Glycative Stress Research
Online edition : ISSN 2188-3610
Print edition : ISSN 2188-3602
Received : March 24, 2015
Accepted : April 16, 2015
Published online : June 30, 2015

Original article

Peel extract of water chestnut (Trapa bispinosa Roxb.) inhibits glycation,
degradesα-dicarbonyl compound,and breaks advanced glycation end product crosslinks
Shouko Takeshita 1), Masayuki Yagi 2), Tomohiro Uemura 1), Michio Yamada 1), Yoshikazu Yonei 2)1) Hayashikane Sangyo Co., Ltd. Shimonoseki-shi,Yamaguchi, Japan2) Anti-Aging Medical Research Center and Glycation Stress Research Center, Graduate School of Life and Medical Sciences,
Doshisha University, Kyoto, Japan

Abstract

Objective: We evaluated the anti-glycation and glycation product degrading activities of peel extract of the water chestnut(Trapa bispinosa Roxb.), and studied the potential of this extract as a food ingredient to combat glycation stress.
Method: Glycation product production inhibiting activity was evaluated in an in vitro test system using a glycated protein
model established by incubating glucose and human serum albumin (HSA) for 30 h at 60°C. A mixture of glucose, HSA, andwater chestnut peel extract (TBE) was reacted, the amount of produced glycated proteins was compared with that of a control
system not supplemented withTBE, and the production inhibition rate was calculated. In evaluating the amount of produced
glycated proteins, in addition to measuring fluorescence levels characteristic of advanced glycation end products (AGEs), theamount of pentosidine and carboxymethyllysine (CML) produced were measured by ELISA, and the amount of the produced
intermediate 3-deoxyglucosone (3DG) was measured by HPLC. α-Dicarbonyl compound cleavage activity was evaluated using
HPLC by measuring the amount of benzoic acid produced by the degradation of α-dicarbonyl model compound 1-phenyl1, 2-propanedione (PPD) after addition of TBE. For evaluation of the AGE crosslink cleavage activity, a model system wasestablished wherein AGE crosslinks formed on a collagen-coated plate were broken by adding TBE, and the quantity of broken
AGE crosslinks was measured by ELISA and evaluated.
Results: The TBE exhibited high anti-glycation activity, with an IC 50 value of 2.53 µg/mL. This value is approximately 1/50thof that of aminoguanidine. TBE also exhibited a dose-dependent production inhibitory activity against pentosidine, CML, and
3DG. Furthermore, TBE degraded α-dicarbonyl compounds and AGE crosslinks in a dose-dependent manner, and the cleavage
rates were higher than those of the positive control N-phenacylthiazolium bromide.
Conclusion: The results indicate high activity of TBE in inhibition of glycation product synthesis, degradation of α-dicarbonylcompounds, and breaking of AGE crosslinks. This suggests that TBE may inhibit production of AGEs and their intermediates
and degrade α-dicarbonyl compounds and AGE crosslinks in vivo, and we believe that it may alleviate glycation stress.

KEY WORDS: glycative stress, anti-glycation, AGE breaker, water chestnut (Trapa bispinosa)
Introduction

In recent years, glycation in organisms has been
positioned as a major risk factor for accelerated aging process,and the concept of glycation stress has gained attention 1).
The concept of glycation stress is that the reaction of reducing
sugars, aldehydes, etc. with proteins and amino acids produces
advanced glycation end products (AGEs), and the accumulationof these substances overloads cells and tissues and disrupts
the functioning of functional proteins as well as causes a
series of reactions such as induction of inflammation 2).
The glycation reaction at the core of glycation stress isa reaction in which proteins and reducing sugars bond nonenzymatically to produce glycation products. Glycation
products that have been identified include the AGEs
pentosidine, N ε - (carboxymethyl ) lysine (CML), andpyraline, and their intermediates 3-deoxyglucosone (3DG),
glyoxal (GO), and methylglyoxal (MGO) 3). Bioaccumulationof these glycation products is said to be involved in kidneydisease and eye disorders associated with progression ofdiabetes 4), arteriosclerosis 5), osteoporosis 6), Alzheimer’s
disease 7), infertility 8), and sclerema 9). Based on these facts,

