Chebulic Acid Prevents Methylglyoxal-Induced Mitochondrial Dysfunction in INS-1 Pancreatic ß-Cells.

To investigate the anti-diabetic properties of chebulic acid (CA) associated with the prevention of methyl glyoxal (MG)-induced mitochondrial dysfunction in INS-1 pancreatic ß-cells, INS-1 cells were pre-treated with CA (0.5, 1.0, and 2.0 µM) for 48 h and then treated with 2 mM MG for 8 h. The effects of CA and MG on INS-1 cells were evaluated using the following: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay; glyoxalase 1 (Glo-1) expression via Western blot and enzyme activity assays; Nrf-2, nuclear factor erythroid 2-related factor 2 protein expression via Western blot assay; reactive oxygen species (ROS) production assay; mRNA expression of mitochondrial dysfunction related components (UCP2, uncoupling protein 2; VDAC1, voltage-dependent anion-selective channel-1; cyt c, cytochrome c via quantitative reverse transcriptase-PCR; mitochondrial membrane potential (MMP); adenosine triphosphate (ATP) synthesis; glucose-stimulated insulin secretion (GSIS) assay. The viability of INS-1 cells was maintained upon pre-treating with CA before exposure to MG. CA upregulated Glo-1 protein expression and enzyme activity in INS-1 cells and prevented MG-induced ROS production. Mitochondrial dysfunction was alleviated by CA pretreatment; this occurred via the downregulation of UCP2, VDAC1, and cyt c mRNA expression and the increase of MMP and ATP synthesis. Further, CA pre-treatment promoted the recovery from MG-induced decrease in GSIS. These results indicated that CA could be employed as a therapeutic agent in diabetes due to its ability to prevent MG-induced development of insulin sensitivity and oxidative stress-induced dysfunction of ß-cells.

Anti-hyperglycemic and hypolipidemic effects of black ginseng extract containing increased Rh4, Rg5, and Rk1 content in muscle and liver of type 2 diabetic db/db mice.

Black ginseng (BG), which is produced by repeated steaming and drying of fresh ginseng, has various pharmacological and therapeutic properties. This study investigated the anti-hyperglycemic and hypolipidemic effects of BG ethanolic extract in type 2 diabetic db/db mice. The levels of fasting blood glucose, HbA1c, insulin levels and thiobarbituric acid reactive substances values were decreased in the groups fed BG extract (BG) (100 and 900 mg/kg BW/day), compared to the control group (CON). In the BG compared with the CON, hepatic steatosis in the liver and the size of adipocytes in muscle tissue were improved. The administration of BG regulated the glucose transporter type (GLUT) 4 and 2, and peroxisome proliferator-activated receptor (PPAR) α and γ in muscle and liver. Moreover, ginsenosides (Rh4, Rg5, and Rk1), which profiled by HPLC, regulated the markers for lipid metabolism and glucose metabolism; PPARs and GLUTs in muscle and C2C12 rather than liver cells and tissue. These findings suggested that ginsenosides (Rh4, Rg5, and Rk1) from BG extract can ameliorate type 2 diabetes through their anti-hyperglycemic and hypolipidemic effects.

Inhibitory Effect of Chebulic Acid on Alveolar Epithelial to Mesenchymal Transition in Response to Urban Particulate Matter Using Co-treatment and Post-treatment Exposure.

Urban particulate matter (UPM) is atmospheric particulate samples obtained from industrialized urban areas. It is known that pulmonary fibrosis can result directly or indirectly from particulate matter. In this study, the protective effect of chebulic acid (CA) against UPM-induced epithelial-mesenchymal transition (EMT) in the pulmonary alveolar epithelial (PAE) cells were investigated. Our findings revealed that PAE cells were changed from the epithelial phenotype to mesenchymal one after exposure to UPM. Furthermore, co-treatment and post-treatment of CA inhibited EMT progression. Especially the key epithelial marker, E-cadherin, was down-regulated by UPM and recovered by CA. Also, gelatin zymogram showed that the activity of matrix metalloproteinase (MMP)-2 and MMP-9 was decreased by co-treatment and post-treatment of CA. Further investigation revealed that CA attenuated UPM-stimulated PAE cells invasion ability. These data showed that UPM promoted PAE cells invasion, reactive oxygen species-mediated extracellular matrix degradation and CA reduced the potential health risks associated with UPM.

