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Chemistry rooted in cultural knowledge : unearthing the links between antimicrobial properties and traditional… Bannister, Kelly Patricia 2006

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Chemistry Rooted in Cultural Knowledge: Unearthing the Links Between Antimicrobial Properties and Traditional Knowledge In Food and Medicinal Plant Resources of the Secwepemc (Shuswap) Aboriginal Nation  by Kelly Patricia Bannister  B.Sc, The University of Victoria, 1988 M.Sc, The University of Victoria, 1993  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Botany)  THE UNIVERSITY OF BRITISH COLUMBIA July 2000 © Kelly Patricia Bannister, 2000  ABSTRACT The role of phytochemicals as ecological mediators of interrelationships between humans and plants was explored. Specifically, antimicrobial properties of plants were examined in the context of traditional plant use as food and/or medicine by the Secwepemc (Shuswap) Aboriginal peoples of south central British Columbia. The research was conducted in collaboration with the Secwepemc Cultural Education Society (Kamloops) as part of a larger ethnobotanical research program. The first component of the study involved the screening for antimicrobial activities in vitro of sixty-eight plant species used by the Secwepemc to treat microbial-based conditions. Extracts of eighty-eight percent of plant species examined had antibacterial activity, seventy-five percent had antifungal activity and twenty-five percent had antiviral activity. Based on the screening results and additional ethnobotanical information, Balsamorhiza sagittata (Pursh) Nutt. (Asteraceae), commonly called balsamroot, was selected for further characterisation. In the second part of the study, the phytochemistry of aerial and underground parts of balsamroot was examined within the cultural context of traditional plant preparation and use as food and medicine. The effect of differential heat-processing for food and medicine on antimicrobial compounds in roots was assessed. A biologically active compound known to occur in roots (thiophene E) was used as a 'marker' to compare the bioactivity and localisation of antimicrobial compounds in pitcooked roots (prepared as food) with boiled roots (prepared as medicine). Only the edible portion of roots was devoid of antimicrobial activity. Bioactivityguided isolation lead to the purification and identification of a known phytosterone (16R, 23Rdihydroxycycloartenone) and an unreported phytosterol (16R, 23R-dihydroxycycloartenol) from roots. The effect on antimicrobial compounds of drying leaves for medicine also was measured. Three antimicrobial compounds were present in fresh leaves but absent in dried leaves. One of these was purified; using spectroscopic techniques its structure was determined as a previously unreported sesquiterpene lactone (guaianolide), designated 2-deoxy-pumilin-8-0-acetate. The cultural relevance of the findings was discussed in terms of the antimicrobial activity, potential allergenicity and localisation to glandular trichomes of sesquiterpene lactones. The integration of phytochemical data and Secwepemc cultural knowledge of balsamroot underscored the sophistication in Secwepemc botanical knowledge. It served also as an instructive case for the third component of this study, which moved beyond chemistry to raise and discuss some important ethical and legal issues concerned with research involving the cultural knowledge and traditional resources of Aboriginal peoples. The main issues discussed were associated with research obligations, direct and indirect impacts of research, and the dissemination and control of knowledge in ethnobotanical and related investigations.  Ill  TABLE OF CONTENTS Page  Abstract  ii  Contents  iii  List of Tables  vii  List of Figures  ix  Abbreviations  xi  Acknowledgements  xiv  Dedication  xix  1  General Introduction  1  1.1  Plants, Peoples and Phytochemicals  1  1.2  Research Collaboration with the Secwepemc Cultural Education Society and the Secwepemc Aboriginal Nation through the Secwepemc Ethnobotany Project  .  6  1.3  Ethnobotany or Economic Botany?  8  1.4  Research Objectives and Thesis Organisation  8  2  A History of Microbial Diseases in Secwepemc Culture and the Contemporary Antimicrobial Properties of Secwepemc Medicinal Plants  10  2.1  Introduction  10  2.1.1  S ecwepemc Culture and Traditional Territory  10  2.1.2 Secwepemc History: Two Levels of Colonisation  13  2.1.3 Contemporary Studies of Traditional Medicines: Unveiling Past Ironies  15  2.1.4 Contemporary Studies of Traditional Medicines: Moving Forward  17  2.1.5 Translation, Interpretation and Other Biases Involved in this Study  18  2.1.6 Assessing Antimicrobial Activity  21  2.2  Research Objectives  22  2.3  Materials and Methods  23  2.3.1 Ethnobotanical Information  24  2.3.2 Plant Collections  25  2.3.3 Preparation of Crude Plant Extracts  27  2.3.4 Microorganisms  28  2.3.5 Antibacterial and Antifungal Disk Diffusion Assays  29  2.3.6 Antiviral Assays  30  2.4  Results  31  2.5  Discussion  34  3  What Are the People Really Taking? Antimicrobial Properties of  Balsamroot as Food and Medicine  3.1  Introduction  40  3.2  Balsamroot  41  3.2.1  Balsamroot as Food  43  3.2.2  Balsamroot as Medicine  45  3.3  Research Objectives and Rationale  48  3.4  Materials and Methods  49  3.4.1  Ethnobotanical Information  49  3.4.2  Plant Collections  49  3.4.3  Plant Extracts  50  3.4.4  Antibacterial and Antifungal Disk Diffusion Assays  52  3.4.5  Bacterial Overlay Spot Tests  52  3.4.6  Thin Layer Chromatography (TLC) and TLC Agar Overlays  53  3.4.7  Minimum Inhibitory Concentration (MIC)  54  3.4.8  Antiviral Assays  55  3.4.9  Bioactivity-guided Isolation of Antimicrobial CompoundsfromBalsamroot  56  3.4.10 High Performance Liquid Chromatography  61  3.4.11 Mass Spectrometry (MS)  62  3.4.12 Gas Chromatography-Mass Spectroscopy (GC-MS)  62  3.4.13 Proton Nuclear Magnetic Resonance ( H NMR) !  6 2  3.4.14 Carbon Nuclear Magnetic Resonance ( C NMR)  6 3  3.4.15 X-ray Crystallography  63  13  13  V  3.5  Results  63  3.5.1 Roots of Balsamroot as Medicine  64  3.5.2 Roots of Balsamroot as Food  75  3.5.3 Leaves of Balsamroot as Medicine  81  3.6  Discussion  97  3.6.1  Properties of Roots  97  3.6.2  Properties of Leaves  106  3.6.3  Toward a Deeper Understanding of Human-Plant Interrelationships  113  4  Biocultural Issues In Ethnobotany  116  4.1  Introduction: Ethnobotany at a Crossroads  4.2  Organisation and Objectives of this Chapter  119  4.3  Stakeholders and Research Obligations  120  116  4.3.1 Academic Research Policy and Ethical Standards: The Tri-Council Statement on Ethical Conduct for Research Involving Humans  121  4.3.2 International Law and Policy Relating to Protection of Cultural Knowledge and Traditional Resources: The Convention on Biological Diversity  125  4.3.3 Legal Approaches to Protection of Cultural Knowledge and Traditional Resources: The Canadian Intellectual Property Rights System  127  4.3.4 Assessing the Implications: Some of the 'Wrongs' with Intellectual Property Rights  130  4.3.5 Moral Approaches to Protection of Cultural Knowledge and Traditional Resources: The Role of International Initiatives, Local Initiatives and 4.4  Professional Societies  135  Impacts of Research  140  4.4.1 The Role of Ethnobotany in the Search for Medicines  141  4.4.2 The Publication Dilemma  146  4.4.3 A Precautionary Approach to Publication  148  4.5  Conclusions: Ethnobotany—Where Many Crossroads Intersect  153  vi  5  General Discussion  157  5.1  Chemical and Other Complexities of Human-Plant Interrelationships  157  5.2  Antimicrobials in Plant Foods and Medicines  159  5.3  Necessary Tensions in Ethnobotanical Research  162  5.4  Summary and Concluding Remarks  164  Literature Cited  167  Appendix A: Letter of Consent/Research Agreement  187  Appendix B: Chapter 2 Summary of Plant Use and Antimicrobial Screening Data  190  Appendix C: Chapter 3 Supplementary Data  204  Appendix D: The St'at'imc Nation Statement of Proprietary Rights  217  The Statement of the Union of British Columbia Indian Chiefs' Protecting Knowledge: Traditional Resource Rights in the New Millennium Conference  Appendix E: The International Society of Ethnobiology Code of Ethics  220  vii LIST O F T A B L E S  Table  Title  2.1  Secwepemc medicinal plants grouped according to medicinal use.  2.2  Species list and voucher numbers of the 68 Secwepemc plants collected for antimicrobial activity screening.  2.3  29 29  Summary of antimicrobial activity screening results of 68 Secwepemc medicinal plant extracts.  3.1  25  Fungal species tested in inhibition assays using Secwepemc medicinal plant extracts.  2.5  24  Bacterial species tested in inhibition assays using Secwepemc medicinal plant extracts.  2.4  Page  33  Summary of antibacterial and antifungal activity profiles of methanolic extracts of raw and pitcooked balsamroot samples and boiled pitch from raw and pitcooked roots, based on disk diffusion assays.  3.2  Results of bacterial overlay spot assays using a methanolic extract of raw outer roots.  3.3  3.9  81  Results of bacterial overlay spot assays using dichloromethane surfaceextracts of dried leaves.  3.8  77  Results of disk diffusion assays of methanolic extracts of dried leaves, and dichloromethane surface-extracts offreshand dried leaves.  3.7  76  Results of bacterial overlay spot assays using a methanolic extract of pitcooked inner roots.  3.6  76  Results of bacterial overlay spot assays using a methanolic extract of raw inner roots.  3.5  76  Results of bacterial overlay spot assays using a methanolic extract of pitcooked outer roots.  3.4  75  86  Results of bacterial overlay spot assays using dichloromethane surfaceextracts of fresh leaves.  86  Results of bacterial overlay spot assays using purified band 2.  86  viii 3.10  ' H N M R spectral data of band 2.  95  C N M R spectral data of band 2.  96  3.11  1 3  B. l  Plant names, medicinal indications and antimicrobial activity of the Secwepemc plant extracts examined in this study.  191  C. l  X-ray crystallographic data summary for crystal I.  208  C.2  Mass intensity table of the electron impact mass spectra of band 2.  209  C.3  Mass intensity table of the electron impact mass spectra of 4-acetoxyisopruteninone.  C.4  C.5  210  Comparison of fragmentation patterns of known products from Balsamorhiza sagittata leaves.  211  Interpretations of the fragmentation pattern for band 2.  213  ix LIST O F F I G U R E S  Figure  Title  Page  2.1  Map of Secwepemc traditional territory.  12  3.1  Balsamroot.  42  3.2  The structure of inulin.  43  3.3  The structure of thiophene E.  46  3.4  Nucleophilic addition of a thiol group across the a-methylene moiety of the y-butyrolactone ring in a sesquiterpene lactone.  3.5  47  A schematic summary of procedural steps taken for the bioactivity-guided isolation of thiophene E and the purification and identification of crystal I from boiled balsamroot pitch.  3.6  59  T L C overlay series showing fractions A to X derived from bioactivity-guided partitioning of boiled balsamroot pitch on silica column chromatography.  3.7  Thin layer chromatography overlays comparing antibacterial activity of thiophene E standard and crude thiophene E from boiled balsamroot pitch.  3.8  68  GC-MS chromatograms of thiophene E standard compared with crude thiophene E isolated from boiled balsamroot pitch.  3.9  67  69  GC-MS chromatograms of the major component of fraction R at 16.97 minutes.  70  3.10  Crystal I purified from fraction I of balsamroot pitch.  72  3.11  T L C and T L C overlays of fraction I and crystal I using S. aureus.  73  3.12  Structure of crystal I (C30O3H48) based on X-ray crystallography.  74  3.13  Mass spectra and GC-MS chromatograms of thiophene E standard compared with methanolic extracts of raw and pitcooked root samples.  3.14  79  Thin layer chromatography overlays comparing antibacterial activity of thiophene E standard and methanolic extracts of raw and pitcooked roots.  80  3.15  T L C plates and T L C overlay comparing dichloromethane surface-extracts o f fresh and dried leaves.  3.16  83  H P L C profiles comparing fresh and dried leaf extracts for the presence o f band 2.  85  3.17  Bacterial overlay spot tests for purified band 2.  87  3.18  Electron impact mass spectrum o f band 2 purified from a dichloromethane surface-extraction o f fresh leaves o f balsamroot.  3.19  89  Mass spectrum o f band 2, mass spectrum o f 4-acetoxyisopruteninone and the difference between the two mass spectra.  90  3.20  Structure o f 4-acetoxyisopruteninone.  91  3.21  Proposed mass spectrum fragmentation for 4-acetoxyisopruteninone.  91  3.22  Proposed structure for band 2 (2-deoxy-pumilin-8-0-acetate).  94  3.23  Proposed mass spectrum fragmentation for band 2.  94  3.24  Structural comparison o f 16R,23R-dihydroxycycloartenone and 16R,23R-dihydroxycycloartenol.  101  C. 1  Summary o f ' H N M R spectral data o f crystal I.  205  C.2  Summary o f *H N M R spectral data o f 16R,234-dihydroxycycloartenone.  205  C.3  G C - M S o f crystal I.  206  C.4  Interpretation o f the fragmentation pattern o f crystal I based on combined G C - M S and X - r a y crystallographic data.  207  C.5  Ultraviolet spectrum o f band 2.  214  C.6  !  C.7  1 3  H N M R spectrum o f band 2. C N M R spectrum o f band 2.  215 216  xi  ABBREVIATIONS AND DEFINITIONS £,  undetermined configuration at an asymmetric carbon atom  ACS  American Chemical Society  ATCC  American Type Culture Collection  AU  absorbance units  1 3  CNMR  13  carbon nuclear magnetic resonance  CAS R N  Chemical Abstracts Service Registry Number  CBD  Convention on Biological Diversity  CHCI3  chloroform  cfu  colony-forming units  chloroform-di  deuterium labeled chloroform  cm  centimetre  CO2  carbon dioxide  d  doublet  de novo  starting anew  DMEM  Dulbecco's modified Eagles Medium  DNA  deoxyribonucleic acid  ex situ  removed from its original place  FBS  foetal bovine serum  FW  formula weight  g  gram  GC-MS  gas chromatography-mass spectrometry  'FI N M R  proton nuclear magnetic resonance  HPLC  high performance liquid chromatography  HSV1  '  Herpes simplex virus type 1  in situ  'on site'; in its original place  in vitro  'in glass'; outside of the body or performed in an artificial environment  in vivo  taking place within the body or within a living organism  int  intensity in counts  IPR  intellectual property rights  IUPAC  International Union of Applied Chemistry  J m  coupling constant metre  r^g  microgram  1*1 um  microlitre micrometre  [M] or [M-H] +  +  molecular ion peak  m  multiplet  m/z  mass/charge ratio  MBC  minimum bactericidal concentration  mg  milligram  MHA  Mueller Hinton agar  MHB  Mueller Hinton broth  MHz  megahertz  MIC  minimum inhibitory concentration  ml  millilitre  mm  millimetre  MRC  Medical Research Council of Canada  MS  mass spectrum  MTT  methylthiazolyltetrazolium chloride  NIST MS  National Institute of Standards and Technology Mass Spectral Library  nm  nanometre  NSERC  Natural Science and Engineering Council of Canada  °C  degrees Celsius  pfu  plaque-forming units  ppm  parts per million  %BP  percentage of base peak  q  quartet  R  rectus or right-handed  Rf  migration distance (from origin) of a substance divided by (-H) the migration distance of solvent front  s  singlet  S  sinister or left-handed  SCES  Secwepemc Cultural Education Society  SSHRC  Social Science and Humanities Council of Canada  thiophene E  7,10-epithio-7,9-tridecadiene-3,5,ll-triyne-l,2-diol or 2-(l-propynyl)-5-(5,6-dihydroxyhex-3-yn-l-ynyl)-thiophi  TLC  thin layer chromatography  TRR  traditional resource rights  UV  ultraviolet  v/v  volume/volume  VSA  vanillin sulphuric acid  XIV  ACKNOWLEDGEMENTS It is with deep gratitude that I offer thanks to the many individuals (and other entities) who have contributed to this dissertation, directly or indirectly and knowingly or unknowingly, in various ways. This section may be rather lengthy but for this I offer no apologies as each person noted below has played an important role in the development of this dissertation. There are, of course, others who have also assisted with the process, whom I have not mentioned by name, but my gratitude extends to them as well. First, for the opportunity to be a part of the Secwepemc Ethnobotany Project and conduct my field research in Shuswap country, I acknowledge the Shuswap Nation Tribal Council, the Secwepemc Cultural Education Society, and members of the Secwepemc Nation. I thank especially: Chief Ron Ignace who has always supported my research and enriched my fieldwork. One summer he even broadened my ethnobotanical experiences to those 'ethnozoological' in nature with an unforgettable moose hunt—and he offered support and a hearty laugh at my inability to pull the trigger when I had the only clear shot; Marianne Ignace who has been an inspiration and provided me with much 'food for thought' over the years (and I hope has forgiven me for not pulling that trigger); the lovely Ignace children who have shared with me their Skeetchestn home and many words of wisdom on numerous occasions; (the late) Elder Nellie Taylor who inspired me to study soapberry, and whose mesmerising stories and special twinkle will always be remembered fondly; Elder Sarah Denault who first challenged me to reconsider the objectives and broader implications of my research; and Elder Mary Thomas who inspired me to study balsamroot and who (along with her family) unceasingly has shared her knowledge, insights, home, and more—Mary's pride for Secwepemc culture, concern for the environment and fearless thirst for knowledge shall surely serve as timeless inspiration to all peoples. I thank Neil Towers for sharing his fascination and enthusiasm for phytochemistry and his wealth of entertaining stories, and for providing laboratory space, materials and practical assistance in the form of research chemists. I am greatly indebted to chemists Leoncio Rene Orozco and Nikolay Stoynov for their generous assistance and unceasing patience that made the phytochemical component of the balsamroot research possible. I also thank Srinivas Malladi, Jens Pedersen, Hanawa Fujinori and Hector Barrios Lopez for assistance with research on  XV  soapberry that was not included in this dissertation. I thank Jim Hudson for generously sharing his facilities and expertise for the antiviral research component. I have appreciated the contributions of the Towers' Lab members over the years, and in particular, I would like to thank : Robin Taylor for plenty of helpful tips in my initial years; Jon Page for providing inspiration and for many thoughtful suggestions both in the lab and in my early writings; Kevin Usher for helping make sense of the mysteries of secondary metabolism in our early years, collaborative work on balsamroot leaf chemistry in our later years, and many necessary pints of beer spread over all of the years; Fiona Cochrane for late night (or all-night) comradeship and innumerable favours (especially in the last few months when I fled the big city to resume Island life); and the lovely and irreplaceable Zyta Abramowski, miracle-worker and the heartbeat of the Towers' Lab, who has generously assisted me in so many ways. The Botany office is staffed with angels who not only have divine patience but good senses of humour as well. For their assistance, I thank Fiona Craig, Judy Heyes, Veronica Oxtoby, and especially Lebby Balakshin. The other important angel without whom this dissertation would never have made it into electronic form is my gifted computer guru and friend Nitin Verma. The research process was financially assisted by the Social Science and Humanities Research Council (grant to N. J. Turner), the Natural Science and Engineering Research Council (grants to G. H. N. Towers and J. B. Hudson), a University of British Columbia Graduate Fellowship (1997-99), the Canadian Bacterial Diseases Network (graduate student research stipend, 1999), the Craig Adams Sandercock Memorial Scholarship (1999) and a grant from Global Forest Science (graduate student research stipend, 2000) Catalogue Number GF-18-200073. I am particularly grateful to Reese Halter of Global Forest for providing financial support at a crucial time, and in so doing, acknowledging that humans are an important part of forest ecology. My Ph. D. supervisory committee may have been the largest and most diverse in the history of the Botany department. I thank them all for their assistance and for graciously rising to the various extra challenges that presented themselves en route due to the multidisciplinary nature of my work. These individuals are: Nancy Turner, Iain Taylor, Jim Hudson, Neil Towers, Terry Pearson, Edward Ishiguro and George Nicholas.  xvi Several of my committee members have gone beyond the call of duty on numerous occasions. Iain Taylor, Terry Pearson and George Nicholas provided Herculean editorial assistance with numerous conference papers, book chapters or journal articles, as well as with this dissertation (with that said, however, I still lay claim to all errors herein). Collectively, I thank this scholarly triumvirate for sharing with me their literary brilliance, but individually, I must acknowledge them as well. First, I thank Iain Taylor for clearing a chair for me on the many occasions that I sought his 'sage wisdom' in his role as department Head. Second, I thank George Nicholas for opening up to me the archaeological world, for invaluable discussions about the parallels between current archaeology and ethnobotany, for usually understanding all the 'important stuff whether or not I was able to articulate it, and for sharing his beautiful Kamloops home with the entire 'Ethnobotany Team' on numerous occasions. My third 'thanks' provides a challenge; as Lulu said (or more accurately, sang), it isn't easy to "thank someone who has taken you from crayons to perfume". It is difficult, indeed, to articulate fully my appreciation of Terry  Pearson who has been a mainstay of support, encouragement, and inspiration throughout my entire graduate career. While his role has evolved from mentor to colleague, the role model that he represents shall always be one to which I aspire. I am indebted also to Michael Macdonald for his interest in the ethnobotanical applications of research ethics that have been raised in my research. I am grateful for his helpful comments and the thoughtful and thought-provoking course materials that he has brought to my attention. I am encouraged and inspired also by his significant efforts toward restructuring of ethical policy with the Tri-Council. Throughout the research process, my understanding has benefited greatly from others who have offered alternative positions. In particular, I extend my appreciation to Michael Brown for helpful criticism and discussion in the area of heritage protection, and Fidel Fogarty for support amid frank (if not edifying) discussions related to some of the political and economic realities of ethnobotanical and phytochemical research. The best part about my dissertation research was that it was part of a larger project, which provided a mixture of perspectives, tremendous collaborative opportunities, and fieldwork that could always be adjusted to 'feed several pigeons with one bean'. For field assistance, stimulating conversations, and heaps of fun 'ethnobotanising' in Shuswap country, I thank the members of the Secwepemc Ethnobotany Project 'research team', especially: Nancy Turner,  XVII  Chief Ron Ignace, Marianne Ignace, Sandra Peacock, George Nicholas, Dawn Loewen, Gladys Baptiste, and Darrell Eustache. I also thank Sandra Peacock for our balsamroot collaborative research (and writings)—that little 'side project' that unexpectedly turned into a fascinating and major undertaking, for which I am most grateful. For legal assistance and help acquiring copies of those hard-to-get type documents, I thank the three nicest lawyers on the planet, Howard Mann, David Stephenson, and Maui Solomon. I am deeply grateful to Maui Solomon also for sharing with me many a special waiata and karakii, and much about his Maori and Moriori heritage, in ways that have inspired and influenced me beyond words. There are four individuals who have been the corner posts that have grounded my research orientation and provided the foundation upon which I have built my understandings. In person and through their writings, each has inspired, encouraged and challenged me tremendously. For this, I am deeply grateful to Timothy Johns, Richard Ford, Darrell Posey, and George Nicholas. For four and a half years, I have traveled back and forth each weekend from Vancouver to my home in Victoria and later to my home on Thetis Island, which added up to over 500 ferry trips, thousands of bus or car trips, and of course, thousands of dollars in travel. I am greatly indebted to those Vancouverites who generously provided me with a home away from home, as well as their friendship and support during this time, including: Tanya and Eduardo Jovel, George and Nora Treit, Harold and Julie Lindgaard, Kevin and Kathy Usher, Christine Park, Allen and Zoe Sinell, and my wonderful cousin Sheilah MacDonald. My parents, Kathie and Tom Richardson, my parents-in-law, Helen and Bill Bannister, and the rest of my family have patiently awaited the day that my time as a student would formally come to an end. I offer my thanks to them, with the qualification that 'thank you' is particularly inadequate to express my appreciation for their many forms of support over the years that I have been in graduate school. A person who has played an important role in this work is Katherine Barrett. I am thankful to her for inspiration, for company in meeting challenges that have stoked the creative fire (not precariously, but 'precautiously'), for sharing 'craaazy' ideas over coffee during graduate school at UBC, and for teaming up with me to put the best of those ideas into action  XV111  during our upcoming postdoctoral years at UVic with the Eco-Research of Environmental Law and Policy. Another special person that I wish to thank is the one who got me into this area in the first place—quite by accident, and what seems like several lifetimes ago—Glen Rutledge. I first learned of ethnobotany at the Astrophysical Observatory in Victoria, through Glen's insistence that I read an article in Discover magazine (written by Paul Cox) that was in the lunchroom. "It's perfect for you, Kel", he'd said. "Sure", I'd thought at the time, "from microbiology to ethnobotany—nice thought, but not very likely". Some months later, I wandered out of the Biochemistry/Microbiology Department and into the School of Environmental Studies to meet Nancy Turner. It was a case of being in therightplace at therighttime—I was offered the opportunity to do some microbiologial work within the Secwepemc Ethnobotany Project, and Glen is still chuckling at his foresight. Working with Nancy Turner has been the highlight of my doctoral degree. I am greatly indebted and eternally grateful that she took a gamble on 'the likes of me'—well-meaning but certainly naive and inexperienced outside of the relatively safe confines of the laboratory. Nancy opened unto me an entirely new world, that has offered many experiences, challenges and privileges that I could never have anticipated. The greatest of these privileges, however, has been to have Nancy as my Ph. D. supervisor, collaborator, mentor and friend. Finally, I search for words worthy of honouring the one person who has supported me, in his quiet but unceasing way, through each of the numerous and often unexpected challenges that this journey has presented. Ma te rakau te hau e pupuri (By the trees is the wind tamed)  Ma te kohatu te ngaru e whati (By the rock is the wave broken) —Maori saying, shared by Maui Solomon  I thank my husband, Ron, whose support has been as a forest—enabling me to bend with the wind but not break, and as an island—securing my stand amid rough seas. At last, my love, I am coming home.  XIX  DEDICATION  This work is dedicated to the memory of my two grandmothers:  For Gran—my father's mother, Kathleen Richardson—whose athletic and other life accomplishments have challenged and greatly inspired me, and whose Olympian endurance, discipline and determination (lodged somewhere in my genes, I reason) were gifts for me to draw upon over the past five years or so.  For Nan—my mother's mother, Mary Archer—in loving memory of amply-rewarded hours in her berry patch, wanderings through her jungle garden and that little book of cure-alls and homemade remedies that she trusted more than any doctor. Her ingenuity, pioneering spirit, and unconditional love inspire and honour us still.  1 1  General Introduction We are all the guests of the green plants around us.  —Ismail Serageldin (1995:5)  The eloquence of Serageldin's statement above is a humble reminder that our reliance on plants is far deeper than mere resource-dependence for nutritional, medicinal, spiritual and material necessities or pleasures. The immobile, non-predatory nature of (most) plants requires the ultimate self-sufficiency—the biochemical manufacturing of an energy source through photosynthesis—and this, either directly or indirectly, assures the existence of most life on earth (Demmig-Adams and Adams 2000). The chemical components of plants produced by metabolism (i.e., phytochemicals), and certain interrelationships with humans that are mediated by these chemicals, are a primary focus of this dissertation. A second major aspect of this dissertation moves beyond the chemistry of plant-human interactions into the current ethical, political and legal debate on contemporary use of the cultural knowledge of Indigenous peoples. The issues are of global interest and concern, at local and international levels, but have largely remained outside the realm of academic research in the natural sciences. The combined field and laboratory research undertaken for this dissertation provides a unique opportunity and a valuable context to present these complex issues to a wider audience. 1.1  Plants, Peoples and Phytochemicals  The following two (seemingly unrelated) definitions of the word "plant" are found in the Oxford dictionary (1983:498): (i) "a living organism that makes its own food from inorganic substances and has neither the power of movement nor special organs of sensation and digestion"; and (ii) "a factory or its machinery and equipment". The amalgamation of these two definitions—plants as organisms viewed as miniature chemical producing factories—provides an accurate description of the perspective that has guided this research.  2  Plants synthesise a vast array of chemicals, which are often categorised as primary and secondary metabolites. This distinction is not rigorous because of multiple roles for some metabolites, and shared precursors that interconnect metabolic pathways. Hence, while products of primary metabolism often serve as precursors for secondary metabolism, it is an oversimplification to envision primary and secondary metabolism as a one-way connection. In general, primary metabolites (e.g., sugars, amino acids, common fatty acids, nucleotides, and derived polymers) are essential for normal cellular functions (i.e., for survival of the plant at the individual level), and largely contribute to the nutritional value of plants as a food source for other organisms (Mann, 1987). In contrast, many products of secondary metabolism are considered to be non-essential for the survival of individual plants, but to confer advantages for survival and reproduction, and to mediate a wide variety of interactions between plants and their environments, such as attraction of pollinators or protection from herbivory and microbial pathogenesis (Mann, 1987; Harborne 1993: Ryan and Jagendorf 1995: Harborne 1997). While the distinction between primary and secondary metabolites may be somewhat hazy, it provides a suitable framework for the applications of plant chemistry to this dissertation. The term phytochemical will be used when referring collectively to both primary and secondary  metabolites, or when a distinction between the two is not possible or appropriate for the purposes of this research. Recognition of the ecological importance of secondary compounds in mediating interactions with other species is a relatively recent development (Harborne 1993; Mann 1987). Once they were thought of "as the useless detritus produced by obscure metabolic pathways" (Mann 1987:ix), but now it is widely acknowledged that many secondary metabolites have profound influences on the interactions of species with one another (Mann 1987). The study of secondary metabolites as ecological mediators is fundamental to the field of chemical ecology (also called ecological biochemistry), which seeks to identify not only the chemical interactions between organisms but also the co-evolutionary adaptations to these interactions, and the resultant ecological effects of these interactions and adaptations (Mann 1987). Chemical ecology provides a fascinating and valuable perspectivefromwhich to view the chemistry of natural products within biological systems, and the co-evolutionary significance of phytochemicals for all organisms, including humans.  3 The extensive chemical repertoire of plant secondary metabolites has been widely accepted as the result of co-evolutionary adaptations to plant-herbivore, plant-pathogen and plant-plant interactions, and responses to changes in the physical environment such as climate or geology (Mann 1987). In oversimplified terms, if the only evolutionary consequences to adverse conditions are adaptation, emigration or extinction, then the diversity of secondary metabolites found in plants may be explained by the fact that plants can't run away. From the perspective of chemical ecology, it is not surprising that many phytochemicals have a variety of biological activities that can exert significant physiological effects in situ or ex situ on other organisms, including humans. In fact, phytochemicals can account for many  beneficial or detrimental effects of plants on humans, some of which are therapeutic or medicinal. Delaveau (1981:399) suggested that a "food-poison-remedy" sequence largely explains the discovery of medicinal plants by ancient cultures, and that bad taste and bitterness in particular were directly related to the activity of a given remedy. Indeed, plants have been exploited for their chemical properties by humans for millennia (albeit with varying degrees of chemical awareness), and extracts of plants have remained a significant component of the pharmacopoeiae of cultures worldwide, especially in Aboriginal (or Indigenous) and local societies embodying traditional lifestyles . As noted by Mann (1987:1): "Primitive man found 1  these extracts efficient as medicines for relief of pain or alleviation of the symptoms of disease, as poisons for use in warfare and hunting, as effective agents for euthanasia and capital punishment, and as narcotics, hallucinogens, or stimulants to relieve the tedium, or alleviate the fatigue and hunger in his life. He must also surely have used the more odiferous and spicy compounds to obscure the odour of unwashed humanity, and to disguise the putrid or bland flavour of his food." An ecological emphasis for ethnobotanical research is grounded in the work of the late Volney Jones, who, as Ford (1994a) pointed out, acknowledged a two-way cause-and-effect relationship in the human exploitation of plants (i.e., not only do plants affect humans but humans affect plants), and underscored "the reciprocal and dynamic aspects" of plant-human In this dissertation, the terms "Aboriginal" and "Indigenous" are used interchangeably. According to Cassidy and Longford (n.d.:preface) the term "Aboriginal" is commonly used in Canada to include Indians, Metis and Inuit as in the Constitution Act of 1982 while the term "Indigenous" is used in international treaties worldwide in the context of "Indigenous and local societies embodying traditional lifestyles". The term "Aboriginal is now commonly capitalised in federal documents and I have chosen to follow this format. The term "Indigenous" is rarely 1  4 interactions (Ford 1994a:31). In a similar manner, Johns (1990:15) viewed the traditional use of plants as food and medicine from an evolutionary perspective as "an integrated response to the chemical environment with both biological and cultural components". In his proposed model of human chemical ecology, Johns (1990, 1997) depicted a triangular interrelationship between humans, parasites and plant chemicals. The phytochemical composition of a plant is seen largely as a biological response to factors such as parasitic disease, herbivory and plant domestication. This phytochemical repertoire, in turn, is an exploitable resource of nutritional and medicinal value to humans, and of particular importance in limiting infectious disease. Johns' (1990:213) model further depicted specific interrelationships between nutritional status, infectious disease and toxicity. "Balanced nutritional status" (as a factor in "health") is seen as directly related to "diet", which includes both "nutrients" and "allelochemicals" (i.e., secondary metabolites). Johns' model is consistent with Scrimshaw's (1995) view that the aetiology of disease (whether it is an infectious organism, a toxin, a nutritional imbalance, or other) is multifactorial and can only be understood in the broader context of human ecology that considers all of the factors involved. Scrimshaw (1995) suggested that the effect of a diseasecausing agent on a given host will be influenced by synergistic interactions involving the host, the agent and the environment. Indeed, it is widely accepted that nutrition and immunity are connected (Chandra 1988; Grimble 1995; and Harbige 1996) and that nutritional status can influence susceptibility to infectious disease. In addition, nutritional status can affect the capacity for detoxication, i.e., the inherent physiological or biochemical mechanisms that protect an organism from toxic compounds (Johns 1990). It is also clear that some phytochemicals in the diet can cause toxicity, a property with both positive and negative potential for influencing health status. For example, in addition to acute toxicity, chronic exposure to some plant toxins can depress the immune system (Colwell and Patz 1998). In other cases, toxicity can be used advantageously, such as in the use of plants as antimicrobial agents (i.e., in the treatment of infections caused or exacerbated by bacteria, fungi or viruses). The latter type of application is particularly relevant to this dissertation. The biological effect of a phytochemical may or may not be selective in nature. For example, in the case of plants exploited for their antimicrobial properties by humans, the capitalised but I have chosen to capitialise it as well since it is equivalent to "Aboriginal" in my usage. My rationale is that both terms substitute for "Indian", which is a proper noun.  5 biological activity of a phytochemical might lead to inhibition or eradication of a specific microorganism or might be generally inhibitory or toxic to many kinds of living cells, including those of the human host. In the latter case, therapy is obviously effective only if the human is able to withstand the treatment while the microbe is not. While numerous detoxification strategies (i.e., physiological mechanisms and physical methods to remove toxicity) exist to lessen the undesirable effects of phytochemicals (Johns and Kubo 1988), Johns (1990) claimed that it is not possible to avoid them completely in the diet. This led Johns (1990:xv) to suggest that human adaptation to these has, in an evolutionary sense, linked them with our "internal ecology" such that phytochemicals are essential for basic metabolism (Johns 1997). As many of the properties of plants that make them unpalatable or toxic also make them pharmacologically useful, Johns (1990) proposed that the medicinal use of some plants lies in the fact that humans can consume and tolerate moderate levels of most toxins. Furthermore, Johns (1997) proposed that toxic plants provide nutrients in a diet that would otherwise be deficient, and he made the intriguing statement that: "in a chemical-ecological sense, obtaining adequate nutrition and responding to allelochemicals are two sides of the same coin" (Johns 1990:251). From a human chemical-ecology perspective, then, the distinction between food and medicine becomes increasingly blurred, and parallels the sometimes hazy distinction between primary and secondary metabolites indicated previously. Johns (1990) suggested that interactions between humans and plant chemicals are more appropriately understood as a concentration-related (or situation-related) continuum that includes foods, beverages, condiments, medicines, stimulants, psychoactive agents, and toxins. Thompson elder Hilda Austin (Lytton) described it this way: "Our food is our medicine. If you eat it often, that's a medicine" (Turner et al. 1990:43). In this respect, the existing botanical (and other ecological) knowledge of Indigenous peoples and traditional societies offers a precious link between the past and present importance of plants as food and medicinal resources. From a chemical perspective, such cultural knowledge provides an invaluable opportunity to expand our understanding and appreciation of plant choice for a particular purpose, since among any repertoire of available plants presumably there are those that are inedible, unpalatable, toxic or ineffective as medicines. These plants are not necessarily avoided as foods and medicines, but are processed by appropriate technologies designed to increase digestibility, improve taste, detoxify and/or otherwise alter or eliminate undesirable properties while enhancing those that are desirable.  6  Furthermore, while the presence of some phytochemicals is relatively constant throughout the plant life-cycle, the presence of others is subject to significant variations in concentrations, due not only to life-cycle stage but also to various environmental stresses (Mann, 1987; Harborne 1997). Thus, an understanding of the phytochemistry underlying plant selection, harvesting, processing methods, and differential uses, provides an essential perspective for understanding human-plant interrelationships in the context of plant use for food and medicine. The pursuit of such an understanding has been a motivating force behind the research described within this dissertation.  1.2  Research Collaboration with the Secwepemc Cultural Education Society and the Secwepemc (Shuswap) Aboriginal Nation through the Secwepemc Ethnobotany Project We are not presenting a culture under glass. We want people to understand we are very much alive.  —Leona Thomas, Secwepemc Heritage Park (as quoted in Coull 1996:137) The aspiration to understand the chemically-mediated plant-human interrelationships described previously is by no means accompanied by the right to do so. Thus, I have been privileged with the opportunity to address my research questions in a research collaboration with the Secwepemc Cultural Education Society (SCES) and the Secwepemc (Shuswap) Nation as 2  part of a large, interdisciplinary ethnobotanical research program focussing on Secwepemc plants, language and culture. The Secwepemc Ethnobotany Project was initiated in 1991 in consultation with and with 3  the support of the Shuswap Nation Tribal Council , the Secwepemc Cultural Education Society, 4  and Secwepemc elders and researchers from various Secwepemc communities. The initial focus  The Secwepemc Cultural Education Society is a non-profit organisation that was formed in 1983. It arose from a commitment to cultural renewal embodied by the Shuswap Cultural Declaration, which was signed in 1982. The Secwepemc Cultural Education Society is devoted to preserving and perpetuating the Secwepemc language, culture and history. Principal investigator: N. J. Turner (UVic); Co-investigators: M . B. Ignace (SFU), G. P. Nicholas (SFU), H. V. Kuhnlein (CINE, McGill), and Chief R. Ignace (Skeetchestn Band, Secwepemc). Shuswap Nation Tribal Council Chiefs Council Meeting, motion #04/10/93 of support. 2  3  4  7  of the Secwepemc Ethnobotany Project (1991-1994) involved comparison of Secwepemc plant 5  names and associated knowledge of plants from different ecological and dialect regions of Secwepemc territory. Subsequently (1994-1998) , the project expanded its focus to include the 6  role of plant resources in the lifeways of Secwepemc peoples, past and present, by incorporating a combination of anthropological, archaeological, ecological, linguistic, nutritional, and biochemical approaches. The research described here is part of the biochemical research on Secwepemc food and medicinal plants. The combined data and experiences resulting from the Secwepemc Ethnobotany Project were meant to provide a base of information and training in applications such as community education, language and cultural programs, resource management, biodiversity research, traditional use studies, treaty negotiations involving land and resources, and healthcare regimes for Secwepemc peoples. There are two forthcoming manuscripts based on this research that will be co-published by the Secwepemc Cultural Education Society (Turner and Ignace in preparation; Turner et al. in preparation). Research within the Secwepemc Ethnobotany Project has taken place with the approval of the appropriate governing bodies of the Secwepemc Nation. As is required for all research involving humans conducted in Canada, individual academic researchers underwent formal ethical review by their university authorities for their research proposals , including approval of a 7  written Letter of Consent. In Chapter 4,1 discuss the ethical review process and other academic policies that have governed my dissertation research. A copy of my Letter of Consent is included in Appendix A. There are a number of important practical and philosophical issues related to the preparation and implementation of such letters and agreements, especially within the broader scope of research involving the cultural knowledge of Indigenous societies. These are discussed in Chapter 4.  Secwepemc Plant Knowledge: More Than the Sum of the Parts. SSHRC Grant 410-91-0550. Secwepemc (Shuswap) Ethnobotany: Expanding Horizons. SSHRC Grant 410-94-1555. Ethical standards and guidelines are set out by the Tri-Council Policy Statement: Ethical Research Conduct Involving Humans, which governs all such academic research at Canadian institutions, as well as federal funding supported by the Social Sciences and Humanities Research Council (SSHRC), the Natural Sciences and Engineering Research Council (NSERC) and the Medical Research Council (MRC).  5 6  7  8  1.3  Ethnobotany or Economic Botany? Since this research was conducted as part of a larger ethnobotanical study, I think that it  is important to distinguish between two fields of inquiry that are sometimes considered to be synonymous, namely ethnobotany and economic botany. Ford (1994b:44) defined ethnobotany as "the study of direct interrelations between humans and plants" and emphasised that it is "concerned with the totality of the place of plants in a culture". He distinguished ethnobotany from economic botany, which focuses on the uses of plants and the utility of their incorporation into another culture (i.e., most often mainstream society), thereby leading to indirect plant contact by the "benefactors" through plant "by-products" (Ford, 1994b:44). In my interpretation, Ford's distinction between ethnobotany and economic botany relies partly on an emphasis of process over product. While I hope that the plant 'by-products' that were 'discovered' through  this research are useful in any number of ways, it is a deeper understanding and appreciation of the interactive process of plant-people relationships within Secwepemc culture to which this research is primarily meant to contribute. This point is addressed more fully in Chapter 4. 1.4  Research Objectives and Thesis Organisation This research is an investigation of the antimicrobial properties of traditional and  contemporary plant resources of the Secwepemc First Nation, and the specific plant-human interactions that are mediated by these antimicrobial compounds. Ethnobotanical information on Secwepemc medicinal plants (based on Turner and Ignace in preparation) directed the initial plant collections. I assumed that Secwepemc use of plants to treat conditions likely caused by pathogenic bacteria, fungi or viruses was an indicator of antimicrobial properties of the plants. I confirmed antimicrobial activities in many of these plants using assays in vitro (Chapter 2). The results of the antimicrobial assays, combined with additional ethnographic information, led to the selection of one plant for in-depth chemical analysis and antimicrobial characterisation. The indepth analyses helped to clarify the relationship between Secwepemc botanical knowledge (relating to plant selection and differential processing for food and medicine) and phytochemical composition (relating to the solubility, localisation and biological activity of antimicrobial plant  9  compounds in processed plants). The plant selected was Balsamorhiza sagittata (Pursh) Nutt. (Asteraceae), commonly called balsamroot (Chapter 3). A current study involving the cultural knowledge or traditional resources of Indigenous peoples would be remiss without acknowledging the ethical and legal issues of rights to intellectual property and traditional resources. Chapter 4 outlines some of the specific challenges encountered through this research and from the research outcomes, and places the issues within the broader contexts of biocultural diversity research and bioethical aspects of research involving humans. Chapter 5 concludes with a general discussion, a summary, some concluding comments, and suggestions for future research in this area.  10 2  A History of Microbial Diseases in Secwepemc Culture and the Contemporary Antimicrobial Properties of Secwepemc Medicinal Plants To see things in their right perspective, we need to understand the past as well as the present.  -Secwepemc Elder Mary Thomas (pers. comm. 1997)  2.1 2.1.1  INTRODUCTION Secwepemc Culture and Traditional Territory  The Secwepemc (Shuswap) Nation, presently composed of 17 bands, is the largest of the Interior Salish-speaking Aboriginal groups in south central British Columbia. "Shuswap" is the 8  anglicised word for "Secwepemc" or sex epemx (Ignace 1998:203), which can be translated as w  the "spilled people" or the "spread-out people" (Ignace 1998:217). It is an appropriate name considering that Secwepemc traditional territory extends over approximately 180,000 km in the Interior Plateau region , bordered by the Coast Mountain Range on the west and the Rocky 9  Mountain Range on the east (Figure 2.1). The territory has considerable botanical diversity, and includes nine biogeoclimatic zones: Bunchgrass, Ponderosa Pine, Interior Douglas-fir, Interior Cedar - Hemlock, Montane Spruce, Sub-Boreal Pine - Spruce, Engelmann Spruce - Subalpine Fir, Sub Boreal Spruce, and Alpine Tundra (Meidinger and Pojar 1991). As in most Aboriginal societies, Secwepemc traditional subsistence strategies varied and paralleled the diversity in landscape, climate, and floral and faunal resources. They included fishing (e.g., salmon and trout), hunting (e.g., ungulates, black bear, small game and birds), and harvesting of an extensive number of wild plants. Annual cycles were based on seasonal availability of resources, beginning in Spring with trout fishing, plant shoot and tree cambium harvesting, and digging of bulb and root foods. Summer months included salmon fishing, berrypicking and further bulb and root harvesting, and Fall was the main hunting season (Ignace 1998). In winter, people survived mainly on preserved foods, so that prior collection, processing The Interior Salish consist of the Stl'atl'imx (Lillooet), the Nlaka'pamux (Thompson), the Okanagan and the Secwepemc (Shuswap). Interior Plateau Aboriginal peoples include the Interior Salish (see footnote 8) as well as the Kutenai of south eastern B.C., the Chilcotin, the Carrier, and the now extinct Nicola (McMillan 1988). 8  9  11 and food storage were vital to survival. Over 200 plant species were traditionally used by the Secwepemc and other Interior Salish peoples for foods, medicines, materials and spiritual purposes (Ignace 1998). A successful subsistence lifestyle requires sustainable relationships with land and resources. In this respect, Secwepemc traditional resource management was linked with social and spiritual values, and a belief in kinship ties between humans, animals and the natural world. This kinship was manifested in the active management of resources that included: selective harvesting of plants and animals in appropriate seasons, taking only what was needed, using all parts of an animal, controlled burning regimes, bark harvesting methods that would not kill the tree, pruning of berry bushes, and re-planting of underground crop propagules as part of the harvesting process, among other strategies (Coffey et al. 1990; Ignace 1998; Peacock and Turner 1999; Turner, Ignace and Ignace in press). Like relationships with the land in many other Aboriginal societies, traditional approaches to resource management in Secwepemc culture were "permeated with rules and rituals designed to ensure the collective health of the people" (Kelm 1998:86) and embodied a conservation ethic that was based on respect for nature and all living things—including "rocks, fire, water and other natural phenomena [that] were believed to have a soul" (Ignace 1998:213). However, the lifeways of the Secwepemc were significantly altered with European contact. As noted by Coffey et al. (1990:7), "the balanced lifestyle depended on traditional Indian skills and knowledge handed down through the ages by word of mouth. This all changed with the appearance of the fur traders, missionaries, gold miners and settlers". Some of the historical changes that have particular relevance to my research are outlined in the following section.  