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Phytomedicine 19 (2012) 1072– 1076

Contents lists available at SciVerse ScienceDirect

Phytomedicine

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hort communication

he sterols isolated from Evening Primrose oil modulate the release ofroinflammatory mediators

ergio Montserrat-de la Paz ∗, Ángeles Fernández-Arche, María Ángel-Martín,aría Dolores García-Giménez

epartment of Pharmacology, School of Pharmacy, University of Seville. C/Profesor García González 2, 41012 Seville, Spain

r t i c l e i n f o

eywords:vening Primrose oilenothera biennisterolsacrophagesitric oxide

nflammationytokine

a b s t r a c t

Evening Primrose oil is a natural product extracted by cold-pressed from Oenothera biennis L. seeds. Theunsaponifiable matter of this oil is an important source of interesting minor compounds, like long-chainfatty alcohols, sterols and tocopherols. In the present study, sterols were isolated from the unsaponifiablematter of Evening Primrose oil, and the composition was identified and quantified by GC and GC–MS.The major components of sterols fraction were �-Sitosterol and campesterol. We investigated the abil-ity of sterols from Evening Primrose oil to inhibit the release of different proinflammatory mediatorsin vitro by murine peritoneal macrophages stimulated with lipopolysaccharide. Sterols significantly anddose-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 alsoreduced tumor necrosis factor-�, interleukine 1� and tromboxane B2. However, sterols did not reduceprostaglandin E2. The reduction of eicosanoid release was related to the inhibition of cyclooxygenase-2expression. These results showed that sterols may have a protective effect on some mediators involvedin inflammatory damage development, suggesting its potential value as a putative functional componentof Evening Primrose oil.

ntroduction

Oil obtained from the seeds of Evening Primrose (Oenotheraiennis L., Onagraceae; EPO) is widely used as a dietary supplementue to its high content of polyunsaturated fatty acids, in particu-

ar, �-linolenic acid (18:3n-6) whose beneficial effects have beeneported in rheumatic and arthritic conditions, atopic dermatitis,remenstrual and menopausal syndrome, and diabetic neuropa-hy (Mahady et al. 2001). The �-6 fatty acids are precursors oficosanoids of the 1-series and exert an inhibitory effect on leu-otriene synthesis (Belch and Hill 2000).

In contrast, little effort has been expended to characterize theon triglyceridic constituents of EPO. The non-saponifiable por-ion, about 1.5–2% of the oil, contains elevated amounts of sterols,ocopherols, hydrocarbons and alcohols (Hudson 1984). Sterolsaven been studied both for their cholesterol lowering effects andheir anticancer properties (Bradford and Awad 2007). In addi-

ion to these properties, phytosterols have been suggested toossess anti-inflammatory, antibacterial and antifungical activitiesAkihiso et al. 2000).

∗ Corresponding author. Tel.: +34 605383541.E-mail address: delapaz@us.es (S. Montserrat-de la Paz).

944-7113/$ – see front matter © 2012 Elsevier GmbH. All rights reserved.ttp://dx.doi.org/10.1016/j.phymed.2012.06.008

© 2012 Elsevier GmbH. All rights reserved.

Nitric oxide (NO) has been shown to play a central role ininflammatory and immune reaction activities. Macrophages appearto be the main cellular source of NO, since these cells signifi-cantly contribute to inducible NO synthetase (iNOS) induction afterlipopolysaccharide (LPS) incubation (Salkowski et al. 1997). NOis also able to enhance the production of tumor necrosis factor-� (TNF-�) and interleukin 1� cytokines, which participate in themacrophage-dependent inflammation (Marcinkiewiez et al. 1995).Activation of macrophages also leads to cyclooxygenase 2 (COX-2)stimulation with consequent prostaglandin E2 (PGE2) overproduc-tion, which plays a key role in the pathogenesis of inflammatoryprocesses (Kang et al. 1996). Another eicosanoid of particularimportance is thromboxane A2 (TXA2), which is produced by theaction of thromboxane synthase on the prostaglandine endoper-oxide H2 (PGH2) resulting from the enzymatic transformation ofarachidonic acid by the COX-2. TXA2 is a potent inducer of plateletaggregation, vasoconstriction and bronchoconstriction, and hasbeen involved in series of major pathophysiological conditions(Dognè et al. 2006).

Arachidonic acid is released from phospholipids by the action of

the secretory phospholipase A2 (sPLA2), thereby providing the sub-strate for the biosynthesis of proinflammatory eicosanoids. Thus,sPLA2 might play important roles in the initiation and amplificationof inflammatory reaction (Triggiani et al. 2005).