Protective effects of chebulic acid from Terminalia chebula Retz. against t-BHP-induced oxidative stress by modulations of Nrf2 and its related enzymes in HepG2 cells.

Although chebulic acid isolated from Terminalia chebular has diverse biological effects, its effects on the expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and the expression of downstream genes have not been elucidated. The purpose of this research is to investigate the hepatoprotective mechanism of chebulic acid against oxidative stress produced by tert-butyl hydroperoxide (t-BHP) in liver cells. The treatment with chebulic acid attenuated cell death in t-BHP-induced HepG2 liver cells and increased intracellular glutathione content, upregulated the activity of heme oxygenase-1, and also increased the translocation of Nrf2 into the nucleus and Nrf2 target gene expression in a dose-dependent manner. The exposure of chebulic acid activated the phosphorylation of mitogen-activated protein kinases. The overall result is that chebulic acid has cytoprotective effect on t-BHP-induced hepatotoxicity in HepG2 cells through Nrf2-mediated antioxidant enzymes.

Anti-inflammatory effect of glucose-lysine Maillard reaction products on intestinal inflammation model in vivo.

Inflammatory bowel diseases (IBDs) are chronic disorders that are characterized by intestinal epithelial inflammation and injury. Currently, the most employed therapies are antibiotics and anti-inflammatory drugs; however, the side effects limit long-term effectiveness. We evaluated the impact of glucose-lysine Maillard reaction products (Glc-Lys MRPs) on colitis, induced in rats by an administration of 5% dextran sulfate sodium (DSS) in drinking water. Glc-Lys MRPs ameliorate DSS-induced colitis, as determined by a decrease in disease index activity, colon weight/length ratio, nitric oxide levels in serum, recovery of body weight loss, colon length and serum lysozyme levels. Furthermore, Glc-Lys MRPs increase the glutathione content and the activity of glutathione peroxidase, superoxide dismutase and catalase, and inhibit lipid peroxidation and myeloperoxidase activity in colon tissues. In particular, Glc-Lys MRPs suppress the mRNA level of the inflammatory cytokines and nuclear factor-κB in colon tissues. This study suggests the potential of Glc-Lys MRPs in preventing or treating IBDs.

Protective effects of chebulic acid on alveolar epithelial damage induced by urban particulate matter.

BACKGROUND: Chebulic acid (CA) isolated from T. chebula, which has been reported for treating asthma, as a potent anti-oxidant resources. Exposure to ambient urban particulate matter (UPM) considered as a risk for cardiopulmonary vascular dysfunction. To investigate the protective effect of CA against UPM-mediated collapse of the pulmonary alveolar epithelial (PAE) cell (NCI-H441), barrier integrity parameters, and their elements were evaluated in PAE. METHODS: CA was acquired from the laboratory previous reports. UPM was obtained from the National Institutes of Standards and Technology, and these were collected in St. Louis, MO, over a 24-month period and used as a standard reference. To confirm the protection of PAE barrier integrity, paracellular permeability and the junctional molecules were estimated with determination of transepithelial electrical resistance, Western Blotting, RT-PCR, and fluorescent staining. RESULTS: UPM aggravated the generation of reactive oxygen species (ROS) in PAE and also decreased mRNA and protein levels of junction molecules and barrier integrity in NCI-H441. However, CA repressed the ROS in PAE, also improved barrier integrity by protecting the junctional parameters in NCI-H411. CONCLUSIONS: These data showed that CA resulted in decreased UPM-induced ROS formation, and the protected the integrity of the tight junctions against UPM exposure to PAE barrier.

Plantamajoside from Plantago asiatica modulates human umbilical vein endothelial cell dysfunction by glyceraldehyde-induced AGEs via MAPK/NF-κB.

