Glycative Stress Research
Online edition : ISSN 2188-3610
Print edition : ISSN 2188-3602
Received : September 2, 2016
Accepted : November 21, 2016
Published online : December 31, 2016

Original Article

Anti-glycation and skin beautification properties from ingestion of mixed herb
extract: A placebo-controlled, double-blind, randomized, parallel-group study.
Hiroshige Kawai 1), Masako Shoshihara 1), Hirosato Kawakami 1), Junko Naito 2),
Umenoi Hamada 2, 3), Masayuki Yagi 3), Keiji Ohbayashi 4)1) Karada Lab, ARKRAY, Inc., Kyoto, Japan
2) Life and Medical Sciences Inspection Center, A-Kit, Inc., Gifu, Japan3) Antiaging Medical Research Center and Glycative Stress Research Center, Faculty of Life and Medical Sciences,
Doshisha University, Kyoto, Japan
4) Ohbayashi Clinic, Kyoto, Japan

Abstract

Purpose: The anti-glycation properties of mixed herb extract (MHE) composed of Chamaemelum nobile (roman chamomile),
Crataegus laevigata (hawthorne berries), Houttuynia cordata (dokudami) and Vitis vinifera (grape leaves) have been reported
in various clinical trials. However, these studies used high dosages and composite products that combine other functional
ingredients, so this new study evaluated the anti-glycation and skin beautification properties in low doses of a single product.
Methods: The subjects were 24 Japanese women (12 in the MHE group, 12 in the placebo group) 40 to 64 years of age
who had high amounts of advanced glycation end products (AGEs) deposits on the skin as measured by an AGE Reader TM.
A placebo-controlled, double-blind, randomized, parallel-group comparative study was conducted using MHE as the test food,
which was administered in capsules of 100 mg per day for 12 weeks. The subjects underwent blood tests, skin-related
measurements and medical interviews before administration as well as after 8 and 12 weeks of administration. Note that thisstudy has been conducted upon approval by an ethics review committee (Japan Clinical Trial Registry ID: UMIN000015242).
Results: In all cases, significant reductions were observed in the MHE group compared to the placebo group for both
Melanin Index (MHE group: - 0.04, placebo group: + 0.01, p = 0.009) and color difference b* (yellow) (MHE group: - 0.66,
placebo group: + 0.16, p = 0.025) for the skin of the upper arm. Also, a significant reduction in Brown Spots on the face wasobserved for the MHE group after 12 weeks’ administration compared to before administration (48.39 → 4 6.49, p = 0.005).
For the subgroup of healthy subjects that excluded 2 subjects suspected of having diabetes, (10 in the MHE group, 12 in
the placebo group), significant reductions in 3-deoxyglucosone (3DG) were observed in the MHE group when 3DG levels
after 12 weeks were compared to pre-administration levels (16.96 → 13.73 ng/mL, p = 0.043). Likewise for the hemoglobin
oxygen saturation index (Hb SO 2 Index) (an index for complexion) in the cheek skin, a significant increase was observed in
the MHE group compared to the placebo group (MHE group: +7.38, placebo group: + 4.36, p = 0.020).
Conclusion: MHE suppressed the yellowing and browning of skin color. This is possibly caused by a reduction incarbonylation due to suppression of 3DG generation. Furthermore, the improvement in complexion could possibly be
attributed to the herb constituents.

KEY WORDS: Mixed herb extract, anti-glycation, skin beautification, advanced glycation end products (AGEs),
3-deoxyglucosone (3DG)

Corresponding author: Hiroshige Kawai
Yousuien-nai, 59 Gansuin-cho, Kamigyo-ku, Kyoto, 602-0008, Japan
Karada Lab, ARKRAY, Inc.
Phone: 050 - 5830 - 0973 Fax: 075 - 431- 1253 Email: kawaih@arkray.co.jp
Co-authors: Shoshihara M, shoshiharam@arkray.co.jp ;Kawakami H, kawakamih@arkray.co.jp ;Naito J, j-naito@a-kit.co.jp ;Hamada U, hamada@yonei-labo.co.jp ;Yagi M,: myagi@mail.doshisha.ac.jp ;Ohbayashi K, keiji-ob@grape.plala.or.jp_ 23 6 _Glycative Stress Research 2016; 3 (4): 236- 245(c) Society for Glycative Stress ResearchGlycative Stress Research

Introduction

It is reported that the carbonylation of skin proteins is
involved in yellowing and reduced clarity of the skin 1). Also,
the mixed herb extract composed of Chamaemelum nobile(roman chamomile), Crataegus laevigata (hawthorne berries),
Houttuynia cordata (dokudami) and Vitis vinifera (grape
leaves) in in vitro experiments suppresses the generation of
3-deoxyglucosone (3DG) 2), glyoxal, methylglyoxal and othercompounds that cause carbonylation.
MHE has been reported to have various anti-glycation
properties in clinical trials, but these evaluations involved highdosages or composite products that contain other functional
ingredients 3 - 6). Therefore, we evaluated anti-glycation andskin beautification properties of a single product at low doses.

MethodSubjects

The subjects for this study were Japanese women 40 to
64 years of age, and to achieve a sample size of 10 or more
women in a group, the target size was set to 24 people toaccount for possible drop-outs (12 in the MHE group, 12 in
the placebo group). A screening test was applied to a pool ofapplicants who had passed the selection criteria and exclusion
criteria, to select subjects with relatively higher levels of skin
AGEs deposits as measured by an AGE Reader TM (Diagnoptics
Technologies B.V., Groningen, Netherlands). The group
allocator used random numbers to create the allocation table.
In the allocation, highest priority was given to matching theskin AGEs deposit levels, and then to ensure an even
distribution of the average and standard deviation of age andBMI ( Table 1 ).

