A )8) Blurred vision when watchingsomething ( Column B )8) Blurred vision when watchingsomething ( Column A )7) Difficulty opening eyes
( Column B )7) Difficulty opening eyes
( Column A )6) Redness in eyes
( Column B)

6) Redness in eyes
( Column A )5) Heavy sensation in eyelids( Column B)

5) Heavy sensation in eyelids( Column A )4) Ocular fatigue( Column B)

4) Ocular fatigue( Column A )3) Painful or sore eyes
( Column B)

3) Painful or sore eyes
( Column A )2) Dry sensation in eyes
( Column B)

2) Dry sensation in eyes
( Column A )1) Foreign body sensation( Column B)

Test foodPlacebo

Test foodPlacebo

Test foodPlacebo

Test foodPlacebo

Test foodPlacebo

Test foodPlacebo

Test foodPlacebo

Test foodPlacebo

Test foodPlacebo

Test foodPlacebo

Test foodPlacebo

Test foodPlacebo

Test foodPlacebo

Test foodPlacebo

Test foodPlacebo

Test foodPlacebo

Test food

2.3 ± 0.4

1.4 ± 0.3
1.7 ± 0.3

2.5 ± 0.2
2.6 ± 0.2

1.9 ± 0.3
1.9 ± 0.2

2.3 ± 0.3
2.4 ± 0.3

0.9 ± 0.2
1. 5 ± 0.3

1.7 ± 0.3
1.9 ± 0.3

1. 5 ± 0.3
1.1 ± 0.2

2.3 ± 0.3
2. 5 ± 0.2

2.1 ± 0.3
2.1 ± 0.4

2.8 ± 0.3
3.4 ± 0.2

3.4 ± 0.2
3.4 ± 0.2

2.2 ± 0.3
3.0 ± 0.3

1.4 ± 0.3
1.3 ± 0.3

2.9 ± 0.2
3.3 ± 0.2

2.9 ± 0.1
2.9 ± 0.3

2.3 ± 0.2
2. 5 ± 0.3

0.457

0.704

0.926

0.631

0.179

0.370

0.347

0.628

0.875

0.078 §

0.884

0.054 §

0.735

0.089 §

0.450

0.379

0.404

1.9 ± 0.3
2.1 ± 0.2

Test food1) Foreign body sensation( Column A )Placebop value( between-groupcomparison)

Before

Group

Item

Table 3. Results of item (DEQS).

1.8 ± 0.4

0.9 ± 0.3
0.7 ± 0.3

1.9 ± 0.2
2.3 ± 0.2

1.5 ± 0.3
1.0 ± 0.2

1.8 ± 0.3
2.3 ± 0.4

0.6 ± 0.2
0.9 ± 0.3

1.5 ± 0.2
1.3 ± 0.3

0.8 ± 0.3
0.4 ± 0.2

1.7 ± 0.3
2.1 ± 0.3

1.4 ± 0.3
1.1 ± 0.3

2.3 ± 0.2
2.3 ± 0.2

2.2 ± 0.3
2.5 ± 0.3

2.1 ± 0.3
2.0 ± 0.3

0.9 ± 0.3
0.9 ± 0.2

1.9 ± 0.2
2.2 ± 0.2

1.9 ± 0.3
2.1 ± 0.2

1.9 ± 0.2
1.6 ± 0.2

1.3 ± 0.2
0.9 ± 0.22 weeks

0.180

0.011 *

0.121

0.096 †

0.096 †

0.010 *

0.328

0.317

0.083 †

0.011 *

0.103

0.317

0.458

0.041 *

0.067 †

0.257

0.086 †

0.012 *

0.093 †

0.003 **

0.124

0.004 **

0.003 **

0.066 †

1.000

0.164

0.107

0.005 **

0.005 **

0.032 *

0.011 *

0.003 **

0.236

0.002 **

0.101

0.981

0.239

0.169

0.389

0.595

0.453

0.352

0.278

0.437

0.825

0.617

0.713

0.816

0.389

0.475

0.348

0.273

p valuep value( between-group(vs. Before)
comparison)

