Glycative Stress Research
Online edition : ISSN 2188-3610
Print edition : ISSN 2188-3602
Received : December 15, 2015
Accepted : February 1, 2016
Published online : March 31, 2016

Original article

An open-label clinical trial of Geranium dielsianum extract administered for
12 weeks: Anti-glycative actions, skin quality, and intestinal environment
Yoshikazu Yonei 1), Wakako Takabe 1), Masayuki Yagi 1),
Katsumi Takahashi 2), Misaki Ito 2), Hiroko Morii 2)
1) Anti-Aging Medical Research Center / Glycative Stress Research Center, Graduate School of Life and Medical Sciences,
Doshisha University, Kyoto, Japan
2) TOWA CORPORATION Ltd., Tokyo, Japan

Abstract

Objectives: Numerous in vitro and in vivo studies of the functionality of Geranium dielsianum extract (GDE:MISKAMISKAT M ) have found many novel functions, including improvements in the intestinal environment. The present
study was conducted to evaluate the effects of GDE in preventing glycation, maintaining beautiful skin, and improving theintestinal environment in healthy women, and to verify its safety in an uncontrolled open-label clinical trial using a test
material based on GDE.
Methods: Thirty-three women, mainly those aged 30 to < 50 years who had a constipation tendency and rough skin problems,and preferred eating sweets and carbohydrates in their daily diet, were recruited. From among these 33, the 13 women having
the highest values of the glycative stress index skin AGE deposition (Auto Fluorescence: AF) as determined using the AGE
Reader ® (DiagnOptics), were selected. One woman dropped out of the study; the remaining 12 women (mean age: 41.5 ± 5.0
years) received the test material for 12 weeks. The daily consumption of the primary ingredients of the test material was 1,200
mg of GDE and 800 mg of each crystalline cellulose and calcium stearate. Before taking the test material (Week 0), andat Weeks 8 and 12 of its ingestion, blood chemistry, a lifestyle survey using the Anti-Aging QOL Common Questionnaire(AAQOL), a questionnaire-based survey on the skin, and a questionnaire-based survey on bowel movement were performed.
Skin AF was determined as a glycative stress index. As dermatological endpoints, skin elasticity (R2, R7), water content
of the stratum corneum, and transepidermal water loss were measured using the Cutometer (Courage & Khazaka),
Corneometer, and Tewameter, respectively. Blood oxidative stress (bOS) and blood antioxidant power (bAP) in plasma
samples were measured as oxidative stress indexes using Spotchem (Arklay).
Results: The skin questionnaire survey revealed that the subjective symptom items ‘Dry skin’, ‘Make-up becomes runny’,‘Dull skin’, and ‘Skin smoothness’ improved significantly (p < 0.05) at Week 12 of ingestion of the test material. ‘Skin
moisture’ improved significantly (p< 0.05) at Weeks 8 and 12 of ingestion. The AAQOL survey showed that ‘Constipation’
symptoms were significantly relieved, and the defecation frequency began to increase significantly at Week 1 of ingestion,and continued to increase up to Week 12 (p< 0.001). Blood chemistry showed that HDL cholesterol increased significantly
after ingestion of the test material (baseline value, 66.3 ± 14.7 mg/dL; 7.8%, p = 0.034), and that blood oxidative stress (bOS),
an oxidative stress index, did not change, but another index, blood antioxidant power (bAP), increased significantly (14.5%,
p = 0.013). Skin moisture retention test revealed a significant improvement (14.0%, p= 0.040) in the water content of thestratum corneum in the right upper arm at Week 12 of ingestion, and a significant reduction in transepidermal water loss in
the right upper arm at Week 8 of ingestion (−18.9%, p= 0.020) and at Week 12 (−25.3%, p= 0.002).
Subclass analysis was performed in two groups according to pre-ingestion skin AF: those with a relatively high value of≥ 2.19 (median) and those with a relatively low value of <2.19. In the subjects with higher skin AF values, the AF value andfasting blood glucose did not change, but HbA1c decreased significantly at Week 12 (baseline value, 5.45 ± 0.22; −2.2%,
p=0.034). Supplementary analysis was performed to determine the relationship between skin AF value and actual age. The
AF value from actual age was calculated. In 3 of the 4 subjects with a skin AF value of ≥25% higher than the baseline actual
age, the AF value decreased at Week 12. No serious adverse events were found during or after the end of the study period.
Conclusion: The 12-week uncontrolled open-label clinical trial with the GDE-based test material demonstrated bowel
movement improvement and skin moisture-retaining effects in support of the previously reported in vitro and in vivo studies.
No definite data demonstrating the anti-glycation action of GDE were obtained in the present study. However, since in vitro
studies revealed potent anti-glycation action and carbohydrate absorption suppressing action, and since supplementary analysisof selected subjects with high age-corrected AF values revealed AF value reductions, the anti-glycation action of GDE is
expected to be demonstrated through a double-blind study with limited subject conditions in the future. The safety of ingested
GDE in humans was established.

