Glycative Stress Research
Online edition : ISSN 2188-3610
Print edition : ISSN 2188-3602
Received : February 13, 2015
Accepted : March 24, 2015
Published online : June 30, 2015

Original article

Cleaving effect of pomegranate (Punica granatum ) extract on crosslink derived
from advanced glycation endproducts
Masayuki Yagi 1), Riko Mitsuhashi 1), Ai Watanabe 1), Yu Moritani 1), Junko Naito 1),
Parengkuan Lanny 1), Hideyuki Ito 2), Akifumi Nagatomo 3), Norihisa Nishida 3), Yoshikazu Yonei 1)
1) Anti-Aging Medical Research Center and Glycation Stress Research Center, Graduate School of Life and Medical Sciences,
Doshisha University, Kyoto, Japan
2) Faculty of Health and Welfare Science, Department of Natural Science, Okayama Prefectural University, Okayama, Japan
3) Morishita Jintan Co., Ltd. Osaka, Japan

Abstract

Objectives: The accumulation of advanced glycation endproducts (AGEs) is a risk factor of glycation stress for age-related
deterioration and diseases. AGE degeneration and excretion is a method reducing glycation stress. The purpose of the present
study is to elucidate whether pomegranate (Punica granatum) can be a functional food which ruduces glycation stress. The
AGE-derived and collagen-derived crosslink cleaving reactions were evaluated in pomegranate extracts and ellagitannins
isolated and purified from pomegranates.
Methods: For evaluation of AGE-derived and collagen-derived crosslink cleaving activity, 22 pomegranate-related samples
were used and compared with N-phenacylthiazolium bromide (PTB) as a positive control and 6 hydroxybenzene compounds.
To evaluate the AGE crosslink activity, by HPLC analysis we measured the amount of benzoic acid which was formed as aresult of C-C binding breaking of α-diketone structure in 1-phenyl-1,2-propanegione (PPD), a model compound for AGE
degeneration. For evaluation of collagen crosslink cleaving activity, using collagen-coated 96-well microplates, AGE-formed
bovine serum albumin (AGE-BSA) was added to the wells and reacted with collagen, thus forming collagen crosslinks,
followed by measuring residual AGE-BSA amount by ELISA. AGE-derived and collagen-derived crosslink cleaving ratio was
calculated as a relative ratio with the ratio of PTB assumed to be 100.
Results: AGE crosslink cleaving activity of 6 pomegranate extracts was higher than that of 0.4 mmol/L PTB. The activity
ratio was highest in gallic acid (26.02%) and lowest in ellagic acid (3.24%) among the 16 pomegranate-derived compounds.
Regarding the hydroxybenzene compounds, the activity ratio in trihydroxybenzene was higher in hydroxyquinol (51.62%) andpyrogallol (42.13%) than in phloroglucinol (6.06%), and the activity ratio in dihydroxybenzene was higher in hydroquinone(15.73%) and pyrocatechol (14.70%) than in resorcinol (6.88%).
Collagen crosslink cleaving activity was noted in 4 pomegranate extracts from pomegranate juice powder (78.50%),
condensed and dried polyphenols (I) (24.99%), ellagitannins (74.75%), and leaf (62.44%). Also, 12 of 16 pomegranate-derived
compounds showed collagen crosslink cleaving activity which was significantly correlated with AGE crosslink cleaving activity.
The activity was not noted in delphinidin chloride, delphinidin 3,5-diglucoside chloride, gallic acid, and cyanidin 3-glucoside.
All of the samples of pomegranate extracts and related compounds showed AGE crosslink cleaving activity, and some ofthem showed collagen crosslink cleaving activity. A significantly positive correlation was noted between two actions (r = 0.635,
p = 0.034). These findings indicate that the trihydroxybenzene structure of ellagitannins may be involved in the AGE crosslink
cleaving activity of pomegranate.
Conclusions: Pomegranate extracts and related compounds are confirmed to show AGE crosslink cleaving activity in addition
to anti-oxidation, α-glucosidase inhibition and the prevention of AGE formation as previously reported, indicating that they can
reduce glycation stress in various ways.

