Food Chemistry 129 (2011) 17211728

Contents lists available at ScienceDirect

Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Anti-inammatory effect of avonoids isolated from Korea Citrus aurantium L.

on lipopolysaccharide-induced mouse macrophage RAW 264.7 cells by blocking
of nuclear factor-kappa B (NF-jB) and mitogen-activated protein kinase (MAPK)
signalling pathways
Sang Rim Kang a,1, Kwang Il Park a,1, Hyeon Soo Park a, Do Hoon Lee a, Jin A Kim b, Arulkumar Nagappan a,
Eun Hee Kim c, Won Sup Lee d, Sung Chul Shin e, Moon Ki Park f, Dae Yong Han a, Gon Sup Kim a,
a

Research Institute of Life Science, College of Veterinary Medicine, Gyeongsang National University, Gazwa, Jinju 660-701, Republic of Korea
Korea National Animal Research Resource Center, Korea National Animal Bio-Resource Bank, Gyeongsang National University, Gazwa, Jinju 660-701, Republic of Korea
c
Department of Nursing Science, International University of Korea, Jinju 660-759, Republic of Korea
d
Department of Internal Medicine, Institute of Health Sciences, Gyeongsang National University School of Medicine, Gyeongnam Regional Cancer Center,
Gyeongsang National University Hospital, Jinju 660-702, Republic of Korea
e
Department of Chemistry, Research Institute of Life Science, Gyeongsang National University, Jinju 660-701, Republic of Korea
f
Department of Herbal Pharmaceutical Engineering, Daegu Haany University, 290 Yugok-dong, Gyeongbuk 712-715, Republic of Korea
b

a r t i c l e

i n f o

Article history:
Received 26 January 2011
Received in revised form 14 April 2011
Accepted 15 June 2011
Available online 26 June 2011
Keywords:
Citrus aurantium L.
Anti-inammation
Cyclooxygenase-2
Inducible nitric oxide synthase
Nuclear factor-kappa B (NF-jB)
Mitogen-activated protein kinase (MAPK)

a b s t r a c t
Citrus fruits are an abundant source of various avonoids, which have been used as a traditional herbal
medicine in Korea and China. Most avonoids are known to have anti-oxidant, anti-bacterial and analgesic properties. In this study, it was examined whether avonoids (nobiletin, naringin and hesperidin) isolated from Korea Citrus aurantium L. inhibited the pro-inammatory mediators including cytokines by
blocking nuclear factor-kappa B (NF-jB) and mitogen-activated protein kinase (MAPK) signalling in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. The avonoids suppressed mRNA and protein
expression of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) in LPS-induced macrophages. The molecular mechanism was associated with inhibition of the degradation/phosphorylation
of I-jB-a and nuclear translocation of the NF-jB p-65 as well as phosphorylation of MAPK by avonoids.
These results suggest that avonoids have anti-inammatory effects by suppressing expression of COX-2,
iNOS and cytokines by blocking the NF-jB and MAPK signalling in RAW 264.7 macrophages.
2011 Elsevier Ltd. All rights reserved.

1. Introduction
Flavonoids are part of a family of naturally occurring polyphenolic compounds characterised by a common benzo-c-pyrone chemical structure. They are naturally rich in fruits and vegetables
including the genus Citrus (family Rutaceae) (Nogata et al., 2006).
Especially, citrus plants are an abundant source of various
avonoids such as hesperidin, naringin, nobiletin (Fig. 1) and
polymethoxylated avones (PMFs). Flavonoids have various
pharmacological properties such as anti-oxidant, anti-cancer and
anti-inammatory activities. Of these biological activities, the
anti-inammatory potential of avonoids has long been used in
Chinese medicine as a form of crude plant extracts. A wide variety
of avonoid molecules possess anti-inammatory activity on

Corresponding author. Tel.: +82 55 751 5823; fax: +82 55 751 6648.
1

E-mail address: gonskim@gnu.ac.kr (G.S. Kim).

The rst two authors contributed equally to the completion of this study.

0308-8146/$ - see front matter 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodchem.2011.06.039

various animal models of inammation (Kim, Son, Chang, & Kang,

2004).
Hesperidin inhibits inammatory cytokine production from
mast cells and suppresses infection-induced endotoxin shock in
mice (Choi et al., 2007). Nobiletin reduces the inammatory actions on human synovial broblasts, mouse macrophages and
phorbol ester-induced skin inammation and oxidative stress in
mice (Murakami et al., 2000). Naringin also suppresses inammatory signalling such as COX-2, iNOS and cytokines in macrophage
cells (Kanno et al., 2006).
Mitogen-activated protein kinases (MAPKs) have important
functions such as the regulation of cell differentiation, cell growth
and cellular responses to cytokines. The MAPK cascades are
important signalling pathways in the immune system and include
p38 MAPK, extracellular signal-regulated kinase (ERK) and c-jun
N-terminal kinase (JNK) (Seger & Krebs, 1995). They are activated
by kinase cascades such as MAPK kinase (MAPKK or MEK) and
MAPKK/MEK kinase (MAPKKK or MEKK). Once activated by
kinases, MAPK can phosphorylate the transcription factors such

