Journal of Ethnopharmacology 149 (2013) 162–168

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Journal of Ethnopharmacology
journal homepage: www.elsevier.com/locate/jep

A fraction of stem bark extract of Entada africana suppresses

lipopolysaccharide-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, Germany
b
Laboratory of Pharmacology and Toxicology, University of Yaoundé I, Cameroon
c
Medical Chemistry, Department of Pharmacy & Biochemistry, Eberhard-Karls-Universität Tübingen, Auf der Morgenstelle 8, 72076 Tübingen, Germany

art ic l e i nf o a b s t r a c t

Article history: Ethnopharmacological relevance: Entada africana is a plant used in African traditional medicine for the
Received 20 March 2013 treatment of stomachache, fever, liver related diseases, wound healing, cataract and dysentery.
Received in revised form Aims of the study: This study aimed at evaluating the anti-inflammatory activity of fractions of the stem
12 June 2013
bark extract of the plant using lipopolysaccharide (LPS)-induced inflammation in RAW 264.7
Accepted 12 June 2013
Available online 21 June 2013
macrophages model.
Materials and methods: The crude extract was prepared using the mixture CH2Cl2/MeOH (1:1, v/v) and
Keywords: fractionated by flash chromatography using solvents of increasing polarity to obtain five different
Entada africana fractions. The effects of the fractions on the cells viability were studied by the 3-(4,5-dimethylthiazol-2-
Baicalin yl)-2,5-diphenyltetrazolium bromide (MTT) assay and their inhibitory activity against LPS-induced nitric
Macrophages
oxide (NO) production screened by Griess test. The most active fraction was further investigated for its
Cytokines
effects on reactive oxygen species (ROS) production using flux cytometry, the expression of inducible
p38 MAPK kinase
nitric 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 after
24 h incubation. The CH2Cl2/MeOH 5% (Ea5) fraction was found to be the most potent in inhibiting NO
production with a half inhibition concentration (IC50)¼ 18.36 μg/ml, and showed the highest inhibition
percentage (89.068%) in comparison with Baicalin (63.34%), an external standard at 50 μg/ml. Ea5, as well
as Baicalin significantly (P o0.05) inhibited the expression of TNFα, IL6 and IL1β mRNA, attenuated mRNA
expression of inducible NO synthase in a concentration-dependent manner, stimulated the expression of
anti-inflammatory cytokines (IL10 and IL13), and showed a 30% inhibition of the activity of p38 MAPK
kinase.
Conclusion: The results of the present study indicate that the fraction Ea5 of Entada africana possesses
most potent in vitro anti-inflammatory activity and may contain compounds useful as a therapeutic
agent in the treatment of inflammatory related diseases cause by over-activation of macrophages.
& 2013 Elsevier Ireland Ltd. All rights reserved.

1. Introduction including reactive oxygen species (ROS), nitric oxide (NO) and
cytokines such as IL1β, TNFα and IL6 are known to be involved in
Inflammation is considered to be the major cause of most the development of inflammation (Cui et al., 2012). Macrophages
chronic diseases like diabetes, Alzheimer's disease, asthma, and are major immune cells that act as a first line of defense against
atherosclerosis (Yu et al., 2012). Many biological molecules invading agents (bacteria, virus, and fungi), and they respond to
pathogen attack by release of ROS and NO, known as cellular
signaling molecules and antimicrobial agents (Blazovics et al.,
Abbreviations: Ea5, Entada africana fraction CH2CL2/MeOH 5%; iNOS, inducible 2004). However, an exaggerated production of inflammatory
nitric oxide synthase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium mediators by macrophage defensive systems can cause damage
bromide; NO, nitric oxide; ROS, reactive oxygen species; MAPK, mitogen activated to the host (Huo et al., 2012).
protein kinase. Macrophages ROS are, for example, important contributors to
n
Corresponding author at: Division of Immunopathology of the Nervous System,
Department of Neuropathology, Institute of Pathology, University of Tübingen,
the manifestation of allergic inflammation (Varga et al., 2013) and
Germany. Tel.: +49 15156219006; fax: +49 70 7129 484. also involved in TNFα (Haddad and Land, 2002) and IL1β produc-
E-mail address: [email protected] (B. Ayissi Owona). tion in LPS stimulated macrophages (Ryan et al., 2004). During

