Phytochemical profile, antioxidant, antimicrobial and antipancreatic lipase activities of fermented Camellia japonica L leaf extracts

Purpose: To investigate the probable antioxidant, antimicrobial and  antipancreatic lipase effects of fermented Camellia japonica leaf extracts.Methods: Camellia japonica leaves fermented with Nuruk were extracted using methanol and ethanol. Total phenolic, flavonoid, carotenoid and L-ascorbic acid contents were determined by UV-visible spectrophotometry. The antioxidant activities of these extracts were determined by free radical scavenging, ferrous ion chelating and reducing power assays. Their  antimicrobial properties against Gram-positive Staphylococcus epidermidis and Bacillus subtilis, and Gram-negative Klebsiella pneumonia and Escherichia coli bacteria were evaluated by disc diffusion method. Inhibition of pancreatic lipase was measured based on the hydrolytic reaction of p-nitrophenyl butyrate with pancreatic lipase.Results: The ethanol extracts of fermented Camellia japonica leaves exhibited higher phenolic (32274mg GAE/100 g) and flavonoid (20519 mg RE/100 g) contents with higher superoxide (IC50 = 0.23  mg/mL), hydrogen peroxide (IC50 = 0.28 mg/mL) radical scavenging and ferrous ion chelating (IC50 = 0.21 mg/mL) activities than those of methanol. These ethanol extracts also showed higher antimicrobial activities against all bacterial strains tested with higher inhibitory effects on pancreatic lipase than methanol extracts.Conclusion: The results highlight the possible use of fermented Camellia japonica leaf extracts as a source of antioxidant, antibacterial and antiobesity agents. Ethanol is recommended as solvent for extracting antioxidants, antibacterial and antiobesity agents from this plant.Keywords: Antioxidant activity, Antimicrobial activity, Fermented Camellia japonica extracts, Pancreatic lipase inhibitio

GENOTOXICITY OF N-HYDROXY AND AMINOPHENOL METABOLITES OF 2,6- AND 3,5-DIMETHYLANILINE AT THE HYPOXANTHINEGUANINE PHOSPHORIBOSYLTRANSFERASE LOCUS IN TK6 CELLS

Objective: The objective of this study as to characterize the genotoxicity of reactive metabolites of 2,6-dimethylaniline (2,6-DMA) and 3,5-DMA in the hypoxanthineguanine phosphoribosyltransferase (HPRT) gene of human lymphoblastoid TK6 cells.Methods: Cultures were exposed to N-hydroxylamine and aminophenol metabolites of 2,6- and 3,5-DMA for 1 h in serum-free medium. Cell survival 24 h after exposure was determined by trypan blue exclusion. Cells were then subcultured for 7–10 days to allow to phenotypic expression of HPRT mutants. After the expression period, cells were plated in the presence of 2 μg/ml 6-thioguanine for the selection of HPRT mutants. Plating efficiency was determined and mutant fraction calculated. Electron paramagnetic resonance (EPR) was also used to determine whether 3,5- dimethylaminophenol (DMAP) produced reactive oxygen species (ROS).Results: All of the metabolites tested were cytotoxic to these cells but exhibited a considerable variation in potency. The aminophenol metabolites of 2,6- and 3,5-DMA were considerably more toxic than the corresponding N-hydroxylamines. Furthermore, each metabolite of 3,5-DMA was more toxic than its 2,6-DMA counterpart; N-OH-3,5-DMA and 3,5-DMAP were clearly mutagenic at a level of 50 μM. EPR studies showed intracellular oxidative stress induced under 3,5-DMAP treatment.Conclusions: Our findings suggest that genotoxic responses of 2,6- and 3,5-DMA are mediated through the generation of ROS by hydroxylamine and/ or aminophenol metabolites.Â

