Med Chem Res
DOI 10.1007/s00044-016-1611-1
ORIGINAL RESEARCH
Protective effect of 5-hydroxy-3′,4′,7-trimethoxyflavone against
inflammation induced by lipopolysaccharide in RAW 264.7 macrophage:
in vitro study and in silico validation
Arumugam Sudha1 Jeyaraman Jeyakanthan1 Pappu Srinivasan1,2
● ●
Received: 25 January 2016 / Accepted: 11 June 2016
© Springer Science+Business Media New York 2016
Abstract The herb Lippia nodiflora L. (Verbenaceae) has
been documented to exhibit anti-inflammatory, antipyretic,
antitussive, antidiabetic, anticancer, and antimelanogenesis
properties. In the present study, we aimed at evaluating the
anti-inflammatory activity of 5-hydroxy-3′,4′,7-trimethoxy-
flavone, a flavonoid from L. nodiflora, using lipopoly-
saccharide induced inflammation in RAW 264.7
macrophages. 5-hydroxy-3′,4′,7-trimethoxyflavone signifi-
cantly inhibited nitric oxide production and demonstrated
slight reduction in prostaglandin-E2 level at tested con-
centrations. The production of pro-inflammatory cytokines,
such as TNF-α, IL-6, and IL-1β, were obviously reduced by
5-hydroxy-3′,4′,7-trimethoxyflavone in a concentration-
dependent manner. Moreover, 5-hydroxy-3′,4′,7-trimethox-
yflavone significantly induced reduction in the mRNA
expressions of inducible nitric oxide synthase and cycloox-
ygenase-2, representing that inhibition occurs at the tran-
scriptional level. In addition, we performed molecular
docking and molecular dynamic simulations to study the
interaction of 5-hydroxy-3′,4′,7-trimethoxyflavone with
inflammatory mediators such as inducible nitric oxide syn-
thase and cyclooxygenase-2. Docking study showed its
hydrogen bond interactions with key residues in the active
site of inducible nitric oxide synthase and cyclooxygenase-2,
enlightening its possible binding mode at the molecular level.
The results of molecular dynamic simulations showed the
* Pappu Srinivasan
sri.bioinformatics@gmail.com
1
Department of Bioinformatics, Alagappa University, Karaikudi
630 004 Tamilnadu, India
2
Department of Animal Health and Management, Alagappa
University, Karaikudi 630 004 Tamilnadu, India
MEDICINAL
CHEMISTRY
RESEARCH
stability of complexes and their interactions. Taken together,
these findings envisage 5-hydroxy-3′,4′,7-trimethoxyflavone
as a potential candidate molecule for the progress of ther-
apeutic strategy against inflammation-related diseases.
Keywords 5-hydroxy-3′,4′,7-trimethoxyflavone ●
Inflammation Cytokines COX-2 iNOS Molecular docking
● ● ● ●
Introduction
Inflammation is a host defense mechanism that is produced
in response to tissue injury, harmful chemicals, or infection
caused by pathogens (Galli et al., 2008; Ashley et al., 2012).
Macrophages play major roles in innate immunity and they
recognize the pathogen-associated molecules such as lipo-
polysaccharide (LPS, a bacterial endotoxin) and trigger
innate immune response through toll like receptor (TLR)
signaling process (Morgensen, 2009; Chatterjee et al.,
2012). Due to the binding of LPS to TLR4 receptor, an
intracellular tyrosine kinase system is upregulated, which in
turn leads to stimulation of the transcriptional factor NF-κB
and ultimately results in the production of various inflam-
matory mediators like nitric oxide (NO), prostaglandins
(PG), tumor necrosis factor (TNF)-α, interleukin (IL)-1β
and IL-6 (Cohen, 2002). The formation of NO and PG are
catalyzed by inducible forms of nitric oxide synthase
(iNOS) and cyclooxygenase (COX-2), which are induced
by cytokines, growth factors, and other inflammatory sti-
muli (Vane et al., 1998). The release of inflammatory
mediators in macrophage contribute to various inflamma-
tion related diseases such as arthritis, atherosclerosis, can-
cer, diabetes, obesity, metabolic syndrome, allergies, and