0378-8741/$ - see front matter & 2013 Elsevier Ireland Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.jep.2013.06.016
 B. Ayissi Owona et al. / Journal of Ethnopharmacology 149 (2013) 162–168 163

infection, LPS acts on cellular Toll like receptor 4 (TLR4), activates The crude extract was subjected to flash chromatography to yield a
different pathways in various cells (macrophages, Kuppfer cells total of five fractions, namely EaChl (CH2Cl2), Ea5 (CH2Cl2/MeOH
and hepatocytes) (Chen et al., 2001) and induces nitric oxide 5%), Ea10 (CH2Cl2/MeOH 10%), Ea25 (CH2Cl2/MeOH 25%), and
synthase (iNOS) as well as the production of pro-inflammatory EaMet (MeOH).
agents, including cytokines (IL1β, TNFα, and IL6), prostaglandins
and NO (Glushkova et al., 2012). On the other hand, activated
macrophages also produce anti-inflammatory cytokines such as 2.2. Chemicals
IL10 and IL13, which are useful to slow down and terminate the
inflammatory response (Abuarqoub et al., 2006). Fetal bovine serum (FBS), antibiotics (streptomycin/penicillin),
Many pathways have been described to explain the effect of and RPMI medium were purchased from Gibco (Grand Island, NY,
anti-inflammatory compounds on macrophages. It is the case of USA). Escherichia coli-LPS and 3-(4,5-dimethylthiazol-2-yl)-2,5-
the mitogen activated protein kinases (MAPKs), a specific class of diphenyltetrazolium bromide (MTT) were purchased from
serine/threonine kinases, which respond to extracellular signals Sigma-Aldrich (St. Louis, MO, USA). Baicalin (99%) was purchased
(LPS, virus and cytokines). In the field of inflammation, the p38 from Carbosynth Ltd. (Compton, Berkshire, UK). Mouse cytokines
MAPK kinases are described as stress-activated protein kinases primers (iNOS, TNFα, IL6, IL1β, IL13 and IL10) were supplied by
(Correa and Eales, 2012) and many inhibitors of these kinases are Santa Cruz Biotechnology (Santa Cruz, CA, USA).
more and more investigated for their anti-inflammatory effects
(Chao et al., 2007; Rafiee et al., 2009).
2.3. In vitro cell culture
The development of modulators such as Baicalin (BA), a
flavonoid compound purified from the medicinal plant Scutellaria
The primary mouse macrophage cell line RAW 264.7 was used
baicalensis Georgi for targeting the LPS signaling cascade in
to determine the effects of Entada africana on macrophage in vitro.
macrophages, is an attractive strategy for the therapy of inflam-
The cells were cultured in RPMI medium (Life Technologies)
matory diseases (Hu et al., 2012). BA has been shown to possess
containing penicillin (100 U/ml), and 10% fetal bovine serum. Cells
anti-inflammatory activity in many in vitro models of inflamma-
were cultured at 37 1C in a humidified incubator in an atmosphere
tion (Li et al., 2012; Liu et al., 2008; Zhu et al., 2012) and it has
of 5% CO2. RAW 264.7 cells were grown in 12-well plates at a
been reported to inhibit NF-kappaB activation in cigarette smoke
density of approximately 1  105 cells per well. The plant com-
induced inflammatory models (Lixuan et al., 2010). Medicinal
pounds were dissolved in dimethylsulfoxide (DMSO) and filtered
plants are therefore an important potential source of anti-
through 0.45 μm cellulose membranes.
inflammatory compounds.
Entada africana is a plant used in African traditional medicine
for the treatment of stomachache, wound dressing, prevention of 2.4. MTT assay for measuring cell proliferation
suppuration, treatment of fever (Obidike and Emeje, 2011), liver
related diseases, wound healing, skin-eruptions, rheumatism, The cytotoxic effects of the crude extract and fractions were
cataract, fevers and dysentery (Karou et al., 2011). Studies on evaluated by a MTT assay. Cells were seeded in 12-well plates
Entada africana report its antimicrobial, antiplasmodial, antioxi- (1  105 cells/well) and incubated for 24 h. After this incubation
dant (Karou et al., 2011), anti-proliferative (Cioffi et al., 2006), period, cells were treated with various concentrations of Entada
complement fixing (Diallo et al., 2001), fungistatic and fungicidal africana (0.01, 0.1, 1, 10, and 100 μg/ml), Baicalin (5 μg/ml) and LPS
activities (Fabry et al., 1996). Recent publications from our research (1 μg/ml) at 37 1C in 5% CO2 for 24 h. For different plant fractions,
group report the hepatoprotective and antioxidant effects of the the same test was realized at plant final concentrations of 100 μg/
stem bark of Entada africana (Njayou et al., 2013) as well as its ml. After treatment, 100 μL of MTT (5 mg/ml) dissolved in RPMI
immunomodulatory activity on rat peritoneal macrophages was added to each well, followed by incubation for 3 h. The
(Owona et al., 2013). medium was aspirated, and the formazan crystals were dissolved
To the best of our knowledge, anti-inflammatory and macro- in 500 μL of DMSO for 15 min. The optical density of each well was
phage regulation of NO, ROS, and cytokines mRNA expression by measured at 540 nm in a microplate reader.
Entada africana have not been reported. The present study demon-
strated the effect of Entada africana on the release of inflammatory
mediators. We also showed the effect of the most active fraction 2.5. Determination of nitric oxide production
on the activity of p38 MAPK kinase, with Baicalin as reference
anti-inflammatory compound. Production of NO was determined by measuring the accumu-
lated level of nitrite, an indicator of NO in the supernatant after
24 h of LPS treatment with or without different concentrations of
2. Material and methods plant 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 and
2.1. Plant materials and solvent extraction 100 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 at
Entada africana was harvested in 2010, in the West region of 37 1C in 5% CO2 for 24 h. The quantity of nitrite in the culture
Cameroon. Voucher specimens were deposited at the National medium was measured as an indicator of NO production. Amounts
Herbarium, Yaoundé, Cameroon (52661 YA). Dried Entada africana of nitrite, a stable metabolite of NO, were measured using Griess
barks were air dried, cut into small pieces and ground. One reagent (1% sulfanilamide and 0.1% naphthyl ethylene diamine
kilogram of the powder was immersed and extracted in methylene dihydrochloride in 2.5% phosphoric acid). Briefly, 50 μl of cell
chloride/methanol 1/1 v/v at room temperature for 7 days. After culture medium was mixed with 100 μl of Griess reagent. Subse-
the mixture was filtered, the filtered cakes were extracted and quently, the mixture was incubated at room temperature for
filtered three more times to increase the extraction yield. The 10 min and the absorbance at 570 nm was measured in a micro-
procedure was repeated until the solvent present a clear color. The plate reader. Fresh culture medium was used as a blank in every
filtrate was concentrated under reduced pressure, and the crude experiment. The quantity of nitrite was determined from a sodium
extract obtained was freeze-dried, and stored at 4 1C until used. nitrite standard curve.
164 B. Ayissi Owona et al. / Journal of Ethnopharmacology 149 (2013) 162–168

