Preventive effects of chebulic acid isolated from Terminalia chebula ...

Journal of Ethnopharmacology 131 (2010) 567–574
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Journal of Ethnopharmacology
journal homepage: www.elsevier.com/locate/jethpharm
Preventive effects of chebulic acid isolated from Terminalia chebula on advanced
glycation endproduct-induced endothelial cell dysfunction
Hyun-Sun Lee a , Yoon-Chang Koo d , Hyung Joo Suh b , Kyung-Yong Kim c , Kwang-Won Lee d,∗
a Institute of Health Science, College of Health Science, Korea University, Seoul 136-703, Republic of Korea
b Department of Food and Nutrition, College of Health Science, Korea University, Seoul 136-703, Republic of Korea
c Department of Anatomy, College of Medicine, Chung-Ang University, Seoul 156-756, Republic of Korea
d Division of Food Bioscience & Technology, College of Life Science and Biotechnology, Korea University, 5-ga, Anam-dong, Sungbuk-ku, Seoul 136-701, Republic of Korea
article info
Article history:
Received 15 April 2010
Received in revised form 15 July 2010
Accepted 16 July 2010
Available online 24 July 2010
Keywords:
Terminaila chebula
Chebulic acid
Diabetic complications
Advanced glycation endproducts
Endothelial dysfunction
1. Introduction
abstract
The dried ripe fruits of Terminalia chebula Retzius (T. chebula
Retz.) (Combretaceae), which is a plant native plant to India and
Southeast Asia, are commonly known as black Myroblans in English
and Harad in Hindi (Rao and Nammi, 2006), and have traditionally
been used as a popular folk medicine for alternative, astringement,
denrifrice, purgative, stomachic, tonic, antiseptic, cardiotonic and
laxative purposes. This fruit is also useful for burns, digestive disorders,
diabetes, eye diseases, weak eye sight, fever, skin diseases
and kidney dysfunction along with other herbs (Saleh et al., 1952;
Rao and Nammi, 2006). Terminalia chebula has exhibited in vitro
antioxidant and free radical scavenging activities (Naik et al., 2005).
In addition, its antimicrobial (Sato et al., 1997), antiviral (Badmaev
and Nowakowski, 2000), anticancer (Saleem et al., 2002), antianaphylaxis
(Shin et al., 2001) and anti-diabetic (Sabu and Kuttan,
∗ Corresponding author. Tel.: +82 2 3290 3027; fax: +82 2 953 0737.
E-mail address: kwangwon@korea.ac.kr (K.-W. Lee).
0378-8741/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.jep.2010.07.039
Aim of the study: The aqueous extract of Terminalia chebular fruits was reported to have antihyperglycemia
and anti-diabetic complication effects. The present study therefore investigated the
protective mechanism of chebulic acid, a phenolcarboxylic acid compound isolated from the ripe fruits of
Terminalia chebula against advanced glycation endproducts (AGEs)-induced endothelial cell dysfunction.
Materials and methods: To investigate the protective mechanism of chebulic acid against vascular endothelial
dysfunction human umbilical vein endothelial cells (HUVEC) were treated with chebulic acid in the
presence/absence of glyceraldehyde-related AGEs (glycer-AGEs).
Results: HUVEC incubated with 100 �g/ml of glycer-AGEs had significantly enhanced reactive oxygen
species formation, whereas the treatment of chebulic acid dose-dependently reduced glycer-AGEinduced
formation to 108.2 ± 1.9% for 25 �M versus 137.8 ± 1.1% for glycer-AGEs treated alone. The
transendothelial electrical resistance (TER) value of the glycer-AGEs group was dramatically decreased to
76.9 ± 2.2% compared to the control, whereas chebulic acid treatment prevented glycer-AGE-induced TER
change with a value of 91.3 ± 5.3%. The incubation of confluent HUVEC with 100 �g/ml of glycer-AGEs for
24 h remarkably increased the adhesion of human monocytic THP-1 cells compared to non-stimulated
HUVEC. These increases in HUVEC adhesiveness were dose-dependently reduced by chebulic acid.
Conclusions: The present study shows the effects of chebulic acid against the progression of AGE-induced
endothelial cell dysfunction suggesting that this compound may constitute a promising intervention
agent against diabetic vascular complications.
© 2010 Elsevier Ireland Ltd. All rights reserved.
2002; Rao and Nammi, 2006; Murali et al., 2007) activities have
been reported.
