POTENTIAL FOR USING INSECTS TO GUIDE THE SEARCH FOR MEDICINALLY-ACTIVE CHEMICAL COMPOUNDS IN PLANTS Ciara Raudsepp-Heame Department of Plant Science - NEO McGill University, Montreal Submitted July 2003 A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements of the degree of Masters of Science ©Ciara Raudsepp-Heame 2003 Library and Bibliothèque et 1+1 Archives Canada Archives Canada Published Heritage Direction du Branch Patrimoine de l'édition 395 Wellington Street 395, rue Wellington Ottawa ON K1A ON4 Ottawa ON K1A ON4 Canada Canada Your file Votre référence ISBN: 0-612-98724-8 Our file Notre référence ISBN: 0-612-98724-8 NOTICE: AVIS: The author has granted a non­ L'auteur a accordé une licence non exclusive exclusive license allowing Library permettant à la Bibliothèque et Archives and Archives Canada to reproduce, Canada de reproduire, publier, archiver, publish, archive, preserve, conserve, sauvegarder, conserver, transmettre au public communicate to the public by par télécommunication ou par l'Internet, prêter, telecommunication or on the Internet, distribuer et vendre des thèses partout dans loan, distribute and sell theses le monde, à des fins commerciales ou autres, worldwide, for commercial or non­ sur support microforme, papier, électronique commercial purposes, in microform, et/ou autres formats. paper, electronic and/or any other formats. The author retains copyright L'auteur conserve la propriété du droit d'auteur ownership and moral rights in et des droits moraux qui protège cette thèse. this thesis. Neither the thesis Ni la thèse ni des extraits substantiels de nor substantial extracts from it celle-ci ne doivent être imprimés ou autrement may be printed or otherwise reproduits sans son autorisation. reproduced without the author's permission. ln compliance with the Canadian Conformément à la loi canadienne Privacy Act some supporting sur la protection de la vie privée, forms may have been removed quelques formulaires secondaires from this thesis. ont été enlevés de cette thèse. While these forms may be included Bien que ces formulaires in the document page cou nt, aient inclus dans la pagination, their removal does not represent il n'y aura aucun contenu manquant. any loss of content from the thesis. ••• Canada ABSTRACT Tropical insects are weIl known for their diversity of defensive secondary compounds, most ofwhich originate in the insect's host plant. These have similar structures to compounds that are responsible for most pharmacological activity in plants and that are mined by pharmaceutical companies for drug development. There is a potential for using bioprospecting projects in the biodiverse tropics as an economic incentive to preserve the forests that are the source of potentially lucrative and life-saving new medicines. This thesis investigates the possibility of using aposematic insects as guides to plants that contain pharmacologically-active compounds. Various plants that were found to have preliminary activity against cancer, leishmaniasis, malaria or Chagas' disease were investigated to determine whether they were associated with an aposematic insect. Active plants that were found to have an interesting ecological relationship with at least one herbivorous insect were subjected to bioassay-guided fractionation using HPLC and standard chromatographic techniques in order to isolate the active chemical component of the plant. Plants were monitored within national parks in the Republic of Panama over a period of six months and aIl insects feeding on them were collected and raised in captivity. The insects were then extracted and analyzed to œtermine how they were treating toxic chemical compounds in their host plant. Two principle plants were investigated with their associated insects: (1) Vismia baccifera and (2) Mikania guaco. One generalist and one specialist Lepidopteran species were found to sequester vismione B from their host plant Vismia baccifera, a cytotoxic compound active against three cancer celllines. Two specialist Coleopterans were found to sequester the novel compound Guacanone, isolated by the primary author from the vine Mikania guaco and active against Trypanosoma cruzi, the causative agent of Chagas' disease. A generalist Coleopteran was found to not sequester this compound. RÉSUMÉ Les insectes tropicaux sont reconnus pour la diversité de leurs composés chimiques dont la plupart proviennent des plantes hôtes. Ces composés chimiques de défense ont des structures similaires aux composés chimiques dans les plantes qui sont exploités par l'industrie pharmaceutique pour la mise au point de nouveaux médicaments. Les projets de bioprospection dans les tropiques, riches en biodiversité, pourraient d'ailleurs inciter la préservation des forêts. Ces