Glycative Stress Research
Online edition : ISSN 2188-3610
Print edition : ISSN 2188-3602
Received : January 19, 2022
Accepted : February 1, 2022
Published online : March 31, 2022
doi:10.24659/gsr.9.1_7

Original article

Suppression of glycated protein cross-linking formation and cross-linking
cleavage reaction of edible purple Chrysanthemum flower extract
Masayuki Yagi 1), Hideo Hara 2, 3), Ryusuke Mifuru 3), Yoshiro Ishikura 3),
Chieko Sakiyama 1), Yoshikazu Yonei 1)
1) Anti-Aging Medical Research Center and Glycative Stress Research Center,
Graduate School of Life and Medical Sciences, Doshisha University, Kyoto, Japan
2) Graduate School of Life Science, Hokkaido University, Sapporo, Japan
3) Unial Co., ltd., Tokyo, Japan

Abstract

The accumulation of advanced glycation end products (AGEs) in the body due to glycative stress is a factor in thedevelopment and progression of aging and lifestyle-related diseases. In particular, the formation of protein cross-links
associated with the formation of AGEs hardens tissues, resulting in the deterioration of biological functions. In this study, we
examined the usefulness of the edible purple Chrysanthemum flower from the viewpoint of reducing cross-link formationin the glycated proteins, by examining its inhibitory effect on this cross-link formation, its degradation of glycated protein
cross-linking, its inhibition of glycated reaction intermediate formation, its degradation of AGE cross-linking, and its
enhancement of oxidized protein hydrolase (OPH) activity. A 50 % ethanol extract (EE) of edible purple chrysanthemum(Chrysanthemum morifolium) flowers was used as a sample. EE inhibited the formation of lysozyme dimer and trimer
at sample concentrations of 0.01 to 1.1 mg/mL. EE inhibited the formation of fluorescent AGEs (F-AGEs) and glycationintermediates in a human serum albumin (HSA)-glucose glycation model. This effect was similar to that of aminoguanidine,
an inhibitor of glycation reaction. Furthermore, EE enhanced the enzymatic activity of OPH, which is an AGEs degrading
agent, by cleaving AGE cross-links using the α-diketone bond of 1-phenyl-1, 2-propanedione (PPD) as a model. The
inhibitory effect of EE on the formation of glycated protein cross-links was suggested to be related to the inhibition of theformation of glycation intermediates and AGEs, and the degradation of glycated protein cross-links was suggested to berelated to the involvement of various substances in Asteraceae plants and the pigment component of purple chrysanthemum
flowers. EE may have an effect on repairing protein dysfunction by degrading and removing cross-linked proteins byglycation. There is a possibility that EE may have the ability to repair protein dysfunction by removing cross-linked proteinsthrough glycation.

KEY WORDS: advanced glycation end products (AGEs), cross-link formation in the glycated protein,		cross-link cleavage in the glycated protein, Chrysanthemum morifolium

Introduction

intermediate, promotes the formation of protein crosslinks 3, 4). Inhibition of glycative stress in vivo is called
anti-glycation. Anti-glycation includes inhibition of
postprandial hyperglycemia, inhibition of glycation
reactions, and degradation of AGEs. From the viewpoint ofglycated-protein cross-link (GPCL) formation, inhibitionof postprandial hyperglycemia and inhibition of glycation
reaction are preventive measures, while degradation of
AGEs is a restorative measure.

The accumulation of advanced glycation end products(AGEs) in the body due to glycative stress is a factor in thedevelopment of skin aging, diabetic complications, andosteoporosis 1, 2). In particular, the formation of protein crosslinks associated with the AGE formation causes tissue
hardening, leading to a decline in biological functions.
Furthermore, 3- deoxyglucosone (3DG), a glycation
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: + 81-774-65-6394 e-mail: myagi@mail.doshisha.ac.jp
Co-authors; Hara H, h-hara@unial.co.jp; Mifuru R, mifuru@unial.co. jp;
Ishikura Y, ishikura@unial.co.jp; Sakiyama C, csakiyam@mail.doshisha.ac.jp;
Yonei Y, yyonei@mail.doshisha.ac.jp_ 7 _Glycative Stress Research 2022; 9 (1) : 7-14(c) Society for Glycative Stress ResearchSuppression of Glycated Protein Cross-linking and Cross-linking Cleavage of Purple Chrysanthemum Flower

