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International Journal of CURRENT PHARMACEUTICAL RESEARCH ISSN- 0975-1491 Vol 2, Issue 4, 2010 Research Article EVALUATION OF ANTHELMINTIC AND ANTI­INFLAMMATORY ACTIVITY OF AMARANTHUS SPINOSUS LINN MANIK BARAL1, SUBRATA CHAKRABORTY2, PRANABESH CHAKRABORTY3 Gupta College of Technological Sciences, Ashram More, Asansol­1, WB. 2B. C. Roy College of Pharmacy and AHS, Bidhannagar, Durgapur, WB.3 Supreme Education Foundation, Chandannagor, Hooghly, India. Email: m_baral@rediffmail.com 1 Received: 26 Jun 2010, Revised and Accepted: 24 July 2010 ABSTRACT Water extracts of whole plant of Amaranthus spinosus Linn was evaluated for anthelmintic on adult Indian earthworms (Pheritima posthuma) and Tubifex tubifex ,using piperazine citrate as reference standard. Aqueous extract showed anthelmintic activity in dose‐dependent manner giving shortest time of paralysis(P) and death(D) with 50 mg/ml concentration, for both the worms. Extract shows more potent activity (15 mg/ml) against Tubifex tubifex. Extract shows more potent activity (15 mg/ml) against Tubifex tubifex . The petroleum ether and ethanolic extracts of whole plant of Amaranthus spinosus Linn. were tested for anti‐inflammatory activity at the dosed of 250,500 and 750 mg/kg body weight.The extract produced dose dependent and significant inhibition of carrageenan induced paw oedema. The exhibited anti‐inflammatory activity of this plant was comparable with the standard drug Ibuprofen.The presence of steroids, alkaloids & flavonoids in the extracts may be contributory to its anti‐ inflammatory activity. Key words: Anthelmintic, Anti‐inflammatory, Carrageenan, Ibuprofen, Piperazine citrate INTRODUCTION Preparation of extract Helminth infections are most widely founds in those human beings particularly who are in low poverty level & does not maintain hygienic condition.Helminths are generally restricted to tropical regions and cause serious problem to health and contribute to the prevalence of undernourishment, anaemia, eosinophilia and pneumonia1. Worm diseases cause serious morbidity & affect population in endemic areas2.The gastro‐intestinal helminthes becomes resistant to currently available anthelmintic drugs so the treatment of this disease is a problem3.The plant exhibits cooling and alexeteric properties.4 The genus shows emollient and anthelmintic properties.5 Aqueous extract (Maceration method) The leaves and stems contains α‐spinasterol and hentriacontane.The roots contain α‐spinasterols octacosanoate(C57H102O2,mp 85‐860) and saponin,viz. saponin of oleanolic acid. 6The plant has a wide reputation among natives of being curative for intestinal‐worm infections.This plant is being used by the tribals of Purulia district as an anthelmintic in the form of extract,prepared by dissolving the powdered material in water for 4‐5 hours.This extract is used to treat intestinal‐worm infections. The benzene and alcoholic extract shown wound healing activity using excision, incision and dead space wound models7. The anti‐inflammatory property of methanolic extract of A. spinosus leaves was studied in different animal models8. As no scientific data on the anti‐inflammatory activity of petroleum and ethanolic extract of whole plant has been reported, hence the present study was done to evaluate scientifically the usefulness of this whole plant parts.So an attempt has been made to evaluate the anthelmintic potential and anti‐inflammatory activity of Amaranthus spinosus Linn. MATERIALS AND METHODS Plant material The whole plants of Amaranthus spinosus Linn were procured from different places of Purulia, District, WB and authenticated by the Botanical survey of India, Shibpur, Howrah, WB and also by Botany Department,B.B.College,Asansol,WB, India. A voucher specimen (NO‐ CHN/I‐I/2008/Tech. II/) was retained in our laboratory for further references. The plant material was dried in sunlight, pulverized, passed through sieve no.40 and stored in air tight container and used for further extraction. Powdered plant material (whole plant) 200 gm was macerated with 1000 ml of distilled water for 24 hrs.The extract was double filtered by using muslin cloth and Whatman no.1 filter paper and concentrated by evaporation on water bath. The extract was dried and used as a powder.The percentage yield of extract was 3.58 percent. Petroleum ether and ethanol extract (soxhlet ) For anti‐inflammatory activity freshly collected whole plant (200gm) were washed thoroughly and kept to dry in shade,were powdered to 40 mesh size with light petroleum ether(40‐600C) and then with ethanol in a soxhlet extractor for 72 hrs. In both the cases the menstrum was removed and the extract was concentrated in vaccum at 400C.The percentage of petroleum ether and ethanol soluble extractives were calculated with reference to air dried plant material and the yield was found to be 4.12±0.25% w/w and 5.58±0.62%w/w respectively. Animals Adult earthworms(Pheretima posthuma) and aquarium worm (Tubifex tubifex) were used to evaluate anthelmintic activity in vitro .Earthworms were collected near the swampy water from santuri ,purulia and aquarium worm from aquarium fish food supplier.They all were collected and kept in normal saline solution.The average size of earthworm was 6‐8 cm and aquarium worm was 1‐2 cm.They all were identified from the Dept.of Zoology,B.B.College,Asansol,WB. Albino wistar rats of either sex weighing 140‐160 gm were used in the screening experiments. Selected animals were maintained under standard laboratory conditions.The