Glycative Stress Research
Online edition : ISSN 2188-3610
Print edition : ISSN 2188-3602
Received : July 31, 2021
Accepted : September 1, 2021
Published online : September 30, 2021
doi:10.24659/gsr.8.3_156

Original article

Antiglycative effect of genipin and crocetin
Masayuki Yagi 1), Chieko Sakiyama 1), Yuusuke Miyata 2), Soichiro Kamiya 2), Yoshikazu Yonei 1)
1) Anti-Aging Medical Research Center and Glycative Stress Research Center,
Graduate School of Life and Medical Sciences, Doshisha University, Kyoto, Japan2) Riken vitamin Co., Ltd., Osaka, Japan

Abstract

Accumulation of advanced glycation end products (AGEs) in the body due to glycative stress is a factor in the onset of aging
and lifestyle-related diseases. Suppression of glycative stress is called anti-glycation. Anti-glycation includes suppressionof postprandial hyperglycemia, suppression of glycation reaction, and decomposition and excretion of AGEs. Sinceaminoguanidine (AG), a substance having an AGE production inhibitory effect, has side effects, functional foods having a
glycation reaction inhibitory activity are desired. In this study, for evaluating the anti-glycation effect of gardenia (Gardenia
jasminoides Ellis) extract on the skin proteins, the glycation reaction inhibitory activity and AGE cross-linking breaking
activity of genipin and crocetin, purified from gardenia, was measured. Keratin, collagen, and elastin were used as model
proteins for the glycation reaction. Genipin and crocetin were found to suppress the production of fluorescent AGEs (F-AGEs),
pentosidine, N ε- carboxymethyllysine (CML) and 3-deoxyglucosone (3DG). The activity of genipin on F-AGEs was strong
with the half maximal inhibitory concentration (IC 50) lower than that of crocetin. Its IC 50 was 2.4 to 31.6 times lower thanthat of AG, a positive control. The AGE cross-linking breaking activity was 2.8 times stronger with genipin than withcrocetin. These findings indicate that application of a preparation containing genipin and crocetin to the skin may improve
the skin condition by suppressing the AGE production from skin proteins and by breaking the AGE cross-linking.

KEY WORDS: advanced glycation end products (AGEs), inhibition of AGE formation, AGE-crosslinks breaking ability,		gardenia (Gardenia jasminoides Ellis)

Introduction
Accumulation of advanced glycation end products (AGEs)
in the body due to glycative stress is a factor in the onset of
aging and lifestyle-related diseases. Suppression of glycative
stress is called anti-glycation. Anti-glycation includes
suppression of postprandial hyperglycemia, suppressionof glycation reaction, and decomposition and excretion of
AGEs 1, 2). Aminoguanidine (AG) is known as a substancehaving an AGE production inhibitory effect 3, 4). However,ingestion of AG has side effects such as anemia, liver damage,and vitamin B6 deficiency, and has not been put into practicaluse. Meanwhile, tea /herbal tea 5), ​​vegetables/herbs 6), andfruits 7) have been reported to have an inhibitory effect on

glycative reaction. AGE production inhibitory ingredientscontained in natural products include chamaemeloside(Roman chamomile) 8), luteolin (edible purple chrysanthemum
flower) 9), polymethoxy flavonoid (black galangal or black
ginger) 10, 11), cyanidin 3-O-galactoside (red water pepper) 12),
caffeoyl glucose (cherry blossom) 13), and rhodanthenone B(mangosteen's peel) 14). Furthermore, N-phenacylthiazolium
bromide (PTB) has been reported as a substance that may
have a decomposing effect on AGEs 15). PTB cleaves the
α-diketone structure, which is a type of cross-linked
structure of AGE-modified proteins 16). Similar effects havebeen observed with rosmarinic acid 17), ellagitannin 18), andflavonoids 19).