Table 1 mean 7SD. Statistical significance was determined by one-way

Primers used for RT-PCR analysis (F: forward and R: reverse). analysis of variance (ANOVA) using Graph Pad Prism 4.0 for
windows. For all statistical analyses, significance levels were set
Gene Primer sequences
at Po 0.05.
TNFα F5′-TTGACCTCAGCGCTGAGTTG-3′
R5′-CCTGTAGCCCACGTCGTAGC-3′
IL1β F5′-CAGGATGAGGACATGAGCACC-3′
3. Results
R5′-CTCTGCAGACTCAAACTCCAC-3′
IL6 F5′-GTACTCCAGAAGACCAGAGG-3′
R5′-TGCTGGTGACAACCACGGCC-3′ 3.1. Effect of Entada africana crude extract and fractions on the
iNOS F5′-CCCTTCCGAAGTTTCTGGCAGCAGC-3′ viability of RAW 264.7 cells
R5′-GGCTGTCAGAGCCTCGTGGCTTTGG-3′
β-actin F5′-CCGTCTTCCCCTCCATCGT-3′
R5′-ATCGTCCCAGTTGGTTACAATGC-3′
Examination of the cytotoxicity on RAW 264.7 cells by MTT
IL10 F5′-CTGTGAAAACAAGAGCAAGGC-3′ assay indicated that up to 100 μg/ml for 48 h of incubation, the
R5′-GAAGCTTCTGTTGGCTCCC-3′ plant crude extract and fractions did not affect the viability of the
IL13 F5′-GGAGCTGGTCAACATCACCC-3′ cells and likewise, the fractions presented no significant effect on
R5′-CGTTGATCAGGGATTCCAGG-3′
the viability of RAW 264.7 at 100 μg/ml after 24 h incubation (data
not shown).

