UHI Thesis - pdf download summary Quantitative analysis of active compounds found in Arnica montana and Arnica chamissonis in relation to varied provenance, environmental and agronomic factors. Barron-Majerik, Elizabeth DOCTOR OF PHILOSOPHY (AWARDED BY OU/ABERDEEN) Award date: 2011 Awarding institution: The University of Edinburgh Link URL to thesis in UHI Research Database General rights and useage policy Copyright,IP and moral rights for the publications made accessible in the UHI Research Database are retained by the author, users must recognise and abide by the legal requirements associated with these rights. This copy has been supplied on the understanding that it is copyright material and that no quotation from the thesis may be published without proper acknowledgement, or without prior permission from the author. 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A thesis presented for the degree of Doctor of Philosophy at the University of Aberdeen June 2010 Elizabeth J. Barron BSc (Hons) Plant Science, University of Edinburgh Arnica montana L. Inverness College UHI Longman Campus Inverness College Highlands IV1 1SA Declaration I, Elizabeth Jane Barron, confirm that I composed the thesis, that it has not been accepted in any previous application for a degree, that the work is my own, and that all quotations have been distinguished by quotation marks and the sources of information specifically acknowledged. Signature: Date: i Acknowledgements Firstly, thanks go to my family. Without their unconditional support, love and wine, none of this would have been possible. Thank you also to my friends who have supported me throughout this project. Particularly, Katie, Gordon, Wendy, Matthew and Chantel: I owe you. Jan, you were right about the film, but thanks for sticking by me nonetheless. This was a part-time PhD, which meant it took a lot longer than most projects to be completed. Credit is due to my very patient supervisors, Melanie and Peter (and Paddy who retired before I could finish), who miraculously maintained enthusiasm for this project, even when mine was waning. Thank you to the Agronomy Team at Orkney, particularly John Wishart whose help was indispensable to this project. Thank you to Kenny Boyd and Sandy Gray for your advice and guidance and thanks also to my very understanding line manager Janet Mahon, who expertly found the perfect balance between support, encouragement and blatant threats. But above all this is for my dad, who isn’t here to see me finish, but it is thanks to him that I did. ii Abstract The northern hemisphere plant Arnica montana L. can be found across Europe on high alpine slopes with acidic soils. The flowers are well documented as a source of natural products, particularly, active compounds associated with anti- inflammatory properties. The high demand for this extract has meant that wild plant numbers have reduced dramatically and now in many countries, it is illegal to harvest A. montana from the wild. As a result, flowers from the non-endangered A. chamissonis Less. have been employed for the production of extracts with similar properties. A. montana and A. chamissonis were both grown during the period 2004 to 2007 in the North of Scotland (Orkney) in order to determine whether this environment was condusive to high yield. The qualitative and quantitative effects of weeding, fertiliser, mulch, seed source and species on yield were also studied. This research found that A. montana had high quality extract but was prone to crown rot and was less robust than initial trials suggested. Conversely, while A. chamissonis grew vigorously, the extract contained approximately a third of the sesquiterpene lactone content. iii Contents iv Figures xi Tables xxi List of Abbreviations xxv 1. Introduction 1 1.1 Research context 1 1.2 Arnica extract 2 1.3 The market 7 1.4 The taxonomy of Arnica 9 1.4.1 Arnica montana 12 1.4.1.1 