Glycative Stress Research
Online edition : ISSN 2188-3610
Print edition : ISSN 2188-3602
Received : June 24, 2016
Accepted : August 20, 2016
Published online : September 30, 2016

Original Article

Effect of supplement containing Silybum marianum extract, soy extract, collagen
peptide, bifidobacteria and apple extract on skin: A randomized placebo-controlled, double-blind, parallel group comparative clinical study.
Yuri Ishii 1), Yumika Okada 1), Sayuri Matsuoka 1), Manzhen Shen 1), Kei Yui 1),
Masayuki Yagi 2), Yoshikazu Yonei 2)1) FANCL Research Institute, FANCL Corporation, Yokohama, Kanagawa
2) Anti-Aging Medical Research Center/Glycative Stress Research Center, Graduate School of Life and Medical Sciences,
Doshisha University, Kyotanabe, Kyoto

Summary

Objective and Methods: Healthy Japanese women aged from 41 to 69 years old with saggy skin and winkles were thesubjects of a randomized, placebo-controlled, double-blind clinical study on improvements in skin condition after ingestinga test food for 12 weeks. The active food contained 462 mg Silybum marianum extract (138 mg silybin), 70 mg soy extract(10 mg saponin B and 25 mg isoflavone), 900 mg collagen peptide, 32 mg bifidobacteria (2 billion or more bifidobacteria
BB536), and 83 mg apple extract (65 mg apple polyphenol) in 6 tablets taken daily. The control food was a placebo formulation.
Fifty-six subjects (51.5 ± 8.0 years) were randomly divided into an active food group (n = 28) and a placebo food group(n = 28). Before and after ingesting the test food, we measured skin parameters (skin viscoelasticity, wrinkle replica analysis,color difference, stratum corneum moisture content and transepidermal water loss), oxidative stress markers (in blood andstratum corneum), glycative stress markers (skin and serum), and inflammatory markers (blood). As a subjective assessment
measure, we implemented an anti-aging quality of life (AAQoL) questionnaire and a feelings questionnaire (only after theuse of supplements).
Results: In the active food group, an improvement in wrinkles and suppression of the decline in skin viscoelasticity wereconfirmed. Regarding oxidative stress markers, the decrease in the level of the deglycation protein DJ-1 in the stratum corneum
was alleviated, but the results were not superior in the active food group for glycative stress markers or an inflammatory
marker. During the test period, there were no serious adverse events.
Conclusions: The study showed that the active food could support the maintenance of skin elasticity and improve winkles
in the skin of middle-aged women.

KEY WORDS: supplements, wrinkles, elasticity, silybin, apple polyphenol

Introduction

Physiological changes in the body occur with age ineveryone. Unlike the internal organs, photo-aging by UV
rays is known to have an impact on aging of the skin in
addition to physiological aging 1, 2). With chronic exposure to
sunlight and UV rays, reactive oxygen species are generated
in the skin and damage the DNA. At the same time, inhibitionof tyrosine-phosphatase oxidation 3) ultimately promotes theinduction of the transcription factor AP-1 and the transfer ofmatrix metalloproteinase (MMP) 4). Collagen production in
fibroblasts decreases, and the collagen and elastin that are thestructural components of the dermis become decomposed 5, 6).

As a result, collagen becomes denatured and loses its elasticity
through cross linking, and elastin forms an irregular elastin
fiber network; therefore, wrinkles form with the loss offunction and structure of the dermis that is gradually causedby chronic irradiation with UV rays. In order to protect the
body from damage by UV rays, the generation of melaninin pigment cells is promoted in skin that has been exposed
to UV light through the actions of melanocyte stimulating
hormone (α-MSH) and adrenocorticotropic hormone (ACTH).
These are secreted by epidermal keratinocytes 7), and melaninthat has not been metabolized remains as a discoloration. In
recent years, the usefulness of stratum corneum DJ-1 protein

Corresponding author: Yuri Ishii
Clinical Research Group, Health Science Research Center,
FANCL Research Institute, FANCL Corporation
12-13 Kamishinano, Totsuka-ku, Yokohama-shi, Kanagawa 244-0806, Japan
TEL: +81-45-820-3419, E-mail: yuishii@fancl.co.jp
Contact for Co-author: Okada Y, yumika1403@fancl.co.jp;
Matsuoka S, matsuoka@fancl.co.jp; Shen M, mashin@fancl.co.jp; Yui K, ke-yui@fancl.co.jp;
Yagi M, myagi@mail.doshisha.ac.jp; Yonei Y, yyonei@mail.doshisha.ac.jp_ 15 6 _Glycative Stress Research 2016; 3 (3): 156- 171(c) Society for Glycative Stress ResearchGlycative Stress Research

as an index of anti-oxidative defenses from UV rays has beenreported in a study on stratum corneum markers. There havebeen many studies on photo-aging in various fields 8). Under
these circumstances, a type of advanced glycation end product(AGE) named N ε- carboxymethyl-lysine (CML) is generated
in a non-enzymatic oxidation reaction between carbonylcompounds and sugars such as glucose, which modifies
proteins. This has been demonstrated in elastic fibers in solar
elastosis degeneration, and attention has been focused on
glycative stress as a factor associated with aging of the skin 9).
As such, aging of the skin can be said to occur because ofboth oxidation and glycation 10-12).
For women, aging of the skin not only changes theirappearance, but impacts on their mental state and lowers thequality of life (QOL). From this perspective, the developmentof an anti-aging technology for the skin has been an important
issue both medically and cosmetically. Presently, the techniquesthat are provided as medical treatments include dermatologicaltreatments (external preparations, peeling, laser, hyaluronic
acid injection), cosmetic surgery, medicines taken internally(such as hormone treatments), and regenerative medicines(injections of autologous cultured fibroblasts, isolagen).
These techniques have been developed and used in practicalsettings. On the other hand, foods that have anti-oxidantor anti-glycation effects and lead to improvements in skincondition may be used as common measures, in addition to
the prevention and improvements achieved through sunscreensand cosmetics. From the various food materials for whichsuch effects are expected, we focused on five: Silybum
marianum extract, soy extract, collagen peptide, bifidobacteria,
and apple extract. Silybum marianum is widely found insouthern Europe, northern Africa, and Asia, and its seeds
contain flavonolignans such as silybin. Anti-oxidant effects 13),anti-glycation effects 14), production of collagen in the dermis 15),
and retinoic acid-like activity 16, 17) have been indicated with
its use. Furthermore, a 16-week long placebo-controlled
double-blind parallel group comparative study showed thatthe intake of 462 mg Silybum marianum extract ( 138 mg
silybin) had a wrinkle-improvement effect 15). Soy extracts
containing components such as saponin and isoflavone havealso been reported to have anti-oxidant effects 18), inhibitory
effects on increases in blood glucose 19), a lowering effect onprotein carbonyl formation through proteasome activation,
and wrinkle-improvement effects in middle-aged women
after a 10 -week course of 55 mg saponin B 20-22). Soy
isoflavone not only has anti-glycation effects 23) and antioxidant effects 24, 25), but has also been reported to improve
the amount of collagen in the dermis and increase the amountof elastin in a test that targeted 30 perimenopausal women(a six-month course of 100 mg isoflavone daily) 26), andimproved wrinkles and skin elasticity in a test that targeted
women in their late 30s to early 40s (a 12 week course of 4 0
mg isoflavone daily) 27). Collagen peptides are derived fromhydrolyzed gelatin and include tripeptides with a molecularweight of about 300 Da. These have been reported to promotecollagen synthesis more effectively than non-degradable
collagen 20). Bifidobacteria refers to the genus Bifidobacteriumthat is found in the intestine and decomposes lactose andoligosaccharides to produce lactic and acetic acid, lowering
the pH in the intestine significantly and adjusting theenvironment of the intestine 28), improving bowel movements 29),
and relieving allergy symptoms such as hay fever 30). As forthe skin, the ingestion of drinks that contain two billion
bifidobacteria (BB536) has been reported to improve

