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Ethnopharmacology of western North American plants with special focus on the genus Artemisia L. McCutcheon, Allison R. 1996

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ETHNOPHARMACOLOGY OF WESTERN NORTH AMERICAN PLANTS WITH SPECIAL FOCUS ON THE GENUS ARTEMISIA L. \ by ALLISON R. McCUTCHEON B'.Sc, The Univeristy of British Columbia, 1987 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENT FOR THE DEGREE OF DOCTOR OF PHILOSOPHY . in , THE FACULTY OF GRADUATE STUDIES (Department of Botany) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA 1996 © Allison R. McCutcheon In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department The University of British Columbia Vancouver, Canada DE-6 (2/88) ABSTRACT This thesis is comprised of a series of investigations into the pharmacological activities of plants from western North America. In the first phase of the research, one hundred methanolic plant extracts were screened for: antibiotic, antifungal, anti-mycobacterial and antiviral activity. Eighty-nine of these extracts exhibited antibiotic activity and eighty-one exhibited antifungal activity. Nineteen extracts also showed anti-mycobacterial activity. There was a correlation (0.945) between anti-mycobacterial activity and strong activity against the fast growing, non-pathogenic Mycobacterium phlei which was used in the antibiotic screening. Twelve extracts were each active against one of the seven viruses screened. Several interesting observations arose from the analyses of the phase one screening results. There was a significant correlation between anti-mycobacterial activity and the specific usage of the plants to treat tuberculosis. Significantly higher percentages of active plants were found among those categorized as potential antibiotics and antifungals based on their traditional usage. There appeared to be correlations between activity and the taxa to which the active plants belonged and the habitats they were collected from. The phase two screening of one hundred eighty-five extracts was designed to further test these apparent correlations. In these phase two screenings, 77% of the extracts exhibited antibiotic activity. Seventy-five percent (75%) of the plants which were used medicinally were active while only 22% of the non-medicinal plants were active. Of the plants which were classified as potential antibiotics based on their traditional uses, 91% were active. The taxa with the highest percentage of active extracts were the Filicinae and the Gymnospermae. Fifty-nine percent (59%) of the extracts exhibited significant activity in the phase two antifungal screening. The taxon with the largest percentage of active extracts was the Gymnospermae (100% active). There was a great difference in the percentage of active extracts among the traditional plant medicines (32% active) compared to the non-medicinal plants (5% active). Seventy-five percent (75%) of the plants classified as potential antifungals based on their traditional uses were found to have significant activity. Throughout these phase one and two screenings, the members of the genus Artemisia L. assayed were particularity noteworthy for their broad spectrum of activity. Therefore, this genus was chosen for more extensive research on the anti-infectious properties of 74 additional samples from 30 Artemisa taxa. All of the Artemisia I l l samples exhibited antibiotic and antifungal activity. In the antiviral assays, a total of 18 extracts inhibited the virally induced cytopathic effects. A total of twenty-nine extracts exhibited activity in the anti-mycobacterial assays. There were representative samples from each of the four Artemisia subgenera among the active extracts in each of the four screens, although it was noted that the extracts with the strongest activity in the anti-mycobacterial assays were all members of the subgenera Dracunculus and Tridentatae. In all of the Artemisia assays, there was as much variation in activity among samples of a taxa (species or subspecies) as there was between taxa. Samples of the Artemisia species which were most frequently cited in the ethnobotanical literature (A. dracunculus, A. frigida, A. ludoviciana and A. tridentata) were among the most active extracts in all of the assays. IV Table of contents Abstract ii. List of Tables viii. Acknowledgements x. Foreword xii. Dedication xiv. 1.0 General introduction 1. 2.0 Introduction to phase one research 3. 2.1 Phase one antibiotic screening 6. 2.1.1 Introduction 6. 2.1.2 Methods 6. 2.1.3 Results 9. 2.1.4 Discussion and conclusions 23. 2.2 Phase one antifungal screening 26. 2.2.1 Introduction 26. 2.2.2 Methods 27. 2.2.3 Results 29. 2.2.4 Discussion and conclusions 44. 2.3 Phase one anti-mycobacterial screening 46. 2.3.1 Introduction 46. 2.3.2 Methods 47. 2.3.3 Results 48. 2.3.4 Discussion and conclusions 50. 2.4 Phase one antiviral screening 2.4.1 Introduction. 2.4.2 Methods 2.4.3 Results 2.4.4 Discussion and conclusions 2.5 Phase one ethnopharmacological analyses 2.5.1 Introduction 2.5.2 Methods 2.5.3 Results 2.5.4 Discussion and conslusions 2.6 Conclusions from phase one research 2.6.1 Introduction 2.6.2 Leads on potential antimicrobial agents 2.6.3 Conclusions from ethnopharmacological analyses 2.6.4 Other factors which may be correlated with antimicrobial activity 3.0 Introduction to phase two research 3.1 Phase two antibiotic screening 3.1.1 Introduction 3.1.2 Methods 3.1.3 Results 3.1.4 Discussion and conclusions 3.2 Phase two antifungal screening 2.2.1 Introduction 2.2.2 Methods 2.2.3 Results 2.2.4 Discussion and conclusions 4.0 Conclusions from ethnopharmacological screenings 54. 54. 55. 56. 58. 61. 61. 61. 64. 67. 69. 69. 69. 73. 74. 76. 80. 80. 80. 84. 107. 112. 112. 113. 116. 136. 139. VI 5.0 Introduction to Artemisia research 5.1 Antibiotic screening of Artemisia species 5.1.1 Introduction 5.1.2 Methods 5.1.3 Results 5.1.4 Discussion and conclusions 5.2 Antifungal screening of Artemisia species 5.2.1 Introduction 5.2.2 Methods 5.2.3 Results 5.2.4 Discussion and conclusions 5.3 Anti-mycobacterial screening of Artemisia species 5.3.1 Introduction 5.3.2 Methods 5.3.3 Results 5.3.4 Discussion and conclusions 5.4 Antiviral screening of Artemisia species 5.4.1 Introduction 5.4.2 Methods 5.4.3 Results 5.4.4 Discussion and conclusions 5.5 Conclusions to Artemisia research 6.0 Appendixes 6.1 Appendix 1 - Annotated list of phase one voucher specimens 6.2 Appendix 2 - Annotated list of phase two voucher specimens 6.3 Appendix 3 - Summary of Artemisia taxonomic treatments 6.4 Appendix 4 - Annotated list of Artemisia voucher specimens 140. 143. 143. 143. 144. 154. 157. 157. 157. 158. 167. 169. 169. 169. 170. 176. 178. 178. 179. 181. 194. 198. 203. 204. 227. 258. 262. 6.5 Appendix 5 - Taxonomic references 279. 6.6 Appendix 6 - Ethnopharmacological summary of phase one plants 281. 6.7 Appendix 7 - Ethnopharmacological summary of phase two plants 345. 6.8 Appendix 8 - Ethnopharmacological summary of Artemisia species 411. 6.9 Appendix 9 - Ethnobotanical references 422. 7.0 Glossary 434. V l l l List of tables Table 1 - Phase one antibiotic screening results 11. Table 2 - Phase one antibiotic screening results summarized by taxa 21. Table 3 - Ethnopharmacological analysis of phase one antibiotic screening results 22. Table 4 - Phase one antifungal screening results 32. Table 5 - Phase one antifungal screening results summarized by taxa 42. Table 6 - Ethnopharmacological analysis of phase one antifungal screening results 43. Table 7 - Phase one anti-mycobacterial screening results 49. Table 8 - Phase one antiviral screening results 57. Table 9 - Correlations between anti-mycobacterial activity and infectious disease categories 65. Table 10 - Correlations between anti-mycobacterial activity and pharmacological categories 65. Table 11 - Correlations between antiviral activity and infectious disease categories 65. Table 12 - Correlations between antiviral activity and pharmacological categories 66. Table 13 - Phase two antibiotic screening results 86. Table 14 - Phase two antibiotic screening results summarized by taxa 103. Table 15 - Phase two antibiotic screening results for methanol, water and petroleum ether extracts 104. Table 16 - Ethnopharmacological analysis of phase two antibiotic screening results 106. Table 17 - Phase two antibiotic screening results summarized by habitat 107. Table 18 - Phase two antifungal screening results 117. Table 19 - Phase two antifungal screening results summarized by taxa 133. Table 20 - Ethnopharmacological analysis of phase two antifungal screening results 134. Table 21 - Phase two antifungal screening results summarized by habitat 135. Table 22 - Artemisia antibiotic screening results 146. Table 23 - Artemisia antifungal screening results 159. Table 24 - Artemisia anti-mycobacterial screening results 171. Table 25 - Artemisia cytotoxicity assay results 183. Table 26 - Artemisia preliminary antiviral screening results Table 27 - Polio plaque assay results Table 28 - Coxsackie plaque assay results Table 29 - Sindbis plaque assay results Table 30 - Artemisia M.I.C. against polio virus Table 31 - Artemisia antiviral screening results Acknowledgements x This research project was based upon the medicinal lore of the North American First Nations peoples published over the last three hundred years. The accumulated wisdom of hundreds of individuals preserved in the historical literature was central to both the selection of the plants to investigate and the ethnopharmacological analyses. I gratefully acknowledge the tremendous contribution of all these people who had so generously shared their knowledge. I would like to acknowledge two individuals whose support was crucial to this research, Dr. G.H.N. Towers (Department of Botany, U.B.C.) and Dr. R.E.W. Hancock (Department of Microbiology, U.B.C. and scientific director of the Canadian Bacterial Diseases Network). I am very grateful for the support of my graduate supervisor, Dr. G.H.N. Towers and his steadfast committment to this investigation. My thanks to Dr. Towers for his expert advice, support and encouragement throughout the many phases of this work. It was Dr. R.E.W. Hancock's visionary discernment of the inherent value of this research which enabled its successful realization. I would like to thank Dr. Hancock for all his expert advice, support and encouragement. I would also like to acknowledge the key role Dr. Hancock played as scientific director of the Canadian Bacterial Diseases Network, in facilitating the research colloborations with other network members. Financial support for this research was provided by the Canadian Bacterial Diseases Network and through grants to Dr. G.H.N. Towers from the Natural Science and Engineering Research Council. I am grateful for the opportunity to collaborate with Dr. R. Stokes and L. Thorson of the Department of Pediatrics, Division of Infectious and Immunological Diseases, U.B.C; and Dr. L. Babiuk, Dr. T. Roberts and E. Gibbons at the Veterinary Infectious Diseases Organization (V.I.D.O.) Saskatoon. I would like to thank these exceptional researchers for providing their time and their expertise to conduct the anti-mycobacterial and antiviral screenings. I was very privileged to work under Dr. J. Hudson, Department of Medical Microbiology, U.B.C, to conduct the Artemisia L. antiviral assays. I would like to acknowledge and thank Dr. Hudson for all of the time and expert advice he so generously contributed to this project. I would also like to thank Dr. Hudson, his technican E. Graham and colleague L. Yip for their expert tutoring in antiviral techniques and assays. xi I would like to acknowledge all of the contributions of my advisory committee members; Dr. J. Maze and Dr. B. Bohm (Department of Botany, U.B.C.). Their continual support and encouragement, as well their expert advice, greatly facilitated the completion of this project. Special thanks to Dr. Maze for all his assistance with the statistical analyses. I would also like to thank the U.B.C. faculty members who graciouly lent their expertise to authenticate voucher specimens: Dr. W. Schofield, Dr. J. Maze, Dr. G. Straley and J. Oliviera. I would like to acknowledge the contributions of my colleague Shona Ellis who was my research partner in the phase one plant collection, extraction and antibiotic testing. This work could not have been accomplished without Shona's tremendous enthusiasm, energy and hard work. I would also like to thank Zyta Abramowski for her technical assistance. I am very grateful for all the efforts of our summer students Cheng-Han Lee, Lehli Pour and Jen Sung who undertook the grinding task of preparing the phase two plant materials. Finally, I would like to acknowledge the person who has been my spiritual mainstay, my partner Klaus Michels. It was the strength of Klaus's love, understanding and encouragement which sustained me through all the phases of this project. Xll Foreword Portions of chapter two were previously published by the author in the Journal of Ethnopharmacology. These papers were all written by the thesis author and the full citations for these publications are as follows: McCutcheon, A.R., Ellis, S.M., Hancock, R.E.W. and Towers, G.H.N. (1992) Antibiotic screening of medicinal plants of the British Columbian native peoples. Journal of Ethnopharmacology 37: 213-233. McCutcheon, A.R., Ellis, S.M., Hancock, R.E.W. and Towers, G.H.N. (1994) Antifungal screening of medicinal plants of the British Columbian native peoples. Journal of Ethnopharmacology 44: 157-169. McCutcheon, A.R., Roberts, T.E., Gibbons, E., Ellis, S.M., Hancock, R.E.W. and Towers, G.H.N. (1995) Antiviral screening of British Columbian medicinal plants. Journal of Ethnopharmacology 49: 101-110. McCutcheon, A.R., Stokes, R.W., Thorson, L.M., Ellis, S.M., Hancock, R.E.W. and Towers, G.H.N. (1996) Anti-mycobacterial screening of British Columbian medicinal plants. Manuscript prepared for publication, unsubmitted as of the date of this writing (February 5, 1996). Co-author S.M. Ellis was an equal partner in the plant collection and extract preparation for the research cited above. Ms. Ellis was also a research partner in the screening of these extracts for antibiotic activity. The thesis author was entirely responsible for the analyses of the antibiotic screening results. The antifungal screening of the extracts and the analyses of the results were conducted by the thesis author alone. The screening of the extracts for antiviral activity was performed by T.E. Roberts and E. Gibbons in the research facilities of L. Babiuk at the Veterinary Infectious Diseases Organization (VIDO) in Saskatoon. The screening methods and raw data were supplied by T.E. Roberts. T.E. Roberts and L. Babiuk both contributed to the editing of the antiviral manuscript. xiv DEDICATION This theses is dedicated to the North American First Nations peoples and to Klaus, for his extraordinary patience. 1 1.0 General introduction In recent years, there has been a groundswell of public interest in ethnopharmacology. The Aryuvedic, Chinese and European systems of traditional medicine from the Old World are the most well known and have been the focus of most ethnopharmacological research to date. Of the New World traditional medicines, the attention of both the public and the scientific community has been captured by the exotic appeal of South America's indigenous peoples and their threatened flora which still holds many unexplored mysteries. Perhaps due to the historical devaluation of the North American First Nations cultures, relatively little attention has been directed toward their equally rich and endangered cultural heritages, in spite of the significant benefits humankind has already reaped from the few native plants which have been investigated. By far the greatest bulk of the work done on the North American flora has been screens for potential cancer drugs which have already yielded three new therapeutic agents: etoposide derived from the mayapple, Podophyllum peltatum; taxol, isolated from the yew tree, Taxus brevifolia; and betulinic acid from the birch tree, Betula alba. Even though the example set by these cancer screens is extremely positive, there has only been a handful of other types of pharmacological screenings conducted. The ethnopharmacological screenings which comprise the first half of this thesis are the first comprehensive evaluations of the anti-infectious properties of traditional plant medicines from western North America. This work barely scratches the surface of the large pharmacopeia of the First Nations peoples and many other types of pharmacological activities have yet to be investigated as only antibiotic, antifungal, anti-Mycobacterium and antiviral assays were conducted. Nonetheless, this research represents a positive step forward in the efforts to recognize and assess some of the important contributions of native cultures in the context of western knowledge. These screenings not only validate the efficacy of First Nations' traditional medicines and provide new leads for clinical therapeutics, they also give insights into some of the factors which may influence pharmacological activity and hence the means to improve the effectiveness of future screenings. The first phase screening of one hundred extracts pointed.to several different factors which may correlate with pharmacological activity. Therefore, the second phase screening was designed to obtain the necessary data to evaluate the impact of factors such as medicinal versus non-medicinal plants, the specific traditional medicinal uses, plant habitat, 2 and the higher plant taxa. Throughout all of the screenings, the members of the genus Artemisia assayed were particularily outstanding for their potent and broad spectrum anti-infectious activity. A review of the North American ethnopharmacological literature on Artemisia revealed the significant role these plants had in native medicine. The treatment of infectious diseases and infectious disease symptoms figured prominently among the traditional usages of Artemisia species by First Nations peoples throughout North America. These two factors made this group an obvious selection for more intensive research. Therefore, the second half of this thesis is devoted to a more in depth investigation of the genus, focused on the antibiotic, antifungal, anti-mycobacterial and antiviral activities of Artemisia species. 3 2.0 Introduction to phase one screenings There is an increasingly urgent need for novel antibiotic compounds as drug resistance is rapidly becoming a major obstacle in the treatment of bacterial infections. Until recently, the search for antibiotic compounds was focused on soil microrganisms. Now that the most promising leads from this source have been investigated, some researchers have once again turned their focus to the plants which were human's primary antibacterial agent before the advent of modern antibiotics. Considering the many other types of therapeutic compounds that scientists have derived from traditional medicines, ethnopharmacological screenings provide a rational approach to the search for new antibiotic compounds. Based on this rationale, this research project was originally conceived of as an antibiotic screening of traditional medicines of the British Columbian First Nations peoples to identify new leads on antibacterial therapeutics. It was due to the very encouraging results of this initial antibiotic screen that the project was subsequently expanded to include other types of infectious organisms for which new drugs are also urgently needed. The increasing numbers of immunosuppressed and immunocompromised patients stricken with life threatening fungal infections has resulted in a dramatic increase in the demand for systemic antifungals. Drug resistance has also begun to be an obstacle to the successful treatment of these patients, underscoring the need for novel chemical structures which can successfully be administered as therapeutics. Exploring traditional treatments for fungal infections provides one promising route to the discovery of such drugs. c Similarly, tuberculosis, an ancient disease which most people believed had been conquered by modern antibiotics, has staged a resurgence in recent years due to the emergence of multiple drug-resistant strains. In western countries, the impact of this resurgence has been greatly heightened by the large pool of super-susceptible immunocompromised patients facilitating the dissemination of the multiple drug resistant strains. In order to stave off this potentially epidemic situation, new anti-mycobacterial drugs are desperately required. Again, investigating the many plant medicines which were formerly used to treat this disease may provide the needed solutions. Pharmacologists have recorded the fewest successes in the field of antiviral development. Until recently, there was little impetus for antiviral research because few people in western countries died from viral infections and it was believed that the great similarities in viral and human biochemistry were prohibitive to the development of safe therapeutics. Antiviral research has been greatly accelerated in the face of the current AIDS 4 epidemic. However there are still relatively few drugs available and viral resistance to these compounds has already become a serious obstacle to treatment. Ethnopharmacological screening presents one promising avenue of approach to the discovery of new antivirals as there are hundreds, if not thousands, of traditional remedies which have yet to be investigated for their therapeutic potential in modern medicine. The traditional medicines of the British Columbian First Nations peoples in particular had not been scientifically investigated for any type of pharmacological activity prior to this study. There was a substantial body of literature on their medicinal uses of plants, largely due to the research of the preeminent British Columbian ethnobotanist Dr. N.J. Turner. This literature provided the basis for the selection of the medicinal plant species examined in this study. A list of those plants used medicinally by the native peoples of this province was compiled from Dr. Turner's reports for use in the field as a selection guide for the plant species and type of material to be collected. The main focus of the plant collection was on plants whose traditional uses suggested that they may have been used to treat infections, although a few plants with other uses such as general tonics were also collected. Out of the hundreds of medicinal plants referred to in Thompson Ethnobotany (Turner, 1990), Ethnobotany of the Okanagan-Colville Indians (Turner, 1980) and Plant Taxonomy and Systematics of Three Contemporary Indian Groups of the Pacific Northwest (Turner, 1974), one hundred plant samples were collected. A list of the botanical names of the plant species collected and collection details for each plant is given in Appendix 1. Methanolic extracts of these plant samples were prepared and then subjected to the antibiotic, antifungal, anti-mycobacterial and antiviral screens which are reported in the following chapters. For each screening, an ethnopharmacological analysis was also conducted, based on the abbreviated summaries of the traditional medicinal uses of these plants compiled in Appendix 6. These summaries were based on an extensive review of all the available ethnobotanical literature. 