Effects of Chebulic Acid on Advanced Glycation Endproducts-Induced Collagen Cross-Links

Abstract

Advanced glycation end-products (AGEs) have been implicated in the development of diabetic complications. We report the antiglycating activity of chebulic acid (CA), isolated from Terminalia chebula on breaking the cross-links of proteins induced by AGEs and inhibiting the formation of AGEs. Aminoguanidine (AG) reduced 50% of glycated bovine serum albumin (BSA) with glycolaldehyde (glycol-BSA)-induced cross-links of collagen at a concentration of 67.8±2.5 mM, the level of CA required for exerting a similar antiglycating activity was 38.8±0.5 µM. Also, the breaking activity on collagen cross-links induced by glycol-BSA was potent with CA (IC₅₀=1.46±0.05 mM), exhibiting 50-fold stronger breaking activity than with ALT-711, a well-known cross-link breaker (IC₅₀=72.2±2.4 mM). IC₅₀ values of DPPH· scavenging activity for CA and ascorbic acid (AA) were 39.2±4.9 and 19.0±1.2 µg dry matter (DM) mL⁻¹, respectively, and ferric reducing and antioxidant power (FRAP) activities for CA and AA were 4.70±0.06 and 11.4±0.1 mmol/FeSO₄·7H₂O/g DM, respectively. The chelating activities of CA, AG and ALT711 on copper-catalyzed oxidation of AA were compared, and in increasing order, ALT-711 (IC₅₀ of 1.92±0.20 mM)<CA (IC₅₀ of 0.96±0.07 mM)<AG (0.47±0.05 mM). Thus, CA could be a breaker as well as an inhibitor of AGE cross-linking, the activity of which may be explained in large part by its chelating and antioxidant activities, suggesting that CA may constitute a promising antiglycating candidate in intervening AGE-mediated diabetic complications.

Non-enzymatic glycation is initiated by a nucleophilic reaction between a carbonyl group from reducing sugars and an amino group from amino acids to form a Schiff base.¹⁾ This reaction is reversible, and is dependent on the concentration of sugar.²⁾ The labile Schiff base rearranges to more stable Amadori products, which can undergo a series of dehydration rearrangements to form advanced glycation end products (AGEs).³⁾ A number of studies have shown that the hyperglycemia has an important role in the pathogenesis of diabetic complications by increasing protein glycation and the gradual formation of AGEs in tissues.⁴⁾ The AGEs-induced cross-links of proteins such as collagen have been correlated with the severity of diabetic complications.⁵⁾

As preventive or therapeutic approaches for screening antiglycating agents, there are considerable interests in compounds inhibiting or breaking AGEs-protein cross-links. A variety of pharmacological compounds have been studied for their potential to prevent AGE formation or to break AGEs.⁶⁾ Aminoguanidine (AG) is a well-known inhibitor of AGEs formation.⁷⁾ However, Phase III clinical studies with AG were discontinued because of its side-effects of gastrointestinal disturbance and abnormalities in liver function tests.⁸⁾ On the other hand, because the inhibitors of AGEs formation cannot usually affect the AGEs-induced cross-links of proteins once formed, the agents that can break AGEs-induced cross-links would have the potential to attenuate such AGEs-related disease consequences. It is known that ALT-711 is an important protein cross-link breaker.⁶⁾ ALT-711 bonds with two carbons are centrally located in an AGE-derived cross-links which makes the complex unstable leading eventually to breakage of bonds and separation of protein.⁹⁾ In series of studies in various models of diabetic complications, ALT-711 improved arterial and ventricular function in older rhesus monkeys¹⁰⁾ and reverses large artery stiffness in diabetic rats,¹¹⁾ but ineffectiveness in blood pressure has been reported in hypertential trials.¹²⁾ Thus, the continual searches for new antiglycating agents with higher levels of efficacy and safety in humans are pursued.

