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Effects of virgin olive oil phenolics on scavenging of reactive nitrogen species and upon nitrergic neurotransmission.

The major phenolics from the polar fraction of virgin olive oil (caffeic acid, oleuropein, tyrosol and hydroxytyrosol) have well-established antioxidant activities but their effects on reactive nitrogen species and nitrergic neurotransmission have not been fully investigated. The three catechol compounds were active as scavengers of nitric oxide generated spontaneously from the decomposition of sodium nitroprusside (approximately 50% inhibition achieved at 75 microM), and had similar ability to scavenge chemically generated peroxynitrite, as determined by an alpha1-antiproteinase inactivation assay (67.2%-92.4% reduction when added at 1 mM). Tyrosol was less active in these tests, but does not possess the catechol functionality. Despite their ability to interact with chemically prepared nitric oxide, neither oleuropein nor hydroxytyrosol at 5 microM altered NO*-mediated relaxations of the nerve-stimulated rat anococcygeus preparation, but this may be because the nitrergic transmitter is protected from the effects of externally applied scavengers. In conclusion, the phenolics found in virgin olive oil possess ability to scavenge reactive oxygen and nitrogen species that are implicated in human pathologies, but their impact may be restricted to those species present in the extracellular environment.

Effect of minor components of virgin olive oil on topical antiinflammatory assays.

Abstract Interest in the health-promoting effects of virgin olive oil, an important part of the “Mediterranean diet”, prompted us to determine the antiinflammatory effects of erythrodiol, β-sitosterol and squalene, identified as major components of the so-called “unsaponifiable fraction” of virgin olive oil, as well as of the phenolic compounds from the “polar fraction”: oleuropein, tyrosol, hydroxytyrosol and caffeic acid. Their activities were compared to those of both, total unsaponifiable and polar fractions. This study was designed to analyse the antiinflammatory effect of these specific compounds from virgin olive oil on edema in mice induced by either arachidonic acid (AA ) or 12-O-tetradecanoylphorbol acetate (TPA). The inhibition of the myeloperoxidase (MPO), marker enzyme of the accumulation of neutrophils in the inflamed tissue, was also investigated by the TPA model. The topical application of the olive oil compounds (0.5 mg/ear) produced a variable degree of antiinflammatory effect with both assays. In the auricular edema induced by TPA, β-sitosterol and erythrodiol from the unsaponifiable fraction of the oil showed a potent antiedematous effect with a 61.4% and 82.1% of inhibition respectively, values not very different to that of the reference indomethacin (85.6%) at 0.5 mg/ear. The four phenolics exerted a similar range of inhibition (33- 45%). All compounds strongly inhibited the enzyme myeloperoxidase, indicating a reduction of the neutrophil influx in the inflamed tissues. The strongest inhibitor of AA edema was the total unsaponifiable fraction which inhibition was 34%, similar to that obtained by the reference drug dexamethasone at 0.05 mg/ear. Among the phenolics, oleuropein also produced an inhibition of about 30% with the same dose, but all the other components were found less active in this assay. The anti-inflammatory effects exerted by both unsaponifiable and polar compounds might contribute to the potential biological properties reported for virgin olive oil against different pathological processess.

Cytotoxic effect of the pentacyclic oxindole alkaloid mitraphylline isolated from uncaria tomentosa bark on human ewing's sarcoma and breast cancer cell lines

Preparations from Uncaria tomentosa, a South American Rubiaceae, have been used in the Peruvian traditional medicine for the treatment of infective, inflammatory and tumoral processes. In this study, the pentacyclic oxindole alkaloid mitraphylline was isolated from the dried inner bark of this plant species, and its structure elucidated by analysis of NMR spectroscopic data. Mitraphylline was differentially identified from its stereoisomeric pair isomitraphylline by (15)N-NMR. Its antiproliferative and cytotoxic effects have been tested on human Ewings sarcoma MHH-ES-1 and breast cancer MT-3 cell lines, using cyclophosphamide and vincristine as reference controls. A Coulter counter was used to determine viable cell numbers, followed by the application of the tetrazolium compound MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy phenyl)-2-(4-sulfophenyl)-2H-tetrazolium] an inner salt. A colorimetric method was employed to evaluate cell viability in this cytotoxic assay. Micromolar concentrations of mitraphylline (5 microM to 40 microM) inhibited the growth of both cell lines in a dose-dependent manner. The IC (50) +/- SE values were 17.15 +/- 0.82 microM for MHH-ES-1 and 11.80 +/- 1.03 microM for MT-3 for 30 hours, smaller than those obtained for the reference compounds. This action suggests that the pentacyclic oxindole alkaloid mitraphylline might be a new promising agent in the treatment of both human sarcoma and breast cancer.