Fig. 1 Chemical structure of 5-hydroxy-3′,4′,7-trimethoxyflavone
(HTMF) (Sudha and Srinivasan, 2014)
other autoimmune disorders (Moro et al., 2012; Vogl et al.,
2013). The inhibition of adverse macrophage activation or
selectively neutralizing the overproduction of macrophage
products was recommended as a promising therapeutic
route against diverse inflammatory conditions (Duffield,
2003). Since, the treatment based on steroidal and non-
steroidal anti-inflammatory drugs exists to alleviate
inflammation have several adverse effects (McGettigan and
Henry, 2011), the search for new anti-inflammatory drugs
still represents an important field in drug discovery process.
Medicinal plants represent a possible alternative to the
discovery of new and safer bioactive compounds (Gautam
and Jachak, 2009). In fact, natural products engage a
momentous role related to the prevention and treatment of
inflammatory conditions through modulation of various
inflammatory mediators. Consequently, plant constituents
used in herbal and traditional medicine was considered as
one of the ongoing research candidates (Nam, 2006;
Abdelwahab et al., 2012). Among several compounds that
have exposed anti-inflammatory activity, phenolic com-
pounds have concerned great attention owing to both their
large distribution and the array of biological activities that
they exhibit (Garcia-Lafuente et al., 2009). We have recently
reported the isolation of flavonoid compound 5-hydroxy-
3′,4′,7-trimethoxyflavone (HTMF) (Fig. 1) from the medic-
inal plant, Lippia nodiflora and it is known to exhibit free
radical scavenging and antilipoxygenase properties (Sudha
and Srinivasan, 2014, 2015). Various studies have been
undertaken in the present scenario to better understand the
molecular mechanism of inflammatory processes. The anti-
inflammatory effect of drugs or natural products was eval-
uated using the LPS-stimulated RAW 264.7 macrophage cell
line as widely accepted in vitro model (Hu et al., 2008; Ha
et al., 2012; Moro et al., 2012). Hence, in the present study,
the anti-inflammatory potential of HTMF was elucidated for
the first time, using LPS-activated RAW 264.7 macrophage
cells by investigating its inhibitory effects on diverse
Med Chem Res
inflammagens. Further, the mechanism of the anti-
inflammatory effect of HTMF on iNOS and COX-2
expression was also validated by in silico analysis.
Materials and methods
In vitro anti-inflammatory study
Reagents
Dulbecco’s modified eagle’s medium (DMEM), fetal
bovine serum (FBS), and phosphate buffered saline were
purchased from Invitrogen (Carlsbad, USA). LPS (Escher-
ichia coli, serotype 026:B6), 3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazoliumbromide (MTT), Griess reagent,
and sodium nitrite were purchased from Sigma-Aldrich Co.
(St. Louis, MO, USA). All other chemicals and reagents
used in this study were of analytical grade.
Cell culture and culture conditions
RAW 264.7, a mouse macrophage cell line was obtained
from National Centre for Cell Science (NCCS, Pune, India).
RAW 264.7 cells were cultured in DMEM supplemented
with 10 % heat-inactivated FBS, 1 mM sodium pyruvate,
2 mM glutamine, 100 µg/ml streptomycin, and 50 units/ml
penicillin. The cells were incubated at 37 °C in the presence
of 5 % CO2 and were subcultured every 2 days.
Cell viability assay
The viability of the cells was evaluated by MTT colori-
metric assay (Mosmann, 1983). The RAW 264.7 cells
were seeded into 96-well culture plate at a density of
5 × 104 cells/well and incubated overnight at 37 °C and 5 %
CO2 for attachment. The cells were then treated with var-
ious concentrations of HTMF with (1 µg/ml) or without LPS
(Shen et al., 2002; Moro et al., 2012; Syam et al., 2014) and
incubated for 24 h. After incubation, the culture medium
was removed and 100 µl of fresh DMEM and 20 µl of MTT
(5 mg/ml in PBS) solution were added to each well. Fol-
lowing 4 h incubation in dark, the media was discarded
again and 100 µl of DMSO was added to each well for the
solubilization of formazan deposits. The optical density of
the cells at 570 nm was measured using an enzyme-linked
immunosorbent assay plate reader (Bio-Rad Laboratories,
CA, USA) and the experiment was carried out in triplicate.
Assay for NO production
To assess the effects of HTMF on NO production, the cell
culture supernatant was examined using Griess reaction
Med Chem Res
Table 1 Primers used for Gene Primer sequences
RT–PCR analysis (F: forward
and R: reverse) iNOS F: 5′-TTTCCAGAAGCAGAATGTGACC-3′; R: 5′-AACACCACTTTCACCAAGACTC-3′
COX-2 F: 5′-GAAGTCTTTGGTCTGGTGCCTG-3′; R: 5′-GTCTGCTGGTTTGGAATAGTTGC-3′
GAPDH F: 5′-TCATCCCAGAGCTGAACG-3′; R: 5′-GGGAGTTGCTGTTGAAGTC-3′
(Granger et al., 1996). The RAW 264.7 cells were seeded
at a density of 2 × 104 cells/well in 24-well culture plates
and incubated for 2 h at 37 °C and 5 % CO2 for adherence.
The adhered cells were incubated for 24 h, which indicated
the concentrations of HTMF (1–40 µg/ml) in the absence
or presence of LPS (1 µg/ml). The accumulation of nitrite
in the culture supernatant was measured as an indicator of
NO production using Griess reagent. The culture super-
natant (100 µl) was mixed with an equal volume of Griess
reagent (1 % (w/v) sulfanilamide, 0.1 % (w/v) naphthy-
lethylenediamine dihydrochloride, dissolved in 2.5 %
H3PO4) and the mixture was incubated for 10 min at
room temperature. The absorbance of mixture was read at
540 nm spectrophotometrically and fresh culture medium
was used as a blank. The assay was performed in triplicate
and the results were expressed as µM.
Assay for PGE2 production
To examine the effect of HTMF on PGE2 levels in LPS
stimulated cells, RAW 264.7 cells (2 × 104 cells/well) were
seeded on 24-well culture plates. The cells were treated with
the indicated concentrations of HTMF (1–40 µg/ml) in the
absence or presence of LPS (1 µg/ml) and incubated for 24 h
at 37 °C in a 5 % CO2 incubator. The concentration of PGE2
in the culture supernatants of RAW 264.7 cells was quan-
tified using an enzyme immunoassay (EIA) kit (Cayman
Chemical Company, USA) according to the manufacturer’s
instructions. The experiments were performed in triplicate
and PGE2 production was calculated relative to that of the
control value.
Measurement of pro-inflammatory cytokines (TNF-α, IL-1β,
and IL-6) production
To investigate the effect of HTMF on the production of cyto
kines from LPS-stimulated cells, RAW 264.7 cells (1 ×
106 cells/well) were plated onto 24-well culture plates. The
cell culture supernatants were harvested after 24 h of incu-
bation of cells with LPS (1 µg/ml) and HTMF (1–40 µg/ml).
These supernatants were tested for quantitation of pro-
inflammatory cytokines (TNF-α, IL-1β, and IL-6) using
mouse specific EIA kits (BD Biosciences, USA) according to
the manufacturer’s instructions. The concentrations were
determined for three wells for each cytokines and values were
derived from the standard curve and expressed as pg/ml.
RNA isolation and RT (Reverse Transcriptase)–PCR
analysis
The RAW 264.7 cells were seeded at a density of
1 × 106 cells/well in six-well culture plates and incubated
for 24 h at 37 °C and 5 % CO2 for cells attachment. The
cells were treated with HTMF at the adequate concentration
(40 µg/ml) in the absence or presence of LPS (1 µg/ml) and
the culture was incubated for 24 h. After incubation, to
examine the effects of HTMF on mRNA expression levels
of iNOS and COX-2, total RNA was isolated using the
RNeasy Mini kit (Qiagen, Hilden, Germany), as described
by the manufacturer. The total RNA samples were stored at
−80 °C until the further use. The total RNA was reverse-
transcribed using Omniscript Reverse Transcription kit
(Qiagen, Hilden, Germany). According to the manu-
facturer’s instructions, 1 μg of total RNA was converted to
complementary DNA (cDNA) by using 10X RT buffer,
oligo-dT primers (10 µM), dNTPs mix (each 5 mM), RNase
inhibitor (10 U/µl), reverse transcriptase, and RNase-free
water and made up to a final volume of 20 µl. The reaction
mixture was incubated at 37 °C for 1 h. The synthesized
cDNA was used in RT–PCR. RT–PCR was performed in
96-well plates in triplicate on an ABI Prism 7900HT
sequence detection system using the comparative ΔCt
method according to ABI manual (SYBR Green PCR
Master Mix Protocol, Applied Biosystems, Foster City, CA,
USA). The reaction mixture (20 µl) consisted of SYBR
Green PCR Master Mix (10 µl), cDNA sample (1 µl), and
forward and reverse primers. The primer sequences were
used as previously reported (Zhang et al., 2007) and are
listed in Table 1. The PCR protocol consisted of an initial
activation at 95 °C for 20 s, 40 amplification cycles at
95 °C for 15 s (denaturation), followed by annealing and
extension at 60–65 °C for 20 s. The relative gene expression
levels were evaluated and normalized relative to that of
glyceraldehyde 3-phosphate dehydrogenase (GAPDH)
housekeeping gene. The data were expressed as fold
change and calculated using the 2−ΔΔCT method (Livak and
Schmittgen, 2001).
Statistical analysis
The results were represented as mean ± SD. Data were
statistically analyzed using one-way analysis of variance
(ANOVA). p value o 0.05 was considered statistically

