Rhodiola Rosea L.-An Evaluation of Safety and Efficacy in the Context of a Neurological Disorder, Alzheimer Disease
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Rhodiola rosea L.-An evaluation of safety and efficacy in the context of a neurological disorder, Alzheimer Disease Fida Al Noor Ahmed Thesis submitted to the Faculty of Graduate and Postdoctoral Studies in partial fulfillment of the requirements for the Doctorate in Philosophy degree in Biology Department of Biology Faculty of Science University of Ottawa © Fida Al Noor Ahmed, Ottawa, Canada, 2015 ABSTRACT This thesis examined the safety and efficacy of Rhodiola rosea L. (Crassulaceae), a medicinal plant used traditionally by the Inuit of Nunavik, Québec, for the maintenance of mental and physical health. To assess the effects of Nunavik R. rosea on the central nervous system, a phytochemically characterized extract was tested in behavioural assays of anxiety with rats. Significant changes in behaviour were observed, particularly in the conditioned emotional response test. R. rosea was not a potent modulator of the benzodiazepine site of the GABAA receptor, indicating possible involvement of other neurotransmitters implicated in the neurobiology of anxiety. Safety of Nunavik R. rosea, its marker phytochemicals, and additional R. rosea products was assessed by evaluating the risk of drug interaction potential. Inhibitory capacity was tested on major human drug metabolizing enzymes, the cytochrome P450s. Further, effects on the metabolism of repaglinide, an anti-diabetic drug, were examined in human liver microsomes. While the overall risk of interactions was low, variable impacts of R. rosea products on the formation of glucuronide metabolites of repaglinide necessitate caution. In the TgCRND8 model of Alzheimer disease, R. rosea chronic administration led to modest improvements in the survival of male transgenic mice, which exhibit accelerated rates of mortality. Effects on learning and memory performance in the Morris water maze were limited to alterations in the patterns of use of search strategies as determined by our automated scoring algorithm, MWM Visual; changes in escape latencies were not observed. Nunavik R. rosea administration resulted in elevated plasma levels of anandamide (20:1, n-9), a member of the endocannabinoid family, tentatively identified using an untargeted metabolomics approach via ultra-performance liquid chromatography-quadrupole-time-of-flight- ii mass spectrometry. R. rosea phytochemicals were either eliminated via the renal pathway, or transformed into potential metabolites. Collectively, the anxiolytic activity of Nunavik R. rosea in rats, its protective effects on high background mortality in an aggressive Alzheimer disease model, low risk of inhibition of major enzymes involved in drug metabolism, and its ability to induce detectable changes in in vivo metabolic pathways are supportive scientific evidence for the use of this traditional medicine for general well-being by the Inuit. iii RÉSUMÉ Cette thèse a évalué la sûreté et l’efficacité de Rhodiola rosea L. (Crassulaceae), une plante médicinale traditionnelle utilisée par le peuple Inuit du Nunavik, Québec, pour le maintien de leur santé mentale et physique. Afin d’évaluer les effets de R. rosea sur le système nerveux central, un extrait phytochimiquement caractérisé a été testé par l’entremise de tests de comportement standardisé chez les rats. Des changements significatifs au niveau du comportement furent observés, particulièrement lors de tests de réponses émotionnelles conditionnées. R. rosea était un faible modulateur du site benzodiazépine du récepteur GABAA, indiquant l’implication potentielle d’autres réseaux de neurotransmetteurs impliqués dans la neurobiologie de l’anxiété. La sûreté de R. rosea du Nunavik, ses marqueurs phytochimiques, ainsi qu’autres produits naturels contenant R. rosea furent évalués en jaugeant leur risque d’interactions potentielles. L’effet inhibitoire de ces extraits furent testés sur les cytochromes P450, communément utilisées dans le métabolisme de drogues, ainsi que sur des enzymes impliquées dans le métabolisme du répaglinide, une drogue antidiabétique, dans des microsomes du foie humain. Quoique le risque d’interactions des produits de R. rosea soit minime, l’impact variable des produits de R. rosea sur la formation de métabolites glucuronidés du répaglinide nécessite des précautions. Le modèle TgCRND8 de l’Alzheimer a démontré que l’administration chronique de R. rosea a mené à une amélioration modeste de la survie de souris mâles transgéniques, qui exhibent des taux accélérés de mortalité. Les effets sur l’apprentissage et la mémoire dans le labyrinthe de Morris furent limités à des modifications de stratégies de recherche, tel que iv déterminé par MWM Visual, notre algorithme automatisé. Des réductions