Ethnobiological and Chemical Investigations of Selected Amazonian Plants
ETHNOBIOLOGICAL AND CHEMICAL INVESTIGATIONS OF SELECTED AMAZONIAN PLANTS
by
WILLIAM DONALD MACRAE
B.Sc, The University of Victoria, 1974 M.Sc, The University of British Columbia, 1978
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March 1984
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< Date ^ ,>1 Abstract The ethnobotanical literature of Amazonian South America has been surveyed, compiled and organized. This information allowed the identification of certain taxonomic groups and plants with specific uses which seem promising for further research. The use of Just ic ia pectoralis as an additive to hallucinogenic Virola based snuffs has been investigated. No alkaloidal compounds could be detected in the plant and the pharmacological effects of an extract on mice were not indicative of the presence of strongly psychoactive constituents. Nor did the J. pectoralis extracts have any inhibitory or synergistic effect upon the responses of mice to 5-methoxy-N,N-dimethyltryptamine, the psychoactive constituent of Vi rola bark. Extracts of the plant caused the relaxation of smooth muscle and this activity was shown to result from the presence of coumarin and umbelliferone. Betaine, which was also present, was observed to elicit smooth muscle contraction at high concentrations. Strong inhibitory activity of the extract towards four dermatophytic fungi was observed and may explain the use of the plant in the treatment of certain infections. Coumarin was shown to be wholly responsible for the aroma. The possibility that the hypnotic effects of coumarin may play a role in its use as a snuff constituent is considered. The use of Vi rola elongata as both an hallucinogenic snuff and an arrow poison was examined. Extracts of the bark were evaluated for their effects on mouse behavior. A non- alkaloidal fraction caused a reduction in spontaneous motor activity in mice while the alkaloidal fraction from the same amount of plant material had no significant effect. The non- alkaloidal fraction was examined and some of its biological activity was attributed to the presence of polyphenolic compounds. Eleven compounds were isolated from the non-polar part of this extract. In addition to j3-sitosterol, two isomeric stilbenes, two neolignans, four bis-tetrahydrofuran lignans and two tetrahydrofuran lignans, were identified. The bis-tetrahydrofuran lignans were shown to reduce spontaneous motor activity and isolation induced aggression in mice. The possibility that they are at least partly responsible for the use of Virola elongata as an arrow poison is' raised. Thirty-four species of Amazonian Euphorbiaceae were screened for inhibitory activity towards Escherichia coli, Staphylococcus aureus, two yeasts, four dermatophytic fungi, two animal viruses, tumour formation in potato discs and toxicity to brine shrimp. A large proportion of the extracts were active against S. aureus, the dermatophytes, at least one of the viruses, the potato tumours and the brine shrimp. The biological activities observed are discussed with respect to the use of certain species in Amazonian ethnomedicine. The antiviral activity of one of the plants screened, a species of Amanoa was examined. The inhibitory activity towards infection by murine cytomegalovirus was found to i v result from the presence of a single compound, identified as the lignan, a- ( - )-peltatin. At low doses(lO ng/ml for two hours), this compound prevented the replication of viruses in already infected cells. It's activity was observed to be very similar to that of another naturally occurring lignan, podophyllotoxin. Lignans of a variety of structural types were examined for antiviral activity but anti-murine cytomegalovirus activity of compounds other than the podophyllotoxin type was not observed. V Table of Contents Abstract i i List of Tables x List of Figures xii Acknowledgement xiv CHAPTER I GENERAL INTRODUCTION 1 1. ETHNOBIOLOGY; AN INTERDISCIPLINARY APPROACH 1 2. OBJECTIVES OF ETHNOBIOLOGY 6 3. IMPORTANCE OF ETHNOBIOLOGY IN THE TROPICS 8 4. APPROACH OF THIS STUDY 11 LITERATURE CITED 25 CHAPTER II Justicia pectoralis : A STUDY OF THE BASIS FOR ITS USE AS A Virola SNUFF ADMIXTURE 30 1. INTRODUCTION 30 2. MATERIALS AND METHODS .31 a. Plant material 31 b. Chromatography and spectroscopy 32 c. Behavior experiments 33 d. Rat stomach strip experiments 33 e. Antimicrobial tests 34 f. Antiviral tests 35 3. RESULTS 35 vi a. Examination of Justicia pectoralis for alkaloids 35 b. Compound 1 36 c. Behavioral effects of Just ic ia pectorali s 37 d. Effect of Justic ia pectoralis extracts on 5-MeODMT induced behavioral responses 39 e. Effect of 5-MeODMT on mouse activity 41 f. Gross behavioral effects of 5-MeODMT 44 g. Effect of co-injections of 5-MeODMT and Justicia pectoralis extracts 44 h. Effect of Justic ia pectorali s extracts on smooth muscle 47 i. Analysis of aromatic constituents 51 j. Quantification of coumarins of J. pectoralis ....53 k. Examination of J. pectoralis for lignans 55 1. Screening for other biological activities 57 4. DISCUSSION 59 LITERATURE CITED 63 CHAPTER III AN ETHNOPHARMACOLOGICAL EXAMINATION OF Virola elongata BARK, A SOUTH AMERICAN ARROW POISON 69 INTRODUCTION 69 PART A. ISOLATION AND IDENTIFICATION OF THE MAJOR NON- POLAR CONSTITUENTS OF Virola elongata BARK 73 1. INTRODUCTION 7 3 2. EXPERIMENTAL 73 vii a. Extraction of plant material 73 b. Chromatography 74 3. COMPOUNDS ISOLATED 7 4 4. RESULTS 80 a. 3,4',5-trimethoxy-cis-stilbene and 3,4',5- trimethoxy-trans-stilbene 83 b. Eusiderin and virolongin 87 c. Epi-sesartemin, sesartemin, epi-yangambin and yangambin 88 d. Dihydrosesartemin and 0-yangambin 91 PART B. EXAMINATION OF THE BIOLOGICAL ACTIVITY OF Virola elongata BARK EXTRACTS 100 1. INTRODUCTION 100 2. MATERIALS AND METHODS 100 a. Preparation of extracts 100 b. Fractionation of extracts 101 c. Chromatographic analysis of extracts 102 d. Assay of spontaneous motor activity 103 e. Assay of anti-aggressive activity 104 3. RESULTS 105 a. Examination of the aqueous fraction for toxicity 106 b. Examination of the diethyl ether extract for depression of spontaneous motor activity 109 c. Quantification of major constituents of diethyl ether extract 114 d. Effect of bis-tetrahydrofuran lignans on viii isolation induced aggression 116 4. DISCUSSION 119 LITERATURE CITED 127 CHAPTER IV STUDIES ON THE PHARMACOLOGICAL ACTIVITY OF AMAZONIAN EUPHORBIACEAE 134 PART A. MULTIPLE SCREENING OF AMAZONIAN EUPHORBIACEAE FOR BIOLOGICAL ACTIVITIES 134 1 . INTRODUCTION 134 2. MATERIALS AND METHODS 139 a. Plant material 139 b. Preparation of plant extracts 140 c. Antimicrobial screening 140 d. Antiviral activity 141 e. Potato disc tumour assay 143 f. Toxicity to brine shrimp 144 g. Analysis of data 144 3. RESULTS a. Antimicrobial activity 145 b. Antiviral activity 150 c. Inhibition of potato tumour formation 153 d. Toxicity to brine shrimp 156 e. Correlation between biological assays 156 4. DISCUSSION 167 a. Antimicrobial activity 167 ix b. Antiviral activity 170 c. Antitumour activity 172 d. Toxicity to brine shrimp 172 5. CONCLUSION 173 PART B. a-( - )- PELTATIN, THE ANTIVIRAL CONSTITUENT OF Amanoa sp 176 1 . INTRODUCTION 176 2. MATERIALS AND METHODS 176 a. Plant material 176 b. Antiviral assays 177 c. Chromatography 177 3. RESULTS AND DISCUSSION 178 PART C. THE ANTIVIRAL ACTION OF LIGNANS 183 1 . INTRODUCTION 183 2. MATERIALS AND METHODS ...184 a. Chemicals 184 b. Cells and viruses 184 c. Antiviral screening of lignans 186 d. Effect of time of treatment 187 3. RESULTS 188 4. DISCUSSION 193 LITERATURE CITED 201 APPENDIX A - LIST OF AMAZONIAN.ANGIOSPERMS OF ETHNOBOTANICAL INTEREST 207 BIBLIOGRAPHY: APPENDIX A 231 APPENDIX B - SP4100 COMPUTER PROGRAM(BASIC) 236 X List of Tables I. Phylogenetic distribution of species of Amazonian angiosperms having documented etnobotanical use ..14 II. Effect of Just ic ia pectoralis extracts on spontaneous locomotor actrvity in mice 40 III. Effect of co-administration of 5-MeODMT and Justicia pectoralis extracts on spontaneous locomotor activity of mice 46 IV. Levels of coumarin and umbelliferone in different samples of Just ic ia pectoralis 56 V. 'H-NMR spectra of bis-tetrahydrofuran lignans isolated from Vi rola elongata bark 81 VI. ' 13C-NMR spectra of bis-tetrahydrofuran lignans isolated from Vi rola elongata bark -82 VII. Gross behavioral responses of Swiss mice to administration of alkaloidal and non-alkaloidal extracts of Vi rola elongata bark 107 VIII. Effect of purified compounds of Vi rola elongata bark on spontaneous locomotor activity of Swiss mice 113 IX. Quantitative analysis of thirteen most common constituents of diethyl ether extract of Vi rola elongata bark 115 X. Effect of epi-sesartemin on three behavioral parameters related to aggressiveness of mice ....118 XI. Effect of bis-tetrahydrofuran lignans on xi aggressiveness in mice 120 XII. Antimicrobial screening of extracts of Euphorbiaceous plants ...146 XIII. Screening of Euphorbiaceous plants for anti-dermatophytic fungus activity 148 XIV. Antiviral screening of extracts of Euphorbiaceous plants 151 XV. Screening of extracts of Euphorbiaceous plants for inhibition of Agrobacteriurn induced tumour formation 154 XVI. Screening of extracts of Euphorbiaceous plants for their toxicity to brine shrimp, Artemia salina 157 •XVII. Summary of biological screening of extracts of Euphorbiaceous plants 159 XVIII. 2 X 2 Contingency tables for agreement between each pair of the 14 assays used to screen the 85 plant extracts 164 XIX. Values of Fisher's exact test for contingency tables of Table XVIII 165 XX. Summary of ethnobotanical information and biological activity of the 34 species of Euphorbiaceae tested 168 XXI. Examination of lignans for their effect on replication of Sindbis virus and MCMV 189 XXII. Effect of time of lignan treatment upon inhibition of Sindbis virus infection 192 xii List of Figures 1. Distribution Amazonian angiosperms with documented ethnobotanical uses 18 2. Structures of betaine (1), coumarin (2) and umbelliferone (3) 38 3. Effect of 5-MeODMT on spontaneous locomotor activity of mice 43 4. Effect of a) serotonin(l ng/ml) and b) betaine (50 jug/ml) on smooth muscle 48 5. Effect of (a) coumarin ( 10 Mg/ml) and (b) (umbelliferone) (10 Mg/ml) on smooth muscle 52 6. HPLC chromatogram of the organic fraction of leaves of J. pectoralis 54 7. Structures of stilbenes and neolignans isolated from Vi rola elongata 84 8. Structures of bis-tetrahydrofuran lignans isolated from Vi rola elongata 85 9. Structures of tetrahydrofuran lignans isolated from Vi rola elongata 86 10. Scheme of fragmentation of bis-tetrahydrofuran lignans, epi-sesartemin and sesartemin by mass spectrometry 92 11. Scheme of fragmentation of bis-tetrahydrofuran lignans, epi-yangambin and yangambin by mass spectrometry 93 12. Scheme of fragmentation of tetrahydrofuran lignan, xi i i dihydrosesartemin, by mass spectrometry 95 13. Scheme of fragmentation of tetrahydrofuran lignan, /3-dihydroyangambin by mass spectrometry 96 14. Example of transducer output used to record spontaneous motor activity 111 15. Effect of bis-tetrahydrofuran lignans, epi-sesartemin and epi-yangambin on spontaneous locomotor activity of mice 112 16. Chromatotron elution profile of ethyl acetate fraction of Amanoa sp. leaves 179 17. Structure of a-( - )- peltatin isolated from Amanoa sp .181 18. Structures of lignans tested for antiviral activity 185 19. Effect of time of lignan treatment on inhibition of murine cytomegalovirus infection 191 x i v Acknowledgement An ethnobotanical or ethnobiological approach is a strongly interdisciplinary one. This study is no exception and I am indebted, to an especially large degree, to the work of my predecessors as well as to many contemporary advisors, contributors and informants. I owe special thanks to Prof. G.H.N. Towers, who supplied unlimited enthusiasm and support for this research and gave generously of his time. I was fortunate to be able to work in cooperation with Dennis McKenna, both during the field work and, following that, in the laboratory. Our free exchange of information was beneficial to many aspects of this work. His interest in Just ic ia pectorali s and Vi rola sp. was especially helpful and he kindly made plant material available for analysis. The ethnobotanical fieldwork of Prof. R.E. Schultes has influenced the course of this study in many ways. This is evident from the number of times his name is cited and from the important role he played in describing the uses of both Justicia pectoralis and Virola elongata. I wish to acknowledge the personal communication of Prof. R.E. Schultes, Professor and Director, Harvard University Botanical Museum concerning the ethnobotany and taxonomy of Justicia pectoralis and also Dr. G.T. Prance, Director of Botanical Research, New York Botanical Garden, for helpful information concerning the ethnobotany of both Justicia XV pectoralis and Vi rola elongata. A great many people may be credited with assisting in the collection of plant material. Tim Plowman, Chicago Field Museum, generously provided advice and assistance and arranged for the identification of much of the plant material collected. All of the species of Euphorbiaceae collected were kindly identified by Dr. M.J. Huft of the Chicago Field Museum. Dr. Ramon Ferryra, Director of the Museo Historia Naturale de Peru, made time available for consultation and assisted in the procurement of the necessary authorization for plant collection and export. Sr. Puricaca of the Departamento de Agricultura y Alimentacion was also helpful in this regard. Dr. Franklin Ayala, Universidad Nacional de Amazonia Peruana, kindly made herbarium facilities and equipment available. I am grateful to Dick Rutter and Wes Thiessen of the Summer Institute of Linguistics, Pucallpa, for their helpful information. Adriana Lyoaza and Nicole Maxwell graciously offered both information and hospitality. Nicole Maxwell is cited as the source of several personal communications concerning medicinal plant use. I owe special thanks to the Witoto and Bora Indians of Puco Urquillo and the Boras of Brillo Nuevo for their hospitality. Juan Ruiz Macedo, Andres Cdrdoba, Enrique Donez, and Mario Cordoba provided valuable assistance in plant collecting. Among the many Indian and mestizo informants contacted, may be numbered Don Juan Mozombite of Pucallpa, Don Juan Peso of Nina Rumi, and Don Ramiro Diaz of Balsas. These are just a few of the many xvi Peruvians who, in a spirit of friendship and a common interest, offered information on plant uses. The laboratory phase of this research was also interdisciplinary in nature. Bob Kantymir provided assistance in the greenhouse. Terry Crawford and Pete Drummond, U.B.C, generously made equipment available for recording smooth muscle contraction and mouse locomotor activity. Sam Gopaul provided advice concerning animal care and behavior. All of the NMR spectra and the high resolution mass spectra were obtained through the services of the Chemistry Department, U.B.C. Infrared spectra and optical rotations were recorded on the instruments of Drs. Pierce and Drummond, Chemistry, U.B.C The interpretation of spectral data was facilitated by many helpful discussions with Felipe Balza. I am grateful to Dr. M.P. Gordon, Dept. of Biochemistry, U. of Washington, for providing the Aqrobacterium tumefaciens and to Dr. Barbara Dill, Dept of Medical Microbiology, U.B.C, for the sporulating strains of dermatophytic fungi. I would like to thank Dr. J.B. Hudson, Dept. of Medical Microbiology, U.B.C, for allowing me to carry out the studies on antiviral activity in his lab. Some of the lignans tested were provided by Dr. E. Swan, Forintek, Vancouver, and Dr. G.H. Sheriha, El Fateh University, Libya. Finally, the financial support of the University of British Columbia, in the form of a Graduate Fellowship, and the Natural Science and Engineering Research Council of Canada for a Postgraduate Fellowship is gratefully acknowledged. 1 I. GENERAL INTRODUCTION 1. ETHNOBIOLOGY: AN INTERDISCIPLINARY APPROACH Ethnobiology, ethnobotany and ethnopharmacology are overlapping terms for the study of the use of the earth's biotic resources by man. Its students and practitioners are not numerous and they come from different disciplines and backgrounds, bringing with them varying points of view. There are, perhaps, as many definitions of "ethnobiology" as there are ethnobiologists. The terms, ethnobiology, ethnobotany and ethnopharmacology have been loosely applied to the many types of scientifically sanctioned observations, identifications, descriptions and experimental investigations of plants, and their principals, used by indigenous peoples (Efron e_t al, 1967; Holmstedt and Bruhn, 1983; Schultes, 1963; Schultes and Swain, 1976). Bruhn and Holmstedt (1982) have succinctly defined ethnopharmacology as " the interdisciplinary scientific exploration of biologically active agents traditionally employed or observed by man." Ethnobotany or ethnobiology may be similarly defined. The basic disciplines involved are ethnology or anthropology, pharmacology and botany (or zoology in the case of animal products). The only term which encompasses all situations is ethnobiology. It is used in the present work for this reason. 2 My conception of this field of study is not unique, having been influenced by many previous workers. Neither does it coincide with some imaginary concensus view. In an attempt to clarify the objectives of this study, there follows a general consideration of the role, objectives and importance of ethnobiology in modern science. The recent rapid advances in science have broadened the conceptual gap between western industrialized and primitive societies. Acceptance of the value of simple technologies or belief systems different from our own is not common. There is a widespread tendency in our society not only to accept the primacy of the scientific method, but to adopt the corollary that information gained in other ways is necessarily trivial or false. The interface between the scientific method and the beliefs of primitive peoples, which is an essential part of ethnobiology, is not always a smooth one. The scientific view is that the use of plants by man has emerged slowly, as the result of empirical testing by generations of inquisitive primitives and that it has been sustained by the oral tradition. When one queries an Indian or mestizo about how they came to know of a plant use, one sometimes gets the appropriate answer; that the information was obtained from a friend, a relative, an elder or from a neighboring community. If, on the other hand, one asks a native specialist who has been trained in the shamanic tradition, the answer seldom varies; that it was learned from the plant, itself. According 3 to the shaman, the process is one of summoning up the spirit of the plant, establishing contact and listening to its explanation. The Peruvian mestizo shaman routinely refers to certain plants as doctores or vegetales que ensenan (plants that teach) (Luna, 1983). They are the subjects of invocations made during trance like states induced, or regulated by, the use of hallucinogenic ayahuasca and potent tobacco. The truly amazing thing about this practice is that it is common, in similar forms, to the tribes, not only of the Amazon, but of all the Americas and much of Eurasia (Eliade, 1964; Metraux, 1944). The beliefs can be traced in the cultures of the Fuegians of Cape Horn as well as the barren land Eskimos (Browman and Schwartz, 1979; Chine, 1976). The implications of plants as teachers are not easily reconciled with scientific thought. It is ironic, however, that if the concept is taken figuratively, it indicates remarkable insight. For example, until approximately ten years ago, modern science knew very little of fundamental importance about pain. Since then, it has been discovered that morphine, an alkaloid from the poppy, prevents pain by binding to specific receptors on neurons of the central nervous system and that peptides are present in the human body which also bind to the same receptors (Costa and Trabucchi, 1978). A major rethinking in neurophysiology has ensued. The provocative discovery of the normal peptides, or endorphins, was dependent upon the availability of the compound morphine and, of course, the poppy plant. The euphoric properties of 4 Papaver somniferum were, coincidentally, known to the Sumerians some 6000 years ago (Swain, 1972). Many similar examples may be cited. Picrotoxin, a convulsant from Anamirta cocculus (Menispermaceae), is currently used widely in investigations of neurotransmission (Hoffmeister and Stille, 1980). Phorbol esters, from species of the Euphorbiaceae, are tumour promoters and important tools in experimental cancer research (Evans and Soper, 1978; Weinstein et a_l, 1977). Recently, they have been found to be potent inducers of interferon in mammalian cells (Yip e_t al, 1981). For many years, animal physiologists have distinguished neurons based upon whether they were excited by nicotine (from Nicotiana tabacum) or muscarine (from Amanita muscaria). Tubocurarine (from plants of the Loganiaceae) and atropine (Atropa belladonna) are both used extensively in studies of neuromuscular transmission, and rotenone (Derris spp.) is commonly used as a metabolic inhibitor. Colchicine (Colchicum autumnale) is used routinely by most cytogeneticists and several hundred papers have been written on the effects of caffeine (Caffea sp. and Thea sp.) in the inhibition of DNA repair processes. The list could be continued. Looking at the Merck Index, it is difficult to pick a page on which at least one citation to a scientifically useful or economically important constituent produced by a plant or microorganism, does not appear. It is instructive to imagine what modern biology and medicine might have been like had the organic constituents of plants or microorganisms never been available 5 to us. Knowledge in these areas would probably be reduced to a small fraction of what it is now. Without biologically active, naturally occurring compounds as models, very few antibiotics or chemotherapeutic agents would yet have been synthesized by chemi sts. Even such a relatively straightforward matter as the economic importance of plants to medicine is not widely recognized. Educated estimates are that approximately one-half of the economically important compounds continue to be obtained from plants (Oldfield, 1981). Farnsworth and Morris (1976) have presented figures which indicate that, between 1959 and 1973, 25% of all the prescriptions filled in the U.S. contained plant products. The value, to the consumer, was estimated to be $3.0 billion, annually. In 1980, the percentage was unchanged but the dollar value had climbed to in excess of $8.0 billion (Farnsworth and Loub, 1982). The wholesale value of medicinal plants imported to the U.S. from tropical forests, alone, has been estimated to be $25 million (U.S. Interagency Task Force, 1980). Given sufficient space, the argument that modern science and medicine are deeply indebted to the information obtained from biologically active plant compounds, could be expanded further. The degree of respect for plants held by the primitive American societies has already .been contrasted with our own society's general lack of acknowledgment of the importance of plants to knowledge. The irony of this situation is tempered by the observation that the "most useful drugs 6 derived from higher plants have been 'discovered' as a result of the scientific enquiry into alleged folkloric claims of therapeutic effects" (Farnsworth and Loub, 1982). Many microbial products have been discovered as a result of a random screening program. For various reasons, higher plants are not nearly as amenable to suchr an approach. Moreover, since they are macroscopic, they are an important part of the material world of primitive man. As a result, a rich history of plant use by man is available to draw upon. In the past, the investigation of plants used by primitive man has been the preferred approach and has led to the development of such drugs as atropine, digitalis and reserpine. Less commonly carried out today, such studies still lead to the discovery of constituents with novel biological activity (Gran, 1973) . Some random screening programs of higher plants have been carried out. The most extensive of these has been conducted by the U.S. National Cancer Institute (Suffness and Douros, 1982). Plants were screened for antitumour activity. In an interesting follow-up study, Spjut and Perdue (1976) presented evidence that the occurrence of antitumour activity was significantly higher in plants having a history of folk use than those selected at random. 2. OBJECTIVES OF ETHNOBIOLOGY The foregoing discussion has developed the idea that one 7 of the primary objectives of ethnobiology is the discovery of naturally occurring constituents with novel biological activity. A natural extension of this aim is a desire to describe and explain the distribution of biologically active constituents in the plant kingdom. Swain (1972) has pointed to the use of medieval plants in medicine as being responsible for the origin of modern taxonomy. There has followed a gradual accumulation of information on the chemical constituents of various plant taxa in the related fields of phytochemistry, natural products chemistry and chemotaxonomy. The task of synthesizing this information so as to gain a broad perspective on how various biologically active compounds are distributed in the plant kingdom is an immense one and has been attempted by only a few (Farnsworth and Loub, 1982; Farnsworth, et al, 1974; Hegnauer, 1962; Karrer, 1958). This type of descriptive information may eventually provide valuable insights into the role of various constituents in evolution. Moreover, it would be expected to provide predictive information on the presence of specific compounds, or possibly even compounds with specific biological activity, in taxa not previously examined chemically (Barclay and Perdue, 1976). Ethnobotanical information, too, is considered by some to be a useful tool in predicting the occurrence of certain biologically active constituents (Spjut and Perdue, 1976). The applicability of the computer in handling large data bases of this sort, containing both ethnobotanical and chemical information on plants, is currently being 8 investigated (Farnsworth and Bingel, 1977; Farnsworth and Loub, 1982; Farnsworth et al, 1981). There is another basic objective in ethnobiological work that should not be overlooked. The subjective nature of the approach is the aspect of ethnobiology most often criticized. Yet this property can also be regarded as a unique advantage. A single statement concerning the use of a plant by man is unlikely to contribute directly to basic science. It can, on the other hand, summarize an aspect of the relationship between man and the plant kingdom in an historical context in a way not possible using only a reductive scientific approach. The extent of the interactions between man and plants is declining as the industrialized world expands and the endurance of ethnobotanical information, if unrecorded, is predictably low. This will result in the loss of information, not only on specific cultures, but also on habitats and even complete ecosystems since these, too, are inevitably altered as a result of economic development. One of the roles of ethnobotany, therefore, is the documentation of plant use and the identification of the vegetal materials. This activity continues to be of importance as the existence of little known groups of the world's biota, including many human cultures, face the certainty of drastic change. 3. IMPORTANCE OF ETHNOBIOLOGY IN THE TROPICS The question of the future of tropical ecosystems of the 9 world is an especially pertinent one for biologists at this time in history. Although ethnobiological study can be carried out anywhere that people are using plants, the tropics has been, and continues to be, an especially fertile region for this type of work. It is not clear why so many of the important drugs of natural origin were identified from tropical plants (Oldfield, 1981). The diversity in species of all kingdoms of organisms and the enormous potential for intra- and inter-specific interactions are certainly important factors. Evidence has been presented which indicates that diversity in chemical compounds synthesized parallels this increase in species diversity (Raffauf, 1970). An equally compelling reason for studying ethnobiology, or any branch of biology, in the tropics is the uncertain future facing this region. Tropical forests account for almost 70% of global forest productivity (Bruning, 1977). Covering only 7% of the earth's surface, they contain between 40 and 50% of the world's estimated 5 to 10 million species (Meyers, 1981). It is agreed that it is the least known of the planet's biomes, with estimates of the extent of the species already described ranging between 10 and 16 % (Meyers, 1981; Raven et al, 1971). Of these, we possess only the most rudimentary information (Raven et a_l, 1971). The most often cited prediction of the future of the tropical moist forest is that of Richards (1952; 1973) who forecast that 10 "The tropical forest ecosystem as we know it will virtually disappear from the face of the earth by the end of the 20th century." Other predictions are generally less pessimistic but not much less dramatic. There is broad agreement among specialists that, by the year 2000, very large segments of the world's tropical moist forests will be reduced to remnants (Farnworth and Golley, 1974; Golley and Medina,1975; Gomez-Pompa et al, 1972; Lanly and Clement, 1979; Meyers, 1980a and b; Persson, 1974, 1975, and 1977; Poore, 1976; Richards, 1973; Sommer, 1976; UNESCO, 1978). The vast watershed of the Amazon River system accounts for approximately 40% of the world's moist tropical forests .(Goodland, 1980). Predicted rates of deforestation are representative of the global forecasts cited above (Fearnside, 1979). The major disruptive factors have been identified as the pattern of shifting cultivation practiced by the expanding population, timber harvesting and cattle ranching (Denevan, 1980; Shane, 1980). The emerging evidence points towards the limited ability of this ecosystem to resist change (Gentry, 1977). The magnitude of the contribution of the seasonally inundated areas, a relatively small fraction of the total area, to the overall productivity has been realized quite recently (Meggers, 1971). This, however, is the most vulnerable to modification and the ability of this complex ecosystem to tolerate such pressures has not been established. The importance of the relationships between the trees of the 11 inundated regions and the many species of fruitiferous fish has recently been documented (Goulding, 1980). Fish are also the major source of protein for the enlarging population. Any likely scenario for the future of the moist tropical forests in the next century, a very short interval on an evolutionary scale, is of a drastic reduction in the extent of the planet's biota (Meyers, 1981). In preparation for this seemingly unavoidable situation, certain priorities for research have been proposed (Research Priorities in Tropical Biology, 1980; Tropical Deforestation, 1980). These are aimed at accumulating as much information on the biology of tropical organisms as possible in the limited time allowed. Descriptive ethnobotany is considered to be a minor, but valued contribution (Raven et_ al, 1971; Research Priorities in Tropical Biology, 1980). The longstanding relationship of the indigenous peoples with their biota is considered likely to have resulted in many biological insights which may be obtained by scientific methods only after years of research. 4. APPROACH OF THIS STUDY The objectives of the present study were several-fold. The basic approach was to select instances of traditional plant use, to examine the plants by biological assays for the presence of biologically active constituents and to attempt the isolation and identification of the active constituents. It was expected that this process would lead to conclusions on the general efficacy of the plant and provide some insight 1 2 into its use by a relatively primitive culture. Moreover, it was hoped that either the biological activity or the chemical structure (preferably both) of the active constituent(s) would be novel and that a contribution to basic science might be made. The first stage in this approach involved the selection of plants of ethnobotanical interest upon which to work. Many criteria came into play at this level. Preference was given to plants with well established and relatively clearly defined uses which were amenable to assay in the laboratory. The potential for obtaining new information was assessed. The plant's taxonomic position and ethnobotanical and chemical data concerning related plants was also used to assess the probability of active constituents being present. It was assumed that the likelihood of a plant containing such compounds would be greater in a plant belonging to a taxon: a) with many documented ethnobotanical uses b) which had not already been studied chemically The ethnobotanical data on South American plants has not been arranged systematically. Pertinent information is present in a wide variety of locations including the research notes and monographs of botanists, anthropologists and pharmacologists, the works of explorers, travellers and missionaries and herbarium labels. As much of this information as possible was collected and organized according to the classification system of Cronquist (1981). This is presented 1 3 as Appendix A. The information has been summarized, according to the distribution of species with ethnobotanical documentation at the order and family level, in Table I. It has also been arranged in Figure 1 according to Dahlgren (1980), whose system of classification consists of a two dimensional scheme of the orders of angiosperms. This has the advantage of allowing, not only the main phylogenetic relationships between the orders, but the relative size of each group, in numbers of species described, to be easily perceived. The information gathered is inherently subjective. It arises from the biases of the authors whose data was selected for compilation. However, combining the information in this way made the survey more extensive and provided a higher degree of verification of observations. It is evident from Table I and Figure T, that the Angiosperms are widely represented among the medicinal and poisonous plants known to indigenous Amazonians. The Class Magnoliopsida is, however, much better represented than the Class Liliopsida. Within the Magnoliopsida, the Subclasses Rosidae and Asteridae are especially rich in number of species with ethnobotanical uses. The families that are the most important, according to numbers of species used, are the Leguminosae, Apocynaceae, Euphorbiaceae, Rubiaceae and Solanaceae with 95, 53, 47, 46 and 45 species, respectively. It can be seen from Figure 1 that the number of species of ethnobotanical interest in each group is generally a 1 4 Table I - Phylogenetic distribution of species of Amazonian angiosperms having documented ethnobotanical use. Taxa are arranged according to the classification scheme of Cronquist (1981). Table I is a summary of Appendix A. Taxon Number of species with documented use Division: Maqnoliophyta 793 Class: Maqnoliopsida 727 Subclass I. Magnoliidae 1 04 Order: Magnoliales 42 Family: Annonaceae 1 2 Family: Myristicaceae 30 Order: Laurales 6 Family: Monimiaceae 2 Family: Lauraceae 4 Order: Piperales 29 Family: Piperaceae 29 Order: Aristolochiales 8 Family: Aristolochiaceae 8 Order: Ranunculales 18 Family: Menispermaceae 18 Order: Papaverales 1 Family: Papaveraceae 1 Subclass II. Hamamelidae 1 7 Order: Urticales 1 7 Family: Moraceae 10 Family: Cecropiaceae 7 Subclass III. Caryophyllidae 22 Order: Caryophyllales 20 Family: Phytolaccaceae 3 Family: Nyctaginaceae 2 Family: Cactaceae 4 Family: Chenopodiaceae 1 Family: Amaranthaceae 5 Family: Portulacaceae 3 Family: Basellaceae 1 Family: Caryophyllaceae 1 Order: Polygonales 2 Family: Polygonaceae 2 15 Table I(continued) Taxon No. spec ies Subclass IV. Dilleniidae 80 Order: Dilleniales 3 Family: Dilleniaceae 3 Order: Theales 18 Family: Caryocaraceae 4 Family: Marcgraviaceae 3 Family: Quiinaceae 1 Family: Clusiaceae 10 Order: Malvales' 19 Family: Tiliaceae 3 Family: Sterculiaceae 4 Family: Bombacaceae 2 Family: Malvaceae 10 Order: Violales 31 Family: Flacourtiaceae 1 4 Family: Bixaceae 3 Family: Violaceae 2 Family: Turneraceae 1 Family: Passifloraceae 1 Family: Cucurbitaceae 9 Family: Begoniaceae 1 Order: Salicales 1 Family: Salicaceae 1 Order: Ericales 3 Family: Ericaceae 3 Order: Primulales 5 Family: Theophrastaceae 1 Family: Myrsinaceae 4 Subclass V. Rosidae 230 Order: Rosales 4 Family: Connaraceae 4 Order: Fabales 95 Family: Leguminosae 95 Order: Myrtales 1 1 Family: Lythraceae 1 Family: Thymelaeaceae 3 Family: Onagraceae 1 Family: Melastomataceae 3 Family: Combretaceae 3 Order: Santalales 8 Family: Olacaceae 2 Family: Loranthaceae 5 Family: Balanophoraceae 1 16 Table I(continued) Taxon No. spec ies Order: Celastrales 5 Family: Celastraceae 2 Family: Icacinaceae 2 Family: Dichapetalaceae 1 Order: Euphorbiales 47 Family: Euphorbiaceae 47 Order: Linales 1 0 Family: Erythroxylaceae 2 Family: Humiriaceae 8 Order: Polygalales 22 Family: Malpighiaceae 1 7 Family: Vochysiaceae 5 Order: Sapindales 27 Family: Sapindaceae 6 Family: Burseraceae 5 Family: Anacardiaceae 6 Family: Simaroubaceae 3 Family: Meliaceae 2 Family: Rutaceae 5 Order: Geraniales 1 Family: Oxalidaceae 1 Subclass VI. Asteridae 247 Order: Gentianales 68 Family: Loganiaceae 8 Family: Gentianaceae 4 Family: Apocynaceae 53 Family: Asclepiadaceae 3 Order: Solanales 49 Family: Solanaceae 45 Family: Convolvulaceae 3 Family: Hydrophyllaceae 1 Order: Lamiales 29 Family: Boraginaceae 8 Family: Verbenaceae 8 Family: Lamiaceae 1 3 Order: Scrophulariales 44 Family: Scrophulariaceae 4 Family: Gesneriaceae 4 Family: Acanthaceae 18 Family: Pedaliaceae 1 Family: Bignoniaceae 1 7 Order: Campanulales 4 Family: Campanulaceae 4 Order: Rubiales 46 Family: Rubiaceae 46 Order: Asterales 1 7 Family: Asteraceae 1 7 1 7 Table I(continued) Class: Liliopsida 66 Subclass I: Alismatidae 2 Order: Alismatales 2 Family: Alismataceae 2 Subclass II: Arecidae 29 Order: Arecales 5 Family: Arecaceae 5 Order: Cyclanthales 1 Family: Cyclanthaceae 1 Order: Arales 23 Family: Araceae 23 Subclass III: Commelinidae 8 Order: Commelinales 1 Family: Commelinaceae 1 Order: Cyperales 7 Family: Cyperaceae 2 Family: Poaceae 5 Subclass IV: Zingiberidae 1 3 Order: Bromeliales 1 Family: Bromeliaceae 1 Order: Zingiberales 12 Family: Musaceae 4 Family: Zingiberaceae • 1 Family: Costaceae 4 Family: Cannaceae 1 Family: Marantaceae 2 Subclass V. Liliidae 1 4 Order: Liliales 8 Family: Pontederiaceae 1 Family: Liliaceae 1 Family: Iridaceae 2 Family: Agavaceae 1 Family: Smilacaceae 1 Family: Dioscoreaceae 2 Order: Orchidales 6 Family: Orchidaceae 6 Figure 1- Distribution of Amazonian angiosperms with documented ethnobotanical uses, arranged according to the classification scheme of Dahlgren (1980). Numbers refer to species that have at least one documented ethnobotanical use. Data are summarized from Appendix A. 19 reflection of the number of species in that group. A notable exception is the order, Orchidales, which seems not to be utilized in proportion to its size. This may be because the majority of its species are rare, small in size and not easily obtained. The broad perspective permitted by this ethnobotanical list played a significant role in my selection of ethnobotanical problems for investigation. The studies finally selected were investigations of the chemical and biological bases for the uses of the Justicia pectoralis and Vi rola elongata and a screening program of various biological activities of members of the family, Euphorbiaceae. Just ic ia pectorali s is an Acanthaceous herb used as an additive to the Vi rola based hallucinogenic snuffs of the Yanomamo. Its use in unacculturated tribes has been well documented by anthropologists and botanists. The reasons for its inclusion in the snuff, and also for its use alone as a snuff, however, have never been subjected to scientific scrutiny. In addition to this being a well identified ethnobotanical problem, other considerations indicated that its study might yield interesting results. The family, Acanthaceae, is moderately important ethnobotanically. Eighteen Amazonian species with ethnobotanical uses are known (Table I). The subclass, Asteridae, in which it is placed, \s very well represented in Table I. Related families (Rubiaceae, Asteraceae, Apocynaceae, Solanaceae and Bignoniaceae) are well known for their ability to produce complex biologically active 20 chemical constituents. A number of Asian species of Justic ia are used medicinally and a variety of compounds with interesting biological activities have been isolated. These are lignans, primarily of the bis-tetrahydrofuran class. They have recently been shown to display antidepressant action (Ghosal and Banerjee, 1979). Finally, the South American members of the family Acanthaceae are little known chemically. No reports of the genus, Justicia, from the Americas have been made. The use of Virola species as hallucinogens is also a well established phenomenon in the ethnobotanical literature. The dual uses of Virola elongata and Virola theiodora as both an arrow poison and an hallucinogen are especially intriguing. The family, Myristicaceae, is important in the Amazonian flora. It is moderately well utilized, ethnobotanically (30 species), and relatively well studied chemically. In addition to a number of psychotropic tryptamine and /3-carboline alkaloids, a wide variety of lignans and neolignans are also known (Gottlieb, 1979). Some of these possess interesting biological activities. The species, V. elongata, has been studied chemically only with respect to its indole alkaloids. The question of whether the presence of hallucinogenic alkaloids could explain the use of the bark resin as an arrow poison or if some other compound with toxic, or otherwise marked biological activity, was responsible was considered an especially interesting one. It was assumed to be readily amenable to experimental analysis. 