The majority of diabetic complications are related to pathophysiological
alterations in the vasculature, with macro- and
microvascular disease being the most common cause of morbidity
and mortality in patients with diabetes mellitus (Calcutt et
al., 2009). Clinical and animal model data indicate that chronic
hyperglycemia appears to be the central initiating factor for all
types of diabetic microvascular disease (Zannad, 2008; Calcutt et
al., 2009). Diabetes is also associated with accelerated atherosclerotic
macrovascular disease affecting arteries that supply the
heart, brain and lower extremities. As a result, patients with diabetes
have a much higher risk of myocardial infarction, stroke
and limb amputation (Laakso and Kuusisto, 1996). A hypothesis
for the initial lesion of macrovascular disease such as
atherosclerosis is endothelial dysfunction, defined pragmatically
as changes in the concentration of chemical messengers produced
by the endothelial cells and/or by blunting of the nitric
oxide-dependent vasodilatory response to hyperemia (Cohen,
2007).

568 H.-S. Lee et al. / Journal of Ethnopharmacology 131 (2010) 567–574
Advanced glycation endproducts (AGEs) accumulate in the vessel
wall, where they may perturb cell structure and function. AGEs
have been implicated in both the microvascular and macrovascular
complications of diabetes (Brownlee, 1995). The broad consequences
of the receptor of AGEs (RAGE)–ligand interaction for
cellular properties are emphasized by a spectrum of signaling
mechanisms. One such consequence of this interaction can lead
to the generation of reactive oxygen species (ROS). Other consequences
include the production of growth factors and cytokines,
chronic inflammatory responses, and cellular and vascular dysfunction
associated with diabetic complications. The adhesion
of leukocytes to the activated endothelium, their migration into
the vessel wall, subsequent infiltration, and differentiation into
macrophages are key events in the development of atherosclerotic
cardiovascular disease, which is the leading cause of premature
death in patients with diabetes (Renier et al., 2003).
Murali et al. (2007) reported that oral administration of Terminalia
chebula extract once daily for two months reduced elevated
blood glucose, and significantly reduced increases in glycosylated
hemoglobin (HbA 1c). Shaila et al. (1998) also reported that the
aortas of Terminalia chebula treated rabbits showed decreased
athermatous plaque formation compared to control group. In previous
studies, we identified chebulic acid isolated from Terminalia
chebula, and presented data on its antioxidant activity (Lee et al.,
2007). In the present study, the anti-diabetic complication properties
of chebulic acid were evaluated by assaying anti-glycation
activity, ROS scavenging activity, normalized activity, and antiadhesion
activity against AGEs.
2. Materials and methods
2.1. Chemicals
dl-Glyceraldehyde, 2-deoxy-d-ribose, bovine serum albumin
(BSA, low-endotoxin, fatty acid free), 2 ′ ,7 ′ -bis-(2-carboxyethyl)-5-
(and-6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM), and
2 ′ ,7 ′ -dichlorofluorescein diacetate (DCFH-DA) were obtained from
Sigma Chemical Co. (St. Louis, MO, USA). An endothelial cell basal
medium-2 (EBM-2) bullet kit was purchased from Clonetics (San
Diago, CA, USA), and all other tissue culture reagents were obtained
from GIBCO-BRL (Gaithersburg, MD, USA). All reagents were of analytical
grade.
2.2. Preparation of chebulic acid isolated from Terminalia chebula
The dried ripe fruits of Terminalia chebula cultivated in Taiwan
were purchased from a local market (Kyungdong Herb-Market,
Seoul, Korea), and were identified by Prof. B.W. Kang (College
of Life Sciences and Biotechnology, Korea University). The isolation
procedures for chebulic acid were followed as described
previously (Lee et al., 2007). Briefly, the dried fruits of Terminalia
chebula were extracted twice with 97% ethanol. The extract was
combined, lyophilized, resuspended in H 2O, and then extracted
successively with n-hexane, chloroform, ethyl acetate (EtOAc), and
n-butanol. The EtOAc-soluble portion was applied to a column
of silica gel, and eluted by a gradient with increasing amounts
of methanol in ethyl acetate. The active fraction of EtOAc and
methanol (70:30, v/v) was further purified by Sephadex TM LH-20
(Amersham Biosciences, Uppsala, Sweden) column chromatography
using methanol as the eluent, followed by PR-�-BondaPak
C18 column (300 mm × 3.9 mm, 10 �m) (Waters, Milford, MA, USA)
chromatography. The purified compound was identified as chebulic
acid (Fig. 1) using NMR spectra, which were interpreted by Prof.