Trial Design

The clinical trial design was a placebo-controlled,
double-blind, randomized, parallel-group comparative study.
The test food was MHE supplied in capsules, 100 mg
per day. Over 12 weeks, the trial subjects generally ingested
a placebo or MHE once per day on an empty stomach.
Blood tests, physical tests and medical interviews wereconducted prior to administration, and after 8 and 12 weeks
of administration for a total of 3 times. Furthermore, in
addition to the above, the questionnaire portion only was
conducted after the fourth week by mail.
The trial subjects fasted on the day prior to testing,ingesting only water from 10 pm until the end of testing on
the next day. On the day of testing, blood and stratum corneum
samples were collected from the subjects, who also completed
all other tests from 9 am to 2 pm.
The primary endpoints were related to the anti-glycation
properties: skin AGEs deposit levels, skin elasticity, stratum
corneum carboxymethyl lysine (CML) and blood test results(CML, 3DG).
Secondary endpoints were skin color differences, imaging
diagnosis of skin using VISIA T M, blood test results (glucose,hemoglobin [Hb] A1c and insulin), and medical interviews(Anti-Aging QOL Common Questionnaire and a skinquestionnaire).
Other blood tests were conducted as safety endpoints:
total bilirubin, aspartate aminotransferase (AST) (glutamic
oxaloacetic transaminase [GOT]), alanine aminotransferase(ALT) (Glutamic Pyruvic Transaminase [GPT]), alkaline
phosphatase (ALP), lactate dehydrogenase (LDH),
γ -glutamyltransferase ( γ -GTP), creatine phosphokinase(CPK), total protein, albumin, albumin/globulin (A/G)
ratio, creatinine, urea nitrogen, uric acid, triglyceride, totalcholesterol, high-density lipoprotein (HDL) cholesterol, low-

Table 1. Baseline demographic and clinical characteristics.

MHE group

Placebo groupp-value

1212(t-test)51.5 ± 6.3

51.4 ± 5.5

p = 0.973

Height (cm)155.40 ± 5.15

158.38 ± 4.68

p = 0.153

Weight (kg)55.00 ± 6.81

56.76 ± 4.99

p = 0.478

Body fat (%)

29.38 ± 3.51

28.60 ± 3.62

p = 0.596

BMI (kg/m 2 )22.77 ± 2.46

22.64 ± 2.07

p = 0.894

Skin AGEs deposit level2.43 ± 0.17

2.41 ± 0.18

p = 0.791

Glucose (mg/dL)

88.8 ± 11.0

83.7 ± 6.3

p = 0.173

HbA1c [NGSP] (%)

5.71 ± 0.59

5.58 ± 0.24

p = 0.473

Insulin (μU/mL)

5.77 ± 1.65

4.83 ± 1.86

p = 0.203

Target sizeAge (years of age)

Measured value: Average ± standard deviation. MHE, mixed herb extract; BMI, body mass index; AGEs, advanced glycation end
products; NGSP, National Glycohemoglobin Standardization Program.

_ 237 _

Anti-Glycation and Skin Beautification Properties of Mixed Herb Extract
density lipoprotein (LDL) cholesterol, Na, K, Cl, Ca andFe, as well as hematology tests (white blood cell counts, redblood cell counts, hemoglobin levels, hematocrit, platelet
counts and hemograms).
For basic measurements, height (only on first time),body weight, body fat ratio, fat mass, lean body mass, BMI,
basal metabolic rate, blood pressure and pulse rate. The
examinations were conducted by physicians.
Trial subjects recorded ingestion status of the test food,any harmful events, and any changes to lifestyle (diet, exercise,alcohol ingestion, etc.) in their diaries.
The test period was from September 25 to December
19, 2014. Physician interviews, blood pressure measurement
and blood collection was administered at the Senrigaoka
Kyoritsu Clinic, and skin-related tests were conducted at
the A-Kit, Inc. Life and Medical Sciences Inspection Center
directly afterwards.

Test Food

The test food was a food product in capsule form
containing the MHE (product name: AG Herb MIX) (100 mg/
capsule) or a placebo capsule. To the extent possible, the placebo
was made to be indiscernible from the MHE food product inappearance, taste and odor. The test food composition andnutritional constituents are listed in Table 2 and Table 3.

Testing Methods

Subjective Symptoms (Questionnaire Items)
The evaluation of subjective symptoms was mainly
divided into physical symptoms and mental symptoms. The
Anti-Aging QOL Common Questionnaire (AAQol) was used,and evaluations were made on a five-point scale similar

