Advances in Biological Chemistry, 2013, 3, 541-548 ABC Published Online December 2013 (
Molecular basis of the anti-inflammatory potential of
a diarylheptanoid in murine macrophage RAW 264.7 cells
Bharathi Raja Rajaganapathy1, Karthikeyan Thirugnanam1, Muthusamy Velusamy Shanmuganathan1,2,
Anand Singaravelu1, Lakshmi Baddireddi Subadhra1
1Tissue Culture and Drug Discovery Lab, Centre for Biotechnology, Anna University, Chennai, India
2National Institute of Nutrition (Indian Council of Medical Research), Hyderabad, India
Email: *
Received 9 November 2013; revised 29 November 2013; accepted 5 December 2013
Copyright © 2013 Bharathi Raja Rajaganapathy et al. This is an open access article distributed under the Creative Commons Attribu-
tion License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
Natural products play a significant role in human
health in relation to the prevention and treatment of
inflammatory disorders. In this study, we examined
the molecular basis of the anti-inflammatory poten-
tial of a diarylheptonoid (DAH) isolated from Alp-
inia officinarum hexane extract (AOHE) with special
emphasis on their ability to modulate the nuclear
factor-ĸB (NF-кB) signaling involved in the inflam-
matory response. Measurement of Nitrite by Griess
reaction which revealed the effect of DAH in RAW
264.7 macrophages showed an inhibition in the nitric
oxide production through the suppression of induc-
ible nitric oxide synthase (iNOS) gene level expression.
NF-кB reporter gene assay suggests inhibition of NF-
кB transcriptional activity, thus inhibiting LPS-in-
duced phosphorylation and degradation of IкBα and
a downregulation of NF-кB protein expression con-
firms the immunomodulatory effect of DAH. Further-
more, downregulation in the gene level expression of
NF-кB signaling markers such as IL-1β, TNF-α and
COX-2 suggests the anti-inflammatory potential of
DAH via inhibition of NF-кB activation.
Keywords: Diarylheptanoid; NF-кB;
Lipopolysaccharide; TNF-α; COX-2; iNOS; Cytokines
Macrophages, vital components of the innate immune
system, perform a major role in acute inflammatory re-
sponses. Lipopolysaccharide (LPS) stimulated macro-
phages generate a variety of inflammatory mediators,
such as nitric oxide (NO), prostaglandin E2 (PGE2), in-
terleukin 1β (IL-1β) and tumor necrosis factor alpha
(TNF-α) [1]. NO generation plays a critical role in a va-
riety of pathophysiological conditions, including in-
flammation and carcinogenesis [2]. iNOS, an isoform of
NO synthase, is expressed in the response to a variety of
inflammatory stimuli and generates high levels of NO in
macrophages during the inflammatory condition [3].
Therefore, NO production might be reflective of the in-
flammation process and may provide a measure to assess
the effect of anti-inflammatory drugs.
NF-кB is one of the most ubiquitous transcription fac-
tors and regulates the genes involved in cellular prolif-
eration, inflammatory responses and cell adhesion. Func-
tionally, active NF-кB exists mainly as a heterodimer
comprised of subunits of the Rel family p50 and p65,
which is normally sequestered in the cytosol as an inac-
tive complex due to its binding with inhibitors of кB
(IкBs) in unstimulated cells [4]. The activation of NF-кB
involves in the phosphorylation of IкBs at two critical
serine residues (Ser 32, Ser 36) via the IкB kinase (IKK)
signalosome complex [5]. Once IкBs have been phos-
phorylated, they are ubiquitinated and degraded by 26S
proteosome. The resulting free NF-кB is then translo-
cated to the nucleus, where it binds to кB-binding sites in
the promoter regions of target genes and induces the
transcription of proinflammatory mediators [6]. The in-
appropriate activation of NF-кB and the systemic in-
flammatory response has been attributed to TNF-α and
its superfamily members such as IL-6 and IL-8 [7]. TNF-
α plays a key role in induction and perpetuation of in-
flammation due to autoimmune reactions by activating T
cells and macrophages by up-regulating the other proin-
flammatory cytokines [8]. iNOS and COX-2 expression
are also regulated by NF-кB and COX2 produces large
quantities of pro-inflammatory PGs at the inflammatory
site and formed PGs mediate pain and inflammation [9].
*Corresponding author.
