STUDY OF ANALGESIC, ANTI-INFLAMMATORY AND ANTIARTHRITIC ACTIVITY OF INDIAN MEDICINAL PLANT IN LABORATORY ANIMALS
A THESIS SUBMITTED TO BHARATI VIDYAPEETH UNIVERSITY, PUNE FOR AWARD OF DEGREE OF DOCTOR OF PHILOSOPHY IN PHARMACOLOGY UNDER THE FACULTY OF PHARMACEUTICAL SCIENCES
SUBMITTED BY Mr. GOPAL V. BIHANI M.Pharm
UNDER THE GUIDANCE OF Dr. SUBHASH L. BODHANKAR
RESEARCH CENTRE BHARATI VIDYAPEETH DEEMED UNIVERSITY, POONA COLLEGE OF PHARMACY, ERANDWANE, PUNE- 411 038, INDIA
FEBRUARY 2016
CERTIFICATE
This is to certify that the work incorporated in the thesis entitled “Study of Analgesic, Anti-inflammatory and Antiarthritic activity of Indian medicinal plant in laboratory animals” for the degree of ‘Doctor of Philosophy’ in the subject of Pharmacology under the faculty of Pharmaceutical sciences has been carried out by Mr. Gopal Vijaykumar Bihani in the Department of Pharmacology, Bharati Vidyapeeth Deemed University, Poona College of Pharmacy, Pune during the period from November 2012 to January 2016, under the guidance of Dr. S. L. Bodhankar, Professor and Head, Department of Pharmacology, Poona College of Pharmacy, Pune.
Place: Pune
Date:
Prof. K. R. Mahadik Professor and Principal Poona College of Pharmacy, Bharati Vidyapeeth Deemed University, Pune - 411038
CERTIFICATE
This is to certify that the work incorporated in the thesis entitled “Study of Analgesic, Anti-inflammatory and Antiarthritic activity of Indian medicinal plant in laboratory animals” Submitted by Mr. Gopal Vijaykumar Bihani for the degree of ‘Doctor of Philosophy’ in the subject of Pharmacology under the faculty of Pharmaceutical sciences has been carried out in the Department of Pharmacology, Bharati Vidyapeeth Deemed University, Poona College of Pharmacy, Pune during the period from November 2012 to January 2016, under my direct supervision/guidance.
Place: Pune
Date:
Dr. S. L. Bodhankar Professor and Head Dept. of Pharmacology Poona College of Pharmacy, Bharati Vidyapeeth Deemed University, Pune - 411038
DECLARATION BY THE CANDITATE
I hereby declare that the thesis entitled “Study of Analgesic, Anti-inflammatory and Antiarthritic activity of Indian medicinal plant in laboratory animals” submitted by me to the Bharati Vidyapeeth University, Pune for the degree of Doctor of Philosophy (Ph.D.) in Pharmacology under the faculty of “Pharmaceutical Sciences” is original piece of work carried out by me under the supervision of Dr. S. L. Bodhankar. I further declare that it has not been submitted to this or any other university or institution for the award of any degree or diploma. I also confirm that all the material which I have borrowed from other sources and incorporated in this thesis is duly acknowledged. If any material is not duly acknowledged and found incorporated in this thesis, it is entirely my responsibility. I am fully aware of the implications of any such act which might have been committed by me advertently or inadvertently.
Place: Pune
Date:
Mr. Gopal V. Bihani Research student Acknowledgement
AAACKNOWLEDGEMENTACKNOWLEDGEMENT
The path from dreams to success does exist, may you have the vision to find it, the courage to get on to it.
A journey is easier when you travel together. Interdependence is certainly more valuable than independence. This thesis is the result of three years of work whereby I have been accompanied and supported by many people. It is a pleasant aspect that I have now the opportunity to express my gratitude for all of them. This thesis would not appear in its present form without the kind assistance and support of the following individuals and organizations.
I thank the almighty for showering infinite bounties and grace upon me and for being my constant companion.
I am thankful to our Vice-Chancellor Dr. S. S. KadamKadam and Principal Dr. K. R.
Mahadik for providing proper resources and infrastructure to carry out the work of this stature.
I would like to thank my Guide Dr. S. L. Bodhankar, the best advisor and guide I could have wished for. During these years I have known him as a sympathetic and principle-centered person. Their enthusiasm and integral view on research and mission for providing 'only high-quality work and not less', has made a deep impression on me. I owe him lots of gratitude for having me shown this way of research. He taught me to look for solutions to problems rather than focus on the problem. I learned to believe in my future, my work and myself only because of him.
I am deeply indebted to Dr. S. R. RojatkarRojatkar, Director, R & D centre for Pharmaceutical Sciences and Applied chemistry, Pune for extended support, encouragement and fruitful discussion during execution of this work. It is my pleasant duty to thank, extending a helping hand and supporting me throughout my research work. A special thanks to Dr. P. R. RajRajaaaamohanan,mohanan, Head NMR
Acknowledgement facility group and Dr. K. B. SonawSonawane,ane, Scientist E1, Dept. of Organic Chemistry, National Chemical Laboratory, Pune for his help in generating valuable data during research work.
I wish to express my sincere and respectful thanks to Dr. A. P. Pawar, Dean and Vice Principal, Bharati Vidyapeeth Deemed University, Poona College of
Pharmacy and Mr. G. N. ZambareZambare, Dr. Arulmozhi and Dr. Balasaheb Siraskar for their constant support and valuable suggestions. I would also like to thank
Dr. Suryawanshi for his timely suggestions.
It is said that ‘accomplishment must be credited to those who have put up the foundation of the particular chore’. I would like to thank my parents, Mummy and Papa who were always ready to help me at all times now I can only show you my extreme appreciation for your support by being true to all the ideals and values that you tried to teach me thank you forever and standing by me, I love and appreciate you forever. Also I would like to thank my bbbrother brother Mr.
Radheshyam and bhabhi Mrs. Trupti for their parent like care and blessings have driven my performance and success. Their support has helped me to complete this work successfully. Also I thank my beloved cute niece Sachi and nephew Swarit for the laughter gifted to me with their cute and tender voice.
Also thanks to my wife Renuka you have been a never-ending source of love, encouragement and motivation. I look forward to our lifelong journey.
It would be unfair if I don’t mention the help showered on me by my senior and friend DDDr.Dr. Parag Kadam. I sincerely thank and express my profound gratitude towards him for providing invaluable insight.
It gives me immense pleasure to express my thanks to my colleagues and seniors
Dr. Vishal Mali, Dr. Smeeta Mohod, DDDr.Dr. Hemant Kamble, Mr. Sameer Sawant,
Mr. Amit Kandhare, Mr. Amol Muthal, andandand Mrs. Bhagyshri Atre who believed in constantly motivating me and supported me in achieving my goals. I am also thankful to my other department colleagues DDDr. Dr. Ashwin Kuchekar, DDDr. Dr. Sujit
Acknowledgement
Bhansali, Dr. Prashant Bhondave, Mr. Vijay Kale, Mr. Pravin Pawar,
Mr. Prakash Jadhav, Mr. Sumit DeoDeore,re, Mrs. Anjali, Mrs. Sampada Sawant and
Mrs. Sabina.
I am also thankful to Botanical Survey of IndiaIndia, Pune, for extending help in getting my plant authenticated. I am also thankful to Dhande laboratorylaboratory, Pune for interpretation of histopathological data. I would like to thank Mr. BBhhhhatlekaratlekaratlekar,
Incharge, National Institute of Biosciences Pune for providing animals towards this project. I am thankful to all non-teaching staff especially Mr. D. J. Joshi for extending help and cooperation.
I am deeply indebted to all animals whose lives were sacrificed during this research work. I hope that precious life of the animals used during this project would not be in vain; it would contribute to the development of the science in one way or another.
Beside this there are several other people who have knowingly or unknowingly helped me in the successful completion of this project. I thank all the people for every ounce of efforts they contributed.
Date:
Place: Pune
Mr. Gopal V. Bihani
Dedicated to my Beloved Family, Guide & God INDEX
INDEX
Title Page No. Title page Certificate of Principal Certificate of Guide Declaration Acknowledgement Index Contents List of Tables List of Figures List of Abbreviations Chapter 1 Introduction 01 – 06 Chapter 2 Review of Literature 07 – 71 Chapter 3 Aims and Objectives 72 – 73 Chapter 4 Materials and Methods 74 – 107 Chapter 5 Results 108 – 228 Chapter 6 Discussion 229 – 241 Chapter 7 Summary and Conclusion 242 – 247 Chapter 8 Highlights of the work 248 – 253 Chapter 9 Bibliography 254 – 267 Chapter 10 Publications 268 Errata 269 – 271
INDEX
CONTENTS
Chapter 1 Introduction 01 – 06 Chapter 2 Review of literature 07 – 71 2.1 Inflammation 07 – 35 2.1.1 Definition and causes 07 2.1.2 Signs of inflammation 07 2.1.3 Types of inflammation 08 2.1.4 Chemical mediators of inflammation 20 2.1.5 Regulation of inflammation 26 2.1.6 Inflammatory cells 27 2.1.7 Pre-clinical screening methods for anti-inflammatory agents 30 (In vivo methods) 2.1.8 Plants reported to have anti-inflammatory activity 33 2.2 Arthritis: Historical Background & Epidemiology 36 – 66 2.2.1 Rheumatoid arthritis (RA) 37 2.2.2 Epidemiology and Etiology 37 2.2.3 Pathophysiology 38 2.2.4 Comorbidities associated with RA 40 2.2.5 Clinical manifestations 41 2.2.6 Diagnosis 42 2.2.7 Investigations 43 2.2.8 Pre-clinical screening methods for antiarthritic agents (In vivo 44 methods) 2.2.9 Treatment 48 2.2.10 RA and pregnancy 65 2.3 Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze 67 – 71 2.3.1 Taxonomic Classification 67 2.3.2 Botanical description and Vernacular name 67 2.3.3 Morphology 68 2.3.4 Occurrence and Distribution 68 INDEX
2.3.5 Phytoconstituents 68 2.3.6 Pharmacological activities reported of Cyathocline purpurea 69 (Buch-Ham ex D. Don.) Kuntze Chapter 3 Aims and Objectives 72 – 73 3.1 Aims 72 3.2 Objectives 72 3.2.1 Part A: (Analgesic, Anti-inflammatory and Antiarthritic 72 activity of Cyathocline purpurea extracts) 3.2.2 Part B: (Isolation and characterization) 73 3.2.3 Part C: (Antiarthritic activity of isolated compound) 73 Chapter 4 Materials and methods 74 – 107 4.1 Materials 74 – 75 4.1.1 Plant material 74 4.1.2 Identification and authentication of plant material 74 4.2 Methods: Part A 76 – 89 (Analgesic, Anti-inflammatory and Antiarthritic activity of Cyathocline purpurea extracts) 4.2.1 Drugs and Chemicals 76 4.2.2 Preparation of extracts 76 4.2.3 Storage of extracts 76 4.2.4 Phytochemical analysis of PECP, MECP and AECP 76 4.2.4.1 Test for Carbohydrates 76 4.2.4.2 Test for Proteins 77 4.2.4.3 Test for Steroids 77 4.2.4.4 Test for Volatile oils 77 4.2.4.5 Test for Glycosides 77 4.2.4.6 Test for Saponins 78 4.2.4.7 Test for Tannins and Phenolic compounds 78 4.2.4.8 Test for presence of Flavonoids 78 4.2.4.9 Test for Alkaloids 78 4.2.5 Pharmacological study 79 INDEX
4.2.5.1 Chemicals and drugs 79 4.2.5.2 Apparatus 79 4.2.5.3 Instruments used 79 4.2.5.4 Preparation of dosage form 79 4.2.5.5 Storage conditions 80 4.2.5.6 Volume of extract solution 80 4.2.5.7 Route of administration 80 4.2.5.8 Experimental animals 80 4.2.5.9 Approval of research protocol 81 4.2.5.10 Acute oral toxicity (AOT) study 81 4.2.5.11 Analgesic activity 81 4.2.5.11.1 Hot plate test in mice 81 4.2.5.11.2 Acetic acid induced writhing in mice 82 4.2.5.12 Anti-inflammatory activity 82 4.2.5.12.1 Carrageenan induced paw edema in rats 82 4.2.5.12.2 Cotton pellet induced granuloma in rats 83 4.2.5.13 Antiarthritic activity 83 4.2.5.13.1 Measurement of change in paw volume 84 4.2.5.13.2 Measurement of change in joint diameter 84 4.2.5.13.3 Measurement of pain threshold 84 4.2.5.13.4 Measurement of paw withdrawal latency 85 4.2.5.13.5 Measurement of mechanical nociceptive threshold 85 4.2.5.13.6 Body weight recording 85 4.2.5.13.7 Radiological analysis of ankle joints 85 4.2.5.13.8 Haematological and serum parameters 86 4.2.5.13.9 Biochemical parameters 86 4.2.5.13.10 Anti-oxidant parameters 86 4.2.5.13.11 Histopathological analysis of ankle joints 89
INDEX
4.3 Methods: Part B 90 – 97 (Isolation and characterization) 4.3.1 Chemicals and reagents 90 4.3.2 Apparatus and instruments 90 4.3.3 Experimental animals 90 4.3.4 Approval of research protocol 90 4.3.5 Liquid – solid separation chromatographic technique 91 4.3.5.1 Petroleum ether fraction (F – 1) 91 4.3.5.2 10 % acetone in petroleum ether fraction (F – 2) 91 4.3.5.3 20 % acetone in petroleum ether fraction (F – 3) 91 4.3.5.4 30 % acetone in petroleum ether fraction (F – 4) 91 4.3.5.5 50 % acetone in petroleum ether fraction (F – 5) 92 4.3.5.6 Methanol fraction (F – 6) 92 4.3.6 Anti-inflammatory activity of fractions (F – 1 to F – 6) in 92 carrageenan induced paw edema. 4.3.7 Column chromatography of most active anti-inflammatory 93 fraction i.e. 30% acetone in petroleum ether fraction (F – 4) 4.3.8 Anti-inflammatory activity of pools (P – 1 to P – 10) 95 collected from 30% acetone in petroleum ether fraction (F – 4) in carrageenan induced paw edema 4.3.9 Preparative TLC of most active anti-inflammatory pool i.e. P 96 – 8 4.3.10 Spectral characterization of isolated compound (P – 8) 97 4.3.11 Docking study 97 4.4 Methods (Part C) 98 – 107 (Antiarthritic activity of isolated compound) 4.4.1 Measurement of change in paw volume 98 4.4.2 Measurement of change in joint diameter 99 4.4.3 Measurement of pain threshold 99 4.4.4 Measurement of paw withdrawal latency 99 4.4.5 Measurement of mechanical nociceptive threshold 99 INDEX
4.4.6 Body weight recording 100 4.4.7 Radiological analysis of ankle joints 100 4.4.8 Haematological and serum parameters 100 4.4.9 Biochemical parameters 100 4.4.10 Cytokine measurement by ELISA 100 4.4.10.1 Measurement of serum TNF-α 100 4.4.10.2 Measurement of serum IL-1β 101 4.4.10.3 Measurement of serum IL-6 102 4.4.11 Anti-oxidant parameters 104 4.4.12 Histopathological analysis of ankle joints 107 4.5 Statistical analysis 107 Chapter 5 Results 108 – 228 5.1 Part A 108 – 157 (Analgesic, Anti-inflammatory and Antiarthritic activity of Cyathocline purpurea extracts) 5.1.1 Phytochemical analysis 108 5.1.1.1 Percent yield and characteristics of different extracts of 108 Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze 5.1.1.2 Qualitative phytochemical analysis of PECP, MECP and 108 AECP 5.1.2 Acute toxicity test (AOT) 110 5.1.3 Pharmacological assessment 111 5.1.3.1 Analgesic activity 111 5.1.3.1.1 Effect of oral administration of Cyathocline purpurea (Buch- 111 Ham ex D. Don.) Kuntze. on hot plate test in mice 5.1.3.1.2 Effect of oral administration of Cyathocline purpurea (Buch- 113 Ham ex D. Don.) Kuntze. on acetic acid induced writhing in mice 5.1.3.2 Anti-inflammatory activity 114 5.1.3.2.1 Effect of oral administration of Cyathocline purpurea (Buch- 114 Ham ex D. Don.) Kuntze. on carrageenan induced paw edema INDEX
in rats 5.1.3.2.2 Effect of oral administration of Cyathocline purpurea (Buch- 116 Ham ex D. Don.) Kuntze. on cotton pellet induced granuloma in rats 5.1.3.2.2.1 Gastric ulcerogenic effect of oral administration of 117 Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze. in cotton pellet induced granuloma in rats 5.1.3.3 Antiarthritic activity 118 5.1.3.3.1 Effect of oral administration of methanol extract of 118 Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze. on change in paw volume in arthritic rats 5.1.3.3.2 Effect of oral administration of methanol extract of 120 Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze. on change in joint diameter in arthritic rats 5.1.3.3.3 Effect of oral administration of methanol extract of 122 Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze. on pain threshold in arthritic rats 5.1.3.3.4 Effect of oral administration of methanol extract of 124 Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze. on paw withdrawal latency in arthritic rats 5.1.3.3.5 Effect of oral administration of methanol extract of 126 Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze. on mechanical nociceptive threshold in arthritic rats 5.1.3.3.6 Effect of oral administration of methanol extract of 128 Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze. on body weight in arthritic rats 5.1.3.3.7 Radiological analysis of ankle joints 130 5.1.3.3.8 Haematological parameters 131 5.1.3.3.8.1 Effect of oral administration of methanol extract of 131 Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze. on blood haemoglobin count in arthritic rats INDEX
5.1.3.3.8.2 Effect of oral administration of methanol extract of 133 Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze. on blood WBC count in arthritic rats 5.1.3.3.8.3 Effect of oral administration of methanol extract of 135 Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze. on blood RBC count in arthritic rats 5.1.3.3.8.4 Effect of oral administration of methanol extract of 137 Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze. on blood platelet count in arthritic rats 5.1.3.3.8.5 Effect of oral administration of methanol extract of 139 Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze. on serum CRP level in arthritic rats 5.1.3.3.8.6 Effect of oral administration of methanol extract of 141 Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze. on RF value in arthritic rats 5.1.3.3.9 Biochemical parameters 143 5.1.3.3.9.1 Effect of oral administration of methanol extract of 143 Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze. on serum AST level in arthritic rats 5.1.3.3.9.2 Effect of oral administration of methanol extract of 145 Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze. on serum ALT level in arthritic rats 5.1.3.3.9.3 Effect of oral administration of methanol extract of 147 Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze. on serum alkaline phosphatase level in arthritic rats 5.1.3.3.9.4 Effect of oral administration of methanol extract of 149 Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze. on serum total protein level in arthritic rats 5.1.3.3.10 Antioxidant parameters 151 5.1.3.3.10.1 Effect of oral administration of methanol extract of 151 Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze. on INDEX
liver MDA level in arthritic rats 5.1.3.3.10.2 Effect of oral administration of methanol extract of 153 Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze. on liver SOD level in arthritic rats 5.1.3.3.10.3 Effect of oral administration of methanol extract of 155 Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze. on liver GSH level in arthritic rats 5.1.3.3.11 Histopathology of ankle joint 157 5.2 Part B 158 – 180 (Isolation and characterization) 5.2.1 Liquid-solid separation chromatographic technique 158 5.2.1.1 Anti-inflammatory activity of fractions (F – 1 to F – 6) on 159 carrageenan induced paw edema in rats. 5.2.2 Column chromatography of most active anti-inflammatory 161 fraction i.e. 30% acetone in petroleum ether fraction (F – 4) 5.2.2.1 Anti-inflammatory activity of pools (P – 1 to P – 10) 162 collected from 30% acetone fraction (F – 4) in carrageenan induced paw edema 5.2.3 Preparative TLC of most active anti-inflammatory pool i.e. (P 164 – 8) 5.2.4 Spectral characterization of isolated compound (P – 8) 164 5.2.5 Structure assigned to isolated compound (P – 8) 176 Docking study 177 5.3 Part C 181 – 228 (Antiarthritic activity of isolated compound, Isoivangustin) 5.3.1 Effect of oral administration of isoivangustin on change in 181 paw volume in arthritic rats 5.3.2 Effect of oral administration of isoivangustin on change in 183 joint diameter in arthritic rats
INDEX
5.3.3 Effect of oral administration of isoivangustin on pain 185 threshold in arthritic rats 5.3.4 Effect of oral administration of isoivangustin on paw 187 withdrawal latency in arthritic rats 5.3.5 Effect of oral administration of isoivangustin on mechanical 189 nociceptive threshold in arthritic rats 5.3.6 Effect of oral administration of isoivangustin on body weight 191 in arthritic rats 5.3.7 Radiological analysis of ankle joints 193 5.3.8. Haematological parameters 194 5.3.8.1 Effect of oral administration of isoivangustin on blood 194 haemoglobin count in arthritic rats 5.3.8.2 Effect of oral administration of isoivangustin on blood WBC 196 count in arthritic rats 5.3.8.3 Effect of oral administration of isoivangustin on blood RBC 198 count in arthritic rats 5.3.8.4 Effect of oral administration of isoivangustin on blood 200 platelet count in arthritic rats 5.3.8.5 Effect of oral administration of isoivangustin on blood ESR 202 count in arthritic rats 5.3.8.6 Effect of oral administration of isoivangustin on serum CRP 204 level in arthritic rats 5.3.8.7 Effect of oral administration of isoivangustin on RF value in 206 arthritic rats 5.3.9 Biochemical parameters 208 5.3.9.1 Effect of oral administration of isoivangustin on serum AST 208 level in arthritic rats 5.3.9.2 Effect of oral administration of isoivangustin on serum ALT 210 level in arthritic rats 5.3.9.3 Effect of oral administration of isoivangustin on serum 212 alkaline phosphatase level in arthritic rats INDEX
5.3.9.4 Effect of oral administration of isoivangustin on serum total 214 protein level in arthritic rats 5.3.10 Cytokine measurement 216 5.3.10.1 Effect of oral administration of isoivangustin on serum TNF- 216 α in arthritic rats 5.3.10.2 Effect of oral administration of isoivangustin on serum IL-1β 218 in arthritic rats 5.3.10.3 Effect of oral administration of isoivangustin on serum IL-6 220 in arthritic rats 5.3.11 Antioxidant parameters 222 5.3.11.1 Effect of oral administration of isoivangustin on liver MDA 222 level in arthritic rats 5.3.11.2 Effect of oral administration of isoivangustin on liver SOD 224 level in arthritic rats 5.3.11.3 Effect of oral administration of isoivangustin on liver GSH 226 level in arthritic rats 5.3.12 Histopathology of ankle joint 228 Chapter 6 Discussion 229 – 241 Chapter 7 Summary and Conclusion 242 – 247 Chapter 8 Highlights of the work 248 – 253 Chapter 9 Bibliography 254 – 267 Chapter 10 Publications 268 Errata 269 – 271
INDEX
List of Tables
Sr. No. Table Page No. Table 1 Mediators of acute inflammation 08 Table 2 Mediators of chronic inflammation 09 Table 3 Comparison between acute and chronic inflammation 09 Table 4 Naturally occurring compounds with anti-inflammatory 33 properties Table 5 Cytokines involved in the pathogenesis of RA 39 Table 6 Summary for diagnosis of RA 43 Table 7 Common and shared side effects of NSAIDs 50 Table 8 The percent yield and characteristics of different extracts of 108 Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze Table 9 Qualitative phytochemical analysis of PECP, MECP and 109 AECP Table 10 Acute toxicity test of PECP, MECP and AECP 110 Table 11 Effect of oral administration of Cyathocline purpurea (Buch- 112 Ham ex D. Don.) Kuntze. on hot plate test in mice Table 12 Effect of oral administration of Cyathocline purpurea (Buch- 113 Ham ex D. Don.) Kuntze. on acetic acid induced writhing in mice Table 13 Effect of oral administration of Cyathocline purpurea (Buch- 115 Ham ex D. Don.) Kuntze. on carrageenan induced paw edema in rats Table 14 Effect of oral administration of Cyathocline purpurea (Buch- 116 Ham ex D. Don.) Kuntze. on cotton pellet induced granuloma in rats Table 15 Effect of oral administration of methanol extract of 119 Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze. on change in paw volume in arthritic rats
INDEX
Table 16 Effect of oral administration of methanol extract of 121 Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze. on change in joint diameter in arthritic rats Table 17 Effect of oral administration of methanol extract of 123 Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze. on pain threshold in arthritic rats Table 18 Effect of oral administration of methanol extract of 125 Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze. on paw withdrawal latency in arthritic rats Table 19 Effect of oral administration of methanol extract of 127 Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze. on mechanical nociceptive threshold in arthritic rats Table 20 Effect of oral administration of methanol extract of 129 Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze. on body weight in arthritic rats Table 21 Effect of oral administration of methanol extract of 132 Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze. on blood haemoglobin count in arthritic rats Table 22 Effect of oral administration of methanol extract of 134 Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze. on blood WBC count in arthritic rats Table 23 Effect of oral administration of methanol extract of 136 Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze. on blood RBC count in arthritic rats Table 24 Effect of oral administration of methanol extract of 138 Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze. on blood platelet count in arthritic rats Table 25 Effect of oral administration of methanol extract of 140 Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze. on serum CRP level in arthritic rats
INDEX
Table 26 Effect of oral administration of methanol extract of 142 Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze. on RF value in arthritic rats Table 27 Effect of oral administration of methanol extract of 144 Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze. on serum AST level in arthritic rats Table 28 Effect of oral administration of methanol extract of 146 Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze. on serum ALT level in arthritic rats Table 29 Effect of oral administration of methanol extract of 148 Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze. on serum alkaline phosphatase level in arthritic rats Table 30 Effect of oral administration of methanol extract of 150 Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze. on serum total protein level in arthritic rats Table 31 Effect of oral administration of methanol extract of 152 Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze. on liver MDA level in arthritic rats Table 32 Effect of oral administration of methanol extract of 154 Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze. on liver SOD level in arthritic rats Table 33 Effect of oral administration of methanol extract of 156 Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze. on liver GSH level in arthritic rats Table 34 Fractions (F – 1 to F – 6) prepared from MECP 158 Table 35 Anti-inflammatory activity of fractions (F – 1 to F – 6) on 160 carrageenan induced paw edema in rats. Table 36 Pools (P – 1 to P – 10) collected from 30% acetone in 161 petroleum ether fraction (F – 4) based on TLC Table 37 Anti-inflammatory activity of pools (P – 1 to P – 10) 163 collected from 30% acetone fraction (F – 4) in carrageenan INDEX
induced paw edema Table 38 1H-NMR and 13C-NMR with DEPT values of isolated 165 compound P – 8 (200 MHz and 50 MHz, respectively,
CDCl3, TMS as internal standard) Table 39 Docking score of native ligand IH6 on active site of TACE 178 Table 40 Docking score of diclofenac on active site of TACE 179 Table 41 Docking score of isoivangustin on active site of TACE 180 Table 42 Effect of oral administration of isoivangustin on change in 182 paw volume in arthritic rats Table 43 Effect of oral administration of isoivangustin on change in 184 joint diameter in arthritic rats Table 44 Effect of oral administration of isoivangustin on pain 186 threshold in arthritic rats Table 45 Effect of oral administration of isoivangustin on paw 188 withdrawal latency in arthritic rats Table 46 Effect of oral administration of isoivangustin on mechanical 190 nociceptive threshold in arthritic rats Table 47 Effect of oral administration of isoivangustin on body weight 192 in arthritic rats Table 48 Effect of oral administration of isoivangustin on blood 195 haemoglobin count in arthritic rats Table 49 Effect of oral administration of isoivangustin on blood WBC 197 count in arthritic rats Table 50 Effect of oral administration of isoivangustin on blood RBC 199 count in arthritic rats Table 51 Effect of oral administration of isoivangustin on blood 201 platelet count in arthritic rats Table 52 Effect of oral administration of isoivangustin on blood ESR 203 count in arthritic rats Table 53 Effect of oral administration of isoivangustin on serum CRP 205 level in arthritic rats INDEX
Table 54 Effect of oral administration of isoivangustin on RF value in 207 arthritic rats Table 55 Effect of oral administration of isoivangustin on serum AST 209 level in arthritic rats Table 56 Effect of oral administration of isoivangustin on serum ALT 211 level in arthritic rats Table 57 Effect of oral administration of isoivangustin on serum 213 alkaline phosphatase level in arthritic rats Table 58 Effect of oral administration of isoivangustin on serum total 215 protein level in arthritic rats Table 59 Effect of oral administration of isoivangustin on serum TNF- 217 α in arthritic rats Table 60 Effect of oral administration of isoivangustin on serum IL-1β 219 in arthritic rats Table 61 Effect of oral administration of isoivangustin on serum IL-6 221 in arthritic rats Table 62 Effect of oral administration of isoivangustin on liver MDA 223 level in arthritic rats Table 63 Effect of oral administration of isoivangustin on liver SOD 225 level in arthritic rats Table 64 Effect of oral administration of isoivangustin on liver GSH 227 level in arthritic rats
INDEX
List of Figures
Sr. No. Figure Page No. Figure 1 The components of acute and chronic inflammatory 10 responses Figure 2 ‘Triple response’ elicited by firm stroking of skin of 12 forearm with a pencil (A). Diagrammatic view of microscopic features of triple response of the skin (B) Figure 3 The major local manifestations of acute inflammation, 14 compared to normal. (1) Vascular dilation and increased blood flow (causing erythema and warmth), (2) Extravasation and deposition of plasma fluid and proteins (edema), and (3) Leukocyte emigration and accumulation in the site of injury Figure 4 Outcomes of acute inflammation: resolution, healing by 17 fibrosis, or chronic inflammation Figure 5 Chemical mediators of inflammation 25 Figure 6 Comparison of NSAIDs binding sites of COX-1 and COX-2 53 Figure 7 Structural differences in active sites of cyclooxygenase 54 (COX)-1 and COX-2 Figure 8 Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze 67 Figure 9 Column chromatography of 30% acetone in petroleum ether 93 fraction (F – 4) (A) Silica gel adsorbed with 30% acetone in petroleum ether fraction (F – 4) (B) Column loaded with 30% acetone in petroleum ether fraction (F – 4) (C) 30% acetone in petroleum ether fraction (F – 4) uniformly spread fraction in column after addition of mobile phase (D) 30% acetone in petroleum ether fraction uniformly spread fraction in column continued (E) Elution collection started. Figure 10 TLC of pools (P – 1 to P – 10) collected from 30% acetone 95 in petroleum ether fraction (F – 4). (A) TLC of pool P – 1 to INDEX
P – 6 (B) TLC of pool P – 7 to P – 10. Figure 11 Histopathology of stomach in cotton pellet induced 117 granuloma in rats Figure 12 Effect of oral administration of methanol extract of 118 Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze. on change in paw volume in arthritic rats Figure 13 Effect of oral administration of methanol extract of 120 Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze. on change in joint diameter in arthritic rats Figure 14 Effect of oral administration of methanol extract of 122 Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze. on pain threshold in arthritic rats Figure 15 Effect of oral administration of methanol extract of 124 Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze. on paw withdrawal latency in arthritic rats Figure 16 Effect of oral administration of methanol extract of 126 Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze. on mechanical nociceptive threshold in arthritic rats Figure 17 Effect of oral administration of methanol extract of 128 Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze. on body weight in arthritic rats Figure 18 Radiological analysis of ankle joints. (A) Healthy control 130 (B) Arthritic control (C) Diclofenac 5 mg/kg treated (D) MECP 100 mg/kg treated (E) MECP 200 mg/kg treated (F) MECP 400 mg/kg treated Figure 19 Effect of oral administration of methanol extract of 131 Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze. on blood haemoglobin count in arthritic rats Figure 20 Effect of oral administration of methanol extract of 133 Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze. on blood WBC count in arthritic rats INDEX
Figure 21 Effect of oral administration of methanol extract of 135 Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze. on blood RBC count in arthritic rats Figure 22 Effect of oral administration of methanol extract of 137 Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze. on blood platelet count in arthritic rats Figure 23 Effect of oral administration of methanol extract of 139 Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze. on serum CRP level in arthritic rats Figure 24 Effect of oral administration of methanol extract of 141 Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze. on RF value in arthritic rats Figure 25 Effect of oral administration of methanol extract of 143 Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze. on serum AST level in arthritic rats Figure 26 Effect of oral administration of methanol extract of 145 Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze. on serum ALT level in arthritic rats Figure 27 Effect of oral administration of methanol extract of 147 Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze. on serum alkaline phosphatase level in arthritic rats Figure 28 Effect of oral administration of methanol extract of 149 Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze. on serum total protein level in arthritic rats Figure 29 Effect of oral administration of methanol extract of 151 Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze. on liver MDA level in arthritic rats Figure 30 Effect of oral administration of methanol extract of 153 Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze. on liver SOD level in arthritic rats
INDEX
Figure 31 Effect of oral administration of methanol extract of 155 Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze. on liver GSH level in arthritic rats Figure 32 Histopathological analysis of ankle joints stained with 157 H&E. (A) Healthy control (B) Arthritic control (C) Diclofenac 5 mg/kg treated (D) MECP 100 mg/kg treated (E) MECP 200 mg/kg treated (F) MECP 400 mg/kg treated Figure 33 Infra-Red spectrum of isolated compound (P – 8) 166 Figure 34 1H-NMR spectrum of isolated compound (P – 8) 167 Figure 35 First elaborated 1H-NMR spectrum of isolated compound (P 168 – 8) Figure 36 Second elaborated 1H-NMR spectrum of isolated compound 169 (P – 8) Figure 37 Third elaborated 1H-NMR spectrum of isolated compound 170 (P – 8) Figure 38 13C-NMR spectrum of isolated compound (P – 8) 171 Figure 39 First elaborated 13C-NMR spectrum of isolated compound 172 (P – 8) Figure 40 Second elaborated 13C-NMR spectrum of isolated 173 compound (P – 8) Figure 41 DEPT spectrum of isolated compound (P – 8) 174 Figure 42 Mass spectrum of isolated compound (P – 8) 175 Figure 43 Isoivangustin 176 Figure 44 3D binding of native ligand IH6 on active site of TACE 178 Figure 45 3D binding of diclofenac on active site of TACE 179 Figure 46 3D binding of isoivangustin on active site of TACE 180 Figure 47 Effect of oral administration of isoivangustin on change in 181 paw volume in arthritic rats Figure 48 Effect of oral administration of isoivangustin on change in 183 joint diameter in arthritic rats
INDEX
Figure 49 Effect of oral administration of isoivangustin on pain 185 threshold in arthritic rats Figure 50 Effect of oral administration of isoivangustin on paw 187 withdrawal latency in arthritic rats Figure 51 Effect of oral administration of isoivangustin on mechanical 189 nociceptive threshold in arthritic rats Figure 52 Effect of oral administration of isoivangustin on body 191 weight in arthritic rats Figure 53 Radiological analysis of ankle joints. (A) Healthy control 193 (B) Arthritic control (C) Diclofenac 5 mg/kg treated (D) Isoivangustin 2.5 mg/kg treated (E) Isoivangustin 5 mg/kg treated (F) Isoivangustin 10 mg/kg treated Figure 54 Effect of oral administration of isoivangustin on blood 194 haemoglobin count in arthritic rats Figure 55 Effect of oral administration of isoivangustin on blood 196 WBC count in arthritic rats Figure 56 Effect of oral administration of isoivangustin on blood RBC 198 count in arthritic rats Figure 57 Effect of oral administration of isoivangustin on blood 200 platelet count in arthritic rats Figure 58 Effect of oral administration of isoivangustin on blood ESR 202 count in arthritic rats Figure 59 Effect of oral administration of isoivangustin on serum CRP 204 level in arthritic rats Figure 60 Effect of oral administration of isoivangustin on RF value 206 in arthritic rats Figure 61 Effect of oral administration of isoivangustin on serum AST 208 level in arthritic rats Figure 62 Effect of oral administration of isoivangustin on serum ALT 210 level in arthritic rats
INDEX
Figure 63 Effect of oral administration of isoivangustin on serum 212 alkaline phosphatase level in arthritic rats Figure 64 Effect of oral administration of isoivangustin on serum total 214 protein level in arthritic rats Figure 65 Effect of oral administration of isoivangustin on serum 216 TNF-α in arthritic rats Figure 66 Effect of oral administration of isoivangustin on serum IL- 218 1β in arthritic rats Figure 67 Effect of oral administration of isoivangustin on serum IL-6 220 in arthritic rats Figure 68 Effect of oral administration of isoivangustin on liver MDA 222 level in arthritic rats Figure 69 Effect of oral administration of isoivangustin on liver SOD 224 level in arthritic rats Figure 70 Effect of oral administration of isoivangustin on liver GSH 226 level in arthritic rats Figure 71 Histopathological analysis of ankle joints stained with 228 H&E. (A) Healthy control (B) Arthritic control (C) Diclofenac 5 mg/kg treated (D) Isoivangustin 2.5 mg/kg treated (E) Isoivangustin 5 mg/kg treated (F) Isoivangustin 10 mg/kg treated
INDEX
List of Abbreviations
0C Degree centigrade 13C-NMR 13Carbon-nuclear magnetic resonance 1H-NMR 1Proton-nuclear magnetic resonance Å Angstrom AECP Aqueous extract of Cyathocline purpurea ANA Antinuclear antibodies AIA Adjuvant induced arthritis ALP Alkaline phosphatase ALT Alanine transaminase AOT Acute oral toxicity AP-1 Activator protein-1 APR Acute phase reactant AST Aspartate transaminase CCP Citrullinated peptide cm Centimeter
CDCl3 Deuterated chloroform CIA Collagen induced arthritis CNS Central nervous system COMP Cartilage oligomeric matrix protein COX Cyclooxygenase CPCSEA Committee for the Purpose of Control and Supervision on Experiments of Animals CRP C-reactive protein d Doublet dd Doublet of doublet DA Dark agouti DDA Dioctadecyldiammonium DEPT Distortion-less enhancement by polarization transfer DRG Dorsal root ganglia INDEX
DTNB 5,5’-dithiobis (2-nitro benzoic acid) DMARDs Disease modifying anti-rheumatic drugs DNA Deoxyribonucleic acids EDTA Ethylene diamine tetraacetic acid ENA Extractable nuclear antigens ELISA Enzyme linked immunosorbent assay ESR Erythrocyte sedimentation rate FCA Freund’s complete adjuvant FAP Familial adenomatious polyposis g Gram g/l Gram/liter GI Gastrointestinal GIT Gastrointestinal tract GSH Glutathione GC-MS Gas chromatography-Mass spectrometry GC-FID Gas chromatography with flame ionization detector GM-CSF Granulocyte macrophage-colony stimulating factor HPETE Hydroperoxyeicosatetraenoic acid HACA Human anti-chimeric antibodies HLA Human leukocyte antigens H & E Haematoxylin and eosin HIV Human immunodeficiency virus Hb Haemoglobin HT Hydroxytryptamine HSP Heat shock protein HUVECs Human umbilical vein endothelial cells h hour i.p. Intraperitoneal i.v. Intravenous IAEC Institutional animal ethics committee IFN Interferon INDEX
IFA Incomplete freund’s adjuvant IL Interleukin Ig Immunoglobulin Ile Isoleucine iNOS Inducible nitric oxide synthase IR Infrared IU International unit J Coupling constant kg Kilogram LEW Lewis LPS Lipopolysaccharide LT Leukotriene LX Lipoxins M Molar MECP Methanol Extract of Cyathocline purpurea MS Mass spectroscopy m Multiplates ml Milliliter MDA Malondialdehyde mg/dl Milligram/ deciliter mg/kg Milligram /kilogram mg/ml Milligram/ milliliter MALT Mucosa associated lymphoid tissue MBC Minimal bactericidal concentration MHC Major histocompatibility complex Min. Minutes mm Millimeter MMP Matrix metalloproteinases MPO Myeloperoxidase MRI Magnetic resonance imaging MS Mass spectroscopy INDEX ng Nanogram nm Nanometer NF-κB Nuclear factor kappa B NMR Nuclear magnetic resonance NK Natural killer NO Nitric oxide NSAIDs Non steroidal anti-inflammatory drugs OA Osteoarthritis OIA Oil induced arthritis OECD Organization for economic co-operation and development p.o. Per oral pg/ml Picograms per millilitre PAF Platelet activating factor PDGF Platelet derived growth factor PIA Pristane induced arthritis PPI Proton pump inhibitors PLT Platelets ppm Parts per million PBS Phosphate buffer saline PECP Petroleum ether extract of Cyathocline purpurea PECAM-1 Platelet endothelial cell adhesion molecule-1 PG Prostaglandin PMN Polymorphonuclear neutrophils RA Rheumatoid arthritis RBC Red blood cell RF Rheumatoid factor ROS Reactive oxygen species rpm Rotations per minute RMSD Rheumatic musculoskeletal disorder RT Room temperature RNA Ribonucleic acids INDEX
Sec Seconds SCW Streptococcal cell wall s/c Subcutaneous S.E.M. Standard error mean SOD Superoxide dismutase SRS-As Slow reacting substances of anaphylaxis TACE TNF-alpha converting enzyme TGF-α Transforming growth factor α TLC Thin layer chromatography TCA Trichloroacetic acid TBA Thiobarbituric acid TMB Tetramethylbenzidine TMS Tetramethylsilane TNF-α Tumour necrosis factor-α TXA Thromboxane UV Ultraviolet val Valine VCAM-1 Vascular cell adhesion molecule-1 VEGF Vascular endothelial growth factor VIP Vasoactive intestinal polypeptide w/v Weight/volume WBC White blood cell WHO World Health Organization µg/ml Microgram/milliliter µl Microlitre µm Micrometer µg Microgram
Introduction Introduction
By definition, the word natural is an adjective referring to something that is present in or produced by nature and not artificial or man-made. The term natural products today is quite commonly understood to refer to herbs, herbal concoctions, dietary supplements, traditional Chinese medicine, or alternative medicine (Holt and Chandra, 2002). Alternative medicine for the treatment of various diseases is getting more popular. Many medicinal plants provide relief of symptoms comparable to that of conventional medicinal agents (Verpoorte, 1999). Plants are natural and traditional sources of medication in large parts of the world. Herbs have been used since ancient times by physicians and also by layman to treat a great variety of human diseases. A wide variety of herbs singly and in mixture have been extensively investigated in basic biological sciences to evaluate their chief as well as supplementary, complementary and synergistic action in health and diseases (Rajeshwari and Andallu, 2012). Herbal products are receiving increasing public interest, and herbal treatment is now the most popular alternative therapy (Zhang et al., 2009). The exploration of traditional knowledge for cure to common diseases is attractive since antiquity. The medicinal plants are responsible for the most of the medicine and food used in modern society. It is estimated that an amount of 20,000 species from several families are useful for these purposes.
The World Health Organization (WHO) estimates that approximately 80 percent of the world’s population relies primarily on traditional medicines as sources for their primary health care (WHO, 1996; Farnsworth et al., 1985). Over 100 chemical substances that are considered to be important drugs that are either currently in use or have been widely used in one or more countries in the world have been derived from different plants. Approximately 75 percent of these substances were discovered as a direct result of chemical studies focused on the isolation of active substances from plants used in traditional medicine (Cragg and Newman, 2001). Indeed, if one looks at new drugs from an indication perspective over the same period of time, over 60 percent of antibacterials and antineoplastics were again either natural products themselves or based on structures of natural products. One of the earliest treatises of Indian medicine, the Charaka Samhita (1000 B.C.) mentions the use of over 2000 herbs for medicinal purpose (Meenal et al., 2010). Plants continue to serve as possible
Study of Analgesic, Anti-inflammatory and Antiarthritic activity of Indian medicinal plant in laboratory animals 1
Introduction sources for new drugs and chemicals derived from various parts of plants (Srivastav et al., 2011). The natural products derived from medicinal plants have proven to be an abundant source of biologically active compounds, many of which have been the basis for the development of new lead chemicals for pharmaceuticals.
The costs of drug discovery and drug development continue to increase at astronomical rates, yet despite these expenditures, there is a decrease in the number of new medicines introduced into the world market. Despite the successes that have been achieved over the years with natural products, the interest in natural products as a platform for drug discovery has waxed and waned in popularity with various pharmaceutical companies. Plants today are most likely going to continue to exist and grow to become even more valuable as sources of new drug leads. This is because the degree of chemical diversity found in plants is broader than that from any other source, and the degree of novelty of molecular structure found in plants is greater than that determined from any other source (Cragg et al., 1997; Harvey, 2001). Higher plants have been over time an extremely popular source of natural products (O’Keefe, 2001).
Plants chosen for drug development are most commonly selected based on their use in traditional medicine. A plant becomes medicinal only when its biological activities suggested by ethnobotany have been reported or scientifically investigated and established. The isolation of the constituents of medicinal plants has been carried out now for almost two hundred years. It commenced with the isolation of opium alkaloids in the early 19th century which has resulted in the discovery of a large number of compound with a very wide diversity of structures and biological activity (Deraniyagala et al., 2003). The last fifty years has seen much activity in this area and many thousands of novel compounds have been isolated and characterized. The research for drugs from plants should focus on tropical countries, because over 50 percent of the estimated plants species found on earth come from tropical forests, which are currently being destroyed at very alarming rate, to give way for construction and or mineral development or exploitation.
Study of Analgesic, Anti-inflammatory and Antiarthritic activity of Indian medicinal plant in laboratory animals 2
Introduction
After centuries of empirical use of herbal preparation, the first isolation of active principles – alkaloids such as morphine, strychnine, quinine etc. – in the early 19th century marked a new era in the use of medicinal plants and the beginning of modern medicinal plant research. Emphasis shifted away from plant derived drugs with the tremendous development of synthetic pharmaceutical chemistry and microbial fermentation after 1945. Plant metabolites were mainly investigated from a phytochemical and chemotaxonomic viewpoint during this period. Consumption of medicinal plants has almost doubled in Western Europe during that period. Ecological awareness, the efficacy of a good number of phyto pharmaceutical preparations, such as ginkgo, garlic or valerian and increased interest of major pharmaceutical companies in higher plants as sources for new lead structures has been the main reasons for this renewal of interest. Phytomedicine almost went into extinction during the first half of the 21st century due to the use of the ‘more powerful and potent synthetic drug’. However, because of the numerous side effects of these drugs, the value of medicinal plants is being rediscovered as some of them have proved to be as effective as synthetic medicines with fewer or no side effects and contraindications. It has been proved that although the effects of natural remedies may seem slower, the results are sometimes better on the long run especially in chronic diseases (Akunyili, 2003).
Of the 252 essential drugs selected by WHO, 11% comes from plants and 8.7% from animal kingdom (Marques, 1997). The industrial revolution and development of modern chemical and engineering technology has facilitated the preference for synthetic products for pharmacological treatment. But the impact of these products has some negative effects like toxicity, etc. and for this reason; the researchers are once again showing their interests in the natural products for the pharmacological treatment. There are several reports that the toxicity of natural compounds is much lesser than the synthetic compounds. 74% of all plant-derived drugs in clinical use worldwide have been discovered through follow-up investigation of their ethno medical uses (Soejarto, 1996). The clinical applications of taxol, etoposide and artemisinin have helped to revive an interest in higher plants as sources of new drugs (Phillipson, 1999). It is estimated that around 2,50,000 species of higher plants are
Study of Analgesic, Anti-inflammatory and Antiarthritic activity of Indian medicinal plant in laboratory animals 3
Introduction existing and the majority of these have not been examined in detail for their pharmacological activities (Park and Ernst, 2005; Mamtani and Mamtani, 2005) whereas some plants have been tested with promising results (Aggarwal et al., 2007), so still there is great potential for discovering novel bioactive compounds. There is a worldwide belief that herbal remedies are safer and less damaging to the human body than synthetic drugs. Therefore laboratories around the world are engaged in screening of plants for biological activities with therapeutics potential. The traditional Indian medicinal system mentions herbal remedies for the treatment of variety of diseases. Numerous drugs have entered the International Pharmacopoeia via the study of Ethnopharmacology and traditional medicine. Traditional medicines can offer a more holistic approach to drug design and myriad possible targets for scientific analysis. Powerful new technologies such as automated separation techniques, high- throughput screening and combinatorial chemistry are revolutionizing drug discovery. Traditional knowledge can serve as powerful search engine, which will greatly facilitate intentional, focused and safe natural product drug discovery and help to rediscover the drug discovery process.