2.0 ± 0.4

0.8 ± 0.2
0.5 ± 0.2

2.1 ± 0.3
2.0 ± 0.3

1.3 ± 0.3
1.2 ± 0.3

1.8 ± 0.4
2.2 ± 0.4

0.4 ± 0.2
0.7 ± 0.3

1.6 ± 0.4
1.6 ± 0.3

0.6 ± 0.3
0.6 ± 0.2

1.8 ± 0.3
2.0 ± 0.4

1.2 ± 0.3
0.9 ± 0.3

2.0 ± 0.3
2.1 ± 0.3

2.1 ± 0.3
2.1 ± 0.3

2.0 ± 0.4
2.0 ± 0.2

0.9 ± 0.3
0.8 ± 0.3

1.7 ± 0.2
1.9 ± 0.2

1.6 ± 0.3
1.7 ± 0.3

1.9 ± 0.3
1.9 ± 0.2

1.1 ± 0.3
1.0 ± 0.2

4 weeks

0.180

0.007 **

0.146

0.075 †

0.180

0.038 *

0.146

0.103

0.257

0.008 **

0.083 †

0.564

0.157

0.071 †

0.026 *

0.103

0.121

0.006 **

0.016 *

0.003 **

0.039 *

0.002 **

0.004 **

0.046 *

0.414

0.203

0.114

0.003 **

0.012 *

0.004 **

0.004 **

0.014 *

0.317

0.087 †

0.003 **

p value(vs. Before)

0.362

0.781

0.801

0.546

0.631

1.000

0.805

0.778

0.342

0.699

0.892

1.000

0.868

0.659

0.874

0.927

0.819

Effects of Low-Molecular Hyaluronic Acid and Collagen Peptides on Ocular Dryness

_ 117 _Placebo

QOL score
51.3 ± 4.8

2.3 ± 0.3
46.3 ± 3.5

1.3 ± 0.3
2.2 ± 0.4

0.7 ± 0.3

3.0

0.1 ± 0.1
2.0

2.8 ± 0.2
0.1 ± 0.1

1.6 ± 0.3
2.4 ± 0.3

2.8 ± 0.2
1.9 ± 0.3

1.9 ± 0.3
2.7 ± 0.2

2.9 ± 0.3
2.4 ± 0.2

2.7 ± 0.3
2.4 ± 0.2

2.5 ± 0.3
2.5 ± 0.2

1.7 ± 0.3
2.3 ± 0.3

2.1 ± 0.2
1.6 ± 0.3

1.7 ± 0.3
2.3 ± 0.4

2.6 ± 0.2
1.4 ± 0.3

1.9 ± 0.2
2.5 ± 0.2

2.4 ± 0.3
1.9 ± 0.3

1. 5 ± 0.3
2.3 ± 0.3

0.678

0.791

0.129

0.317

1.000

0.219

0.598

0.742

0.504

0.185

0.470

0.707

0.716

0.547

0.457

0.704

0.926

0.631

30.5 ± 4.2

2.3 ± 0.4
29.8 ± 5.0

1.0 ± 0.3
0.8 ± 0.3
1.8 ± 0.2

0.0 ± 0.0
2.0

1.7 ± 0.2
0.1 ± 0.1

0.9 ± 0.2
2.4 ± 0.3

2.2 ± 0.3
1.0 ± 0.3

1.3 ± 0.3
2.4 ± 0.2

2.4 ± 0.2
1.3 ± 0.3

1.8 ± 0.3
1.9 ± 0.2

2.1 ± 0.2
1.6 ± 0.3

1.1 ± 0.3
1.9 ± 0.3

1.6 ± 0.3
0.9 ± 0.3

0.7 ± 0.3
1.8 ± 0.4

2.3 ± 0.2
0.9 ± 0.3

1.0 ± 0.2
1.9 ± 0.2

2.3 ± 0.4
1.5 ± 0.3

0.9 ± 0.3
1.8 ± 0.3

Data are shown as the mean ± SEM※ Comparisons with "before" were made using Wilcoxon's test; †p < 0.10, *p < 0.05, **p < 0.01, ***p < 0.001.
※ Comparisons between groups at each time point were made by Mann-Whitney's U test; §p < 0.10, # p < 0.05.

