Mouse mammary epithelial cell strain, SCp2 cells (kindly provided as a gift by P.Y. Desprez, Geraldine Brush Cancer Research Institute, California Pacific Medical Center, San Francisco, CA, USA) were cultured as previously described by Saliba et al. (2009) . Briefly, cells were grown on 100-mm tissue culture plates (BD Falcon, Franklin Lakes, NJ, USA) in growth medium (GM) consisting of Dulbecco's Modified Eagle's Medium Nutrient Mixture/F12 Ham (DMEM/F12; Gibco, Paisley, Scotland) supplemented with 5% heat-inactivated fetal bovine serum (FBS; Gibco), insulin (5 μg/ml; Sigma, St. Louis, MO, USA) and 1% penicillin/streptomycin mixture (Gibco) in a humidified incubator (95% air 5% CO2; VWR Scientific, West Chester, PA, USA) at 37°C. Upon confluency, cells were detached by trypsinization and replated in GM either on 100-mm tissue culture plates for maintenance or on 6-well plates (BD Falcon) at 1 × 106 cells/well to be used in different experiments. On the second day after plating, cells were shifted to differentiation medium (DM) consisting of DMEM/F12 supplemented with insulin (5 μg/ml), hydrocortisone (1 μg/ml; Sigma), ovine prolactin (3 μg/ml; Sigma) and 1% penicillin/streptomycin. On the third day, unless otherwise indicated, plant and ET treatments were performed in triplicates.
Murine intestinal epithelial cell type Mode-K cells were cultured as previously described by Homaidan et al. (2003) . Briefly, cells were maintained in DMEM containing 1 g/L glucose and 10 mM sodium pyruvate (Invitrogen, Carlsbad, CA, USA) and supplemented with 10% FBS (Invitrogen), 1% non-essential amino acids (Invitrogen) and 0.5% penicillin/streptomycin (Invitrogen). At 70-80% confluency, cells were detached by trypsinization and replated on tissue culture flasks (BD Falcon) for maintenance or on 6-well plates at 2 × 105 cells/well to be used in different experiments. On the second day after plating, cells were starved. Plant and IL-1 treatments were performed on the second and third days.
Extraction of plant material
Collection and drying
Ranunculus constantinopolitanus was collected from Yanta, Lebanon, located at an altitude of 1395 m. A voucher specimen of the plant (voucher number: 72) was deposited at the Post Herbarium of the American University of Beirut, Beirut, Lebanon. Following collection, the aerial part (stems, leaves and flowers) of R. constantinopolitanus was dried by leaving the plant sample in the shade for two weeks before grinding it into approximately 10-mm pieces using a blender. Ground samples were subjected to solvent extraction using methanol (MeOH) or else they were stored at -20°C for later use.
Methanol extraction and fractionation
The crude MeOH extract was subjected to further solvent fractionation as previously described by Saliba et al. (2009) . In brief, the dried plant material was subjected to extraction through soaking in MeOH (1:10 w/v) for 16 hr. Incubation on a shaker at 20°C occurred in the first 2 hr, and then the plant sample was left in MeOH for the remaining time. The MeOH extract was filtered using a cheese cloth (sterile gauze sponges 30 × 30 cm) to give a solid phase (R-I) and a filtrate numbered "I" and referred to as "crude MeOH extract". R-I was soaked in ethyl acetate (EtOAc) at a ratio of 10:1 (w/v). It was then separated by filtration into a solid phase and a filtrate numbered "I.1". The crude MeOH extract (I) was evaporated to 1/10 of its volume at less than 40°C, acidified to pH 2 by concentrated H2SO4 and then separated into an aqueous and an organic layer using a mixture of CHCl3 and H2O (2:1 v/v). The organic layer was collected and labelled as "I.2". The aqueous layer was basified to pH 10 by the addition of concentrated NH4OH and was then resuspended in a mixture of CHCl3 and MeOH (3:1 v/v) (the total volume of CHCl3:MeOH is equal to four times the volume of the aqueous layer) to be later separated into an organic layer and an aqueous layer labelled as "I.3" and "I.4", respectively. I.1, I.2, I.3 and I.4 were evaporated to dryness under vacuum, weighed, dissolved in 100% ethanol (EtOH) and stored in dark bottles at -20°C. The fractions were subsequently screened for potential biological activities, and the active ones were selected for further purification.
