Glycative Stress Research
Online edition : ISSN 2188-3610
Print edition : ISSN 2188-3602
Received : November 29, 2022
Accepted : February 21, 2023
Published online : March 31, 2023
doi:10.24659/gsr.10.1_27

Original article

Verification of improvement of periorbital wrinkles by using Asakado Skin Care
Yoshikazu Yonei, Masayuki Yagi
Anti-Aging Medical Research Center / Glycative Stress Research Center,
Faculty of Life and Medical Sciences, Doshisha University, Kyoto, Japan

Abstract

The present study conducted a trial for a skin application of a test cosmetic product with 34 healthy women aged
between 35 to 55 years of age (43.4 ± 0.6) who satisfied the classification of 1, 2, and 3 in the standards for the wrinkle
grade assessment in accordance with “Guidelines for Evaluation of Cosmetic Functions”. In a half face method for 8 weeks,
the test cosmetic sample was applied to one lateral periorbital lines of a face (applied side) twice a day and no treatment wasapplied over the other periorbital lines (non-applied side). The test product was a skin-care cosmetic that contained diverse
constituents such as Ceratonia siliqua L. fruit extract, Polygonum tinctorium leaf/stem extract, and ribosomal constituents.
In test results after 8 weeks for the primary endpoint of grade assessments of periorbital wrinkles, the grade on the appliedside indicated significant improvements through a visual assessment during a medical examination and a visual assessment ofobserving case photographic images by medical specialists. However, results of the primary endpoint on the non-applied side
also indicated significant improvements. There was no significant difference between two sides. Replica analysis examining
periorbital wrinkles did not suggest any significant changes between on applied or non-applied side as well as between before
and after test product application. Secondary endpoint of skin viscoelasticity (R2) significantly improved on the applied andnon-applied sides after 8 weeks. There was no significant difference of changes in quantity. In the process of the trial, noadverse event was reported and the safety of the test product was confirmed. We conducted a further investigation to explore
possibilities that diverse functional ingredients contained in this product were absorbed through percutaneous absorption withan assistance of ribosomal constituents, and exerted effects toward the other side of the face. To verify this hypothesis, further
examinations are required to confirm blood concentrations of active components after tests.

KEY WORDS: cosmetic products, periorbital wrinkles, skin elasticity, half face method, Ceratonia siliqua

Introduction
Skin wrinkles start to appear on the face around 30
years of age. The major causative factor is recognized tobe quantitative and qualitative alternations in proteins,
elastic fibers, and collagen, which are produced by dermal
fibroblasts 1, 2). Another causative factor for shallow facial
wrinkles is structural alternations in epidermis due toepidermal dryness. Faces and necks, which are constantly
exposed to the sunlight, have deep wrinkles. Many womenare concerned about periorbital wrinkles (crow's feetwrinkles), whose appearance is noticeable. There is an
expectation for developments in effective periorbital wrinkle
amelioration of cosmetics in the cosmetic dermatology field.

The purpose of the present study is to verify
improvement effects on wrinkles at the corners of the eyes byusing the test cosmetic product (Asakado skin care cosmetics,
Nabocul Cosmetics, Tokyo, Japan). Research participants
were healthy women aged between 35 to 55 years of age
who satisfied the classification of 1, 2, and 3 in the standards
for the wrinkle grades 3). A comparative verification was
conducted to verify improvement effectiveness on periorbital
wrinkles of the test cosmetic product, employing a halfface method between the test product-applied side and nonapplied side.

Contact Address: Professor Yoshikazu Yonei, MD, PhD
Anti-Aging Medical Research Center /Glycative Stress 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; Yagi M, myagi@mail.doshisha.ac.jp

Glycative Stress Research 2023; 10 (1): 27- 42(c) Society for Glycative Stress Research_ 27 _

Improvement of Periorbital Wrinkles by Skin Care

Methods
TargetPotential participants in research were recruited: 63
healthy women aged between 35 to 55 years of age, who
satisfied classifications in pictures of 1, 2, and 3 in thestandards for the wrinkle grades in accordance with
“ Guidelines for Evaluation of Cosmetic Functions” (http://
www. jcss. jp/journal/contents_guideline5.pdf ) 3). These
potential participants underwent screening tests andpre-consumption tests (SCR & Visit-1). Among them, 34
participants who satisfied the inclusion criteria, didn’t matchwith the exclusion criteria, and were judged as appropriate tothis study by a responsible doctor, were selected as subjects
for this test according subject selection standards.
Key inclusion criteria were as follows:
1) Japanese females aged 35- 54 years, when they agreed toparticipate in the test
2) Individuals who do not have chronic physical illnesses,
including skin disorders
3) Individuals whose wrinkles at the corners of the eyes are
judged to be wrinkle grade 1-34) Individuals who have wrinkles at the corners of the eyesthat are almost symmetrical5) Individuals who were fully informed regarding the
purpose and contents of the test, has an ability ofconsent, voluntarily applied for participation with a fullunderstanding, and agreed to participate in the test withwritten informed consent
6) Individuals who can come on the designated examination
date and receive the examination
7) Individuals judged appropriate for the study by theprincipal
Key exclusion criteria were as follows:
Individuals (who)1) using medical products for diseases
2) under treatment or with history of mental disorders,sleep disorders, hypertension, diabetes, lipid metabolism
abnormality, or other serious disorders
3) have a history of serious diseases (hepatic, renal,
cardiovascular, respiratory, hematologic, etc.)
4) have a serious history and/or contract digestive diseaseand comorbidities
5) with a history of cosmetic troubles or current medical
history6) with skin symptoms such as atopic dermatitis, perioral
dermatitis, and rosacea-like dermatitis7) with experience in cosmetology and aesthetic medicine
that affect the measuring area8) have performed or received the following acts within thepast 3 monthsa. Hormone replacement therapy
b. Use of cosmetics (eye cream, sealer, patch, etc.) thathave strong moisturizing effect and wrinkle-related
effectc. Regular use of medicines, quasi-drugs, foods anddrinks for the purpose of improving the skin9) have an inflammation or a scar on a measuring area10) receiving/received medical drug treatment for the past
one month except for temporary relief medication forheadache, menstrual pain, common cold, etc.
11) drink more than 60 g/day on average in terms of purealcohol12) with possible changes of life style during test periods(night work, long-term trip, etc.)13) will develop seasonal allergy symptoms, such as pollinosis,
use an anti-allergic drug14) cannot avoid excessive direct sunlight exposure, such as
sunburn, during test periods.
15) are currently or are possibly pregnant, or are breastfeeding16) are participating and/or had participated in other clinicalstudies within the last 3 months
17 are judged as not appropriate to this study by a
responsible doctor

Trial Design

This trial conducted a parallel-group trial with ashielding test, employing a half face test, to examine
improvement effects on wrinkles at the corners of the eyesby using the test cosmetic product between the applied sideof the face and the non-applied side.
Research participants, after morning and evening skincare for 8 weeks, dispensed one push of the test product(approximately 0. 5 mL) into the palm of their hand froma container and applied it around the corner of their eye,which was assigned, allowing it to blend into the skin of thehalf of face. The other side is left untreated. The numberof observations was three times: before the test for usage,
4 weeks after, and 8 weeks after the commencement of thetest. The following was performed: visual assessment of
medical examinations for grades in wrinkles of periorbitallines by medical specialists, visual assessment for gradesin periorbital wrinkles using photographic images byspecialized doctors, photographic images of periorbital
wrinkle parts (Visia, Canﬁeld Scientiﬁc, Parsippany, N J,
USA), replica analysis of periorbital wrinkle, measurementsof skin viscoelasticity, medical interview by doctors, andjudgments for the presence or absence of occurrence ofadverse reactions/adverse events. For photographic images
at periorbital wrinkle parts (Visia), analysis of periorbital
wrinkle replicas, and measurements of skin viscoelasticity,
the test was performed in an environmental test chamberwith 21 ± 1 ℃ of temperature and 50 ± 5 % of moisture.
Research participants recorded a living diary during the test
period with usage conditions of the test cosmetic product,
alternations in skin conditions and physical conditions (aninvestigation of adverse events), the presence or absence ofalternations in living conditions, conditions of medication
usage, conditions of dietary supplementation, and thepresence or absence of menstruation.
In this trial, 34 research participants started to undergo
the test and all the participants completed the test. Age of
34 female subjects was 43. 4 ± 0.6 and visually inspectedassessment of periorbital wrinkle was 2.265 ± 0.122 by aspecialized doctor at the time of the assignment.

Test Product

The test product is produced by Nabocul Cosmetics co.,ltd. as a brand-new product of Asakado Project (Nabocul
Cosmetics). This company with an approximate 40 years old_ 28 _Glycative Stress Researchhistory, has developed cosmetics of high performance andhigh quality based on cutting-edge dermatology focusing on
the effectiveness of natural constituents existing in nature,
based on their success in the usage of natural materials withexcellent effectiveness, established Asakado Project, as acollaboration between an in-house research team and an
outside research development institution.
Compounding ingredients are as follows: Ceratonia
siliqua L. fruit extract, polygonum tinctorium leaf/stem
extract, chamomilla recutita flower extract (Anthemis
nobilis), glucosyl hesperidin, glucosyl rutin, herb Robert
extract, hydroxyproline, coenzyme A, chondroitin sulfate
sodium, saccharomyces lysate extract, sodium hyaluronate,beta glucan, sodium polyaspartate, octyldodecyl myristate,ubiquinone (oxidized coenzyme Q10), hydroxyethyl cellulose,adenosine triphosphate disodium, arginine, aloe vera leaf
extract, glycosyl trehalose, glucosylglycerol, dipotassium
glycyrrhizinate, hydroxyphenyl propamidobenzoic acid,ascorbyl palmitate, niacinamide, retinol palmitate, cornoil, hydrogenated starch hydrolysate, licorice extract,hydrogenated lecithin, xanthan gum, carbomer, BG,
EDTA-2Na, glycerin, PEG-60 hydrogenated castor oil,hydroxyacetophenone, pentylene glycol, and aqua. The main
ingredient is a new compound with a molecular formula of
C 23 H 24 O13 that was separated and identified from a shellof Ceratonia siliqua; Ceratonia siliqua is derived from theMediterranean coast. The structural formula is shown in
Fig. 1 4).
OH

OH

HO
O

O

O
O

O

HO

OH
O
OH

OH
Fig. 1. Novel compound that is extracted from Ceratonia
siliqua.

