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UBC Theses and Dissertations

Ethnobiological and chemical investigations of selected Amazonian plants MacRae, W. Donald 1984

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ETHNOBIOLOGICAL AND CHEMICAL INVESTIGATIONS OF SELECTED AMAZONIAN PLANTS by WILLIAM DONALD MACRAE B . S c , The U n i v e r s i t y of V i c t o r i a , 1974 M . S c , The U n i v e r s i t y o f B r i t i s h C o l u m b i a , 1978 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n THE FACULTY OF GRADUATE STUDIES ' B i o l o g y Department We a c c e p t t h i s t h e s i s as c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA Ma r c h 1984 © W i l l i a m D o n a l d MacRae, 1984 I n p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r an a d v a n c e d d e g r e e a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e a n d s t u d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by t h e h e a d o f my d e p a r t m e n t o r b y h i s o r h e r r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . D e p a r t m e n t o f The U n i v e r s i t y o f B r i t i s h C o l u m b i a 1956 Main Ma l l V a n c o u v e r , Canada V6T 1Y3 D a t e ^ ,><1<SH' i i A b s t r a c t The e t h n o b o t a n i c a l l i t e r a t u r e o f A m a z o n i a n S o u t h A m e r i c a h a s been s u r v e y e d , c o m p i l e d and o r g a n i z e d . T h i s i n f o r m a t i o n a l l o w e d t h e i d e n t i f i c a t i o n o f c e r t a i n t a x o n o m i c g r o u p s a n d p l a n t s w i t h s p e c i f i c u s e s w h i c h seem p r o m i s i n g f o r f u r t h e r r e s e a r c h . The use o f J u s t i c i a p e c t o r a l i s a s an a d d i t i v e t o h a l l u c i n o g e n i c V i r o l a b a s e d s n u f f s h a s been i n v e s t i g a t e d . No a l k a l o i d a l compounds c o u l d be d e t e c t e d i n t h e p l a n t a n d t h e p h a r m a c o l o g i c a l e f f e c t s o f an e x t r a c t on m i c e were n o t i n d i c a t i v e o f t h e p r e s e n c e o f s t r o n g l y p s y c h o a c t i v e c o n s t i t u e n t s . Nor d i d t h e J . p e c t o r a l i s e x t r a c t s have any i n h i b i t o r y o r s y n e r g i s t i c e f f e c t upon t h e r e s p o n s e s o f m i c e t o 5 - m e t h o x y - N , N - d i m e t h y l t r y p t a m i n e , t h e p s y c h o a c t i v e c o n s t i t u e n t o f V i r o l a b a r k . E x t r a c t s o f t h e p l a n t c a u s e d t h e r e l a x a t i o n o f s m o o t h m u s c l e a n d t h i s a c t i v i t y was shown t o r e s u l t f r o m t h e p r e s e n c e o f c o u m a r i n and u m b e l l i f e r o n e . B e t a i n e , w h i c h was a l s o p r e s e n t , was o b s e r v e d t o e l i c i t s m ooth m u s c l e c o n t r a c t i o n a t h i g h c o n c e n t r a t i o n s . S t r o n g i n h i b i t o r y a c t i v i t y o f t h e e x t r a c t t o w a r d s f o u r d e r m a t o p h y t i c f u n g i was o b s e r v e d and may e x p l a i n t h e use o f t h e p l a n t i n t h e t r e a t m e n t o f c e r t a i n i n f e c t i o n s . C o u m a r i n was shown t o be w h o l l y r e s p o n s i b l e f o r t h e a r o m a . The p o s s i b i l i t y t h a t t h e h y p n o t i c e f f e c t s o f c o u m a r i n may p l a y a r o l e i n i t s u s e a s a s n u f f c o n s t i t u e n t i s c o n s i d e r e d . The u s e o f V i r o l a e l o n g a t a a s b o t h an h a l l u c i n o g e n i c snu f f and an arrow po i son was examined. E x t r a c t s of the bark were e va lua t ed fo r t h e i r e f f e c t s on mouse b e h a v i o r . A non-a l k a l o i d a l f r a c t i o n caused a r e d u c t i o n in spontaneous motor a c t i v i t y in mice whi le the a l k a l o i d a l f r a c t i o n from the same amount of p l a n t m a t e r i a l had no s i g n i f i c a n t e f f e c t . The non-a l k a l o i d a l f r a c t i o n was examined and some of i t s b i o l o g i c a l a c t i v i t y was a t t r i b u t e d to the presence of p o l y p h e n o l i c compounds. E l even compounds were i s o l a t e d from the non-polar pa r t of t h i s e x t r a c t . In a d d i t i o n to j 3 - s i t o s t e r o l , two i somer i c s t i l b e n e s , two n e o l i g n a n s , four b i s - t e t r a h y d r o f u r a n l i g n a n s and two t e t r a h y d r o f u r a n l i g n a n s , were i d e n t i f i e d . The b i s - t e t r a h y d r o f u r a n l i g n a n s were shown to reduce spontaneous motor a c t i v i t y and i s o l a t i o n induced a g g r e s s i o n i n m i ce . The p o s s i b i l i t y that they are at l e a s t p a r t l y r e s p o n s i b l e fo r the use of V i r o l a e longa ta as an arrow po i son i s ' r a i s e d . T h i r t y - f o u r s p e c i e s of Amazonian Euphorb iaceae were screened fo r i n h i b i t o r y a c t i v i t y towards E s c h e r i c h i a c o l i , S t aphy lococcus aureus , two y e a s t s , four dermatophyt i c f u n g i , two an imal v i r u s e s , tumour fo rmat ion in po ta to d i s c s and t o x i c i t y to b r i n e shr imp. A l a rge p r o p o r t i o n of the e x t r a c t s were a c t i v e a g a i n s t S. au reus , the dermatophytes , at l e a s t one of the v i r u s e s , the po ta to tumours and the b r i n e sh r imp . The b i o l o g i c a l a c t i v i t i e s observed are d i s c u s s e d w i th r espec t to the use of c e r t a i n s p e c i e s in Amazonian e thnomed i c ine . The a n t i v i r a l a c t i v i t y of one of the p l a n t s s c r eened , a s p e c i e s of Amanoa was examined. The i n h i b i t o r y a c t i v i t y towards i n f e c t i o n by murine c y tomega lov i rus was found to i v r e s u l t from t h e p r e s e n c e o f a s i n g l e compound, i d e n t i f i e d a s t h e l i g n a n , a- ( - ) - p e l t a t i n . At low d o s e s ( l O ng/ml f o r two h o u r s ) , t h i s compound p r e v e n t e d t h e r e p l i c a t i o n of v i r u s e s i n a l r e a d y i n f e c t e d c e l l s . I t ' s a c t i v i t y was o b s e r v e d t o be v e r y s i m i l a r t o t h a t o f a n o t h e r n a t u r a l l y o c c u r r i n g l i g n a n , p o d o p h y l l o t o x i n . L i g n a n s of a v a r i e t y of s t r u c t u r a l t y p e s were examined f o r a n t i v i r a l a c t i v i t y but a n t i - m u r i n e c y t o m e g a l o v i r u s a c t i v i t y of compounds o t h e r t h a n t h e p o d o p h y l l o t o x i n t y p e was n o t o b s e r v e d . V Tab le of Contents A b s t r a c t i i L i s t of Tab l e s x L i s t of F i g u r e s x i i Acknowledgement x i v 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 J u s t i c i a p e c t o r a l i s : A STUDY OF THE BASIS FOR ITS USE AS A V i r o l a SNUFF ADMIXTURE 30 1. INTRODUCTION 30 2. MATERIALS AND METHODS .31 a . P l an t m a t e r i a l 31 b. Chromatography and spec t roscopy 32 c . Behav ior exper iments 33 d . Rat stomach s t r i p exper iments 33 e. A n t i m i c r o b i a l t e s t s 34 f . A n t i v i r a l t e s t s 35 3. RESULTS 35 v i a . Examinat ion of J u s t i c i a p e c t o r a l i s f o r a l k a l o i d s 35 b. Compound 1 36 c . B ehav i o r a l e f f e c t s o f Jus t i c i a p e c t o r a l i s 37 d . E f f e c t of J u s t i c i a p e c t o r a l i s e x t r a c t s on 5-MeODMT induced b e h a v i o r a l responses 39 e. E f f e c t of 5-MeODMT on mouse a c t i v i t y 41 f . Gross b e h a v i o r a l e f f e c t s of 5-MeODMT 44 g . E f f e c t of c o - i n j e c t i o n s of 5-MeODMT and J u s t i c i a p e c t o r a l i s e x t r a c t s 44 h. E f f e c t of J u s t i c i a p e c t o r a l i s e x t r a c t s on smooth muscle 47 i . A n a l y s i s of a romat ic c o n s t i t u e n t s 51 j . Q u a n t i f i c a t i o n of coumarins of J . p e c t o r a l i s . . . . 5 3 k. Examinat ion of J . p e c t o r a l i s fo r l i g n a n s 55 1. Sc reen ing fo r o ther b i o l o g i c a l a c t i v i t i e s 57 4. DISCUSSION 59 LITERATURE CITED 63 CHAPTER III AN ETHNOPHARMACOLOGICAL EXAMINATION OF V i r o l a e l onga ta BARK, A SOUTH AMERICAN ARROW POISON 69 INTRODUCTION 69 PART A. ISOLATION AND IDENTIFICATION OF THE MAJOR NON-POLAR CONSTITUENTS OF V i r o l a e longa ta BARK 73 1. INTRODUCTION 7 3 2. EXPERIMENTAL 73 v i i a. E x t r a c t i o n of p l a n t m a t e r i a l 73 b. C h r o m a t o g r a p h y 74 3. COMPOUNDS ISOLATED 7 4 4. RESULTS 80 a. 3 , 4 ' , 5 - t r i m e t h o x y - c i s - s t i l b e n e and 3,4',5-t r i m e t h o x y - t r a n s - s t i l b e n e 83 b. E u s i d e r i n and v i r o l o n g i n 87 c . E p i - s e s a r t e m i n , s e s a r t e m i n , e p i - y a n g a m b i n and yangambin 88 d. D i h y d r o s e s a r t e m i n and 0-yangambin 91 PART B. EXAMINATION OF THE BIOLOGICAL ACTIVITY OF V i r o l a e l o n g a t a BARK EXTRACTS 100 1. INTRODUCTION 100 2. MATERIALS AND METHODS 100 a. P r e p a r a t i o n of e x t r a c t s 100 b. F r a c t i o n a t i o n of e x t r a c t s 101 c . C h r o m a t o g r a p h i c a n a l y s i s o f e x t r a c t s 102 d. A s s a y of s p o n t a n e o u s motor a c t i v i t y 103 e. A s s a y of a n t i - a g g r e s s i v e a c t i v i t y 104 3. RESULTS 105 a. E x a m i n a t i o n of t h e aqueous f r a c t i o n f o r t o x i c i t y 106 b. E x a m i n a t i o n o f t h e d i e t h y l e t h e r e x t r a c t f o r d e p r e s s i o n o f s p o n t a n e o u s motor a c t i v i t y 109 c . Q u a n t i f i c a t i o n of major c o n s t i t u e n t s o f d i e t h y l e t h e r e x t r a c t 114 d. E f f e c t of b i s - t e t r a h y d r o f u r a n l i g n a n s on v i i i i s o l a t i o n i n d u c e d a g g r e s s i o n 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 A C T I V I T I E S 134 1 . INTRODUCTION 134 2. MATERIALS AND METHODS 139 a. P l a n t m a t e r i a l 139 b. P r e p a r a t i o n of p l a n t e x t r a c t s 140 c. A n t i m i c r o b i a l s c r e e n i n g 140 d. A n t i v i r a l a c t i v i t y 141 e. P o t a t o d i s c tumour a s s a y 143 f . T o x i c i t y t o b r i n e s h r i m p 144 g. A n a l y s i s of d a t a 144 3. RESULTS a. A n t i m i c r o b i a l a c t i v i t y 145 b. A n t i v i r a l a c t i v i t y 150 c . I n h i b i t i o n of p o t a t o tumour f o r m a t i o n 153 d. T o x i c i t y t o b r i n e s h r i m p 156 e. C o r r e l a t i o n between b i o l o g i c a l a s s a y s 156 4. DISCUSSION 167 a. A n t i m i c r o b i a l a c t i v i t y 167 i x b. A n t i v i r a l a c t i v i t y 170 c. A n t i t u m o u r a c t i v i t y 172 d. T o x i c i t y t o b r i n e s h r i m p 172 5. CONCLUSION 173 PART B. a-( - ) - PELTATIN, THE ANTIVIRAL CONSTITUENT OF Amanoa sp 176 1 . INTRODUCTION 176 2. MATERIALS AND METHODS 176 a. P l a n t m a t e r i a l 176 b. A n t i v i r a l a s s a y s 177 c. C h r o m a t o g r a p h y 177 3. RESULTS AND DISCUSSION 178 PART C. THE ANTIVIRAL ACTION OF LIGNANS 183 1 . INTRODUCTION 183 2. MATERIALS AND METHODS ...184 a. C h e m i c a l s 184 b. C e l l s and v i r u s e s 184 c. A n t i v i r a l s c r e e n i n g o f l i g n a n s 186 d. E f f e c t o f t i m e o f t r e a t m e n t 187 3. RESULTS 188 4. DISCUSSION 193 LITERATURE CITED 201 APPENDIX A - L I S T OF AMAZONIAN.ANGIOSPERMS OF ETHNOBOTANICAL INTEREST 207 BIBLIOGRAPHY: APPENDIX A 231 APPENDIX B - SP4100 COMPUTER PROGRAM(BASIC) 236 X L i s t of Tab l e s I. P h y l ogene t i c d i s t r i b u t i o n of s p e c i e s of Amazonian angiosperms hav ing documented e t n o b o t a n i c a l use . . 14 I I . E f f e c t of Jus t i c i a p e c t o r a l i s e x t r a c t s on spontaneous locomotor a c t r v i t y in mice 40 I I I . E f f e c t of c o - a d m i n i s t r a t i o n of 5-MeODMT and J u s t i c i a p e c t o r a l i s e x t r a c t s on spontaneous locomotor a c t i v i t y of mice 46 IV. L e v e l s of coumarin and u m b e l l i f e r o n e in d i f f e r e n t samples of Just i c i a p e c t o r a l i s 56 V. 'H-NMR s p e c t r a of b i s - t e t r a h y d r o f u r a n l i g n a n s i s o l a t e d from V i r o l a e l onga ta bark 81 V I . ' 1 3C-NMR s p e c t r a of b i s - t e t r a h y d r o f u r a n l i g n a n s i s o l a t e d from V i r o l a e l onga ta bark -82 V I I . Gross b e h a v i o r a l responses of Swiss mice to a d m i n i s t r a t i o n of a l k a l o i d a l and n o n - a l k a l o i d a l e x t r a c t s of V i r o l a e l onga ta bark 107 V I I I . E f f e c t of p u r i f i e d compounds of V i r o l a e l onga t a bark on spontaneous locomotor a c t i v i t y of Swiss mice 113 IX. Q u a n t i t a t i v e a n a l y s i s of t h i r t e e n most common c o n s t i t u e n t s of d i e t h y l e ther e x t r a c t of V i r o l a  e l onga t a bark 115 X. E f f e c t of ep i - sesa r t em in on three b e h a v i o r a l parameters r e l a t e d to agg re s s i v enes s of mice . . . . 1 1 8 X I . E f f e c t of b i s - t e t r a h y d r o f u r a n l i g n a n s on x i a g g r e s s i v e n e s s i n mice 120 X I I . A n t i m i c r o b i a l s c r e e n i n g of e x t r a c t s of E u p h o r b i a c e o u s p l a n t s ...146 X I I I . S c r e e n i n g of E u p h o r b i a c e o u s p l a n t s f o r a n t i - d e r m a t o p h y t i c f u n g u s a c t i v i t y 148 XIV. A n t i v i r a l s c r e e n i n g o f e x t r a c t s of E u p h o r b i a c e o u s p l a n t s 151 XV. S c r e e n i n g of e x t r a c t s of E u p h o r b i a c e o u s p l a n t s f o r i n h i b i t i o n o f A g r o b a c t e r i u r n i n d u c e d tumour f o r m a t i o n 154 XVI. S c r e e n i n g of e x t r a c t s of E u p h o r b i a c e o u s p l a n t s f o r t h e i r t o x i c i t y t o b r i n e s h r i m p , A r t e m i a s a l i n a 157 •XVII. Summary of b i o l o g i c a l s c r e e n i n g o f e x t r a c t s of E u p h o r b i a c e o u s p l a n t s 159 X V I I I . 2 X 2 C o n t i n g e n c y t a b l e s f o r agreement between e a c h p a i r of t h e 14 a s s a y s u s e d t o s c r e e n t h e 85 p l a n t e x t r a c t s 164 XIX. V a l u e s o f F i s h e r ' s e x a c t t e s t f o r c o n t i n g e n c y t a b l e s o f T a b l e X V I I I 165 XX. Summary of e t h n o b o t a n i c a l i n f o r m a t i o n and b i o l o g i c a l a c t i v i t y o f t h e 34 s p e c i e s o f E u p h o r b i a c e a e t e s t e d 168 XXI. E x a m i n a t i o n o f l i g n a n s f o r t h e i r e f f e c t on r e p l i c a t i o n o f S i n d b i s v i r u s and MCMV 189 X X I I . E f f e c t of t i m e o f l i g n a n t r e a t m e n t upon i n h i b i t i o n o f S i n d b i s v i r u s i n f e c t i o n 192 x i i L i s t of F i g u r e s 1. D i s t r i b u t i o n Amazonian a n g i o s p e r m s w i t h documented e t h n o b o t a n i c a l u s e s 18 2. S t r u c t u r e s of b e t a i n e ( 1 ) , c o u m a r i n (2) and u m b e l l i f e r o n e (3) 38 3. E f f e c t of 5-MeODMT on s p o n t a n e o u s l o c o m o t o r a c t i v i t y of mice 43 4. E f f e c t o f a) s e r o t o n i n ( l ng/ml) and b) b e t a i n e (50 jug/ml) on smooth m u s c l e 48 5. E f f e c t o f (a) c o u m a r i n ( 10 Mg/ml) and (b) ( u m b e l l i f e r o n e ) (10 Mg/ml) on smooth m u s c l e 52 6. HPLC chromatogram o f t h e o r g a n i c f r a c t i o n o f l e a v e s of J . p e c t o r a l i s 54 7. S t r u c t u r e s of s t i l b e n e s and n e o l i g n a n s i s o l a t e d from V i r o l a e l o n g a t a 84 8. S t r u c t u r e s of b i s - t e t r a h y d r o f u r a n l i g n a n s i s o l a t e d f r o m V i r o l a e l o n g a t a 85 9. S t r u c t u r e s of t e t r a h y d r o f u r a n l i g n a n s i s o l a t e d f r o m V i r o l a e l o n g a t a 86 10. Scheme o f f r a g m e n t a t i o n of b i s - t e t r a h y d r o f u r a n l i g n a n s , e p i - s e s a r t e m i n and s e s a r t e m i n by mass s p e c t r o m e t r y 92 11. Scheme o f f r a g m e n t a t i o n of b i s - t e t r a h y d r o f u r a n l i g n a n s , e p i - y a n g a m b i n and yangambin by mass s p e c t r o m e t r y 93 12. Scheme o f f r a g m e n t a t i o n of t e t r a h y d r o f u r a n l i g n a n , x i i i d i h y d r o s e s a r t e m i n , by mass s p e c t r o m e t r y 95 13. Scheme o f f r a g m e n t a t i o n of t e t r a h y d r o f u r a n l i g n a n , /3-dihydroyangambin by mass s p e c t r o m e t r y 96 14. Example o f t r a n s d u c e r o u t p u t u s e d t o r e c o r d s p o n t a n e o u s motor a c t i v i t y 111 15. E f f e c t o f b i s - t e t r a h y d r o f u r a n l i g n a n s , e p i - s e s a r t e m i n and e p i - y a n g a m b i n on s p o n t a n e o u s l o c o m o t o r a c t i v i t y of m i c e 112 16. C h r o m a t o t r o n e l u t i o n p r o f i l e o f e t h y l a c e t a t e f r a c t i o n o f Amanoa s p . l e a v e s 179 17. S t r u c t u r e of a-( - )- p e l t a t i n i s o l a t e d f r o m Amanoa sp .181 18. S t r u c t u r e s of l i g n a n s t e s t e d f o r a n t i v i r a l a c t i v i t y 185 19. E f f e c t o f t i m e of l i g n a n t r e a t m e n t on i n h i b i t i o n of m u r i n e c y t o m e g a l o v i r u s i n f e c t i o n 191 x i v Acknowledgement An e t h n o b o t a n i c a l or e t h n o b i o l o g i c a l approach i s a s t r o n g l y i n t e r d i s c i p l i n a r y one. T h i s study i s no excep t i on and I am i n d e b t e d , to an e s p e c i a l l y l a r g e degree , to the work of my p r edecesso r s as we l l as to many contemporary a d v i s o r s , c o n t r i b u t o r s and i n fo rman t s . I owe s p e c i a l thanks to P r o f . G .H .N . Towers, who s u p p l i e d u n l i m i t e d enthus iasm and support f o r t h i s r e s e a r c h and gave generous l y of h i s t ime . I was f o r t u n a t e to be ab l e to work in c o o p e r a t i o n w i th Dennis McKenna, both du r i ng the f i e l d work and , f o l l o w i n g t h a t , in the l a b o r a t o r y . Our f r e e exchange of i n f o r m a t i o n was b e n e f i c i a l to many aspec t s of t h i s work. H i s i n t e r e s t in J us t i c i a p e c t o r a l i s and V i r o l a sp . was e s p e c i a l l y h e l p f u l and he k i n d l y made p l a n t m a t e r i a l a v a i l a b l e fo r a n a l y s i s . The e t h n o b o t a n i c a l f i e l dwork of P r o f . R.E. S chu l t e s has i n f l u e n c e d the course of t h i s study in many ways. T h i s i s ev iden t from the number of t imes h i s name i s c i t e d and from the important r o l e he p l ayed in d e s c r i b i n g the uses of both J u s t i c i a p e c t o r a l i s and V i r o l a e l o n g a t a . I wish to acknowledge the p e r s o n a l communicat ion of P r o f . R.E. S c h u l t e s , P r o f e s s o r and D i r e c t o r , Harvard U n i v e r s i t y B o t a n i c a l Museum conce rn ing the ethnobotany and taxonomy of J u s t i c i a p e c t o r a l i s and a l s o D r . G .T . P rance , D i r e c t o r of B o t a n i c a l Resea rch , New York B o t a n i c a l Garden , f o r h e l p f u l i n f o r m a t i o n conce rn ing the ethnobotany of both J u s t i c i a X V p e c t o r a l i s and V i r o l a e l o n g a t a . A g rea t many people may be c r e d i t e d wi th a s s i s t i n g in the c o l l e c t i o n of p l a n t m a t e r i a l . Tim Plowman, Ch icago F i e l d Museum, gene rous l y p r o v i d e d adv i c e and a s s i s t a n c e and ar ranged fo r the i d e n t i f i c a t i o n of much of the p l an t m a t e r i a l c o l l e c t e d . A l l of the s p e c i e s of Euphorb iaceae c o l l e c t e d were k i n d l y i d e n t i f i e d by Dr . M.J. Huft of the Ch icago F i e l d Museum. D r . Ramon F e r r y r a , D i r e c t o r of the Museo H i s t o r i a Na tu r a l e de Pe ru , made time a v a i l a b l e fo r c o n s u l t a t i o n and a s s i s t e d in the procurement of the necessary a u t h o r i z a t i o n fo r p l a n t c o l l e c t i o n and e x p o r t . S r . P u r i c a c a of the Departamento de A g r i c u l t u r a y A l i m e n t a c i o n was a l s o h e l p f u l in t h i s r e g a r d . D r . F r a n k l i n A y a l a , U n i v e r s i d a d Nac i ona l de Amazonia Peruana, k i n d l y made herbar ium f a c i l i t i e s and equipment a v a i l a b l e . I am g r a t e f u l to Dick Ru t te r and Wes Th i e s sen of the Summer I n s t i t u t e of L i n g u i s t i c s , P u c a l l p a , f o r t h e i r h e l p f u l i n f o r m a t i o n . Ad r i ana Lyoaza and N i c o l e Maxwell g r a c i o u s l y o f f e r e d both i n f o r m a t i o n and h o s p i t a l i t y . N i c o l e Maxwell i s c i t e d as the source of s e v e r a l p e r s o n a l communicat ions c o n c e r n i n g m e d i c i n a l p l an t u se . I owe s p e c i a l thanks to the Wi to to and Bora Ind ians of Puco U r q u i l l o and the Boras of B r i l l o Nuevo fo r t h e i r h o s p i t a l i t y . Juan Ruiz Macedo, Andres Cdrdoba , En r ique Donez, and Mar io Cordoba p r o v i d e d v a l u a b l e a s s i s t a n c e in p l a n t c o l l e c t i n g . Among the many Ind ian and mes t i zo in fo rmants c o n t a c t e d , may be numbered Don Juan Mozombite of P u c a l l p a , Don Juan Peso of Nina Rumi, and Don Ramiro D i az of B a l s a s . These are j u s t a few of the many x v i P e r u v i a n s who, i n a s p i r i t o f f r i e n d s h i p and a common i n t e r e s t , o f f e r e d i n f o r m a t i o n on p l a n t u s e s . The l a b o r a t o r y phase o f t h i s r e s e a r c h was a l s o i n t e r d i s c i p l i n a r y i n n a t u r e . Bob K a n t y m i r p r o v i d e d a s s i s t a n c e i n t h e g r e e n h o u s e . T e r r y C r a w f o r d and P e t e Drummond, U . B . C , g e n e r o u s l y made equipment a v a i l a b l e f o r r e c o r d i n g smooth m u s c l e c o n t r a c t i o n and mouse l o c o m o t o r a c t i v i t y . Sam G o p a u l p r o v i d e d a d v i c e c o n c e r n i n g a n i m a l c a r e and b e h a v i o r . A l l o f t h e NMR s p e c t r a and t h e h i g h r e s o l u t i o n mass s p e c t r a were o b t a i n e d t h r o u g h t h e s e r v i c e s o f t h e C h e m i s t r y D e p a r t m e n t , U . B . C . I n f r a r e d s p e c t r a and o p t i c a l r o t a t i o n s were r e c o r d e d on t h e i n s t r u m e n t s of D r s . P i e r c e and Drummond, C h e m i s t r y , U . B . C The i n t e r p r e t a t i o n o f s p e c t r a l d a t a was f a c i l i t a t e d by many h e l p f u l d i s c u s s i o n s w i t h F e l i p e B a l z a . I am g r a t e f u l t o Dr. M.P. Gord o n , D e p t . o f B i o c h e m i s t r y , U. of W a s h i n g t o n , f o r p r o v i d i n g t h e A q r o b a c t e r i u m t u m e f a c i e n s and t o D r . B a r b a r a D i l l , Dept of M e d i c a l M i c r o b i o l o g y , U . B . C , f o r t h e s p o r u l a t i n g s t r a i n s o f d e r m a t o p h y t i c f u n g i . I would l i k e t o thank D r . J.B. Hudson, D e p t . of M e d i c a l M i c r o b i o l o g y , U . B . C , f o r a l l o w i n g me t o c a r r y o u t t h e s t u d i e s on a n t i v i r a l a c t i v i t y i n h i s l a b . Some of t h e l i g n a n s t e s t e d were p r o v i d e d by D r . E. Swan, F o r i n t e k , V a n c o u v e r , and D r . G.H. S h e r i h a , E l F a t e h U n i v e r s i t y , L i b y a . F i n a l l y , t h e f i n a n c i a l s u p p o r t o f t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , i n t h e form o f a G r a d u a t e F e l l o w s h i p , and t h e N a t u r a l S c i e n c e and E n g i n e e r i n g R e s e a r c h C o u n c i l o f Canada f o r a P o s t g r a d u a t e F e l l o w s h i p i s g r a t e f u l l y a c k n o w l e d g e d . 1 I. GENERAL INTRODUCTION 1. ETHNOBIOLOGY: AN INTERDISCIPLINARY APPROACH E t h n o b i o l o g y , ethnobotany and ethnopharmacology are o v e r l a p p i n g terms fo r the study of the use of the e a r t h ' s b i o t i c r e sou r ces by man. I t s s tudents and p r a c t i t i o n e r s are not numerous and they come from d i f f e r e n t d i s c i p l i n e s and backgrounds , b r i n g i n g w i th them v a r y i n g p o i n t s of v iew. There a r e , pe rhaps , as many d e f i n i t i o n s of " e t h n o b i o l o g y " as there are e t h n o b i o l o g i s t s . The te rms , e t h n o b i o l o g y , ethnobotany and ethnopharmacology have been l o o s e l y a p p l i e d to the many types of s c i e n t i f i c a l l y s anc t i oned o b s e r v a t i o n s , i d e n t i f i c a t i o n s , d e s c r i p t i o n s and expe r imen ta l i n v e s t i g a t i o n s of p l a n t s , and t h e i r p r i n c i p a l s , used by ind igenous peop les ( E f ron e_t a l , 1967; Holmstedt and Bruhn, 1983; S c h u l t e s , 1963; S chu l t e s and Swain, 1976). Bruhn and Holmstedt (1982) have s u c c i n c t l y d e f i n e d ethnopharmacology as " the i n t e r d i s c i p l i n a r y s c i e n t i f i c e x p l o r a t i o n of b i o l o g i c a l l y a c t i v e agents t r a d i t i o n a l l y employed or observed by man." Ethnobotany or e thnob io l ogy may be s i m i l a r l y d e f i n e d . The b a s i c d i s c i p l i n e s i n v o l v e d are e thno logy or a n t h r o p o l o g y , pharmacology and botany (or zoo logy in the case of an imal p r o d u c t s ) . The on l y term which encompasses a l l s i t u a t i o n s i s e t h n o b i o l o g y . I t i s used in the p resen t work fo r t h i s r eason . 2 My concep t i on of t h i s f i e l d of study i s not un ique , hav ing been i n f l u e n c e d by many p r e v i o u s workers . Ne i the r does i t c o i n c i d e w i th some imaginary concensus v iew. In an attempt to c l a r i f y the o b j e c t i v e s of t h i s s tudy , there f o l l o w s a gene ra l c o n s i d e r a t i o n of the r o l e , o b j e c t i v e s and importance of e thnob io l ogy in modern s c i e n c e . The recent r a p i d advances in s c i e n c e have broadened the concep tua l gap between western i n d u s t r i a l i z e d and p r i m i t i v e s o c i e t i e s . Acceptance of the va lue of s imple t e c h n o l o g i e s or b e l i e f systems d i f f e r e n t from our own i s not common. There i s a widespread tendency in our s o c i e t y not on ly to accept the pr imacy of the s c i e n t i f i c method, but to adopt the c o r o l l a r y tha t i n f o r m a t i o n ga ined in other ways i s n e c e s s a r i l y t r i v i a l or f a l s e . The i n t e r f a c e between the s c i e n t i f i c method and the b e l i e f s of p r i m i t i v e p e o p l e s , which i s an e s s e n t i a l par t of e t h n o b i o l o g y , i s not always a smooth one. The s c i e n t i f i c view i s tha t the use of p l a n t s by man has emerged s l o w l y , as the r e s u l t of e m p i r i c a l t e s t i n g by gene r a t i ons of i n q u i s i t i v e p r i m i t i v e s and tha t i t has been s u s t a i n e d by the o r a l t r a d i t i o n . When one q u e r i e s an Ind ian or mes t i zo about how they came to know of a p l a n t use , one sometimes ge ts the a p p r o p r i a t e answer; tha t the i n f o r m a t i o n was o b t a i n e d from a f r i e n d , a r e l a t i v e , an e l d e r or from a n e i g h b o r i n g community. I f , on the o ther hand, one asks a n a t i v e s p e c i a l i s t who has been t r a i n e d in the shamanic t r a d i t i o n , the answer seldom v a r i e s ; tha t i t was l e a rned from the p l a n t , i t s e l f . A c c o r d i n g 3 t o t h e shaman, t h e p r o c e s s i s one of summoning up t h e s p i r i t o f t h e p l a n t , e s t a b l i s h i n g c o n t a c t and l i s t e n i n g t o i t s e x p l a n a t i o n . The P e r u v i a n m e s t i z o shaman r o u t i n e l y r e f e r s t o c e r t a i n p l a n t s as d o c t o r e s or v e g e t a l e s que e n s e n a n ( p l a n t s t h a t t e a c h ) (Luna, 1983). They a r e t h e s u b j e c t s of i n v o c a t i o n s made d u r i n g t r a n c e l i k e s t a t e s i n d u c e d , or r e g u l a t e d by, t h e use of h a l l u c i n o g e n i c a y a h u a s c a and p o t e n t t o b a c c o . The t r u l y a m a z i n g t h i n g a b o u t t h i s p r a c t i c e i s t h a t i t i s common, i n s i m i l a r f o r m s , t o t h e t r i b e s , n o t o n l y of t h e Amazon, but o f a l l t h e A m e r i c a s and much of E u r a s i a ( E l i a d e , 1964; M e t r a u x , 1944). The b e l i e f s can be t r a c e d i n t h e c u l t u r e s of t h e F u e g i a n s o f Cape Horn a s w e l l as t h e b a r r e n l a n d E s k i m o s (Browman and S c h w a r t z , 1979; C h i n e , 1976). The i m p l i c a t i o n s o f p l a n t s as t e a c h e r s a r e n o t e a s i l y r e c o n c i l e d w i t h s c i e n t i f i c t h o u g h t . I t i s i r o n i c , however, t h a t i f t h e c o n c e p t i s t a k e n f i g u r a t i v e l y , i t i n d i c a t e s r e m a r k a b l e i n s i g h t . F o r example, u n t i l a p p r o x i m a t e l y t e n y e a r s ago, modern s c i e n c e knew v e r y l i t t l e of f u n d a m e n t a l i m p o r t a n c e a b o u t p a i n . S i n c e t h e n , i t has been d i s c o v e r e d t h a t m o r p h i n e , an a l k a l o i d from t h e poppy, p r e v e n t s p a i n by b i n d i n g t o s p e c i f i c r e c e p t o r s on n e u r o n s o f t h e c e n t r a l n e r v o u s s y s t e m and t h a t p e p t i d e s a r e p r e s e n t i n t h e human body w h i c h a l s o b i n d t o t h e same r e c e p t o r s ( C o s t a and T r a b u c c h i , 1978). A m a j o r r e t h i n k i n g i n n e u r o p h y s i o l o g y has e n s u e d . The p r o v o c a t i v e d i s c o v e r y o f t h e n o r m a l p e p t i d e s , o r e n d o r p h i n s , was d e p e n d e n t upon t h e a v a i l a b i l i t y o f t h e compound m o r p h i n e and, o f c o u r s e , t h e poppy p l a n t . The e u p h o r i c p r o p e r t i e s of 4 Papaver somniferum were, c o i n c i d e n t a l l y , known to the Sumerians some 6000 years ago (Swain, 1972). Many s i m i l a r examples may be c i t e d . P i c r o t o x i n , a convu l san t from Anamir ta c o c c u l u s (Menispermaceae) , i s c u r r e n t l y used w ide ly in i n v e s t i g a t i o n s of n e u r o t r a n s m i s s i o n (Ho f fme i s t e r and S t i l l e , 1980). Phorbo l e s t e r s , from s p e c i e s of the Euphorb iaceae , are tumour promoters and important t o o l s in expe r imen ta l cancer r e sea r ch (Evans and Soper , 1978; We ins te in et a_l, 1977). R e c e n t l y , they have been found to be potent i nduce r s of i n t e r f e r o n in mammalian c e l l s (Y ip e_t a l , 1981). For many y e a r s , an imal p h y s i o l o g i s t s have d i s t i n g u i s h e d neurons based upon whether they were e x c i t e d by n i c o t i n e (from N i c o t i a n a tabacum) or muscar ine (from Amanita m u s c a r i a ) . Tubocu ra r i ne (from p l a n t s of the Logan iaceae ) and a t r o p i n e (Atropa be l l adonna ) are both used e x t e n s i v e l y in s t u d i e s of neuromuscular t r a n s m i s s i o n , and rotenone ( D e r r i s spp. ) i s commonly used as a metabo l i c i n h i b i t o r . C o l c h i c i n e (Co lch icum  autumnale) i s used r o u t i n e l y by most c y t o g e n e t i c i s t s and s e v e r a l hundred papers have been w r i t t e n on the e f f e c t s of c a f f e i n e (Ca f f ea s p . and Thea sp . ) in the i n h i b i t i o n of DNA r e p a i r p r o c e s s e s . The l i s t c o u l d be c o n t i n u e d . Look ing at the Merck Index, i t i s d i f f i c u l t to p i c k a page on which at l e a s t one c i t a t i o n to a s c i e n t i f i c a l l y u s e f u l or e conomi ca l l y important c o n s t i t u e n t produced by a p l an t or m i c roo rgan i sm, does not appea r . I t i s i n s t r u c t i v e to imagine what modern b i o l o g y and medic ine might have been l i k e had the o rgan i c c o n s t i t u e n t s of p l a n t s or mic roorgan isms never been a v a i l a b l e 5 to u s . Knowledge in these areas would p robab l y be reduced to a sma l l f r a c t i o n of what i t i s now. Without b i o l o g i c a l l y a c t i v e , n a t u r a l l y o c c u r r i n g compounds as models , very few a n t i b i o t i c s or chemotherapeut i c agents would yet have been s y n t h e s i z e d by chemi s t s . Even such a r e l a t i v e l y s t r a i g h t f o r w a r d matter as the economic importance of p l a n t s to med ic ine i s not w ide ly r e c o g n i z e d . Educated es t imates are that app rox ima te l y one-ha l f of the e c o n o m i c a l l y important compounds con t i nue to be ob t a i ned from p l a n t s ( O l d f i e l d , 1981). Farnswor th and M o r r i s (1976) have p resen ted f i g u r e s which i n d i c a t e t h a t , between 1959 and 1973, 25% of a l l the p r e s c r i p t i o n s f i l l e d in the U.S. c on t a i ned p l a n t p r o d u c t s . The v a l u e , to the consumer, was es t ima ted to be $3.0 b i l l i o n , a n n u a l l y . In 1980, the percentage was unchanged but the d o l l a r va lue had c l imbed to in excess of $8.0 b i l l i o n (Farnsworth and Loub, 1982). The who lesa le va lue of m e d i c i n a l p l a n t s imported to the U.S. from t r o p i c a l f o r e s t s , a l o n e , has been es t ima ted to be $25 m i l l i o n (U.S. In te ragency Task F o r c e , 1980). G iven s u f f i c i e n t space , the argument tha t modern s c i e n c e and medic ine are deep ly indebted to the i n f o r m a t i o n ob ta ined from b i o l o g i c a l l y a c t i v e p l a n t compounds, c o u l d be expanded f u r t h e r . The degree of r e spec t f o r p l a n t s h e l d by the p r i m i t i v e American s o c i e t i e s has a l r e a d y .been c o n t r a s t e d w i th our own s o c i e t y ' s gene ra l l a ck of acknowledgment of the importance of p l a n t s to knowledge. The i r ony of t h i s s i t u a t i o n i s tempered by the o b s e r v a t i o n tha t the "most u s e f u l drugs 6 d e r i v e d from h ighe r p l a n t s have been ' d i s c o v e r e d ' as a r e s u l t of the s c i e n t i f i c enqu i r y i n t o a l l e g e d f o l k l o r i c c l a i m s of t h e r a p e u t i c e f f e c t s " (Farnsworth and Loub, 1982). Many m i c r o b i a l p roduc ts have been d i s c o v e r e d as a r e s u l t of a random s c r e e n i n g program. For v a r i ous r easons , h i ghe r p l a n t s are not n e a r l y as amenable to suchr an approach . Moreover , s i n ce they are mac ros cop i c , they are an important pa r t of the m a t e r i a l wor ld of p r i m i t i v e man. As a r e s u l t , a r i c h h i s t o r y of p l a n t use by man i s a v a i l a b l e to draw upon. In the p a s t , the i n v e s t i g a t i o n of p l a n t s used by p r i m i t i v e man has been the p r e f e r r e d approach and has l e d to the development of such drugs as a t r o p i n e , d i g i t a l i s and r e s e r p i n e . Less commonly c a r r i e d out today , such s t u d i e s s t i l l l e ad to the d i s c o v e r y of c o n s t i t u e n t s w i th nove l b i o l o g i c a l a c t i v i t y (Gran, 1973) . Some random s c r een ing programs of h ighe r p l a n t s have been c a r r i e d o u t . The most e x t e n s i v e of these has been conducted by the U.S. N a t i o n a l Cancer I n s t i t u t e ( Su f fness and Douros , 1982). P l an t s were screened f o r ant i tumour a c t i v i t y . In an i n t e r e s t i n g fo l l ow-up s tudy , Sp jut and Perdue (1976) p resen ted ev idence tha t the occur rence of ant i tumour a c t i v i t y was s i g n i f i c a n t l y h ighe r in p l a n t s hav ing a h i s t o r y of f o l k use than those s e l e c t e d at random. 