significant. All calculations were performed using Graph-
Pad Prism Software v 5.01 (GraphPad Software Inc., San
Diego, CA).
Computational studies
All the computational analyses were carried out on a HP PC
operated with CentOS V6.2 Linux operating platform run-
ning with Intel core i5 processor and 4 GB of RAM speed.
Ligand structure preparation
Drawing of ligand (HTMF) is performed in ChemSketch
version 11.01 (http://www.acdlabs.com) and imported
into Maestro user interface (Maestro, 2013). The 2D
structure was transformed into 3D structure using ligprep
module embedded in Schrodinger software (LigPrep,
2013). The preparation of ligand includes addition of
hydrogen atoms, bond order fixation, neutralization of
charged groups, and generation of probable tautomers.
Finally, the ligand was optimized using OPLS_2005 force
field.
Protein preparation
The X-ray crystal structures of iNOS complexed with
2-[(1R)-3-amino-1-phenyl-propoxy]-4-chloro-benzonitrile
and COX-2 complexed with SC-558 were retrieved from
Protein Databank (http://www.rcsb.org/pdb) with their
accession codes 2Y37 and 1CX2, respectively (Cheshire
et al., 2011; Kurumbail et al., 1996). The iNOS protein
consists of two identical chains, each complexed with H4B
and heme group. But the COX-2 receptor consists
of four identical chains, each binding a SC-558 molecule
with other small molecules (HEM and NAG). Therefore,
one chain of iNOS and three chains of COX-2 with
other chemical components were deleted from the protein.
The crystal structures were prepared by a multi-step process
through protein preparation wizard (Schrodinger Suite,
2013). The right bond orders as well as charges were
assigned to the protein atoms and hydrogen atoms were
added to the crystal structure. The crystallographic
water molecules were removed and optimized at neutral pH.
The optimized structure was then subjected to energy
minimization using the OPLS_2005 force field with implicit
solvation. The minimization was carried out by setting
maximum heavy atom root mean square deviation (RMSD)
to 0.30 Å. After the cleaning of ligand and protein struc-
tures, the receptor-binding site was generated as a grid-
enclosing box, centered to co-crystallized ligand molecule
in each protein, to enclose the residues within 10 Å from
their centroid. The generated grid of each protein was used
for docking calculations.
Med Chem Res
Molecular docking analysis and binding free energy
calculation
Molecular docking analysis is performed to find the possi-
ble interactions involved in the binding of HTMF to active
sites of iNOS and COX-2. To evaluate the docking analysis,
the bounded ligand H4B and SC-558 were removed and
redocked into the binding site of respective target proteins.
The docking methodology is reliable, as the RMSD value of
the docked ligand is found to be less than 2.0 Å. The pre-
pared ligand molecule was docked into the active sites of
each protein by using Glide module (Glide, 2013) in extra
precision (XP) mode (Friesner et al., 2004) which uses
MCSA (Monte Carlo-based simulated algorithm) based
minimization. Glide XP mode predicts all practical con-
formations for each low-energy conformer in the selected
binding site. Glide XP score was used to find the best
conformer with optimum binding affinity. The best con-
former of the HTMF molecule along with the receptors
iNOS and COX-2 were further subjected to binding free
energy calculation by Prime/MM-GBSA method in Prime
module (Prime, 2013) using OPLS-2005 force field and
GB/SA continuum solvent model.
Molecular dynamics (MD) simulations of ligand-bound
complexes
The protein-ligand complex obtained from Glide’s XP
docking was subjected to MD simulations using Desmond
molecular dynamics system, Version 3.8 (D. E. Shaw
Research, New York) (Desmond, 2014) with OPLS-2005
force field. The complex was solvated in an orthorhombic
box containing SPC water molecules and was neutralized
using a suitable number of salt counter-ions. The distance
between the box wall and protein-ligand complex was set
to be more than 10 Å to evade direct interactions with its
own periodic image. The energy minimization of the
prepared systems was carried out up to a maximum of
2000 steps using a steepest descent method until a gra-
dient threshold at 25 kcal mol−1 Å−1 was reached and
equilibrated using the default protocol of Desmond.
This equilibrated system was further simulated at a con-
stant temperature of 300 K and constant pressure of 1 atm
with a time step of 2 fs for a time period of 3 ns simulation
for iNOS and COX-2 protein. The long-range electro
static interactions were calculated using smooth
Particle–Mesh–Ewald method during MD simulations. A
9 Å cutoff radius value was used for Coulombic short-
range interaction cutoff method. The trajectory frames
were captured at each 1.2 ps time step. The RMSD and
hydrogen bonding interactions of the protein-ligand
complex was calculated for the entire simulations trajec-
tory with reference to their first frame.
Med Chem Res
Fig. 2 The effects of HTMF on cell viability of RAW 246.7 macro-
phage cells. Data were expressed as the mean ± SD of three
experiments
Results and discussion
Effects of HTMF on cell viability
The precise determination of cytotoxicity related with pro-
longed incubation of the cells is the prerequisite for accu-
rately assessing the biological properties of individual
molecules or complex mixtures (Ayissi Owona et al., 2013).
MTT assay is used to examine and validate the effect of
HTMF on cell growth in RAW 264.7 cells. It was observed
that the exposure of RAW 264.7 cells to the increasing
concentrations (1–40 µg/ml) of HTMF did not exhibit any
signs of cytotoxicity after 24 h incubation. The compound
HTMF inhibited the cell growth by 2.2, 4.7, 3.9, 4.4, and
8.3 % at doses of 1, 5, 10, 20, and 40 µg/ml, respectively.
As a result, the cell viability was confirmed to be 490 % at
all tested concentrations (Fig. 2) and HTMF was considered
appropriate at these concentrations for further assays.
Effects of HTMF on LPS-induced NO production
NO is an important signaling molecule produced by the
iNOS and exhibits a pivotal role in numerous body func-
tions at very low concentrations. However, overproduction
of NO especially in macrophages may direct to cytotoxicity,
inflammation, and autoimmune disorders (Sarkar et al.,
2008; Heo et al., 2012). As LPS is considered to be the most
effective macrophage activation factor, it can elicit iNOS-
stimulated NO production in various cells (macrophages,
smooth muscle cells, and hepatocytes), to respond to
inflammation, sepsis, and stroke (Nathan, 1992; Shen et al.,
2002). Thus, NO production by LPS may perhaps reveal the
degree of inflammation and used as a marker to evaluate the
potential of drugs or phytochemicals on inflammatory
Fig. 3 Effects of HTMF on NO production in LPS-stimulated RAW
264.7 cells. The production of NO was assayed in the culture medium
of cells stimulated with LPS (1 µg/ml) for 24 h in the presence of
HTMF (1–40 µg/ml). Data were expressed as mean ± SD of three
experiments. #p o 0.05 represents a significant difference compared
with control group that was not treated with LPS and *p o 0.05
represents a significant difference compared with cells treated with
LPS alone
progressions. Therefore, NO inhibitors are necessary for
prevention of inflammatory-related diseases and regulation
of NO production is essential for human health. In the
present study, the nitrite level in culture supernatant of
RAW 264.7 cells was found to be increased significantly
(p o 0.05) from 2.5 to 21.4 µM, after 24 h LPS treatment as
shown in Fig. 3. NO produced due to LPS treatment was
inhibited as 7.3, 18.2, 27.4, 47.6, and 63.2 % by the addition
of HTMF at doses of 1, 5, 10, 20, and 40 µg/ml, respec-
tively, and the IC50 was calculated as 23.2 µg/ml. The
results showed that HTMF significantly (p o 0.05) inhib-
ited LPS-induced NO production in a dose-dependent
manner and the inhibitory effect was reported not due to
any cytotoxic action on RAW 264.7 cells, as suggested
from the cell viability experiment. The obtained result
necessitates that prominent inhibitory activity of HTMF on
NO production could be partly due to its phenolic nature. In
addition to the inhibition of NO generation, the compound
HTMF also renders a direct scavenging effect on NO, and
might serve as a effective therapeutic agent for regulating
the pathological conditions caused by excessive generation
of NO.
Effects of HTMF on LPS-induced PGE2 production
PGs are a group of AA-derived eicosanoids and are con-
sidered as one of the powerful modulators in inflammation
as well as immune responses. The enhanced PGE2 pro-
duction is observed in immune cells, due to relatively ele-
vated levels of AA (precursor) in the immune cell
membrane phospholipids along with the initiation of
COX-2 in response to cellular stimulation. Several studies

