original article the effect of edible purple …anti-aging.gr.jp/english/pdf/2012/9(2)6174.pdf62...

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IntroductionA variety of degenerative changes occur with age, and

our current research focuses on factors that increase the risk of harmful body changes. Representative risk factors include mental, physical and oxidative stress, and we are particularly interested in glycation stress 1).

Glycation is a process in which reducing sugars, such as glucose and fructose, and proteins react non-enzymatically to form glycated proteins. Further reactions lead to the formation of advanced glycation endproducts (AGEs), which are deposited in tissues and bind to receptors, such as RAGE (receptor for AGEs), and impair tissue function. These reactions and the accumulation of AGEs in tissues are referred to as glycation stress 2).

In skin, AGEs may accumulate with aging 3), and advanced glycation of collagen decreases skin-elasticity and is involved in the formation of wrinkles 4). Ages accumulation also induces photo-aging 5,6). Tissue aging may be reduced by inhibiting glycation, and several authors have investigated the anti-glycation activity of various edible plants and herbs 7-9).

Original Article

The Effect of Edible Purple Chrysanthemum Extract on Advanced Glycation End Products Generation in Skin: a Randomized Controlled Clinical Trial and in vitro Study

Masayuki Yagi, Keitaro Nomoto, Mio Hori, Takahiro Kitano, Hiroyuki Yabukita, Mari Ogura, Umenoi Hamada, Yoshikazu Yonei

Anti-Aging Medical Research Center and Glycation Stress Research Center, Graduate School of Life and Medical Sciences, Doshisha University

AbstractPurpose: We examined the effect of edible purple chrysanthemum (Chrysanthemum morifolium) powder in an in vitro model on the generation of advanced glycation end products (AGEs), and the effect of oral intake of chrysanthemum on skin and serum AGEs in a randomized controlled clinical trial (RCT).Method: We measured fluorescent AGEs (F-AGEs) generation for 3-deoxyglucosone (3DG), pentosidine, Nε-(carboxymethyl)lysine (CML), in a glycation model using human serum albumin. Different concentrations of edible purple chrysanthemum powder (EPC) and aminoguatin (AG) control were tested. In the RCT, 35 women (48.1 ± 6.1 years, BMI 25.7 ± 1.4) participated in a double blind test over eight weeks. The subjects were divided into placebo control (n = 12), low dose (LD: 50 mg, n = 12) and high dose (HD: 150 mg, n = 12) groups. One patient in the HD group dropped out, after developing pimples.Results: Addition of EPC powder reduced fluorescence of three F-AGEs in vitro by 50% (0.039mg/mL, 3DG, pentosidine and CML: < 0.01mg/mL) to less than or equal to the level produced by AG (F-AGEs: 0.077mg/mL, pentosidine: > 1.0mg/mL, CML: 0.708mg/mL). In the RCT, subjective symptoms, physical condition, skin AGEs fluorescence and blood biochemistry were similar at the beginning and end of the eight-week trial period. An intergroup significant difference was not observed in a skin elasticity. The intake dependent tendency was seen by the skin elasticity of the left cheek at the rate of change of the indices R2 as a result of the subclass analyses which compared test period before and after. It did not accept the adverse event by eight-week series uptake of trial products.Conclusions: The in vitro experiment confirmed that chrysanthemum extracts have an anti-glycation effect. The results of the RCT were inconclusive, however. skin-elasticity may be influenced by post-translation modification of collagen or other proteins. The test product was safe for human consumption.

KEY WORDS: glycation stress, advanced glycation end products (AGEs), polyphenol, luteolin, chlorogenic acid

Received: Oct 17, 2011 Accepted: Feb. 24, 2012Published online: Mar. 30, 2012

Anti-Aging Medicine 9 (2) : 61-74, 2012(c) Japanese Society of Anti-Aging Medicine

Prof. Yoshikazu Yonei, M.D., Ph.D.Anti-Aging Medical Research Center, Graduate School of Life and Medical Sciences Doshisha University

1-3, Tatara Miyakodani, Kyotanabe city, Kyoto Prefecture 610-0321, JapanTel: +81-774-65-6382 / Fax: +81-774-65-6394 / E-mail: [email protected]

One such herb, the edible purple chrysanthemum (EPC), Chrysanthemum morifolium, has been used in traditional Chinese medicine as a medication for detoxification, as an antipyretic and antiphlogistic, and for the treatment of some eye problems, such as blurred vision, itching, loss of vision, tired eyes or redness of the eyes 9). Chrysanthemum contains N1,N5,N10,N14-tetra-coumaroyl spermine, which is reported to increase the production of intracellular glutathione 10) and inhibit the formation of AGEs

9,11). Out of the various types of EPC, a variety called “Enmeiraku” was found to contain high amounts of polyphenol, luteolin and chlorogenic acid 9).

In the present study, we tested the effect of EPC “Enmeiraku” powder on AGEs production in vitro, and completed a double blind study on the effect EPC ingestion in healthy women. The study ran for eight weeks and investigated the effect of high and low doses of EPC on the levels of AGEs in blood serum or plasma and skin.

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Effect of Chrysanthemum Extract on AGEs Generation and Skin

Method

I. In vitro experimentTest product

The edible purple chrysanthemum, C. morifolium powder “Enmeiraku” (EPC) was provided by UNIAL Co. Ltd. (Itabashi-ku, Tokyo, Japan). The test product was obtained after drying, sterilizing, crushing and sieving the powder through a 120-mesh sieve. The profile of test EPC is presented in Table 1 9). Aminoguanidine (AG; Wako Pure Chemical Industries Co. Ltd., Osaka-city, Osaka, Japan) was used as a positive control. All test products were prepared as 10mg/mL (w/v) suspensions or solutions.

AGEs measurement(1) F-AGEs (fluorescent AGEs)

The AGEs-derived f luorescence was measured using a microplate reader ARVO MX 1420 Multilabel Counter (Perkin-Elmer Japan, Yokohama-city, Kanagawa, Japan), at an excitation wave-length 370 nm and fluorescent wave-length 440 nm 7,9). F-AGEs inhibition rate was calculated from:

F-AGEs inhibition rate (%) = {1-(A - B)/(C - D)} × 100

where:A = Fluorescence of test sample containing EPC or AG with glucose solution;B = Fluorescence of test sample containing EPC or AG with distilled water instead of glucose solution; C = Fluorescence of test sample without EPC or AG with glucose solution added and incubated;D = Fluorescence of test sample without EPC, AG or glucose solution.

The f luorescence of EPC and AG was tested at three concentrations (1.0mg/mL, 0.1mg/mL, 0.01mg/mL). A graph of the F-AGEs inhibition rate plotted, and the half maximal inhibitory concentration (IC50) was calculated

(2) 3-deoxyglucosone (3DG)The concentration of 3DG was measured by HPLC following

Kitano et al. 9).

(3) PentosidinePentosidine was measured by ELISA using a commercial

kit “FSK Pentosidine” (Fushimi Medicine Manufacture Place, Marugame-city, Kagawa, Japan) following Yonei et al. 7), Kitano et al. 9) and the manufacturer’s instructions.

(4) Nε-(carboxymethyl) lysine (CML)CML (Nε-(carboxymethyl) lysine) was measured by ELISA

using a commercial kit “CircuLex CML/Nε-(carboxymethyl) lysine ELISA Kit” (CyLex, Ina-city, Nagano, Japan).

Table 1 Profile of test sample

ORAC

Scavenging activity on superoxide *

Polyphenol

Luteolin

Chlorogenic acid

Unit Amount or activity

µmol TE/g

Unit/g

g/100g

mg/100g

g/100g

1900

4300

4.44

15

0.33

ORAC: oxygen radical absorbance capacity. *Measured by electron paramagnetic resonance (ESR) spectrometry using superoxide generation model with hypoxanthine and xanthine oxidase 9).

In vitro inhibition test of AGEs generationThe glycation (Maillard reaction) in vitro was modeled

following previously published methods 7,9). In brief, 0.05 mol/L phosphate buffer solution (PBS) (pH7.4), 8 mg/mL human serum albumin (HSA, Sigma Chemical, MO, USA) and 0.2 mol/L glucose solution were incubated at 60 °C for 40 hours. Under these conditions, the amount of AGEs generated is approximately as same as the amount generated after incubation for 60 days at 37 °C (Fig. 1).

