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Journal of Ethnopharmacology 129 (2010) 203–207

Contents lists available at ScienceDirect

Journal of Ethnopharmacology

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ffect of an extract of Andrographis paniculata leaves on inflammatory andllergic mediators in vitro

.V. Chandrasekaran ∗, Anumita Gupta, Amit Agarwalepartment of Cellular Assay, R&D Centre, Natural Remedies Pvt. Ltd., Bangalore, India

r t i c l e i n f o

rticle history:eceived 10 October 2009eceived in revised form 8 February 2010ccepted 13 March 2010vailable online 20 March 2010

eywords:ndrographis paniculata

a b s t r a c t

Aim of study: Andrographis paniculata has been known to possess widespread traditional application inthe treatment of allergy and inflammatory diseases. In the current study, we sought to examine theeffects of an extract of Andrographis paniculata leaves on inhibition of lipopolysaccharide (LPS) induced[nitric oxide (NO), prostaglandin E2 (PGE2), interleukin-1beta (IL-1 beta), and interleukin-6 (IL-6)] andcalcimycin (A23187) induced [leukotriene B4 (LTB4), thromboxane B2 (TXB2) and histamine] mediatorsin diverse cell based models.Materials and methods: Effect of an extract of Andrographis paniculata leaves (AP) was studied on inhibition

ro-inflammatory mediators (NO, IL-1 betand IL-6)nflammatory mediators (PGE2 and TXB2)llergic mediators (LTB4 and histamine)

of LPS induced NO, PGE2, IL-1 beta and IL-6 in J774A.1 murine macrophages; A23187 induced LTB4 andTXB2 in HL-60 promyelocytic leukemic cells and histamine in RBL-2H3 rat basophilic leukemia cells.Results and conclusion: AP illustrated significant alleviation of pro-inflammatory, inflammatory, andallergic mediators. However, no inhibition was observed against histamine release. This outcome hasbeen summed up to deduce that AP is fairly potent in attenuating the inflammation by inhibiting pro-inflammatory (NO, IL-1 beta and IL-6), inflammatory (PGE2 and TXB2) and allergic (LTB4) mediators.

. Introduction

Andrographis paniculata (Acanthaceae) is a well-known medici-al plant and has long been used in Chinese official herbal medicinegainst wide spectrum of ailments (Coon and Ernst, 2004). Ourecent study has reported the safety of Andrographis paniculataKalmColdTM) in a battery of genotoxic tests and also the LD50alue was determined to be more than 5 g/kg rat body weight in ancute oral toxicity study (Chandrasekaran et al., 2009). In a random-zed double blind placebo controlled clinical trial, AP (KalmColdTM)emonstrated significant relief of common cold symptoms inuman populations (Saxena et al., 2010). Andrographis panicu-

ata has been reported to have anti-inflammatory (Sheeja anduttan, 2008), anti-allergic (Madav et al., 1998), immunostimula-

ory (Iruretagoyena et al., 2005) activity. The anti-inflammatory

ction of the plant is attributed to andrographolide, the majorctive principle of the plant (Madav et al., 1996; Abu-Ghefreht al., 2009). However, little is known about the pharmacologi-al mechanisms underlying these actions, although some of its

∗ Corresponding author at: Plot No. 5B, Veerasandra Indl. Area, 19th K.M. Stone,osur Road, Bangalore 560100, Karnataka, India. Tel.: +91 80 40209999;

ax: +91 80 40209817.E-mail addresses: cvc@naturalremedy.com, cvctox@gmail.com

C.V. Chandrasekaran).

378-8741/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.jep.2010.03.007

© 2010 Elsevier Ireland Ltd. All rights reserved.

anti-inflammatory effects have been investigated (Shen et al.,2002).