Drugs developed from plants: Less than 100 years ago, therapeutic agents from medicinal plants have been incorporated into orthodox medicine and examples include the sesquiterpene endoperoxide, artemisinin from the Chinese plant Artemisia annua for the treatment of malaria; and taxol from Taxus brevifolia, used in metastatic breast cancer (Lenaz and Furial, 1993). Other classical examples include atropine, an alkaloid from Atropa belladonna as ophthalmics and synthetic spasmolytics, morphine and paraverine from Papaver somniferum for the synthesis of analgesic and spasmolytics: quinine and quinidine from Cinchona succirubra bark, for malaria and antiarrythma, and digitoxin, most important cardiotonic drug in orthodox medicine from Digitalis purpurea for semi-synthetic cardiac glycosides. Cocaine from Erythroxylum coca for synthetic local aenesthetics. Ephedrine from Ephedra sinica for synthetic sympathomimetics; Reserpine, ajmaline and other alkaloids from Rauwolfia serpentina for synthetic antihypertensives and antiarrythmics. Strophantin from Since 1961 many compounds derived from plants have been approved in the United States
Study of Analgesic, Anti-inflammatory and Antiarthritic activity of Indian medicinal plant in laboratory animals 4
Introduction
(Lee, 1999). These drugs include vinblastine, etoposide, paclitaxel, vincristine, topotecan, and irinotecan. Strophantus seeds, emetine from ipecac, anthraquinone glycosides from cassia leaf, etc are also used clinically. A phorbol derivative prostratin, was discovered as possessing good anti-HIV activity following the lead of a plant, samoan plant Homalanthus nutans used locally for yellow fever.
Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease (Gomes et al., 2010). It is a common disease affecting millions of people (Zhang et al., 2009). Although various drugs have been used to control RA, there are numerous reports regarding the side effects of these drugs (Paval et al., 2009). RA is a major syndrome among the aged people that has been in this world since the beginning of civilization, an oldest disease of the universe. Arthritis has been mentioned in the ancient Hindu and Greek mythology (Sturrock and Sharma, 1977). The first written reference on arthritis was found in the Indian holistic book Chakra Samhita where it has been described as swollen painful joints, initially occurring in hands, feet, causing loss of appetite and occasionally related with fever (Fornaciari et al., 2009). Although RA can start at any age, the peak onset is between 25 and 55 years. RA primarily affects the synovial joints of all extremities and is pathologically characterized by severe inflammation and progressive destruction of cartilage and subchondral bone (Michel et al., 2007). When chronic inflammation occurs in RA, it involves the actions of large numbers of lymphocytes, macrophages and polymorphonuclear cells in the inflamed joint (Haynes et al., 1998). The inflammatory process of RA is reportedly associated with an increase of pro-inflammatory cytokines TNF-α and IL-1β (Fan et al., 2005). The prevalence of RA in Indian subcontinent is 1.5-2 % of population. The epidemiological ratio of arthritis in female: male is 3:1 and the prevalence is 1% of the world population (Mishra et al., 2011). Adjuvant- induced arthritis is a good laboratory model for studying RA (Tanaka et al., 1996; Nagakura et al., 2003). In this model the clinical and pathological changes are comparable to with those observed in human RA (Noguchi et al., 2005; Geetha and Varalakshmi, 1999). The aim for the treatment of this disease is to reduce pain and to minimize the changes occurring during RA. Physiotherapy, acupuncture, physical exercise and analgesics are often prescribed by the rheumatologists. Non-steroidal anti-inflammatory drugs (NSAIDs)
Study of Analgesic, Anti-inflammatory and Antiarthritic activity of Indian medicinal plant in laboratory animals 5
Introduction are the first line of defense against arthritis. But these NSAIDs have certain side effects like gastro-intestinal tract irritation; inhibit PG biosynthesis, shows problem in platelets aggregation (Tastekin et al., 2007; Deguchi et al., 2011). Glucocorticoid therapy also shows some side effects like immune suppression, muscular breakdown, pubertal delay etc. Disease modifying anti rheumatic drugs (DMARDS) now days are mostly advised but these drugs have side effects like sepsis, pulmonary tuberculosis etc. Since the available therapeutics have their own limitations so there is a dramatic increase in the use of alternative therapy.
This present research work is an effort to reveal the analgesic, anti-inflammatory and antiarthritic activity of Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze Fam. Asteraceae in experimental animal models. It is expected that the information may open a new dimension in alternative therapeutic management of pain, inflammation and arthritis in the near future.
Study of Analgesic, Anti-inflammatory and Antiarthritic activity of Indian medicinal plant in laboratory animals 6
Review of Literature Review of Literature
2.1 INFLAMMATION 2.1.1 Definition and causes Inflammation is defined as the local response of living mammalian tissues to injury due to any agent. It is a body defense reaction in order to eliminate or limit the spread of injurious agent, followed by removal of the necrosed cells and tissues. The agents causing inflammation may be as under: 1. Infective agents like bacteria, viruses and their toxins, fungi, parasites. 2. Immunological agents like cell-mediated and antigen antibody reactions. 3. Physical agents like heat, cold, radiation, mechanical trauma. 4. Chemical agents like organic and inorganic poisons. 5. Inert materials such as foreign bodies. Thus, inflammation is distinct from infection—while inflammation is a protective response by the body to variety of etiologic agents (infectious or non-infectious), while infection is invasion into the body by harmful microbes and their resultant ill- effects by toxins. Inflammation involves 2 basic processes with some overlapping, viz. early inflammatory response and later followed by healing. Though both these processes generally have protective role against injurious agents, inflammation and healing may cause considerable harm to the body as well e.g. anaphylaxis to bites by insects or reptiles, drugs, toxins, atherosclerosis, chronic rheumatoid arthritis, fibrous bands and adhesions in intestinal obstruction (Harshmohan, 2010).
2.1.2 Signs of inflammation The Roman writer Celsus in named the famous 4 cardinal signs of inflammation as: V Rubor (redness); V Tumor (swelling); V Calor (heat); and V Dolor (pain). To these, fifth sign functio laesa (loss of function) was later added by Virchow. The word inflammation means burning. This nomenclature had its origin in old times but now we know that burning is only one of the signs of inflammation.
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Review of Literature
2.1.3 Types of inflammation (Harshmohan, 2010) Depending upon the defense capacity of the host and duration of response, inflammation can be classified as acute and chronic. A. Acute inflammation Is of short duration (lasting less than 2 weeks) and represents the early body reaction, resolves quickly and is usually followed by healing. The main features of acute inflammation are: 1. Accumulation of fluid and plasma at the affected site; 2. Intravascular activation of platelets; and 3. Polymorphonuclear neutrophils as inflammatory cells. Sometimes, the acute inflammatory response may be quite severe and is termed as fulminant acute inflammation.
Table 1: Mediators of acute inflammation (Harshmohan, 2010) Mediators Vasodilation Vascular permeability Chemotaxis Pain Histamine ++ +++ - - Serotonin + + - - Bradykinin +++ + - +++ Prostaglandin +++ + +++ + Leukotrienes - +++ +++ - (+++ severe, ++ moderate, + mild, - absent)
B. Chronic inflammation Is of longer duration and occurs either after the causative agent of acute inflammation persists for a long time, or the stimulus is such that it induces chronic inflammation from the beginning. A variant, chronic active inflammation is the type of chronic inflammation in which during the course of disease there are acute exacerbations of activity. The characteristic feature of chronic inflammation is presence of chronic inflammatory cells such as lymphocytes, plasma cells and macrophages, granulation tissue formation, and in specific situations as granulomatous inflammation. In some instances, the term subacute inflammation is used for the state of inflammation between acute and chronic.
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Review of Literature
Table 2: Mediators of chronic inflammation (Harshmohan, 2010) Mediators Sources Primary effects IL-1, IL-2, Macrophages, T-lymphocytes Lymphocyte activation, IL-3 prostaglandin production GM-CSF T-lymphocytes, endothelial cells, Macrophages and granulocyte fibroblast activation TNF-α Macrophages Prostaglandin Interferons Macrophages, endothelial cells, T- Many lymphocytes PDGF Macrophages, endothelial cells, Fibroblast chemotaxis, fibroblast, platelets proliferation
Table 3: Comparison between acute and chronic inflammation (Harshmohan, 2010) Acute inflammation Chronic inflammation
Causative Pathogens, injured tissues Persistent acute inflammation due to non- agent degradable pathogens, persistent foreign bodies, or autoimmune reactions
Major cells Neutrophils, mononuclear Mononuclear cells (monocytes, involved cells (monocytes, macrophages, lymphocytes, plasma cells), macrophages) fibroblasts
Primary Vasoactive amines, IFN-γ and other cytokines, growth mediators eicosanoids factors, reactive oxygen species, hydrolytic enzymes
Onset Immediate Delayed
Duration Few days Up to many months, or years
Outcomes Healing, abscess Tissue destruction, fibrosis formation.
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Figure 1: The components of acute and chronic inflammatory responses (Vinay et al., 2007)
A. Acute inflammation Acute inflammatory response by the host to any agent is a continuous process but for the purpose of discussion, it can be divided into following two events: I. Vascular events. II. Cellular events.
I. Vascular events Alteration in the microvasculature (arterioles, capillaries and venules) is the earliest response to tissue injury. These alterations include 1. Haemodynamic changes 2. Changes in vascular permeability
1. Haemodynamic changes The earliest features of inflammatory response result from changes in the vascular flow and calibre of small blood vessels in the injured tissue. The sequence of these changes is as under: 1. Irrespective of the type of injury, immediate vascular response is of transient vasoconstriction of arterioles. With mild form of injury, the blood flow may Study of Analgesic, Anti-inflammatory and Antiarthritic activity of Indian medicinal plant in laboratory animals 10
Review of Literature
be re-established in 3-5 seconds while with more severe injury the vasoconstriction may last for about 5 minutes. 2. Next follows persistent progressive vasodilatation which involves mainly the arterioles, but to a lesser extent, affects other components of the microcirculation like venules and capillaries. This change is obvious within half an hour of injury. Vasodilatation results in increased blood volume in microvascular bed of the area, which is responsible for redness and warmth at the site of acute inflammation. 3. Progressive vasodilatation, in turn, may elevate the local hydrostatic pressure resulting in transudation of fluid into the extracellular space. This is responsible for swelling at the local site of acute inflammation.
4. Slowing or stasis of microcirculation follows which causes increased concentration of red cells, and thus, raised blood viscosity.
5. Stasis or slowing is followed by leucocytic margination or peripheral orientation of leucocytes (mainly neutrophils) along the vascular endothelium. The leucocytes stick to the vascular endothelium briefly, and then move and migrate through the gaps between the endothelial cells into the extravascular space. This process is known as emigration.
The features of haemodynamic changes in inflammation are best demonstrated by the Lewis experiment. Lewis induced the changes in the skin of inner aspect of forearm by firm stroking with a blunt point. The reaction so elicited is known as triple response or red line response consisting of the following,
i) Red line appears within a few seconds following stroking and is due to local vasodilatation of capillaries and venules.
ii) Flare is the bright reddish appearance or flush surrounding the red line and results from vasodilatation of the adjacent arterioles.
iii) Wheal is the swelling or oedema of the surrounding skin occurring due to transudation of fluid into the extravascular space.
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These features thus elicit the classical signs of inflammation— redness, heat, swelling and pain.
Figure 2: ‘Triple response’ elicited by firm stroking of skin of forearm with a pencil (A). Diagrammatic view of microscopic features of triple response of the skin (B) (Harshmohan, 2010)
2. Changes in vascular permeability Pathogenesis In and around the inflamed tissue, there is accumulation of oedema fluid in the interstitial compartment which comes from blood plasma by its escape through the endothelial wall of peripheral vascular bed. In the initial stage, the escape of fluid is due to vasodilatation and consequent elevation in hydrostatic pressure. This is transudate in nature. But subsequently, the characteristic inflammatory oedema, exudate, appears by increased vascular permeability of microcirculation.
The appearance of inflammatory oedema due to increased vascular permeability of microvascular bed is explained on the basis of Starling’s hypothesis. In normal circumstances, the fluid balance is maintained by two opposing sets of forces:
i) Forces that cause outward movement of fluid from microcirculation are intravascular hydrostatic pressure and colloid osmotic pressure of interstitial fluid.
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Review of Literature
ii) Forces that cause inward movement of interstitial fluid into circulation are intravascular colloid osmotic pressure and hydrostatic pressure of interstitial fluid.
Mechanisms of increased vascular permeability
In acute inflammation, normally non-permeable endothelial layer of microvasculature becomes leaky.
1) Contraction of endothelial cells This is the most common mechanism of increased leakiness that affects venules exclusively while capillaries and arterioles remain unaffected. The endothelial cells develop temporary gaps between them due to their contraction resulting in vascular leakiness. It is mediated by the release of histamine, bradykinin and other chemical mediators. The response begins immediately after injury, is usually reversible, and is for short duration (15-30 minutes). Example of such immediate transient leakage is mild thermal injury of skin of forearm.
2) Retraction of endothelial cells In this mechanism, there is structural re-organisation of the cytoskeleton of endothelial cells that causes reversible retraction at the intercellular junctions. This change too affects venules and is mediated by cytokines such as interleukin-1 (IL-1) and tumour necrosis factor (TNF)-α. The onset of response takes 4-6 hours after injury and lasts for 2-4 hours or more (somewhat delayed and prolonged leakage). The example of this type of response exists in vitro experimental work only.
3) Direct injury to endothelial cells Direct injury to the endothelium causes cell necrosis and appearance of physical gaps at the sites of detached endothelial cells. Process of thrombosis is initiated at the site of damaged endothelial cells. The change affects all levels of microvasculature (venules, capillaries and arterioles). The increased permeability may either appear immediately after injury or last for several hours or days (immediate sustained leakage), or may occur after a delay of 2-12 hours and last for hours or days (delayed prolonged leakage). The examples of immediate sustained leakage are severe bacterial
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Review of Literature infections while delayed prolonged leakage may occur following moderate thermal injury and radiation injury.
4) Endothelial injury mediated by leucocytes Adherence of leucocytes to the endothelium at the site of inflammation may result in activation of leucocytes. The activated leucocytes release proteolytic enzymes and toxic oxygen species which may cause endothelial injury and increased vascular leakiness. This form of increased vascular leakiness affects mostly venules and is a late response. The examples are seen in sites where leucocytes adhere to the vascular endothelium e.g. in pulmonary venules and capillaries.
5) Leakiness in neovascularisation In addition, the newly formed capillaries under the influence of vascular endothelial growth factor (VEGF) during the process of repair and in tumours are excessively leaky. Figure 3: The major local manifestations of acute inflammation, compared to normal. (1) Vascular dilation and increased blood flow (causing erythema and warmth), (2) Extravasation and deposition of plasma fluid and proteins (edema), and (3) Leukocyte emigration and accumulation in the site of injury (Vinay et al., 2007)
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II. Cellular events The cellular phase of inflammation consists of 2 processes: 1. Exudation of leucocytes 2. Phagocytosis
1. Exudation of Leucocytes The escape of leucocytes from the lumen of microvasculature to the interstitial tissue is the most important feature of inflammatory response. In acute inflammation, polymorphonuclear neutrophils (PMNs) comprise the first line of body defense, followed later by monocytes and macrophages. 1. Changes in the formed elements of blood In the early stage of inflammation, the rate of flow of blood is increased due to vasodilatation. But subsequently, there is slowing or stasis of bloodstream. With stasis, changes in the normal axial flow of blood in the microcirculation take place. The normal axial flow consists of central stream of cells comprised by leucocytes and RBCs and peripheral cellfree layer of plasma close to vessel wall. Due to slowing and stasis, the central stream of cells widens and peripheral plasma zone becomes narrower because of loss of plasma by exudation. This phenomenon is known as margination. As a result of this redistribution, the neutrophils of the central column come close to the vessel wall; this is known as pavementing.
2. Rolling and adhesion Peripherally marginated and pavemented neutrophils slowly roll over the endothelial cells lining the vessel wall (rolling phase). This is followed by the transient bond between the leucocytes and endothelial cells becoming firmer (adhesion phase).
3. Emigration After sticking of neutrophils to endothelium, the former move along the endothelial surface till a suitable site between the endothelial cells is found where the neutrophils throw out cytoplasmic pseudopods. Subsequently, the neutrophils lodged between the endothelial cells and basement membrane cross the basement membrane by damaging it locally with secreted collagenases and escape out into the extravascular space; this is known as emigration. The damaged basement membrane is repaired almost
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Review of Literature immediately. As already mentioned, neutrophils are the dominant cells in acute inflammatory exudate in the first 24 hours, and monocyte-macrophages appear in the next 24-48 hours. However, neutrophils are short-lived (24-48 hours) while monocyte-macrophages survive much longer. Simultaneous to emigration of leucocytes, escape of red cells through gaps between the endothelial cells, diapedesis, takes place. It is a passive phenomenon—RBCs being forced out either by raised hydrostatic pressure or may escape through the endothelial defects left after emigration of leucocytes. Diapedesis gives haemorrhagic appearance to the inflammatory exudate.
4. Chemotaxis The chemotactic factor-mediated transmigration of leucocytes after crossing several barriers (endothelium, basement membrane, perivascular myofibroblasts and matrix) to reach the interstitial tissues is called chemotaxis. The concept of chemotaxis is well illustrated by Boyden’s chamber experiment. In this, a millipore filter (3 µm pore size) separates the suspension of leucocytes from the test solution in tissue culture chamber. If the test solution contains chemotactic agent, the leucocytes migrate through the pores of filter towards the chemotactic agent.
2. Phagocytosis Phagocytosis is defined as the process of engulfment of solid particulate material by the cells (cell-eating). The cells performing this function are called phagocytes. There are 2 main types of phagocytic cells: i) Polymorphonuclear neutrophils (PMNs) which appear early in acute inflammatory response, sometimes called as microphages. ii) Circulating monocytes and fixed tissue mononuclear phagocytes, commonly called as macrophages. Neutrophils and macrophages on reaching the tissue spaces produce several proteolyitc enzymes—lysozyme, protease, collagenase, elastase, lipase, proteinase, gelatinase, and acid hydrolases. These enzymes degrade collagen and extracellular matrix. The microbe undergoes the process of phagocytosis by polymorphs and macrophages and involves the following 3 steps, 1. Recognition and attachment
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2. Engulfment 3. Killing and degradation
Outcomes of acute inflammation The acute inflammatory process can culminate in one of the following outcomes, 1. Resolution: It means complete return to normal tissue following acute inflammation. This occurs when tissue changes are slight and the cellular changes are reversible e.g. resolution in lobar pneumonia. 2. Healing: Healing by fibrosis takes place when the tissue destruction in acute inflammation is extensive so that there is no tissue regeneration. But when tissue loss is superficial, it is restored by regeneration. 3. Suppuration: When the pyogenic bacteria causing acute inflammation result in severe tissue necrosis, the process progresses to suppuration. Initially, there is intense neutrophilic infiltration. Subsequently, mixture of neutrophils, bacteria, fragments of necrotic tissue, cell debris and fibrin comprise pus which is contained in a cavity to form an abscess. The abscess, if not drained, may get organised by dense fibrous tissue, and in time, get calcified. 4. Chronic inflammation: Persisting or recurrent acute inflammation may progress to chronic inflammation in which the processes of inflammation and healing proceed side by side (Harshmohan, 2010).
Figure 4: Outcomes of acute inflammation: resolution, healing by fibrosis, or chronic inflammation (Vinay et al., 2007)
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B. Chronic inflammation Definition and causes Chronic inflammation is defined as prolonged process in which tissue destruction and inflammation occur at the same time. Chronic inflammation can be caused by one of the following 3 ways:
1. Chronic inflammation following acute inflammation: When the tissue destruction is extensive, or the bacteria survive and persist in small numbers at the site of acute inflammation e.g. in osteomyelitis, pneumonia terminating in lung abscess.
2. Recurrent attacks of acute inflammation: When repeated bouts of acute inflammation culminate in chronicity of the process e.g. in recurrent urinary tract infection leading to chronic pyelonephritis, repeated acute infection of gallbladder leading to chronic cholecystitis.
3. Chronic inflammation starting de novo: When the infection with organisms of low pathogenicity is chronic from the beginning e.g. infection with Mycobacterium tuberculosis.
General features of chronic inflammation Though there may be differences in chronic inflammatory response depending upon the tissue involved and causative organisms, there are some basic similarities amongst various types of chronic inflammation. Following general features characterise any chronic inflammation: 1. Mononuclear cell infiltration: Chronic inflammatory lesions are infiltrated by mononuclear inflammatory cells like phagocytes and lymphoid cells. Phagocytes are represented by circulating monocytes, tissue macrophages, epithelioid cells and sometimes, multinucleated giant cells. The macrophages comprise the most important cells in chronic inflammation. These may appear at the site of chronic inflammation from: i) Chemotactic factors and adhesion molecules for continued infiltration of macrophages;
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Review of Literature ii) Local proliferation of macrophages; and iii) Longer survival of macrophages at the site of inflammation. The blood monocytes on reaching the extravascular space transform into tissue macrophages. Besides the role of macrophages in phagocytosis, they may get activated in response to stimuli such as cytokines (lymphokines) and bacterial endotoxins. On activation, macrophages release several biologically active substances e.g. acid and neutral proteases, oxygen-derived reactive metabolites and cytokines. These products bring about tissue destruction, neovascularisation and fibrosis. Other chronic inflammatory cells include lymphocytes, plasma cells, eosinophils and mast cells. In chronic inflammation, lymphocytes and macrophages influence each other and release mediators of inflammation. 2. Tissue destruction or necrosis: Tissue destruction and necrosis are central features of most forms of chronic inflammatory lesions. This is brought about by activated macrophages which release a variety of biologically active substances e.g. protease, elastase, collagenase, lipase, reactive oxygen radicals, cytokines (IL-1, IL-8, TNF-α), nitric oxide, angiogenesis growth factor etc. 3. Proliferative changes: As a result of necrosis, proliferation of small blood vessels and fibroblasts is stimulated resulting in formation of inflammatory granulation tissue. Eventually, healing by fibrosis and collagen laying takes place.
Systemic effects of chronic inflammation Chronic inflammation is associated with following systemic features: 1. Fever: Invariably there is mild fever, often with loss of weight and weakness. 2. Anaemia: Chronic inflammation is accompanied by anaemia of varying degree. 3. Leucocytosis: As in acute inflammation, chronic inflammation also has leucocytosis but generally there is relative lymphocytosis in these cases. 4. ESR: ESR is elevated in all cases of chronic inflammation. 5. Amyloidosis: Long-term cases of chronic suppurative inflammation may develop secondary systemic amyloidosis.
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Types of chronic inflammation Conventionally, chronic inflammation is subdivided into 2 types: 1. Non-specific: When the irritant substance produces a nonspecific chronic inflammatory reaction with formation of granulation tissue and healing by fibrosis e.g. chronic osteomyelitis, chronic ulcer.
2. Specific: When the injurious agent causes a characteristic histologic tissue response e.g. tuberculosis, leprosy, syphilis. However, for a more descriptive classification, histological features are used for classifying chronic inflammation into 2 corresponding types: 1. Chronic non-specific inflammation: It is characterised by non-specific inflammatory cell infiltration e.g. chronic osteomyelitis, lung abscess. A variant of this type of chronic inflammatory response is chronic suppurative inflammation in which infiltration by polymorphs and abscess formation are additional features e.g. actinomycosis.
2. Chronic granulomatous inflammation: It is characterised by formation of granulomas e.g. tuberculosis, leprosy, syphilis, actinomycosis, sarcoidosis etc.
2.1.4 Chemical mediators of inflammation (Harshmohan, 2010)
1. Vasoactive amines Two important pharmacologically active amines that have role in the early inflammatory response (first one hour) are histamine and 5- hydroxytryptamine (5- HT) or serotonin; another recently added group is of neuropeptides. i) Histamine It is stored in the granules of mast cells, basophils and platelets. Histamine is released from these cells by various agents as under: a) Stimuli or substances inducing acute inflammation e.g. heat, cold, irradiation, trauma, irritant chemicals, immunologic reactions etc.
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Review of Literature b) Histamine-releasing factors from neutrophils, monocytes and platelets. c) Interleukins. The main actions of histamine are: vasodilatation, increased vascular (venular) permeability, itching and pain. Stimulation of mast cells and basophils also releases products of arachidonic acid metabolism including the release of slowreacting substances of anaphylaxis (SRS-As). The SRS-As consist of various leukotrienes (LTC4, LTD4 and LTE4).
ii) 5-Hydroxytryptamine (5-HT or serotonin) It is present in tissues like chromaffin cells of GIT, spleen, nervous tissue, mast cells and platelets. The actions of 5-HT are similar to histamine but it is a less potent mediator of increased vascular permeability and vasodilatation than histamine.
iii) Neuropeptides Another class of vasoactive amines is tachykinin neuropeptides, such as substance P, neurokinin A, vasoactive intestinal polypeptide (VIP) and somatostatin. These small peptides are produced in the central and peripheral nervous systems. The major proinflammatory actions of these neuropeptides is as follows: a) Increased vascular permeability. b) Transmission of pain stimuli. c) Mast cell degranulation.
2. Arachidonic acid metabolites (eicosanoids) Arachidonic acid metabolites or eicosanoids are the most potent mediators of inflammation, much more than oxygen free radicals. Arachidonic acid is a fatty acid, eicosatetraenoic acid; Greek word ‘eikosa’ means ‘twenty’ because of 20 carbon atom composition of this fatty acid. Arachidonic acid is a constituent of the phospholipid cell membrane, besides its presence in some constituents of diet. Arachidonic acid is released from the cell membrane by phospholipases. It is then activated to form arachidonic acid metabolites or eicosanoids by one of the following 2 pathways: via cyclo-oxygenase pathway and via lipo-oxygenase pathway:
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i) Metabolites via cyclo-oxygenase pathway: Prostaglandins, thromboxane A2, prostacyclin. The name ‘prostaglandin’ was first given to a substance found in human seminal fluid but now the same substance has been isolated from a number of other body cells. Prostaglandins and related compounds are also called autocoids because these substances are mainly auto- and paracrine agents. The terminology used for prostaglandins is abbreviation as PG followed by suffix of an alphabet and a serial number e.g. PGG2, PGE2 etc. Cyclo-oxygenase (COX), a fatty acid enzyme present as COX-1 and COX-2, acts on activated arachidonic acid to form prostaglandin endoperoxide (PGG2). PGG2 is enzymatically transformed into PGH2 with generation of free radical of oxygen. PGH2 is further acted upon by enzymes and results in formation of the following 3 metabolites, a) Prostaglandins (PGD2, PGE2 and PGF2-α). PGD2 and PGE2 act on blood vessels to cause increased venular permeability, vasodilatation and bronchodilatation and inhibit inflammatory cell function. PGF2-α induces vasodilatation and bronchoconstriction. b) Thromboxane A2 (TXA2). Platelets contain the enzyme thromboxane synthetase and hence the metabolite, thromboxane A2, formed is active in platelet aggregation, besides its role as a vasoconstrictor and broncho-constrictor. c) Prostacyclin (PGI2). PGI2 induces vasodilatation, bronchodilatation and inhibits platelet aggregation. d) Resolvins are a newly described derivative of COX pathway. These mediators act by inhibiting production of pro-inflammatory cytokines. Thus, resolvins are actually helpful—drugs such as aspirin act by inhibiting COX activity and stimulating production of resolvins. It may be mentioned here that some of the major antiinflammatory drugs act by inhibiting activity of the enzyme COX; e.g. non-steroidal anti-inflammatory drugs (NSAIDs), COX-2 inhibitors.
ii) Metabolites via lipo-oxygenase pathway: 5-HETE, leukotrienes, lipoxins. The enzyme, lipo-oxygenase, a predominant enzyme in neutrophils, acts on activated arachidonic acid to form hydroperoxy eicosatetraenoic acid (5-HPETE) which on further peroxidation forms following 2 metabolites,
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Review of Literature a) 5-HETE (hydroxy compound), an intermediate product, is a potent chemotactic agent for neutrophils. b) Leukotrienes (LT) are so named as they were first isolated from leucocytes. Firstly, unstable leukotriene A4 (LTA4) is formed which is acted upon by enzymes to form LTB4 (chemotactic for phagocytic cells and stimulates phagocytic cell adherence) while LTC4, LTD4 and LTE4 have common actions by causing smooth muscle contraction and thereby induce vasoconstriction, bronchoconstriction and increased vascular permeability; hence they are also called as slowreacting substances of anaphylaxis (SRS-As). c) Lipoxins (LX) are a recently described product of lipooxygenase pathway. Lipooxygenase-12 present in platelets acts on LTA4 derived from neutrophils and forms LXA4 and LXB4. Lipoxins act to regulate and counterbalance actions of leukotrienes.
3. Lysosomal components The inflammatory cells—neutrophils and monocytes, contain lysosomal granules which on release elaborate a variety of mediators of inflammation. These are as under: i) Granules of neutrophils. Neutrophils have 3 types of granules: primary or azurophil, secondary or specific, and tertiary. a) Primary or azurophil granules are large azurophil granules which contain functionally active enzymes. These are myeloperoxidase, acid hydrolases, acid phosphatase, lysozyme, defensin (cationic protein), phospholipase, cathepsin G, elastase, and protease. b) Secondary or specific granules contain alkaline phosphatase, lactoferrin, gelatinase, collagenase, lysozyme, vitamin-B12 binding proteins, plasminogen activator. c) Tertiary granules or C particles contain gelatinase and acid hydrolases. Myeloperoxidase causes oxidative lysis by generation of oxygen free radicals, acid hydrolases act within the cell to cause destruction of bacteria in phagolysosome while proteases attack on the extracellular constituents such as basement membrane, collagen, elastin, cartilage etc. However, degradation of extracellular components like collagen, basement membrane, fibrin and cartilage by proteases results in harmful tissue destruction
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Review of Literature which is kept in check by presence of antiproteases like α1-antitrypsin and α2- macroglobulin. ii) Granules of monocytes and tissue macrophages. These cells on degranulation also release mediators of inflammation like acid proteases, collagenase, elastase and plasminogen activator. However, they are more active in chronic inflammation than acting as mediators of acute inflammation.
4. Platelet activating factor (PAF) It is released from IgE-sensitised basophils or mast cells, other leucocytes, endothelium and platelets. Apart from its action on platelet aggregation and release reaction, the actions of PAF as mediator of inflammation are: V increased vascular permeability; V vasodilatation in low concentration and vasoconstriction otherwise; V bronchoconstriction; V adhesion of leucocytes to endothelium; and V chemotaxis.
5. Cytokines Cytokines are polypeptide substances produced by activated lymphocytes (lymphokines) and activated monocytes (monokines). These agents may act on ‘self’ cells producing them or on other cells. Although over 200 cytokines have been described, major cytokines acting as mediators of inflammation are: interleukin-1 (IL- 1), tumour necrosis factor (TNF)-α and β, interferon (IFN)-γ, and chemokines (IL-8, PF-4). IL-1 and TNF-α are formed by activated macrophages while TNF-β and IFN-γ are produced by activated T cells. The chemokines include interleukin 8 (released from activated macrophages) and platelet factor-4 from activated platelets, both of which are potent chemoattractant for inflammatory cells and hence their name.
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6. Free radicals: oxygen metabolites and nitric oxide Free radicals act as potent mediator of inflammation: i) Oxygen-derived metabolites are released from activated neutrophils and macrophages. These oxygen-derived free radicals have the following action in inflammation: V Endothelial cell damage and thereby increased vascular permeability. V Activation of protease and inactivation of antiprotease causing tissue matrix damage. V Damage to other cells. ii) Nitric oxide (NO) was originally described as vascular relaxation factor produced by endothelial cells. Now it is known that NO is formed by activated macrophages during the oxidation of arginine by the action of enzyme, NO synthase. NO plays the following role in mediating inflammation: V Vasodilatation V Anti-platelet activating agent V Possibly microbicidal action (Harshmohan, 2010).
Figure 5: Chemical mediators of inflammation (Vinay et al., 2007)
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2.1.5 Regulation of inflammation The onset of inflammatory responses outlined above may have potentially damaging influence on the host tissues as evident in hypersensitivity conditions. Such self- damaging effects are kept in check by the host mechanisms in order to resolve inflammation (Harshmohan, 2010). These mechanisms are as follows:
i) Acute phase reactants A variety of acute phase reactant (APR) proteins are released in plasma in response to tissue trauma and infection. Their major role is to protect the normal cells from harmful effects of toxic molecules generated in inflammation and to clear away the waste material. APRs include the following: i) Certain cellular protection factors (e.g. α1-antitrypsin, α1- chymotrypsin, α2-antiplasmin, plasminogen activator inhibitor): They protect the tissues from cytotoxic and proteolytic damage. ii) Some coagulation proteins (e.g. fibrinogen, plasminogen, von Willebrand factor, factor VIII): They generate factors to replace those consumed in coagulation. iii) Transport proteins (e.g. ceruloplasmin, haptoglobin): They carry generated factors. iv) Immune agents (e.g. serum amyloid A and P component, C-reactive protein): CRP is an opsonising agent for phagocytosis and its levels are a useful indictor of inflammation in the body. v) Stress proteins (e.g. heat shock proteins—HSP, ubiquitin): They are molecular chaperons who carry the toxic waste within the cell to the lysosomes. vi) Antioxidants (e.g. ceruloplasmin are active in elimination of excess of oxygen free radicals. The APR are synthesised mainly in the liver, and to some extent in macrophages. APR along with systemic features of fever and leucocytosis is termed ‘acute phase response’. Deficient synthesis of APR leads to severe form of disease in the form of chronic and repeated inflammatory responses.
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ii) Glucosteroids The endogenous glucocorticoids act as anti-inflammatory agents. Their levels are raised in infection and trauma by self-regulating mechanism. iii) Free cytokine receptors The presence of freely circulating soluble receptors for cytokines in the serum correlates directly with disease activity. iv) Anti-inflammatory chemical mediators PGE2 or prostacyclin has both pro-inflammatory as well as anti-inflammatory actions.
2.1.6 Inflammatory cells (Harshmohan, 2010) 1. Polymorphonuclear Neutrophils (PMNs) Commonly called as neutrophils or polymorphs, these cells along with basophils and eosinophils are known as granulocytes due to the presence of granules in the cytoplasm. These granules contain many substances like proteases, myeloperoxidase, lysozyme, esterase, aryl sulfatase, acid and alkaline phosphatase, and cationic proteins. The diameter of neutrophils ranges from 10 to 15 µm and are actively motile. These cells comprise 40-75% of circulating leucocytes and their number is increased in blood (neutrophilia) and tissues in acute bacterial infections. These cells arise in the bone marrow from stem cells. The functions of neutrophils in inflammation are as follows: i) Initial phagocytosis of microorganisms as they form the first line of body defense in bacterial infection. The steps involved are adhesion of neutrophils to vascular endothelium, emigration through the vessel wall, chemotaxis, engulfment, degranulation, killing and degradation of the foreign material. ii) Engulfment of antigen-antibody complexes and nonmicrobial material. iii) Harmful effect of neutrophils in causing basement membrane destruction of the glomeruli and small blood vessels.
2. Eosinophils These are larger than neutrophils but are fewer in number, comprising 1 to 6% of total blood leucocytes. Eosinophils share many structural and functional similarities with neutrophils like their production in the bone marrow, locomotion, phagocytosis, lobed
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Review of Literature nucleus and presence of granules in the cytoplasm containing a variety of enzymes, of which major basic protein and eosinophil cationic protein are the most important which have bactericidal and toxic action against helminthic parasites. However, granules of eosinophils are richer in myeloperoxidase than neutrophils and lack lysozyme. High level of steroid hormones leads to fall in number of eosinophils and even disappearance from blood. The absolute number of eosinophils is increased in the following conditions and, thus, they partake in inflammatory responses associated with these conditions: i) Allergic conditions; ii) Parasitic infestations; iii) Skin diseases; and iv) Certain malignant lymphomas.
3. Basophils (Mast Cells) The basophils comprise about 1% of circulating leucocytes and are morphologically and pharmacologically similar to mast cells of tissue. These cells contain coarse basophilic granules in the cytoplasm and a polymorphonuclear nucleus. These granules are laden with heparin and histamine. Basophils and mast cells have receptors for IgE and degranulate when cross-linked with antigen. The role of these cells in inflammation is: i) in immediate and delayed type of hypersensitivity reactions; and ii) release of histamine by IgE-sensitised basophils.
4. Lymphocytes Next to neutrophils, these cells are the most numerous of the circulating leucocytes (20-45%). Apart from blood, lymphocytes are present in large numbers in spleen, thymus, lymph nodes and mucosa-associated lymphoid tissue (MALT). They have scanty cytoplasm and consist almost entirely of nucleus.
5. Plasma cells These cells are larger than lymphocytes with more abundant cytoplasm and an eccentric nucleus which has cart-wheel pattern of chromatin. Plasma cells are normally not seen in peripheral blood. They develop from B lymphocytes and are rich
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Review of Literature in RNA and γ-globulin in their cytoplasm. There is an interrelationship between plasmacytosis and hyperglobulinaemia. These cells are most active in antibody synthesis. Their number is increased in the following conditions: i) Prolonged infection with immunological responses e.g. in syphilis, rheumatoid arthritis, tuberculosis; ii) Hypersensitivity states; and iii) Multiple myeloma.
6. Mononuclear-Phagocyte System (Reticuloendothelial System) This cell system includes cells derived from 2 sources with common morphology, function and origin. These are as under: Blood monocytes: These comprise 4-8% of circulating leucocytes. Tissue macrophages: These include the following cells in different tissues: i) Macrophages in inflammation. ii) Histiocytes which are macrophages present in connective tissues. iii) Kupffer cells are macrophages of liver cells. iv) Alveolar macrophages (type II pneumocytes) in lungs. v) Macrophages/histiocytes of the bone marrow. vi) Tingible body cells of germinal centres of lymph nodes. vii) Littoral cells of splenic sinusoids.
7. Giant cells A few examples of multinucleate giant cells exist in normal tissues (e.g. osteoclasts in the bones, trophoblasts in placenta, megakaryocytes in the bone marrow). However, in chronic inflammation when the macrophages fail to deal with particles to be removed, they fuse together and form multinucleated giant cells. Besides, morphologically distinct giant cells appear in some tumours also. A. Giant cells in inflammation: i) Foreign body giant cells. These contain numerous nuclei (up to 100) which are uniform in size and shape and resemble the nuclei of macrophages. These nuclei are scattered throughout the cytoplasm. These are seen in chronic infective granulomas, leprosy and tuberculosis.
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Review of Literature ii) Langhans’ giant cells. These are seen in tuberculosis and sarcoidosis. Their nuclei are like the nuclei of macrophages and epithelioid cells. These nuclei are arranged either around the periphery in the form of horseshoe or ring, or are clustered at the two poles of the giant cell. iii) Touton giant cells. These multinucleated cells have vacuolated cytoplasm due to lipid content e.g. in xanthoma. iv) Aschoff giant cells. These multinucleate giant cells are derived from cardiac histiocytes and are seen in rheumatic nodule. B. Giant cells in tumours: i) Anaplastic cancer giant cells. These are larger, have numerous nuclei which are hyperchromatic and vary in size and shape. These giant cells are not derived from macrophages but are formed from dividing nuclei of the neoplastic cells e.g. carcinoma of the liver, various soft tissue sarcomas etc. ii) Reed-Sternberg cells. These are also malignant tumour giant cells which are generally binucleate and are seen in various histologic types of Hodgkin’s lymphomas. iii) Giant cell tumour of bone. This tumour of the bones has uniform distribution of osteoclastic giant cells spread in the stroma (Harshmohan, 2010).
2.1.7 Pre-clinical screening methods for anti-inflammatory agents (In vivo methods) (Vogel and Vogel, 2002)
Vascular permeability This test is useful to evaluate inhibitory activity of drugs against increased vascular permeability, which is induced by phlogistic substance. After inflammation, histamine, prostaglandins and other mediators of inflammation are released this leads to dilation of arterioles and increased vascular permeability. Histamine liberator compound 48/80 is used to increase vascular permeability and this can be recognized by infiltration of injected sites of the skin with Evans blue. Evaluation is based on measurement of area, which is penetrated by the dye.
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Oxazolone induced ear edema in mice It is a model of delayed contact hypersensitivity that permits the quantitative evaluation of the topical & systemic anti-inflammatory activity following topical administration. Average values of the increase of the weight are calculated for each treated group and compared statistically with the control group. This method is useful to detect anti-inflammatory activity of both steroidal as well as non-steroidal drugs.
Croton-oil ear edema in rats and mice This method is useful for assessment of antiphlogistic and thymolytic activities of topically applied steroids. The composition of irritant is 1 part croton oil, 10 parts ethanol, 20 parts pyridine and 69 parts ethyl ether. Evaluation is based on comparison of weight of treated and control animals. Rabbits are also useful for testing the drugs.
Pleurisy test Pleurisy is well known phenomenon of exudative inflammation in man. Pleurisy can be induced by using histamine, bradykinin, dextran, enzymes, antigen, turpentine, and carrageenan. Carrageenan induced pleurisy is an excellent acute inflammatory model in which fluid extravasation leukocyte migration and various biochemical parameters can be studied which are associated with inflammation. Evaluation is based on following parameter: · Measurement of WBC · Determination of lysosomal enzyme activities · Determination of fibronectin · Determination of PGE2
Granuloma pouch technique In this method carrageenan or croton oil are used to produce aseptic inflammation, which results in large volumes of hemorrhage exudate. Evaluation is based on volume of exudate fluid and total leukocyte count in control and test animals.
Urate crystal induced synovitis Deposition of urate crystal and sodium urate is important in gout and gouty tophi. It has been found that injection of urate crystals in synovial fluid of dog results in severe
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Review of Literature pain resembling gout. This is the excellent method to study anti-inflammatory activity and it closely resembles pathological condition. For evaluation a scoring system is adopted in which inflammatory symptoms ranging from tenderness, limping, occasional legged gait to complete 3-legged gait are scored from 1+ to 4+.
Glass rod granuloma This method is used to develop chronic proliferative inflammation. In contrast to other methods, glass rod granuloma measures the late proliferative phase of inflammation. Since, the newly formed connective tissue is not contaminated with the irritant, biochemical analysis can be performed. The peculiar feature is the possibility of newly formed proliferative connective tissue.
Carrageenan induced rat paw edema (Winter et al., 1962) This method was first proposed by Winter et al (1962). They injected carrageenan (an extract of chondrus) 1%, 0.1ml in 0.9% sterile NaCl. They found that carrageenan probably does not release histamine or serotonin since relatively large doses of strong inhibitors of serotonin or histamine are ineffective in this test. Thus, this model has distinct advantage over other edema models which employ brewer’s yeast, formalin, dextran, egg white as phlogistic material. Vinegar et al (1987) found that development of edema after carrageenan injection is biphasic. The 1st phase begins immediately after injection of irritant and diminishes in an hour. The 2nd phase begins immediately at the end of the 1st phase. All the steroidal and non-steroidal anti-inflammatory drugs inhibit the edema formation in dose dependent manner.
Chronic inflammation (cotton pellet induced granuloma) The cotton pellet induced method was first introduced by (Swingle and Shideman, 1972) to study the effect of local and systemic application of cortisone upon developing granulation tissue. Since then this method has been extensively used for evaluation of anti-inflammatory agents. This method can be divided into three phases namely exudation, granulation, and consolidation. Exudation phase: Three days after implantation, there is marked rise in the wet weight of the normal pellets attributable to accumulation of visible fluid exudate. The fluid is protein rich and contains numerous polymorphnuclear leukocytes largely neutrophils.
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Granulation phase: At seventh day, the granulation tissue is loosely adherent to the normal cotton pellets. The granulation layer thickens during the following week and its capillary net work become more and more complex with noticeable budding of new vessels towards the pellet. Fibroblast and macrophage numbers markedly increase with a thickening of the web of collagen fiber. Consolidation phase: In this phase there is a decrease in dry weight of deposing material. This is accompanied by further decrease in wet weights. Histological changes occurring during this time are further penetration of fibroblast and collagen fiber to the centers of pellets and some giant cell formation around the cotton fibers. There is rapid increase in weight of cotton pellets granulomas between 0-2 days of the implantation.
2.1.8 Plants reported to have anti-inflammatory activity Recent data from literature demonstrate the anti-inflammatory activity of many plant derived compounds. The mechanism of action is attributed to their ability to inhibit cytokine, chemokine or adhesion molecule synthesis (Calixto et al., 2004). The naturally occurring compounds with anti-inflammatory properties are summarized in Table 4. Plant phytochemicals such as polysaccharides, terpenes, alkaloids, etc. have been reported to be useful in alleviating inflammatory diseases including arthritis, rheumatism, acne skin allergy and ulcers. Plants containing polysaccharides are reported to be the most potent in curing inflammatory diseases.
Table 4: Naturally occurring compounds with anti-inflammatory properties Sr. Class of Botanical name Active chemical Mechanism of action No. compound constituent 1 Flavonoids Silybum Silymarin Prevent TNF-α induced NF- marianum κB activation in human (Astaraceae) histocytic lymphoma cells 2 Scutellaria Baicalin, Inhibit LPS induced NO baicalensis Baicalein, production and iNOS gene Georgi Wogonin expression and antioxidant (Lamiaceae) properties
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3 Ginkgo biloba L Quercetin Suppresses the activation of the transcription factor AP-1 4 Hypericum hypericin Inhibited IL-12 production perforatum on LPS activated (hypericaceae) macrophages 5 Curcoma Procurcumenol Inhibited TNF-α secretion in zedoaria L LPS activated macrophages (Zingiberaceae) 6 Polyphenols Curcoma longa L Curcumin Block IL-12 mediated T cell (Zingiberaceae) proliferation. Down regulate the TNF-α induced NF-κB inhibition 7 Caesalpinia Hematein Effectively reduced TNF-α sappan Linn induced VCAM-1 (Leguminosae) expression in HUVECs 8 Lignans Coptis japonica Pinoresinol, Inhibition of TNF-α Mankino Woorenoside V (Ranunculaceae) 9 Morina chinensis Morinols A and B Inhibtion of cytokines (Dipsacaceae) 10 Terpenes Tripterygium Celastrol Inhibited mRNA synthesis wifordii Hook F and protein expression of (Cellastraceae) MMP-3 and MMP-13, indued by the proinflammatory cytokines IL-17, TNF-α 11 Panax ginseng Ginsenosides Rb1 Inhibition of TNF-α (Araliaceae) and Rb2 12 Terpenic Kalopanax pictus Kalopanaxasapon Inhibition of TNF-α saponins Nakai in A (Araliaceae)
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13 Alkaloids Radix stephaniae Tetrandrine Prevented integrin mediated tetrandrae neutrophil adhesion and (Menispermaceae) transmigration induced by leukotriene B4 14 Piper kadsura Piperlactum S Inhibition of TNF-α (piperaceae)
Thus, it is quite clear that many plant derived compounds present significant anti- inflammatory effects. Plants appear to be a major source for potential molecules for the development of new drugs, especially designed for the treatment or control of chronic inflammatory states such as rheumatism. In fact, pharmaceutical industries are currently making tremendous efforts in order to identify new, relevant therapeutic molecules capable of modulating cytokine activated responses. These agents would be useful not only for the treatment of inflammatory disorders, but also for the control of some other diseases which have an inflammatory origin, such as atherosclerosis and alzheimer’s disease. Thus, this potential of vast medicinal flora needs to be explored. In this context, the development of therapeutic agents based on plant derived compounds that present anti-inflammatory activities would have clear benefits. Plant derived agents could be used alone or in association with other available anti- inflammatory drugs, allowing a reduction in costs and side effects and possibly an increase in effectiveness. Therefore, the plant Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze Fam. Asteraceae was selected after detailed review to establish the relevance of folk claims in developing modern drugs.
