A synthetic curcuminoid derivative inhibits nitric oxide and proinflammatory cytokine synthesis

Article abstract of DOI:10.1016/j.ejphar.2009.11.053

Curcumin is a highly pleiotropic molecule with significant regulatory effects upon inflammation and inflammatory related diseases. However curcumin has one major important limitation in which it has poor bioavailability. Design of synthetic structural derivatives of curcumin is but one approach that has been used to overcome its poor bioavailability while retaining, or further enhancing, its drug-like effects. We have synthesized a series of curcumin analogues and describe the effects of 2,6-bis-4-(hydroxyl-3-methoxy-benzylidine)-cyclohexanone or BHMC upon nitric oxide and cytokine synthesis in cellular models of inflammation. BHMC showed a significant dose-response inhibitory action upon the synthesis of NO and we have shown that this effect was due to suppression of both iNOS gene and enzyme expression without any effects upon scavenging of nitrite. We also demonstrated that BHMC has a very minimal effect upon iNOS activity with no effect at all upon the secretion of PGE₂ but has a strong inhibitory effect upon MCP-1 and IL-10 secretion and gene expression. Secretion and gene expression of TNF-α and IL-6 were moderately inhibited whereas IL-8 and IL-1β were not altered. We conclude that BHMC selectively inhibits the synthesis of several inflammatory mediators. BHMC should be considered a promising drug lead for preclinical and further pharmacological studies.

Full text of DOI:10.1016/j.ejphar.2009.11.053

European Journal of Pharmacology 628 (2010) 247–254
Contents lists available at ScienceDirect
European Journal of Pharmacology
journal homepage: www.elsevier.com/locate/ejphar
Immunopharmacology and Inflammation
A synthetic curcuminoid derivative inhibits nitric oxide and proinflammatory
cytokine synthesis
Chau Ling Tham a, Choi Yi Liew a, Kok Wai Lam b, Azam-Shah Mohamad a, Min Kyu Kim a,
Yoke Kqueen Cheah a, Zainul-Amirudin Zakaria a, Mohd-Roslan Sulaiman a, Nordin H. Lajis b, Daud A. Israf a,
⁎
a
Department of Biomedical Science, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia
b
Institute of Bioscience, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 27 July 2009
Received in revised form 27 October 2009
Accepted 23 November 2009
Available online 1 December 2009
Curcumin is a highly pleiotropic molecule with significant regulatory effects upon inflammation and
inflammatory related diseases. However curcumin has one major important limitation in which it has poor
bioavailability. Design of synthetic structural derivatives of curcumin is but one approach that has been used
to overcome its poor bioavailability while retaining, or further enhancing, its drug-like effects. We have
synthesized a series of curcumin analogues and describe the effects of 2,6-bis-4-(hydroxyl-3-methoxy-
benzylidine)-cyclohexanone or BHMC upon nitric oxide and cytokine synthesis in cellular models of
inflammation. BHMC showed a significant dose–response inhibitory action upon the synthesis of NO and we
have shown that this effect was due to suppression of both iNOS gene and enzyme expression without any
effects upon scavenging of nitrite. We also demonstrated that BHMC has a very minimal effect upon iNOS
activity with no effect at all upon the secretion of PGE2 but has a strong inhibitory effect upon MCP-1 and IL-
10 secretion and gene expression. Secretion and gene expression of TNF-α and IL-6 were moderately
inhibited whereas IL-8 and IL-1β were not altered. We conclude that BHMC selectively inhibits the synthesis
of several inflammatory mediators. BHMC should be considered a promising drug lead for preclinical and
further pharmacological studies.
Keywords:
Cytokine
Chemokine
iNOS
Curcuminoid
Macrophage
© 2009 Elsevier B.V. All rights reserved.