subjective symptoms such as firmness and pore size 31). Apple
extract contains a large amount of polyphenols such asprocyanidin, chlorogenic acid, and phlorizin, and has beenshown to have anti-oxidant effects 32, 33), anti-glycation
effects 34 -36), and inhibitory actions on melanin production 37).
In addition, the suppression of wrinkle generation by UV
irradiation has been reported in an experiment that used
hairless mice. The mechanism was by the suppression of
singlet oxygen generation by UV irradiation, and the
suppression of a series of phenomena promoted by oxidative
stress (oxidative carbonyl formation, a decrease in proteasome
activity, transcription factor AP-1 activation, increased
production of MMP-1, and decreased production of TIMP1) 38). In a human experiment that used both 2.6 g collagen
peptide containing a large amount of tripeptide and 83 mg
apple extract, improvements in the skin condition were
expected, such as improved elasticity 38). As we have discussed
so far, there have been many reports on the usefulness ofthese raw food materials for improving the skin condition andtheir anti-oxidant and anti-glycation effects through in vitro
studies, animal experiments, or clinical studies; however,there has not yet been a report on the effectiveness of theiruse in combination. Thus, we developed a supplement inwhich these raw materials were combined, and evaluatedchanges in the skin condition along with changes in oxidativeand glycative stress markers by using middle-aged women as
subjects. In this manner, we examined the usefulness of the
supplement contained present foodstuffs in a randomized
placebo-controlled double-blind parallel group comparative
clinical study.

Methods

1. Subjects

Subjects were healthy Japanese women between 41 and69 years old who were concerned by sagging skin andwrinkles. However, we excluded those who met the following
exclusion criteria: 1) those who were allergic to soy, collagen,dairy products, and apples, 2) those who were taking
supplement(s) that contain Silybum marianum, soy saponin,
soy isoflavone, collagen, lactic acid bacteria, and/or apple
extract, 3) those who were being treated or seen by a physician
for liver disease or kidney disease, 4) those who were being
treated or seen by a physician for type II diabetes, 5) thosewith atopic dermatitis, 6) those with a serious medical
history, 7) those who were being treated for a disease, 8)
those who were participating in other clinical studies when
this study began, 9) those who had difficulties maintaining
their life style (diet, exercise, sleep, skin care), 10) those who
had difficulty making entries in a journal and completing adiet questionnaire, 11) those who worked at a company thatdevelops, manufactures, or sells health food or whose family
members work at such a company, and 12) those who weredeemed unfit as subjects by the investigating physician.
During the test period (from screening to the completionof the final test), subjects were required to adhere to thefollowing: 1) avoid excessive eating, extreme exercise, andlack of sleep, 2) fast from 10 pm the night before until theend of the test process (water was allowed), 3) abstain fromalcohol starting from the day before until the end of the test
process, 4) sleep at least six hours during the night before thetest day, 5) avoid smoking on the test day from the time the_ 157 _

Skin Improvement Effects of Anti-aging Supplement
subject wakes up until the test process is completed, 6) avoid
changes in life style (such as quitting or beginning exercise,starting or quitting drinking), 7) avoid excessive exposure
to the sun or traveling to places with strong UV rays, 8) do
not start taking new health foods (including vitamins andnutritional drinks), 9) do not change skin care products
and methods, 10) avoid the use of new bath products andbody care products, 11) if moisturizing cream is needed for
extremely dry skin, avoid its use on the measured area (faceand arms), and make sure to enter such use in the journal,
12) avoid activities that impact the quality of the skin suchas peeling, visiting beauty salons, scrubbing, and 13) avoid
shaving the face or treating excess hair for two days before
the test day.

2. Test food

The test food containing 462 mg/day Silybum marianum
extract (138 mg/day silybin), 70 mg/day soy extract (10 mg/day
saponin B and 25 mg/day isoflavone), 900 mg/day collagen
peptide, 32 mg/day bifidobacteria ( > 2 billion Bifidobacterium
BB536), and 83 mg/day apple extract (65 mg/day polyphenol)
was used as active food. The control food that appeared thesame but did not include any of the active ingredients(placebo). Each dose was six tablets once daily, taken withwater following dinner. Table 1 shows the formulation of thetest food.

3. Procedure
3.1 Screening
To screen those who provided informed consent, we
examined the background information (age, smoking, drinking,exercise, diet, use of health food products, bowel movements,
sleep, medications, allergies, hay fever, menstruation,nightshift work, medical history, skin problems, andexposure to UV rays), and measured AGE deposition in theskin (hereafter, the AF value). The principal investigator
examined the content of the background survey, and selected
those who met the selection criteria and not the exclusion
criteria. From the selected group, the 56 subjects with thehighest AF values were selected as the subjects of this study.
3.2 The test
The test design was a randomized placebo-controlled
double-blind parallel group comparative clinical study. The
person responsible for group assignment randomly divided
the 56 subjects into two groups: a group that ingested theactive food (active food group) and a group that ingested theplacebo (placebo group). The assignment table was sealed
and stored until unlocked. The period of ingesting the test
food was 12 weeks, and skin measurements, examination ofoxidative stress, glycative stress, and inflammatory markers,
blood tests (safety), basic measurements (body composition
and blood pressure measurements), an interview with a

Table 1. Composition of test foodcontent(active ingredient)quantity (mg/day)ActivePlaceboSilybum marianum extract(silibin)

462
（138）

0

Soy extract(sapnin B, isoflavon)

70（10, 25）

0Collagen peptide

9000Bifidobacteria(Bifidobacterium longum BB536)

32（ > 2 billion cell)0Apple extract(apple polyphenol)

83
(65)

0

excipient(cellulose, trehalose, erythritol,
scallop shell powder, calcium stearate)

853

2,400

total

2,400

2,400

_ 158 _Glycative Stress Researchphysician, and anti-aging QOL questionnaire were performed
before taking the supplement (0w) and at the end of the study(12w). In addition, subjects kept a journal every day during
the test period, and completed a feelings questionnaire on
the skin and physical condition at the end of the period during
which they took the supplement.