Aminoguanidine (AG) is a substance that inhibits theformation of AGEs 5, 6). However, AG has side effects, i.e.,anemia, liver dysfunction, and vitamin B6 deficiency. On
the other hand, inhibitory effects on glycation reactions havebeen observed in various food materials such as herbal teas7),
vegetables 8), and fruits 9). N- phenacylthiazolium bromide(PTB) has been reported as a substance with GPCL
degradation activity 10). PTB cleaves α-diketone bonds, atype of cross-linkage structure in the glycated protein 11).
The GPCL degradation of has been reported to be inhibitedby substances found in plants, including rosmarinic acid 12),ellagitannins 13), and flavonoids 14).
Oxidized protein hydrolase (OPH) is a type of proteasefound in various tissues, i.e., blood, liver, and skin, and has
been reported to degrade AGEs as well as release N-terminal
acyl groups of acylated proteins 15). The activity- enhancing
effect of OPH has been observed in many herbal extracts,
including Job's tears (Coix lacryma-jobi var. ma-yuen), kuma
bamboo grass (Sasa veitchii), and fenugreek (Trigonella
foenum-graecum) 16, 17).
There are about 25,000 species of Asteraceae in theworld, and they are used as agricultural crops since they cangrow in various regions 18). Lettuce (Lactuca sativa), burdock(Arctium lappa), and chamomile (Matricaria recutita) are
typical Asteraceae crops, and their leaves, rhizomes, andflowers are edible. Edible purple chrysanthemum is a varietyof Chrysanthemum (Chrysanthemum morifolium) and iscalled "kaki no moto" in Niigata prefecture, "motte no hoka"
or "enmei raku" in Yamagata prefecture, and is eaten in avariety of ways such as soaked, namasu, tempura, and soup.
There are two main edible varieties of chrysanthemum
flowers: one with reddish-purple petals, such as Enmeiraku,
and the other with yellow petals, such as Abokyu.
Chrysanthemum flowers have been reported to contain 46
flavonoids and 17 caffeic acid derivatives 19). Edible purple
chrysanthemum flowers also contain pigment components
such as delphinidin and cyanidin. It has already been reported
that chrysanthemum flowers inhibit AGE production 20, 21),
have antioxidant effects 22), and have anti-photoaging effects 23).
In a study in which 11 healthy Japanese women took 150
mg of edible purple chrysanthemum powder per day for
8 weeks, it was found that the material was safe and thatthe skin elasticity index of the left cheek increased before
and after intake 20). These results verified the usefulnessof chrysanthemum flowers in preventing AGE formation.
In this study, we examined the usefulness of edible purple
chrysanthemum flowers from the viewpoint of preventing
and repairing the deterioration of protein function caused
by glycation by examining their effects on inhibiting GPCL
formation, degrading GPCL, inhibiting glycation reaction
intermediate formation, degrading AGE cross-linking, andenhancing OPH activity.

solution; MGO) from Sigma-Aldrich Japan (Meguro -ku,
Tokyo, Japan). 1-phenyl-1, 2-propanedione (PPD), benzoic acid,lysozyme (from Egg White), aminoguanidine hydrochloride(AG), 2, 3- diaminonaphthalene 2, 3 - diaminonaphthalene(DAN) and glyoxal (4 0 % glyoxal solution; GO) wereobtained from Fujifilm Wako Pure Chemical Industries(Osaka, Japan). 3-deoxyglucosone (3DG) was obtained from
Dojin Chemical Research Institute ( Kamimashiki-gun,
Kumamoto, Japan). 2-4 0 % Mini-Protean TGX Precast gel isfrom Bio-Rad (Hercules, California, USA). Acetyl-alanine
p-nitroanilide (AAPA) is from Bachem (Bubendorf,
Switzerland). The oxidized protein hydrolase (OPH) is an
acylamino-acid releasing enzyme from Takara Bio (Otsu,
Shiga, Japan). Other reagents of special grade or HPLC
grade were purchased from Fujifilm Wako Pure Chemical
Industries or Nacalai Tesque (Kyoto, Japan) and used.

2) Sample
The sample used was edible purple Chrysanthemum
flower extract (EE), which was obtained by 50 % ethanol
extraction from edible purple chrysanthemum flowers (solid
content 22.7 %). This sample was provided by Unial (Tokyo,
Japan).