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2.2 ARTHRITIS: HISTORICAL BACKGROUND & EPIDEMIOLOGY Rheumatic musculoskeletal disorder (RMSD) more popularly known as “Arthritis” or “Rheumatism” is a primitive bone and joint disorder mostly affecting senior citizens around the world. Arthritis, a pathophysiological conditions of joint most common and major socio-economic problem now days. Arthritis is a general term used to describe many connective tissue disorders that affect bone and joints. The name “Arthritis” derived from Greek ‘arthro’ meaning joints and ‘itis’ meaning inflammation; indicates a group of conditions involving damage to the joints of the body. Arthritis is the oldest disease of the universe and has been in this world since the beginning of civilization. The first known traces of human arthritis date back as far as 4500 BC in the fossils of native Americans, found in Tennessee, USA. The first written reference on arthritis was found in Indian holistic medicinal book Charaka Samhita, where it was described as swollen painful joints, initially occurring in hands, feet, causing loss of appetite and occasionally related with fever. The ancient classic Ayurvedic text has described painful deforming polyarthritis called “amavata” and “sandhighatvata” that bears resemblance to rheumatoid arthritis and inflammatory arthritis (Chopra and Doiphode, 2000). It is an estabilished fact that the dinosaurs suffered from arthritis and there is ample proof to show that our earliest ancestors also suffered from chronic aches and pains. Remains of a herd of Iguanadons, small (three- ton) dinosaurs, around 85,000,000 BC were found in Brussels, Belgium. From their remains, we find that they had ankle osteoarthritis (OA). This is a rare phenomenon, since not many dinosaurs show symptoms of primary OA, but many show symptoms of secondry OA from injuries or congenital defects. According to researchers, due to difference in joint structure, dinosaurs that weighed several tons did not suffer from OA. Between 30,000 BC and 28,000 BC, a relative of modern man, Neanderthal man, made his first appearance. According to his remains, individuals of this time developed secondary OA due to injuries and the difficulties of daily life. In 4500 BC, arthritis was first discovered in human beings. It was seen in the skeletal remains of native Americans of Tennessee and parts of modern day Olathe, Kansas, America. In fact, arthritis was evidenced in ancient, a mummy in 3000 BC. Ã-tzi was the name given to a mummy, popularly known as the Iceman, who crossed the Alps near the border of Italy and Austria. Though he was not successful in his venture, the
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2.2.1 Rheumatoid arthritis Rheumatoid arthritis (RA) is one of the most common inflammatory disorders affecting the population worldwide. It is a systemic inflammatory disease which affects not only the joints but a wide range of extra-articular organs. The disease, if not treated early, will lead to progressive joint deformity and increased morbidity and mortality. RA is a potentially fatal illness, with mortality increased twofold and an average decrease in life expectancy of 7-10 years. Patients with RA have an increased prevalence of other serious illnesses. The predominant conditions leading to this increased co-morbidity and mortality include infections, renal impairment, cardiovascular disease and lymphomas. The incidence of lymphoma is twofold higher than expected before taking into account the disease-modifying immunosuppressant drugs used in treating RA (Roger and Cate, 2012).
2.2.2 Epidemiology and Etiology RA affects approximately 1% of the population worldwide. RA arises from an immunologic reaction, and there is speculation that it is in response to a genetic or infections antigen. Risk factors associated with the development of RA include: V Female gender (3:1 females to males) V The prevalence of RA increases with age in both sexes; nearly 5% of women and 3% of men over the age of 65 years are affected by the disease. V The peak age of incidence is about 30-50 years in women and slightly older in men. V RA also affects young children and its classification and treatment differs slightly from adults. V Current tobacco smoking. Studies have identified a direct relationship between tobacco use and RA disease severity.
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V Family history of RA. Genetic studies demonstrate a strong correlation between RA and the presence of major histo-compatibility complex class II human leukocyte antigens (HLA), especially HLA-DR1 and HLA-DR4. HLA is a molecule associated with the presentation of antigens to T lymphocytes. V Potential environmental exposures. The number of RA cases has increased during industrialization, although a specific link to environmental factors has not been determined. V Oral contraceptive use and high ingestion of vitamin D and tea are associated with a decreased risk of RA (Marie et al., 2006).
2.2.3 Pathophysiology The characteristics of a synovium affected by RA are, V The presence of a thickened, inflamed membrane lining called pannus. V The development of new blood vessels. V An influx of inflammatory cells in the synovial fluid, predominantly T lymphocytes. The pathogenesis of RA is driven by T lymphocytes, but the initial catalyst causing this response is unknown. Understanding specific components of the immune system and their involvement in the pathogenesis of RA will facilitate understanding of current and emerging treatment options for RA. The components of most significance are T lymphocytes, cytokines, and B lymphocytes (Marie et al., 2006).
T lymphocytes The development and activation of T lymphocytes are important to maintain protection from infection without causing harm to the host. Activation of mature T lymphocytes requires two signals. The first is the presentation of antigen by antigen- presenting cells to the T lymphocyte receptor. Second, a ligand receptor complex on antigen presenting cells binds to CD28 receptors on T lymphocytes. Once a cell successfully passes through all the stages, the inflammatory cascade is activated. Activation of T lymphocytes (1) stimulates the release of macrophages or monocytes, which subsequently causes the release of inflammatory cytokines, (2) activates osteoclasts, (3) activates the release of matrix metalloproteinases or enzymes
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Cytokines An imbalance of proinflammatory and inflammatory cytokines in the synovium leads to inflammation and joint destruction. Proinflammatory cytokines including IL-1, TNF-α, IL-6 and IL-12 are found in high concentrations in synovial fluid. Cytokines are low-molecular weight regulatory proteins or glycoprotein’s secreted by white blood cells and various other cells in the body in response to a number of stimuli. These proteins assist in regulating the development of immune effector cells, and some cytokines possess direct effector functions of their own. Cytokines are involved in a staggeringly broad array of biological activities including innate immunity, adaptive immunity, inflammation, and hematopoiesis. Table 5: Cytokines involved in the pathogenesis of RA (Marie et al., 2006) Cytokines Secreted by Targets and effects Interleukin 1 Monocytes, Vasculature (inflammation); hypothalamus (IL-1) macrophages, (fever); liver (induction of acute phase endothelial cells, proteins) epithelial cells Tumor Macrophages Vasculature (inflammation); liver Necrosis (induction of acute phase proteins); loss of Factor-α (TNF- muscle, body fat (cachexia); induction of α) death in many cell types; neutrophil activation Interleukin-12 Macrophages, Natural killer (NK) cells; influences (IL-12) dendritic cells adaptive immunity Interleukin- 6 Macrophages, Liver (induces acute phase proteins); (IL-6) endothelial cells influences adaptive immunity (proliferation and antibody secretion of B cell lineage) Interferon- α Macrophages Induces an antiviral state in most nucleated (IFN-α) cells; increases MHC class I expression; activates NK cells
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Interferon- β Fibroblasts Induces an antiviral state in most nucleated (IFN-β) cells; increases MHC class I expression; activates NK cells
B Lymphocytes In addition to serving as antigen-presenting cells to T lymphocytes, B lymphocytes may produce proinflammatory cytokines and antibodies. Antibodies of significance in RA are rheumatoid factors and antibodies against cyclic citrullinated peptide (CCP). Rheumatoid factors are not present in all patients with RA, but their presence is indicative of disease severity and likelihood of extraarticular manifestations. CCPs are produced early in the course of disease. High levels of anti-CCP antibodies are indicative of aggressive disease and a greater likelihood of poor outcomes. Monitoring anti-CCP antibodies may be useful to predict the severity of disease and match aggressive treatment appropriately.
2.2.4 Comorbidities associated with RA (Marie et al., 2006) RA reduces a patient’s average life expectancy by 3 to 10 years, but RA alone rarely causes death. Instead, specific comorbidities contribute to premature death independent of safety issues surrounding the use of immunomodulating medications. The comorbidities with the greatest impact on morbidity and mortality associated with RA are (1) cardiovascular disease, (2) infections, (3) malignancy, and (4) osteoporosis.
Cardiovascular Half of all deaths in RA patients are cardiovascular related. Because a patient with RA experiences inflammation and swelling in his or her joints, it is likely that there is inflammation elsewhere, such as in blood vessels, termed vasculitis. C-reactive protein (CRP), a nonspecific marker of inflammation, is associated with an increased risk of cardiovascular disease; CRP is elevated in patients with RA. Chronic systemic inflammation may contribute to the relationship between RA and cardiovascular disease, but the exact mechanism is still under investigation.
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Infection RA alone leads to changes in cellular immunity and causes a disproportionate increase in pulmonary infections and sepsis. Because medications that alter the immune system are linked to an increased risk of infections, it is difficult to distinguish between an increased risk of infection secondary to RA and the medications used to treat RA. Patients and clinicians must pay close attention to signs and symptoms of infection because of this increased risk.
Malignancy Patients with RA have an increased risk of developing lymphoproliferative malignancy (e.g., lymphoma, leukemia, and multiple myeloma) and a decreased risk of developing cancer of the digestive tract. The relationship between RA and cancer is not clear. To confound the issue, medications for the treatment of RA may contribute to cancer risk. Patients presenting with new onset of symptoms (e.g., fevers, night sweats, chills, or anorexia) out of proportion with disease activity and patients not responding to conventional RA treatment should be evaluated further for lymphoproliferative malignancy.
Osteoporosis Osteoporosis associated with RA follows a multifaceted pathogenesis, but the primary mechanism likely is mediated by osteoclast activity. The cytokines involved in the inflammatory process directly stimulate osteoclast and inhibit osteoblast activity. Additionally, arthritis medications can lead to increased bone loss. Bone mineral density should be evaluated at baseline and routinely using dual-energy x-ray absorptiometry.
2.2.5 Clinical manifestations (Roger and Cate, 2012) There are different patterns of clinical presentation of rheumatoid arthritis. The disease may start as polyarticular arthritis with a gradual onset, intermittent or migratory joint involvement, or monoarticular manifestations may be present. Extraarticular features occur in approximately 75% of seropositive patients and are often associated with a poor prognosis.
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Disease onset is usually insidious with the predominant symptoms being pain, stiffness and swelling. Typically, the metacarpophalangeal and proximal interphalangeal joints of the fingers, interphalangeal joints of the thumbs, the wrists, and metatarsophalangeal joints of the toes are affected during the early stages of the disease. Other joints of the upper and lower limbs, such as the elbows, shoulders and knees, are also affected. Morning stiffness may last for 30 min to several hours, and usually reflects the severity of joint inflammation. Up to one-third of patients also suffer from prominent myalgia, fatigue, low-grade fever, weight loss and depression at disease onset. Rheumatoid arthritis shows a marked variation of clinical expression in individual patients both in number and pattern of joints affected, disease progression and the rapidity of joint damage. Disease activity may not abate in 10-20% of cases. Remission has been reported in a small proportion of patients but usually is very rare without DMARDs.
Extra-articular features of RA V Amyloidosis V Carpal tunnel syndrome V Episcleritis V Felty’s Syndrome V Fever V Lymphadenopathy V Nodules; may be subcutaneous or within the lungs, eyes or heart V Osteoporosis V Pericarditis V Pleural and pericardial effusions V Vasculitis
2.2.6 Diagnosis (Roger and Cate, 2012) A clinical diagnosis of RA is made based on the patient’s history, presenting symptoms and clinical findings. Family history is useful, as well as investigations including blood tests, ultrasound for the presence of synovitis and X-rays. The latter is
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Table 6: Summary for diagnosis of RA Arthritis Patients History Physical Exam Tests Rheumatoid -Pain duration > 6 -Synovitis Radiologic arthritis weeks -Joint involvement, -Erosions on X- -Morning stiffness symmetrical Ray or MRI (lasting > 30 minutes) -Joint destruction -Synovitis noted -Systemic symptoms -Extra-articular by ultrasound (e.g. anorexia, fatigue) manifestations -ESR or CRP -Anti-CCP -Rheumatoid factor
2.2.7 Investigations Inflammatory markers, including C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR), are usually but not always elevated in RA and are useful for monitoring response to treatment. Rheumatoid factor (RF) is an autoantibody directed against the host immunoglobulin and is most commonly found in RA. Routinely performed tests only detect immunoglobulin M rheumatoid factor (IgM RF) which is present in 75-80% of patients with RA (termed seropositive disease) and 5% of normal subjects. Those patients who do not have a detectable RF are said to be ‘seronegative’. RF is not specific to RA and is also present in patients with chronic lung and liver disease, other connective tissue diseases, neoplasia, infections (particularly bacterial endocarditis) and cryoglobulinaemia. Anti-cyclic citrullinated peptide antibodies (anti-CCP antibody) are a more specific of 90-96% compared with the specificity of IgM RF of 85%. They are more useful for the early detection of RA in a patient with inflammatory arthritis. The sensitivity of both anti-CCP antibody and IgM RF is approximately 70%. Antinuclear antibodies (ANA) and extractable nuclear antigens (ENA) are useful for establishing the differential diagnosis, such as other connective tissue diseases
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Review of Literature presenting or associated with arthritis. ANA is almost universally positive in systemic lupus erythematosus and only positive in 20% of patients with RA. Other abnormal laboratory tests include an elevated alkaline phosphatase, an elevated platelet count, a decreased serum albumin and a normochromic, normocytic anaemia. White cell count, particularly neutrophils, is elevated in patients with infected joints and is also elevated whilst the patient is on steroids (Roger and Cate, 2012).
2.2.8 Pre-clinical screening methods for antiarthritic agents (In vivo methods) Rat and mouse models of experimental autoimmune arthritis provide powerful alternative approaches to evaluate potential etiopathogenetic mechanisms in human RA. The major rat models of experimental erosive arthritis can be classified into three general groups (Wilder et al., 1999; Crofford and Wilder, 1997). V The first group is induced by hyperimmunization of genetically susceptible rat strains with antigens such as native type II collagen (collagen-induced arthritis, CIA) or cartilage oligomeric matrix protein (COMP-induced arthritis) in incomplete Freund’s adjuvant (IFA). V The second group is induced by intradermal administration of various oil- based adjuvants, of which heat-killed Mycobacterium tuberculosis emulsified in IFA is the most widely studied (Mtb-adjuvant-induced arthritis, AIA). Chronic erosive arthritis has also been induced with other oil-based adjuvants including avridine in IFA (avridine-induced arthritis), pristane (pristane- induced arthritis, PIA), and IFA alone (oil-induced arthritis [OIA]). Additionally, newer models of adjuvant arthritis have recently been described that are induced by exogenous chemicals such as dioctadecyldiammonium
bromide (DDA=C38H80NBr), heptadecane (C17H36) and even endogenous
lipids such as the cholesterol precursor squalene (C30H50) (Lorentzen, 1999). V The third group of rat models includes various forms of bacterial cell wall peptidoglycanpolysaccharide-induced arthritis (Wilder et al., 1999; Simelyte et al., 1999). The streptococcal cell wall (SCW) arthritis model is the best characterized of the third group. Additional arthritogenic cell-wall structures from bacteria and yeast such as beta-glucan, lipopolysaccharide, and
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trehalosedimycolate, have been identified that induce arthritis in susceptible strains of rats (Lorentzen, 1999). In mice, CIA, PIA and proteoglycan-induced arthritis are the major experimentally induced models. Mice are relatively resistant to classic AIA (Yoshino et al., 1998) and SCW arthritis (Wilder et al., 1999).
Rat models of erosive arthritis Collagen-induced arthritis The collagen-induced arthritis (CIA) model in rats is typically induced by intradermal injections of native, heterologous (non-rat) type II collagen (CII) in IFA, followed by a booster injection on day 7. CII is highly arthritogenic in DA and LEW rats, but not in F344 inbred rats. Homologous (rat) CII is also arthritogenic in DA, but not other strains of rats. Autoimmunity to type IX collagen (CIX), in contrast to CII and type XI collagen (CXI), is not directly pathogenic but may contribute to joint injury provided arthritis is initiated by an independent disease process (Cremer et al., 1998). Erosive polyarthritis typically develops 10 to 14 days after the primary immunization. Similar to RA, female DA rats tend to be more susceptible than males (Wilder et al., 1999). Pathogenesis of CIA depends on autoreactive T cells as well as B cells that produce antibodies to type II collagen. TNF-α is also important for disease progression. A multivalent guanylhydrazone (CNI-1493) that inhibits TNF-α production by suppressing its translational efficiency, when injected intraperitoneally either before the onset of arthritis or after the establishment of clinical disease suppresses the severity of arthritis in DA rats (Kerlund et al., 1999). IL-1 is also of major importance in mediating disease progression in rat CIA. Bendele and coworkers have demonstrated that sustained blood levels of IL-1 receptor antagonist (IL-1Ra) result in dose dependent suppression of joint inflammation, pannus for mation, cartilage damage, and bone lesions (Bendele et al., 1999). Neutrophil elastase, among other hydrolytic enzymes induced in CIA, contributes to cartilage degradation in CIA, and elastase inhibitors reduce the clinical scores and joint swelling (Janusz and Durham, 1997).
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Cartilage oligomeric matrix protein–induced arthritis Immunization with both native and denatured rat COMP in IFA induces severe arthritis in susceptible rat strains. Although the peripheral joint arthritis in this newly described model clinically resembles RA, COMP induced arthritis does not result in permanent destruction of the joints. Disease development appears to be dependent on an immune response to autologous COMP and not on cross-reactivity to rat cartilage collagens (Carlson et al., 1998).
Mycobacteria-adjuvant induced arthritis in rats An intradermal injection at the base of the tail with heat killed mycobacteria (Mtb) in IFA results in destructive arthritis within 14 days in susceptible DA or LEW inbred rats. AIA can also be induced with cell walls from other bacterial types in IFA, although the arthritogenicity varies. The disease progresses rapidly over several weeks in what appears clinically as a monophasic process. Increased synthesis of TNF-α, IL-1 and IL-6 is detected as early as day 4 after adjuvant injection. As in CIA, IL appears to be very important in mediating the bone resorption that occurs in rat AIA (Wilder et al., 1999). Granulocytes and autoreactive CD4+ cells play major roles in the disease.
Avridine-induced arthritis Injection of avridine (N, N-dioctadecyl-N, N'-bis (2-hydroxyethyl) propanediamine/ CP-20961), emulsified in IFA, at the base of the tail is potentially arthritogenic in susceptible rat strains (DA and LEW) (Wilder et al., 1999). As in RA, females develop more severe disease than males. Experimental data also suggest that sex chromosomes regulate the gender differences in avridine-induced arthritis severity, but gonadal hormones also exert regulatory effects. Since avridine is a synthetic adjuvant devoid of properties that elicit classical T cell or B cell immunological responses, this model has focused investigations on the role of adjuvants and the innate immune system in the development of erosive arthritis. T cells, however, are critical to the development of the disease because avridine-induced arthritis does not develop in athymic nude rats.
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Pristane-induced arthritis Intradermal injection of 2, 6, 10, 14-tetramethylpentadecane (pristane), another synthetic immunological adjuvant, at the base of the tail, induces arthritis in susceptible rats (DA and LEW) that resembles RA (Wilder et al., 1999). PIA develops about 2 to 3 weeks after injection and progresses with a relapsing course that persists for months. T cells are required for disease development. Bone erosions, beginning subchondrally, are evident about two days after the onset of clinical arthritis. Arthritic (E3 x DA) F2 rats have increased serum concentrations of COMP on days 35 and 49 after pristane injection (Vingsbo-Lundberg et al., 1998). COMP levels correlate with the severity of macroscopically detectable arthritis at both time points r > 0.9). Rats with a chronic disease course are distinguished by higher serum concentrations of COMP during the acute stage than animals with similar early clinical scores but with resolving arthritis. Serum analysis of COMP offers promise for monitoring tissue involvement in experimental arthritis.
Oil-induced arthritis This unique rat arthritis model develops in DA but not other inbred rat strains, after a single subcutaneous injection of IFA, which is a very weak adjuvant. The onset of OIA is around day 14. Joint inflammation is milder than in other rat arthritis models. Interestingly, autoreactive T cells, expressing high levels of IL-2, interferon-g and TNF-α, mediate OIA. In contrast to CIA, antibodies to type II collagen are not produced in OIA (Wilder et al., 1999).
Streptococcal cell wall-induced arthritis A single intraperitoneal injection of an aqueous suspension of cell wall peptidoglycan-polysaccharide fragments from group A streptococci and several other types of Eubacteria including E. aerofaciens, E. contortum, and E. lentum, into susceptible rat strains, such as LEW, induces severe erosive arthritis (Wilder et al., 1999; Simelyte et al., 1999). An acute, thymic-independent, complement-dependent phase develops within 24 hours. This primary acute arthritic phase is followed by a chronic, secondary, thymic-dependent phase, which begins about 14 days after injection and is characterized by a fluctuating course similar to that observed in
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Review of Literature patients with RA. In affected joints, this chronic phase is associated with the production of high levels of proinflammatory cytokines, growth factors, metalloproteinases, cyclooxygenase-2 and nitric oxide. Administration of plasmid DNA encoding human TGF-b1 intramuscularly to rats with SCW induced arthritis suppressed progression of inflammation and joint destruction (Song et al., 1998). Intra-articular injection of streptococcal cell wall antigen followed by intravenous challenge (reactivation) results in destructive, lymphocyte-dependent monoarticular arthritis. To define further the role of immune mechanisms in this model, antibodies to Th1- and Th2-related cytokines were evaluated (Schimmer et al., 1998). Anti–IL-4, but not anti-IL-10 or anti-IFN-g, is effective in lowering joint swelling in the reactivation model of streptococcal cell wall-induced arthritic rats, suggesting that Th2 mechanisms, to some extent, may be operative in this model of arthritis (Schimmer et al., 1998). Alternatively, IL-4 may play a role in the development of Th-1 related inflammation. TNF-α plays a major role in the development of joint swelling, whereas IL-1 is dominant in mediating cartilage destruction and inflammatory cell influx (Kuiper et al., 1998). As in RA patients, the transcription factor NF-κB is activated in the synovium of rats with “reactivation” SCW-induced arthritis (Miagkov et al., 1998). In vivo suppression of NF-κB in the synovium of rats with SCW- and pristane-induced arthritis by either proteasomal inhibitors or intra- articular adenoviral gene transfer of super-repressor kBa profoundly enhances apoptosis. Activation of NF-κB protects synovial cells against apoptosis and thus provides a potential link between inflammation and synovial hyperplasia. Intra- articular administration NF-κB decoys not only prevents the recurrence of SCW arthritis in treated joints but in addition reduces the severity of arthritis in the untreated joints (Miagkov et al., 1998).
2.2.9 Treatment The goals of the treatment in RA are V To reduce or eliminate pain V To protect articular structures V To control systemic complications V To prevent loss of joint functions V To improve or maintain quality of life.
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Non-pharmacologic therapy All patients should receive education about the non-pharmacologic and pharmacologic measures to help manage RA. Empowered patients take an active role in care by participating in therapy-related decisions. Certain forms of non- pharmacologic therapy benefit all levels of severity, whereas others (i.e. surgery) are reserved for severe cases only. Occupational and physical therapy may help patients to preserve joint function, extend joint range of motion and strengthen joints and muscles through strengthening exercises. Patients with joint deformities may benefit from the use of mobility or assistive devices that help to minimize disability and allow continued activities of daily living. In situations where the disease has progressed to a severe from with extensive joint erosions, surgery to replace or reconstruct the joint may be necessary (Marie et al., 2006).
Pharmacologic therapy The pharmacological management of RA is evolving rapidly as more advanced therapies become available. The advent of biological therapies has brought new technologies which target different cytokine pathways involved in the pathogenesis of RA and have revolutionized disease management. There are four main categories of drugs employed in RA: non-steroidal anti- inflammatory drugs (NSAIDs) including cyclooxygenase (COX)-II inhibitors, glucocorticoids, DMARDs and biological therapies. Simple analgesia also has a small role to play in basic symptom relief and includes paracetamol, codeine and opiate combination products. These analgesics do not have any anti-inflammatory effect and will not aid disease modification. The aim of analgesia is to achieve symptom relief and reduce the need for long term use of NSAIDs, COX-II inhibitors and glucocorticoids (Marie et al., 2006).
Non-steroidal anti-inflammatory drugs (Roger and Cate, 2012) The analgesic and anti-inflammatory properties of NSAIDs are used to reduce joint pain and swelling. However, as with simple analgesics, these drugs provide only symptomatic relief to improve joint functions, and should always be used in combination with other agents which modify the disease process.
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The COX enzyme coverts arachidonic acid into prostaglandins and thromboxanes. These prostanoids have a variety of physiological functions, and are also believed to be responsible for causing pain and swelling in inflammatory conditions. There are two main isoforms of the COX enzyme: COX-I produces prostaglandins required for homeostatic functions, such as maintaining the gastric mucosa, support of renal function and platelet function. COX-II is responsible for the production of inflammatory prostanoids. NSAIDs vary in their selectivity for the COX-I and COX-II isoforms, and are categorized as either non-selective NSAIDs or selective COX-II inhibitors, otherwise known as the coxibs. Non-selective NSAIDs generally block COX-I and COX-II, whereas the coxibs have higher selectivity for the COX-II isoforms. However, COX- II selectivity in NSAIDs varies according to the dose of drug given, which is demonstrated by the dose-related toxicity exhibited by some agents such as ibuprofen. The three most commonly used non-selective NSAIDs have differing levels of COX-I or COX-II selectivity: diclofenac is COX-II ‘preferential’, whereas ibuprofen and particularly naproxen preferentially inhibit COX-I. Originally, inhibition of COX-II was thought to be involved solely with the anti-inflammatory, anti-pyretic and analgesic properties of NSAIDs. However, it is possible that COX-II inhibition may also impair endothelial health, cause a prothrombotic state and promote cardiovascular disease.
Table 7: Common and shared side effects of NSAIDs (Laurence et al., 2011) System Manifestations GI (side effects decreased with COX-2– Abdominal pain selective drugs) Nausea Anorexia Gastric erosions/ulcers Anemia GI hemorrhage Perforation Diarrhea
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Renal Salt and water retention Edema, worsening of renal function in renal/cardiac and cirrhotic patients Decreased effectiveness of antihypertensive medications Decreased effectiveness of diuretic medications Decreased urate excretion (especially with aspirin) CNS Headache Vertigo Dizziness Confusion Depression Lowering of seizure threshold Hyperventilation (salicylates) Platelets (side effects absent with COX- Inhibited platelet activation 2–selective drugs) Propensity for bruising Increased risk of hemorrhage Uterus Prolongation of gestation Inhibit labor
Cyclooxygenase enzyme Since the discovery of the mechanism of the non-steroidal anti-inflammatory drugs by Vane (1971), it has been widely accepted that the inhibition of prostaglandin (PG) synthesis through cyclooxygenase (COX) blockade is responsible for both their therapeutic and side effects. More recently, a second inducible COX has been characterized as a distinct isoform, named COX-2, and is encoded by a gene different form that producing the constitutive isoform, COX-1 (Trummlitz and Van Ryn, 2002). COX-1, a constitutive enzyme located in most tissues, for example, the platelets, endothelium, stomach, kidney, smooth muscles and lumen of endoplasmic reticulum and performs a housekeeping function to synthesize PGs with normal cell regulatory activity. It is a membrane bound haem and glycoprotein with a molecular Study of Analgesic, Anti-inflammatory and Antiarthritic activity of Indian medicinal plant in laboratory animals 51
Review of Literature weight of 71 KDa with 599 amino acid residues. The protein contained both the cyclooxygenase and endoperoxidase activities required to form PGG2 and PGH2 respectively. COX-2 is an immediate early gene product, with a molecular weight of 70 KDa with 604 amino acid residues. It is expressed, as inducible form, considerably after exposure to inflammatory mediators like fibroblasts, cytokines etc. Levels of COX-2 protein increase in parallel with overproduction of prostaglandins in many cells and tissue in chronic inflammation. COX-1 is the only isoform in the normal gastric mucosa and platelets and is responsible primarily for the biosynthesis of eicosanoids involved in gastrointestinal mucosal cytoprotection and the maintenance of platelet function. COX-2, on the other hand, is involved in many physiologic responses, but mainly in the amplification of inflammation and pain.
Structure and Functions of COX-1 and COX-2 (Willoughby et al., 2000) The structure of COX protein consists of three different domains the N terminal epidermal growth factor domain, a membrane binding motif, and a C-terminal catalytic domain that contains the COX and peroxidase active sites. Both isoforms consists of cassette of 17 amino acids and 18 amino acids sequence near the N- terminal of COX-1 and COX-2 respectively. The catalytic domain is a globular structure containing the cyclooxygenase and peroxidase active sites.
The peroxidase active site The peroxidase activity has two functions: it reduces the PGG2 produced by the cyclooxygenase step and activates the cyclooxygenase reaction. This enzyme collectively termed prostaglandin synthase actually has two different active sites. On one side, it has the cyclooxygenase active site and on the opposite side, is has an entirely separate peroxidase site, which is needed to activate the haem group that participate in the cyclooxygenase reaction. The enzyme complex is a dimmer of identical subunits; so altogether, there are two cyclooxygenase active sites and two peroxidase active sites in close proximity. Each subunit has a small carbon-rich knob, pointing downward in this illustration. These knobs anchor the complex to the membrane of the endoplasmic reticulum, shown in light blue at the bottom of the picture. The cyclooxygenase active site is buried deep within the protein, and is reachable by a tunnel that opens out in the middle of the knob. This acts like a funnel,
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Review of Literature guiding arachidonic acid out of the membrane and into the enzyme for processing. In the structure shown here, cyclooxygenase enzyme, a drug (yellow and green) is blocking the active site in both subunits. The haem groups are also shown above the drug in each subunit.
The cyclooxygenase active site The cyclooxygenase active site consists of a long, narrow channels extending from the outer surface of the membrane-binding motif. Tyr-385 is found at the apex of the COX-1 enzymes and Tyr-371 in case of COX-2 represent together with the haem group at the catalytic center. X-ray crystallography of 3D structures of COX-1 and COX-2 enzymes as well as complexes with NSAIDs has provided insight onto the mechanism of action. COX-1 and COX-2 are very similar enzymes consisting of a long narrow channel with a hairpin bend at their end and both are membrane associated. Arachidonic acid released from damaged membranes adjacent to the opening of the enzymes channel, mostly hydrophobic is sucked in twisted around the hairpin bend and subjected to chemical reactions, resulting in the formation of the cyclopenta ring of PGs. Epidermal Growth factor like domain (blue), amphopathic membrane binding motif (red), catalytic globular domain (gray), haem groups (brown) and arachidonic acid in its binding site (yellow).
Figure 6: Comparison of NSAIDs binding sites of COX-1 and COX-2 (Rang et al., 2007)
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Difference between binding sites of COX-1 and COX-2 (Flower, 2003) After isolation in the early 1990’s of the COX-2 isozyme its genetic structure and regulation of expression were characterized and compared with COX-1. Both enzymes are encoded by separate genes on different chromosomes: COX-1 is on chromosome 9; COX-2 is on chromosome 1. The COX-2 gene contains regions characteristic of early response genes, allowing a rapid up regulation in response to inflammatory stimuli as well as rapid turnover and diminished expression in the absence of continued stimulation. Meanwhile, the COX-1 gene is expressed in almost all normal tissues and regulated by inflammatory stimuli (constitutive expression). Although both isozymes are 60% homologous there are small differences in the amino acid sequence lining the COX active sites. The active site is preponderantly hydrophobic in nature with two internal hydrophilic pockets I and II, both of which have a valine (val) in COX-2 and an isoleucine in COX-1 (positions 523 and 89) at the opening of the pocket, leading to the constriction of this pocket in COX-1. The accessibility of these pockets is controlled by a valine in COX-2 as against isoleucine in COX-1, at position 523. The side chain of residue at 523 packs against phenylalanine (Phe) 518, which forms a molecular gate that extends to the hydrophilic pocket l. In COX-1, this gate is closed because of the bulkier side chain whereas in COX-2, with the less voluminous Val at 523, the gate has room to swing open, allowing the entry of inhibitor. Figure 7: Structural differences in active sites of cyclooxygenase (COX)-1 and COX-2 (Harvey et al, 2009)
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Physiological function of COX-1 and COX-2 (Flower, 2003) Gastrointestinal Tract: - The clearest function is in the gastrointestinal (GI) tract, where prostaglandins clearly play a protective role, as evidenced by the superior safety of COX-2 selective agents and the protective effects of misoprostal on the GI lining. The primary drawback of traditional nonselective agents is in the gastric system where reduction of protective prostaglandins causes ulceration, bleeding and occasionally death. Cardiovascular systems: - Cardiovascular effects of prostaglandins are more complex. The coagulation system is clearly modulated by platelet derived thromboxanes, which have pro- coagulation effects and the anticoagulative effects of endothelial cell- derived prostacyclin. Thromboxanes are clearly COX-1 derived because platelets do not express COX-2. The source of endothelial cell prostacyclin production is less clear with both enzymes expressed and mixed opinions on the relative contribution of the two enzymes. Kidney: - Prostaglandins regulate renin-angiotensin secretion and thus glomerular filtration rate and sodium homeostasis. These effects appear to be COX-2 driven. The kidney is a rare organ, one that expresses COX-2 under nonpathological situations. Expression in the loop of Henle apparently drives prostaglandin formation in the kidney and the subsequent physiological responses. Thus a selective agent would likely have similar negative effects on kidney function as those of the nonselective NSAIDs. Cancer: - COX-2 is over expressed in cancerous lesions in the colon and the degree of its expression has been related to survival. COX-2 is also over expressed in the precancerous lesions of patients with familial adenomatious polyposis (FAP). The precise role of COX-2, and specifically its interplay with COX-1 in carcinogenesis, not clears. Atherosclerosis: - Expression of both COX isoforms is detectable in atherosclerotic lesions in humans. In summary, COX-2 expressed, along with COX-1 in atheroscletrotic plaques. However, the predominant prostaglandins formed vary with cell type and have divergent effects on disease progression and perhaps on plaque stability. Neurological disease: - The place of COX-2 inhibitors in neurological diseases, such as Alzheimer’s disease, Parkinson’s disease and seizure disorders continues to attract
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Review of Literature basic and clinical investigation. Initial studies in Alzheimer’s disease failed to support the hypothesis that COX-2 inhibition would slow the development of the disease. This hypothesis had been configured on the presence of increased expression of COX-2 in Alzheimer’s disease lesions (along with COX-1 in some cases); exacerbation of amyloid deposition following over expression of neuronal COX-2. Rheumatoid arthritis: - Rheumatoid arthritis is an autoimmune disease in which there is joint inflammation, synovial proliferation and destruction of articular cartilage. Immune complexes composed of IgM activate complement and release factors, which are chemotactic for neutrophils. These inflammatory cells release lysosomal enzymes, which damage cartilage and erode bone, while PGs produced in the process cause vasodilatation and pain.
Safety In 2004, rofecoxib, selective COX-II inhibitor, was withdrawn from the worldwide market due to evidence of an increased risk of confirmed serious thrombotic events that included myocardial infarction and stroke, following long term use. In the following years, similar evidence against the other COX-II inhibitors and also against some of the non-selective NSAIDs accumulated. At present, the exact cardiovascular risk for individual selective COX-II inhibitors and NSAIDs is not known. Evidence from clinical trials of COX-II inhibitors suggests that about 3 additional thrombotic events per 1000 patients/year may occur in the general population. A dose-dependent increase in cardiovascular risk is associated with use of celecoxib, high-dose diclofenac (150 mg/day) and high-dose ibuprofen (2400 mg/day). There does not appear to be an increased risk of myocardial infarction in association with low-dose ibuprofen (<1200 mg/day). Naproxen is associated with a lower risk of arterial thrombotic events than COX-II inhibitors. There may be some increased cardiovascular risk in all patients receiving any NSAID, irrespective of their baseline risk or duration of therapy. The key message is that patients should use the lowest effective dose and the shortest duration of treatment necessary to control symptoms to minimize the risk of adverse events. The most common adverse events of NSAIDs are those that predominantly inhibit COX-I and cause adverse gastro-intestinal effects. These range from minor
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Review of Literature symptoms, including dyspepsia, nausea and diarrhoea, to more serious events, such as gastric erosion, bleeding and duodenal and gastric ulceration. Patients are at higher risk of serious gastro-intestinal complications if they are over 65 years of age, have a previous history of gastro-intestinal ulceration/bleeding or peptic ulcer disease, or are taking concomitant anti-platelet, anti-coagulation or steroid therapy. There are several gastro-protective agents available who may be used to reduce adverse events, including H2 antagonists; misoprostol and proton pump inhibitors (PPIs). PPIs, such as omeprazole and lansoprazole, have shown to be particularly effective at preventing gastric and duodenal ulcers with NSAIDs. All patients taking a non-selective NSAID or COX-II inhibitor should receive concomitant treatment with a PPI to minimize gastro-intestinal adverse effects. Aspirin inhibits the COX enzyme irreversibly through a different mechanism of action to the NSAIDs. Therefore, there is an increased risk of gastro-intestinal toxicity if aspirin and non-selective NSAIDs are used concomitantly, and the gastro-intestinal advantage of using selective COX-II inhibitor is severely reduced. Low-dose aspirin should only be co-prescribed with NSAIDs where absolutely necessary. All NSAIDs may potentially cause adverse cardio-renal effects such as edema, hypertension and heart failure. The distribution of COX-I and COX-II differs in the kidney, but there is no evidence to suggest differing degrees of isoforms inhibition have an impact on the severity of cardio-renal adverse effects. Pharmacokinetic parameters, such as half-life and metabolism, may affect both thrombotic and cardio- renal properties of NSAIDs (Roger and Cate, 2012).
Choice of agent Evidence suggests that all non-selective NSAIDs and COX-II inhibitors are of similar efficacy, but vary in their toxicity profiles. However, there is individual patient variability in terms of response to a given NSAID, and so some patient may need to try several agents before reaching an effective and well tolerated agent. Non selective NSAIDs or COX-II inhibitors should be used at the lowest effective dose for the shortest possible period of time. There are no recommendations on which agent to use first-line as all NSAIDs have analgesic effects of similar magnitude. However, as these drugs vary in terms of potential gastro-intestinal, liver and cardio-renal toxicity,
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Disease-modifying anti-rheumatic drugs (Roger and Cate, 2012) Joint damage is known to occur early in RA and is largely irreversible. The need for early intervention with DMARDs as part of an aggressive approach to minimize disease progression has become standard practice and is associated with better patient outcome. Early introduction of DMARDs also results in fewer adverse reactions and withdrawals from therapy. The DMARDs that are commonly used for RA and have clear evidence of benefit are methotrexate, sulphasalazine, leflunomide and intramuscular gold. There is less compelling evidence for the use of hydroxyl-chloroquine, d-penicillamine, oral gold, ciclosporin and azathioprine, although these agents do improve symptoms and some objective measures of inflammation. The exact mechanism of action of all these drugs is unknown. All DMARDs inhibit the release or reduce the activity of inflammatory cytokines, such as TNF-α, IL-1, IL-2 and IL-6. Activated T-lymphocytes have been implicated in the inflammatory process, and these are inhibited by methotrexate, leflunomide and ciclosporin. Patients should be made aware that the DMARDs all have a slow onset of action. They must be taken for at least 8 weeks before any clinical effect is apparent, and it may be months before an optimal response is achieved. Whilst early initiation of DMARDs is crucial, it is important to ensure the patient is maintained on therapy to maintain disease suppression. This itself is a challenge, due to the toxicity profiles of the majority of these drugs. The majority of the DMARDs require blood monitoring. Guidelines are available on the action to take in the event of abnormal blood results. Patients with a new diagnosis of RA should be offered combination DMARD therapy as first-line therapy as soon as possible, ideally within 3 months of the onset of persistent disease symptoms. The combination therapy should include methotrexate and at least one DMARD usually sulphasalazine and/or hydroxychloroquine. Evidence suggests that combination therapy appears to be superior in terms of benefits to symptoms, quality of life, remission rates and slowing of joint damage, when compared to monotherapy. Once sustained and satisfactory levels of disease
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Review of Literature control have been achieved, the doses of drugs should be cautiously reduced to levels that continue to maintain disease control. In patients where combination therapy is not appropriate, for example where there are contraindications to a drug due to existing co-morbidities, DMARD monotherapy should be started, placing greater emphasis on fast escalation to a clinically effective dose rather than on choice of agent. There are many factors that influence the choice of DMARD: relative efficacy severity of disease, convenience, monitoring requirements, patient co-morbidities, cost, time period to benefit, prescriber’s experience and success rates with the drug, side effects and patient adherence. Studies have shown that methotrexate has the best benefit to toxicity ratio. Both sulphasalazine and hydroxychloroquine alone does not slow radiological damage. Most patients started on a DMARD will not be taking it 3- 4 years later because of adverse reactions or lack of efficacy. Despite promising results initially, some patients experience disease reactivation at a later stage and become unresponsive to treatment.
Methotrexate Methotrexate is recognized as the gold standard DMARD in the management of RA. It is given as once weekly dose and can be given orally or parenterally via the intramuscular or subcutaneous routes. Patients usually begin on oral therapy; parenteral methotrexate may be considered in those who do not respond adequately to the maximum tolerated oral dose, or in those who suffer from gastro-intestinal side effects. Doses used, whether administered by the parenteral or oral route, are similar, although bioavailability is greater with parenteral administration. Methotrexate is primarily excreted unchanged by the kidneys and so elderly patients or those with renal impairment may require lower dose. Methotrexate is a folic acid antagonist and acts by reversibly inhibiting dihydrofolate reductase, the enzyme that reduces folic acid to tetrahydrofolic acid. Concomitant administration of oral folic acid reduces adverse effects of methotrexate and improves continuation of therapy and adherence. Doses used range from 5mg weekly to 5mg daily except on the day of methotrexate administration. Methotrexate is associated with lung, liver and bone marrow toxicities. As a consequence, strict monitoring is advised and alcohol intake should be minimized.
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Methotrexate pneumonitis is usually seen within the first year of treatment, but can sometimes occur after long-term therapy. Myelosuppression can cause significant falls in blood cell counts. It is more likely to occur in the elderly, patients with renal impairments or patients who are also taking anti-folate drugs. A clinically significant drop in cell counts calls for immediate withdrawal of methotrexate, and folinic acid rescue therapy. Patients should be counseled to report any of the following warning symptoms immediately to a healthcare professional: blood disorders, for example sore throat, bruising, mouth ulcers, liver toxicity, for example nausea, vomiting, abdominal discomfort, dark urine, and respiratory effects, for example shortness of breath, persistent dry cough. Methotrexate tablets are available as 2.5mg and 10mg strengths; most pharmacies will dispense the 2.5mg strength only for non-chemotherapy indications such as RA.
Sulphasalazine Sulphasalazine has been shown to slow joint erosions and suppress inflammatory activity in RA. Blood dyscrasias usually occur within the first 3-6 months of treatment, therefore necessitating close monitoring in the initiation period. Patients should also be counseled to report warning symptoms of unexplained bleeding, bruising, purpurea, sore throat, fever or malaise. Enteric-coated tablets are available to minimize gastro-intestinal side effects.
Hydroxychloroquine Hydroxychloroquine is significantly less effective than other DMARDs and historically was reserved for milder cases of RA. It still has a place in therapy, particularly in combination with other DMARDs, as it seems to give some symptomatic relief to patients and is the least toxic of the DMARDs. It has also been used relatively safely in pregnancy. Regular visual assessment for retinopathy is recommended as long term use of anti-malarial agents has been linked to ocular toxicity.
Leflunomide Leflunomide has a long half-life of approximately 2 weeks, and consequently a loading dose may be given to achieve therapeutic drug levels more quickly. However,
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Review of Literature in practice, the loading dose is often omitted due to intolerable gastro intestinal side effects such as diarrhoea. Leflunomide is associated with hepato- and haemato- toxicity, and should be used with caution if co-prescribed with drugs which also cause these adverse effects. Washout procedures using colestyramine or activated charcoal may be necessary when switching to other DMARDs, in the event of a serious adverse effect or before conception in females.
Gold therapy Gold compounds can be given via intramuscular injection as sodium aurothiomalate, or orally as auranofin. Intramuscular gold is more effective than oral. These drugs can be used over a long period of time provided the patient does not experience side effects such as proteinuria, blood disorders, rashes, gastro-intestinal side effects or bleeding.
Other DMARDs D-Penicillamine is less commonly used, as side effects such as rashes, taste loss and vomiting, are common. It can be effective in some patients, but doses above 750mg daily are associated with a high incidence of adverse effects. Azathioprine and ciclosporin can be used in refractory RA, but use is limited due to monitoring requirements and high incidence side effects.
Glucocorticoids Steroids can be given via the oral, intramuscular or intra-articular routes. They act by inhibiting cytokine release and give rapid relief of symptoms and decrease inflammation. Prednisolone is the most commonly used oral steroid. Intra-articular injections, such as triamcinolone or methyl-prednisolone, are administered into inflamed joints for local anti-inflammatory action, pain relief and to reduce deformity. The effects of the injection tend to last for approximately 4 weeks and should generally not be repeated more than three times a year into an affected joint. Intramuscular and, less commonly intravenous, injections are used as high dose pulse therapy to control aggressive disease flares. Steroids are also used as a bridging therapy and are particularly useful when introducing DMARDs which may take several months to take effect. There are
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Review of Literature various studies which demonstrate steroids are disease modifying in slowing radiological damage over 2 years. Doses of prednisolone 7.5 mg daily have been suggested to reduce the rate of joint destruction in moderate to severe RA of less than 2 years duration. Ideally, steroids should be reserved for short-term use in new-onset RA because of their long-term complications and adverse effects. However, because they exert such a potent anti-inflammatory effect, it may be difficult in some patients to withdraw therapy as the disease tends to flare with dose reductions. Gradual reducing regimens should be used with the aim to reach the lowest possible maintenance dose. Steroids can induce osteoporosis, which is a known complication associated with RA itself. Prophylactic therapy, such as calcium and vitamin D supplementation and bisphosphonates, should be considered in patients on steroids at a high dose or for an extended period of time. Gastroprotection may also be necessary in the form of H2 antagonist or proton pump inhibitors. Other adverse effects associated with steroids are diabetes, increased risk of infection, hypertension and weight gain.
Biological therapies Over the past decade, there have been significant advances in the treatment of RA due to emerging biological therapies. The so-called biologics in the RA are genetically engineered monoclonal antibodies which selectively target different parts of the inflammatory pathways. Activated T-cells release pro-inflammatory cytokines including TNF-α, IL-1 and IL-6. Adalimumab, etanercept, golimumab, infliximab and certolizumab pegol target TNF-α, anakinra and tocilizumab target the interleukins, whilst abatacept and rituximab act on T-cells and B-cells, respectively. In current practice, biologics are used after a patient has failed DMARDs, although there is emerging evidence to suggest they should be used earlier in the disease. Combination DMARD therapy is increasingly advocated and may lead to earlier use of biologics. For example, a patient may now be trialed on two DMARDs, including methotrexate, over a period of 6 months and if the response is inadequate could be eligible for an anti-TNF agent within a year of diagnosis.
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Anti-TNF agents There are five anti-TNF agents available: adalimumab, etanercept, golimumab, infliximab and cetrolizumab pegol. All inhibit TNF-α which is a inflammatory cytokine found in high concentrations within the joint synovium of RA patients. Infliximab was the first anti-TNF agent licensed for the treatment of RA. It is a chimeric human-murine monoclonal antibody that binds with high affinity to TNF-α, thereby neutralizing its activity. Infliximab is the only anti-TNF agent which is given by intravenous infusion and must be given concomitantly with methotrexate. It is usually well tolerated, with the most common adverse effects being mild infusion reactions, such as headache and urticaria. Anaphylaxis and delayed hypersensitivity reactions have also been rarely reported. As infliximab is part murine monoclonal antibody, it is thought to carry a higher risk of developing human anti-chimeric antibodies (HACAs). HACAs are associated with an increased frequency of infusion- related reactions and can be minimized by administering with an immunomodulating therapy. Adalimumab is a recombinant human monoclonal antibody that binds to and neutralizes TNF-α. Etanercept is a human TNF fusion protein that binds to TNF cell surface receptors, thereby inhibiting interactions of TNF-α with its receptors. Cetrolizumab pegol is a pegylated antibody fragment which binds and neutralizes TNF-α and is thought to have a relatively more rapid onset of action. Golimumab is the most recent addition to this family of agents and has the advantage of having a less frequent dosing schedule. Optimum clinical benefit is achieved when these drugs are used in combination with methotrexate. However, adalimumab, etanercept and cetrolizumab pegol can be used alone as monotherapy in patients for whom methotrexate is not tolerated.
