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.     

Inducible nitric-oxide synthase attenuates vasopressin-dependent Ca2+ signaling in rat hepatocytes

Increases in both Ca(2+) and nitric oxide levels are vital for a variety of cellular processes; however, the interaction between these two crucial messengers is not fully understood. Here, we demonstrate that expression of inducible nitric-oxide synthase in hepatocytes, in response to inflammatory mediators, dramatically attenuates Ca(2+) signaling by the inositol 1,4,5-trisphosphate-forming hormone, vasopressin. The inhibitory effects of induction were reversed by nitric oxide inhibitors and mimicked by prolonged cyclic GMP elevation. Induction was without effect on Ca(2+) signals in response to AlF(4)(-) or inositol 1,4,5-trisphosphate, indicating that phospholipase C activation and release of Ca(2+) from inositol 1,4,5-trisphosphate-sensitive Ca(2+) stores were not targets for nitric oxide inhibition. Vasopressin receptor levels, however, were dramatically reduced in induced cultures. Our data provide a possible mechanism for hepatocyte dysfunction during chronic inflammation.     

Components of Unsaponifiable Matter of Rice Bran Oil

Components of the unsaponifiable matter of crude rice bran oil were examined. A new compound having melting point of 157~158°C was obtained. Molecular weight was determined and ultraviolet and infrared analyses were carried out. Squalene and ferulic acid ester were confirmed to exist in the unsaponifiablc matter of crude rice bran oil. Quantitative relation of components was calculated and found to be 42% sterols, 24% higher alcohols. 20% ferulic acid esters, 10% hydrocarbons and 2% unreported compound.     

Interferon-γ and Nitric Oxide Down-Regulate Lipopolysaccharide-Induced Prostanoid Production in Cultured Rat Microglial Cells by Inhibiting Cyclooxygenase-2 Expression

Abstract: We have used purified microglial cultures obtained from neonatal rat brains to study some aspects of the regulation of prostanoid production in these cells. We found that nitric oxide, which is released into the culture medium along with prostanoids when the cells are exposed to lipopolysaccharide (1–100 ng/ml), can down-regulate prostanoid biosynthesis. Indeed, the abrogation of endogenous nitric oxide production, obtained by depleting the medium of the precursor l -arginine or by blocking the activity of nitric oxide synthase by the specific inhibitor N^G-monomethyl-l -arginine, led to a remarkable increase in lipopolysaccharide-induced prostanoid release. Moreover, the nitric oxide-generating compound 3-morpholinosydnonimine caused a substantial reduction of prostanoid formation, in the absence of endogenous nitric oxide, suggesting that both endogenous and exogenous nitric oxide may inhibit the induced prostanoid production. We also found that interferon-γ potentiated the effect of lipopolysaccharide on nitrite accumulation as previously described by others and depressed the lipopolysaccharide-evoked production of prostaglandin E₂, prostaglandin D₂, and thromboxane. It is interesting that the inhibitory effect of interferon-γ on prostanoid production did not appear to depend on the potentiation of NO release, as it was present also when the endogenous synthesis of nitric oxide was abrogated. Additional experiments showed that interferon-γ and nitric oxide act mainly by down-regulating the lipopolysaccharide-induced enzymatic activity and expression (analyzed by western blot) of cyclooxygenase-2. Our data indicate that microglial prostanoid biosynthesis induced by proinflammatory stimuli, such as lipopolysaccharide, is tightly regulated by nitric oxide. Interferon-γ appears to affect the balance between these local mediators by favoring nitric oxide production and inhibiting the prostanoid cascade and may thus contribute to the modulation of inflammation, local immune reactivity, and neuronal damage.     

Phytochemical characterization of potential nutraceutical ingredients from Evening Primrose oil (Oenothera biennis L.)