KEY WORDS: advanced glycation endproducts (AGEs), pomegranate (Punica granatum), ellagitannins,
AGE-derived crosslink cleaving reaction, collagen crosslink cleaving reaction

Contact Address: Associate Professor Masayuki Yagi, PhD
Glycation Stress Research Center, Graduate School of Life and Medical Sciences, Doshisha University
1-3, Tataramiyakodani, Kyotanabe-shi, Kyoto, 610-0321 Japan
Phone/Fax: +81-774-65-6394 E-mail: yagi@yonei-labo.com
Co-authors: Mitsuhashi R, dmn2011@mail4.doshisha.ac.jp; Watanabe A, bml2098@mail4.doshisha.ac.jp;
Moritani Y, bmk2055@mail4.doshisha.ac.jp; Naito J, j-naito@a-kit.co.jp;
Lanny P, lannyparengkuan@ymail.com; Ito H, hito@fhw.oka-pu.ac.jp; Nishida N, nishida@jintan.co.jp;
Nagatomo A, a-nagatomo@jintan.co.jp; Yonei Y, yyonei@mail.doshisha.ac.jp_ 58 _

Glycative Stress Research 2015; 2 (2): 58-66(c) Society for Glycation Stress ResearchGlycative Stress Research

Introduction

High glycation stress conditions cause formation andaccumulation of advanced glycation endproducts (AGEs)
in the body, and thus play a role as a risk factor in thepathogenesis of diseases such as diabetic complications,atheroscrerosis, osteoporosis, infertility, or Alzheimer’s
disease, and age-related conditions like elasticity reduction
and loss of clearness in the skin 1). AGE formation causes ayellow-brownish change of tissue and physical deterioration,
especially in the collagen tissue of the skin, bone andcartilage, thus it is a topic in the field of aesthetics, cosmetics
and health promotion. The methods for reducing glycationstress include prevention of postprandial hyperglucosemia,
inhibition of AGE formation, and promotion of AGE
regeneration and excretion 2).
N-phenacylthiazolium bromide (PTB) is known as acleaving material against AGE-derived crosslink. PTB
recognizes crosslink proteins and α-diketone structure
in AGEs and degenerates C-C bonds. PTB is reported tocontribute to the prevention of AGE deposit and the treatment
of diabetic complications in the vascular vessels 3). Extracts ofmugwort (Artemisia indica) and rooibos tea leaves (Aspalathus
linearis) are known to possess a same action as PTB 4).
The extracts from these plants are confirmed to show antiglycation effect and AGEs formation prevention in the in vitro
glucose/albumin reaction 5).
Pomegranates (Punica granatum) are rich in ellagitannins.
Ellagic acid, a degeneration product of ellagitannins, isreported to have anti-oxidative effects 6) and tyrosinase
inhibition activity 7). Recently, pomegranate ext ract andellagitannins have been confirmed to prevent AGE formationin the in vitro glucose/albumin reaction 8, 9).
The present study was conducted to elucidate the effectof pomegranate extract and ellagitannin, isolated and purified
from pomegranate, on the AGE-derived crosslink cleaving
reaction and the collagen crosslink breaking reaction, in orderto evaluate the potential action of pomegranate as a functional
food which reduces glycation stress.

actions, 6 hydroxybenzene compounds were used as follows;hydroxyquinol, pyrogallol, phloroglucinol, hydroquinone,
pyrocatechol, and resorcinol. PTB was used as a positive
control for AGE-derived crosslink cleaving actions. Samples
were dissolved in distilled water, 100% or 50% ethanol. PTB
was used as a positive control for evaluation of AGE-derived
crosslink cleaving activity and collagen crosslink cleaving
activity.