2. OBJECTIVES OF ETHNOBIOLOGY The f o r e g o i n g d i s c u s s i o n has deve loped the idea tha t one 7 of the pr imary o b j e c t i v e s of e thnob io l ogy i s the d i s c o v e r y of n a t u r a l l y o c c u r r i n g c o n s t i t u e n t s w i th nove l b i o l o g i c a l a c t i v i t y . A n a t u r a l ex tens ion of t h i s aim i s a d e s i r e to d e s c r i b e and e x p l a i n the d i s t r i b u t i o n of b i o l o g i c a l l y a c t i v e c o n s t i t u e n t s in the p l an t kingdom. Swain (1972) has p o i n t e d to the use of medieva l p l a n t s in medic ine as be ing r e s p o n s i b l e fo r the o r i g i n of modern taxonomy. There has f o l l owed a g radua l accumula t ion of i n f o rma t i on on the chemica l c o n s t i t u e n t s of v a r i o u s p l an t taxa in the r e l a t e d f i e l d s of phy tochemi s t r y , n a t u r a l p roduc t s chemis t r y and chemotaxonomy. The task of s y n t h e s i z i n g t h i s i n f o rma t i on so as to ga in a broad p e r s p e c t i v e on how v a r i o u s b i o l o g i c a l l y a c t i v e compounds are d i s t r i b u t e d in the p l a n t kingdom i s an immense one and has been at tempted by on ly a few (Farnsworth and Loub, 1982; Fa rnswor th , et a l , 1974; Hegnauer, 1962; K a r r e r , 1958). T h i s type of d e s c r i p t i v e i n fo rma t i on may e v e n t u a l l y p rov i de v a l u a b l e i n s i g h t s i n t o the r o l e of v a r i o u s c o n s t i t u e n t s in e v o l u t i o n . Moreover , i t would be expected to p rov ide p r e d i c t i v e i n f o rma t i on on the presence of s p e c i f i c compounds, or p o s s i b l y even compounds w i th s p e c i f i c b i o l o g i c a l a c t i v i t y , in taxa not p r e v i o u s l y examined c h e m i c a l l y ( Ba rc l ay and Perdue, 1976). E t h n o b o t a n i c a l i n f o r m a t i o n , t o o , i s c o n s i d e r e d by some to be a u s e f u l t o o l in p r e d i c t i n g the occu r rence of c e r t a i n b i o l o g i c a l l y a c t i v e c o n s t i t u e n t s (Spjut and Perdue , 1976). The a p p l i c a b i l i t y of the computer in hand l i ng l a r g e data bases of t h i s s o r t , c o n t a i n i n g both e t h n o b o t a n i c a l and chemica l i n f o rma t i on on p l a n t s , i s c u r r e n t l y be ing 8 i n v e s t i g a t e d (Farnsworth and B i n g e l , 1977; Farnsworth and Loub, 1982; Farnsworth et a l , 1981). There i s another b a s i c o b j e c t i v e in e t h n o b i o l o g i c a l work tha t shou ld not be o v e r l o o k e d . The s u b j e c t i v e na ture of the approach i s the aspect of e thnob io l ogy most o f t e n c r i t i c i z e d . Yet t h i s p rope r t y can a l s o be regarded as a unique advantage . A s i n g l e statement conce rn ing the use of a p l a n t by man i s u n l i k e l y to c o n t r i b u t e d i r e c t l y to b a s i c s c i e n c e . I t c an , on the o ther hand, summarize an aspec t of the r e l a t i o n s h i p between man and the p l an t kingdom in an h i s t o r i c a l contex t in a way not p o s s i b l e us ing on ly a r e d u c t i v e s c i e n t i f i c approach . The extent of the i n t e r a c t i o n s between man and p l a n t s i s d e c l i n i n g as the i n d u s t r i a l i z e d wor ld expands and the endurance of e t h n o b o t a n i c a l i n f o r m a t i o n , i f un reco rded , i s p r e d i c t a b l y low. T h i s w i l l r e s u l t in the l o s s of i n f o r m a t i o n , not on ly on s p e c i f i c c u l t u r e s , but a l s o on h a b i t a t s and even complete ecosystems s i n ce t hese , t o o , are i n e v i t a b l y a l t e r e d as a r e s u l t of economic deve lopment . One of the r o l e s of e thnobotany , t h e r e f o r e , i s the documentat ion of p l a n t use and the i d e n t i f i c a t i o n of the v e g e t a l m a t e r i a l s . T h i s a c t i v i t y c o n t i n u e s to be of importance as the e x i s t e n c e of l i t t l e known groups of the w o r l d ' s b i o t a , i n c l u d i n g many human c u l t u r e s , face the c e r t a i n t y of d r a s t i c change. 3. IMPORTANCE OF ETHNOBIOLOGY IN THE TROPICS The q u e s t i o n of the f u tu r e of t r o p i c a l ecosystems of the 9 w o r l d i s an e s p e c i a l l y p e r t i n e n t one f o r b i o l o g i s t s a t t h i s t i m e i n h i s t o r y . A l t h o u g h e t h n o b i o l o g i c a l s t u d y c a n be c a r r i e d o u t anywhere t h a t p e o p l e a r e u s i n g p l a n t s , t h e t r o p i c s has been, and c o n t i n u e s t o be, an e s p e c i a l l y f e r t i l e r e g i o n f o r t h i s t y p e of work. I t i s n o t c l e a r why so many of t h e i m p o r t a n t d r u g s of n a t u r a l o r i g i n were i d e n t i f i e d from t r o p i c a l p l a n t s ( O l d f i e l d , 1981). The d i v e r s i t y i n s p e c i e s o f a l l kingdoms o f o r g a n i s m s and t h e enormous p o t e n t i a l f o r i n t r a - and i n t e r - s p e c i f i c i n t e r a c t i o n s a r e c e r t a i n l y i m p o r t a n t f a c t o r s . E v i d e n c e has been p r e s e n t e d w h i c h i n d i c a t e s t h a t d i v e r s i t y i n c h e m i c a l compounds s y n t h e s i z e d p a r a l l e l s t h i s i n c r e a s e i n s p e c i e s d i v e r s i t y ( R a f f a u f , 1970). An e q u a l l y c o m p e l l i n g r e a s o n f o r s t u d y i n g e t h n o b i o l o g y , o r any b r a n c h o f b i o l o g y , i n t h e t r o p i c s i s t h e u n c e r t a i n f u t u r e f a c i n g t h i s r e g i o n . T r o p i c a l f o r e s t s a c c o u n t f o r a l m o s t 70% o f g l o b a l f o r e s t p r o d u c t i v i t y ( B r u n i n g , 1977). C o v e r i n g o n l y 7% o f t h e e a r t h ' s s u r f a c e , t h e y c o n t a i n between 40 and 50% o f t h e w o r l d ' s e s t i m a t e d 5 t o 10 m i l l i o n s p e c i e s ( M e y e r s , 1981). I t i s a g r e e d t h a t i t i s t h e l e a s t known of t h e p l a n e t ' s b i o m e s , w i t h e s t i m a t e s o f t h e e x t e n t o f t h e s p e c i e s a l r e a d y d e s c r i b e d r a n g i n g between 10 and 16 % ( M e y e r s , 1981; Raven e t a l , 1971). Of t h e s e , we p o s s e s s o n l y t h e most r u d i m e n t a r y i n f o r m a t i o n (Raven e t a_l, 1971). The most o f t e n c i t e d p r e d i c t i o n of t h e f u t u r e o f t h e t r o p i c a l m o i s t f o r e s t i s t h a t of R i c h a r d s (1952; 1973) who f o r e c a s t t h a t 10 "The t r o p i c a l f o r e s t e c o s y s t e m as we know i t w i l l v i r t u a l l y d i s a p p e a r f r o m t h e f a c e of t h e e a r t h by t h e end of t h e 2 0 t h c e n t u r y . " O t h e r p r e d i c t i o n s a r e g e n e r a l l y l e s s p e s s i m i s t i c b u t not much l e s s d r a m a t i c . T h e r e i s b r o a d agreement among s p e c i a l i s t s t h a t , by t h e y e a r 2000, v e r y l a r g e segments o f t h e w o r l d ' s t r o p i c a l m o i s t f o r e s t s w i l l be r e d u c e d t o remnants ( F a r n w o r t h and G o l l e y , 1974; G o l l e y and Medina,1975; Gomez-Pompa e t a l , 1972; L a n l y and C l e m e n t , 1979; M e y e r s , 1980a and b; P e r s s o n , 1974, 1975, and 1977; P o o r e , 1976; R i c h a r d s , 1973; Sommer, 1976; UNESCO, 1978). The v a s t w a t e r s h e d of t h e Amazon R i v e r s y s t e m a c c o u n t s f o r a p p r o x i m a t e l y 40% o f t h e w o r l d ' s m o i s t t r o p i c a l f o r e s t s .(Goodland, 1980). P r e d i c t e d r a t e s o f d e f o r e s t a t i o n a r e r e p r e s e n t a t i v e of t h e g l o b a l f o r e c a s t s c i t e d above ( F e a r n s i d e , 1979). The major d i s r u p t i v e f a c t o r s have been i d e n t i f i e d a s t h e p a t t e r n of s h i f t i n g c u l t i v a t i o n p r a c t i c e d by t h e e x p a n d i n g p o p u l a t i o n , t i m b e r h a r v e s t i n g and c a t t l e r a n c h i n g (Denevan, 1980; Shane, 1980). The e m e r g i n g e v i d e n c e p o i n t s t o w a r d s t h e l i m i t e d a b i l i t y o f t h i s e c o s y s t e m t o r e s i s t c h ange ( G e n t r y , 1977). The m a g n i t u d e o f t h e c o n t r i b u t i o n o f t h e s e a s o n a l l y i n u n d a t e d a r e a s , a r e l a t i v e l y s m a l l f r a c t i o n of t h e t o t a l a r e a , t o t h e o v e r a l l p r o d u c t i v i t y has been r e a l i z e d q u i t e r e c e n t l y (Meggers, 1971). T h i s , however, i s t h e most v u l n e r a b l e t o m o d i f i c a t i o n and t h e a b i l i t y o f t h i s complex e c o s y s t e m t o t o l e r a t e s u c h p r e s s u r e s has not been e s t a b l i s h e d . The i m p o r t a n c e of t h e r e l a t i o n s h i p s between t h e t r e e s o f t h e 11 i n u n d a t e d r e g i o n s and t h e many s p e c i e s of f r u i t i f e r o u s f i s h h as r e c e n t l y been documented ( G o u l d i n g , 1980). F i s h a r e a l s o t h e m a j o r s o u r c e of p r o t e i n f o r t h e e n l a r g i n g p o p u l a t i o n . Any l i k e l y s c e n a r i o f o r t h e f u t u r e of t h e m o i s t t r o p i c a l f o r e s t s i n t h e n e x t c e n t u r y , a v e r y s h o r t i n t e r v a l on an e v o l u t i o n a r y s c a l e , i s o f a d r a s t i c r e d u c t i o n i n t h e e x t e n t o f t h e p l a n e t ' s b i o t a ( M e y e r s , 1981). I n p r e p a r a t i o n f o r t h i s s e e m i n g l y u n a v o i d a b l e s i t u a t i o n , c e r t a i n p r i o r i t i e s f o r r e s e a r c h have been p r o p o s e d ( R e s e a r c h P r i o r i t i e s i n T r o p i c a l B i o l o g y , 1980; T r o p i c a l D e f o r e s t a t i o n , 1980). T h e s e a r e aimed a t a c c u m u l a t i n g as much i n f o r m a t i o n on t h e b i o l o g y of t r o p i c a l o r g a n i s m s as p o s s i b l e i n t h e l i m i t e d t i m e a l l o w e d . D e s c r i p t i v e e t h n o b o t a n y i s c o n s i d e r e d t o be a m i n o r , b ut v a l u e d c o n t r i b u t i o n (Raven et_ a l , 1971; R e s e a r c h P r i o r i t i e s i n T r o p i c a l B i o l o g y , 1980). The l o n g s t a n d i n g r e l a t i o n s h i p o f t h e i n d i g e n o u s p e o p l e s w i t h t h e i r b i o t a i s c o n s i d e r e d l i k e l y t o have r e s u l t e d i n many b i o l o g i c a l i n s i g h t s w h i c h may be o b t a i n e d by s c i e n t i f i c methods o n l y a f t e r y e a r s o f r e s e a r c h . 4. APPROACH OF THIS STUDY The o b j e c t i v e s o f t h e p r e s e n t s t u d y were s e v e r a l - f o l d . The b a s i c a p p r o a c h was t o s e l e c t i n s t a n c e s o f t r a d i t i o n a l p l a n t u s e , t o examine t h e p l a n t s by b i o l o g i c a l a s s a y s f o r t h e p r e s e n c e o f b i o l o g i c a l l y a c t i v e c o n s t i t u e n t s and t o a t t e m p t t h e i s o l a t i o n and i d e n t i f i c a t i o n o f t h e a c t i v e c o n s t i t u e n t s . I t was e x p e c t e d t h a t t h i s p r o c e s s would l e a d t o c o n c l u s i o n s on t h e g e n e r a l e f f i c a c y of t h e p l a n t and p r o v i d e some i n s i g h t 1 2 i n t o i t s use by a r e l a t i v e l y p r i m i t i v e c u l t u r e . Moreover , i t was hoped tha t e i t h e r the b i o l o g i c a l a c t i v i t y or the chemica l s t r u c t u r e ( p r e f e r a b l y both) of the a c t i v e c o n s t i t u e n t ( s ) would be nove l and that a c o n t r i b u t i o n to b a s i c s c i e n c e might be made. The f i r s t stage in t h i s approach i n v o l v e d the s e l e c t i o n of p l a n t s of e t h n o b o t a n i c a l i n t e r e s t upon which to work. Many c r i t e r i a came i n t o p l ay at t h i s l e v e l . P r e f e rence was g iven to p l a n t s w i th we l l e s t a b l i s h e d and r e l a t i v e l y c l e a r l y d e f i n e d uses which were amenable to assay in the l a b o r a t o r y . The p o t e n t i a l f o r o b t a i n i n g new i n f o rma t i on was a s s e s s e d . The p l a n t ' s taxonomic p o s i t i o n and e t h n o b o t a n i c a l and chemica l data conce rn ing r e l a t e d p l a n t s was a l s o used to a s sess the p r o b a b i l i t y of a c t i v e c o n s t i t u e n t s be ing p r e s e n t . I t was assumed tha t the l i k e l i h o o d of a p l a n t c o n t a i n i n g such compounds would be g r ea t e r in a p l a n t be long ing to a t axon : a) w i th many documented e t h n o b o t a n i c a l uses b) which had not a l r e ady been s t u d i e d c h e m i c a l l y The e t h n o b o t a n i c a l data on South American p l a n t s has not been a r ranged s y s t e m a t i c a l l y . P e r t i n e n t i n f o rma t i on i s p resen t in a wide v a r i e t y of l o c a t i o n s i n c l u d i n g the r e s e a r c h notes and monographs of b o t a n i s t s , a n t h r o p o l o g i s t s and p h a r m a c o l o g i s t s , the works of e x p l o r e r s , t r a v e l l e r s and m i s s i o n a r i e s and herbar ium l a b e l s . As much of t h i s i n f o rma t i on as p o s s i b l e was c o l l e c t e d and o rgan i zed a c c o r d i n g to the c l a s s i f i c a t i o n system of C ronqu i s t (1981) . T h i s i s p resen ted 1 3 as Appendix A. The i n fo rma t i on has been summarized, a c c o r d i n g to the d i s t r i b u t i o n of spec i e s w i th e t h n o b o t a n i c a l documentat ion at the order and f am i l y l e v e l , in Tab le I. I t has a l s o been a r ranged in F i gu re 1 a c c o r d i n g to Dahlgren (1980), whose system of c l a s s i f i c a t i o n c o n s i s t s of a two d imens iona l scheme of the o rde rs of ang iosperms . T h i s has the advantage of a l l o w i n g , not on l y the main p h y l o g e n e t i c r e l a t i o n s h i p s between the o r d e r s , but the r e l a t i v e s i z e of each group, in numbers of spec i e s d e s c r i b e d , to be e a s i l y p e r c e i v e d . The i n f o r m a t i o n ga thered i s i n h e r e n t l y s u b j e c t i v e . I t a r i s e s from the b i a s e s of the au thors whose data was s e l e c t e d fo r c o m p i l a t i o n . However, combin ing the i n f o rma t i on in t h i s way made the survey more ex t ens i v e and p r o v i d e d a h ighe r degree of v e r i f i c a t i o n of o b s e r v a t i o n s . It i s e v iden t from Tab le I and F i g u r e T, that the Angiosperms are w ide ly r ep resen ted among the m e d i c i n a l and po isonous p l a n t s known to ind igenous Amazonians. The C l a s s Magno l i ops ida i s , however, much b e t t e r r ep resen ted than the C l a s s L i l i o p s i d a . W i th in the M a g n o l i o p s i d a , the Subc l asses Rosidae and A s t e r i d a e are e s p e c i a l l y r i c h in number of s p e c i e s wi th e t h n o b o t a n i c a l u ses . The f a m i l i e s tha t are the most impor tan t , a c c o r d i n g to numbers of s p e c i e s used , are the Leguminosae, Apocynaceae, Euphorb iaceae , Rubiaceae and Solanaceae w i th 95, 53, 47, 46 and 45 s p e c i e s , r e s p e c t i v e l y . I t can be seen from F i g u r e 1 tha t the number of s p e c i e s of e t h n o b o t a n i c a l i n t e r e s t in each group i s g e n e r a l l y a 1 4 T a b l e I - P h y l o g e n e t i c d i s t r i b u t i o n o f s p e c i e s o f Amazonian a n g i o s p e r m s h a v i n g documented e t h n o b o t a n i c a l u s e . T a x a a r e a r r a n g e d a c c o r d i n g t o t h e c l a s s i f i c a t i o n scheme of C r o n q u i s t ( 1 9 8 1 ) . T a b l e I i s a summary of A p p e n d i x A. Taxon Number o f s p e c i e s w i t h documented use D i v i s i o n : M a q n o l i o p h y t a 793 C l a s s : M a q n o l i o p s i d a 727 S u b c l a s s I . M a g n o l i i d a e 1 04 O r d e r : M a g n o l i a l e s 42 F a m i l y : Annonaceae 1 2 F a m i l y : M y r i s t i c a c e a e 30 O r d e r : L a u r a l e s 6 F a m i l y : M o n i m i a c e a e 2 F a m i l y : L a u r a c e a e 4 O r d e r : P i p e r a l e s 29 F a m i l y : P i p e r a c e a e 29 O r d e r : A r i s t o l o c h i a l e s 8 F a m i l y : A r i s t o l o c h i a c e a e 8 O r d e r : R a n u n c u l a l e s 18 F a m i l y : M e n i s p e r m a c e a e 18 O r d e r : P a p a v e r a l e s 1 F a m i l y : P a p a v e r a c e a e 1 S u b c l a s s I I . Hamamelidae 1 7 O r d e r : U r t i c a l e s 1 7 F a m i l y : M o r a c e a e 10 F a m i l y : C e c r o p i a c e a e 7 S u b c l a s s I I I . C a r y o p h y l l i d a e 22 O r d e r : C a r y o p h y l l a l e s 20 F a m i l y : P h y t o l a c c a c e a e 3 F a m i l y : N y c t a g i n a c e a e 2 F a m i l y : C a c t a c e a e 4 F a m i l y : C h e n o p o d i a c e a e 1 F a m i l y : A m a r a n t h a c e a e 5 F a m i l y : P o r t u l a c a c e a e 3 F a m i l y : B a s e l l a c e a e 1 F a m i l y : C a r y o p h y l l a c e a e 1 O r d