Journal of Ethnopharmacology 145 (2013) 598–607

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

Methanol extract of Hopea odorata suppresses inflammatory responses

via the direct inhibition of multiple kinases
Yanyan Yang a,1, Tao Yu a,1, Yong Gyu Lee b,1, Woo Seok Yang a, Jueun Oh a, Deok Jeong a, Sukchan Lee a,
Tae Woong Kim c, Yung Chul Park d, Gi-Ho Sung e, Jae Youl Cho a,n
a
Department of Genetic Engineering, Sungkyunkwan University, Suwon 440–746, Republic of Korea
b
College of Biomedical Sciences, Kangwon National University, Chuncheon 200–701, Republic of Korea
c
Department of Biochemistry, Kangwon National University, Chuncheon 200–701, Republic of Korea
d
Department of Forest Environment Protection, Kangwon National University, Chuncheon 200–701, Republic of Korea
e
Department of Herbal Crop Research, National Institutes of Horticultural & Herbal Science, Rural Development Administration, Suwon 441–707, Republic of Korea

a r t i c l e i n f o a b s t r a c t

Article history: Ethnopharmacological relevance: Hopea odorata Roxb. (Dipterocarpaceae) is a representative Thai
Received 4 October 2012 ethnopharmacological herbal plant used in the treatment of various inflammation-related diseases.
Received in revised form In spite of its traditional use, systematic studies of its anti-inflammatory action have not been
15 November 2012
performed.
Accepted 18 November 2012
Available online 7 December 2012
Materials and methods: The inhibitory activities of a Hopea odorata methanol extract (Ho-ME) on the
production of nitric oxide (NO), tumour necrosis factor (TNF)-a, and prostaglandin E2 (PGE2) in
Keywords: RAW264.7 cells and peritoneal macrophages were investigated. The effects of Ho-ME on the gastritis
Hopea odorata Roxb symptoms induced by HCl/EtOH and on ear oedemas induced by arachidonic acid were also examined.
Dipterocarpaceae
Furthermore, to identify the immunopharmacological targets of this extract, nuclear fractionation, a
Transcription factor
reporter gene assay, immunoprecipitation, immunoblot analysis, and a kinase assay were employed.
Inflammatory mediator
Target enzyme Results: Ho-ME strongly inhibited the release of NO, PGE2, and TNF-a in RAW264.7 cells and peritoneal
Gastritis macrophages stimulated by lipopolysaccharide (LPS). Ho-ME also clearly suppressed the gene expres-
sion of pro-inflammatory cytokines and chemokines, such as interferon (IFN)-b, interleukin (IL)-12, and
monocyte chemotactic protein-1 (MCP-1). By analysing the inhibited target molecules, Syk and Src
were found to be suppressed in the inhibition of nuclear factor (NF)-kB pathway. In addition, the
observed downregulation of activator protein (AP)-1 and cAMP response element-binding (CREB) was
due to the direct inhibition of interleukin-1 receptor-associated kinase (IRAK)1 and IRAK4, which was
also linked to the suppression of c-Jun N-terminal kinase (JNK) and p38. In agreement with the in vitro
observations, this extract also ameliorated the inflammatory symptoms in EtOH/HCl-induced gastritis
and arachidonic acid-induced ear oedemas in mice.
Conclusion: Ho-ME has potential as a functional herbal remedy targeting Syk- and Src-mediated anti-
inflammatory mechanisms. Future pre-clinical studies will be needed to investigate this possibility.
& 2012 Elsevier Ireland Ltd. All rights reserved.

1. Introduction

Abbreviations: PG, prostaglandin; NO, nitric oxide; COX, cyclooxygenase; iNOS,

When bacteria or fungi infect the body, phagocytic inflamma-
inducible NO synthase; (TNF)-a, tumour necrosis factor; ERK, extracellular
signal-related kinase; TLR, Toll-like receptor; MAPK, mitogen activated protein tory cells such as macrophages and neutrophils participate in
kinase; NF-kB, nuclear factor-kB; AP-1, activator protein-1, JNK, c-Jun N-terminal removing the pathogens. During this response, various chemo-
kinase; EIA, enzyme immunoassay; ELISA, enzyme-linked immunosorbent assay; kines and cytokines, such as monocyte chemoattractant protein
MTT, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a tetrazole;
(MCP)-1 and tumour necrosis factor (TNF)-a, and various inflam-
PI3K, phosphoinositide 3-kinase; LPS, lipopolysaccharide; RT-PCR, reverse
transcription-polymerase chain reaction; DSS, dextran sulphate sodium; PMA,
matory mediators, such as reactive oxygen species (ROS), pros-
phorbol 12-myristate 13-acetate; MyD88, myeloid differentiation primary taglandin (PG)E2, and nitric oxide (NO), mediate the activation of
response gene (88); IkBa, inhibitor of kappa B alpha; Ikk, IkB kinase; Syk, spleen inflammatory cells (Toltl et al., 2008). If inflammatory inducers
tyrosine kinase; CREB, cAMP response element-binding; CMC, sodium are not fully neutralised, chronic inflammation is generated and
carboxymethylcellulose; PEI, Polyethylenimine
n sustained without a strong inflammatory response. Tissues and
Corresponding author. Tel.: þ82 31 290 7868; fax: þ82 31 290 7870.
E-mail addresses: jaecho@skku.edu, jaecho@kangwon.ac.kr (J. Youl Cho). organs can be damaged by the inflammatory mediators released
1
These authors equally contributed to this work. during chronic inflammation, leading to the loss of normal