As natural products have generally been proven to be relatively safe for human consumption, as compared to synthetic compounds, there has been an increasing interest in the use of natural plant compounds, such as anti-glycating agents.¹³⁾ Our research group has attempted to identify compounds having such activity from a variety of plant sources. One of the candidate compounds is chebulic acid (CA), which was isolated form the dried ripe fruit of T. chebula.¹⁴⁾ This plant is used extensively in Ayurveda, and is widely distributed throughout India, Burma and Sri Lanka, and has been used as a traditional medicinal herb.¹⁵⁾ In our previous study, T. chebula extract was determined to have a protective effect against liver damage by monitoring reactive oxygen species production both in vivo and in vitro.¹⁶⁾ The protective role of CA against AGEs-induced endothelial cell dysfunction was reported.¹⁷⁾ However, to the best of our knowledge there has been no previous report on the inhibitory activity of CA against AGEs-induced cross-link and breaking. Hence, the objective of this study is to evaluate inhibitory and breaking activities of CA on AGEs-induced cross-links of collagen.

MATERIALS AND METHODS

Plant Materials and Preparation of CA

The medicinal dried ripe fruit of T. chebula was purchased from a local market (Kyungdong Herb-Market, Seoul, Korea). Voucher specimens were deposited in the Herbarium at College of Life Sciences and Biotechnology, Korea University (registration number H-358). The isolation procedures for CA were followed as described previously.¹⁴⁾ The dried ripe fruits of T. chebula were ground with a mortar, and were extracted with ethanol at 80°C for 3 h. The extract was lyophilized, resuspended in H₂O, and then extracted successively with n-hexane, chloroform and ethyl acetate (EtOAc). The EtOAc-soluble portion was further purified by Sephadex™ LH-20 (Amersham Biosciences, Uppsala, Sweden) column chromatography, as a standard single compound, using 20% methanol as the eluent. HPLC analysis with a SymmetryPrep™ C18 column (300×7.8 mm, 7 µm) (Waters, Milford, MA, U.S.A.) was conducted to determine the concentrations of the standard compound, CA (Faces Biochemical Co., Wuhan, China).

Preparation of Glycolaldehyde-Glycated Bovine Serum Albumin (Glycol-BSA)

AGEs were prepared by incubating 10 mg/mL of BSA, 10 mM glycolaldehyde, and 1 mM diethylenetriaminepentaacetic acid (DTPA) in 0.1 M potassium phosphate buffer in the presence of 0.02% sodium azide (pH 7.4) at 37°C for 7 d. After 7 d, free glycolaldehyde and low molecular reactants were removed by extensive dialysis against 0.1 M potassium phosphate buffer.

Enzyme-Linked Immunosorbent Assay (ELISA)

The inhibitory activity on Glycol-BSA-induced collagen cross-links was measured according to a previously reported method with some modifications. Glycol-BSA (1 mg/mL) was incubated in either the presence or absence of AG or CA in 1.5 mg/mL of rat tail tendon type I collagen for 24 h at 37°C. In detail, horseradish peroxidase (HRP)-labeled Glycol-BSA was prepared using a peroxidase labeling kit-NH₂ Unit (Dojindo Molecular Technologies, Inc., Tokyo, Japan) according to the manufacturer’s instruction. Glycol-BSA was incubated in either the presence or absence of AG or CA in 0.45 mg/mL collagen coated 96-well plates for 4 h at 37°C. The unattached AGEs-BSA was washed with 0.05% PBST buffer. Glycol-BSA-induced collagen cross-links were detected using tetramethylbenzidine (TMB) substrate. Inhibition of Glycol-BSA induced collagen cross-links was expressed as the percentage decrease in optical density of 450 nm. We calculated the IC₅₀ concentration as 50% inhibition of Glycol-BSA-induced collagen cross-links, in which the cross-link inhibition percent was calculated as follows:

  