to previous reports 3). Also, a Skin Questionnaire wasadministered at the same time.
Body Measurements and Physical Testing
Body fat ratio, lean body mass, and other items in addition
to height, body weight, blood pressure and heart rate were
measured by bioelectrical impedance analysis, using a WellSCAN50 0 (Canon Lifecare Solutions, Inc.: Bunkyo- ku,
Tokyo, Japan) body composition analyzer.
Blood Testing
For the blood tests, 3DG was tested at SRL, Inc.
(Tachikawa, Tokyo, Japan), and the rest were done at LSI
Medience Corporation (Chiyoda-ku, Tokyo, Japan).
Skin-Related Testing
The skin-related tests below were started after washing
the face and cleansing the measurement area, thenacclimatizing to an environment with constant temperatureand humidity (21 ± 1°C, 50 ± 5%) for 20 minutes.
1) Skin AGEs deposit levels
The skin AGEs deposit levels were measured using an
AGE Reader T M. Following a previous report 7), the measured
area for the determination was the inner side of the upper
right arm (about 10 cm from the olecranon toward theshoulder).
2) Skin elasticity
Skin elasticity was measured using a Cutometer MPA580
Dual (Courage + Khazaka electronic GmbH, Cologne,
Germany) skin elasticity measurement device. The
measurement area for the determination was the left cheek(the median position between the bottom of the ear lobe
and the corner of the mouth) as well as the inner part of theupper right arm.

Table 2. Test food composition (Compounded amount: mg/capsule).

MHE food

PlaceboDextrin

117.5

217.5

MHE (as AG Herb MIX)

100.0

0.0

Starch25.0

25.0

Calcium stearate

7.5

7.5

250.0

250.0

Total
MHE, mixed herb extract.

Table 3. Test food nutritional constituents (Administration amount per day: per capsule).

MHE food

Placebo

Energy (kcal)

1.2

1.2

Protein (g)

0.003

0

Lipid (g)

0.001

0

Carbohydrate (g)

0.276

0.287

Sodium (mg)

0.243

0.139

MHE, mixed herb extract.

_ 23 8 _Glycative Stress Research3) Stratum corneum CML
Following a previous report 8), the stratum corneum CML
was measured. An adhesive film was applied to the skin tocollect the stratum corneum (tape stripping method). This
test was conducted three times in the same location, and thesamples were stored frozen for measuring CML levels in thestratum corneum. The collection area was the inner part of
the upper right arm. Measurements were conducted at A-Kit,
Inc. (Ogaki, Gifu, Japan).
4) Skin color difference
Skin color difference was measured using a CM-600d
spectrophotometer and the CM-SA (Konica Minolta, Inc.,
Chiyoda-ku, Tokyo, Japan) skin analysis software package.
The measurement area was the left cheek and the inner partof the upper right arm.
5) Imaging analysis of the facial skin
Imaging analysis of the facial skin was conducted byusing the VISIA T M Evolution (Canfield Imaging Systems,
Inc., NJ, USA) system.

Statistical Analysis

Analysis was done using the IBM SPSS Statistics 22(Japan IBM Corporation, Chuo-ku, Tokyo, Japan) package.
Dunnett’s multiple comparison test was used as the assay
method for before and after comparisons of the measured
items prior to administration, and after the 8 weeks and 12
weeks of administration. For inter-group comparisons, at test (two- group comparison) was used. Friedman’s multiple
comparison test was used as the assay method for before
and after comparisons of questionnaire items prior toadministration, and after 4 weeks, 8 weeks and 12 weeks ofadministration. The Mann-Whitney test was used for intergroup comparisons. The significance level was a relative risk
below 5 % for all two-tailed tests.

Ethical Standards

The study complied with the ethical principles set
forth in the Helsinki Declaration and the Japan Personal
Information Protection Act, and was conducted inaccordance with the Japan Ministerial Ordinance on
Good Clinical Practice for Drugs (GCP) (Ordinance ofthe Ministry of Health and Welfare No. 28 of March 27,1997). Review documents were submitted to the Kenshokai
Ethics Committee (Fukushima-ku, Osaka, Japan), andwere reviewed and approved on August 27, 2014. Informed
consent was obtained from trial subjects prior to screening
tests, and the subjects sufficiently understood the content of
the testing plan and voluntarily expressed their willingness
to participate in the test by submitting the consent form. The
Clinical Trial Registry ID was UMIN000015242 for thisstudy.

Results

Target Group for Analysis

Fig. 1 shows the tracking flow diagram for trial subjects.
The solicitation for trial subjects was conducted between
August 28 to September 7, 2014, and screening tests wereconducted on the 9th and 10th of September. 24 people
participated in the trial with twelve people in the MHEgroup and twelve people in the placebo group allocated in arandom manner. All subjects completed the trial, and none
dropped out. Also, no subjects were deemed unsuited foranalysis, and all were included in the analysis. In addition,
the changes in physical testing are shown in Table 4.

Baseline Data

Table 1 shows baseline demographic and clinicalcharacteristics for each group. Furthermore, participants
were limited to the Asian race (Japanese) and female sex.

Primary Endpoints

For the primary endpoints of skin AGEs deposit levels,
skin elasticity, stratum corneum CML and blood test results(CML, 3DG), significant differences were not observed forboth between the MHE group and the placebo group andwithin the MHE group in changes from before administration
to the 12 th week of administration ( Table 5 and Table 6 ).
However, parameters identified to have undergone changes
opposite to improvement were listed under the safety
endpoints described later on in the paper.
Furthermore, changes in the blood 3DG levels for the
subgroup of healthy subjects (excluding 2 persons who showed
HbA1c values higher than the standard range) are shown in
Fig. 2. There was a significant reduction in the MHE group(before administration 16.96 ± 2.92 → 12 th week 13.73 ±4.00 ng/mL [ p = 0.043]), whereas the placebo group did
not show any significant change (before administration
15.09 ± 3.59 → 12 th week 13.15 ± 3.63 ng/mL [ p = 0.191]).
In an inter-group comparison, no significant inter-groupdifferences were noted ( p = 0.456).