1722

S.R. Kang et al. / Food Chemistry 129 (2011) 17211728

Fig. 1. Chemical structure of avonoids isolated from Korea Citrus aurantium L.

as NF-jB and activator protein-1 (AP-1), or transcriptionally

co-regulate or phosphorylate downstream kinases that leads to
the expression of pro-inammatory mediators of extracellular
stimuli (Collart, Baeuerle, & Vassalli, 1990). LPS activates all three
types of MAPKs. Activation of MAPKs, especially p38, suppresses
the production of iNOS and COX-2 in mouse macrophages (Chen,
Chen, & Lin, 1999). Also, phosphorylation of MAPKs is related to
pro-inammatory cytokines and transcriptional activation of
NF-jB in LPS-induced marcrophages (Carter, Knudtson, Monick,
& Hunninghake, 1999).
NF-jB is a transcription factor that plays a principal role in the
expression of genes in immune and inammatory responses. The
most commonly inducible form of NF-jB is a heterodimer consisting of p50 and p65, which functions principally as a transcriptional
activator. In normal conditional cells, inactivated NF-jB dimers are
bound to inhibitor of jB (I-jB) in the cytoplasm (Karin, 1999). Signal cascades triggered by proinammatory cytotokines such as
TNF-a and IL-1b, bacterial endotoxin (e.g., LPS) and oxidative stress
leads to NF-jB activation through the phosphorylation of I-jB by
cytoplasmic IjB-kinase (IKK) complexes. On activation of NF-jB,
I-jB is phosphorylated by IKK and then is degraded via an ubiquitination-proteasome pathway. Thereby, NF-jB is enabled to translocate into the nucleus where it binds to the cis-acting jB enhancer
element of target genes, which leads to the transcription of target
genes such as the pro-inammatory mediators cyclooxygenase
(COX)-2 and inducible nitric oxide synthase (iNOS); various cytokines including TNF-a, IL-1b and IL-6; chemokines and adhesion
molecules (Lawrence, Gilroy, Colville-Nash, & Willoughby, 2001).
The inammatory response plays a crucial role in host defence
against stimuli such as pathogen invasion or endotoxin exposure,
and ultimately leads to the restoration of normal cell structure
and function. Normal inammatory responses are self-limited by
a process that involves the down-regulation of pro-inammatory
mediators and increase of anti-inammatory mediators (Kim
et al., 2008). However, chronic inammation causes the increase
of pro-inammatory mediators including iNOS, COX-2 and various
cytokines including TNF-a, IL-1b and IL-6. iNOS and COX-2, which
are responsible for increased level of nitric oxide (NO) and prostaglandins (PGs), respectively, are active in the pathogenesis of various chronic inammatory diseases such as multiple sclerosis,
Parkinsons disease, Alzheimers disease and colon cancer (Heiss,
Herhaus, Klimo, Bartsch, & Gerhauser, 2001).
The present study was undertaken to investigate the hypothesis
that avonoids (nobiletin, naringin and hesperidin) isolated from
Korea Citrus aurantium L. can suppress LPS-induced inammatory
responses in mouse macrophage RAW 264.7 cells.

2. Materials and methods

2.1. Materials and chemicals
Escherichia coli O111:B4 LPS and (3-(4,5-dimethyl-2yl)-2,5diphenyltetrazolium bromide) (MTT) were purchased from SigmaAldrich (St. Louis, MO, USA). Foetal bovine serum (FBS) and
antibiotics (streptomycin/penicillin) were obtained from Gibco
(Grand Island, NY, USA) and Dulbeccos modied Eagles medium
(DMEM) was purchased from Hyclone (Logan, UT, USA). Antibody
to COX-2 and iNOS were purchased from Santa Cruz Biotechnology
(Santa Cruz, CA, USA) and antibody to b-actin was purchased from
Chemicon (Temecula, CA, USA). An antibody sampler kit of
phospho-ERK1/2, p38, JNK MAPKs and phospho-IjB and IjB were
obtained from Cell Signalling Technology (Danvers, MA, USA).
2.2. Isolation of avonoids from Korea Citrus aurantium L
Matured Korea Citrus aurantium L. fruits having a homozygous
genetic background were obtained in November 2009 from the Citrus Genetic Resources Bank, Jeju National University. Voucher
fruits were deposited in the herbarium of Jeju National University.
The fruits were washed with water and stored at 70 C until
extraction and inoculation experiments. The fruit peel was nely
ground after lyophilisation in a PVTED50A apparatus (Ilsin Bio
Base, Yangju, Korea). The powder of the fruit peel (600 g) was extracted using a model PT-MR 2100 apparatus (Kinematica, Lucerne,
Switzerland) in 2 l of 70% aqueous methanol for 24 h. After three
extractions, the extracts were combined and ltered using a Bchner funnel. The ltrate was concentrated to 140 ml using an Eyela
NVC-2100 rotary evaporator (Tokyo Rikakikai, Tokyo, Japan). Aqueous 5% NaOH (60 ml) was added to the concentrate, which was extracted with 200 ml of ethyl acetate. After three extraction cycles,
the organic layer was neutralised with brine and dried over anhydrous MgSO4 to yield 3.0 g of avonoid components.
2.3. Cell culture and treatment
RAW 264.7 mouse macrophage cells were purchased from the
Korea Cell Line Bank (KCLB, Seoul, Korea). The cells were maintained in DMEM supplemented with 10% FBS and penicillin
(100 U/ml)/streptomycin (100 lg/ll) at 37 C in a 5% CO2 humidied incubator. Flavonoids isolated from Korea Citrus aurantium L.
were dissolved in dimethylsulphoxide (DMSO) and were used for
the treatment of cells. Cells grown to 80% conuence were