Evening Primrose oil (EPO) is a natural product extracted by cold-pressed from Oenothera biennis L. seeds. EPO is widely used as a dietary supplement from which beneficial effects have been reported in rheumatic and arthritic conditions, atopic dermatitis, psoriasis, premenstrual and menopausal syndrome, and diabetic neuropathy. The beneficial effects of EPO are thought to be due to its γ-linolenic acid content; in contrast, little effort has been expended to characterize the non-triglyceridic constituents of EPO. In order to evaluate its potential as source of functional food ingredients our aim in this work has been identified and quantified the different components of EPO by different techniques (GC–MS and HPLC). The lipid profile showed that oleic (7%), linoleic (74%) and γ-linolenic (9%) were the most abundance fatty acids. Unsaponifiable matter and subfractions were obtained by CEE/2568/91. Separation of the compounds under study was achieved giving a reasonable analysis time and good resolution. A yield (1.82–1.95%) of unsaponifiable matter was obtained and levels of saturated hydrocarbons (0.291.97 ± 14.85 mg) were noticed. β-Sitosterol (7952.00 ± 342.25 mg/kg oil) and campesterol (883.32 ± 0.45 mg/kg oil) were predominant in phytosterol fraction (9573 mg/kg oil), while tetracosanol (236.93 ± 2.32 mg/kg oil) and hexacosanol (289.92 ± 3.41 mg/kg oil) in linear aliphatic alcohol fraction (798.04 ± 5.66 mg/kg oil). In the phenolic fraction (55.49 ± 2.76 mg/kg oil), ferulic acid (25.23 ± 2.64 mg/kg oil) was the major component. From the results obtained, it can be suggested that the Evening Primrose oil can be considered an interesting alimentary source of substances of nutraceutical value.     

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

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, beta-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, beta-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 processes.     

Inhibition of 26S protease regulatory subunit 7 (MSS1) suppresses neuroinflammation

Recently, researchers have focused on immunosuppression induced by rifampicin. Our previous investigation found that rifampicin was neuroprotective by inhibiting the production of pro-inflammatory mediators, thereby suppressing microglial activation. In this study, using 2-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS), we discovered that 26S protease regulatory subunit 7 (MSS1) was decreased in rifampicin-treated microglia. Western blot analysis verified the downregulation of MSS1 expression by rifampicin. As it is indicated that the modulation of the ubiquitin-26S proteasome system (UPS) with proteasome inhibitors is efficacious for the treatment of neuro-inflammatory disorders, we next hypothesized that silencing MSS1 gene expression might inhibit microglial inflammation. Using RNA interference (RNAi), we showed significant reduction of IkBα degradation and NF-kB activation. The production of lipopolysaccharides-induced pro-inflammatory mediators such as inducible nitric oxide synthase (iNOS), nitric oxide, cyclooxygenase-2, and prostaglandin E(2) were also reduced by MSS1 gene knockdown. Taken together, our findings suggested that rifampicin inhibited microglial inflammation by suppressing MSS1 protein production. Silencing MSS1 gene expression decreased neuroinflammation. We concluded that MSS1 inhibition, in addition to anti-inflammatory rifampicin, might represent a novel mechanism for the treatment of neuroinflammatory disorders.     

Isolation and identification of new constituents in milk fat

After removal of cholesterol on a digitonin column, the unsaponifiable matter of milk fat was examined for alcoholic substituents. Derivatization with pyruvic acid chloride 2,6-dinitrophenylhydrazone and fractionation of the derivatives gave four main fractions. The second, the hexane-benzene fraction, was shown by thin-layer chromatography to have a mobility similar to many common sterols. The hexane-benzene fraction was saponified and gave rise to free alcohols, which were then analyzed on a combination gas-liquid chromatograph-mass spectrometer. Dihydrolanosterol, previously unreported in milk fat, and lanosterol, previously identified but never confirmed, were characterized. The sterols which were precipitated on the digitonin column, were removed, and by the use of the combination gas-liquid chromatograph-mass spectrometer beta-sitosterol was identified. In addition, lanosterol and dihydrolanosterol were isolated from the unsaponifiable matter by chromatography on Florisil.     

Effect of olive oil minor components on oxidative stress and arachidonic acid mobilization and metabolism by macrophages RAW 264.7

Minor components of virgin olive oil may explain the healthy effects of the Mediterranean diet on the cardiovascular system and cancer development. The uncontrolled production of reactive oxygen species (ROS) and arachidonic acid (AA) metabolites contributes to the pathogenesis of cardiovascular disease and cancer, and inflammatory cells infiltrated in the atheroma plaque or tumor are a major source of ROS and eicosanoids. We aimed to determine the effects of squalene, beta-sitosterol, and tyrosol, which are representative of the hydrocarbons, sterols, and polyphenols of olive oil, respectively, on superoxide anion (O2(-)), hydrogen peroxide (H2O2), and nitric oxide (*NO) levels. We also studied AA release and eicosanoid production by phorbol esters (PMA)-stimulated macrophages RAW 264.7. beta-Sitosterol and tyrosol decreased the O2(-) and H2O2 production induced by PMA, and tyrosol scavenged the O2(-) released by a ROS generating system. These effects were correlated with the impairment of [3H]AA release, cyclooxygenase-2 (COX-2) expression, and prostaglandin E(2)/leukotriene B(4) synthesis in RAW 264.7 cultures stimulated by PMA. beta-Sitosterol exerted its effects after 3-6 h of preincubation. Tyrosol inhibited the [3H]AA release induced by exogenous ROS. beta-Sitosterol and tyrosol also reduced the *NO release induced by PMA, which was correlated with the impairment of inducible nitric oxide synthase (iNOS) levels. This may be correlated with the modulation of NF-kappaB activation. Further studies are required to gain more insight into the potential healthy effects of minor components of extra virgin olive oil.     