Bong-Sik Yun (Chonbuk National University, Korea).
Fig. 1. Structure of chebulic acid isolated from Terminalia chebula.
2.3. Quantitative assay for cross-linking and AGE formation
The quantitative assay for cross-linking was followed as
described previously (Lee et al., 1998; Lehman and Ortwerth, 2001)
with several modifications. Reaction mixtures were prepared containing
10 mM threose, fatty acid free bovine serum albumin (BSA;
5 mg/ml), 2.5 �Ci [ 14 C]lysine (1.0 mCi/mmol), 1.0 mM diethylenetriaminepentaacetic
acid (DTPA), and 0.1 M phosphate buffer (pH
7.4) and various concentrations of chebulic acid or aminoguanidine
as a positive control. Each reaction mixture was filter sterilized into
a 2.0 ml sterile Nunc tube, capped, and incubated at 37 ◦ C for 4 days.
Aliquots of 50 �l were spotted on 2.5 cm × 3 mm filter paper discs
suspended on pins. After 30 s, each filter was placed in a basket
suspended in 300 ml of ice-cold 10% trichloroacetic acid (TCA) to
precipitate the protein within the paper disc. At the end of sampling,
each group of discs was washed in a 5% TCA solution with
stirring at room temperature for 15 min, followed by 5% TCA at
70 ◦ C for 15 min and ethanol:ether (2:1) for 15 min to remove the
TCA and water from the discs. The discs were dried under an incandescent
lamp, and protein-bound radioactivity was determined in
a liquid scintillation counter (Beckman LS 5000TD, Beckman Instruments
Inc., Canada). Reaction mixtures for AGE formation were
prepared with 5 mg/ml of BSA, 10 mM threose, and 1 mM DTPA
in 1.0 ml of 0.1 M and incubated at 37 ◦ C for 7 days in an incubator.
Triplicate 100 �l aliquots were removed, and their fluorescence
intensity was measured at an excitation of 370 nm and an emission
of 440 nm with a spectrofluorometer (VICTOR3 TM , PerkinElmer,
USA). The percentage of inhibition of glycation was calculated by
the following equation (Choi et al., 2008):
Inhibition (%)
�
�
result of sample mixture − result of no sugar mixture
= 1 −
result of glycation mixture − result of no sugar mixture
×100
The calculated IC 50 values denote the concentration of sample
required to inhibition 50% of glycation.
2.4. Preparation of glyceraldehyde-induced AGEs (glycer-AGEs)
Glycer-AGEs were prepared as described previously (Takeuchi
et al., 2000). BSA was incubated with 0.1 M dl-glyceraldehyde,
10 mg/ml BSA, and 5 mM DTPA in 0.2 M phosphate buffer (pH 7.4)
in sterile conditions in the dark at 37 ◦ C for 7 days. Before incubation,
the solution was sterile filtered by passing it through a 0.2 �m
filter. Unmodified BSA was treated under the same conditions without
glyceraldehyde as a control. At the end of the incubation period,
unincorporated glyceraldehyde and low molecular reactants were
removed by dialysis against 0.1 M phosphate buffer. We chose a
concentration of 100 �g/ml of glycer-AGEs to treat the cultured
cells as described previously (Yamagishi et al., 2002).

Table 1
Anti-glycation activity of chebulic acid.
50% of inhibitory
concentration on
cross-linking (mM) a
H.-S. Lee et al. / Journal of Ethnopharmacology 131 (2010) 567–574 569
50% of inhibitory
concentration on
formation of AGEs (mM) b
Chebulic acid 17.1 ± 0.7 1.32 ± 0.10
Aminoguanidine 21.3 ± 1.4 2.37 ± 0.09
a The effect of inhibitors on the cross-linking of [ 14 C]lysine to BSA proteins. Reaction
mixtures contained 5.0 mg/ml of BSA, 5.0 mM l-threose, 2.5 �Ci [ 14 C]lysine,
10 mM inhibitor, and 1.0 mM DTPA in 0.1 M phosphate buffer, pH 7.0, and were
incubated at 37 ◦ C for 4 days. Protein-bound radioactivity was determined in a liquid
scintillation counter.
b The effects of inhibitors on the formation of AGEs. The reaction mixtures for
AGE formation contained 5.0 mg/ml of BSA, 5.0 mM l-threose, 10 mM inhibitor, and
1.0 mM DTPA in 0.1 M phosphate buffer, pH 7.0, and were incubated at 37 ◦ C for 7
days. The intensity of AGE formation was measured at an excitation of 370 nm and
an emission of 440 nm with a spectrofluorometer.
