Angeles Fernandez-Arche

Supressive effect of maslinic acid from pomace olive oil on oxidative stress and cytokine production in stimulated murine macrophages.

The pentacyclic triterpene maslinic acid (MA) is a natural compound present in the non glyceride fraction of pomace olive oil, also called orujo olive oil. This compound has previously demonstrated antioxidant properties against lipid peroxidation in vitro, but its effects on reactive oxygen and nitrogen-derived species and pro-inflammatory cytokines generated by a cell system have not yet been investigated. In this study, we have tested the effect of MA upon oxidative stress and cytokine production using peritoneal murine macrophages. MA significantly inhibited the enhanced production of nitric oxide (NO) induced by lypopolysaccharide (LPS) when it was measured by the nitrite production with an inhibitory concentration 50% value (IC50) of 25.4 μM. This inhibiting effect seems to be consequence of an action at the level of the LPS-induction of the inducible nitric oxide synthethase (iNOS) gene enzyme expression rather than to a direct inhibitory action on enzyme activity. The secretion of the inflammatory cytokines interleukine-6 and TNF-α from LPS-stimulated murine macrophages was also significantly reduced (p < 0.05 and 0.01) by 50 and 100 μM of MA. In addition, reactive oxygen species were measured after stimulation with phorbol-12-myristate-13-acetate (PMA). Thus, pre-treatment with MA reduced the generation of hydrogen peroxide from stimulated macrophages in a dose-dependent manner (IC50: 43.6 μM) as assayed by the oxidation of the peroxidase enzyme. However, no inhibitory effect on superoxide release, measured by the reduction of ferricytochrome c, was observed after the pretreatment with MA in the culture medium. These results suggest a potential biopharmaceutical use of this hydroxy-pentacyclic triterpene derivative, present in orujo olive oil, on preventing oxidative stress and pro-inflammatory cytokine generation.

Long-chain fatty alcohols from pomace olive oil modulate the release of proinflammatory mediators

Pomace olive oil is a by-product of olive oil extraction that is traditionally produced and consumed in Spain. The nonglyceride matter of this oil is a good source of interesting minor compounds, like long-chain fatty alcohols, which are present free or as part of waxes. In the present study, long-chain fatty alcohols were isolated from the nonglyceride fraction of pomace olive oil, and the composition was identified and quantified. The major components of long-chain fatty alcohols were tetracosanol, hexacosanol and octacosanol. We investigated the ability of long-chain fatty alcohols from pomace olive oil to inhibit the release of different proinflammatory mediators in vitro by cells involved in inflammatory processes. Long-chain fatty alcohols significantly and dose-dependently decreased nitric oxide production by RAW 264.7 murine macrophages stimulated with lipopolysaccharide. Western blot analysis showed that nitric oxide reduction was a consequence of the inhibition of inducible nitric oxide synthetase expression. Long-chain fatty alcohols also reduced tumor necrosis factor-alpha and prostaglandin E(2) production, although the potency of inhibition for the latter was lower. On the other hand, long-chain fatty alcohols significantly reduced thromboxane A(2) production in rat peritoneal neutrophils stimulated with the calcium ionophore A-23187. The reduction of eicosanoid release was related to the inhibition of phospholipase A(2) enzyme activity by long-chain fatty alcohols, reaching an inhibitory concentration 50% value of 6.2 microg/ml. These results showed that long-chain fatty alcohols may have a protective effect on some mediators involved in the inflammatory damage development, suggesting its potential value as a putative functional component of pomace olive oil.

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.

The sterols isolated from Evening Primrose oil modulate the release of proinflammatory mediators

Evening Primrose oil is a natural product extracted by cold-pressed from Oenothera biennis L. seeds. The unsaponifiable matter of this oil is an important source of interesting minor compounds, like long-chain fatty alcohols, sterols and tocopherols. In the present study, sterols were isolated from the unsaponifiable matter of Evening Primrose oil, and the composition was identified and quantified by GC and GC-MS. The major components of sterols fraction were β-Sitosterol and campesterol. We investigated the ability of sterols from Evening Primrose oil to inhibit the release of different proinflammatory mediators in vitro by murine peritoneal macrophages stimulated with lipopolysaccharide. Sterols significantly and dose-dependently decreased nitric oxide production. Western blot analysis showed that nitric oxide reduction was a consequence of the inhibition of inducible nitric oxide synthetase expression. Sterols also reduced tumor necrosis factor-α, interleukine 1β and tromboxane B₂. However, sterols did not reduce prostaglandin E₂. The reduction of eicosanoid release was related to the inhibition of cyclooxygenase-2 expression. These results showed that sterols may have a protective effect on some mediators involved in inflammatory damage development, suggesting its potential value as a putative functional component of Evening Primrose oil.

Topical anti-inflammatory effect of tirucallol, a triterpene isolated from Euphorbia lactea latex

Latex from Euphorbia lactea (Euphorbiaceae), a native Dominican medicinal plant, is claimed to be useful in the treatment of inflammation. Topical application of tirucallol, a tetracyclic triterpene isolated from Euphorbia lacteal latex, suppressed ear edema in the mouse model in a dose-dependent manner, as well as affecting the influx of polymorphonuclear cells in response to topical application of 12-O-tetradecanoylphorbol-acetate (TPA) in the mouse ear. In addition, the effect of tirucallol, on some macrophage functions was analyzed in vitro. Non-toxic concentrations of tirucallol potently inhibited nitrite production in lipopolysaccharide-stimulated macrophages. Western blot analysis showed that nitric oxide reduction was a consequence of the inhibition of inducible nitric oxide synthetase expression although tirucallol slightly affected to prostaglandin E(2) (PGE(2)) generation. The results of the study revealed that tirucallol (0.3%), present in Euphorbia lactea latex, exerts a topical anti-inflammatory effect in vivo, via a mechanism of action related to the neutrophil migration. On the other hand, it can be deduced that the mechanism of the anti-inflammatory activity of this triterpene is related to the control of the production of NO and its effect on the expression of iNOS.

Validation and additional support for an experimental animal model of fibromyalgia.

Abstract Objectives. In the current study, we have evaluated the intermittent cold stress (ICS) induction in mice, in order to validate and optimize its utility as a fibromyalgia-like model. Methods. Twenty-four mice of 5-week old, female Swiss, weighing 18–22 g were used for the experiments. These mice were divided into three groups of eight animals per group [health control group (control), ICS group (ICS), and Gabapentin group (GBP)]. When in-vivo tests were completed, we proceeded to isolation and culture of peritoneal macrophages in order to determine the effects of the ICS on the release of proinflammatory mediators. Results. The results showed that this model is suitable to induce mechanical allodynia, thermal allodynia, and hyperalgesia. It is also able to reproduce behavioral changes related to cognitive disturbances, anxiety, and depression. Besides, ICS model might increase the inflammatory response in LPS-macrophages stimulated from stressed mice. Conclusions. Our results show that ICS is a useful animal model to assess hypothesis about underlying mechanisms involved in the development of fibromyalgia as well as to evaluate possible future therapies.

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.