Contact Address: Shouko Takeshita
Hayashikane Sangyo Co., Ltd.
2-4-8 Yamato-machi, Shimonoseki City, Yamaguchi 750-8608, Japan
Phone&Fax: 083-267- 0094, 083-267-0192 E-mail: stakesita@hayashikane.co.jp
Co-authors: Yagi M, yagi@yonei-labo.com, Uemura T, tuemura@hayashikane.co.jp,
Yamada M, myamada@hayashikane.co.jp, Yonei Y, yyonei@mail.doshisha.ac.jp_ 72 _

Glycative Stress Research 2015; 2 (2): 72-79(c) Society for Glycation Stress ResearchGlycative Stress Research

we believe that inhibiting the production and accumulationof glycation products and stimulating the degradation andmetabolism of previously produced glycation products willlead to an increased life expectancy, improved quality of life,and disease prevention.
The plant we focused on in this study, the water chestnut(Trapa bispinosa Roxb.), is an annual aquatic plant belonging
to the family Trapaceae. The peel of the fruit has been used in
medicinal teas and other herbal medicines. Dried peel extractof the water chestnut has been reported to have beneficial
properties such as antioxidant 10) and antibacterial activity 11).
However, there are no reports concerning its anti-glycation
activity or its degradation action against glycation products.
Thus, we conducted a detailed study on its anti-glycation
activity and degradative action against glycation products,
which are expected to prevent diseases.

Materials and Methods

Pentosidine Measurement

Preparation of Water chestnut (T. bispinosa)
Peel Extract (TBE)

Water chestnut peel was dried, sterilized, and crushed,
and then extracted using hot water (approximately six-fold theweight of the water chestnut peel). Dextrin was added to thissolution at a ratio of 67:33 of dry weight, and the resulting
solution was spray-dried to obtain TBE. Sample solutions for
subsequent measurements were prepared by dissolving the
TBE in distilled water.

Glycated Protein Sample Preparation

To synthesize glycated proteins, a glycation reaction
system of glucose and human serum albumin (HSA) wasused according to previously described method. 12) A glycatedprotein solution was obtained by mixing 500 µL of 0.1 mol/
L phosphate buffered saline (PBS) (pH 7.4), 200 µL of 4.0
mg/mL HSA (Sigma-Aldrich, St. Louis, MO, USA), 100µL of 2.0 mol/L glucose aqueous solution, and 180 µL of
distilled water, to which 20 µL of sample solution of arbitrary
concentration or distilled water was added, and the mixture
was incubated for 30 h at 60°C. Additionally, aminoguanidine
sulfate (AG) (Wako Pure Chemical Industries, Ltd., Osaka,
Japan) instead of sample solution was dissolved in distilledwater and this was used as a positive control.
The glycated protein solution was used for themeasurements of anti-glycation activity, pentosidine, CML,and 3DG.

Anti-glycation Activity Measurement

as a sample system solution, and the post-reaction glucose(-)
solution was used as sample blank. The solutions obtainedby adding distilled water instead of the sample to glucose(+)
and glucose(-) solutions were used as control and blank,
respectively.
A total of 200 µL of post-reaction solution was loaded
into a 96-well black assay plate (BD Biosciences, San Jose,
CA, USA), and fluorescence was measured at an excitation
wavelength of 370 nm and emission wavelength of 440 nm
using a Tecan Infinite 200 microplate reader (Tecan Japan
Co., Ltd., Kanagawa, Japan).
The inhibition rate of fluorescence production in samples
was calculated by comparing the fluorescence with that of
the control, and 50% inhibitory concentration ( IC 50 ) was
calculated as anti-glycation activity.
Anti-glycation activity (%) = 100 × (1- ([S] - [SB]/[C] - [B])),
where [S], [SB], [C], and [B] indicate the fluorescence levelsof the sample system solution, sample blank, control, andblank, respectively.