Table 2
Cytotoxicity of glycer-induced AGEs on HUVEC.
Concentration (�g/ml) BSA Glycer-AGEs
0 100.0 ± 2.0 a 100.0 ± 2.7 a
100 98.3 ± 2.5 a 84.5 ± 1.3 b
250 99.4 ± 2.3 a 79.8 ± 1.7 c
500 99.0 ± 4.3 a 78.9 ± 3.4 c
750 102.7 ± 5.1 a 82.7 ± 0.6 b
1000 103.9 ± 1.9 a 85.3 ± 1.4 b
Glycer-induced AGEs were prepared by incubating BSA (10 mg/ml) with 0.1 M glyceraldehyde
for 7 days. HUVEC incubated with various concentrations of glycer-AGEs
or BSA for 24 h. Cell viability was determined by the MTT assay. Results were
expressed as means ± S.D. (n = 3 or more). Means with different superscript letters
are significantly different at p < 0.05 by Duncan’s multiple range tests.
Table 3
Cytotoxicity of chebulic acid with or without glyceraldehyde-induced AGEs on
HUVEC.
Chebulic acid
levels (�M)
Chebulic acid Chebulic acid with
100 �g/ml of glycer-AGEs
0 100.0 ± 2.2 a 94.4 ± 1.3 c
1 101.0 ± 0.5 a 95.9 ± 1.2 b
5 100.0 ± 2.0 a 97.4 ± 0.7 b
10 100.1 ± 2.8 a 99.4 ± 1.2 a
25 101.0 ± 2.3 a 101.4 ± 1.2 a
50 86.2 ± 4.5 d –
100 69.6 ± 3.3 e –
250 66.6 ± 9.8 e –
Cells were incubated with various concentrations of chebulic acid with/without
100 �g/ml of glycer-induced AGEs for 24 h. Cell viability was determined by the
MTT assay. Results are expressed as means ± S.D. (n = 3 or more). Means with different
superscript letters are significantly different at p < 0.05 by Duncan’s multiple
range tests.
2.5. Cell culture
Human umbilical vein endothelial cells (HUVEC) were purchased
from Biobud Co. (Seoul, Korea), were grown on 1.5%
gelatin-coated culture dishes or plates in EBM-2, and used for the
experiments within the first 5–6 passages. The cells were supplemented
at 37 ◦ C in a humidified atmosphere of 5% CO 2. Cells of the
human acute monocytic leukemia THP-1 cell line (ATCC TIB202)
were obtained from the American Type Culture Collection (ATCC;
Rockville, MD, USA), and were cultured in RPMI-1640 medium
supplemented with 10% (v/v) heat-inactivated fetal bovine serum
(FBS), 100 U/ml penicillin, and 100 �g/ml streptomycin.
Fig. 2. Intracellular reactive oxygen species (ROS) production by glyceraldehyde-induced AGEs (glycer-AGEs) on human umbilical vein endothelial cells (HUVEC). Dichlorofluorescein
diacetate (DCFH-DA)-loaded HUVEC were exposed to BSA (A), 50 �g/ml of glycer-AGEs (B), 100 �g/ml of glycer-AGEs (C), and 200 �g/ml of glycer-AGEs (D)
for 24 h. Data are expressed as a histogram of the fluorescence peak channel by flow cytometry. The horizontal axis represents the intensity of cellular fluorescence (FL1-H),
the vertical axis represents the number of cells detected for a given fluorescent value. Given the fixed intervals of FL1-H, the relative cell distributions are indicated for each
treatment.

570 H.-S. Lee et al. / Journal of Ethnopharmacology 131 (2010) 567–574
Fig. 3. ROS scavenging activities of chebulic acid against glycer-AGEs. DCFH-DA-loaded HUVEC were exposed to BSA (A), 100 �g/ml of glycer-AGEs (B), 100 �g/ml of
glycer-AGEs + 5 �M chebulic acid (C), 100 �g/ml of glycer-AGEs + 10 �M chebulic acid (D), and 100 �g/ml of glycer-AGEs + 25 �M chebulic acid (E) for 24 h. Data (A–E) were
expressed as a histogram of the fluorescence peak channel by flow cytometry, and their fluorescence intensities were assayed with fluorescence spectrometry (F).