B. R. Rajaganapathy et al. / Advances in Biological Chemistry 3 (2013) 541-548
Thus inhibitions in the overproduction of NO production
in macrophages through inhibiting iNOS and COX-2
expressions or their activities may have therapeutic po-
tential in the development of anti-inflammatory drugs
[10]. LPS-stimulated macrophages also activate several
intracellular signaling pathways, including the NF-ĸB
pathway, mitogen-activated protein kinase (MAPK) path-
ways and c-Jun N-terminal kinase (JNK) [11].
Alpinia ofcinarum is a traditional medicinal plant
used for treating inammatory and gastrointestinal dis-
orders. It belongs to the Zingiberaceae family, which
includes other important medicinal plants such as Cur-
cuma longa and Zingiber ofcinale with well-documented
medicinal properties. Diarylheptanoids from Alpinia of-
cinarum are phenolic compounds that exhibit anti-in-
ammatory, anti-cancer and anti-bacterial properties [12].
Herein, we report an alternative approach to the devel-
opment of novel therapeutics based on the endogenous
mediators and mechanisms that switch off inflammation.
Therefore, we have evaluated the anti-inflammatory ef-
fects of DAH isolated from the rhizome part of Alpinia
officinarum hexane extract on the production of NO in
LPS-induced murine RAW 264.7 macrophages. Our re-
sults demonstrate that DAH inhibits the production of
NO and downregulates the inflammatory mediators through
inhibition of NF-ĸB transactivation.
2.1. Cell Culture
The murine macrophage cell line RAW 264.7 were pur-
chased from NCCS, Pune, India and cultured in Dul-
becco’s modified Eagle’s medium (DMEM, GIBCO Inc,
NY, USA) 10% Fetal bovine serum (FBS) and RPMI
1640 medium containing 10% FBS supplemented with
penicillin (120 units/mL), streptomycin (75 g/mL),
gentamycin (160 g/mL) and amphotericin B (3 g/mL).
Cells were maintained in a humidified atmosphere with
5% CO2 at 37˚C and were sub-cultured in every three
2.2. Extraction, Fractionation, Structural
An authenticated, dried, pulverized rhizome powder of
Alpinia officinarum (100 g) was subjected to sequential
extraction using organic solvents of increasing polarity
such as hexane, ethyl acetate and methanol by Soxhlet
extraction technique. The extracts obtained were design-
nated as Alpinia officinarum hexane extract (AOHE),
Alpinia officinaru methyl acetate extract (AOEAE) and
Alpinia officinarum methanol extract (AOME). The ex-
tracts were then subjected to TLC analysis using hexane:
ethyl acetate (8:2) as mobile phase. Based on the preli-
minary results AOHE (5 g) was subjected to solvent-
solvent fractionation by dissolving it in 70% methanol.
The soluble portion of AOHE in 70% methanol was par-
titioned with hexane and chloroform. Based on the anti-
inflammatory activity, the hexane soluble fraction was
selected for column purification which was performed
using a gradient mixture of hexane: ethyl acetate as the
mobile phase using silica gel (60-120 mesh size) as ad-
sorbent. As a result of column fractionation, sub fractions
were collected and based on the anti-inflammatory activity,
sub fraction 3 on further purification yielded a pure mo-
The mass spectra confirmed the elemental composi-
tion of the compound. The structure of the active mole-
cule was determined by 1H, 13C nuclear magnetic reso-
nance spectroscopy. The structural characterization de-
tails are as follows: 1H NMR (CDCl3, 500 MHz): d 2.50
(2H, H-6), 2.74 (H-7), 2.78 (2H, H-2), 2.82 (2H, H-1),
4.58 (br s, 1H, OH), 6.08 (1H,H-4), 6.72 (2H, H-3’,
H-5’), 6.81 (1H, H-5), 7.02 (d, H-2’ , H-6’), 7.15 (d,
2H,H-2’’, H-6’’), 7.19 (br, 1H, H-4’’), 7.27 (br, 2H,
H-3’’ , H-5’’); 13C NMR (CDCl3, 100 MHz): d 29.2
(C-1), 34.0 (C-6), 34.3 (C-7), 41.9 (C-2), 115.2 (C-3’,
C-5’), 126.1 (C-4’’), 128.2 (C-2’’, C-6’’), 128.4 (C-3’’,
C-5’’), 129.4 (C-2’, C-6’), 130.6 (C-4),133.3 (C-1’),
140.6 (C-1’’), 146.2 (C-5), 153.8 (C-4’), 199.6 (C-3);
ESMS (+ve): [M+H]+; calculated for C19H20O2+H,
280.36. The mass spectra confirmed the elemental com-
position of the compound.