Secondary Endpoints

For the secondary endpoints of skin color differences,imaging diagnosis of the skin using VISIA T M ( Table 6 ),blood testing (glucose, HbA1c and insulin) ( Table 5 ) andmedical interview (Anti-Aging QOL Common Questionnaire,skin questionnaire) ( data not shown ), improvements were
demonstrated in the three items 1) to 3) below. However,items showing changes opposite to improvement were listed
under the safety endpoints described later in the paper.
Furthermore, the subgroup analysis is shown in 4).
1) Skin (upper arm) Melanin Index
Changes in the Melanin Index in the skin of the upperarm are shown in Fig. 3.
There were significant reductions in the MHE group(before administration 0.70 ± 0.09 → 12 th week 0.66 ± 0.10
ng/mL [ p = 0.001]), whereas the placebo group did not show
any significant change (before administration 0.70 ± 0.06 →12th week 0.70 ± 0.09 ng/mL [p = 0.727]). In an inter-groupcomparison, the MHE group showed significant reductions
in contrast to the placebo group ( p = 0.009).
2) Skin (upper arm) color difference b* (yellow)
Fig. 4 shows the color difference b* (yellow) for theskin of the upper arm. There were significant reductions in
the MHE group (before administration 14.84 ± 1.31 → 12 th
week 14.18 ± 1.72 ng/mL [ p = 0.005]), whereas the placebogroup did not show significant change (before administration
14.78 ± 1.31 → 12 th week 14.93 ± 1.42 ng/mL [ p = 0.819]).
In an inter-group comparison, the MHE group showedsignificant reductions in contrast to the placebo group ( p =0.025).

_ 23 9 _Anti-Glycation and Skin Beautification Properties of Mixed Herb Extract

Fig. 1. Tracking flow diagram for trial subjects.
MHE, mixed herb extract.

Table 4. Physical testing.

Parameter

Unit

Weightkg

Body fat%Fat mass

kg

Lean body mass

kg

Body
composition

Bloodpressure
Pulse rate

BMI

kg/m 2

Basal metabolic ratekcal/daysystolic

mmHg

diastolic

mmHg
bpm

Group

0W

8W

12W

MHE
Placebo
MHE
Placebo
MHE
Placebo
MHE
Placebo
MHE
Placebo
MHE
Placebo
MHE
Placebo
MHE
Placebo
MHE
Placebo55.0 ± 6.7
57.0 ± 5.1
29.9 ± 3.0
28.7 ± 3.6
16.5 ± 3.3
16.4 ± 3.1
38.5 ± 3.9
40.5 ± 3.3
22.8 ± 2.4
22.7 ± 2.1
1072 ± 64
1097 ± 52
121 ± 14124 ± 1781 ± 1076 ± 678 ± 1170 ± 1155.4 ± 6.7
57.0 ± 5.2
29.8 ± 3.9
29.1 ± 3.3
16.7 ± 3.9
16.7 ± 3.0
38.8 ± 3.7
40.4 ± 3.1
22.9 ± 2.4
22.7 ± 2.1
1074 ± 62
1095 ± 49
121 ± 17128 ± 1679 ± 1080 ± 8 #74 ± 867 ± 855.1 ± 6.9
57.2 ± 5.2
28.9 ± 4.3
28.2 ± 3.3
16.0 ± 3.7
16.2 ± 2.8
39.1 ± 4.5
41.0 ± 3.6
22.8 ± 2.5
22.8 ± 2.0
1076 ± 68
1101 ± 50
122 ± 12125 ± 1879 ± 1180 ± 8 #74 ± 1168 ± 12

Subjects: n = 12 (MHE group), n = 12 (Placebo group). Values: means ± standard deviation. * p < 0.05, ** p < 0.01 : Comparison with the Placebo group( t-test ). # p < 0.05, ## p < 0.01: Comparison with before administration (Dunnett). MHE, mixed herb extract; BMI, body mass index.

_ 240 _Glycative Stress Research

Table 5. Clinical chemistry testing.

Reference
range

Parameter

Unit

CML in stratum corneum

μg/mg
protein

CML

μg/mL

−

3-deoxyglucosone

ng/mL

3.76 - 18.14

Glucosemg/dL

70 - 109%

4.6 - 6.2

Insulin

μU/mL

1.7 - 10.4

Total protein

g/dL

6.7 - 8.3

Albuming/dL

3.8 - 5.2

HbA1c/NGSP

A/G ratio−

1.1 - 2.0

CPK

U/L

40 - 150

AST (GOT)

U/L

10 - 40

ALT (GPT)