Measurement of AGE-derived crosslink cleaving
activity

The AGE -derived crosslink cleaving activity wasevaluated by the modified method of Vasan et al. 3). 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 (HC l ) 200 µL, followed by centrifugation
at 10,000 rpm ( 9,170 g ) for 2 minutes. The benzoic acidamount in the supernatant was measured by high performanceliquid chromatography ( HPLC ).
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. Analysis conditions
were as follows: eluate, 0.2% acetic acid/acetonitrile (70/30: volum/volum) 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 at 270 nm; injection volume, 50µL.

Calculation of AGE crosslink cleaving activity ratio

Methods
Materials

In total, we used 22 samples of pomegranate extract andisolates and purified materials from the extract in the present
study as follows; 6 pomegranate extracts were pomegranate
juice residue extract, pomegranate juice powder, condensed
and dried product of pomegranate polyphenols ( I and II ),pomegranate ellagitannins, and pomegranate leaf extract.
The 16 purified components were gallic acid, punicalagin,
eucalbanin B, pomegraniin A, punicalin, delphinidin
3, 5-diglucoside chloride, delphinidin chloride, eucarpanin
T 1 , oenothein B, penta-O-galloylglucouse, granatin B,
granatin A, cyanidin chloride, cyanidin 3-glucoside chloride,
pomegralignan, and ellagic acid ( Fig. 1 ).
These pomegranate samples were provided from
Morishita Jintan Co., Ltd. (Chuo-ku, Osaka, Japan), except
for pomegranate ellagitannins which were provided from
Professor Hideyuki Ito, Okayama Prefectural University (Soja,
Okayama, Japan).
For the comparison of AGE-derived crosslink cleaving

When 1 mol of PPD breaks down, 1 mol of benzoic acidis formed 3). The AGE crosslink cleaving ratio was defined asa value when the benzoic acid amount in the reaction solution
was divided by the PPD amount added to the reaction solution
which was measured by HPLC. The benzoic acid amount ineach sample, measured similarly, was pre-excluded from thevalue of benzoic acid amount in the reaction solution. The
AGE crosslink cleaving activity was expressed by calculation
as a relative ratio with the activity by 0.4 mmol/L PTB
assumed to be 100.

Preparation of AGE-BSA

AGEs combined with bovine serum albumin (AGEBSA) were prepared as a crosslink model between collagenand proteins. Briefly, 40 mg/mL BSA 4 mL were mixed
with 0.1 mol/L PBS (pH 7.4) 10 mL, 2 mol/L glucose 2 mL
and distilled water 4 mL, and then incubated at 60°C for 40
hours. After the reaction, AGE-BSA solutions were purified
using a disposable column (PD-10; GE Healthcare Japan,
Hino, Tokyo, Japan), followed by adjusting of the protein
concentrations to attain 10 µg/mL.

_ 59 _

AGEs-derived Crosslink Cleaving Effect by Pomegranate

Fig 1. Chemical structure of compounds and purified materials in pomegranate.
_ 60 _Glycative Stress Research

Measurement of collagen crosslink cleaving activity

Collagen crosslink cleaving activity was evaluated by
the modified method of Vasan et al. 3). Prepared AGE-BSA
was added to each well of a 96-well microplate coatedwith collagen (BioCoat Collagen I 96 -well plate; Corning
International, Minato-ku, Tokyo, Japan) and incubated at 37°C
for 4 hours, followed by 3 minutes of shaking after adding
0.05% polyethylene (20) sorbitan monolaurate (Tween20)/
phosphate buffered saline (PBS) (-). Wells were washed 3times by filling and wasting PBS. Then, each sample 100 µL
was added to a well and incubated at 37°C for 18 hours. Afterwashing wells, the primary antibody (anti-albumin bovine
serum rabbit – polyclonal, 1/20,000 dilution) (Anti-Albumin
Bovine Serum Rabbit-Poly; Rockland, Limerick, PA, USA)100 µg/well was added and reacted at room temperature for
30 minutes. After washing the wells, the secondary antibody(Goat anti-rabbit IgG horseradish peroxidase conjugate; BioRad, Shinagawa-ku, Tokyo, Japan) 100 µg/well was added
and reacted at room temperature for 30 minutes.
After washing each well, 100 µg/well of TMB (3, 3’, 5, 5’tetramethylbenzidine) One Component HRP ( horseradish
peroxidase) Microwell Substrate (SurModics, Eden Prairie,
MN, USA) and 50 µL/well of 1.5 mg/mL polyethylene glycol
6000 (PEG6000) were added and reacted for 20 minutes
in a dark condition. After the reaction, 1 N HCl 100 µL/
well, stop solution, was added to each well, followed by themeasurement of absorbance in each well by using a microplate
reader (SpectraMax Paradigm Multi-Mode Microplate Reader;
Molecular Devise, Sunnyvale, CA, USA) at dual wavelengthsof 450 nm (main) / 630 nm (sub).
The AGE-BSA amount in each well was calculated byusing the AGE-BSA standard curve in which reaction was
conducted without adding samples. The AGE-BSA collagencrosslink cleaving ratio (%) was calculated by using theformula below. The relative ratio was also calculated with themeasured value by 10 mmol/L PTB assumed to be 100.
Cleaving ratio (%) = { (added AGE-BSA amount – remaining
AGE-BSA amount) / added AGE-BSA amount} × 100