en latences d’échappement n’ont pas été observées. L’administration de R. rosea du Nunavik a entrainé une augmentation d’anandamide (20 :1, n-9), un membre de la famille des endocannabinoïdes, dans le plasma sanguin tel que déterminé par une analyse du métabolome via UPLC-Q-TOF. Les composés phytochimiques de R. rosea ont soit été éliminés par la voie rénale, ou transformés en métabolites potentiels. Collectivement, ces activités biologiques apportent un support scientifique en ce qui a trait à l’utilisation de cette plante médicinale traditionnelle pour le bien-être du peuple Inuit. v ACKNOWLEDGEMENTS It has been nearly 7 years since the beginning of my experience as a graduate student, and what an amazing journey it has been! I am deeply grateful to our project collaborators, namely the Makivik Corporation and its subsidiary organization, Nunavik BioSciences, the Inuit communities of Nunavik and Nunatsiavut, as well as Dr. Alain Cuerrier, for introducing me to the world of Inuit traditional knowledge, for sharing his ethnobotanical expertise and for meeting my never-ending demands for roseroot samples. I am honoured to have been a part of this multi- disciplinary project, and to contribute, in a small way, to the rich fabric of Inuit knowledge, “a picker-up of shells on the shores of the great unknown ocean,” as it were. Indeed, I have taken away much more than I was able to give; the Inuit worldview of mental, physical and spiritual health as one has deeply touched my own outlook towards life and well-being. Most importantly, I am thankful for my two co-supervisors, Dr. John T. Arnason and Dr. Steffany A. L. Bennett – no student could ever ask for more knowledgeable, enthusiastic and supportive mentors, not only in the context of graduate studies, but also, the life beyond. I am grateful for the advice and guidance of my research committee members, Dr. Vance Trudeau, Dr. William Willmore, and Dr. Claude Messier. I was incredibly fortunate to be able to work alongside several senior mentors over the course of my degree, without whose expertise and help this project would not have been completed. To Dr. Ammar Saleem, thank you for your guidance and knowledge, and for all those snowy weekends you gave up so I could “play” on the UPLC- Q-TOF. I would like to extend my gratitude to Dr. Martine Bertrand and Graeme P. Taylor for their enthusiasm, support, and technical guidance during the design and testing of MWM Visual and Searcher. Dr. Brian Foster, thank you for sharing your insider tips on the workings of natural health product regulatory agencies, and for encouraging me to constantly assess the applicability vi of my laboratory findings in the “real world”. My special thanks go to Dr. Alexandra Pettit for undertaking the herculean task of training me on the cryostat, and in immunofluorescence techniques. I would like to thank Jacky Liang and Matthew W. Granger for welcoming me to the Messier lab and guiding me through the process of conversion of video files for the development of MWM Visual. I would also like to thank Christian Cayer for the memorable experience of conducting my first “pharmacokinetic study”. My deepest thanks go to my dear colleagues, and my friends, in the Arnason and Bennett laboratories for their assistance and support over the years. I would like to thank Dr. Ammar Saleem and Rui Liu for their help with phytochemical analyses. I am especially grateful for my “feeding study” accomplices- Dr. Carolina Cieniak, Matthew W. Granger, Matthew W. Taylor, Dr. Hongbin Xu, Bettina Franko, Shelby Allison, and Emily Gardiner, many of whom also participated in the manual scoring “marathons” for MWM Visual. A big thank you to Ashleigh McLean and Vian Peshdary for introducing me to brain dissection techniques, Stephanie Fowler for training me in tissue culture methods, Alexandre P. Blanchard, and Graeme S. McDowell for acquainting me with lipid extraction protocols and aiding in the beta-testing of MWM Visual, and to Matthew W. Granger- those glucose tolerance tests would not have been the same without you. My heartfelt gratitude goes to Dr. Chieu Anh Kim Ta for her critical review of this manuscript, and to Marc Rosembert- merci beaucoup for translating my abstract into French. Additionally, I would like to acknowledge the financial support received for this project from Nunavik BioSciences, the University of Ottawa, the National Sciences and Engineering Research Council (NSERC), and the CIHR Training Program in Neurodegenerative Lipidomics. Above all, I would like to dedicate this work to my family. To my parents, thank you for believing in me and pushing me to achieve greater heights; to my siblings, thank you for the vii endless pep-talks and the 5 AM rides to work. Your unconditional love and prayers have made possible the realization of this dream. viii TABLE OF CONTENTS ABSTRACT