3.2. Inhibition of LPS-induced NO production by Entada africana

2.6. ROS determination
crude extract and fractions
The level of intracellular ROS was determined based on the
After treatment with LPS (1 μg/ml) for 24 h, nitrite concentra-
change in fluorescence resulting from oxidation of the fluorescent
tions in the medium increased remarkably by about two fold (data
probe H2DCFDA, as described by Lee et al. (2007). Briefly, 5  105
not shown). When RAW 264.7 cells were treated with different
cells/wells were incubated with extracts or Baicalin for 30 min.
concentrations of the samples together with LPS (1 μg/ml) for 24 h,
After this time, cells were then incubated with LPS (1 μg/ml) as an
a significant (P o0.05) concentration-dependent inhibition of
inducer of ROS production at 37 1C for 6 h. After a final incubation
nitrite production was detected in the medium. The IC50 values
with H2DCFDA (50 μM) at 37 1C for 1 h, the intracellular ROS level
of the crude extract and fractions in inhibiting LPS-induced NO
was determined using flow cytometry.
production is presented in Table 2. For further experiments, Ea5
with an IC50 ¼18.36 μg/ml has been used.
2.7. RNA extraction and reverse transcription-polymerase chain
reaction (RT-PCR)
3.3. Inhibitory effect of the fraction Ea5 on LPS-induced ROS
RAW 264.7 cells were treated with Baicalin, plant fractions and production in RAW 264.7 cells
LPS (1 μg/ml) for 24 h. Total RNA from LPS-treated RAW 264.7 cells
was prepared using the innuPREP RNA Mini kit, according to the Since ROS are known to be representative toxic and pro-
manufactures protocol. cDNA (1 μg/ml) was used to perform RT- inflammatory mediators in many different acute and chronic
PCR. The primer sequences used for quantification of iNOS, TNF α, inflammatory diseases, as well as in normal defense reactions,
IL1 β, IL6, IL10, IL13 and β-actin and the PCR conditions are we addressed whether Ea5 modulated the production of ROS in
described in Table 1. The PCR products were separated by 1.5% LPS treated RAW 264.7 cells. As shown in Fig. 1, Ea5 inhibited the
agarose gel electrophoresis and visualized by ethidium bromide LPS induced intracellular production of ROS at 0.05, 0.5 and 5 μg/
(EtBr) staining. The gels were then analyzed with ultraviolet trans- ml (P o0.05). However, no significant modulation was observed at
illumination. The quantity of each mRNA was calculated using 50 μg/ml.
ImageJ software and normalized to the amount of the house-
keeping β-actin gene. 3.4. Inhibitory effects of the fraction Ea5 on pro-inflammatory
cytokines gene expression
2.8. p38 MAPK kinase inhibition assay
We next investigated whether Ea5 suppresses the production
The inhibition of p38 MAPK kinase was realized as described by of pro-inflammatory cytokines such as IL6, IL1β and TNFα in LPS-
Goettert et al. (2012). Briefly, 96-well plates were coated with ATF- stimulated RAW 264.7 cells. It was observed that 1 μg/ml LPS
2, overnight, at 4 1C. Blocking buffer was added at room tempera- significantly increased TNFα, IL1β and IL6 mRNA levels in RAW
ture and the plates were incubated for 30 min before the addition 264.7 cells. Ea5 significantly (P o0.001) inhibited IL6, TNFα and
of the kinase reaction mix containing the enzyme, with or without IL1β mRNA expression at 0.5, 5 and 50 μg/ml for both former
test compounds. The p38α reaction was carried out by using kinase cytokines and at all tested concentrations for the later, respectively
(12 ng per well), ATP (100 μM), for 45 min at 37 1C. The ATF-2
phosphorylation was detected with a specific anti-phospho ATF-2 Table 2
(Thr69/71) antibody (60 min at 37 1C). After each incubation time, Inhibition of LPS-induced production of nitric oxide by Entada africana fractions
the plate was washed three times with double distilled water. The and IC50 values in μg/ml.
optical density was measured after the addition of the substrate at
Fraction number IC50 in μg/ml % NO inhibition at 50 μg/ml
450 nm, by ELISA. The p38 MAPK kinase inhibitor SB203580 was
used as a reference compound. 1-EaChl 61.25 58.5
2-Ea5 18.36 89.068
2.9. Statistical analysis 3-Ea10 42 58.07
4-Ea25 54.66 69.23
5-EaMet 35.75 64.82
All experiments were reiterated at least three times in tripli- Baicalin – 63.34
cate. The results of multiple observations are expressed as the
 B. Ayissi Owona et al. / Journal of Ethnopharmacology 149 (2013) 162–168 165