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The aim of our study was to analyze and identify the compo-ition of the sterols’ fraction, and to examine its ability to inhibithe release of some proinflammatory mediators by cells involvedn inflammation like macrophages. With this purpose, we investi-ated the effect of sterols to inhibit in vitro NO, sPLA2, PGE2, TXB2,NF-� and IL 1-� generation in LPS-stimulated murine peritonealacrophages.

aterials and methods

ample preparation

The unsaponifiable matter of EPO was isolated following con-entional procedures and its components were analyzed followinghe IUPAC method (Paquot 1992). For sterols analysis by GC, thesolated sterols were transformed into trimethylsilyl ethers.

eneral experimental procedures

Sterols were analyzed with a Chrompack (Middelburg, Theetherlands) CP900 gas chromatography equipped with a capil-

ary column SGL-5. The MS analyses were performed using KratosS 80 mass spectrometer equipped with a NBSLIB2 data system.

eagents

sPLA2, PGE2 and TXB2 EIA Kits (Cayman Chemical Company, Annrbor, MI, USA), Mouse IL 1-� ELISA (eBioscience, Vienna, Austria),ouse TNF-� ELISA (Thermo Scientific, Rockford, IL, USA), Thiogly-

olate (Scharlau Chemie S.A. Barcelona, Spain). The rest of reagentsere purchased from Sigma Aldrich Chem. (St. Louis, MO, USA).

tock solutions of compounds were prepared in DMSO and laterissolved in ethanol. The final concentration of DMSO or ethanol inhe culture medium did not significantly influence cell response.

solation and culture of murine peritoneal macrophages

Peritoneal exudate cells (1 × 106 cells/well) from thioglycolate-nduced mice were collected from the peritoneal cavities ofemale Swiss mice and were suspended in culture medium RPMI640 supplemented with 10% fetal bovine serum (FBS), peni-illin (100 units/ml) and streptomycin (100 �g/ml). They werere-cultured in 24-well plates at 37 ◦C in 5% CO2 in air for 2 h.onadherent cells were removed and adherent cells were cultured

n 1 ml of fresh medium (5% FBS) containing 5 �g/ml of E. coliSerotype 0111:B4) LPS at 37 ◦C for 24 h in the presence of sterolsraction at different doses (25, 50 or 100 �g/ml) or vehicle.

ell viability

The mitochondrial-dependent reduction of 3-(4,5-imethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) toormazan was used to asses possible cytotoxic effect on the murineeritoneal macrophage (Mosmann 1983).

easurement of nitrite production

Nitrite, as index of NO generation, was determinated by a fluo-imetric method (Griess reaction method) (Green et al. 1982). Theirect scavenging ability of each of the compounds was determined

y incubating 5 mM sodium nitropusside (SNP) with the test com-ounds at 25 ◦C for 120 min (Marcocci et al. 1994), following which00 �l of incubation solution was withdrawn and nitrite contenteterminated with Griess reagent in 96-well plates.

edicine 19 (2012) 1072– 1076 1073

sPLA2, PGE2, TXB2, IL-1 ̌ and TNF- ̨ production in murineperitoneal macrophages

sPLA2, PGE2, TXB2, IL 1-� and TNF-� levels’ production werequantified by sandwich immunoassay (Moroney et al. 1988; Choiet al. 2001).

Western blot analysis of iNOS and COX-2 expression

Cells (1 × 106 cells/ml) were rinsed, scraped off and collectedin ice-cold PBS containing a cocktail of protease and phosphataseinhibitors. Protein concentration was measured according to theBradford method (Bradford 1976), using �-globuline as a stan-dard. Equal amounts of cell protein extract (30 �g) were loadedper well on 7–10% SDS-polyacrylamide gel electrophoresis andtransferred on to nitrocellulose membranes and incubated in theappropriate blocking solution (5% nonfat dry milk, 1% Twen-20in 20 mM Tris-buffered saline, pH 7.6) for 2 h at room tempera-ture, and then incubated in primary antibody-containing blockingsolutions rabbit anti-INOs (Cayman, Spain) (1:100.000), rabbit anti-COX-2 (Cayman, Spain) (1:100.000); overnight at 4 ◦C and washed3 times. After rinsing, the membranes were incubated with ahorseradish peroxidase-labeled (HRP) secondary antibody anti-rabbit (Cayman, Spain) at dilution 1:50.000 containing blockingsolution for 1–2 h at room temperature. Immunodetection wasperformed using enhanced chemioluminiscence light-detecting kit(Pierce, Spain). The signals were then captured using LAS-3000Imaging System from Fuji Densitometric and data were studiedfollowing the normalization to the housekeeping protein �-actin(Sigma–Aldrich, Spain) and assessed by Image J software.

