Glycative Stress Research
Online edition : ISSN 2188-3610
Print edition : ISSN 2188-3602
Received : December 15, 2015
Accepted : January 18, 2016
Published online : March 31, 2016

Original article

Cleaving effect of melatonin on crosslinks in advanced glycation end products
Wakako Takabe, Riko Mitsuhashi, Lanny Parengkuan, Masayuki Yagi, Yoshikazu Yonei
Anti-Aging Medical Research Center and Glycative Stress Research Center, Graduate School of Life and Medical Sciences,
Doshisha University, Kyoto, Japan

Abstract

Objective: The cleavage of AGE crosslinks represents a mechanism for the degradation of advanced glycation end products(AGEs) of proteins. This study was conducted to evaluate the effects of melatonin (a sleep-related hormone) on AGE degradation,in comparison with representative plant-containing components.
Methods: Melatonin (0.4 nmol/L) was chosen as the test material, with N-phenacylthiazolium bromide (PTB) as a positive
control, and 28 plant-containing flavonoids (0.4 nmol/L) and 6 tea plant-containing catechins (10 mg/mL) as reference controls.
The evaluation of AGE crosslink cleaving activity was based on the phenomenon in which an equimolar amount of benzoic
acid is produced upon degradation of 1-phenyl-1,2-propanegione (PPD). After the reaction of PPD and each sample solution
or PTB (0.4 and 10 nmol/L), the benzoic acid released was quantified using high performance liquid chromatography. [Allmeasurements are expressed herein as mean ± standard deviation (n=3).]
Results: AGE crosslink degradation activity was determined to be 15.76 ± 0.38% for melatonin, 6.37 ± 2.71% for PTB (0.4nmol/L), and 23.00 ± 7.50% for PTB (10 nmol/L); melatonin was found to be more potent than the same amount of PTB.
Of the plant-containing flavonoids examined, urolithin B (14.89 ± 0.01%) and carnosic acid (9.01 ± 0.93%) were more active
than the same amount of PTB, but less active than melatonin. Of the catechins (10 mg/mL) examined, (-)-epigallocatechingallate (8.78 ± 0.05%) and (-)-epigallocatechin (8.45 ± 0.64%) were highly active, but less active than melatonin (0.4 nmol/L).
Conclusion: Since melatonin proved to be markedly effective in AGE crosslink degradation, it may be biologically active in
reducing AGE accumulation in living organisms.

KEY WORDS: advanced glycation endproducts (AGEs) , melatonin, N-phenacylthiazolium bromide (PTB),		Introduction

catechin, AGE breaker,

Accelerated degenerative changes in senescence due tothe production and accumulation of advanced glycation end
products (AGEs) of protein in living organisms have recently
been recognized to be problematic in view of the concept ofglycation stress 1, 2). The AGE production process is mediatedby a wide variety of reactions, including oxidation andcrosslinking, and it is thought to be influenced by lifestyle
habits such as diet, exercise, sleep, and stress 3 - 6).
Our laboratory previously quantified skin fluorescent
AGEs in healthy humans in a non-invasive manner, andreported that the AGE content increased with aging, and that
individual differences widened accordingly 7). The increased
individual differences were shown to be related to lifestylefactors such as smoking, drinking, and lack of sleep 8).
Melatonin, a representative sleep-related hormone, hasbeen investigated from a broad range of perspectives 9-14);
however, only a few studies have been focused on glycative
stress.
One study showed that melatonin is weakly active insuppressing AGE production 15). On the other hand, to facilitate
AGE degradation in living organisms, and thus contributeto the prevention of senescence and disease, studies were
undertaken to determine substances that promote AGE
degradation. Based on these studies, pomegranate 16) , water
chestnut 17) , and rosemary 18) extracts were found to be such
substances. The present study was conducted, with a focus
on crosslink-cleaving action as a mechanism for AGE
degradation 19) , to evaluate the AGE crosslink cleaving
effects of melatonin.