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, Tataramiyakodani, Kyotanabe-shi, Kyoto, 610 - 0321 Japan
Phone/Fax: +81-774 -65-6394 Email: yyonei@mail.doshisha.ac.jp
Co-authors: Takabe W, wtakabe@mail.doshisha.ac.jp; Yagi M, yagi@yonei-labo.com;
Takahashi K, k.takahashi@towacorp.co.jp; Ito M, misaki@towacorp.co.jp; Morii H, hmorii@towacorp.co.jp_ 44 _

Glycative Stress Research 2016; 3 (1): 44-55(c) Society for Glycation Stress ResearchGlycative Stress Research

KEY WORDS: Geranium dielsianum, oxidative stress, glycative stress, constipation, moisturizing action
Introduction

A number of plants belonging to the family Geraniaceae,
such as Geranium dielsianum, Geranium lecheri Knuya,
and Geranium ayabacense Wild ex HBK, are native tothe highlands in the Central Andean Mountains in Peru.
In particular, Geranium dielsianum (GD) is reportedly
indigenous exclusively to the Peruvian Andes as its only
habitat in the world, and has traditionally been recognized
as being highly effective against diabetes mellitus 1, 2). In
Peru, GD is infused to make a popular herbal tea.
To elucidate the useful bioactivities of GD, we haveconducted chemical and physiological investigations using
MISKAMISKAT M, a Geranium dielsianum extract powder(GDE). To date the functionality of GDE has been studied
with a focus on in vitro or in vivo experimentation 3, 4), withfew human studies reported; therefore data to clarify thebioactivities of Geranium dielsianum is lacking.
The present study was conducted as an uncontrolled
open-label clinical trial in women who had a constipation
tendency and rough skin problems, and preferred eating
sweets, to evaluate the anti-glycation effect, skin-conditioning
effect, and intestinal environment-improving effect of GDE
taken for 12 consecutive weeks.

Methods
Subjects

Thirty-three Japanese healthy women aged 30 to < 50years meeting all the following three criteria were recruited:i) preferring eating sweets and carbohydrates, ii) suffering
constipation, and iii) having rough skin problems. Fromamong them, 13 women with large amounts of advanced

glycation end products (AGEs) deposited in the skin 5 ) as
determined using the AGE Reader ® (DiagnOptics, Groningen,
The Netherlands), were enrolled in the study. One subject
experienced cystitis during the study period, and was excluded
from the analysis set at the investigator’s discretion, leaving
12 subjects for data analysis [age, 41.50 ± 5.02 years; height,
158.38 ± 2.92 cm; body weight, 54.94 ± 10.32 kg; body massindex (BMI), 21.95 ± 4.34].

Study design

The present study was conducted as an uncontrolled
open-label clinical trial. The test material was a tablet
formulation containing 150 mg of Geranium dielsianum
extract powder (GDE). Each subject took 8 tablets every day(GDE dose of 1,200 mg/day) in two divided portions (4 tablets
taken after breakfast and 4 tablets after dinner) for 12 weeks.
Even when no food was taken, each subject took the test
material alone. The test material was supplied by Towa
Corporation (Tokyo, Japan).
Subjective symptoms were checked, and somatometry,blood chemistry, and oxidative stress tests were performed
before and at 8 and 12 weeks after the start of the study. Eachsubject recorded in the life diary the presence/absence andseverity of adverse events, test material ingestion status,
lifestyle habits, and dietary and exercise habits during thestudy period from January 8, 2015 to April 27, 2015.

Properties of the test material

The nutritive components and composition of the test
material are shown in Tables 1 and 2.

Table 1. Nutrition information of the test material per 8 tablets.
Item

AmountCalorie7.96 Kcal

Protein14.0 mg

Lipid

56.0 mg

Carbohydrate

1.85 mg

Sodium0.06 mg

Table 2. The composition of the test materials per 8 tablets.
Use

AmountActive ingredient

Geranium dielsianum extract (GDE)