Statistical analyses

Results are expressed as mean ± standard deviation(SD) after 3 times repeated measurement (n = 3). Pearson’s
correlation coefficient test was used to compare relationships
between AGE-derived crosslink cleaving action and collagencrosslink cleaving action.
The significance level was set at less than 5%. All analyses
were performed using SPSS II software (IBM Japan, Chuoku, Tokyo, Japan).

Results

AGE crosslink cleaving activity

When the AGE crosslink cleaving activity in 6 kindsof pomegranate extracts was compared, the activity ratio of
pomegranate ellagitannins (31.39%) and pomegranate leaf
extract (31.22%) was higher than in the 4 kinds of pomegranate
fruit extracts ( Table 1 ).
These values of AGE crosslink cleaving activity werehigher than that of 0.4 mmol/L PTB. The added concentrationof pomegranate juice extract was even 10 times higher,
however, it showed less activity than those of pomegranate
ellagitannins and pomegranate leaf extract ( Table 2 ).

Table 1. AGE crosslink cleaving activity of pomegranate extracts.
SampleConcentration

Pomegranate juice residue extract 1)

10 mg/mL

29.92

±

1.02

Condensed and dried pomegranate polyphenols ( II ) 2 )10 mg/mL

27.88

±

3.29

Condensed and dried pomegranate polyphenols ( I ) 2)

10 mg/mL

25.05

±

1.04

Pomegranate juice powder 3)

10 mg/mL

11.77

±

0.53

Pomegranate ellagitannins

1 mg/mL

31.39

±

0.02

Pomegranate leaf extract

1 mg/mL

31.22

±

0.40

1) Polyphenols in hot water extract from squeezed pomegranate residue are condensed and dried.
2) Pomegranate juice polyphenols are filtrated and condensed using resin columns, and the dried.
3) Pomegranate juice is dried and powdered.
AGE, advanced glycation endproduct. Results are expressed as means ± standard deviation (n = 3).

_ 61 _AGE crosslink cleaving ratio (%)

AGEs-derived Crosslink Cleaving Effect by Pomegranate
Table 2. AGE crosslink cleaving activity of pomegranate derived compounds.
SampleAGE crosslink cleaving ratio (%)

Relative ratio 1)

Gallic acid

26.02

±

0.07

409

Punicalagin

15.32

±

2.57

241

Eucalbanin B

13.93

±

0.31

219

Pomegraniin A

13.51

±

0.08

212

Punicalin

12.68

±

2.02

199

Delphinidin 3, 5-diglucoside chloride

12.66

±

0.32

199

Delphinidin chloride

12.38

±

0.77

194

Eucarpanin T 1

12.32

±

0.24

194

Oenothein B

11.35

±

0.06

178

Penta- O-galloylglucose

9.49

±

0.04

149

Granatin B

8.07

±

0.18

127

Granatin A

7.66

±

0.38

120

Cyanidin chloride

7.36

±

4.85

116

Cyanidin 3 -glucoside chloride

3.85

±

0.04

60

Pomegralignan

3.48

±

0.20

55

Ellagic acid

3.24

±

0.10

51

PTB (positive control)

6.37

±

2.71

100

1) Relative ratio of AGE crosslink cleaving activity when the ratio of 0.4 mmol/L PTB is assumed to be 100.
Each sample concentration: 0.4 mmol/L. AGE, advanced glycation endproduct; PTB, N-phenacylthiazolium bromide. Results are
expressed as means ± standard deviation (n = 3).