S.R. Kang et al. / Food Chemistry 129 (2011) 17211728

pretreated with avonoids (6.25, 12.5, 25 and 50 lg/ml) for 1 h and

then treated with LPS (1 lg/ml).
2.4. Cell viability
The growth inhibitory effect of avonoids on RAW 264.7 cells
was measured by a MTT assay. The cells were cultured in wells
of 12-well plates at density of 1  104 cells/well. After 24 h, the
cells were pretreated with 10, 50, 100 and 150 lg/ml of avonoid
and then treated with LPS (1 lg/ml) for 24 h at 37 C. MTT solution
was added to each well and further incubated for 3 h at 37 C. The
medium was discarded and DMSO was added to dissolve the formazan dye. The optical density was determined at 540 nm. All
experiments were carried out in triplicate.
2.5. Western blot
The treated cells were washed with ice-cold PBS and incubated
on ice for 10 min in a lysis buffer containing 50 mM TrisHCl (pH
8.0), 150 mM NaCl, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulphate (SDS), 1% NP-40, protease inhibitor cocktail, 0.5 M
EDTA, and phosphatase inhibitor. Cell debris was removed by centrifugation at 13 000 rpm for 30 min and then the protein concentration of each cell lysate was determined with a Bradford assay kit
(Bio-Rad, Hercules, CA, USA). The cell lysates were separated by
10% SDSpolyacrylamide gel electrophoresis (SDSPAGE) and the
resolved proteins were transferred to a polyvinylidene uoride
(PVDF) membrane (Immunobilon-P, 0.45 mm; Millipore, Billerica,
MA, USA) using the TE 77 Semi-Dry Transfer Unit (GE Healthcare
Life Sciences, Buckinghamshire, UK). The membranes were blocked
with 5% fat-free skim milk in Tris-buffered saline containing 0.5%
Tween-20 (TBS-T, pH 7.4) at room temperature for 30 min. Membranes were probed with the antibodies to iNOS, COX-2, phospho-I-jB, NF-jB p65, phospho-ERK1/2, phospho-JNK or phosphop38 overnight at 4 C. The blots were washed four times with
TBS-T for 10 min each and incubated with a 1:2000 dilution of
horseradish peroxidase-conjugated secondary antibody for 1 h at
room temperature. Following ve further washings in TBS-T. Each
bands of protein were visualised by enhanced chemiluminescence
(GE Healthcare Life Sciences) and exposed to X-ray lm (Fuji,
Tokyo, Japan). Each band was quantitatively determined using Image J (http://rsb.info.nih.gov). The density ratio represented the relative intensity of each band against those of b-actin as a control in
each experiment.
2.6. Reverse transcription-polymerase chain reaction (RT-PCR)
Total RNA was prepared from RAW 264.7 macrophages using
TRIzol reagent (GeneALL Biotechnology, Seoul, Korea) according
to the manufacturers instructions. One microgramme of total
RNA was reverse-transcribed into cDNA using commercially available cDNA synthesis kits (iScript cDNA Synthesis Kit; Bio-Rad).
The tubes were incubated at 25 C for 5 min and then at 42 C for
30 min, and the reactions were stopped by heating at 85 C for
5 min. The sample was then stored at 70 C until use. RT-PCR conditions were the same as previously described (Kang et al., 2011).
After reverse transcription, the PCR was performed with initial
heat denaturation at 95 C for 4 min and the PCR cycles were repeated 3035 times under the following conditions: denaturation
at 95 C for 30 s, annealing at 5255 C for 1 min and extension
at 72 C for 1 min. PCR primers used in this study are listed below
and were purchased from Bioneer (Seoul, Korea): COX-2 (Sense:
50 -CCCAGAGCTCCTTTTCAACC-30 , Antisense: 50 -ATTTGGCACATTTCT
TCCCC-30 ), iNOS (Sense: 50 -CTCCCCTCTCTCCCTTTCCT-30 , Antisense:
50 -TGGAAATTGGGGTAGGAAGG-30 ), TNF-a (Sense:50 -AGCACAGAA
AGCATGATCCG-30 , Antisense: 50 - GTTTGCTACGACGTGGGCTA-30 ),