Safety The anti-TNF agents are generally well tolerated, with the main side effects being injection site reaction with the subcutaneous agents, and infusion-related reactions with infliximab. There are fewer monitoring requirements compared to the DMARDs and less frequent dosing, making these drugs potentially more appealing. However, the long-term safety of these drugs is being monitored in the UK by the British Society of Rheumatology Biologics Registry. This database collects data on efficacy
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Rituximab Rituximab is a chimeric human-murine monoclonal anti-body which binds to the C20 antigen on B-lymphocytes to mediate B-cells lysis. It causes depletion of peripheral B-cells which play a vital role in the pathogenesis of RA. Recovery of B-cells appears to occur 6 months after treatment, with some patients showing prolonged B-cell depletion persisting up to 2 years after treatment. Rituximab in combination with methotrexate is licensed for the treatment of severe active RA in patients who have had an inadequate response or intolerance to other DMARDs including one or more anti-TNF agent. Rituximab is also licensed for non- Hodgkin’s disease and chronic lymphocytic leukemia, both using a different dosing schedule to that of RA. A course of Rituximab consists of two intravenous infusions administered as a day case in hospital: 1000-mg infusion followed by a second 1000- mg infusion 2 weeks later. The course may be repeated every six months depending on patient response. Disease response varies between patients in that some achieve disease remission after one course and do not require re-treatment, whilst others require further repeat infusions every 6-12 months. Rituximab is generally well tolerated, and the most common adverse effects are infusion-related reactions including fever, changes in blood pressure and rash. Minor infusion related side effects can be managed by reducing the rate of infusion and giving treatment for relief of symptoms such as paracetamol. As with the anti-TNF agents, Rituximab increases the risk of infections and should not be used in the presence of active or severe infections. Use has been associated with progressive multifocal leukoencephalopathy. It may also exacerbate existing cardiac conditions such as angina pectoris and atrial fibrillation. Patients with known history of cardiac disease should be closely monitored during treatment administration for changes in blood pressure and pulse. Anti-hypertensive may be omitted 12 hr prior to the infusion.
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Abatacept Abatacept acts by blocking the full activation of T-cells thereby inhibiting the release of inflammatory cytokines. It is licensed for use in combination with methotrexate in the treatment of moderate to severe active RA in adults who have had an insufficient response or intolerance to other DMARDs including at least one anti-TNF agent and who cannot receive Rituximab, or when Rituximab is withdrawn due to an adverse event.
Anakinra Anakinra blocks the binding of IL-1 to its receptor, thus inhibiting the inflammatory effects of IL-1. The evidence of its benefit in RA is weak and it is considered modestly effective. There are no trials directly comparing Anakinra with other biologic agents. Anakinra is not cost effective in the treatment of RA and availability for routine use in the NHS has not been supported.
Tocilizumab Tocilizumab acts by binding to IL-6 receptors. IL-6 is a pro-inflammatory cytokine produced by a variety of cells including T and B-cells, and has been implicated in the pathogenesis of RA and other inflammatory diseases. Tocilizumab is licensed for use in combination with methotrexate in the treatment of moderate to severe active RA in adults who have had an insufficient response or intolerance to other DMARDs including at least one anti-TNF agent. It is given as a monthly intravenous infusion at a dose of 8 mg/kg and requires regular monitoring of liver function tests and full blood count. Tocilizumab is recommended for the treatment of RA in patients who fulfill the following criteria; V The patients has not responded adequately to one or more anti-TNF agents V The patients cannot receive Rituximab due to contraindication V The patients has not responded adequately to Rituximab treatment
2.2.10 RA and pregnancy (Roger and Cate, 2012) The management of RA during pregnancy is a common challenge, with disease activity improving in approximately 70-80% of patients. Disease activity usually
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Review of Literature decreases in the first trimester, and this lasts for a number of weeks to months into the postpartum period. Subsequently, 90% of patients will then experience a flare usually during the first 3 months. Although case control studies of pregnancy outcome demonstrate a slight increase in spontaneous abortion in women with RA, most reports have failed to show an increase in fetal morbidity. Medication may potentially increase the risk, for example, steroids may restrict intrauterine growth. Women with active RA or other types of inflammatory arthritis may have children with lower birth weights. None of the available drug treatments for RA are absolutely safe in pregnancy. A prescriber must carefully assess the risks and benefits of treatment in consultation with the patient. In patients with active RA during pregnancy, prednisolone is recommended at the lowest dose (below 20mg daily if possible) to control the disease. Sulphasalazine and hydroxychloroquine are considered safe to prescribe by most obstetric physicians.
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2.3 Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze
Figure 8: Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze
2.3.1 Taxonomic Classification Kingdom: Plantae Phylum: Magnoliophyta Class: Magnoliopsida Order: Asterales Family: Asteraceae Genus: Cyathocline Species: Cyathocline purpurea Part used: Whole plant
2.3.2 Botanical description and Vernacular name Cyathocline purpurea is one of the most valuable plant in traditional system of medicine from ancient time. Synonyms of Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze are Tanacelum purpureum, Cyathochine lyrata. Cyathocline purpurea is also called as, Bandhaniya in Hindi, Gangotra in Marathi, Gal Phulle in Nepali, Hong Hao Zhi in Chinese.
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2.3.3 Morphology (Joshi, 2013) Cyathocline is a genus of flowering plants in the daisy family i.e. Asteraceae. The family Asteraceae includes 1100 genera and 30000 species. In India, the family is represented by 167 genera and 980 species. In majority of the members of the family sesquiterpene lactones are present which are known for their anti-infective property (anticancer, antifungal, anti-inflammatory, antibacterial, antiviral, antimalarial and antiageing activity).
Cyathocline purpurea is an erect, strongly aromatic, glandular hairy annual or biennial herb, upto 60 cm high. The leaves are sessile, variable in size and shape. Leaf segments are irregularly serrate. Heads are small, in panicles, dark purple. Stem is branched grooved. Flower heads 0.3 to 0.6 cm across in terminal corymbose panicles; acute, hairy on margins. The stems of this plant are usually reddish or purplish tinged, branched from the base and glandular-pubescent. Whole plant is aromatic and is commonly observed as undergrowth in open spaces especially near wet surface; rice fields or on the banks of river streams.
2.3.4 Occurrence and Distribution Cyathocline purpurea is a rare existence Indian medicinal plant which is not popular among the local in habitants and commonly found in moist habitats such as along water courses and in rice fields throughout most of peninsular and northern India at an elevation of 1300m. It is also available in the western and central Himalayas. Cyathocline is a small genus with two or three species and distributed in tropical Asia. The genus was described in 1829 by Cassini as Cyathocline purpurea (Buch – Ham. ex D. Don.) Kuntze. Cyathocline purpurea is native to India and south- western China and is a traditional medicine.
2.3.5 Phytoconstituents Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze is reported to contain guaianolide (Chintalwar et al., 1991), eudesmanolide, sesquiterpene lactones, isoivangustin, santamarine, 9 B-acetoxycostunolide and 9 B-acetoxyparthenolide (Nagasampagi et al., 1981).
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Review of Literature
Sesquiterpene lactones are secondary metabolites that belong to the group of C15 terpenoids. They are formed from three isoprene units. This is a large group of secondary metabolites. So far, over 90% of identified lactones were isolated from the plant family Asteraceae. Sesquiterpene lactones were also isolated from the more primitive representatives of families such as Magnoliaceae and Lauraceae, but also from the more derivative groups such as Apiaceae. Sesquiterpene lactones were formed in a pathway from isoprene C5 units. All of them are modifications of germacranolide, a compound that forms from germacradien. Germacranolides are precursors for most of the more derivative lactones, such as guaianolides, pseudoguaianolides, eudesmanolides etc. The first identified lactones were artemisinin and costunolide, which do not belong to guaianolides. They were both isolated from the plant family Asteraceae.
2.3.6 Pharmacological activities reported of Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze Anticancer (Guoyi et al., 2009) Cyathocline purpurea is used in traditional Chinese medicine as an herbal remedy for human tuberculosis, malaria and bleeding (Yu et al., 1993). The traditional medicinal practitioners of the Hani ethnic minority in Yunnan, China also commonly use this plant to treat various cancers. Three sesquiterpene lactones, santamarine, 9β- acetoxycostunolide and 9β-acetoxyparthenolide were isolated from Cyathocline purpurea by bioactivity-guided fractionation (Li et al., 2006). Sesquiterpene lactones are a class of natural sesquiterpenes which are chemically distinct from other members of the group through the presence of a γ-lactone system and have a wide range of biological activities including mutagenic, genotoxic, cytotoxic and antitumour actions (Rodriguez et al 1976; Picman, 1986). Many sesquiterpene lactones have shown significant antineoplastics effects (Lee et al., 1971). Santamarine and 9β-acetoxycostunolide are sesquiterpene lactones with the α-methylene-γ-lactone moiety in their structure. There have been no reports of cytotoxicity with new compounds of 9β-acetoxycostunolide and 9β-acetoxyparthenolide.
Santamarine and 9 β -acetoxycostunolide inhibited mitosis and reduced thymidine uptake in L1210 cells. The mechanism of action of santamarine and 9 β -
Study of Analgesic, Anti-inflammatory and Antiarthritic activity of Indian medicinal plant in laboratory animals 69
Review of Literature acetoxycostunolide might be related to suppression of microtubular protein formation and activation of caspase 3, induction of cell cycle blockage and apoptosis. It has been mentioned that Cyathocline purpurea has been traditionally used to treat various diseases related to inflammation including cancers for many years without any reports of toxicity to humans (Yu et al., 1993), suggesting that it is not harmful to humans. This investigation provides pharmacological support to its use in cancers.
Antibacterial (Joshi, 2013) The essential oils from the roots of Cyathocline purpurea were screened in vitro for antibacterial activity against eight human pathogenic bacteria. The essential oil of roots was analyzed by using GC–FID and GC–MS. The antibacterial activity of oil was tested against four Gram-positive and four Gram-negative bacteria and antibacterial activity was determined by the tube dilution method. The main constituents of the oil were thymohydroquinone dimethyl ether (57.4%) and β- selinene (14.0%), among twenty five identified compounds, which represented 90.1% of the total oil. The oil was found to be active against Gram-positive bacteria with minimal bactericidal concentration (MBC) values in the range of 0.26–0.57 mg/mL. The observation of MBC assay suggested that the Gram positive microorganisms were susceptible to essential oil, while oil was found to be resistant against Gram- negative bacteria, and the oil has bactericidal property.
The roots of Cyathocline purpurea are used to relieve stomach pains (Parrotta, 2001). This plant releases an essential oil that is reportedly owns antimicrobial, anthelmintic and hypotensive properties (Parrotta, 2001). Cyathocline purpurea is used to treat pulmonary tuberculosis (Qiang et al., 2006).
Therefore, based on the primary information available on this plant, further series of studies like isolation and identification of active constituents, pharmacological standardization of extracts and activities on isolated compounds as well as clinical and toxicological efficacy is still remained to explore so far. Sesquiterpene lactones isolated from other plants have been found to possess good anti-inflammatory activities (Hall et al., 1980). As the literature survey shows that Cyathocline purpurea may show anti-inflammatory activity due to presence of sesquiterpene lactones and be
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Review of Literature utilized for treating pathological states like arthritis (Joshi et al., 2010) and up till now there were no studies reported on in-vivo activity of this plant as anti-inflammatory and antiarthritic; therefore the objective of the present study was to determine the efficacy of Cyathocline purpurea (whole plant) as analgesic, anti-inflammatory and antiarthritic in different animal models.
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Aims and Objectives Aims and Objectives
3.1 AIMS: The aim of the present work was to carry out phytochemical and pharmacological investigation of selected plant for analgesic, anti-inflammatory and antiarthritic activity in animals. The medicinal plant was selected by carrying out literature survey of plant mentioned for the treatment of analgesic, anti-inflammatory and antiarthritic activity in traditional system of Indian medicine. Based on the literature survey plant Cyathocline purpurea (Buch-Ham ex D.Don) Kuntze Fam. Asteraceae was selected for the study.
3.2 OBJECTIVES:
3.2.1 Part A: (Analgesic, anti-inflammatory and antiarthritic activity of Cyathocline purpurea extracts) 1. To procure and authenticate the whole plant Cyathocline purpurea. 2. To prepare three extracts of Cyathocline purpurea of different polarity, petroleum ether extract of Cyathocline purpurea (PECP), methanolic extract of Cyathocline purpurea (MECP) and aqueous extract of Cyathocline purpurea (AECP). 3. To carry out preliminary qualitative phytochemical analysis of all the three extracts. 4. To carry out acute oral toxicity studies of all the three extracts as per Organization for Economic Co-operation and Development (OECD) guidelines no. 425 in mice. 5. To investigate analgesic activity of all the three extracts using hot-plate test and acetic acid induced writhing in mice. 6. To investigate anti-inflammatory activity of all the three extracts using carrageenan induced paw edema and cotton pellet induced granuloma model in rats. 7. To investigate antiarthritic activity of extract showing superior analgesic and anti-inflammatory activity using Freund’s Complete Adjuvant (FCA) induced arthritis model in rats.
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Aims and Objectives
3.2.2 Part B: (Isolation and characterization) 1. To perform liquid-solid separation chromatography of most active extract to prepare various fractions. 2. To investigate the anti-inflammatory activity of all the fractions by carrageenan induced paw edema in rats. 3. To fractionate most active fraction further by column chromatography and to collect different pools based on thin layer chromatography (TLC). 4. To investigate the anti-inflammatory activity of all the pools collected from column chromatography by carrageenan induced paw edema in rats. 5. To isolate compound by preparative TLC technique from most active pool. 6. To characterize the isolated compound by IR, 1H-NMR, 13C-NMR, DEPT and MS. 7. To determine structure of isolated compound. 8. To carry out docking studies of isolated compound on the active sites of TNF- alpha converting enzyme (TACE).
3.2.3 Part C: (Antiarthritic activity of isolated compound) 1. To evaluate the antiarthritic activity of isolated compound using FCA induced arthritis model in rats and to unravel its mechanism of action.
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Materials and Methods Materials and Methods
4.1 MATERIALS
4.1.1 Plant material Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze Fam. Asteraceae was collected in the month of January 2013 from Mulshi, Pune, Maharashtra.
4.1.2 Identification and authentication of plant material Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze was identified and authenticated by J. Jayanthi, Scientist C, Botanical Survey of India, Pune and voucher specimen (No. BSI/WRC/Tech/2013/1094) was deposited at that institute.
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Materials and Methods
4.1.2a Authentication certificate of Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze
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Materials and Methods
4.2 METHODS: Part A (Analgesic, anti-inflammatory and antiarthritic activity of Cyathocline purpurea extracts)
4.2.1 Drugs and Chemicals Petroleum ether (Merck) and Methanol (Molychem) were purchased from authorized vendor. The reagents for phytochemical identification were obtained from the freshly prepared stock used in pharmacogonosy and pharmaceutical chemistry laboratories of the college.
4.2.2 Preparation of extracts The whole plant was shade dried and powdered. Dried powder (500 g) was subjected to successive extractions by maceration using petroleum ether followed by methanol and then distilled water. The extracts were filtered and concentrated on a rotary evaporator (Medica Instrument, India) and stored in desiccator. The percentage yields of petroleum ether extract of Cyathocline purpurea (PECP), methanol extract of Cyathocline purpurea (MECP) and aqueous extract of Cyathocline purpurea (AECP) were 3.3 %, 6.7 % and 7.7 % respectively.
4.2.3 Storage of extracts The PECP, MECP and AECP were stored in tightly closed amber color glass bottles in refrigerator.
4.2.4 Phytochemical analysis of PECP, MECP and AECP Phytochemical analysis of PECP, MECP and AECP were performed using standard procedures to identify the constituents present in them (Khandelwal, 2010). Preparation of extract solutions: The extracts PECP, MECP and AECP were dissolved in petroleum ether, methanol and water, respectively to obtained solution of respective extract (100 mg/ml) for phytochemical analysis.
4.2.4.1 Test for Carbohydrates Molisch’s test: Solution of extract (2 ml) and α-napthol solution (2-3 drops) were mixed in test tube, shaken for few min and concentrated H2SO4 (1 ml) was added
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Materials and Methods from the side of test tube. A deep violet colored ring at the junction of two layers indicated the presence of sugars.
4.2.4.2 Test for Proteins Biuret test: Solution of extract (3 ml), (sodium hydroxide) 4% NaOH (1 ml) and
(copper sulphate) 1% CuSO4 (1 ml) were mixed in test tube. The change in color of solution to violet or pink indicates presence of proteins. Millon’s test: Solution of extract (3 ml) and Millon’s reagent (5 ml) were mixed in test tube and observed for the appearance of white precipitate changing to brick red and appearance of red color to solution on heating indicates presence of proteins.
4.2.4.3 Test for Steroids
Salkowski reaction: Solution of extract (2 ml), chloroform (2 ml) and H2SO4 (2 ml) were mixed in test tube, shaken well. The change of chloroform layer to red and acid layer to greenish yellow fluorescence indicates presence of steroids. Liebermann-Burchard reaction: Solution of extract (2 ml), chloroform (2 ml), acetic anhydride (2 ml) were mixed in test tube. Concentrated H2SO4 (2 drops) was added from the side of test tube. The change in color first red, then blue and finally green indicates presence of steroids. Liebermann’s reaction: Solution of extract (3 ml) and acetic anhydride (3 ml) were mixed in test tube. Heated the mixture and cooled. Concentrated H2SO4 (2-3 drops) was added from the side of test tube. Appearance of blue color indicates presence of steroids.
4.2.4.4 Test for Volatile oils Odour test: Characteristic odour of extract indicates presence of volatile oil. Solubility test: Solubility in 90% alcohol indicates presence of volatile oil.
4.2.4.5 Test for Glycosides
Keller-Killani test: Solution of extract (2 ml), glacial acetic acid (1 ml), 5% FeCl3 (3 drops) and concentrated H2SO4 were mixed in test tube and observed for the appearance of reddish-brown color at the junction of two layers and bluish green in the upper layer indicates presence of cardiac glycosides.
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Materials and Methods
Borntrager’s test: Solution of extract (2 ml) and dilute H2SO4 (2 ml) was mixed in test tube, boiled for 5 min and filtered. In filtrate equal volume of chloroform was added and mixed well. Organic layer was separated and ammonia was added to it. Pink-red color of the ammonia layer indicates presence of anthraquinone glycosides.
4.2.4.6 Test for Saponins Foam test: Extract (1 g) was shaken vigorously with water and observed for persistent foam indicating presence of saponins.
4.2.4.7 Test for Tannins and Phenolic compounds The following reagents were added to the 3 ml of solution of extract. 1. 5% Ferric chloride (3 ml): The blue- black color indicates presence of tannins or phenols. 2. Lead acetate (3 ml): The occurrence of white precipitate indicates presence of tannins or phenols. 3. Potassium permanganate (3 ml): The discoloration of potassium permanganate solution indicates presence of tannins or phenols. 4. Acetic acid solution (3 ml): The red color indicates presence of tannins or phenols.
4.2.4.8 Test for presence of Flavonoids Shinoda test: In the solution of extract (5 ml), 95% ethanol (5 ml), few drops of HCl and magnesium tunings (0.5 g) were added. The appearance of pink color indicates presence of flavonoids.
4.2.4.9 Test for Alkaloids Extract (10 g) and dilute hydrochloric acid were mixed in test tube, shaken and filtered. With filtrate following tests were performed. Dragendorff’s test: Extract solution filtrate (3 ml) and Dragendorff’s reagent (1 ml) were mixed in test tube. The appearance of orange brown precipitate indicates presence of alkaloids. Mayer’s test: Extract solution filtrate (3 ml) and Mayer’s reagent (1 ml) were mixed in test tube. The appearance of precipitate indicates presence of alkaloids.
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Materials and Methods
Wagner’s test: Extract solution filtrate (3 ml) and Wagner’s reagent (1 ml) were mixed in test tube. The appearance of reddish brown precipitate indicates presence of alkaloids. Hager’s test: Extract solution filtrate (3 ml) and Hager’s reagent (1 ml) were mixed in test tube. The appearance of yellow precipitate indicates presence of alkaloids.
4.2.5 Pharmacological study 4.2.5.1 Chemicals and drugs Carrageenan (Sigma-Aldrich, St. Louis, USA), Freund’s Complete Adjuvant (FCA) (Sigma-Aldrich, St. Louis, USA), Acetic acid (Pure Chem. Ltd., India), Acetylsalicylic acid (Cipla Pharmaceuticals, India), Pentazocine (Fortwin, Ranbaxy), Tween 80 (Research Lab, India), ELISA kits for determination of serum TNF-α, IL- 1β, and IL-6 (Raybiotech), Biochemical diagnostic kits for determination of Aspartate aminotransferase (AST), Alanine aminotransferase (ALT), Total protein and Alkaline phophatase (ALP) (Accurex biomedical Pvt. Ltd) were purchased from authorized vendors. Diclofenac was obtained as a gift sample from Emcure pharmaceuticals Ltd., Pune. All other chemicals and solvents used were of analytical grade purchased from authorized vendors.
4.2.5.2 Apparatus Microcapillary tubes, microtips (Tarson, India), test tubes, centrifuging tubes (Tarson, India), tissue paper, micropipettes were purchased from authorized vendors.
4.2.5.3 Instruments used Plethysmometer (Model: 7140, UGO Basile, Italy), Digital vernier caliper (Model: CD-6 CS, Mitutoyo Corp., Japan), Thermal planter tester (Model: 37360, UGO Basile, Italy), Von Frey Hairs (Model: 2888, Almemo, Germany), Hot plate (Model: DS-37, UGO Basile, Italy), UV/visible spectrophotometer (Model: Jasco V-530, Japan), Eppendorff’s cryocentrifuge machine (Model: 5810 R, Germany).
4.2.5.4 Preparation of dosage form Dosage forms of individual extracts were prepared as per the following procedures.
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Materials and Methods
Petroleum ether extract: The petroleum ether extract of Cyathocline purpurea was emulsified with tween 80 (2%) in a glass mortar with gradual addition of distilled water to make up the required volume. Methanolic extract: The methanolic extract of Cyathocline purpurea was suspended with tween 80 (2%) in a glass mortar with gradual addition of distilled water to make up the required volume. Aqueous extracts: Aqueous extract of Cyathocline purpurea was dissolved in distilled water to make up the required volume. Drugs Accurately weighed quantity of acetylsalicylic acid and diclofenac were suspended in distilled water to make volume. Vehicles Vehicle was prepared by adding 2% tween 80 into distilled water, without addition of extracts or drugs.
4.2.5.5 Storage conditions All the dosage forms of extracts and drug solutions were prepared freshly on the day of dosing and stored in airtight amber colored vials to protect from exposure to sunlight during the experiments.
4.2.5.6 Volume of extract solution The volume of extract solution was calculated based upon the body weight of animal.
4.2.5.7 Route of administration The extract solution was administered per orally.
4.2.5.8 Experimental animals Female Swiss albino mice (25-30 g) and female Wistar rats (180-220 g) were purchased from National Institute of Biosciences, Pune, India. Animals were housed in an air-conditioned room at a temperature of 25 ± 1 °C and relative humidity of 45
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Materials and Methods to 55% under 12-h light: 12-h dark cycle. The animals had free access to food pellets (Manufactured by Pranav Agro Industries Ltd., Sangli, India) and water ad libitum.
4.2.5.9 Approval of research protocol The experimental protocol was approved by the Institutional Animal Ethics Committee (IAEC) constituted in accordance with the rules and guidelines of the Committee for the Purpose of Control and Supervision on Experimental Animals (CPCSEA), India (Protocol approval number was CPCSEA/28/2014 and CPCSEA/29/2014).
4.2.5.10 Acute oral toxicity (AOT) study Healthy female Swiss albino mice of 25-30 g were used in acute toxicity studies as per OECD guidelines-425. The animals were fasted overnight and divided into 3 groups with 5 mice in each group. Extracts (PECP, MECP and AECP) were administered at dose of 2000 mg/kg, p.o. body weight. The mice were observed continuously for behavioral and autonomic profiles for 2 hrs and for any signs of toxicity or mortality up to 48 hrs (OECD-425, 2001).
4.2.5.11 Analgesic activity 4.2.5.11.1 Hot plate test in mice Female Swiss albino mice (25 – 30 g) were treated according to the method described by Eddy and Leimback, 1953. Mice were screened by placing them on hot plate (UGO Basile, Italy. Model No. DS-37) maintained at 55 ± 1 °C and the reaction time was recorded in seconds. The time for paw licking or jumping on the hot plate was considered as a reaction time. The responses were recorded before and after 30, 60, 90, 120, 150 and 180 min of the administration of PECP, MECP, AECP and pentazocine. A cut-off time of 15s was used to avoid injury to the animals.
The mice were divided into eleven groups with six mice in each group. Group 1: - Vehicle control (2% Tween 80). Group 2: - Standard (Pentazocine 5 mg/kg, s.c.). Group 3, 4 and 5: - PECP (100, 200 and 400 mg/kg, p.o.), respectively. Group 6, 7 and 8: - MECP (100, 200 and 400 mg/kg, p.o.), respectively.
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Materials and Methods
Group 9, 10 and 11: - AECP (100, 200 and 400 mg/kg, p.o.), respectively.
4.2.5.11.2 Acetic acid induced writhing in mice Female Swiss albino mice (25 – 30 g) were treated according to the method described by Collier et al, 1963. Mice were pretreated orally with PECP, MECP, AECP and acetylsalicylic acid, 60 min before administration of acetic acid solution at a dose of 10 ml/kg (0.6%, i.p.). The number of abdominal constrictions (full extension of both hind paws) was cumulatively counted over a period of 15 min. The mice were divided into eleven groups of six mice each. Group 1: - Vehicle control (2% Tween 80). Group 2: - Standard (Acetylsalicylic acid 100 mg/kg p.o.). Group 3, 4 and 5: - PECP (100, 200 and 400 mg/kg, p.o.), respectively. Group 6, 7 and 8: - MECP (100, 200 and 400 mg/kg, p.o.), respectively. Group 9, 10 and 11: - AECP (100, 200 and 400 mg/kg, p.o.), respectively. The percent inhibition of writhing was calculated as follows: % Inhibition = (VC-VT/VC) * 100 Where, VT, number of writhes in drug treated mice. VC, number of writhes in control group mice.
4.2.5.12 Anti-inflammatory activity 4.2.5.12.1 Carrageenan induced paw edema in rats Female Wistar rats (180 – 220 g) were treated according to the method described by Winter et al, 1962. Inflammation was produced by injecting 0.1ml of 1% lambda carrageenan in sterile normal saline into the sub plantar region of the right hind paw of the rat. Rats were pretreated orally with PECP, MECP, AECP and diclofenac 1h before the carrageenan injection. The paw volume was measured from 0-6 h, at an hourly interval using plethysmometer (Ugo Basile, Italy, Model No. 7140). The mean changes in injected paw volume with respect to initial paw volume were calculated. Female Wistar rats were divided into eleven groups of six rats each. Group 1: - Carrageenan control (2% Tween 80). Group 2: - Standard (Diclofenac 10 mg/kg p.o.). Group 3, 4 and 5: - PECP (100, 200 and 400 mg/kg, p.o.), respectively. Group 6, 7 and 8: - MECP (100, 200 and 400 mg/kg, p.o.), respectively.
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Materials and Methods
Group 9, 10 and 11: - AECP (100, 200 and 400 mg/kg, p.o.), respectively. Percentage inhibition of paw volume between treated and control group was calculated by the following formula, % Inhibition = (VC-VT / VC *100) Where, VT and VC are the mean increase in paw volume in treated and control groups, respectively.
4.2.5.12.2 Cotton pellet induced granuloma in rats Method described by D’Arcy et al, 1960 was followed. Chronic inflammation was produced by implanting the pre-weighed sterile cotton pellets (50 mg) in the axilla region of the each rat through a small incision. PECP, MECP, AECP and diclofenac were administered orally for seven consecutive days after the cotton pellet implantation. Before implanting the cotton pellets, rats were anaesthetized with anesthetic ether. On the eight day animals were sacrificed by cervical dislocation and stomach was removed for histopathology study and cotton pellets were removed from animal’s body, freed from the extraneous tissues, dried in oven at 60 °C for 24 h and weighed. Female Wistar rats weighing (180 – 220 g) were divided into eleven groups of six rats each. Group 1: - Vehicle control (2% Tween 80). Group 2: - Standard (Diclofenac 10 mg/kg p.o.). Group 3, 4 and 5: - PECP (100, 200 and 400 mg/kg, p.o.), respectively. Group 6, 7 and 8: - MECP (100, 200 and 400 mg/kg, p.o.), respectively. Group 9, 10 and 11: - AECP (100, 200 and 400 mg/kg, p.o.), respectively.
4.2.5.13 Antiarthritic activity Freund’s complete adjuvant induced arthritis in rats Arthritis was induced by single intra-dermal injection of 0.1 ml of Freund’s complete adjuvant into a foot pad of the left hind paw of female Wistar rats (180-220 gm) rats. Each ml contains 1 mg of Mycobacterium tuberculosis (Strain H37Ra, ATCC-25177) (Patel et al., 2012). The female Wistar rats weighing (180 – 220 g) were divided into six groups consisting of six animals per group:
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Materials and Methods
Group I – Healthy control Group II – Arthritic control Group III –Standard, Diclofenac 5 mg/kg, p.o. Group IV – MECP 100 mg/kg, p.o. Group V – MECP 200 mg/kg, p.o. Group VI –MECP 400 mg/kg, p.o. Anti-arthritic activity of MECP on the injected paw was evaluated on the following parameters change in paw volume, change in joint diameter, pain threshold, paw withdrawal latency, mechanical nociceptive threshold and body weight on day 0, 1, 4, 8, 12, 16, 20, 24, and day 28 (Kumar et al., 2006). On day 28 the animals were anaesthetized with anesthetic ether and the blood was withdrawn by retro-orbital puncture. Serum was used for the estimation of biochemical parameters (AST, ALT, Alkaline phosphatase, and Total protein), CRP and RF value. Whole blood was used for estimation of hematological parameters (WBC, RBC, Hb and Platelets) and isolated liver was used for estimation of antioxidant parameters (SOD, MDA and GSH). Radiological and histopathological analyses of ankle joints were also done.
4.2.5.13.1 Measurement of change in paw volume Change in paw volume was measured using a Plethysmometer (UGO Basile, Italy) on day 0 before FCA injections and thereafter on day 1, 4, 8, 12, 16, 20, 24, and day 28 (Lee et al., 2009). The change in paw volume was calculated as the difference between the final and initial paw volume.
4.2.5.13.2 Measurement of change in joint diameter Change in joint diameter was measured using a digital Vernier caliper (Mitutoyo, Japan) on day 0 before FCA injections and thereafter on day 1, 4, 8, 12, 16, 20, 24, and day 28 (Banchet et al., 2009). The change in joint diameter was calculated as the difference between the final and initial joint diameter.
4.2.5.13.3 Measurement of pain threshold (Randall Selitto) Pain threshold was measured using Randall-Selitto analgesiometer (UGO Basile, Italy) on day 0 just before FCA injections and thereafter on day 1, 4, 8, 12, 16, 20, 24, and day 28. The hind paw was placed between the flat surface and blunt pointer and
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Materials and Methods was applied with increasing pressure. The cut-off pressure was 450 g. The pain threshold was determined when rat attempted to remove the hind paw from the apparatus (Authier et al., 2003).
4.2.5.13.4 Measurement of paw withdrawal latency (Thermal hyperalgesia) Paw withdrawal latency was measured using a radiant heat apparatus (UGO Basile, Italy) on day 0 just before FCA injections and thereafter on day 1, 4, 8, 12, 16, 20, 24, and day 28. The paw was placed on the heat radiator with infrared intensity of lamp was set at 40. A cut of latency of 15 s was used to avoid tissue damage (Ramteke et al., 2009).
4.2.5.13.5 Measurement of mechanical nociceptive threshold (Tactile allodynia) Mechanical nociceptive threshold was determined by measuring paw withdrawal following probing of the plantar surface with a series of calibrated fine filaments (Von Frey hairs, Almemo, Germany) of increasing gauge (Jalalpure et al., 2011; Pepys and Hirschfield, 2003). The rats were allowed to acclimatize for 10 min in the Perspex box and Von Frey hairs (0.6 to 12.6 g) were applied to plantar surface of left hind paw. A series of three stimuli were applied to paw for each hair within a period 2–3 s. The lowest weight of Von Frey hair to evoke a withdrawal from the three consecutive applications was considered to indicate the threshold. Lifting of the paw was recorded as a positive response (Mali et al., 2011).
4.2.5.13.6 Body weight recording Body weight was recorded on day 0 just before FCA injections and thereafter on day 1, 4, 8, 12, 16, 20, 24, and day 28 (Asquith et al., 2009).
4.2.5.13.7 Radiological analysis of ankle joints On day 28, rats were anesthetized and radiographs of the adjuvant injected hind paws were taken using X-ray (AGFA CR 30-X unit, Germany). Radiographic analysis of hind paws were taken at 55 kV peak, 50 mA and the exposure time was 5 s.
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Materials and Methods
4.2.5.13.8 Haematological and serum parameters On day 28, haematological parameters like red blood cell (RBC) count, white blood cell (WBC) count, haemoglobin (Hb), and platelets (PLT) were determined by usual standardized laboratory method (Mythilypriya et al., 2008). Serum C-reactive protein (CRP) and Rheumatoid factor (RF) level was also measured (Mehta et al., 2012).
4.2.5.13.9 Biochemical parameters On day 28, blood of the rats was withdrawn by retro-orbital puncture and centrifuged at 7000 rpm at 4°C for 15 minutes. Serum was used for the estimation of AST, ALT, alkaline phosphatase and total protein levels (Mythilypriya et al., 2008).
4.2.5.13.10 Antioxidant parameters The rats were sacrificed on day 28 by cervical dislocation, the levels of malondialdehyde (MDA), reduced glutathione (GSH) and superoxide dismutase (SOD) in liver were estimated as biomarkers of inflammation. 4.2.5.13.10.1 Removal and processing of tissue for estimation of tissue parameters Reagents Phosphate Buffered Saline Ph (7.4) Disodium ethylene diamine tetra acetic acid (1.38 gm), 0.19 gm of potassium dihydrogen phosphate and 8 gm of sodium chloride were dissolved in 900 ml of distilled water and pH was adjusted using dilute hydrochloric acid. The volume was adjusted to 1000 ml using distilled water. Sucrose solution (0.25 M) 85.58 gm of sucrose was dissolved in 200 ml of water and diluted to 1000 ml with distilled water. Tris hydrochloric buffer (10mM, pH 7.4) 1.21 gm tris was dissolved in 900 ml of distilled water and the pH was adjusted to 7.4 with 1M hydrochloric acid. The resulting solution was diluted to 1000 ml with distilled water. Procedure The rats were sacrificed after blood collection; liver was dissected and quickly transferred to ice-cold phosphate buffered saline (pH 7.4). It was blotted free of blood and tissue fluids, weighed on electronic Balance. The liver were cross-chopped
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Materials and Methods with surgical scalpel into fine slices, suspended in chilled 0.25M sucrose solution and quickly blotted on a filter paper. The tissues were then minced and homogenized 1 min. in chilled tris hydrochloride buffer (10mM, pH 7.4) to a concentration of 10% w/v. Homogenization under hypotonic condition was carried out to disrupt as far as possible the structure of the cells so as to release soluble proteins. The homogenate was centrifuged at 7000 rpm at 25 minutes using Remi C-24 high speed cooling centrifuge. The clear supernatant was used for the determination of MDA, GSH, and SOD concentration.
4.2.5.13.10.2 Assay of lipid Peroxidation (MDA content) Reagents Thiobarbituric acid (0.67% w/v) Thiobarbituric acid 0.67 gm was dissolved in 50 ml of distilled water and the final volume was made up to 100 ml with distilled water. Trichloroacetic acid (10% w/v) Trichloroacetic acid 10 gm was dissolved in 60 ml of distilled water and the final volume was made up to 100 ml with distilled water. Standard Malondialdehyde stock solution (50mM) A standard malondialdehyde stock solution was prepared by mixing 25µ of 1,1,3,3-Tetraethoxypropane up to 100 ml with distilled water. 1.0 ml of this stock solution was diluted up to 10 ml to get solution containing 23µ of malondialdehyde/ml. One ml of this stock solution was diluted up to 100 ml to get a working standard solution containing 23 ng of malondialdehyde/ml. Procedure Tissue homogenate (supernatant) 2.0 ml was added to 2.0 ml of freshly prepared 10% w/v trichloroacetic acid (TCA) and the mixture was allowed to stand in an ice bath for 15 minutes. After 15 minutes, the precipitate was separated by centrifugation and 2.0 ml of clear supernatant solution was mixed with 2.0 ml of freshly prepared thiobarbituric acid (TBA). The resulting solution was heated in a boiling water bath for 10 minutes. It was then immediately cooled in an ice bath for 5 minutes. The color developed was measured at 532nm against reagent blank by U.V spectrophotometer. Different concentrations (0-23nM) of standard malondialdehyde were processed as
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Materials and Methods above for obtaining standard graph. The values were expressed as nM of MDA/mg protein (Slater and Sawyer, 1971).
4.2.5.13.10.3 Assay of endogenous antioxidant (reduced glutathione i.e. GSH) Reagents Trichloroacetic acid (20% w/v) Trichloroacetic acid 20 gm was dissolved in sufficient quantity of distilled water and the final volume was made up to 100 ml with distilled water. Phosphate Buffer (0.2M, pH 8.0) Sodium phosphate 0.2 M was prepared by dissolving 30.2 gm sodium phosphate in 600 ml of distilled water, the pH was adjusted to 8.0 with 0.2M sodium hydroxide solution and the final volume was adjusted up to 1000 ml with distilled water. 5,5-dithiobis-2-nitrobenzoic acid (DTNB) reagent (0.6mM) DTNB reagent 60 mg was dissolved in 50 ml of buffer and the final volume was adjusted to 100 ml with buffer. Standard glutathione (100 µg/ml) 10 mg of glutathione was dissolved in 60 ml of distilled water and the final volume was made up to 100 ml with distilled water. Procedure Equal volumes of tissue homogenate (supernatant) and 20% TCA were mixed. The precipitated fraction was centrifuged at 2500 rpm at 4°C for 15 min and 2.0 ml of DTNB reagent was added to 0.25 ml of supernatant. The final volume was made up to 3.0 ml with phosphate buffer. The color developed was read at 412 nm against reagent blank. Different concentrations (10-50 µg) of standard glutathione were prepared and processed as above for standard graph. The amount of reduced glutathione is expressed as µg of GSH/mg protein (Morgon et al., 1979).
4.2.5.13.10.4 Assay of Superoxide Dismutase (SOD) Reagents Carbonate buffer (0.05 M, pH 10.2) Sodium bicarbonate 16.8 gm and 22 gm of sodium carbonate were dissolved in 500 ml of distilled water and the volume was made up to 1000 ml with distilled water. Ethylene diamine tetra acetic acid (EDTA) solution (0.4 M)
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EDTA 1.82 gm was dissolved in 200 ml of distilled water and the volume was made up to 1000 ml with distilled water.
Hydrochloric acid (0.1 N) Concentrated hydrochloric acid 8.5 ml was mixed with 500 ml of distilled water and the volume was made up to 1000 ml with distilled water. Epinephrine solution (3mM) Epinephrine bitartarate 0.99 gm was dissolved in 100 ml of 0.1N hydrochloric acid and the volume was adjusted to 1000 ml with 0.1N hydrochloric acid. Superoxide dismutase standard (10 U/ml) SOD 1 mg (1000 U/mg) from bovine liver was dissolved in 100 ml of carbonate buffer. Procedure Liver tissue homogenate (0.5 ml) was diluted with (0.5 ml) distilled water to which 0.25 ml of ice-cold ethanol and 0.15 ml of ice-cold chloroform were added. The mixture was mixed well using cyclo mixer and centrifuged at 2500 rpm at 4°C for 15 min. To 0.5 ml of supernatant, 1.5 ml of carbonate buffer and 0.5 ml of EDTA solution were added. The reaction was initiated by the addition of 0.4 ml of epinephrine and the change in optical density/min was measured at 480 nm against reagent blank. Calibration curve was prepared by using 10-125 units of SOD. Change in optical density per minute at 50% inhibition of epinephrine to adrenochrome transition by the enzyme is taken as the enzyme unit. SOD activity is expressed as units/mg protein (Misra and Fridovich., 1972).
4.2.5.13.11 Histopathological analysis of ankle joints On day 28, ankle joints were separated from the hind paw and immersed in 10% buffered formalin for 24 h followed by decalcification in 5% formic acid, processed for paraffin embedding sectioned at 5µ thickness. The sections were stained with haematoxylin-eosin and evaluated under light microscope with 10X magnifications for the presence of inflammatory cells, hyperplasia of synovium, pannus formation and destruction of joint space (Patil et al., 2012).
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Materials and Methods
4.3 METHODS: Part B (Isolation and characterization)
4.3.1 Chemicals and reagents Petroleum ether, ethyl acetate, acetone, methanol, ethanol (Merck) of GR grades, column grade silica (60-120 mesh) (Merck Specialties Private Limited, Mumbai) and thin layer chromatography (TLC) silica gel plates 60 F254 (Merck, Germany) were purchased from respective vendors.
4.3.2 Apparatus and instruments Borosil glass column (height: 60 cm; diameter: 3 cm) was purchased from Ajay scientific enterprises, Pune, India. Precoated TLC silica gel plates (Merck, Kieselgel 60, F-254, 0.2 mm, Germany) were used for analytical TLC. IR spectra were recorded using KBr pellets on JASCO FT-IR 5300 spectrophotometer. 1H-NMR and 13C-NMR spectra were recorded on 200 MHz and 50 MHz spectrometer, respectively (Bruker,
Germany). Deuterated chloroform (CDCl3) was used for recording NMR and tetramethylsilane (TMS) was used as an internal standard. Chemical shifts were reported as δ (ppm). The coupling constants (J) were reported as Hz. Mass spectrum was obtained on a Thermo Finigen Surveyor MSQ spectrometer.
4.3.3 Experimental animals Female Wistar rats (180-220 g) were purchased from National Institute of Biosciences, Pune, India. Animals were housed in an air-conditioned room at a temperature of 25 ± 1 °C and relative humidity of 45 to 55% under 12-h light: 12-h dark cycle. The animals had free access to food pellets (Manufactured by Pranav Agro Industries Ltd., Sangli India) and water ad libitum.
4.3.4 Approval of research protocol The experimental protocol was approved by the Institutional Animal Ethics Committee (IAEC) constituted in accordance with the rules and guidelines of the Committee for the Purpose of Control and Supervision on Experimental Animals (CPCSEA), India (Protocol approval number was CPCSEA/PCL/07/2014-2015).
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4.3.5 Liquid – solid separation chromatographic technique 15 g of MECP (most active extract) was mixed with 30 ml of acetone and 30 g of silica gel. Sample were mixed thoroughly and dried in oven at 1100C for 10 min and following fractions were prepared.
4.3.5.1 Petroleum ether fraction (F – 1) 100 ml of petroleum ether was added into the mixture and shaken properly. Then the mixture was allowed to settle, two layers were formed. Upper layer was separated into the separate flask and labeled as petroleum ether fraction. Same procedure was repeated twice with 100 ml of petroleum ether.
4.3.5.2 10% acetone in petroleum ether fraction (F – 2) 100 ml of 10% acetone in petroleum ether was added into the residue and shaken properly. Then the mixture was allowed to settle, two layers were formed. Upper layer was separated into the separate flask and labeled as 10% acetone in petroleum ether fraction. Same procedure was repeated twice with 100 ml of 10% acetone in petroleum ether.
4.3.5.3 20% acetone in petroleum ether fraction (F – 3) 100 ml of 20% acetone in petroleum ether was added into the residue and shaken properly. Then the mixture was allowed to settle, two layers were formed. Upper layer was separated into the separate flask and labeled as 20% acetone in petroleum ether fraction. Same procedure was repeated twice with 100 ml of 20% acetone in petroleum ether.
4.3.5.4 30% acetone in petroleum ether fraction (F – 4) 100 ml of 30% acetone in petroleum ether was added into the residue and shaken properly. Then the mixture was allowed to settle, two layers were formed. Upper layer was separated into the separate flask and labeled as 30% acetone in petroleum ether fraction. Same procedure was repeated twice with 100 ml of 30% acetone in petroleum ether.
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4.3.5.5 50% acetone in petroleum ether fraction (F – 5) 100 ml of 50% acetone in petroleum ether was added into the residue and shaken properly. Then the mixture was allowed to settle, two layers were formed. Upper layer was separated into the separate flask and labeled as 50% acetone in petroleum ether fraction. Same procedure was repeated twice with 100 ml of 50% acetone in petroleum ether.
4.3.5.6 Methanol fraction (F – 6) 100 ml of methanol was added into the residue and shaken properly. Then the mixture was allowed to settle, two layers were formed. Upper layer was separated into the separate flask and labeled as methanol fraction. Same procedure was repeated twice with 100 ml of methanol.
4.3.6 Anti-inflammatory activity of fractions (F – 1 to F – 6) in carrageenan induced paw edema in rats. Inflammation was produced by injecting 0.1ml of 1% lambda carrageenan (Sigma Chemical Co., USA) in sterile normal saline into the sub plantar region of the right hind paw of the rat (Winter et al., 1962). Rats were pretreated by test substance orally 1h before the carrageenan injection. The paw volume was measured from 0-6 h, at an hourly interval using plethysmometer (Ugo Basile, Italy, Model No. 7140). The mean changes in injected paw volume with respect to initial paw volume were calculated. Percentage inhibition of paw volume between treated and control group was calculated by the following formula, % Inhibition = (VC-VT / VC *100) Where, VT and VC are the mean increase in paw volume in treated and control groups, respectively. Female Wistar rats weighing 180-220 g were divided in following groups (n=6) viz; Group 1: - Carrageenan control. Group 2: - Standard, diclofenac 10 mg/kg, p.o. Group 3: - Petroleum ether fraction (F – 1), 100 mg/kg, p.o. Group 4: - 10% acetone in petroleum ether fraction (F – 2), 100 mg/kg, p.o. Group 5: - 20% acetone in petroleum ether fraction (F – 3), 100 mg/kg, p.o. Group 6: - 30% acetone in petroleum fraction (F – 4), 100 mg/kg, p.o.
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Group 7: - 50% acetone in petroleum fraction (F – 5), 100 mg/kg, p.o. Group 8: - Methanol fraction (F – 6), 100 mg/kg, p.o.
4.3.7 Column chromatography of most active anti-inflammatory fraction i.e. 30% acetone in petroleum ether fraction (F – 4)
Figure 9- Column chromatography of 30% acetone in petroleum ether fraction (F – 4) (A) Silica gel adsorbed with 30% acetone in petroleum ether fraction (F – 4) (B) Column loaded with 30% acetone in petroleum ether fraction (F – 4) (C) 30% acetone in petroleum ether fraction (F – 4) uniformly spread in column after addition of mobile phase (D) 30% acetone in petroleum ether fraction uniformly spread in column continued (E) Elution collection started. Column was packed with activated silica (60-120#). The 30% acetone in petroleum ether fraction (F – 4) was adsorbed on silica gel and the dried silica was added to top Study of Analgesic, Anti-inflammatory and Antiarthritic activity of Indian medicinal plant in laboratory animals 93 Materials and Methods of the packed silica in column (Figure 9). After stabilization column was eluted with mobile phase (petroleum ether: ethyl acetate, initially starting with lower proportion of ethyl acetate and then increasing the proportion of ethyl acetate). Fractions were collected and analyzed by TLC. Fractions showing similar bands were pooled together and labeled as pool P – 1 to P – 10.
Activation of silica: Column grade silica (60 – 120 mesh) was placed in oven at 1100C overnight (12 h) to remove all moisture content present in it. This activated silica was packed in the column. Preparation of mobile phase: The solvents petroleum ether and ethyl acetate were used for the preparation of mobile phase. The composition of mobile phase was petroleum ether: ethyl acetate, with successive increase in percentage of ethyl acetate. Packing of column: A clean and dry borosil glass column (height: 60 cm; diameter: 3 cm) was aligned in a vertical position with the help of clamps attached to metal stand. A piece of cotton soaked in mobile phase was placed at the bottom of the column and gently tapped down with a glass rod. The column was slowly and evenly filled to about 5/6 capacity with gradual addition of silica. Side of the column was gently tapped with a cork during the packing process to compact silica column. Application of sample: Weighed quantity (2.5 g) of the 30% acetone in petroleum ether fraction (F – 4) was dissolved in 5 ml of acetone and was adsorbed on 5 gm of silica gel (particle size: 60- 120 mesh) to prepare slurry, acetone was evaporated from the slurry and the dried sample was added to top of the packed silica in column. A thin disc (column diameter) of cotton soaked in mobile phase was placed on top of the bed to prevent disturbing the sample layer after addition of mobile phase. Stopcock was opened to drain excess mobile phase until it reaches top of sample. Column was filled to the top with the mobile phase and allowed to stand overnight (~12 h) to develop bands. Elution:- Elution was carried out by gravity at the flow rate of 2 ml/min. Mobile phase added to top of column was petroleum ether: ethyl acetate. Polarity of mobile phase was
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Materials and Methods increased by increasing the proportion of ethyl acetate and fractions (25 ml in each 50 ml tube) were collected in tubes. Remaining loaded material in the column which could not be eluted with mobile phase was eluted with methanol and collected as methanol fraction and completed the column chromatography. All fractions were analyzed by TLC and fractions showing similar bands were pooled together and labeled as pool P – 1 to P – 10 (Figure 10).