Anti-glycation activity of the test substance was measured
as previously described 12) by incubating HSA in the presence(glucose[+]) or absence (glucose[-]) of glucose for 30 h at
60°C. The glucose(+) reaction solution was obtained bymixing 500 µL of 0.1 mol/L PBS (pH 7.4), 200 µL of 4.0 mg/
mL HSA, 100 µL of 2.0 mol/L glucose aqueous solution, and180 µL of distilled water. The glucose(-) reaction solution was
obtained by mixing 500 µL of 0.1 mol/L PBS (pH 7.4), 200µL of 4.0 mg/mL HSA, and 280 µL of distilled water. Afteradding 20 µL of sample solution of arbitrary concentration to
glucose(+) and glucose(-) solutions, they were incubated for
30 h at 60°C. The post-reaction glucose(+) solution was used

Pentosidine was measured by enzyme-linked
immunosorbent assay (ELISA) as previously described 13),
using an FSK pentosidine ELISA kit (Fushimi PharmaceuticalCo., Ltd., Kagawa, Japan). Fifty-microliters of each glycatedprotein solution or 50 µL of isotonic saline for enzyme
blank measurement was added to 100 µL of pronase solution
included in the kit, and 150 µL of each of these mixtures
was incubated for 90 min at 55°C, after which they were
heated for 15 min in boiling water to inactivate the pronase.
Fifty microliters of 0.2 M disodium edetate was added toeach mixture as a stabilizer to produce samples for ELISA
measurements.
A total of 50 µL of sample or pentosidine standard
solution was added to 96-well microplate with a solid phaseof pentosidine-keyhole limpet hemocyanin antigen includedin the kit, and 50 µL of anti-pentosidine rabbit polyclonal
antibody solution included in the kit was added as a primary
antibody to each well and mixed. The plate was hermeticallysealed, and after incubation for 60 min at 37°C, the reaction
solution in the wells was discarded and the wells were
washed thrice with 0.0025% Tween-20 solution. Afterwashing, 100 µL of peroxidase-labeled anti-rabbit IgG goatpolyclonal antibody solution included in the kit was added asa secondary antibody to each well, and after incubating for
60 min at room temperature, the wells were washed in thesame manner as described above. After washing, 100 µL of asolution containing 3, 3’, 5, 5’- tetramethylbenzidine (TMB)
was added as a color developer to each well. It was incubatedfor 10 min in the dark, and then 100 µL of a stop solution
was added to stop color development. Obtained reaction
solution was subjected to absorbance measurement at primary
wavelength of 450 nm and reference wavelength of 630 nm
using a Tecan Infinite 200 microplate reader. The pentosidine
concentration in each of the measurement samples wascalculated from a pentosidine standard calibration curve andused to calculate the production inhibition rate in the samples
versus the control.

CML Measurement

CML was measured by ELISA using a CircuLex CML/
N ε- (Carboxymethyl)lysine ELISA Kit (CycLex Co., Ltd.,
Nagano, Japan) as previously described 14 ). The glycated_ 73 _

Effect of Water Chestnut on AGE formation and Crosslink Breaking
protein solution was diluted 4- fold using dilution buffer
solution included in the kit, and the resulting solution wasused as the sample.
Sixty microliters of anti-CML monoclonal antibody
solution included in the kit was added to 60 µL each of the
included CML-HSA standard solution, blank solution, or
sample and mixed well. Hundred microliters of each mixture
was added to each well of the CML-bovine serum albumin(BSA) solid phase microplate included in the kit, and the plate
was incubated while shaking for 1 h at room temperature.
The solution was removed from the wells, and after washingeach well four times with 200 µL of washing buffer solution
containing 0.2% Tween-20, 100 µL of horseradish peroxidase(HRP)-labeled anti-mouse IgG polyclonal antibody includedin the kit was added to each well as a secondary antibody andincubated while shaking for 1 h at room temperature. Afterthe completion of the reaction, the wells were washed using
the same procedure as described above. Solution (100 µL)
containing TMB included in the kit was added to each well,and incubated while shaking for 1 h at room temperature. The
reaction was terminated by adding stop solution containing
1.0 N H 2 SO 4 to each well and the absorbance was measured
at a primary wavelength of 450 nm and reference wavelengthof 540 nm using a Tecan Infinite 200 microplate reader. The
CML concentration in each of the samples was calculated
from a CML-HSA standard calibration curve and then used to
calculate production inhibition rate of the samples versus thecontrol.

