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In Vitro Cellular & DevelopmentalBiology - Animal ISSN 1071-2690 In Vitro Cell.Dev.Biol.-AnimalDOI 10.1007/s11626-014-9813-7

Antioxidant and anti-inflammatory effectsof Ruta chalepensis L. extracts on LPS-stimulated RAW 264.7 cells

Mohamed Kacem, Gaëlle Simon,Raphael Leschiera, Laurent Misery,Abdelfattah ElFeki & NicolasLebonvallet

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Antioxidant and anti-inflammatory effects of Ruta chalepensis L.extracts on LPS-stimulated RAW 264.7 cells

Mohamed Kacem & Gaëlle Simon & Raphael Leschiera &

Laurent Misery & Abdelfattah ElFeki &Nicolas Lebonvallet

Received: 23 February 2014 /Accepted: 18 August 2014 / Editor: T. Okamoto# The Society for In Vitro Biology 2014

Abstract Ruta chalepensis L. is used in the traditionalherbal treatment of various diseases. The aim of thiswork is to investigate the effect of different extracts ofR. chalepensis L. on inducible nitric oxide synthase(iNOS) and cyclooxygenase-2 (COX-2) gene expres-sions and their antioxidant capacity on murine RAW264.7 macrophage challenged with lipopolysaccharide(LPS). In fact, this study shows that the ethanol andethyl acetate extracts of R. chalepensis L. considerablydecreased the nitric oxide (NO) production in murineRAW 264.7 macrophages stimulated with lipopolysac-charide. Thus, the treatment with both extracts signifi-cantly suppressed the levels of iNOS and COX-2 geneexpressions through the inhibition of the nuclearfactor-κB (NF-κB) activation. The preincubation ofRAW 264.7 cells with various concentrations of ethanoland ethyl acetate extracts decreased the production ofthiobarbituric acid-reactive substances (TBARS) in a

dose-dependent manner. It also increased the activitiesof antioxidative enzymes, including superoxide dismut-ase (SOD), catalase (CAT), and glutathione peroxidase(GPx) in LPS-stimulated macrophages, compared tothose in the cells treated only with LPS. Besides, the1H NMR spectra of both extracts have demonstrated thepresence of aromatic signals, thus confirming the exis-tence of phenolic compounds such as flavonoids andpolyphenols. So, the ethanol and ethyl acetate extractsof R. chalepensis L. have been shown to possessenough antioxidant and anti-inflammatory activities toprevent LPS-induced oxidative stress and inflammationin RAW 264.7 macrophages.

Keywords COX-2 . iNOS . Lipopolysaccharide . Oxidativestress . Ruta chalepensis L.

Introduction

For several years, special attention has been paid tooxidative stress and the state of an excessive productionof reactive oxygen species (ROS) in the organism. ROSare constantly formed as a byproduct of normal meta-bolic reactions, and their formation is accelerated by theaccidental exposure to lipopolysaccharide (LPS). Thisprototypical endotoxin derived from the cell wall ofGram-negative bacteria plays a dominant role in theprocess of sepsis (Fu et al. 2008). LPS may inducemediators of oxidative stress leading to the generationof ROS, which themselves induce cytokine production.The oxidative stress leads to cytokine induction via theactivation of nuclear factor-κB (NF-κB), which is amajor transcription factor for tumor necrosis factor-alpha (TNF-α), interleukin-6, and interleukin-8 (Yokoo

M. Kacem (*) :A. ElFeki (*)Laboratory of Animal Ecophysiology, Life Sciences Department,Sfax Faculty of Science, University of Sfax, BP1171, 3000 Sfax,Tunisiae-mail: mohamedkacem2008@yahoo.fre-mail: Abdelfattah.ElFeki@fss.rnu.tn

G. SimonLaboratory of Nuclear Magnetic Resonance, University of WesternBrittany—U.E.B., Brest, France

R. Leschiera : L. Misery :N. LebonvalletEA 44685, Laboratory of Neurosciences of Brest (LNB), Universityof Western Brittany—U.E.B., Brest, France

N. LebonvalletDepartment of Biochemistry and Pharmaco-Toxicology, hôpital de laCavale-Blanche, CHRU, Boulevard Tanguy-Prigent, 29 609 Brestcedex, France

In Vitro Cell.Dev.Biol.—AnimalDOI 10.1007/s11626-014-9813-7

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and Kitamura 1996; Bhat-Nakshatri et al. 1998). In theoxidative stress status, the balance between the radicalformation and protection, which is normally present inthe cells, is disturbed. This leads to the oxidative dam-age of all the biological molecules present in our body,such as nucleic acids, proteins, lipids, and the initiationor aggravation of diverse pathological states (Harborneand Williams 2000; Heim et al. 2002; Valko et al.2006).

Nitric oxide (NO), which is one of the major inflam-matory mediators, is synthesized from L-arginine by NOsynthases (NOS), with inducible NOS (iNOS) markedlyupregulated in inflammatory disorders (Kleinert et al.2004). The exposure to inflammatory stimuli such asreactive oxygen species (ROS), microbial products suchas LPS, and proinflammatory cytokines such asinterleukin-1 (IL-1), TNF-α, and interferon-γ (IFN-γ)leads to the fact that inhibitory kappa B (IκBα) isphosphorylated and released from NF-κB complexwhich subsequently forms dimers that are capable ofpassing through the nuclear membrane. In the translo-cation to the nucleus, NF-κB promotes the transcriptionof a wide variety of genes such as inducible nitric oxidesynthase (iNOS) and cyclooxygenase-2 (COX-2) (Allenand Tresini 2000; Alderton et al. 2001).

Recently, there has been considerable interest in find-ing natural antioxidants from plant materials as alterna-tives to synthetic ones. Actually, antioxidants are sub-stances that neutralize both free radicals and their neg-ative effects. They act at different stages (prevention,interception, and repair), using various mechanisms asreducing agents by donating hydrogen, quenching sin-glet oxygen, acting as chelators, and trapping free rad-icals (Devasagayam et al. 2004).