1. Introduction
signaling pathways, gene expression and also inducible enzyme activity
(Aggarwal and Sung, 2009). Of the host of inflammatory related
Curcumin (diferuloylmethane) or 1,7-bis-(4-hydroxy-3-methox-
yphenyl)-1, 6-heptadiene-3,5-dione], a member of the curcuminoid
family, is the major active component of turmeric, a yellow compound
originally isolated from the plant Curcuma longa L. Over the last two
decades many studies have shown that curcumin has a number of
biological and pharmacological activities such as anti-carcinogen
(Aggarwal et al., 2004; Cen et al., 2009; Yoysungnoen et al., 2008),
anti-malarial (Mishra et al., 2008), antioxidant, anti-mutagenic, anti-
bacterial (Parvathy et al., 2009), anti-angiogenic (Yoysungnoen et al.,
2008), immune-modulatory (Allam, 2009), chemo-preventive (Chan
et al., 1998), and anti-inflammatory (Chan et al., 1998; Liang et al.,
2009, in press).
Curcumin is a highly pleiotropic molecule with significant regulatory
effects upon inflammatory, proliferative and oxidative tissue damage. It
is not surprising that preclinical studies have shown that curcumin can
prevent neurodegenerative, respiratory, cardiovascular, neoplastic,
metabolic and autoimmune diseases (Aggarwal and Harikumar,
2009). Curcumin has been shown to regulate a diverse array of cellular
mediators, enzymes, and transcription factors, curcumin has shown
strong inhibitory effects on proinflammatory cytokines such as tumor
necrosis factor (TNF)-α, Interleukin (IL)-1, IL-6; prostaglandin, induc-
ible enzymes such as inducible nitric oxide synthase (iNOS) and
cyclooxygenase-2 (COX-2), protein kinases, transcription factors such
as NF-κB, STAT-3 and AP-1 to name a few (Aggarwal and Sung, 2009).
Curcumin has entered many clinical trials and proven to be safe and
effective in the prevention and treatment of mainly enteric diseases.
Despite the effective modulatory activity of curcumin upon many
cellular targets linked to various diseases, one of the most important
limitations with curcumin is its bioavailability (Anand et al., 2007).
Pharmacokinetic studies have shown that orally administered curcumin
undergoes hepatic conjugation leading to the formation of glucoronides
and sulphates (Anand et al., 2007; Garcea et al., 2005). Systemic
administration of curcumin causes it to undergo reduction. In any case,
the products following biotransformation are clearly biologically
inactive (Sandur et al., 2007). Numerous approaches have been adopted
in an effort to increase the bioavailability of this promising natural
molecule. These approaches attempt to either interfere with glucur-
onidation, protect curcumin with liposomal formulations and nanopar-
ticles, or design synthetic structural derivatives of curcumin (Aggarwal
and Sung, 2009).
⁎
Corresponding author. Tel./fax: +603 8947 2337.
E-mail address: daud@medic.upm.edu.my (D.A. Israf).
0014-2999/$ – see front matter © 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.ejphar.2009.11.053

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C.L. Tham et al. / European Journal of Pharmacology 628 (2010) 247–254
Our group has adopted the chemical synthesis of curcumin
analogues as an approach to overcome the limitation of bioavailabil-
ity. The presence of the β-diketone moiety renders curcumin to be
rapidly metabolized by aldo-keto reductase in the liver, therefore
limiting the potential therapeutic or prophylactic effect of curcumin
on many types of disease. The phenolic OH has been shown to be
essential for its antioxidant activity (Parvathy et al., 2009). Taking into
account these structural functionalities, a synthetic diarylpentanoid,
BHMC was synthesized based on the chemical structure of curcumin,
by eliminating the unstable β-diketone moiety and modifying it into
conjugated double bonds while preserving the phenolic OH group.
Preliminary screening of a panel of synthetic derivatives of curcumin
for nitric oxide inhibitory activity has enabled us to select BHMC for
further in vitro studies. We report here the effect of BHMC upon both
nitric oxide and proinflammatory cytokine synthesis in cellular
models of inflammation.
Fig. 1. Chemical structure of (A) BHMC and (B) curcumin.
Griess assay. We observed consistent results between different
batches with variation not exceeding 3%. BHMC was dissolved in
100% DMSO as a stock at 100 mmol/l and diluted to appropriate
concentrations for assays. The final concentration of DMSO in all
assays was kept constant at 0.1%.