Fig. 4 Effects of HTMF on LPS-stimulated PGE2 production in RAW
264.7 cells. The production of PGE2 was assayed in the culture
medium of cells stimulated with LPS (1 µg/ml) for 24 h in the presence
of HTMF (1–40 µg/ml). HTMF showed a tendency to inhibit PGE2
production. Data were expressed as mean ± SD of three experiments.
#p o 0.05 represents a significant difference compared with control
group that was not treated with LPS and *p o 0.05 represents a sig-
nificant difference compared with cells treated with LPS alone
have reported that the production of PGE2 by LPS in mac-
rophages is primarily correlated with the transcriptional acti-
vation of COX-2 expression (Lee et al., 1992; Reddy and
Herschman, 1994; Kao et al., 2012). PGE2 receives para-
mount importance among the PGs and is a foremost target for
anti-inflammation and anticancer research activities (Zhong
et al., 2012). Hence, natural products that can efficiently
suppress the unusual production of PGE2 might act as
potential anti-inflammatory and cancer chemopreventive
agents. In this work, the PGE2 levels in unstimulated RAW
264.7 cells are very low and are found to be 174.07 ± 8.06 pg/
ml. But, the stimulation of RAW 264.7 cells by LPS treat-
ment provoked remarkable increase in PGE2 production of
about 1021.3 ± 18.12 pg/ml (Fig. 4). However, LPS treatment
along with HTMF involvement decreased the production of
PGE2 to 1007.3 ± 6.13, 957.3 ± 24.69, 905.3 ± 6.14, 853.14
± 20.63, and 748.6 ± 29.58 pg/ml at doses of 1, 5, 10, 20, and
40 μg/ml, respectively. Thus, HTMF demonstrated 1.3, 6.2,
11.3, 16.4, and 26.7 % inhibition at various tested con-
centrations, compared to cells treated with LPS alone. Even
though HTMF showed inhibitory effect on PGE2 production
in a dose-dependent manner, the result was not as strong as
that displayed in the inhibition of NO production. It was
stated that flavonoids demonstrate differential effects on PGE2
production in macrophages owing to the presence of hydroxyl
moieties in their A-ring (i.e., the 5,7-dihydroxyl groups pos-
sess higher activity as in case of quercetin than the dihydroxyl
groups in other positions) (Wu et al., 2008). The present result
supports the above statement; as the examined compound
HTMF possesses only one hydroxyl group (5-OH) in its A-
ring, it might be the cause for its lower inhibitory activity on
PGE2 production.
Med Chem Res
Effects of HTMF on pro-inflammatory cytokines
production
The pro-inflammatory cytokines are notorious to be impli-
cated in the pathogenesis of inflammation, in addition to
NO. In this series, production of the pivotal pro-
inflammatory cytokines such as TNF-α, IL-6, and IL-1β
along with COX-2 has been well established through the
activation of macrophage cells during inflammation, and
their excess generation can be related to progress of chronic
diseases like atherosclerosis and rheumatoid arthritis
(Takaki et al., 2003). Recently, the association between
iNOS induction and its dependence on the pro-
inflammatory cytokine such as TNF-α signaling has been
reported (Medeiros et al., 2007). Therefore, in inflammatory
diseases, controlling the cytokines such as TNF-α, IL-6, and
IL-1β had predominantly guided to the greatest advances in
medicine (Moller and Villiger, 2006). Hence, the suppres-
sive effect of HTMF on TNF-α, IL-6, and IL-1β were also
evaluated by quantifying the amount of respective cytokine
production in the LPS-induced cells after 24 h via enzyme-
linked immunoassay. The RAW 264.7 cells treated with
LPS demonstrated an appreciable increase in TNF-α
(4.5-fold), IL-6 (7.4-fold), and IL-1β (16.3-fold) levels in
the culture supernatants, compared to unstimulated control
group (p o 0.05). Based on the observation, it was noticed
that the production of those cytokines was significantly
(p o 0.05) inhibited by HTMF in a dose-dependent man-
ner. Among them, HTMF has moderately reduced TNF-α
production by 19 and 31 % at concentrations of 20 and
40 µg/ml, respectively (Fig. 5a).
On contrary, reduction in IL-6 and IL-1β levels pursued a
similar pattern, with close inhibition range. At 20 µg/ml,
HTMF caused 42 and 45 % inhibition of IL-6 and IL-1β pro-
duction whereas the highest concentration of HTMF (40 µg/ml)
caused 54 % (IL-6), and 58 % (IL-1β) inhibition (Fig. 5b, c).
These results revealed that compound HTMF remarkably
attenuated the production of more than one inflammatory
cytokine from LPS-induced cells and thereby signifying its
valuable role in controlling inflammatory responses. Likewise,
Crouvezier et al. (2001) stated that only a part of polyphenols
in green tea was important in inhibiting LPS-induced IL-1β in
human whole blood cultures, whereas all the other polyphenols
were found unsuccessful to hold up IL-6 and TNF-α produc-
tion. Similarly, the phenolic compounds, namely p-coumaric
acid, syringic acid, homovanillic acid, kaempferol, tyrosol,
caffeic acid, and oleuropein glycoside, did not demonstrate
inhibitory effect on TNF-α production at a dose from 10−7 to
10−4 M (Miles et al., 2005). From these observations, it was
inferred that the inhibition of TNF-α formation might be
influenced by the presence of hydroxyl groups on B ring of
flavonoids, with two hydroxyl groups at the 3′ and 4′ position
being the most efficient, pursued by single hydroxyl group and
Med Chem Res
Fig. 5 Effects of HTMF on pro-inflammatory cytokine production in
LPS-stimulated RAW 264.7 cells. Culture supernatants from treated
cells were immunoassayed for TNF-α (a), IL-6 (b), and IL-1β (c)
production by ELISA kit. Data were expressed as mean ± SD of three
absence of hydroxyl group (Comalada et al., 2006). To note,
the structural characteristics of HTMF might be insufficient in
reducing TNF-α formation in the present finding, since the
compound HTMF possesses no hydroxyl group on its B ring.
Collectively, the examined effect on pro-inflammatory cyto-
kines production suggests that HTMF is competent of dis-
rupting key signal transduction pathways stimulated by LPS in
RAW 264.7 cells, consequently preventing the production of
pro-inflammatory mediators.
Effects of HTMF on mRNA expressions of iNOS and
COX-2
Macrophage-derived NO is an important intracellular and
intercellular signaling molecule that is involved in the reg-
ulation of various physiological and pathophysiological
mechanisms in immunological systems (Bogdan, 2001;
MacMicking et al., 1997). Moreover, COX-2, the key
enzyme implicated in the synthesis of PGE2, was induced
by various stimuli such as LPS, cytokines, and growth
factors and in turn collectively enhances the inflammatory
experiments. #p o 0.05 represents a significant difference compared
with control group that was not treated with LPS and *p o 0.05
represents a significant difference compared with cells treated with
LPS alone
responses (Rossi et al., 2002; Federico et al., 2007). The
mRNA expression levels for the key enzymes are the
foremost indicators of anti-inflammatory activity and thus
inhibition of iNOS and COX-2 can afford a valuable
strategy for inhibiting the inflammation. Therefore, the
effect of HTMF on iNOS and COX-2 gene expression was
further studied by RT–PCR analysis, to reveal the
mechanism concerned in the inhibitions of NO and PGE2
generation by HTMF in LPS-induced RAW 264.7 cells.
The RAW 246.7 cells were treated with LPS and
40 µg/ml of HTMF for mRNA expression studies, since
HTMF showed the best possible anti-inflammatory activity
at that particular dose in earlier findings. The mRNA
expressions of iNOS and COX-2 were scarcely noticeable
in the control group without LPS and HTMF. On the other
hand, the iNOS and COX-2 on mRNA expression levels
was found to be markedly increased in RAW 264.7 cells
exposed to LPS after 24 h treatment, and HTMF sig-
nificantly (p o 0.05) suppressed the upregulated gene
expression of iNOS (∼40 %) and COX-2 (∼35 %) at
40 µg/ml (Fig. 6a, b). The production pattern of NO and