AGE Crosslink Cleavage Activity Measurement

3DG Measurement

3DG was measured by fluorescent high performanceliquid chromatography (HPLC) as previously reported 15) .
A volume of 12.5 µL of 0.005% 2,3-butanedione (Wako)
was added to 250 µL of each glycated protein solutions
and mixed by stirring. Then, 250 µL of 6.0% perchloric
acid (Wako) was added to remove proteins, the resulting
solution was centrifuged for 20 min at 3,000 rpm, and500 µL of supernatant was recovered. The recovered
supernatant was neutralized by adding 500 µL of saturated
sodium hydrogen carbonate and then, 25 µL of 1.0 mg/
mL 2,3-diaminonaphthalene (DAN) (Dojindo Laboratories,
Kumamoto, Japan) was added to the solution. After stirring,the mixture was incubated overnight at 4°C in the dark and1.0 mL of ethyl acetate was added to the solution and stirred
well. The resulting solution was centrifuged for 10 min at
3,000 rpm, and 800 µL of the supernatant was recovered,dried under vacuum, and the dried solid was redissolved in
50 µL of 50% methanol. The resulting solution was filteredusing a 0.45-µm Millex-HV filter unit (Merck Millipore Ltd.,
Billerica, MA, USA) to produce an HPLC sample.
A TSKgel ODS-80T column, 150 × 6.0 mm I.D. (Tosoh
Bioscience LLC, Tokyo, Japan) was used in the analysis.
The eluent was prepared with 50 mM phosphoric acid, 100%
acetonitrile, and 100% methanol in the ratio 14:3:3, the flowrate was 1.0 mL/min, and the UV detection wavelength was
268 nm.
A calibration curve was constructed using 3DG-DAN
standard solution (Dojindo), which was then used to calculate
the 3DG concentration in each sample and the production
inhibition rate versus the control.

α-Dicarbonyl Bond Cleavage Activity Measurement

measuring benzoic acid concentration obtained by degradationof the α-dicarbonyl compound, 1-phenyl-1, 2-propanedione(PPD), by using HPLC as previously described 16).
A PPD solution was prepared by dissolving 4.2 µL of
98% PPD solution (Sigma-Aldrich Japan, Tokyo, Japan) in
25 mL of 50 mM PBS (pH 7.4)/50% methanol to obtain a
final concentration of 1.0 mM. Hundred microliters of TBE
solution of arbitrary concentration was added to 900 µL of the
prepared PPD solution, mixed, and incubated for 4 h at 37°C
while shaking. The reaction was terminated by adding 200 µL
of 2.0 N HCl and the resulting solution was filtered through a
0.45-µm filter to produce an HPLC sample.
A TSKgel ODS-80T, 150 × 6.0 mm I.D. (Tosoh) column
was used in the analysis. PBS (50 mM, pH 2.2) was used as
the eluent, the flow rate was 1.0 mL/min, and the detection
wavelength was 230 nm UV.
The benzoic acid concentration in each measurement
sample was calculated from a calibration curve constructedusing solutions of benzoic acid ( Wako) of various
concentrations, and based on the assumption that 1.0 mM
benzoic acid is obtained from 1.0 mM PPD, dicarbonyl bondbreaking activity in the PPD structure was determined using
the formula below.
Dicarbonyl bond breaking activity = 100 × benzoic acidconcentration (mM)/1 mM
N-phenacylthiazolium bromide (PTB) (Prime Organics,
Inc., Woburn, MA, USA) was used as a positive control.

Dicarbonyl bond breaking activity was determined by

For AGE crosslink breaking activity, crosslinks formed
between type I collagen and AGE-BSA were measured by
ELISA as previously reported 16), and the crosslink breaking
rate was calculated.
Hundred microliters of 10 µg/mL AGE-BSA (Medical &Biological Laboratories Co., Ltd., Nagoya, Japan) was added
to a BD BioCoat Collagen I 96-well micro test plate (BD) andincubated for 4 h at 37°C. The plate was washed five times
with 0.05% Tween-20/PBS(-), and 100 µL of reagent of eachconcentration dissolved in PBS(-) was added and incubatedfor 20 h at 37°C. After the incubation period, the wells were
washed thrice with 0.05% Tween-20/PBS(-), and100 µL of 1.0µg/mL anti-bovine serum albumin rabbit polyclonal antibody
solution (Rockland Immunochemicals Inc., Limerick, PA,USA) was added as a primary antibody and the mixture wasincubated for 30 min at room temperature. After incubation,
the wells were washed five times with 0.05% Tween-20/
PBS(-), and 100 µL of 1.0 µg/mL HRP-labeled anti-rabbit
IgG antibody (Funakoshi Co., Ltd., Tokyo, Japan) solution
was added to each well as a secondary antibody and incubatedfor 30 min at room temperature. The wells were washed thricewith 0.05% Tween-20/PBS(-), and after washing, 100 µL of
TMB Microwell Peroxidase Substrate (Kirkegaard & Perry
Laboratories, Inc., Gaithersburg, MD, USA) was added andthe wells were incubated for 15 min at room temperature.
The reaction was terminated by adding 100 µL of 1N H 2 SO 4
to each well and absorbance at 450 nm was measured usinga Tecan Infinite 200 microplate reader. Using the measured
values of the sample and control groups, the AGE-BSA
crosslink breaking rate was calculated from the following
formula.
AGE-BSA crosslink breaking rate (%) = 100 × ( 1 absorbance of sample group/absorbance of control group).

