antiasthmatic activity of methanolic extract of sphaeranthus indicus

Available online on www.ijppr.comInternational Journal of Pharmacognosy and Phytochemical Research 2010; 2(3): 15-19Research ArticleISSN: 0975 4873Antiasthmatic Activity of Methanolic Extract of Sphaeranthus indicusM. S. Prajapati*, M. B. Shah, A. K. Saluja, U. D. Shah, S. K. ShahDept. of Pharmacognosy and Phytochemistry, A. R. College of Pharmacy & G. H. Patel Institute of Pharmacy,Vallabh vidyanagar -388120 (Gujarat)ABSTRACTSphaeranthus indicus auct. Non L. (Asteraceae) commonly known as Gorkhmundi. It is used for various ailments inIndian system of traditional medicines. Methanolic extract of aerial parts was evaluated for antiasthmatic activity.Histamine induced bronchospasm in guinea pigs and egg albumin induced bronchospasm in guinea pig model wereperformed and various parameters were investigated like PCD time, serum bicarbonate level, differential leukocytescount and histopathological changes in lung. Biochemical estimation like glutathione (GSH) level, malondialdehyde(MDA) level and total proteins were also checked. The result showed that it significantly increased preconvulsiondyspnoea (PCD) time, decreased differential leukocyte counts and serum bicarbonate level. It decreased the oxidativeburden by reducing the MDA level and increasing the GSH level which was imbalanced in asthmatic condition, alsoshown significant action on total protein level. The drug also reduced the inflammation and dilated the bronchioles. Allthese data suggested that S. indicus is having promising antiasthmatic activity.Key words: S. indicus, Histamine, Antiasthmatic, Asteraceae and BronchospasmINTRODUCTIONAsthma is a chronic inflammatory disorder of theairways in which many cells and cellular elements play arole, in particular, mast cell, eosinophils, T-lymphocytes,macrophages, neutrophils, and epithelial cells 1 .Thepathophysiological changes occur in asthma is likeairway obstruction due to combination of various factorsinclude spasm of airway smooth muscle, edema ofairway mucosa, increased mucus secretion, cellularinfiltration of the airway walls and injury anddesquamation of the airway epithelium 2,3 .Sphaeranthus indicus auct. Non L. (Asteraceae)commonly called as Gorkhmundi is found to be grown inrice fields, dry waste places and cultivated throughoutIndia in rainy season, Sri Lanka, Africa and Australiafrom sea level to 1200m altitude 4 . It is a much branched,strongly scented annual erect herb most commonly growas a weed. The plant flowers in October- January andfruits are formed from December – April 5 . According toAyurveda, this herb is hot, laxative, digestible, tonic,fattening, alterative, anthelmintic and alexipharmic. It isused in tuberculosis, bronchitis, anemia, piles, asthma,leucoderma, etc. All the parts of the plant have medicinalvalue. The juice of the plant is styptic and said to beuseful in liver and gastric disorders 6 . The sesquiterpeneglycoside isolated from S.indicus is havingimmunostimulant effect 7 . The plant is reported to possessanti-inflammatory, antimicrobial activity and woundhealing property 8, 9 . Considering the ayurvedic uses andreported activities of the drug, it is proposed toinvestigate its antiasthmatic potential using variousexperimental models.MATERIAL AND METHODSAll chemicals and solvents used were of analyticalgrades. Ketotifen fumarate was obtained as a gift samplefrom FDC Ltd., Mumbai. Histamine hydrochloride waspurchased from Chitichem, Baroda. Egg albumin,sodium dodecyl sulfate, acetic acid, hydrochloric acid,thiobarbituric acid (TBA), trichloroacetic acid, disodiumhydrogen phosphate, 5, 5’-dithiobis-2-nitrobenoic acid,sodium-potassium tartrate, sodium hydroxide, sodiumcarbonate, Folin-phenol reagent were of analytical grade.Plant material and ExtractionFully grown, fresh entire herb of Spharaenthus indicusauct. Non L. was collected in the month of Decemberfrom Vallabh Vidyanagar. The herb was authenticatedby a taxonomist at the Department of Bioscience, SardarPatel University, and Vallabh Vidyanagar. A voucherspecimen (no argh-7) was deposited in the Departmentof Pharmacognosy, A. R. College of Pharmacy, VallabhVidyanagar. The aerial parts were cleaned, dried at roomtemperature, powdered to coarse powder (60 #) andstored in air tight container. 5 kg of dried aerial parts ofSphaeranthus indicus was exhaustively extracted bysubjecting to reflux below 80 0 C (3 X 5 L). Thecombined methanol extract was distilled and evaporatedto dryness (25 %w/w). A weighed quantity of the extractwas taken and suspension was prepared using Tween 80and was used for further study.AnimalsGuinea pigs (450-600 gms) were procured from AnimalVaccine Institute, Gandhinagar. All animals were housedat ambient temperature (21±10C) and relative humidity(55±5 %) with fixed 12h/12h light/dark cycle. Animalshad free access to standard pellet diet and water given ad*Author for correspondence: manishasprajapati@gmail.com

