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Evaluation of the Anticonvulsant Activity of 80% Methanol Leaf Extract and Solvent Evaluation of the anticonvulsant activity of 80% methanol leaf extract and solvent fractions of Buddleja polystachya Fresen. (Buddlejacea) in Mice By: Tewodros Agedew A thesis submitted to the Department of Pharmacology and Clinical Pharmacy, School of Pharmacy, College of Health Sciences Presented in partial fulfilment of the requirements for the Degree of Master of Science in Pharmacology March, 2018 Addis Ababa University Addis Ababa, Ethiopia Addis Ababa University School of Graduate Studies This is to certify that the thesis prepared by Tewodros Agedew, entitled “Evaluation of the anticonvulsant activity of 80% methanol leaf extract and solvent fractions of Buddleja polystachya Fresen. (Buddlejacea) in Mice" and submitted in partial fulfillment of the requirements for the Degree of Master of Sciences in Pharmacology complies with the regulations of the University and meets the accepted standards with respect to originality and quality. Signed by the examining committee Name Signature Date Examiner (internal): ______________ ______________ Examiner (external): _____________ _______________ Adviser: Teshome Nedi (PhD) ______________ _______________ Adviser: Workineh Shibeshi (PhD) ______________ _______________ ______________________________________________________ Chair, Department or Graduate Program Coordinator i | P a g e Abstract Evaluation of the anticonvulsant activity of 80% methanol leaf extract and solvent fractions of Buddleja polystachya Fresen. (Buddlejacea) in mice. Tewodors Agedew Addis Ababa University, 2018 Buddleja polystachya is used in Ethiopian traditional medicine for the treatment of epilepsy. Since this claim has not been investigated scientifically, this study was conducted to evaluate the anticonvulsant activity of 80% methanol leaf extract and solvent fractions of Buddleja polystachya in mice. The leaves of plant were extracted using maceration technique; butanol and chloroform were used for fraction. Anticonvulsant activity was evaluated by using maximal electroshock (MES) and Pentylenetetrazol (PTZ) model. Motor coordination effect was assessed by using rotarod test. Mice were randomly assigned to five groups (n=6 per group). The test groups received 100 mg/kg, 200 mg/kg and 400 mg/kg of cure extract and solvent fraction. The positive control groups received phenytoin 25mg/kg for MES test, valproate 200mg/kg for PTZ test and diazepam 5mg/kg for rotarod test. The negative control groups received distilled water or 2% Tween80, 10 ml/kg. The crude extract exhibited significant anticonvulsant effect in both MES test (p<0.001 for all doses) and PTZ test (p<0.001 for 200 mg/kg and 400 mg/kg, p<0.05 for 100mg/kg) compared with control. A similar effect was observed with butanol fraction in both models. Whereas, the chloroform fraction showed significant (p<0.001) anticonvulsant effect relative to control only in PTZ test at doses of 200mg/kg and 400mg/kg. The aqueous fraction was devoid of anticonvulsant activity in both seizure models. No significant changes in motor coordination were detected in all doses of the crude extract and solvent fractions. The plant extract contained flavonoids, phenols, tannins, steroids, terpenoids and saponins. This study indicated that the plant has a promising anticonvulsant activity, and it could be considered as a potential source to develop new anti-epileptic drug. Key words: Epilepsy, Seizure, Anticonvulsant, Buddleja polystachya ii | P a g e Table of Contents Abstract ........................................................................................................................................... ii Acknowledgements ......................................................................................................................... v List of Abbreviations and Acronyms ............................................................................................. vi List of Tables ............................................................................................................................... viii List of Figures ................................................................................................................................ ix 1. Introduction ............................................................................................................................. 1 1.1. Overview of Epilepsy ....................................................................................................... 1 1.1.1. Epidemiology of Epilepsy......................................................................................... 3 1.2. Seizure Initiation and Propagation ................................................................................... 3 1.3. Pathophysiology of Epilepsy ............................................................................................ 4 1.4. Management of Epilepsy .................................................................................................. 9 1.4.1. Non-pharmacological Treatment .............................................................................. 9 1.4.2. Pharmacological Treatment .................................................................................... 10 1.4.3. Herbal Treatment .................................................................................................... 12 1.5. Overview of the Experimental Plant .............................................................................. 13 1.6. Rational for the Study .................................................................................................... 16 2. Objectives .............................................................................................................................. 17 2.1. General Objective ........................................................................................................... 17 2.2. Specific Objectives ......................................................................................................... 17 3. Material and Methods ............................................................................................................ 18 3.1. Drug and Chemicals ....................................................................................................... 18 3.2. Collection of Plant Materials.......................................................................................... 18 3.3. Experimental Animals .................................................................................................... 18 3.4. Extraction and Fractionation of Plant Material .............................................................. 19 iii | P a g e 3.5. Acute Oral Toxicity Test ................................................................................................ 20 3.6. Grouping and Dosing of Animals .................................................................................. 21 3.7. Anticonvulsant Activity Tests ........................................................................................ 21 3.7.1. Pentylenetetrazol Induced Seizure Test .................................................................. 21 3.7.2. Maximal Electroshock Induced Seizures Test ........................................................ 22 3.8. Rotarod Test ............................................................................................................... 24 3.9. Preliminary Phytochemical Screening Tests .................................................................. 24 3.10. Data Analysis .............................................................................................................. 26 4. Result ..................................................................................................................................... 27 4.1. Acute Oral Toxicity Test ................................................................................................ 27 4.2. Anticonvulsant Activity in Pentylenetetrazol Induced Seizure...................................... 27 4.3. Anticonvulsant Activity in Maximal Electroshock Induced Seizure ............................. 30 4.4. The Rotarod Test ............................................................................................................ 32 4.5. Preliminary Phytochemical Screening ........................................................................... 33 5. Discussion .............................................................................................................................. 34 5.1. Limitation of the study ................................................................................................... 39 6. Conclusion ............................................................................................................................. 40 7. Recommendations ................................................................................................................. 41 8. References ............................................................................................................................. 42 iv | P a g e Acknowledgements First of all, I would like to thank the almighty God who has given me the strength and patience since the start of the study. I am so grateful to thank my advisors Dr. Teshome Nedi and Dr. Workineh Shibeshi for their encouragement, guidance and constructive comments from the beginning to the end of the project. My honest thanks also goes to Dr. Samson Salile, Mrs. Fantu Assefa and Mr. Kalkidan for their cooperation in providing chemical and materials as well as
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