Contact address: Professor Masayuki Yagi, PhD
Anti-Aging Medical Research Center / Glycative Stress Research Center,
Graduate School of Life and Medical Sciences, Doshisha University
1-3, Tatara Miyakodani, Kyotanabe-shi, Kyoto, 610- 0394 Japan
TEL & FAX: +81-774-65-63943 e-mail: myagi@mail.doshisha.ac.jp
Co-authors: Sakiyama C, csakiyam@mail.doshisha.ac.jp;
Miyata Y, yuu_miyata@rike-vita.co. jp; Kamiya S, sou_kamiya@rike-vita.co.jp;
Yonei Y, yyonei@mail.doshisha.ac. jp_ 15 6 _Glycative Stress Research 2021; 8 (3): 156-161(c) Society for Glycative Stress ResearchGlycative Stress Research

Gardenia (Gardenia jasminoides Ellis) is a woody plantof the Rubiaceae family. Gardenia fruit contains geniposide,a type of iridoid glycoside, and crocetin, a type of carotenoid,
and is used as a yellow colorant and as a crude drug forsedation, anti-inflammatory, hemostatic, and antipyretic
effects 20). When geniposide is converted to aglycone by
β-glucosidase, it becomes the blue pigment genipin. It has
been reported that genipin has a breaking action on the crosslinking of proteins, i.e., collagen, and a suppressing action on
the activity of uncoupling protein 2 (UCP2) 21). Crocetin has
been reported to have various functions in vivo, including
anti-cancer effect 22), fatigue recovery effect 23), sleep-improving
effect 24), and retinal ischemic injury-suppressing effect 25).
In this study, for the purpose of evaluating the antiglycation effect of the components contained in gardenia on
skin proteins, we verified the inhibitory activity on glycation
reaction and breaking activity on AGE cross-linking ofgenipin and crocetin purified from gardenia.

Genipin

Crocetin
Fig. 1. Structure of genipin and crocetin.

Figure 1.

Materials and methods
1) Reagent

The reagents used were purchased from the following
manufacturers and used; Human serum albumins ( HSA,
lyophilized powder, ≥ 96 %, agarose gel electrophoresis) andN-phenacylthiazolium bromide (PTB) are from Sigma-Aldrich
Japan (Meguro-ku, Tokyo, Jpam). Collagen Type I (collagentype I, bovine skin, pepsin-solublilized) is from Nippi(Adachi-ku, Tokyo). Elastin peptide (P-Elastin) derived from
porcine blood vessels is from Nippon Ham (Osaka, Japan).
Aminoguanidine hydrochloride (AG), epigallocatechingallate (EGCg), 2, 3-diaminonaphthalene (DAN), 1-phenyl1, 2-propanedione (1-phenyl) -1, 2-propanedione (PPD) andbenzoic acid are from Fuji Film Wako Pure Chemical
Industries (Osaka). 3-deoxyglucosone (3DG) is from the Dojin
Chemical Research Institute (Kamimashiki-gun, Kumamoto,Japan). CircuLex CML /N ε- (Carboxymethyl) Lysine) ELISA
Kit is from MBL (Nagoya, Aichi, Japan). Other reagents ofspecial grade or HPLC grade were purchased from Fuji Film
Wako Pure Chemical Industries or Nacalai Tesque (Kyoto,
Japan).

2) Sample
As samples, a genipin preparation and a crocetin
preparation purified from gardenia (Japanese name
Kuchinashi) were used ( Fig. 1). Genipin is formulated with
1, 3-butanediol. Crocetin is solubilized by amine compounds
and glycerin. These samples were provided by Riken Vitamin(Shinjuku-ku, Tokyo).

3) Protein-glucose glycation model
The protein-glucose glycation model was used for theverification of the glycation reaction inhibitory activity withreference to the previously reported 26). The composition of
the reaction solution was prepared by adding 1/10 amount of
the sample solution to 0.1 mol/L phosphate buffer (pH 7.4)
containing protein and glucose. The protein and glucose
concentrations were as follows; keratin 2.5 mg/mL and glucose

0.6 mol/ L, collagen 1. 2 mg/mL and glucose 0.4 mol/L, andelastin 6 mg/mL and glucose 0.2 mol/L.
For the glycation reaction, a solution (A) in which allof the phosphate buffer, protein solution, glucose solution,and sample solution are added, a solution (B) in which
purified water is added instead of the glucose solution ofA, a solution (C) to which purified water was added instead
the sample solution of A, and a solution (D) to which
purified water was added instead of the glucose solutionof C were prepared and incubated at 60 °C for 10 days. The
concentrations of AGEs and glycation reaction intermediates
in the solution after incubation were measured. AG and
EGCg were used as positive controls for the inhibitory
activity on glycation reaction.