2.6. Cytotoxicity of glycer-AGEs and chebulic acid
The cytotoxicities of the glycer-AGEs and various concentrations
(1–100 �M) of chebulic acid were analyzed by MTT colorimetric
assays (Mosmann, 1983). The cells were incubated with chebulic
acid with/without 100 �g/ml of glycer-AGEs for 24 h. At the end of
the treatment time, all the wells were aspirated, refilled with MTT
solution, and incubated for 3 h (Roffey et al., 2007).
2.7. Detection of ROS in HUVEC
The production of intracellular ROS was determined by DCFH-
DA by fluorescence spectrophotometry (Shukla et al., 2006), and
flow cytometry. DCFH-DA was dissolved in dimethyl sulfoxide
(DMSO) as a 10 mM stock solution, and kept frozen at −20 ◦ C. To
load the cells, DCFH-DA from the stock solution was mixed with
M199 medium. Next, 20 �M DCFH-DA-loaded HUVEC were incubated
for 30 min, washed twice with M199, and exposed to various
concentrations of chebulic acid and/or 100 �g/ml of glycer-AGEs.
After 24 h, the cells were harvested by trypsinization, and washed
with cold phosphate buffered saline (PBS) twice. ROS were measured
by flow cytometry (FACS Calibur, Becton Dickinson, NJ, USA)
at a level of 10,000 events for each test. Fluorescence was obtained
by a histogram of the peak channel using the CellQuest program
(Becton Dickinson), and the fluorescence intensity was determined
with excitation of 485 nm and emission of 530 nm using a plate
reader (VICTOR3 TM , PerkinElmer, MA, USA).
2.8. Assessment of transendothelial electrical resistance (TER)
The effects of glycer-AGEs on TER were measured in HUVEC
according to the method of Kazakoff et al. (1995). The HUVEC were
seeded at 3 × 10 5 cells per inset on a type 1 rat tail collagen coated
membrane (pore size, 0.4 �m) contained in the apical chamber of
a 12-well trans-well system (Corning, NY, USA). After monolayer
confluence was achieved (>120 cm 2 ), the HUVEC were treated
with chebulic acid and glycer-AGEs. TER was measured every 4 h
over 24 h. The measurements were taken in triplicate, and the data
were expressed as relative percentages.
2.9. Adhesion assay of THP-1
The monocyte adhesion assay was performed as described previously
(Hiraoka et al., 2004). Briefly, THP-1 cells were prepared

y combining the cells with BCECF-AM, and then the cells were
washed three times with 1% FBS in PBS. The HUVEC grown to confluence
in a 24-well plate were pretreated with chebulic acid and
glycer-AGEs at 37 ◦ C for 24 h for the adhesion assay. The BCECF-AM
labeled THP-1 cells were co-incubated with the HUVEC for 60 min at
37 ◦ C. After incubation, nonadherent cells were removed by washing
each well three times with 1% FBS in PBS. The attached cells
were examined via confocal microscopy (LSM 510; Carl Zeiss Micro-
Imaging, Inc., NY, USA). The fluorescence intensity of each well
was measured using a fluorescence multiwell plate reader (Wallac
1420, VICTOR3 TM ) at excitation and emission wave lengths of
485 and 530 nm, respectively.
2.10. Statistical analysis
All statistical analyses were performed using the Statistical
Package for Social Sciences (SPSS) version 12.0 (SPSS Inc., IL, USA).
The differences among groups were evaluated by one-way analysis
of variance (ANOVA) and Duncan’s multiple range tests. All data
were reported as means ± standard deviation (S.D.).
3. Results
3.1. Anti-glycation activity of chebulic acid
Table 1 shows that chebulic acid had IC 50 values of 17.1 mM for
protein cross-linking and 1.32 mM for AGE formation. As a positive
control, aminoguanidine had IC 50 values of 21.3 mM and 2.37 mM,
respectively. However, based on these millimolar levels of chebulic
acid, it seemed to have very low potency regarding with inhibitory
activities on glycation-induced protein cross-linking and AGE formation.