Legend •  Present Shuswap Bands  •  Extinct Shuswap Bands Traditional Shuswap Territory  flz/  Territory occupied by the Chilcotin after 1880  =^=i  Territory shared with other Indian nations  'flfl  Figure 2.1. Map of Secwepemc traditional territory (Coffey et al. 1997:9).  13 2.1.2  Secwepemc History: Two Levels of Colonisation As in the histories of many Aboriginal societies, the relationships between Secwepemc  peoples, their land and their traditional resources were altered dramatically by the arrival of European settlers. Despite the changes that accompanied first contact and the subsequent fur 10  trade that was established, the initial decades were recorded as both peaceful and mutually beneficial (Laurier Memorial, 1910; Coffey et al. 1990). The influx of non-aboriginal miners that coincided with the gold rush in the 1850's, however, swiftly led to conflicts, largely related to access and ownership disputes over gold, water, plant and animal resources, and land (Coffey et al. 1990; Ignace 1998). As significant and often violent as the battles were, however, they paled in comparison to the assault on Aboriginal populations at the microbial level. This tragic aspect of history is one shared by all Indigenous peoples of the Americas (or the so-called New World) as explained by Diamond (1999:211-212): "The main killers were Old World germs to which Indians had never been exposed, and against which they therefore had neither immune nor genetic resistance. Smallpox, measles, influenza, and typhus competed for top rank amongst the killers. As if these had not been enough, diphtheria, malaria, mumps, pertussis, plague, tuberculosis, and yellow fever came up close behind" . 11  As the newcomers increased in numbers, the Secwepemc populations declined drastically through a series of "virgin soil epidemics" (Crosby 1986:196), beginning with whooping cough 12  infections in 1827, and a deadly epidemic (probably) of diphtheria during the winter of 1842-43 (Coffey et al. 1990). For the Secwepemc, however, the worst of the "Eurasian germs [that] played a key role in decimating native peoples" (Diamond 1999:213) was the smallpox epidemic of 1862 that swept through the entire province of B. C. and left no part of the Secwepemc Nation untouched (Coffey et al. 1990). Of the 30 original Bands, 17 survived, only to be assaulted further by subsequent measles, influenza, whooping cough and tuberculosis infections. In just over 50 years (1850-1903), it was estimated that almost 70 percent of the Secwepemc population  The first European to make contact with the Secwepemc (in the northwestern part of the territory, with the Soda Creek Band, near Williams Lake, B. C.) was Alexander Mackenzie of the North West Company (which later became the Hudson's Bay Company) in 1793 (Coffey et al. 1990; Ignace 1998). Whether or not one accepts the New World/Old World disease theory is largely irrelevant to the fact that large numbers of Aboriginal peoples did not have immunity to the species (or strains) of pathogens that accompanied Europeans to North America. defined by Crosby (1986:196) as the "rapid spread of pathogens among people whom they have never infected before". 10  11  12  14 fell victim to disease (Coffey et al. 1990), devastating Secwepemc culture and society: "With the loss of so many elders, the Shuswap lost many centuries of accumulated oral history, skills, and knowledge. With the loss of so many great leaders, the Shuswap were less able to defend their lands and their culture from permanent change at the hands of the missionaries, settlers, and government officials. With the death of their young, the very survival of the Shuswap people was threatened" (Coffey et al. 1990:37). In her historical account of Aboriginal health in B. C. from 1900-1950, Kelm (1998:3) described the first half of the twentieth century as "the cusp of a major epidemiological transition through which the First Nations had not yet passed". She also linked the impact of colonisation through microbial disease to broader effects on culture, noting that Aboriginal Elders saw the historic epidemics of disease "as harbingers of the devastation that was to come" (Kelm (1998:xv). In fact, Kelm (1998:xv) pointed out that the physical bodies of Aboriginal peoples "have been central players in the drama of colonization in British Columbia" (Kelm (1998:xv), a point that is elaborated below. Historically, it is impossible to separate disease (i.e., the microbial colonisation of individual bodies) from that of the broader imperial colonisation of Aboriginal cultures as a whole. Although it may not have been intended, the devastating introduction of microbial disease significantly assisted with a colonial agenda in at least two ways. First, it weakened Aboriginal populations (in terms of both numbers and infrastructure), thereby eroding a physical ability to withstand colonial settlement and expansion. Second, the introduction of disease then warranted the provision of cures, further justifying colonisation in what Kelm (1998:101) referred to as "perfectly circular logic", indicated by the following: "Within the imperial worldview of the late nineteenth century, bringing medicine to the Natives helped lay the moral basis for colonization. ... Thus, 'humanitarianism' became integral to the colonial project, not in some cynical self-aggrandizing way but in a sincere fashion that saw 'doing good' as inextricably linked with racial superiority and the right to rule. Medicine provided the practice and the symbols for that 'humanitarian' domination" (Kelm 1998:101). At the same time, medicine was seen by missionaries as an effective vehicle for spreading the Gospel, since "healed bodies frequently led to impressionable minds" (Kelm 1998:104). Here, however, Kelm (1998) noted that the goal was not only spiritual conversion. Missionaries and government alike were optimistic that "evidence of Euro-Canadian medical  15 superiority would work to disrupt the relationship between Aboriginal people and their leaders, especially traditional healers" (Kelm 1998:104). In a symbiotic way, then, Euro-Christian belief systems upheld the Euro-scientific explanation for death and disease over the 'superstition' associated with malignant spirits, while Euro-scientific medicine exposed the 'quackery' involved in traditional healing practices, thereby undermining the power and prestige of traditional and spiritual healers who were opponents of assimilation (Whitehead 1988; Kelm 1998). The promise of medical assistance (or the threat of withholding it) apparently also was used to manipulate the 'converted' into abandoning their traditional ways of healing and gratefully embracing assimilation (Whitehead 1988; Kelm 1998). Furthermore, as the culpability for these new deadly diseases was recognised by Aboriginal leaders, the Aboriginal demand for 'white' cures to treat 'white' diseases increased (Kelm 1998). Thus, the "perfectly  circular logic" (Kelm 1998:101) apparently worked perfectly.  2.1.3  Contemporary Studies of Traditional Medicines: Unveiling Past Ironies Although missionaries and government officials frowned on the practice of Aboriginal medicine, they encouraged individuals to dress up in regalia to have their pictures taken. -Mary-Ellen Kelm (1998: 95)  When viewed within the broader context of the history outlined above, there is an irony apparent in conducting a contemporary study of traditional Aboriginal plant medicines. Traditional medicines and healing regimes of Indigenous societies—at one time apparently despised and aggressively subverted by many—are now highly esteemed under the rubrics of 'holistic health' and 'environmental consciousness'. The irony is exacerbated in that Aboriginal plant medicine, once deemed "unscientific" (Kelm 1998:106) and viewed as "at best fraudulent and at worst satanic" (Kelm 1998:126) is now valued within the scientific search for new drugs and other plant products of benefit to humanity (King and Tempestra 1994; Cox 1994; King et al. 1996; Towers et al. 1997; Carlson et al. submitted). While the current respect afforded to the cultural knowledge of Indigenous peoples is generally viewed in a positive light, it is important  16  to note that various international statements and declarations by Indigenous peoples (discussed in Chapter 4) clearly identify the concern that persisting colonial attitudes may lead history to repeat, i.e., what was taken away before will be taken away again (albeit inversely). In other words, cultural knowledge—at one time denigrated by some to "quackery and superstition" (Kelm 1998:105)—has now become a key target for  appropriation.  Furthermore, from a co-evolutionary perspective, a contradiction emerges in the examination of traditional Aboriginal plant medicines for potential disease remedies. This discrepancy relates to a potentially flawed rationale for screening programs that use traditional plant knowledge as a basis for plant selection to seek cures for recently-introduced diseases. If such screening programs are based on the notion that Indigenous plant knowledge can pinpoint plants with a higher than random probability of harbouring new drugs, then this presumes that Aboriginal peoples have developed an efficacious pharmacopoeia for disease treatment over centuries or millennia. For some diseases, this rationale is reasonable; the origin of human medicine is largely accepted as the result of co-evolutionary relationships between humans and plants (Johns 1990). This is based on the reasoning that cultural knowledge will only be retained over many generations if it is useful. However, we must also consider how long it takes for such relationships to co-evolve. Old World diseases were brought to the northwestern New World only in the last two •  13  centuries, which is a very short period in evolutionary terms . More significantly, however, these Old World diseases both devastated Aboriginal populations (including many elderly individuals who would presumably have been the primary custodians and teachers of cultural healing practices) and accompanied the myriad of ensuing cultural changes brought by colonisation that fragmented Aboriginal lifeways and belief systems. Amid such a complex tapestry of change in the cultural fabric, it is impossible to deduce much about the efficacy of traditional medicines in ameliorating the new diseases. If 70 percent of the Secwepemc population succumbed to the first smallpox epidemic, for example, was this because Aboriginal medicines were not effective? Or did 30 percent of the Secwepemc survive because they were? Such questions (while oversimplified) highlight the potential divergence in perspectives and the uncertainty in making claims about the past whilst situated in the present. Furthermore, Here again, I suggest that this argument holds whether the newly introduced diseases consist of new species of pathogens or new virulent strains of existing species. 13  17 traditional ways essential to the transmission of cultural knowledge (including belief systems, economies, and language) were dramatically altered by assimilative colonial legacies, such as the residential school system. Therefore, although many custodians of cultural knowledge may have endured the epidemiological transition, many may have not have withstood the cultural transition associated with assimilation. Based on a combination of historical fact and historical uncertainties about the state and transmission of cultural plant knowledge, it could be argued that a contemporary search for Old World disease cures in Aboriginal cultures of the New World may be 'looking for cures in all the wrong places'. A similar assessment for endemic diseases does provide more theoretical support for a co-evolutionary basis to the development of efficacious human medicines based on plants. Again, it is not possible to deduce the full impacts of colonisation on the transmission of cultural knowledge related to healing. However, the knowledge of plants used in the treatment of common ailments (i.e., relatively non-specialised knowledge) presumably would be relatively wide-spread in Aboriginal societies and thus would be more likely to be passed on as part of cultural healing regimes. Available ethnographies, plant usages, and disease symptoms treated lead me to suggest that many of the plants examined for antimicrobial properties in my dissertation research belong in the latter category of widely known, general medicines with histories of use that largely survived colonial change.  2.1.4  Contemporary Studies of Traditional Medicines: Moving Forward  Culture is seen as a complex of learned traditions and customs that govern beliefs and behaviour (Kottak 1991) that is adaptive (or maladaptive) over time. It is useful to consider Secwepemc cultural knowledge of plants not merely as an archive of preserved data, but as a living, dynamic, and integrated compilation of both the knowledge that was transmitted from previous centuries of plant-human interrelationships, and that which Secwepemc peoples are discovering and practicing today in their investigation of plants. In his 1997 public address to the graduates of the Secwepemc Cultural Education Society/Simon Fraser University  18 (SCES/SFU) program, Chief Ron Ignace (Skeetchestn) underscores this point: I remember some of our Elders, people like Mary Thomas and the late Nellie Taylor, telling us about their years of research with their own Elders and teaching this information to the younger generation, and also sharing it with ethnobotanists, ethnographers, linguists, and others, some of whom in turn have become our allies in getting this knowledge out to younger people, even among our own Elders. Our Elders to this day thirst for knowledge, and I learned from [our] own work with them in social research that they do not fear to investigate and experiment, to think and reflect on the past, on natural phenomena and causes, in searching for medicinal cures, for social and economic cures. We, too, must adopt their thirst for knowledge and their courage to investigate and experiment to find cures for our social, medical, and cultural ills (reprinted in Murphy et al. 1999:Preface). I suggest that the potential value offered by contemporary research on Secwepemc (or other Aboriginal) plant medicines hinges on "an understanding of the past as well as the present" (Mary Thomas, pers. comm. 1997). Essential too, however, are acknowledgement of and respect for the wishes, goals, and concerns of Secwepemc peoples of the present, who are not just looking back in time but forward as well, to what the future holds for their culture. With mutual respect as the fulcrum, there is an opportunity to move forward together.  2.1.5  Translation, Interpretation and Other Biases Involved in this Study  Several aspects of this study of the antimicrobial properties of Secwepemc medicinal plants required interpretation and thus have introduced researcher and/or cultural bias. As outlined below, these biases are related to understanding the healing role of plants in Secwepemc traditional medicine, and consequences of 'transforming' or 'translating' cultural knowledge into scientific perceptions and practices. For example, while phytochemicals can account for many medicinal or other effects of plants on humans, medicines can certainly be more than just biologically active chemicals. Indeed, it may be impossible to separate out the healing  19 components of traditional medicine. This important point is eloquently described by Salmon (1996:72): The drama that surrounds healing plants is symbolic of our worldviews as indigenous people. Each plant, the healer's visit with the patient, traditional concepts of disease, and the treatment are all microcosms of our culture and how we understand life. An indigenous plant/healing paradigm will locate a plant at the center of a circle: Cycling around the plant are four aspects of the plant/human relationship. Central to these aspects are mental, physical, social and spiritual relationships to the plant. These aspects are anchored by history, identity, language, land base, and the beliefs of the culture. The healer must tie all these aspects and anchors to each other and to the plant and patient in order for healing to be successful. Even when the physical aspect of healing is considered alone, only some physiological effects will directly influence the cause of disease. Others may have more subtle, or indirect therapeutic effects mediated by the body's immunological defenses (immunomodulatory effects) or by psychoneuro-immunological effects, reflecting the mind-body interaction of interrelated physiology. While such investigations are beyond the scope of this study, an awareness of the multifactorial nature or the medicinal ecology of healing is essential in any medicinal plant analysis and subsequent interpretation. There are also problems inherent in 'translating' medicinal plant information from the cultural context of its significance and use to a method for assessing chemical properties and biological activities. Such translations occur at many levels: in information sharing, in assigning plants to categories, in collecting and preparing plants for assays or analyses, and in both the design and interpretation of the assays themselves. Potentially, assay results may have little or no meaning to the traditional use of a plant, or to the cultural group and the peoples' relationship with plants. However, it would be unrealistic (and thus inappropriate) to ignore the numerous logistical and technical challenges in using the cultural context of plant use as the sole basis for the biological context of assessing medicinal plants. While there is much to be learned through analyses of traditional plant medicines, we should openly acknowledge that much is likely to be 'lost in the translation'. As an example, plants are routinely grouped according to medicinal use before deciding which plants to assess for which properties and to facilitate further investigation. Problems involved in translating Indigenous disease concepts into Euro-scientific disease concepts, or vice  20 versa, have been discussed elsewhere (Elisabetsky 1994; Salmon 1996) and are at least partly related to differences in concepts of disease. This may lead to difficulties or inaccuracies in placing some traditional medicines into Euro-scientific disease categories. Categorisation of plants by medicinal use, based on symptoms treated, allows the indirect implication of a causative agent in many of the diseases, and aids the subsequent choice of assay for detection of biological activity. However, as traditional medicines are also likely to meet expectations related to the cultural perception of disease, it may be unimportant whether the plant cures the disease or simply removes the symptoms. Thus, from ethnobotanical information, it is often difficult to deduce whether a medicine is specifically used to eliminate the causative agent or to provide relief of symptoms. This somewhat indirect (and often ambiguous) relationship between medicinal use and aetiological agent must be kept in mind in the experimental design of bioassays in vitro and in vivo, as well as in interpretation of the resulting data—especially if claims relating to efficacy in the context of 'traditional use' are made. Moerman (1998:12) suggested that a medicine that "works" meets expectations of an anticipated effect, but expectations can be based on different concepts of health and healing. As he put it, ".. .definitions of health and well-being are often cultural matters; they are rarely simple matters of fact" (Moerman 1998:12). With an awareness of the above, I have examined the ethnographic information on Secwepemc medicinal indications (Turner and Ignace in preparation) and grouped the Secwepemc medicinal plants investigated in this study into the 13 general categories delineated by Turner et al. (1990) (see Table 2.1), to provide a general overview of the prevalence of diseases and disease remedies recognised in the Secwepemc culture, and to aid in the selection of those plants with potential antimicrobial properties that are of specific interest in this study (listed in Table 2.2). It should be noted that, due to multiple uses for many Secwepemc medicinal plants, some plants in Table 2.1 are assigned to more than one category. Also, the category referred to as 'miscellaneous or unspecified medicines' includes some plants and their uses that, as discussed previously, can not be translated from a traditional cultural context into the conceptual framework imposed by Euro-centric views of disease. While there is a spiritual aspect to all plant medicines that is beyond the ability of laboratory science to acknowledge adequately, some of the plants placed in the latter category are considered particularly sacred by  21 the Secwepemc. An assessment outside of their cultural context seemed inappropriate for these so they were not examined in this study. Several other aspects of the 'translation' process discussed above also require mention. For example, the method of preparation of plant extracts for chemical analysis brings into question how closely the extract prepared and tested in the lab resembles what is used by Indigenous peoples. Solvent choice, extraction method, heat and light exposure, storage conditions and other parameters can influence the solubility or stability of phytochemicals and therefore the phytochemical composition and biological activity of plant extracts (to be discussed further in Chapter 3). I chose methanol as the standard extraction solvent because it is a reasonable compromise between high polarity (although not as high as water, which was the most frequent solvent used in traditional extractions) and logistical considerations, such as ease of evaporation for crude extract preparation and maintenance of crude extract sterility (water extracts are extremely susceptible to fungal contamination, even under refrigeration). Most traditional medicines are administered as mixtures of many compounds (from a single plant or from multiple species). Interactions between 'drugs' are possible, and it is likely that some mixtures affect the bioavailability of pharmacologically active principles (Farnsworth 1994a; Holmstedt and Bruhn 1995). Medicinal plant mixtures have not routinely been investigated due to difficulties and drawbacks inherent to analysis of complex mixtures rather than those that are chemically defined. However, there is increasing interest in the analysis of such mixtures (or 'formulations') on the part of both industry and government, and federal quality control standards are currently being developed (Volpe 1998). Given that biological activity in a crude extract may be the result of a single chemical compound, or the result of multiple compounds interacting in an additive, synergistic or even antagonistic fashion, these elaborate chemical mixtures merit further investigation to "analyse what the indigenous peoples are really taking" (Prance 1994:2). However, an initial understanding of the properties and chemical constituents of single plants (or plant parts) is prerequisite to more complex analyses. 2.1.6  Assessing Antimicrobial Activity Testing the effects of plant extracts on microorganisms using assays in vitro is an initial  step in establishing the chemical basis of antimicrobial activity. Most antimicrobial assays in  22 vitro are designed to assess a direct action of plant chemicals on the microbe that lead either to  direct killing of the microbe or to an interference with its ability to replicate. Although this is a valuable and necessary preliminary step, in the context of an infection in vivo, the situation is far more complex. An infecting microbe must surmount several obstacles to colonize and establish an infection in its host. These include gaining entry, multiplication, and evading host immune responses (Mims et al. 1995). In fact, the majority of infections do not lead to disease and are quickly resolved by the normal functions of host barriers, including the immune system (Brody et al. 1994). In more severe infections, chemotherapy (e.g., antibiotics in the case of a bacterial  infection) may help to limit the number of microbes in the body, but even so, the body's defenses must resolve the majority of the infection (Brody et al. 1994; Janeway and Travers 1996). Thus, extrapolation of results of bioassays in vitro is limited, except perhaps in cases of certain topically applied medicines or treatments where the active plant chemicals can more readily come into direct contact with the target microorganisms. Examination of the 'antimicrobial profile' of a plant may still provide clues as to the microbial target(s) of active phytochemicals, and thus serve as a basis for further research.  2.2  RESEARCH OBJECTIVES The research described in this chapter was designed to provide an initial assessment of  the antimicrobial properties of Secwepemc plant medicines for two main reasons, namely, for educational and other uses by the Secwepemc Nation, and for subsequent selection of one or more plants for in-depth chemical analysis and antimicrobial characterisation (Chapter 3). The specific objectives of the research were: 1) to identify Secwepemc medicinal plants that are likely to have antimicrobial properties, based on disease symptoms treated and traditional uses; 2) to collect, extract and assay these plants for antibacterial, antifungal and antiviral activities in vitro; and  23 3) to examine the antimicrobial profiles of plant extracts for obvious trends in antimicrobial activity that might contribute to an understanding of the underlying chemical nature of antimicrobial phytochemicals.  2.3  MATERIALS AND METHODS  2.3.1  Ethnobotanical Information  The selection of plants for antimicrobial activity screening was based on the collective ethnobotanical information shared by Secwepemc elders and other knowledgeable persons with researchers who were part of the Secwepemc Ethnobotany Project. This ethnobotanical information is presently unpublished but a manuscript is in preparation for co-publication by the Secwepemc Cultural Education Society (Turner and Ignace in preparation) . From the 14  ethnobotanical information, I selected 68 plant species (listed in Table 2.2 and Appendix B, Table B.l) to screen for antimicrobial activity, based on the criteria that the documented traditional or contemporary uses (as described in English) implied antimicrobial activity. The type of information used for collection of plants included: plant species and plant part, medicinal and nutritional uses, and methods of preparation as food and medicine. When specified, traditional collecting locations, season of harvest and harvest methods were also taken into account. Ethnobotanical information related to the antimicrobial potential of each plant species is summarised in Appendix B, Table B.l. In addition, more detailed ethnobotanical information relating specifically to one plant that was chosen for further antimicrobial and phytochemical analyses is included in Chapter 3, with the permission of the Secwepemc Cultural Education Society and the primary investigators of the Secwepemc Ethnobotany Project.  Unfortunately, there has been an unanticipated delay in publication of the ethnobotanical information (documented through the Secwepemc Ethnobotany Project) that formed the basis of my plant selection. An abbreviated version of the plant uses related to my dissertation research was approved for inclusion here and is found in Appendix B, Table B . l . The information is taken from the most recent draft of Turner and Ignace (in preparation) and is correct to the best of my knowledge at the time of submission of this dissertation. 14  24 Plants were selected for antimicrobial screening based on the criteria that their recorded Secwepemc use implied the treatment of conditions caused or exacerbated by bacterial, fungal or viral microorganisms. These included plants used to treat the following: • colds, coughs, tuberculosis, influenza and other respiratory tract infections; • kidney and urinary ailments; • stomach and/or digestive tract disorders; • sexually transmitted diseases; • eye infections; and • skin infections, burns, sores, and wounds. Plants used as tonics or general medicines were also investigated.  T a b l e 2.1.  Secwepemc medicinal plants grouped according to medicinal use. N u m b e r of plant  Plant Use Category  species  Tonics and General Medicines  26  Purgatives, Laxatives, Emetics  4  b  Colds, Coughs, Tuberculosis, Influenza, Other Respiratory Ailments  34  Poultices, Salves or Washes for Wounds, Infections, Bums, Sores  40  Arthritis, Rheumatism, Muscular Aches and Pains  23  Kidney and Urinary Ailments  8  b  Sexually Transmitted Diseases  3  b  Eye Medicines  13  Stomach and/or Digestive Tract Medicines  16  Medicines for Women (especially at childbirth)  12  Medicines for "Cancer" (or disorders perceived as cancer)  2  Medicines for Circulatory System  8  Miscellaneous or Unspecified Uses  53  Approximately 110 plant species described by Turner and Ignace (in preparation) assigned to multiple categories. "Categories from which plants listed in Table 2.2 were selected. a  3  b  b  b  b  are included, with some plants  25 2.3.2  Plant Collections Plants were collected, under the permission guidelines of the Secwepemc Ethnobotany  Project, at various locations in Secwepemc territory between May 1995 and August 1999 and voucher specimens were prepared for verification and deposit at the herbarium of the University of British Columbia, Vancouver (see Table 2.2 for a complete list of plant species collected and voucher numbers assigned). Whole plants or specified plant parts were collected at the life cycle stages and seasons specified by Secwepemc ethnobotanical information (e.g., roots of Balsamorhiza sagittata were collected in mid summer, after flowering, when the medicinal  quality of the root is said to be highest). Plants were air-dried in the field or dried in a plant drier at a maximum temperature of 40 °C, and stored at room temperature. Samples of tree pitch were collected in glass vials and then stored at room temperature. Samples of latex were collected using a sterile plastic syringe and stored in glass vials at 4 °C.  Table 2.2. Species list ' and voucher specimen collection numbers of the 68 species of Secwepemc a b  plants collected for antimicrobial activity screening.  Abies lasiocarpa (Hook.) Nutt. (Pinaceae) S-35 Acer glabrum Torr. (Aceraceae) S-17 Achillea millefolium L. (Asteraceae) S-43 Allium cernuum Roth (Liliaceae) S-7 Alnus incana (L.) Moench (Betulaceae) S-22 Amelanchier alnifolia Nutt. (Rosaceae) S-50 Apocynum cannabinum L. (Apocynaceae) S-27 Aralia nudicaulis L. (Araliaceae) S-71 Arctostaphylos uva-ursi (L.) Spreng. (Ericaceae) S-33 Arnica cordifolia Hook. (Asteraceae) S-36 Artemisia dracunculus L. (Asteraceae) S-19 Artemisia frigida Willd. (Asteraceae) S-18 Artemisia tridentata Nutt. (Asteraceae) S-55  Asclepias speciosa Torr. (Asclepiadaceae) S-54 Aster conspicuus Lindl. (Asteraceae) S-10 Balsamorhiza sagittata (Pursh) Nutt. (Asteraceae) S-24 Bryoria fremontii Tuck. (Usneaceae, Lichen) S-14 Calvatia gigantea (Batsch: Pers.) Lloyd. (Lycoperdaceae, Fungi) S-15 Ceanothus velutinus Dougl. (Rhamnaceae) S-68 Cirsium undulatum (Nutt.) Spreng. (Asteraceae ) S-73 Clematis columbiana (Nutt.) T. & G. (Ranunculaceae) S-40 Cornus stolonifera Michx. (Cornaceae) S-45 Epilobium angustifolium L. (Onagraceae) S-67 Equisetum hyemale L. (Equisetaceae) S-3 Erythronium grandiflorum Pursh (Liliaceae) S-29 Fragaria vesca L. (Rosaceae) S-60 Fragaria virginiana Duchesne (Rosaceae) S - l l Gaillardia aristata Pursh (Asteraceae) S-51 Geum macrophyllum Willd. (Rosaceae) S-66 Heracleum lanatum Michx. (Apiaceae) S-3 8 Heuchera cylindrica Dougl. (Saxifragaceae) S-46 Inonotus obliquus (Ach.ex Pers.) Pil. (Hymenochaetaceae, Fungi) S-28 Juniperus communis L. (Cupressaceae) S-44 Juniperus scopulorum Sarg. (Cupressaceae) S-9 Ledum glandulosum Nutt. (Ericaceae) S-77 Ledum groenlandicum Oeder (Ericaceae) S-34 Lilium columbianum Hanson (Liliaceae) S-75 Lithospermum ruderale Dougl. (Boraginaceae) S-53 Lomatium macrocarpum (Nutt.) Coult. & Rose (Apiaceae) S-74 Lomatium nudicaule (Pursh) Coult. & Rose (Apiaceae) S-6 Lonicera ciliosa (Pursh) DC. (Caprifoliaceae) S-64 Lonicera involucrata (Rich.) Banks (Caprifoliaceae) S-59 Mahonia aquifolium Pursh (Berberidaceae) S-56 Matricaria matricarioides (Less.) Porter (Asteraceae) S-78 Mentha arvensis L. (Lamiaceae) S-12 MonardafistulosaL. (Lamiaceae) S-32 Picea engelmannii Parry (Pinaceae) S-16  27 Pinus contorta Dougl. var. latifolia Engelm. (Pinaceae) S-63 Pinus ponderosa Dougl. (Pinaceae) S-48 Plantago major L. (Plantaginaceae) S-62 Populus balsamifera L. (Salicaceae) S-31 Populus tremuloides Michx. (Salicaceae) S-37 Prunus virginiana L. (Rosaceae) S-49 Pseudotsuga menziesii (Mirbel) Franco var. glauca (Beissn.) Franco (Pinaceae) S-41 Pyrola asarifolia Michx. (Ericaceae) S-13 Ribes lacustre (Pers.) Poir. (Grossulariaceae) S-21 Rosa acicularis Lindl. (Rosaceae) S-8 Rosa woodsii Lindl. (Rosaceae) S-72 Rubus idaeus L. (Rosaceae) S-20 Shepherdia canadensis (L.) Nutt. (Elaeagnaceae) S-42 Spiraea betulifolia Pall. (Rosaceae) S-61 Symphoricarpos albus (L.) Blake (Caprifoliaceae) S-79 Symphoricarpos occidentalis Hook. (Caprifoliaceae) S-26 Taxus brevifolia Nutt. (Taxaceae) S-65 Thuja plicata Donn. (Cupressaceae) S-57 Valeriana sitchensis Bong. (Valerianaceae) S-70 Verbascum thapsus L. (Scrophulariaceae) S-47 Zigadenus venenosus Wats. (Liliaceae) S-l nomenclature as in Hitchcock and Cronquist (1973) for vascular plants and Maries et al. (2000) for lichens and fungi. a  2.3.3  Preparation of Crude Plant Extracts All solvents used in extractions were standard American Chemical Society (ACS) grade  and purchased from Fisher Scientific (Fair Lawn NJ) unless otherwise stated. All extractions were carried out at room temperature unless otherwise noted. Crude plant extracts for antimicrobial screening were prepared as follows. Dried plant material was ground in a Wiley grinder with a 2 mm diameter mesh. Typically, 20 g of the  28 ground plant material were extracted 3 times with 200 ml methanol over a period of 48 hours at room temperature. The resulting methanolic extracts were filtered through Whatman® No. 1 filter paper (Maidstone, England) to remove particulate matter, then rotary-evaporated to dryness in a tared rotary flask at 30-37 °C in a waterbath. The dried material was weighed in the flask and then reconstituted in a minimum volume of between 50-100 % methanol (addition of distilled water was required in several instances to facilitate solubilisation of dried crude extracts). A minimum volume of solvent was used to facilitate subsequent detection of any active plant compounds present in low proportions of the total crude extract. Concentrations of crude plant extracts (indicated in Appendix B, Table B.l) were calculated based on the weight of dried, extracted material per volume of solvent. Chloroform, petroleum ether and methanol were used as solvents for dissolving tree pitches, as required. Latex collected from plants was diluted to 10 % (v/v) in sterile distilled water and was filter sterilized using a 0.2 pm Acrodisc® syringe filter (Pall Corporation, Ann Arbor MI). All crude plant extracts were stored at 4 °C until use. 2.3.4  Microorganisms A variety of bacteria (Table 2.3) and fungi (Table 2.4) were selected for assays in vitro  to examine the antimicrobial properties of crude extracts madefromselected Secwepemc medicinal plants. The microorganisms were selectedfromavailable laboratory collections and represent a standard range of morphological and physiological characteristics and thus a range of potential targets for antimicrobial action of the phytochemicals within the extracts. All bacteria and fungi were originally wild type strains (unless otherwise noted) that were maintained in culture,fromthe laboratory collection of G. H. N. Towers (Department of Botany, UBC).  29 Table 2.3. Bacterial species tested in inhibition assays using Secwepemc medicinal plant extracts.  Species  Morphological Characteristics  Bacillus subtilis  Gram positive, bacilli  Staphylococcus aureus K147  Gram positive, cocci  Enterococcus faecalis  Gram positive, cocci  Escherichia coli DC-2  Gram negative, bacilli (enteric)  Pseudomonas aeruginosa 187  Gram negative, bacilli (non enteric)  Mycobacterium phlei  Acid fast, bacilli  Table 2.4. Fungal species tested in inhibition assays using Secwepemc medicinal plant extracts. Species  Classification  Aspergillus fumigatus  filamentous, systemic (opportunistic)  Microsporum gypseum  filamentous, superficial (dermatophyte)  Trichophyton mentagrophytes  filamentous, superficial (dermatophyte)  Candida albicans  yeast, superficial or systemic (opportunistic)  Saccharomyces cerevisiae  yeast (non pathogenic)  Herpes simplex virus type 1 (HSV1), a double-stranded DNA virus of the Herpesvirus family that causes cold sores in humans, was used in screening assays to test inhibition of viral cytopathic effects on Vero cell monolayers (African green monkey tissue-culture cell line). Both virus and cell line werefromthe laboratory collection of J. B. Hudson (Department of Pathology and Laboratory Medicine, Faculty of Medicine, UBC). The Vero cell line was originally obtained from the American Type Culture Collection (ATCC; Rockville MD). HSV1 was originally obtained from Dr. Peter Middleton (B. C. Centre for Disease Control, Provincial Laboratory, Vancouver). 2.3.5  Antibacterial and Antifungal Disk Diffusion Assays  The following assays were qualitative rather than quantitative in this application. Plant extracts were assessed at maximum concentrations (i.e., not a standard concentration) for the  30 presence or absence of antimicrobial activity. Antibacterial and antifungal activity of 87 crude plant extracts derived from the 68 Secwepemc plant species indicated in Table 2.2 were assessed using the disk diffusion assay (Lennette 1985). Assays were performed under sterile conditions in a certified biosafety hood. Sterilized 7 mm filter paper disks (Schleicher & Schuell, Keene NH) were impregnated with 10-20 ul of crude plant extract (concentrations ranging from approximately 20-1000 mg/ml) so that disks contained between 0.4-10.0 mg dry weight crude extract. The solvent was given time (at least 30 minutes) to evaporate at room temperature. Freshly grown bacterial (Mueller-Hinton agar, BBL® Becton Dickinson & Co., Cockeysville MD) or fungal (Sabouraud agar, BBL® Becton Dickinson & Co., Cockeysville MD) cultures were swabbed on appropriate media in amounts suitable for formation of a uniform 'lawn' of growth. The impregnated disks were placed on the inoculated plates, which were subsequently incubated at 37 °C (bacteria) or 28 °C (fungi) for 18-36 hours, producing a confluent lawn except where inhibition of microbial growth occurred. Assays were performed in triplicate using the bacterial organisms listed in Table 2.3 on Mueller-Hinton agar, with the antibiotic gentamicin (Sigma Chemical Co., St. Louis MO) as a positive control and methanol as a negative control, and using the fungal organisms listed in Table 2.4 on Sabouraud Agar, with the antifungal agent nystatin (Sigma Chemical Co., St. Louis MO) as a positive control and methanol as a negative control. Zones of inhibition of bacterial or fungal growth were recorded and extracts that inhibited microbial growth (i.e., as shown by the presence of a zone of inhibition larger than the diameter of the impregnated disk) were considered positive for the presence of antimicrobial activity. 2.3.6  Antiviral Assays Antiviral activity of 87 crude plant extracts derivedfromthe 68 Secwepemc medicinal  plants indicated in Table 2.2 was tested using HSV1 infection of Vero cell monolayers and a combination protocol (virus and extract added simultaneously), essentially as described by Hudson (1994) and Kim et al. (1997). Vero cells were cultivated in a 5 % C O 2 atmosphere at 37 °C in Dulbecco's modified Eagles Medium (DMEM; GIBCO-Life Sciences, Burlington, ON) containing 5 % foetal bovine serum (FBS; GIBCO-Life Sciences, Burlington, ON) and 25 ug/ml  31 gentamicin sulfate (Sigma Chemical Co., St. Louis MO). Assays were performed under sterile conditions in a certified biosafety hood. To perform the assays, duplicate series of 2-fold serial dilutions (from 400 ug/ml to 3 ug/ml) of crude plant extracts, diluted in DMEM containing 1 % FBS and sterilized using a 0.2 um filter (Nalgene® syringe filters, Rochester NY), were incubated with Vero cell monolayers (previously grown until confluent in 96-well tissue culture trays and then aspirated to remove growth medium) for 1 hour in a 5 % C O 2 in air atmosphere at 37 °C. One hundred plaqueforming units (pfu) of HSV1 (100 ul of 1000 pfu/ml) were added to each well and the trays were exposed to a combination of fluorescent and long-wave ultraviolet light (320-600 nm) for 30 minutes with gentle shaking, then incubated for 3-4 days at 37 °C in a 5 % C O 2 in air incubator to allow viral infection to occur. Control wells were also set up to monitor the course of viral infection (i.e., virus was added to cell monolayers in the absence of any extract), to rule out antiviral effects due to solvent alone (i.e., virus was added to cell monolayers in the presence of appropriate concentrations of solvent), and to assess cell mortality or other cytotoxic effects due to the crude extracts alone (i.e., crude extracts were added to cells in absence of virus). Wells were visually inspected for  extract-induced cytotoxicity and viral-induced cytopathic effects using an inverted light microscope at a magnification of 150 X. An absence of cytopathic effects in test cells (i.e., healthy Vero cell monolayers) compared with uninfected cells indicated the presence of antiviral activity at a given concentration of extract.  2.4  RESULTS Sixty-eight plant species (see Table 2.2) were selected for antimicrobial analysis based  on their availability and on their reported use as Secwepemc medicines. Plants or specified plant parts were collected,froma number of locations in Secwepemc territory, at various times of the year, coinciding with information or recommendations from Secwepemc elders and others participating in the Secwepemc Ethnobotany Project. In several cases, plants were separated into two or more parts (i.e., aerial parts, roots, fruit, pitch and/or latex), crude extracts were prepared from each part, and these were assayed separately at maximum concentrations.  32 In total, 87 crude extracts derived from 68 different plant species were assayed in vitro for the ability to inhibit the growth of six species of bacteria (Table 2.3), five species of fungi (Table 2.4) and one virus. A list of the plants tested, their relevant indications, and their antimicrobial activity profiles is tabulated in Appendix B, Table B.l. This chapter includes a general summary and discussion of the data, which contributed to the selection of one plant (balsamroot) that was chosen for more detailed analyses (Chapter 3). Seventy-seven of the 87 crude plant extracts derived from 60/68 plant species (88 % of plant species) assayed for antibacterial activity inhibited the growth of at least one bacterial species. The majority of these active extracts inhibited bacteria of more than just one morphological class. Twelve of the 87 crude extracts derived from 10/68 plant species (15 % of plant species) inhibited growth of all six species of bacteria. These extracts werefrom:Abies lasiocarpa (subalpine fir); Pseudotsuga menziesii var. glauca (interior Douglas-fir); Amelanchier alnifolia (saskatoon berry); Arctostaphylos uva-ursi (kinhickinnick); Ceanothus velutinus  (buckbrush); Juniperus communis (common juniper); Rosa woodsii (wild rose); Erythronium grandiflorum (yellow avalanche lily); Fragaria vesca (wild common strawberry); Geum macrophyllum (large-leaved avens). Sixty-four of 87 crude plant extracts derivedfrom51/68  plant species (75 % of plant species) assayed for antifungal activity inhibited the growth of at least one fungal species. Of these active extracts, the majority inhibited only the growth of one or both of the dermatophytes, M. gypseum and T. mentagrophytes. Eight of the 87 extracts derived from seven plant species (10 % of plant species) showed activity against all five species of fungi. These extracts werefrom:Cornus stolonifera (red osier dogwood); Mahonia aquifolium (tall Oregon-grape); Rosa woodsii (wild rose); Epilobium angustifolium (fireweed); Fragaria vesca (wild common strawberry); Geum macrophyllum (large-leaved avens); Heracleum lanatum (cow parsnip). Only five of the 87 crude extracts derivedfromfour plant  species (6 % of plant species) had activity against all six species of bacteria and all five species of fungi. These plant species were Geum macrophyllum; Cornus stolonifera; Rosa woodsii; and Fragaria vesca.  The antiviral activity of 87 crude extracts derivedfrom68 plant species was assessed using HSV1 (combination protocol). Twenty of the 87 crude extracts derivedfrom19/68 plant species (28 % of plant species) inhibited the ability of the virus to infect and/or replicate in Vero host cells without accompanying cytotoxic effects. The antiviral activity of 19/87 crude extracts  33  derived from 17/68 plant species (25 % of plant species) could not be assessed accurately, due to adverse effects on the host cells which interfered with interpretation of the results; these were designated as "cytotoxic" based on morphological alterations (e.g., cell rounding, cell elongation, enlarged nuclei, etc.). Interestingly, all four tree pitch samples tested were cytotoxic in these assays, i.e., Abies lasiocarpa (subalpine fir); Picea engelmannii (Engelmann spruce); Pinus contorta var. latifolia (lodgepole pine); and Pseudotsuga menziesii var. glauca (interior Douglas-  fir). In four cases, the crude extract of one part of a plant was cytotoxic while the crude extract of another part of the same plant was not. Indeed, not all parts of a given plant had identical antimicrobial profiles (see Appendix B, Table B.l). In total, 16 crude extracts derived from 15 different plant species (22 % of plant species) showed antibacterial, antifungal, and antiviral activities. These extracts were from: Pinus ponderosa (ponderosa pine); Pseudotsuga menziesii var. glauca (interior Douglas-fir); Acer glabrum (rocky mountain maple); Alnus incana (mountain alder); Populus tremuloides (trembling aspen); Ceanothus velutinus (buckbrush); Cornus stolonifera (red osier dogwood); Ledum glandulosum (swamp tea); Ribes lacustre  (swamp gooseberry); Rosa acicularis and Rosa woodsii (wild roses); Shepherdia canadensis (soapberry); Fragaria vesca and Fragaria virginiana (wild strawberries); and Pyrola asarifolia (pink wintergreen). Among these, however, only two (i.e., Rosa woodsii and Fragaria vesca) had activity against all of the species of microorganisms tested.  Table 2.5. Summary of antimicrobial activity screening results of 68 Secwepemc medicinal plant extracts.  Proportion of  % of Plant Species with  Antimicrobial Assay  Active Plants/Total Plants  Activity in vitro  Antibacterial Activity  60/68  88%  Antifungal Activity  51/68  75%  •inhibition ofHSVl  19/68  28%  • cytotoxic  17/68  25%  Antiviral Activity 3  'Cytotoxicity in Vero cells observed even in the absence of virus.  34  2.5  DISCUSSION As part of a collaborative, multidisciplinary project centered on Secwepemc ethnobotany,  and as a preliminary investigation into the role of chemical mediators in plant-people interactions, a selection of Secwepemc medicinal plants was examined for antibacterial, antifungal and antiviral activities using assays in vitro. Based on descriptions of traditional use and disease symptoms treated, plants were selected for their apparent role in the treatment of diseases caused or exacerbated by microorganisms, and were then examined for antimicrobial properties. Assays were qualitative rather than quantitative in this application. Dried crude plant extracts were solubilised in minimal amounts of solvent and assessed at the highest possible dissolved concentrations to maximise detection of antimicrobial activity. This procedure resulted in a range of crude extract concentrations assayed, and these are indicated for each crude plant extract in Appendix B, Table B.l. Six bacterial, five fungal and one viral species were selectedfromavailable laboratory collections and used as 'model' microorganisms for the assays in vitro. The rationale for selection of bacteria and fungi was to provide a range of morphological (and thus physiological) characteristics as potential target sites for antimicrobial action. This rationale is based on an accepted classification scheme for commercial antimicrobial agents, in which agents are classified into five groups according to the point in cellular biochemical pathways (i.e., cell wall, membrane, nucleic acid synthesis/metabolism, protein synthesis, and energy metabolism) at which the agent exerts its primary action (Neu 1994). The five bacterial species used here represent three general categories based on cell wall differences (referred to as Gram positive, Gram negative and acid-fast bacteria). Bacterial species in all three categories have a cytoplasmic membrane, which theoretically could provide an impermeable barrier to some compounds. For example, antimicrobial compounds which target nucleic acids, protein synthesis  35 or intermediary metabolism must first successfully cross the cytoplasmic membrane to exert their effect. However, antimicrobial compounds that affect later stages of cell wall biosynthesis may find their targets exposed externally on the cytoplasmic membrane surface, freely available as a substrate for antimicrobial action. In addition to the cytoplasmic membrane, Gram negative organisms have an outer membrane that prevents passage of many other compounds. Based on the above rationale, one might expect to observe pronounced trends in the antimicrobial activity profiles of plant extracts. For example, effects specific to Gram positive bacteria might be expected if an active compound targeted peptidoglycan cell wall synthesis but was excluded from its target site in Gram negative bacteria by virtue of the Gram negative outer membrane, whereas a broader spectrum of activity would be expected if all microorganisms shared an exposed common site of action. No clear trends were observed in the antimicrobial profiles of crude plant extracts derived from Secwepemc medicinal plants, although a number of the extracts (largely derived from gymnosperms) did exhibit identical antimicrobial profiles. While it would be an oversimplification to think that any antibiotic is active against all Gram positive or all Gram negative organisms, the general lack of specific activity observed suggests that many of the crude extracts harbour multiple phytochemicals with antimicrobial activity. Further research to examine this prediction on two plants, namely Balsamorhiza sagittata (balsamroot) (Chapter 3), and Shepherdia canadensis (soapberry) (data not included), has not only shown this to be the case, but has underscored the importance of more detailed chemical research for understanding the complex antimicrobial repertoire of a given plant. Numerous reports on the chemical elucidation of antimicrobial compounds in crude plant extracts have already indicated the presence of many antimicrobial compounds. For example, Saxena et al. (1995b) reported the isolation of two antibacterial compoundsfromAlnus rubra Bong, (red alder), while Kobaisy et al. (1997) identified five polyyne compounds from the inner bark of Oplopanax horridus (T. & G.) Miq. (devil's club) with antibacterial and antifungal activities. As most studies are undertaken for the purpose of 'discovering' new products, however, the isolation and identification of only the compound(s) deemed 'most active' are  36  usually pursued in a given extract, leading to only a sampling of the antimicrobial properties of the plant, and often biasing the chemical isolations towards the main active compounds (i.e., most abundant or most potent) or toward those active compounds that are most easily isolated. Consistent with the multitude of published studies of plants screened for antimicrobial properties based on traditional use, a high proportion of crude plant extracts derived from Secwepemc medicinal plants did cause growth inhibition of one or more species of bacteria, fungi or virus. The majority of crude plant extracts had antibacterial activity (derivedfrom88 % of plant species assayed), and/or antifungal activity (derived from 75 % of plant species assayed), while far fewer extracts (derived from 28 % of plant species assayed) showed antiviral activity in assays using HSV1.' Only three crude plant extracts assayed, derivedfromInonotus obliquus (cinder conk), Allium cernuum (wild nodding onion) and Lithospermum ruderale  (stoneseed), had no detectable antimicrobial activity. Interestingly, comparison of this study with similar published studies (McCutcheon et al. 1992; McCutcheon et al. 1994) show a marked difference in antibacterial and antifungal profiles of some of the crude plant extracts. In these previously published disk diffusion assay results (McCutcheon et al. 1992), which used five of the same bacterial species as used in this study and 22 of the same plant species (and comparable plant parts), only 5/22 plant species had identical antibacterial activity profiles as reported here. Similarly, in published assay results (McCutcheon et al. 1994) usingfiveof the same fungal species as used in this study and 24 of the same plant species (and comparable plant parts), only 7/24 plant species had identical antifungal activity profiles as reported here. While the reasons for these discrepancies are unknown, differences in collecting locations and seasons are evident (e.g., in this study plants were collected at various life cycle stages, according to information specified by Secwepemc Elders whereas, in the aforementioned published studies plants were collected in the flowering stage only). Such differences between studies highlight the significant variability that can exist in medicinal plant analyses due to multiple factors. Indeed, fluctuations of secondary metabolites within a given plant, or even  37 within plant populations, are recognised but, to date, remain largely unexplained (Harborne 1997). As an example, a comparison of the flavonoid patterns in leaves of two distinct populations of Balsamorhiza sagittata (balsamroot) showed that only a singleflavonoidwas isolated from the surface of balsamroot leaves collected in Princeton, B.C. (Bohm et al. 1989), while six different leaf surface flavonoids were reported from balsamroot collected in California (Robson and McCormick 1988). In addition to potential qualitative and quantitative differences in phytochemicals within a given plant species, the phytochemical composition of crude plant extracts may be affected by differences in plant storage, processing and extract preparation. Such phytochemical variability is important to consider in the interpretation of any plant analysis, and highlights one of the weaknesses in the standard approach to antimicrobial activity screening used here, i.e., a given plant species collected or processed under different conditions may not have a consistent antimicrobial profile. Susceptibility testing in vitro of antiviral compounds differs substantiallyfromthat of antibacterial or antifungal compounds since viruses require host cells to replicate. While many compounds have activity in vitro against a variety of viruses, few have proven effective clinically. Cytotoxicity would be expected to limit clinical effectiveness in many cases since viruses and their hosts share many targets for antiviral action. Drug distribution, timing of infection, and problems with administration also can limit the effectiveness of many agents, and the various antiviral assays cannot accurately reproduce all in vivo situations (Havlichek 1994). For research designed to screen plants for new antiviral agents, HSV (the causative agent in herpes infections) has proven to be a popular choice for assays in vitro, and some 'ethnodirected' research has proven successful in finding new drug candidates (King et al. 1994; and as outlined in Cotton 1996). Characterising phytochemicals for their mechanisms and specificity in antiviral properties relating to medicinal uses (rather than looking for antiviral drugs, per se), however, requires a strategy using a combination of structurally and biochemially distinct viruses and a combination of assay protocols. A careful selection of virus models combined with variations in  38  the timing of addition of crude plant extracts to the host cells (i.e., pre-infection, co-infection and post-infection with virus) has proven valuable in identifying stages in the viral replication cycle and potential mechanisms of action of various antiviral compounds from plants (Hudson 1990; Hudson 1994). It was not feasible to pursue such research (to elucidate stages in viral replication blocked by plant extracts) for all extracts. This strategy was successful in determining that the antiviral activities of extracts of branches, leaves and roots of Shepherdia canadensis (soapberry) are virucidal in nature, i.e., viral killing is the result of direct interaction of antiviral compounds with viral particles (data not included). However, balsamroot, the specific plant example described in Chapter 3, did not exhibit antiviral activity with the viruses assayed. As with any preliminary screening study, it is not clear what the results mean in terms of medicinal value of Secwepemc medicinal plants. The microorganisms used in the assays in vitro can be viewed as laboratory 'models' for assessing the effects of plant extracts on similar species or strains of pathogenic or opportunistic microorganisms. This type of approach is common in studies designed to find new therapeutic agents from plants. However, from an ethnobotanical perspective, the above assumption merits caution as there is no simple correlation between the results of bioassays of plant extracts for antibacterial, antifungal or antiviral activity in vitro and the efficacy of plant medicines as antibacterial, antifungal or antiviral agents in vivo (as discussed previously). If a plant extract does have activity in vitro, it still may not be effective as an antimicrobial agent in vivo. On the other hand, if a plant extract does not have activity in vitro, it cannot be assumed ineffective in vivo. As discussed already, many factors must be  considered. This point is essential to consider, especially given the various aspects of interpretation and 'translation' of Secwepemc plant knowledge involved in this study (as outlined previously), i.e., in many cases it is unclear if a plant is used for its antimicrobial properties specifically, or for some other biological activity and/or spiritual purpose that alleviates the symptoms being treated. To claim efficacy (or inefficacy) in vivo of a plant medicine based solely on assays in vitro is invalid scientifically and could have detrimental connotations for the indigenous culture involved. In some cases, it may not be possible to  39 validate scientifically plant medicines that are known to be essential parts of traditional healing regimes—not because they are ineffective, but because of limits on the design of biologicallyand culturally-relevant systems for testing. As Moerman (1998:13) suggested, at least to some degree, "the 'effectiveness' of the treatment probably comes from the context within which the 'cure' takes place". This research was not designed to prove efficacy, however, through assays in vitro, I have confirmed the presence of antimicrobial activity in a majority of the 68 crude extracts that are derived from plants with traditional uses that imply antimicrobial potential. I did not specifically test the predictive value of using cultural information as an indicator of antimicrobial activity, but the presence of antimicrobial agents in plants employed to combat microbial infections does support a chemical aspect to plant-people interrelationships, and a phytochemical awareness (albeit, not necessarily the same perspective as presented here) in human choice of plants for specific medicinal applications within Secwepemc culture. While antimicrobial screening assays in vitro are important as a preliminary step in the investigation of antimicrobial activity of plant medicines, further research to characterise the activity is essential before making broader conclusions. In the following chapter, the antimicrobial activity of one of the 68 plant species assayed is examined in more detail, to illustrate this point.  