The data from both scientific reports and laboratorystudies show that plants contain a wide range of sub-stances that possess antioxidant activities (Chanwitheesuket al. 2005). The natural antioxidant commonly found infruits, vegetables, and grains provides chemoprotectiveeffects to combat oxidative stress in the body and main-tain balance between oxidants and antioxidants in order toimprove human health (Hsu 2006). It is in this contextthat the Ruta chalepensis L. most commonly known asFijel is studied. In fact, R. chalepensis L. (Rutaceae) is anative herb of the Mediterranean region (Gonzalez-Trujano et al. 2006) but widely diffused in many partsof the world mainly in temperate and tropical countries(Zeichen de Sa et al. 2000). It is characterized by thepresence of alkaloids, flavonoids, phenols, amino acids,furocoumarins, saponins coumarins, tannins, volatile oil,glycosides, sterols, and triterpenes as possible active con-stituents (Hnatyszyn et al. 1974; Grundon and Okely1979; Ulubelen et al. 1986; Al-Said et al. 1990; Kostova

et al. 1999; Chen et al. 2001). R. chalepensis L. is used intraditional medicine for the treatment of a variety ofdiseases such as rheumatism, mental disorders, dropsy,fever, neuralgia, menstrual problems, convulsion, andother nervous disorders (Iauk et al. 2004). Flavonoidsbelong to a large class of natural polyphenolic com-pounds, occurring in fruits and vegetables regularlyconsumed by humans. These compounds have receivedmuch attention due to their anti-inflammatory and anti-oxidant activities (Middleton et al. 2000; Beecher2003). Rue contains different active compounds, amongwhich is rutin, a flavonoid known to have nitric oxidescavenging activity (Vanacker et al. 1995). A previousresearch study of Khlifi et al. (2013) has reported thatmethanolic extract inhibits iNOS expression gene inRAW 264.7. However, the present study investigatesfor the first time the action of the different extracts ofR. chalepensis L. on iNOS and cyclooxygenase-2 geneexpressions at different concentrations. Hence, the aimof the present work is to evaluate not only their effecton iNOS and cyclooxygenase-2 gene expressions butalso their antioxidant capacity on murine macrophageRAW (264.7) cells challenged with LPS.

Materials and Methods

Plant material. The aerial parts (stem and leaves) ofR. chalepensis L. were collected in March 2011 fromChebba region (Mahdia, Tunisia, latitude 35.23° andlongitude 11.11°). The plant was identified and authen-ticated by Pr. Mohamed Chaieb (Department of Botany,Faculty of Science, University of Sfax, Tunisia), accord-ing to the Flora of Tunisia (Pottier-Alaptite 1979;Chaieb and Boukhris 1998). The voucher specimenwas deposited at the herbarium of the Department ofBotany in the cited institute. The aerial parts were shadeair dried at 25°C for 2 wk, powdered with a blender for15 min, and stored in dry, dark, and controlled condi-tions (25°C) for extraction by maceration at room tem-perature (22°C) for 3 d.

Chemical and biological materials. Dulbecco’s modifiedEagle’s medium (DMEM) (BE12-604F/U1, Lonza, Ba-sel, Switzerland), fetal bovine serum (FBS) (DE14-801E, Lonza), LPS (Sigma-Aldrich, St. Louis, MO),dimethylsulfoxide (DMSO) (Sigma-Aldrich), Dulbecco’sphosphate buffered saline (DPBS) 0.0095 (PO4) withoutCa and Mg (Lonza), MYCOZAP antibiotics (Lonza),and 3-(4,5-dimethyl-2yl)-2,5-diphenyltetrazolium bro-m i d e (MTT ) ( S i gma ) we r e u s e d a nd 4 - ( 2 -hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES),phenylmethylsulfonyl fluoride (PMSF), and protease

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inhibitor cocktail were purchased from Sigma Chemicals(Perth, Australia).

Preparation of R. chalepensis L. extracts.Aqueous extract

The ground sample was soaked in distilled water withintermittent shaking and extracted three times at 4°C for3 d. The extract was then separated by filtration andsubsequently concentrated at low temperature by lyoph-ilization. The residue was designated as aqueous extract(AE) and kept at −20°C in the dark until further analysis.Ethanol extract

The powder plant material was soaked in ethanol for3 d with intermittent shaking and extracted three times atroom temperature. Then, the obtained solution was fil-tered and the solvents were vacuum evaporated. Theextract was designated as ethanol extract (EE) and storedat a temperature of 4°C until future use.Hexane extract

To obtain the hexane extract (HE) ofR. chalepensis L.,the powder plant material was soaked in hexane for 3 dwith intermittent shaking and extracted three times atroom temperature. After filtration, the extract was con-centrated to dryness using a rotary evaporator attached toa vacuum pump and stored at a temperature of 4°C untiluse.Ethyl acetate extract

The ground aerial parts of R. chalepensis L.were soaked in ethyl acetate for 3 d with intermit-tent shaking and extracted three times at roomtemperature. The ethyl acetate extract (EAE) ofR. chalepensis L. was then filtered, lyophilizedusing a freeze drier, and stored at a temperatureof 4°C until use.

NMR spectroscopy. All the acquisitions were recorded on aBruker AVANCE 500 equipped with an inverse 5 mm TCI1H/13C/15N cryoprobe. 1H NMR spectra were phased, base-line corrected, and calibrated (TSP at 0.0 ppm). The spectrawere performed with a 30° pulse, with a 2 s delay. Thetemperature was controlled at 25°C.

Determination of total phenolic content. The total phenoliccontent (TPC) in the different extracts of R. chalepensiswas determined calorimetrically using the Folin-Ciocalteu method (Singleton and Rossi 1965). Briefly,50 μl diluted extract solution was mixed with 250 μl ofFolin reagent and 500 μl of sodium carbonate (Na2CO3)(20%, w/v). The mixture was vortexed and diluted withwater to a final volume of 5 ml. After incubation for30 min at room temperature, the absorbance was read at727 nm using a spectrophotometer. The analysis was

performed in triplicate and the total phenolics contentwas expressed in milligrams of gallic acid equivalents(GAEs) per gram of each dry extract, using a calibrationcurve of a freshly prepared gallic acid solution. For thegallic acid, the curve absorbance versus concentration isdescribed by the equation y=0.001x+0.014(R2=0.999).