1723

IL-6 (Sense: 50 - CGATGATGCACTTGCAGAAA-30 , Antisens: 50 TGGAAATTGGGGTAGGAAGG-30 ) and GAPDH (Sense: 50 - AAGGGTCATCATCTCTGCCC-30 , Antisense: 50 -GTGATGGCATGGACTGTGGT30 ). After amplication, products of the PCR reaction were analysed
on an ethidium bromide-stained 1.5% agarose gel. The amount of
mRNA was evaluated by Image J. The signal intensity of the specic
mRNAs were normalised by a comparison with that of GAPDH and
calculated as the relative amounts.

2.7. Statistical analysis

The experimental results are presented as the mean SD values
at least three replications. One-way analysis of variance (ANOVA)
procedures were used for comparisons of multiple group means
using SPSS version 10.0 for Windows (SPSS, Chicago, IL, USA). A
probability <0.05 was considered as signicant.

3. Results
3.1. Isolation of avonoids from Korea Citrus aunratium L.
In the present study, avonoid were isolated from Korea Citrus
aurantium L. by using LC chromatogram at department of chemistry, Gyeongsang national university (Professor Shin, Sung Chul). As
indicated in Fig. 2, according to the peaks of LC chromatogram, it
could be conrmed that there were the avonoid components in
the extract at 280 nm. These avonoid compounds are the essential components present in Korea Citrus aurantium L.

3.2. Effect of avonoids on cell viability

Fig. 3 indicates the cell viability at 10, 50, 100 and 150 lg/ml of
the avonoids in the presence of LPS (1 lg/ml). After 24 h of incubation, concentration of 150 lg/ml signicantly decreased cell viability to about 90% (p < 0.05), whereas concentration of avonoids
ranging from 10 to 100 lg/ml did not exhibit any cytotoxic effect.
Therefore, concentrations of avonoids were selected from 6.25 to
50 lg/ml for study on anti-inammatory effect.

3.3. Flavonoids decrease LPS-induced mRNA expression of proinammatory cytokines in RAW 264.7 macrophages
As shown in Fig 3B and C, avonoids potently inhibited proinammatory cytokines in LPS-induced macrophage cell. The
mRNA level of IL-6 and TNF-a was increased signicantly by the
LPS treatment when compared with untreated control group. However, the level of LPS-induced IL-6 and TNF-a gene expression was
signicantly decreased in the presence of 12.5, 25 and 50 lg/ml of
the avonoids.

3.4. Flavonoids decrease protein and mRNA levels of COX-2 and iNOS
in LPS-stimulated RAW 264.7 macrophages
The mRNA expressions of COX-2 and iNOS were signicantly increased upon LPS treatment and this induction was effectively
inhibited in a dose-dependent manner by avonoids treatment
(Fig. 4A and B). Similar was the case of protein expression of
COX-2 and iNOS. LPS-activated macrophages increased the proteins expression of COX-2 and iNOS when compare with untreated
control group. However, avonoids prominently suppressed the
LPS-induced COX-2 and iNOS protein expression in a dose-dependent manner (Fig. 4C and D).

1724

S.R. Kang et al. / Food Chemistry 129 (2011) 17211728

Fig. 2. Chromatogram of Korea Citrus aurantium L.

Fig. 3. Effect of avonoids on cell cytotoxicity and production of the LPS-induced pro-inammatory cytokines TNF-a and IL-6 in RAW 264.7 macrophages. (A) Macrophages
were incubated with avonoids in the presence of LPS for 24 h. The results are reported as a percentage compared to the untreated controls. (B, C) RAW 264.7 cells were
pretreated with avonoids (6.25, 12.5, 25 and 50 lg/ml) for 1 h, and then stimulated with LPS (1 lg/ml). After 6 h, the total mRNA was isolated, and the mRNA levels of (B)
TNF-a and (C) IL-6 were examined by RT-PCR.  indicates an increase in mRNA expression relative to the control (p < 0.05).  indicates a decrease in mRNA expression
relative to the LPS group (p < 0.05).

S.R. Kang et al. / Food Chemistry 129 (2011) 17211728

1725

Fig. 4. Suppressive effects of avonoids on LPS-induced mRNA and proteins expression of COX-2 and iNOS in RAW 264.7 macrophages. RAW 264.7 cells were pretreated with
avonoids for 1 h and then incubated with LPS for 6 h. The total mRNA was isolated, and the mRNA levels of (A) COX-2 and (B) iNOS were ascertained by RT-PCR. RAW 264.7
cells were pretreated with avonoids for 1 h and then incubated with LPS for 24 h. The cells were lysed, and the lysates were examined by Western blot for (C) COX-2 and (D)
iNOS.  indicates an increase in mRNA expression relative to the control (p < 0.05).  indicates a decrease in mRNA expression relative to the LPS group (p < 0.05).

