A fraction of stem bark extract of Entada africana suppresses lipopolysaccharide-induced inflammation in RAW 264.7 cells

$Page 1: A fraction of stem bark extract of Entada africana suppresses lipopolysaccharide-induced inflammation in RAW 264.7 cells$
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Journal of Ethnopharmacology

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A fraction of stem bark extract of Entada africana suppresseslipopolysaccharide-induced inflammation in RAW 264.7 cells

Brice Ayissi Owona a,b,n, Nico Frederic Njayou b, Stefan Laufer c, Paul Fewou Moundipa b,Hermann J. Schluesener a

a Division of Immunopathology of the Nervous System, Department of Neuropathology, Institute of Pathology, University of Tübingen, Germanyb Laboratory of Pharmacology and Toxicology, University of Yaoundé I, Cameroonc Medical Chemistry, Department of Pharmacy & Biochemistry, Eberhard-Karls-Universität Tübingen, Auf der Morgenstelle 8, 72076 Tübingen, Germany

a r t i c l e i n f o

Article history:Received 20 March 2013Received in revised form12 June 2013Accepted 12 June 2013

Keywords:Entada africanaBaicalinMacrophagesCytokinesp38 MAPK kinase

41/$ - see front matter & 2013 Elsevier Irelanx.doi.org/10.1016/j.jep.2013.06.016

viations: Ea5, Entada africana fraction CH2CLide synthase; MTT, 3-(4,5-dimethylthiazol-2-; NO, nitric oxide; ROS, reactive oxygen speckinase.esponding author at: Division of Immunopathent of Neuropathology, Institute of Patholoy. Tel.: +49 15156219006; fax: +49 70 7129 48ail address: [email protected] (B. Ayissi O

e cite this article as: Ayissi Owona,ced inflammation in RAW 264.7 cell

a b s t r a c t

Ethnopharmacological relevance: Entada africana is a plant used in African traditional medicine for thetreatment of stomachache, fever, liver related diseases, wound healing, cataract and dysentery.Aims of the study: This study aimed at evaluating the anti-inflammatory activity of fractions of the stembark extract of the plant using lipopolysaccharide (LPS)-induced inflammation in RAW 264.7macrophages model.Materials and methods: The crude extract was prepared using the mixture CH2Cl2/MeOH (1:1, v/v) andfractionated by flash chromatography using solvents of increasing polarity to obtain five differentfractions. The effects of the fractions on the cells viability were studied by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and their inhibitory activity against LPS-induced nitricoxide (NO) production screened by Griess test. The most active fraction was further investigated for itseffects on reactive oxygen species (ROS) production using flux cytometry, the expression of induciblenitric oxide synthase (iNOS), pro-and anti-inflammatory cytokines (IL1β, TNFα, IL6, IL10 and IL13) by RT-PCR, and the activity of the enzyme p38 MAPK kinase by enzyme-linked immunosorbent assay (ELISA).Results: The fractions presented no significant effect on the viability of macrophages at 100 μg/ml after24 h incubation. The CH2Cl2/MeOH 5% (Ea5) fraction was found to be the most potent in inhibiting NOproduction with a half inhibition concentration (IC50)¼18.36 μg/ml, and showed the highest inhibitionpercentage (89.068%) in comparison with Baicalin (63.34%), an external standard at 50 μg/ml. Ea5, as wellas Baicalin significantly (Po0.05) inhibited the expression of TNFα, IL6 and IL1βmRNA, attenuated mRNAexpression of inducible NO synthase in a concentration-dependent manner, stimulated the expression ofanti-inflammatory cytokines (IL10 and IL13), and showed a 30% inhibition of the activity of p38 MAPKkinase.Conclusion: The results of the present study indicate that the fraction Ea5 of Entada africana possessesmost potent in vitro anti-inflammatory activity and may contain compounds useful as a therapeuticagent in the treatment of inflammatory related diseases cause by over-activation of macrophages.

& 2013 Elsevier Ireland Ltd. All rights reserved.

1. Introduction

Inflammation is considered to be the major cause of mostchronic diseases like diabetes, Alzheimer's disease, asthma, andatherosclerosis (Yu et al., 2012). Many biological molecules

d Ltd. All rights reserved.