Assessment Items for Effectiveness

Visual inspection assessment of periorbital wrinkle
grades by specialized doctors

Scoring for periorbital wrinkles was performed at the
beginning of the trial based on the wrinkle grade assessment 3)
in accordance with “Guideline for Evaluation of Anti-wrinkle
Products for acquisition of new effectiveness”, which was
stipulated by Japanese Cosmetic Science Society (http://www.
jcss.jp/ ).
At the time of each observation for each measurement
point, referring to photographs of the beginning and theprevious measurement point, scoring was performed basedon the wrinkle grade assessment. Values of 50 % and 25%
were employed for scoring such as 2.5 or 2.25 when there
was no standard picture grade for assessment.

Visual Assessment of Periorbital Wrinkle Gradesby Specialized Doctors Using Case Photographic
ImagesAt the beginning of the trial, the scoring for periorbital
wrinkles using case photographic images of outer canthus was
performed in accordance with the wrinkle grade assessment 3).
At the time of each observation for each measurement
point, referring to photographs at the beginning and theprevious measurement point, scoring was performed basedon the wrinkle grade assessment. Values of 50 % and 25 %were employed for scoring such as 2.5 or 2.25 when there
was no standard picture grade for assessment.

Replica Analysis of Periorbital Wrinkle
Replicas were prepared from the corners of eyes, fromthe area 5 mm away from the edges of the left and right eyesto the area of fine wrinkles with reticulations. The sample
was analyzed by using a reflective replica analysis system,the 3D Skin Roughness Analysis Measurement System ASA03RXD (Asahi Biomed Co., Tokyo, Japan). The range ofsample analysis was 1) area of 1.0 cm × 1.0 cm, approximately
5 – 7 mm away from the corner of the eye, and 2) area of 1.0
cm × 1.0 cm, approximately 10 – 12 mm away from thecorner of the eye. Analysis was performed in terms of both 0
degrees and 180 degrees, and the mean values were adopted.
Assessment items were as follows:
1) 5 – 7 mm away from the corner of the eye
area ratio of wrinkles/volume fraction of wrinkles/
average depth of total wrinkles/average depth of maximum
wrinkles/maximum depth of maximum wrinkle/thenumber of wrinkles2) 10 –12 mm away from the corner of the eye
area ratio of wrinkles/volume fraction of wrinkles/
average depth of total wrinkles/maximum depth of total
wrinkles/the number of wrinkles

Secondary Endpoint
Measurement of Skin Viscoelasticity

Employing a cutometer, Cutometer MPA580 ® (CourageKhazaka, Köln, Germany), the following area of the left
and right faces was measured five times: the center part ofconnected lines from the bottom of earlobe and the edge ofthe lip. With the standard R2 values, maximum and minimum
values were eliminated and the mean value of measurements,
which were performed three times, was obtained.

Other Assessment Item

Photographic Images of Periorbital Wrinkle Area
(VISIA)

Employing VISIA-Evo (Canﬁeld Scientiﬁc), the left andright faces were photographed and periorbital wrinkle areas
were trimmed to extract.

_ 29 _

Improvement of Periorbital Wrinkles by Skin Care

Safety Assessment Item

Results

Medical Interview by a Medical Specialist for the
Presence or Absence of Occurrence of Adverse
Reactions/Adverse Events

Effectiveness Assessment Items

At screening tests and pre-consumption tests, backgroundquestionnaires were performed. At the time of observation
after the commencement of tests, subjective symptoms ofresearch participants and occurrence of adverse events werechecked, and medical recoding, interviews, and appropriate
treatments were conducted.

Statistical Analysis
Items of target analysis were visual inspection
assessment of periorbital wrinkle grades by specialized
doctors, visual assessment of periorbital wrinkle grades byspecialized doctors using case photographic images, replica
analysis of periorbital wrinkles and skin viscoelasticity
measurements. Values of examination results were compiled
to a grand total table using Microsoft Office Excel 2016
(Microsoft Corp., Redmond, WA, USA). As fundamental
statistics, the mean values, standard deviation, standard error,
maximum values, and minimum values were calculated.
Values used in the statistical analysis were measured values
and amount of change from the commencement of usage. All
examination items obtained as category data were compiledat each examination point. Statistical analysis was performed
using appropriate statistical software such as SAS (SAS 9.4,
SAS Institute Japan, Tokyo) or SPSS (Statistics 26, IBM
Japan, Tokyo). For all tests, significance level was 5 % in
two-tailed tests.
Statistical data of each examination point wereanalyzed in a paired t-test (two-tailed test) with comparison
between groups, the test cosmetic product application sideand the non-treatment side. Scores which were obtained byvisual inspection assessment of periorbital wrinkle gradesby specialized doctors were treated as non-parametric,and Wilcoxon signed-rank sum test was conducted forcomparison between groups.
Statistical data of each examination were analyzed in apaired t-test (two-tailed test) with comparison among groups(time comparison), the time before the commencement ofusage and each observation time after the commencementof usage. Scores which were obtained by visual inspection
assessment of periorbital wrinkle grades by specialized
doctors was treated as non-parametric, and Wilcoxon signedrank sum test was conducted for time comparison.

Ethical Examination
The present study was conducted in compliance withethical principles based on the Declaration of Helsinki(Amendment, Fortaleza, October 2013) and “Ethicalprinciples for medical research involving human subjects(Partial amendment, 2017). Ethical approval was obtained
from the committee of medical research involving humansubjects of “general incorporated association: Society for
Glycative Stress Research (GSE #2022-001). Pre-registration
for a clinical trial was conducted (UMIN #000046645).

Visual inspection assessment of periorbital wrinkle
grades by specialized doctors (Table 1)
On the applied side, before usage (2.27 ± 0.12) → 4
weeks after the commencement of usage (2.20 ± 0.12) →8 weeks after the commencement of usage (2.10 ± 0.12).
Wrinkle grade decreased. Compared with before usage, 4weeks after the commencement of usage (p = 0.005) and 8
weeks after the commencement of usage (p < 0.001). Wrinkle
grade significantly improved (Fig. 2).
On the non-applied side, before (2.27± 0.12) → 4 weeks(2.21 ± 0.12) → 8 weeks (2.10 ± 0.12). Wrinkle grade
decreased. Compared with before usage, 4 weeks ( p = 0.029)
and 8 weeks ( p < 0.001). Wrinkle grade significantly
improved.
There was no significant difference between the twosides.

Visual assessment of periorbital wrinkle grades byspecialized doctors using case photographic images(Table 1)
On the applied side, before (2.27 ± 0.12) → 4 weeks(2.18 ± 0.12) → 8 weeks (2.08 ± 0.12). Wrinkle grade
decreased. Compared with before usage, 4 weeks (p < 0.001)
and 8 weeks (p < 0.001). Wrinkle grade significantly improved(Fig. 3 ).
On the non-applied side, before ( 2.27 ± 0.12) → 4
weeks (2.19 ± 0.12) → 8 weeks (2.06 ± 0.12). Wrinkle grade
decreased. In chronological comparison by Wilcoxon signedrank sum test, compared with before usage, 4 weeks (p =0.003) and 8 weeks (p < 0.001). Wrinkle grade significantly
improved.
There was no significant difference between two sides.

Replica Analysis of Periorbital Wrinkles (Table 2)

No items regarding the following suggested any
significant improvement on the applied side: wrinkles 5 mmfrom the corner of the eye in area ratio of wrinkles/volume
fraction of wrinkles/average depth of total wrinkles/average
depth of maximum wrinkles/maximum depth of maximum
wrinkle/the number of wrinkles, and wrinkles 10 –12mm from the corner of the eye in area ratio of wrinkles/
volume fraction of wrinkles/average depth of total wrinkles/
maximum depth of total wrinkles/the number of wrinkles.
There was no significant difference between both sides.

Secondary Endpoint Items

Skin Viscoelasticity Measurement (Table 3)

R2 on the test-product-applied side changed as follows:
before usage (0.788 ± 0.012) → after 4 weeks (0.780 ±0.011) → after 8 weeks (0.809 ± 0.009). The chronologicalcomparison by the paired t-test indicated that wrinkles
significantly improved at 8 weeks after the commencementof usage (p = 0.004) in comparison with before usage (Fig. 4 ).
R2 on the non-applied side changed as follows: before_ 30 _Glycative Stress Research

usage (0.792 ± 0.010) → after 4 weeks (0.803 ± 0.009) →after 8 weeks (0.815 ± 0.007). The chronological comparisonby the paired t-test indicated that wrinkles significantly
improved at 8 weeks after usage (p < 0.001) in comparison
with before usage.

