Anti-inflammatory activity of Pupalia lappacea L.Juss

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A.Tamil Selvan etal / Int. J. of Allied Med. Sci. and Clin. Research Vol-22 2014 97-101 Corresponding author: A.Tamil Selvan E-mail address: 97 IJAMSCR |Volume 2 | Issue 2 | April - June - 2014 Research article Anti-inflammatory activity of Pupalia lappacea L.Juss A.Tamil Selvan M.Rama Devi N.Siva Subramanian L.Srinivas K.Prathyusha K.Sri Silam T.Lalappa Usha Yadav Department of Pharmacology Teegala Krishna Reddy College of Pharmacy Meerpet Saroor nagar Hyderabad Andhra Pradesh. ABSTRACT Pupalia lappacea L Juss is an erect shrub used in folklore medicine to treat bone fractures and in inflammatory conditions. Methanolic extract of aerial parts shown is claimed in traditional medicine that the leaves of the plant are used in the treatment of inflammation. In the present study the methanolic extract of Pupalia lappacea was screened for its anti-inflammatory activity using carageenan induced rat paw edema egg white induced paw oedema models. The methanolic extract at the dose of 200 mg/kg p.o exhibited significant anti-inflammatory activity in carrageenan induced paw edema model p0.01. In egg white induced model methanolic extract at the dose of 200 mg/kg inhibited paw oedema significantly p0.01 indicating that both test samples inhibit the increase in number of fibroblasts and synthesis of collagen and mucopolysaccharides during prostaglandin formation during the inflammation. These experimental results have established a pharmacological evidence for the folklore claim of the drug to be used as an anti inflammatory agent. HPTLC analysis of the extract shows the presence of gallic acid 1.24 g/ml ferulic acid 2.00 g/ml chlorogenic acid 46.25 g/ml and rutin 7.02 g/ml of the extract which were responsible for the claimed anti-inflammatory action in the animal models studied. Keywords: Pupalia lappacea Methanolic extract Aerial parts Diclofenac sodium Carrageenan Egg white HPTLC Rutin Gallic acid Ferulic acid and Chlorogenic acid. INTRODUCTION Herbal medicines are becoming more and more popular now a days. Among the entire flora 35000 to 70000 species have been used for medicinal purposes. This is the highest proportion of medicinal plants known for their medicinal purposes in any country of the world for the existing flora of that respective country. Pupalia lappacea belongs to the family Amaranthaceae is commonly known as Forest Burr or Creeping cock’s comb. It is an erect or straggling under shrub found in the hedges of fields fruit orchards dry scrub forests and waste places of Kashmir to Kauman at an altitude of 300-1050 m and in all states of India 1 . The leaf paste of Pupalia lappacea with edible oil Sesamum or Carthamus is an effective and inexpensive treatment of bone fracture for human beings as well as cattle. Stem is used as tooth brush for treating toothache. Poultice of the fresh leaves is used in treatment of boils new and chronic wounds. A decoction of the black powder of the plant is drunk to cure piles and for enema fever and malaria 2 . In this present work the methanolic extract of Pupalia lappacea was preclinically evaluated for anti-inflammatory activity by invivo carrageenan and egg white induced paw edema method and by invitro heat induced haemolytic method and protein inhibition method. MATERIALS AND METHODS Collection International Journal of Allied Medical Sciences and Clinical Research IJAMSCR

