Journal of Ethnopharmacology 129 (2010) 203207

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Journal of Ethnopharmacology
j our nal homepage: www. el sevi er . com/ l ocat e/ j et hphar m
Effect of an extract of Andrographis paniculata leaves on inammatory and
allergic mediators in vitro
C.V. Chandrasekaran

, Anumita Gupta, Amit Agarwal

Department of Cellular Assay, R&D Centre, Natural Remedies Pvt. Ltd., Bangalore, India
a r t i c l e i n f o
Article history:
Received 10 October 2009
Received in revised form 8 February 2010
Accepted 13 March 2010
Available online 20 March 2010
Keywords:
Andrographis paniculata
Pro-inammatory mediators (NO, IL-1 beta
and IL-6)
Inammatory mediators (PGE
2
and TXB
2
)
Allergic mediators (LTB
4
and histamine)
a b s t r a c t
Aim of study: Andrographis paniculata has been known to possess widespread traditional application in
the treatment of allergy and inammatory diseases. In the current study, we sought to examine the
effects of an extract of Andrographis paniculata leaves on inhibition of lipopolysaccharide (LPS) induced
[nitric oxide (NO), prostaglandin E
2
(PGE
2
), interleukin-1beta (IL-1 beta), and interleukin-6 (IL-6)] and
calcimycin (A23187) induced [leukotriene B
4
(LTB
4
), thromboxane B
2
(TXB
2
) and histamine] mediators
in diverse cell based models.
Materials andmethods: Effect of anextract of Andrographis paniculata leaves (AP) was studiedoninhibition
of LPS induced NO, PGE
2
, IL-1 beta and IL-6 in J774A.1 murine macrophages; A23187 induced LTB
4
and
TXB
2
in HL-60 promyelocytic leukemic cells and histamine in RBL-2H3 rat basophilic leukemia cells.
Results and conclusion: AP illustrated signicant alleviation of pro-inammatory, inammatory, and
allergic mediators. However, no inhibition was observed against histamine release. This outcome has
been summed up to deduce that AP is fairly potent in attenuating the inammation by inhibiting pro-
inammatory (NO, IL-1 beta and IL-6), inammatory (PGE
2
and TXB
2
) and allergic (LTB
4
) mediators.
2010 Elsevier Ireland Ltd. All rights reserved.
1. Introduction
Andrographis paniculata (Acanthaceae) is a well-known medici-
nal plant and has long beenused inChinese ofcial herbal medicine
against wide spectrum of ailments (Coon and Ernst, 2004). Our
recent study has reported the safety of Andrographis paniculata
(KalmCold
TM
) in a battery of genotoxic tests and also the LD
50
value was determined to be more than 5g/kg rat body weight in an
acute oral toxicitystudy(Chandrasekaranet al., 2009). Ina random-
ized double blind placebo controlled clinical trial, AP (KalmCold
TM
)
demonstrated signicant relief of common cold symptoms in
human populations (Saxena et al., 2010). Andrographis panicu-
lata has been reported to have anti-inammatory (Sheeja and
Kuttan, 2008), anti-allergic (Madav et al., 1998), immunostimula-
tory (Iruretagoyena et al., 2005) activity. The anti-inammatory
action of the plant is attributed to andrographolide, the major
active principle of the plant (Madav et al., 1996; Abu-Ghefreh
et al., 2009). However, little is known about the pharmacologi-
cal mechanisms underlying these actions, although some of its

Corresponding author at: Plot No. 5B, Veerasandra Indl. Area, 19th K.M. Stone,
Hosur Road, Bangalore 560100, Karnataka, India. Tel.: +91 80 40209999;
fax: +91 80 40209817.
E-mail addresses: cvc@naturalremedy.com, cvctox@gmail.com
(C.V. Chandrasekaran).
anti-inammatory effects have been investigated (Shen et al.,
2002).