21 Although the subject of the use and effects of hallucinogenic plant constituents is peripheral to the theme of this thesis, some clarification of the subject is in order. It is intriguing that geographically and culturally distinct groups of South American Indians adopted the custom of using materials containing indole alkaloids as ceremonial drugs. This is especially so when it is considered that at least four major groups of source plants are involved: Anadenanthera peregrina (Leguminosae), Virola spp. (Myristicaceae), Psychotria spp. (Rubiaceae) and Banisteriopsis spp. (Malpighiaceae). Many of the indole constituents have been examined pharmacologically and found to have a range of actions. Indole compounds of both the tryptamine and j3- carboline class have been detected in many of the plants of this group. These indole compounds comprise two of the seven classes of compounds distinguished as having hallucinogenic or psychotomimetic activity (Hollister, 1982). Although these terms overlap in meaning and are often used synonomously, the former refers to a subjective interpretation, the latter to an objective observation that the agent produces behavioral effects which mimic a psychosis. Problems are associated with use of either term. Hallucinogenic activity is largely a function of the subject, highly dependent upon dose and impossible to quantitate. Moreover, hallucinogenic activity is only one of the potential pharmacological effects that an indole compound may have. The other pharmacological actions 22 may also result in behavioral alterations. The precise nature of the biological effects of such compounds is therefore difficult to define. This is particularly true when animals are used as models to quantify the pharmacological response. The term, psychotropic, refers to the ability to alter mood or cognitive processes. As defined by Lewin (1927), it includes agents with hypnotic, inebriant, excitant, euphoriant and hallucinogenic activity. Because it is a more general term for describing agents with activity on the central nervous system, its use is appropriate when the nature of the biological activity under investigation is not completely defined (Hoffmeister and Stille, 1982). It is used in this thesis to describe the effects of the administration of 5- methoxy-N,N-dimethyltryptamine and extracts of J. pectoralis and V. elongata to mice for this reason. The third, and final, part of this research project was somewhat different in approach. While in Peru, I became aware of the importance of species of the family, Euphorbiaceae in folk medicine there. This is the third ranking family in terms of number of ethnobotanical uses (Table I). Its species are known to contain a great diversity of biologically active constituents. The most widely distributed compounds are probably diterpenes of the phorbol ester class. These possess a variety of biological activities, foremost among which are complex regulatory actions on mammalian tissues (Evans and Soper, 1978). The Amazonian species of Euphorbiaceae have been examined with respect to their chemical constituents or 23 pharmacological activity in only a very small number of cases. A significant proportion of the Euphorbiaceous species of ethnobotanical interest are used by the Indian or mestizo population o.f Peru in the treatment of wounds, ulcers, infections and cancerous conditions. Their mechanism of action may be hypothesized to involve either the inhibition of replication of microorganisms or viruses, or the regulation of some aspect of cellular proliferation. These biological activities are especially amenable to analysis by bioassay. A variety of Euphorbiaceous plants, some with documented ethnobotanical uses, others without, were examined for their ability to inhibit the replication of bacteria, yeasts, dermatophytic fungi, animal viruses and potato tumours and toxicity to brine shrimp. The aim of the study was to provide descriptive information on the biological activities of these little known species. It was hoped that the information obtained would allow some insight into the mechanism of action of the medicinally used species and also the relative likelihood of detecting activity in plants that did, and did not, have a documented ethnobotanical use. As in the case of the studies of J. pectoralis and Vi rola elongata, the identification of chemical constituents with novel biological activity was also an objective. The marked antiviral activity of an extract of Amanoa sp. was pursued and the compound responsible was identified. 24 These three projects: i) the study of J. pectoralis, ii) the analysis of Vi rola elongata resin, and iii) the screening of 34 species of Euphorbiaceae for biological activities and the subsequent analysis of the antiviral constituent of one of these, a species of Amanoa, are dealt with as independent chapters in this thesis. 25 LITERATURE CITED Chapter I_ Barclay, A.S. and Perdue, R.E. (1976) Distribution of anticancer activity in higher plants. Cancer Treat. Rep. 60, 1081-1113. Bruning, E.F. (1977) The tropical rain forest- a wasted asset or an essential biosphere resource? Ambio 6, 187-191. Browman, D.L. and Schwartz, R.A. (eds.) (1979) Spirits, Shamans and Stars: Perspectives from South America . Monton Publishers, The Hague. Bruhn, J.G. and Holmstedt, B. (1982) Ethnopharmacology: objectives, principles and perspectives. In: Natural Products as Medicinal Agents (Reinhard, E. and Beal, J.L., edsTT Hippokrates, Stuttgart, pp. 405-430. Chine, F.J., Jr. (1976) Witchcraft, the shaman and an active pharmacopoeia. In: Medical Anthropology (Grollig, F.X. and Haley, H.B., eds.) Mouton Publishers, the Hague, pp. 5-16. Costa, E. and Trabucchi, M. (eds.) (1978) The Endorphins . Raven Press, New York. Cronquist, A. (1981) An Integrated System of Classification of Flower ing Plants . Columbia University Press, New York. Dahlgren, R.M.T. (1980) A revised system of classification of the angiosperms. Bot. J. Linn. Soc. 80, 91-124. Denevan, W.M. (1980) Swiddens and cattle versus forest: the imminent demise of the Amazon rainforest reexamined. In: Where are all the Flowers Gone: Deforestation in the Third World , Studies in Third World Societies No. 13, pp. 25-44. Efron, D.H., Holmstedt, B. and Kline, N.S. (eds.) (1967) Ethnopharmacolacologic Search for Psychoactive Drugs . U.S. Gov't Printing Office, Washington, D.C, Public Health Service Publ. No. 1645. Eliade, M. (1964) Shamanism: Archaic Techniques of Ecstasy . Pantheon, New York. Evans, F.J. and Soper, CJ. (1978) The tigliane, daphnane and ingenane diterpenes, their chemistry, distribution and biological activities. A review. Lloydia 41, 193-233. Farnsworth, N.R. and Bingel, A.S. (1977) Problems and 26 prospects of discovering drugs from higher plants by pharmacological screening. In: New Natural Products and Plant Drugs with Pharmacological, Biological or Therapeutic Activity. (Wagner, ST and Wolff, P., eds.) Springer-Verlag, Berlin, pp. 1-22. Farnsworth, N.R. and Loub, W.D. (1982) Information gathering and data bases that are pertinent to furthering the economic potential of plants. Manuscript. Farnsworth, N.R. and Morris, D.J. (1976) Higher plants: the sleeping giants of drug development. Am. J. Pharm. 148, 46-52. Farnsworth, N.R., Bloomster, R.N., Quimby, M.W. and Schermerhorn, J.W., eds. (1974) The Lynn Index: A Bibliography of Phytochemistry . Norman R. Farnsworth Publ., U. of Illinois, Chicago, Illinois. Farnsworth, N.R., Loub, W.D., Soejarto, D.D., Cordell, G.A., Quinn, M.L. and Mulholland, K. (1981) Computer services for research on plants for fertility regulation. Korean J. Pharmacognosy 12, 98-110. Farnworth, F.B. and Golley, F.G. (eds.) (1974) Fragile Ecosystems: Evolution of Research and Application in the Neotropics , Springer-Verlag, New York. Fearnside, P.M. (1979) The development of the Amazon rain forest: priority problems for the formulation of guidelines. Interciencia 4, 220-223. Gentry, A.H. (1977) Endangered plant species and habitats of Ecuador and Amazonian Peru. In: Extinction is Forever (Prance, G.T and Elias, T.S., eds.) New York Botanical Garden, Bronx, New York, pp. 136-149. Ghosal, S. and Banerjee, S. (1979) Prostalidins A, B, C, and retrochinensin, a new antidepressant: 4~aryl-2,3- napthalide lignans from Justicia postrata . Chem. Ind. Dec. 854-855. Golley, F.G. and Medina, F.A. (eds.) (1975) Tropical Ecological Systems , Springer-Verlag, New York. Gomez-Pompa, A., Vasquez-Yanes, C. and Guevara, S. (1972) The tropical i;ain forest: a renewable resource. Science 1977, 762-765. Goodland, R.J.A. (1980) Environmental ranking of Amazonian development projects in Brazil. Environ. Conserv. 7, 9- 26. 27 Gottlieb, O.R. (1979) Chemical studies on medicinal Myristicaceae from Amazonia. J. Ethnopharmacol. 1, 309- 323. Goulding, M. (1980) The Fishes and the Forests: Explorations in Amazonian Natural History . University of California Press, Berkeley. Gran, G.L. (1973) The uteroactive principle of "kalata-kalata" ( Oldenlandia aff inis DC.) Dissertation, University of Bergen. Hegnauer, R. (1962) Chemotaxonomie der Pflanzen . Birkhauser Verlag, Basel. Hoffmeister, F. and Stille, G. (eds.) (1982) Psychotrophic Agents I . Antipsychotics and Ant idepressants. Handbook of Experimental Pharmacology, Vol 55/1 . Springer-Verlag, Berlin. Hollister, L.E. (1982) Pharmacology and toxicology of psychotomimetics. In: Psychotropic Agents III. (Hoffmeister, F. and Stille, G., eds!) Handbook of Experimental Pharmacology, Vol 55/111, Springer-Verlag, Ber1 in. Holmstedt, B. and Bruhn, J.G. (1983) Ethnopharmacology-a challenge. J. Ethnopharmacol. 8, 251-256. Karrer, W. (1958) Konstitution und Vorkommen der Organischen Pflanzenstoffe. Birkhauser Verlag, Basel. Lanly, J.P. and Clement, J. (1979) Present and Future Forest and Plantation Areas in the Tropics. FAO, Rome, Italy, FO:MISC,79,1. Lewin, L. (1931) Phantastica, Narcotic and Stimulating Drugs, Their Uses and Abuse . Routledge and Kegan Paul, London (translated from the second German edition, 1927). Luna, L.E. (1983) The concept of plants as teachers among four mestizo shamans of Iquitos, northeast Peru. Paper presented at the Symposium on Shamanism, Xlth International Congress of Anthropological and Ethnological Sciences, Phase 2, Vancouver, B.C., August 20-23, 1983. Meggers, B.J. (1971) Amazonia: Man and Culture in a Counterfeit Paradise . AHM Publishing Corporation, Arlington Heights, Illinois. Metraux, J. (1944) Le Shamanism chez les Indiens de l'Amerique du Sud tropicale. Acta Americana (Mexico) 11, 320-332. 28 Meyers, N. (1980a) Conversion of Tropical Moist Forests . Report to National Academy of Sciences, National Research Council, Washington, D.C. Meyers, N. (1980b) The present status and future prospects of tropical moist forests. Environmental Conservation 7, 101-114. Meyers, N. (1981) Deforestation in the tropics: who gains, who looses? In Where Have All the Flowers Gone 7. Deforestation in the Third World . Studies in Third World Series, Publ. No. 13, 1-21. Oldfield, M.L. (1981) Tropical deforestation and genetic resources conservation. In Blowing in the Wind; Deforestat ion and Long Range Implicat ions . Studies in Third World Societies, Publ. No. 14, 277-345. Persson, R. (1974) World Forest Resources . Royal college of Forestry, Stockholm, Sweden, Research Notes No. 17, iii. Persson, R. (1975) Forest Resources of Africa, Part I. Country Descriptions . Department of Forest Survey, Royal College of Forestry, Stockholm, Sweden, Research Notes No. 18. Persson, R. (1977) Forest Resources of Africa, Part II. Regional Analysis . Department of Survey, Royal College of Forestry, Stockholm, Sweden, Research Notes No. 22. Poore, M.E.D. (1976) Ecological Guidelines for Development in Tropical Rainforests . International Union for Conservation of Nature and Natural Resources, Morges, Switzerland. Raffauf, R.F. (1970) Some notes on the distribution of alkaloids in the Plant Kingdom. Econ. Bot. 24, 34-38. Raven, P.H., Berlin, B. and Breedlove, D.H. (1971) The origins of taxonomy. Science, 174, 1210-1213. Research Priorities in Tropical Biology (1980) Committee on Research Priorities in Tropical Biology, National Research Council, National Academy of Sciences, Washington, D.C. Richards, P.W. (1952) The Tropical Rainforest . Cambridge University Press, Cambridge. Richards, P.W. (1973) The Tropical Rainforest . Sci. Amer. 229, 58-68. Schultes, R.E. (1963) The widening panorama of medical botany. 29 Rhodora 65, 97-120. Schultes, R.E. and Swain, T. (1976) The plant kingdom: a virgin field for new biodynamic constituents. In: The Recent Chemistry of Natural Products, Including Tobacco (Fina, N.J., ed.) Proceedings of the Second Phillip Morris Science Symposium, New York, pp. 133-171. Shane, D.R. (1980) Hoofprints on the forest: an enquiry into the beef cattle industry in the tropical forest areas of Latin America. Report of Office of Environmental Affairs, Department of State, Washington, D.C. Sommer, A. (1976) Attempt at an assessment of the world's tropical moist forests. Unasylva 28, 5-24. Spjut, R.W. and Perdue, R.E. (1976) Plant folklore: a tool for predicting sources of antitumour activity among higher plants. Cancer Treat. Rep. 60, 979-985. Suffness, M. and Douros, J.D. (1982) Current status of NCI plant and animal program. J. Nat. Prod. 45, 1-14. Swain, T. (1972) The significance of comparative phytochemistry in medical botany. In: Plants in the Development of Modern Medicine (Swain T., ed.TT Harvard University Press, Cambridge, Mass. Tropical Deforestation (1980) Hearings before the Subcommittee on International Organizations, Committee of Foreign Affairs, House of Representatives, U.S. Govt. Printing Office, Washington, D.C. U.S. Interagency Task Force on Tropical Forests (1980) The World's Tropical Forests: A Pol icy, Strategy and Program for the United States . U.S. Department of State, Washington, D.C. UNESCO (1974) International Working Group on Project I. Ecological Effects of Increasing Human Activities on Tropical and Sub-tropical Forest Ecosystems . Final Report, Man and the Biosphere (MAB) Report Series No. 16, UNESCO, Paris, France. Weinstein, I.B., Wigler, M. and Pietropaolo,C. (1977) The action of tumour promoting agents in cell culture. Cold Spring Harbour Conference of Cell Proliferation, Vol. 4, Book A. Cold Spring Harbour, New York. Yip, Y.K., Pang, R.H.L., Urban, C. and Vilcek, J. (1981) Partial purification and characterization of human a (immune) interferon. Proc. Natl. Acad. Sci. USA 78, 1601- 1605. 30 II. Justicia pectoralis: A STUDY OF THE BASIS FOR ITS USE AS A Virola SNUFF ADMIXTURE 1. INTRODUCTION The widespread use of tryptamine based hallucinogenic snuffs in the Orinoco and northern Amazon drainage areas has been well documented during the past century (Wassen, 1967). Bark resin of species of Virola (Myristicaceae) was reported as one of the sources of these snuffs as early as 1909 (Koch- Griinberg, 1909) and their uses are now well established (Schultes and Holmstedt, 1968). Not until 1953, however, was it known that there existed other important constituents of Virola based snuffs. The missionary, Barker (1953), and the anthropologist, Zerries (1960), described the addition of an herbaceous additive to the pulverized resin snuff. This plant was later identified as Justicia pectoralis var. stenophylla (Acanthaceae) (Schultes and Holmstedt, 1968). The addition of its dry, pulverized leaves to Vi rola snuffs is now well established as a custom which is widespread amongst the tribes of the Yanomamo group (Brewer-Carias and 'Steyermark, 1976; Chagnon et al, 1971; Prance, 1972; Schultes, 1978; Seitz, 1967). The reason for the inclusion of J. pectoralis in Virola snuffs is not understood. Several Indian informants have suggested that it is added because of its aroma (Prance, 1972; 31 Schultes and Holmstedt, 1968; Seitz, 1967). There is evidence, however, that it may possess other properties (Schultes and Holmstedt, 1968). The reports that J. pectoralis constitutes the sole ingredient of a snuff and that it produces a state of intoxication (Brewer-Carias and Steyermark, 1976; Chagnon e_t al, 1971; Prance, G.T., pers. comm.) are especially interesting. The chemistry and pharmacology of the active constituents of Vi rola snuffs have been well studied (Holmstedt e_t al, 1980). In contrast, nothing is known of the chemistry of J. pectoralis. Moreover, no information on the chemical constituents of other American species of this genus appears to be available. This study of the possible physiological basis for the use of J. pectoralis was therefore undertaken. 2- MATERIALS AND METHODS a. Plant material The plant used in this study was collected in Pucallpa, Peru in 1981. It was identified as Just ic ia pectoralis var. stenophylla by Dr. Timothy Plowman of the Chicago Field Museum. Voucher specimens (D. McKenna No. 1) have been deposited at UBC herbarium, Chicago Field Museum, University of San Marcos herbarium, Lima and UNAP herbarium, Iquitos, Peru. It has been pointed out (Schultes, R.E., pers. comm.) that Just ic ia pectoralis var. stenophylla is probably a growth form of J. pectoralis and it is unlikely that the difference 32 in name used reflects a genetic difference. In keeping with Prof. Schultes suggestion, the varietal distinction is not considered in the subsequent report. Plants were propagated vegetatively and grown under greenhouse conditions. Freshly cut leaves, flowers and stems of mature plants (1150 g) were washed, placed in boiling 100% ethanol and homogenized. The homogenate was extracted four times and the filtrates were combined and concentrated by rotary evaporation i_n vacuo. Distilled water (500 ml) was added to the residue which was heated on a steam bath for 20 minutes and filtered through celite. The aqueous fraction was extracted continuously for 48 hours with diethyl ether, then ethyl acetate. The diethyl ether, ethyl acetate and aqueous fractions accounted for 0.931, 0.478 and 42.3 g, respectively, of the original 1150 g fresh weight, b. Chromatography and spectroscopy Thin layer chromatography (TLC) was carried out using Polygram Silica gel G UV25a (Brinkman) and cellulose (Eastman) precoated plates. High performance liquid chromatography (HPLC) separations were performed on a Varian MCH-10 reverse phase column and Varian Model 5000 HPLC with Varian Series 634 variable wavelength UV detector. Gas chromatography/mass spectrometry (GC/MS) data were obtained with a Finnegan 1020 automated GC/MS. Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker FT-100 instrument and infrared (IR) spectra were obtained using a Perkin Elmer 710B Infrared 33 Spectrophotometer. c. Behavior experiments Locomotor activity was measured in mice using an adapted version of a "jiggle cage" (Robbins, 1977). Female Swiss mice which had been bred and maintained by the Animal Care Centre, U.B.C. were used. The test substance was administered by intraperitoneal injection and the mouse placed in an 18 cm cubic wire mesh cage which was suspended from an isometric force transducer by elastic bands. The transducer signal was amplified 1000 times and monitored using a Spectrophysics SP 4100 integrator. A computer program which sampled the input voltage approximately every 100 milliseconds, calculated the difference between successive signals and summed the absolute value of that difference over a five minute period was written. This apparatus produced an average value for the degree of movement of the mouse, in arbitrary units, at five minute intervals. It was sensitive to both locomotor activity and to fine body movements, although the former produced a proportionately larger signal. All extracts and compounds were administered to.mice by intraperitoneal injection. Because of its ability to solubilize both hydrophilic and lipophilic constituents and its low toxicity (Budden et a_l, 1979), Tween 80 (Eastman Kodak Co.) was used as a vehicle for the substances administered. One hundred nl of a 25% (v/v) aqueous solution of Tween 80 was used in each case. d. Rat stomach strip experiments The procedure used for evaluating the activity of the 34 extracts on smooth muscle was based on that described by Vane (1957). Briefly, a male Wistar rat was sacrificed by a blow to the head, the stomach was removed and the fundic region cut into a 10 to 15 cm long by 1 to 2 mm wide strip as described by Vane (1957). The stomach strip was suspended vertically in a 5 ml capacity bath containing Kreb's solution (composition in g/1: NaCl = 6.9; KC1 = 0.35; CaCl2-2H20 = 0.36; MgSO„-7H20 = 0.29; KH2PO„ = 0.16; NaHC03 = 2.1; dextrose = 1.0) through which was bubbled continuously a mixture of 95% oxygen and 5% carbon dioxide. The lower end of the strip was fixed in position while the uppermost one was attached by a thread to the lever of a Harvard Isotonic Force Transducer (Model 363). A Harvard Apparatus Recorder (Model 350) and Harvard Chart. Recorder (Model 480) were used to record the length of the muscle strip. The muscle preparations were perfused continuously from below at a flow rate of 2.5 ml/min. Extracts, or compounds to be tested, were dissolved in Kreb's solution and used to perfuse the muscle strip for a period of 30 or 60 seconds. This was followed by a 5 to 10 minute period of perfusion with Kreb's solution during which the muscle returned to a stable length prior to beginning the next assay. e. Antimicrobial tests A simple paper disc, spot test was used to test for antimicrobial activity. Aqueous suspensions of Escherishia coli, Staphylococcus aureus, Candida albicans and Saccharomyces cerevisiae were spread on Difco Bacto agar and 35 Sabouraud's dextrose agar plates, respectively, using cotton swabs. Sterile filter paper discs, to which the extract to be tested had been applied, were placed on the agar surface. The plates were examined for inhibition of growth around the filter paper discs after 24 and 48 hours, f. Antiviral tests Extracts were tested for their ability to inhibit the formation of plaques by Sindbis virus and murine cytomegalovirus (MCMV) in cultured mouse cells. Extracts were included in the agarose overlay applied to virus infected cell monolayers and the plaques which developed were counted 2 and 5 days later for the Sindbis and MCMV, respectively. Complete details of the assay are presented in Chapter III. 3. RESULTS a. Examination of Justicia pectoralis for alkaloids The reports that J.' pectoralis is used, not only as an admixture to Vi rola, but as the sole ingredient of a snuff (Brewer-Carias and Steyermark, 1976; Chagnon et a_l, 1971; Prance, G.T., pers. comm.) suggest that this plant alone may have psychotropic or even hallucinogenic activity. The most potent psychotropic agents are alkaloids (Hoffmeister and Stille, 1982; Schultes, 1970). This fact, combined with preliminary evidence indicating that J. pectoralis may contain N,N-dimethyltryptamine (Schultes, 1970; Schultes, 1972) indicated that the pharmacological activity of J. pectoralis 36 may be attributable to the presence of indole or other classes of alkaloids. The three J. pectoralis extracts were examined for alkaloids by TLC. Samples were applied to Silica gel G plates, developed with ethyl ether/2-butanone/6 N NH4OH (5/4/1) (upper phase) and sprayed with Ehrlich's reagent (McKenna et al, 1984). N,N-Dimethyltryptamine, 5-OH-N,N-dimethyltryptamine, and 5-methoxy-N,N-dimethyltryptamine were included as standards. No Ehrlich's positive compound could be detected in any of the three extracts. Each extract was tested for the presence of alkaloids using Valser's, Meyer's and Dragendorff's reagents for precipitation (Martello and Farnsworth, 1962). Only the aqueous fraction was positive, yielding a precipitate with each of the reagents. This fraction was chromatographed on cellulose thin layer plates using n-propanol/0.3 N NHttOH (19/1) as a developer. A single spot with Rf value 0.46 was observed after spraying with Dragendorff's reagent. It produced a distinct salmon-pink colour when sprayed with the Bregoff-Delwiche modification of Dragendorff's reagent which is specific for quaternary ammonium compounds (Stahl, 1969) and caused the deposition of platinum, forming a gray spot with iodoplatinate reagent for alkaloids (Stahl, 1969). b. Compound J_ The compound (1) was purified by column chromatography on silica using n-propanol/0.3 N. NHi,OH (3/1) as eluant and was 37 crystallized from aqueous methanol. The following spectral data were obtained. Compound J_ (beta ine) UV X max11 : 215 nm. IR v max cm"1: 3250br, 2900, 1650, 1480, 1400, 1330, 980, 930 and 880. 1H-NMR (100 MHz, D20) 5 : 3.9 (2H, s), 3.31 (9H, s). The IR spectrum was identical to that reported for betaine (carboxymethyl-trimethylammonium hydroxide inner salt) (Poucert, 1981) and the 100 MHz 1H-NMR spectrum matched that of an authentic sample (Sigma). Compound 1 co-chromatographed with betaine both on cellulose TLC and by HPLC (retention time = 4.7 min. using 2% acetonitrile/H20 with a flow rate of 1 ml/min.). On the basis of this evidence, it was concluded that compound 1 was betaine(Figure 2). c. Behavioral ef fects of Just ic ia pectorali s The first approach to the ethnopharmacology of J. pectoralis established that psychotropic alkaloids did not account for the use of this plant as a snuff. This did not eliminate the possibility that a non-alkaloidal psychotropic compound might be present. To approach this question, behavioral changes in mice injected with J. pectoralis extracts were observed. A variety of behavioral changes are known to accompany the administration of psychotropic agents to rodents(Hoffmeister and Stille, 1982). Alterations in 38 Figure 2 - Structures of betaine (1), coumar in (2) and umbelliferone (3), compounds isolated from Just ic ia pectoralis. 39 spontaneous locomotor activity are commonly observed (Hollister, 1982). The aqueous, ethyl acetate and diethyl ether extracts of J. pectoralis were administered intraperitoneally to mice at a dose of 250 mg/kg. Behavioral changes were observed and locomotor activity was recorded. The results of the spontaneous locomotor recording are presented in Table II. Each extract caused a reduction in activity. Mice adopted a huddled posture, often with eyes closed, and displayed piloerection. The recording of activity level provides a quantitative estimate of the reduction in activity. The ethyl acetate fraction possessed the strongest of the relatively weak activities of the three extracts. None of the responses observed were indicative of the presence of psychotropic activity. d. Ef fect of Just ic ia pectoralis extracts on 5-MeODMT induced behavioral responses Since no evidence was obtained to indicate that J. pectoralis, alone, has psychotropic activity, its role when used in combination with Vi rola snuff was considered. There is strong evidence that the use of Virola based snuffs is based on the hallucinogenic activity of their indole constituents. N,N-Dimethyltryptamine (DMT) and 5-methoxy-N,N- dimethyltryptamine (5-MeODMT) are, quantitatively, the most important indoles in most Vi rola barks (Agurell et al, 1969; Holmstedt et al, 1980; McKenna et al, 1984). At least one of these two constituents is known from all of the Vi rola species used in the manufacture of snuffs and their presence in the 40 % normal activity leveKmin. post injection) Extract t 5 1 0 1 5 20 25 30 35 40 45 50 55 60 Injected aqueous 93 91 1 04 92 95 86 85 88 77 79 75 77 ethyl 96 1 07 101 64 52 61 58 44 53 57 51 59 acetate diethyl 92 94 98 91 94 87 83 88 77 82 75 88 ether 25% Tween 80 91 96 1 1 0 1 04 92 94 109 1 1 2 95 99 1 1 4 91 ( 1 00 /il) Table II - Effect of Just ic ia pectoralis extracts on spontaneous locomotor activity in mice. f Extracts were injected intraperitoneally at doses of 250 mg/kg dissolved in 100 M1 of 25% Tween 80. 41 snuffs themselves is well documented (Agurell et_ aj^, 1969; Holmstedt and Lindgren, 1967). Five-MeODMT is approximately ten times aspotent a psychotomimetic as DMT (Shulgin, 1982). It would, therefore, be expected to account for most of the pharmacological effects of the snuffs. This tryptamine was chosen as a model compound for co-administration studies designed to test the hypothesis that some component of J. pectoralis was responsible for modulating the effects of the tryptamines. Five-MeODMT is known to cause a number of characteristic behavioral changes in rats injected intraperitoneally. Doses of 1 mg/kg produce excitation, hyperactivity, tremor and some salivation (Ahlborg et al., 1968; Grahame-Smith, 1971). The tremor produced by this compound is dose-dependent in the range 1-10 mg/kg (Ahlborg e_t a_l, 1968). Changes in activity levels produced in rats by 5-MeODMT (Ahlborg et al, 1968; Grahame-Smith, 1971) and in mice by DMT (Shah and Hedden, 1978) have been quantified in devices for measuring animal activity levels. The responses have been shown to be reproducible and to vary in a dose-dependent manner. This method was therefore chosen to examine the possibility that J. pectoralis contains some constituent that alters the behavioral response to 5-MeODMT. e. Effect of 5-MeODMT on mouse activity Solutions of 5-MeODMT (Sigma) were dissolved in 2% aqueous Tween 80 (100 MD and administered intraperitoneally. Activity levels were monitored at 5 minute intervals for 60 42 minutes after injection. The results presented in Figure 3 show that all doses of 5-MeODMT tested (0.31 to 12.5 mg/kg) produced an initial period of hyperactivity lasting 10 to 20 minutes. The degree of hyperactivity varied in a dose- dependent manner. In the case of a low (0.31 mg/kg) or a high (2.5 or 12.5 mg/kg) dose of 5-MeODMT, the activity levels returned almost to normal after 30 minutes post injection. Administration of intermediate doses (1.25 and 0.625 mg/kg; data not shown), in contrast, resulted in significant reductions in activity which persisted until at least 60 minutes post injection. Ahlborg et a_l (1968) and Grahame-Smith (1971) observed very similar increases in activity in rats injected with 3 and 1 mg/kg of 5-MeODMT, respectively. The reduction in activity observed in the present study (50% normal activity level after 1.25 mg/kg and 70% normal activity level after 0.625 mg/kg 5-MeODMT; data not shown) have not previously been reported as an effect of 5-MeODMT administration. Dimethyltryptamine, however, has been shown to produce a similar pattern of gradually decreasing activity levels following the administration of 2.5 and 25 mg/kg (Grahame-Smith, 1971). The reduction in activity was observed by them to last for approximately 60 and 120 minutes, respectively. The results presented here indicate that low doses of 5-MeODMT produce a similar reduction in motor activity but that, at high doses, activity levels increase markedly (3-fold at 12.5 mg/kg). 43 "1 1 1 1 1 T 10 20 30 i.0 50 60 TIME AFTER INJECTlON(minutes) Figure 3 - Effect of 5-MeODMT on spontaneous locomotor activity of mice. Mice were injected with 5-MeODMT at time 0 and locomotor activity was recorded during five minute intervals thereafter. Administration of vehicle alone (control) is not shown: maximum deviation from normal activity level observed was 22%. Values are means, N=3; SEMs are not drawn but were all less than 5%. 44 f. Gross behavioral effects of 5-MeODMT Changes in behavior induced by 5-MeODMT correlate well with the effects on motor activity presented in Figure 3. A dose of 0.31 mg/kg resulted in an initial period of hyperactivity lasting approximately 10 minutes, followed by a period of roughly 60 minutes during which the animal lay quietly, displaying a flattened posture. As Shah and Hedden (1978) reported for DMT, fright responses were elicited when the animal was disturbed. At still higher doses (2.5 and 12.5 mg/kg), the mice adopted an extremely flattened posture with extension of the hindlegs and showed a series of multiple sudden backwards movements. This behavior was periodically and briefly interrupted when the animals crouched and pawed their noses. Gross locomotor activity was reduced but the number of fine body movements were greatly increased, compared to an untreated animal. Jerkiness, head twitching, rigidity and trembling, characteristically produced in mice by DMT (Shah and Hedden, 1978), were also observed. After 15-20 minutes post injection, all of these behavioral effects gradually began to be reduced in frequency and severity. Normal activity levels were slowly regained. g. Effect of co-injections of 5-MeODMT and Justicia pectoralis extracts A dose of 5-MeODMT which would produce a marked elevation in activity level was selected to examine the J. pectoralis extracts for synergistic effects. Injections of 5 mg/kg 5- 45 MeODMT, combined with 250 mg/kg of each of the three Just ic ia extracts, were made and the activity levels recorded. The results are presented in Table III. Despite the fact that alone, each extract caused slight reductions in activity level, there was no significant effect of any of the Justicia extracts upon 5-MeODMT induced hyperactivity. The observations of the gross behavioral responses were in agreement with the locomotor measurements. All of the previously mentioned behavioral effects of high doses of 5-MeODMT were observed after administration of 5 mg/kg 5-MeODMT, either alone, or in combination with any of the three Just ic ia extracts. From this series of experiments it is concluded that the J. pectoralis extracts do not contain any constituent with a pharmacological activity that is comparable to-that of the tryptamine hallucinogens. Nor do they contain compounds which are responsible for modulating the most obvious behavioral changes induced by 5-MeODMT. Constituents which inhibit the enzyme, monoamine oxidase (Bhattacharya ejb a_l, 1976; Holmstedt et al, 1980) and mixed function oxidases (Brattsten, 1979) are known to occur in higher plants. Although the existing data indicate that the former is the most important enzyme system for converting tryptamines to inactive metabolites (Grahame- Smith, 1971), there is evidence to suggest that the latter also plays a role (Ahlborg, 1968). Plant constituents with the ability to inhibit either enzyme system could possibly potentiate the physiological activity of tryptamines. The results obtained in the behavior experiments do not support 46 Per cent Normal t Spontaneous Locomotor Activity (Minutes Post Injection) Sample Injected 5 1 0 1 5 20 25 30 5 mg/kg 5-MeODMT + 250 1 76 204 204 1 28 1 08 1 05 mg/kg aqueous extract 5 mg/kg 5-MeODMT + 250 1 55 194 204 126 101 95 mg/kg ethyl acetate ext. 5 mg/kg 5-MeODMT + 250 1 64 198 215 1 34 1 18 1 04 mg/kg ethyl ether ext. 5 mg/kg 5-MeODMT 1 70 214 217 121 1 09 1 08 100 M1 25% Tween 80 +• 92 104 100 95 107 101 Table III - Effect of co-administration of 5-MeODMT and Just ic ia pectoralis extract on spontaneous motor activity of mice. t Normal spontaneous locomotor activity was established for each mouse by measuring activity for 10 minutes prior to injection. +• Control. 47 this hypothesis for J. pectoralis. Any increase in the half life of 5-MeODMT would be expected to be manifested in the behavioral responses observed. Neither the magnitude nor the duration of 5-MeODMT induced behavioral changes was affected by any of the three J. pectoralis extracts, h. Effeet of J. pectoralis extracts on smooth muscle Dimethyltryptamine and other tryptamine derivatives are known to antagonize the action of 5-hydroxytryptamine (serotonin) on smooth muscle (Barlow and Khan, 1959). This pharmacological activity correlates with hallucinogenic activity, suggesting that serotonin receptor affinity is central to the mechanism of action of these agents as hallucinogens (Glennon et_ al, 1980). The possibility that J. pectoralis could exert some subtle behavioral effect by either acting directly on the serotonin receptors or by competing for them with tryptamines, was considered. The rat stomach strip assay for serotonin receptors afforded a relatively easy means of testing this hypothesis. A strip of smooth muscle prepared from the fundic region of a rat stomach responds to low concentrations of 5- hydroxytryptamine by contracting (Vane, 1957). Figure 4a shows a tracing of the length of such a rat stomach strip (RSS) exposed to 1 ng/ml 5-hydroxytryptamine (serotonin). Within two minutes of treatment with serotonin, the muscle reaches its minimum length (corresponding to a 2% reduction in total length). The muscle relaxes completely within two minutes of removal of serotonin. This response is quite reproducible and 48 Figure 4 - Effect of (a) serotonin (ing/ml) and (b) betaine (50 yg/ml) on smooth muscle. Increase in elevation of tracing corresponds to contraction muscle. Interval of perfusion of compound is delimited arrows: chart speed= 6 mm/min: amplification= 16X. 49 the degree of contraction varies in a dose-dependent manner. Muscle preparations were perfused with 1 ng/ml 5- hydroxytryptamine combined with one of the three J. pectoralis extracts at a concentration of .1 mg/ml. The ether extract completely eliminated the 5- hydroxytryptamine induced contraction and produced a marked relaxation of the muscle. When the RSS was exposed to the ether extract alone, however, a similar relaxation was observed. In contrast, the aqueous extract alone elicited a strong and long lasting contraction at a concentration of 1 mg/ml. The spontaneous activities observed in the ether and aqueous fractions rendered an examination of these extracts for specific 5-hydroxytryptamine antagonism impractical. The ethyl acetate fraction produced no effect on the response of the RSS to 5-hydroxytryptamine. A knowledge of the nature of the constituents responsible for the activity on the smooth muscle preparations may shed light on the pharmacological activity of J. pectoralis. The aqueous and diethyl ether fractions were fractionated further in an attempt to isolate and identify the active compounds. Aqueous fraction The aqueous fraction was fractionated as described in section 2a. It was observed that contraction inducing activity was retained only by those fractions containing the quaternary ammonium compound, betaine, which had already been identified because of its reaction with reagents for alkaloid detection. 