Fig. 6 Effects of HTMF on the mRNA expression of iNOS and COX-2
in LPS-stimulated RAW 264.7 cells. The relative expression levels of
iNOS and COX-2 were determined by RT–PCR relative to the GAPDH
gene and the data were expressed as fold of change from LPS alone treated
PGE2 was well correlated with the alterations in the mRNA
levels of iNOS and COX-2, quantified through RT–PCR
analysis. These results denote that the downregulation of
iNOS and COX-2 gene expression might have contributed to
the inhibition of NO and PGE2 production, as iNOS and
COX-2 are the enzymes responsible for the synthesis of NO
and PGE2. This finding also corroborates the fact that iNOS
suppression might, in turn, have an inhibitory role on induced
pro-inflammatory cytokines TNFα and IL-1β and vice versa
(Fig. 5a, c). In a similar study, Balakrishnan et al. (2010)
reported that the L. nodiflora extract and isolated sterol CPP
significantly downregulated LPS induced expression of the
pro-inflammatory cytokines (TNF-α, IL-6, and IL-1β) in
PBMC and other important inflammatory mediators (iNOS
and COX-2) in RAW 264.7 macrophages. Therefore, com-
pounds that inhibit iNOS and COX-2 gene expression by
transcriptional downregulation have appreciable therapeutic
effect in the treatment of all major inflammatory and infec-
tious diseases (Fischer et al., 1999). Several studies reported
the suppressive effect of natural products on the production of
pro-inflammatory mediators (NO and PGE2) and cytokines
(Oh et al., 2008; Syam et al., 2014; Heo et al., 2012;
Abdelwahab et al., 2012). Since there is no report on anti-
inflammatory activity of HTMF, the present findings indicates
that HTMF might mediate anti-inflammatory role in either an
iNOS- or a COX-2-dependent manner. Therefore, further
studies are desired to make obvious the mechanisms by which
HTMF shows different levels of anti-inflammatory effect.
Computational studies
Binding mode analysis of HTMF with iNOS and COX-2
Molecular docking and molecular dynamics studies further
validated the in vitro anti-inflammatory profiles of HTMF.
Med Chem Res
group. Data were expressed as mean ± SD of three independent experi-
ments. #p o 0.05 represents a significant difference compared with con-
trol group that was not treated with LPS and *p o 0.05 represents a
significant difference compared with cells treated with LPS alone
iNOS and COX-2 are considered as the most significant
enzymatic systems for inflammation and with the aim to
validate the experimental results, as HTMF is active in
controlling the expression of both iNOS and COX-2 at
transcriptional level, a molecular modeling study was con-
ducted (Syam et al., 2014; Priscilla et al., 2011; Zhang et al.,
2012). The docking study demonstrates the binding pattern
of HTMF within the active site of iNOS and COX-2 (Figs. 7
and 8). From Fig. 7, it was noticed that the predicted pose of
HTMF compound bind in the major cavity related to the
active site of the iNOS and the aromatic moiety of HTMF is
placed almost perpendicular to the heme group. In their
active pocket, the HTMF is surrounded by hydrophobic
residues, such as Met368, Tyr367, Trp366, Phe363, Pro344,
Ala345, Val346, Trp457, and Tyr485. It can be concluded
that there are hydrophobic interactions and desolvation
effects in ligand binding. The binding mode analysis shows
that HTMF makes two hydrogen bond interactions with
aromatic Trp366 residue (1.77 Å) and with negatively
charged Glu371 residue (1.82 Å), both of which exhibit a
crucial role in substrate binding of murine iNOS (Tiperciuc
et al., 2013). Hence, these residues are reported as key
residues in the design of selective murine iNOS inhibitors in
addition to Met368, Gly365, Tyr367, Phe363, Pro344,
Gln257, Val346, Asn364, Arg382, Arg 260, Met349,
Thr370, and Tyr485. The docking score of interaction
obtained was −7.75 kcal/mol (Table 2) and in general, a
negative value (kcal/mol) signifies lowest possible energy at
which high potency of binding between receptor and ligand
is accomplished. The hydrogen bond interaction formed by
HTMF with key residues might prove that this compound has
potent activity against iNOS. Moreover, the binding to the
active site of iNOS is achieved by the contribution of
5-hydroxyl and 3′-OCH3 group from the A and B ring of
HTMF, respectively. Based on the docking result, it is also
Med Chem Res

Fig. 7 Binding mode and molecular interaction of HTMF with the iNOS binding site. In the left side of the figure, heme and HTMF is shown in
red and green color stick models, meanwhile the right side of the figure depicts the 2D interaction of HTMF with iNOS

Fig. 8 Binding mode and molecular interaction of HTMF with the COX-2 binding site. In the left side of the figure, HTMF was shown in pink
color stick model, meanwhile the right side of the figure depicts the 2D interaction of HTMF with COX-2

sensible to deduce that inhibition of iNOS expression is an In case of COX-2, it could be exemplified that HTMF
additional possible mechanism by which the compound put forms hydrogen bonds with two aromatic residues in the
forth an anti-inflammatory and antioxidative action. active sites of COX-2, i.e. with Tyr355 and Tyr385.
 Med Chem Res

Table 2 Docking score, glide Target Docking score Glide energy ΔGbind Active site residues H-bond
energy, and binding free energy (kcal/mol) (kcal/mol) (kcal/mol) interaction (D…H–A) length (Å)
results of HTMF with iNOS and
COX-2 predicted through XP iNOS −7.75 −41.18 −62.48 CH3 (O)… OH (Glu371) 1.82
docking — — — OH…O (Trp366) 1.77
COX-2 −9.10 −32.08 −58.04 OH (Tyr385)…O(CH3) 2.04
— — — OH…O (Tyr355) 1.58

Fig. 9 The root mean-square

deviation (RMSD, in Å) vs. MD
simulation time of the backbone
atoms for the iNOS/HTMF (a)
and COX-2/HTMF (b)
complexes

