Phytochemical Evaluation and Screening of Cardiotonic, Antibacterial and Anthelmintic Activities of Sida cordifolia L.

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1 2574 Int J Pharm Sci Nanotech Vol 7;Issue 3 July September 2014 International Journal of Pharmaceutical Sciences and Nanotechnology Volume 7 Issue 3 July September 2014 Research Paper MS ID: IJPSN NATHANIEL Phytochemical Evaluation and Screening of Cardiotonic, Antibacterial and Anthelmintic Activities of Sida cordifolia L. Sarella Prakash Nathaniel Kumar*, L.K. Kanthal, S. Durga, D. Achutha Rama Raju and K. Satyavati Koringa College of Pharmacy, Korangi , Tallarevu Mandal, East Godavari, Andhra Pradesh, India. Received December 9, 2013; accepted March 16, 2014 ABSTRACT Several herbs are traditionally used in the treatment of a variety of ailments particularly in the rural areas, where herbal medicine is mainly the source of health care system. Many of these herbs havenot been assessed for safety or toxicity to tissue or organs of the mammalian recipients. An attempt is made to prove the efficacy of Sida cordifolia Linn., (a traditional medicinal plant chosen on the basis of ethnomedical knowledge) for its Cardiotonic, Antibacterial and Anthelmintic activities. Sida cordofolia Linn., is used traditionally, inter alia, in the treatment of various infections, asthma, diarrhoea, heart and stomach disorders. Cardiotonicactivity is performed using isolated Frog Heart Perfusion Technique. Antibacterial activity of the whole plant extracts were assessed by Agar well diffusion method against the strains of Bacillus subtilis, Escherichia coli, and Staphylococcus aureus. Anthelmintic activity was studied against Pheretima posthuma. Phytochemical screening of powdered plant material revealed thepresence of some secondary metabolites such as alkaloids, saponins, tannins, glycosides and flavonoids. Results indicated that methanol, chloroform, aqueous extracts have significant cardiotonic activity but less than standard drugs. Methanol and Aqueous extracts showed high antibacterial activity and anthelmintic activity than the standard drugs. In a nutshell, we can conclude that the methanol and aqueous fractions of Sida cordifolia Linn., had a profound antibacterial and anthelmintic effect eventhough it possessed very significant cardiotonic activity. This validates its continuous usage in ethnomedicine. This plant could be developed into cheap, safe and culturally acceptable standardized herbal products and may serve as asource of new molecules for broad-spectrum antimicrobial and anthelmintic agent. KEYWORDS: Sida cordifolia Linn.; Ethnomedicines; Phytochemical screening; Cardiotonic Activity; Antibacterial Activity; Anthelmintic Activity. Introduction Traditional Medicine (TM) implies generally to those medical and health care systems which are practiced in a traditional manner and not presently considered to be part of conventional Western medicine. The World Health Organization (WHO) defines Traditional Medicine as: The health practices, approaches, knowledge and beliefs incorporating plant, animal and mineral-based medicines, spiritual therapies, manual techniques and exercises, applied singularly or in combination to treat, diagnose and prevent illness or maintain well-being. This discipline has devolved over years by drawing on the religious beliefs and social structures of numerous indigenous people, by exploiting natural products, and bydeveloping and validating therapeutic and preventive approaches using the scientific method (Mukherjee and Houghton, 2009). Cardiovascular diseases are the secondary cause deaths in many parts of the world, although modern drugs are effective in preventing the disorders, their use is often limited because of their side effects and adverse reactions. A wide array of plants and its active principles, with minimal side effects, provide an alternate therapy for Ischemic heart disease (Janardan, 2011). The development of drug resistance in human pathogens against commonly used antibiotics has necessitated a search for new antimicrobial substances from other sources including plants. Plants are known to produce a variety of compounds to protect themselves against a variety of their pathogens and therefore considered as potential source for different classes of antimicrobial substances (Pelczer et al., 1998). Helmenthic and microbial infections are among the common infections in humanbeings, affecting a large proportion of the world s populationin developing countries they pose a large threat to public health and contribute to prevalence of anemia, malnutrition, eosinophilia and pneumonia. Ideally an anthelmintic agent should have broad spectrum of action, high percentage of cure with a single therapeutic dose, free from toxicity to the host and should be cost effective. None of the synthetic drug available meets these requirements. It necessitates research for new nature based drugs which increased efficacy and a broad therapeutic index. Sida cordifolia L. is a small, erect, downy shrub (Harbone, 1973). The leaves of the plant are 2574