Quantification of the isolated active molecule was
done by HPTLC: The stock solution of AOHE (100
mg/mL) was used for TLC identification/quantification.
10 mg of active moleculewas weighed and dissolved in
10 mL of methanol. 0.1 mg/ml solution was prepared by
serial dilution from the above stock solution of active
molecule standard (1 mg/ml) for TLC quantification. The
following chromatographic conditions were applied. Sta-
tionary phase: Silica gel GF254; Mobile phase: n-Hexane:
Ethyl acetate (8: 2 v/v); Chamber saturation time: 1 hr;
Instrument: HPTLC (Camag–version 1.3.4); Applicator:
Linomat V; Scanner: Camag TLC scanner III; Develop-
ing chamber: Twin trough glass chamber (20 X 10 cm);
Developing mode: Ascending mode (multiple develop-
ment); Detection scanning wavelength: 280 nm; Experi-
mental condition: 25 + 28˚C; Temp/RH: 55% - 65%. The
limit of detection (LOD) and limit of quantification
(LOQ) were estimated using the active compound. Quan-
titative analysis of AOHE was chromatographed and
scanned at 546 nm. The amount of active molecule pre-
sent in AOHE was calculated by comparing the absorp-
tion units (AU) of standard active molecule.
2.3. Measurement of Nitrite by Griess Reaction
RAW 264.7 cells were seeded at 5 x 105 cells/well in 24-
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B. R. Rajaganapathy et al. / Advances in Biological Chemistry 3 (2013) 541-548 543
well plates and incubated with or without LPS (1 μg/mL)
and with various concentrations (5, 10, 25, 50 and 100
μg/mL) of AOHE and DAH for 24h. Nitrite levelswere
determined using the Griess reaction [13]. Briefly, 100 μl
of cell culture medium was mixed with 100μl ofGriess
reagent (1% sulfanilamide and 0.1% NEDD in 2.5% or-
tho phosphoric acid) and incubated at room temperature
for 10 min. The nitric oxide concentration was estimated
using standard curve plotted against known quantity of
sodium nitrite. Results were expressed in μM obtained
from the mean OD of triplicate wells. The standard curve
was plotted using different concentrations of sodium
nitrite (1 to 100 μM) with absorbance of 570 nm.
2.4. Isolation of PBMC
Heparinised venous blood was taken from 3 healthy hu-
uman volunteers with their mutual consent. Mononuclear
cells were isolated in a Ficoll-Hypaque (Pharmacia, Pis-
cataway, NJ density gradient using standard procedures
which separated PBMCs from whole blood. The buffy
coat containing PBMCs was removed carefully follow-
ing centrifugation and washed twice in RPMI 1640 me-
dium containing 10% FCS. Cells were counted and as-
sessed for viability using MTT assay.
2.5. Assessment of Cytotoxicity
Cell viability was measured with the conventional MTT-
reduction assay. Briefly, PBMC cells were seeded in
96-well plates in 200 μL of RPMI with 10% FBS. Then
the culture supernatant was removed and replaced with
phenol red free RPMI containing various concentrations
(5 - 100 µg/mL) of AOHE and DAHwhich was incu-
bated for 24 h. Treatment of 10 ul Triton-x-100/ well,
served as positive control for cytotoxicity. After the in-
cubation time, MTT reagents were added and incubate
for 4 h. After incubation add 100 μL of DMSO and read
the plates at 490 nm on a scanning multi-well spectro-
photometer and expressed as % of vehicle treated control
2.6. Western Blot Analysis
RAW 264.7 cells were incubated with or without LPS (1
μg/mL) and with the optimized dose of AOHE and DAH,
the cells were collected and washed twice with ice cold
PBS (phosphate buffered Saline). The lysis of cells were
done in a lysis buffer [50 mMTris-HCl (pH 7.5), 150
mMNaCl, 1% Nonidet P- 40, 2 mMEDTA, 1 mM EGTA,
1 mM NaVO3, 10 mMNaF, 1 mMdithiothreitol, 1 Mm
phenylmethylsulfonyl fluoride, 25 μg/mL aprotinin, 25
μg/mL leupeptin] and kept on ice for 30 min. The cell
lysates were centrifuged at 12,000 rpm at 4˚C for 30 min
and the supernatant were stored at 70˚C. Protein con-
centration was measured by Bradford’s method. Aliquots
of the lysates (100 μg of protein) were separated on a
10% SDS-polyacrylamide gel and transferred onto a ni-
trocellulose membrane with a glycine transfer buffer
[192 mM glycine, 25 mMTris- HCl (pH 8.8), 20%
MeOH (v/v)]. After blocking the nonspecific site with
5% nonfat dried milk with PBS, the membrane was in-
cubated with specific primary and secondary antibody.