Separation and identification of subfraction Y2+3
Biologically active fraction I.2 (6 g) was re-dissolved in a minimum volume of petroleum ether (P.ether):CHCl3:EtOAc mixture (2:2:1) and was applied to a column chromatography consisting of 800 g of silica gel. A gradient elution was performed using P.ether:CHCl3:EtOAc (2:2:1) (6500 ml), followed by P.ether:CHCl3:EtOAc (1:3:1) (4000 ml), CHCl3:EtOAc:MeOH (3:3:1) (4200 ml) and MeOH, successively. Subfraction Y2+3 was collected using P.ether:CHCl3:EtOAc (2:2:1) as a mobile phase.
Y2+3, which had the highest biological activity, was purified via solid phase extraction (SPE). Spectroscopic data using infrared (IR) spectroscopy and nuclear magnetic resonance (NMR) showed that Y2+3 was a mixture of FAs. The mixture was consequently converted to FA methyl esters (FAMEs) and resolved into individual components using gas chromatography-mass spectrometry (GC-MS). GC analysis was performed using a Trace™ gas chromatograph equipped with HP-5 capillary column (30 m long, 250 μm i.d and 0.25 μm film thickness), Helium as a carrier at a flow rate of 1 ml/min. The maximum temperature was 350°C. The column was heated from 35°C to 290°C. The injector temperature was set at 300°C in a splitless mode. Results were recorded as percent of total peak areas. The mass spectrometer employed in the GC-MS analysis was a Polarization Q series mass selective detector in the electron impact (EI) ionization mode (70 eV). Using appropriate reference standards of FAMEs, Y2+3 was identified as a mixture of four FAs: palmitic acid (C16:0), isomers of C18:2 and C18:1 and stearic acid (C18:0) in the corresponding proportion 1:5:8:1.
Treatment of cells with plant extracts and fatty acids
Plant extracts, fish oil, CLA, the FAs: palmitic, linoleic (cis-9, cis-12-octadecadienoic acid), oleic (cis-9-octadecenoic acid) and stearic acid and a mix of the four FAs (1:5:8:1 ratio) were all diluted in 100% EtOH and stored at -20°C. On day 3 after plating, SCp2 cells were treated with plant extracts or other FA compounds at different concentrations in DM supplemented with 1% FBS up to a final volume of 1 ml/well. Following treatment, cells were incubated at 37°C for different time points (24 or 48 hr) to assess cytotoxicity or for 30 min (short-term treatment) before ET treatment. For other experiments, media were supplemented with plant extracts or other FA compounds at different concentrations for 3 days as of the plating day (long-term treatment), and cells were treated with ET on day 3.
For Mode-K cells, on day 2 after plating, cultures were pretreated with Y2+3 or a synthetic FA mix at different concentrations in the absence of FBS up to a final volume of 1 ml/well. Following treatment, cells were incubated at 37°C for 8 or 12 hr before IL-1 treatment on day 3. Another method involved the cotreatment of Mode-K cells with Y2+3 or a synthetic FA mix and IL-1 on day 3 for 8 hr under similar conditions.
Induction of inflammation
Endotoxin treatment of SCp2 cells
ET treatment was performed as previously described by Saliba et al. (2009) . Salmonella typhosa ET (Sigma) was dissolved in DM, filter-sterilized using 0.2 μm non-pyrogenic sterile-R filter and stored at -20°C. On day 3 after plating, cells were treated with ET at 10 μg/ml and then incubated at 37°C for 9 hr.
