INTERNATIONAL JOURNAL OF UNIVERSAL PHARMACY AND BIO SCIENCES

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1 International Journal of Universal Pharmacy and Bio Sciences 2(4): July-August 2013 INTERNATIONAL JOURNAL OF UNIVERSAL PHARMACY AND BIO SCIENCES IMPACT FACTOR 1.89*** ICV 3.00*** Pharmaceutical Sciences Research Article!!! Received: ; Accepted: ANTICONVULSANT ACTIVITY OF AEGLE MARMELOS LEAF AQUEOUS EXTRACT USING MAXIMAL ELECTROSHOCK INDUCED SEIZURE MODEL IN MICE Mr. Rejeesh.E.P *, Mrs. Lovelyn Joseph, Dr. S.N Rao Department of Pharmacology, Yenepoya Medical College, Yenepoya University, Derlakkatte, Mangalore, India ABSTRACT KEYWORDS: Maximal electroshock seizure (MES), Aegle marmelos, Phenytoin, Anticonvulsant. For Correspondence: Rejeesh E.P.* Address: Department of Pharmacology, Yenepoya Medical College, Yenepoya University, Derlakkatte, Mangalore, India Many patients with epilepsy fail to experience adequate control of their seizures, despite the optimal use of available anti-epileptic drugs marketed; other patients do so only at the expense of significant toxic side effects. Leaves of Aegle marmelos is used for anticonvulsant activities in Ayurvedic medicine. In this study we have screened antiepileptic activity of the aqueous extract of Aegle marmelos leaf in Maximal electroshock seizure model in mice. Animals were divided into four groups, administered with aqueous extract (300 mg/kg, 600mg/k, 900mg/kg), Phenytoin (25 mg/kg, as positive control), saline (10 ml/kg, as control). The limit test for acute toxicity at 2000mg/kg/orally didn t show any mortality or severe adverse events within the 48 hrs of observation period. The phytochemical analysis of aqueous extract of leaves of A. marmelos reveal a high content of Steroids, Flavonoids, Tannins, Saponins, Glycosides, Carbohydrates, Proteins and Alkaloids. Aqueous extract produced dose-dependent anticonvulsant effect against Hind limb tonic extensor phase (HLTE) of Maximal electroshock (MES) model in Mice compared with control. Duration of HLTE phase among different treatment groups was recorded. A dose dependant reduction in duration of HLTE was observed compared to control group [significant (P <0.001)]. Percentage reduction in anticonvulsant activity was calculated, a log dose response curve was plotted, linear regression analysis and ANOA (Significant P<0.05) was done and the ED50 was calculated as 853.1mg/kg. The leaf aqueous extract of A. marmelos possesses protective effect against experimental seizures induced by MES, but the exact mechanism and the active compounds involved in these effects need to be clarified in future studies. 110

2 INTRODUCTION: Many patients with epilepsy fail to experience adequate control of their seizures, despite the optimal use of available anti-epileptic drugs marketed; other patients do so only at the expense of significant toxic side effects. [1] The word epilepsy derives from the Greek epilambanein, meaning to be seized, to be overwhelmed by surprise. [2, 3] Epilepsy is one of the most common serious disorders of the brain, affecting at least 50 million people worldwide. It knows no geographical, racial or social boundaries. Epilepsy accounts for 1% of the global burden of disease, determined by the number of productive life years lost as a result of disability or premature death. Among primary disorders of the brain, this burden ranks with depression and other affective disorders, Alzheimer s disease and other dementias, and substance abuse. Among all medical conditions, it ranks with breast cancer in women and lung cancer in men. Eighty per cent of the burden of epilepsy is in the developing world, where 80 90% of people with epilepsy receive no treatment at all [2, 4]. This indicates development of new drug candidates and evaluation of our ethnic medicinal recipes which will be cheap and easily available. The plant Aegle marmelos Corr. (AM) belongs to the family Rutaceae and is known as Vilvam in Tamil, Bael in Hindi, Bilwa or Sriphal in Sanskrit and Bael tree in English [5] Leaves of Aegle marmelos is used for anticonvulsant activities in Ayurvedic medicine. In this study we have screened antiepileptic activity of the aqueous extract of Aegle marmelos leaf (AmAE) in Maximal electroshock seizure model in mice. MATERIALS AND METHODS Plant Materials Leaves of A. marmelos were collected locally from Mangalore. The plant sample was identified and authenticated by Dr.Krishna Kumar G, Taxonomist and Professor of Botany, Mangalore University, India. Drugs Phenytoin sodium (Akums drugs and pharmaceuticals Ltd) was dissolved in saline that was alkalinized slightly with 0.1mM potassium hydrochloride. Phenytoin was administered intraperitoneally (i.p.) in volume of 0.1 ml/10g of mice body weight. The extract was dissolved in distilled water and administered orally. Extracts Preparation The collected leaves of A.marmelos were washed, air-dried, powdered and extracted with Soxhlet apparatus, in portions of 200g, with distilled water at 90 o c temperature for 3 days. After exhaustive extraction, the collected aqueous extract was dried and kept under refrigeration. The final weight of crude extract was 25g. The extract was maintained at 4 0 C throughout experiments. 111