Prajapati et.al./ Antiasthmatic activity of methanolic extract…libitum. The experimental protocol was approved byInstitutional Animal Ethical Committee as per theguidance of committee for the purpose of Control andSupervision of Experiments on Animals (CPCSEA),Ministry of Social Justice and Empowerment,Government of India.Table: 1 Effect of Sphaeranthus indicus on PCDtime in guinea pigs after acute treatment.Treatment group % increase in time ofPCDNormal control group 5.17 ± 2.54Ketotifen group (1mg/kg) 87.35±3.80 @MSI group (150mg/kg)MSI group (300mg/kg)64.26±2.62 @36.34±4.8 @(sensitized group) and Group III received methanolicextract of Sphaeranthus indicus (MSI group)(150mg/kg).Normal groupMSI group (450mg/kg) 29.63±3.9 @@ Significantly different from control group (p < 0.05)MSI- Methanolic extract of aerial part of S. indicusIn vivo antiasthmatic activityHistamine induced bronchospasm in guinea pigThe guinea pigs (Animal breeding centre) were kept in aclosed chamber and exposed to an aerosol of 0.1 percenthistamine hydrochloride and time for preconvulsiondyspnoea was noted. As soon as preconvulsion dyspnoea(PCD) commenced, animals were removed from thechamber and placed in fresh air to recover. This time forPCD was taken as basal value. After 15 days of wash outperiod the same animals were randomly divided into fivegroups each containing six animals. Group I receiveddistilled water (normal control); Group II receivedKetotifen (1 mg/kg, P.O.) (Standard control) and GroupIII, IV and V received methanolic extract of S. indicus(MSI group) in divided doses 150, 300 and 450 mg/kgrespectively. The suspension of drugs was administeredorally. Two hours after the drugs treatment, animalswere exposed to histamine aerosol and times for PCDwere noted. The effect of drug was calculated by thefollowing formula 10% increase in PCD time = [1 – T1/T2] X 100Where, T1 = time for PCD onset on day 0, T2 = time forPCD onset after drug treatment.Egg albumin induced bronchospasm in consciousGuinea pigsHartley strain Guinea pigs of either sex weighing 400-600 gm were selected. The animals were examined forthe following parameters:Eosinophil and macrophages count in thebronchoalveolar fluidSerum bicarbonate levelMeasurement of MDA levelMeasurement of GSH levelMeasurement of total protein levelHistopathologyThe animals were randomly divided into three groupseach containing six animals. Group I received distilledwater (normal control), Group II received egg albuminSensitized groupMSI groupFig. 1 Histopathological studiesEosinophil and macrophages count in thebronchoalveolar fluidThe suspension of drugs was administered orally. Theguinea pigs of all the groups except Group I weresensitized with egg albumin (1 ml, 10% w/v).Theanimals of Group III was dosed once daily for fifteendays with the suspension of drug. Two hours after thelast dose of drug administration on fifteen days all theanimals except Group I animals were challenged withegg albumin (0.5 ml, 2% w/v) through saphenous vein.After 3 hours of the challenge of the egg albumin or justprior to death of animals, whichever was earlier, thetracheobronchial tree was lavaged with 10 ml of salineand the fluid so collected was centrifuged at 2000 rpmfor 5 minutes and the pellet was resuspended in 0.5 mlsaline. 0.2 ml of geimsa stain in buffered saline (pH 6.5)was added to it. After 5 minute the number of each typeof leukocytes in 0.5 ml fluids was determined under themicroscope 450 X magnifications. The results obtainedwere compared with unsensitized and untreated eggalbumin sensitized guinea pig. 11Serum bicarbonate levelSerum is suitable specimens for measurement ofbicarbonate level. The whole blood should be collectedand handled anaerobically to minimize exposure to air.Serum bicarbonate is stable for one hour when storedunder anaerobic conditions in an ice bath. Lable one testIJPPR September - November 2010, Volume 2, Issue 3 (15-19) 16

Prajapati et.al./ Antiasthmatic activity of methanolic extract…tube or cuvette for a reagent blank. Calibrator, controisand unknown sample. Add 1 ml of completely dissolvedreagent to each tube or Cuvette. Add 0.01 ml of H 2 O tothe reagent blank and 0.01 ml of calibrator, controls andunknown sample to the appropriately labeled tube. Mixand incubate for 5min and measure the absorbance at380 nm. 12Table: 2 Effect of MSI on differential leukocytecounts of the bronchoalveolar lavage (BAL) fluidNormalcontrolgroupSensitizedgroupMSI group(150mg/kg)TLC/cmm7134.16± 13440.16 11422.5 ±101.79 ± 315.9 # 537.18 $Polymorphs 20.5 ± 37.74 ±count/cmm 0.83 0.93 # 34.09 ± 1.29 $Lymphocyte 49.79 ± 58.86 ±count/cmm 0.66 0.76 # 52.18 ± 1.44 $Eosinophil 6.21 ± 14.05 ±count/cmm 0.31 0.62 # 8.98 ± 0.57 $Monocytecount/cmm4.83±0.3313.41 ±0.87 # 7.01 ± 0.91 $# Significantly different from control (p

Prajapati et.al./ Antiasthmatic activity of methanolic extract…in diagnosis and treatment. Lloyd-lake London 16. Fireman, P. Allergy Asthma Proc. 2003; 24 2: 79-831979; chapter IV: 78-113. 17. Hulya Ercan, Esra Birben, Evrim A. Dizdar, Ozlem13. J. A. Buege, S. D. Aust, Microsomal lipidperoxidation method. Enzymology 1978; 52; 302–Keskin, Cagatay Karaaslan, Ozge U. Soyer, RaziyeDut, Cansin Sackesen, Tanju Besler, and Omer310.Kalayci, “Oxidative stress and genetic and14. C. W. Owens, R.V. Belcher, Biochem. J. 1965; 94,705–711.15. Busse, W. W. and Lemanske, R. F. Jr. Asthma Nepidemiologic determinants of oxidant injury inchildhood asthma”, J allergy clin immunol. 2006;18 (5), 1097-1104Engl. J. Med. 2001; 344: 350-362IJPPR September - November 2010, Volume 2, Issue 3 (15-19) 19