5 2.0.1 Methods Plant collection The plant collecting was carried out from May-July 1991, in five areas of the province: the Wyndel region in the Kootenay mountains, the Princeton-Penticton region in the interior, Haida Gwaii (the Queen Charlotte Islands), the Fraser River canyon, and the Lower Mainland. From the several hundred plant species on the ethnobotanical list, 100 samples were collected. In order to ensure accurate botanical identifications, only plants which were in flower were collected, introducing a seasonal bias into the selection. A voucher specimen was made for each collection and these vouchers have been filed in the University of British Columbia Herbarium, U.B.C.. See Appendix 1 for a complete listing. Extract preparation The plant material was air dried and then ground in a Wiley grinder with a 2-mm diameter mesh. Twenty g of the ground material were extracted in 100 ml of methanol with three washes of 100 ml, over 24 hours. The crude methanolic extract was first filtered through cheesecloth and cotton wool, then through a Biichner funnel with a No. 4 paper filter. The filtrate was rotoevaporated to dryness and then reconstituted with 10 ml of methanol. The prepared extracts were refrigerated up until the time they were used. Acknowledgements I would like to acknowledge the contributions of my colleague Shona Ellis who was my research partner in the plant collection and extraction. References Turner, N.J., Bouchard, R. and Kennedy, D.I.D. (1980) Ethnobotany of the Okanagan-Colville Indians of British Columbia and Washington. British Columbia Provincial Museum No. 21. Occasional Papers Series. British Columbia Provincial Museum, Victoria, British Columbia, pp. 156. Turner, N.J., Thompson, L.E., Thompson, M.T. and York, A.Z. (1990) Thompson Ethnobotany: Knowledge and Usage of Plants by the Thompson Indians. Royal British Columbia Museum Memoir No. 25. Royal British Columbia Museum, Victoria, British Columbia, pp. 321. Turner, N.J. (1974) Plant Taxonomy and Systematics of Three Contemporary Indian Groups of the Pacific Northwest. Ph.D. Thesis, University of British Columbia. 6 2.1 Phase one antibiotic screening Abstract One hundred methanolic plant extracts were screened for antibiotic activity against 11 bacterial strains. Eighty-nine per cent were found to have significant antibiotic activity. Ninety-four per cent of the plants categorized as potential antibiotics based on their traditional usage were found to exhibit significant antibiotic activity. Seventy-five were found to be active against methicillin resistant Staphylococcus aureus, 46 were active against an antibiotic supersusceptible strain of Pseudomonas aeruginosa and 18 of these were also active against a wild type strain. The extracts with the broadest spectra of activity were prepared from: Alnus rubra bark and catkins, Fragaria chiloensis leaves, Moneses uniflora aerial parts, and Rhus glabra branches. 2.1.1 Introduction The First Nations peoples used plants extensively in their medical practice. Several hundred of these medicinal plants have been identified and their usage documented in the ethnobotanical literature (see Appendix 6). However, only in a few cases have the pharmacological properties of these traditional remedies been investigated. This study constitutes the first antibiotic screening of British Columbian medicinal plants as well as the initial attempt at an ethnopharmacological analysis of the results. In screening these plants for antibiotic activity, it is hoped that the data obtained will not only provide useful leads towards the discovery of new antibiotics but also encourage further interest and research in North American ethnobotany and ethnopharmacology. 2.1.2 Methods Microorganisms Eleven bacterial strains were used in the screening: Bacillus subtilis, Enterobacter aerogenes, Escherichia coli DC2, Klebsiella pneumoniae, Mycobacterium phlei, Pseudomonas aeruginosa Z61, Pseudomonas aeruginosa K799, Serratia marcescens, Staphylococcus aureus meths, Staphylococcus aureus methR P00017, and Salmonella typhimurium TA98. The two P. aeruginosa strains were obtained from the 7 laboratory of R.E.W. Hancock. The Z61 strain is an antibiotic supersusceptible strain and the K799 strain is a wild type strain. The methicillin resistant strain of S. aureus was provided by Dr. A. Chow, Department of Medical Microbiology, U.B.C. The remaining bacterial cultures were those from the collection of G.H.N. Towers. An inoculum of each bacterial strain was suspended in 3 ml of nutrient broth and incubated overnight at 37°C. The overnight cultures were diluted 1/10 with nutrient broth before use. To ensure that the density of the diluted cultures were all within the range of 107"8 CFU/ml, serial dilution plate counts were also made for each culture. Antibiotic assays The disc diffusion assay (Lennette, 1985) was used to screen for antibiotic activity. Paper discs (1/4") were impregnated with 20 ul of extract, the equivalent of 40 mg of dried plant material, and the solvent allowed to evaporate at room temperature. One hundred ul of the diluted bacterial culture was spread on sterile Mueller-Hinton agar plates before placing the extract impregnated paper discs on the plates. For each extract, three replicate trials were made against each bacteria screened. Gentamicin was used as a positive control and methanol as a negative control. The plates were incubated for 18 h at 37°C, with the exception of M. phlei which was incubated for 36 h. The diameter of the zone of inhibition around each disc was measured and recorded at the end of the incubation period. Data analysis The average zone of inhibition was calculated for the 3 replicates. A clearing zone of 8 mm or greater was used as the criteria for designating significant antibiotic activity. The overall trial average for each assay was used for the classification of results in Table 1. For each of the major taxonomic divisions (Eumycota, Thallophyta, Bryopsida, Sphenopsida, Lycopsida, Filicinae, Gymnospermae and Angiospermae), the total number and percentage of active extracts was calculated, as well as the percentage of active extracts excluding those with only slight (1+) activity against the susceptible organisms M. phlei and P. aeruginosa Z61. The ethnopharmacological data collated in Appendix 6 summarizing the traditional medicinal uses of each plant was used as the basis for the ethnopharmacological classifications. Each extract was assigned to the 8 highest numbered category it fit into. The three ethnopharmacological categories used were: (1) potential antibiotics, (2) possible antibiotics, and (3) tonics. The one plant for which there were no references to medicinal usage was categorized as 4) non-medicinal plant. Extracts which were used to treat specific ailments caused by bacterial organisms were assigned to category 1: potential antibiotics. The specific bacterial ailments included in category 1 were: abcesses, acne, bladder or kidney infections, blood poisoning, boils, consumption, diptheria, dysentery, food poisoning, gonorrhea, infected wounds or sores, ptomaine poisoning, rheumatic fever, scarlet fever, sepsis, syphilis, tooth abscess, tuberculosis, venereal disease, and whooping cough. The infected wounds or sores classification included the descriptors: inflamed wounds/sores, discharge from wounds/sores, wounds/sores with pus, feverish wounds/sores, etc. Plants traditionally used as disinfectants or antiseptics were also assigned to this category. Plants which were compounded for these applications were assigned to category 2, as there is a degree of uncertainty about the actual role of a particular plant in a mixture. Extracts of plants traditionally used to treat ailments and symptoms which were possibly caused by bacterial infections were assigned to category 2: possible antibiotics. Ethnopharmacological descriptors included in this category were: bladder or kidney disease/problems/troubles, burns, coughs, cuts, diarrhea, fever, gastroenteritis, lung trouble, lung hemorrhage, pneumonia, scrofula, sores, sore gums, sore throat, stomachache, stomach ailments/disease/problems, stomach/intestinal flu, too frequent urination, toothache, tonics and wounds. Plants which were not used for any of the applications listed above but were reported to have been used as physics, tonics or general medicines were assigned to category 3: tonics. One plant for which there was no recorded medicinal use of that genus in the literature surveyed nor in the Napralert database was assigned to category 4: non-medicinal plant. The total number of active plant extracts in each category was calculated, as well as the number of active extracts excluding those with only slight (1+) activity against the super-susceptible organisms E. coli, M. phlei and P. aeruginosa Z61. The statistical test "chi squared goodness-of-fit" with a significance level of 0.01 was used to analyze the percentage of active extracts in each category to determine if the observed values exceeded those expected from a random sampling. 9 2.1.3 Results One hundred crude methanolic extracts of plants, 99 of which were used medicinally by First Nations peoples were screened for antibiotic activity against 11 bacterial strains. The overall results of the screening are presented in Table 1, alphabetically by family. The degree of activity of all the extracts are comparable since a standard amount of dried plant material, as well as standard extraction and test procedures were used. Eighty-nine of the extracts assayed (89%) demonstrated significant antibiotic activity. Seventy of the extracts exhibited activity against both Gram negative and Gram positive organisms. Nine extracts had activity against Gram positive organisms only and five extracts had activity against Gram negative organisms only. Four extracts had activity against the super-susceptible M. phlei only. Eleven extracts exhibited no significant activity against any of the bacteria tested. Forty-six extracts exhibited activity against P. aeruginosa Z61 (antibiotic supersusceptible strain) and 18 of these also showed significant activity against P. aeruginosa K799 (wild type). Fifty-one extracts were active against S. aureus meths and 75 were active against S. aureus methR. All the extracts which were active against the methicillin sensitive strain were also active against the methicillin resistant strain. Thirteen extracts exhibited activity against S. marcescens and only six extracts had activity against K. pneumoniae. Several other important observations may be summarized from the data in Table 1: 1. The extracts which exhibited the broadest spectrum of activity (activity against at least 10 bacteria) were: Alnus rubra bark and catkins, Fragaria chiloensis leaves, Moneses uniflora aerial parts, and Rhus glabra branches. The extracts of Arctostaphylos uva-ursi branches and roots, Artemisia ludoviciana var. latiloba aerial parts, Balsamorhiza sagittata aerial parts and roots, Cornus canadensis aerial parts, Geum macrophyllum roots, Heuchera cylindrica roots, Juniperus communis branches, Larix occidentalis branches, Lomatium dissectum roots, and Ribes sanguineum branches were active against nine of the bacteria. 2. The extracts with the greatest activity against P. aeruginosa K799 (normal strain) were: Alnus rubra catkins, Argentina egedii aerial parts, Artemisia ludoviciana var. latiloba, Cornus canadensis aerial parts, Fragaria chiloensis leaves, Juniperus communis branches, Polystichum munitum rhizomes, Ribes sanguineum branches and Rhus glabra branches. 10 3. The extracts with the greatest activity against the methicillin resistant S. aureus strain were: Alnus rubra bark, Ambrosia chamissonis aerial parts, Lomatium dissectum roots, Nuphar lutea rhizomes, and Rhus glabra branches. 4. The families with the largest number of species screened were Compositae and Rosaceae. All 13 of the species from the Compositae exhibited significant antibiotic activity, 11 with activity against both Gram-positive and Gram-negative bacteria and 2 with Gram-positive activity only. Of the 14 species from the Rosaceae screened, 12 exhibited activity against both Gram-positive and Gram-negative bacteria while one had Gram-negative activity only and one exhibited no significant antibiotic activity. The screening results are presented summarized by taxa in Table 2. The taxa with the greatest degree of antibiotic activity were the Filicinae (ferns) and the Gymnospermae (conifers). The results of the ethnopharmacological data analysis are summarized in Table 3. Ninety-four per cent of the extracts designated as having potential antibiotic activity based on their traditional usage, exhibited activity. This value significantly exceeds the percentage of active extracts expected from a random sampling. 11 Table 1 - Phase one antibiotic screening results3 Family Cat.b Partc B.s.d E.a. E.e. K.p. M.p. Z61 K799 S.m. S.a.S S.a.R S.t. Species (Voucher No.) Controls Methanol - - 0 0 0 0 0 0 0 0 0 0 0 Gentamycin (10 ug) - - 5 + 4 + 5+ 5+ 5+ 5+ 4+ 4+ 5+ 5+ 5+ Anacardiaceae Rhus glabra (P-23) 1 Br 2+ 2+ 5+ 2+ 5+ 5+ 2+ 2+ 5+ 3+ 3+ Aracaceae Lysichiton americanum (Q-26) I R t O 0 0 0 0 0 0 0 0 0 0 Araliaceae Oplopanax horridus (Q-14) 1 lb 1+ 0 0 0 4+ 2+ 0 0 4+ 1+ 1+ Aristolochiaceae Asarum caudatum (W-12) l W h O O 0 0 1+ 0 0 0 0 1+ 1+ Berberidaceae Mahonia aquifolium (W-2) 1 Rt 1+ 0 3+ 0 5+ 0 0 0 1+ 1+ 0 Betulaceae Alnus rubra (Q-l) 1 Bk 1+ 3+ 3+ 1+ 5+ 3+ 2+ 2+ 3+ 3+ 3+ Family Cat." Part* B.s.d E.a. E.e. K.p. M.p. Z61 K799 S.m. S.a.S S.a.R S.t. Species (Voucher No.) Alnus rubra (Q-2) 1 Ck 2+ 1+ 5+ 1+ 5+ 3+ 2+ 2+ 2+ 3+ 2+ Betula papyrifera (P-38) 1 Br 1+ 0 3+ 0 3+ 0 0 0 2+ 1+ 1+ Cactaceae Opuntia frag His (F-4) 2 A e 0 0 0 0 1 + 0 0 0 0 0 0 Caprifoliaceae Lonicera ciliosa (F-2) 2 Br 0 0 2+ 0 0 0 0 0 1+ 1 + 0 Lonicera involucrata (F-5) I B r O 0 0 0 0 0 0 0 0 0 0 Sambucus cerulea (P-16) 1 Br 2+ 0 0 0 2+ 1+ 1+ 0 1+ 2 + 0 Sambucus racemosa (Q-21) l B k O 0 0 0 1+ 0 0 0 0 1 + 0 Symphoricarpos albus (P-26) 1 Br 0 0 1+ 0 1+ 1+ 0 0 0 1 + 0 Compositae Achillea millefolium (P-10) 1 Wh 0 0 3+ 0 2+ 0 0 0 1+ 2+ 1+ Ambrosia chamissonis (Q-29) 1 Ae 1+ 0 1+ 0 3+ 1+ 0 0 3+ 3+ 2+ Antennaria microphylla (P-21) 2 Wh 0 0 0 0 2+ 1+ 0 0 0 1+ 0 Arnica cordifolia (P-31) 2 A e 0 0 0 0 1+ 0 0 0 0 0 1+ Arnica sororia (P-7) 2 Ae 1+ 0 1+ 0 2+ 0 0 0 2+ 2+ 1+ 13 Family Cat." Partc B.s.d E.a. E.e. K.p. M.p. Z61 K799 S.m. S.a.S S.a.R S.t. Species (Voucher No.) Artemisia ludoviciana (W-5) 1 Ae 1+ 1+ 3+ 1+ 2+ 2+ 2+ 2 + 0 2 + 0 Artemisia michauxiana (P-29) 2 Ae 0 0 0 0 2+ 0 0 0 1+ 2 + 0 Artemisia tridentata (W-19) 1 Br 0 0 2+ 0 2+ 0 0 0 2+ 2+ 1+ Balsamorhiza sagittata (W-18) 1 Ae 0 0 1+ 0 2+ 0 0 0 2+ 2+ 1+ Balsamorhiza sagittata (P-2) 1 Rt 1+ 1+ 5+ 0 3+ 2+ 1+ 0 1+ 2+ 0 Chaenactis douglasii (P-3) 1 Wh 2+ 0 3+ 0 3+ 0 0 0 2+ 2+ 1+ Chrysothamnus nauseosus (P-25) 1 Br 0 1+ 0 0 1+ 0 0 0 0 1 + 0 Erigeron filifolius (P-9) 2 Ae 0 0 2+ 0 2+ 0 0 0 1+ 2 + 0 Gaillardia aristata (P-8) 1 Ae 2+ 0 3+ 0 2+ 2+ 0 0 2+ 2 + 0 Conocephalaceae [Bryophyta] Conocephalum conicum (Q-28) 2 T h 0 0 0 0 1 + 0 0 0 0 1 + 0 Cornaceae Cornus canadensis (Q-12) 2 Ae 2+ 2+ 4+ 0 3+ 2+ 2+ 1+ 1+ 2+ 0 Comas sericea (W-6) I B r 0 0 0 0 1 + 0 0 0 0 0 0 Crassulaceae Sedum lanceolatum (P-20) 2 W h 0 0 0 0 0 0 0 0 0 0 0 14 Family Cat." Part1 B.s.d E.a. E.e. K.p. M.p. Z61 K799 S.m. S.a.S S.a.R S.t. Species (Voucher No.) Cruciferae Capsella bursa-pastoris (W-8) 1 Wh 0 0 0 0 1+ 0 0 0 0 1 + 0 Cardamine angulata (Q-16) 2 Rt 0 0 0 0 1+ 0 0 0 0 1+ 0 Cupressaceae [Gymnospermae] Juniperus communis (Q-25) 1 Br 2+ 0 3+ 0 2+ 2+ 2+ 0 2+ 2+ 2+ Elaeagnaceae Shepherdia canadensis (W-16) 1 Wh 0 1+ 2+ 0 1+ 3+ 0 0 0 0 0 Empetraceae Empetrum nigrum (Q-17) 1 Br 2+ 0 2+ 0 5+ 2+ 0 0 2+ 2+ 2+ Equisetaceae [Sphenopsida] Equisetum arvense (W-3) 2 A e O 0 0 0 0 0 0 0 0 0 0 Equisetum hyemale (P-37) l A e O 0 0 0 0 0 0 0 0 0 0 Ericaceae Arctostaphylos uva-ursi (P-42a) 1 Br 2+ 1+ 5+ 0 3+ 4+ 1+ 0 2+ 2+ 1+ Arctostaphylos uva-ursi (P-42b) 1 Rt 0 0 3+ 0 1+ 0 0 0 0 1 + 0 Kalmia microphylla (Q-5) 1 Br 1+ 0 2+ 0 2+ 1+ 0 0 2+ 2+ 2+ 15 Family Cat.b Partc B.s.d E.a. Ex. K.p. M.p. Z61 K799 S.m. S.a.S S.a.R S.t. Species (Voucher No.) Ledum groenlandicum (Q-4) 2 Br 1+ 0 2+ 0 2+ 1+ 0 0 1+ 2+ 2+ Moneses uniflora (Q-8) 1 Ae 2+ 2+ 4+ 1+ 5+ 2+ 1+ 1+ 5+ 4+ 3+ Monotropa uniflora (P-19) 2 Wh 1+ 2+ 0 0 0 0 0 0 1+ 1 + 0 Grossulariaceae Ribes sanguineum (P-18) 1 Br 2+ 1+ 3+ 0 2+ 2+ 1+ 0 2+ 2+ 2+ Hydrangeaceae Philadelphus lewisii (P-22) I B r O 0 0 0 1+ 0 0 0 1 + 0 0 Hylocomiaceae [Bryophyta] Hylocomium splendens (Q-9) 2 G a 0 0 0 0 0 0 0 0 0 0 0 Hypericaceae Hypericum perforatum (P-30) 1 Ae 2+ 0 3+ 0 3+ 2+ 1+ 0 1+ 2 + 0 Leguminosae Lupinus sericeus (P-12) 1 Ae 0 0 2+ 0 1+ 0 0 0 0 1 + 0 Liliaceae Disporum trachycarpum (W-ll) 2 W h 0 0 0 0 0 0 0 0 0 0 0 Maianthemum racemosa (W-17) 2 R h O 0 0 0 0 0 0 0 0 0 0 16 Family Cat.b Partc B.s.d E.a. E.e. K.p. M.p. Z61 K799 S.m. S.a.S S.a.R S.t. Species (Voucher No.) Maianthemum stellata (W-13) 1 Rh 0 0 0 0 1+ 0 0 0 0 0 0 Lobariaceae [Eumycota] Lobaria oregana (Q-ll) 2 T h 1+ 0 0 0 3+ 1+ 0 0 0 0 0 Lycopodiaceae [Lycopsida] Lycopodium clavatum (Q-6) 2 B r 0 0 0 0 2+ 1 + 0 0 0 2 + 0 Menyanthaceae Fauna crista-galli (Q-19) 3 Ae 0 0 1+ 0 2+ 1+ 0 0 1+ 2 + 0 Nymphaeaceae Nuphar lutea (Q-3b) 1 Rt 2+ 0 2+ 0 3+ 2+ 0 0 2+ 2+ 0 Nuphar lutea (Q-3c) 1 Rh 3+ 0 5+ 0 5+ 1+ 0 0 5+ 3 + 0 Onagraceae Epilobium minutum (P-l) 2 W h O 0 3+ 0 1+ 0 0 0 0 1 + 0 Pinaceae [Gymnospermae] Larix occidentalis (W-15) 1 Br 1+ 0 3+ 0 2+ 1+ 1+ 1+ 1+ 2+ 1+ Pinus contorta (Q-18) 1 Br 1+ 0 2+ 0 3+ 2+ 0 0 2+ 2+ 1+ Pinus ponderosa (W-20) 1 Br 1+ 0 2+ 0 4+ 2+ 0 0 2+ 2+ 1+ 17 Family Cat." Partc B.s.d E.a. E.e. K.p. M.p. Z61 K799 S.m. S.a.S S.a.R S.t. Species (Voucher No.) Plantaginaceae Plantago major (Q-22) Polemoniaceae Ipomopsis aggregata (P-13a) Ipomopsis aggregata (P-13b) Polygonaceae Eriogonum heracleoides (P-11) Eriogonum heracleoides (P-17) Polypodiaceae [Filicinae] Polypodium glycyrrhiza (Q-27) Polystichum munitum (Q-15) Polyporaceae [Eumycota] Ganoderma applanatum (Q-10) Ranunculaceae Clematis ligusticifolia (P-14) Delphinium nuttallianum (P-33) Wh Ae Rt Ae Rt Rh Rh Wh Ae Wh 1+ 0 1+ 2+ 0 0 0 0 0 2+ 0 0 0 1+ 0 0 3+ 0 3+ 3+ 2+ 3+ 0 0 0 0 0 0 2+ 1+ 2+ 2+ 1+ 3+ 0 0 1+ 2+ 1+ 2+ 0 0 0 0 0 2+ 0 0 0 0 0 0 0 0 1+ 1+ 0 2+ 1+ 0 2+ 2+ 0 2+ 0 0 2+ 2+ 1+ 2+ 0 0 0 0 0 0 1+ 0 1+ 1+ 0 2+ 0 0 0 0 0 0 2+ 1+ 0 18 Family Species (Voucher No.) Cat.b Parf B.s.d E.a. E.e. K.p. M.p. Z61 K799 S.m. S.a.S S.a.R S.t. Rhamnaceae Ceanothus velutinus (P-39) Rosaceae Amelanchier alnifolia (P-6) Amelanchier alnifolia (P-35) Argentina egedii (Q-20) Aruncus dioicus (F-l) Crataegus douglasii (W-14) Fragaria chiloensis (Q-7) Fragaria vesca (W-l) Geum macrophyllum (Q-23) Holodiscus discolor (F-3) Potentilla arguta (W-7) Prunus virginiana (W-9) Prunus virginiana (P-40) Rosa nutkana (P-5) 2 2 2 Br Br Br Br Br Br Lf Lf Rt Br Rt Br Br Br 1+ 1+ 0 1+ 0 0 2+ 2+ 2+ 0 2+ 0 1+ 0 0 0 1+ 0 0 2+ 2+ 2+ 0 1+ 0 0 0 3+ 1+ 1+ 4+ 2+ 1+ 4+ 4+ 4+ 1+ 4+ 2+ 2+ 3+ 0 0 0 0 0 0 1+ 0 0 0 0 0 0 2+ 2+ 1+ 2+ 0 1+ 3+ 2+ 3+ 0 3+ 0 2+ 1+ 1+ 0 0 0 0 0 2+ 2+ 3+ 1+ 3+ 0 0 0 0 0 2+ 0 0 1+ 0 1+ 0 1+ 0 0 0 0 0 1+ 0 0 1+ 0 0 0 0 0 0 0 1+ 0 0 0 1+ 0 2+ 0 1+ 0 2+ 0 0 0 2+ 1+ 1+ 2+ 2+ 1+ 2+ 2+ 2+ 0 2+ 1+ 1+ 0 2+ 0 1+ 1+ 1+ 0 1+ 0 1+ 0 1+ 0 0 0 19 Family . Cat." Partc B.s.d E.a. E.e. K.p. M.p. Z61 K799 S.m. S.a.S S.a.R S.t. Species (Voucher No.) Rubus parviflorus (P-28) 1 Br 0 0 3+ 0 1+ 0 0 0 0 2 + 0 Spiraea betulifolia (P-41) I B r O 0 0 0 0 0 0 0 0 0 0 Spiraea pyramidata (P-l5) 3 Br 0 0 0 0 2+ 2+ 0 0 0 1+ 0 Salicaceae Populus tremuloides (P-34) 1 Br 0 0 1+ 0 1+ 0 0 0 0 0 0 Salix bebbiana (P-36) 2 B r 0 0 1 + 0 0 0 0 0 0 0 0 Saxifragaceae Heuchera cylindrica (W-4) 1 Rt 2+ 1+ 4+ 0 2+ 2+ 0 1+ 2+ 2+ 1+ Scrophulariaceae Penstemon fruticosus (P-4) 1 Br 0 0 4+ 0 2+ 1+ 0 0 0 2+ 2+ Verbascum thapsus (P-24) l L f 0 0 1+ 0 1 + 0 0 0 0 2 + 0 Umbelliferae Glehnia littoralis (Q-13) 1 Rt 1+ 0 1+ 0 4+ 1+ 0 0 3+ 2+ 2+ Heracleum maximum (P-32a) 1 Ae 0 0 0 0 2+ 1+ 0 0 2+ 2+ 2+ Heracleum maximum (P-32b) 1 Rt 1+ 0 2+ 0 4+ 0 0 0 1+ 1+ 2+ Lomatium dissectum (W-10) 1 Rt 3+ 2+ 3+ 0 4 + 3 + 0 1+ 2+ 3+ 2+ 20 Family Cat." Parf B.s.d E.a. E.e. K.p. M.p. Z61 K799 S.m. S.a.S S.a.R S.t. Species (Voucher No.) Osmorhiza purpurea (Q-24) 2 R t 0 0 1 + 0 2+ 0 0 0 0 2 + 0 Urticaceae Urtica dioica ssp. gracilis (P-27) 2 A e O 0 0 0 0 0 0 0 0 0 0 Total number of active extracts 42 20 66 6 85 46 18 13 51 75 42 Key to Table 1 a Classification of results: 0 = no zone or zone of inhibition < 8.0 mm; 1+ = zone of inhibition 8.0-10.0 mm; 2+ = 10.1-15.0 mm; 3+ = 15.1-20.0 mm; 4+ = 20.1-25.0 mm; 5+ = > 25.0 mm. b Cat. = Ethnobotanical category: 1 = Potential antibiotic, 2 = Possible antibiotics, 3 = Tonics, 4 = Non-medicinal plant. 0 Part Extracted: Ae = Aerial, Bk = Bark, Br = Branch, Ck = Catkin, Ga = Gametophyte, lb = Inner Bark, Lf = Leaf, Rh = Rhizome, Rt = Root, Th = Thallus, Wh = Whole plant. d Bacteria: B.s. = Bacillus subtilis (Gm+); E.a. = Enterobacter aerogenes (Gm"); E.e. - Escherichia coli DC2 (Gm"); K.p. = Klebsiella pneumoniae (Gm"); M.p. = Mycobacterium phlei, Gm ( + ), non-acid fast; Z61 = Pseudomonas aeruginosa Z61 (Gm); K799 = Pseudomonas aeruginosa K799 (Gm); S.m. = Serratia marcescens (Gm); S.a.S. = Staphylococcus aureus methicillin sensitive (Gm*); S.a.R. = Staphylococcus aureus methicillin resistant (Gm+); S.t. = Salmonella typhimurium TA98 (Gm). 