In order to extend the understanding of its mechanism on anti-
inammatory activity, we investigated inhibitory activity of AP on
LPS induced NO, PGE
2
, IL-1 beta and IL-6 levels; A23187 activated
LTB
4
, TXB
2
, and histamine levels using mammalian cell lines in
vitro. We used murine macrophages (J774A.1) as cellular model,
since they represent population of macrophages that can be stim-
ulated in vitro by LPS. In the current study, we have utilized human
promyelocytic leukemic (HL-60) cells and rat basophilic leukemic
cells RBL-2H3 cells in order to study the anti-inammatory activity
of AP. These cell lines have been proved as an appropriate cell assay
system to study the anti-inammatory/anti-allergic nature of the
compounds (Ikawati et al., 2001; Zaitsu et al., 2002).
2. Materials and methods
2.1. Source of materials
Lipopolysaccharide (LPS), calcimycin (A23187), acetyl salicylic
acid, MTT [1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan],
ketotifen fumarate, dexamethasone (D-2915), 1400W dihy-
drochloride, sulphanilamide, naphthyl ethylene diamine
dihydrochloride (NEDD) and captopril were purchased from
SigmaAldrich, Inc. (St. Louis, MO, USA). Iscoves modied Dul-
0378-8741/$ see front matter 2010 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.jep.2010.03.007
204 C.V. Chandrasekaran et al. / Journal of Ethnopharmacology 129 (2010) 203207
beccos media (IMDM), Earles minimum essential media (EMEM)
and Dulbeccos modied Eagles medium (DMEM) were supplied
by Gibco Life Technologies (Grand Island, NY). Fetal bovine serum
(FBS) was purchased from Hyclone (Logan, USA).
2.2. Plant material
Andrographis paniculata (Burm.f.) Wall. ex. Nees (part used
leaves) belonging to the family Acanthaceae was collected from
regions of West Bengal, India. Botanical identication was car-
ried out at NISCAIR (National Institute of Science Communication
And Information Resources). A voucher specimen (No. 110) was
deposited in our herbarium.
2.3. Preparation of extract of Andrographis paniculata (AP)
Coarse ground leaves of Andrographis paniculata were extracted
with methanol (4 times of the raw material quantity) for 3h, in
a stainless steel jacketed extractor tted with reux condenser.
The liquid extract was removed and the remaining raw material
was re-extracted two more times with methanol in a similar man-
ner. The resulting extracts were combined, concentrated and dried
under vacuum (at <55

C). The yield of the dried extract was 6%

(w/w). After extraction with methanol, the left over raw material
was extracted with water (4 times of the rawmaterial quantity) for
3h under reux conditions. This water extract was separated and
concentrated under vacuum at around 75

C until the total solid

content in the liquid reached about 15% to 20% (w/v), followed by
spray drying. Two parts of methanolic and one part of successive
water extract were blended to get AP, which was analyzed for phy-
toconstituents by HPLC as described earlier by us (Chandrasekaran
et al., 2009).
AP was found to contain andrographolide (31.3%, w/w),
isoandrographolide (0.4%, w/w), neoandrographolide (3.2%, w/w),
andrograpanin (0.6%, w/w), 14-deoxy-11,12-didehydroandro-
grapholide (2.8%, w/w), skullcapavone-I (0.05%, w/w) and 7-O-
methylwogonin (0.05%, w/w). This herbal extract is indexed as
KalmCold
TM
, manufactured by Natural Remedies Pvt. Ltd., Banga-
lore.
AP was solubilized in DMSOand lter sterilized through 0.2M
positively charged nylon DMSO compatible lter to remove the
endotoxins. Thelteredsolutionwas aliquotedandstoredat 80

C
for further use in all the assays. DMSOwas used as a solvent control
in all the assays up to a maximum concentration of 0.2%. DMSO up
to 0.2% did not inuence the stimulant induced release of any of
the inammatory mediators. All the assays were well controlled by
using respective reference standards.
2.4. Cell lines and culture conditions
J774A.1 murine macrophage cell line (TIB-67
TM
), HL-60 human
promyelocytic leukemia cell line (CCL-240
TM
) and RBL-2H3 rat
basophilic leukemia cell line (CRL-2256
TM
) were procured from
American Type Culture Collection (ATCC) (Rockville, MD, USA). The
cells were cultured in appropriate ATCC recommended medium
and maintained at 37

C under 5% CO
2
humidied air.