0378-8741/$ - see front matter & 2012 Elsevier Ireland Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.jep.2012.11.041
 Y. Yang et al. / Journal of Ethnopharmacology 145 (2013) 598–607 599

functions. It is generally accepted that chronic inflammation is a 2.3. Preparation of peritoneal macrophages
silent killer, causing serious diseases such as cancer, diabetes,
atherosclerosis, and arthritis (McGeer and McGeer, 2008). There- Peritoneal exudates were obtained from C57BL/6 male mice by
fore, there have been recent efforts to develop safe, potent anti- lavage 4 days after the intraperitoneal injection of 1 ml of sterile
inflammatory drugs for the treatment of chronic inflammation 4% thioglycollate broth (Difco Laboratories, Detroit, MI) as
(Massarotti, 2008). One of the best sources of safe anti- reported previously (Lee et al., 2010). After washing with RPMI
inflammatory remedies is ethnomedicinal herbal plants, which 1640 medium containing 2% FBS, the peritoneal macrophages
have been in use for many years (Lukhoba et al., 2006). (1  106 cells/ml) were plated in 100-mm tissue culture dishes for
Hopea odorata Roxb. (known as Tkhian Thong, Dipterocarpaceae) 4 h at 37 1C in a 5% CO2 humidified atmosphere.
is a representative medicinal plant used in the Khok Pho District
(Pattani Province) of Thailand (Mahady, 2002; Wiyakrutta et al., 2.4. Cell culture
2004) to treat various inflammation-related diseases. The tannin-
rich bark and leaves of this plant have been used for treating The primary macrophages, RAW 264.7, and HEK293 cells were
paralysis, haemorrhoids, diarrhoea, gum inflammation, and urinary cultured in RPMI 1640 medium supplemented with 10% heat-
incontinence (Chuakul, 2005; Gardner et al., 2000). The stem bark of inactivated FBS, glutamine, and antibiotics (penicillin and strep-
Hopea odorata has also been traditionally used to treat neck pains in tomycin) at 37 1C under 5% CO2. For each experiment, the cells
the North Andaman Islands, India (Prasad et al., 2008). This plant were detached with a cell scraper. Under our experimental cell
contains various resveratrol derivatives, including hopeaphenol, density (2  106 cells/ml), the proportion of dead cells was less
vaticanol B, hemsleyanol B, stemonoporal A, e-viniferin, and laevi- than 1% according to Trypan blue dye exclusion tests.
fonol, which show radical scavenging activity as well as other
important biological properties, such as anti-inflammatory, anti-
2.5. NO, TNF-a, and PGE2 production
bacterial, and anti-cancer activity (Hasan et al., 2009; Zain et al.,
2010, 2011). Although Hopea odorata has been used in traditional
A stock solution (350 mg/ml) of Ho-ME was prepared in 100%
herbal medicine, detailed studies on its anti-inflammatory actions
dimethyl sulfoxide (DMSO) and diluted to 0–200 mg/ml with
have not yet been performed. Therefore, in this study, we aimed to
media for the in vitro assays with cell lines or suspended with
investigate the anti-inflammatory potential of Hopea odorata under
1.5% sodium carboxymethylcellulose (CMC) for the in vivo
both in vitro and in vivo inflammatory conditions and to examine the
experiments.
molecular targets involved in its anti-inflammatory pharmacology.
After the pre-incubation of the RAW264.7 cells or peritoneal
macrophages (1  106 cells/ml) for 18 h, the cells were pre-
treated with Ho-ME (0 to 40 mg/ml) for 30 min and then further
2. Materials and methods incubated with LPS (1 mg/ml) for 24 h. The inhibitory effect of
Ho-ME on NO, TNF-a, and PGE2 production was determined by
2.1. Materials analysing the levels of NO, TNF-a, and PGE2 using the Griess
reagent, an enzyme immunoassay kit (Amersham, Little Chalfont,
A 99% methanol extract (Code No.: FBM018-051) of whole Buckinghamshire, UK), and an enzyme-linked immunosorbent
parts (leaves and bark) of Hopea odorata Roxb. (Dipterocarpaceae) assay kit (Amersham), as described previously (Cho et al., 2000;
was purchased from the Plant Extract Bank of the Plant Diversity Green et al., 1982).
Research Center (http://extract.pdrc.re.kr/extract/f.htm, Daejeon,
Korea). Resveratrol, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylte-
2.6. Cell viability test
trazolium bromide, tetrazole (MTT), and lipopolysaccharide (LPS,
E. coli 0111:B4) were purchased from Sigma Chemical Co. (St.
After the pre-incubation of the RAW264.7 cells and peritoneal
Louis, MO, USA). SB203580, SP600125, PP2, and piceatannol were
macrophages (1  106 cells/ml) for 18 h, the cells were incubated
obtained from Calbiochem (La Jolla, CA, USA). The luciferase
with Ho-ME (0 to 40 mg/ml) for 24 h. The cytotoxic effects of
constructs containing binding promoters for NF-kB, CREB, and
Ho-ME were then evaluated using a conventional MTT assay, as
AP-1 were gifts from Prof. Chung, Hae Young (Pusan National
reported previously (Gerlier and Thomasset, 1986; Yoo et al.,
University, Pusan, Korea) and Man Hee Rhee (Kyungpook National
2011). At 3 h prior to culture termination, 10 ml of an MTT
University, Daegu, Korea). Foetal bovine serum and RPMI1640
solution (10 mg/ml in phosphate buffered saline, pH 7.4) was
were obtained from GIBCO (Grand Island, NY, USA). The
added, and the cells were cultured until the termination of the
RAW264.7 cells, a BALB/c-derived murine macrophage cell line
experiment. Incubation was halted by the addition of 15% sodium
(ATCC No.: TIB-71), and HEK293 cells, a human embryonic kidney
dodecyl sulphate to each well, solubilising the formazan (Do Kim
cell line (ATCC No.: CRL-1573), were purchased from ATCC
et al., 2010). The absorbance at 570 nm (OD570–630) was measured
(Rockville, MD, USA). All other chemicals were purchased from
using a Spectramax 250 microplate reader.
Sigma. The phospho-specific or total antibodies for p65, p50,
c-Fos, ATF-2, c-Jun, PDK1, Src, Syk, IkBa, lamin A/C, and b-actin
were obtained from Cell Signaling (Beverly, MA, USA). 2.7. mRNA analysis by real-time polymerase chain reaction (PCR)

