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Investigation on the Pharmacoprinciples of a Pergularia Daemia (Forssk.) Chiov

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Investigation on the Pharmacoprinciples of a Pergularia Daemia (Forssk.) Chiov Investigation on the Pharmacoprinciples of a Pergularia daemia (Forssk.) Chiov. Thesis Submitted to the BHARATHIDASAN UNIVERSITY, TIRUCHIRAPPALLI for the award of the Degree of DOCTOR OF PHILOSOPHY IN BIOTECHNOLOGY By P. Vinoth Kumar, M.Sc. (Reg. No. 021817/Ph.D. 2/Biotechnology/Full-time/January 2008) Under the Guidance of Dr. N. Ramesh, M.Sc., M.Phil., Ph.D. DEPARTMENT OF BIOTECHNOLOGY J.J. COLLEGE OF ARTS AND SCIENCE (AFFILIATED TO BHARATHIDASAN UNIVERSITY) PUDUKKOTTAI 622 422, TAMIL NADU AUGUST 2013 DECLARATION I do hereby declare that the Thesis entitled “Investigation on the Pharmacoprinciples of a Pergularia daemia (Forssk.) Chiov. ” submitted to Bharathidasan University, Tirucharapalli, Tamil Nadu, has been carried out by me under the supervision of Dr. N. Ramesh, Assistant Professor, Department of Biotechnology, J.J.College of Arts and Science, Pudukkottai for award of Degree of DOCTOR OF PHILOSOPHY IN BIOTECHNOLOGY. I also declare that this Thesis is a result of my own effort and has not been submitted earlier for the award of any Diploma, Degree, Associateship, Fellowship or other similar title to any candidate other university. Place: Pudukkottai (P. VINOTH KUMAR) Date: ACKNOWLEDGEMENT I prostrate before the God for his blessing, which guided me in taking up this project and gave me the confidence and ability to complete it successfully. I express my personal indebtness and greatfulness to my Research Guide Dr. N. Ramesh, Asst. Prof., Department of Biotechnology, J.J. College of Arts and Science, Pudukkottai, for his sustained guidance and encouragement throughout the course of this project. I would like to express my sense of gratitude to Mr. N. Subramanian, Secretary, J.J. College of Arts and Science, Dr. Kavitha Subramanian, Management Trustee, J.J. College of Arts and Science and Dr. J. Parasuraman, Principal, J.J. College of Arts and Science, Pudukkottai for allowing me to under take this project work. I extend my sincere gratitude to my Doctoral Committee Member Dr. M.B. Viswanathan, Prof. Department of Plant Science, Bharathidasan University, Tirchurappalli, for valuable suggestions and encouragement. I am grateful to Mr. B. Venkatesh, Head, Department of Biotechnology, J.J. College of Arts and Science, Pudukkottai Mr. G. Manigandan, Asst. Prof., Department of Biotechnology, J.J. College of Arts and Science, Pudukkottai, Mr. V. Jeyaraman, Asst. Prof., Department of Biotechnology, J.J. College of Arts and Science, Pudukkottai and Ms. P. Priyadharshini, Asst. Prof., Department of Biotechnology, J.J. College of Arts and Science, Pudukkottai for their continued support. My deep since of gratitude is extended Prof. S. Navaneethan, Department of English, J.J. College of Arts and Science, Pudukkottai, and Dr.S. Siva Subramaniyan, Head, Department of Botany, J.J. College of Arts and Science, Pudukkottai for timely helpful and valuable suggestions during the course of this study. I deem it as a great previlage to thank sincerely and whole heartedly to the research scholars Mr. R. Ravi Kumar, Mr. A. Kalidhass, Mr. P. Muthu, Mr. A. Venkata Ramana for their timely help and constant encouragement throughout the course of this study. I thank the Lab assistants Ms. S. Usha, Ms. K. Thangamani, Ms. P. Hemalatha and Ms. R. Rajeswari for their help. I owe special thanks to my family and my Friends for successful completion of this thesis. P. Vinoth Kumar CONTENT Chapter-1 INTRODUCTION 1 1.1 Background of the Study 1 1.2 Diabetes Mellitus 3 1.3 Classification of Diabetes Mellitus 3 1.4 Demand for Medicinal Plants 4 1,5 Micropropagation and Callus Induction 5 1.6 Pergularia daemia – an Ethnomedicinal Plant 6 1.7 Ethnomedicinal uses 6 1.8 Epidemiological Studies 7 1.9 Pharmacological Studies of Pergularia daemia 8 1.10 Objectives and Scope of Research work 11 Chapter-2 REVIEW OF LITERATURE 12 2.1 Trials for Anti-diabetes Potential of Medicinal Plants 13 2.2 Phytochemical and Pharmacology of Pergularia species 14 2.2.1 Pergularia daemia Root- Phytochemical Constituents 14 2.2.1.1 Pergularia daemia Root-Biological Activity 14 2.2.2 Pergularia daemia Stem-Phytochemical Constituents 14 2.2.2.1 Pergularia daemia Stem-Biological Activity 15 2.2.3 Pergularia daemia Leaves-Biological Activity 15 2.2.4 Pergularia daemia Aerial Part-Phytochemical Constituents 15 2.2.4.1 Pergularia daemia Aerial Part-Biological Activity 16 2.2.5 Pergularia daemia Whole Plant-Phytochemical Constituents 16 2.2.5.1 Pergularia daemia Whole Plant-Biological Activity 16 2.2.5.2 