Journal of Ethnopharmacology 147 (2013) 584–594

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Journal of Ethnopharmacology

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Atropa acuminata Royle Ex Lindl. blunts production ofpro-inflammatory mediators eicosanoids., leukotrienes, cytokinesin vitro and in vivo models of acute inflammatory responses

Albeena Nisar a, Akhtar H. Malik b, Mohammed Afzal Zargar a,n

a Department of Biochemistry, University of Kashmir, Srinagar, J&K 190006, Indiab Department of Botany, University of Kashmir, Srinagar, J&K 190006, India

a r t i c l e i n f o

Article history:Received 4 October 2012Received in revised form21 February 2013Accepted 11 March 2013Available online 22 March 2013

Keywords:Atropa acuminataNOPGE2LTB4TNF-αIL-1β

41/$ - see front matter & 2013 Elsevier Irelanx.doi.org/10.1016/j.jep.2013.03.038

viations: AA, Arachidonic acid; COX-1, Cyclooxygenase-2; AAEE, Ethanolic extract of Atropaase; GR, Glutathione reductase; g, Grams; H2Ole nitric oxide synthase; IL-6, Interleukin-6; ILkin-β; LTB4, Leukotriene B4; LOX, Lipoxygenase; NO, Nitric oxide; NF-κB, Nuclear factor-κB; Osaline; PGE2, Prostaglandin E2; ROS, Reactivedical; SOD, Super oxide dismutase; TNF-α, Tuesponding author. Tel.: þ91 941946 3306.ail addresses: clinicalbiochemalb@gmail.com (ar@gmail.com (A.H. Malik), zargarafzal01@gm

a b s t r a c t

Ethnopharmacological relevance: Atropa acuminata Royle Ex Lindl. has been widely used in folk medicinefor several inflammatory disorders such as arthritis, asthma, conjunctivitis, encephalitis, pancreatitis,peritonitis, acute infections and neuroinflammatory disorders.Aim of the study: Our aim was to evaluate Atropa acuminata for its anti-inflammatory properties and todelineate its possible mechanism of action on the modulation of the inflammatory mediators.Materials and methods: We investigated the inhibitory action of ethanolic extract of Atropa acuminata(AAEE) on production of NO, TNF-α and IL-1β in lipopolysaccharide (LPS)-stimulated RAW264.7 cells andalso assayed it for COX 1/2 and 5-LOX inhibitory activities. Next AAEE was tested in acute inflammatoryanimal models., carragenean induced rat paw edema, carragenean induce pleurisy in rats and vascularpermeability in mice and the effects on NO, PGE2 and LTB4 production in the pleural fluid and pawexudates were evaluated. In addition the effects on leukocyte migration and exudation and vascularpermeability were also observed.Results: Our findings summarized novel anti-inflammatory mechanisms for Atropa acuminata based ondual in vitro cyclooxygenase 1/2/ and 5-Lipoxygenase inhibitory activities and also significant down-regulation of nitric oxide (NO) and pro-inflammatory cytokin (TNF-α and Il-1 β) release inLPS-stimulated RAW 246.7 macrophage cell line. In acute inflammatory models in vivo (carrageneaninduced edema, carragenean induced pleurisy in rats and vascular permeability in mice), AAEE exhibitedan extensive diverse mechanism for anti-inflammatory properties. This was indicated on the basis ofdose dependent suppression of multi targeted inflammatory mediators., NO, TNF-α and IL-1β, eicosa-noids., PGE2 and leukotrienes., LTB4 along with significantly decreased leucocyte migration, exudationand decreased vascular permeability. These effects were more potent and prolonged than traditionalNSAIDS, thereby indicating fewer side effects. AAEE was found to be safe for long term administration, asconfirmed by the results of acute toxicity studies and MTT assay. The complex mode of action of theherbs was attributed possibly due to the high polyphenolic, flavanol and flavonoid content present in theextracts as observed by means of quantitative screening for phytochemicals.Conclusion: Our study provides scientific evidence to support the traditional anti-inflammatory usesof Atropa acuminata and is probably due to inhibitory effects on multiple inflammatory mediatorswhich indicates a promising potential for the development of a strong anti-inflammatory agent fromthis plant.

& 2013 Elsevier Ireland Ltd. All rights reserved.

d Ltd. All rights reserved.

ygenase-1; COX-2,acuminata; GPX, Glutathione2, Hydrogen peroxide; iNOS,-8, Interleukin-8; IL-1β,e; mg, Milligram; ml,H, Hydroxyl; PBS, Phosphateoxygen species; O2

-, Supermour necrosis factor-α.

A. Nisar),ail.com (M.A. Zargar).

1. Introduction

Atropa acuminata Royle Ex Lindl. belongs to the family Solana-ceae and is a tall perennial sub-alpine plant native to Asia. It is anIndian medicinal plant, widely distributed across North-WesternHimalayas (Dhar and Kachroo, 1983). The rhizome of Atropaacuminata has been traditionally used since ages for the treatmentof arthritis related inflammatory disorders, muscle and joint pain,muscle spasms (Kaul, 1997; Wealth of India, 1952). Aerial parts of

A. Nisar et al. / Journal of Ethnopharmacology 147 (2013) 584–594 585

this plant have also been used in traditional medicine to treatinnumerable ailments such as acute infections, anxiety, asthma,chicken pox (Bettermann et al., 2001; Kumar et al., 1997) con-junctivitis, fever, encephalitis (Ceha et al., 1997; Duncan andCollison, 2003), muscle and joint pain, acute inflammation, pan-creatitis, peritonitis, scarlet fever, Parkinson's disease and neuoroinflammatory disorders (Kahn et al., 1991; King, 1966). The roots ofthe plant have also been used as a sedative (Rhodes et al.,1978)and also against sore throat, ulcerative colitis (Shanafelt et al.,2002) and whooping cough (Walach et al., 2001). ChemicallyAtropa acuminata has been found to contain tropane alkaloidsand highly oxygenated triterpenes (Mehmood et al., 2002). Pre-viously these alkaloids were found to have anti-cholinergic andanti-spasmodic activity (Tyler et al., 1988).

The present study was intended to investigate the modulatoryeffect of ethanolic extract of Atropa acuminata (AAEE) on in vitroand in vivo acute inflammatory models for its possible abilityas an anti-inflammatory agent and also to explore the under-lying mechanism in view of the inhibitory effects on variousinflammagens., eicosanoid metabolites (prostaglandins and leuko-trienes), nitric oxide and cytokines. This report is the first ofits kind concerning the pharmacological properties of Atropaacuminata.