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A.Tamil Selvan etal / Int. J. of Allied Med. Sci. and Clin. Research Vol-22 2014 97-101 98 The aerial parts were purchased from Dr.Madhava Chetty Sri Venkateswara University Tirupathi and was air dried until free from moisture. Then they were subjected to size reduction to get coarse powder of desired particle size. The powdered drug was subjected to extraction with petroleum ether and methanol in a Soxhlet extractor temperature was maintained on an electric heating mantel with thermostat control. The extracts were then concentrated to 3/4th of their original mass using rotary vapour apparatus. The concentrated extract were then transferred to a china dish and evaporated on a thermostat controlled water bath till it formed a thick paste. The thick mass was vacuum dried in a dessicator till it is free from moisture. Phytochemical test Phytochemical tests on the extract was performed using standard procedures 3 . HPTLC analysis Chromatography was performed on silica gel F254 HPTLC pre-coated plates. Samples were applied on the plates as band of 7mm width using a Camag Linomat V sample applicator at the distance of 14mm from the edge of the plates. The mobile phase was constituted of ethyl acetate-acetic acid- formic acid-water 100:11:11:27 V/V/V/V 4 5 . After development plates were dried and derivatised in NP-PEG reagent. The finger prints were evaluated at 366 nm in fluorescence mode with WinCats and VideoScan software. Extracts Peaks Rf values Peak height and Peak area were given in Table - . The HPTLC chromatogram was given as Fig – 1. Animals Normal healthy male wistar albino rats 180-240g were housed under standard environmental conditions at temperature 25±2º C and light and dark 12: 12 h. They were fed with standard pellet diet and water ad libitum. Toxicity and Pharmacological studies were carried out with approval of Reg.No.GNIP TKR/CPCSEA /IAEC /2013/06 IAEC members for the title method selected animal species and parameters to be evaluated. Toxicity study The acute toxicity study was performed to assess the acute toxic effects and to determine the minimum lethal dose of the plant extract as per the guideline OECD 423 acute toxic class oral method. The methanolic extract of Pupalia lappacea was administered orally to different groups of overnight fasted mice at the dose 5 50 300 and 2000mg/kg of body weight. After the administration of the extracts animals were observed continuously for 24hrs for any toxic manifestation. Further the animals were kept under investigation upto a period of one week. 6 Pharmacological screening Anti-inflammatory activity – Carrageenan induced paw oedema For carrageenan – induced paw edema model healthy rats 100-190gm n6 of either sex were grouped into three groups containing six animals per group. The animals were fasted over night prior to the start of the experiment and water ad libitum. First group control negative control received normal saline solution 10ml/kg and second group received test drug Pupalia lappacea methanolic extract 200mg/kg and third group received standard drug diclofenac sodium 4.5mg/kg 1ml dissolved in normal saline orally with the help of an oral catheter respectively. After 1h the rats were challenged with 0.2ml of 1 W/V solution of carrageenan into the sub plantar side of the right hand paw. The paw was marked with ink at the level of lateral malleous and immersed in mercury up to the mark. A plethysmometer was used for the measurement of rat paw edema volume 7 . The paw volume was measured immediately after injection 0 h and then every hour till 3 hour after injection of carrageenan to each group. The difference between the initial and subsequent reading gave the actual edema volume. The average paw swelling in the group of drug treated rats was compared with untreated control rats and the percent of inhibition of the edema formation was determined using formula: Percentage inhibition 100X 1- Vt/Vc Where ‘Vc’ represents edema volume in control ‘Vt’ represents edema volume in the group treated with test drug. Fresh egg white induced paw edema Swiss albino rats were divided into three groups. Each consists of 6 animals First group served as negative control. The second group received diclofenac sodium 4.5mg/kg. The third group received methanolic extract of Pupalia lappacea 200mg/kg. Edema was induced by administration of 0.75ml of undiluted fresh egg white in the sub-

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A.Tamil Selvan etal / Int. J. of Allied Med. Sci. and Clin. Research Vol-22 2014 97-101 99 plantar region 3. The paw volume was measured at 0 min – 60 min after the injection of undiluted fresh egg white using plethysmograph 8 . Then percentage inhibition of edema is calculated by using formula: Percentage inhibition of edema mean of control – mean of test / mean of control X100 RESULTS The percentage yield of the methanolic extract was 20 W/V of methanol. The phytochemical evaluation showed the presence of alkaloids terpenoids flavonoids carbohydrates tannins phenolic compounds saponins and phytosterols. Toxicity study was done upto 2000mg/kg and the animals were monitored for 24 hrs to 72 hours. The animals didn’t show any toxic symptoms and the abnormal behaviour after extract administration. From this the extract was found to be safe even up to 2000mg/kg and the LD 50 was calculated as 200mg/kg as the therapeutic dose 1/10 LD 50 ED 50 . Invivo anti-inflammatory activity was screened at the dose of 200mg/kg. The methanolic extract at the dose of 200 mg/kg p.o exhibited significant anti-inflammatory effect in carrageenan induced paw edema model p0.01 Table - 2. It was a well known fact that carrageenan induced paw edema model was commonly employed as an experimental model for evaluating anti- inflammatory activity of natural products the extract inhibited the inflammatory mediators and prostaglandin synthesis there by exhibited anti- inflammatory action. Also the extract inhibited the paw edema formation and few volumes in egg white induced few edema model. The inflammatory is physiological characteristic of vascular tissue damage which was inhibited by the extract significantly p0.01 in egg white induced paw inflammation Table – 3. DISCUSSION In spite of the tremendous development in the field of synthetic drug during recent era. They are found to have some are other side effects where as plants still hold their own unique place by the way of having no side effects. Therefore a systematic approach should be made to find out the efficacy plants against inflammation so as to explants them as herbal anti-inflammatory agents. It is well known that carrageenan induced paw oedema is characterised by biphasic events which involves of different inflammatory mediators. In the first phase chemical mediators such as histamine and serotonin play a role while in second phase 3 – 4 hour after carrageenan injection kinin and prostaglandins are involved. The results revealed that administration of methanolic extract inhibited the oedema starting from the first hour and during all phases of inflammation which probably inhibition of different aspects in chemical mediators of inflammation. The anti-inflammatory effect may be due to their content of tannins flavonoids alkaloids saponins and carbohydrates. Diclofenac sodium is a cyclo-oxygenase inhibitor and can be said to the inhibit the cyclooxgenase enzyme but lipooxygenase inhibitors also posses significant antiinflammatory activity against carrageenan induced paw edema. So inhibition of carrageenan induced paw oedema by the crude extract could also be due to its inhibitory activity on the lipooxygenase activity 910 . Exudation which is a consequence of vascular permeability at various times after injury. Chemically induced permeability can causes an immediate reaction and its inhibition suggests that the extract may effectively suppress the exudative phase of acute inflammation induced by undiluted fresh egg white. The results also shows the effect of extracted compound on a oedematous response to fresh egg white paw oedema provoking an inhibitory effect equal to that of standard. From the above studies it was quite apparent that the methanolic extract of Pupalia lappacea possesses significant anti-inflammatory activity. The study justifies its use in inflammation as suggested in the folklore medicine. Table - 1HPTLC estimation methanolic extract of Pupalia lappacea Flavonoids Rf value Peak height Peak area Concentration gm/ml Gallic acid 0.06 38.672 343.048 1.24 Ferulic acid 0.08 42.469 1436.64 2.00 Chlorogenic acid 0.78 837.28 837.28 46.25 Rutin 0.92 946.229 946.229 7.02