Parameter(1) Skin conditions
Skin measurements included skin viscoelasticity, wrinkle
replica analysis, stratum corneum moisture content,transepidermal water loss and color difference. Measurementsof the face were made after washing the face and habituationfor 20 minutes in a room with constant temperature andhumidity (21 ± 1 °C and 50% ± 5%, respectively). Skinviscoelasticity, stratum cor neum moisture content,transepidermal water loss, and color difference were measured
five times around the right cheek bone in a supine position,and the mean of three measurements was used after excluding
the highest and lowest values.
Skin viscoelasticity was measured with a cutometer(Courage+khazaka CT580) using a single-suction method.
The evaluated parameters were R2 and R7. R2 indicatesthe recovery rate of the skin length after elongation andconstriction (Ua1/Uf1) while R7 indicates the ratio of elastic
part during constriction (Ur1/Uf1). Both R2 and R7 indicate
higher elasticity as the value approaches 1.00.
The stratum corneum moisture content was measured
with a corneometer (Courage+khazaka CM825), whiletransepidermal water loss was measured with a Tewameter(Courage+khazaka TM300).
Color differences were measured with aspectrocolorimeter (Konica Minolta CM-600d), and theevaluation parameters were the Melanin Index, Hb Index,
HbSO2 Index, and L*, a*, b* values.
For the wrinkle replica analysis, a replica silicon casting
solution was applied in a mold with a silicon coagulating
liquid while keeping the eyes lightly closed in the supine
position, and a replica was prepared of a 10 mm × 10 mm or
wider area approximately 5 mm away from the corner of theright eye. The replica was analyzed using three-dimensional
image analysis (GFMesstechnik GmbH PRIMOS lite 18 ×
13 mm), and the wrinkle area rate, average wrinkle depth(total wrinkle), average wrinkle depth (maximum wrinkle),
maximum wrinkle depth, and total wrinkle volume were
calculated.
(2) Marker
The concentrations of the oxidative stress markers, lipidhydroperoxide (LPO) in blood and DJ-1 in the stratum corneum
were measured. The stratum corneum was sampled bystripping cells from the right cheekbone with tape. The DJ-1
concentration was measured using an enzyme-linked
immunosorbent assay (ELISA) according to a previous report 8.
The glycative stress markers, AF values, 3-deoxyglucosone(3-DG), CML, and glyceraldehyde-derived AGEs (Toxic
AGEs : TAGE) were measured in the blood.
For AF values, we used an AGE Reader (DiagnOptics
B.V. AGE Reader SU) to measure the inside of the rightupper arm three times (10 cm above the olecranon toward
the shoulder while seated), and their mean value was used.

In addition, we measured Tumor Necrosis Factor α(TNF-α) as an inflammatory marker. Blood sampling was
performed by a qualified person based on standard blood
sampling guidelines, and 24 mL was sampled each time.
The 3-DG, CML, and TNF-α levels were measured by LSI
Medience Corporation, TAGE was measured by Trans Genic
Inc., LPO was measured by Japan Institute for the Control of
Aging, NIKKEN SEIL Co., Ltd, and DJ-1 was measured by
FANCL Corporation.
(3) Anti-aging QOL questionnaire (subjective symptoms)
The survey of subjective symptoms evaluated physicalsymptoms (30 items) and mental symptoms (21 items) using
a five-point scale (score 1: none, score 2: few, score 3: alittle, score 4: some, score 5: quite a few) with an Anti-AgingQOL Common Questionnaire (AAQoL). Subjects wereasked to note sleeping habits, eating habits, exercise habits,
and time spent at visual display terminals.
(4) Feelings questionnaire
Changes in skin and physical condition after taking thetest food were self-evaluated on a four-point scale (2 points:
improved, 1 point: somewhat improved, 0 points: no change,-1 point: deteriorated). Evaluation parameters were as follows:
dry skin (moisture), sag of circumoral skin (nasolabial fold),sag under the eyes, texture, pores, wrinkles and fine lines,
dullness of skin (brightness of the skin), spots, make-up
condition, shine and strength of the hair, tendency to tire
(frequency of fatigue), heaviness in the head, feelings offatigue (intensity of fatigue), awakening (how fresh one feels
when waking up), sleep quality, and defecation (constipation
or loose stools).
(5) Blood tests (safety)
As safety parameter, glucose, glycated hemoglobin(HbA1c), glycoalbumin, triglycerides, total cholesterol, highdensity lipoprotein cholesterol (HDL-C), low-density
lipoprotein cholesterol (LDL-C), gamma-glutamyl transferase(γ -GTP), aspartate transaminase (AST), alanine transaminase(ALT), total bilirubin, total protein, albumin (ALB), albumin/
globulin (A/G) ratio, alkaline phosphatase, lactate
dehydrogenase (LDH), creatine phosphokinase (CPK),
creatinine, uric acid, and blood urea nitrogen were measured.
Measurements were outsourced to LSI Medience Corporation.
(6) Body composition and blood pressure
For body composition, we measured height, weight,
BMI, body fat percentage, and basal metabolic rate. Items
other than height were measured using a body composition
analyzer (WELL-SCAN500, Canon Lifecare Solutions,Inc.). Blood pressure was measured with an automatedsphygmomanometer (UDEX-I, Canon System & SupportInc.) while resting (three or more minutes in a sitting
position, after taking three deep breaths). Measurements
were taken twice on the right arm, and the mean value wasused.
(7) Adverse event
During the test period, subjects were asked to record
their intake of the test food, the amount of daily activity, thelength of sleep, stress conditions, bowel movements, use of
medications or supplements other than the test food and thereason for its use, and as a survey of the diet, the time andamount of three meals on a four point scale (a lot, as usual,_ 159 _

Skin Improvement Effects of Anti-aging Supplement
less, no meal), the details of the main dish and side dish, andalcohol consumption every day. The contents of this journal
were assessed every four weeks, and it was confirmed that
there were no major fluctuations in the lifestyle habits of thesubjects.

4. Evaluations

We used skin viscoelasticity and wrinkle replica
analysis results as the main endpoints. Other skin evaluation
items (AF values, stratum corneum moisture content,transepidermal water loss, and color difference), oxidative
stress markers, glycative stress markers, the inflammatory
marker, the anti-aging QOL common questionnaire, and the
feelings questionnaire were used as the secondary endpoints.
Safety was evaluated through general biochemical analyses,
body composition, fluctuations in blood pressure, and thepresence/absence of adverse events was determined by theprincipal investigator.

5. Statistical analysis

Data for the background characteristics of the subjects
are presented as mean ± standard deviation, and evaluation
items are presented as mean ± standard error. We consideredsubjects who were included in the test and began the use of
the test food as the Full Analysis Set (FAS), and the group of
subjects remaining after excluding those who dropped out
from the FAS due to serious protocol breaches was considered
to be the group that was suited to the test implementation plan
(Per-Protocol Set; PPS). To evaluate the safety of the subjects,
we used FAS analysis, and the analysis of effectiveness wasmade using PPS analysis. The test method was a paired t test
(AF values, color difference, skin viscoelasticity, bodycomposition, and blood pressure) or Wilcoxon signed-rank
test (for items other than those listed above) to compare items
before and after the intake of the test food, and comparisons
between groups were made using Student’s t test (AF values,
color difference, skin viscoelasticity, body composition, andblood pressure) or Mann-Whitney U test (for items otherthan those listed above). Statistical analysis was conductedusing JMP11.1.1 (SAS), and the significance level was lessthan 5% in a two-tailed test.