e r : P o l y g o n a l e s 2 F a m i l y : P o l y g o n a c e a e 2 1 5 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 T a b l e I ( c o n t i n u e d ) Taxon No. s p e c i e s O r d e r : C e l a s t r a l e s 5 F a m i l y : C e l a s t r a c e a e 2 F a m i l y : I c a c i n a c e a e 2 F a m i l y : D i c h a p e t a l a c e a e 1 O r d e r : E u p h o r b i a l e s 47 F a m i l y : E u p h o r b i a c e a e 47 O r d e r : L i n a l e s 1 0 F a m i l y : E r y t h r o x y l a c e a e 2 F a m i l y : H u m i r i a c e a e 8 O r d e r : P o l y g a l a l e s 22 F a m i l y : M a l p i g h i a c e a e 1 7 F a m i l y : V o c h y s i a c e a e 5 O r d e r : S a p i n d a l e s 27 F a m i l y : S a p i n d a c e a e 6 F a m i l y : B u r s e r a c e a e 5 F a m i l y : A n a c a r d i a c e a e 6 F a m i l y : S i m a r o u b a c e a e 3 F a m i l y : M e l i a c e a e 2 F a m i l y : R u t a c e a e 5 O r d e r : G e r a n i a l e s 1 F a m i l y : O x a l i d a c e a e 1 S u b c l a s s V I . A s t e r i d a e 247 O r d e r : G e n t i a n a l e s 68 F a m i l y : L o g a n i a c e a e 8 F a m i l y : G e n t i a n a c e a e 4 F a m i l y : A p o c y n a c e a e 53 F a m i l y : A s c l e p i a d a c e a e 3 O r d e r : S o l a n a l e s 49 F a m i l y : S o l a n a c e a e 45 F a m i l y : C o n v o l v u l a c e a e 3 F a m i l y : H y d r o p h y l l a c e a e 1 O r d e r : L a m i a l e s 29 F a m i l y : B o r a g i n a c e a e 8 F a m i l y : V e r b e n a c e a e 8 F a m i l y : L a m i a c e a e 1 3 O r d e r : S c r o p h u l a r i a l e s 44 F a m i l y : S c r o p h u l a r i a c e a e 4 F a m i l y : G e s n e r i a c e a e 4 F a m i l y : A c a n t h a c e a e 18 F a m i l y : P e d a l i a c e a e 1 F a m i l y : B i g n o n i a c e a e 1 7 O r d e r : C a m p a n u l a l e s 4 F a m i l y : Campanulaceae 4 O r d e r : R u b i a l e s 46 F a m i l y : R u b i a c e a e 46 O r d e r : A s t e r a l e s 1 7 F a m i l y : A s t e r a c e a e 1 7 1 7 T a b l e I ( c o n t i n u e d ) C l a s s : L i l i o p s i d a 66 S u b c l a s s I : A l i s m a t i d a e 2 O r d e r : A l i s m a t a l e s 2 F a m i l y : A l i s m a t a c e a e 2 S u b c l a s s I I : A r e c i d a e 29 O r d e r : A r e c a l e s 5 F a m i l y : A r e c a c e a e 5 O r d e r : C y c l a n t h a l e s 1 F a m i l y : C y c l a n t h a c e a e 1 O r d e r : A r a l e s 23 F a m i l y : A r a c e a e 23 S u b c l a s s I I I : C o m m e l i n i d a e 8 O r d e r : C o m m e l i n a l e s 1 F a m i l y : Commelinaceae 1 O r d e r : C y p e r a l e s 7 F a m i l y : C y p e r a c e a e 2 F a m i l y : Poaceae 5 S u b c l a s s IV: Z i n g i b e r i d a e 1 3 O r d e r : B r o m e l i a l e s 1 F a m i l y : B r o m e l i a c e a e 1 O r d e r : Z i n g i b e r a l e s 12 F a m i l y : Musaceae 4 F a m i l y : Z i n g i b e r a c e a e • 1 F a m i l y : C o s t a c e a e 4 F a m i l y : Cannaceae 1 F a m i l y : M a r a n t a c e a e 2 S u b c l a s s V. L i l i i d a e 1 4 O r d e r : L i l i a l e s 8 F a m i l y : P o n t e d e r i a c e a e 1 F a m i l y : L i l i a c e a e 1 F a m i l y : I r i d a c e a e 2 F a m i l y : A g a v a c e a e 1 F a m i l y : S m i l a c a c e a e 1 F a m i l y : D i o s c o r e a c e a e 2 O r d e r : O r c h i d a l e s 6 F a m i l y : O r c h i d a c e a e 6 F i g u r e 1- D i s t r i b u t i o n of Amazonian angiosperms with documented e t h n o b o t a n i c a l uses, arranged a c c o r d i n g to the c l a s s i f i c a t i o n scheme of D a h l g r e n (1980). Numbers r e f e r to s p e c i e s that have at l e a s t one documented e t h n o b o t a n i c a l use. Data a r e summarized from Appendix A. 19 r e f l e c t i o n o f t h e number o f s p e c i e s i n t h a t g r o u p . A n o t a b l e e x c e p t i o n i s t h e o r d e r , O r c h i d a l e s , w h i c h seems not t o be u t i l i z e d i n p r o p o r t i o n t o i t s s i z e . T h i s may be b e c a u s e t h e m a j o r i t y of i t s s p e c i e s a r e r a r e , s m a l l i n s i z e and not e a s i l y o b t a i n e d . The b r o a d p e r s p e c t i v e p e r m i t t e d by t h i s e t h n o b o t a n i c a l l i s t p l a y e d a s i g n i f i c a n t r o l e i n my s e l e c t i o n of e t h n o b o t a n i c a l p r o b l e m s f o r i n v e s t i g a t i o n . The s t u d i e s f i n a l l y s e l e c t e d were i n v e s t i g a t i o n s of t h e c h e m i c a l and b i o l o g i c a l b a s e s f o r t h e u s e s of t h e J u s t i c i a p e c t o r a l i s and V i r o l a  e l o n g a t a and a s c r e e n i n g p r o g r a m of v a r i o u s b i o l o g i c a l a c t i v i t i e s o f members of t h e f a m i l y , E u p h o r b i a c e a e . J u s t i c i a p e c t o r a l i s i s an A c a n t h a c e o u s h e r b u s e d as an a d d i t i v e t o t h e V i r o l a b a s e d h a l l u c i n o g e n i c s n u f f s o f t h e Yanomamo. I t s use i n u n a c c u l t u r a t e d t r i b e s has been w e l l documented by a n t h r o p o l o g i s t s and b o t a n i s t s . The r e a s o n s f o r i t s i n c l u s i o n i n t h e s n u f f , and a l s o f o r i t s use a l o n e as a s n u f f , however, have n e v e r been s u b j e c t e d t o s c i e n t i f i c s c r u t i n y . In a d d i t i o n t o t h i s b e i n g a w e l l i d e n t i f i e d e t h n o b o t a n i c a l p r o b l e m , o t h e r c o n s i d e r a t i o n s i n d i c a t e d t h a t i t s s t u d y m i g h t y i e l d i n t e r e s t i n g r e s u l t s . The f a m i l y , A c a n t h a c e a e , i s m o d e r a t e l y i m p o r t a n t e t h n o b o t a n i c a l l y . E i g h t e e n Amazonian s p e c i e s w i t h e t h n o b o t a n i c a l u s e s a r e known ( T a b l e I ) . The s u b c l a s s , A s t e r i d a e , i n w h i c h i t i s p l a c e d , \s v e r y w e l l r e p r e s e n t e d i n T a b l e I . R e l a t e d f a m i l i e s ( R u b i a c e a e , A s t e r a c e a e , A p o c y n a c e a e , S o l a n a c e a e and B i g n o n i a c e a e ) a r e w e l l known f o r t h e i r a b i l i t y t o p r o d u c e complex b i o l o g i c a l l y a c t i v e 20 c h e m i c a l c o n s t i t u e n t s . A number of A s i a n s p e c i e s o f J u s t i c i a a r e u s e d m e d i c i n a l l y and a v a r i e t y of compounds w i t h i n t e r e s t i n g b i o l o g i c a l a c t i v i t i e s have been i s o l a t e d . T h e s e a r e l i g n a n s , p r i m a r i l y of t h e b i s - t e t r a h y d r o f u r a n c l a s s . They have r e c e n t l y been shown t o d i s p l a y a n t i d e p r e s s a n t a c t i o n ( G h o s a l and B a n e r j e e , 1979). F i n a l l y , t h e S o u t h A m e r i c a n members o f t h e f a m i l y A c a n t h a c e a e a r e l i t t l e known c h e m i c a l l y . No r e p o r t s of t h e genus, J u s t i c i a , f r o m t h e A m e r i c a s have been made. The use o f V i r o l a s p e c i e s as h a l l u c i n o g e n s i s a l s o a w e l l e s t a b l i s h e d phenomenon i n t h e e t h n o b o t a n i c a l l i t e r a t u r e . The d u a l u s e s of V i r o l a e l o n g a t a and V i r o l a t h e i o d o r a as b o t h an ar r o w p o i s o n and an h a l l u c i n o g e n a r e e s p e c i a l l y i n t r i g u i n g . The f a m i l y , M y r i s t i c a c e a e , i s i m p o r t a n t i n t h e Amazonian f l o r a . I t i s m o d e r a t e l y w e l l u t i l i z e d , e t h n o b o t a n i c a l l y (30 s p e c i e s ) , and r e l a t i v e l y w e l l s t u d i e d c h e m i c a l l y . In a d d i t i o n t o a number of p s y c h o t r o p i c t r y p t a m i n e and / 3 - c a r b o l i n e a l k a l o i d s , a wide v a r i e t y of l i g n a n s and n e o l i g n a n s a r e a l s o known ( G o t t l i e b , 1979). Some o f t h e s e p o s s e s s i n t e r e s t i n g b i o l o g i c a l a c t i v i t i e s . The s p e c i e s , V. e l o n g a t a , has been s t u d i e d c h e m i c a l l y o n l y w i t h r e s p e c t t o i t s i n d o l e a l k a l o i d s . The q u e s t i o n o f whether t h e p r e s e n c e o f h a l l u c i n o g e n i c a l k a l o i d s c o u l d e x p l a i n t h e use of t h e b a r k r e s i n a s an ar r o w p o i s o n o r i f some o t h e r compound w i t h t o x i c , o r o t h e r w i s e marked b i o l o g i c a l a c t i v i t y , was r e s p o n s i b l e was c o n s i d e r e d an e s p e c i a l l y i n t e r e s t i n g one. I t was assumed t o be r e a d i l y amenable t o e x p e r i m e n t a l a n a l y s i s . 21 A l t h o u g h t h e s u b j e c t of t h e use and e f f e c t s of h a l l u c i n o g e n i c p l a n t c o n s t i t u e n t s i s p e r i p h e r a l t o t h e theme of t h i s t h e s i s , some c l a r i f i c a t i o n o f t h e s u b j e c t i s i n o r d e r . I t i s i n t r i g u i n g t h a t g e o g r a p h i c a l l y and c u l t u r a l l y d i s t i n c t g r o u p s of S o u t h A m e r i c a n I n d i a n s a d o p t e d t h e c u s t o m of u s i n g m a t e r i a l s c o n t a i n i n g i n d o l e a l k a l o i d s a s c e r e m o n i a l d r u g s . T h i s i s e s p e c i a l l y so when i t i s c o n s i d e r e d t h a t a t l e a s t f o u r m ajor g r o u p s of s o u r c e p l a n t s a r e i n v o l v e d : A n a d e n a n t h e r a  p e r e g r i n a ( L e g u m i n o s a e ) , V i r o l a s p p. ( M y r i s t i c a c e a e ) , P s y c h o t r i a spp. ( R u b i a c e a e ) and B a n i s t e r i o p s i s s pp. ( M a l p i g h i a c e a e ) . Many o f t h e i n d o l e c o n s t i t u e n t s have been examined p h a r m a c o l o g i c a l l y and f o u n d t o have a range o f a c t i o n s . I n d o l e compounds of b o t h t h e t r y p t a m i n e and j3-c a r b o l i n e c l a s s have been d e t e c t e d i n many o f t h e p l a n t s o f t h i s g r o u p . These i n d o l e compounds c o m p r i s e two of t h e sev e n c l a s s e s of compounds d i s t i n g u i s h e d as h a v i n g h a l l u c i n o g e n i c o r p s y c h o t o m i m e t i c a c t i v i t y ( H o l l i s t e r , 1982). A l t h o u g h t h e s e t e r m s o v e r l a p i n meaning and a r e o f t e n u s e d s y n o n o m o u s l y , t h e f o r m e r r e f e r s t o a s u b j e c t i v e i n t e r p r e t a t i o n , t h e l a t t e r t o an o b j e c t i v e o b s e r v a t i o n t h a t t h e a g e n t p r o d u c e s b e h a v i o r a l e f f e c t s w h i c h mimic a p s y c h o s i s . P r o b l e m s a r e a s s o c i a t e d w i t h use of e i t h e r t e r m . H a l l u c i n o g e n i c a c t i v i t y i s l a r g e l y a f u n c t i o n o f t h e s u b j e c t , h i g h l y d e p e n d e n t upon dose and i m p o s s i b l e t o q u a n t i t a t e . M o r e o v e r , h a l l u c i n o g e n i c a c t i v i t y i s o n l y one of t h e p o t e n t i a l p h a r m a c o l o g i c a l e f f e c t s t h a t an i n d o l e compound may h a v e . The o t h e r p h a r m a c o l o g i c a l a c t i o n s 22 may also result in behavioral a l t e r a t i o n s . The precise nature of the b i o l o g i c a l e f f e c ts of such compounds is therefore d i f f i c u l t to define. This is p a r t i c u l a r l y true when animals are used as models to quantify the pharmacological response. The term, psychotropic, refers to the a b i l i t y to a l t e r mood or cognitive processes. As defined by Lewin (1927), i t includes agents with hypnotic, inebriant, excitant, euphoriant and hallucinogenic a c t i v i t y . Because i t is a more general term for describing agents with a c t i v i t y on the central nervous system, i t s use i s appropriate when the nature of the b i o l o g i c a l a c t i v i t y under investigation i s not completely defined (Hoffmeister and S t i l l e , 1982). It is used in th i s thesis to describe the effects of the administration of 5-methoxy-N,N-dimethyltryptamine and extracts of J. p e c t o r a l i s and V. elongata to mice for this reason. The t h i r d , and f i n a l , part of this research project was somewhat d i f f e r e n t in approach. While in Peru, I became aware of the importance of species of the family, Euphorbiaceae in folk medicine there. This is the t h i r d ranking family in terms of number of ethnobotanical uses (Table I ) . Its species are known to contain a great d i v e r s i t y of b i o l o g i c a l l y active constituents. The most widely d i s t r i b u t e d compounds are probably diterpenes of the phorbol ester c l a s s . These possess a variety of b i o l o g i c a l a c t i v i t i e s , 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 p h a r m a c o l o g i c a l a c t i v i t y i n o n l y a v e r y s m a l l number o f c a s e s . A s i g n i f i c a n t p r o p o r t i o n o f t h e E u p h o r b i a c e o u s s p e c i e s o f e t h n o b o t a n i c a l i n t e r e s t a r e u s e d by t h e I n d i a n o r m e s t i z o p o p u l a t i o n o.f P e r u i n t h e t r e a t m e n t o f wounds, u l c e r s , i n f e c t i o n s a n d c a n c e r o u s c o n d i t i o n s . T h e i r m e c h a n i s m o f a c t i o n may be h y p o t h e s i z e d t o i n v o l v e e i t h e r t h e i n h i b i t i o n o f r e p l i c a t i o n o f m i c r o o r g a n i s m s o r v i r u s e s , o r t h e r e g u l a t i o n o f some a s p e c t o f c e l l u l a r p r o l i f e r a t i o n . T h e s e b i o l o g i c a l a c t i v i t i e s a r e e s p e c i a l l y a m e n a b l e t o a n a l y s i s by b i o a s s a y . A v a r i e t y o f E u p h o r b i a c e o u s p l a n t s , some w i t h d o c u m e n t e d e t h n o b o t a n i c a l u s e s , o t h e r s w i t h o u t , were e x a m i n e d f o r t h e i r a b i l i t y t o i n h i b i t t h e r e p l i c a t i o n o f b a c t e r i a , y e a s t s , d e r m a t o p h y t i c f u n g i , a n i m a l v i r u s e s a nd p o t a t o t u m o u r s a n d t o x i c i t y t o b r i n e s h r i m p . The a i m o f t h e s t u d y was t o p r o v i d e d e s c r i p t i v e i n f o r m a t i o n on t h e b i o l o g i c a l a c t i v i t i e s o f t h e s e l i t t l e known s p e c i e s . I t was h o p e d t h a t t h e i n f o r m a t i o n o b t a i n e d w o u l d a l l o w some i n s i g h t i n t o t h e m e c h a n i s m o f a c t i o n o f t h e m e d i c i n a l l y u s e d s p e c i e s a n d a l s o t h e r e l a t i v e l i k e l i h o o d o f d e t e c t i n g a c t i v i t y i n p l a n t s t h a t d i d , a n d d i d n o t , have a d o c u m e n t e d e t h n o b o t a n i c a l u s e . As i n t h e c a s e o f t h e s t u d i e s o f J . p e c t o r a l i s a nd V i r o l a e l o n g a t a , t h e i d e n t i f i c a t i o n o f c h e m i c a l c o n s t i t u e n t s w i t h n o v e l b i o l o g i c a l a c t i v i t y was a l s o an o b j e c t i v e . The m a r k e d a n t i v i r a l a c t i v i t y o f an e x t r a c t o f Amanoa s p . was p u r s u e d and t h e compound r e s p o n s i b l e was i d e n t i f i e d . 24 T hese t h r e e p r o j e c t s : i ) t h e s t u d y of J . p e c t o r a l i s , i i ) t h e a n a l y s i s of V i r o l a e l o n g a t a r e s i n , and i i i ) t h e s c r e e n i n g of 34 s p e c i e s of E u p h o r b i a c e a e f o r b i o l o g i c a l a c t i v i t i e s and t h e s u b s e q u e n t a n a l y s i s o f t h e a n t i v i r a l c o n s t i t u e n t of one o f t h e s e , a s p e c i e s of Amanoa, a r e d e a l t w i t h as i n d e p e n d e n t c h a p t e r s i n t h i s t h e s i s . 25 LITERATURE CITED  C h a p t e r I_ B a r c l a y , A.S. and P e r d u e , R.E. (1976) D i s t r i b u t i o n o f a n t i c a n c e r a c t i v i t y i n h i g h e r p l a n t s . C a n c e r T r e a t . Rep. 60, 1081-1113. B r u n i n g , E . F . 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(1974) W o r l d F o r e s t R e s o u r c e s . R o y a l c o l l e g e of F o r e s t r y , S t o c k h o l m , Sweden, R e s e a r c h N o t e s No. 17, i i i . P e r s s o n , R. (1975) F o r e s t R e s o u r c e s o f A f r i c a , P a r t I . C o u n t r y  D e s c r i p t i o n s . Department o f F o r e s t S u r v e y , R o y a l C o l l e g e o f F o r e s t r y , S t o c k h o l m , Sweden, R e s e a r c h N o t e s No. 18. P e r s s o n , R. (1977) F o r e s t R e s o u r c e s of A f r i c a , P a r t I I . R e g i o n a l A n a l y s i s . Dep a r t m e n t of S u r v e y , R o y a l C o l l e g e o f F o r e s t r y , S t o c k h o l m , Sweden, R e s e a r c h N o t e s No. 22. P o o r e , M.E.D. (1976) E c o l o g i c a l G u i d e l i n e s f o r Development i n  T r o p i c a l R a i n f o r e s t s . I n t e r n a t i o n a l U n i o n f o r C o n s e r v a t i o n of N a t u r e and N a t u r a l R e s o u r c e s , Morges, S w i t z e r l a n d . 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(1976) The p l a n t kingdom: a v i r g i n f i e l d f o r new b i o d y n a m i c c o n s t i t u e n t s . I n : The  R e c e n t C h e m i s t r y of N a t u r a l P r o d u c t s , I n c l u d i n g T o b a c c o ( F i n a , N .J., ed.) P r o c e e d i n g s of t h e S e c o n d P h i l l i p M o r r i s S c i e n c e Symposium, New Yo r k , pp. 133-171. Shane, D.R. (1980) H o o f p r i n t s on t h e f o r e s t : an e n q u i r y i n t o t h e b e e f c a t t l e i n d u s t r y i n t h e t r o p i c a l f o r e s t a r e a s of L a t i n A m e r i c a . R e p o r t o f O f f i c e of E n v i r o n m e n t a l A f f a i r s , D e partment o f S t a t e , W a s h i n g t o n , D.C. Sommer, A. (1976) A t t e m p t a t an a s s e s s m e n t o f t h e w o r l d ' s t r o p i c a l m o i s t f o r e s t s . U n a s y l v a 28, 5-24. S p j u t , R.W. and P e r d u e , R.E. (1976) P l a n t f o l k l o r e : a t o o l f o r p r e d i c t i n g s o u r c e s of a n t i t u m o u r a c t i v i t y among h i g h e r p l a n t s . C a n c e r T r e a t . Rep. 60, 979-985. S u f f n e s s , M. and D o u r o s , J.D. (1982) C u r r e n t s t a t u s of NCI p l a n t and a n i m a l program. J . N a t . P r o d . 