Interleukin-1 treatment of Mode-K cells
IL-1 treatment was performed as previously described by Homaidan et al. (2003) . IL-1α (US Biological, Swampscott, MA, USA) was dissolved in 1% bovine serum albumin (BSA; Invitrogen) and stored at -20°C. On day 3 after plating, cells were treated with IL-1 at 10 ng/ml and then incubated at 37°C for 6 or 8 hr.
Trypan blue exclusion method
Twenty-four or 48 hr post plant treatment, viable and dead SCp2 cells were counted using the trypan blue exclusion method. It involves the trypsinization of the attached cells and washing them using the same treatment medium, which contains dead cells, to form a suspension of the total treated cells. An aliquot of 50 μl is taken from this suspension and mixed with an equal volume of trypan blue (Gibco). Dead cells stain blue, while viable cells appear bright. The percentage of viability is calculated relative to the control.
Enzyme-linked immunosorbent assay (ELISA)
Media of SCp2 cells were sampled from triplicate wells 9 hr post ET treatment. Forty μl of complete protease inhibitors [one tablet dissolved in 2 ml of double distilled water (DDW); Roche Diagnostics GmbH, Mannheim, Germany] was added to each 1 ml of sample. Samples were stored at -80°C until the day of the assay.
A two-site (sandwich) ELISA was performed for the quantitative determination of mouse IL-6 (mIL-6) present in SCp2 cell culture media, using mIL-6 ELISA immunoassay kit (BioSource International, Inc., Camarillo, CA, USA). The IL-6 assay was performed according to manufacturers' instructions. All standards and samples were run in duplicates on high-binding 96-well microtiter plates (Thermo Labsystems, Philadelphia, PA, USA). The optical density was measured at a wavelength of 450 nm by an ELISA microplate reader (Multiskan Ascent, Thermo Labsystems). Concentrations were calculated using the Ascent software and were expressed in pg/ml.
Mode-K cells were washed twice with phosphate-buffered saline (PBS) and scraped in 2× electrophoresis sample buffer (SB) containing 0.25 M Tris-HCl (pH 6.8; Amersham Biosciences, San Diego, CA, USA), 4% w/v sodium dodecyl sulfate (SDS; Amersham Biosciences), 20% w/w glycerol (Amersham Biosciences), 0.1% bromophenol blue and 40 μl/ml protease inhibitor cocktail (Biomol, Plymouth Meeting, PA, USA). Samples were then collected in microfuge tubes, boiled for 5 min, centrifuged and the supernatant representing total soluble protein extract was collected and stored at -80°C.
Total protein extracts were run on a 12% SDS-polyacrylamide gel (BioRad, Hercules, CA, USA), and the gels were transferred to polyvinylidene difluoride (PVDF) membranes (Amersham Biosciences) overnight at 4°C. Following transfer, membranes were washed once with TPBS wash buffer (PBS containing 0.1% Tween 20) and then blocked in 5% non-fat dry milk for 2 hr at room temperature. Rabbit polyclonal COX-2 antibody (Cayman Chemical, Ann Arbor, MI, USA) was then added to the membranes and incubated for 2 hr at room temperature. Unbound antibodies were washed three times with TPBS. Horse-raddish peroxidase (HRP)-conjugated anti-rabbit IgG (Santa Cruz Biotechnology, Santa Cruz, CA, USA) was added at 1:5000 dilution for 1 hr at room temperature. Membranes were washed, incubated with luminol reagents (Santa Cruz Biotechnology) and directly exposed to autoradiography.
Data were expressed as mean ± S.D. The effectiveness of plant treatments was analyzed by one-way analysis of variance (ANOVA). To check for treatments with similar effects, Student-Newman-Keuls (SNK) and Tukey HSD tests were performed. The effect of each treatment, if any, was then compared to the control using two-sample t-test or Dunnett t-tests for multiple comparisons. All statistical analyses were carried out using statistical program for social sciences (SPSS) 11.5, except for t-test, which was performed using Excel. Statistical probability of P < 0.05 was considered significant.