21 Table 2 - Phase one antibiotic screening results summarized by taxa Excluding 1+ super-suscept." Taxa Number in Number Percent Number Percent Category (N) Active (N) Active (%) Active (N) Active (%) NON-FLOWERING PLANTS Lower plants Eumycota 2 1 50 1 50 Bryopsida 2 1 50 1 50 Lycopsida 1 1 100 1 100 Sphenopsida 2 0 0 0 0 Lower plants sub-total 7 3 43 3 43 Higher plants Filicinae 2 2 100 2 100 Gymnospermae 4 4 100 4 100 Higher plants sub-total 6 6 100** 6 100** NON-FLOWERING Sub-total 13 9 69 9 69 FLOWERING Sub-total 87 80 92 73 84 GRAND TOTALS 100 89 89 82 82 a Number active (N) calculated excluding those extracts with only slight (1+) activity against the super-susceptible organisms E. coli, M. phlei and P. aeruginosa Z61. ** Percentage of active extracts in category statistically significant, p < 0.01 22 Table 3 - Ethnopharmacological analysis of phase one antibiotic screening results Category Number in Category (N) Number Active (N) Percent Active (%) Excluding 1+ Super-susc.a Number Percent Active (N) Active (%) Potential antibiotics 69 65 94* 60 87* Possible antibiotics 27 21 77 19 70 Tonics 3 3 100 3 100 Subtotal medicinal 99 89 90 83 86 No medicinal use 1 0 0 0 0 Grand Totals 100 89 89 82 82 a Number active (N) calculated excluding those extracts with only slight (1+) activity against the super-susceptible organisms E. coli, M. phlei and P. aeruginosa Z61. Percentage of active extracts in category statistically significant, p < 0.05 23 2.1.4 Discussion and Conclusions Many antibiotic screening studies use a relatively small number of bacteria as their screen. From the data in Table 1, it can be seen that if the results from only five of the test organisms (B. subtilis, E. aerogenes, E. coli, S. aureus and S. typhininium) are considered, the number of extracts reported as inactive would more than double from 11 to 23. Moreover, some of the extracts which were active against the clinically important organisms P. aeruginosa and methicillin resistant S. aureus would then have been reported as inactive and many of the active extracts would have been reported as having more limited spectra. Clearly, reducing the size of the bacterial screen would have resulted not only in the failure to retrieve valuable information but, more importantly, in reporting misleading negative data. However, the number of organisms used in a screening often must be restricted due to resource limitations. The efficacy of a small bacterial screen can be greatly enhanced by the inclusion of a supersensitive organism such as M. phlei. In this study, 85 of the 89 extracts which were active against M. phlei were also active against at least one other organism. Based on these results, a screening using M. phlei and only three other organisms would still have identified all of the active extracts. In classifying the activity of the antibiotic extracts as Gram positive or Gram negative, it would generally be expected that a much greater number would be active against Gram positive organisms than against Gram negative. However, in this study, a large number of the extracts (70) were active against both Gram positive and Gram negative bacteria while only a relatively low number (9) were active against Gram positive bacteria only. The activity against both types of bacteria may be indicative of the presence of broad spectrum antibiotic compounds or simply general metabolic toxins. The therapeutic potential of each of the broad-spectrum extracts will have to be evaluated individually to determine which merit further investigation. Given the small number of known antibiotic compounds with a high Therapeutic Index which are effective against Gram negative organisms, all nine extracts which exhibited only Gram negative activity also merit further investigation. The gram negative organisms K. pneumoniae, S. marcescens, and P. aeruginosa are particularly resistant to current antibiotic therapy. The extracts which were found to be active against these organisms especially merit further investigation of their therapeutic potential. The analysis of the results by taxa showed that a higher percentage of the flowering plants exhibited 24 antibiotic activity than did the non-flowering plants (see Table 2). Among the non-flowering plants though, the Gymnospermae and the Filicinae were particularly noteworthy in that all the species tested exhibited antibiotic activity. Unfortunately, the sample size of the non-flowering plants was too small to draw any definitive conclusions. However, these results certainly suggested that it would worthwhile to collect a larger sample of non-flowering plants for assessment in the next phase of screening. While the high percentage of extracts which were found to exhibit antibiotic activity in this study may be attributed in part to the relatively large number of bacterial species screened, it may also be partly attributed to the accuracy of the ethnobotanical data used as selection criterion. The results of the ethnopharmacological analysis show that a significant percentage of the traditional medicines used to treat bacterial infections exhibited antibiotic activity (see Table 3). Overall, 89% of the plants which had been documented as being used medicinally by First Nations peoples were found to have antibiotic activity. More specifically, 94% of the plants designated as potentially antibiotic and 77% of the plants designated as possible antibiotics based on their traditional usage, exhibited antibiotic activity. The sample sizes in the other categories were too small to draw any definitive conclusions. These results suggest that the selection criterion used was effective in targeting a high percentage of plants with antibiotic activity, however, a larger control group (non-medicinal plants) is needed to make a comparison with. The results of this study also have some much broader implications. The results suggest that at least some of the herbal medicines of British Columbian First Nations peoples may have been efficacious, there remains hundreds more which have yet to be investigated. The present results lend weight to the argument that British Columbian ethnobotany, in particular, and North American ethnobotany, in general, is worthy of further research. Acknowledgements I would like to acknowledge the contributions of my research partner Shona Ellis in conducting the antibiotic screening. I would like to thank Ms. Z. Abramowski for the training and technical assistance she provided. 25 Reference Lennette, E. H. (1985) Manual of Clinical Microbiology (4th ed.) American Association for Microbiology. Washington, D.C, pp. 978-987. 26 2.2 Phase one antifungal screening Abstract One hundred methanolic plant extracts were screened for antifungal activity against nine fungal species. Eighty-one were found to have antifungal activity and 30 extracts showed activity against four or more of the fungi assayed. One hundred percent of the plants classified as potential antifungals based on their traditional usage exhibited activity compared to 33% of the plants with other types of medicinal usages. All of the ferns and gymnosperms assayed exhibited antifungal activity. The extracts with the greatest fungal inhibition were prepared from: Alnus rubra catkins, Artemisia ludoviciana aerial parts, A. tridentata aerial parts, Geum macrophyllum roots, Mahonia aquifolium roots, and Moneses uniflora aerial parts. In addition to these, extracts prepared from the following also exhibited antifungal activity against all nine fungi: Asarum caudatum whole plant, Balsamorhiza sagittata roots, Empetrum nigrum branches, Fragaria chiloensis leaves, Glehnia littoralis roots, Heracleum maximum roots, Heuchera cylindrica roots, Ipomopsis aggregata aerial parts and roots, and Rhus glabra branches. 2.2.1 Introduction Until recently, there was very little antifungal research being conducted. However, with the upsurge in the number of immuno-suppressed and immuno-compromised patients succumbing to fungal infections, the demand for new antifungal compounds has risen dramatically. Given the growing need for effective antifungal therapeutics and the encouraging results of the antibiotic screening, it was deemed worthwhile to conduct an antifungal screening of the traditional medicines of the British Columbian First Nations peoples. Since ethnopharmacologists rely so heavily on ethnobotanical information for research leads, an attempt was made to evaluate the screening results in the context of the background ethnobotanical literature to determine how such information may best be used to guide future research projects. 27 2.2.2 Methods Microorganisms Nine fungal strains were used in the screening; Aspergillus flavus, Aspergillus fumigatus, Candida albicans, Fusarium tricuictum, Microsporum cookerii, Microsporum gypseum, Saccharomyces cerevisiae, Trichoderma viridae and Tricophyton mentagrophytes. All of the above cultures were from the U.B.C. collection of G.H.N. Towers. Antifungal assays The disc diffusion assay (Lennette, 1985) was used to screen for antifungal activity. Sterile paper discs (1/4 ") were impregnated with 20 ul of methanolic extract and the methanol allowed to evaporate at room temperature. Sterile Yeast Morphology Agar (Difco) plates were inoculated with fungal spores before placing the extract impregnated paper discs on the plates. For each extract, four replicate trials were conducted against each fungus. Nystatin was used as a positive control and methanol as a negative (solvent) control. The temperature and length of incubation used for each fungus were as follows: A. flavus, A. fumigatus, C. albicans, S. cerevisiae and T. viridae were all incubated at 37°C for 18 h; F. tricuictum cultures were incubated at 20°C for 36 h; and M. cookerii, M. gypseum and T. mentagrophytes were incubated at 30°C for 72 h. The diameters of the zones of inhibition around each disc were measured and recorded at the end of the incubation period. Data analysis The average zone of inhibition was calculated for the four replicates. A clearing zone of 8 mm or greater was used as the criterion for designating significant antifungal activity. In trials where there was germination of a few spores within a very distinctive zone of inhibition, the zone measurement was annotated with the letter "i" to indicate that the inhibition was incomplete. The total number of fungi against which an extract exhibited significant activity was calculated. In order to provide a more rigorous assessment of the results, the total number of fungi which an extract inhibited was also calculated, excluding those extracts which showed only slight activity (1+) against the susceptible dermatophytes Microsporum cookerii, M. gypseum and Tricophyton mentagrophytes. For each of the major taxa (Eumycota, Bryophyta, Lycopsida, Sphenopsida, Filicinae, Gymnospermae and Angiospermae), the percentage of active extracts was calculated, as well as the percentage of active extracts 28 excluding those with only slight activity (1+) against the dermatophytes M. cookerii, M. gypseum and T. mentagrophytes. The ethnopharmacological classifications were based on the data collated from the ethnobotanical literature which was summarized in Appendix 6. Based on this list of traditional uses, each plant extract was assigned to the highest numbered category it fit into. The numerical breakdown of the classification which resulted was as follows: (1) potential antifungals - 23 extracts, (2) possible antifungals - 36 extracts, (3) other skin ailments - 24 extracts, (4) tonics - 7 extracts, (5) other medicinal uses - 9 extracts, (6) no known medicinal use - 1 extract. There were very few references to specific fungal ailments such as thrush or diaper rash found in the literature survey. The majority of the references described the treatment of ailments in colloquial, symptomatic terms. As it was far beyond the author's expertise to assess whether a particular symptom might be indicative of a fungal infection, the aid of a panel of eight medical practitioners was solicited, which included both family physicans and dermatologists. From the background ethnobotanical literature search, a list of all the terms and descriptions that referred to skin ailments or possible yeast infections was compiled. For each of the terms, the physicans were asked to give their opinion as to the probability that the symptom or description was indicative of a fungal infection on a scale of one to three, with one being very likely and three being very unlikely. Those descriptions which the majority of the physicans assessed as very likely to be indicative of a fungal infection were assigned to category 1: potential antifungals. The descriptors assigned to this category were: athlete's foot, baby's coated tongue, use of baby powder, salve or talc, dandruff, diaper rash, leucorrhea, scaly skin, split skin between the toes, the whites, thrush and wash for baby's bottom. Those descriptions assessed as very unlikely to be indicative of a fungal infection, were assigned to category 3: other skin ailments. The dermatological descriptors assigned to this category were: acne, blisters, boils, bruises, carbuncles, chancres, corns, eczema, erysipelas, festering sores, hair tonic, healing sores, heat rash, pimples, poison ivy or poisoning of the skin, psoriasis, prickly rash, scrofula, skin eruptions, skin pustules, skin sores, skin ulcers, sores that would not heal, tetters, to draw blisters, ulcers and warts. 29 The remaining descriptors which may have described a fungal infection although not necessarily so were assigned to category 2: possible antifungals. The descriptors in this category included: body sores, broken skin, chafed skin, chapped lips, chapped hands, chapped skin, cracked skin, dry skin, disinfecting or antiseptic wash for itch, disinfecting or antiseptic wash for newborns, female complaints, female medicine, female tonic, foot soak, hair wash, head wash, irritated scalp, irritated skin, itch, itchy scalp, rash, raw spots on baby, running sores, scabby skin, scabs, scalp disease, skin ailments, skin disease and sores of the feet. Plants whose traditional uses were not included in categories 1-3 but were cited as being used medicinally as blood purifiers, general medicines, physics, and tonics were categorized as 4: tonics. The remaining medicinal plants with usages other than those listed above were categorized as 5: other medicinal uses. One plant for which there was no recorded medicinal use of that genus in the literature summary nor in the Napralert database was assigned to category 6: non-medicinal plant. For each category, the percentage of extracts which exhibited activity was calculated, as well as the percentage of active extracts excluding those with only slight activity (1+) against the dermatophytes M. cookerii, M. gypseum and T. mentagrophytes. The statisitical test chi squared goodness-of-fit was used to determine whether the percentage of active extracts in each category was significantly non-random. 2.2.3 Results One hundred crude methanolic extracts of plants, 99 of which were used medicinally by First Nations peoples, were screened for antifungal activity against nine fungal strains. The overall results of the screening are presented in Table 4, alphabetically by family. The degrees of activity of all the extracts were comparable since a standard amount of dried plant material, as well as standard extraction and test procedures were used. Eighty-one (81%) of the extracts assayed demonstrated antifungal activity against at least one of the fungal strains assayed. If those extracts which had only slight activity against the more susceptible dermatophytes M. cookerii, M. gypseum and T. mentagrophytes were excluded, 57 of the extracts exhibited significant activity. Sixteen of these exhibited activity against all nine fungi tested. The extract prepared from the aerial parts of Moneses uniflora had the greatest antifungal activity, giving a zone of inhibition greater than 10 mm against every fungi assayed and the largest zone of inhibition 30 against A. flavus, A. fumigatus and F. tricuictum of all the extracts tested. The extracts prepared from Alnus rubra catkins, Artemisia ludoviciana aerial parts, A. tridentata aerial parts, Geum macrophyllum aerial parts and Mahonia aquifolium roots were also active against all nine fungi, with zones of inhibition greater than 10 mm against all the organisms except A. flavus. The extracts of Asarum caudatum, Balsamorhiza sagittata roots, Empetrum nigrum branches, Fragaria chiloensis leaves, Glehnia littoralis roots, Heracleum maximum roots, Heuchera cylindrica roots, Ipomopsis aggregata aerial parts and roots, Rhus glabra branches were also active against all nine fungi. Several other important observations may be summarized from the data in Table 4: 1. The extracts of Alnus rubra catkins and Geum macrophyllum aerial parts gave zones of inhibition comparable to that of the positive control (Nystatin) against Aspergillus fumigatus. The.extract prepared from Moneses uniflora aerial parts gave zones of inhibition greater than 25 mm against A. fumigatus, more than double that of the positve control. Of the 20 extracts that were active against the related species A. flavus, only the extracts of Asarum caudatum and Moneses uniflora gave a zone of inhibition comparable to that of the positive control. 2. Of the 30 extracts which were active against S. cerevisiae, only the extracts of Moneses uniflora and Philadelphus lewisii gave zones of inhibition comparable to that of Nystatin. The extracts of Ipomopsis aggregata aerial parts and roots both gave larger zones of inhibition than that of the positive control. 3. Only five extracts exhibited a strong inhibitory effect on T. viridae: Alnus rubra catkins, Artemisia ludoviciana, A. tridentata, Mahonia aquifolium and Moneses uniflora. The extract prepared from Mahonia aquifolium roots gave a zone of inhibition greater than that of the positive control. Table 5 summarizes the percentage of active extracts in each of the following taxonomic groups: Eumycotia, Bryophyta, Lycopsida, Sphenopsida, Filicinae, Gymnospermae and Angiospermae. Among the non-flowering plants (Eumycota, Lycopsida, Sphenopsida, Filicinae and Gymnospermae cumulatively), 92% of the 31 extracts exhibited antifungal activity. Among the flowering plants (Angiospermae), 79% of the extracts were active. These figures dropped to 69% and 55% respectively when extracts which exhibited only slight activity (1+) against the dermatophytes were excluded from the calculations. The results of the ethnopharmacological data analysis are summarized in Table 6. Based on the overall totals, 100% of the extracts designated as potential antifungals and 83% of extracts designated as possible antifungals exhibited antifungal activity. The percentage of extracts which exhibited antifungal activity in both category 1 (potential antifungals) and category 2 (possible antifungals), was significantly higher than any of the other categories. The statisitical test of chi squared supported the hypothesis that the percentage of active extracts in these categories was non-random. The more stringent evaluation of the results, i.e., totals calculated, excluding extracts which exhibited only slight activity (1+) against the susceptible dermatophytes, shows a clear trend in the percentage of active extracts with the highest percentage found in category 1 and the lowest percentage in category 5. 32 Table 4 - Phase one antifungal screening resultsa Family Species (Voucher No.) Cat.b Part" A.f.d A.fu. Ca. F.t. M.c. M.g. S.c. T.v. T.m. Number5 Number1 active excluding +1 Derm. Controls Methanol Nystatin (50 ug) ANACARDIACEAE Rhus glabra (P-23) ARACEAE Lysichiton americanum (Q-26) ARALIACEAE Oplopanax horridus (Q-14) ARISTOLOCHIACEAE Asarum caudatum (W-12) BERBERIDACEAE Mahonia aquifolium (W-2) BETULACEAE Alnus rubra (Q-l) 0 0 0 0 0 0 0 0 0 2+ 3+ 4+ 3+ 4+ 3+ 3+ 3+ 3+ 2 Br 1+i 1+ 1+ 2+ 5+ 4+ 1+ 1+ 5+ 3 Rt 0 0 0 0 0 0 0 0 0 1 lb 0 1+ 1+i 1+ 5+ 3+ 1+ 1+ 5+ 2 Wh 2 + 2 + 1+i 2+ 4+ 4+ 1+i 1+ 2+ 2 Rt 1+i 2+ 2+ 3+ 5+ 5+ 2+ 4+ 2+ 2 Bk 0 0 0 0 3+ 3+ 0 0 4+ 0 0 9 9 0 0 33 Family Species (Voucher No.) Cat.b Part0 A.f.d A.fu. Ca. F.t. M.c. M.g. S.c. T.v. T.m. Number6 Numbed active excluding +1 Derm. Alnus rubra (Q-2) Betula papyrifera (P-38) CACTACEAE Opuntia fragilis (F-4) CAPRIFOLIACEAE Lonicera ciliosa (F-2) Lonicera involucrata (F-5) Sambucus caerulea (P-l6) Sambucus racemosa (Q-21) Symphoricarpos albus (P-26) COMPOSITAE Achillea millefolium (P-10) Ambrosia chamissonis (Q-29) Antennaria microphylla (P-21) Arnica cordifolia (P-31) Arnica sororia (P-7) 2 Ck 1+i 3+ 2+ 3+ 5+ 3+ 3+i 2+ 5+ 1 Br 0 0 0 0 3+ 2+ 0 0 4+ 3 A e 0 0 0 0 0 0 0 0 0 4 2 2 2 1 2 1 5 3 3 Br Br Br Bk Br Wh Ae Wh Ae Ae 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1+i 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1+ 1+ 0 1+ 1+ 1+ 2+ 0 1+ 0 1+ 1+ 0 1+ 1+ 1+ 2+ 0 1+ 0 0 0 0 0 0 0 2+i 0 0 0 1+ 2+ 0 0 0 1+ 0 0 1+ 0 3 3 0 2 2 3 4 0 3 0 0 1 0 0 0 0 4 0 0 0 34 Family Species (Voucher No.) Cat." Part0 A.f.d A.fu. Ca. F.t. M.c. M.g. S.c. T.v. T.m. Number0 Numbed active excluding + 1 Derm. Artemisia ludoviciana (W-5) 1 Ae Artemisia michauxiana (P-29) 2 Ae Artemisia tridentata (W-19) 1 Br Balsamorhiza sagittata (W-18) 2 Ae Balsamorhiza sagittata (P-2) 3 Rt Chaenactis douglasii (P-3) 2 Wh Chrysothamnus nauseosus (P-25) 3 Br Erigeron filifolius (P-9) 3 Ae Gaillardia aristata (P-8) 2 Ae CONOCEPHALACEAE [BRYOPHYTA] Conocephalum conicum (Q-28) 2 Th CORNACEAE Cornus canadensis (Q-12) 3 Ae Cornus sericea (W-6) 2 Br CRASSULACEAE Sedum lanceolatum (P-20) 5 Wh 1+ 0 1+ 0 1+i 0 0 0 0 2+ 0 2+ 0 2+ 0 0 0 0 2+ 0 2+ 0 2+ 0 0 0 0 2+ 0 2+ 0 1+ 0 0 0 0 3+ 1+ 4+ 1+ 4+ 2+ 2+ 1+ 1+ 2+ 1+ 3+ 1+ 3+ 1+ 1+ 1+ 1+ 2+ 0 2+ 0 2+ 0 0 0 0 2+ 0 2+ 0 1+ 0 0 0 0 2+ 0 2+ 1+ 3+ 1 + 1+ 1 + 1+ 0 1+ 0 0 0 2+ 0 0 2+ 1+i 0 0 1 + 2 + 3 + 0 1+ 5+ 0 0 0 0 0 1+ 0 0 1+ 0 0 0 0 0 0 0 0 0 9 2 9 3 9 3 3 3 3 9 0 9 0 9 1 1 0 0 6 0 35 Cat.1 Parf A.f.d A.fu. Ca. F.t. Family Species (Voucher No.) M.c. M.g. S.c. T.v. T.m. Numbere Numbed active excluding +1 Derm. CRUCIFERAE Capsella bursa-pastoris (W-8) Cardamine angulata (Q-16) CUPRESSACEAE [Gymnospermae] Juniperus communis (Q-25) E L A E A G N A C E A E Shepherdia canadensis (W-16) EMPETRACEAE Empetrum nigrum (Q-17) EQUISETACEAE [Lycopsida] Equisetum arvense (W-3) Equisetum hyemale (P-37) ERICACEAE Arctostaphylos uva-ursi (P-42a) Arctostaphylos uva-ursi (P-42b) Kalmia microphylla (Q-5) 5 W h 0 0 0 0 0 0 0 0 0 3 Rt 0 0 0 0 0 0 1+ 0 0 1 Br 0 0 0 0 1+ 2+ 0 0 1+ 2 F r 0 0 0 0 0 0 0 0 0 5 Br 1+ 2+ 2+i 1+i 5+ 5+ 1+ 1+ 5+ 2 Ae 0 0 0 0 0 0 0 0 0 2 Ae 0 0 0 1+ 1+ 1+ 1+i 0 0 2 Br 0 0 0 0 3+ 2+ 0 1+ 5+ 2 Rt 0 0 0 0 0 2+ 0 0 1+ 2 Br 0 0 0 0 2+ 2+ 0 0 1+ 0 1 0 4 4 2 3 0 1 0 2 4 1 2 36 Family Species (Voucher No.) Cat.b Partc A.f.d A.fu. Ca. F.t. M.c. M.g. S.c. T.v. T.m. Number6 Numbed active excluding + 1 Derm. Ledum groenlandicum (Q-4) 1 Br Moneses uniflora (Q-8) 3 Ae Monotropa uniflora (P-l9) 2 Wh G R O S S U L A R I A C E A E Ribes sanguineum (P-l8) H Y D R A N G E A C E A E Philadelphus lewisii (P-22) H Y L O C O M I A C E A E [ B R Y O P H Y T A ] Hylocomium splendens (Q-9) H Y P E R I C A C E A E Hypericum perforatum (P-30) L E G U M I N O S A E Lupinus sericeus (P-l2) L I L I A C E A E Disporum trachycarpum (W-ll) 5 Wh Maianthemum racemosa (W-17) 2 Rh 0 0 0 0 2+ 2+ 0 0 0 3+ 5+ 2+ 5+ 5+ 5+ 3+ 2+ 5+ 0 0 0 0 0 0 0 0 0 2 Br 0 0 0 0 2+ 1+ 0 0 2+ 2 Br 0 0 0 0 0 0 3+ 0 0 3 Ga 0 0 0 0 1+ 1+ 0 0 0 3 Ae 0 0 0 0 1+ 1+ 0 0 0 5 Ae 0 0 0 0 1+ 1+ 0 0 1+ 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 2 9 0 2 9 0 0 0 37 Cat." Parf A.f.d A.fu. Ca. F.t. M.c. M.g. S.c. T.v. T.m. Number6 Numbed Family active excluding Species (Voucher No.) +1 Derm. Maianthemum stellata (W-13) l R h 0 0 0 0 4+ 3+ 0 0 5+ 3 3 LOBARIACEAE [ETJMYCOTA] Lobaria oregana (Q-ll) 3 T h 0 0 0 0 2+ 2+ 0 0 0 2 2 LYCOPODIACEAE [Lycopsida] Lycopodium clavatum (Q-6) 2 Br 0 0 0 1+ 2+ 2+ 1+ 0 0 4 4 MENYANTHACEAE Fauria crista-galli (Q-19) 4 Ae 0 1+ 1+ 1+ 1+ 1+ 1+ 0 0 6 4 NYMPHAEACEAE Nuphar lutea (Q-3b) 2 Rt 0 0 0 0 0 1+ 0 0 2+ 2 1 Nuphar lutea (Q-3c) 2 Rh 0 0 0 0 2+i 2+ 0 0 5+ 