Figure 10- TLC of pools (P – 1 to P – 10) collected from 30% acetone in petroleum ether fraction (F – 4). (A) TLC of pool P – 1 to P – 6 (B) TLC of pool P – 7 to P – 10
4.3.8 Anti-inflammatory activity of pools (P – 1 to P – 10) collected from 30% acetone in petroleum ether fraction (F – 4) in carrageenan induced paw edema Pools (P – 1 to P – 10) collected from 30% acetone in petroleum ether fraction (F – 4) were subjected further for anti-inflammatory activity. Female Wistar rats weighing 180-220 g were divided in following groups (n=6) viz; Group 1: - Carrageenan control. Group 2: - Standard, diclofenac (10 mg/kg, p.o) Group 3 to 12: - P – 1 to P – 10, respectively (10 mg/kg, p.o)
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Inflammation was produced by injecting 0.1ml of 1% lambda carrageenan (Sigma Chemical Co., USA) in sterile normal saline into the sub plantar region of the right hind paw of the rat (Winter et al., 1962). Rats were pretreated by test substance orally 1h before the carrageenan injection. The paw volume was measured from 0-6 h, at an hourly interval using plethysmometer (Ugo Basile, Italy, Model No. 7140). The mean changes in injected paw volume with respect to initial paw volume were calculated. Percentage inhibition of paw volume between treated and control group was calculated by the following formula, % Inhibition = (VC-VT / VC *100) Where, VT and VC are the mean increase in paw volume in treated and control groups, respectively.
4.3.9 Preparative TLC of most active anti-inflammatory pool i.e. P – 8 Application of sample: The sample of pool P – 8 was applied by streaking across the full length of the plate by glass capillary. Mobile phase: The solvents petroleum ether and ethyl acetate were used for the preparation of mobile phase. The composition of mobile phase was petroleum ether: ethyl acetate (70:30). Preconditioning of chamber (saturation): Chromatogram was developed in a saturated twin trough chambers. A sufficient quantity (approximately 10 ml) of mobile phase was poured along the side into the chamber to saturate the chamber. Chamber was then closed and allowed to stand for at least 30 min at room temperature (RT). Development of chromatogram: The plate was marked 10 mm below the upper edge. Plate was placed vertically into the chamber ensuring that the points of application were above the surface of the mobile phase. Chamber was closed and mobile phase was allowed to ascend to the specific distance at the room temperature. Plate was removed; the position of mobile phase front was marked. Mobile phase was allowed to evaporate at room temperature and dried under hot air.
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Observation and elution of compound: Chromatogram was observed in daylight under ultra violate (UV) light at 254 and 366 nm wavelength. Area was marked and scrapped off with sharp blade. With minimum volume of mobile phase the components from scrapped material was eluted. Scrapped material and mobile phase were homogenized in vortex mixer to ensure complete elution then filtered by Whatman filter paper and filtrate was allowed to evaporate.
4.3.10 Spectral characterization of isolated compound (P – 8) The chemical structure of isolated compound was elucidated by IR, 1H-NMR, 13C- NMR, DEPT and MS spectroscopy.
4.3.11 Docking study Glide (Glide, 2009) was used for docking study to examine the binding mode of isolated compound with TNF-alpha converting enzyme (TACE) (PDB: 1ZXC). The ligands were prepared using LigPrep (LigPrep, 2009). The protein was refined using the protein preparation wizard present in Maestro 9.0 (Maestro, 2009). All the water molecules were deleted. Hydrogen atoms were added to the protein, including the protons necessary to define the correct ionization and tautomeric states of the amino acid residues. Prime interface module incorporated in Maestro 9.0 was used to add the missing residues of the side chain. Each structure minimization was carried out with the impact refinement module to alleviate steric clashes potentially existing in the structures. Minimization was terminated when the energy converged or the root mean square deviation reached a maximum cutoff of 0.30 Å. To find out active site grid was prepared using grid generation panel of glide with the default settings. Grid is prepared for defining the binding site of native ligand on the receptor. The ligand was selected to define the position and size of the active site (Friesner et al., 2004; Halgren et al., 2004; Bhansali and Kulkarni, 2014). Glide XP docking was used for docking purposes.
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4.4. METHODS: Part C (Antiarthritic activity of isolated compound, Isoivangustin)
Freund’s complete adjuvant induced arthritis in rats Arthritis was induced by single intra-dermal injection of 0.1 ml of Freund’s complete adjuvant into a foot pad of the left hind paw of female Wistar rats (180-220 gm) rats. Each ml contains 1 mg of Mycobacterium tuberculosis (Strain H37Ra, ATCC-25177). The rats were anesthetized with ether inhalation during adjuvant injection as the viscous nature of adjuvant exerts difficulty while injecting (Patel et al., 2012). The animals were divided into six groups consisting of six animals per group: Group I – Healthy control Group II – Arthritic control Group III –Standard, diclofenac 5 mg/kg, p.o. Group IV – Isoivangustin 2.5 mg/kg, p.o. Group V – Isoivangustin 5 mg/kg, p.o. Group VI – Isoivangustin 10 mg/kg, p.o. Anti-arthritic activity of Isoivangustin was evaluated on the following parameters change in paw volume, change in joint diameter, pain threshold, paw withdrawal latency, mechanical nociceptive threshold and body weight on day 0, 1, 4, 8, 12, 16, 20, 24, and day 28 (Kumar et al., 2006). On day 28 the animals were anaesthetized with anesthetic ether and the blood was withdrawn by retro-orbital puncture and centrifuged. Serum was used for the estimation of cytokines (TNF-α, IL-1β, and IL- 6), biochemical parameters (AST, ALT, Alkaline phosphatase, and Total protein), CRP and RF value. Whole blood was used for estimation of hematological parameters (WBC, RBC, ESR, Hb and Platelets) and isolated liver was used for estimation of antioxidant parameters (SOD, MDA and GSH). Radiological and histopathological analyses of ankle joints were also done on last day.
4.4.1 Measurement of change in paw volume Change in paw volume was measured using a Plethysmometer (UGO Basile, Italy) on day 0 before FCA injections and thereafter on day 1, 4, 8, 12, 16, 20, 24, and day 28 (Lee et al., 2009). The change in paw volume was calculated as the difference between the final and initial paw volume.
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4.4.2 Measurement of change in joint diameter Change in joint diameter was measured using a digital Vernier caliper (Mitutoyo, Japan) on day 0 before FCA injections and thereafter on day 1, 4, 8, 12, 16, 20, 24, and day 28 (Banchet et al., 2009). The change in joint diameter was calculated as the difference between the final and initial joint diameter.
4.4.3 Measurement of pain threshold (Randall Selitto) Pain threshold was measured using Randall-Selitto analgesiometer (UGO Basile, Italy) on day 0 just before FCA injections and thereafter on day 1, 4, 8, 12, 16, 20, 24, and day 28. The hind paw was placed between the flat surface and blunt pointer and was applied with increasing pressure. The cut-off pressure was 450 g. The pain threshold was determined when rat attempted to remove the hind paw from the apparatus (Authier et al., 2003).
4.4. 4 Measurement of paw withdrawal latency (Thermal hyperalgesia) Paw withdrawal latency was measured using a radiant heat apparatus (UGO Basile, Italy) on day 0 just before FCA injections and thereafter on day 1, 4, 8, 12, 16, 20, 24, and day 28. The paw was placed on the heat radiator with infrared intensity of lamp was set at 40. A cut of latency of 15 s was used to avoid tissue damage (Ramteke et al., 2009).
4.4.5 Measurement of mechanical nociceptive threshold (Tactile allodynia) Mechanical nociceptive threshold was determined by measuring paw withdrawal following probing of the plantar surface with a series of calibrated fine filaments (Von Frey hairs, Almemo, Germany) of increasing gauge (Jalalpure et al., 2011; Pepys and Hirschfield, 2003). The rats were allowed to acclimatize for 10 min in the Perspex box and Von Frey hairs (0.6 to 12.6 g) were applied to plantar surface of left hind paw. A series of three stimuli were applied to paw for each hair within a period 2–3 s. The lowest weight of Von Frey hair to evoke a withdrawal from the three consecutive applications was considered to indicate the threshold. Lifting of the paw was recorded as a positive response (Mali et al., 2011).
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4.4.6 Body weight recording Body weight was recorded on day 0 just before FCA injections and thereafter on day 1, 4, 8, 12, 16, 20, 24, and day 28 (Asquith et al., 2009).
4.4.7 Radiological analysis of ankle joints On day 28, rats were anesthetized and radiographs of the adjuvant injected hind paws were taken using X-ray (AGFA CR 30-X unit, Germany). Radiographic analysis of hind paws was performed at 55 kV peak, 50 mA and the exposure time was 5 s.
4.4.8 Haematological and serum parameters On day 28, haematological parameters like red blood cell (RBC) count, white blood cell (WBC) count, haemoglobin (Hb), platelets (PLT) and erythrocyte sedimentation rate (ESR) were determined by usual standardized laboratory method (Mythilypriya et al., 2008). Serum C-reactive protein (CRP) and Rheumatoid factor (RF) level was also measured (Mehta et al., 2012).
4.4.9 Biochemical parameters On day 28, blood of the rats was withdrawn by retro-orbital puncture and centrifuged at 7000 rpm at 4°C for 15 minutes. Serum was used for the estimation of serum AST, ALT, ALP and total protein levels (Mythilypriya et al., 2008).
4.4.10 Cytokine measurement by ELISA On day 28, serum was used for estimation of levels of TNF-α, IL-1β and IL-6.
4.4.10.1 Measurement of serum TNF-α Reagent preparation: Sample dilution: 100 µl serum was diluted with 200 µl of Assay Diluent A Standard preparation: The vial of Item C (Recombinant Rat TNF-α) was briefly spin and 400 µl of Assay Diluent A was added to it with gentle mixing. Then 100 µl of TNF-α standard from the vial of Item C was added into a tube with 400 µl Assay Diluent A to prepare a 20,000 pg/ml stock standard solution. 200 µl of these prepared solution was diluted serially into more six tubes containing 400 µl Assay Diluent A to prepare different concentrations.
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Wash buffer solution preparation: 20 ml of wash buffer concentrate was diluted with 400 ml of distilled water. Biotin antibody solution preparation: Detection Antibody TNF-α (Item F) was briefly spin before use. 100 µl of 1x Assay Diluent B was added into the vial to prepare a detection antibody concentrate. The prepared detection antibody concentrate was again diluted 80 fold with 1x Assay Diluent B. Streptavidin solution preparation: 50 µl of HRP-Streptavidin concentrate was added into a tube with 10 ml 1x Assay Diluent B. Procedure: 100 µl of standard and samples were added to each well and incubated for 2.5 hr at room temperature and then washed 4 times with wash buffer solution.
Then 100 µl of prepared biotin antibody was added to each well and incubated for 1 hr at room temperature and then washed 4 times with wash buffer solution.
Then added 100 µl of prepared Streptavidin solution and incubated for 45 minutes at room temperature and then washed 4 times with wash buffer solution.
Then added 100 µl of TMB (tetramethylbenzidine) to each well, the blue color was developed in proportion to the amount of TNF-α bound and incubated for 30 minutes.
Finally 50 µl of Stop solution was added to each well, the color changed from blue to yellow and reading was noted at 450 nm immediately. Calculations: The TNF-α protein was quantified by comparing the sample to the standard curve generated. The results were expressed as cytokine concentrations (e.g., pg/ml protein).
4.4.10.2 Measurement of serum IL-1β Reagent preparation: Sample dilution: 100 µl serum was diluted with 200 µl of Assay Diluent A Standard preparation: The vial of Item C (Recombinant Rat IL-1β) was briefly spin and 400 µl of Assay Diluent A was added to prepare a 50,000 pg/ml stock standard
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Materials and Methods solution. 130 µl of these prepared solution was diluted serially into more seven tubes containing 260 µl Assay Diluent A to prepare different concentrations. Wash buffer solution preparation: 20 ml of wash buffer concentrate was diluted with 400 ml of distilled water. Biotin antibody solution preparation: Detection Antibody IL-1β (Item F) was briefly spin before use. 100 µl of 1x Assay Diluent B was added into the vial to prepare a detection antibody concentrate. The prepared detection antibody concentrate was again diluted 80 fold with 1x Assay Diluent B. Streptavidin solution preparation: 50 µl of HRP-Streptavidin concentrate was added into a tube with 10 ml 1x Assay Diluent B. Procedure: 100 µl of standard and samples were added to each well and incubated for 2.5 hr at room temperature and then washed 4 times with wash buffer solution.
Then 100 µl of prepared biotin antibody was added to each well and incubated for 1 hr at room temperature and then washed 4 times with wash buffer solution.
Then added 100 µl of prepared Streptavidin solution and incubated for 45 minutes at room temperature and then washed 4 times with wash buffer solution.
Then added 100 µl of TMB to each well, the blue color was developed in proportion to the amount of IL-1β bound and incubated for 30 minutes.
Finally 50 µl of Stop solution was added to each well, the color changed from blue to yellow and reading was noted at 450 nm immediately.
Calculations: The IL-1β protein was quantified by comparing the sample to the standard curve generated. The results were expressed as cytokine concentrations (e.g., pg/ml protein).
4.4.10.3 Measurement of serum IL-6 Reagent preparation: Sample dilution: 100 µl serum was diluted with 200 µl of Assay Diluent C
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Standard preparation: The vial of Item C (Recombinant Rat IL-6) was briefly spin and 500 µl of Assay Diluent C was added to prepare a 10,000 pg/ml stock standard solution. 200 µl of these prepared solution was diluted serially into more seven tubes containing 300 µl Assay Diluent C to prepare different concentrations. Wash buffer solution preparation: 20 ml of wash buffer concentrate was diluted with 400 ml of distilled water. Biotin antibody solution preparation: Detection Antibody IL-6 (Item F) was briefly spin before use. 100 µl of 1x Assay Diluent B was added into the vial to prepare a detection antibody concentrate. The prepared detection antibody concentrate was again diluted 80 fold with 1x Assay Diluent B. Streptavidin solution preparation: 30 µl of HRP-Streptavidin concentrate was added into a tube with 12 ml 1x Assay Diluent B. Procedure: 100 µl of standard and samples were added to each well and incubated for 2.5 hr at room temperature and then washed 4 times with wash buffer solution.
Then 100 µl of prepared biotin antibody was added to each well and incubated for 1 hr at room temperature and then washed 4 times with wash buffer solution.
Then added 100 µl of prepared Streptavidin solution and incubated for 45 minutes at room temperature and then washed 4 times with wash buffer solution.
Then added 100 µl of TMB to each well, the blue color was developed in proportion to the amount of IL-1β bound and incubated for 30 minutes.
Finally 50 µl of Stop solution was added to each well, the color changed from blue to yellow and reading was noted at 450 nm immediately. Calculations: The IL-6 protein was quantified by comparing the sample to the standard curve generated. The results were expressed as cytokine concentrations (e.g., pg/ml protein).
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Materials and Methods
4.4.11 Antioxidant parameters The rats were sacrificed on day 28 by cervical dislocation, the levels of malondialdehyde (MDA), reduced glutathione (GSH) and superoxide dismutase (SOD) in liver were estimated as biomarkers of inflammation.
4.4.11.1 Removal and processing of tissue for estimation of tissue parameters Reagents Phosphate Buffered Saline Ph (7.4) Disodium ethylene diamine tetra acetic acid (1.38 gm), 0.19 gm of potassium dihydrogen phosphate and 8 gm of sodium chloride were dissolved in 900 ml of distilled water and pH was adjusted using dilute hydrochloric acid. The volume was adjusted to 1000 ml using distilled water. Sucrose solution (0.25 M) 85.58 gm of sucrose was dissolved in 200 ml of water and diluted to 1000 ml with distilled water. Tris hydrochloric buffer (10mM, pH 7.4) 1.21 gm tris was dissolved in 900 ml of distilled water and the pH was adjusted to 7.4 with 1M hydrochloric acid. The resulting solution was diluted to 1000 ml with distilled water. Procedure The rats were sacrificed after blood collection; liver was dissected and quickly transferred to ice-cold phosphate buffered saline (pH 7.4). It was blotted free of blood and tissue fluids, weighed on electronic Balance. The liver were cross-chopped with surgical scalpel into fine slices, suspended in chilled 0.25M sucrose solution and quickly blotted on a filter paper. The tissues were then minced and homogenized 1 min. in chilled tris hydrochloride buffer (10mM, pH 7.4) to a concentration of 10% w/v. Homogenization under hypotonic condition was carried out to disrupt as far as possible, the structure of the cells so as to release soluble proteins. The homogenate was centrifuged at 7000 rpm at 25 minutes using Remi C-24 high speed cooling centrifuge. The clear supernatant was used for the determination of MDA, GSH, and SOD concentration.
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4.4.11.2 Assay of lipid Peroxidation (MDA content) Reagents Thiobarbituric acid (0.67% w/v) Thiobarbituric acid 0.67 gm was dissolved in 50 ml of distilled water and the final volume was made up to 100 ml with distilled water.
Trichloroacetic acid (10% w/v) Trichloroacetic acid 10 gm was dissolved in 60 ml of distilled water and the final volume was made up to 100 ml with distilled water. Standard Malondialdehyde stock solution (50mM) A standard malondialdehyde stock solution was prepared by mixing 25µ of 1,1,3,3-Tetraethoxypropane up to 100 ml with distilled water. 1 ml of this stock solution was diluted up to 10 ml to get solution containing 23µ of malondialdehyde/ml. One ml of this stock solution was diluted up to 100 ml to get a working standard solution containing 23ng of malondialdehyde/ml. Procedure Tissue homogenate (supernatant) 2.0 ml was added to 2.0 ml of freshly prepared 10% w/v trichloroacetic acid (TCA) and the mixture was allowed to stand in an ice bath for 15 minutes. After 15 minutes, the precipitate was separated by centrifugation and 2.0 ml of clear supernatant solution was mixed with 2.0 ml of freshly prepared thiobarbituric acid (TBA). The resulting solution was heated in a boiling water bath for 10 minutes. It was then immediately cooled in an ice bath for 5 minutes. The color developed was measured at 532nm against reagent blank by U.V spectrophotometer. Different concentrations (0-23nM) of standard malondialdehyde were processed as above for obtaining standard graph. The values were expressed as nM of MDA/mg protein (Slater and Sawyer, 1971).
4.4.11.3 Assay of endogenous antioxidant (reduced glutathione i.e. GSH) Reagents Trichloroacetic acid (20% w/v) Trichloroacetic acid 20 gm was dissolved in sufficient quantity of distilled water and the final volume was made up to 100 ml with distilled water.
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Phosphate Buffer (0.2M, pH 8.0) Sodium phosphate 0.2 M was prepared by dissolving 30.2 gm sodium phosphate in 600 ml of distilled water, the pH was adjusted to 8.0 with 0.2M sodium hydroxide solution and the final volume was adjusted up to 1000 ml with distilled water. 5,5-dithiobis-2-nitrobenzoic acid (DTNB) reagent (0.6mM) DTNB reagent 60 mg was dissolved in 50 ml of buffer and the final volume was adjusted to 100 ml with buffer. Standard glutathione (100 µg/ml) 10 mg of glutathione was dissolved in 60 ml of distilled water and the final volume was made up to 100 ml with distilled water. Procedure Equal volumes of tissue homogenate (supernatant) and 20% TCA were mixed. The precipitated fraction was centrifuged at 2500 rpm at 4°C for 15 min and 2.0 ml of DTNB reagent was added to 0.25 ml of supernatant. The final volume was made up to 3.0 ml with phosphate buffer. The color developed was read at 412 nm against reagent blank. Different concentrations (10-50 µg) of standard glutathione were prepared and processed as above for standard graph. The amount of reduced glutathione is expressed as µg of GSH/mg protein (Morgon et al., 1979).
4.4.11.4 Assay of Superoxide Dismutase (SOD) Reagents Carbonate buffer (0.05 M, pH 10.2) Sodium bicarbonate 16.8 gm and 22 gm of sodium carbonate were dissolved in 500 ml of distilled water and the volume was made up to 1000 ml with distilled water. Ethylene diamine tetra acetic acid (EDTA) solution (0.4 M) EDTA 1.82 gm was dissolved in 200 ml of distilled water and the volume was made up to 1000 ml with distilled water. Hydrochloric acid (0.1 N) Concentrated hydrochloric acid 8.5 ml was mixed with 500 ml of distilled water and the volume was made up to 1000 ml with distilled water. Epinephrine solution (3mM) Epinephrine bitartarate 0.99 gm was dissolved in 100 ml of 0.1N hydrochloric acid and the volume was adjusted to 1000 ml with 0.1N hydrochloric acid.
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Superoxide dismutase standard (10 U/ml) SOD 1 mg (1000 U/mg) from bovine liver was dissolved in 100 ml of carbonate buffer. Procedure Liver tissue homogenate (0.5 ml) was diluted with (0.5 ml) distilled water, to which 0.25 ml of ice-cold ethanol and 0.15 ml of ice-cold chloroform, were added. The mixture was mixed well using cyclo mixer and centrifuged at 2500 rpm at 4°C for 15 min. To 0.5 ml of supernatant, 1.5 ml of carbonate buffer and 0.5 ml of EDTA solution were added. The reaction was initiated by the addition of 0.4 ml of epinephrine and the change in optical density/min was measured at 480 nm against reagent blank. Calibration curve was prepared by using 10-125 units of SOD. Change in optical density per minute at 50% inhibition of epinephrine to adrenochrome transition by the enzyme is taken as the enzyme unit. SOD activity was expressed as units/mg protein (Misra and Fridovich., 1972).
4.4.12 Histopathological analysis of ankle joints On day 28, ankle joints were separated from the hind paw and immersed in 10% buffered formalin for 24 h followed by decalcification in 5% formic acid, processed for paraffin embedding sectioned at 5µ thickness. The sections were stained with haematoxylin-eosin and evaluated under light microscope with 10X magnifications for the presence of inflammatory cells, hyperplasia of synovium, pannus formation and destruction of joint space (Patil et al., 2012).
4.5 Statistical analysis The data of pharmacological experiments were expressed as mean ± standard error mean (SEM). Data analysis was performed using Graph Pad Prism 5.0 software (Graph Pad, San Diego, CA, USA). Data of change in paw volume, change in joint diameter, pain threshold, mechanical nociceptive threshold, paw withdrawal latency and body weight were analyzed by Two-way analysis of variance (ANOVA) followed by Bonferroni post hoc test. Data of hematological, serum, biochemical and antioxidant parameters were analyzed using One way analysis of variance (ANOVA) followed by Dunnett’s test. A value of P<0.05 was considered to be statistically significant.
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5.1 Part A (Analgesic, anti-inflammatory and antiarthritic activity of Cyathocline purpurea extracts)
5.1.1 Phytochemical analysis 5.1.1.1 Percent yield and characteristics of different extracts of Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze The percent yields of petroleum ether extract of Cyathocline purpurea (PECP), methanol extract of Cyathocline purpurea (MECP) and aqueous extract of Cyathocline purpurea (AECP) were 3.3 %, 6.7 % and 7.7 % respectively (Table 8).
Table 8- The percent yield and characteristics of different extracts of Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze. Sr. Plant Extracts Appearance Consistency Yield % No. 1. Cyathocline PECP Pale yellow Semisolid 3.3 2. purpurea (Buch- MECP Dark Green Semisolid 6.7 Ham ex D. Don.) 3. AECP Dark Brown Semisolid 7.7 Kuntze.
5.1.1.2 Qualitative phytochemical analysis of PECP, MECP and AECP The qualitative phytochemical analysis indicated that the PECP contained alkaloids, flavonoids, tannins and phenolic compounds. The MECP contained glycosides, steroids, alkaloids, flavonoids, tannins, saponins, phenolic compounds and triterpenoids. The AECP contained carbohydrates, proteins, amino acids, glycosides, alkaloids, and phenolic compounds (Table 9).
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Table 9- Qualitative phytochemical analysis of PECP, MECP and AECP
Sr. Test PECP MECP AECP No.
1 Glycosides - + + 2 Proteins - - + 3 Carbohydrates - - + 4 Amino acids - - + 5 Steroids - + - 6 Alkaloids + + + 7 Flavonoids + + - 8 Tannins + + - 9 Triterpenoids - + - 10 Phenols + + + 11 Saponins - + - (+ present, - absent)
PECP- Petroleum ether extract of Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze MECP- Methanolic extract of Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze AECP- Aqueous extract of Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze
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5.1.2 Acute toxicity test (AOT) Administration of 2000 mg/kg, p.o. of all the three extracts of Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze. did not produce any behavioral abnormalities and mortality (Table 10). So the dose selected for further study was 100, 200 and 400 mg/kg, p.o. for each extracts.
Table 10- Acute toxicity test of PECP, MECP and AECP
Extracts No. of animals Sr. No. 2000 mg/kg, p.o. dead/survived 1. PECP 0/5 2. MECP 0/5 3. AECP 0/5
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5.1.3 Pharmacological assessment
5.1.3.1 Analgesic activity 5.1.3.1.1 Effect of oral administration of Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze. on hot plate test in mice In hot plate test, pentazocine (5 mg/kg, s.c.) significantly (p<0.001) increased the paw withdrawal latency at 60 and 90 minutes. Onset of action was observed at 60 minutes of administration of pentazocine. However, all the extracts (PECP, MECP and AECP) did not inhibit pain produced by thermal means at the doses of 100, 200 and 400 mg/kg (Table 11).
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Table 11- Effect of oral administration of Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze. on hot plate test in mice
Paw withdrawal latency (Sec) Treatment 150 180 Groups 0 min 30 min 60 min 90 min 120 min min min Vehicle 6.02 ± 5.45 ± 5.50 ± 4.83 ± 5.65 ± 5.83 ± 5.68 ± Control 0.45 0.44 0.62 0.21 0.55 0.41 0.58 Pentazocine 5.65 ± 5.90 ± 9.15 ± 11.33 ± 7.98 ± 6.08 ± 5.87 ± (5 mg/kg) 0.50 0.38 0.50*** 0.36*** 0.40** 0.58 0.40 PECP (100 5.95 ± 5.03 ± 4.63 ± 6.10 ± 4.35 ± 5.27 ± 5.10 ± mg/kg) 0.58 0.38 0.67 0.49 0.21 0.58 0.35 PECP (200 4.73 ± 5.58 ± 4.60 ± 5.30 ± 5.77 ± 5.82 ± 5.78 ± mg/kg) 0.53 0.57 0.49 0.24 0.58 0.38 0.21 PECP (400 6.50 ± 4.98 ± 5.63 ± 6.15 ± 5.92 ± 5.55 ± 5.22 ± mg/kg) 0.40 0.51 0.46 0.51 0.32 0.37 0.60 MECP (100 4.90 ± 5.43 ± 5.42 ± 5.80 ± 5.38 ± 5.80 ± 5.97 ± mg/kg) 0.39 0.54 0.39 0.44 0.27 0.38 0.69 MECP (200 5.22 ± 5.35 ± 5.35 ± 5.80 ± 6.40 ± 5.20 ± 5.27 ± mg/kg) 0.38 0.40 0.49 0.51 0.47 0.30 0.54 MECP (400 5.38 ± 5.85 ± 5.12 ± 5.62 ± 5.62 ± 5.17 ± 5.72 ± mg/kg) 0.43 0.45 0.36 0.57 0.57 0.33 0.47 AECP (100 5.18 ± 4.55 ± 5.02 ± 5.65 ± 5.28 ± 5.55 ± 5.60 ± mg/kg) 0.58 0.53 0.34 0.37 0.60 0.26 0.36 AECP (200 5.30 ± 5.55 ± 6.02 ± 5.32 ± 6.02 ± 5.17 ± 5.33 ± mg/kg) 0.40 0.30 0.34 0.26 0.47 0.30 0.41 AECP (400 5.62 ± 5.32 ± 5.32 ± 5.15 ± 5.68 ± 5.30 ± 5.55 ± mg/kg) 0.44 0.40 0.25 0.30 0.45 0.23 0.51
Values are expressed as mean ± S.E.M.; n=6 mice per group. Two way ANOVA followed by Bonferroni post hoc test when compared with vehicle control **p<0.01, ***p<0.001.
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5.1.3.1.2 Effect of oral administration of Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze. on acetic acid induced writhing in mice MECP (200 and 400 mg/kg) significantly (p<0.05 and p<0.001, respectively) reduced the number of wriths induced by 0.6% acetic acid at the dose of 10 ml/kg. Also PECP (400 mg/kg) showed a significant (p<0.05) reduction in number of wriths when compared to vehicle control group. While MECP (100 mg/kg), PECP (100 and 200 mg/kg) and AECP (100, 200 and 400 mg/kg) showed non-significant reduction in writhing. The number of wriths in the acetic acid vehicle control group was found to be 68 ± 1.5. Acetylsalicylic acid (100 mg/kg) appears to be more effective in reducing the number of wriths, it significantly (p<0.001) reduced the number of wriths by 64.71%. (Table 12).
Table 12- Effect of oral administration of Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze. on acetic acid induced writhing in mice Number of Percentage Treatment groups writhing inhibition Vehicle control 68 ± 1.5 -
Acetyl salicylic acid (100 mg/kg) 24 ± 2.1*** 64.71
PECP (100 mg/kg) 67 ± 2.0 1.47
PECP (200 mg/kg) 64 ± 3.7 5.88
PECP (400 mg/kg) 57 ± 3.4* 16.18
MECP (100 mg/kg) 62 ± 2.6 8.82
MECP (200 mg/kg) 56 ± 2.2* 17.65
MECP (400 mg/kg) 44 ± 3.3*** 35.29
AECP (100 mg/kg) 67 ± 2.0 1.47
AECP (200 mg/kg) 65 ± 2.5 4.41
AECP (400 mg/kg) 65 ± 3.0 4.41
Values are expressed as mean ± S.E.M.; n=6 mice per group. One way ANOVA followed by Dunnett’s test when compared with vehicle control *p<0.05, ***p<0.001.
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5.1.3.2 Anti-inflammatory activity
5.1.3.2.1 Effect of oral administration of Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze. on carrageenan induced paw edema in rats There was a gradual increase in paw volume of rats in the carrageenan control group. In the test groups, the MECP (200 and 400 mg/kg) showed a significant (p<0.001) reduction in paw volume in a dose dependent manner at 3rd and 5th h. The inhibitory effect of the MECP at (400 mg/kg) was found to be 40.92% at 3rd h and 51.47% at 5th h. However, PECP (400 mg/kg) showed significant (p<0.001) inhibition in paw volume at 5th h with 22.76% inhibition when compared to carrageenan control group. On treatment with AECP there was no significant inhibition at all the doses when compared to carrageenan control group. Diclofenac (10 mg/kg) caused significant (p<0.001) inhibition of increase in paw volume at 3rd and 5th h. The inhibitory effect of the diclofenac at 10 mg/kg was 46.84% at 3rd h and 53.99% at 5th h (Table 13).
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Table 13- Effect of oral administration of Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze. on carrageenan induced paw edema in rats
Change in paw volume (ml) Treatment 1 h 3 h 5 h
Carrageenan control 1.43 ± 0.24 2.56 ± 0.10 2.78 ± 0.07
1.10 ± 0.11 1.36 ± 0.04*** 1.28 ± 0.04*** Diclofenac (10 mg/kg) (23.14) (46.84) (53.99) 1.40 ± 0.04 2.47 ± 0.11 2.67 ± 0.11 PECP (100 mg/kg) (2.44) (3.45) (3.90) 1.34 ± 0.04 2.25 ± 0.12 2.37 ± 0.12* PECP (200 mg/kg) (6.63) (12.30) (14.59) 1.19 ± 0.05 2.09 ± 0.08* 2.14 ± 0.07*** PECP (400 mg/kg) (16.98) (18.48) (22.76) 1.23 ± 0.06 2.15 ± 0.10* 2.25 ± 0.09** MECP (100 mg/kg) (14.30) (15.94) (18.98) 1.22 ± 0.05 1.82 ± 0.09*** 1.62 ± 0.07*** MECP (200 mg/kg) (15.23) (28.82) (41.50) 1.12 ± 0.06 1.51 ± 0.04*** 1.35 ± 0.05*** MECP (400 mg/kg) (21.63) (40.92) (51.47) 1.39 ± 0.16 2.55 ± 0.18 2.74 ± 0.17 AECP (100 mg/kg) (3.02) (0.65) (1.14) 1.38 ± 0.09 2.56 ± 0.11 2.68 ± 0.12 AECP (200 mg/kg) (3.84) (0.13) (3.48) 1.40 ± 0.04 2.53 ± 0.12 2.66 ± 0.13 AECP (400 mg/kg) (2.44) (1.24) (4.20)
Values are expressed as mean ± S.E.M.; n=6 rats per group. Two way ANOVA followed by Bonferroni post hoc test when compared with carrageenan control *p<0.05, **p<0.01, ***p<0.001. The figures in parenthesis indicate the percent inhibition.
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5.1.3.2.2 Effect of oral administration of Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze. on cotton pellet induced granuloma in rats MECP (200 and 400 mg/kg) significantly (p<0.001) inhibited the granuloma formation in a dose dependent manner with (21.18% and 48.24% inhibition, respectively), when compared to vehicle control group. PECP (400 mg/kg) also significantly (p<0.01) inhibited the granuloma formation with 15.29% inhibition. However there was no significant inhibition in granuloma formation on treatment with AECP at all the doses tested. Diclofenac (10 mg/kg) also significantly (p<0.001) inhibited granuloma formation with maximum inhibition of 65.88% (Table 14)
Table 14- Effect of oral administration of Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze. on cotton pellet induced granuloma in rats
Increase in weight Treatment groups Percent inhibition of cotton pellet (mg) Vehicle Control 85 ± 3.1 - Diclofenac (10 mg/kg) 29 ± 1.2*** 65.88 PECP (100 mg/kg) 84 ± 3.5 1.18 PECP (200 mg/kg) 81 ± 1.7 4.71 PECP (400 mg/kg) 72 ± 2.5** 15.29 MECP (100 mg/kg) 79 ± 2.1 7.06 MECP (200 mg/kg) 67 ± 2.8*** 21.18 MECP (400 mg/kg) 44 ± 2.0*** 48.24 AECP (100 mg/kg) 84 ± 2.5 1.18 AECP (200 mg/kg) 85 ± 2.4 0.00 AECP (400 mg/kg) 84 ± 3.1 1.18
Values are expressed as mean ± S.E.M.; n=6 rats per group. One way ANOVA followed by Dunnett’s test when compared with vehicle control **p<0.01, ***p<0.001.
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5.1.3.2.2.1 Gastric ulcerogenic effect of oral administration of Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze. in cotton pellet induced granuloma in rats Histopathology of stomach of vehicle control group rats showed intact gastric mucosa, with no ulceration and no congestion. All the rats treated with (PECP, MECP and AECP) at dose of (400 mg/kg) showed less ulcer and absence of congestion when compared to the standard group treated with diclofenac. Diclofenac (10 mg/kg) treated rats showed ulceration and congestion (Figure 11).
Figure 11- Histopathology of stomach in cotton pellet induced granuloma in rats
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5.1.3.3 Antiarthritic activity 5.1.3.3.1 Effect of oral administration of methanol extract of Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze. on change in paw volume in arthritic rats There was significant (p<0.001) increase in paw volume of all the rats treated with FCA compared to healthy control. MECP (200 and 400 mg/kg) significantly (p<0.001) lowered the paw volume from day 20 onwards as compared to arthritic control group with maximum inhibition of (44.09% and 62.72%), respectively on day 28. MECP lower dose (100 mg/kg) was less effective, it significantly (p<0.01) lowered paw volume on day 28 with 9.97% inhibition on last day. Diclofenac (5 mg/kg) showed significant (p<0.001) reduction in paw volume from day 16 onwards with 75.32% inhibition on day 28. The change in paw volume of MECP treated (400 mg/kg; 1.42 ± 0.18) and (200 mg/kg; 2.13 ± 0.07) was evident as compared to arthritic control group (3.81 ± 0.10) on day 28 (Figure 12, Table 15) Figure 12- Effect of oral administration of methanol extract of Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze. on change in paw volume in arthritic rats
Values are expressed as mean ± SEM for six animals and analysed by Two way ANOVA followed by Bonferroni post-hoc test, **p<0.01, ***p<0.001 when compared to arthritic control #p<0.001 when compared to healthy control.
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Table 15- Effect of oral administration of methanol extract of Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze. on change in paw volume in arthritic rats
Day 1 4 8 12 16 20 24 28
Healthy 0.00 ± 0.00 ± 0.01 ± 0.00 ± 0.01± 0.01 ± 0.01 ± 0.01 ±
Control 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 Arthritic 2.82 ± 3.51 ± 3.48 ± 3.51 ± 3.66 ± 3.72 ± 3.81 ± 3.81 ±
Control 0.09# 0.11# 0.10# 0.11# 0.10# 0.10# 0.11# 0.10#
Diclofenac 2.82 ± 3.49 ± 3.45 ± 3.50 ± 3.04 ± 2.28 ± 1.48 ± 0.94 ± 5 mg/kg 0.08 0.08 0.08 0.09 0.07*** 0.09*** 0.07*** 0.06***
MECP 2.79 ± 3.50 ± 3.47 ± 3.50 ± 3.61 ± 3.65 ± 3.55 ± 3.43 ±
100 mg/kg 0.07 0.06 0.06 0.06 0.06 0.06 0.07 0.06** MECP 2.80 ± 3.50 ± 3.47 ± 3.53 ± 3.53 ± 3.22 ± 2.76 ± 2.13 ±
200 mg/kg 0.04 0.04 0.04 0.04 0.03 0.04*** 0.05*** 0.07***
MECP 2.78 ± 3.49 ± 3.47 ± 3.50 ± 3.25 ± 2.78 ± 2.05 ± 1.42 ± 400 mg/kg 0.06 0.06 0.07 0.07 0.08** 0.12*** 0.19*** 0.18***
Values are expressed as mean ± SEM for six animals and analysed by Two way ANOVA followed by Bonferroni post-hoc test, **p<0.01, ***p<0.001 when compared to arthritic control #p<0.001 when compared to healthy control.
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5.1.3.3.2 Effect of oral administration of methanol extract of Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze. on change in joint diameter in arthritic rats There was significant (p<0.001) increase in joint diameter of rats of all the groups treated with FCA compared to healthy control. MECP (200 and 400 mg/kg) significantly (p<0.01 and p<0.001, respectively) decreased the joint diameter from day 20 as compared to arthritic control group with maximum inhibition of (37.09% and 58.78%, respectively) on day 28. MECP (100 mg/kg) showed no significant decrease in joint diameter. Diclofenac (5 mg/kg) showed significant (p<0.01) decrease in joint diameter from day 16 onwards with 65.57% inhibition on day 28. The change in joint diameter of MECP treated rats (400 mg/kg; 1.39 ± 0.19) and (200 mg/kg; 2.12 ± 0.06) was evident as compared to arthritic control group (3.37 ± 0.14) on day 28 (Figure 13, Table 16) Figure 13- Effect of oral administration of methanol extract of Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze. on change in joint diameter in arthritic rats
Values are expressed as mean ± SEM for six animals and analysed by Two way ANOVA followed by Bonferroni post-hoc test, **p<0.01, ***p<0.001 when compared to arthritic control #p<0.001 when compared to healthy control. Study of Analgesic, Anti-inflammatory and Antiarthritic activity of Indian medicinal plant in laboratory animals 120
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Table 16- Effect of oral administration of methanol extract of Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze. on change in joint diameter in arthritic rats
Day 1 4 8 12 16 20 24 28
Healthy 0.01 ± 0.00 ± 0.02 ± 0.01 ± 0.01 ± 0.01 ± 0.02 ± 0.01 ±
Control 0.01 0.00 0.01 0.00 0.00 0.01 0.01 0.00 Arthritic 3.23 ± 3.28 ± 3.27 ± 3.32 ± 3.34 ± 3.36 ± 3.37 ± 3.37 ± Control 0.12# 0.12# 0.13# 0.14# 0.13# 0.14# 0.14# 0.14#
Diclofenac 3.21 ± 3.26 ± 3.25 ± 3.29 ± 2.92 ± 2.38 ± 1.90 ± 1.16 ± 5 mg/kg 0.05 0.05 0.05 0.05 0.05** 0.11*** 0.03*** 0.06*** MECP 3.18 ± 3.25 ± 3.24 ± 3.27 ± 3.29 ± 3.32 ± 3.31 ± 3.23 ±
100 mg/kg 0.09 0.09 0.09 0.09 0.09 0.09 0.09 0.10
MECP 3.20 ± 3.25 ± 3.25 ± 3.29 ± 3.29 ± 2.94 ± 2.60 ± 2.12 ± 200 mg/kg 0.06 0.06 0.06 0.05 0.06 0.05** 0.05*** 0.06***
MECP 3.22 ± 3.28 ± 3.28 ± 3.31 ± 3.13 ± 2.66 ± 2.13 ± 1.39 ±
400 mg/kg 0.08 0.10 0.10 0.10 0.10 0.10*** 0.13*** 0.19***
Values are expressed as mean ± SEM for six animals and analysed by Two way ANOVA followed by Bonferroni post-hoc test, **p<0.01, ***p<0.001 when compared to arthritic control #p<0.001 when compared to healthy control.
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5.1.3.3.3 Effect of oral administration of methanol extract of Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze. on pain threshold in arthritic rats The pain threshold of the paw in the FCA administered rats decreased progressively till day 12. MECP (200 and 400 mg/kg) significantly (p<0.001) increased the pain threshold from day 24 and day 20 respectively with maximum increase in pain threshold of 54.48% and 72.41%, respectively on day 28, where as MECP (100 mg/kg) was less effective, it significantly (p<0.01) increased the pain threshold on day 28 with increase of 29.65%. Diclofenac (5 mg/kg) used as standard significantly (p<0.001) increased pain threshold form day 20 with increase in pain threshold of 84.82% on day 28. The pain threshold of MECP (400 mg/kg; 250 ± 15.2) and (200 mg/kg; 224 ± 4.7) was evident as compared to arthritic control group (145 ± 7.9) on day 28 (Figure 14, Table 17) Figure 14- Effect of oral administration of methanol extract of Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze. on pain threshold in arthritic rats
Values are expressed as mean ± SEM for six animals and analysed by Two way ANOVA followed by Bonferroni post-hoc test, *p<0.05, **p<0.01, ***p<0.001 when compared to arthritic control #p<0.001 when compared to healthy control.
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Table 17- Effect of oral administration of methanol extract of Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze. on pain threshold in arthritic rats
Day 0 1 4 8 12 16 20 24 28
Healthy 298 ± 300 ± 307 ± 306 ± 311 ± 292 ± 292 ± 297 ± 300 ± Control 11.30 7.85 8.13 7.79 7.46 9.90 11.88 10.62 11.47 Arthritic 298 ± 195 ± 186 ± 185 ± 169 ± 165 ± 154 ± 148 ± 145 ± Control 10.46 6.19# 6.88# 7.96# 4.90# 7.07# 9.70# 7.61# 7.96# Diclofenac 294 ± 187 ± 181 ± 178 ± 173 ± 203 ± 222 ± 248 ± 268 ± 5 mg/kg 10.60 4.94 4.36 4.23 4.23 5.11** 4.94*** 5.88*** 9.89*** MECP 301 ± 190 ± 183 ± 180 ± 176 ± 173 ± 179 ± 182 ± 188 ± 100 mg/kg 11.21 7.53 6.16 5.63 4.73 5.43 5.39 7.03* 7.49** MECP 296 ± 189 ± 182 ± 175 ± 170 ± 173 ± 182 ± 202 ± 224 ± 200 mg/kg 11.58 7.79 5.11 7.16 8.06 9.20 8.82 6.28*** 4.73*** MECP 300 ± 197 ± 188 ± 181 ± 164 ± 181 ± 207 ± 223 ± 250 ± 400 mg/kg 11.40 6.01 7.39 6.38 6.38 9.44 8.43*** 10.94*** 15.22***
Values are expressed as mean ± SEM for six animals and analysed by Two way ANOVA followed by Bonferroni post-hoc test, *p<0.05, **p<0.01, ***p<0.001 when compared to arthritic control #p<0.001 when compared to healthy control.
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5.1.3.3.4 Effect of oral administration of methanol extract of Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze. on paw withdrawal latency in arthritic rats There was significant (p<0.001) decrease in paw withdrawal latency of all the rats treated with FCA compared to healthy control. MECP (200 and 400 mg/kg) significantly (p<0.01) increased the paw withdrawal latency from day 24 and day 20 respectively, where as MECP (100 mg/kg) showed very minute effect on day 28, it significantly (p<0.05) increased the paw withdrawal latency on day 28. Diclofenac (5 mg/kg) also caused a significant (p<0.001) increase in paw withdrawal latency from day 20 onwards. The paw withdrawal latency of MECP (400 mg/kg; 7.32 ± 0.25) and (200 mg/kg; 6.17 ± 0.36) was evident as compared to arthritic control group (2.90 ± 0.18) on day 28 (Figure 15, Table 18). Figure 15- Effect of oral administration of methanol extract of Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze. on paw withdrawal latency in arthritic rats
Values are expressed as mean ± SEM for six animals and analysed by Two way ANOVA followed by Bonferroni post-hoc test, *p<0.05, **p<0.01, ***p<0.001 when compared to arthritic control #p<0.001 when compared to healthy control.
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Table 18- Effect of oral administration of methanol extract of Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze. on paw withdrawal latency in arthritic rats
Day 0 1 4 8 12 16 20 24 28
Healthy 8.63 ± 8.70 ± 8.57 ± 8.80 ± 8.80 ± 8.62 ± 8.92 ± 8.62 ± 8.80 ± Control 0.62 0.35 0.65 0.58 0.59 0.84 0.43 0.35 0.57 Arthritic 8.67 ± 6.12 ± 5.45 ± 5.40 ± 4.20 ± 3.67 ± 3.13 ± 2.93 ± 2.90 ± Control 0.43 0.25# 0.33# 0.32# 0.18# 0.17# 0.17# 0.15# 0.18# Diclofenac 8.78 ± 6.32 ± 5.35 ± 5.32 ± 4.18 ± 5.07 ± 5.75 ± 6.93 ± 7.90 ± 5 mg/kg 0.56 0.36 0.38 0.36 0.39 0.33 0.42*** 0.37*** 0.39*** MECP 8.57 ± 6.48 ± 5.52 ± 5.43 ± 4.25 ± 3.95 ± 4.03 ± 4.23 ± 4.82 ± 100 mg/kg 0.56 0.46 0.49 0.51 0.49 0.61 0.41 0.39 0.43* MECP 8.80 ± 6.52 ± 5.50 ± 5.27 ± 4.22 ± 4.25 ± 4.78 ± 5.23 ± 6.17 ± 200 mg/kg 0.39 0.42 0.46 0.49 0.54 0.48 0.48 0.40** 0.36*** MECP 8.75 ± 6.47 ± 5.47 ± 5.32 ± 4.27 ± 4.65 ± 5.47 ± 6.25 ± 7.32 ± 400 mg/kg 0.30 0.32 0.33 0.30 0.24 0.25 0.40** 0.29*** 0.25***
Values are expressed as mean ± SEM for six animals and analysed by Two way ANOVA followed by Bonferroni post-hoc test, *p<0.05, **p<0.01, ***p<0.001 when compared to arthritic control #p<0.001 when compared to healthy control.