4) Measurement of AGEs

For fluorescent AGEs (F-AGEs), 200 µL of the glycation
reaction solution was placed in a black microplate and thefluorescence derived from AGEs (excitation wavelength
370 nm/fluorescence wavelength 440 nm) was measured
according to the previously reported 26). Pentosidine wasmeasured by HPLC after mixing 50 µL of the reaction
solution and 6 N hydrochloric acid and hydrolyzing at 110 °C
for 18 hours according to the previously reported 27). CML
was measured using 30 µL of reaction solution using CircuLex
CML/N ε- (Carboxymethyl) lysine ELISA Kit.

5) Measurement of glycation reaction intermediates
The glycation reaction intermediate was measured by
HPLC after deproteinizing 200 µL of the solution withperchloric acid, adding DAN under alkaline conditions, andlabeling according to previously reported 28, 29).

6) Calculation of glycation reaction inhibitory
activity

According to the previously reported 26), inhibitory
activity on the glycation reaction was calculated by thefollowing formula for the production inhibition ratio (%) of
AGEs and glycation reaction intermediates.

_ 157 _

Anti-glycative Effect of Genipin and Crocetin

Results

The inhibition ratio (%) = {1 − (A − B) / (C − D) } × 100
Furthermore, a half maximal inhibitory concentration(IC 50 in mg/mL) was calculated from the production inhibition
ratio at three concentrations 26, 30). The smaller the IC 50 value,
the stronger the inhibitory activity.

7) Measurement of AGE cross-linking breaking
activity

Evaluation of the AGE cross-linking breaking activity
was carried out by mixing the sample solution, 10 mmol/L
PPD, and 0.2 mol/L phosphate buffer (pH 7.4) at a ratio of 5:
1: 4 at 37 °C, according to previously reported 15, 18, 19). Afterreacting for 8 hours, 0.7 N hydrochloric acid was further
added to stop the reaction, and then the amount of benzoic
acid cleaved from PPD was measured by HPLC. PTB wasused as a positive control for cross-linking breaking activity.
When the α-diketone structure of PPD, which is a model
substance, is cleaved, 1 mol of benzoic acid is produced
from 1 mol of PPD. Using this principle, the cross-linking
breaking ratio was calculated by the following formula.
The breaking ratio = { (A − B) / C } × 100
A; Amount of benzoic acid in the reaction solution, B; Amountof benzoic acid in the sample, C; Amount of PPD used in
the reaction

Statistical analysis

120

Keratin100

Elastin

The inhibitory activity of genipin and crocetin on F-AGEs
production was found to be concentration-dependent in all
three reaction models using keratin, collagen, and elastin asmodel proteins (Fig. 2). The inhibitory activity on F-AGEs
production at a genipin concentration of 0.4 mg/mL wasstronger in the order of elastin (inhibition ratio: 96.9 %),keratin (83.6 %), and collagen (71.1%). Similarly, the activityof crocetin was stronger in the order of keratin (80.1%),
collagen (61. 5 %), and elastin (12. 2 %). The F-AGE
production inhibition ratio of genipin and crocetin were 1.0fold different for keratin, 1. 2-fold for collagen, and 7.9 -foldfor elastin. IC 50 values ​​for keratin, collagen, and elastin werelower for genipin than for crocetin ( Table 1 ). The IC 50 values​​of genipin and crocetin were lower than those of AG in all
combinations except the IC 50 value of crocetin for elastin.