To evaluate the cytokine mRNA expression levels, RAW264.7

2.2. Animal experiments cells pre-treated with Ho-ME (200 mg/ml) for 30 min were incu-
bated with LPS (1 mg/ml) for 6 h. The total RNA from the cells was
Male ICR and C57BL/6 mice (6–8 weeks old, 17–21 g) were then isolated using the TRIzol Reagent (Gibco BRL) according to
obtained from Daehan Biolink (Chungbuk, Korea) and maintained in the manufacturer’s instructions. The total RNA was stored at
plastic cages under standard conditions. Water and food pellets  70 1C until use. The mRNA was quantified with real-time
(Samyang, Daejeon, Korea) were supplied ad libitum. All studies RT-PCR using SYBR Premix Ex Taq according to the manufac-
were performed in accordance with the guidelines established by turer’s instructions (Takara, Japan) and a real-time thermal cycler
the Institutional Animal Care and Use Committee at Sungkyunkwan (Bio-Rad, USA), as reported previously (Khorolragchaa et al.,
University (Suwon, Korea). 2010). The results are expressed as the ratio of the optimal
600 Y. Yang et al. / Journal of Ethnopharmacology 145 (2013) 598–607

density relative to GAPDH. The primers (Bioneer, Seoul, Korea) b-galactosidase (0.25 mg/ml) using the polyethylenimine (PEI)
used are listed in Table 1. method, as reported previously (Shen et al., 2011). After 24 h, the
transfected cells were treated with Ho-ME in the presence or absence
of PMA (100 nM for NF-kB- and AP-1-Luc assays) or forskolin
2.8. Luciferase reporter gene activity assay (100 nM for CERB-Luc assay), and the cells were harvested and lysed
to determine the luciferase activity one day later. The luciferase
HEK293 cells (1  106 cells/ml) were transfected in 12-well plates assays were performed using the Luciferase Assay System (Promega)
with NF-kB-Luc, AP-1-Luc, or CREB-Luc (each 1 mg/ml) as well as as reported previously (Kim et al., 2010b). The luciferase activity was
normalised to the b-galactosidase activity.
Table 1
Real-time PCR primers.
2.9. Preparation of total lysates and nuclear extracts,
Name Sequence (50 to 30 )
immunoblotting, and immunoprecipitation
iNOS
F GGA GCC TTT AGA CCT CAA CAG A RAW264.7 cells (5  106 cells/ml) were washed three times in
R TGA ACG AGG AGG GTG GTG cold PBS with 1 mM sodium orthovanadate and lysed by sonica-
IFN-b
F TCC AAG AAA GGA CGA ACA TTC G
tion in lysis buffer (20 mM Tris–HCl, pH 7.4, 2 mM EDTA, 2 mM
R GAG GCC ATT TGG GAA CTT CT ethyleneglycotetraacetic acid, 50 mM b-glycerophosphate, 1 mM
COX-2 sodium orthovanadate, 1 mM dithiothreitol, 1% Triton X-100, 10%
F CACTACATCCTGACCCACTT glycerol, 10 mg/ml aprotinin, 10 mg/ml pepstatin, 1 mM benzi-
R ATGCTCCTGCTTGAGTATGT
mide, and 2 mM PMSF) for 30 min with rotation at 4 1C. The
MCP-1
F GAAGGAATGGGTCCAGACAT lysates were clarified by centrifugation at 16,000  2g for 10 min
R ACGGGTCAACTTCACATTCA at 4 1C and stored at 20 1C until needed as reported previously
TNF-a (Yoo et al., 2011).
F TGCCTATGTCTCAGCCTCTT The nuclear lysates were prepared in a three-step procedure
R GAGGCCATTTGGGAACTTCT
IL-12p40
(Byeon et al., 2008). After treatment, the cells were collected with a
F TGAACTGGCGTTGGAAGC rubber policeman, washed with 1  PBS, and lysed in 500 ml of lysis
R GCGGGTCTGGTTTGATGA buffer containing 50 mM KCl, 0.5% Nonidet P-40, 25 mM HEPES (pH
GAPDH 7.8), 1 mM phenylmethylsulfonyl fluoride, 10 mg/ml leupeptin,
F CAA TGA ATA CGG CTA CAG CAA C
20 mg/ml aprotinin, and 100 mM 1,4-dithiothreitol (DTT) on ice for
R AGG GAG ATG CTC AGT GTT GG
4 min. The cell lysates were then centrifuged at 19,326  g for 1 min