3 Animals Male Swiss albino mice weighing 20-25g were used. The animals were obtained institutional animal house, department of pharmacology, Yenepoya University, Mangalore. The animals were housed in standard cages with free access to food (standard laboratory rodent chow) and water adlibitum. The animal house temperature was maintained at 23 ± 3.0 0C with a 12-h light/dark cycle. The ethical guidelines for the investigation of experimental seizure in conscious animals were followed in all tests. All efforts were made to minimize animal suffering and to reduce the number of animals used. All the experimental procedures and protocols used in this study were reviewed by the Institutional Animal Ethical Committee and were in accordance with the guidelines of CPCSEA. Acute Toxicity Study Limit test at 2000mg/kg (OECD Guidelines) The acute toxicity study of the AmAE was performed. [6] Adult Swiss albino mice weighing 20-25g of both sex in equal ratio was used in a group containing six animals. Each sex was caged separately. A dose of 2000 mg/kg body wt orally was employed for the test drug. About 48 h later, the number of dead animals in a group was recorded. The data were tabulated. Phytochemical Screening The test sample was subjected to phytochemical analysis in order to find out the presence of phytochemical constituents. The phytochemical tests employed for Alkaloids and Tannins [7, 8, and 9], Cardiac glycosides, Saponins and Flavonoids [7, 10] and terpenoids [7, 11]. Test for alkaloids. 20 mg of extract was dissolved in 2ml of methanol. Few drops of 1% HCl added to it. Then the mixture was heated, kept in steam and after cooling. Then the mixture was treated with few drops of Wagner s reagent. The sample was observed for turbidity or precipitation. Test for tannins: 20mg of extract was dissolved in 1ml of distilled water in a test tube and 1-3 drops of Ferric chloride were added to the solution. Then the mixture was observed for blue or green colour. Test for cardiac glycosides: 20mg of extract was dissolved in 1ml of glacial acetic acid and 1-2 drops of ferric chloride solution was added. 0.5ml of concentrated sulphuric acid was slowly added along the sides of the test tube. A brown ring at the interface indicated a deoxysugar characteristic of cardenolides. Test for saponins: 20mg of extract was dissolved with 5ml of distilled water and shaken vigorously till a stable persistent froth was obtained. The froth was mixed with 3 drops of olive oil and shaken vigorously and then observed for emulsion. 112

4 Test for Flavonoids: International Standard Serial Number (ISSN): mg of extract was dissolved in 1ml of distilled water. 0.5ml of dilute ammonia solution was added to it. Conc. Sulphuric acid was added later. A yellow colour indicated the presence of Flavonoids. The yellow colour disappeared on allowing the solution to stand. Test for terpenoids: 20mg of extract was dissolved in 1ml of chloroform and 1ml of concentrated sulphuric acid was added to it. A reddish brown discolouration at the interface showed the presence of terpenoids. Test for carbohydrates: Fehling's test: Few drops of extract heated with Fehling's A and B solution. Appearance of orange red precipitate indicates presence of carbohydrates. Test for lactones: Baljets test: Treat extract with sodium picrate solution. Appearance of yellow to orange colour indicates presence of lactone ring. Test for proteins: Biuret test: Add 2ml of Biuret reagent to 2ml of extract. Shake well and warm it on water bath. Appearance of red or violet colour indicates presence of proteins. Test for Fixed oils and fatty acid: Spot test: Prepare spot on the filter paper with the test solution and oil staining on the filter paper indicated the presence of fixed oil & fats. Tests for steroids: Salkowski Tests: Chloroform solution of the extract when shaken with concentrated sulphuric acid and on standing yields red colour. Anticonvulsant Activity - MES-Induced Seizures: Animals were divided into four groups each consisting of six animals, administered with AmAE (300 mg/kg, 600mg/k, 900mg/kg), Phenytoin (25 mg/kg, as positive control), saline (10 ml/kg, as control), and after 30 minutes various phases of convulsion i.e. tonic flexion, tonic extensor, clonic convulsion, stupor and recovery were studied after applying electrical stimulus of 48 ma 50 Hz, 0.2s duration with cornel electrode and noted time spent in each phase. Abolition of the hind limb tonic extensor (HLTE) component indicates the test compound's ability to inhibit MES-induced seizure spread. [12, 13] Data Analysis The statistical analysis of data obtained from convulsion tests were carried out using one-way analysis of variance (ANOVA) followed by Dennett s multiple comparison test. All the results obtained in the study were compared with the vehicle control group. Percentage reduction in 113