DPPH radical scavenging activity. The radical scavengingactivity of R. chalepensis L. extracts was determinedusing 2,2-diphenyl-1-picrylhydrazyl (DPPH) reagent ac-cording to the method of Kirby and Schmidt (1997).Briefly, 1 ml of 4% (w/v) solution of DPPH radical inmethanol was mixed with 1 ml of the sample solutionsin methanol at different concentrations. The mixture wasshaken vigorously and incubated for 20 min in the darkat room temperature. The absorbance of the samples andcontrol solutions were measured at 517 nm against ablank and the inhibition of free radical DPPH in percent(I%) was calculated in the following way:

%I ¼ Acontrol−Asample

Acontrol� 100

where Acontrol is the absorbance of the control reaction(containing all reagents except the test compound), and Asample

is the absorbance of the test compound. The ascorbic acid wasused as a control.

Cells and cell culture. RAW 264.7 mouse macrophage cellswere purchased from the American Type Culture Col-lection (TIB-71, Molsheim, France). The cells werecultured in DMEM containing a 25 mM HEPES buffer,4.5 g/l D-glucose, 0.2% sodium bicarbonate, 1 mM so-dium pyruvate, and 2 mM L-glutamine, supplementedwith 10% heat-inactivated FBS and completed withMYCOZAP antibiotics (Lonza). Cultures were main-tained in 75 cm2 culture flasks in a humidified atmo-sphere with 5% CO2 at 37°C. The culture was allowedto grow to confluence and used for further experiments.Viability was determined by trypan blue (0.2% trypanblue in sterile water) exclusion method.

Cell viability assay. The effect of R. chalepensis L. ex-tracts on cell viability was determined by MTT assay.RAW 264.7 cells were mechanically scraped and platedat 5×104 cells/well in 96-well plates containing 100 μlof DMEM medium with 10% heat-inactivated FBS andincubated overnight. R. chalepensis L. extracts weredissolved in DMSO whose concentrations in all assaysdid not exceed 0.2%. After overnight incubation, thetest material was added and the plates were incubatedfor 18 h. Following an 18 h incubation period with or

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without plant extracts, the medium was removed and thecells were further incubated in the presence of 50 μl ofFBS-free medium containing MTT 5 mg/ml. After 4 hof incubation at 37°C, the medium was removed fromeach well and 100 μl DMSO was added to each well tosolubilize the formazan produced in the cells. The opti-cal density (OD) of the formazan product in solutionwas measured with a microplate reader at 540 nm. Thepercentage of cell viability was calculated asAx�Abg

Ac�Abg� 100

h i, where Ax, Abg, and Ac are the mean

absorbance values of the n wells containing the cellsunder study (x), devoid of cells (background, bg), andcontrol cells without treatment (c), respectively.

Griess nitrite assay. RAW 264.7 cells (2.5×105 cells/well)were plated in six-well plates and preincubated withdifferent concentrations of the test extracts (0, 30, 100,200, 300, and 500 μg/ml) for 2 h and were thenstimulated with 1 μg/ml LPS. The culture supernatantswere collected 16 h after the LPS stimulation, and theconcentrations of NO were measured according to theGriess reaction. Fifty microliters of supernatant sampleswere mixed with 50 μl of sulfanilamide solution (1%sulfanilamide in 5% phosphoric acid) and 50 μl of theNED solution (0.1% N-1-napthylethylenediaminedihydrochloride in water). The mixture was then incu-bated at room temperature for 10 min in darkness. Theabsorbance at 550 nm was measured in a microplatereader. In all experiments, fresh culture medium wasused as the blank and sodium nitrite was used as thestandard.

Total RNA isolation and quantitative real-time PCR for de-tecting messenger RNA of COX-2 and iNOS. RAW 264.7cells (2.5×105 cells/well) were plated in 24-well plates andpreincubated with different concentrations (0, 100, 300, and500 μg/ml) of ethanol and ethyl acetate extracts ofR. chalepensis L. for 2 h and were then stimulated with1 μg/ml LPS for 4 h.

The total RNA from the LPS-stimulated RAW 264.7cells was extracted using TRIzol reagent (AppliedBiosystems, Foster City, CA) according to the manufac-turer’s recommendations. Chloroform was added and thetotal RNA was collected in the aqueous phase aftercentrifugation. Finally, RNA was precipitated byisopropanol, then washed with 75% ethanol, andredissolved in RNAse-free water. The total RNA extractwas stored at −80°C before use. The concentrations ofRNA samples were measured at A260 nm with a spec-trophotometer (Nanodrop 2000, Thermo Scientific, Wil-mington, DE). The purity was verified by the calcula-tion of A260/A280 ratio before PCR assays.

The RNA samples were reverse transcribed into com-plementary DNA (cDNA) using commercially availablecDNA synthesis kits (High-capacity cDNA ReverseTranscription Kits; Applied Biosystems). The tubes wereincubated at 25°C for 10 min and then at 37°C for120 min, and the reactions were stopped by heating at85°C for 5 min. The sample was then stored at −20°Cuntil future use. The quantitative real-time PCR wasperformed for different genes in separate wells of 96-well plate using the Sybr Green procedure and an ABIPrism 7000 Sequence Detection System (AppliedBiosystems). The used primers are listed below andwere purchased from the Oligo Centre (Eurogentec,Seraing, Belgium):

– Cyclooxygenase-2 (COX-2):

& Forward: 5′-AAAGGTTCTTCTACGGAGAGAGTTCA-3′

& Reverse: 5′-TGGGCAAAGAATGCAAACATC-3′

– Inducible nitric oxide synthase (iNOS):

& Forward: 5′-GCAGCTGGGCTGTACAAA-3′& Reverse: 5′-AGCGTTTCGGGATCTGAAT-3′

– Glyceraldehyde-3-phosphate dehydrogenase (GAPDH):

& Forward: 5′-TGTGTCCGTCGTGGATCTGA-3′& Reverse: 5′-CCTGCTTCACCACCTTCTTGAT-3′

The expression of messenger RNA (mRNA) valueswas calculated using the threshold cycle (Ct) value, thenumber of PCR cycles at which the fluorescent signalduring the PCR reaches a fixed threshold. For each sam-ple, ΔCt sample was calculated by subtracting the Ctvalue of GAPDH, a housekeeping gene, from that of eachgene of interest to normalize the data. The expressionlevels relative to control were estimated by calculatingΔΔCt (ΔCt sample−ΔCt control) and subsequently usingthe 2−ΔΔCt method.