2.5. Preparation of Ringers buffer
The components of Ringers buffer were 118mM NaCl, 4.6mM
KCl, 1.0mM CaCl
2
, 1.0mM KH
2
PO
4
, 1.10mM MgSO
4
, 24.9mM
NaHCO
3
, 5.0mM HEPES, 0.1% BSA and 11.1mM d-glucose. The pH
was adjusted to 7.4.
2.6. Cytotoxicity assay
Cytotoxic effect of AP was checked in the respective cell lines
by using MTT. Based on the cell viability results, different non-
cytotoxic concentrations were selected for each study. All the
experiments were conducted in quadruplicates per treatment
using 96-well tissue culture plates.
2.7. NO scavenging and PGE
2
inhibition assay
J774A.1 murine macrophages (passage number between 10
and 25) were seeded at a density of 110
5
cells/well and incu-
bated overnight. The macrophages were then pretreated with AP
at different non-cytotoxic concentrations (1.2530g/mL for NO
and 1.650g/mL for PGE
2
) and incubated for 1h, thereafter, LPS
(5g/mL) was added followed by further incubation for 24h. The
reference standards used for NO scavenging and PGE
2
inhibition
assay were 1400W dihydrochloride and dexamethasone respec-
tively. The cell supernatant was collected for nitrite and PGE
2
estimation.
Nitrite production, an indicator of NO synthesis, was measured
in the supernatant of cultured macrophages by Griess reaction.
Briey, equal volumes of treated culture supernatant and Griess
reagent (1% sulphanilamide, 0.1% NEDD and 5% orthophosphoric
acid) were mixed and incubated at room temperature for 5min,
and then the absorbance was measured at 540nm in a microplate
reader. The amount of nitrite in the sample was determined using
sodium nitrite standard curve.
PGE
2
levels in macrophage supernatants were quantied as per
the method described by the kit manufacturer [Homogenous Time
Resolved Fluorescence (HTRF) kit, CisBio, France].
2.8. IL-1 beta inhibition assay
J774A.1 murine macrophages were seeded at a density of
110
5
cells/well and incubated overnight. The macrophages were
then pretreated with AP at different non-cytotoxic concentrations
(540g/mL) and incubated for 1h, thereafter, LPS (5g/mL) was
added followed by further incubation for 6h. The treated cells were
lysedusing cell lysis buffer [0.1%TritonX-100 andprotease cocktail
inhibitor (1)] in combination with repeated freeze thaw cycles.
The plates were centrifuged and the supernatant was collected for
estimating the levels of IL-1 beta (ELISA kit, R&D Systems, Min-
neapolis, USA). Dexamethasone was used as a reference standard.
2.9. IL-6 inhibition assay
J774A.1 murine macrophages were seeded at a density of
110
5
cells/well and incubated overnight. The macrophages were
then pretreated with AP at different non-cytotoxic concentrations
(2.540g/mL) and incubated for 1h, thereafter, LPS (0.1g/mL)
was added followed by overnight incubation. The supernatant was
collected and used to estimate the levels of IL-6 as per the method
described by the kit manufacturer (ELISA kit, OptEIA
TM
, BD Bio-
sciences, USA). Dexamethasone was used as a reference standard.
2.10. LTB
4
and TXB
2
inhibition assay
HL-60 promyelocytic leukemia cells (passage number between
2 and 10) were rst allowed to undergo differentiation into
metamyelocytes and neutrophils, as this enabled the cells to pro-
duce higher levels of LTB
4
and TXB
2
. Cell differentiation was
attained by seeding the cells at a density of 510
5
cells/mL in
IMDM enriched with 20% FBS and 1.3% DMSO in a T-25 ask
followed by incubation for 5 days (Collins, 1987). The Giemsa
stained cells were examined under microscope to visualize the dif-
ferentiated granulocytes. These differentiated cells were seeded
at a density of 110
5
cells/well in Ringers buffer. The cells
were pretreated with AP at different non-cytotoxic concentrations
(1.2540g/mLfor LTB
4
and2.540g/mLfor TXB
2
) andincubated
C.V. Chandrasekaran et al. / Journal of Ethnopharmacology 129 (2010) 203207 205
for 1h, followed by stimulation with A23187 (5M) for 15min.