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 -0394 Japan
Phone/Fax: +81-774 -65-6394 E-mail: yyonei@mail.doshisha.ac.jp
Co-authors: Takabe W, wtakabe@mail.doshisha.ac.jp ; Mitsuhashi R, mitsuha@yonei-labo.com ;Parengkuan L, lannyparengkuan@ymail.com ; Yagi M, yagi@yonei-labo.com_ 38 _

Glycative Stress Research 2016; 3 (1): 38-43(c) Society for Glycation Stress ResearchGlycative Stress Research

Methods

Test materials

The main compound tested was melatonin and thecomparison included 28 kinds of flavonoids as follows;
apigenin, 7, 4’-dihydroxyflavone, liquiritigenin, chrysin,
pinocembrin, 5 - hydroxyflavone, 7- hydroxyflavanone,
naringenin, flavone, 4’-hydroxflavanone, 7-hydroxyflavone,luteolin, scutellarein, quercetagetin, apigenin-7-O-glucoside,daidzein, genistein, acacetin, carnosic acid, kaempferol,
corosoric acid, diosmetin, pinitol, maslinic acid, rutin,
rubusoside, rosmarinic acid, urolithin B. remove it These
compounds were dissolved and with distilled water, 100%
ethanol or 100% dimethyl sulfoxide (DMSO) and were
adjusted to a concentration of 0.4 mmol/L. Furthermore, 6
kinds of catechins, which are known compounds in tea leaf
(Camelia sinensis) 20) were used for comparison; (+)-catechin,(-)-epicatechin, (-)-epicatechin gallate, (-)-epigallocatechin,(-)-epigallocatechin gallate, (-)-gallocatehin. Catechins were
adjusted to a concentration of 10 mg/mLwith distilled water.
As a positive control for AGE-derived crosslink cleaving
activity, 10 mmol/L N-phenacylthiazolium bromide (PTB)
were used.

Evaluation for AGE-derived crosslink cleaving
activity

The AGE-derived crosslink cleaving activity wasevaluated by the modified method 16) of Vasan et al. 19).
Briefly, 1-phenyl-1, 2-propane dione (PPD), dissolved in 50%
acetonitrile, was used as a reactive substrate in the AGE
crosslink model. For the measurement of AGE-derived
crosslink cleaving activity, the samples (500 μL) were mixed
with 10 mmol/L PPD 100 μL and 0.2 mol/L phosphatebuffered saline (PBS) 400 μL, and then incubated at 37°C
for 8 hours. The reaction was stopped by adding 2 mol/Lhydrochloric acid (HCl) 200 μL, followed by centrifugation
at 10,000 rpm (9,170g) for 2 minutes. The benzoic acid amountin the supernatant was measured by high performance liquid
chromatography (HPLC).
An HPLC (LC-10A; Shimadzu, Nakagyo-ku, Kyoto,
Japan) equipped with Cadenza CD- C18 75 x 4.6 mmID
(Imtakt, Shimogyo-ku, Kyoto, Japan) was used. Analyticalconditions were as follows: eluate, 0.2 % acetic acid/
acetonitrile (70/30) containing 2 mmol/L ethylenediamineN, N, N’, N’- tetraacetic acid - disodium salt ( EDTA-2Na) dehydrate; flow rate, 1.0 mL/min; column temperature,
40°C; detection wave length, UV 270 nm; injection volume,
50 μL.
When 1 mol of PPD breaks down, 1 mol of benzoic acidis formed 3). Calculation of AGE crosslink cleaving activity
ratio was conducted after measuring bezoic acid by HPLC
as previously reported 16). The measurement was conductedin triplets (n = 3) and the results were expressed as mean ±standard deviation.

Results

The AGE-derived crosslink cleaving activity is presented
in Table 1. Among the 29 kinds of compounds examined, thecleaving activity was the highest in melatonin (15.76 ± 0.38%).
The cleaving activity was higher in melatonin, urolithin

B and carnosic acid of 0.4 mmol/L than that in a positive
control PTB of the same concentration. The activity of(-)-epigallocatechin gallate and (-)-epigallocatechin (10 mg/mL) was higher than that in 0.4 mmol/L PTB.