In order to extend the understanding of its mechanism on anti-inflammatory activity, we investigated inhibitory activity of AP onLPS induced NO, PGE2, IL-1 beta and IL-6 levels; A23187 activatedLTB4, TXB2, and histamine levels using mammalian cell lines invitro. 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 humanpromyelocytic leukemic (HL-60) cells and rat basophilic leukemiccells RBL-2H3 cells in order to study the anti-inflammatory activityof AP. These cell lines have been proved as an appropriate cell assaysystem to study the anti-inflammatory/anti-allergic nature of thecompounds (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 diaminedihydrochloride (NEDD) and captopril were purchased fromSigma–Aldrich, Inc. (St. Louis, MO, USA). Iscove’s modified Dul-

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ecco’s media (IMDM), Earle’s minimum essential media (EMEM)nd Dulbecco’s modified Eagle’s medium (DMEM) were suppliedy Gibco Life Technologies (Grand Island, NY). Fetal bovine serumFBS) was purchased from Hyclone (Logan, USA).

.2. Plant material

Andrographis paniculata (Burm.f.) Wall. ex. Nees (part used –eaves) belonging to the family Acanthaceae was collected fromegions of West Bengal, India. Botanical identification was car-ied out at NISCAIR (National Institute of Science Communicationnd Information Resources). A voucher specimen (No. 110) waseposited in our herbarium.

.3. Preparation of extract of Andrographis paniculata (AP)

Coarse ground leaves of Andrographis paniculata were extractedith methanol (4 times of the raw material quantity) for 3 h, instainless steel jacketed extractor fitted with reflux condenser.

he liquid extract was removed and the remaining raw materialas re-extracted two more times with methanol in a similar man-er. The resulting extracts were combined, concentrated and driednder vacuum (at <55 ◦C). The yield of the dried extract was 6%w/w). After extraction with methanol, the left over raw materialas extracted with water (4 times of the raw material quantity) forh under reflux conditions. This water extract was separated andoncentrated under vacuum at around 75 ◦C until the total solidontent in the liquid reached about 15% to 20% (w/v), followed bypray drying. Two parts of methanolic and one part of successiveater extract were blended to get AP, which was analyzed for phy-

oconstituents by HPLC as described earlier by us (Chandrasekarant al., 2009).

AP was found to contain andrographolide (31.3%, w/w),soandrographolide (0.4%, w/w), neoandrographolide (3.2%, w/w),ndrograpanin (0.6%, w/w), 14-deoxy-11,12-didehydroandro-rapholide (2.8%, w/w), skullcapflavone-I (0.05%, w/w) and 7-O-ethylwogonin (0.05%, w/w). This herbal extract is indexed as

almColdTM, manufactured by Natural Remedies Pvt. Ltd., Banga-ore.

AP was solubilized in DMSO and filter sterilized through 0.2 �Mositively charged nylon DMSO compatible filter to remove thendotoxins. The filtered solution was aliquoted and stored at −80 ◦Cor further use in all the assays. DMSO was used as a solvent controln all the assays up to a maximum concentration of 0.2%. DMSO upo 0.2% did not influence the stimulant induced release of any ofhe inflammatory mediators. All the assays were well controlled bysing respective reference standards.

.4. Cell lines and culture conditions

J774A.1 murine macrophage cell line (TIB-67TM), HL-60 humanromyelocytic leukemia cell line (CCL-240TM) and RBL-2H3 ratasophilic leukemia cell line (CRL-2256TM) were procured frommerican Type Culture Collection (ATCC) (Rockville, MD, USA). Theells were cultured in appropriate ATCC recommended mediumnd maintained at 37 ◦C under 5% CO2 humidified air.

.5. Preparation of Ringer’s buffer

The components of Ringer’s buffer were 118 mM NaCl, 4.6 mMCl, 1.0 mM CaCl2, 1.0 mM KH2PO4, 1.10 mM MgSO4, 24.9 mMaHCO3, 5.0 mM HEPES, 0.1% BSA and 11.1 mM d-glucose. The pHas adjusted to 7.4.