EPC or AG were added to this model at 0.01, 0.1, 1.0mg/mL (w/v) concentrations. F-AGEs (AGEs-derived fluorescence), 3-deoxyglucosone (3DG), pentosidine, Nε-(carboxymethyl)lysine (CML) were measured and inhibition ratio of AGEs generation calculated.

Fig. 1. The amount of in vitro AGEs generated at different temperatureThe amount of AGEs is expressed as the intensity of AGEs-derived fluorescence (n = 3).

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II. Clinical trialSubjects

Seventy healthy Japanese female volunteers, who had expressed concern about “dull skin” or “slackened skin”, age 40 – 60 years, with a body mass index (BMI) 24 – 28, were selected .

Volunteers with any of the following conditions were excluded:

1) Any disease, such as diabetes mellitus, renal dysfunction or gastrointestinal disease requiring abdominal operation.2) Any patients taking drugs or supplements containing anti-oxidants.3) Any patients whom the investigators determined to be unsuitable for the study.

The skin-elasticity and skin-AGE f luorescence of all participants were tested. The skin-elasticity of the left cheek was measured by Cutometer™ (MPA580; Courage+Khazaka Electronic, GmbH, Kern, Germany), and AGEs-derived fluorescence (AF) of the left upper arm was measured by AGE reader™ (DiagnOptics, Groningen, Netherlands) 12). Of the 70 volunteers, 35 who presented with relatively low levels of skin-elasticity R2 index and high intensity of AF were selected to participate in the study (mean age 48.1 ± 6.1 years, mean BMI 25.7 ± 1.4 kg/m2)

Participants were divided into three groups at random: control (n = 12), low-dose test group (LD group; n = 12), and high-dose test group (HD group; n = 12). One patient in the HD group dropped out after developing pimples. All participants provided informed consent before joining the program.

Test productsEPC and the placebo were provided in tablet form (Table 2).

Both tablets were sweet and odorless, and similar in appearance or smell.

The tablets were tested for bacteria and heavy metals by Japan Food Research Laboratories, Shibuya-ku, Tokyo, Japan and for residual pesticides by Manufacture Analysis Scientific International Systems, Hirosaki-city, Aomori, Japan.

Study designThe study was a randomized controlled clinical trial

(RCT). Participants took a total of three tablets once daily with water after breakfast for eight weeks. Subjective symptoms, anthropometry, blood biochemistry and skin function were examined before the trial (0W), at four weeks (4W) and eight weeks (8W).

Intake ratio of test product was 96.6% in control, 99.2% in LD group, 98.4% in HD group and 98.1% in average.

Participants were instructed to avoid excessive eating, demanding exercise and lack of sleep during the trial, and to continue usual alcohol consumption habits. Each participant maintained a “daily diary” to record their health, any adverse effects, lifestyle-related habits, diet, exercise and compliance with instructions of the study.

The study was completed between January 31 and April 4, 2011 at DRC (Data Research Coordination) examination laboratory (DRC, Inc., Sakai-city, Osaka, Japan).

Subjective symptoms and anthropometryPhysical and mental symptoms were recorded and

rated following, on a 5-grade scale using the Anti-Aging QOL Common Questionnaire (AAQol) 8,12). The following anthropometric parameters were recorded: height (cm), body weight (kg), blood pressure (systolic/diastolic: mmHg), pulse rate (pulse/min) and body composition using a body composition meter, BC118D (Tanita Corporation, Itabashi-ku, Tokyo, Japan).

Biochemical parametersBlood samples (10 mL) were taken from the subjects at

0W, 4W and 8W. Biochemical measurements for the following parameters were completed at the Mitsubishi Chemical Medicine Corporation (Minato-ku, Tokyo, Japan): total bilirubin (mg/dL), aspartate aminotransferase (AST) (IU/L), alanine transaminase (ALT) (IU/L), lactate dehydrogenase (LDH) (IU/L), γ-glutamyltranspeptidase (γ-GTP) (IU/L), creatinine phosphokinase (CPK) (IU/L), uric acid (mg/dL), BUN (mg/dL), creatinine (mg/dL), highly-sensitive C-reactive protein (CRP) (mg/dL), dehydroepiandrosterone-sulfate (DHEA-s) (μg/dL), insulin-like growth factor-I (IGF-I) (ng/mL), cortisol (μg/dL), adiponectin (μg/dL), for sugar and lipid metabolism, fasting plasma glucose (FPG) (mg/dL), hemoglobin A1c (HbA1c) (%), glycoalbumin (%), immuno-reactive insulin (IRI) (μU/mL), total cholesterol (TC) (mg/dL), high-density lipoprotein-cholesterol (HDL-C) (mg/dL), low density lipoprotein-cholesterol (LDL-C) (mg/dL), triglyceride (TG) (mg/dL), atherogenic index for protein metabolism, total protein (g/dL), albumin (g/dL), albumin /globulin (A/G) ratio, and the electrolytes, sodium (Na), potassium (K), chlorine (Cl) (mEq/L), calcium (Ca) (mg/dL), iron (Fe) (μg/dL).

Serum pentosidine, CML, and plasma 3DG were measured using the methods described above.

Skin functionA quantitative evaluation of facial pigmentation and

wrinkles was made using a face imaging shot apparatus (VISIATM Evolution, Fairfield, NJ, USA) 13). This method distinguishes the difference in a color temperature of melanine (brown spot), hemoglobin (red spot) and silverfish (spot).

Skin moisture was measured using a Corneometer™ (CM825; Courage+Khazaka Electronic) 14). Transepidermal water loss (TEWL) in the cheek and the dorsal surface of the upper arm was measured using a TewameterTM TM210 (Courage+Khazaka Electric) 15). This method measures the vapor tension at two

Table 2 Tablet composition for EPC (HD and LD) and AG (amount per 3 tablets)

Chrysanthemum powder

Cellulose

Silicon dioxide

Calcium stearate

Caramel color

Total

UnitMaterial

mg

mg

mg

mg

mg

mg

HD

150

280

10

10

0

450

LD

50

362

10

10

18

450

Control (placebo)

0

410

10

10

20

450

HD = high dose; LD = low dose.

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points several millimeters from a skin surface and computes the water loss (g/h · m2). After washing face, the measurements were taken after 20 minute habituation at room temperature (25°C) and 60% humidity.

The level of melanin in the skin and degree of erythema were measured using a Mexameter™ (MX18; Courage+Khazaka Electronic), which irradiates the skin with a specific wavelength light, and measures the reflected light using a diode. The meter calculates melanin and erythema indices 16).

Skin-elasticity was evaluated using a Cutometer™(MPA580; Courage+Khazaka Electronic) 17,18). Briefly, a probe was placed on the skin surface, and an area of skin was drawn up into the probe using negative pressure; the length of skin drawn into the probe was then measured using a glass prism. The R2 index is the ratio of skin length recovery after elongation and constriction (Ua1/Uf1); a ratio of 1.0 indicates an ideal elastic, and the R2 value for normal skin ranges from 0.3 – 0.5. The R7 index is a ratio of skin-elasticity during constriction (Ur1/Uf1); the R7 value for elastic skin is close to 1.00. A previous study found the R2 and R7 are the most reliable indices derived from a Cutometer, and that these indices decrease with aging and are lower in patients with diabetes mellitus 19). The skin-elasticity of each participant was measured four times and the highest and lowest values removed before calculating the average skin-elasticity.

The skin color difference (L*a*b*) was measured using a CM-2600d portable spectrophotometer (Konica Minolta, Sakai-city, Osaka, Japan) 20).

Skin AGEs auto-fluorescenceThe amount of AGEs in a patient’s skin was measured using

an AGE Reader™ (DiagnOptics, Groningen, Netherlands) 21,22). In the present study, subjects were asked to rest their elbow

on the AGE Reader. Measurements were taken at the outer upper right arm, 10 cm away from the edge of the elbow. After wiping the measuring area using a cotton swab and alcohol, AF intensity was measured three times at the same point, and the results were expressed as the mean of three values.