1,200 mg

Excipient

Crystalline cellulose, calcium stearate

800 mgTotal

2,000 mg_ 45 _

Open Label Clinical Trial of Geranium dielsianum Extract

Endpoints(1) Subjective symptoms
Subjective symptoms were evaluated in two categories:
physical symptoms and mental symptoms. Their scores wererated according to five grades ( points 1 to 5) as previously
reported 6 -9) using the Anti-Aging QOL Common Questionnaire(AAQOL). A questionnaire-based survey on skin symptoms
was performed using a previously reported questionnaire
form (24 items) 8 ) . The number of defecations per day wascalculated from the records in their diary.
(2) Somatometry
Somatometry was performed for height (cm), bodyweight (kg), body fat ratio (%), body mass index (BMI),
basal metabolic rate (kcal), systolic and diastolic bloodpressure (mmHg), and pulse rate (/min). The body composition
was determined using a body composition analyzer (WELLSCAN500; Canon Lifecare Solutions Inc., Tokyo, Japan).
(3) Blood chemistry
The following hematology parameters were measured:
white blood cell count (WBC) ( /μL), red blood cell count(RBC) (x 10 4/μL), hemoglobin (Hb) (g/dL), hematocrit (Hct)(%), platelet count (Plt) (x 10 4/μL), MCV (fL), MCH (pg),
MCHC (%), and differential WBC counts (neutrophils,
lymphocytes, monocytes, eosinophils, basophils: %). Bloodchemistry parameters consisted of total protein (g/dL),
albumin (g/dL), A/G ratio, CPK (IU/L), AST (GOT, IU/L),
ALT (GPT, IU/L), LDH (IU/L), ALP (IU/L), γ - GTP (IU/L),
creatinine (mg/dL), uric acid (mg/dL), blood urea nitrogen
BUN (mg/dL), fasting blood glucose (mg/dL), HbA1c [NGSP](%), total cholesterol (TC) (mg/dL), HDL cholesterol (HDL-C)(mg/dL), LDL cholesterol (LDL-C) (mg/dL), triglycerides(TG) (mg/dL), serum electrolytes (Na, K, Cl (mEq/L), Ca(mg/dL), Fe (μg/dL)), and total bilirubin (mg/dL). Bloodchemistry testing was performed by LSI Medience
Corporation (Tokyo, Japan).
(4) Evaluation of glycative stress and skin functions
The measurements shown below were started after
cleaning the measuring sites of the body following 20-minuteacclimation in a constant-temperature constant-humidity
room (21 ± 1°C, 50 ± 5%). Tests were performed at A-KIT
Corporation (Ibaraki, Osaka, Japan).
a) Amount of AGEs deposited on the skin (glycative stressindex)As a glycative stress index, AGE-derived fluorescenceintensity was measured three times at one site of the bodyusing the AGE Reader ® 5) as previously reported 8, 9) . The
mean skin auto fluorescence (AF) value was adopted as thetest value. The measuring site of the body was the medial
part of the right upper arm.
b) Water content of the stratum corneum
The water content of the stratum corneum was measured
five times at the center of each measuring site of the bodyusing the Corneometer (CM825; Courage +khazaka, Cologne,
Germany) 11, 12) as previously reported 9, 10). The mean of threemeasurements (excluding the data of the maximum andminimum values, was adopted as the test value. The measuring
sites of the body were the left cheek (midpoint between theearlobe and lip margin) and the medial part of the right upperarm (approx. 10 cm from the olecranon toward the shoulder).
c) Transepidermal water loss
Transepidermal water loss was measured five times
at the center of each measuring site of the body using the
Tewameter (TM300; Courage+khazaka) 13) as previously
reported 9, 10). The mean of three measurements (excluding thedata of the maximum and minimum values) was adopted as
the test value. The measuring sites of the body were the sameas stated above in b), the left cheek and the right upper arm.
d) Measurement of color differences
Color differences were measured five times at eachmeasuring site of the body using a spectrophotometer (CM600d; Konica Minolta, Inc., Osaka, Japan) as previously
reported 8, 10). The mean of three measurements (excluding
the data of the maximum and minimum L* values) was
adopted as the test value of each parameter. The evaluation
parameters consisted of Melanin Index, Hb Index, HbSO2
Index, and L*, a*, and b* values. The measuring sites of the
body were the same as stated above, the left cheek and theright upper arm.
e) Measurement of skin viscoelasticity
Skin viscoelasticity was measured five times aroundeach measuring site of the body using a single suction methodwith a Cutometer (CT580; Courage +khazaka) 14) as previously
reported 8 - 10). The mean of three measurements (excluding
the data of the maximum and minimum R2 values) was
adopted as the test value of each parameter. The evaluation
parameters consisted of R2 and R7. The measuring sites ofthe body were the same as stated above, the left cheek andthe right upper arm.
f ) Diagnostic imaging of the skin
Image analysis was performed using VISIA (VISIA®
Evolution; Canﬁeld Scientiﬁc, Fairfield, NJ, USA), as
previously reported 10) to obtain data on pores, freckles, andother findings. Data for the left face were recorded.
(5) Evaluation of oxidative stressPhysical oxidative stress was evaluated on the basis ofmeasurements of blood oxidative stress (bOS) and blood
antioxidant power (bAP) using the i-Pack Oxystress Test
(Spotchem; Arkray, Kyoto, Japan) 15). The correlation betweenbOS and derivatives of reactive oxygen metabolite (d-ROM)
was determined to be r = 0.973, and the correlation betweenbAP and biological antioxidant potential (BAP) to ber = 0.975 16).
Statistical analyses
Data were statistically analyzed by performing paired-t
test, two-way repeated measure ANOVA, or Dunnett’s test
using the Dr.SPSSII statistical analysis software (IBM SPSS
Japan, Tokyo, Japan). A significance level of ≤ 5% wasconsidered to indicate a significant difference.
●

Ethical review
After the ethics and rationality of the study werereviewed and approved at a human study ethics committee
meeting by the Ethics Committee of Tokyo Synergy Clinic(Chuo-ku, Tokyo, Japan), the present study was started
and implemented in accordance with the study protocol
approved thereby.
●_ 46 _Glycative Stress Research

Results

Subjective symptoms

The score improved significantly for 3 of the 32 itemsof physical symptoms in the Anti-Aging QOL Common
Questionnaire: ‘Lethargy’, ‘Skin problems’, and ‘Constipation’( Table 3 ). The score did not change significantly in any of
the 21 items of mental symptoms.
The Week-12 score improved significantly for 5 of the
24 items of skin symptoms: ‘Dry skin’, ‘Make-up becomesrunny, ‘Dull skin’, ‘Skin smoothness’, and ‘Skin moisture’(p<0.05) ( Table 4 ). The score tended to improve for the items‘Concerned about pores’, ‘Rough skin’, ‘Corners of eyessagging’, and ‘Make-up does not stay on’ (p < 0.01).
Defecation frequency analysis results are shown in Table 5.
The defecation frequency began to increase significantly at
Week 1 of GDE ingestion, and this increase continued up to
Week 12 (p< 0.001).
Somatometry and physical test results
Results of somatometry and physical test (bodycomposition) are shown in Table 6. Blood pressure rosesignificantly after ingestion of the test material. All of thechanges in systolic (6.2%, p < 0.001) and diastolic blood●pressure (9.1%, p < 0.001) fell within the respective rangesof reference values. No significant change was found in any
other parameter.
Blood chemistry
Data analysis results for blood chemistry are shown in
Table 7. HDL cholesterol increased significantly at Week 12
of ingestion (7.8%, p = 0.034). Although significant changes
were also found in white blood cell count (8.8%, p = 0.022),
monocyte count (−18.1%, p=0.019), and fasting blood glucose(5.3%, p = 0.040), all these changes fell within the respective
ranges of reference values.
●

Evaluation of skin functions ( Table 8 )The color difference test revealed significant changes
in L* ( baseline value, 70.22 ± 3.55; 1.3%, p = 0.014) and a*(baseline value, 5.88 ± 1.52; −8.8%, p< 0.050) at Week 12,
with no changes found in any other parameters.
Skin elasticity test revealed no significant change in theupper arm. In the cheek, however, the R2 (baseline value,
0.80 ± 0.04; −5.0%, p< 0.001) and R7 (baseline value, 0.45
± 0.06; −6.7%, p = 0.027) were significantly decreased at
Week 12.
●

Table 3. Physical symptoms by AAQOL.
0W

8W

12W

Lethargy

2.67 ± 1.15

2.08 ± 0.90

Skin problems

2.83 ± 0.72

Constipation

3.50 ± 0.80

p values
8W

12W

1.50 ± 0.52

0.581

0.021

2.25 ± 0.62

1.83 ± 0.72

0.798

0.043

2.50 ± 0.80

2.25 ± 0.97

0.239

0.034

Anti-Aging QOL Common Questionnaire: (AAQOL) 6 -9) is used and results are expressed as mean ± standard deviation, Dunnett’ s test vs. 0W, n = 12.