The breaking assay on Glycol-BSA induced cross-links to collagen was performed according to the method by Kim et al.¹⁸⁾ with some modifications. To confirm the breaking activity on Glycol-BSA induced cross-links to collagen, 1 mg/mL of Glycol-BSA was pre-incubated on 1.5 mg/mL of collagen for 24 h, and was then incubated in either the presence or absence of ALT-711 or CA at 37°C for 16 h. In detail, HRP labeled Glycol-BSA was pre-incubated on collagen-coated 96-well plates for 4 h, and the Glycol-BSA induced complexes with collagen were incubated in either the presence or absence of ALT-711 (Suchem Pharma Co., Wenzhou, China) or CA at 37°C for 16 h. After the unattached Glycol-BSA was washed in 0.05% PBST, breaking levels were detected using TMB substrate. Breaking of AGEs-induced cross-links was expressed as the percentage decrease in optical density. The breaking percentage was calculated according to the above equation.

Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) Separation

Also, AGE formation induced by Glycol-BSA was accessed by SDS-PAGE. SDS-PAGE was carried out according to the procedure of Laemmli, as described previously.¹⁹⁾ The protein determinations were performed using the Bradford method (Biorad, Hercules, CA, U.S.A.). Samples were dissolved in sample buffer (60 mM Tris–HCl, pH 6.8; 2% (w/v) SDS; 10% (v/v) glycerol; 5% (v/v) 2-mercaptoethanol; 0.01% (w/v) bromophenol blue) and were denatured at 100°C for 5 min. The samples were separated on 10% polyacrylamide gels. The equipment employed was a mini gel from Amersham (Amersham Biosciences, Uppsala, Sweden). The gels were stained with Coomassie Brilliant Blue.

Western Blotting

We performed Western blotting of Glycol-BSA induced cross-links of collagen. Each sample was fractionated of a 10% polyacrylamide gel, after which the proteins were transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, MA, U.S.A.) using MIGHTY SMALL TRANSPHOR (Amersham Biosciences, Uppsala, Sweden). The membrane was then incubated in blocking buffer (5% skim milk in Tris-buffered saline with Tween-20) to reduce non-specific binding. Membranes were then exposed to primary anti-AGEs monoclonal antibody (Trans Genic Inc., Kobe, Japan) overnight at 4°C. Thereafter, the blot was washed, incubated in HRP-conjugated secondary antibody (Sigma Chemical Co., St. Louis, MO, U.S.A.) and finally, membranes were detected using ECL solution (iNtRON Biotechnology, Seoul, Korea). The intensity of bands on images was measured using Image J software (Research Service Branch, National Institutes of Mental Health, Bethesda, MD, U.S.A.).

2,2-Diphenyl-1-picrylhydrazyl Radical (DPPH·) Free Radical Scavenging and Ferric Reducing/Antioxidant Power (FRAP) Assay

The scavenging activity was estimated by using DPPH· as a free radical model and reducing power method adapted from Lee et al.¹⁶⁾ FeSO₄·7H₂O concentration was used for calibration of FRAP assay.

Measurement of Inhibition of Metal-Catalyzed Oxidation of Ascorbic Acid (AA)

The chelation activity was performed in chelex-treated 50 mM phosphate buffer, pH 7.4, in water bath at 30°C as previously reported,²⁰⁾ with a slight modification in which 2.0 µM CuCl₂ instead of 0.5 µM CuCl₂ was added into the chelex-treated buffer to obtain a linear relationship between time and the logarithmic percentage value of AA remaining.

Statistical Analysis

All data were expressed as mean values±S.D. Statistical significance for the difference between the groups was assessed using one-way ANOVA and Duncan’s multiple range tests. SAS software version 9.2 (SAS Institute, NC, U.S.A.) was used to perform all statistical tests. Values of p<0.05 were considered to indicate a significant difference.