Ethical Considerations and conflict of interestThe study was performed at a third party institution in

compliance with the ethical principles of the Declaration of Helsinki and the Personal Information Protection Law, and in accordance with “Ordinance regarding the Good Clinical Practice (GCP)” (MHW Ordinance No. 28, 1997). The ethical committee for clinical studies at DRC approved the study protocol.

The present study complies with the “conflict of interest“ instruction of Japanese Society of Internal Medicine. We declare that the Glycation Stress Research Center is supported in part by A-kit Co. Ltd. (Ogaki-city, Gifu, Japan).

Statistical AnalysisThe mean value ± standard error of mean (SEM) for

measurements were calculated with SPSS II software (IBM Japan, Minatoku-ku, Tokyo, Japan). The means of parameters before and after treatment were compared by Wilcoxon’s signed rank test or Dunnett test; Mann-Whitney or Tukey test was used for inter-group and sub class analysis. Results were considered significant at < 5% level. Patient safety was evaluated based on prevalence of adverse effects in individual subjects.

Results

I. In vitro experimentEPC had a greater inhibition effect on F-AGEs, 3DG,

pentosidine and CML production by the model, than AG at all three concentrations (Fig. 2). In consequence the inhibition ratio of AGEs generation for EPC was much lower than that for AG. The IC50 for EPC was less than or equal to AG for all AGEs tested (Table 3). The IC50 of EPC on 3DG was 1/3 or less than that for AG, the IC50 for pentosidine was 1/100, and for CML generation was 1/70 of that produced by AG. AGEs was not contained in the EPC itself.

II. Results of RCTObjective symptoms

For the 32 physical symptoms assessed by the AAQOL, the scores of three symptoms (diarrhea, p = 0.016; frequent urination, p = 0.020; hot flushes, p = 0.026) increased in the HD group (Table 4).

Although the diarrhea score differed between the HD and control groups (p = 0.030), the change in score between 0W (1.1 ± 0.1) and 8W (1.6 ± 0.1) was small. None of the patients reported loose intestines or the loose passage. “Eye pain” (p = 0.026), “palpitations” (p = 0.010), and “thirst” (p = 0.028) all increased in the LD group, but the inter-group difference was not significant

For the 21 mental symptoms assessed, the scores of “daily life is not enjoyable” increased in the HD group (p = 0.047), and the score for “pessimism” (p = 0.035) decreased in the LD group. However the scores for the LD and HD groups did not differ from the control.

In the HD group, for the 23 skin symptoms assessed, the score for “itchy skin” increased in the (p = 0.033); none of the skin symptom scores changed in the LD group, neither group differed from the control

None of the scores for the eight lifestyle-related behaviors assessed changed in either group or the control.

Somatometry and physiological examination (Table 5)In the HD group, three somatometric measures increased

between 0W and 8W: body weight (+ 0.8%, p = 0.016), BMI (+ 0.8%, p = 0.021). These changes are small (< 1.0%) and did not differ significantly between LD, HD groups and the control. Blood pressure did not change significantly in any group.

In the LD group, did not differ significantly from the control.

Biochemical examination In the HD group between 0W and 8W, total protein,

albumin levels and Ca significantly decreased. Indicators of sugar metabolism (glycoalbumin, IGF-I, IRI) significantly increased (Table 6). In the LD group, Na and Cl significantly decreased, and glycoalbumin, IGF-I, significantly increased. In the control group, glycoalbumin, IGF-I, significantly increased. However, none of the between-group tests (HD vs. control and LD vs. control) were significant.

For AGEs concentration, pentosidine decreased and 3DG increased in the HD group, pentosidine decreased, 3DG and CML increased in the LD group and the control. However, none of the between-group tests (HD vs. control and LD vs. control) were significant (Table 6).

Measurements of sugar metabolism found glycoalbumin and IGF-I increased in both the HD and LD groups, but the difference was not significantly different from the control.

For AGEs, the serum CML levels in the control group significantly increased at 4W (+83.7%, p = 0.004) and at 8W

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Table 3 IC50 in in vitro AGEs generation model

EPC

AG

PentosidineSample

0.039

0.077

F-AGEs

< 0.01

0.027

3DG CML

< 0.01

> 1.0

< 0.01

0.708

AGEs = advanced glycation end products; F-AGEs = fluorescent AGEs; 3DG = 3-deoxyglucosone; CML = Nε-(carboxymethyl)lysine; EPC = edible purple chrysanthemum; AG = aminoguanidine; IC50 = half maximal (50%) inhibitory concentration. Units: mg/mL.

Fig. 2. Inhibition ratio in the in vitro AGEs generation modelAG = aminoguanidine; AGEs = advanced glycation end products; F-AGEs = fluorescent AGEs; 3DG = 3-deoxyglucosone; CML = Nε-(carboxymethyl)lysine.

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Table 4 Physical and mental symptoms, lifestyle-related behaviors

Physical symptoms Tired eyes Blurry eyes Eye pain Stiff shoulders Muscular pain/stiffness Palpitations Shortness of breath Tendency to gain weight Weight loss; thin Lethargy Lack of sense of wellness Thirst Skin problems Anorexia Early satiety Epigastralgia Liable to catch colds Coughing and sputum Diarrhea Constipation Hair loss Gray hair Headache Dizziness Tinnitus Lumbago Arthralgia Edematous Easily breaking into a sweatFrequent urination Hot flashes Cold skin

n=11

2.8 2.4 1.5 3.5 2.4 1.6 1.6 3.5 1.2 1.9 1.7 1.8 2.5 1.3 1.3 1.3 1.6 1.5 1.1 1.7 2.2 3.2 2.1 1.4 1.4 2.3 2.4 1.9 1.8 1.6 1.2 1.9

0.3 0.2 0.3 0.3 0.4 0.2 0.2 0.2 0.1 0.3 0.3 0.4 0.3 0.1 0.2 0.1 0.2 0.2 0.1 0.2 0.3 0.3 0.3 0.2 0.2 0.3 0.5 0.3 0.3 0.3 0.1 0.3

±±±±±±±±±±±±±±±±±±±±±±±±±±±±±±±±

3.0 2.9 1.8 3.5 2.6 1.7 1.8 3.7 1.5 2.1 2.0 2.1 2.9 1.3 1.3 1.4 1.9 2.0 1.6 2.0 2.3 3.4 2.3 2.0 1.6 2.6 2.5 2.2 2.0 2.1 1.6 2.1

0.3 0.2 0.2 0.4 0.3 0.2 0.2 0.2 0.2 0.3 0.3 0.3 0.3 0.1 0.1 0.1 0.2 0.3 0.1 0.3 0.3 0.3 0.3 0.3 0.2 0.2 0.3 0.2 0.2 0.3 0.1 0.2

±±±±±±±±±±±±±±±±±±±±±±±±±±±±±±±±

0.194 0.046 0.301 1.000 0.343 0.792 0.206 0.216 0.102 0.631 0.474 0.438 0.263 1.000 1.000 0.453 0.197 0.039 0.016 0.140 0.785 0.102 0.301 0.072 0.197 0.161 0.595 0.167 0.399 0.020 0.026 0.260

HD group0W

n=114W + 8W avarage

Wilcoxonp value

n=12

2.9 2.2 1.4 3.7 2.6 1.3 1.6 4.0 1.2 2.3 1.6 1.5 2.3 1.4 1.5 1.6 1.9 1.5 1.6 2.2 2.3 2.8 2.0 1.4 1.6 2.5 1.8 2.3 2.3 1.8 1.8 2.6

0.3 0.2 0.1 0.3 0.3 0.1 0.2 0.3 0.1 0.2 0.1 0.2 0.2 0.1 0.2 0.2 0.2 0.2 0.1 0.2 0.2 0.4 0.2 0.1 0.2 0.3 0.2 0.3 0.3 0.2 0.2 0.3

±±±±±±±±±±±±±±±±±±±±±±±±±±±±±±±±

3.1 2.5 1.9 3.6 2.9 1.8 1.8 3.6 1.1 2.3 1.8 2.0 2.3 1.5 1.8 1.5 1.9 1.9 1.8 2.5 2.4 2.8 2.3 1.5 1.5 2.5 2.1 2.1 2.5 1.5 1.9 2.5