Inflammation is an overt or covert component of severalconditions and illnesses affecting humans. Although inflammationis a process protecting the integrity of organisms against physical,chemical and infective insults, yet the inflammatory response toseveral insults erroneously leads to damaging of normal tissues and awide range of chronic diseases such as arthritis, asthma, inflamma-tory bowel disease and many others (Ross, 1999; Rostasy, 2005).Acute inflammatory response has been demonstrated to be amulti-step process characterized by increased sequestration ofleukocytes to target sites and over production of complex inflam-matory mediators by macrophages and monocytes (Zedler andFaist, 2006). Among the key mediators of inflammation are thearachidonic acid (AA) derived eicosanoids (prostaglandins andleukotrienes), NO and inflammatory cytokines., TNF-α and IL-1βrecruited by the activated monocytes and macrophages. Prosta-glandin E2 (PGE2), the lipid mediator derived from cyclooxygenasepathway of AA metabolism produces the classical signs of inflam-mation in the joints: redness, swelling, and pain. Leukotriene B4

(LTB4) is another inflammatory mediator derived from AA via thelipoxygenase (LOX) pathway which promotes production of TNF-αand IL-1β and also acts as a chemo attractant for leukocytes toexacerbate the inflammatory process (Kanaoka and Boyce, 2004).Of late, dual inhibition of biosynthesis of prostaglandins andleukotrienes has evolved as a promising anti-inflammatory ther-apeutic approach (Martel-Pelletier et al., 2003). Such agents havebeen found to be effective for reducing the deleterious side effectsby balancing the AA metabolism and several plant extractshave been reported to exhibit such dual inhibitory activities(Li et al., 2004). Nitric oxide (NO) is another ubiquitous mediatorof inflammation which reacts with the super oxide radical (O2

−) toform peroxynitrate anion and stimulates the COX enzymes. It alsoenhances the production of a variety of other inflammatorymediators such TNF-α and IL-1β (Marcinkiewicz et al., 1995).Cytokines (IL-1β, TNF-α, etc.), in addition are considered to play apivotal role in the pathogenesis of arthritis and exhibit pleiotropiceffects that contribute to the inflammatory process. TNF-α andIL-1β both escalate the pro-inflammatory cellular actions likestimulating the production of other cytokines IL-6, IL-8 andchemokines which further perpetuate the inflammatory cascade(Moser et al., 1989).

In this report we summarize novel anti-inflammatory mechan-isms mediated by AAEE based on the inhibitory effects on suchaforementioned multiple inflammatory mediators.

2. Materials and methods

2.1. Chemicals

Growth medium, DMEM, fetal calf serum (FCS), penicillin,streptomycin, di-methyl sulphoxide (DMSO), glutamine, pyruvate,carrageenan, ibuprofen (IBP), Evans blue, phenylmethylsulfonylfluoride (PMSF), aprotinin and Tween 20 (Sigma Chemicals). Gumacacia (Hi Media), COX1/2, 5-LOX ELISA kits (Cayman chemicals),PGE2, LTB4, TNF-α and IL-1β ELISA kits (R&D Systems). All otherchemicals were purchased from Hi-Media and Merck, India.Standard drugs were purchased from Sigma-Aldrich chemicalsco. (St. Louis O., USA) and all solvents used were of HPLC grade(Ranbaxy Chemicals Ltd., Mohali, and Punjab, India).

2.2. Test material

2.2.1. Collection of plantAtropa acuminata (whole plant) was collected from the higher

reaches of Kangdoori, Khilanmarg, Afarwarth areas of Gulmargand Thajwas glaciers of Sonamarg. The plant was correctlyidentified by Centre of Biodiversity and Toxonomy, University ofKashmir, Srinagar, India and authenticated by a botanist, Dr. IrshadWanchoo. A voucher sample is retained and deposited at CentralHerbarium, Department of Botany, University of Kashmir, India as1758 KASH (16/05/2009). The material was ensued to be free frompathogens, aflatoxins, pesticidal residues and heavy metals accor-ding to WHO (1998).

2.2.2. Preparation of extractsThe authentically identified whole part of the plant material

was shade dried and then powdered. About 1.6 kg of Atropaacuminata was subjected to Soxhlet extraction separately withabsolute ethanol (EtOH). The solvent was removed under reducedpressure on a vacuum rotary evaporator to get a darkish greenextract for Atropa acuminata referred from here as AAEE (yield:32.2%). The crude extract was stored at 4 1C for experimental use.

2.3. Determination of total polyphenolic content

2.3.1. Total phenolic content in the test materialThe concentration of total phenolic compounds in the test

material was determined by the Folin–Ciocalteau method with afew modifications (Singleton and Rossi, 1965). The test materialAAEE was evaluated at a final concentration of 0.5 mg/ml. To analiquot of the extract (0.5 ml) in duplicates 2.5 ml of reagent(0.124 μg/ml) and 2 ml sodium carbonate (7.5%) was added.The reaction mixtures were allowed to stand for 1 h protectedfrom light and the absorbance at 675 nm was measured byHevlett–Packard UV visible spectrophotometer. The concentrationof total phenolic compounds for AAEE was calculated on the basisof standard curve using Gallic acid.

2.3.2. Total flavonoids in the test materialThe concentration of flavonoids was determined according to

the method of Ordon ez. et al., 2006. To 0.5 ml of extract 0.5 ml of2% AlCl3 ethanol solution was added followed by 1 h incubation at37 1C. The absorbance of yellow colour (indicating the presence offlavonoids) was measured at 420 nm. Extract sample was evalu-ated at a final concentration of 0.5 mg/ml. Total flavonoid cont-ent was calculated as Quercetin equivalants (mg/g) of the testmaterial.

A. Nisar et al. / Journal of Ethnopharmacology 147 (2013) 584–594586

2.3.3. Total flavanols in the test materialTotal flavanols in the test material was estimated on the

method of Kumaran and Karunakaran, 2006. An aliquot of thetest material and standard, 2 ml of 2% AlCl3 and 3 ml sodiumacetate solution (50 g/l) were added. The samples were allowed tostand for 24 h at 20 1C followed by measuring absorbance at440 nm. The total flavanol content was determined based asstandard curve using Quercitin.

2.4. In vitro COX 1/2 and 5-LOX inhibition assay

The in vitro COX 1/2 and LOX was determined using ELISA kit(Cayman chemicals company).

2.4.1. Assessment of cyclooxygenase (COX 1 and COX 2) inhibitoryactivity

COX inhibitory activity was measured using an enzyme immu-noassay (EIA) screening assay kit (Cat. No.: 560131, CaymanChemical Company, USA). The assay kit was used to measure theCOX 1/2 derived PGH2 produced in the COX reaction and theprostanoid product is quantified using a broadly specific antiserumthat binds to all major prosatglandins. All samples and positivecontrols were added as ethanol solutions to assay solutions.All procedures were performed as indicated in the assay kitinstructions. Absorbance of assay buffer (160 μl of 100 mM TrisHCL (pH 8.0), 10 μl of Heme and 10 μl of Methanol) and assaybuffer with COX enzyme was measured as background and 100%Initial Activity.