2/MeOH 5%; iNOS, inducibleyl)-2,5-diphenyltetrazoliumies; MAPK, mitogen activated

ology of the Nervous System,gy, University of Tübingen,4.wona).

B., et al., A fraction of stems. Journal of Ethnopharmaco

including reactive oxygen species (ROS), nitric oxide (NO) andcytokines such as IL1β, TNFα and IL6 are known to be involved inthe development of inflammation (Cui et al., 2012). Macrophagesare major immune cells that act as a first line of defense againstinvading agents (bacteria, virus, and fungi), and they respond topathogen attack by release of ROS and NO, known as cellularsignaling molecules and antimicrobial agents (Blazovics et al.,2004). However, an exaggerated production of inflammatorymediators by macrophage defensive systems can cause damageto the host (Huo et al., 2012).

Macrophages ROS are, for example, important contributors tothe manifestation of allergic inflammation (Varga et al., 2013) andalso involved in TNFα (Haddad and Land, 2002) and IL1β produc-tion in LPS stimulated macrophages (Ryan et al., 2004). During

bark extract of Entada africana suppresses lipopolysaccharide-logy (2013), http://dx.doi.org/10.1016/j.jep.2013.06.016i

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infection, LPS acts on cellular Toll like receptor 4 (TLR4), activatesdifferent pathways in various cells (macrophages, Kuppfer cellsand hepatocytes) (Chen et al., 2001) and induces nitric oxidesynthase (iNOS) as well as the production of pro-inflammatoryagents, including cytokines (IL1β, TNFα, and IL6), prostaglandinsand NO (Glushkova et al., 2012). On the other hand, activatedmacrophages also produce anti-inflammatory cytokines such asIL10 and IL13, which are useful to slow down and terminate theinflammatory response (Abuarqoub et al., 2006).

Many pathways have been described to explain the effect ofanti-inflammatory compounds on macrophages. It is the case ofthe mitogen activated protein kinases (MAPKs), a specific class ofserine/threonine kinases, which respond to extracellular signals(LPS, virus and cytokines). In the field of inflammation, the p38MAPK kinases are described as stress-activated protein kinases(Correa and Eales, 2012) and many inhibitors of these kinases aremore and more investigated for their anti-inflammatory effects(Chao et al., 2007; Rafiee et al., 2009).

The development of modulators such as Baicalin (BA), aflavonoid compound purified from the medicinal plant Scutellariabaicalensis Georgi for targeting the LPS signaling cascade inmacrophages, is an attractive strategy for the therapy of inflam-matory diseases (Hu et al., 2012). BA has been shown to possessanti-inflammatory activity in many in vitro models of inflamma-tion (Li et al., 2012; Liu et al., 2008; Zhu et al., 2012) and it hasbeen reported to inhibit NF-kappaB activation in cigarette smokeinduced inflammatory models (Lixuan et al., 2010). Medicinalplants are therefore an important potential source of anti-inflammatory compounds.

Entada africana is a plant used in African traditional medicinefor the treatment of stomachache, wound dressing, prevention ofsuppuration, treatment of fever (Obidike and Emeje, 2011), liverrelated diseases, wound healing, skin-eruptions, rheumatism,cataract, fevers and dysentery (Karou et al., 2011). Studies onEntada africana report its antimicrobial, antiplasmodial, antioxi-dant (Karou et al., 2011), anti-proliferative (Cioffi et al., 2006),complement fixing (Diallo et al., 2001), fungistatic and fungicidalactivities (Fabry et al., 1996). Recent publications from our researchgroup report the hepatoprotective and antioxidant effects of thestem bark of Entada africana (Njayou et al., 2013) as well as itsimmunomodulatory activity on rat peritoneal macrophages(Owona et al., 2013).

To the best of our knowledge, anti-inflammatory and macro-phage regulation of NO, ROS, and cytokines mRNA expression byEntada africana have not been reported. The present study demon-strated the effect of Entada africana on the release of inflammatorymediators. We also showed the effect of the most active fractionon the activity of p38 MAPK kinase, with Baicalin as referenceanti-inflammatory compound.