140 Peritoneal macrophages

RAW 264.7 cells
NO production (% of control)

120 120
production (% of control)
Inflammatory mediator

100 100
80 80
60 60
*
40 NO 40
Cell viability
20 PGE 20
TNF -α
0 0
0 0.3 0.6 1.25 2.5 5 10 20 40 Ho-ME (μg/ml)- - 0.62 1.25 2.5 5 10 20 40
Ho-ME (μg/ml) LPS (1μg/ml) - + + + + + + + +

160 Peritoneal macrophages

140
Cell viability (% of control)

120
100
80
60
40
20
0
0 0.62 1.25 2.5 5 10 20 40
Ho-ME (μg/ml)

Fig. 1. The in vitro anti-inflammatory effects of Ho-ME in RAW264.7 cells and peritoneal macrophages. ((A) and (B)) The levels of NO, TNF-a, and PGE2 were determined
from the culture supernatants of RAW264.7 cells or peritoneal macrophages treated with Ho-ME and LPS (1 mg/ml) for 24 h. (C) The cell viability of RAW264.7 cells was
determined using an MTT assay. *P o 0.05 and **Po 0.01 compared to the control.
 Y. Yang et al. / Journal of Ethnopharmacology 145 (2013) 598–607 601

in a microcentrifuge. In the second step, the pellet (the nuclear where DET ¼the ear thickness of the drug-treated group, NET ¼the
fraction) was washed once in washing buffer, which was the same ear thickness of the normal untreated group, and CET ¼the ear
buffer as the lysis buffer but without Nonidet P-40. In the final step, thickness of the control group.
the nuclei were treated with extraction buffer (lysis buffer contain-
ing 500 mM KCl and 10% glycerol). The nuclei/extraction buffer
2.13. HPLC analysis of Ho-ME
mixture was frozen at  80 1C and then thawed on ice and
centrifuged at 19,326  g for 5 min. The supernatant was collected
The phytochemical characteristics of Ho-ME and the standard
as a nuclear extract. The soluble cell lysates were immunoblotted,
compound resveratrol were identified using high performance liquid
and the protein levels were visualised as previously described (Kim
chromatography (HPLC) analysis (Almela et al., 2006; Starkenmann
et al., 2010a). For immunoprecipitation, cell lysates containing equal
et al., 2006). The system was equipped with a KNAUER (Wellchrom)
amounts of protein (500 mg) from RAW264.7 cells (1  107 cells/ml)
K-1001 HPLC pump, a Wellchrom K-2600 fast scanning spectro-
treated with or without LPS (1 mg/ml) for 2.5 min were pre-cleared
photometer, and a K-500 4-channel degasser. The elution solvents
with 10 ml protein A-coupled Sepharose beads (50% v/v) (Amersham,
used were solvent A (0.1% H3PO4 in H2O) and solvent B (acetoni-
UK) for 1 h at 4 1C. The pre-cleared samples were incubated with 5 ml
trile). The gradient step of the solvent was solvent A to solvent
of the antibodies for p38, Syk, TBK1 (TANK-binding kiase I), toll like
B/min, and a Phenomenex Gemini C18 ODS (5 mm) column was used.
receptor (TLR)4, or IkB kinase (IKK)e overnight at 4 1C. The immune
complexes were mixed with 10 ml protein A-coupled Sepharose beads
(50% v/v) and rotated for 3 h at 4 1C. 2.14. Statistical analysis