50 A commercial sample of betaine hydrochloride (Sigma) was assayed for activity on the RSS. It induced a strong and prolonged contraction. This effect was observed at relatively high concentrations (100 Mg/ml) (Figure 4b). Contractions were elicited at concentrations as low as 10 Mg/ml, but at levels lower than this, the contractions observed were slight and not reproducible. The concentration of betaine present in the aqueous extract of J. pectoralis was estimated, by comparison of TLC spot size with measured amounts of pure betaine, to be 2.4%. All of the smooth muscle contraction induced by the aqueous extract was attributable to the presence of this amount of betaine. Ether fraction An aliquot of the ether fraction was fractionated by column chromatography on silica using a chloroform/methanol gradient as the eluate. Smooth muscle relaxing (spasmolytic) activity was observed to be concentrated in two fractions. Crystalline compounds were obtained from these fractions. When applied to the RSS, each compound, at concentrations between 10 and 100 Mg/ml, caused a marked increase in the length of the muscle. The RSS rapidly returned to its resting length following the removal of the compounds from the medium (Figures 5a and b). The following spectral data were obtained for the two compounds isolated from the diethyl ether fraction. 51 Compound 2 (coumarin). Mp 67-69 °C. UV X max nm (log e): 310 (3.70), 273 (3.70). MS m/z (rel.int): 146[M+] (72), 118 (100), 90 (52), 89 (53), 64 (12), 63 (41), 62 (16), 51 (13), 50 (12). MeOH Compound 3 (umbelliferone). Mp 223-225 °C. UV Xmax nm (log 1 e): 325 (4.22), 254 (sh), 242 (sh). IR v ^x cm" : 3200 br, 1720, 1620, 1575, 1420, 1330, 1240, 1150, 910, 850. 1H- NMR (100 MHz, acetone-D-6) 5 : 7.86 (IH, d, J = 12 Hz, H-4), 7.52 (1H, d, J = 8 Hz, H-5), 6.82 (2H, m, H-6 and H-8). MS m/z (rel int): 162[M+] (67), 134 (74), 105 (39), 97 (18), 95 (14), 91 (14), 85 (13), 83 (14), 78 (35), 69 (38), 57 (32), 55 (40), 51 (32), 43 (100). On the basis of the melting points and the spectral data obtained, Compounds 2 and 3 were identified as coumarin and 7- hydroxycoumarin (umbelliferone), respectively (Figure 2). They co-chromatographed with authentic standards on silica gel (toluene/acetone, 1/1) and by high performance liquid chromatography (50% aqueous acetonitrile), confirming their identity. i. Analysis of aromatic constituents A possible explanation for the inclusion of J. pectoralis in Virola snuff is that it contributes a desirable aroma (Prance, 1972; Schultes and Holmstedt, 1968; Seitz, 1967). A fragrant nature is indicated by one of its common names, "Jamaica garden balsam" (Schultes and Holmstedt, 1968). To Figure 5 - Effect of (a) coumarin (10 Mg/ml) and (b) umbel1iferone (10 ug/ml) on smooth muscle. Interval of perfusion of compounds is delimited by arrows: chart speed= 6 mm/min: amplification= 16X. 53 establish the nature of the volatile constituents responsible for its aroma, samples of fresh and dried leaves and stems of J. pectoralis were subjected to steam distillation for 2 hours. The distillate was analyzed by GC/MS. In the case of each sample, only a single constituent was present. This was identified, by its mass spectrum, as coumarin. The aroma of J. pectoralis appears to be a result of the single constituent, coumarin. j. Quanti f ication of coumarins of J. pectoralis The ether extract contained, as primary constituents, coumarin and umbelliferone. To provide quantitative information on the presence of these two important metabolites, various samples of J. pectoralis were analyzed by HPLC. Samples of young, green leaves, mature, pigmented leaves and flowers were extracted exhaustively with methanol. A sample of leaves collected from the plant while it was growing in Peru was also analysed. The methanolic extracts were evaporated ir\ vacuo and partitioned between water and dichloromethane. The latter fraction was evaporated, dissolved in methanol and analysed by HPLC. The sample was separated using 50% aqueous methanol (flow rate = 1ml/min) and the absorbance was monitored at 250 nm. The leaf extracts contained as major UV absorbing constituents, coumarin and umbelliferone (Figure 6). Samples were quantified by comparison with peak heights of chromatograms of authentic samples of coumarin (J.T. Baker Company) and umbelliferone (K and K Laboratories, Ltd.). The 54 Figure 6 - HPLC chromatogram of the organic fraction (methanolic extract) of Just ic ia pectorali s . Flow rate Iml/min, isocratic, 50% MeOH, detection at 254 nm, chart speed= 0.5 cm/min. Compound with retention time of 5.91 is umbelliferone; that with retention time of 8.62 is coumarin. 55 results are presented in Table IV. The importance of coumarin and umbelliferone as secondary metabolites of this species of Justicia is evident. Not only are they present at concentrations several orders of magnitude higher than the other UV absorbing compounds of the extract, but they appear, from this limited sampling, to be"sequestered in the plant and reach higher levels in more mature leaves, k. Examination of J. pectoralis for lignans Of the seven species of Just ic ia that have previously been analysed chemically, six are known to contain lignans as major constituents (Ghosal and Banerjee, 1979; Ghosal e_t al, 1980; Munakata et al, 1965; Ohta and Munakata, 1970; Okigawa et al, 1970; Olaniyi, 1982; Olaniyi and Powell, 1980). All but one of the 21 lignans identified from Just ic ia species contain" the methylenedioxyphenyl moiety. To determine whether lignans containing this moiety were present in J. pectoralis, the chromotrophic acid spray reagent described by Beroza (1963) was used to examine TLC separations of the three Justic ia fractions. Only one compound, present in the diethyl ether fraction, reacted with this reagent. It was isolated and spectral data were obtained. Its melting point (132-135 °C), 'H-NMR, UV and mass spectrum were identical to those obtained for an authentic sample of /3-sitosterol (Sigma). Furthermore, the behavior of the unknown compound on two chromatographic systems ( TLC: Rf=0.45 on Silica gel developed with chloroform/acetone (95/5) and HPLC: Retention time = 6.10 min eluted with 25% aqueous methanol, flow rate = 1 ml/min.) was 56 Concentration in Per Cent Dry Weight t Sample Analyzed Coumar in Umbelli ferone flowers N.D. t- N.D. young leaves 0.42(0.04) 0.30(0.04) mature leaves 1 . 18(0.08) 0.58(0.05) leaves(Peru) 0.93(0.05) 0.44(0.04) T Table IV - Levels of coumarin and umbelliferone in different samples of Just ic ia pectoralis. t Figures are the average of three determinations; standard deviation of mean in brackets. $ N.D. = Not Detectable 57 identical to that of an authentic sample of 0-sitosterol (S i gma). The diethyl ether and ethyl acetate fractions of J. pectoralis were examined for the presence of justicidin B and diphyllin, lignans known from several other Just ic ia species. Samples were chromatographed on Silica gel G using toluene/acetone (1/1) as developer. Diphyllin and justicidin B (gifts of Dr. G.H. Sheriha, El Fateh University) had Rf values of 0.57 and 0.79, respectively. Both displayed a strong blue fluorescence and reacted with chromotrophic acid reagent. No evidence for the presence of either compound in the J. pectoralis extracts was obtained. The chemistry of J. pectorali s appears to be quite different from that of any of the other species in this genus that have been examined. The apparent absence of lignans is of note. This difference may reflect phylogenetic distance since all of the species previously studied are native to Asia or Africa. 1. Screening for other biological activities Although J. pectoralis is best known as a snuff admixture, there is considerable ethnobotanical information indicating that it has multiple uses. In the Colombian states of Vaupes and Amazonas, it is known to the Puinave Indians as yakayu and is used by them in the form of a decoction to treat pulmonary infections and pneumonia (Schultes, 1978). It has also been said to be effective in the treatment of infections of the nasal cavity (Maxwell,N., pers. comm.). An herbarium 58 specimen collected by Jose J. Triana is accompanied by an herbarium label stating that it is widely used in Meta, Colombia as a decoction to treat children afflicted with rickets (Schultes, 1978). Just ic ia pectorali s is a relatively well known medicinal plant in Central America and the Caribbean (Morton, 1977). It is used as a pectoral tea to relieve coughs (Wong, 1976), an expectorant (Roig y Mesa, 1945) and as a wound poultice (Stehle and Stehle, 1962). It has also been reported to have sudorific and aphrodisiac effects (Morton, 1977). It is possible that the use of J. pectoralis in the treatment of chest infections or wounds is dependent upon antimicrobial activity. This hypothesis was examined by testing extracts of the plant for inhibitory activity against a variety of microorganisms. The three fractions were tested against the gram positive bacterium, Staphylococcus aureus, the gram negative bacterium, Escherishia coli, the yeasts, Candida albicans and Saccharomyces cerevisiae and the dermatophytic fungi, Microsporum canis, M. gypseum, M. fulvum and Trichophyton gallinae. None of the extracts had any effect upon the growth of the bacteria or yeasts tested. The diethyl ether extract, however, exerted a strong inhibitory effect upon the growth of each of the dermatophytic fungi. Their growth was completely inhibited at a concentration of 1 Mg/ml. Coumarin and umbelliferone were tested separately on these fungi. No inhibitory activity was observed at doses as high as 59 50 Mg/ml, indicating that the antifungal activity of the diethyl ether extract is not attributable to either of these major constituents. The three extracts of J. pectoralis were also tested for their ability to inhibit the replication of murine cytomegalovirus and Sindbis virus (methods described in chapter III). No effects were observed from any of the extracts at concentrations as high as 10 Mg/ml. 4. DISCUSSION No evidence, from either the chemical or the biological studies, that the samples of J. pectoralis analysed possessed chemical constituents capable of eliciting hallucinogenic activity, was found. Nor did J. pectoralis extracts have any detectible effects upon the behavioral responses of mice to 5- MeODMT. Three compounds with biological activity were isolated and identified: betaine, coumarin and umbelliferone. Betaine is widely distributed in both plants and animals (Guggenheim, 1951). Its only known function is as a source of methyl groups for the remethylation of.homocysteine to methionine via the enzyme betaine-homocysteine methyl transferase (Shapiro and Schlenk, 1965). Few studies of a pharmacological nature have been published concerning betaine. The first report was that of Hunt and Renshaw (1929) who examined the effects of a series of derivatives of betaine on the autonomic nervous system. Most derivatives were observed to have a muscarinic effect; i.e., to reduce blood pressure 60 and heart rate. Their consideration of the parent compound, betaine, is brief and restricted to the statement that it is "practically without action on the autonomic nervous system." A fraction of an extract of the plant, Pluchea lanceolata (Compositae), containing betaine as a major constituent, has been reported to induce contractions in isolated smooth muscle preparations (Dasgupta, 1967; Dasgupta et_ a_l, 1968; Prasad et al, 1965). Furthermore, the extract potentiated barbiturate induced hypnosis in rats (Prasad e_t a_l, 1965) and possessed anti-inflammatory activity (Prasad e_t a_l, 1966). The smooth muscle contraction inducing effect of the crude extract is in close agreement with the results presented in the present study on pure betaine. To what extent the other properties of the crude extract can be attributed to betaine is not known. More recently, it has been reported that betaine is a non• specific anti-convulsive agent (Freed e_t §_1, 1979). At high doses, it is capable of blocking convulsions induced by electric shock as well as chemical convulsants. Coumarin and umbelliferone, like betaine, are distributed widely. They are present in bacteria, fungi and a broad range of plants (Karrer, 1958). A smooth muscle relaxing, or spasmolytic, action has been reported previously for coumarin (Patyra, 1963a and b) as well as umbelliferone (Achterrath- Tuckermann et al, 1980) and this appears to be a feature that is common to many coumarin derivatives (Lee and Fujiwara, 1971; Ojewole and Adesina, 1983a and b). This general spasmolytic action, which affects many smooth muscles, 61 apparently results in a lowering of heart rate and blood pressure in animals treated with the coumarin, scopoletin (Ojewole and Adesina, 1983a and 1983b). Coumarin, and some of its derivatives, possess the ability to reduce body temperature (Kitagawa and Iwaki, 1958). At doses approaching the toxic level, coumarin has sedative and hypnotic activity (Ito et al, 1951; Kitagawa, 1956a and b; Kreitmair, 1949). Both of these actions could possibly be related to the ability of these compounds to inhibit mitochondrial respiration (Kitagawa, 1956a; Pai e_t al, 1975). Coumarin is also effective as an anti-inflammatory agent, acting on macrophages to stimulate phagocytosis (Koh and Willoughby, 1979). Finally, coumarin is a volatile and sweetly aromatic compound and has many commercial applications as an aromatic additive (Kirk and Othmer, 1979). It is the sole ingredient responsible for the aroma of J. pectoralis. The contribution of these varied biological activities to the overall effect of J. pectoralis snuff is not clear. The sedative and hypnotic activity observed for coumarin is interesting in this respect. Administered parenterally to small animals, it produces sedation and analgesia at doses of 50 mg/kg. Whereas it is not reasonable to expect that such high doses may be achieved during snuff taking, it is possible that localized regions of the head may be exposed to pharmacologically active levels as a result of its absorption across the nasal mucosa. Local analgesia or sedative/hypnotic effects could possibly be induced at lower doses than if 62 administered parenterally. The ability of scopoletin to reduce blood pressure (Ojewole and Adesina, 1983b) is also of interest. Tryptamine admininistration is often accompanied by transient increases in blood pressure (Szara, 1956). It is not known whether this effect is a direct one or whether it is mediated by psychological changes. The possibility that coumarin or umbelliferone could lower blood pressure and thereby reduce one of the stressful aspects of the tryptamines, is intriguing. In conclusion, no evidence to support the belief that J. pectoralis is a hallucinogenic plant was obtained. Nor does its administration to m„ice appear to affect directly the action of 5-meODMT, the hallucinogenic tryptamine of Vi rola, with which it is combined as a snuff. The possibility that coumarin and umbelliferone contribute to the pharmacological effects of the snuff has been raised. 63 LITERATURE CITED Chapter 11 Achterrath-Tuckermann, U., Kunde, R., Flaskamp, E., Isaac, 0. and Thiemar, K. (1980) Pharmacologic investigations of chamomile. V. Investigations on the spasmolytic effects of compounds of chamomile and kamillosan on the isolated guinea pig ileum. Planta Med. 39, 38-50. Agurell, S., Holmstedt, B., Lindgren, J.E. and Schultes, R.E. (1969) Alkaloids in certain species of Virola and other South American plants of ethnopharmacologic interest. Acta. Chem. Scand. 23, 903-916. Ahlborg, U., Holmstedt, B. and Lindgren, J.E. (1968) Fate and metabolism of some hallucinogenic indolealkylamines. Adv. Pharmacol. 6B, 213-229. Barker, J. (1953) Memoria sobre la cultura de los Guaika. Bol. Indog. Venez. 1, 433-489. Barlow, R.B. and Khan, I. (1959). Actions of some analogues of 5-hydroxytryptamine on the isolated rat uterus and. the rat fundus strip preparations. Brit. J. Pharmacol. 14, 265-272. Beroza, M. (1963) Identification of 3,4-methylenedioxyphenyl synergists by thin layer chromatography. Agr. Food Chem. 11, 51-54. Bhattacharya, S.K., Reddy, P.K.S.P., Ghosal, S., Singh, A.K. and Sharma, P.V. (1976) Chemical constituents of Gentianaceae. XIX: CNS-depressant effects of swertiamerin. J. Pharm. Sci. 65, 1547-1551. Brattsten, L.B. (1979) Biochemical defense mechanisms in herbivores against plant allelochemicals. In: Herbivores, their Interaction with Secondary Metabolites (Rosenthal, G.A. and Janzen, D,H., eds.) Academic Press, New York, pp. 199-270. Brewer-Carias, C. and Steyermark, J.A. (1976) Hallucinogenic snuff drugs of the Yanomamo Caburiwe-Teri in the Cauaburi River, Brazil. Econ. Bot. 30, 57-66. Budden, R., Kuhl, U.G. and Bahlsen, J. (1979) Experiments on the toxic, sedative and muscle relaxant potency of various drug solvents in mice. Pharmac. Ther. 5, 467-474. Chagnon, N.A., LeQuesne, P. and Cook, J.M. (1971) Yanomamo hallucinogens: anthropological, botanical and chemical findings. Curr. Anthopol. 12, 72-74. 64 Dasgupta, B. (1967) Chemical investigation of Pluchea lanceolata I. Isolation of a new quaternary base, pluchine. Experientia 15, 989-991. Dasgupta, B., Bas, K. and Dasgupta, S. (1968). Chemical investigation of Pluchea lanceolata II. Identity of pluchine with betaine hydrochloride. Experientia 24, 882- 883. Freed, W.J.. Gillin, J.C. and Wyatt, R.J. (1979) Anticonvulsant properties of betaine. Epilepsia 20, 209- 213. Ghosal, S. and Banerjee, S. (1979) Prostalidins A, B, C and retrochinensin, a new antidepressant; 4-aryl-2,3- napthalide lignans from Just ic ia prostrata. Chem. Ind., Dec, 854-855. Ghosal, S., Banerjee, S. and Jaiswal, D.K. (1980) New furofurano lignans from Just ic ia simplex. Phytochem. 18, 503-505. Glennon, R.A., Young, R., Rosecranz, J.A. and Kallman, M.J. (1980) Hallucinogenic agents as discriminative stimuli: a correlation with serotonin receptor affinities. Psychopharm. 68, 155-158. Grahame-Smith, D.G. (1971) Inhibitory effect of chlorpromazine on the syndrome of hyperactivity produced by L-tryptophan or 5-methoxy-N,N,-dimethyltryptamine in rats treated with a monoamine oxidase inhibitor. Br. J. Pharmacol. 43, 856- 864. Guggenheim, M. (1951) Die Bioqenen Amine , 4th Edition, S. Karger, Basel. Hoffmeister, F. and Stille, G. (eds.) (1982) Psychotropic agents I. Antipsychotics and Antidepressants . Handbook of Experimental Pharmacology, Vol 55/1. Springer-Verlag, Berlin. Hollister, L.E. (1982) Pharmacology and toxicology of psychotomimetics. In: Psychotropic Agents, Handbook of Experimental Pharmacology , Vol. 55, Part III (Hoffmeister, F. and Stille, G., eds) Springer-Verlag, Berlin, pp. 31-44. Holmstedt, B. and Lindgren, J.E. (1967) Chemical constituents and pharmacology of South American snuffs. In: Ethnopharmacolacologic Search for Psychoactive Drugs (Efron, D.H., ed.) U.S. Health Serv. Publ. No 1645, 339- 373. 65 Holmstedt, B., Lindgren, J.E., Plowman, T., Rivier, L., Schultes, R.E. and Tovar, 0. (1980) Indole alkaloids in Amazonian Myristicaceae: field and laboratory research. Bot. Mus. Leafl. Harv. Univ. 28, 215-234. Hunt, R. and Renshaw, R.R. (1929) Some effects of betaine esters and analogous compounds on the autonomic nervous system. J. Pharm. Exper. Therap. 29, 17-34. Ito, Y., Kitagawa, H., Hiramori, T., Suzuki, Y. and Yamagata, M. (1951) Coumarin derivatives for medicinal purposes. II. Toxicity of coumarin, 2-thiocoumarin and of 4- methylcoumarin. J. Pharm. Soc. Japan 71, 686-689. Karrer, W. (1958) Konstitution und Varkommen der Organischen Pflanzenstoffe . Birkenhauser Verlag, Basel. Kirk, R.E. and Othmer, D.F. (eds) (1979) Encyclopedia of Clinical Technology , 3rd Edition, Vol. 7, 196-206. Kitagawa, H. (1956a) Coumarin derivatives for medicinal purposes. X. Relationship between hypnotic action and physicochemical properties and distribution into brain and liver _in vivo . Yakugaku Zasshi 76, 582-587. Kitagawa, H. (1956b) Coumarin derivatives for medicinal purposes. IV. Hypnotic potency ratio and safety index of 4-methylcoumarin, 3-coumarin carboxylic acid diethyl amide and barbital. J. Pharm. Soc. Japan 76, 588-591. Kitagawa, H. and Iwaki, R. (1958) Coumarin derivatives for medicinal purposes. J. Pharm. Soc. Japan 78, 491-497. Koch-Grunberg, T. (1909) Zwei Jahre unter den Indianern Ernst wasmauth ,Berlin, 1", 298. Koh, M.S. and Willoughby, D.A. (1979) A comparison of coumarin and levaminasole on parameters of the inflammatory response. Agents and Actions 9, 284-288. Kreitmair, H. (1949) Asperula odorata -der Waldmeister. Pharmazie 4, 140-142. Lee, W.-H. and Fujiwara, M. (1971) Spasmolytic action of 4- methylumbelliferone in isolated guinea pig's gallbladder. Japan J. Pharmacol. 12, 827-829. Martello, R. and Farnsworth, N.R. (1962) Observations on the sensitivity of several common alkaloid precipitating reagents. Lloydia 25, 176-185. McKenna, D.J., Abbott, F.S. and Towers, G.H.N. (1984) 66 Monoamine oxidase inhibitors in South American hallucinogenic plants II. Constituents of orally active Myristicaceous hallucinogens.. J. Ethnopharmacol. (submitted). Morton, J.F. (1977) Some folk remedy plants of Central American markets. Quart. J. Crude Drug Res. 15, 165-192. Munakata, K., Marumo, S. and Ohta, K. (1965) Justicin A and B, the fish-killing components of Just ic ia hayatai var. decumbens . Tetr. Lett. 47, 4167-4170. Ohta, K. and Munakata, K. (1970) Justicidin C and D, the 1- methoxy-2,3-napthalide lignans isolated from Justic ia procumbens L. Tetr. Lett. 12, 923-925. Ojewole, J.A.O. and Adesina, S.K. (1983a) Mechanism of the hypotensive effect of scopoletin isolated from the fruit of Tetrapleura tetraptera . Planta Med. 49, 46-50. Ojewole, J.A.O. and Adesina, S.K. (1983b) Cardiovascular and neuromuscular actions of scopoletin from fruit of Tetrapleura tetraptera . Planta Med. 49, 99-102. Okigawa, M., Maeda, T. and Kawano, N. (1970) The isolation and structures of three new lignans from Just ic ia procumbens Linn. var. leucantha Honda. Tetr. 26, 4301-4305. Olaniyi, A.A. (1982) Two new arylnapthalide lignans from Justic ia flava roots. Planta Med. 4, 154-156. Olaniyi, A.A. and Powell, J.W. (1980) Lignans from Just ic ia flava . J. Nat. Prod. 43, 482-486. Pai, M.R., Bai, N.J., Venkitasubramanian, T.A. and Murthy, V.V.S. (1975) Effects of coumarins on mitochondrial function. Environ. Physiol. Biochem. 5, 184-188. Patyra, S. (1963a) Pharmacodynamic properties of coumarin. I. Influence of coumarin on the action of isolated frog hearts. Ann. Univ. Mariae-Curie, Lubin, DD, 18, 69-78. Patyra, S. (1963b) Pharmacodynamic properties of coumarin. II. Influence of coumarin on the motor behavior of the frog, mouse and rabbit. Ann. Univ. Mariae Curie, Lubin, DD, 18, 79-90. Pouchert, C.J. (Ed.) (1981) The Aldrich Library of Infrared Spectra , Edition III, Aldrich Chem. Co. Prance, G.T. (1972) An ethnobotanical comparison of four tribes of Amazonian indians. Acta Amazonica 2, 7-28. 67 Prasad, D.N., Bhattacharya, S.K. and Das, P.K. (1966) A study of anti-inflammatory acivity of some indigenous drugs in albino rats. Ind. J. Med. Res. 54, 582-590. Prasad, D.N., Gode, K.D., Sinha,P.S., and Das, P.K. (1965) Preliminary phytochemical studies on Pluchea lanceolata Linn. Ind. J. Med. Res. 53, 1062-1068. Robbins, T.W. (1977) A critique of the methods available for the measurement of spontaneous motor activity. In Handbook of Psychopharmacology: Pr inc iples of Behavioral Pharmacology (Iversen, L.L., Iversen, S.D. and Snyder, S.H., eds). Vol. 7, Plenum Press, New York, pp. 37-82. Roig y Mesa, J.T. (1945) Plantas medic inales, aromat icos y_ venenosas de Cuba . Cultural, S.A., Havana. Schultes, R.E. (1970) The botanical and chemical distribution of hallucinogens. Ann. Rev. PI. Phys. 21, 571-598. Schultes, R.E. (1972) De plantis toxicariis e mundo novo tropicale commentationes XI. The ethnotoxicological significance of additives to New World hallucinogens. Pi. Sc. Bull., Dec., 34-40. Schultes, R.E. (1978) De plantis toxicariis e mundo novo tropicale commentationes XXV. Biodynamic Acanthaceous plants of the northwest Amazon. Bot. Mus. Leafl. Harv. Univ. 26, 267-275. Schultes, R.E. and Holmstedt, B. (1968) De plantis toxicariis e mundo novo tropicale commentationes II. The vegetal ingredients of the myristicaceous snuffs of the northwest Amazon. Rhodora 70, 113-160. Seitz, G.J. (1967) Epena, the intoxicating snuff powder of the Waika Indians and the Tucano medicine man, Agostino. In Ethnopharmacologic Search for Psychoactive Drugs (Efron, D., Ed.) U. S. Health Serv. Publ. No. 1645, 315-338. Shah, N.S. and Hedden, M.P. (1978) Behavioral effects and metabolic fate of N,N-dimethyltryptamine in mice pretreated with /3 -diethylaminoethyl- diphenylpropylacetate (SKF 525-A), iproniazid and chlorpromazine. Pharmacol. Biochem. Behav. 8, 351-356. Shapiro, S.K. and Schlenk, F. (eds.) (1965) Transmethylation and Methionine Biosynthesis . University of Chicago Press, Chicago. Shulgin, A.T. (1982) Chemistry of psychotomimetics. In Handbook of Experimental Pharmacology , Vol. 55, Part III, pp. 3-29. 68 Stahl, E. (1969) Handbook of Thin-Layer Chromatography , 2nd Edition, Springer-Verlag, New York. Stehle, H. and Stehle, M. (1962) Flora medicinale illustree (Flore Agr. Antil. Francaises Vol. IX ). Imrimerie Parisienne, Pointe-a-pitre, Guadeloupe. Szara, S. (1956) Dimethyltryptamine: its metabolism in man; the relation of its psychotic effect to the serotonin metabolism. Experientia 12, 441-442. Vane, J.R. (1957) A sensitive method for the assay of 5- hydroxytryptamine. Br. J. Pharmacol. 12, 344-349. Wassen, S.H. (1967) Anthropologic survey of the use of South American Snuffs. In: Ethnopharmacologic Search for Psychoactive Drugs (Efron, D.H., ed.) U.S. Health Serv. Publ. No. 1645, 233-289. Wong, W. (1976) Some folk medicinal plants from Trinidad. Econ. Bot. 30, 103-142. Zerries, 0. (1960) Medizinmannwesen und Geisterlaube der Waika-Indianer des oberen Orinoco. Ethnologia, N.F. 2, 485-507. 69 III. AN ETHNOPHARMACOLOGICAL EXAMINATION OF Virola elongata BARK, A SOUTH AMERICAN ARROW POISON INTRODUCTION Chemical and pharmacological investigations of arrow poisons used by indigenous peoples have lead, in many instances, to the discovery of potent pharmacologically active compounds. The bis-isoquinoline alkaloid, tubocurarine, and related compounds from species of the Menispermaceae, indole alkaloids, such as strychnine, from Strychnos (Loganiaceae) and cardiac glycosides from a variety of members of the Apocynaceae and Moraceae, are some of the best known examples (Lewis and Elvin-Lewis, 1977). Less well known are the diterpene alkaloids of Aconitum sp. used in China (Bisset, 1981), the batrachotoxins (triterpene alkaloids), pumilotoxins (cis-decahydroquinoline alkaloids) and histrionicotoxins (spiropiperidine alkaloids) from Colombian arrow poison frogs of the Dendrobatidae (Daly, 1982) and the phorbol ester, huratoxin, from Hura crepitans (Sakata et a_l, 1971). Some of the active constituents of arrow poisons have proven valuable as medicinal agents (tubocurarine and cardiac glycosides) while others have been applied as tools in research (batrachotoxins and others). Spjut and Perdue (1976) have shown that sources of arrow poisons are five times more likely to possess antitumour activity than higher plants selected at random. The use of Virola species as arrow poisons is 70 interesting, not only ethnologically and anthropologically, but pharmacologically. Reports on the use of bark resin of Vi rola theidora and Virola elongata as an hallucinogenic snuff by the Yanomamo (Waika) of southern Venezuela and northern Brazil were accompanied by observations that the same material was applied alternately as an arrow poison. Schultes and Holmstedt(1971) cite Salathe (1931) and Becher (i960) as the first to describe the use of a tree bark as an arrow poison amongst the Waika group. This source plant is readily distinguished from the lianas of the Menispermaceae and Loganiaceae which are also reported to have been used in the preparation of dart-tip arrow poisons by the Waika tribes (Bauer, 1965). Although it has been reported that some tribes may use a mixture of curare and Vi rola resin in the preparation of their arrow poison (Biocca, 1965; Biocca et al, 1966), the use of Virola sp. as the sole ingredient has also been reported as a well established practice (Schultes and Holmstedt, 1968; Prance, 1970). Schultes and Holmstedt (1968) carefully describe the manner of preparation of arrow tips by the Waikas of the Rio Tototobi. Strips of bark of Vi rola theiodora are heated over a fire and the resin which flows from the bark is applied repeatedly to bamboo arrow tips, followed by heating over the fire. Prance (1970) has observed an almost identical procedure by the Sanama tribe (closely related to the Waikas), living more than 300 kilometers north of the Rio Tototobi. Vi rola theiodora and V. elongata have been considered to 71 be synonomous by authorities in the taxonomy of this group (Rodrigues, 1980; Smith and Wodehouse, 1937). Schultes prefers to distinguish V. theiodora as a separate species on the basis of the ease with which it can be recognized in the field (Schultes and Holmstedt, 1968). Prance (G.T., pers. comm.) has pointed out that the two varieties are generally found in the same locality, V. theiodora occurring on tierra firme and V. elongata growing close to the watercourses and that they are both used in the preparation of snuff and arrow poison. Vi rola species have been studied extensively with respect to their alkaloidal constituents. Both V. theiodora and V. elongata have been examined. Although there was considerable variation between samples, the indole alkaloids, N- methyltryptamine, N,N-dimethyltryptamine and 5-methoxy-N,N- dimethyltryptamine were usually the major alkaloids present (Agurell et al, 1969; Holmstedt et al, 1980; McKenna et al, 1984). These, and other indole alkaloids, are known to have a number of physiological activities in animals, foremost among which is their psychotomimetic activity and their ability to elicit hallucinations in man (Szara, 1956). N,N- dimethyltryptamine and 5-methoxy-N,N-dimethyltryptamine have been shown to disrupt normal behavioral responses of rodents at low doses (Gessner and Page, 1962; Gessner e_t a_l, 1961; Gessner et al, 1968; Grahame-Smith, 1971; Ho et al, 1970; Shah and Hedden, 1978). It has been suggested that indole alkaloids are the biologically active constituents of Vi rola arrow poison that are responsible for incapacitating the wounded 72 animal. Yanomamo darts have been examined chemically and found to contain a single alkaloid, 5-methoxy-N,N-dimethyltryptamine in high concentration (8% by weight) (Galeffi e_t al, 1983). These workers attribute the effectiveness of the arrow poison to the presence of this indole constituent. In the present study, an experimental approach to this ethnopharmacological problem has been taken. Extracts of Vi rola elongata were administered to mice and the effects on behavior observed and quantified. It was intended, by this approach, to determine if toxic constituents were present or if the tryptamines caused behavioral disruption of sufficient severity to explain the use of the bark as an arrow poison. 73 PART A. ISOLATION AND IDENTIFICATION OF THE MAJOR NON-POLAR CONSTITUENTS OF Virola elongata BARK 1. INTRODUCTION During the course of the studies dealing with the pharmacological effects of Vi rola extracts, some difficulty was encountered in determining the relative potency of the biological effects of certain fractions. This was a function of the difficulty involved in quantifying locomotor activity in large numbers of mice and the nature of the sample being fractionated. As a result, it was decided that a better approach would be to isolate and purify the major compounds first and then to measure their biological activity. This section describes the isola'tion and identification of these compounds. 2. EXPERIMENTAL a. Extract ion of Plant Material The bark was collected near the village of Brillo Nuevo on the Rio Ampiyacu, a Peruvian tributary of the Amazon. Voucher specimens (D. McKenna No. 59) were deposited at UNAP herbarium in Iquitos, San Marcos Herbarium in Lima, the Chicago Field Museum and the UBC herbarium. The identification was carried out by Dr. W.A. Rodrigues, INPA, Manaus, Brazil. The dried bark (1.3 kg) was milled and extracted four times at room temperature with diethyl ether. 74 b. Chromatography After evaporation i_n vacuo the residue was subjected to chromatography on a Chromatotron (Harrison Research Associates) utilizing preparative (4 mm) plates coated with Silica gel PF25, (Merck). Repeated chromatography with petroleum ether/ethyl ether/acetonitrile (12/12/1; 6/6/1; 3/3/1) yielded each of the following compounds in sufficient purity for spectroscopy. They are presented in order of elut ion. 3. COMPOUNDS ISOLATED 0 -sitosterol. (425 mg) Mp 133-135 °C (from Et20), PMR and MS data agree with data obtained for an authentic sample (Sigma). Chromatography on TLC (silica) using heptane/chloroform/ethanol (25/ 25/1) and petroleum ether/ethyl ether/acetonitrile (12/12/1) was identical to that of an authentic sample (Sigma). 3,4',5-trimethoxy-cis-stilbene 1a. (23 mg) Mp 73-74 °C (from Et20). UV X cyclohex.( lo3g e). 283 (4.50), 235sh (4.64), 214 max 1 (4.97). H-NMR (400 MHz) (CDC13): 5 3.65 (6H, s, 2 X OCH3), 3.78 (3H, s, OCH3), 6.32 (1H, t, J = 2 Hz, H-4), 6.43 (2H, d, J = 2 Hz, H-2, H-6), 6.43 (1H, d, J = 12 Hz, vinyl), 6.52 (1H, d, J = 12 Hz, vinyl), 6.77 (2H, d, J = 9 Hz, H-3', H-5'), 7.21 (2H, d, J = 9 Hz, H-2', H-6'). MS m/z (rel int) 270[M+] (100), 239 (12), 224 (12), 212 (8), 196 (11), 195 (13), 181 (10), 169 75 (10), 165 (10), 153 (13), 152 (19), 149 (12), 141 (9), 135 (9) , 127 (8), 115 (15), 104 (10), 95 (11), 91 (17), 85 (11), 83 (11), 81 (15), 71 (15), 69 (22) . 3,4',5-trimethoxy-trans-stilbene 1b. (41 mg) Mp 56-57 0 C lohex (from Et20). UV X^ ' (log e): 335sh (4.45), 318 (4.68), 303 (4.72), 235sh (4.42), 216 (4.66). 1H-NMR (400 MHz) (CDC13): 6 3.81 (9H, s, 3 X OCH3), 6.38 (IH, d, J = 2 Hz, H- 4), 6.65 (2H, d, J = 2 Hz, H-2, H-6), 6.90 (2H, d, J = 9 Hz, H-3', H-5'), 6.90 (1H, d, J = 16 Hz, vinyl), 7.04 (1H, d, J = 16 Hz, vinyl), 7.44 (2H, d, J = 9 Hz, H-2', H-6'). MS m/z (rel int) 270[M+] (100), 239 (14), 224 (11), 212 (8), 196 (9), 195 (10) , 181 (7), 169 (7 ) 167 (13), 165 (8), 153 (9), 152 (13), 149 (37), 141 (7), 135 (7), 128 (8), 115 (12), 104 (7), 91 (12), 83 (9), 76 (11), 71 (25), 70 (23), 69 (27). 0 Eusiderin 2. (28 mg) Mp 93-95 C (from Et20). UV XMeOH (log ^ max r e): 271 (3.21), 234sh (4.24), 230 (4.78). [a] -10.40 in 1 CHC13. H-NMR (80 MHz) (CDC13): 5 1.26 (3H, d, J = 6Hz, Me-3), 3.28 (2H, d, J = 7Hz, CH2), 3.85 (3H, s, OCH3), 3.88 (9H, s, 3 X OCH3), 3.9-4.3 (IH, m, H-3), 4.56 (1H, d, J = 8Hz, H-2), 4.9-5.25 (2H, m, =CH2), 5.7-6.25 (1H, m, CH=), 6.37 (1H, d, J = 2Hz, H-6), 6.48 (1H, d, J = 2Hz, H"8), 6.58 (2H, s, H-2', H- 6*). MS m/z (rel int)': 386[M+] (22), 372 (5), 344 (3), 343 (3), 312 (3), 311 (4), 302 (5), 210 (10), 209 (72), 208 (100), 205 (13), 195 (5), 194 (22), 193 (75), 192 (6), 191 (28), 181 (11) , 179 (15), 178 (12), 177 (9), 168 (5), 165 (15), 164 (8), 76 163 (9), 161 (6), 151 (10), 150 (18), 149 (48), 148 (7), 147 (7), 137 (13), 135 (19), 133 (18), 132 (8), 123 (11), 121 (14), 119 (10), 107 (19), 105 (23), 104 (11), 103 (10), 97 (13) , 95 (13), 91 (50), 85 (17), 83 (18), 81 (14), 79 (28). Virolongin 3. (34 mg). Colourless oil. UV X^|°H (log e) 268 0 1 (3.35), 227 (4.04). [a]jp -12.4 in CHC13. H-NMR (80 MHz) (CDC13) 6 1.22 (3H, d, J = 6Hz, Me-9), 1.87 (3H, d, J = 6Hz, Me-9'), 2.71 (1H, dd, J = 13, J = 8.0 Hz, H-7), 3.10 (1H, dd, J = 14, J = 5.5 Hz, H-7), 3.78 (3H, s, OCH3), 3.80 (6H, s, 2 X OCH3), 3.83 (6H, s, 2 X OCH3), 4.2-4.5 (IH, m, H-8), 6.05-6.25 (1H, m, H-8'), 6.2-6.5 (1H, H-7'), 6.43 (2H, s, H-2, H-6), 6.53 (2H, s, H-3', H-5'). MS m/z (rel int) 402[M+] (27), 211 (5), 210 (40), 209 (100), 208 (73) , 195 (19), 194 (87),193 (55), 192 (7), 191 (6), 182 (7), 181 (51), 179 (30), 178 (39), 177 (12), 169 (4), 168 (28), 167 (6), 166 (7), 165 (16), 164 (7), 163 (17), 162 (10), 161 (7), 153 (11), 151 (23), 150 (14) , 149 (14), 148 (11), 147 (18), 138 (5), 137 (16), 136 (10), 135 (22), 134 (9), 133 (23), 131 (11), 125 (6), 123 (9), 122. (7), 121 (19), 120 (8), 119 (19), 118 (9), 115 (9), 109 (10), 107 (19), 105 (25), 103 (15), 95 (12), 93 (10), 91 (40), 85 (11), 83 (12), 81 (13), 79 (33), 78 (13), 77 (34), 71 (18), 70 (11), 69 (25). 77 0 Epi-sesartemin 4. (97 mg). 114-115 C (from Et20). C23H2808 (found 430.1622 for 430.1628 by HR-MS). UV X ^??SH (log *): 270 0 (3.40), 235sh (4.14), 210 (4.92). [a] 25 +108 in CHC13. IR i>KBr u max cm "1 : 2900, 2820, 1625, 1585, 1540, 1500, 1450, 1410, 1360, 1320, 1230, 1200, 1125, 1080, 1040, 1000, 925, 830. 'H- NMR (see Table V) . 13 ONMR (see Table VI). MS m/z (rel int): 430 [M+] (40), 249 (6), 233 (10), 224 (18), 219 (15), 209 (8), 208 (24), 207 (40), 206 (17), 205 (7), 203 (9), 197 (42), 196 (16), 195 (23), 194 (15), 192 (13), 191 (34), 182 (20), 181 (60), 180 (28), 179 (100), 178 (15), 177 (8), 176 (15), 175 (7) , 169 (55), 168 (13), 167 (12), 166 (36), 165 (78), 161 (19), 154 (14), 153 (13), 152 (21), 151 (24) 139 (7), 138 (15), 133 (13), 125 (11), 115 (10), 95 (15), 93 (13), 91 (13), 81 (15), 79 (15). Sesartemin 5. (36 mg). Mp 115-116 °C (from Et20). C23H2808 (found 430.1627 for 430.1628 by HR-MS). UV X Me0H (log e): max J 5 0 1 270 (4.11), 211 (4.83). [a]J + 50 in CHC13. H-NMR (see Table V).13C~NMR (see Table VI). MS m/z (rel int): 430[M+] (47), 265 (5), 249 (9), 235 (7), 234 (8), 233 (11), 224 (18), 222 (6), 219 (17), 209 (7), 208 (24), 207 (51), 206 (15), 205 (8) , 203 (11), 197 (36), 196 (21), 195 (38), 194 (21), 193 (10) , 192 (14), 191 (46), 190 (9), 189 (9), 182 (23), 181 (72), 180 (33), 179 (100), 178 (22), 177 (11), 176 (19), 175 (9) , 169 (42), 168 (15), 167 (12), 166 (35), 165 (89), 161 (26), 154 (10), 153 (18), 152 (26), 151 (26), 138 (11), 135 (11) , 133 (16), 125 (13), 121 (8), 115 (11), 110 (10), 105 (10) , 95 (18), 93 (15), 91 (15), 81 (18), 79 (15). 78 Epi-yanqambin 6. (127 mg). Mp 119-120 °C (from Et20). UV X^eOH (log e): 272 (3.51), 238sh (4.11), 210 (4.80). [a] 25 0 1 13 +122 in CHC13. H-NMR (see Table V). C-NMR (see Table VI). MS m/z (rel int): 446[M+] (37), 265 (6), 250 (10), 249 (9), 235 (12), 224 (26), 223 (9), 219 (12), 208 (13), 207 (51), 206 ( 10) , 1 97 (32), 1 96 (24), 195 (68) , 1 94 (18) , 191 (10), 189 (8) , 182 (39) , 181 (100), 179 (12) , 177 (12) , 176 (21), 169 (42) , 1 68 (16), 1 67 (13) , 166, (10), 1 65 (21 ), 154 (11), 1 53 (14), 151 (20), 138 (13), 125 (14), 1 10 (10), 95 (9), 93 (13), 91 (11) , 81 (15), 79 (9) . 0 Yanqambin 7. (115 mg) Mp 119-121 (from Et20) UV X MeOH (log e): 270 (3.52), 235 (4.14), 210 (4.93). [a]J5 +45.1 0 in CHCI3. 1H-NMR (see Table V).13C-NMR (see Table VI). MS m/z (rel int): 446[M+] (27), 265 (6), 250 (8), 249 (8), 235 (10), 224 (19), 223 (8), 219 (9), 208 (12), 207 (48), 206 (8), 197 (20) , 196 (28), 195 (51), 194 (20), 193 (10), 191 (11), 190 (8), 189 (9), 182 (27), 181 (82), 179 (12), 177 (13), 176 (21) , 169 (25), 168 (15), 167 (12), 166 (8), 165 (17), 163 (8), 154 (8), 153 (17), 152 (9), 151 (18), 149 (13), 145 (9), 138 (11), 137 (13), 136 (8), 135 (12), 131 (11), 128 (8), 125 (21), 123 (14), 121 (12), 119 (15), 117 (9), 115 (13), 111 (13), 110 (19), 109 (19), 107 (11), 105 (15), 97 (20), 96 (13), 95 (33), 93 (20), 91 (24), 85 (16), 83 (23), 82 (17), 81 (36), 79 (19). 79 Dihydrosesartemin 8. (21 mg) UV X (log e): 282 (3.25), 0 1 232sh (3.87), 218 (4.08). [a] 25 +11.8 in CHC13. H-NMR (400 MHz) (CDC13): 6 1.56 (1H, br s, exchangeable with D20, OH), 2.40 (1H, m, J = 5 Hz, H-3), 2.55 (1H, dd, J = 14, 11 Hz, ArCH2), 2.73 (1H, m, H-4), 2.92 (1H, dd, J = 14, 5 Hz, ArCH2), 3.73-4.08 (4H, H-5, CH2OH), 3.83 (3H, s, OCH3), 3.84'(6H, s, 2 X OCH3), 3.89 (3H, s, OCH3), 4.78 (1H, d, J = 6.3 Hz, H-2), 5.95 (2H, s, OCH20), 6.40 (2H, s, H-2', H-6'), 6.53 (1H, s, H- 2" or H-6"), 6.54 (IH, s, H-2" or H-6"). MS m/z (rel int): 432[M+] (23), 414 (1.2), 383 (2.4), 368 (1.3), 353 (1.1), 249 (6), 233 (17), 224 (14), 219 (18), 208 (21), 207 (12), 206 (12), 195 (22), 193 (14), 183 (10), 182 (82) , 181 (100), 180 (18), 179 (53), 169 (10), 167 (22), 166 (21), 165 (45), 153 (18), 152 (13), 151 (38), 148 (18), 137 (14), 136 (12), 123 (23), 105 (12), 95 (27), 93 (12), 91 (22), 79 (18). j3-Dihydroyangambin 9. (13 mg) UV X MeOH (log e): 280 (3.75), max 5 0 1 227sh (4.25), 213 (4.58). [a] J +15.1 in CHC13. H-NMR (400 MHz) (CDC13) 6 1.78 (1H, br s, exchangeable with D20, OH), 2.03 (1H, m, J = 5 Hz, H-3), 2.52 (1H, dd, J = 14, 7 Hz, ArCH2), 2.8 (1H, dd, J = 14, 5 Hz, ArCH2), 2.88 (1H, m, H-4), 3.70-4.05 (4H, m, H-5, CH2OH), 3.84 (6H, s, 2 X OCH3), 3.89 (6H, s, 2 X OCH3), 3.94 (6H, s, 2 X OCH3), 4.70 (IH, d, J = 8.5 Hz, H-2), 6.36 (1H, s, H-2"),.6.63 (1H, s, H-5"), 6.66 (2H, s, H-2', H-6'). MS m/z (rel int): 448[M+] (29), 399 (6), 265 (3), 263 (5), 249 (18), 240 (21), 235 (16), 233 (9), 224 (42), 223 (23), 222 (45), 221 (12), 219 (14), 210 (20), 208 80 (14) , 207 (18) , 205 (11), 198 (11), 197 (27) , 196 (32), 195 (83) , 1 93 (18), 191 (13), 189 (10) , 183 (17) , 1 82 (77) , 181 (100) , 179 (18) , 177 (12), 1 76 (13) , 1 69 (48) , 168 (21 ) , 167 (46) , 165 (20), 161 (11), 1 54 (19) , 1 53 (19) , 1 52 (22), 151 (50) , 1 49 (12), 1 48 (22) , 1 39 (20) , 1 38 (28) , 1 37 (27) , 1 36 (16) , 1 35 (12) , 1 33 (10) , 1 25 (15) , 124 (12) , 1 23 (13) , 1 22 (12), 121 (15), 1 10 (12), 109 (18), 1 07 (15) , 1 06 (11), 105 (17) , 95 (20) , 93 (17), 92 (12), 91 (29) , 81 (23) , 79 (22). 