3 3 ONAGRACEAE Epilobium minutum (P-l) 5 W h 0 0 0 0 0 0 0 0 0 0 0 PINACEAE [Gymnospermae] Larix occidentalis (W-15) 3 Br 1+i 0 0 0 1+ 1+ 0 0 1+ 4 1 Pinus contorta (Q-18) 1 Br 0 0 0 0 3+ 2+ 0 0 1+ 3 2 P/'/IMJ ponderosa (W-20) 1 Br 0 0 0 0 2+ 1+ 0 0 2+ 3 2 38 Family Species (Voucher No.) Cat.b Parf A.f.d A.fu: Ca. F.t. M.c. M.g. S.c. T.v. T.m. Number6 Number* active excluding +1 Derm. PLANTAGINACEAE Plantago major (Q-22) POLEMONIACEAE Ipomopsis aggregata (P-13a) Ipomopsis aggregata (P-13b) POLYGONACEAE Eriogonum heracleoides (P-11) Eriogonum heracleoides (P-17) POLYPODIACEAE [Filicinae] Polypodium glycyrrhiza (Q-27) Polystichum munitum (Q-15) POLYPORACEAE [EUMYCOTA] Ganoderma applanatum (Q-10) RANUNCULACEAE Clematis ligusticifolia (P-14) Delphinium nuttallianum (P-33) 2 Wh 0 0 1+i 1+ 0 1+ 1+i 0 0 1 Ae 1+i • 1+i 2+ 1+ 5+ 4+ 4+ 0 5+i 1 Rt 1+i 1+i 2+ 1+ 5+ 4+ 4+ 1+i 5+i 3 Ae 0 0 0 0 1+ 1+ 0 0 0 3 Rt 0 0 0 0 1+ 1+ 0 0 0 1 Rh 0 0 0 0 2+ 3+ 0 0 5+ 1 Rh 0 0 0 0 3+ 3+ 0 1+ 3+ 6 W h 0 0 0 0 1+ 1+ 0 0 0 1 Ae 0 0 0 0 1+ 1+ 0 0 0 4 W h 0 0 0 0 0 0 0 0 0 2 2 3 4 0 0 3 4 2 0 0 0 39 Family Species (Voucher No.) Cat." Partc A.f.d A.fu. Ca. F.t. M.c. M.g. S.c. T.v. T.m. Number6 Numbed active excluding +1 Derm. RHAMNACEAE Ceanothus velutinus (P-39) ROSACEAE Amelanchier alnifolia (P-6) Amelanchier alnifolia (P-35) Argentina egedii (Q-20) Aruncus dioicus (F-l) Crataegus douglasii (W-14) Fragaria chiloensis (Q-7) Fragaria vesca (W-l) Geum macrophyllum (Q-23) Holodiscus discolor (F-3) Potentilla arguta (W-7) Prunus virginiana (W-9) Prunus virginiana (P-40) Rosa nutkana (P-5) 3 3 "3 2 4 3 1 2 3 5 2 2 1 Br Br Br Br Br Br Lf Lf Rt Br Rt Br Br Br 0 0 1+i 0 0 1+i 1+i 1+i 0 0 0 0 0 1+ 0 0 2+ 0 0 2+i 0 3+i 0 0 0 0 0 2+ 0 0 2+i 0 0 1+i 0 2+i 0 1+i 0 0 0 2+ 0 0 0 0 0 2+ 1+i 2+ 0 0 0 0 0 4+ 0 0 2+ 1+ 0 3+ 2+ 3+ 1+ 3+ 0 1+ 1+ 2+ 0 0 1+ 1+ 0 2+ 2+ 2+ 1+ 1+ 0 1+ 1+ 1+ 0 0 2+i 0 0 2+i 2+i 2+i 0 1+i 0 0 0 0 0 1+ 0 0 1+i 1+i 2+i 0 0 0 0 0 1+ 0 0 3+ 1+ 0 4+ 2+ 5+ 1+ 2+ 0 0 0 0 0 8 3 0 9 7 9 3 5 0 2 2 0 0 7 0 0 9 7 9 0 4 0 0 0 40 Cat." Parf A.f.d A.fu. Ca. F.t. M.c. M.g. S.c. T.v. T.m. Family Species (Voucher No.) Number6 Number* active excluding +1 Derm. Rubus parviflorus (P-28) Spiraea betulifolia (P-41) Spiraea pyramidata (P-15) SALICACEAE Populus tremuloides (P-34) Salix bebbiana (P-36) SAXIFRAGACEAE Heuchera cylindrica (W-4) SCROPHULARIACEAE Penstemon fruticosus (P-4) Verbascum thapsus (P-24) UMBELLIFERAE Glehnia littoralis (Q-13) Heracleum maximum (P-32a) Heracleum maximum (P-32b) Lomatium dissectum (W-10) 3 5 4 4 2 1 1 Br Br Br Rt Ae Rt Rt 0 0 0 0 0 0 1+ 0 0 2+ 0 1+ 2+ 0 1+ 1+ 0 2+i 0 2+ 1+ 2+ 0 1+ 0 1+ 0 1+ 0 3+ 0 5+ 1+ 5+ 0 4+ 1+ 3+ 0 0 0 0 1+ 0 1+ 0 0 0 0 1+ 0 1+ 0 3+ 0 2+ 3 Br 0 1+ 1+ 2+ 3+ 2+ 1+i 1+ 1+ 3 Br 0 0 0 0 1+ 1+ 0 0 0 1 Rt 1+ 1+ 1+ 1+ 1+ 1+ 1+ 1+ 2+ 2 Br 0 0 0 0 1+ 1+ 0 0 0 1 Lf 0 0 0 0 1+ 1+ 0 0 0 4+ 1+ 1+ 3+i 4 0 3 4 0 1 7 0 0 0 9 1 41 Cat." Paif A.f.d A.fu. Ca. F.t. M.c. M.g. S.c. T.v. T.m. Number6 Number* Family active excluding Species (Voucher No.) +1 Derm. Osmorhiza purpurea (Q-24) 4 Rt 0 0 0 1+ 0 1+ 1+i 0 0 3 2 URTICACEAE Urtica dioica (P-27) 2 Ae 0 0 0 0 1+ 1+ 1+i 0 0 3 1 Total number of active extracts 20 24 23 27 72 78 30 23 56 81 57 Key to Table 4 a Classification of results: 0 = no inhibition or zone of inhibition < 8.0 mm; 1+ = zone of inhibition 8.0-10.0 mm; 2+ = 10.1-15.0 mm; 3+ = 15.1-20.0 mm; 4+ = 20.1-25.0 mm; 5+ = > 25.0 mm; i = incomplete inhibition, some spores germinated within clearing zone. b Cat. = Ethnobotanical category: 1 = Potential antifungals; 2 = Possible antifungals; 3 = Other skin problems; 4 = Other medicinal uses; 5 = Related species; 6 = Non-medicinal plant. c Part Extracted: Ae = Aerial; Bk - Bark; Br = Branch; Ck = Catkin; Fr = Fruit; Ga - Gametophyte; lb - Inner Bark; Lf = Leaf; Rh = Rhizome; Rt = Root; Th = Thallus; Wh = Whole plant. d Fungi: A.f. = Aspergillus flavus; A.fu. = Aspergillus fumigatus; Ca. = Candida albicans; F.t. = Fusarium tricuictum; M.c. = Microsporum cookerii; M.g. = Microsporum gypseum; S.c. = Saccharomyces cerevisiae; T.v. = Trichodermd viridae; T.m. = Tricophyton mentagrophytes e Total number of fungi the extract was active against. f Total number of fungi the extract was active against, excluding +1 activity against M. cookerii, M. gypseum, and T. mentagrophytes. 42 Table 5 - Phase one antifungal screening results summarized by taxa Excluding 1+ super-suscept.a Taxa Number in Number Percent Number Percent Category (N) Active (N) Active (%) Active (N) Active (%) NON-FLOWERING PLANTS Lower plants Eumycota 2 2 100 1 50 Bryopsida 2 2 100 1 50 Lycopsida 1 1 100 1 100 Sphenopsida 2 1 50 0 0 Lower plants sub-total 7 6 86 3 43 Higher plants Filicinae 2 2 100 2 100 Gymnospermae 4 4 100 4 100 Higher plants sub-total 6 6 100 6 100 NON-FLOWERING Sub-total 13 12 92 9 69** FLOWERING Sub-total 87 69 79 48 55 GRAND TOTALS 100 81 81 57 57 " Number active (N) calculated excluding those extracts with only slight (1+) activity against the super-susceptible organisms M. cookerii, M. gypseum and T. mentagrophytes. " Percentage of active extracts in category statistically significant, p < 0.01 43 T a b l e 6 - Ethnopharmaco log ica l analysis of phase one antifungal screening results Category Number in Category (N) Number Active (N) Percent Active (%) Excluding 1+ Super-susc.a Number Percent Active (N) Active (%) Potential antifungals 23 23 100** 19 §3** Possible antifungals 36 30 83** 21 58 Other skin problems 24 19 80 11 46 Tonics 7 5 71 4 57 Other medicinal uses 9 3 33 2 22 Subtotal medicinal 99 80 80 57 57 No medicinal use 1 1 100 0 0 Grand Totals 100 81 81 57 57 a Number active (N) calculated excluding those extracts with only slight (1+) activity against the super-susceptible organisms M. gypseum and T. mentagrophytes. " Percentage of active extracts in category statistically significant, p < 0.01 44 2.2.4 Discussion and Conclusions Since fungal infections rarely pose any serious health problems, especially in temperate climates, there are very few specific references to their treatment in the ethnobotanical literature. Consequently, it would seem doubtful that such literature could provide much assistance in the search for new antifungal compounds. However, in consideration of the growing need for new systemic antifungals, it has become important to evaluate the results of this screening in the context of the ethnobotanical literature to determine how this type of information could be of future assistance in identifying potential new antifungal medicaments. The screening results show a fairly high correlation between traditional medicinal use and antifungal activity (see Table 6). Eighty percent of the plants which have been documented as being used medicinally by First Nations peoples were found to have antifungal activity. This figure drops to 57% when those extracts which were only slightly active against the more susceptible dermatophytes (M. cookerii, M. gypseum and T. mentagrophytes) are excluded from the calculation. Given the fairly ambiguous nature of the descriptions on which the ethnopharmacological classifications were based, the results of the ethnopharmacological analysis far exceeded expectations. The percentage of active extracts in category 1 was significantly higher than in any other group. These results certainly suggest that it would be most profitable for future antifungal screenings to focus on plants used specifically to treat fungal infections. The analysis of results by taxa (see Table 5) suggests that it may be worthwhile to screen more non-flowering plants in future studies. It should be pointed out that the vast majority of plants used medicinally among the British Columbian First Nations belong to the Gymnospermae and Angiospermae. Despite the fact that British Columbia has a very rich diversity mosses and fungi due to its cool, moist climate, very few of these organisms were utilized by the aboriginal peoples as medicines. As the ethnopharmacological analysis demonstrated, the selection of specimens based on traditional usages appears to increase the probability of selecting plants with antifungal activity. Hence a larger screening study of randomly selected non-medicinal lower plants may not find a high percentage of active extracts within these taxa. In general, those extracts found to have antifungal activity in this screening correlate fairly well with those found to have antibiotic activity with a few notable exceptions. While the extracts of Arctlostaphylos uva-45 ursi, Juniperus communis, Lomatium dissectum, Nuphar lutea and Ribes sanguineum all exhibited good antibiotic activity, they had fairly poor antifungal activity, inhibiting only the sensitive dermatophytes. Conversely, while the extracts of Asarum caudatum and Ipomopsis aggregata exhibited fairly poor antibiotic activity, they inhibited the growth of all nine fungi in this antifungal screening study. The remaining 13 extracts which had antifungal activity against all nine fungi, also had exhibitied good antibiotic activity. It is also interesting to note that while the extracts of both the catkins and bark of Alnus rubra had very good antibiotic activity, only the catkin extract exhibited broad spectrum antifungal activity. The Rhus glabra extract, which had the strongest antibiotic activity, was only moderately inhibitory of the fungi although it exhibited a broad spectrum of activity. In addition to providing promising new leads in the ongoing search for new antimicrobial compounds, the data analysis from this screening has also suggested how future screenings may be improved. The results reported here seem to support the assertion that the North American flora is worthy of further pharmacological investigation and that the ethnobotanical literature can be useful in guiding this research. Acknowledgements I would like to thank the physicans of Student Health Services at the University of British Columbia for their assistance in evaluating the ethnobotanical information. I would also like to thank Mrs. Z. Abramowski for the training and technical assistance she provided. References Lennette, E . H. (1985) Manual of Clinical Microbiology (4th ed.). American Association for Microbiology, Washington, D.C, pp. 978-987. 46 2.3 Phase one anti-mycobacterial screening Abstract One hundred methanolic plant extracts were screened for antibiotic activity against Mycobacterium tuberculosis and an isoniazid resistant strain of Mycobacterium avium. Nineteen extracts exhibited activity against M. tuberculosis and 16 extracts showed activity against M. avium. Thirteen of these 19 active extracts were traditionally used to treat tuberculosis. There was a significant correlation (0.945) between anti-mycobacterial activity and activity against the bacteria M. phlei used in the general antibiotic screening (see chapter 2.1). Extracts made from Heracleum maximum (Umbelliferae) roots, Moneses uniflora (Ericaceae) aerial parts and Oplopanax horridus (Araliaceae) inner bark completely inhibited the growth of both organisms at a concentration equivalent to 20 mg of dried plant material per disc. Extracts of Alnus rubra (Betulaceae) bark and catkins, Empetrum nigrum (Empetraceae) branches, Glehnia littoralis (Umbelliferae) roots and Lomatium dissectum (Umbelliferae) roots completely inhibited the growth of both organisms at a concentration equivalent to 100 mg of dried plant material per disc. 2.3.1 Introduction In the western world, tuberculosis is commonly considered to be a disease of the past, a disease which has long since been conquered by the miracles of modern antibiotics. Along with the general public, many medical practitioners have come to consider tuberculosis as a disease that no longer posed a serious public health problem. Consequently, the scientific community has been fairly slow to respond to the growing evidence that the incidence of tuberculosis in North America and Europe is increasing. Some epidemiologists have warned that AIDS and multiple drug-resistant tuberculosis have the potential to precipitate the most disastrous public health crises since the bubonic plague (Stanford, 1991). In addition to Mycobacterium tuberculosis, two other species, M. avium and M. intracellulare (commonly referred to as MAC, M. avium complex) cause human disease, particularity in immunocompromised hosts. These two species have also emerged as important pathogens of humans because of the increased incidence associated with AIDS and their natural resistance to the common 47 anti-mycobacterial drugs. It is clear that public health measures alone cannot contain the threat of multiple drug resistant tuberculosis. Potent new anti-mycobacterial drugs are desperately, needed not only for AIDS patients but also for the health care workers and members of the general public who are being striken by these often fatal bacterial infections. In Canada, the incidence of tuberculosis is significantly higher (10 x) among aboriginal populations than in the general public (Young, 1988). Many people have assumed that this was due to the fact that tuberculosis was newly introduced into the native population by European settlers. However, there is strong archaeological evidence that tuberculosis was present in Pre-Columbian America (Bulkstra, 1981; Clark, 1987; Pfeiffer, 1984) and it is therefore reasonable to assume that the North American aboriginal peoples have an equally long history of seeking out a cure for this disease. Given the pressing need for new anti-mycobacterial drugs, it was deemed worthwhile to examine the potential of these traditional remedies as modern therapeutics. 2.3.2 Methods Microorganisms Mycobacterium tuberculosis (strain Erdman, Trudeau Mycobacterial Collection [TMC] # 107; American Type Culture Collection [ATCC] # 35801) and M. avium (TMC # 724; ATCC # 25291) were grown, stored and assessed for viability as previously described (Stokes et al., 1993). Assay protocol A standard drug sensitiviity testing method for mycobacteria was employed. Ten ul of plant extract (representing 20 mg of dried plant material) was applied to a 0.25 inch diameter blank paper disc (Becton Dickinson, Cockeysville, MD) and allowed to air dry. Discs were placed in quadrant plates (Becton Dickinson) and five mis of molten (56°C) Middlebrook 7H10 agar + oleic acid, dextrose complex (Becton Dickinson) was plated onto each quadrant. After setting, plates were incubated overnight at 4°C to allow for diffusion of the compounds. Control discs were loaded with 10 ul methanol or 10 ul of 10 mg/ml isoniazid (one of the first choice drugs for treatment of M. tuberculosis). To each quadrant 100 ul of bacterial suspension was added which contained approximately 1.5 x 106 M. tuberculosis or 2 x 103 M. avium. Plates were incubated for 3 weeks 48 in sealed bags at 37"C after which bacterial growth was assessed. To confirm the activity of those extracts which showed only slight inhibition, the assay was repeated using 50 ul/disc (the equivalent of lOOmg of dried plant material). An arbitrary scale was used to score the anti-mycobacterial activity of each extract. Extracts scored as "-" had no discernable effect on the bacterial growth. Extracts scored as "+" caused a small zone of clearing or a zone of inhibition with a few resistant colonies within it, though colonies were too numerous to count. Extracts scored as "++" greatly inhibited the growth of the mycobacteria, to the extent that less than 50 colonies were present. Extracts scored as "+++" completely inhibited all growth. The Pearson correlation coefficient between the results of this study and the activity these extracts exerted against M. phlei in the general antibiotic screening (chapter 2.1) was calculated using the computer program SYSTAT (Wilkinson, 1988). 2.3.3 Results The anti-mycobacterial screening results for those plant extracts which exhibited activity are given in Table 7. Nineteen of the 100 methanolic plant extracts screened exhibited some antibiotic activity against M. tuberculosis and 16 of the extracts were active against M. avium. Thirteen of these 19 active extracts were traditionally used to treat tuberculosis and another four were used in the treatment of coughs. The extracts of Heracleum maximum roots, Moneses uniflora aerial parts and Oplopanax horridus inner bark completely inhibited the growth of both M. tuberculosis and M. avium at a concentration equivalent to 20 mg of dried plant material. The extracts of Alnus rubra bark and catkins, Empetrum nigrum branches, Glehnia littoralis roots and Lomatium dissectum roots completely inhibited the growth of both test organisms at a concentration equivalent to 100 mg of dried plant material. Three extracts inhibited the growth of M. tuberculosis but did not affect the growth of M. avium. These active extracts were made from: Balsamorhiza sagittata roots, Fragaria vesca leaves and Geum macrophyllum roots. The correlation between anti-mycobacterial activity and antibiotic activity against M. phlei was calculated to be 0.945. 49 Table 7 - Phase one anti-mycobacterial assay results" Organisms assayed against Equivalent of dried plant material/disc M. tuberculosis 20 mg 100 mg M. avium 20 mg 100 mg Positive control (Isoniazid) Alnus rubra (Betulaceae) Q-l Bark T b Alnus rubra (Betulaceae) Q-2 Catkins T Balsamorhiza sagittata (Compositae) P-2 T Chaenactis douglasii (Compositae) P-3 T Empetrum nigrum (Empetraceae) Q-l7 T Fragaria vesca (Rosaceae) W-l T Geum macrophyllum (Rosaceae) Q-23 P Glehnia littoralis (Umbelliferae) Q-l3 M Heracleum maximum (Umbelliferae) P-32b T Hypericum perforatum (Hypericaceae) P-30 C Juniperus communis (Cupressaceae) Q-25 T Lomatium dissectum (Umbelliferae) W-10 T Moneses uniflora (Ericaceae) Q-8 C Nuphar lutea (Nymphaceae) Q-3c T Oplopanax horridus (Araliaceae) Q-l4 T Pinus contorta (Pinaceae) Q-l8 T Polystichum munitum (Polypodiaceae) Q-l5 C Populus tremuloides (Salicaceae) P-34 T Rosa nutkana (Rosaceae) P-5 C +++ ++ ++ +++ +++ +++ +++ +++ +++ ++ +++ +++ + +++ +++ +++ + ++ +++ +++ +++ +++ +++ + +++ ++ +++ ++ ++ +++ + +++ +++ +++ +++ +++ +++ ++ + +++ +++ + + + Total 15 19 16 a Key to scoring: -, no inhibition; +, zone of inhibition with a few resistant colonies within it or small zone of clearing (colonies too numerous to count) ; ++, large zone of clearing or greatly inhibited growth (less than 50 colonies present) ; +++, complete inhibition. b Traditional usage: C = coughs, M = unspecified medicinal plant, P = physic, T = tuberculosis medicine. 50 2.3.4 Discussion and Conclusions Tuberculosis's infamy is due not only to the fact that it it the greatest killer in human history, responsible for over a billion deaths in the last two centuries alone (Ryan, 1992) but also because each death was preceded by a prolonged, painful decline in health. Tuberculosis is contracted simply by inhaling the airborne bacteria, making every human, regardless of race, sexual preference or economic status, susceptible to the disease. In the majority of people that come in contact with the bacteria, the immune system is able to successfully contain the organism and disease symptoms do not develop. It is thought that the unusual waxy coat of Mycobacterium sp. protects them from the killing action of macrophages' proteolytic enzymes, allowing them to survive and multiply within white blood cells. Unable to eradicate the bacteria, the immune system walls off the bacteria within a granuloma to contain the infection. The bacteria remain as a latent threat within these tubercules, capable of reactivating if the host's immune system is compromised in any way. It has been estimated that there are approximately 1.7 billion people or roughly one third of the world's population infected with tuberculosis (Sudre, 1992). In approximately 10% of these infections, the body fails to contain the bacteria, resulting in disease. Left unchecked the bacteria eventually infiltrate and infect every organ, causing permanent scarring of the lungs, grotesque and painful abcessess of the skin and soft tissues (scrofulous sores), excruciating inflammation of the internal organs and gouging cankerous cavities in the bone. An estimated 2.9 million people died from tuberculosis in 1990, making this disease the largest cause of death from a single pathogen in the world (Murray, 1991). The HIV virus destroys the very white blood cells which enable most people to fight off Mycobacterium infections. AIDS patients are therefore extremely vunerable to contracting tuberculosis from either inhaling the bacteria or through the reactivation of latent disease. The HIV virus also makes patients susceptible to infections of M. avium and M. intracellular (MAC), though it is thought that infection with the MAC species occurs through the gastrointestinal route and leads to a very different pathenogenesis from that usually associated with M. tuberculosis. Another reason for the resurgence of mycobacterial infections in the developed world is thought to be the rapidly increasing incidence of multiple drug-resistant strains of M. tuberculosis and M. avium. The innate capacity of Mycobacterium to develop resistance to a drug was observed in trials of the first antibiotics against 51 tuberculosis. Resistance was also observed to emerge when only two drugs are used. Therefore the standard treatment for tuberculosis became a combination of drugs, typically isoniazid, rifampin and pyrazinamide. During the 1980's, the number of reports of multiple drug resistant (MDR) tuberculosis began to increase. More alarmingly, many of these MDR strains were resistant not only to the first line antibiotics but also many of the secondary drugs, some strains being resistant to seven of the most effective tuberculosis drugs available (Iseman, 1985) and as virulent as the wild type strains (Rosenthal, 1992). The increasing incidence of MDR strains worldwide emphasizes the desperate need for new anti-mycobacterial drugs. The search for tuberculosis therapeutics however, is both far more difficult and dangerous than most antibiotic development programs. The virulence of these airborne pathogens necessitates extraordinary containment facilties and specially trained personnel in order to safely conduct research in this area. Other genera of bacteria cannot reliably substitute for anti-mycobacterial screenings as the unusual waxy coat that makes Mycobacterium impervious to the digestive enzymes of white blood cells is also an inpenetrable barrier to many antibiotics and some compounds with anti-mycobacterial activity have no effect against other bacterial species. Therefore, leads towards the discovery of new drugs and findings which may improve the efficacy of the search are of value. In both of these contexts, the results of this antibiotic screening of 100 methanolic plant extracts against M. tuberculosis and M. avium appear promising. Nineteen extracts showed activity against M. tuberculosis and 16 extracts were active against M. avium. Of these active extracts, six extracts were particularily outstanding in their ability to completely inhibit the growth of both organisms: Empetrum nigrum, Glehnia littoralis, Heracleum maximum, Lomatium dissectum, Moneses uniflora, and Oplopanax horridus. Chemical isolation work to identify the active constituents of Oplopanax horridus is still in progress at this writing. It is noteworthy that 3 of these very active extracts were made from members of the same plant family, the Umbelliferae: G. littoralis, L. dissectum and H. maximum. A pair of unstable tetronic acids were identified as the antimicrobial constituents of L. dissectum (Cardellina and Vanwagenen, 1985; Vanwagenen and Cardellina, 1986) but it is not known if these compounds are also responsible for this plant's anti-mycobacterium activity. The family Umbelliferae is well known for its cytotoxic furano-coumarin constituents and these compounds may be responsible for the anti-mycobacterial activity observed in these family members. 