Isoliquiritigenin isolated from the roots of Glycyrrhiza uralensis inhibits LPS-induced iNOS and COX-2 expression via the attenuation of NF-κB in RAW 264.7 macrophages

Article abstract of DOI:10.1016/j.ejphar.2008.01.032

In this study, the anti-inflammatory effects of flavonoids isolated from the roots of Glycyrrhiza uralensis (Leguminosae), namely, isoliquiritin (the glycoside of isoliquirigenin) and isoliquiritigenin (the aglycone of isoliquiritin) were evaluated on lipopolysaccharide (LPS)-treated RAW 264.7 macrophages. Isoliquiritigenin (ILG) more potently inhibited LPS-induced nitric oxide (NO) and prostaglandin E₂ (PGE₂) production than isoliquiritin (ILT). Consistent with these findings, ILG reduced the LPS-induced expressions of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) at the protein and mRNA levels in a concentration-dependent manner, as determined by Western blotting and RT-PCR, respectively. In addition, the release of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), and the mRNA expression levels of these cytokines were reduced by ILG in a dose-dependent manner. Moreover, ILG attenuated the LPS-induced DNA binding activity and the transcription activity of nuclear factor-kappa B (NF-κB), and this was associated with a decrease in inhibitory kappa B-α (IκB-α) phosphorylation and in the subsequent blocking of p65 and p50 protein translocations to the nucleus. Furthermore, ILG suppressed the phosphorylations of IκB kinase (IKK), ERK1/2, and p38, whereas the phosphorylation of JNK1/2 was unaffected. These results suggest that the anti-inflammatory properties of ILG are caused by iNOS, COX-2, TNF-α, and IL-6 down-regulation due to NF-κB inhibition via the suppression of IKK, ERK1/2 and p38 phosphorylation in RAW 264.7 cells.

Full text of DOI:10.1016/j.ejphar.2008.01.032

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European Journal of Pharmacology 584 (2008) 175–184
www.elsevier.com/locate/ejphar
Isoliquiritigenin isolated from the roots of Glycyrrhiza uralensis inhibits
LPS-induced iNOS and COX-2 expression via the attenuation
of NF-κB in RAW 264.7 macrophages
Ji-Yeon Kim a,b, Seung Jae Park a,b, Kyung-Jin Yun a,b, Young-Wuk Cho b,
Hee-Juhn Park c, Kyung-Tae Lee a,b,
⁎
a
Department of Pharmaceutical Biochemistry, College of Pharmacy, Kyung-Hee University, Dongdaemun-ku, Hoegi-Dong, Seoul 130-701, South Korea
b
Department of Biomedical Science, College of Medical Science, Kyung-Hee University, Seoul 130-701, South Korea
c
Department of Botanical Resources, Sangji University, Wonju 220-702, South Korea
Received 3 September 2007; received in revised form 3 January 2008; accepted 22 January 2008
Available online 5 February 2008
Abstract
In this study, the anti-inflammatory effects of flavonoids isolated from the roots of Glycyrrhiza uralensis (Leguminosae), namely, isoliquiritin
(the glycoside of isoliquirigenin) and isoliquiritigenin (the aglycone of isoliquiritin) were evaluated on lipopolysaccharide (LPS)-treated RAW
264.7 macrophages. Isoliquiritigenin (ILG) more potently inhibited LPS-induced nitric oxide (NO) and prostaglandin E2 (PGE2) production than
isoliquiritin (ILT). Consistent with these findings, ILG reduced the LPS-induced expressions of inducible nitric oxide synthase (iNOS) and
cyclooxygenase-2 (COX-2) at the protein and mRNA levels in a concentration-dependent manner, as determined by Western blotting and RT-
PCR, respectively. In addition, the release of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), and the mRNA expression levels of these
cytokines were reduced by ILG in a dose-dependent manner. Moreover, ILG attenuated the LPS-induced DNA binding activity and the
transcription activity of nuclear factor-kappa B (NF-κB), and this was associated with a decrease in inhibitory kappa B-α (IκB-α) phosphorylation
and in the subsequent blocking of p65 and p50 protein translocations to the nucleus. Furthermore, ILG suppressed the phosphorylations of IκB
kinase (IKK), ERK1/2, and p38, whereas the phosphorylation of JNK1/2 was unaffected. These results suggest that the anti-inflammatory
properties of ILG are caused by iNOS, COX-2, TNF-α, and IL-6 down-regulation due to NF-κB inhibition via the suppression of IKK, ERK1/2
and p38 phosphorylation in RAW 264.7 cells.
© 2008 Published by Elsevier B.V.
Keywords: Isoliquiritigenin; Isoliquiritin; NF-κB; iNOS; COX-2
1. Introduction
response to infectious pathogens, whereas chronic inflammation
is an undesirable persistent phenomenon that can lead to the
developments of inflammatory diseases (Kaplanski et al., 2003).
Prolonged inflammation contributes to the pathogenesis of many
inflammatory diseases, such as, bronchitis (Vernooy et al., 2002),
gastritis (Sakagami et al., 1997), inflammatory bowel disease
(Fichtner-Feigl et al., 2005), multiple sclerosis (Klotz et al., 2005),
and rheumatoid arthritis (Ponchel et al., 2002).