Table 4. Skin symptoms.
0W

8W

12W

Concerned about pores

3.25 ± 1.06

2.50 ± 0.90

Dry skin3.25 ± 1.14

Make-up becomes runnyp values
8W

12W

2.42 ± 0.90

0.106

0.055

2.50 ± 1.09

2.08 ± 0.90

0.197

0.005

3.17 ± 1.11

2.50 ± 1.00

2.17 ± 0.83

0.683

0.027

Rough skin3.08 ± 0.90

2.25 ± 0.75

2.00 ± 0.74

0.239

0.055

Corners of eyes sagging

2.67 ± 1.07

1.92 ± 0.67

1.75 ± 0.75

0.239

0.086

Dull skin3.08 ± 1.08

2.33 ± 0.78

2.00 ± 0.85

0.347

0.012

Make-up does not stay on

2.67 ± 1.07

2.08 ± 0.90

1.75 ± 0.75

1.000

0.086

Skin smoothness

3.58 ± 1.16

2.75 ± 1.06

2.33 ± 0.89

0.161

0.007

Skin moisture

3.42 ± 1.08

2.33 ± 0.65

1.92 ± 0.51

0.027

0.001

Skin Symptom Questionnaire 8) is used and results are expressed as mean ± standard deviation, Dunnett’ s test vs. 0W, n = 12.

_ 47 _

Open Label Clinical Trial of Geranium dielsianum Extract

Table 5. Change of bowel movement.
Time course

Stool frequency per day

p value

0W

0.40 ± 0.11

1W

0.74 ± 0.32

< 0.0012W

0.82 ± 0.40

< 0.0013W

0.85 ± 0.28

< 0.001

4W

0.90 ± 0.37

< 0.0015W

0.80 ± 0.28

< 0.001

6W

0.85 ± 0.28

< 0.001

7W

0.82 ± 0.28

< 0.0018W

0.77 ± 0.25

< 0.001

9W

0.83 ± 0.28

< 0.001

10W

0.87 ± 0.38

< 0.001

11W

0.85 ± 0.38

< 0.00112W

0.80 ± 0.35

< 0.001

Stool frequency per day is calculated by using the data of test diary. Results are expressed as mean ± standard
deviation, Dunnett’ s test vs. 0W, n = 12.

Table 6. Physical information.
0W

8W

12W

p values
Two-way ANOVA

8W

12W

Heightcm

158.38 ± 2.92

Weightkg

54.94 ± 10.32

54.88 ± 10.76

55.12 ± 10.43

0.659

0.978

0.826

Body fatkg

24.78 ± 6.55

24.43 ± 6.87

24.38 ± 5.97

0.569

0.601

0.521

BMI.

–

21.95 ± 4.34

21.92 ± 4.52

22.02 ± 4.41

0.623

0.953

0.827

BMR

kcal/kg

1144.75 ± 47.36

1144.67 ± 52.08

1147.17 ± 53.37

0.570

0.999

0.568

Systolic BP

mmHg

102.25 ± 13.75

106.33 ± 13.57

108.58 ± 11.46

0.019

0.112

0.011

Diastolic BP mmHg

69.42 ± 9.63

73.83 ± 9.18

75.71 ± 6.46

0.001

0.013

0.001

Puls

69.75 ± 7.40

71.29 ± 6.89

71.96 ± 7.24

0.413

0.562

0.330

/min

Data are expressed as mean ± standard deviation, two-way repeated measure ANOVA and Dunnett’ s test vs. 0W, n = 12. BMI, body mass index; BMR, basal
metabolic rate; BP, blood pressure.

_ 48 _Glycative Stress Research

Table 7. Blood chemistry test
0W

12W

p values

WBC/μL

3300 – 9000

4558.33 ± 1427.30

4958.33 ± 1465.02

0.022

RBC

x10 4/μL

380 – 500

431.00 ± 39.44

433.92 ± 34.73

0.525

Hb

g/dL

11.5 – 15.0

11.67 ± 2.27

11.73 ± 2.21

0.610

Hct

%

34.8 – 45.0

37.06 ± 5.35

37.22 ± 4.91

0.699

Plt

x10 4/μL

14.0 – 34.0

31.86 ± 5.41

31.01 ± 7.00

0.317

MCV

fL

85 – 10285.67 ± 9.74

85.83 ± 9.93

0.689

MCH

Pg

28.0 – 34.0

27.03 ± 4.51

27.04 ± 4.73

0.929

MCHC

%

30.2 – 35.1

31.25 ± 2.06

31.30 ± 2.20

0.751

Neutrophil%

40.0 – 75.0

61.66 ± 8.34

61.58 ± 8.33

0.970

Lymphocyte%

18.0 – 49.0

28.46 ± 7.62

29.58 ± 7.23

0.477

Monocytes%

2.0 – 10.0

6.37 ± 1.07

5.22 ± 1.12

0.019

Eosinophil%

0.0 – 8.0

2.95 ± 1.37

2.97 ± 2.02

0.973

Basophil%

0.0 – 2.0

0.57 ± 0.27

0.65 ± 0.46

0.628

Total protein

g/dL

6.7 – 8.3

6.93 ± 0.34

6.95 ± 0.25

0.870Albuming/dL

3.8 – 5.2

4.18 ± 0.27

4.18 ± 0.21

1.000

1.1 – 2.0

1.54 ± 0.20

1.54 ± 0.18

1.000

A /G ratio
CPK

U/L

40 – 150

81.00 ± 30.47

74.58 ± 21.67

0.495

AST (GOT)