Furthermore, in a comparison among groups by a paired
t-test, the applied side was significantly lower than the nonapplied side at the point of 4 weeks after usage (p = 0.027).
There was no significant difference between two sides at 8
weeks.

Table 1. Periorbital wrinkle grades assessed by specialized doctors.

VisualPhotographic
imagesComparison
between groups

Time Comparison

Periorbital
wrinkle grades
AppliedNon-applied
AppliedNon-applied

Before4W

P value

8W

P value

2.27 ± 0.12

2.20 ± 0.12

0.005

2.10 ± 0.12

< 0.001

2.27 ± 0.12

2.21 ± 0.12

0.029

2.10 ± 0.12

< 0.001

2.27 ± 0.12

2.18 ± 0.12

0.001

2.08 ± 0.12

< 0.001

2.27 ± 0.12

2.19 ± 0.12

0.003

2.06 ± 0.12

< 0.001

p value (4W) p value (8W)
0. 527

1.000

0.782

0. 597

Periorbital wrinkle grades are assessed by visual inspection and photographic images. Results are expressed as mean values ± SEM, n = 34. Wilcoxon
signed-rank sum test is used for analysis of the time comparison with before usage and the comparison between applied side and non-applied side. SEM,
standard error mean.

Table 2. Replica analysis of periorbital wrinkles.

Comparison
between groups

Time Comparison

Replicaanalysis

BeforeArea ratio of
wrinkles (5 mm)

μm 2/mm 2/100

Volume fraction of
wrinkles (5 mm)

μm 3/mm 2/100

Average depth of
total wrinkles(5 mm)

μm

Average depth of
maximum wrinkles(5 mm)

μm

Maximum depth of
maximum wrinkle(5 mm)

μm

Number ofwrinkles (5 mm)

N/mm

Area ratio of
wrinkles (10 mm)

μm 2/mm 2/100

Volume fraction of
wrinkles (10 mm)

μm 3/mm 2/100

Average depth of
total wrinkles(10 mm)μm

Maximum depth of
total wrinkle(10 mm)μm

Number ofwrinkles (10 mm)

N/mm

Applied2.73 ± 0.18

Non-applied

2.66 ± 0.16

Applied39.39 ± 2.70

Non-applied

38.22 ± 2.37

Applied149.90 ± 1.74

Non-applied 150.17 ± 1.62
Applied152.22 ± 2.10

Non-applied 152.84 ± 1.70
Applied401.12 ± 9.80

4W

P value

8W

3.00 ± 0.19

0.015

2.88 ± 0.18

3.10 ± 0.19

0.088

0.008

43.51 ± 3.05

2.98 ± 0.18

0.019

0.017

41.56 ± 2.74

46.12 ± 3.43

0.097

0.010

150.57 ± 1.63

43.35 ± 2.70

0.661 152.79 ± 1.72

0.051

0.255 151.77 ± 1.23

0.197

0.461 156.45 ± 2.50

0.030

0.302 156.14 ± 1.47

0.035

151.68 ± 1.56
153.44 ± 1.96
154.47 ± 1.50
410.30 ± 14.13

Non-applied 402.40 ± 13.26 399.63 ± 10.06
Applied0.55 ± 0.02

Non-applied

0.54 ± 0.02

Applied2.79 ± 0.25

Non-applied

2.70 ± 0.22

Applied41.04 ± 3.98

Non-applied

39.45 ± 3.41

Applied155.19 ± 2.85

Non-applied

155.12 ± 2.44

Applied

0.315 432.84 ± 14.97 0.006
0.718 426.13 ± 15.23

0.107

0.027

0.021

0.56 ± 0.02

0.58 ± 0.02

0.374

0.016

3.23 ± 0.29

0.57 ± 0.02

0.001

0.017

2.89 ± 0.25

3.10 ± 0.24

0.226

0.027

47.97 ± 4.85

2.98 ± 0.23

0.002

0.019

42.51 ± 3.87

45.83 ± 4.05

0.202

0.032

155.92 ± 2.82

43.68 ± 3.67

0.682 156.09 ± 2.58

0.584

0.056 155.97 ± 2.34

0.442

402.70 ± 11.18 409.28 ± 12.87 0.277 413.55 ± 14.60

Non-applied 400.44 ± 8.46
Applied0.49 ± 0.03

Non-applied

0.49 ± 0.03

0.013

0.58 ± 0.02

157.48 ± 2.73
399.15 ± 7.91

0.53 ± 0.03
0.49 ± 0.03

p value p value(8W)

P value (4W)

0.185

0.817 382.77 ± 7.16

0.025

0.011

0.52 ± 0.02

0.025

0.981

0.51 ± 0.03

0.049

0.730 0.750
0.746 0.564
0.474 0.379
0.706 0.629
0.365 0.642
0.307 0.880
0.084 0.787
0.090 0.745
0.309 0.980
0.343 0.007
0.036 0.992

Periorbital wrinkle is analyzed at 5 mm and 10-12 mm from the corner of the eye. Results are expressed as mean values ± SEM, n = 34. Paired t test is used foranalysis of the time comparison with before usage and the comparison between applied side and non-applied side. SEM, standard error mean.

_ 31 _

Improvement of Periorbital Wrinkles by Skin Care

Periorbital wrinkle grades (visual)

2.4
2.3
2.2

Applied side(n = 34)
Non-applied side (n = 34)
2.27
2.27

2.21

*

2.20

**

2.1

2.10
2.10 ***

***

2.0
1.9
1.8

Before4 weeks

8 weeks

Fig. 2. Visual inspection assessment of periorbital wrinkle grades by a specialized doctor.

The mean value ± SEM, n = 34. Applied side, test-product topically applied for 8 weeks. *p < 0.05, **p < 0.01,***p < 0.001 vs before usage, Wilcoxon signed-rank sum test. SEM, standard error mean.

Applied side(n = 34)
Non-applied side (n = 34)

Periorbital wrinkle grades (images)

2.4
2.3
2.2

2.27
2.27

2.19 **
2.18

***

2.1

2.08 ***
2.06 ***

2.0
1.9
1.8

Before4 weeks

8 weeks

Fig. 3. Visual assessment of periorbital wrinkle grades by specialized doctors using casephotographic images.
The mean value ± SEM, n = 34. Applied side, test-product topically applied for 8 weeks. **p < 0.01, ***p < 0.001
vs before usage, Wilcoxon signed-rank sum test. SEM, standard error mean.

_ 32 _Glycative Stress Research

Table 3. Skin Viscoelasticity Measurement.

R0
R2
R5
R6
R7

Comparison
between groups

Time Comparison

Skinelastisity

Before4W

P value

8W

P value

Applied0. 32 ± 0.01

0. 34 ± 0.01

0. 001

0. 33 ± 0.01

0.024

Non-applied

0. 31 ± 0.01

0. 33 ± 0.01

0. 004

0. 32 ± 0.01

0.165

Applied0.79 ± 0.01

0.78 ± 0.01

0.320

0. 81 ± 0.01

0.004

0. 82 ± 0.01

0.001

Non-applied

0.79 ± 0.01

0. 80 ± 0.01

0.179

Applied0. 58 ± 0.02

0. 51 ± 0.02

0.000

0. 56 ± 0.02

0.163

Non-applied

0. 58 ± 0.02

0. 55 ± 0.01

0. 001

0. 56 ± 0.01

0.055

Applied0. 37 ± 0.01

0. 34 ± 0.01

0. 0230. 35 ± 0.01

0.342

Non-applied

0. 37 ± 0.01

0. 34 ± 0.01

0. 0080. 35 ± 0.01

0.021

Applied0. 42 ± 0.01

0. 39 ± 0.01

0. 0000. 42 ± 0.01

0.351

Non-applied

0. 42 ± 0.01

0. 41 ± 0.01

0. 0220. 42 ± 0.01

0.264

p value (4W) p value (8W)
0.479

0.552

0.089

0.589

0.031

0.805

0.747

0.803

0.052

0.948

Skin elasticity is Measured by Cutometer. Results are expressed as mean values ± SEM, n = 34. Paired t test is used for analysis of the time comparison with
before usage and the comparison between applied side and non-applied side. R0, Uf; R2, biological elasticity (Ua1/Uf1); R5, net elasticity (Ur/Ue); R6,
viscoelasticity (Uv/Ue); R7: elastic recovery (Ur1/Uf1); SEM, standard error mean.

0.84

Applied side(n = 34)
Non-applied side (n = 34)

Skin elasticity (R2)

0.82
0.803

0.80

#

0.815 ***
0.809 **

0.792
0.788

0.78

0.780

0.76

0.74

Before4 weeks

Fig. 4. Skin elasticity changes.

8 weeks

The mean value ± SEM, n = 34. Applied side, test-product topically applied for 8 weeks. **p < 0.01, ***p < 0.001
vs before usage, # p < 0.05 vs non-applied side, paired-t test. SEM, standard error mean.

_ 33 _

Improvement of Periorbital Wrinkles by Skin Care

SafetyNo adverse reaction which was considered to be inducedby the test product was reported by any research participants.