3) Inhibition of GPCL formation
The lysozyme-glucose GPCL cross-link model was used
for the inhibition of GPCL formation based on previous
reports 24, 25). The reaction solution was prepared by adding
1/10 of the sample solution in 0.1 mol phosphate buffer (pH7.4 ) containing 5 mg/mL lysozyme and 0.5 mol/L glucose.
The reaction solutions were made by adding all of thephosphate buffer, lysozyme solution, glucose solution, andsample solution; (A), a solution with purified water added
instead of the glucose solution in A; (B), a solution withsample dissolution solution added instead of the sample
solution in A; (C), a solution with purified water added
instead of the glucose solution in C; (D) were prepared andincubated at 60 °C for 40 hours.
After the reaction was completed, the reaction solution
was centrifuged using a 3 kDa ultrafiltration membrane(Amicon Ultra - 0.5 mL centrifugal filters Ultracel - 3 K;
Merck, Darmstadt, Germany) to remove low- molecular weight substances. The reaction solution was then subjected
to 4-20 % polyacrylamide gel electrophoresis (SDS-PAGE).
The gel after electrophoresis was stained with CBB Stain
One (Nacalai Tesque, Nakagyo-ku, Kyoto, Japan), and theimages were captured with Pharos FX System (Bio-Rad),and the intensities of the lysozyme dimer (25.8 kDa) andtrimer (4 0. 5 kDa) bands were analyzed with ImageJ (NIH,
ImageJ ( NIH, Maryland, USA) was used for analysis. AG
was used as a positive control substance for the inhibition ofglycated protein cross-link formation.

Materials and Methods

The inhibition rate of protein cross-link formation was
calculated based on the following equation.

1) ReagentsThe reagents used were purchased from the following
manufacturers: N-phenacylthiazolium bromide (PTB), humanserum albumins (HSA, lyophilized powder, ≥ agarose gelelectrophoresis), and methylglyoxal (4 0 % methylglyoxal

Inhibition rate of protein cross-link formation (%)= {1 − (A − B) / (C − D) } × 100_ 8 _Glycative Stress Research

A - D ; band intensity of lysozyme dimer or trimer in SDSPAGE gel staining image of each reaction solution

Cross-linkage cleavage rate (%) = { ( 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 thereaction4) Cleavage of GPCL
The cross-link cleavage action of glycated lysozyme wasmeasured by preparing glycated lysozyme and measuring
the degradation rate of the lysozyme dimer by the sample.
Glycated lysozyme was prepared by reacting 1 mg/mL
lysozyme with 0.1 mol/L phosphate buffer (pH 7.4) containing
0.2 mol/L glucose at 37 °C for 16 hours, and then removing
low-molecular-weight substances by ultrafiltration (3 kDa).
The reaction solutions were prepared by adding 1/ 2 volumeof the sample solution in 0.05 mmol/L phosphate buffer
containing 0.5 mg/mL glycated lysozyme; (A), and by adding
the sample dissolution solution instead of the sample solution
in A; (B). The reaction solution was then incubated at 37 °C
for 16 hours, after which the reaction solution was centrifugedthrough an ultrafiltration membrane (3 kDa) to remove lowmolecular-weight substances. The reaction solution was thensubjected to SDS-PAGE in the same manner as described in
the previous section, and after staining and imaging the gel,the band intensity of lysozyme dime in the reaction solution
was analyzed. PTB was used as a positive control substancefor glycated protein cross-link cleavage.