_ 74 _Glycative Stress ResearchComparative analysis was carried out using PTB as apositive control.

Statistical Analysis

Results were expressed as mean ± standard deviation. The
significance of the results was calculated using Student’s t-testor Dunnett’s test. The significance level was set at p < 0.05
(two tailed).

Results

Figure 1 shows the anti-glycation activity results for
TBE, and Figure 2 shows the rate of production inhibitionof pentosidine, CML, and 3DG by the TBE samples. The
IC 50 values of each parameter, calculated for comparing thepotency, are shown in Table 1. According to these results,
TBE exerted an anti-glycation activity with an IC 50 value of
2.53 µg/mL, which is lower than the IC 50 of AG (128.13 µg/mL). The TBE also inhibited the synthesis of pentosidine,

CML, and 3DG in a dose-dependent manner, at IC 50 valuesof 1.23 µg/mL, 12.76 µg/mL, and 14.70 µg/mL, respectively.
AG inhibited the synthesis of pentosidine, CML, and 3DG at
IC 50 values of 1.76 µg/mL, 115.70 µg/mL, and 338.31 µg/
mL, respectively, which were all higher than those of the
TBE.
Figure 3 shows the results of α-dicarbonyl compoundcleavage activity. The α-dicarbonyl compound cleavage rates
caused by the addition of the TBE were 7.13% (100 µg/mL,
p < 0.01) and 32.49% (1,000 µg/mL, p < 0.01), increasing
in a dose-dependent manner. The α-dicarbonyl compoundcleavage rates due to addition of PTB were 4.88% (100 µg/
mL, p < 0.01) and 28.37% (1,000 µg/mL, p < 0.01), whichwere lower than those of the TBE.
Figure 4 shows the results of the AGE crosslink cleavage
rate. The AGE crosslink cleavage rates due to the TBE were50.66% (10 µg/mL, p < 0.01) and 81.90% (100 µg/mL, p <0.01), increasing in a dose-dependent manner. The inhibition
rates after PTB addition were 18.63% at 8.5 µg/mL and21.35% at a concentration of 85 µg/mL, demonstrating that
the AGE crosslink cleavage rate of the TBE was higher thanthat of PTB.

100

inhibitory activity (%)

80

60

40

20

TBE

0

AG

- 20
0

20

40

60

80

100

Concentration (µg/mL)

Fig 1. Inhibitory activity of TBE and AG. against fluorescent AGE formation.

AGE, advanced glycation end product; TBE, water chestnut (T. bispinosa) peel extract; AG, aminoguanidine.

_ 75 _

120

Effect of Water Chestnut on AGE formation and Crosslink Breaking

100

Inhibitory activity (%)

80

60

40

20

0

- 20
1

2

5

1

2

TBE

5

1

AG

10

100

1

TBE

Pentsidine

10

100

2

AG

10

20

2

TBE

CML

10

20

AG
3DG

Concentration (µg/mL)

Fig 2. Inhibitory activity of TBE and AG against pentosidine, CML and 3DG formation.

TBE, water chestnut (T. bispinosa) peel extract; AG, aminoguanidine; CML, carboxymethyllysine; 3DG, 3-deoxyglucosone.