.6. Cytotoxicity assay

Cytotoxic effect of AP was checked in the respective cell linesy using MTT. Based on the cell viability results, different non-

opharmacology 129 (2010) 203–207

cytotoxic concentrations were selected for each study. All theexperiments were conducted in quadruplicates per treatmentusing 96-well tissue culture plates.

2.7. NO scavenging and PGE2 inhibition assay

J774A.1 murine macrophages (passage number between 10and 25) were seeded at a density of 1 × 105 cells/well and incu-bated overnight. The macrophages were then pretreated with APat different non-cytotoxic concentrations (1.25–30 �g/mL for NOand 1.6–50 �g/mL for PGE2) and incubated for 1 h, thereafter, LPS(5 �g/mL) was added followed by further incubation for 24 h. Thereference standards used for NO scavenging and PGE2 inhibitionassay were 1400W dihydrochloride and dexamethasone respec-tively. The cell supernatant was collected for nitrite and PGE2estimation.

Nitrite production, an indicator of NO synthesis, was measuredin the supernatant of cultured macrophages by Griess reaction.Briefly, equal volumes of treated culture supernatant and Griessreagent (1% sulphanilamide, 0.1% NEDD and 5% orthophosphoricacid) were mixed and incubated at room temperature for 5 min,and then the absorbance was measured at 540 nm in a microplatereader. The amount of nitrite in the sample was determined usingsodium nitrite standard curve.

PGE2 levels in macrophage supernatants were quantified as perthe method described by the kit manufacturer [Homogenous TimeResolved Fluorescence (HTRF) kit, CisBio, France].

2.8. IL-1 beta inhibition assay

J774A.1 murine macrophages were seeded at a density of1 × 105 cells/well and incubated overnight. The macrophages werethen pretreated with AP at different non-cytotoxic concentrations(5–40 �g/mL) and incubated for 1 h, thereafter, LPS (5 �g/mL) wasadded followed by further incubation for 6 h. The treated cells werelysed using cell lysis buffer [0.1% Triton X-100 and protease cocktailinhibitor (1×)] in combination with repeated freeze thaw cycles.The plates were centrifuged and the supernatant was collected forestimating 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 of1 × 105 cells/well and incubated overnight. The macrophages werethen pretreated with AP at different non-cytotoxic concentrations(2.5–40 �g/mL) and incubated for 1 h, thereafter, LPS (0.1 �g/mL)was added followed by overnight incubation. The supernatant wascollected and used to estimate the levels of IL-6 as per the methoddescribed by the kit manufacturer (ELISA kit, OptEIATM, BD Bio-sciences, USA). Dexamethasone was used as a reference standard.

2.10. LTB4 and TXB2 inhibition assay

HL-60 promyelocytic leukemia cells (passage number between2 and 10) were first allowed to undergo differentiation intometamyelocytes and neutrophils, as this enabled the cells to pro-duce higher levels of LTB4 and TXB2. Cell differentiation wasattained by seeding the cells at a density of 5 × 105 cells/mL inIMDM enriched with 20% FBS and 1.3% DMSO in a T-25 flaskfollowed 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 seededat a density of 1 × 105 cells/well in Ringer’s buffer. The cellswere pretreated with AP at different non-cytotoxic concentrations(1.25–40 �g/mL for LTB4 and 2.5–40 �g/mL for TXB2) and incubated

f Ethnopharmacology 129 (2010) 203–207 205

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Fig. 1. (A) Effect of AP on NO production in LPS stimulated J774A.1 murinemacrophages. Cells were incubated with LPS (5 �g/mL) and indicated concentra-tions of AP for 24 h. The nitrite content of culture media was analyzed by the Griessreaction assay and expressed as a percentage of the control (LPS alone). Data are rep-resented as mean ± S.E.M. values. **P < 0.01 compared with the LPS alone. (B) Effectof AP on PGE2 production in LPS stimulated J774A.1 murine macrophages. Cells werepretreated with indicated concentrations of AP for 1 h, and then stimulated with LPS(5 �g/mL) for 24 h. The PGE2 levels were markedly decreased dose-dependently by