The reference standards used for LTB
4
and TXB
2
inhibition assay
were captopril and acetyl salicylic acid respectively. The aliquots
removed from the conditioned medium were used for the quan-
tication of LTB
4
(HTRF kit, CisBio, France) and TXB
2
(ELISA kit,
Sapphire Bioscience, Australia) levels according to the instructions
by the kit manufacturer.
2.11. Histamine release assay
The RBL-2H3 cells (passage number between 8 and 14) were
seeded at a density of 510
4
cells/well and incubated overnight.
Cells were then subjected to pre-treatment with AP at various
non-cytotoxic concentrations for 1h in Ringers buffer, thereafter,
A23187 (5M) was added, followed by further incubation for
30min. Ketotifenfumaratewas usedas areferencestandard. Super-
natant was collected from the conditioned medium and used for
quantication of histamine as directed by the manufacturer of the
kit (HTRF kit, CisBio, France).
2.12. Data analysis
Data are expressed as meanstandard error mean (S.E.M.) of
four replicates. The mean of the LPS/A23187 control was normal-
ized to 100% and mean of cell control was normalized to zero.
Thereafter, one-way ANOVA was performed on the results fol-
lowedby a Dunnetts test for multiple comparisons using GraphPad
Prism 5.0 (GraphPad Software, Inc., San Diego, CA) statistical soft-
ware package. The signicance level was chosen at P<0.050.01
for all statistical analyses. The half maximal inhibitory concentra-
tion (IC
50
) was calculated using median effect plot (Wolfe and Liu,
2007).
3. Results
3.1. Cytotoxicity of test substances
Cytotoxic effect of AP was assessed at different concentra-
tions by MTT assay in J774A.1, HL-60 and RBL-2H3 cells. AP did
not demonstrate any signicant toxicity up to a concentration of
50g/mL under our experimental conditions (data not shown).
Hereafter, the non-cytotoxic concentrations were chosen for the
further experiments.
3.2. Suppression of LPS induced NO production in cultured
macrophages
Effect of AP was determined in LPS (5g/mL) stimulated NO
in J774A.1 cells. AP showed a signicant dose-dependent decline
in the levels of NO at concentrations ranging from 10g/mL
to 30g/mL (Fig. 1A). The IC
50
value obtained was 20g/mL,
with a maximum inhibition of 69% observed at 30g/mL
of AP.
3.3. Suppression of LPS induced PGE
2
production in cultured
macrophages
Inhibition of LPS (5g/mL) induced PGE
2
production in cul-
tured macrophages was examined at various concentrations of AP.
AP signicantly inhibited PGE
2
levels at the highest concentration
of 50g/mL (Fig. 1B), where a maximum inhibition of 53% was
attained.
3.4. Inhibition of LPS induced interleukin-1 beta levels in cultured
macrophages
AP displayed inhibition of LPS (5g/mL) induced IL-1 beta
levels in J774A.1 cells in a dose-dependent manner at concen-
Fig. 1. (A) Effect of AP on NO production in LPS stimulated J774A.1 murine
macrophages. Cells were incubated with LPS (5g/mL) and indicated concentra-
tions of AP for 24h. The nitrite content of culture media was analyzed by the Griess
reactionassay andexpressedas a percentage of the control (LPS alone). Data are rep-
resented as meanS.E.M. values. **P<0.01 compared with the LPS alone. (B) Effect
of APonPGE2
productioninLPS stimulatedJ774A.1murine macrophages. Cells were
pretreated with indicated concentrations of AP for 1h, and then stimulated with LPS
(5g/mL) for 24h. The PGE
2
levels were markedly decreased dose-dependently by
AP and the values are expressed as a percentage of the control (LPS alone). Data are
represented as meanS.E.M. **P<0.01 compared with the LPS alone.
trations ranging from 20g/mL to 40g/mL (Fig. 2A). Maximum
inhibition of 27% was observed at 30g/mL and 40g/mL
of AP.