RESULTS

Inhibitory Effect of CA on Glycol-BSA-Induced Cross-Links to Collagen

We investigated the inhibitory effects of CA on the Glycol-BSA-induced cross-links to collagen using ELISA assay and gel electrophoresis (Fig. 1). CA markedly reduced this Glycol-BSA-induced cross-links to collagen in a dose-dependent manner. CA has 1750-fold stronger inhibition activity on AGEs-BSA induced cross-links than AG (Figs. 1A, B; IC₅₀ value of CA, 38.8±0.5 µM vs. IC₅₀ value of AG, 67.8±2.5 mM). In addition, the extent of Glycol-BSA-induced cross-links to collagen was examined by SDS-PAGE under denaturing condition (Fig. 1C). Referring to BSA control in lane 2, Glycol-BSA increased Glycol-BSA band (band a, about 70 kDa) as well as additional bands including band c with lower electrophoretic mobility (Fig. 1C). Whereas AG had little effect on the formations of these bands, the treatment of CA in the incubation mixture showed band (band b) with slightly lower mobility compared with band a, indicating the loss of the positive charge in the Glycol-BSA during the glycation reaction. In addition, less formation with lower electrophoretic mobility on PAGE gel was observed in the reaction mixture with CA.

Fig. 1. Inhibitory Effect of Chebulic Acid (CA) on Glycolaldehyde-Glycated BSA (Glycol-BSA) Induced Cross-Links to Collagen in Vitro

The inhibitions of Glycol-BSA induced cross-links to collagen with CA and aminoguanidine (AG) were measured by ELISA (A) and (B), respectively. All data were expressed as mean±standard deviation of triplicate experiments. ** p<0.01 vs. non-treated group (n=3). The inhibitory modulation of Glycol-BSA induced collagen cross-links was revealed by SDS-PAGE (C).

Breaking Effect of CA on Glycol-BSA-Induced Collagen Cross-Links

To investigate the breaking activities of CA on Glycol-BSA induced collagen cross-links, the strength of breaking cross-links at various concentrations of CA was measured by ELISA (Fig. 2). The treatment with CA resulted in a concentration-dependent decrease in the collagen cross-links. CA (Fig. 2A, IC₅₀=1.46±0.05 mM) exhibited 50-fold stronger breaking activity on Glycol-BSA binding to collagen than ALT-711 (Fig. 2B, IC₅₀=72.2±2.4 mM). In addition, these breaking activities of inhibitors on the Glycol-BSA-induced cross-links of collagen were analyzed by SDS-PAGE (Fig. 2C). A molecular band (band c) present at the top of the resolving gel was increased in the Glycol-BSA which was cross-linked with collagen in the absence of CA or ALT-77, whereas 2.5 mM CA and ALT-711 remarkably decreased the formation of this band. Glycol-BSA band (band a) was decreased with ALT-711 and CA in dose-dependent manners. Furthermore, in order to confirm whether band a (about 70 kDa), which was formed during glycation with glycolaldehyde, was altered, a monoclonal antibody (anti-AGEs) was used in Western blotting. Results showed that the density of Glycol-BSA was decreased dose-dependently with inhibitors in which the most reduced band density was observed with 5 mM CA (Fig. 2D). It was confirmed that band a of Fig. 2C and that of Fig. 2D have the same mobility. These results suggest that the band a of Fig. 1C, the band a of Fig. 2C and the band a of Fig. 2D are the same one.

Fig. 2. Breaking Effect of CA on Glycol-BSA Induced Cross-Links to Collagen in Vitro

The breakings of Glycol-BSA induced cross-links to collagen with CA and ALT-711 (alagebrium) were measured by ELISA (A) and (B), respectively. All data were expressed as mean±standard deviation of triplicate experiments. ** p<0.01 vs. non-treated group (n=3). The breaking modulation of Glycol-BSA induced collagen cross-links was revealed by SDS-PAGE (C). Western blotting of Glycol-BSA with anti-AGEs monoclonal antibody (D). * p<0.05 vs. non-treated group (n=3).