2.4.2. Assessment of 5-lipoxygenase inhibitory activityThe assay kit measures the hydroperoxides generated from the

incubation of a lipoxygenase (5-LOX) with either arachidonic orlinoleic acid. LOX inhibitory activity was measured using colori-metric (LOX) inhibitor screening assay kit (Cat No.760700, Caymanchemical company, USA). The assay kit was used to detect andmeasure the hydroperoxides produced in the lipoxygenationreaction using a purified LPO. All samples and positive controlswere added as ethanol solutions to assay solutions. All procedureswere performed as indicated in the assay kit instructions.The hydroperoxide product thus formed was measured using animmunoreader at 540 nm. Absorbance of assay buffer (90 μl LOX,10 μl assay buffer) was measured as positive control. This assay kitprovides an accurate and convenient method for screening lipox-ygenase inhibitors. Lornoxicam, standard LOX inhibitory drug wasused as standard control at a concentration of 100 μg/ml.

2.5. In vitro effect on NO and pro-inflammatory cytokines

2.5.1. Cell cultureRAW 246.7 a murine macrophage cell line was grown in DMEM

medium supplemented by 100 U/ml penicillin, 100 μg/ml of strep-tomycin and 10% FCS, 2 mM glutamine, 1 mM pyruvate. Cells weremaintained at 37 1C in 5% CO2 incubator.

2.5.2. Cell viability assayCell viability was assessed by using MTT based assay. Cells were

plated at a density of 1�104 cells per well in a 24 well cell cultureplate and incubated overnight at 37 1C and 5% CO2 and then thecells were exposed to various concentrations of 6.25–100 μg/ml ofAAEE with or without LPS (1 ug/ml). At 24 h after treatment, MTTsolution in PBS was added to the wells at a final concentration of1 mg/ml. The medium was aspirated carefully and the formazonecrystals were dissolved in 100 μl DMSO. Absorbance was measuredat 520 nm using a micro plate reader to quantitate the extent ofreduction of MTT (Ha et al., 2006).

2.5.3. Nitrite assayRAW 246.7 cells were plated at a density of 2�104 cells per

well in 24 well cell culture plates and then incubated with orwithout LPS 1 μg/ml and solvent alone (DMSO) or the plantextract, AAEE at various concentrations (6.25 μg/ml, 12.5 μg/ml,25 μg/ml, 50 μg/ml, 100 μg/ml). The cells were incubated for 24 h.DMSO concentration did not exceed 0.1% of the media. Twocontrols were incubated in each treatment, media and DMSO(Vehicle Control) and media, LPS and DMSO (LPS Control).The nitrite accumulation in the assay was determined by theGriess reaction (Minghetti et al.,1997). To 100 μl of culture super-natant collected an equal volume of Griess reagent (1% sulphani-lamide and 0.1% naphthyl-ethylenediamine dihydrochloride in 5%phosphoric acid) was added followed by 10 min incubation at37 1C. Optical density at 550 nm was measured and calculatedusing a standard curve obtained by series concentrations ofsodium nitrite (NaNO3).

2.5.4. Determination of TNF-α, IL1-β production in RAW 246.7macrophages

Cell supernatants collected on ice and stored in a −70 1C freezerwere used for cytokine estimation. TNF-α and IL1-β was deter-mined in the culture supernatant using specific ELISA kit accordingto the manufacturer's instructions.

2.6. Animal treatment

Studies were carried out using Male Wistar Albino rats of eithersex weighing 150–180 g and Swiss Albino mice (24–30 g) of eithersex. They were procured from IIIM, Jammu and kept in animalhouse, KU. The animals were grouped in polypropylene cages withnot more than six animals per cage and maintained understandard laboratory conditions (temperature 2572 1C, relativehumidity 60% 715% and with dark and light circle 12 h/14 h).They were allowed free access to standard dry pellet (Hindustanlever, Kolkata, India) and water ad libitum (CPCSEA, 2003).The animals were acclimatized to laboratory conditions beforecommencement of the experiment. All animal experiments wereapproved by the University animal ethical committee vides No: 80/03/CA/[F-IAEC(Pharm.Fc.)APPROVAl/01].

2.7. Acute toxicity studies

The effect of test extracts on general behaviour and safety wasevaluated in mice using the up and down procedure (Organizationof Economic Co-operation and Development (OECD), Guideline No.423, 1996). Mice of either sex (three females); weight: 20–25 g;age: 4–6weeks) were administered graded doses of AAEE (singledose) upto 2500 mg/kg orally by gavage. The animals wereobserved for toxic symptoms continuously for the first 4 h afterdosing. Finally, the number of survivors was noted after 24 h andthese animals were then maintained for a further 13 days withobservations made daily. The animals were observed for anychanges in general behaviour, weight, mortality or other physio-logical activities.

2.8. Carregenean induced rat paw edema

Anti-inflammatory activity was assayed using the methoddescribed by Winter and Nuss, 1962. Edema was induced byinjection of 1% suspension of carrageenan (100 μl) in 0.9% sterilesaline solution into the right plantar region of the rat. The animalswere divided into groups of 6 animals each. Group I, (Naïvecontrol), received 1% gum acacia, Group II, received 1% gum acacia(Cg control) (Negative control); Group III: AAEE (62.5 mg/kg b.w);

A. Nisar et al. / Journal of Ethnopharmacology 147 (2013) 584–594 587

Group IV: AAEE (125 mg/kg b.w); Group V: AAEE (250 mg/kg b.w);Group VI: AAEE (500 mg/kg b.w); Group VII: Ibuprofen (100 mg/kgb.w). The doses were worked out for the animals based on theresults of the in vitro assays on COX 1/2 and 5-LOX enzymes and onobserved effects of AAEE on NO, TNF-and IL-1 on RAW 246.7 cells.All the test extracts worked out at different graded doses andstandard drugs were dissolved in 1% guma acacia. One hour afteroral administration of respective groups edema was induced in alltreatment groups except group I (Naïve Control) by injection of 1%suspension of carrageenan (100 μl) in 0.9% sterile saline solutioninto the right plantar region of the rat. The paw edema wasmeasured using a plethysmograph apparatus. Before oral admin-istration of the drugs, the average volume of the right hind paw ofeach animal was measured twice (Vc) by the plethysmographapparatus. The volume of the right hind paw was determinedagain at 0.5 h, 1 h, 2 h and 3 h after carrageenan treatment (Vt) toeach group. The difference between the initial and subsequentreading gave the actual edema volume. The anti-inflammatoryactivity was expressed in terms of “percent inhibition” (Suleymanet al., 1991) as described at the end of this section.