0.2 0.2 0.2 0.3 0.2 0.2 0.2 0.3 0.1 0.2 0.2 0.2 0.2 0.2 0.2 0.1 0.2 0.3 0.2 0.3 0.3 0.3 0.3 0.2 0.2 0.2 0.2 0.2 0.3 0.2 0.3 0.3

±±±±±±±±±±±±±±±±±±±±±±±±±±±±±±±±

0.380 0.071 0.026 0.713 0.053 0.010 0.319 0.226 0.785 0.792 0.361 0.028 0.875 0.792 0.123 0.380 1.000 0.162 0.160 0.143 0.739 0.829 0.194 0.408 0.725 0.527 0.121 0.931 0.480 0.102 0.832 1.000

LD group0W

n=124W + 8W avarage

Wilcoxonp value

n=12

2.6 1.9 1.4 3.1 2.0 1.3 1.6 3.3 1.1 1.8 1.5 1.8 2.0 1.4 1.7 1.8 1.8 1.7 1.4 2.2 2.4 3.6 1.8 1.7 1.4 2.3 1.8 2.3 2.3 1.8 1.7 2.7

0.3 0.3 0.1 0.3 0.2 0.1 0.2 0.3 0.1 0.3 0.2 0.2 0.2 0.2 0.3 0.2 0.3 0.3 0.1 0.2 0.4 0.3 0.2 0.1 0.1 0.3 0.2 0.3 0.2 0.2 0.3 0.4

±±±±±±±±±±±±±±±±±±±±±±±±±±±±±±±±

2.6 1.9 1.8 3.3 2.4 1.5 1.8 3.6 1.3 2.1 1.9 2.0 2.3 1.4 1.9 2.0 1.8 1.9 1.4 2.3 2.5 3.3 2.0 1.6 1.7 2.5 1.8 2.5 2.4 2.0 2.0 2.7

0.3 0.3 0.2 0.3 0.2 0.2 0.2 0.3 0.1 0.2 0.2 0.2 0.3 0.1 0.3 0.3 0.2 0.3 0.1 0.3 0.4 0.3 0.2 0.1 0.2 0.3 0.2 0.4 0.3 0.2 0.2 0.3

±±±±±±±±±±±±±±±±±±±±±±±±±±±±±±±±

0.726 1.000 0.123 0.206 0.061 0.059 0.429 0.340 0.102 0.071 0.054 0.334 0.260 1.000 0.167 0.414 0.829 0.518 1.000 0.453 0.783 0.288 0.221 0.480 0.098 0.234 0.317 0.194 0.669 0.301 0.038 0.748

Control Inter-group analysis0W

n=124W + 8W avarage

Wilcoxonp value

0.998 0.381 0.874 0.374 0.985 0.670 0.957 1.000 0.521 0.922 0.993 0.226 0.973 0.889 0.866 0.567 0.834 0.761 0.030 0.601 0.780 0.450 0.998 0.080 1.000 0.323 0.330 0.491 0.373 0.554 0.296 0.680

HD vs. Controlp value by Dunnet

0.994 0.318 0.913 0.502 0.532 0.507 0.987 0.290 0.615 0.375 0.532 0.277 0.672 0.978 0.906 0.922 0.755 0.973 0.763 0.737 0.883 0.267 0.991 0.831 0.494 0.999 0.634 0.999 0.890 0.094 0.258 0.814

Mental symptoms IrritabilityEasily angeredLoss of motivationNo feeling of happinessNothing to look forward to in lifeDaily life is not enjoyableLose confidenceReluctance to talk with othersDepressedFeeling of uselessnessShallow sleepDifficulty in falling asleepPessimismLapse of memory Inability to concentrateInability to solve problemsInability to make judgments readilyInability to sleep because of worriesA sense of tensionFeeling of anxiety for no special reasonVague feeling of fear

1.7 2.0 1.7 1.6 1.5 1.3 1.5 1.6 1.3 1.5 1.6 1.5 1.6 2.5 1.9 1.5 1.6 1.5 1.8 1.4 1.5

0.2 0.3 0.3 0.2 0.2 0.1 0.2 0.2 0.1 0.2 0.2 0.2 0.2 0.3 0.2 0.2 0.2 0.2 0.3 0.2 0.2

±±±±±±±±±±±±±±±±±±±±±

2.0 2.0 1.7 1.6 1.5 1.7 1.7 1.5 1.7 1.7 2.1 2.2 1.9 2.8 2.2 1.9 2.0 1.8 2.2 1.8 1.7

0.2 0.2 0.2 0.2 0.1 0.1 0.1 0.2 0.2 0.1 0.3 0.4 0.2 0.3 0.3 0.2 0.2 0.2 0.3 0.2 0.2

±±±±±±±±±±±±±±±±±±±±±

0.236 1.000 1.000 1.000 0.589 0.047 0.251 0.713 0.098 0.167 0.126 0.102 0.197 0.167 0.170 0.272 0.236 0.131 0.306 0.096 0.336

2.7 2.4 2.2 2.3 2.1 2.3 2.2 2.2 2.2 2.1 1.9 2.2 2.3 2.5 2.0 1.8 1.9 1.9 1.8 1.7 1.4

0.3 0.2 0.2 0.2 0.2 0.2 0.3 0.3 0.3 0.2 0.3 0.3 0.3 0.2 0.1 0.1 0.2 0.2 0.2 0.2 0.1

±±±±±±±±±±±±±±±±±±±±±

2.6 2.4 2.2 2.0 1.9 2.0 2.0 2.0 1.9 2.0 2.0 2.0 2.0 2.5 2.0 1.8 1.9 1.8 1.9 1.8 1.5

0.3 0.3 0.2 0.2 0.2 0.3 0.2 0.3 0.2 0.2 0.2 0.3 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.1

±±±±±±±±±±±±±±±±±±±±±

0.885 0.783 0.865 0.301 0.476 0.317 0.396 0.470 0.084 0.774 0.851 0.260 0.035 1.000 0.931 0.557 1.000 0.666 0.482 0.317 0.564

2.2 1.8 1.5 1.5 1.5 1.3 1.4 1.3 1.3 1.3 1.7 1.3 1.3 1.9 1.5 1.5 1.5 1.6 1.8 1.5 1.3

0.2 0.2 0.2 0.2 0.2 0.1 0.1 0.1 0.1 0.1 0.2 0.1 0.1 0.2 0.2 0.2 0.2 0.1 0.2 0.2 0.1

±±±±±±±±±±±±±±±±±±±±±

2.0 2.0 1.6 1.5 1.5 1.5 1.5 1.6 1.5 1.5 1.8 1.7 1.7 2.3 1.9 1.7 1.6 1.7 1.7 1.6 1.3

0.2 0.2 0.2 0.2 0.1 0.1 0.1 0.2 0.2 0.1 0.2 0.2 0.2 0.2 0.1 0.2 0.2 0.2 0.2 0.2 0.1

±±±±±±±±±±±±±±±±±±±±±

0.336 0.234 0.414 0.783 0.890 0.194 0.458 0.084 0.238 0.102 0.257 0.102 0.024 0.030 0.026 0.334 0.334 0.458 0.276 0.480 1.000

0.140 0.669 0.939 0.921 0.892 0.370 0.665 0.492 0.364 0.794 0.245 0.766 0.968 0.943 0.864 0.666 0.530 0.357 0.075 0.127 0.613

0.960 0.214 0.981 0.354 0.845 0.321 0.811 0.378 0.197 0.513 1.000 0.376 0.060 0.364 0.164 0.904 0.997 0.994 0.523 1.000 0.990

Skin symptoms Slackened skinConcerned about dull skinConcerned about poresDry skinFrequent pimplesItchy skinConcerned about spots or frecklesMake-up runs easilyPoor complexionOily faceCoarse skinNot elastic, not glossyConcerned about crows feetConcerned about rough skinBags under eyesCorner of eyes saggingCorners of mouth saggingThinning eyebrowsConcerned about slackened chinDull skinThinning hairSlow growing nailsDull, fragile nails