40 3  What are the people really taking?  Antimicrobial Properties of Balsamroot as Food and Medicine ...the product used as a medicine by local peoples is usually not what is tested in the laboratory. ...We tend to collect the individual plants, dry them, take them back and then see what chemicals they contain. ... how can we analyse what the people are really taking? - G . T. Prance (1994:2)  3.1  INTRODUCTION A deceptively simple yet often overlooked question in the analysis of traditional plant  medicines of Indigenous societies is that of biological and cultural relevance—how does the analysis relate to the biological and cultural contexts upon which it initially was based? In the search for new drugs from plants, seeking to maintain biological and cultural context may or may not assist in the drug discovery process, depending upon reliability of information and the type of disease treatment sought (Cragg et al. 1994; King and Tempestra 1994). However, if chemical analyses of medicinal plants are to further an understanding and appreciation of plant-human interactions, and/or to be of value to the original Indigenous holders of medicinal knowledge, a contextual assessment is essential. In the previous chapter, standard laboratory extraction and assay methods were used to assess Secwepemc plants for antimicrobial activity in vitro. While it may be appropriate (and indeed, necessary) to use standardised, time-efficient and cost-effective methodology (e.g., methanol extracts of dried specimens) in large-scale screening of plants for biologically active compounds, it is unclear how the activity of the resulting extracts in vitro compares with the activity of plant medicines prepared and used in situ. There are significant challenges in maintaining cultural and biological contexts of plant use in scientific studies. However,froman ethnobotanical perspective, retaining such contexts is essential to understand the meaning of plants within a culture. In keeping with the ethnobotanical goals of this research, this chapter expands upon results of Chapter 2, and evaluates the underlying phytochemistry of the antimicrobial properties of one plant species in the context of its processing and use as a traditional Secwepemc food and medicine.  41 3.2  BALSAMROOT In Secwepemctsin , it is called tset'selq which translates as "the head, or the chief 15  16  (Marianne Ignace, pers. comm. 1996 to Peacock, as cited in Peacock 1998:145). In botanical Latin, it is known as Balsamorhiza  sagittata  (Pursh) Nutt., referring to the balsam-like aroma of  its root and the sagittate or arrow-shape of its leaves (Hitchcock and Cronquist 1973; Parish et al. 1996). Some know it as "sunflower", or "spring sunflower" (Turner and Ignace in  preparation),  although it is not a true sunflower in the botanical sense (i.e., Helianthus spp.) but it is assigned to the same tribe (Heliantheae) in the aster plant family (Asteraceae). More commonly though, the plant that is the focus of this chapter is simply called balsamroot (Figure 3.1). Balsamroot is widespread in the hot, arid climate of the Interior Plateau region of British Columbia. It is often abundant on dry, south-facing grassy hillsides or in open forests at mid to low elevations, and it also occurs on warm, dry slopes at subalpine elevations. Its yellow, singly stalked flower heads (composed of both disk and ray flowers) bloom as early as April at lower elevations, adorning many hillsides in the southern interior of B. C. with an intensely golden glow that serves as a harbinger of Spring. The numerous, large leaves and stems are particularly hairy—covered with a thick network of whitish trichomes that give a silvery tinge to the aerial parts. The deep-growing taproot of this herbaceous perennial with its thick, woody bark-like outer layer seems well-adapted to growth on dry soils (Hitchcock and Cronquist 1973; Parish et al.  1996; Turner and Ignace in  preparation).  Balsamroot is known as an important food and medicine to the Secwepemc, as well as other Interior Salish Aboriginal peoples. In fact, it has been referred to as "one of the most versatile food plants used by the peoples of the southern interior" due to its edible roots, root crowns, young shoots, and seeds (Turner 1997:93; Kuhnlein and Turner 1991), and the late Secwepemc Elder Mary Palmantier (Dog Creek Band) has called it "the plant to end all plants" (Turner and Ignace in  preparation).  Secwepemctsin is the Secwepemc language. Note that Turner and Ignace (in preparation) list this plant as "tsets'elq", as similar to "ts'alt" which means "bitter" and is thought to refer to the root. l5  16  42  Figure 3.1. Balsamroot, tset'selq, or Balsamorhiza sagittata.  A. Flower. B. Young shoots. C. Flowering stalks. D. Root. E. Arrow-shaped leaf.  43 3.2.1  Balsamroot as Food In the past, the root was an important food staple for Secwepemc and other Interior Salish  peoples. Atfirstglance, however, the root appears to be a paradoxical choice for a staple food source. The established taproots require a significant effort to dig, are both difficult and timeconsuming to peel, and are mainly composed of the carbohydrate inulin and related oligofructans (Kuhnlein and Turner 1991; Mullin et al. 1998). It is this latter feature that is most perplexing, for the following reasons. Inulins (Figure 3.2) are long-chain, linear polymers of P(2-l)-linked D-fructose units with an ct(l-2)-linked D-glucose as the terminal sugar, while oligofructans are lower molecular weight (i.e., shorter chain) versions of the same (Wang and Gibson 1993; Gibson et al. 1995). These glycosidic linkages cannot be cleaved by the hydrolytic enzymes normally found in the small intestine (Roberfroid 1993; Wang and Gibson 1993; Gibson et al. 1995; Avigad and Dey 1997), and the raw roots are therefore indigestible by humans.  HO-CH  2  Figure 3.2. The structure of inulin (Avigad and Dey 1997:170).  An examination of the traditional processing methods of balsamroot provides a biochemical explanation for the apparent contradiction posed by inulin-containing roots serving as a food source. Prior to consumption, the roots are subjected to a long, slow cooking process (  in an earth oven, a technology known as 'pitcooking' that dates back millennia (Pokotylo and Froese 1983; Peacock 1988). Recently, Peacock (1998) and Mullin et al. (1998) demonstrated  44 that pitcooking of inulin-containing roots in earth ovens increases their digestibility for humans. Pitcooking can result in the hydrolytic cleavage of much of the inulin into simple sugars (i.e., fructose and glucose), thus making the root both more palatable and readily available for digestion and energy metabolism. Peacock's (1998) re-constructions of traditional Interior Salish roasting pits also indicated that careful attention to certain details of the traditional 'recipes' is required for successful chemical conversion. The essential conditions that need to be met include: • adequate temperature for a sustained period of time, provided by fire-heated rocks; • adequate moisture, provided by steam from water added at a specified time point in the cooking process; and • adequate acidity, provided by volatile organic acids emitted by moistened plant stuffs added to the pit, such as boughs of Pseudotsuga menziesii var. glauca (interior Douglasfir) and leafy branches of Rubus parviflorus (thimbleberry), and Rosa spp. (wild roses) (based on Darrell Eustache, pers. comm. 1997; John Mullin, pers. comm. 1998; Peacock 1998). Subsequent carbohydrate analyses of raw and pit-cooked roots by high performance liquid chromatography (HPLC) indicated that the pitcooking process converted well over half of the inulin to fructose or oligofructans (i.e., 65 % inulin in raw roots compared with 20-30 % in cooked roots), and led to a nearly 10-fold increase in fructose and a 4-fold increase in glucose in cooked roots (Mullin et al. 1998; Peacock 1998). Based on this study, Peacock (1998) calculated that the potential energy available through consumption of digestible carbohydrate was increased by 250 % through pitcooking. Pitcooking has become a special event rather than a daily activity (Ignace 1998) and the traditional role of balsamroot as a staple food has now been superseded by foods easier and faster to harvest and process—understandably, since the pitcooking process alone typically took 24-48 hours. It must be noted also that the roasting time is in addition to the time required for earth oven construction, root harvesting (which could take 4 or 5 days for a sizable harvest, according to the late Secwepemc Elder Lilly Harry of the Dog Creek Band), and preparation of roots and other food stuffs for cooking (Turner and Ignace in preparation). However, balsamroot still remains an important Secwepemc medicine. Interestingly, as in traditional processing of balsamroot for food, the preparation of roots for medicine also requires heat.  45  3.2.2  Balsamroot as Medicine As Secwepemc medicine, both the leaves and the resinous roots of balsamroot are used to  treat a variety of skin ailments, including infections (Mary Thomas, pers. comm. 1996; Turner and Ignace in preparation). For making root medicine, Secwepemc Elder Mary Thomas (Neskonlith Band, Salmon Arm) recommends digging large-sized roots (i.e., greater than 2 cm across the top of the root crown) after flowering, in mid-summer, when the medicinal qualities of the root are believed to be highest. The roots are then boiled in water and left to stand until a layer of pitch forms at the surface. The pitch is collected and applied directly to sores or skin infections while the cooled pitchy water can be used as a soaking solution or wash for infected areas (Mary Thomas, pers. comm. 1996, 1997, 1998; Turner and Ignace in preparation). Sometimes Mary Thomas mixes the pitch with mashed Plantago major (plantain) to make a healing salve (Mary Thomas, pers. comm. 1996, 1997, 1998). The leaves of balsamroot can be dried in the oven and then crumpled into a fine powder and sprinkled on top of wounds, sores or infections that will not heal, such as "weeping" poison ivy rash (Mary Thomas, pers. comm. 1996, 1997; Turner and Ignace in preparation). These medicinal applications of balsamroot suggest that the plant might have antimicrobial properties. Consistent with this, both antibacterial and antifungal activity were previously reported in standard laboratory bioassays of methanolic extracts of dried leaves and dried, raw roots (McCutcheon et al. 1992; McCutcheon et al. 1994). Furthermore, a sulphurcontaining polyacetylenic antibacterial compound, referred to as thiophene E (Arnason et al. 1980; Page, 1997), was isolated and purified from the raw roots (Matsuura et al. 1996). The chemical structure of thiophene E is shown in Figure 3.3. The chemical name used for this compound by Matsuura et al. (1996) is 7,10-epithio-7,9-tridecadiene-3,5,l l-triyne-l,2-diol while the name assigned by Balza et al. (1989) and used by Balza and Towers (1993) is 2-(lpropynyl)-5-(5,6-dihydroxyhex-3-yn-l-ynyl)-thiophene. For simplicity, however, only the common name (i.e., thiophene E) will be used here.  46  9 11  lO/v'  8  V\7  3  4  6  13  H C  .OH  3  OH  OH  B.  A.  Figure 3.3. The structure of thiophene E. Duplicate structures indicate the 2 different numbering systems used in assigning chemical names. A (left) indicates the numbering for 7,10-epithio-7,9tridecadiene-3,5,1 l-triyne-l,2-diol (Matsuura et al. 1996). B (right) indicates the numbering for 2-(lpropynyl)-5-(5,6-dihydroxyhex-3-yn-l-ynyl)-thiophene (Balza et al. 1989).  These published data seem consistent with the traditional use of the roots as a topical treatment for skin infections, except for the fact that the antimicrobial activities and the presence of thiophene E were detected in methanol extracts ofrawroot samples, whereas Secwepemc instructions on the medicinal preparation of balsamroot involve heat treatment in the form of boiling in water. As the solubility properties and stability to heat treatment of antimicrobial compounds including thiophene E were not determined, their contribution in Secwepemc medicinal preparations and uses was not known. In addition to thiophenes and other polyacetylenes (also called polyynes), biologically active compounds that are commonly found in members of the Asteraceae include a class of terpenoids called sesquiterpene lactones (Wagner 1977; Fischer 1990). Indeed, a number of sesquiterpene lactones and their derivatives have been reported in the leaves of balsamroot (Bohlmann et al. 1985), although none was isolated based on biological activity. However, in addition to a wide range of biological effects (e.g., cytotoxic, antimicrobial, phototoxic, antineoplastic and others), some sesquiterpene lactones are known to cause allergic eczematous contact dermatitis, or type IV hypersensitivity (Wagner 1977; Mitchell 1980; Kuby 1997). Interestingly, both the antibacterial activity and the allergenicity of sequiterpene lactones are thought to be mediated by the same functional group, i.e., the a-methylenebutyrolactone moiety. As depicted in Figure 3.4, these moieties can interact with biological nucleophiles such as sulfhydryl groups on proteins. The thiol (R-S-H) can add across the double bond of the  47 methylene moiety (R-C=CH ) that is conjugated to the lactone of the y-butyrolactone ring. The 2  antimicrobial activity is thought to be mediated by reaction of this methylene group with the sulfhydryl group of cysteine residues in enzymes or other proteins, thereby resulting in loss of function of the protein (Wagner 1977). The allergic reaction caused by sesquiterpene lactones is a cell-mediated response that occurs when these compounds similarly bind to (and thus modify) host proteins on the skin. The skin proteins consequently are recognised as 'foreign' by the immune system, thereby leading to sensitisation of T-lymphocytes and release of cytokines that cause a net accumulation and activation of macrophages. The macrophages release lytic enzymes that result in damage to local tissues (Subba Rao et al. 1978; Mitchell 1980; Janeway and Travers, 1996; Kuby 1997). The presence of an ct-methylene group is a fundamental requirement of the allergenicity of these compounds since allergenicity is lost if the a-methylene is reduced (i.e., R-C=CH -> R-C-CH ) or blocked by conjugation with certain other 2  3  compounds, such as cysteine residues on proteins (Subba Rao et al. 1978). However, the presence of the a-methylene group, though necessary to complex with skin proteins, is not sufficient for allergenicity (Mitchell 1980), and it has been suggested that the remaining part of the molecule mediates recognition by the sensitised lymphocytes (Subba Rao et al. 1978).  Figure 3.4. Nucleophilic addition of a thiol group (HS-R) across the a-methylene moiety of the ybutyrolactone ring in a sesquiterpene lactone. For simplicity, only the reactive parts of the sesquiterpene lactone and the thiol-containing protein are shown.  48 3.3  RESEARCH OBJECTIVES AND RATIONALE The research on balsamroot was guided by a series of five related objectives. The first two aims were: 1) to determine whether or not thiophene E and/or other antimicrobial compounds were present in balsamroot 'the medicine' (i.e., in the pitch and/or in the cooled pitchy water prepared by boiling, according to Elder Mary Thomas' instructions); and if so, 2) to determine whether or not thiophene E (and/or other antimicrobial compounds) retained activity after heat processing for medicine. Thiophenes are generally noted for their wide-ranging biological toxicity to a number of  different organisms (e.g., Champagne et al. 1986; Hudson et al. 1986; Towers et al. 1997b; Dojillo-Mooney et al. 1999). The toxicity of thiophene E, combined with the nutritional and medicinal duality of balsamroot, raised a third question. If antimicrobial root compounds were found to be stable to heat treatment by boiling, would they also be stable to similar temperatures required for pitcooking and thus be consumed (alongside the carbohydrate and other nutrients) in traditional balsamroot-containing diets? The routine consumption of antibiotic-like and/or toxic compounds would presumably have a significant impact on the ecology of intestinal microflora and be of relevance to general health status. Therefore, the third objective was: 3) to assess the antimicrobial properties of balsamroot 'the food' (i.e., roots heatprocessed by pitcooking in earth ovens). Another aspect of this research also evolved from a concern about the presence of potentially undesirable antimicrobial compounds that might impact traditional uses of the plant—this time in the leaves of balsamroot. Thus, the fourth andfifthobjectives were: 4) to determine whether or not antimicrobial activity in leaves is due to potentially allergenic sesquiterpene lactones; and if so,  49  5) to determine if drying alters the presence or activity of these sesqiterpene lactones when leaves are processed for medicinal use.  3.4  MATERIALS AND METHODS  3.4.1  Ethnobotanical Information  Ethnographic information on balsamroot was provided by Secwepemc Elders and other community members who participated in the Secwepemc Ethnobotany Project and this information is documented in Turner and Ignace (in preparation). The collection, preparation and importance of roots and leaves of balsamroot in medicinal applications were also described to me by Elder Mary Thomas over the period of 1995-1998, in 'informal open-ended interviews' (i.e., discussions shared over tea or meals, during walks or while travelling). Conversations were  sometimes documented by written notes or photographs, but many times it was most appropriate simply to listen. Information pertaining to balsamroot subsequently was confirmed with Mary Thomas for accuracy and for permission to be cited here. 3.4.2  Plant Collections  Balsamroot root samples were collected from the upper slopes of a Secwepemc traditional root digging ground known as xk'emkendtkwa or Komkanetkwa, translated as "the place where the waters meet" (Peacock and Turner 1995; Peacock 1998:1) on Kamloops Indian Reserve #1, near Kamloops, British Columbia. Raw root samples werefirstcollected in July of 1996 with the assistance of Nancy Turner, Sandra Peacock,and Darrell Eustache (Kamloops Band), and collected again in July of 1998 with the help of Sandra Peacock. Roots (approximately 10-15 cm long by 1.5-3.0 cm wide) were harvested after the plants had flowered and then air-dried in paper bags for several days. Plants were stored at room temperature until further use.  50 Pitcooked root samples were provided by Sandra Peacock from a collaborative pitcooking reconstruction that took place at the UBC research station near Clearwater, B.C.. The following is a brief summary of the collection and preparation of the samples (further details are found in Peacock 1998). Balsamroot samples were collected for nutritional studies (Mullin et al. 1997; Peacock 1998) in July of 1996. Roots (approximately 10-12 cm long by 1-2 cm wide) were harvested after the plants had flowered and were subsequently refrigerated and then frozen prior to pitcooking. Unpeeled balsamroot samples (wrapped in gauze) were roasted in a traditional Interior Salish pitcooking reconstruction. Roasting pit temperatures were monitored hourly with the aid of temperature probes. Over the total 20 hour cooking session, a maximum temperature of 99 °C was achieved and sustained for 5 hours, after which the temperature gradually declined to approximately 60 °C. Immediately after cooking, samples were refrigerated and then frozen at -20 °C until further use. Balsamroot leaves were collected during flowering stage with the assistance of Darrell Eustache in June of 1996 from south facing slopes on Mount Lolo (Kamloops Indian Reservation #1), and post-flowering stage in July of 1998 from the upper slopes of Komkanetkwa. Leaves were air-dried in paper bags for several days and stored at room temperature until further use. A small number of fresh leaves (~20) were collected in July 1999 from plants at postflowering stage growing on a southwest-facing grassy slope along the highway between Boston Bar and Lytton. A larger sample of fresh leaves was collected by Kevin Usher from plants at post-flowering stage (September 1999) growing in a sub-alpine meadow on a south facing slope near Spruce Lake (Chilcotin mountain range, elevation -1200 m). The fresh leaves were kept on ice for several hours during transport, prior to extraction. 3.4.3  Plant Extracts  All solvents used in extractions were American Chemical Society (ACS) grade and purchased from Fisher Scientific (Fair Lawn NJ) unless otherwise stated. All extractions were carried out at room temperature unless otherwise noted. All plant extracts were stored at 4 °C until use.  51  Boiling Water Extractions of Roots  Boiling water extractions were used to isolate the pitch from raw root samples, on two separate occasions, following the instructions of Elder Mary Thomas. In the first extraction, approximately 180 g (dry weight) of raw roots were boiled in 1.0 1 of distilled water for 2 hours and the mixture was allowed to cool to room temperature. Droplets of hot pitch in the form of yellow oil beads were retrieved from the surface of the water using a glass Pasteur pipette, allowed to harden into a sticky pitch-like substance, and rotary-evaporated to remove water. This pitch (800 mg) was dissolved in methanol to a final concentration of 100 mg/ml. After boiling, roots were removed and the remaining 'pitchy water' was cooled and filtered through Whatman® No. 1 filter paper (Maidstone, England) to remove particulate matter, rotaryevaporated to dryness in a tared rotary flask at 30-37 °C in a waterbath, and re-suspended in aqueous methanol (50 %) to a final concentration of 500 mg/ml. In the second extraction, approximately 1 kg (dry weight) of raw roots was boiled in 10 1 of distilled water for 2 hours. Hot pitch (3 g) was collected from the surface of the boiling water as indicated above. A further 4 g of hardened pitch were retrieved from the cooled pitchy water after refrigeration at 4 °C. The pitch was dissolved in ethylacetate and vacuum filtered through Whatman® glass fiber filters (Clifton NJ) to remove particulate matter, then rotary-evaporated to remove solvent and weighed (total pitch weight = 7 g). Pitcooked root samples were also subjected to a boiling water extraction for pitch isolation. Approximately 90 g (dry weight) of pitcooked roots were boiled for 1 hour in 500 ml of distilled water and then allowed to cool to room temperature. Approximately 90 mg of pitch were retrieved from the surface of the water and dissolved in methanol to a final concentration of 100 mg/ml. The cooled, pitchy water was filtered, evaporated and then dissolved in aqueous methanol (50 %) to a final concentration of 500 mg/ml. The boiled, pitcooked roots were dried for subsequent methanol extraction, as described below.  Methanol Extractions of Roots  The bark was removed from dried samples of raw roots, pitcooked roots and pitcooked/boiled roots. For each of these three samples, both the bark and the peeled roots were  52 ground separately in an electric coffee grinder, and then extracted in 3 x 300 ml methanol over several hours. For each of the resulting 6 extracts, the methanol washes were combined, evaporated to dryness and then dissolved in methanol to a final concentration of 200 mg/ml. 3.4.4  Antibacterial and Antifungal Disk Diffusion Assays  All methods and materials for disk diffusion assays, including microorganisms, were the same as those described in Chapter 2. Antibacterial and antifungal activity in  vitro  were assessed  for methanolic extracts of raw and pitcooked balsamroot samples, boiling water-extracted pitch samples, methanolic extracts of dried leaf samples, and dichloromethane extracts of both dried and fresh leaf samples using the standard disk diffusion method (Lennette 1985) and the microorganisms listed in Tables 2.3 and 2.4. Sterile filter paper disks were impregnated with approximately 2 mg of root extracts and 1 mg of leaf extracts. 3.4.5  Bacterial Overlay Spot Tests  A simple and semi-quantitative method for comparing the relative potency of antibacterial activity in balsamroot extracts was developed using a bacterial agar overlay assay, which is a modification of the agar diffusion assay (Hewitt and Vincent 1989), and employs the same rationale as in the thin layer chromatography agar overlay (Saxena et al. 1995). As this method circumvents the radial diffusion requirement of the disk diffusion assay, it increases the likelihood of detecting antibacterial activity of less polar (i.e., lipophilic or non-water soluble) compounds. In general, two-fold serial dilutions of plant samples (diluted in methanol and mixed by vortexing) were spotted (2.5-5 ul per spot) onto quadrants of two 8 x 8 cm silica gel type 60 F254 alumina backed TLC plates (EM Science, Gibbstown NJ) and solvent was evaporated. The TLC plates were placed in 9 x 9 cm Falcon® petri dishes (Becton Dickinson & Co., Franklin Lakes NJ) and overlayed with 10 ml molten Muller Hinton agar (50 °C) containing 0.002 % phenol red indicator (Sigma Chemical Co., St. Louis MO) and 10 -10 cfu/ml bacteria (10 pi of a 2ml 4  5  overnight culture grown Muller Hinton broth at 37 °C with shaking in a 14 ml sterile polypropylene tube). The molten agar was poured over the TLC plates to form a layer  53 approximately 1 mm thick and allowed to solidify. One plate was exposed to U V - A light (254 nm) for 30 minutes to test for light-activated activity, and then both plates were inverted and incubated overnight at 37 °C. The plates were sprayed with a tetrazolium-containing salt, which was an aqueous solution of methylthiazolyltetrazolium chloride (MTT) 5 mg/ml (Sigma Chemical Co., St. Louis MO), to darken selectively the areas of bacterial growth and aid in visualisation of zones of growth inhibition due to enzymatic conversion of the normally colourless tetrazolium to a deep red coloured insoluble formazan (Hamburger and Cordell 1987). Zones of bacterial growth inhibition indicated the minimum amounts of extract (in pg) that were active. Crude extracts of roots, crude extracts of leaves, and purified band 2 (a sesquiterpene lactone-based compound that was purified from crude extracts of fresh leaves) were all assayed using the general procedure described above, with the following differences. Two-fold serial dilutions (in methanol) of 200 ug/ml crude methanol extracts of raw and pitcooked roots were assayed (3 pl/spot) using cultures of B. subtilis, S. aureus K147 (methicillin sensitive) and S. aureus POO 17 (methicillin resistant) at 600 u,g -> 9 pg crude extract. Two-fold serial dilutions (1:1 in methanol) of 200 ug/ml dichloromethane crude extracts of both fresh and dried balsamroot leaves were assayed (5 ul/spof) using cultures of P. aeruginosa, E.faecalis, S. aureus (methicillin sensitive) and S. aureus (methicillin resistant) at 1000 ug -> 16 ug crude extract. Two-fold serial dilutions (1:1 in methanol) of a 20 ug/ml sample of band 2 (purified from fresh balsamroot leaves) were assayed (2.5 ul/spot) using cultures of P. aeruginosa, E.faecalis, S. aureus (methicillin sensitive) and S. aureus (methicillin resistant) at 25 pg -> 0.4 pg compound.  3.4.6  Thin Layer Chromatography ( T L C ) and T L CA g a r Overlays  Antibacterial compounds in methanolic extracts of the raw, pitcooked, and boiled/pitcooked balsamroot samples were compared using the thin layer chromatography (TLC) agar overlay technique as described by Saxena, et al. (1995). Approximately 200 u,g of each extract, along with purified thiophene E standard (provided by Jon Page, Department of Botany, UBC), were spotted on 5 identical 8 x 8 cm silica T L C plates, solvent was evaporated and the plates were placed in a chromatography chamber containing a 1:1 solvent mixture of benzene and ethylacetate. The samples were developed (7 cm), removed from the chamber and residual  54  solvent was evaporated for 3 hours in a fumehood. One plate was viewed under U V light (254 nm and 366 nm), then sprayed with vanillin-sulphuric acid ( V S A ) reagent (0.5 g vanillin dissolved i n 100 m l sulphuric acid-ethanol, 40:10) for detection. T w o o f each o f the other plates were overlayed with B. subtilis or S. aureus (methicillin sensitive), and one each o f these was exposed to 30 minutes o f U V - A , then all were incubated at 37 °C overnight and visualized by spraying with M T T , as described i n the.previous section. Antibacterial compounds were evident as lighter zones o f growth inhibition against a darker purple background o f bacterial growth. Dichloromethane extracts o f fresh and dried balsamroot leaves, purified band 2 and parthenolide standard (Sigma Chemical Co., St. Louis M O ) were chromatographed on 8 x 8 cm T L C plates (as above) i n chloroform: acetone (9:1). After solvent evaporation, one plate was viewed under U V light (254 n m and 366 nm), then briefly immersed i n a solution o f ammonium molybdate (Sigma Chemical Co., St Louis M O ) in 10 % sulphuric acid (20 g ammonium molybdate dissolved in 180 m l distilled water followed by addition o f 20 m l concentrated sulphuric acid), followed by heating at 125 °C until blue spots developed (due to reduction o f the strongly oxidising ammonium molybdate). The other plates were overlayed with B. subtilis or S. aureus (methicillin sensitive), incubated and visualised with M T T as described above.  3.4.7  Minimum Inhibitory Concentration (MIC)  The minimum concentrations necessary to inhibit the growth o f S. aureus (methicillin sensitive) were assessed for dichloromethane extracts o f fresh and dried balsamroot leaves, and for purified band 2 using the microdilution broth assay method (Jones et al. 1985). Crude dichloromethane extracts were evaporated under N gas to remove dichloromethane (which kills 2  bacterial cells) and resuspended i n methanol (100 mg/ml). The resultant suspensions were diluted 1:50 in Muller Hinton broth (to reduce the total concentration o f methanol to 2 %, which is below levels toxic to bacterial cells). Quadruplicate two-fold serial dilutions o f the 4 mg/ml methanol/broth suspension (100 p i suspension + 100 p i broth) were made i n 96-well polystyrene trays (Becton Dickinson & C o . Franklin Lakes N J ) and mixed well by pipetting before transferring diluted suspension to the adjacent well (such that dilutions added to the wells ranged from 4000 ug/ml -> 32 ug/ml). To duplicate test wells, 100 ul o f bacterial culture (10 -10 5  6  cfu/ml made from a 10" dilution o f an overnight broth culture grown i n Mueller Hinton broth i n 3  55  an angled test tube with gentle shaking at 37 °C) was added. For comparison, no bacterial culture was added to the remaining duplicate series of wells (identical to the test wells) to serve as negative growth controls for monitoring turbidity. As positive growth controls, 100 ul diluted bacterial culture were added to wells containing Mueller Hinton broth only (no extract), and to wells containing an identically prepared two-fold serial dilution series of 2 % methanol. Trays were incubated at 37 °C for 24 hours and then visually examined for bacterial growth. The minimum inhibitory concentration (MIC) was recorded as the lowest concentration of extract that showed a lack of growth (indicated by a lack of turbidity in broth culture) compared with the control. Wells in which growth was not observed, or in which growth was noticeably less than that of the equivalent positive control were plated (10 pi) on Mueller Hinton agar and incubated for 24 h at 37 °C to determine the minimum bactericidal concentration (MBC). Purified band 2 was assessed using the same method as described but starting with a 20 mg/ml concentration diluted 1:50 in MHB (such that dilutions added to the wells ranged from 400 ug/ml -> 3 pg/ml). To confirm original concentrations of bacterial cultures used, plate counts of each overnight bacterial culture were made. Ten-fold serial dilution of each culture were made in Mueller Hinton broth and mixed well by vortexing. In duplicate, 100 pi aliquots of the 10" and 10" 6  7  dilutions were plated on Muller Hinton agar and incubated for 24 h at 37 °C. Colony counts were recorded and used to confirm original bacterial concentrations. 3.4.8  Antiviral Assays The combination protocol (as described in Chapter 2) was used to assess antiviral  activity, using HSV1 or Sindbis virus propagated in Vero cell monolayers, for the balsamroot extracts listed below at concentrations ranging from 200 pg/ml to 3 pg/ml: • methanolic extract of raw, dried whole roots • methanolic extract of raw, dried outer roots (bark) • methanolic extract of raw, dried inner roots • methanolic extract of pitcooked, dried whole roots • methanolic extract of pitcooked, dried outer roots (bark) • methanolic extract of pitcooked, dried inner roots • methanolic extract of dried leaves  56 Assays were conducted in the both presence and absence of a 60 minute UV-A light exposure. HSV1 (ds linear DNA, enveloped) and Vero cells were as described in Chapter 2. Sindbis virus (ssRNA, +ve strand, enveloped) was from the laboratory collection of J. B. Hudson (Department of Pathology and Laboratory Medicine, Faculty of Medicine, UBC), originally obtained from the American Type Culture Collection (ATCC; Rockville MD). 3.4.9  Bioactivity-guided Isolation of Antimicrobial Compounds from Balsamroot  All solvents were ACS grade (unless otherwise noted) and purchased from Fisher Scientific (Fair Lawn NJ). Column chromatography was carried out using silica gel type 60, 70230 mesh size (BDH Chemicals Ltd, Poole, England) purchased in bulk from the Chemistry Department Stores, University of British Columbia. Analytical TLC was carried out using 8x8 cm silica gel type 60 F254 alumina backed TLC plates (EM Science, Gibbstown NJ). Preparative TLC was carried out using silica gel type 60 F254 precoated glass plates (20 x 20 cm) of 250 pm thickness (EM Science, Gibbstown NJ). For simplicity, the following abbreviations are used to describe developing solvents for chromatography: acetone (A); benzene (B); chloroform (C); diethyl ether (DE); ethylacetate (E); hexanes (H); methanol (M); and aqueous or water (W).  Balsamroot Pitch  Balsamroot pitch (3 g) was dissolved in H:C (2:1) and the solution was applied to an open chromatography column consisting of silica (30 g) packed with H:C (2:1). Twenty x 30 ml fractions were collected with this solvent and a further 30 x 30 ml fractions were collected after increasing the solvent polarity (H:C:M 10:5:1). Afinalset of 10 x 30 ml fractions was eluted with a methanol wash. All 60 fractions were examined by TLC (H:C:M 10:5:1) and fractions of similar chemical composition were combined and re-chromatographed on TLC for subsequent agar overlays with B. subtilis. Unfortunately, separation of pitch-derived compounds by this method was inadequate for further purification, so a second separation attempt was made, as described below.  57 Balsamroot pitch (6 g) was extracted with hexanes (3x30 ml) and the hexane soluble "fraction was rotary-evaporated and weighed (3.4 g). The hexanes insoluble fraction (2.3 g) was dissolved in 10 ml ethylacetate. To the ethylacetate solution, 100 ml water were added and the solution was mixed well and left to separate overnight. One drop of concentrated hydrochloric acid was added to acidify the solution (pH 3) to assist in separation of the two layers. The ethylacetate soluble fraction (1 g) was mixed with silica (5 g) in hexanes and applied to an open chromatography column consisting of silica (150 g) packed with hexanes. Sixty fractions (200-400 ml each) were collected as the polarity of developing solvent was increased by gradual addition of ethylacetate as follows: 100 % H; H:EA (100:1; 50:1; 25:1; 25:2; 25:4; 25:8; 25:16; 1:1; 1:2); 100 % EA; followed by a methanol wash. The fractions were examined by TLC (developing solvent FLEA 1:1) and visualised with VSA reagent (plus heat), and fractions with similar chemical profiles were combined. Antibacterial properties of the fractions, as well as of thiophene E standard, were assessed using TLC agar overlays with B. subtilis in the presence and absence of 30 min UV-A light exposure. One fraction (fraction R) displayed antibacterial activity, Rf value (Rf = 0.16 in ILEA 1:1) and UV spectral characteristics similar to those of thiophene E and so was subjected to GC-MS for verification of the presence of thiophene E. UV-A light-mediated antibacterial activity was also evident in fraction I. This fraction was eluted from the silica column in H:EA (25:16) and left at room temperature for several days in a closed glass vial. Small amounts of hexane were periodically added to the vial to replace solvent lost by evaporation. Large, clear crystals began to form immediately and these continued to crystalise over the course of two weeks. The crystals were removedfromthe mother liquid, washed in acetone and dissolved in FLEA (2:1). Upon re-crystalisation, the crystals were carefully removed to a clean vial and weighed (25 mg). A small quantity of crystal was dissolved in FLEA (2:1) for analysis by TLC (B:EA 1:1; Rf = 0.2) and TLC overlays using B. subtilis (+/- UV-A light). The purified crystal compound (crystal I) was submitted for GC-MS,  'HNMR, CNMRandX -ray crystallographic analyses for structural elucidation. Figure 3.5 13  summarises the bioactivity-guided isolation of thiophene E and the purification of crystal I from balsamroot pitch. TLC analysis was used to determine where in the roots crystal I was localised. A small amount of crystal I was spotted on an analytical TLC plate, along with methanolic extracts of  58  raw and pitcooked outer roots (bark), and raw and pitcooked inner roots. The TLC plate was developed in B:EA (1:1). After solvent evaporation, the plate was sprayed with Dinitrophenylhydrazine reagent (0.1 g 2,4-dinitrophenylhydrazine [Sigma Chemical Co., St Louis MO] dissolved in 100 ml methanol, followed by addition of 1 ml of 36 % hydrochloric acid) to visualise ketones and aldehydes (Wagner and Bladt 1996).  59  balsamroot pitch (6 g) hexanes (3 x 30 ml)  T hexanes insolubles  hexanes solubles (3.4 g)  (2.3 g)  ethylacetate: water (1:10)  water solubles  ethvlacetate solubles (1 g)  silica column chromatography (1 g)  v  v  v  v  v  v  v  v | | ^ | | i r j r v  A B C D E F G H  crystalisation in H:EA (2:1) ^  crystal I GC-MS  |  NMR  |  X-ray crystallography  v  ir  I J K L M N O P Q R  |  |  v  |  v  v  S T U V W X  ^  TLC agar overlays (+/- UVA)  ^  UV spectroscopy  I  GC-MS  thiophene E  ^  structural identification of crystal I  Figure 3.5. A schematic summary of procedural steps taken for the bioactivity-guided isolation of thiophene E and the purification and identification of crystal I from boiled balsamroot pitch.  60  Balsamroot Leaves Both fresh and dried balsamroot leaves were immersed briefly (-10 seconds) in  dichloromethane to selectively extract surface-held leaf compounds (specifically, any sesquiterpene lactones stored in trichomes). Approximately 1.0 g crude dried extract was obtainedfromeither dichloromethane extractions offreshleaves (37 gfreshweight) or dichloromethane extractions of dried leaves (80 g dry weight). The extract compositions and antibacterial properties were compared by TLC (developing solvent C.A 9:1) with VSA reagent (plus heat) for detection, and TLC agar overlays with B. subtilis and S. aureus (methicillin sensitive). Parthenolide (Sigma Chemical Co., St. Louis MO) was used as a sesquiterpene lactone standard. Three antibacterial compounds, which were present in the fresh leaf extracts but absent in the dried leaf extracts (referred to as band 1 Rf = 0.62; band 2 Rf = 0.5; and band 3 Rf = 0.37, respectively), were isolated by preparative TLC using the same developing solvent as above. One of these compounds (band 2) was sufficiently pure (single spot on TLC, visualised by VSA reagent plus heating) and in high enough yield (0.03 mg) to submit for mass determination by electron impact mass spectroscopy (Department of Chemistry, UBC). Further analyses of this compound, however, required additional material. A large-scale surface extraction of fresh balsamroot leaves (518 gfreshweight) was made by Kevin Usher (Department of Botany, UBC), following the method outlined above, and resulting in 3.0 g of crude dried extract. The dried extracted material was dissolved in 50 ml methanol with sonication and then centrifuged (Sorval GLC-1 bench top centrifuge at 2656 rpm or 1234 x g) to pellet the undissolved waxy layer. The top layer of clear material was removed carefully, evaporated to dryness and weighed (1.6 g), then re-dissolved in EA:C:M (8:1:2). Additional quantities of the purified antibacterial compound of interest (band 2) were obtained by a combination of two sequential preparative TLC methods as follows. The first series of preparative TLC plates was developed in C:A (9:1) as above. However, in this crude extract an additional compound (fluorescent blue when visualised under UV366 nm) co-migrated with the band of interest. Therefore, silica containing both the fluorescent blue band (Rf = 0.5) and band 2 (Rf = 0.5) was scrapedfromthe glass, eluted in ethylacetate, concentrated under a stream of nitrogen gas, and chromatographed a second time on a new set of preparative TLC  61 plates in H:EA (1:1) as developing solvent. Purified band 2 (8.5 mg) was successfully obtained from the second set of TLC plates (Rf = 0.5) as a pale yellow oil. The purity of band 2 was assessed using 2-dimensional analytical TLC as follows: Direction 1:  C:A9:1  Rf=0.5  Direction 2:  H:E 1:1  Rf=0.5  A single spot (yellow) was observed by observation at 366 nm, a single spot (quenching) was observed at 254 nm, and a single spot (blue-green turning dark green with heat) was observed using VSA (plus heating) for visualisation. 3.4.10  High Performance Liquid Chromatography (HPLC) High Performance Liquid Chromatography (HPLC) on band 2 from dichloromethane  extracts of fresh leaves and dichloromethane extracts of dried leaves was performed by Kevin Usher (Department of Botany UBC) using a Waters™ (Mississauga) 717 plus Autosampler Photodiode Array Detector and a Waters™ Nova-pak® Ci8 column (3.9 x 150 mm) with a HPLC precolumn (Guard-Pak™ Inserts uBondapak™Ci8; Miford MA). Samples were run in water: acetonitrile using an increasing solvent gradient from 15 % -> 65 % over 40 minutes, followed by a 5 minute wash in 100 % acetonitrile as below: Time (minutes):  Flow (ml):  % Water:  % Acetonitrile:  0.00  1.00  85.0  15.0  40.00  1.00  35.0  65.0  45.00  1.00  0.0  100.00  47.00  2.00  0.0  100.00  52.00  2.00  0,0  100.00  54.00  1.00  85.0  15.0  65.00  1.00  85.0  15.0  62 3.4.11  Mass Spectrometry (MS)  Low resolution electron impact mass spectrometry of band 2 was performed by the Mass Spectrometry Facility (Department of Chemistry, UBC). The mass spectrum was recorded using Kratos MS 50 and MS 80 mass spectrometers. Chemical ionization mass spectrometry was performed by Nikolay Stoynov (Department of Chemistry, UBC) using ammonia-methane mixture as the carrier gas. The mass spectrum was recorded using a Saturn 2000 GC-MS (Varian) including autosampler 8200, gas chromatograph 3800, mass spectrometer 2000, Saturn system control and SatView processing program (as described below). 3.4.12  Gas Chromatography-Mass Spectrometry (GC-MS)  Balsamroot pitch and root extracts, thiophene E standard, band 2 and crystal I were analysed by Nikolay Stoynov (Department of Chemistry, UBC) using GC-MS. Each sample was dissolved to ~1 mg/ml in toluene-dichloromethane (8:2). The analysis was performed using a Saturn 2000 GC-MS (Varian) including autosampler 8200, gas chromatograph 3800, mass spectrometer 2000, Saturn system control and SatView processing program. The analyses were performed under the following conditions: injection volume 1 ul, injector temperature 300 °C, split ratio 10, capillary column VA-5MS, 30 m x 0.25 mm, particle size 25 um (5 % dimethylpolysiloxane, 95 % diphenylpolysiloxane, low bleed), carrier gas helium, constant pressure 25 psa, column temperature 100 °C from 0.00 to 3.00 min; 100 °C->150 °C (80 °C/min) from 3.0 to 3.62 min, 150 °C from 3.63 to 8.63 min, 150 °C^280 °C (40 °C/min) from 8.63 to 11.88 min, 280 °C from 11.88 to 46.88 min, transfer line temperature 170 °C, ion trap mass spectrometer temperature 230 °C, electron multiplier voltage 1900 V, and scanned range m/z 40650. 3.4.13  Proton Nuclear Magnetic Resonance ( H NMR) J  'rl NMR spectral analysis on the two balsamroot compounds, band 2 (8.5 mg) and crystal 1(10 mg), were run by Nikolay Stoynov (Department of Chemistry, UBC) at 200 MHz using a  63 Bruker WH 200 spectrometer. Compounds were dissolved in chloroform-di (Cambridge Isotope Laboratories Inc, Andover MA). All signals were recorded in ppm (8 scale). 3.4.14  13  13  Carbon Nuclear Magnetic Resonance ( C NMR) 13  C NMR spectral analysis on balsamroot compounds band 2 (8.5 mg) and crystal I (10  mg) were run by Nikolay Stoynov (Department of Chemistry, UBC) at 50 MHz using a Bruker WH 200 spectrometer. Samples were dissolved in chloroform-di (Cambridge Isotope Laboratories Inc, Andover MA). All signals were recorded in ppm (8 scale). 3.4.15  X-ray Crystallography X-ray crystallographic analysis and interpretation was performed by Brian Patrick  (Department of Chemistry, UBC). A clear needle crystal of crystal I (C30O3H48) having the approximate dimensions of 0.50 x 0.30 x 0.20 mm was mounted on glass fiber. All measurements were made on a Rigaku/ADSC CCD area detector with graphite monochromated Mo-Kct radiation. The data were collected at a temperature of-100 ± 1 °C to a maximum 29 value of 50.5°. Data were collected at 0.50° oscillations with 35.0 second sweeps. A sweep of data was done using (j) oscillations from 0.0 to 190.0° at % = 0° and a second sweep was performed using co oscillations between -19.0 and 23.0° at % = -90.0°. The crystal-to-detector distance was 40.41 mm. The detector swing angle was -5.55°. All calculations were performed using the teXan Crystal Structure Analysis Package, Molecular Structure Corporation, versions 1982 and 1985 (Patrick 2000).  3.5  RESULTS The results of this chapter are subdivided into the following three parts: subsection 3.5.1  summarises antimicrobial properties and the results of chemical analyses of roots related to their medicinal uses, including the structural elucidation of cycloartenone- and cycloartenol-derived phytosterols; subsection 3.5.2 summarises the results of parallel antimicrobial analyses on roots  64 prepared as food; and subsection 3.5.3 summarises antimicrobial properties of leaves, and presents the results of chemical analyses of leaf extracts, including spectral data and the structural elucidation of a novel sesquiterpene lactone-derived compound from fresh leaves. 