U/L

5 - 45LDH

U/L

120 - 240

ALP

U/L

100 - 325

γ- GTP

U/L

30 以下

Creatinine

mg/dL

0.47 - 0.79

Uric acidmg/dL

2.5 - 7.0

Urea nitrogen

mg/dL

8.0 - 20.0

TG ( Triglyceride)

mg/dL

30 - 149

Total cholesterol

mg/dL

120 - 219

HDL cholesterol

mg/dL

40 - 95

LDL cholesterol

mg/dL

65 - 139

SodiummEq/L

137 - 147

Potassium

mEq/L

3.5 - 5.0

ChloridemEq/L

98 - 108

Calcium

mg/dL

8.4 - 10.4

Serum iron

μg/dL

40 - 180

Total bilirubin

mg/dL

0.2 - 1.2

Group

0W

8W

12W

MHE
Placebo
MHE
Placebo
MHE
Placebo
MHE
Placebo
MHE
Placebo
MHE
Placebo
MHE
Placebo
MHE
Placebo
MHE
Placebo
MHE
Placebo
MHE
Placebo
MHE
Placebo
MHE
Placebo
MHE
Placebo
MHE
Placebo
MHE
Placebo
MHE
Placebo
MHE
Placebo
MHE
Placebo
MHE
Placebo
MHE
Placebo
MHE
Placebo
MHE
Placebo
MHE
Placebo
MHE
Placebo
MHE
Placebo
MHE
Placebo
MHE
Placebo36.7 ± 4.9
39.0 ± 16.0
0.65 ± 0.05
0.71 ± 0.23
18.0 ± 3.9
15.1 ± 3.6
88.8 ± 11.0
83.7 ± 6.3
5.71 ± 0.59
5.58 ± 0.24
5.77 ± 1.65
4.83 ± 1.86
7.3 ± 0.4
7.3 ± 0.3
4.3 ± 0.3
4.4 ± 0.3
1.48 ± 0.23
1.57 ± 0.27
67.3 ± 42.0
100.2 ± 33.4
18.0 ± 3.7
19.2 ± 3.8
15.3 ± 7.9
15.9 ± 5.3
175.0 ± 24.7
181.1 ± 35.4
207.3 ± 69.9
182.8 ± 59.0
26.3 ± 15.0
20.8 ± 10.5
0.65 ± 0.08
0.69 ± 0.07
4.66 ± 1.26
4.98 ± 0.95
11.8 ± 2.5
10.9 ± 3.1
124.1 ± 50.1
96.8 ± 43.3
248.3 ± 47.3
218.9 ± 29.3
61. 3 ± 12.8
64.1 ± 8.6
159. 3 ± 45.0
133.4 ± 27.3
141.3 ± 1.4
142.2 ± 0.8
4.25 ± 0.39
4.12 ± 0.20
104.7 ± 1.5
105.0 ± 1.7
9.53 ± 0. 39
9.47 ± 0. 39
93.9 ± 42.0
83.1 ± 4 4.4
0.79 ± 0.24
0.69 ± 0.17−−−−
14.6 ± 3.8 #15.0 ± 3.0
84.6 ± 10.3
80.0 ± 5.7 ##5.60 ± 0.44
5.52 ± 0.22
5.27 ± 1.48
4.84 ± 1.56
7.2 ± 0.3
7.3 ± 0.3
4.3 ± 0.3
4.4 ± 0.3
1.50 ± 0.21
1.55 ± 0.19
82.1 ± 64.2
121.1 ± 54. 3 #21.5 ± 4.4 ##20.5 ± 3.8
19.3 ± 10.4
16.0 ± 3.9
176.4 ± 27. 3
186.2 ± 32. 3
212.7 ± 70.4
187.0 ± 58.2
27.7 ± 17.7
20.3 ± 8.7
0.63 ± 0.10
0.66 ± 0.07 #4.49 ± 1.19
4.68 ± 0.68
10.9 ± 2.4
11.5 ± 3.1
108.7 ± 44.4
93.0 ± 41. 3
235.7 ± 36.9
222.3 ± 28.8
61.6 ± 12.3
68.5 ± 11.3
149.0 ± 38.4
134.7 ± 26.3
14 0.7 ± 1.6
141. 3 ± 1.2 #4.23 ± 0.36
4.28 ± 0.16
10 4.1 ± 1.6
10 4.6 ± 1.6
9.33 ± 0.31
9.53 ± 0.32
83.2 ± 4 6.5
94.7 ± 49.3
0.78 ± 0.34
0.68 ± 0.12

37.8 ± 14.9
42.7 ± 11.0
0.88 ± 0.16 ##0.96 ± 0.32 ##15.1 ± 5.1
13.1 ± 3.6
88.3 ± 13.0
81.0 ± 6.1 #5.64 ± 0.46
5.57 ± 0.17
5.63 ± 1.58
3.59 ± 1.05 #7.3 ± 0.2
7.2 ± 0.3
4.3 ± 0.2
4.4 ± 0.3
1.47 ± 0.21
1.57 ± 0.27
64.2 ± 23.7
112.2 ± 57.1
19.6 ± 3.9
20.5 ± 3.7
15.9 ± 7.0
15.4 ± 3.7
190. 3 ± 28.8 #196. 3 ± 29.9 ##223.8 ± 69.7 #
184.2 ± 58.0
26.7 ± 18.7
22.5 ± 17.0
0.63 ± 0.10
0.65 ± 0.09 ##4.60 ± 1.15
4.74 ± 0.72
12.7 ± 2.8
11.6 ± 3.1
114.2 ± 52.6
86.0 ± 36.5
243.2 ± 43.4
216.4 ± 28.7
63.4 ± 11.5
68. 5 ± 11.1
154.2 ± 41.6
127.0 ± 26.2
140.8 ± 2.0 ＊140.5 ± 1.5 ##4.50 ± 0.49 ##4.48 ± 0.26 ##104.5 ± 1.6
104.1 ± 1.6
9.47 ± 0.38
9.38 ± 0.24
73.8 ± 39.2 # ＊88.9 ± 38.9
0.68 ± 0.16 ＊0.71 ± 0.17

Subjects: n = 12 (MHE group), n = 12 (Placebo group). Values: means ± standard deviation. * p < 0.05, ** p < 0.01 : Comparison with the Placebo group( t-test ). # p < 0.05, ## p < 0.01 : Comparison with before administration (Dunnett [3 times], paired t-test [twice]). MHE, mixed herb extract; CML,
carboxymethyl lysine; NGSP, National Glycohemoglobin Standardization Program; A/G, albumin/globulin; CPK, creatine phosphokinase; AST (GOT),
aspartate aminotransferase (glutamic oxaloacetic transaminase); ALT (GPT), alanine aminotransferase; LDH, lactate dehydrogenase; ALP, alkaline
phosphatase; γ-GTP, γ-glutamyltransferase; HDL, high-density lipoprotein; LDL, low-density lipoprotein.