Bacterial pathogens, like LPS, activate cytokine networks by
inducing many pro-inflammatory genes. Moreover, this induc-
tion is mediated via the activations of inducible transcription
factors (Liu and Malik, 2006). NF-κB is one of the pivotal
regulators of pro-inflammatory gene expression, and aberrant
the regulation of NF-κB activity has been implicated in the
Inflammation is the result of host response to pathogenic
challenges or tissue injuries, and ultimately leads to the restoration
of a normal tissue structure and function. Normal inflammatory
responses are self-limited by a process that involves the down-
regulations of pro-inflammatory proteins and the up-regulations
of anti-inflammatory proteins (Lawrence et al., 2002). Thus, acute
inflammation is a limited beneficial process, particularly in
⁎
Corresponding author. Department of Pharmaceutical Biochemistry, College
of Pharmacy, Kyung-Hee University, Dongdaemun-Ku, Hoegi-Dong, Seoul,
130-701, South Korea. Tel.: +82 2 961 0860; fax: +82 2 966 3885.
E-mail address: ktlee@khu.ac.kr (K.-T. Lee).
0014-2999/$ - see front matter © 2008 Published by Elsevier B.V.
doi:10.1016/j.ejphar.2008.01.032

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pathogenesis of many diseases, which include immune
deficiencies and disorders, neurodegenerative disorders, auto-
immune and inflammatory diseases, ischemia reperfusion
injury, and cancer (Liu and Malik, 2006). NF-κB proteins in
cytoplasm are associated with inhibitory proteins known as
IκBs. Moreover, the main activated form of NF-κB is a
heterodimer composed of p65 and p50 subunits (Li and Verma,
2002). NF-κB induction in response to pro-inflammatory
stimuli involves the phosphorylation of IκBs at two critical
serine residues (Ser32, Ser36) by IKK signalosome complex
(O'Conell et al., 1996). After IκB has been phosphorylated, it is
ubiquitinated and degraded by 26S proteosome (Sanchez-perez
et al., 2002), and the resulting free NF-κB is translocated to the
nucleus, where it binds to κB binding sites in the promoter
regions of target genes, and induces the transcriptions of pro-
inflammatory mediators, such as, iNOS, COX-2, TNF-α and
IL-1β, -6 and -8 (Surh et al., 2001; Lappas et al., 2002).
iNOS catalyzes the oxidative deamination of L-arginine to
produce NO, a potent pro-inflammatory mediator (Ohshima and
Bartsch, 1994). Moreover, iNOS expression is significantly
induced by LPS or cytokines in a variety of immune cells,
including macrophages. COX-2 is another enzyme that plays a
pivotal role in the mediation of inflammation, and catalyzes the
rate-limiting step in prostaglandin (PG) biosynthesis.
Fig. 1. The chemical structures of isoliquirigenin (A) and isoliquiritin (B).
the anti-inflammatory potential of natural compounds, we
investigated the anti-inflammatory effects of ILG isolated from
the roots of G. uralensis in LPS-induced macrophage model.
2. Materials and methods
2.1. Chemicals
There are two isoforms of COX, designated COX-1 and
COX-2 (O'Neill and Hutchinson, 1993). Although both
isoforms are involved in the formation of PG endoperoxides
(Marnett et al., 1999), they are likely to have fundamentally
different biological roles. COX-1 is a housekeeping enzyme and
is constitutively expressed in most mammalian tissues. In
contrast, COX-2 is barely detectable under normal physiologi-
cal conditions, but like other early response gene products, it
can be rapidly and transiently induced by pro-inflammatory
mediators and mitogenic stimuli, which include cytokines,
endotoxins, growth factors, oncogenes, and phorbol esters.
Thus, there is considerable research interest in the identification
of new anti-inflammatory agents from plants used in traditional
medicine. Licorice (Glycyrrhiza uralensis) has been used for more
than 4 millennia as a flavoring agent in foods, beverages, and
tobacco, and to treat individuals with gastric or duodenal ulcers
(Fintelmann, 1991), sore throats, coughs, bronchitis, arthritis
(Kamei et al., 2004), adrenal insufficiency, and allergies (Haggag
et al., 2003). Moreover, many studies have revealed that several
licorice-derived compounds, i.e., glycyrrhizin, isoliquiritigenin
(ILG), licochalcone, and glabridin, have a variety of pharmaceu-
tical effects (Fukai et al., 2003; Yokota et al., 1998; Inoue et al.,
1986; Zhou et al., 2004). In addition, ILG (Fig. 1), a simple
chalcone-type flavonoid, has been evaluated in terms of its anti-
oxidative effects (Haraguchi et al., 1998), anti-platelet aggregation
effects (Tawata et al., 1992), and estrogenic properties (Tamir et al.,
2001). Moreover, several reports have addressed the anti-
carcinogenic activity of ILG. In particular, ILG was found to
suppress COX-2 protein in RAW 264.7 cells, and the proliferation
in colon cancer cells by inducing apoptosis (Takahashi et al.,
2004). However, no report has been issued on the molecular
aspects of its anti-inflammatory effect in RAW 264.7 cells.