U/L

10 – 40

17.75 ± 5.12

16.42 ± 3.15

0.223

ALT (GPT)U/L

5 – 4513.50 ± 6.29

12.08 ± 5.32

0.328

LDH

U/L

120 – 240

170.33 ± 24.55

161.92 ± 17.27

0.123

ALP

U/L

100 – 325

156.58 ± 54.78

157.08 ± 43.77

0.957

γ - GTP

U/L

30 以下

17.92 ± 8.93

17.33 ± 9.62

0.510

Creatinine

mg/dL

0.47 – 0.79

0.66 ± 0.08

0.63 ± 0.07

0.093

Uric acidmg/dL

2.5 – 7.0

4.41 ± 1.25

4.33 ± 1.21

0.658

BUN

mg/dL

8.0 – 20.0

13.49 ± 3.77

12.06 ± 3.99

0.067

FPG

mg/dL

70 – 109

82.92 ± 8.86

87.33 ± 9.87

0.040

%

4.6 – 6.2

5.47 ± 0.23

5.41 ± 0.30

0.206

TG

mg/dL

30 – 149

76.25 ± 54.87

61.58 ± 26.05

0.157

TC

mg/dL

120 – 219

190.33 ± 32.40

192.17 ± 27.86

0.622

HDL-C

mg/dL

40 – 95

66.33 ± 14.66

71.50 ± 12.72

0.034

LDL-C

mg/dL

65 – 139108.25 ± 21.71

106.42 ± 18.98

0.602

Na

mEq/L

137 – 147

139.33 ± 1.50

139.58 ± 1.24

0.600

K

mEq/L

3.5 – 5.0

4.14 ± 0.28

4.28 ± 0.28

0.281

Cl

mEq/L

98 – 108104.33 ± 1.56

104.50 ± 1.31

0.674

Ca

mg/dL

8.4 – 10.4

9.24 ± 0.36

9.25 ± 0.28

0.889

Fe

μg/dL

40 – 180

67.83 ± 40.68

73.58 ± 46.81

0.594

Total bilirubin

mg/dL

0.2 – 1.2

0.68 ± 0.23

0.69 ± 0.34

0.836

HbA1c [NGSP]

Data are expressed as mean ± standard deviation, paired-t test, n = 12.

_ 49 _

Open Label Clinical Trial of Geranium dielsianum Extract

Table 8. Skin test.
0W

8W

12W

0.70 ± 0.20

0.70 ± 0.18

1.01 ± 0.32

p values
Two-way ANOVA

8W

12W

0.70 ± 0.19

0.869

0.972

0.924

0.87 ± 0.30

0.88 ± 0.20

0.051

0.054

0.071

51.18 ± 12.07

53.48 ± 10.15

52.96 ± 8.85

0.567

0.494

0.646

L*

70.22 ± 3.55

70.98 ± 3.30

71.14 ± 2.94

0.018

0.046

0.014

a*

5.88 ± 1.52

5.23 ± 1.53

5.36 ± 1.15

0.017

0.013

0.050

b*

14.74 ± 1.99

15.05 ± 1.74

15.18 ± 1.80

0.294

0.463

0.222

0.99 ± 0.16

0.96 ± 0.14

1.00 ± 0.14

0.013

0.058

0.593

1.14 ± 0.32

1.06 ± 0.30

1.13 ± 0.32

0.249

0.209

0.947

55.18 ± 5.28

56.61 ± 7.58

55.47 ± 5.70

0.269

0.219

0.927

L*

67.90 ± 2.63

68.57 ± 2.58

67.86 ± 2.53

0.045

0.063

0.987

a*

8.62 ± 1.94

8.06 ± 1.54

8.60 ± 1.76

0.131

0.135

0.998

b*

17.65 ± 1.91

17.65 ± 1.62

17.84 ± 1.94

0.711

1.000

0.690

Elasticity

R20.90 ± 0.02

0.89 ± 0.02

0.88 ± 0.02

0.147

0.670

0.098

(Upper arm)

R7

0.69 ± 0.04

0.69 ± 0.04

0.67 ± 0.03

0.129

0.958

0.114

Elasticity

R20.80 ± 0.04

0.79 ± 0.05

0.76 ± 0.04

＜0.001

0.515＜0.001(Cheek)R70.45 ± 0.06

0.45 ± 0.06

0.42 ± 0.05

0.022

0.996

0.027

Stratum

Right upper

corneum waterarm

21.73 ± 6.18

24.11 ± 8.19

24.78 ± 6.38

0.053

0.117

0.040

33.94 ± 12.08

36.47 ± 9.30

36.38 ± 11.27

0.307

0.298

0.319

8.42 ± 2.05

6.83 ± 1.44

6.29 ± 1.40

0.003

0.020

0.002

13.61 ± 2.72

12.66 ± 3.23

13.44 ± 4.33

0.282

0.236

0.945

Score (%)

43.83 ± 8.35

44.40 ± 8.26

44.69 ± 8.02

0.297

0.491

0.229

Pores

Score (%)

22.09 ± 11.52

21.82 ± 11.11

22.59 ± 12.26

0.610

0.942

0.819

Image

Porphyrin

Score (%)