Among the 16 types of pomegranate-derived compounds,
AGE crosslink cleaving activity was highest in gallic acid(26.02%), and lowest in ellagic acid (3.24%), the difference
was approximately 8-fold ( Table 3 ).
AGE crosslink cleaving activity was compared amonghydroxybenzene compounds. Regarding trihydroxybenzene,
the activity was higher in hydroxyquinol (51.62%)
and pyrogallol (42.13%) than in phloroglucinol (6.06%)( Fig. 2 ). For dihydroxybenzene, the activity was higher inhydroquinone (15.73%) and pyrocatechol (14.70%) than inresorcinol (6.88%).
The AGE crosslink cleaving activity of two compounds,hydroxyquinol and pyrogallol, were higher than that of PTB.

delphinidin chloride, delphinidin 3, 5-diglucoside chloride,
gallic acid, and cyanidin 3-glucoside. The activity ratio washighest in eucalbanin B, that of which was 3.8 times higher
than that of punicalagin with the lowest activity.

Correlation between AGE crosslink cleaving activity
and collagen crosslink cleaving activity

Two kinds of pomegranate extracts and 9 kinds ofpomegranate-derived compounds showed both AGE crosslink
cleaving activity and collagen crosslink cleaving activity.
There was a significant positive correlation ( r = 0.635, p =0.034) among these samples ( Fig. 3 ).

Collagen crosslink cleaving activity

Results of collagen crosslink cleaving activity in 6 kinds ofpomegranate extracts were as follows. Activity was high in 4
extracts; pomegranate juice powder (78.50%), condensed anddried pomegranate polyphenol ( I ) (24.99%), pomegranate
ellagitannins (74.75%), and pomegranate leaf extract (62.44%)( Table 4 ). Pomegranate ellagitannins and pomegranate
leaf extract showed high activity although their sample
concentrations were lower than that of the rest.
Results of the collagen crosslink cleaving activity in
13 kinds of pomegranate derived compounds showed thatthe activity was generally noted except for in 4 compounds,

Discussion

AGE crosslink cleaving activity by pomegranate

From the results of the present experiment, AGE
crosslink cleaving activity was higher in pomegranate extract,in which ellagitannins are rich, than in all four extracts frompomegranate juice. In the 12 compounds with higher AGE
crosslink cleaving activity, the trihydroxybenzene structure
was recognized as a common structure, but not noted in theremaining 4 compounds. These findings indicate that the_ 62 _Glycative Stress Research

Table 3. Collagen crosslink cleaving activity of pomegranate extracts.
SampleRelative ratio 1)

Cleaving ratio (%)

Concentration

Pomegranate juice powder 4)

5 mg/mL

78.50

±

5.55

130

Condensed and dried pomegranate polyphenols ( I ) 2)

5 mg/mL

24.99

±

7.90

41

Condensed and dried pomegranate polyphenols ( II ) 2)

5 mg/mL- 14.84

±

25.18

NE

Pomegranate juice residue extract 3 )5 mg/mL- 78.44

±

52.88

NE

Pomegranate ellagitannins

1 mg/mL

74.75

±

0.21

124

Pomegranate leaf extract

1 mg/mL

62.44

±

1.52

103

PTB (positive control)