3.5. Inhibition of LPS induced interleukin-6 levels in cultured
macrophages
Inhibition of LPS (0.1g/mL) induced IL-6 levels in J774A.1
cells was determined by testing the non-cytotoxic concentra-
tions of AP. A signicant dose-dependent reduction in the
levels of IL-6 was monitored at concentrations ranging from
20g/mL to 40g/mL (Fig. 2B). The IC
50
value obtained was
27.5g/mL with a maximum inhibition of 73% observed at
40g/mL.
3.6. Inhibition of A23187 induced LTB
4
release in HL-60
promyelocytic leukemia cells
Inhibition of A23187 (5M) induced LTB
4
production in HL-60
cells was studied using various concentrations of AP. AP dis-
played signicant inhibition at concentrations of 20g/mL and
40g/mL (Fig. 3A). The IC
50
value obtained was 30g/mL with
206 C.V. Chandrasekaran et al. / Journal of Ethnopharmacology 129 (2010) 203207
Fig. 2. (A) Effect of AP on IL-1 beta production in LPS stimulated J774A.1 murine
macrophages. Cells were pretreated with the indicated concentration of AP for 1h.
Thecells werethenstimulatedwithLPS(5g/mL) for 6h. Thelevels of IL-1betawere
decreased dose-dependently by AP and the values are expressed as a percentage of
the control (LPS alone). Data are represented as meanS.E.M. **P<0.01 compared
with the LPS alone. (B) Effect of AP on IL-6 production in LPS stimulated J774A.1
murine macrophages. Cells were pretreated with the indicated concentration of AP
for 1h. The cells were then stimulated with LPS (0.1g/mL) for 24h. The levels of
IL-6 were decreased dose-dependently by AP and the values are expressed as a per-
centage of the control (LPS alone). Data are represented as meanS.E.M. **P<0.01
and *P<0.05 compared with the LPS alone.
a maximum inhibition of 69% at the highest concentration of
40g/mL.
3.7. Inhibition of A23187 induced TXB
2
release in HL-60
promyelocytic leukemia cells
HL-60 cells were stimulated by calcimycin (A23187) (5M)
to augment the production of TXB
2
. Pre-treatment of AP, sig-
nicantly inhibited the levels of TXB
2
at concentrations ranging
from 10g/mL to 40g/mL (Fig. 3B). AP treatment produced
a maximum inhibition of 99% at the highest concentration of
40g/mL with an IC
50
value of 12g/mL.
3.8. Inhibition of A23187 induced histamine release in RBL-2H3
rat basophilic leukemic cells
RBL-2H3 cells were stimulated with A23187 (5M) to enhance
the production of histamine. AP was tested for its ability to inhibit
the levels of histamine. However, AP was found to be ineffective
in alleviating the release of histamine up to a concentration of
50g/mL (data not shown).
Fig. 3. (A) Effect of AP on LTB
4
production in A23187 stimulated HL-60 cells. Dif-
ferentiated HL-60 cells were pretreated with indicated concentrations of AP for
1h. After stimulation with A23187 (5M) for 15min, the levels of LTB
4
in the
medium were quantied. AP dose-dependently decreased the LTB
4
production and
the values are expressed as a percentage of the control (A23187 alone). Data are
represented as meanS.E.M. **P<0.01 compared with the A23187 alone. (B) Effect
of AP on TXB
2
production in A23187 stimulated HL-60 cells. Differentiated HL-60
cells were pretreated with indicated concentrations of AP for 1h. After stimulation
with A23187 (5M) for 15min, the levels of TXB
2
in the medium were quantied.
AP dose-dependently decreased the TXB
2
production and the values are expressed
as a percentage of the control (A23187 alone). Data are representedas meanS.E.M.
**P<0.01 compared with the A23187 alone.
4. Discussion
In this study, we report the inhibitory effects of AP on the
production of inammatory and allergic mediators. AP exhib-
ited signicant dose-dependent decrease of LPS induced NO
release. Previous studies suggested suppression of LPS induced NO
production by andrograpanin and neoandrographolide via down-
regulation of phosphorylation of p38MAPKs signaling pathway
(Liu et al., 2008). Similar studies on andrographolide reported the
inhibition of NO release by reduction of iNOS protein in the post-
transcriptional stage (Chiou et al., 2000). These ndings suggest
that AP possibly exhibited the ameliorating effect on NOrelease by
a synergistic effect causedby all of the phytochemicals present init.