Table 1. Inhibitory activity against formation of fluorescent AGEs and each glycated products.
Sample name

Fluorescent AGEs

Pentosidine

CML

3DG

IC 50 of AG ( µg/mL）

128.13

1. 76

115.70338. 31

IC 50 of TBE ( µg/mL）

2. 53

1. 23

12.76

14.70

AGEs, advanced glycation end products; IC50, 50% inhibitory concentration; CML, carboxymethyllysine; 3DG, 3-deoxyglucosone; AG,
aminoguanidine; TBE, water chestnut (T. bispinosa) peel extract.

_ 76 _Glycative Stress Research
35

32.49 ＊＊

Cleavage rate of α- dicarbonyl (%)

30

28.37 ＊＊

25

20

15

10
7.13 ＊＊
4.88 ＊＊

5
1.65

2.20

DW

10 µg/ml

2.05

0100 µg/ml

Control

1000 µg/ml

10 µg/ml

TBE

100 µg/ml

1000 µg/ml

PTB

Concentration (µg/mL)

Fig 3. Cleavage activity of TBE and PTB against α-dicarbonyl structure in PPD.

Results are expressed as means ± standard deviation. ** p < 0.01, vs. control, Student’s t test. DW, distilled water; TBE, water chestnut (T. bispinosa)
peel extract; PTB, N-phenacylthiazolium bromide; PPD, 1-phenyl-1,2-propanedione. PTB has been proposed the mechanism for cleaving C-C bond.

90

81.90 ＊＊

80

Cleavagerate (%)

70
60

50.66 ＊＊

50
40
18.63

30

＊＊
21.35

20
10.11

5.55

10
0
1

10

100

0.85

TBE

8.5

85

PTB

Concentration (µg/mL)

Fig 4. Cleavage rate of TBE and PTB against AGE-BSA-collagen crosslinks.

Results are expressed as means ± standard deviation. ** p < 0.01, vs. control, Dunnet’s test. TBE, water chestnut (T. bispinosa) peel extract; PTB,
N-phenacylthiazolium bromide; AGE, advanced glycation end product; BSA, bovine serum albumin..

_ 77 _

Effect of Water Chestnut on AGE formation and Crosslink Breaking

Discussion
The water chestnut fruit used as the subject of this study
is a large fruit among plants in the Trapaceae family 17). Its
flesh is consumed in Thailand and China, while in Japan, thepeel is dried to produce tea and is distributed as a specialty of
Fukuoka and Saga Prefectures. Because there is a long historyof consumption of this fruit in local diets, we expect that thefruit can be used as a safe plant-based ingredient. However,
the efficacy of ingesting water chestnuts is mostly anecdotal,
with only few scientific reports. Thus, we studied theinhibition of glycation product synthesis and the degradative
activity against glycation product by the TBE using an invitro test system, and observed that the TBE has a strong
anti-glycation activity. In particular, by comparing the IC 50
values, we found that the anti-glycation activity of the TBE
was approximately 50-fold higher than that of AG, and theactivity of the TBE against production of pentosidine, CML,and 3DG were nearly equal, nine-fold, and 23-fold stronger,
respectively, than those of AG. Based on these results, we
believe that TBE is a potent anti-glycation ingredient, whichacts at a much lower concentration than AG.
AG binds to the keto group of Amadori compounds and
3DG, which are intermediates of the glycation reaction, andinhibits further progress of the reaction 18). In addition to areport concerning inhibition of glycation reaction in vitro 19), it
has also been suggested to have effects on kidney disease 20, 21)
and inhibit the onset or progression of diabetic, neurological,
and vascular disorders 22, 23) in diabetic rats. The results of thisstudy show that TBE has a more potent anti-glycation action
than AG, and we expect that the TBE will exhibit efficacy invivo when ingested.
Next, we ascertained that the TBE has α-dicarbonylcompound cleavage action and AGE crosslink cleavage action,
which is higher than that exerted by positive control PTB. The
α-dicarbonyl structure is thought to be present in some AGEs
as well as the highly reactive glycation intermediates 3DG,
GO, and MGO 3). It is also believed that the AGE crosslink
structure with collagen influences the degree of diabetic
complications 24). For this reason, degradation of previously
produced α-dicarbonyl compounds and AGE crosslinks in the
body leads to improvement of disease severity, and researchon “AGE breakers” has progressed. In addition to PTB 25),
which was used as a positive control in this study, AGE
crosslink cleavage activity has also been reported for phenyl4, 5-dimethylthiazolium chloride (ALT-711), which is a morestabilized version of PTB, and 3-benzyloxycarbonylmethyl4-methyl-thiazol-3-ium bromide (C36), which has a different
structure 26, 27 ). The possibility of ALT-711 breaking AGE
crosslinks formed in α-crystallin derived from a human lensin an in vitro test system has been suggested 28), and in ahuman clinical study, a pulse pressure improvement effectwas observed after consumption of 210 mg/day of ALT-711
for 8 weeks in men and women aged 50 years and older withcardiovascular sclerosis 29). Based on those studies, we believethat ingredients that are capable of degrading α-dicarbonylcompounds and breaking AGE crosslinks in an in vitro test
system may also be involved in solubility and mobility ofstructural proteins in vivo. Additionally, since TBE has acarbonyl bond cleavage activity similar to PTB and a morepotent AGE crosslink cleavage activity than PTB, we believe
the TBE may act as an AGE breaker in vivo.
Other plants from the genus Trapa are also expected
to be functional food ingredients. For example, peel extractof the related species Trapa japonica Flerov 30). is reported