The chromogenic substrate NBT/BCIP was used for the
band development.
2.7. Transient Transfection and Luciferase
RAW 264.7 cells were seeded into 24-well plates (5 x
105 cells/well) and allowed to grow up to 50% - 70% con-
fluence. The pNF-кB-luciferase plasmid was transfected
with Lipofectamine Plus TM reagent (Invitrogen, Carls-
bad, CA, USA) in accordance with the manufacturer’s
instructions. The cells were pretreated for 1 h with vari-
ous concentrations of AOHE and DAH and were stimu-
lated for 6 h with 20 ngof TNF-α. The cells were then
collected and disrupted via sonication in lysis buffer (25
mMTris–phosphate, pH 7.8, 2 mM EDTA, 1% Triton X-
100, and 10% glycerol) and the cell lysates were assayed
for luciferase activity with a luminometer (Promega) in
accordance with the manufacturer’s instructions.
2.8. Isolation of Total RNA and
Semi-Quantitative Reverse
Transcription-Polymerase Chain Reaction
RAW 264.7 cells were incubated with or without LPS (1
μg/mL) and with the optimized dose of AOHE and DAH
incubated for 24 h. Cells were homogenized using TRI-
zol® reagent and RNA was isolated by phenol-chloro-
form extraction. The aqueous phase containing RNA was
then precipitated by adding an equal volume of isopropyl
alcohol. The RNA obtained was then converted to cDNA
by reverse transcription using MMLV reverse transcript-
tase enzyme and subjected to PCR with specific primers
(Table 1). PCR products were run on 1.2% agarose gels,
stained with ethidium bromide and photographed. PCR
products were consistent with the predicted sizes and
analysed on ethidium bromide-stained agarose gels.
2.9. Statistical Analysis
Statistical analysis was performed using GraphPad Prism,
4.03 (San Diego). One way analysis of variance (ANO-
VA) followed by Dunnett’s post hoc was used for the
other parameters. The data are expressed as mean ±
S.E.M and P < 0.05 was considered to be statistically
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B. R. Rajaganapathy et al. / Advances in Biological Chemistry 3 (2013) 541-548
Table 1. List of gene sequences and the predicted product size
Primer Sequence
size (bp)
3.1. Bioassay Guided Fractionation, Purification
and Quantification of DAH in AOHE
The rhizome of Alpinia officinarum was sequentially ex-
tracted successively using hexane, ethyl acetate and
methanol. The extraction yield was found to be 0.224%
w/w, 0.512% w/w, 0.28% w/w respectively. After solvent-
solvent fractionation and based on the anti-inflammatory
activity, the hexane soluble fraction was subjected to
column chromatography leading to the isolation of a pure
molecule. Structural characterization using NMR and
mass spectral analysis led to the identification of the
major chemical constituent from the active fraction to
be1-(4-Hydroxyphenyl)-7-phenyl-hept-4-en-3-one (DAH)
with the molecular formula C19 H20 O2 (molecular weight
- 280.36) (Figure 1). The characteristic color of the com-
pound is yellow and was observed to be oily in nature.
The chromatographic analysis of AOHE and isolated
pure molecule (DAH) was carried out at 546 nm and
photographed. The purity of the DAH isolated from
AOHE was confirmed by comparing the UV absorption
spectra at the start, middle, and end position of the bands.
The identity of the bands of DAH in the AOHE was con-
firmed by overlaying their UV absorption spectra with
those of the isolated DAH. The amount of DAH present
in AOHE was found to be 0.81 %w/w (dry wt.basis).