Therefore, as a part of our on-going screening program to evaluate
Dulbecco's modified Eagle's minimum essential medium
(DMEM), fetal bovine serum (FBS), penicillin, and streptomy-
cin were obtained from Life Technologies Inc. (Grand Island,
NY, USA). iNOS, COX-2, p65, p50, p-IκB-α, IκB-α, p-IKK-α/
β, IKK-α, IKK-β, p-ERK, ERK, p-p38, p38, p-JNK, JNK and
β-actin monoclonal antibodies and peroxidase-conjugated
secondary antibody were purchased from Santa Cruz Biotech-
nology, Inc. (Santa Cruz, CA, USA). The enzyme immunoassay
(EIA) kits for PGE2, TNF-α and IL-6 were obtained from R&D
Systems (Minneapolis, MN, USA). Random oligonucleotide
primers and M-MLV reverse transcriptase were purchased from
Promega (Madison, WI, USA). dNTP Mix and ex Taq were
obtained from TaKaRa (Seoul, Korea). iNOS, COX-2, TNF-α,
IL-6, and β-actin oligonucleotide primers were purchased from
Bioneer (Seoul, Korea). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphe-
nyl tetrazolium bromide (MTT), sulfanilamide, aprotinin,
leupeptin, phenylmethylsulfonylfluoride (PMSF), dithiothreitol
(DTT), L-N6-(1-iminoethyl)lysine (L-NIL), NS-398, lipopoly-
saccharide (LPS) (Escherichia coli, serotype 0111:B4), Triton
X-100, and all other chemicals were purchased from the Sigma
Chemical Co. (St. Louis, MO, USA).
2.2. Plant material and the isolation of ILG and ILT
The roots of cultivated G. uralensis were collected on a farm in
Pyongchang-kun, Gangwon province, Korea, during July 2004
and identified by Professor Sang-Cheol Lim at the Department of
Botanical Resources, Sangji University (voucher specimen
(#natchem-29) has been deposited at the same university). Dried
roots of G. uralensis (2.5 kg) were extracted with methanol three
times under reflux. The combined extract was filtered and
evaporated under reduced pressure to give a viscous methanol
extract (220 g). A portion of this methanol extract (200 g) was

J.-Y. Kim et al. / European Journal of Pharmacology 584 (2008) 175–184
177
suspended in water (1.0 l) and successively partitioned three times
with chloroform (3×800 ml), ethylacetate (3×800 ml) and n-
butanol (3×800 ml). The ethylacetate- and n-butanol-soluble
fractions were evaporated under reduced pressure to yield the
ethylacetate fraction (49 g) and a n-butanol fraction (87 g),
respectively. A portion of the butanol extract (20 g) was subjected
to silica gel column chromatography (7×60 cm, 650 g) using
chloroform–methanol–water (65:35:10) as a mobile phase.
Collected fractions were checked on TLC and combined to pro-
duce six fractions (GUB-1–GUB-6). The third fraction, GUB-3
(1.5 g), was again purified using Medium Pressure Liquid
Chromatography (MPLC) system with the column (SiO2,
3×21.5 cm, flash column Biotago SF 25+M0712-2, Biotago.
Inc., USA) and with the solvent chloroform–methanol–water
(7:3:1, lower phase) to yield isoliquiritin (320 mg), the structure of
which was identified by comparing its 1H, 13C NMR and FAB-MS
spectra with those previously reported in the literature (Lee et al.,
2003). The purity of the isoliquiritin so obtained was greater than
98.0% by HPLC.
absence or presence of various concentrations (0.4, 0.8, or
1.6 µM) of ILG and ILT for 24 h. Nitrite levels in culture media
were determined using the Griess reaction and presumed to
reflect NO levels (Won et al., 2006). Briefly, 100 µl of cell
culture medium was mixed with 100 μl of Griess reagent [equal
volumes of 1% (w/v) sulfanilamide in 5% (v/v) phosphoric acid
and 0.1% (w/v) naphtylethylenediamine-HCl], and incubated at
room temperature for 10 min. Absorbance was then measured at
540 nm using a microplate reader (Perkin Elmer Cetus, Foster
City, CA, USA). Fresh culture media were used as blanks in all
experiments. Nitrite levels in samples were read off a standard
sodium nitrite curve.
2.7. PGE2, TNF-α and IL-6 assays
RAW 264.7 cells were pretreated with ILG or ILT for 1 h and
then stimulated with LPS (1 μg/ml) for 24 h. Levels of PGE2,
TNF-α and IL-6 in the culture media were quantified using EIA
kits (R&D Systems, Minneapolis, MN. USA).
2.3. Acid hydrolysis of isoliquiritin
2.8. Western blot analysis
Isoliquiritin (500 mg) was dissolved in 100 ml of 5% hydro-
chloric acid and refluxed for 5 h. After cooling at room tem-
perature, the reaction mixture was extracted with ethylacetate
three times. The combined organic phase was then washed with
distilled water twice and concentrated in vacuo. The residue so
obtained was dissolved in methanol and stood overnight to afford
isoliquiritigenin as yellowish needles. Its structure was identified
by comparing its 1H, 13C NMR and FAB-MS spectra with those
previously reported in the literature (Lee et al., 2003). The purity
of the isoliquiritigenin obtained was greater than 98.0% by HPLC.