4.0 4.0 3.8 3.1 1.7 1.5 3.6 3.1 3.0 2.0 2.3 4.0 3.3 2.6 2.4 3.0 3.5 2.3 3.4 3.8 3.6 2.2 2.7

0.2 0.2 0.4 0.3 0.2 0.2 0.4 0.3 0.4 0.3 0.2 0.3 0.3 0.3 0.3 0.3 0.3 0.4 0.4 0.2 0.3 0.4 0.4

±±±±±±±±±±±±±±±±±±±±±±±

3.5 3.6 3.5 3.2 2.1 2.1 3.2 3.0 3.1 2.1 2.5 3.6 3.3 2.6 2.1 2.9 3.3 2.5 3.4 3.3 3.0 2.3 2.6

0.2 0.3 0.3 0.3 0.1 0.3 0.4 0.2 0.3 0.3 0.2 0.2 0.3 0.3 0.3 0.3 0.3 0.3 0.4 0.4 0.4 0.3 0.4

±±±±±±±±±±±±±±±±±±±±±±±

0.077 0.159 0.168 0.672 0.099 0.033 0.068 0.902 0.716 0.603 0.774 0.135 1.000 0.915 0.832 0.726 0.450 0.258 1.000 0.120 0.072 0.726 0.722

3.3 3.5 3.3 2.7 2.3 2.1 3.9 3.1 2.6 2.4 2.3 3.4 3.3 2.4 2.3 2.8 3.3 2.1 3.5 3.3 3.4 2.1 2.5

0.2 0.2 0.3 0.2 0.3 0.2 0.2 0.3 0.2 0.3 0.2 0.1 0.3 0.3 0.3 0.3 0.4 0.3 0.3 0.2 0.4 0.2 0.2

±±±±±±±±±±±±±±±±±±±±±±±

3.3 3.5 3.3 2.6 2.1 2.2 3.8 3.0 2.6 2.4 2.5 3.3 3.2 2.8 2.1 3.0 3.5 2.1 3.6 3.5 3.4 2.2 2.5

0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.3 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.3 0.3 0.2 0.1 0.3 0.3 0.3

±±±±±±±±±±±±±±±±±±±±±±±

1.000 0.666 0.891 0.414 0.470 0.454 0.732 0.492 0.902 0.903 0.435 0.557 0.773 0.098 0.558 0.398 0.301 0.886 0.798 0.374 0.792 0.732 0.861

3.9 3.7 3.6 3.0 1.8 1.8 3.3 2.8 2.4 1.8 2.0 3.8 3.5 2.7 2.3 3.3 3.5 2.4 3.5 3.3 2.8 1.7 2.6

0.2 0.2 0.3 0.2 0.3 0.3 0.3 0.3 0.3 0.2 0.2 0.2 0.3 0.4 0.4 0.4 0.3 0.3 0.3 0.3 0.3 0.2 0.4

±±±±±±±±±±±±±±±±±±±±±±±

3.5 3.5 3.4 2.8 2.0 2.0 3.3 2.7 2.3 1.9 1.8 3.3 3.2 2.5 2.2 3.1 3.3 2.4 3.5 3.4 3.1 2.2 2.8

0.3 0.3 0.3 0.3 0.2 0.2 0.3 0.3 0.3 0.2 0.2 0.3 0.3 0.4 0.4 0.4 0.4 0.3 0.3 0.3 0.3 0.2 0.3

±±±±±±±±±±±±±±±±±±±±±±±

0.026 0.339 0.059 0.163 0.096 0.288 0.752 0.671 0.599 0.470 0.574 0.006 0.131 0.717 0.435 0.480 0.675 0.725 0.595 0.807 0.491 0.018 0.272

0.989 0.930 0.831 0.217 0.356 0.670 0.403 0.921 0.752 0.834 0.626 0.370 0.366 0.955 1.000 0.994 0.989 0.429 0.967 0.241 0.111 0.427 0.490

0.152 0.718 0.641 0.725 0.510 0.528 0.590 0.975 0.924 0.663 0.870 0.100 0.184 0.381 0.980 0.480 0.397 0.769 0.842 1.000 0.518 0.325 0.486

Lifestyle-related behaviors SmokingConverted into ethanolAlcohol consumptionFrequency of alcohol drinking ExerciseSleeping hoursWater consumptionVDT working hours

Cigarettes/ day mL/daymL/day

times/weekdays/weekhours/day

L/dayhours/day

2.7 5.7

245.5 1.5 1.0 6.5 1.1 5.5

1.9 3.3 92.3 0.7 0.6 0.3 0.1 1.1

±±±±±±±±

3.2 6.1

262.5 1.6 1.5 6.4 1.1 6.1

2.2 3.3 90.5 0.8 0.7 0.3 0.1 1.1

±±±±±±±±

0.317 0.345 0.500 0.655 0.285 0.526 0.865 0.159

3.3 5.6

183.3 0.9 0.4 6.5 1.3 4.2

1.9 3.6 81.7 0.4 0.2 0.2 0.1 0.5

±±±±±±±±

3.3 6.0

184.4 1.0 0.5 6.6 1.3 4.1

1.9 4.2 89.2 0.5 0.3 0.3 0.1 0.5

±±±±±±±±

1.000 0.686 1.000 0.197 0.528 1.000 0.592 0.798

2.9 2.5

112.5 1.1 0.7 6.6 1.2 4.7

2.0 1.1 51.5 0.5 0.4 0.3 0.2 0.7

±±±±±±±±

2.9 1.9

110.4 0.9 1.4 6.4 1.1 4.9

2.0 0.9 49.2 0.4 0.6 0.3 0.1 0.6

±±±±±±±±

1.000 0.068 0.655 0.257 0.141 0.203 0.726 0.437

0.355 0.078 0.510 0.473 0.410 0.949 0.687 0.552

1.000 0.169 0.886 0.099 0.869 0.666 0.920 0.930

LD vs. Control

Mean ± SEM.

67


67

Mean ± SEM.

Table 5 Anthropometry

HeightWeightPercentage body fatBMISystolic blood pressureDiastolic blood pressureHeart Rate

cmkg%-

mmHgmmHg

bpm

n=11

155.42 61.85 33.51 25.58

121.27 74.55 67.36

1.75 1.70 0.96 0.47 3.25 3.05 1.14

±±±±±±±

62.34 34.21 25.78

125.09 75.73 67.50

1.67 1.05 0.45 3.90 2.42 1.88

±±±±±±

0.016 0.062 0.021 0.306 0.450 0.767

HD group0W

n=114W + 8W avarage

Wilcoxonp value

Inter-group analysis


LD vs. Controln=12

156.51 63.93 34.81 26.08

124.92 75.25 68.17

1.55 1.47 0.48 0.32 2.40 1.43 0.63

±±±±±±±

64.11 34.91 26.14

121.96 73.29 73.38

1.55 0.45 0.35 2.64 2.07 2.40

±±±±±±

0.476 0.456 0.433 0.182 0.248 0.054

LD group0W

n=124W + 8W avarage

Wilcoxonp value

n=12

158.58 63.63 33.41 25.31

121.83 73.17 68.08

1.73 1.31 0.50 0.37 3.52 2.03 1.42

±±±±±±±

63.54 33.91 25.26

124.08 73.79 67.21

1.40 0.52 0.35 4.01 1.97 2.44

±±±±±±

0.666 0.060 0.754 0.286 0.695 0.814

0.094 0.859 0.094 0.857 0.922 0.942

0.580 0.509 0.580 0.307 0.561 0.147

Control0W

n=124W + 8W avarage

Wilcoxonp value

Mean ± SEM.