3.5.1  Roots of Balsamroot as Medicine  There was antimicrobial activity in a methanolic extract of raw, dried roots of balsamroot. Results of disk diffusion assays in vitro (~2 mg/disk), showed antibacterial activity against two Gram positive species (B. subtilis and S. aureus), and one acid-fast species (M. phlei). Similarly, antifungal activity was detected using two dermatophytic fungi (M. gypseum  and T. mentagrophytes). No activity was found in assays using two Gram negative species of bacteria (P. aeruginosa and E. coli), nor in assays with two opportunistic fungal pathogens (C. albicans and A. fumigatus). No antiviral activity was detected in methanolic extracts of  balsamroot leaves or roots in assays using HVS1 or Sindbis virus (combination protocol, in the presence and absence of UV light), although cytotoxicity in vero cell monolayers, possibly due to acidity of the extracts, was evident at extract concentrations of 100 pg/ml and higher. No further antiviral testing of balsamroot extracts (using additional viruses or alternative protocols) was pursued. The same antibacterial and antifungal profiles as above were found for balsamroot pitch, which was extracted from roots by boiling and then dissolved in methanol. The cooled pitchywater in which the roots were boiled was active against B. subtilis, M. gypseum and T. mentagrophytes only. Antiviral activity was not tested for the pitch or cooled pitchy-water. The  above results are tabulated in Table 3.1 for direct comparison with disk diffusion assay results for other extracts. Figure 3.6 shows a triplicate series of 3 TLC plates and TLC overlays of boiled pitch and the final 24 fractions (A -> X) resultingfrombioactivity-guided partitioning of the boiled pitch (described in Section 3.4). Figure 3.6A (upper series) shows TLC plates developed in benzene: ethyl acetate (1:1) and sprayed with VSA reagent (plus heat) for detection. Figure 3.6B (middle series) shows the antibacterial activity of each of thefractions(using B. subtilis) without UV-A light exposure prior to incubation, and Figure 3.6C (lower series) shows the antibacterial activity of the fractions (using B. subtilis) with UV-A light exposure. The presence of multiple  65 antibacterial compounds is evident by the lighter zones of growth inhibition against the darker stained bacterial lawn (refer to Figure 3.6B and C). For some of these compounds (e.g., fractions I -> R), antibacterial activity appears to be either enhanced or dependent on exposure to UV light (compare Figure 3.6B with C). Results of TLC and TLC overlays indicated that thiophene E was most likely present in fractions Q and R, based on comparisons of the Rf values and the characteristic UV-enhanced activity of thiophene E (Matsuura et al. 1996). However, TLC plates visualised by a strongly oxidizing agent (ammonium molybdate reagent) indicated thatfractionR was the more pure of the two fractions and thus likely to contain a higher concentration of the putative thiophene E, so fraction R (Figure 3.7) was chosen for further analysis by GC-MS. Note that the TLC overlay without UV-A light exposure (Figure 3.7B) indicates that a second antibacterial compound (independent of UV-A light for activity) may also be present infractionR. GC-MS analysis of fraction R and thiophene E standard (Figure 3.8) confirmed the presence of thiophene E in fraction R, and thus in balsamroot pitch. Both mass spectra show mass ion peaks of the same relative intensity at mass/charge ratio (m/z) = 170, 126, and 93 (Mass Spectrum 1 compared with Mass Spectrum 2), and identical elution peaks at 9.2 minutes (Chromatogram 1 compared with Chromatogram 2). The expected molecular ion peak at m/z - 231 [M«H] or m/z = 230 [M] for thiophene E +  (C13H10SO2)  +  was not observed in either of these two spectra under the conditions that they were  run, presumably due to pyrolysis (i.e., decomposition by heating) occurring before the compound entered the mass spectrometer. In the procedure used, the sample evaporates (at 300 °C) and passes through a capillary column (containing non-polar silanized silica gel) for about 1 minute before it enters the mass spectrometer. In this procedure, pyrolysis resulting in loss of the intact molecule is not uncommon (Nikolay Stoynov, pers. comm. 1999). The prominent signal at m/z = 170 likely corresponds to loss of the terminal ethane diol (R-OH-CH2-CH2-OH) to give an ethene diol fragment (OH-CH=CH-OH). Comparison of the selected ion chromatogram at m/z =170 (Chromatogram 2) and the total ion chromatogram (Chromatogram 3) for fraction R clearly shows that thiophene E is not the major compound in this fraction. The electron impact and chemical ionization mass spectra and the corresponding GC-MS chromatograms of the major compound in fraction R (eluting at 16.97 minutes) are shown in Figure 3.9. The identity of this compound could not be determined  66  from mass spectral data and remains unknown. However, the results of chemical ionization MS (Mass Spectrum 2) indicate that the molecular mass is 354 while the fragmentation pattern observed with electron impact MS (Mass Spectrum 1) is consistent with a branched aliphatic acid (Nikolay Stoynov, pers. comm. 1999).  67  A  w pi A B C D C F G H  t  pll J K L M N O P  """""W  - •  •'••••-••••••••••••Mm-  ptQRSTuVWX  Figure 3.6. TLC overlay series showing fractions A to X derived from bioactivity-guided partitioning of boiled balsamroot pitch on silica column chromatography. A. (upper series) TLC plates visualised with VSA reagent (plus heat). B. (middle series) TLC plates overlayed with 5. subtilis and incubated without exposure to UV-A light. C. (lower series) TLC plates overlayed with B. subtilis and exposed to UV-A light for 30 min prior to incubation, in order to observe UV light-dependent or UV light-enhanced antibacterial activity. The first lane in each plate is crude pitch (pt).  68  Figure 3.7. Thin layer chromatography overlays (using S. aureus) comparing antibacterial activity (-/+ UV-A light exposure) of thiophene E standard (lane 1) and crude thiophene E from fraction R (lane 2) isolated from boiled balsamroot pitch. A. TLC plate visualised by ammonium molybdate reagent (plus heating). B. TLC overlay incubated without exposure to UV-A light. C. TLC overlay exposed to UV-A light for 30 minutes. The arrow indicates the location of thiophene E.  69  Mass Spectrum 1 100%-  9 2 2 1  J S RIC: 1757833 EBC  S c a n : 5 5 4C h a n : 1 l o n :9 4 9  BP 170 (471528=100%) thfc>e-phy23wl1 .sms  0  1  75%50%25%-  126 50  ^  69  ^  ^  ^  ,  1  ,44  J  7  0%-  Mass Spectrum 2  a 2 1 6m i n S c a n  . 554  C h a n  |l  ., .  lon 1 2 7 5  BP170(12835=100%) fr-r-phy23wl1 .sms  187  195  211  230  239  7 us RIC: 46910BC  ro  1  100%75%50%126  25%0%-  50  93 ,  74 in  5  0  i  d  ...  .1  .  1 1 U  o  1  .  1 5  1  .  4  11  4  i  1  0  5  194  208  248  9  200 m/z  MCounts  RIC all thio-e-phy23wl1 .sms  Chromatogram f  1.5-  1.0-  0.5-  0.0kCounts_  Jo. Chromatogram 2  lon: 170 all fr-r-phy23wl1.sms  Chronlatogram 3 ^  RIC all fr-r-phy23wl1 .sms kCounts 300-  200-  100-  0-  ib  2b  3b  40  sb minutes  Figure 3 . 8 . GC-MS chromatograms of thiophene E standard compared with crude thiophene E isolated from boiled balsamroot pitch (fraction R). The total ion chromatogram of thiophene E (m/z = 40 to m/z = 250) is shown in Chromatogram 1. The selected ion chromatogram at m/z =170 for fraction R is shown in Chromatogram 2 while the total ion chromatogram for fraction R (m/z = 40 to m/z = 250) is shown in Chromatogram 3. A comparison of the mass spectra at 9.2 minutes confirms the presence of thiophene E in boiled balsamroot pitch.  70  Figure 3.9. GC-MS chromatograms of the major component of fraction R at 16.97 minutes. Mass Spectrum 1 and Chromatogram 1 (total ion chromatogram) are from electron impact MS while the Mass Spectrum 2 and Chromatogram 2 (total ion chromatogram) are from chemical ionization MS. The fragmentation pattern in Mass Spectrum 1 is consistent with a branched aliphatic and the molecular mass is likely m/z = 354[M] based on Mass Spectrum 2 showing m/z = 355[M*H] . +  +  71  Figure 3.10 shows the clear crystalline compound (crystal I) that was purified from fraction I by re-crystalisation at room temperature in H:EA (2:1). Although fraction I exhibited UV-A light-enhanced antibacterial activity, TLC overlays using B. subtilis showed that crystal I (Rf = 0.2 in B:EA 5:1; purple colour when visualised with VSA plus heating) was not one of the antibacterial components (Figure 3.11). Nevertheless, the purified compound was submitted for GC-MS, ' H NMR, C NMR and X -ray crystallography. The X-ray crystallographic data alone 1 3  (Figure 3.12) was sufficient to deduce the structure of crystal I (Patrick 2000). In fact, satisfactory interpretations of the NMR spectra (Appendix C, Figures C.l and C.2) and the GC-MS data required the structural information from X-ray crystallography. For example, the GC-MS chromatogram indicated the presence of two major compounds (the flagged peaks at 27.17 and 27.95 minutes, respectively), suggesting possible isomers, with almost identical mass spectra and a likely mass ion peak of m/z = 438 (Appendix C, Figure C.3). However, it is likely that these two compounds (m/z = 438) were dehydration products resulting from pyrolysis of the original compound (Nikolay Stoynov, pers comm. 2000). The major ion peak observed (m/z = 286) is consistent with the suggested fragmentation pattern of these compounds (Appendix C, Figure C.4)  A clear needle crystal of approximate dimensions 0.50 x 0.30 x 0.20 mm was used for Xray crystallography which led to the deduction of a mixture of two related compounds (9:1 ratio). The major compound was a cycloartenone-related structure (B. Patrick, pers. comm 2000) of molecular formula C3o03H and FW = 456 (Figure 3.12). This compound was previously 48  reported in balsamroot by Bohlmann et al. (1985) as 16R,23^-dihydroxycycloartenone, and is catalogued as 19-cyclolanost-24-en-3-one, 16,23-dihydroxy-, (16 beta)- (9CI) under Chemical Abstracts Service Registry Number (CAS RN) 99297-50-4 9. My NMR and MS data are consistent with those reported for this compound in Bohlmann et al. (1985). The minor compound was the cycloartenol-related alcohol derviative of the major compound, of molecular formula C 3 0 O 3 H 5 0 and FW = 458. Its structure has not been reported previously. TLC analysis indicated that crystal I was not localised to pitch, but also found throughout the inner and outer heat processed root.  Figure 3.10. Crystal I (~10 X magnification) purified from fraction I of balsamroot pitch.  7 3  Figure 3.11. TLC and TLC overlays of Fraction I (lane 1) and Crystal 1 (lane 2) using S. aureus. A. TLC overlay without exposure to UV light. B. TLC plate with VSA (plus heating). C. TLC overlay with 30 minute exposure to UV-A light.  74  02 H48  Figure 3.12.  H49  Structure of crystal I (C oH4 0 ) based on X-ray crystallography. A tabular summary of the X-ray crystallographic data for crystal I is included in Appendix C, Table C.l. Abbreviations are as follows: oxygen (O), carbon (C), and hydrogen (H). 3  8  3  75  3.5.2  Roots of Balsamroot as Food  The antimicrobial profiles of methanolic extracts of both raw and pitcooked balsamroot samples (whole roots, inner roots, and outer roots) are summarized in Table 3.1. The antimicrobial profiles of balsamroot pitch (extracted from raw and pitcooked roots by boiling water and then dissolved in methanol) and the cooled, pitchy-water (described in section 3.3) are also shown for comparison. Extracts of all samples tested, except for the edible portion of the cooked root (i.e., inner root, pitcooked), inhibited the growth of both dermatophytic fungi (M. gypseum and T. mentagrophytes), the acid fast bacteria (M. phlei) and one or both of the Gram  positive bacteria (B. subtilis and S. aureus), based on disk diffusion assays. No activity in any of the extracts was found in assays against the two Gram negative bacteria, E. coli and P. aeruginosa, the opportunistic yeast C. albicans, nor the opportunistic fungal pathogen A. fumigatus (data not included in table).  Table 3.1. Summary of antibacterial and antifungal activity profiles of methanolic extracts of raw and  pitcooked balsamroot samples and boiled pitch from raw and pitcooked roots, based on disk diffusion assays. A "+" indicates inhibition of bacterial growth and a "-" indicates no inhibition. Note that UV light exposure was not included in these assays.  Microorganism' Sample  Bs  Sa  Mp  Mg  Tm  Whole root (raw) Inner root (raw) Outer root (raw) Pitch (raw, boiled) Pitchy water (raw, boiled) Whole root (pitcooked) Inner root (pitcooked) Outer root (pitcooked) Pitch (pitcooked, boiled) Pitchy water (pitcooked, boiled) Inner root (pitcooked, boiled) Outer root (pitcooked, boiled)  + + + + + +  + + + +  +  + + +  -  -  +  +  + +  + + + + + +  -  -  -  -  -  + + +  + + +  -  -  +  +  + nt nt nt nt  + nt nt nt nt  + nt nt nt nt  b  + +  c  Bacillus subtilis, S a = Staphylococcus aureus, M p = Mycobacterium phlei, M g = Microsporum gypseum, T m = Trichophyton mentagrophytes " A b b r e v i a t i o n s o f m i c r o o r g a n i s m s are as f o l l o w s : B s = " S a m p l e s w e r e a s s a y e d at ~2 m g p e r d i s k c  nt = not tested  76  A comparision of the antibacterial activity of raw and pitcooked roots by bacterial overlay spot assays indicated that the highest concentration of activity was in the bark.  3.2, 3.3, 3.4  and  3.5  summarise the results of these assays  (methicillin sensitive) and  Tables  in vitro using B. subtilis, S. aureus  S. aureus (methicillin resistant).  Table 3.2. Results of bacterial overlay spot assays using a methanolic extract of raw outer roots. The amount of extract (ug) that inhibited growth is indicated for each bacterial species. Lack of antibacterial activity is indicated as while "(+)" indicates a hazy (rather than a clear) zone of inhibition. Note that UV light exposure was not included in these assays.  Zone of inhibition (mm) Microorganism  600 ug 300 ug  150 ug  75 ug  38 g  19 ug  9ug  M  B. subtiltis  9  8  8  7  5  (3)  (3)  S. aureus (methicillin sensitive)  8  6  6  4  (3)  -  -  S. aureus (methicillin resistant)  7  5  5  4  (3)  -  -  Table 3.3. Results of bacterial overlay spot assays using a methanolic extract of pitcooked outer roots. Abbreviations are the same as in Table 3.2.  Zone of inhibition (mm) 600 ug  300 ug  150 ug  75 ug  38 ^  19 ug  9ug  B. subtiltis  9  7  6  5  3  (2)  (+)  S. aureus (methicillin sensitive)  6  5  5  4  +  -  S. aureus (methicillin resistant)  5  4  4  (3)  (+)  (+) (+)  Microorganism  -  Table 3.4. Results of bacterial overlay spot assays using a methanolic extract of raw inner roots. Abbreviations are the same as in Table 3.2.  Zone of inhibition (mm) Microorganism B. subtiltis  600 Mg 4  S. aureus (methicillin sensitive)  (+)  S. aureus (methicillin resistant)  (+)  300 Mg 3  -  150 Mg .  75 Mg .  -  38 Mg .  -  19 Mg .  -  .  -  9 Mg  77 Table 3.5. Results of bacterial overlay spot assays using a methanolic extract of pitcooked inner roots. Abbreviations are the same as in Table 3.2.  Zone of inhibition (mm) Microorganism B. subtiltis  600 ug 300 ug -  -  150 pg  75 pg  38 ug  19 ug  9 pg  .-  S. aureus (methicillin sensitive)  .  .  .  .  .  .  .  S. aureus (methicillin resistant)  .  .  .  .  .  .  .  Mass spectra and GC-MS chromatograms of methanolic extracts of the inner and outer portions of both raw, dried roots and pitcooked roots (Figure 3.13) confirm that thiophene E is present in all root samples except the pitcooked inner root (i.e., the part of the root that is consumed as food). The elution peak at 9.1-9.2 minutes (i.e., the 'flagged' peak in GC-MS Chromatogram 1) with mass ion peak m/z =170 characteristic of thiophene E standard (Mass Spectrum 1) is also found in GC-MS Chromatogram 2 and Mass Spectrum 2 of the raw outer roots, GC-MS Chromatogram 3 and Mass Spectrum 3 of the pitcooked outer roots, and GC-MS Chromatogram 4 and Mass Spectrum 4 of the raw inner roots. However, the elution peak at 9.19.2 minutes with mass ion peak m/z = 170 is not observed in GC-MS Chromatogram 5 and Mass Spectrum 5 of the pitcooked inner roots. Figure 3.14 confirms that thiophene E retains antibacterial activity in pitcooked roots. This figure shows a comparision of thiophene E standard (lane 1) with methanolic extracts of raw and pitcooked root samples (lanes 2-5) by TLC overlays (using S. aureus) in the presence and absence of UV-A light exposure. The overlay atright(Figure 3.14B) shows the UV-A light-activated antibacterial activity of thiophene E (indicated by an arrow) in the outer portions (i.e., bark) of both raw (lane 2) and pitcooked (lane 3) roots, as well as in the inner portion of raw  roots (lane 4). However, there is no antibacterial activity detected in the inner portion of pitcooked roots (lane 5), which is the part of the root considered edible. A second antibacterial compound (i.e., located above thiophene E on the TLC plate) also occurs in both the raw and pitcooked outer portion (i.e., bark) of the roots (Figure 3.14A and B, lanes 2 and 3). Comparison of the two overlays indicates that the antibacterial activity of this  78  compound is not UV-A light-dependent. The identity of this compound is presently unknown but its purification and identification are priorities for further research on balsamroot.  79  Mass Spectrum 1 100% 75% 50% 25% -  50  62  ' • • -  86  126 |  1 7  1  144  |  167  195  211 •  219  RAW OUTER ROOT (BAF IK)  ml  ..MI. 1,1.  ..7  9.117 min., scan #548  1  T.  187  203  218  PITCOOKED OUTER ROCDT (BARK) 170  93  79  II.l  ,, l. l, 11 I. ...l u l l  M J l ,  Mass Spectrum 4  .  ..Ml,.™  lll.li...  1  ,T  9.150 min, scan # 550  187  203  2  „  RAW INNER ROOT 170  -  S3 III , •  1  Mass Spectrum 5 43  5  68 |  76  1 ll  .1  '  •3  1 2 6  11  6 3  I  ,  3  .„  ,  1.  1  ' *  S  ,1  1....  PITCOOKED INNER ROOT  «  " '  97  ill I , , , ,lll  1  1 1 7  II . ,, II , ,. g 13;! min., sc<an # 549  "  '50  I,  ,  75  1 l,l,ll, 100  133  105  i  GC-MS Chromatogram 1  1500  |  93  ill ,,,.?, .. ,J,i. ..ill,,.. L i  25% -  -  no  170  Mass Spectrum 3  100% 75% 50% 25%  «  THIOPHENE E  9.115 min., scan # 548 79  100% " 75% 50% -  100% 75% 50% 25% 0%  1 min., scan # 554  69  Mass Spectrum 2 100% 75% 50% 25%  2 2  g  I,I . .,  I  I llllll..ll ,  _  1 U I, il  125  1  , ll 1.  T  *  ^  1 7 7  150  1  'I  7  2 0 7  '175  1  2 2 4  200  m/z  1  THIOPHENE E  1000  500  RAW OUTER ROOT (BARK)  GC-MS Chromatogram 2 1000  750 500  .  250 Counll  1250 •  1.111 L . l .  JAJVJ*._.  GC-MS Chromatogram 3  1 ..  /iA^.. — d . — „ . — ^ A A ^ .  n  Li  1000  750  u  500 C0UN  " 700 600 500 400  R  PITCOOKED OUTER ROCT(BA RK)  ill IjllAJuLj  GC-MS Chromatogram 4  RAW INNER ROOT  GC-MS Chro natogram 5  PITCOOKED INNER ROO"r  200 100 500 400 300 200 100  0-  »MLI.  'i  '10  'l5  '20  '25  '30  '35^  Figure 3.13. Mass spectra and GC-MS chromatograms of thiophene E standard compared with methanolic extracts of raw and pitcooked root samples. The total ion chromatogram of thiophene E is shown in Mass Spectrum 1 while the selected ion chromatograms at m/z =170 are shown in Mass Spectra 2-5. The GC-MS chromatograms at 9.1-9.2 minutes confirm the presence of thiophene E in all samples except the edible portion of the pitcooked root (pitcooked inner root).  80  Figure 3.14. Thin layer chromatography overlays (using S. aureus) comparing antibacterial activity of Thiophene E standard (lane 1) and methanolic extracts of raw and pitcooked roots (lanes 2-5), with and without UV-A light exposure. A. The overlay without exposure to UV light. B . The overlay with a 30 minute exposure to UV light. Antibacterial activity due to Thiophene E (indicated by the arrows) is observed in all samples except the edible portion of the pitcooked root (pitcooked inner root) in lane 5. Lane Assignments are as follows: 1. Thiophene E standard 2. raw outer root (bark) 3. pitcooked outer root (bark) 4. raw inner root 5. pitcooked inner root  81 3.5.3  Leaves of Balsamroot as Medicine  Antibacterial and antifungal properties of balsamroot leaf extracts assayed in vitro are summarised in Table 3.6.  Table 3.6. Results of disk diffusion assays of methanolic extracts of dried leaves, and dichloromethane surface-extracts of fresh and dried leaves. Extracts that inhibited microbial growth are indicated as "+" and those that did not inhibit growth are indicted as Note that UV light exposure was not included in these assays.  Microorganism  1  Sample  Bs  Sa  +  +  Dried leaf-dichloromethane  d  +  Fresh leaf-dichloromethane  e  +  13  Dried leaf-methanol  c  Ec  Pa Mg Tm +  Af  Sc  Ca  -  -  -  f  -  -  -  nt  -  +  +  +  +  nt  +  +  "Abbreviations of microorganisms are as follows: Bs = Bacillus subtilis, Sa = Staphylococcus aureus, Ec = Escherichia coli, Pa = Pseudomonas aeruginosa, Mg = Microsporum gypseum, Tm= Trichophyton mentagrophytes, Af = Aspergillus fumigatus,Sc = Saccharomyces cerevisiae, Ca = Candida albicans Samples were assayed at ~1 mg per disk dried leaves extracted in methanol dried leaves surface-extracted in dichloromethane fresh leaves surface-extracted in dichloromethane not tested b c  d e  f  The chemical profile and antibacterial activity of dichloromethane surface extractions of fresh (lane 2) and dried (lane 3) balsamroot leaves were compared by TLC and TLC overlays using S.  aureus  (Figure 3.15). The bands outlined in Figure 3.15A indicate all compounds that  exhibited quenching (i.e., appeared dark against a light green fluorescent background) when visualised under UV light (254 nm). Figure 3.15A also shows the colour reactions of the same bands when visualised by VSA reagent (plus heating). Note that the fresh and dried leaf-surface extracts are similar in composition except for three bands (bands 1, 2 and 3, indicated by arrows) that are present in fresh leaves (lane 2) but absent in dried leaves (lane 3). The antibacterial activity of the two more lipophilic of these bands (band 1 and band 2) is obvious in the overlay shown in Figure 3.15B. Band 2 (lane 2) co-migrates with purified parthenolide (lane 1), which  82 is a sesquiterpene lactone with known antibacterial activity, included here as a standard for comparison. The initial VSA colour reaction of band 2 was blue-green, turning to dark green with heating. The initial VSA colour reaction of parthenolide was blue with a pink halo, turning to a uniform deeper pink/purple with heating.  <— Band 1 <— Band 2 <—Band 3  1  2  3  1  2  3  Figure 3.15. TLC plate and TLC overlay (using S. aureus) comparing dichoromethane surface-extracts of fresh and dried leaves. A. TLC plate visualised by VSA (plus heating) B. TLC overlay. The areas circled indicate bands that are visible at 254 nm. The arrows indicate the locations of Bands 1. 2 and 3. Lane assignments are as follows: 1. Parthenolide (sesquiterpene lactone standard) 2. Dichloromethane surface-extract of fresh leaves 3. Dichloromethane surface-extract ofdned leaves  84 After purification by preparative TLC, the purity of band 2 was confirmed by the presence of a single spot on 2-dimensional TLC (chloroform:acetone 9:1; Rf = 0.5 then hexanes:ethylacetate 1:1; Rf = 0.5) using VSA (plus heating) for visualisation. Dichloromethane surface extracts of fresh and dried leaves were assessed for the presence of band 2 using HPLC (with UV detector) and a sample of semi-purified band 2. The elution peak corresponding to band 2 was observed at 32.087 minutes. The UV spectrum of band 2 is shown in Appendix C, Figure C.5. A peak corresponding to band 2 (with an identical UV spectrum) was observed at 32.135 minutes in thefreshleaf extract but not in the dried leaf extract (Figure 3.16). A summary of the relative antibacterial activities of dichloromethane surface extracts of fresh and dried leaves and purified band 2, assessed using bacterial overlay spots tests, is shown in Tables 3.7, 3.8 and 3.9. Based on these solid media assays, antibacterial activity was observed in these samples for three species of Gram positive bacteria at concentrations between 2 ug and 25 pg, and these results (using methicillin sensitive S. aureus, methicillin resistant S. aureus, and E.faecalis) are shown in Figure 3.17. No antibacterial activity was observed in  these samples using the one species of Gram negative bacteria (P. aeruginosa) assayed. The MICs for dichloromethane surface extracts offreshand dried leaves were greater than 2000 ug/ml based on microdilution broth assays using S. aureus (methicillin sensitive). The MIC of purified band 2 using S. aureus (methicillin sensitive) was 100 ug/ml but the MBC was greater than 200 ug/ml.  Figure 3.16. HLPC profiles comparing dried and fresh leaf extracts for the presence of band 2. A. Dichloromethane surface-extraction of dried leaves (showing absence of band 2). B. Dichloromethane surface-extraction of fresh leaves (band 2 retention time = 32.1 minutes). C. Semi-purified band 2 (Retention time = 32.1 minutes).  86  Table 3.7. Results of bacterial overlay spot assays using dichloromethane surface-extracts of dried leaves. The amount of extract (pg) that inhibited growth is indicated for each bacterial species. Lack of antibacterial activity is indicted as while "(+)" indicates a hazy (rather than a clear) zone of inhibition. Note that UV light exposure was not included in these assays. Zone of inhibition (mm) Microorganism  1000 ng 500 ug 250 ug  125 ug  63 ug  S. aureus (methicillin sensitive)  16  15  (+)  (+)  -  S. aureus (methicillin resistant)  10  5  (+)  (+)  -  E. faecalis  7  6  4  4  (+)  32 ug  16 ug  P. aeruginosa  Table 3.8. Results of bacterial overlay spot assays using dichloromethane surface-extracts of fresh leaves. All abbreviations are the same as in Table 3.7. Zone of inhibition (mm) Microorganism  1000 ug 500 ug 250 ug  125 ug  63 ug  32 ug  16 ug  S. aureus (methicillin sensitive)  16  15  12  10  9  -  -  S. aureus (methicillin resistant)  10  9  9  (+)  -  -  -  E. faecalis  9  8  7  6  (+)  (+)  (+)  P. aeruginosa  Table 3.9. Results of bacterial overlay spot assays using purified band 2 (see also Figure 3.17). A l l abbreviations are the same as in Table 3.7. Zone of inhibition (mm) 25 ug  12 ug  6 ug  3 ug  1.5 ng  0.8 ^ig  0.4 ^g  S. aureus (methicillin sensitive)  12  10  6  5  3  (+)  (+)  S. aureus (methicillin resistant)  6  4  4  -  -  E. faecalis  5  4  (+)  -  -  Microorganism  P. aeruginosa  ng  S. aureus (MS)  E.faecalis  Figure 3.17. Bacterial overlay spots tests for purified band 2 (see also Table 3.9). Abbreviations are as follows: MS = methicillin sensitive; MR = methicillin resistant.  88 Purified band 2 was subjected to further analysis for structural determination. Figure 3.18 shows the electron impact mass spectrum of this compound. The mass spectrum of band 2 shows no similarity to hydrocarbons or phytosterols isolatedfromvarious plant species (Nikolay Stoynov, pers. com 1999). The predicted molecular mass of band 2fromthis spectrum is 402, although there are weak signals at m/z = 416, 418, and 440. A search using the 1997 National Institute of Standards and Technology Mass Spectral Library (NIST MS Library) to match mass spectra for compounds with molecular mass in the range of 402 to 440 atomic mass units (amu) gave 28 results. However, only the spectrum of (Z)-6a,8,ll-trihydroxy-(8S,llR)-guaia-3,10(14)-dien-12-oic acid, 12,6-lactone,8-acetate 11-(2methylcrotonate), alternative name 4-acetoxyisopruteninone, FW = 402, molecular formula C22H26O7,  and Chemical Abstracts Service Registry Number (CAS RN) 33439-67-7 was  sufficiently similar to the mass spectrum of the unknown compound band 2 for consideration. The search results are summarised in Figure 3.19, along with the mass spectrum of band 2, the mass spectrum of 4-acetoxyisopruteninone and the difference between the two spectra. The mass intensity tables for the mass spectra of band 2 and 4-acetoxyisopruteninone are shown in Appendix C (Tables C.4 and C.5, respectively). Comparisons of the mass spectra and mass intensity tables indicate enough differences between the two compounds to eliminate 4acetoxyisopruteninone as a match for band 2. However, this information was useful in generatingfragmentationpatterns for the two compounds to assist in prediction of the structure of band 2. An examination of the structure of 4-acetoxyisopruteninone (Figure 3.20) and its mass spectrum (Figure 3.19) led to the proposed mass spectrumfragmentationpattern for 4acetoxyisopruteninone which is shown in Figure 3.21.  89 Mass Spectrum of BAND 2 BP 83 (830=100%)  342  185 111.  I • I  1  H u II  11  III.  I  ll  Hi  nn  274 J.ll. ,1  374 I.  Figure 3.18. Electron impact mass spectrum (m/z = 60 to 440) of band 2 purified from a dichloromethane surface-extraction of fresh leaves of balsamroot. The spectrum contains a prominent signal at m/z = 83. From this spectrum, the molecular mass of the compound is most likely m/z = 402, although there are weak signals at m/z = 416, 418, and 440.  90  Saturn Fit S e a r c h Results 28  Hits F o u n d : P r e - S e a r c h Hits F o u n d :  278  Saturn Fit S e a r c h Parameters Threshold:  500  Target lon R a n g e :  35 - 600 402 - 440 65 - 440 (Acquired R a n g e of Target)  Library M W R a n g e : Library lon R a n g e : Local Normalization:  Off 250 200  Requested P r e - S e a r c h : Requested Final S e a r c h :  A . c:\saturnws\satlib\nist98m.lbr B. c:\saturnws\satlib\nist98r.Ibr C . c:\saturnws\satlib\user230°c.lbr  S e a r c h 3 Libraries:  Hit 3 Fit: 6 5 3 RFit: 383 Purity: 304 T a r g e t : M a s s S p e c t r u m of BAND 2 BP 83 (830=100%) 100% 75%50% 25% 163  302  185  342  • M]|L,,I  0%  Hit: (z)-6.alpha.,8,11-trihydroxy-(8S,11R)-Guaia-3,10(14)-dien-12-oic acid,12,6-lactone, 8-acetate, 11-(2-methylcrotonate) BP 242 (100000=100%) 91746 in nist98m.lbr  C A S No. 33439-67-7, C22H2607, MW402  100% 75%-  402  50%171  199  25%-  I  0%  III I I III  HH '  302 _ J  Target - Hit Difference 100% 50%  0%  I III  M  I «''  -50% -  -100% -  Figure 3.19. Mass spectrum of band 2, mass spectrum of 4-acetoxyisopruteninone and the difference between the two mass spectra.  Figure 3.21. Proposed mass spectrum fragmentation for 4-acetoxyisopruteninone (Nikolay Stoynov, pers. comm. 1999). In the scheme below, "+" refers to a positive charge while "-" refers to loss of a group/fragment.  92 The scheme for fragmentation of 4-acetoxyisopruteninone (Figure 3.21) shows a fragment peak at m/z = 227, but does not explain the observed fragment for band 2 at m/z = 224. Furthermore, 4-acetoxyisopruteninone lacks an a-methylene moiety conjugated to the ketone on the lactone ring, which is not consistent with other structures of sesquiterpene lactones that have antibacterial activity, thus this structure does not easily account for the antibacterial activity observed for band 2. The mass spectrum of band 2 shows some resemblance to the mass spectra described for heliangolides, germacranolides and guaianolides previously isolated from balsamroot (Bohlmann et al. 1985). A tabular summary of the mass spectra described for these known compounds is provided in Appendix C, Table C.7 to aid in the following comparison of the unknown compound band 2 with 12 known compounds from balsamroot (note that the 12 known compounds are numbered from 2-13 simply for consistency with the numbering system used in Bohlmann et al. 1985). From the mass spectrum, the molecular mass of band 2 is most likely m/z = 402, however there are weak signals at m/z = 416, 418, and 440 that can not be disregarded without further consideration. A strong molecular ion signal (m/z = 402) for band 2 is inconsistent with the weak or unreported signals for Compounds 2-9, 12, or 13 in Appendix C, Table C.7. Apparently the 10-membered ring (germacranolide) compounds (i.e., Compounds 2-7) easily fragment, thus they do not produce strong molecular ion signals (Nikolay Stoynov, pers. comm. 1999). The prominent signal at m/z = 83 in the spectrum of band 2 bears similarity to compounds 2-7, however this signal could be due to a higher temperature of the MS analysis for band 2 (Nikolay Stoynov, pers. comm. 1999). The fragmentation pattern of band 2 reveals the presence of the following: • hydroxyl group (M-18); • acetate (M-60); • C4H7COOH  ester of angelate or senecioate or tiglate (M-100);  • C4H7COOH  ester plus hydroxyl group (M-l 18);  • acetate plus C 4 H 7 C O O H ester group (M-l60); and • acetate plus C 4 H 7 C O O H ester plus hydroxyl group (M-l78). The only signal that suggests the presence of epoxidized C 4 H 7 O C O O H acid (as found in Compounds 9, 10, 12 and 13) is m/z = 227, but alternatively this peak could be due to loss of  93 acetic acid plus C 4 H 7 C O O H acid plus a methyl group (M-l75). There is no other evidence in the mass spectrum of band 2 that supports the presence of an epoxide group (Nikolay Stoynov, pers. comm. 1999). The possibility that the molecular mass ion peak of band 2 could be any one of 402, 416, 418, 426, or 440 (based on weak signals in the mass spectrum) leads to the interpretations shown in Appendix C, Table C.8. The results for FW = 418 are reasonable, however, of the 6 major fragments for band 2 (i.e., m/z = 224, 227, 242, 302, 342, and 360) only 3 can be explained (i.e., m/z = 224, 242, and 302) if the molecular mass is assigned as 418. As indicated in Appendix C, Table C.8, the results for FW = 416, FW = 426 and FW = 440 do not correspond to the observed fragmentation of band 2 as the major mass ion peaks cannot be explained. Thus, 402 is the most reasonable assignment for the molecular mass ion peak of band 2. Based on the comparisons outlined above, the overall fragmentation pattern of band 2 suggests that this compound is none of the 12 known compounds previously described in balsamroot by Bohlmann et al. (1985). However, if the molecular mass of the unknown compound band 2 is assigned as 402, the most probable structure is the non-epoxidized analogue of Compound 12 (17, 18-epoxy-2-deoxy-pumilin-8-0-acetate). This prediction would correspond to the structure depicted in Figure 3.22, and following the scheme in Bohlmann et al. (1985), the name of this compound is 2-deoxy-pumilin-8-0-acetate. A name consistent with the nomenclature of the International Union of Applied Chemistry (IUPAC) is 9-(carboxyethyl-2methylene)-1,4,7,8,9,10-hexadehydro-1,7,8,10-tetrahydroxy-2,6-dimethylazulene 9', 10-lactone 7-(2-methylbutan-2,3-enoate) 8-acetate (Nickolay Stoynov, pers. comm. 2000). However, for simplicity, the designation band 2 will be used for the remainder of this dissertation. The fragmentation pattern generated for the proposed structure of band 2 (Figure 3.23) explains how the fragment m/z = 224 is obtained. Furthermore, this structure is consistent with the antibacterial activity observed in band 2 that would result from the presence of an ocmethylenebutyrolactone group.  94  Figure 3.22. Proposed structure for band 2 (2-deoxy-pumilin-8-0-acetate). The duplicate structure at right shows the numbering of the carbon skeleton assigned by Bohlmann et al. (1985).  Figure 3.23. Proposed mass spectrum fragmentation for band 2 (Nickolay Stoynov, pers. comm. 1999).  95 The H N M R spectral data of band 2 (Table 3.6) is consistent with the structure proposed !  in Figure 3.22. For confirmation of the angelate side chain function, a comparison with the nonepoxidized germacranolide structures (Compounds 2-7 in Table C.7) reported by Bohlmann et al. (1985) was helpful since senecioate is reported to give signals at 5 5.70qq, 2.14c? and 1.90c? while angelate gives signals at 5 6.\2>qq, 2.00dq and l.90dq and the latter signals are observed in band 2. Note that H-2 and H-13 indicate C H . 2  Table 3.10 'H NMR spectral data of band 2 (400 MHz, CDC1 ). The NMR spectrum was interpreted by N. Stoynov (Department of Chemistry, UBC) and is included in Appendix C, Figure C.6. Abbreviations are as follows: .s=singlet; ^doublet; m=multiplet; g=quartet. 3  Proton (H) # 2 2' 3 6 7 8 9 13 13' 14 15 CH COO (Oacetate) CH CH=(CH )COO CH CH=(CH )COO CH CH=(CH )COO 3  3  3  3  3  3  3  8 (ppm) 2.99 2.91 5.66 3.99 4.10 5.33 6.31 6.15 5.49 1.59 1.91 2.10 6.13 2.00 1.90  Multiplicity d d m d m dd d d d d m  s m m m  Number of protons 1H 1H 1H 1H 1H 1H 1H 1H 1H 3H 3H 3H 1H 3H 3H  96 Table 3.11. C NMR spectral data of band 2 (75 MHz, CDC1 ). The NMR spectrum was interpreted by N. Stoynov (Department of Chemistry, UBC) and is included in Appendix C, Figure C.7. 13  3  O  Predicted C shift 8 (ppm)  Carbon atom CH CH=C(CH )COO 14 15 CH CO CH CH=C(CH )COO 7 8 9 6 5 13 3 CH CH=C(CH )COO CH CH=C(CH )COO 10 1 4 12 2 11 CH CO CH CH=C(CH )COO unassigned 3  3  3  3  3  3  3  3  3  3  3  3  9.9 11.3 14.5 17.6 17.9 26.6 74.6 77.4 82.7 85.9 118.3 120.6 130.6 132.3 134.1 137.9 143.0 147.3 150.5 165.0 171.0 174.5  Determined C shift 5 (ppm) 1 3  , 3  a  Type  b  13.85 15.75 15.96 20.48 20.48 35.81 69.58 72.57 83.26 81.83 120.51 127.57 141.03 126.71 130.00 137.04 138.75 142.22 167.12 168.93 169.81 91.3,45.64  c  CH CH CH CH CH CH CH CH CH C CH CH C CH C C C C C C=0 C=0 C=0 3  3 3  3 3  2  "Predictions were made using ChemDraw (Cambridge Soft Corporation 1999 version 3.5.2). Determinations were based on Bruker-200 at 50 MHz in CDC1 . Type was deduced from APT and the structure of band 2. Note: 103.13 is from the NMR spectrophotometer; 76.36, 77.00 and 77.63 are from the solvent. b  3  97 3.6  Discussion The assistance of members of the Secwepemc Nation and other investigators involved in  the Secwepemc Ethnobotany Project has made it possible to consider the biological and cultural contexts related to plant collection, differential processing methods, and multiple uses of balsamroot in the phytochemical and antimicrobial analyses presented here. As a result, I suggest that these analyses better approximate "what the people are really taking [or using]" (Prance 1994:2) in balsamroot-containing diets or medical regimes, thereby increasing the relevance and hopefully the usefulness of this research to Secwepemc peoples over standard antimicrobial activity screening and phytochemical analyses. The antimicrobial properties of balsamroot are far more complex than were anticipated at the onset of this research, based on previous analyses (McCutcheon et al. 1992; McCutcheon et al. 1994; Matsuura et al. 1996). Furthermore, it is clear that what has been characterised here describes only a fraction of the total antimicrobial compounds (and these are a fraction of the total biologically active compounds) that exist in the plant. However, the results of this research indicate that traditional Secwepemc processing methods of balsamroot for food and medicine do affect some of the underlying phytochemical composition and biological activities of the plant to which the Secwepemc are exposed. This point is especially interesting given the nutritional and medicinal duality of balsamroot as a plant resource in Secwepemc and other Interior Salish cultures. These findings highlight also a need for cautious extrapolation of the biological activities of plants from assays in vitro to plant use in situ, especially if assays in vitro are based on standard plant collection and extraction procedures designed to maximise product discovery, rather than on traditional processing methods designed to understand plant use within a culture. 3.6.1  Properties of Roots Thiophene E and Antibacterial Activities: The results of assays in vitro confirmed that  the pitch of balsamroot that is used as a treatment for skin infections, does have antibacterial and antifungal activity, as does the cooled pitchy-water used as a soaking solution for wound healing—even after exposure to extreme heat treatment by boiling. Furthermore, it was shown conclusively that the previously identified antibacterial compound thiophene E is present and  98 active in the pitch, along with an undetermined number of other (as yet unidentified)  antimicrobial compounds. These results suggest that thiophene E may indeed play a role in the medicinal properties of (heat processed) balsamroot to treat skin infections, as described by Elder Mary Thomas. This result is somewhat unexpected as, according to Bohlmann et al. (1980), most acetylenes, especially polyacetylenes, are thermally unstable. However, these results also indicate that the antimicrobial nature of the pitch is chemically complex, so that the antimicrobial activity cannot be attributed solely to thiophene E. While the identities of the other antimicrobial compounds in the pitch remain unknown, the possibility exists that some of them are also polyynes, as these compounds tend to co-occur in plants (Towers et al. 1997). Without an ethnographic reference to purposeful exposure of the pitch to ultraviolet light (e.g., sunshine), however, it is difficult to assess the contribution of thiophene E to the overall  antimicrobial properties of the pitch when used in the traditional medicinal context. Matsuura et al. (1996) showed that the MIC of thiophene E is in the order of 50-100 pg/ml for S. aureus and B. subtilis without light exposure, and 25 pg/ml in the presence of UV light. Although the  antibacterial activity of crude thiophene E isolated from roots was not quantified in this study, the activity observed by TLC overlays suggests that the difference between the UV-A lightexposed and the non-UV-A light-exposed compound may be greater than the two- to four-fold difference reported by Matsuura et al. (1996), as negligible activity was observed for thiophene E in the absence of UV-A light exposure. Other studies have shown that many thiophenes are completely inactive in the absence of UV irradiation (Constable and Towers 1989; Hudson and Towers 1991). For example, antibacterial assays in vitro of thiophene A, a structually-related monoterthienyl (one sulphur heterocycle) thiophene that differs from thiophene E only in the 1,2 carbon (i.e., thiophene A is a diene, H-C=C-H, rather than a diol, HO-C-C-OH), indicated an absolute UV-A requirement for activity (Constable and Towers 1989). The light-mediated or "photodynamic" (Towers et al. 1997:395) biological activities of thiophenes involve an oxidative process that leads to the generation of singlet oxygen, which may damage a number of cellular molecules such as unsaturated lipids, proteins and nucleic acids. The main targets of thiophenes are believed to be cell membranes (Hudson and Towers 1991; Towers et al. 1997). However, with or without UV-A exposure, no antiviral activity was detected for thiophene E in assays in vitro using HSV1, nor for balsamroot pitch using HSV1 or Sindbis virus, which are both membrane-containing viruses. Cytotoxicity in the tissue culture  99 cells used to propagate the viruses was observed at pitch concentrations of higher than 100 Lig/ml,  thus the presence of antiviral activity at these concentrations could not be determined. It  is possible that the changes in cellular morphology induced by cytotoxic components of the extracts obscured the visual observation of antiviral activity. If this were the case, the activity would have been greater than 100 ug/ml crude extract. It is also possible that serum or other compounds in the assay medium interfered with the antiviral activity of thiophene E, a phenomenon that has been observed with thiophenes in similar assays (Towers et al. 1997; Hudson et al. 1999). Antiviral activity (albeit "weak") was reported for thiophene A using another membrane-containing virus (Hudson et al. 1986), and a non-membrane-containing virus (Hudson et al. 1987; Hudson and Towers 1991:198), but apparently none of the other thiophenes that are structurally similar to thiophene E has been tested in this regard. Analysis of pitcooked roots revealed that antibacterial activity is present, but it is diminished to undetectable levels, based on assays in vitro, by removal of the outer bark-like covering after pitcooking. Observations of pitcooked roots indicated that cooking draws the pitch out of the edible portion of the root where it hardens onto the inner wall of the outer bark, which must be removed prior to consumption. Consistent with this observation, analysis by GCMS confirmed that thiophene E is present in pitcooked roots, but is completely localised to the bark-like covering. From the combined results, it can be concluded that the edible portion of the root isfreeof detectable antibacterial properties when prepared for consumption following traditional Secwepemc cooking methods. In this case, pitcooking and peeling may be considered forms of detoxification. The specific concerns raised by the routine consumption of the potentially toxic compound thiophene E, as well as other antibiotic-like compounds, in traditional balsamroot-containing diets were largely alleviated by this study. Crystal I:  The second compound from boiled root pitch that was examined in this study  (crystal I) was, in fact, a ketone:alcohol mixture of two compounds related to cycloartenol, ' which is a common phytosterol (i.e., a triterpene steroid alcohol found in plant cell membranes). Sterols are important for plant growth, and their role in plant membranes is analogous to that of cholesterol in animal cells, although the other functions of phytosterols are less well understood than their counterparts in animals (Brielmann 1999). Only about 300 phytosterols or closely  100  related compounds are known to occur in plants, and the factors involved in regulating which sterols occur in a given plant species are not well characterised (Bramley 1997). The structure of crystal I was deduced by X-ray crystallography alone since the NMR and MS data did not provide conclusive structural information for this compound, largely due to the complexity of the NMR spectra in key regions and pyrolysis of the intact compound during the GC-MS analysis that lead to erroneous conclusions. Crystal I was a 90:10 mixture of 16R,23Rdihydroxycycloartenone (C30H48O3; FW = 456) and 16R,23R-dihydroxycycloartenol (C30H50O3; FW = 458). The major compound differed from the parent cycloartenol compound  (C30H50O;  FW = 426) in only three respects, namely the C-3 hydroxyl (C-OH) moiety was converted to a ketone (C=0) in ring A, an hydroxyl group was added to C-16 in ring D, and a second hydroxyl group was added to the alkyl tail at C-23. The minor compound differed only in the two hydroxylations (Figure 3.24). The majority of plant steroids are sterols, hydroxylated at the C-3 position (Brielmann 1999). Fatty acid esterification of the C-3 hydroxyl group in phytosterols is relatively common (Bramley 1997) but the conversion of the alcohol to a ketone is not, thus cycloartenone and derivatives are much less common than cycloartenol and derivatives. The addition of the two hydroxyl groups (at C-16 and C-23) to cycloartenone makes 16R,23Rdihydroxycycloartenone an unusual derivative. The structure of the dihydroxycycloartenone compound was previously reported (as 16R,23c^-dihydroxycycloartenone) from a diethylether-petrol (1:1) extraction of dried raw roots of balsamroot (Bohlmann et al. 1985). Six other closely-related monohydoxy- or dihydroxycycloartenone derivatives were also isolated (Bohlmann et al. 1985). No biological activities were investigated in his study but Bohlmann et al. (1985) suggested that these unusual compounds may be of chemotaxonomic importance as they were not known to occur in other Asteraceae. To date, 16R,23R-dihydroxycycloartenone has not been reported elsewhere in the chemical literature based on a search of Chemical Abstract Services 1986 (bound version) and CD ROM version (including 12 and 13 collective indices; 1997; 1998 and 1999 Vol 1-17) th  th  covering the period of 1987-1999 (Oct). This is the first report of the X-ray crystallographic structure of this compound based on a search of the Cambridge Structural Database, October 1999 version (Cambridge Crystallographic Data Centre, Cambridge). The X-ray structure showed that that the configuration (i.e., orientation of groups around an asymmetric carbon) of the hydroxyl group at C-23 was R (rectus, or right-handed), a detail that was not previously  101 determined by Bohlmann et al. (1985). The minor compound, 16R,23R-dihydroxycycloartenol  has not been reported previously in the chemical literature so this research likely constitutes the first structural identification of this compound.  Figure 3.24. Structural comparision of 16R, 23R-dihydroxycycloartenone and 16R, 23R-dihydroxycycloartenol purified from balsamroot pitch.  Antibacterial activity was not detected in crystal I, although the column fraction (fraction I) from which it was crystalised did have antibacterial activity. It is possible that crystal I has other interesting and/or medically-relevant biological activities since these compounds have structual similarity to the active compound in the fungus Inonotus obliquus Pers.: Fr.  102 (Polyporaceae) that has been examined for cancer treatment (Yokoyama et al. 1975; Bohlmann et al 1985; Hobbs, 1995). Anticancer and other biological activities have been reported for cycloartenol, numerous cycloartenol-related compounds and other phytosterols. For example, Yashukawa et al. (1998) reported inhibition of carcinogenic tumor promotion and tumor-induced inflammation by cycloartenol ferulate from a methanol extract of rice bran in the skin of mice, while Ahumada et al. (1997) reported anti-inflammatory action in vivo of cycloartenolfroma hexane extraction of Crataegus monogyna Jacq. (Rosaceae), which lowered oedema and inhibited peritoneal leucocyte infiltration in rats. Other phytosterols, such as P-sitosterol have anti-inflammatory and cholesterol-lowering properties and semi-synthetic derivatives of Psitosterol are gaining attention for their serum cholesterol-lowering properties (Johns and Chapman 1995; Fraser 1994). Calculations of the yield (dry weight compound/dry weight roots) of 16R,23£,dihydroxycycloartenone chemically extracted from dried roots (90 mg/100 g s 0.09 %) as described in Bohlmann et al. (1985) compared with that extractedfromboiled root pitch (25 mg/1000 g = 0.003 %) in this study showed that the chemical extraction process is more efficient than the boiling water extraction for this compound. The yield from boiled pitch was higher (25 mg/6 mg = 0.42 %) but TLC analysis with dinitrophenylhydrazine reagent for visualisation (specific for ketones and aldehydes) indicated that crystal I is not localised to the pitch within the root. However, as this compound also appears to be structurally-stable to heat exposure, it does likely comprise part of the chemical cocktail of the boiled pitch that is used as traditional Secwepemc medicine, thereby impling the existence of other potential health benefits of the roots when prepared and used in a traditional context. Furthermore, as these compounds are present in the pitcooked root consumed as food, they certainly would be ingested as part of traditional balsamroot-containing diets. In medicinal preparations, the contemporary additions of other plant species to the boiled pitch, such as mashed Plantago major (plantain) for making healing salves (Mary Thomas, pers. comm. 1996; 1997; 1998), further complicate chemical and biological analyses and interpretations, and provide additional challenges for understanding the medicinal properties of the root as used in Secwepemc culture. Such mixtures were not investigated in this research. However, I believe that the accommodation and incorporation of these kinds of dynamic and adaptive elements of cultural knowledge into further laboratory investigations may be key to  103 stimulating highly productive and meaningful research that also more fully acknowledges the innovation, and intellectual contributions of Aboriginal societies, past and present, to the research process and outcomes. A Broader Assessment: The comparison of the antimicrobial and chemical properties  of  the roots of balsamroot prepared as food and medicine has contributed to a deeper understanding of the importance of heat as a differential processing method—essentially creating multiple uses for a single plant part. In this case, heat is crucial in both nutritional and medicinal applications, albeit apparently for different reasons. In the nutritional context, heat and other factors are required to increase the availability of carbohydrate by a process of chemical degradation, which relies on the heat-, acid- and moisture-sensitive nature of inulin (Peacock 1998). In the medicinal context, however, heat applied by boiling makes available water-soluble compounds (in the pitchy-water) and water-insoluble compounds (in the pitch), both of which must be chemically stable (in terms of their biological activity) to extreme heat exposure. An understanding of the utility of balsamroot as both food and medicine, and the 'discoveries' of antibacterial properties of the processed bark and pitch would not have been possible without the guidance of Secwepemc Elders who shared knowledge of their traditional preparation methods and uses. Thus, cultural knowledge has played a key role in these research findings, and this research in return, has underscored the sophistication and utility of past and present Secwepemc plant knowledge. It is interesting to note that recent studies on certain health-promoting functions of inulin and dietary fructans suggest that the inulin remaining in pitcooked roots may also have beneficial roles. These include addition of fiber to the diet, a sweet taste with low calorific value, and the ability to stimulate colonic health by serving as a preferential substrate for the growth of 'beneficial' intestinal bacteria {e.g., Bifidobacterium spp. which subsequently out-compete potentially pathogenic species (e.g., Escherichia coli) in microbial colonisation of the human colon (Wang and Gibson 1993; Gibson et al. 1995; Van Loo et al. 1995). The presence of selective bacterial growth-promoting properties of inulin in balsamroot provide an interesting contrast to the growth-inhibiting properties of the antibacterial compounds found in the root. Indeed, at least initially, these properties seem ironic, especially considering Bifidobacterium spp. are Gram positive, and thus may be susceptible to thiophene E and other compounds that are  104 inhibitory to the Gram positive species of bacteria assayed in vitro. However, these opposing properties of the root may now be considered in the context of balsamroot processed as food, i.e., in terms of the properties of the edible portion of the pitcooked root, which appears to befreeof antibacterial compounds. While it can safely be assumed that the vast majority of the roots would have been cooked prior to eating, there are ethnographic references to consumption of the raw roots (e.g., by Leslie Jules as recorded in Turner and Ignace in preparation). Here, however, the strong and distinctive flavour, the fibrous texture, and the resulting flatulencefromtrying to digest inulin presumably would have limited intake to small amounts or to times of necessity. One interesting question left unanswered from ethnographic information is whether the outer bark-like covering of the root was typically removed prior to pitcooking for food and/or boiling the root for medicine, or whether it was left intact during heat processing—an historical detail that could have implications for the chemistry underlying balsamroot processing. Elder Mary Thomas indicated that the roots could be peeled before or after cooking, while Elder Lilly Harry recalled that the roots were dug and beaten to remove the outer covering prior to cooking, and the late Elder Aimee August's description is one of peeled roots skewered on a stick (Turner and Ignace in preparation). This study has shown that the prior removal of bark from the root is not necessary to collect pitch by boiling, although it may affect the quantity of pitch released from the root. Prior removal of the bark also would eliminate much of the dirt (presumably undesirable), which would otherwise collect in the cooled pitchy water used as a wash; Elder Mary Thomas does indicate that if the root is left unpeeled, then the bark should be cleaned well (Turner and Ignace in preparation). As previously mentioned, when the root is used in food preparation, the bark is significantly easier to remove after pitcooking. While it has been proposed that acid is essential for hydrolysis of inulin to fructose, and that volatile organic acids are provided by addition of other plant stuffs to the earth oven (Peacock 1998; Mullin, pers. comm. 1998), resultsfromthis study indicate it likely that unpeeled balsamroot itself is sufficiently acidic to catalyse the hydrolytic cleavage, since the pH of boiling water extracts of root (unconcentrated) was recorded as pH 5. The outer bark would also help to retain both heat and moisture once the core of the root reached the minimum temperature required for inulin conversion—a situation likened to "a self-basting turkey, roasting in its own juices" (Bannister and Peacock 1998:11). Peacock (1998) found no significant difference between peeled and unpeeled roots in conversion of inulin to  105 fructose or oligofructose. However, in addressing this question, more than just chemical and energetic efficiencies of food preparation should be considered; for example, factors such as flavour may have played a role. Perhaps the root just tastes better (or at least tastes better to some) when it is cooked without the bark, as its flavour would be more influenced by other plants in the earth oven. Likewise, beliefs and rituals may have governed root preparation to some degree. Turner et al. (1990:177) note a number of rituals that were observed by the neighbouring Nlaka'pamux (Thompson) peoples (located to the south-west of Secwepemc territory) as recorded by ethnographer James Teit in 1900. These early ethnographic records suggest that the plant was very highly esteemed, as indicated by a prayer addressed to "the Sunflower-Root" by young people partaking in their first plant products of the season: "I inform thee that I intend to eat thee. May thou always help me to ascend, so that I may always be able to reach the tops of mountains, and may I never be clumsy! I ask this from thee, Sunflower-Root. Thou art the greatest of all in mystery" (Teit 1900, as cited in Turner et al. 1990:177). Omission of this prayer was said to "make the person partaking of the food lazy..." (Teit 1900, as cited in Turner et al. 1990:177). Certainly participating in the labour-intensive process of root harvesting would have likely staved off laziness, although it is unclear if the root would have been as difficult to dig in the past as it is today from places like Komkanetkwa—where soil compaction and extensive turf build-up resulting from relatively recent factors such as cattle grazing, introduced grass species, and lack of regular seasonal root harvesting have presumably altered the harvesting experience. While it may no longer be possible to corroborate some of the historical details of balsamroot harvesting and processing with certainty, the widespread references to its past use certainly suggest that the plant was worth the significant effort of preparation, and clearly recognised for both its nutritional and medicinal importance. As indicated by the late Elder Lilly Harry in an excerpt from a direct translation by Mona Jules of Lilly Harry's narrative Re Stq 'elsem ("Openpit Cooking"): "The balsam root is very hard work. The lichen is easy.. .but the balsam roots require many things! 