Control and ILG-treated RAW 264.7 cells were collected by
centrifugation and washed once with phosphate-buffered saline
(PBS). Washed cell pellets were resuspended in extraction lysis
buffer (50 mM HEPES pH 7.0, 250 mM NaCl, 5 mM EDTA, 0.1%
Nonidet P-40, 1 mM PMSF, 0.5 mM DTT, 5 mM NaF and 0.5 mM
sodium orthovanadate) containing 5 µg/ml of leupeptin and
aprotinin, respectively, and incubated for 20 min at 4 °C. Cell
debris was removed by microcentrifugation, and supernatants
were rapidly frozen. Protein concentrations were determined using
Bio-Rad protein assay reagent, according to the manufacture's
instruction. Forty micrograms of cellular proteins from treated or
untreated cell extracts were separated on 10% SDS-polyacryla-
mide gels and electroblotted onto nitrocellulose membranes,
which were incubated overnight with blocking solution (5% skim
milk) at 4 °C, and then with primary antibody for 4 h. Blots were
then washed four times with Tween 20/Tris-buffered saline
(TTBS), incubated with a 1:1000 dilution of horseradish
peroxidase-conjugated secondary antibody for 1 h at room
temperature, rewashed three times with TTBS, and then developed
by enhanced chemiluminescence (Amersham Life Science).
2.4. Cell culture and sample treatment
The RAW 264.7 macrophage cell line was obtained from the
Korea Cell Line Bank (Seoul). Cells were grown at 37 °C in
DMEM medium supplemented with 10% FBS, penicillin (100
units/ml), and streptomycin sulfate (100 µg/ml) in a humidified
5% CO2 atmosphere. Cells were incubated with tested samples
(ILG or ILT) at concentrations of 0.4, 0.8, or 1.6 µM, or with
positive controls (L-NIL or NS-398) and then stimulated with
LPS 1 µg/ml for the indicated time.
2.9. RNA preparation and RT-PCR (reverse transcriptase
polymerase chain reaction)
2.5. MTT assay for cell viability
RAW 264.7 cells were plated at a density of 105 cells/well in
96-well plates. To determine the appropriate concentration not
toxic to cells, cytotoxicity studies were performed 24 h after
treating cells with various concentrations of ILG and ILT.
Viabilities were determined using colorimetric MTT assays, as
described previously (Won et al., 2006).
Total cellular RNAwas isolated using Easy Blue® kits (Intron
Biotechnology, Seoul, Korea). From each sample, 1 µg of RNA
was reverse-transcribed (RT) using MuLV reverse transcriptase,
1 mM deoxyribonucleotide triphosphate (dNTP), and oligo
(dT12–18) 0.5 µg/µl. PCR was performed on aliquots of the
cDNA preparations to detect iNOS, COX-2, TNF-α, IL-6 and
β-actin (the internal standard) gene expressions using a thermal
cycler (Perkin Elmer Cetus, Foster City, CA). Reactions were
carried out in a volume of 25 μl containing; 1 unit of Taq DNA
polymerase, 0.2 mM dNTP, ×10 reaction buffer, and 100 pmol
of 5′ and 3′ primers. After an initial denaturation for 2 min at
2.6. Nitrite determination
RAW 264.7 cells were plated at 4×105 cells/well in 24-well
plates and then incubated with or without LPS (1 μg/ml) in the

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95 °C, 30 amplification cycles were performed for iNOS (1 min
at 95 °C denaturation, 1 min at 60 °C annealing, and 1.5 min at
72 °C extension), COX-2 (1 min at 94 °C, 1 min at 60 °C, and
1 min at 72 °C), TNF-α (1 min at 95 °C, 1 min at 55 °C, and
1 min at 72 °C), and IL-6 (1 min at 94 °C, 1 min at 56 °C, and
1 min at 72 °C). The PCR primers used in this study are listed
below and were purchased from Bioneer (Seoul, Korea): sense
strand iNOS, 5′-AAT GGC AAC ATC AGG TCG GCC ATC
ACT-3′, anti-sense strand iNOS, 5′-GCT GTG TGT CAC
AGA AGT CTC GAA CTC-3′; sense strand COX-2, 5′-GGA
GAG ACT ATC AAG ATA GT-3′, anti-sense strand COX-2,
5′-ATG GTC AGT-AGA CTT TTA CA-3′; sense strand TNF-
α, 5′-ATG AGC ACA GAA AGC ATG ATC-3′, anti-sense
strand TNF-α, 5′-TAC AGG CTT GTC ACT CGA ATT-3′;
sense strand IL-6, 5′-GAG GAT ACC ACT CCC AAC AGA
CC-3′, anti-sense strand IL-6, 5′-AAG-TGC ATC ATC GTT
GTT CAT ACA-3′; sense strand β-actin, 5′-TCA TGA AGT
GTG-ACG TTG ACA TCC GT-3′, anti-sense strand β-actin,
5′-CCT AGA AGC ATT TGC-GGT GCA CGA TG-3′. After
amplification, portions of the PCR reactions were electro-
phoresed on 2% agarose gel, and visualized under UV after
ethidium bromide staining.
Biosciences, San Jose, CA, USA), according to the manufac-
turer's instructions. Cells were incubated for 2 h before adding
5 ml of DMEM containing 10% FBS. At 48 h after the start of
transfection, cells were pretreated with ILG for 1 h and then
stimulated with LPS (1 μg/ml). After 3 h of stimulation, cells were
lysed and luciferase activity was determined using the Promega
luciferase assay system (Promega, Madison, CA, USA) and a
luminometer (Perkin Elmer Cetus, Foster City, CA, USA).