animals were fed with standard pellets and water ad libitum. The animal experimental protocol was approved by the Institutional Animal Ethics Committee. (955/A/06/CPC SEA). Drugs and chemicals Piperazine citrate (Glaxo Smithkline) was used during the experimental protocol. Carrageenan(Sigma Chemicals Co.,USA) was used for inflammation. Ibuprofen from local medicine shop was used as standard in anti‐ inflammatory activity. Baral et al. Int J Curr Pharm Res, Vol 2, Issue 4, 44­47 Methods Ghosh18. The anthelmintic assay was carried as per the method of Ajaiyeoba et al9. with minor modifications. The assay was performed on the aquarium worm, Tubifex tubifex, and Pheretima posthuma because they belong to same group of Annelida. They were used owing to its anatomical and physiological similarity with intestinal roundworm parasites of human beings for evaluation of anthelmintic activity.10,11,12. 20 ml formulations containing three different concentrations, of crude aqueous extract (15, 25 and 50 mg/ml in double distilled water) were prepared and six earthworms (same type) and a lump of Tubifex worms were placed in each nine cm petri dish containing 20 ml of above test solution of extracts. Piperazine citrate(10 mg/ml) was used as reference standard and distilled water as control13,14,15,16.All the test solution and standard solution were prepared freshly before starting experiments. Time for paralysis was noted when no movement of any sort could be observed except when the worms were shaken vigorously. Time for death of worms were recorded after ascertaining that the worms neither moved when shaken vigorously nor when dipped in warm water at 500C. All the results were shown in Table.1 and expressed as a mean ± SEM of six worms in each group. In all groups, acute inflammation was recorded by sub planter injection of 0.1ml of 1% w/v suspension of carrageenan(Sigma Chemicals Co.,USA) in normal saline in the right hind paw of the rats and paw volume was measured plethysmometrically at 1 , 2 and 4hr after carrageenan injection. The negative control groups were pre‐ treated with vehicle (2 % of w/v Tween‐80) at a dose of 10ml/kg body weight . The positive control group with ibuprofen at a dose of 10mg/kg body weight .The remaining six groups received the petroleum ether and ethanolic extracts at dosage of 250,500,750 mg/kg body weight .All of the treatments were given orally 2hr before the carrageenan injection. The measurement of paw volume was accomplished immediately by displacement technique using the plethysmograph before the carrageenan injection and at 1, 2 & 4 hr after the carrageenan injection.Oedema was expressed as the increment in paw volume due to carrageenan administration. In inflammation there is an initial release of histamine and 5‐ hydroxytryptamine(5‐HT) producing an increased vascular permeability followed by release of kinins further contributing to the increased vascular permeability and finally, the prostaglandins and slow reacting substanmce(SRS) are released to maintain the increased vascular permeability produced by histamine,5‐HT and kinins19. In anti‐inflammatory activity, a dose level of 250mg/kg, 500mg/kg and 750mg/kg were prepared in both petroleum ether and ethanol extract by suspending the extract in 2 % of w/v Tween‐80 and reference control ibuprofen (10mg/kg) was also suspended in same vehicle. The wistar albino rats of either sex were divided into eight groups comprising six animals in each group .Male or female albino rats with body weight of 140‐160 gm were selected for the study. Carrageenan induced paw odema17 is the simplest and most widely used model for studying the anti‐inflammatory activity of new compounds.Paw volume was measured immediately,and then at pre‐determined intervals by plethysmometric method of Singh and RESULTS AND DISCUSSION From the result it can be concluded that higher concentration of extract produced paralytic effect much earlier and the time to death was shorter for both the worms.Aqueous extract showed anthelmintic activity in dose‐dependent manner giving shortest time of paralysis(P) and death(D) with 50 mg/ml concentration, for both the worms. Extract shows more potent activity (15 mg/ml) against Tubifex tubifex.Anthelmintic evaluation was compared with reference standard Piperazine citrate (Table 1). Table 1: Result of Anthelmintic Activity ­ Table representing the anthelmintic activity of aqueous extract. Group Control Aqueous extract Piperazine Citrate Concentration (mg/ml) ___ 15 25 50 10 Pheretima posthuma(Earthworm) Time taken for Time taken for paralysis(P) in death(D) in min.(Mean & SEM) min.(Mean & SEM) Tubifex tubifex Time taken for paralysis(P) in min.(Mean & SEM) Time taken for death(D) in min.(Mean & SEM) 49 ± 0.43 33 ± 0.47 18 ± 0.59 22.66 ± 1.12 36 ± 1.23 23 ± 1.21 11.35 ± 0.48 11 ± 1.43 49 ± 1.21 42 ± 0.86 35 ± 0.56 31 ± 0.38 70 ± 1.25 55 ± 1.19 33 ± 1.38 45.33 ± 0.56 Each value represents mean ± SEM (N = 6) Table 2: Anti­inflammatory activity of petroleum ether & ethanol extract of Amaranthus spinosus.Linn. Group Group 1 Group II Group III Group IV Group V Group VI Group VII GroupVIII Dose(mg/kg body weight) ‐‐‐ 10 250 500 750 250 500 750 1 hr 0.35±0.60 0.18±0.24 0.39±0.26 0.43±0.25 0.31±0.03 0.41±0.04 0.39±0.01 0.21±0.07 Average volume of mercury displaced (ml) 2 hr 0.64±0.05 1.00±0.03 0.22±0.24* 0.31±0.23* 0.56±0.24 0.77±0.28 0.52±0.23* 0.74±0.26* 0.41±0.04* 0.62±0.06* 0.52±0.03 0.73±0.02 0.49±0.01* 0.71±0.02* 0.37±0.01* 0.56±0.01* 4 hr Values are expressed as mean ± SEM. Number of animal used 6 in each group;*P