Table 6 Blood biochemistry

Total proteinAlbuminA/G ratioASTALTLDHDirect bilirubinγ-GTPCPKBUNCreatinineUric acidNaClKCaFeTCLDL-CHDL-CTGFPGHbA1cGlycoalbuminAtherogenic indexIGF-ICortisolDHEA-sIRIAdiponectinhs-CRPCMLPentosidine3DG

g/dLg/dL-

IU/LIU/LIU/L

mg/dLIU/LIU/L

mg/dLmg/dLmg/dLmEq/LmEq/LmEq/Lmg/dLμg/dLmg/dLmg/dLmg/dLmg/dLmg/dL

%%-

ng/mLμg/dLμg/dLμU/mLμg/mLmg/dLμg/mLng/mLμg/mL

n=11

7.79 4.55 1.43

18.64 18.09

185.73 0.70

31.27 77.73 11.23 0.61 4.46

141.18 104.91

4.25 9.93

92.27 234.09 134.45 70.73

134.18 83.36 5.20

13.80 2.47

184.91 7.80

123.73 4.60

10.35 0.08 0.86

32.12 0.09

0.19 0.09 0.06 2.29 2.95 10.16 0.03 5.36 5.76 0.93 0.02 0.22 0.48 0.44 0.09 0.18 9.86 15.45 14.90 4.60 31.54 2.06 0.10 0.28 0.34 11.16 0.63 13.72 0.57 1.59 0.02 0.10 5.29 0.01

±±±±±±±±±±±±±±±±±±±±±±±±±±±±±±±±±±

7.48 4.44 1.48

18.23 17.09

188.14 0.72

30.14 84.41 11.84 0.60 4.39

140.36 104.05

4.20 9.72

90.50 223.14 126.91 69.95

125.50 83.77 5.17

14.09 2.41

211.95 8.05

114.64 5.26

10.20 0.12 0.99

18.60 0.12

0.14 0.07 0.06 1.40 2.12 8.98 0.05 5.41 6.31 0.79 0.02 0.22 0.27 0.41 0.09 0.15 10.23 12.30 12.03 5.26 31.80 2.03 0.09 0.29 0.34 11.42 0.36 11.29 0.42 1.56 0.04 0.05 1.87 0.01

±±±±±±±±±±±±±±±±±±±±±±±±±±±±±±±±±±

0.007 0.050 0.079 0.878 0.859 0.422 0.719 0.655 0.182 0.155 0.120 0.563 0.106 0.085 0.507 0.036 0.534 0.247 0.286 0.656 0.213 0.906 0.496 0.018 0.673 0.006 0.575 0.075 0.047 0.398 0.247 0.213 0.008 0.003

HD group0W

n=114W + 8W avarage

Wilcoxonp value



LD vs. Controln=12

7.55 4.54 1.52

25.08 29.75

180.58 0.65

46.25 123.75

11.93 0.62 4.75

141.33 104.92

4.14 9.78

86.83 228.42 137.00 62.67

114.33 88.00 5.23

13.29 2.83

153.87 7.83

120.75 6.63 8.22 0.11 0.89

51.49 0.09

0.14 0.09 0.04 2.37 4.17 6.78 0.06 10.24 41.74 0.74 0.01 0.33 0.36 0.45 0.10 0.13 10.56 14.04 11.55 4.66 14.33 4.50 0.10 0.31 0.32 13.32 0.59 24.03 0.66 1.17 0.04 0.32 7.97 0.01

±±±±±±±±±±±±±±±±±±±±±±±±±±±±±±±±±±

7.63 4.54 1.48

22.58 25.67

182.58 0.65

40.92 80.33 11.11 0.61 4.72

140.21 103.46

4.18 9.83

98.46 231.54 142.29 62.63

105.96 85.79 5.25

13.62 2.85

174.84 6.85

107.25 6.59 8.50 0.10 1.15

23.29 0.12

0.13 0.08 0.05 1.87 2.84 7.57 0.05 7.30 6.58 0.66 0.02 0.24 0.44 0.38 0.10 0.09 8.85 12.71 11.84 4.27 9.92 3.42 0.08 0.31 0.30 14.13 0.53 14.57 0.83 1.20 0.03 0.19 2.99 0.01

±±±±±±±±±±±±±±±±±±±±±±±±±±±±±±±±±±

0.284 0.959 0.170 0.138 0.125 0.530 0.892 0.194 1.000 0.410 0.479 0.753 0.020 0.006 0.824 0.421 0.480 0.388 0.147 0.555 1.000 0.289 0.402 0.009 0.623 0.031 0.182 0.724 1.000 0.109 0.480 0.034 0.003 0.017

LD group0W

n=124W + 8W avarage

Wilcoxonp value

n=12

7.58 4.37 1.36

16.83 13.83

166.00 0.60

21.17 83.08 12.43 0.62 4.57

140.75 104.42

4.13 9.79

85.92 224.92 130.83 69.33

112.17 78.42 5.08

13.30 2.40

163.17 7.50

114.42 4.53

10.08 0.05 0.69

47.35 0.09

0.13 0.07 0.04 0.84 1.02 7.59 0.05 3.60 6.21 0.82 0.02 0.23 0.37 0.58 0.11 0.13 8.94 11.29 10.45 4.26 20.82 1.58 0.07 0.21 0.30 8.52 0.66 19.82 0.60 1.30 0.01 0.09 5.57 0.01

±±±±±±±±±±±±±±±±±±±±±±±±±±±±±±±±±±

7.55 4.36 1.38

16.21 13.96

171.71 0.62

22.42 75.83 12.03 0.62 4.46

140.54 103.17

4.26 9.71

95.00 222.13 130.75 67.42 92.67 79.54 5.07

13.64 2.46

194.79 7.70

99.33 4.53 9.70 0.12 1.10

22.14 0.11

0.09 0.05 0.04 0.76 1.17 8.26 0.04 4.84 7.62 0.76 0.03 0.26 0.41 0.25 0.09 0.08 8.88 10.38 10.17 3.70 11.97 1.65 0.06 0.20 0.32 10.85 0.68 13.78 0.38 1.16 0.03 0.15 1.63 0.01

±±±±±±±±±±±±±±±±±±±±±±±±±±±±±±±±±±

0.624 0.894 0.495 0.285 0.788 0.158 0.888 0.323 0.091 0.875 0.878 0.722 0.238 0.040 0.348 0.270 0.146 0.666 0.859 0.366 0.158 0.289 0.798 0.009 0.448 0.007 0.969 0.239 0.753 0.169 0.099 0.005 0.004 0.038

0.074 0.314 0.644 0.714 0.961 0.777 0.799 0.642 0.089 0.496 0.366 0.994 0.400 0.771 0.266 0.542 0.828 0.644 0.647 0.994 0.950 0.939 0.864 0.808 0.716 0.715 0.984 0.998 0.645 0.767 0.159 0.312 0.636 0.948

0.564 1.000 0.197 0.951 0.448 0.660 0.724 0.523 0.964 0.865 0.758 0.817 0.130 0.908 0.613 0.447 0.596 0.636 0.599 0.785 0.579 0.437 0.685 0.989 1.000 0.644 0.317 0.974 0.946 0.110 0.162 0.899 0.652 0.996

Control0W

n=124W + 8W avarage

Wilcoxonp value

(+35.1%, p = 0.034), however did not change significantly in either the LD or the HD groups (Fig. 3a). There was no significant inter-group difference.

A subclass analysis of difference in the change ratio from pre-value to average value of 4W and 8W was lower (+56.0 ± 15.5%) in LD+HD group than that in control (+68.6 ± 16.7%), however there was no significant inter-group difference (p = 0.394) (Fig.4).

Serum pentosidine decreased by 78.0% by 8W in the HD group (p = 0.003), 46.4% by 4W in the LD group (p = 0.023) and 64.0% by 8W (p = 0.003), however there was no significant inter-group difference (Fig. 3b).

Plasma 3DG increased 47.0% by 4W (p = 0.008) and 28.7% by 8W (p = 0.013) in the HD group, and 52.9% by 4W (p = 0.015) in the LD group , however there was no significant inter-group difference (Fig. 3c).

Skin functionSkin moisture and TEWL did not change in any group

but the melanin spots did not change in the HD (+ 0.3%, p > 0.05) and declined in the control (–6.7%, p = 0.020) with inter-group difference (p = 0.021) and erythema spots did not change between the beginning and the end of the study (Table 7). Skin image analysis by VISIA found no differences in skin color. Similarly, skin-elasticity changes varied: R5 and R7 increased in the HD group, eight measures changed significantly in the LD group, and seven in the control group. AGEs auto-fluorescence increased in the control group, but not in the HD or LD groups.