2.8.1. Quantification of PGE2, LTB4 in paw tissueBefore homogenization frozen paws containing bony tissue was

weighted using an extraction buffer containing 1 mM phenylmethyl sulphonyl fluoride (PMSF), 1 μM/ml Apoprition and 0.05%TWEEN in PBS (4 ml/g of tissue). The paw tissues were homo-genized using polytron homogenizer. The homogenate wascentrifuged at 5000 g for 15 min (Magari et al., 2004) and thesupernatants stored at −80 1C analysis for PGE2 and LTB4 analysis.PGE2 and LTB4 were estimated using commercially available(Cayman chemicals) according to the manufacturer's instructions.

2.8.1.1. Measurement of anti oxidant enzymes. The experimentalanimals of the carragenean induced model from the respectivegropus were sacrificed. Liver was isolated and whole tissue afterrinsing in ice cold normal saline was immediately placed innormal saline (double this volume) and homogenized at 4 1C.The homogenate was centrifuged at 12,000 rpm for 5 min and thesupernatant stored at −20 1C was used for assay of several anti-oxidant enzymes. The different biochemical parameters assessedwere as follows.,

SOD activity—Beauchamp and Fridovich (1971)). GPX activity—Sharma et al. (2001)). GR activity—Carlberg and Mannervik (1985).

2.9. Carragenean induced pleurisy (in vivo)

Pleurisy was induced in male Wistar rats by injecting 0.5 ml(1% w/v) in sterilized normal saline into the pleural cavity follow-ing the procedure of Meacock and Kitchen et al. (1979). The extractAAEE at different graded doses (6.25 mg/kg b.w, 125 mg/kg b.w,250 mg/kg b.w and 500 mg/kg b.w) or standard drug (Ibuprofen-100 mg/kg), was administered 1 h before and 6 h after carragen-ean injection. After 24 h, the rats were anaesthetized with diethylether. The chest was carefully opened to collect the pelural fluid.Total leukocyte and differential count of the exudate was calcu-lated. Total leucocytes count was performed in a Neubauerchamber after diluting the pleural fluid with Turk solution(1:200) and cytospin preparations of pleural wash were stainedwith May-Grunwald Giemsa for the differential leucocytes count,which was performed under an oil immersion objective. The cellfree supernatant was assayed for PGE2, LTB4 and nitrite/nitrateconcentration. Assay for PGE2 and LTB4 was done based onmanufacturer's instructions of ELISA kits from Cayman chemicals.Nitrite/nitrate was measured in the exudate as previouslydescribed on RAW 246.7 macrophage cell line.

2.10. Vascular permeability in mice

In order to study the effects of AAEE on the altered vascularpermeability in the inflammatory process, an in vivo study wascarried on mice where in evaluation of the effect of AAEE onvascular permeability was based on the method of Whittle (1964).Mice were orally administered with AAEE at different doses (62.5–500 mg/kg b.w) and Ibuprofen (100 mg/kg b.w.). After 30 min theanimals were injected intravenously with 0.2% solution of Evan'sblue dye (0.25% w/v normal saline). Later mice were injected intra-peritoneally (1 ml/100 g of body-weight) with 0.6% acetic acid(v/v) in normal saline. Thirty minutes later the mice weresacrificed and the peritoneal cavities were washed with 5 mlnormal saline into heparinised tubes and the wash was centri-fuged for 10 min (3000 g). The dye content was measured at610 nm using a UV visible spectrophotometer.

2.11. Data analysis and statistical considerations

Data is expressed as mean 7S.E.M. Statistical significance ofdifferences was assessed by One way ANOVA followed by Bonfer-roni test for multiple comparisons. The level of significance was setat Po0.05. IC50 values were calculated by regression analysis usingGraph Pad Instat software. Percent inhibitory activity in differentexperimental groups was derived using following method:

#Percent inhibition of inflammation was calculated using theformula,

% inhibition ¼ 100 1−Vt

Vc

� �

where, “Vc”, represents edema volume in control; “Vt”, representsedema volume in group treated with test extract.

3. Results

3.1. Total polyphenolic content

The total phenolic content in AAEE was found to be fairly high.The total polyphenolic content in AAEE was found to be 104.19 μgGallic acid equivalents (GAE)/gm of dry plant material. In addition,the flavanoid and total flavanol content was found to be 1.19 mgand 0.97 mg quercetin (QRC)/g of dry plant material, respectively.

3.2. Effects on COX 1/COX 2 enzymes

AAEE was assayed at graded concentrations from 6.25 μg/ml to100 μg/ml and compared to the standard reference drugs, Aspirin,Celecoxib and Indomethancin at 40 μM, 30 μM and 30 μM, respec-tively. AAEE at doses of 6.25 μg/ml, 12.5 μg/ml, 25 μg/ml, 50 μg/mland 100 μg/ml showed 9.23%, 23.4%, 52.7%, 86% and 89% percen-tage inhibition in COX-1 activity, respectively. Aspirin, celecoxiband indomethacin exhibited 80.9%, 12.6% and 97.2% percentageinhibition in COX-1 activity. At the concentration of 100 μg/ml,COX 1 inhibitory activity of AAEE was significantly comparable tothat of aspirin and indomethacin, standard COX 1 inhibitors.

The COX 2 inhibitory activity also increased with increase in theconcentration of the extract, AAEE. At doses of 6.25 μg/ml, 12.5μg/ml, 25 μg/ml,50 μg/ml and 100 μg/ml AAEE demonstrated 3.67%,12.2%, 34.8%, 67.2% and 69% percentage inhibition, respectively, inCOX-2 activity. Standard reference, celecoxib demonstrated an inhi-bition of 92.8% in this case. Aspirin and indomethacin inhibited COX-2 activity by 26.2% and 51.2%, respectively. Maximal COX- 2 inhibitionwas observed for AAEE at a concentration of 100 μg/ml. The IC50

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Fig. 1. Effects Of AAEE on cell viability (a), on production of NO (b) and on TNF-α & IL-1β production (c) on macrophage cell line after LPS stimulation. RAW macrophageswere pre incubated with indicated concentrations of AAEE for 1 h and then activated for 24 h with LPS 1 μg/ml. All values were expressed as Mean7SD (n¼6). Statisticalsignificance: nPo0.05, nnPo0.01, nnnPo0.001, versus LPS control., δPo0.001 versus vehicle control (see Section 4.1) (One way ANNOVA followed by Bonferroni Multiplecomparison test).

A. Nisar et al. / Journal of Ethnopharmacology 147 (2013) 584–594588

values of AAEE for COX 1/COX 2 were found to be 23.76 μg/ml and30.76 μg/ml, respectively.