The compound HTMF could successfully dock into the
COX-2 active site and the docking score of interaction
obtained was −9.10 kcal/mol (Table 2). The bond distances
between 5-OH of HTMF and OH of Tyr 355 is 1.58 Å,
while between 3′-OCH3 of HTMF and OH of Tyr385 is
2.04 Å (H…O) (Fig. 8). The hydroxyl (act as donor) and
oxygen-bound methyl group (act as acceptor) of HTMF
have played very significant roles in ligand-receptor inter-
action for the formation of hydrogen bonds. The score
might be recognized to its one hydrogen bonding interaction
with Tyr385 and it is further accountable for showing good
inhibitory activity on COX-2 gene expression, as Tyr385
was reported to be implicated in the abstraction of hydrogen
from C-13 of arachidonate (Thuresson et al., 2001).
Med Chem Res
The hydrophobic and π–π stacking interactions were also
observed between HTMF and the surrounding residues in
the binding site. The π–π stacking interaction was
observed between HTMF and Arg120 with distance of
5.17 Å and angle of 70.34°. It was well predictable from
the literature that the active site of COX-2 holds three
important regions, viz. a hydrophobic pocket portrayed by
the occurrence of Tyr385, Trp387, Phe518, Ala201,
Tyr248, and Leu352. The second key region is char-
acterized by three hydrophilic amino acid residues
(Arg120, Glu524, and Tyr355) which are present at the
entrance of the active site whereas the third region is a
side pocket associated with the existence of His90,
Arg513, and Val523 residues (Priscilla et al., 2011).
From the docking result, it was noted that the bonding of
HTMF was in the close vicinity of the hydrophobic pocket.
The docking of some COX-2 inhibitors like celecoxib,
diclofenac, ibuprofen, and naproxen has revealed their
hydrogen bonding interactions with Tyr324, Tyr385,
Ser530, Arg120, and Tyr355 residues (Kurumbail et al.,
1996; Llorens et al., 2002; Dilber et al., 2008; Krishna et al.,
2013). Moreover, in another study, the virtual screening of
flavonoids against COX-2 protein exposed that the hydroxyl
groups present in C5 or C7 or in both could bind with
Ile517, Phe518, Ser 355, Leu352, and Gln192, respectively.
In addition, it should be noted that the C4 carbonyl and the
substituent moiety at C3′ and C4′ could interact with
Tyr355 and Tyr385, by which they enhance the inhibitory
effect on COX-2 (Li et al., 2011). The current examination
with the comparative analysis of literature data further
specified that the target compound HTMF might persuade
the COX-2 protein’s active site confirmation through
interaction with the active site and thereby provokes the
anti-inflammatory activity.
MD simulations of HTMF with iNOS and COX-2
MD simulations were carried out to check the stability of
the iNOS-HTMF and COX-2-HTMF complexes. The
comparative RMSDs along the trajectories of both iNOS-
HTMF and COX-2-HTMF complexes have been deter-
mined and plotted in Fig. 9. It is obvious that the coordi-
nates of active site residues of HTMF bound to iNOS was
altered and the analysis of Fig. 9a indicates that the RMSD
values steadily increase from 0 to 600 ps, and reached
equilibration after 1500 ps time period. The RMSD was
about 3.2 Å after 1.5 ns and it pointed out that the complex
structure is stable after 1.5 ns of simulation. Similarly, the
conformation of COX-2-HTMF complex changes to some
extent, particularly from 0–1000 ps equilibration. But the
complex attained a stable conformation after 1000 ps
simulation time and the RMSD value was found to be
∼2.7 Å (Fig. 9b). Furthermore, the RMSD gets lowered
during the last 2500–3000 ps. This indicates that binding of
the compound HTMF with COX-2 has increased the con-
formational stability of the protein at these time intervals.
The root mean square fluctuation (RMSF) of residues in
the trajectory of both iNOS-HTMF and COX-2-HTMF
complexes was presented in Fig. 10. The iNOS protein con-
sists of residues numbered 77–496, whereas COX-2 protein
includes 33–584 residues. It is known that the bigger change
in RMSF value takes place due to the softer residues. The four
major flexible protein segments corresponding to residues
145–155, 370–380, 395–410, 460–465 were noticed in iNOS-
HTMF complex and the active site residues exhibit a small
range of fluctuations (Fig. 10a). Further, the RMSF of residues
in the active site also shows wavy impression, which could be
due to the movement of the heme during MD simulation.
However, the RMSF values of the COX-2-HTMF complex
shows larger fluctuations to residue 50–100, 210–220,
264–278, and 400–420 (Fig. 10b). These residues changed
greatly, which proves that the complex is flexible at those
positions. Moreover, the fluctuations in the inhibitor binding
site residues (Tyr355 and Tyr385) are small. Hence, the result
shows that the small range of RMSDs reproduced minor
structural movement in the docking complex during simula-
tion time. These observations go in hand with the experimental
results that the interaction of proteins with ligand frequently
occurs with an increase in protein stability and structural order
(Latypov et al., 2008; Celej et al., 2003). From the trajectory
analysis, it was noticed that HTMF formed stable hydrogen
bonding interactions with Trp366 and Glu371 residues of
iNOS. In the COX-2-HTMF complex, amino acids Tyr385
and Tyr355 also showed highly stable interactions with the
ligand throughout MD simulations. The results acquired from
the computational approach will be useful for the structure-
based lead optimization. Overall, these findings indicate that
HTMF exhibits potential anti-inflammatory properties, that
can act on iNOS and COX-2 and thus it can be developed as
herbal anti-inflammatory agent.
Conclusion
The present study shows that HTMF possesses potent anti-
inflammatory effects in LPS-induced macrophage cell line
mediated by inhibition of release of inflammatory media-
tors, NO, PGE2, and pro-inflammatory cytokines. The
inhibitory effect is mainly correlated to the ability of the
compound concerned to downregulate iNOS and COX-2
expression at transcriptional level. The mechanism of
inhibition exerted by HTMF also seems to be associated
with the attenuation of TNF-α, IL-6, and IL-1β formation.
Moreover, molecular docking was employed to contribute
more useful information of HTMF with iNOS and COX-2,
such as active site, binding mode, and important residues.
 Med Chem Res