Discussion

Etiological associations between sleep time and lifestylerelated diseases with severe glycation stress, such as obesity,
dyslipidemia, and type 2 diabetes mellitus have recently been
attracting attention.
In these days many people have a wide variety of sleeprelated problems, including short sleeping hours due tolengthy commutes and long working hours and time-shift
work, sleep time reductions, and decreased quality of sleep;
the number of people with such problems has recently been
increasing year by year 21, 22). Several lines of evidence 23 -27)
indicate that such sleep problems represent a risk factor for
diabetes mellitus and obesity; a recent study even insist uponadvocating sound sleep as a third-choice treatment for diabetes
mellitus 28).
A U-shaped relationship exists between sleep time andlifestyle-related disease, with a negative correlation foundbetween too long or short of a sleep time and lifestyle-relateddisease 29). The percentage complication of diabetes mellitus
in people with a sleep time of ≤ 5 hr is approximately 2.5
times higher than in those with a sleep time of 7 to 8 hr 30).
In fact, many diabetic patients have sleep disorders as adiabetic complication 31). Sleep environment deterioration canfurther worsen blood glucose control in diabetic patients 31).
In a cohort study of lifestyle-related disease in 8, 218
children, known as the Toyama Study, sleep disorders
such as a late bedtime and short sleeping hours were shown
to be associated with conditions such as overweight andobesity 32, 33). Since sleep disorders represent a causal factor
for childhood obesity, and can progress to type 2 diabetes
mellitus with increased insulin resistance, diabetes mellitus
is often preceded by sleep problems. Our past study revealed
sleep time shortening to be a factor for increasing the skinaccumulation of fluorescent AGEs 8) ; sleep disorders arethought to intensify glycation stress.
Sleep disorders cause alteration of the hormone balancein the body. Abnormalities of the balance among eatingrelated hormones such as leptin and ghrelin due to sleep timeshortening, secondary increased insulin resistance due tointensified obesity, and increased anti-insulin hormones suchas cortisol secretion in nighttime are involved in the onsetof type 2 diabetes mellitus 30). Increased secretion of theadrenal medullary hormones adrenalin and noradrenalin
is involved in hypertension 34). Growth hormones secreted
during sleep, classified in a class of anti-insulin hormones,
have blood glucose elevating effects; however, their secretioncan be rather decreased with sleep time shortening, and seemto be little involved in the onset of diabetes mellitus.
Although the pineal hormone melatonin is expected
to be largely influenced by sleep disorders, much remains
unknown. In most regions in the Northern Hemisphere,
diabetic patients have seasonal changes in their blood glucose
levels irrespective of the type of diabetes. HbA1c values
change seasonally in 12-month cycles, tending to minimize inspring and summer and maximize in winter, with a variationof about 0.2 to 0.6% 35) . Melatonin secretion increases in the_ 39 _

Cleaving Effect of Melatonin on AGE Crosslinks

Table 1. AGE-derived crosslink cleaving activity.

SampleCleaving activity (%)

Relative ratio 1)

Relative ratio 2)

Melatonin

15.76 ± 0.38

69

248

Carnosic acid9.01 ± 0.93

Urolithin B

Rosmarinic acid
Quercetagetin
Scutellarein

7-Hydroxyflavone

7,4'-Dihydroxyflavone
Apigenin
Flavone14.89 ± 0.01
1.93 ± 0.11
1.29 ± 0.13
1.20 ± 0.06
0.82 ± 0.89
0.65 ± 0.55
0.06 ± 0.42
0.00 ± 0.28

Apigenin-7-O-glucoside-0.07 ± 0.07

Naringenin-0.23 ± 0.05

4'-Hydroxflavanone
Luteolin

5-Hydroxyflavone

7-Hydroxyflavanone
Pinocembrin-0.18 ± 0.03
-0.24 ± 0.06
-0.28 ± 0.13
-0.28 ± 0.20
-0.29 ± 0.11

Chrysin-0.38 ± 0.08

Rubusoside-0.70 ± 0.17

Rutin

-0.93 ± 0.17

Liquiritigenin-1.09 ± 0.96

Pinitol-1.32 ± 0.07

Acacetin-1.46 ± 0.08(-)- epigallocatechin gallate

8.78 ± 0.05(-)-gallocatechin

5.77 ± 0.04(-)- epicatechin

3.57 ± 0.06

PTB (10 mmol/L)