Inhibitory activity on production of AGEs andintermediates in keratin-glucose glycation model

Inhibitory activity of 0.4 mg/mL genipin and 0.4 mg/mL
crocetin was examined on production of F-AGEs, pentosidine,
CML, and 3DG in the keratin-glucose glycation model( Table 2). The inhibitory activity of genipin was stronger
than that of crocetin in all the verified items. In particular,
the inhibition ratio of pentosidine production (97.1%) of
genipin was 2.1 times that of crocetin.

Breaking activity on AGE cross-linking

The AGE cross-linking breaking activity of genipin(18.6 %) was 2.8 times stronger than crocetin (6.7%, Fig. 3 ).
The breaking ratio of 2 mg/mL genipin was 0.7 times that of
5 mmol/mL PTB (breaking ratio 25.5 %), a positive control.

Collagen

Inhibition ratio (%)

Inhibition ratio (%)

The measured values ​​are expressed as mean ± standard
deviation. Tukey's test or t-test was used to compare themeasured values. As a result of statistical analysis, asignificance level of less than 5% was considered significant.

Inhibitory activity on F-AGE production in three
types of protein-glucose glycation model

80
60
40
20
0

120

Keratin100

Elastin

Collagen

80
60
40
20

0.04

0.4

1.0

Genipin

0

0.3

AG

0.04

0.4

0.76

Crocetin

Concentration (mg /mL)

0.03

AG

Concentration (mg /mL)

Fig. 2. Inhibitory activity of genipin and crocetin on F-AGEs formation.

Results are expressed as mean ± SD, n = 3; * p < 0.05 by Tukey's test. AG, aminoguanidine; F-AGEs, fluorescent advanced glycation
end products; SD, standard deviation.

_ 158 _Glycative Stress Research

Table 1. IC50 on the protein-glucose glycation model.
Model protein

Genipin(mg/mL)

Keratin

0.041

Elastin

0.086

Collagen

Crocetin(mg/mL)

Aminoguanidine 1)(mg/mL)

0.145

0.135

0.013

1.0 >

0.275

0.423

0.207

1) Positive control of glycation inhibitor. IC 50, Half maximal inhibitory concentration.

Table 2. AGE inhibition ratio on the keratin-glucose glycation model.
AGE compound
Fluorescent AGEs 2)

Genipin 1)(%)

Crocetin 1)(%)

83.6 ± 0.1

80.1 ± 0.05

71.0 ± 1. 8

50. 5 ± 0.3

Pentosidine

97.1 ± 0. 5

4)46.9 ± 0. 2

CML
3DG

3)

45.4 ± 5. 5

27.9 ± 0.1

1) Sample concentration: 0.4 mg/mL, mean ± SD (n = 3). 2) Characteristic fluorescence (excitation/emission = 370/440 nm).
3) Nε-carboxymethyl lysine. 4) 3-deoxyglucosone. AGEs, advanced glycation end products; SD, standard deviation.

35

Breaking ratio (%)

30
25
20
15
10
5
0

Genipin

Crocetin

PTB

Fig. 3. AGE cross-link breaking activity.

Results are expressed as mean ± SD, n = 3; * p < 0.05 by
Tukey’ s test. PTB, 5 mmol/L N-phenacylthiazolium bromide;
AGE, advanced glycation end product; SD, standard deviation.

Discussion

Inhibitory activity of genipin and crocetin on
glycation reaction

Iridoid glycosides, i. e. genipin, are contained inmany medicinal plants other than gardenia 31). Noni
(Morinda citrifolia L.) fruits contain iridoids such as
deacetylasperulosidic acid and loganic acid, and have beenreported to have an AGE production inhibitory action 32).
Persons who take noni fruit juice daily have less accumulationof AGEs in their skin 33). In addition, carotenoids, i.e. crocetin,