3.2. Effects on the formation of AGEs and toxicity
The effects of the AGEs and toxicity were assessed using various
sugars such as ribose (a pentose), glyceraldehydes, glycoaldehyde
(a short chain sugar), and glyoxal (a dicarbonyl compound) for the
glycation of HUVEC. Ribose and glyoxal produced few AGEs, and
did not induce cell death at the tested concentrations of 10–50 mM
(data not shown). Glycoaldehyde and glyceraldehydes caused
more potent cytotoxicity in a concentration-dependent manner.
Overall, glyceraldehyde caused the fastest rate of AGE formation
in a concentration-dependent manner in the HUVEC (data not
shown).
3.3. Glyer-AGEs cytotoxicity and ROS formation
As shown in Table 2, glycer-AGEs caused significant cytotoxicity
in a dose-dependent manner up to 500 �g/ml of glycer-AGEs. These
levels of glycer-AGEs were used for intracellular ROS generation,
and its ROS occurred in a dose-dependent manner (Fig. 2). In previous
in vitro studies (Twigg et al., 2002; Ohashi et al., 2004; Xu et al.,
2004), the concentration of AGEs was used mostly at 50–200 �g/ml.
Therefore, based on these results, the concentration of glycer-AGEs
at 100 �g/ml was used in our next studies.
3.4. Cytotoxicity by chebulic acid with or without Glyer-AGEs
Chebulic acid had slight toxic effects on the HUVEC above a concentration
of 50 �M. Therefore, 25 �M chebulic acid was used for
the subsequent experiments (Table 3). In addition, the results from
Tables 2 and 3 suggest that the apparent cytotoxic effect of chebulic
acid may reflect the variability of the effect of glycer-AGEs.
H.-S. Lee et al. / Journal of Ethnopharmacology 131 (2010) 567–574 571
Fig. 4. Changes in permeability by glycer-AGEs and chebulic acid. HUVEC were
seeded at 3 × 10 5 cells per inset on a type 1 rat tail collagen coated membrane
contained in the apical chamber of a 12-well trans-well system. After monolayer
confluence was achieved (>120 cm 2 ), the HUVEC were treated with chebulic acid
and/or glycer-AGEs. Changes in transendothelial electric resistance (TER) were measured
every 4 h over 24 h. Measurements were taken in triplicate and the data are
expressed as relative percentages. *Significant difference compared to glycer-ACEs
only treated group at p < 0.05.
3.5. Intracellular ROS scavenging activities
Intracellular ROS was estimated by flow cytometry using DCFH
as a probe. Levels of ROS were low in the unstimulated HUVEC
(Fig. 3(A)), and the HUVEC treated with 100 �g/ml of glycer-
AGEs (Fig. 3(B)) significantly increased ROS formation. As shown
in Fig. 3F, chebulic acid dose-dependently prevented glycer-
AGE-induced ROS generation, which was significantly reduced to
122.0 ± 3.9% for 5 �M, to 120.0 ± 3.3% for 10 �M, and to 108.2 ± 1.9%
for 25 �M versus 137.8 ± 1.1% for the control treated with glycer-
AGEs alone (p < 0.01 versus glycer-AGEs alone).
3.6. Preventive effects on TER against AGEs
As shown in Fig. 4, the control showed no significant
changes in TER during 24 h. But the TER value of the glycer-
AGEs group dramatically decreased in 4 h, and remained at
98.0 ± 2.8 cm 2 (76.9 ± 2.2%) after 24 h as compared to the TER
value of the control group (116.0 ± 2.8 cm 2 , 97.9 ± 2.4%). However,
the treatment of chebulic acid dose-dependently recovered
TER to 114.7 ± 8.7 cm 2 (91.3 ± 5.3%).
3.7. Reduction of adhesion THP-1 cells on HUVEC
The incubation of confluent HUVEC with 100 �g/ml of glycer-
AGEs for 24 h caused a significant increase in the adhesion of THP-1
monocytic cells compared to non-stimulated HUVEC (Fig. 5A versus
Fig. 5B). This increased cell adhesion was dose-dependently
reduced by chebulic acid treatments (Fig. 5C and D). As shown
in Fig. 5E, chebulic acid reduced glycer-AGE-induced fluorescence
intensity to 23600 ± 6600 for 5 �M, 22600 ± 36000 for 10 �M, and
to 134000 ± 4100 �M for 25 �M versus 269000 ± 37000 �M for the
control treated with glycer-AGEs alone.