3.3. In vitro 5-lipoxygenase inhibitory activities

The 5-LOX inhibitory activity was also quantified at differentconcentrations 6.25–100 μg/ml. AAEE showed LOX inhibition at allthe concentrations tested and a concentration dependant suppres-sion of LOX activity was observed with IC50 value of 27.61 μg/ml.AAEE at doses of 6.25 μg/ml, 12.5 μg/ml, 25 μg/ml, 50 μg/ml and100 μg/ml showed 11.8%, 20.9%, 48.2%, 88.2% and 89.16% percen-tage inhibition in LOX activity, respectively. Maximal LOX inhibi-tory activity for AAEE was observed at a concentration of 100 μg/ml and the activity was comparable to the standard LOX inhibitor,Lornoxicam which demonstrated 78.53% inhibition. In contrast,Indomethacin exhibited LOX inhibitory activity of 30.94%.

3.4. In vitro studies on RAW 246.7 macrophage cell line

3.4.1. Effect of AAEE on cell viabilityTo test and verify the effect of AAEE on cell growth in RAW

246.7 cells, we used the MTT assay as shown in Fig. 1(a). It was

observed that AAEE did not impair cell viability at concentrationsupto 100 μg/ml. This concentration range was within the concen-tration range exhibiting COX 1/2 and 5-LOX enzyme inhibition.

3.4.2. Effect on NO release in LPS stimulated cell linesAfter discovering the effectiveness of test extracts on COX 1/2

and 5-LOX enzyme pathway we examined whether or not AAEEcould suppress activation of macrophages, induced by LPS. LPS isone of the most potent macrophage activation factors. The activitywas measured by quantifying the release of NO in the presence ofLPS and our test extracts. The effects of AAEE on NO production inunstimulated and LPS stimulated RAW macrophages is as shownin Fig. 1(b). Low levels of nitrate 2.99 μM were seen in the unsti-mulated macrophages. Cells activated with LPS (1 μg/ml) after 24 hcaused sustainable increase in nitrate production (25.8 μM). Co-treatment of macrophages with AAEE 6.25 to 100 μg/ml and LPS(1 μg/ml) for 24 h evoked significant decrease in nitrate produc-tion and in a concentration dependent manner. At 100 μg/ml AAEEnitrate levels maximally decreased to 4.08 μM demonstrating84.1% inhibitory effect. This indicated for strong concentrationdependent anti-inflammatory effects for AAEE. However, AAEE at

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AAEE IbuprofenControlCg

± ±

Fig. 2. (A) Effect of graded doses of AAEE on carragenean induced paw edema. Data represent the Mean7SEM (n¼6). Statistical significance: nPo0.05, nnPo0.01,nnnPo0.001, versus with respect to Cg control group (One way repeated measures ANNOVA followed by Bonferroni Multiple comparison test). (B) Effects Of AAEE on PGE2(i) and LTB4 (ii) levels in paw exudates in the carragenean rat paw model. Data represent the Mean7SEM (n¼6). Statistical significance: *Po0.05, nnPo0.01, nnnPo0.001,versus with respect to Cg control group (One way repeated measures ANNOVA followed by Bonferroni Multiple comparison test).

A. Nisar et al. / Journal of Ethnopharmacology 147 (2013) 584–594 589

100 μg/ml alone did not show any appreciable increase inNO production. The IC50 value for AAEE here was found to be25.91 μg/ml.

3.4.3. Effect on the production of pro inflammatory cytokines., TNF-αand IL-1β

The levels of pro inflammatory cytokines are important inregulating the extent of inflammation. To elucidate the effect ofAAEE on LPS stimulated TNF-α and IL-1β release, the supernatantfrom the activated macrophages was assayed using ELISA kits. Itwas observed that concurrent treatment with LPS and AAEEinhibited the release of both cytokines, TNF-α and IL-1β in aconcentration dependent manner. The inhibitory effect was82.14% for TNF-α and 74.14% for IL-1β, when AAEE concentrationwas 100 μg/ml. See Fig. 1(c).

3.5. Acute safety studies

There was no noticeable change in behaviour nor any mortalitywas observed in mice observed upto a single maximum dose of2500 mg/kg b.w dose of AAEE over a period of 14 days. No adverseeffects or mortality was observed in mice, which hint that doses ofAAEE used in the study are tolerable, without any noticeable sideeffects and was safe to use at high doses also.

3.6. Selection of doses

No toxic effects or mortality were observed in mice upto 2500 g(LD-0 g). Evaluation of the test material was initiated by giving tothe animals one-tenth of this dose (250 mg/kg b.w) as well aslower (62.5 mg/kg b.w and 125 mg/kg b.w) and higher dose(500 mg/kg b.w).

Table 1Effect of AAEE on antioxidant enzymes after carragenean challenge. Data represent the Mean7SEM (n¼6). Statistical significance: nPo0.05, nnPo0.01, nnnPo0.001, versuswith respect to Cg control group (One way repeated measures ANNOVA followed by Bonferroni Multiple comparison test).

Concentration (mg/kg b.w.) SOD (U/mg protein) GPX (U/mg protein) GPR (U/mg protein)

Ibuprofen 100 35.2372.73 2.3570.16 0.09270.005AAEE 500 30.5272.05 1.8770.12 0.08170.003AAEE 250 25.0173.18 1.4570.14** 0.06770.006*AAEE 125 15.3971.56*** 0.8970.18*** 0.04570.007***Cg Control 8.1772.54*** 0.3370.16*** 0.02370.004***AAEE 62.5 12.7572.21*** 0.6570.15*** 0.03870.003***

Table 2Effect of AAEE on leucocytes migration and exudation in pleural fluid. Datarepresent the Mean7SEM (n¼6). Statistical significance: nPo0.05, nnPo0.01,nnnPo0.001, versus with respect to Cg control group (One way repeated measuresANNOVA followed by Bonferroni Multiple comparison test).

Concentration(mg/kg)

Dye Concentration(mg/kg)

% Inhibition(against control)

Control (Naive) – – –

Control – 0.8470.04 –

AAEE 62.5 0.75 70.03 10.71AAEE 125 0.6870.04** 19.04AAEE 250 0.5770.04*** 31.33AAEE 500 0.3670.01*** 51.4Ibuprofen 100 0.5170.02*** 39.28

Mea

n ±

SE

M(p

g/m

l)

0

200

400

600

800

1000

AAEE Ibuprofen

NC Cg Control 62.5 mg/kg 250 mg/kg125 mg/kg 100 mg/kg

38.02 762.23 685.12 541.9 396.4 238.5 381.11

20.17 561.24 409.67 196.43493.8 282.3 286.77

500 mg/kg

Fig. 3. Data represent the Mean7SEM (n¼6) Statistical test employed is One WayANNOVA followed by post Bonferroni test. with N¼6 in each group. nPo0.05,nnPo0.01, nnnPo0.001, Comparisons were made between Cg Control group andother experimental groups.