Discussion

Overview of Results

The present study verified improvement effectiveness ofthe test cosmetic product on periorbital wrinkles conducting
a parallel-group comparison shield study (a half face method)with 34 healthy women aged 35 to 55 years of age whose
periorbital wrinkles met the classification of 1, 2, and 3 inthe standard photographs 3) for the wrinkle grade assessment.
Visual assessments on periorbital wrinkle graded bya medical examination of specialized doctors, and visualassessment by doctors observing case photographic images,
which were effectiveness assessment items of this test,
indicated significant improvements on the test-productapplied side. However, assessments on the non-applied side
also indicated significant improvements. There were no
significant differences between both sides. Regarding replica
analysis, there was no difference. Though specialized doctors
visually detected fine alternations in shallow wrinkles during
before-after examinations, there was a possibility of somedifficulties in detecting an alternation via replica analysis.
Skin viscoelasticity measurements, which were
the secondary endpoint assessment item, indicated asignificant improvement on the applied side at the point of
8 weeks. However, improvements were also observed on
the non-applied side. There were no recognized significant
differences between the two sides.
This test cosmetic product contained diverse types offunctional ingredients as well as emulsifying agents andribosomal constituents, which enhance skin permeability. In
the half face method, a possibility cannot be eliminated thatabsorbed functional ingredients via skin application resulted
in effects on the other side of the face.
For further investigations, we collected bibliographic
information regarding the contained ingredients and exploredpossibilities of effects on wrinkles and percutaneousabsorption. Furthermore, it was discussed how further
research would verify if ingredients affected wrinkles on theother side via percutaneous absorption.

Components of the Test ProductTo begin, we explain main components that are involvedin the functions of this product.

• Ceratonia siliqua L. fruit extract

Ceratonia siliqua L. is in the legume family and native
to the Mediterranean region. Legume of Ceratonia siliqua,
in other words, pod and fruit, is designated as a carob.
Carob has been used as food and as a food ingredient for
generations. Ripe pods are approximately 10 –25 cm longand taste mildly sweet. The carob contains a large amountof polysaccharide, cellulose and mineral and a small amountof non-hydrocarbon low molecular weight compounds, suchas antioxidant polyphenols 5), and protein. Studies on new
functions of carob have been developed in recent years.
Extract of Ceratonia siliqua bark has an inhibitory effect ontyrosinase, whose substrate is L-tyrosine. In a clinical trial,
application on the skin for 28 days improved skin tone by 3 %to even out skin with ultraviolet-induced hyperpigmentation 6).
Previous studies reported that pod extracts of Ceratonia
siliqua, carob, had effects of the prevention against and the
medical treatments for digestive diseases such as ulcerative
colitis and gastric ulcer 7, 8). Carob contains a chemicalcompound with ester linkages as a novel polyphenolic
compound, where both isoferulic acid and gallic acid bindwith glucose ( Fig. 1 ), as is reported. Carob seed extracts haveinhibitory effects against α-glucosidase to suppress weightincrease 4). Possibilities based on effective assessments havereported that “OLANDU” promotes the expression of collagen
COL1A1 gene, enhances the formation of collagen, andameliorates wrinkles 9).
Retinoid compounds (retinol palmitate) have beenutilized as a topical medication effective for the treatmentof photo-aging skin for decades. Niacinamide is a quasipharmaceutical product that has effectiveness for wrinkle
improvement. A trial, which verified retinoid response geneof keratinocyte, indicated that activities of retinyl propionate,Vitamin A ester, were re-enforced by niacinamide (Nam)and Ceratonia siliqua L. fruit extract 10). The test cosmetic
product can be expected to have this type of synergy.

• Polygonum tinctorium leaf/stem extract

Polygonum tinctorium, which is designated as
inagomame in Japan, is a plant in the knotweed family,
polygonaceae. The kanji character of ai is “ 藍 ”. This isalso called tadeai, indigo plant. This plant has been mainly
utilized in dyes. Other than that, effects of detoxication andanti-pyresis were known. Recent investigations discovered
diverse functions and effects such as anti-tumor action,
antioxidation, anti-inflammatory, anti-virus, anti-allergy 11),and anti-tumor action 12). Tryptanthrine, which is one of themain components extracted from Polygonum tinctorium,
strongly inhibits the formation of hepatocyte growth factor(HGF), which is stimulated by diverse inducers in humanskin fibroblasts. It is confirmed that HGF not only arranges
normal cell functions but also is involved in malignantcell-related transformations and cancer-cell-related
multiplications, invasions, and metastasis. It is supposed thatthe inhibition of HGF formation has a suppressive effect on
the transformation of malignant cells and the progressionof tumors 12). An experimental study using mice, which had
2, 4-dinitrofluorobenzene (DNFB)-induced atopic dermatitis,
reported that extracts of Polygonum tinctorium suppressed
serum concentration of IgE and interleukin-4, inhibited theexpression of caspase-1 which was derived from mast cellsof skin lesions, and improved clinical conditions 11). It has
been reported in cosmetic fields that Japanese indigo extracts
have diverse effects such as moisturizing effects, antiinflammatory effects, skin whitening effects, antioxidation
effects and wrinkle improvement effects with enhanced skinresilience 13).
Our research lab reported that extracts of Polygonumhydropiper had an intensive inhibitory effect on theformation of advanced glycation end products (AGEs) with_ 34 _Glycative Stress Researchin vitro testing, and an inhibitory effect of the formation ofaldehydes (3-DG, GO, and MGO) 14). To determine whether
or not extracts from wild relatives of Japanese indigo(Polygonum tinctorium) also have anti-glycation abilities,
further verifications have yet to be completed.

• Chamomile recutita flower extract (Anthemis nobilis)

Kamitsure (commonly used name: German chamomile,scientific name: Anthemis nobilis) is native to Europe. This
plant started to be utilized in herbal remedies around thefirst century B.C. In the Middle Ages, it was documented
that chamomile had effects for the mitigation of digestive
disorders and abdomen enlarged feeling and effects thatfacilitate good sleep. Presently, chamomile is characterized
as a typical European medical herb. Its evaluation covers
diverse effects such as the mitigation and amelioration ofanxiety, muscular paralysis, skin problems and digestive
disorders for a broad range of age groups from infants toadults 15, 16). Flowers have a rich, mellow, fruity and apple-likearoma. This aroma provides relaxing effects and chamomile
herb tea is consumed all over the world 17). Main ingredientsare sesquiterpene, α-bisabolol, chamazulene, which are interpenoids, and apigenin, quercetin, patuletin, and luteolin,which are in flavonoids, as well as glucosides 18, 19).
Clinical trials of mixed herbs such as kamitusre flower(Anthemis nobilis, Chamomile: flower, AN), Crataegus
oxyacantha (Hawthor: berry, CO), Houttuynia coradata(Doku-dami: whole plants, HC), and Vitis vinifera (Grape:
leaf, VV), suggested as follows: An open test indicatedthat HerbMix product 20) ingestion of 600 mg/day for 12
weeks led to the decreases of CML and 3DG in blood, andthe improvement in skin viscoelasticity (elevation of skinelasticity, R2 and R7). A double-blind test indicated thatthrough the product ingestion of 3,000 mg/day for 8 weeks,
decreasing trends of CML and 3DG (p < 0.1) in blood, andincreased improvement score of subjective symptoms arerecognized 21). Another double-blind test for 100 mg/day ofthe product ingestion for 12 weeks demonstrated the decreaseof 3DG in blood and the reduction of melanin index andcolor difference b* (index for yellow) of upper arm skin 22).
Therefore, stable anti-glycative effects were exerted. There
was a possibility that the ingredients of the test productcontributed to the improvement of skin viscoelasticity.

• Hydroxyproline

Collagen, which is the most abundant protein in thehuman body, is an essential protein to maintain normal
structure and strength of connective tissues such as bones,
skin, cartilage, and blood vessels. Glycine, proline, andhydroxyproline account for 57 % of total amino acid (AA) of
collagen 23). Hydroxyproline, which is an abundant amino acidin type I collagen of the skin, is produced via a translational
modification of proline by an enzyme prolyl hydroxylase,
and this synthesis is irreversible. Hydroxyproline exists in theposition of “Y ” of a repetitive sequence (Gly-X-Y) in type I
collagen, playing an important role in synthesis of collagen,and to thermodynamically stabilize the triple helicalstructures of collagen and related tissues 24, 25). Hydroxylationof proline requires ascorbic acid (vitamin C ). Therefore,
deficient vitamin C leads to lower ratio of hydroxyproline incollagen and consequently, stability deteriorates and scurvy
is induced.
It is reported that through ingestion of hydroxyproline,
collagen synthesis of the skin increases and theproliferation of epidermal keratinized cells is promoted.
Acetylhydroxyproline, which is a hydroxyproline derivative,
is known to have a vulnerary promoting action andmoisturizing effects with increased moisture content ofhorny cell layer 26).
This cosmetic test product, other than 5-hydroxyproline,
contains lecithin, rutin derivative (glucosylrutin), and ascorbyl
palmitate. Composite material, RonaCare VTA of Merck(Merck KGaA, Germany) 27, 28) is designed to be produced asfollows; Hyaluronic acid is protected from degrading enzyme
and simultaneously, synthesis of glucosaminoglycan, which isan extracellular matrix, is promoted, where liposome, whichcontains hydroxyproline, rutin derivative, and magnesium
ascorbyl phosphate, penetrate the skin. There is an expectationthat the test product has similar effects.

