Ligusticum Canbyi Coult. &A

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Ligusticum Canbyi Coult. &A The Novel Use of Metabolomics as a Hypothesis Generating Technique for Analysis of Medicinal Plants: Ligusticum canbyi Coult. & Rose and Artemisia tridentata Nutt. by Christina Turi M.Sc., University of Kent, Canterbury, UK 2009 B.Sc., University of British Columbia 2007 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE COLLEGE OF GRADUATE STUDIES (Biology) THE UNIVERSITY OF BRITISH COLUMBIA (Okanagan) June 2014 © Christina Turi, 2014 Abstract In response to the environment, plants produce a phytochemical arsenal to communicate and to withstand abiotic and biotic pressures. The average plant tissue contains upwards of 30,000 phytochemicals. The vast majority of approaches used to study plant chemistry are reductionist and only target specific classes of compounds which can be easily isolated or detected. Metabolomics is the qualitative and quantitative analysis of all metabolites present in a biological sample. By providing researchers with a phytochemical snapshot of all existing metabolites present in a sample, metabolomics has allowed researchers to study plant primary and secondary metabolism in ways that were never done before. The first objective of this thesis is to identify candidate species for studying plant neurochemicals. Statistical analysis using residual, bayesian and binomial analysis was applied to the University of Michigan’s Native American Ethnobotany Database and revealed that the genera Artemisa and Ligusticum are used most frequently during ceremony and ritual. Plant melatonin, serotonin, γ-aminobutyric acid, and acetylcholine were quantified in Artemisia tridentata Nutt. and Ligusticum canbyi Coult. & Rose. Significant variability was observed between tissue types, germplasm line and species. Manipulation of cholinergic signalling in A. tridentata led to changes in auxin, melatonin and serotonin levels, and suggests cross-talk between cholinergic and indoleamine pathways could be occurring in plants. The second objective of this thesis is to develop novel statistical and biochemical tools for analyzing metabolomic datasets and hypothesis generation. Datasets were generated using ultra performance reverse phase chromatography with time of flight mass spectrometry detection. Between 16,000 and 40, 000 metabolites were detected in L. canbyi and A. tridentata root and leaf tissues respectively. Principal component analysis, synthetic biotransformation, significant ion generation, putative identification and logical algorithms were applied in order to develop hypotheses and research approaches for pthalide biosynthesis, plant responses to stress, and the biological activity of smoke. Metabolomics, as a field of study, is still in its infancy. Thus approaches to effectively mine datasets are still ii needed, and provide researchers with new ways to examine the processes which regulate the production of primary and secondary metabolites in vivo. iii Preface A version of Chapter 1 has been published in the following manuscripts: Turi, C.E, Murch, S.J., 2011. The genus Ligusticum in North America: An ethnobotanical review with special emphasis upon species commercially known as ‘Osha’. HerbalGram 89, 40-51. I conducted the literature review and wrote most of the manuscript. Turi, C.E., Shipley, P.R., Murch, S.J., 2014a. North American Artemisia species from the subgenus Tridentatae (Sagebrush): A phytochemical, botanical and pharmacological review. Phytochemistry. I conducted the literature review and wrote most of the manuscript. The section on phytochemistry was revised by Dr. Paul Shipley, all structures were drawn by him. A version of chapter 2 has been published. Turi, C.E., Murch, S.J., 2013b. Spiritual and ceremonial plants in North America: An assessment of Moerman's Ethnobotanical Database comparing residual, binomial, bayesian and imprecise dirichlet model (IDM) analysis. J. Ethnopharmacol. 148, 386-394. doi: 10.1016/j.jep.2013.03.029. I accessed Moerman’s database and conducted all the reported statistical analysis. A majority of the manuscript was written by me. A version of chapter 3 has been published. Turi, C.E., Murch, S.J., 2013a. Targeted and untargeted phytochemistry of Ligusticum canbyi: Indoleamines, phthalides, antioxidant potential, and use of metabolomics as a hypothesis-generating technique for compound discovery. Planta Med. 79, 1370-1379. doi: 10.1055/s-0033-1350618. I conducted targeted, untargeted and antioxidant analysis and wrote the majority of the manuscript. A version of chapter 4 has been published. Turi, C.E., Axwik, K.E., Murch, S.J., 2014b. In vitro conservation, phytochemistry, and medicinal activity of Artemisia tridentata Nutt.: metabolomics as a hypothesis-generating tool for plant tissue culture. Plant Growth Regulation. Doi: 10.1007/s10725-014-9915-y. I wrote the majority of this manuscript and conducted both untargeted and anti-AchE analysis. Germplasm lines were established with assistance from Katie Axwik. A version of chapter 5 has been published. Turi, C.E., Axwik, K.E., Smith, A., Jones, M.P., Saxena, P.K., Murch, S.J., 2014c. Doi: 10.4161/psb.28645. Galanthamine an anticholinesterase drug effects plant growth and development in Artemisia tridentata Nutt. through modulation of auxin and neutrotransmitter signaling. Plant Signaling and Behavior. I performed all targeted and untargeted analysis. Katie Axwik helped to keep the germplasm lines viable for the duration of the experiment, while Anderson Smith provided technical assistance for the instruments. Dr. A.M.P Jones kindly ran the standard auxin bioassay at the University of Guleph. iv Chapter 6 is based on work I conducted in Dr. Susan Murch’s lab at UBC Okanagan. All experimental work and data analysis was performed by me. v Table of Contents Abstract .................................................................................................................................................. ii Preface ................................................................................................................................................... iv List of Tables......................................................................................................................................... xi List of Figures ......................................................................................................................................xiii List of Abbreviations ........................................................................................................................... xvii Glossary ............................................................................................................................................. xviii Acknowledgements .............................................................................................................................. xix Chapter 1 Introduction ........................................................................................................................... 1 1.1 Phytochemistry ............................................................................................................................. 1 1.2 Ethnobotany, Ethnopharmacology and Drug Discovery .............................................................. 2 1.2.1 Traditional Approaches for the Study of Medicinal Plants ................................................... 2 1.2.2 Plant Collection and Extraction ............................................................................................. 1 1.2.3 Bioassay ................................................................................................................................ 1 1.2.4 Chromatography and Elucidation of Structures .................................................................... 2 1.3 North American Biodiversity as a Source for Drugs ................................................................... 2 1.3.1 Spiritual and Ceremonial Plants as a Source for Drugs ........................................................ 3 1.3.2 Ceremonial Smokes as a Source for Drugs ........................................................................... 4 1.4 Model Species 1: Ligusticum ....................................................................................................... 4 1.4.1 Botany and Taxonomy .......................................................................................................... 9 1.4.2 Ethnobotany ........................................................................................................................ 11 1.4.3 Medicinal Chemistry ........................................................................................................... 13 1.4.4 Bioassay .............................................................................................................................. 15 1.5 Model Species II: Artemisia ....................................................................................................... 16 1.5.1 Evolution, Botany and Taxonomy ...................................................................................... 23 1.5.2 Phytochemistry .................................................................................................................... 24 1.5.3 Bioassay .............................................................................................................................. 34 1.6 Metabolomics, Metabonomics, and the Plant Metabolome ....................................................... 36 1.6.1 Targeted vs Untargeted
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