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 (IC(50)) of 25.4 microM. 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-a from LPS-stimulated murine macrophages was also significantly reduced (p < 0.05 and 0.01) by 50 and 100 microM 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 (IC(50): 43.6 microM) 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.

Many studies have shown that the nature of the lipid consumed in the diet significantly affects the prevalence of coronary, inflammatory or autoimmune diseases (1,2) . The present study was conducted to compare the impact of feeding mice on diets enriched with edible oils (150 g/kg diet; fish oil (FO), olive oil (OVO) and orujo olive oil (ORO)) with that of a basal diet (BD) enriched with 20 g maize oil/kg on the ability to modulate oxidative reactive species and pro-inflammatory mediator generation by stimulated murine macrophages. Swiss male mice were fed on the different diets for 8 weeks. Diets were formulated according to American Institute of Nutrition (AIN) recommendations (3) . Peritoneal macrophages were isolated from these mice and stimulated. Reactive oxygen (O 2 -and H 2 O 2 ) and nitrogen (NO 2 -) species, PGE 2 , TNFa and IL-6 were measured in the supernatant fractions from 10 6 cells. All test diets down regulated NO generation compared with the BD (Fig.1); in contrast, FO increased H 2 O 2 generation whereas OVO and ORO group diets significantly inhibited this ROS production compared with the BD. In addition, ORO was able to decrease O 2 -formation compared with the BD group (Fig.1). Finally, both OVO and FO groups significantly decreased PGE 2 and cytokine production (Fig. 2). These results are in agreement with those of other authors in that a diet enriched in olive oil was found to show a protective effect against oxidative stress and inflammation (4,5) and they confirm the preventive anti-inflammatory properties of FO (6) . Moreover, the results provide important additional data about the ability of ORO to prevent oxidative damage to cells.

Olive pomace oil ('orujo' oil) is an olive oil product suitable for human consumption that is traditionally produced in Spain (1) . The nonacylglycerol component of this oil is a good source of interesting minor components, e.g. triterpenes (2) , or fatty alcohols, derived from waxy materials. Tetracosanol (C 24 OH; 30 %), hexacosanol (C 26 OH; 37 %) and octacosanol (C 28 OH; 15 %) are the major constituents of the long-chain fatty alcohol (LCFA) fraction isolated from orujo olive oil (3) . A similar mixture of long-chain alcohols, termed 'policosanol' and purified from waxy materials of different sources such as sugar cane, bees wax, rice bran or spinach, have shown many beneficial physiological activities (4,5) . The present study focused on the effect of LCFA isolated from orujo olive oil on NO, PGE 2 and TNFa release by a lipopolysaccharide (LPS)-stimulated murine macrophage cell line (RAW-264.7) as well as the effect on thromboxane B 2 (TXB 2 ) generation by A-23187-stimulated rat peritoneal neutrophils (PMN). Nitrite (as an index of NO generation) levels were determined by a fluorometric method. PGE 2 , TNFa and TXB 2 production were quantified by sandwich immunoassay.LCFA significantly and dose-dependently decreased the NO production in LPS-stimulated RAW-264.7 cell line macrophages (Fig. 1). Western-blot analysis for inducible NO synthase (iNOS) showed that NO reduction was a consequence of the 100% inhibition of iNOS expression at a dose of 100 mg/ml (Fig. 2). By contrast, LCFA scarcely affected PGE 2 levels ( Fig.