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5.1.3.3.5 Effect of oral administration of methanol extract of Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze. on mechanical nociceptive threshold in arthritic rats The mechanical nociceptive threshold was observed to be the lowest on day 12 after FCA injection. Administration of MECP (200 and 400 mg/kg) significantly (p<0.001) improved the mechanical nociceptive threshold from day 20 when compared to arthritic control group. However, there was little improvement observed in MECP (100 mg/kg) which significantly (p<0.01) increased mechanical nociceptive threshold on day 28. Diclofenac (5 mg/kg) showed significant (p<0.001) improvement in mechanical nociceptive threshold from day 16 onwards. The mechanical nociceptive threshold of MECP (400 mg/kg; 59.82 ± 1.01) and (200 mg/kg; 52.58 ± 1.76) was evident as compared to arthritic control group (24.97 ± 0.98) on day 28 (Figure 16, Table 19) Figure 16- Effect of oral administration of methanol extract of Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze. on mechanical nociceptive threshold in arthritic rats
Values are expressed as mean ± SEM for six animals and analysed by Two way ANOVA followed by Bonferroni post-hoc test, **p<0.01, ***p<0.001 when compared to arthritic control #p<0.001 when compared to healthy control.
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Table 19- Effect of oral administration of methanol extract of Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze. on mechanical nociceptive threshold in arthritic rats
Day 0 1 4 8 12 16 20 24 28
Healthy 65.07 ± 66.48 ± 64.50 ± 67.23 ± 65.77 ± 66.43 ± 66.98 ± 65.95 ± 66.15 ± Control 2.76 2.08 1.59 1.92 2.65 2.60 1.77 2.76 2.72 Arthritic 66.58 ± 28.38 ± 27.08 ± 27.05 ± 26.42 ± 25.53 ± 25.32 ± 25.27 ± 24.97 ± Control 2.12 1.34# 0.99# 0.90# 0.84# 0.77# 0.80# 0.83# 0.98# Diclofenac 67.12 ± 28.40 ± 27.85 ± 27.87 ± 26.75 ± 35.78 ± 46.20 ± 55.42 ± 63.50 ± 5 mg/kg 2.83 1.35 1.28 0.78 0.68 1.21*** 1.47*** 1.99*** 2.22*** MECP 65.48 ± 29.58 ± 28.48 ± 28.57 ± 27.53 ± 27.15 ± 27.08 ± 28.18 ± 32.83 ± 100 mg/kg 1.99 1.17 0.82 0.83 0.88 0.95 1.00 0.98 0.98** MECP 64.55 ± 30.50 ± 29.35 ± 29.02 ± 27.60 ± 27.92 ± 34.58 ± 45.83 ± 52.58 ± 200 mg/kg 2.86 0.55 0.46 0.37 0.44 0.43 1.00*** 1.92*** 1.76*** MECP 66.20 ± 28.98 ± 28.18 ± 28.02 ± 26.77 ± 33.35 ± 42.43 ± 53.77 ± 59.82 ± 400 mg/kg 2.68 0.91 0.91 0.83 0.80 1.56** 1.64*** 1.29*** 1.01***
Values are expressed as mean ± SEM for six animals and analysed by Two way ANOVA followed by Bonferroni post-hoc test, **p<0.01, ***p<0.001 when compared to arthritic control #p<0.001 when compared to healthy control.
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5.1.3.3.6 Effect of oral administration of methanol extract of Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze. on body weight in arthritic rats The rats in the arthritic control group lost body weight as compared with the MECP and diclofenac treated group. The body weight of MECP (400 mg/kg; 209 ± 4.10) and (200 mg/kg; 201 ± 5.08) was evident as compared to arthritic control group (168 ± 3.50) on day 28. The results indicate that MECP (200 and 400 mg/kg) increased the body weight by 18.93% and 23.66% respectively on day 28, while diclofenac (5 mg/kg) increased the body weight by 25.44% as compared to arthritic control group. (Figure 17, Table 20) Figure 17- Effect of oral administration of methanol extract of Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze. on body weight in arthritic rats
Values are expressed as mean ± SEM for six animals and analysed by Two way ANOVA followed by Bonferroni post-hoc test, **p<0.01, ***p<0.001 when compared to arthritic control #p<0.001 when compared to healthy control.
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Table 20- Effect of oral administration of methanol extract of Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze. on body weight in arthritic rats
Day 0 1 4 8 12 16 20 24 28
Healthy 207 ± 208 ± 211 ± 215 ± 219 ± 225 ± 228 ± 232 ± 238 ± Control 3.85 3.64 3.75 3.55 3.75 2.54 2.89 2.91 2.70 Arthritic 203 ± 202 ± 196 ± 189 ± 185 ± 181 ± 177 ± 173 ± 169 ± Control 4.06 4.03 3.58 4.00# 3.94# 4.12# 4.28# 3.53# 3.50# Diclofenac 204 ± 203 ± 198 ± 193 ± 188 ± 190 ± 199 ± 205 ± 212 ± 5 mg/kg 6.04 6.20 6.37 5.95 6.15 5.94 5.49** 5.81*** 6.01*** MECP 201 ± 200 ± 196 ± 191 ± 186 ± 182 ± 179 ± 180 ± 185 ± 100 mg/kg 4.94 5.19 5.11 5.00 5.08 4.78 5.00 4.97 5.16 MECP 209 ± 207 ± 202 ± 197 ± 191 ± 191 ± 193 ± 197 ± 201 ± 200 mg/kg 4.82 4.82 4.65 4.50 4.79 4.98 5.34 5.17** 5.08*** MECP 203 ± 201 ± 197 ± 192 ± 188 ± 189 ± 194 ± 200 ± 209 ± 400 mg/kg 3.30 3.39 3.32 3.35 3.63 3.62 3.71 3.79*** 4.10***
Values are expressed as mean ± SEM for six animals and analysed by Two way ANOVA followed by Bonferroni post-hoc test, **p<0.01, ***p<0.001 when compared to arthritic control #p<0.001 when compared to healthy control.
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5.1.3.3.7 Radiological analysis of ankle joints As shown in Figure 18, FCA injected rats had developed definite joint space narrowing of the inter-tarsal joints, diffuse soft tissue swelling, cystic enlargement of bone and extensive erosions. Arthritic control group rats (Figure 18B) suffered from more pronounced bone destruction than MECP (400 mg/kg) (Figure 18F) and diclofenac (5 mg/kg) (Figure 18C) treated groups. MECP (200 mg/kg) showed moderate effect (Figure 18E), while MECP (100 mg/kg) showed no obvious effect (Figure 18D).
Figure 18- Radiological analysis of ankle joints. (A) Healthy control (B) Arthritic control (C) Diclofenac 5 mg/kg treated (D) MECP 100 mg/kg treated (E) MECP 200 mg/kg treated (F) MECP 400 mg/kg treated.
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5.1.3.3.8 Haematological parameters 5.1.3.3.8.1 Effect of oral administration of methanol extract of Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze. on blood haemoglobin count in arthritic rats The challenge with FCA (0.1ml) showed significant (p<0.001) decrease in the haemoglobin count when compared to healthy control which was found to be 9.4 ± 0.28 gm/dl. The treatment with diclofenac (5 mg/kg) showed significant (p<0.001) increase in the haemoglobin count by 50.00% as compared to arthritic control group. MECP (200 and 400 mg/kg) showed significant (p<0.001) increase in the haemoglobin count by 29.78% and 40.42%, respectively. MECP (100 mg/kg) also showed significant (p<0.05) elevation in the haemoglobin count by 14.89% when compared to arthritic control group. (Figure 19, Table 21)
Figure 19- Effect of oral administration of methanol extract of Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze. on blood haemoglobin count in arthritic rats
20
15 *** *** *** # * 10
Hb Hb (gm/dl) 5
0 l o l tr ro g g n t /k g g /k o n g k /k g C o / g y C m g m h c 5 m m 00 lt ti c 0 0 4 a ri a 0 20 e h n 1 P H rt fe P P C o C C E A l E E M ic M D M
Values are expressed as mean ± S.E.M.; n=6 rats per group. One way ANOVA followed by Dunnett’s test when compared with arthritic control *p<0.05, ***p<0.001 and when arthritic control compared with healthy control #p<0.001.
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Table 21- Effect of oral administration of methanol extract of Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze. on blood haemoglobin count in arthritic rats
Groups Hb (gm/dl) Healthy control 14.3 ± 0.23 Arthritic control 9.4 ± 0.28# Diclofenac 5 mg/kg 14.1± 0.35*** MECP 100 mg/kg 10.8 ± 0.40* MECP 200 mg/kg 12.2 ± 0.26*** MECP 400 mg/kg 13.2 ± 0.39***
Values are expressed as mean ± S.E.M.; n=6 rats per group. One way ANOVA followed by Dunnett’s test when compared with arthritic control *p<0.05, ***p<0.001 and when arthritic control compared with healthy control #p<0.001.
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5.1.3.3.8.2 Effect of oral administration of methanol extract of Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze. on blood WBC count in arthritic rats The challenge with FCA (0.1ml) showed significant (p<0.001) elevation in the WBC count when compared to healthy control group which was found to be 14.2 ± 0.25 thousands/mm3. Diclofenac (5 mg/kg) showed significant (p<0.001) decrease in the WBC count by 40.42% when compared to arthritic control group. The treatment with MECP (200 and 400 mg/kg) showed significant (p<0.001) decrease in the WBC count by 19.01% and 31.90% respectively, when compared to arthritic control group. However, MECP (100 mg/kg) showed non-significant decrease in WBC count. (Figure 20, Table 22)
Figure 20- Effect of oral administration of methanol extract of Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze. on blood WBC count in arthritic rats
20 ) 3
15 # *** *** 10 ***
5
WBC (thousands/mm 0 l l o o tr tr g g n n /k g g /k o o g /k /k g C C m g g m y c 5 m m 0 h ti c 0 0 0 lt ri a 0 0 4 a h n 1 2 P e rt fe P P C H o C C E A l E E M ic M D M
Values are expressed as mean ± S.E.M.; n=6 rats per group. One way ANOVA followed by Dunnett’s test when compared with arthritic control ***p<0.001 and when arthritic control compared with healthy control #p<0.001.
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Table 22- Effect of oral administration of methanol extract of Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze. on blood WBC count in arthritic rats
Groups WBC (thousands/mm3) Healthy control 7.38 ± 0.33 Arthritic control 14.2 ± 0.25# Diclofenac 5 mg/kg 8.46 ± 0.25*** MECP 100 mg/kg 13.4 ± 0.64 MECP 200 mg/kg 11.5 ± 0.47*** MECP 400 mg/kg 9.67 ± 0.24***
Values are expressed as mean ± S.E.M.; n=6 rats per group. One way ANOVA followed by Dunnett’s test when compared with arthritic control ***p<0.001 and when arthritic control compared with healthy control #p<0.001.
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5.1.3.3.8.3 Effect of oral administration of methanol extract of Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze. on blood RBC count in arthritic rats The challenge with FCA (0.1ml) showed significant (p<0.001) decrease in the RBC count when compared to healthy control group which was found to be 3.50 ± 0.20 million/mm3. Diclofenac (5 mg/kg) showed significant (p<0.001) elevation in the RBC count by 74.28% when compared to arthritic control group. The treatment with MECP (200 and 400 mg/kg) showed significant (p<0.01 and p<0.001, respectively) elevation in the RBC count by 42.85% and 65.71%, respectively as compared to arthritic control group. However, MECP (100 mg/kg) showed non-significant increase in RBC count. (Figure 21, Table 23)
Figure 21- Effect of oral administration of methanol extract of Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze. on blood RBC count in arthritic rats
8
) *** 3 6 *** ** 4 #
2 RBC (millions/mm 0 l l o o tr tr g g n n /k g g /k o o g /k /k g C C m g g m y c 5 m m 0 h ti c 0 0 0 lt ri a 0 0 4 a h n 1 2 P e rt fe P P C H o C C E A l E E M ic M D M
Values are expressed as mean ± S.E.M.; n=6 rats per group. One way ANOVA followed by Dunnett’s test when compared with arthritic control **p<0.01, ***p<0.001 and when arthritic control compared with healthy control #p<0.001.
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Table 23- Effect of oral administration of methanol extract of Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze. on blood RBC count in arthritic rats
Groups RBC (millions/mm3) Healthy control 6.80 ± 0.26 Arthritic control 3.50 ± 0.20# Diclofenac 5 mg/kg 6.10 ± 0.26*** MECP 100 mg/kg 3.80 ± 0.09 MECP 200 mg/kg 5.00 ± 0.34** MECP 400 mg/kg 5.80 ± 0.42***
Values are expressed as mean ± S.E.M.; n=6 rats per group. One way ANOVA followed by Dunnett’s test when compared with arthritic control **p<0.01, ***p<0.001 and when arthritic control compared with healthy control #p<0.001.
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5.1.3.3.8.4 Effect of oral administration of methanol extract of Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze. on blood platelet count in arthritic rats The challenge with FCA (0.1ml) showed significant (p<0.001) elevation in the platelet count when compared to healthy control group which was found to be 1734 ± 40 thousands/mm3. Diclofenac (5 mg/kg) showed significant (p<0.001) decrease in the platelet count by 37.08% when compared to arthritic control group. The treatment with MECP (200 and 400 mg/kg) also showed significant (p<0.001) decrease in the platelet count by 19.78% and 31.37% respectively when compared to arthritic control group. However, MECP (100 mg/kg) showed no significant change in platelet count when compared to arthritic control group. (Figure 22, Table 24)
Figure 22- Effect of oral administration of methanol extract of Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze. on blood platelet count in arthritic rats
2000 )
3 # *** 1500 *** *** 1000
500
Platelets (thousand/mm Platelets 0 l l o o tr tr g g n n /k g g /k o o g /k /k g C C m g g m y c 5 m m 0 h ti c 0 0 40 lt ri a 0 0 a h n 1 2 P e rt fe P P C H o C C E A l E E M ic M D M
Values are expressed as mean ± S.E.M.; n=6 rats per group. One way ANOVA followed by Dunnett’s test when compared with arthritic control ***p<0.001 and when arthritic control compared with healthy control #p<0.001.
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Table 24- Effect of oral administration of methanol extract of Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze. on blood platelet count in arthritic rats
Platelets Groups (thousands/mm3) Healthy control 903 ± 20 Arthritic control 1734 ± 40# Diclofenac 5 mg/kg 1091 ± 49*** MECP 100 mg/kg 1626 ± 57 MECP 200 mg/kg 1391 ± 46*** MECP 400 mg/kg 1190 ± 49***
Values are expressed as mean ± S.E.M.; n=6 rats per group. One way ANOVA followed by Dunnett’s test when compared with arthritic control ***p<0.001 and when arthritic control compared with healthy control #p<0.001.
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5.1.3.3.8.5 Effect of oral administration of methanol extract of Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze. on serum CRP level in arthritic rats The serum CRP level of the healthy control group was 1.65 ± 0.14 mg/lit, which was significantly (p<0.001) increased in arthritic control group and found to be 7.05 ± 0.26 mg/lit. Diclofenac (5 mg/kg) showed significant (p<0.001) decrease in serum CRP level by 59.14% when compared to arthritic control group. Treatment with MECP (200 and 400 mg/kg) exhibited a significant (p<0.001) decrease in serum CRP level by 27.65% and 44.39% respectively when compared to arthritic control group. MECP (100 mg/kg) showed significant (p<0.05) decrease in serum CRP level by 14.04%. (Figure 23, Table 25)
Figure 23- Effect of oral administration of methanol extract of Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze. on serum CRP level in arthritic rats
8 # * 6 ***
4 *** ***
CRP CRP (mg/lit) 2
0 l l ro ro g g t t /k k kg g n n g / / /k o o g g g C C m m m y c 5 0 0 m h ti c 0 0 0 t i a 1 2 0 al r n P 4 th e P P e r f C C C H A lo E E E ic M M M D
Values are expressed as mean ± S.E.M.; n=6 rats per group. One way ANOVA followed by Dunnett’s test when compared with arthritic control *p<0.05, ***p<0.001 and when arthritic control compared with healthy control #p<0.001.
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Table 25- Effect of oral administration of methanol extract of Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze. on serum CRP level in arthritic rats
Groups CRP (mg/lit) Healthy control 1.65 ± 0.14 Arthritic control 7.05 ± 0.26# Diclofenac 5 mg/kg 2.88 ± 0.17*** MECP 100 mg/kg 6.03 ± 0.31* MECP 200 mg/kg 5.10 ± 0.31*** MECP 400 mg/kg 3.92 ± 0.30***
Values are expressed as mean ± S.E.M.; n=6 rats per group. One way ANOVA followed by Dunnett’s test when compared with arthritic control *p<0.05, ***p<0.001 and when arthritic control compared with healthy control #p<0.001.
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5.1.3.3.8.6 Effect of oral administration of methanol extract of Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze. on RF value in arthritic rats The serum RF value of the arthritic control group was 57 ± 1.20 IU/ml. Diclofenac (5 mg/kg) showed significant (p<0.001) decrease in RF value by 40.35% when compared to arthritic control group. Treatment with MECP (200 and 400 mg/kg) exhibited a significant (p<0.001) decrease in RF value by 15.78% and 26.31% respectively when compared to arthritic control group. MECP (100 mg/kg) also showed significant (p<0.05) decrease in RF value by 7.01%. (Figure 24, Table 26)
Figure 24- Effect of oral administration of methanol extract of Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze. on RF value in arthritic rats
80
# 60 * *** *** 40 *** RF (IU/ml) 20
0 l o tr g g g n /k g k /k o g /k / g C m g g m 5 m m 0 ic c 0 0 0 it a 0 0 4 r n 1 2 P th e P P C r f C C E A lo E E ic M M D M
Values are expressed as mean ± S.E.M.; n=6 rats per group. One way ANOVA followed by Dunnett’s test when compared with arthritic control *p<0.05, ***p<0.001.
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Table 26- Effect of oral administration of methanol extract of Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze. on RF value in arthritic rats
Groups RF (IU/ml) Arthritic control 57 ± 1.20 Diclofenac 5 mg/kg 34 ± 0.91*** MECP 100 mg/kg 53 ± 1.10* MECP 200 mg/kg 48 ± 1.30*** MECP 400 mg/kg 42 ± 0.81***
Values are expressed as mean ± S.E.M.; n=6 rats per group. One way ANOVA followed by Dunnett’s test when compared with arthritic control *p<0.05, ***p<0.001.
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5.1.3.3.9 Biochemical parameters 5.1.3.3.9.1 Effect of oral administration of methanol extract of Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze. on serum AST level in arthritic rats The serum AST level of the healthy control group was 42 ± 2.3 U/L, which was significantly (P<0.001) increased in arthritic control group and found to be 127 ± 4.4 U/L. Treatment with diclofenac (5 mg/kg) caused significant (p<0.001) decrease in serum AST level by 53.54% when compared to arthritic control group. Treatment with MECP (200 and 400 mg/kg) exhibited a significant (p<0.01 and p<0.001, respectively) decrease in serum AST level which was 18.89% and 37.79% respectively when compared to arthritic control group. However, MECP (100 mg/kg) showed no significant decrease in serum AST level. (Figure 25, Table 27)
Figure 25- Effect of oral administration of methanol extract of Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze. on serum AST level in arthritic rats
150 #
* ** 100 *** ***
AST (U/L) AST 50
0 l l o ro g tr t k g n n / kg /k g o o g / g /k C C m g m g y c 5 m 0 m h ti c 0 0 0 lt ri a 0 2 0 a h n 1 P 4 e t e P P r f C C C H A lo E E E ic M D M M
Values are expressed as mean ± S.E.M.; n=6 rats per group. One way ANOVA followed by Dunnett’s test when compared with arthritic control **p<0.01, ***p<0.001 and when arthritic control compared with healthy control #p<0.001.
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Table 27- Effect of oral administration of methanol extract of Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze. on serum AST level in arthritic rats
Groups AST (U/L) Healthy control 42 ± 2.3 Arthritic control 127 ± 4.4# Diclofenac 5 mg/kg 59 ± 3.3*** MECP 100 mg/kg 110 ± 6.8 MECP 200 mg/kg 103 ± 2.6** MECP 400 mg/kg 79 ± 5.5***
Values are expressed as mean ± S.E.M.; n=6 rats per group. One way ANOVA followed by Dunnett’s test when compared with arthritic control group **p<0.01, ***p<0.001 and when arthritic control group compared with healthy control group #p<0.001.
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5.1.3.3.9.2 Effect of oral administration of methanol extract of Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze. on serum ALT level in arthritic rats The serum ALT level of the healthy control group was 42 ± 1.7 U/L, which was significantly (p<0.001) increased in arthritic control group and found to be 173 ± 4.8 U/L. Treatment with diclofenac (5 mg/kg) caused significant (p<0.001) decrease in serum ALT level by 67.05% when compared to arthritic control group. Treatment with MECP (200 and 400 mg/kg) exhibited a significant (p<0.001) decrease in serum ALT level which was 28.32% and 63.58% respectively when compared to arthritic control group. However, MECP (100 mg/kg) showed non-significant decrease in serum ALT level. (Figure 26, Table 28)
Figure 26- Effect of oral administration of methanol extract of Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze. on serum ALT level in arthritic rats
200 #
150 *** 100 *** ALT (U/L) ALT *** 50
0 l l o ro g tr t k g g n n / k /k g o o g / g /k C C m g m g y c 5 m 0 m h ti c 0 0 0 lt ri a 0 2 0 a h n 1 P 4 e t e P P r f C C C H A lo E E E ic M D M M
Values are expressed as mean ± S.E.M.; n=6 rats per group. One way ANOVA followed by Dunnett’s test when compared with arthritic control ***p<0.001 and when arthritic control compared with healthy control #p<0.001.
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Table 28- Effect of oral administration of methanol extract of Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze. on serum ALT level in arthritic rats
Groups ALT (U/L) Healthy control 42 ± 1.7 Arthritic control 173 ± 4.8# Diclofenac 5 mg/kg 57 ± 2.0*** MECP 100 mg/kg 162 ± 2.7 MECP 200 mg/kg 124 ± 3.5*** MECP 400 mg/kg 63 ± 2.3***
Values are expressed as mean ± S.E.M.; n=6 rats per group. One way ANOVA followed by Dunnett’s test when compared with arthritic control ***p<0.001 and when arthritic control compared with healthy control #p<0.001.
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5.1.3.3.9.3 Effect of oral administration of methanol extract of Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze. on serum alkaline phosphatase level in arthritic rats The serum ALP level of the healthy control group was 73 ± 3.3 U/L, which was significantly (p<0.001) increased in arthritic control group and found to be 475 ± 16 U/L. Treatment with diclofenac (5 mg/kg) showed significant (p<0.001) decrease in serum ALP level by 73.26% when compared to arthritic control group. MECP (200 and 400 mg/kg) also exhibited a significant (p<0.001) decrease in serum ALP level by 26.94% and 57.26% respectively when compared to arthritic control group. (Figure 27, Table 29)
Figure 27- Effect of oral administration of methanol extract of Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze. on serum alkaline phosphatase level in arthritic rats
600 #
400 ***
200 *** ***
Alkaline Phosphatase (U/L) Phosphatase Alkaline 0 l l o ro g tr t k g n n / kg /k g o o g / g /k C C m g m g y c 5 m 0 m h ti c 0 0 0 lt ri a 0 2 0 a h n 1 P 4 e t e P P r f C C C H A lo E E E ic M D M M
Values are expressed as mean ± S.E.M.; n=6 rats per group. One way ANOVA followed by Dunnett’s test when compared with arthritic control ***p<0.001 and when arthritic control compared with healthy control #p<0.001.
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Table 29- Effect of oral administration of methanol extract of Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze. on serum alkaline phosphatase level in arthritic rats
Alkaline Groups phosphatase (U/L) Healthy control 73 ± 3.3 Arthritic control 475 ± 16# Diclofenac 5 mg/kg 127 ± 6.7*** MECP 100 mg/kg 444 ± 19 MECP 200 mg/kg 347 ± 14*** MECP 400 mg/kg 203 ± 5.1***
Values are expressed as mean ± S.E.M.; n=6 rats per group. One way ANOVA followed by Dunnett’s test when compared with arthritic control ***p<0.001 and when arthritic control compared with healthy control #p<0.001.
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5.1.3.3.9.4 Effect of oral administration of methanol extract of Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze. on serum total protein level in arthritic rats The serum total protein level of the healthy control group was found to be 6.6 ± 0.07 g/dl, arthritic control group showed significant (p<0.001) decrease which was found to be 5.1 ± 0.04 g/dl. Diclofenac (5 mg/kg) showed significant (p<0.001) increase by 25.49% and MECP (200 and 400 mg/kg) also showed significant (p<0.01 and p<0.001, respectively) increase by 5.88% and 17.64% in serum total protein level when compared with arthritic control group. MECP (100 mg/kg) showed no significant increase in serum total protein level. (Figure 28, Table 30)
Figure 28- Effect of oral administration of methanol extract of Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze. on serum total protein level in arthritic rats
8 *** *** 6 # **
4
2 Total Protein (g/dl) Total
0 l l o ro g tr t k g n n / kg /k g o o g / g /k C C m g m g y c 5 m 0 m h ti c 0 0 0 lt ri a 0 2 0 a h n 1 P 4 e t e P P r f C C C H A lo E E E ic M D M M
Values are expressed as mean ± S.E.M.; n=6 rats per group. One way ANOVA followed by Dunnett’s test when compared with arthritic control **p<0.01, ***p<0.001 and when arthritic control compared with healthy control #p<0.001.
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Table 30- Effect of oral administration of methanol extract of Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze. on serum total protein level in arthritic rats
Total Groups protein (g/dl) Healthy control 6.6 ± 0.07 Arthritic control 5.1 ± 0.04# Diclofenac 5 mg/kg 6.4 ± 0.05*** MECP 100 mg/kg 5.2 ± 0.02 MECP 200 mg/kg 5.4 ± 0.06** MECP 400 mg/kg 6.0 ± 0.05***
Values are expressed as mean ± S.E.M.; n=6 rats per group. One way ANOVA followed by Dunnett’s test when compared with arthritic control **p<0.01, ***p<0.001 and when arthritic control compared with healthy control #p<0.001.
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5.1.3.3.10 Antioxidant parameters 5.1.3.3.10.1 Effect of oral administration of methanol extract of Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze. on liver MDA level in arthritic rats The liver MDA level of the healthy control group was found to be 2.00 ± 0.03 n mole MDA/mg which was significantly (p<0.001) increased in arthritic control group and found to be 3.60 ± 0.03 n mole MDA/mg. Diclofenac (5 mg/kg) caused significant (p<0.001) decrease in liver MDA level by 22.22% when compared to arthritic control group. Treatment with MECP (200 and 400 mg/kg) exhibited a significant (p<0.01 and p<0.001, respectively) dose dependent decrease in liver MDA level by 5.55% and 11.11% respectively when compared to arthritic control group. However, MECP (100 mg/kg) showed no change in liver MDA level when compared to arthritic control group. (Figure 29, Table 31)
Figure 29- Effect of oral administration of methanol extract of Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze. on liver MDA level in arthritic rats
4 # ** *** 3 ***
2
1 MDA (nmole MDA/mg) (nmole MDA 0 l l o ro g tr t k g g n n / k /k g o o g / g /k C C m g m g y c 5 m 0 m h ti c 0 0 0 lt ri a 0 2 0 a h n 1 P 4 e t e P C P H r f C C A lo E E E ic M D M M
Values are expressed as mean ± S.E.M.; n=6 rats per group. One way ANOVA followed by Dunnett’s test when compared with arthritic control **p<0.01, ***p<0.001 and when arthritic control compared with healthy control #p<0.001.
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Table 31- Effect of oral administration of methanol extract of Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze. on liver MDA level in arthritic rats
Groups MDA (n mol MDA/mg) Healthy control 2.00 ± 0.03 Arthritic control 3.60 ± 0.03# Diclofenac 5 mg/kg 2.80 ± 0.04*** MECP 100 mg/kg 3.50 ± 0.04 MECP 200 mg/kg 3.40 ± 0.02** MECP 400 mg/kg 3.20 ± 0.05***
Values are expressed as mean ± S.E.M.; n=6 rats per group. One way ANOVA followed by Dunnett’s test when compared with arthritic control **p<0.01, ***p<0.001 and when arthritic control compared with healthy control #p<0.001.
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5.1.3.3.10.2 Effect of oral administration of methanol extract of Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze. on liver SOD level in arthritic rats The liver SOD level of the healthy control group was 4.80 ± 0.06 mU/mg protein which was significantly (p<0.001) decreased in arthritic control group and found to be 2.50 ± 0.03 mU/mg protein. Diclofenac (5 mg/kg) caused significant (p<0.001) increase in liver SOD level by 48.00% when compared to arthritic control group. Treatment with MECP (200 and 400 mg/kg) exhibited a significant (p<0.001) increase in liver SOD level by 20.00% and 32.00%, respectively when compared to arthritic control group. MECP (100 mg/kg) also showed a significant (p<0.05) increase in liver SOD level by 8.00% when compared to arthritic control group. (Figure 30, Table 32)
Figure 30- Effect of oral administration of methanol extract of Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze. on liver SOD level in arthritic rats
6
4 *** *** *** # * 2 SOD (mU/mg SOD protein) (mU/mg 0 l l o ro g tr t k g g n n / k /k g o o g / g /k C C m g m g y c 5 m 0 m h ti c 0 0 0 lt ri a 0 2 0 a h n 1 P 4 e t e P C P H r f C C A lo E E E ic M D M M
Values are expressed as mean ± S.E.M.; n=6 rats per group. One way ANOVA followed by Dunnett’s test when compared with arthritic control *p<0.05, ***p<0.001 and when arthritic control compared with healthy control #p<0.001.
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Table 32- Effect of oral administration of methanol extract of Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze. on liver SOD level in arthritic rats
Groups SOD (mU/mg protein) Healthy control 4.80 ± 0.06 Arthritic control 2.50 ± 0.03# Diclofenac 5 mg/kg 3.70 ± 0.03*** MECP 100 mg/kg 2.70 ± 0.04* MECP 200 mg/kg 3.00 ± 0.07*** MECP 400 mg/kg 3.30 ± 0.04***
Values are expressed as mean ± S.E.M.; n=6 rats per group. One way ANOVA followed by Dunnett’s test when compared with arthritic control *p<0.05, ***p<0.001 and when arthritic control compared with healthy control #p<0.001.
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5.1.3.3.10.3 Effect of oral administration of methanol extract of Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze. on liver GSH level in arthritic rats The liver GSH level of the healthy control group was 74 ± 2.00 mU/mg protein which was significantly (p<0.001) decreased in arthritic control group and found to be 45 ± 1.80 mU/mg protein. Treatment with diclofenac (5 mg/kg) exhibited a significant (p<0.001) increase in liver GSH level by 42.22% when compared to arthritic control group and MECP (200 and 400 mg/kg) exhibited a significant (p<0.01 and p<0.001, respectively) increase in liver GSH level by 22.22% and 35.55% when compared to arthritic control group, whereas MECP (100 mg/kg) showed non-significant increase in liver GSH level when compared to arthritic control group. (Figure 31, Table 33)
Figure 31- Effect of oral administration of methanol extract of Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze. on liver GSH level in arthritic rats
80 *** *** 60 ** # 40
20 GSH (µmol/mg protein) GSH (µmol/mg 0 l l o ro tr t kg g n n / kg /k g o o g / g /k C C m g m g y c 5 m 0 m h ti c 0 0 0 lt ri a 0 2 0 a h n 1 P 4 e rt e P C P H f C C A lo E E E ic M D M M
Values are expressed as mean ± S.E.M.; n=6 rats per group. One way ANOVA followed by Dunnett’s test when compared with arthritic control *p<0.05, **p<0.01, ***p<0.001 and when arthritic control compared with healthy control #p<0.001.
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Table 33- Effect of oral administration of methanol extract of Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze. on liver GSH level in arthritic rats
Groups GSH (µmol/mg protein) Healthy control 74 ± 2.00 Arthritic control 45 ± 1.80# Diclofenac 5 mg/kg 64 ± 2.20*** MECP 100 mg/kg 51 ± 2.10 MECP 200 mg/kg 55 ± 1.40** MECP 400 mg/kg 61 ± 2.90***
Values are expressed as mean ± S.E.M.; n=6 rats per group. One way ANOVA followed by Dunnett’s test when compared with arthritic control *p<0.05, **p<0.01, ***p<0.001 and when arthritic control compared with healthy control #p<0.001.
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5.1.3.3.11 Histopathology of ankle joint Histopathology of ankle joint of healthy control group rats showed no inflammation, with intact synovial lining and no necrosis of bone (Figure 32A). Arthritic control group rats showed massive influx of inflammatory cells, necrosis of bone, cartilage destruction and disturbed synovial lining (Figure 32B). In contrast to these pathological changes the rats treated with MECP (400 mg/kg) and diclofenac (5 mg/kg) showed significant protection against necrosis of bone with low influx of inflammatory cells and little cartilage destruction (Figure 32F and Figure 32C, respectively). MECP (200 mg/kg) treated rats showed moderate necrosis of bone with little presence of inflammatory cells and cartilage destruction (Figure 32E) and MECP (100 mg/kg) treated rats showed cartilage destruction, influx of inflammatory cells with evidence of disturbed synovial lining and necrosis of bone (Figure 32D).
Figure 32- Histopathological analysis of ankle joints stained with H&E. (A) Healthy control (B) Arthritic control (C) Diclofenac 5 mg/kg treated (D) MECP 100 mg/kg treated (E) MECP 200 mg/kg treated (F) MECP 400 mg/kg treated.
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5.2 Part B (Isolation and characterization)
5.2.1 Liquid-solid separation chromatographic technique During liquid-solid separation chromatographic technique of the 15 g of MECP, 6 fractions (F – 1 to F – 6) were prepared. Physical characteristics of these fractions were studied and presented in Table 34.
Table 34- Fractions (F – 1 to F – 6) prepared from MECP Sr. Volume Solvents ratio Label Weight No. used Petroleum ether Petroleum ether fraction 1. 100 ml x 3 0.9 gm (100 %) (F – 1) Petroleum ether: 10% acetone in petroleum 2. 100 ml x 3 1.2 gm Acetone (90:10 %) ether fraction (F – 2) Petroleum ether: 20% acetone in petroleum 3. 100 ml x 3 1.8 gm Acetone (80:20 %) ether fraction (F – 3) Petroleum ether: 30% acetone in petroleum 4. 100 ml x 3 2.9 gm Acetone (70:30 %) ether fraction (F – 4) Petroleum ether: 50% acetone in petroleum 5. 100 ml x 3 3.1 gm Acetone (50:50 %) ether fraction (F – 5) Methanol Methanol fraction 6. 100 ml x 3 3.5 gm (100 %) (F – 6)
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5.2.1.1 Anti-inflammatory activity of fractions (F – 1 to F – 6) on carrageenan induced paw edema in rats. In the fraction study (F – 1 to F – 6), the paw edema of the rats increased progressively after carrageenan injection. Fractions F – 4 and F – 5 reduced carrageenan induced inflammation significantly (p<0.001) at 3rd and 5th h. Fractions F – 2 and F – 3 also significantly (p<0.001) reduced paw edema at 5th h as compared to carrageenan control group. Fraction F – 4 was found to exert the highest anti- inflammatory activity, i.e. 30.77% inhibition at 3rd h and 47.49% inhibition of inflammation at 5th h as compared to carrageenan control group. On treatment with fraction F – 1 there was no significant inhibition while fraction F – 6 showed significant (p<0.001) anti-inflammatory activity at 5th h. All the fractions produced lower effects than that of standard drug diclofenac (Table 35)
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Table 35- Anti-inflammatory activity of fractions (F – 1 to F – 6) on carrageenan induced paw edema in rats.
Dose Change in paw volume (ml) Treatment (mg/kg, Groups p.o.) 1 h 3 h 5 h
Carrageenan control - 1.31 ± 0.10 2.30 ± 0.10 2.69 ± 0.09
1.19 ± 0.04 1.35 ± 0.04*** 1.25 ± 0.05*** Diclofenac 10 (9.29) (41.15) (53.68) F – 1 (petroleum 1.29 ± 0.04 2.22 ± 0.07 2.56 ± 0.05 100 ether fraction) (1.53) (3.34) (5.02) F – 2 (10 % acetone 1.27 ± 0.05 2.06 ± 0.07* 2.28 ± 0.03*** in petroleum ether 100 (3.18) (10.45) (15.36) fraction) F – 3 (20 % acetone 1.25 ± 0.04 2.01 ± 0.07** 2.17 ± 0.08*** in petroleum ether 100 (4.45) (12.41) (19.57) fraction) F – 4 (30 % acetone 1.22 ± 0.04 1.59 ± 0.09*** 1.41 ± 0.07*** in petroleum ether 100 (6.62) (30.77) (47.49) fraction) F – 5 (50 % acetone 1.25 ± 0.03 1.78 ± 0.06*** 1.62 ± 0.06*** in petroleum ether 100 (4.58) (22.57) (39.81) fraction) F – 6 (methanol 1.24 ± 0.03 1.99 ± 0.07** 2.11 ± 0.07*** 100 fraction) (5.34) (13.21) (21.73)
Values are expressed as mean ± SEM for six animals and analysed by Two way ANOVA followed by Bonferroni post-hoc test, *p<0.05, **p<0.01 and ***p<0.001 when compared to carrageenan control. The figures in parenthesis indicate the percent inhibition.
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5.2.2 Column chromatography of most active anti-inflammatory fraction i.e. 30% acetone in petroleum ether fraction (F – 4) During column chromatographic separation of the 2.5 g of 30% acetone in petroleum ether fraction (F – 4), 118 fractions were collected (25 ml in each 50 ml glass tube). TLC of each fraction was carried out after column chromatography. Fractions showing close resemblance were pooled together and labeled as pool P – 1 to P – 10. The pooled fractions were P – 1 (1 – 14), P – 2 (15 – 41), P – 3 (42 – 54), P – 4 (55 – 63), P – 5 (64 – 70), P – 6 (71 – 76), P – 7 (77 – 84), P – 8 (85 – 102), P – 9 (103 – 110), and P – 10 (111 – 118). Pools P – 1 to P – 9 were obtained by the eluting column with mobile phase (petroleum ether: ethyl acetate, with successive increase in percentage of ethyl acetate), whereas pool P – 10 was the remaining mass which could not be eluted with mobile phase and therefore eluted by methanol. (Table 36)
Table 36- Pools (P – 1 to P – 10) collected from 30% acetone in petroleum ether fraction (F – 4) based on TLC.
Sr. Mobile phase Fractions Pools Label Weight (mg) No. 1. 1 – 14 Pool 1 P – 1 71 2. 15 – 41 Pool 2 P – 2 83 3. Petroleum ether: 42 – 54 Pool 3 P – 3 69 4. ethyl acetate, with 55 – 63 Pool 4 P – 4 32 5. successive increase 64 – 70 Pool 5 P – 5 20 6. in percentage of 71 – 76 Pool 6 P – 6 19 7. ethyl acetate. 77 – 84 Pool 7 P – 7 21 8. 85 – 102 Pool 8 P – 8 68 9. 103 – 110 Pool 9 P – 9 25 10. Methanol 111 – 118 Pool 10 P – 10 23
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5.2.2.1 Anti-inflammatory activity of pools (P – 1 to P – 10) collected from 30% acetone in petroleum ether fraction (F – 4) in carrageenan induced paw edema The anti-inflammatory effect of pools P – 1 to P – 10 obtained from column of fraction 30% acetone in petroleum ether (F – 4) revealed that pool P – 8 was found to be the most active in reducing the inflammation (29.00% inhibition at 3rd h and 47.56% inhibition at 5th h). It significantly (p<0.001) reduced the paw edema at 3rd and 5th h. Pools P – 1 and P – 2 were also found to be active in reducing the inflammation but less active than pool P – 8. Pools P – 1 and P – 2 significantly (p<0.001) reduced paw edema at 5th h. Pools P – 7 and P – 9 significantly (p<0.001) reduced paw edema at 5th h. Treatment with other pools (P – 3, P – 4, P – 5, P – 6, P – 10) showed no significant inhibition of paw edema as compared to carrageenan control group (Table 37)
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Table 37- Anti-inflammatory activity of pools (P – 1 to P – 10) collected from 30% acetone in petroleum ether fraction (F – 4) in carrageenan induced paw edema
Change in paw volume (ml) Treatment groups 1 h 3 h 5 h
Carrageenan 1.29 ± 0.03 2.16 ± 0.04 2.53 ± 0.04 control Diclofenac (10 1.20 ± 0.03 1.41 ± 0.04*** 1.22 ± 0.03*** mg/kg) (7.60) (34.57) (51.84) 1.25 ± 0.04 1.91 ± 0.07* 1.81 ± 0.05*** P – 1 (10 mg/kg) (3.22) (11.45) (28.46) 1.25 ± 0.04 1.88 ± 0.06** 1.73 ± 0.07*** P – 2 (10 mg/kg) (3.35) (12.68) (31.62) 1.25 ± 0.01 2.02 ± 0.04 2.33 ± 0.04 P – 3 (10 mg/kg) (3.74) (6.26) (7.91) 1.27 ± 0.05 2.11 ± 0.05 2.46 ± 0.04 P – 4 (10 mg/kg) (1.80) (2.24) (2.77) 1.27 ± 0.04 2.09 ± 0.09 2.45 ± 0.08 P – 5 (10 mg/kg) (1.55) (3.09) (3.10) 1.25 ± 0.05 2.07 ± 0.11 2.39 ± 0.09 P – 6 (10 mg/kg) (3.35) (3.87) (5.47) 1.27 ± 0.03 1.91 ± 0.09* 1.76 ± 0.07*** P – 7 (10 mg/kg) (2.19) (11.45) (30.30) 1.24 ± 0.03 1.53 ± 0.04*** 1.33 ± 0.05*** P – 8 (10 mg/kg) (4.25) (29.00) (47.56) 1.28 ± 0.06 1.97 ± 0.05 1.90 ± 0.04*** P – 9 (10 mg/kg) (1.03) (8.74) (25.10) 1.27 ± 0.02 2.11 ± 0.06 2.45 ± 0.06 P – 10 (10 mg/kg) (1.93) (2.32) (3.10)
Values are expressed as mean ± SEM for six animals and analysed by Two way ANOVA followed by Bonferroni post-hoc test, *p<0.05, **p<0.01 and ***p<0.001 when compared to carrageenan control. The figures in parenthesis indicate the percent inhibition.
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5.2.3 Preparative TLC of most active anti-inflammatory pool i.e. P – 8 The pool P – 8 was subjected to preparative TLC. It was further scrapped and mixed with acetone and homogenized in vortex mixer to ensure complete elution and then filtered by Whatman filter paper; filtrate was allowed to evaporate. The pure isolated compound was labeled as P – 8 and the quantity obtained was 42 mg.
This compound was further isolated in bulk quantity to study its antiarthritic activity in Freund’s complete adjuvant induced arthritic model. Total 389 mg of pure compound was obtained from 120 gm of MECP by repeated column chromatography by the same procedure discussed above.
5.2.4 Spectral characterization of isolated compound P – 8 The chemical structure of isolated compound P – 8 was elucidated by IR, 1H-NMR, 13C-NMR, DEPT and MS. The IR (Figure 33), 1H-NMR (Figure 34), 13C-NMR (Figure 38), DEPT (Figure 41) and MS (Figure 42), of isolated compound suggested that sample is pure and contained only one compound.
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Table 38- 1H-NMR and 13C-NMR with DEPT values of isolated compound P – 8
(200 MHz and 50 MHz, respectively CDCl3, TMS as internal standard)
1H-NMR 13C-NMR with DEPT
δH Value (ppm), Proton No. Carbon No. δC Value (ppm) (Coupling Constant) H-1 3.46 dd (J=6.0,10.0 Hz) C-1 75.31 d H-2α 1.96 m C-2 36.19 t H-2β 2.25 m C-3 120.45 d H-3 5.34 br.s C-4 132.97 s H-5 1.98 m C-5 40.19 d H-6α 1.32 m C-6 26.49 t H-6β 1.96 m C-7 43.54 d H-7 3.03 m C-8 76.93 d 4.62 ddd (J=5.0,1.5,10.0 H-8 C-9 31.04 t Hz) H-9α 1.47 dd (J=6.0,1.5 Hz) C-10 35.83 s H-9β 2.59 dd (J=16.0,1.5 Hz) C-11 141.59 s H-13a 6.15 br.d (J=1.5 Hz) C-12 171.12 s H-13b 5.67 br.d (J=1.0 Hz) C-13 120.56 t H-14 0.85 s C-14 20.34 q H-15 1.62 d (J=1.5 Hz) C-15 10.46 q
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Figure 33- Infra-Red spectrum of isolated compound (P – 8)
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Figure 34- 1H-NMR spectrum of isolated compound (P – 8)
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Figure 35- First elaborated 1H-NMR spectrum of isolated compound (P – 8)
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Figure 36- Second elaborated 1H-NMR spectrum of isolated compound (P – 8)
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Figure 37- Third elaborated 1H-NMR spectrum of isolated compound (P – 8)
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Figure 38- 13C-NMR spectrum of isolated compound (P – 8)
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Figure 39- First elaborated 13C-NMR spectrum of isolated compound (P – 8)
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Figure 40- Second elaborated 13C-NMR spectrum of isolated compound (P – 8)
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Figure 41- DEPT spectrum of isolated compound (P – 8)
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Figure 42- Mass spectrum of isolated compound (P – 8)
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5.2.5 Structure assigned to isolated compound (P – 8) -1 P – 8 (C15H20O3): IR νmax (cm ) (KBr): 3630 (-OH stretching), 2996 (Aliphatic), 1772 (-C=O stretching), 1153 (C-O stretching) (Figure 33). MS, m/z: 271.03 [M-23]-. (Figure 42). 1 H-NMR (CDCl3, 200 MHz): δ 3.46 (1H, dd, J=6.0, 10.0 Hz, H-1), 1.96 (1H, m, H- 2α), 2.25 (1H, m, H-2β), 5.34 (1H, br.s, H-3), 1.98 (1H, m, H-5), 1.32 (1H, m, H-6α), 1.96 (1H, m, H-6β), 3.03 (1H, m, H-7), 4.62 (1H, ddd, J=5.0, 1.5, 10.0 Hz, H-8), 1.47 (1H, dd, J=6.0, 1.5Hz, H-9α), 2.59 (1H, dd, J=16.0, 1.5 Hz, H-9β), 6.15 (1H, br.d, J=1.5 Hz, H-13a), 5.67 (1H, br.d, J=1.0 Hz, H-13b), 0.85 (3H, s, H-14), and 1.62 (3H, d, J=1.5 Hz, H-15). (Figure 34). 13 C-NMR and DEPT (CDCl3, 50 MHz): δ 75.31 (d, C-1), 36.19 (t, C-2), 120.45 (d, C- 3), 132.97 (s, C-4), 40.19 (d, C-5), 26.49 (t, C-6), 43.54 (d, C-7), 76.93 (d, C-8), 31.04 (t, C-9), 35.83 (s, C-10), 141.59 (s, C-11), 171.12 (s, C-12), 120.56 (t, C-13), 20.34 (q, C-14), and 10.46 (q, C-15). (Figure 38 and 42). The structure and stereochemistry was established on the basis of above spectral data and comparison with the reported spectral data in the literature (Nagasampagi et al., 1981). This compound was found to be Isoivangustin, a sesquiterpene lactone (Figure 43).
8 9 1 7
2 6 3 5 4
Figure 43- Isoivangustin
IUPAC name: (3aS,4aS,8R,8aR,9aR)-8-hydroxy-5,8a-dimethyl-3-methylene- 3,3a,4,4a,8,8a,9,9a-octahydronaphthol[2,3-b]furan-2(7H)-one
Molecular formula: C15H20O3 Category: Sesquiterpene lactone Melting point: 139°C - 140°C.
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5.2.6 Docking study Docking studies were carried out to study the binding mode of the isolated compound, isoivangustin on the active site of TACE. The docking score of native ligand IH6, diclofenac and isoivangustin was found to be -7.432, -7.358 and -5.341 respectively. TACE converts membrane bound pro-TNF-α to mature and soluble TNF-α. The native ligand IH6 was successfully docked into the active site of TACE. Hydroxamate group of compound IH6 forms van der Waals interaction with zinc, the co-catalytic metal ion in the active site of the enzyme (Figure 44). The compound IH6 actively takes part in forming hydrogen bond interaction with the key amino acids Gly349 and Leu348 in the enzyme protein. The phenyl ring forms an interaction with amino acid His405 by П-П stacking. Furthermore, the compound is surrounded with residues, such as Ala439, Leu348, Val434, Tyr436, His415, Ilu438 and Pro437 in the enzyme and makes contacts through van der Waals interactions with these amino acids and the docking score of compound IH6 with TACE was -7.432. Also, the binding studies of diclofenac with TNF-α were studied and it was found that it forms the van der Waals interaction with zinc and show П-П stacking with amino acid HIS405. The docking score for diclofenac with TACE was -7.358 (Figure 45) Docking analysis of isoivangustin at the active site of TACE showed hydrogen binding with amino acid Gly349 and Leu348 like in native ligand IH6 which showed hydrogen bonding with same amino acids. Also, the octahydronapthyl ring fits into hydrophobic pocket formed by amino acid His405 in the enzyme. The docking score of isoivangustin with TACE enzyme was -5.341, which shows that it has good binding interaction with active site of TACE (Figure 46) Validation of docking procedure: In order to validate our docking procedure, we eliminated the co-crystallized ligand IH6 from the active site, and redocked within the inhibitor binding cavity of TACE enzyme. In this study, the root mean square deviation value was below 2Å, showing that our docking method is valid for the inhibitors studied.