4. RESULTS Eleven of the major constituents of a diethyl ether extract of V. elongata bark were isolated as described in the Experimental section-and identified by spectroscopic means. 0- Sitosterol was the only phytosterol identified. The stilbenes, 3, 4', 5-trimethoxy-trans-stilbene (1a) and its cis isomer were found in approximately equal proportions. Only the former has been described as a naturally occurring constituent. Eusiderin (2) and a hitherto undescribed compound, virolongin (3), comprised the neolignans. Four bi s-tetrahydrof .uran lignans were identified: epi-sesartemin (4), sesartemin (5), epi-yangambin (6) and yangambin (7). Finally, two new tetrahydrofuran compounds, dihydrosesartemin (8) and 0- dihydroyangambin (9) were identified. 81 Epi- Epi- Protons sesartemin Sesartemin yangambi n Yangambin (4) (5) (6) (7) 1H 2.91(1H),m 3.05(1H),m 2.95(1H),m 3.08(1H),m 2H 4.85(1H),d 4.70(1H),d 4.84(1H),d 4.75(1H),d J = 5.5 J=4.0 J=5.0 J=5. 5 4aH 3.70-3.95 4. 15-4.50 3.70-4.00 4.20-4.43 ( 1H) ,m (1H),m (1H),m (1H),m 4j3H 3.15-3.5 3.70-4.10 3.20-3.55 3.82-3.97 (IH),M (1H),m (IH),m (1H),m 5H 3.15-3.50,m 3.05(1H),m 3.20-3.55 3.08(1H),m 6H 4.41(1H),d 4.70(1H),d 4.43(1H),d 4.75(1H),d J = 7.0 J = 4.0 J=7.0 J=5. 5 8aH 4.00-4.25 4.15-4.50 4.00-4.25 4.20-4.43 (1H),m (1H),m (1H),m (1H),m 8/3H 3.70-3.95 3.70-4.10 3.70-4.00 3.82-3.97 (1H),m (1H),m (1H),m (1H),m 2 ' , 2 " 6.56(2H),s 6.55(4H),s 6.57(4H),s 6.58(4H),s 6* , 6" 6.53(2H),s OCH3 3.90(3H),s 3.91(3H),s 3.88(12H),s 3.86(12H),s 3.87(3H),s 3.87(3H),s 3.83(6H),s 3.83(6H),s 3.85(3H),s 3.82(3H),s OCH2 0 5.95(2H),s 5.95(2H),s - - Table V - 1H-NMR spectra of bis-tetrahydrofuran lignans isolated from Vi rola elongata bark. Data are presented(in order) as chemical shift(6, relative to. TMS); integral value(number of protons); multiplicity of peaks; coupling constant(in Hz). Spectra were recorded in CDC13 at 80 MHz. 82 Carbon Epi- Sesartemin Epi- Yangambin Number Sesartemin yangambin (4) (5) (6) (7) 1 54.52 54.41 54.36 54.29 2 87.59 85.85(c) 87.66 85.88 4 71 .03 71.87(d) 71 .00 71 .91 5 49.96 54.41 49.90 54.29 6 82.11 86.02(c) 82.06 85.88 8 69.99 72.00(d) 69.77 71.91 1 ' 1 35.88 135.86 136.69 136.66 1 " 133.97 136.77 133.89 136.66 2' 105.73(a) 105.73(e) 103.00 102.98 2" 102.73 1 02.94(e) 102.69 1 02.98 3' 153.21 153.29(f) 153.32 153.39 3" 153.21 153.48(f) 153. 1 3 153.39 4' 149.06(b) 149.20(g) 137.60 137.64 4" 137.09 136.77 137.00 137.64 5' 143.62(b) 143.72(g) 153.32 153.39 5" 153.21 153.48 153. 1 3 153.39 6' 101.39(a) 101.52(e) 103.00 102.98 6" 102.73 102.94(e) 102.69 102.98 OCH3 56.13,56.66, 56.24,57.51 56.07,60.68 56.16,60.73 60.77 60.86 OCH2 0 1 00. 1 1 100.09 - - Table VI - 13C-NMR spectra of bis-tetrahydrofuran lignans isolated from Virola elongata bark. Chemical shifts are given in S(ppm) relative to TMS; 6(TMS)= 5(CDC13) + 77.0 ppm; recorded at 20 MHz in CDC13 . Values followed by letters are interchangeable. 83 a. 3,4'-5-trimethoxy-cis-stilbene and 3,4',5-trimethoxy-trans- stilbene Compounds 1a and 1b (Figure 7) were present in approximately equal amounts and behaved similarly on TLC using several solvent systems. Both formed a pink colour upon spraying with H2SO„. They were separated using the chromatotron and crystallized from diethyl ether. The UV spectrum of 1b displayed bands of approximately equal intensity at 319 and 305 nanometers, corresponding to Band I and II, respectively, that are characteristic of stilbenes (Hillis and Ishikura, 1966). The spectral data obtained for 1b are in agreement with those presented for 3,4',5-trimethoxy- trans-stilbene (Blair et aJL, 1969). The signals of the vinyl protons are reported by Blair et a_l (1969) to be broad singlets in a 60 MHz 1H-NMR spectrum. The better resolution of the 400 MHz spectrum obtained here showed the vinyl proton signals to be doublets with a coupling constant of 16 Hz. This value is characteristic of unsymmetrically substituted trans stilbenes and compound 1b was thus assigned. The UV spectrum of 1a, a single band at 283 nm, is suggestive of a cis-stilbene derivative (Hillis and Ishikura, 1966). The mass spectrum is very similar to that of 1b, both displaying a parent ion of mass 270. In the 400 MHz 'H-NMR spectrum, the aromatic AB pattern of C-3',5';C- 2',6', the signals of the two vinyl protons and the C-2, C-4 and C-6 proton signals are all present but shifted upfield somewhat from those of 1b. The vinyl proton signals appear as doublets 84 Figure 7 - Structures of stilbenes and neolignans isolated from Virola elongata bark. 85 Figure 8 - Structures of bis-tetrahydrofuran lignans isolated from Vi rola elongata bark. 86 Figure 9 - Structures of tetrahydrofuran lignans isolated from Virola elongata bark. 87 with a coupling constant of 12 Hz, a value that is characteristic of unsymmetrically substituted cis stilbenes. On the basis of these data compound 1a is assigned as the cis isomer of 1b. The occurrence of this compound is noteworthy since stilbenes are normally present naturally in the more stable trans form (Drewes and Fletcher, 1974). b. Eusiderin and Virolongin Compounds 2 and 3 (Figure 7) demonstrated similar behavior by TLC. Both displayed a pink colour upon spraying with H2SOa. Compound 2 was found to have a molecular weight of 386 by MS. Proton and methoxyl counts by NMR and the MS revealed the formula C18H1302 (OMe)„ and the MS fragment m/z = 208 suggested the presence of the system (CH30)3C6H2CHCHCH3. Further analysis showed the spectral data to be identical to that reported for the benzodioxan neolignan, eusiderin (Fernandes et al, 1980; Gottlieb et al, 1976; Hobbs and King, 1960). The precise structure of that compound has been established definitively by lanthanide induced shift data (Braz Filho e_t al, 1976). The chemical shift of Me~3 ( 5 1.26) and the value for J (8 Hz) by 'H-NMR are evidence for the trans relationship of the Ar-2/Me-3 groups (Fernandez et al, 1980) and support the identification of compound 2 as eusiderin. Eusiderin has previously been reported from Virola guggenheimi i and Vi rola pavonis (Fernandes e_t a_l, 1980) and various genera of the family Lauraceae (Gottlieb et al, 1976). Compound 3 bears considerable resemblance to eusiderin (2), based upon its UV, 1H-NMR spectrum and mass spectrum. The 88 presence of the MS fragments with m/z = 209 and 208 suggest the presence of the groups (CH30)3C6H2CH2CHCH3 and (CH30)3C6H2CHCHCH3, respectively. This, combined with the appearance of two sets, each of two equivalent aromatic protons and five methoxy groups in the 1H-NMR spectrum is indicative of the presence of an 8-0-4'-neolignan. The 1H-NMR spectrum closely ressembled that reported for 1 -(3,4,5-trimethoxyphenyl)-2-allyl-2,6-dimethoxyphenoxy)- propane, a compound isolated from seeds of Myristica fragrans (Isogai §_t §_1, 1973), also of the Myristicaceae. The only differences are in the occurrence of the signals attributed to the aromatic allylic substituent as a doublet (3H, J = 6 Hz) at 6 1.87 and multiplets between 6 6.05 (1H) and 6.25 and 5 6.2 and 6.5 (1H) in the spectrum of compound 3. These three signals would, instead, be expected to result from an aromatic propenyl group (Fernandes et a_l, 1980). Double resonance experiments further supported the assignments. Irradiation at 6 1.9 produced the collapse of the signal at 5 6.05-6.25 and irradiation at 6 6.2 caused the doublet at 5 1.87 to appear as a singlet. c. Epi-sesartemin, Sesartemin, Epi-yanqambin and Yanqambin Compounds 4 and 5 (Figure 8) behaved similarly in various chromatographic systems. Two spots, forming a brown colour upon spraying with H2SO<,, were distinguished by TLC. Both compounds were found to have formulae C23H2608 by HR-MS and the 1H-NMR clearly indicated the presence of four methoxy and one methylenedioxy group and two pairs of equivalent aromatic 89 protons. Furthermore, signals between 5 3.0 and 4.5 indicated the presence of a bis-tetrahydrofuran ring. These compounds were concluded to be 2,6-diaryl-3,7-dioxabicyclof3,3,0]-octane type lignans. The C-1, C~5 bond of naturally occurring bis- tetrahydrofuran lignans is characteristically in the cis configuration and the 1H-NMR and 13C-NMR spectra obtained (Tables V and VI) indicate that this is also true of compounds 4 and 5. The aryl substituents of C-2 and C-6 can be either axial or equatorial, allowing for three types of stereoisomers. Compound 4 was concluded to be an axial- equatorial isomer on the basis of the following features of its 1H-NMR spectrum (Pelter and Ward, 1978; Russel and Fenemore, 1973): 1) a difference in C-1 and C-5 methine proton chemical shifts (5 2.91 and 6 3.15-3.5, respectively), 2) a difference in the chemical shifts of the benzylic protons at C-2 and C-6 ( 6 4.85 and 6 4.41, respectively), 3) the presence of one axial (C-4) methylene proton upfield ( 6 3.15-6 3.5) from the "normal" position ( 6 3.8 to 5 4.0) due to shielding by the axial aromatic ring and an equatorial methylene proton (C-8) downfield between 6 4.0 and 6 4.25, due to deshielding by the equatorial aromatic ring. Chiba et al (1980) used 13C-NMR to establish the positions of aryl groups of unsymmetrically substituted bis- tetrahydrofuran lignans and Greger and Hofer(l980) based their assignment of the stereochemistry on data obtained using the lanthanide induced shift technique. In the present study, the 90 relative configurations were assigned on the basis of 13C~NMR spectra. The chemical shifts of carbon atoms 1' and 1" are particularly useful in determining the stereochemistry of attachment of the phenyl group to the bis-tetrahydrofuran skeleton (Pelter and Ward, 1978). Compounds 6 and 7 (Figure 8) are known, symmetrically substituted bis-tetrahydrofuran lignans and were identified on the basis of their melting points, optical rotations and UV, IR, MS and 'H-NMR spectral data which agreed closely with published information (Abe et al, 1974; Briggs et al, 1968; Chen et al, 1976; Jeffries et al, 1961; Lai e_t al, 1973). The 1 3C-NMR data of compounds 6 and 7 were compared with those of the unsymmetrically substituted compounds 4 and 5 and the assignments made on this basis. The 1' and 1" carbons of the diequatorially substituted compound, yangambin (7), have chemical shifts of 136.66 ppm, a value that compares well with those published by Pelter and Ward (1978) for similar lignans. The axial 3,4,5- trimethoxyphenyl substituent of epi-yangambin (6), which has a chemical shift of 133.89 ppm (Table VI), is easily distinguished from the equatorial one. The similarity between this value (133.89 ppm) and the signal seen in the spectrum of compound 4 (133.97 ppm) is taken as evidence that the 3,4,5- trimethoxy phenyl substituent is in the axial position in compound 4. The remaining C-1 signal (135.88 ppm) was assigned to the 3-methoxy-4,5-methylenedioxyphenyl substituent which was already known, from the 1H-NMR spectrum, to be in the equatorial position, by default. The 13C-NMR spectrum of the 91 diequatorial compound 5 supports these assignments since it has signals at 135.86 ppm and 136.77 ppm, values which correspond closely to those assigned above for equatorial 3- methoxy-4,5-methylenedioxyphenyl and 3,4,5-trimethoxy phenyl substituents, respectively. All other 13C-NMR signals showed close agreement with the spectra reported for similar bistetrahydrofuran lignans (Pelter and Ward, 1978). The mass spectra of compounds 4 and 5 are almost identical and bear close resemblance to those of compounds 6 and 7 which are also almost identical. The molecular formulae for all of the fragments described for compounds 4 and 5 were obtained from mass measurements by high resolution mass spectrometry. Corresponding structures are drawn in Figure 10. The basic fragmentation pathways described by Pelter (1967) and Duffield (1967) are easily identifiable. The fragmentation pattern of compounds 6 and 7 has also been included (Figure 11) for comparison. As has been observed by others (Pelter, 1967; Taniguchi, 1972), reliable differences in the structure of isomers are not identifiable, d. Dihydrosesartemin and 0 -Dihydroyanqambin Compounds 8 and 9 (Figure 9) are the most polar constituents of the ether extract. They are separated easily by TLC, compound 8 giving a salmon-pink coloured spot with H2SOa and compound 9 a grey coloration. The mass spectra of these two compounds were especially informative since they produced parent ions with masses 432 and 448, respectively. These values are just 2 mass units greater than the parent F i gure 10 - Scheme of fragmentation of bis-tetrahydrofuran lignans, epi-sesartemin and sesartemin, by mass spectrometry Figure 11 - Scheme of fragmentation of bis-tetrahydrofuran lignans, epi-yangambin and yangambin, by mass spectrometry 94 ions of sesartemin and yangambin, respectively. Further similarities in the mass spectra of all four compounds were apparent. The mass spectrum of dihydrosesartemin (8) showed two series of fragments: 181, 169, 168 and 165, 153, 152, indicating the presence of both 3~methoxy-4,5- methylenedioxyphenyl and 3,4,5-trimethoxyphenyl substituents. The greater abundance of the peak at m/z = 181 than that at m/z = 165, was taken as evidence that the trimethoxy substituted aromatic group was a benzyl, rather than a phenyl, substituent. The remaining features of the mass spectrum resemble those expected from a substituted tetrahydrofuran. The fragmentation pattern has been interpreted according to the schemes presented by Pelter e_t a_l (1966) and Pelter ( 1 967) (Figure 12). The 1H-NMR spectrum supported the proposal that the compound was a substituted tetrahydrofuran and provided information on its relative configuration. Hall (1964) and Hall ejt al (1972) have discussed the applicability of NMR data in assigning the conformation of furanoses in solution. At least 20 conformers are recognized. The relative importance of each in the distribution of all possible configurations will vary depending upon the nature of the substituents and the solvent used. Unfortunately, the variety of tetrahydrofuran lignans presently known is not great and detailed NMR spectra have been reported in only a limited number of cases. Few data are therefore available for comparison. From the existing Figure 12 - Scheme of fragmentation of tetrahydrofuran lignan. dihydrosesarternin, by mass spectrometry 97 data, it appears that the feature that can be most easily determined is the relative configuration at the benzyl carbon. A cis orientation of constituents about the C-2-C-3 bond will result in deshielding of the benzyl proton and a shift downfield in its resonance to approximately 6 5.5 (Birch and Smith, 1964; Birch et al, 1967; Inoue et al, 1981; Sarkanen and Wallis, 1973a and b; Smith, 1963). A trans orientation results in the C-2 proton having a chemical shift of approximately 6 4.7. The corresponding signal observed in the spectrum of compound 8 is a doublet, 6 4.78. This value agrees well with the assignment of the trans configuration. The coupling constant may be expected to vary considerably, depending upon the nature of the substituents and the favoured conformation of the furan ring. The published data are insufficient to allow the use of coupling constant as a definitive criterion for assigning configuration. The measured value, J = 6.3 Hz is, however, in line with several values previously reported for the trans configuration about the C-2- C-3 bond of a substituted tetrahydrofuran (Sarkanen and Wallis, 1973a; Smith, 1963). The relative configuration of the tetrahydrofuran lignan, (+)-lariciresinol has been established by chemical methods. Its optical rotation has been reported as [a] = + 17.5 0 (Weinges, 1961). The optical rotation of compound 8 was found to be [a] = + 11.8 °. These two values appear to be sufficiently similar to form the basis for assigning the relative configuration of the remaining carbon of compound 8. 98 The configuration is therefore, 2S, 3R,.4R, identical to that of (+)-lariciresinol. The mass spectrum of compound 9 (Figure 13) is analogous to that of dihydrosesartemin (8) (Figure 12) suggesting that it, too, is biosynthetically related to a bis-tetrahydrofuran lignan; differing only in that one furan ring is opened, leaving a free hydroxyl group. When the 1H-NMR spectrum is examined, however, it is apparent that a difference exists in the type of aromatic substituents present in compound 9 and yangambin or epi-yangambin. Instead of two singlets, each arising from two equivalent aromatic protons, one singlet integrating for 2 protons (6 = 6.66) and two single proton singlets at 8 6.63 and 6.36 are observed. This pattern is similar to the singlets at 6 6.84 and 6 6.43 which were observed for the 3,4-methylenedioxy-6- methoxyphenyl group observed by Russel and Fenemore (1973). Although the positioning of the aromatic groups cannot be unambiguously defined on the basis of only the 1H-NMR spectrum, the structure has been tentatively assigned on the basis of a comparison of the chemical shifts with data from related compounds. In the 'H-NMR spectrum of dihydrosesartemin (8), the chemical shift of the C-21 and C-6' protons of the methoxy-methylenedioxybenzyl substituent, which are resolved into two peaks at 6 6.53' and 6.54, are downfield from the signal of the corresponding C-2" and C-6" protons of the 3,4,5-trimethoxy-phenyl substituent opposite (6 6.40). Assuming the same relationship to exist in the 1H-NMR spectrum 99 of /3-dihydroyangambin, the lower field signal (a 2 proton singlet, 5 = 6.66) is assigned to the protons of the benzyl substituent. This would correspond to the two equivalent protons of the 3,4,5~trimethoxy-benzyl group. The remaining two aromatic singlets (integrating at 1 proton each), with chemical shifts 5 6.63 and 6 6.36 are assigned to the C-5" and C-2" protons, respectively. These chemical shifts are in agreement with 1H-NMR data published for other lignans (Russel and Fenemore, 1973; Taniguchi and Oshima, 1972a and b). The optical rotation of compound 9, [a] = +15.1 0 is similar to that of (+)- lariciresinol, [a] = +17.5 0 (Weinges, 1961), suggesting similar configurations at carbons 2,3 and 4. Although the chemical shift, 5 = 4.75, is indicative of a trans configuration about C2-C3, the coupling constant measured from the 400 MHz spectrum, J = 8.5, is larger than that of compound 8, J = 6.3, and other values published for tetrahydrofuran lignans with analogous configurations. The 1H- NMR data for compounds 8 and 9 are sufficiently different to suggest that their relative configurations are also different and no further attempt was made to assign the stereochemistry of compound 9. 100 PART B. EXAMINATION OF THE BIOLOGICAL ACTIVITY OF Virola elongata EXTRACTS 1. INTRODUCTION This section describes the series of biological experiments that were carried out in an effort to determine if a toxic constituent was present in the bark resin and to try to elucidate the nature of the effects observed in mice injected with extracts of this material. The methods used to prepare the extracts were somewhat different from those used to obtain purified compounds for spectroscopic analysis. Full details have been included in the following section. 2. MATERIALS AND METHODS The collection and identification of plant material has been described in Part A, 2a. Except where indicated otherwise, the bark samples which had been preserved in methanol immediately after being collected were used in the preparation of extracts for biological testing. The extracts obtained from bark which had been dried and that which had been preserved in methanol were indistinguishable by TLC and HPLC. a. Preparation of extracts Alkaloidal and non-alkaloidal extracts Virola elongata bark (28 g dry weight) was milled and extracted exhaustively at 20 0 C with 100% methanol. The 278 mg of material extracted was suspended in water, acidified (to 101 pH 3 with HCl), and extracted with diethyl ether. The aqueous phase was next basified (pH 12 with NaOH) and extracted with dichloromethane. This formed the alkaloidal fraction (12.5 mg). The remaining aqueous and diethyl ether fractions were combined and neutralized to form the non-alkaloidal fraction (260 mg). Aqueous extract Dried V. elongata bark (30 g) was milled and extracted with distilled water at 20 0 C for three, eight hour periods. The mixture was agitated constantly during extraction. After filtration of the combined extracts, the solution was evaporated in vacuo and stored at 4 0 C until use. Diethyl ether extract The methanol preserved bark was dried, milled and extracted five times with dry diethyl ether. The extract was filtered and concentrated by rotary evaporation in. vacuo and the brown, syrupy residue stored at -30 0 C until use. b. Fract ionat ion of extracts Aqueous extract This extract was fractionated by ion exchange chromatography. A column was prepared from thoroughly washed Dowex 50WX8 (2-50 mesh, H+ form) resin. The sample was dissolved in 1N HCl and applied to the column. It was eluted with aqueous solutions of pH 6, 8, 10 and I2(adjusted using NaOH). Fractions were neutralized immediately after collection. The fractions were monitored for the presence of 1 02 indole alkaloids and amino acids by TLC using Ehrlich's and ninhydrin reagents, respectively. Diethyl ether extract The separation of this extract was similar to that described in Part B, 2b of this chapter, c. Chromatographic analysis of extracts HPLC Quantitative analysis of extracts was carried out using a Varian MCH-10 reverse phase column on a Varian HPLC with a Varian variable wavelength detector. The sample was eluted with methanol/water by gradually increasing the methanol concentration from 50% to 100% over a 20 minute period. The UV absorbing substances, were detected at 250 nm. Quantification was performed by comparing peak heights of the sample chromatogram with the values from a standard curve prepared using known quantities of pure compounds. TLC Because the four major constituents of the diethyl ether extract were not readily resolved by HPLC, thin layer chromatography systems were used to estimate the relative amounts of these constituents. Polygram Silica gel UV25a TLC plates were used with either petroleum ether/ diethyl ether/ acetonitrile (6/6/1) or hexane/ chloroform/ ethanol (25/25/1) as developing solvents. 1 03 d. Assay of spontaneous motor activity The apparatus used to measure spontaneous locomotor activity is a modification of the "jiggle cage" (Kinnard and Watzman, 1966). The same procedure was used to measure spontaneous motor activity of mice in the previously described study on the biological activity of J. pectoralis. Complete details of the apparatus and the method for recording spontaneous motor activity are provided in Chapter II, section 2a. The spontaneous motor activity was measured in arbitrary units. The measuring device was sensitive not only to gross behavioral activity, but also to more subtle behavior such as grooming or even breathing. The signals produced were roughly proportional in magnitude to the type of activity observed. Female Swiss mice (30-35 g) were used in this study to examine the effects of the extracts. All animals were of comparable ages and were maintained under standard conditions of animal care. Extracts were injected intraperitoneally in aliquots of either 50 or 100 ML Distilled water was used as a vehicle for the aqueous extracts. Because of its solubility properties and low toxicity (Budden et al, 1979; Worthley and Schott, 1966), Tween 80 (Sigma) in water at a concentration of 10% (v/v) was used as a vehicle for administering the diethyl ether extracts. Recording of locomotor activity was begun immediately after injection and carried out for 60 minutes. Because of the effect of such variables as lighting (Walsh and Cummins, 1976), noise (Inglis, 1975), the presence 1 04 or absence of an observer (Norton, 1980), or time in the animals' circadian rhythm (Hughes e_t a_l, 1978), these conditions were standardized. Locomotor activity was measured under ambient fluorescent light and in the presence of a constant machine noise of low intensity. All assays were carried out between 7 and 10 PM in the presence of a single observer. e. Assay of anti-aggressive activity Lignans were examined for their ability to inhibit isolation induced aggression. Male Swiss mice were isolated by housing them singly in cages for a period of at least six weeks. This procedure resulted in distinct behavioral responses in most mice. Two types of behavior were distinguished when an isolated mouse was placed in a cage with a mouse not previously isolated (socialized mouse). Some of the isolated mice lost the behaviorisms that normally accompany socialization. Instead, they resisted any attempts at contact made by the socialized mouse, becoming nervous, assuming a defensive posture and emitting sharp squeaks. Mice which responded in this fashion were used in a study of the effect of the lignan, epi-sesartemin, on this antisocial behavior. Most of the remainder of the isolated mice were openly aggressive when placed in the same cage as a socialized mouse. After a relatively brief period, during which the mice explored each other's oral and anogenital regions, the isolated mouse generally attacked and fighting ensued. The four bis-tetrahydrofuran lignans isolated from V. elongata 1 05 bark were examined for their ability to reduce the aggressiveness observed in isolated mice. 3. RESULTS The only physiologically active substances known from the genus Vi rola are the anti-schistosomal neolignans, surinamensin and virolin from Virola surinamensis (Barata e_t al, 1978), the anti-fungal neolignan, (+)-guaiacin from Virola carinata (Gottlieb, Maia and Ribeiro, 1976) and tryptamine and /3-carboline derivatives from various species (Agurell e_t al, 1969; Holmstedt et al, 1980). It has been proposed that the potent hallucinogenic activity of the tryptamines, 5-MeODMT and DMT accounts for the use of Virola bark resin as an arrow poison (Galeffi et al, 1983; Maia and Rodrigues, 1976). This hypothesis was examined. The alkaloids of the V. elongata bark used in this study had been analysed and shown to consist of a single tryptamine, 5-MeODMT, at a concentration of 0.245 mg/g dry weight (McKenna et a_l, 1984). Five-MeODMT was the only tryptamine detected by Galeffi e_t a_l (1983) in their authentic sample of the arrow poison. The question of the importance of this compound to the pharmacological effects of the bark resin was approached by preparing a methanolic extract of the bark and separating it into alkaloidal and non-alkaloidal fractions (described in Materials and Methods, section 2a). The effects of the extracts were compared with respect to the gross behavioral responses observed in mice after intraperitoneal injection. 106 The results are summarized in Table VII. The results clearly indicate that the animals' gross behavior was altered to a greater extent by the non-alkaloidal extract than by the alkaloidal extract. Opposite effects were elicited by the two extracts. Whereas the alkaloidal extract induced a mild degree of hyperactivity at the highest doses tested, a comparable dose of the non-alkaloidal fraction resulted in a marked reduction in activity and a state of stupor lasting several hours. a. Examinat ion of the aqueous fraction for toxic ity The lack of toxicity of the methanolic extract of V. elongata raised the possibility that a toxic constituent not extracted by this solvent might be present. Mebs e_t aJL (1982) have reported the toxic nature of a polypeptide of an aqueous extract of the larvae of Diamphida nigrornata, a Bushman arrow poison. To examine whether V. elongata contained a toxin which was either insoluble in, or inactivated by organic solvents, an aqueous extract of the bark was examined for pharmacological activity. This extract was administered intraperitoneally to female Swiss mice in doses of 40, 100 and 200 mg/kg. Within two minutes of injecting 200 mg/kg frequent writhing was observed. The animals became somewhat uncoordinated and, within five minutes of injection, motor activity was significantly reduced. The animals lay prone and an increased rate of respiration, accompanied by piloerection, was observed. After a period of two hours, normal behavior gradually resumed. 1 07 Dose administered Behavioral . response Amt. bark Amt. 5-MeODMT Non- represented in alkaloid alkaloidal Alkaloidal by extract f ract ion fraction fraction injected(mg) m g mg/kg siight 5 (20)t 0.05 1 .0 reduct ion no effect in activity 1 0(40 ) 0.09 1 .9 reduction in no effect activity 20 (80) 0.19 3.8 inactivity:1 hr no effect durat ion 40 (160) 0.36 7.5 inactivity:2 hr si hyperact: durat ion 10 min 80 (320) 0.74 15.0 inactivity:>3hr si hyperact: durat ion 15 min Table VII - Gross behavioral responses of Swiss mice to administration of alkaloidal and non-alkaloidal extracts of Vi rola elongata bark. Three mice per dose were observed: extracts.were administered by intraperitoneal injection. t numbers in brackets refer to dose (in mg/kg) of non-alkaloidal fraction 108 Lower doses of this extract (100 and 40 mg/kg) produced similar behavioral changes, but of lesser intensity and shorter duration. Three mice were injected with each dose and complete recovery after 3 hours was observed in all cases. An attempt was made to concentrate the biological activity of this extract by fractionating it using ion exchange chromatography. All of the fractions obtained, however, caused writhing and reduced activity levels when injected into mice. Each fraction was examined for the presence of polar alkaloids or amino acids by spraying thin layer chromatograms of the fractions with Ehrlich's, iodoplatinate and ninhydrin reagents. Only negative results were obtained. The possibility that the irritant activity observed in the aqueous extract resulted from the presence of a complex phenolic mixture was considered. Tannic acid (J. L. Baker) was injected at a dose of 20 mg/kg. It produced a response which was indistinguishable from that of the aqueous extract of the Virola bark. The mice responded by writhing within two minutes of the injection. Piloerection, a prone posture and a reduction in activity lasting approximately three hours were also observed. 109 b. Examination of the diethyl extract for depression of spontaneous motor activity Having determined that strongly toxic constituents were absent from both the aqueous and the methanol fractions and that the non-alkaloidal fraction of the methanol extract possessed the strongest effect when administered to mice, this fraction was examined further. Preliminary fraction of the non-alkaloidal fraction by TLC on silica gel yielded non-polar fractions which still possessed the ability to reduce the activity levels of mice injected peritoneally. More than one of the fractions demonstrated this activity. Because of this, and the time consuming nature of and variability in the bioassay, the approach of fractionation, guided by bioassay," was abandoned. Instead, the separation and purification of the major constituents of the active diethyl ether extract of the bark was undertaken. The biological activities of the major compounds were then examined at carefully measured doses. A modified version of the "jiggle cage" was used to record locomotor activity. This approach allowed a quantitative estimate of the compound's effectiveness in reducing locomotor activity. Thirteen compounds were isolated and identified from the diethyl ether extract. These were: the ubiquitous phytosterol, ^-sitosterol; two stilbene derivatives, 3,4',5-trimethoxy-cis- stilbene (1a) and its trans isomer (1b); the neolignans, eusiderin (2) and virolongin (3); the bis-tetrahydrofuran 110 lignans, epi-sesartemin (4), sesartemin (5), epi-yangambin (6) and yangambin (7); and the tetrahydrofuran lignans dihydrosesartemin (8) and /3-dihydroyangambin (9). In addition, two unidentified aromatic constituents, compounds X and Y, were isolated and tested. The isolation and identification of these compounds by spectroscopic means has been described in Part A of the present chapter. The ability of each of these compounds to suppress spontaneous motor activity in mice was tested. A pure sample of each compound was injected intraperitoneally in a dose of 25 mg/kg. Some of the compounds tested caused marked reduction in spontaneous locomotor activity. This was especially obvious in the case of the bis-tetrahydrofuran lignans. The reduction in motor activity caused by epi-sesartemin is evident in the diminished electrical output of the force transducer (Figure 14). This signal decreased steadily between 10 and 60 minutes following injection and gradually began to recover between 80 and 90 minutes post-injection. Typical time courses for the reduction in motor activity induced by epi-sesartemin and epi- yangambin can be observed in Figure 15. All of the compounds isolated were tested in this way. The average of the 24 measurements of locomotor activity made (at 5 minute intervals) during the 2 hour period following injection was calculated in each case. The results of this testing are presented in Table VIII. Ten of the thirteen compounds tested produced a significant reduction in activity Ill Figure 14 - Example of output of "jiggle cage" force transducer used to measure spontaneous motor activity of mice. a) recorded during normal motor activity, b) recorded after injection of epi-sesartemin (25 mg/kg): Chart speed= 10 cm/hr. 112 0 Epi-sesartemin (25 mg/kg) n—i—i—i—i—i—i—i—i—i—r* 20 AO 60 80 100 120 TIME AFTER INJECTION(min.) Figure 15 - Effect of bis-tetrahydrofuran lignans, epi- sesartemin and epi-yangambin on spontaneous locomotor activity of mice. Values are means, N= 3: SEMs are omitted from graph but were all less than 15%. 1 1 3 Compound Dose(mg/kg) Reduction in activity t- t % Control level SEM(n=4) ^-sitosterol 25 98 8 1a 25 46 7 1b 25 62 6 2 25 86 7 3 25 46 8 4 25 41 10 12.5 54 9 6.3 94 8 5 25 51 8 6 25 43 9 7 25 54 9 8 25 48 8 9 25 83 9 X 25 101 8 Y 25 104 9 control Tween 80 1 00 7 Table VIII - Effect of purified compounds of V. elongata bark on spontaneous locomotor activity of Swiss mice. t Compounds were injected in 50 M1 of 25% aqueous Tween 80. t- Values are averages of 4 injections per compound: SEM = standard error of mean. 1 1 4 level. 3,4',5-trimethoxy-cis-stilbene (1a), virolongin (3), epi-sesartemin (4), sesartemin (5), epi-yangambin (6), yangambin (7) and dihydrosesartemin (8) all produced approximately a 50% reduction in activity at the dose administered. 3,4',5-Trimethoxy-trans-stilbene (1b), eusiderin (2) and 0-dihydroyangambin (9) showed lesser, but still significant activity. Only the unknown compounds (X and Y) and /3-sitosterol were inactive. c. Quantification of major constituents of diethyl ether extract Because such a large percentage of the compounds tested possessed significant inhibitory activity on mice, it became of importance to determine the concentrations of each of the compounds in the extracts. Only then could the relative importance of each constituent to the overall pharmacological response of the bark resin be estimated accurately. High performance liquid chromatography was utilized to quantify the UV absorbing compounds of the extract. The results of this analysis are presented in Table IX. It can be seen from Table IX that the four bis- tetrahydrofuran lignans are, quantitatively, the most important constituents. Together, they comprise approximately 38%, by weight, of the thirteen compounds quantified. More importantly, with respect to the present study, is the fact that they represent approximately 87% of the constituents that were active in suppressing locomotor activity. 1 1 5 Compound RT(min) Rf Concentration 1 2 Mg/g dry wt bark 1a 18.80 0.75 0.89 1 .5 1b 18.45 0.68 0.77 3.0 2 15.94 0.63 0.53 3.2 3 15.22 0.59 0.46 1 .6 4 13.33 0.37 0.19 50 t 5 13.33 0.31 0.18 50 f 6 14.47 0.26 0.14 26 f 7 14.47 0.21 0.11 26 f 8 1 1 .99 0.15 0.05 1 .2 9 11.22 0.09 0.Q3 1 .3 X 24.00 0.56 0.10 1 1 Y 22.71 0.53 0.06 8 /3-sitosterol 0.75 0.33 95 t- Table IX - Quantitative analysis of thirteen most common constituents of diethyl ether extract of V. elongata bark. solvent 1 = pet ether/ethyl ether/acetonitrile(6/6/1). solvent 2 = hexane/chloroform/ethanol(25/25/1). t amounts estimated, based upon the relative size of UV absorbing spots on TLC. t- amount estimated, based upon amount isolated from 1500 g of dried bark. 1 1 6 d. Effect of bis-tetrahydrofuran 1iqnans on isolation induced aggression After the administration of compounds 4 to 7, it was observed that the treated mouse often behaved in an unusually passive manner. At doses lower than those required to suppress locomotor activity, distinct behavioral changes could be discerned. Normal exploratory activity was very much reduced and a general hesitancy was apparent. The mice were especially timid and when handled showed little resistance. They were extremely reluctant to defend themselves. These observations led to the hypothesis that the bis- tetrahydrof uran lignans possessed anti-aggressive activity and that this may play a role in the use of V. elongata bark as an arrow poison. This hypothesis was tested by examining the effect of these four compounds upon aggression induced in mice by isolation. The induction of aggression in mice by some sort of isolation procedure has been used extensively as an experimental model of aggression (Brain and Jones, 1982; Scott and Fredericson, 1951). A variety of parameters have been measured as indicators of aggressiveness. In the present study, isolated mice were selected for experimentation, based upon either overt aggressiveness or their nervousness upon introduction to a group-housed mouse. These two groups of mice displayed different behavior. Consequently, different measurements were made in either case. The less aggressive group of isolated mice were used to 1 1 7 examine the effect of different doses of the most abundant bis- tetrahydrofuran lignan, epi-sesartemin (4) on aggression related behavior. These mice were observed to interact with newly introduced group-housed mice in a characteristic manner. Each isolated mouse resisted attempts at socialization by the group-housed mouse, adopting a defensive posture and emitting a series of sharp squeaks. When not confronted by the group- housed mouse, they displayed exploratory behavior, combined with pursuit and anogenital investigation of the group-housed mouse. This latter activity has been correlated with aggressive behavior and is predictive of it (Simon et al, 1983). As indicators of behavior, these three activities were measured during a 5 minute interval beginning 15 minutes after injection of lignan. The results are presented in Table X. The results indicate that epi-sesartemin had a dose- dependent effect upon the behavior observed in previously isolated mice. Not only was the time spent in exploratory behavior reduced upon administration of this lignan, but the defensive behavior observed in response to social contact by the group-housed mouse, was attenuated significantly. Moreover, the time spent in actively pursuing the group housed mouse was markedly reduced, even at the lowest dose tested (3.1 mg/kg). This is especially suggestive of a reduction in aggressiveness. To verify this initial observation, isolated mice which demonstrated overtly aggressive behavior were used to test the effect of the four bis-tetrahydrofuran lignans. Each was 1 18 Dose of Behavior observed in 5 minute period Epi-sesartemin Number of % time in % time in pursuit (mg/kg) squeaks explorat ion plus anogenital invest igat ion 25 4 1 1 0 12.5 3 9 0 6.3 6 21 1 3. 