2.12. Statistical analysis
Results are expressed as the means S.D. of triplicate
experiments. Statistically significant values were compared
2.10. Nuclear extraction and electrophoretic mobility shift
assay (EMSA)
RAW 264.7 macrophages cells were plated in 100-mm dishes
(1×106 cells/ml), and treated with ILG (0.4, 0.8, 1.6 µM),
stimulated with LPS for 1 h, washed once with PBS, scraped into
1 ml of cold PBS, and pelleted by centrifugation. Nuclear extracts
were prepared as described previously (Won et al., 2006). Cell
pellets were resuspended in hypotonic buffer (10 mM HEPES, pH
7.9, 1.5 mM MgCl2, 10 mM KCl, 0.2 mM PMSF, 0.5 mM DTT,
10 µg/ml aprotinin) and incubated on ice for 15 min. Cells were
then lysed by adding 0.1% Nonidet P-40 and vortexed vigorously
for 10 s. Nuclei were pelleted by centrifugation at 12,000 ×g for
1 min at 4 °C and resuspended in high salt buffer (20 mM HEPES,
pH 7.9, 25% glycerol, 400 mM KCl, 1.5 mM MgCl2, 0.2 mM
EDTA, 0.5 mM DTT, 1 mM NaF, 1 mM sodium orthovanadate).
Nuclear extracts (10 µg) were mixed with double-stranded NF-κB
oligonucleotide. 5′-AGTTGAGGGGACT-TTCCCAGGC3′ end-
labeled with [γ-32P] dATP (underlying indicates a κB consensus
sequence or a binding site for NF-κB/cRel homodimeric or
heterodimeric complex). Binding reactions were performed at
37 °C for 30 min in 30 µl of reaction buffer containing 10 mM
Tris–HCl, pH 7.5, 100 mM NaCl, 1 mM EDTA, 4% glycerol, 1 µg
of poly (dI–dC) and 1 mM DTT. The specificity of binding was
examined by competition with the 80-fold unlabeled oligonucleo-
tide. DNA–protein complexes were separated from the unbound
DNA probe on native 5% polyacrylamide gels at 100 V in 0.5×
Tris Boric acid EDTA (TBE) buffer. Gels were vacuum dried for
1 h at 80 °C and exposed to X-ray film at −70 °C for 24 h.
Fig. 2. The effects of ILG and ILT on LPS-induced NO (A) and PGE2 (B)
production and cell viability (C) in RAW 264.7 cells. Cells were treated with
different concentrations of ILG or ILT (0.4, 0.8, 1.6 µM) for 1 h, then with LPS
(1 µg/ml), and incubated for 24 h. Control (Con) values were obtained in the
absence of LPS, ILG and ILT. L-N6-(1-iminoethyl) lysine (L-NIL) was used as a
positive control at a concentration of 10 µM (A). Samples were treated as
described in the legend of Fig. 2A. NS-398 (10 µM) was used as a positive
control. Values are means S.D. of three independent experiments. #pb0.05 vs.
the control group; pb0.05, pb0.01 vs. the LPS-treated group; significances
between treated groups were determined using ANOVA and Dunnett's post-hoc
test (B). Cells were exposed to ILG or ILT (from 0.1 µM to 6 µM), and cell
viabilities were assessed using assay (C).
2.11. Transient transfection and luciferase assay
⁎
⁎⁎
RAW 264.7 cells were transfected using Superfect reagent
(Qiagen GmbH, Germany) and NF-κB-Luc reporter plasmid (BD

J.-Y. Kim et al. / European Journal of Pharmacology 584 (2008) 175–184
179
Fig. 3. The effects of ILG on LPS-induced iNOS (A) and COX-2 (B) protein and their mRNA expressions in RAW 264.7 cells. Lysates were prepared from control or
24 h LPS (1 µg/ml) stimulated cells or from LPS plus ILG (0.4, 0.8, 1.6 µM). Total cellular proteins (40 µg) were resolved by SDS-PAGE, transferred to nitrocellulose
membranes, and detected using specific antibodies, as described in Materials and methods. A representative immunoblot of three separate experiments is shown. Total
RNA was prepared for RT-PCR of iNOS and COX-2 gene expressions from RAW 264.7 macrophages stimulated with LPS (1 µg/ml) with/without ILG at 0.4, 0.8, or
1.6 µM for 4 h. iNOS-specific sequences (807 bp) and COX-2-specific sequences (721 bp) were detected by agarose gel electrophoresis, as described in Materials and
methods. PCR of β-actin was performed to confirm that the initial cDNA contents of samples were similar. The experiment was repeated three times and similar results
#
⁎
⁎⁎
were obtained. Values shown are means S.D. of three independent experiments. pb0.05 vs. the control group; pb0.05, pb0.01 vs. the LPS-treated group;
significant differences between groups were determined using ANOVA and Dunnett's post-hoc test (A), (B).
Fig. 4. The effects of ILG on LPS-induced TNF-α (A) and IL-6 (B) release and their mRNA expressions (C) in RAW 264.7 cells. Cells were treated with different
concentrations (0.4, 0.8, 1.6 µM) of ILG for 1 h, LPS (1 µg/ml) was then added, and cells were incubated for a further 24 h. Control (Con) values were obtained in the
absence of LPS or ILG. Samples were treated as described in the legend of Fig. 2A. Values represent means S.D. of three independent experiments. #pb0.05 vs. the
⁎
⁎⁎
control group; pb0.05, pb0.01 vs. the LPS-treated group; significant differences between groups were determined using ANOVA and Dunnett's post-hoc test (A),
(B). Total RNA was prepared for RT-PCR of TNF-α and IL-6 gene expressions from RAW 264.7 cells pretreated with different concentrations (0.4, 0.8, 1.6 µM) of
ILG for 1 h, and then with LPS (1 µg/ml) for 4 h. TNF-α-specific sequences (351 bp) and IL-6-specific sequences (142 bp) were detected by agarose gel
electrophoresis, as described in Materials and methods. PCR for β-actin was performed to confirm that initial cDNA contents were similar. The experiment was
repeated three times with similar results (C).