_ 2 41 _Anti-Glycation and Skin Beautification Properties of Mixed Herb Extract
Table 6. Skin-related testing.
Parameter
Skin AGEs
deposit level

AF

Skin
viscoelasticity( Upper arm )

R2

Skin
viscoelasticity( Cheek )

Unit

R7
R2
R7
Melanin Index
Hb IndexColor
difference( Upper arm )Hb SO 2 Index

%

L*
a*
b*
Melanin Index
Hb IndexColor
difference( Cheek )Hb SO 2 Index

%

L*
a*
b*

Imaginganalysisby VISIA

Brown Spots

Score × 100

Pores

Score × 100

Porphyrin

Score × 100

Red

Score × 100

Spots

Score × 100

TextureScore × 100

UV Spots

Score × 100

WrinklesScore × 100

Group

0W

8W

MHE
Placebo
MHE
Placebo
MHE
Placebo
MHE
Placebo
MHE
Placebo
MHE
Placebo
MHE
Placebo
MHE
Placebo
MHE
Placebo
MHE
Placebo
MHE
Placebo
MHE
Placebo
MHE
Placebo
MHE
Placebo
MHE
Placebo
MHE
Placebo
MHE
Placebo
MHE
Placebo
MHE
Placebo
MHE
Placebo
MHE
Placebo
MHE
Placebo
MHE
Placebo
MHE
Placebo
MHE
Placebo2.36 ± 0.21
2.45 ± 0.21
0.90 ± 0.02
0.90 ± 0.02
0.66 ± 0.04
0.68 ± 0.05
0.72 ± 0.07
0.75 ± 0.08
0.37 ± 0.07
0.41 ± 0.08
0.70 ± 0.09
0.70 ± 0.06
0.77 ± 0.13
0.79 ± 0.17
56.4 ± 9.1
57.8 ± 7.2
70.0 ± 1.4
70.1 ± 1.5
4.82 ± 0.61
5.04 ± 0.90
14.8 ± 1.3
14.8 ± 1.3
1.11 ± 0.12
1.15 ± 0.13
1.08 ± 0.22
1.01 ± 0.15
51.7 ± 7.0
54.5 ± 4.5
6 4.6 ± 2.6
65.1 ± 2.0
8.4 4 ± 1.07
8.53 ± 0.85
18.5 ± 1.5
19.2 ± 1.6
48.4 ± 4.9
48.1 ± 6.6
18.4 ± 9.9
17.7 ± 9.0
10.7 ± 8.5
8.5 ± 9.2
25.9 ± 5.1
26.8 ± 5.1
38.9 ± 6.3
39.2 ± 8.8
12.2 ± 5.8
13.6 ± 5.3
30.4 ± 9.2
32.3 ± 3.7
11.6 ± 8.8
16.8 ± 10.2

2.29 ± 0.18
2. 37 ± 0.23
0.88 ± 0.0 3 #0.88 ± 0.03 #0.65 ± 0.06
0.66 ± 0.05 #0.73 ± 0.05
0.73 ± 0.07
0.4 0 ± 0.05
0.39 ± 0.07
0.68 ± 0.09
0.69 ± 0.09
0.84 ± 0.19
0.92 ± 0.27
52.7 ± 7.4
53.9 ± 10.6
70.1 ± 1.5
69.6 ± 1.8
4.95 ± 0.87
5.43 ± 0.91
14.7 ± 1.4
14.7 ± 1.6
1.06 ± 0.13 ##1.11 ± 0.14 #1.06 ± 0.27
1.06 ± 0.23
56.2 ± 7.1 ##59.1 ± 5.0 ##65.8 ± 2.4 ##65.6 ± 2.1
8.40 ± 1.55
8.80 ± 0.99
18.2 ± 1.6
18.8 ± 1.947.2 ± 4.8
47.4 ± 8.2
20.0 ± 10.8
18.1 ± 8.5
8.9 ± 7.6
6. 2 ± 7.4
26.4 ± 6.0
29.1 ± 6.7
39.4 ± 7.2
38.8 ± 10.0
12.1 ± 5.5
12.5 ± 6.0
32.0 ± 9.4
33.1 ± 2.8
11.7 ± 7.2
13.8 ± 16.4