BACKGROUND: Plantago asiatica has been traditionally used for traditional medicine around East Asia. Plantamajoside (PM), which is isolated from this plant, is known for biological properties including anti-inflammation and antioxidant activity. To demonstrate the biological activity of PM against endothelial dysfunction induced by advanced glycation end-products (AGEs), a cellular inflammatory mechanism system was evaluated in human umbilical vein endothelial cells (HUVECs). METHODS: We obtained PM through previous research in our laboratory. We formed the AGEs from bovine serum albumin with glyceraldehyde in the dark for seven days. To confirm the modulation of the inflammatory mechanism in endothelial dysfunction, we quantified the various pro-inflammatory cytokines and endothelial dysfunction-related proteins in the HUVECs with Western blotting and with real-time and quantitative real-time polymerase chain reactions. RESULTS: Co-treatment with PM and AGEs significantly suppressed inflammatory cytokines and adhesion molecule expression. Moreover, the PM treatment for down-regulated inflammatory signals and blocked monocyte adhesion on the HUVECs. CONCLUSIONS: Theses results demonstrated that PM, as a potential natural compound, protects AGE-induced endothelial cells against inflammatory cellular dysfunction.

Chebulic acid prevents hepatic fibrosis induced by advanced glycation end-products in LX-2 cell by modulating Nrf2 translocation via ERK pathway.

Advanced glycation end-products (AGEs) are formed during normal aging, and at an accelerated rate in metabolic syndrome patients. Nonalcoholic steatohepatitis (NASH) can be caused by the AGEs in plasma, while glyceraldehyde-derived AGEs (glycer-AGEs) are significantly higher in the serum of NASH patients. In this study, we investigated the molecular mechanisms of chebulic acid, isolated from Terminalia chebula Retz., in the inhibition of glycer-AGEs induced production of reactive oxygen species (ROS) and collagen accumulation using the LX-2 cell line. Chebulic acid significantly inhibited the induction of ROS and accumulation of collagen proteins by glycer-AGEs. ERK phosphorylation and total nuclear factor E2-related factor 2 (Nrf2) protein expression were induced by chebulic acid in a dose-dependent manner. Chebulic acid was also found to induce translocation of Nrf2 into the nucleus, which was attenuated by inhibition of ERK phosphorylation through treatment with PD98059. Following translocation of Nrf2, chebulic acid induced the protein expressions of catalytic subunit of γ-glutamylcysteine synthetase and glutathione synthesis. Collagen accumulation was also significantly reduced by chebulic acid treatment. The observed effects of chebulic acid were all inhibited by PD98059 treatment. Taken together, these results suggest that chebulic acid prevents the glycer-AGEs-induced ROS formation of LX-2 cells and collagen accumulation by ERK-phosphorylation-mediated Nrf2 nuclear translocation, which causes upregulation of antioxidant protein production.

Pheophorbide a from Capsosiphon fulvescens Inhibits Advanced Glycation End Products Mediated Endothelial Dysfunction.

During hyperglycemia, the first step toward the formation of advanced glycation end products is the nonenzymatic glycation between the carbonyl group of a sugar and the primary amino group of a protein. Advanced glycation end products are then produced through more complex reactions. Reactive oxygen species derived from advanced glycation end products may play a key role in inflammation of the endothelium, leading to the complications seen in diabetes. Glycolaldehyde-induced advanced glycation end products have been reported to express proinflammatory cytokines, such as tumor necrosis factor-α and interleukin-1ß. This study focused on Capsosiphon fulvescens, a Capsosiphonaceae type of green algae that has shown potential as a functional food material. Pheophorbide a, an anti-glycation compound, was isolated from C. fulvescens by extraction using a mixture of ethanol and water, followed by column fractionation of the resulting extract. The compound separated from C. fulvescens was identified by means of high-performance liquid chromatography combined with mass spectrometry. Pheophorbide a showed scavenging activity of the intracellular reactive oxygen species as well as monocyte adhesiveness inhibitory activity on the human myelomonocytic cell line (THP-1) and human umbilical vein endothelial cells cocultivation system. The mRNA levels of inflammation-related genes such as monocyte chemoattractant protein-1 and interleukin-6 were significantly decreased by pheophorbide a, and advanced glycation end products-stimulated tumor necrosis factor-α and interleukin-1ß were downregulated as well. These results indicate that pheophorbide a has significant reactive oxygen species-scavenging activity, monocyte adhesive inhibitory activity, and downregulatory activity of cytokines related to inflammation affecting the endothelium. Pheophorbide a could therefore be a promising candidate for modulating endothelial cell dysfunction.