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using ANOVA and Dunnett's post-hoc test, and p-values of less
than 0.05 were considered statistically significant.
assays (EMSA) and luciferase assays were used to determine
whether ILG suppresses NF-κB activation. Accordingly, a
DNA binding assay of NF-κB was carried out using nuclear
extracts obtained from RAW 264.7 cells stimulated with LPS in
the presence or absence of ILG. Treatment with LPS (1 µg/ml)
was found to increase NF-κB-DNA binding, and pretreating
cells with ILG prior to LPS decreased NF-κB-DNA binding in a
concentration-dependent manner (Fig. 5A). In an additional
study, we examined the effects of ILG on NF-κB-dependent
reporter gene expression induced by LPS. In this experiment,
we used a pNF-κB-Luc plasmid, which was generated by
inserting four spaced NF-κB binding sites into pLuc-promoter
vector. Accordingly, RAW 264.7 cells were transiently
transfected with pNF-κB-Luc plasmid and then stimulated
with 1 µg/ml LPS either in the presence or absence of ILG. ILG
treatment at 0.4, 0.8, or 1.6 μM significantly reduced LPS-
3. Results
3.1. Effects of ILG or ILT on LPS-induced NO and PGE2
production and cell viability
To compare the effects of ILG and ILT on LPS-induced NO
and PGE2 production in RAW 264.7 cells, culture media were
harvested and nitrite and PGE2 levels were measured. ILG more
significantly inhibited LPS-induced NO and PGE2 production
than ILT, and did so in a dose-dependent manner (Fig. 2A and B).
Moreover, ILG potently reduced NO production with an IC50
value of 1.3 μM, whereas ILT had less effect at the same
concentration. In addition, treatments with ILG or ILT at 1.6 μM
reduced LPS-induced PGE2 production by 86% and 49%,
respectively. L-NIL (10 µM) and NS-398 (10 µM) were used as
positive NO and PGE2 production controls, respectively.
Furthermore, the cytotoxic effects of ILG and ILTwere evaluated
in RAW 264.7 cells using MTT assays, but neither affected cell
viabilities at the concentrations used (0.4, 0.8, 1.6 μM) to inhibit
NO and PGE2 production (Fig. 2C).
3.2. Effects of ILG on LPS-induced iNOS and COX-2 protein
and mRNA expressions
Since ILG was found to inhibit NO and PGE2 production, we
used Western blotting and RT-PCR to determine whether these
inhibitory effects were related to iNOS and COX-2 modulation.
iNOS and COX-2 protein levels were markedly up-regulated in
response to LPS, and ILG strongly inhibited these protein
expression in a concentration-dependent manner. Moreover, RT-
PCR analysis showed that the expressions of iNOS and COX-2
mRNA correlated with their protein levels (Fig. 3A and B).
However, ILG had no effect on the expression of the house-
keeping gene β-actin.
3.3. Effects of ILG on LPS-induced TNF-α and IL-6 release
and their mRNA expressions
Since our data showed that ILG inhibited the pro-
inflammatory mediators, such as NO and PGE2, we further
examined its effects on the LPS-induced TNF-α and IL-6
release by enzyme immunoassay and RT-PCR. It was found that
pretreatment of cells with ILG considerably reduced the
production of TNF-α and IL-6 (Fig. 4A and B) and their
mRNA expressions (Fig. 4C) in a dose-dependent manner.
Moreover, changes in TNF-α and IL-6 production were similar
to the observed reduction in their mRNA expressions.
Fig. 5. The inhibitory effects of ILG on LPS-induced NF-κB-DNA binding
(A) and luciferase activity (B). Nuclear extracts were prepared from control or
pretreated with different concentrations (0.4, 0.8, 1.6 µM) of ILG for 1 h, then
with LPS (1 µg/ml) for 1 h, and analyzed for NF-κB binding by EMSA. The
arrow indicates the position of the NF-κB band. The data shown are
representative of three independent experiments (A). Cells were transiently
cotransfected with pNF-κB-luc reporter and then either left untreated (Con) or
were pretreated with different concentrations (0.4, 0.8, 1.6 µM) of ILG for 1 h.
LPS (1 µg/ml) was then added and cells were further incubated for 3 h. Cells
were then harvested and luciferase activities were determined using a Promega
luciferase assay system and a luminometer. The values shown are means S.D.
3.4. Effects of ILG on LPS-induced NF-κB-DNA binding and
NF-κB transcriptional activity
Since NF-κB activation is critically required for the
expressions of iNOS, COX-2, TNF-α and IL-6 by LPS (Surh
et al., 2001; Lappas et al., 2002), electrophoretic mobility shift
of three independent experiments. #pb0.05 vs. the control group; pb0.05,
⁎
⁎⁎
pb0.01 vs. the LPS-treated group. Significant differences between treated
groups were determined using ANOVA and Dunnett's post-hoc test (B).