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Table 39- Docking score of native ligand IH6 on active site of TACE
Compound ID Docking score Native ligand IH6 -7.432
Figure 44- 3D binding of native ligand IH6 on active site of TACE
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Table 40- Docking score of diclofenac on active site of TACE
Compound ID Docking score Diclofenac -7.358
Figure 45- 3D binding of diclofenac on active site of TACE
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Table 41- Docking score of isoivangustin on active site of TACE
Compound ID Docking score Isoivangustin -5.341
Figure 46- 3D binding of isoivangustin on active site of TACE
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5.3 Part C (Antiarthritic activity of isolated compound, Isoivangustin)
5.3.1 Effect of oral administration of isoivangustin on change in paw volume in arthritic rats There was significant (p<0.001) increase in paw volume of all the rats treated with FCA compared to healthy control. Isoivangustin (5 and 10 mg/kg) significantly (p<0.001) lowered the paw volume with 43.09% and 73.48%, respectively on day 28. Isoivangustin (2.5 mg/kg) was less effective, it significantly (p<0.01) lowered paw volume on day 28 with 6.90%. Diclofenac (5 mg/kg) showed most significant (p<0.001) reduction in paw volume from day 16 onwards with 74.03% reduction on day 28. The change in paw volume of isoivangustin treated (10 mg/kg; 0.96 ± 0.07) and (5 mg/kg; 2.06 ± 0.04) was evident as compared to arthritic control group (3.62 ± 0.09) on day 28 (Figure 47, Table 42). Figure 47- Effect of oral administration of isoivangustin on change in paw volume in arthritic rats
Values are expressed as mean ± SEM for six animals and analysed by Two way ANOVA followed by Bonferroni post-hoc test, **p<0.01, ***p<0.001 when compared to arthritic control #p<0.001 when compared to healthy control.
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Table 42- Effect of oral administration of isoivangustin on change in paw volume in arthritic rats
Day 1 4 8 12 16 20 24 28
Healthy 0.00 ± 0.00 ± 0.00 ± 0.01 ± 0.01 ± 0.01 ± 0.01 ± 0.01 ±
control 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
Arthritic 2.74 ± 3.31 ± 3.29 ± 3.38 ± 3.45 ± 3.52 ± 3.59 ± 3.62 ± control 0.08# 0.09# 0.09# 0.09# 0.10# 0.10# 0.09# 0.09#
Diclofenac 2.75 ± 3.33 ± 3.32 ± 3.40 ± 2.98 ± 2.19 ± 1.36 ± 0.94 ±
5 mg/kg 0.06 0.04 0.04 0.04 0.05*** 0.05*** 0.03*** 0.07*** Isoivangustin 2.73 ± 3.31 ± 3.30 ± 3.40 ± 3.45 ± 3.49 ± 3.45 ± 3.37 ±
2.5 mg/kg 0.05 0.03 0.03 0.03 0.03 0.03 0.03 0.03**
Isoivangustin 2.71 ± 3.29 ± 3.28 ± 3.38 ± 3.28 ± 3.00 ± 2.51 ± 2.06 ± 5 mg/kg 0.05 0.03 0.03 0.03 0.03 0.04*** 0.04*** 0.04***
Isoivangustin 2.71 ± 3.30 ± 3.29 ± 3.39 ± 2.98 ± 2.19 ± 1.35 ± 0.96 ±
10 mg/kg 0.04 0.06 0.05 0.05 0.06*** 0.06*** 0.05*** 0.07***
Values are expressed as mean ± SEM for six animals and analysed by Two way ANOVA followed by Bonferroni post-hoc test, **p<0.01, ***p<0.001 when compared to arthritic control #p<0.001 when compared to healthy control.
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5.3.2 Effect of oral administration of isoivangustin on change in joint diameter in arthritic rats There was significant (p<0.001) increase in joint diameter of rats of all the groups treated with FCA compared to healthy control group. Isoivangustin (5 and 10 mg/kg) significantly (p<0.001) decreased the joint diameter with 38.10% and 65.54%, respectively on day 28 as compared to arthritic control group. Isoivangustin (2.5 mg/kg) showed no significant decrease in joint diameter as compared to arthritic control group. Diclofenac (5 mg/kg) showed significant (p<0.001) decrease in joint diameter with 65.85% inhibition on day 28. The change in joint diameter of isoivangustin treated (10 mg/kg; 1.13 ± 0.07) and (5 mg/kg; 2.03 ± 0.10) was evident as compared to arthritic control group (3.28 ± 0.02) on day 28 (Figure 48, Table 43) Figure 48- Effect of oral administration of isoivangustin on change in joint diameter in arthritic rats
Values are expressed as mean ± SEM for six animals and analysed by Two way ANOVA followed by Bonferroni post-hoc test, ***p<0.001 when compared to arthritic control #p<0.001 when compared to healthy control.
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Table 43- Effect of oral administration of isoivangustin on change in joint diameter in arthritic rats
Day 1 4 8 12 16 20 24 28
Healthy 0.00 ± 0.00 ± 0.00 ± 0.01 ± 0.01 ± 0.01 ± 0.01 ± 0.01 ±
control 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
Arthritic 3.10 ± 3.17 ± 3.17 ± 3.22 ± 3.24 ± 3.27 ± 3.28 ± 3.28 ± control 0.03# 0.03# 0.03# 0.02# 0.02# 0.02# 0.02# 0.02#
Diclofenac 3.11 ± 3.17 ± 3.17 ± 3.23 ± 2.83 ± 2.36 ± 1.77 ± 1.12 ±
5 mg/kg 0.05 0.06 0.06 0.05 0.05*** 0.06*** 0.05*** 0.06*** Isoivangustin 3.10 ± 3.16 ± 3.16 ± 3.21 ± 3.24 ± 3.26 ± 3.20 ± 3.12 ± 2.5 mg/kg 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06
Isoivangustin 3.11 ± 3.16 ± 3.16 ± 3.22 ± 3.08 ± 2.88 ± 2.63 ± 2.03 ± 5 mg/kg 0.06 0.06 0.06 0.06 0.06 0.04*** 0.06*** 0.10*** Isoivangustin 3.12 ± 3.17 ± 3.17 ± 3.23 ± 2.86 ± 2.38 ± 1.79 ± 1.13 ±
10 mg/kg 0.05 0.05 0.05 0.05 0.02*** 0.04*** 0.05*** 0.07***
Values are expressed as mean ± SEM for six animals and analysed by Two way ANOVA followed by Bonferroni post-hoc test, ***p<0.001 when compared to arthritic control #p<0.001 when compared to healthy control.
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5.3.3 Effect of oral administration of isoivangustin on pain threshold in arthritic rats The pain threshold of the paw in the FCA administered rats decreased progressively till day 12. Isoivangustin (5 and 10 mg/kg) significantly (p<0.01) increased the pain threshold from day 20 with 63.63% and 93.00%, respectively on day 28, where as isoivangustin (2.5 mg/kg) was less effective, it significantly (p<0.05) increased the pain threshold with 31.46%. Diclofenac (5 mg/kg) showed maximum increase in pain threshold with 93.70% as compared to arthritic control group. The pain threshold of isoivangustin (10 mg/kg; 276 ± 8.89) and (5 mg/kg; 234 ± 2.39) was evident as compared to arthritic control group (143 ± 7.38) on day 28 (Figure 49, Table 44) Figure 49- Effect of oral administration of isoivangustin on pain threshold in arthritic rats
Values are expressed as mean ± SEM for six animals and analysed by Two way ANOVA followed by Bonferroni post-hoc test, *p<0.05, **p<0.01, ***p<0.001 when compared to arthritic control #p<0.001 when compared to healthy control.
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Table 44- Effect of oral administration of isoivangustin on pain threshold in arthritic rats
Day 0 1 4 8 12 16 20 24 28
Healthy 302 ± 300 ± 296 ± 289 ± 290 ± 298 ± 304 ± 301 ± 301 ± control 5.27 3.65 3.00 3.52 2.89 4.23 5.39 4.73 4.17 Arthritic 303 ± 211 ± 193 ± 192 ± 177 ± 166 ± 156 ± 148 ± 143 ± control 6.15 7.79# 7.39# 7.82# 8.03# 7.46# 6.51# 6.29# 7.38# Diclofenac 303 ± 208 ± 193 ± 193 ± 177 ± 198 ± 227 ± 251 ± 277 ± 5 mg/kg 5.44 6.91 7.15 7.16 7.26 8.72** 10.54*** 11.14*** 11.52*** Isoivangustin 301 ± 208 ± 191 ± 192 ± 178 ± 167 ± 164 ± 175 ± 188 ± 2.5 mg/kg 5.39 5.73 7.12 6.54 5.88 6.28 5.54 5.00* 4.96*** Isoivangustin 300 ± 207 ± 190 ± 188 ± 174 ± 181 ± 194 ± 214 ± 234 ± 5 mg/kg 4.47 3.33 2.89 2.47 2.39 2.01 2.39*** 3.00*** 2.39*** Isoivangustin 298 ± 207 ± 193 ± 192 ± 173 ± 197 ± 223 ± 250 ± 276 ± 10 mg/kg 4.61 2.79 3.35 4.94 4.23 4.22** 4.94*** 6.32*** 8.89***
Values are expressed as mean ± SEM for six animals and analysed by Two way ANOVA followed by Bonferroni post-hoc test, *p<0.05, **p<0.01, ***p<0.001 when compared to arthritic control #p<0.001 when compared to healthy control.
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5.3.4 Effect of oral administration of isoivangustin on paw withdrawal latency in arthritic rats There was significant (p<0.001) decrease in paw withdrawal latency of all the rats treated with FCA compared to healthy control group. Isoivangustin (10 mg/kg) significantly (p<0.01) increased the paw withdrawal latency from day 20, where as isoivangustin (5 mg/kg) significantly (p<0.05) increased the paw withdrawal latency from day 24. Diclofenac (5 mg/kg) showed significant (p<0.01) increase in paw withdrawal latency from day 20 onwards. The paw withdrawal latency of isoivangustin (10 mg/kg; 6.60 ± 0.58) and (5 mg/kg; 5.65 ± 0.48) was evident as compared to arthritic control group (2.77 ± 0.31) on day 28 (Figure 50, Table 45) Figure 50- Effect of oral administration of isoivangustin on paw withdrawal latency in arthritic rats
Values are expressed as mean ± SEM for six animals and analysed by Two way ANOVA followed by Bonferroni post-hoc test, *p<0.05, **p<0.01, ***p<0.001 when compared to arthritic control #p<0.001 when compared to healthy control.
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Table 45- Effect of oral administration of isoivangustin on paw withdrawal latency in arthritic rats
Day 0 1 4 8 12 16 20 24 28
Healthy 8.77 ± 8.60 ± 8.53 ± 8.60 ± 8.52 ± 8.65 ± 8.75 ± 8.85 ± 8.78 ± control 0.38 0.39 0.23 0.29 0.56 0.47 0.25 0.44 0.21 Arthritic 8.60 ± 6.42 ± 5.57 ± 5.45 ± 4.30 ± 3.70 ± 3.18 ± 2.97 ± 2.77 ± control 0.26 0.38# 0.32# 0.32# 0.37# 0.56# 0.45# 0.41# 0.31# Diclofenac 8.50 ± 6.50 ± 5.47 ± 5.28 ± 4.22 ± 4.85 ± 5.53 ± 6.17 ± 6.98 ± 5 mg/kg 0.48 0.51 0.48 0.46 0.43 0.44 0.41** 0.41*** 0.48*** Isoivangustin 8.57 ± 6.43 ± 5.67 ± 5.57 ± 4.50 ± 3.70 ± 3.32 ± 3.50 ± 4.22 ± 2.5 mg/kg 0.38 0.40 0.51 0.45 0.53 0.49 0.45 0.48 0.47 Isoivangustin 8.35 ± 6.45 ± 5.70 ± 5.47 ± 4.43 ± 4.38 ± 4.37 ± 4.98 ± 5.65 ± 5 mg/kg 0.59 0.60 0.61 0.58 0.56 0.53 0.52 0.51* 0.48*** Isoivangustin 8.67 ± 6.63 ± 5.73 ± 5.57 ± 4.57 ± 4.93 ± 5.38 ± 5.93 ± 6.60 ± 10 mg/kg 0.46 0.39 0.46 0.45 0.50 0.50 0.49** 0.49*** 0.58***
Values are expressed as mean ± SEM for six animals and analysed by Two way ANOVA followed by Bonferroni post-hoc test, *p<0.05, **p<0.01, ***p<0.001 when compared to arthritic control #p<0.001 when compared to healthy control.
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5.3.5 Effect of oral administration of isoivangustin on mechanical nociceptive threshold in arthritic rats The mechanical nociceptive threshold was observed to be the lowest on day 12. Administration of isoivangustin (5 and 10 mg/kg) significantly (p<0.001) improved the mechanical nociceptive threshold from day 24 and day 20, respectively when compared to arthritic control group. However, there was little improvement observed with isoivangustin (2.5 mg/kg) which significantly (p<0.01) increased mechanical nociceptive threshold on day 28. Diclofenac (5 mg/kg) showed significant (p<0.001) improvement in mechanical nociceptive threshold from day 20 onwards. The mechanical nociceptive threshold of isoivangustin (10 mg/kg; 60.01 ± 3.09) and (5 mg/kg; 46.30 ± 1.08) was evident as compared to arthritic control group (25.14 ± 1.35) on day 28 (Figure 51, Table 46) Figure 51- Effect of oral administration of isoivangustin on mechanical nociceptive threshold in arthritic rats
Values are expressed as mean ± SEM for six animals and analysed by Two way ANOVA followed by Bonferroni post-hoc test, *p<0.05, **p<0.01, ***p<0.001 when compared to arthritic control #p<0.001 when compared to healthy control.
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Table 46- Effect of oral administration of isoivangustin on mechanical nociceptive threshold in arthritic rats
Day 0 1 4 8 12 16 20 24 28
Healthy 64.45 ± 64.31 ± 65.32 ± 64.52 ± 64.66 ± 64.50 ± 63.86 ± 63.38 ± 64.80 ± control 2.53 1.98 1.74 2.07 2.16 2.44 3.42 2.12 1.65 Arthritic 65.51 ± 30.44 ± 29.30 ± 29.07 ± 28.15 ± 27.10 ± 26.34 ± 25.67 ± 25.14 ± control 2.14 1.49# 1.35# 1.50# 1.38# 1.38# 1.36# 1.32# 1.35# Diclofenac 65.39 ± 29.30 ± 28.54 ± 28.46 ± 27.54 ± 34.45 ± 41.49 ± 49.92 ± 59.36 ± 5 mg/kg 2.38 1.41 1.35 1.29 1.25 1.38* 1.09*** 1.18*** 1.79*** Isoivangustin 66.50 ± 30.41 ± 29.63 ± 29.26 ± 28.34 ± 27.27 ± 27.65 ± 29.58 ± 33.24 ± 2.5 mg/kg 2.71 0.58 0.57 0.51 0.60 0.64 0.86 0.91 0.78** Isoivangustin 64.80 ± 29.18 ± 28.24 ± 27.88 ± 26.98 ± 28.17 ± 32.01 ± 38.70 ± 46.30 ± 5 mg/kg 2.32 1.05 0.93 0.93 1.00 0.81 1.16 1.20*** 1.08*** Isoivangustin 66.17 ± 29.89 ± 29.08 ± 28.19 ± 27.16 ± 33.59 ± 40.87 ± 50.09 ± 60.01 ± 10 mg/kg 1.54 0.71 0.80 0.76 0.74 0.98* 0.58*** 2.61*** 3.09***
Values are expressed as mean ± SEM for six animals and analysed by Two way ANOVA followed by Bonferroni post-hoc test, *p<0.05, **p<0.01, ***p<0.001 when compared to arthritic control #p<0.001 when compared to healthy control.
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5.3.6 Effect of oral administration of isoivangustin on body weight in arthritic rats The rats in the arthritic control group lost body weight as compared with the isoivangustin and diclofenac treated group. Isoivangustin (5 and 10 mg/kg) increased the body weight by 16.85% and 22.47%, respectively on last day of the study. Isoivangustin (2.5 mg/kg) showed no significant increase in body weight as compared to arthritic control group. The body weight of isoivangustin (10 mg/kg; 218 ± 4.22) and (5 mg/kg; 208 ± 4.28) was evident as compared to arthritic control group (178 ± 2.72) on day 28. (Figure 52, Table 47) Figure 52- Effect of oral administration of isoivangustin on body weight in arthritic rats
Values are expressed as mean ± SEM for six animals and analysed by Two way ANOVA followed by Bonferroni post-hoc test, *p<0.05, **p<0.01, ***p<0.001 when compared to arthritic control #p<0.001 when compared to healthy control.
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Table 47- Effect of oral administration of isoivangustin on body weight in arthritic rats
Day 0 1 4 8 12 16 20 24 28
Healthy 211 ± 212 ± 214 ± 217 ± 223 ± 228 ± 233 ± 236 ± 240 ± control 3.61 3.59 3.20 3.08 3.25 3.31 3.09 3.12 2.96 Arthritic 212 ± 211 ± 208 ± 204 ± 198 ± 192 ± 187 ± 183 ± 178 ± control 2.15 2.04 1.71 1.78 1.86# 1.92# 2.14# 2.51# 2.72# Diclofenac 212 ± 211 ± 207 ± 204 ± 197 ± 200 ± 207 ± 213 ± 220 ± 5 mg/kg 5.38 5.24 5.36 5.67 5.74 5.41 5.08* 5.22*** 5.21*** Isoivangustin 213 ± 213 ± 209 ± 205 ± 198 ± 193 ± 189 ± 190 ± 195 ± 2.5 mg/kg 6.01 5.90 6.01 5.56 5.68 5.51 5.81 6.01 6.36 Isoivangustin 211 ± 210 ± 206 ± 202 ± 197 ± 198 ± 202 ± 205 ± 208 ± 5 mg/kg 4.23 4.27 4.15 4.23 4.30 4.22 4.01 4.26** 4.28*** Isoivangustin 214 ± 213 ± 208 ± 205 ± 198 ± 200 ± 205 ± 211 ± 218 ± 10 mg/kg 4.50 4.32 4.30 4.11 4.08 3.89 3.98* 4.00*** 4.22***
Values are expressed as mean ± SEM for six animals and analysed by Two way ANOVA followed by Bonferroni post-hoc test, *p<0.05, **p<0.01, ***p<0.001 when compared to arthritic control #p<0.001 when compared to healthy control.
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5.3.7 Radiological analysis of ankle joints Radiography of rats in arthritic control group (Figure 53B) revealed intense periarticular inflammation, soft tissue swelling, bone resorption and joint erosion. These changes were reverted back to near normal upon treatment with isoivangustin (10 mg/kg) (Figure 53F) and diclofenac (5 mg/kg) (Figure 53C), a significant decrease in inflammation and soft tissue swelling was observed. In rats treated with isoivangustin (5 mg/kg) showed moderate effect (Figure 53E), while isoivangustin (2.5 mg/kg) (Figure 53D) showed no obvious effect.
Figure 53- Radiological analysis of ankle joints. (A) Healthy control (B) Arthritic control (C) Diclofenac 5 mg/kg treated (D) Isoivangustin 2.5 mg/kg treated (E) Isoivangustin 5 mg/kg treated (F) Isoivangustin 10 mg/kg treated
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5.3.8 Haematological parameters 5.3.8.1 Effect of oral administration of isoivangustin on blood haemoglobin count in arthritic rats The challenge with FCA (0.1ml) showed significant (p<0.001) decrease in the haemoglobin count in arthritic control group which was found to be 8.5 ± 0.27 gm/dl. The treatment with diclofenac (5 mg/kg) showed significant (p<0.001) increase in the haemoglobin count by 64.70% as compared to arthritic control group. Isoivangustin (5 and 10 mg/kg) also showed significant (p<0.001) increase in the haemoglobin count by 29.41% and 64.70%, respectively. Isoivangustin (2.5 mg/kg) showed no significant increase in the haemoglobin count when compared to arthritic control group. (Figure 54, Table 48)
Figure 54- Effect of oral administration of isoivangustin on blood haemoglobin count in arthritic rats
20
15 *** *** *** 10 #
Hb (gm/dl) 5
0 l l o ro g kg g g tr t /k / k /k n n g g / g o o m g m C C m 5 m y c 5 . 5 0 ti c 2 1 th i a n in n al r n ti t ti th e s s s e r f u u u H A lo g g g c n n n i a a a D v iv v i o i o s o Is I Is
Values are expressed as mean ± S.E.M.; n=6 rats per group. One way ANOVA followed by Dunnett’s test when compared with arthritic control ***p<0.001 and when arthritic control compared with healthy control #p<0.001.
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Table 48- Effect of oral administration of isoivangustin on blood haemoglobin count in arthritic rats
Groups Hb (gm/dl) Healthy control 15.0 ± 0.16 Arthritic control 8.50 ± 0.27# Diclofenac 5 mg/kg 14.0 ± 0.37*** Isoivangustin 2.5 mg/kg 8.80 ± 0.14 Isoivangustin 5 mg/kg 11.0 ± 0.19*** Isoivangustin 10 mg/kg 14.0 ± 0.35***
Values are expressed as mean ± S.E.M.; n=6 rats per group. One way ANOVA followed by Dunnett’s test when compared with arthritic control ***p<0.001 and when arthritic control compared with healthy control #p<0.001.
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5.3.8.2 Effect of oral administration of isoivangustin on blood WBC count in arthritic rats The challenge with FCA (0.1ml) showed significant (p<0.001) elevation in the WBC count when compared to healthy control group which was found to be 14.0 ± 0.41 thousands/mm3. Diclofenac (5 mg/kg) showed significant (p<0.001) decrease in the WBC count by 40.00% when compared to arthritic control group. The treatment with isoivangustin (5 and 10 mg/kg) showed significant (p<0.001) decrease in the WBC count which was 21.42% and 39.28%, respectively when compared to arthritic control group. However, isoivangustin (2.5 mg/kg) showed no significant decrease in WBC count. (Figure 55, Table 49)
Figure 55- Effect of oral administration of isoivangustin on blood WBC count in arthritic rats
20 ) 3
15 # *** 10 *** ***
5
WBC (thousands/mm 0 l l o ro g kg g g tr t /k / k /k n n g g / g o o m g m C C m 5 m y c 5 . 5 0 ti c 2 1 th i a n in n al r n ti t ti th e s s s e r f u u u H A lo g g g c n n n i a a a D v iv v i o i o s o Is I Is
Values are expressed as mean ± S.E.M.; n=6 rats per group. One way ANOVA followed by Dunnett’s test when compared with arthritic control ***p<0.001 and when arthritic control compared with healthy control #p<0.001.
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Table 49- Effect of oral administration of isoivangustin on blood WBC count in arthritic rats
Groups WBC (thousands/mm3) Healthy control 7.30 ± 0.30 Arthritic control 14.0 ± 0.41# Diclofenac 5 mg/kg 8.40 ± 0.29*** Isoivangustin 2.5 mg/kg 13.0 ± 0.25 Isoivangustin 5 mg/kg 11.0 ± 0.25*** Isoivangustin 10 mg/kg 8.50 ± 0.28***
Values are expressed as mean ± S.E.M.; n=6 rats per group. One way ANOVA followed by Dunnett’s test when compared with arthritic control ***p<0.001 and when arthritic control compared with healthy control #p<0.001.
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5.3.8.3 Effect of oral administration of isoivangustin on blood RBC count in arthritic rats The challenge with FCA (0.1ml) showed significant (p<0.001) decrease in the RBC count when compared to healthy control group which was found to be 3.50 ± 0.17 million/mm3. Diclofenac (5 mg/kg) showed significant (p<0.001) increase in the RBC count by 82.85% when compared to arthritic control group. The treatment with isoivangustin (5 and 10 mg/kg) showed significant (p<0.01 and p<0.001, respectively) increase in the RBC count by 28.57% and 71.42%, respectively as compared to arthritic control group. However, isoivangustin (2.5 mg/kg) showed non-significant increase in RBC count. (Figure 56, Table 50)
Figure 56- Effect of oral administration of isoivangustin on blood RBC count in arthritic rats
8 ) 3 *** 6 *** ** 4 #
2 RBC (millions/mm 0 l l o ro g kg g g tr t /k / k /k n n g g / g o o m g m C C m 5 m y c 5 . 5 0 ti c 2 1 th i a n in n al r n ti t ti th e s s s e r f u u u H A lo g g g c n n n i a a a D v iv v i o i o s o Is I Is
Values are expressed as mean ± S.E.M.; n=6 rats per group. One way ANOVA followed by Dunnett’s test when compared with arthritic control **p<0.01, ***p<0.001 and when arthritic control compared with healthy control #p<0.001.
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Table 50- Effect of oral administration of isoivangustin on blood RBC count in arthritic rats
Groups RBC (millions/mm3) Healthy control 7.10 ± 0.07 Arthritic control 3.50 ± 0.17# Diclofenac 5 mg/kg 6.40 ± 0.26*** Isoivangustin 2.5 mg/kg 3.60 ± 0.21 Isoivangustin 5 mg/kg 4.50 ± 0.25** Isoivangustin 10 mg/kg 6.00 ± 0.20***
Values are expressed as mean ± S.E.M.; n=6 rats per group. One way ANOVA followed by Dunnett’s test when compared with arthritic control **p<0.01, ***p<0.001 and when arthritic control compared with healthy control #p<0.001.
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5.3.8.4 Effect of oral administration of isoivangustin on blood platelet count in arthritic rats The challenge with FCA (0.1ml) showed significant (p<0.001) elevation in the platelet count when compared to healthy control group which was found to be 1643 ± 36 thousands/mm3. Diclofenac (5 mg/kg) showed significant (p<0.001) decrease in the platelet count by 39.25% when compared to arthritic control group. The treatment with isoivangustin (5 and 10 mg/kg) showed significant (p<0.01 and p<0.001, respectively) decrease in the platelet count by 12.47% and 37.12%, respectively when compared to arthritic control group. However, isoivangustin (2.5 mg/kg) showed no significant decrease in platelet count when compared to arthritic control group. (Figure 57, Table 51)
Figure 57- Effect of oral administration of isoivangustin on blood platelet count in arthritic rats
2000 ) 3 # 1500 **
*** 1000 ***
500
Platelets (thousand/mm Platelets 0 l l g ro ro g k g kg t t /k / /k / n n g g g g o o m m C C m 5 m y c 5 . 5 0 h ti c 2 1 lt i a n in n a r n ti t ti th e s s s e r f u u u H A o g g g cl n n i an a a D v iv v i o i o s o Is I Is
Values are expressed as mean ± S.E.M.; n=6 rats per group. One way ANOVA followed by Dunnett’s test when compared with arthritic control **p<0.01, ***p<0.001 and when arthritic control compared with healthy control #p<0.001.
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Table 51- Effect of oral administration of isoivangustin on blood platelet count in arthritic rats
Platelets Groups (thousands/mm3) Healthy control 898 ± 40 Arthritic control 1643 ± 36# Diclofenac 5 mg/kg 998 ± 37*** Isoivangustin 2.5 mg/kg 1618 ± 38 Isoivangustin 5 mg/kg 1438 ± 38** Isoivangustin 10 mg/kg 1033 ± 27***
Values are expressed as mean ± S.E.M.; n=6 rats per group. One way ANOVA followed by Dunnett’s test when compared with arthritic control **p<0.01, ***p<0.001 and when arthritic control compared with healthy control #p<0.001.
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5.3.8.5 Effect of oral administration of isoivangustin on blood ESR count in arthritic rats The challenge with FCA (0.1ml) showed significant (p<0.001) elevation in the ESR count when compared to healthy control group which was found to be 14.0 ± 0.27 mm/h. Diclofenac (5 mg/kg) showed significant (p<0.001) decrease in the ESR count by 35.00% when compared to arthritic control group. The treatment with isoivangustin (5 and 10 mg/kg) showed significant (p<0.01 and p<0.001, respectively) decrease in the ESR count by 14.28% and 33.57%, respectively when compared to arthritic control group. However, isoivangustin (2.5 mg/kg) showed no significant decrease in ESR count when compared to arthritic control group. (Figure 58, Table 52)
Figure 58- Effect of oral administration of isoivangustin on blood ESR count in arthritic rats
15 # ** 10 *** ***
5 ESR ESR (mm/h)
0
l g /k /kg tro g mg/kg m mg y con 10 h 2.5 in alt tin t e rthritis control s s H A gu ngu an Diclofenaca 5 v iv i Isoivangustin 5 mg/kg Iso Iso
Values are expressed as mean ± S.E.M.; n=6 rats per group. One way ANOVA followed by Dunnett’s test when compared with arthritic control **p<0.01, ***p<0.001 and when arthritic control compared with healthy control #p<0.001.
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Table 52- Effect of oral administration of isoivangustin on blood ESR count in arthritic rats
ESR Groups (mm/h) Healthy control 6.80 ± 0.23 Arthritic control 14.0 ± 0.27# Diclofenac 5 mg/kg 9.10 ± 0.44*** Isoivangustin 2.5 mg/kg 13.0 ± 0.44 Isoivangustin 5 mg/kg 12.0 ± 0.42** Isoivangustin 10 mg/kg 9.30 ± 0.43***
Values are expressed as mean ± S.E.M.; n=6 rats per group. One way ANOVA followed by Dunnett’s test when compared with arthritic control **p<0.01, ***p<0.001 and when arthritic control compared with healthy control #p<0.001.
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5.3.8.6 Effect of oral administration of isoivangustin on serum CRP level in arthritic rats The serum CRP level of the healthy control group was 1.30 ± 0.04 mg/lit, which was significantly (p<0.001) increased in arthritic control group and found to be 7.90 ± 0.34 mg/lit. Diclofenac (5 mg/kg) showed significant (p<0.001) decrease in serum CRP level by 63.29% when compared to arthritic control group. Treatment with isoivangustin (5 and 10 mg/kg) exhibited a significant (p<0.001) decrease in serum CRP level by 36.70% and 62.02%, respectively when compared to arthritic control group. Isoivangustin (2.5 mg/kg) showed no significant decrease in serum CRP level. (Figure 59, Table 53)
Figure 59- Effect of oral administration of isoivangustin on serum CRP level in arthritic rats
10 # 8
6 *** 4 *** *** CRP (mg/lit) 2
0 l l o ro g kg g g tr t /k / k /k n n g g / g o o m g m C C m 5 m y c 5 . 5 0 ti c 2 1 th i a n in n al r n ti t ti th e s s s e r f u u u H A lo g g g c n n n i a a a D v iv v i o i o s o Is I Is
Values are expressed as mean ± S.E.M.; n=6 rats per group. One way ANOVA followed by Dunnett’s test when compared with arthritic control ***p<0.001 and when arthritic control compared with healthy control #p<0.001.
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Table 53- Effect of oral administration of isoivangustin on serum CRP level in arthritic rats
Groups CRP (mg/lit) Healthy control 1.30 ± 0.04 Arthritic control 7.90 ± 0.34# Diclofenac 5 mg/kg 2.90 ± 0.24*** Isoivangustin 2.5 mg/kg 7.30 ± 0.18 Isoivangustin 5 mg/kg 5.00 ± 0.27*** Isoivangustin 10 mg/kg 3.00 ± 0.13***
Values are expressed as mean ± S.E.M.; n=6 rats per group. One way ANOVA followed by Dunnett’s test when compared with arthritic control ***p<0.001 and when arthritic control compared with healthy control #p<0.001.
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5.3.8.7 Effect of oral administration of isoivangustin on RF value in arthritic rats The serum RF value of the arthritic control group was 55 ± 1.50 IU/ml. Diclofenac (5 mg/kg) showed significant (p<0.001) decrease in RF value by 38.18% when compared to arthritic control group. Treatment with isoivangustin (5 and 10 mg/kg) exhibited a significant (p<0.001) decrease in RF value by 21.81% and 38.18%, respectively when compared to arthritic control group. Isoivangustin (2.5 mg/kg) showed no significant decrease in RF value. (Figure 60, Table 54)
Figure 60- Effect of oral administration of isoivangustin on RF value in arthritic rats
60 #
*** 40 *** ***
20 RF (IU/ml)
0 l o g tr kg /k g g n / g /k /k o g m g g C m m c 5 .5 m ti c 2 5 0 i a n n 1 r n i ti n th e st s i r f u u st A lo g g u ic n n g D a a n iv iv a o o iv Is Is o Is
Values are expressed as mean ± S.E.M.; n=6 rats per group. One way ANOVA followed by Dunnett’s test when compared with arthritic control ***p<0.001.
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Table 54- Effect of oral administration of isoivangustin on RF value in arthritic rats
Groups RF (IU/ml) Arthritic control 55 ± 1.50 Diclofenac 5 mg/kg 34 ± 1.50*** Isoivangustin 2.5 mg/kg 52 ± 1.60 Isoivangustin 5 mg/kg 43 ± 1.70*** Isoivangustin 10 mg/kg 34 ± 0.97***
Values are expressed as mean ± S.E.M.; n=6 rats per group. One way ANOVA followed by Dunnett’s test when compared with arthritic control ***p<0.001.
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5.3.9 Biochemical parameters 5.3.9.1 Effect of oral administration of isoivangustin on serum AST level in arthritic rats The serum AST level of the healthy control group was found to be 43 ± 2.80 U/L, which was significantly (p<0.001) increased in arthritic control group and found to be 126 ± 3.20 U/L. Treatment with diclofenac (5 mg/kg) caused significant (p<0.001) decrease in serum AST level by 50.00% when compared to arthritic control group. Treatment with isoivangustin (5 and 10 mg/kg) also exhibited a significant (p<0.001) decrease in serum AST level by 27.77% and 47.61%, respectively when compared to arthritic control group. However, isoivangustin (2.5 mg/kg) showed no significant decrease in serum AST level. (Figure 61, Table 55)
Figure 61- Effect of oral administration of isoivangustin on serum AST level in arthritic rats
150 # * 100 *** *** ***
AST (U/L)AST 50
0 l l o ro g kg g g tr t /k / k /k n n g g / g o o m g m C C m 5 m y c 5 . 5 0 ti c 2 1 th i a n in n al r n ti t ti th e s s s e r f u u u H A lo g g g c n n n i a a a D v iv v i o i o s o Is I Is
Values are expressed as mean ± S.E.M.; n=6 rats per group. One way ANOVA followed by Dunnett’s test when compared with arthritic control ***p<0.001 and when arthritic control compared with healthy control #p<0.001.
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Table 55- Effect of oral administration of isoivangustin on serum AST level in arthritic rats
Groups AST (U/L) Healthy control 43 ± 2.80 Arthritic control 126 ± 3.20# Diclofenac 5 mg/kg 63 ± 4.10*** Isoivangustin 2.5 mg/kg 122 ± 3.80 Isoivangustin 5 mg/kg 91 ± 3.10*** Isoivangustin 10 mg/kg 66 ± 1.50***
Values are expressed as mean ± S.E.M.; n=6 rats per group. One way ANOVA followed by Dunnett’s test when compared with arthritic control ***p<0.001 and when arthritic control compared with healthy control #p<0.001.
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5.3.9.2 Effect of oral administration of isoivangustin on serum ALT level in arthritic rats The serum ALT level of the healthy control group was found to be 39 ± 1.50 U/L, which was significantly (p<0.001) increased in arthritic control group and found to be 171 ± 6.70 U/L. Treatment with diclofenac (5 mg/kg) caused significant (p<0.001) decrease in serum ALT level by 67.25% when compared to arthritic control group. Treatment with isoivangustin (5 and 10 mg/kg) exhibited a significant (p<0.001) decrease in serum ALT level by 43.85% and 66.08%, respectively when compared to arthritic control group. Treatment with isoivangustin (2.5 mg/kg) showed significant (p<0.05) decrease in serum ALT level by 11.11%. (Figure 62, Table 56)
Figure 62- Effect of oral administration of isoivangustin on serum ALT level in arthritic rats
200 # * 150
100 ***
ALT (U/L)ALT *** *** 50
0 l l o ro g kg g g tr t /k / k /k n n g g / g o o m g m C C m 5 m y c 5 . 5 0 ti c 2 1 th i a n in n al r n ti t ti th e s s s e r f u u u H A lo g g g c n n n i a a a D v iv v i o i o s o Is I Is
Values are expressed as mean ± S.E.M.; n=6 rats per group. One way ANOVA followed by Dunnett’s test when compared with arthritic control *p<0.05, ***p<0.001 and when arthritic control compared with healthy control #p<0.001.
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Table 56- Effect of oral administration of isoivangustin on serum ALT level in arthritic rats
Groups ALT (U/L) Healthy control 39 ± 1.50 Arthritic control 171 ± 6.70# Diclofenac 5 mg/kg 56 ± 4.20*** Isoivangustin 2.5 mg/kg 152 ± 3.20* Isoivangustin 5 mg/kg 96 ± 3.10*** Isoivangustin 10 mg/kg 58 ± 3.80***
Values are expressed as mean ± S.E.M.; n=6 rats per group. One way ANOVA followed by Dunnett’s test when compared with arthritic control *p<0.05, ***p<0.001 and when arthritic control compared with healthy control #p<0.001.
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5.3.9.3 Effect of oral administration of isoivangustin on serum alkaline phosphatase level in arthritic rats The serum alkaline phosphatase level of the healthy control group was found to be 72 ± 3.10 U/L, which was significantly (p<0.001) increased in arthritic control group and found to be 439 ± 3.70 U/L. Treatment with diclofenac (5 mg/kg) showed significant (p<0.001) decrease in serum ALP level by 68.33% when compared to arthritic control group. Isoivangustin (5 and 10 mg/kg) also exhibited a significant (p<0.001) decrease in serum alkaline phosphatase level by 34.85% and 68.79%, respectively when compared to arthritic control group. Isoivangustin (2.5 mg/kg) showed no significant effect (Figure 63, Table 57)
Figure 63- Effect of oral administration of isoivangustin on serum alkaline phosphatase level in arthritic rats
500 # 400
300 ***
200 *** *** 100
Alkaline Phosphatase (U/L) Phosphatase Alkaline 0 l l o ro g kg g g tr t /k / k /k n n g g / g o o m g m C C m 5 m y c 5 . 5 0 ti c 2 1 th i a n in n al r n ti t ti th e s s s e r f u u u H A lo g g g c n n n i a a a D v iv v i o i o s o Is I Is
Values are expressed as mean ± S.E.M.; n=6 rats per group. One way ANOVA followed by Dunnett’s test when compared with arthritic control ***p<0.001 and when arthritic control compared with healthy control #p<0.001.
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Table 57- Effect of oral administration of isoivangustin on serum alkaline phosphatase level in arthritic rats
Alkaline Groups phosphatase (U/L) Healthy control 72 ± 3.10 Arthritic control 439 ± 3.70# Diclofenac 5 mg/kg 139 ± 4.50*** Isoivangustin 2.5 mg/kg 426 ± 4.70 Isoivangustin 5 mg/kg 286 ± 4.20*** Isoivangustin 10 mg/kg 137 ± 2.40***
Values are expressed as mean ± S.E.M.; n=6 rats per group. One way ANOVA followed by Dunnett’s test when compared with arthritic control ***p<0.001 and when arthritic control compared with healthy control #p<0.001.
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5.3.9.4 Effect of oral administration of isoivangustin on serum total protein level in arthritic rats The serum total protein level of the healthy control group was found to be 6.5 ± 0.38 g/dl, arthritic control group showed significant (p<0.001) decrease which was found to be 4.5 ± 0.22 g/dl. Diclofenac (5 mg/kg) showed significant (p<0.001) increase by 40.00% and isoivangustin (5 and 10 mg/kg) showed significant (p<0.05 and p<0.01, respectively) increase by 22.22% and 33.33%, respectively in serum total protein level when compared with arthritic control group. Isoivangustin (2.5 mg/kg) showed no significant increase in serum total protein level. (Figure 64, Table 58)
Figure 64- Effect of oral administration of isoivangustin on serum total protein level in arthritic rats
8 *** 6 * ** # 4
2 Total Total Protein (g/dl)
0 l l o ro g kg g g tr t /k / k /k n n g g / g o o m g m C C m 5 m y c 5 . 5 0 ti c 2 1 th i a n in n al r n ti t ti th e s s s e r f u u u H A lo g g g c n n n i a a a D v iv v i o i o s o Is I Is
Values are expressed as mean ± S.E.M.; n=6 rats per group. One way ANOVA followed by Dunnett’s test when compared with arthritic control *p<0.05, **p<0.01, ***p<0.001 and when arthritic control compared with healthy control #p<0.001.
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Table 58- Effect of oral administration of isoivangustin on serum total protein level in arthritic rats
Total Groups protein (g/dl) Healthy control 6.5 ± 0.38 Arthritic control 4.5 ± 0.22# Diclofenac 5 mg/kg 6.3 ± 0.31*** Isoivangustin 2.5 mg/kg 4.7 ± 0.26 Isoivangustin 5 mg/kg 5.5 ± 0.21* Isoivangustin 10 mg/kg 6.0 ± 0.12**
Values are expressed as mean ± S.E.M.; n=6 rats per group. One way ANOVA followed by Dunnett’s test when compared with arthritic control *p<0.05, **p<0.01, ***p<0.001 and when arthritic control compared with healthy control #p<0.001.
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5.3.10 Cytokine measurement 5.3.10.1 Effect of oral administration of isoivangustin on serum TNF-α in arthritic rats The serum TNF-α level of the healthy control group was found to be 30 ± 2.1 pg/ml which was significantly (p<0.001) increased in arthritic control group and found to be 117 ± 6.7 pg/ml. Diclofenac (5 mg/kg) caused significant (p<0.001) decrease in serum TNF-α level by 40.17 % when compared to arthritic control group. Treatment with isoivangustin (5 and 10 mg/kg) exhibited a significant (p<0.001) dose dependent decrease in serum TNF-α level by 21.37% and 36.75%, respectively when compared to arthritic control group. However, isoivangustin (2.5 mg/kg) showed no significant decrease in serum TNF-α level when compared to arthritic control group. (Figure 65, Table 59)
Figure 65- Effect of oral administration of isoivangustin on serum TNF-α in arthritic rats
150 #
100 *** *** ***
50 TNF TNF -(pg/ml) alpha 0
g kg k ntrol g/ m Control 5 mg/ s Co 10 mg/kg ti in ri t in h enac 5 s t rt f gu Healthy A an Diclo iv ivangustin 2.5 mg/kg Iso Iso Isoivangus
Values are expressed as mean ± S.E.M.; n=6 rats per group. One way ANOVA followed by Dunnett’s test when compared with arthritic control ***p<0.001 and when arthritic control compared with healthy control #p<0.001.
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Table 59- Effect of oral administration of isoivangustin on serum TNF-α in arthritic rats
Groups TNF-α (pg/ml) Healthy control 30 ± 2.1 Arthritic control 117 ± 6.7# Diclofenac 5 mg/kg 70 ± 4.6*** Isoivangustin 2.5 mg/kg 111 ± 4.0 Isoivangustin 5 mg/kg 92 ± 3.1*** Isoivangustin 10 mg/kg 74 ± 3.0***
Values are expressed as mean ± S.E.M.; n=6 rats per group. One way ANOVA followed by Dunnett’s test when compared with arthritic control ***p<0.001 and when arthritic control compared with healthy control #p<0.001.
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5.3.10.2 Effect of oral administration of isoivangustin on serum IL-1β in arthritic rats The serum IL-1β level of the healthy control group was found to be 60 ± 3.1 pg/ml which was significantly (p<0.001) increased in arthritic control group and found to be 452 ± 8.7 pg/ml. Diclofenac (5 mg/kg) caused significant (p<0.001) decrease in serum IL-1β level by 44.69 % when compared to arthritic control group. Treatment with isoivangustin (5 and 10 mg/kg) exhibited a significant (p<0.001) dose dependent decrease in serum IL-1β level by 25.22% and 44.91%, respectively when compared to arthritic control group. Isoivangustin (2.5 mg/kg) showed no significant decrease in serum IL-1β level when compared to arthritic control group. (Figure 66, Table 60)
Figure 66- Effect of oral administration of isoivangustin on serum IL-1β in arthritic rats
500 #
400 *** 300 *** *** 200
100 IL -IL 1 beta (pg/ml)
0 l l o o g g tr tr /k /k n n g g g/kg o m m C 5 5 m 10 y c 2. th a n in l n ti t a fe s s u He Arthritis Co g gu n an Diclo a ivangustinv 5 mg/kg iv o Is Iso Isoi
Values are expressed as mean ± S.E.M.; n=6 rats per group. One way ANOVA followed by Dunnett’s test when compared with arthritic control ***p<0.001 and when arthritic control compared with healthy control #p<0.001.
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Table 60- Effect of oral administration of isoivangustin on serum IL-1β in arthritic rats
Groups IL-1β (pg/ml) Healthy control 60 ± 3.1 Arthritic control 452 ± 8.7# Diclofenac 5 mg/kg 250 ± 10*** Isoivangustin 2.5 mg/kg 428 ± 3.4 Isoivangustin 5 mg/kg 338 ± 9.8*** Isoivangustin 10 mg/kg 249 ± 6.4***
Values are expressed as mean ± S.E.M.; n=6 rats per group. One way ANOVA followed by Dunnett’s test when compared with arthritic control ***p<0.001 and when arthritic control compared with healthy control #p<0.001.
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5.3.10.3 Effect of oral administration of isoivangustin on serum IL-6 in arthritic rats The serum IL-6 level of the healthy control group was found to be 71 ± 3.6 pg/ml which was significantly (p<0.001) increased in arthritic control group and found to be 405 ± 7.6 pg/ml. Diclofenac (5 mg/kg) caused significant (p<0.001) decrease in serum IL-6 level by 25.93 % when compared to arthritic control group. Treatment with isoivangustin (10 mg/kg) exhibited a significant (p<0.01) decrease in serum IL-6 level by 10.86 % when compared to arthritic control group. However, isoivangustin (2.5 and 5 mg/kg) showed no significant decrease in serum IL-6 level when compared to arthritic control group. (Figure 67, Table 61)
Figure 67- Effect of oral administration of isoivangustin on serum IL-6 in arthritic rats
500 # 400 ** 300 ***
200 IL- IL- 6 (pg/ml) 100
0
g kg k ntrol g/ m Control 5 mg/ s Co 10 mg/kg ti in ri t in h enac 5 s t rt f gu Healthy A an Diclo iv ivangustin 2.5 mg/kg Iso Iso Isoivangus
Values are expressed as mean ± S.E.M.; n=6 rats per group. One way ANOVA followed by Dunnett’s test when compared with arthritic control **p<0.01, ***p<0.001 and when arthritic control compared with healthy control #p<0.001.
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Table 61- Effect of oral administration of isoivangustin on serum IL-6 in arthritic rats
Groups IL-6 (pg/ml) Healthy control 71 ± 3.6 Arthritic control 405 ± 7.6# Diclofenac 5 mg/kg 300 ± 10*** Isoivangustin 2.5 mg/kg 403 ± 8.9 Isoivangustin 5 mg/kg 378 ± 7.0 Isoivangustin 10 mg/kg 361 ± 8.4**
Values are expressed as mean ± S.E.M.; n=6 rats per group. One way ANOVA followed by Dunnett’s test when compared with arthritic control **p<0.01, ***p<0.001 and when arthritic control compared with healthy control #p<0.001.