Preparation of nuclear extracts. The murine macrophagecells were preincubated for 2 h with various concentra-tions (0, 100, 300, and 500 μg/ml) of EE and EAE ofR. chalepensis L. and then stimulated with LPS(1 μg/ml) for 30 min at 37°C in a humidified atmo-sphere containing 5% CO2. Nuclear extract was pre-pared according to the previous method described by(Schreiber et al. 1990). Briefly, the cells were thenwashed with phosphate buffered saline (PBS), dislodgedand pelleted by centrifugation, resuspended in the celllysis buffer [10 mM HEPES, pH 7.5, 10 mM KCl,

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0.1 mM EDTA, 1 mM dithiothreitol (DTT), 0.5%Nonidet‐40, and 0.5 mM PMSF along with the proteaseinhibitor cocktail (Sigma)], and allowed to swell on icefor 15–20 min with intermittent mixing. Tubes werevortexed to disrupt cell membranes and then centrifugedat 12,000×g at 4°C for 10 min. The pellets containingcrude nuclei were resuspended in the nuclear extractionbuffer containing 20 mM HEPES (pH 7.5), 400 mMNaCl, 1 mM EDTA, 1 mM DTT, and 1 mM PMSFwith protease inhibitor cocktail and then incubated for30 min in ice. The samples were centrifuged at12,000×g for 15 min at 4°C to obtain the supernatantcontaining nuclear extract. The protein concentration ofthe nuclear extract was estimated using Bradford reagent(Bio-Rad, Hercules, CA). The extract is either immedi-ately used or stored at −70°C until use.

Western blot analysis. Nuclear extracts from macrophagecells were separated on a 12% sodium dodecyl sulfate(SDS) polyacrylamide gel and electrotransferred to anitrocellulose membrane in 25 mM Tris, 192 mM gly-cine, and 20% methanol for 45 min at 15 V using asemidry transfer apparatus (Bio-Rad). Nonspecific bind-ing sites were then blocked by incubating the membranein 1% bovine serum albumin (BSA) in PBS at roomtemperature for 2 h. The membrane was washed twicewith PBST (PBS with 0.05% Tween-20) buffer and thenincubated with primary antibody (polyclonal anti-NF-κBp65 subunit raised in rabbit) (Santa Cruz Biotechnology,Dallas, TX; diluted 1:500 in PBS containing 0.25%BSA) or primary antibody (polyclonal anti actin raisedin rabbit) (Sigma, A2066; 1:1,000) for 3 h at roomtemperature. The membrane was washed with PBSTbuffer thrice and further incubated for 1 h at roomtemperature with the anti-rabbit IgG horseradish perox-idase conjugated (Santa Cruz Biotechnology; 1:1,000diluted in PBS containing 0.25% BSA). After washingthe membrane with PBST buffer, the blots were exposedto peroxidase substrate (15 mg 4-chloronaphthol in 5 mlmethanol and 20 ml PBS buffer containing 50 μl of 3%hydrogen peroxide).

Lipid peroxidation. RAW 264.7 cells (2.5×105 cells/well)in six-well plates were first incubated with and withoutthe indicated concentrations of the ethanol and ethylacetate extracts of R. chalepensis L. for 2 h and thenincubated with LPS (1 μg/ml). The culture supernatantswere collected after 16 h of LPS stimulation. The con-centration of malondialdehyde (MDA) in the samples asan index of lipid peroxidation was determined spectro-photometrically according to the method of Draper andHadley (1990). Briefly, 0.5 ml of each medium super-natant was mixed with 1 ml of 30% trichloroacetic acid

(TCA) solution and centrifuged at 3,500×g for 10 min.One milliliter of the new supernatants was then mixedwith 1 ml of a solution containing thiobarbituric acid(TBA), and the mixture was heated at 90°C during10 min and then cooled. The absorbance of the TBA-MDA complex was determined at 532 nm using aspectrophotometer. The TBARS values were expressedas nanomoles of malondialdehyde equivalents, using1,1,3,3-tetraethoxypropane as standard.

Antioxidant enzyme assays. RAW 264.7 cells (7.5×106

cells/dish) in 100 mm dishes were preincubated withand without the indicated concentrations of test extracts(EE and EAE of R. chalepensis L.) for 2 h and thenincubated with LPS (1 μg/ml) for 16 h. The mediumwas removed and the cells were washed twice withDPBS. One milliliter of ice-cold Tris buffered saline(TBS) with pH 7.4 was added and cells were scraped.Cell suspensions were sonicated three times for 5 s eachtime on ice. The sonicated cells were centrifuged at10,000×g for 20 min at 4°C. The obtained supernatantswere then used for antioxidant enzyme activities. Theprotein concentration of the cell supernatants was mea-sured using the method of Bradford (1976) withgamma-globulin as standard.

– Determination of glutathione peroxidase activityThe glutathione peroxidase (GPx) activity was assayed

in cell supernatants according to the method of Paglia andValentine (1967). In fact, cell homogenate was mixedwith 400 μl of 0.1 mM glutathione (GSH) and 200 μlof 67 mM of KNaHPO4 (pH=7.8). After 5 min ofpreincubation at 25°C, 200 μl of 1.3 M of H2O2 wasadded. After 10 min, the mixture was treated with 1 ml of1% TCA and centrifuged at 3,000×g at 4°C during10 min.

Supernatants were homogenized with 0.32 M ofNa2HPO4 and 1 mM of 5,5′-dithiobis(2-nitrobenzoicacid) (DTNB). The enzyme activity was spectropho-tometrically measured at 412 nm and expressed asmicromoles of reduced GSH per minute per milli-gram of protein.