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A.Tamil Selvan etal / Int. J. of Allied Med. Sci. and Clin. Research Vol-22 2014 97-101 100 Figure – 1 HPTLC Chromatogram of methanolic extract of Pupalia lappacea Table –2 Anti-inflammatory activity of Pupalia lappacea methanolic extract by carrageenan induced paw oedema method n6 values are expressed in mean ±SEMP0.05 P0.01 Assessed using One way ANOVA followed by Tukey’s multiple comparison test. Figure –2 Anti-inflammatory activity of Pupalia lappacea methanolic extract by carrageenan induced paw oedema method Control Diclofenac sodium PL 0.0 0.5 1.0 1.5 0 min 30 min 60 min 120 min 240 min Treatment Paw volume mm Table – 3 Anti-inflammatory activity of Pupalia lappacea methanolic extract by Egg white induced paw oedema method Treatment 0 min 15 min 30 min 45 min 60 min Control 0.20±0.00 0.60±0.00 0.40±0.00 0.40±0.00 0.40±0.00 PL extract 200mg/kg 0.20±0.00 0.40±0.00 0.40±0.00 0.30±0.00 0.20±0.00 Diclofenac sodium 4.5mg/kg 0.30±0.00 0.70±0.00 0.30±0.00 0.30±0.00 0.10±0.00 n6 Values are expressed in Mean±SEMp0.01 P0.001 Assessed using One way ANOVA followed by Tukey’s multiple comparison test. Treatment 0 min 30 min 60 min 120 min 240 min Control 0.48 ±0.01 0.31 ±0.01 0.36±0.02 0.36±0.02 0.38 ±0.01 PL extract 200mg/kg 1.05±0.02 0.75±0.02 0.36±0.02 0.150±0.02 0.05±0.00 Diclofenac sodium 4.5mg/kg 0.068±0.02 0.51±0.01 0.26±0.08 0.02±0.02 0.01±0.02

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A.Tamil Selvan etal / Int. J. of Allied Med. Sci. and Clin. Research Vol-22 2014 97-101 101 Figure –3 Anti-inflammatory activity of Pupalia lappacea methanolic extract by Egg white induced paw oedema method 0 min 15 min 30 min 45 min 60 min 0.0 0.5 1.0 1.5 Control PL Extract 200mg/kg Diclofenac 4.5mg/kg Treatment time Paw volume mm REFERENCES 1 Anonymous The Wealth of India A Dictionary of Indian raw materials and industrial products Council of Scientific and Industrial Research New Delhi India 1950 Vol.3 324. 2 Reddy S.C. Reddy K.N. Murthy E. N. and Raju U. S. Traditional Medicinal plants in Seshachalam Hills Andhra Pradesh India Journal of Medicinal Plants Research 3 2009 408-412. 3 Kokate C. K. Practical Pharmacognosy Vallabh Prakashan New Delhi India fourth ed. 1994 107- 109. 4 Harborne J. B. Phytochemical Methods A guide to modern techniques of plant analysis Springer Pvt Ltd New Delhi India third ed. 2007 45. 5 Armatu et al Evaluation of antioxidant and free radical scavenging potential of some Lamiaceae species growing in Romania Vol.15 No.3 2010 5274 5280. 6 Ecobichon D.J The Basis of Toxicology Testing. CRC Press New York 199743–86. 7 Winter C.A. Porter C.A. Effect of alterations in side chain upon anti-inflammatory and liver glycogen activities of hydrocortisone esters. J. Am. Pharm. Assoc. 46 1957 515–519. 8 Winter C.A. Risley E.A. Nuss G.W. 1962. Carrageenan-induced edema in hind paws of rats as an assay for anti-inflammatory drugs. Proc. Soc. Exp. Biol. Med. 11 544–547. 9 Savitree M. Isara P. Nittaya S.L. Worapan S. Radical Scavenging Activity and Total Phenolic Content of Medicinal Plants Used in Primary Health Care. Journal of Pharm. Science 91 2004 32- 35. 10 Zhishen J Mengcheng T. Jianming W The determination of flavonoid contents in mulberry and their scavenging effects on superoxide radicals. Food Chemistry 644 1999 555-559.

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