C.V. Chandrasekaran et al. / Journal o

or 1 h, followed by stimulation with A23187 (5 �M) for 15 min.he reference standards used for LTB4 and TXB2 inhibition assayere captopril and acetyl salicylic acid respectively. The aliquots

emoved from the conditioned medium were used for the quan-ification of LTB4 (HTRF kit, CisBio, France) and TXB2 (ELISA kit,apphire Bioscience, Australia) levels according to the instructionsy the kit manufacturer.

.11. Histamine release assay

The RBL-2H3 cells (passage number between 8 and 14) wereeeded at a density of 5 × 104 cells/well and incubated overnight.ells were then subjected to pre-treatment with AP at variouson-cytotoxic concentrations for 1 h in Ringer’s buffer, thereafter,23187 (5 �M) was added, followed by further incubation for0 min. Ketotifen fumarate was used as a reference standard. Super-atant was collected from the conditioned medium and used foruantification of histamine as directed by the manufacturer of theit (HTRF kit, CisBio, France).

.12. Data analysis

Data are expressed as mean ± standard error mean (S.E.M.) ofour replicates. The mean of the LPS/A23187 control was normal-zed to 100% and mean of cell control was normalized to zero.hereafter, one-way ANOVA was performed on the results fol-owed by a Dunnett’s test for multiple comparisons using GraphPadrism 5.0 (GraphPad Software, Inc., San Diego, CA) statistical soft-are package. The significance level was chosen at P < 0.05–0.01

or all statistical analyses. The half maximal inhibitory concentra-ion (IC50) was calculated using median effect plot (Wolfe and Liu,007).

. Results

.1. Cytotoxicity of test substances

Cytotoxic effect of AP was assessed at different concentra-ions by MTT assay in J774A.1, HL-60 and RBL-2H3 cells. AP didot demonstrate any significant toxicity up to a concentration of0 �g/mL under our experimental conditions (data not shown).ereafter, the non-cytotoxic concentrations were chosen for the

urther experiments.

.2. Suppression of LPS induced NO production in culturedacrophages

Effect of AP was determined in LPS (5 �g/mL) stimulated NOn J774A.1 cells. AP showed a significant dose-dependent declinen the levels of NO at concentrations ranging from 10 �g/mLo 30 �g/mL (Fig. 1A). The IC50 value obtained was 20 �g/mL,ith a maximum inhibition of ∼69% observed at 30 �g/mL

f AP.

.3. Suppression of LPS induced PGE2 production in culturedacrophages

Inhibition of LPS (5 �g/mL) induced PGE2 production in cul-ured macrophages was examined at various concentrations of AP.P significantly inhibited PGE2 levels at the highest concentrationf 50 �g/mL (Fig. 1B), where a maximum inhibition of 53% wasttained.

.4. Inhibition of LPS induced interleukin-1 beta levels in culturedacrophages

AP displayed inhibition of LPS (5 �g/mL) induced IL-1 betaevels in J774A.1 cells in a dose-dependent manner at concen-

AP and the values are expressed as a percentage of the control (LPS alone). Data arerepresented as mean ± S.E.M. **P < 0.01 compared with the LPS alone.

trations ranging from 20 �g/mL to 40 �g/mL (Fig. 2A). Maximuminhibition of 27% was observed at 30 �g/mL and 40 �g/mLof AP.

3.5. Inhibition of LPS induced interleukin-6 levels in culturedmacrophages

Inhibition of LPS (0.1 �g/mL) induced IL-6 levels in J774A.1cells was determined by testing the non-cytotoxic concentra-tions of AP. A significant dose-dependent reduction in thelevels of IL-6 was monitored at concentrations ranging from20 �g/mL to 40 �g/mL (Fig. 2B). The IC50 value obtained was∼27.5 �g/mL with a maximum inhibition of ∼73% observed at40 �g/mL.

