572 H.-S. Lee et al. / Journal <strong>of</strong> Ethnopharmacology 131 (2010) 567–574 Fig. 5. Effects <strong>of</strong> <strong>chebulic</strong> <strong>acid</strong> on THP-1 cell adhesion to HUVEC stimulated by glycer-AGEs. HUVEC treated with BSA (A), 100 �g/ml <strong>of</strong> glycer-AGEs (B), 100 �g/ml <strong>of</strong> glycer-AGEs + 10 �M <strong>chebulic</strong> <strong>acid</strong> (C), and 100 �g/ml <strong>of</strong> glycer-AGEs + 25 �M <strong>chebulic</strong> (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 <strong>of</strong> 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 <strong>of</strong> their glycemic level, blood pressure, and lipid pr<strong>of</strong>ile, and such vascular diseases are an important cause <strong>of</strong> morbidity and mortality in diabetic patients (Laakso and Kuusisto, 1996; Cohen, 2007; Calcutt et al., 2009). The authors <strong>of</strong> one particular diabetes complications and control trial: The Epidemiology <strong>of</strong> Diabetes Interventions and Complications Trial, suggested that the influence <strong>of</strong> early glucose control on the development <strong>of</strong> macrovascular events may become more obvious with a longer follow-up time (The Diabetes Control and Complications Trial/Epidemiology <strong>of</strong> Diabetes Interventions and Complications Research Group, 1993, 2005; Writing Team for the Diabetes Control and Complications Trial/Epidemiology <strong>of</strong> Diabetes Interventions and Complications Research Group, 2003). These findings suggest that advanced protein glycation, a pro-
cess involving the nonenzymatic modification <strong>of</strong> tissue proteins by physiological sugars in vivo, appears to play a central role in the pathogenesis <strong>of</strong> diabetic complications; this is because the concept <strong>of</strong> ‘glycemic memory’ is well-fitting to the in vivo formation and accumulation <strong>of</strong> 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 <strong>from</strong> sugar autoxidation and other metabolic pathways (Takeuchi and Yamagishi, 2004). Inside cells, these can much readily accelerate AGE formation with amino groups <strong>of</strong> 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 <strong>of</strong> diabetic complications. AGEs mediate their <strong>effects</strong> 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 <strong>of</strong> 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 <strong>of</strong> the redox-sensitive transcription factor nuclear factor-kB in vascular wall cells and the expression <strong>of</strong> a variety <strong>of</strong> 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 <strong>of</strong> 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 <strong>effects</strong> such as gastrointestinal disturbance (Thornalley, 2003). Alternatively, the search for AGE inhibitors, by examining naturally occurring compounds used traditionally was started. In this regard, <strong>chebulic</strong> <strong>acid</strong> <strong>isolated</strong> <strong>from</strong> <strong>Terminalia</strong> <strong>chebula</strong>r has been investigated by our research group (Lee et al., 2007). This compound belongs to the phenolcarboxylic <strong>acid</strong> group, and its structure is simpler compared to other compounds such as <strong>chebula</strong>nin, <strong>chebula</strong>gic <strong>acid</strong>, and casuarinin (Juang et al., 2004). We previously reported that the antioxidant activity <strong>of</strong> <strong>chebulic</strong> <strong>acid</strong> 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, <strong>chebulic</strong> <strong>acid</strong> 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 <strong>of</strong> [ 14 C]lysine into BSA. Also, <strong>chebulic</strong> <strong>acid</strong>’s IC 50 value (mM) for inhibition <strong>of</strong> AGE formation was 1.8-fold-lower (1.32 ± 0.10 mM) than the IC 50 <strong>of</strong> aminoguanidine (2.37 ± 0.09 mM). Whiles such millimolar concentrations <strong>of</strong> <strong>chebulic</strong> <strong>acid</strong> seem to indicate relatively low potency with respect to inhibitory <strong>effects</strong> against cross-linking and AGE formation, effective levels <strong>of</strong> <strong>chebulic</strong> <strong>acid</strong> 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 <strong>of</strong> intracellular reactive oxygen species H.-S. Lee et al. / Journal <strong>of</strong> Ethnopharmacology 131 (2010) 567–574 573 (ROS) and subsequently evoke inflammatory responses leading to the development <strong>of</strong> vascular complications in diabetes (Basta, 2008). This increase in ROS in turn activates a complex cascade <strong>of</strong> signal transduction pathways, and subsequently enhances the expression <strong>of</strong> 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 <strong>of</strong> 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 <strong>of</strong> ehtanolic extract <strong>of</strong> <strong>Terminalia</strong> <strong>chebula</strong>r containing <strong>chebulic</strong> <strong>acid</strong> had significantly reduced mRNA levels <strong>of</strong> 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 <strong>chebulic</strong> <strong>acid</strong> had activity for normalizing the permeability <strong>of</strong> endothelial cells and reduced the adhesion <strong>of</strong> 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 <strong>of</strong> AGEs. At this point, the activities <strong>of</strong> <strong>chebulic</strong> <strong>acid</strong> are not yet fully understood. However, the present results suggest that <strong>chebulic</strong> <strong>acid</strong> <strong>from</strong> <strong>Terminalia</strong> <strong>chebula</strong> may act as an anti-diabetic complication agent through the extracellular inhibition <strong>of</strong> AGE formation as well as intracellular ROS scavenging in endothelial cells. 5. Conclusion Inhibitors <strong>of</strong> AGE formation to reduce tissue accumulation <strong>of</strong> AGEs in diabetes have potentially beneficial <strong>effects</strong> in innate chronic atherosclerosis. Alternatively, interference on RAGE-AGE axis may be a promising novel therapeutic strategy for the treatment <strong>of</strong> acute vascular injury. Our in vitro results with <strong>chebulic</strong> <strong>acid</strong> <strong>isolated</strong> <strong>from</strong> <strong>Terminalia</strong> <strong>chebula</strong> may constitute a promising intervention agent in these two setting relevant to diabetic vascular complications. Further in vivo studies are required to prove whether <strong>chebulic</strong> <strong>acid</strong> could inhibit the risk <strong>of</strong> diabetic vascular complications beyond blood glucose lowering effect. Acknowledgements This work supported by grant number PJ007100201003 <strong>from</strong> the BioGreen 21 program, Rural Development Administration, Republic <strong>of</strong> 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. References Badmaev, V., Nowakowski, M., 2000. Protection <strong>of</strong> epithelial cells against influenza A virus by a plant derived biological response modifier Ledretan-96. Phytotherapy Research 14, 245–249. Basta, G., 2008. Receptor for advanced glycation endproducts and atherosclerosis: <strong>from</strong> basic mechanisms to clinical implications. Atherosclerosis 196, 9–21. Brownlee, M., 1995. Advanced protein glycosylation in diabetes and aging. Annual Review <strong>of</strong> Medicine 46, 223–234.
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