2. Materials and methods
2.1. Materials
Antibiotics (5000 U/ml penicillin and 5000 μg/ml streptomycin),
Amphotericin B (0.25 μg/ml), Fetal bovine serum (FBS), Dulbecco's
Modified Eagle Medium (DMEM), and Roswell Park Memorial
Institute medium (RPMI)-1640 were purchased from Hyclone
(Utah, USA). TNF-α, IL-1β, IL-6, IL-8, IL-10 and monocyte chemotactic
protein (MCP)-1 assay kits were purchased from BD Pharmingen (San
Diego, CA, USA). Prostaglandin E2 EIA monoclonal kit and rabbit anti-
mouse iNOS polyclonal antibody were purchased from Cayman
Chemicals, USA. HRP-conjugated donkey anti-rabbit Ig-G and HRP-
conjugated mouse anti-mouse β-actin were purchased from Santa
Cruz Biotechnology (Santa Cruz, CA, USA). All the other reagents of
Western blot analysis, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tet-
razolium bromide (MTT), bovine serum albumin (BSA) and dimethyl
sulfoxide (DMSO) were purchased from Amresco (Solon, Ohio). The
polyvinylidene fluoride (PVDF) membrane was purchased from
Milipore (Bredford, MA, USA). Super Signal West Femto Maximum
Sensitivity Substrate and Bicinchoninic acid (BCA) protein determi-
nation kit (Cat No. 23225) were purchased from Pierce (Rockford, IL,
USA). Phorbol-12–Myristate-13–Acetate (PMA), NS-398, E. coli
lipopolysaccharide (LPS) 055:B5, sulphanilamide, naphthylenedia-
mine, sodium nitroprusside (SNP), 2-phenyl-4,4,5,5,-tetramethylimi-
dazoline-1-oxyl-3-oxide (PTIO), and Nω-nitro-L-arginine-methyl
ester hydrochloride (L-NAME) were purchased from Sigma Chemical
co. (St. Louis, MO, USA). One-step RT-PCR kit and RNeasy Extraction
kit were purchased from Qiagen (Valencia, CA, USA). Benzonase
nuclease and 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-
one (PD98059) were purchased from Calbiochem (San Diego, CA,
USA). Gene and protein ladder were purchased from Fermentas (Glen
Burnie, MD, USA).
2.3. Cell culture
U937 and RAW 264.7 were purchased from the American Type
Culture Collection (ATCC). U937 were cultured in RPMI-1640
supplemented with 10% FBS, 4.5 g/l glucose, sodium pyruvate
(1 mmol/l), L-glutamine (2 mmol/l), amphotericin B, streptomycin
(50 μg/ml) and penicillin (50 U/ml). RAW 264.7 were cultured in
DMEM supplemented with 10% FBS, 4.5 g/L glucose, sodium pyruvate
(1 mmol/l), L-glutamine (2 mmol/l), amphotericin B, streptomycin
(50 μg/ml) and penicillin (50 U/ml). Cells were split when cell
confluency reached 80–90%.
2.4. Differentiation of U937 monocytes into macrophages
The human macrophage U937 cells at logarithmic phase of growth
were harvested, adjusted to 2×106 cells/ml in the presence of 10 nM
PMA, and incubated at 37 °C in 5% CO2 incubator for 24 h to
differentiate into adherent macrophages. After 24 h incubation, cells
were washed twice with phosphate buffered saline (PBS) to remove
non-adherent cells. Cell counting was carried out each time before
and after the cell differentiation process to make sure that there was
consistency in differentiation. The variation in the differentiation
process was minimal with more than 90% of differentiated cells
remaining attached and viable. Remaining adherent cells were
quiesced in fresh RPMI medium in the absence of PMA for 24 h
before stimulation with 10 μg/ml of LPS.