23.00 ± 7.50

Corosoric acid

-0.68 ± 0.56

Kaempferol-0.70 ± 0.19

Maslinic acid

-0.93 ± 0.07

Diosmetin-1.31 ± 0.03

Daidzein-1.34 ± 0.37

Genistein-2.23 ± 0.34(-)- epigallocatechin

8.45 ± 0.64(+)-catechin

5.12 ± 0.59(-)- epicatechin gallate

1.95 ± 0.01

PTB (0.4 mmol/L)

6.37 ± 2.71

65
39
8
6
5
4
3
0
0
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
38
37
25
22
15
8
100
−

234
142
30
20
19
13
10
1
0
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
138
133
91
80
56
31
−

100

1) : Relative ratio of AGE crosslink cleaving activity when the ratio of 1 0 mmol/L PTB is assumed to be 100.
2) : Relative ratio of AGE crosslink cleaving activity when the ratio of 0.4 mmol/L PTB is assumed to be 100.
Concentrations of components are 0.4 mmol/L except catechins of which concentration are 10 mg/mL. Results are expressed as mean ± standard
deviation, n = 3. AGE, advanced glycation end product; NE, not effective; PTB, N-phenacylthiazolium bromide．

_ 40 _Glycative Stress Researchlong-night season of winter, and may someway respond toincreased blood glucose levels in the season.
As stated above, patients with a lack of sleep as a diabetic
complication are severely affected by glycative stress 8) ,and can therefore be at increased risk of disease caused byglycative stress. We hypothesized that melatonin, a sleeprelated hormone, lessened glycative stress. Methods of
reducing glucose-associated glycative stress include thefollowing: i) glucose consumption reduction and glucose
absorption retardation, ii ) AGE production inhibition,iii) promotion of AGE degradation, and iv) inhibition of
AGEs/RAGE binding/activation 1, 2 ). In addition, since AGE
production is in part related to oxidizing reactions,antioxidants act to reduce AGE production. Melatonin has
antioxidant action, and in this regard, it may have some
contribution to the suppression of AGE production; however,melatonin did not suppress AGE production in an in vitro
glucose/human serum albumin reaction model 15). In thepresent study, melatonin was found to have AGE degradation
promoting effects. The mechanism is based on the cleavageof AGE crosslinks 19). This may account, in part, for the factthat skin deposition of AGEs is relatively common among
persons with lack of sleep 8).
AGEs are degraded mainly in two ways: cleavage of the
α-diketone structure in crosslinked AGEs 19) and the actionof oxidized protein hydrolase (OPH), an enzyme that
preferentially degrades oxidized and glycated proteins 36).
AGE crosslink degradation activity is attracting attention
in attempt to determine a therapeutic approach to degradeand eliminate AGEs by cleaving AGE crosslinks, and
it is suggested to suppress AGE accumulation in bloodvessels, and contribute to the treatment of diabetic vascularcomplications 37). PTB, when ingested, can pose safetyissues, including adverse reactions 38). Accordingly, researchis ongoing to identify natural substances that can suppress
AGE accumulation with little influence on the body.
Substances reported to cleave AGE crosslinks include