2. Material and methods

2.1. Plant materials and solvent extraction

Entada africana was harvested in 2010, in the West region ofCameroon. Voucher specimens were deposited at the NationalHerbarium, Yaoundé, Cameroon (52661 YA). Dried Entada africanabarks were air dried, cut into small pieces and ground. Onekilogram of the powder was immersed and extracted in methylenechloride/methanol 1/1 v/v at room temperature for 7 days. Afterthe mixture was filtered, the filtered cakes were extracted andfiltered three more times to increase the extraction yield. Theprocedure was repeated until the solvent present a clear color. Thefiltrate was concentrated under reduced pressure, and the crudeextract obtained was freeze-dried, and stored at 4 1C until used.

Please cite this article as: Ayissi Owona, B., et al., A fraction of steminduced inflammation in RAW 264.7 cells. Journal of Ethnopharmaco

The crude extract was subjected to flash chromatography to yield atotal of five fractions, namely EaChl (CH2Cl2), Ea5 (CH2Cl2/MeOH5%), Ea10 (CH2Cl2/MeOH 10%), Ea25 (CH2Cl2/MeOH 25%), andEaMet (MeOH).

2.2. Chemicals

Fetal bovine serum (FBS), antibiotics (streptomycin/penicillin),and RPMI medium were purchased from Gibco (Grand Island, NY,USA). Escherichia coli-LPS and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased fromSigma-Aldrich (St. Louis, MO, USA). Baicalin (99%) was purchasedfrom Carbosynth Ltd. (Compton, Berkshire, UK). Mouse cytokinesprimers (iNOS, TNFα, IL6, IL1β, IL13 and IL10) were supplied bySanta Cruz Biotechnology (Santa Cruz, CA, USA).

2.3. In vitro cell culture

The primary mouse macrophage cell line RAW 264.7 was usedto determine the effects of Entada africana on macrophage in vitro.The cells were cultured in RPMI medium (Life Technologies)containing penicillin (100 U/ml), and 10% fetal bovine serum. Cellswere cultured at 37 1C in a humidified incubator in an atmosphereof 5% CO2. RAW 264.7 cells were grown in 12-well plates at adensity of approximately 1�105 cells per well. The plant com-pounds were dissolved in dimethylsulfoxide (DMSO) and filteredthrough 0.45 μm cellulose membranes.

2.4. MTT assay for measuring cell proliferation

The cytotoxic effects of the crude extract and fractions wereevaluated by a MTT assay. Cells were seeded in 12-well plates(1�105 cells/well) and incubated for 24 h. After this incubationperiod, cells were treated with various concentrations of Entadaafricana (0.01, 0.1, 1, 10, and 100 μg/ml), Baicalin (5 μg/ml) and LPS(1 μg/ml) at 37 1C in 5% CO2 for 24 h. For different plant fractions,the same test was realized at plant final concentrations of 100 μg/ml. After treatment, 100 μL of MTT (5 mg/ml) dissolved in RPMIwas added to each well, followed by incubation for 3 h. Themedium was aspirated, and the formazan crystals were dissolvedin 500 μL of DMSO for 15 min. The optical density of each well wasmeasured at 540 nm in a microplate reader.

2.5. Determination of nitric oxide production

Production of NO was determined by measuring the accumu-lated level of nitrite, an indicator of NO in the supernatant after24 h of LPS treatment with or without different concentrations ofplant material and Baicalin. After pre-incubation of cells (1�105

cells) for 24 h, Baicalin, the plant crude extract (0. 01, 0.1, 1, 10 and100 μg/ml) or fractions (0.05, 0.5, 5, and 50 μg/ml) were added,together with LPS (1 μg/ml). The cells were further incubated at37 1C in 5% CO2 for 24 h. The quantity of nitrite in the culturemediumwas measured as an indicator of NO production. Amountsof nitrite, a stable metabolite of NO, were measured using Griessreagent (1% sulfanilamide and 0.1% naphthyl ethylene diaminedihydrochloride in 2.5% phosphoric acid). Briefly, 50 μl of cellculture medium was mixed with 100 μl of Griess reagent. Subse-quently, the mixture was incubated at room temperature for10 min and the absorbance at 570 nm was measured in a micro-plate reader. Fresh culture medium was used as a blank in everyexperiment. The quantity of nitrite was determined from a sodiumnitrite standard curve.





Table 1Primers used for RT-PCR analysis (F: forward and R: reverse).