J.-Y. Kim et al. / European Journal of Pharmacology 584 (2008) 175–184
181
Fig. 6. Inhibition of the nuclear translocation of NF-κB (A) and of the phosphorylation of IκB-α (B) by ILG. RAW 264.7 macrophages were treated with LPS (1 µg/ml)
alone or with LPS (1 µg/ml) plus different concentrations (0.4, 0.8, 1.6 µM) of ILG for 1 h. Nuclear extracts were prepared for the Western blotting of p65 and p50 of
NF-κB using specific anti-p65 and p50 NF-κB monoclonal antibodies. PARP and β-actin were used as internal controls. The data shown are representative of three
independent experiments (A). Cells were pretreated with the indicated concentrations of ILG for 1 h and then with LPS (1 µg/ml) for 15 min. Total cellular proteins
were then prepared and Western blotted for IκB-α and p-IκB-α using specific IκB-α and p-IκB-α antibodies (B).
induced increases in NF-κB-dependent luciferase activity
LPS-induced IκB-α phosphorylation in a concentration-depen-
(Fig. 5B).
dent manner.
3.5. Inhibitory effects of ILG on the LPS-induced nuclear
translocation of NF-κB and the phosphorylation of IκB-α
3.6. Effects of ILG on LPS-induced phosphorylation of IKK and
the activations of mitogen-activated protein (MAP) kinases
Here, we investigated whether ILG prevents the transloca-
tion of the subunits of NF-κB, i.e., p65 and p50, from the
cytosol to the nucleus after its release from IκBs. It was found
that pretreatment with ILG prior to LPS attenuated p65 and p50
levels in nuclear fractions and recovered p65 and p50 levels in
cytosolic fractions (Fig. 6A). β-actin and PARP were used as
internal controls. We also explored whether ILG inhibits the
LPS-stimulated degradation of IκB-α in RAW 264.7 cells by
Western blotting with anti-IκB-α antibody. Fig. 6B shows that
LPS-induced IκB-α degradation was significantly blocked by
ILG pretreatment. Furthermore, to determine whether this IκB-
α degradation was related to IκB-α phosphorylation, we
examined the effect of ILG on the LPS-induced p-IκB-α by
Western blotting, and found that ILG also significantly reduced
Since IKK-α and -β are upstream kinases of IκB in the NF-
κB signal pathway, and in particular, phosphorylate IκB-α and
-β (Surh et al., 2001), we examined the effects of ILG on LPS-
induced IKK-α/β activation by immunoblotting using IKK-α,
-β, and phosphorylated IKK-α/β antibodies. RAW 264.7 cells
were pretreated with ILG (0.4, 0.8, 1.6 μM) for 1 h and then
stimulated with LPS (1 μg/ml). It was found that LPS strongly
induce IKK-α/β phosphorylation, whereas ILG markedly
reduced this LPS-induced phosphorylation (Fig. 7A). ILG did
not affect the total amounts of IKK-α and IKK-β. β-actin was
used as internal control.
MAP kinases play critical roles in the regulation of cell growth
and differentiation, and control cellular responses to cytokines
and stresses (Vandenberghe et al., 1998). In addition, they are also

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J.-Y. Kim et al. / European Journal of Pharmacology 584 (2008) 175–184
Fig. 7. The effects of ILG on the LPS-induced phosphorylations of IKK-α/β (A) and MAP kinases (B) in RAW 264.7 cells. RAW 264.7 macrophages were treated with
LPS (1 µg/ml) in the absence or presence of the indicated concentrations of ILG for 5 min. Lysates were analyzed by Western blotting using specific p-IKK-α/β, IKK-α,
and IKK-β antibodies. β-Actin was used as an internal control (A). RAW 264.7 macrophages were treated with LPS (1 µg/ml) alone or with LPS (1 µg/ml) plus the
indicated concentrations of ILG for 15 min. Whole cell lysates were analyzed by Western blotting using antibodies against the activated MAP kinases. The data shown are
representative of three independent experiments (B).
known to be important for the activation of NF-κB (Vanden-
berghe et al., 1998). To investigate whether the inhibition of NF-
κB activation by ILG is mediated through the MAP kinases
pathway, we examined the effect of ILG on the LPS-stimulated
phosphorylations of ERK1/2, JNK1/2, and p38 MAP kinases in
RAW 264.7 cells by Western blotting. As shown in Fig. 7B, ILG
suppressed the LPS-induced activations of ERK1/2 and p38 MAP
kinases, but did not affect the phosphorylation of JNK1/2. Non-
phosphorylated ERK, JNK, and p38 kinase expressions were
unaffected by LPS or by LPS plus ILG.
2005). Thus, RAW 264.7 macrophages provide us with an
excellent model for anti-inflammatory drug screening and for
subsequently evaluating the inhibitors of the pathways that
leads to the induction of pro-inflammatory enzymes and to the
production of pro-inflammatory cytokines.
During our continued search for novel anti-inflammatory
agents from natural products, we found that ILG (isoliquir-
itigenin) and ILT (isoliquiritin) isolated from the roots of G.
uralensis potently inhibit LPS-induced NO and PGE2 produc-
tion in RAW 264.7 macrophages. In addition, we also found
that ILG has a greater inhibitory effect on LPS-induced NO and
PGE2 production than ILT, which is possibly due to their
different affinities for cellular membranes, because affinity for
cellular membranes appears to play an important role in the
cellular uptakes of lipophilic compounds by passive diffusion,
and because ILT possesses a sugar moiety, which would reduce
its lipophilicity and compatibility with cellular membranes.