In the skin-elasticity test, the R2 the cheek in both sides did not change significantly in HD group (+ 5.8%) or control (+ 0.4%), but did improve in the LD group (+ 9.2%, p = 0.009) and did not differ between groups (Fig. 5a).

The R5 of the cheek improved in HD group (+ 20.4%, p= 0.001) and LD group (+ 18.5%, p < 0.001), but did not differ between groups (Fig. 5b).

68


Fig. 4. Change ratio of serum CML by subclass analysisComparison between control (n = 12) and LD+HD group (n = 23). CML = Nε-(carboxymethyl)lysine. Average ± standard error of mean (SEM).

Fig. 3. Serum AGEs concentration (average ± standard error of mean) at 0W, 4W and 8Wa: CML; b:pentosidine; c: 3DG. Comparison between control (n = 12), LD group (n = 12) and HD group (n = 11). AGEs = advanced glycation end products; 3DG = 3-deoxyglucosone, CML = Nε-(carboxymethyl)lysine. * p<0.05, ** p<0.01, *** p<0.001 vs. 0W.

69


69

Mean ± SEM.

Table 7 Skin analysis

MoistureMoisture levelTEWL

－g/h/m2

n=11

57.95 14.55

3.45 1.56

±±

53.91 16.25

3.00 1.96

±±

0.082 0.211

HD group0W

n=114W + 8W avarage

Wilcoxonp value



LD vs. Controln=12

56.13 11.08

4.16 1.11

±±

60.08 12.87

2.21 1.47

±±

0.153 0.009

LD group0W

n=124W + 8W avarage

Wilcoxonp value

n=12

60.25 11.68

3.62 1.26

±±

61.16 13.18

2.57 1.51

±±

0.742 0.056

0.322 0.942

0.236 0.916

Skin image analysis(Mexameter)MelaninErythema(Skin color difference)Melanin IndexHb IndexHb SO2 Index(%)ValueChromaL*a*b*(VISIA)SpotsPoresWrinklesTextureUV spotsPorphyrinsBrown spotsRed areas

－－

－－%－－－－－－－－－－－－－－

170.55 258.30

1.15 1.28

53.59 6.25 3.60

64.12 10.02 18.14

36.73 206.23

9.73 215.82 60.18 70.59

111.05 36.95

9.30 9.32

0.05 0.05 1.30 0.07 0.08 0.68 0.37 0.48

4.16 23.77 2.16 34.79 8.93 27.86 4.33 4.27

±±

±±±±±±±±

±±±±±±±±

171.00 298.89

1.15 1.30

54.94 6.25 3.64

64.13 10.35 18.07

37.91 206.59 10.07

230.23 58.50 67.93

110.25 37.39

11.03 8.91

0.04 0.06 1.51 0.06 0.06 0.62 0.30 0.46

4.32 24.61 2.16 27.50 8.34 26.93 4.93 4.36

±±

±±±±±±±±

±±±±±±±±

0.673 < 0.001

0.871 0.709 0.036 0.733 0.355 0.758 0.306 0.833

0.487 0.758 0.875 0.381 0.603 0.426 0.782 1.000

161.57 259.58

1.06 1.41

53.71 6.34 3.54

64.95 10.37 17.11

39.46 216.29

6.17 239.71 102.13 59.58

110.08 34.46

5.95 13.94

0.05 0.09 1.29 0.09 0.09 0.91 0.57 0.51

3.27 39.73 1.17 48.76 7.18 20.35 4.99 4.87

±±

±±±±±±±±

±±±±±±±±

147.85 301.16

1.03 1.36

56.41 6.41 3.49

65.70 10.23 16.88

39.54 222.56

6.00 245.19 99.96 65.73

108.08 35.33

6.58 15.56

0.04 0.07 1.21 0.08 0.08 0.78 0.49 0.45

3.03 40.09 1.23 50.33 7.05 19.86 4.19 4.75

±±

±±±±±±±±

±±±±±±±±

0.001 < 0.001

0.061 0.137

< 0.001 0.024 0.189 0.022 0.568 0.209

0.796 0.637 0.732 0.966 0.189 0.100 0.198 0.354

156.93 245.72

1.07 1.28

56.13 6.41 3.52

65.67 9.99

17.40

37.08 194.17

7.38 200.17 88.04 16.00

111.88 33.71

9.18 11.58

0.05 0.07 1.08 0.07 0.09 0.68 0.43 0.64

5.02 31.56 1.84 37.02 12.16 6.27 4.77 5.30

±±

±±±±±±±±

±±±±±±±±

146.47 294.54

1.06 1.32

57.28 6.40 3.54

65.62 10.33 17.13

37.48 211.46

6.06 220.42 91.27 16.94

111.27 34.31

11.57 12.49

0.06 0.08 1.22 0.08 0.10 0.76 0.51 0.63

5.11 29.60 1.23 33.87 12.56 6.00 5.10 5.17

±±

±±±±±±±±

±±±±±±±±

0.020 < 0.001

0.668 0.219 0.081 0.732 0.432 0.775 0.145 0.042

0.685 0.067 0.529 0.236 0.191 0.287 0.679 0.775

0.021 0.513

0.634 0.952 0.939 0.950 0.731 0.951 0.952 0.701

0.918 0.080 0.557 0.573 0.719 0.779 0.999 0.969

0.886 0.500

0.491 0.179 0.082 0.032 0.315 0.033 0.376 0.963

0.988 0.320 1.000 0.263 0.770 0.618 0.913 0.877

Skin Elasticity(Left cheek)R2R5R7(Mouth corner)R2R5R7

－－－

－－－

0.61 0.41 0.26

0.18 0.22 0.26

0.03 0.02 0.01

0.01 0.01 0.01

±±±

±±±

0.65 0.49 0.32

0.19 0.23 0.26

0.02 0.02 0.01

0.01 0.01 0.01

±±±

±±±

0.088 0.001

< 0.001

0.626 0.590 0.909

0.59 0.42 0.27

0.20 0.24 0.24

0.02 0.02 0.01

0.01 0.01 0.01

±±±

±±±

0.64 0.50 0.32

0.19 0.23 0.25

0.02 0.02 0.01

0.01 0.01 0.01

±±±

±±±

0.009 < 0.001 < 0.001

0.338 0.440 0.664

0.63 0.40 0.27

0.19 0.23 0.25

0.03 0.02 0.01

0.01 0.02 0.01

±±±

±±±

0.63 0.49 0.32

0.19 0.22 0.24

0.02 0.02 0.01

0.01 0.01 0.01

±±±

±±±

0.689 < 0.001 < 0.001

0.455 0.603 0.011

0.404 1.000 0.835

0.578 0.634 0.231

0.164 0.862 0.882

0.969 0.999 0.106

AGEs measurementAuto fluorescenceReflectance value

－－

1.90 0.14

0.06 0.01

±±

2.02 0.15

0.11 0.01

±±

0.328 0.182

2.00 0.16

0.07 0.01

±±

2.05 0.17

0.07 0.01

±±

0.209 0.308

1.93 0.16

0.10 0.01

±±

2.06 0.16

0.09 0.01

±±

0.050 0.937

0.916 0.351

0.485 0.898

Control0W

n=124W + 8W avarage

Wilcoxonp value

Fig. 5. Change of skin-elasticity in the cheeka:R2 (Ua/Uf); b: R5 (Ur/Ue); c: R7 (Ur/Uf).The change ratio of index from pre-values (0W) to the average values of 4W and 8W was compared among control (n = 12), LD group (n = 12) and HD group (n = 11).* p<0.05, *** p<0.001 vs. 0W. Average ± standard error of mean (SEM).

70


The R7 of the cheek significantly improved in HD group (+ 20.3%, p < 0.001) and LD group (+ 19.1%, p < 0.001), while there was no significant inter-group difference (Fig. 5c).

In the corners of the mouth (average of both sides), there was no significant difference in changes of R2, R5 or R7.

A subclass analysis found the change of R2 of the left cheek was dose-dependent +10.6% in HD group, +5.0% in LD group and –5.2% in control, but did not differ between groups (Fig. 6).

Sk in AF of upper-ar ms measured by AGE reader significantly increased at 8W in HD group (+10.6%, p = 0.037) and LD group (+8.1%, p = 0.009), however there was no significant inter-group difference (Fig. 7).