2.5. Induction of RAW 264.7 and U937 cells
2.2. Compound synthesis
Cells at a confluency of 80–90% were centrifuged at 120 g at 4 °C
for 7 min. The concentration was adjusted to 2×106 cells/ml and cell
viability was always more than 90%, as determined by trypan blue dye
exclusion. A total of 50 μl of cell suspension was dispensed into wells
of a tissue-culture-grade 96-well plate (10×104 cells/well) and
incubated for 2 h at 37 °C, 5% CO2 to attach the cells. Unattached
cells were discarded gently after 2 h. The attached cells were induced
with 10 μg/ml of Escherichia coli LPS (Strain 055:B5) in the presence or
absence of sample at a final volume of 100 μl/well. The sample stock
was serially diluted in DMSO to decreasing concentrations ranging
from 12.5 to 0.4 μmol/l, where the final concentration of DMSO in
media was maintained at 0.1%. Untreated cells and drug controls were
stimulated with LPS and also had the same amount of DMSO in culture
medium. Cells were then incubated for 24 h at 37 °C in 5% CO2.
BHMC or 2,6-bis-4-(hydroxyl-3-methoxy-benzylidine)-cyclohex-
anone was chemically synthesized at the Institute of Bioscience,
Universiti Putra Malaysia. Briefly, a mixture of vanillin (20 mmol, 2
equiv) and cyclohexanone (10 mmol, 1 equiv) was dissolved in 20 ml
of absolute ethanol. Concentrated HCl (2.0 ml) was added drop wise
over 5 min to the stirred mixture. The mixture was further refluxed
for 1 h. One hundred milliliters of distilled water was then poured into
the dark viscous solution and extracted with ethyl acetate. The ethyl
acetate layer was collected and subjected to further purification using
column chromatography. The structure was determined by nuclear
magnetic resonance and mass spectrometry (Fig. 1A). BHMC is 99.9%
pure as determined by HPLC. Our preliminary screening of several
synthetic derivatives involved the testing of several batches with the

C.L. Tham et al. / European Journal of Pharmacology 628 (2010) 247–254
249
2.6. Cell viability assay
prior to assay. PGE2 concentrations were determined with an enzyme
immunoassay kit (Cayman Chemicals, USA, Cat No. 514010) according
to the manufacturer's instructions. NS-398 was used at 50 µM as a
drug control.
Cell viability was assessed by the MTT method after the removal of
spent media. In the MTT assay a total of 100 μl of DMEM or RPMI-1640
containing 5% FBS was added to each well followed by 20 μl of MTT
(5 mg/ml). The formazan crystals were dissolved with 100 μl of 100%
DMSO per well after 4 h. The absorbance was measured at λ 570 nm
with a microplate reader (UVM 340, ASYS Hitech GmbH, Austria,
Europe) by using a reference wavelength of 650 nm. Cell viability was
determined as the percentage of untreated stimulated cells.
2.11. Cytokine immunoassay
For evaluation of secreted cytokines level, U937 cells were
differentiated and stimulated as described above. Following 18 h
incubation with varying concentrations of BHMC spent media was
collected for cytokine immunoassay. Supernatants of spent cell
culture media were stored at −80 °C prior to assay. Spent media
was analyzed for TNF-α, IL-1β, IL-6, IL-8, IL-10 and MCP-1 by enzyme-
linked immunosorbent assay (ELISA) using commercial kits (BD
Pharmingen, USA, Cat No. 555212, 557953, 555220, 555244, 555157,
555179 BD OptEIA) according to the manufacturer's instructions.
2.7. Measurement of nitric oxide production
To evaluate the inhibitory activity of test materials on nitric oxide
(NO) production, culture media was assayed using Griess reaction.
RAW 264.7 macrophages were incubated with LPS treatment with the
presence or without compound simultaneously. A concentration of
10 µg/ml of E. coli LPS were treated in RAW 264.7 cells which were
seeded in a 96-well plate for 24 h. Briefly, cell culture supernatants
were mixed with an equal volume of Griess reagent [1%sulfanilamide/
0.1%N-(1-naphtyl)-ethylene diamide dihydrochloride in 2.5% H3PO4],
and absorbance was read at 550 nm with microplate reader (Asys
HiTech UVM 340, USA). The amount of nitrite in the sample was
calculated from a sodium nitrite standard curve.
