PHYTOCHEMICAL AND BIOLOGICAL STUDIES OF SELECTED MEDICINAL PLANTS Farsetia hamiltonii (BRASSICACEAE) AND Neurada procumbens (NEURADACEAE) FROM CHOLISTAN DESERT (PAKISTAN) Thesis submitted in the fulfilment of the degree of Ph.D. Pharmacy (Pharmaceutical Chemistry) By Muhammad Munawar Hayat Roll No. 04-PhDC-12 Session 2012-2015 FACULTY OF PHARMACY BAHAUDDIN ZAKARIYA UNIVERSITY MULTAN-PAKISTAN (In the name of ALLAH, Who is Most Merciful and Most Beneficent) II Al-Quran Read! With the name of thy Allah, Who created you” (Al Alaq; 1). III IV V BRIEF CONTENTS Sr. No. Description Page No. 1 Title i 2 List of contents vii-xi 3 Abstract xv-xvii 4 List of tables xviii 5 List of figures xix-xxi 6 Chapter No.1: Introduction 01 7 Aims and objective of study 14 8 Chapter No.2: Literature review 16 9 Chapter No.3: Material and methods 31 10 Chapter No.4: Results and discussion 49 11 Discussion 98 12 Conclusion 108 13 References 110 14 Appendix (Publications and CV of scholar) 122 VI LIST OF CONTENTS Sr. No. Description Page No. 1 Title i 2 Bismillah ii 3 Al-Quran iii 4 Attendance sheet of Viva iv 5 Examiners report for PhD Viva-Voce / Public defense v 6 Brief contents vi 7 List of contents vii-xi 8 Dedication xii 9 Acknowledgements xiii 10 Certificate xiv 11 Abstract xv-xvii 12 List of tables xviii 13 List of figures xix-xxi 14 List of abbreviations xxii Chapter No. 1: Introduction 01 1 Medicinal plants 02 1.1 Cholistan desert 03 1.2 Botanical description of family Brassicaceae 03 1.3 Botanical description of genus Farsetia 04 1.4 Farsetia hamiltonii Royle 06 1.5 Botanical description of family Neuradaceae 09 1.6 Botanical description of genus Neurada 09 1.7 Neurada procumbens L. 10 1.8 Aims and objectives of study 14 Chapter No. 2: Literature Review 16 2 Literature review on selected plants 17 2.1 Literature review on Farsetia hamiltonii Royle 17 2.1.1 Literature survey on the phytochemical studies of family 17 Brassicaceae 2.1.1.1 Flavonoids 17 VII Sr. No. Description Page No. 2.1.1.2 Hydroxycinnamic acids 18 2.1.1.3 Vitamins 18 2.1.1.4 Minerals 19 2.1.1.5 Phytoalexins 19 2.1.1.6 Glucosinolates 22 2.1.2 Phytochemical studies of the genus Farsetia 22 2.1.3 Studies on the biological activities of the genus Farsetia 26 2.1.3.1 Cytotoxic activity 26 2.1.3.2 Antimicrobial activity 26 2.1.3.3 Antioxidant activity 26 2.1.4 Folk uses of genus Farsetia 27 2.2 Literature review on Neurada procumbens L. 28 2.2.1 Literature survey on the phytochemical studies family 28 Neuradaceae 2.2.2 Phytochemical studies of Neurada procumbens L. 28 2.2.3 Biological activities of Neurada procumbens L. 29 2.2.3.1 Effect on Blood Pressure 29 2.2.3.2 Antibacterial activity 29 2.2.3.3 Antifungal activity 29 2.2.4 Folk uses of Neurada procumbens L. 30 Chapter No.3: Material and Methods 31 3.1 Plant collection 32 3.2 Chemicals and reagents 32 3.3 Instrumentation 32 3.4 Detection of secondary metabolites 35 3.4.1 Detection of alkaloids 35 3.4.2 Detection of cardiac glycosides 35 3.4.3 Detection of anthraquinones 35 3.4.4 Detection of flavonoids 36 3.4.5 Detection of saponins 36 3.4.6 Detection of terpenoids 36 VIII Sr. No. Description Page No. 3.4.7 Detection of sterols 36 3.5 Determination of mineral contents 36 3.5.1 Atomic absorption spectroscopy 37 3.6 Extraction 37 3.7 Preparation of reagents 38 3.7.1 Mayer‟s reagent 38 3.7.2 Dragendorff‟s reagent 38 3.7.3 Wagner‟s reagent 38 3.7.4 Hager‟s reagent 38 3.7.5 Godin‟s reagent 38 3.8 Preparation of Solutions 38 3.8.1 Dilute sulphuric acid (10%) 38 3.8.2 Dilute ammonia solution 38 3.8.3 Dilute hydrochloric acid 39 3.8.4 Sodium hydroxide (10M) solution 39 3.8.5 Ferric chloride solution (10%) 39 3.8.6 Ferric chloride solution (3.5%) 39 3.8.7 Gelatin (1%) solution 39 3.8.8 Lead sub-acetate solution 39 3.9 Total flavonoid contents determination 39 3.10 Total phenol contents determination 40 3.11 Gas chromatography-mass spectrophotometry 40 3.12 Biological and enzyme inhibition activities 40 3.12.1 Antioxidant activity 40 3.12.2 Cholinesterase inhibition activities 41 3.12.3 Lipoxygenase inhibition activity 42 3.12.4 Chymotripsin inhibition activity 43 3.12.5 α-glucosidase inhibition activity 43 3.12.6 Urease inhibition activity 43 3.12.7 Tyrosinase inhibition activity 44 3.12.8 Carbonic anhydrase II inhibition activity 44 IX Sr. No. Description Page No. 3.12.9 Phosphodiesterase I inhibition activity 44 3.12.10 Antibacterial activity 45 3.13 Experiments on isolated