6. Ethics

This study was reviewed by the ethics board of Tokyo
Synergy Clinic from the perspective of ethics, science, andmedical validity, and was implemented according to theapproved test protocol (October 28, 2014). The study was
implemented according to the ethical doctrine of the
Declaration of Helsinki, and respect was paid to the ordinanceon the criteria for the implementation of clinical trials of
drugs (Good Clinical Practice, GCP; Ministry of Health andWelfare Ordinance No. 28, March 27, 1997). The principal
investigator of this study provided sufficient explanation to
the subjects prior to the commencement of the study using
informed consent documents with the support of the CRC.
Once subjects fully understood the content, we obtained
informed written consent based on their free will prior tocommencement of the study. This study was implemented
during the period from November 2014 to April 2015 at
A-kit Corporation Skin Lab.

Results

Of the 131 people who voluntarily agreed to participate
and provided written informed consent, 12 were excluded
based on the background survey. Of the remaining 119 people,
one person was excluded as his/her AF value was an outlier.
Among the remaining 118 people, we registered the 56 people
with the highest AF values. Background characteristics ofthe selected subjects are shown in Table 2.
All 56 subjects in this test completed the project,
but two subjects in the active food group met the dropout
criteria (subjects did not take the test food for three or moreconsecutive days), and thus the PPS analysis was performedon 26 subjects in the active food group and 28 subjects inthe placebo group, totaling 54 subjects. The flow diagram ofthis test is shown in Fig. 1.

[Effectiveness]

Skin conditions

Results of skin measurements and stress indicators areshown in Table 3. Compared with the R7 of the placebogroup, which dropped significantly from 0.32 ± 0.01 to 0.28
± 0.01, R7 in the active food group remained at 0.31 ± 0.01
and did not show any decrease with the intake of the active
food. Therefore, the placebo group showed a significant
decrease after the 12 weeks of the test period. On the other
hand, R2 dropped significantly in both groups after taking
the test, with no difference between the two groups. In thewrinkle replica analysis, the wrinkle area rate decreased
significantly in the active food group vs placebo group at 12
weeks. The average wrinkle depth (total wrinkle), average
wrinkle depth (maximum wrinkle), and total wrinkle volume
decreased significantly only in the active food group after
the test. None of the parameters for color difference showed
any difference between the two groups, but on comparing
the before and after test results, the melanin index, Hb index,
and a* value decreased significantly in both groups, while
the L* value increased significantly. In addition, the b* value
increased significantly in the placebo group. Stratum corneum
moisture content did not show any difference between orwithin groups, and the transepidermal water content showeda significant decrease only in the placebo group.

MarkerTable 4 shows stratum corneum and skin markers inthe blood. Among the oxidative stress markers, blood LPO
decreased significantly in both groups, while the stratum
corneum DJ-1 levels decreased significantly only in theplacebo group but there was no difference between the groups
after 12 weeks. Among the glycative stress markers, the AF
value and blood CML increased significantly in both groups,
while blood TAGE significantly decreased in both groups.
On the other hand, the glycation reaction intermediate 3-DG
decreased significantly only in the placebo group, but there
was no difference between the groups at 12 weeks. The bloodinflammatory marker, TNF-α decreased significantly inboth groups, but there was no difference between the groups.

_ 16 0 _Glycative Stress Research

Table 2. Backgroundunit

mean ± SD

Median

Min

Max

n

-

56

-

-

-

Age

year

51.5 ± 8.0

49.5

41

68

R2

-

0.59 ± 0.08

0.60

0.35

0.77

R7

-

0.32 ± 0.06

0.32

0.19

0.48

Area rate%

20.9 ± 4.9

21.4

8.2

31.5

Average depth (total wrinkle)

μm

43.1 ± 16.9

37.5

24

113

Average depth (max wrinkle)

μm

80.3 ± 57.5

59.0

26

368

Max depth (max wrinkle)

μm

487.3 ± 508.1

275.0

51

2199

Volume (total wrinkle)

mm 30.91 ± 0.40

0.85

0.21

2.16

Melanin Index-

1.19 ± 0.13

1.18

0.98

1.56

Hb Index-

1.69 ± 0.37

1.70

0.76

2.68

Color difference

Hb SO 2 Index (%)%

57.0 ± 5.3

56.7

44.2

76.0

analysisL*-62.1 ± 2.2

62.2

57.5

68.8

a*

-

12.9 ± 2.1

12.9

7.4

17.7

b*

-

16.7 ± 1.9

16.7

12.0

20.2

Moisture content%

46.6 ± 11.0

44.7

20.0

72.8

Transepidermal water loss

g/m 2・h19.5 ± 4.5

19.2

12.1

30.3

LPO (serum)

nmol/mL

3.1 ± 0.5

3.0

2.2

4.1

DJ-1 (straum corneum)

pg/ug protein

4.3 ± 2.1

3.8

1.4

12.8

Skin AGE fluorescence-

2.14 ± 0.24

2.12

1.72

2.72

3-DG (serum)

ng/mL

15.1 ± 3.9

14.6

7.7

27.6

CML (serum)

μg/mL

1.12 ± 1.39

0.91

0.70

11.3

TAGE (serum)

U/mL

10.8 ± 4.1

10.1

3.3

28.6

TNF-α (serum)

pg/mL

1.09 ± 0.56

0.99

0.55

3.04

base

Parameter

Skin viscoelasticity

Wrinkle Replicas using

Skin function

3D Image Analysis

markerOxidative stress marker

Glycative stress marker

Inflammatory marker

SD, standard deviation; 3D, 3 dimension; Hb, hemoglobin; SO 2, oxygen saturation; LPO, lipid peroxide; AGE, advanced glycation endproduct;
3-DG, 3-deoxyglucosone; CML, carboxymethyl-lysine; TAGE, toxic AGE; TNF, tumor necrosis factor.

_ 161 _

Skin Improvement Effects of Anti-aging Supplement

Enrollment

Assessed for eligibility( n = 131)

Excluded (n=75)
・Did not meet inclusion criteria ( n = 12)・Other reasons ( n = 63)

Intervention

Test food( n = 28)

Placebo( n = 28)

Analysis

Allocation

Randomized( n = 56)

FAS Analyzed( n = 28)

FAS Analyzed( n = 28)
Excluded ( n = 2)
・withdrawal ( n = 0)・dropout ( n = 2)
PPS Analyzed( n = 28)

PPS Analyzed( n = 26)

Fig. 1. Flow diagram.

FAS, full analysis set; PPS, per protocol set.