10 mmol / L

60.4

±

11.62

100

1) Relative ratio of collagen crosslink cleaving activity when the ratio of 10 mmol/L PTB is assumed to be 100.
2) Pomegranate juice polyphenols are filtrated and condensed using a resin column, and then dried.
3) Polephenols in hot water extract from squeezed pomegranate residue are condensed and dried.
4) Pomegranate juice is dried and powdered.
PTB, N-phenacylthiazolium bromide; NE, no effect. Results are expressed as means ± standard deviation (n = 3).

trihydroxybenzenehydroxyquinol(1, 2,4-trihydroxybenzene)

pyrogallol(1, 2, 3-trihydroxybenzene)

phloroglucinol(1, 3, 5-trihydroxybenzene)

OH

OH

OH
OH

HO

OH
HO

OH
51.62 ± 0.02 %42.13 ± 0.25 %OH
6.06 ± 0.06 %dihydroxybenzenehydroquinone(1, 4-dihydroxybenzene)

pyrocatechol(1, 2-dihydroxybenzene)OH
HO

resorcinol(1, 3-dihydroxybenzene)

HO

OH

OH
OH
15.73 ± 0.18 %14.7 ± 0.02 %6.88 ± 0.03 %Fig 2. Chemical structure and AGE crosslink cleaving activity of hydroxybenzene compounds.
Concentration of each sample; 10 mg/mL. Results are expressed as means ± standard deviation (n = 3).

_ 63 _

AGEs-derived Crosslink Cleaving Effect by Pomegranate

Table 4. Collagen crosslink cleaving activity of pomegranate-derived compounds.
SampleCleaving ratio (%)

Eucalbanin B

78.49

±

0.25

Cyanidin chloride

45.18

±

11.43

Pomegralignan

45.09

±

3.98

Punicalin

30.74

±

7.39

Granatin B

29.23

±

12.00

Penta - O -galloylglucose

26.71

±

5.81

Granatin A

25.23

±

4.73

Ellagic acid

21.06

±

13.18

Punicalagin

20.67

±

5.63

Delphinidin chloride- 3.18±4.93

Delphinidin 3, 5 - diglucoside chloride- 8.76±

18.39

Gallic acid

- 59.06±

6 4.99Cyanidin 3 - glucoside chloride- 175.54±

52.18

Concentration of each sample: 0.4 mmol/L. Results are expressed as means ± standard deviation (n = 3).

90

Collagen crosslink cleaving ratio (%)

80

70

60

50

40

30

20

10

0
0

5

10

15

20

25

30

35

AGE crosslink cleaving ratio (%)

Fig 3. Relationship between AGE crosslink cleaving activity and collagen crosslink cleaving activity
Regression formula: y = 1.3824x + 23.704, r = 0.635, p = 0.036, n = 11, Pearson’s regression analysis.

_ 64 _Glycative Stress Researchtrihydroxybenzene structure in ellagitannins may be involvedin the mechanism of AGE crosslink cleaving activity.
PTB, which was used as a positive control, recognizes thedicarbonyl structure in the Amadori-protein-ene-dion-derived
protein crosslinks which are formed by glycation stress andbreak the protein-protein crosslink 3). Regarding the crosslink
cleaving activity in natural products, there is one reportof terpinen-4-ol, one of monoterpene alcohols, containedin yuzu (Citrus junos), a species of Asian citrus fruits(Rutaceae) 10). The mechanism of protein crosslink cleaving
activity by terpinen-4-ol has been speculated as follows; First
step is nucleophilic substitution caused by hydroperoxide,
then carboxylate ester is formed by a similar reaction to
Baeyer-Villiger oxidation, finally reaching the hydrolysis ofcarboxylate esters.
In the present study comparing the AGE crosslink
cleaving activity of hydroxybenzene compounds, the activity
was higher in trihydroxybenzene than in dihydroxybenzene.
Furthermore, the activity was higher in the compoundswith larger deviation in substitute positions. For example,
the activity was high in compounds with a hydroquinone
structure in which hydroxyl groups are located at 1, 2, 4moiety, in a benzene structure or with a pyrocatechol structurein which hydroxy groups are located at 1, 2, 3 moiety. Thesefindings indicate the involvement of nucleophilicity in thetrihydroxybenzene structure of ellagitannins with AGE
crosslink cleaving activity caused by pomegranate.

