Duangdeun Meksuriyen

Effect of nicotine and polyaromtic hydrocarbons on cerebral endothelial cells

The present study was designed to investigate the effect of nicotine and polyaromatic hydrocarbon compounds on cerebral endothelial cells (CECs). Nicotine treatments from 15 min to 5 h did not cause any changes in the expression and localization of principal junctional proteins. One day of treatment with a relatively high concentration of nicotine induced a decrease in the expression of the tight junction protein ZO‐1, occludin, and the adherens junction protein, cadherin. Treatment with 3 × 10−5 M phenanthrene for 24 h caused a redistribution of occludin from the Triton X‐100 insoluble to the Triton X‐100 soluble fraction. Transendothelial electrical resistance was not significantly affected by 24 h treatments with nicotine, methylanthracene or phenanthrene. However, 24 h nicotine treatment increased transendothelial permeability in CECs exposed to oxidative stress. Both nicotine and phenanthrene were able to regulate the expression of a large number of proteins as revealed by 2D electrophoresis. Our experiments suggest that tobacco smoking may affect the junctional complex of CECs, and that this effect is enhanced by oxidative stress.

Cissus quadrangularis ethanol extract upregulates superoxide dismutase, glutathione peroxidase and endothelial nitric oxide synthase expression in hydrogen peroxide-injured human ECV304 cells

ETHNOPHARMACOLOGICAL RELEVANCE Cissus quadrangularis has been widely used in traditional medicine for the treatment of hemorrhoid. However, the detailed mechanism of antioxidant defense of C. quadrangularis in endothelial cells under oxidative stress remains unclear. AIM OF THE STUDY The present study aims to elucidate the protective role of ethanol extract of C. quadrangularis (CQE) including its constituents, quercetin and resveratrol, on hydrogen peroxide (H(2)O(2))-injured human umbilical vein endothelial ECV304 cells. MATERIALS AND METHODS Viability, genotoxicity and protein expression of ECV304 cells were analyzed by MTT, alkaline comet and Western blot, respectively. Production of intracellular reactive oxygen species (ROS) was determined using dichlorofluorescein fluorescence dye. RESULTS After exposing cells to CQE containing quercetin and resveratrol, DNA damage was not observed. CQE including quercetin and resveratrol significantly attenuated ROS in H(2)O(2)-injured ECV304 cells in a concentration-dependent manner. The protein expression of superoxide dismutase (Cu/Zn-SOD, Mn-SOD), glutathione peroxidase (GPx) and endothelial nitric oxide synthase (eNOS) increased in the cells treated with CQE, quercetin or resveratrol prior to H(2)O(2) exposure, as compared with control. CONCLUSIONS The results provide a molecular mechanism of C. quadrangularis, which could be partially related to quercetin and resveratrol, in restoring ROS in endothelial cells through the upregulation of Cu/Zn-SOD, Mn-SOD, GPx and eNOS.

Canarosine: A new guanidine alkaloid from Canavalia rosea with inhibitory activity on dopamine D1 receptors

A new acyclic guanidine alkaloid, canarosine (1), together with five known compounds, β-sitosterol (2), stigmasterol (3), daucosterol (4), epi-inositol 6-O-methyl ether (5), and rutin (6), were isolated from the aerial parts of Canavalia rosea. Their structures were established on the basis of their spectroscopic data. In the radioligand receptor binding assay, canarosine (1), at a concentration of 100 μg/ml, caused 91% inhibition of the dopamine D1 receptor binding with an IC50 value of 39.4 ± 5.8 μM.

Tamarind seed coat extract restores reactive oxygen species through attenuation of glutathione level and antioxidant enzyme expression in human skin fibroblasts in response to oxidative stress

OBJECTIVE To investigate the role and mechanism of tamarind seed coat extract (TSCE) on normal human skin fibroblast CCD-1064Sk cells under normal and oxidative stress conditions induced by hydrogen peroxide (H2O2). METHODS Tamarind seed coats were extracted with boiling water and then partitioned with ethyl acetate before the cell analysis. Effect of TSCE on intracellular reactive oxygen species (ROS), glutathione (GSH) level, antioxidant enzymes such as superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase activity including antioxidant protein expression was investigated. RESULTS TSCE significantly attenuated intracellular ROS in the absence and presence of H2O2 by increasing GSH level. In the absence of H2O2, TSCE significantly enhanced SOD and catalase activity but did not affected on GPx. Meanwhile, TSCE significantly increased the protein expression of SOD and GPx in H2O2-treated cells. CONCLUSIONS TSCE exhibited antioxidant activities by scavenging ROS, attenuating GSH level that could protect human skin fibroblast cells from oxidative stress. Our results highlight the antioxidant mechanism of tamarind seed coat through an antioxidant enzyme system, the extract potentially benefits for health food and cosmeceutical application of tamarind seed coat.

Barakol-induced apoptosis in P19 cells through generation of reactive oxygen species and activation of caspase-9.