METABOLIC ACTIVATION OF 2,6-DIMETHYLANILINE: MUTATIONAL SPECIFICITY IN THE GPT GENE OF AS52 CELLS

Objective: The purpose of the current work was to characterize the mechanisms of cytotoxicity and mutagenesis of a potential human bladder carcinogen 2,6-dimethylaniline (2,6-DMA).Methods: Chinese hamster ovary (CHO) AS52 cells were exposed to either human S9 activated 2,6-DMA for 6 h or its N-hydroxylamine and aminophenol metabolites for 1 h in serum-free medium. Cell survival was determined by trypan blue exclusion 24 h after treatment, and 6-thioguanine-resistant mutants at the xanthine-guanine phosphoribosyl transferase (gpt) gene locus were assessed with doses, of which relative survival is 30% or more. Nested polymerase chain reaction-based deletion analysis was also performed.Results: AS52 cells exhibited a dose-dependent increase in cytotoxicity and mutant fraction on treatment of 2,6-DMA and its metabolites but show a considerable variation in potency with aminophenol metabolites having the highest potency and parent compound at least; at the highest 2,6-dimethylaminophenol dose (10 μM), the mutant fraction in AS52 cells was 8-fold (13.2×10−5) greater than the spontaneous fraction of 1.62×10−5. Total deletion of the gpt gene sequences was found in 1 (4%) spontaneous and 2 (6%) the 6-thioguanine mutants generated by N-hydroxy-2,6-DMA.Conclusions: These findings indicate the mutagenicity of 2,6-DMA at the gpt gene, which is mediated through hydroxylamine and aminophenol metabolites, and contribute to the elucidation of mechanisms through which 2,6-DMA may exert its effects in vivo

Simplified Analytical Method for Optimized Initial Shape Analysis of Self-Anchored Suspension Bridges and Its Verification

A simplified analytical method providing accurate unstrained lengths of all structural elements is proposed to find the optimized initial state of self-anchored suspension bridges under dead loads. For this, equilibrium equations of the main girder and the main cable system are derived and solved by evaluating the self-weights of cable members using unstrained cable lengths and iteratively updating both the horizontal tension component and the vertical profile of the main cable. Furthermore, to demonstrate the validity of the simplified analytical method, the unstrained element length method (ULM) is applied to suspension bridge models based on the unstressed lengths of both cable and frame members calculated from the analytical method. Through numerical examples, it is demonstrated that the proposed analytical method can indeed provide an optimized initial solution by showing that both the simplified method and the nonlinear FE procedure lead to practically identical initial configurations with only localized small bending moment distributions

Modulation of A375 human melanoma cell proliferation and apoptosis by nitric oxide

The present study aimed to assess the effect of NO• on melanoma A375 cell growth and apoptotic cell death. Trypan blue exclusion assay was employed to detect the cytotoxicity induced by controlled steady-state concentrations (given in µM • min) of NO•. The characteristics of the cellular cell cycle and apoptosis in NO•-treated A375 cells were also analyzed by Annexin V/PI and DNA fragmentation assays. Western blotting was applied to detect the expression of apoptosis-related proteins (p53, Bax, Fas, DR5, caspase-3 and -9, and PARP). When exposed to preformed 100% NO• for 8 h reactor system, a cumulative dose of 3360 μM • min reduced the viability by 22% 24 h after treatment and promoted apoptosis, 2.9- and 12.2-folds 24 and 48 h after treatment higher than the argon control, respectively. Cell cycle analysis 48 h after treatment revealed S-phase arrest in cells treated with 3360 μM • min NO•. It was also observed that the expression of p53, DR5, caspase 9 and PARP increased significantly upon NO• treatment. In addition, the present study assessed the inhibitory effects of endogenous NO• on the proliferation of human melanoma cells by employing specific (AMG, 1400W and/or SMTC) and nonspecific (NMA) NO• synthase (NOS) inhibitors resulting in melanoma cell growth inhibition; the highest cytotoxic effect was seen when inducible NOS inhibition by 1 mM 1400W treatment. Collectively, the present data suggest that NO• is involved in a key mechanism limiting melanoma proliferation and apoptosis, which may play in improving the efficacy of melanoma treatment