4. Discussion
Diabetic patients have a 2- to 4-fold increased risk of subsequently
developing microvascular complications such as renal,
neuronal and retinal diseases, as well as macrovascular problems
such as atherosclerosis and coronary heart disease (Calcutt et al.,
2009). Unfortunately, these complications may develop in both

572 H.-S. Lee et al. / Journal of Ethnopharmacology 131 (2010) 567–574
Fig. 5. Effects of chebulic acid on THP-1 cell adhesion to HUVEC stimulated by glycer-AGEs. HUVEC treated with BSA (A), 100 �g/ml of glycer-AGEs (B), 100 �g/ml of
glycer-AGEs + 10 �M chebulic acid (C), and 100 �g/ml of glycer-AGEs + 25 �M chebulic (D) for 24 h to the adhesion assay. After stimulation, the HUVEC were incubated with
BCECF-AM-labeled THP-1. The adherent cells were collected by treatment with 0.1% SDS and measured using a fluorescence multiwell plate reader (Wallac 1420, Germany)
at excitation and emission wave lengths of 485 and 530 nm, respectively, and the fluorescence intensity was expressed as a histogram (E). Shared letters represent means
that were not significantly different (p < 0.05).
type 1 and type 2 diabetic patients even with careful control of
their glycemic level, blood pressure, and lipid profile, and such vascular
diseases are an important cause of morbidity and mortality in
diabetic patients (Laakso and Kuusisto, 1996; Cohen, 2007; Calcutt
et al., 2009). The authors of one particular diabetes complications
and control trial: The Epidemiology of Diabetes Interventions and
Complications Trial, suggested that the influence of early glucose
control on the development of macrovascular events may become
more obvious with a longer follow-up time (The Diabetes Control
and Complications Trial/Epidemiology of Diabetes Interventions
and Complications Research Group, 1993, 2005; Writing Team
for the Diabetes Control and Complications Trial/Epidemiology of
Diabetes Interventions and Complications Research Group, 2003).
These findings suggest that advanced protein glycation, a pro-

cess involving the nonenzymatic modification of tissue proteins
by physiological sugars in vivo, appears to play a central role in
the pathogenesis of diabetic complications; this is because the
concept of ‘glycemic memory’ is well-fitting to the in vivo formation
and accumulation of AGEs and their roles (Brownlee, 1995).
In addition, one must take into consideration that AGE formation
can arise not only with glucose, but also with short chain sugars
such as glycolaldehyde, and dicarbonyl compounds such as methylglyoxal
derived from sugar autoxidation and other metabolic
pathways (Takeuchi and Yamagishi, 2004). Inside cells, these can
much readily accelerate AGE formation with amino groups of proteins
as compared to glucose alone (Takeuchi et al., 2000). Thus,
controlling AGE formation as well as blood glucose levels are essential
aspects in diabetes treatment for the prevention of diabetic
complications.
AGEs mediate their effects directly and via their engagement
with receptors for AGEs (RAGE). It was reported that AGE crosslinking
to collagen can decrease vascular elasticity, and increases
vascular stiffness in the vessel wall, as well as glomerulosclerosis
in the kidney and vascular hyperpermeability in the retina
(Wendt et al., 2003). The binding of AGEs to RAGE results in
intracellulatr ROS generation, which seems to be linked to the
NAD(P)H-oxidase system (Wautier et al., 2001), along with subsequent
activation of the redox-sensitive transcription factor nuclear
factor-kB in vascular wall cells and the expression of a variety
of athrosclerosis-related genes, including intercellular adhesion
molecule-1 (ICAM-1), vsasular cell adhesion molecule-1 (VCAM-
1), monocyte chemoattractant protein-1 (MCP-1), plasminogen
activator inhibitor-1 (PAI-1), vascular endothelial growth factor
(VGEF) and RAGE (Basta, 2008; Yamagishi et al., 2008). As a
candidate inhibitor for AGE formation, aminoguanidine, a nucleophilic
hydrazine compound that confines reactive carbonyls
during glycation reactions thus inhibiting the conversion of these
intermediates into AGEs, was reported to enhance vascular elasticity
and decrease vascular permeability in diabetic rats (Huijberts
et al., 1993). However, clinical trials for aminoguanidine were
stopped due to reported side effects such as gastrointestinal disturbance
(Thornalley, 2003). Alternatively, the search for AGE
inhibitors, by examining naturally occurring compounds used
traditionally was started. In this regard, chebulic acid isolated
from Terminalia chebular has been investigated by our research