– Determination of superoxide dismutase activityThe superoxide dismutase (SOD) activity was

determined by following the manufacturer’s protocolusing SOD determination kit (cat. no. 19160) pur-chased from Sigma-Aldrich. Indeed, the cell homog-enate (20 μl) was added to 200 μl of the kitworking solution. The mixture was incubated at37°C for 20 min after gentle shaking and adding20 μl of the kit enzyme working solution. Theabsorbance of the mixtures was measured spectro-photometrically at 450 nm using a microplate reader

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and the SOD activity was calculated using the fol-lowing equation:

% SOD activity ¼ Ablank1−Ablank3ð Þ− Asample−Ablank2

� �Ablank1−Ablank3

� 100

where blank 1 was a mixture of the workingsolution, enzyme working solution, and ddH2O;blank 2 was a mixture of the cell homogenate,working solution, and dilution buffer; and blank 3was a mixture of the working solution, ddH2O, anddilution buffer.

– Determination of catalase activityThe catalase activity in the cell supernatants was mea-

sured by following the manufacturer’s protocol using theAmplex Red Catalase Assay kit (Molecular Probes, Eu-gene, OR).

Statistical analysis. Data is reported as mean±SEM. TheStatistical analyses were carried out on an XLSTAT 2012.One-way ANOVA with Duncan’s multiple range tests wasused to examine the difference between groups. p values<0.05 were considered significant.

Results

1H NMR spectra results. The 1H NMR spectrum (a) of HE ofR. chalepensis L. shows that lipids are the major organiccompounds in this extract (characteristic profiles between0.5 and 2.5 ppm), whereas the proton spectrum (d) of theaqueous extract of R. chalepensis L. essentially reveals thepresence of carbohydrates observed between 3.0 and 5.5 ppm(characteristic signals of the α-glucose anomeric proton: δ=5.22 and β-glucose anomeric proton: δ=4.60 ppm). What isworthy to note is the very low presence of aromatic signals(δ=5.5 to 9 ppm) which indicate the existence of phenoliccompounds such as flavonoids and polyphenols in these twoextracts of R. chalepensis L.

The 1H NMR spectra of the EE (b) and EAE (c) ofR. chalepensis L. confirm the presence of three major groupswhich are as follows: lipids (δ=0.5 to 2.5 ppm), carbohydrates(δ=2.5 to 5.5 ppm), and aromatic compounds (δ=5.5 to9 ppm). Both extracts contain very less quantities of lipidand carbohydrates than the HE and AE, respectively. Howev-er, the aromatic peaks confirming the presence of phenoliccompounds such as flavonoids and polyphenols are higher inEE and EAE than in the two other extracts (Fig. 1).

Total phenolic contents. As shown in Table 1, the TPCs of thedifferent extracts of R. chalepensis L. were examined. This

compound showed differences in their total contents depend-ing on the solvents’ polarities. The highest content of the totalphenolics was found in the EE (178 mg GAEs/g dry extract)followed by the EAE (142.5 mg GAEs/g dry extract) and theAE (120.5mgGAEs/g dry extract). The content obtained withHE was much smaller (15 mg GAEs/g dry extract).

DPPH radical scavenging activity. The DPPH assay consti-tutes a rapid and low cost method that has frequently beenused for the evaluation of the antioxidative potential of variousnatural products (Bouaziz et al. 2005).

A high percentage of radical scavenging indicated a strongantioxidant activity in the tested sample. As shown in Fig. 2,the DPPH test reveals that the increase in extract concentrationresults in the increase in free radical scavenging activity in adose-dependent manner. Then, this low concentration andhigh inhibition show a high scavenging activity. Indeed, at aconcentration of the order of 500 μg/ml, these extracts wereable to discolor DPPH and the free radical scavenging poten-tials of these extracts were found to be in the following order:EE (84.83±0.29%)>EAE (58.63±3.96%)>AE (46.10±0.14%)>HE (19.75±0.36%). These activities for differentextracts of R. chalepensis L. are significantly lower than theascorbic acid (85.34±0.70). It can be inferred from the ob-tained results that these extracts of R. chalepensis L. reducethe radical DPPH when it reacts with the hydrogen ionsreleased from the samples containing antioxidants.

Cell toxicity of R. chalepensis L. extracts. The MTT assayshowed that the EE, EAE, and AE of R. chalepensis L. werenot cytotoxic for murine macrophage cells (RAW 264.7).Indeed, Fig. 3 demonstrates that up to a concentration of500 μg/ml, these extracts did not affect the cell viability after18 h of incubation. On the other hand, HE presented a toxiceffect on murine macrophage cells (RAW 264.7).Thus, celltreatment with increasing concentrations (0, 30, 100, 200,300, and 500 μg/ml) of R. chalepensis L. hexane extract for18 h exhibited a significant dose-dependent decrease in cellviability. This reduction can reach up to 61% at 500 μg/ml ofHE of R. chalepensis L.

R. chalepensis L. extracts inhibit LPS-induced NO productionin macrophages. Because NO is known to be a proinflamma-tory mediator in many different acute and chronic inflamma-tory diseases (Payne 2003), we investigated whether the dif-ferent extracts of R. chalepensis L. such as EE, EAE, AE, andHE inhibited NO production from the RAW 264.7 that werestimulated with LPS. As shown in Fig. 4, the NO concentra-tion increased sharply after LPS stimulation in murine mac-rophage cells (RAW264.7). This significant increase of NO inthe group of cells treated with LPS without plant extracts wasfound to reach 30±0.58 μM. To assess the effects ofR. chalepensis L. extracts on NO production in RAW 264.7

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cells, cell culture mediumwas harvested and the production ofnitrite was measured using the Griess method. Figure 4 showsan inhibitory effect on LPS-induced NO production in a dose-dependent manner concerning the EE and EAE ofR. chalepensis L. The treatment with the ethanol and ethylacetate extracts of R. chalepensis L. at 300 μg/ml suppressedNO production to 58 and 63% of LPS-treated control, respec-tively. It was also shown (Fig. 4) that the NO production in thecells stimulated with LPS decreases when using the hexaneextracts. In addition, the 1H NMR spectra of R. chalepensis L.hexane extract has shown that lipids are the major organiccompounds in this extract. However, the aromatic com-pounds, confirming the presence of phenolic compounds suchas flavonoids and polyphenols, are present in minor amounts.Thus, the decrease of NO in RAW 264.7 cells is related to thedecrease of cell viability with the hexane extract. On the otherhand, the aqueous extract of R. chalepensis L. has no inhibi-tory effect on the NO production in the RAW264.7 stimulatedwith LPS. This result may be explained by the fact that thisextract contains a very small quantity of aromatic compounds.