Antioxidant and Chelating Activities of CA

The beneficial effects of test compounds for treatment of cardiovascular and renal malfunctions in diabetes and aging can be largely explained, if not completely, by their antioxidant and chelating activities.^12,20) The antioxidant activities of CA were evaluated based on the FRAP value and DPPH· SC₅₀ value, showing that the activities of this compound were effective and comparable to those of ascorbic acid, showing DPPH· scavenging activity (39.2±4.9 vs. 19.8±1.2 µg/dry matter (DM) mL⁻¹) and FRAP (4.70±0.06 vs. 11.4±0.1 mmol FeSO₄·7H₂O·/g DM) (Table 1). In addition, we measured the kinetics of oxidation of AA of CA, and ALT711 and AG as positive controls. These compounds inhibited the oxidation of AA in dose-dependent manners (Figs. 3A–C). Normalized rates of AA oxidation were plotted as functions of the square roots of the concentrations of CA, ALT711 and AG. (Fig. 3D). The concentrations that produced a 50% inhibitory effect (IC₅₀) of three compounds on AA oxidation are shown in Fig. 3D. The compounds displayed IC₅₀ values of 0.96±0.07 mM for CA, 1.92±0.20 mM for ALT711 and 0.47±0.05 mM for AG.

Table 1. Antioxidant Activities of Chebulic Acid

	DPPH· SC50a) (µg/DMb) mL−1)	FRAPc) (mmol FeSO4·7H2O/g DM)
Chebulic acid	39.2±4.9	4.70±0.06
Ascorbic acid	19.0±1.2	11.4±0.1

Each value is mean±S.D. of three replicate experiments (n=3). a) Amount of sample necessary to decrease the initial 2,2-diphenyl-1-picrylhydrazyl radical (DPPH·) concentration by 50%. b) DM, dry matter. c) Ferric-reducing antioxidant power.

Fig. 3. Kinetics of Oxidation of Ascorbic Acid (AA) with and without Different Levels of AG (A), ALT 711 (B) and CA (C), and Their Effect on the Kinetic of Copper-Catalyzed Oxidation of AA (D)

Reaction mixtures contained 0.5 mM AA, 1.0 µM CuCl₂ and different concentrations of AG, ALT-711 and CA. Data points represent means±S.D. of triplicate experiments.

DISCUSSION

Many reports have alluded to the potential role of AGEs in the development of diabetic complications.²¹⁾ The AGEs-induced cross-links of collagen have been correlated with the severity of diabetic complications as well as in certain pathophysiological states commonly seen in aging.²²⁾ In the present study, CA significantly inhibited 1750-fold more Glycol-BSA induced cross-links to collagen than AG (IC₅₀, 38.8±0.5 µM versus 67.8±2.5 mM; Fig. 1) based on cross-links to collagen, as determined using an ELISA system where the HRP labeled AGEs-BSA induced cross-links of collagen-coated plates was measured in either the presence or absence of CA or AG. However, our previous study showed that the inhibitory activity of CA on glycation-induced protein cross-links was comparable to that of AG, showing that CA and AG had an IC₅₀ values of 17.1±0.7 mM and 21.3±1.4 mM, respectively, based on cross-linking assays, which were carried out using a system measuring the incorporation of [¹⁴C]lysine into BSA.²³⁾ The reaction conditions for inducing protein cross-links may explain the discrepancy between the above results on cross-link inhibitory activities. In the present study, BSA glycated with glycolaldehyde, Glycol-BSA was prepared, and was then employed to induce cross-links to collagen, which was measured using an ELISA system.¹⁸⁾ Glycated BSA with glycolaldehyde in the absence of collagen was employed to induce AGEs including cross-links, and the incorporation of [¹⁴C]lysine into BSA was used to measure cross-link activity in our previous study. CA showed significantly potent activity in inhibiting cross-links to collagen compared with AG in the present assay, whereas AG as a positive control gave similar magnitude of potency against cross-links in ELISA assay (IC₅₀, 67.8±2.5 mM) and the previously reported radioactivity assay (IC₅₀, 21.3±1.4 mM).²³⁾ To the best of our knowledge, it is the first report that such micromolar concentrations of CA have a highly inhibitory effect against AGEs-BSA induced cross-links to collagen, suggesting that CA may constitute a promising intervention agent in diabetic complications.

