Interleukin-6 and Cyclooxygenase-2 downregulation by fatty-acid fractions of Ranunculus constantinopolitanus

Abstract

Background: Medicinal plants represent alternative means for the treatment of several chronic diseases, including inflammation. The genus Ranunculus, a representative of the Ranunculaceae family, has been reported to possess anti-inflammatory, analgesic, antiviral, antibacterial, antiparasitic and antifungal activities, possibly due to the presence of anemonin and other. Different studies have shown the occurrence of unusual fatty acids (FAs) in Ranunculaceae; however, their therapeutic role has not been investigated. The purpose of this study is to characterize potential anti-inflammatory bioactivities in Ranunculus constantinopolitanus D'Urv., traditionally used in Eastern Mediterranean folk medicine.

Methods: The aerial part of R. constantinopolitanus was subjected to methanol (MeOH) extraction and solvent fractionation. The bioactive fraction (I.2) was further fractionated using column chromatography, and the biologically active subfraction (Y2+3) was identified using infrared (IR) spectroscopy, nuclear magnetic resonance (NMR) and gas chromatography-mass spectrometry (GC-MS). The effects of I.2 and Y2+3 on cell viability were studied in mouse mammary epithelial SCp2 cells using trypan blue exclusion method. To study the anti-inflammatory activities of I.2 and Y2+3, their ability to reduce interleukin (IL)-6 levels was assessed in endotoxin (ET)-stimulated SCp2 cells using enzyme-linked immunosorbent assay (ELISA). In addition, the ability of Y2+3 to reduce cyclooxygenase (COX)-2 expression was studied in IL-1-treated mouse intestinal epithelial Mode-K cells via western blotting. Data were analyzed by one-way analysis of variance (ANOVA), Student-Newman-Keuls (SNK), Tukey HSD, two-sample t-test and Dunnett t-tests for multiple comparisons.

Results: The chloroform fraction (I.2) derived from crude MeOH extract of the plant, in addition to Y2+3, a FA mix isolated from this fraction and containing palmitic acid, C18:2 and C18:1 isomers and stearic acid (1:5:8:1 ratio), reduced ET-induced IL-6 levels in SCp2 cells without affecting cell viability or morphology. When compared to fish oil, conjugated linoleic acid (CLA) and to individual FAs as palmitic, linoleic, oleic and stearic acid or to a mix of these FAs (1:5:8:1 ratio), Y2+3 exhibited higher potency in reducing ET-induced IL-6 levels within a shorter period of time. Y2+3 also reduced COX-2 expression in IL-1-treated Mode-K cells.

Conclusion: Our studies demonstrate the existence of potential anti-inflammatory bioactivities in R. constantinopolitanus and attribute them to a FA mix in this plant.

Figure 1

Ranunculus constantinopolitanus. Ranunculus constantinopolitanus (Arabic name: Hawdhan fa'ri) is an Eastern Mediterranean plant that grows in Aintab and extends from Hasbayya to Jazzin. Description: "Villous below, appressed-hairy above. Root-fibers descending directly from neck. Root-leaves triangular-ovate, ternate, with obovate, cut, and coarsely toothed lobes. Carpels large, ovate, striate, smooth, ending abruptly in a very short, hooked beak". Its flowering season falls between April and June [41]. The plant has been identified by Dr. Nada Sinno-Saoud, and a voucher specimen (voucher number: 72) was deposited at the Post Herbarium of the American University of Beirut, Beirut, Lebanon. Photos courtesy of Mr. Khaled Sleem (2004), Crop Production and Protection Department, Faculty of Agricultural and Food Sciences, American University of Beirut, Beirut, Lebanon.

Figure 2

Exposure of SCp2 cells to R. constantinopolitanus fraction I.2 at noncytotoxic concentrations reduces, in a concentration-dependent manner, ET-induced IL-6 levels. SCp2 cells were treated on day 3 of culture with EtOH (Ctrl) or I.2 at different concentrations in DM supplemented with 1% FBS: (A) Twenty-four hr later, trypan blue assay was performed. (B) Thirty min later, cells were treated with ET at 10 μg/ml and their media collected 9 hr post-ET. The values depicted are the means (± S.D.) of a triplicate treatment. Means with the same letter are not significantly different. Statistical significance of the difference from Ctrl+ET is with *P < 0.05, **P < 0.01 or ***P < 0.001. (Ctrl, control; ET, endotoxin; IL-6, interleukin-6). -ET (white square); +ET (black square).