3.2. Effect of AOHE and DAH on Nitrite
Nitric oxide release was assessed in RAW 264.7 macro-
phages to check the effect of AOHE and DAH on pro
inflammatory mediators. The cells were stimulated with
LPS (1 μg/mL) and treated with different concentrations
of AOHE and DAH ranges from 5 µg/mL to100 µg/mL
to check the anti-inflammatory effect. The results sug-
gested that AOHE and DAH showed a significant de-
crease in the nitric oxide production when compared to
LPS treated control cells at 24 h. The IC50 of AOHE was
found to be 25 µg/mL and for DAH 10 µg/mL respec-
tively (Figure 2). The IC50 values were used for the fur-
ther molecular mechanism studies.
3.3. Cytotoxicity Effect of AOHE and DAH
LDH release was measured quantitatively in PBMC at 24
h using a cytotox 96 assay kit and the results are ex-
pressed as % cytotoxicity with respect to control (un-
treated cells) and Triton-X-100 was used as positive con-
trol. Treatment of AOHE and DAH with RAW264.7 cells
at various concentrations showed a minimal LDH release
and the cytotoxicity was less than 20% at the highest
concentration tested. The results clearly showed that
AOHE and DAH have no toxic effects to RAW 264.7
cells (Figure 3).
3.4. Effect of AOHE and DAH on NF-κB
Inhibition and IκBα Degradation
To investigate whether AOHE and DAH could affect the
nuclear translocation of NF-кB, western blot analysis
was conducted to check the NF-кB expression and IкBα
degradation in RAW264.7 cell lysates. The amount of
NF-кB p65 was markedly increased upon exposure to
LPS treated cells, whereas AOHE and DAH inhibited
NF-кB activation by downregulating the protein level
expression of NF-кB (Figure 4). Furthermore, AOHE
and DAH significantly upregulated IкBα expression sug-
gests that inhibition of NF-кB activation by IкBα degra-
3.5. Effect of AOHE and DAH on NF-кB
Transcriptional Activity in RAW 264.7
To understand the effect of AOHE and DAH on NF-кB
transcriptional activity, RAW264.7 cells were transiently
transfected with a plasmid harboring four tandem copies
of the NF-кB consensus sequence linked to the luciferase
gene. Luciferase assays were performed by using the
Dual-Luciferase Reporter System (Promega), in which
relative luciferase activities were calculated by normal-
izing transfection efficiency according to the Renilla
luciferase activitiy. NF-кB transcriptional activity was
measured as luciferase activity with a luminometer. Inhi-
bition in the TNF-α(20 ng)induced promoter activation of
NF-кB confirms the potential inhibitory effect of AOHE
and DAH by showing an inhibition in the reporter activity
when compared with TNF-α (6 h) exposed cells (Figure
5). These results indicate that AOHE and DAH inhibits
NF-кB transactivation which reflects in the down regula-
tion of the pro-inflammatory mediators.
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B. R. Rajaganapathy et al. / Advances in Biological Chemistry 3 (2013) 541-548 545
Figure 1. Structure of the bioactive compound chemically
characterized as 1-(4 Hydroxyphenyl)-7-phenyl-hept-4-en-3-
one with molecular formula C19 H
20 O
2 (molecular weight:
280.36) and identified as Diarylheptanoid (DAH).
Figure 2. Dose response analysis of AOHE and DAH using
Griess reaction. Cells were stimulated with 1 µg/mL of LPS
and after 24 hours incubation the effect of AOHE and DAH on
NO production was evaluated. Nitrite concentrations were re-
duced in a dose response manner and are expressed in μM. The
results are mean ± SEM of 3 independent experiments.
Figure 3. Cytotoxicity profiling on PBMC. Cells were incu-
bated with the IC50 value of AOHE and DAH for 24 h. Cyto-
toxicity was assessed based on the lactate dehydrogenase re-
leased into the supernatant and measured at 490 nm. Triton-
x-100 served as the positive control. LDH was measured by
using the formula: % LDH release = (OD Sample – OD con-
trol)/(OD TritonX-OD control) × 100. Cytotoxicity was ex-
pressed as % of LDH release. Data presented as mean ± SEM
from three separate experiments for each data point.
Figure 4. Analysis of AOHE and DAH on LPS induced IкBα
degradation and NF-кB p65 inhibition in RAW264.7 macro-
phages (Lane 1-LPS control, lane 2-Control, Lane 3-AOHE,
Lane 4-DAH). The densitometric band intensities have been
represented as normalized IDV values.