45, 1-14. Swain, T. (1972) The s i g n i f i c a n c e of c o m p a r a t i v e p h y t o c h e m i s t r y i n m e d i c a l b o t a n y . I n : P l a n t s i n t h e Development of Modern M e d i c i n e (Swain T., e d . T T H a r v a r d U n i v e r s i t y P r e s s , C ambridge, Mass. T r o p i c a l D e f o r e s t a t i o n (1980) H e a r i n g s b e f o r e t h e Subcommittee on I n t e r n a t i o n a l O r g a n i z a t i o n s , Committee o f F o r e i g n A f f a i r s , House of R e p r e s e n t a t i v e s , U.S. G o v t . P r i n t i n g O f f i c e , W a s h i n g t o n , D.C. U.S. I n t e r a g e n c y T a s k F o r c e on T r o p i c a l F o r e s t s (1980) The W o r l d ' s T r o p i c a l F o r e s t s : A P o l i c y , S t r a t e g y and Program  f o r t h e U n i t e d S t a t e s . U.S. Department o f S t a t e , W a s h i n g t o n , D.C. UNESCO (1974) I n t e r n a t i o n a l W o r k i n g Group on P r o j e c t I . E c o l o g i c a l E f f e c t s of I n c r e a s i n g Human A c t i v i t i e s on T r o p i c a l and S u b - t r o p i c a l F o r e s t E c o s y s t e m s . F i n a l R e p o r t , Man and t h e B i o s p h e r e (MAB) R e p o r t S e r i e s No. 16, UNESCO, P a r i s , F r a n c e . W e i n s t e i n , I.B., W i g l e r , M. and P i e t r o p a o l o , C . (1977) The a c t i o n of tumour p r o m o t i n g a g e n t s i n c e l l c u l t u r e . C o l d S p r i n g H a r b o u r C o n f e r e n c e o f C e l l P r o l i f e r a t i o n , V o l . 4, Book A. C o l d S p r i n g H a r b o u r , New Y o r k . Y i p , Y.K., Pang, R.H.L., Urban, C. and V i l c e k , J . (1981) P a r t i a l p u r i f i c a t i o n and c h a r a c t e r i z a t i o n o f human a (immune) i n t e r f e r o n . P r o c . N a t l . A c a d . S c i . USA 78, 1601-1605. 30 I I . J u s t i c i a p e c t o r a l i s : A STUDY OF THE BASIS  FOR ITS USE AS A V i r o l a SNUFF ADMIXTURE 1. INTRODUCTION The w i d e s p r e a d use o f t r y p t a m i n e b a s e d h a l l u c i n o g e n i c s n u f f s i n t h e O r i n o c o and n o r t h e r n Amazon d r a i n a g e a r e a s has been w e l l documented d u r i n g t h e p a s t c e n t u r y (Wassen, 1967). Bark r e s i n of s p e c i e s o f V i r o l a ( M y r i s t i c a c e a e ) was r e p o r t e d as one o f t h e s o u r c e s o f t h e s e s n u f f s a s e a r l y a s 1909 (Koc h -G r i i n b e r g , 1909) and t h e i r u s e s a r e now w e l l e s t a b l i s h e d ( S c h u l t e s and H o l m s t e d t , 1968). Not u n t i l 1953, however, was i t known t h a t t h e r e e x i s t e d o t h e r i m p o r t a n t c o n s t i t u e n t s o f V i r o l a b a s e d s n u f f s . The m i s s i o n a r y , B a r k e r ( 1 9 5 3 ) , and t h e a n t h r o p o l o g i s t , Z e r r i e s ( 1 9 6 0 ) , d e s c r i b e d t h e a d d i t i o n o f an h e r b a c e o u s a d d i t i v e t o t h e p u l v e r i z e d r e s i n s n u f f . T h i s p l a n t was l a t e r i d e n t i f i e d as J u s t i c i a p e c t o r a l i s v a r . s t e n o p h y l l a ( A c a n t h a c e a e ) ( S c h u l t e s and H o l m s t e d t , 1968). The a d d i t i o n of i t s d r y , p u l v e r i z e d l e a v e s t o V i r o l a s n u f f s i s now w e l l e s t a b l i s h e d as a c u s t o m w h i c h i s w i d e s p r e a d amongst t h e t r i b e s o f t h e Yanomamo g r o u p ( B r e w e r - C a r i a s and 'Steyermark, 1976; Chagnon e t a l , 1971; P r a n c e , 1972; S c h u l t e s , 1978; S e i t z , 1967). The r e a s o n f o r t h e i n c l u s i o n of J . p e c t o r a l i s i n V i r o l a s n u f f s i s n o t u n d e r s t o o d . S e v e r a l I n d i a n i n f o r m a n t s have s u g g e s t e d t h a t i t i s added b e c a u s e o f i t s aroma ( P r a n c e , 1972; 31 S c h u l t e s and H o l m s t e d t , 1968; S e i t z , 1967). T h e r e i s e v i d e n c e , however, t h a t i t may p o s s e s s o t h e r p r o p e r t i e s ( S c h u l t e s and H o l m s t e d t , 1968). The r e p o r t s t h a t J . p e c t o r a l i s c o n s t i t u t e s t h e s o l e i n g r e d i e n t of a s n u f f and t h a t i t p r o d u c e s a s t a t e o f i n t o x i c a t i o n ( B r e w e r - C a r i a s and S t e y e r m a r k , 1976; Chagnon e_t a l , 1971; P r a n c e , G.T., p e r s . comm.) a r e e s p e c i a l l y i n t e r e s t i n g . The c h e m i s t r y and p h a r m a c o l o g y of t h e a c t i v e c o n s t i t u e n t s of V i r o l a s n u f f s have been w e l l s t u d i e d ( H o l m s t e d t e_t a l , 1980). In c o n t r a s t , n o t h i n g i s known of t h e c h e m i s t r y o f J . p e c t o r a l i s . M o r e o v e r , no i n f o r m a t i o n on t h e c h e m i c a l c o n s t i t u e n t s o f o t h e r A m e r i c a n s p e c i e s of t h i s genus a p p e a r s t o be a v a i l a b l e . T h i s s t u d y o f t h e p o s s i b l e p h y s i o l o g i c a l b a s i s f o r t h e use of J . p e c t o r a l i s was t h e r e f o r e u n d e r t a k e n . 2 - MATERIALS AND METHODS a. P l a n t m a t e r i a l The p l a n t u s e d i n t h i s s t u d y was c o l l e c t e d i n P u c a l l p a , P e r u i n 1981. I t was i d e n t i f i e d a s J u s t i c i a p e c t o r a l i s v a r . s t e n o p h y l l a by D r . T i m o t h y Plowman o f t h e C h i c a g o F i e l d Museum. V o u c h e r s p e c i m e n s (D. McKenna No. 1) have been d e p o s i t e d a t UBC h e r b a r i u m , C h i c a g o F i e l d Museum, U n i v e r s i t y of San M a r c o s h e r b a r i u m , Lima and UNAP h e r b a r i u m , I q u i t o s , P e r u . I t has been p o i n t e d o u t ( S c h u l t e s , R.E., p e r s . comm.) t h a t J u s t i c i a p e c t o r a l i s v a r . s t e n o p h y l l a i s p r o b a b l y a g r o w t h form o f J . p e c t o r a l i s and i t i s u n l i k e l y t h a t t h e d i f f e r e n c e 32 i n name u s e d r e f l e c t s a g e n e t i c d i f f e r e n c e . In k e e p i n g w i t h P r o f . S c h u l t e s s u g g e s t i o n , t h e v a r i e t a l d i s t i n c t i o n i s n o t c o n s i d e r e d i n t h e s u b s e q u e n t r e p o r t . P l a n t s were p r o p a g a t e d v e g e t a t i v e l y and grown under g r e e n h o u s e c o n d i t i o n s . F r e s h l y c u t l e a v e s , f l o w e r s and stems of mature p l a n t s (1150 g) were washed, p l a c e d i n b o i l i n g 100% e t h a n o l and h o m o g e n i z e d . The homogenate was e x t r a c t e d f o u r t i m e s and t h e f i l t r a t e s were com b i n e d and c o n c e n t r a t e d by r o t a r y e v a p o r a t i o n i_n v a c u o . D i s t i l l e d w a ter (500 ml) was a d d e d t o t h e r e s i d u e w h i c h was h e a t e d on a steam b a t h f o r 20 m i n u t e s and f i l t e r e d t h r o u g h c e l i t e . The aqueous f r a c t i o n was e x t r a c t e d c o n t i n u o u s l y f o r 48 h o u r s w i t h d i e t h y l e t h e r , t h e n e t h y l a c e t a t e . The d i e t h y l e t h e r , e t h y l a c e t a t e and aqueous f r a c t i o n s a c c o u n t e d f o r 0.931, 0.478 and 42.3 g, r e s p e c t i v e l y , o f t h e o r i g i n a l 1150 g f r e s h w e i g h t , b. C h r o m a t o g r a p h y and s p e c t r o s c o p y T h i n l a y e r c h r o m a t o g r a p h y (TLC) was c a r r i e d o u t u s i n g P o l y g r a m S i l i c a g e l G U V 2 5 a (B r i n k m a n ) and c e l l u l o s e (Eastman) p r e c o a t e d p l a t e s . H i g h p e r f o r m a n c e l i q u i d c h r o m a t o g r a p h y (HPLC) s e p a r a t i o n s were p e r f o r m e d on a V a r i a n MCH-10 r e v e r s e p h a s e column and V a r i a n M odel 5000 HPLC w i t h V a r i a n S e r i e s 634 v a r i a b l e w a v e l e n g t h UV d e t e c t o r . Gas c h r o m a t o g r a p h y / m a s s s p e c t r o m e t r y (GC/MS) d a t a were o b t a i n e d w i t h a F i n n e g a n 1020 a u t o m a t e d GC/MS. N u c l e a r m a g n e t i c r e s o n a n c e (NMR) s p e c t r a were r e c o r d e d on a B r u k e r FT-100 i n s t r u m e n t and i n f r a r e d (IR) s p e c t r a were o b t a i n e d u s i n g a P e r k i n E l m e r 710B I n f r a r e d 33 Spec t rophotomete r . c . Behav ior exper iments Locomotor a c t i v i t y was measured in mice us ing an adapted v e r s i o n of a " j i g g l e cage " (Robbins , 1977). Female Swiss mice which had been bred and ma in ta ined by the Animal Care C e n t r e , U .B .C . were used . The t e s t substance was a d m i n i s t e r e d by i n t r a p e r i t o n e a l i n j e c t i o n and the mouse p l a c e d in an 18 cm c u b i c wire mesh cage which was suspended from an i s o m e t r i c f o r c e t r ansduce r by e l a s t i c bands. The t r ansduce r s i g n a l was a m p l i f i e d 1000 t imes and moni tored us i ng a S p e c t r o p h y s i c s SP 4100 i n t e g r a t o r . A computer program which sampled the input v o l t a g e approx imate l y every 100 m i l l i s e c o n d s , c a l c u l a t e d the d i f f e r e n c e between s u c c e s s i v e s i g n a l s and summed the abso lu t e va lue of that d i f f e r e n c e over a f i v e minute p e r i o d was w r i t t e n . T h i s appara tus produced an average va lue fo r the degree of movement of the mouse, in a r b i t r a r y u n i t s , at f i v e minute i n t e r v a l s . I t was s e n s i t i v e to both locomotor a c t i v i t y and to f i n e body movements, a l though the former produced a p r o p o r t i o n a t e l y l a r g e r s i g n a l . A l l e x t r a c t s and compounds were a d m i n i s t e r e d t o .m i ce by i n t r a p e r i t o n e a l i n j e c t i o n . Because of i t s a b i l i t y to s o l u b i l i z e both h y d r o p h i l i c and l i p o p h i l i c c o n s t i t u e n t s and i t s low t o x i c i t y (Budden et a_l, 1979), Tween 80 (Eastman Kodak Co . ) was used as a v e h i c l e fo r the subs tances a d m i n i s t e r e d . One hundred nl of a 25% (v/v) aqueous s o l u t i o n of Tween 80 was used in each c a s e . d . Rat stomach s t r i p exper iments The procedure used f o r e v a l u a t i n g the a c t i v i t y 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  c o l i , Staphylococcus aureus, Candida albicans and Saccharomyces cerevisiae were spread on Difco Bacto agar and 35 Sabouraud 's dex t rose agar p l a t e s , r e s p e c t i v e l y , u s i ng c o t t o n swabs. S t e r i l e f i l t e r paper d i s c s , to which the e x t r a c t to be t e s t e d had been a p p l i e d , were p l a c e d on the agar s u r f a c e . The p l a t e s were examined fo r i n h i b i t i o n of growth around the f i l t e r paper d i s c s a f t e r 24 and 48 hou r s , f . A n t i v i r a l t e s t s E x t r a c t s were t e s t e d fo r t h e i r a b i l i t y to i n h i b i t the fo rmat ion of p laques by S i n d b i s v i r u s and murine c y tomega lov i rus (MCMV) in c u l t u r e d mouse c e l l s . E x t r a c t s were i n c l uded in the agarose o ve r l a y a p p l i e d to v i r u s i n f e c t e d c e l l monolayers and the p laques which deve loped were counted 2 and 5 days l a t e r f o r the S i n d b i s and MCMV, r e s p e c t i v e l y . Complete d e t a i l s of the assay are p resen ted in Chapter I I I . 3. RESULTS a . Examinat ion of J u s t i c i a p e c t o r a l i s fo r a l k a l o i d s The r e p o r t s tha t J . ' p e c t o r a l i s i s used , not on ly as an admixture to V i r o l a , but as the so l e i n g r e d i e n t of a snu f f (Brewer-Car ias and Steyermark, 1976; Chagnon et a_l, 1971; P rance , G . T . , p e r s . comm.) suggest tha t t h i s p l a n t a lone may have p s y c h o t r o p i c or even h a l l u c i n o g e n i c a c t i v i t y . The most potent p s y c h o t r o p i c agents are a l k a l o i d s (Ho f fme i s t e r and S t i l l e , 1982; S c h u l t e s , 1970). T h i s f a c t , combined w i th p r e l i m i n a r y ev idence i n d i c a t i n g tha t J . p e c t o r a l i s may c o n t a i n N ,N-d imethy l t ryptamine ( S c h u l t e s , 1970; S c h u l t e s , 1972) i n d i c a t e d tha t the pha rmaco log i c a l a c t i v i t y of J . p e c t o r a l i s 36 may be a t t r i b u t a b l e t o t h e p r e s e n c e of i n d o l e or o t h e r c l a s s e s o f a l k a l o i d s . The t h r e e J . p e c t o r a l i s e x t r a c t s were examined f o r a l k a l o i d s by TLC. Samples were a p p l i e d t o S i l i c a g e l G p l a t e s , d e v e l o p e d w i t h e t h y l e t h e r / 2 - b u t a n o n e / 6 N NH 4OH (5/4/1) ( u p p e r p h a s e ) and s p r a y e d w i t h E h r l i c h ' s r e a g e n t (McKenna e t a l , 1984). N , N - D i m e t h y l t r y p t a m i n e , 5 - O H - N , N - d i m e t h y l t r y p t a m i n e , and 5 - m e t h o x y - N , N - d i m e t h y l t r y p t a m i n e were i n c l u d e d as s t a n d a r d s . No E h r l i c h ' s p o s i t i v e compound c o u l d be d e t e c t e d i n any of t h e t h r e e e x t r a c t s . E a c h e x t r a c t was t e s t e d f o r t h e p r e s e n c e of a l k a l o i d s u s i n g V a l s e r ' s , M e y e r ' s and D r a g e n d o r f f ' s r e a g e n t s f o r p r e c i p i t a t i o n ( M a r t e l l o and F a r n s w o r t h , 1962). O n l y t h e aqueous f r a c t i o n was p o s i t i v e , y i e l d i n g a p r e c i p i t a t e w i t h e a c h of t h e r e a g e n t s . T h i s f r a c t i o n was c h r o m a t o g r a p h e d on c e l l u l o s e t h i n l a y e r p l a t e s u s i n g n - p r o p a n o l / 0 . 3 N NHttOH (19/1) as a d e v e l o p e r . A s i n g l e s p o t w i t h Rf v a l u e 0.46 was o b s e r v e d a f t e r s p r a y i n g w i t h D r a g e n d o r f f ' s r e a g e n t . I t p r o d u c e d a d i s t i n c t s a l m o n - p i n k c o l o u r when s p r a y e d w i t h t h e B r e g o f f - D e l w i c h e m o d i f i c a t i o n o f D r a g e n d o r f f ' s r e a g e n t w h i c h i s s p e c i f i c f o r q u a t e r n a r y ammonium compounds ( S t a h l , 1969) and c a u s e d t h e d e p o s i t i o n o f p l a t i n u m , f o r m i n g a g r a y s p o t w i t h i o d o p l a t i n a t e r e a g e n t f o r a l k a l o i d s ( S t a h l , 1969). b. Compound J_ The compound (1) was p u r i f i e d by column c h r o m a t o g r a p h y on s i l i c a u s i n g n - p r o p a n o l / 0 . 3 N. NHi,OH (3/1) a s e l u a n t and was 37 c r y s t a l l i z e d f r o m aqueous m e t h a n o l . The f o l l o w i n g s p e c t r a l d a t a were o b t a i n e d . Compound J_ ( b e t a i n e ) UV X max11 : 215 nm. IR v max cm" 1: 3250br, 2900, 1650, 1480, 1400, 1330, 980, 930 and 880. 1H-NMR (100 MHz, D 20) 5 : 3.9 (2H, s ) , 3.31 (9H, s ) . The IR s p e c t r u m was i d e n t i c a l t o t h a t r e p o r t e d f o r b e t a i n e ( c a r b o x y m e t h y l - t r i m e t h y l a m m o n i u m h y d r o x i d e i n n e r s a l t ) ( P o u c e r t , 1981) and t h e 100 MHz 1H-NMR s p e c t r u m matched t h a t o f an a u t h e n t i c sample ( S i g m a ) . Compound 1 c o - c h r o m a t o g r a p h e d w i t h b e t a i n e b o t h on c e l l u l o s e TLC and by HPLC ( r e t e n t i o n t i m e = 4.7 min. u s i n g 2% a c e t o n i t r i l e / H 2 0 w i t h a f l o w r a t e of 1 m l / m i n . ) . On t h e b a s i s of t h i s e v i d e n c e , i t was c o n c l u d e d t h a t compound 1 was b e t a i n e ( F i g u r e 2 ) . c. B e h a v i o r a l e f f e c t s o f J u s t i c i a p e c t o r a l i s The f i r s t a p p r o a c h t o t h e e t h n o p h a r m a c o l o g y of J . p e c t o r a l i s e s t a b l i s h e d t h a t p s y c h o t r o p i c a l k a l o i d s d i d n o t a c c o u n t f o r t h e use of t h i s p l a n t a s a s n u f f . T h i s d i d n o t e l i m i n a t e t h e p o s s i b i l i t y t h a t a n o n - a l k a l o i d a l p s y c h o t r o p i c compound m i g h t be p r e s e n t . To a p p r o a c h t h i s q u e s t i o n , b e h a v i o r a l c h a n g e s i n mi c e i n j e c t e d w i t h J . p e c t o r a l i s e x t r a c t s were o b s e r v e d . A v a r i e t y o f b e h a v i o r a l c h a n g e s a r e known t o accompany t h e a d m i n i s t r a t i o n of p s y c h o t r o p i c a g e n t s t o r o d e n t s ( H o f f m e i s t e r and S t i l l e , 1982). A l t e r a t i o n s i n 38 F i g u r e 2 - S t r u c t u r e s of b e t a i n e and u m b e l l i f e r o n e ( 3 ) , J u s t i c i a p e c t o r a l i s . ( 1 ) , coumar i n (2) compounds i s o l a t e d from 39 spontaneous locomotor a c t i v i t y are commonly observed ( H o l l i s t e r , 1982). The aqueous, e t h y l a ce t a t e and d i e t h y l e ther e x t r a c t s of J . p e c t o r a l i s were a d m i n i s t e r e d i n t r a p e r i t o n e a l l y to mice at a dose of 250 mg/kg. B e h a v i o r a l changes were observed and locomotor a c t i v i t y was r e c o r d e d . The r e s u l t s of the spontaneous locomotor r e c o r d i n g are p resen ted in Tab le I I . Each e x t r a c t caused a r e d u c t i o n in a c t i v i t y . Mice adopted a huddled p o s t u r e , o f t e n wi th eyes c l o s e d , and d i s p l a y e d p i l o e r e c t i o n . The r e c o r d i n g of a c t i v i t y l e v e l p rov i des a q u a n t i t a t i v e es t imate of the r e d u c t i o n in a c t i v i t y . The e t h y l a ce t a t e f r a c t i o n possessed the s t r onges t of the r e l a t i v e l y weak a c t i v i t i e s of the th ree e x t r a c t s . None of the responses observed were i n d i c a t i v e of the presence of p s y c h o t r o p i c a c t i v i t y . d . Ef f e c t of J us t i c i a p e c t o r a l i s e x t r a c t s on 5-MeODMT  induced b e h a v i o r a l responses S ince no ev idence was ob t a i ned to i n d i c a t e tha t J . p e c t o r a l i s , a l o n e , has p s y c h o t r o p i c a c t i v i t y , i t s r o l e when used in combina t ion wi th V i r o l a snu f f was c o n s i d e r e d . There i s s t rong ev idence that the use of V i r o l a based s n u f f s i s based on the h a l l u c i n o g e n i c a c t i v i t y of t h e i r i ndo l e c o n s t i t u e n t s . N ,N-Dimethy l t ryptamine (DMT) and 5-methoxy-N,N-d ime thy l t r yp t am ine (5-MeODMT) a r e , q u a n t i t a t i v e l y , the most important i n d o l e s in most V i r o l a barks ( A g u r e l l et a l , 1969; Holmstedt et a l , 1980; McKenna et a l , 1984). At l e a s t one of these two c o n s t i t u e n t s i s known from a l l of the V i r o l a s p e c i e s used i n the manufacture of s n u f f s and t h e i r presence in the 40 % n o r m a l a c t i v i t y l e v e K m i n . p o s t i n j e c t i o n ) E x t r a c t t I n j e c t e d 5 1 0 1 5 20 25 30 35 40 45 50 55 60 aqueous 93 91 1 04 92 95 86 85 88 77 79 75 77 e t h y l a c e t a t e 96 1 07 101 64 52 61 58 44 53 57 51 59 d i e t h y l e t h e r 92 94 98 91 94 87 83 88 77 82 75 88 25% Tween 80 ( 1 00 / i l ) 91 96 1 1 0 1 04 92 94 109 1 1 2 95 99 1 1 4 91 T a b l e II - E f f e c t of J u s t i c i a p e c t o r a l i s e x t r a c t s on s p o n t a n e o u s l o c o m o t o r a c t i v i t y i n m i c e . f E x t r a c t s were i n j e c t e d i n t r a p e r i t o n e a l l y a t d o s e s o f 250 mg/kg d i s s o l v e d i n 100 M1 o f 25% Tween 80. 