52 A comparison of the results of the general antibiotic screening reported in chapter 2.1 and the anti-mycobacterial screening results suggest that an extract's ability to strongly inhibit the growth of the related organism Mycobacterium phlei (a fast growing, non-pathogenic bacteria) may provide a good selection criterion for anti-mycobacterial screening candidates. There was a significant correlation (0.945) between those extracts which had an inhibitory effect on M. phlei and those extracts which were active in the present study. The six extracts found to be most active in this study were also in the group of ten extracts which were the most active against M. phlei. These results seem to support the assertion that the inclusion of M. phlei in general antibiotic screenings is quite useful, as very strong activity against this organism may be indicative of activity against other species of Mycobacterium. Can the traditional usage of a plant to treat tuberculosis also be used as an effective selection criterion for anti-mycobacterial screenings? In this study, a comparison of the ethnobotanical literature and the screening results shows that 13 of the 19 active extracts (68%) were prepared from plant species which were specifically reported to have been used for the treatment of tuberculosis. These active tuberculosis remedies are indicated with a letter "T" in Table 7. There were no reports that the extracts of the six other plant species which exhibited anti-mycobacterial activity were used specifically to treat tuberculosis, however, four of these plants were reported to have been used to treat coughs (these extracts are indicated by a letter "C" in Table 7). Of the 100 extracts screened, 37 samples were prepared from plant species which were reported to have been used to treat tuberculosis or consumption, and an additional 16 were reported to have been used to treat scrofula, lung hemorrhage or blood spitting (see Appendix 6 for ethnobotanical references). These results suggest that there may be a correlation between traditional usage in the treatment of tuberculosis and anti-mycobacterial activity. Acknowledgements This anti-mycobacterial screening was conducted by research collaborators Dr. R. Stokes and L. Thorson, Department of Pediatrics, Division of Infectious and Immunological Diseases, U.B.C.. 53 References Bulkstra, J.E. and Cook, D.C. (1981) Pre-Columbian tuberculosis in west-central Illinois: Prehistoric disease in biocultural perspective. In: Bulkstra, J.E. (ed.) Prehistoric tuberculosis in the Americas. Northwestern University Archeology Program. Evanston, 111. Cardellina, J.H. and Vanwagenen, B.C. (1985) Antifungal agents from Lomatium dissectum. Abstracts of the International Research Congress on Natural Products. University of North Carolina, Chapel Hill, N.C. Abstract 211. Clark, G.A., Kelley, M.A., Grange, J.M. and Hill, M.C. (1987) The evolution of mycobacterial disease in human poulations. Current Anthropology 28: 45-62. Iseman, M.D. and Madsen, L.A. (1989) Drug resistant tuberculosis. Clinics in Chest Medicine 10 (3): 341-353. - Murray, J.F. (1991) An emerging global programme against tuberclosis: agenda for research, including the impact of HIV infection. Bulletin of the International Union for Tubercular Lung Disease 66: 199-201. Pfeiffer, S. (1984) Paleopathology in an Iroquian ossuary, with special references to tuberclosis. American Journal of Physical Anthropology 65: 181-189. Ryan, F. (1992) The Forgotten Plague: How the battle against tuberculosis was won - and lost. Little, Brown and Co., Boston, pp. 460. Stanford, J.L., Grange, J.M. and Pozniak, A. (1991) Is Africa lost? Lancet 338: 557-558. Vanwagenen, B.C. and Cardellina, J.H. (1986) Native American food and medicinal plants 7. Antimicrobial Tetronic Acids. Tetrahedron 42(4): 1117-1122. Wilkinson, L. (1988) SYSTAT: the system for statistics. SYSTAT Inc., Evanston, 111. Young, T.K. and Casson, R.I. (1988) The decline and persistence of tuberculosis in a Canadian Indian population: implications for control. Canadian Journal of Public Health 79: 302- 305. See also: Enarson, D.A. and Grzybowski, S. (1986) Incidence of active tuberculosis in native populations of Canada. Canadian Medical Association Journal 134: 1149. 5 4 2.4 Phase one antiviral screening Abstract One hundred methanolic plant extracts were screened for antiviral activity against seven viruses. Twelve extracts were found to have antiviral activity at the non-cytotoxic concentrations tested. The extracts of Rosa nutkana and Amelanchier alnifolia, both members of the Rosaceae, were very active against an enteric coronavirus. A root extract of another member of the Rosaceae, Potentilla arguta, completely inhibited respiratory syncytial virus. A Sambucus racemosa branch tip extract was also very active against respiratory syncytial virus while the inner bark extract of Oplopanax horridus partially inhibited this virus. An extract of Ipomopsis aggregata demonstrated very good activity against parainfluenza virus type 3. A Lomatium dissectum root extract completely inhibited the cytopathic effects of rotavirus. In addition to these, extracts prepared from the following plants exhibited antiviral activity against herpesvirus type 1: Cardamine angulata, Conocephalum conicum, Lysichiton americanum, Polypodium glycyrrhiza, and Verbascum thapsus. 2.4.1 Introduction The search for selective antiviral agents, principally focused on anti-human immunodefiency virus (HIV) agents, has been vigorous in recent years (De Clercq, 1988) but progress in the development of useful new antivirals has been painstakingly slow (Galasso, 1988). Meanwhile, frequencies of viral resistance to the relatively few anti-viral drugs currently used are increasing (De Clercq, 1993) and the problem of viral latency, the greatest obstacle to treatment of some viral infections, remains unsolved. The increasingly urgent need to find effective therapeutics justifies not only an accelerated search for new agents but also a broader scope to such research. Ethnopharmacological screenings provide scientists with an alternative avenue to discovery from the current mainstream approach of attempting to design narrow spectrum drugs for specific molecular targets. The ethnopharmacological approach has equal potential for identifying new antiviral compounds, yet relatively few antiviral screenings of plant ethnomedicines have been conducted to date. "In view of the significant proportion of plant extracts that have yielded positive results in these screenings, it seems reasonable to conclude that there 55 are probably numerous types of antiviral compounds in these materials. Further characterization of the active ingredients of some of these plants should reveal some useful compounds" (Hudson, 1990). It seems prudent, if not imperative, that researchers continue to investigate these sources before the knowledge or the plants themselves are lost. In this chapter, the results of an antiviral screening of 100 methanolic plant extracts against seven viruses are presented. 2.4.2 Methods Viruses and Cell lines The effect of the methanolic plant extracts on the replication of seven selected viruses representing a spectrum of viral families was assayed. The viruses selected were: bovine coronavirus (BCV, Coronaviridae), bovine herpesvirus type 1 (BHV1, Herpesviridae), bovine parainfluenza virus type 3 (BPI3, Paramyxoviridae), bovine rotavirus (BRV, Reoviridae), bovine respiratory syncytial virus (BRSV, Paramyxoviridae), vaccinia virus (Poxviridae) and vesicular stomatitis virus (VSV, Rhabdoviridae). Viruses were propagated in established cell lines which were maintained in vitro as monolayer cultures using Eagle minimal essential medium (MEM) supplemented with fetal bovine serum (10% v/v) and gentamicin (lOug/ml). The cells were incubated at 37"C in a humidified environment containing 5% C0 2 . BCV, BHV1 and VSV were grown in Madin-Darby bovine kidney (MDBK) cells; BRV and vaccinia virus in African green monkey kidney (MA104) cells; BRSV in Georgia bovine kidney (GBK) cells; and BPI3 in African green monkey kidney (Vero) cells. Antiviral assays The abilities of dilute plant extracts to inhibit virus-specific cytopathic effects were used as a measure of antiviral activities. Near-confluent 0.3 cm2 cell monolayers in 96-well plates (Flow Laboratories) were rinsed with serum-free MEM then each was treated with 0.2 ml of a plant extract diluted in serum-free MEM. The extracts were tested at dilutions ranging from 1 x 10'1 through 1 x 10"7. Antiviral activities were scored using cell cultures treated with extracts diluted sufficiently (usually 1 x 10"4) to eliminate any microscopically observable 56 toxic effects. Two samples (Ql and Q2) demonstrated residual toxicity at that level, hence they were scored after application at a dilution of 2.5 x 10"5. After 12 hours of treatment at 37°C, the medium was removed and the cultures were infected with stock preparations containing approximately 100 plaque-forming units (PFU) of the respective infectious virus in 0.1 ml of MEM. Mock-infected controls received sterile cell-culture medium. After a one hour absorption period, the innoculum was removed, the cells were washed twice with MEM then overlaid with 0.2 ml of fresh diluted plant extract. Plates were incubated at 37"C for two to seven days, depending upon the virus-cell combination used. Cytopathic effects were scored after microscopic observation. Each treatment (+/- plant extract, +/- virus) was performed in triplicate and the entire regimen was repeated at least once for each extract tested. 2.4.3 Results One hundred crude methanolic extracts of plants, 96 of which were used medicinally by British Columbian native peoples were screened for antiviral activity against seven viruses. Twelve plant extracts each demonstrated some antiviral activity against one virus. Scores of the degrees of inhibition of virus-induced cytopathic effects caused by treatment with these extracts are presented in Table 8. Results for vaccinia virus and VSV are not shown as none of the plant extracts was observed to inhibit the cellular cytopathology induced by these viruses at the extract dilutions used. 57 Table 8 - Phase one antiviral assay results3 Viruses assayed againstb corona- herpes- para- RSV rota-virus virus influenza virus Amelanchier alnifolia (Rosaceae) P-6 ++ - - -Cardamine angulata (Cruciferae) Q-l6 - + Conocephalum conicum (Conocephalaceae) Q-28 - + - -Ipomopsis aggregata (Polemoniaceae) P-13 - - ++ Lomatium dissectum (Umbelliferae) W-10 . . . - ++ Lysichiton americanum (Araceae) Q-26 - - + -Oplopanax horridus (Araliaceae) Q-l4 - - - + Polypodium glycyrrhiza (Polypodiaceae) Q-27 - + - -Potentilla arguta (Rosaceae) W-7 - - - + + -Sambucus racemosa (Caprifoliaceae) Q-21 - - - + + -Rosa nutkana (Rosaceae) P-5 ++ - - -Verbascum thapsus (Scrophulariaceae) P-24 - + - -a Classification of results: +, partial inhibition of virus-induced CPE; ++, complete inhibition of virus-induced CPE. b Viruses assayed against: coronavirus, bovine herpesvirus type 1, parainfluenza virus type 3, respiratory syncytial virus, rotavirus. 58 2.4.4 Discussion and Conclusions One of the inherent drawbacks of in vitro antiviral testing is the environmental sensitivity of animal cells in culture. Preparations which exert antiviral effects in vivo may not be detected in in vitro assays because of the extremely low concentrations of extract tolerated by cells in the artifical system. Even with this limitation, 12 of the 100 methanolic plant extracts screened exhibited some antiviral activity. Six of these active extracts completely inhibited virus induced cytopathic effects at the non-cytotoxic concentrations tested. As has been found in previous antiviral screenings (see Hudson, 1990 for overview), none of the extracts exhibited broad spectrum activity. Each active extract was effective against only one of the seven viruses screened. Three of the most active extracts in this study were members of the same plant family. The extracts made from Rosa nutkana and Amelanchier alnifolia, both members of the Rosaceae, completely inhibited the cytopathic effects of an enteric coronavirus. The extract of another member of the Rosaceae, Potentilla arguta, completely inhibited respiratory syncytial virus. Coronavirus and respiratory syncytial virus are similar in that they are both single-stranded RNA viruses which infect mucosal surfaces. A branch tip extract of Sambucus racemosa (Caprifoliaceae) also completely inhibited the cytopathic effects of respiratory syncytial virus while an inner bark extract of Oplopanax horridus (Araliaceae) exhibited partial inhibition. The extract of Ipomopsis aggregata (Polemoniaceae) completely inhibited cytopathology induced by parainfluenza virus type 3, another single-stranded RNA virus which causes respiratory disease. None of the extracts was effective against the fourth single-stranded RNA virus used in the screening, vesicular stomatitis virus. Rotavirus is a double-stranded RNA virus that causes gastroenteritis, one of the major infectious diseases in the world today, as judged by mortality statistics (Vesikari, 1988). The only extract which exhibited activity against this serious pathogen was a Lomatium dissectum (Umbelliferae) root extract which completely inhibited the cytopathic effects. Two double-stranded DNA viruses were used in this screening, herpesvirus type 1 and vaccinia virus. Herpesviruses cause respiratory, genital, conjunctival or encephalitic infections which become latent in the trigeminal ganglion. There is also a growing body of evidence that Kaposi's sarcoma is caused by a newly discovered type of herpesvirus (Chang, 1994; Cohen, 1995; Chang, 1995). Five of the plant extracts were found 59 to partially inhibit the cytopathic effects of herpesvirus: Cardamine angulata (Cruciferae), Conocephalum conicum (Conocephalaceae), Lysichiton americanum (Araceae), Polypodium glycyrrhiza (Polypodiaceae) and Verbascum thapsus (Scrophulariaceae). None of the extracts exhibited activity against vaccinia virus at the non-cytotoxic concentrations tested. Given the pressing need for new antiviral agents and the inherent limitations of in vitro antiviral testing for such agents, the results of this screening were promising. It is possible that the elucidation of the active constituents in these plants may provide useful leads in the development of antiviral therapeutics. It is interesting to note that 10 of these 12 active plant species were traditionally used to treat what are now known as viral ailments. Eight of the active plants were used to treat the specific diseases or symptoms caused by the virus that they exhibited activity against. These observations suggest that there may be a useful correlation between antiviral activity and traditional usage. They also suggest that some of these traditional remedies may have been efficacious. Acknowledgements This antiviral screening was conducted by research collaborators Dr. L. Babiuk, Dr. T. Roberts and E. Gibbons of the Veterinary Infectious Disease Organization (VIDO), Saskatoon. References Chang, Y., Cesarman, E., Pessin, M.S., Lee, F., Culpepper, J., Knowles, D.M. and Moore, P.S. (1994) Identification of Herpesvirus-like DNA sequences in AIDS-associated Kaposi's sarcoma. Science 266: 1865-1869. Chang, Y. (1995) Letters. Science 268: 1078. Cohen, J. (1995). AIDS mood upbeat - for a change. Science 267: 959-960. De Clerq, E. (1988) Recent advances in the search for selective antiviral agents. Advances in Drug Research 17: 1-59. De Clerq, E. (1993) Antiviral agents: characteristic activity spectrum depending on the molecular target with which they interact. Advances in Virus Research 43: 1-55. Galasso, G.J. (1988) Promises to keep: clinical use of antiviral drugs. In: De Clerq, E. (ed.) Clinical Use of Antiviral Drugs. Martinus Nijhoff Publishing, Boston, pp. 413. Hudson, J.B. (1990) Antiviral Compounds from Plants. CRC Press, Boca Raton, FL, pp. 200. 60 Vesikari, T., Isolauri, E., Ruuska, T., Delem, A. and Andre, F.E. 1988. Rotavirus: new vaccine and vaccination. In: Kurstak, E., Marusyk, R.G., Murphy, F.A. and Van Regenmortel, M.H.V. (eds.) Applied Antiviral Research (Volume 1): New Vaccines and Chemotherapy. Plenum Medical Book Co., New York. pp. 306. 61 2.5 Ethnobotanical analyses 2.5.1 Introduction The ethnopharmacological analyses conducted on the antibiotic and antifungal screening results demonstrated that a significantly higher percentage of plants that were used specifically to treat bacterial or fungal ailments exhibited activity compared to plants that were used for other types of ailments. A review of the ethnobotanical literature on the species which were active in the antiviral and anti-mycobacterial screenings suggested that there may be a correlation between the traditional usage of a plant to treat tuberculosis or viral infections and pharmacological activity. Therefore this study was designed to analyze the data sets to determine whether there were statistically significant correlations. The results of the antiviral and anti-mycobacterial screenings were easily converted to numerical values. However, it was not readily apparent what method should be used to classify the ethnobotanical data. Therefore three different classification methods were used and the correlations between each of these classifications and the screening results calculated. 2.5.2 Methods Data classification The traditional medicinal applications of phase one plants summarized in Appendix 6 were used as the basis for the ethnobotanical classifications. The three different methods used to categorize these references were a "systematic" classification, a "pharmacological" classification, and an "infectious disease" classification system. In the systematic classification, each referenced usage was classified as to the physiological system the medicine was purported to effect. The categories used were as follows: C = cardiovascular, D = dermal and mucosal, G = gastrointestinal, L = liver, M = muscular-skeletal, N = neurological, O = ophthalmic and otic, P = respiratory, R = reproductive, S = systemic, U = urinary. The cardiovascular category included all medicines for: the heart, blood circulation and hemostats but not blood tonics. The dermal-mucosal category included remedies for: topical abscesses, boils, cuts, eczema, itches, rashes, skin ailments/diseases/problems, all types of sores, wounds, etc. The gastrointestinal category 62 included all references to digestive ailments and their treatment: carminatives, cathartics, diarrhea, dysentery, dyspepsia, emetics, flatulence, hemorrhoids or piles, indigestion, purgatives, stomachache, stomach problems/ailments/diseases/ulcers, etc. References to treatments purported to affect the liver were placed in the liver category. The muscular-skeletal category included references to: arthritis, broken or aching bones, muscular aches and swellings, rheumatism, sprains, stiffness, etc. The neurological category included: analgesics, headache, narcotics, pain, sedatives, soreness, stimulants, etc. The ophthalmic and otic category included all eye and ear medicines, the majority of which were remedies for sore and/or inflamed eyes. The respiratory category included all medicines purported to affect the respiratory system and included: cold and cough remedies, decongestants, expectorants, throat medicines, pulmonary complaints, pneumonia, bronchitis, tuberculosis or consumption, lung ailments or sickness, etc. The reproductive category included: abortifacients, childbirth medicines, female or women's medicines and tonics, lacteal stimulants, medicines affecting fertility, menstruation, sterility, male impotence and virility. The urinary medicines category included references to: bladder, kidney or urinary ailments/diseases/problems, diuretics, too frequent urination, failure to urinate, etc. The systemic category included treatments for systemic ailments or symptoms such as: antipyretics, diaphoretics, fever, all infectious diseases, blood tonics, tonics, physics, panaceas and general "good for everything" medicines. The second classification system was based on the traditional western system of classifying medicines by their pharmacological effect. Each reported usage was categorized as to the purported effect that it was thought to exert or the symptom it was used to treat. Definitions of these traditional terms are provided in the glossary. There were a total of 53 categories used in this pharmacological classification system, designated as follows: 1 = Abortifacient, 2 = Analgesic, 3 = Antidiarrheal, 4 = Antiemetic, 5 = Antihelminthic, 6 = Antinflammatory, 7 = Antipyretic, 8 = Antirheumatic, 9 = Antiscorbutic, 10 = Antiseptic, 11 = Antispasmotic, 12 = Antisyphilitic, 13 = Antitussive, 14 = Astringent, 15 = Carminative, 16 = Cathartic, 17 = Cholagogue, 18 = Colds, 19 = Decongestant, 20 = Diaphoretic, 21 = Digestive, 22 = Diuretic, 23 = Emetic, 24 = Emmenogogue, 25 = Expectorant, 26 = Febrifuge, 27 = Hair growth, 28 = Heart, 29 = Hemorrhoids, 30 = Hemostat, 31 = Insect bites, 32 = Lacteal stimulant, 33 = Laxative, 34 = Liver, 35 = Opthalmic, 36 = Other, 37 - Oxytocic, 38 = Purgative, 39 = Sedative, 40 = Sore throat, 41 = Stimulant, 42 = Stomachic, 43 = Tonic, 44 = Urinary System, 45 = Women's medicines, 46 = Wounds (vulnary), 47 = Other skin ailments, 48 = Other pulmonary complaints, 49 = 63 Viral infections, 50 = Diabetes, 51 = Cancer, 52 = Rubefacients and counterirritants, 53 = Anti-venom, poison antidotes. The final classification system used was a modification of the systems used in chapters 2.1 and 2.2, specifically focusing on the reports of traditional usage to treat specified infectious diseases. All medicines which were used to treat a specified bacterial infection or disease were categorized as B = bacterial infections. References to treatments for bacterial infection symptoms and ailments which may be caused by bacterial infections were classified as B2 = bacterial infection symptoms. This B2 category included: ague, headache, fever, diarrhea, digestive ailments and diseases, stomach and intestinal ailments/diseases/problems, sore throat, running sores, ulcers, scrofulous sores, swollen glands, toothache, etc. Similarly, treatments for specified fungal infections were classified as F = fungal infections. The descriptors from the literature designated to this category were: athlete's foot, baby's coated tongue, baby powder or talc, baby's rashes, dandruff, diaper rash, leucorrhea, scaly skin, split skin between the toes, the whites, wash for baby's bottom, thrush, and yeast infections. Treatments for symptoms of fungal infections were categorized as F2 = fungal symptoms. Descriptors for this category included: body sores, broken skin, chafed skin, chapped lips, chapped hands, chapped skin, cracked skin, dry skin, disinfecting or antiseptic wash for itch, disinfecting or antiseptic wash for newborns, female complaints, female medicine, female tonic, foot soak, irritated scalp, irritated skin, itch, itchy scalp, rash, raw spots on baby, running sores, scabby skin, scabs, scalp disease, skin ailments, skin disease and sores of the feet. References to the treatment of tuberculosis or consumption were classified as T = tuberculosis and references to tubercular symptoms such as spitting or coughing blood and chronic coughs were categorized as T2 = tubercular symptoms. Similarly, treatments for specified viral infections were classified as V = viral infections and treatments for viral infection symptoms were classified as V2 = viral symptoms. A third category, V3, was used for the numerous references to cold and cough medicines. All blood tonics, blood remedies, physics, tonics, panaceas and medicines for changing or purifying the blood were classified as P = physics. All remaining ailments and symptoms which were not included in any of the preceding categories were classified as O = other medicines. The anti-mycobacterial data from Table 7 (chapter 2.3) was converted by assigning each entry a value equivalent to the number of "+" in the data table (- = 0, + = 1, ++ = 2, +++ = 3). Similarly, the antiviral data 64 from Table 8 (chapter 2.4) was converted by assigning each entry a value related to number of "+" in the data table (- = 0, + = 1, +++ = 2). Data analyses The computer program "SYSTAT" (Wilkinson, 1988) was used to analyze the data sets for correlations between each of the two types of pharmacological activities screened for and each of the ethnopharmacological classification systems used. The Pearson product correlations between the screening results and the ethnopharmacological classifications were calculated, using a Bonferroni adjustment as the basis for statistical significance. 