1 6 28 1 2 0 53 27 24 Table X - Effect of epi-sesartemin on three behavioral parameters related to aggressiveness of isolated mice. Values are averages of three injections of each dose. 1 19 injected at doses of 25 mg/kg and the effect on the readiness of the isolated mouse to attack the group-housed mouse was observed. Five minutes after injection, the mice were placed together and the time elapsed before the first attack by the isolated mouse was measured. The data of Table XI indicate that each of the lignans tested significantly suppressed the tendency of the previously isolated mouse to attack. The mice treated in this way were particularly passive, offering little resistance to the social contact initiated by the group housed partner. They responded by remaining quietly in one corner of the cage and huddling with eyes partially closed, when contacts were made. 4. DISCUSSION The results of this study indicate that the arrow poison prepared from Virola bark may well be unique among aboriginal arrow poisons. The physiological effects observed in mice were subtle and restricted to behavior. No serious disruption of normal physiology could be distinguished and no mortality was observed in any of the mice tested, despite the administration of relatively large doses of bark extract. The Yanomamo darts analysed by Galeffi et auL (1983) were reported to each carry approximately 150 mg of resin. Laboratory mice readily survived doses of 200 mg/kg of V. elongata bark extract. It seems unlikely that wild game, which would be expected to weigh in excess of 1 kg and would, therefore, receive a significantly lesser dose than that 1 20 Compound Latency to attack: time(min.) epi-sesartemin(4) >30 sesartemin(5) >30 epi-yangambin(6) >30 yangambi n(7) >30 control 3.7 Table XI - Effect of bis-tetrahydrofuran lignans on aggressiveness of mice. Latency to attack refers to the time between the introduction the isolated mouse (to which lignan had been administered) to the socialized mouse and the time of the first attack of the isolated mouse. 121 administered to the mice in this study, would suffer any mortality from administration of the resinous portion of the darts. The suggestion that the tryptamine constituents of the bark are responsible for interrupting the normal responses of injured game, in this way facilitiating its capture, is an intriguing one. If true, it is an interesting ethnobiological phenomenon. There seems to be no preparation with comparable effects amongst the many types of arrow poisons. The primary alkaloid of the majority of the bark samples of Virola elongata that have been analysed is 5-MeODMT (Agurell et al, 1969; 1980; Galeffi et al, 1983; Holmstedt et al, 1980; McKenna e_t al, 1984). When tested for ability to bind to serotonin receptor sites (Glennon and Gessner, 1975), behavioral effects in rodents (Gessner and Page, 1962; Glennon et al, 1980; Ho et a_l, 1970) and psychotomimetic activity in man (Shulgin, 1978), 5-MeODMT is consistently shown to be the most potent naturally occurring tryptamine. However appealing this hypothesis is, the data presented here offer no support for it. The non-alkaloidal and alkaloidal fractions from equivlent amounts of V. elongata bark were compared and the non-alkaloidal fraction was clearly observed to have a greater effect on the behavior of the mice to which they were administered (Table VII). The injection of the non-alkaloidal fraction at a dose of 3.8 mg/kg was responsible for a reduction in activity lasting several hours, while the alkaloidal fraction from an equivalent amount of 1 22 bark (0.19 mg/kg) produced no observable change in behavior. Behavioral changes in mice in response to 5-MeODMT administration have been detected at doses as low as 0.5 mg/kg (Ho et al, 1970; Chapter II, this thesis). It is necessary to consider the possibility that the level of 5-MeODMT in the elongata bark sample used to prepare the extracts for these experiments was unusually low. Galeffi et a_l (1983) report that 5-MeODMT comprised some 8% (by weight) of the resin coating the tips of the Yanomamo darts analysed by them. Although the bark resin, itself, has not been used in this study, it is likely that a methanolic extract of the bark bears a close resemblance to the resin with respect to type and amount of chemical constituents. The alkaloidal fraction (which consisted entirely of 5-MeODMT) comprised 4.5% (by weight) of the methanolic extract. Although this figure is slightly less than that reported by Galeffi e_t al (1983), the difference is not sufficient to explain the relative lack of activity observed from the alkaloidal fraction in these experiments. The reduction in locomotor activity observed by the non- alkaloidal extract is likely the result of the complex mixture of phenolics present. The identity of the red-brown constituent which forms upon exposure of the resin to air is not known. It has been suggested that it is the result of the oxidation of flavonoid constituents and perhaps the formation of pigmented dimers (Gottlieb, 1979). The contribution of the non-polar constituents identified 1 23 from the diethyl ether extract to the biological activity of the non-alkaloidal fraction is difficult to assess. The results of the locomotor activity studies carried out show that compounds 1 to 9 are, to varying degrees, effective in reducing spontaneous motor activity at moderately high doses ( 1 0-25 mg/kg). It is unclear, however, whether the concentrations of these constituents is high enough in the resin to make a significant contribution to its overall biological effects. The bis-tetrahydrofuran lignans epi-sesartemin (4), sesartemin (5), epi-yangambin (6) and yangambin (7), because of their high concentrations compared to the other constituents of the extract, would be expected to contribute proportionately more to the total biological activity. The bi s-tetrahydrof uran lignan, pinoresinol |3-D-glucoside has been shown to have hypotensive activity and syringaresinol 0-D-glucoside is claimed to increase the performance of animals under stress (reviewed by MacRae and Towers, 1984). The mechanism by which these two lignans exert their effect is not understood. Similarly, the mechanism by which compounds 4 to 7 reduce isolation induced aggression is completely unknown. Many agents are now known which affect aggression in isolated mice. Among those which have been shown to reduce it are antipsychotic drugs such as chlorpromazine (Kreiskott, 1981) or reserpine (Lai e_t al, 1975), the anxiolytic drugs such as barbiturates or benzodiazepines (Haefely e_t a_l, 1981), the 1 24 alkaloids morphine and muscimol (Poshivalov, 1982), certain opiate peptides such as met-enkephalin and neo-endorphin and certain hypothalamic-pituitary peptides such as somatostatin (Poshivalov, 1982). The.doses required for an effect to be observed range between 1 and 25 mg/kg for all but the anxiolytic drugs, for which much higher doses are required. The bis-tetrahydrofuran lignans, epi-sesartemin, sesartemin, epi-yangambin and yangambin are, by comparison, moderately effective anti-aggressive agents. It is possible that the neolignans, eusiderin and virolongin, as well as the tetrahydrofuran lignans dihydrosesartemin and 0-dihydroyangambin, may also play a minor role in the pharmacological activity of the resin. The ether extract of Magnolia obovata bark, a Chinese medicinal agent, has been shown to have sedative effects in mice (Watanabe e_t a_l, 1973). The major constituents are neolignans (Fujita e_t a_l, 1972) not unlike eusiderin and virolongin. The available evidence indicates that V. elongata bark does not contain a highly toxic constituent which might explain its use as an arrow poison. Toxic compounds have not previously been reported from the family Myristicaceae. None was detected during a thorough screening of V. elongata bark. Moreover, methanol extracts of V.. pavonis, V. sebifera and V. calophylla have also been tested by administering them to mice at a dose of 100 mg/ml and found no evidence of toxicity or mortality. It can be concluded that the use of Vi rola sp. as an arrow poison is either based on a more subtle biological 1 25 activity than toxicity or, alternatively, that it is without a physiological basis. It should be pointed out that several important uncertainties remain regarding the use of V. elongata as an arrow poison. The first concerns the exact means of preparing the resin. The present study may be criticized because resin prepared in the manner of the Yanomamos has not been used. Since the method of preparing the resin is straightforward and involves nothing more than collecting and warming the bark over a fire, it seems improbable that the dart tip resin differs significantly from an extract of the bark. The possibility exists, however, that the application of heat promotes the formation of some active constituent and this should, therefore, be borne in mind. The source of the Virola sample is also an important consideration. The bark used in this study was collected in Peru, while the material used in the preparation of authentic Yanomamo arrow poison is obtained from trees growing in northern Brazil. The possibility that geography may contribute to qualitative or quant itiative variation in chemical constituents must be considered. Finally, there is some doubt as to the relationship between V. elongata and V. theiodora, the two sources of Yanomamo arrow poison. Virola theiodora is considered a separate species by Schultes (1969) and most of the sources of arrow poison have been attributed to this species. Specialists in the taxonomy of this genus have generally regarded V. 1 26 theiodora and V. elongata to be synonomous (Rodrigues, 1980; Smith and Wodehouse, 1937) and in the latest monograph, only the latter name is recognized (Rodrigues, 1980). Although the species concept is not yet completely resolved, there is general agreement that two types can be distinguished in the field. Both are used as a snuff and arrow poison (Prance, G.T., pers. comm.). The present study is the first report of the non-alkaloidal constituents of either tree. A comparative study of the chemistry of these two types is now in order. Gaps exist in our understanding of the ethnological aspects of Vi rola resin arrow poison. Of particular importance is the way in which it is used for hunting. Salathe (1931) has reported that it is used for hunting monkeys and birds. Becher (1960), Prance (1970) and Schultes and Holmstedt (1968) have pointed out that this is a slow acting arrow poison and that the wounded animal must be followed for some time so as to allow the substance to take effect. It is conceivable that assaying the resin by intraperitoneal injection in mice is hot an appropriate experimental system. Either the mode of administration or the response of the particular animal used may introduce unsuspected variables. Of particular importance from this point of view, is the type of animals hunted with this arrow poison. Primate behavior may be more easily disrupted by 5-MeODMT than that of rodents. Clearly, more complete descriptions of field observations of the use and effects of the arrow poison will be needed if these questions are ever to be fully answered. 127 LITERATURE CITED Chapter III Abe, F., Yakara, S., Kubo, K., Nonaka, G., Okabe, H. and Nishioka, I. (1974) Studies on Xanthoxylum spp. II. Constituents of the bark of Xanthoxylum piper iturn DC. Chem. Pharm. Bull. 22, 2650-2655. Agurell, S., Holmstedt, B., Lindgren, J.E. and Schultes, R.E. (1969) Alkaloids in certain species of Vi rola and other South American plants of ethnopharmacologic interest. Acta. Chem. Scand. 23, 903-916. Barata, L.E.S., Baker, P.M., Gottlieb, O.R. and Ruveda, E.A. (1978) Neolignans of Virola surinamensis . Phytochem. 17, 783-786. Bauer, W.P. (1965) Der curare-giftkreis im lichte neuer chemischer unter suchunger. Baessler Archiv. Neue Folge 13, 207-234. Becher, H. (i960) Die Surara und Pakidai, zwei Yanonami-Stamme in nordwestbrasilien. Mitteil. Mus. Volkerkunde Hamburg 26, 1-138. Biocca, E. (1966) Viaggi tra gli Indi , Vol. 2, C.N.R., Roma. Biocca, E., Bovet, C, Galeffi, C. and Martini Bettolo, G.B. (1965) Sui curaro Yanoama. Un nuovo tipo di curaro indigeno "Curaro di torrefazione e percolazione." Atti Accad. Naz. Lincei. Rend. CI. Sci. Fis. Mat. Nat. 38, 34- 38. Birch, A.J. and Smith, M. (1964) The constitution of gmelinol. Part IV. Stereochemistry and relationships to other lignans. J. Chem. Soc. 2705-2708. Birch, A.J., MacDonald, P.L. and Pelter, A. (1967) A revised structure for neogmelinol: determinations of configurations in tetrahydrofuranoid lignans. J. Chem. Soc. (C), 1968-1972. Birch, A.J., Moore, B., Smith, E. and Smith, M. (1964) The conversion of gmelinol into neogmelinol. J. Chem. Soc, 2709-2712. Bisset, N.G.(1981) Arrow poisons in China. Part II. Aconitum- botany, chemistry and pharmacology. J. Ethnopharmacol. 4, 247-336. Blair, G.E., Cassady, J.M., Robbers, J.E., Tyler, V.E. and Raffauf, R.F. (1969) Isolation of 3,4',5-trimethoxy- 1 28 trans-stilbene, otobaene and hydroxyotobaene from Vi rola cuspidata. Phytochem. 8, 497-500. Brain, P.F. and Jones, S.E. (1982) Assessing the unitary nature of agression by using strains of laboratory mice. Agress. Behav. 2, 108-111. Braz Filho, R. , Mourao, J.C, Gottlieb, O.R. and Maia, J.G.S. (1976) Lanthanide induced shifts as an aid in the structural elucidation of eusiderins. Tetr. Lett. 15, 1157-1160. Briggs, L.H. Cambie, R.C. and Crouch, R.F. (1968) Lirioresinol-C dimethyl ether, a diaxially substituted 3,7-dioxabicyclo[3,3,0] octane lignan from Macropiper excelsum (Forst. f.) Miq. J. Chem. Soc. (C) 3042-3045. Budden, R., Kuhl, U.G. and Bahlsen, J. (1979) Experiments on the toxic, sedative and muscle relaxant potency of various drug solvents in mice. Pharmac. Ther. 5, 467-474. Chen, C.L., Chang, H.M. and Cowling, E.B. (1976) Aporphine alkaloids and lignans in heartwood of Liriodendron tulipi fera . Phytochem. 15, 547-550. Chiba, M., Hisada, S., Nishibe, S. and Thieme, H. (1980) 13C- NMR analysis of symplocosin and (+)-epipinoresinol* glucoside. Phytochem. 19, 335-336. Daly, J.W.(1982) Alkaloids of Neotropical poison frogs (Dendrobatidae). Forsch. Chem. Organ. Naturstoffe 41, 205-340. Drewes, S.E. and Fletcher,'J.P. (1974) Polyhydroxystilbenes from the heartwood of Schotia brachpetala . J. Chem. Soc. Perkin I, 961-962. Duffield, A.M. (1967) Mass spectrometric fragmentation of some lignans. J. Heterocycl. Chem. 4, 16-22. Fernandes, J.B., Nilce de S. Ribeiro, M., Gottlieb, O.R. and Gottlieb, H.E. (1980) Eusiderins and 1,3-diarylpropanes from Virola species. Phytochem. 19, 1523-1525. Fujita, M., Itokawa, H., and Sashida, Y. (1972) Honokiol, a new phenolic compound isolated from the bark of Maqnolia obpvata Thunb. Chem Pharm. Bull. 20, 212-213. Galeffi, C, Messana, I. and Marini Bettolo, G.B. (1983) N,N- dimethyl-5-methyoxytryptamine, a component of a dart poison of the Yanoama Indians. J. Nat. Prod. 46, 586-587. Gessner, P.K. and Page, I.H. (1962) Behavioral effects of 5- 129 methoxy-N,N-dimethyltryptamine, other tryptamines and LSD. Am. J. Physiol. 203, 167-172. Gessner, P.K., Godse, D.D., Krull, A.H. and McMullan, J.M. (1968) Structure activity relationships among 5-methoxy- N,N-dimethy1tryptamine, 4-hydroxy-N,N-dimethyltryptamine (psilocin) and other substituted tryptamines. Life Sci. 7, 267-277. Gessner, P.K., Mclsaac, W.M. and Page, I.H. (1961) Pharmacological actions of some methoxyindolealkylamines. Nature 4771, 179-180. Glennon, R.A. and Gessner, P.K. (1975) Correlations between the activity of N,N-dimethyltryptamines as 5- hydroxytryptamine antagonists and their quantum chemical parameters. Pharmacologist 17, 259-268. Glennon, R.A., Young, R., Rosecranz, J.A. and Kallman, M.J. (1980) Hallucinogenic agents as discriminative stimuli: a correlation with serotonin receptor affinities. Psychopharmacol. 68, 155-158. Gottlieb, O.R. (1979) Chemical studies on medicinal myristicaceae from Amazonia. J. Ethnopharmacol. 1, 309- 323. Gottlieb, O.R., Maia, J.G.S. and Mourao, J.C. (1976) Neolignans from a Licaria species. Phytochem. 15, 1289- 1 291 . Gottlieb, O.R., Maia, J.G.S. and Ribeiro, M.N. de S. (1976) The chemistry of Brazilian Myristicaceae. VII. Neolignans from Virola carinata . Phytochem. 15, 773-774. Grahame-Smith, D.G. (1971) Inhibitory effect of chlorpromazine on the syndrome of hyperactivity produced by L-tryptophan or 5-methoxy-N,N-dimethyltryptamine in rats treated with a monoamine oxidase inhibitor. Br. J. Pharmacol. 43, 856- 864. Greger, H. and Hofer, 0. (1980) New unsymmetrically substituted tetrahydrofuran lignans from Artemesia absinthium . Tetrahedron 36, 3551-3558. Haefely, W., Pieri, L., Pole, P. and Schaffner, R. (1981) General pharmacology and neuropharmacology of benzodiazepine derivatives. In Psychotropic Agents• Part II. (Hoffmeister, F. and Stille, G., eds), Springer- Verlag, Berlin, pp. 13-262. Hall, L.D. (1964) Nuclear magnetic resonance, Adv. Carb. Res., 19, 51-94. 1 30 Hall, L.D., Black, S.A., Slessor, K.N. and Tracey, A.S. (1972) Conformation studies on the 1,2;5,6-Di-O-isopropylidene- D-hexoses. Can. J. Chem. 50, 1912-1924. Hillis, W.E. and Ishikura, N. (1966) Chromatographic and spectral properties of stilbenes. J. Chromatog. 32, 323- 336. Ho, B.T., Mclsaac, W.M. An, R., Harris, R.T., Walker, K.E., Kralil, P.M. and Airaksinen, M.M. (1970) Biological activities of some 5-substituted N,N-dimethyltryptamines, a -methyltryptamines and gramines. Psychopharmacologia 16, 385-394. Hobbs, J.J. and King, F.E. (1960) The chemistry of extractives from hardwoods. Part XXIX. Eusiderin, a possible byproduct of lignin synthesis in Eusidroxylon zwazeri . J. Chem. Soc. 4732. Holmstedt, B., Lindgren, J.E., Plowman, T., Rivier, L., Schultes, R.E. and Tovar, 0. (-1980) Indole alkaloids in Amazonian Myristicaceae; Field and laboratory research. Bot. Mus. Leafl. Harv. Univ. 28, 215-234. Hughes C.W., Schneider, B.F. and Norton, S. (1978) Circadian and sex differences in hyperactivity produced by amphetamine in rats. Physiol. Behav. 22, 47-51. Inglis, T.R. (1975) Enriched sensory experience in adulthood increases subsequent exploratory behaviour. Animal Behav. 23, 932-940. Inoue, K., Inoue, H. and Chen, C.-C. (1981) A napthoquinone and a lignan from the wood of Kiqelia pinnata . Phytochem. 20, 2271-2276. Isogai, A., Murakoshi, S., Suzuki, A. and Tamura, S. (1973) Structures of new dimeric phenylpropanoids from Myristica fragrans Houtt. Agr. Biol. Chem. 37, 1479-1486. Jeffries, P.R., Knox, J.R. and White, D.E. (1961) The occurrence of lirioresinol-/3-dimethyl ether in Eremophila glabra R.Br. Aust. J. Chem. 14, 175-177. Kinnard, W.J. and Watzman, N. (1966) Techniques utilized in the evaluation of psychotropic drugs on animal activity. J. Pharm. Sci. 55, 995-1012. Kreiskott, H. (1981) Behavioral pharmacology of antipsychotics. In Psychotropic Agents. Part I. (Hoffmeister, F. and Stille, G., eds), Springer-Verlag, Berlin, pp. 59-88. 131 Lai, A., Tin-Wa, M., Mika, E.S., Persinos, G.J. and Farnsworth, N.R. (1973) Phytochemical investigation of Vi rola peruviana, a new hallucinogenic plant. J. Pharm Sci. 62, 1561-1563. Lai, H., Gianutsos, G., and Puri, S.K. (1975) Comparison of narcotic analgesics with neuroleptics on behavior measures of dopinaminergic activity. Life Sci. 17, 29-34. Lewis, W. H. and Elvin-Lewis, M.P.F.(1977) Medical Botany , Wiley Interscience, John Wiley and Sons, New York. Maia, J.G.S. and Rodrigues, W.A. (1976) Vi rola theiodora como alucinogena e toxica. Acta Amazonica 6, 21-23. McKenna, D.J., Abbott, F.S. and Towers, G.H.N. (1984) Monoamine oxidase inhibitors in South American plants Part II: Constituents of orally active Myristicaceous hallucinogens. J. Ethnopharmacol. (submitted). Mebs, D., Briining, F. and Pfaff, N. (1982) Preliminary studies on the chemical properties of the toxic principal from Diamphidia nigroornata larvae, a source of Bushman arrow poison. J. Ethnopharmacol. 6, 1-12. Norton, S. (1980) Behavioral toxicology: a critical appraisal. In The Scientific Basis of Toxicity Assessment (ed. Witschi" H.R. ) Elsevier Press, New York, pp. 91-107. Pelter, A. (1967) The mass spectra of oxygenated heterocycles. Part IV. The mass spectra of some complex lignans. J. Chem. Soc. (C), 1376-1380. Pelter, A. and Ward, R.S. (1978) Substituted furofurans. In Chemistry of Lignans (Rao, C.B.S., ed), Andhra University Press, India, pp. 227-275. Pelter, A., Stainton, A.P. and Barber, M. (1966) The mass spectra of oxygen heterocycles. (III). An examination of simple lignans. J. Heterocycl. Chem. 3, 191-197. Poshivalov, V.P. (1982) Ethological analysis of neuropeptides and psychotropic drugs: effects on intraspecies agression an sociability of isolated mice. Agress. Behav. 8, 355- 370. Prance, G.T. (1970) Notes on the use of plant hallucinogens in Amazonian Brazil. Econ. Bot. 24, 62-68. Rodrigues, W.A. (1980) Revisao taxonomica das especies de Vi rola Aublet (Myristicaceae) do Brazil. Acta Amazonica 10, Suppl. 1-127. 1 32 Russel, G.B. and Fenemore, P.G. (1973) New lignans from leaves of Macropiper excelsum . Phytochem. 12, 1799-1803. Sakata, K., Kawazu, K. and Mitsui, O. (1971) Studies on a piscicidal constituent of Hura crepitans. Part I. Isolation and characterization of huratoxin and its piscicidal activity. Agr. Biol. Chem. 35, 1084-1091. Salathe, G. (1931 ) Les indiens Karime. Rev. Inst. Ethnol. Univ. Nac. Tucuman 2, 297-316. Sarkanen, K.V. and Wallis, F.A. (1973a) Oxidative dimerizations of (E)- and (Z)-Isoeugenol (2-methoxy-4- propenylphenol) and (E)- and (Z)-2,6-Dimethoxy-4- propenylphenol. J. Chem. Soc. Perkin I, 1869-1878. Sarkanen, K.V. and Wallis, F.A. (1973b) PMR analysis and conformation of 2,5-bis-(3,4,5-trimethoxyphenyl)-3,4- dimethyltetrahydrofuran isomers (I). J. Heter. Chem. 10, 1025-1027. Schultes, R.E. and Holmstedt, B. (1968) The vegetal ingredients of the Myristicaceous snuffs of the Northwest Amazon. Rhodora 70, 113-160. Schultes, R.E. and Holmstedt, B. (1971) De plantis toxicariis e mundo novo tropicale commentationes. VIII. Miscellaneous notes on Myristicaceous plants of South America. Lloydia 34, 61-78. Scott, J.P. and Fredericson, E. (1951) The cause of fighting in mice and rats. Physiol. Zool. 24, 273-309. Shah, N.S. and Hedden, M.P. (1978) Behavioral effects and metabolic fate of N,N-dimethyltryptamine in mice pretreated with 0 -diethylaminoethyl- diphenylpropylacetate (SKF 525-A), iproniazid and chlorpromazine. Pharmacol. Biochem. Behav. 8, 351-356. Shulgin, A.T. (1978) Characterization of three new psychotomimetics. In The Pharmacology of Hallucinogens (Stillman , R.C. and Willette, R.E., eds), Pergamon Press, New York, pp. 74-83. Simon, N.G., Gray, J.L. and Gandelman, R. (1983) An empirically derived scoring system for intermale aggression in mice. Aggress. Behav. 9, 157-166. Smith, A.C. and Wodehouse, R.P. (1937) The american species of Myristicaceae. Brittonia 2, 393-510. Smith, M. (1963) The structure of ( - ) -olivil. Tetr. Lett. 133 15, 991-992. Spjut, R.W. and Perdue, R.E. (1976) Plant folklore: a tool for predicting sources of antitumour activity. Cancer Treat. Rep. 60, 979-985. Szara, S. (1956) Dimethyltryptamine: its metabolism in man; the relation of its psychotic effect to the serotonin metabolism. Experientia 12, 441-442. Taniguchi, E. (1972) The mass spectra of phrymaroline, 1,2- dioxygenated-3,7-dioxabicyclot3.3.0] octane lignans. Agr. Biol. Chem. 36, 1497-1503. Taniguchi, E. and Oshima, Y. (1972a) New gmelinol-type lignan, leptostachyol acetate. Tetr. Lett. 8, 653-656. Taniguchi, E. and Oshima, Y. (1972b) Structure of phrymarolin- II. Agric. Biol. Chem. 36, 1489-1496. Walsh, R.N. and Cummins, R.A. (1976) Mechanisms mediating the production of environmentally induced brain changes. Psychol. Bull. 82, 986-1000. Watanabe, K., Goto, Y. and Yoshitomi, K. (1973) Central depressant effects of the extracts of Magnolia cortex. Chem. Pharm. Bull. 21, 1700-1708. Weinges, K. (1961) Uber einige neue Lignane und stereochemische Zusammenhange in der Lignangruppe. Chem. Ber. 94, 2522-2532. Worthley, E.G. and Schott, CD. (1966) Pharmacotoxic evaluation of nine vehicles administered intraperitoneally. to mice. Lloydia 29, 123-129. 1 34 IV. STUDIES ON THE PHARMACOLOGICAL ACTIVITY OF AMAZONIAN EUPHORBIACEAE PART A. MULTIPLE SCREENING OF AMAZONIAN EUPHORBIACEAE FOR BIOLOGICAL ACTIVITIES 1. INTRODUCION The family, Euphorbiaceae, is well represented among the plants used by indigenous South Americans (Appendix A). It is the third most important plant family in the Amazon region, with respect to numbers of species used as medicinal plants by the local inhabitants (Chapter I). It comprises some 6% of the almost 800 species of medicinal plants enumerated (Appendix A).- The range of pharmacological actions observed is broad and includes preparations effective against wounds, skin infections, malignancies, inflammations and used as tonics and aphrodisiacs. Many species are poisonous and their use is probably based on this property. Certain species are used widely as medicinals by the large mestizo population. They are frequently prescribed by herbalists and can be purchased in the market places. These include Alchornea castaneifolia, Phyllanthus urinaria and Croton lechleri..Alchornea castaneifolia is a small tree that grows, partly submerged during the period when the rivers are high, along the banks of the Amazon's major tributaries. Its bark is highly esteemed for the treatment of rheumatic 1 35 conditions and its leaves are taken as a decoction to stimulate, or rejuvenate, the older man. Phyllanthus urinaria, and perhaps also Phyllanthus amarus, are used to treat a number of ailments of the kidney and are believed to be effective in causing the fragmentation and/or expulsion of kidney and gall stones. Croton lechleri is tree of the tierra f irme whose thick red bark resin is collected and used as a wound treatment and for internal injuries and malignancies. From personal experience, the claims concerning its abilities to promote healing of superficial wounds seem not to be greatly exaggerated. Two species, Jatropha curcas and Jatropha gossypiifolia, are dooryard cultivars. They are both used widely in the treatment of burns and skin infections and occasionally to treat malignancies. In Jonathan Hartwell's survey of plants used against cancer, the family Euphorbiaceae is very well represented (Hartwell, 1969). The genera Acalypha (9 species), Croton (9 species, including C. palanostigma, Chamaesyce (both C. hyssopi folia and C. thymi folia, among others), Jatropha (9 species, including J. curcas and J.. gossypi i f ol ia) , Manihot (Manihot esculenta), and Phyllanthus (3 species, including P. urinaria) are already represented in the literature. Only a few of the plants listed above have been examined previously for their biologically active chemical constituents. Of those that have, Jatropha curcas has been found to contain a compound, jatrophone, that possesses antitumour activity (Kupchan et. a_l, 1976). Similarly, 1 36 Phyllanthus brasiliensis, a plant that is closely related to P. urinaria and P. amarus, has been shown to contain the antitumour substance, phyllanthocin (Kupchan, 1977). One of the species, Croton lechleri, has been found to contain an alkaloid, taspine, that is not only anti• inflammatory (Persinos-Perdue et a_l, 1979), but also possesses the ability to inhibit viral reverse transcriptase (Sethi, 1977). Major constituents of several other species have been found to be lignans: dimers of phenylpropanoid units. Recently, Jatropha gossypi i folia has been shown to contain the lignan, 2-piperonyl-3-veratryl-3R-7-butyrolactone (Chatterjee et al, 1981). Phyllanthus urinaria has been studied intensively from the point of view of its chemistry and five lignans have been identified: phyllanthin, hypophyllanthin, niranthin, nirtetralin, and phyltetralin (Anjaneyulu, 1973). These compounds are similar in structure to the podophyllotoxin group of lignans. Podophyllotoxin, and related compounds, have recently been shown to have antiviral activity (Bedows and Hatfield, 1982). The variety in structure of secondary products from Euphorbiaceous species is already known to be great. The predominant biological activity observed for compounds produced by plants of this family is toxicity (Kinghorn, 1979). The possibility that the presence of toxic compounds could explain the widespread uses of Euphorbiaceous species in the Amazon was considered. It is apparent, from what little we know of the diseases 1 37 of primitive Amazonian societies, that infections and infectious diseases have always figured prominently (Black, 1975; Black et al, 1978; Buck et al, 1968; Larrick et al, 1979). Infections of the eye, ear and mouth are not uncommon and the tendency for injuries or insect bites to become infected is always great (Larrick et a_l, 1979). The incidence of exposure to the fungal pathogens Histoplasma capsulatum or Paracoccidiodes brasiliensis is high (Larrick e_t §_1,1979; Mok and Netto, 1978). The viral diseases, herpes simplex, mononucleosis (Epstein-Barr virus), chicken pox, hepatitis B and cytomegalovirus diseases have been shown to be endemic to all the Brazilian tribes examined (Baruzzi et aJL, 1971; Black, 1975; Neel et a_l, 1968). Amazonians are regularly exposed to a variety of protozoan and helminthic parasites (Larrick et. al, 1979) . It is reasonable to expect that a proportionately large fraction of the medicinal plants used by indigenous Amazonians would be utilized in the treatment of infectious diseases of this nature. The toxic properties of Euphorbiaceous species may be applied in such a way. These observations suggested that the screening of Amazonian Euphorbiaceous species for a variety of pharmacological activities related to toxicity or growth regulation, would constitute an interesting study. Thirty-four species from this family were selected at random and samples of leaf, bark (or the aerial portions of herbaceous species) were collected. Sixteen of these 34 species had some 1 38 ethnobotanical use. Extracts of the plant material were prepared and examined for their ability to inhibit the growth of bacteria, yeasts, dermatophytic fungi, viruses and potato tumours, as well as their toxicity to brine shrimp. Antibacterial activity against the gram negative organism, E. coli and the gram positive bacterium, S. aureus was examined. The yeasts tested included brewer's yeast, S. cerevisiae and the pathogen, C. albicans. Four dermatophytic fungi were tested: Microsporum canis, M. fulvum, M. gypseum and Trichophyton gallinae. Two animal viruses for which laboratory techniques for handling are available were examined for their sensitivity to the extracts. These were Sindbis virus, a membrane encapsulated, single stranded RNA virus and murine cytomegalovirus, a membrane encapsulated, double stranded DNA virus. The ability of the 34 Euphorbiaceous extracts to inhibit the growth of tumours elicited by Agrobacterium tumefaciens in potato tuber discs was also studied. This biological assay has been shown to be predictive of an extract's ability to inhibit the growth of 3PS leukemia cells i_n vivo (Ferrigni et al, 1982) . Finally, the general toxicity of extracts was assessed using the brine shrimp assay (Meyer et a_l, 1982). Toxicity to brine shrimp (Artemia salina) has been used to monitor the presence of a variety of compounds including pesticides, pollutants, mycotoxins, anesthetics, dinoflagellate toxins, morphine analgesics, cocarcinogens and carcinogens (Meyer et 1 39 al, 1982). It is also capable of detecting phorbol esters, the growth regulatory compounds that are widespread in the family Euphorbiaceae (Kinghorn et a_l, 1977). The present study is intended to provide a description of certain of the biological activities of an ethnobotanically important, yet chemically little known, group of plants. It was hoped that this approach would yield general information on the types and potencies of the pharmacological activities among the species tested which will, perhaps, help explain their ethnobotanical uses. It may be possible to gain some appreciation of the extent to which the biological activities of this group of plants is known to indigenous Amazonians (or what has been transmitted into the ethnobotanical literature). This information may lead to the eventual isolation and identification of novel, biologically active constituents. 2. MATERIALS AND METHODS a. Plant material Plants were collected in Peru in 1981. Voucher specimens have been deposited at UNAP (Iquitos, Peru), San Marcos (Lima, Peru), Chicago Field Museum and the UBC herbarium. Determinations were carried out by Dr. M.J. Huft, Chicago Field Museum. Samples were preserved in methanol soon after collection. An extract of podophyllin, the resin of Podophyllum sp. rhizome was included in each of the assays as a positive control. It is known to contain podophyllotoxin, as well as a 1 40 number of other lignans, with antitumour, antiviral and toxic activity (MacRae and Towers, 1984). b. Preparation of plant extracts Leaf, bark, or the aerial part of herbaceous plants, was homogenized in a Waring blender and extracted exhaustively with methanol at 20 °C. The extracts were concentrated by evaporation i_n vacuo and partitioned between water and ethyl acetate. The fractions were separated, dried by evaporation in vacuo and dissolved in either 95% or 50% aqueous ethanol for the ethyl acetate and aqueous fractions, respectively. The final concentration of the extracts was 100 mg/ml. They were stored at -30 °C. c. Antimicrobial screening Bacteria and yeasts The extracts were examined for their ability to inhibit the growth of the bacteria, E. coli and S. aureus and the yeasts S. cerevisiae and C. albicans. The paper disc assay method was used to assess antimicrobial activity. Overnight cultures of the bacteria and yeasts were applied to agar containing nutrient broth (Difco) or Sabouraud's dextrose broth (Difco), respectively, using a sterile cotton swab. Plant extracts (100 mg/ml) were applied in 10 nl aliquots (img) to sterile 6 mm diameter discs of Whatman No. 1 filter paper. These were air dried and applied to the agar plates to which bacteria or yeast had been added. They were incubated at 30 °C until a distinct "lawn" was visible (24 to 48 hours). 141 The zone of inhibition around any of the discs was noted and its diameter recorded. Assays were carried out in triplicate. Dermatophytic fungi Sporulating cultures of M. canis, M. gypseum, M. fulvum and T. gallinae were obtained from the Department of Medical Microbiology, U.B.C. The method used to assay the extracts for inhibition of growth was a modification of that reported by Brancato and Golding (1953). Spore suspensions were prepared by adding 1 ml of sterile distilled water to a mature culture on an agar slant and vortexing it briefly. Using a sharp wire tip, a small amount of the spore suspension was transferred to a plate of Sabouraud's agar, in which had been included various amounts of plant extract and 1% ethanol. The plates were left at 20 °C for 14 days. The diameter of the colonies formed was measured. The assay was carried out in triplicate, d. Ant ivi ral activity Antiviral activity was assayed by determining the effects of extracts on plaque forming ability of Sindbis virus and murine cytomegalovirus (MCMV). Sindbis virus is a single stranded RNA virus in the family Togaviridae while MCMV is a double stranded DNA virus of the herpes virus group. In each case, the effect of the extracts on virus prior to infection and on cells already infected with virus, was measured. Viruses and cells Sindbis virus (obtained originally from Dr. D.E. Vance) had been grown in BHK-21 cells and purified by the technique 1 42 described previously (Mosmann and Hudson, 1973). It was stored in tissue culture medium at -70 °C prior to use. Murine cytomegalovirus (Smith strain) had been propagated in ME cells, purified and stored in the same manner. Both viruses were assayed by their effect on mouse (3T3 strain) embryo cells (passages 18 to 21). Cells were cultivated in Dulbecco's modified Eagle's medium (Gibco) containing 0.37% sodium bicarbonate, 10% fetal bovine serum (Gibco), 100 Units/ml penicillin G (Sigma), 100 Mg/ml' streptomycin sulfate (Sigma) and 2 Mg/ml econazole (Cilag- Chemie AG). They were incubated at 37 °C in an atmosphere of 95% air: 5% carbon dioxide. Plaque forming assay The assays were carried out similarly for S.indbis virus and MCMV. They differed only in the dilution of the virus prepared initially and in the size of the petri dishes on which the mouse embryo cells were grown. In the case of Sindbis virus, the plaques are relatively large (4-5 mm) and 60 mm diameter dishes were used; MCMV produces smaller plaques (approx 1 mm) and 35 mm diameter dishes were used. The viruses were exposed to plant extracts either prior to infection (pre-infection) or after infection (post• infection) of cells. In the treatment pre-infection assay, appropriate dilutions of virus were exposed to the plant extract in Dulbecco's modified Eagle medium (MEM) containing 1% ethanol. Concentrations of 1, 10 and 100 Mg/ml extract were 143 tested. After 2 hours incubation at 37 °C, the virus/plant extract mixture was added to a monolayer of mouse embryo cells and incubated for another 2 hours at 37 °C. Finally, the virus/ plant extract mixture was removed and the monolayer of cells overlayed with Dulbecco's MEM containing 5% FBS and 0.5% agarose. After the plates had solidified, they were incubated at 37 °C until the plaques were sufficiently well developed to be counted. The treatment post-infection assay was carried out by first infecting monolayers of mouse embryo cells with appropriate amounts of virus in Dulbecco's MEM for 2 hours. The virus suspension was then removed and the plates were overlayed with Dulbecco's MEM containing 5% FBS, the plant extract in ethanol (final concentration 1%), and 0.5% agarose. After the overlay had solidified, the plates were incubated at 37 °C until they were ready for counting. Plaques were counted against a dark background using the unaided eye. e. Potato disc tumour assay The procedure used was that of Ferrigni e_t a_l (1982), with some minor modifications. Potatoes (Solanum tuberosum, red Russet variety) were surface sterilized by immersing in 20% bleach for 15 minutes. Sterile discs of 15 mm diameter and 4 mm thickness were prepared from the tubers using a cork hole borer and a knife containing razor blades. The discs were rinsed three times with sterile distilled water, drained and applied to a layer of 1% agar (Difco) in petri dishes. 1 44 Twenty M1 of an overnight culture of Agrobacterium tumefaciens, strain B-6 (obtained from Dr. M.P. Gordon, University of Washington) in nutrient broth (Difco) was applied evenly to each disc. After 2 hours, the plant extracts, dissolved in 10% (v/v) aqueous dimethylsulfoxide (DMSO) (Eastman Kodak Co.), were added to the discs in aliquots of 20 M1 and spread evenly. The discs were incubated at 25 °C in the dark for 12 days. The tumours, which were between 0.25 and 1.5 mm in diameter by this time, were illuminated from the side and counted with the aid of a dissecting microscope. f. Toxic ity to br ine shr imp The method used has been described by Meyer e_t al (1982). Brine shrimp (Artemia salina) eggs (New Technology, Ltd.) were incubated in artificial seawater (Marine Enterprises, Ltd.) for 48 hours at 20 °C. The larvae were counted into groups of 10 and placed in 5 ml of artificial seawater, to which had been added the plant extract in DMSO (final concentration 1%, v/v). Concentrations of 10, 100 and 1000 Mg/ml extract were tested. Survival was measured after 24 hours incubation at 20 °C. g. Analysis of data The screening for inhibition of growth of bacteria and yeasts allowed some degree of quantification, based upon the width of the zone of inhibition surrounding the extract saturated filter paper disc. In the case of the assays for anti-dermatophytic fungus activity, the diameter of the colony 1 45 was measured after growth in the presence of different concentrations of plant extract. Results were expressed as the minimum concentration required to reduce the diameter of the colony to 25% of that of untreated colonies. Three-fold dilutions of plant extract were tested and the minimum dose required to satisfy this criterion was recorded. The anti-viral, anti-potato tumour and brine shrimp toxicity assays involved the collection of a more easily quantified set of data. Data exhibiting easily discernible dose-response relationships were subjected to logit transformation and the LD50 calculated by least mean squares analysis (Ashton, 1972; Hafner and Noack, 1977). 