41 s n u f f s t h e m s e l v e s i s w e l l documented ( A g u r e l l et_ aj^, 1969; H o l m s t e d t and L i n d g r e n , 1967). Five-MeODMT i s a p p r o x i m a t e l y t e n t i m e s a s p o t e n t a p s y c h o t o m i m e t i c a s DMT ( S h u l g i n , 1982). I t would, t h e r e f o r e , be e x p e c t e d t o a c c o u n t f o r most of t h e p h a r m a c o l o g i c a l e f f e c t s of t h e s n u f f s . T h i s t r y p t a m i n e was c h o s e n as a model compound f o r c o - a d m i n i s t r a t i o n s t u d i e s d e s i g n e d t o t e s t t h e h y p o t h e s i s t h a t some component of J . p e c t o r a l i s was r e s p o n s i b l e f o r m o d u l a t i n g t h e e f f e c t s o f t h e t r y p t a m i n e s . Five-MeODMT i s known t o c a u s e a number o f c h a r a c t e r i s t i c b e h a v i o r a l c h a n g e s i n r a t s i n j e c t e d i n t r a p e r i t o n e a l l y . Doses of 1 mg/kg p r o d u c e e x c i t a t i o n , h y p e r a c t i v i t y , t r e m o r and some s a l i v a t i o n ( A h l b o r g e t al . , 1968; Grahame-Smith, 1971). The t r e m o r p r o d u c e d by t h i s compound i s d o s e - d e p e n d e n t i n t h e ra n g e 1-10 mg/kg ( A h l b o r g e_t a_l, 1968). Changes i n a c t i v i t y l e v e l s p r o d u c e d i n r a t s by 5-MeODMT ( A h l b o r g e t a l , 1968; Grahame-Smith, 1971) and i n mice by DMT (Shah and Hedden, 1978) have been q u a n t i f i e d i n d e v i c e s f o r m e a s u r i n g a n i m a l a c t i v i t y l e v e l s . The r e s p o n s e s have been shown t o be r e p r o d u c i b l e and t o v a r y i n a d o s e - d e p e n d e n t manner. T h i s method was t h e r e f o r e c h o s e n t o examine t h e p o s s i b i l i t y t h a t J . p e c t o r a l i s c o n t a i n s some c o n s t i t u e n t t h a t a l t e r s t h e b e h a v i o r a l r e s p o n s e t o 5-MeODMT. e. E f f e c t of 5-MeODMT on mouse a c t i v i t y S o l u t i o n s of 5-MeODMT (Sigma) were d i s s o l v e d i n 2% aqueous Tween 80 (100 M D and a d m i n i s t e r e d i n t r a p e r i t o n e a l l y . A c t i v i t y l e v e l s were m o n i t o r e d a t 5 m i n u t e i n t e r v a l s f o r 60 42 m i n u t e s a f t e r i n j e c t i o n . The r e s u l t s p r e s e n t e d i n F i g u r e 3 show t h a t a l l d o s e s of 5-MeODMT t e s t e d (0.31 t o 12.5 mg/kg) p r o d u c e d an i n i t i a l p e r i o d o f h y p e r a c t i v i t y l a s t i n g 10 t o 20 m i n u t e s . The d e g r e e of h y p e r a c t i v i t y v a r i e d i n a d o s e -d e p e n d e n t manner. In t h e c a s e of a low (0.31 mg/kg) o r a h i g h (2.5 or 12.5 mg/kg) dose of 5-MeODMT, t h e a c t i v i t y l e v e l s r e t u r n e d a l m o s t t o normal a f t e r 30 m i n u t e s p o s t i n j e c t i o n . A d m i n i s t r a t i o n o f i n t e r m e d i a t e d o s e s (1.25 and 0.625 mg/kg; d a t a n o t shown), i n c o n t r a s t , r e s u l t e d i n s i g n i f i c a n t r e d u c t i o n s i n a c t i v i t y w h i c h p e r s i s t e d u n t i l a t l e a s t 60 m i n u t e s p o s t i n j e c t i o n . A h l b o r g e t a_l (1968) and Grahame-Smith (1971) o b s e r v e d v e r y s i m i l a r i n c r e a s e s i n a c t i v i t y i n r a t s i n j e c t e d w i t h 3 and 1 mg/kg of 5-MeODMT, r e s p e c t i v e l y . The r e d u c t i o n i n a c t i v i t y o b s e r v e d i n t h e p r e s e n t s t u d y (50% n o r m a l a c t i v i t y l e v e l a f t e r 1.25 mg/kg and 70% n o r m a l a c t i v i t y l e v e l a f t e r 0.625 mg/kg 5-MeODMT; d a t a n ot shown) have n o t p r e v i o u s l y been r e p o r t e d a s an e f f e c t o f 5-MeODMT a d m i n i s t r a t i o n . D i m e t h y l t r y p t a m i n e , however, has been shown t o p r o d u c e a s i m i l a r p a t t e r n o f g r a d u a l l y d e c r e a s i n g a c t i v i t y l e v e l s f o l l o w i n g t h e a d m i n i s t r a t i o n o f 2.5 and 25 mg/kg (Grahame-Smith, 1971). The r e d u c t i o n i n a c t i v i t y was o b s e r v e d by them t o l a s t f o r a p p r o x i m a t e l y 60 and 120 m i n u t e s , r e s p e c t i v e l y . The r e s u l t s p r e s e n t e d h e r e i n d i c a t e t h a t low d o s e s o f 5-MeODMT p r o d u c e a s i m i l a r r e d u c t i o n i n motor a c t i v i t y b u t t h a t , a t h i g h d o s e s , a c t i v i t y l e v e l s i n c r e a s e m a r k e d l y ( 3 - f o l d a t 12.5 mg/kg). 43 "1 1 1 1 1 T 10 20 30 i.0 50 60 TIME AFTER INJECTlON(minutes) F i g u r e 3 - E f f e c t o f 5-MeODMT on s p o n t a n e o u s l o c o m o t o r a c t i v i t y of m i c e . M i c e were i n j e c t e d w i t h 5-MeODMT a t t i m e 0 and l o c o m o t o r a c t i v i t y was r e c o r d e d d u r i n g f i v e m i n u t e i n t e r v a l s t h e r e a f t e r . A d m i n i s t r a t i o n of v e h i c l e a l o n e ( c o n t r o l ) i s not shown: maximum d e v i a t i o n from n o r m a l a c t i v i t y l e v e l o b s e r v e d was 22%. V a l u e s a r e means, N=3; SEMs a r e not drawn b ut were a l l l e s s t h a n 5%. 44 f . G r o s s b e h a v i o r a l e f f e c t s of 5-MeODMT Changes i n b e h a v i o r i n d u c e d by 5-MeODMT c o r r e l a t e w e l l w i t h t h e e f f e c t s on motor a c t i v i t y p r e s e n t e d i n F i g u r e 3. A dose of 0.31 mg/kg r e s u l t e d i n an i n i t i a l p e r i o d of h y p e r a c t i v i t y l a s t i n g a p p r o x i m a t e l y 10 m i n u t e s , f o l l o w e d by a p e r i o d of r o u g h l y 60 m i n u t e s d u r i n g w h i c h t h e a n i m a l l a y q u i e t l y , d i s p l a y i n g a f l a t t e n e d p o s t u r e . As Shah and Hedden (1978) r e p o r t e d f o r DMT, f r i g h t r e s p o n s e s were e l i c i t e d when t h e a n i m a l was d i s t u r b e d . At s t i l l h i g h e r d o s e s (2.5 and 12.5 mg/kg), t h e mi c e a d o p t e d an e x t r e m e l y f l a t t e n e d p o s t u r e w i t h e x t e n s i o n of t h e h i n d l e g s and showed a s e r i e s o f m u l t i p l e sudden backwards movements. T h i s b e h a v i o r was p e r i o d i c a l l y and b r i e f l y i n t e r r u p t e d when t h e a n i m a l s c r o u c h e d and pawed t h e i r n o s e s . G r o s s l o c o m o t o r a c t i v i t y was r e d u c e d b u t t h e number of f i n e body movements were g r e a t l y i n c r e a s e d , compared t o an u n t r e a t e d a n i m a l . J e r k i n e s s , head t w i t c h i n g , r i g i d i t y and t r e m b l i n g , c h a r a c t e r i s t i c a l l y p r o d u c e d i n mi c e by DMT (Shah and Hedden, 1978), were a l s o o b s e r v e d . A f t e r 15-20 m i n u t e s p o s t i n j e c t i o n , a l l o f t h e s e b e h a v i o r a l e f f e c t s g r a d u a l l y began t o be r e d u c e d i n f r e q u e n c y and s e v e r i t y . Normal a c t i v i t y l e v e l s were s l o w l y r e g a i n e d . g. E f f e c t of c o - i n j e c t i o n s of 5-MeODMT and J u s t i c i a p e c t o r a l i s  e x t r a c t s A d ose o f 5-MeODMT w h i c h w o u l d p r o d u c e a marked e l e v a t i o n i n a c t i v i t y l e v e l was s e l e c t e d t o examine t h e J . p e c t o r a l i s e x t r a c t s f o r s y n e r g i s t i c e f f e c t s . I n j e c t i o n s of 5 mg/kg 5-45 MeODMT, combin e d w i t h 250 mg/kg o f e a c h of t h e t h r e e J u s t i c i a e x t r a c t s , were made and t h e a c t i v i t y l e v e l s r e c o r d e d . The r e s u l t s a r e p r e s e n t e d i n T a b l e I I I . D e s p i t e t h e f a c t t h a t a l o n e , e a c h e x t r a c t c a u s e d s l i g h t r e d u c t i o n s i n a c t i v i t y l e v e l , t h e r e was no s i g n i f i c a n t e f f e c t of any o f t h e J u s t i c i a e x t r a c t s upon 5-MeODMT i n d u c e d h y p e r a c t i v i t y . The o b s e r v a t i o n s of t h e g r o s s b e h a v i o r a l r e s p o n s e s were i n agreement w i t h t h e l o c o m o t o r measurements. A l l of t h e p r e v i o u s l y m e n t i o n e d b e h a v i o r a l e f f e c t s of h i g h d o s e s of 5-MeODMT were o b s e r v e d a f t e r a d m i n i s t r a t i o n of 5 mg/kg 5-MeODMT, e i t h e r a l o n e , o r i n c o m b i n a t i o n w i t h any of t h e t h r e e J u s t i c i a e x t r a c t s . From t h i s s e r i e s of e x p e r i m e n t s i t i s c o n c l u d e d t h a t t h e J . p e c t o r a l i s e x t r a c t s do not c o n t a i n any c o n s t i t u e n t w i t h a p h a r m a c o l o g i c a l a c t i v i t y t h a t i s c o m p a r a b l e t o - t h a t o f t h e t r y p t a m i n e h a l l u c i n o g e n s . Nor do t h e y c o n t a i n compounds w h i c h a r e r e s p o n s i b l e f o r m o d u l a t i n g t h e most o b v i o u s b e h a v i o r a l c h a n g e s i n d u c e d by 5-MeODMT. C o n s t i t u e n t s w h i c h i n h i b i t t h e enzyme, monoamine o x i d a s e ( B h a t t a c h a r y a ejb a_l, 1976; H o l m s t e d t e t a l , 1980) and mixed f u n c t i o n o x i d a s e s ( B r a t t s t e n , 1979) a r e known t o o c c u r i n h i g h e r p l a n t s . A l t h o u g h t h e e x i s t i n g d a t a i n d i c a t e t h a t t h e f o r m e r i s t h e most i m p o r t a n t enzyme s y s t e m f o r c o n v e r t i n g t r y p t a m i n e s t o i n a c t i v e m e t a b o l i t e s (Grahame-S m i t h , 1971), t h e r e i s e v i d e n c e t o s u g g e s t t h a t t h e l a t t e r a l s o p l a y s a r o l e ( A h l b o r g , 1968). P l a n t c o n s t i t u e n t s w i t h t h e a b i l i t y t o i n h i b i t e i t h e r enzyme s y s t e m c o u l d p o s s i b l y p o t e n t i a t e t h e p h y s i o l o g i c a l a c t i v i t y of t r y p t a m i n e s . The r e s u l t s o b t a i n e d i n t h e b e h a v i o r e x p e r i m e n t s do n o t s u p p o r t 4 6 Per cent Normal t Spontaneous Locomotor A c t i v i t y (Minutes Post I n j e c t i o n ) Sample I n j e c t e d 5 1 0 1 5 2 0 2 5 3 0 5 mg/kg 5-MeODMT + 2 5 0 mg/kg aqueous e x t r a c t 1 7 6 2 0 4 2 0 4 1 2 8 1 0 8 1 0 5 5 mg/kg 5-MeODMT + 2 5 0 mg/kg e t h y l a ce t a t e e x t . 1 5 5 1 9 4 2 0 4 1 2 6 1 0 1 9 5 5 mg/kg 5-MeODMT + 2 5 0 mg/kg e t h y l e ther e x t . 1 6 4 1 9 8 2 1 5 1 3 4 1 1 8 1 0 4 5 mg/kg 5-MeODMT 1 7 0 2 1 4 2 1 7 1 2 1 1 0 9 1 0 8 1 0 0 M1 2 5 % Tween 8 0 +• 9 2 1 0 4 1 0 0 9 5 1 0 7 1 0 1 Tab le III - E f f e c t of c o - a d m i n i s t r a t i o n of 5-MeODMT and Jus t i c i a p e c t o r a l i s e x t r a c t on spontaneous motor a c t i v i t y of m ice . t Normal spontaneous locomotor a c t i v i t y was e s t a b l i s h e d fo r each mouse by measur ing a c t i v i t y f o r 10 minutes p r i o r to i n j e c t i o n . +• C o n t r o l . 47 t h i s h ypo thes i s f o r J . p e c t o r a l i s . Any i n c r ease in the h a l f l i f e of 5-MeODMT would be expected to be man i f e s t ed in the b e h a v i o r a l responses obse r ved . Ne i t he r the magnitude nor the d u r a t i o n of 5-MeODMT induced b e h a v i o r a l changes was a f f e c t e d by any of the three J . p e c t o r a l i s e x t r a c t s , h. E f f e e t of J . p e c t o r a l i s e x t r a c t s on smooth muscle D imethy l t r yp tamine and other t r yp tamine d e r i v a t i v e s are known to an tagon ize the a c t i o n of 5-hydroxytryptamine ( s e ro ton in ) on smooth muscle (Barlow and Khan, 1959). T h i s p h a r m a c o l o g i c a l a c t i v i t y c o r r e l a t e s wi th h a l l u c i n o g e n i c a c t i v i t y , sugges t ing that s e r o t o n i n r ecep to r a f f i n i t y i s c e n t r a l to the mechanism of a c t i o n of these agents as h a l l u c i n o g e n s (Glennon et_ a l , 1980). The p o s s i b i l i t y that J . p e c t o r a l i s c o u l d exer t some s u b t l e b e h a v i o r a l e f f e c t by e i t h e r a c t i n g d i r e c t l y on the s e r o t o n i n r e c e p t o r s or by compet ing fo r them wi th t r yp t am ines , was c o n s i d e r e d . The ra t stomach s t r i p assay fo r s e r o t o n i n r e c e p t o r s a f f o r d e d a r e l a t i v e l y easy means of t e s t i n g t h i s h y p o t h e s i s . A s t r i p of smooth muscle p repared from the f und i c r eg ion of a ra t stomach responds to low c o n c e n t r a t i o n s of 5-hydroxy t ryp tamine by c o n t r a c t i n g (Vane, 1957). F i g u r e 4a shows a t r a c i n g of the l eng th of such a ra t stomach s t r i p (RSS) exposed to 1 ng/ml 5-hydroxytryptamine ( s e r o t o n i n ) . W i th in two minutes of t reatment wi th s e r o t o n i n , the muscle reaches i t s minimum l eng th ( co r respond ing to a 2% r e d u c t i o n in t o t a l l e n g t h ) . The muscle r e l a x e s comp le t e l y w i th i n two minutes of removal of s e r o t o n i n . T h i s response i s q u i t e r e p r o d u c i b l e and 48 F i g u r e 4 - E f f e c t of (a) s e r o t o n i n ( i n g / m l ) and (b) b e t a i n e (50 yg/ml) on smooth m u s c l e . I n c r e a s e i n e l e v a t i o n of t r a c i n g c o r r e s p o n d s t o c o n t r a c t i o n m u s c l e . I n t e r v a l o f p e r f u s i o n of compound i s d e l i m i t e d a r r o w s : c h a r t speed= 6 mm/min: a m p l i f i c a t i o n = 16X. 49 the degree of c o n t r a c t i o n v a r i e s in a dose-dependent manner. Muscle p r e p a r a t i o n s were pe r fused w i th 1 ng/ml 5-hydroxy t ryp tamine combined wi th one of the th ree J . p e c t o r a l i s e x t r a c t s at a c o n c e n t r a t i o n of .1 mg/ml. The e ther e x t r a c t comp le te l y e l i m i n a t e d the 5-hydroxy t ryp tamine induced c o n t r a c t i o n and produced a marked r e l a x a t i o n of the musc l e . When the RSS was exposed to the e ther e x t r a c t a l o n e , however, a s i m i l a r r e l a x a t i o n was obse r ved . In c o n t r a s t , the aqueous e x t r a c t a lone e l i c i t e d a s t rong and long l a s t i n g c o n t r a c t i o n at a c o n c e n t r a t i o n of 1 mg/ml. The spontaneous a c t i v i t i e s observed in the e ther and aqueous f r a c t i o n s rendered an examinat ion of these e x t r a c t s fo r s p e c i f i c 5-hydroxytryptamine antagonism i m p r a c t i c a l . The e t h y l a c e t a t e f r a c t i o n produced no e f f e c t on the response of the RSS to 5-hydroxyt ryptamine . A knowledge of the nature of the c o n s t i t u e n t s r e s p o n s i b l e fo r the a c t i v i t y on the smooth muscle p r e p a r a t i o n s may shed l i g h t on the pha rmaco log i c a l a c t i v i t y of J . p e c t o r a l i s . The aqueous and d i e t h y l e ther f r a c t i o n s were f r a c t i o n a t e d f u r t h e r in an attempt to i s o l a t e and i d e n t i f y the a c t i v e compounds. Aqueous f r a c t i o n The aqueous f r a c t i o n was f r a c t i o n a t e d as d e s c r i b e d in s e c t i o n 2a . It was observed that c o n t r a c t i o n i nduc i ng a c t i v i t y was r e t a i n e d on l y by those f r a c t i o n s c o n t a i n i n g the qua te rnary ammonium compound, b e t a i n e , which had a l r e ady been i d e n t i f i e d because of i t s r e a c t i o n wi th reagents fo r a l k a l o i d d e t e c t i o n . 