All data presented in this paper are the mean7SD of an

2.10. Enzyme assay experiment performed with six (Figs. 1, 2, 3A, 4H, and 7C) or
three (Fig. 4G) samples for in vitro experiments and seven
To evaluate the ability of the extract to inhibit the kinase activity (gastritis and ear oedema) mice for the in vivo tests. For statistical
TM
of purified IRAK1, IRAK4, Syk, and Src, a kinaseProfiler service
(http://www.millipore.com/life_sciences/flx4/ld_kinases&Table1=3 RAW264.7 cells (1 h incubation)
140
#order) from Millipore (Billerica, MA) was used. In a final reaction
volume of 25 ml, IRAK1, IRAK4, Src, or Syk (human) (1–5 mU) was
120
incubated with the reaction buffer. The reaction was initiated by the
mRNA level (% of control)

addition of MgATP. After incubation for 40 min at room tempera-

100
ture, the reaction was stopped by the addition of 5 ml of 3%
phosphoric acid solution. Ten microliters of the reaction product
80
was then spotted onto a P30 filtermat and washed three times for
iNOS
5 min in 75 mM phosphoric acid and once in methanol prior to
60 TNF-α
drying and scintillation counting.
COX-2
40 MCP-1
2.11. EtOH/HCl-induced gastritis IL-12p40
20
IFN-β
Stomach inflammation was induced with EtOH/HCl according
to a previously published method (Lee et al., 2010; Okabe et al., 0
0 5 10 15 20
1986). Fasting ICR mice (n ¼7) were orally treated with Ho-ME
(0 to 100 mg/kg) twice per day for three days. The in vivo dosage Ho-ME (μg/ml)
of Ho-ME was selected based on previous in vivo tests in which 50
to 300 mg/kg doses were administered to mice (Abu-Ghefreh RAW264.7 cell line (6 h incubation)
et al., 2009). Thirty minutes after the final administration of 120
Ho-ME, 400 ml of 60% ethanol in 150 mM HCl was administered
orally. Each animal was anaesthetised and sacrificed using 100
mRNA level (% of control)

urethane 1 h after the administration of necrotising agents. The

stomach was then excised and gently rinsed under running tap
80
water. After opening the stomach along the greater curvature and
spreading it out on a board, the area (mm2) of erosive mucosal
lesions was measured using a pixel-counter by a technician that 60 iNOS
was blind to the treatment conditions. TNF
TNF-α
40 COX-2
MCP-1
2.12. Arachidonic acid-induced mouse ear oedema
20 IL-12p40
ICR mice (n ¼7) were orally pre-treated with Ho-ME (50, 100, IFN
IFN-β
and 200 mg/kg) or indomethacin (1 mg/kg) for 7 days. After the 0
final treatment, arachidonic acid [2% (w/v)] was applied to the 0 5 10 15 20
ears of the mice (25 ml/ear), as described previously (Koshihara Ho-ME (μg/ml)
et al., 1988). The thickness of the oedema was measured with a
Fig. 2. The effect of Ho-ME on the expression of inflammatory genes in LPS-
constant-pressure thickness gauge 1 h after treatment with ara-
treated RAW264.7 cells. ((A) and (B)) RAW264.7 cells (5  106 cells/ml) were
chidonic acid. The inhibitory effect of Ho-ME on the ear oedema incubated with Ho-ME in the presence or absence of LPS (1 mg/ml) for 1 or 6 h. The
was calculated as follows: level of mRNA expression for iNOS, COX-2, TNF-a, IL-12p40, MCP-1, and IFN-b was
determined using real-time PCR. The results shown are representative of three
% of controlðoedema inducer aloneÞ ¼ ½ðDET NET Þ=ðC ET NET Þ  100; independent experiments.
602 Y. Yang et al. / Journal of Ethnopharmacology 145 (2013) 598–607

comparisons, these results were analysed using an ANOVA et al., 2012; Yu et al., 2011), Ho-ME displayed an inhibitory activity
followed by Scheffe’s post-hoc test and the Kruskal–Wallis/ on the production of inflammatory mediators such as NO and PGE2
Mann–Whitney test. A P-value of o0.05 was considered statis- that was 10 to 20 times stronger than that of other compounds.
tically significant. All statistical tests were carried out using the To understand the molecular mechanism of Ho-ME inhibition,
SPSS computer program (SPSS Inc., Chicago, IL). Similar experi- we investigated its effects in terms of transcriptional control. To
mental data were also observed by an additional independent set do this, we measured the mRNA levels of inflammatory genes
of in vitro and in vivo experiments performed with the same using real-time PCR. As Fig. 2 illustrates, the LPS-induced upre-
numbers of samples or mice. gulation of iNOS, TNF-a, COX-2, MCP-1, IL-12, and IFN-b was
strongly suppressed by treatment with Ho-ME for 1 and 6 h,
indicating that the inhibition of inflammatory mediator produc-
3. Results and discussion tion can be observed at the transcriptional level. Given that
several major transcription factors, such as NF-kB, AP-1, and
To determine whether Ho-ME is able to suppress the CREB, have been reported to participate in transcriptional regula-
macrophage-mediated inflammatory responses, we first examined tion (Wang et al., 1995), we examined which transcription factors
the inhibitory effect of this extract on the production of TNF-a, NO, were targeted by Ho-ME using a reporter gene assay, nuclear
and PGE2 in LPS-treated RAW264.7 cells. As shown in Fig. 1A, Ho-ME fractionation, and immunoblotting analysis. As shown in Fig. 3A,
potently suppressed the release of these molecules in a dose- Ho-ME strongly suppressed all of the enhanced luciferase activ-
dependent manner without altering the viability of the RAW264 ities that were mediated by NF-kB, AP-1, and CREB in a dose-
cells. This effect was confirmed by analysis with primary macro- dependent manner. In agreement with these observations, the
phages. The dose-dependent suppression of NO production from translocation of p65 at 5, 30, and 90 min, c-Jun at 5 and 30 min,
peritoneal macrophages induced by LPS (Fig. 1B) without evidence c-Fos at 5, 30, and 90 min, and phospho-CREB at 5 min was clearly
of cytotoxicity (Fig. 1C) suggests that Ho-ME can block macrophage- diminished following exposure to Ho-ME (Fig. 3B), suggesting
mediated inflammatory responses by suppressing the release of that the pathways regulating the translocation of these transcrip-
inflammatory mediators. Compared with other anti-inflammatory tion factors could be directly or indirectly targeted by this extract.
extracts, such as extracts of Osbeckia stellata, Aralia continentalis, On the basis of the current knowledge regarding upstream
Archidendron clypearia, Polygonum hydropiper, and Phaseolus angu- signalling events in LPS-treated macrophages, we next sought to
laris (Seok Yang et al., 2012; Yang et al., 2012a, 2012b; Yoon Jeong define the exact molecular target of Ho-ME. First, the ability of