2 Kumar et al: Phytochemical Evaluation and Screening of Cardiotonic, Antibacterial and Anthelmintic activities of cordate-oblong or ovate-oblong and fruits with a pair of awns on each carpel. Roots of the plant which constitute a drug are 5-15 cm long with few lateral roots of smaller size. It is almost odorless with slightly bitter taste (Mukherjee, 2002). It grows well through the plains of India, especially, in damp climates. The shrub grows up to meters in height. The leaves are cm long and cm broad, with 7-9 veins. They are heart shaped, serrate and truncate. The flowers are small, yellow or white in color, solitary and axillaries. The fruits are moong-sized, 6-8 mm in diameter. It is useful in blood, throat, urinary system related troubles, piles, phthisis, and insanity (Vogar. 1984). Franco et al., tested that rather than being a stimulant, Sida cordifolia actually acts as a depressant and decreases CNS activity (Nirmal et al., 2007). It acts as a weight loss product is through its hypoglycemic (blood sugar lowering) activity (Ansari, 2001). Fumaric acid isolated from S. cordifolia was reported to be hepatoprotective (Khandelwal, 2002). The roots of Sida cordifolia have been re-ported to possess astringent, diuretic and tonic properties. The drug has also demonstrated antibacterial, antiplaque and antifungal activities. Also it is shown to contain phytoconstituents such as pseudoephedrine, asparagin, ephedrine, vasicinone, vascine, vascinol. In the light of these observations, the present study was undertaken to review its pharmacognostical features and evaluate its cardiotonic, antibacterial and anthelmintic activity. Materials and Methods Collection of plant materials: The whole plant of Sida cordifolia L., free from disease were collected from Indrapalem, Kakinada, East Godavari District, Andhra Pradesh, India on July15 th of It was identified and authenticated with specimen number LKK-04 by Mr.P.V.Prasanna, Scientist E-In-Charge, Botanical Survey of India, Deccan Regional Centre, Hyderabad, India.The leaves were washed thoroughly 2-3 times with running water and once with sterile distilled water. Leaf, bark and root material was then air-dried on sterile blotter under shade. Preparation of Sida cordifolia extracts: The whole plant parts of Sida cordifolia L were shade dried at room temperature, powdered and passed through 60 mesh size sieves. 100 gm of powdered plant parts were weighed accurately and extracted with solvents selecting from non-polar to polar such as hexane, chloroform, methanol, and aqueous extracts using cold maceration method. Thus obtained extracts were filtered through whatman No.1 filter paper and the filtrate was concentrated. The extracts were transferred to sterile screw cap bottles, labeled and stored in refrigerator (4 o C) until use. Final yield of the extracts obtained were; 1. Hexane Extract gm 2. Chloroform extract-6.8 gm, 3. Methanol Extract-8.4 gm, 4. Aqueous Extract-7.3 gm. Hexane, chloroform, methanol, aqueous extracts were diluted to 1 mg/ml for antibacterial activity using dimethylformamide (DMF) and the same extracts were diluted to 25 mg/ml, 50 mg/ml and 100 mg/ml using 5% DMF in saline solution as vehicle for anthelmintic activity. For the evaluation of cardiotonic activity 0.25 mg/ml, 0.5 mg/ml and 1 mg/ml dilutions of each extract in water were used. Phytochemical evaluation All the extracts were subjected to qualitative chemical tests for the identification of various phytochemical constituents (Kumar, 2010). Small quantities of different extracts and powder were treated in distilled water separately and filtered. The extracts were taken for various tests systematically to detect the presence of chemical constituents. Anti bacterial activity Bacteria tested: (a) Escherichia coli (ATCC-2593) b) Bacillus subtilis (UC-564) c) Staphylococcus aureus (NCTC-8325). Study protocol The cultures of Bacillus subtilis, Escherichia coli, and Staphylococcus aureus are collected from Rangaraya Medical College, Microbiology department, Kakinada, East Godavari District, Andhra Pradesh. The cultures are sub-cultured for every ten days on nutrient agar slants, and maintained at cool temperature (Parekh and Chanda, 2007a; 2007b). The antimicrobial test was performed using agar well diffusion method. The test organisms were inoculated by using pour plate method. On nutrient agar, wells of 5 mm diameter were made by using sterile borer. The cut agar was carefully removed by using sterile forceps. To each well the dilution of plant extracts were added, control experiment with DMF (Dimethylformamide) was done on the same agar plate. The petriplates were incubated over night at 37 o C. The antibacterial spectrum of each extract then determined in terms of zone sizes [Inhibition Zone Diameter]. Preparation of nutrient agar The weighed amount of sodium chloride (5 gm), peptone (10 gm), beef extract (10 gm) are dissolved in 1000 ml of the water, then agar (20 gm) is added slowly on heating with continues stirring until agar is completely dissolved and ph is adjusted to 7.2 to 7.4. This nutrient agar medium is then sterilized by moist heat sterilization method using autoclave at temperature of 120 o C at 15 lb pressure maintained for 15 minutes. Anthelmintic activity Animals: Pheretima posthuma Earthworms are also called megadriles (or big worms), as opposed to the microdriles (or small worms) in the family s Tubificidae, Lumbriculidae, and Enchytraeidae, among others. The megadriles are characterized by having a distinct clitellum and a vascular system with true capillaries. Collection of worms: Indian earthworms Pheretima posthuma were collected from water logged area of soil, Indrapalem, E.G. Dt and washed with normal saline solution then stored in Tyrode solution.