(Fig. 2A–C). The same effects were observed with Baicalin, the reported to slow down and terminate the inflammatory response.
standard reference. As shown in Fig. 3A and B, IL10 and IL13 cytokines mRNA
expression were significantly increased, the later after treatment
with Ea5 at 50 μg/ml (P o0.001) and the former at all concentra-
3.5. Inhibitory effects of the fraction Ea5 on anti-inflammatory tions tested (P o0.01). The same effect was observed with Baicalin,
cytokines gene expression concerning the increase of IL13 mRNA expression.

The effect of Ea5 on the expression of anti-inflammatory

cytokines mRNA was also examined, as these cytokines are 3.6. Inhibitory effect of the fraction Ea5 on LPS induced iNOS
expression in macrophages

We carried out RT-PCR to investigate the question whether

inhibition of NO production was associated with decreased levels
of 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 (P o0.001) inhibited the mRNA expression
of iNOS at all the tested concentrations, as did Baicalin.

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

inhibition

Fig. 1. Effect of Entada africana fraction CH2CL2/MeOH 5% on the production of Ea5 and Baicalin were tested for their ability to inhibit p38
reactive oxygen species in LPS-activated RAW 264.7 cells. RAW 264.7 cells (5  105) MAPK kinase and the p38 MAPK inhibitor SB203580
were treated with various concentrations of Entada africana and Baicalin in the
(IC50 ¼0.048 70.01 μM) was used as a reference compound. As
presence or absence of LPS (1 μg/ml) for 30 min. The level of generated ROS was
then determined by flow cytometry analysis as described in the Materials and indicated in Fig. 5, Ea5 showed a moderate inhibitory effect on the
Methods section. *Po 0.05, **Po 0.01 and ***P o0.001 compared to LPS enzyme over the concentration range tested (0.1–100 μg/ml). A
treated group. prominent inhibition was obtained with Baicalin.

Fig. 2. Effect of Entada africana fraction CH2CL2/MeOH 5% on LPS-induced expression of pro-inflammatory cytokines in macrophages. RAW 264.7 cells were pre-treated with
different concentrations of Ea5 and/or Baicalin, with or without LPS for a stimulation period of 24 h. Total RNAs were isolated, and mRNA levels of IL1β, TNFα, and IL6 were
measured by RT-PCR. β-actin expression was used as an internal control. (A) IL6, (B) IL1β, and (C) TNFα. *P o0.05, **Po 0.01 and ***P o0.001 compared to LPS treated group.
166 B. Ayissi Owona et al. / Journal of Ethnopharmacology 149 (2013) 162–168

Fig. 5. Effect of Entada africana fraction CH2CL2/MeOH 5% and Baicalin on the

inhibition of p38 MAPK kinase activity. The inhibitory potency of the fraction and
Baicalin was evaluated by using p38α ELISA assay containing BSA (0.01%) in the
kinase buffer. SB203580 was used as reference compound.