2.12. Reverse-transcriptase-polymerase chain reaction (RT-PCR)
Total RNA from cell monolayers was isolated using Qiagen RNeasy
Mini Extraction kit (Qiagen, USA, Cat No. 74106) as suggested by the
manufacturer. RNA concentration was determined and 100 ng of RNA
was used for Qiagen One-Step RT-PCR kit (Qiagen, USA, Cat No.
210212) according to the manufacturer's instructions. RNA integrity
was examined by formaldehyde agarose gel electrophoresis and
concentrations were determined by UV spectrophotometry (DU 530
Life Science UV/Visible Spectrophotometer, Fullerton, CA). The master
mix was performed in an Eppendorf thermal cycler for reverse
transcription at 50 °C for 30 min, initial PCR activation at 95 °C for
15 min, final extension at 72 °C for 10 min. The reaction products from
PCR were examined by 1.8% agarose gel electrophoresis and stained
with ethidium bromide. Band intensities were quantified by Image J
Java-based image processing program (NIH, USA) and normalized by
comparison to the RT-PCR products of glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) mRNA. PCR product for each gene was run
in parallel as a molecular size marker (providing bands at 1000 bp,
900 bp, 800 bp, 700 bp, 600 bp, 500 bp, 400 bp, 300 bp, 200 bp and
100 bp) except for iNOS gene, which was run in parallel with another
molecular size marker (providing bands at 766 bp, 500 bp, 350 bp,
300 bp, 250 bp, 200 bp, 150 bp, 100 bp, 75 bp, 50 bp and 25 bp).
Primers used in this study were adapted from published gene
sequences (Table 1).
2.8. Nitrite-scavenging activity
In an attempt to determine whether BHMC was scavenging
secreted nitrite in culture media, we subjected the compound to a
chemical scavenging assay. Briefly, 50 μl of serially-diluted BHMC was
pipetted into a 96-well flat-bottomed plate. Next, 50 μl of 20 mM
sodium nitroprusside dissolved in PBS (SNP, 10 mM final concentra-
tion) was added into each well and the plate was then incubated at
room temperature for 150 min. A control experiment was conducted
to determine NO scavenging effect of 0.1% DMSO in media. PTIO, a
nitrite scavenger, was used as a positive control. After incubation, an
equal volume of Griess reagent was added into each well in order to
measure the nitrite content. The absorbance of these solutions was
measured at 550 nm against the corresponding blank solutions. Fresh
culture medium was used as the blank in all the experiments. The
amount of nitrite in the samples was calculated from a sodium nitrite
standard curve (0–100 μM) freshly prepared in deionized water.
2.9. Indirect determination of iNOS activity
2.13. Western blot analysis
Induction of cells into an inflammatory state prior to treatment
with BHMC enables the synthesis of intracellular iNOS and accumu-
lation of high levels with corresponding enhanced synthesis and
secretion of NO. Since the induction with LPS will induce iNOS
synthesis, it is suggested that variations in the secretion of NO
following treatment with BHMC point to an effect upon iNOS activity
rather than gene or enzyme expression. RAW 264.7 cells were placed
in a 96-well plate and treated with LPS for 12 h. The cells were washed
three times with PBS and incubated in the presence of serial dilutions
of BHMC without LPS in medium for a further 12 h. L-NAME was used
as a specific inhibitor of iNOS enzyme activity (positive control) and
0.1% DMSO as a solvent control. The supernatants were removed and
assayed for nitrite using the Griess assay as described above.
RAW 264.7 cells were stimulated with 10 μg/ml of LPS as described
earlier and treated with BHMC for 18 h for iNOS protein detection.
Whole cell protein extract was used to analyze iNOS protein. Protein
sample was extracted by using an equal volume of sample buffer
(125 mM, 4% SDS, 20% glycerol, 0.004% bromophenol blue) with
benzonase nuclease (25 U/ml) was added in to digest nucleic acid.