In fact, it is formed mainly by carbohydrates which have noeffect on the reduction of NO production. Therefore, we havefocused on the R. chalepensis L. ethanol and ethyl acetateextracts since they are the only noncytotoxic extracts having asignificant inhibitory effect on NO production.

Inhibition of iNOS gene expression in macrophages RAW264.7 by plant extracts. The quantitative real-time PCR anal-ysis was performed to determine whether the inhibitory effectof the R. chalepensis L. ethanol and ethyl acetate extracts onthese inflammatory mediators such as NO was related to amodulation of iNOS enzymes. The cells were pretreated for2 h with various concentrations of EE and EAE ofR. chalepensis L. and then stimulated with LPS (1 μg/ml)for 4 h at 37°C in a humidified atmosphere containing 5%CO2. In the untreated control group (negative control withoutLPS treatment), iNOS mRNA expression was not detectable.The mRNA expression of iNOS was significantly increasedupon LPS treatment (p<0.001), and this induction wasinhibited in a dose-dependent manner by both R. chalepensisL. extracts (Fig. 5).

Inhibition of COX-2 gene expression in macrophages RAW264.7 by plant extracts. The quantitative real-time PCR anal-ysis was performed to determine whether R. chalepensis L.ethanol and ethyl acetate extracts inhibited the COX-2 geneexpression in murine macrophage RAW 264.7 cells. The cellswere pretreated for 2 h with various concentrations of EE andEAE of R. chalepensis L. and then stimulated with LPS(1 μg/ml) for 4 h at 37°C in a humidified atmosphere con-taining 5% CO2. Thus, in unstimulated RAW 264.7 cells,

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Figure 1. 1H NMR spectra:hexane extract (HE) (a), ethanolextract (EE) (b), ethyl acetateextract (EAE) (c), aqueous extract(AE) (d), of R. chalepensis L.

Table 1. Total phenol content (TPC) of different extracts ofR. chalepensis L.

Extracts TPC (mg GAEs/g dry extract)a

Ethanol extract (EE) 178±0.00

Ethyl acetate extract (EAE) 142.5±6.36

Aqueous extract (AE) 120.5±4.95

Hexane extract (HE) 15±0.01

a Values expressed are means±SD of three parallel measurements

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COX-2 mRNAwas not detectable. In response to LPS, COX-2 mRNA production was inhibited in a dose-dependent man-ner by both of the R. chalepensis L. extracts (Fig. 6).

NF-κB activity. NF-κB activation, an important transcriptionfactor for inflammatory mediation, was measured to evaluatethe effect of EE and EAE ofR. chalepensis L. on LPS-inducedinflammation. The results showed that NF-κB p65 was hardlydetected in the untreated group (DMEM only), but increasedin the LPS-treated control group. The preincubation of RAW264.7 cells in the presence of ethanol and ethyl acetate extractsof R. chalepensis L. at different concentrations (0, 100, 300,

and 500 μg/ml) showed an inhibitory effect on NF-κB p65activity. These results suggest that both extracts ofR. chalepensis L. were associated with the downregulationor degradation of NF-κB p65 protein in LPS-stimulated mac-rophages (Fig. 7).

Status of oxidative stress and antioxidative enzymeactivities. To assess the defensive function of EAE and EEof R. chalepensis L. against oxidative stress, the markers ofoxidative stress status, such as the levels of TBARS andantioxidative enzyme activities, were investigated. As shownin Fig. 7, the exposure of RAW 264.7 cells to LPS induced a

Figure 2. Free radicalscavenging capacities of differentextracts of R. chalepensis L. andascorbic acid as positive controlmeasured by DPPH assay. Valuesexpressed are means±SEM ofthree parallel measurements.

Figure 3. Effect of differentextracts of R. chalepensis L.(ethanol (a), ethyl acetate (b),aqueous (c), and hexane (d)) onthe viability of RAW 264.7 cells.Cells were incubated withincreasing concentrations (0, 30,100, 200, 300, and 500 μg/ml) ofR. chalepensis L. extract during18 h at 37°C in a humidifiedatmosphere containing 5% CO2.Three independent experimentswere performed, and data areexpressed as the means±SEM.#p<0.05 and ###p<0.001compared to normal cell.

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significant increase of TBARS level (p<0.001) in thesupernatant medium. After the pretreatment of RAW264.7 cells with various concentrations (0, 30, 100, 200,300, and 500 μg/ml) of EE and EAE of R. chalepensis L.for 2 h, followed by 16 h of LPS (1 μg/ml) incubation at37°C, the TBARS production was inhibited in a dose-dependent manner by these extracts, suggesting that lipidperoxidation was attenuated in LPS-stimulated macro-phages by R. chalepensis L. extract. This decrease ofTBARS level can reach 1.55±0.09 and 1.02±0.24 nmolfor EE and EAE, respectively. With regard to the antiox-idant enzymes, Fig. 8 shows that the treatment of murinemacrophage cells (RAW 264.7) with LPS induced a sig-nificant decrease in antioxidant enzyme (catalase (CAT),GPx, and SOD) activities compared to the untreated con-trol group. These enzyme levels are enhanced significant-ly (p<0.05) in LPS-treated RAW 264.7 cells preincubatedwith various concentrations (30, 100, 200, 300, and500 μg/ml) of the ethanol and ethyl acetate extracts ofR. chalepensis L. when compared with those determinedin LPS-only-treated cells (Fig. 9).