mugwort and rooibos extracts 39), as well as pomegranate 16),
water chestnut 17), and rosemary 18) extracts.
The positive control N-phenacylthiazolium bromide (PTB)
is a compound that cleaves the C-C bonds of the α-diketone
structure in AGEs to degrade the AGEs 19). Here, melatonin
had potent AGE crosslink cleaving effects which were equal
to or higher than those of PTB at the same concentrations. A
comparison with flavonoid polyphenol controls revealed thatmelatonin possessed higher activity than 28 representative
flavonoids contained in vegetables and herbs.
Measurements of the cleaving activity of catechinscontained in tea revealed (-)-epigallocatechin gallate 40)
to be the most potent single component, suggesting that(-)-epigallocatechin gallate in tea is associated with AGE
crosslink cleaving effects. Tea also contains (-)-epigallocatechinand (-)-gallocatechin 41), which were ranked second to(-)-epigallocatechin gallate in cleaving activity. Hence,catechins with a benzenetriol structure are expected to have
AGE crosslink cleaving effects. Although it was difficult
to accurately compare prepared concentrations, 0.4 mmol/
L melatonin was found to be more potent than tea plantderived catechins (10 mg/mL) in terms of AGE crosslink
degradation activity.
Among various fruit extracts, pomegranate extracts
possess relatively high activity to suppress AGE production 42) ;a pilot study in human subjects reported an improvementof serum HbA1c with pomegranate extracts 43). A study toevaluate the AGE crosslink cleaving effects of pomegranate
components revealed that gallic acid (0.4 nmol/L) was had
an extremely high activity of 26.02 ± 0.07%, followed bypinicalagin (0.4 nmol/L) at 15.32 ± 2.57%, eucalbanin B at
13.93 ± 0.31%, and pomegraniin A at 13.51 ± 0.08%, withother components accounting for less than 13% each ( 0.4
nmol/L concentration each) 1 6 ) . Although its activity was
weaker than that of gallic acid, melatonin was found to be as
active as other components or higher.
Among various extracts from vegetables, root crops,
and nuts/berries, water chestnut extracts possess a relatively
high activity to suppress AGE production 4 4 ) . With regard to
AGE crosslink cleaving effects, water chestnut extracts had
activities of 7.2% (100μg/mL) and 32.49% (1 mg/mL), whichwere equal to or higher than the activity of the positive
control PTB 17). When comparing the activity at a dose of 0.4
nmol/L, melatonin is approximately 2.5 times more potent
than PTB, and it is comparable to water chestnut extracts interms of activity potency.
Taken toether, the above results suggest that melatonin
is biologically active to prevent AGE accumulation in the
body via its more potent AGE crosslink degradation effects
compared to those of the positive control PTB andrepresentative plant components. Melatonin secretion
decreases with aging and qualitative deteriorations of sleep,
including sleep under bright conditions and sleep timeshortening. This situation is likely to retard AGE degradation,
causing AGE accumulation, and increasing the risk of onsetof glycation stress-induced disease.