Data analysis

The results are presented as mean ± SEM. Statically significantdifferences were evaluated by analysis of variance (ANOVA) fol-lowed by Dunnet’s t test for multiple comparisons. A p < 0.05 wasconsidered significant.

Results and discussion

Analysis of the sample composition

Sterols were isolated from the EPO unsaponifiable matter andrepresented a 49.40% from this fraction and almost a 1% fromEPO (0.98%). This high proportion shows that EPO is one of therichest natural products in phytosterols versus others vegetal oilsfrequently used in the diet, like those coming from corn (0.95%)(Ostlund et al. 2002) or from sunflower (0.73%). Even the famousolive oil only has a 0.17% of phytosterols (Weihrauch and Gardner1978).

The identification of the different phytosterols has been madeby GC and GC–MS comparing their RRt values and their MS frag-ments with literature values. Chromatographic analyses are shownin Fig. 1. We consider very significant the proportion of campes-terol (9.07%) present in EPO as in other oils, like olive oil wherethe content reaches about 4%. Since this compound has an advan-tage as a functional component, as its bioavailability in the bodyis 20% compared with 7% showing sitosterol (Bhattacharyya 1981;Ostlund et al. 2002; Duan et al. 2004).

Effect of sterols on cell viability

Viability of cells treated with sterols was not significantlyreduced at sterols concentrations 100 and 50 �g/ml (data notshown).

1074 S. Montserrat-de la Paz et al. / Phytomedicine 19 (2012) 1072– 1076

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ig. 1. (A) Gas chromatogram of the sterols fraction from Evening Primrose oil. 17-Avenasterol. (B) Composition of the sterols fraction from EPO analyzed by gas

hytosterols of EPO.

itrite production by LPS-stimulated murine peritonealacrophages

The effect of sterols on the release of inflammatory media-ors is depicted in Fig. 2. Co-incubation with sterols significantlyeduced nitrite production, in a dose-dependent manner (Fig. 2A).he sterols from EPO at all doses did not decrease nitrite production

n vitro NO scavenging (SNP test), indicating was not a consequencef direct scavenging of this radical (Fig. 2B).

PLA2, PGE2, TXB2, IL-1 ̌ and TNF- ̨ production by LPS-stimulatedurine peritoneal macrophages

Likewise, sPLA2, TXB2, IL-1� and TNF-� generation were also

educed by sterols at all doses assayed. In all the cases, the highestnhibition was found at 100 �g/ml (Fig. 2C, D, F and G). In con-rast, sterols were not able to inhibit PGE2 generation (Fig. 2E). Oneeason could be that the specific thromboxane inhibition, would

olestanol, 2. campesterol, 3. �-Sitosterol, 4. Sitostanol, 5. �5-Avenasterol, and 6.atograph. (C) Fragmentation ions obtained in the identification of TMS-derivates

redirect arachidonic acid metabolism increasing the formation ofthe prostanoids (Fiddler and Lumley 1990; Brownli et al. 1993).

iNOS and COX-2 expression

Western blot assay was performed to study possible effects oniNOS and COX-2 enzyme gene expression. Fig. 2H shows that sterolsat 100 �g/ml caused an inhibition of the LPS-induced iNOS expres-sion (61%). This effect was similar to dexamethasone, the referencecompound (58%). Sterols also reduced COX-2 enzyme gene expres-sion although the potency of inhibition for the latter was lower (50%at 100 �g/ml, Fig. 2I).

EPO has attracted much interest in particular due to the high

content in GLA. Supplementation with other oils rich in GLA, suchas borage seed oil (Borrago officinalis) or blackcurrant (Ribes nigrum)was less effective in various inflammatory disorders, at the samedoses, suggesting that there are probably other constituents, such

S. Montserrat-de la Paz et al. / Phytomedicine 19 (2012) 1072– 1076 1075

Fig. 2. Effect of sterols fraction at 25, 50 and 100 �g/ml on release of proinflammatory mediators in LPS activated macrophages. (A) Nitrite generation, (B) radical scavengingby SNP test, (C) PLA2, (D) TXB2, (E) PGE2, (F) IL-1�, and (G) TNF-�. (H) Densitometric analysis of sterols fraction at 50 and 100 �g/ml on murine macrophages iNOS expression.( value(

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I) COX-2 expression, the figure is representative of three similar experiments. Each1 �M); caffeic acid (50 �M) (*p < 0.05; ***p < 0.001 vs LPS).

s sterols, that might be involved in the beneficial effects of EPOEngler 1993; Belch and Hill 2000).

In conclusion, we consider that EPO is an interesting source ofunctional components, especially rich in sterols that may exert aignificant protective effect against the release of proinflammatoryediators.

cknowledgment

We are grateful to Dr. Carmen Pérez-Camino (CSIC) who collab-rated in the identified components in this study.

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