Influence of abiotic factors on growth and biosynthesis of secondary plant components in Duboisia species Zur Erlangung des akademischen Grades eines Dr. rer. nat. von der Fakultät Bio- und Chemieingenieurwesen der Technischen Universität Dortmund genehmigte Dissertation vorgelegt von Sophie Friederike Ullrich aus Mainz, Deutschland Tag der mündlichen Prüfung: 09.12.2016 1. Gutachter: Prof. Dr. Oliver Kayser 2. Gutachter: Prof. Dr. Bernd Honermeier Dortmund 2016 “Look deep into nature, and then you will understand everything better.” Albert Einstein I Acknowledgements Acknowledgements I would like to express my sincere appreciation and gratitude to all the people who accompanied and supported me in finishing my thesis. At first, I would like to thank my supervisor Prof. Oliver Kayser (Department of Biochemical and Chemical Engineering, Technical Biochemistry, TU Dortmund) for his support within working on my PhD thesis. His valuable input helped me in all the time of research as well as in writing of my thesis. I am very grateful for the trust he has placed in me over the past years. I am thankful to Boehringer Ingelheim Pharma GmbH & Co.KG for funding as well as for proving all the plant material I worked with. Great thanks go to my supervisor Dr. Hansjörg Hagels (Boehringer Ingelheim Pharma GmbH & Co.KG, Ingelheim, Germany) for promoting personal development, his valuable scientific thoughts and guidance on various aspects of my research work. Moreover, I also sincerely thank my supervisor Andreas Rothauer (Boehringer Ingelheim Pharma GmbH & Co.KG, Ingelheim, Germany) for his encouragement in taking my research forward and on his versatile input on different aspects of my scientific work. Thanks to Prof. Bernd Honermeier (Institute for Plant Production and Plant Breeding I, Plant production, Justus-Liebig-University of Gießen, Germany) for his input within our “Duboisia Kolloquiums” as well as for being part of the board of examiners and providing the second opinion on this thesis. I would also like to thank Prof. Young Hae Choi for offering me the opportunity to work in his lab at the University of Biology in Leiden and to learn a lot from him in the field of NMR- based plant metabolomics. Furthermore, thanks to Dr. Leonardo Castellanos, who took a lot of time for instructing me and assisted me in NMR data processing and analysis. Special thanks go to Des Crane and Andrew Hole from the Boehringer Ingelheim Pty Limited Duboisia Farms in Australia for taking care of the cultivation of some of my experimental plants and Nils Averesch from the University of Brisbane for doing the harvesting and further sample work-up. Thank you all for the nice, inter-continental cooperation. I warmly thank all my colleagues at the Chair of Technical Biochemistry in Dortmund for their warm welcome, the good cooperation within the team as well as for all the fun we have had in the last years; especially Eva-Maria Gruchattka, Friederike Degenhardt, Kathleen Pamplanyil, Torsten Arndt and Bastian Zirpel, who accompanied me over all the time in Dortmund, not only at work, but also during my free time. Thanks go to all nice colleagues at the Phyto Center in Ingelheim always helping me to make progress, especially to Julia Sparke II Acknowledgements and her team. I am in particular grateful to my Duboisia research mates Laura Kohnen, Rosa Hiltrop and Sabine Oster for the great time we spent together within the past years. I really appreciate your support, our good conversations, our joint travel to conferences and I do also gladly remember our enjoyable long weekend together in Zeeland. I would also like to thank my parents as well as to my brother and sisters for all of their confidence, encouragement and support within my whole life. Furthermore, thanks to all of my friends, especially Mira Oswald, Sabine Bellers and Laura Tretzel for always having an open ear for me and for the nice time we spent together within yet more than 10 years. At the end I would like to express my deepest appreciation to my boyfriend Michael Eckbauer who is always there for me, encourages me and makes my life more beautiful! III Table of Contents Table of Contents Abstract ................................................................................................................................... VII Zusammenfassung ................................................................................................................. VIII Chapter 1: Scope of the thesis ............................................................................................. 1 1.1. Aims and Objectives .................................................................................................... 2 Chapter 2: Introduction ....................................................................................................... 3 2.1. Abstract ........................................................................................................................ 4 2.2. Introduction .................................................................................................................. 5 2.3. Botanical origin ........................................................................................................... 5 2.4. Pharmacology .............................................................................................................. 7 2.4.1. Ethnopharmacological use ................................................................................... 7 2.4.2. Contemporary therapeutical use ........................................................................... 9 2.4.3. Mechanism of action .......................................................................................... 11 2.4.4. Pharmacokinetics ................................................................................................ 12 2.4.5. Toxicity .............................................................................................................. 12 2.4.6. Abuse as “Truth serum” ..................................................................................... 13 2.5. Biosynthesis ............................................................................................................... 13 2.5.1. Plant biosynthetic pathway ................................................................................. 13 2.5.2. Structure elucidation and full chemical synthesis .............................................. 16 2.6. Production .................................................................................................................. 17 2.6.1. Industrial production .......................................................................................... 17 2.6.2. Agricultural cultivation ...................................................................................... 18 2.6.3. Biotechnological Production .............................................................................. 19 2.7. Conclusions................................................................................................................ 27 2.8. References .................................................................................................................. 30 Chapter 3: Discrimination of wild types and hybrids of Duboisia myoporoides and Duboisia leichhardtii at different growth stages using 1H NMR-based metabolite profiling and tropane alkaloids-targeted HPLC-MS analysis ........................................................................ 43 IV Table of Contents 3.1. Abstract ...................................................................................................................... 44 3.2. Introduction ................................................................................................................ 45 3.3. Methods and Materials .............................................................................................. 48 3.3.1. Plant material ...................................................................................................... 48 3.3.2. Chemicals and reagents ...................................................................................... 48 3.3.3. NMR analysis ..................................................................................................... 49 3.3.4. HPLC-MS ........................................................................................................... 50 3.4. Results and discussion ............................................................................................... 52 3.4.1. 1H NMR- based metabolite profiling ................................................................. 52 3.4.2. Comparison of different genotypes .................................................................... 55 3.4.3. Comparison of different growth stages .............................................................. 57 3.4.4. Impact of growth stage and genotype on the metabolite distribution ................ 59 3.4.5. Comparison of different cultivation conditions .................................................. 61 3.4.6. HPLC-MS method validation for quantification of tropane alkaloids ............... 62 3.4.7. Tropane alkaloid profile ..................................................................................... 64 3.5. Conclusion ................................................................................................................. 66 3.6. References .................................................................................................................. 68 Chapter