Gene Primer sequences

TNFα F5′-TTGACCTCAGCGCTGAGTTG-3′R5′-CCTGTAGCCCACGTCGTAGC-3′

IL1β F5′-CAGGATGAGGACATGAGCACC-3′R5′-CTCTGCAGACTCAAACTCCAC-3′

IL6 F5′-GTACTCCAGAAGACCAGAGG-3′R5′-TGCTGGTGACAACCACGGCC-3′

iNOS F5′-CCCTTCCGAAGTTTCTGGCAGCAGC-3′R5′-GGCTGTCAGAGCCTCGTGGCTTTGG-3′

β-actin F5′-CCGTCTTCCCCTCCATCGT-3′R5′-ATCGTCCCAGTTGGTTACAATGC-3′

IL10 F5′-CTGTGAAAACAAGAGCAAGGC-3′R5′-GAAGCTTCTGTTGGCTCCC-3′

IL13 F5′-GGAGCTGGTCAACATCACCC-3′R5′-CGTTGATCAGGGATTCCAGG-3′

Table 2Inhibition of LPS-induced production of nitric oxide by Entada africana fractionsand IC50 values in μg/ml.

Fraction number IC50 in μg/ml % NO inhibition at 50 μg/ml

1-EaChl 61.25 58.52-Ea5 18.36 89.0683-Ea10 42 58.074-Ea25 54.66 69.235-EaMet 35.75 64.82Baicalin – 63.34

B. Ayissi Owona et al. / Journal of Ethnopharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎ 3

2.6. ROS determination

The level of intracellular ROS was determined based on thechange in fluorescence resulting from oxidation of the fluorescentprobe H2DCFDA, as described by Lee et al. (2007). Briefly, 5�105

cells/wells were incubated with extracts or Baicalin for 30 min.After this time, cells were then incubated with LPS (1 μg/ml) as aninducer of ROS production at 37 1C for 6 h. After a final incubationwith H2DCFDA (50 μM) at 37 1C for 1 h, the intracellular ROS levelwas determined using flow cytometry.

2.7. RNA extraction and reverse transcription-polymerase chainreaction (RT-PCR)

RAW 264.7 cells were treated with Baicalin, plant fractions andLPS (1 μg/ml) for 24 h. Total RNA from LPS-treated RAW 264.7 cellswas prepared using the innuPREP RNA Mini kit, according to themanufactures protocol. cDNA (1 μg/ml) was used to perform RT-PCR. The primer sequences used for quantification of iNOS, TNF α,IL1 β, IL6, IL10, IL13 and β-actin and the PCR conditions aredescribed in Table 1. The PCR products were separated by 1.5%agarose gel electrophoresis and visualized by ethidium bromide(EtBr) staining. The gels were then analyzed with ultraviolet trans-illumination. The quantity of each mRNA was calculated usingImageJ software and normalized to the amount of the house-keeping β-actin gene.

2.8. p38 MAPK kinase inhibition assay

The inhibition of p38 MAPK kinase was realized as described byGoettert et al. (2012). Briefly, 96-well plates were coated with ATF-2, overnight, at 4 1C. Blocking buffer was added at room tempera-ture and the plates were incubated for 30 min before the additionof the kinase reaction mix containing the enzyme, with or withouttest compounds. The p38α reactionwas carried out by using kinase(12 ng per well), ATP (100 μM), for 45 min at 37 1C. The ATF-2phosphorylation was detected with a specific anti-phospho ATF-2(Thr69/71) antibody (60 min at 37 1C). After each incubation time,the plate was washed three times with double distilled water. Theoptical density was measured after the addition of the substrate at450 nm, by ELISA. The p38 MAPK kinase inhibitor SB203580 wasused as a reference compound.

2.9. Statistical analysis

All experiments were reiterated at least three times in tripli-cate. The results of multiple observations are expressed as the


mean7SD. Statistical significance was determined by one-wayanalysis of variance (ANOVA) using Graph Pad Prism 4.0 forwindows. For all statistical analyses, significance levels were setat Po0.05.

3. Results

3.1. Effect of Entada africana crude extract and fractions on theviability of RAW 264.7 cells

Examination of the cytotoxicity on RAW 264.7 cells by MTTassay indicated that up to 100 μg/ml for 48 h of incubation, theplant crude extract and fractions did not affect the viability of thecells and likewise, the fractions presented no significant effect onthe viability of RAW 264.7 at 100 μg/ml after 24 h incubation (datanot shown).