Lipids of Algae

A fresh cake of Scenedesmus. cells was extracted with hot acetone, and the extracts were saponified. The yield of the unsaponifiable matter was 4.5% of the dry matter. Hydrocarbons and alcohols were extracted with petroleum ether from this unsaponifiable matter. The petroleum ether extracts were fractionated by the vacuum distillation. Each of the fractions was further separated by column chromatography or/and by the solubility difference of the urea adducts. Phytol was the major component of the unsaponifiable matter. Small amounts of n-hexadecane, n-hexacosene and n-eicosanol were isolated. On the other hand, the residue of the petroleum ether extraction was extracted with ether to separate sterols, of which the major component was found to be chondrillasterol.     

Effects of ambient air particles on nitric oxide production in macrophage cell lines

We assessed the in vitro toxicity of various particles on three murine macrophage cell lines, (J774A.1, WR19M.1, RAW264.7). The cells were exposed to aqueous suspensions (0-100 microg/30 mm2 well) of urban particulate matter (SRM-1648, SRM-1649, EHC-93), fine particulate matter (PM2.5), titanium dioxide (SRM-154b), and respirable cristobalite (SRM-1879) for 2 h and were then stimulated with lipopolysaccharide (LPS, 100 ng/ml) and recombinant interferon-gamma (IFN, 100 U/ml). After overnight incubation with the particles and LPS/IFN, nitric oxide production was estimated from culture supernatant nitrite. Cell viability was determined by monitoring the rate of AlamarBlue reduction. The dose-effect relationships for nitrite and viability were modeled as a power function (Fold change = [Dose+1]beta), where beta represents the slope of the dose-response curve. Potency was defined as the rate of change in nitrite production corrected for cell viability (beta(POTENCY) = beta(NITRITE) - beta(VIABILITY)). Overall, the urban particles decreased nitric oxide production (beta(POTENCY) < 0), while exposure of the cells to fine particulate matter or cristobalite increased the production of nitric oxide (beta(POTENCY) > 0). Titanium dioxide (TiO2) was essentially inactive (beta(POTENCY) approximately to 0). The decrease in nitric oxide production seen in cells exposed to the urban particles was directly correlated to a decrease in the expression of inducible nitric oxide (iNOS) as determined by Western blot analysis. The results indicate that particles are modulators of nitric oxide production in murine macrophages and may directly disrupt expression of iNOS during concomitant pathogen exposure. Pathways leading to enhanced NO production causing cell injury, and to decreased NO release resulting in lower bacterial clearance, may both be relevant to the health effects of ambient particles.     

Green tea polyphenol epigallocatechin-3-gallate inhibits the IL-1 beta-induced activity and expression of cyclooxygenase-2 and nitric oxide synthase-2 in human chondrocytes

We have previously shown that green tea polyphenols inhibit the onset and severity of collagen II-induced arthritis in mice. In the present study, we report the pharmacological effects of green tea polyphenol epigallocatechin-3-gallate (EGCG), on interleukin-1 beta (IL-1 beta)-induced expression and activity of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) in human chondrocytes derived from osteoarthritis (OA) cartilage. Stimulation of human chondrocytes with IL-1 beta (5 ng/ml) for 24 h resulted in significantly enhanced production of nitric oxide (NO) and prostaglandin E(2) (PGE(2)) when compared to untreated controls (p <.001). Pretreament of human chondrocytes with EGCG showed a dose-dependent inhibition in the production of NO and PGE(2) by 48% and 24%, respectively, and correlated with the inhibition of iNOS and COX-2 activities (p <.005). In addition, IL-1 beta-induced expression of iNOS and COX-2 was also markedly inhibited in human chondrocytes pretreated with EGCG (p <.001). Parallel to these findings, EGCG also inhibited the IL-1 beta-induced LDH release in chondrocytes cultures. Overall, the study suggests that EGCG affords protection against IL-1 beta-induced production of catabolic mediators NO and PGE(2) in human chondrocytes by regulating the expression and catalytic activity of their respective enzymes. Furthermore, our results also indicate that ECGC may be of potential therapeutic value for inhibiting cartilage resorption in arthritic joints.     

