EVALUATION OF ANALGESIC AND ANTICONVULSANT ACTIVITY OF HYDROALCOHOLIC EXTRACT OF COSTUS PICTUS LEAVES IN WISTAR ALBINO RATS Dissertation Submitted to THE TAMILNADU Dr. M.G.R MEDICAL UNIVERSITY In partial fulfilment of the requirements for the award of the degree of M.D. PHARMACOLOGY Branch VI APRIL 2016 EVALUATION OF ANALGESIC AND ANTICONVULSANT ACTIVITY OF HYDROALCOHOLIC EXTRACT OF COSTUS PICTUS LEAVES IN WISTAR ALBINO RATS Dissertation Submitted to THE TAMILNADU Dr. M.G.R MEDICAL UNIVERSITY In partial fulfilment of the requirements for the award of the degree of M.D. PHARMACOLOGY Branch VI APRIL 2016 CERTIFICATE This is to certify that this dissertation entitled “Evaluation of Analgesic and Anticonvulsant Activity of Hydroalcoholic Extract of Costus pictus Leaves in Wistar Albino Rats” is a bonafide record of the work done by Dr. Anandhalakshmi. A under my guidance and supervision in the Department of Pharmacology during the period of her postgraduate study for M.D. Pharmacology [Branch – VI] from 2013-2016. Dr. Rema Menon N, M.D., Dr. Madhavrao, M.D., [Guide] [Co-Guide] Professor and Head, Assistant Professor, Department of Pharmacology, Department of Pharmacology, Sree Mookambika Institute of Medical Sree Mookambika Institute of Medical Sciences, Kulasekharam, Sciences, Kulasekharam, Kanyakumari District, Kanyakumari District, Tamil Nadu – 629 161. Tamil Nadu – 629 161. Mr. Sarath Babu Kurra, M. Sc., Dr. Rema. V. Nair, M.D., D.G.O., [Co-Guide] Director Assistant Professor, Sree Mookambika Institute of Medical Department of Pharmacology, Sciences, Kulasekharam, Sree Mookambika Institute of Medical Kanyakumari District, Sciences, Kulasekharam, Tamil Nadu – 629 161. Kanyakumari District, Tamil Nadu – 629 161. DECLARATION I, Dr. Anandhalakshmi. A hereby submit the dissertation entitled “Evaluation of Analgesic and Anticonvulsant Activity of Hydroalcoholic Extract of Costus pictus Leaves in Wistar Albino Rats” done in partial fulfilment of M.D. Pharmacology [Branch-VI] in Sree Mookambika Institute of Medical Sciences, Kulasekharam. This is an original work done by me under the guidance and supervision of Dr. Rema Menon N. Dr. Rema Menon N, M.D., Dr. Anandhalakshmi. A Professor and Head Post graduate Department of Pharmacology Department of Pharmacology Sree Mookambika Institute of Sree Mookambika Institute of Medical Sciences Medical Sciences Kulasekharam [K.K District] Kulasekharam [K.K District] Acknowledgement I express my heartfelt gratitude to Dr. Rema Menon N, Professor and Head of Department of Pharmacology and my guide for her valuable suggestions, critical views and constant motivation throughout the study period. She lent her full support in times of difficulties that I encountered during this study period and I thank her for all the help rendered to me in completing my study. I sincerely thank her for the valuable help, suggestions and supervision throughout the study. I express my gratitude for her constructive criticisms and pains she had taken for completion of this work. I sincerely thank my Co-guide Dr. Madhavrao for his tremendous help and guidance throughout the study. His constant encouragement has been indispensible to the completion of the work. I once again thank him for his full support and patience he had shown in times of obstacles that I encountered during the study. I also thank him for helping me with the statistical analysis of the study. I express my gratitude to Mr. Sarath Babu Kurra my Co-guide who gave his valuable suggestions, critical views and comments at each stage of my dissertation work. I acknowledge with gratitude the immense help and support he showed to me for completing my study. I extend my thanks to my Professors Dr. Reneega Gangadhar and Dr. V. P. Bhalla and Associate Professor Dr. Ganesh. V, for their motivation and support. I also thank my Assistant professors Dr. Shruthi R and Dr. V. M. Sandeep for their guidance and suggestions during the study. I extend my sincere and heartfelt thanks to our Chairman Dr. Velayuthan Nair and Director Dr. Rema V Nair for giving me an opportunity to conduct the study. I humbly thank them for providing me the facilities to carry out my study and also for being a source of inspiration. I would also like to thank the Principal of the Institution Dr. Padmakumar for his support in accomplishment of my dissertation work. I express my heartfelt thanks to Mr. Muthusamy for the timely authentication of my plant specimen. I specially thank my colleague, Dr. Prathab Asir. A, for his immense help, comments and suggestions during each