Test foodPlacebo

Test foodPlacebo

Test foodPlacebo

Test foodPlacebo

Test foodPlacebo

Test foodPlacebo

Test foodPlacebo

Test foodPlacebo

Test foodPlacebo

Test foodPlacebo

Test foodPlacebo

Test foodPlacebo

Test foodPlacebo

Test foodPlacebo

Test foodPlacebo

Test foodPlacebo

Test foodPlacebo

Test foodPlacebo15) Feeling depressed because ofeye symptoms ( Column B )15) Feeling depressed because ofeye symptoms ( Column A )14) Not feeling like going out becauseof eye symptoms ( Column B )14) Not feeling like going out becauseof eye symptoms ( Column A )13) Eye symptoms affect work
( Column B )13) Eye symptoms affect work
( Column A )12) Feeling distracted because ofeye symptoms ( Column B )12) Feeling distracted because ofeye symptoms ( Column A )10) Problems with eyes whenreading ( Column B )11) Problems with eyes when watching
television or looking at a computeror cell phone (Column A)11) Problems with eyes when watching
television or looking at a computeror cell phone (Column B)

10) Problems with eyes whenreading ( Column A )9) Sensitivity to bright light( Column B )9) Sensitivity to bright light( Column A )8) Blurred vision when watchingsomething ( Column B )8) Blurred vision when watchingsomething ( Column A )7) Difficulty opening eyes
( Column B )( Column A )0.001 ***

0.002 **

0.713

0.157

0.021 *

0.234

N/A

1.000

0.317

1.000

0.008 **

0.103

0.026 *

0.020 *

0.039 *

0.034 *

0.104

0.002 **

0.046 *

0.167

0.006 **

0.015 *

0.129

0.578

0.040 *

0.079 †

0.084 †

0.180

0.011 *

0.121

0.096 †

0.096 †

0.010 *

0.328

0.317

0.083 †

0.011 *

0.810

0.310

0.539

N/A

0.301

0.038 #

0.926

0.438

0.909

0.145

0.704

0.494

0.691

0.637

0.981

0.239

0.169

0.389

26.5 ± 4.4

26.4 ± 4.7

2.3 ± 0.5
1.9 ± 0.3

0.5 ± 0.3
0.7 ± 0.2

0.0 ± 0.0
1.0 ± 0.0

2.0 ± 0.5
0.1 ± 0.1

0.5 ± 0.2
1.9 ± 0.4

2.0 ± 0.4
0.9 ± 0.3

0.9 ± 0.2
2.1 ± 0.1

1.8 ± 0.2
1.0 ± 0.3

1.5 ± 0.3
2.1 ± 0.2

2.1 ± 0.3
1.6 ± 0.3

0.9 ± 0.3
1.7 ± 0.3

1.6 ± 0.4
0.9 ± 0.3

0.5 ± 0.2
2.0 ± 0.4

2.0 ± 0.3
0.8 ± 0.2

1.2 ± 0.3
2.1 ± 0.3

2.2 ± 0.4
1.3 ± 0.3

0.7 ± 0.3
1.8 ± 0.4

0.001 ***

0.004 **

0.257

0.706

0.020 *

0.450

N/A

0.317

0.317

0.317

0.257

0.063 †

0.006 **

0.014 *

0.020 *

0.034 *

0.024 *

0.003 **

0.017 *

0.527

0.004 **

0.022 *

0.198

0.083 †

0.018 *

0.098 †

0.194

0.180

0.007 **

0.146

0.075 †

0.180

0.038 *

0.146

0.103

0.257

0.008 **

0.948

0.418

0.419

N/A

0.136

0.937

0.153

0.302

0.677

0.370

0.822

0.325

0.981

0.478

0.362

0.781

0.801

0.546

Glycative Stress Research_ 118 _Placebo

Test foodPlacebo

Test foodPlacebo

Test foodPlacebo

Test foodPlacebo

Test foodPlacebo

Test foodPlacebo

Test foodPlacebo

Test food51.1 ± 7.2

55.4 ± 7.2

51.1 ± 8.3

56.9 ± 7.2

61. 3 ± 8.9

73.1 ± 4.5

60.8 ± 5.0

50.0 ± 7.3

84.1 ± 4.8

73.4 ± 3.5

64.0 ± 6.6

59.1 ± 6.0

42.8 ± 9.3

37.6 ± 6.7

50.1 ± 6. 2

53.4 ± 5. 3

0.677

0.604

0.257

0.229

0.083 §

0.591

0.661

0.698

0.280

p value( betweengroupcomparison)

33. 5 ± 6. 2

55. 5 ± 4.8

32.6 ± 6. 5

37.9 ± 7. 1

4 7.9 ± 8. 1

64.4 ± 5.0

43.1 ± 6.0

49.6 ± 5.1

64.9 ± 6.4

57.0 ± 5.0

53. 5 ± 5.6

52.1 ± 5.3

40. 3 ± 7.0

33.7 ± 6.4

32.7 ± 5.8

48.4 ± 4.9

0.045 *

0.981

0.002 **

0.024 *

0.029 *

0.042 *

0.006 **

0.953

0.002 **

0.006 **

0.088 †

0.269

0.719

0.629

0.003 **

0.511

0.020 *

0.001 **

48.9 ± 3.8

54.7 ± 4.9

p value(vs. Before)