NF- κB
120
luciferase activity (% of control)

AP-1
CREB
100

80

60

40

20

0
0 0 5 10 20 40
Ho-ME (μg/ml)

RAW264.7 cells : Nuclear fraction

5 min 15 min 30 min 90 min

LPS (1 μg/ml) - + + + + + + + +
Ho-ME (20 μg/ml) - - + - + - + - +

p65

c-Jun

c-Fos

p-CREB

γ-Tubulin

Fig. 3. The effect of Ho-ME on the activation of transcription factors. (A) HEK293 cells co-transfected with the plasmid constructs for b-gal (as a transfection control) and
NF-kB-Luc, CREB-1-Luc, or AP-1-Luc (each 1 mg/ml) were treated with Ho-ME in the presence or absence of PMA (100 nM) or forskolin (100 nM). The luciferase activity
was measured using a luminometer. (B) The total or phospho-protein levels of NF-kB (p65), AP-1 family (c-Jun and c-Fos), CREB, and g-tubulin in the nuclear fractions
were determined via immunoblotting analysis with total or phospho-antibodies. *Po 0.05 and **P o0.01 compared to the control.
 Y. Yang et al. / Journal of Ethnopharmacology 145 (2013) 598–607 603

2 min 3 min 5 min 10 min

LPS (1 μg/ml) - + + + + + + + +
(μg ) -
Ho-ME (μg/ml) - 20 - 20 - 20 - 20

5 min
i 15 min
i 30 min
i 90 min
i p-p85
LPS (1 μg/ml) - + + + + + + + + p85
Ho-ME (20 μg/ml) - - + - + - + - +
p-PDK1
p-IκBα PDK1
p-AKT
AKT
Total-IκBα
AKT
β-actin
p IKK /β
p-IKKα/β

IKKβ
β
β-actin

2 min IP: Syk IP: Src

3 min
LPS (1 μg/ml) - + + + + 2 min 2 min
Ho-ME (μg/ml) - - 20 - 20 LPS (1 μg/ml) - + + LPS (1 μg/ml) - + +
Ho-ME (μg/ml) - - 20 Ho-ME (μg/ml) - - 20
p-Src
Src p-p85
p p85 p-p85
p p85
y
p-Syk
p Syk Src
Syk H.C. H.C.

5 min 15 min 30 min 60 min

LPS (1 μg/ml) - + + + + + + + + 2 min 3 min 5 min 10 min
LPS (1 μg/ml) - + + + + + + + +
Ho-ME (20 μg/ml) - - + - + - + - +
Ho-ME (μg/ml) - - 20 - 20 - 20 - 20
p-Erk p-MKK3/6
MKK3
Erk
p-MKK4/7
p-JNK1/2
MKK4/7
JNK2 p-TAK1
TAK1

TAK1
p-p38
IRAK1
p38
β-actin
β-actin

120
120
Kinase activity ( % of control)

NO
100
NO and PGE2 production

100 PGE

80
(% of control)

80

60 60
**
40 40 ** **
**
** **
20 ** 20 **
**
**
0 0
Control IRAK1 IRAK4 Src Syk Vehicle Pic PP2 SB SP
Ho-ME (20 μg/ml )