• Niacinamide

Niacinamide (commonly used name: nicotinic acidamide, vitamin B3) is a type of vitamin B3, and is containedin fish, meat, beans, and grains. Niacinamide is approved asa quasi-pharmaceutical product.
The facilitation of ceramide synthesis in vitro is known
for niacinamide. Different concentrations of niacinamide
were added into normal human keratinocytes culture(keratinocytes), and ceramide synthesis was measured withuptake of 14-C serine to ceramide. Addition of niacinamide
with concentrations over 1 μM significantly increased
ceramide synthesis 29).
Niacinamide of 10 μM was added to a keratinocytes
culture, and synthesis of ceramide, which is a structuralcomponent of intercellular lipid, fatty acid, and cholesterol,
were measured with uptake of 14- C acetic acid to eachlipid. The addition of niacinamide of 10 μM significantly
promoted synthesis of not only ceramide but also fatty acidand cholesterol ( p < 0.01) 29).
Kawada Akira, Professor of Department of Dermatology,
Kinki University School of Medicine et al. verified usefulnessof niacinamide on human skin wrinkles 30). They conducteda clinical examination based on a double-blind trial, with 28
Japanese female subjects who had fine periorbitalwrinkles(31– 49 year-old, the mean age: 39. 4). Oil-in-Water (O/W)
moisturizing agent of 4 % niacinamide mixture was applied
on a half face and on the other side of the face, (O/W)
moisturizing agent without niacinamide was applied for
8 weeks. According to guidelines of the Japan Cosmetic
Industry Association, effectiveness was assessed at the pointsof 4 weeks and 8 weeks, through two researchers’ observationand photographic images. Experimental results using fivegrade evaluation, “remarkably significant ”, “significant”,“somewhat significant”, “no change” and “change for theworse”, suggested that the application of moisturizing
agent of 4 % niacinamide mixture exerted the middle orhigher level of effects of wrinkle improvement to 18 out of
28 at 8 weeks. This result indicated that niacinamide was
effective at wrinkle improvement. There are possibilities thatpermeability improvement of niacinamide by a cosmetic
base could contribute to it 30).

_ 35 _

Improvement of Periorbital Wrinkles by Skin Care

Mechanism of anti-wrinkle effects in niacinamide has not
yet to be identified. Judging the findings that the addition of
niacinamide to keratinocytes cultures, which did not havesufficient collagen, significantly increased synthetic quantityof collagen 31); however, there are peculiarities, to someextent, in the promotion of collagen synthesis. Contributionof this action could be a key role. Furthermore, there areother possibilities of complex actions such as cell activationthat niacinamide possesses. We await further ongoing
examinations.

• Mechanism of wrinkle formation

The major purpose of the present study is the preventionof wrinkle formation. We start by reviewing the mechanismof wrinkle formation.
Skin wrinkles start to appear on the face around 30
years of age. The major causative factor is recognized tobe quantitative and qualitative alternations in proteins,
elastic fibers, and collagen, which are produced by dermal
fibroblasts 32 -34). Another causative factor for shallow facial
wrinkles is structural alternations in epidermis due toepidermal dryness. Faces and necks, which are constantly
exposed to the sunlight, have deep wrinkles. Farmers
have chronic and severe sun exposure and thus, they havecutis rhomboidalis nuchae, which is deep triangle-shaped
wrinkles on the neck, around 50 years of age. The skinsurface is yellowish, grooved, and stiff. Histopathologically,on the cutis rhomboidalis nuchae, antibody against CML
positive material is massively deposited in upper and middle
layers of derma 35). Briefly, a type of AGEs is accumulatedon the skin. This alternation does not appear on the face skinbefore 20 years of age, and older people who are not exposed
to ultraviolet radiation do not have cutis rhomboidalis nuchae.
Cutis rhomboidalis nuchae is age-related alternation on theskin with chronic exposure to ultraviolet light. The relation
to wrinkles is unknown.
Components of derma, which supports epiderma frombelow, are classified into two categories, cell constituent andinterstitial component forming fibrillar tissues (extracellularmatrix component). Interstitial components, principal
components, consist of collagen fibers comprising collagen,elastic fibers elastin comprising elastine, and substrates
fulfilling space between these. Fibroblasts, which produce
these, are sparsely located as collagen fibers among them 1, 36, 37).
Collagen is configured in triple helical structure ofthree peptide chains, and the amino acid composition ofthese peptide chains have a repeated structure; a repetitive
sequence consisting of three elements, which includes
glycine, represents (glycine-amino acid X-amino acid Y).
It is known that proline and hydroxyproline account for
approximately 21%, and alanine accounts for approximately
11% of amino acid X and amino acid Y 38 - 41).
Collagen, which accounts for most of the interstitial
component, is colloidal fibers, which are produced via achemical association with a certain ratio of Type I collagen(80 – 85 %) and Type III collagen (10 –15 %); a chemical
association means an aggregation of atoms or molecules
into larger units held together by relatively weak forces andbehaves as if it is one molecule or one ion 42). Type I collagen
exists in the skin and bones in a large quantity and functions
to enhance strength and resilience and to support structuresof tissues. Type III collagen consists of thin fibers andhas functions for resilience and flexibility. Both collagens
maintain supple skins 1, 36, 43). Furthermore, fibroblasts, cellularcomponents, which are dispersed in the dermis, are cells
where collagen fibers, elastic fibers, and mucopolysaccharide
are produced. Fibroblasts are activated, when necessary, toform these compounds smoothly.
Impacts of ultraviolet exposure have been gradually
elucidated on wrinkle-formation-related collagen fibers,
protein in basal membranes of dermoepidermal junction.
Ultraviolet B stimulates epidermal keratinocytes, andproduces and releases inflammatory cytokines IL-1α, IL-6
and TNFα. These cytokines stimulate epidermal fibroblasts
and keratinocytes in an autocrine manner, and increase levels
of mRNA and proteins such as enzyme MMP-1, MMP3, and MMP-9, which cleave collagen and elastic fibers,
accelerating activities. It is considered that the cleavagesof fibers progress, leading to wrinkles 44). At an animal
experiment level, it is suggested that by the inhibition ofelastase activities, which cleave elastic fibers, ultravioletinduced wrinkles are remarkably suppressed. Wrinkle
formation is mainly induced by quantitative and qualitative
alternation in elastic fibers, as is suggested 34). However, there
is a strong opinion that the decrease of collagen is a cause of
wrinkle formation, as the expression of MMP-1 is apparent
to cleave collagen fibers.
It is considered that Ultraviolet A directly acts ondermal fibroblasts, and induces the increased levels ofmRNA and protein of enzyme MMP-1 and others, whichcleave fiber proteins. Consequently, this results in wrinkle
formation 45). Involvements of active enzymes are a trigger of
the acceleration of mRNA of MMMP-1 by Ultraviolet B and
A. Recently, it is clarified that infrared light, like ultraviolet
light, is involved, via active oxygen, in the acceleration ofactivities of MMP-1 46). Mitogen-activated protein kinase(MAPK) is involved in its intracellular activation pathway 47).
In proportion to frequency of exposure to ultraviolet
light, impacts on interstitial components become stronger.
Aging phenomenon of the promotion of wrinkle formationand pigmentation gradually progress 48). It is reported thatthe synthetic ability of collagen decreases due to ultraviolent
exposure 49). A cause of photoaging is an unbalance of
production and degradation of extracellular matrix components
induced by long-term ultraviolet exposure 50). From thesefindings, it is significant for the suppression of ultravioletinduced photoaging to promote the synthesis of collagens,whose synthetic quantity is decreased by ultraviolet exposure.
Antioxidant agents is recognized to be effective on wrinkle
formation.
In some fibroblasts of wound healing areas, p75
neurotrophin receptors (p75NTR), which are a marker ofmesenchymal stem cells, are expressed. In a cellular culture
system, fibroblasts, which are attached to collagen fibers,
stop growth even under the existence of growth factorsof proteins, and emulate the resting state of fibroblasts ofnormal tissues. Fibroblasts of wound healing areas proliferate_ 36 _Glycative Stress Research

and are surrounded by collagen fibers. This demonstrates
that in fibroblasts attached to collagen fibers of woundhealing areas, new growth initiation factors exist, and prolylhydroxyproline (Pro-Hyp), which is a collagen-derived
peptide, is produced. Pro-Hyp induces the proliferationof p75NTR positive fibroblasts, which are cultivated in acollagen gel culture, but does not induce the proliferation of
p75NTR negative fibroblasts. Pro-Hyp is a growth initiation
factor with a low molecular weight for a specific fibroblast,and is involved in the wound healing process 51).
Prolyl-hydroxyproline (Pro-Hyp) and hydroxyprolylGlycine (Hyp-Gly) appear in human blood by the ingestion ofcollagen hydrolysate, promote the proliferation of fibroblasts
attached to collagen gel, and promote the formation ofcollagen. Therefore, Pro-Hyp and Hyp-Gly are related tothe effectiveness of collagen hydrolysate ingestion such as
improvements of symptoms of the skin and articulations 52).
Pro-Hyp is supplied to tissues via oral administration ofgelatin and/or collagen hydrolysate. Therefore, throughsupply of gelatin and/or collagen hydrolysate, the therapeutic
effect on chronic wounds can be expected. In animal
experiments and human clinical trials, ingestion of gelatin
and/or collagen hydrolysate promote therapeutic effectsof treatments for decubitus of animals and humans, andimprove protracted wound healing of animals with diabetes.