1 week

0.010 §

0.586

0.099 §

0.412

0.348

0.858

0.493

0.049 #

0.357

p value( betweengroupcomparison)

Data are shown as the mean ± SEM.
※ Comparisons with "before" were made with a paird t-test; †p < 0.10, *p < 0.05, **p < 0.01, ***p < 0.001.
※ Comparisons between groups at each time point were made with an unpaired t-test; §p < 0.10, #p < 0.05.

9. Sleep depth

8. Headache

7. Stiff shoulders

6. Visual clarity

5. Eye fatigue

4. Blurred vision

3. Eye pain

2. Eye irritation

65.7 ± 2.8

71.3 ± 4.1

Test food

1. Eye dryness

Placebo

Before

Group

Item

Table 4. Visual analogue scale.

33.3 ± 7.0

48.9 ± 4.9

32.9 ± 6.7

34.4 ± 7.3

38.4 ± 8.3

59.0 ± 5.5

41.5 ± 6.4

44.5 ± 5.5

60.4 ± 5.7

50.8 ± 6.0

52.8 ± 4.8

47.4 ± 6.4

38.2 ± 6.9

31.9 ± 7.2

34.5 ± 5.0

44.2 ± 5.7

48.1 ± 5.7

52.2 ± 7.1

2 week

0.017 *

0.323

0.022 *

0.003 **

0.010 *

0.008 **

0.002 **

0.371

0.001 **

0.003 **

0.188

0.095 †

0.599

0.527

0.012 *

0.178

0.025 *

0.011 *

p value(vs. Before)

0.085 §

0.887

0.050 §

0.731

0.256

0.503

0.532

0.207

0.663

p value( betweengroupcomparison)

29.1 ± 6.6

37. 9 ± 7. 3

25. 9 ± 8.0

31.1 ± 7.2

32.8 ± 8.5

50.6 ± 6.4

34.4 ± 5.4

41. 3 ± 6. 3

53. 0 ± 6.2

45. 9 ± 6.1

43. 3 ± 5.9

42. 9 ± 6. 5

35. 3 ± 6.8

29.8 ± 7.2

35.9 ± 6.6

43.4 ± 6.2

4 1.1 ± 5.1

4 9. 9 ± 7.2

3 week

0.024 *

0.107

0.003 **

0.021 *

0.005 **

0.003 **

0.0 02 **0.213

0.001 ***

0.003 **

0.022 *

0.029 *

0.4 47

0.4 010.04 5 *0.241

0.012 *

0.001 **

p value(vs. Before)

0.382

0.638

0.109

0.411

0.422

0.963

0.581

0.417

0.336

p value( betweengroupcomparison)

24.3 ± 7.7

35.6 ± 6.7

23.9 ± 8.0

28.6 ± 8.4

38. 5 ± 8.9

53.2 ± 7.1

28.7 ± 6.6

39.7 ± 7. 4

47.1 ± 7. 3

44.8 ± 7. 5

31.9 ± 6.8

39. 2 ± 7. 5

23.1 ± 5.6

24.4 ± 7.0

25. 5 ± 5. 5

33.1 ± 7.6

43. 5 ± 7. 0

4 4.2 ± 6.1

4 week

0.008 **

0.041 *

0.002 **

0.009 **

0.019 *

0.005 **

0.000 ***

0.153

0.000 ***

0.005 **

0.000 ***

0.029 *

0.011 *

0.154

0.000 ***

0.045 *

0.000 ***

0.009 **

p value(vs. Before)

0.276

0.687

0.211

0.279

0.824

0.473

0.885

0.419

0.940

p value( betweengroupcomparison)

Effects of Low-Molecular Hyaluronic Acid and Collagen Peptides on Ocular DrynessGlycative Stress Research

Table 5. BUT (Tear film break-up time)

4 weekp value(vs. Before)

p value(betweengroupcomparison)
0. 752

Item

Unit

Group

Beforep value(betweengroupcomparison)