tissues 45 3.13.1 Search for spasmolytic activity 46 3.13.2 Search for bronchorelaxant activity 45 3.14 In vivo study 46 3.14.1 Screening for hypoglycaemic activity 46 3.14.1.1 Induction of hyperglycemia 47 3.14.1.2 Administration of plants extracts 47 3.14.1.3 Protocol of study 48 Chapter No. 4: Results and Discussion 49 4.1 Phytochemical screening of secondary metabollites 50 4.2 Results of atomic absorption spectroscopy (AAS) 51 4.3 Extraction yields 51 4.4 Determination of total flavonoid and phenol contents 52 4.5 GC-MS analysis results of dichloromethane and methanol 53 extracts of different parts of Farsetia hamiltonii Royle and Neurada procumbens L. 4.5.1 GC-MS results of dichloromethane extract of aerial part of 53 Farsetia hamiltonii Royle 4.5.2 GC-MS results of methanol extract of aerial part of 58 Farsetia hamiltonii Royle 4.5.3 GC-MS results of dichloromethane extract of root part of 62 Farsetia hamiltonii Royle 4.5.4 GC-MS results of methanol extract of root part of 68 Farsetia hamiltonii Royle 4.5.5 GC-MS results of dichloromethane extract of Neurada 71 procumbens L. 4.5.6 GC-MS results of methanol extract of Neurada 78 procumbens L. 4.6 Biological and enzymatic activities of extracts of selected 83 medicinal plants. X Sr. No. Description Page No. 4.6.1 Antioxidant activity 83 4.6.2 Cholinesterase inhibition activity 84 4.6.3 Lipoxygenase inhibition activity 84 4.6.4 Chymotripsin inhibition activity 85 4.6.5 Alpha glucosidase inhibition activity 85 4.6.6 Urease inhibition activity 86 4.6.7 Tyrosinase inhibition activity 86 4.6.8 Carbonic anhydrase II inhibition activity 87 4.6.9 Phosphodiesterase I inhibition activity 87 4.6.10 Antibacterial activity (%age inhibition) 88 4.7 Biological activities of selected medicinal plants on 89 isolated tissues 4.7.1 Spasmolytic activity 89 4.7.2 Brochorelaxant activity 94 4.8 In vivo hypoglycaemic activity of F. hamiltonii 96 4.9 Discussion 98 4.10 Conclusion 108 5 References 110 6 Appendix (Publications from Ph D work) 122 6.1 Research article-1: Published in Journal of Ethanopharmacology 6.2 Research article-2: Published in Journal of Chemical Society of Pakistan 6.3 Research article-3: Accepted in Biomedical Research Journal 6.4 CV of Ph D scholar XI DEDICATION The study is dedicated to my beloved family, whose support; encouragement and prayers have always been medium of devotion and power for the achievement of this objective. XII ACKNOWLEDGEMENTS all praises for ,ﷻI begin by bowing my heart to ALMIGHTY ALLAH the most merciful, the most beneficent, Who helped and ,ﷻALMIGHTY ALLAH blessed me all the way along till the completion of the research work. Without His grace and mercy, I am nothing. Then I would pay gratitude to our beloved Prophet .who guided the humanity to the right path ,ﷺHazrat MUHAMMAD In the first place with great pleasure, I express my most cordial and humble thanks to my eminent, respected guide and supervisor Prof. Dr. Muhammad Uzair, Chairman, Department of Pharmaceutical Chemistry, Faculty of Pharmacy, B. Z. University, Multan. His real time guide line, supportive attitude and motivation made me able to complete the research work. His nice, humble and motivated behaviour is exemplary for the whole period of lab work. I express my deep gratitude and special thanks to Chairman, Department of Pharmacy and Dean, Faculty of Pharmacy Prof. Dr. Bashir Ahmed Chaudhary for his valuable guidance and enduring support during my research work. I express my most cordial and humble thanks to my eminent, respected guide and teacher Prof. Dr. Shazia Anjum, Department of Chemistry, The Islamia University of Bahawalpur for providing her valuable guidance, inspiration, supervision, keen interest and moral support from the very early stage of my research work till the end of my dissertation work. Her encouraging words were source of constant motivation to act with quality and precision and they enriched my growth as a student. I am indebted to her for her constant support. I am indebted to my FATHER, for his care, support and love. I have no suitable words that can fully describe his everlasting love to me. It is his dream that I get excellence in my studies. I cannot ask for more from my MOTHER as she is simply perfect. I have no suitable words that can fully describe her everlasting prayers and love to me. I remember many sleepless nights with her accompanying me, giving me strength and praying for my success.