Discussion

NO is produced by activated macrophages as a result ofinduction by several stimuli, including TNF-α, IFN-γ,and LPS. The NO may react with superoxide to formperoxynitrite, which contributes to the etiology of cardio-vascular diseases and cancer by promoting oxidativestress and inflammation processes (Gobert et al. 2002;Murakami et al. 2003). In the present study, we haveshown that NO production increased considerably inLPS-treated cells compared to LPS-untreated negativecontrol, which is in accordance with the results found bymany researchers (Kang et al. 2000; Ippouchi et al. 2002;Wang and Mazza 2002). In addition, the LPS-treated cellsexhibited an increase in the levels of the mRNA expres-sion of iNOS and COX-2 compared to the untreated cells.These results were confirmed by Park et al. (2005). Ac-tually, LPS, which is an endotoxin, plays a key role in theappearance and development of a shock state during in-fections with Gram-negative bacteria. In fact, the inflam-matory response to infection is initiated by the binding of

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- + + + + + + - + + + + + +Figure 4. Effect of different extracts of R. chalepensis L. (ethanol (a),ethyl acetate (b), aqueous (c), and hexane (d)) on LPS-induced NOproduction in macrophages RAW (264.7). Cells were preincubated withdifferent concentrations (0, 30, 100, 200, 300, and 500 μg/ml) ofR. chalepensis L. extract for 2 h and then incubated with LPS (1 μg/ml)

for 16 h at 37°C in a humidified atmosphere containing 5%CO2. NOwasmeasured by the Greiss reaction. Three independent experiments wereperformed, and data are expressed as the means±SEM. *p<0.05,**p<0.01, and ***p<0.001 versus LPS alone. ###p<0.001 comparedto normal cell.

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microbial products such as the bacterial cell wall lipo-polysaccharide components to Toll-like receptor-4 (TLR-4). Subsequently, the NF-κB will be activated due to thecleavage of the NF-κB complex and the release of theinhibitory kappa B (IκB). The activated NF-κB is capableof passing through the nuclear membrane. The transloca-tion of NF-κB to the nucleus induces the transcription of awide variety of genes and leads to the expression ofsignificant circulating levels of proinflammatory mole-cules. Such molecules include cell adhesion moleculessuch as intercellular adhesion molecule 1 (ICAM-1); cy-tokines such as IL-1β, TNF-α, and IL-6; chemokinessuch as regulated upon normal T-cell expressed and se-creted protein (RANTES), monocytes chemoattractantprotein-1 (MCP-1), IL-8; and other enzymes such asiNOS and COX-2 (Blackwell and Christman 1997;Christman et al. 1998, 2000; Lee et al. 2003; Liu andMalik 2006; Carré and Singer 2008; O’Sullivan et al.2009). The production of prostaglandins in LPS-treatedmacrophages is catalyzed mainly by the transcriptional

activation of the COX-2 gene. This represents a crucialstep in the inflammatory process (Lee et al. 1992; Reddyand Herschman 1994).

The defense against the negative effect of LPS consists ofan antioxidant synthesized in the tissues and an exogenousantioxidant supplied by medicinal plants. In this context, theaerial part of R. chalepensis L. was used in the present study toreduce the oxidant status and inflammation, based on thepharmacological activity of its extracts onmurinemacrophageRAW264.7 cells challengedwith LPS.We have demonstratedthat the R. chalepensis L. ethanol and ethyl acetate extractssignificantly inhibited the nitrite level and intracellular oxida-tive stress in LPS-stimulated murine macrophage (RAW264.7) cells without showing any cell toxicity. Furthermore,the inhibitory action of the R. chalepensis L. ethanol and ethylacetate extracts on LPS-induced NO production appears toinvolve the inhibition of iNOS gene expression. It is worthnoting that both plant extracts have also shown a decrease ofCOX-2 gene expression. These results could be explained bythe fact that these two R. chalepensis L. extracts rich in

Figure 5. Effects of ethanolextract (EE) (a) and ethyl acetateextract (EAE) (b) ofR. chalepensis L. on LPS-inducediNOS mRNA expression. RAW264.7 cells were pretreated withvarious concentrations of testextracts (0, 100, 300, and500 μg/ml) for 2 h and thenchallenged with 1 μg/ml LPS for4 h at 37°C in a humidifiedatmosphere 5% CO2. Threeindependent experiments wereperformed, and data are expressedas the means±SEM. *p<0.05 and**p<0.01 versus LPS alone.###p<0.001 compared to normalcell.

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Figure 6. Effects ofR. chalepensis L. ethanol extract(EE) (a) and ethyl acetate (EAE)extract (b) on LPS induced COX-2 mRNA expression. RAW 264.7cells were pretreated with variousconcentrations of test extracts (0,100, 300, and 500 μg/ml) for 2 hand then challenged with 1 μg/mlLPS for 4 h at 37°C in ahumidified atmosphere 5% CO2.Three independent experimentswere performed, and data areexpressed as the means±SEM.*p<0.05 and **p<0.01 versusLPS alone. ###p<0.001compared to normal cell.

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phenolic compounds (as shown in 1H NMR spectra) possessan important antioxidant activity revealed by the DPPH test.

This result accords well with that reported in literature. Infact, the photochemical screening of the aerial parts ofR. chalepensis L. characterizes the presence of coumarins,alkaloids (Ulubelen et al. 1986), as well as flavonoids (Al-Said et al. 1990). The pharmacological investigations clearlyindicated that plant flavonoids have various properties such asantioxidant (Ng et al. 2000), anticancer, and anti-inflammatory activities (Raghav et al. 2006) and inhibit lipidperoxidation in biological membranes (Maridonneau-Pariniet al. 1986). Flavonoids show strong inhibitory effects on

the expression of COX-2 (Takano-Ishikawa et al. 2006) andiNOS (Kim et al. 1999) in macrophage cells due to their C2-C3 double bond and 4-oxo functional group of the C-ring,which are important factors for a high level of inhibitoryactivities. Rutin, one of the flavonoid constituents of Rue, iswell known to have nitric oxide scavenging activity (Vanackeret al. 1995). According to the available literature (Fleminget al. 2000), the R. chalepensis L. plant contains approximate-ly 2–5% of rutin. Recently, several studies have reported thatrutin decreases nitric oxide with the reduction in the iNOSprotein in BALB/c mice pretreated with LPS (Shen et al.2002). In the same vein, Iauk et al. (2004) have demonstratedthat R. chalepensis L. extract inhibits LPS-induced nitricoxide production in BALB/c mice.