A

B
HO

O

OH

O

H

H
O

HO

O

CH3

_ 162 _O

Glycative Stress Research

Table 3. Skin function

Skinviscoelasticity

Parameter

R2
R7

Wrinkle Replicas using
3D Image Analysis

Area rate
Average depth(total wrinkle)
Average depth(max wrinkle)
Max depth(max wrinkle)
Volume(totall wrinkle)
Melanin Index

Color difference analysis

Hb Index
Hb SO 2 Index
L*a*b*
Moisture content
Transepidermal
water loss

Group
ActivePlacebo
ActivePlacebo
ActivePlacebo
ActivePlacebo
ActivePlacebo
ActivePlacebo
ActivePlacebo
ActivePlacebo
ActivePlacebo
ActivePlacebo
ActivePlacebo
ActivePlacebo
ActivePlacebo
ActivePlacebo
ActivePlacebomean ± SE

unit

%
μm
μm
μm
mm 3
%
%
g/m 2・hinter-group
analysis0w

12w

0.59 ± 0.02

0.53 ± 0.02

0.59 ± 0.02

0.50 ± 0.01

0.31 ± 0.01

0.31 ± 0.01

0.32 ± 0.01

0.28 ± 0.01

19.9 ± 1.0

19.7 ± 1.0

21.6 ± 0.9

21.9 ± 0.8

41.9 ± 3.2

38.8 ± 3.2

43.1 ± 3.1

41.8 ± 2.3

75.3 ± 9.0

67.9 ± 9.0

82.8 ± 12.6

74.2 ± 8.2

426.8 ± 81.9

362.7 ± 81.9

494.6 ± 94.1

465.3 ± 91.7

0.86 ± 0.08

0.79 ± 0.08

0.93 ± 0.06

0.92 ± 0.06

1.18 ± 0.03

1.14 ± 0.02

1.19 ± 0.02

1.14 ± 0.02

1.72 ± 0.07

1.45 ± 0.06

1.67 ± 0.07

1.37 ± 0.05

58.1 ± 0.8

57.0 ± 1.2

55.8 ± 1.1

55.8 ± 1.0

62.2 ± 0.4

64.0 ± 0.5

61.8 ± 0.5

64.1 ± 0.4

13.2 ± 0.4

11.4 ± 0.3

12.6 ± 0.4

10.8 ± 0.4

16.5

0.4

17.0

0.4

16.7

0.3

17.4

0.2

46.6

2.0

49.7

2.0

46.6

2.2

49.9

1.7

19.6

0.9

18.9

0.9

19.4

0.9

17.9

0.9

Bold font, p < 0.05; SE, standard error; 3D, 3 dimension; Hb, hemoglobin; SO 2, oxygen saturation.

_ 163 _

P-value

0w

12w

0.770

0.052

0.751

0.030

0.158

0.035

0.406

0.141

0.585

0.275

0.527

0.169

0.345

0.076

0.841

0.940

0.612

0.296

0.110

0.427

0.537

0.859

0.300

0.224

0.767

0.395

0.769

0.842

0.697

0.550

Intra-group
analysis0.002< 0.001
0.519
0.003
0.449
0.726
0.003
0.803
0.025
0.371
0.091
0.299
0.019
0.767
0.012
0.009
< 0.001< 0.001
0.199
0.993< 0.001< 0.001< 0.001< 0.001
0.119
0.005
0.068
0.237
0.666
0.001

Skin Improvement Effects of Anti-aging Supplement
Table 4.

Inflammatory
markerGlycative stress marker

Oxydative
stress marker

Parameter

LPO(serum)DJ-1(straum corneum)
Skin AGE
fluorescence
3-DG(serum)CML(serum)TAGE(serum)TNF-α(serum)Group

ActivePlacebo
ActivePlacebo
ActivePlacebo
ActivePlacebo
ActivePlacebo
ActivePlacebo
ActivePlacebomean ± SE

unit

nmol/mL
pg/ug protein
ng/mL
μg/mL
U/mL
pg/mL

0w

12w

3.2 ± 0.1

2.7 ± 0.1

3.0 ± 0.1

2.6 ± 0.1

3.51 ± 0.25

3.54 ± 0.25

5.10 ± 0.47

4.32 ± 0.35

2.13 ± 0.05

2.26 ± 0.05

2.15 ± 0.05

2.31 ± 0.04

14.9 ± 0.8

14.9 ± 0.8

15.2 ± 0.7

12.9 ± 0.6

1.4 ± 0.4

1.5 ± 0.4

0.9 ± 0.0

1.1 ± 0.0

11.0 ± 0.8

7.6 ± 0.8

10.6 ± 0.8

6.9 ± 0.6

1.2 ± 0.1

0.9 ± 0.1

1.0 ± 0.1

0.9 ± 0.1

P-valueinter-group
analysis
0w

12w

0.443

0.715

0.005

0.106

0.727

0.488

0.622

0.091

0.008

0.332

0.431

0.243

0.488

0.662

Intra-group
analysis0.001< 0.001
0.561
0.039< 0.001< 0.001
0.796
0.002
0.001
< 0.001< 0.001< 0.001
0.002
0.047

Bold font, p < 0.05; SE, standard error; LPO, lipid peroxide; AGE, advanced glycation endproduct;
3-DG, 3-deoxyglucosone; CML, carboxymethyl -lysine; TAGE, toxic AGE; TNF, tumor necrosis factor.

Anti-aging QOL questionnaire (subjective symptoms)

Results of the anti-aging QOL common questionnaire areshown in Table 5. “Difficulty falling asleep” did not show asignificant fluctuation before and after the test in both groups,
but at week 12, the active food group had a significantly higher
value. In addition, “easily breaking into a sweat” increased
significantly in both groups, “dry mouth” and “swelling”
increased significantly only in the active food group, “tendency
to gain weight” only decreased significantly in the placebogroup, and “difficulty falling asleep” increased significantlyin the placebo group. However, there was no difference
between the groups.

Feelings questionnaire

Results of the feelings questionnaire are shown in Fig. 2.
On all items, there was no difference between the groups,
but “dullness (brightness of skin)” was 0.58 ± 0.11 for theactive food group and 0.35 ± 0.09 for the placebo group,
showing a greater tendency towards an improvement in theactive food group (p < 0.1).

[Safety]

Body composition, blood pressure measurement,
and general biochemistry

Results for body composition, sphygmomanometry
measurements, and general biochemistry analyses are shownin Table 6. On all items, there was no difference between thegroups at weeks 0 and 12. There was a significant difference
before and after the test period for the following five: diastolicblood pressure (the active food group), HDL-C, ALB,
ALP (the placebo group), and total protein (both groups).
Fluctuations were all within the normal range. In addition, it
was confirmed that individual fluctuations in subjects were
not abnormal and would not be a clinical problem.

Adverse events and others

During the test period, there were multiple adverse
events in both groups, but all cases were examined and were
determined to be within normal limits for symptoms andseverity and have no causal relationship with the test food.
In addition, on all observed items, there was no problem
with safety.

_ 16 4 _Glycative Stress Research

Table 5-1. AAQoL.

