Chronic toxicity study of neosaxitoxin in rats.

Neosaxitoxin (NeoSTX) is a specific reversible blocker of voltage gated sodium channels on excitable cells. In the last decade, it has been tested in a number of interesting clinical trials, however there is still little information available on mammalian toxicity. Rats were treated for 12 weeks with doses of 1, 3 or 6 µg/kg of subcutaneous NeoSTX. At weeks 12 and 17, animals were sacrificed and blood samples collected for hematological and biochemical analysis. Organs were harvested for weight determination and histopathological assessments. The lowest acute toxicity via the intraperitoneal (ip) route was (30.35 µg/kg) and there was no significant difference between intramuscular and subcutaneous routes (11.4 and 12.41 µg/kg). The NeoSTX adiministration did not produce lethality at week 12 and after five weeks of suspension. NeoSTX 6 µg/kg ip produced reductions (p < 0.05) in body weight and food intake, and increased blood level of total and direct bilirubin, GGT and SGOT at week 12; all of these were reversed in the recovery period. NeoSTX 1 and 3 µg/kg ip did not show significant changes with the control group. Histopathological presentations were normal in all groups. This study revealed that NeoSTX is safe in vivo, giving a reliable security margin for its use like a local anesthetic.

Honey flavonoids inhibit Candida albicans morphogenesis by affecting DNA behavior and mitochondrial function.

AIM: Candida albicans is a pathogenic yeast, which forms a range of polarized and expanded cell shapes. We aimed to determine the correlation between honey extract (HFE) activity and changes in C. albicans cell cycle, morphology and subcellular organelles. MATERIALS & METHODS: HFE anticandidal properties were investigated using flow cytometry and scanning electron microscopy. RESULTS: Flow cytometry and scanning electron microscopy analyses indicated that HFE may inhibit the growth of the three phenotypes displayed by C. albicans and reduce infection by affecting membrane integrity. HFE affects hyphal transition by reducing the G0/G1 phase and increasing the G2/M phase. Conversely, yeast and pseudohyphae do not appear to be affected. Modifications of vacuolization and mitochondrial activity, during yeast-hypha transition establish the involvement of vacuole and mitochondria. CONCLUSION: HFE improved mitochondrial functionality and reduced the vacuolization, modifying the branching process associated with virulence. It is hypothesized that HFE induces changes in cell cycle progress, membrane integrity, mitochondrial function and biogenesis.

Rice bran enzymatic extract-supplemented diets modulate adipose tissue inflammation markers in Zucker rats.

OBJECTIVE: Chronic low-grade inflammation in obesity is characterized by macrophage accumulation in white adipose tissue and adipokine production deregulation. Obesity also is characterized by oxidative stress related to inflammatory signaling. The aim of this study was to analyze whether dietary supplementation with a rice bran enzymatic extract (RBEE), rich in bioactive compounds with antioxidant and hypocholesterolemic properties, would ameliorate the inflammatory state existing in visceral adipose tissue of obese Zucker rats. METHODS: Obese Zucker rats and their littermate controls, lean Zucker rats ages 8 wk, were daily fed an enriched diet with either 1% or 5% RBEE supplementation over 20 wk. Measurement of adipocyte size and mRNA expression of proinflammatory molecules from visceral abdominal/epididymal tissue was performed. RESULTS: An RBEE-supplemented diet decreased the overproduction of tumor necrosis factor-α, interleukin (IL)-6, IL-1 ß, and inducible nitric oxide synthase (iNOS), as well as the overproduction of IL-6 and iNOs in visceral abdominal adipose tissue and visceral epididymal adipose tissue, respectively. An RBEE-supplemented diet modified the adipocyte-size distribution pattern in both abdominal and epididymal adipose tissue, shifting it toward smaller cell sizes. CONCLUSIONS: Chronic administration of a novel water-soluble RBEE, rich in polyphenols, tocotrienols and γ-oryzanol, could be a suitable treatment to ameliorate the obesity-associated proinflammatory response.

Rice bran enzymatic extract restores endothelial function and vascular contractility in obese rats by reducing vascular inflammation and oxidative stress.