have been reported to be useful for anti-oxidant, antiinflammatory, and ocular disease protection 34). The antiglycation effect of carotenoids has been reported for
β-carotene 35) and astaxanthin 36). Crocin, which has twomolecular bindings of crocetin, has been verified to have an
inhibitory effect on AGE production on crystallin in the rat
eye lens 37). Also, the IC 50 value of the AGE production
inhibitory activity of silibinin, a flavonolignan, was 0.0702
mg/mL, which was 4.8 times lower than that of AG, a positive
control 38). A gel formulation of 1% milk thistle (Silybum
marianum (L.) Gaertn) flower extract, which contains 0.24%
silibinin, prevented the decrease in network formation offibulin-1 and reduced CML expression in human skin explantmodel treated with methylglyoxal. The anti-glycation effect ofsilibinin is thought to be involved in the suppression of protein
carbonylation. A similar effect has been observed in akebi(Akebia quinata Decaisne) fruit extract (AQFE), which has
the same level of inhibitory activity on the F-AGE production
as AG 39).
In this study, we examined the AGE production
inhibitory activity using skin protein, i.e. keratin, collagen,and elastin, as model proteins for the purpose of verifying
the anti-glycation effect of genipin and crocetin containedin gardenia. As a result, genipin and crocetin were found to
suppress the production of F-AGEs, pentosidine, CML, and
3DG in all three types of glycation reaction models. These
effects were stronger with genipin than with crocetin. A
strong inhibitory activity of genipin was observed and its
IC 50 of F-AGEs in the three models was 0.013 to 0.086 mg/
mL, which was 2.4 to 31.6 times lower than that of AG.
AG suppresses the AGE formation by blocking glycation
reaction intermediates with carbonyl groups 3). Combinedwith these facts, skin application of a gel containing genipin
and crocetin may suppress the AGE production in the skinby blocking the carbonyl group of the intermediates and byanti-oxidative action as in silibinin and AQFE.

_ 159 _

Anti-glycative Effect of Genipin and Crocetin

Conclusion

Breaking activity on AGE cross-linking of genipin
and crocetin
Genipin and crocetin were found to have an AGE crosslinking breaking activity. The PTB, a positive control in thisstudy, cleaves the dicarbonyl bond in the PPD molecule toproduce benzoic acid, indicating the possibility of cleaving
the dicarbonyl structure formed by glycation cross-linking ofproteins 15). It has also been reported that PTB reduces AGEs
in rat arterial walls 40). However, another study showed that
PTB did not reduce in AGE cross-linking of tail collagenin rats 41). Further verification is required for the effect ofdegrading AGEs in human tissues by ingestion of AGE crosslinking breaking substances. On the other hand, the AGE
decomposing effect of the cross-linking breaking component
by skin application has been reported in the extracts ofmugwort (YAC extract) and Astragalus sinicus L 42, 43). In a
study in which a solution containing ethanol extract of mugwort(1% YAC extract) was added and incubated dropwise to aplate coated with AGE-formed collagen gel, the AGEs on thecollagen gel surface almost disappeared. In a clinical study inwhich 38 healthy middle-aged women used a skin care productcontaining 1 % YAC extract for 6 months, the elasticity
reduction index (RRT values) and sagging index (Sagging
index) in the central part of the left cheek improved. Since
genipin and crocetin have an AGE cross-linking breaking
activity like YAC extract, skin application of these components
may lead to reduction of skin AGEs and improvement of skincondition.

For the purpose of verifying the anti-glycation effect ofgenipin and crocetin contained in gardenia on skin proteins,
inhibitory activity on AGE production and breaking activity
on AGEs cross-linking were verified in the glycation modelusing keratin, collagen, and elastin as model proteins. Genipin
and crocetin were found to have inhibitory actions on
production of F-AGEs, pentosidine, CML, and 3DG. Thesefindings indicate that topical application of a preparation
containing genipin and crocetin may improve the skincondition by suppressing the AGE production and by breaking
the AGE cross-linking.

Conflict of interest declaration
This research was funded by Riken Vitamin Co., Ltd.

Acknowledgments
The publication of this study was supported by the
Research Foundation of Crude Drug gained from the same
source of Medicine & Food. (IDF#21008).