Conclusion

Melatonin proved to be potently effective in AGE
crosslink degradation in vitro, and was suggested to bebiologically active in reducing AGE accumulation in livingorganisms. Decreased melatonin secretion with aging andsleep disorders may intensify glycative stress.

Acknowledgements

This work was supported by the Japanese Council
for Science, Technology and Innovation, SIP (Project ID
14533567), “Technologies for creating next-generation
agriculture, forestry and fisheries” (funding agency: Biooriented Technology Research Advancement Institution,
NARO) and JSPS KAKENHI Grant Number 26350917.

Conflict of Interest Statement

The authors state that the performance of this study
entailed no issues representing a conflict of interest.

_ 41 _

Cleaving Effect of Melatonin on AGE Crosslinks

References
1) Nagai R, Mori T, Yamamoto Y, et al. Significance ofadvanced glycation end products in aging-related disease.
Anti-Aging Medicine. 2010; 7: 112-119.
2) Ichihashi M, Yagi M, Nomoto K, et al. Glycation stressand photo-aging in skin. Anti-Aging Medicine. 2011; 8:
23-29.
3) Vlassopoulos A, Lean ME, Combet E. Oxidative stress,
protein glycation and nutrition--interactions relevant tohealth and disease throughout the lifecycle. Proc Nutr Soc.
2014; 73:430-438.
4) Prasad C, Imrhan V, Marotta F, et al. Lifestyle and advanced
glycation end products (AGEs) burden: Its relevance tohealthy aging. Aging Dis. 2014; 5:212-217.
5) Couppé C, Svensson RB, Grosset JF, et al. Life-longendurance running is associated with reduced glycationand mechanical stress in connective tissue. Age (Dordr).
2014; 36(4):9665. doi: 10.1007/s11357-014-9665-9.
6) Turner DP. Advanced glycation end-products: a biologicalconsequence of lifestyle contributing to cancer disparity.
Cancer Res. 2015; 75:1925-1929.
7) Nomoto K, Yagi M, Arita S, et al. A survey of fluorescencederived from advanced glycation end products in theskin of Japanese: Differences with age and measurement
location. Anti-Aging Medicine. 2012; 9: 119-124.
8) Nomoto K, Yagi M, Arita S, et al. Skin accumulation ofadvanced glycation end products and lifestyle behaviors
in Japanese. Anti-Aging Medicine. 2012; 9: 165-173.
9) Oba S, Nakamura K, Sahashi Y, et al. Consumption ofvegetables alters morning urinary 6-sulfatoxymelatoninconcentration. J Pineal Res. 2008; 45:17-23.
10) Yonei Y, Hattori A, Tsutsui K, et al. Effects of melatonin:
Basics studies and clinical applications. Anti-Aging
Medicine. 2010; 7: 85-91.
11) Mihara T, Nakamura N, Ka K, et al. Effects of melatoninpremedication to prevent emergence agitation after generalanaesthesia in children: A systematic review and metaanalysis with trial sequential analysis. Eur J Anaesthesiol.
2015; 32: 862-871.
12) Kozaki T, Kubokawa A, Taketomi R, et al. Effects of daytime exposure to different light intensities on light-induced
melatonin suppression at night. J Physiol Anthropol. 2015;
34(1):27. doi: 10.1186/s40101-015-0067-1.
13) Obayashi K, Saeki K, Iwamoto J, et al. Physiological levelsof melatonin relate to cognitive function and depressive
symptoms: The Heijo-Kyo Cohort. J Clin Endocrinol
Metab. 2015; 100: 3090-3096.
14) Kawada T. Sleep parameters by actigraphy and relationshipbetween plasma melatonin and intestinal permeability inalcoholics. Am J Physiol Gastrointest Liver Physiol. 2015
Aug 15; 309(4):G279. doi: 10.1152/ajpgi.00153.2015.
15) Moniruzzaman M, Takabe W, Yonei Y. Melatonin is nota carbonyl scavenger. Glycative Stress Research. 2016; 3:00-00 (in press)
16) Yagi M, Mitsuhashi R, Watanabe A, et al. Cleaving effectof pomegranate (Punica granatum) extract on crosslink
derived from advanced glycation endproducts. Glycative
Stress Research. 2015; 2: 58-66.