Fig. 4. The effect of Ho-ME on the activation of upstream signalling enzymes for NF-kB, AP-1, and CREB translocation. ((A), (B), (C), (E), and (F)) The phospho-protein or total protein
levels of the upstream signalling enzymes for the translocation of NF-kB, CREB, AP-1 and b-actin from RAW264.7 cells were determined using phospho-specific or total protein
antibodies. (D) RAW264.7 cells (5  106 cells/ml) were incubated with Ho-ME in the presence or absence of LPS (1 mg/ml) for 0 or 2 min. After preparing the total lysates, the binding
levels of phospho-p85/PI3K to Syk or Src were identified using immunoprecipitation with antibodies for Syk or Src and immunoblotting with antibodies for p-p85. (G) The kinase
activities of IRAK1, IRAK4, Syk, and Src were determined with a direct kinase assay using purified enzymes. The activity for each enzyme (IRAK1, IRAK4, Src, or Syk) in the control,
which received vehicle treatment only, was considered to be 100%. (H) The culture supernatants prepared from LPS-treated RAW264.7 cells pre-treated with standard Src, Syk, JNK,
and p38 inhibitors [PP2 (25 mM), piceatannol (pic: 25 mM), SP600125 (SP: 20 mM), and SB203580 (SB: 20 mM)] were assayed for NO and PGE2. H.C.: Heavy chain. **Po0.01
compared to the control.
604 Y. Yang et al. / Journal of Ethnopharmacology 145 (2013) 598–607

this extract to suppress IkBa phosphorylation was examined. As

Fig. 4A illustrates, Ho-ME strongly reduced the early induction of
IkBa phosphorylation at 5 min, which was also observed in the
Normal
case of the Syk inhibitor piceatannol (Lee et al., 2009). In
agreement with these results, the Ho-ME extract clearly inhibited
the upstream signalling cascade of IKK, AKT, PDK1, and p85/PI3K
between 2 and 10 min (Fig. 4B), suggesting that the upstream Ho-ME (mg/kg)
kinases that activate PI3K could be target molecules. Given that
two kinases, Syk and Src, have been reported to be the enzymes
responsible for this action (Beitz et al., 1999; Hung et al., 2007),
the ability of Ho-ME to suppress the activation of these enzymes
was examined. Interestingly, the extract not only blocked the

EtOH/HCl
auto-phosphorylation of both Src and Syk at 2 and 3 min (Fig. 4C)
but also suppressed the kinase activities of these enzymes
0 12.5
(Fig. 4G). Furthermore, the inhibition of these enzymes by
Ho-ME also altered the binding patterns between phospho-p85
and Syk or Src (Fig. 4D), suggesting that the suppression of Src or
Syk activation by this extract could be linked to the reduction of
signalling complex formation for NF-kB activation. Recently, we
found that extracts from plants such as Osbeckia stellata, Aralia
continentalis, Archidendron clypearia, Polygonum hydropiper, and
25 50 100
Phaseolus angularis, which have traditionally been used to treat or
prevent inflammatory diseases, have inhibitory functions against 120
the activation of Syk and Src (Seok Yang et al., 2012; Yang et al.,

Gastritis level (% of control)

2012a, 2012b; Yoon Jeong et al., 2012; Yu et al., 2011). In addition, 100
numerous studies have reported that Syk and Src can be used as
target enzymes for the treatment of various inflammatory dis- 80
eases (Ho et al., 2005; Nag and Chaudhary, 2009; Sanderson et al.,
2009). These enzymes have been shown to be highly active in TLR 60
*
activation conditions, and the inhibition of these enzymes by the
specific inhibitors piceatannol (for Syk) and PP2 (for Src) blocked 40
*
the release of NO and PGE2 under the same conditions (Fig. 4H) *
(Yang et al., 2012a, 2012b). The knockdown or knockout of Syk 20
has been reported to alter cytokine production levels (Lee et al.,
2009; Sedlik et al., 2003). In addition, immunoprecipitation 0
experiments have demonstrated that these molecules are Normal 0 12.5 25 50 100
strongly associated with TLR4 and its adaptor molecules (Wang Ho-ME (mg/kg)
et al., 2010).
To define the AP-1 signalling mechanism, we evaluated the Fig. 5. Effect of Ho-ME on EtOH/HCl-induced gastritis. (A). Mice administered Ho-
phosphorylation pattern of MAPK under LPS stimulation. As ME orally for 3 days were treated orally with EtOH/HCl. After 1 h, gastric lesions in
the stomach were measured using a pixel counter. The images were taken with a
shown in Fig. 4E, the phosphorylation of JNK and p38 was clearly
camera (B). The incidence of gastric lesions after the inducer alone was 100%.
suppressed by Ho-ME treatment at 5 to 30 min, indicating that
these two enzymes could be targeted by Ho-ME. To identify the
upstream target participating in the activation of these enzymes,
the phosphorylation patterns of MKK3/6, MKK4/7, and TAK1 were
measured. Interestingly, this extract strongly blocked the phos-
140
AA-induced ear edema (% of control)