forêts abritent les sources de médicaments éventuellement lucratifs et profitables pour la médecine. Ce mémoire de maîtrise étudie la possibilité d'utiliser les insectes aposématiques comme indicateurs pour identifier les plantes qui contiennent des éléments pharmacologiques actifs. Plusieurs plantes sont connues pour leurs activités préliminaires contre le cancer, la leishmaniose, la malaria ou la maladie de Chagas. Celles-ci ont été étudiées afin de déterminer si elles sont liées à des insectes aposématiques. Les plantes actives qui ont une relation écologique intéressante avec au moins un insecte herbivore ont été extraites, fractionnées et testées par bioessai dans le but d'isoler l'élément chimique actif. Les insectes qui utilisent ces plantes comme plantes hôtes ont été collectionnés dans les forêts de la République de Panama. Par la suite, les insectes ont été analysés pour savoir comment ils métabolisaient les molécules toxiques de la plante. Dans cette étude, deux plantes ont été principalement étudiées avec leurs insectes correspondants: (1) Vismia baccifera et (2) Mikania guaco. L'étude d'une espèce générale et d'une espèce spécifique de lépidoptères a révélé qu'ils séquestrent, absorbent et concentrent la vismione B de leur plante hôte, la Vismia baccifera. Cette molécule est un élément cytotoxique actif contre trois lignées de cellules cancéreuses. L'étude de deux types spécifiques de coléoptères a révélé qu'ils séquestrent une nouvelle molécule, la guacanone, à partir de la vigne Mikania guaco. Cet élément a été isolé par l'auteur de cette étude. Il s'est révélé actif contre le Trypanosoma cruzi, l'agent responsable de la maladie de Chagas. Un type général de coléoptère ne s'est cependant pas révélé avoir les mêmes propriétés. ii ACKNOWLEDGEMENTS 1 thank my supervisors Todd Capson and Tim Johns for guidance and encouragement. 1 thank Don Smith who served in my thesis advisory committee. 1 am grateful to Catherine Potvin for support in Panama. 1 thank Donald Windsor and Annette Aiello for their knowledge about insect ecology, collecting and rearing, and for the identification of several insects. 1 thank Ahmed Hussein for the e1ucidation of the structure of Guacanone and also for guidance in chemistry techniques. 1 am grateful to Maria HelIer and Erika Garibaldo for technical support at STRI and within the ICBG and Carolyn Bowes for administrative support from the department of Plant Science. 1 thank Rafael Aizprua, Nayda Flores, Blanca Arauz, for botanical assistance in the collection and identification of plant specimens. 1 thank Nivia Rios for assistance in the chemistry lab and Johant Lakey for instruction on HPLC techniques. 1 thank Eduardo Ortega, Luz Ramiro, Mahabir Gupta and their laboratories for running the disease bioassays. 1 thank Kerry McPhail at the University of Oregon for running Mass spectrometry for my samples. Financial support for this project was provided by the National Institute of Health (NIH), the Organization of American States (OAS), and the Office Qœbec• Amériques pour la Jeunesse (OQAJ). 1 am extremely grateful to Dana, Enn, Karl, Rory as well as Scott Smith, Andrew Moeser, Neilan Kuntz and Javier Barrios. iii CONTRIBUTIONS OF AUTHORS The manuscripts were written by the first author under the guidance of the supervisor, Todd Capson (co-supervisor Tim Johns offered input but is not listed as an author on either paper). Ahmed Hussein contributed to the work described in the first manuscript as the chemist who isolated and elucidated the structures of the chemical compounds described therein. Ahmed Hussein contributed to the second manuscript by elucidating the structure of the compound isolated by the first author from the plant Mikania guaco. AlI other experiments were performed by the first author using materials, equipment and facilities supplied by the primary supervisor in Panama. iv TABLE OF CONTENTS ABSTRACT .................................................................................... i RÉSUMÉ ....................................................................................... .ii ACKNOWLEDGEMENTS .................................................................. .iii CONTRIBUTIONS OF AUTHORS ....................................................... .iv LIST OF FIGURES ........................................................................... vii 1. INTRODUCTION ...................................................................... 1 1.l.Insects in bioprospecting ........................................................... 1 L2.Relevance ofproject to science .................................................... 2 1.3. Broader relevance ofproject: health and conservation ....................... .3 1.3.l.Irreplaceable sources ofnovel