A subclass analysis found the change ratio of AF was lower in LD+HD group (4W: –0.3 ± 2.6%, 8W: +9.4 ± 2.4%) than that in control (4W: +0.4 ± 4.4%, 8W: +12.4 ± 3.6%), however there was no significant inter-group difference (4W: p = 0.509, 8W: p = 0.385) (Fig. 7b).

The change ratio of AF was lower in LD + HD group (4W + 8W: + 4 .7 ± 2.3%) than that in control (4W + 8W: + 8.2 ± 3.7%), however there was no significant inter-group difference (p = 0.424) (Fig. 7c).

Fig. 6. Change of skin-elasticity in the left cheekThe change ratio of index R2 (Ua/Uf) from pre-values (0W) to the average values of 4W and 8W was compared among control (n = 12); LD group (n = 12) and HD group (n = 11).* p<0.05; ** p<0.01 vs. 0W. Average ± standard error of mean (SEM).

Fig. 7. Intensity of AGEs-derived AF measured from arm skina: comparison among control (n = 12), LD group(n = 12) and HD group (n = 11). * p<0.05, ** p< 0.01 vs. 0W. b: subclass analysis: comparison between control (n = 12) and LD + HD group (n = 23).　c: subclass analysis: comparison of change ratio between pre-values (0W) and average values of 4W and 8W. Average ± standard error of mean (SEM).

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Discussion

Anti-glycation effectHere we found EPC powder reduced 3DG generation in the

in vitro glycation model more than AG. However, no differences were found in serum levels of CML, pentosidine, 3DG and the AGEs-derived fluorescence in the clinical trial after eight weeks oral ingestion of the test product. Several measures of skin-elasticity improved in the LD group, and these improvements may be related to the anti-glycation action EPC.

However, no clear improvements were observed during the RCT. Here we demonstrated that EPC controlled AGEs generation more than AG in vitro. Skin-elasticity also improved in vivo but there was no improvement in serum AGEs concentrations or skin AGEs fluorescence after eight weeks. No adverse effects were observed during the test and we suggest that edible purple chrysanthemum powder may be taken as a safe antioxidant.

Our colleagues have reported oral administration of mixed herb extracts decreased blood serum concentrations of 3DG level in 8 weeks, and CML in about 12 weeks 23). This suggests that the observation period of the present study may not have been long enough to detect decrease CML and pentosidine. In order to evaluate an effect on the in blood AGEs of an anti-glycation substance, it will be necessary to construct the test protocol for 12 weeks or more.

Some anti-glycation products, such as AG 24-28), have been developed to control AGEs generation, and in the present study, AG was used to evaluate the comparable effect of the EPC.

Astragaloside, derived from Astragalus radix, is known to control CML and the pentosidine generation in vitro 29). An in vitro study and experiment using diabetic rats found a mixture Anthemis nobilis, Crataegus laevigata, C. oxyacantha, Houttuynia cordata, and Vitis vinifera controlled the generation of 3DG, pentosidine, and CML 7). As chrysanthemum contains luteolin and chlorogenic acid, which are known to act on AGEs both in vivo and in vitro 9,11), we expected EPC to have a similar effect.

LuteolinLuteolin (Fig. 8) is a plant f lavonoid, used as a yellow

coloring matter and found various food plants, including Perilla frutescens var. frutescens, Perilla frutescens var. crispa, Chrysanthemum morifolium, Capsicum annuum, Mentha plants, Rosmarinus officinalis, Apium graveolens, Malus pumila, and Matricaria recutita. Luteolin is an aglycone and does not

combine with sugar but often exists in glycoside forms, such as luteolin-7-o-glucoside and luteolin-4-O-glucoside, that are not easily digested and absorbed. Luteolin and its derivatives have an antioxidation effect 30-32), anti-inflammation action 33), anti-allergic 34-36), antitumor activities 37-39), and adepressor effect 40), anti-bacterium activity 41), repress blood-pressure originating in paroxysmal atrial fibrillation (PAF) 42) and repress osmotic cataracts 43). Other reports found luteolin derived from Phyllostachys nigra hampers aldose reductase and generation of AGEs 44), and in consequence, this substance has been used as a therapy for diabetic complications.

Any food that includes high concentrations of polyphenols, such as luteolin, anthocyanidin, and chlorogenic acid are likely to inhibit AGEs generation.

Chlorogenic acidChlorogenic acid (5-caffeoylquinic acid) is a compound

in which the carboxyl of a caffeic acid and the hydroxy of the quinic acid are condensed (Fig. 9). It is found in the seeds and leaves of many dicotyledon plants including coffee. Chlorogenic acid is unstable in heat, breaks down easily into caffeic and quinic acids and is reported to be an antioxidant 45-47) and andtihypotensive 48,49). In addition, this substance is reported to control post prandial blood sugar 50), lipid hyperoxidation of the liver caused by carbon tertrachloride 51), amylase activation in the pancreas and sputum 52), N-methyl-N-nitrosourea (MNU) caused by stomach gland cancer 53), and reduce the activity of blood angiotensin by converting the enzymes ACE 54) and ACTH 55). These actions occur after meals, and thus blood sugar may be controlled by chlorogenic acid blocking α-glucosidase action, thus inhibiting glucose absorbtion in the small intestine 50). As a result, it is possible that chrysanthemum constituents, by controlling blood sugar and the inhibition action of luteolin, may help prevent diabetic complications, by inhibiting AGEs generation .

The act ive ing redients d if fer between species of chrysanthemum: C. indicum contains less polyphenol than C. morifolium and, as a result, C. morifolium may have higher anti-glycation activity and pentosidine inhibition effect than other chrysanthemum species 11). Activity may also differ by variety: C. morifolium Enmeiraku was reported to have a higher ORAC, superoxide erasure activity, polyphenol, luteolin and chlorogenic acid activity than the C. morifolium Aboukyu variety 9).

Previous studies have found chrysanthemum decreases oxidized LDL 56), elevates glutathione formation 10), and has anti-inflammatory 57), anti-oxidation 58,59), anti-tumor 60,61), anti-virus 62,63), and anti- tuberculosis bacteria effects 64), and represses

Fig. 9. Structure of chlorogenic acidFig. 8. Structure of luteolin

72


blood sugar elevation 65). A constituent of chrysanthemum component controls the formation of CML, pentosidine 11) and 3DG 9) in vitro and since C. morifolium also effects sugar metabolism in the small intestine by inhibiting α-glucosidase, the extract may also inhibit blood sugar rise 65).

Nevertheless, the present study found little evidence of changes, (in vivo) in biochemical glycation measures or skin ageing measures during the trial period. No differences between groups were observed for serum AGEs concentration or skin fluorescence, although several measures of skin-elasticity (for example, left cheek R2) did improve.

The biochemical pathway that generates and degrades AGEs is complex and divergent. It is possible that the regulation of one pathway by single product will not regulate the product of AGEs as an alternative pathway for AGEs may exist 8).

SafetyAn evaluation of the Natural Medicine Comprehensive

Database found that although C. morifolium may increase blood flow and insulin receptivity, there is insufficient scientific data to draw firm conclusions on pharmaceutical effects of EPC 66). Although scores for “diarrhea” and “itchy of the skin” rose in the HD group, the difference between groups was not significant and we consider that neither of these symptoms could be attributed to EPC consumption. Moreover, chrysanthemum has been used as a food in China and Japan for more than 150 years, and there have been no reports of any detrimental or side effects. This would indicate a toxicity test is not required 67).

Nevertheless, C. morifolium may increase photosensitivity 66), and as with other herbs and herb extracts, hypersensitive persons, pregnant women and nursing mothers should be warned of potential allergic reactions.

ConclusionHere we demonstrated that EPC controlled AGEs generation

more than AG in vitro. Skin-elasticity also improved in vitro but there was no improvement in serum AGEs concentrations or skin AGEs fluorescence after eight weeks. No adverse affects were observed during the test and we suggest that edible purple chrysanthemum powder may be taken as a safe antioxidant.

Conflict of interest statement:The authors declare no financial or other conf licts of

interest in the writing of this paper.

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73

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