3.5. Flavonoids inhibit degradation and phosphorylation of I-jB and

nuclear translocation of NF-jB p65 in LPS-stimulated RAW 264.7
macrophages
Un-stimulated RAW 264.7 cells did not detectable form of phosphorylated I-jB, whereas addition of 1 lg/ml LPS caused phosphorylation of I-jB. However, pre-treatment with any of the
avonoids prominently inhibited the LPS-induced I-jB phosphorylation in a dose dependent-manner. In contrast, the level of I-jB
signicantly increased in a dose-dependent manner by avonoids
(Fig. 5A and B). NF-jB p65 protein was less expressed in the cytosol fraction and strongly expressed in the nuclear fraction after
stimulation with 1 lg/ml LPS. However, NF-jB p65 in the cytosol
fraction and nuclear fraction were increased and decreased by
avonoids, respectively (Fig. 5C and D). The results were consistent
with the view that the avonoids isolated from Korea Citrus
aurantium L. may block the LPS-induced translocation of NF-jB
p65 from the cytosol to the nucleus by blocking I-jB phosphorylation and degradation.

3.6. Flavonoids affect phosphorylation of MAPK in LPS-stimulated RAW

264.7 macrophages
LPS stimulation rapidly caused the phosphorylation of ERK, JNK
and p38 MAPK within 15 min in macrophage RAW 264.7 cells.
However, avonoids signicantly suppressed the phosphorylation
of ERK and p38 MAPK, and slightly reduced the phosphorylation
of JNK MAPK in LPS-activated macrophages (Fig. 6). These results
show that MAPK signal transduction might be effectively blocked
by avonoids in stimulated macrophage RAW 264.7 cells.

4. Discussion
Previously, it was reported that Korea Citrus aurantium L. methanol extract suppressed LPS-induced pro-inammatory mediators
via the NF-jB signal pathway in RAW 264.7 macrophages (Kang
et al., 2011). In the present study the effect of avonoids (nobiletin,
naringin and hesperidin) involved in Korea Citrus aurantium L.
were investigated. The present results demonstrate that the avonoids isolated from Korea Citrus aurantium L. inhibit inammatory
responses against LPS-induced RAW 264.7 macrophages. The inhibition appears to be due to the quelling of LPS-induced iNOS and
COX-2 expression by the decreased NF-jB and MAPK activities.
Herbal medicinal plants or crude extracts have been used as an
alternative therapy and a traditional medicine to treat various diseases. The crude extracts of citrus had the anti-inammation and
anti-tumour effects by suppressing IFN-c and histamine in human
keratinocytes and inhibiting TGF-b and IL-6 in a murine renal cell
carcinoma model, respectively (Cardile, Frasca, Rizza, Rapisarda,
& Bonina, 2010; Lee et al., 2011). Especially, the avonoids isolated
from citrus have reported the pharmaceutical activities such as
anti-inammatory, anti-cancer, anti-oxidative, anti-viral, and
anti-allergic (Benavente-Garcia & Castillo, 2008). Also, the avonoids such as quercetin, curcumin, ()-epigallocatechin gallate,
apigenin and morin reduce the inammatory response through
the inhibition of COX or iNOS activities (Soliman & Mazzio, 1998).
Inammation is a host response to foreign pathogens or tissue
injury by the organism to eliminate harmful stimuli, such as infection and noxious stimuli, as well as to initiate the healing and repair process of the damaged tissue (Mariathasan & Monack,
2007). In this process, activated macrophages play an important
role for the maintenance of homoeostatic function such as the

1726

S.R. Kang et al. / Food Chemistry 129 (2011) 17211728

Fig. 5. Flavonoids suppresses LPS-induced phosphorylation and degradation of I-jB as well as translocation of NF-jB in RAW 264.7 macrophages. The cells were pretreated
with avonoids at various concentrations for 1 h and then with LPS for 30 min. The cell lysates were analysed by Western blot for the detection of the (A) degradated and (B)
phosphorylated forms of I-jB as well as (C) the cytosolic and (D) nucleic NF-jB.  indicates an increase in protein relative to the control (p < 0.05).  indicates a decrease in
protein relative to the LPS group (p < 0.05).