Nephroprotection of plantamajoside in rats treated with cadmium.

Cadmium (Cd), an environmental and industrial pollutant, generates free radicals responsible for oxidative stress. Cd can also lead to various renal toxic damage such as the proximal tubules and glomerulus dysfunction. Plantamajoside (PMS), a major compound of Plantago asiatica (PA), was reported to have the antioxidant effects. In this study, we investigated the protective effects of PMS on Cd-induced renal damage in the NRK-52E cell and rat kidney tissue. Cd exposure increased the ROS generation, lipid peroxidation, serum biochemical values of renal damage, and mRNA and protein expressions of KIM-1 in vitro and in vivo. The significant reduction in glutathione (GSH)/glutathione disulfide (GSSG) ratio and activities of antioxidant enzymes were also observed in the rats treated with Cd. PMS significantly decreased the ROS generation and lipid peroxidation, thus enhancing GSH/GSSG ratio, antioxidant enzyme activities in the cells and rats, and improved histochemical appearances, indicating that PMS has protective activities against Cd-induced renal injury.

5-α Reductase inhibitory effect and astringent activity of green apple rind extract on human keratinocytes and fibroblast cells.

Unripe green apples contain condensed tannins at 10 times higher levels than ripe apples. Tannin not only has strong antioxidant activity, but also an astringent property. In this study, we investigated the effects of green apple rind (GAR) extracts in reducing facial pores and sebum secretion. Among the GAR extracts, the 70% ethanol GAR extract showed the highest antioxidant activity and tannin content. Hence, it was further fractionated with different solvents. Among these rind solvent fractions, the ethyl acetate fraction of the extract (GAR-E) showed astringent activity. Additionally, it exhibited inhibitory effects on 5-α reductase, and induced type 1 collagen and involucrin synthesis. These results suggest that GAR-E can be applied in cosmetics to reduce facial pore size and sebum secretion.

The hepatoprotection of caffeic acid and rosmarinic acid, major compounds of Perilla frutescens, against t-BHP-induced oxidative liver damage.

Perilla frutescens leaves are often used in East Asian gourmet food. In this study, we investigated the hepatoprotective effects of caffeic acid (CA), rosmarinic acid (RA), and their combination. P. frutescens contains 1.32µg CA/mg dry material (DM) and 26.84µg RA/mg DM analyzed by HPLC-DAD and HPLC-MS. CA remarkably reduced the oxidative damage than rosmarinic acid in an in vitro study. Oral intubation with CA or RA alone for five days was conducted prior to treatment with a single dose of tert-butyl hydroperoxide (0.5mmol/kg b.w., i.p.), which led to a significant reduction of indicators of hepatic toxicity, such as aspartate aminotransferase, alanine aminotransferase, oxidized glutathione, lipid peroxidation and enzyme activities related to antioxidant such as catalase, glutathione peroxidase and superoxide dismutase. Interestingly, compared to treatment with CA or RA alone, a combination of both compounds more increased the endogenous antioxidant enzymes and glutathione (GSH) and decreased lipid peroxidation in livers. These results suggest that CA from perilla leaves plays a role in the increased hepatic GSH concentration, and shows an additive hepatic protection with RA against oxidative hepatic damage.

Separation of the antioxidant compound quercitrin from Lindera obtusiloba Blume and its antimelanogenic effect on B16F10 melanoma cells.