Figure 5. TNFα-induced NF-кB-dependent reporter gene ex-
pression was observed to be inhibited by AOHE and DAH.
RAW264.7 cells were transiently transfected with NF-кB-con-
taining plasmid and treated with AOHE and DAH for 12 h.
Thereafter, cells were activated for NF-кB activation using 20
ng of TNFα as indicated for 6 h. After 24 h the cell lysate was
collected and assayed. Results are expressed as fold activity of
the vector control activity. Data presented as mean ± SEM from
three separate experiments for each data point.
3.6. Inhibitory Effect of AOHE and DAH on
Pro-Inflammatory Cytokines
The release of pro-inflammatory cytokines is an important
mechanism by which the immune cells regulate the in-
flammatory responses and contribute to various inflam-
matory and autoimmune disorders. Hence we checked the
ability of the AOHE and DAH in LPS induced TNFα and
IL-1β gene level expression in RAW 264.7 cells. The
results suggest, that TNFα and IL-1β showed a decrease
in the gene expression after treatment with AOHE and
DAH when compared to the LPS induced control cells.
Generally, COX-2 was hardly detectable in resting mac-
rophages, but pronounced amounts of COX-2 production
can be attained when cells were induced upon exposure to
LPS. Incubation with AOHE and DAH showed down-
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B. R. Rajaganapathy et al. / Advances in Biological Chemistry 3 (2013) 541-548
Copyright © 2013 SciRes.
regulation in the gene level expression of LPS induced
COX-2 synthesis. Nitric oxide production is directly
proportional to the expression of iNOS and we observed a
downregulation in the gene level expression of iNOS after
the cells were exposed with AOHE and DAH confirms the
potential of AOHE and DAH which can regulates the
pro-inflammatory mediators in response to inflammatory
signals (Figure 6).
The inflammatory process as a sequence of events occurs
in response to noxious stimuli, trauma or infection which
is orchestrated by a highly modulated interaction be-
tween mediators of inflammation and inflammatory cells
[15]. A large number of compounds of varied chemical
structures isolated from medicinal plants had been shown
to possess anti-inflammatory activity and modulates
most of the inflammatory signaling. Although the etiol-
ogy of most chronic inflammatory diseases remains un-
known, the initiation of certain types of chronic in-
flammation has been associated with development of
autoimmune response, which progresses to a sustained,
self-perpetuated inflammation [16]. Various anti-inflam-
matory drugs, including antioxidants, glucocorticoids,
non-steroidal anti-inflammatory drugs (NSAIDs), im-
munosuppressants and various plant compounds act as
inhibitors of the NF-ĸB pathway suggesting that sup-
pression of NF-ĸB dependent transcription is an essential
part of their anti-inflammatory activity [17].
Experimental evidences suggest that flavonoids have
been reported to be potentially inhibits or regulate the
activation of transcription factors including NF-κB, both
at the stage of initiation and perpetuation of chronic in-
flammation [18]. Curcumin, a dietary supplement with
potent anti-inflammatory and anti-tumor activities, inhib-
its NF-ĸB activation by acting on upstream pathways
controlling IKK activation [19]. In this study, DAH we
have isolated from Alpinia officinarum structurally re-
sembles as curcumin analog inhibits the transcriptional
activeity of NF-ĸB and its mediators which confirms
DAH as a potential anti-inflammatory agent.
NO is a signaling molecule involved in a broad spec-
trum of pathophysiological processes such as inflamma-
tion, apoptosis, the regulation of enzyme activity and
gene expression. High levels of NO are generated in re-
sponse to inflammatory stimuli and mediate pro-in-
flammatory effects. Therefore, NO production may re-
flect the process of inflammation and may provide a
measure for assessing the effects of drugs on the in-
flammatory process [20]. In this study, we have induced
the cells with LPS (1µg/mL) and treated with different
concentration of AOHE and DAH. We have observed a
significant decrease in the NO production at 24 h when
compared with the LPS treated control cells and the IC50
value is found to be 10 µg/mL for AOHE and for DAH 5
µg/mL respectively. The cytotoxicity data suggests that
AOHE and DAH possessed no cytotoxicity effect at the
maximum concentration AOHE and DAH tested in
RAW264.7 macrophages. Further, the molecular level
studies were conducted with the IC50 values of AOHE
and DAH.