A limitation of AGE inhibitors is that they cannot affect pre-existing AGE cross-links, and so, another way to reduce levels of AGEs is through the use of the so-called ‘cross-link breakers,’ such as alagebrium chloride and ALT-711, which have been investigated with hypertension trials (Phase II), although those have been terminated due to lack of effectiveness in blood pressure.¹²⁾ We performed the breaking assay in which the HRP-labeled AGEs-BSA induced cross-links to collagen was incubated for measuring breaking activity in the presence of ALT-711 and CA. The significantly strong activity of CA compared to that of ALT-711 (Figs. 2A, B, IC₅₀, 1.46±0.05 mM vs. 72.2 ±2.4 mM) was confirmed by SDS-PAGE and Western blotting analysis. Thiazolium salts, such as ALT-711 and N-phenacylthiazolium bromide, are known to selectively add a dicarbonyl AGE cross-links, followed by an internal rearrangement to cleave the dicarbonyl bond, breaking the cross-links.⁶⁾ However, not a single AGE-cross-link structure, which contains a dicarbonyl structure prone to the proposed action of AGE breakers, has been identified in tissue proteins to date.²²⁾ Recently, Nagai et al.²⁴⁾ proposed that the activities of AGE inhibitors and breakers of the AGEs formation and development of diabetes complications may be accounted at large by their chelating activity. The reported IC₅₀ values for of ALT-711 and AG, for the inhibition of copper-catalyzed oxidation of AA are 80 µM and 2.5 mM, respectively.^20,22) However, IC₅₀ values for inhibiting metal-catalyzed oxidation were determined as 1.92±0.20 mM for ALT711, and 0.47±0.05 mM for AG as well as 0.96±0.07 mM for CA, in this study (Fig. 3). Although such discrepancies of IC₅₀ values remains to be explained, in our experiment we added 2.0 µM CuCl₂ instead of 0.5 µM CuCl₂ to 50 mM chelex-treated phosphate buffer to obtain a linear relationship between time and the logarithmic percentage value of AA remaining. The chelating activities of CA as well as AG and ALT711 on copper-catalyzed oxidation of AA were compared based on our experimental condition, and in increasing order, ALT-711<CA<AG. In addition, the antioxidant activities of CA were comparable to those of AA, showing DPPH· scavenging activity (IC₅₀ values; 39.2±4.9 vs. 19.0±1.2 µg/dry matter mL⁻¹) and FRAP activity (4.70±0.06 vs. 11.4±0.1 mmol FeSO₄·7H₂O/g DM). On the other hand, the values of ALT-711 and AG for DPPH· scavenging and FRAP activities were below the threshold of determination. Because CA lacks functional groups which can cleave dicarbonyl compounds or react with carbonyl intermediates, the high potency of CA on AGEs-BSA induced cross-link breaking can be ascribed to its chelating and antioxidant activities. It is interesting to note that citrate, a nonspecific chelator at the same dose as triethylenetetraamine (0.1 g/100 mL of drinking water) yielded comparable protection against cardiac structural and functional changes in the Zuker type 2 diabetic rat, and that citrate inhibited the formation of a well-known AGE, carboxymethyl lysine in the lens.²⁴⁾

Our present in vitro studies showed that CA is a more potent inhibitor cross-linking and beaker of collagen cross-linking than the control compounds, such as AG and ALT-711. These results suggest that CA could be a breaker as well as an inhibitor of AGE cross-linking, and the activity may be explained in large part by its chelating and antioxidant activities. However the mechanism of action of CA is not clear yet. In vivo studies are needed for the more practical use of CA as an anti-diabetic agent and for use in therapy of diabetic complications.

Acknowledgment

This research was supported by High Value-added Food Technology Development Program No. 111137-03-1-HD120, Ministry of Agriculture, Food and Rural Affairs. The authors also thank the Korea University-CJ Food Safety Center (Seoul, South Korea) for providing equipment and facilities.

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