Parameter

Tired eyes
Blurry eyes
Eye pain
Stiff shoulders
Muscular pains/stiffness
Palpitations
Shortness of breath
Tendency to gain weight

Physical symptoms

Weight loss, thin
Lethargy
Lack of sense of wellness
ThirstSkin problems
Anorexia
Early satiety
Epigastralgia
Liability to catch colds
Coughing and sputum
Diarrhea
Constipation
Hair loss
Gray hair
HeadacheDizziness
Tinnitus
LumbagoArthralgiamean ± SE

Group
ActivePlacebo
ActivePlacebo
ActivePlacebo
ActivePlacebo
ActivePlacebo
ActivePlacebo
ActivePlacebo
ActivePlacebo
ActivePlacebo
ActivePlacebo
ActivePlacebo
ActivePlacebo
ActivePlacebo
ActivePlacebo
ActivePlacebo
ActivePlacebo
ActivePlacebo
ActivePlacebo
ActivePlacebo
ActivePlacebo
ActivePlacebo
ActivePlacebo
ActivePlacebo
ActivePlacebo
ActivePlacebo
ActivePlacebo
ActivePlacebo

2.58
2.36
2.15
1.96
1.62
1.54
2.96
2.86
2.35
2.39
1.54
1.61
1.58
1.75
2.62
2.96
1.35
1.36
1.81
1.93
1.92
1.82
1.77
1.68
2.23
2.07
1.46
1.39
1.54
1.54
1.46
1.36
1.85
1.68
1.62
1.71
1.38
1.64
2.27
2.00
2.15
2.11
3.23
3.18
1.96
2.11
1.50
1.57
1.31
1.54
1.88
2.04
1.58
1.54

0w
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±

0.20
0.18
0.16
0.15
0.16
0.13
0.23
0.24
0.21
0.19
0.14
0.12
0.15
0.16
0.24
0.21
0.11
0.09
0.16
0.18
0.15
0.19
0.14
0.17
0.17
0.16
0.13
0.09
0.13
0.14
0.13
0.11
0.14
0.13
0.16
0.17
0.10
0.12
0.23
0.15
0.17
0.17
0.17
0.19
0.15
0.23
0.13
0.15
0.09
0.16
0.17
0.17
0.13
0.16

12w
2.65
2.39
2.23
2.07
1.85
1.61
3.15
2.71
2.46
2.21
1.65
1.54
1.58
1.79
2.65
2.64
1.38
1.46
2.12
2.11
2.12
1.93
2.04
1.89
2.12
2.18
1.38
1.46
1.73
1.50
1.69
1.36
1.81
1.71
1.92
1.71
1.50
1.54
2.27
1.89
2.23
2.11
3.19
3.14
2.19
2.25
1.62
1.82
1.38
1.64
2.12
2.21
1.77
1.89

±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±

0.19
0.16
0.18
0.16
0.15
0.14
0.21
0.22
0.19
0.19
0.14
0.12
0.15
0.14
0.23
0.23
0.15
0.13
0.18
0.17
0.15
0.16
0.14
0.15
0.14
0.18
0.12
0.10
0.15
0.12
0.15
0.11
0.12
0.12
0.21
0.13
0.11
0.12
0.23
0.17
0.18
0.18
0.18
0.14
0.16
0.23
0.14
0.15
0.11
0.17
0.19
0.17
0.15
0.17

Bold font, p < 0.05; AAQoL, Anti-Aging QOL Common Questionnaire; QOL, quality of life; SE, standard error.

_ 165 _

P-valueinter-group
analysis
Intra-group
analysis
0w
12w
0.540

0.265

0.373

0.541

0.800

0.239

0.803

0.165

0.878

0.302

0.575

0.537

0.446

0.232

0.408

0.915

0.792

0.518

0.775

0.964

0.338

0.363

0.410

0.475

0.463

0.948

0.879

0.378

0.837

0.287

0.584

0.091

0.406

0.575

0.666

0.661

0.127

0.906

0.489

0.280

0.698

0.678

0.978

0.948

0.941

0.840

0.890

0.375

0.323

0.314

0.576

0.683

0.492

0.679

0.838
1.000
0.800
0.613
0.285
0.807
0.392
0.463
0.510
0.283
0.581
0.754
1.000
1.000
0.960
0.045
1.000
0.531
0.116
0.273
0.180
0.641
0.039
0.291
0.494
0.816
0.781
0.688
0.180
1.000
0.183
1.000
1.000
1.000
0.063
0.914
0.549
0.375
1.000
0.453
0.727
1.000
1.000
1.000
0.070
0.367
0.590
0.145
0.688
0.375
0.148
0.307
0.063
0.051

Skin Improvement Effects of Anti-aging Supplement
Table 5-2. AAQoL.

Parameter

Physical symptoms

Edematous
Easily breaking
into a sweat
Frequent urination
Hot flashes
Cold skin
Irritability
Easily angered
Loss of motivation
Unhappy
Nothing to look forward
to in my life
Daily life is not enjoyable
No confidence
Reluctance to talkwith others

Mental symptoms

Depressed
Feeling useless
Shallow sleep
Difficulty falling asleep
Memory lapse
Pessimism
Inability to concentrate
Inability to solve problems
Inability to readily makejudgments
Inability to sleep due toworries
Feeling tense
Feeling anxious for no
particular reason
Vague feeling of fearmean ± SE

Group
ActivePlacebo
ActivePlacebo
ActivePlacebo
ActivePlacebo
ActivePlacebo
ActivePlacebo
ActivePlacebo
ActivePlacebo
ActivePlacebo
ActivePlacebo
ActivePlacebo
ActivePlacebo
ActivePlacebo
ActivePlacebo
ActivePlacebo
ActivePlacebo
ActivePlacebo
ActivePlacebo
ActivePlacebo
ActivePlacebo
ActivePlacebo
ActivePlacebo
ActivePlacebo
ActivePlacebo
ActivePlacebo
ActivePlacebo

1.77
1.86
1.96
2.07
1.96
1.86
1.85
1.54
2.50
2.11
2.15
2.07
1.92
1.89
1.85
1.75
1.54
1.57
1.50
1.57
1.50
1.57
1.65
1.46
1.54
1.43
1.50
1.32
1.58
1.57
1.77
1.68
1.85
1.54
1.92
1.68
2.62
2.21
1.88
1.71
1.62
1.54
1.58
1.57
1.54
1.43
1.92
1.79
1.54
1.43
1.31
1.29

0w
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±

0.17
0.16
0.17
0.21
0.16
0.19
0.15
0.17
0.23
0.20
0.16
0.14
0.17
0.14
0.16
0.12
0.11
0.11
0.11
0.11
0.10
0.12
0.12
0.10
0.16
0.11
0.13
0.09
0.11
0.10
0.17
0.15
0.15
0.12
0.17
0.13
0.18
0.17
0.19
0.12
0.12
0.11
0.13
0.12
0.11
0.10
0.17
0.15
0.11
0.10
0.09
0.09

12w
2.08
2.07
2.31
2.54
2.27
2.00
1.88
1.68
2.42
1.96
2.04
2.14
1.96
1.93
1.92
1.86
1.65
1.64
1.54
1.54
1.77
1.64
1.81
1.68
1.65
1.50
1.62
1.46
1.81
1.57
2.00
1.75
1.88
1.43
1.85
1.79
2.54
2.36
2.04
1.89
1.81
1.57
1.81
1.64
1.58
1.71
1.96
1.93
1.62
1.57
1.46
1.32

±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±

0.21
0.16
0.19
0.22
0.21
0.15
0.18
0.12
0.22
0.18
0.14
0.13
0.14
0.14
0.18
0.14
0.14
0.12
0.11
0.10
0.13
0.12
0.17
0.12
0.17
0.11
0.12
0.11
0.16
0.10
0.18
0.17
0.16
0.12
0.16
0.14
0.19
0.16
0.19
0.14
0.16
0.11
0.15
0.11
0.11
0.13
0.16
0.16
0.12
0.11
0.10
0.09

Bold font, p < 0.05; AAQoL, Anti-Aging QOL Common Questionnaire; QOL, quality of life; SE, standard error.