3.2. Inhibition of LPS-induced NO production by Entada africanacrude extract and fractions

After treatment with LPS (1 μg/ml) for 24 h, nitrite concentra-tions in the medium increased remarkably by about two fold (datanot shown). When RAW 264.7 cells were treated with differentconcentrations of the samples together with LPS (1 μg/ml) for 24 h,a significant (Po0.05) concentration-dependent inhibition ofnitrite production was detected in the medium. The IC50 valuesof the crude extract and fractions in inhibiting LPS-induced NOproduction is presented in Table 2. For further experiments, Ea5with an IC50¼18.36 μg/ml has been used.

3.3. Inhibitory effect of the fraction Ea5 on LPS-induced ROSproduction in RAW 264.7 cells

Since ROS are known to be representative toxic and pro-inflammatory mediators in many different acute and chronicinflammatory diseases, as well as in normal defense reactions,we addressed whether Ea5 modulated the production of ROS inLPS treated RAW 264.7 cells. As shown in Fig. 1, Ea5 inhibited theLPS induced intracellular production of ROS at 0.05, 0.5 and 5 μg/ml (Po0.05). However, no significant modulation was observed at50 μg/ml.

3.4. Inhibitory effects of the fraction Ea5 on pro-inflammatorycytokines gene expression

We next investigated whether Ea5 suppresses the productionof pro-inflammatory cytokines such as IL6, IL1β and TNFα in LPS-stimulated RAW 264.7 cells. It was observed that 1 μg/ml LPSsignificantly increased TNFα, IL1β and IL6 mRNA levels in RAW264.7 cells. Ea5 significantly (Po0.001) inhibited IL6, TNFα andIL1β mRNA expression at 0.5, 5 and 50 μg/ml for both formercytokines and at all tested concentrations for the later, respectively






(Fig. 2A–C). The same effects were observed with Baicalin, thestandard reference.

3.5. Inhibitory effects of the fraction Ea5 on anti-inflammatorycytokines gene expression

The effect of Ea5 on the expression of anti-inflammatorycytokines mRNA was also examined, as these cytokines are

Fig. 1. Effect of Entada africana fraction CH2CL2/MeOH 5% on the production ofreactive oxygen species in LPS-activated RAW 264.7 cells. RAW 264.7 cells (5�105)were treated with various concentrations of Entada africana and Baicalin in thepresence or absence of LPS (1 μg/ml) for 30 min. The level of generated ROS wasthen determined by flow cytometry analysis as described in the Materials andMethods section. *Po0.05, **Po0.01 and ***Po0.001 compared to LPStreated group.

Fig. 2. Effect of Entada africana fraction CH2CL2/MeOH 5% on LPS-induced expression of pdifferent concentrations of Ea5 and/or Baicalin, with or without LPS for a stimulation pemeasured by RT-PCR. β-actin expression was used as an internal control. (A) IL6, (B) IL1β


reported to slow down and terminate the inflammatory response.As shown in Fig. 3A and B, IL10 and IL13 cytokines mRNAexpression were significantly increased, the later after treatmentwith Ea5 at 50 μg/ml (Po0.001) and the former at all concentra-tions tested (Po0.01). The same effect was observed with Baicalin,concerning the increase of IL13 mRNA expression.

3.6. Inhibitory effect of the fraction Ea5 on LPS induced iNOSexpression in macrophages

We carried out RT-PCR to investigate the question whetherinhibition of NO production was associated with decreased levelsof iNOS expression. Results are displayed in Fig. 4 show that 1 μg/ml LPS significantly increased iNOS mRNA levels after 24 h treat-ment. Ea5 significantly (Po0.001) inhibited the mRNA expressionof iNOS at all the tested concentrations, as did Baicalin.