Dominant eye

secondTest foodPlacebo

5.1 ± 0.5
5.2 ± 0.8

0.951

6.3 ± 1.1
5.9 ± 0.8

0.319
0.552

Nondominant eye

secondTest foodPlacebo

5.6 ± 0.4
6.3 ± 1.0

0. 5247.3 ± 0.7
6.1 ± 0.9

0.039 *

Binocular mean

secondTest foodPlacebo5.36 ± 0.44
5.73 ± 0.80

0. 6896.79 ± 0.87
6.00 ± 0.74

0. 496All eyessecondTest foodPlacebo

5.4 ± 0.3
5.7 ± 0.6

0. 5946.8 ± 0.6
6.0 ± 0.6

0.123
0.782
0.038 *
0.710

0. 378p value(betweengroupcomparison)

0.889

0. 351

Data are shown as the mean ± SEM.
※ Comparisons with "before" were made with a paird t- test.* : p < 0.05
※ Comparisons between groups at each time point were made with an unpaired t-test.
※ all eyes Test food n = 28, Placebo n = 30

Table 6. Schirmer's test.
Item

Unit

Group

Beforep value(betweengroupcomparison)

Dominant eyemm

Test foodPlacebo

7.8 ± 1. 3
7.6 ± 0.7

0.898

15.1 ± 2.9
9.8 ± 2. 0

0.019 *
0.175

0.132

Nondominant eyemm

Test foodPlacebo

7.9 ± 1. 2
7.1 ± 0. 8

0.582

13.6 ± 2. 8
9. 0 ± 1. 3

0.046 *
0.136

0.133

Binocular meanmm

Test foodPlacebo7.86 ± 1. 07
7.37 ± 0.68

0.698

14. 39 ± 2.78
9. 40 ± 1. 54

0.026 *
0.136

0.121

All eyesmm

Test foodPlacebo

7.9 ± 0. 8
7.4 ± 0. 5

0.623

14. 4 ± 2. 0
9. 4 ± 1. 2

0.002 **
0.042 *

0.030 #

4 weekp value(vs. Before)

Data are shown as the mean ± SEM.
※ Comparisons with "before" were made with a paird t-test; *p > 0.05, **p < 0.01.
※ Comparisons between groups at each time point were made with an unpaired t - test; #p < 0.05.
※ All eyes; Test food (n = 28), Placebo (n = 30).

Secondary endpoints
There were no significant differences recognized
between the test product group and the control group in thevisual acuity test and the tonometry test. Assessments in
AAQOL at four weeks compared with before ingestion
showed as follows: in the test product group, three items whereconditions were significantly improved were “eye fatigue,”“stiff shoulders,” and “muscle pains and stiffness” (all items:
p < 0.05). In the control group, five significantly improved
items were “eye fatigue” (p < 0.01), “cloudy and hazy vision”(p < 0.05), “stiff shoulders” (p < 0.01), “bad conditions of theskin” (p < 0.05), and “stomachache” (p < 0.05). The control
group showed significantly lower values in comparison withthe test product group. For two items of quantity changes atfour weeks: “bad conditions of the skin” and “being inclined
to catch a cold” were recorded (p < 0.05).

Assessment in safety
No abnormal findings in safety assessments werereported for both the test product group and the control
product group. The following four cases were observed:“ itchy eyes,” “subconjunctival bleeding,” “sinus condition,”and “slightly throbbing stomach pain”. However, all of these
symptoms were minor and there was no factors indicating a
causal relationship with test products. From the above, thesafety of the test food product ingestion during the four-week
trial was confirmed.

_ 119 _

Effects of Low-Molecular Hyaluronic Acid and Collagen Peptides on Ocular Dryness

Discussion

Overview of results

The present study, with the purpose of examinations on
the effectiveness and the safety for eye moisture via the test
food product ingestion, conducted a double-blind randomized
controlled clinical trial with parallel-group comparison.
Research participants were healthy males and females aged
between 20 and 59 years old who experienced dry eyes or
eye fatigue on a daily basis. The test food product was orally
ingested for four weeks. Effectiveness on eye moisturization
was examined regarding related indexes. Primary endpoints
were DEQS, VAS, BUT, and Schirmer’ test. Assessments ofsecondary endpoints were visual acuity test, tonometry test,
and AAQOL.
Safety of the present study was validated, examining
ophthalmoscopy, slit lamp microscopy test, refraction test,
blood pressure/pulse, consultation with a doctor, and absence/
presence of occurrence of adverse medical reactions andadverse events.
Data results of the trial showed significant increasein the secretion of tears (all eyes) for the test group, incomparison with the control group (significant difference
between two groups, p = 0.030). For BUT (all eyes), no
significant difference between the two groups was observed.
However, the break-up time was significantly extended
only in the test group. Regarding assessments in subjective
symptoms, improvements in effects (placebo effects) wereshown in the control group (DEQS, VAS, and AAQOL).
There was no significant difference between groups.
Schirmer’ test and BUT, which were the primary endpoints
for dry eye, were objective indexes. Therefore, these results
suggest that the test food product had positive impacts on
ocular dryness.