50 A c o m m e r c i a l sample of b e t a i n e h y d r o c h l o r i d e (Sigma) was a s s a y e d f o r a c t i v i t y on t h e RSS. I t i n d u c e d a s t r o n g and p r o l o n g e d c o n t r a c t i o n . T h i s e f f e c t was o b s e r v e d a t r e l a t i v e l y h i g h c o n c e n t r a t i o n s (100 Mg/ml) ( F i g u r e 4 b ) . C o n t r a c t i o n s were e l i c i t e d a t c o n c e n t r a t i o n s as low as 10 Mg/ml, but a t l e v e l s l o w e r t h a n t h i s , t h e c o n t r a c t i o n s o b s e r v e d were s l i g h t and n o t r e p r o d u c i b l e . The c o n c e n t r a t i o n of b e t a i n e p r e s e n t i n t h e aqueous e x t r a c t o f J . p e c t o r a l i s was e s t i m a t e d , by c o m p a r i s o n o f TLC s p o t s i z e w i t h measured amounts of p u r e b e t a i n e , t o be 2.4%. A l l o f t h e smooth m u s c l e c o n t r a c t i o n i n d u c e d by t h e aqueous e x t r a c t was a t t r i b u t a b l e t o t h e p r e s e n c e of t h i s amount of b e t a i n e . E t h e r f r a c t i o n An a l i q u o t o f t h e e t h e r f r a c t i o n was f r a c t i o n a t e d by column c h r o m a t o g r a p h y on s i l i c a u s i n g a c h l o r o f o r m / m e t h a n o l g r a d i e n t as t h e e l u a t e . Smooth m u s c l e r e l a x i n g ( s p a s m o l y t i c ) a c t i v i t y was o b s e r v e d t o be c o n c e n t r a t e d i n two f r a c t i o n s . C r y s t a l l i n e compounds were o b t a i n e d from t h e s e f r a c t i o n s . When a p p l i e d t o t h e RSS, e a c h compound, a t c o n c e n t r a t i o n s between 10 and 100 Mg/ml, c a u s e d a marked i n c r e a s e i n t h e l e n g t h of t h e m u s c l e . The RSS r a p i d l y r e t u r n e d t o i t s r e s t i n g l e n g t h f o l l o w i n g t h e r e m o v a l of t h e compounds from t h e medium ( F i g u r e s 5a and b ) . The f o l l o w i n g s p e c t r a l d a t a were o b t a i n e d f o r t h e two compounds i s o l a t e d f r o m t h e d i e t h y l e t h e r f r a c t i o n . 51 Compound 2 ( c o u m a r i n ) . Mp 67-69 °C. UV X max nm ( l o g e ) : 310 ( 3 . 7 0 ) , 273 ( 3 . 7 0 ) . MS m/z ( r e l . i n t ) : 146[M +] ( 7 2 ) , 118 ( 1 0 0 ) , 90 ( 5 2 ) , 89 ( 5 3 ) , 64 ( 1 2 ) , 63 ( 4 1 ) , 62 ( 1 6 ) , 51 ( 1 3 ) , 50 ( 1 2 ) . MeOH Compound 3 ( u m b e l l i f e r o n e ) . Mp 223-225 °C. UV Xmax nm ( l o g e ) : 325 ( 4 . 2 2 ) , 254 ( s h ) , 242 ( s h ) . IR v ^ x cm" 1: 3200 b r , 1720, 1620, 1575, 1420, 1330, 1240, 1150, 910, 850. 1H- NMR (100 MHz, a c e t o n e - D - 6 ) 5 : 7.86 ( I H , 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 ( r e l i n t ) : 162[M +] ( 6 7 ) , 134 ( 7 4 ) , 105 ( 3 9 ) , 97 ( 1 8 ) , 95 ( 1 4 ) , 91 ( 1 4 ) , 85 ( 1 3 ) , 83 ( 1 4 ) , 78 ( 3 5 ) , 69 ( 3 8 ) , 57 ( 3 2 ) , 55 ( 4 0 ) , 51 ( 3 2 ) , 43 ( 1 0 0 ) . On t h e b a s i s o f t h e m e l t i n g p o i n t s and t h e s p e c t r a l d a t a o b t a i n e d , Compounds 2 and 3 were i d e n t i f i e d as c o u m a r i n and 7-h y d r o x y c o u m a r i n ( u m b e l l i f e r o n e ) , r e s p e c t i v e l y ( F i g u r e 2 ) . They c o - c h r o m a t o g r a p h e d w i t h a u t h e n t i c s t a n d a r d s on s i l i c a g e l ( t o l u e n e / a c e t o n e , 1/1) and by h i g h p e r f o r m a n c e l i q u i d c h r o m a t o g r a p h y (50% aqueous a c e t o n i t r i l e ) , c o n f i r m i n g t h e i r i d e n t i t y . i . A n a l y s i s of a r o m a t i c c o n s t i t u e n t s A p o s s i b l e e x p l a n a t i o n f o r t h e i n c l u s i o n of J . p e c t o r a l i s i n V i r o l a s n u f f i s t h a t i t c o n t r i b u t e s a d e s i r a b l e aroma ( P r a n c e , 1972; S c h u l t e s and H o l m s t e d t , 1968; S e i t z , 1967). A f r a g r a n t n a t u r e i s i n d i c a t e d by one of i t s common names, " J a m a i c a g a r d e n b a l s a m " ( S c h u l t e s and H o l m s t e d t , 1968). To F i g u r e 5 - E f f e c t of (a) c o u m a r i n (10 Mg/ml) and (b) u m b e l 1 i f e r o n e (10 ug/ml) on smooth m u s c l e . I n t e r v a l of p e r f u s i o n of compounds i s d e l i m i t e d by a r r o w s : c h a r t speed= 6 mm/min: a m p l i f i c a t i o n = 16X. 53 e s t a b l i s h the nature of the v o l a t i l e c o n s t i t u e n t s r e s p o n s i b l e fo r i t s aroma, samples of f r e s h and d r i e d l eaves and stems of J . p e c t o r a l i s were sub j e c t ed to steam d i s t i l l a t i o n fo r 2 h o u r s . The d i s t i l l a t e was ana l yzed by GC/MS. In the case of each sample, on ly a s i n g l e c o n s t i t u e n t was p r e s e n t . T h i s was i d e n t i f i e d , by i t s mass spect rum, as coumar in . The aroma of J . p e c t o r a l i s appears to be a r e s u l t of the s i n g l e c o n s t i t u e n t , coumar in . j . Quant i f i c a t i o n of coumarins of J . p e c t o r a l i s The e ther e x t r a c t c o n t a i n e d , as pr imary c o n s t i t u e n t s , coumarin and u m b e l l i f e r o n e . To p rov i de q u a n t i t a t i v e i n f o r m a t i o n on the presence of these two important m e t a b o l i t e s , v a r i o u s samples of J . p e c t o r a l i s were ana l yzed by HPLC. Samples of young, green l e a v e s , mature, p igmented l eaves and f l owers were e x t r a c t e d e x h a u s t i v e l y wi th methano l . A sample of l eaves c o l l e c t e d from the p l a n t whi le i t was growing in Peru was a l s o a n a l y s e d . The methano l i c e x t r a c t s were evapora ted ir\ vacuo and p a r t i t i o n e d between water and d i ch lo rome thane . The l a t t e r f r a c t i o n was e vapo ra t ed , d i s s o l v e d in methanol and ana l y sed by HPLC. The sample was separa ted us i ng 50% aqueous methanol ( f low ra te = 1ml/min) and the absorbance was mon i tored at 250 nm. The l e a f e x t r a c t s c o n t a i n e d as major UV abso rb ing c o n s t i t u e n t s , coumarin and u m b e l l i f e r o n e ( F igu re 6 ) . Samples were q u a n t i f i e d by compar ison w i th peak h e i g h t s of chromatograms of a u t h e n t i c samples of coumarin ( J .T . Baker Company) and u m b e l l i f e r o n e (K and K L a b o r a t o r i e s , L t d . ) . The 54 F i gu re 6 - HPLC chromatogram of the o rgan i c f r a c t i o n (methanol i c e x t r a c t ) of Just i c i a p e c t o r a l i s . Flow ra te Iml/min, i s o c r a t i c , 50% MeOH, d e t e c t i o n at 254 nm, cha r t speed= 0.5 cm/min. Compound with r e t e n t i o n time of 5.91 i s u m b e l l i f e r o n e ; that wi th r e t e n t i o n time of 8.62 i s coumar in . 55 r e s u l t s are p resen ted in Tab le IV. The importance of coumarin and u m b e l l i f e r o n e as secondary m e t a b o l i t e s of t h i s s p e c i e s of J u s t i c i a i s e v i d e n t . Not on ly are they present at c o n c e n t r a t i o n s s e v e r a l o rde r s of magnitude h igher than the o ther UV abso rb ing compounds of the e x t r a c t , but they appear , from t h i s l i m i t e d samp l i ng , to be " seques t e r ed in the p l a n t and reach h igher l e v e l s in more mature l e a v e s , k. Examinat ion of J . p e c t o r a l i s fo r l i g n a n s Of the seven spec i e s of Jus t i c i a that have p r e v i o u s l y been ana l y sed c h e m i c a l l y , s i x are known to c o n t a i n l i g n a n s as major c o n s t i t u e n t s (Ghosal and Baner jee , 1979; Ghosa l e_t a l , 1980; Munakata et a l , 1965; Ohta and Munakata, 1970; Okigawa et a l , 1970; O l a n i y i , 1982; O l a n i y i and Powe l l , 1980). A l l but one of the 21 l i g n a n s i d e n t i f i e d from Jus t i c i a s p e c i e s con t a i n " the methy lened ioxypheny l mo ie t y . To determine whether l i g n a n s c o n t a i n i n g t h i s moiety were present in J . p e c t o r a l i s , the chromot roph ic a c i d spray reagent d e s c r i b e d by Beroza (1963) was used to examine TLC s e p a r a t i o n s of the th ree J u s t i c i a f r a c t i o n s . Only one compound, p resen t in the d i e t h y l e ther f r a c t i o n , r eac t ed wi th t h i s r eagen t . I t was i s o l a t e d and s p e c t r a l da ta were o b t a i n e d . I t s m e l t i n g po in t (132-135 ° C ) , 'H-NMR, UV and mass spectrum were i d e n t i c a l to those ob ta ined f o r an a u t h e n t i c sample of /3-s i tos te ro l (S igma). Fu r the rmore , the behav io r of the unknown compound on two chromatograph ic systems ( TLC : Rf=0.45 on S i l i c a g e l deve loped w i th ch lo ro fo rm/ace tone (95/5) and HPLC: Re ten t i on time = 6.10 min e l u t e d w i th 25% aqueous methano l , f low ra te = 1 ml/min. ) was 5 6 Sample Ana l yzed C o n c e n t r a t i o n in Per Cent Dry Weight t Coumar in Umbe l l i fe rone f lowers N.D. t- N.D. young l eaves 0 . 4 2 ( 0 . 0 4 ) 0 . 3 0 ( 0 . 0 4 ) mature l eaves 1 . 1 8 ( 0 . 0 8 ) 0 . 5 8 ( 0 . 0 5 ) l eaves (Pe ru ) 0 . 9 3 ( 0 . 0 5 ) 0 . 4 4 ( 0 . 0 4 ) T Tab le IV - L e v e l s of coumar in and u m b e l l i f e r o n e in d i f f e r e n t samples of Jus t i c i a p e c t o r a l i s . t F i g u r e s are the average of th ree d e t e r m i n a t i o n s ; s tandard d e v i a t i o n of mean in b r a c k e t s . $ N.D. = Not D e t e c t a b l e 57 i d e n t i c a l to that of an au then t i c sample of 0 - s i t o s t e r o l (S i gma). The d i e t h y l e ther and e t h y l a c e t a t e f r a c t i o n s of J . p e c t o r a l i s were examined fo r the presence of j u s t i c i d i n B and d i p h y l l i n , l i g n a n s known from s e v e r a l o ther Jus t i c i a s p e c i e s . Samples were chromatographed on S i l i c a ge l G us ing to luene/ace tone (1/1) as d e v e l o p e r . D i p h y l l i n and j u s t i c i d i n B ( g i f t s of D r . G .H . S h e r i h a , E l Fa teh U n i v e r s i t y ) had Rf va lues of 0.57 and 0 .79 , r e s p e c t i v e l y . Both d i s p l a y e d a s t rong b lue f l u o r e s c e n c e and r eac ted wi th chromotroph ic a c i d r eagen t . No ev idence f o r the presence of e i t h e r compound in the J . p e c t o r a l i s e x t r a c t s was o b t a i n e d . The chemis t r y of J . p e c t o r a l i s appears to be q u i t e d i f f e r e n t from that of any of the o ther s p e c i e s in t h i s genus tha t have been examined. The apparent absence of l i g n a n s i s of n o t e . T h i s d i f f e r e n c e may r e f l e c t p h y l o g e n e t i c d i s t a n c e s i n c e a l l of the s p e c i e s p r e v i o u s l y s t u d i e d are n a t i v e to A s i a or A f r i c a . 1 . S c reen ing fo r o ther b i o l o g i c a l a c t i v i t i e s A l though J . p e c t o r a l i s i s best known as a snu f f admix tu re , the re i s c o n s i d e r a b l e e t h n o b o t a n i c a l i n f o rma t i on i n d i c a t i n g tha t i t has m u l t i p l e u se s . In the Colombian s t a t e s of Vaupes and Amazonas, i t i s known to the Puinave Ind ians as yakayu and i s used by them in the form of a d e c o c t i o n to t r e a t pulmonary i n f e c t i o n s and pneumonia ( S c h u l t e s , 1978). It has a l s o been s a i d to be e f f e c t i v e in the t reatment of i n f e c t i o n s of the nasa l c a v i t y (Maxwe l l ,N . , p e r s . comm.). An herbar ium 58 specimen c o l l e c t e d by Jose J . T r i a n a i s accompanied by an herbar ium l a b e l s t a t i n g that i t i s w ide ly used in Meta, Colombia as a d e c o c t i o n to t r e a t c h i l d r e n a f f l i c t e d wi th r i c k e t s ( S c h u l t e s , 1978). Jus t i c i a p e c t o r a l i s i s a r e l a t i v e l y we l l known m e d i c i n a l p l a n t in C e n t r a l Amer ica and the Car ibbean (Morton, 1977). I t i s used as a p e c t o r a l tea to r e l i e v e coughs (Wong, 1976), an expec to ran t (Roig y Mesa, 1945) and as a wound p o u l t i c e ( S teh le and S t e h l e , 1962). It has a l s o been r e p o r t e d to have s u d o r i f i c and a p h r o d i s i a c e f f e c t s (Morton, 1977). It i s p o s s i b l e tha t the use of J . p e c t o r a l i s in the t reatment of ches t i n f e c t i o n s or wounds i s dependent upon a n t i m i c r o b i a l a c t i v i t y . T h i s h y p o t h e s i s was examined by t e s t i n g e x t r a c t s of the p l an t f o r i n h i b i t o r y a c t i v i t y a g a i n s t a v a r i e t y of m i c roo rgan i sms . The th ree f r a c t i o n s were t e s t e d a g a i n s t the gram p o s i t i v e b a c t e r i u m , S t aphy lococcus au reus , the gram nega t i ve b a c t e r i u m , E s c h e r i s h i a c o l i , the y e a s t s , Candida a l b i c a n s and Saccharomyces c e r e v i s i a e and the dermatophy t i c f u n g i , Microsporum c a n i s , M. gypseum, M. fu lvum and T r i chophy ton g a l l i n a e . None of the e x t r a c t s had any e f f e c t upon the growth of the b a c t e r i a or y eas t s t e s t e d . The d i e t h y l e the r e x t r a c t , however, exe r t ed a s t r ong i n h i b i t o r y e f f e c t upon the growth of each of the dermatophyt i c f u n g i . T h e i r growth was comp le t e l y i n h i b i t e d at a c o n c e n t r a t i o n of 1 Mg/ml. Coumarin and u m b e l l i f e r o n e were t e s t e d s e p a r a t e l y on these f u n g i . No i n h i b i t o r y a c t i v i t y was observed at doses as h igh as 59 50 M g / m l , i n d i c a t i n g tha t the a n t i f u n g a l a c t i v i t y of the d i e t h y l e ther e x t r a c t i s not a t t r i b u t a b l e to e i t h e r of these major c o n s t i t u e n t s . The th ree e x t r a c t s of J . p e c t o r a l i s were a l s o t e s t e d fo r t h e i r a b i l i t y to i n h i b i t the r e p l i c a t i o n of murine c y tomega lov i rus and S i n d b i s v i r u s (methods d e s c r i b e d in chapte r I I I ) . No e f f e c t s were observed from any of the e x t r a c t s at c o n c e n t r a t i o n s as h igh as 10 M g / m l . 4. DISCUSSION No e v i dence , from e i t h e r the chemica l or the b i o l o g i c a l s t u d i e s , tha t the samples of J . p e c t o r a l i s ana l y sed possessed chemica l c o n s t i t u e n t s capab le of e l i c i t i n g h a l l u c i n o g e n i c a c t i v i t y , was found . Nor d i d J . p e c t o r a l i s e x t r a c t s have any d e t e c t i b l e e f f e c t s upon the b e h a v i o r a l responses of mice to 5-MeODMT. Three compounds wi th b i o l o g i c a l a c t i v i t y were i s o l a t e d and i d e n t i f i e d : b e t a i n e , coumarin and u m b e l l i f e r o n e . Be ta ine i s w ide ly d i s t r i b u t e d in both p l a n t s and an imals (Guggenheim, 1951). I t s on ly known f u n c t i o n i s as a source of methyl groups fo r the r e m e t h y l a t i o n o f . homocys t e ine to meth ion ine v i a the enzyme beta ine-homocys te ine methyl t r a n s f e r a s e (Shapi ro and Sch l enk , 1965). Few s t u d i e s of a p h a r m a c o l o g i c a l nature have been p u b l i s h e d conce rn i ng b e t a i n e . The f i r s t r e p o r t was tha t of Hunt and Renshaw (1929) who examined the e f f e c t s of a s e r i e s of d e r i v a t i v e s of be ta ine on the autonomic nervous sys tem. Most d e r i v a t i v e s were observed to have a musca r i n i c e f f e c t ; i . e . , to reduce b lood p ressu re 60 and hear t r a t e . T h e i r c o n s i d e r a t i o n of the parent compound, b e t a i n e , i s b r i e f and r e s t r i c t e d to the statement that i t i s " p r a c t i c a l l y w i thout a c t i o n on the autonomic nervous s y s t em . " A f r a c t i o n of an e x t r a c t of the p l a n t , P luchea l a n c e o l a t a (Compos i tae ) , c o n t a i n i n g be ta ine as a major c o n s t i t u e n t , has been r epo r t ed to induce c o n t r a c t i o n s in i s o l a t e d smooth muscle p r e p a r a t i o n s (Dasgupta, 1967; Dasgupta et_ a_l, 1968; Prasad et a l , 1965). Fu r the rmore , the e x t r a c t p o t e n t i a t e d b a r b i t u r a t e induced hypnos i s in r a t s (Prasad e_t a_l, 1965) and possessed an t i - i n f l ammato r y a c t i v i t y (Prasad e_t a_l, 1966). The smooth muscle c o n t r a c t i o n i nduc ing e f f e c t of the c rude e x t r a c t i s in c l o s e agreement wi th the r e s u l t s p resen ted in the present study on pure b e t a i n e . To what ex tent the o the r p r o p e r t i e s of the crude e x t r a c t can be a t t r i b u t e d to be ta ine i s not known. More r e c e n t l y , i t has been r e p o r t e d that be ta ine i s a non -s p e c i f i c a n t i - c o n v u l s i v e agent (Freed e_t §_1, 1979). At h igh doses , i t i s capab le of b l o c k i n g c o n v u l s i o n s induced by e l e c t r i c shock as we l l as chemica l c o n v u l s a n t s . Coumarin and u m b e l l i f e r o n e , l i k e b e t a i n e , are d i s t r i b u t e d w ide l y . They are p resent in b a c t e r i a , fung i and a broad range of p l a n t s ( K a r r e r , 1958). A smooth muscle r e l a x i n g , or s p a s m o l y t i c , a c t i o n has been r e p o r t e d p r e v i o u s l y fo r coumarin ( P a t y r a , 1963a and b) as we l l as u m b e l l i f e r o n e (Ach te r r a th-Tuckermann et a l , 1980) and t h i s appears to be a f ea tu re that i s common to many coumarin d e r i v a t i v e s (Lee and F u j i w a r a , 1971; Ojewole and A d e s i n a , 1983a and b ) . T h i s gene ra l spasmo l y t i c a c t i o n , which a f f e c t s many smooth musc l es , 61 appa r en t l y r e s u l t s in a lower ing of hear t r a t e and b l ood p ressu re in an imals t r e a t e d w i th the coumar in , s c o p o l e t i n (Ojewole and A d e s i n a , 1983a and 1983b). Coumar in , and some of i t s d e r i v a t i v e s , possess