17) Takeshita S, Yagi M, Uemura T, et al. Peel extract of water

chestnut (Trapa bispinosa Roxb.) inhibits glycation,
degrades α-dicarbonyl compound, and breaks advanced
glycation end product crosslinks. Glycative StressResearch. 2015; 2: 72-79.
18) Jean D, Pouligon M, Dalle C. Evaluation in vitro of AGEcrosslinks breaking ability of rosmarinic acid. Glycative
Stress Research. 2015; 2: 204-000.
19) Vasan S, Zhang X, Zhang X, et al. An agent cleaving
glucose-derived protein crosslinks in vitro and in vivo.
Nature. 1996; 382: 275-278.
20) Otake K, Yagi M, Takabe W, et al. Effect of tea (Camelliasinensis) and herbs on advanced glycation endproductformation and the influence of post-fermentation. Glycative
Stress Research. 2015; 2: 156-162.
21) Okawa M. Sleep disturbance and diabetes mellitus.
Diabetes Frontier. 2011; 22: 119-128. (in Japanese)22) Mishima K. Sleep medicine for the treatment andprevention of life style disease. Journal of Clinical andExperimental Medicine ( Igaku No Ayumi). 2011; 236:
5-10. (in Japanese)23) Kondo F, Nakajima T, Suzuki A, et al. Actigraph:
Application for clinical situation Amount of activity
increase during sleep in type-2 diabetes mellitus patients.
Journal of International Society of Life Information
Science. 2006; 24: 435-443.
24) Nakajima H, Kaneita Y, Yokoyama E, et al. Insomnia
symptoms associated with hyperglycemia. Sleep and
Biological Rhythms. 2010; 8: 203-211.
25) Hsieh SD, Muto T, Murase T, Tsuji H, Arase Y. Associationof short sleep duration with obesity, diabetes, fatty liverand behavioral factors in Japanese men. Intern Med.
2011; 50: 2499-2502.
26) Fuse K, Yoshimine F, Kasai A, et al. Sleep disturbances inthe diabetic patients. Japanese Journal of Psychosomatic
Medicine. 2011; 51: 799-806. (in Japanese)27) Tanaka M, Kaneko R, Kamata N, et al. The relation of
the sleep indicator by Actigraph to fasting blood glucose
and HbA1c: A study in the laborer. Occupational Mental
Health. 2012; 20: 250-258. (in Japanese)28) Kawamori R. Sleep and diabetes mellitus. The Journal of
Adult Diseases. 2010; 40: 433-440. (in Japanese)29) Nakagome S, Kaneita Y. Relationship of sleeping time toobesity and lifestyle-related diseases. Sleep and Clinical
Practice. 2014; 7: 5-7. (in Japanese)30) Yamamoto N, Otsuka K, Kim G, et al. Insomnia anddiabetes mellitus. Journal of Chronobiology. 2010; 16: 4247. (in Japanese)31) Yamada S, Inaba M. Diabetes mellitus and sleep. Sleep
and Clinical Practice. 2014; 7: 8-11. (in Japanese)32) Numata N, Yamagami T, Soukejima S, et al. Physiquechanges from infancy to childfood: Lifestyle factors
causing overweight. Journal of the Japanese Association
for Cerebro- Cardiovascular Disease Control. 2000; 35:
35-43. (in Japanese)
33) Sekine M, Kanayama H, Kagamimori S. Formation andmaintenance of exercise custom in infants and influence ofthe social family background: Epidemiological approachon the life course. Research-Aid Report. 2007; 22: 62-69.
(in Japanese)_ 42 _Glycative Stress Research34) Fujii M, Akamine Y, Kuroda K, et al. Effect of melatoninon the pathogenesis of hypertension in the SpontaneousHypertensive Rats (SHR). Japanese Journal of Applied
Physiology. 1993; 23: 525-532. (in Japanese)35) Mineyama T, Noda M. Diabetes mellitus and seasonalchanges. Japanese Journal of Sleep Medicine. 2014; 8:219-224. (in Japanese)36) Fujino T, Tada T, Beppu M, et al. Purification andcharacterization of a serine protease in erythrocyte cytosolthat is adherent to oxidized membranes and preferentiallydegrades proteins modified by oxidation and glycation. J
Biochem. 1998; 124: 1077-1085.
37) Cooper ME, Thallas V, Forbes J, et al. The cross-link
breaker, N-phenacylthiazolium bromide prevents vascularadvanced glycation end-product accumulation. Diabetologia.
2000; 43: 660-664.
38) Uniwersytet J, Olszanecki R, Bujak-Giżycka B, et al. The
new application of 2-pyrrolidone derivatives. Int. Patent:
WO 2011-049475: 2011-04-28.
39) Tada A. An evaluation and the material choice of thenatural ingredient for anti-glycation cosmetics. Cosmetic
Stage. 2011; 5: 33-38. (in Japanese)40) Yoshino K, Hara Y. Determination of catechins andtheaflavins in infusions by high-performance liquid
chromatography. Numazu College of Technology. 1993;
27: 87-91. (in Japanese)41) Huang S, Inoue K, Li Y, et al. Analysis of catechins in
autoclaved tea leaves and drinks. Jpn J Food Chem. 2004;
11: 99-102. (in Japanese)
42) Parengkuan L, Yagi M, Matsushima M, et al. Anti-glycation
activity of various fruits. Anti-Aging Medicine. 2013; 10:
70 -76.
43) Yagi M, Parengkuan L, Sugimura K, et al. Anti-glycation
effect of pomegranate (Punica granatum L) extract: An
open clinical study. Glycative Stress Research. 2014; 1:
60-67.
44) Ishioka Y, Yagi M, Ogura M, et al. Antiglycation effect ofvarious vegetables: Inhibition of advanced glycation end
product formation in glucose and human serum albumin
reaction system. Glycative Stress Research 2015; 2: 22-34.

_ 43 _