Reference1) Ichihasi M, Yagi M, Nomoto K, et al. Glycation stress andphoto-aging in skin. Anti-Aging Med. 2011; 8: 23-29.
2) Yagi M, Yonei Y. Glycative stress and anti-aging: 1. Whatis glycative stress? Glycative Stress Res. 2016; 3: 152-155.
3) Brownlee M, Vlassara H, Kooney A, et al. Aminoguanidine
prevents diabetes-induced arterial wall protein cross-linking.
Science. 1986; 232: 1629-1632.
4) Bolton WK, Cattran DC, Williams ME, et al. Randomized
trial of an inhibitor of formation of advanced glycation
end products in diabetic nephropathy. Am J Nephrol.
2004; 24: 32-40.
5) Hori M, Yagi Y, Nomoto K, et al. Inhibition of advanced
glycation end product formation by herbal teas and its
relation to anti-skin aging. Anti-Aging Med. 2012; 9: 135148.
6) 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 Res. 2015; 2: 22-34.
7) Parengkuan L, Yagi M, Matsushima M, et al. Antiglycation activity of various fruits. Anti-Aging Med. 2013;
10: 70-76.
8) Yagi M, Matsuura N, Yonei Y. The effectivity for antiaging medicine of the chamameloside which is containedin Roman chamomile. Aromatopia. 2007; 16: 26-29. (inJapanese)

9) Yagi M, Nomoto K, Hori M, et al. The effect of edible

purple Chrysanthemum extract on advanced glycation
end products generation in skin: A randomized controlled
clinical trial and in vitro study. Anti-Aging Med. 2012; 9:61-74.
10) Yagi M, Tateiwa Y, Inoue K, et al. Antiglycative effect of
Kaempferia parviflora Wall. Ex. Baker (Zingiberaceae):
Prevention of advanced glycation end product formation.
Glycative Stress Res. 2018; 5: 163-170.
11) Nakata A, Koike Y, Matsui H, et al. Potent SIRT1
enzyme-stimulating a nd a nti-glycation activity
ofpolymethoxyflavonoids from Kaempferia parviflora.
Nat Prod Commun. 2014; 9: 1291-1294.
12) Takabe W, Yamaguchi T, Hayashi H, et al. Identificationof antiglycative compounds in Japanese red water pepper(red leaf variant of the Persicaria hydropiper sprout).
Molecules. 2018; 23: 2319.
13) Shimoda H, Nakamura S, Morioka M, et al. Effect of
cinnamoyl and flavonol glucosides derived from cherry
blossom flowers on the production of advanced glycation
end products (AGEs) and AGE- induced fibroblast
apoptosis. Phytother Res. 2011; 25: 1328-1335.
14) Ohno R, Moroishi N, Sugawa H, et al. Mangosteen
pericarp extract inhibits the formation of pentosidine andameliorates skin elasticity. J Clin Biochem Nutr. 2015;57: 27-32.