ETHNOPHARMACOLOGICAL RELEVANCE Barakol, an anxiolytic agent isolated from Senna siamea leaves which has been traditionally used for producing natural sleep, has been described as toxic to patients. AIM OF THE STUDY The aim of current study was to investigate the molecular mechanism of barakol-induced toxicity in mouse embryonal carcinoma P19 cell model. MATERIALS AND METHODS XTT assay was used to determine cell viability in P19 cells treated with barakol. Apoptotic cells were detected by Hoechst 33342 staining. Intracellular reactive oxygen species (ROS) generation was analyzed by flow cytometry using a fluorescent dye, DCFH-DA. Detection of apoptotic protein expression in P19 cells was performed by Western blot analysis. Caspase-9 activity was measured using a fluorescent immunosorbent enzyme assay kit. RESULTS Treatment with barakol decreased cell viability in a concentration- and time-dependent manner with an IC(50) value of 1.5mM in 24-h treated cells. A Hoechst 33342 assay revealed that barakol cytotoxicity was due to a significant increase in the number of apoptotic cells. Different scavengers to characterize ROS were utilized and revealed that hydroxyl radicals played a major role in ROS-induced apoptosis in barakol-treated cells. Western blot analysis demonstrated that barakol-induced apoptosis was mediated by the increase in expression ratio of Bax/Bcl-2. Furthermore, increase in caspase-9 activity after exposure to barakol for 24h was also observed. Pretreatment of cells with N-acetyl-l-cysteine (NAC) attenuated intracellular ROS generation, the Bax/Bcl-2 protein expression, and apoptosis. CONCLUSIONS The mechanism of barakol-mediated toxicity in P19 cells is mainly associated with the ROS generation, followed by the imbalance of the Bax/Bcl-2 ratio, and caspase-9 activation leading to apoptotic cell death. Pretreatment of cells with NAC could antagonize the toxicity produced by barakol.

Synergistic antioxidant action of Phikud Navakot ameliorates hydrogen peroxide-induced stress in human endothelial cells

Background Phikud Navakot (PN), a combination of nine herbs, has been used traditionally in Thai medicinal formulas to relieve circulatory disorder. The present study aimed to compare the synergistic antioxidant efficacy and toxicity of the hydroethanolic and water extracts of PN at cellular level. Methods PN and its nine herbs were extracted with either 50% ethanol or water. All extracts were tested for in vitro antioxidant potential using standard antioxidant assays. Evaluation of cytotoxicity, genotoxicity, and intracellular reactive oxygen species were performed using human endothelial ECV304 cells. Results Antioxidant assays in cell-free systems showed that the hydroethanolic extract of PN scavenged superoxide, hydroxyl, nitric oxide radicals, and hydrogen peroxide more effectively than its water extract. Combination indices were calculated to show that the ingredients of the hydroethanolic extract acted synergistically to exhibit antioxidant activities against all tested radicals, whereas, in the case of water extract, this effect was observed only against 2,2-diphenyl-1-picrylhydrazyl, superoxide, and hydroxyl radicals. A cell-based assay also revealed that the hydroethanolic extract concentration-dependently attenuated hydrogen peroxide-induced stress more effectively than the water extract. At the antioxidant and cytotoxic concentrations of both extracts, no genotoxicity was found. Conclusion Our findings demonstrate that the synergistic antioxidant action of PN ameliorates endothelial stress, which may provide some clues for understanding the traditional use of PN for the treatment of circulatory disorder. Additionally, the selection of a suitable solvent for the extraction of PN herbal combination is essential for maximal efficacy and safety.

Azole-synergistic anti-candidal activity of altenusin, a biphenyl metabolite of the endophytic fungus Alternaria alternata isolated from Terminalia chebula Retz.

In this study, a tropical endophytic fungus, Alternaria alternata Tche-153 was isolated from a Thai medicinal plant Terminalia chebula Rezt. The ethyl acetate extract prepared from the fermentation broth exhibited significant ketoconazole-synergistic activity against Candida albicans. Bioassay-directed fractionation of the ethyl acetate extract led to the isolation of altenusin (1), isoochracinic acid (2), and altenuic acid (3) together with 2,5-dimethyl-7-hydroxychromone (4). Using the disc diffusion method and the microdilution chequerboard technique, only altenusin (1) in combination with each of three azole drugs, ketoconazole, fluconazole or itraconazole at their low sub-inhibitory concentrations exhibited potent synergistic activity against C. albicans with the fractional inhibitory concentration index range of 0.078 to 0.188. This first discovery of altenusin (1) as a new azole-synergistic prototype possessing a biphenyl structure is of significance for further development of new azole-synergists to treat invasive candidiasis.

Stability of Barakol under Hydrolytic Stress Conditions and its Major Degradation Product

The aim of the present study was to investigate the stability of barakol, an anxiolytic constituent extracted from leaves of Senna siamea (Lam.) Irwin & Barneby (syn. Cassia siamea Lam.), under the International Conference on Harmonisation suggested conditions using HPLC with photodiode array detection. Extensive degradation of barakol was found to occur under alkaline conditions through base-catalyzed hydrolysis. Mild degradation of barakol was observed under thermal and oxidative stress while it was stable under acidic conditions. The reaction rate constants (Kobs) of barakol degradation under alkaline conditions at pHs 12 and 13 were 3.0x10(-5) and 9.6x10(-3) min(-1), respectively. The activation energy according to the Arrhenius plot was calculated to be 26.9+/-3.3 kcal/mol at pH 13 and temperatures between 12 and 51 degrees C. The major degradation product of barakol under both alkaline and thermal stress conditions was characterized by LC-MS and NMR as cassiachromone.