'Medicine plants for pit-cooking' is what the old people called it" (SCES Language Department, 1994:33-41 as cited in Peacock 1998:147).  106 3.6.2  Properties of Leaves Biological Activities:  Although many questions are left unanswered about the various  uses of aerial parts of balsamroot for food and medicine, the traditional Secwepemc information that is available suggests an interesting plant-people relationship at the chemical level. Many members of the Asteraceae contain sesquiterpene lactones as part of their battery of phytochemical defenses, and these are often found abundantly in the aerial parts but largely sequestered in trichomes to avoid auto toxicity (Duke 1994). The wide-ranging biological activities of many of these compounds have been the focus of numerous investigations over the last few decades. For example, much research has been conducted on parthenolide, a common sesquiterpene lactone found in Tanacetum parthenium (L.) Schultz-Bip. (feverfew), which is commonly used for alleviation of migraine headaches (Murphy et al. 1988). Along with antimicrobial and other biological effects, however, sesquiterpene lactones such as parthenolide are the cause of a delayed contact dermatitis reaction. This reaction can result in redness, swelling, itching, blistering sores or eruptions with oozing and crusting, particularly on exposed parts of the body, especially the eyelids, neck, face, fronts of elbows and backs of knees (Towers et al. 1977; Mitchell 1980). Mitchell (1980) reports that the condition can lead to secondary infection and considerable disability. Furthermore, individuals sensitised to one sesquiterpene lactone can exhibit cross-sensitivity to different sesqiterpene lactones found in many other species of Asteraceae, or in other plant families (Mitchell 1980). The significant health hazards that such allergenic sesquiterpene lactones can pose is illustrated by an example involving the introduced weed Parthenium hysterophorus Linn. (Asteraceae) in parts of India. This plant produces the highly allergenic pseudoguaianolide compounds parthenin and ambrosin (Rodriguez et al. 1976) and serious outbreaks of allergic contact dermatitis were attributed to these compounds in the 1970s (Krishnamurthy et al. 1975; Towers et al. 1977; Mitchell 1980). Among the many clinical examples described that illustrate the potency of these compounds is the case of a woman at Malleswaram, Bangalore: "According to her statement, all that she did was to uproot the parthenium plants in her compound. She is suffering for over one year from cracking of the skin in the palm and foot, e[r]ruptions of the skin and rashes on hands and legs" (Krishnamurthy et al. 1975:170). Leaf-surface extraction and  107 chemical analysis showed that the highest concentration of these compounds in P. hysterophorus is found in the glandular trichomes of flower heads and leaves (Rodriguez et al. 1976). Investigations of cross-sensitivity by parthenin to its diasteriomer hymenin indicated that there is some stereospecificity involved in the immunological activity governing the allergic response. Patch tests of a limited number of individuals showed that sensitisation to parthenin does not cause cross-sensitivity to hymenin (Subba Rao et al. 1978). Similar stereospecificity in allergenic response has been documented for other plant compounds, such as usnic acid found in some lichens (Subba Rao et al. 1978; Mitchell 1980; Neil Towers, pers. comm. 2000). The leaves of balsamroot, which exhibited both antibacterial and antifungal activity in vitro when dried and extracted with methanol, were examined further to see if the antimicrobial  activity was due to the presence of potentially allergenic sequiterpene lactones, since a number of these compounds was previously reported in balsamroot (Bohlmann et al. 1985). Numerous antibacterial compounds were detected in methanol extracts of dried leaves, and in dichloromethane surface-extracts of both fresh and dried leaves based on TLC agar overlays using S. aureus. The brief immersion of leaves in a solvent such as chloroform or dichloromethane collapses the subcuticular space of glandular trichomes without damaging other tissues (Duke et al. 1994; Tellez et al. 1999) so this method is selective in extracting relatively lipophilic surface-held leaf compounds. The three most lipophilic antibacterial compounds extracted were present only in the fresh leaves. Purification and structural elucidation of one of these compounds (referred to as band 2) confirmed that the structure was based on a sesquiterpene lactone-skeleton and contained a typical a-methylenebutyrolactone, which is the functional group involved in mediating both antimicrobial and allergenic activities. Gram positive antibacterial activity in vitro was confirmed for purified band 2 but assessment of potential allergenicity was not attempted. The antibacterial activity of band 2 was qualitatively and quantitatively similar to that observed for commercial grade parthenium, the sesquiterpene lactone used as a standard in this research. Structure of Band 2:  A comparison with sesquiterpene lactones and the closely related  compounds isolatedfrombalsamroot (Bohlmann et al. 1985) indicated that this C22H26O7 guaianolide (FW = 402) has not been previously reported in this plant. However, Bohlmann et al. (1985) did isolate a structurally-similar epoxide (17,18-epoxy-2-deoxy-pumilin-8-0-acetate).  108 No biological activity was reported for the epoxide, and likely biological activity was not tested as the research by Bolmann et al. (1985) was apparently conducted for chemotaxonomic purposes. The predicted structure for band 2 has not been reported in any of the chemical literature based on a search of Chemical Abstract Services CD ROM version (including 12 and 13 th  th  collective indices; 1997; 1998 and 1999 Vol 1-17) covering the period of 1987-1999 (Oct). Aside from the compounds previously reported in balsamroot (Bohlmann et al. 1985), the most structurally similar guaianolides were from other plant species of the Asteraceae, including two Argentinean species of Stevia (Hernandez et al. 1994; and Hernandez et al. 1996), two species of Hymenoxys (Zdero et al. 1991; and Gao et al. 1991) and a species of Tetraneuris (Diaz et al.  1992). It appears that this study constitutes the first report of the compound referred to as band 2, as well as the first report of antibacterial activity of a guaianolide isolated from balsamroot leaves. As indicated previously, this newly identified antibacterial compound would be called 2deoxy-pumilin-8-O-acetate following the naming scheme of Bohlmann et al. (1985). Following the nomenclature of the International Union of Applied Chemistry (IUPAC), however, it would be called 9-(carboxyethly-2-methylene)-l,4,7,8,9,10-hexadehydro-l,7,8,10-tetrahydroxy-2,6dimethylazulene 9',10-lactone 7-(2-methylbutan-2,3-enoate) 8-acetate (Nickolay Stoynov, pers. comm. 2000). Interestingly, band 2 was not detected in dried leaves, which can be interpreted as indirect support that this compound is mainly localised to the glandular trichomes (i.e., modified epithelial cells on plant surfaces, commonly called hairs) of leaf surfaces, as appears to be the case for sesquiterpene lactones of feverfew (Kevin, Usher, pers. comm. 2000; Usher in progress), and as established in other Asteraceae, such as Artemisia annua L. for the sesquiterpene lactone artemisinin (Duke et al. 1994; Tellez et al. 1999). If this proposal is correct, it is possible that the glandular trichomes are physically lost or lose their structural integrity upon drying of the leaf. Biologically active sesquiterpene lactones released from glandular trichomes could be susceptible to oxidative, enzymatic or other environmental degradation. Confirmation of this hypothesis, however, awaits a new season of fresh leaves.  109 Experimental Limitations:  As band 2 was the only compound purified and identified  from balsamroot leaves in this study, it remains unknown if any of the compounds isolated by Bohlmann et al. (1985) are also present in these leaf extracts. Unfortunately, none of these previously isolated leaf compounds could be obtained for direct comparison with my extracts, which would have led to conclusive answers to the questions relating to the presence and antimicrobial activity of these compounds, using TLC bacterial overlays and GC-MS or HPLC analyses. Significant qualitative and quantitative variability is said to exist in sesquiterpene lactone and other secondary metabolite production in plants (Harborne 1997) so the presence of any given compound from a given plant species can not necessarily be assumed. This may be especially true for sesquiterpene lactones. It is possible that ecological or genetic variation leading to a slight alteration in sesquiterpene lactone biosynthesis could create distinct chemical compounds without compromising on plant defensive (or other physiological) functions  in situ.  . As with any phytochemical isolated in the laboratory, there is also the possibility of unintentionally altering the structural and/or biological properties through standard laboratory extraction and purification procedures, thereby leading to final analysis of an 'artifact'. Temperature, light, oxygen, pH or other exposures, as well as loss of stabilising co-factors or susceptibility to enzymatic degradation are some of the factors that can all potentially affect the structure and activity of the final product isolated in the lab. For unknown compounds, the effects of these variables can only be determined empirically. It could be argued that the presence of an isolated compound should be re-confirmed in the plant and/or synthesised to ensure that it is not simply an artifact of the isolation procedure. Typically, the initial isolation of an unknown compound is based on a small-scale plant collection and extraction, thus, largerscale collection and extraction often are required to obtain adequate yield of the compound for structural and biological characterisation. Such was the case for band 2, which was initially isolated from a small collection of fresh leaves and was subsequently isolated and purified for structural analyses from a second, larger collection. Thus, while it was not disproven that band 2 is an artifact, the isolation and purification of band 2 from fresh balsamroot leaves was reproducible. The variables that can affect the structure of a compound potentially can affect its biological activity also. Given that the yield of an isolated compound may be close to the minimal amount required for structural analyses, a quantitative assessment of the relevant  110  biological activities of the purified compound may have to wait until the structural analyses are completed. Such was the case for band 2, as its yield was sufficient for NMR and other structural analyses at the facilities available but insufficient for simultaneous quantification of its antibacterial activity (i.e., the structural analyses are largely non-destructive so most of the compound can be recovered for subsequent use). The quantitative antibacterial analyses had to wait until all structural analyses were completed, data were analysed and a reasonable structure was proposed (i.e., several months in total). During this time, a darkening of the yellow colour of band 2 was noted, even though between analyses it was evaporated under nitrogen gas and stored sealed at 4 °C. There is potential also to introduce impurities to a sample through the analyses themselves (e.g., multiple solvent and glassware exposures), or to lose sample (e.g., physical losses through multiple glassware transfers, or degradative losses by exposure to solvents required for the analyses). All of these factors combined suggest that the antibacterial activity of band 2 reported here could reasonably be considered an underestimation of the activity in vivo (and in situ). Interpretations: The concerns for potential allergenicity raised by traditional use of  balsamroot leaves in treatment of wounds cannot be alleviated by the single compound identified in this investigation. However, this study does suggest that if dried rather than fresh leaves are applied directly to skin, and if sesquiterpene lactones are localised to glandular trichomes, then the potential for delayed hypersensitivity contact dermatitis induced by certain sesquiterpene lactone compounds may be decreased. It should be noted also that the antibacterial potential of the dried leaves is less than that of fresh leaves. However, several more hydrophilic antibacterial compounds are still present in dried leaves, and the general antimicrobial profile does not change (Table 3.6). Of course, the possibility also exists that the leaves used in wound treatment have additional properties to those of an antimicrobial nature—an intriguing point to be discussed shortly. In proposing that sesquiterpene lactones produced in balsamroot may be localised to glandular trichomes and thus may pose less of an allergenic 'threat', especially if leaves are used in dry form, it is important to mention that glandular trichomes containing sesquiterpene lactones and other phytochemicals occur commonly on most aerial surfaces of the plant. There are ethnographic references to consumption of non-leaf aerial parts of balsamroot. For example, the  Ill  bud stalks of balsamroot are noted by Turner and Ignace (in preparation) as an important food for Secwepemc peoples, although they are eaten only when young, and peeled before consumption. Nlaka'pamux Elder Mabel Joe (Shulus) also indicated that shoots were peeled before eating (Turner et al. 1990). The tender inner shoots are certainly more pleasant to eat when peeled as the outer layers are tough and stringy in texture and somewhat bitter in taste (personal observation), so presumably this practice of prior peeling would be widespread. There is no specific mention of rashes or allergies associated with handling or consumption of fresh aerial parts of balsamroot by Secwepemc peoples, which suggests a number of possible explanations: the glandular trichomes of young shoots may not yet produce or contain allergenic sesquiterpene lactones; fortuitously the compounds may not make skin contact because stalks were peeled (i.e., removal of the epidermis would remove the trichomes); or, unlike the potent allergen parthenin, it is possible that the particular sesquiterpene lactones (or the specific isomers) produced by balsamroot may not cause an allergic reaction at all. However, as the onset of the dermatitis caused by exposure or re-exposure to sesquiterpene lactones is delayed by 24-48 hours, it is also possible that any allergic reactions experienced are/were simply not associated with balsamroot. Fighting Fire with Fire?  Finally, it is feasible that balsamroot leaves may be used for  medicine because of their potentially allergenic compounds rather than despite them. The use of balsamroot leaves for "weeping" poison ivy rash and wounds that will not heal (Mary Thomas, 1997 pers. comm.; Turner and Ignace in preparation) could be interpreted as a treatment for certain hypersensitivity reactions, implying that the leaves may be used as a form of countertreatment (immune desensitisation) and/or for anti-inflammatory properties. One consequence of a delayed-type hypersensitivity reaction is the nonspecific destruction of cells due to the activity of cytotoxic T-lymphocytes and the accumulation of lytic enzymes produced by activated macrophages at the sight of antigenic exposure (Kuby 1997). Thus, repeated antigenic exposure or chronic allergic reaction can lead to substantial localised tissue damage and interference with healing. It is possible that the purposeful application of other extrinsic allergens in such cases can alter the immune reaction in a net positive fashion—by leading to a net decrease in inflammation and thereby assisting in the healing process.  112 Numerous studies have documented anti-inflammatory activity in sesquiterpene lactones (Hall et al. 1979; Hall et al. 1980; Ysrael and Croft 1990; Akihisa et al. 1996; Lyss et al. 1997; Mazor et al. 2000), and Wagner (1989) suggested that the anti-inflammatory activity of some sesquiterpene lactones may be due to their immunomodulating activities on complement or Tlymphocytes, giving them anti-allergenic properties. Related to this, Taniguchi et al. (1995) found that the sesquiterpene lactones costunolide and dehydrocostus lactone inhibited the killing activity of cytotoxic T-lymphocytes, and Lee et al. (1999) showed that dehydrocostus lactone inhibited the expression of both nitric oxide and tumour necrosis factor alpha in lipopolysaccharide-activated macrophages. Based on studies with parthenolide, Hwang et al. (1996) proposed that sesquiterpene lactones may have therapeutic value in the treatment of septic shock induced by lipopolysaccharide from Gram negative bacteria, as well as in the treatment of other acute inflammatory diseases. This prediction is based on studies that showed that parthenolide and other sesquiterpene lactones inhibited the production of proinflammatory cytokines in rat alveolar macrophages in a dose-dependent fashion (Hwang et al. 1996). It was shown that this inhibition was mediated through conjugation of the a-methylenebutyrolactone ring to sulfhydryl groups on target proteins (either through direct binding with protein tyrosine kinases or indirect binding with other proteins that interact with the kinases) since the inhibitory action was abolished by prior saturation of the double bond between C-l 1 and C-l 3. Interestingly, structure-function studies comparing parthenolide with the structurally similar sesquiterpene lactone costulide (which differs from parthenolide only by the absence of an epoxide between C-4 and C-5), showed that the presence of the epoxide increases the inhibitory activity of parthenolide (Hwang et al. 1996). Interestingly, the capacity to bind and thus interfere with the function of proteins also is linked with anti-inflammatory activity and neutralisation of snake venoms. The role of a wide range of phytochemicals in neutralising the actions of snake venoms was documented by Pereira et al. (1994). No sesquiterpene lactones were examined specifically, but many flavonoids (which are compounds also previously reported in balsamroot leaves) did protect against the lethal effects of jararaca  (Bothrops jararaca)  snake venom in mice (Pereira et al. 1994). The  anti-snakebite activity was linked to the enzyme-inhibiting properties of flavonoids, specifically the inhibition of phospholipases, which are important components of snake venoms (Mors et al., n.d.). Anti-snakebite activity was related also to the metal-binding capacity of flavonoids, as the  113 enzymes in some snake venoms are zinc-containing metalloproteinases (Mors et al., n.d.). Interestingly, anti-allergenic properties were mentioned among the various other biological activities of flavonoids, and these properties were thought to be related to their macromolecularbinding capacity (Mors et ah, n.d.). It is worth noting also that numerous phytosterols (including sitosterol from a number of sources and cycloartenol from Linum usitatissimum L.) also had antisnake venom and anti-inflammatory activities (Pereira et al. 1994; Mors et al, n.d). It would be interesting to subject the balsamroot extracts and the purified compounds examined in my study to such analyses. Without further research, it is only possible to speculate on the possible relevance and value of the balsamroot compounds characterised in this study. However, it is interesting to observe the wide variety of biological properties that have been investigated for similar compounds to date, some of which may relate directly or indirectly to traditional medicinal uses. With any given compound, however, the proof 'lies in the pudding' such that no activity should be assumed without supporting empirical evidence. Furthermore, the significant 'leap' required from biological activities in vitro to biocultural relevance in vivo must also be kept in mind. The results of the more in-depth analyses of balsamroot presented in this chapter certainly attest to the inadequacy of relying soley on standard screening procedures in vitro for understanding the biological affects of plants used as traditional food and medicine. Obtaining a complete understanding of the medicinal and other valued properties of plants relating to their biological and cultural contexts may be beyond the limits of laboratory analyses, but a combination of more tailored investigations can make vital contributions to increasing our understanding in this regard. 3.6.3  Toward a Deeper Understanding of Human-Plant Interrelationships  The antimicrobial analyses presented here have provided a new perspective at the chemical level on the relationship between Secwepemc peoples and balsamroot as a food and medicinal plant resource. Moreover, the analyses have elucidated some of the chemistry underlying Secwepemc traditional plant knowledge, such as the use of different plant parts for different purposes, and differential heat processing of a single part for multiple uses. Heat is one of many well-recognised technologies for altering the character of food and expanding food  114 resources, for example, by making a plant more palatable or more digestible, or by eliminating toxic or unpalatable plant constituents (Johns and Kubo 1988; Johns 1990). Interestingly, the processing of roots of balsamroot as food and medicine provides an opportunity to observe all of the above. Upon examining this 'altered character' of processed balsamroot, a clear distinction between food and medicine can no longer be made. Ford (1994a:30) claimed that "the distinction between food and medicine is an artifact of Western specialization. ... Most cultures, in fact, classify all plants (and many animals) taken internally into a unified taxonomy. Illness may result from overindulgence of one, the exclusion of another, or the consumption of almost any substance under culturally inappropriate circumstances". For this reason, he suggested that it is appropriate to incorporate gastronomy into ethnobotanical (especially ethnomedicinal) studies, and indeed such a study on processed and unprocessed roots of balsamroot would be of tremendous value in assessing the net effects of balsamroot-containing diets on gut microflora, and thus on their human hosts. The importance of such an approach is further underscored by increasing interest in the nutritional value, possible health benefits, and commercial potential (at both the local and international levels) of inulin-containing foods such as balsamroot. Inulin-containing foods are beginning to receive attention in North America as "functional foods" and already have established markets in Japan and other parts of Asia and Europe (Mullin et al. 1998; John Mullin, pers. comm. 1998). Researchers at Agriculture Canada are currently investigating the potential of balsamroot as an "exotic native vegetable" or as a possible commercial source of short chain inulin (John Mullin, pers. comm. 1998). A large-scale, commercially-motivated 'balsamroot revival' would, of course, have significant ramifications for Secwepemc and other Aboriginal peoples who have been guardians of the knowledge of balsamroot processing for food and medicine—without which balsamroot might be viewed as just another pretty flower on the dry south-facing slopes of Komkanetkwa and other Interior Plateau locales. If all stakeholders are appropriately acknowledged, however, this situation may hold significant promise for mutually-beneficial collaborations between local communities, government and industry for co-management and development of balsamroot as a multi-functional, renewable natural resource. Biological and cultural issues related to acknowledgement of stakeholders in  115 research, and consideration of the consequences of research are topics that are discussed at length in the following chapter. Finally, although the focus of this research has been on understanding the process of plant-human interrelationships, rather than seeking products per se, some products have resulted. These include the isolation and identification of a previously unknown antibacterial compound from balsamroot leaves (i.e., band 2, or 2-deoxy-pumilin-8-0-acetate), the isolation and identification of a previously unknown compound in the root (16R,23Rdihydroxycycloartenonol), the further biological activity, solubility and structural characterisation of two known compounds in the root (i.e., thiophene E and 16R,23Rdihydroxycycloartenone), and the observation that numerous as of yet unidentified antimicrobial compounds are present in both leaves and roots. While the utility of any these compounds as 'products' has yet to be fully determined, their 'discovery' has assisted in addressing some intriguing questions about the nutritional and medicinal uses of balsamroot. In the end, however, more questions have been created than answered. This dissertation research represents but the 'tip of the root crown' in unearthing a new level of plant-people interactions and deepening our collective understanding of the meaning of balsamroot in Secwepemc and other Interior Salish cultures.  116  4  Biocultural Issues in Ethnobotany  ...biodiversity gets too much emphasis compared with cultural conservation. We should be asking ourselves: how can our work in ethnobotany help to maintain cultural identity? ... This leads on to the issue of ethics. What have indigenous peoples gained from the use of their knowledge by Western culture?  —G. T. Prance (1994:1)  4.1  INTRODUCTORY COMMENTS: ETHNOBOTANY AT A CROSSROADS  In the previous two chapters, I have presented the main field and laboratory findings of my dissertation research. The purpose of this chapter is to depart from the biological and chemical data to explore some of the ethical issues that have arisen during the course of the research. It was not my original intention to devote a significant amount of this dissertation to an examination of social, political and legal aspects of the research. However, the importance of these aspects, combined with a general lack of awareness of related research issues (particularly in the natural sciences), have become apparent through my work. From my perspective, the wide gap in awareness that exists between the natural and social sciences is a major problem in ethnobotanical and related research. This chapter is a contribution toward increasing crossdisciplinary awareness and bridging this divide. In recent years, ethnobotany has achieved a new level of recognition—as Ford (1994c:viii) proclaimed in his opening statement of The Nature and Status of Ethnobotany, " E T H N O B O T A N Y IS IN!" At least in some circles, it would be difficult today to overstate such an observation. Amid the current and seemingly insatiable public interest in Indigenous cultures and plant-based resources, ethnobotanical study—especially that involving human diet and medicine—has surely found its niche. B y all accounts, ethnobotany certainly is "in". Or is it? Is it a search for the meaning of plants within a culture that guides current widespread interest in ethnobotany, or is it more accurately a search for plant products of use to other cultures? As 17  will be explored in this chapter, the difference here is not merely semantic, but may be rife with  17  According to Ford (1994c), the former is ethnobotany while the latter is economic botany.  117 ethical, legal, political, and economic implications for current ethnobotanical research, and may have a bearing on biological diversity and cultural identity. While it is controversial whether or not research conducted explicitly to 'discover' useful plant products from the botanical knowledge of Indigenous cultures can be considered ethnobotany (based on Ford 1994), such research is occurring under the guise of ethnobotany. This raises important concerns and issues about the appropriation and control of knowledge and related resources (to be discussed further in a later section). The implicit question about ethnobotanical research (above) posed by Prance (1994) is how might Indigenous peoples benefit ? This question is fundamental to current 'ethical' research practices in ethnobotany; it 1  also has been and continues to be a focus for dialogue that has challenged and strengthened the discipline in many ways (Elizabetsky 1986; Chadwick and Marsh 1994; Brush and Stabinsky 1996; Salmon 1996; Posey 1999; Gyllenhaal 2000; Carlson et al. submitted; Laird in press). An equally important (but less popular) question to consider, however, is how might Indigenous peoples be harmed?  The potential for undesirable cultural impacts of research has already fueled decades of debate amongst anthropologists (Brush 1993; Nicholas and Andrews 1997; Brown 1998; Sillitoe 1998; Posey 1999). As noted, this contrasts with the natural sciences where such discussions seem to have remained largely outside the awareness or interest of many researchers who are engaged (directly or indirectly) in ethnoscientific study. While widespread concerns have been expressed about the loss of biological diversity through habitat erosion due to development or through over-exploitation (Cordell 1995; Borris 1997; Hostettmann et al. 1997; Kingston et al. 1997), these concerns have rarely been associated with corresponding cultural losses—at least not beyond losses of those aspects of cultural knowledge that are seen as useful resources to mainstream society (Bruhn and Helmstedt 1981; Kingston et al. 1997). The dangerous corollary of saving the knowledge because it is of value to us, however, is that if we do not see its value then it may not seem worth saving. Linden's (1991) "Lost Tribes, Lost Knowledge" cover story article in Time magazine stimulated just such responsesfrommany readers: "I do not favor the idea of studying tribal headmen as geniuses or the practices of the indigenous peoples because of economic value. Very soon the West will exhaust the utility of the natives and forget them" (Wan-kan Chin 1991:3); "If the tribal people disappear, our loss will be great, but that is not the issue. ... Culture must be preservedfromwithin" Schmidt 1991:6  118 emphasis mine); and "diversity of life is the true legacy of humankind. We should be no less concerned with the loss of these lifeways than we are with the extinction of plant and animal species" (Nicholas 1991:6). As with the inextricable link between biological and cultural diversity, so too are issues of biological relevance linked with issues of cultural relevance, and vice versa. However, as Etkin (1994:14) observed: "If conservation of what has reached a  bewildered adolescence, conservation for whom is barely in its infancy". Since the inception of the term ethnobotany, which was popularised by botanist John Harshberger in the mid-1890s (Anonymous 1895; Ford 1994c), a utilitarian emphasis on the uses of plants by humans has strongly influenced ethnobotanical approaches to research within the natural sciences. Ford (1994a:31) pointed out, however, that an emphasis on uses contributes to a misleading definition of the field and "denies the intellectual life of non-Western people". Anthropologists who have acknowledged the essentiality of cultural cognition within ethnobotanical research have assisted in expanding the objectives of ethnobotany from those based mainly on scientific categorisation and use to include those that encompass the importance of Indigenous understandings, perspectives and ways of "ordering the universe" (Ford 1994b:39). Accordingly, Indigenous peoples and others who were once the 'subjects' of past ethnobotanical inquiry are now increasingly acknowledged as key contributors and essential partners in the research process. Furthermore, the explicit acknowledgement of the intellectual contributions of Indigenous societies to ethnobotanical research is increasingly accompanied by a concomitant recognition of Indigenous rights to traditional resources and intellectual properties (Ford 1994c; Brush 1996; Posey and Dutfield 1996; International Society of Ethnobiology, 1998; Dutfield 1999). This recognition is particularly pertinent to current research in British Columbia, within the complex milieu of Aboriginal land claims and treaty negotiations. At least here in British Columbia, as Ford (1994c:xiv) has put it, "the days of 'hit and run' ethnobotany are over". Ford (1994c:xx) acknowledged also that ethnobotany today finds itself at an "intellectual crossroads". He advised that the key to progress of the discipline as a whole lies in a "return to one of its more productive historic roots, the meaning of plants" (Ford 1994c:xxi; emphasis mine). What will a return to these disciplinary roots require? I suggest, first, that it will require a grounding in the past—knowing where ethnobotany has come from, how the discipline has evolved. Second, it will require a critical assessment of where ethnobotany is today—we need to  119 understand the complexities presented by ethnobotanical research because (as Prance 1994:2 diplomatically put it) it is "an area that can easily be misunderstood", and because it is research that can have significant impacts, both positive and negative, not only on humans and plants but on ecology in general. Third, a 'return to these roots' will require making the necessary time and space to cultivate understanding and mutual respect, to redefine research relationships and objectives that acknowledge the concerns and support the aspirations of Indigenous collaborators, and to honor the contributions of Indigenous peoples—who, indisputably, hold a vital role in deciphering the meaning of plants within their own cultures.  4.2  ORGANISATION AND OBJECTIVES OF THIS C H A P T E R  The main purpose of this chapter is to raise and discuss some important biocultural issues that have emerged in contemporary ethnobotanical research, especially when conducted in an academic setting. I attempt to show the relevance of these issues to my dissertation research by drawing upon some examples from my research experiences over the last several years. It is my hope that the issues and examples discussed here will both serve as a useful resource to others who may face similar situations, and stimulate wider thought on the practical and philosophical aspects of ethnobotanical and other research involving cultural knowledge. I suggest that some of these issues confronting researchers in ethnobotany and related fields stemfromat least two sources: (i) multiple and sometimes conflicting obligations to stakeholders in the research that can lead to difficult ethical decisions. Here, I identify available resources to assist in decisionmaking, and I examine some of the inconsistencies and conflicts in these that can complicate the decision-making process, particularly in decisions relating to research involving the cultural knowledge of Indigenous societies; and (ii) lack of clarity on the foreseeable impacts of the research or research outcomes, especially on Indigenous communities whose members may have different value systems and priorities than those members of the academic community engaged in the research. In particular, I assess impacts of disseminating cultural knowledge through academic publication. The two areas of concern above form the two main sections of this chapter.  120 4.3  STAKEHOLDERS AND RESEARCH OBLIGATIONS  Ethnobotany, as a multidisciplinary field of inquiry, spans disciplines that may not otherwise have obvious interconnections—this is the field's greatest strength as well as one of its greatest challenges. With multiple disciplines come not only richness in viewpoints and a variety of tools to shape new questions and forge new solutions at both theoretical and practical levels, but also different approaches and perceptions about how and why ethnobotanical research ought (or ought not) to be conducted. Some of these differences parallel philosophical differences between the social and natural sciences, between pure and applied research, and between so-called traditional and scientific knowledge systems, while others relate to how researchers prioritise their research obligations to the various stakeholders in the research, as discussed below. If plant-people interrelationships are the basis of ethnobotanical inquiry, as noted by Ford (1994b) and as I have argued in Chapter 1, then the protection of these relationships, including the biological and cultural diversity upon which the relationships depend, is essential to the discipline . Therefore, an ethnobotanist has both professional and moral obligations to uphold 18  the best interests of the human and biological populations that are fundamental to the research. If the research is also part of an academic program, then the researcher has additional legal and moral obligations to the sponsoring institution (e.g., compliance with academic research and funding policies), and to the academic community and society in general (e.g., publication, dissemination of research findings and other contributions to the 'common knowledge' base). In these times of cutbacks and budget restrictions, scarce research funding has led to the increasingly common trend of academic research alliances with industrial or other private sponsors. Such alliances not only confer further obligations, opportunities and incentives on researchers, but often favour applied, economically-valued and/or patentable research. It is clear that multiple stakeholders in ethnobotanical studies culminate in multiple obligations for those who are engaged in the research. What happens when these obligations are in conflict? Whose interests take precedence? How does one decide? These questions may not have clear answers, but it is important that they are asked, and they comprise a recurring theme in this chapter.  This is intended as a pragmatic observation rather than a value-laden statement. Without a diversity of plants, peoples, and interactions between them, ethnobotany (by definition) simply could not exist.  121  These issues involve not only the research itself, but the outcomes of research as well. Typically, institutions and corporate or governmental sponsors have policies that clearly specify their interests in and rights to any products of research conducted with their support. For example, U.B.C. Policy #88 (Patents and Licensing) explicitly states that rights to the 19  protection or licensing of an invention or discovery that was made with university funds or facilities must be assigned to the university. The interests of other potential stakeholders in the research, such as non-governmental organisations, societies, or local communities are often less clearly specified, or assumed subsidiary to institutional or corporate interests. Indeed, at many universities, academic policies have largely co-evolved as 'corporate-friendly', encouraging industrial collaborations and accommodating corporate rights to the research outcomes. The same academic policies, however, do not necessarily support or accommodate community-based interests, rights and concerns about research. This suggests that those who conduct ethnobotanical research involving local communities within an academic setting may be faced with some difficult ethical choices or compromises in meeting obligations to the multiple stakeholders in their research. The following subsections outline some of the academic, legal, and moral policies or guidelines that have influenced my dissertation research, and challenged me to explore the questions related to research obligations that I have indicated above.  4.3.1  Academic Research Policy and Ethical Standards: The Tri-Council Statement on Ethical Conduct for Research Involving Humans  In Canada, the current Tri-Council Policy Statement: Ethical Conduct for Research Involving Humans can be seen as one response to the many ethical concerns raised by research  that involves people. This policy governs all research funded by the Natural Sciences and Engineering Research Council (NSERC), the Social Sciences and Humanities Research Council (SSHRC) and the Medical Research Council (MRC). While recognising the numerous benefits of research involving humans to individuals, groups and society as a whole, the Tri-Council U.B.C. Policy # 88 (Patents and Licensing) states that "If any member of faculty or staff, any student, or anyone connected with the University proposes to protect or license an invention or discovery in which the university facilities or funds administered by the University were used, a disclosure must be made to the University and the rights assigned to the University." U.B.C. Policy and Procedure Handbook, http://www.policy.ubc.ca/policy88.htm 19  122 Policy Statement requires commitment of researchers to respect some fundamental ethical  principles of research and human rights. These include: respect for human dignity; respect for free and informed consent; respect for vulnerable persons; respect for privacy and confidentiality; respect for justice and inclusiveness; and balancing of harms and benefits (i.e., minimising harm and maximising benefit). One section of the Tri-Council Policy Statement specifically deals with research involving Aboriginal peoples, although it has been noted explicitly that this section is "in abeyance" pending further discussions with Aboriginal representatives and affected communities (Tri-Council Policy Statement: Ethical Conduct for Research Involving Humans 1998; Michael McDonald, pers. comm. 2000 to Bannister, as cited in Bannister in press). Despite its shortcomings, this section of the Tri-Council Policy Statement acknowledges that research with Aboriginal communities involves added complexities, and suggests incorporation of "additional requirements [rather than separate standards] to ensure that the rights and interests of the community as a whole are respected". Some of these requirements have particular relevance to this chapter and they include: consideration of past harms to individuals and communities incurred by expropriation of cultural properties; respect for the culture, traditions and knowledge of the Aboriginal group; conceptualisation of the research as a partnership with the Aboriginal group; adjustment of the research to address the needs and concerns of the Aboriginal peoples involved; willingness to deposit data and other research outcomes in an agreed-upon repository; and the opportunity for the community to react and respond to research findings and publications . 20  As indicated in C h a p t e r 1, the Tri-Council Policy Statement also requires that proposals for research involving humans at Canadian academic institutions be subject to the approval of 21  an ethical review board. This ethical review process is intended to protect some fundamental 22  rights of the researchers, their sponsoring institutions, the "subjects" involved in the research  Tri-Council Policy Statement: Ethical Conduct for Research Involving Humans: Section 6: Research Involving Aboriginal Peoples, http://www.nserc.ca/programs/ethics/english/sec06.htm Specifically, ethical review is required for "activities involving human subjects in questionnaires, interviews, observations, testing, video, audio tapes, etc." (Behavioural Sciences Screening Committee for Research Involving Human Subjects 1995:1). "Subjects" is the antiquated language of the ethical review process and is not necessarily a term deemed appropriate in referring to current ethnobotanical research in which Indigenous peoples who are engaged in the research process are usually considered research participants or partners. It is noteworthy that one of the recommendations to the Tri-Council in the Draft Code of Ethical Conduct for Research Involving Humans (1997) 21  22  123 and the wider public for whose benefit publicly-funded research is ultimately conducted. As were the other academic researchers participating in the Secwepemc Ethnobotany Project, it was required that my research proposal undergo such an ethical review process. At U.B.C, the requirement for ethical review for non-clinical research and other studies involving humans is stipulated by Policy # 87 (Research) and governed by the Behavioural Sciences Screening 23  Committee for Research Involving Human Subjects. Along with other requirements, the U.B.C. ethical review process includes submission of a written Letter of Consent that must outline various items of concern and establish that the research will proceed with the informed consent  24  of the "subjects" of the study. In the case of my dissertation research, the "subjects" of my study were individual participants in the Secwepemc Ethnobotany Project and the Secwepemc Cultural Education Society (as the sponsoring organisation within the community). The wording of the Letter of Consent for my research was developed jointly with co-investigators of the Secwepemc Ethnobotany Project and the president of the Secwepemc Cultural Education Society. The process of drafting this Letter of Consent provided us an opportunity to discuss some of the positive and negative impacts of past research within Secwepemc territory, some general concerns about medicinal plant research and biological prospecting endeavours, and some specific concerns about how the data generated from my research would be used and by whom. While it was impossible to accurately predict my research outcomes in advance, and while the discovery of new products was not the objective of my research per se, based on my proposed methods and objectives we could reasonably assume that the data would incorporate some of the traditional and contemporary intellectual 'know-how' of community members, as well as some of the physical plant resources obtainedfromSecwepemc traditional territory. Proprietary rights over land and resources (which are inextricably tied to the cultural knowledge that governs land and resource use) historically have been held in common by Secwepemc people (Ignace 1998). It was in a spirit of protection of this generally egalitarian ethic of Secwepemc culture that the 25  was to replace research 'subjects' with 'participants'. However, this recommendation was not implemented in the Tri-Council Policy Statement. U.B.C. Policy and Procedure Handbook, http://www.policy.ubc.ca/policy87.htm Also known as "informed choice" which is defined by the National Council on Bioethics in Human Research (1996) as follows: "A morally valid choice concerning research participation is made: (1) by a competent person; (2) on the basis of adequate information concerning the nature and foreseeable consequences of the research (as these are known at the time the request is made) and all available alternatives; and (3) without controlling influences such as 'force, fraud, deceit, duress, over-reaching, or other ulterior forms of constraint or coercion' (Nuremberg Code)". 1 acknowledge that not all Aboriginal societies have (or had) an egalitarian ethic and that within any egalitarian society there may be (or may have been) differential access to resources or knowledge. 23  24  25  124 issues of proprietary rights to intellectual properties and natural resources as part of Secwepemc heritage were negotiated for my research. It was mutually agreed by the contracting parties 26  that the Letter of Consent governing my research on Secwepemc plants and peoples would serve dually as a research agreement, and include an explicit statement of ownership of Secwepemc knowledge and provisions for future use as follows: "the Secwepemc Nation has control over access to the traditional plant knowledge, as well as to potential development of any marketable products (such as drugs or pharmaceuticals) that may be discovered as a result of the traditional knowledge shared during the course of this research" (Bannister and the Secwepemc Cultural Education Society 1997:2; see Appendix A). Immediately apparent in this statement of ownership is its inconsistency with therightsof the University, as outlined in U.B.C. Policy #88 (Patents and Licensing) and as noted previously. It was interesting to me that the ethical review committee did not highlight this as a point of contention for the University. However, my statement of ownership is not inconsistent with the requirements and recommendations of the Tri-Council Policy Statement for research involving Aboriginal peoples (also noted previously), which forms the basis of the principles and guidelines underlying the ethical review process required by U.B.C. Policy # 87 (Research). Thus, some of the confusion stemming from multiple research obligations in ethnobotanical research conducted in an academic environment begins here, with potential contradictions in university research policies themselves, a point that will be explored in more detail in a subsequent section. In part, such contradictions can be understood as a consequence of transitions that are occurring in existing research policy and guidelines—transitions at institutional, local, national and international levels. It is important that the statement of ownership noted above and my research agreement in general are understood within the broader context of the current multilevel debate on protection of cultural knowledge and traditional resources of Indigenous peoples, outlined in the following sections. While current interpretations of 'heritage' are somewhat controversial, a definition with widespread international use is that put forward by Madame Erica-Irene Daes, Chairperson-Rapporteur of the United Nations Working Group on Aboriginal Populations: " 'Heritage' is everything that belongs to the distinct identity of a people and which is theirs to share, if they wish, with other peoples. It includes all of those things which international law regards as the creative production of human thought and craftsmanship, such as songs, stories, scientific knowledge and artworks. It also includes inheritances from the past and from nature, such as human remains, the natural features of the landscape and naturally-occurring species of plants and animals with which a people has long been connected" (as cited in Sanders, 1994:36). 