overproduction of pro-inammatory cytokines and inammatory

mediators (Walsh, 2003). However, chronic inammation causes
the increase of pro-inammatory mediators including iNOS, COX2 and various cytokines including TNF-a, IL-1b and IL-6. iNOS
and COX-2, which are responsible for increased levels of nitric
oxide (NO) and prostaglandins (PGs), respectively. These are active
in the pathogenesis of various chronic inammatory diseases such
as multiple sclerosis, Parkinsons disease, Alzheimers disease and
colon cancer (Heiss et al., 2001). NO is a major product regulated
by three distinct NOS isoforms: neuronal (n) NOS, inducible (i)
NOS and endothelial (e) NOS. iNOS not only exists in healthy tissues, but is also expressed after exposure to specic stimulants
such as LPS and cytokines. The iNOS produces NO until the enzyme
is decomposed (MacMicking, Xie, & Nathan, 1997).
PGs play benecial roles in almost every system and regulate
different physiological processes including cell growth, ovulation,
immunity, nerve growth and development and bone metabolism
(Gupta & Dubois, 2001). There are two major isoforms of cyclooxygenase: COX-1 and COX-2. COX-1 is expressed constitutively in
many tissues and is associated with the synthesis of PGs involved
in normal kidney and gastrointestinal function (Fletcher, 1993).
COX-2 is not detected in normal tissues, but is excessively induced
by a variety of physiopathological conditions affecting tissues, such
as growth factors, inammatory stimuli, oncogenes and other ligands (Herschman, 1996). 4-Methoxyhonokiol isolated from the
stem bark of Magnolia obovata suppresses the expression of iNOS
and COX-2 in RAW 264.7 macrophages via NF-jB, JNK and p38
MAPK inactivation (Zhou et al., 2008). Poncirin isolated from the
fruit of Poncirus trifoliata (Rutaceae) inhibits LPS-induced

expression of iNOS, COX-2 and cytokines through the inactivation

of NF-jB in RAW 264.7 macrophages (Kim et al., 2007). Thus, the
regulation of iNOS and COX-2 is important in the inammatory response. The present study examined the effect of avonoids on the
expression of iNOS and COX-2 at the protein and mRNA levels. The
results show that avonoids dose-dependently suppress the
expression of COX-2 and iNOS at both the protein and mRNA levels
in LPS-stimulated RAW 264.7 macrophages.
The MAPK pathway is one of the important intracellular signalling cascades in pro-inammatory responses. MAPKs comprise
three subfamilies: p38 MAPK, JNK/stress activated protein kinase
and ERK (Chen et al., 2006). The precise signalling pathways
amongst the three MAPKs are still unclear. In this study, avonoids
inhibited the expression of all three MAPKs in a dose-dependent
manner in LPS-stimulated RAW 264.7 macrophages. Similarly, a
previous study reported the anthocyanidin-mediated suppression
of LPS-induced COX-2 by the reduced activation of p38, JNK and
ERK (Hou, Yanagita, Uto, Masuzaki, & Fujii, 2005). In contrast, the
differential regulation of MAPKs on LPS-induced NO signalling
has been described. For example, only p38 amongst the three types
MAPKs has been linked with the LPS-induced NO production in
murine macrophages (Chen & Wang, 1999). Also, 4-methoxyhonokiol inhibited LPS-induced iNOS and COX-2 expression in RAW
264.7 macrophages via JNK and p38 MAPK, but not ERK1/2 (Zhou
et al., 2008).
NF-jB is a major factor regulating the expression of inammation-induced enzymes and cytokines such as iNOS, COX-2, TNF-a
and IL-6, which include the NF-jB binding sites in their promoters,
and has attracted attention as a new target for treating

1727

S.R. Kang et al. / Food Chemistry 129 (2011) 17211728

Fig. 6. Flavonoids suppress LPS-induced phosphorylation of MAPKs signal pathways in RAW 264.7 macrophages. The cells were pretreated with avonoids at various
concentrations for 1 h and then with LPS for 15 min. The proteins were analysed by Western blot for the detection of phosphorylation of ERK1/2, JNK and p38 MAPK. 
indicates an increase in protein relative to the control (p < 0.05).  indicates a decrease in protein relative to the LPS group (p < 0.05).

inammatory diseases (Lawrence et al., 2001). Therefore, the suitable regulation of NF-jB may be benecial in treating many
inammatory disorders. Recent studies have demonstrated that
various natural compounds including curcumin, green tea polyphenols, resveratrol and lactones inhibit NF-jB activation. Curcumin suppresses NOS by decreasing IKK and NF-jB activation in
LPS-induced macrophages (Pan, Lin-Shiau, & Lin, 2000). Green tea
polyphenols and resveratrol inhibit NF-jB activation by suppressing IKK (Holmes-McNary & Baldwin, 2000). The root of Panax notogingeng inhibits LPS-induced inammatory mediators, including
iNOS and COX-2 by blocking I-jB degradation in the cytosol and
the nuclear translocation of the NF-jB p65 subunit (Jung, Seo,
Kim, Leem, & Park, 2009). The present results showed that avonoids inhibited signicantly the LPS-induced activation and nuclear translocation of NF-jB p65 at various concentrations. Moreover,
in RAW 264.7 macrophages pretreated with avonoids, the phosphorylation of I-jB was inhibited in a dose-dependent manner.
The collective ndings suggest that avonoids suppressed NF-jB
activation and nuclear translocation in LPS-stimulated RAW
264.7 macrophages by blocking the phosphorylation of I-jB.