• Prevention of wrinkle formation

The most significant causes of wrinkle are UV-exposureinduced photoaging and oxidative stress 1). Thus, antioxidant
materials are effective to the prevention of wrinkle formation.
Compounding ingredients of the test product such as
Ceratonia siliqua L. fruit extract 53), Polygonum tinctorium
leaf/stem extract 54), flower extract of Roman chamomile(Chamaemelum nobile, former Anthemis nobilis) 55),
Geranium robertianum L. extract 56, 57), and ubiquinone haveantioxidant effects.
Niacinamide ( it is also called nicotinamide) and nicotinicacid (it is commonly called niacin) are converted by eachother, and thus, they have the same effects as a vitamin. These
two materials are collectively called Vitamin B3. Niacinamide
is a structural component of an important coenzyme
related to the delivery and acceptance of hydrogen 58) Two
codehydrogenases, nicotinamide adenine dinucleotide (NAD)and nicotinamide adenine dinucleotide phosphate (NADP),
play a central and important role. External application of
niacinamide has effects to stabilize the epidermal barrier
functions including the decrease of transepidermal water loss
and the improvement in water content of stratum corneum.
Niacinamide induces an increase in synthesis of proteinssuch as keratin, the promotion of ceramide synthesis, thepromotion of keratinocytes differentiation, and the increaseof cellular NADP level. For aged skin, a topical applicationof niacinamide improves surface structures, and smooths
wrinkles 58).
Rutin is a quercetin glycoside and a type of bioflavonoid,
which is known for an antioxidative effect. A double-blind
clinical trial, where cream containing rutin was applied onhuman skin for 4 weeks with 40 subjects between 30 to 50
years of age was conducted to examine effects of rutin in thehuman body. Experimental data were analyzed after 2 weeks
and 4 weeks, with examinations of dermal density, skinelasticity, length and area of crow’s feet wrinkles, and thenumber of periorbital wrinkles of a group with the applicationof the control cream and a group with the application of
cream containing rutin. Results of assessments confirmedthat rutin enhanced skin elasticity and decreased length, area,
and the number of wrinkles. Effectiveness of the inhibitionof wrinkle formation was confirmed 59). It confirms that rutinincreases the expression of mRNA of collagen type I and
α1 in human skin fibroblasts and decreases the expressionof mRNA of matrix metallopeptidase 1. Furthermore, it wasverified that scavenging activities on active enzyme of rutin
were stimulated in a dose-dependent manner. This finding
confirmed that protective effects of rutin were exerted under
the conditions of oxidative stress 59).
Hydroxyproline is involved in wrinkle formation. An
experimental study on mice models with UVB-induced
photoaging has suggested that UV radiation induces theincreased activities of catalase and in particular, superoxide
dismutase of the skin, and the decreased malondialdehyde
content, the formation of matrix metalloproteinases(MMP-1 and MMP-3), and the decrease of hydroxyproline 60).
Histological evaluations confirm the infiltration of
inflammatory cells, the degradation of collagen, and theabnormality of elastin. These findings are related to wrinkle
formation. It is suggested that the decrease of hydroxyproline
is involved in wrinkle formation.
Any research regarding wrinkle improvement effects ofsole administration of hydroxyproline has not been reported
yet. However, there are some reports regarding a peptide
containing Hyp. With the purpose of verification of effective
treatments of collagen peptide supply, a double-blind
randomized placebo-controlled trial was conducted to assess
effects on moisture, elasticity, and wrinkle improvement of
human skin. Sixty-four subjects were randomly assigned
to undergo the ingestion of 1,000 mg, once a day, ofeither placebo or low-molecular-weight collagen peptides(LMWCP) with tripeptide (Gly -X-Y ) (content rate: 15 or
higher percent), which contains Gly-Pro-Hyp (3%) for 12
weeks. Parameters of skin moisture, wrinkles, and elasticity
were assessed at the base line, after 6 weeks, and 12 weeks.
Compared with the placebo group, in the LMWCP group,
hydration values of the skin were significantly high after
6 weeks and 12 weeks, and visual assessment scores andthree parameters of wrinkles were significantly improved.
Regarding skin elasticity in the LMWCP group; one item ofthree significantly improved from the base line to 12 weeks,
and two items of three significantly improved after 12 weeks.
These findings suggested possibilities of LMWCP usage asa health functional food material for moisture, elasticity, andwrinkles of the skin 61).
The present study infers that these compounds, whichwere contained in the test product, contributed to theimprovement of wrinkle formation, creating synergy.

• Involvement of glycative stress in skin aging

Reducing sugar such as glucose forms aldehyde groups
and ketone groups in alkaline solution. β-oxidation andhydrogen peroxide also form aldehyde groups and ketone_ 37 _

Improvement of Periorbital Wrinkles by Skin Caregroups. Glycative stress is a state of excessive formationsof aldehyde groups and ketone groups. Aldehydes arecharacterized by having a carbonyl group. Electrons ofcarbonyl groups shift towards oxygen atoms with a highelectronegativity, which is polarization. Carbon atoms
readily undergo nucleophilic attacks. These reactive groups
uncontrollably react to a biological material, proteins,which induces post-modifications (protein denaturation),
accumulations of waste materials, degradations of functional
protein, functional disorders of TCA cycle, activations ofintercellular signals, and tissue disorders. This is a major
cause for aging-related degenerative change 62). Maillard
reaction, in a limited definition, is a non-enzymatic andirreversible reaction between reducing sugars and proteins.
The process from the formation of Schiff bases, via Amadori
rearrangements, through diverse intermediates, to theformation of AGEs, is Maillard reaction.
Glycative stress is a significant risk factor 62, 63). Glycative
stress induces the denaturation of collagen and elastin,which comprise the skin, and nonelastic or sagging skins arecaused. The accumulation of AGEs causes yellowish skintone . Glycation of keratin and filaggrin (natural moisturizing
ingredients) increases water evaporation from the skin, anddecreases moisturizing function. The decrease of filaggrin
triggers the destruction of skin barrier function, and raises
the onset risk of atopic dermatitis 64). Furthermore, glycative
stress accelerates the melanin formation in melanocyte 65).

• Improvement of skin viscoelasticity,
Secondary endpoint

Elasticity of the skin is reduced along with aging 2).
Dermal collagen type I is configured in a triple helix
structure and plays a role with elastin fibers to maintain skin

A previous clinical trial regarding skin viscoelasticity
with R2 index is introduced. The test, which verified
improvement effectiveness of periorbital wrinkles and skinquality with usage of a serum containing multiple types ofbeauty ingredients, reported improvement effects on not only
periorbital wrinkles but also skin elasticity 68). Assessmentof R5 by Cutometer (the elastic portion of the suction phaseversus the elastic portion of the relaxation phase) showed
an increasing ( improving) trend on the test product appliedside from before the application 0. 51 ± 0.07 to after 4 weeks
0. 56 ± 0.14 (p = 0.076). Assessment of R2 (the ratio of
the skin resistance to the suction phase and ability to return
during the relaxation step) showed on the applied side
significantly improved from before 0.62 ± 0.07 to after 4
weeks 0.70 ± 0.09 ( p = 0.002). Assessment of R7 (the ratioof elastic recovery to the total deformation) on the appliedside significantly improved from before 0.31 ± 0.04 to after
4 weeks after 0.38 ± 0.14 ( p = 0.018).

b)
0.70

0.55

0.65

0.50

Skin elasticity (R7)

Skin elasticity (R2)a)

elasticity. The formation of extracellular matrix component
such as fibronectin from fibroblasts decreases due toaging. Formations of collagen and elastin also decrease.
Furthermore, via glycative stress, collagen fibers 66) areglycated to AGEs and undergo crosslinking. Consequently,
flexibility of fibers reduces, which is a significant causeof decreased skin resilience. Lysine and arginine residues,
which comprise collagen protein, readily undergo glycation
reaction, and form crosslinking via AGE glycation. Thus,
collagen loses mobility 66). Aging-related alternation in humanskin elasticity is shown in Fig. 5 20). In patients with diabetes,
who have strong glycative stress, elasticity index curvesof R2 and R7 shift downward in comparison with healthy
persons. Skin AGE fluorescence volume measured by an
AGE Reader has a positive correlation with skin stiffness 67).

0.60
0.55
0.50

Control (n = 77)

0.45
0.40
0.35
0.30

0.45
0.40

T2DM (n = 76)

35

40

45

50

55

60

65

70

0.25

75

Age (years)

35

40

45

50

55

60

65

70

75

Age (years)

Fig. 5. Transitional change by age of skin elasticity and impacts of glycative stress.
a) R2, b) R7. The mean value ± SEM, n = 77, healthy persons; n = 76, patients with type 2 diabetes (T2DM). Measurement of
the inside of an upper arm using Cutometer (Courage & Khazaka). R2, biological elasticity (Ua1/Uf1); R7, elastic recovery (Ur1/
Uf1); SEM, standard error mean.

_ 38 _Glycative Stress Research

The present trial showed no significant differences in
R5 and R7 examinations. R2 significantly decreased. No
improvement effects on skin elasticity of this test productwere confirmed.
Research reports have been published regarding effects
of skin elasticity improvements by oral administration of
mixed herb 69), mangosteen extracts 70), mangosteen pericarp
extract-containing black vinegar drink 71), and trapa japonica
extract 72).
Among components of the test cosmetic product, it wasconsidered that Kamitsure (Anthemis nobilis) flower extract,
glucosyl hesperidin, and glucosylrutin contain inhibitory
effects of the formation of AGEs. However, it is consideredthat these did not reach to improvements of skin elasticity.