In Vitro Investigation of the Immunomodulatory Potential of Probiotic Lactobacillus casei

The current study investigated the immunomodulatory potential of ethyl acetate soluble supernatant of Lactobacillus casei (LC-EAS) in vitro. The effect of LC-EAS on nitric oxide release was analyzed in RAW 264.7 cells, wherein, an inhibition in nitric oxide production through suppression of inducible nitric oxide synthase mRNA expression was observed. Evaluation of LC-EAS on LPS-induced peripheral blood mononuclear cells showed a down-regulation in TNF-α and IL-6 genes and an upregulation of IL-10. An inhibition in the protein expression of NF-κB, ERK1/2 and STAT3 phosphorylation confirms the immunomodulatory potential of LC-EAS. The effect of LC-EAS on in vitro intestinal epithelial cells was investigated using HT-29 human colon adenocarcinoma cancer cells. LC-EAS exhibited an inhibition of NF-κB and ERK1/2 phosphorylation, whereas STAT3 phosphorylation was unregulated. To evaluate the downstream target of STAT3 upregulation, expression of the intestinal trefoil factor TFF3 which is a NF-κB regulator and STAT3 downstream target was studied. LC-EAS was observed to elevate TFF3 mRNA expression. Overall the study shows that the anti-inflammatory potential of LC-EAS is through inhibition of NF-κB in different cell types.     

Phorbol esters induce nitric oxide synthase activity in rat hepatocytes. Antagonism with the induction elicited by lipopolysaccharide.

The incubation of primary cultures of rat hepatocytes with lipopolysaccharide (LPS) or biologically active phorbol esters promotes the release of nitric oxide to the incubation medium. This process is the result of the induction of the Ca(2+)-and calmodulin-independent form of nitric oxide synthase. Both the release of nitric oxide to the incubation medium and the expression of nitric oxide synthase activity exhibited a lag period of about 45-60 min after cell stimulation. Exposure of hepatocytes to both stimuli produced an antagonistic effect on nitric oxide release, with a half-maximal inhibition obtained with 14 nM phorbol 12,13-dibutyrate at saturating concentration of LPS. Incubation of cells with alpha-phorbol 12,13-didecanoate failed to counteract the effect of LPS or to induce nitric oxide synthase, suggesting that activation of protein kinase C was involved in this process.     

Coronary vascular endothelium-myocyte interactions in protection of the heart by ischaemic preconditioning.

Because ischaemia preconditioning is a general phenomenon - it is present in almost all tissues and organs - we must look for a mechanism which involves cell types common to all organs. The hypothesis outlined below is that endothelial cells, in response to a brief ischaemic stimulus, release protective mediators which (in the heart) diffuse to cardiac myocytes to increase resistance to a subsequent ischaemic stress. These substances include nitric oxide, generated through initial bradykinin release, and prostacyclin. The evidence includes measurement of mediator release and the pharmacological attenuation of the antiarrythmic effects of preconditioning by blockade of bradykinin receptors and inhibition of nitric oxide production.     

Modulation of inflammatory responses by diterpene acids from Helianthus annuus L

Fractionation of a petroleum ether extract of Helianthus annuus L. led to the isolation of three diterpene acids: grandiflorolic, kaurenoic and trachylobanoic acids. These compounds were studied for potential anti-inflammatory activity on the generation of inflammatory mediators in lipopolysaccharide (LPS)-activated RAW 264.7 macrophages. At non-toxic concentrations, these compounds reduced, in a concentration-dependent manner nitric oxide (NO), prostaglandin E₂ (PGE₂) and tumor necrosis factor (TNF-α) production, as well as expression of inducible nitric oxide synthase (NOS-2) and cyclooxygenase-2 (COX-2).All diterpenoids displayed significant in vivo anti-inflammatory activity and suppressed the 12-O-tetradecanoylphorbol-13-acetate (TPA)-mouse ear edema. In addition, inhibition of myeloperoxidase (MPO) activity, an index of cellular infiltration, was observed.In summary, our results suggest that the inhibition of the expression of NOS-2, COX-2 and the release of inflammatory cytokines, is responsible for the anti-inflammatory effects of the diterpenoids isolated from H. annuus L. which likely contributes to the pharmacological action of sunflower.