Fig. 10 RMSF of the backbone

atoms of (a) iNOS and (b)
COX-2 protein in the presence
of HTMF at 300 K

The results of molecular docking studies showed that
HTMF has more favorable interaction with better docking
score, docking energy, and binding free energy. MD
simulations of iNOS-HTMF and COX-2-HTMF complexes
indicated that HTMF displays stable hydrogen bonds. The
findings of in vitro and in silico studies suggested that
HTMF is a capable lead compound that may have future
prospect in progress of anti-inflammatory therapeutics.
Acknowledgments We gratefully acknowledge the financial
assistance given by Department of Science and Technology
(DST WOS-A), New Delhi, for granting fellowship to A. Sudha
(Grant No. SR/WOS-A/LS-237/2010). The authors thank Dr.
B. Kadalmani, Department of Animal Science, Bharathidasan
University, Tiruchirapalli for providing help in in vitro studies.
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
References
Abdelwahab SI, Koko WS, Taha MME, Mohan S, Achoui M,
Abdulla MA, Mustafa MR, Ahmad S, Noordin MI, Yong CL,
Sulaiman MR, Othman R, Hassan AA (2012) In vitro and
in vivo anti-inflammatory activities of columbin through the
inhibition of cycloxygenase-2 and nitric oxide but not
the suppression of NF-κB translocation. Eur J Pharmacol
678:61–70
Med Chem Res
Ashley NT, Weil ZM, Nelson RJ (2012) Inflammation: mechanisms,
costs, and natural variation. Annu Rev Ecol Evol Syst
43:385–406
Ayissi Owona B, Njayou NF, Laufer S, Moundipa PF, Schluesener HJ
(2013) A fraction of stem bark extract of Entada africana sup-
presses lipopolysaccharide-induced inflammation in RAW 264.7
cells. J Ethnopharmacol 149:162–168
Balakrishnan G, Janakarajan L, Balakrishnan A, Lakshmi BS (2010)
Molecular basis of the anti-inflammatory property exhibited by
cyclo-pentanophenanthrenol isolated from Lippia nodiflora.
Immunol Invest 39:713–739
Bogdan C (2001) Nitric oxide and the immune response. Nat Immunol
2:907–916
Celej MS, Montich CG, Fidelio GD (2003) Protein stability induced
by ligand binding correlates with changes in protein flexibility.
Protein Sci 12:1496–1506
Chatterjee N, Das S, Bose D, Banerjee S, Das S, Chattopadhyay D,
Saha KD (2012) Exploring the anti-inflammatory activity of a
novel 2-phenylquinazoline analog with protection against
inflammatory injury. Toxicol Appl Pharmacol 264:182–191
Cheshire DR, Aberg A, Andersson GM, Andrews G, Beaton HG,
Birkinshaw TN, Boughton-Smith N, Connolly S, Cook TR,
Cooper A, Cooper SL, Cox D, Dixon J, Gensmantel N, Hamley
PJ, Harrison R, Hartopp P, Kack H, Leeson PD, Luker T, Mete A,
Millichip I, Nicholls DJ, Pimm AD, St-Gallay SA, Wallace AV
(2011) The discovery of novel, potent and highly selective inhi-
bitors of inducible nitric oxide synthase (iNOS). Bioorg Med
Chem Lett 21:2468–2471
Cohen J (2002) The immunopathogenesis of sepsis. Nature 420:885–891
Comalada M, Ballester I, Bailon E, Sierra S, Xaus J, Galvez J, de
Medina FS, Zarzuelo A (2006) Inhibition of pro-inflammatory
markers in primary bone marrow-derived mouse macrophages by
naturally occurring flavonoids: analysis of the structure-activity
relationship. Biochem Pharmacol 72:1010–1021
Crouvezier S, Powell B, Keir D, Yaqoob P (2001) The effects of
phenolic components of tea on the production of pro- and anti-
inflammatory cytokines by human leukocytes in vitro. Cytokine
13:280–286
Desmond Molecular Dynamics System (2014) Version 3.8. D. E.
Shaw Research, New York
Dilber SP, Dobric SL, Juranic ZD, Markovic BD, Vladimirov SM,
Juranic IO (2008) Docking studies and anti-inflammatory activity
of beta-hydroxy-betaarylpropanoic acids. Molecules 18:603–615
Duffield JS (2003) The inflammatory macrophage: a story of Jekyll
and Hyde. Clin Sci (Lond) 104:27–38
Federico A, Morgillo F, Tuccillo C, Ciardiello F, Loguercio C (2007)
Chronic inflammation and oxidative stress in human carcino-
genesis. Int J Cancer 121:2381–2386
Fischer LG, Horstman DJ, Hahnenkamp K, Kechner NE, Rich GF
(1999) Selective iNOS inhibition attenuates acetylcholine- and
bradykinin-induced vasoconstriction in lipopolysaccharide-
exposed rat lungs. Anesthesiology 91:1724–1732
Friesner RA, Banks JL, Murphy RB, Halgren TA, Klicic JJ, Mainz
DT, Repasky MP, Knoll EH, Shaw DE, Shelley M, Perry JK,
Francis P, Shenkin PS (2004) Glide: A new approach for rapid,
accurate docking and scoring. 1. Method and assessment of
docking accuracy. J Med Chem 47:1739–1749
Galli SJ, Grimbaldeston M, Tsai M (2008) Immunomodulatory mast
cells: negative, as well as positive regulators of immunity. Nat
Rev Immunol 8:478–486
Garcia-Lafuente A, Guillamon E, Villares A, Rostagno MA, Martinez
JA (2009) Flavonoids as anti-inflammatory agents: implications
in cancer and cardiovascular disease. Inflamm Res 58:537–552
Gautam R, Jachak SM (2009) Recent developments in anti-
inflammatory natural products. Med Res Rev 29:767–820
Glide (2013) Version 6.1. Schrödinger, LLC, New York
Granger DL, Taintor RR, Boockvar KS, Hibbs Jr. JB (1996) Mea-
surement of nitrate and nitrite in biological samples using nitrate
reductase and griess reaction. Methods Enzymol 268:142–151
Ha SK, Park HY, Eom H, Kim Y, Choi I (2012) Narirutin fraction
from citrus peels attenuates LPS-stimulated inflammatory
response through inhibition of NF-Band MAPKs activation. Food
Chem Toxicol 50:3498–3504
Heo SJ, Yoon WJ, Kim KN, Oh C, Choi YU, Yoon KT, Kang DH,
Qian ZJ, Choi IW, Jung WK (2012) Anti-inflammatory effect of
fucoxanthin derivatives isolated from Sargassum siliquastrum in
lipopolysaccharide-stimulated RAW 264.7 macrophage. Food
Chem Toxicol 50:3336–3342
Hu XD, Yang Y, Zhong XG, Zhang XH, Zhang YN, Zheng ZP, Zhou
Y, TangW, Wang YF, Hu LH, Zuo JP (2008) Anti-inflammatory
effects of Z23 on LPS-induced inflammatory responses in
RAW264.7 macrophages. J Ethnopharmacol 120:447–451
Impact (2013) Version 6.1. Schrödinger, LLC, New York
Kao TH, Huang CW, Chen BH (2012) Functional components in
Luffa cylindrica and their effects on anti-inflammation of mac-
rophage cells. Food Chem 135:386–395
Krishna PS, Vani K, Prasad MR, Samatha B, Bindu NS, Charya MA,
Reddy Shetty P (2013) In-silico molecular docking analysis of
prodigiosin and cycloprodigiosin as COX-2 inhibitors. Spring-
erplus 2:172
Kurumbail RG, Stevens AM, Gierse JK, McDonald JJ, Stegeman RA,
Pak JY, Gildehaus D, Miyashiro JM, Penning TD, Seibert K,
Isakson PC, Stallings WC (1996) Structural basis for selective
inhibition of cyclooxygenase-2 by anti-inflammatory agents.
Nature 19:644–648
Latypov RF, Liu DJ, Gunasekaran K, Harvey TS, Razinkov VI,
Raibekas AA, Lee CC, Lin CP, Lee YL, Wang GC, Cheng YC,
Liu HE (2008) Meisoindigo is a promising agent with in vitro and
in vivo activity against human acute myeloid leukemia. Leuk
Lymphoma 51:897–905
Lee SH, Soyoola E, Chanmugam P, Hart S, Sun W, Zhong H, Liou S,
Simmons D, Hwang D (1992) Selective expression of mitogen-
inducible cyclooxygenase in macrophages stimulated with lipo-
polysaccharide. J Biol Chem 267:25934–25938
Li YD, Frenz CM, Chen MH, Wang YR, Li FJ, Luo C, Liang N, Yang
H, Bohlin L, Wang CL (2011) Primary virtual and in vitro
bioassay screening of natural inhibitors from flavonoids against
COX2. Chin J Nat Med 9:156–160
LigPrep (2013) Version 2.8. Schrödinger, LLC, New York
Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression
data using real-time quantitative PCR and the 2(-Delta Delta C
(T)) method. Methods 25:402–408
Llorens O, Perez JJ, Palomer A, Mauleon D (2002) Differential
binding mode of diverse cyclooxygenase inhibitors. J Mol Graph
Model 20:359–371
MacMicking J, Xie QW, Nathan C (1997) Nitric oxide and macro-
phage function. Annu Rev Immunol 15:323–350
Maestro (2013) Version 9.6. Schrodinger, LLC, New York
Maestro-Desmond Interoperability Tools (2014) Version 3.8. Schrö-
dinger, New York
McGettigan P, Henry D (2011) Cardiovascular risk with non-steroidal
anti-inflammatory drugs: Systematic review of population-based
controlled observational studies. PLoS Med 8:e1001098
Medeiros R, Prediger RD, Passos GF, Pandolfo P, Duarte FS, Franco
JL, Dafre AL, Di Giunta G, Figueiredo CP, Takahashi RN,
Campos MM, Calixto JB (2007) Connecting TNF-alpha signaling
pathways to iNOS expression in a mouse model of Alzheimer’s
disease: relevance for the behavioral and synaptic deficits induced
by amyloid beta protein. J Neurosci 27:5394–5404
Miles EA, Zoubouli P, Calder PC, Phil D (2005) Differential anti-
inflammatory effects of phenolic compounds from extra virgin olive
oil identified in human whole blood cultures. Nutrition 21:389–394