Mea

n ±

SE

M(μ

M)

0

10

20

30

40

50

NC

IbuprofenAAEE

NC Cg Contro l62.5 mg/kg 250 mg/kg125 mg/kg 100 mg/kg500 mg/kg

1.68 40.7 35.8 29.54 22.38 12.61 19.74

Fig. 4. Data represent the Mean7SEM (n¼6) Statistical test employed is One WayANNOVA followed by post Bonferroni test. with N¼6 in each group. nPo0.05,nnPo0.01, nnnPo0.001, Comparisons were made between Cg Control group andother experimental groups

A. Nisar et al. / Journal of Ethnopharmacology 147 (2013) 584–594590

3.7. Inhibition of carragenean induced rat paw edema

Intraperitoneal injection of carragenean to rats caused aninflammatory reaction. These effects were observed at four time

points (0.5 h, 1 h, 2 h and 3 h). In carregenean administeredanimals, the severe paw swelling observed reached maximum at3rd h. At the end of 3 h groups treated with AAEE 62.5 mg/kg b.w,125 mg/kg b.w, 250 mg/kg b.w and 500 mg/kg b.w showed inhi-bitory effects of 14.6%, 31.7%, 48.7% and 68.2%, respectively.The percent reduction in paw edema was highly significant inthe group treated with 500 mg/kg and was more potent thanIbuprofen which showed percent inhibition of 49.8% at the end of3 h (Fig. 2A).

3.7.1. Downregulation of PGE2 and LTB4 in tissue exudatesAt 500 mg/kg b.w, AAEE demonstrated 68% inhibitory effect on

PGE2 production and 75.4% inhibitory on LTB4 production. Undersame experimental conditions Ibuprofen reduced the levels to50.67% and 50.01% in case of PGE2 and LTB4, respectively (Fig. 2B).

3.7.2. Effect on level of the anti-oxidant enzymesThe liver tissues of the animals used for the study were

analysed for anti-oxidant enzyme activities (SOD, GPX and GR).As is seen in Table 1 treatment with carragenean caused a distinctdecrease in the level of anti-oxidant enzymes. Pre-treatment ofAAEE followed by carrageenan injection was accompanied withsignificant dose dependant increase in the levels of anti oxidantenzymes. AAEE at 6.25 mg/kg b.w., 125 mg/kg b.w., 250 mg/kg b.w.and 500 mg/kg b.w. up regulated SOD, GPX and GR levels in a dosedependent manner.

Table 3Effect of graded doses of AAEE on vascular permeability. Data represent the Mean7SEM (n¼6). Statistical significance: nPo0.05, nnPo0.01, nnnPo0.001, versus with respectto Cg control group (One way repeated measures ANNOVA followed by Bonferroni Multiple comparison test).

Concentration (mg/kg) Total cells (�103/mm3) Nuetrophils (�103/mm3) Mononuclear cells (�103/mm3) Exudate volume (ml)

NC – 1.8270.41 1.5270.13 0.7 070.01 0.3170.01Cg Control – 68.197 3.91 56.7274.21 12.427 0.65 3.0170.34AAEE 62.5 60.8 74.09 50.872.12 1270.50 2.770.18AAEE 125 55.0973.86 42.2072.96nnn 11.4770.69 2.2870.19AAEE 250 38.5074.51nnn 30.4571.23nnn 10.8670.42 1.5670.10nn

AAEE 500 22.0273.98nnn 18.3171.94nnn 9.6670.50nn 0.9670.2nnn

Ibuprofen 100 3671.02nnn 24.9272.29nnn 10.0870.35n 1.3070.21nnn

A. Nisar et al. / Journal of Ethnopharmacology 147 (2013) 584–594 591

3.8. Effect on carragenean induced pleurisy in rats

The pleurisy model in rats serves as an acute model whereinflammatory response is characterized by the accumulation offluids in the pleural cavity with a large number of polymorpho-nuclear leukocytes and elevated levels of pro-inflammatorymediators.

3.8.1. Quantification of leukocyte migration and exudate volumeAs depicted in Table 2 leukocytes and neutrophil count was

significantly increased in carrageenan induced group, which how-ever got reduced in a dose dependent manner with test extractsand ibuprofen. Treatment with 500 mg/kg b.w, AAEE demon-strated maximal reduction in leukocyte and neutrophil count.No appreciable effect was seen on mononuclear cells at any doseadministered. Exudate volume was decreased by AAEE from 10%(62.5 mg/kg b.w) to 70% (500 mg/kg b.w) against the carragenean(Cg) control group. Ibuprofen at the dose 50 mg/kg showed 46.94%and 56% inhibition of exudate volume and total leukocytes,respectively. Consistent to results of carregenean paw model theinhibitory effects of AAEE were more pronounced than Ibuprofen.

3.8.2. Suppression of arachidonic acid metabolites (PGE2 and LTB4)and NO

Following carragenean injection into the pleural fluid, thelevels of PGE2, LTB4 and NO got elevated. The group treated withIbuprofen showed a significant inhibition of 49.9% on PGE2 (Fig. 3)and 48.9% on NO at a dose of 100 mg/kg b.w (Fig. 4). Compara-tively AAEE at doses ranging from 62.5 to 500 mg/kg b.w downregulated both PGE2 and LTB4 levels in the pleural fluid and in adose dependant manner. At a dose of 500 mg/kg b.w PGE2 andLTB4 levels were decreased maximally to 14% and 12% by AAEE.Also, AAEE at 500 mg/kg b.w. inhibited NO production by 69%.These effects were higher than Ibuprofen.

3.9. Effect of AAEE on vasuclar permeability in mice

Acetic acid-induced vascular permeability in a mouse model isa commonly used vascular permeability assay. The inflammatoryresponse is a physiological characteristic of vascularized tissue.In this model, AAEE at all graded doses significantly decreasedvascular permeability in a dose dependent manner. Ibuprofenreduced the dye concentration with 39.2% inhibitory effect incomparison to control. Under same experimental conditions, AAEEdecreased vascular permeability with 10.11%, 19.04%, 32.14% and57.14% inhibitory effect at 62.5 mg/kg b.w, 125 mg/kg b.w, 250 mg/kg b.w and 500 mg/kg b.w dosages as shown in Table 3. Interest-ingly the effect of AAEE at 500 mg/kg b.w was more potent whencompared with the Ibuprofen treated group.

4. Discussion

Inflammatory response is basically a dynamic process thatinvolves a complex set of interactions among inflammagens thatarise in any tissue in response to traumatic, infectious, post–ischemic, toxic or auto immune injury (Zedler and Faist, 2006).The present study was intended to investigate the modulatoryeffects of Atropa acuminata on various such inflammagens in vitroand in vivo acute inflammatory models.