3 2576 Int J Pharm Sci Nanotech Vol 7; Issue 3 July September 2014 Preparation of Tyrode solution: Accurate amounts of Sodium Chloride (8 gm), Potassium Chloride (0.2 gm), Calcium Chloride (0.18 gm), Sodium Dihydrogen Phosphate (0.1 gm), Magnesium Chloride (0.1 gm), Glucose (1 gm) and Sodium Bicarbonate (1 gm) are weighed. Mix all the ingredients in small amount of water until it dissolves, and finally make up the volume to 1000 ml. Study protocol: Earthworms of 5 to 8 cm in length and 0.2 to 0.3 cm in width were used. Animals were divided into 5 groups containing 5 earthworms in each group (Manoj et al., 2008; Martin, 1985; Sollmann, 1918). Three different concentrations [25 mg/ml, 50 mg/ml, and 100 mg/ml] of all the four extracts (Hexane, Chloroform, Methanol and Aqueous) were prepared. 50 ml of the standard drug piperazine citrate having the concentrations of 25 mg/ml, 50 mg/ml and 100 mg/ml were prepared by using 5% DMF in saline solution which was used as control. Sixteen petridishes of equal sizes were taken,earthworms are added and numbered. 50 ml of different concentrations of all the four extracts were placed in 12 petridishes. Three different concentrations of piperazine citrate were placed in other three petridishes. Control was maintained in one petridish. All petridishes were maintained at room temperature. The time of paralysis was noted when no moment of any sort could be observed except when the worms were shaken vigorously and the time for death recorded after ascertaining that worms neither moved when shaken vigorously or when they are dipped in warm water [50 o C]. Evaluation of cardiotonic activity: Cardiotonic activity was evaluated as per previous reports (Dama et al., 2010; Muthumani et al., 2010; Kulkarni, 1993; Bheemachari, 2005; Kumar, 2005; Dama et al., 2011; Mohireetal., 2007; Pai et al., 2009; Muralidharan et al., 2004). Frogs were pithed and the heart was exposed. The inferior venacava was traced and cannulated for perfusing the heart with the frog ringer s solution (Sodium Chloride-6.5 gm, Potassium Chloride-0.14 gm, Calcium Chloride-0.03 gm, Sodium Bicarbonate- 0.2 gm, Glucose- 2 gm, Distilled Water upto 1000 ml). The basal cardiac contraction was recorded on kymograph after the administration of frog ringer s solution and tween80 (1%). The administration of tween80 was done to see that it did not contribute to the effect of extracts. The drugs and extracts are administered through cannula. The extracts were diluted with water and three dilutions are prepared mg/ml, 0.5 mg/ml and 1 mg/ml. Digoxin (25 µg/ml, 50 µg/ml) and Adrenaline (10 µg/ml) are used as standard drugs and the responses of each dilution was noted on the kymograph. The amplitude of force of contraction and heart rate was recorded. Results and Discussion The whole plant of Sida cordifolia L was subjected to cold maceration. After the maceration all the extracts were subjected to qualitative chemical tests for the identification of various phytochemical constituents. This revealed the presence of pharmacologically important carbohydrates, proteins, volatile oils, steroids, flavonoids, glycosides, alkaloids, tannins, enzymes, terpenoids and vitamins. The results are reported in Table 1. TABLE 1 Phytochemical evaluation of SIDA CORDIFOLIA L. Test for carbohydrates Test for proteins and amino acids Test for fixed oils and fats Test for alkaloids Test for glycosides Test for flavonoids Test for tannins Test for steroids Name of the test H.E CF.E M.E AQ.E Molisch s Test Fehling s Test Benedict s Test Barfoed s Test Test for Starch Ninhydrin Test Biuret Test Millon s Test Xanthoproteic Test Tannic Acid With Heavy Metals Spot Test Saponification Test Dragendroff