to inhibit glucose metabolizing enzymes amylase andglucosidase as well as inhibit the postprandial hyperglycemia
in rats 31), whereas the extract of a related species, Trapa
natans, is reported to reduce blood glucose concentration in
streptozotocin-induced diabetic rats.
The glycation response inhibiting activity and glycation
product degradation activity of the TBE confirm its production
inhibitory effect against AGEs and its effect in reducing theamount of accumulated AGEs by degradation of previously
produced AGEs. Further, since activities other than antiglycation have been demonstrated, we expect TBE to havewide applications as an anti-aging ingredient. We plan to
study its efficacy in humans in the future.

Conclusion

The study verified TBE to inhibit glycation productsynthesis, degrade α-dicarbonyl compound, and breakthe AGE crosslinks. The above results suggest that TBE
may inhibit production of AGEs and their intermediates
and degrade existing α-dicarbonyl compounds and AGE
crosslinks in vivo. We expect that TBE may be suitable toreduce glycation stress.

Conflicts of interest statement

Part of this work was supported by Hayashikane Sangyo
Co., Ltd.

_ 78 _Glycative Stress Research

References

1) Nomoto K, Yagi M, Arita S, et al. Skin accumulation ofadvanced glycation end products and lifestyle behaviors inJapanese. Anti-Aging Medicine. 2012; 9: 165-173.
2) Nagai R, Jinno M, Ichihashi M, et al. Advanced glycation
end products and their receptors as risk factors for aging.
Anti-Aging Medicine. 2012; 9: 108-113.
3) Ulrich P, Cerami A. Protein glycation, diabetes, and aging.
Recent Prog Horm Res. 2001; 56: 1-21.
4) Nagai R, Mori T, Yamamoto Y, et al. Significance ofadvanced glycation end products in aging-related disease.
Anti-Aging Medicine. 2010; 7: 112-119.
5) Shapiro B, Owan T, Mohammed S, et al. Advanced
glycation end products accumulate in vascular smoothmuscle and modify vascular but not ventricular properties
in elderly hypertensive canines. Circulation. 2008; 118:
1002-1010.
6) Saito M, Marumo K. Bone quality in diabetes. Front
Endocrinol (Lausanne). 2013; 4: 72.
7) Barić N. Role of advanced glycation end products in
Alzheimer’s disease. Glycative Stress Research. 2014; 1:
68-83.
8) Jinno M, Takeuchi M, Watanabe A, et al. Advanced
glycation end-products accumulation compromises
embryonic development and achievement of pregnancy by
assisted reproductive technology. Hum Reprod. 2011; 26:604-610.
9) Ichihashi M, Yagi M, Nomoto K, et al. Glycation stress andphoto-aging in skin. Anti-Aging Medicine. 2011; 8: 23-29.
10) Kim B, Kim J, Kim H, et al. Biological screening of 100
plant extracts for cosmetic use (II): Anti-oxidative activity
and free radical scavenging activity. Int J Cosmet Sci. 1997;
19: 299-307.
11) Rahman M, Wahed M, Biswas M, et al. In vitro antibacterial
activity of the compounds of Trapa bispinosa Roxb.
Sciences. 2001; 1: 214-216.
12) Parengkuan L, Yagi M, Matsushima M, et al. Anti-glycation
activity of various fruits. Anti-Aging Medicine. 2013; 10:70-76.
13) Taneda S, Monnier V. ELISA of pentosidine, an advanced
glycation end product, in biological specimens. Clin Chem.
1994; 40: 1766-1773.
14) Hori M, Yagi M, Nomoto K, et al. Experimental models foradvanced glycation end product formation using albumin,collagen, elastin, keratin and proteoglycan. Anti-Aging
Medicine. 2012; 9: 125-134.
15) Yamada H, Miyata S, Igaki N, et al. Increase in 3deoxyglucosone levels in diabetic rat plasma. J Biol Chem.
1994; 269: 20275-20280.
16) 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.
17) Arima S. Studies on growth and yield performance ofwater chestnut ( Trapa bispinosa Roxb.). Bulletin of
the Experimental Farm, Faculty of Agriculture, Saga
University. 1994; 76: 1-79. (in Japanese)18) Edelstein D, Brownlee M. Mechanistic studies of advanced
glycation end product inhibition by aminoguanidine.
Diabetes. 1992; 41: 26-29.
19) Matsuura N, Aradate T, Sasaki C, et al. Screening system
for the Maillard reaction inhibitor from natural productextracts. Journal of Health Science. 2002; 48: 520-526.