BACKGROUND: Rice bran enzymatic extract (RBEE) used in this study has shown beneficial activities against dyslipidemia, hyperinsulinemia and hypertension. Our aim was to investigate the effects of a diet supplemented with RBEE in vascular impairment developed in obese Zucker rats and to evaluate the main mechanisms mediating this action. METHODS AND RESULTS: Obese Zucker rats were fed a 1% and 5% RBEE-supplemented diet (O1% and O5%). Obese and their lean littermates fed a standard diet were used as controls (OC and LC, respectively). Vascular function was evaluated in aortic rings in organ baths. The role of nitric oxide (NO) was investigated by using NO synthase (NOS) inhibitors. Aortic expression of endothelial NOS (eNOS), inducible NOS (iNOS), tumor necrosis factor (TNF)-α and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunits and superoxide production in arterial wall were determined. Endothelial dysfunction and vascular hyperreactivity to phenylephrine in obese rats were ameliorated by RBEE treatment, particularly with 1% RBEE. Up-regulation of eNOS protein expression in RBEE-treated aortas should contribute to this activity. RBEE attenuated vascular inflammation by reducing aortic iNOS and TNF-α expression. Aortas from RBEE-treated groups showed a significant decrease of superoxide production and down-regulation of NADPH oxidase subunits. CONCLUSION: RBEE treatment restored endothelial function and vascular contractility in obese Zucker rats through a reduction of vascular inflammation and oxidative stress. These results show the nutraceutical potential of RBEE to prevent obesity-related vascular complications.

Anti-apoptotic activity of hydroxytyrosol and hydroxytyrosyl laurate.

Hydroxytyrosol (HyT) is a polyphenol primarily released in olive mill wastewater and in olive oil. In animal and cell model studies, HyT and its metabolites have strong antioxidant and antimicrobial activities, as well as beneficial effects on the cardiovascular system and in several human diseases. Differently, many researchers reported that HyT down-regulates tumor cell viability and cell cycle progression, and induces reactive oxygen species (ROS) production and apoptosis. In this study we have investigated the effects of HyT and the corresponding ester hydroxytyrosyl laurate in U937 cells, a human monocytoid cell line, and in C2C12 myoblasts, a murine proliferating muscle cell model, after apoptotic death induction. Inverted, light and transmission electron microscopy have been utilized to characterize cell death patterns. H2O2, at the concentrations known to induce apoptosis, was utilized as cell death trigger. The results obtained show that laur-HyT has a protective antioxidant effect against H2O2 treatment, greater than HyT, so having a role in the prevention of apoptotic death in normal and tumor cells. These data suggest these compounds as good candidate for novel therapeutic strategies.

Anti-inflammatory activity of a honey flavonoid extract on lipopolysaccharide-activated N13 microglial cells.

Neuroinflammation is an important contributor to pathogenesis of age-related neurodegenerative disorders such as Alzheimer's or Parkinson's disease. Accumulating evidence indicates that inhibition of microglia-mediated neuroinflammation may become a reliable protective strategy for neurodegenerative processes. Flavonoids, widely distributed in the vegetable kingdom and in foods such as honey, have been suggested as novel therapeutic agents for the reduction of the deleterious effects of neuroinflammation. The present study investigated the potential protective effect of a honey flavonoid extract (HFE) on the production of pro-inflammatory mediators by lipopolysaccharide-stimulated N13 microglia. The results show that HFE significantly inhibited the release of pro-inflammatory cytokines such as TNF-α and IL-1ß. The expressions of iNOS and the production of reactive oxygen intermediates (ROS) were also significantly inhibited. Accordingly, the present study demonstrates that HFE is a potent inhibitor of microglial activation and thus a potential preventive-therapeutic agent for neurodegenerative diseases involving neuroinflammation.

Mitochondria accumulate large amounts of quercetin: prevention of mitochondrial damage and release upon oxidation of the extramitochondrial fraction of the flavonoid.

Quercetin uptake in Jurkat cells is extremely rapid and associated with a remarkable accumulation of the flavonoid, dependent on its binding to intracellular components. Cell-associated quercetin is biologically active, quantitatively consumed to promote survival in the presence of reactive species, such as peroxynitrite (ONOO(-)), or reduction of extracellular oxidants via activation of plasma membrane oxidoreductases. In alternative, quercetin is very slowly released upon post-incubation in drug-free medium, an event significantly accelerated by extracellular albumin. Quercetin uptake is also observed in isolated mitochondria, resulting in an enormous accumulation of the flavonoid, consumed under conditions associated with prevention of lipid peroxidation induced by ONOO(-). Interestingly, remarkable quercetin accumulation is also detected in the mitochondria isolated from quercetin-pre-loaded cells, and exposure to either ONOO(-) or extracellular oxidants caused the parallel loss of both the mitochondrial and cytosolic fractions of the flavonoid. In conclusion, Jurkat cells accumulate large amounts of quercetin and even larger amounts of the flavonoid further accumulate in their mitochondria. Intramitochondrial quercetin appears to be functional for prevention of mitochondrial damage as well as for redistribution to the cytosol, when the fraction of the flavonoid therein retained is progressively consumed either by cell-permeant oxidants or by activation of plasma membrane oxidoreductases.