Causes of dry eye symptoms
Functions of tears are to prevent dryness on the ocularsurface, to supply oxygen and nutrients to ocular tissues,and to prevent invasion and infection of foreign bodies andbacteria. Moreover, tears lubricate and smooth the surfacesof cornea, which enables a clear vision to be sent to the brain.
Therefore, the decrease of tears and the deterioration of its
functions could induce the decline of functional visual acuity(values of one-minute continuous measurement of visualacuity).
Precorneal tear film is composed of three layers, themucin layer, the water layer and the lipid layer (from the deeppart of the surface of corneal epithelium).
The mucin layer: Mucins are produced by conjunctival
goblet cells. A destroyed or damaged mucin layer may causea deficiency of tear function.
The water (Aqueous) layer: The lacrimal glands produce
the middle layer, the main component of tear film. The waterlayer accounts for 95 % of tear film and contains a variety ofcomponents such as protein and hyaluronic acid (HA).
The lipid layer: The meibomian glands produce the oilrich component. The lipid layer creates the smooth surface oftears, prevents tears from evaporation, and plays an importantrole in the protection from infection. 12)
The tear film, composed of these three layers, obtains
driving force to flow due to nictation or blinking, moisturizes

the ocular surface with the flow in the one direction, andthen, flow into the lacrimal punctum in upper and lower
parts inside the lid margin and through upper and lower
punctum, lacrimal sac, nasolacrimal duct, and nasal cavity.
The following mechanism is recognized according to Dry
Eye Diagnostic Guidelines 8).
Instability of precorneal tear film, due to diverse
risk factors in the upper stream, causes corneal epithelium
disorder via dry stress. Consequently, the deteriorationof wettability due to damage to mucin on the surface ofthe epithelium triggers a core mechanism that reduces thestability of precorneal tear film. As the result of this coremechanism, inflammations are initiated, which exacerbate
the epithelium disorder.

Roles of hyaluronic acid (HA)
Hyaluronic acid (HA), which is widely distributed
throughout the body, is the main component of synovial fluid
produced from synovial membranes, and is a constituent
of articular cartilage aggrecan. Hyaluronic acid (HA) has
diverse important functions such as water retention 9, 10),
lubrications of joints 11), and intercellular adhesion 12, 13). As acomponent of tears, HA serves as a function of preventing
ocular dryness 14, 15).
The level of hyaluronic acid in the blood is clinicallyapplied to indicators for articular diseases such as
osteoarthritis (OA) ahd rheumatoid arthritis (RA), and as
well as hepatic fibrosis.
Blood concentration level of HA differs substantiallybetween individuals: Matsuura et al. 30.1 ± 16 ng/mL 16),
Nakazono et al. 42.2 ± 45.6 ng/mL 17), and Yamada et al.
33.7 ± 24.2 ng/mL 18), according to previous reports. Patientswith Osteoarthritis (OA) and rheumatoid arthritis (RA) havehigher levels of HA. Synovial fluid concentrations of HA
in healthy adults are lower than that of blood concentrations(3.4 ng/mL) 19). Tears concentrations of HA are slightly
higher than 18. 9 ± 12.6 ng/mg protein of healthy adults (inthe range of 3.2- 45.0 ng/mg protein) were slightly higher
than blood concentraitions 20). There is no difference for tears
concentration in spite of wearing contact lenses. However,there would be a possible risk factor for the aged to wear
contact lenses, because lacrimal fluid concentration of HA
are lowered due to aging 21) and aged eyes tend to become dryeasily.
Eye drops which contain HA are frequently used
as ophthalmic drugs (eye drop formulation) for dry eyes.
However, its mechanisim of action is not well known. A
previous study with animal experiences reported that eye
drops with HA improved a function to repair the ectocornea
after ectocornea desquamation 22). Matrix metalloproteinases
is related to diverse pathophysiology of corneal disorders.
Against this enzyme, HA has inhibitory effects 23 -26). It is
assumed that HA has anti-inflammatory effects on lacrimal
glands and corneas. Surveys on treatment patterns forpatients with dry eyes syndrome in ophthalmology clinics inJapan have indicated that ophthalmic solutions containing
HA is regarded as a major agent 27).
When glycative stress is added and advanced glycation
endproducts (AGEs) increase in the blood and the skin tissues,
via receptor for AGEs (RAGE) stimulation, the formation andthe secretion of inflammatory cytokine increases, and MMP_ 12 0 _Glycative Stress Research