3.7. Inhibitory effect of the fraction Ea5 on p38 MAPK kinaseinhibition

Ea5 and Baicalin were tested for their ability to inhibit p38MAPK kinase and the p38 MAPK inhibitor SB203580(IC50¼0.04870.01 μM) was used as a reference compound. Asindicated in Fig. 5, Ea5 showed a moderate inhibitory effect on theenzyme over the concentration range tested (0.1–100 μg/ml). Aprominent inhibition was obtained with Baicalin.

ro-inflammatory cytokines in macrophages. RAW 264.7 cells were pre-treated withriod of 24 h. Total RNAs were isolated, and mRNA levels of IL1β, TNFα, and IL6 were, and (C) TNFα. *Po0.05, **Po0.01 and ***Po0.001 compared to LPS treated group.





Fig. 3. Effect of Entada africana fraction CH2CL2/MeOH 5% on LPS-induced expres-sion of anti-inflammatory cytokines in macrophages. RAW 264.7 cells were pre-treated with different concentrations of Ea5 and/or Baicalin, with or without LPSfor a stimulation period of 24 h. Total RNAs were isolated, and mRNA levels of IL10and IL13 were measured by RT-PCR. β-actin expression was used as an internalcontrol. (A) IL10 and (B) IL13.*Po0.05, **Po0.01 and ***Po0.001 compared to LPStreated group.

Fig. 4. Entada africana fraction CH2CL2/MeOH 5% and Baicalin reduced LPS-inducediNOS gene expression in macrophage cell line. RAW 264.7 cells were incubatedwithout or with LPS 1 μg/ml together with the indicated amounts of the extract orBaicalin. After 24 h iNOS mRNA was quantified by qRT-PCR. *Po0.05, **Po0.01and ***Po0.001 compared to LPS treated group.

Fig. 5. Effect of Entada africana fraction CH2CL2/MeOH 5% and Baicalin on theinhibition of p38 MAPK kinase activity. The inhibitory potency of the fraction andBaicalin was evaluated by using p38α ELISA assay containing BSA (0.01%) in thekinase buffer. SB203580 was used as reference compound.



4. Discussion

Nitric oxide is synthesized by the inducible nitric oxidesynthase from L-arginine using NADPH and molecular oxygen bymany inflammatory cells including macrophages, neutrophils andneuronal cells. NO mediates a variety of biological effects includingvasodilatation, neurotransmission, inhibition of platelet adherenceand aggregation, as well as killing of pathogens (Abuarqoub et al.,2006; Oh et al., 2008). It has been demonstrated that induction ofiNOS produces a large amount of NO under inflammatory condi-tions (Chen et al., 2001). Therefore, compounds that inhibit iNOSexpression and NO production may be good candidates in thetreatment of diseases caused by an overproduction of NO (Fischeret al., 1999).

In this study, we explored the effect of Entada africana crudeextract and fractions on NO production by RAW 264.7 cells. Wethen examined the effect of the most active fraction on intracel-lular ROS production by RAW 264.7 cells, as well as on theexpression of iNOS mRNA in these cells. Besides, we exploredthe effect of Ea5 fraction on pro and anti-inflammatory cytokinesmRNA expression, and finally we observed its effect on p38 MAPKkinase inhibition.

Prerequisite for properly evaluating the biological properties ofindividual molecules or complex mixtures is the exact determina-tion of cytotoxicity associated with prolonged incubation of thecells. Thus, the effect of the plant crude extract and fractions oncell viability was assayed, using the MTT assay. The plant extractand fractions presented no significant toxicity on cells viability,thereby indicating that the modulatory effect of the tested sampleson RAW 264.7 cells was not simply due to cytotoxic effects.

The excessive production of NO by macrophages is implicatedin the pathogenesis of many diseases, including Alzheimer disease,cancer, and diabetes. The exposure of RAW 264.7 cells to LPS for24 h was associated with an accumulation of nitrite in themedium, suggesting an enhanced production of NO upon LPStreatment. This overproduction of NO induced by LPS was sig-nificantly inhibited by Entada africana crude extract and fractionsas testify by Baicalin, the reference compound. Based on theirrespective IC50 values, the potency of tested samples might beranked as follows Ea54EMet4Ea104Ea254EaChl (Table 2).Since the fraction Ea5 was the most potent, its activity was furtherstudied.

In murine macrophages, LPS stimulation has been demon-strated to induce iNOS transcription with a corresponding increasein NO production (Yang et al., 2009). Therefore, drug screeningusing nitric oxide and effect on iNOS mRNA expression appear tobe an excellent approach to discover new anti-inflammatorydrugs. Ea5 as well as Baicalin significantly inhibited (Fig. 4) iNOS






mRNA expression in RAW 264.7 cells and from the results it mightbe suggested that the fraction Ea5 exhibit its anti-inflammatoryaction through the inhibition of iNOS as it is reported aboutBaicalin (Hao et al., 2012; Zhu et al., 2012).