9.50 ± 10.03

10.67 ± 13.83

11.05 ± 15.39

0.550

0.697

0.541

analysisRed

Score (%)

22.37 ± 4.32

22.75 ± 3.32

24.00 ± 4.90

0.198

0.883

0.154

by VISIA

Spots

Score (%)

34.55 ± 6.81

35.05 ± 7.09

34.74 ± 6.14

0.875

0.828

0.972

TextureScore (%)

9.84 ± 2.70

10.16 ± 3.26

10.82 ± 3.73

0.270

0.819

0.200

UV Spots

Score (%)

33.45 ± 10.57

33.65 ± 10.92

31.72 ± 9.80

0.266

0.982

0.308

WrinklesScore (%)

7.20 ± 6.50

9.53 ± 10.34

7.30 ± 5.86

0.242

0.229

0.996

Melanin
Index
Color

Hb Indexdifference

Hb SO 2

(Upper arm)

Index (%)

MelaninIndex
Hb Index(Cheek)content

Hb SO 2
Index (%)

Left cheek
Right upper

TEWL

arm
Left cheek
Brown
Spots

Data are expressed as mean ± standard deviation, two-way repeated measure ANOVA and Dunnett’ s test vs. 0W, n = 12.

_ 50 _Glycative Stress Research

Skin moisture retention test revealed a significant
increase in the water content of the stratum corneum (14.0%,
p = 0.040) in the upper arm at Week 12 of ingestion, andsignificant decreases in transepidermal water loss in the upperarm at Week 8 (−18.9%, p = 0.020) and Week 12 (−25.3%,
p = 0.002) of GDE ingestion. In the cheek, no significant
change was found. Diagnostic imaging with VISIA revealed
that no parameters that changed significantly.
Glycative stress index ( Table 9 )The amount of AGEs deposited on the skin, a glycative
stress index, increased significantly from 2.14 ± 0.30 atbaseline to 2.26 ± 0.22 at Week 12 (5.6%, p = 0.028).

Safety evaluation

No serious adverse events were found during or after theend of the study period. Hematology and blood chemistry
revealed significant elevations of fasting blood glucose and
HDL cholesterol within the respective ranges of physiologicalchanges, but no clinically problematic variation was found;
there was no problematic finding concerning the safety ofthe test material.

●

Oxidative stress index ( Table 10 )Blood antioxidant power (bAP), an oxidative stress index,
increased significantly from 5,166 ± 835 μmol/L at baseline
to 5,917±527 μmol/L at Week 12 (14.5%, p = 0.013). Anotheroxidative stress index, blood oxidative stress (bOS), did notchange significantly during the study period.
●

Subclass analysis
Subclass analysis on the 12 subjects was performed in
two groups divided by the amount of AGE deposited on theskin (AF value): those with higher values (6 subjects) andthose with lower values (6 subjects) ( Table 11 ). In the subjects
with an AF value of ≥ 2.19 (median of the measurements),
fasting blood glucose did not change significantly, but HbA1cdecreased significantly from 5.45 ± 0.22% at baseline to
5.33 ± 0.23% at Week 12 (−2.2%, p = 0.034). In the subjects
with an AF value of < 2.19, neither fasting blood glucose nor
HbA1c changed significantly.
In the subjects with higher AF values, the AF value did
not change. In the subjects with lower AF values, the AF value
increased significantly from 1.88 ± 0.13 at baseline to 2.09 ±0.12 at Week 12 (11.2%, p= 0.013), whereas the transepidermal
water loss (right upper arm) was markedly decreased at
Week 12 (−35.4%, p = 0.001).
●

Discussion

GDE, the primary component of the test material, is an
extract from Geranium dielsianum (GD), a plant of the family
Geraniaceae reportedly indigenous exclusively to the Peruvian
Andes. GD, traditionally used as a herb tea, has been said tobe pharmacologically effective against diabetes mellitus andother conditions. Recent studies have focused on the effects of
GDE for preventing inflammation, adjusting intestinal
function (mitigation of diarrhea symptoms) 2), controlling
diabetes mellitus 1, 3), and improving lipid metabolism 16 - 18),
with anti-glycation action to suppress AGE production, as
demonstrated in an in vitro experimental system 19). In a
human serum albumin (HSA)/glucose reaction system, GDE
more potently suppresses the production of fluorescent AGEs
than aminoguanidine. In a collagen/glucose reaction system as
well, GDE suppresse the production of fluorescent AGEs andcarboxymethyllysine (CML), and these activities are moremarked than those of aminoguanidine.
As the functionality of GDE has been mainly investigatedin vitro or in vivo, but not in humans, we conducted apreliminary study to elucidate the likely useful bioactivities
of GDE in humans. We evaluated the effects of a GDEcontaining test material in preventing glycation, maintaining
beautiful skin, and improving the intestinal environment in
women, and verifying its safety. Thirty-three women aged 30

Table 9. Skin AF

Skin AF0W

12W

p value

2.14 ± 0.30

2.26 ± 0.22

0.028

Skin AGE deposit is evaluated as AF measured by AGE Reader ®. Data are expressed as mean ± standard deviation,paired-t test vs. 0W, n = 12. AGEs, advanced glycation end products; AF, skin auto fluorescence.

Table 10. Oxidative stress.
0W

12W

p valuebOS

μmol/L

13.43 ± 2.69

12.64 ± 2.66

0.136

bAP

μmol/L

5.166 ± 835

5.917 ± 527

0. 013bOS and bAP values are measured by i-Pack Oxystress Test (Spotchem). Data are expressed as mean ± standard
deviation, paired-t test vs. 0W, n = 12. bOS, blood oxidative stress; bAP, blood antioxidant power.