expresses. Consequently, contents of collagen and HA
decrease. Therefore, to improve these conditions, it is effective
to orally ingest products with a functional component that
promotes the degaradation of AGEs. This product decreases
the amout of AGEs, inflammatory cytokine, and theexpression of MMP, which results in increasing contents ofcollagen and HA in the skin 28). With a similar mechanism,
it is assumed that glycative stress induced by diabetes would
worsen dry eyes symdrome.

Role of collagenWe have assumed that endogenous collagen in corneal
epithelial cells plays a promotive role for wound healing of
the corneal epithelial 29). Patients with underlying medical
conditions such as diabetes, glaucoma, and aqueous-deficiency
dry eyes, have fragile corneal tissues. It is assumed that there
is a possibility of delayed cell adhesion in the corneal wound.
In lacrimal gland extracellular matrix of patients withchronic dacryoadenitis, type I, III, and V collagen werepositive, fibronectin was positive in acinar cells and fibrous
tissues. Type IV collagen was positive only in basal
membranes 30). Via administration of transforming growth
factor β (TGFβ), only TGFβ1 became positive in acinar cells
and fibrous tissues. Therefore, a possibility was suggested
that acinar-cell derived TGFβ1 was related to the formationand deposition of extracellular matrix for chronic
dacryoadenitis.
As is a unique case, chronic Graft Versus Host Disease(cGVHD), which is a major complication of hematopoietic
stem cell transplant, has dry eyes syndrome as an ocularcondition. To treat this syndrome, other than the control
of inflammatory reaction, up-regulations of type Iα1 andIIIα1 collagens and nuclear factor-kB (NF-kB) in lacrimal
glands are important 31). Furthermore, under pathologicalcondition, MMP, which has effects of collagen degradation,is synthesized from corneal epithelial cells, corneal stromalcells, and inflammatory cells. The expression of MMP
exacerbates tear function 32).
The destruction of diverse network such as allergic
reaction and inflammation of upper palpebral conjunctiva
could trigger corneal epithelium disorder and delayed
cell adhesion in the corneal wound. It is assumed that
cornea epithelium, which is a barrier against the external
environment, is destroyed, and then, the expression of
corneal fibroblasts is activated by inflammatory cytokine
in tears and cellular infiltrate, inducing the acceleration of
MMP activation and excessive degradation of collagen 33).
A therapy currently drawing attention is a method ofthermosensitive atelocollagen injection using a punctureneedle for plug 34, 35). This method could solve dry eye problems.
Even though the amount of tear secreted from lacrimal
glands is insufficient, tears are drained through openingof lacrimal punctums. The insufficient tear volume results
in a dry ocular surface. This therapy, by setting a kind ofplug, aims to maintain the sufficient amount of tears on
ocular surface. Thermosensitive atelocollagen is liquid ata low temperature, and becomes a jelly-like substance atbody temperature. By injecting this type of collagen witha puncture needle into the lacrimal duct connected fromlacrimal canaliculus, the drainage of tears can be controlled.
Under normal circumstances, up-regulation of collagenformation in lacrimal grands is required 31). Normal state ofextracellular matrix structure such as collagen is desirable
at the lacrimal duct which is located downstream of thelacrimal canaliculus.

