Chemical and Biological Studies of Medicinal Plants Used by the Yaegl Aboriginal Community of Australia Tarannum Naz Bachelor of Pharmacy (Honours) Master of Pharmacy A thesis submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy Department of Chemistry and Biomolecular Sciences Macquarie University Sydney, Australia 2013 TABLE OF CONTENTS TABLE OF CONTENTS ............................................................................................................. i LIST OF TABLES .................................................................................................................... vii LIST OF FIGURES ................................................................................................................... ix ABSTRACT ............................................................................................................................. xiii DECLARATION ...................................................................................................................... xv ACKNOWLEDGEMENTS ..................................................................................................... xvi LIST OF ABBREVIATIONS ................................................................................................ xviii CHAPTER ONE .............................................................................................................................. 1 1. Introduction ......................................................................................................................... 1 Overview ..................................................................................................................................... 2 1.1. Brief history of use of plants as medicines ...................................................................... 2 1.2. Natural products as a source of valuable modern medicines ........................................... 4 1.3. Ethnopharmacological approach - an effective avenue for drug discovery ..................... 6 1.4. The Aboriginal people in Australia ................................................................................. 8 1.4.1. The Yaegl Aboriginal community ............................................................................ 8 1.4.2. Collaboration with the Yaegl Aboriginal community .............................................. 9 1.4.3. Best ethical practices with the Yaegl community .................................................. 10 1.5. Necessity of searching for new drugs/leads for the treatment of skin infections and wounds ...................................................................................................................................... 10 1.5.1. Antimicrobials ........................................................................................................ 13 1.5.2. Anti-inflammatory agents ....................................................................................... 15 1.5.3. Antioxidants ........................................................................................................... 17 1.6. Objectives of this study .................................................................................................. 18 1.7. Thesis overview ............................................................................................................. 19 CHAPTER TWO ........................................................................................................................... 21 2. Selection of plants and methods for screening and isolation of bioactive compounds ..... 21 2.1. Introduction .................................................................................................................... 22 i 2.2. Review of literature of promising medicinal plants ....................................................... 25 2.3. Selection of two medicinal plants for detailed chemical and biological studies ........... 30 2.4. Lophostemon suaveolens ................................................................................................ 31 2.4.1. Description of Lophostemon suaveolens ................................................................ 32 2.4.2. Medicinal uses of Lophostemon suaveolens ........................................................... 34 2.4.3. Previous chemical and biological studies on Lophostemon suaveolens ................. 34 2.4.4. Studies on other species of Lophostemon ............................................................... 34 2.5. Alphitonia excelsa .......................................................................................................... 35 2.5.1. Description of Alphitonia excelsa ........................................................................... 36 2.5.2. Medicinal uses of Alphitonia excelsa ..................................................................... 37 2.5.3. Previous chemical studies on Alphitonia excelsa ................................................... 38 2.5.4. Previous biological studies on Alphitonia excelsa .................................................. 39 2.5.5. Previous chemical, biological studies and uses of other species of Alphitonia ...... 39 2.6. Selection of methods for biological testing of extracts and for isolation of bioactive compounds ................................................................................................................................ 41 2.6.1.1. Antimicrobial activity study ............................................................................ 41 2.6.1.2. Anti-inflammatory activity study .................................................................... 44 2.6.1.3. Antioxidant assay (ORAC assay) .................................................................... 50 2.6.2. Selection of methods for chemical studies ............................................................. 52 2.7. Concluding remarks ....................................................................................................... 52 CHAPTER THREE ........................................................................................................................ 53 3. Chemical and biological studies on Lophostemon suaveolens .......................................... 53 3.1. Introduction .................................................................................................................... 54 3.2. Experimental .................................................................................................................. 54 3.2.1. General experimental procedures ........................................................................... 54 3.2.2. Plant material .......................................................................................................... 55 3.2.3. Preparation of extracts ............................................................................................ 55 3.2.4. Bioassays: methods and materials .......................................................................... 56 3.2.4.1. Selection of microorganisms for antibacterial study ....................................... 56 3.2.4.2. Disc diffusion assay for antibacterial activity ................................................. 57 3.2.4.3. TLC bioautography ......................................................................................... 58 3.2.4.4. MTT microdilution (turbidity and MTT) assay .............................................. 58 3.2.4.5. Anti-inflammatory and antioxidant assays ...................................................... 60 3.2.5. Preliminary phytochemical screening ..................................................................... 64 ii 3.2.5.1. Preparation of staining agents for TLC plates ................................................. 64 3.2.6. GC-MS analysis ...................................................................................................... 65 3.2.7. Chemical study methods and materials .................................................................. 65 3.2.7.1. Isolation of bioactive compounds from LS-DCM extract ............................... 65 3.2.7.2. Isolation of bioactive compounds from LS-Hex extract ................................. 68 3.3. Results and discussion ................................................................................................... 69 3.3.1. Preliminary screening and extraction of Lophostemon suaveolens leaves ............. 69 3.3.2. Antibacterial screening of extracts of Lophostemon suaveolens ............................ 72 3.3.2.1. Disc diffusion assay ........................................................................................ 72 3.3.2.2. MTT microdilution assay ................................................................................ 73 3.3.3. Phytochemical screening ........................................................................................ 76 3.3.4. GC-MS analysis of LS-Hex extract of Lophostemon suaveolens .......................... 77 3.3.5. Bioassay guided isolation of bioactive compounds from LS-Hex and LS-DCM .. 82 3.3.5.1. Optimisation of TLC bioautography ............................................................... 82 3.3.5.2. Isolation of bioactive fractions and compounds from LS-DCM ..................... 86 3.3.5.3. Isolation of bioactive compounds from LS-Hex extract ................................. 91 3.3.6. Summary of bioassay guided studies ..................................................................... 94 3.3.7. Characterisation of bioactive