s Test Mayer s Test Wagner s Test Hager s Test Legal s Test Baljet s Test Borntrager s Test Keller-Killiani s Test Shinoda s Test With Lead Acetate % FeCl3 solution With Lead acetate Acetic acid Solution Dil. HNO Salkowski Test Liebermann- Burchard Test Test for enzymes With H2O Test for saponins Foam Test Test for phenolic compounds Potassium Ferricyanide+ Ammonia solution + Present - Absent H.E-Hexane Extract, CF.E- Chloroform Extract, M.E- Methanol Extract, AQ.E- Aqueous Extract The cardiotonic activity was performed using Isolated Frog Heart perfusion technique. The extracts were diluted with water and three dilutions are prepared mg/ml, 0.5 mg/ml and 1 mg/ml. Digoxin (25 µg/ml, 50 µg/ml) and Adrenaline (10 µg/ml) are used as standard drugs and the responses of each dilution was noted on the kymograph. The amplitude of force of contraction and heart rate was recorded. Results indicated lower cardiotonic activity than the standard drugs but can be taken into account for having less toxic effects than the synthetic drugs. Results were reported in Table 2.

4 Kumar et al: Phytochemical Evaluation and Screening of Cardiotonic, Antibacterial and Anthelmintic activities of TABLE 2 Effect of different extracts of Sida cordifolia on isolated frog heart perfusion. S.No. Drug/ Extract used 1. Hexane extract 2. Chloroform extract 3. Methanol Extract Concentration 0.25 mg/ml 0.50 mg/ml 1.00 mg/ml 0.25 mg/ml 0.50 mg/ml 1.00 mg/ml 0.25 mg/ml 0.50 mg/ml 1.00 mg/ml Dose (ml) HR (Heart rate Beats/min) HFC in mm (Height of force of contraction) Cardiac output: HR HFC Normal Normal Normal Normal Normal Normal Normal Normal Normal Aqueous Extract 0.25 mg/ml Normal mg/ml Normal mg/ml Normal Adrenaline 10 µg/ml Normal Digoxin 25 µg/ml Normal µg/ml Normal

5 2578 Int J Pharm Sci Nanotech Vol 7; Issue 3 July September 2014 The methanol and aqueous extracts showed antibacterial activity more than the other extracts. All the extracts showed less activity against Escherichia coli (Gram ve organism). They showed more activity on Bacillus subtilis and Staphylococcus aureus (Gram +ve organism) compared to standard (Table 3). Totally all the extracts of Sida cordifolia showed good anti bacterial activity. TABLE 3 Antimicrobial activity of Sida cordifolia L. S.No. EXTRACTS Zones of Inhibition(mm) E.coli B. subtilis S. aureus 1 Hexane-1 mg/ml 6 mm 8 mm 7 mm 2 Chloroform-1 mg/ml 10 mm 19 mm 17 mm 3 Methanol-1 mg/ml 16 mm 22 mm 24 mm 4 Aqueous-1 mg/ml 15 mm 20 mm 19 mm 5 Cefotaxime-1 mg/ml 16 mm 15 mm 17 mm 6 Control (DMF) The anthelmintic activity was performed on the Indian earth worms (Pheretima posthuma) and the results are reported in Table 4. The methanol and aqueous extracts showed more activity compared to the other extracts. All the extracts were found to possess anthelmintic activity. TABLE4 Anthelmintic activity of Hexane, Chloroform, Methanol, Aqueous Extracts of Sida cordifolia L. S. No. Extracts Used 1 Piperazine Citrate Time taken for Paralysis (P) and Conc. Death(D) of worms in minutes: mg/ml (P) (D) ± ± ± ± ± ± Hexane ± ± ± ± ± ± Chloroform ± ± ± ± ± ± methanol ± ± ± ± ± ± Aqueous ± ± ± ± ± ± Control ( 5% - DMF in Saline solution) DMF = Dimethylformamide Value are mean ± SEM; n=5 worms in each group p< 0.05 is considered when compared with standard. Sida cordifolia L. is chosen based on the ethnomedical knowledge and the traditional history of its usage in treatment of various ailments. This is verified on the grounds of modern sciences in systematic manner both qualitatively and quantitatively. The grounded whole plant powder material of the whole plant of Sida cordifoliais successively extracted with hexane, chloroform, methanol and distilled water by cold