Pergularia daemia Whole Plant-Phytochemical Constituents and 17 Biological Activity 2.2.6 Pergularia daemia Latex-Biological Activity 17 2.2.7 Pergularia pallida Whole Plant-Phytochemical Constituents 17 2.2.8 Pergularia tomentosa Whole Plant-Phytochemical Constituents 17 2.2.9 Pergularia tomentosa Whole Plant-Biological Activity 17 Chapter-3 MATERIALS AND METHODS 18 3.1 Plant Material 18 3.2 Plant Description 18 3.3 Strategy of Work Plan 19 3.4 Micropropagation and Callus Induction 19 3.4.1 Explant Collection 19 3.4.2 Sterilization of Explants 19 3.4.3 Preparation of Medium 20 3.4.4 Preparation of Stock Solution 20 3.4.5 Culture Medium and Conditions for Plant Regeneration 20 3.4.6 Effects of Basal Medium Strength on Multiple Shoot Induction 21 3.4.7 Effect and Concentration of Auxin for In Vitro Rooting 21 3.4.8 Hardening of In Vitro Grown Plant 21 3.5 Phytochemistry 22 3.5.1 Qualitative Analysis on Phytochemical Constituents 22 3.5.1.1 Test for Tannins 22 3.5.1.2 Test for Flavonoids 22 3.5.1.3 Test for Terpenoids - Puncal D test 22 3.5.1.4 Test for Alkaloids - Dragendorff’s test 23 3.5.1.5 Test for Glycosides 23 3.5.1.6 Test for Steroids 23 3.5.1.7 Test for Carbohydrates 23 3.5.2 Gas Chromatography –Mass Spectroscopic Analysis 23 3.6 Pharmacology 24 3.6.1 Animals 24 3.6.2 Chemicals 24 3.6.3 Acute Toxicity Study 24 3.6.4 Experimental Induction of Diabetes 25 3.6.5 Experimental Design 25 3.6.6 Preparation of Erythrocyte Lysate 26 3.6.7 Preparation of Tissue Homogenate 26 3.6.8 Biochemical Estimation 26 3.6.8.1 Determination of Blood Glucose 26 3.6.8.2 Estimation of Total Cholesterol 27 3.6.8.3 Estimation of Triglycerides 27 3.6.8.4 Estimation of Phospholipids 28 3.6.8.5 Estimation of Plasma Insulin 29 3.6.8.6 Estimation of Glycosylated Haemoglobin 29 3.6.8.7 Assay of Hexokinase 30 3.6.8.8 Assay of Glucose 6-phosphatase 30 3.6.8.9 Assay of Fructose 1, 6-bisphosphatase 31 3.6.8.10 Estimation of Tissue Protein 31 3.6.8.11 Estimation of Haemoglobin 32 3.6.8.12 Estimation of Albumin 32 3.6.8.13 Estimation of Urea 33 3.6.8.14 Estimation of Uric Acid 33 3.6.8.15 Estimation of Creatinine 34 3.6.8.16 Estimation of Glutathione Peroxidase 34 3.6.8.17 Estimation of Reduced Glutathione 34 3.6.8.18 Estimation of Glutathione-S-Transferase 35 3.6.8.19 Assay of Superoxide Dismutase 35 3.6.8.20 Estimation of Catalase 36 3.6.8.21 Estimation of Hydroperoxides 37 3.6.8.22 Estimation of Thiobarbituric Acid Reactive Substances 37 3.6.8.23 Estimation of Ascorbic Acid 38 3.6.8.24 Estimation of α–Tocopherol 39 3.6.8.25 Assay of Alkaline Phosphatase 39 3.6.8.26 Assay of Acid Phospatase 40 3.6.8.27 Assay of Aspartate Aminotransferase 40 3.6.8.28 Assay of Alanine Aminotransferase 41 3.6.9 Histopathological Studies 42 3.6.10 Statistical Analysis 42 Chapter-4 RESULTS AND DISCUSSION 43 4.1 Micropropagation and Callus Induction 43 4.2 Phytochemistry 47 4.3 Pharmacology 80 Chapter-5 SUMMARY 107 REFERENCES 112 PUBLICATIONS ABBREVIATIONS BAP _ 6-Benzylamino purine CAT _ Catalase D.W _ Dry weight F.W _ Fresh weight GPX _ Glutathione peroxidase GSH _ Reduced glutathione GST _ Glutathione-s-transferase H Hour H2O2 _ Hydrogen peroxide Hb _ Haemoglobin HbA1c _ Glycosylated haemoglobin IAA _ Indole 3-acetic acid IBA _ Indole 3-butyric acid IDDM _ Insulin dependent diabetes mellitus KIN _ Kinetin MS _ Murashige and Skoog Medium Min _ Minutes NIDDM _ Non Insulin dependent diabetes mellitus PGR _ Plant growth regulators SOD _ Superoxide dismutase STZ _ Streptozotocin TBARS _ Thiobarbituric acid reactive substances 2,4-D _ 2,4-Dichlorophenoxcy acetic acid α-NAA _ Naphthyl acetic acid Chapter 1 INTRODUCTION 1.1. Background of the Study Medicinal plants have been in use in traditional health care systems since ancient times and are still the most important health care source for the vast majority of the population around the world (Heinrich, 2000; Leaman, 2006; Teklehaymanot and Giday, 2007). At present, 80% of the population in developing countries rely largely on plant based drugs for their primary health care needs, and the world market for herbal products based on traditional knowledge was estimated to be worth US $ 60 million (WHO, 2002). Thirty percent of the drugs sold worldwide contain compounds derived from plant material. Among the 4,00,000 plant species on the earth, only a small percentage have been phytochemically investigated and the fraction submitted to biological or pharmacological screening is even smaller (Hostettmann et al., 1998). Only 1,00,000 secondary plant products have so far been characterized (Harborne and Williams, 2000). The plant kingdom thus represents an enormous reservoir of pharmacologically valuable materials to be discovered (Potterat and Hostettmann, 1995).
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