Ethnobiological and Chemical Investigations of Selected Amazonian Plants
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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 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES ' Biology Department We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA March 1984 © William Donald MacRae, 1984 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 < Date ^ ,>1<SH' i i 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.