_ 16 6 _P-valueinter-group
analysis
Intra-group
analysis
0w
12w
0.683

0.870

0.905

0.431

0.467

0.391

0.701

0.501

0.217

0.136

0.752

0.591

1.000

0.865

0.813

0.955

0.814

0.938

0.623

0.913

0.798

0.474

0.289

0.740

0.856

0.696

0.346

0.379

0.945

0.345

0.763

0.301

0.143

0.026

0.361

0.889

0.149

0.494

0.671

0.711

0.690

0.327

0.992

0.478

0.531

0.545

0.561

0.826

0.531

0.868

0.870

0.301

0.022
0.146
0.031
0.001
0.375
0.388
1.000
0.432
0.796
0.344
0.453
0.754
1.000
1.000
0.688
0.590
0.375
0.727
1.000
1.000
0.065
0.754
0.219
0.070
0.508
0.727
0.375
0.289
0.109
1.000
0.109
0.781
1.000
0.375
0.688
0.549
0.727
0.325
0.289
0.188
0.188
1.000
0.217
0.625
1.000
0.008
1.000
0.439
0.688
0.359
0.219
1.000

Glycative Stress Research

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

dry skin (moisture)
circumoral sagging（nasolabial fold）
sagging under eyes
texturepores
wrinklesp=0.096(vs placebo)dullnessspots
make-up condition
hair condition
nail condition
tiring easily
heaviness in the headfeeling fatigued

ActivePlacebofeelings on waking up
sleep quality
constipation or loose stools

Fig. 2. Feelings questionnaire on skin or healthmean ± SE (standard error)_ 167 _

Skin Improvement Effects of Anti-aging Supplement
Table 6. Safety

blood chemical analysis

sphygmomanometry

body composition

Parameter

unit

referencevalue

mean ± SE

Group

Activekg
Weightplacebo
Active%Body Fat percentage
placebo
Activekg/m 2
BMI
placebo
Activekcal/daybasal metabolic rate
placebo
Activesystolic blood pressure mmHg
placebo
Activediastolic blood pressure mmHg
placebo
Activebpm
pulse
placebo
Activeng/dL
70 - 109
glucose (FPG)
placebo
Active%4.6 - 6.2
HbA1c (NGSP)
placebo
Active%12.3 - 16.5
glycoalbuminplacebo
Activemg/dL
30 - 149
Triglyceride
placebo
Activemg/dL 120 - 219
Total cholesterol
placebo
Activemg/dL
40 - 95
HDL-C
placebo
Activemg/dL
65 - 139
LDL-C
placebo
ActiveU/Lγ - GT＜ 30
placebo
ActiveU/L
10 - 40
AST
placebo
ActiveU/L
5 - 45
ALT
placebo
Activemg/dL
0.2 - 1.2
Total bilirubinplacebo
Activeg/dL
6.7 - 8.3
Total protein
placebo
ActiveALB
g/dL
3.8 - 5.2
(improved BCP method)
placebo
Active1.1 - 2.0
A/G (0w)
placebo
Active100 - 325
U/L
ALP (0w)
placebo
Active120 - 240
U/L
LD (LDH)placebo
Active40 - 150
U/L
CK
placebo
Activemg/dL 0.47 - 0.79
Creatinine
placebo
Active2.5 - 7.0
mg/dLUA
placebo
Active8.0 - 20.0
mg/dL
UN
placebo

52.8
55.2
25.9
26.1
21.5
21.6
1074.51098.3
112.0
112.0
72.6
73.0
72.9
72.1
85.6
84.8
5.5
5.5
14.8
14.7
81.6
82.0
222.4
208.3
73.5
72.1
131.0
118.6
26.5
18.9
21.4
18.8
17.6
14.1
0.8
0.7
7.1
7.2
4.4
4.4
1.6
1.6
206.4
185.1
179.9
174.7
87.7
88.0
0.6
0.6
4.2
4.5
12.3
13.4

0w

± 1.5
± 1.7
± 1.1
± 1.0
± 0.6
± 0.6
± 11.6± 13.3
± 2.9
± 2.7
± 2.0
± 1.8
± 1.9
± 2.0
± 3.0
± 2.0
± 0.1
± 0.1
± 0.3
± 0.2
± 8.7
± 7.8
± 9.9
± 8.5
± 3.9
± 3.1
± 8.2
± 7.7
± 7.3
± 1.9
± 2.1
± 1.0
± 3.9
± 1.2
± 0.1
± 0.0
± 0.1
± 0.1
± 0.0
± 0.0
± 0.0
± 0.0
± 14.3± 10.3
± 6.7
± 6.6
± 7.2
± 8.5
± 0.0
± 0.0
± 0.2
± 0.2
± 0.6
± 0.7

53.2
55.1
26.3
25.9
21.6
21.5
1073.6
1097.0
114.3
111.6
76.2
73.9
73.2
70.8
87.4
86.0
5.5
5.5
14.9
14.8
76.6
78.1
218.7
201.9
72.0
67.3
130.0
116.9
27.5
18.5
21.8
18.2
19.9
14.1
0.8
0.7
7.0
7.1
4.3
4.3
1.6
1.6
207.3
175.1
181.7
174.8
88.2
90.9
0.6
0.6
4.2
4.4
13.0
13.6

12w

± 1.5
± 1.7
± 1.0
± 0.9
± 0.6
± 0.6
± 11.8± 13.4
± 2.8
± 3.1
± 2.3
± 2.0
± 2.0
± 1.9
± 3.0
± 1.5
± 0.1
± 0.1
± 0.4
± 0.3
± 5.9
± 7.3
± 8.8
± 9.3
± 3.1
± 3.1
± 7.9
± 7.8
± 7.5
± 2.2
± 2.7
± 1.1
± 4.7
± 1.8
± 0.1
± 0.0
± 0.1
± 0.1
± 0.0
± 0.0
± 0.0
± 0.0
± 16.6± 10.2
± 7.3
± 6.6
± 6.4
± 8.8
± 0.0
± 0.0
± 0.1
± 0.1
± 0.5
± 0.7

P-valueinter-group
analysis
Intra-group
analysis
0w
12w
0.298

0.402

0.932

0.780

0.909

0.929

0.183

0.194

0.996

0.516

0.885

0.449

0.779

0.406

0.828

0.663

0.734

0.746

0.812

0.792

0.971

0.874

0.285

0.196

0.779

0.288

0.273

0.246

0.323

0.260

0.258

0.226

0.399

0.256

0.469

0.303

0.286

0.356

0.433

0.574

0.676

0.955

0.234

0.104

0.579

0.483

0.977

0.806

0.781

0.911

0.194

0.259

0.238

0.504

0.135
0.513
0.094
0.446
0.207
0.545
0.556
0.476
0.193
0.848
0.004
0.406
0.819
0.328
0.090
0.199
0.708
0.611
0.091
0.080
0.401
0.353
0.436
0.063
0.451
< 0.001
0.791
0.545
0.216
0.681
0.692
0.281
0.073
0.966
0.696
0.746
0.028
0.007
0.096
0.026
0.811
0.372
0.840
0.023
0.605
0.972
0.944
0.447
0.511
0.531
0.837
0.296
0.159
0.721

Bold font, p < 0.05; SE, standard error; BMI, body mass index; FPG, fasting plasma glucose; NGSP, National Glycohemoglobin Standardization Program;
HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; γ-GT, γ-glutamyl aminotransferase; AST, aspartate transaminase;
ALT, alanine aminotransferase; ALB, albumin; BCP, bromcresol purple; A/G, albumin globulin ratio; ALP, alkaline phosphatase; LDH, lactate dehydrogenase;
CK, Creatine kinase; UA uric acid; UN, urea nitrogen.