Important molecules produced by immune cells during inflam-mation are ROS. These molecules contribute to pathogen andinvader elimination during phagocytosis (Guido et al., 2012).However, an excessive production of ROS may amplify inflamma-tion (Dumont and Beal, 2011). We therefore examined ROSscavenging activity of Ea5 and observed its strong inhibitory action(Fig. 1) on LPS-induced ROS production at the tested concentra-tions. Qualitative and quantitative analysis have revealed thepresence of polyphenols in Entada africana plant crude extract(Njayou et al., 2013, Owona et al., 2013). The activity shown in thisstudy might be due in part to these free radical scavengingproperties of polyphenols.

The effect of Ea5 on the expression of pro and anti-inflammatorycytokines mRNA, including IL1β, TNFα, IL6, IL13 and IL10 was alsoinvestigated. These cytokines amplify inflammation (proinflamma-tory) or attenuate (anti-inflammatory) inflammation, and anycompound that can suppress the excessive production of proin-flammatory cytokines and promote the production of anti-inflammatory cytokines in vitro, may be a good candidate for thediscovery of new anti-inflammatory drugs. As shown in Figs. 3 and4, Ea5, as well as Baicalin significantly suppressed the cytokinesmRNA expression induced by LPS in RAW 264.7 cells and stimulatedIL13 and IL10 mRNA expression, respectively. This observationsupports the anti-inflammatory and the immunomodulatory effectof Ea5. This fraction could therefore contain prominent compoundswith strong anti-inflammatory effects.

The MAPK pathway is responsible for the induction of path-ways leading to the production of proinflammatory cytokines incells. In this assay, the compounds compete with ATP for thebinding site of the kinase and inhibit enzyme activity. P38α MAPKphosphorylates the substrate ATF-2 and the amount of phosphor-ylation reflects the enzyme activity. A 30% inhibition was obtainedwith Ea5 at concentrations ranging from 0.1 to 100 μg/ml (Fig. 5).These results suggest that the inhibition of LPS-induced inflam-mation may partly be due to the inhibition of the p38 MAPKkinase.

Phenolic compounds were detected in high amounts in Entadaafricana (Karou et al., 2011). Qualitative analysis done with Entadaafricana crude extract also revealed the presence of polyphenols inthis extract (Njayou et al., 2013). The biological effects of poly-phenols have been reported to be related to their ability toinfluence cell signaling pathways and to modulate pro and anti-inflammatory gene expression (Lacour et al., 2009; Tominari et al.,2012). Therefore, we suggest that the phenolic compounds presentin Entada africana may be responsible for the observed effects onRAW 264.7 cells.

In conclusion, the present study has revealed that Entadaafricana treatment of mouse macrophages cells inhibited LPS-induced NO production by suppressing iNOS mRNA expression.Ea5 inhibited the production of pro-inflammatory cytokines TNFα,IL6 and IL1β, by suppressing their transcriptional activity. Theexpression of anti-inflammatory cytokines mRNA IL13 and IL10was also significantly stimulated by Ea5. The fraction Ea5 alsoinhibited the activity of p38 MAPK kinase. Therefore, Entadaafricanamight have substantial therapeutic potential for treatmentof inflammation and inflammatory related diseases. In prospect tothis work, the effect of the active fraction on different cytokinesand iNOS protein levels needs to be done to confirm the effect ofEa5 on RAW 264.7 cells. Further studies are going on to isolate andidentify the pure active compounds present in the fraction Ea5 anddeeply explore the signaling pathways responsible for theobserved effects.


Acknowledgments

This research was supported by the DAAD and the chemistrypart co-funded by the International Foundation for Sciences (IFS),Stockholm, Sweden and the Organisation for the Prohibition ofChemical Weapons (OPCW), through Grant no. F/4223-2 awardedto Dr. Njayou Frederic Nico.

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A fraction of stem bark extract of Entada africana suppresses lipopolysaccharide-induced inflammation in RAW 264.7 cells