3.6. Inhibition of A23187 induced LTB4 release in HL-60promyelocytic leukemia cells

Inhibition of A23187 (5 �M) induced LTB4 production in HL-60cells was studied using various concentrations of AP. AP dis-played significant inhibition at concentrations of 20 �g/mL and40 �g/mL (Fig. 3A). The IC50 value obtained was ∼30 �g/mL with

206 C.V. Chandrasekaran et al. / Journal of Ethnopharmacology 129 (2010) 203–207

Fig. 2. (A) Effect of AP on IL-1 beta production in LPS stimulated J774A.1 murinemacrophages. Cells were pretreated with the indicated concentration of AP for 1 h.The cells were then stimulated with LPS (5 �g/mL) for 6 h. The levels of IL-1 beta weredecreased dose-dependently by AP and the values are expressed as a percentage ofthe control (LPS alone). Data are represented as mean ± S.E.M. **P < 0.01 comparedwith the LPS alone. (B) Effect of AP on IL-6 production in LPS stimulated J774A.1murine macrophages. Cells were pretreated with the indicated concentration of APfor 1 h. The cells were then stimulated with LPS (0.1 �g/mL) for 24 h. The levels ofIca

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Fig. 3. (A) Effect of AP on LTB4 production in A23187 stimulated HL-60 cells. Dif-ferentiated HL-60 cells were pretreated with indicated concentrations of AP for1 h. After stimulation with A23187 (5 �M) for 15 min, the levels of LTB4 in themedium were quantified. AP dose-dependently decreased the LTB4 production andthe values are expressed as a percentage of the control (A23187 alone). Data arerepresented as mean ± S.E.M. **P < 0.01 compared with the A23187 alone. (B) Effectof AP on TXB2 production in A23187 stimulated HL-60 cells. Differentiated HL-60cells were pretreated with indicated concentrations of AP for 1 h. After stimulationwith A23187 (5 �M) for 15 min, the levels of TXB2 in the medium were quantified.

L-6 were decreased dose-dependently by AP and the values are expressed as a per-

entage of the control (LPS alone). Data are represented as mean ± S.E.M. **P < 0.01nd *P < 0.05 compared with the LPS alone.

maximum inhibition of 69% at the highest concentration of0 �g/mL.

.7. Inhibition of A23187 induced TXB2 release in HL-60romyelocytic leukemia cells

HL-60 cells were stimulated by calcimycin (A23187) (5 �M)o augment the production of TXB2. Pre-treatment of AP, sig-ificantly inhibited the levels of TXB2 at concentrations ranging

rom 10 �g/mL to 40 �g/mL (Fig. 3B). AP treatment producedmaximum inhibition of 99% at the highest concentration of

0 �g/mL with an IC50 value of ∼12 �g/mL.

.8. Inhibition of A23187 induced histamine release in RBL-2H3at basophilic leukemic cells

RBL-2H3 cells were stimulated with A23187 (5 �M) to enhance

he production of histamine. AP was tested for its ability to inhibithe levels of histamine. However, AP was found to be ineffectiven alleviating the release of histamine up to a concentration of0 �g/mL (data not shown).

AP dose-dependently decreased the TXB2 production and the values are expressedas a percentage of the control (A23187 alone). Data are represented as mean ± S.E.M.**P < 0.01 compared with the A23187 alone.

4. Discussion

In this study, we report the inhibitory effects of AP on theproduction of inflammatory and allergic mediators. AP exhib-ited significant dose-dependent decrease of LPS induced NOrelease. Previous studies suggested suppression of LPS induced NOproduction by andrograpanin and neoandrographolide via down-regulation of phosphorylation of p38MAPKs signaling pathway(Liu et al., 2008). Similar studies on andrographolide reported theinhibition of NO release by reduction of iNOS protein in the post-transcriptional stage (Chiou et al., 2000). These findings suggestthat AP possibly exhibited the ameliorating effect on NO release bya synergistic effect caused by all of the phytochemicals present in it.