2.5.3 Results With the "systematic" classification, there were no significant correlations found between any of the physiological categories and any of the screening results (antibiotic, antifungal, anti-Mycobacterial or antiviral). There were significant correlations between the "infectious disease" classifications and both anti-mycobacterial and anti-viral activity. Most notably, the traditional usage of a plant to treat tuberculosis was significantly correlated with anti-mycobacterial activity (Table 9). Usage to treat specified viral infections was correlated with antiviral activity (Table 12). There were also significant correlations between a few of the symptomatic categories and both anti-mycobacterial activity and antiviral activity. Traditional usage to treat rheumatism was correlated with anti-mycobacterial activity (Table 10). Traditional usage to treat pulmonary ailments other than colds and coughs, and specified viral ailments were correlated to antiviral activity (Table 11). There were also correlations between traditional usage as an emetic or purgative and antiviral activity. Only the statistically significant correlations are shown in Tables 9 to 12. 65 Table 9 - Correlation between anti-mycobacterial screening results and infectious disease categories M. tuberculosis M. avium Dried plant material (mg/ml) 20 100 20 100 Tuberculosis medicine 0.451 0.411 0.405 0.483 Table 10 - Correlations between anti-mycobacterial activity and pharmacological categories Dried plant material (mg/ml) M. tuberculosis 20 100 M. avium 20 100 (8) Rheumatism 0.434** 0.311 0.471** 0.340 (50) Diabetes 0.477** 0.277 0.524** 0.333 (51) Cancer 0.288** 0.232 0.413** 0.268 ** Statistically significant correlations Table 11 - Correlations between antiviral activity and pharmacological categories Activity against RSVa Rotavirus Pharmacological category: (23) Emetics 0.587** 0.016 (38) Purgatives 0.606** -0.032 (48) Other pulmonary ailments 0.010 0.552** (49) Viral infections 0.045 0.460** a Respiratory syncytial virus Statistically significant correlations 66 Table 12 - Correlations between antiviral screening results and infectious disease categories Activity against RSVa Rotavirus Infectious disease category B - bacterial diseases 0.500** 0.273 B2 - bacterial symptoms 0.557** 0.195 F2 - fungal symptoms 0.435** 0.234 V - viral diseases 0.348* 0.475** V2 - viral symptoms 0.583** 0.249 V3 - colds and coughs 0.476** 0.191 T - tuberculosis 0.598** 0.412** T2 - tuberculosis symptoms 0.436** 0.019 0 - other medicines 0.591** 0.078 a Respiratory syncytial virus Statistically significant correlations 67 2.5.4 Discussion and Conclusions The analyses based on both the "infectious disease" and the "pharmacological" classifications revealed several correlations with anti-mycobacterial and antiviral activity. As a visual inspection of the anti-mycobacterial screening results suggested, there was a statistically significant correlation between traditional usage of a plant as a tuberculosis medicine and anti-mycobacterial activity (Table 9). This result would seem to imply that it would be worthwhile to specifically target plants that had been used as tuberculosis medicines for future anti-mycobacterial screenings. However, this analysis was based on a fairly small sample (37 were used as tuberculosis medicines). Analysis of a larger sampling of tuberculosis medicines would provide a greater degree of confidence in the significance of this correlation. There were also "statistically significant" correlations between some of the pharmacological classifications and anti-mycobacterial activity (Table 10). As there were less than five reports in both the diabetes and cancer medicine categories, these correlations must be treated with a great deal of skepticism. There were over one hundred reports in the rheumatism medicine category so the correlation with this category may be more reliable. Since there is no obvious scientific connection between rheumatism medicines and tuberculosis medicines, it would be quite interesting to see if this apparent correlations stands up to more rigorous testing with a larger sample of plants. There were also "statistically significant" correlations between the pharmacological categories of emetics, purgatives, other pulmonary ailments and medicines for viral infections, and antiviral activity. However, it is highly questionable whether these findings have any real significance as each of these categories were correlated with activity against either respiratory syncytial virus or rotavirus and there was only one extract active against each of these viruses. Similarly, the correlations between the infectious disease categories and antiviral activity (Table 12) are suspect for the same reason. Clearly, these findings would have to hold up under an analysis of a much larger data set before the significance of these results could be given much weight. 68 References Wilkinson, L. (1988) SYSTAT: the system for statistics. SYSTAT Inc., Evanston, 111. Acknowledgements I would like to acknowlege and thank Dr. J. Maze for lending his expertise and advice on the conduction of these analyses and his greatly appreciated assistance in using the Systat program. 69 2.6 Conclusions from phase one research 2.6.1 Introduction The phase one screenings provided a great deal of valuable information. Many new leads on potential anti-infectious agents were obtained. The results of the ethnobotanical analyses supported the hypothesis that the North American ethnobotanical literature can be used as an effective tool for identifying plants with anti-infectious activity and provided some insights on how future screenings may be improved. The data collected in these screenings also gave rise to several other hypothesis regarding other factors which may be correlated to pharmacological activity. 2.6.2 Leads on potential antimicrobial agents Several of these leads have already been followed up on with more detailed chemical analysis. From the antibiotic screening, eight plants were targeted for further investigation: Rhus glabra, Alnus rubra, Balsamorhiza sagittata, Ceanothus velutinus, Empetrum nigrum and Glehnia littoralis. Several of these plants exhibited activity in one or more of the other screenings as well. The extract of Rhus glabra exhibited the strongest broad spectrum antibiotic activity of all the extracts screened although it did not display significant activity in any of the other screens. Three antibiotic compounds were isolated from R. glabra using activity guided fractionation: 3,4,5-trihydroxybenzoic acid (methyl gallate), 4-methoxy-3,5-dihydroxybenzoic acid and gallic acid (Saxena, 1994). These compounds are all tannins, a class of compounds which are common constituents found in great abundance in members of the Rhus genus and are the basis of usage of Rhus species in the tanning industry. The first two compounds, reported for the first time from R. glabra, exhibited fairly low minimum inhibitory concentrations (MIC), 12.5 ug/ml and 25 ng/ml respectively, compared to the MIC of the ubiquitous gallic acid which was > 1000 ug/ml. These MIC do not compare favorably to that of commercial antibiotics which range from 0.3-1 ug/ml (Farmer, 1992) and the propensity of tannins to cross-link protein make these compounds unsuitable for intravenous use. Alnus rubra exhibited strong antibiotic and antifungal activity as well as anti-mycobacterial activity. Two active compounds were isolated from the bark of this plant; diarylheptanone and oregonin (Saxena, 1995a). 70 The lowest MIC these compounds exerted was 31.2 ug/ml against Staphylococcus aureus, values which also do not compare favorably against commercial antibiotics. The extract of Balsamorhiza sagittata exhibited very good antifungal and antibiotic activity. A known thiophene, 7,10-epithio-7,9-tridecadien-3,5,ll-triyn-l,2-diol was the main active compound subsequently isolated from B. sagittata (Matsuura, 1995b). This compound exhibited antibacterial activity against Staphylococcus aureus and Bacillus subtilis which was slightly enhanced by exposure to long wave ultraviolet light 320-400 nm. Again the MIC against these organisms (25 ug/ml) did not compare favorably with that of commerical antibiotics. Although the crude extract of B. sagittata exhibited activity against the gram negative organisms Escherichia coli and Pseudomonas aeruginosa, this thiophene was not active against these organisms, suggesting the presence of another antibacterial constituent which was not isolated. The extract of Ceanothus velutinus also exhibited strong antifungal activity and moderate antibiotic activity in the screenings. Four antibiotic compounds were later isolated from Ceanothus velutinus: octacosanoic acid, 4',5-dihydroxy-3',7-dimethoxyflavone (velutin), 5-hydroxy-3',4',7'-trimethoxyflavone (4'-0-methylvelutin) and 2-formyl-3-methoxy-A(l)-norlup-20 (29)-en-28-oic acid (Matsuura, 1995c). The last compound which exhibited the lowest MIC of these four compounds (25ug/ml), was a novel triterpene that was isolated for the first time in the course of this research. The crude extract of Empetrum nigrum exhibited good antibiotic, antifungal and anti-mycobacterial activity, prompting the selection of this plant for further investigation. Four antimicrobial compounds were subsequently isolated: batatasin, 4'-0-methylbatatasin, 3-O-methylbatatasin and 7-hydroxy-2,4-dimethoxy-9,10-dihydrophenanthrene (Matsuura, 1995d). However the MIC of these compounds were all relatively poor ( >200 ug/ml) and none of these compounds showed anti-mycobacterial activity. Catechin and an unidentified triterpene were the antibiotic compounds isolated from Geum macrophyllum (Matsuura, 1995e). Although the crude extract of this plant exhibited strong broad spectrum antibiotic and antifungal activity, both of these isolated compounds had quite high MIC ( > 270 ug/ml and > 420 ug/ml respectively). Activity guided fractionated resulted in the identification of seven active constituents from Glehnia littoralis (Matsuura, 1995a). Three of these compounds were known furanocoumarins (psoralen, bergapten and 71 xanthotoxin) which did not exhibit any activity under 400 ug/ml. A fourth constituent, falcarindiol, had been previously reported as an antibiotic by Muir (1982). The three other constituents identified were novel compounds, an unstable fatty acid, (8E, 10Z) 7-hydroxy-8, 10-octadienoic acid, and two polyyne compounds; (9Z) l,9-heptadecen-4, 6-diyn-3, 8, 11-triol and (10E) 1, 10-heptadecen-4, 6-diyn-3, 8, 9-triol. The latter two polyne compounds had weak inhibitory effects against both the bacteria and fungi tested (MIC ~ 200-400 ug/ml). The instability of the fatty acid prohibited MIC determination for this compound and attempts to form active stable derivatives were unsuccessful. It has yet to be determined whether any of these antimicrobial compounds isolated from G. littoralis (Umbelliferae) are also responsible for the anti-mycobacterial activity that the crude G. littoralis extract exhibited. Two other members of the Umbelliferae, Heracleum maximum and Lomatium dissectum also exhibited very strong anti-mycobacterial activity (as well as antibiotic and antifungal activity), suggesting that these members of the Umbelliferae may share a common constituent with anti-mycobacterial activity. H. maximum was considered a second priority candidate for chemical investigation because it is known to contain a number of toxic constituents, including psoralen (Foster, 1990). L. dissectum was previously reported to exert antimicrobial activity (Cardellina and Vanwagenen, 1985). Vanwagenen and Cardellina (1986) identified the active compounds as a pair of unstable, homologous tetronic acids (2-alkenyl-3-hydroxy-penta-2,4-dien-4-olides). It is not known if these compounds are also responsible for the anti-mycobacterial and antiviral activity that the crude L. dissectum extract exhibited in these screenings. The fact that unstable acids were reported as the active antimicrobial compounds in both G. littoralis and L. dissectum leads to the speculation that a similar compound may also be responsible for the strong antimicrobial activity exhibited by the H. maximum extract. The fact that the L. dissectum extract exhibited antiviral activity at the non-cytotoxic concentrations tested while the G. littoralis and H. maximum extracts did not, does not rule out the possibility of a common type of anti-infectious constituent in this family. The absence of demonstrable antiviral activity in the assays of the G. littoralis and H. maximum extracts was most likely due to higher concentrations of cytotoxic constituents. This point however, will only be of any real interest to chemtaxonomists if pharmaceutical chemists cannot succeed in stabilizing these antimicrobial compounds without loss of activity. Two plants which demonstrated strong antifungal activity, Ipomopsis aggregata and Moneses uniflora, were also subjected to in depth chemical investigations. M. uniflora exhibited good antibiotic activity including strong anti-mycobacterial activity, while /. agreggata exhibited strong activity against parainfluenza virus. A novel chloroquinone (8-chloro-chimaphilin) was isolated from M. uniflora in addition to the known antimicrobial compound chimaphilin and its derivative 3-hydroxy-chimaphilin (Saxena, 1995b). The 8-chloro-chimaphilin had the lowest MIC of these three compounds, 12.5 ug/ml against S. aureus. This compound was also found to be responsible for the anti-mycobacterial activity exhibited by the crude extract of M. uniflora. Four active compounds were isolated from /. aggregata: giliacoumarin, cucurbitacin B, resorcinol, and hydroquinone glucoside (Saxena, 1995c). Resorcinol exhibited the lowest MIC of these four compounds, 25 ug/ml against S. aureus. As with all of the other chemical constituents referred to above, the MIC of these compounds did not compare favorably to that of commercial antibiotics. The relatively poor activity of all these isolated pure active compounds appears to be at odds with the strong activity exhibited by the crude extracts of the plants they were isolated from. Particularily in the plants with many active compounds, synergistic interactions may account for this difference in activity, however this possibility has not been explored. Clearly, in the cases of G. macrophyllum and B. sagittata where the active compounds did not exert the same range of activity as their respective crude extracts, some active compounds have been broken down or lost in the isolation process. Oplopanax horridus exhibited the most promising activity in the anti-mycobacterial assays. A novel antimicrobial compound was isolated from this plant (Saxena, 1995d). O. horridus also exhibited mild antiviral activity against bovine herpesvirus however the constituent responsible for this activity has not been identified. In addition to the O. horridus, I. aggregata and L. dissectum extracts discussed above, the extracts of Amelanchier alnifolia, Potentilla arguta, Rosa nutkana and Sambucus racemosa also exhibited strong antiviral activity. It is noteworthy that three of these antiviral extracts were prepared from plant species belonging to the Rosaceae (Amelanchier alnifolia, Potentilla arguta and Rosa nutkana) and all three were active against viruses that infect mucosal surfaces. Although these plants did not perform similarly in the other antimicrobial screenings, it is possible that they possess a common antiviral compound since antimicrobial and antiviral activity are not necessarily correlated. 73 Tannins are commonly found in large quantities in many members of the Rosaceae, making this type of compound a logical candidate for the common antiviral constituent. Two other facts however, argue strongly against this. Tannins have been reported as the active antimicrobial compounds in many members of the Rosaceae, of which the G. macrophyllum discussed above is one example and Potentilla another (Selenina, 1973; Makarenko and Chaika, 1974). The samples of these genera exhibited significant antimicrobial activity but none of these plants exhibited antiviral activity. More importantly, A. alnifolia, P. arguta and R. nutkana exhibited antiviral activity at non-cytotoxic concentrations, demonstrating that any tannin constituents must be present in very low concentrations. It was therefore considered to be worthwhile to attempt to identify the antiviral constituents in these plants. The chemical isolation work is still in progress at this writing. With the exception of O. horridus, none of these chemical investigations has led to commercially viable anti-infectious compounds although the research is not complete and several promising plants have not been explored yet. These chemical investigations have contributed to our knowledge of plant constituents, particularity with the isolation of several novel compounds whose range of pharmacological activities have not been fully researched. The discovery of even one new potential therapeutic agent and the promise of more discoveries yet to come more than justify the continuation of this type of research. 2.6.3 Conclusions from ethnopharmacological analyses The results of the ethnopharmacological analysis of both the antibiotic and antifungal screening data support the hypothesis that the North American ethnobotanical literature provides an effective tool for targeting plants with anti-infectious activity. These results, along with the results of the anti-mycobacterial screening, further suggest that the specific traditional usages of a medicinal plant may be used as an indicator of the specific type(s) of pharmacological activity a plant possesses. Therefore, future screening studies may be able to identify a higher percentage of active plants if the specific traditional usages are used as selection criteria (ie. selecting plants whose traditional usage implied the treatment of bacterial infections for antibiotic screenings). However, the analyses also suggest that non-flowering plants and plants used as tonics should not be excluded from examination either. 74 2.6.4 Other factors which may be correlated with antimicrobial activity The summary of the antifungal results by taxa suggests the hypothesis that more of the lower plants exert antifungal activity than do the higher plants (see Table 4). However, since the selection criterion used in the plant collection for this screening was not designed to test this notion, the sample size of the lower plant group is too small to lend much statistical evidence in support of this. Furthermore, the selection of lower plants was quite biased towards the tiny fraction of lower plants which were used medicinally by the British Columbian First Nations peoples. A much larger, balanced sampling of the lower plants would be required to obtain adequate data to test this hypothesis. The screening data gives one the impression that the most active antifungal plants were those collected from arid habitats. It would seem much more logical to hypothesize that plants from wet and moist habitats would exhibit the greatest degree of antifungal activity. This intriguing contradiction suggested that it would be very interesting to analyze the degree of activity in relation to plant habitat in future studies. References Cardellina, J.H. and Vanwagenen, B.C. (1985) Antifungal agents from Lomatium dissectum. Abstracts of the International Research Congress on Natural Products. University of North Carolina, Chapel Hill, N.C. Abstract 211. Foster, S. and Duke, J.A. (1990) A Field Guide to Medicinal Plants. Houghton Mifflin Company, Boston, pp. 366. Farmer, S.W., Li, Z. and Hancock, R.E.W. (1992) Influence of outer membrane mutations on susceptibility of Escherichia coli to the dibasic macrolide azithromycin. Journal of Antimicrobial Chemotherapy 29: 27-33. Matsuura, H., Saxena, G., Farmer, S.W., Hancock, R.E.W. and Towers, G.H.N. (1995a) Antibacterial and antifungal compounds from Glehnia littoralis F. Schmidt. Phytochemistry (submitted March, 1995). Matsuura, H., Saxena, G., Farmer, S.W., Hancock, R.E.W. and Towers, G.H.N. (1995b) An antibacterial thiophene from Balsamorhiza sagittata (Pursh) Nutt. Planta Medica (submitted March, 1995). Matsuura, H., Saxena, G., Farmer, S.W., Hancock, R.E.W. and Towers, G.H.N. (1995c) Antibacterial compounds from Ceanothus velutinus Dougl. Phytochemistry (submitted January, 1995). Matsuura, H., Saxena, G., Farmer, S.W., Hancock, R.E.W. and Towers, G.H.N. (1995d) Antimicrobial constiuents of Empetrum nigrum L. Planta Medica (submitted April, 1995). Matsuura, H., Saxena, G., Farmer, S.W., Hancock, R.E.W. and Towers, G.H.N. (1995e) Antibacterial constituents of Geum macrophyllum Willd (unpublished manuscript). 