7) OPH activity enhancement
In accordance with our previous report 16), the OPH
activity-enhancing effect was determined by incubating 250µL of the reaction solution (0.1 mol/L Tris-HCl (pH 7.4),
2 mmol/L Acetyl-alanine p-nitroanilide (AAPA), 1 mU/mL
OPH, and sample extract) with 1/25 volume of the sample
solution at 37 °C for 60 min. Incubated at 37 °C for 60
min, the amount of p-nitroanilide ( pNA) released by OPH
degradation of AAPA was measured absorbance at 4 05 nm(S). The OPH activation rate (%) was calculated by using
the following equation, where the amount of p NA released
during 60 min from the beginning of reaction (R) ( 0 min)was taken as 100 %.
OPH activation rate (%) = { (S60 - S0 ) / ( R60 - R0 ) } x 100
S ; pNA concentration of reaction solution with sample solution
R ; pNA concentration of reference reaction solution
60 ; After 60 minutes
0 ; Immediately after start of reaction (0 min)5) Inhibition of glycation reaction
The amount of F-AGEs and glycation intermediates wasdetermined by measuring their concentrations in the reaction
solution after incubation. The concentrations of F-AGEs andglycation intermediates were measured in the reaction solution
after incubation. AG was used as a positive control substancefor the inhibition of glycation reaction, and AGE-derived
fluorescence (excitation wavelength: 370 nm /fluorescencewavelength: 440 nm) was measured for F-AGEs according toour previous report 26). For glycation intermediates, 200 µL of
glycation reaction solution was deproteinized with perchloric
acid and labeled with DAN under alkaline conditions, and
3DG, GO, and MGO were measured by HPLC according toprevious reports 27, 28). The inhibition rate (%) of the formationof F-AGEs and glycation intermediates was calculated
according to our previous report 26). In addition, the 50 %inhibitory concentration (IC 50; mg/mL) was calculated fromthe inhibition rates of three concentrations of the sample
solutions 26, 29). The smaller the IC 50 value, the stronger the
inhibitory effect of the glycation reaction.

Statistical analysis
Measurements were expressed as mean ± standard
deviation (SD) of triplicate measurements. Tukey's test orDunnett's test was used for comparison of measurements.
Statistical analysis results were considered significant at arisk rate of less than 5 %.

Results

1) Inhibition of GPCL formation
EE inhibited the formation of lysozyme dimer andtrimer in a concentration-dependent manner at sample
concentrations of 0.01-1.1 mg/mL ( Table 1, Fig. 1). 0.1
mg/mL EE and AG inhibited GPCL formation at 37.1 ±3.4 % (EE), 24.9 ± 8.8% (AG), and 78.3 ± 8.1 % (EE) and61.7 ± 14.1 % (AG), respectively. The inhibition rate of
GPCL formation by lysozyme dimer was 37.1 ± 3.4% (EE),
24.9 ± 8.8 % (AG), and by lysozyme trimer was 78.3 ± 8.1%
(EE), 61.7 ± 14.1% (AG), showing no difference in theeffects of EE and AG.

6) Measurement of AGE cross-link cleavage
The cleavage of the α-diketone bond of PPD by the
sample was measured using PPD as a model material for
AGE cross-linking, according to previous reports 30, 31). The
reaction solution was a mixture of sample solution, 10 mmol/L
PPD, and 0.2 mol/L phosphate buffer (pH 7.4) in the ratio of
5 : 1 : 4, and the reaction was carried out at 37 °C for 8 hours.
The amount of benzoic acid released was measured under
the same HPLC conditions as previously reported 31).
PTB was used as a positive control substance for AGE
cross-link cleavage. 1 mol of PPD generates 1 mol ofbenzoic acid when the α-diketone bond of PPD molecule iscleaved.

2) Cleavage of GPCL
EE degraded lysozyme dimers at sample concentrations
ranging from 11. 4 to 56.8 mg/mL ( Table 2, Fig. 2). 41. 2 ±9.9 % of the protein cross-link cleavage rate of EE at 56.8
mg/mL did not differ from that of PTB at 5 mmol/L (39.6 ±4.6 %).

3) Inhibition of glycation reaction
EE inhibited the formation of F-AGEs and glycation_ 9 _Suppression of Glycated Protein Cross-linking and Cross-linking Cleavage of Purple Chrysanthemum Flower(Fig. 3 ). The AGE cross-link cleavage rate of EE at 5.68
mg/mL (14.2 ± 0.02 %) was 0.4 times higher than that of 5mmol/mL PTB (cleavage rate 4 0. 5 ± 0.2 %, p < 0.01).

intermediates (3DG, GO, and MGO) in the HSA-glucose
glycation reaction model ( Table 3 ). The IC 50 values of
EE for inhibiting the formation of F -AGEs and glycationintermediates ranged from 0.076 to 0.157 mg/mL. Values ofAG ranged from 0.064 to 0.172 mg/mL, which were close toeach other.