5 anticonvulsant activity was calculated using the formula given in fig.2 and a log dose response curve was plotted (Fig. 2). Linear regression analysis and ANOA was done and the ED50 was calculated. P values < 0.05 were considered to be statistically significant. RESULTS: Acute Toxicity: The limit test of AmAE at 2000mg/kg/orally didn t show any mortality or severe adverse events within the 48 hrs of observation period. Phytochemical Analysis: The phytochemical analysis of AmAE reveals a presence of Steroids, Flavonoids, Tannins, Saponins, Glycosides, Carbohydrates, Proteins and Alkaloids. Anticonvulsant Activity - MES-Induced Seizures: The AmAE produced dose-dependent anticonvulsant effect in Maximal electroshock seizure model (Fig 1) when compared with control. ED50 of AmAE derived from log dose response curve was 853.1mg/kg Figure 1: Duration of Hind Limb Tonic Extensor Phase among different treatment groups. A dose dependant reduction in duration of HLTE was observed compared to control group, ANOVA and Dunnett Multiple comparison test were done and were found to be statistically significant (P< 0.001). 114

6 Figure 2 : Percentage reduction in anticonvulsant activity of AmAE was calculated using the formula. % reduction in HLTE duration = (HLTE control - HLTE test / HLTE control ) X 100 Percentage reduction in duration of HLTE was plotted against log dose and regression analysis was done and a log dose response curve was plotted. Linear regression analysis and ANOA (Significant P<0.05) was done and the ED50 of AmAE was calculated as 853.1mg/kg. 115

7 Figure 3 : Onset of HLTE and duration of different phases of MES in mice, the last bar diagram shows duration of stupor in different treatment groups. DISCUSSION AND CONCLUSION: A publication by World Health Organization Atlas: Epilepsy Care in the World, clearly states the severity of epileptic disorders, the shortcomings of current therapeutic strategy and shortages or unaffordability of drugs to the majority of the world population. Ethnopharmacology is one of the approaches to search for new drugs. The investigation of traditional antiepileptic agents has an 116

8 excellent potential for invention of new structural classes or therapeutic targets. In addition, on scientific validation of their properties, the traditional preparations could be cheap alternatives to reduce the dose of the standard drugs by using them in combination or may completely substitute the standard drug to render the treatment affordable and viable for the developing and underdeveloped countries. In traditional medicine, A. marmelos is used in treatment of epilepsy and other mental diseases. In the present study we have evaluated the effect of A. marmelos leaf aqueous extract against seizures induced by maximal electroshock (MES) in mice. The results indicate that the aqueous extract produced dose-dependent anticonvulsant effect against MES induced seizure. The Maximal electroshock seizure identifies those compounds which prevent seizure spread. Abolition of the hind limb tonic extensor (HLTE) component (Fig 1) indicates the test compound's ability to inhibit MES-induced seizure spread [12-15]. The model is highly reproducible with consistent endpoints. The behavioral and electrographic seizures generated in this model are consistent with the human disorder [12]. Drugs that inhibit voltage-dependent Na+ channels, such as Phenytoin, Valproate and Lamotrigine, GABA mimetic agents like Phenobarbitone and Benzodiazepines, agents that antagonize Glutamate (NMDA) system and some studies even show glycine and opioid agonists to have protective effect in MES. The leaf aqueous extract of A.marmelos possesses protective effect against experimental seizures induced by MES, but the exact mechanism and the active compounds involved in these effects need to be clarified in future studies. ACKNOWLEDGEMENTS: This work was done at the Ethnopharmacology lab, Department of pharmacology, Yenepoya medical college, Yenepoya University, Mangalore. REFERENCES: 1. James P. Stables and Harvey J. Kupferberg, The NIH Anticonvulsant Drug Development (ADD) Program: preclinical anticonvulsant screening project, Chapter 16, Epilepsy Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892, USA. 2. Atlas: Epilepsy Care in the World, World Health Organization, Programme for Neurological Diseases and Neuroscience Department of Mental Health and Substance Abuse, ISBN , Page no: Engel J Jr, Pedley TA, eds. (1997), Epilepsy: a comprehensive textbook. Vols 1, 2 and 3. Philadelphia, PA, Lippincott-Raven Publishers. 117

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