_ 16 0 _Glycative Stress Research15) 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.
16) Furber JD. Extracellular glycation crosslinks: prospects
for removal. Rejuvenation Res. 2006; 9: 274-278.
17) Jean D, Pouligon M, Dalle C. Evaluation in vitro of AGEcrosslinks breaking ability of rosmarinic acid. Glycative
Stress Res. 2015; 2: 204-207.
18) Yagi M, Mitsuhashi R, Watanabe A, et al. Cleaving effectof pomegranate (Punica granatum) extract on crosslink
derived from advanced glycation endproducts. Glycative
Stress Res. 2015; 2: 58-66.
19) Takabe W, Mitsuhashi R, Parengkuan L, et al. Cleaving
effect of melatonin on crosslinks in advanced glycation
end products. Glycative Stress Res. 2016; 3: 38-43.
20) Xiao W, Shiming Li, Wang S, et al. Chemistry andbioactivity of Gardenia jasminoides. J Food Drug Anal.
2017; 25: 43-61.
21) Zhang CY, Parton LE, Ye CP, et al. Genipin inhibits
UCP2-mediated proton leak and acutely reverses obesityand high glucose-induced b cell dysfunction in isolated
pancreatic islets. Cell Metab. 2006; 3: 417-427.
22) Gutheil WG, Reed G, Ray A, et al. Crocetin: An agent
derived from saffron for prevention and therapy forcancer. Curr Pharm Biotechnol. 2012; 13: 173-179.
23) Mizuma H, Tanaka M, Nozaki S, et al. Daily oral
administration of crocetin attenuates physical fatigue inhuman subjects. Nutr Res. 2009; 29: 145-150.
24) Umigai N, Takeda R, Mori A. Effect of crocetin on
quality of sleep: A randomized, double-blind, placebocontrolled, crossover study. Complement Ther Med. 2018;
41: 47-51.
25) Ishizuka F, Shimazawa M, Umigai N, et al. Crocetin, acarotenoid derivative, inhibits retinal ischemic damage inmice. Eur J Pharmacol. 2013; 703: 1-10.
26) Hori M, Yagi M, Nomoto K, et al. Experimental models
for advanced glycation end product formation using
albumin, collagen, elastin, keratin and proteoglycan. AntiAging Med. 2012; 9: 125-134.
27) Yagi M, Isshiki K, Takabe W, et al. Measurement ofpentosidine in human plasma by the high performanceliquid chromatography. Glycative Stress Res. 2018; 5: 119128.
28) Kusunoki H, Miyata S, Ohara T, et al. Relation between
serum 3-deoxyglucosone and development of diabetic
microangiopathy. Diabetes Care. 2003; 26: 1889-1894.
29) Yagi M, Yonei Y. Glycative stress and anti-aging: 2. The
Evaluation of glycative stress: Measurement of blood
glucose, glycated proteins and intermediates. Glycative
Stress Res. 2016; 3: 205-209.
30) Yagi M, Yonei Y. Glycative stress and anti-aging. 4:
The evaluation of glycative Stress: Evaluation for antiglycative effect. Glycative Stress Res. 2017; 4: 87-92.
31) Dinda B, Debnath S, Harigaya Y. Naturally occurring
iridoids. A review, part 1. Chem Pharm Bull (Tokyo).
2007; 55: 159-222.
32) Abe Y, Yagi M, Uwaya A, et al. Effect of iridoid(containing plants) on AGE formation and degradation.
Glycative Stress Res. 2016; 3: 56-64.
33) West BJ, Uwaya A, Isami F, et al. Antiglycation activityof iridoids and their food sources. Int J Food Sci. 2014;
2014: 276950.

34) Milani A, Basirnejad M, Shahbazi S, et al. Carotenoids:

Biochemistry, pharmacology and treatment. Br J Pharmacol.
2017; 174: 1290-1324.
35) Bodiga VL, Eda SR, Veduruvalasa VD, et al. Attenuationof non-enzymatic thermal glycation of bovine serum
albumin (BSA) using β-carotene. Int J Biol Macromol.
2013; 56: 41-48.
36) Sun Z, Liu J, Zeng X, et al. Astaxanthin is responsible
for antiglycoxidative properties of microalga Chlorella
zofingiensis. Food Chem. 2011; 126: 1629-1635.
37) Bahmani F, Bathaie SZ, Aldavood SJ, et al. Inhibitory
effect of crocin(s) on lens α-crystallin glycation andaggregation: Results in the decrease of the risk of diabetic
cataract. Molecules. 2016; 21: 143.
38) Shin S, Lee JA, Kim M, et al. Anti-glycation activitiesof phenolic constituents from Silybum marianum (milkthistle) flower in vitro and on human explants. Molecules.
2015; 20: 3549-3564.
39) Shin S, Son D, Kim M, et al. Ameliorating effect of Akebia
quinata fruit extracts on skin aging induced by advanced
glycation end products. Nutrients. 2015; 7: 9337-9352.
40) 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.
41) Yang S, Litchfield JE, Baynes JW. AGE-breakers cleave
model compounds, but do not break Maillard crosslinks
in skin and tail collagen from diabetic rats. Arch Biochem
Biophys. 2003; 412: 42-46.
42) Ohshima H, Tada A, Kanamaru A, et al. Relevance ofthe directionality of skin elasticity to aging and sagging
ofthe face. Skin Res Technol. 2011; 17: 101-107.
43) Tada A. An evaluation and the material choice of thenatural ingredient for anti-glycation cosmetics. Cosmetic
Stage. 2011; 5: 34-38. (in Japanese)_ 161 _