Cycoloxygenase 1 and 2 and 5-lipoxygenase are the rate limitingenzymes that catalyze the formation of two multifunctional inflam-matory mediators, prostaglandins and leukotrienes (Simmons et al.,2004). In order to screen and evaluate AAEE for its anti-inflammatoryproperties, we first aimed to examine its inhibitory effects on bothCOX 1/2 and 5-LOX pathways in vitro. COX exists in two isoforms,COX 1 and COX 2. COX 1 is a constitutive isoform expressed inplatelets, kidney, GIT and liver whereas COX 2 is an inducible isoform(Vane et al., 1998). Our in vitro study revealed that AAEE exhibiteddose dependant inhibitory effects on COX 1 as well as COX 2 enzymeswith maximal inhibitory effects observed at a concentration of100 μg/ml. This concentration related inhibitory activities werehighly comparable to indomethacin and Celecoxib, the standardCOX1 and COX2 inhibitors, respectively. Several plant extracts havebeen reported for in vitro inhibitory effects on COX enzymes (Albertoet al., 2009; Farahbakhsh et al., 2011; Li et al., 2004). Manycompounds like procumentin, gossyptein and chrysin isolated fromdifferent plants have demonstrated significant COX 2 enzyme inhibi-tion. Many flavanoids and phenolic components have been reportedto inhibit COX enzymes via iNOS mechanism (Mada et al., 2009). Ithas been reported that in macrophages, LPS induces co-expression ofinducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2)resulting in the production of pro-inflammatory mediators., nitricoxide and prostaglandins at the inflammatory site (Lee et al., 1992).The COX inhibitory activities of AAEE could be attributed to itsregulation at the level of iNOS and COX 2 gene like many otherplants. For example, Rosa Rugosa reported anti-inflammatory effectshave been shown to suppress both iNOS and COX 2 expression (Anet al., 2011).

5-LOX, a non heme iron containing dioxygenase is implicated toplay a critical role in cardiovascular, asthma and various associatedinflammatory disorders (Peters-Golden, 2008). For 5 –LOX path-way, AAEE afforded concentration dependant inhibition withmaximal inhibition at 100 μg/ml and this effect was significantlycomparable to standard drugs, indomethacin and lornoxicam.It has been reported that several whole plant extracts like thatof Tinospora smilancan and Cissus quadrangularis act as potentinhibitors of LOX (Bhujade et al., 2012; Li et al., 2004). Further,many Boswellic acid derivatives from Boswellia seratta have alsobeen found to act as novel and specific inhibitors for LOX (Ammon,1992). We know that 5-LOX is the main enzyme catalyzing theproduction of LTB4 (Back,2008; Noguchi and Okubo, 2011). AAEEexhibited dual inhibitory effects on both COX 1/2 and 5-LOXenzymes. Interestingly the effects of AAEE on 5 –LOX pathway

A. Nisar et al. / Journal of Ethnopharmacology 147 (2013) 584–594592

were higher as compared to COX enzymes. In recent years, severalinvestigations have suggested that both traditional NSAIDS andnewer Celecoxibs inhibit the COX enzymes but in turn shunt thearachidonic acid metabolism towards LTB4 biosynthesis (Burnettand Levy, 2012) consequently leading to renal dysfunction(Perazella and Eras, 2000) and GIT side effects (Hinz and Brune,2002). Therefore the use of “dual inhibitory agents”, which couldinhibit not only COX 1 and COX 2 enzymes but also leukotrienebiosynthesis via 5-LOX pathway has evolved as a vital target foranti-inflammatory therapies (Brune,2004). Such agents tend tobalance the AA metabolism in the body and are therefore con-sidered to be particularly effective in minimizing the gastric andcardiovascular side effects (Leone et al., 2007). Our in vitro dataindicated AAEE exhibiting potential for similar dual inhibitoryeffects.

To gain more information on the underlying mechanisms of theanti-inflammatory effects of our test extracts, we extended studieson murine macrophage cell line, RAW 246.7 for the effects on NO,IL-1β and TNF-α production in the presence of LPS, a stimulant.Our results demonstrated that AAEE markedly down regulated theLPS induced NO production along with the pro inflammatorycytokines in RAW cells. Parallel to the findings on COX 1, COX2 and 5 –LOX enzymes, macrophages when treated with variousconcentrations of AAEE revealed concentration dependentdecrease in NO, TNF and IL-1β production. Maximal inhibitoryeffects were observed for AAEE at 100 μg/ml. To rule out anycytotoxicity induced anti inflammatory effects of AAEE on RAWcells, MTT assay was carried out in absence of LPS. Henceforth itwas evident that AAEE inhibited the production of NO and pro-inflammatory cytokines in activated macrophages without anytoxic effects. Macrophages secrete NO and a series of cytokinesparticularly—TNF-α, IL-1β (Qureshi et al., 2012) and aberrant oroverproduction of these mediators has been implicated in thepathogenesis of several chronic inflammatory diseases such asarthritis, multiple sclerosis, Parkinson's disease and Alzheimer's(Takahashi et al., 1997). NO is reported to activate both theconstitutive COX 1 as well as the inducible COX 2 (Salveminiet al., 1993), thus the in vitro inhibition of COX enzymes by AAEEcould be attributed to suppressive effects on NO production,presumably a consequence of iNOS inhibition at transcriptionlevel. iNOS is the key enzyme involved in NO production and LPSactivated macrophages have been reported to co-induce iNOS, COX1 and COX 2 enzymes. In addition, both TNF-α and IL1-β synergis-tically are involved in chronification of inflammatory reactionsthrough induction of COX 2 expression in activated immune cellsand also enhances the production of PGE2 (eicosanoids) (Raz et al.,1988). Therefore, inhibition of COX 2 activity by AAEE could alsointurn modulate local and systemic cytokine responses. Hence wecould possibly relate the COX enzyme inhibitory activities of AAEEand suppression of NO production in vitro to the down regulation ofpro inflammatory cytokines. Our findings that AAEE inhibits for-mation of NO, pro inflammatory cytokines, COX 1, COX 2 and 5-LOXenzyme activities point to their role in the anti-inflammatory effectsof our test extracts.

The data of test extracts obtained from in vitro studies wastested further by carrying out various in vivo studies in differentacute models of inflammation. In all the animal models, AAEEexhibited potent anti inflammatory activity when administeredorally. Upto 2500 mg/kg b.w. no signs of mortality nor anytoxicological consequences were observed for AAEE indicatingabout its safe usage even at higher doses.