maceration method. Phytochemical studies revealed the presence of various pharmacologically important alkaloids, flavonoids, glycosides, phenolic compounds, saponnins and tannins along with general constituents of carbohydrates, fats and proteins. All the extracts are screened for the in vitro cardiotonic activity on isolated frog heart. These extracts are found to contain significant cardiotonic activity comparing to the standards (Fig.1and2). Further, the documented reports indicate that, the saponins and cardiac glycosides might be responsible for the positive inotropic effect. The chloroform, methanol and aqueous extracts under study contain all afore mentioned phytoconstituents. Hence, it may be concluded that, the Sida cordifolia is endowed with significant positive inotropic effect with beneficial properties. The antibacterial activities of the extracts were screened by using Bacillus subtilis, Escherichia coli and Staphylococcus aureusas experimental models (Graph 3). The demonstration of activity against the test microorganisms provides scientific basis for the local usage of these plants in the treatment of various ailments. The fact that the extracts were active against both gram-negative and gram-positive bacteria tested may indicate a broad spectrum of activity, the property which is very significant because of the possibility of developing therapeutic substances that will be active against multidrug-resistant organisms. Presence of alkaloids, flavanoids, phenols and tannins which may either individual or in combination may be attributed to the antibacterial activity of Sida cordifolia whole plant extracts. The soil transmitted gastro-intestinal helminthes becomes resistant to currently available anthelmintic drugs therefore there is a foremost problem in treatment of helminthes diseases. Hence, there is an increasing demand towards natural anthelminthics. Plant based anthelmintic drugs have enormous therapeutical potential as they can serve the purpose with less number of side effects than that are often associated with synthetic anthelmintic drugs. The anthelmintic activity was screened by taking Pheretima posthuma as experimental model. The extracts are having good anthelmintic activity (Fig.4). The phytochemical screening reports revealing the presence of flavonoids and tannins may be responsible for the anthelmintic activity. Based on the result in the study, it was concluded that the whole plant extracts of Sida cordifolia were found to be having good cardiotonic, antibacterial and anthelmintic activities. Further studies are required to identify specific active principles of this plant for these significant pharmacological effects.it is present need to conserve the plant for medicinal usage. Tissue culture techniques may be more useful in the conservation point of view and to make the drug available throughout the year.

6 Kumar et al: Phytochemical Evaluation and Screening of Cardiotonic, Antibacterial and Anthelmintic activities of Fig. 1. Change in heart rate by inducing different dilutions of two extracts. Fig. 2. Change in force of contraction by inducing different dilutions of different extracts. H1: 0.25 mg/ml Hexane Extract M2: 0.50 mg/ml Methanol Extract H2: 0.50 mg/ml Hexane Extract M3: 1.00 mg/ml Methanol Extract H3: 1.00 mg/ml Hexane Extract A1: 0.25 mg/ml Aqueous Extract C1: 0.25 mg/ml Chloroform Extract A2: 0.50 mg/ml Aqueous Extract C2: 0.50 mg/ml Chloroform Extract A3: 1.00 mg/ml Aqueous Extract C3: 1.00 mg/ml Chloroform Extract Adr: 10 µg/ml solution of Adrenaline M1: 0.25 mg/ml Methanol Extract Dig1: 25 µg/ml solution of Digoxin Fig. 2: 50µg/ml solution of Digoxin Fig. 3. Antimicrobial activity of Sida cordifolia L.