4. Discussion

Nitric oxide is synthesized by the inducible nitric oxide

synthase from L-arginine using NADPH and molecular oxygen by
many inflammatory cells including macrophages, neutrophils and
neuronal cells. NO mediates a variety of biological effects including
vasodilatation, neurotransmission, inhibition of platelet adherence
and aggregation, as well as killing of pathogens (Abuarqoub et al.,
2006; Oh et al., 2008). It has been demonstrated that induction of
iNOS produces a large amount of NO under inflammatory condi-
tions (Chen et al., 2001). Therefore, compounds that inhibit iNOS
expression and NO production may be good candidates in the
treatment of diseases caused by an overproduction of NO (Fischer
et al., 1999).
In this study, we explored the effect of Entada africana crude
extract and fractions on NO production by RAW 264.7 cells. We
then examined the effect of the most active fraction on intracel-
lular ROS production by RAW 264.7 cells, as well as on the
expression of iNOS mRNA in these cells. Besides, we explored
Fig. 3. Effect of Entada africana fraction CH2CL2/MeOH 5% on LPS-induced expres- the effect of Ea5 fraction on pro and anti-inflammatory cytokines
sion of anti-inflammatory cytokines in macrophages. RAW 264.7 cells were pre- mRNA expression, and finally we observed its effect on p38 MAPK
treated with different concentrations of Ea5 and/or Baicalin, with or without LPS
for a stimulation period of 24 h. Total RNAs were isolated, and mRNA levels of IL10
kinase inhibition.
and IL13 were measured by RT-PCR. β-actin expression was used as an internal Prerequisite for properly evaluating the biological properties of
control. (A) IL10 and (B) IL13.*P o0.05, **P o 0.01 and ***Po 0.001 compared to LPS individual molecules or complex mixtures is the exact determina-
treated group. tion of cytotoxicity associated with prolonged incubation of the
cells. Thus, the effect of the plant crude extract and fractions on
cell viability was assayed, using the MTT assay. The plant extract
and fractions presented no significant toxicity on cells viability,
thereby indicating that the modulatory effect of the tested samples
on RAW 264.7 cells was not simply due to cytotoxic effects.
The excessive production of NO by macrophages is implicated
in the pathogenesis of many diseases, including Alzheimer disease,
cancer, and diabetes. The exposure of RAW 264.7 cells to LPS for
24 h was associated with an accumulation of nitrite in the
medium, suggesting an enhanced production of NO upon LPS
treatment. This overproduction of NO induced by LPS was sig-
nificantly inhibited by Entada africana crude extract and fractions
as testify by Baicalin, the reference compound. Based on their
respective IC50 values, the potency of tested samples might be
ranked as follows Ea54 EMet4Ea10 4Ea254 EaChl (Table 2).
Since the fraction Ea5 was the most potent, its activity was further
studied.
In murine macrophages, LPS stimulation has been demon-
strated to induce iNOS transcription with a corresponding increase
Fig. 4. Entada africana fraction CH2CL2/MeOH 5% and Baicalin reduced LPS-induced in NO production (Yang et al., 2009). Therefore, drug screening
iNOS gene expression in macrophage cell line. RAW 264.7 cells were incubated
without or with LPS 1 μg/ml together with the indicated amounts of the extract or
using nitric oxide and effect on iNOS mRNA expression appear to
Baicalin. After 24 h iNOS mRNA was quantified by qRT-PCR. *P o0.05, **Po 0.01 be an excellent approach to discover new anti-inflammatory
and ***P o0.001 compared to LPS treated group. drugs. Ea5 as well as Baicalin significantly inhibited (Fig. 4) iNOS
 B. Ayissi Owona et al. / Journal of Ethnopharmacology 149 (2013) 162–168 167

mRNA expression in RAW 264.7 cells and from the results it might Acknowledgments
be suggested that the fraction Ea5 exhibit its anti-inflammatory
action through the inhibition of iNOS as it is reported about This research was supported by the DAAD and the chemistry
Baicalin (Hao et al., 2012; Zhu et al., 2012). part co-funded by the International Foundation for Sciences (IFS),
Important molecules produced by immune cells during inflam- Stockholm, Sweden and the Organisation for the Prohibition of
mation are ROS. These molecules contribute to pathogen and Chemical Weapons (OPCW), through Grant no. F/4223-2 awarded
invader elimination during phagocytosis (Guido et al., 2012). to Dr. Njayou Frederic Nico.
However, an excessive production of ROS may amplify inflamma-
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