75 Muir, A.D., Cole, A.L.J, and Walker, J.R.A. (1982) Planta Medica 44: 128-133. Saxena, G., McCutcheon, A.R., Farmer, S., Towers, G.H.N, and Hancock, R.E.W. (1994) Antimicrobial constituents of Rhus glabra L. Journal of Ethnopharmacology 42: 95-99. Saxena, G., Farmer, S., Hancock, R.E.W. and Towers, G.H.N. (1995a) Antimicrobial compounds from Alnus rubra Bong. International Journal of Pharmacognosy 33 (1): 33-36. Saxena, G., Farmer, S., Towers, G.H.N, and Hancock, R.E.W. (1995b) Antimicrobial compounds from Moneses uniflora (L.) A. Gray. Phytochemistry (in press). Saxena, G., Farmer, S., Towers, G.H.N, and Hancock, R.E.W. (1995c) Antimicrobial constituents of Ipomopsis agreggata (Pursh) Spreng. Phytochemistry (in press). Saxena, G., Farmer, S., Towers, G.H.N, and Hancock, R.E.W. (1995d) Antimicrobial constituents of Oplopanax horridus (unpublished manuscript). Vanwagenen, B.C. and Cardellina, J.H. (1986) Native American food and medicinal plants 7. Antimicrobial tetronic acids. Tetrahedron 42(4): 1117-1122. 76 3.0 Introduction to phase two screening In light of the positive results in the phase one screenings, the decision was made to undertake another round of plant collection and screenings. The primary objective of this second phase was also to screen traditional plant medicines for anti-infectious activity in order to identify promising leads on new therapeutics. The first phase of research also raised a number of interesting questions and possible correlations for which there was insufficient data to draw any definitive conclusions. Therefore, the phase two screening study was designed so that several parallel objectives could also be met. For this second screening, the range of ethnobotanical literature used as the main plant selection criteria was expanded to include all of North America and the range of plant collection expanded to that of western North America. The plant collection for this phase of the research was planned so as to facilitate analyses of the correlation between antimicrobial activity and groupings based on four other factors: (1) medicinal versus non-medicinal plants; (2) the specific medicinal use of the plants; (3) taxa; and (4) plant habitat. Also, sufficient plant material was collected to allow for a comparison of the differences in activity due to the solvent of extraction. The difference in the degree of activity among medicinal versus non-medicinal plants was one of the most prominent questions raised by the ethnopharmacological analyses of the phase one results. The answer to this question may vary significantly, depending upon the criteria used to designate a plant as non-medicinal. One must assume that there may be any number of medicinal plants classified as non-medicinal simply because their traditional usage has not been recorded in the literature. This problem can not be overcome but the number of plants misclassified can be reduced by excluding plants belonging to medicinal genera from the non-medicinal plant category. There are a number of strong arguments supporting the use of this non-medicinal plant selection criterion, as was done in this study. First of all, the literature abounds with examples of members of a medicinal genus being used for the same medicinal purposes by disparate cultures around the world. Two good examples of this are the genera Artemisia and Rhus. The indigenous Artemisia species are commonly used to treat infection and inflammation while other Rhus species are commonly used to treat diarrhea in China, India, Africa, Europe, North and Central America. This pattern suggests that when there are records that several members of a genus were used as a medicine, related species may also have been used medicinally in other regions. 77 A much more tangible argument is that the ethnobotanical literature contains many taxonomic uncertainties at the species level and most likely errors in identification as well. There are numerous examples of plants identified by their common name or generic name only. It can not simply be assumed that First Nations peoples always made the same species differentiations as botanists. Nor can it be assumed that ethnobotanist's plant identifications were always correct. There are a number of genera whose members are extremely difficult to identify at the species level unless one is an expert in that genus. It seems most reasonable to suggest that some specimens belonging to taxonomically difficult genera such as Aster or Carex may have been misidentifed in the literature. For all of these reasons, the decision was made to classify "related species" as medicinal plants. This "related species" group was comprised of plants for which there were generic references to medicinal usage but no specific references. The previous ethnopharmacological analyses also suggested that the specific medicinal usages of a plant may be used as an indicator of the specific types of pharmacological activity the plant possesses. A larger data set which included appropriate control groups was required to conduct a more rigorous statistical analysis of these apparent correlations. Therefore a larger sampling of plants whose traditional uses did not suggest antimicrobial activity, as well as non-medicinal plants were collected for this study. Similarily, an effort was also made to collect a greater number of samples belonging to the lower plant taxa so that a more robust analysis of the differences in activity between taxa could be made. Careful detailing of the habitat from which each sample was collected was made at the time of collection and verified against information in the relevent flora so that the plants could be accurately classified according to their habitat. This information was collected so that the screening data could later be analyzed to determine if there were any significant correlations between plant habitats and antimicrobial activity. 3.0.1 Methods Plant collection Moerman's bibliography Medicinal Plants of Native America (1986) and the British Columbian ethnobotanical literature (Turner et al., 1980, 1990) was surveyed to compile a representative list of those plants used medicinally by the native peoples of western North America. Medicines used to treat abcesses, burns, 78 infected sores and wounds, skin ailments, tuberculosis and yeast infections were the primary focus. The list was used in the field as a selection guide for the plant species and type of material to be collected. From the several hundred plant species on the ethnobotanical list, 142 samples were collected. In addition to these, 18 plants with no reported medicinal use were collected. The material from 25 of these 160 plant species was separated into constituent parts (aerial, roots, etc.) which resulted in a grand total of 185 plant samples. The collecting was carried out during the period from May, 1994 to September, 1994 in five general areas of western North America: northern California and Oregon, the U.B.C. Malcolm Knapp Research Forest in Maple Ridge, B.C., northern British Columbia, the Princeton-Penticton region in the interior of B.C., and Vancouver Island, B.C.. Details on the plant's habitat were also recorded at the time of collection. Identification authentications were obtained for the plant species whose identification was beyond the taxonomic expertise of the author. In order to ensure accurate botanical identifications of the angiosperms, only plants which were in flower were collected, introducing a seasonal bias into the selection. A voucher specimen was made for each collection and these vouchers have been filed in the University of British Columbia Herbarium. An annotated list of the voucher specimens for the plants collected including their full botanical names and synonyms is located in Appendix 2. An abbreviated summary of the traditional uses of each of these plants compiled from the literature is given in Appendix 7. Extract preparation The plant material was air dried and then ground in a Wiley grinder with a 2 mm diameter mesh. Forty g of the ground material were extracted in 200 ml of methanol with three washes of 200 ml, over 3 hours. The crude methanolic extract was first filtered through cheesecloth and cotton wool, then through a Buchner funnel with a No. 4 paper filter. The filtrate was rotoevaporated to dryness and then reconstituted with 20 ml of methanol. The extracts were refrigerated until the time of use. 79 Acknowledgements I would like to thank our summer students Cheng-Han Lee, Lehli Pour and Jen Sung for their assistance in preparing the plant materials for the screenings. For the taxonomic expertise provided to verify the identifications of some difficult taxa, my appreciation and thanks to: Dr. J. Maze (Cyperaceae and Juncaceae), Dr. W. Schofield (Bryidae and Eumycota), Dr. G. Strayley (Iris and Horkelia) and J. Olivera (Thallophyta). Financial support for this research was provided by the Canadian Bacterial Diseases Network and Natural Sciences and Engineering Research Council. References Moerman, D.E. (1986) Medicinal Plants of Native America. Research Reports in Ethnobotany, Contribution 2. University of Michigan Museum of Anthropology, Technical Reports, Number 19. Ann Arbor, Mich, pp. 534. Turner, N.J., Bouchard, R. and Kennedy, D.I.D. (1980) Ethnobotany of the Okanagan-Colville Indians of British Columbia and Washington. British Columbia Provincial Museum No. 21. Occasional Papers Series. British Columbia Provincial Museum, Victoria, British Columbia, pp. 156. Turner, N.J., Thompson, L.E., Thompson, M.T. and York, A.Z. (1990) Thompson Ethnobotany: Knowledge and Usage of Plants by the Thompson Indians. Royal British Columbia Museum Memoir No. 25. Royal British Columbia Museum, Victoria, British Columbia, pp. 321. 80 3.1 Phase two antibiotic screening Abstract Methanolic extracts of 185 samples of western North American plants were screened for antibiotic activity against 7 bacterial strains. One hundred forty-three (77%) exhibited significant antibiotic activity. There was a great difference in the degree of antibiotic activity between the traditional plant medicines (75% active) and the non-medicinal plants (22% active). Ninety-one percent (91%) of the plants classified as potential antibiotics based on their traditional usage were found to have antibiotic activity. The taxa with the highest percentage of active plants were the Filinicae (ferns) and Gymnospermae (conifers) of which 100% were active, followed by the Angiospermae (flowering plants) of which 89% were active. The most active broad spectrum antibiotics were made from Abies grandis branches, Elliottia pyroliflorus branches, Geum triflorum roots, Horkelia fusca roots, Paxistima myrsinites branches, Paeonia brownii roots, Phyllodoce empetriformis aerial parts, Picea sitchensis inner bark and Pseudotsuga menzesii branches. 3.1.1 Introduction In the phase one screenings of British Columbian medicinal plants, the results of the data analyses suggested that the specific applications of plant medicines could be used to select species which have a higher probability of exhibiting antimicrobial activity than medicinal plants in general. Therefore in addition to identifying plants with promising antibiotic activity, this study was designed to test this hypothesis more rigorously as well as to analyze whether other factors such as plant habitat may be used as indicators of antibiotic activity. 3.1.2 Methods Extract preparation In addition to the 185 methanol extracts prepared, 30 samples were randomly chosen for extraction with boiling water. These water extracts were prepared using the same procedure as for the methanolic extracts, with the substitution of boiling water for the solvent instead of methanol. Each water extract was refrigerated 81 immediately after preparation and filter sterilized before use. Ten samples which were traditionally prepared as salves or liniments were also extracted with petroleum ether. These extracts were prepared using the same procedure as for the methanolic extracts with the substitution of petroleum ether as the solvent. Microorganisms The clinically important pathogens Escherichia coli UB1002, Enterococcus faecalis, Pseudomonas aeruginosa K799 (wild type), multiple drug resistant Staphylococcus aureus P00017 and multiple drug resistant Staphylococcus epidermidis were used for this screening. The antibiotic super-susceptible strains of Mycobacterium phlei and P. aeruginosa Z61 were also used because they are sensitive indicator organisms which may detect the activity of compounds present in concentrations which are too low to exert an observable effect against the hardier wild type pathogens. Cultures of the two P. aeruginosa strains and E. coli UB1002 were from the collection of R.E.W. Hancock. The Z61 strain was an antibiotic supersusceptible strain and the K799 strain was a wild type strain. The cultures of Enterococcus faecalis, multiple drug resistant S. aureus P00017 and multiple drug resistant S. epidermidis were clinical isolates provided by Dr. A. Chow, Department of Medical Microbiology, U.B.C. The culture of M. phlei was from the collection of G.H.N. Towers. An inoculum of each bacterial strain was suspended in 3 ml of nutrient broth and incubated overnight at 37°C. The overnight cultures were diluted 1/10 with nutrient broth before use. To ensure that the density of the diluted cultures were all within the range of 107"8 CFU/ml, serial dilution plate counts were also made for each culture. Antibiotic assays The disc diffusion assay (Lennette, 1985) was used to screen for antibiotic activity. Paper discs (1/4") were impregnated with 20 ul of extract, the equivalent of 40 mg of dried plant material, and the solvent allowed to evaporate at room temperature. One hundred ul of the diluted bacterial culture was spread on sterile Mueller-Hinton agar plates before placing the extract impregnated paper discs on the plates. For each extract, three 82 replicate trials were made against each bacterial species screened. Gentamicin was used as a positive control and methanol as a negative control. The plates were incubated for 18 h at 37HC, with the exception of M. phlei which was incubated for 36 h. The diameter of the zone of inhibition around each disc was measured and recorded at the end of the incubation period. Data analysis The average zone of inhibition was calculated for the three replicates. A clearing zone of 8 mm or greater was used as the criterion for designating significant antibiotic activity. In cases where there were a few colonies growing within the zone of inhibition, the activity rating was annotated with the letter "i" for incomplete inhibition. The overall trial average for each assay was used for the classification of results in Table 13. For each of the major taxonomic divisions (Eumycota, Thallophyta, Bryopsida, Sphenopsida, Lycopsida, Filicinae, Gymnospermae and Angiospermae), the total number and percentage of active extracts was calculated, as well as the percentage of active extracts excluding those with only slight (1+) activity against the susceptible organisms M. phlei and P. aeruginosa Z61. The ethnopharmacological data collated in Appendix 7 summarizing the traditional medicinal uses of each plant was used as the basis for the ethnopharmacological classifications. Each extract was assigned to the highest numbered category it fit into. The five ethnopharmacological categories used were: (1) potential antibiotics, (2) possible antibiotics, (3) tonics, (4) other medicinal uses, (5) related species, and (6) non-medicinal plants. Extracts which were used to treat specific ailments caused by bacterial organisms were assigned to category 1: potential antibiotics. The specific bacterial ailments included in category 1 were: abcesses, acne, bladder or kidney infections, blood poisoning, boils, consumption, diptheria, dysentery, food poisoning, gonorrhea, infected wounds or sores, pneumonia, ptomaine poisoning, rheumatic fever, scarlet fever, scrofula, sepsis, syphilis, tooth abcess, tuberculosis, venereal disease, and whooping cough. The infected wounds or sores classification included the descriptors: inflamed wounds/sores, discharge from wounds/sores, wounds/sores with pus, feverish wounds/sores, etc. Plants traditionally used as disinfectants or antiseptics were also assigned to this category. 83 Extracts of plants traditionally used to treat ailments and symptoms which were possibly caused by bacterial infections were assigned to category 2: possible antibiotics. Ethnopharmacological descriptions included in this category were: bladder or kidney disease/problems/troubles, burns, coughs, cuts, diarrhea, fever, gastroenteritis, lung trouble, lung hemorrhage, sores, sore gums, sore or inflamed eyes, sore throat, stomachache, stomach ailments/disease/problems, stomach/intestinal flu, too frequent urination, toothache and wounds. Plants whose traditional usages did not include those listed for category one or two which were used as tonics or physics were designated to category 3: tonics. The remaining plants with specific references that did not suggest treatment of a bacterial infection or use as a tonic, were assigned to category 4: other medicines. The descriptors included in this category were: abortifacents, arthritis, biliousness, broken bones, bruises, cancer, cathartics, childbirth, constipation, emenagogues, emetics, flatulence, gas, hair tonics, hair washes, heart disease/problems/ailments, indigestion, insect bites, laxatives, liver disease/problems/ailments, purgatives, rheumatism, sprains, swellings and women's medicines. Plants for which there was no recorded medicinal use under the botanical species name but for which there were generic references in the literature were assigned to category 5: related species. Most of the plants in this category were either referred to generically or by common name only in the literature and/or belonged to taxonomically difficult genera (ie, Carex). As errors and uncertainties in taxonomic identification may occur in the ethnobotanical literature, particularly in older works using common names, discrimination for the non-medicinal category was made at the generic level. Plants for which there was no recorded medicinal use of that genus in the literature cited above nor in the Napralert database were assigned to category 6: non-medicinal plants. The total number of active plant extracts in each category was calculated, as well as the number of active extracts excluding those with only slight (1+) activity against the super-susceptible organisms M. phlei and P. aeruginosa Z61. The plants were also categorized according to the habitat in which they were collected. These classifications were verified with habitat descriptions in the relevant floras; Hitchcock and Conquist (1973), Hickman (1993), MacKinnon (1992) and Pojar and MacKinnon (1994). The habitat categories used were: saltwater, coastal, freshwater wetlands (included aquatic plants, plants of bogs, swamps, lake and stream 84 margins), moist, temperate, dry, arid (pine scrub and sagebrush scrub) and subalpine (elevations over 5,000 feet). As in the phase one analyses, the total number of active plant extracts in each category was calculated, as well as the number of active extracts excluding those with only slight (1+) activity against the super-susceptible organisms M. phlei and P. aeruginosa Z61. In each analysis, the statisitical significance of the percentage of active extracts in each category was evaluated using the chi squared goodness-of-fit test. 3.1.3 Results The results of the antibiotic screening are summarized in Table 13, alphabetically by family. A total of 158 extracts (85%) exhibited some antibiotic activity. Excluding those extracts with only slight (1+) activity against the super-susceptible organisms M. phlei and P. aeruginosa Z61 from the calculations, 143 extracts were active (77%). The most active broad spectrum antibiotics were made from the plants: Abies grandis, Elliottia pyroliflorus, Geum triflorum, Horkelia fusca, Paxistima myrsinites, Paeonia brownii, Phyllodoce empetriformis, Picea sitchensis, and Pseudotsuga menziesii. The antibiotic assay results summarized by taxa are given in Table 14. There was a significant difference in the percentage of lower non-flowering plants (54%), higher non-flowering plants (100%) and flowering plants (89%) which were active. Calculated excluding those extracts with only slight activity against the super-susceptible organisms M. phlei and P. aeruginosa Z61, only 29% of the lower non-flowering plants were active while 100% of the higher non-flowering plants (ferns and conifers) and 83% of the flowering plants were active. Table 15 shows the antibiotic activity of selected methanolic extracts compared to water and petroleum ether extracts of the same plants. The differences in antibiotic activity between the methanol extracts and the water extracts were mostly quantitative. None of the petroleum ether extracts was active with the lone exception of M-4 which was slightly active against S. aureus. The results of the ethnopharmacological analysis are shown in Table 16. There was a significant difference in the percentage of active medicinal plants (83%) compared to the non-medicinal plants (22%). Among the medicinal plants, the highest percentage of active extracts were those classified as potential antibiotics (91%) and those classified as possible antibiotics (79%) based on their traditional uses. The results summarized by plant habitat are shown in Table 17. The habitat from which the highest percentage of active plants was collected was the sub-alpine (100%). 86 Table 13 - Phase two antibiotic screening results3 Family Species (Voucher No.) Catb Partc Habd E.c.e E.f. M.p. Z61 K799 S.a. S.e. Totalf Total8 active excl. S.S. Controls Methanol Gentamicin (10 ug) ACERACEAE Acer macrophyllumM-na 2 Acer macrophyllum M-17b 2 AIZOACEAE Carpobrotus edulis Ca-3 3 ALISMATACEAE Alisma trivalis N-40a 2 Alisma trivalis N-40b 2 Sagittaria latifolia N-11 APOCYNACEAE Apocynum andwsaemifolium P-47a 2 Apocynum androsaemifolium P-47b 2 Lf T lb T Ae C Ae W Rt W 1 Wh W Ae D Rt D 0 0 0 0 0 0 0 4+ 2+i 5+ 5 + 4 + 0 0 0 0 2+ 2+ 1+ 2+ 2+ 1+i 0 3+ 3+ 1+i 2+ 2+ 0 0 0 0 0 0 0 0 0 0 2+ 1+ 2+ 1+ 1+i 1+ 1+ 0 0 0 2+ 0 0 0 0 1+i 0 3+ 0 2+ 2+ 2+ 1+ 2+ 0 0 0 2+ 1+ 0 5 5 6 1 2 2 5 3 0 5 5 6 0 2 1 4 2 87 Family Species (Voucher No.) Cat" Partc Habd E.c.e E.f. M.p. Z61 K799 S.a. S.e. Total* Total* • active excl. S.S. ARALIACEAE Aralia nudicaulis N-3a Aralia nudicaulis N-3b ASCLEPIADACEAE Asclepias speciosa P-45 BALSAMINACEAE Impatiens capensis M-18 BERBERIDACEAE Achlys triphylla V-20 BETULACEAE Corylus cornuta N-41 BORAGINACEAE Lithospermum ruderale P-43 Mertensia paniculata N-5 1 Ae M 1 Rt M 1 Wh W 2 Wh D 5 Wh M 0 0 2+ 1+ 0 0 0 0 0 2+ 1+i 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 2 Ae M 0 0 1+ 0 0 1+ 1+ 2 Ae M 0 0 0 0 0 0 1+i 1 1 2 Br T 1+i 0 2+ 2+ 1+ 2+ 3+ 0 0 0 0 88 Family Cat" Parf Habd E.c.e E.f. M.p. Z61 K799 S.a. S.e. Totalf Total* Species (Voucher No.) active excl. S.S. BRYBDAE Homalothecium nevadense P-51 . 