Mechanism of action
The patent publications of No. 6435072 in Japan and of
No. US-2019-0307788-A1 in the U. S. A., which referred tothe patent obtained by Laimu Co., Ltd. regarding INJUV®,a food product containing a rooster comb enzymatic
degradation product, mentioned that test results of the humanfibroblasts, which INJUV® was added, suggested significant
proliferative effect of fibroblasts and significant promotive
effect of collagen formation, in comparison with the control
group (non-addition of the test products. This was in-vitro
findings. However, it was suggested that low molecularweight hyaluronic acid (HA) and collagen peptide, whichwere contained in INJUV®, after intestinal absorption,
proliferated fibroblasts in extracellular matrix, which led tothe promotion of HA and collagen formation.
The major component of the test food product usedin the present study was the same low molecular weighthyaluronic acid (HA) and collagen peptide as INJUV®.
CRISTA® is a product where INJUV® is condensed at a highconcentration. This product is characterized for the extremely
low molecular weight (molecular weight: 380 –5,000) of HA.
An open-label pilot trial was conducted and suggested thatsignificant improvements were indicated in not only subjective
symptoms but also items of tea film break-up tests as well as
in tonometry and visual acuity tests. Absorbed low molecularweight HA in the body exerted some type of effects on
fibroblasts (proliferation activities and actions of activating
synthetic enzyme for HA and collagen). Mechanisms of action
in HA functions have not yet been clarified. Further studies
are required. We believe that the findings of improvements
in subjective and objective symptoms via oral ingestion of
HA is promising. The previous report suggested that via
the ingestion of INJUV®, symptoms of arthralgia of knees
and lumbago were improved 3). Improvements in dry eye
syndromes would be similarly explained as the processes ofarthralgia and lumbago.
Currently obtained findings indicated how collagen
peptide exerts beneficial effects. The ingestion of collagenhydrolysate induces the expression of prolyl-hydroxyproline( Pro-Hyp) and hydroxypropyl-glycine (Hyp-Gly) in humanblood, and the proliferation of fibroblasts as well as thepromotion of HA synthesis 36, 37). Consequently, the long-term
ingestion of collagen hydrolysate on an everyday basis improves
conditions of the skin and joints 38). Pro-Hyp, as a proliferative
factor, plays an important role in wound healing 39).
These findings suggested the following possibility:
Collagen, a test product, was orally ingested, and absorbed.
Collagen-derived dipeptides (Pro-Hyp, Hyp-Gly) are expressedin blood, and theyaffected the fibroblasts around thelacrimal glands. Furthermore, there was another suggested
possibility of the contribution to the rise of HA concentrationin blood and tears. For lacrimal tissues, HA, via the inhibitionof MMP activation, acts as an anti-inflammatory, and
HA had beneficial effects on functions of tears formation,
as is expected. Collagen-derived dipeptide promoted theformation of collagen and HA in fibroblast cells. The_ 121 _

Effects of Low-Molecular Hyaluronic Acid and Collagen Peptides on Ocular Dryness

anti-inflammatory activities (MMP activation inhibition)of endogenous and exogenous collagen maintained andimproved lacrimal environment. These could lead to theincrease in tears formation. Furthermore, another possibility
was suggested that the increase of HA in tears enhanced the
moisturizing function of tears, which led to the prevention of
decrease of tears due to evaporation.

Research limitation
The present study did not measure tears concentrationsof HA and collagen-derived dipeptides, and was unable toobtain information whether or not a functional component,
which was contained in the test food product, reallytransferred into the tears. Further examinations are required.
There are various sizes of HA. It is recognized that highmolecular weight type HA is effective in tear production 14).
There is no information regarding activities of low molecularweight HA. Further examination is eagerly required. As thenumber of cases is limited in the present study, we intend to
conduct larger-scale tests to verify data results. The present
study employed data obtained in all eyes test. However, dataof both eyes of one subject are corelated with each other.
Therefore, it was necessary to take it into consideration.

Conclusionsof a test food product, which contains a rooster comb
enzymatic degradation in a high concentration to evaluate
the effectiveness on eye moisturization and safety. The test
food products were orally ingested for four weeks. The
Schirmer’ test showed a significant increase in lacrimal
secretion volume four weeks after the commencementof ingestion. Thus, the effectiveness of the test products
on eye moisturization was verified. Furthermore, the test
food products were confirmed to be safe throughout thecontinuous four-week ingestion. It is expected that this test
product is effective for the prevention of ocular dryness andsafe as an oral-administration functional food.

Research fundings
The present study was financially supported with all
expenses and test food for the research by Laimu Co., Ltd.

Conflict of interest declaration
This trial was appropriately performed by third-party
institutions, Anti Aging Bank Co., Ltd. (Tokyo) and TES
Holdings Co., Ltd. (Tokyo). Authors of the present study
included an employee of Laimu Co., Ltd. Other than that,
there are no personal interests to declare.

We conducted a double-blind randomized controlled
clinical trial with parallel-group comparison for the ingestion

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