Institut für Pharmazeutische Wissenschaften Bereich Pharmakognosie Karl-Franzens-Universität Graz United States Department of Agriculture - Agricultural Research Service Natural Products Utilization Research Unit University of Mississippi Diplomarbeit Bio-guided isolation of phytotoxic coumarin derivatives of Thamnosma montana Torr. & Frém., a Rutaceae native to the southwestern United States Zur Erlangung des akademischen Grades eines Magisters der Pharmazie an der Naturwissenschaftlichen Fakultät der Karl-Franzens-Universität Graz vorgelegt von Aaron Sallegger Graz, Juni 2009 This thesis was composed between August 2008 and June 2009 at the United States Department of Agriculture - Agricultural Research Service, Natural Products Utilization Research Unit, University of Mississippi and the Institute of Pharmaceutical Sciences - Department of Pharmacognosy, University of Graz. First of all, I want to thank Dr. Wolfgang Schühly for being a great and motivated supervisor. Without him, this project would not have taken place and I would never have had the chance to experience working in the United States of America. I also want to thank Dr. Charles Cantrell for taking over supervision in Oxford and Dr. Stephen Duke for coordinating the bioassays. Dr. Cantrell was a great mentor and friend, always ready to guide me through this interesting project and help me to get set up in Oxford. Furthermore, my thanks go to Amber Callahan Reichley, Solomon Green III and Bob Johnson for being the best technicians I ever had the honor to work with and for giving me an amazing time in Oxford. I will never forget you guys! Very special thanks goes to my family for being with me all the time, emotionally, mentally and financially. Without them, I would not have been able to achieve my goals. Moreover, I want to thank the “Büro für Internationale Beziehungen” and the “Naturwissenschaftliche Fakultät” for the generous funding. I also wish to thank Rolf Muertter, Aaron Schusteff and “jrdnz” for granting me permission to use their beautiful pictures of Thamnosma montana , which gave my thesis a colorful touch. Last but not least, I want to mention my numerous friends and all the people, who helped me along my way, making me the person I am. Thank y'all! Table of Contents Table of Contents 1 Introduction.........................................................................................1 2 Literature review..................................................................................2 2.1 Rutaceae.......................................................................................2 2.2 Thamnosma ...................................................................................2 2.2.1 Classification...........................................................................3 2.3 Thamnosma montana .....................................................................4 2.3.1 Botanical characteristics............................................................4 2.3.2 Distribution.............................................................................5 2.3.3 Ethnobotanical use...................................................................6 2.3.4 Constituents............................................................................7 2.4 Coumarins.....................................................................................7 2.4.1 Classification...........................................................................8 2.4.1.1 Simple coumarins..............................................................8 2.4.1.2 Furanocoumarins...............................................................8 2.4.1.3 Pyranocoumarins...............................................................8 2.4.2 Biosynthesis............................................................................9 2.4.2.1 Simple coumarins..............................................................9 2.4.2.2 Furanocoumarins...............................................................9 2.4.3 Furanocoumarins and phototoxicity..........................................10 2.5 Bioassays....................................................................................10 2.5.1 Bioassay-guided fractionation..................................................11 3 Materials and methods.........................................................................13 3.1 Chromatography...........................................................................13 3.1.1. Analytical thin-layer chromatography.......................................13 3.1.2 Preparative TLC......................................................................13 3.1.3 Column chromatography.........................................................14 3.1.4 Analytical and preparative high-performance liquid chromatography (HPLC)..........................................................................................14 3.1.5 GC-MS..................................................................................14 3.1.6 LC-MS...................................................................................15 I Table of Contents 3.2 NMR............................................................................................15 3.3 Instrumentation...........................................................................16 3.3.1 TLC and preparative TLC.........................................................16 3.3.2 Column chromatography.........................................................16 3.3.3 HPLC and preparative HPLC.....................................................16 3.3.4 GC-MS..................................................................................17 3.3.5 High resolution LC-MS.............................................................17 3.3.6 NMR.....................................................................................17 3.4 Plant material...............................................................................17 3.5 Extraction....................................................................................18 3.6 Fractionation................................................................................18 3.7 Bioassays against A. stolonifera and L. sativa ...................................19 3.8 Bioassay guided fractionation.........................................................20 3.8.1 Fraction V6............................................................................21 3.8.2 Fraction V7............................................................................22 3.8.3 Fraction V7ppt.......................................................................24 3.8.4 Fraction V8ppt.......................................................................24 3.8.5 Fraction V9............................................................................24 3.8.6 Fraction V10..........................................................................26 3.8.7 Fraction V11..........................................................................28 3.9 Bioassay against Lemna paucicostata L...........................................29 3.9.1 General.................................................................................29 3.9.2 Conduction of L. paucicostata bioassay.....................................30 4 Results and discussion.........................................................................32 4.1 Structure elucidation.....................................................................32 4.2 AS1WS37_V6_comp1 (Isoimperatorin)............................................33 4.3 AS1WS37_V6_comp2 (Suberosin)..................................................35 4.4 AS1WS37_V7_B and AS1WS37_V7_ppt (Swietenocoumarin B)..........38 4.5 AS1WS37_V8_ppt (Bergapten).......................................................45 4.6 AS1WS37_V9_A_P2 (Psoralen).......................................................48 4.7 AS1WS37_V9_C (Dehydrogeijerin).................................................50 4.8 AS1WS37_V10_C_cry (Isopimpinellin)............................................53 II Table of Contents 4.9 AS1WS37_V10_E_B1 (Epoxysuberosin)...........................................55 4.10 AS1WS37_V10_E_B2 (Alloimperatorin methyl ether epoxide)...........58 5 Summary...........................................................................................61 6 Appendix............................................................................................63 6.1 Bibliography.................................................................................63 6.2 Index of Figures...........................................................................67 6.3 Index of Tables............................................................................68 III Introduction 1 Introduction Secondary metabolites from various higher plants are of high interest to both the biologist, who aimes to understand the reason for their production and the pharmacologist looking out for potential sources of substances useful for human beings either as pharmaceuticals, dietary supplements or pesticides. To date, out of the ten thousands of known natural products, many have found their way into an application in either medicine or agriculture. The use of natural products as pesticides dates back to ancient Greece and to the Roman Empire. Humans always searched for methods to extinguish weeds, insects or other herbivores in order to optimize crop yields. Modern agricultural practices rely on chemicals for pest control, but concerns about a potential impact of these pesticides on the environment and