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5.3.11 Antioxidant parameters 5.3.11.1 Effect of oral administration of isoivangustin on liver MDA level in arthritic rats The liver MDA level of the healthy control group was found to be 2.00 ± 0.03 n mole MDA/mg which was significantly (p<0.001) increased in arthritic control group and found to be 3.50 ± 0.03 n mole MDA/mg. Diclofenac (5 mg/kg) caused significant (p<0.001) decrease in liver MDA level by 22.85% when compared to arthritic control group. Treatment with isoivangustin (5 and 10 mg/kg) exhibited a significant (p<0.001) decrease in liver MDA level by 14.28% and 22.85%, respectively when compared to arthritic control group. However, isoivangustin (2.5 mg/kg) showed no significant decrease in liver MDA level when compared to arthritic control group. (Figure 68, Table 62)
Figure 68- Effect of oral administration of isoivangustin on liver MDA level in arthritic rats
4 # *** 3 *** ***
2
1 MDA (nmole MDA/mg) (nmole MDA 0 l l o o kg g tr tr kg / g /k n / g /k g n o g m g o C m m C 5 .5 m 0 y ic c 2 5 1 th it a n n n l r n ti ti ti a th e s s s e r f u u u H A lo g g g c n n n i a a a D iv iv iv o o o Is Is Is
Values are expressed as mean ± S.E.M.; n=6 rats per group. One way ANOVA followed by Dunnett’s test when compared with arthritic control ***p<0.001 and when arthritic control compared with healthy control #p<0.001.
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Table 62- Effect of oral administration of isoivangustin on liver MDA level in arthritic rats
Groups MDA (n mol MDA/mg) Healthy control 2.00 ± 0.03 Arthritic control 3.50 ± 0.03# Diclofenac 5 mg/kg 2.70 ± 0.03*** Isoivangustin 2.5 mg/kg 3.40 ± 0.04 Isoivangustin 5 mg/kg 3.00 ± 0.04*** Isoivangustin 10 mg/kg 2.70 ± 0.03***
Values are expressed as mean ± S.E.M.; n=6 rats per group. One way ANOVA followed by Dunnett’s test when compared with arthritic control ***p<0.001 and when arthritic control compared with healthy control #p<0.001.
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5.3.11.2 Effect of oral administration of isoivangustin on liver SOD level in arthritic rats The liver SOD level of the healthy control group was 4.90 ± 0.08 mU/mg protein which was significantly (p<0.001) decreased in arthritic control group and found to be 2.30 ± 0.03 mU/mg protein. Diclofenac (5 mg/kg) caused significant (p<0.001) increase in liver SOD level by 60.86% when compared to arthritic control group. Treatment with isoivangustin (5 and 10 mg/kg) exhibited a significant (p<0.001) increase in liver SOD level by 34.78% and 60.86%, respectively when compared to arthritic control group. Isoivangustin (2.5 mg/kg) showed no significant increase in liver SOD level when compared to arthritic control group. (Figure 69, Table 63)
Figure 69- Effect of oral administration of isoivangustin on liver SOD level in arthritic rats
6
4 *** *** *** # 2 SOD (mU/mg protein) SOD (mU/mg 0 l l o ro kg g tr t kg / g /k n n / g /k g o o g m g C C m m m c 5 .5 0 y i c 2 5 1 th it a n n n l r n ti ti ti a th e s s s e r f u u u H A lo g g g c n n n i a a a D iv iv iv o o o Is Is Is
Values are expressed as mean ± S.E.M.; n=6 rats per group. One way ANOVA followed by Dunnett’s test when compared with arthritic control ***p<0.001 and when arthritic control compared with healthy control #p<0.001.
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Table 63- Effect of oral administration of isoivangustin on liver SOD level in arthritic rats
Groups SOD (mU/mg protein) Healthy control 4.90 ± 0.08 Arthritic control 2.30 ± 0.03# Diclofenac 5 mg/kg 3.70 ± 0.02*** Isoivangustin 2.5 mg/kg 2.40 ± 0.05 Isoivangustin 5 mg/kg 3.10 ± 0.03*** Isoivangustin 10 mg/kg 3.70 ± 0.04***
Values are expressed as mean ± S.E.M.; n=6 rats per group. One way ANOVA followed by Dunnett’s test when compared with arthritic control ***p<0.001 and when arthritic control compared with healthy control #p<0.001.
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5.3.11.3 Effect of oral administration of isoivangustin on liver GSH level in arthritic rats The liver GSH level of the healthy control group was 76 ± 2.30 mU/mg protein which was significantly (p<0.001) decreased in arthritic control group and found to be 46 ± 2.30 mU/mg protein. Treatment with diclofenac (5 mg/kg) exhibited a significant (p<0.001) increase in liver GSH level by 36.95% when compared to arthritic control group and isoivangustin (5 and 10 mg/kg) exhibited a significant (p<0.01 and p<0.001, respectively) increase in liver GSH level by 23.91% and 36.95%, respectively when compared to arthritic control group, whereas isoivangustin (2.5 mg/kg) showed no significant increase in liver GSH level when compared to arthritic control group. (Figure 70, Table 64)
Figure 70- Effect of oral administration of isoivangustin on liver GSH level in arthritic rats
100
80 *** *** 60 ** # 40
20
GSH (µmol/mg protein) GSH (µmol/mg 0 l l o o kg g tr tr kg / g /k n / g /k g n o g m g o C m m C 5 .5 m 0 y ic c 2 5 1 th it a n n n l r n ti ti ti a th e s s s e r f u u u H A lo g g g c n n n i a a a D iv iv iv o o o Is Is Is
Values are expressed as mean ± S.E.M.; n=6 rats per group. One way ANOVA followed by Dunnett’s test when compared with arthritic control **p<0.01, ***p<0.001 and when arthritic control compared with healthy control #p<0.001.
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Table 64- Effect of oral administration of isoivangustin on liver GSH level in arthritic rats
Groups GSH (µmol/mg protein) Healthy control 76 ± 2.30 Arthritic control 46 ± 2.30# Diclofenac 5 mg/kg 63 ± 1.00*** Isoivangustin 2.5 mg/kg 50 ± 0.82 Isoivangustin 5 mg/kg 57 ± 2.30** Isoivangustin 10 mg/kg 63 ± 1.90***
Values are expressed as mean ± S.E.M.; n=6 rats per group. One way ANOVA followed by Dunnett’s test when compared with arthritic control **p<0.01, ***p<0.001 and when arthritic control compared with healthy control #p<0.001.
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5.3.12 Histopathology of ankle joints Histopathological analysis of ankle joint of healthy control group rats did not reveal any visible signs of inflammatory infiltration, with intact synovial lining, and no joint destruction (Figure 71A). Rats in arthritic control group showed high degree of inflammatory infiltration, with cartilage and bone destruction, severe necrosis of bone and disturbed synovial lining (Figure 71B). Treatment with isoivangustin (10 mg/kg) and diclofenac (5 mg/kg) led to decrease in the degree of inflammatory infiltration with significant protection against necrosis of bone and cartilage destruction (Figure 71F and Figure 71C, respectively). Treatment with isoivangustin (5 mg/kg) showed moderate effect with minute presence of inflammatory cells, little cartilage destruction and little disturbance in synovial lining (Figure 71E) and isoivangustin (2.5 mg/kg) treated rats showed disturbed synovial lining, with presence of inflammatory cells; severe cartilage and bone destruction (Figure 71D).
Figure 71- Histopathological analysis of ankle joints stained with H&E. (A) Healthy control (B) Arthritic control (C) Diclofenac 5 mg/kg treated (D) Isoivangustin 2.5 mg/kg treated (E) Isoivangustin 5 mg/kg treated (F) Isoivangustin 10 mg/kg treated.
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Discussion Discussion
Pain and inflammation are associated with pathophysiology of various diseases like arthritis, cancer and vascular diseases. A number of natural products are used in various traditional medicinal systems to relief symptoms of pain and inflammation (Ashok Kumar et al., 2010). Alternative medicines for the treatment of rheumatoid arthritis are getting more popular. Many medicinal plants provide relief of symptoms in rheumatoid arthritis whose effects are comparable to that of available conventional medicinal agents (Verpoorte, 1999). Over the centuries number of medicinal plants has been exploited for the treatment of the disorders associated with the inflammatory conditions or for the control of inflammatory aspects of diseases. These medicinal plants owe their activities due to the phytoconstituents and may exert anti- inflammatory effect by interfering generally with the inflammatory pathways or especially with certain components of the pathway, such as release of pro- inflammatory mediators, migration of leukocytes under inflammatory stimulus with consequent release of the cytoplasmic contents at inflammatory sites (Otari et al., 2010).
Throughout the evolutions, the importance of natural products for medicine and health has been enormous. Since our earliest ancestors used certain herbs to relieve pain or wrapped leaves around wounds to improve healing, natural products have often been the sole means to treat disease and injuries. In fact, it has been during past decades that natural products taken a secondary role in drug discovery and drug development, after the advent of molecular biology and combinatorial chemistry made possible the rational design of chemical compounds to target specific molecules. The past few years, however have seen a renewed interest in the use of natural products and more importantly their role as a basis for drug development. Numerous useful drugs are developed from lead compounds discovered from medicinal plants. In addition, the elucidation of the molecular structure of many natural products allowed chemists to synthesize them, rather than isolating them from natural sources, which markedly lowered the cost of drug production. Subsequently, a large number of well known natural compounds were identified, analyzed and synthesized. The structural analysis of natural compounds and the ability to synthesize them allowed chemists to modify them in order to suppress or enhance certain characteristics such as solubility, efficiency, or stability in human body. Newman and Cragg (2008) estimated that
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Discussion about 60% of the drugs that are now available such as artemisinin, camptothecin, lovastatin were either directly or indirectly derived from natural products. Moreover, natural products have also been an invaluable source of inspiration for organic chemists to synthesize novel drug candidates (Beghyn et al., 2008).
It has been predicted that arthritis especially rheumatoid arthritis would rank fourth for the leading cause of disability by 2020. Arthritis is a global problem that will increase in significance with the growing elderly population. The condition affects both sexes and all races. This disease is characterized by inflammation of one or more joints, pain, wear and tear of joint and muscle strains. The traditional therapy recommended for the treatment of arthritis includes non-steroidal anti-inflammatory drugs like diclofenac, indomethacin etc, glucocorticoid therapy, disease modifying anti rheumatic drugs like methotreaxate, cyclosporine A, stem cell therapy, anti TNF- α blockers etc. but it is well known that the therapeutic managements have several side effects as a result of which the past decades or two have seen a dramatic increase and growing interest in the use of alternative treatments and herbal therapies in arthritis.
Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze is reported to contain chemical constituents which may exert analgesic and anti-inflammatory effect; however till now there were no investigations supporting the pharmacological properties of this plant. Therefore the present investigation was designed to evaluate the use of Cyathocline purpurea in pain, inflammation and arthritis. Three extracts of different polarities i.e. petroleum ether extract of Cyathocline purpurea (PECP), methanol extract of Cyathocline purpurea (MECP) and aqueous extract of Cyathocline purpurea (AECP) were prepared and tested for their analgesic and anti-inflammatory activities.
Acute oral toxicity study performed at the dose of 2000 mg/kg, p.o. revealed the non- toxic nature of all the three extracts PECP, MECP and AECP. There were no toxic reactions or mortality found with these extracts. Therefore the doses selected for the pharmacological studies were 100, 200 and 400 mg/kg, p.o. Phytochemical analysis of this extracts has mainly demonstrated the presence of flavonoids, steroids,
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Discussion alkaloids, phenols, tannins and saponins. Steroids and alkaloids have been reported to have analgesic and anti-inflammatory activity. Flavonoids and phenolic compounds have multiple biological effects such as antioxidant activity (Zeashan et al., 2009). Flavonoids have also been reported to have anti-inflammatory effect (Ilavarasan et al., 2006). Steroids can decrease inflammation and reduce the activity of the immune system, while triterpenoids impairs histamine release from mast cells and exerts anti- inflammatory effects (Mehta et al., 2012).
The peripheral analgesic effect may be mediated through inhibition of cyclooxygenase and/or lipooxygenases, while central analgesic action may be mediated through inhibition of central pain receptors (Shulan et al., 2011). Therefore peripheral (acetic acid induced writhing) and central (hot plate test) models were selected to observe the analgesic effect of PECP, MECP and AECP. Acetic acid induced writhing test is a simple, reliable and affords rapid evaluation of analgesic drugs (Ishola et al., 2011). The intraperitoneal injection of acetic acid elicited writhing (a syndrome characterized by a wave of abdominal musculature contraction followed by extension of the hind limbs). The intraperitoneal administration of agents that irritate serous membranes provokes a stereotypical behavior in mice which is characterized by abdominal contractions, movements of the body as a whole, twisting of dorsoabdominal muscles, and a reduction in motor activity and coordination (Perazzo et al., 2005). The abdominal constrictions induced in mice results from an acute inflammatory reaction with production of prostaglandins E2 and F2 in the peritoneal fluid (Ramachandran et al., 2011). MECP (400 mg/kg) significantly (p<0.001) inhibited the number of wriths with 35.29% inhibition. PECP (400 mg/kg) and MECP (200 mg/kg) also significantly (p<0.05) inhibited the number of wriths compared to vehicle control group. Acetyl salicylic acid (100 mg/kg) showed maximum activity with 64.71% inhibition. It has been reported that NSAID’s prevent prostaglandin production, thus sensitization of pain receptors by prostaglandin at the inflammatory site is inhibited (Dhara et al., 2000). The mechanism of peripheral analgesic action of MECP, likewise other NSAID’s, could probably be due to the blockade of effect or due to the release of endogenous substances that excite pain nerve endings. The hot plate model has been found to be suitable for the evaluation of centrally acting analgesics (Bhandare et al., 2010). Hence, the hot plate test was
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Discussion performed to check if PECP, MECP and AECP would have any central analgesic effect. There were no significant results obtained in these test with PECP, MECP and AECP. On the other hand pentazocine (5 mg/kg, s.c.) showed a significant result by elevating the pain threshold. Hence it can be assumed that PECP, MECP and AECP had no effect on central nervous system.
The anti-inflammatory activity of PECP, MECP and AECP in this study was investigated using the carrageenan induced paw edema and cotton pellet induced granuloma models. Carrageenan is a family of linear sulphated polysaccharides extracted from the red seaweed marine alga Chondrus crispus. Lambda carrageenan is used in animal models of inflammation to test analgesics because dilute carrageenan solution (1-2%) injection causes swelling and pain (Costa et al., 2004). Inflammation induced by carrageenan is an acute and highly reproducible inflammatory model. Carrageenan has been widely used as an inflammagen capable of inducing experimental inflammation (William et al., 2010). This model has frequently been used to evaluate the anti-inflammatory agents (Panthong et al., 2007). The induction of edema by using carrageenan is believed to be biphasic in nature. The first phase involved within 1 h of carrageenan administration is associated with the release of histamine and serotonin from mast cells. The second phase starts after 1 h and is characterized by an increased release of prostaglandins (PGs) in the inflammatory area. During the second phase, the macrophages are known to release the large amounts of interleukin-1 (IL-1) which led to the increased accumulation of polymorphic nuclear cells (PMNs) to the site of inflammation. The activated PMNs then release the lysosomal enzymes and active oxygen species to destroy connective tissue and induce paw swelling (Marzouk et al., 2010). Statistical analysis revealed that MECP (200 and 400 mg/kg) significantly (p<0.001) inhibited the development of paw edema induced by carrageenan from 3 h onwards. Therefore it may be assumed that MECP is associated with inhibition of later phase. PECP (400 mg/kg) also showed a significant (p<0.001) inhibition at 5 h, but was less active than MECP. Moreover, diclofenac (10 mg/kg) exhibited an enhanced effect of inhibiting the paw edema than MECP with 53.99% inhibition at 5 h.
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Discussion
Sub acute inflammation or proliferative phase is measured by methods for testing granuloma formation such as cotton pellet induced granuloma (Begum et al., 2010). The cotton pellet induced granuloma model is a widely used method to evaluate the transudative and proliferative components of chronic inflammation (Gupta et al., 2005). The repair phase of inflammation starts as proliferation of fibroblasts, as well as multiplication of small blood vessels. Such proliferating cells penetrate the exudates producing a highly vascularised reddened mass known as granulation tissue (Sanmugapriya et al., 2005). The fluid absorbed by the pellet greatly influences the wet weight of the granuloma and the dry weight correlates with the amount of granulomatous tissue formed (Babu et al., 2009). MECP (200 and 400 mg/kg) were significantly (p<0.001) effective in both the models of inflammation, i.e. carrageenan induced rat paw edema as well as cotton pellet induced granuloma, therefore it can be assumed that it is effective in all phases of inflammation i.e. acute, sub acute, and proliferative phases.
Evaluation of the ulcerogenic effect of the three extracts (PECP, MECP and AECP) on the rat stomach revealed a lesser ulceration of the gastric mucosa and absence of congestion as compared to diclofenac. Ulceration of the gastric mucosa by anti- inflammatory drugs is a common side effect which usually indicates that prostaglandin synthesis inhibition may be involved in their mechanisms of action. Inhibition of the synthesis of prostaglandin, a group of prostanoid mediators of inflammation and intact gastric mucosa is largely responsible for the anti- inflammatory and gastric ulceration effects of NSAIDs.
Thus the experimental findings in the study demonstrated the peripheral analgesic, and anti-inflammatory activity of Cyathocline purpurea extracts. PECP and AECP showed weak analgesic and anti-inflammatory effect when compared with MECP. MECP (200 and 400 mg/kg) was found to be highly effective. The results suggested that the mechanism of action of MECP seems to be similar to NSAID’s rather than to steroidal drugs. One of the factors responsible for anti-inflammatory activity of MECP is solubility of the active constituent in the solvent system used for preparation of methanolic extract. The absence of anti-inflammatory activity in the AECP may be due to poor solubility of the active ingredients in the water. MECP showed most
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Discussion significant anti-inflammatory activity; therefore it was further selected for determination of its antiarthritic potential in FCA induced arthritis model.
RA is a chronic inflammatory disease affecting about 1% of the population in developed countries (Amresh et al., 2007). Limb swelling, inflammatory cell infiltrations, proliferative synovitis, erosion of the bone are clinical findings common to human arthritis and adjuvant-induced arthritis rat. Owing to this similarity in pathologic features, the adjuvant-induced arthritis rat is a widely used model of RA in evaluating the efficacy of anti-arthritic drugs (Noguchi et al., 2005). Freund’s complete adjuvant arthritis is a well established model that has been used in numerous studies for identifying the potential therapeutic targets. In experimental arthritis animal model female Wistar rats were used because animal model provides more uniform experimental data and allow for extensive testing of potential therapies. Adjuvant arthritic is very similar to human RA both in pathological and serological changes, including the involvement of inflammatory mediators in the arthritic etiology (Gao et al., 2008). The acute stage of arthritis is characterized by signs of hyperalgesia, lack of mobility and pause in body weight gain; during the acute period, hind paw and fore paw joint diameter increase. In the later stage of disease (day 12+), rats with adjuvant arthritis are often relatively immobile due to severity of paw swelling (Amresh et al., 2007).
In the present study, MECP (200 and 400 mg/kg) treatment showed anti-arthritic effect in all the inflammatory parameters. It significantly decreased the inflammation compared to the arthritic control group as observed by decreased paw volume and joint diameter. The present study revealed that paw volume and joint diameter increases with ankle stiffness in FCA challenged rats. Paw swelling is one of the major factors in assessing the degree of inflammation and curative efficacy of drugs. Intraplantar injection of inflammatory agents, such as carrageenan, lipopolysaccharide (LPS), bacterial endotoxin or FCA produce mechanical or thermal hyperalgesia associated with an upregulation of IL-1β and other inflammatory cytokines in the inflamed tissue and in the dorsal root ganglia (DRG) (Ren and Richard, 2009). The analgesic effect of MECP (200 and 400 mg/kg) in rats with adjuvant arthritis is also marked as evident by the increase in pain threshold, paw withdrawal latency and
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Discussion mechanical nociceptive threshold. Von Frey filaments elucidate degree of mechanical nociceptive threshold where the mechanical nociceptive threshold is measured in response to increasing pressure stimuli applied to the plantar surface of paw by Von Frey filaments. Whereas tail flick unit throws IR beam on the inflamed paw bearing hyperalgesic response. Rats with severe arthritis in the arthritic control group demonstrated low paw withdrawal threshold. The animals treated with MECP (200 and 400 mg/kg) were able to bear significantly higher pressure whereas the animals in the arthritic control group were able to bear the minimum weight due to severe arthritic condition. The decrease in body weight during inflammation is due to reduced absorption of nutrients through the intestine (Patil and Suryavanshi, 2007). Therefore the restoration of the body weight in rats by MECP treatment may involve improvement in the absorption of the nutrients through the intestine of rats.
It has been reported that a moderate rise in the WBC count occurs in arthritic conditions due to an IL-1β mediated rise in the respective colony stimulating factors and reduction in Hb count in arthritis results from reduced erythropoietin levels, a decreased response of the bone marrow erythropoietin and premature destruction of red blood cells (Jalalpure et al., 2011). MECP and diclofenac treatments significantly decreased the WBC count and increased the Hb level. In addition to this, other characteristic haematological alterations such as the decreased RBC and increased platelet count were also significantly restored by the MECP and diclofenac.
MECP treatment significantly reduced the levels of RF and CRP dose dependently. RF could be a marker of RA, characterized by a significant increase in the incidence of distal interphalangeal arthritis (Patel et al., 2012). Also a persistent high serum level of CRP is recognized as strong indicator of RA (Pepys and Hirschfield, 2003). Challenge with FCA (0.1 ml) significantly (p<0.001) elevated the serum AST, ALT and ALP level and decreased the total protein level. Assessment of the serum levels of AST, ALT and ALP provides an excellent and simple tool to measure the anti- arthritic activity of the drug. The activities of aminotransferases and ALP increases significantly in arthritic rats, since these are good indices of liver and kidney impairment which is also considered a feature of adjuvant arthritis. Serum AST and ALT has been reported to play a vital role in the formation of biologically active
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Discussion chemical mediators such as bradykinins in inflammatory process (Mythilypriya et al., 2008). The administration of MECP dose dependently decreased the level of AST, ALT and ALP and increased the level of total protein which confirms the anti-arthritic effect.
The role of oxidative stress in arthritis is not surprising since reactive oxygen species serve as mediators of tissue damage. An antioxidant can be defined as any substance that when present in low concentrations compared to that of an oxidisable substrate significantly delays or inhibits the oxidation of that substrate (Halliwell, 1991). The physiological role of antioxidant as the definition suggest is to prevent damage to cellular components arising as a consequence of chemical reactions involving free radicals. It is well recognized that free radicals are critically involved in various pathological conditions like cancer, arthritis, inflammation and liver diseases (Vijayakumar et al., 2012). Lipid peroxidation is a critical mechanism of the injury that occurs during RA, which is often measured by analysis of tissue MDA. The large amount of MDA in arthritic control group is consistent with the occurrence of damage mediated by free radicals (Arulmozhi et al., 2011). Treatment with MECP (200 and 400 mg/kg) produced a significant reduction of MDA level. GSH reflect the endogenous defense against damage caused by ROS and organic peroxides as they act as an intracellular reductant in oxidation reduction processes. The decreased levels of GSH in liver of arthritic rats might be due to the excessive consumption of GSH by the system to defend oxidative damage (Hemshekhar et al., 2013). The production of oxygen free radicals that occurs with the development of arthritis leads to decreased GSH and SOD levels as a consequence of their consumption during oxidative stress and cellular lysis (Kizilntuc et al., 1998; Hassan et al., 2001) which is evident by decreased levels of GSH and SOD in arthritic control group. Oral administration of MECP to the rats significantly re-established the depleted levels of GSH and SOD, probably by competing for scavenging of free radicals.
From the histopathological studies of the ankle joint, it is evident that the inflammation of the connective tissue is controlled by treatment with MECP. Bone destruction, which is a common feature of adjuvant arthritis, was examined by radiological analysis (Patel et al., 2012). X-ray studies of the rat paws showed that
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Discussion treatment with diclofenac and MECP inhibited the arthritis associated joint alterations.
Thus the study revealed that MECP (200 and 400 mg/kg) possess significant anti- arthritic activity which is mediated by its analgesic and anti-inflammatory effects on different parameters like decrease in paw volume and joint diameter; increase in pain threshold, mechanical nociceptive threshold, and paw withdrawal latency. The anti- arthritic activity of MECP was also supported by haematological, biochemical, anti- oxidant, radiological and histopathological parameters. All this results thus predict that MECP provide pharmacological rationale for the traditional use of the plant against inflammatory conditions like RA.
Based upon the antiarthritic activity of MECP it was further subjected to fractionation by liquid-solid separation chromatographic technique followed by column chromatography and isolation of active compound was done. MECP was fractionated by liquid-solid separation chromatographic technique and the dose for screening the active anti-inflammatory fraction was reduced to 100 mg/kg, p.o. The anti- inflammatory activity was screened by carrageenan induced paw edema model in rats. In the fraction study (F – 1 to F – 6), the paw edema of the rats increased progressively after carrageenan injection. 30% acetone in petroleum ether fraction (F – 4) was found to exert the highest anti-inflammatory activity compared to other fractions, 30.77% inhibition of inflammation at 3rd h. Ten pools (P – 1 to P – 10) were collected from fraction F – 4 through column chromatography based on TLC, which were then screened for anti-inflammatory activity by carrageenan induced paw edema model in rats and the dose was reduced to 10 mg/kg, p.o. Pool (P – 8) showed most significant anti-inflammatory activity compared to other pools and was further subjected to preparative TLC for removal of impurities. Compound was scratched from the preparative TLC and subjected for structural elucidation using IR, 1H-NMR, 13C-NMR, DEPT and MS. Based on the spectral data obtained and its comparison with the reported spectral values in the literature (Nagasampagi et al., 1981) the compound P – 8 was identified as Isoivangustin, a known Sesquiterpene lactone. It had a melting point of 139 – 140 0C. Docking studies were carried out to study the binding mode of the isolated compound, isoivangustin on the active site of TNF-alpha
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Discussion converting enzyme (TACE). TACE converts membrane bound pro-TNF-α to mature and soluble TNF-α. The native ligand IH6 was successfully docked into the active site of TACE. Hydroxamate group of compound IH6 forms van der Waals interaction with zinc, the co-catalytic metal ion in the active site of the enzyme (Figure 44). The compound IH6 actively takes part in forming hydrogen bond interaction with the key amino acids Gly349 and Leu348 in the enzyme protein. The phenyl ring forms an interaction with amino acid His405 by П-П stacking. Furthermore, the compound is surrounded with residues, such as Ala439, Leu348, Val434, Tyr436, His415, Ilu438 and Pro437 in the enzyme and makes contacts through van der Waals interactions with these amino acids and the docking score of compound IH6 with TACE was - 7.432. Also, the binding studies of diclofenac with TNF-α were studied and it was found that it forms the van der Waals interaction with zinc and show П-П stacking with amino acid HIS405. The docking score for diclofenac with TACE was -7.358 (Figure 45). Docking analysis of isoivangustin at the active site of TACE showed hydrogen binding with amino acid Gly349 and Leu348 like in native ligand IH6 which showed hydrogen bonding with same amino acids. Also, the octahydronapthyl ring fits into hydrophobic pocket formed by amino acid His405 in the enzyme. The docking score of isoivangustin with TACE enzyme was -5.341, which shows that it has good binding interaction with active site of TACE (Figure 46). Validation of docking procedure: In order to validate our docking procedure, we eliminated the co-crystallized ligand IH6 from the active site, and redocked within the inhibitor binding cavity of TACE enzyme. In this study, the root mean square deviation value was below 2Å, showing that our docking method is valid for the inhibitors studied.
Therefore it was thought necessary to evaluate the antiarthritic activity of isoivangustin by FCA induced arthritis in rats and to find out its probable mechanism of action. The doses selected for the study were 2.5, 5 and 10 mg/kg, p.o. In the investigation of FCA induced RA, it was observed that swelling developed over twenty four hour period in the foot injected with FCA. Our results showed that isoivangustin, a sesquiterpene lactone isolated from methanol extract of Cyathocline purpurea showed anti-arthritic effect in all the inflammatory parameters. Isoivangustin (5 and 10 mg/kg) significantly inhibited development of paw volume
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Discussion and joint diameter dose dependently. Analgesic effect of isoivangustin (5 and 10 mg/kg) were observed as evident by the increase in pain threshold, thermal hyperalgesia and mechanical nociceptive threshold. The loss of body weight in the arthritic control animals could be due to reduced absorption of glucose and leucine in rat intestine in arthritic condition (Babu et al., 2009). Isoivangustin (10 mg/kg) restored the body weight in FCA injected rats; therefore it may also improve the absorption process.
Arthritic control rats showed a significant increase in WBC and platelet count. Treatment with isoivangustin tends to normalize the WBC and platelet count dose dependently. It has been reported that moderate rise in WBC count occurs in arthritic conditions due to an IL-1β; mediated rise in the respective colony stimulating factors. The result of haematological parameters reveals that isoivangustin increased the Hb level and RBC count dose dependently, supporting the anti-arthritic activity of isoivangustin. It has been reported that reduction in the Hb level during arthritis results from reduced erythropoietin levels, a decreased response of the bone marrow erythropoietin and premature destruction of RBC (Jalalpure et al., 2011). ESR is influenced by an increase in the plasma concentration of acute-phase reactant proteins in response to inflammation (Talwar et al., 2011). Isoivangustin treatment restored the ESR count by decreasing its level dose dependently.
Significant decrease in levels of RF and CRP by treatment with isoivangustin indicates the anti-arthritic potential. The highest levels of RF are usually found in RA, also CRP is a marker for inflammation and its level rises dramatically during inflammation (Mehta et al., 2012). The animals on exposure to FCA (or mycobacteria) in the early phases induces the release of cytokines such as TNF-α, IL- 1-β, IL-6, IFN-γ and several chemokines (Billiau and Matthys, 2001). It is also well known that leucocytes produce pro-inflammatory cytokines such as TNF-α and IL-1β which play important role in RA (Fan et al., 2005). TNF-α and IL-1 β originate from the activated macrophages, and TNF- α is also produced by antigen-primed helper T cells. These cytokines have been documented as critically important in RA in rats as well as in human. They contribute too many features of arthritic inflammation, including synovial tissue inflammation, synovial proliferation and cartilage and bone
Study of Analgesic, Anti-inflammatory and Antiarthritic activity of Indian medicinal plant in laboratory animals 239
Discussion damage (Zhang et al., 2009). The key mediators of RA are TNF-α, IL-1 and IL-6 that drive inflammation in RA (Eric and Lawrence, 1996). Isoivangustin significantly decreased the elevated levels of the pro-inflammatory cytokines TNF-α and IL-1 β in dose dependent manners that were induced by FCA. Isoivangustin was less effective in reducing the elevated levels of IL-6. Therefore the mechanism of isoivangustin for the anti-arthritic activity may be via modulation of cytokines TNF-α and IL-1β which have been associated with the pathogenesis of RA.
Assessment of the serum levels of AST, ALT and ALP provides an excellent and simple tool to measure the anti-arthritic activity. The activities of aminotransferases and ALP increases significantly in arthritic rats, since these are good indices of liver and kidney impairment which is also considered a feature of adjuvant arthritis (Mythilypriya et al., 2008). Serum AST and ALT has been reported to play a vital role in the formation of biologically active chemical mediators such as bradykinins in inflammatory process (Mali et al., 2011). The treatment with isoivangustin significantly decreased the serum levels of AST, ALT and ALP and increased the level of total protein which confirms its anti-arthritic activity.
There are many studies which have reported the important participation of reactive oxygen species in RA pathophysiology. In these studies, free radicals have been reported to increase in joint cavity first, and then start its effects on the vessel wall with a consequent origination of edema (Tastekin et al., 2007). Lipid peroxidation is a critical mechanism of the injury that occurs during RA, which is often measured by analysis of tissue MDA. The large amount of MDA in arthritic control group is consistent with the occurrence of damage mediated by free radicals. The production of oxygen free radicals that occurs with the development of arthritis in the articular cartilage leads to decreased GSH and SOD levels as a consequence of their consumption during oxidative stress and cellular lysis, which is evident by decreased levels of SOD and GSH in arthritic control group (Arulmozhi et al., 2011). Treatment with isoivangustin significantly decreased the MDA level and increased the depleted levels of GSH and SOD, probably by competing for scavenging of free radicals.
Study of Analgesic, Anti-inflammatory and Antiarthritic activity of Indian medicinal plant in laboratory animals 240
Discussion
Bone destruction is a common feature of RA which is examined by radiological analysis. Radiographic observation of the rat paw showed that treatment with isoivangustin and diclofenac inhibited arthritis associated joint alterations. From the histopathological studies of ankle joints of arthritic rats, there was destruction of joint, due to the continued migration of lymphocytes, monocytes, into the synovium and joint fluid, connective tissue proliferation and necrosis, all of which produce inflammatory cytokines. Treatment with isoivangustin and diclofenac inhibited this leukocyte migration, connective tissue proliferation and necrosis in arthritis which may have beneficial effects for joint preservation.
The study thus revealed that isoivangustin possess anti-arthritic activity which is mediated by its analgesic and anti-inflammatory effects on various parameters evaluated. The anti-arthritic activity is also supported by its effects on haematological, biochemical, anti-oxidant, radiological and histopathological parameters. The mechanism for its anti-arthritic effect is via suppression of cytokines TNF-α and IL- 1β.
Study of Analgesic, Anti-inflammatory and Antiarthritic activity of Indian medicinal plant in laboratory animals 241
Summary and Conclusion Summary and Conclusion
‹ In the present investigation the Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze. (whole plant) Fam. Asteraceae was selected for evaluation of analgesic, anti-inflammatory and antiarthritic activity. ‹ Three extracts of Cyathocline purpurea were prepared from petroleum ether, methanol and water and labeled as PECP, MECP and AECP, respectively. ‹ Phytochemical analysis of the extracts revealed presence of flavonoids, alkaloids, phenols, tannins in PECP and MECP along with saponins, steroids, and triterpenoids in MECP only. Glycosides, proteins, carbohydrate and amino acids were found to be present in AECP. ‹ Acute oral toxicity studies performed according to OECD guideline- 425 revealed that all the three extracts were safe at the dose of 2000 mg/kg. ‹ Analgesic activity of the three extracts was investigated using hot-plate test and acetic acid induced writhing model in Swiss albino mice. ‹ In hot plate test, all the extract didn’t show increase in pain latency, while standard drug pentazocine showed significant increase in pain latency. ‹ In acetic acid induced writhing model, MECP (400 mg/kg) showed significant decrease in number of writhings than PECP, while AECP showed no significant activity. ‹ Results of analgesic activity suggest that, among all the three extracts MECP has highest peripheral analgesic potential than the PECP and AECP. ‹ Anti-inflammatory activity of all the three extracts were investigated using carrageenan induced rat paw edema model and cotton pellet induced granuloma model in Wistar rats. ‹ In carrageenan induced rat paw edema model, MECP dose dependently showed significant decrease in paw volume. PECP was found to be less active in decreasing paw volume, while AECP showed no activity. ‹ In cotton pellet induced granuloma model, MECP (200 and 400 mg/kg) showed significant inhibition of granuloma formation. ‹ Histopathology of stomach was also performed in cotton pellet induced granuloma model to assess ulcerogenic property of all the extracts and the standard drug diclofenac. Diclofenac showed ulceration and congestion in
Study of Analgesic, Anti-inflammatory and Antiarthritic activity of Indian medicinal plant in laboratory animals 242
Summary and Conclusion
stomach. In comparison to that all the three extracts showed lesser ulceration and congestion. ‹ Results of anti-inflammatory activity in carrageenan-induced rat paw edema model as well as cotton pellet induced granuloma model suggested that, MECP had good anti-inflammatory activity than PECP and AECP. ‹ MECP showed superior analgesic and anti-inflammatory activity than PECP and AECP. So MECP was further selected for determination of its antiarthritic activity. ‹ Antiarthritic activity of MECP was investigated using FCA induced arthritis in female Wistar rats. ‹ Antiarthritic activity of MECP was assessed by regular parameters such as, paw volume, joint diameter, pain threshold, mechanical nociceptive threshold, paw withdrawal latency and body weight. Last day parameters include measurement of haematological, serum, biochemical, and antioxidant levels. Radiological and histopathology of ankle joint were also assessed. ‹ In case of paw volume and joint diameter, MECP (200 and 400 mg/kg) treated group showed significant inhibition of increase in paw volume and joint diameter as compared to arthritic control group. ‹ MECP (200 and 400 mg/kg) treated group showed significant increase in pain threshold, mechanical nociceptive threshold, paw withdrawal latency and body weight as compared to arthritic control group. This result confirms the analgesic activity of MECP. ‹ In case of haematological parameters, MECP (200 and 400 mg/kg) treated group showed significant increase in Hb and RBC count and significant decrease in WBC and platelet count as compared to arthritic control group. ‹ In case of serum parameters, CRP level and RF value was significantly decreased dose dependently by MECP (200 and 400 mg/kg) treatment as compared to arthritic control group. ‹ In measurement of biochemical parameters, MECP (200 and 400 mg/kg) treated group showed significant decrease in AST, ALT and alkaline phosphatase level, whereas total protein level was significantly increased as compared to arthritic control group.
Study of Analgesic, Anti-inflammatory and Antiarthritic activity of Indian medicinal plant in laboratory animals 243
Summary and Conclusion
‹ In case of antioxidant parameters, treatment with MECP (400 and 200 mg/kg) showed significant increase in SOD and GSH level and significant decrease in MDA level as compared to arthritic control group. ‹ In radiological analysis of ankle joints arthritic control group rats had developed definite joint space narrowing of the inter-tarsal joints, diffuse soft tissue swelling, cystic enlargement of bone and extensive erosions as compared to MECP (400 mg/kg) treated group. ‹ In histopathological studies, arthritic group showed massive influx of inflammatory cells, necrosis of bone, cartilage destruction and disturbed synovial lining. Treatment with MECP (400 mg/kg) showed significant protection against necrosis of bone with low influx of inflammatory cells and little cartilage destruction. ‹ Based upon these pharmacological data, we concluded that MECP possess peripheral analgesic, anti-inflammatory and antiarthritic activities in animal models. ‹ So, based upon these results MECP was further subjected to fractionation by liquid-solid separation chromatographic technique followed by column chromatography and isolation of active compound was done. ‹ By using liquid-solid separation chromatographic technique 6 fractions (F – 1 to F – 6) were prepared from MECP and labeled as petroleum ether fraction (F – 1), 10% acetone in petroleum ether fraction (F – 2), 20% acetone in petroleum ether fraction (F – 3), 30% acetone in petroleum ether fraction (F – 4), 50% acetone in petroleum ether fraction (F – 5) and methanol fraction (F – 6). ‹ Anti-inflammatory activity of all the fractions (F – 1 to F – 6) were investigated using carrageenan induced rat paw edema in rats. ‹ Result suggested that 30% acetone in petroleum ether fraction (F – 4) showed more significant inhibition of increase in paw volume compared to other fraction. ‹ Therefore 30% acetone in petroleum ether fraction (F – 4) was further selected for column chromatography to isolate active compound.
Study of Analgesic, Anti-inflammatory and Antiarthritic activity of Indian medicinal plant in laboratory animals 244
Summary and Conclusion
‹ By using column chromatography, 10 pools (P – 1 to P – 10) were collected from fraction F – 4. All the pools (P – 1 to P – 10) were analyzed by TLC. ‹ Anti-inflammatory activity of pools (P – 1 to P – 10) was investigated using carrageenan induced rat paw edema model. ‹ Result suggested that pool P – 8 showed more significant inhibition of increase in paw volume than the other pools, whose effect was comparable to standard drug diclofenac. ‹ Pool P – 8 was further subjected to preparative TLC to isolate active compound. Scrapped material was mixed with acetone and homogenized in vortex mixer, and filtered; filtrate was collected and allowed to evaporate. The compound was labeled as P – 8. ‹ The structure of isolated compound (P – 8) was elucidated by IR, 1H- NMR, 13C-NMR, DEPT and MS spectral analysis. ‹ Based upon the results of spectral analysis, the structure of the isolated compound P – 8 was found to be isoivangustin, a known sesquiterpene lactone. ‹ The docking study of isoivangustin was done to examine its binding mode with TNF-alpha converting enzyme (TACE) the score of which indicated that it has good binding with TACE. ‹ Therefore isoivangustin was isolated in bulk quantity and screened for antiarthritic activity in FCA induced arthritis model at the dose of 2.5, 5 and 10 mg/kg. ‹ Antiarthritic activity of isoivangustin was assessed by various parameters such as, paw volume, joint diameter, pain threshold, mechanical nociceptive threshold, paw withdrawal latency and body weight. On the last day, haematological, serum, biochemical, antioxidant parameters, radiological and histopathology of ankle joint were also assessed. ‹ Measurement of paw volume and joint diameter in isoivangustin treated group dose dependently showed decrease in paw volume and joint diameter as compared to arthritic control group. The results of isoivangustin (10 mg/kg) were comparable to standard drug diclofenac (5 mg/kg). This result confirmed the anti-inflammatory activity of isoivangustin.
Study of Analgesic, Anti-inflammatory and Antiarthritic activity of Indian medicinal plant in laboratory animals 245
Summary and Conclusion
‹ In measurement of pain threshold, mechanical nociceptive threshold, paw withdrawal latency and body weight, isoivangustin (10 mg/kg) treated group showed significant increase as compared to arthritic control group. These results were comparable to standard drug diclofenac which confirms its analgesic activity. ‹ In case of haematological parameters, isoivangustin (10 mg/kg) treated group showed significant increase in Hb and RBC level as compared to arthritic control group. Isoivangustin also showed significant decrease in WBC, ESR and platelet count as compared to arthritic control group. ‹ In case of serum parameters, the levels of CRP and RF value was significantly decreased by isoivangustin treatment. ‹ In case of cytokine measurement (TNF-α, IL-1β and IL-6) treatment with isoivangustin (10 mg/kg) showed significant decrease in level of TNF-α and IL-1β, whose effects were comparable to diclofenac. The levels of these cytokines were significantly increased in arthritic control group. ‹ In case of biochemical parameters, isoivangustin (10 mg/kg) treated group showed significant decrease in AST, ALT and alkaline phosphatase level, whereas total protein level was significantly increased as compared to arthritic control group. ‹ In case of antioxidant parameters, isoivangustin (10 mg/kg) treated group showed significant increase in SOD and GSH level as compared to arthritic control group. Isoivangustin (10 mg/kg) treated group showed significant decrease in MDA level as compared to arthritic control group. ‹ Radiographic observation of the rat paw showed that treatment with isoivangustin and diclofenac inhibited arthritis associated joint alterations. ‹ From the histopathological studies of ankle joints of arthritic rats, there was destruction of joint, due to the continued migration of lymphocytes, monocytes, into the synovium and joint fluid, connective tissue proliferation and necrosis, all of which produce inflammatory cytokines. Treatment with isoivangustin and diclofenac inhibited this leukocyte migration, connective tissue proliferation, and necrosis in arthritis which may have beneficial effects for joint preservation.
Study of Analgesic, Anti-inflammatory and Antiarthritic activity of Indian medicinal plant in laboratory animals 246
Summary and Conclusion
‹ It is thus concluded that, methanol extract of Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze possesses a significant anti-inflammatory and antiarthritic activity which is mainly due to isoivangustin, a sesquiterpene lactone. The mechanism of action appears to be due to inhibition of cytokines TNF-α and IL-1β.
Study of Analgesic, Anti-inflammatory and Antiarthritic activity of Indian medicinal plant in laboratory animals 247
Highlights of the work Highlight of the work
Symbols and its significance
Significant decrease (p<0.001)
Significant decrease (p<0.01)
Significant decrease (p<0.05)
No change
Significant increase (p<0.001)
Significant increase (p<0.01)
Significant increase (p<0.05)
Study of Analgesic, Anti-inflammatory and Antiarthritic activity of Indian medicinal plant in laboratory animals 248 Highlight of the work
Analgesic, anti-inflammatory and antiarthritic activities Cyathocline purpurea extracts
Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze Extracts PECP MECP AECP 100 200 400 100 200 400 100 200 400 Dose mg/ mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg kg Analgesic activity
Hot plate
Acetic acid induced
writhing Anti-inflammatory activity Carrageenan induced paw edema Cotton pellet granuloma
Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze MECP Dose 100 mg/kg 200 mg/kg 400 mg/kg Antiarthritic activity Change in paw volume
Change in joint diameter
Pain threshold
Mechanical nociceptive threshold
Paw withdrawal latency
Body
weight AST
ALT
Study of Analgesic, Anti-inflammatory and Antiarthritic activity of Indian medicinal plant in laboratory animals 249 Highlight of the work
Alkaline phosphatase
Total protein
CRP
RF
Haemoglobin
RBC
WBC
Platelets
MDA
SOD
GSH
MECP MECP MECP Histopathological Healthy Arthritic Diclofenac 100 200 400 analysis control control 5 mg/kg mg/kg mg/kg mg/kg Synovial lining - ++ - ++ - -
Influx of inflammatory - +++ + +++ ++ ++ cells Necrosis of bone - +++ - ++ ++ -
Cartilage destruction - +++ + ++ ++ +
- No change + Mild ++ Moderate +++ Severe
Study of Analgesic, Anti-inflammatory and Antiarthritic activity of Indian medicinal plant in laboratory animals 250
Highlight of the work
Anti-inflammatory activity by carrageenan induced paw edema of fractions and pools
Fractions Change in paw Volume F – 1 (100 mg/kg)
F – 2 (100 mg/kg)
F – 3 (100 mg/kg)
F – 4 (100 mg/kg)
F – 5 (100 mg/kg)
F – 6 (100 mg/kg)
Pools Change in paw Volume P – 1 (10 mg/kg)
P – 2 (10 mg/kg)
P – 3 (10 mg/kg)
P – 4 (10 mg/kg)
P – 5 (10 mg/kg)
P – 6 (10 mg/kg)
P – 7 (10 mg/kg)
P – 8 (10 mg/kg)
P – 9 (10 mg/kg)
P – 10 (10 mg/kg)
Study of Analgesic, Anti-inflammatory and Antiarthritic activity of Indian medicinal plant in laboratory animals 251
Highlight of the work
Antiarthritic activity of isoivangustin
Dose of 2.5 mg/kg 5 mg/kg 10 mg/kg Isoivangustin Change in paw volume
Change in joint diameter
Pain threshold
Mechanical nociceptive threshold
Paw withdrawal latency
Body weight AST
ALT
Alkaline phosphatase
Total protein
CRP
RF
Haemoglobin
RBC
WBC
Platelets
ESR
TNF-α
IL-1β
Study of Analgesic, Anti-inflammatory and Antiarthritic activity of Indian medicinal plant in laboratory animals 252 Highlight of the work
IL-6
MDA
SOD
GSH
Histopatholo Isoivangu Isoivangus Isoivangu Healthy Arthritic Diclofenac gical stin 2.5 tin 5 stin 10 control control 5 mg/kg analysis mg/kg mg/kg mg/kg Synovial lining - +++ - ++ + -
Influx of inflammatory - +++ + +++ ++ + cells Necrosis of bone - +++ + ++ ++ +
Cartilage destruction - +++ + ++ + +
- No change + Mild ++ Moderate +++ Severe
Study of Analgesic, Anti-inflammatory and Antiarthritic activity of Indian medicinal plant in laboratory animals 253
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Publications Publications
List of publications (International)
1. Gopal V. Bihani, Supada R. Rojatkar, Subhash L. Bodhankar. Anti-arthritic activity of methanol extract of Cyathocline purpurea (whole plant) in Freund’s complete adjuvant induced arthritis in rats. Biomedicine & Aging Pathology 2014; 4: 197-206.
2. Gopal V. Bihani, Supada R. Rojatkar, Subhash L. Bodhankar. Investigation of in-vivo analgesic and anti-inflammatory activity in rodents and in-vitro antioxidant activity of extracts of whole plant of Cyathocline purpurea. International Journal of Pharmacy and Pharmaceutical Sciences 2014; 6(4): 492-498.
3. Gopal Bihani, Supada Rojatkar, Sujit Bhansali, Vithal Kulkarni, Revansiddha Katte, Kiran Sonawane, Subhash Bodhankar. Spectral characterization, docking and in-vivo anti-inflammatory activity of isoivangustin, a constituent isolated from methanol extract of Cyathocline purpurea (Buch-Ham ex D. Don.) Kuntze. Der Pharmacia letter 2015; 7(4): 115-121.
4. Gopal V. Bihani, Supada R. Rojatkar, Subhash L. Bodhankar. Isoivangustin a sesquiterpene lactone attenuates pro-inflammatory cytokines levels in Freund’s complete adjuvant induced arthritis in Wistar rats. (Communicated to Pharmaceutical Biology. Manuscript No. NPHB-2015-1359).
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