26  125  4.3.2  International Law and Policy relating to Protection of Cultural Knowledge and Traditional Resources: The Convention on Biological Diversity  Recently, issues involving the protection of Aboriginal heritage have come to the forefront of both international law and Canadian law and policy. The heightened interest in and awareness of these issues may be attributed in part to the 1992 United Nations Conference on Environment and Development (the Rio Earth Summit) of which the Convention on Biological Diversity (CBD) was one of several outcomes. The objectives of the CBD are stated in Article 1 as follows: "the conservation of biological diversity, the sustainable use of its components and the fair and equitable sharing of the benefits arising out of the utilization of genetic resources, including by appropriate access to genetic resources and by appropriate transfer of relevant technologies, taking into account all rights over those resources and to technologies, and by appropriate funding" (Convention on Biological Diversity, 1992). Canada, as a signatory to the CBD, is legally and morally bound to implementation of all 42 articles comprising the CBD, but two of these articles (i.e., Article 8 "In-situ Conservation" and Article 10 "Sustainable Use of Components of Biological Diversity") are seen as particularly relevant to the protection of Aboriginal heritage. Article 8 (j) acknowledges: the importance of respecting, preserving and maintaining the knowledge, innovations and practices of Indigenous and local communities embodying traditional lifestyles for the conservation and sustainable use of biological diversity; promoting their wider application with the approval and involvement of the holders of such knowledge, innovations and practices; and encouraging the equitable sharing of benefits arising from the utilisation of such knowledge, innovations and practices. Article 10 (c) acknowledges the importance of protecting and encouraging customary use of biological resources in accordance with traditional cultural practices that are compatible with conservation or sustainable use requirements. The implementation of these articles has been fraught with problems, partly linked to lack of some fundamental definitions. The tasks of clarification and implementation have largely been left to subsequent CBD working groups and other initiatives . For example, the Canadian 27  For example, recommendations in the recent Report of thefirstmeeting of the Ad Hoc Open-ended InterSessional Working Group on the Implementation of Article 8 (j) and Related Provisions of the CBD  27  126 Working Group on Article 8 (j) has been addressing definitions of "indigenous knowledge"  28  and identifying potential protection strategies in Canada (Augustine 1997; Ford 1997; Lambrou 1997; Mann 1997a: 1). To assist with these goals, Mann (1997a) undertook a critical assessment of the use of Intellectual Property Rights (IPR) mechanisms in Canada for the protection of biodiversity and the cultural knowledge of Indigenous peoples. Mann's assessment included a series of six case studies (Mann 1997b), which included and critiqued my research agreement  29  as part of a "contractual approach to protecting knowledge in a knowledge building environment" (Mann 1997:133). These case studies were submitted to the Executive Secretary of the Ad Hoc Open-Ended Inter-sessional Working Group on Article 8 (j) and Related Provisions of the CBD, and have been integrated formally as part of the Canadian list of •J A  submissions within this Working Group (Executive Secretary of the Ad Hoc Open-Ended Intersessional Working Group on Article 8 (j) and Related Provisions of the Convention on Biological Diversity, 2000a). Mann's legal assessment was extremely informative. It pointed out a number of strengths and weaknesses in my research agreement and drew my attention to some important concerns about current research involving the cultural knowledge of Indigenous societies, in particular, the issue of third-party use of the research data once it is published. Mann (1997b) advised that without formal links to legal mechanisms of IPR protection (such as patent, trademark, copyright and licensing arrangements), after publication of my dissertation research, therightsof (UNEP/CBD/COP/5/5) include the following calls on other CBD Parties, governments, international organisations and organisations representing Indigenous peoples: to examine ways and means to establish guidelines for equitable sharing of benefits arising from the utilisation of knowledge, innovations and practices of Indigenous and local communities; to provide opportunities for Indigenous and local communities to identify their capacity needs; to include funding to build the capacity of communications in proposals and plans for projects carried out in Indigenous and local communities; to strengthen and build the capacity for communication among and between Indigenous peoples, local communities and Governments at local, national, regional and international levels; to use clearing-house mechanisms with direct participation and responsibility of Indigenous and local communities; to increase the use of local language for communication; and to provide case studies on methods and approaches for recording the knowledge, innovations and practices of Indigenous and local communities and for controlling such records. Mann (1997a: 1) suggested that "indigenous knowledge as a concept concerns information, understanding and knowledge that reflects symbiotic relationships between individuals, communities, generations, the physical environment and other living creatures, and the spiritual relationships of a people. [Indigenous knowledge] evolves as ecosystem and other factors change, but remains grounded in the more enduring aspects of identity, culture, generations and spirituality". Cassidy and Longford (n.d:30) defined traditional knowledge as "the expression of the human soul in all its aspects, as well as the foundation for economic, social and spiritual growth". The research agreement was included in Mann's (1997b) analysis with my consent and the consent of the Secwepemc Cultural Education Society. 28  2 9  127 ownership of the Secwepemc First Nation as stipulated in my research agreement were left open to definition by third-parties. It stated further that: "a considerable reliance on the acknowledgement by others of Aboriginal rights in the subsequent uses of traditional knowledge is implicit in this provision for its full operation to be effective in the manner it appears to have been intended" and concluded that "the longer term impacts of these arrangements will unfold in time" (Mann 1997:37). Mann's (1997) analysis compelled me to consider whether formal links to legal mechanisms of IPR protection were feasible in this case, and if so, were they appropriate? The following section summarises my assessment of these queries. 4.3.3  Legal Approaches to Protection of Cultural Knowledge and Traditional Resources: The Canadian Intellectual Property Rights System  The term 'intellectual property' refers to precisely defined kinds of knowledge that can be protected by law. The expression of an idea, or the embodiment of an idea in a process or product can be protected but the idea itself cannot. Intellectual property laws are seen as investments in new innovation; the main purposes of these laws are to promote creativity and the exchange of ideas. Creators and innovators are encouraged and rewarded through protection of ownership and commercial investment, but the rights of creators and innovators are weighed against those of consumers and other stakeholders (Cassidy and Longford n.d.). According to Coombe (1998:207), IPR mechanisms are "premised on a social bargain" that aims to strike a balance between granting ofrightsand imposing of responsibilities in exercising these rights. Patents are probably the most common means of intellectual property protection for chemical compounds of value from plants. In Canada, patents can be obtained for new technologies that include processes, structures and/or functions. The new technology must meet three basic criteria: 1) novelty (i.e., the invention must be new); 2) utility (i.e., it must be both functional and operative); and 3) inventive ingenuity (i.e., it must not be obvious to someone skilled in the trade). How do these patent criteria relate to my research? Without further analysis to chemically define the compositions of the crude extracts, the antimicrobial activity screening data (Chapter 2) likely would not qualify for patent protection, although if unique  Specific reference to my Letter of Consent is found in the CBD Document UNEP/CBD/WG8J/1/INF/2, paragraph 85.  3 0  128 traditional preparation methods were incorporated, some might merit a processual patent. Publication of the screening data, however, would make it freely available for further research that could result in patentable products. If this were the case, there would be no legal obligation to acknowledge the role of Secwepemc cultural knowledge in such findings, nor would the researchers have a legal obligation to inform the Secwepemc of their 'discoveries'. Compounds such as thiophene E, 16R, 23R-dihydrocycloartenone or inulinfrombalsamroot (Chapter 3) would not qualify for patent protection because they had previously been identified, thus they would fail the test of 'novelty'. On the other hand, compounds such as band 2 and 16R, 23Rdihydrocycloartenol (and perhaps some of the still unidentified antibacterial or other bioactive compounds found in the pitch, outer root bark, or leaves of balsamroot), likely would meet basic Canadian patent criteria. However, even if the tri-criteria for obtaining a patent in Canada were met for one or more of these compounds, then a patent would only be valid if the 'invention' was not made public before the patent application was filed . Thus, publication of the chemical structures, functions and/or the processes for obtaining them (e.g., such as submission of a doctoral thesis or a journal article) prior to patent application would preclude obtaining patent protection for these compounds (WWLIA 1995). Interestingly, precisely because of the stipulation of non-disclosure prior to patent application, publication itself is seen as a strategy that could be used to block others from obtaining a monopoly through patent protection—a process termed 'defensive publication' (Crucible Group 1994; Posey and Dutfield 1996). Public disclosure of the invention (at a level sufficient for reproduction of the invention) subsequent to acquiring a patent is a mandatory requirement of obtaining patent protection. This stipulation is to ensure that patented inventions serve as starting points for future innovation, and it has been estimated that approximately 90 % of patents are for improvements to existing patented inventions rather than for new inventions (WWLIA 1995). Thus, public disclosure of data (be it as a requirement of patent protection, through defensive publication to block patent protection, or by other means) still serves as a valuable resource for exploitation by third-parties, a point that I will return to subsequently.  In Canada, there is a one-year exception to this clause if the inventor, or someone who learned of the invention from the inventor, publicly discloses the invention then a patent application can still be filed within one year (WWLIA, 1995); however in practice, this one-year exception is considered a 'grey' area and better avoided if possible. 31  129 Trade secrets have been proposed as a means of protection of formulae, methods, techniques and other commercially valuable know-how related to medicinal plant knowledge (Barton 1994; Posey and Dutfield 1996; Peteru 1997; Stephenson 1999). Confidentiality is a key component in obtaining trade secret protection, however, which implies that even prior to publication, the entire ethnobotanical knowledge base already shared amongst researchers and  community members within the context of the Secwepemc Ethnobotany Project over several years is likely already disqualified from this sort of protection. Peteru (1997:98) commented that "establishing a trade secret may be elusive in a culture that emphasizes common over private property". Indeed, it might appear counter-productive or even antagonistic in an environment where reviving, rebuilding, and celebrating language, culture and traditional lifeways are priorities. Licensing agreements have been suggested as potential means of employing IPR protection for medicinal plant knowledge (Barton 1994; Stephenson 1999) since licensing effectively grant rights without relinquishing ownership. However, this may be of limited utility in practice since establishing a form of IPR (e.g., patent or trade secret) is obviously prerequisite to the licensing of IPR. Apparently the application of licensing arrangements in this context remains largely theoretical (David Stephenson, pers. comm. 2000). Copyright offers protection to subject matter in a physical form, so it is applicable to the physical expression of ideas but not to the protection of knowledge itself. This makes copyright largely irrelevant to the laboratory products of medicinal plant research, but of course it is of importance to the publication of research results. As mentioned previously, the cumulative results of research within the Secwepemc Ethnobotany Project (including results of the screening of Secwepemc plants for antimicrobial activity that was summarised in C h a p t e r 2) will be copublished by the Secwepemc Cultural Education Society (Turner et al. in preparation), and it is my understanding that the copyright of this material will remain with this society. As stipulated in my research agreement, the other publications resulting from my research are subject to review by the Secwepemc Cultural Education Society and so the issue of copyright will be dealt with on a case-by-case basis for these publications, as appropriate.  130  4.3.4  Assessing the Implications: Some of the 'Wrongs' with Intellectual Property Rights  There are several interesting (if unanswerable) questions that arise from my assessment. These questions further highlight the confusion resultingfromexisting research policies. First, relating to patents and licensing, even if all Canadian patent criteria could be met for one or more of the end-products of my research on balsamroot, how would U.B.C. Policy # 88 (Patents and Licensing) be accommodated? Would the rights of the University supersede those of the Secwepemc community, or would it be possible to negotiate a patent with thousands of inventors or owners represented ? Would my research agreement with the Secwepemc Cultural 32  Education Society be respected by the University as a legal contract or would my authority to enter into such an agreement without the consent of the University be called into question and thus invalidate the contract ? Was my authority to enter into an agreement implied by the 33  requirement to submit a Letter of Consent, and likewise, was the consent of the University  implied by approval of my Letter of Consent, given that this Letter of Consent was required by U.B.C. Policy # 87 (Research)? If not (and assuming that the requirement for a Letter of Consent is more than simply lip-service), shouldn't a "delegated signing authority" of the University be required to review, approve and endorse all Letters of Consent for University research? If in the end, a patent was pursued by the Secwepemc Nation, or jointly pursued by U.B.C. and the Secwepemc Nation, what would be the repercussions if the patent was subsequently challenged by a neighbouring Aboriginal group (or several groups) who traditionally used balsamroot in a similar fashion? The University administration may not yet be equipped to deal with the above questions in theory, let alone in practice, however, I suggest that the inconsistencies that I have identified in the policies merit attention. The queries currently are  Who is named as an inventor on a patent is determined by patent law, which requires that "an inventive contribution" be made to the patent. The inventorship of a patent is distinct from the ownership of a patent. The owner can be a person, institution, corporation or any other entity with signing authority (John Proffett, pers. comm. 2000). To meet these legal requirements, Cassidy and Longford (n.d.:9) suggested that an elder or other member of the Aboriginal community "who recorded the knowledge in fixed form" could serve as the designated "creator", while the right of ownership could be assigned to an entity representing the community. Cassidy and Longford (n.d.) also raised the related question of: what obligations to their community do individuals have when they use or share traditional knowledge? Although important, this question raises additional issues that are beyond the scope of the current discussion. U.B.C. research Polcy # 87 states: "Only the University itself has the legal authority to enter into contracts which are binding on the University; such contracts must be executed by a delegated signing officer". 33  131 under investigation by the Technology Transfer Manager for the Life Sciences of the University Industry Liaison Office on my behalf, as apparently there is no precedent at UBC where inconsistencies in policy have led to conflict between Aboriginal cultural knowledge and intellectual property. Furthermore, the University Industry Liaison Office representative indicated that if a patent were pursued, it would be seen as "a valuable opportunity for all parties involved". However, it would be essential that the University "maintained enough control over the invention to facilitate the patent process" (John Proffett, pers. comm. 2000). From this, it seems likely that clarification will require initiation of a specific case that challenges the policies in question. Without such clarification in advance of initiating a case, however, I suggest that it would be a rather risky decision for an Aboriginal community or any other stakeholder to pursue the process. Second, with respect to copyright and community review prior to submission of my publications, there are issues of censorship, suppression of academic freedom, and revisionism to consider. Furthermore, there is no guarantee that the same persons in elected positions of authority within the Secwepemc community who initially approved the research will still be in power when decisions on my research outcomes must be made. I realise that I shall have to deal with these issues should they arise and I accept this as part of fulfilling my research agreement. Amid the many questions outlined above, two conclusions can also be drawn from my assessment. First, publication plays a powerful role in maintaining legalrightsto intellectual property. Second, even if it were feasible to employ IPR protection for Secwepemc traditional plant knowledge and resources, subsequent to publication, these would still be left largely unprotected from potential appropriation, commodification and perhaps monopolisation of derived knowledge by enterprising individuals or organizations who chose not to acknowledge Aboriginal concerns in subsequent uses. Such concerns are, of course, far deeper and more complex than can be portrayed by a discussion limited to specific chemical properties of specific plants, but from my perspective these concerns are not easy to articulate. The Report of the Royal Commission for Aboriginal Peoples (1996) is helpful in this regard as it acknowledged a number of collective concerns of Aboriginal peoples for protection of their intellectual property and identified three general areas in need of appropriate protection in Canada. These are: assurance of appropriate use of Indigenous knowledge; portrayal of authentic identity of originators of the knowledge; and fair compensation for appropriate  132 commercial use of intellectual and cultural property (as in Mann 1997b). Numerous communitybased interests that are related to protection of Indigenous knowledge are also noted in the Report (1996). As summarised in Mann (1997b:27-28), these include: •  "preventing the loss of control over indigenous knowledge that could lead to its commercialization or to the identification of sacred sites by those who do not appreciate their significance;  •  control over revealing spiritual knowledge to outsiders who can destroy its sacredness or twist the meanings of the teachings;  •  inappropriate imitation of indigenous practices which are a misrepresentation of Aboriginal culture and weaken its teachings; protection from imitative works;  •  control over the integrity of [indigenous knowledge] by exercising control over who has access to it and how it can be used;  •  where it is appropriate to use the knowledge in a commercial setting which benefits others, a sharing of the benefits should be required;  •  a greater sense of protection of the sacredness associated with the knowledge; and proper protection of the collective rights as opposed to individual rights, including [indigenous knowledge] that might otherwise be defined to be in the public domain." All of these concerns are relevant (directly or indirectly) to the issues posed by  ethnobotanical research on traditional medicines and publication of resulting data. With respect to my assessment of IPR mechanisms, these concerns imply that the feasibility of employing IPR protection to Secwepemc medicinal plant knowledge and resources is a related but nevertheless separate issue as to whether or not these means are seen as appropriate in the first place. While I can address the former to some degree, I cannot determine the latter, which (to my understanding) has yet to be resolved by Secwepemc people themselves, but undoubtedly it is a difficult question to resolve. The threat of privatisation or inappropriate commercialisation of shared cultural knowledge and teachings deeply mars the spirit of sharing and poses a serious problem—are indigenous peoples being forced into considering IPR protection mechanisms simply as a defensive strategy to prevent others from employing them first?  133 I suggest that many of the fears and frustrations felt at the local level are directly related to misinformation and mistrust. With adequate time and effort, these may be alleviated, although it is apparent that an intercultural chasm exists, for example, as in concepts such as 'ownership' and 'value' of knowledge and resources. The framework underlying the legal and economic protection afforded by IPR regimes is suited to protection of inventions or discrete expressions of intellectual creativity with commercial potential. It is ill-equipped, however, to encompass the long-term, communal concept of guardianship and the concomitant protection of knowledge and resources as integral parts of cultural identity and lifeways. The significance of plants such as balsamroot to the Secwepemc people cannot be measured only in terms of uses and economic potentials for inulin, phytosterols and antibacterial or other interesting compounds—nor do these products alone represent the importance of chemical or other research on traditional processing methods of balsamroot. Any such products can attest to only one of many valuable aspects of the traditional knowledge shared, and the process of sharing by and with Secwepemc peoples. Ironically, it is the products and perhaps certain technical aspects of knowledge that qualify for protection under current legal regimes—even if to the detriment of the underlying relationships, processes and knowledge systems upon which the 'discoveries' are based. This situation presents a challenge that is paralleled in the field of political ecology to "recast[...] our understanding of wealth" (M'Gonigle 1999:22) and create "new political [and legal] contexts that will shift economic activity from linear to circular processes of wealth-generation, at which point economic 'values' will begin to have some relevant, contextual meaning" (M'Gonigle 1999:24). The conceptual and logistical limitations in existing IPR regimes have compelled Posey and Dutfield (1996) to advocate the establishment of a more inclusive set of protective rights that they have called traditional resource rights (TRR). This term builds on the concept of IPR protection and compensation but recognises that traditional resources are both tangible and intangible, and that the latter may be "inalienable or belonging to no human being" (Posey and Dutfield 1996:3). They explained the genesis of TRR as follows: "The term 'traditional' refers to the cherished practices, beliefs, customs, knowledge and cultural heritage of indigenous and local communities who live in close association with the Earth; 'resource' is used in its broadest sense to mean all knowledge and technology, esthetic and spiritual qualities, tangible and intangible sources that together, are deemed  134 by local communities to be necessary to ensure healthy and fulfilling lifestyles for present and future generations; and 'rights' refers to the basic inalienable guarantee to all human beings and the collective entities in which they choose to participate of the necessities to achieve and maintain the dignity and well-being of themselves, their predecessors, and their descendants" " (Posey and Dutfield 1996:3). The application of the term 'property' to the traditional resources of Indigenous communities is described as largely "inappropriate" by current CBD documents, since the concept of ownership and the ability to transfer ownership are contrary to the worldviews of many Indigenous peoples. Hence, the TRR concept has been embraced by the CBD Working Groups as a unifying conceptual approach "that more accurately reflects the views and concerns of indigenous and local communities and yet is entirely compatible with the requirements of the Convention on Biological Diversity, the International Undertaking on Plant Genetic Resources and the [World Trade Organisation] Agreement on Trade-Related Aspects of Intellectual Property Rights" (Executive Secretary of the Ad Hoc Open-Ended Inter-sessional Working Group on Article 8 (j) and Related Provisions of the CBD, 2000b paragraphs 49 and 50). Systems of IPR, as well as modern science and technology, have been described by some as means to perpetuate imperialism and the control and exploitation of lands, territories and resources of Indigenous peoples (Suva Statement 1995). For this reason, Posey (1999) cautioned that neither existing IPR nor the proposal of TRR should be used simply to reduce the cultural knowledge of Indigenous peoples into Eurocentric legal concepts—while IPR and TRR may serve as useful frameworks, to be effective they must be open to modification according to the legal systems and worldviews of Indigenous peoples, which are diverse. Posey (1999) also underscored the important point that an effective regime for the protection of cultural knowledge and resources also must support therightof Indigenous peoples to not have knowledge and resources privatised or commercialised. The commodification of cultural knowledge is  ;  inconsistent with, or even abhorrent to, many Indigenous worldviews. As stated in an international declaration on Indigenous Peoples' Knowledge and Property Rights: "For indigenous peoples, life is a common property which cannot be owned, commercialised and monopolised by individuals. Based on this worldview, indigenous peoples find it difficult to relate intellectual propertyrightsissues to their daily lives. Accordingly, the patenting of any life forms and processes is unacceptable to indigenous peoples" (Sabah Statement 1995; emphasis mine).  135 Brown (1998) offered a skeptical view of employing legal means to control cultural appropriation. He suggested also that there are much broader issues to consider in the development of legal regimes that "impose new limitations on the free exchange of information in the name of protecting ethnic minorities" (Brown 1998:195). He explained that while it may be possible to protect culture to some extent, culture can't be bounded, defined and protected from contamination, or commodified without doing it harm. Aboriginal societies borrow from other Aboriginal societies and from the majority societies in which they live. The concept of heritage protection as "a recovery of some unique authenticity" cannot be implemented without coming into conflict with such equally important human rights as "the right to innovate, communicate and share", nor is it easy to reconcile with the realities of Aboriginal engagement in majority society (Michael Brown, pers. comm. 2000).  4.3.5  Moral Approaches to Protection of Cultural Knowledge and Traditional Resources: The Role of International Initiatives, Local Initiatives and Professional Societies  Due to the many inadequacies and ambiguities in existing policies, contracts and legal mechanisms for the protection of cultural knowledge and resources of Indigenous peoples, in the last decade there has been a heavy reliance on moral imperatives and ethical standards. Numerous statements and international declarations by Indigenous peoples and/or concerned professionals highlight the depth and breadth of concerns relating to the protection of cultural knowledge, resources and Indigenous lifeways. These include, for example, the Declaration of Belem of the International Society of Ethnobiology (1988), the Kari-Oca Declaration and the Indigenous Peoples Earth Charter (1992), the Charter of the Indigenous-Tribal Peoples of the Tropical Forests (1992), the United Nations Draft Declaration on the Rights of Indigenous Peoples (1993), the Bellagio Declaration on Cultural Agency/Cultural Policy (1993), the Mataatua Declaration on Cultural and Intellectual Property Rights of Indigenous Peoples (1993), Recommendations from the Voices of the Earth Congress (1993), the Julayinbul Statement on Indigenous Intellectual Property Rights (1993), the Sabah Statement from the United Nations Development Programme Asian Consultation Workshop on the Protection and Conservation of  136 Indigenous "Knowledge (1995), and the Suva Statementfromthe United Nations Development Programme on Indigenous Peoples' Knowledge and Intellectual Property Rights (1995). While each of these statements and declarations makes unique contributions to the international debate on Indigenous intellectual and cultural property rights, there are many commonalties among them. The need for affirmation of basic human rights and recognition of past injustices are seen as essential starting points, and rights to self-determination are seen as fundamental to more equitable interactions. Recognition and respect for cultural knowledge, language, spirituality and all aspects of traditional lifeways, and protection of these from exploitation is viewed to be of paramount importance. A willingness exists to share cultural and intellectual properties with all humanity, for 'the common good', provided that the past, present and potential contributions of Indigenous peoples are acknowledged, and the fundamental rights of Indigenous peoples to define and control these properties are recognised and protected by the international community. An overriding responsibility to both ancestors and descendants and to continued relationships with the land and its inhabitants is expressed, and customary practices and teachings are seen as the most effective means of fulfilling these responsibilities. The role of Indigenous peoples as guardians, managers and perpetuators of biological diversity over millennia is promoted, and the exploitation of biological diversity for commercial or other purposes without the knowledge and consent of Indigenous peoples is condemned. Apparent in these individual documents.and in their collective overlap is the 'rights-oriented' approach that generates a unifying vocabulary for voicing Indigenous concerns. Saunders (1994:21) pointed out that Indigenous peoples' focus on rights has been criticised "as an inadequateframeworkfor advancing the actual situation of particular groups". However, he argued also that "it is compelling and it is available. The dominant society values rights. The gaining of rights could be followed by other gains" (Saunders 1994:21). Similar sentiments, concerns and rights discourse expressed in these international initiatives are also being asserted in local approaches to cultural protection in British Columbia, as indicated by the Skeetchestn (Deadman's Creek, Secwepemc Nation) Territorial Heritage Conservation Law (1998), the St'at'imc Nation (Mount Currie) Statement of Proprietary Rights (2000), and a recent statement issued by the Union of British Columbia Indian Chiefs (Vancouver) Protecting Knowledge: Traditional Resource Rights in the New Millennium  137 Conference (2000). Copies of the latter two statements are included in Appendix D°\ Essentially, local initiatives such as these are laying out 'the law of the land'—Aboriginal laws that govern conduct within traditional Aboriginal territories—and these statements or promulgations will certainly be key in shaping the general and specific ways that ethnobotanical and other research is conducted with and within Aboriginal communities in British Columbia. As Chief Ron Ignace (Skeetchestn) explained about the Skeetchestn Territorial Heritage Conservation Law during a recent Roundtable discussion on Indigenous Intellectual and Natural Resource Property Rights (hosted by the Society of Ethnobiology) , if researchers, companies or 35  other proponents don't abide by Aboriginal laws when working in traditional territories, "then they won't be working with us" (Ron Ignace, pers. comm. 2000). As part of the same Roundtable discussion, Angelo Joaquin, Jr. (Tohono O'odham), Executive Director of Native Seeds/SEARCH (Tucson AZ), explained that the shift in balance of power occurring in the U.S.A. is largely due to the profits to Native American Bands generatedfromcasinos. The money from "gaming" is giving Native Americans an economic basis for self-sufficiency and the freedom to choose how and with whom they will do business: "Before we had'gaming, we never had that choice. As researchers, you need to know that things are different now—now that we do have a choice" (Angelo Joaquin, Jr., pers. comm. 2000). Collectively, these local, regional, and international statements and declarations indicate that ethnobotanical and other research involving the cultural knowledge and traditional resources of Indigenous peoples is poised on the edge of an historic period of transition. Precisely how future ethnobotanical research perspectives and practices will be altered formally is not yet clear. In the meantime, many researchers are left with uncertainty about how to proceed in the present. It is becoming increasingly obvious that some of the standard ways of 'doing business' are not necessarily okay anymore, but there is little in the way of existing precedent to comfortably guide change—and those who do attempt to meet these new challenges do so with significant risk of criticism (albeit for different reasons) from the various stakeholders in the research, including their institutions, their sponsors, their professions, and (local and international) Indigenous communities. Nicholas and Andrews (1997:226) described a parallel situation in archaeology in British Columbia: "Being on the 'leading edge' of anything may carry some Permission to include a copy of the Skeetchestn Territorial Heritage Conservation Law (1988) was denied as it is not considered a public document by the Skeetchestn Band Council. 34  138 status (real or imagined). However, as many of us have discovered, it can also be a very uncomfortable if not dangerous place to be, and there is the constant danger of falling off, of doing the wrong thing." To assist with resolving these complex issues and ethical dilemmas, many nonprofit organisations, professional societies, and research institutes (including botanical gardens ) have 36  begun to develop (or revise) Codes of Conduct and/or Codes of Ethics that reflect the earnest need for standards of practice that promote the conservation of biological and cultural diversity, and are acceptable and sensitive to the current concerns of Indigenous and local societies, as well as to other stakeholders in the research. Embodying these objectives in a written form that is acceptable to all those involved in the research process, however, is not necessarily an easy or straightforward task. The ten-year negotiation process involving hundreds of people in developing and ratifying a Code of Ethics for the International Society of Ethnobiology (1998) attests to this fact. Furthermore, the very concept of developing an ethical code has met with resistance in some professional societies (e.g., the American Society of Pharmacognosy and the Society of Economic Botany), as some researchers claimed not to see the relevance of ethics to their practical field work while others were opposed to the imposition of authoritarian or inflexible rules, or limits on their academic freedom (as noted in Laird and Posey in press). Others have suggested that the process of discussing, developing and revising acceptable research guidelines and professional Codes of Ethics is just as important for individual researchers and for the evolution of academic disciplines as the resulting end product (Cassell and Jacobs 1987; Laird and Posey in press). In this respect, I can affirm the significant impact on my research perspectives and practices of being involved in discussing and drafting the final version of the Code of Ethics for the International Society of Ethnobiology (1998). While the end product may read as a unified collection of 15 principles chosen to guide ethnobiological research conduct, even after ten years in progress, the final wording of this Code of Ethics was agreed upon only after several more days of deliberation by a large group of diverse people who had to push through many differences in opinions and perspectives to find their commonalty— the commitment that they shared to improving ethnobiological research. This Code of Ethics  The 23 Annual Conference of the Society of Ethnobiology, Ann Arbor, MI. March 29, 2000. As many botanical gardens have established bioprospecting collaborations, policies have been needed to establish the proprietary rights of source countries that provided the botanical materials (Dove 1998). 35 36  rd  139 will no doubt continue to evolve as it is revisited at future meetings of the Society. A copy of the Code of Ethics is included in Appendix E. Involvement in this type of process is also invaluable from the perspective of compliance, since enforcement of most (if not all) professional Codes of Ethics is logistically impossible. Some societies, such as the American Anthropological Association, that attempted to hear grievances and enforce compliance were cautioned against this approach due to legal implications and excessive costs that would be incurred (Catherine Fowler, pers. comm. 1999; Laird and Posey in press). Thus, the objectives of most professional Codes of Ethics are education-oriented, and implementation relies largely on the awareness and voluntary compliance of individuals, while drawing also on the power of moral suasion. However, here again, compliance can be complicated by the multidisciplinary nature of ethnobotany—when one belongs to multiple professional societies with Codes of Ethics that are not necessarily consistent, to which should one adhered In the following section, I suggest that examining the impacts of research (existent and potential) may be of assistance in resolving some of the  questions that have been raised in this section. As a preface to the following section, it should be noted that my focus here is more on the negative and unknown consequences of ethnobotanical research than on its benefits. For this reason, I would like to acknowledge and express my utmost admiration and appreciation for the many researchers and local peoples, both Indigenous and non-indigenous, who have engaged respectfully in ethnobotanical and related research, worked unceasingly for biological and cultural conservation, and brought the discipline of ethnobotanyfromobscurity to high regard in both public awareness and academic contributions. Ethnobotanical research has been an important and effective medium for inter- and intra-cultural conservation efforts. For example, Elder Mary Thomas expressed that her motivation to share her plant knowledge with others comes from her deep concerns about the environment. As she explained one day while we were on an outing to investigate the status of local populations of Erythronium grandiflorum (yellow avalanche lily): "The way I see it, the more people who see and admire one of these [pointing to a lily], the better chance of preserving it" (Mary Thomas, pers. comm. 1997). The large body of information that exists about the cultural knowledge, languages, traditional beliefs, customs and lifeways of past and present Indigenous peoples, in locations through out the world, represents decades of dedication and commitment by countless  140  individuals. The documentation of such knowledge has been an invaluable resource to many Indigenous and non-indigenous peoples alike, especially at a time in history when in many cases there are few individuals, often aged, who have managed to retain fluency in their language and an integrated understanding of traditional lifeways. The material in the following section is not intended as a critique of these invaluable research contributions, and while some material necessarily draws upon research of the past to assess the present impacts of ethnobotanical and related research, this should by no means be taken as a criticism of that research done in the past. 4.4  IMPACTS OF RESEARCH  We want the maximum good per person, but what is good? To one person it is wilderness, to another it is ski lodges for thousands. To one it is estuaries to nourish ducks for hunters to shoot; to another it is factory land. Comparing one good with another is, we usually say, impossible because goods are incommensurable. Incommensurables cannot be compared.  —Garrett Hardin (1968:1243)  The primary difficulty in meeting obligations to different stakeholders in ethnobotanical research may not lie in the potentially conflicting policies, protocols or ethical standards that govern research, although as I have indicated, some of the inconsistencies in these are sufficiently disconcerting. The greater challenge may be in developing a full understanding of the existent and potential impacts of research and research outcomes—an understanding that takes into account the positive, negative and unknown consequences that are experienced or perceived (sometimes in very different ways) by the various stakeholders. These various perceptions, perspectives and experiences suggest, however, that in many cases, weighing the impacts of research is like comparing Hardin's (1968:1243) "incommensurables". When faced with multiple and perhaps divergent perspectives, a unifying place to begin is with the commonalties. A common thread that is woven through all of the institutional, international, local, professional, ethical and legal resources to guide decision-making that have been mentioned so far is the principle of informed consent. All of these resources agree that full disclosure of the impacts of research—including known benefits, harms and risks, along with the prediction of their potentialities—is necessary to ensure that research will proceed with the  1 4 1  informed consent of those people directly involved . However, exactly which evidence is 37  required to establish that informed consent has been given is not necessarily agreed—and related to this is a question raised by Nicholas and Andrews (1997:8): "who has the right [or authority] to speak for whom, and of what"? As an adequate discussion of this important aspect is beyond the scope of this chapter, I will instead focus on an equally important (and controversial) issue that tends to receive less attention, the question of what constitutes a 'benefit' or 'harm'. Since one individual's (or society's) benefit may be another individual's (or society's) harm, this makes disclosure or prediction of these subject to both perspective and context (Bannister and Barrett in press). A critical question arises in considering the impacts of research: how are benefits and harms defined and by whom?  Too often, it seems that benefits and harms are projected by researchers who are not members of the affected community, and for obvious reasons, research proposals tend to emphasise benefits over harms. As a consequence, definitions of harm may be limited to those directly foreseeable or precedented, grounded in singular cultural perspectives, or not recognised or articulated at all. The multidisciplinary nature of ethnobotany likely accentuates this problem, as researchers from different academic backgrounds may have varying degrees of experience, awareness or interest in the biocultural issues related to these deficits.  4.4.1  The Role of Ethnobotany in the Search for Medicines Let us not forget that progress is an optional goal, not an unconditional commitment... Let us also remember that a slower progress in the conquest of disease would not threaten society ...but that society would indeed be threatened by the erosion of those moral values whose loss, possibly caused by too ruthless a pursuit of scientific progress, would make its most dazzling triumphs not worth saving. —Hans Jonas (1970:28)  While the issues raised by my questions above are relevant to a number of different research areas, current applications for ethnobotanical research on medicinal plants provide an The Draft Code of Ethical Conduct for Research Involving Humans (1997) identified informed consent as a process that continues throughout the research project. 3 7  142 opportunity to explore the pertinence of these issues to my dissertation research. In particular, I return to the example of product-oriented research conducted as ethnobotany or the use of ethnobotanical research in biological prospecting endeavours, practices that have caught public attention and somewhat polarised the discipline of ethnobotany. Such programs can claim potential benefits for general society in areas of medicine (i.e., finding new cures), economic development (i.e., stimulating new market economies) and ecological integrity (i.e., providing new rationales for keeping forest ecosystems intact). However, some ethnobotanists have expressed concern about the involvement of ethnobotany in the "economically motivated search for new drugs" (Berlin and Berlin 1994:247) since drug discovery "cannot be divorcedfromthe broader objective of promoting the survival of biological and cultural diversity" (Martin 1994:228; emphasis mine). Others have highlighted advantages for local communities such as capacity-building and benefit-sharing (King et al. 1996; Carlson et al. submitted). Some of the reservations about biological prospecting in general may be related to violations of what Longino (1983:9) referred to as "folk traditions in science" that are "connected with the constitutive ideal of truth as well as with considerations of justice". According to Longino's explanation of these folk traditions, "scientists do not or ought not profit commerciallyfromtheir scientific activity [as] no one scientist should profitfromdiscoveries made possible by the work of others". The "epistemological basis" here is that having a stake in the outcome of research might bias research design or interpretation, or (as Longino 1983:14 put it) make the data 'suspect' when: "[t]he lure of discovery and the lure of profit dangle together". Societal and general health benefits aside, biological prospecting often is perceived as a 38  'business' first and foremost, and assumed by many as a potentially lucrative business at that . Understandably, while the association of biological prospecting with ethnobotanical research may be seen as an opportunity by some, it is difficult to reconcile for others. Regardless of existing qualms, ethnobotanical-based selection has gained in popularity as one of several means to identify which plants among the 250,000-500,000 existing species have a higher than random probability of containing medically-relevant compounds of interest. Issues related to over-harvesting, ecosystem degradation, and access to resources are relevant to all of  Here, I emphasise the widespread perception of biological prospecting endeavors but I acknowledge also that not all biological prospecting initiatives involve commercial objectives.  143 the various methods of plant selection used , but there are additional considerations with the ethnobotanical approach to drug discovery. This approach uses medicinal plant knowledge of Indigenous cultures, either directly (through ethnobotanical research) or indirectly (through access to published ethnobotanical data) as a means of "prescreening" plants for relevant compounds (Wills and Lipsey 1999:31). Ethnobotanical research conducted explicitly for this purpose would be subject to an assessment (such as an ethical review) to satisfy the governing authorities that the research was proceeding with the awareness and consent of the appropriate people or communities involved. In contrast, use of the published ethnobotanical literature (e.g., the use of my antimicrobial screening data by other researchers after it is published) by would not be subject to any regulation, as there are no restrictions on use of information in the public domain. Wills and Lipsey (1999:31) claimed that whether the information is obtained directly from Indigenous sources or indirectlyfromthe public domain is "irrelevant to the legal claim of proprietary first nations' rights in traditional knowledge". They argued that when the ethnobotanical literature is used as the basis for producing, patenting and selling products based on Indigenous knowledge, the proponents "are appropriating and using this knowledge in the same way people use pirated computer software" (Wills and Lipsey 1999:31). However, ethical and legal approaches to issues may not lead to the same conclusions. While Wills and Lipsey's (1999) argument may hold weight on moral grounds, under current federal law there is no legal basis for such a claim if the knowledge is not formally protected by patent or other forms of IPR (as discussed in Chapter 3). The public domain, simply put, isfreegame forfreeenterprise. The unprecedented interest in medicinal plant knowledge of Indigenous societies and the consequent rise in ethnobotanical literature-based plant screening programs have been cause for alarm by Indigenous peoples and researchers worldwide. Concerns about loss of control over cultural knowledge and related biological resources have escalated as 'Indigenous knowledge' (reified) has become a much sought-after component of research, development and commercialisation efforts by non-indigenous peoples. Indeed, in British Columbia, "the In addition to ethnobotanical or ethno medicinal selection (i.e., using the medicinal plant knowledge of Indigenous peoples to predict which species will be of interest), methods of plant selection generally include: random or empirical selection (i.e., collecting what is readily available); taxonomic selection (i.e., sampling a cross-section of representatives from the entire plant kingdom and then concentrating efforts on taxa related to the species of interest); and phytochemical or chemotaxonomic selection (i.e., focussing on a group of related plant compounds) (Cordell 1995; Bonis 1997). 39  144 tremendous interest in and demand for [natural health products] and the need to accommodate both the industry and consumers in this area", as noted by Volpe (1998:3) have led some to promote provincial regulations for harvesting of non-timber forest products and federal regulations for the use and commercialisation of natural health products (De Geus 1995; Wills and Lipsey 1999; Volpe 1998) . It is notable that the Shuswap Nation Tribal Council has 40  intervened with the provincial government for acknowledgement of Aboriginal rights to control and manage the non-timber forest resources within their traditional territory (Natural Resources Coordinator for the Shuswap Nation Tribal Council, pers. comm. 1999). If some of the most significant rights of Indigenous peoples derive from physical control over traditional plant resources and the knowledge relating to these plants (Barton 1994), then publication of plant lists, plant locations, and traditional knowledge related to medicinal preparation and use may significantly decrease this control (Bannister and Barrett in press). The ethnobotanical literature (i.e., in particular, journal articles, databases, and field collections) serves as a major source of information and ideas for researchers and industries with commercial objectives (Posey and Dutfield 1996). Often, as Wills and Lipsey (1999) indicated, these are third-parties that have had no direct contact with the Indigenous communities whose knowledge they are appropriating, which brings up additional concerns about accuracy of information, appropriateness of use, and adequate acknowledgement of the Indigenous sources. Posey and Dutfield (1996) note also that access to such information is typically not conditional on recognition of the rights to intellectual property of Indigenous peoples, nor is any form of permission or compensation legally required. Ironically, some have criticised contributors to the ethnobotanical literature for not providing enough details on specific uses and preparations in the reporting of traditional medicinal plant information (Farnsworth, 1994b; and as discussed in Cotton, 1996). Kaplan (2000) pointed out that recording, publishing and making the knowledge available to those who share an interest in it is a form of respect by the scientific community. He suggested also that "given the low probability of finding a new plant remedy with commercial potential, .. .the So far, it is unclear how government regulations will accommodate issues of Aboriginal access, use and control. For example, the recent Report of the Standing Committee on Health (Volpe 1998:66) recommended that a regulatory framework for natural health products should "not apply to products prepared by Aboriginal Healers where the product was prepared for an individual patient", while the legislation would apply in any attempt at commercialisation of traditional remedies. However, situations falling between the extremes of single patient treatment and commercialisation (i.e., presumably common situations) were not addressed. 