After that, sample was boiled for 10 min to denature protein. Equal
amounts of protein (50 μg) were electrophoresed on a 7% SDS-
polyacrylamide gel and blotted on a 0.45 μm PVDF membrane. The
membrane was blocked with 5% BSA in Tris buffered saline (TBS)–
Tween 20 (0.05%) for 1 h and incubated overnight at 4 °C with rabbit
anti-mouse polyclonal iNOS antibody (1:1000) in TBS–Tween
containing 5% BSA. After washing three times with TBS–Tween, the
membrane was hybridized with HRP-conjugated donkey anti-rabbit
secondary antibody (1:5000) for 2 h and washed three times with
TBS–Tween. The same membrane was stripped and re-probed with
HRP-conjugated mouse anti-mouse β-actin (1:10,000). The mem-
brane was incubated with ECL reagent for 2 min and viewed by
chemiluminescence. Band intensities were quantified by Image J Java-
based image processing program and normalized by comparison to β-
actin.
2.10. Prostaglandin E2 (PGE2) immunoassay
In order to determine the effect of BHMC on prostaglandin
production, U937 cells were differentiated and stimulated as
described earlier. Following 24 h incubation with varying concentra-
tions of BHMC spent media was collected for prostaglandin immuno-
assay. Supernatants of spent cell culture media were stored at −80 °C

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C.L. Tham et al. / European Journal of Pharmacology 628 (2010) 247–254
Table 1
Primer sequences, operating conditions, and PCR product sizes.
Gene name
Oligonucleotide sequences (5′–3′)
Denaturing temperature
and time (°C, s)
Annealing temperature
and time (°C, s)
Elongation temperature
and time (°C, s)
Cycle number
Product size
(bp)
hTNF-α
hIL-1β
hIL-6
F-GAGCACTGAAAGCATGATCCGGGAC
R-TTGGTCTGGTAGGAGACGGCGATGC
F-GCATCCAGCTACGAATCTCC
94 (60)
94 (60)
94 (60)
94 (60)
94 (60)
94 (60)
94 (60)
94 (60)
94 (60)
67 (45)
72 (60)
72 (60)
72 (60)
72 (60)
72 (60)
72 (60)
72 (60)
25
25
23
25
25
24
26
495
700
450
430
444
297
139
62 (45)
R-CCACATTCAGCACAGGACTC
F-CACCGGGAACGAAAGAGAAG
R-AGCAGGCTGGCATTTGTGGT
F-CATCAGGGTGGCGACTCTAT
R-CCCAAGCCCAGAGACAAGAT
F-ACCGGAAGGAACCATCTCACT
R-GCATCTGGCAACCCTACAACA
F-GCTCATAGCAGCCACCTTCATTC
R-TGCAGATTCTTGGGTTGTGGAG
F-GAATCTTGGAGCGAGTTGTGG
R-AGGAAGTAGGTGAGGGCTTGG
F-CCCTGTTGCTGTAGCCGTAT
66 (45)
hIL-10
62 (45)
hIL-8
69 (45)
hMCP-1
miNOS
mGAPDH
hGAPDH
69 (45)
66 (45)
Depends on target gene
Depends on target gene
R-TGTTCCTACCCCCAATGTGT
F-TGAAGGTCGGAGTCAACGGATTTGGT
R-CATGTGGGCCATGAGGTCCACCAC
hTNF-α: human tumor necrosis factor-alpha; mGAPDH: murine glyceraldehyde-3-phosphate dehydrogenase.
2.14. Statistical analysis
3. Results
Data was presented as means S.E.M of three separate experi-
ments performed in triplicate. The IC50 values were calculated using
one parameter model [y=100/(1+(a/x)] using GraphPad Prism
software. SPSS version 15.0 was used to determine the differences
between groups by using one-way analysis of variance (ANOVA)
followed by post hoc comparison using Dunnett test. P-value of less
than 0.05 was considered to be significant.
3.1. Cytotoxicity
Fig. 2 shows the effect of BHMC upon both RAW 264.7 and U937
cell viability. Treatment with BHMC caused a reduction in cell viability
at 25 µM and above in both cell lines. Therefore BHMC was used at
12.5 µM and below for further assays.