3. RESULTS a. Antimicrobial activity The results of the antibacterial and antifungal screening are presented in Tables XII and XIII. The two species of bacteria tested differed markedly in their response to the extracts. The growth of E. coli was inhibited by extracts of only two of the 34 species of Euphorbiaceae (6%) while S. aureus was inhibited by extracts of 26 (or 76%) of the species tested (Table XII). C. albicans was completely resistant to all the extracts and S. cerevisiae was inhibited by the extracts of only two species. The four dermatophytic fungi tested were more sensitive to the presence of the extracts (Table XIII). Microsporum canis was the most sensitive of the dermatophytes, being Table XII Spec i es Part Extract* Zone of inhib i t i on(w i dth, in mm) E. co1i S. aureus C. a 1b i cans S. cerevisiae Acalypha benensis leaf o - - - - a - 2 - - bark o - 4 - - a - 4 - - A. diversifolia 1 eaf o - 2 - - a - 2 - - A. macrostachya bark o - - - - A. stachyura bark o - 3 - - a - 2 - - Alchornea castaneifolia bark o - - - - a - A. di scolor bark 0 - 1 - a - 2 - - A . triplinervia bark o - 1 - - a •Amanoa aff. oblonqifolia leaf - - - o 1 a - 1 - - bark o - - - - a Aparisthmium cordatum leaf - 2 - - o a bark - 2 - - o a - 2 - - Apodandra loretensis bark o - 2 - - a - 2 - - Caryodendron orinocense bark o - 1 - - a Chamaesyce hyssopifolia aer i a 1 - 1 - - o a - 1 - - C. thym i foli a aer i al o - 1 - - Cnidoscolus peruvianus leaf o - - - - a bark - 3 - - o a Conceveibastrum martianum bark - 1 - - o a - 1 - - Croton cuneatus leaf o - 1 - 2 a - 2 - - bark o - - - - a C . 1echler i bark o - 2 - - a C . pa 1anost igma bark - 2 - - o a - 2 - - C. trinitat i s aer i a 1 o - 1 - - a Didymocistus chrysadenius leaf - o - - - bark o - 1 - 4 a Hevea brasi1iensis leaf - - - - o a - - - - Jatropha curcas aer i a 1 o - - - - a - - - - J. qossypi ifolia aer i al o - - - - J. weberbaueri leaf o - - - - a bark o - - - - a Mabea maynens i s leaf - 3 - - o a - 3 - - M . ni t i da bark o 3 1 - - a - - - - Manihot esculenta aer i a 1 o - 1 - - a - - - - Maprouna guianensis bark o - 1 - - a - 1 - - Phyllanthus amarus aer i a 1 o - - - - a - 3 - - P. orbiculatus aer i a 1 o 5 3 - - a - - - - P. pseudo-conami aer ial o - - - a - - - - P. ur i nar i a aer ial o - - - - a - 4 - - Podocalyx loranthoides leaf o - - - - a - 1 - - bark o - - - - a Securinecja congesta aer i a 1 - - - o Podophy11i n t res i n o - - - a - 2 Table XII - Antimicrobial screening of extracts of Euphorbiaceous plants. Results are expressed as absence(-) or presence(width of zone of inhibition in mm) of antimicrobial activity. * o = organic fraction; a = aqueous fraction t Podophyllin resin extract is included as a positive control Table XIII Concentration producing >75% nh i b i t i on of growth( „g/ml ) Spec i es Part Extract* M. can is M. gypseum M. fulvum T. ga11i nae Acalypha benensis leaf o < 0.125 0 5 0 25 <0.125 a 0. 25 - 2 0 0 5 bark o <0.125 1 0 1 0 0 25 a <0.125 2 0 1 0 0 5 A. diversifolia leaf o <0.125 0 25 0 25 0 25 a <0.125 - 2 0 0 5 A. macrostachya bark o <0.125 0 5 0 5 0 25 A. stachyura bark o <0.125 0 25 0 5 0 25 a <0.125 1 0 1 0 0 25 Alchornea castaneifolia bark o 1 .0 0 25 0 5 0 25 a 1 .0 - - - A. discolor bark o <0.125 0 25 1 0 0 25 a 2.0 - - - A. tr i p1i nerv i a bark o 0. 25 0 25 1 0 0 5 a 1 .0 1 0 1 0 1 0 Amanoa aff. oblonqifolia leaf o 0. 25 2 . 0 2 0 2 0 a 0.5 2 0 2 0 1 0 bark o <0.125 <0.125 2 0 0 5 a 2.0 2 0 2 0 2 0 Apar i sthm i urn cordatum leaf o <0.125 0. 5 1 0 0 5 a 1 .0 2 .0 2 0 1 0 bark o <0.125 0. 5 0 5 0 25 a - 2 0 2 0 1 0 Apodandra loretensis bark o 0. 25 <0.125 0 25 0 25 a 0. 25 2 . 0 2 0 0 25 Caryodendron orinocense bark o 1 .0 1 .0 2 0 2 0 a - 2 . o - 2 0 2 0 Chamaesyce hyssopifolia aer ial o 0.5 1 0 0 5 0 5 a <0.125 2 . 0 - 2 0 C. thym i foli a aer ial o <0.125 <0.125 1 0 0 25 a 0.5 - - 1 0 Cnidoscolus peruvianus leaf 0 <0.125 e 1 0 0 5 a bark <0.125 <0.125 <0.125 <0.125 o a bark 0. 25 0. 5 1 0 25 Conceveibastrum martianum o o a 0. 25 - - 2 0 Croton cuneatus leaf o <0.125 0. 5 0 5 0 25 a 2.0 2 - - bark o <0.125 0 5 0 5 0 5 a 2.0 2 0 2 0 2 0 C. lechleri bark o <0.125 <0.125 - 0 25 a C. pa 1anostiqma bark <0.125 <0.125 0 .5 <0.125 o a <0.125 - 2 .0 1 0 C. trinitatis aer i al o <0.125 0 . 5 1 0 0 125 a Didymocistus chrysadenius leaf <0.125 - - 2 0 o a - - - 1 0 bark o - - - 2 0 a 1 .0 2 .0 2 0 2 0 Hevea brasiliensis leaf o 0. 25 2 .0 2 0 2 0 a Jatropha curcas aer i a 1 <0.125 - - - o a <0.125 1 .0 - - J . cjossyp i i f ol i a aer i a 1 o 1 .0 - - - a 1 .0 - - - J. weberbaueri 1 eaf o 0.5 2 .0 2 0 2 0 a bark <0.125 2 0 2 0 1 0 o a Mabea maynensis leaf 1 .0 1 0 1 0 1 0 o a <1 . 25 2 0 1 0 0 5 M. ni t ida bark o <0. 125 . 25 0 5 0 25 a Manihot esculenta aer i al <0.125 <0.125 <0.125 <0.125 o a 1 .0 - - - Maprouna guianensis bark o <0.125 0 25 0 25 0 25 Phyllanthus amarus aer i a 1 o <0.125 2 0 - 0 25 a 0.5 2 0 2 0 1 0 P. orbicu1atus aer i al o <0.125 0 25 0 5 <0.125 a <0.125 2 0 - 2 0 P. pseudo-conami aer i a 1 o <0.125 0 5 2 0 1 0 a 0. 25 - 1 0 - P. urinaria aerial o o.25 - - 0 25 a <0.125 2 0 2 0 1 0 Podocalyx loranthoides leaf o 0.5 0 5 0 25 0 25 a <0.125 2 0 2 0 2 0 bark o <0.125 1 0 1 0 0 5 a Securineqa conqesta aer i al <0.125 0 5 1 0 1 0 o Podophy11i n t res i n o 0.5 0 5 0 5 0. 25 Table XIII - Screening of Euphorbiaceous plants for anti-dermatophytic fungus activity. The values recorded refer to the dose of extract required to inhibit colony growth by at least 75%. * o = organic fraction; a = aqueous fraction t Podophyllin resin extract is included as a positive control 150 inhibited to some degree by at least one of the extracts of each of the 34 species tested. Microsporum gypseum was inhibited by 97% of the species, although 6 of the active extracts were effective only at high concentrations (>2 mg/ml). Microsporum fulvum was inhibited by 91% of the Euphorbiaceous species tested, 21% of these being of low activity. Finally, 94% of the extracts inhibited T. gallinae and 12% of these were of low potency. The extract of podophyllin, known to contain podophyllotoxin lignans with antiviral activity, was moderately effect ive in inhibiting the growth of each species of dermatophyte, b. Antiviral activity The percentage reduction in number of viral plaques formed in response to treatment of Sindbis virus and murine cytomegalovirus, both before and after infection, is presented in Table XIV. The extracts are particularly effective in inactivating both viruses when applied to them prior to infection of the cells. With the exception of the bark of Didymocistus chrysadenius, which inactivated MCMV but not Sindbis virus, at least one of the extracts of each of the species tested was effective in inhibiting infection by both viruses. The organic fractions were generally more effective than the aqueous ones. Forty of the 42 organic fractions tested inactivated Sindbis virus and all inac.tivated MCMV. The aqueous fractions were slightly less effective. Forty-eight per cent inactivated Sindbis virus while 91% inactivated MCMV. When the extracts were applied to cells which had already Table XIV Inhibition of Plaque Forming Ability S i ndb s virus Murine cytomegalovirus Spec i es Part Ext. Treatment Treatment Treatment Treatment * Pre-i nfecti on Post -i nfect i on Pre-i nfect i on Post -i nfect i on 1 100 1 10 100 LCs o 1 10 100 LC5 o 1 10 100 LCs o 10 LCs o 1/9/ uQ/ 1/9/ 1/9/ 1/9/ n9/ c9/ «/9/ ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml Acalypha benensis leaf o 100 100 100 <1 1 2 2 - 100 100 100 <1 0 0 1 - a 0 0 0 - 2 0 1 - 89 100 100 < 1 0 0 0 - bark o 100 100 100 <1 2 2 2 - 100 100 100 <1 0 0 tox - a 33 69 89 3.0 1 2 1 - 100 100 100 < 1 0 0 tox - A. d i vers i foli a leaf o 100 100 100 <1 0 24 tox 16 98 100 100 <1 0 0 0 - a 100 100 100 <1 0 0 1 - 100 100 100 < 1 0 0 0 - A. macrostachya bark o 100 100 100 <1 0 0 6 - 99 100 100 <1 0 0 0 - a 0 0 o - 0 0 3 - 0 0 99 - 0 0 0 - A. stachyura bark o 100 100 100 <1 0 0 13 - 98 100 100 <1 0 0 0 - a 100 100 100 < 1 0 0 0 - 100 100 100 <1 0 0 0 - Alchornea castaneifolia bark o 96 100 100 <1 1 0 6 - 65 100 100 .30 0 0 0 - a 0 0 0 - O 0 0 - 72 100 100 . 22 0 O 0 - A. discolor bark o 14 56 100 3.9 17 41 tox 20 60 100 tox .86 0 0 tox - a 0 0 0 - 0 0 0 - 100 100 100 <1 0 0 0 - A. tr ipli nervi a bark o 100 100 100 <1 5 37 64 35 72 100 100 . 22 0 0 0 - a 100 100 100 < 1 0 21 tox 17 100 100 tox < 1 0 0 tox - Amanoa aff. oblonqifolia 1 eaf o 100 100 tox < 1 100 100 tox < 1 100 100 tox <1 100 100 tox <1 a 100 100 tox <1 23 95 tox 2.0 100 100 tox <1 100 100 tox <1 bark o 92 100 100 <1 43 69 95 1 . 9 100 100 100 < 1 100 100 100 <1 a 0 0 0 - 0 0 0 - 0 91 100 7 . 7 0 0 100 270 Aparisthmiurn cordatum leaf 0 87 88 99 .08 0 0 tox - 26 99 100 1 . 3 0 0 tox - a 22 58 79 8.0 0 0 0 - 82 100 100 < 1 0 0 0 <1 bark o 63 71 84 . 15 9 24 100 3.8 96 100 100 <1 0 0 0 - a 0 0 . 0 - 0 6 29 147 92 100 100 <1 0 0 0 - Apodandra loretensis bark o 100 100 100 <1 0 6 75 51 57 100 100 .91 0 0 0 - a 41 60 79 2.9 13 17 tox - 100 100 100 < 1 0 0 0 - Caryodendron orinocense bark o 0 0 0 - 0 0 0 - 65 100 100 . 30 0 o 0 - a 100 100 100 <1 0 0 tox - 100 100 tox <1 0 0 tox - Chamaesyce hyssopifolia aer i al o 100 100 100 <1 0 12 18 - 97 100 100 <1 0 0 0 - a 0 0 0 - 0 0 0 - 64 99 100 . 53 0 0 0 - C. thym i fo1i a aer i a 1 0 100 100 100 <1 7 41 tox 15 100 100 100 <1 0 0 tox - a 0 0 0 - 0 0 0 - 0 0 0 - 0 0 0 - Cnidoscolus peruvianus leaf o 100 100 100 <1 100 100 tox < 1 82 100 100 <1 0 0 tox - a 0 0 0 - 0 0 51 208 0 0 98 41 0 0 0 - bark o 1 1 26 60 53 27 35 79 12 0 0 100 22 0 0 0 - a 12 25 63 47 36 38 40 6 . 1 100 100 100 <1 0 0 0 - Conceveibastrum martianum bark o 99 100 100 <1 0 0 48 226 100 100 100 <1 0 0 0 - a 100 100 100 <1 0 12 25 40 100 100 100 < 1 0 0 0 - Croton cuneatus leaf o 100 100 100 <1 0 0 0 - 58 99 100 .63 0 0 0 - a 100 100 100 <1 0 0 0 - 100 100 100 < 1 0 0 0 C. cuneatus bark o 94 99 100 <1 0 0 tox - 100 100 100 <1 0 0 tox - a 100 100 100 < 1 0 0 0 - 100 100 100 < 1 o 0 0 - C. lechleri bark o 100 100 100 <1 0 0 tox - 66 100 100 . 28 0 0 tox - a 0 0 0 - 0 0 5 - 0 0 0 - 0 0 26 487 C. palanostiqma bark o 34 41 67 14 20 32 tox 155 52 98 100 . 86 0 0 tox - a 100 100 100 <1 0 0 69 391 100 100 100 <1 0 25 tox 15 C. tr i nitat i s aerial 0 61 78 94 .50 0 36 tox 12 23 99 100 1 .4 0 0 tox - a 0 0 0 - 0 0 0 - 0 34 100 1 1 0 0 0 - Didymocistus chrysadenius leaf o 53 88 100 1 . 3 15 23 72 31 0 100 100 4.6 0 0 69 133 a 0 0 0 - 0 0 0 - 6 1 100 100 . 34 0 0 0 - bark o 0 0 0 - 0 0 0 - 0 73 100 8.9 0 0 0 - a 0 0 0 - 0 23 100 1 1 100 100 100 <1 0 0 0 - Hevea bras i1i ens i s 1 eaf o 100 100 100 <1 0 0 13 - 60 99 100 .60 100 100 100 < 1 a 0 0 0 - 0 0 0 - 98 100 100 <1 0 0 0 - Jatropha curcas aer i a 1 o 100 100 100 <1 0 0 84 88 0 96 100 7.0 0 0 0 - a 100 100 100 <1 7 1 7 78 32 0 0 100 22 0 0 0 - J. gossyp i i fo1i a aer i a 1 o 100 100 100 < 1 0 25 75 37 2 1 99 100 1 . 5 0 0 0 - a 100 100 100 <1 0 0 25 512 22 98 100 1 . 7 0 0 tox - J. weberbaueri 1 eaf o 100 100 100 <1 13 73 91 6.0 87 100 100 .08 0 0 0 - a 0 0 0 - 0 0 0 - 0 0 ' 99 34 0 0 0 - bark o 100 100 100 < 1 14 43 97 6 . 7 58 99- 100 .64 0 0 tox - a 0 0 0 - 0 0 0 - 0 0 0 - o 0 0 - Mabea maynensis leaf o 100 100 100 <1 6 13 31 724 100 100 100 < 1 0 0 0 - a 96 99 100 < 1 0 0 0 - 100 100 100 < 1 0 0 0 - M. ni t i da bark o 99 99 98 < 1 0 0 tox - 98 100 100 < 1 0 0 0 - a 0 0 0 - 15 43 77 15 100 100 100 < 1 100 100 100 < 1 Manihot esculenta aer i a 1 o 37 51 78 5.2 41 53 63 6 . 1 81 100 100 . 14 0 0 0 - a 20 32 69 26 39 43 99 3 . 2 58 94 100 . 18 0 0 0 - Maprouna pjuianensis bark o 100 100 100 <1 0 0 0 - 99 100 100 <1 o 0 0 - a 0 0 0 - 0 0 0 - 91 99 100 . 10 0 0 0 - Phyllanthus amarus aer ial o 100 100 100 < 1 8 56 tox 8 . 1 0 98 99 8 . 1 0 0 tox - a 0 0 0 - 4 6 33' 614 98 100 100 <1 0 0 0 - P. orbiculatus aer i a 1 o 100 100 100 <1 0 0 tox - 97 100 100 <1 0 0 0 - a 0 0 0 - 6 34 48 74 58 100 100 . 37 0 0 0 - P. pseudo-conami aer ial o 100 100 100 < 1 100 100 tox <1 57 99 100 .65 0 0 tox - a 0 0 0 - 0 5 60 40 0 12 99 18 0 0 0 - P. ur inar i a aer i a 1 o 100 100 100 < 1 0 46 tox 1 1 95 100 100 <1 o 0 tox - a 0 0 0 - 0 1 1 31 1 16 98 100 100 <1 0 0 0 - Podocalyx loranthoides leaf o 100 100 100 <1 0 0 18 782 100 100 100 <1 0 0 0 - a 100 100 100 <1 0 0 0 - 100 100 100 <1 0 0 0 - bark o 100 100 100 <1 0 0 0 - 91 99 100 . 10 0 0 0 - a 94 96 99 <1 0 0 0 - 100 100 100 <1 0 0 0 - Securineqa conqesta aer ial o 91 95 99 .01 28 100 100 .81 55 98 100 .81 62 100 100 . 33 a 0 0 0 - 0 0 100 22 22 98 100 1 . 7 0 0 100 22 Podophy11i n t res i n o 98 100 tox <1 0 0 tox - 1 1 12 tox - 100 100 tox <1 Table XIV - Antiviral screening of extracts of Euphorbiaceous plants. Results are expressed as per cent reduction in number of plaques observed in the controls. * o = organic extract; a = aqueous extract t Podophyllin resin extract is included as a positive control 1 53 been infected with virus (treatment post-infection), a much smaller percentage of extracts produced a reduction in the number of viral plaques formed. Sindbis virus was susceptible to 23 (68%) and MCMV to only 8 (24%) of the 34 species tested. Both leaf and bark material demonstrated antiviral activity. Little difference between the number of organic and aqueous fractions that possessed activity (29 and 25, respectively) was observed. No correlation between antiviral activity resulting from pre-infect ion and post-infection application was evident. The strong antiviral action of the Amanoa sp. tested was of note. The organic and aqueous fraction of the bark prevented infection by both Sindbis virus and MCMV completely at a concentration of 1 ag/ml. The extracts were effective when applied•according to either the pre-infection or post• infection protocols. An extract of the antiviral podophyllin resin was effective against both viruses when applied prior to infection but inhibited the replication of only MCMV when applied as for the post-infection protocol. It inhibited the infection completely at a concentration of 1 jug/ml. c. Inhibition of potato tumour formation The results of the screening for antitumour activity were also characterized by a very high proportion of active extracts (Table XV). Thirty-one (91%) of the species tested were effective in inhibiting the formation of tumours. This represented 53 of the 84 organic and aqueous extracts. Table XV Per cent inhibitior i of tumour formation(SEM) Spec i es Part Extract* 0.3 eg/ml 1 .0 ^g/ml 3.0 yg/ml ED 5 o ( j,g/ml ) Acalypha benensis leaf o 70( 6) 91(2) 99( 1 ) 1 7 a 87 4) 94(2) 98( 1 ) 0 44 bark 0 94 2) 81(4) 100(1) 0 16 a 68 9) 85(6) 100(0) 0 35 A. d i vers i foli a leaf o 21 19) 45(17 ) 53(9) 2 03 a 12 3) 17(6) 11(4) A. macrostachya bark o 26 13) 91(5) 100(0) 0 50 a 2 2) 0( 13) 0(11) A. stachyura bark o 85 9) 99( 1 ) 100(0) 0 17 a 45 6) 60(5) 94( 1 ) 0 51 Alchornea castaneifolia bark o 83 6) 90(4 ) 99( 1 ) 0 14 a 36 4) 74(7) 94(3) 0 52 A. discolor bark o 76 7) 83(5) 87(6) 0 01 a 0( 10) 64(7) 66(5) 1 75 A. tr i p1i nerv i a bark o 28( 9) 4 3(11) 100(0) 0 62 a 0( 11) 0( 12) 0( 14) Amanoa aff. oblonqifolia leaf 0 84 6) 93(3) 96(2 ) 0 03 a 15 8) 13(8) 26(13) bark o 1 1 11) 74(6) 69(8) 0 09 a 0 12) 0(9) 7(6) Aparisthmium cordatum leaf o 23 12) 57(10) 78(7 ) 0 93 a 0 10) 17(11) 0(11) bark o 17 7) 60(6) 87(2) 0 89 a 0 15) 70(8) 93(2) 1 28- Apodandra loretensis bark o 0 12) 0( 14) 0(11) a 1 1 6( ) 11(3) 8 1(18) 1 76 Caryodendron orinocense bark o 45 8) 74(8) 89(3) 0 41 a ' 121 6) 8(4) 11(8) Chamaesyce hyssopifolia aer i a 1 o 53( 5) 72(5) 79(4) 0 25 a 34( 9) ' 47(5) 87(5) 0 75 C. thym i foli a aer ial o 38 5) 72(5) 95( 1 ) 0 51 a 0 10) 0(12) 9(7) Cnidoscolus peruvianus leaf o 3 6) 7(7) 0(7) a 8 7) 0(5) 4(3) bark o 0 5) 49(8) 100(0) 1 03 a 19 7) 26(7) 28(8) Conceveibastrum martianum bark o 601 8) 88(4) 97(2) 0 57 a 66 10) 79(5) 81(4) 0 05 Croton cuneatus leaf o 72( 8) 85(4) 91(6) 0. 08 a 36( 9) 62(10) 89(4) 0 60 bark o 0( 10) 2(4 ) 5(9) a 0( 8) 0(11) 0( 1 1 ) C. lechleri bark o 0 11) 30(7) 81 (4) 1 75 a 0 9) 0(7) 0 (11) C. pal anost i cjma bark o 79 5) 85(5) 91 (3) 0 02 a 0 9) 62(5) 8 1 (3) 1 57 C. tr i ni tat i s aer ial o 17 9) 74(13) 87 (9) 0 78 a 0 11) 0(9) 0 (13) Didymocistus chrysadenius leaf o 72 9) 74(9) 91 (8) 0 1 1 a 0 4) 0( 10) 0 (9) bark o 0 13) 64(12) 77 (9) 1 56 a 4 9) 0( 12 ) 0 13) Hevea brasiliens is leaf o 0 13) 7(8) 6 5) a 3 5) 0(7) 1 5) Jatropha curcas aer i a 1 o 0 15) 81(7) 82 8) 1 43 a 14 12) 0( 13) 4 9) J. qossyp i i foli a aer i a 1 o 5 12) 0( 10) 0 9) a 2 5) 0( 13) 0 12) J. weberbaueri leaf o 9 11) 12(13) 5 9) a 0 11) 10(11) 7 9) bark 0 0 13) 66(13) 83 9) 1 52 a 5( 11) 4(10) 9 12) Mabea maynensis leaf 0 0( 15) 65(7) 94 7) 1 33 a 0( 13) 0(9) 87 6) . 2 64 M. n i t i da bark o 16( 13) 70(11) 79( 10) 0 91 a 0( 14) 17(12) 83 11) 0 60 Manihot esculenta aer i a 1 o 77( 11) 64( 15) 87( 8) 0 03 a 57( 13) 62( 13) 87( 10) 0 32 Maprouna quianensis bark o 62( 15) 74( 13) 75( 13) 0 05 a 0( 15) 0( 13) 12( 11) Phyllanthus amarus aer i a 1 o 81 ( 6) 79(8) 94 ( 5) 0 05 a 0( 10) 83(7) 90 ( 5) 1 31 P. orbiculatus aer i a 1 o 76( 5) 98(3) 100( 0) 0 24 a 0( 11) 38(10) 89( 8) 1 55 P. pseudo-conami aer i a 1 o 89( G) 96(5) 100( 1 ) 0 17 a 74( 9) 77(8) 100( 3) 0 34 P. ur i nar i a aer i a 1 o 0( 11) odo) 85( 5) 2 73 a 32( 8) 55(7) 96( 4) 0 64 Podocalyx loranthoides leaf o 23( 12) 34(10) 80( 8) 1 17 a 0( 10) 0(7) 0( 9) bark o 0( 11) 0(8) 0( 12) a 0( 8) 0(11) 5( 7) Securineqa conqesta aer i a 1 o 19( 12) 49(9) 91 ( 7) 0 88 a 9( 12) 0(11) 0( 9) Podophy1 lint res i n o 4( 12) 72(9) 93( 5) 0 98 Table XV - Screening of extracts of Euphorbiaceous plants for inhibition of Agrobacter i um induced tumour formation. * o = organic fraction; a = aqueous fraction t Podophyllin resin extract is included as a positive control 1 56 Extracts of both leaf and bark showed antitumour activity and no difference in the activities of the aqueous and organic fractions was apparent. The antitumour activities observed were generally quite strong: the ED50s ranged between 0.01 and 3.0 Mg/ml. Podophyllin was moderately effective in inhibiting tumour formation. d. Toxic ity to br ine shr imp A large percentage of the extracts examined were toxic to brine shrimp (Table XVI). The range of toxicities observed was great and the LC50 varied between less than 1 Mg/ml and 1 mg/ml, the highest dose tested. Extracts with an LC50 less than 1 mg/ml were classed as active. Twenty-seven of the 34 Euphorbiaceous species (79%) produced a toxic response, by this criterion. Approximately three times as many organic fractions (32) as aqueous fractions (10) were toxic. Podophyllin demonstrated a high degree of toxicity (LC50 << 1 Mg/ml) towards brine shrimp. e. Correlat i on between biological assays The data sets of Tables XII to XVI are detailed and contain much information. A large number of.extracts with activities in each of the biological assays is the predominant trend. This information has been summarized in Table XVII. Although each of the assays measures a somewhat different aspect of the biological activity of the extract, certain assays are more closely related, in terms of the nature of the activity detected, than others. The assays measuring antibacterial activity (Nos. 3 and 4), for example, would be Table XVI Per cent mortcilit y of brine shrimp Species Part Extract* 10 100 1000 LCs o i/O/ml „g/ml eg/ml »,g/ml Acalypha benensis 1 eaf o 0 20 100 1 16 a 0 0 56 - bark o 0 0 100 214 a 0 0 20 - A. divers i foli a leaf o 0 0 24 - a 0 0 0 - A. macrostachya bark o 0 40 100 104 a 0 0 0 - A. stachyura bark o 0 0 100 214 a 0 0 0 - Alchornea castaneifolia bark o 8 72 100 41 a 0 0 4 - A. discolor bark o- 0 24 72 399 a 0 0 4 - A . tr i pii nerv i a bark o 0 28 100 1 10 a 0 68 100 92 Amanoa aff. oblonqifolia leaf o 76 92 100 6 . 1 a 52 84 100 14.3 bark o 0 8 32 - a 0 8 44 875 Aparisthmiura cordatum leaf o 0 60 100 95 a 0 0 12 - bark o 0 8 96 254 a 0 0 0 - Apodandra loretensis bark o 0 4 100 142 a 0 0 0 - Caryodendron orinocense bark o 72 96 100 5.8 a 8 56 96 76 Chamaesyce hyssopifolia aer i a 1 o 0 0 0 - a 0 0 0 - C. thymi folia aer i a 1 o 76 100 100 1 . 8 a 0 0 0 - Cnidoscolus peruvianus leaf o 0 4 44 - a 0 0 0 - bark o 0 68 100 92 a 0 0 4 - Conceveibastrum martianum bark o 12 88 100 30 a 4 8 12 - Croton cuneatus leaf o 100 100 100 <<1 a 0 16 100 120 bark o 68 100 100 2.6 a 0 8 100 130 C. lechleri bark o 40 92 100 15 a 0 0 0 - C. palanostiqma bark o 92 96 100 1 . 3 a 0 8 24 - C. tr i n i tat i s aer i a 1 o 24 76 100 27 a 0 0 0 - Didvmocistus chrysadenius leaf o 0 20 52 615 a 0 0 0 - bark o 40 68 96 30 a 0 0 100 2 14 Hevea brasiliens is leaf o 0 0 76 1 106 a 0 0 4 - Jatropha curcas aer i a 1 o 20 28 32 - a 8 4 12 - J. qossypi i foli a aer i a 1 o 24 28 36 - a 0 36 32 - J. weberbaueri leaf o 0 4 88 401 a 0 0 0 - bark 0 0 20 84 326 a 0 0 12 - Mabea maynensis leaf o 0 0 24 - a 0 0 0 - M. n i t i da bark o 0 16 100 1 20 a 0 8 96 254 Manihot esculenta aer i a 1 o 0 8 16 - a 0 0 28 - Maprouna quianensis bark o 24 64 100 30 a 0 0 24 Phyllanthus amarus aer i a 1 o 1G 24 92 ' 14 a 0 0 0 - P. orbiculatus aer i a 1 o 4 48 100 57 a 0 0 28 - P. pseudo-conami aer i a 1 o 48 84 100 15 a 0 0 100 214 P. ur i nar i a aer i a 1 o 0 40 100 104 a 0 0 4 - Podocalyx loranthoides leaf o 4 0 4 - a 0 0 0 - bark o 4 36 84 193 a 0 0 8 - Securineqa conqesta aer i a 1 o 96 100 100 0.09 a 0 0 92 642 Podophy1 lint res i n o 100 100 100 <<1 Table XVI - Screening of extracts of Euphorbiaceous plants for their toxicity to brine shrimp, Artemia sali na. Extracts having a LCso < 1000 ^g/ml were classified as active. * o = organic fraction; a = aqueous fraction t Podophyllin resin is included as a positive control. Table XVII * Biological Assay t Spec i es Part Ext 1 2 3 4 5 6 7 8 9 10 1 1 12 13 14 _ Acalypha benensis leaf o <0. 13 0.5 0. 25 <0. 13 <1 .0 <1 .0 1 7 1 16 a - + 2 - - 0. 25 - 2.0 0.5 - - <1 .0 - 0 44 - bark o - +4 - - <0. 13 1 .0 1 .0 0. 25 <1 .0 - <1 .0 - 0 16 214 a - + 4 - - <0. 13 2.0 1 .0 0.5 3.0 - <1 .0 - 0 35 - A. di vers i foli a leaf o - + 2 - - <0. 13 0. 25 0. 25 0. 25 <1 .0 16 <1 .0 - 2 0 - a - + 2 - - <0. 13 - 2.0 0.5 <1 .0 - <1 .0 - - - A. macrostachya bark o - - - - <0. 13 0.5 0.5 0. 25 <1 .0 - <1 .0 - 0 5 104 A. stachyura bark o - + 3 - - <0. 13 0. 25 0.5 0. 25 <1 .0 - <1 .0 - 0 17 214 a - + 2 - - <0. 13 1 .0 1 .0 0. 25 <1 .0 - <1 .0 - 0 51 - Alchornea castaneifolia bark o - - - - 1 .0 0. 25 0.5 0. 25 <1 .0 - 0.30 - 0 14 4 1 a - - - - 1 .0 - - - - - 0.22 - 0 52 - A. discolor bark 0 - + 1 - - <0. 13 0. 25 1 .0 0. 25 3.9 20 0.86 - 0 01 399 a - + 2 - - 2.0 - - - - - <1 .0 - 1 75 - 'A. tr i p 1 i nerv i a bark o - + 1 - - 0. 25 0. 25 1 .0 0.5 <1 .0 35 0. 22 - 0 62 1 10 a - - - - 1 .0 1 .0 1 .0 1 .0 <1 .0 17 <1 .0 - - 92 Amanoa aff. oblonqifolia 1 eaf o - + 1 - - 0.25 2.0 2.0 2.0 <1 .0 <1 .0 <1 .0 <1 .0 0 03 6 . 1 a - + 1 - - 0.5 2.0 2.0 1 .0 <1 .0 2.0 <1 .0 <1 .0 - 14.3 bark o - - - - <0. 13 <0. 13 2.0 0.5 <1 .0 1 .9 <1 .0 <1 .0 0 09 - a - - - - 2.0 2.0 2.0 2.0 <1 .0 - 7 . 7 270 - 875 Aparisthmium cordatum leaf o - + 2 - - <0. 13 0.5 1 .0 0.5 0.08 - 1 . 3 - 0 93 95 a - - - - 1 .0 2.0 2.0 1 .0 8.0 - <1 .0 - - - bark o - + 2 - - <0. 13 0.5 0.5 0. 25 0. 15 3 . 8 - - 0 89 254 a - + 2 - - - 2.0 2.0 1 .0 - 147 <1 .0 - 1 28 - Apodandra loretensis bark o - + 2 - - 0. 25 <0. 13 0. 25 0. 25 <1 .0 51 0.91 - - 142 a - + 2 - - 0. 25 2.0 2.0 0. 25 2.9 - <1 .0 - 1 76 - Caryodendron orinocense bark o - + 1 - - 1 .0 1 .0 2.0 2.0 - - 0. 30 - 0 41 5 . 8 a - - - - - 2.0 2.0 2.0 <1 .0 - <1 .0 - - 76 Chamaesyce hyssopifolia aer . o - + 1 - - 0.5 1 .0 0.5 0.5 <1 .0 - <1 .0 - 0 25 - a - + 1 - - <0. 13 2.0 - 2.0 - - 0.53 - 0 75 - C. thym i fo1i a aer . o - + 1 - - <0 . 13 <0. 13 1 .0 0. 25 <1 .0 15 <1 .0 - 0 5 1 1 . 8 a - - - - 0.5 - - 1 .0 ------Cnidoscolus peruvianus 1 eaf o - - - - <0. 13 0.5 1 .0 0.5 <1 .0 <1 .0 <1 .0 - - - a ------208 41 - - bark o - + 3 - - <0. 13 <0. 13 <0. 13 <0. 13 5 . 3 12.1 22 - 1 0 92 a ------47 6 . 1 <1 .0 - - - Conceveibastrum martianum 1 eaf o - + 1 - - 0. 25 0.5 1 .0 0. 25 <1 .0 23 <1 .0 - 0 57 30 a - + 1 - - 0. 25 - - 2.0 <1 .0 40 <1 .0 - 0 05 - Croton cuneatus leaf o - + 1 - + 2 <0. 13 0.5 0.5 0. 25 <1 .0 - 0.63 - 0 08 <1 .0 a - + 2 - - 2.0 - - - <1 .0 - <1 .0 - 0 6 120 bark o - - - - <0. 13 0.5 0.5 0.5 <1 .0 - <1 .0 - - 2 . 2 a - - - - 2.0 0.2 2.0 2.0 <1 .0 - <1 .0 - — 130 C. lechleri bark o + 2 _ _ <0. 13 <0. 13 0.25 <1 .0 - 0. 28 - 1 8 15.2 a ------487 - - C. palanostiqma bark o - + 2 - - <0. 13 <0. 13 0 5 <0. 13 14 155 0.86 - 0 02 1 . 3 a - + 2 - - <0. 13 - 2 0 1 .0 <1 .0 391 <1 .0 15 1 6 - C. tr i ni tat i s aer . o - + 1 - - <0. 13 0.5 1 0 0.13 0.5 12 1 .4 ~ 0 78 27 \ 1 a D i dymoc i stus chrysadenius leaf - - - - <0. 13 - - 2.0 1 . 3 31 4 . 6 133 0 1 1 615 o a ------1 .0 - - 0. 34 - - - bark o - + 1 - + 4 - - - 2.0 - - 8.9 - 1 6 30 a - - - - 1 .0 2.0 2 0 2.0 - 1 1 <1 .0 - - 214 Hevea brasiliensis leaf o - - - - 0. 25 2.0 2 0 2.0 <1 .0 - 0.6 <1 .0 - - a ------<1 .0 - - - Jatropha curcas aer . o - - - - <0. 13 - - - <1 .0 88 7.0 - 1 4 - a ------< 1 .0 32 22 - - - J. qossypi i foli a aer . o - - - - <0. 13 - - - <1 .0 37 1 . 5 - - - a - - - - <0. 13 1 .0 - - <1 .0 512 1 . 7 - - - J. weberbaueri leaf o - - - - 1 .0 - - - <1 .0 6.0 0.08 - - 401 a - - - - 1 .0 - - - - - 34 - - - bark o - - - - <0. 13 2.0 2 0 1 .0 <1 .0 6 . 7 0.64 - 1 5 326 a Mabea maynens i s leaf - + 3 - - 1 .0 1 .0 1 0 1 .0 <1 .0 724 <1 .0 - 1 3 - o a - + 3 - - <0. 13 2.0 1 0 0.5 <1 .0 - <1 .0 - 2 6 - M . n i t i da bark o + 3 + 1 - - <0. 13 0. 25 0 5 0. 25 <1 .0 - <1 .0 - o 91 120 a ------15 <1 .0 <1 .0 0 60 254 Manihot esculenta aer . o - + 1 - - <0. 13 <0. 13 <0. 13 <0. 13 5 . 2 6 . 1 0.14 - 0 03 2390 a - - - - 1 .0 - - - 26 3 . 2 0.18 - 0 32 - Maprouna quianensis bark o - + 1 - - <0. 13 0. 25 0 25 0. 25 <1 .0 - <1 .0 - 0 05 30 a - + 1 ------0. 10 - - 5380 Phyllanthus amarus aer . o - - - - <0. 13 2.0 - 0. 25 <1 .0 8 . 1 8 . 1 - 0 05 1 14 a - + 3 - - 0.5 2.0 2 0 1 .0 1 .0 614 <1 .0 - 1 3 - P. orbiculatus aer . o + 5 + 3 - - <0. 13 0. 25 0 5 <0. 13 <1 .0 - <1 .0 - 0 24 57 a - - - - <0. 13 2.0 - 2.0 - 74 0. 37 - 1 6 4430 P. pseudo-conami aer . o - - - - <0. 13 0.5 2 0 1 .0 <1 .0 <1 .0 0.65 - 0 17 15 a - - - - 0.25 - 1 0 - - 40 18 - 0 34 214 P. urinaria aer . o - - - - 0.25 - - 0.25 <1 .0 1 1 <1 .0 - 2 7 104 a - + 4 - - <0. 13 2.0 2 0 1 .0 - 1 16 <1 .0 - 0 64 - Podocalyx loranthoides leaf o - - - - 0.5 0.5 0 25 0. 25 <1 .0 782 <1 .0 - 1 2 - a - + 1 - - <0. 13 2.0 2 0 2.0 <1 .0 - <1 .0 - - - bark o - - - - <0. 13 1 .0 1 0 0.5 <1 .0 - 0. 10 - - 193 a ------<1 .0 - <1 .0 - - - Securineqa conqesta aer . o - + 1 - <0. 13 0.5 1 0 1 .0 0.01 0.81 0.81 0. 33 0 88 0.09 a ------22 1 . 7 22 - 642 Podophy11i n + res. o - + 2 - - 0.5 0.5 0 5 0.25 <1 .0 - 4 . 3 <1 .0 0 98 <1 .0 Table XVII - Summary of bi ological screening of extracts of Euphorbiaceous plants Legend for Tab!e XVII t Bioassays to which numbers refer: 1 == inhibition of growth of E_. col i 2 == inhibition of growth of S . aureus = inhibition of growth of S . cereviseae 3 == inhibition of growth of C. alb i cans 4 = 5 == inhibition of growth of M. cani s 6 == inhibition of growth of M . gypseum 7 == inhibition of growth of M. fu1vum 8 == inhibition of growth of T . ga11inae 9 =; anti-Sindbis virus activity : pre-infection 10 == anti-Sindbis virus activity: post-infection = anti-MCMV activity: post- i nfect i on 1 1 == anti-MCMV activity: pre-infection 12 = = toxicity to Artemia salina 13 == inhibition of Aqrobacteriurn induced potato tumours 14 = * Podophyllin resin extract is included as a positive control. * o = organic fraction; a = aqueous fraction 162 expected to resemble each other more closely, with respect to the results obtained, than would two assays utilizing distantly related organisms. Data has been presented which indicates that the potato disc tumour assay is predictive of in vivo antileukemic activity (Ferrigni et a_l, 1982) and it is being considered as a preliminary screening assay for antineoplastic agents (Cassady e_t al, 1981). Toxicity to brine shrimp, while not specific for antitumour activity, shows some degree of correlation with results of assays for cytotoxicity (Galsky et al, 1980 and 1981; Meyer et al, 1982). Both assays have been used only relatively recently and more descriptive information on the degree of association between the activities measured is needed. The results of Tables XII to XVI have been summarized in the form of 2 X 2 contingency tables in Table XVIII. Each of the sets of four numbers represents a comparison of the results of the two bioassays indicated. The format for each of these is illustrated in the following table. 1 63 Test A positive negat ive positive a b Test B negat ive c d The letter (a) refers to the number of extracts in which both Test A and Test B produced a positive result; (b) indicates the number having a negative result in Test A and a positive result in Test B, etc. These data were subjected to statistical analysis utilizing Biomedical Data Packages program P:4F to compute values for Fisher's exact test. These statistics (for a two tailed test) have been summarized in Table XIX. The values observed for this statistic range from less than 0.0001, indicating a close association of results, to 1.0,. indicating no association of results. Since no inhibitory activity towards C. albicans was observed in any of the extracts, associations between bioassay No. 3 and any of the other bioassays could not be calculated. The corresponding columns of Table XIX are blank. Certain interesting trends can be distinguished in these data. The values of 0.4941 for Fisher's exact test applied to 2 41 2 0 42 0 0 0 0 3 2 83 43 42 0 2 2 0 0 2 4 2 81 41 42 0 83 2 67 40 29 0 69 1 68 5 0 16 3 13 0 16 1 15 2 53 22 20 0 55 1 54 53 2 6 0 30 8 35 0 30 1 29 16 14 2 52 36 18 0 54 1 53 52 2 50 4 7 0 31 7 24 0 31 1 30 15 14 5 26 2 61 40 23 0 63 2 61 59 4 54 9 53 10 8 0 22 3 19 0 22 0 22 10 12 1 21 1 21 2 60 35 27 0 62 1 61 58 4 49 13 48 14 52 9 9 0 23 8 15 0 23 1 22 1 1 12 6 17 6 17 10 13 0 43 20 22 0 42 0 42 36 6 28 14 27 15 31 1 1 34 8 10 2 40 23 20 0 43 2 41 33 10 27 16 27 16 32 1 1 28 15 2 78 42 38 0 80 2 78 67 13 54 26 53 27 6 1 19 61 19 4 1 39 1 1 0 5 1 4 0 5 0 5 2 3 1 4 1 4 2 3 1 4 1 4 0 12 5 7 0 12 0 12 9 3 7 5 8 3 9 3 9 3 8 4 1 1 1 12 2 7 1 38 35 0 73 2 7 1 60 13 48 25 47 27 54 19 53 20 34 39 69 4 2 52 38 16 0 54 2 52 51 3 41 13 40 14 47 7 43 1 1 30 24 53 1 6 47 13 0 31 5 26 0 31 . 0 31 18 13 14 17 14 17 16 15 19 12 12 19 27 4 6 26 2 41 24 19 0 43 2 41 20 4 34 9 34 4 38 5 37 6 23 20 42 1 8 35 31 12 14 0 42 19 23 0 42 0 42 30 12 2 1 21 20 22 35 17 25 17 19 23 38 4 4 38 23 19 10 1 1 12 13 Table XVIII - 2 X 2 Contingency tables for agreement between each pair of the 14 assays used to screen the 85 plant extracts. Data are summarized from Table XVII. 0.4941 - - 1 .00 0.4941 - 1 .00 0.0056 - 0.3429 0.5378 0.0015 - 1 .00 0.0000 0.531 1 0.0001 - • 1 .00 0.0000 0.0000 1 .00 0.0001 - 1 .00 0.0000 0.0000 0.0000 1 .000 0.0913 - 0.4703 0.0000 0.0000 0.0000 0.0002 0.2412 1 .000 - 0.4941 0.4065 0.82 13 1 .00 1 .00 0.1429 1 .00 0.2020 - 1 .00 0.0440 0.0503 0.0570 0.1065 00178 0.3600 1 .00 0.5486 - 1 .00 0.6899 0.7464 0.7393 1 .00 1 .00 0.2278 0.5421 0.531 1 0.0000 - 0.5310 0.0001 0.0088 0.0104 0.0006 0.0800 0.1774 0.0570 0.0347 0.4941 1 .000 - 0.4941 0.0283 0.0066 0.0034 0.0030 0.0074 1 .000 0.2020 0. 3512 0.1178 1 23456789 10 11 12 13 Table XIX - Values of Fisher's exact test for contingency tables of Table XVIII 1 66 the results of the screening of gm negative and gm positive bacteria indicates no appreciable degree of association between these results. Nor did the results of the inhibition of E. coli growth significantly resemble the results of any of the other bioassays. The results obtained for S. aureus, however, were shown to be quite closely associated with the data for inhibition of growth of each of the dermatophytic fungi tested. The similarity in the results obtained in the screening for anti-dermatophyte activity is supported by the observation that the corresponding value for Fisher's exact test is less than 0.0001 in all cases. It is interesting that the results of the anti-Sindbis virus (pre-infection treatment) screening were also closely associated with the results of the anti-dermatophytic fungi screening. This was not the case for any of the other three anti-viral tests carried out. No association between the pre- infection and post-in feetion treatment of either Sindbis virus (p= 0.1429) or MCMV (p= 0.5421) was indicated. The results of the assays of the pre-infection treatment of Sindbis virus and MCMV were somewhat similar (p= 0.0178) while the post• infection treatment results showed no significant association (p= 0.2278) . The results of the potato disc tumour inhibition assay were associated with the results of the inhibition of S. aureus (p= 0.0001), each of the anti-dermatophytic fungi assays and anti-Sindbis virus (pre-infection), as well as anti-MCMV (pre- and post-infect ion) assays to some extent (p= 167 0.035-0.08). The pattern of association of the brine shrimp toxicity results with the results of the other bioassays is similar to that of the anti-tumour results. The single exception is that anti- S. aureus activity and brine shrimp toxicity show no association. The value of Fisher's statistic for the comparison of antitumour activity and brine shrimp toxicity is 0. 1 1 78. 4. DISCUSSION a. Antimicrobial activity The percentage of Euphorbiaceous species exhibiting antimicrobial activity is comparable to that of plants selected from all families. Khan et a^L (1980) reported that 98% and 18% of 60 species of African medicinal plants inhibited the growth of S. aureus and E. coli, respectively. Ieven et al (1979) observed 78% and 31% of 100 species of higher plants with medicinal uses to inhibit the growth of these two species. It is apparently common for S. aureus to be more sensitive than E. coli to antimicrobial agents from plants. With the exception of Jatropha curcas and J. gossypiifolia, all of the species of Euphorbiaceae whose medicinal use is concerned with the treatment of wounds or skin conditions, were inhibitory to S. aureus (Table XX). Few of the Euphorbiaceous extracts exhibited any inhibitory activity towards yeasts. None prevented the growth Table XX Inh i b i tory Activity or Toxicity Towards: Spec i es Col 1 . Folk Use gm-ve gm+ve dermato potato No. bacter i a bacter i a yeast phyt i c v i rus tumour Artern i a fungus Acalypha benensis 1 14 - + - + + + + A. d ivers i folia 63 - + - + + + - A. macrostachya 91 - - - + - + + A. stachyura 1 13 - + - + - + + Alchornea castaneifolia 95 rheumatism, tonic - - - + - + + A. discolor 81 - + - + + + + A. tr i pii nerv i a 31 - + - + + + + Amanoa aff. oblonqifolia 70 - + - + + + + Aparisthmium cordatum 29 - + - + + + + Apodandra loretensis 56 - + - + + + + Carvodendron orinocense 84 skin diseases - + - + - + + Chamaesyce hyssopifolia 65 wounds, cancer - + - + - + - C. thym i fo1i a 38 wounds, cancer - + - + + + + Cnidoscolus peruvianus 133 anti-aphrodisiac - - - + + + + Conceveibastrum martianum 101 - + - + + + + Croton cuneatus 93 - + + + - + + C. lechleri 62 wounds, cancer + + + + + C. pa 1 anost i gma 76 wounds, cancer - + - + + + + C. tr i n i tat i s 48 sore throat, chest ailments - + - + + + + Didymocistus chrysadenius 27 - + + + + + + Hevea brasiiiens is 54 - - - + + - - Jatropha curcas 67 purgative,cancer,skin cond. - - - - + + - J . pjossyp i i f o 1 i a 68 purgative,cancer,skin cond. - - • - - + - - J. weberbauer i 136 aphrod i s i ac - - - - + - + Mabea maynens i s 79 - + - + + + - M. ni t ida 53 skin conditions + + - + + + + Manihot esculenta 61 burns, skin infections - + - + + + - Maprouna guianensis 73 - + - + - + + Phyllanthus amarus 37 kidney ailments, liver - + - + + + + P. orbiculatus 49 + + - + + + + P. pseudo-conami 112 - - + + + + P. ur i nar i a 36 kidney ailments, liver - + - + + + + Podocalyx loranthoides 89 - + - + + + + Securineqa conqesta 30 + + + + + Frequency +ve 6 76 6 91 79 91 79 Result(%) Table XX - Summary of ethnobotanical information and biological activity of the 34 species of Euphorbiaceae tested. Data are summarized from Tables XII to XVIII. 1 70 of C. albicans, while 2 out of 34 species were active against S. cerevisiae. Screening studies of all families of higher plants, by comparison, have detected a much higher percentage (10-42%) of extracts inhibitory towards yeasts (Farnsworth e_t al, 1966; Fong et al, 1972; Ieven et al, 1979). The percentage of extracts inhibiting the growth of dermatophytic fungi (91%) is high compared to that observed in other studies: 2.6% (Farnsworth et_ a_l, 1966); 2.1% (Fong et al, 1972); and 65% (Ieven et al, 1979). As in the case of anti-S. aureus activity, 8 out of the 10 species of Euphorbiaceae that were used in the treatment of skin conditions are inhibitory towards the dermatophytic fungi tested. b. Antiviral activity It is somewhat surprising that the extracts were so uniformly effective in inactivating both Sindbis virus and MCMV when applied to them at low concentrations. The ED50s of most of the active extracts was less than 1 jug/ml (Table XVII). The inactivation of viruses by direct application of a wide variety of plant extracts has been reported previously (Konowalchuk and Speirs, 1976 and 1978). This has been attributed to the presence of phenolic compounds in the .aqueous extracts applied to the viruses (Cheo and Lindner, 1964; John and Mukundan, 1978; Takechi and Tanaka, 1981). Among the phenolic compounds with activity is the ubiquitous compound, tannic acid (Konowalchuk and Speirs, 1978). These 171 sorts of compounds would be expected to occur predominantly in an aqueous extract. The virus inactivating activity of the aqueous extracts tested in the present study may be the result of phenolic compounds. A large percentage of the organic fractions, however, were also highly effective in inactivating •both Sindbis virus and MCMV and it is difficult to attribute this activity to phenolic constituents. It is known that saponins can also inactivate certain viruses (Ragetli and Weintraub, 1974; Ragetli, 1975) and it is possible that the presence of this, or other classes of lipophilic compounds with the ability to disrupt the structure of biomembranes may be responsible. Although direct viral inactivation is a potentially important biological activity, an agent which inhibits virus replication in already infected cells would seem to be more useful as a therapeutic agent. Of the 34 species of Euphorbiaceae tested, 23 (68%) inhibited infection by Sindbis virus and 8 (24%) were effective against MCMV. These are very high percentages compared with what has been observed from the higher plant screening programs carried out by Farnsworth e_t al (1966) ( 3% of species active), Fong et al (1972) (8.3% species active) and Van den Berghe e_t a_l (1979) (8% species active). It is interesting to note, again, that the results of the anti-Sindbis virus and anti-MCMV assay were not associated, statistically, indicating that the requirements for, and compounds involved in, the inhibition of infection by each virus are quite different. 