The effect of tyrosol, hydroxytyrosol and oleuropein on the non-enzymatic lipid peroxidation of rat liver microsomes

The effects of the polyphenolic compounds from virgin olive oil: tyrosol, hydroxytyrosol and oleuropein on the non-enzymatic lipid peroxidation induced by ascorbate-Fe2+ of rat liver microsomes were examined. The inhibition of light emission (maximal induced chemiluminescence) by oleuropein was concentration dependent. Hydroxytyrosol showed a substantial degree of inhibition against ascorbate-Fe2+ induced lipid peroxidation in rat liver microsomes that was at least 6 times higher than that observed in the presence of oleuropein. Inhibition of lipid peroxidation by tyrosol was not observed. In rat liver microsomes incubated alone or in the presence of tyrosol, the fatty acid composition was profoundly modified when subjected to in vitro peroxidation mediated by ascorbate-Fe2+, with a considerable decrease of 18:2n6 and 20:4n6; however, changes in fatty acid composition were not observed when microsomes were incubated with hydroxytyrosol. When oleuropein was used at low concentration (5, 15 μM) a considerable decrease of 20:4n6 was observed, but 18:2n6 was not modified; at higher concentration (30, 60 μM) changes in fatty acid composition were not observed. There was a very good correlation between the presence of oxidized phospholipids and the changes in polyunsaturated fatty acids previously observed. Thus, hydroxytyrosol showed the highest protection again oxidized phospholipid formation. The presence of oleuropein at low concentration (5, 15 μM) does not prevent the formation of oxidized phospholipids (8.02 ± 1.22 and 1.22 ± 1.22) but concentration higher than 30 μM avoids completely the formation of this molecules whereas tyrosol at any concentration assayed was found to be ineffective and allows the formation not only of oxidized phospholipids but also of oxidized cholesterol.

Influence of dietary fat on oxidative stress and inflammation in murine macrophages.

OBJECTIVE Many studies have shown that the nature of the lipid consumed in the diet significantly affects the development of inflammatory diseases. In this study, we compared the effect of diets supplemented with 15% by weight of fish oil (FO), refined olive oil (ROO), and pomace olive oil (POO) with that of a low-fat diet, 2% by weight of corn oil, considered as the basal diet (BD), on the ability to modify reactive oxidative species and proinflammatory mediator generation by stimulated murine macrophages. METHODS Mice were fed the different oil-enriched diets for 8 wk. Peritoneal macrophages were isolated from these mice and subsequently stimulated. Reactive oxygen species and proinflammatory mediators were measured in the corresponding supernatants. Data were statistically treated by one-way analysis of variance and Tukeys multiple comparison post hoc test. RESULTS The ROO and POO significantly reduced the hydrogen peroxide production compared with BD, whereas FO stimulated its production. Moreover, the generation of nitric oxide was significantly prevented in all the experimental oil-enriched dietary groups. The ROO and FO groups showed significantly reduced cytokine (tumor necrosis factor-alpha, interleukin-1beta, interleukin-6) and prostaglandin E(2) production. CONCLUSION These results confirm the prevention action on proinflammatory mediator generation exerted by FO and demonstrate the protective antioxidant properties not only of olive oil but also of POO. The consumption of these olive oils may help to prevent cellular oxidative stress and inflammation.

Sucrose esters from Physalis peruviana calyces with anti-inflammatory activity.