7 2580 Int J Pharm Sci Nanotech Vol 7; Issue 3 July September 2014 Fig. 4. Time taken for death different extracts using Sida cordifolia L. (S.C) different extracts for evaluation of anthelmintic acivity. Acknowledgements We wish to express our deep sense of gratitude to Prof. T.Veerraju, Academic Director, Koringa College of Pharmacy for his constant encouragement throughout the entire period, which helped us in successful completion of this work. With great pleasure we acknowledge our profound and sincere thanks to the management of Koringa College of Pharmacy for providing all the facilities and support during our academic period of the study. References Ansari HS (2001).Essentials of Pharmacognosy, Nirali Prakashan, Delhi, pp Bheemachari J (2010). Effect of ArqGauzaban - a Unanipathy product on the isolated frog heart. ActaPharmaceuticaTurcica 47: Dama G.Y et al., (2010). Comparative Cardiotonic activity of AegleMarmelos juice with digoxin on isolated frog heart. Int J Drug Dev. & Res 2(4): Dama. G. Y et al., (2011). Comparative cardiotonic activity of Heldina cardifolia with degoxin on isolated frog heart. IntJ Current Pharm Res 3(1): J. B. Harbone (1973). Phytochemical Methods: A Guide to Modern Techniques of Analysis, Chapman and Hall Publishers, London, pp Janardan et al., (2011).Cardiotonic activity of Petrolium Ether and alcoholic extract of Seeds of Cassia Tora Linn. Pharmacologyonline 3: Khandelwal KR (2002). Practical Pharmacognosy, Ninth Edition, Nirali Prakashan, Delhi, pp Kulkarani SK (1993). Hand book of experimental pharmacology, second edition, page no: 85. Kumar P (2010). Evaluation of cardiac activity of some traditionally used backyard Indian medicinal plants. Research Journal of Pharmaceutical, Biological and Chemical Sciences 1(4): Manoj A, AswarUrmrila, Watkar Bhagyashri, Vyas Meenakshi, WaghAkshaya, Gujar N.kishore (2008). Anthelmentic activity of Ficusbenghalensis. Ind J Green Pharmacy 2(3): Martin RJY (1985). Aminobutyic acid and piperazine activated single channel currents from Ascaris sum body muscle. British J Pharmacol.Edn 84(2): Mohire NC et al., (2007).Cardiotonic activity of aqueous extract of heart wood of Pterocarpus marsupium. Ind J Exp Biology 45: Mukherjee PK (2002). Quality Control of Herbal Drugs, 1 st Edition, Bussiness Horizon, Pharmaceutical Publishers, New Delhi, pp Mukherjee P.K, Houghton P.J (2009). The worldwide phenomenon of increased ues of herbal products: opportunity and threats, in Houghton P.J, Mukherjee P.K (Eds), Evaluation of Herbal medicinal products-perspectives on Quality, Safety and Efficacy. Pharmaceutical Press, Royal Pharmaceutical Society of Great Britain, pp Muralidharan A, et al., (2004). Cardiac stimulant activity of Ocimum basilicum Linn extracts. Ind J Pharmacol 36(3): Muthumani P et al., (2010). Anti-diarrhoeal and cardiotonic activity of extracts of Elephantopus scaber linn in experimental animals. Res J Pharm, Biological and Chemical Sci 1(3): 1-4. Nirmal SA, G. Malwadkar and R. B. Laware (2007). Anthelmintic activity of Pongamia glabra. Songklanakarin J Science Tech 29(3): Pai PT, et al., (2009). Evaluation of cardiotonic activity of leaves Vitex negundo. Linn. Int J Green Pharmacy 2: Parekh J and Chanda S (2007a). Antibacterial and phytochemical studies on twelve species of Indian medicinal plants. African Journal Biology Res Edn 10: Parekh J and Chanda S (2007b). In-vitro screening of antibacterial activity of aqueous and alcoholic extracts of various Indian plant species against selected pathogens from Enterobacteriaceae. Afr. J Microbiology Res 1(6): Pelczer MJ, Chan ECS, Noel RK (1998). Microbiology, fourth edition, Tata McGraw Hill, pp SollmannT (1918). Anthelmentics: Their efficiency as tested on earthworms. J.Pharmacol.Exp.Ther 18: Thorn GW, R. D. Adams, E. Braunwald, K. J. Issel-bacher and R. G. Peterdrof. Harrison s Principles of Internal Medicine. McGraw Hill Co., New York, pp Vigar Z (1984). Atlas of Medicinal Parasitology. PG Publication House, Singapore, pp Address correspondence to: Sarella Prakash Nathaniel Kumar, Koringa College of Pharmacy, Korangi , Tallarevu Mandal, East Godavari, Andhra Pradesh, India. Mob: , sarellaprakash@gmail.com