COX-2 is an inducible enzyme catalyzing the conversionof arachidonic acid to prostaglandins which play a crucial roleas mediators of inflammatory responses (Fu et al. 1990). Thepresent study has also demonstrated that the R. chalepensis L.ethanol and ethyl acetate extracts significantly decreased theCOX-2 gene expression, which is confirmed by several re-searchers. Indeed, Shen et al. (2002) have shown that querce-tin (a derivative of rutin devoid of a glycan called rutinose)decreases in prostaglandin level associated with a decrease inCOX-2 protein expression in vitro in LPS-treated murinemacrophages but failed in vivo. Similarly, Ashour et al.(2011) have demonstrated that the ethanolic extract ofR. chalepensis decreases the activity of COX in the monocyteextracted from hypercholesteromic rats. The inhibition of bothiNOS and COX-2 gene expressions by the present extract

Figure 7. Effects of R. chalepensis L. ethanol extract (EE) and ethylacetate extract (EAE) on LPS-induced NF-κB p65 in RAW 264.7 cells.Cells were pretreated with different concentrations (0, 100, 300, and500 μg/ml) of ethanol extract (EE) and ethyl acetate extract (EAE) ofR. chalepensis L. for 2 h and then stimulated with 1 μg/ml of LPS orwithout LPS for 30 min at 37°C in a humidified atmosphere containing5% CO2. The NF-κB p65 protein was analyzed by Western blot. Eachexperiment was repeated two times.

Figure 8. Effects of ethanol extract (EE) (a) and ethyl acetate extract(EAE) (b) of R. chalepensis L. on TBARS generation in LPS-stimulatedRAW 264.7 macrophages. Cells were preincubated with different con-centrations (0, 30, 100, 200, 300, and 500 μg/ml) of ethanol extract (EE)and ethyl acetate extract (EAE) of R. chalepensis L. extract for 2 h and

then incubated with LPS (1 μg/ml) for 16 h at 37°C in a humidifiedatmosphere containing 5% CO2. Three independent experiments wereperformed, and data are expressed as the means±SEM. *p<0.05 and**p<0.01 versus LPS alone. ###p<0.001 compared to normal cell.

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reveals the possible involvement of the NF-κB, which is acommon activator of iNOS and COX-2 promoters (Nakaoet al. 2000). In an in vitro model, Ruta graveolens L., a relatedplant, has been found to reduce nitric oxide production inmurine macrophage cell line (J-774) challenged with LPSdue to the reduction of iNOS gene transcription through theinhibition of NF-κB activation (Raghav et al. 2006).

In macrophages and invading immune cells, the highamount of NO produced by the upregulated iNOS in responseto LPS plays a crucial role in oxidative stress. Indeed, theproduced high NO level reacts rapidly with superoxide (O2

−)to form cytotoxic oxidant peroxynitrite (ONOO−), which con-tributes to the damage of many biological molecules includingDNA, lipid, and protein and can lead to amplification ofinflammation and tissue injury (Yen and Lai 2002). Theoxidative stress-induced damage may disrupt cellular functionand membrane integrity, thus leading to cell death (Shieh et al.2010). Our data have shown that the R. chalepensis L. ethanol

and ethyl acetate extracts ameliorate LPS-induced oxidativestress, as indicated by the suppressed MDA concentrations,through the elevation of antioxidative enzyme activities, suchas catalase, SOD, and GPx. Therefore, the superoxide dismut-ase forms the first line of defense against superoxide radicals(first toxic species formed from oxygen) to form hydrogenperoxide (H2O2) and O2 and hence against oxidative stress(Scott and Malcolm 2008). Catalase mediates its function bythe removal of H2O2 generated by the auto-oxidation of lipidsand the oxidation of organic substances (Sharma et al. 1991).Glutathione peroxidase is an enzyme that needs glutathioneand selenium to function properly. Its main role is to eliminatelipid peroxides resulting from the effect of oxidative stress onthe polyunsaturated fatty acids. Our study has revealed thatGPx, SOD, and catalase activity was significantly decreasedby LPS treatment. However, R. chalepensis L. ethanol andethyl acetate extracts restored the activities of antioxidativeenzymes compared to LPS-treated controls. The elevation of

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Figure 9. Effects of ethanol extract (EE) (a) and ethyl acetate extract(EAE) (b) of R. chalepensis L. on enzyme activities in LPS-stimulatedRAW264.7. Cells were preincubated with different concentrations (0, 30,100, 200, 300, and 500 μg/ml) of ethanol extract and ethyl acetate extractofR. chalepensisL. extract for 2 h and then incubated with LPS (1 μg/ml)

for 16 h at 37°C in a humidified atmosphere containing 5% CO2. Threeindependent experiments were performed, and data are expressed as themeans±SEM. *p<0.05, **p<0.01, and ***p<0.001 versus LPS alone.#p<0.05 and ##p<0.01 compared to normal cell.

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antioxidative enzyme activity may be responsible for thesuppression of oxidative stress in LPS-stimulated RAW264.7 cells. In an in vivo model, R. graveolens L., a relatedplant has been found to reduce oxidative stress by decreasingTBARS level and increasing the activities of antioxidantenzymes such as SOD, CAT, and GPx in the liver and heartin hypercholesteromic rats (Ratheesh et al. 2011). Similarly,Ashour et al. (2011) have shown that the treatment with theethanolic extract of R. chalepensis L. could reduce oxidativestress as well as inflammation in hypercholesteromic rats. Inin vitro experiments, it was shown that a low concentration ofR. graveolens extract was able to scavenge hydroxyl radicaland inhibit lipid peroxidation (Preethi et al. 2006). It has beenreported that plant extracts containing high total phenol con-centrations show strong antioxidative capacity (Velioglu et al.1998).

Conclusion

It can be concluded that the ethanol and ethyl acetate extractsof R. chalepensis L. inhibit the secretion of NO inflammatorymediator, suppress the expression of iNOS and COX-2 genesthrough the inhibition of NF-κB activation, and protectagainst oxidant stress in RAW 264.7 macrophage cells stim-ulated with LPS. Therefore, these anti-inflammatory and an-tioxidative properties of R. chalepensis L. seem to be impor-tant enough to suggest that this plant may be used as avaluable, safe, and effective therapy for a variety of diseasescaused by oxidative stress and inflammation.

Acknowledgments This work was supported by the Tunisian Ministryof Higher Education and Scientific Research. We are grateful to CMazière and Cathy Gomila for kindly providing RAW 264.7 cells. Weextend our thanks to all the members of the Laboratory of Neurosciencesof Brest (LNB) for being an important source of support and to Mr.Hafedh Bjaoui, an English teacher at the Sfax Faculty of Sciences, forproofreading this paper.

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