The carragenean induced paw edema has been commonly usedas an experimental model to assess the contribution of mediatorsinvolved in vascular changes associated with acute inflammation(Winter and Nuss, 1962). The development of edema in rat hindpaw following an injection of carragenean has been categorized as a

biphasic event in which various mediators operate in a sequence toproduce inflammatory response. The initial rapid phase (0–1 h) ofthe inflammation has been attributed to the release of histamine,serotonin, bradykinin and 5-hydroxytryptamine and similar sub-stances (Di Rosa et al., 1971). The latter accelerating but sustainedphase of swelling (upto 3 h) is sustained by proteases, lysozymes,prostaglandin release, leukotrienes and NO by polymorphonuclearcells and has been attributed to the induction of inducible cyclox-ygenase and iNOS (Sidhapuriwala et al., 2007). AAEE at a dose of500 mg/kg reduced edema to a greater extent than standard drug,ibuprofen. NSAIDS like ibuprofen are reported not to be effective inthe later phase of such edema formation (Boughton-Smith et al.,1993). In our study, dose dependent suppressive effects on pawedema by AAEE were observed in both the phases, howeversignificant inhibitory effect was seen only at 3 h suggesting a morepotent inhibition on PGE2 release than histamine or serotonin.Consistent to these reports, the production of eicosanoids., PGE2and leukotrienes., LTB4 in paw exudates was also significantlyreduced and the effects seen, were more potent than ibuprofen.

The carragenean induced inflammatory response has also beenstrongly linked to the release of neutrophil dervived mediators likeH2O2, O2

−, OH radicals. ROS produced for the neutrophils andmacrophages contribute to macromolecular based tissue injuriesduring inflammatory reactions. There has been large body ofevidence to link the production of free radical species (ROS) tothe pathophysiolgy of tissue damage associated with inflamma-tory response. In various, in vivo models it has been revealed thatmany of the anti inflammatory agents mediate their effectsthrough the inhibition of free radical generation by activatedneutrophils (Winrow et al., 1993). Among the compensatoryresponses against ROS generation, SOD, GPX and GRX are theenzymes of particular interest. In our findings, we observed asignificant dose dependent increase in these antioxidant enzymesby the test plant extracts. AAEE upregulated the levels ofglutathione peroxidase (GPX) and glutathione reductase (GR).The effects were comparable with Ibuprofen.

Carregenean injection in the pleural fluid promoting an acuteinflammatory response resulted in increased total leukocytecounts in particular the neutrophil counts and also increasedexudation volume (Di Paola et al., 2004). Normally the pleuralfluid contains a few lymphocytes not more than 100 cells/ml(Light, 1990). However carregenean injection in the pleural fluidcaused noted exudation, increased neutrophil and total leukocytecount. In contrast, treatment with Ibuprofen and test plantextracts decreased the cell count and also the exudate volume,with maximal effects seen at 500 mg/kg body weight. Thedecrease in exudate volume by AAEE may be attributed tosuppressive effects of AAEE on vascular permeability, since AAEEalso decreased acetic acid induced vascular permeability in micemodel. Acetic acid unleashes the release of several mediators suchas bradykinin, substance P and prostaglandins, as well as cytokinessuch as IL-1β, TNF-α and IL-8 (Sekiya et al., 1982). Total decreasedleukocyte count could be attributed to significant inhibition ofneutrophils. Neutrophils have been implicated for potential tissuedamage in chronic inflammatory diseases and are also involved inthe synthesis of leukotrienes and thromboxane A2 and in severalinflammatory conditions (Kitz et al., 2006). AAEE was observed tohave potent capacity to significantly reduce the migration ofneutrophils to the site of inflammation.

PGE2 is synthesized in substantial amounts at target sites ofinflammation to potentiate vasodilation, increase blood flow,cause increased vascular permeability and potentiate the effectof pro-inflammatory cytokines (IL-1β) (Legler et al., 2010). LTB4 is astrong chemoattractant for neutrophils and implicated in cardio-vascular asthmatic diseases. Taken together, these cause vascularleakage and extravasation of fluid and cause pain, erythema and

A. Nisar et al. / Journal of Ethnopharmacology 147 (2013) 584–594 593

swelling, all symptoms of acute inflammation (Sadik and Luster,2012). We observed reduced production of eicosanoids and leuko-trienes for AAEE possibly from downregulation of COX 1 and COX2 and 5-LOX in both acute inflammatory responses.

Our findings also demonstrated decreased NO production aftertreatment with AAEE in the pleural fluid which could be respon-sible for the decrease in PGE2 production. NO maintains thecarragenean evoked inflammatory response by activation of COXpathway (Salvemini et al., 1993). It can be speculated that COXpathway might not have been activated in absence of activationsignals by NO after treatment with AAEE and which in turn causedthe suppression of PGE2 production and the subsequent inhibitionof inflammation. The significant suppression of NO and acutetissue damage with increased dosage of AAEE indicated for diverserange of effects for Atropa acuminata. Since NO activity has beenrelated to activated leukocytes, cell migration and exudation, lowNO levels after oral AAEE administration could be explained bydecreased influx of leukocytes. In addition, NO promotes entry ofphagocytes into inflamed areas by increasing permeability of postcapillary venules (Bogdan, 2001). Also NO and PGE2 production isreported to be regulated by NO synthase and cytokines. Thisindicates for an extensive range of action for Atropa acuminata.The significant decrease of NO and PGE2 could be attributed to thedownregulation of cytokines and the consequent decreased ofexpression of iNOS and COX enzymes.

Many of such naturally occurring compounds such as poly-phenolics, diterpenoids, triterpenoids are reported to ameliorateinflammatory response. Experimental data has revealed significantcorrelation between the high content of phenolic compounds andthe pharmacological activities of natural products (Spada et al.,2008). The qualitative and quantitative screening of phytochem-icals showed AAEE to have significant high phenolic content. Totalflavonoid and flavanol concentration was also significantly high.Flavonoids also have biochemical effects including inhibition ofenzymes such as lipoxygenase, cycloxygenase, etc. and have beenfound to have anti-inflammatory activity in both proliferative andexudative phases of inflammation. The diverse mode of action ofAtropa acuminata maybe attributed possibly due to the highpolyphenolic and flavonoid content present in the extract.

5. Conclusion

Collectively our results with Atropa acuminata demonstratedhighly potent anti-inflammatory effects mediated by suppressionof release of inflammatory mediator, NO, prostaglandins, leuko-trienes and pro-inflammatory cytokines. Since pro inflammatorycytokines upregulate the expression of such genes (NF-kB) that areinvolved in inflammatory response and cause co- induction ofiNOS and COX2, the two pivotal enzymes related to NO andeicosanoid production in inflamed tissues, Atropa acuminata canbe presumed to act through inhibition of NF-κB signaling pathwayleading to reduced expression of interdependent cytokine and NOproduction and in turn lead to decreased COX enzyme activity.

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