NF-κB, a major transcription factor which regulates
the expression of inflammation induced enzymes and
cytokines and has attracted as a new target for treating
Figure 6. Effect of AOHE and DAH on IL-1β, TNF-α, COX-2 and iNOS gene expression in RAW264.7 cells at 24 h. Lane 1- DNA
ladder (100 bp), Lane 2- Control, Lane 3- LPS Control, Lane 4- AOHE, Lane 5- DAH. The graph shows the IDV values ratio of den-
sity of the genes expression to that of endogenous control GAPDH and represents mean±SEM of three replicates when compared to
untreated control.
B. R. Rajaganapathy et al. / Advances in Biological Chemistry 3 (2013) 541-548 547
inflammatory diseases [21]. Inactive NF-кB is localized
in the cytoplasm by the inhibitor of кB (IкB) and gets
stimulated with LPS, TNF-α or cellular stress. Once IкB
is degraded, NF-кB will be translocated from the cytosol
to the nucleus, and binds to its cognate DNA binding
which activates several intracellular signaling markers
Therefore, the suitable regulation of NF-κB may be
beneficial in treating many inflammatory disorders [22].
The present study demonstrates the potential of AOHE
and DAH in the LPS-induced phosphorylation and deg-
radation of IκB and the activation and translocation of
NF-κB p65 in LPS-stimulated RAW264.7 cells. The
western blot analysis results revealed the overexpression
of IкBα and downregulation of NF-κB confirms AOHE
and DAH exhibits anti-inflammatory effect. Evidence for
the inhibition of NF-κB signaling by AOHE and DAH in
distinct mechanisms can be determined directly using a
reporter gene assay. Through this concerted mechanism,
AOHE and DAH blocked TNF-α induced NF-κB de-
pendent transcription in RAW264.7 cells. The cells
transfected with a plasmid containing the NF-κB luci-
ferase coding region showed an inhibitory effect on
RAW264.7 cells, indicates that NF-κB transactivation
was suppressed when compared with the control which
clearly shows that AOHE and DAH inhibited TNF-α
induced NF-кB transactivation.
Pro-inflammatory cytokines such as IL-1β, TNFα, and
IL-6 were involved in amplification and initiation of the
inflammatory process. Stimulating the cells with LPS
leads to a cascade of intracellular signaling events that
ultimately result in production and secretion of cytokines
and other inflammatory mediators that constitute the
pro-inflammatory response [23]. Hence, treatment with
AOHE and DAH to LPS stimulated RAW 264.7 cells
showed downregulation in the expression of TNFα and
IL-1β. TNFα and IL-1β also have been shown to directly
regulate NO release through iNOS gene expression. Thus
the inhibition of these pro-inflammatory mediators has
proven to have tremendous therapeutic value of DAH.
Studies revealed that nitric oxide appears to mediate or
augment the synthesis of TNFα, IL-1β and chemokines
and a reduced gene level expression of IL-1β have been
observed after inhibition of iNOS activation [24]. iNOS
is expressed in response to a variety of inflammatory
stimuli and generates high levels of NO in macrophages
during the inflammatory process which was regulated by
NF-κB [25]. Our results revealed that LPS induced re-
ducetion in the NO production and downregulation of
iNOS gene expression confirms the NF-κB inhibition by
AOHE and DAH. COX-2, the rate-limiting enzyme in
prostaglandin synthesis, is induced in many cells by in-
flammatory mediators [26]. Similarly observations made
on AOHE and DAH treated cells demonstrated the anti-
inflammatory effect of AOHE and DAH was contributed
by the down regulation of COX-2 expression.
This study revealed the anti-inflammatory activity of
AOHE and DAH inhibited the LPS-induced expression
of TNF-α, IL-1β, iNOS and COX-2 at gene level in
RAW 264.7 cells. These suppressive effects of AOHE
and DAH are mediated by inhibiting the NF-κB tran-
scriptional activity. Thus, inhibition of the overproduc-
tion of NO and inhibition of NF-κB transcriptional activ-
ity of DAH can have a therapeutic potential in the de-
velopment of anti-inflammatory drug against inflamma-
tory diseases.
The work was supported financially by the National Medicinal Plants
Board, Department of Ayurvedha, Yoga & Naturopathy, Unani, Siddha
and Homeopathy (AYUSH), Government of India (Grant no: GO/TN-
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