_ 16 8 _Glycative Stress Research

Discussion

In this study of with women aged between 41 and 69 who
were concerned by sagging skin and wrinkles, we examinedthe effectiveness of the test food on the skin condition, changesin oxidative and glycative stress markers, and subjective
symptoms by having the subjects take the active food orplacebo for 12 weeks. The results showed that the R7 indicatorof skin elasticity decreased significantly (deteriorated) in
the placebo group between the periods before and after theintake. In the active food group, the R7 value before the intake
was maintained throughout the test, and showed a significant
difference between the groups at 12 weeks. It confirmed the
suppressive effect of the active food on the decrease in skinelasticity. In addition, in the replica analysis of the areaaround the eyes, the wrinkle area rate after the test showedsignificantly low values in the active food group. Furthermore,
the average wrinkle depth (total wrinkle), average wrinkle
depth (maximum wrinkle), and total wrinkle volume showedsignificant improvements only in the active food groupbetween the periods before and after the intake; and thus, on
all items, wrinkles were considered to have improved in theactive food group. In addition, in the feelings questionnaire
implemented after the intake, the level of improvement was
higher in the active food group on items that asked about
improvements in “dullness (brightness of skin)” ( p < 0.1),
although it was not significant. Five of the raw materials usedin the active food were expected to maintain or improve theskin condition, and each of them has been tested on its own
or as a combination of two; however, their effectiveness in a
combination more than two had not yet been examined. In
this study, we used an active food that combined these five
raw materials. The results of the randomized controlled trial(RCT) with a placebo as the control confirmed a suppressionof the decrease in elasticity and an improvement in wrinkles,
and an improvement in the skin condition could be attributed
to the active food.
The present test was implemented using women living
in Kyoto, Osaka, and Hyogo during November to April as
subjects. The UV index (estimated value) from January to
April in Kyoto in 2015 gradually increased: 1.5 (January),
2.1 (February), 3.1 (March), and 4.2 (April) 39). On the other
hand, there was a significant decrease in the melanin index
and a significant increase in the L* value when comparing
the measurements taken before and after the intake in bothgroups. These facts indicate that the subjects’ protection
against UV rays during the test period was sufficient, andthat exposure to UV rays was equivalent in both groups.
In other words, subjects were protected from the oxidative
stress of UV rays during the intake period, and this was thereason for the significant decrease in the oxidative stressmarker LPO in both groups. There was a positive correlation
between the protective effect on skin cells from oxidative
stress by UV-B irradiation, and the stratum corneum Total
Anti-oxidative Capacity (TAC). Stratum corneum DJ-1 8) isa useful marker for anti-oxidation in the skin, and is known
to increase with UV irradiation and oxidative stress. As
mentioned earlier, oxidative stress from stimulation by UV
rays during the test period was similar in both groups, andthe stress level was estimated to be low; therefore, it waspredicted that stratum corneum DJ-1 would either decreaseor be maintained in both groups. In fact, it only decreased
significantly in the placebo group between the periods before
and after the intake. This indicates the possibility that DJ-1

production was promoted or its metabolism and decomposition
were suppressed in the active food group. The impact offood intake on DJ-1 production and metabolism has not beenstudied, but if DJ-1 production could be promoted without
stimulation from UV rays and oxidative stress, it would be
useful for protecting the skin from oxidative stress.
There are various approaches to reducing glycative stressboth upstream and downstream of AGE production. Loweringhigh blood sugar levels, and delaying and inhibiting sugar
absorption are upstream of AGE formation and lead to a
reduction in AGE generation downstream. Silybum marianum
extract in the active food has a reducing effect on blood sugar
and HbA1c 40, 41), and its mechanism involves peroxisome
proliferator - activated receptor - γ ( PPARγ ) 42, 43). There arealso reports of apple extract suppressing sugar absorption in
the small intestine 44, 45), improving α-glucosidase inhibition
and insulin resistance 46), and suppressing postprandial
hyperglycemia 47). The saponin B in soy extract has an
inhibitory effect on increases in blood glucose 48), while
aglycone-type isoflavone has been reported to improve
insulin resistance 49). In addition, in an experiment showing
inhibition of 3-DG generation in reactions between humanserum albumin and glucose, the IC 50 of the active food andplacebo food were 1.5 mg/mL and >10 mg/mL, respectively(unpublished data), and the active food was expected todecrease 3-DG generation and reduce 3-DG levels in the
blood. However, in this study, the levels of the glycation
reaction intermediate 3-DG were significantly decreased in
the placebo group, but there was no change in the active foodgroup. Unlike the prediction, a reduction in glycative stressmarkers was not confirmed through the use of the active food.
If the changes observed in the placebo group were seasonalchanges, a decrease should be observed in the active foodgroup as well. This was not observed in this study, perhaps
because the active food has a promoting effect on 3-DG
generation or suppresses its metabolism. The former isunlikely based on the results of an experiment investigating
the inhibition of 3-DG generation. If anything, 3-DG
metabolism was suppressed, or in other words, the conversion
from the intermediate product to the final metabolite. To
verify this possibility, 3-DG derived AGEs such as pyrraline
and imidazoline need to be measured. After including theseinvestigations, the effect of the active food on glycation
should be re-examined.
In the AAQoL common questionnaire, “difficulty falling
asleep” was different between groups after the test period, butsince there was no change within the group, the deterioration
was not considered to be due to the test food, and there is
no issue with safety. All the materials included in the active
food have been commonly used as foodstuffs or functional
food materials in the past, and are considered highly safe. In
the present test, there were no serious adverse events arisingfrom the components, and it was determined that there wasno problem with the safety of the active food.

Conclusions

The active food was formulated as a complex of foodmaterials that were expected to improve the skin condition,
reduce oxidative and glycative stress, suppress the decreasein elasticity observed in the skin of middle-aged women,
and improve winkles in a randomized placebo-controlled_ 169 _

Skin Improvement Effects of Anti-aging Supplement
double-blind clinical study. In terms of subjective symptoms,
the results showed the possibility that dullness of the skin
may be improved. On the other hand, in terms of oxidative
stress markers, a decrease in stratum corneum DJ-1 protein
was not observed. There were no clear results for glycative
stress markers, and this it is an issue for future research,in addition to elucidating the mechanism for improving
for the condition of skin. There were no safety issues with
consumption of the active food, and thus it is expected thatthe present active food could maintain skin elasticity in
middle-aged women and improve wrinkles.

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