Physalis peruviana is a native plant from the South American Andes and is widely used in traditional Colombian medicine of as an anti-inflammatory medicinal plant, specifically the leaves, calyces, and small stems in poultice form. Previous studies performed by our group on P. peruviana calyces showed potent anti-inflammatory activity in an enriched fraction obtained from an ether total extract. The objective of the present study was to obtain and elucidate the active compounds from this fraction and evaluate their anti-inflammatory activity in vivo and in vitro. The enriched fraction of P. peruviana was purified by several chromatographic methods to obtain an inseparable mixture of two new sucrose esters named peruviose A (1) and peruviose B (2). Structures of the new compounds were elucidated using spectroscopic methods and chemical transformations. The anti-inflammatory activity of the peruvioses mixture was evaluated using λ-carrageenan-induced paw edema in rats and lipopolysaccharide-activated peritoneal macrophages. Results showed that the peruvioses did not produce side effects on the liver and kidneys and significantly attenuated the inflammation induced by λ-carrageenan in a dosage-dependent manner, probably due to an inhibition of nitric oxide and prostaglandin E2, which was demonstrated in vitro. To our knowledge, this is the first report of the presence of sucrose esters in P. peruviana that showed a potent anti-inflammatory effect. These results suggest the potential of sucrose esters from the Physalis genus as a novel natural alternative to treat inflammatory diseases.

Bioactive Constituents from “Triguero” Asparagus Improve the Plasma Lipid Profile and Liver Antioxidant Status in Hypercholesterolemic Rats

We have previously shown that the Andalusian-cultivated Asparagus officinalis L. “triguero” variety produces hypocholesterolemic and hepatoprotective effects on rats. This asparagus is a rich source of phytochemicals although we hypothesized there would be some of them more involved in these functional properties. Thus, we aimed to study the effects of asparagus (500 mg/kg body weight (bw)/day) and their partially purified fractions in flavonoids (50 mg/kg bw/day), saponins (5 mg/kg bw/day) and dietary fiber (500 mg/kg bw/day) on oxidative status and on lipid profile in rats fed a cholesterol-rich diet. After 5 weeks treatment, plasma lipid values, hepatic enzyme activities and liver malondialdehyde (MDA) concentrations were measured. With the exception of the saponin fraction (SF), the administration of lyophilized asparagus (LA), fiber fraction (FF), and flavonoid fraction (FVF) to hypercholesterolemic rats produced a significant hypolipidemic effect compare to a high-cholesterol diet (HCD). In addition, the LA and FVF groups exhibited a significant increase in enzyme activity from multiple hepatic antioxidant systems including: superoxide dismutase, catalase, and gluthatione reductase/peroxidase as well as a decrease in MDA concentrations compared to HCD group. These results demonstrate that “triguero” asparagus possesses bioactive constituents, especially dietary fiber and flavonoids, that improve the plasma lipid profile and prevent hepatic oxidative damage under conditions of hypercholesterolemia.

Mitraphylline inhibits lipopolysaccharide-mediated activation of primary human neutrophils

BACKGROUND Mitraphylline (MTP) is the major pentacyclic oxindolic alkaloid presented in Uncaria tomentosa. It has traditionally been used to treat disorders including arthritis, heart disease, cancer, and other inflammatory diseases. However, the specific role of MTP is still not clear, with more comprehensivestudies, our understanding of this ancient herbal medicine will continue growing. HYPOTHESIS/PURPOSE Some studies provided its ability to inhibit proinflamatory cytokines, such as TNF-α, through NF-κB-dependent mechanism. TNF-α primes neutrophils and modulates phagocytic and oxidative burst activities in inflammatory processes. Since, neutrophils represent the most abundant pool of leukocytes in human blood and play a crucial role in inflammation, we aimed to determine the ability of MTP to modulate neutrophil activation and differentially regulate inflammatory-related cytokines. METHODS To determine the mechanism of action of MTP, we investigated the effects on LPS-activated human primary neutrophils responses including activation surface markers by FACS and the expression of inflammatory cytokines, measured by real time PCR and ELISA. RESULTS Treatment with MTP reduced the LPS-dependent activation effects. Activated neutrophils (CD16(+)CD62L(-)) diminished after MTP administration. Moreover, proinflamatory cytokines (TNF-α, IL-6 or IL-8) expression and secretion were concomitantly reduced, similar to basal control conditions. CONCLUSION Taken together, our results demonstrate that MTP is able to elicit an anti-inflammatory response that modulates neutrophil activation contributing to the attenuation of inflammatory episodes. Further studies are need to characterize the mechanism by which MTP can affect this pathway that could provide a means to develop MTP as new candidate for inflammatory disease therapies.