Reason that no differences were observed between
the applied side and non-applied side

As the findings of the present study are described,
from the perspectives of characteristics of compounding
ingredients of the test product, effects of the ameliorationsof wrinkle formation and the improvements of skin elasticity
can be explained. However, the point is that no differenceswere observed between the applied side and non-applied sideof the face.
Manner of process of aging are individually different
73 -75). There are diverse skin types: a person with dry skin,a person who is concerned about blemishes, wrinkles andyellowish tone, and a person with reduced elasticity. Visualparameters for assessment on skin have been developed as
quantitative index; skin hydration measurement 63, 76) for skinage of moisture, image analysis 76) for skin age of stain andwrinkle, color-difference meter and skin AGE fluorescencemeasurement 77) for skin age of yellowing, and elasticity test
20, 63, 78)
for soft skin age (supple).
Risk factors are common in the skin and the body.
When an antioxidant supplement is ingested, not only theskin but also the body are affected. This is the same as antiglycation supplements.
The test product used in the present study was a topicalapplication on the skin. This product adopts emulsifying
agents and liposomal components as well as naturalingredients. High permeability is expected. A possibility wassuggested that this could result in influence appearances on
not only the applied side but also the non-applied side. We
consider that this finding would be verified by examining
blood concentration changes of effective components after
an application of the test product on the skin.

Conclusions
We conducted a trial to verify effects of the test productapplication on the skin with a target of women who wereconcerned with periorbital wrinkles. Due to collective
actions of diverse functional components, improvement
effects of periorbital wrinkles were confirmed by two typesof assessment methods (medical examination of visualassessment in wrinkle grade and visual assessment in wrinkle
grade using photographic images). However, improvements
were observed on the non-applied side of the face and there
was no statistically significant difference between the appliedside and the non-applied side. During the trial, no adverse
event was reported and the safety of the trial was confirmed.
The test product contained diverse types of functional
components. The test product is designed to enhance skinpermeability. Therefore, after the percutaneous absorption ofeffective components, it is possible that the components could
move to the other side of the face. However, the examinationof the present study cannot confirm that this occurs. To verify
this inference, further examinations are required to confirm
that blood concentrations of effective components would
increase by a percutaneous absorption after an application on
the skin of the test product.

Conflict of Interest
The present study was supported by Nabocul Cosmetics
Co., Ltd.

SafetyAs the results of safety assessment, no adverse reaction
effects induced by the test products were reported. Regarding
the safety of the test product, we judged that there was noproblem.

_ 39 _

Improvement of Periorbital Wrinkles by Skin Care

References
1) Ichihashi M, Ando H, Yoshida M, et al. Photoaging of theskin. Anti-Aging Med. 2009; 6: 46-59
2) Ichihashi M, Yagi M, Nomoto K, et al. Glycation stressand photo-aging in skin. Ant-Aging Med. 2011; 8: 23-29.
3) Japanese Cosmetic Science Society. Wrinkle grade
standard photo of periorbital wrinkles. Guidelines forcosmetic function evaluation methods. (in Japanese)
http://www.jcss.jp/journal/contents_guideline5.pdf
4) Tsim K, Yashiro T, Dong T, et al. New polyphenol
compound. Patent JP6976014B1, 2021.
5) Zulim Botega D, Bastida S, Marmesat S, et al. Carob
fruit polyphenols reduce tocopherol loss, triacylglycerol
polymerization and oxidation in heated sunflower oil.
Journal of the American Oil Chemists’ Society. 2009; 86:
419-425.
6) Lall N, Kishore N, Momtaz S, et al. Extract from
Ceratonia siliqua exhibits depigmentation properties.
Phytother Res. 2015; 29: 1729-1736.
7) Rtibi K, Jabri MA, Selmi S, et al. Preventive effect ofcarob (Ceratonia siliqua L.) in dextran sulfate sodiuminduced ulcerative colitis in rat. RSC Advances. 2016; 6:19992-20000.
8) Rtibi K, Selmi S, Grami D, et al. Chemical constituentsand pharmacological actions of carob pods and leaves(Ceratonia siliqua L.) on the gastrointestinal tract: A review.
Biomed Pharmacother. 2017; 93: 522-528.
9) Nabocul Cosmetics Co., Ltd. Developed a new brand"Asakado" and announced "OLANDU", the latest
technology chemical compound in the aging care field.
Press Distribution, Jan 19, 2022. (in Japanese)
https://kyodonewsprwire.jp/prwfile/release/M106981/202
201196276/_prw_PR1fl_LS85a7Fd.pdf
10) Lam ECS, Li R, Rodrigues MR, et al. Enhanced retinoid
response by a combination of the vitamin A ester retinyl
propionate with niacinamide and a flavonoid containing
Ceratonia siliqua extract in retinoid responsive in vitro
models. Int J Cosmet Sci. 2021; 43: 102-106.
11) Han NR, Kang SW, Moon PD, et al. Genuine traditional
Korean medicine, Naju Jjok (Chung-Dae, Polygonum
tinctorium) improves 2,4-dinitrofluorobenzene-induced
atopic dermatitis-like lesional skin. Phytomedicine. 2014;
21: 453- 460.
12) Motoki T, Takami Y, Yagi Y, et al. Inhibition of hepatocyte
growth factor induction in human dermal fibroblasts bytryptanthrin. Biol Pharm Bull. 2005; 28: 260-266.
13) Cella Cosmetics. Persicaria tinctoria leaf/stem extract.
“Ingredient Dictionary,” Cella Cosmetics. (in Japanese)
https://cella.jp/ingredient/detail.php?p_id=1370 (Accessedat Feb 7, 2023)
14) Takabe W, Yamaguchi T, Hayashi H, et al. Identificationof antiglycative compounds in Japanese red water pepper(red leaf variant of the Persicaria hydropiper Sprout).
Molecules. 2018; 23: 2319.
15) Johnson LR, et. German chamomile. “Guide to Medicinal
Herbs”, Nikkei National Geographic Inc, Tokyo, pp145147, 2014. (in Japanese)16) Miraj S, Alesaeidi S. A systematic review study oftherapeutic effects of Matricaria recuitta chamomile(chamomile). Electron Physician. 2016; 8: 3024-3031.

17) Nagashima T. German chamomile. “ Herbal Tea: The

Healing of Science”, Flagrance Journal, Tokyo, pp42-43,
2010. (in Japanese)18) McKay DL, Blumberg JB. A review of the bioactivity andpotential health benefits of chamomile tea (Matricaria
recutita L.). Phytother Res. 2006; 20: 519-530.
19) Hayashi S. German chamomile. “The Encyclopedia ofMedical Herb”, Tokyodo Shuppan Co. Ltd, Tokyo, pp7475, 2016. (in Japanese)20) Kubo M, Yagi M, Kawai H, et al. Anti-glycation effects of
mixed-herb-extracts in diabetes and pre-diabetes. J Clin
Biochem Nutr. 2008; 43(Suppl. 1): 66-69.
21) Yonei Y, Miyazaki R, Takahashi Y, et al. Anti-glycation
effect of mixed herbal extract in individuals with prediabetes mellitus: A double-blind, placebo-controlled,parallel group study. Anti-Aging Med. 2010; 7: 26-35.
22) Kawai H, Shoshihara M, Kawakami H, et al. Anti-glycationand skin beautification properties from ingestion of
mixed herb extract: A placebo-controlled, double-lind,randomized, parallel-group study. Glycative Stress Res.
2016; 3: 236-245.
23) Li P, Wu G. Roles of dietary glycine, proline, andhydroxyproline in collagen synthesis and animal growth.
Amino Acids. 2018; 50: 29-38.
24) Gordon MK, Hahn RA. Collagens. Cell Tissue Res. 2010;339: 247-257.
25) Srivastava AK, Khare P, Nagar HK, et al. Hydroxyproline:
A potential biochemical marker and its role in thepathogenesis of different diseases. Curr Protein Pept Sci.
2016; 17: 596-602.
26) Cosmetic ingredients online. Basic information, purposeof formulation, and safety of hydroxyproline. Cosmetic
Ingredient Journal, the latest update at July 11, 2022. (inJapanese)https://cosmetic-ingredients.org/skin-conditioningmiscellaneous/1271/
27) Matsumoto Trading Co., Ltd. Ronacare VTA. “Databaseof Cosmetic Ingredients,” Sept 1, 2009. (in Japanese)
https://matsumoto-trd.com/product/pdf/concept/h02.pdf28) Matsumoto Trading Co.,Ltd. Ronacare ASCⅢ &Ronacare VTA. “Database of Cosmetic Ingredients,” Oct
1, 2009. (in Japanese)
https://matsumoto-trd.com/product/pdf/02/vo2/05.pdf
29) Tanno O. Improvement epidermal permeability barrier
by increasing de novo synthesis of ceramides. Fragrance
Journal. 1999; 27(10); 23-28. (in Japanese)30) Kawada A, Konishi N, Oiso N, et al. Evaluation of antiwrinkle effects of a novel cosmetic containing niacinamide.
J Dermatol. 2008; 35: 637-642.
31) Matts PJ, Oblong JE, Bissett DL. A review of the rangeof effects of niacinamide in human skin. International
Federation of Societies of Cosmetic Chemists (IFCC)
Magazine. 2002; 5: 285-289.
32) Fisher GJ, Datta SC, Talwar HS, et al. Molecular basisof sun-induced premature skin ageing and retinoid
antagonism. Nature. 1996; 379(6563): 335-339.
33) Chung JH, Seo JY, Choi HR, et al. Modulation of skincollagen metabolism in aged and photoaged human skinin vivo. J Invest Dermatol. 2001; 117: 1218-1224.