5. Conclusions
Flavonoids (nobiletin, naringin and hesperidin) substantially
suppressed the pro-inammatory mediators such as COX-2 and
iNOS as well as various cytokines (IL-6 and TNF-a) in stimulated
murine macrophage RAW 264.7 cells by blocking NF-jB and the
MAPK signalling pathway. In conclusion, these ndings proved that
there is an anti-inammatory property of avonoids isolated from
Korea Citrus aurantium L. and suggest that avonoids might be

promising
diseases.

chemotherapeutic

agents

against

inammatory

Acknowledgments
This work was supported by the National Research Foundation
(NRF) of Korea grant funded by the Korean government (MEST)
(No. 2009-0084454) and the National R&D Program for Cancer
Control, Ministry for Health, Welfare and Family affairs, Republic
of Korea (No. 0820050).
References
Benavente-Garcia, O., & Castillo, J. (2008). Update on uses and properties of citrus
avonoids: New ndings in anticancer, cardiovascular, and anti-inammatory
activity. Journal of Agricultural and Food Chemistry, 56, 61856205.
Cardile, V., Frasca, G., Rizza, L., Rapisarda, P., & Bonina, F. (2010). Antiinammatory
effects of a red orange extract in human keratinocytes treated with interferongamma and histamine. Phytotherapy Research: PTR, 24, 414418.
Carter, A. B., Knudtson, K. L., Monick, M. M., & Hunninghake, G. W. (1999). The p38
mitogen-activated protein kinase is required for NF-kappaB-dependent gene
expression. The role of TATA-binding protein (TBP). The Journal of Biological
Chemistry, 274, 3085830863.
Chen, C., Chen, Y. H., & Lin, W. W. (1999). Involvement of p38 mitogen-activated
protein kinase in lipopolysaccharide-induced iNOS and COX-2 expression in
J774 macrophages. Immunology, 97, 124129.
Chen, T. H., Kao, Y. C., Chen, B. C., Chen, C. H., Chan, P., & Lee, H. M. (2006).
Dipyridamole activation of mitogen-activated protein kinase phosphatase-1
mediates
inhibition
of
lipopolysaccharide-induced
cyclooxygenase-2
expression in RAW 264.7 cells. European Journal of Pharmacology, 541, 138146.
Chen, C. C., & Wang, J. K. (1999). P38 but not p44/42 mitogen-activated protein
kinase is required for nitric oxide synthase induction mediated by
lipopolysaccharide in RAW 264.7 macrophages. Molecular Pharmacology, 55,
481488.
Choi, I. Y., Kim, S. J., Jeong, H. J., Park, S. H., Song, Y. S., Lee, J. H., et al. (2007).
Hesperidin inhibits expression of hypoxia inducible factor-1 alpha and

1728

S.R. Kang et al. / Food Chemistry 129 (2011) 17211728

inammatory cytokine production from mast cells. Molecular and Cellular

Biochemistry, 305, 153161.
Collart, M. A., Baeuerle, P., & Vassalli, P. (1990). Regulation of tumor necrosis factor
alpha transcription in macrophages: Involvement of four kappa B-like motifs
and of constitutive and inducible forms of NF-kappa B. Molecular and Cellular
Biology, 10, 14981506.
Fletcher, J. R. (1993). Eicosanoids. Critical agents in the physiological process and
cellular injury. Archives of Surgery (Chicago, Ill.: 1960), 128, 11921196.
Gupta, R. A., & Dubois, R. N. (2001). Colorectal cancer prevention and treatment by
inhibition of cyclooxygenase-2. Nature Reviews. Cancer, 1, 1121.
Heiss, E., Herhaus, C., Klimo, K., Bartsch, H., & Gerhauser, C. (2001). Nuclear factor
kappa B is a molecular target for sulforaphane-mediated anti-inammatory
mechanisms. The Journal of Biological Chemistry, 276, 3200832015.
Herschman, H. R. (1996). Prostaglandin synthase 2. Biochimica et Biophysica Acta,
1299, 125140.
Holmes-McNary, M., & Baldwin, A. S. Jr., (2000). Chemopreventive properties of
trans-resveratrol are associated with inhibition of activation of the IkappaB
kinase. Cancer Research, 60, 34773483.
Hou, D. X., Yanagita, T., Uto, T., Masuzaki, S., & Fujii, M. (2005). Anthocyanidins
inhibit cyclooxygenase-2 expression in LPS-evoked macrophages: Structureactivity relationship and molecular mechanisms involved. Biochemical
Pharmacology, 70, 417425.
Jung, H. W., Seo, U. K., Kim, J. H., Leem, K. H., & Park, Y. K. (2009). Flower extract of
Panax notoginseng attenuates lipopolysaccharide-induced inammatory
response via blocking of NF-kappaB signaling pathway in murine
macrophages. Journal of Ethnopharmacology, 122, 313319.
Kang, S. R., Han, D. Y., Park, K. I., Park, H. S., Cho, Y. B., Lee, H. J., et al. (2011).
Suppressive effect on lipopolysaccharide-induced proinammatory mediators
by Citrus aurantium L. in macrophage RAW 264.7 cells via NF-kappaB signal
pathway. Evidence-based Complementary and Alternative Medicine: eCAM.
248592p. Epub 2010 Sep. 21.
Kanno, S., Shouji, A., Tomizawa, A., Hiura, T., Osanai, Y., Ujibe, M., et al. (2006).
Inhibitory effect of naringin on lipopolysaccharide (LPS)-induced endotoxin
shock in mice and nitric oxide production in RAW 264.7 macrophages. Life
Sciences, 78, 673681.
Karin, M. (1999). How NF-kappaB is activated: The role of the IkappaB kinase (IKK)
complex. Oncogene, 18, 68676874.
Kim, J. B., Han, A. R., Park, E. Y., Kim, J. Y., Cho, W., Lee, J., et al. (2007). Inhibition of
LPS-induced iNOS, COX-2 and cytokines expression by poncirin through the NFkappaB inactivation in RAW 264.7 macrophage cells. Biological & Pharmaceutical
Bulletin, 30, 23452351.
Kim, J. Y., Park, S. J., Yun, K. J., Cho, Y. W., Park, H. J., & Lee, K. T. (2008).
Isoliquiritigenin isolated from the roots of Glycyrrhiza uralensis inhibits LPS-