stage of my study. His valuable and timely help made me complete my study on time. His constant encouragement was an immense source of motivation during the hardships I faced during the study. I am grateful to the other postgraduates in my department Dr. Biacin Babu, Dr. Parvathy RL, Dr. Shanthi M, Dr. Navaneeth A, Dr. Arjun G Nair, Dr. Sushmita Ann S. J and Dr. Suhaina. A, for their help in various technical aspects of my study. I also thank my department office staff Mrs. Florence Vimala and Miss. Sangeetha for the support they have extended to me. I am also thankful for the staff at the animal house for all their timely help. Table of contents TABLE OF CONTENTS S. No Contents Page No 1. INTRODUCTION 1 2. HYPOTHESIS AND JUSTIFICATION 4 3. AIMS AND OBJECTIVES 6 4. REVIEW OF LITERATURE 7-54 4.1 Pharmacognosy of Costus pictus 7 4.1.1 Taxonomical hierarchy 8 4.1.2 Habitat and distribution 9 4.1.3 Growth and propagation 9 4.1.4 Morphological features of Costus pictus 9-10 4.1.4.1 Macroscopic features of Costus pictus leaf 10 4.1.4.2 Microscopic features of Costus pictus leaf 10 4.1.5 Phytochemistry 11 4.1.6 Medicinal properties 14 4.1.7 Toxicity studies 14 4.1.8 Pharmacological screening done with Costus pictus 15-27 4.1.8.1 Analgesic and anti-inflammatory activity 15 4.1.8.2 Antioxidant activity 16 4.1.8.3 Antidiabetic activity 21 4.1.8.4 Hypolipidemic activity 22 4.1.8.5 Anthelminthic activity 23 4.1.8.6 Antimicrobial activity 24 4.1.8.7 Anticancer activity 26 4.2 Oxidative stress 27-30 4.2.1 Role of oxidative stress in pain 28 4.2.2 Role of oxidative stress in epilepsy 29 4.3 Methods of screening for analgesic activity 30-45 4.3.1 In vivo models for testing central analgesic activity 33 4.3.1.1 Models using mechanical stimulus 33-34 4.3.1.1.1 Haffner’s tail clip method 33 4.3.1.2 Models using thermal stimulus 34-37 4.3.1.2.1 Hot plate method 35 4.3.1.2.2 Tail-Flick test 35 4.3.1.2.2.1 Radiant heat method 36 4.3.1.2.2.2 Tail immersion method 36 4.3.1.3 Models using electrical stimulus 37-42 4.3.1.3.1 Grid shock test 37 Table of contents 4.3.1.3.2 Electrical stimulation of tail 39 4.3.1.3.3 Tooth pulp stimulation test 39 4.3.1.3.4 Monkey shock titration test 41 4.3.1.4 Models using chemical stimulus 42-43 4.3.1.4.1 Formalin test in rats 42 4.3.2 In vivo models for testing peripheral analgesic activity 43-45 4.3.2.1 Writhing test 43 4.3.2.2 Randall-selitto test 44 4.4 Methods of screening for anticonvulsant activity 45-54 4.4.1 Models for screening antiepileptic drugs 47 4.4.2 Characteristics of an ideal model of epilepsy 47 4.4.3 In vivo models 48-53 4.4.3.1 Electrically induced seizure models 48-51 4.4.3.1.1 MES model 48 4.4.3.1.2 Threshold models 49 4.4.3.1.3 Kindling models 50 4.4.3.2 Chemically induced seizure models 51-52 4.4.3.2.1 Pentylenetetrazole (PTZ) test 51 4.4.3.3 Focal lesions induced seizure models 52 4.4.3.4 Models of Status Epilepticus 52 4.4.3.5 Genetic models 53 4.4.4 Widely used models in antiepileptic drug discovery 53 5. MATERIALS AND METHODS 55-68 6. RESULTS 69-83 7. DISCUSSION 84-88 8. CONCLUSION 89 9. SUMMARY 90-91 10. REFERENCES I-VII 11. ANNEXURES LIST OF TABLES Table. Title Page No No 1. Grouping of animals for evaluating analgesic activity 61 2. Grouping of animals for evaluating anticonvulsant activity 61 Table of contents LIST OF FIGURES Figure Title Page No No 1. Models for evaluating analgesic activity 32 2. Models for evaluating central analgesic activity 33 Bar diagram showing the reaction time [seconds] in tail clip model of 3. 74 central analgesic activity among different groups in Wistar Albino rats Bar diagram showing the reaction time [seconds] in hot plate model of 4. 75 central analgesic activity among different groups in Wistar Albino rats Bar diagram showing the total number of writhes in 30 minutes in 4% 5. NaCl induced model of peripheral analgesic activity among different 76 groups in Wistar Albino rats Line diagram showing the percent inhibition of total number of writhes 6. in 30 minutes in 4% NaCl induced model of peripheral analgesic 77 activity among different groups in Wistar Albino rats Bar diagram showing the scores of seizures in Maximal Electroshock 7. Seizure [150 mA current at frequency of 60 Hz, for duration of 0.2 sec] 78 electrical model in Wistar Albino rats Bar diagram showing the percentage of protection of Tonic Hind Limb Extension [THLE] in Maximal Electroshock Seizure [150 mA current at 8. 79 frequency of 60 Hz, for duration of 0.2 sec] electrical model in Wistar Albino rats Bar diagram showing the onset of seizures in a chemical model in 9.