1 72 c. Antitumour activity The percentage of the species tested which exhibited anti-potato tumour activity was very high. The extracts of 31 of 34 (or 91% of species) had an ED50 less than 5 nq/ml. Ferrigni et a_l (1982) observed 27% of their sample of Euphorbiaceous seeds to elicit demonstrable antitumour activity. This difference may be a result of the methodology applied or may reflect chemical differences between the vegetative material used in this study and the seeds used by Ferrigni e_t al (1982). It is reasonable to expect that the strain of A_;_ tumefaciens and type of potato used would introduce some variability although there is insufficient information available to discuss this possibility further at this time. d. Toxicity to brine shrimp Thirty-one of the 34 species tested were toxic to brine shrimp. This indicates that toxic constituents are prevalent in the species of this family. Meyer e_t a_l (1982) found that the extracts of the seeds of 18 of 41 (43%) species of Euphorbiaceae were toxic to Artemia. In their study, toxicity was defined on the basis of an LD50 of less than 1000 Mg/ml. If a similar criterion is applied to the present study, the number of active species is reduced to 27 (79%). The greater proportion of toxic species detected in this study supports the idea that the vegetative plant material used in this study is different, with respect to biologically active compounds present, from the seeds used by Meyer et a_l (1982). Kinghorn 173 et_ al (1977) examined a series of phorbol esters for their toxicity to brine shrimp. Artemia were sensitive to many of the phorbol esters tested and this assay should, therefore, be useful for examining the Euphorbiaceae in which phorbol esters are widespread. Many of the extracts examined in the present study exhibited a degree of toxicity to brine shrimp that was as high as some of the pure compounds examined by Kinghorn et_ al (1977). The recent identification of irritant phorbol derivatives from seeds of Jatropha curcas and J. gossypiifolia (Adolf et a_l, 1984) raise the question of whether these, or similar constituents, are also present in vegetative material of these plants. 5. CONCLUSION On the basis of the comparison with information in the literature, the species of Euphorbiaceae sampled appear to be especially rich in agents which inhibit the growth of dermatophytic fungi, certain viruses and tumours on potato discs and in displaying general toxicity. Whether these diverse biological activities are different manifestations of the same compounds or are produced separately by different compounds is not known. Any, or all, of these biological activities could form the basis for the use of these species of Euphorbiaceae in Amazonian ethnomedicine. Approximately 80% of the species used in the treatment of skin infections were active in each of the four above mentioned bioassays. (Table XX). Ten of the 16 174 species with medicinal uses are used in the treatment of conditions that are likely to be the result of either infectious organisms or malignancies. The numbers of species active against S. aureus, the dermatophytic fungi, at least one of the viruses tested, potato tumours and brine shrimp are 26, 31, 27, 31 and 31, respectively. The majority of the species active in these bioassays have no documented use as a medicinal agent. This could be a result either of their properties never having been discovered by aboriginal peoples or the incomplete nature of the ethnobotanical information available. Such considerations involve the assumption that the assays used are predictive only of the sorts of biological activities that might, in practice, be useful therapeutically. This is not the case. A certain percentage of false positive results have been observed using the potato disc tumour assay (Ferrigni et. al, 1982) and limitations in predictability are inherent in any biological assay. In this respect, it is interesting to note some of the quantitative aspects of the data. The potencies of the biological activities of medicinally used species are, on the average, no greater than those of the active species with no recorded'ethnobotanical use. Some of the strongest activities, for example the antiviral activity of Amanoa sp., are present in species having no known ethnobotanical use. If the objective of this screening program had been the attainment of positive results in any of the biological tests carried out, there would, in retrospect, have been no 175 advantage obtained from selecting plants with uses in ethnomedicine. It should be emphasized that this conclusion is not intended to apply to other screening programs for biological activity. It is restricted to the application of these specific bioassays to the Amazonian species of Euphorbiaceae. Nor does it imply that ethnobotanical information concerning individual species of Euphorbiaceae will not be helpful in discovering significant new biologically active constituents. It is intended as a summary statement for this descriptive study. 176 PART B. a 2i Z Iz PELTATIN, THE ANTIVIRAL CONSTITUENT OF Amanoa sp. 1 . INTRODUCTION In a screening project of the Amazonian flora, extracts from the bark and leaves of certain species of Euphorbiaceae were observed to inhibit the development of Sindbis virus and murine cytomegalovirus in tissue culture cells. A sample of Amanoa sp. exhibited the strongest antiviral activity of the 34 species examined. Extracts of both the leaves and bark were highly effective in inhibiting the formation of plaques by both viruses. Both the organic and aqueous fractions were active against the viruses and the former displayed significantly greater activity. The leaf extract was selected for a project aimed at the isolation of the active constituent(s). 2. MATERIALS AND METHODS a. Plant material The plant material was collected near Iquitos, Peru. Voucher specimens(D. MacRae No. 70) have been deposited at UNAP(lquitos), San Marcos(Lima), Chicago Field Museum and UBC Herbarium. The identification of the plant, Amanoa aff. oblongifolia Muell. Arg., was carried out by Dr. M.J. Huft, Chicago Field Museum. Plant material was preserved in methanol prior to extraction. The leaf (41 g dry weight) sample was homogenized in 177 methanol and extracted exhaustively with that solvent at 20 °C. The combined extracts were filtered, evaporated in vacuo and partitioned between ethyl acetate and distilled water. The resulting organic and aqueous extracts were evaporated in vacuo and dissolved in 95% and 50% ethanol, respectively and stored at -30 °C. b. Antiviral assays The method for measuring antiviral activity is described, in detail in Part C of this chapter. Briefly, murine cytomegalovirus (MCMV) was applied to petri dishes containing an almost confluent monolayer of mouse (3T3) embryo cells. After a period of incubation, which allowed for infection by the virus, the medium was removed and replaced with a solid medium (containing 0.5% agarose) in which the plant extracts were dissolved. Cells were incubated at 37°C until the viral plaques were sufficiently well developed for counting. They were counted with the unaided eye and per cent inhibition of plaque formation was calculated. c. Chromatography The extract was resolved using a rotary TLC device, the Chromatotron (Harrison Research Associates). A 2 mm plate prepared from Silica Gel PF25« (Merck) was used exclusively. Elution was carried out using heptane/chloroform/ethanol (25/25/3) at a flow rate of 3 ml/min. Fractions were collected at two minute intervals. The eluate was monitored by recording its absorbance at 254 nanometers. 1 78 3. RESULTS AND DISCUSSION The initial separation of the organic fraction of the Amanoa leaves (350 mg extract) yielded three major U.V. absorbing peaks, each consisting of several components. The elution profile is seen in Figure 16. The polar component of the extract, which did not move in the solvent system heptane/chloroform/ethanol (25/25/3), was eluted with acetone and collected as three large fractions. The fractions collected were each reduced to 3 ml volume by evaporation and 2 M1 diluted 100 times with tissue culture medium (Dulbecco's modified minimum essential medium containing 5% fetal bovine serum). The sample was diluted a further 5 times with minimum essential medium containing 5% fetal bovine.serum (5%-MEM) and 0.5% agarose which was overlayed on a monolayer of mouse embryo cells which had just been infected with MCMV. The medium was allowed to solidify and the plates were incubated at 37 °C until plaques had developed. The assay was carried out in duplicate. Active fractions were identified by their ability to reduce the number, of MCMV plaques by more that 50%. Fractions 19 through 26 were all classified as active (Figure 16). It is clear from the figure that these fractions correspond to the elution of a strong U.V. absorbing peak. The fractions were monitored by TLC, combined and subjected to further purification by identical chromatographic procedures. The antiviral activity was attributable to a 179 TIME(min.) gure 16 - Chromatotron elution profile of ethyl acetate fraction of Amanoa sp. leaves. Fractions with anti-viral activity are blocked out. 180 single compound. The following spectral data were obtained for this active compound. a-(- )-Peltatin. Mp 229-230 °C. [a] 25 (CHC13) -118 °. U.V. X 1 MeOH. 274, 240sh, 214. H-NMR. (80 MHz, CDC13) 5 6.35 (s, 2H, H-2', H-6'), 6.21 (s, 1H, H-8), 5.93 (s, 2H, 0"CH2-0), 4.4 - 4.6 (1H, H-1), 4.4 - 4.6 (1H, H-11), 3.8 - 3.9 (1H, H-11), 3.78 (s, 6H, 2XOCH3), 3.1 - 3.5 (m, 1H, H-4), 2.6 - 2.75 (3H, H-2, H-3, H-4). MS m/z (rel. int.) 400[M+] (100), 355 (12), 341 (8), 340 (7), 323 (6), 315 (5), 309 (6), 291 (4), 285 (6), 283 (5), 279 (4), 255 (8), 253 (5), 247 (13), 246 (69), 234 (23), 228 (26), 202 (23), 201 (73), 200 (12), 190 (23), 189 (66), 188 (65), 184 (12), 181 (13), 172 (10), 168 (il), 167 (51), 165 (14), 155 (17), 154 (39), 153 (13), 152 (12), 151 (19), 139 (17), 131 (13), 115 (22), 103 (10), 91 (14). The U.V. and 1H-NMR spectra indicated that the compound might be a dibenzobutyrolactone lignan. The characteristic chemical shift of the C-8 proton and the equivalence of the C- 2' and C-6' protons suggested that it may be a-peltatin. This was corroborated by a comparison of its mass spectrum with that published for this compound (Duffield, 1967). a-Peltatin exists as (+) and (-) diastereomers, depending upon the relative configuration at C-1. Based upon the characteristic chemical shift (5 4.58) and the optical rotation [a] (CHC13) = -118 °, which were measured for the compound isolated, it was determined to be the (-) isomer (Figure 17). 181 OH H »-(-)-Peltatin Figure 17 - Structure of a-( - ) -peltatin isolated from Amanoa sp. 1 82 a-Peltatin has recently been reported to reduce significantly the cytopathic effect resulting from infection by herpes simplex virus-1 (HSV-l) (Markkanen e_t a_l, 1981a). The related compound, podophyllotoxin, a well known constituent of the medicinal resin of Podophyllum sp., was observed to produce a very similar effect (Markkanen e_t al, 1981b). Bedows and Hatfield (1982) have observed that podophyllotoxin is effective in inhibiting both the cytopathic effect and infectivity produced by measles virus and HSV-1. They observed a-peltatin, however, to have only a slight antiviral effect. By comparison, Farnsworth e_t a_l ( 1 966) detected no antiviral activity of an extract of Podophyllum peltatum against vaccinia virus, poliovirus type III and pseudorabies virus. In comparing those results with the ones obtained in this study, the differences in the assays used should be born in mind. Measles virus is a single stranded RNA virus (paramyxovirus group), while HSV-1 and MCMV are double stranded DNA viruses of the genus herpesvirus. Moreover, it is not stated which isomer of a-peltatin was used. The antiviral activity we observed from a- ( - ) -peltatin may be specific to that isomer. a- ( - )- Peltatin is the first lignan to be reported from the genus Amanoa and the first lignan of it's class (cyclolignan-9'-9-1ignanolide) to be reported from the family Euphorbiaceae. 183 PART C. THE ANTIVIRAL ACTIVITY OF LIGNANS 1. INTRODUCTION The cyclolignanolide, a-( - ) -peltatin, was identified as the potent antiviral agent from an Amazonian species of Amanoa. This observation, combined with recent reports (Bedows and Hatfield, 1982; Markkanen et al, 1981a and b) that other cyclolignanolides also produce antiviral effects stimulated this comparative study on the effects of a variety of lignans on virus infected cells. Cyclolignanolides display a diversity of biological effects which could form the basis for the mechanism of their antiviral action. Some derivatives of podophyllotoxin, the best known example of this class of compounds, can damage DNA directly (Loike and Horowitz, 1976a), while others are known to interfere with nucleoside uptake and nucleic acid metabolism (Loike and Horowitz, 1976b). Still others cause fundamental alterations in cellular metabolism (Waravdekar e_t al, 1953). Finally, certain podophyllotoxin derivatives bind to tubulin (Brewer et al, 1979) interfering with its polymerization to form microtubules and, in this way, inhibiting mitosis. To provide further information on the mode of antiviral action of lignans, the following studies were carried out: 1) a wide variety of lignans were tested for their possible effects on virus infected cells; 2) the antiviral actions of podophyllotoxin and a- 184 peltatin against two viruses, Sindbis virus and murine cytomegalovirus (MCMV) were compared; 3) different times during the process of infection were examined to determine which stage was sensitive to lignan action. 2. MATERIALS AND METHODS a. Chemicals The lignans used were obtained from a variety of sources. Podophyllotoxin was purified from a sample purchased from Sigma. a-Peltatin was isolated from Amanoa sp. as described in Part A. Justicidin B and diphyllin, as well as its apioside and acetylated apioside were provided by Dr. G. H. Sheriha, Dept. of Chemistry, Al Fateh University, Libya. Plicatic acid, dihydroferulic acid, matairesinol, a-conidendrin, and dimethylretrodendrin were a gift of Dr. Eric Swan, Forintek, Vancouver. Arctiin was isolated from Arctium lappa and sesartemin, episesartemin, yangambin, epiyangambin, dihydrosesartemin and 0-dihydroyangambin were isolated from V. elongata (Chapter II, this thesis). The structures of these compounds are presented in Figure 18. b. Cells and vi ruses The preparation of viruses and procedures for maintaining cells has been described in Part A, this chapter. 185 Podophyllotoxin co-(-)-Peltatin DiphyllinR CH,0 OH oo -Conidendrin Dimethyl-OD-ret rodendri n Justicidin B Figure 18 - Structures of lignans tested for antiviral activity. 186 c. Antiviral screen ing of 1ignans The samples were dissolved in 95% ethanol (10% ethanol in the case of plicatic acid). These were diluted with tissue culture medium such as to allow for a final ethanol concentration of 1%. The assays were carried out by allowing the compound to remain in contact with the cells during the period of exposure to the virus and also after the virus had been removed and while the cells were being incubated for several days to allow for the development of viral plaques. Nearly confluent layers of mouse (3T3) embryo cells (between passage 15 and 22) were bathed in Dulbecco's minimum essential medium (MEM) containing either Sin,dbis virus or murine cytomegalovirus (MCMV) at a titer calculated to allow for a countable number of plaques and the compound to be tested. Podophyllotoxin, a-peltatin, justicidin B, and diphyllin and its apiosides were tested at concentrations of 0.01, 0.10 and 1.0 Mg/ml, while the remaining compounds were tested at 1.0, 10 and 100 Mg/ml. After 2 hours of incubation at 37 °C, the solution containing virus and lignan was removed and the plates were overlayed with Dulbecco's MEM containing 5% fetal bovine serum (FBS), 0.5% agarose and the appropriate lignan at the same concentration. After the plates had solidified, they were incubated until the plaques had developed sufficiently for counting. 187 d. Effect of time of treatment Three stages in the process of infection were examined for sensitivity of the virus to the presence of the antiviral lignans, podophyllotoxin and a-peltatin: the pre-infection period; the period during infection; and the period after infect ion: Pre-infect ion treatment Suspensions of virus were exposed to the various concentrations of each compound in Dulbecco's MEM, containing 0.1% ethanol for a period of 2 hours. Aliquots were added to monolayers of mouse embryo cells, diluting the virus and lignan mixture 25,000 times. After incubating the cells with virus at 37 °C for a further 2 hours, the medium was removed and the cells overlayed with MEM containing 5% FBS and 0.1% agarose. The plates were incubated until plaques had developed. Treatment dur ing infect ion Virus was combined with either podophyllotoxin or a- peltatin in Dulbecco's MEM (1% ethanol final concentration), which was then applied to a monolayer of mouse cells and the plates were incubated for 2 hours at 37 °C. The medium was removed and replaced with a solid overlay as described above. Treatment post-infection Mouse embryo cell monolayers were exposed to virus suspended in Dulbecco's MEM for 2 hours at 37 °C. The virus was removed and a solid overlay was added. This contained, in 188 addition to Dulbecco's MEM with 5% FBS and 0.5% agarose, either podophyllotoxin or a-peltatin and 1% ethanol. The plates were incubated until plaques were visible. 3. RESULTS Podophyllotoxin and a-peltatin are highly effective in preventing the development of MCMV plaques in mouse embryo cells (Table XXI). Both compounds are approximately equal in potency, reducing the number of plaques formed by almost 50% at a concentration of 10 ng/ml. Neither compound, however, had any effect on the formation of Sindbis virus plaques in mouse embryo cells, even at a dose as high as 1 Mg/ml. The arylnapthalene compounds, justicidin B and diphyllin, as well as its apioside and acetylated apioside, were effective in reducing the number of Sindbis virus plaques and, to some extent, also the MCMV plaques. Clear dose-response were not evident. None of the other lignans tested showed any antiviral activity, even at doses as high as 100 Mg/ml. To attempt to gain further information on the mode of action of the antiviral lignans podophyllotoxin and a- peltatin, the compounds were applied at various times during the process of viral infection. To determine whether these lignans had any direct effect on the viruses, virus was incubated with lignan for two hours prior to being used to infect the cells (pre-infection treatment). To test whether early or late stages of the infective process were affected, cells were infected with virus in the presence of lignan % Inhibition of Plaque Formation Lignan Tested S i ndb i s v i rus MCMV 0.01 0. 10 1 .00 10 100 0.01 0. 10 1 .00 10 100 „g/ml „/ml ///ml Podophyllotoxin 8 3 4 . nt t nt 35 74 100 nt nt a-peltat i n 0 6 7 nt nt 49 85 100 nt nt Justicidin B 42 . 74 tox * nt nt 13 14 tox nt nt D iphyl1i n 7 18 84 nt nt 7 1 1 '19 nt nt D i phy11i n 8 14 100 nt nt 26 10 9 nt nt ap i os i de D iphyl1i n 20- 96 95 nt nt 33 4 25 nt nt ap i os ide-OAc D i methylretrodendr i n nt nt 3 7 tox nt nt 1 6 tox a-Coni dendr i n nt nt. 7 0 5 nt nt 2 5 0 PIicatic acid nt . nt 1 6 0 nt nt 2 1 3 Mata i res i no 1 nt nt 7 4 tox nt nt 4 0 tox Arct i i n nt nt 4 5 3 nt nt 3 4 3 D i hydroferu1i c nt nt 4 4 0 nt nt 1 3 2 ac i d Episesartemin nt nt 2 7 2 nt nt 0 3 5 Sesartemi n nt nt 0 6 4 nt nt 4 7 6 Ep i yangamb i n nt nt 2 0 6 nt nt 3 1 6 Yangamb i n nt nt 3 6 2 nt nt 7 0 6 D i hydrov i ro1ong i n nt nt 7 4 3 nt nt 1 5 5 0-d ihydroyangamb i n nt nt 2 6 2 nt nt 5 2 4 Table XXI - Examination of lignans for their effect on replication of Sindbis Virus and MCMV in mouse cells. Results are the average of an experiment carried out in duplicate. t nt = not tested. $ tox = toxic to cells; plaques were not counted. 190 (treatment during infection) and cells already infected with virus were exposed to lignan for the complete duration of the culture period (post-infection treatment). The effects of lignans on MCMV infection are presented in Figure 19 while th,e data concerning Sindbis virus can be found in Table XXII. Figure 19 supports the initial finding that podophyllotoxin and a-peltatin are highly effective in inhibiting the formation of plaques by MCMV when applied continuously to infected cells. As shown in Table XXI, a- peltatin is slightly more potent than podophyllotoxin. When the cells are infected with virus and exposed to podophyllotoxin or a-peltatin concurrently, the lignans are only slightly less effective in inhibiting plaque formation. This is somewhat surprizing since the duration of exposure to lignan is only two hours; compared with several days (assuming that it is not metabolized by the cells) in the case of the post-infection treatment. When the virus is exposed directly to podophyllotoxin, no effect was detected at the highest concentration tested (40 Mg/ml). a-Peltatin, on the other hand, caused a dose-dependent reduction in the number of plaques formed from treated viruses. This effect was approximately three orders of magnitude weaker than that produced by treatment during or after infection by virus. The number of plaques were reduced by almost 50% after two hours pre-infection treatment with a- peltatin. Sindbis virus responded quite differently to treatment 191 LOG CONCENTRATlON(ng/ml) Figure 19 - Effect of time of lignan treatment on inhibition of murine cytomegalovirus infection in mouse embryo cells. Solid figures refer to a-( - ) -peltatin; open ones to podophyllotoxin. Squares = pre-infection treatment; triangles = treatment during infection; circles = post-infection treatment. Compound % Reduction in Plaques' Treatment 0.005 0.01 0. 1 10 40 Protoco1 „g/ml l/g/ml „g/ml „g/ml eg/ml podophyllotoxin 0 1 0 1 0 prior to infection a-peltat i n 2 1 0 1 0 podophyl1otox i n 0 1 0 13 58 during infection a-peltat i n 0 1 2 0 52 podophyl1otox i n 1 0 1 0 0 after infection a-peltat i n 0 2 0 1 1 Table XXII - Effect of time of lignan treatment upon inhibition of Sindbis Virus infection in mouse embryo cells. Results are the average of an experiment carried out in triplicate. 1 93 with podophyllotoxin or a-peltatin (Table XXII). Neither pre- infection nor post-infection treatment with either lignan had any effect, even at concentrations as high as 40 Mg/ml. Exposing the cells to lignan at the same time as they are being infected with Sindbis virus, however, resulted in a dose-dependent reduction in the number of viral plaques ultimately formed. This effect, too, is much weaker (1/1000 times as potent) as that observed for lignan treatment during or after infection by MCMV. 4. DISCUSSION Although the number of compounds tested was small, the results indicate that antiviral activity is specific to certain classes of lignans. The tetrahydrofuran lignans, dihydrosesartemin and /3-dihydroyangambin, and the bis- tetrahydrof uran lignans, episesartemin, sesartemin, epiyangambin and yangambin, were all without effect. Bis- tetrahydrof uran lignans are known to have a number of biological effects including the ability to inhibit cyclic AMP phosphodiesterase (Nikaido et_ a_l, 1981), anti-stress activity (Brekhman and Dardymov, 1969), hypotensive activity (Sih e_t al, 1976) and behavioral effects (Chapter III, this thesis). Neither of the butanolide lignans tested, matairesinol or arctiin, displayed any antiviral activity. Matairesinol, like certain bis-tetrahydrofuran lignans, is an inhibitor of the enzyme, cyclic AMP phosphodiesterase (Nikaido et, a_l, 1981). Plicatic acid is a reactive compound and is responsible for 194 producing asthma and eliciting an allergic response in man (Chan-Yeung et al, 1973). It, too, was without effect on the replication of the two viruses tested. Dimethylretrodendrin and a-conidendrin, like podophyllotoxin and a-peltatin, are cyclolignanolides. Neither had any antiviral activity. The effect of the arylnapthalene lignans, justicidin B and diphyllin and its apiosides in inhibiting Sindbis virus infection is interesting. Although these compounds are not known to have any effect on microtubules or upon nucleoside transport, justicidin B is highly toxic to fish (Munakata et al, 1965). The mechanism of action of this piscicidal compound is completely unknown and it is worthwhile considering that the antiviral and piscicidal activities of these compounds share some aspects regarding their mode of action. The experiments in which virus is exposed to podophyllotoxin or a-peltatin before, during or after infection yielded some general, though quite interesting conclusions. The anti-MCMV activity of these compounds cannot be explained by a direct effect upon the virus. Podophyllotoxin had no effect at the doses tested, while a- peltatin caused a slight inactivation of MCMV virus which was not of sufficient magnitude to explain the anti-MCMV activity. The ability of a-peltatin to damage the virus directly is consistent with previous reports that this, and other podophyllotoxin type lignans having a 4'-hydroxyl group, possess the ability to cause DNA fragmentation (Loike and Horowitz, 1976a). It is noteworthy that a-peltatin had no 195 corresponding effect upon the single stranded RNA containing Sindbis virus. A strong anti-MCMV effect of podophyllotoxin and a~ peltatin is observed only when cells are treated during or after infection. Because treatment after infection was the most effective regimen, it can be concluded that neither attachment nor penetration of the virus are stages which are disrupted by these lignans. This statement should be qualified by noting that infection by Sindbis virus was inhibited to some extent by relatively high concentrations of both podophyllotoxin and a-peltatin (Table XXII). It appears that these compounds do have a weak, but significant, inhibitory effect on the early stages of the Sindbis virus infection. It is significant that the anti-MCMV effect is evident whether the cells are treated for two hours during infection or for several days after infection. From Figure 19, it appears that the anti-MCMV activity of the post-treatment protocol was approximately three times more potent than that of the treatment during infection protocol. This difference in antiviral potency is disproportionately small relative to the difference in the dose of lignan applied in each protocol. The most obvious explanations suggested by this result are either that the effect of the two lignans is irreversible and the time of exposure is unimportant or that some stage of virus development affected by them is transient and, if interrupted, the process is not continued. Such a stage would need to exist both during and after the first two hours of virus infection. 1 96 It has been suggested that the antiviral activity of podophyllotoxin type lignans is a result of their affinity for tubulin and their ability to interrupt its aggregation (Bedows and Hatfield, 1982; Markkanen et al, 1981b). Certain drugs which affect tubulin have been studied with respect to their antiviral activities and the results are, by no means, conclusive. The early observations that colchicine or the Vinca alkaloids could suppress viral infections were encouraging from the clinical viewpoint. Weinstein and Chang (i960) found that colchicine was effective in suppressing the early stages of infection of mice by influenza and encephalomyocarditis .viruses. The colchicine derivative, demecolcine, demonstrated antiviral activity against vaccinia, polio, ECHO and Coxsackie B viruses in tissue culture and-Newcastle disease virus in the developing chick embryo (Katsilambros, 1962). Colchicine and demecolcine also suppressed infection of rabbit cornea cells and rabbit tissue culture cells by herpes simplex virus (Tokumaru and Avitabile, 1971). The Vinca alkaloid, vinblastine, increased the survival of mice infected by mengo virus (Johnston, 1965) and vincristine was active against Friend and Rauscher leukemia viruses in mice (Chirigos, 1965). Vinblastine and vincristine were also effective against herpes simplex virus infection in rabbit cornea or rabbit kidney tissue culture cells (Tokumaru and Avitabile, 1971). In a significant number of studies, however, these antimitotic substances have been found to have no effect upon 1 97 viral replication. Colchicine was observed to be ineffective against poliovirus (Kovacs, 1962), REO virus (Dales, 1963) and vaccinia virus (Solovyov and Mentkevich, 1965) in tissue culture. Vincristine had no effect on multiplication of mengo virus (Johnston, 1965) or vaccinia, polyoma, Rous sarcoma or encephalomyocarditis viruses (Freeman e_t a_l, 1965). Several studies have provided evidence that the cessation of RNA synthesis that normally accompanies the metaphase state, and also colchicine induced metaphase arrest, is responsible for the interruption of viral infection. In the case of poxvirus, it was shown that uncoating of the virus was inhibited due to the absence of a cellular protein produced in response to infection (Joklik, 1964). Newcastle disease virus did undergo normal attachment, penetration and eclipse in colchicine treated cells but further replication of the virus was prevented, apparently due to the absence of cellular RNA synthesis (Marcus and Robbins, 1963). Evidence has been presented which indicates that mitotic inhibitors also inhibit the final stage of viral assembly. Colchicine reduced the extracellular production of Semliki Forest virus by 75 to 90% and the effect was attributed to the depolymerization of microtubules (Richardson and Vance, 1978). In comparing this result with the data reported here, it is important to note that, in the study of Richardson and Vance (1978), the concentration of colchicine used was 1000 fold greater than that required to inhibit mitosis. From the results reported here, it is evident that podophyllotoxin 198 inhibits MCMV infection at the minimum dose required to prevent mitosis. An opposing effect of colchicine on viral infections has also been reported. Infectivity of herpes simplex virus type 2 DNA to rabbit kidney cells was enhanced 5 to 7 fold by exposing cells to colchicine, colcemid or vinblastine prior to infection (Farber and Eberle, 1976). Again, the doses used were considerably greater than those required to inhibit mitosis. The available evidence indicates that microtubules play a role in viral infections. Judging from the variation in the results, it appears that the nature of that role may vary a great deal, depending upon the specific virus, the specific cell type and the stage of the process of infection. The results presented here concerning the antiviral activity of a- peltatin and podophyllotoxin' do not support the hypothesis that the inhibition of microtubules by these two compounds is responsible for their antiviral activity. Two pieces of evidence may be cited: 1) these lignans exert an antiviral action against murine cytomegalovirus but not Sindbis virus. 2) the antiviral effect of the lignans against MCMV was not observed to be readily reversible. It seems unlikely that microtubules are a requirement for the multiplication of MCMV but not Sindbis virus. The irreversibility of the antiviral activity is not consistent with what is known of the antimitotic behavior of these 199 compounds (Dustin, 1978). Another cellular activity known to be inhibited by podophyllotoxin is nucleoside transport. Although the concentration required to elicit a detectible effect is somewhat greater than that resulting in inhibition of mitosis, the effect is strong and quite specific (Loike and Horowitz, 1976b; Mizel and Wilson, 1972). The studies of both Loike and Horowitz (1976a and b) and Mizel and Wilson (1972) have provided convincing evidence that the inhibition of tubulin aggregation and nucleoside uptake by podophyllotoxin, as well as by colchicine, are quite independent phenomena. For these reasons, it is worthwhile considering that the mode of the antiviral action of podophyllotoxin and a-peltatin involves an effect upon nucleoside transport. The same arguments cited above against the hypothesis that the antiviral activity is the result of tubulin binding may be applied here: i.e., there is no available explanation for the sensitivity of only MCMV or for the lack of reversibility of the effect. Inhibition of nucleoside uptake by podophyllotoxin has clearly been shown to be readily reversible (Loike and Horowitz, 1976a). Moreover, the capacity for nucleoside uptake is not absolutely required for cell growth and it is not clear why virus replication should be affected under these conditions. The synthesis of RNA, DNA and protein has been shown to be unaffected by treatment with either podophyllotoxin or colchicine (Loike and Horowitz, 1976a: Mizel and Wilson, 1972). 200 It must be concluded that the site of action of podophyllotoxin and a-peltatin which is responsible for their antiviral activity is unknown. If it involves inhibition of either tubulin aggregation or nucleoside transport then it appears that the susceptible site is a critical stage which, if interrupted during the first two hours following virus infection, ultimately prevents the normal development of the replicative cycle. More studies are needed to characterize further the nature of the specific biochemical alteration which is responsible for the inhibition of proliferation of certain viruses. It is possible that these compounds may prove to be useful tools for dissecting the molecular basis of the process of infection. 201 LITERATURE CITED Chapter IV Adolf, W., Opferkuch, H.J. and Hecker, E. (1984) Irritant phorbol derivatives from four Jatropha species. Phytochemistry 129-132. Anjaneyulu, A.S.R., Rao, K., Row, L.R. and Subrahmanyam, C. (1973) Crystalline constituents of Euphorbiaceae XII. isolation and structural elucidation of three new lignans from the leaves of Phyllanthus niruri Linn. Tetr. 29, 1291-1298. Ashton, W.A. (1972) The Logit Transformation: With Special Reference to its use in Bioassay . Griffin, London. Baruzzi, R.G., Rodrigues, M.D., Carvalho, R.P.S. and Souza Diaz, L.L. (1971) Study of neutralizing antibodies against measles virus in Indians of upper Xingu River, Central Brazil. Rev. Inst. Med. Trop. Sao Paulo 13, 356- 562. Bedows, E. and Hatfield, G.M. (1982). An investigation of the antiviral activity of Podophyllum peltatum . J. Nat. Prod. 45, 725-729. Black, F.L. (1975) Infectious disease in primitive societies. Science 187, 515-518. Black, F.L., Pinheira, F., Oliva, 0., Hierholzer, W.J., Lee, R.V., Briller, J.E. and Richards, V.A. (1978) Birth and survival patterns in numerically unstable proto- agricultural societies in the Brazilian Amazon. Medical Anthropol. 2, 95-127. Brancato, F.P. and Golding, N.S. (1953) The diameter of the mold colony as a reliable measure of growth. Mycologia 45, 848-864. Brekhman, I.I. and Dardymov, I.V. (1969). Pharmacological investigation of glycosides from Ginseng and Eleutherococcus . Lloydia 32, 46-51. Brewer, C.F., Loike, J.D., Horowitz, S.B., Sternlicht, H. and Gensler, W.J. (1979). Conformational analysis of podophyllotoxin and its congeners. Structure-activity relationship in microtubule assembly. J. Med. Chem. 22, 215-221. Buck, A.A., Sasaki, T.T. and Anderson, R.I. (1968) Health and Disease in Four Peruvian Villages: Contrasts in Epidemiology . John Hopkins University Press, Baltimore. 202 Cassady, J.M., Chang, C.-J. and McLaughlin, J.L. (1981) Recent advances in the isolation and structural elucidation of antineoplastic agents of higher plants. In: Natural Products as Medicinal Agents ( Beal, J.L. and Reinhard, E., eds.). Hippokrates Verlag, Stuttgart. Chan-Yeung, M., Barton, G.M., MacLean, L. and Grzybowski, S. (1973). Occupational asthma and rhinitis due to western red cedar ( Thuja piicata ). Am. Rev. Resp. Dis. 108, 1094-1102. Chatterjee, A., Das, B., Pascard, C. and Prange, T. (1981) Crystal structure of a lignan from Jatropha gossypi i folia . Phytochem. 20, 2047-2048. Cheo, P.C. and Lindner, R.C. ( 1964) I_n vitro and in vivo effcts of commercial tannic acid and geranium tannin on tobacco mosaic virus. Virology 24, 414-425. Chirigos, M.A. (1965). Utility of leukemia viruses for testing antiviral agents ir\ vivo . Ann. N.Y. Acad. Sci. 130, 56- 64. Dales, S. (1963). Association between the spindle apparatus and reovirus. Proc. Natl. Acad. Sci. USA. 50, 26^-275. Duffield, A.M. (1967). Mass spectronomic fragmentation of some lignans. J. Heterocyclic Chem. 4, 16-22. Dustin, P. (1978) Microtubules , Springer-Verlag, Berlin. Farber, F.E. and Eberle, R. (1976) Effects of cytochalasin and alkaloid drugs on the biological expression of herpes simplex virus type 2 DNA. Exptl. Cell. Res. 103, 15-22. Farnsworth, N.R., Henry, L.K., Svoboda, G.H., Bloomster, R.N., Yates, M.J. and Enler, K.L. (1966) Biological and phytochemical evaluation of plants. I. Biological test procedures and results from two hundred accessions. Lloydia 29, 101-122. Ferrigni, N.R., Putnam, J.E., Anderson, B., Jacobsen, L.B., Nichols, D.E., Moore, D.S., McLaughlin, J.L., Powell, R.G. and Smith, CR. (1982) Modification and evaluation of the potato disc assay and antitumour screening of Euphorbiaceous seeds. J. Nat. Prod. 45, 679-686. Fong, H.H.S., Farnsworth, N.R., Henry, L.K., Svoboda, G.H. and Yates, M.J. (1972) Biological and phytochemical evaluation of plants. X. Test results from a third two hundred accessions. Lloydia 35, 35-48. Freeman, G., Kuehn, A. and Sultanian, L. (1965). Tumorigenic viruses and cell responses to biologically active 203 compounds. II. Pharmacologic specificity in cell-virus relationships. Ann. N.Y. Acad. Sci. 130, 330-338. Galsky, A.G., Kozimor, R., Piotrowski, D. and Powell, R.G. (1981) The crown gall potato disc assay as a primary screen for compounds with antitumour activity. J. Natl. Cancer Inst. 67, 689-692. Galsky, A.G., Wilsey, J.P. and Powell, R.G. (1980) The crown gall tumour disc assay: a possible aid in the detection of compounds with antitumour activity. Plant Physiol. 65, 184-185. Hafner, D. , and Noack, E. (1977) Mathematical analysis of concentration response relationships. Arzneim-Forsch. 27, 1871-1873. Farnsworth, N.R., Svoboda, G.H. and Bloomster, R.N. (1968). Antiviral activity of selected Catharanthus alkaloids. J. Pharm. Sci. 57, 2174-2175. Hartwell, J.L. (1969) Plants used against cancer: a survey. Lloydia 32, 157-176. Ieven, M., Van den Berghe, D.A., Mertens, F., Vlietinck, A. and Lammens, E. (1979) Screening of higher plants for biological activities. I. Antimicrobial activity. Planta Medica 36, 311-321 . John, T.J. and Mukundan, P. (1978) Antiviral property of tea. Curr. Sci. 47, 159-160. Johnston, I.S. (1965). Observations on antiviral screening. Ann. N. Y. Acad. Sci. 130, 52-^55. Joklik, W.K. (1964). The intracellular uncoating of poxvirus DNA. II. The molecular basis of the uncoating process. J. Mol. Biol. 8, 277-288. Katsilambros, L. (1962) Colchicine and its derivatives against viral diseases. Rev. Med. Moyen Orient 19, 318-320. Kelleher, J.K. (1978). Correlation of tubulin binding and antitumour activities of podophyllotoxin analogues. Cancer Treat. Rep. 62, 1443-1447. .Khan, M.R., Ndaalio, G., Nkunya, M.H.H., Wever, H. and Sawhney, A.N. (1980) Studies on African medicinal plants. Part I. Preliminary screening of medicinal plants for antibacterial activity. Planta Medica, Suppl. 91-97. Kinghorn, A.D. (1979) Cocarcinogenic irritant Euphorbiaceae. In: Toxic Plants ( Kinghorn, A.D., ed.) Columbia -University Press, New York, pp. 137-1 60. 204 Kinghorn, D.A., Harjes, K.K., and Doorenbos, N.J. (1977) Screening procedures for phorbol esters using brine shrimp( Artemia salina ) larvae. J. Pharrn. Sci. 66 1362- 1 363. Konowalchuk, J. and Speirs, J.L. (1976) Antiviral effect of fruit extracts. J. Food Sci. 41, 1013-1017. Konowalchuk, J. and Speirs, J.L. (1978) Antiviral effect of commercial juices and beverages. Appl. Environ. Microbiol. 35, 1219-1220. Kovacs, E. (1962). Poliovirus production in HeLa cells in presence of colchicine. Experientia 18, 70-71. Kupchan, S.M., LaVoie, E.J., Branfman, A.R., Fei, B.Y., Bright , W.M., and Bryan, R.F. (1977) Phyllanthocin, a novel bisabolene aglycone from the antileukemic glycoside, phyllanthoside. J. Amer. Chem. Soc. 99, 3199-3201 Kupchan, S.M., Sigel, C.W., Matz, M.J., Gilmore, C.J. and Bryan, R.F. (1976) Stucture and stereochemistry of jatrophone, a novel macrocyclic diterpenoid tumour inhibitor. J. Amer. Chem. Soc. 98, 2295-2300. Larrick, J.W., Yost, J.A., Kaplan, J., King, G. and Mayhall, J. (1979) Patterns of health and disease among the Waorani Indians of eastern Ecuador. Medical Anthropol. Loike, J.D. and Horowitz, S.B. (1976a). Effect of VP-16-213 on the intracellular degradation of DNA in HeLa cells. Biochem. 15, 5443-5448. Loike, J.D. and Horowitz, S.B. (1976b). Effects of podophyllotoxin and VP-16-213 on microtubule assembly in vitro and nucleoside transport in HeLa cells. Biochem. 