6 Wh T 0 0 0 0 0 0 0 0 0 Hookeria lucens V-37 6 W h M 0 0 0 0 0 0 0 0 0 Isothecium stoloniferum V-22 6 W h M 0 0 2+ 1+i 0 1+i 1+ 4 3 Plagiothecium undulatum M - l l 5 Wh M 0 0 1+ 0 0 1+i 0 2 1 Pogonatum contortum M-14 6 W h M 0 0 0 0 0 0 0 0 0 Polytrichum commune V-13 5 W h W 0 0 1 + 0 0 1+ 0 2 1 Racomitrium elongatum V-18 6 W h T 0 0 0 0 0 0 0 0 0 Rhizomnium glabrescens M-13 2 W h M 0 0 1+i 0 0 0 0 1 0 Rhytidiadelphus loreus V-23 6 W h M 0 0 1+ 0 0 0 0 1 0 Scapania bolanderi M-12 6 Wh M 0 0 1+ 1+i 0 0 0 2 0 Sphagnum henryense V-9 6 W h W 0 0 0 0 0 0 0 0 0 CAPRIFOLIACEAE Linnaea borealis V-15 2 Wh M 0 0 1+ 0 0 1+i 0 2 1 Viburnum edule N-4 1 Br M 0 0 2+ 1+ 0 1+ 1+ 4 3 89 Family Species (Voucher No.) Cat" Parf Habd E.c.e E.f. M.p. Z61 K799 S.a. S.e. Total* Total5 active excl. S.S. CELASTRACEAE Paxistima myrsinites N-38 COMPOSITAE Adenocaulon bicolor N-19 Anaphalis margaritacea V-40a Anaphalis margaritacea V-40b Anaphalis margaritacea V-40c Artemisia cana F-6 Artemisia douglasiana E-31 Artemisia pycnocephala E-29 Artemisia tripartita E-30 Aster modestus N-7 Grindelia integrifolia V-38 Grindelia nana N-l Hieracium albiflorum V-16 Leucanthemum vulgare N-21a 5 2 2 2 1 Br Ae L f Rt FI Ae Ae Ae Ae Wh Wh Wh Wh FI D M D D D S T C A M C A D D 1+ 0 0 0 0 0 0 0 0 0 0 0 0 0 1+ 0 1+i 0 0 0 1+i 0 1+i 0 1+ 1+ 0 0 3+ 1+i 3+ 3+ 3+ 2+ 2+ 3+ 3+ 2+ 5+ 3+ 1+ 2+ 2+ 0 1+i 2+ 1+ 3+ 5+ 1+i 3+ 0 2+ 2+ 0 1+ 1+i 0 1+ 0 1+i 0 1+i 1+i 1+i 0 0 0 0 0 2+ 1+ 2+ 3+ 2+ 2+ 2+ 1+ . 3+ 0 2+ 2+ 1+i 2+i 4+ 0 2+ 3+ 3+ 2+ 2+ 1+i 2+ 0 2+ 2+ 0 1+ 2 6 4 5 4 6 5 6 1 5 5 2 4 1 5 4 4 4 6 4 6 0 5 5 1 3 90 Family Catb Partc Habd E.c.e E.f. M.p. Z61 K799 S.a. S.e. Total* Total8 Species (Voucher No.) active excl. S.S. C O M P O S I T A E - continued Leucanthemum vulgare N-21b 5 Vg D 0 0 2+ 2+ 0 0 0 2 2 Matricaria discoidea N-36 l W h D 0 0 2+ 0 0 1+i 0 2 2 Petasites frigidus N-2a 2 Lf M 0 0 1+ 0 0 0 0 1 0 Petasites frigidus N-2b 1 Rh M 0 0 1+ 0 0 1+i 0 2 1 Solidago canadensis N-35 2 Wh D 0 0 2+ 1+ 0 1+i 1+ 4 3 Solidago spathulata N-lOa 1 Ae D 0 0 3+ 3+ 0 2+i 1+i 4 4 Solidago spathulata N-lOb 1 Rt D 0 0 2+ 0 0 1+ 0 2 2 Tragopogon pratensis Ca-20a 1 Ae D 0 0 0 0 0 0 0 0 0 Tragopogon pratensis Ca-20b 1 Rt D 0 0 0 0 0 0 0 0 0 Wyethia mollis Ca-15a 2 Ae A 0 1 + 4 + 2+ 1+i 3 + 3 + 6 6 WyerAia mo/fo Ca-15b 2 Rt A 1+i 0 3+ 2+ 1+i 2 + 2 + 6 6 C R U C I F E R A E Lepidium virginicum N-25 l W h D 0 0 0 0 0 0 0 0 0 Cakile edentulaV-l 6 W h C 0 0 0 0 0 0 0 0 0 91 Family Species (Voucher No.) Catb Partc Habd E.c. e E.f. M.p. Z61 K799 S.a. S.e. Totalf Total* active excl. S.S. CUPRESSACEAE Thuja plicata M-2 CYPERACEAE Car ex aquatilis M-8 Carex lyngbyei V-6 Carex muricata M-7 Scirpus cyperinus V-30 DROSERACEAE Drosera rotundifolia V-8 EPHEDRACEAE Ephedra nevadensis Ca-13 EQTJISETACEAE Equisetum fluviatile N-48 Equisetum pratense V - l l Equisetum scirpoides N-46 Equisetum variegatum P-46 5 2 5 4 lb M Wh Ae Wh Wh Br Ae Wh Ae Wh W C W w Wh W A W W M W 0 0 0 0 2+ 0 0 0 0 1+ 0 0 0 0 0 0 0 2+ 2+ 2+ 1+ 2+ 2+ 2+ 1+i 2+ 0 0 2+ 2+ 2+ 2+ 0 2+ 2+ 1+i 1+ 0 0 0 0 0 0 1+ 2+ 0 0 0 0 2+ 2+ 2+ 1+ 1+ 3+ 4+ 1+i 2+ 0 0 2+ 2+ 2+ 1+ 1+ 4+ 3+ 1+i 2+ 0 0 5 4 4 3 4 4 0 0 2 3 0 0 92 Family Cat" Part" Habd E.c.e E.f. M.p. Z61 K799 S.a. S.e. Totalf Total8 Species (Voucher No.) active excl. S.S. ERICACEAE Arbutus menziesii V-39a 2 Lf D 1+i 0 3+ 3+ 1+i 3 + 3 + 6 6 Arbutus menziesii V-39b 2 lb D 1+i 0 2+ 1+i 1+i 2 + 3 + 6 6 Arbutus menziesii V-39c 2 Ob D 0 0 2+ 1+i 0 2+ 2+ 4 3 Arctostaphylos patula Ca-19 1 Br D 1+i 0 2+ 2+ 2+ 3 + 3 + 6 6 Cassiope mertensiana N-32 1 Ae S 0 0 2+ 0 0 2+ 3+ 3 3 Chimaphila umbellata Ca-18 1 Wh T 1 + 0 3 + 2 + 1 + 4 + 3 + 6 5 Elliottia pyroliflorus P-55 3 Br S 1+ 1+ 2+ 2+ 1+i 3 + 3 + 7 7 Gaultheria shallon V-35 2 Br M 0 0 2+ 1+ 0 2+ 2+ 4 3 Phyllodoce empetriformis N-33 1 Ae S 1+ 1+ 5+ 1+ 1+ 4 + 4 + 7 6 Pyrola picta N-15 2 Wh M 0 0 3+ 1+ 0 2+ 2+ 4 3 EUMYCOTA Peltigera brittanica V-19 5 Wh D 0 0 1+ 1+ 0 1+i 1+i 4 2 GENTIANACEAE Gentianella amarella N-6 5 W h M 0 0 2+ 0 0 1+i 1+i 3 3 93 Family Catb Parf Habd E.c.c E.f. M.p. Z61 K799 S.a. S.e. Total* Total8 Species (Voucher No.) active excl. S.S. GRAMESfAE Ammophila arenaria V-3 6 A e C 0 0 0 0 0 0 0 0 0 HIPPOCASTANACEAE Aesculus californica Ca-8 2 Br D 0 0 2+ 2+ 0 1+ 1+ 4 4 HYDROPHYLLACEAE Phacelia ramosissima Ca-17a 1 Ae D 0 0 0 0 0 0 0 0 0 Phacelia ramosissima Ca-17b 1 Rt D 0 0 0 2+ 1+i 2+ 1+ 4 3 HYPERICACEAE Hypericum anagalloides V-7 5 Wh W 0 1 + 5 + 2+ 1+i 3 + 2 + 6 6 IRIDACEAE Iris tenuissima Ca-25 5 Wh S 0 0 3+ 2+ 1+ 2+ 2+ 5 5 JUNCACEAE Juncus bufonius V-14 3 Wh W 0 0 1+ 0 0 0 1+i 2 1 Juncus effusus var. gracilis V-4 3 Ae W 0 0 1+ 1+ 0 1+ 0 3 1 Juncus effusus var. pacificus V-5 3 A e W 0 0 1 + 0 0 0 0 1 0 Juncus falcatus V-12 5 Wh W 0 0 2+ 1+i 0 2+ 1+i 4 3 94 Family Species (Voucher No.) Cat" Partc Habd E.c.e E.f. M.p. Z61 K799 S.a. S.e. Total* Total8 active excl. S.S. Juncus lesuerii V-2 LABIATAE Prunella vulgaris V-17 Stachys bullata Ca-7 Stachys ciliata V-31 LILIACEAE Clintonia uniflora N-9 Lilium columbianum P-50a Lilium columbianum P-50b Lilium philadelphicum N-23a Lilium philadelphicum N-23b Trillium ovatum M-20 Veratrum viride N-28 LINACEAE Linum lewisii Ca-22 5 Ae C 1 Wh M 1 Ae C Ae Wh Ae Bu Ae Bu Wh Wh Wh M M T T T T M S 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1+ 1+ 1+ 1+ 0 1+ 1+ 0 1+ 3+ 2+ 1+ 1+ 1+ 0 0 1+ 0 0 0 1+ 0 0 0 0 0 0 0 0 0 0 0 1+ 1+ 1+ 1+ 0 0 0 0 2+ 0 0 1+i 2+ 0 0 0 1+i 0 0 0 0 0 4 3 2 0 3 1 0 2 2 1 2 1 1 0 1 0 0 1 1 1 95 Family Species (Voucher No.) Cat" Parf Habd E.c.e E.f. M.p. Z61 K799 S.a. S.e. Totalf Total8 active excl. S.S. LYCOPODIACEAE Lycopodium annotinum N-22 M A L V A C E A E Malva neglecta N-20 MENYANTHACEAE Menyanthes trifoliata V-32 ONAGRACACEAE Camissonia brevipes Ca-12 Epilobium angustifolium V-36a Epilobium angustifolium V-36b Oenothera villosa N-14a Oenothera villosa N-14b Oenothera villosa N-24 ORCHDJACEAE Goody era oblongifolia N-16 Platanthera dilatata P-53 3 Ae M 2 Wh D 2 Wh W Wh Ae Rt Ae Rt Wh A T T T T T 1 Wh T 3 Ae W 0 1+i 1+i 1+i 1+i 1+i 0 0 0 0 0 0 0 0 0 0 1+ 1+ 1+ 0 5+ 3+ 2+ 2+ 3+ 2+ 2+ 1+ 0 2+ 1+ 2+ 2+ 2+ 0 0 0 2+ 2+ 2+i 2+i 2+i 0 0 1+i 0 2+ 2+ 2+ 2+ 3+i 1+i 2+ 1+i 0 3+ 3+ 3+ 3+ 3+ 0 2+ 0 6 6 6 6 6 2 3 0 6 6 6 6 6 2 3 96 Family Catb Parf Habd E.c. e E.f. M.p. Z61 K799 S.a. S.e. Total* Total8 Species (Voucher No.) active excl. S.S. ORCHIDACEAE - continued Platanthera orbiculata N-17 2 W h M 0 0 1+ 0 0 2+ 0 2 1 OXALIDACEAE Oxalis oregana Ca-2 1 Wh M 0 0 2+ 2+ 0 2+ 1+ 4 4 PAEONIACEAE Paeonia brownii Ca-21a 2 Ae A 2+ 1+ 3+ 3+ 2+ 3 + 2 + 7 7 Paeonia brownii Ca-21b 1 Rb A 2+ 1+ 3+ 3+ 2 + 3 + 2 + 7 7 Paeonia brownii Ca-21c 1 Rc A 2+ 1+ 3+ 3+ 2+ 3 + 3 + 7 7 PAPAVERACEAE Argemone munita Ca-14a 2 Ae A 0 0 1+ 0 0 0 0 1 0 Argemone munita Ca-14b 3 Rt A 0 0 1+ 0 0 0 0 1 0 Eschscholzia californica Ca-1 1 Rt D 0 1+ 4+ 1+i 0 3 + 2 + 5 4 Platystemon californicus Ca-5 6 W h C 0 0 2+ 0 0 1+i 0 2 2 PINACEAE Abies grandis N-42 1 Br M 1+i 1+ 3+ 2+ 1+ 2 + 2 + 7 7 Picea sitchensis M-3 1 lb M 1+i 1+i 3+ 3+ 1+i 3+ 4+ 7 7 97 Family Cat" Parf Habd E.c.e E.f. M.p. Z61 K799 S.a. S.e. Totalf Total8 Species (Voucher No.) active excl. S.S. PINACEAE - continued Picea sitchensis M-4 1 Br M 1+i 0 1+ 1+ 0 2 + 2 + 5 3 Picea sitchensis V-10 1 Br M 0 0 1+ 0 0 1+ 1+ 3 2 Pseudotsuga menziesii N-43 1 Br M 1+i 1+i 5+ 1+ 1+ 2+ 2+ 7 6 Tsuga heterophylla M- l 1 lb M 0 1+i 3+ 3+ 1+ 3 + 3 + 6 6 PLUMBAGDMACEAE Armeria maritima Ca-4 3 Wh C 1+i 1+ 2+ 1 + 0 3 + 2 + 6 5 POLEMONIACEAE Polemonium pulcherrimum N-27 3 W h D 0 0 2+ 1 + 0 0 0 2 1 POLYGONACEAE Eriogonum umbellatum Ca-24 1 WTi S 1 + 0 1+ 2+ 1+ 2+ 2+ 6 5 Oxyria digyna N-34 1 Wh S 0 0 2+ 0 0 2+ 2+ 3 3 Polygonum amphibium N-12 3 Wh W 0 0 2+ 2+ 0 1+ 2+ 4 4 POLYPODIACEAE Adiantum pedatum V-25 1 Ae M 0 0 2+ 1+i 0 1+ 1+ 4 4 Blechnum spicant M-6 2 W h M 0 0 1+ 0 0 2+ 2+ 3 3 98 Family Catb Partc Habd E.c.e E.f. M.p. Z61 K799 S.a. S.e. Totalf Total8 Species (Voucher No.) active excl. S.S. POLYPODIACEAE - continued Cryptogramma acrostichoides N-39 4 Wh D 0 0 2+ 1+i 0 1+ 2+ 4 4 Dryopteris campyloptera M-5 1 Ae M 0 0 2+ 1+i 0 2+ 2+ 4 4 Gymnocarpium dryopteris P-52 6 Ae M 0 0 1+ 0 0 1+ 1+ 3 3 Onoclea sensibilis N-45 1 Ae M 0 0 2+ 1+i 0 1+ 2+ 4 4 Polypodium scouleri V-33a 1 Ae C 0 1+i 3+ 2 + 0 2 + 3 + 5 5 Polypodium scouleri V-33b 1 Rh C 0 1 + 4 + 4 + 0 2 + 3 + 5 5 Pteridium aquilinum M-15 1 Ae M 0 0 1+ 0 0 1+ 1+ 3 , 3 Woodsia scopulina N-26 5 Wh D 0 0 2+ 1+i 0 2+ 2+ 4 4 PORTTJLACACEAE Claytonia sibirica V-29 2 Wh M 0 0 2+ 1+ 0 0 2+i 3 3 POTAMOGETONACEAE Potamogeton richardsonii N-13 6 W h W 0 0 1+ 0 0 0 0 1 0 RANUNCULACEAE Actaea rafcra P-56a 2 Ae M 0 0 0 0 0 0 0 0 0 Actaea rubra P-56b 1 Rt M 0 0 1+ 1+i 0 1+ 1+ 4 4 99 Family Catb Part0 Habd E.c.e E.f. M.p. Z61 K799 S.a. S.e. Total* Total8 Species (Voucher No.) active excl. S.S. RANUNCULACEAE - continued Anemone multifida N-8a 3 Ae M 0 0 2+ 1 + 0 0 0 2 2 Anemone multifida N-8b 4 Rt M 0 . 0 2+ 1+ 0 0 1+ 3 3 Aquilegia formosa P-49a 2 Ae M 0 0 2+ 0 0 0 0 1 1 Aquilegia formosa P-49b 1 Rt M 0 0 1+ 0 0 1+i 1+i 3 2 Trauvetteria caroliniensis V-28 2 Wh M 0 0 1+ 2+i 0 0 0 2 1 ROSACEAE Geum triflorum Ca-23a 1 Ae A 1+i 1+ 2+ 2+ 1+ 2 + 2 + 7 7 Geum triflorum Ca-23b 2 Rt A 1+i 2+ 3+ 3+ 2+ 3 + 3 + 7 7 Horkelia fusca Ca-26a 5 Ae S 1+i 0 3+ 2+ 1+i 2 + 3 + 6 6 Horkelia fusca Ca-26b 5 Rt S 1+i 1+ 3+ 2+ 1+ 2 + 3 + 7 7 Lweffcea pectinata N-31 2 Wh S 0 0 2+ 0 0 2+ 1+ 3 3 Malus fusca N-41 1 Br M 0 0 4+ 2+ 0 5+i 2+ 4 4 Physocarpus capitatus M-10 2 Br W 0 0 1+ 1+i 0 1 + 1 + 4 2 Potentilla norwegica N-18 1 Wh W 1+i 0 2+ 2+ 2+i 1+i 2 + 6 6 Purshia tridentata Ca-10 1 Br A 1 + 0 3+ 2+ 1+ 3 + 2 + 6 6 100 Family Species (Voucher No.) Cat" Part* Habd E.c.c E.f. M.p. Z61 K799 S.a. S.e. Totalf Total8 active excl. S.S. ROSACEAE - continued Rosa canina Ca-11 Rosa woodsii P-48 Sorbus sitchensis P-54 RUBIACEAE Galium trifidum M-9 Galium triflorum V-21 SAXIFRAGACEAE Boykinia occidentalis N-44 Leptarrhena pyrolifolia V-24 Mitella brewerii N-30 Tiarella trifoliata V-27 SCROPHULARIACEAE Castilleja affinis Ca-27 Castilleja thompsonii P-44 Euphrasia stricta M-16 3 1 2 2 2 1 2 5 2 Br Br Br Wh Br Wh Wh 1 Wh 2 Ae D D S Wh M Wh M S S S M C A Wh M 1+i 1+i 0 0 0 1+i 1+i 1+i 1+i 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 2+ 2+ 2+ 1+i 1+i 3+ 3+ 4+ 2+ 2+ 0 1+ 3+ 3+ 1+ 0 1+i 2+ 1+ 3+ 2+ 2+ 0 2+ 1+ 1+ 0 0 0 1+i 1+i 2+ 1+i 0 0 0 2+ 2+ 2+ 1+i 2+ 2+ 3+ 3+ 2+ 2+ 1+ 1+ 2+ 2+ 2+ 1+i 4+ 3+ 3+ 3+ 3+ 4+ 1+ 3+ 6 6 4 3 4 6 6 6 6 4 2 4 6 6 3 2 2 6 5 6 6 4 2 3 101 Family Species (Voucher No.) Catb Paif Habd E.c.e E.f. M.p. Z61 K799 S.a. S.e. Total* Total8 active excl. S.S. SCROPHULARIACEAE - continued Mimulus guttatus V-26 SELAGINACEAE Selaginella wallacei N-37 THALLOPHYTA (Algae) Enteromorpha clathrata V-41 Fucus gardneri V-34 Mazzaella splendens V-45 Laminaria saccharina V-43 Nereocystis luetkeana V-42 Ulva fenestrata V-44 TYPHACEAE Typha latifolia M-19 VALERIANACEAE Plectritis congesta Ca-6 Valeriana sitchensis N-29a 6 2 6 6 2 4 Wh W Ae Wh Wh Wh Wh Wh Wh D N N N N N N 2 Fr W 6 Ae M 2 Ae S 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1+ 0 1+ 0 0 0 0 0 0 0 0 1+ 0 2+ 0 2+ 1+ 0 0 0 0 0 0 0 1+ 0 0 0 0 0 1+ 0 0 0 0 0 0 0 0 0 0 0 2+i 0 0 2 0 0 0 0 102 Family Catb Partc Habd E.c.e Species (Voucher No.) VALERIANACEAE - continued Valeriana sitchensis N-29b 2 Rt S 0 VIOLACEAE Viola glabella Ca-16 5 Wh W 0 E.f. M.p. Z61 K799 S.a. S.e. Totalf Total8 active excl. S.S. 0 2+ 0 0 1+i 2+ 3 3 0 0 0 0 0 0 0 0 Total number active 37 23 148 101 45 118 110 158 143 Key to Table 13 a Classification of results: 0 = no inhibition or zone of inhibition < 8.0 mm; 1+ = zone of inhibition 8.0-10.0 mm; 2+ = zone of inhibition 10.1-15.0 mm; 3+ = zone of inhibition 15.1-20.0 mm; 4+ = zone of inhibition 20.1-25.0 mm; 5+ - zone of inhibition > 25.1 mm; i = incomplete inhibition, some colonies within the clearing zone. b Cat. = Ethnopharmacological category: 1 = potential antibiotics; 2 = possible antibiotics; 3 - other medicinal uses; 4 = related species; 5 = Not used medicinally. 0 Part extracted: Ae = Aerial; Bk = Bark; Br = Branch; Bu - Bulb; FI = flowers; Fr = Fruit; lb = Inner bark; Lf = leaf; Ob = Outer bark; Rb = Root bark; Rc - Root cortex; Rh - Rhizome; Rt = Root; Wh - Whole plant. d HabHabitat: A - arid; C = coastal; D - dry; M - moist; N = saltwater; S = subalpine; T = temperate; W = wet. e Bacteria: E.e. = Escherichia coli; E.f. = Enterococcus faecalis; M.p. = Mycobacterium phlei; Z61 = Pseudomonas aeruginosa Z61 (antibiotic super-susceptible); K799 = Pseudomonas aeruginosa K799 (wild type); S.a.R. = Staphylococcus aureus P00017 multiple drug resistant strain; S.e.R. = Staphylococcus epidermidis multiple drug resistant strain. f Total number of bacteria the extract was active against. 8 Total number of bacteria the extract was active against, excluding 1+ activity against the super-susceptible organisms M. phlei and P. aeruginosa Z61. 103 Table 14 - Phase two antibiotic screening results summarized by taxa Excluding 1+ super-suscept." Taxa Number in Number Percent Number Percent Category (N) Active (N) Active (%) Active (N) Active (%) NON-FLOWERING PLANTS Lower plants Eumycota 1 1 100 1 100 Thallophyta 6 2 33 1 17 Bryopsida 11 6 55 3 27 Lycopsida 2 2 100 0 0 Sphenopsida 4 2 50 2 50 Lower plants sub-total 24 13 54 7 29 Higher plants Filicinae 10 10 100 10 100 Gymnospermae 8 8 100 8 100 Higher plants sub-total 18 18 100 18 100** NON-FLOWERING Sub-total 42 31 74 25 60 FLOWERING Sub-total 143 127 89 118 83 GRAND TOTALS 185 158 85 143 77 a Number active (N) calculated excluding those extracts with only slight (1+) activity against the super-susceptible organisms M. phlei and P. aeruginosa Z61. " Percentage of active extracts statistically significant, p < 0.01 104 Table 15 - Phase two antibiotic screening results for methanolic, water and petroleum ether extracts3 Bacteriab E. coli Solvent0 M W P Z61 M W P K799 M W P S. aureus M W P S. epi. M W P Sample No.d Ca-1 Ca-2 Ca-4 Ca-5 Ca-7 Ca-10 Ca-11 Ca-13 Ca-14b Ca-15a Ca-18 Ca-19 Ca-20a Ca-21c Ca-22 Ca-23a Ca-24 Ca-25 Ca-26a Ca-26b E-29 M-l M-2 M-3 9 10 na na 8 10 na na 8 na 9 9 na 10 8 na 11 8 na na na 9 - na 8 11 na na na 12 10 na na 9 12 na na 8 na na 8 10 10 17 21 15 13 16 12 na na 11 10 11 na na 9 11 na 15 12 na 16 17 na 15 12 na na 11 10 na 12 10 na 13 16 na na 18 10 na na 13 17 na 11 11 11 na 11 11 na 15 14 na na na na - 11 na na 13 na 8 14 na 8 16 na 13 9 na na 9 13 na 10 - na 13 15 na na 11 - na na 10 15 na 10 10 na 9 - na 10 10 na 9 8 10 8 11 na 9 16 18 15 13 20 21 na na 18 11 16 15 na 9 - na 10 15 na 17 17 na 13 19 na 25 14 na 7 na 17 10 na 21 12 na 18 19 na na 12 20 na 14 13 15 na 15 15 na 15 16 na na 15 9i 16 22 12 15 24 31 na na 15 20 na na - 20 na 14 22 na 12 25 na 20 15 na na 18 14 na 16 15 na 17 27 na na 16 11 na 16 14 na na 13 23 na 15 18 15 na 16 18 na 17 19 na na 105 Bacteria6 E. coli Z61 K799 S. aureus S. epi. Solvent" M W P M W P M W P M W P M W P Sample No.d M-4 8 8 - 9 9 - - 9 - 14 14 8 14 23 -M-6 - - na - 8 na - - na 12 10 na 11 10 na M-17b 8 na - 16 na - 8 na - 13 na - 15 na -N-7 - na - - na - - na - - na - - na -N-20 - na - - na - - na - - na - - na -P-43 - - na - - na - - na - - na - - na P-44 - - na - - na - - na 10 9 na 10 12 na P-45 - - na - - na - - na - - na - - na P-47 - - na 11 8 na 9 8 na 13 11 na 12 9 na P-48 9 9 na 16 15 na 10 11 na 14 16 na 14 17 na P-50 - - na 8 - na - 8 na - - na 9i - na Key to Table 15 a Values shown are the average zone of inhibition diameters, "-" represents no zone of inhibition; na = not applicable (extract not prepared). b Bacteria screened against: E.e. = E. coli; Z61 = P. aeruginosa Z61; K799 = P. aeruginosa K799; S. aureus P00017; S. epi. = S. epidermidis. c Solvent of extraction: M = methanol; W = water; P = petroleum ether. d Sample No. = Plant collection sample number. 106 Table 16 - Phase two ethnopharmacological analysis of antibiotic screening results Category Number in Category (N) Number Active (N) Percent Active (%) Excluding 1+ Super-susc.a Number Percent Active (N) Active (%) Potential antibiotics 68 62 91 62 91** Possible antibiotics 57 51 90 45 79 Tonics 6 6 100 5 83 Other medicines 13 11 85 9 70 Related species 23 20 87 18 78 Subtotal medicinal 167 150 90 139 83 No medicinal use 18 8 43 4 22 Grand Totals 185 158 85 143 77 a Number active (N) calculated excluding those extracts with only slight (1+) activity against the super-susceptible organisms M. phlei and P. aeruginosa Z61. ** Percentage of active extracts statistically significant, p < 0.01 107 Table 17 - Phase two antibiotic screening results summarized by habitat Category Number in Category (N) Number Active (N) Percent Active (%) Excluding 1+ Super-susc." Number Percent Active (N) Active (%) Saltwater 6 2 33 1 17 Coastal 13 11 85 11 85 Wetlands (freshwater) 28 23 82 19 68 Moist 55 49 89 43 78 Temperate 17 14 82 13 76 Dry 33 28 85 26 79 Arid 16 14 88 13 81 Sub-alpine 17 17 100 17 100** Total 185 158 85 143 77 a Number active (N) calculated excluding those extracts with only slight (1+) activity against the super-susceptible organisms M. phlei and P. aeruginosa Z61. ** Percentage of active extracts statistically significant, p < 0.01 i 108 3.1.4 Discussion and Conclusions The primary objective of this screening was to examine the antibiotic activity of North American plants and in this regard the results were very promising. The secondary objective of this study was to obtain the necessary data to answer some fundamental questions regarding the design of ethnopharmacological screenings. Data analysis of the phase one screening results suggested that there may be differences in antimicrobial activity based on: whether or not the plants were used in traditional medicine; whether the specific medicinal applications of a plant remedy suggested potential antibiotic activity; and whether the extracts were made from flowering or non-flowering plants. Other variables which were considered important to examine were plant habitat and solvent of extraction. Therefore, the results of the screening were also analyzed in relation to all of these factors. Overall, 85% of the methanolic plant extracts exhibited antibiotic activity (see Table 13). Out of the 158 active extracts, 50 extracts showed strong broad spectrum activity (active against a minimum of 5 bacteria) which was equal to or better than the performance of the positve control, gentamicin. Extracts from 9 plant species were active against all seven bacteria: Abies grandis, Elliottia pyroliflorus, Geum triflorum, Horkelia fusca, Paxistima myrsinites, Paeonia brownii, Phyllodoce empetriformis, Picea sitchensis and Pseudotsuga menziesii. In addition to these, the extract of Ephedra nevadensis was particularly noteworthy for its strong activity against the gram negative pathogens E. coli and P. aeruginosa. Extracts of Arctostaphylos patula, Epilobium angustifolium, Mitella breweri, Oenothera villosa, and Potentilla norwegica also exhibited strong activity against the clinically important organism P. aeruginosa. At the family level, 3 families were outstanding for the comprehensive antibiotic activity their members exhibited. Extracts of all 8 species from the Ericaceae, all 10 species from the Polypodiaceae and all 10 species from the Rosaceae were active against a minimum of 3 bacteria. The known antibiotic compounds chimaphilin and arbutin are common constituents among the Ericaceae and these compounds are likely responsible for the activity observed in the members of this family. The candidates for the antibiotic constituents of the Polypodiaceae and the Rosaceae are not as clear cut however. A number of tannins and flavonoids from the Rosaceae have been reported as antibiotic constituents and these types of compounds may be responsible for the activity observed in this study. However, very little work has been done on the Polypodiaceae and the identity of 109 their antibiotic constituents are largely unknown. An analysis of the results categorized by major taxa also provided some interesting observations (see Table 14). The percentage of active extracts among the flowering plants (89%) was higher than that among the non-flowering plants (74%). Among the non-flowering plants, there was a striking division between the percentage of active extracts among the Filicinae and Gymnospermae (100%) and that of the lower plants (54%). This difference was even more apparent when the percentage of active extracts was calculated excluding those extracts with only slight (1+) activity against the super-susceptible M. phlei and P. aeruginosa Z61. Under this more rigorous evaluation, 100% of the Filicinae and 100% of the Gymnospermae were active while only 29% of the lower plants were active. These results were similar to those observed in the phase one analysis of antibiotic activity by taxa. Most ethnopharmacologists prefer the relative ease of organic solvent extraction compared to the time consuming and contamination-prone method of water extraction. However, this practice provides an opening for the criticism that such assays do not truly examine traditional medicines if traditional methods of preparation were not followed. To explore the possibility that some antibiotic activity may be overlooked due to the method of extraction, boiling water and petroleum ether extracts of some samples were also made. A comparison of selected antibiotic activities of extracts made with these solvents are shown in Table 15. There were slight quantitative differences between the methanol and water extracts but there were only a few cases where there were qualitative differences between them. Overall, these differences did not appear significant as there was only one sample (Ca-7) whose antibiotic activity could possibly have been missed entirely in a narrow screening of methanol extracts. The plants selected for extraction with petroleum ether were those which were traditionally prepared as salves or liniments made with grease, fat, lard, or oil. With one minor exception, none of these non-polar extracts exhibited any antibiotic activity although many of the methanolic extracts of the same material were active. Ethnopharmacologists tacitly assume that by following ethnobotanical leads they are much more likely to identify clinically useful phytochemicals and the results of the ethnopharmacological data analysis provide evidence to support the validity of this assumption (see Table 16). Excluding those extracts which had only slight (1+) activity against the super-susceptible M. phlei