4 0  145 cultural value of recording.. .knowledge in the published record is likely to be greater for an indigenous people than is any economic value resulting from the disposition of intellectual property rights" (Kaplan 2000:9). However, others support the view that Indigenous peoples, as research participants, should have the right to decide what is made public and what is not (Cunningham, 1996). This highlights important issues surrounding publication—which may be viewed as a 'double-edged sword'. On one side, publication serves both as a means for public acknowledgement of the source of cultural knowledge and resources, and for sharing information that may directly or indirectly benefit humankind. On the other side, publication also aids the exploitation and appropriation of knowledge and resources as they are sufficiently divorced from their cultural context, such that the research may be seen as ".. .a vehicle for appropriation—not protection—of indigenous knowledge" (Posey 1998:242). The issues raised by this situation have had a significant impact on my dissertation research and publication decisions. The issues also have significant consequences for ongoing relationships between ethnobotanical researchers and Indigenous community members, as well as for the future of the discipline. Ethnobotanical studies that detail information on plant species, preparation methods and medicinal uses by Indigenous societies usually do so as part of establishing a broader, context-dependent framework that is necessary to understand the often complex reciprocal relationships that exist between plants and humans (such as those explored in Chapter 3 for balsamroot as a food and medicine in the Secwepemc culture). It is widely agreed that the cultural knowledge of Indigenous societies cannot be divorcedfromits cultural context without concurrent losses in meaning that, in turn, may lead to culturally inappropriate uses. Inclusion of cultural and biological contexts is essential to the understanding of and respect for the "comprehensiveness of indigenous knowledge as a knowledge system" (Mann 1997a:29; emphasis mine), and may assist in generating awareness that "to preserve the world's traditional knowledge" (Linden 1991:54) is not an ex situ pursuit but is utterly dependent on the existence of the cultures that embody the knowledge. Given that no knowledge or information in the public domain is exempt from third-party exploitation, however, after research is published, its original intention may be largely irrelevant to its net contribution in creating "a colonizing archive of data" (Kelm, 1998:120), and thus opportunities for subsequent exploitation. Posey (1999:225) summarised some of the repercussions for ethnoscientists as follows: "Scientific data banks have become the 'mines' for 'biodiversity prospecting.' The publishing of information,  146 traditionally the hallmark of academic success, has become a superhighway for transporting restricted (or even sacred) information into the unprotectable 'public domain'. As a result, ethnoecologists are increasingly seen by indigenous, traditional, and local communities not as allies but as instruments of corporate interests." Obviously, this has significant implications for future research. Brouwer (1998) suggested that part of the problem lies in the separation of technical knowledge of Indigenous cultures from broader Indigenous knowledge systems. Salmon (1996) articulated the problem as follows: "Western scientists accept plant knowledge as something that first must be elevated to raw data that can then be analyzed and synthesized. The problem is that, removed from its source, the knowledge is incomplete. It is only a whisper of its former self." Salmon commented further that: "Such knowledge undergoes changes as it is separated from its source. Both indigenous peoples and others have yet to fully comprehend the effects of appropriating knowledge" (Salmon, 1996:70). This problem is compounded when Indigenous peoples from whom the knowledge originated are unaware of the development of the knowledge or related resources, as is often the case when cultural knowledge is appropriated by third-parties (Bannister and Barrett in press). Thus, it is clear that the indirect impacts of research outcomes are as important to this debate as the direct impacts of the research itself. Some of the implications and issues related to publication in particular are discussed further in the next subsection. 4.4.2  The Publication Dilemma In order to secure important collective goods—scientific knowledge and advances in medicine—individuals are put in harms way. The moral challenge is to protect the rights and interests of these individuals while enabling and encouraging the advancement ofscience.  —Ruth R. Faden (1996:xxi) The so-called 'publish or perish' predicament is a familiar part of academic culture. Publication is a fundamental tenet of the scientific ethos and forms part of the primary basis for academic reward and advancement. Sharing data and conclusions with the scientific and wider • communities is, in fact, a central criterion for 'ethical' scientific research, and essential to the  147 expansion of the "idea-space in which we all move" called the public domain (Michael Brown, pers. comm. 2000). I suggest that more difficult still is the added 'publish and perish' dilemma that ethnobotanical and other researchers find themselves in today when faced with the decision of publishing data that is considered controversial or culturally-sensitive, such as medicinal plant data (e.g., data in Chapters 2 and 3). In this case, publication may lead to endangerment of the trust underlying the very basis of the research relationship, and to the neglect of moral and professional obligations and the rights of Indigenous peoples (as previously outlined). However, not publishing may curtail an academic career, and may result in disregard for legal obligations to one's institution and moral obligations to society. The tension created by this dilemma is intensified in the absence of adequate guidelines for decision-making. Moral justification undoubtedly can lead in either direction. We may recognise that scientific 'progress' involves the taking of risks, and that these risks can fall on individuals who don't stand to benefit directlyfromthe research or knowledge gained (Faden 1996). However, we must realise also that this rationale can serve as rationalisation. A utilitarian-based approach to maximising benefits and minimising harms will always decide in favour of 'the greater good', i.e., dominant society, because by definition, a minority group can never constitute a majority so utilitarianism is always at the expense of the best interest of the minority group if that group's interest is not consistent with the majority. Jonas (1970) offers an alternative perspective that is not based on maximum benefits per se but on what society can or cannot afford  (e.g.,  'averting a disaster' carries greater weight than 'promoting a good' and  'saving society' is of higher rank than 'improving society'): "Our descendants have a right to be left an unplundered planet; they do not have a right to new miracle cures. We have sinned against them, if by our doing we have destroyed their inheritance—which we are doing at full blast; we have not sinned against them, if by the time they come around arthritis has not yet been conquered" (Jonas 1970:14). I would argue that, if, as Nicholas (1991:6) claimed, "diversity of life is the true legacy of humankind" and if cultural and biological diversity are considered as part of the "inheritance" that Jonas' (1970:14) noted, then it is in the best interest of society to protect the integrity of these, rather than to exploit them, if exploitation is to their detriment. The decision-making process, however, is not limited simply to considering benefits and harms; risks and uncertainties also must be are factored in. The issues and dilemmas raised by publication of Indigenous medicinal plant knowledge are intensified significantly by the  148 uncertainties about effects of knowledge dissemination on Indigenous communities whose value systems, priorities, expectations, and time frames may differ from those of the academic community engaged in the research. It is accepted that the impacts of research, be they positive or negative, can not always be foreseen when the research is initiated, but it is expected that a reasonable effort will be made to foresee, forestall and disclose adverse effects. More difficult, if not impossible, is prediction of all downstream effects that might arise as a direct or indirect consequence of the original research, and problems that do arise are likely to be case specific. In this regard, Brown (1998:200) pointed out that "the Law of Unintended Consequences reigns supreme; there will always be unforeseen effects, both good and bad, when information enters the public domain." Perhaps the question here that needs to be addressed is: where does researcher responsibdity end? It seems this is a question largely left to the individual to  determine. I suggest that rather than serving as rationalisation for maintaining the status quo, Brown's (1998) observation above can be taken as an invitation to acknowledge and address the powerful role that publication can play in issues of control. This is particularly pertinent as mounting evidence suggests that Indigenous peoples are increasingly uncomfortable with the sharing of cultural knowledge outside their communities due to uncertainty in what dissemination of that knowledge will mean in the longer term. In fact, the potential of harm being sustained by Indigenous communities may justify renewed consideration of the norms of scientific publication. Certainly guidelines are needed to acknowledge the social, political and economic complexities involved in dissemination of cultural knowledge, and to assist in ethical decision making in light of the uncertainties presented by publication (Bannister and Barrett in press). 4.4.3  A Precautionary Approach to Publication The Precautionary Principle has been suggested as one such framework for guiding  decision-making given the uncertainty in ethnobotanical research and publication (Bannister and Barrett in press). This ethical and legal principle was originally founded in German environmental policies (Boehmer-Christiansen 1994) but has since gained widespread  149 international application and support in areas of biodiversity and human health (RIS et al. 41  1999) biological conservation (Backes and Verschuuren 1998), ecological restoration (Noss 1998), agricultural biotechnology (Barrett 1999) and in ethnobiological research (Bannister and Barrett in press; Code of Ethics of the International Society of Ethnobiology 1998). The Precautionary Principle embodies a 'when in doubt, err on the side of caution' approach wherein measures to prevent damage or harm should not be deferred on the grounds that insufficient scientific evidence exists about the effects of a particular activity (Backes and Verschuuren 1998). Various interpretations of the principle have incited much controversy, and Foster et al. (2000) suggested that this variability presents the greatest challenge to use of the Precautionary Principle as a policy tool. On the other hand, its wide-spread use is a direct reflection of the adaptability of the principle, which, if carefully clarified for a given application, offers a valuable perspective for consideration. The Precautionary Principle may have relevance in the area of ethnobotanical research and publication of cultural knowledge, in light of uncertainties and perceived risks in dissemination of knowledge, and consequences of these for formal Indigenous claims to IPR or general concerns about knowledge and resource protection. The Precautionary Principle does not lay out a set of specific rules to be followed; rather it offers a framework to assist in decision-making, based on a set of guiding principles that generally includes: (i) protection of the environment and recognition of intrinsic value; (ii) recognition of uncertainty; (iii) proactive and anticipatory action; (iv) reversal in burden of proof; and sometimes (v) cost-effectiveness. An in-depth application of these principles to ethnobiological research and publication issues has been discussed elsewhere (Bannister and Barrett in press). However, the essence of points (ii) and (iii) may be of particular relevance to the publication issues raised, namely that while the consequences of our actions can not always be predicted, the potential of harmful outcomes compels us to consider proactive approaches for preventing harm rather than to rely strictly on compensatory measures after harm has occurred. 41  Some examples of international applications of the Precautionary Principle include the following (as cited in Bannister and Barrett in press): Protocol on Substances that Deplete the Ozone Layer/Montreal Protocol (1987); Nordic Council's International Conference on Pollution of the Seas (1989); Bergen Declaration on Sustainable Development (1990); Rio Declaration on Environment and Development (1992); United Nations Framework Convention on Climate Change (1992); Treaty on the European Union (Maastricht Treaty) (1994); Cartagena Protocol on Biosafety (2000); and  150 Thus, while benefit-sharing and up-front compensation are important considerations in ethnobotanical research, these are "reactive" rather than "proactive" measures that may even prove inadequate in the long-term given the uncertainty of research and publication impacts (Bannister and Barrett in press). Furthermore, an obligation to share benefits accrued from research based on cultural knowledge typically results only when direct links between local communities and researchers exist. Research based on published ethnomedicinal studies is often perceived to be free of obligation, even if cultural knowledge was key to subsequent findings, since the knowledge was already in the public domain. In this way, publication serves to circumvent the spirit and obligations of benefit-sharing. Similarly, publication circumvents third-party accountability and just compensation for harm. Without engaging in direct interaction with Indigenous communities, in many cases it is likely that a third-party would not even be aware of the 'kinds of harm' that could stem from knowledge appropriation. While harm cannot always be accurately predicted in advance of initiating research, a commitment to ongoing interactive dialogue would be invaluable in better understanding the forms that harm can take, and thus would assist in prediction and prevention of similar harms in future (Bannister and Barrett in press). Affected communities ought to have a key role in co-defining harm since perceptions of harm are socially and culturally influenced. Stevenson (1996:283) stressed the importance of having the Indigenous communities that are most directly affected by proposed research or developments "identify what is important to them and why". Soliciting the experiences and insights of Aboriginal peoples who have already experienced such impacts would assist in prediction, assessment and management of specific and cumulative research impacts on others. Stevenson observed that (1996:283) "[b]y documenting their concerns, aboriginal people participate directly and effectively in impact prediction and assessment", which grants Aboriginal peoples a positive right and confers an active responsibility. Participatory and inclusive processes may assist in reducing uncertainties that resultfromsingular or narrow perspectives. If part of the current backlash regarding appropriation of cultural knowledge has resultedfroman accumulation of many harms unattended, perhaps the cumulative impact of foreseeing and addressing potential harms in future will likewise forestall future injustices. Such undertakings will, however, require adequate time and resources, thus success will require that externally imposed research agendas and timelines the main tenant is stated in the preamble of the Convention on Biological Diversity (1992).  151  be adjusted, and resources to enable active community participation be included in research budgets. A central concern about a precautionary approach to ethnobotanical research and publication may be the implication that no research or publication on traditional plant medicines should proceed unless or until there is conclusive proof that there are no adverse effects, be they direct or indirect—the principle of in dubio pro natura . Taken to an extreme, of course, such 42  an interpretation of the Precautionary Principle could lead to a form of paralysis, and be reasonable grounds for dismissal (i.e., while we debate the pros and cons of our actions and inactions, the world waits for much needed medicines and other physical, social and economic remedies). Obviously there can never be such conclusive proof, and further still, the criteria for 'proof may change over time. In some cases the reverse argument likely would be true, i.e., cultural harm may stem from not promoting and disseminating cultural knowledge. However, I should re-emphasise that principles are not rules but worthy considerations with considerable flexibility, to be taken into account in making decisions. The merit of the approach suggested here (following Bannister and Barrett in press) is in the #c//o«-oriented nature of the Precautionary Principle, which first compels us to acknowledge problems or limitations and then challenges us to seek out alternative approaches for redress (i.e., in the interest of the greater good, the price for new medicines need not, and ought not be born unevenly by Indigenous peoples). The point is not to halt medicinal plant research but to adjust the research approach so that it acknowledges and attempts to prevent both existing and potential problems. What forms might alternative approaches take? As indicated, biological and cultural  contexts are essential for an appreciation of the broader knowledge systems of Indigenous societies. However, many journals that publish medicinal plant research allocate little or no space for such background material. Addressing the dilemma presented by publication of traditional medicinal knowledge could begin with editorial boards allocating adequate space for publications to include relevant biological and cultural information (even insisting that these be included). Likewise, editorial boards could require mandatory full disclosure of the sources of any cultural information cited in articles considered for publication, whether or not the information was obtained directly through research or indirectly through the published literature  152 (unless, of course it was clear that permission for publication was contingent on confidentiality or anonymity). This would help to ensure that full credit is given to Indigenous individuals and their communities, i.e., a level of recognition consistent with academic citation and credit. It would also facilitate contact with communities who wished to be consulted if further use of the data or resources is sought. Such credit also could be extended to field collections and literaturebased databases. Posey and Dutfield (1996) recommended the inclusion of prominent statements in publications that inform readers of the origins of the data and moral obligations arising from further use, such as exemplified by Elisabetsky and Posey (1994). Such statements may raise significant awareness among the academic community of the concerns of Indigenous and local peoples. As national legislation regulating the use of biological resources and traditional knowledge is developed by concerned countries, Laird and Posey (in press) predict that a lack of awareness of such broader biocultural implications of research may soon not only constitute a moral infringement but a legal one as well. Appropriate acknowledgement of sources for credit and to ensure accuracy of information are fundamental, but perhaps Indigenous contributions also merit co-authorship. With or without co-authorship, Indigenous partners in research should be entitled and encouraged to review and comment on not only publications but research grant proposals, and research records. Alternative avenues for publication that enable the local communities participating in research to access material and to maintain a comfortable level of control over its dissemination ought to be considered. As indicated in Chapter 2, researchers within the Secwepemc Ethnobotany Project have chosen to support an alternative publication avenue that enables review, co-publication and copyright by the Secwepemc Cultural Education Society (Turner et al. in preparation). I have chosen to submit the results of the antimicrobial activity screening research (Chapter 2 and Appendix B) in its entirety for this publication, rather than to divide it into smaller units to submit to multiple academic journals. I believe this will better enable a more appropriate level of control of the data by the Secwepemc Nation . In 43  my view, this community-based approach to publication addresses many of the ethical issues that  This Latin phrase is translated in wildlife law applications as: whenever there are doubts about the consequences of a certain action on wildlife, the decision must always be in favour of the interests of wildlife ecology (Backes and Verschuuren 1998). 0riginally I had requested that the antimicrobial screening data (Appendix B) be omitted from this dissertation, but this request was denied by the dissertation examination committee. Thus, to address concerns expressed by members of the Secwepemc community about access and control of the data, arrangements have been made to place this dissertation in restricted access for the maximum time period allowed by U.B.C. 43  153  have been raised here, and sets an important precedent for ethnobotanical and related research. It is clear that supporting Indigenous rights to protect cultural knowledge will require challenging some academic policies that inadvertently encourage exploitation of cultural knowledge—such as rewards based on publication, industrial partnerships that focus on applied or patentable research, and university ownership policies. It will also require challenging university administrations to examine and amend inconsistencies in their policies, and anticipating the need for new policies in research, such as those related to Indigenous intellectual property and cultural heritage rights. Many academics still publish based on the concept of free exchange of information, without regard for the current corporate assault on the public domain. However, until legal means exist for supporting Indigenous rights to not have knowledge and resources privatised (Posey 1999), publication in such ignorance may serve to further disadvantage protection of the knowledge. As Barton puts it, "the question is one of drawing a rational line between a scientific world of free exchange and a proprietary world of controlled exchange" (Barton 1994:220), and it is a question that, as academics, we all ought to consider. We ought to be informed and honest about the state of the public domain so that Indigenous peoples can truly make an informed choice about what it means today to share cultural knowledge with the wider public. 4.5  CONCLUDING COMMENTS: ETHNOBOTANY—WHERE  MANY CROSSROADS INTERSECT  While the discipline of ethnobotany may be at an "intellectual crossroads" as Ford (1994c:xx) suggested, given the diverse interests and obligations in current ethnobotanical and related research, individual researchers today may feel like they face a 'cloverleaf junction' where several roads are crossing simultaneously, and at multiple levels. Researchers in ethnobotany are poised at the privileged yet precarious point of contact between the natural and social sciences, between academic and local communities, and between Indigenous and nonindigenous worldviews and systems of knowledge. However, the order within each of these realms today appears to be just as dynamic as the intersections between them. As a new  154 equilibrium is established, the ensuing changes undoubtedly will confer significant challenges to present and future research. The tremendous potential in ethnobotanical research lies in the synergy possible through interconnecting these different approaches and ways of knowing. Synergy involves combining individual components in such a way as to enhance or potentiate their contributions. Perhaps the situation is somewhat similar to the potential synergy of a chemical mixture. A major challenge to understanding the net effects of the mixture is identifying the exact combination of individual ingredients, in specific ratios, that potentiate synergistic rather than antagonistic effects. This usually requires defining each constituent, and assessing its individual contribution before the combined effects can be accurately predicted or understood as a whole. In this chapter, I have taken a similar approach to tease apart an understanding of the effects of multiple stakeholders and obligations on ethnobotanical research and research outcomes. The only clear conclusion is that the situation is complex, and while there is much to learn from the past and present, there is no set formula that can be derived for proceeding into the future. Many of the challenges will be case specific, i.e., dependant on the particular 'mixture' of stakeholders involved. One thing for certain, however, is that understanding or predicting the combined effects of the whole will require that the interests and contributions of all of the stakeholders are considered. If ethnobotany involves the study of reciprocal and dynamic relationships between humans and plants (Ford 1994b), then as these relationships evolve, so too must the discipline— but has it? Almost a century ago, Boas (1940[1920j:331) launched an urgent appeal for research on the cultural knowledge of Aboriginal peoples: "With the energetic economic progress of Canada, primitive life is disappearing with ever-increasing rapidity; and, unless work is taken up at once and thoroughly, information on the earliest history of this country, which has at the same time a most important bearing upon the general problems of anthropology, will never be obtained". He continued: "... May we hope that it may be seized upon, and that the aborigines of the Dominion may be studied before it is too late?" (Boas 1940[1910]:343). In Boas' time, the inevitable loss of traditional Aboriginal cultures was a foregone conclusion. Have things changed? A similar 'salvage' approach seems to drive much of the research today in ethnobotany and related fields, i.e., that we need to document the cultural knowledge of Indigenous societies before it is 'too late'. While the documentation of cultural knowledge is sure to be of value in cultural conservation efforts, it is questionable if documentation alone can  155 conserve knowledge. Furthermore, conserving cultural knowledge does not in itself conserve culture. Thus, if we deduce anything from the state of ethnobotany today, surely it is the need for a greater emphasis on protection of and support for the living relationships that embody cultural knowledge rather than preservation of the knowledge or information itself for subsequent use. To achieve this, however, likely will require that researchers relinquish the 'apron strings' that have justified their past presence and control in research with Indigenous societies. It was explained to me recently that, admirable as it is have a desire to help, this does not confer a right to impose your version of 'helping' on others (Angelo Joaquin, Jr., pers. comm. 2000). If ethnobotany is to 'help' Indigenous societies to maintain their cultural identity and safeguard biological diversity, then surely it is imperative that researchers understand what constitutes 'help' from the perspectives of the Indigenous community members, and then advocate these perspectives to the other stakeholders involved in the research. While we may not know exactly what the 'right thing to do' is, if projects are co-developed with the interested communities, then mutual concerns and interests will more likely be addressed. One of the concerns that I have discussed at length in this chapter is the control of knowledge and the powerful role that academic publication plays in this regard. It is important to note that the implications here are not limited to the cultural knowledge of Indigenous societies, rather the issues are fundamental to the current state of the public domain and the general "flow of knowledge" (Laird and Posey in press). The tendency has been for scientific knowledge to be disseminated mainly among other scientists, through journal articles, conferences and databases; local knowledge either has tended not to reach the scientific communities, or has been overlooked as nonscientific. It is important that the flow between scientific and local knowledge is encouraged, and ethnobotanical research can be an important vehicle to facilitate this exchange. It is imperative, however, that this flow is not impeded by third-party interests in the knowledge, and this is where a precautionary approach to publication has merit. Publication is a major contributor to proprietary interests, hence the role of ethnobotanical publication (intentional or not) in the appropriation and industrial commodification of traditional botanical knowledge must be acknowledged by researchers. I argue that as both a defensive response and a proactive measure to prevent perpetuating this situation, a precautionary approach to publication is a powerful tool that can be used in "vigorous defense of the concept of a public  156 domain" (Brown 1998:205), the survival of which "is of vital significance to us all" (Brown 1998:206). A final point that warrants reiteration is that the research relationships entered into with Indigenous and local communities are not initiated de novo; they are necessarily embedded in histories amid the complex web of injustices that already has been spun. The concerns expressed by Indigenous peoples about use of traditional plant knowledge and resources are deeply linked to both the past and the present. Addressing the present therefore necessitates acknowledgement of historical events and the significance of their impacts (as I argued in Chapter 2). In British Columbia, for example, the legacy left by colonisation has been particularly challenging in that the complex issues of land title questions and negotiating Aboriginal treaties are only now being addressed. This leads to significant uncertainties in what the future holds for all peoples of British Columbia, and how present actions will affect the future. Thus, fears for loss of control of traditional knowledge and access to resources, which seem to be at the heart of concerns about appropriation and commodification, must be understood in a broader context than simply economic. An understanding will require a careful unraveling of the inner-weavings of this broader context, which among other things will require two things that can not be legislated: time and mutual respect. At this point, however, the current interest in ethnobotany and the wide audience presently captivated by plant medicines of Indigenous societies may offer a unique forum to bring forward the important complex of issues that have been discussed in this chapter. With optimism, I suggest that perhaps this may be used as "a step in the transformative process of decolonizing our history, a start toward undoing the colonial legacies that still characterize the relationships between Native and non-Native people in this country. Too often researchers have heard these stories without listening, listened without acting, and acted without listening again. It is time to break that cycle" (M.-E. Kelm 1998:xxiii). In light of current biocultural issues in ethnobotanical and related research, perhaps clarifying research obligations, developing a clearer understanding of impacts of research, and rethinking the role of publication—tools that are in the hands of each individual researcher—are means to break this cycle.  157 5  General Discussion We live in an age when economics and the social conscience dictate that to be worthy of study, a problem should be relevant to our present situation, and of potential utility to Mankind; it is no longer sufficient that it is interesting. The still unknown ecological interactions which are mediated by natural products are manifold...an increased awareness of the chemical complexities of our environment can do nothing but good. —John Mann (1987:328)  5.1  Chemical and other Complexities of Human-Plant Interrelationships  Chemistry is the basis of life on earth, and chemical interactions between organisms are important driving forces in generating diversity, yet as noted above by Mann (1987:328), we still understand relatively little about the "chemical complexities" of our biological surroundings. Mann (1987:ix) suggested also that a clearer understanding of the role of chemicals as mediators of complex interactions between different species would help us "to ensure that we do not disturb the environment to our detriment or to that of the species with which we coexist". As described in the previous chapters, the first part of my dissertation research involved an investigation of the chemical complexities in human-plant interrelationships relating to antimicrobial compounds in food and medicinal plant resources. The second part of my research explored some of the sociopolitical complexities stemmingfroman investigation of this type (i.e., issues of ownership and rights to cultural knowledge and traditional resources).  As many biologically active plant compounds are part of the chemical defense system of plants, phytochemical composition is influenced by ecological interactions between plantherbivore, plant-plant, and plant-microorganism (Mann 1987; Harborne 1993). Humans have 'borrowed' and exploited many of the phytochemical by-products of these interactions for therapeutic purposes over centuries or millennia. However, the medicinal use of plants is not necessarily a passive exercise; humans also can alter directly the phytochemical composition and properties of plants. I suggested in Chapter 1 that cultural traditions and technologies such as plant selection, harvesting seasons and methods, processing, and differential uses all have the potential to alter qualitatively and/or quantitatively the phytochemical repertoire of a given plant. My research on the traditional processing and use of balsamroot as a Secwepemc food and  158 medicine (Chapter 3) supports this claim; pitcooking and peeling eliminated antimicrobial compounds in roots prepared as food, and served to "favorably alter the nutrient/toxin ratio" (Johns 1990:243); boiling made available antimicrobial and other biologically active compounds in roots prepared as medicine; and drying decreased exposure to biologically active sesquiterpene lactones in leaves used for medicine. I did not choose to take a comparative approach to analysing the balsamroot data, although this plant does have widespread documented use as food and/or medicine in many North American Aboriginal cultures (Moerman 1998). I purposely chose to look more closely at some of the combined cultural, biological and chemical aspects of balsamroot as a food and medicine within a single culture, with the intention of unearthing a deeper level of human-plant interrelationship than is typically found in ethnomedicinal studies. I believe that my dissertation research has been successful in this regard. Furthermore, I suggest that the research supports the position that unless traditional technologies are incorporated into chemical investigations of medicinal plants, it is unlikely that a full appreciation will emerge of the significant alterations that these and other aspects of cultural knowledge can bring about in the chemistry of a plant. However, to design experiments that incorporate elements of cultural knowledge—i.e., beyond simply basing selection on lists of plant species and disease symptoms treated—is to assume a level of commitment and community acknowledgement that may present significant challenges and complexities to research conducted under current academic polices, as discussed in Chapter 4.  Understandably, it may be easier to keep things simple (e.g., use informationfromthe  literature rather than work directly with communities), but I suggest that many genuine leaps in understanding within this area will emerge from the synergy in actively engaging different perspectives, approaches, and knowledge systems in the research process. Such an engagement enables us to consider the "embeddedness" of the research process and resultant data "within larger systems of meaning" (Gragson and Blount 1999:xii), and this enables research that is not only interesting, but (in Mann's 1987:328 words above) both "relevant to our present situation, and of potential utility" to Humankind.  159 5.2  Antimicrobials in Plant Foods and Medicines The existence of antimicrobial activity in plants has been integral to my dissertation  research. Antimicrobial activity is one of many measurable properties that is presumed to be linked to the medicinal value of plants, and balsamroot is only one of many different species with antimicrobial properties that is used as Secwepemc medicine (as shown by the antimicrobial activity screening data in Chapter 2 and Appendix B). Moerman (1998) claimed that, although many plants have traditional uses as both food and medicine in Aboriginal cultures of North America, usually different parts of the plant are used. The intriguing thing about the root of balsamroot is that the antimicrobial aspect of its medicinal character exists alongside its traditional role as a staple food. This initially suggested to me that either Secwepemc people were subjecting themselves (intentionally or not) to substantial amounts of antimicrobial (and presumably other biologically active) compounds in their balsamroot-containing diets, or that the differential processing and food preparation methods somehow eliminated the majority of these compounds from the roots. While my data suggest that the latter more accurately describes the situation for antimicrobial compounds in balsamroot, it may be that the former is the case for other plants used as medicine and consumed as food by the Secwepemc, such as the bulbs of Erythronium grandiflorum (yellow avalanche lily), the seeds and leaves of Lomatium nudicaule  (barestem desert parsley) and the roots of Geum macrophyllum (large-leaved avens), all of which showed antibacterial and antifungal activities in vitro. The roots of Geum macrophyllum offer a particularly interesting case for further study, since my assays showed that they have the broadest spectrum of antimicrobial activity of the extracts tested, and according to Turner and Ignace (in preparation), the roots are cooked and consumed as a tonic. Further research is necessary to determine if the antimicrobial properties of these plants are retained when they are prepared as traditional foods, although my preliminary results indicate that this is likely. If so, this presents an interesting topic for further investigation, and broadens discussion on traditional diets and health, and the origins of human diet and medicine. Johns' (1990) analysis of the origins of human diet and medicine suggested that the human intake of 'food' (i.e., nutrientsfromplants) was at one time closely linked with the intake of 'medicine' (i.e., non-nutrient and even toxic phytochemicals), but that this link was severed by such industrial 'advances' as plant domestication and breeding, food selection, and  160 (over)processing or refining of foods. Food and medicine have since become fairly discrete categories in mainstream North American society. Recently, however, the idea of re-connecting medicinal properties with certain nutritional foodstuffs seems to have caught on, as indicated by current research (and marketing strategies) on 'functional foods' and 'nutraceuticals' (Staples, 44  2000). For example, Forbes Medi-Tech (Vancouver) are creating cholesterol-lowering food ingredients such as the semi-synthetic phytosterol known as Phytrol—and apparently "it was a cinch" for researchers to recruit 100 eager volunteers for the first clinical trial of Belgian chocolate-containing Phytrol (Wigod 1999: A3). The next step is to chemically modify the fatsoluble Phytrol to be more water-soluble and incorporate it into "foods that are more intrinsically healthful" such as juices (Wigod 1999:A4; Nikolay Stoynov, pers. comm. 2000). Such trends suggest that the dose-dependent food-medicine continuum that Johns (1990) used to describe the dietary habits of our ancestors and many traditional societies, and that I discussed in relation to balsamroot (Chapter 3), soon may come back into vogue. However, the existence of a somewhat indiscriminant association of 'medicinal' with 'healthy' is an interesting lay assumption that merits further comment, especially in the context of antimicrobial compounds in foods. While antimicrobial compounds in plants may be effective in limiting microbial diseases and parasites in humans, it is questionable whether or not constant exposure to such compounds can be considered 'healthy'. Many within the medical profession, research communities and society in general are legitimately concerned about past and present overuse or inappropriate use of antibiotics, and the increasing emergence of antibiotic resistant microorganisms (Neu 1992; Travis 1994; Levy 1995; Sanders and Sanders 1995; Davies 1996; Fidler 1998; Huycke et al. 1998). The selective pressure provided by regular exposure to antibiotics, especially at low doses, is a key factor in the development of bacterial drug resistance. Therefore, while it is logical that antimicrobial compounds in plants might be seen as valuable in the treatment of certain microbial-based disease conditions, it is rather perplexing that antimicrobial compounds in foodstuffs would be viewed generally as 'healthy', especially when most of us certainly would not consider a long-term course of antibiotics as such. The widely-known Manuka (Leptospernum scopium J.  R. Et G. Forst.) honey of New Zealand, renowned and indeed  Etkin (1994) defines nutraceuticals as constituents of common foods in North American cuisines for which studies have demonstrated healthful effects.  44  161 marketed for its 'healing' antibiotic properties, is a case in poinf". As a potential consumer, what I have yet to understand is why I would want to eat antibiotic-laden honey (aside from healing an ulcer, perhaps) when I purposely avoid eating antibiotic-raised meat and exposing myself to antibiotics in general. A key question is whether or not there is a distinction between naturally-occurring antibacterial compounds in plants and synthetic antibiotics. Another question is whether or not the antibiotic compounds in Manuka honey or other foodstuffs retain activity in vivo after ingestion—in this case, it is unclear if the 'scientific evidence' referred to in footnote 45 is based on disk diffusion assays alone (likely, and a point to which I will return to shortly). The presence of antimicrobial compounds in plants is interesting and in many cases useful to humans, but I suggest that it is not necessarily positive—in certain circumstances antimicrobial activity may be beneficial while in other cases it could be detrimental, for not all microbes are villains. In fact, the effects on humans of the majority of microorganisms is unknown as most have not yet been identified, let alone cultured in vitro. Of those known, many are not only nonpathogenic to humans but arguably are essential for life and/or normal health maintenance. The presence of antimicrobial compounds in plants, especially those used as food and medicine, merits caution and a 'healthy' level of respect. The effects of antimicrobial compounds on pathogens must be weighed against the effects on normal flora, and it should be kept in mind that antimicrobial activity may be accompanied by other biological activities {e.g., such as allergies, as discussed for sesquiterpene lactones in Chapter 3). Johns (1990; 1994) proposed that biologically active plant compounds in traditional diets may have been efficacious in controlling gastrointestinal infections. It may be that the routine consumption of antimicrobial compounds along with nutrients was essential in diets less hygienic (in the sense of greater exposure to various environmental and food-spoilage microorganisms) than those of typical North American cuisine today. However, an interesting question that arises with the widespread presence of antimicrobial compounds in plants is whether or not continual exposure to these compounds would result in resistance by pathogenic For example, the label on Organic Manuka Honey produced by N.Z. Coromandel Mountains Co. Ltd., Whitianga, reads as follows: "Scientific research reveals the antibiotic qualities of this N . Z. honey. You'll love it!". A pamphlet by Comvita (N.Z.) manufacturers of Manuka Honey states: "It appears that Manuka Honey can offer peptic ulcer sufferers a cure. In scientific tests Maunka Honey successfully destroyed bacteria including: Helicobacter Pylori, which is associated with peptic ulcers, Staphylococcus, Streptococcus and Staphaureus." 45  162  and/or commensalistic/mutualistic microorganisms. If this occurred then it certainly would be an important aspect of research to pursue before promoting plant foods or other consumables with antimicrobial properties. If resistance does not occur, however, it would be interesting and useful to know why not, for example, if a lack of resistance is related to the presence of multiple antimicrobial compounds, perhaps with different modes of action and spectra of activity. . These questions hint at one of the largest gaps remaining in medicinal plant research— the gap between effects in vitro and in vivo. What does the long-term and regular consumption of and/or exposure to antimicrobial (or other innumerable biologically active) plant compounds mean for humans? The general public and perhaps many researchers may not be in a position to interpret adequately the results of disk diffusion and other assays in vitro that are typically used in assessing antibacterial and antifungal activity in plant extracts, nor understand the limits of extrapolating these assays to situations in vivo. A lack of in vivo data forces us still to extrapolate too much and speculate too often, and further underscores the value in opportunities to study plant medicines in a traditional context (in situ), as I have discussed in the previous chapters. 5.3  Necessary Tensions in Current Ethnobotanical Research  The opportunity to work with Indigenous or traditional communities, and to employ their cultural knowledge in research programs comes with additional obligations and responsibilities, as discussed in Chapter 4. It seems that many of these obligations have been overlooked in past decades. As a result, researchers involved in ethnobotany and related fields are finding themselves positioned at the exciting but often uncomfortable frontline of some major transitions that are occurring within and outside of the discipline—transitions that may redefine what it means to do ethnobotany. While we wait to see where ethnobotany is headed, however, some of us may be faced with significant uncertainty and apprehension about how to proceed. Nicholas (1999:3) discussed how the "pressures of a changing world order" have resulted in similar growing pains within the equally multidimensional field of archaeology. He suggested that "the tensions that exist between different flavors of archaeology" ought to be viewed not only as important in the general development of the discipline, but as "particularly productive areas of discourse" between the stakeholders (Nicholas 1999:3). Nicholas (1999) reasoned that  163 the friction or dissention created by these tensions are necessary because they do not allow complacency, but force us to respond to situations or ideas that we might not otherwise bother with. He noted also some important and progressive changes in archaeology that have emerged from areas of conflict, and how some cooperative projects are "stretching the boundaries of the discipline". He continued: "Some archaeologists are involved with indigenous peoples because they are legally required to; some do it because it is politically correct, but others because it is correct, period" (Nicholas 1999:14). I suggest that these observations are applicable to current ethnobotanical research as well. As legal and ethical obligations in research involving the cultural knowledge of Indigenous peoples await clarification and entrenchment, it is imperative that the broader ethnobotanical objectives of protecting cultural and biological diversity are continually emphasised alongside other objectives and applications. Godbole and Eyzaquirre (1997:83) have underscored the importance of "link[ing] ethical issues with researchers' accountability toward the local communities". Such links, combined with an increase in awareness and acknowledgement of Indigenous traditions and systems of knowledge likely will present a challenge to the current concept of 'inventions' that are based on research on traditional plant medicines; in fact, many 'discoveries' might be more appropriately recognised and acknowledged as 'rediscoveries', or as new applications of old ideas. I suggest that the evolution of systems for protection of knowledge, be that the modification of current intellectual property rights regimes or the introduction of new systems of protection, has implications beyond the concerns of Indigenous cultural knowledge. Indeed, no matter where one stands on issues of cultural heritage rights per se, there may be reason to be grateful to the Indigenous voices that have brought forward rights-based concerns to the international community. At a fundamental level, these concerns are further evidence that (as Brown 1998:206 pointed out) it is really the state of the "intellectual and artistic commons" that we refer to as "the public domain" that is in jeopardy. I suggest that this is a situation that ought to merit the concern of all peoples.  164 5.4  Summary and Concluding Remarks Science requires the challenge of the established order; the right to be heard, however outlandish the assertion, subject only to the test of rigorous method. To quote Jacob Bronowski, who wrote The Ascent of Man, "the essence of science" is "ask an impertinent question, and you are on the way to a pertinent answer". The scientist at her lab bench and the farm family in the hinterland must both participate in this process of productive impertinence. So must the senior scientist and his or her aspiring, inquiring, junior colleagues. —Ismail Serageldin (1997:4-5)  My dissertation research has provided an opportunity to work in collaboration with Secwepemc peoples and to increase my cultural awareness while exploring the underlying chemical nature of some fascinating and sophisticated interrelationships that exist between the Secwepemc and their traditional food and medicinal plant resources. In addition, this work has enabled an investigation of some important and controversial ethical issues involved in ethnobotanical and related research. The major findings of my research that have been discussed in the previous chapters include: •  identification of antibacterial, antifungal and/or antiviral activities in vitro in Secwepemc medicinal plants traditionally used to treat conditions likely caused by microorganisms (Chapter 2);  •  determination that traditional technologies such as differential heat processing and drying alter the presence or availability of antimicrobial and other biologically-active phytochemicals in balsamroot prepared for use as Secwepemc food and medicine (Chapter 3);  characterisation of the heat stability, solubility and antimicrobial activity of thiophene E, and its localisation within the root of balsamroot during food and medicinal processing (Chapter 3);  •  isolation, purification and identification of a known phytosterone 16R,23Rdihydroxycycloartenone, determination of the configuration of the hydroxyl group at C23, and localisation of the compound within the root of balsamroot during food and medicinal processing (Chapter 3);  165 •  isolation, purification and identification of a previously unknown phytosterol 16R,23Rdihydroxycycloartenol, and its localisation within the root of balsamroot during food and medicinal processing (Chapter 3);  •  isolation, purification and identification of a previously unknown antibacterial sesquiterpene lactone (guaianolide) 2-deoxy-pumilin-8-0-acetate from fresh balsamroot leaves, characterisation and quantification of its antibacterial activity, and indirect evidence that this compound may be localised to glandular trichomes (Chapter 3);  •  identification and discussion of biocultural issues in current ethnobotanical and related research involving the cultural knowledge of Indigenous societies (Chapter 4), specifically issues involved in: • meeting research obligations to multiple stakeholders, including: • assessment of academic research policies and ethical standards; and • recognition of means of protecting rights to intellectual and cultural properties, such as international law, Canadian IPR law, local initiatives, and professional codes of conduct; • identifying direct and indirect impacts of research, particularly: • prediction of unknown or harmful consequences; and • highlighting the important role of publication in the control of knowledge.  