15, 5435-5442. MacRae, W.D. and Towers, G.H.N. (1984) The biological activities of lignans. Phytochem. (in press). Marcus, P.I. and Robbins, E. (1963) . Viral inhibition in the metaphase-arrest cell. Proc. Natl. Acad. Sci. USA 50, 1156-1164. Markkanen, T. , Makinen, M.L., Maunuksela, E. and Himanen, P. (1981a). Podophyllotoxin lignans under experimental antiviral research. Drugs Exptl. Clin. Res. 7, 711-718. Markkanen, T., Makinen, M.L., Nikoskelainen, J., Ruohonen, J., Nieminen, K., Jokinen, P., Raunio, R., and Hirvonen, T. (1981b). Antiherpetic action from juniper tree (Juniperus communis), its purification, identification, and testing 205 in primary human amnion cell cultures. Drugs Exptl. Clin. Res. 7, 691-697. Meyer, B.N., Ferrigni, N.R., Putnam, J.E., Jacobsen, L.B., Nichols, D.E. and McLaughlin, J.L. (1982) Brine shrimp: a convenient general bioassay for active plant constituents. Planta Med. 45, 31-34. Mizel, S.B. and Wilson, L. (1972). Nucleoside transport in mammalian cells inhibited by colchicine. Biochem. 11, 2573-2578. Mok, W.Y. and Netto, CF. (1978) Paracoccidioidin and histoplasmin sensitivity in Coari(state of Amazonas), Brazil. Amer. J. Trop. Med. Hygiene 27, 808-814. Mosmann, T.R. and Hudson, J.B. (1973) Some properties of the genome of murine cytomegalovirus MCV. Virology 54, 135- 149. Munakata, K., Marumo, S. and Ohta, K. (1965). Justicidin A and B, the fish-killing components of Just ic ia hayatai var. decumbens . Tetr. Lett. 47, 4167-4170. Neel, J.V., Andrade, A.H.P., Brown, G.E., Eveland, W.E., Weinstein, E.D. and Wheeler, A.H. (1968) Further studies of the Xavante Indians IX. Immunologic status with respect to various diseases and organisms. Am. J. Trop. Med. 17, 486-498. Nikaido, T.f Ohmoto, T., Kinoshita, T., Sankawa, U., Nishibe, S. and Hisada, S. (1981). Inhibition of cyclic AMP phosphodiesterase by lignans. Chem. Pharm. Bull. 29, 3586-3592. Persinos-Perdue, G., Bloomster, R.N., Blake, D.A. and Farnsworth, N.R. (1979) South American plants II. taspine isolation and anti-inflammatory activity. J. Pharm/ Sci. 68, 124-126. Ragetli, H.W.J. (1975) The mode of action of natural plant virus inhibitors. Curr. Adv. Pi. Sci. 19, 321-334. Ragetli, H.W.J, and Weintraub, M. (1974) The influence of inhibitors on the reaction of indicator plants. Contribution No. 339, Research Station, Agriculture Canada, Vancouver, B.C., pp. 1-13. Richardson, CD. and Vance, D.E. (1978). The effect of colchicine and dibucaine on the morphogenesis of Semliki Forest virus. J. Biol. Chem. 253, 4584-4589. Sethi, M.L. (1977) Inhibition of RNA directed RNA polymerase activity of RNA tumour viruses by taspine. Can. J. Pharm. 206 Sci. 12, 7-9. Sih, C.J., Ravikumar, P.R., Huang, F.-C, Buckner, C. and Whitlock, H., Jr. (1976). Isolation and synthesis of pinoresinol diglucoside, a major antihypertensive principle of Tu-Cheng ( Eucommia ulmoides , Oliver). J. Amer. Chem. Soc. 98, 5412-5413. Solovyov, V.D. and Mentkevich, L.M. (1965). The effect of colchicine on viral interference and interferon formation. Acta Virol. (Prague) 9, 308-312. Spendlove, R.S., Lennette, E.N., Chin, J.N. and Knight, CO. (1964). Effect of antimitotic agents on intracellular reovirus antigen. Cancer Res. 24, 1826-1833. Takechi, M. and Tanaka, Y. (1981) Purification and characterization of antiviral substances from the bud of Syzygium aromatica• Planta Medica 42, 69-74. Tokumaru, T. and Avitable, A. (1971). Suppression of Herpes Simplex virus infection by antimitotic substances in the rabbit cornea (35505). Proc. Soc. Exp. Biol. Med. 137, 29-34. Van den Berghe, D.A., Ieven, M., Mertens, F.and Vlietinck, A.J. (1978) Screening of higher plants for biological activities. II. Antiviral activity. Lloydia 41, 43-47. Waravdekar, V.S., Paradis, A.D. and Leiter, J. (1953). Enzyme changes induced in normal and malignant tissues with chemical agents. III. Effect of acetylpodophyllotoxin-cj- pyridinium chloride on cytochrome oxidase, cytochrome c, succinoxidase, succinic dehydrogenase and respiration of sarcoma 37. J. Natl. Cancer Inst. 14, 585-592. Weinstein, L. and Chang, T.-W. (1960). The effect of colchicine and some chemically related compounds on experimental viral infections. Antibiot. Chemother. (Washington, D.C.) 10, 180-187. 207 APPENDIX A List of Amazonian Angiosperms of Ethnobotanical Interest Taxon Use Code References Class: Magnoliopsida Subclass I. Magnoliidae Order: Magnoliales Family: Annonaceae Anaxagorea sp. 02 55 Annona ambotay 02 2 A. spinescens 03 42 A. tessmannii T 2 Duguetia riparia 02 42 Guatteria calva 02 53 G. duckeana T 55 G. dura 02 61 Unonopsis veneficiorum G1 02 42,49,51,57,61 Xylopia amazonica K2 55 X. aromatica G5 P5 61 X. benthamii K2 55 Family: Myristicaceae Compsoneura capitellata K6 49 C. debilis A1 A2 49 C. sprucei T 49 Dialyanthera otoba A2 30,31 D. parvifolia C6 38 Iryanthera crassifolia A2 49 I. grandis T 49 I . longi folia A2 49 I. macrophylla R1 64 I. polyneura A2 49 I. tricornis A2 49 I. ulei A2 49 Osteophloem platyspermum D1 D2 55 Virola albidiflora A2 54 V. bicuhyba A2 B2 D2 49 K3 M V. calophylla R1 49 V. calophylloides R1 49,2 V. carinata A4 49 V. cuspidata R1 49 V. elongata A2 02 R1 49 V. flexuosa A2 49 208 V. loretensis T 49 v. melinonii A2 49 V. peruviana K1 46, 49 V. rufula R1 4 V. schultesi i A2 49 V. sebi fera B2 R1 S 2,49 V. surinamensis A2 A8 C8 49 V. theiodora 02 R1 49 Virola sp. G1 4 Order : Laurales Family: Monimiaceae Siparuna quianensis A2 B2 CI 2 S. sp. B2 C1 G2 2, 36 Family: Lauraceae Nectandra pichiirin C5 K2 1 5 N. radiac i P2 1 5 Ocotea venenosa 02 45 Couroupita sp. A2 25 Order : Piperales Family: Piperaceae Peperomia emarqinella N 2, 51 P. glabella JI 51 P. macrostachya F1 P2 55 P. serpens N 51 Piper aduncum A1 A8 C5 E3 2 P. alegreanum K1 2 P. anti rheumat icum B2 2 P. bogotense A2 2 P. caudatum 02 42 P. dactylost igmum K1 51 P. erythroxyloides C7 I 1 K1 1 P. futuri T 2 P. gen iculatum K1 42 P. hispidum 04 P2 U2 2 P. hostmannianum A5 55 P. interitum K5 55 P. iguitosensis T 2 P. marginatum CI 1 1 P. nigrum D3 1 1 P. nubigenum A1 2 P. obliquum 02 2 P. peltatum A2 K1 2 P. schultesi i D3 F1 61 P. serpens N 2 |\ sudisilvestre A2 B2 C5 1 1 E3 F1 G9 P. tingens U 2 P. tuberculatum B2 C6 N 04 1 1 Piper sp. A1 K1 K2 02 2 Pothomorphe umbellata F1 02 55 209 Order: Aristolochiales Family: Aristolochiaceae Aristolochia anquicida N 1 5 A. barbata N 1 5 A. brasiliensis T 1 5 A. grandiflora 12 1 5 A. maxima N 12 1 5 A. medicinalis K6 47 A. pilosa K1 1 5 Aristolochia sp. A2 C2 K7 N T 2, 11, 15 Order: Ranunculales Family: Menispermaceae Abuta caudicans 02 42 A. qrandiflora 02 P2 2, 1 1 A. imene 02 03 1 142,5 , 3 A. macrocarpa 02 1 1 A. rufescens F3 02 1 1 ,4 2 A. splendida 02 53 A. vaupesensis 02 53 A. verruculosa 02 1 1 Andontocarya tripetala C6 53 Anomospermum reticulatum 02 53 Chondrodendron tomentosum B2 F3 14 28, 42 * N 02 P2 C. toxiferum 02 2, 42 Cissampelos andromorpha 02 1 1 C. pareira F1 03 P2 1 1 ,4 2 Cocculus imene 02 42 Curarea tecunarum G1 02 42, Orthomene schomburgkii 01 55 Telitoxicum peruvianum 02 A2 55 Order: Papaverales Family: Papaveraceae Boccocia frutescens A2 C3 C6 04 1 1 Subclass II. Hamamelidae Order: Urticales Family: Moraceae Brosimium utile D2 D3 1 i Chlorophora tinctoria C6 K1 U3 2, 1 1 Ficus anthelminthica 01 42 F. atrox 02 42 F. dulceria C6 1 1 F. qlabrata C6 1 1 Helicostylis scabra A2 54 Naucleopsis mello-barretoi 03 37 Poulsenia armata T 2 Pseudolmedia laeviqata 01 54 21 0 Family: Cecropiaceae Cecropia tolimensis P2 2 Coussapoa cinnamomea 03 54 C. magnifolia T 54 C. orthoneura J3 54 C. trinervia J3 54 Pourouma cucura B2 61 P. schultesii A2 54 Subclass III. Caryophyllidae Order: Caryophyllales Family: Phytolaccaceae Pelteveria alliaceae 1 1 Phytolacca bogotensis A2 C6 M 01 1 1 ,5 3 P. rivinoides A6 C6 M 03 1 1 ,5 3 Family: Nyctaginaceae Boerhaavia coccinea C2 2 Nea parviflora U4 2 Family: Cactaceae Epiphyllum sp. R2 39 Opuntia sp. R1 39 Trichocereus pachanoi R1 39 Neoraimondia macrostibas R2 39 Family: Chenopodiaceae Chenopodium ambrosiodes Family: Amaranthaceae •Alternanthera Lehmanni i R2 47 Amaranthus hybridus CI 2 Gomphrena sp. U1 42 Iresine celosia A2 A8 B2 1 1 Iresine sp. R2 1 1 Family: Portulacaceae Calandrinia caulescens T 2 Portulaca perennis F3 2 Talinum paniculatum T 2 Family: Basellaceae Andredera di f fusa T 2 Family: Caryophyllaceae Drymaria pauciflora T 2 Order: Polygonales Family: Polygonaceae Polygonum punctatum C2 C5 04 1 1 Rumex obtusifolia C3 48 Subclass IV. Dileniidae Order: Dilleniales Family: Dilleniaceae Curatella americana B2 E4 1 1 Davilla lacunosa A2 A8 U2 2 Tetracera rotundiflora hi 2 21 1 Order: Theales Family: Caryocaraceae Caryocar glabrum 03 42 C. gracile 01 47 C. tessmanni i 03 2 Caryocar sp. 03 47 Family: Marcgraviaceae Souroubea crassipetala F3 53 S. guianensis K2 61 S. pachyphylla Jl 61 Family: Quiinaceae Qui ina leptoclada E3 53 Family: Clusiaceae Caraipa parrielliptica A2 J3 55 Clusia amazonica A6 1 1 C_. 1ineolata T 2 C. renggerioides A2 C3 1 1 Clusia sp. R2 39 Kielmeyera rosea A4 2 Symphonia globulifera A1 55 Vismia angusta A2 5 V. ferruginea A2 55 V. tomentosa A2 3 Order: Malvales Family: Tiliaceae Apeiba tibourou T 1 5 Luehea sp. A6 2 Triumfetta lappula A1 2 Family: Sterculiaceae Guazuma ulmi folia A2 1 1 Herronia camargoana V 55, 61 Sterculia apetala F1 K2 1 1 Theobroma subincanum R2 55 Family: Bombaceae Bombax globosum A1 55 Patinoa icthyotoxica 03 47 Family: Malvaceae Abelmoschus esculentus Cl 2 Abutilon vi rgatum P6 2 Hibi scus abelmoschus C4 K6 1 1 Malachra rudis A8 1 1 Malva verticillata A2 2 Malvastrum peruvianum C3 2 Pavonia cancellata Cl 2 P. hookeri T 1 5 Sida glomerata T 1 1 S. setosa T 1 1 21 2 Order: Violales Family: Flacourtiaceae Banara quianensis 01 61 Carpotroche amazonica 01 2 Casearia resinifera 01 2 C. sylvestris A1 A2 1 1 Casearia sp. A4 D1 U2 2, 3 Lunania parviflora 01 47 Mayna amazonica B2 04. 43, 53 M. linquifolia A2 53 M. longifolia C2 53 M. muricida 01 47 M. toxica 01 55 Ryania angustifloia 01 02 55 R. pyrifera 01 2 R. spruceana 01 2 Family: Bixaceae Bixa orellana C1 H1 04 1 1 B. purpura C1 H1 04 1 1 B. urucurana C1 H1 04 1 1 Family: Violaceae Corynostylis volubilis C6 K3 53 Hybanthus lanatus D1 2 Family: Turneraceae Turnera ulmifolia D6 F1 13 Q 1 1 Family: Passifloraceae Passiflora vitifolia C4 K2 P2 R1 1 1 Family: Cucurbitaceae Anguria umbrosa 01 53 Anguria sp. 03 60 Apodanthera herverae C3 1 5 Anisosperma passiflora C1 1 5 Cayaponia opthalmica J1 47 C. racemosa 01 1 Fevillea cordifolia N 1 1 Gurania rufipila 01 53 Luffa operculata C3 P5 1 1 Family: Begoniaceae Begonia rossmanniae J1 2 Order: Salicales Family: Salicaceae Salix humboldtiana C1 C6 P2 11, Order: Ericales Family: Ericaceae Befaria congesta D6 53 B. resinosa D6 11, 53 Pernettya prostrata 01 2 21 3 Order: Primulales Family: Theophrastaceae Clavija poeppiqii 02 2 Family: Myrsinaceae Anthodiscus obovatus 02 03 53, 60 A. peruanas 03 53, 60 Conomorpha citrifolia P2 V 48 C. lithophyta 03 47 Subclass V. Rosidae Order: Rosales Family: Connaraceae Connarus opacus 03 46 C. sprucei 03 47 Rourea cuspidata 01 54 R. qlabra D4 03 10, 47 Order: Fabales Family: Leguminosae Abrus precatorius C3 D6 Jl 04 1 1 Acosmium nitens 02 55 Alexa imperatracis 03 42 Anadenanthera peregrina R1 47 Andira araroba A2 1 5 A. inermis C6 K5 01 1 1 A. retusa T 2 Apurimacia incarum 03 42 A. michelli 03 42 Bauhinia guianensis 03 G1 15, 42 B. inermis K2 1 5 B. splendens 14 P2 1 1 B. tarapotensis B2 C5 1 1 Bowdichia virgiliodes B2 03 1 1 ,4 2 B. ariza E3 1 1 Caesalpinia coriaria C5 1 5 C. pulcherima 03 42 Cajanus cajan A8 2 Campsiandra anqustifolia P2 61 C. laurifolia A2 R2 35, 61 Cassia affinis A2 C3 1 5 C. alata A2 C3 1 1 C. fruticosa K1 61 C. hirsuta 03 42 C. macrophylla 03 42 C. moschata C3 1 1 C. occidentalis C3 D2 P2 1 1 ,1 5 C. ruiziana T 61 C. tora C3 K2 M P2 1 5 Centrosema plumeria 03 42 Chaetocalyx latisiligua A4 2 Clathropis brachypetala 03 42 21 4 Clitoria ternatea C3 F1 1 1 Copaifera guayanensis D6 1 5 C. hymenaefolia K1 K5 03 1 5 Coumarouma odorata A2 D3 V 1 1 Crotolaria pilosa A1 1 1 Crudia amazonica C2 61 Desmodium sp. I 1 1 5 Dipteryx tetraphylla E2 2 Entada polyphylla D1 53 E. scandens N 1 5 Erythrina corallodendron 02 42 Heterostemon mimosoides V 55 Hoffmanseggia gracilis D4 2 Hymenaea courbaril C6 F3 1 1 Indigofera suffruticosa 14 K6 1 1 Inga spectabilis B2 C5 1 1 Leguminosa sp. A6 01 2 Lonchocarpus latifolius C3 03 1 1 L. nicou 03 1 1 L. sericeus C3 02 04 1 1 L. urucu 03 2 Lonchocarpus sp. 03 60 Lupinus mutabilis 04 42 Macrolabium acaciaefolium A1 61 M. multijugum 01 61 Mimosa hostilis P1 1 5 M. inuisa C2 01 Q 1 5 M. pudica Q 1 1 M. verrucosa K5 50 Monopteryx angustifolia C6 53 M. uaucu C6 53 Mucuna rostrata A7 42 Myroxylon balsamum A1 1 1 Ocimum micranthum R2 47 Ormosia coccinea 01 47 0. lignivalvis A1 47 0. macrophylla 02 47 Piptadenia macrocarpa D3 R2 1 1 Pithecellobium laetum R1 35 Prosopis juliflora D3 1 1 Psoralea pubescens F3 2 Pterocarpus rohrii P2 61 Swartzia auriculata 03 48 S. brachyrhachis C6 48 S. cabrerae C6 48 S. conferta C5 48 S. gigantea 01 48 S. longistipitata G5 61 S. microcarpa C5 48 S. pendula 03 48 S. racemosa C5 48 • 21 5 S. recurva Q 61 S. schomburgkii C6 48 S. schultesii 03 48 S. sericea 03 48 S. simplex T 48 Tachiqalia cavipes A2 C2 G1 04 54 T. myrmecophila A2 K1 61 T. paniculata C2 54 T. ptychophysea I 1 K1 61 Tephrosia sinapou A8 03 04 1 1 Tephrosia sp. 03 60 Zornia leptophylla 04 61 Order: Myrtales Family: Lythraceae Cuphea racemosa F1 2, 60 Family: Thymelaeaceae Schoenobiblus peruvianus A2 02 03 52, 54 Styrax tessmannii A2 01 49, 54 S. yapobodensis A5 54 Family: Onagraceae Epilobium denticulatum T 2 Family: Melastomataceae Arthrostemma grandiflorum F1 2 A. volubile J3 P2 1 1 Graffenrieda rupestris A2 61 Family: Combretaceae Combretum alternifolium JI 1 5 C. cacoucia 01 2,53,55 Terminalia catappa C1 C5 D2 1 1 Order: Santalales Family: Olacaceae Heisteria pallida B2 R2 35 Heisteria sp. BI 2 Family: Loranthaceae Gaiadendron punctatum D1 2 Oryctanthus botryostachys A8 E4 1 1 Phoradendron piperoides G5 1 1 Phrygilanthus eugeniodes R2 39 Psittacanthus collum-cygni E3 2 Family: Balanophoraceae Corynaea crassa G8 22 Order: Celastrales Family: Celastraceae Maytenus laevis B2 1 1 M. pseudocasearia C5 2 Family: Icacinaceae Calatola columbiana A9 U4 32 Humirianthera ampla 01 42 Family: Dichapetalaceae Stephanopodium peruvianum P2 35 21 6 Order: Euphorbiales Family: Euphorbiaceae Alchornea castaneifolia B2 22 A. cordatum T 2 Caryodendron orinocense A4 1 1 Cnidoscolus urens C3 2 Codiaeum varieqatum 01 2 Croton cajucara P2 2 C. ferrugineus T 2 C. qlabellus A2 55 C. gossipifolius A1 2 C. lechlefi A1 A2 M 2, 22 C. maqdalensis T 2 C. palanostiqma A2 2, 55 C. polycarpus T 2 C. rhamnifolius T 2 C. scaber T 2 C. trinitatis C2 D3 D4 3 Croton sp. 04 P2 1 5 Euphorbia cotinifolia 02 42 E. cotinoides A6 02 2, 42 E. peplus 01 2 E. pilulifera D2 1 5 E. thymifolia P4 1 5 Euphorbia sp. A1 3, 58 * Guatteria maqalophylla 02 42 G. veneficiorum 02 42 Hippomane mancinella 01 02 42 Hura crepitans A6 03 04 2,1 1 ,42 Jatropha angusti I 1 2 J. ciliata 11 03 15, 42 J. curcas C3 M 01 2,15,42 02 03 J. gossypifolia A6 C3 M 1 5 J. urens A8 F1 12 2, 1 5 Mabea nitida A4 55 Manihot esculenta A2 1 1 Micrandra spruceana E3 J3 54 Nealchornia yapurensis 03 53 Pedilanthus tithymaloides C2 C5 D6 1 11 , 5,42 14 01 Phyllanthus acuminatus 03 2 P. brasiliensis 03 2 P. cladotrichus 03 42 P. conami 03 42 P. niruri F3 P2 2 P. piscatorum 03 04 2, 60 P. pseudo-conami 03 2 P. rosellus F3 2 P. salviaefolius F1 P3 1 5 Sebastiana pachyphylla C3 2 21 7 Order: Linales Family: Erythroxylaceae Erythroxylum coca Cl K1 K3 35 E. novagranatensis C1 K1 K3 35 Family: Humiriaceae Humiria balsamifera A1 A2 49 H. crassifolia A2 49 Humiriastrum piraparanense C2 K1 49 H. villosum C3 49 Sacoglottis ceratocarpa D3 49 Schiekia orinocensis K7 54 Schistostemon macrophyllum D3 D5 G2 49 Vantanea parviflora T 49 Order: Polygalales Family: Malpighiaceae Banisteria leiocarpa T 2 Banisteriopsis caapi R1 2, 47 B. inebriens R1 2, 47 B. martiniana R1 1 1 B. rusbyana R1 2 Byrsonima crassifolia C5 03 P2 1 1 ,4 2 B. lancifolia B2 2 Heteropsis macrostachya A2 C5 52 H. riparia 14 01 47, 52 Hirea apaporiensis Jl 52 H. schultesii Jl 52 Mascagnia glandulifera A2 52 Mezia includens C2 C3 F1 52 Tetrapteris methystica R1 47 T. mucronata 02 R2 2, 52 T. silvatica A2 52 T. styloptera A2 P2 52 Family: Vochysiaceae Qualea acuminata C6 55, 61 Vochysia columbiensis 02 53 V. ferruqinea A2 J3 02 R2 53, 60 V. laxiflora A2 D3 F3 53 V. lomatophylla G1 G4 2, 53 Order: Sapindales Family: Sapindaceae Cardiospermum qrandifolium 03 42 Paullinia alata 03 2 P. emetica C2 53 P. yoco K3 Q P2 2, 1 1 Serjania sp. J3 61 Toulicia bullata P2 2 218 Family: Burseraceae Bursera gumifera C3 D6 14 1 5 K1 P1 Hedwigia balsamifera A1 1 5 Protium heptaphyllum K1 2 P. neqlectum T 2 Protium sp. 02 2 Family: Anacardiaceae Anacardium occidentale A4 A6 C3 1 1 13 01 Q Loxopterygium huasango A7 42 Mauria aurantiodora A7 42 -M. heterophylla A7 2 Rhus sp. A7 42 Spondias mombin A2 G5 14 11,25 Family: Simaroubaceae Quassia cedron U1 42 Simarouba cedron P2 2 S. versicolor U1 42 Family: Meliaceae Carapa guianensis B2 - 1 5 Trichilia oblonga D3 2 Family: Rutaceae Angostura trifoliata P2 Q 1 5 Hortia sp. P2 2 Rauia resinosa P2 2 Ruta qraveolens K1 1 1 Zanthoxylum sp. 02 42 Order: Geraniales Family: Oxalidaceae Oxalis lotoides D4 55 Subclass VI. Asteridae Order: Gentianales Family: Loganiaceae Antonia ovata 03 2, 42 Buddleia americana T 2 Potalia amara C3 14 J1 N 54 Spigelia humboldtiana C6 1 1 Strychnos guianensis 02 42 S. peckii 02 2 S. solimoesana 02 42 S. toxifera 02 42 Family: Gentianaceae Chelonanthus alatus 04 3 Chelonanthus chelonoides 04 2 Gentiana chamuchni T 2 Halenia weddelliana 14 2 21 9 Family: Apocynaceae Allamandra ambetti C2 C3 15 A. cuneata 03 57 A. lopezii A2 02 57 A. markqrafiana P2 57 Aspidosperma discolor P2 2 A. megalocarpa A7 57 A. nitidum A6 P2 2, 57 A. schultesii A2 04 57, 61 Aspidosperma sp. P2 2 Couma macrocarpa A9 K5 3, 57 Hancornia speciosa A5 57 Himatanthus bracteatus N P2 57, 60 H. phagedoenicus N P2 57 H. sucuuba D3 01 2, 57 Himatanthus sp. 03 58 Lacmella sp. K5 57 Macoubea guianensis D3 2, 57 Macrosiphonia longiflora 2 M. velame 14 2 Malouetia duckei 03 2 M. nitida A2 01 11, 53 M. tamaquarina 01 R2 47 Mandevilla anceps A5 57 M. annulariifolia A5 57 M. cuneifolia 01 57 M. neriodes A2 57 M. scabra A9 57 M. steyermarkii A2 C5 I 1 57 M. stephanotidifolia N 57 M. subcarnosa 01 57 M. thevetioides 03 57 M. trianae A2 57 M. vanheurekii A2 01 57 Mandevilla sp. 03 47 Mesechites trifida K2 57 Odontadenia cognata 04 57 0. funigera V. 57 0. neglecta D5 01 57 0. sylvestris K1 04 57 Parahancornia krukovii A1 57 Plumeria tarapotensis B2 2 220 Tabernaemontana amygdalaefolia A5 01 M 1 1 T. grandiflora A2 B2 1 1 T. heterophylla . Q 57 T. muricata K3 57 T. rimulosa K2 57 T. sananho C2 F1 K2 57 01 P2 Q T. stenoloba 01 61 T. tetrastachya B2 K3 57 T. undulata C6 57 Tabernaemontana sp. 03 2 Thevetia peruviana 01 42 Family: Asclepiadaceae Asclepias curassavica C2 C3 C6 2,11,15 E3 14 01 Sarcostemma andinum N 2 S. claucum JI 1 1 Order: Solanales Family: Solanaceae Brunfelsia chiricaspi K5 35 B. guianensis B2 14 01 21 B. grandiflora B2 D3 K1 K5 35 01 P2 B. latifolia B2 2 B. maritima B2 2 B. mire C6 01 35, 41 B. tastevinii R1 41 B. uniflora A8 B2 C3 F1 23,29,66 G1 14 K1 K5 01 PI Brunfelsia sp. N, B2 1,6,7,8,33 Capsicum pendulum 02 42 Cestrum laeviqatum 03 42 C. loretense 01 61 C. ochraceum B2 P1 61 C. reflexum 01 61 Cyphomandra crassifolia C6 54 C. dolichorachis C6 54 C. endopoqon T 54 Datura arborea A8 M 1 5 D. stramonium 02 42 D. suaveolens K5 47 Iochroma fuchsiodes R2 50 Jaltometa procumbens F1 P2 61 221 Juanulloa ochraceae Al R2 54 Markea coccinea C6 Jl 54 Nicotiana tabacum R2 47 Physalis pubescens T 2 Saracha asperolanatum F3 2 S . asperr imum 01 2 S. lycocarpum D1 2 S . phyllanthum A1 2 S . procumbens F1 P2 55 S. spectabile 01 2 S. swartzianum K1 Q 2 Solanum albidum A2 54 S . apaporum 04 54 S . campani forme 04 54 S. crinitipes 01 61 S. jamaicense 04 54 S. lepidotum B2 61 S 04 61 S mammosum n 1 g r urn A2 C4 01 M 1 1 S. scabridum J2 61 S. subinerme T 54 S. topi ro J3 54 S. verbascifolium A7 54 Family: Convolvulaceae Ipomea crassi folia Q 2 Ipomea sp. C3 2 Merremia alata C3 2 Family: Hydrophyllaceae Wigandia caracasana K3 2 Order: Lamiales Family: Boraginaceae Cordia allidora A2 B4 1 1 C. dentata D3 1 1 C. ecalyculata Q 2 C. lutea T 2 C. verbenacea T 2 Heliotropium argentatum K6 2 H. tiaridioides D3 2 Tournefortia brevilobata Q 2 Family: Verbenaceae Callicarpa odorata B2 2 Lantana affinis C5 2 L. camara C1 C4 D6 1 1 P1 P2 L. fucata D1 2 Stachtarpheta cayennensis A2 C5 2 S. straminea A2 C5 2 Verbena litoralis C3 2 Verbenacea sp. C1 2 222 Family: Lamiaceae Hyptis brachiata A2 E3 1 1 H. capitata A2 A4 B4 1 1 H. carpinifolia B2 CI 2 H. mutabilis P2 1 1 H. sinuata D2 D3 1 1 Lepechinia meyeni C1 2 Ocimum micranthum R2 50 Rosmarinus officinalis B2 2 Salvia haenkei T 2 S. macrophylla F3 2 S. palaefolia 04 2 S. pichinchensis K2 2 Satureia tomentosa D1 2 Order: Scrophlariales Family: Scrophulariaceae Alonsoa cuadrialata D2 1 5 Antirrhinum majus D3 1 5 Calceolaria inamoena B3 2 Castilleja communis T 2 Family: Gesneriaceae Besleria drymophila N 1 1 Besleria sp. N 2 Columnea sp. T 2 Nauticalyx sp. N 47 Family: Acanthaceae Acantha viridis T 1 5 • Aphelandra aurantiaca J2 56 A. pilosa D4 56 Fittonia argyroneura K1 56 F. verschaffeltii D4 F3 P2 56 Justicia blackei D7 56 J. cabrerae A2 56 J. chlorastachya A2 56 J. comata 04 56 J. ideogenes K7 56 J. pectoralis A2 R2 25, 56 J. schultesii A2 56 Mendoncia aspera 03 47 Ruellia colorata C2 C6 56 R. humboldtiana C3 1 1 Sanchezia thinophila A9 56 Teliostachya lanceolata R2 56 Trichanthera gigantea C6 E4 F1 1 1 Family: Pedaliaceae Proboscidea peruviana T 2 223 Family: Bignoniaceae Arrabidaea chica A2 A9 1 1 A. xanthophylla J1 47 Bignonia opthalmica JI 1 5 Cremastus sceptrum 14 2 Distictella racemosa 01 02 48 Jacaranda glabra A2 14 1 1 Macfadyena unguis-cati 1 4 N P2 1 1 Martinella obovata 02 P2 47, 48 Mussat ia hyac inthina V 43 Pleonotoma jasminifolium C2 1 1 Pseudocalymma alliaceum D3 B2 2, 55 Pyrostegia venusta 01 2 Tabebuia barbata A2 01 1 1 T. serratifolia M 43 Tabebuia sp. A7 42 Tanaec ium nocturnum R1 1 1 Order: Campanulales Family: Campanulaceae Centropogon calycinus I 1 2 Isotoma longiflora R2 50 Lobelia decurrens 01 2, 42 Siphocampylus corymbi ferus 01 2 Order: Rubi'ales Family: Rubiaceae Calycophyllum spruceanum A2 04 53 Cephaelis barcellana T 61 Chiococca brachiata U1 42 C. racemosa C2 1 5 Coussarea pilosiflora T 2 Diodia hyssopi folia T 2 Duroia hirsuta 01 2, 47 D. kotchubaeoides 01 46, 47 D. pet iolaris 01 47 D. sacc i fera 01 47 D. sprucei 01 46 Exostemma peruviana P2 2 Genipa americana D3 25 Isert ia alba A2 M 1 1 haenkeana B2 M 1 1 hypoleuca G5 53 rosea P2 54 Ladenbergia magnifolia P2 1 1 Manettia divaricata T 2 Pagamea coriacea K7 54 P. macrophylla S 54 Palicourea elongata 01 2 P. qardneriana 01 2 P. riqida Q 2 Palicourea sp. 03 2 224 Psychotria barbiflora 01 2 P. capitata 01 2 P. carthagenensis 01 R 46 P. involucrata 01 46 P. nudiceps 01 46 P. pinularis N 2 P. psychotriaefolia R 47 P. rufescens M P2 1 1 Randia formosa M 1 1 Remijia pedunculata T 1 1 Retiniphyllum concolor D5 54 R. pilosa C6 54 R. schomburgkii C6 54 R. speciosum C6 54 R. truncatum A8 54 Retiniphyllum sp. S 47 Rubiacea sp. 01 2 Rudgea subsessilis 01 2 R. viburnoides Q 2 Sabicea amazonensis R 47 Order: Asterales Family: Asteraceae Acanthospermum australe M 1 1 Ageratum conyzoides C5 P2 1 1 Ambrosia elatior P2 1 5 Andromachya igniaria E3 1 5 Baccharis genistelloides E3 G6 1 5 B. salicifolia A8 D3 14 1 5 B. trinervis T 1 5 Bidens andicola B2 1 5 Clibadium sylvestre A2 1 1 Clibadium sp. 03 60 Eupatorium ayapanoides Ul 42 E. odoratum A8 N P2 Q 1 1 E. scabrum 14 M 1 1 E. sternbergianum CI 1 5 Mikania guaco U1 42 Neurolaena lobata M N 1 1 Spilanthes americana J3 1 1 225 Class: Liliopsida Subclass I: Alismatidae Order: Alismatales Family: Alismataceae Echinodorus grandiflorus Q 2 Echinodorus sp. C8 , 2 Subclass II: Arecidae Order: Arecales Family: Arecaceae Chamaedorea fragrans A7 42 Elaeis quineensis C6 1 1 Geonoma sp. K1 1 5 Jessenia polycarpa D3 1 1 Kuethia montana N 1 5 Order: Cyclanthales Family: Cyclanthaceae Cardulovicia palmata D3 61 Order: Arales Family: Araceae Anthurium crassinervium J2 54 A. eminens T 61 A. jenmanii B3 61 A. scopendrinum D4 61 A. tessmannii G1 46 A. tikunorum J3 47 Aracea sp. J3 2 Caladium bicolor U2 61 Dieffenbachia obliqua 01 61 D. pictac 01 42 D. sequine 02 2,42 Dracontium asperum U1 42 D. longipes N 61 D. trianae C5 N 61 Heteropsis sp. T 2 Philodendron craspedodromum 03 47 P. dyscarpium G1 47 P. haematinium S 47 P. remifolium V 47 Urospatha antisylleptica G1 47 U. somndenta A1 A2 1 5,47 Xanthosoma conspurcatum 01 61 Subclass III: Commelinidae Order: Commelinales Family: Commelinaceae Tradescantia multiflora 226 Order: Cyperales Family: Cyperaceae Cyperus esculentus F3 1 5 Cyperus sp. R2 39 Family: Poaceae Axonopus micay F1 1 1 A. scoparius F3 1 5 Calamaqrostis cuminens F3 2 Chloris distichophylla F1 1 5 Hierochloe redolens R 2, 53 Subclass IV: Zingiberidae Order: Bromeliales Family: Bromeliaceae Ananas anannasoides C1 C6 F1 1 1 Order: Zingiberales Family: Musaceae Heliconia acuminata B3 K7 N 1 1 Musa balbisiana D5 1 1 Heliconia brasiliensis B3 K7 N 1 1 H. cannoidea B3 K7 1 1 Family: Zingiberaceae Hedychium coronarium K1 54 Family: Costaceae Costus amazonicus T 54 C. cylindricus Cl P3 1 1 C. erythrocoryne E3 54. C. villosissimus K1 1 1 Family: Cannaceae Canna sp. A2 2 Family: Marantaceae Calathea veitcheana R2 50 Calathea sp. 2 3 227 Subclass V. Liliidae Order: Liliales Family: Pontederiaceae Pontederia cordata K7 R2 2, 50, 61 Family: Liliaceae Eucharis amazonica C2 54 Family: Iridaceae 2 Eleutherine piicata G8 Sisyrinchium alatum 1 1 Family: Agavaceae C3 D1 Agave americana 42 Family: Smilacaceae 03 Smilax sp. 2 Family: Dioscoreaceae 12 Dioscorea pozucoensis 2 D. trifida T 42 02 Order: Orchidales Family: Orchidaceae Dichaea muricata J1 54 Eriopsis sceptrum J3 53 One idium pusillum A1 54 Phragmipedium ecuadorensis C1 54 Psymorchis pusilla T 54 Vanilla odorata E1 K2 1 1 Species have been arranged according to the classification scheme of Cronquist (1981) with one exception. The family Leguminosae is used in preference to the Mimosaceae, Caesalpiniaceae and Fabaceae. The species name listed is that used by the author(s) cited. 228 List of Ethnobotanical Codes Used LETTER SYSTEM No. Biological Action or Condition Used For ******************************************************* A skin 1 wounds, cuts 2 absesses, boils ulcers, infections 3 burns 4 dermatitis, eczema, psoriasis, etc. 5 warts 6 leprosy 7 causes dermatitis or allergy 8 swelling, inflammation 9 skin colorant or depilatory B skeleto- 1 broken bones muscular . 2 rheumatism, arthritis 3 muscle stiffness 4 bruises C digest ive 1 carminative, digestive 2 emet ic 3 cathartic, purgative, laxative 4 ant i spasmodic 5 ant idiarrheal 6 amebicide, antihelminthic 7 tooth decay 8 colic D respi ratory 1 colds, influenza, coughs, 2 asthma 3 bronchitis, chest infections 4 sore throat 5 tuberculosis 6 expectorant 7 sinusitis E circulatory 1 anemia 2 heart ailments 3 hemostat 4 hypotensive F excretory 1 diuretic 2 calcifications 3 kidney ailments 229 G reproduct ive, 1 contraceptive female 2 fertility promoter 3 ant i-abort ive 4 abort i fac ient 5 menstrual pain 6 emmenogogue 7 galactogogue 8 uterine hemmorage 9 aphrodisiac H reproduct ive, 1 aphrodisiac male I reproduct ive, 1 aphrodisiac both sexes 2 venereal disease 3 anaphrodi siac J sensory 1 eye ailments 2 ear problems 3 gum and mouth diseases K nervous 1 analgesic, anesthetic, toothache, headache 2 sedative 3 st imulant 4 halluc inogen 5 narcot ic 6 convulsions, fits, epilepsy 7 palsy, paralysis L antibiotic M ant itumour, ant icancer N antidotes for bites and stings of animals (primarily :snakes ) 0 poison 1 general 2 arrow poison 3 fish poison 4 repellent or toxic to arthropods P systematic 1 sudorific, diaphoretic 2 febrifuge, malaria 3 diabetes 4 swollen glands 5 cholera 6 shock 230 Q tonic R admixture to hallucinogen S magic T undefined U miscellaneous 1 herbicide 2 veterinary use 3 tooth extraction 4 tooth coloration V flavouring, aromat ic The categories describing medicinal activity or conditions for which the plants are used as treatments have been established to accomodate the information in the form in which it is available. Although it has been organized as systematically as possible, this approach is limited by the fact that the informants concepts of diagnosis and treatment of illnesses differed widely. The scheme is based on that of Morton(1977). 231 BIBLIOGRAPHY Appendix A de Almeida Costa, 0.(1935) Estudio Farmacognostico de Manaca. Revista de Flora Medicinal 1, 345-360. Altschul, S. von Reis(l973) Drugs and Foods from Little Known Plants-Notes in Harvard University Herbarium , Harvard University Press, Cambridge, Mass. Ballick, M.J., unpublished notes on herbarium labels, UNAP Herbarium, Iquitos, Peru. Biocca, E.(1966) Viaggi tra gli indi-Alto Rio Negro-Alto Orinoco , Dischi, Rome. Bodley, J.H. (1978) Preliminary ethnobotany of the Peruvian Amazon. WSU Laboratory of Investigations, No. 55, Washington State University. Brandl, J.(1895) Chemisch-pharmakologische Untersuchung liber die Manaca-Wurzel. Zeitschrift fur Biologie 31, 251 — 264. Caminhoa, J.M.(1971) Das Plantas Toxicas do Brazil . Typographia Perseveranca, Rio de Janeiro. Dragendorff, G.(1898) Die Heilpflanzen der Verschiedenen Volker und Zeiten , Verlag von Ferdinand Enke, Stuttgart. Ernst, J. P.(1881) Memoria botanica sobre el embarbascar 6 sea la pesca por media de plantas venenosus. Ann. Acad. Cienc. Habana, 18, 135-147. Forero, E.A.(1976) A revision of the American species of Rourea subgenus Rourea (Connaraceae). Mem. N.Y. Bot. Gard. 26,1-119. Garcia-Barriga, H.(1976) Flora Medicinal de Colombia. Botanica Medica, Instituto de Ciencias Naturales, Universidad Nacional, Bogota, Colombia. Gibbs, R.D.(197 4) Chemotaxonomy of Flower ing Plants . McGill -Queen's University Press, Montreal. Githens, T.S.(1924) Preliminary report on studies on Mire. J. Am. Pharm. Assoc. 13, 102-124. Goncalves de Lima, 0.(1946) Observaciones sobre o "vinho de jurema" utilazado pelos indios Pancaru de Tacaratu(Pernambuco). Arqu. Inst. Pesq. Agron. 4, 45-56. 232 15. Hirschorn, H.H.(1981) Botanical remedies of South and Central Americas and the Caribbean: an archival analysis, Part 1. J. Ethnopharm., 4, 129-158. 16. Iyer, R.P., Brown, J.K., Chanbal, M.G. and Malone, M.H.(1977) Brunfelsia hopeana I: hippocratic screening and anti-inflammatory evaluation. Lloydia 40, 356-360. 17. Killip, E.P. (1931) The use of fish poisons in South America. Ann. Rep. Smithsonian Inst., 401-408. 18. Krukoff, B.A. and Barneby, R.C.(1970) Supplementary notes on American Menispermaceae, VI. Mem. N.Y. Bot. Gard. 20, 1-80. 19. Krukoff, B.A.(1965) Supplementary notes on the Amazonian species of Strychnos , VII. Mem. N.Y. Bot. Gard., 12, 42- 43. 20. Kunkel, A.J.(1901) Handbuch der Toxikoloqie , Gustav Fisher Verlag, Jena . 21. LeCointe, P.(1947) Arvores e Plantas Uteis, Amazonia Braziliera 3, ser 5. Brasiliana, Companhia Editoria Nacional, Sao Paulo, 251-279. 22. MacRae, unpublished observations recorded on herbarium labels. 23. Martius, C.F.P. von (1843) Systema Meteriae Medicae Vegetabilis Brasiliensis , F. Fleischer, Liepzig. 24. Maxwell, N. (1977) Jungle pharmacy. Americas 29, 6-7. 25. Maxwell, N. (1982) Personal communication. 26. Mihalik, G.J.(1978) Guyanese ethnomedical botany - a folk pharmacopoeia. Ethnomedicine 5, 83-96. 27. Mors, W.B. and Rizzini, C.T. (1966) Useful Plants of Brazil . Holden-Day Inc., San Francisco. 28. Morton, J.F.(1977) Major Medicinal Plants: Botany, Culture and Uses , Charles C. Thomas, Springfield. 29. Peckolt, T. (1909) Heil und Nutzpflanzen Brasiliens. Berichte der Deutschen Pharmazentischen Gesellschaft 19, 307-315. 30. Perez-Arbelaez, E. (1937) Plantas Medicinales y Venenosas de Colombia, Bogota. 31. Perez-Arbelaez, E. (1956) Plantas Utiles de Colombia , 233 Libraria Colombiana-Camacho Roldan, Bogota 32. Pinkley, H.V.(1973) The ethnoecology of the Kofan Indians. Unpuplished PhD thesis, Harvard University, Cambridge, Ma s s. 33. Piso, W.(1648) De Materia Medicina Brasiliensis . F. Hack, Leiden. 34. Plowman, T. (1977) Brunfelsia in ethnomedicine. Bot. Mus. Leafl. Harv. Univ. 25, 289-320. 35. Plowman, T. (1981) Amazonian Coca. J; of Ethnopharmacol. 3, 195-225. 36. Plowman, T. (1981) Personal communication. 37. Prance, G.T.(1972) An ethnobotanical comparison of four tribes of Amazonian indians. Acta Amazonica 2, 7-28. 38. Record, S.J. and Hess, R.W. (1943) Timbers of the New World . Salem University Press, New Haven. 39. Rivier, L. and Lindgren, J.E.(1972) Ayahuasca, the South American hallucinogenic drink: an ethnobotanical and chemical investigation. Econ. Bot. 40. Ruiz, L.H. y Pavon, J.A.(1954-1959) Flora Peruviana et Chilensis, sire descriptiones et icones plantarum peruvianum et chilensium . Vol. I-111, Madrid. 41. Rusby, H.H.(1924) Pharmacodynamics of caapi . J. Am. Pharm. Assoc. 13, 194. 42. Rutter, R. (1976) Plantas Medicinales de la Amazonia Peruana. Datos Etno-linguisticos No. 44, Instituto Linguistico de Verano, Ministerio de Educacion, Lima(Microfiche). 43. Schultes, R.E. and Hofman, A.(1973) The Botany and Chemistry of Hallucinogens . Charles C. Thomas, Springfield. 44. Schultes, R.E. (1949) Plantae Austo- Americae V. De Plantis principaliter Columbiae. Bot. Mus. Leafl. Harv. Univ. 13, 261-293. 45. Schultes, R.E.(1967) De Plantis toxicariis e Mundo Novo tropicale Commentationes I. Bot. Mus. Leafl. Harv. Univ. 21, 265-284. 46. Schultes, R.E.(1969a) De plantis toxicariis e Mundo Novo tropicale Commentationes IV. Bot. Mus. Leafl. Harv. Univ. 234 22, 133-164. 47. Schultes, R.E.(1969b) De plantis toxicariis e Mundo Novo tropicale commentationes VI. Notas etnotoxicologicas acerca de la flora amazonica de Colombia. In Simposio y Foro de Biologia Tropical Amazonica (Idrobo, J.M., Ed.7. Association pro Biologia Tropical, Bogota. 48. Schultes, R.E.(1970) De plantis toxicariis e Mundo Novo tropicale Commentationes VII. Several ethnotoxicological notes from the Colombian Amazon. Bot. Mus. Leafl. Harv. Univ. 22, 345-352. 49. Schultes, R.E.(1971) De Plantis toxicariis e Mundo Novo tropicale Commentationes VIII. Miscellaneous notes on myristicaceous plants of South America. Lloydia 34, 61-78. 50. Schultes, R.E.(1972) De Plantis toxicariis e Mundo Novo tropicale commentationes XI. The ethnotoxicological significance of additives to New World hallucinogens. Pi. Sci. Bull. Dec., 34-42. 51. Schultes, R.E.(1975a) De Plantis toxicariis e Mundo novo tropicale Commentationes XII. Notes on biodynamic piperaceous plants. Rhodora 77 , 165-170. 52. Schultes, R.E.(1975b) De Plantiis toxicariis e Mundo Novo tropicale Commentationes XIII. Notes on poisonous or medicinal malpigiaceous species of the Amazon. Bot. Mus. Leafl. Harv. Univ. 24, 121-131. 53. Schultes, R.E.(1977) De Plantis toxicariis e Mundo Novo tropicale Commentationes XVI. Miscellaneous notes on biodynamic plants of South America. Bot Mus. Leafl. Harv. Univ. 25, 109-130. 54. Schultes, R.E.(1978a) De Plantis toxicariis e Mundo Novo tropicale Commentationes XXII. Notes on biodynamic plants of aboriginal use in Northwestern Amazonia. Bot. Mus. Leafl. Harv. Univ. 26, 177-187. 55. Schultes, R.E.(1978b) De Plantis toxicariis e Mundo Novo tropicale Commentationes XXIII. Ethnopharmacological notes from northern South America. Bot. Mus. Leafl. Harv. Univ. 26, 225-236. 56. Schultes, R.E.(1978c) De Plantiis toxicariis e Mundo Novo tropicale Commentationes XXV. Biodynamic acanthaceous plants of the Northwest Amazon. Bot. Mus. Leafl. Harv. Univ. 26, 267-275. 57. Schultes, R.E.(1979a) De Plantis toxicariis e Mundo Novo tropicale Commentationes XIX. Biodynamic apocynaceous 235 plants of the northwest Amazon. J. of Ethnopharm. 1, 165- 1 92. 58. Schultes, R.E.(1979b) De Plantis toxicariis e Mundo Novo tropicale Commentationes XX. Medicinal and toxic uses of Swartzia in the northwest Amazon. J. of Ethnopharm. 1, 79- 87. 59. Schultes, R.E.(1979c) De Plantis toxicariis e Mundo Novo tropicale Commentationes XXI. Interesting native uses of the Humiriaceae in the northwest Amazon. J. of Ethnopharm. 1, 89-94. 60. Schultes, R.E.(l979d) Indicios la riqueza etnofarmacologica do noroeste da Amazonia. Acta Amazonica 9, 209-213. 61. Schultes, R.E.(1980) De Plantis toxicariis e Mundo Novo tropicale Commentationes XXVI. Ethnopharmacological notes on the flora of Northwestern South America. Bot. Mus. Leafl. Harv. Univ. 28, 1-26. 62. Soukup, J.(1970) Vocabulario de los Nombres Vulqares de la Flora Peruana . Colegio Salesiano, Lima. 63. Spix, J.B. and von Martius, C.F.P.(1831) Reise in Brasien Munchen. 64. Tovar, 0. (1952) Revision de las especias Peruanas del Genero Chuquiraga. Publ. Museo Hist. Nat. Javier Prado, Serie B, Botanica, No. 5, Lima. 65. Webb, L.J.(1948) Guide to Medicinal and Poisonous Plants of Queensland . Council for Scientific and Industrial Research, Melbourne. 66. Wren, R.C.(1956) Potter's New Cyclopedia of Botanical Drugs and Preparations , Pitman and Sons, London. 236 APPENDIX 1 - SP4100 COMPUTER PROGRAM(BASIC) The program takes incoming signals, after amplification X1000, from the force transducer, computes the absolute value of the difference between two successive signals, and prints the average value every 5 minutes over a 2 hour period. A=1: IF TB<3 THEN A=100 ALWAYS IF TB=0 THEN A=1000 POKE #C394, TB POKE $#01C107,1 ACQUIRE 1 C = 0 T=1 X=CLEV M=0 C = 0 B=CLEV M=ABS(B-X)+M X=B C=C+1 IF 2PEEK#C234/500>T THEN GO TO 90 ELSE 65 14:28:18;M/C IF T=24 THEN GO TO 140 ELSE 120 T=T+1 GO TO 50 ACQUIRE 2 END Publications MacRae, W. D., Whiting, R.F. and Stich, H.F. Sister chromatid exchanges induced in cultured mammalian cells by chromate. Chem.-Biol. Interact. 26: 281-286(1979). MacRae, W.D., MacKinnon, E.A. and Stich, H.F. The fate of U.V.-induced lesions affecting SCEs, chromosome aberrations and survival of CHO cells arrested by deprivation of arginine. Chromosoma 72: 15-22(1979). MacRae, W.D.,MacKinnon, E.A. and Stich, H.F. Effects of arginine deprivation upon chromosome aberrations, SCEs and survival of CHO cells treated with mutagenic agents. Mutat. Res. 62: 495-504(1979). MacRae, W.D., MacKinnon, E.A. and Stich, H.F. Induction of sister chromatid exchanges and chromosome aberrations in CHO cells arrested in the cell cycle by arginine deprivation. In Vitro 15: 555-564(1979). MacRae, W.D. and Stich, H.F. Induction of sister chromatid exchanges in Chinese hamster ovary cells by the reducing agents bisulfite and ascorbic acid. Toxicology 13: 167-174(1979). MacRae, W.D. and Stich, H.F. Induction of sister chromatid exchanges in Chinese hamster ovary cells by thiol and hydrazine compounds. Mutat. Res. 68: 351-365(1979) . Yamamoto, E., Wat, C.-K., MacRae, W.D., Towers, G.H.N, and Chan, G.F.Q. Photoinactivation of human erythrocyte enzymes by <=<-terthienyl and phenyl- heptatriyne, naturally occurring compounds in the Asteraceae. FEBS Letters 107: 134-136(1979). Wat, C.-K., MacRae, W.D., Yamamoto, E., Towers, G.H.N, and Lam, J. Phototoxic effects of naturally occurring polyacetylenes and <*-terthienyl on human erythrocytes. Photochem. Photobiol. 32: 167-172(1980). MacRae, W.D., Chan. G.F.Q., Wat, C.-K., Towers, G.H.N, and Lam, J. Examination of naturally occurring polyacetylenes and c<-terthienyl for their ability to induce cytogenetic damage. Experientia 36: 1096-1097(1980). MacRae, W.D., Irwin, D.A.J., Bisalputra, T. and Towers, G.H.N. Membrane lesions in human erythrocytes induced by the naturally occurring compounds <*> -terthienyl and phenylheptatriyne. Photobiochem. Photobiophys. 1: 309-318 (1980) . MacRae, W.D. and Towers, G.H.N. Letter to the editor. J. Ethnopharm. 7: 343- 348(1983). Yamamoto, E., Wat, C.-K., MacRae, W.D., Garcia, F.J. and Towers, G.H.N. Photodynamic hemolysis caused by °N-terthienyl. Planta Med.(in press, Nov., 1983). MacRae, W.D. and Towers, G.H.N. Review: The biological activities of lignans. Phytochem.(In press, Nov., 1983). MacRae, W.D. and Towers, G.H.N. Justicia pectoralis: A study of the basis for its use as Virola snuff admixture(in preparation). MacRae, W.D. and Towers, G.H.N. Non-alkalidal constituents of Virola elongata. Phytochem.(to be submitted). MacRae, W.D., McKenna, D.J. and Towers, G.H.N. An ethnopharacological examination of Virola elongata bark, a South American arrow poison.(in preparation). MacRae, W.D., Hudson, J.B. and Towers, G.H.N. Multi-dimensional pharmacological screening of South Americal Euphorbiaceous plants, (in preparation). MacRae, W.D., Hudson, J.B. and Towers, G.H.N. c< -Peltatin: the antiviral constituent of Amanoa sp. (in preparation). MacRae, W.D., Hudson, J.B. and Towers, G.H.N. Antiviral activities of lignans. (in preparation).