Experimental evidence suggests that the ethanol extract of
roasted licorice root has potent anti-inflammatory effects because
it reduces NO and PGE2 levels, and pro-inflammatory cytokine
production in LPS-stimulated RAW 264.7 cells. However,
constituents responsible have not been identified (Kim et al.,
2006). ILG is a chalcone, and is one of the major active
components in this licorice extract. Earlier comparative studies on
various molecules with a chalcone structure found that ILG
contains a unique structure (4,2′,4′-trihydroxychalcone) and that
4. Discussion
Macrophages play a central role in inflammatory response
and serve as an essential interface between innate and adaptive
immunity (Iontcheva et al., 2004). In particular, they are
responsible for antigen processing and for presenting antigens
to antigen-specific T cells. Following activation, macrophages
modulate the expression of toll-like receptor (TLR) 4 (Iontcheva
et al., 2004), and the stimulation of TLR4 by LPS triggers its
recruitment of cytoplasmic adaptor protein MyD88, which
results in the activation of two distinct downstream signaling
pathways; the NF-κB pathway and the MAP kinase pathway
(Akira and Takeda, 2004). These two pathways induce the
expressions of various inflammatory mediators, which include
NO, PGs, and the pro-inflammatory cytokines (Gomez et al.,

J.-Y. Kim et al. / European Journal of Pharmacology 584 (2008) 175–184
183
this structure could confer considerable anti-inflammatory
activity (Madan et al., 2000; Takano-Ishikawa et al., 2003).
Although (Kumar et al., 2007) recently reported that ILG inhibits
the TNF-α induced adhesion of neutrophils to endothelial
monolayers by blocking the expressions of ICAM-1, VCAM-1
and E-selectin, and that it blocks NF-κB activity by inhibiting IκB
kinase activity and TNF-α induced ROS production in
endothelial cells, the mechanisms underlying the anti-inflamma-
tory action of ILG in macrophages have not been addressed. Thus,
we investigated the effect of ILG on pro-inflammatory molecules
in LPS-treated RAW 264.7 cells. To investigate the inhibitory
effects of ILG on pro-inflammatory mediators, we examined the
expressions of iNOS and COX-2 protein and of iNOS, COX-2,
TNF-α and IL-6 mRNA. We found that the inhibition of iNOS
and COX-2 proteins by ILG paralleled those of their mRNAs in a
concentration-dependent manner. Thus, we considered that the
observed reductions in NO production and PGE2 release by ILG
might have been due to the transcriptional suppressions iNOS and
COX-2. Moreover, the inhibitory effects of ILG on the LPS-
induced expressions of these molecules in RAW 264.7 cells were
not due to the cytotoxicity of ILG, as assessed by MTT assay and
by the expression of β-actin.
Cytokines like TNF-α and IL-6 have been reported to be pro-
inflammatory in vitro and in vivo (Molly et al., 1993), and the
production of TNF-α is known to be crucial for the induction of
NO synthesis in IFN-γ and/or LPS-stimulated macrophages
(Jun et al., 1995). TNF-α elicits a number of physiological
effects that include septic shock, inflammation, cachexia, and
cell death (Aggarwal and Natarajan, 1996), and similarly, IL-6
is also considered to be a pivotal pro-inflammatory cytokine, for
example, it is regarded an endogenous mediator of LPS-induced
fever. Hence, we scrutinized the inhibitory effect of ILG on
TNF-α and IL-6 production and transcription. Our findings
indicate that ILG significantly inhibits the release of TNF-α and
IL-6 to media and their mRNA expressions.
1998). Transient IKK activation or deactivation is physiologi-
cally important because persistent NF-κB activity can result in
deleterious or even fatal conditions, such as, septic shock or
acute inflammation (Karin and Ben-Neriah, 2000). Other
studies have shown that specific inhibitors of the ERK and
p38 MAP kinase pathways block nuclear NF-κB activity and
the transactivation activity of p65 (Beyaert et al., 1996;
Vandenberghe et al., 1998). In the present study, we observed
that ILG inhibited the phosphorylations of IKKα/β and MAP
kinases. Furthermore, our findings indicate that the inhibition of
the phosphorylations of IKKα/β, ERK1/2 and p38 kinase is
involved in NF-κB inactivation. Thus, we suggest that the
inhibition of IKK, ERK1/2 and p38 kinase by ILG underlies its
inhibition of NF-κB activation.
In summary, the present study demonstrates that ILG ef-
fectively inhibits LPS-induced iNOS and COX-2 protein
expressions and iNOS, COX-2, TNF-α and IL-6 transcriptions
by inhibiting the degradation and phosphorylation of IκB-α, and
by blocking the activation of NF-κB in RAW 264.7 macrophages.
The anti-inflammatory effects of ILG may also be accomplished
by reducing NF-κB activation via the IKK and MAP kinases
signal pathways. Thus, our results suggest that ILG should be
considered a potential treatment for inflammatory diseases.
Acknowledgements
This work was supported by grant No. R13-2002-020-01002-
0 from the Korea Science & Engineering Foundation and by
Seoul Research and Business Development Program (10524).
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