Considering the growing evidence of the presence of antioxidant compounds in plant extracts, the objectives of this study were to identify antioxidant compounds in Lindera obtusiloba Blume (Lauraceae) and to evaluate their antimelanogenic activities on B16F10 melanoma cells. Organic solvent fractions were separated from L. obtusiloba extracts (LOE). The ethyl acetate fraction (LOE-E) was significantly active against oxidative damage induced by tert-butyl hydroperoxide in primary rat hepatocytes. Two single purified compounds, quercitrin (quercetin-3-O-α-L-rhamnopyranoside) and afzelin (kaempferol-3-O-α-L-rhamnoside), were identified by HPLC and NMR. These compounds were evaluated for antioxidant activities by 1,1-diphenyl 2-picrylhydrazyl (DPPH) radical scavenging assay and ferric reducing antioxidant power (FRAP) assay, and for their antimelanogenic activities by tyrosinase inhibitory assay melanin formation inhibition assay and Western bolt analysis for the signaling pathway. The significant effects of quercitrin on antioxidant and antimelanogenic activities, and signal modulation of ERK and MITF in B16F10 melanoma cells were observed. This is the first report to identify quercitrin in L. obtusiloba and its whitening effect.

Protective effect of 70% ethanolic extract of Lindera obtusiloba Blume on tert-butyl hydroperoxide-induced oxidative hepatotoxicity in rats.

Lindera obtusiloba Blume, a native plant of East Asia, has traditionally been used as a folk medicine for liver disease. We studied the in vitro antioxidant and in vivo hepatoprotective activities of a 70% ethanolic extract of L. obtusiloba (LOE) containing 62.9% quercitrin and 22.0% afzelin. LOE prevented tert-butyl hydroperoxide (t-BHP)-induced oxidative damage in HepG2 cells. Along with its high antioxidant potency in vitro, our animal study confirmed that pretreatment with LOE (500 or 2000 mg/kg) for 7 days prior to a single dose of t-BHP (i.p.: 0.5 mmol/kg) significantly lowered the serum levels of alanine and aspartate aminotransferases. In addition, glutathione levels were increased in the liver, and lipid peroxidation levels were decreased in a dose-dependent manner. The histopathological examinations of rat livers showed that LOE significantly reduced the incidence of liver lesions induced by t-BHP. Therefore, we concluded that LOE has merit as a potent candidate to protect the liver against oxidative damage.

Anti-glycation effect of gold nanoparticles on collagen.

Gold nanoparticles (GNPs) have been reported to exhibit a variety of biological effects including anti-inflammatory and anti-oxidant activities. The extent of an in vitro glycation reaction mixture of collagen and glycolaldehyde was assayed to investigate the inhibition of glycolaldehye-derived advanced glycation end products (glycol-AGEs) formation with GNPs in collagen, which is a major protein component of the human dermis. GNP-treated collagen showed significantly less glycation (56.3 ± 4.2%) than an untreated glycation control. Moreover, GNP-treated glycation in a collagen lattice model significantly decreased the AGEs distribution in the model system. Taken together, these results suggest that GNPs have the potential for use in the prevention of glycation-induced skin aging.

Mutagenicity and oral toxicity studies of Terminalia chebula.

The fruit of Terminalia chebula Retz. (T. chebula), which is a member of the Combfreetaceae family, is used widely in Asian countries as a traditional folk medicine, and its extract has been reported to be an anticancer, antidiabetic and anticaries agent. In our previous study, chebulic acid isolated from T. chebula extract was confirmed to show antioxidant activity and protective action against endothelial cell dysfunction. In order to support the safety-in-use of the ethyl acetate (EtOAc)-soluble portion of a T. chebula ethanol extract containing 29.4% chebulic acid content, the prepared portion was tested in an in vitro mutagenicity assay, and a single- and 14-day repeated dose oral toxicity study. In the bacterial mutation assay, up to 5000 µg/mL concentration of the EtOAc-soluble portion, the numbers of colonies did not increase whether with or without metabolic activation. In the oral toxicity study, the single oral dose of the extract at 2000 mg/kg did not produce mortality or abnormal lesions in the internal organs of rats. The results of a 14-day orally repeated dose showed that the EtOAc-soluble portion of T. chebula ethanol extracts gave no adverse effects at dosages of 2000 mg/kg in rats in the study.