5) OPH activity enhancement
EE enhanced OPH activity at sample concentrations
ranging from 0.9 to 181.6 µg/mL ( Fig. 4 ). The concentrationof EE at which OPH was most activated was 90.8 µg/mL(173.1 ± 0.6 %).

4) AGE cross-link cleavage
The AGE cross-link cleavage rate of EE increased in aconcentration-dependent manner from 0.06 to 5.68 mg/mL

Table 1. Inhibitory effect of edible purple Chrysanthemum flower extract on
cross-linking formation in the lysozyme-glucose reaction model.
SampleEE
AG

Inhibition ratio (%)

Conc.
(mg/mL)

Dimer

Trimer

0. 01-0.9 ± 8.8 **30.4 ± 16.7 *0.1

37.1 ± 3.4

78.3 ± 8.1

1.1

93.0 ± 6.6 **95. 4 ± 7. 4 *0.1

24.9 ± 8.8

61.7 ± 14.1

5 mg/mL lysozyme were incubated at 60℃ for 4 0 hours. SDS-PAGE was conducted using 2- 40 % acrylamide gels.
Data are expressed as mean ± standard deviation, n = 3; Dimer, 25.8 kDa; Trimer, 40.5 kDa; *p < 0.05, ** p < 0.01
vs AG by Dunnett test; EE, edible purple Chrysanthemum flower extract; AG, aminoguanidine; Conc., concentration.

Ref
MW

(G)

EE
(-)

(G)

AG
(-)

(G)

(-)

Trimer

35kDa

Dimer

25kDa

Monomer

11kDa

Fig. 1. Fig.
Inhibitory
effect ofedibleflower extract
cross-linking
1. Inhibitory
effectof purple
edible Chrysanthemum
purple Chrysanthemum
flower on
extract
on crosslinking
formationinthelysozyme-glucose
reactionmodel.
formation in the lysozyme-glucose reaction model.
5 mg/mL lysozyme were incubated at 60 ℃ for 40 hours. SDS-PAGE was conducted using

5 mg/mL lysozyme
incubatedgels.
at 60℃
40 hours.
SDS-PAGE
2-40%
2 - 40were% acrylamide
MW,formolecular
weightmarkers;was
Ref,conducted
incubationusing
without
E E (withacrylamide gels.50MW,
molecular
markers;
Ref,
incubation
EE (with 50% ethanol);EE,% ethanol);EE, weight
incubation
with 0.1
mg/mL
edible without
purple Chrysanthemum
flower extract;incubation withAG,
0.1mg/mL
edibleChrysanthemum
flowerAG,withincubation
with
0.1mg/mL
incubation
withpurple
0.1 mg/mL
aminoguanidine;(G),extract;incubation
0.5 mol/L
glucose;(-),
incubation
without glucose.
aminoguanidine;(G), incubation
with 0.5mol/L glucose; (-), incubation without glucose
4_ 10 _Glycative Stress Research

Table 2. Cleavage effect of edible purple Chrysanthemum flower extract on lysozyme
dimer in the glycated lysozyme.
SampleConc. (mg/mL)

Cleavage ratio (%)

11. 4-2.9 ± 1.6 **

37.8

10.2 ± 4.7 **56. 8

41.2 ± 9.9

5. 0

39.6 ± 4.6

EEPTB

1 mg/mL glycated lysozyme were incubated at 37℃ for 16 hours. SDS-PAGE was conducted using 2- 40 % acrylamidegels. Data are expressed as mean ± standard deviation, n = 3; lysozyme dimer, 25.8kDa; ** p < 0.01 vs PTB by Dunnett
test; EE, edible purple Chrysanthemum flower extract; PTB, N-phenacylthiazolium bromide; Conc., concentration.