phorylation of these enzymes at 2 and 3 min, while the enzyme

level of IRAK1, an upstream kinase of TAK1 (Cui et al., 2012), was 120
not reduced (Fig. 4F). This result suggests that IRAK levels could
be affected by Ho-ME, resulting in the functional inhibition of 100
AP-1/CREB. Indeed, the kinase activities of IRAK1 and IRAK4 were
80
strongly and directly inhibited by this extract (Fig. 4G), suggesting
that enzymes such as Syk and Src could be direct targets of 60
Ho-ME. Given that no compounds have been reported to be
strong IRAK inhibitors, we evaluated the importance of the IRAK 40
pathway in inflammatory responses by using specific inhibitors of
20
JNK and p38, which are downstream enzymes of the IRAK and
TAK1 pathways. As shown in Fig. 4H, these two inhibitors 0
[SP600125 (SP) and SB203580 (SB)] significantly inhibited the Hopea (mg/kg) - - 50 100 200
production of PGE2 but not NO, indicating that the IRAK/AP-1/ Indomethacin (1 mg/kg) - + - - -
CREB pathways may be primarily involved in COX-2 expression
and the subsequent PGE2 production events. Fig. 6. Effect of Ho-ME on arachidonic acid-treated mouse ear oedemas. The mice
Even though the Ho-ME extract has been used in traditional were orally treated with Ho-ME for 7d. An arachidonic acid (2%) solution was
topically applied (25 ml/ear) to the left ear of ICR mice (n ¼7). The thickness of the
medicine, it is necessary to demonstrate whether this extract is oedema was measured with a constant-pressure thickness gauge 4 h after
orally effective in various in vivo inflammatory models. For treatment. The ear thickness of the inducer alone was 100%. *P o 0.05 and
this purpose, we established HCl/EtOH-induced gastritis and *P o 0.01 compared to the control.
 Y. Yang et al. / Journal of Ethnopharmacology 145 (2013) 598–607 605

arachidonic acid-treated ear oedema models. As shown in Fig. 5, ear swelling up to four-fold. As expected, 25 to 100 mg/kg of Ho-
HCl/EtOH dramatically increased the prevalence of inflamed ME extract ameliorated the gastric symptoms (Fig. 5A and B).
lesions in the stomach of mice, and arachidonic acid increased Similarly, Ho-ME significantly decreased ear swelling by up to

Resveratrol
(0.025 mM) 22.6 min
(6.5)

Absorbance at 306 nm
22.6 min
Ho-ME (7 0)
(7.0)
(50 mg/ml)

22.6 min
(10.5)
Ho-ME
Ho ME + resveratrol

Retention Time
(Peak area)

295 nm

306 nm

430 nm

500 nm

120
Luciferase activity (% of control)

o control))

100
ammatory mediator

100

80 * * 80
uction (% of

**
**
60 60
**
40 40
**
produ
Infla

20 20

0 0
Res (50 μM) - + - + - + Res (50 μM) - + - + - +

NF-κB AP-1 CREB NO PGE2 TNF-α

Fig. 7. Analysis of the phytochemical profile of Ho-ME and the inhibitory activity of resveratrol on the inflammatory responses in HEK293 and RAW264.7 cells. (A and B)
The phytochemical characteristics of Ho-ME were analysed by HPLC. (C Left) HEK293 cells co-transfected with the plasmid constructs for b-gal (as a transfection control)
and NF-kB-Luc, CREB-1-Luc, or AP-1-Luc (each 1 mg/ml) were treated with resveratrol (Res: 50 mM) in the presence or absence of PMA (100 nM) or forskolin (100 nM). The
luciferase activity was measured using a luminometer. (C Right) The levels of NO, TNF-a, and PGE2 were determined from the culture supernatants of RAW264.7 cells
treated with resveratrol and LPS (1 mg/ml) for 24 h. *P o0.05 and **P o 0.01 compared to the control.
606 Y. Yang et al. / Journal of Ethnopharmacology 145 (2013) 598–607

45% at doses of 100 and 200 mg/kg (Fig. 6). These data strongly In summary, we demonstrated that Ho-ME is able to suppress
suggest that Ho-ME maintains its anti-inflammatory action in vivo the production of TNF-a, NO, and PGE2 through the suppression of
and could be applied to other inflammatory symptoms. Syk/Src/NF-kB and IRAK1/IRAK4/JNK/p38/AP-1/CREB, as sum-
The final question that we sought to address was the identi- marised in Fig. 8. This extract also displayed anti-gastritis and
fication of the chemical components responsible for the anti- anti-oedema properties. HPLC analysis strongly indicated that one
inflammatory properties of Ho-ME. Thus far, the compounds of the active components of Ho-ME extract is resveratrol. There-
included in this extract have not been defined. Although a few fore, it is strongly suggested that this extract be investigated as a
studies have reported the chemical composition of Hopea odorata, resveratrol-containing anti-inflammatory remedy. Other pharma-
several papers have suggested that resveratrol and its derivatives cological properties of Ho-ME, such as its anti-cancer and anti-
are the major active components. Based on these papers, we aging activities and a detailed phytochemical profile, will be
investigated whether resveratrol is present in this extract using investigated in future studies.
HPLC. As Fig. 7A shows, we observed a peak at 22.6 min, which is
the same retention time as standard resveratrol, and this peak
was increased by a concurrent injection with resveratrol, Acknowledgements
although the peak pattern was not sharp. Indeed, through
measuring the peak area of standard compound, the content of This paper was supported by a grant (2010–2012) from
resveratrol in this extract was calculated to be 0.013%. However, Kangwon Technopark, Kangwon Province, Korea.
analysis of this extract under different wavelengths strongly
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