12W
2.23 ± 0.26 #2.27 ± 0.23 ##0.88 ± 0.03 ＊0.90 ± 0.02
0.66 ± 0.05
0.69 ± 0.05
0.75 ± 0.06
0.77 ± 0.06
0. 39 ± 0.06
0.43 ± 0.06
0.66 ± 0.10 ##＊＊
0.70 ± 0.09
0.89 ± 0.21
0.87 ± 0.25
53.2 ± 5.7
57.5 ± 8.4
69.9 ± 1.6
70.1 ± 1.9
5.21 ± 1.19
5.45 ± 1.36
14.2 ± 1.7 ##＊14.9 ± 1.4
1.04 ± 0.12 ##1.10 ± 0.10 ##1.14 ± 0.22
1.11 ± 0.23
58.5 ± 7.1 ##58.8 ± 4.2 ##65.8 ± 2.4 ##65.8 ± 2.0 #8.83 ± 1.31
9.09 ± 1.22
17.8 ± 1.5 ##18.5 ± 1.6 #46.5 ± 5.3 ##47.3 ± 7.4
20.6 ± 11.9
18.2 ± 9.5
8.0 ± 6.7
6.6 ± 9.2
28.2 ± 7.1
31.7 ± 10.7
37.7 ± 8.1
38.2 ± 9.8
12.7 ± 7.0
12.5 ± 6.1
31.0 ± 8.2
33.9 ± 5.9
11.0 ± 7.8
15.3 ± 14.1

Subjects: n = 12 (MHE group), n = 12 (Placebo group). Values: means ± standard deviation. * p < 0.05, ** p < 0.01: Comparison with the Placebo group(t-test). # p < 0.05, ## p < 0.01: Comparison with before administration (Dunnett). MHE, mixed herb extract; AGEs, advanced glycation end products; AF, auto
fluorescence; Hb, hemoglobin; SO2, oxygen saturation; UV, ultraviolet.

_ 242 _Glycative Stress Research25

1.0
p = 0.009
p = 0.043

0.8

Melanin Index

3DG（ ng/mL ）

20

15

10

0.6

0.4

5

0

p = 0.001

0.2

MHE（ n = 10 ）0.0

Placebo（ n = 12 ）0W

MHE（ n = 12 ）

12W

0W

Fig. 2. Changes in the blood 3DG levels (Healthy subjects).

Measured value: Average ± standard error mean (SEM).
Analysis: Dunnett (intra-group) and t-test (inter-group). MHE,
mixed herb extract.

25
p = 0.02520
p = 0.005

color diﬀerence b*

12W

Fig. 3. Changes in the skin (upper arm) Melanin Index.

Measured value: Average ± standard error mean (SEM).
Analysis: Dunnett (intra-group) and t-test (inter-group). 3DG,
3-deoxyglucosone; MHE, mixed herb extract.

15

10

5

0

Placebo（ n = 12 ）MHE（ n = 12 ）0W

Placebo（ n = 12 ）12W

Fig. 4. Changes in the skin (upper arm) color difference b* (yellow).

Measured value: Average ± standard error mean (SEM). Analysis: Dunnett(intra-group) and t-test (inter-group). MHE, mixed herb extract.

_ 2 43 _Anti-Glycation and Skin Beautification Properties of Mixed Herb Extract
3) Brown spots on skin (face)Fig. 5 shows the changes in brown spots on the skin (face).
There were significant reductions in the MHE group (before
administration 48.39 ± 4.95 → 12 th week 46.49 ± 5.31 ng/mL[ p = 0.005 ]), whereas the placebo group did not show
significant change (before administration 48.08 ± 6.62 → 12 th
week 47.35 ± 7.41 ng/mL [ p = 0.396 ]). In an inter-groupcomparison, a significant difference in the change in amountbetween the two groups was not recognized ( p = 0.171).
4) Hb SO 2 Index (complexion) in the skin (cheek)
Changes in Hb SO 2 Index in the subgroup of healthy
subjects (excluding the 2 persons who showed HbA1c values
higher than the standard range) are shown in Fig. 6.
There were significant increases in the MHE group(before administration 52.70 ± 7.34 → 12 th week 60.08± 6.68 ng/mL [ p < 0.001]) as well as in the placebo group(before administration 54.45 ± 4.48 → 12 th week 58.81 ±4.15 ng/mL [ p < 0.001]). In an inter-group comparison, theMHE group showed significantly higher changes in contrast
to the placebo group ( p = 0.020).
Hb SO 2 Index is one of the indices used to judge
complexion.

Safety Endpoints

Harmful events that occurred during the trial period
included cold, fever, headache, sinusitis, (suspected) cystitis,
vomiting, diarrhea, soft stool, stomachache, constipationand menstrual pain. However, all events became mild or
recovered during the administration period and therefore thephysicians judged that there was no causal relationship with
MHE.
Test parameters were included in the scope if there werechanges opposite to improvement, regardless of whether they
were efficacy endpoints or not.
For changes in individual subjects, parameter values thatwere not abnormal prior to administration but shifted into thedeviation range after 12 weeks were white blood cell count,
MCHC (mean corpuscular hemoglobin concentration),
neutrophils, lymphocytes, LDH, ALP, γ -GTP, creatinine,
triglyceride, total cholesterol, LDL cholesterol, K and Fe.
However, these might have fortuitously crossed the borderline,or might have been just below the borderline within thestandard range and had crossed over the threshold to fall into
the deviation range due to a slight fluctuation. All were
judged to be within the range of physical change.
Measured items identified to have significant changes
from pre-administration to the 12th week within the MHEgroup or inter-group were the blood CML, LDH, ALP, and K
with significant increases, and upper arm skin elasticity (R2)and Fe with significant decreases ( Table 5 and Table 6 ).
However, either all were relatively small changes that fellwithin the standard range, or the change was smaller thanthat in the placebo group, or there was no significant difference
within the subgroup of healthy subjects. Thus, these were notdeemed to be problematic from the clinical perspective. For
questionnaire items, pessimism, easily breaking into a sweat,
and make-up comes off more readily showed significant
improvement in the placebo group in contrast to the MHEgroup, but there was no deterioration in the MHE group.
Therefore, this was deemed not to be problematic.