induced iNOS and COX-2 expression via the attenuation of NF-kappaB in RAW
264.7 macrophages. European Journal of Pharmacology, 584, 175184.
Kim, H. P., Son, K. H., Chang, H. W., & Kang, S. S. (2004). Anti-inammatory plant
avonoids and cellular action mechanisms. Journal of Pharmacological Sciences,
96, 229245.
Lawrence, T., Gilroy, D. W., Colville-Nash, P. R., & Willoughby, D. A. (2001). Possible
new role for NF-kappaB in the resolution of inammation. Nature Medicine, 7,
12911297.
Lee, S., Ra, J., Song, J. Y., Gwak, C., Kwon, H. J., Yim, S. V., et al. (2011). Extracts from
Citrus unshiu promote immune-mediated inhibition of tumor growth in a
murine renal cell carcinoma model. Journal of Ethnopharmacology, 133,
973979.
MacMicking, J., Xie, Q. W., & Nathan, C. (1997). Nitric oxide and macrophage
function. Annual Review of Immunology, 15, 323350.
Mariathasan, S., & Monack, D. M. (2007). Inammasome adaptors and sensors:
Intracellular regulators of infection and inammation. Nature Reviews.
Immunology, 7, 3140.
Murakami, A., Nakamura, Y., Torikai, K., Tanaka, T., Koshiba, T., Koshimizu, K., et al.
(2000). Inhibitory effect of citrus nobiletin on phorbol ester-induced skin
inammation, oxidative stress, and tumor promotion in mice. Cancer Research,
60, 50595066.
Nogata, Y., Sakamoto, K., Shiratsuchi, H., Ishii, T., Yano, M., & Ohta, H. (2006).
Flavonoid composition of fruit tissues of citrus species. Bioscience, Biotechnology,
and Biochemistry, 70, 178192.
Pan, M. H., Lin-Shiau, S. Y., & Lin, J. K. (2000). Comparative studies on the
suppression of nitric oxide synthase by curcumin and its hydrogenated
metabolites through down-regulation of IkappaB kinase and NFkappaB
activation in macrophages. Biochemical Pharmacology, 60, 16651676.
Seger, R., & Krebs, E. G. (1995). The MAPK signaling cascade. The FASEB Journal:
Ofcial Publication of the Federation of American Societies for Experimental Biology,
9, 726735.
Soliman, K. F., & Mazzio, E. A. (1998). In vitro attenuation of nitric oxide production
in C6 astrocyte cell culture by various dietary compounds. Proceedings of the
Society for Experimental Biology and Medicine. Society for Experimental Biology and
Medicine (New York, N.Y.), 218, 390397.
Walsh, L. J. (2003). Mast cells and oral inammation. Critical Reviews in Oral Biology
and Medicine: An Ofcial Publication of the American Association of Oral Biologists,
14, 188198.
Zhou, H. Y., Shin, E. M., Guo, L. Y., Youn, U. J., Bae, K., Kang, S. S., et al. (2008). Antiinammatory activity of 4-methoxyhonokiol is a function of the inhibition of
iNOS and COX-2 expression in RAW 264.7 macrophages via NF-kappaB, JNK and
p38 MAPK inactivation. European Journal of Pharmacology, 586, 340349.