_ 40 _Glycative Stress Research34) Hachiya A, Kobayashi A, Ohuchi A, et al. The paracrinerole of stem cell factor/c-kit signaling in the activation ofhuman melanocytes in ultraviolet-B-induced pigmentation.
J Invest Dermatol. 2001; 116: 578-586.
35) Mizutari K, Ono T, Ikeda K, et al. Photo-enhanced
modification of human skin elastin in actinic elastosis
by N ε-(carboxymethyl)lysine, one of the glycoxidation
products of the Maillard reaction. J Invest Dermatol.
1997; 108: 797-802.
36) Asada Y. “Mechanism and functions of the dermis.” The
Encyclopedia of Cosmetic Dermatology. Chuo Shoin
Co.,Ltd. Tokyo, pp28-33, 2022. (in Japanese)37) Shimizu H. Dermis.” Textbook of Modern Dermatology.”
Nakayama Shoten Co., Ltd. Tokyo, pp13-20, 2018. (inJapanese)38) Hattori S, Kuwaba K. Cosmetics and Collagen.
“Manufacturing, Application and Development of
Collagens 2. CMC Publishing Co.,Ltd. Tokyo, pp143-166,
2020. (in Japanese)39) Oki M, et. “collagen” Encyclopedic dictionary of chemistry,
Tokyo Kagaku Dojin, 1989; 814. (in Japanese)40) Okuyama K. “Molecular structure of collagen”
Manufacturing, Application and Development of Collagens
II,” CMC Publishing Co.,Ltd. pp1-12, 2020. (in Japanese)41) Seiwa Kasei Co Ltd. Hydrolyzed collagen derived fromfish scale. Patent JP2007326869A. Dec 20, 2007.
42) Keene DR, Sakai LY, Bächinger HP, et al. Type Ⅲ collagencan be present on banded collagen fibrils regardless offibril diameter. J Cell Biol. 1987; 105: 2393-2402.
43) Murakami Y, Adachi H, Sakaida T, et al. The Reduction
mechanism of the type III/I collagen ratio with aging: Agerelated change in meprin, a type III collagen propeptide
cleavage enzyme. Journal the Society of Cosmetic
Chemists of Japan. 2013; 47: 278-284. (in Japanese)44) Woodley DT, Kalebec T, Banes AJ, et al. Adult humankeratinocytes migrationg over nonviable dermal collagenproduce collagenolytic enzymes that degrade type I andtype IV collagen. J Invest Dermatol. 1986; 86: 418-423.
45) Herrmann G, Walscheke M, Lange TS, et al. UVA
irradiation stimulates the synthesis of various matrixmetalloproteinases (MMPs) in cultured human fibroblasts.
Exp Dermatol. 1993; 2: 92-97.
46) Schieke SM, Schroeder P, Krutmann J. Cutaneous
effects of infrared radiation: From clinical observations
to molecular response mechanisms. Photodermatol
Photoimmunol Photomed. 2003; 19: 228-234.
47) Calles C, Schneider M, Macaluso F, et al. Infrared A
radiation influences the skin fibrobrast transcriptome:
Mechanisms and consequences. J Invest Dermatol. 2010;
130: 1524-1536.
48) Asada Y. Acute and chronic skin lesions. “TheEncyclopedia of Cosmetic Dermatology.” Chuo ShoinCo., Ltd., Tokyo, pp195, 2002. (in Japanese)49) Tanaka H, Okada T, Konishi H, et al. The effect ofreactive oxygen species on the biosynthesis of collagenand glycosaminoglycans in cultured human dermal
fibroblasts. Arch Dermatol Res. 1993; 285: 352-355.
50) Obayashi K, Kyotani D, Masuda K, et al. Inhibitory effectsof the plant extracts on matrix proteinases. Journal theSociety of Cosmetic Chemists of Japan. 1998; 32: 272279. (in Japanese)

51) Sato K, Asai TT, Jimi S. Collagen-derived di-peptide,

prolylhydroxyproline (Pro-Hyp): A new low molecularweight growth-initiating factor for specific fibroblasts
associated with wound healing. Front Cell Dev Biol.
2020; 8: 548975.
52) Asai TT, Oikawa F, Yoshikawa K, et al. Food-derived
collagen peptides, prolyl-hydroxyproline (Pro-Hyp), andhydroxyprolyl-glycine (Hyp-Gly) enhance growth ofprimary cultured mouse skin fibroblast using fetal bovine
serum free from hydroxyprolyl peptide. Int J Mol Sci.
2019; 21: 229.
53) Ben Ayache S, Behija Saafi E, Emhemmed F, et al.
Biological activities of aqueous extracts from carob plant(Ceratonia siliqua L.) by antioxidant, analgesic andproapoptotic properties evaluation. Molecules. 2020; 25:3120.
54) Tokuyama-Nakai S, Kimura H, Hirabayashi Y, et al.
Constituents of flavonol O-glycosides and antioxidant
activities of extracts from seeds, sprouts, and aerial partsof Polygonum tinctorium Lour. Heliyon. 2019; 5: e01317.
55) Al-Dabbagh B, Elhaty IA, Elhaw M, et al. Antioxidant andanticancer activities of chamomile (Matricaria recutita
L.). BMC Res Notes. 2019; 12: 3.
56) Ben Jemia M, Aidi Wannes W, Ouchikh O, et al.
Antioxidant activity of Tunisian Geranium robertianum L.
(Geraniaceae). Nat Prod Res. 2013; 27: 2076-2083.
57) Catarino MD, Silva AMS, Cruz MT, et al. Antioxidant andanti-inflammatory activities of Geranium robertianum L.
decoctions. Food Funct. 2017; 8: 3355-3365.
58) Gehring W. Nicotinic acid/niacinamide and the skin. J
Cosmet Dermatol. 2004; 3: 88-93.
59) Choi SJ, Lee SN, Kim K, et al. Biological effects of rutinon skin aging. Int J Mol Med. 2016; 38: 357-363.
60) Zhou Y, He L, Zhang N, et al. Photoprotective effect ofArtemisia sieversiana Ehrhart essential oil against UVBinduced photoaging in mice. Photochem Photobiol. 2021
Nov 12.
61) Kim DU, Chung HC, Choi J, et al. Oral intake of lowmolecular-weight collagen peptide improves hydration,
elasticity, and wrinkling in human skin: A randomized,
double-blind, placebo-controlled study. Nutrients. 2018;
10: 826.
62) Nagai R, Mori T, Yamamoto Y, et al. Significance ofadvanced glycation end products in aging-related disease.
Anti-Aging Med. 2010; 7: 112-119.
63) Yonei Y, Uenaka S, Yagi M, et al. Effects on skin by
dewaxed brown rice: An open label test. Glycative StressRes. 2021; 8: 29-38.
64) Furue M, Chiba T, Tsuji G, et al. Atopic dermatitis:
immune deviation, barrier dysfunction, IgE autoreactivity
and new therapies. Allergol Int. 2017; 66: 398-403.
65) Abe Y, Takabe W, Yagi M, et al. Inhibition of AGEinduced melanogenesis in B16 melanoma cells by iridoidcontaining plants. Glycative Stress Res. 2017; 4: 67-70.
66) Cerami A, Vlassara H, Brownlee M. Glucose and aging.
Sci Am. 1987; 256: 90-96.
67) Ishizaki K, Yagi M, Morita Y, et al. Relationship between
glycative stress markers and skin stiffness. Glycative
Stress Res. 2020; 7: 204-210.
68) Kaneko T, Miyata A, Ishida N. Cosmetic essence makes
both effects which looks younger than the actual age andskin improvement. Medical Consultation & New Remedies.
2016; 53: 807-814. (in Japanese)_ 41 _

Improvement of Periorbital Wrinkles by Skin Care69) Yonei Y, Yagi M, Hibino S, et al. Herbal extracts

inhibit Maillard reaction, and reduce chronic diabetic
complications risk in streptozotocin-induced diabetic rats.
Anti-Aging Med. 2008; 5: 93-98.
70) Ohno R, Moroishi N, Sugawa H, et al. Mangosteen
pericarp extract inhibits the formation of pentosidine andameliorates skin elasticity. J Clin Biochem Nutr. 2015; 57:
27-32.
71) Takabe W, Yagi M, Ogura M, et al. Effect of mangosteen
pericarp extract-containing black vinegar drink on skinquality through anti-glycative actions. Glycative StressRes. 2017; 4: 158-171.
72) Takeshita S, Ishioka Y, Uemura T, et al. Reducing effectof the long term intake of water chestnut (Trapa bispinosaRoxb.) pericap extract on glycative stress in the placebocontrolled double blinded clinical trial and in vitro
inhibitory actions on low-density lipoprotein (LDL)
glycation. Glycative Stress Res. 2017; 4: 299-316.
73) Yonei Y, Mizuno Y. The human dock of tomorrow:
Annual health checkup for anti-aging. Ningen Dock. 2005;
19(6): 5-8.
74) Yonei Y, Takabe W. Aging assessment by anti-aging
medical checkup. Health Evaluation and Promotion.
2015; 42: 459-464.
75) Yonei Y, Takabe W, et al. What does the Anti-Aging
Medical Checkup show?: Data presentation. Health
Evaluation and Promotion. 2017; 44: 600-605.
76) Miyamoto K, Inoue Y, Hsueh K, et al. Characterizationof comprehensive appearances of skin ageing: An 11year longitudinal study on facial skin ageing in Japanese
females at Akita. J Dermatol Sci. 2011; 64: 229-236.
77) Nomoto K, Yagi M, Arita S, et al. A survey of fluorescencederived from advanced glycation end products in theskin of Japanese: Differences with age and measurement
location. Anti-Aging Med. 2012; 9: 119-124.
78) Yo K. The relationship between aging-related changes
in cheek appearance and skin properties. Journal of theJapanese Society for Cutaneous Health. 2012; 34: 108112. (in Japanese)_ 42 _