Protective effect of extracts of Perilla frutescens treated with sucrose on tert-butyl hydroperoxide-induced oxidative hepatotoxicity in vitro and in vivo.

Perilla frutescens leaves are often used in East Asian gourmet food. In this study, we investigated the hepatoprotective effects of P. frutescens leaves grown in different concentrations of sucrose (0, 115, 175 and 235 mM sucrose) leading to four samples of perilla leaf extracts (PLEs). Based on caffeic acid level and antioxidant activities, further experiments were conducted using perilla leaf extracts treated with 6% sucrose compared with non-treated perilla leaf extracts as a control. Oral intubation with non-treated perilla leaf extracts or perilla leaf extracts treated with 6% sucrose (1000 mg/kg b.w. rat) for 5 days was conducted before treatment with a single dose of tert-butyl hydroperoxide (0.5 mmol/kg b.w., i.p.) led to a significant reduction of hepatic toxicity in the perilla leaf extracts treated with 6% sucrose. We demonstrated that P. frutescens with higher contents of caffeic acid was produced, and that sucrose could play a role in the induction of this secondary metabolite. Sucrose-treated perilla leaves, which had better antioxidant activities than untreated leaves, can be used as a potential dietary source.

Oral administration of ethyl acetate-soluble portion of Terminalia chebula conferring protection from streptozotocin-induced diabetic mellitus and its complications.

Terminalia chebula has been widely used in India as a folk medicine. This study investigated the in vivo anti-hyperglycemia and anti-diabetic complication effects of the EtOAc-soluble portion of ethanolic extract of T. chebula fruit (EETC) containing 29.4% chebulic acid. Rats were divided into non-diabetic, untreated diabetic and diabetic groups. Streptozotocin (40 mg/kg body weight (BW))-induced diabetic rats were orally administered the aminoguanidine (100 mg/kg BW), high dose (500 mg/kg BW; HEETC) and low dose (100 mg/kg BW; LEETC) for 13 weeks. HEETC administration reduced the levels of blood glucose and serum lipids, decreased malondialdehyde concentrations of serum and thoracic aorta in diabetic rats, and significantly improved serum biochemical values and the pathomorphological changes of the liver and kidney in diabetic rats. Also, HEETC decreased the advanced glycation end products (AGEs) distribution in testis seminiferous tubules. Therefore, HEETC has a merit to be a potent candidate to control glycemic and diabetic complications.

Plasma caffeic acid is associated with statistical clustering of the anticolitic efficacy of caffeic acid in dextran sulfate sodium-treated mice.

We hypothesized that interindividual variability in the bioavailability of caffeic acid (CA) would influence its anticolitic efficacy and that mice may be appropriate for modeling human gut microbial metabolism of CA, which is thought to influence CA bioavailability. Anaerobic human fecal and mouse cecal sample mixtures were incubated with CA derivatives from Echinacea purpurea and compound disappearance rates were measured, which were similar in both sample types. CA metabolism, including formation of its main metabolite, m-hydroxyphenylpropionate, in the mouse cecum may usefully model human gut metabolism of this compound. Ten-week-old CD-1/IGS female mice were fed 120 mg CA/kg (n = 36) or control diet for 7 d (n = 12); one-half of each group then drank 1.25% dextran sulfate sodium (DSS) in water for 5 d. DSS-treated mice fed CA showed lessened colitic damage than did mice given DSS alone, with longer colons, greater body weight, and colonic Cyp4b1 expression. Cluster analysis of the cecal histopathological score showed that mice with severe cecal damage (mean cecal score = 8.5; n = 11) also had greater myeloperoxidase (MPO) activity and lower plasma CA compared with mice showing mild cecal damage (mean cecal score = 4.5; n = 4) (P < 0.05). Cecal score was positively correlated with colonic MPO activity (r = 0.72; P < 0.05) and negatively correlated with plasma CA (r = -0.57; P < 0.05). These studies indicated that the anticolitic efficacy of CA was related to variability in CA bioavailability, which may be influenced by gut microbial metabolism of this compound.