Collagen crosslink cleaving activity by pomegranate

Vassan et al. used the collagen crosslink cleaving activity
by PTB as an in vitro model of AGE crosslink cleaving
activity 3). In our experiment, all 6 samples of pomegranate
extracts and 13 pomegranate-derived compounds showed
AGE crosslink cleaving activity. However, 4 compounds,delphinidin chloride, delphinidin 3, 5-diglucoside chloride,
gallic acid and cyanidin 3-glucoside chloride, did not show
the collagen crosslink cleaving effect. We cannot find any
relationship between collagen crosslink cleaving activity andthe features in material structures.
On the other hand, both the AGE crosslink cleaving
activity and collagen crosslink cleaving activity were notedin 2 pomegranate extracts and 9 pomegranate-derived
compounds with significant correlation between two activity
markers. These findings support the possibility that the AGEBSA collagen cleaving reaction can be an in vitro model forevaluating AGE crosslink cleaving activity in other samples.

Potential effect of glycation stress reduction bypomegranate-induced AGE crosslink cleaving

In the present study, PTB, namely called “AGE breaker”,
was used as a positive control for evaluating AGE crosslink
cleaving activity. PTB was reported to inhibit AGE crosslink
formation in collagen in diabetic rats to which 10 mg/kg
PTB was orally administered for 4 weeks 3), and showed theAGE degeneration effect in blood vessels 11). Furthermore,
3-phenacyl-4, 5-dimethylthiazorium chloride (ALT-711), a
more water-soluble compound than PTB, was confirmed toprevent atheroscrelosis and to enhance AGE degenerationin blood vessels when administered to diabetic rats 12). In theclinical trial in which ALT-711, 210 mg/day or 420 mg/day

for 8 weeks, was orally administrated to men, improvement
effects were noted in vascular stiffening and uncontrolled
systolic blood pressure 13, 14). On the contrary, another report
showed that PTB had no effect on AGE crosslink cleaving
against skin and tail tendon collagen in diabetic rats 15).
In the open clinical trial in which pomegranate extract (100mg/day) was administerd to healthy subjects for 12 weeks, it
was noted that a reduction of HbA1c at 8 and 12 weeks, yielded
glycated albumin, and 3-deoxyglucosone, an intermediate in theglycation reaction, at 8 week compared with pre-values 16).
Pomegranate extracts and ellagitannins are confirmed
to have a preventive effect of AGE formation 8, 9), inhibitory
effect of α-amylase and α-glucosidase activity 17), and antioxidant action 6 ) in addition to the AGE crosslink cleaving
action in this study. Thus pomegranate can be expected as a
new functional food which reduces glycative stress throughthe various mechanisms.

Conclusions

The present study showed the AGE crosslink cleaving
activity of pomegranate extracts and pomegranate-derived
compounds. Trihydroxybenzene structure in ellagitannins may
be involved with its mechanism. Some of these compounds
also have collagen crosslink cleaving activity which wascorrelated with AGE crosslink cleaving activity.
Coupled with the finding that pomegranate extracts have
AGE crosslink cleaving activity in addition to the previously
reported activities of anti-oxidation, α-glucosidase inhibitionand prevention of AGE formation, pomegranate can be
expected as a functional food which can reduce glycationstress in various mechanical ways.

Acknowledgements

This work was supported by subsidized projects supportedby the Ministry of Agriculture, Forestry and Fisheries “Greenand Water Environment Technology and Innovation Project,
The 6 th Industry Generation for Agriculture, Forestry and
Fisheries Community Program 2014 ” and the JapaneseCouncil for Science, Technology and Innovation (CSTI), andthe Cross-ministerial Strategic Innovation Promotion Program(SIP Project ID 14533567)

Conflicts of interest statementLtd.

_ 65 _Part of this work was supported by Morishita Jintan Co.,

AGEs-derived Crosslink Cleaving Effect by Pomegranate

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