_ 51 _

Open Label Clinical Trial of Geranium dielsianum Extract

Table 11. Subclass analysys divided into 2 groups with high AF and low AF.
Subclass groupAF ≧ 2.19(n = 6)Item(n = 6)

12W

p values

FPG

mg/dL

84.17 ± 6.74

87.17 ± 6.74

0.312

HbA1c/NGSP%

5.45 ± 0.22

5. 33 ± 0.23

0.034

2.43 ± 0.16

2.43 ± 0.15

0.888

Right upper arm

20.93 ± 8.11

25.68 ± 8.67

0.070

Left cheek31.02 ± 13.74

32.55 ± 10.64

0.715

Right upper arm

7.45 ± 2.02

6.51 ± 1.69

0.445

Left cheek13.04 ± 2.00

13.47 ± 3.42

0.824

FPG

mg/dL

81.67 ± 11.11

87.50 ± 12.99

0.091

HbA1c/NGSP%

5.48 ± 0.26

5.48 ± 0.37

1.000

1.88 ± 0.13

2.09 ± 0.12

0.013

Right upper arm

22.52 ± 4.11

23.87 ± 3.53

0.070

Left cheek36.86 ± 10.58

40.22 ± 11.44

0.462

Right upper arm

9.39 ± 1.71

6.07 ± 1.15

0.001

Left cheek14.19 ± 4.02

13.41 ± 5.44

0.636

AF
Stratum corneum water content

TEWL

AF ＜ 2.19

0W

AF
Stratum corneum water content

TEWL

Data are expressed as mean ± standard deviation, paired-t test vs. 0W. AF, autofluorescence measured by AGE Reader ®. FPG, fasting plasma glucose; NGSP,National Glycohemoglobin Standardization Program; TEWL, transepidermal water loss.

to <50 years who had a constipation tendency and rough skinproblems, and preferred eating sweets and carbohydrates intheir daily diet, were recruited. From these 33, 13 women withthe highest values of AGE deposited on the skin were selected
via screening. One dropout was removed; the remaining 12
women took 1,200 mg/day of GDE for 12 weeks in an
uncontrolled open-label clinical trial. To obtain more definite
evidence for the characteristic anti-glycation action of GDE,
we attempted to identify ‘subjects with great glycative stress’
via screening; however, the number of subjects identified
with a high glycative stress index value was insufficientbecause the mean AF value was 2.14 for the mean age of
41.9 years.
Administration of GDE for 12 weeks relieved thesubjective symptom ‘Constipation’, and improved the skin
symptom items ‘Dry skin’, ‘Make-up becomes runny’, ‘Skin
darkening’, ‘Skin smoothness’, and ‘Skin moisture’. Bloodtesting revealed an increased blood antioxidant power (14.5%),
an increased water content of the stratum corneum (upperarm) (14.0%), and a decreased transepidermal water loss
(upper arm) (−25.3%); a marked skin moisturizing effect wasobserved. Skin findings included reductions in elasticity

indexes R2 (−5.0%) and R7 (−6.7%).
However, no definite results were obtained for glycolipid
metabolism and glycative stress. In the present study, bodyweight and body fat did not change significantly, and HDLcholesterol increased (7.8%); however, skin AF increased(5.6%) as a result of an increase in fasting blood glucose level(5.3%).
Taking into consideration the results of the present study,
the digestive tract functions of the subjects and changes intheir dietary habits must be discussed. GDE was shown to act
on the intestinal flora in laboratory animals 2). In rats, orally
administered GDE favorably influenced the intestinal flora
and increased Bifidobacteria and Lactobacilli and decreased
the Clostridium leptum subgroup and Bacteroides group 2).
With its essential effect of improving the intestinal flora, GDE
improves not only bowel movement, but also various digestive
tract functions. When analyzing the diary data, the subjects
seemed to have felt generally healthier; it is likely that
increased physical activity and appetite might have increased
their food consumption. The increase in food consumption
seems to have been such that fasting blood glucose wasincreased by 5.3% with no significant changes induced in_ 52 _Glycative Stress Researchbody weight, LDL cholesterol, or triglyceride levels. The skin
AF exhibited a similar increase (5.6%). The increased HDLcholesterol ( by 7.8%) seems to suggest increased physicalactivity.
Oxidative stress determinations revealed a 14.5% increasein blood antioxidant power (bAP), although there was nochange in blood oxidative stress (bOS). Various methods areavailable for evaluating the blood antioxidant powers of foods,
with oxygen radical absorbance capacity (ORAC) commonlyused as an index of blood antioxidant power in the US andEurope. GDE has a potent anti-oxidative potential with an
ORAC value of 5,100 μmol TE/g, 4) which is considered tohave increased the blood antioxidant power (bAP).
Subclass analysis was performed in two groups: thosewith higher skin AF values and those with lower values.
In the subjects with higher AF values, the AF value andfasting blood glucose level did not change significantly,
however the first-stage glycation product HbA1c decreasedslightly but significantly (−2.2%) ( Table11 ). GDE inhibits α
glucosidase 3), an enzyme that decomposes oligosaccharides,
such as maltose and sucrose. Inhibition of α glucosidase
activity inhibits the digestion of oligosaccharides in the small
intestine, and retards their absorption, which in turn relieves
postprandial hyperglycemia. The α glucosidase inhibitory
activity of GDE may suppress postprandial hyperglycemia
and reduce HbA1c.
In the subjects with lower skin AF values, the AF value
increased significantly by 11.2%, whereas the transepidermal
water loss (right upper arm) improved markedly (−35.4%)( Table 11 ). The water content of the stratum corneum tended
to be lower in the subjects with higher AF values, andhigher in the subjects with lower AF values, whereas thetransepidermal water loss tended to be smaller in the subjects
with higher AF values, and larger in the subjects with lower
AF values. The smaller transepidermal water loss observedin the subjects with higher AF values is attributable to lower
water contents, and thus the lack of transpiring water. In thesubjects with higher AF values as well, both the water content
of the stratum corneum and the transepidermal water loss
tended to improve; however, the differences were statisticallyinsignificant. The action mechanism for the skin moisturizing
effect of GDE remained unclear in this study; further researchis needed.
Next, AF values from actual ages were calculated using
the formula for the relationship between AF value and actualage 5). After the correction for age, the calculated values werecompared with actual measured values, and the subjects withhigh AF values for their age were selected ( Table 12 ). Results
showed that the subject’s baseline value was worse than theactual age value by ≥ 25% in 4 subjects, of whom 3 (ID;1501001, 1501022, and 1501032) had markedly decreased
AF change ratios of 94.1% to 99.0% at Week 12 of ingestionof the test material.
No serious adverse events due to GDE were found during
or after the end of the study period. However, the criticalfindings that blood pressure rose significantly within the rangeof physiological changes, and that skin elasticity indexes R2
and R7 decreased need to be discussed.
Systolic blood pressure rose by 6.2% from 102 ± 14 mmHg
at baseline, and diastolic blood pressure rose by 9.1% from69 ± 10 mmHg at baseline. The study subjects included anumber of hypotensive subjects, 4 with a systolic bloodpressure of <95 mmHg and 4 with a diastolic blood pressureof <61 mmHg. Among them, some had their blood pressureselevated by GDE into the normal range of blood pressure.
When analyzing the data exclusively for normotensive subjects(8 subjects with a systolic blood pressure of 100 to 140 mmHg
and a diastolic blood pressure of ≥60 mmHg), no significant
rise in systolic blood pressure was found ( Table 13 ). The
elevations of diastolic blood pressure also fell within therange of physiological changes, and the change ratio was low(4.1%, p = 0.033). Therefore, the elevations of blood pressure
found in the present study were not considered to be adverse
events.
Generally, skin elasticity indexes R2 and R7 areconsidered to indicate higher elasticity and hence morefavorable as they approach 1.0, i.e., have higher values.
However, this observation is only applicable to cases where
the skin water content is normal or constant. At low skinwater contents, skin elasticity indexes R2 and R7 sometimes
seem apparently high; an improvement of skin water content
could reduce the apparently high R2 and R7 to their truelevels. Therefore, the changes in skin elasticity indexes R2
and R7 found in the present study were not considered to be
adverse events.
Based on the lack of adverse effects, the safety of GDE
in chronic administration has been established in rats 20). No
safety issues were found in the present study as well.