AP demonstrated significant dose-dependent down-regulatoryeffects on LPS stimulated PGE2 levels. A similar study on peritonealcells showed significant inhibition of LPS/IFN-gamma inducedPGE2 production by ethyl acetate fraction of Andrographis panic-

ulata (Chao et al., 2009). Studies conducted in the mesencephalicneuron-glia cultures suggested that andrographolide (0.1–5 �M)attenuated PGE2 levels by enhanced COX-2 protein degrada-tion (Wang et al., 2004). In another study, neoandrographolide

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30–90 �M) showed significant inhibition of LPS induced PGE2elease in murine macrophages (Liu et al., 2007). Hence, thenhibitory effect of AP could be attributed to these two components.

In our study, AP demonstrated statistically significant inhibitionf LPS induced IL-1 beta and IL-6 production in a dose-dependentanner. Andrographis paniculata extract and andrographolide have

een reported to exhibit inhibitory effect on IL-1 beta and IL-6 pro-uction (Sheeja et al., 2007). In another study, andrographolidend its derivatives have been reported to inhibit LPS activated IL-6xpression in murine macrophages (Li et al., 2007). Hence, we plau-ibly hypothesize that the down-regulatory activity of AP on IL-1eta and IL-6 levels is due to the presence of andrographolide andther diterpenoids.

In the present study, AP elicited inhibition of TXB2 releasenduced by calcimycin. Thus, inhibition of both PGE2 and TXB2ndicates that the extract actively inhibits the COX pathway prod-cts. Andrographolide has been reported to block the binding ofF-kappa B to DNA (Hidalgo et al., 2005) thereby decreases COX-expression (Wang et al., 2004). AP inhibitory property on COX

athway could be attributed to andrographolide.Pre-treatment with AP decreased calcimycin stimulated LTB4

roduction in differentiated HL-60 cells. Findings by Amroyan et al.1999) revealed that andrographolide did not influence the biosyn-hesis of any lipoxygenase pathway products such as LTB4, 6E-LTB4tc. Hence, the possible inhibitory effect of AP might be due to aynergistic effect of the phytochemicals present in it.

Previous studies have reported that andrographolide atoncentration range of 30–300 �g/mL exhibited significant dose-ependent inhibition of histamine release in mast cells (Madav etl., 1998). Nonetheless, testing of non-cytotoxic concentrations ofP revealed no significant inhibition on A23187 induced histamineelease in RBL-2H3 cells.

In conclusion, the extract of Andrographis paniculata contain-ng at least seven phytochemical constituents showed significantnti-inflammatory and anti-allergic properties in the models inves-igated in this study. This provides a rationale for its applicationsn traditional medicine as anti-inflammatory and antipyretic drug.onsidering its dual inhibitory activity on inflammatory and pro-

nflammatory mediators, we propose that a combination of thesewo mechanisms is responsible for the overall activity of thextract. Based on its inhibitory activity towards LTB4 and PGE2roducts, AP could be designated as a dual inhibitor. As a novelnding, our study demonstrates that AP inhibits A23187 inducedTB4 production. The extended comprehension of the molecularechanisms involved in the anti-inflammatory and anti-allergic

ffects of this extract and its diterpenoid constituents may lead ton improved phytomedical treatment of inflammatory and aller-ic disorders at an early phase. In addition, the effects of isolatedhytochemical constituents of AP are currently under study. Theesults shall be reported separately.

cknowledgments

We thank Mr. Thiyagarajan, Department of Cellular Assay for hisechnical assistance. The authors are grateful to Dr. M. Deepak and

r. B. Murali, R&D Centre, Natural Remedies Pvt. Ltd., Bangalore,ndia for providing technical details of test substance.

opharmacology 129 (2010) 203–207 207

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