20) Soulis - Liparota T, Cooper M, Papazoglou D, et al.

Retardation by aminoguanidine of development ofalbuminuria, mesangial expansion, and tissue fluorescencein streptozocin-induced diabetic rat. Diabetes. 1991; 40:
1328-1334.
21) Edelstein D, Brownlee M. Aminoguanidine ameliorates
albuminuria in diabetic hypertensive rats. Diabetologia.
1992; 35: 96 -97.
22) Yagihashi S, Kamijo M, Baba M, et al. Effect ofaminoguanidine on functional and structural abnormalities
in peripheral nerve of STZ-induced diabetic rats. Diabetes.
1992; 41: 47-52.
23) Cameron N, Cotter M, Dines K, et al. Effects ofaminoguanidine on peripheral nerve function and polyol
pathway metabokites in streptozotocin-diabetic rats.
Diabetologia. 1992; 35: 946-950.
24) Cooper M. Importance of advanced glycation end products
in diabetes-associated cardiovascular and renal disease. Am
J Hypertens. 2004; 17: 31S-38S.
25) Cooper M, Thallas V, Forbes J, et al. The cross-link breaker,
N-phenacylthiazolium bromide prevents vascular advanced
glycation end-product accumulation. Diabeteologia. 2000;
43: 660-664.
26) Coughlan M, Forbes J, Cooper M. Role of the AGE
crosslink breaker, alagebrium, as a renoprotective agent
in diabetes. Kidney Int Suppl. 2007; 72: 54-60.
27) Cheng G, Wang L, Long L, et al. Beneficial effects of C36,
a novel breaker of advanced glycation endproducts crosslinks, on the cardiovascular system of diabetic rats. Br J
Pharmacol. 2007; 152: 1196 -1206.
28) Hollenbach S, Thampi P, Viswanathan T, et al. Cleavageof in vitro and in vivo formed lens protein cross-links by anovel cross-link breaker. Mol Cell Biochem. 2003; 243: 7380.
29) Vasan S, Foiles P, Founds H. Therapeutic potential ofbreakers of advanced glycation end product-protein
crosslinks. Arch Biochem Biophys. 2003; 419: 89-96.
30) Tanaka T, Nakao S. Tanaka’s Cyclopedia of edible plantsof the world.Keigaku Publishing Co., Ltd. 1976; 731.
31) Yasuda M, Yasutake K, Hino M, et al. Inhibitory effects
of polyphenols from water chestnut (Trapa japonica) husk
on glycolytic enzymes and postprandial blood glucose
elevation in mice. Food Chem. 2014; 165: 42-49.
32) Das P, Bhattacharya S, Pandey J, et al. Antidiabetic activityof Trapa natans fruit peel extract against streptozotocin
induced diabetic rats. Global Journal of Pharmacology.
2011; 5: 186-190.

_ 79 _