Moller B, Villiger PM (2006) Inhibition of IL-1, IL-6, and TNF-alpha
in immune-mediated inflammatory diseases. Springer Semin
Immunopathol 27:391–408
Morgensen TH (2009) Pathogen recognition and inflammatory
signaling in innate immune defenses. Clin Microbiol Rev
22:240–273
Moro C, Palacios I, Lozano M, D’Arrigo M, Guillamon E, Villares A,
Martinez JA, Garcia-Lafuente A (2012) Anti-inflammatory
activity of methanolic extracts from edible mushrooms in LPS
activated RAW 264.7 macrophages. Food Chem 130:350–355
Mosmann T (1983) Rapid colorimetric assay for cellular growth
and survival: application to proliferation and cytotoxicity assays.
J Immunol Methods 65:55–63
Nam NH (2006) Naturally occurring NF-kappaB inhibitors. Mini Rev
Med Chem 6:945–951
Nathan C (1992) Nitric oxide as a secretory product of mammalian
cells. FASEB J 6:3051–3064
Oh JH, Lee TJ, Park JW, Kwon TK (2008) Withaferin A inhibits iNOS
expression and nitric oxide production by Akt inactivation and
down-regulating LPS-induced activity of NF-kappaB in RAW
264.7 cells. Eur J Pharmacol 599:11–17
Prime (2013) Version 3.4. Schrodinger, LLC, New York
Priscilla D, Manoj Kumar G, Urmila J, Preetam S (2011) Modeling of Cox-
2 inhibitory activity of flavonoids. Int J Pharm Pharm Sci 3:33–44
Reddy ST, Herschman HR (1994) Ligand-induced prostaglandin
synthase requires expression of the TIS10/PGS-2 prostaglandin
synthase gene in murine fibroblast and macrophages. J Biol Chem
269:473–480
Rossi A, Ligresti A, Longo R, Russo A, Borrelli F, Sautebin L (2002)
The inhibitory effect of propolis and caffeic acid phenethyl ester
on cyclooxygenase activity in J774 macrophages. Phytomedicine
9:530–535
Sarkar D, Saha P, Gamre S, Bhattacharjee S, Hariharan C, Ganguly S,
Sen R, Mandal G, Chattopadhyay S, Majumdar S, Chatterjee M
(2008) Anti-inflammatory effect of allylpyrocatechol in LPS-
induced macrophages is mediated by suppression of iNOS and
COX-2 via the NF-kappaB pathway. Int Immunopharmacol
8:1264–1271
Schrodinger Suite (2013) Protein Preparation Wizard, Epik (2013)
Version 2.6. Schrödinger, LLC, New York
Shen SC, Lee WR, Lin HY, Huang HC, Ko CH, Yang LL, Chen YC
(2002) In vitro and in vivo inhibitory activities of rutin, wogonin,
and quercetin onlipopolysaccharide-induced nitric oxide and
prostaglandin E2 production. Eur J Pharmacol 446:187–194
Sudha A, Srinivasan P (2014) Bioassay-guided isolation and anti-
oxidant evaluation of flavonoid compound from aerial parts of
Lippia nodiflora L. BioMed Res Int. doi:10.1155/2014/549836
Med Chem Res
Sudha A, Srinivasan P (2015) In vitro, fluorescence-quenching and
computational studies on the interaction between lipoxygenase
and 5-hydroxy-3′,4′,7-trimethoxyflavone from Lippia nodiflora
L. J Recept Sig Transd 35:569–577
Syam S, Bustamam A, Abdullah R, Sukari MA, Hashim NM, Mohan
S, Looi CY, Wong WF, Yahayu MA, Abdelwahab SI (2014)
β Mangostin suppress LPS-induced inflammatory response in
RAW 264.7 macrophages in vitro and carrageenan-induced
peritonitis in vivo. J Ethnopharmacol 153:435–445
Takaki H, Koqanemaru R, Iwakawa Y, Hiquchi R, Miyamoto T
(2003) Inhibitory effect of norditerpenes on LPS-induced TNF-a
production from the Okinawan soft coral, Sinularia sp. Biol
Pharma Bull 26:380–382
Thuresson ED, Lakkides KM, Rieke CJ, Sun Y, Wingerd BA, Micielli
R, Mulichak AM, Malkowski MG, Garavito RM, Smith WL
(2001) Prostaglandin endoperoxide H synthase-1: the functions of
cyclooxygenase active site residues in the binding, positioning,
and oxygenation of arachidonic acid. J Biol Chem
276:10347–10357
Tiperciuc B, Parvu A, Tamaian R, Nastasa C, Ionut I, Oniga O (2013)
New anti-inflammatory thiazolyl-carbonyl-thiosemicarbazides
and thiazolyl-azoles with antioxidant properties as potential
iNOS inhibitors. Arch Pharm Res 36:702–714
Vane JR, Bakhle YS, Botting RM (1998) Cyclooxygenase 1 and 2.
Annu Rev Pharmacol Toxicol 38:97–120
Vogl S, Picker P, Mihaly-Bison J, Fakhrudin N, Atanasov AG, Heiss
EH, Wawrosch C, Reznicek G, Dirsch VM, Saukel J, Kopp B
(2013) Ethnopharmacological in vitro studies on Austria’s folk
medicine, an unexplored in vitro anti-inflammatory activities of
71 Austrian traditional herbal drugs. J Ethnopharmacol
149:750–771
Wu LC, Fan NC, Lin MH, Chu IR, Huang SJ, Hu CY, Han SY (2008)
Anti-inflammatory effect of spilanthol from Spilanthes acmella
on murine macrophage by down-regulating LPS-induced
inflammatory mediators. J Agric Food Chem 56:2341–2349
Zhang XY, Tao JY, Zhao L, Huang ZJ, Xiong FL, Zhang SL, Li CM,
Xiao F (2007) In vitro anti-inflammatory effects of different
solution fractions of ethanol extract from Melilotus suaveolens
Ledeb. Chin Med J (Engl) 120:1992–1998
Zhang H, Zan J, Yu G, Jiang M, Liu P (2012) A combination of 3D-
QSAR, molecular docking and molecular dynamics simulation
studies of benzimidazole-quinolinone derivatives as iNOS inhi-
bitors. Int J Mol Sci 13:11210–11227
Zhong Y, Chiou YS, Pan MH, Shahidi F (2012) Anti-inflammatory
activity of lipophilic epigallocatechin gallate (EGCG) deriva-
tives in LPS-stimulated murine macrophages. Food Chem
134:742–748