Cephalometric predictors of long-term stability in the early treatment of class III malocclusion

The aim of this study was to examine the differences in the early craniofacial morphology of Class III malocclusions. Lateral cephalograms of 45 subjects with a Class III malocclusion and an anterior crossbite in the deciduous or mixed dentition were examined before treatment, after treatment, and during the long-term retention stage. The anterior crossbites of all patients were corrected after a series of orthodontic treatments. After a mean follow-up period of 5.7 years, all the subjects were reevaluated and divided into three groups according to the final occlusal status: good, fair, and poor occlusal stability. Twenty cephalometric variables on the pretreatment lateral cephalograms were analyzed by one-way analysis of variance and discriminant analysis to identify the key determinants for discriminating among the three groups. Among the 20 variables, 11 showed statistical significance. Generally, the subjects with a smaller gonial angle and a more hypodivergent skeletal pattern had good prognosis after the early treatment of Class III malocclusion. When the AB to mandibular plane angle and N-perpendicular to point A were selected in discriminant analysis, the AB to mandibular plane angle was the most significant variable. Discriminant analysis showed a relatively high degree of correct classifications of the patients with early Class III malocclusion. In particular, discriminant analysis showed the highest accuracy (93.3%) when predicting a poor prognosis.This study was supported by a grant from the Korea Health 21 R&D Project, Ministry of Health & Welfare, Republic of Korea (03- PJ1-PG1-CH09-0001)

Onion peel extracts ameliorate hyperglycemia and insulin resistance in high fat diet/streptozotocin-induced diabetic rats

<p>Abstract</p> <p>Background</p> <p>Quercetin derivatives in onions have been regarded as the most important flavonoids to improve diabetic status in cells and animal models. The present study was aimed to examine the hypoglycemic and insulin-sensitizing capacity of onion peel extract (OPE) containing high quercetin in high fat diet/streptozotocin-induced diabetic rats and to elucidate the mechanism of its insulin-sensitizing effect.</p> <p>Methods</p> <p>Male Sprague-Dawley rats were fed the AIN-93G diet modified to contain 41.2% fat and intraperitoneally injected with a single dose of streptozotocin (40 mg/kg body weight). One week after injection, the rats with fasting blood glucose levels above 126 mg/dL were randomly divided into 4 groups to treat with high fat diet containing 0 (diabetic control), 0.5, or 1% of OPE or 0.1% quercetin (quercetin equivalent to 1% of OPE) for 8 weeks. To investigate the mechanism for the effects of OPE, we examined biochemical parameters (insulin sensitivity and oxidative stresses) and protein and gene expressions (pro-inflammatory cytokines and receptors).</p> <p>Results</p> <p>Compared to the diabetic control, hypoglycemic and insulin-sensitizing capability of 1% OPE were demonstrated by significant improvement of glucose tolerance as expressed in incremental area under the curve (<it>P </it>= 0.0148). The insulin-sensitizing effect of OPE was further supported by increased glycogen levels in liver and skeletal muscle (<it>P </it>< 0.0001 and <it>P </it>= 0.0089, respectively). Quantitative RT-PCR analysis showed increased expression of insulin receptor (<it>P </it>= 0.0408) and GLUT4 (<it>P </it>= 0.0346) in muscle tissues. The oxidative stress, as assessed by superoxide dismutase activity and malondialdehyde formation, plasma free fatty acids, and hepatic protein expressions of IL-6 were significantly reduced by 1% OPE administration (<it>P </it>= 0.0393, 0.0237, 0.0148 and 0.0025, respectively).</p> <p>Conclusion</p> <p>OPE might improve glucose response and insulin resistance associated with type 2 diabetes by alleviating metabolic dysregulation of free fatty acids, suppressing oxidative stress, up-regulating glucose uptake at peripheral tissues, and/or down-regulating inflammatory gene expression in liver. Moreover, in most cases, OPE showed greater potency than pure quercetin equivalent. These findings provide a basis for the use of onion peel to improve insulin insensitivity in type 2 diabetes.</p