Figure 3

Exposure of SCp2 cells to Y₂₊₃ at noncytotoxic concentrations reduces, in a concentration-dependent manner, ET-induced IL-6 levels. (A) SCp2 cells were treated on day 3 of culture with EtOH (Ctrl) or Y₂₊₃ at different concentrations in DM supplemented with 1% FBS. Forty-eight hr later, trypan blue assay was performed. SCp2 cells were treated (B) on day 3 of culture 30 min before ET (short-term exposure) or (C) for 3 days (long-term exposure), as of day 1 of culture, with EtOH (Ctrl) or Y₂₊₃ at different concentrations. Cells were treated on day 3 with ET at 10 μg/ml in DM supplemented with 1% FBS and their media collected 9 hr post-ET. The values depicted are the means (± S.D.) of a triplicate treatment. Means with the same letter are not significantly different. Statistical significance of the difference from Ctrl+ET is with **P < 0.01 or ***P < 0.001. (Ctrl, control; ET, endotoxin; IL-6, interleukin-6). -ET (white square); +ET (black square).

Figure 4

Exposure of SCp2 cells to individual FA components of Y₂₊₃ or a synthetic FA mix does not reduce ET-induced IL-6 levels. SCp2 cells were treated (A) on day 3 of culture 30 min before ET (short-term exposure) or (B) for 3 days (long-term exposure), as of day 1 of culture, with EtOH (Ctrl), Y₂₊₃^a, palmitate, linoleate^b, oleate, stearate or a synthetic FA mix at different concentrations. Cells were treated on day 3 with ET at 10 μg/ml in DM supplemented with 1% FBS and their media collected 9 hr post-ET. The values depicted are the means (± S.D.) of a triplicate treatment. Statistical significance of the difference from Ctrl+ET is with ***P < 0.001. (Ctrl, control; -ET, untreated cells; IL-6, interleukin-6). ^aFor short-term exposure (A), cells were treated with Y₂₊₃ at 10 μg/ml. ^bLong-term exposure (B) to linoleate at 5 μg/ml was cytotoxic to cells.

Figure 5

Exposure of SCp2 cells to Y₂₊₃ reduces ET-induced IL-6 levels more potently than fish oil and CLA. SCp2 cells were treated (A and C) on day 3 of culture 30 min before ET (short-term exposure) or (B and D) for 3 days (long-term exposure), as of day 1 of culture, with EtOH (Ctrl), Y₂₊₃^a, fish oil (A and B) or CLA^b (C and D) at different concentrations. Cells were treated on day 3 with ET at 10 μg/ml in DM supplemented with 1% FBS and their media collected 9 hr post-ET. The values depicted are the means (± S.D.) of a triplicate treatment. Statistical significance of the difference from Ctrl+ET is with **P < 0.01 or ***P < 0.001. (Ctrl, control; ET, endotoxin; IL-6, interleukin-6). ^aCells were treated with Y₂₊₃ at 5 or 10 μg/ml for long- (B and D) and short-term (A and C) exposure, respectively. ^bShort- and long-term exposures to CLA at 5 and 3 μg/ml, respectively, were cytotoxic to cells. -ET (white square); +ET (black square).

Figure 6

Exposure of Mode-K cells to Y₂₊₃, but not to a synthetic FA mix, reduces IL-1-induced COX-2 protein levels. Mode-K cells were treated on day 2 of culture 8 or 12 hr before IL-1 (pretreatment) or on day 3 (cotreatment) with (A) Y₂₊₃ at 10 μg/ml or (B) a synthetic FA mix at different concentrations (Left: 10 μg/ml; Right: 60 μg/ml) in FBS-free medium. Cells were treated on day 3 with IL-1 at 10 ng/ml in the absence of FBS and their proteins extracted 6 or 8 hr post-IL-1 for western blot analysis. β-actin or GAPDH was used to demonstrate equal loading. (COX-2, cyclooxygenase-2; IL-1, interleukin-1).