These findings and points of discussion have both drawn upon and contributed to the larger collaborative, multidisciplinary research program on Secwepemc ethnobotany, of which my dissertation research has been a part. I have presented my dissertation research within a framework that is politically, socially, and historically complex. I see the research as both a specific case study and part of a more generalised foundation for broadening discussion on the conduct and dissemination of research involving cultural knowledge. It has evolved (necessarily but somewhat unintentionally) into a multidisciplinary 'adventure', crossing several different fields of inquiry and challenging the status quo, perhaps precariously at times. While the strength of this study lies in its  interdisciplinary approach, any study that seeks such wide-ranging objectives may be vulnerable to criticism from a number of angles. Even so, I believe that identification and discussion of the issues that arose through my dissertation research and have been brought forward here are vitally important.  166  As important as increased awareness and discussion of the issues are, however, there is also a need to begin to move beyond words. The time has come to translate words into actions, to provide alternatives, and to move toward the creation of a framework that requires, encourages and indeed rewards interaction between all parties involved in the research, leads to meaningful dialogue, and holds potential for mutual resolution of the issues. In Serageldin's words (1997:6): "The real challenge is to create an overall system that promotes interaction to help each actor contribute to the best of its comparative advantages so that the whole is much more than the sum of the parts". A suitable framework cannot be imposed by policymakers lacking ground-level experience, nor will it be based on the formula of one-size-fits-all; its evolution will necessarily be rooted in specific cases and subject to negotiation among those people involved—negotiation that begins with commonalties and is based on respect and an equalised decision-making power. It will emerge from dialogue between those who make policy and those who must abide by it— by providing feedback on what does and doesn't 'work' and how things might be modified. I believe that this a responsibility of researchers as they confront the issues, strive to address them within the framework of the governing ethical and other policies, and in the process gain valuable insights on further modifications that may be required to meet the policy goals. It will also emerge from the willingness to attempt, to critically evaluate, and to improve our perceptions and ideas. It is this wUlingness that will enable new foundations for building mutually respectful, and beneficial interactions between and among all peoples to protect the plant-human interrelationships that are part of the biocultural wealth of humanity, and to assist in stewardship of the biological resources that sustain us all. I began this dissertation with the simple yet eloquent words of Serageldin (1995:5): "We are all the guests of the green plants around us". I close with the simple but compelling words of Chief Justice Lamer as he concluded the precedent-setting Delgamuukw Decision, on the topic of reconciliation of the pre-existence of Aboriginal societies with the sovereignty of the Crown (Delgamuukw v. The Queen 1997): "Let us face it, we are all here to stay".  167  LITERATURE CITED Ahumada, C , T. Saenz, D. Garcia, R. De La Puerta, A. Fernandez, and E. Martinez, 1997.  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V2H 1H1  Letter of Consent for the Secwepemc Ethnobotany Project: Ethnopharmacology of Secwepemc Traditional Medicines The undersigned, Kelly Bannister (in conjunction with Dr. Nancy Turner), hereby obtain permission to conduct interviews with members of the Secwepemc Nation in order to document information on the traditional plant knowledge of the Secwepemc peoples for the purpose of collecting plants used as traditional medicines and examining their biochemical and pharmacological properties. The interviews will be informal and open-ended, may include use of audio or videotape, and will be conducted at a location that is convenient for the participant The results of this work will constitute part of therequirementsof a doctoralresearchproject at the University of British Columbia under the supervision of the Faculty Advisor Dr. G. H. N. Towers, Department of Botany. This Ph.D. thesis work will be conducted during the period of September 1995 and August 2000, with the interviews and field work to be conducted mainly between April - August 1996 and April - August 1997. The amount oftimerequired of participants will vary between a few hours and several days over the duration of this research, as determined by the participant Thisresearchwill be conducted under the following conditions: We acknowledge the ownership of their traditional plant knowledge by the Secwepemc peoples and individual elder's names will appear with theirrecordedplant knowledge, unless the participant indicates that he/she wishes his/her identity to be kept confidential. Typed field notes of individual elder interviews will be sent to each elder. Each elder will be given the opportunity, within a two month period, of providing corrections,revisions,deletions, or additions, which will be incorporated into the field notes. Participation is voluntary and the participant has the right torefuseto participate or withdraw at anytimeand without consequence. If the participant is to be audiotaped or videotaped, permission will be obtained beforehand and a description given as to the disposal of therecordings.Any inquiriesrelatingto this work made by participants will be answered to the best of our ability.  We will provide the Secwepemc Nation with one copy of the original draft and final draft of the information documented. This includes original and typed field notes, audiotapes, videotapes, photographs, Ph.D. dissertations, and journal articles. We will notify the Secwepemc Nation before anyresearchfindingsare published and provide them with copies of any manuscripts prior to publication. The Secwepemc will be given the opportunity, within a two month period, of providing corrections,revisions,deletions, or additions, which will be incorporated into the publication. All publications will acknowledge the contribution of the Secwepemc people and individual elders, and will state that the Secwepemc Nation has control over access to the traditional plant knowledge, as well as to potential development of any marketable products (such as drugs or pharmaceuticals) that may be discovered as aresultof the traditional knowledge shared during the course of this research. No monetary compensation will be given for participation, but we will turn over to the Secwepemc any financial profits accruingfromthe publication or use of the original information gathered, to be shared in an appropriate way with individual native elderfs) who contributed information. It is understood that the undersigned, Kelly Bannister (and her above-named colleague), have right of access to the research materials and information for use in teaching and scholarly works (books, lectures, journal articles). Full credit for information used in this way will be given to the members of the Secwepemc Nation who have supplied the information. The Secwepemc Cultural Education Society will be given the option to publish any materials (books, pamphlets, etc.) seen to be useful and beneficial for use by members of the Secwepemc Nation. A copy of this consent form has beenreceivedby the undersigned for the purpose of records.  Kelly Bannister University of British Columbia  Date  Chief Ron Ignace^ President Secwepemc Cultural Education Society  Date  APPENDIX B: Chapter 2 Summary of Plant Use and Antimicrobial Screening Data  NB: The following table includes Secwepemc cultural information on plant names and medicinal indications based on Turner and Ignace (inpreparation). This cultural information has been included for the purposes of this dissertation only with permission from the Secwepemc Cultural Education Society and investigators of the Secwepemc Ethnobotany Project. Any further use of the information requires permission from the Secwepemc Cultural Education Society (Kamloops). All subsequent use of the information should acknowledge the original contributors as indicated in Turner and Ignace (in preparation) rather than this dissertation, and should respect the spirit and conditions under which the information was shared (as discussed in the preceding chapters).  191  Table B.l.  Plant names, medicinal indications, and antimicrobial activity of the Secwepemc plant extracts examined in this study. 3  Secwepemc medicinal indication  Plant part assayed and crude extract reference #  Bryoria fremontii Tuck.  •bandaging  whole plant  (Usneaceae)  •wound dressing  S-14.1.m  Plant names (Botanical Latin , Common and Secwepemc names) b  11  0  Antibacterial activity (mg/disc assayed) 6  Antifungal activity' (mg/disc assayed)  Antiviral activity ' (MIC in mg/ml)  8 11  LICHENS •emergency food B l a c k tree l i c h e n o r  +  +  -  (1.5)  (1.5)  (> 0.20)  Bs  Tm  Mp  "Black Moss"  wile; wila  FUNGI (MUSHROOMS AND TREE FUNGI) Calvatia gigantea (Batsch:  • s p o r e s a p p l i e d t o cuts  Pers.) Lloyd. (Lycoperdaceae)  spores  -  -  +  S-15.m  (3.2)  (3.2)  (0.2)  Giant puffball  tswiweyulecw Inonotus obliquus  •"moxibustion" type  section  -  -  -  (Ach.ex Pers.) Pil. (Hymenochaetaceae)  treatment for p a i n a n d  S-28.m  (0.4)  (0.4)  (> 0.20)  (1.8)  (> 0.20)  other ailments  "Birch Fungus" or Cinder conk  pucwst'ye; welmin; tikwen 'kten; stq 'mxweke  FERNS AND THEIR RELATIVES Equisetum hyemale L.  •diuretic  whole plant  (Equisetaceae)  •childbirth medicine  S-3.1.m  •healing umbilical cord T a l l scouring rush, Branchless horsetail or "Joint-Grass"  xwiyusten'; xwixwyuy 'sten  •constipation  + (1.8) Pa  192  CONIFEROUS TREES AND SHRUBS (GYMNOSPERMS) Abies lasiocarpa (Hook.) Nutt.•cuts, boils, sores (Pinaceae) •insect repellent •laxative, tonic Subalpine or "Balsam" fir •colds, influenza •lung congestion melenllp; meldnllp; menenpellp •circulation •gastrointestinal disorder •sore bones, rheumatism  Juniperus scopulorum Sarg. •disinfectant wash  (Cupressaceae) Rocky mountain juniper P llp un  Picea engelmannii Parry (Pinaceae) Engelmann Spruce  •acne •vapourizer •humidifier •colds, influenza -swelling and soreness •blood purifier •arthritis •diabetes •kidneys  aerial S-35a.l.m  pitch S-35b.2.m  aerial S-9a.l.m  -  +  +  -  (9.4) Bs, Ef, Sa Ec, Pa Mp  (9.4) Mg, Tm  (> 0.20)  +  +  (0.5) Bs Mp  (0.5) Mg, Tm  cytotoxic (>0.10)  +  +  -  (5.4) Bs, Ef, Sa Mp  (5.4) Mg, Tm  (> 0.20)  «cuts, infections «sore throats •whooping cough .colds, influenza  pitch S-16a.2.m  + (0.5) Bs, Ef, Sa Pa Mp  + (0.5) Mg, Tm  cytotoxic (> 0.050)  •TB, coughs 'sores, wounds •tonic, laxative . e throat  bark S-63a.l.m  + (5.5) Bs, Ef, Sa Mp  + (5.5) Mg, Tm  cytotoxic £0.10)  + (0.20) Mg, Tm  cytotoxic (>0.10)  t 'sellp; ts 'allp Pinus contorta Dougl. var.  latifolia Engelm. (Pinaceae) Lodgepole Pine  sor  qwli7t  Pinusponderosa Dougl. (Pinaceae) Ponderosa Pine or Bull Pine s7etqwllp; s7atqwllp  pitch S-63b.l.m  •wash for newborns •arthritis •colds •burns, infections  aerial S-48a.l.m  (0.20) Bs, Ef Mp + (3.0) Bs, Ef, Sa Pa Mp  (3.0) Mg, Tm  + (0.050)  193  Pseudotsuga menziesii (Mirbel) Franco var. glauca (Beissn.) Franco (Pinaceae) Douglas-fir  aerial S-41b.l.m  + (4.6) Bs, Ef, Sa Ec, Pa Mp  + (4.6) Mg, Tm  + (0.050)  pitch S-41a.l.m  + (1.2) Bs, Ef, Sa Ec, Pa Mp  + (1.2) Mg, Tm  cytotoxic (> 0.050)  •colds •chest congestion •cancer •ulcers, sores •antibiotic •"heal all"  aerial S-65a.2.m  + (2.5) Bs, Ef, Sa Mp  + (2.5) Tm  cytotoxic (> 0.050)  •vapourizer •humidifier •prevention of colds  aerial S-57a.l.m  + (5.5) Bs, Ef, Sa, Ec Mp  + (5.5) Tm  •cuts, stings, infections •burns, blisters •hemhorraging •persistent cough  tsq'ellp; tq'7eseke7  Taxus brevifolia Nutt. (Taxaceae) Western Yew tskwinek; kenik  Thuja plicata Donn. (Cupressaceae) Western Red-cedar qweqwetqwellp;  (> 0.20)  estqw  DECIDUOUS TREES; BROAD-LEAVED TREES Acer glabrum Torr. (Aceraceae) Rocky Mountain Maple  •sores •infections •liver, spleen  bark S-17a.l.m  +(2.0) Bs, Ef, Sa Mp  + (2.0) Mg, Tm  + (0.025)  •vapourizer •sores, cuts, infections •tonic (mild) •colds (strong) •high blood pressure  bark S-22a.l.m  + (3.6) Bs, Ef, Sa Pa Mp  + (3.6) Mg, Tm  + (0.05)  •medicinal salve •sores, cuts •earaches •colds, cough •urinary tract  leaf buds S-31a.l.m  + (1.0) Bs, Ef, Sa Ec Mp  + (1.0) Af Mg, Tm  ts 'wellten; ts 'wdllten  Alnus incana (L.) Moench (Betulaceae) Mountain Alder kwle7ellp Populus balsamifera L. (Salicaceae) Black Cottonwood mule; melcqin'  (buds)  (> 0.20)  194 Populus tremuloides Michx. •tonic (Salicaceae) •sores  aerial S-37a.l.m  + (1.0) Bs, Sa Mp  + (1.0) Sc Mg, Tm  + (0.20)  fruit S-49c.l.m  + (6.2) Ef  (6.2)  (> 0.20)  aerial S-49a.l.m  + (1.1) Bs, Ef, Sa Mp  (1.1)  + (0.050)  aerial S-50a.l.m  + (5.4) Bs, Ef, Sa Ec, Pa Mp  + (5.4) Tm  whole plant S-33.1.m  + (3.3) Bs, Ef, Sa Ec, Pa Mp  + (3.3) Mg, Tm  cytotoxic (> 0.20)  aerial S-55a.l.m  + (1.9) Bs, Sa Mp  + (1.9) Mg, Tm  cytotoxic (> 0.013)  aerial S-68a.l.m  + (3.4) Bs, Ef, Sc Ec, Pa Mp  + (3.4) Tm  - + (0.013)  Trembling Aspen or White Poplar meltellp; meltallp Prunus virginiana L . (Rosaceae) Choke Cherry  •health food •diarrhea •fever •colds, cough  tkwlose7ellp (tree); tkwlose7, tkwlosa (fruit)  SHRUBS Amelanchier alnifolia Nutt. (Rosaceae)  •fever •leukemia  Saskatoon Berry; Service Berry stsiqwem (berries); stsiqwemellp (bush) Arctostaphylos uva-ursi (L.) •kidneys, bladder Spreng. (Ericaceae) •diuretic •burns Kinnikinnik or Bear Berry elk, alk (berries); elkellp, alkdllp (plants) Artemisia tridentata Nutt. (Asteraceae) Big Sagebrush  •colds •tuberculosis •soreness •eyewash  (> 0.20)  kewku; kdwku Ceanothus velutinus Dougl. •influenza (Rhamnaceae) •colds •measles, small pox Snowbrush; Buckbrush •general medicine tsewelstdn; tselstdm; qwunllp; tswelstdm  195 Clematis columbiana (Nutt.) •hair rinse T. & G. (Ranunculaceae) •arthritis  aerial S-40a.l.m  Blue Clematis; "Monkey Rope"  +  (1-0) Pa Mp  (1.0)  cytotoxic (>0.20)  stl 'upel 'qw; slept 'upl 'qw Cornus stolonifera Michx. (Cornaceae)  •kidneys •warts •acne Red-osier Dogwood; "Red •tonic, physic Willow" •flu •headaches cpeqpeqeqen 'cen; xpeqpqeqsa; •toothaches stpekemuse; c 'peqp 'q 'axqin;•arthritis taxpa' (berries); •swellings, bruises tseqwtseqweqwelqw; •abcesses, boils st 'ekemusellp (bush)  Juniperus communis L . (Cupressaceae) Common Juniper; Dwarf Juniper  •fumigant •sore eyes •acne •general medicine  tsexts 'ext; tsaxt 'sdxt  Ledum glandulosum Nutt. (Ericaceae)  aerial S-45a.l.m  bark S-45b.l.m  aerial S-44a.l.m  fruit S-44b.l.m  •eye medicine •childbirth medicine •indigestion •poultice •sores, poison ivy •heart medicine  leaves S-77a.l.m  Ledum groenlandicum Oeder •eye medicine (Ericaceae) •childbirth medicine •indigestion "Swamp Tea", "Indian Tea"; •poultice Trapper Tea, or "Hudson's, •sores, poison ivy Bay Tea" •heart medicine  aerial S-34a.l.m  "Swamp Tea", "Indian Tea"; Trapper Tea, or "Hudson's Bay Tea"  +  (3.6) Bs, Ef, Sa Pa Mp  +  (0.8) Bs, Sa Pa Mp  + (3.6) Af Ca, Sc Mg, Tm + (0.8) Mg  -  (> 0.20)  +  (0.025)  +  +  -  (7.9) Bs, Ef, Sa Ec, Pa Mp  (7.9) Mg, Tm  (> 0.20)  +  (4.7) Bs, Sa Ec, Pa Mp +  (2.4) Bs, Ef, Sa Pa Mp  (4.7)  ' +  (2.4) Ca, Sc Mg, Tm  cytotoxic (> 0.025)  +  (0.050)  secwsqeqxe 7ten; secwsqdqxa7ten  secwsqeqxe7ten; secwsqdqxa7ten  +  -  +  (3.0) Bs, Ef, Sa Ec Mp  (3.0)  (0.005)  196 aerial S-59a.l.m  + (1.8) Mp  whole plant S-56.1.m  + (10.5) Bs, Sa Ec, Pa  fruit S-56a.l.m  + (10.0) Bs, Sa Mp  •health food  aerial S-21a.l.m  •disinfectant •diarrhea •general health •eye medicine •bee stings •sores, cuts, burns •colds •disinfectant •diarrhea •general health •eye medicine •bee stings •sores, cuts, burns •colds  Lonicera involucrata (Rich.) •arthritis Banks (Caprifoliaceae) •"watery" eczema •measles Black Twinberry; Twinflower •general medicine Honeysuckle  (1.8)  (> 0.20)  kenkeknem sq 'wlus tsitsen Mahonia aquifolium Pursh (Berberidaceae) Tall Oregon-grape stsal's (berries); stsal'sellp (plant)  Ribes lacustre (Pers.) Poir. (Grossulariaceae)  •eye rinse •physic •blood tonic •stomach cramps •haemorrhaging •birthing  + (10.5) Af Ca, Sc Mg, Tm  (> 0.20)  (10.0)  (> 0.20)  + (4.8) Bs, Ef, Sa Mp  + (4.8) Mg  + (0.050)  aerial S-8a.l.m  + (3.7) Bs, Sa Pa Mp  + (3.7) Mg, Tm  + (0.10)  aerial S-72a.l.m  + (1.6) Bs, Ef, Sa Ec, Pa Mp  + (1.6) Af(i.s.) Sc Mg, Tm  + (0.025)  whole plant S-20.1.m  + (1.7) Bs, Ef, Sa Mp  + (1.7) Mg, Tm  Swamp Gooseberry; Bristly Black Currant tlhts 'dlqwtn Rosa acicularis Lindl. (Rosaceae) Wild Rose k'eple7llp (bush)  Rosa woodsii Lindl. (Rosaceae) Wild Rose k'epleUlp (bush)  Rubus idaeus L. (Rosaceae) •eye medicine syn. Rubus strigosus •stomach •diarrhea Wild Raspberry s7dytsqwem, s7eytsqwem (berries); s7aytsqwmdllp (bush)  (> 0.20)  197 Shepherdia canadensis (L.) •purgative Nutt. (Elaeagnaceae) •blood cleanser •fever Soapberry; Soopolallie •laxative •tonic, physic sxusem, sxusa (berries); •acne, eczema sxusemllp (plant) •child birth medicine •colds  aerial S-42a.l.m  + (2.0) Bs, Ef, Sa Pa Mg  + (2.0) Mg, Tm  + (0.050)  root S-42c.3.mw  + (1.3) Bs, Ef, Sa Mg  + (1.3) Mg, Tm  + (0.050)  Spiraea betulifolia Pall. (Rosaceae)  •diarrhea •stomach problems  whole plant S-61.2.m  + (1.6) Bs, Sa Mp  + (1.6) Mg (i.s.) Tm  (> 0.20)  •broken bones •arthritis •eye medicine  fruit S-79a.l.m  + (7.0) Mp  (7.0)  (> 0.20)  Symphoricarpos occidentalis •broken bones Hook. (Caprifoliaceae) •arthritis •eye medicine Waxberry; Snowberry  aerial S-26a.l.m  + (5.5) Bs, Sa Pa Mp  (5.5)  (> 0.20)  + (1.6) Mp  (1.6)  (> 0.20)  (1.3)  (1.3)  (> 0.20)  Birch-leaved Spirea petspetskilul 'ecw; petspetskllullelcw Symphoricarpos albus (L.) Blake (Caprifoliaceae) Waxberry; Snowberry peqpequqse, peqpequqsa 7 (berry); t'el'cwelltkellp, t'el'cwellp (plant)  peqpequqse, peqpequqsa7 (berry); t'el'cwelltkellp, t 'el 'cwellp (plant)  HERBACEOUS FLOWERING PLANTS  Achillea millefolium L . (Asteraceae) Yarrow qets'uye7e7llp  Allium cernuum Roth (Liliaceae) Wild Nodding Onion qwlewe  •sores, cuts •aching bones •arthritis •painkiller •diarrhea •blood purifier •cough  whole plant S-43.1.m  •swollen throat  root S-7a.l.m  198 Apocynum cannabinum L . •warts (Apocynaceae)  aerial S-27a.l.m  Indian Hemp spet'sen; spetse7; spets 'a7; spets 'i  Aralia nudicaulis L . (Araliaceae)  latex S-27b.l  •colds  whole plant S-71.1.m  Wild Sarsparilla stqwiq 'wiycen Arnica cordifolia Hook. (Asteraceae)  + (5.0) Bs, Ef, Sa Pa Mp  (10 ul of 10% v/v) + (6.0) Bs, Ef Pa Mp  (5.0)  + (10 ul of 10% v/v) Tm + (6.0) Af(i.s.)  + (1.3) Mg, Tm  cytotoxic (> 0.025)  (> 0.1%)  (> 0.20)  cytotoxic (>0.10)  •sore eyes •swellings, bruises •cuts  whole plant S-36.1.m  •arthritis •rheumatism •laxative •swelling, bruises •aching muscles  aerial S-19a.l.m  + (2.3) Bs, Ef, Sa Ps Mp  + (2.3) Mg, Tm  (> 0.20)  •colds, cough •sore throat, laryngitis •influenza Northern Wormwood or •headache "Little Sage" •arthritis •laxative p 'nellp; p 'enellp; penp 'ndnllp  aerial S-18a.l.m  + (3.0) Bs, Ef, Sa Pa Mp  + (3.0) Mg, Tm  (> 0.20)  Asclepias speciosa Torr. (Asclepiadaceae)  + whole plant (pre-fiower) (1.0) S-54.1.m Bs  Heart-leaved Arnica  (1.3)  sklaltn xkwetkwtutl 'stns Artemisia dracunculus L . (Asteraceae) Dragon Sagewort or Wild Tarragon skiklminst; sek'elminst; skek'elminst Artemisia frigida Willd. (Asteraceae)  Showy Milkweed  •warts  latex (in fruit) S-54a.2.w  (10% v/v)  (1.0)  cytotoxic (> 0.10)  (10% v/v)  cytotoxic (> 0.1%)  199  Aster conspicuus Lindl. (Asteraceae)  + (1.8) Tm  cytotoxic (> 0.013)  •wounds •impetigo, acne •colds, pneumonia Showy Aster of Blackfoqt •tonsilitis •toothaches (s-)qweq 'wiycen; sq 'wicen •sore joints  aerial S-lOa.l.m  Balsamorhiza sagittata (Pursh) Nutt. (Asteraceae)  leaves S-24a.l.m  + (2.0) Bs, Ef, Sa Pa  + (2.0) Mg, Tm  (> 0.20)  root S-24b.l.m  + (3.5) Bs, Ef, Sa Mp  + (3.5) Mg, Tm  (> 0.20)  root pitch S-24b.2.p  + (2.0) Bs Mp  + (2.0) Mg, Tm  (> 0.20)  (0.14)  + (0.14) Mg  (> 0.20)  •sores, infections •insect bites  Balsamroot; Spring Sunflower; "Sunflower" tsets 'elq (root); ts 'elqenupye7, ts 'elqenupya?', smukwe7cen (leafy plants)  Cirsium undulatum (Nutt.) Spreng. (Asteraceae) Wavy-leaved Thistle  •rheumatism •arthritis •stomach  plant S-73a.l.m  (1.8)  qelsp'u7 Epilobium angustifolium L . •diarrhea (Onagraceae) •hemorrhoids •eczema Fireweed •sore throat •rheumatism ts 'ixnellp; ts 'ixndllp •poison ivy sores  aerial S-67a.l.m  + (2.2) Bs, Ef, Sa Ec Mp  + (2.2) Af(i.s.) Ca, Sc Mg, Tm  Erythronium grandiflorum Pursh (Liliaceae)  aerial S-29a.l.m  + (6.6) Bs, Ef, Sa Ec, Pa Mp  + (6.6) Mg, Tm  (> 0.20)  root S-29b.l.m  + (4.7) Bs, Sa Ec, Pa Mp  + (4.7) Tm  (> 0.20)  Yellow Avalanche Lily; Yellow Dogtooth Violet; "Yellow Lily"; or Kamara Root scwicw  •colds  cytotoxic (>0.10)  200  Fragaria vesca L . (Rosaceae)  'diarrhea  Wild Common Strawberry  whole plant S-60.1.m  (1.2) Bs, Ef, Sa Ec, Pa Mp  (1.2) A f (i.s.) Ca, Sc Mg, Tm  (0.025)  whole plant S-ll.l.m  + (2.8) Bs, Sa Pa Mp  + (2.8) Mg, Tm  + (0.20)  whole plant S-51.1.m  + (2.1) Bs, Sa Ec Mp  + (2.1) A f (i.s.) Tm  (>0.20)  aerial S-66a.l.m  + (4.4) Bs, Ef, Sa Ec, Pa Mp  + (4.4) A f (i.s.) Ca, Sc Mg, Tm  (> 0.20)  root S-66b.l.m  + (3.8) Bs, Ef, Sa Ec, Pa Mp  + (3.8) A f (i.s.) Ca, Sc Mg, Tm  tq'itq'a (berries); tqet'qe7ellp (plant) Fragaria virginiana Duchesne 'diarrhea (Rosaceae) Wild Blueleaf Strawberry tq 'itq 'a (berries); tqet'qe7ellp (plant) Gaillardia aristata Pursh (Asteraceae)  'dandruff 'venereal disease  Blanketflower; Brown-eyed Susan sqleleten re ckwetkwtut 'stens Geum macrophyllum Willd. (Rosaceae)  'tonic  Large-leaved Avens  Heracleum lanatum Michx. (Apiaceae) Cow-parsnip "Wild Rhubard" xwtellp; xwtdllp; sqelemcwucwpye 7; nuxwenxwuxwpye 7  •scabies •psoriasis •skin problems, sores •bladder  root S-38a.l.m  (3.1) Bs, Mp  + (3.1) Af Ca, Sc Mg,Tm  (>0.20)  (> 0.20)  201 Heuchera cylindrica Dougl. (Saxifragaceae)  aerial S-46a.l.m  + (1.5) Bs, Ef, Sa Pa Mp  + (1.5) Mg, Tm  (> 0.20)  root S-46b.l.m  + (3.6) Bs, Ef, Sa Mp  + (3.6) Tm  (> 0.20)  aerial S-75a.l.m  + (4.2) Mp  + (4.2) Tm  (> 0.20)  text'sin; taxt'sU  root S-75b.l.m  + (4.1) Mp  +' (4.1) Tm  (> 0.20)  Lithospermum ruderale Dougl. •sores (Boraginaceae) •rids germs  whole plant S-53.1.m  (0.53)  (0.53)  (> 0.20)  •for nursing mother  root S-74a.l.m  + (3.0)  + (3.0)  cytotoxic (> 0.20)  •fumigant •deodorizer •disinfectant •tonic •colds, cough •tuberculosis •sore throat  fruit S-6a.l.m  + (1.6) Bs Mp  + (1.6) Mg, Tm  (> 0.20)  root S-6b.l.m  + (3.1) Bs Mp  + (3.1) Mg, Tm  (> 0.20)  leaves S-6c.l.m  + (3.0) Bs Mp  + (3.0) Mg, Tm  (> 0.20)  Round-leaved Alumroot legmin  Lilium columbianum Hanson (Liliaceae)  •sores, wounds •upset stomach •canker sores •diarrhea •sore throat  •health food  Tiger Lily  Stoneseed; Lemonweed; Gromwell tsgyvugwpa Lomatium macrocarpum (Nutt.) Coult. & Rose (Apiaceae) Desert Parsley; Wild Carrot; Hog Fennel qweqwila Lomatium nudicaule (Pursh) Coult. & Rose (Apiaceae) "Wild Celery"; Indian Celery; or Indian Consumption Plant qdq 'ma; k'utse  202 Lonicera ciliosa (Pursh) DC. •venereal disease (Caprifoliaceae)  aerial S-64a.l.m  cytotoxic (> 0.050)  (2.1)  (2.1)  whole plant S-78.1.m  + (2.4) Mp  + (2.4) Af Mg, Tm  whole plant S-12.1.m  + (2.1) Pa Mp  •"mosquito poison" •insect repellant  whole plant S-32a.l.m  + (1.9) Bs, Sa Mp  + (1.9) Af Mg, Tm  •sores, wounds •burns •bee stings  whole plant S-62.1.m  + (4.7) Bs  (4.7)  (> 0.20)  •kidney •bladder  whole plant S-13.1.m  + (4.9) Bs, Ef, Sa Pa Mp  + (4.9) Mg, Tm  + (0.20)  Orange Honeysuckle stept 'upelqw Matricaria matricarioides (Less.) Porter (Asteraceae)  •colds •heart medicine  Pineapple Weed  (> 0.20)  tsektsek'qiq'a; tsektsek'qiqen Mentha arvensis L . (Lamiaceae) Canada Mint; Field Mint  •colds, coughs •consumption •fever  (2.1)  + (0.20)  cwecw7u7cw; cw7ecw7u7cw MonardaflstulosaL . (Lamiaceae) Wild Bergamot  (> 0.20)  cw7ecw7u7cw Plantago major L . (Plantaginaceae) Broad-leaved Plantain; Common Plantain slleq'wqe.neellp; slleq 'wqe.ndka7; slleqwlleq7wqeneneke7 Pyrola asarifolia Michx. (Ericaceae) Pink Wintergreen; Beaver Ears sqeqlewen'e; sqeqlewne  203  Valeriana sitchensis Bong. (Valerianaceae) Mountain Valerian  •fever, coughs •upset stomach •toothache  kikwe; kikwa; skwelk'welt  Verbascum thapsus L . (Scrophulariaceae)  aerial S-70a.l.m  (10.0)  (> 0.20)  (6.0) Bs, Sa Mp  (6.0)  (> 0.20)  leaves S-47a.l.m  + (1.9) Sa Ec, Pa Mp  + (1.9) Tm  cytotoxic (> 0.10)  root S-la.l.m  + (5.6) Bs, Sa Ec, Pa Mp  + (5.6) Mg, Tm  root S-70b.l.m  •earaches •sores  Mullein; "Wild Tobacco" *smenxul Zigadenus venenosus Wats. (Liliaceae) Death Camas; "Poison Onion"  *sore legs or aches  + (10.0) Bs Mp  (>0.20)  yiwdstn; yiwesten "Extracts that inhibited the growth of one or more species of microorganism are designated as "+" and the inhibited microorganisms are listed. Extracts showing no activity against any of the species of microorganism tested are designated as Inhibition of fungal spore formation is indicated as "(i.s.)" following the relevant fungal species. The amount of dried crude extract (in mg) per disc is indicated in parentheses for antibacterial and antifungal assays. The minimum inhibitory concentration (MIC) is indicated in parentheses for antiviral (HSV1) assays. Botanical Latin names are from Hitchcock and Cronquist (1973) for vascular plants and Maries et al.(2000) for lichens and fungi 'Common and Secwepemc names are from Turner and Ignace (in preparation). Note that every effort was made to ensure accuracy but the names are based on a manuscript that is not in its final form. Note also that" " indicates no recorded Secwepemc name. Medicinal indications are from Turner and Ignace (in preparation). Note that every effort was made to ensure accuracy of Secwepemc medicinal uses but the list is based on a manuscript that is not in its final form. Note also that uses listed here are not inclusive and have been abbreviated. The inclusion of this list is strictly for scientific evaluation in relation to the research in this dissertation. The inclusion of this information should in no way be interpreted as a recommendation for self-medication. The information has been included with permission from the Secwepemc Cultural Education Society and investigators of the Secwepemc Ethnobotany Project. Further use of this information should request permission of the Secwepemc Cultural Education Society (Kamloops) and should acknowledge the original contributors as indicated in Turner and Ignace (in preparation) rather than this dissertation. Bacteria used are listed in Table 2.3 and abbreviated as follows: Bs = Bacillus subtilis, Ef= Enterococcus faecalis, Sa = Staphylococcus aureus, Ec = Escherichia coli, Ps = Pseudomonas aeruginosa, Mp = Mycobacterium phlei Fungi used are listed in Table 2.4 and abbreviated as follows: Af= Aspergillus fumigatus, Ca = Candida albicans, Sc = Saccharomyces cerevisiae, Mg = Microsporum gypseum, Tm = Trichophyton mentagrophytes. Virus used: HSV1 = Herpes simplex virus type 1. Antiviral activity could not be assessed accurately when cytotoxicity was present. Cytotoxic concentrations of crude extract are indicated in parenthesis. b  d  e  8  APPENDIX C:  Chapter 3 Supplementary D a t a  205 Figure C . l . Summary of 'H NMR spectral data of crystal I (400 MHz, CDC1 ). Assignments were based on Bohlmann et al. (1985), with the assistance of N. Stoynov (Department of Chemistry, UBC). Abbreviations are as follows: s = singlet; d = doublet; / = triplet; m = multiplet; J = coupling constant. 3  0.55 (d, 1H, J = 4.3 Hz, H-19oc), 0.83 (d, 1H, J = 4.2 Hz, H-19p), 0.86 (s, 3H, CH -30), 0.86 (m, 1H, H-6p), 0.94 (dd, 3H, J = 9.8, 6.5 Hz (?), CH -21), 1.02 (s, 3H, CH -28), 1.08 (s, 3H, CH 29), 1.16 (m, 5H, H-7oc, H-5, CH -18), 1.41 (m, 2H, H-7(3, H-15a), 1.54 (m, 1H, H-lp), 1.59 (m, 1H, H-8), 1.61 (s, 3H, CH -27), 1.64 (m, 1H, H-6a), 1.66 (br. s, 3H, CH -26), 1.84 (m, 1H, Hla), 2.02 (m, 1H, H-17), 2.04 (m, 1H, H-15p), 2.07 (m, 1H, H-20), 2.28 (ddd, 1H, J = 14.0, 4.1, 2.6 Hz, H-2a), 2.69 (ddd, 1H, J = 13.9, 13.7, 6.4 Hz, H-2p), 4.48 (m, 2H, H-23, H-16), 5.37 (t, 1H, J = 8.4, 1.0 Hz, H-24); (H-lla, H-llp, H-12a, H-12P, H-22a andH-22p are obscured). 3  3  3  3  3  3  3  Figure C.2. Summary of H NMR spectral data of 16R,23^-dihydroxycycloartenone (400 MHz, CDC1 ) from Bohlmann et al. (1985:2035). Abbreviations are the same as in Figure C . l . !  3  0.59 (d, 1H, J = 4 Hz, H-19a), 0.80 (d, 1H, J = 4 Hz, H-19p), 0.90 (s, 3H, CH -30), 0.92 (m, 1H, H-6p), 0.98 (d, 3H, J = 7 Hz (?), CH -21), 1.02 (s, 3H, CH -28), 1.08 (s, 3H, CH -29), 1.12 (m, 2H, H-7a, H-5), 1.16 (s, 3H, CH -18), 1.40 (m, 1H, H-7P), 1.43 (dd, 1H, J = 13.5, 5 Hz, H-15a), 1.52 (ddd, 1H, J = 14, 6, 2.5 Hz, H-lp), 1.66 (dd, 1H, J = 13, 4 Hz, H-8), 1.66 (br. s, 3H, CH 27), 1.69 (ddd, 1H, J = 13, 4, 4 Hz, H-6a), 1.75 (br. s, 3H, CH -26), 1.84 (ddd, J = 14, 14, 4 Hz, 1H, H-la), 2.01 (dd, 1H, J = l l , 7 H z (?), H-17), 2.01 (dd, 1H, J = 13.5, 8 Hz, H-15P), 2.05 (m, 1H, H-20), 2.29 (ddd, 1H, J = 14, 4, 2.5 Hz, H-2a), 2.71 (ddd, 1H, J = 14, 14, 6 Hz, H-2p), 4.38 (br. t, 1H, J = ?, H-23), 4.41 (ddd, 1H, J = 8, 7, 5 Hz, H-16), 5.16 (br. /, 1H, J = 7 Hz, H-24); (H11a, H-llp, H-12a, H-12p, H-22a andH-22p are obscured). 3  3  3  3  3  3  3  206  Figure C.3. GC-MS of crystal I. Mass Spectrum 1 is of the first flagged peak at 27.33 minutes in Chromatogram 1. Mass Spectrum 2 is of the second flagged peak at 27.85 minutes in Chromatogram 1.  207  OH  .OH  •-H  ^30^46^2  438.69  C30H46O2  438.69  This is the highest mass in the GC-MS spectrum  This is the most prominent signal in MS (typical for 14-methyl sterols)  Figure C.4. Interpretation of the fragmentation pattern of crystal I, based on combined GC-MS and X ray crystallographic data (Nikolay Stoynov, pers. comm. 2000).  208  Formula FW Colour, habit Crystal size, mm Crystal system Space group a, A  C30H48O3  456.71 colourless, needle 0.50x0.30x0.20 monoclinic P2i  b,k c,  A  v,A z Dcalc, g/cm F(000) Radiation /i, mm" Transmission factors <p oscillation range (%=-90.0) co oscillation range (%= -90.0) 3  1  9A  o  Cmaxj  Total reflections Unique reflections No. of variables Rmerge  R; Rw Goodness of fit Max A/a (final cycle) Residual density, e/A  3  15.295(1) 7.6187(7) 15.960(2) 98.39(1) 1358.7(2) 2 1.116 504.00 Mo-Ka 0.07 0.813 - 1.000 0.0 - 190.0° -18.0-23.0° 50.5° 10548 4546 314 0.069 0.038; 0.109 0.91 0.00 0.22, -0.24  Table C.l. X-ray crystallographic data summary for crystal I (B. Patrick, pers. comm. 2000). This data summary is provided for completeness only. For further details and abbreviations see Patrick (2000).  209  Table C.2. Mass intensity table (from m/z = 67 to 440) of the electron impact mass spectra of band 2 (from Figure 3.18). Abbreviations are as follows: mass/charge rartio (m/z); intensity in counts (Int) and percentage of base peak m/z = 83 (%BP). m/z  Int  %BP m/z  Int  %BP m/z  Int  %BP m/z  Int  %BP m/z  Int  %BP  67  9  1  134  39  5  184  20  2  229  30  4  291  10  1  68  4  0  135  54  7  185  55  7  230  34  4  292  13  2  69  20  2  136  19  2  186  35  4  231  37  4  300  13  2  71  15  2  137  19  2  187  40  5  232  20  2  301  6  1  77  14  2  145  24  3  188  25  3  240  35  4  302  97  12  79  10  1  146  24  3  189  30  4  241  60  7  303  32  4  81  8  1  147  160  19  195  20  2  242  318  38  304  6  1  82  29  3  148  30  4  196  30  4  243  110  13  316  13  2  83  830  100  149  25  3  197  52  6  244  28  3  320  17  2  84  49  6  150  10  1  198  35  4  245  40  5  336  10  1  85  14  2  151  40  5  199  52  6  246  15  2  340  7  1  91  24  3  152  40  5  200  25  3  247  20  2  342  80  10  93  6  1  153  20  2  201  30  4  251  13  2  343  20  2  96  19  2  155  18  2  202  15  2  255  6  1  348  10  1  97  19  2  157  18  2  203  30  4  256  33  4  356  6  1  100  24  3  159  25  3  205  20  2  257  53  6  358  17  2  105  11  1  161  25  3  211  30  4  258  73  9  360  135  16  107  11  1  162  25  3  212  45  5  259  160  19  361  28  3  109  11  1  163  75  9  213  105  13  260  133  16  362  6  1  110  9  1  164  30  4  214  85  10  261  36  4  372  6  1  111  9  1  165  38  5  215  60  7  262  8  1  374  30  4  115  11  1  166  22  3  216  30  4  273  10  1  375  6  1  119  9  1  167  20  2  217  48  6  274  38  5  376  13  2  121  14  2  169  25  3  218  35  4  275  23  3  390  6  1  122  8  1  171  30  4  219  18  2  276  26  3  402  153  18  123  29  3  172  30  4  223  20  2  277  13  2  403  37  4  125  14  2  173  29  3  224  145  17  278  17  2  404  8  1  128  14  2  175  20  2  225  92  11  279  8  1  416  8  1  129  9  1  179  20  2  226  38  5  282  6  1  418  8  1  131  9  1  181  20  2  227  96  12  284  23  3  426  5  1  133  11  1  183  25  3  228  38  5  '285  10  1  440  2  0  210 Table C.3. Mass intensity table (from m/z = 67 to 440) of the electron impact mass spectra of 4acetoxyisopruteninone (from Figure 3.19). Abbreviations are the same as in Table C.2.  Match Spectrum 91746 from nist98m.lbr Library. Name: (Z)-6a,8,l l-trihydroxy-(8S,l lR)-Guaia-3,10(14)-dien-12-oic acid, 12,6-lactone,8-acetate ll-(2methylcrotonate) Alternative Name: 4-acetoxyisopruteninone FW: 402 Formula: C22H26O7 CAS No: 33439-67-7 Int  %BP m/z  Int  %BP  185  2778  28  227  1067  11  11  186  1003  10  242  10000 100  1427  14  188  1066  11  243  9281  93  161  1075  11  196  705  7  260  568  6  18  164  857  9  197  1076  11  262  1287  13  1148  11  169  1072  11  199  3363  34  302  645  6  145  2152  22  171  3221  32  200  919  9  402  6443  64  33  146  1637  16  172  2780  28  213  1065  11  1642  16  147  1422  14  173  2014  20  214  2276  23  2784  28  148  999  10  184  1715  17  215  1069  11  Int  %BP m/z  Int  %BP m/z  105  2075  21  157  1503  15  36  109  1424  14  158  1079  1934  19  129  2364  24  159  81  2838  28  135  4220  42  82  3593  36  143  1782  83  7879  79  144  84  2490  25  91  3275  93 95  m/z  Int  %BP m/z  67  2909  29  69  3555  79  211 Table C.4. Comparison of fragmentation patterns of known products from Balsamorhiza sagittata leaves, based on Bohlmann et al. (1985). Prepared with the assistance of N . Stoynov (Department of Chemistry, UBC). Abbreviations are as follows: mass/charge ratio (m/z); Compound (Cmpd); Acetate (Ac) ; Angelate (Ang) ; Senecioate (Sen) ; Epoxy angelate (Epang) . In the first column, M indicates the molecular mass ion peak; [M-OH] indicates the fragment obtained by loss of OH ( 0+ H =17) from the molecular ion, thus the corresponding entry in the second column is M-l7; and [M-H 0] indicates the fragment obtained by loss of H 0 ( 0 + 'H + 'H = 18), thus the corresponding entry in the second column is M-18. For each of Compounds 2-13, the molecular mass ion peak is indicated at the top of the column (with % of the base peak shown in parentheses) and each observed mass is indicated below (with % of the base peak in parentheses). A "-" indicates a fragment was neither expected nor reported while a "?" indicates that a fragment was expected but not reported (which could be due to a weak signal). An "??" indicates that the reported fragment could not be explained. a  b  0  d  +  +  16  ]  +  2  16  2  YNX-VX  Formula of the detected mass ion peak  m/z  M [M-OH] [M-H 01 TM-OAC1+ [M-HOAc] TM-HOAC-C01+ [M-HOAng] [M-HOSenl +  M-17 M-18 M-59 M-60 M-88 M - l 00 M - l 00  +  +  2  +  +  +  [M-HOAng-H 0] [M-HOSen-H 0] [M-HOSen-2H 01 [M-HOAc-HOAng] rM-HOAc-HOAng-H 01 +  2  +  2  +  2  +  2  rc H coi+ rcH i+ 4  7  4  7  ' r***^'?^  HCT  o  +  M-118 M-118 M-136 M - l 60 M - l 78 83 55  Cmpd 2  Cmpd 3  Cmpd 4  404 (0.7)  362 (0.4)  362 (0.9)  344 (8) 316(5) 304 (1) 304 (1)  344(1.7)  344 (91) 262 (3)  -  244 (25) 226 (7) 83 (100) 55 (98)  262 (3) 244 (6) 226(1.7) 83 (100) 55 (24)  244 (4) 83 (100) 55 (21)  Cmpd 5 362 (0.5) ? 262 (0.8) 244 (2) ? 83 (100) 55 (62)  o  0  o  0  Cmpd 6 404 (0.8) ? 344 (0.8) 316(4) 244 (24) 226 (7) 83 (100) 55 (98)  Cmpd 7 404(1.5) 387 (0.6) ? 345 (1) 344 (1) 244 (4.5) 226(1.2) 83 (100) 55 (44)  212  Table C.4. continued.  0  m/z +  [M-H 01  +  2  [M-H C=C=OC]  +  2  [M-OAcl  +  [M-HOAc]  +  [M-HOAc-C01 [M-HOAng] [M-HOSenl  +  +  +  [M-HOEpang]  +  [M-HOAng-H 01  +  +  [M-HOEpang-H fJ|  +  2  [M-HOSen-2H 01  +  2  fM-HOEpang-H C=CHCH l 2  [M-HOAc-HOAng]  +  3  +  [M-OAc-HOEpangl  +  [M-HOAc-HOEpang]  +  [M-HOAc-HOAng-H 01  +  2  [M-HOAc-HOEpang-H 0]  +  [M-HOAc-HOEpang-C01  [M-HOAc-HOEpang-CO-MeOHl  +  [M-HOAc-HOEpang-2CO-MeOHl [C H CO] 4  +  +  7  ?? rc H i 4  a  7  Acetate  b  Angelate  Cmpd 9 Cmpd 10 Cmpd 11 Cmpd 12 Cmpd 13  -  -  -  -  M-18  -  414(1.2)  -  -  M-42  -  -  -  -  M-59  404 (0.6)  -  -  -  -  -  M-60  -  -  -  -  358 (2.5)  -  M-88  -  -  -  -  -  -  M-100  -  -  -  -  -  -  M-100  -  M - l 16  -  M - l 18  -  M - l 18  -  M - l 34 M-136  -  416(19)  358 (16)  -  418(3)  -  -  -  376 (8)  -  258 (8)  418(0.2)  -  -  -  -  302 (2.5)  -  -  -  -  -  -  -  -  -  298 (1.7)  -  -  -  -  -  -  -  -  M-158  -  -  -  260 (5.5)  M - l 60  -  -  -  -  -  -  241 (100)  -  -  -  316(1)  300(11)  258 (69)  M - l 75  -  M-176  288 (2.6)  M-178  -  -  -  -  M-194  -  -  -  -  256 (4)  M-204  260 (9)  -  -  M-236  228 (7)  -  -  M-264  200(10)  -  -  83  -  . -  -  61  -  -  -  55  +  its  M-17  2  +  Cmpd 8  0  ,A-  HO-It/*—v 1 H | .."  432 (0.4)  2  [M-HOSen-H,0]  O  464 (0.3)  +  [M-OH]  O  n\ °  Formula of the detected mass ion peak M  o  55(100)  c  55 (100)  Senecioate  -  -  224 (20)  224 (4)  -  -  -  -  -  -  -  -  61 (100)  55 (27)  d  242 (8)  83 (100)  -  260 (6)  242 (12)  -  302 (4)  Epoxy angelate  55 (100)  213 Table C.5. Interpretations of the fragmentation pattern for band 2 assuming molecular mass ion peak of m/z = 402, 416, 418, 426, or 440. Prepared with the assistance of N. Stoynov (Department of Chemistry, UBC). Abbreviations are the same as in Table C.4. Values in parentheses indicate % of base peak for the fragment. For convenience, bold face type indicates the major fragments observed.  Formula of the ion M  m/z -  +  [M-OH]  M-17  +  [M-H 0]  +  2  [M-H C=C=01  +  2  [M-OAc]  [M-HOAc"]  [M-HOAc-C01 [M-HOEpang"|  [M-HOAng-H 0] or [M-HOSen-H 0]  416(0.5)  418 (0.5)  426 (0.4)  440 (0.3)  -  -  360 (16)  M-116  +  402 (18)  M-42  M-100  +  FW440  374 (4)  [M-HOAng] or [M-HOSen"] +  FW426  398 (0.2)  M-88  +  FW418  384(1)  M-60  +  FW416  M-18 M-59  +  FW402  -  342 (10) -  302 (12) -  -  356(1)  316(2)  -  -  -  -  376 (2) -  358 (2)  -  -  398 (0.5)  -  -  -  -  -  -  -  -  -  340(1)  300 (2)  302 (12)  -  -  298 (1)  300 (2)  -  -  +  2  M-118  +  284 (3)  2  rM-HOEpang-H 0]  +  2  rM-HOSen-2H 01  +  2  rM-HOEpang-H C=CHCH l 2  [M-HOAc-HOAng1  -  -  284 (3)  M-136  -  -  282 (1)  3  -  292 (2) -  -  -  -  M-160  242 (38)  -  -  -  M-175  227 (12)  -  -  M-158  +  +  [M-OAc-HOEpang]  M-134  -  282 (1) -  +  or [M-HOAc-HOAng-CH ]  +  251 (2)  -  3  [M-HOAc-HOEpang]  M-176  +  [M-HOAc-HOAng-H 01  M-178  +  2  [M-HOAc-HOEpang-H 0]  +  2  [M-HOAc-HOEpang-C01  +  [M-HOAc-HOEpang-CO-MeOH]  +  [M-HOAc-HOEpang-2CO-MeOHl [C H CO] 4  7  +  +  224 (17)  -  242 (38) -  224 (17)  -  -  -  -  M-194  -  -  -  -  M-204  -  -  -  -  -  M-236  -  -  -  -  -  M-264  -  -  -  -  83  83 (100)  152 (5) 83 (100)  83 (100)  83 (100)  83 (100)  (202.0 nm  0.00200'.00  $00'.00  400'.  32.087 minutes,  06  '  '  500'. 00 nm  600.00  700'.00  192 - 797  Figure C.5. Ultraviolet spectrum of band 2 (HPLC elution peak at 32.087 minutes). Abbreviations: A U = Absorbance Units; nm = wavelength.  L  a (u o o to cnroc i •»» a o n o o 0e) 10 01 O «rt O O CO C O O v . . I OOIOIOI0O •OZ30 ni --rfvr^o o>o-«*4 orocuoiv  oo-varum TP10»  one • oaoui «H  a<  o o o ooa.  o-<r o»i_i cu ionm CD  co n m t— CL -S, x cats to com xcva. a cacoocat-cax a. ecu tea:»- u . o a  OH"  Figure C.6. 'H NMR spectrum of band 2.  oo oomo OOOOOOIDOI nuaonoono ar-^vio  f  ni**'** i o v « ro zcw X U OS SS  tnnjx>-«ruMo.rt jnuuii.ii.xan  ox  r©  APPENDIX D:  The St'at'imc Nation Statement of Proprietary Rights Statement of the Union of B. C. Indian Chiefs' Protecting Knowledge: Traditional Resource Rights in the New Millennium Conference  FROM TIME OUT OF MIND  Statement of Proprietary Rights Over All Species on our Traditional Territory St'at'imc Nation Mount Currie, BC Lil'wat Nation From time out of mind, St'at'imc peoples have gone into the forest to gather plants, soils and creatures for food, for healing and for spiritual purposes. As a result of this inherent relationship we have a proprietary interest andrightto all species on our traditional territory, and to our cumulative knowledge of their preparation and use, as part of our property and cultural heritage. Our trees have been stolen, and now we are faced with global interest in our other resources. Therefore in the interests of preservation of biodiversity and the survival of our peoples, we hereby give notice that all gathering and resources extraction activities on our unceded traditional territory fall under the ownership and jurisdiction of the St'at'imc and will be enforceable by the St'at'imc Nation Tribal Police. All collectors and in-field buyers of mushrooms and other nontimber forest resources collected for commercial purposes on these territories will require a permit.  February 22, 2000  PROTECTING  KNOWLEDGE  T R A D I T I O N A L RESOURCE RIGHTS IN T H E N E W M I L L E N N I U M SPIRIT OF T H E C O N F E R E N C E Although we have been subjected to colonial forces for several centuries, we retain and affirm all of our inherent collective rights as sovereign nations. These rights include the right to protect our own survival, in particular, by protecting our cultures, languages, and knowledge systems from expropriation, encroachment, or theft. 1. Indigenous Peoples' own languages, knowledge systems and laws are indispensable to their identity, and are a foundation for self-determination. 2. Indigenous Peoples' knowledge systems are inextricably and inalienably connected with their ancestry and ancestral territories. 3. Indigenous Peoples' heritage is not a commodity, nor the property of the nation-state. The material and intellectual heritage of each Indigenous People is a sacred gift and a responsibility that must be honoured and held for the benefit of future generations. 4. Indigenous rights, individual and collective, are defined by Indigenous laws. The starting-point for any consideration ofrightsto learn, use or transmit Indigenous knowledge therefore must therefore be the laws of the Indigenous peoples concerned. 5. The use of Indigenous Peoples' knowledge or resources is unlawful and illegitimate unless it is done in conformity with the laws of the Peoples concerned. ACTIONS  1. We will take steps within our own communities to ensure that our people, and in particular our children, learn our own laws concerning the acquisition and use of knowledge and resources, and the credentials of knowledge-keepers, so that they can fully enjoy the right to self-determination. 2. We condemn all trade in unlawfully-obtained resources or knowledge, and we will act jointly at the local, regional, national, and international levels to deprive corporations and governments of any profit from such trade through effective international legal, political and economic actions. 3. We will cooperate to establish an effective international network to monitor the activities of corporations in the ancestral territories of Indigenous Peoples worldwide, and to support Indigenous Peoples everywhere in the full exercise of their rights. 4. We will take steps to prevent any assertion of intellectual property rights to the genetic integrity or genetic potential of biotic systems in our ancestral territories. 5. We will press for the ratification and full implementation of all international conventions in the fields of human rights, Indigenous Peoples, and their ecosystems, which we deem applicable. 6. We will work together for the speedy adoption of the Draft Principles and Guidelines on the Heritage of Indigenous Peoples by the United Nations. 7. We will promote the adoption of the Principles and Guidelines by our own Nations and Peoples as an international compact among ourselves, and as a basis for dealing with non-indigenous interests, and we will work together to establish a global registry of Indigenous Nations and Peoples who have agreed to implement the Principles and Guidelines within their own territories. 8. We support all international standard-setting initiatives by Indigenous Peoples that advance these actions. Hosted by the Union Of British Columbia Indian Chiefs with support from Law Foundation Of British Columbia, Legal Services Society Of British Columbia - Native Programs, UBC Museum Of Anthropology & Canadian International Development Agency Vancouver, British Columbia, Canada Wednesday, February 23 to Saturday, February 26*, 2000 rt  APPENDIX E: The International Society of Ethnobiology Code of Ethics  221  International Society of Ethnobiology (ISE) Code of Ethics Preamble This Code of Ethics has its origins in the Declaration of Belem agreed upon in 1988 at the Founding of the international Society of Ethnobiology (in Belem, Brazil). It is acknowledged that much research has been undertaken in the past without the sanction or prior consent of indigenous and traditional peoples and that such research has resulted in wrongful expropriation of cultural and intellectual heritage rights of the affected peoples causing harm and violation of rights. The ISE is committed to working in genuine partnership and collaboration with indigenous peoples, traditional societies and local communities to avoid these past injustices and build towards developing positive, beneficial and harmonious relationships in the field of ethnobiology. The ISE recognises that culture and language are intrinsically connected to land and territory, and cultural and linguistic diversity are inextricably linked to biological diversity. Therefore, the right of Indigenous Peoples to the preservation and continued development of their cultures and languages and to the control of their lands, territories and traditional resources are key to the perpetuation of all forms of diversity on Earth. Purpose The Purpose of this Code of Ethics is: (i)  to optimise the outcomes and reduce as much as possible the adverse effects of research (in all its forms, including applied research and development work) and related activities of ethnobiologists that can disrupt or disenfranchise indigenous peoples, traditional societies and local communities from their customary and chosen lifestyles; and  (ii)  to provide a set of principles to govern the conduct of Ethnobiologists and all Members of the International Society of Ethnobiology (ISE) engaged in or proposing to be engaged in research in all its forms, especially collation and use of traditional knowledge or collections of flora, fauna, or any other element found on community lands or territories.  The ISE recognises, supports and prioritises the efforts of indigenous peoples, traditional societies and local communities to undertake and own their research, collections, databases and publications. This Code is intended to enfranchise indigenous peoples, traditional societies and local communities conducting research within their own society, for their own use.  222  It is hoped that this Code of Ethics will also serve to guide ethnobiologists and other researchers, business leaders, policy makers, and others seeking meaningful partnerships with indigenous peoples, traditional societies and local communities and thus to avoid the perpetuation of past injustices to these peoples. The ISE recognises that, for such partnerships to succeed, all relevant research activities must be collaborative. Consideration must be given to the needs of all humanity, and to the maintenance of robust and vigorous scientific standards. It is desirable that scientists, international citizens and organisations, and indigenous peoples and local communities collaborate to achieve the purpose of this Code of Ethics and the objectives of the ISE. Principles The Principles of this Code are to embrace, support, and embody the many established principles and practices of international law and customary practice as expressed in various international instruments and declarations including, but not limited to, those documents referred to in Appendix 1 of this Code of Ethics. The following Principles are the fundamental assumptions that form this Code of Ethics. 1. Principle of Prior Rights. This principle recognises that indigenous peoples, traditional societies, and local communities have prior, proprietaryrightsand interests over all air, land, and waterways, and the natural resources within them that these peoples have traditionally inhabited or used, together with all knowledge and intellectual property and traditional resource rights associated with such resources and their use. 2. Principle of Self-Determination. This principle recognises that indigenous peoples, traditional societies and local communities have a right to self determination (or local determination for traditional and local communities) and that researchers and associated organisations will acknowledge and respect suchrightsin their dealings with these peoples and their communities. 3. Principle of Inalienability. This principle recognises the inalienablerightsof indigenous peoples, traditional societies and local communities in relation to their traditional territories and the natural resources within them and associated traditional knowledge. Theserightsare collective by nature but can include individualrights.It shall be for indigenous peoples, traditional societies and local communities to determine for themselves the nature and scope of their respective resourcerightsregimes. 4. Principle of Traditional Guardianship. This principle recognises the holistic interconnectedness of humanity with the ecosystems of our Sacred Earth and the obligation and responsibility of indigenous peoples, traditional societies and local communities to preserve and maintain their role as traditional guardians of these ecosystems through the maintenance of their cultures, mythologies, spiritual beliefs and customary practices.  223  5. Principle of Active Participation. This principle recognises the crucial importance of indigenous peoples, traditional societies and local communities to actively participate in all phases of the project from inception to completion, as well as in application of research results. 6. Principle of Full Disclosure. This principle recognises that indigenous peoples, traditional societies and local communities are entitled to be fully informed about the nature, scope and ultimate purpose of the proposed research (including methodology, data collection, and the dissemination and application of results). This information is to be given in a manner that takes into consideration and actively engages with the body of knowledge and cultural preferences of these peoples and communities. 7. Principle of Prior Informed Consent and Veto. This principle recognises that the prior informed consent of all peoples and their communities must be obtained before any research is undertaken. Indigenous peoples, traditional societies and local communities have the right to veto any programme, project, or study that affects them. Providing prior informed consent presumes that all potentially affected communities will be provided complete information regarding the purpose and nature of the research activities and the probable results, including all reasonably foreseeable benefits and risks of harm (be they tangible or intangible) to the affected communities. 8. Principle of Confidentiality. This principle recognises that indigenous peoples, traditional societies and local communities, at their sole discretion, have the right to exclude from publication and/or to have kept confidential any information concerning their culture, traditions, mythologies or spiritual beliefs. Furthermore, such confidentiality shall be guaranteed by researchers and other potential users. Indigenous and traditional peoples also have the right to privacy and anonymity. 9. Principle of Respect. This principle recognises the necessity for researchers to respect the integrity, morality and spirituality of the culture, traditions and relationships of indigenous peoples, traditional societies, and local communities with their worlds, and to avoid the imposition of external conceptions and standards. 10. Principle of Active Protection. This principles recognises the importance of researchers taking active measures to protect and to enhance the relationships of indigenous peoples, traditional societies and local communities with their environment and thereby promote the maintenance of cultural and biological diversity. 11. Principle of Precaution. This principle acknowledges the complexity of interactions between cultural and biological communities, and thus the inherent uncertainty of effects due to ethnobiological and other research. The Precautionary Principle advocates taking proactive, anticipatory action to identify and to prevent biological or cultural harms resulting from research activities or outcomes, even if cause-and-effect relationships have not yet been scientifically proven. The prediction and assessment of such biological and cultural harms must include local criteria and indicators, thus must fully involve indigenous peoples, traditional societies, and local communities.  224  12. Principle of Compensation and Equitable Sharing. This principle recognises that indigenous peoples, traditional societies, and local communities must be fairly and adequately compensated for their contribution to ethnobiological research activities and outcomes involving their knowledge. 13. Principle of Supporting Indigenous Research. This principle recognises, supports and prioritises the efforts of indigenous peoples, traditional societies, and local communities in undertaking their own research and publications and in utilising their own collections and data bases. 14. Principle of The Dynamic Interactive Cycle. This principle holds that research activities should not be initiated unless there is reasonable assurance that all stages of the project can be completedfrom(a) preparation and evaluation, to (b) full implementation, to (c) evaluation, dissemination an