group (Lee et al., 2007). This compound belongs to the phenolcarboxylic
acid group, and its structure is simpler compared
to other compounds such as chebulanin, chebulagic acid, and
casuarinin (Juang et al., 2004). We previously reported that the
antioxidant activity of chebulic acid was evaluated by examining
2,2-diphenyl-1-picrylhydrazyl radical (DPPH • ) scavenging activity,
ferric reducing/antioxidant power (FRAP), and the specific
ESR spectrum (Lee et al., 2007). In the present study, chebulic
acid inhibited glycation-mediated cross-linking more actively
than aminoguanidine (1.2-fold-lower IC 50, 17.1 ± 0.7 mM versus
21.3 ± 1.4 mM; Table 1) based on cross-linking assays, which
were carried out using a system measuring the incorporation of
[ 14 C]lysine into BSA. Also, chebulic acid’s IC 50 value (mM) for inhibition
of AGE formation was 1.8-fold-lower (1.32 ± 0.10 mM) than
the IC 50 of aminoguanidine (2.37 ± 0.09 mM). Whiles such millimolar
concentrations of chebulic acid seem to indicate relatively low
potency with respect to inhibitory effects against cross-linking and
AGE formation, effective levels of chebulic acid in this micromolar
range appear to be markedly more potent in reducing AGE-induced
cytotoxicity, AGE-induced ROS formation, AGE-induced reductions
in endothelial cell electrical resistance and AGE-induced monocyte
adhesion (Tables 2 and 3 and Figs. 2–5).
There is accumulating evidence that RAGE interact with AGEs,
which is described by the term ‘AGE-RAGE-oxidative stress axis’,
and induce increased levels of intracellular reactive oxygen species
H.-S. Lee et al. / Journal of Ethnopharmacology 131 (2010) 567–574 573
(ROS) and subsequently evoke inflammatory responses leading
to the development of vascular complications in diabetes (Basta,
2008). This increase in ROS in turn activates a complex cascade
of signal transduction pathways, and subsequently enhances the
expression of many genes that are highly relevant for inflammation,
immunity, and atherosclerosis, such as cell adhesion
molecules, tissue factors, adhesion molecules (e.g., ICAM-1 and
VCAM-1), cytokines, as well as RAGE (Basta, 2008; Yamagishi
et al., 2008). These up-regulations are associated with endothelial
adherence and the migration of leukocytes, which are the
first steps in atherosclerosis, thus priming and sustaining vascular
wall injury and the inflammatory response and causing diabetic
vascular lesions (Jay et al., 2006). Interestingly, our preliminary
experiment (data not shown), which employed streptozotocininduced
diabetic rats, showed that the group orally supplemented
with the ethylacetate-soluble layer of ehtanolic extract of Terminalia
chebular containing chebulic acid had significantly reduced
mRNA levels of RAGE and ICAM-1 in the thoracic aorta, which
were increased only in the streptozotocin-treated group. However,
it should be noted that our present in vitro results, which
showed chebulic acid had activity for normalizing the permeability
of endothelial cells and reduced the adhesion of monocytes
to endothelial cells treated with AGEs, must be reproduced in
vivo to form the basis for a completely novel anti-diabetic therapy
that not only reduces blood glucose, but also reduces the
action of AGEs. At this point, the activities of chebulic acid are
not yet fully understood. However, the present results suggest that
chebulic acid from Terminalia chebula may act as an anti-diabetic
complication agent through the extracellular inhibition of AGE
formation as well as intracellular ROS scavenging in endothelial
cells.
5. Conclusion
Inhibitors of AGE formation to reduce tissue accumulation
of AGEs in diabetes have potentially beneficial effects in innate
chronic atherosclerosis. Alternatively, interference on RAGE-AGE
axis may be a promising novel therapeutic strategy for the treatment
of acute vascular injury. Our in vitro results with chebulic
acid isolated from Terminalia chebula may constitute a promising
intervention agent in these two setting relevant to diabetic vascular
complications. Further in vivo studies are required to prove
whether chebulic acid could inhibit the risk of diabetic vascular
complications beyond blood glucose lowering effect.
Acknowledgements
This work supported by grant number PJ007100201003 from
the BioGreen 21 program, Rural Development Administration,
Republic of Korea Research Fund. The authors thank the Korea
University-CJ Food Safety Center (Seoul, South Korea) for providing
the equipment and facilities.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.jep.2010.07.039.
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