EE (mg/mL)
MW

Ref

11.4

37.8

56.8

PTB

35kDa
Dimer

25kDa

Monomer
11kDa

Fig. 2. Cleavage effect of edible purple Chrysanthemum flower extract on lysozyme

Fig. 2. Cleavage
effectpurpleChrysanthemum flower extract on lysozyme dimer in the
dimer
in of
theedible
glycatedlysozyme.
glycated lysozyme.
1 mg/mL glycated lysozyme were incubated at 37 ℃ for 16 hours. SDS-PAGE was conductedusing
2- 40 %were
acrylamidegels.atMW,
weightmarkers; Ref,
EE
1 mg/mL glycatedlysozyme
incubated
37℃molecular
for 16 hours.
SDS-PAGE
was incubation
conductedwithout
using 2-40%(with 50 % ethaol); EE, incubation with edible purple Chrysanthemum flower extract; PTB,
acrylamide gels. MW, molecular weight markers; Ref, incubation without EE (with 50% ethaol); EE,
incubation with 5 mmol/L N-phenacylthiazolium bromide.
incubation with edible purple Chrysanthemum flower extract; PTB, incubation with 5mmol/L Nphenacylthiazolium bromide

5

Table 3. Inhibitory effect of edible purple Chrysanthemum flower extract on fluorescent AGE in the HSA-glucose
glycation model.
Glycation products
Fluorescent AGEs
3DG
GO

MGO

Inhibition ratio (%)
0.01 (mg/mL)

0.1 (mg/mL)

1.1 (mg/mL)

IC 50 (EE)
(mg/mL)

8.9 ± 0.7 **65. 3 ± 0.2 **99.9 ± 0.3 **

0.076

0.064

4.0 ± 0.7 **41.8 ± 0.6 **86.8 ± 1.0 **

0.157

0.153

-10.1 ± 1. 2 **88.0 ± 0.1 **94.8 ± 0.1 **

0.081

0.073

2.8 ± 0. 5 **67.8 ± 1. 2 **85.7 ± 0. 9 **0.101

0.17 2

IC 50 (AG)
(mg/mL)

Data are expressed as mean ± standard deviation, n = 3; ** p < 0.01 vs same glycation products by Tukey's test; IC 50, half inhibitory concentration (mg/mL)on the HSA-glucose glycation model; EE, edible purple Chrysanthemum flower extract; AG, aminoguanidine; HSA, human serum albumin; AGE, advanced
glycation end product; IC 50, half-maximal inhibitory concentration.

_ 11 _

Suppression of Glycated Protein Cross-linking and Cross-linking Cleavage of Purple Chrysanthemum Flower

20 0

30
20

**
10
0

**

**

*

**

40

Relative
activity(%)(%)
Relative activity

Cleavage ratio (%)

50

**
0.06

15 0

10 0

50

**
0.57

EE
EE (mg/mL)(mg/mL)

5.68

0

PTB
(5mmol/L)(5mmol/L)

0.9

9.1

90.8

181.6

Ref

EE
g/mL)
EE ((μg/mL)

Fig. 3. Cleavage effect of edible purple
Chrysanthemum flower extract on α-diketone
bond in the PPD reaction model.

Fig. 4. OPH activation effect of edible purple
Chrysanthemum flower extract.

Data are expressed as mean ± standard deviation, n = 3;
* p < 0.05, ** p < 0.01 by Tukey's test; EE, edible purple
Chrysanthemum flower extract; Ref, water; OPH, oxidized
protein hydrolase.

Data are expressed as mean ± standard deviation, n = 3; ** p
< 0.01 by Tukey's test; PPD, 1-phenyl-1, 2-propanedione;
EE, edible purple Chrysanthemum flower extract; PTB,
N-phenacylthiazolium bromide.

Discussion1) Inhibition of GPCL formation and glycation
reaction
There are many types of AGEs produced in vivo, which
are classified as fluorescent and non-fluorescent, or crosslinked and non-cross-linked. F-AGEs include argpyrimidine,vesperlysine, and pentosidine. Cross-linked AGEs include
pentosidine, crossline, and glucosepane. In addition,
dicarbonyls and aldehydes such as 3DG, GO, and MGO areglycation intermediates that promote the formation of many
types of AGEs. 3DG has already been reported to be involvedin protein cross-link formation 3). In GPCL formation, 3DG
acts as a cross-linker through arginine and lysine residues oflysozyme, ovalbumin, and bovine serum albumin (BSA) 33).
EE inhibited GPCL formation in the lysozyme-glucose
glycation reaction model and inhibited the formation of
F-AGEs, 3DG, GO, and MGO in the HSA-glucose glycation
reaction model. These effects were similar to those ofAG, which binds to the dicarbonyl group of glycationintermediates and inhibits protein glycation 34). EE may haveinhibited the formation of F-AGEs and cross-linked AGEs,
induced by glycation, by acting similarly to AG. Edible
purple chrysanthemum contains luteolin, delphinidin, andchlorogenic acid 20). Since these substances have both antiglycation 20) and antioxidant 22) effects, their involvement in
the inhibition of GPCL formation in EE was presumed.