Distribution and growth 12 1.4.1.2 Description 14 1.4.2 Arnica chamissonis 16 1.4.2.1 Distribution and growth 16 1.4.2.2 Description 17 1.5 Pests and grazing 19 1.6 Conservation and cultivation 21 1.7 Alternative agricultural crops and the Highlands and Islands 24 1.8 The chemistry of Arnica 29 1.8.1 Terpenes 31 1.8.2 Sesquiterpenes 33 1.8.3 Sesquiterpene lactones 34 1.8.3.1 Classification of sesquiterpene lactones 35 1.8.3.2 Sesquiterpene lactones and chemosystematics 39 iv 1.8.3.3 Sesquiterpene lactones in Arnica 42 1.8.3.4 The role of sesquiterpene lactones in plants 48 1.8.3.5 The medical uses of sesquiterpene lactones 49 1.8.3.6 Chemical synthesis of sesquiterpene lactones 55 1.8.4 Other constituents of Arnica extract 55 1.8.5 Toxicology 57 1.9 Discussion 59 1.10 Research question 61 1.11 Aim 61 1.12 Objectives for this study 61 2. The extraction, analysis and identification of sesquiterpene lactones 63 2.1 Introduction 63 2.1.1 Flower material 63 2.1.2 Extraction 64 2.1.3 Chemical analysis 73 2.1.4 Compound identification 74 2.1.5 Quantification 80 2.2 Methodology 83 2.2.1 Flower material 83 2.2.2 Extraction methods 84 2.2.2.1 Hydrodistillation 84 2.2.2.2 Solvent extraction 86 2.2.3 GC-MS methodology 87 2.2.4 Compound identification 88 2.2.5 Quantification of sesquiterpene lactones 89 v 2.3 Results 92 2.3.1 Calibration curve 92 2.3.2 Hydrodistillation extraction results 94 2.3.3 Results of essential oil analysis 94 2.3.4 Solvent extraction compound identification of A. montana 102 2.3.4.1 Peak 1 103 2.3.4.2 Peak 2 104 2.3.4.3 Peak 3 106 2.3.4.4 Peak 4 107 2.3.4.5 Peak 5 109 2.3.4.6 Peak 6 110 2.3.4.7 Peak 7 111 2.3.4.8 Peak 8 112 2.3.4.9 Peak 9 113 2.3.4.10 Peak 10 115 2.3.4.11 Peak 11 116 2.3.4.12 Peak 12 117 2.3.4.13 Peak 13 118 2.3.4.14 Peak 14 119 2.3.5 Quantitative analysis of A. montana solvent extract 120 2.3.5.1 Commercial and Orcadian extract 120 2.3.5.2 Extract from different floral stages 124 2.3.6 Solvent extraction compound identification of A. chamissonis 128 2.3.6.1 Peak 15 128 vi 2.3.6.2 Peak 16 131 2.3.6.3 Peak 17 133 2.3.7 Quantitative analysis of A. chamissonis solvent extract 135 2.3.7.1 A. chamissonis floral extract 135 2.3.7.2 Extract from different floral stages 136 2.4 Discussion 138 3. Yield of A. montana and A. chamissonis over 6 years 145 3.1 Introduction 145 3.1.1 Yield: a balance between quality and quantity 145 3.1.2 The established trials 148 3.1.3 Harvesting method 152 3.1.4 The climate 156 3.1.5 The soil 160 3.2 Materials and Methods 162 3.2.1 The site 162 3.2.2 Meteorological data 163 3.2.3 Flower harvesting 164 3.2.4 Extraction 165 3.2.5 Analysis 165 3.2.6 Statistical analysis 165 3.3 Results 166 3.3.1 Soil 166 3.3.2 Climate 170 3.3.3 Yield 175 vii 3.3.4 Competition and herbivory 179 3.3.5 Correlations between yield and environmental conditions 184 3.3.6 Quantitative and qualitative analysis of floral extract 20 5 3.4 Discussion of the yield and floral extract results 219 4. The influence of fertiliser and weeding on yield 227 4.1 Introduction 227 4.2 Materials and Methods 236 4.2.1 The sites 236 4.2.1.1 AM3 236 4.2.1.2.AM5 238 4.2.2 Treatment regime 239 4.2.3 Harvesting 242 4.2.4 Extractions 243 4.2.5 Analysis 243 4.3 Results 245 4.3.1 Plant survival 245 4.3.2 Yield 248 4.3.2.1 AM3 248 4.3.2.2 AM5 250 4.3.3 Essential oil 253 4.3.4 Sesquiterpene lactone production 257 4.4 Discussion 262 5. The influence of soil and planting regime on yield 268 5.1 Introduction 268 5.2 Materials and Methods 275 viii 5.2.1 The sites 275 5.2.1.1 Mulch (AM7) 276 5.2.1.2 Ridges and mulch (ACM1) 278 5.2.1.3 Ridges and seed source (ACM2) 281 5.2.2 Chemical analysis 282 5.2.3 Statistical analysis 282 5.3 Results 283 5.3.1 Plant survival 283 5.3.1.1 AM7 283 5.3.1.2 ACM1 289 5.3.1.3 ACM2 290 5.3.2 Flower production 291 5.3.2.1 AM7 291 5.3.2.2 ACM1 291 5.3.2.3 ACM2 294 5.3.3 Sesquiterpene lactone production 294 5.3.3.1 AM7 294 5.3.3.2 ACM1 – A.