AP demonstrated signicant dose-dependent down-regulatory
effects on LPS stimulated PGE
2
levels. A similar study on peritoneal
cells showed signicant inhibition of LPS/IFN-gamma induced
PGE
2
production by ethyl acetate fraction of Andrographis panic-
ulata (Chao et al., 2009). Studies conducted in the mesencephalic
neuron-glia cultures suggested that andrographolide (0.15M)
attenuated PGE
2
levels by enhanced COX-2 protein degrada-
tion (Wang et al., 2004). In another study, neoandrographolide
C.V. Chandrasekaran et al. / Journal of Ethnopharmacology 129 (2010) 203207 207
(3090M) showed signicant inhibition of LPS induced PGE
2
release in murine macrophages (Liu et al., 2007). Hence, the
inhibitoryeffect of APcouldbe attributedtothese twocomponents.
In our study, AP demonstrated statistically signicant inhibition
of LPS induced IL-1 beta and IL-6 production in a dose-dependent
manner. Andrographis paniculataextract andandrographolide have
been reported to exhibit inhibitory effect on IL-1 beta and IL-6 pro-
duction (Sheeja et al., 2007). In another study, andrographolide
and its derivatives have been reported to inhibit LPS activated IL-6
expressioninmurine macrophages (Li et al., 2007). Hence, we plau-
sibly hypothesize that the down-regulatory activity of AP on IL-1
beta and IL-6 levels is due to the presence of andrographolide and
other diterpenoids.
In the present study, AP elicited inhibition of TXB
2
release
induced by calcimycin. Thus, inhibition of both PGE
2
and TXB
2
indicates that the extract actively inhibits the COX pathway prod-
ucts. Andrographolide has been reported to block the binding of
NF-kappa B to DNA (Hidalgo et al., 2005) thereby decreases COX-
2 expression (Wang et al., 2004). AP inhibitory property on COX
pathway could be attributed to andrographolide.
Pre-treatment with AP decreased calcimycin stimulated LTB
4
production in differentiated HL-60 cells. Findings by Amroyan et al.
(1999) revealed that andrographolide did not inuence the biosyn-
thesis of any lipoxygenase pathway products such as LTB
4
, 6E-LTB
4
etc. Hence, the possible inhibitory effect of AP might be due to a
synergistic effect of the phytochemicals present in it.
Previous studies have reported that andrographolide at
concentration range of 30300g/mL exhibited signicant dose-
dependent inhibition of histamine release in mast cells (Madav et
al., 1998). Nonetheless, testing of non-cytotoxic concentrations of
AP revealed no signicant inhibition on A23187 induced histamine
release in RBL-2H3 cells.
In conclusion, the extract of Andrographis paniculata contain-
ing at least seven phytochemical constituents showed signicant
anti-inammatory andanti-allergic properties inthe models inves-
tigated in this study. This provides a rationale for its applications
in traditional medicine as anti-inammatory and antipyretic drug.
Considering its dual inhibitory activity on inammatory and pro-
inammatory mediators, we propose that a combination of these
two mechanisms is responsible for the overall activity of the
extract. Based on its inhibitory activity towards LTB
4
and PGE
2
products, AP could be designated as a dual inhibitor. As a novel
nding, our study demonstrates that AP inhibits A23187 induced
LTB
4
production. The extended comprehension of the molecular
mechanisms involved in the anti-inammatory and anti-allergic
effects of this extract and its diterpenoid constituents may lead to
an improved phytomedical treatment of inammatory and aller-
gic disorders at an early phase. In addition, the effects of isolated
phytochemical constituents of AP are currently under study. The
results shall be reported separately.
Acknowledgments
We thank Mr. Thiyagarajan, Department of Cellular Assay for his
technical assistance. The authors are grateful to Dr. M. Deepak and
Mr. B. Murali, R&D Centre, Natural Remedies Pvt. Ltd., Bangalore,
India for providing technical details of test substance.
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