60

100
p = 0.005

p = 0.020

50

Hb SO2 Index（ % ）80

Brown Sports

40

30

20

p < 0.001

MHE（ n = 10 ）Placebo（ n = 12 ）

60

40

20

10

0

p < 0.001

MHE（ n = 12 ）0W

0Placebo（ n = 12 ）

0W

12W

Fig. 5. Changes in Brown Spots on the skin (face).

Measured value: Average ± standard error mean (SEM).
Analysis: Dunnett (intra-group) and t-test (inter-group). MHE,
mixed herb extract.

12W

Fig. 6. Changes in Hb SO 2 Index in the skin (cheek)(Healthy subjects).

_ 244 _

Measured value: Average ± standard error mean (SEM).
Analysis: Dunnett (intra-group) and t-test (inter-group). MHE,
mixed herb extract.

Glycative Stress Research

Discussion

The ingestion of food products containing MHE resulted
in all cases in the suppression of yellowing or browning of
the skin. Also, for the subgroup of healthy subjects (excluding
the subjects suspected of having diabetes), there weresignificant reductions in blood 3DG after 12 weeks in contrast
to pre- administration levels.
Prior research reported 3DG generation inhibition by
MHE in vitro for MHE either as each individual herbs or a
mixture of the four types 2). Also, in an open study targetingtype 2 diabetes at a dosage of 600 mg/day (six times thedosage in this study), significant reductions in blood 3DG
were noted after 8 weeks and after 12 weeks in comparison
to pre-administration levels 4). Thus, it is likely that the dosageof 100 mg/day was not sufficient for subjects who have highblood glucose levels.
Significant differences were noted in the skin Melanin
Index before and after administration and in contrast to theplacebo group. The area measured is the inner part of theupper arm, which is normally not exposed and unlikely to be
impacted by solar radiation, so according to the measurement
principle, the value is deemed to include glycation end
products. Therefore, it is possible that a reduction in glycation
end products was expressed in the Melanin Index. Previous
studies have reported that the presence of carbonyl-modified
compounds due to UV radiation and glycation in the skin isrelated to the progressive yellowing of the skin 1, 9).

On the other hand, a comparison of the subgroup of
healthy subjects shows that complexion was significantly
improved compared to the placebo group. The flavonoids
and polyphenols in dokudami 10), hawthorne berries 11) andgrape leaves 12) in the MHE have vasodilator and antioxidant
properties, and thus these constituents were deemed to haveacted to improve blood flow. Also, the result was the sameas that for the previously reported MHE-containing vinegar
beverage 6) and we surmise that the same action has occurredagain in this study.

Conclusion

MHE suppressed yellowing and browning of the skin.
This is possibly caused by a reduction in carbonylation
due to suppression of 3DG generation. The improvement
in complexion could be possibly attributed to the herb
constituents. Additionally, a high degree of safety wasconfirmed, even with continuous ingestion.

Conflict of Interest Declaration

This study was funded by ARKRAY, Inc. and A- Kit,
Inc. was consigned with the implementation.

References
1) Ogura Y, Kuwahara T, Akiyama M, et al. Dermal carbonyl

modification is related to the yellowish color change ofphoto-aged Japanese facial skin. J Dermatol Sci. 2011; 64:
45-52.
2) 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
Medicine. 2008; 5: 93-98
3) 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 Medicine. 2010; 7: 26-35.
4) 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.
5) Tamura T, Yagi M, Nomoto K, et al. Anti-glycation effectof a novel herbal mixture made of mixed herbal extract
and two crude drugs: Short and long term effect. The
Science and Engineering Review of Doshisha University.
2012; 52: 244-252. (in Japanese)
6) Yagi M, Shimoide A, Hamada U, et al. Evaluation of theanti-glycation effect and the safety of a vinegar beveragecontaining indigestible dextrin and a mixed herbal extract:
A placebo-controlled, double-blind study. Glycative stressresearch. 2014; 1: 14-24.

7) Nomoto K, Yagi M, Arita S, et al. A survey of fluorescencederived from advanced glycation end products in the skinof Japanese: Differences with age and measurement
location. Anti-Aging Medicine. 2012; 9: 119-124.
8) Kamitani Y, Yagi M, Nomoto K, et al. Non-invasive
collection of stratum corneum samples by a tape-stripping
technique. Anti-Aging Medicine. 2013; 10: 55-59.
9) Ohshima H, Oyobikawa M, Tada A, et al. Melaninand facial skin fluorescence as markers of yellowish
discoloration with aging. Skin Res Technol. 2009; 15: 496502.
10) Jiangang Fu, Ling Dai, Zhang Lin, et al. Houttuynia cordata
Thunb: A review of phytochemistry and pharmacology andquality control. Chinese Medicine. 2013; 4: 101-123.
11) Dahmer S, Scott E. Health effects of hawthorn. Am Fam
Physician. 2010; 81: 465- 468.
12) European Medicines Agency. Assessment report on Vitis
vinifera L., folium (Final). 2010. http://www.ema.europa.
eu/docs/en_GB/document_library/Herbal_-_HMPC_
assessment_report/2011/01/WC500100390.pdf_ 2 45 _