3.2. BHMC inhibits NO secretion in a dose-dependent manner with
minimal inhibition of iNOS activity
Fig. 3A shows the dose–response of BHMC towards the secretion of
NO. LPS-induced RAW 264.7 cells produced large amounts of nitrite.
Significant inhibition of nitrite was demonstrated at almost all
concentrations tested with an IC50 value of 2.93 0.15 μM. To further
investigate whether the inhibition of NO production by BHMC was
mediated through the inhibition of iNOS enzyme activity, we assessed
the effects of BHMC on iNOS activity by induction of enzyme
expression followed by treatment with BHMC. Fig. 3B shows a slight
reduction in nitrite synthesis following prior induction of iNOS before
treatment and therefore we conclude that BHMC has a minimal effect
upon iNOS activity.
3.3. BHMC inhibits iNOS gene and protein expression
Fig. 4A and B shows the effect of various doses of BHMC upon iNOS
gene and protein expression respectively. Both gene and protein
expression were reduced in a dose-dependent manner.
3.4. BHMC does not scavenge nitrite and does not inhibit PGE2 secretion
All of the tested concentrations of BHMC did not reveal any level of
nitrite-scavenging activity (data not shown). Therefore the inhibitory
effect of BHMC on NO secretion is not caused by its scavenging
activity. BHMC did not inhibit the secretion of PGE2 at all concentra-
tions tested (data not shown).
3.5. BHMC variably inhibits cytokine/chemokine secretion and gene
expression
Fig. 2. Effect of BHMC on RAW 264.7 and U937 cell viability using MTT assay. (A) RAW
264.7 macrophages (2×106 cells/ml) were treated with indicated concentrations of
BHMC for 24 h. (B) Differentiated U937 monocytes (2×106 cells/ml) were treated with
indicated concentrations of BHMC for 24 h. All values are the mean S.E.M. of three
independent experiments. *Pb0.05, **Pb0.01 and ***Pb0.005, significantly different
from the control group.
Table 2 and Fig. 5 shows the IC50 values of BHMC inhibitory effects
upon cytokine secretion and dose–response effects upon cytokine
gene expression respectively.

C.L. Tham et al. / European Journal of Pharmacology 628 (2010) 247–254
251
Fig. 3. Effect of BHMC upon nitric oxide production and iNOS activity by RAW 264.7
macrophages. (A) RAW 264.7 macrophages (2×106 cells/ml) were treated with 10 μg/
ml E. coli LPS and indicated concentrations of BHMC. (B) RAW 264.7 macrophages
(2×106 cells/ml) were treated with 10 μg/ml E. coli LPS for 12 h prior to treatment with
indicated concentrations of BHMC. Nitrite levels were determined by the Griess
reaction after 24 h treatments. All values are the mean S.E.M. of three independent
experiments. *Pb0.05, **Pb0.01 and ***Pb0.005, significantly different from the LPS-
induced control group.
Fig. 4. Effect of BHMC upon iNOS gene and protein expression in LPS-stimulated RAW
264.7 macrophages. (A) Cells were induced with 10 μg/ml E. coli LPS and treated with
increasing concentration of BHMC for 4 h. RNA was extracted and analyzed by Reverse-
Transcriptase-PCR. (B) Cells were induced with 10 μg/ml E. coli LPS and treated with
increasing concentration of BHMC for 18 h. Protein was extracted and probed against
rabbit anti-mouse polyclonal iNOS antibody. All values are the mean S.E.M. of three
independent experiments. *P0.05, **Pb0.01 and ***Pb0.005, significantly different
from the LPS-induced control group. Cur: Curcumin; Dex: Dexamethasone.
BHMC showed a strong inhibition of MCP-1 and IL-10 secretion.