Conclusion

In the present uncontrolled open-label clinical trial, theGDE-based test material taken for 12 weeks relieved subjective
skin symptoms, improved bowel movement, had a skinmoisture retention effect, and improved the anti-oxidative
potential. Safety of GDE in humans was established. Although
no definite data were obtained, the anti-glycation action of
GDE is expected to be demonstrated by conducting a doubleblind study with limited subject conditions in the future.

Acknowledgement

This study was partially supported by the JapaneseCouncil for Science, Technology and Innovation, SIP(Project ID 14533567), “Technologies for creating nextgeneration agriculture, forestry and fisheries” (funding
agency: Bio-oriented Technology Research Advancement
Institution, NARO). Abstract of this study was presented at
The 6th Symposium of Pruvian Herb Functional Research,
November 6, 2015, Tokyo.

Conflict of interest statement_ 53 _A part of this work was supported by Towa Corporation.

Open Label Clinical Trial of Geranium dielsianum Extract

Table 12. Profile of skin AF
0W

8W

12W

predicted *AF

AF (0W) ／AF% change of% change of

41

2.48

2.50

2.33

1.83

1. 3555

101.0%

94.1%

1501002

33

1.76

1.68

1.97

1.69

1.041495.2%

112.0%

1501004

44

1.85

2.18

2.23

1.88

0.9844117.7%

120.6%

1501006

34

1.89

2.01

2.04

1.71

1.1054106. 3%108.0%

1501008

42

1.78

2.06

2.16

1.85

0.9657115.6%

121.2%

1501009

38

2.32

2.52

2.42

1.78

1. 3079108.2%

104.3%

1501015

48

2.13

2.06

2.19

1.95

1.091796.9%

102.6%

1501016

41

1.86

2.09

1.95

1.83

1.0183112.1%

104.6%

1501020

45

2.37

2.23

2.39

1.90

1.249993.7%

100.8%

1501022

48

2.62

2.51

2.59

1.95

1. 3437

95.8%

98.6%

1501028

46

2. 39

2.59

2.60

1.92

1.2451108.4%

109.1%

1501032

38

2.24

2.22

2.22

1.78

1.2615

99.0%

99.0%

ID

Age

1501001

AF

AF

AF

( predicted *)AF 8W

AF 12W

Skin AGE deposit is evaluated as AF measured by AGE Reader ® . * Predicted AF value (y) is calculated, using chronological age (x), by the equation;y=0.0175x+1.1121, as previously reported in reference 5). AGEs, advanced glycation end products; AF, skin auto fluorescence.

Table 13. Profile of blood pressure in the subjects with normal blood pressure.
0W
Age

Blood pressure

8W

12W

p values

P value by ett’s

Two-way ANOVA

8W

12W

years

39.75 ± 4.40

Systolic mmHg

109.44 ± 10.57

113.00 ± 10.92

112.88 ± 10.90

0.357

0.344

0.368

Diastolic mmHg

75.06 ± 5.87

78.75 ± 6.46

78.31 ± 6.35

0.033

0.034

0.049

Data are expressed as mean ± standard deviation, two-way repeated measure ANOVA and Dunnett’ s test, n = 8.

_ 54 _Glycative Stress Research

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