2) Cleavage of GPCL and AGE cross-links
GPCL is caused by glucosupanes, α-diketones, andlysine-dihydropyridinium-lysine 11). PTB has the ability tocleave α-diketone bonds, and this action cleaves GPCL 30).
EE is shown to have cleavage effects both on GPCL and AGE

cross-linking in a concentration-dependent manner. AGE
cross-link cleavage actions were observed with ellagitannin,
cyanidin, delphinidin and their glycosides contained in
pomegranate extract. The involvement of the hydroxybenzene
structure in these effects has been postulated 31).
Delphinidin, a kind of purple component substanceof edible purple chrysanthemum flowers, may have been
involved in the GPCL cleavage in EE. Meanwhile, it has
been reported that PTB was not involved in the cross-linking
of mouse tail tendon collagen 35). The cross-linking of AGEs
using PPD as a model does not necessarily coincide with thecross-linking of proteins. Therefore, it is necessary to verify
the GPCL cleavage effect of EE in vivo.

3) OPH activity enhancement
The OPH activity-enhancing effect has been observedin many kinds of herbal teas, and its effect has also beenreported in those that use leaves and roots as raw materials,
such as chicory (Cichorium intybus) and dandelion(Taraxacum sp.) 16). The enhancement of OPH activity of EE
was presumed to be due to the involvement of substances
commonly found in Asteraceae plants.

4) Inhibition of GPCL formation and GPCL cleavage
potential in edible purple chrysanthemum

Inhibition of glycative stress includes suppression of
postprandial hyperglycemia, suppression of AGE formation,and degradation of AGEs. In addition, oxidative stress is afactor that accelerates the formation of AGEs, and antioxidant
effects also contribute to the suppression of glycative stress 1).
EE was found to inhibit the formation of F-AGEs, 3DG, GO,_ 12 _Glycative Stress Researchand MGO, as well as inhibit the formation of GPCL. These
effects of EE were similar to those of AG, an inhibitor ofglycation reaction, and AG has been reported to improvediabetic nephropathy with continuous intake of 150 -300 mg
per day 16). Furthermore, delphinidins and cyanidins, which
are pigment components of edible purple chrysanthemum,
have antioxidant effects 36, 37). Therefore, the continuous intakeof EE may prevent the formation of GPCL by inhibiting theformation of AGEs as well as AG.
The formation of AGE cross-links due to glycative
stress is a factor in the reduction of protein elasticity 1).
The accumulation of AGEs therefore increases the risk ofbone fractures due to stiffening of bone collagen, reduces
range of joint motion, decreases skin elasticity, and causes
wrinkle formation and tarseness. Glycated proteins are lesslikely to be degraded by proteases, thus slowing down tissue
metabolism 38).
In a study in which healthy women ingested a mixed
herbal extract with both AGE cross-link cleavage actions andOPH activity enhancing effects for 12 consecutive weeks, theblood pentosidine, a cross-linking AGE, and facial wrinkles
decreased 39). In a study of 8 weeks of continuous intake
of edible purple chrysanthemum powder, a trend toward
improvement in skin elasticity was observed 20). EE, a 50 %ethanol extract of edible purple chrysanthemum powder,
showed AGE cross-link cleavage, GPCL cleavage, and OPH
activity enhancement. These results suggest that continuous
intake of EE may degrade and remove proteins cross-linked
by glycation, and prevent and repair protein dysfunction.

Reference

Research limitations
The inhibition of GPCL formation and GPCL cleavage
by EE in this study were in vitro results. Further verificationof these benefits when EE is continuously ingested by humans
is needed.

Conclusion
EE inhibited the GPCL formation and cleaved GPCL. In
addition, EE inhibited the formation of F-AGEs and glycationintermediates, cleaved AGE cross-links, and enhanced OPH
activity, suggesting that EE may prevent and repair thedeterioration of protein function caused by glycative stress.

Acknowledgements
The publication of this study was supported by the
Isyoku-Dogen Research Foundation (IDF#22002).

Declaration of Conflict of Interest
Research funding was provided by Unial Corporation
for this study.

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