TNF-α and IL-6 were also inhibited in a dose-dependent fashion albeit
at a moderate level and minimal effects were noted upon the
secretion of IL-1β and IL-8. Results from RT-PCR (Fig. 5) also showed
that inhibition of MCP-1 and IL-10 mRNA expression was strong. In
fact MCP-1 gene expression was totally abolished at the concentration
of 3.1 µM and above. Effects upon TNF-α and IL-6 gene expression
were moderate and the expression of IL-8 and IL-1β genes were not
affected at all and reflect the lack of effect upon cytokine synthesis and
secretion.
MCP-1 are inhibited (Hidaka et al., 2002; Jain et al., 2009). The
inhibition of curcumin on the production of IL-8, macrophage
inflammatory protein (MIP)-1α, MCP-1, IL-1β and TNF-α from
monocytes and alveolar macrophages is achieved via several
mechanisms. The promoter gene encoding these cytokines contains
sequences for binding several nuclear transcriptional factors including
AP-1 and NF-κB (Siebenlist et al., 1994; Baeuerle and Henkel, 1994).
Furthermore curcumin has beenshown toinhibitthe activation of c-Jun/
AP-1 in osteoblastic cells (Hanazawa et al., 1993) and mouse fibroblasts
4. Discussion
This study shows that our modification of curcumin into BHMC
preserved several inhibitory characteristics upon proinflammatory
mediators, while enabling the compound to be more specific since it
lost its inhibitory effect upon PGE2 secretion and had minimal
inhibitory effect upon IL-1β and IL-8. BHMC showed a strong
inhibitory effect upon NO synthesis, which we have shown to be
due to suppression of both iNOS gene and protein expression rather
than an effect upon iNOS activity or the simple scavenging of nitrite in
the media. An interesting observation is the particularly strong
inhibitory effect upon MCP-1 synthesis.
Although BHMC possessed a prominent suppressive effect on
MCP-1 synthesis, its effect upon IL-8 secretion was minimal with no
effect upon IL-8 gene expression. This finding suggests that the
inhibitory effect of BHMC was geared towards chronic rather than
acute inflammatory networks (Mukaida et al., 1998). In contrast,
curcumin affects chemokines non-specifically whereby both IL-8 and
Table 2
The IC50 of BHMC upon TNF-α, IL-1β, IL-6, IL-8, IL-10 and MCP-1 secretions by LPS-
induced U937 cells. Concentrations of cytokines were determined by immunoassay.
Cytokine/chemokine
IC50 (μM)
TNF-α
IL-1β
IL-6
5.537 0.477
n.d.a
6.542 0.613
IL-8
n.d.a
IL-10
MCP-1
2.448 0.177
0.981 0.241
a
n.d.: not determined (none of the doses tested caused 50% of inhibition).

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Fig. 5. Effect of BHMC on cytokine and chemokine gene expression in LPS-induced U937 cells by using One Step Reverse-Transcriptase-PCR. (A) TNF-α. (B) IL-6. (C) IL-10. (D) MCP-1.
(E) IL-8. (F) IL-1β. All values are the mean S.E.M. of three independent experiments. *Pb0.05, **Pb0.01 and ***Pb0.005, significantly different from the LPS-treated control group.
PD: PD 98059; Dex: Dexamethasone.
(Huang et al., 1991), and of NF-κB in a human monocytic macrophage
cell line (Chan, 1995; Singh and Aggarwal, 1995). Inhibition of MCP-1
gene expression has also been demonstrated in closely related
analogues in which this effect was associated with inhibition of NF-κB
gene expression (Liang et al., in press). Strong inhibition of NO, PGE2,
and proinflammatory cytokine synthesis by curcumin (Jin et al., 2007)
has been ascribed to down-regulation of NF-κB-dependent transcrip-
tion (Chen et al., 2008). Closely related analogues of BHMC have also
been shown to inhibit the secretion of proinflammatory cytokines, iNOS
and COX 2 through inhibition of NF-κB gene expression (Liang et al., in
press). In view of the differences in the inhibitory nature of BHMC upon
cytokine synthesis compared to closely related analogues and

C.L. Tham et al. / European Journal of Pharmacology 628 (2010) 247–254
253
curcumin, further work is warranted to determine whether BHMC
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