the a b i l i t y to reduce body temperature (Kitagawa and Iwak i , 1958). At doses approach ing the t o x i c l e v e l , coumarin has s eda t i v e and hypno t i c a c t i v i t y ( I to et a l , 1951; K i tagawa, 1956a and b; K r e i t m a i r , 1949). Both of these a c t i o n s c o u l d p o s s i b l y be r e l a t e d to the a b i l i t y of these compounds to i n h i b i t m i t o c h o n d r i a l r e s p i r a t i o n (K i tagawa, 1956a; Pa i e_t a l , 1975). Coumarin i s a l s o e f f e c t i v e as an an t i - i n f l ammato r y agent , a c t i n g on macrophages to s t i m u l a t e phagocy tos i s (Koh and W i l l oughby , 1979). F i n a l l y , coumarin i s a v o l a t i l e and sweet ly a romat ic compound and has many commerc ia l a p p l i c a t i o n s as an aromat ic a d d i t i v e (K i rk and Othmer, 1979). It i s the so l e i n g r e d i e n t r e s p o n s i b l e fo r the aroma of J . p e c t o r a l i s . The c o n t r i b u t i o n of these v a r i e d b i o l o g i c a l a c t i v i t i e s to the o v e r a l l e f f e c t of J . p e c t o r a l i s snuf f i s not c l e a r . The s e d a t i v e and hypno t i c a c t i v i t y observed fo r coumarin i s i n t e r e s t i n g in t h i s r e s p e c t . Admin i s t e r ed p a r e n t e r a l l y to sma l l a n i m a l s , i t produces s eda t i on and a n a l g e s i a at doses of 50 mg/kg. Whereas i t i s not reasonab le to expect tha t such h igh doses may be a ch i e ved du r i ng snuf f t a k i n g , i t i s p o s s i b l e tha t l o c a l i z e d r eg ions of the head may be exposed to p h a r m a c o l o g i c a l l y a c t i v e l e v e l s as a r e s u l t of i t s a b s o r p t i o n a c r o s s the nasa l mucosa. L o c a l a n a l g e s i a or s e d a t i v e / h y p n o t i c e f f e c t s c o u l d p o s s i b l y be induced at lower doses than i f 62 a d m i n i s t e r e d p a r e n t e r a l l y . The a b i l i t y of s c o p o l e t i n t o r e d u c e b l o o d p r e s s u r e ( O j e w o l e and A d e s i n a , 1983b) i s a l s o of i n t e r e s t . T r y p t a m i n e a d m i n i n i s t r a t i o n i s o f t e n a c c o m p a n i e d by t r a n s i e n t i n c r e a s e s i n b l o o d p r e s s u r e ( S z a r a , 1956). I t i s not known whether t h i s e f f e c t i s a d i r e c t one or whether i t i s m e d i a t e d by p s y c h o l o g i c a l c h a n g e s . The p o s s i b i l i t y t h a t c o u m a r i n o r u m b e l l i f e r o n e c o u l d l o w e r b l o o d p r e s s u r e and t h e r e b y r e d u c e one o f t h e s t r e s s f u l a s p e c t s of t h e t r y p t a m i n e s , i s i n t r i g u i n g . In c o n c l u s i o n , no e v i d e n c e t o s u p p o r t t h e b e l i e f t h a t J . p e c t o r a l i s i s a h a l l u c i n o g e n i c p l a n t was o b t a i n e d . Nor does i t s a d m i n i s t r a t i o n t o m„ice a p p e a r t o a f f e c t d i r e c t l y t h e a c t i o n o f 5-meODMT, t h e h a l l u c i n o g e n i c t r y p t a m i n e of V i r o l a , w i t h w h i c h i t i s combined a s a s n u f f . 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AN ETHNOPHARMACOLOGICAL EXAMINATION OF V i r o l a e longa ta  BARK, A SOUTH AMERICAN ARROW POISON INTRODUCTION Chemica l and pha rmaco log i c a l i n v e s t i g a t i o n s of arrow po i sons used by ind igenous peop les have l e a d , in many i n s t a n c e s , to the d i s c o v e r y of potent p h a r m a c o l o g i c a l l y a c t i v e compounds. The b i s - i s o q u i n o l i n e a l k a l o i d , t u b o c u r a r i n e , and r e l a t e d compounds from s p e c i e s of the Menispermaceae, i n d o l e a l k a l o i d s , such as s t r y c h n i n e , from S t rychnos (Loganiaceae) and c a r d i a c g l y c o s i d e s from a v a r i e t y of members of the Apocynaceae and Moraceae, are some of the best known examples (Lewis and E l v i n - L e w i s , 1977). Less we l l known are the d i t e r p e n e a l k a l o i d s of Aconitum sp . used in Ch ina ( B i s s e t , 1981), the b a t r a c h o t o x i n s ( t r i t e r p e n e a l k a l o i d s ) , p u m i l o t o x i n s ( c i s - d e c a h y d r o q u i n o l i n e a l k a l o i d s ) and h i s t r i o n i c o t o x i n s ( s p i r o p i p e r i d i n e a l k a l o i d s ) from Colombian arrow po ison f r ogs of the Dendrobat idae (Da ly , 1982) and the phorbo l e s t e r , h u r a t o x i n , from Hura c r e p i t a n s (Sakata et a_l, 1971). Some of the a c t i v e c o n s t i t u e n t s of arrow po i sons have proven v a l u a b l e as m e d i c i n a l agents ( t ubocu ra r i ne and c a r d i a c g l y c o s i d e s ) wh i l e o the r s have been a p p l i e d as t o o l s in r e s e a r c h ( b a t r a c h o t o x i n s and o t h e r s ) . Spjut and Perdue (1976) have shown that sources of arrow po i sons are f i v e t imes more l i k e l y to possess ant i tumour a c t i v i t y than h ighe r p l a n t s s e l e c t e d at random. The use of V i r o l a s p e c i e s as arrow po i sons i s 70 i n t e r e s t i n g , not o n l y e t h n o l o g i c a l l y and a n t h r o p o l o g i c a l l y , but p h a r m a c o l o g i c a l l y . R e p o r t s on t h e use of b a r k r e s i n of V i r o l a t h e i d o r a and V i r o l a e l o n g a t a as an h a l l u c i n o g e n i c s n u f f by t h e Yanomamo (Waika) of s o u t h e r n V e n e z u e l a and n o r t h e r n B r a z i l were a c c o m p a n i e d by o b s e r v a t i o n s t h a t t h e same m a t e r i a l was a p p l i e d a l t e r n a t e l y as an a r r o w p o i s o n . S c h u l t e s and H o l m s t e d t ( 1 9 7 1 ) c i t e S a l a t h e (1931) and B e c h e r ( i 9 6 0 ) as t h e f i r s t t o d e s c r i b e t h e use of a t r e e b a r k as an a r r o w p o i s o n amongst t h e Waika g r o u p . T h i s s o u r c e p l a n t i s r e a d i l y d i s t i n g u i s h e d from t h e l i a n a s o f t h e M e n i s p e r m a c e a e and L o g a n i a c e a e w h i c h a r e a l s o r e p o r t e d t o have been u s e d i n t h e p r e p a r a t i o n of d a r t - t i p arrow p o i s o n s by t h e Waika t r i b e s ( B a u e r , 1965). A l t h o u g h i t has been r e p o r t e d t h a t some t r i b e s may use a m i x t u r e of c u r a r e and V i r o l a r e s i n i n t h e p r e p a r a t i o n of t h e i r a r r o w p o i s o n ( B i o c c a , 1965; B i o c c a e t a l , 1966), t h e use of V i r o l a s p . as t h e s o l e i n g r e d i e n t has a l s o been r e p o r t e d as a w e l l e s t a b l i s h e d p r a c t i c e ( S c h u l t e s and H o l m s t e d t , 1968; P r a n c e , 1970). S c h u l t e s and H o l m s t e d t (1968) c a r e f u l l y d e s c r i b e t h e manner o f p r e p a r a t i o n o f arrow t i p s by t h e Waikas of t h e R i o T o t o t o b i . S t r i p s o f b a r k of V i r o l a  t h e i o d o r a a r e h e a t e d o v e r a f i r e and t h e r e s i n w h i c h f l o w s f r o m t h e b a r k i s a p p l i e d r e p e a t e d l y t o bamboo a r r o w t i p s , f o l l o w e d by h e a t i n g o v e r t h e f i r e . P r a n c e (1970) has o b s e r v e d an a l m o s t i d e n t i c a l p r o c e d u r e by t h e Sanama t r i b e ( c l o s e l y r e l a t e d t o t h e W a i k a s ) , l i v i n g more t h a n 300 k i l o m e t e r s n o r t h o f t h e R i o T o t o t o b i . V i r o l a t h e i o d o r a and V. e l o n g a t a have been c o n s i d e r e d t o 71 be synonomous by a u t h o r i t i e s i n t h e taxonomy o f t h i s g r o u p ( R o d r i g u e s , 1980; S m i t h and Wodehouse, 1937). S c h u l t e s p r e f e r s t o d i s t i n g u i s h V. t h e i o d o r a as a s e p a r a t e s p e c i e s on t h e b a s i s of t h e e a s e w i t h w h i c h i t c a n be r e c o g n i z e d i n t h e f i e l d ( S c h u l t e s and H o l m s t e d t , 1968). P r a n c e (G.T., p e r s . comm.) has p o i n t e d out t h a t t h e two v a r i e t i e s a r e g e n e r a l l y f o u n d i n t h e same l o c a l i t y , V. t h e i o d o r a o c c u r r i n g on t i e r r a f i r m e and V. e l o n g a t a g r o w i n g c l o s e t o t h e w a t e r c o u r s e s and t h a t t h e y a r e b o t h u s e d i n t h e p r e p a r a t i o n o f s n u f f and a r r o w p o i s o n . V i r o l a s p e c i e s have been s t u d i e d e x t e n s i v e l y w i t h r e s p e c t t o t h e i r a l k a l o i d a l c o n s t i t u e n t s . B o t h V. t h e i o d o r a and V. e l o n g a t a have been e x a m i n e d . A l t h o u g h t h e r e was c o n s i d e r a b l e v a r i a t i o n between s a m p l e s , t h e i n d o l e a l k a l o i d s , N-m e t h y l t r y p t a m i n e , N , N - d i m e t h y l t r y p t a m i n e and 5-methoxy-N,N-d i m e t h y l t r y p t a m i n e were u s u a l l y t h e major a l k a l o i d s p r e s e n t ( A g u r e l l e t a l , 1969; H o l m s t e d t e t a l , 1980; McKenna e t a l , 1984). T h e s e , and o t h e r i n d o l e a l k a l o i d s , a r e known t o have a number of p h y s i o l o g i c a l a c t i v i t i e s i n a n i m a l s , f o r e m o s t among whi c h i s t h e i r p s y c h o t o m i m e t i c a c t i v i t y and t h e i r a b i l i t y t o e l i c i t h a l l u c i n a t i o n s i n man ( S z a r a , 1956). N,N-d i m e t h y l t r y p t a m i n e and 5 - m e t h o x y - N , N - d i m e t h y l t r y p t a m i n e have been shown t o d i s r u p t n o r m a l b e h a v i o r a l r e s p o n s e s of r o d e n t s a t low d o s e s ( G e s s n e r and Page, 1962; G e s s n e r e_t a_l, 1961; G e s s n e r e t a l , 1968; Grahame-Smith, 1971; Ho e t a l , 1970; Shah and Hedden, 1978). I t has been s u g g e s t e d t h a t i n d o l e a l k a l o i d s a r e t h e b i o l o g i c a l l y a c t i v e c o n s t i t u e n t s of V i r o l a a r r o w p o i s o n t h a t a r e r e s p o n s i b l e f o r i n c a p a c i t a t i n g t h e wounded 72 a n i m a l . Yanomamo d a r t s have been examined c h e m i c a l l y and f o u n d t o c o n t a i n a s i n g l e a l k a l o i d , 5 - m e t h o x y - N , N - d i m e t h y l t r y p t a m i n e i n h i g h c o n c e n t r a t i o n (8% by w e i g h t ) ( G a l e f f i e_t a l , 1983). T h e s e w o r k e r s a t t r i b u t e t h e e f f e c t i v e n e s s o f t h e a r r o w p o i s o n t o t h e p r e s e n c e o f t h i s i n d o l e c o n s t i t u e n t . In t h e p r e s e n t s t u d y , an e x p e r i m e n t a l a p p r o a c h t o t h i s e t h n o p h a r m a c o l o g i c a l p r o b l e m has been t a k e n . E x t r a c t s o f V i r o l a e l o n g a t a were a d m i n i s t e r e d t o mice and t h e e f f e c t s on b e h a v i o r o b s e r v e d and q u a n t i f i e d . I t was i n t e n d e d , by t h i s a p p r o a c h , t o d e t e r m i n e i f t o x i c c o n s t i t u e n t s were p r e s e n t or i f t h e t r y p t a m i n e s c a u s e d b e h a v i o r a l d i s r u p t i o n o f s u f f i c i e n t s e v e r i t y t o e x p l a i n t h e use o f t h e bark as an arrow p o i s o n . 73 PART A. ISOLATION AND IDENTIFICATION OF THE MAJOR NON-POLAR  CONSTITUENTS OF V i r o l a e longa ta BARK 1. INTRODUCTION Dur ing the course of the s t u d i e s d e a l i n g w i th the p h a r m a c o l o g i c a l e f f e c t s of V i r o l a e x t r a c t s , some d i f f i c u l t y was encountered i n de te rm in ing the r e l a t i v e potency of the b i o l o g i c a l e f f e c t s of c e r t a i n f r a c t i o n s . T h i s was a f u n c t i o n of the d i f f i c u l t y i n vo l v ed in q u a n t i f y i n g locomotor a c t i v i t y in l a r g e numbers of mice and the nature of the sample be ing f r a c t i o n a t e d . As a r e s u l t , i t was dec ided that a b e t t e r approach would be to i s o l a t e and p u r i f y the major compounds f i r s t and then to measure t h e i r b i o l o g i c a l a c t i v i t y . T h i s s e c t i o n d e s c r i b e s the i s o l a ' t i o n and i d e n t i f i c a t i o n of these compounds. 2. EXPERIMENTAL a . E x t r a c t ion of P l an t M a t e r i a l The bark was c o l l e c t e d near the v i l l a g e of B r i l l o Nuevo on the R io Ampiyacu, a Pe ruv ian t r i b u t a r y of the Amazon. Voucher specimens (D. McKenna No. 59) were d e p o s i t e d at UNAP herbar ium in I q u i t o s , San Marcos Herbar ium in L ima, the Ch icago F i e l d Museum and the UBC he rba r ium. The i d e n t i f i c a t i o n was c a r r i e d out by Dr . W.A. Rod r i gues , INPA, Manaus, B r a z i l . The d r i e d bark (1.3 kg) was m i l l e d and e x t r a c t e d four t imes at room temperature wi th d i e t h y l e t h e r . 74 b. Chromatography A f t e r e vapo ra t i on i_n vacuo the r e s idue was sub j e c t ed to chromatography on a Chromatotron (Ha r r i son Research A s s o c i a t e s ) u t i l i z i n g p r e p a r a t i v e (4 mm) p l a t e s coa ted w i th S i l i c a g e l P F 2 5 , (Merck ) . Repeated chromatography wi th pet ro leum e t h e r / e t h y l e t h e r / a c e t o n i t r i l e (12/12/1; 6/6/1; 3/3/1) y i e l d e d each of the f o l l o w i n g compounds in s u f f i c i e n t p u r i t y fo r s p e c t r o s c o p y . They are p resen ted in order of e l u t i o n . 3. COMPOUNDS ISOLATED 0 - s i t o s t e r o l . (425 mg) Mp 133-135 °C (from E t 2 0 ) , PMR and MS data agree w i th data ob ta ined fo r an a u t h e n t i c sample (S igma). Chromatography on TLC ( s i l i c a ) u s i ng hep tane/ch lo ro fo rm/e thano l (25/ 25/1) and pe t ro leum e t h e r / e t h y l e t h e r / a c e t o n i t r i l e (12/12/1) was i d e n t i c a l to that of an a u t h e n t i c sample (S igma). 3 , 4 ' , 5 - t r i m e t h o x y - c i s - s t i l b e n e 1a. (23 mg) Mp 73-74 °C (from E t 2 0 ) . UV X c y c l o h e x . ( l o g e ) . 2 8 3 (4 .50 ) , 235sh ( 4 . 6 4 ) , 214 max 3 ( 4 . 9 7 ) . 1H-NMR (400 MHz) (CDC1 3 ) : 5 3.65 (6H, s, 2 X O C H 3 ) , 3.78 (3H, s, O C H 3 ) , 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, v i n y l ) , 6.52 (1H, d , J = 12 Hz , v i n y l ) , 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 ( r e l i n t ) 270[M + ] (100) , 239 (12) , 224 (12) , 212 (8 ) , 196 (11) , 195 (13) , 181 (10) , 169 75 ( 1 0 ) , 165 ( 1 0 ) , 153 ( 1 3 ) , 152 ( 1 9 ) , 149 ( 1 2 ) , 141 ( 9 ) , 135 (9) , 127 ( 8 ) , 115 ( 1 5 ) , 104 ( 1 0 ) , 95 ( 1 1 ) , 91 ( 1 7 ) , 85 ( 1 1 ) , 83 ( 1 1 ) , 81 ( 1 5 ) , 71 ( 1 5 ) , 69 (22) . 3 , 4 ' , 5 - t r i m e t h o x y - t r a n s - s t i l b e n e 1b. (41 mg) Mp 56-57 0 C ( f r o m E t 2 0 ) . UV X ^ l o h e x ' ( l o g e ) : 335sh ( 4 . 4 5 ) , 318 ( 4 . 6 8 ) , 303 ( 4 . 7 2 ) , 235sh ( 4 . 4 2 ) , 216 ( 4 . 6 6 ) . 1H-NMR (400 MHz) ( C D C 1 3 ) : 6 3.81 (9H, s, 3 X O C H 3 ) , 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, v i n y l ) , 7.04 (1H, d, J = 16 Hz, v i n y l ) , 7.44 (2H, d, J = 9 Hz, H-2', H-6'). MS m/z ( r e l i n t ) 2 7 0 [ M + ] ( 1 0 0 ) , 239 ( 1 4 ) , 224 ( 1 1 ) , 212 ( 8 ) , 196 ( 9 ) , 195 (10) , 181 ( 7 ) , 169 (7 ) 167 ( 1 3 ) , 165 ( 8 ) , 153 ( 9 ) , 152 ( 1 3 ) , 149 ( 3 7 ) , 141 ( 7 ) , 135 ( 7 ) , 128 ( 8 ) , 115 ( 1 2 ) , 104 ( 7 ) , 91 ( 1 2 ) , 83 ( 9 ) , 76 ( 1 1 ) , 71 ( 2 5 ) , 70 ( 2 3 ) , 69 ( 2 7 ) . E u s i d e r i n 2. (28 mg) Mp 93-95 0 C ( f r o m E t 2 0 ) . UV XMeOH ( l o g ^ max r e ) : 271 ( 3 . 2 1 ) , 234sh ( 4 . 2 4 ) , 230 ( 4 . 7 8 ) . [ a ] -10.4 0 i n CHC1 3. 1H-NMR (80 MHz) ( C D C 1 3 ) : 5 1.26 (3H, d, J = 6Hz, Me-3), 3.28 (2H, d, J = 7Hz, C H 2 ) , 3.85 (3H, s, O C H 3 ) , 3.88 (9H, s, 3 X O C H 3 ) , 3.9-4.3 ( I H , m, H-3), 4.56 (1H, d, J = 8Hz, H-2), 4.9-5.25 (2H, m, =CH 2), 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 ( r e l i n t ) ' : 3 8 6 [ M + ] ( 2 2 ) , 372 ( 5 ) , 344 ( 3 ) , 343 ( 3 ) , 312 ( 3 ) , 311 ( 4 ) , 302 ( 5 ) , 210 ( 1 0 ) , 209 ( 7 2 ) , 208 ( 1 0 0 ) , 205 ( 1 3 ) , 195 ( 5 ) , 194 ( 2 2 ) , 193 ( 7 5 ) , 192 ( 6 ) , 191 ( 2 8 ) , 181 (11) , 179 ( 1 5 ) , 178 ( 1 2 ) , 177 ( 9 ) , 168 ( 5 ) , 165 ( 1 5 ) , 164 ( 8 ) , 76 163 ( 9 ) , 161 ( 6 ) , 151 ( 1 0 ) , 150 ( 1 8 ) , 149 ( 4 8 ) , 148 ( 7 ) , 147 ( 7 ) , 137 ( 1 3 ) , 135 ( 1 9 ) , 133 ( 1 8 ) , 132 ( 8 ) , 123 ( 1 1 ) , 121 ( 1 4 ) , 119 ( 1 0 ) , 107 ( 1 9 ) , 105 ( 2 3 ) , 104 ( 1 1 ) , 103 ( 1 0 ) , 97 (13) , 95 ( 1 3 ) , 91 ( 5 0 ) , 85 ( 1 7 ) , 83 ( 1 8 ) , 81 ( 1 4 ) , 79 ( 2 8 ) . V i r o l o n g i n 3. (34 mg). C o l o u r l e s s o i l . UV X ^ | ° H ( l o g e) 268 ( 3 . 3 5 ) , 227 ( 4 . 0 4 ) . [ a ] j p -12.4 0 i n C H C 1 3 . 1H-NMR (80 MHz) (CDC1 3) 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, O C H 3 ) , 3.80 (6H, s, 2 X O C H 3 ) , 3.83 (6H, s, 2 X O C H 3 ) , 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 ( r e l i n t ) 4 0 2 [ M + ] ( 2 7 ) , 211 ( 5 ) , 210 ( 4 0 ) , 209 ( 1 0 0 ) , 208 (73) , 195 ( 1 9 ) , 194 ( 8 7 ) , 1 9 3 ( 5 5 ) , 192 ( 7 ) , 191 ( 6 ) , 182 ( 7 ) , 181 ( 5 1 ) , 179 ( 3 0 ) , 178 ( 3 9 ) , 177 ( 1 2 ) , 169 ( 4 ) , 168 ( 2 8 ) , 167 ( 6 ) , 166 ( 7 ) , 165 ( 1 6 ) , 164 ( 7 ) , 163 ( 1 7 ) , 162 ( 1 0 ) , 161 ( 7 ) , 153 ( 1 1 ) , 151 ( 2 3 ) , 150 (14) , 149 ( 1 4 ) , 148 ( 1 1 ) , 147 ( 1 8 ) , 138 ( 5 ) , 137 ( 1 6 ) , 136 ( 1 0 ) , 135 ( 2 2 ) , 134 ( 9 ) , 133 ( 2 3 ) , 131 ( 1 1 ) , 125 ( 6 ) , 123 ( 9 ) , 122. ( 7 ) , 121 ( 1 9 ) , 120 ( 8 ) , 119 ( 1 9 ) , 118 ( 9 ) , 115 ( 9 ) , 109 ( 1 0 ) , 107 ( 1 9 ) , 105 ( 2 5 ) , 103 ( 1 5 ) , 95 ( 1 2 ) , 93 ( 1 0 ) , 91 ( 4 0 ) , 85 ( 1 1 ) , 83 ( 1 2 ) , 81 ( 1 3 ) , 79 ( 3 3 ) , 78 ( 1 3 ) , 77 ( 3 4 ) , 71 ( 1 8 ) , 70 ( 1 1 ) , 69 ( 2 5 ) . 77 E p i - s e s a r t e m i n 4. (97 mg). 114-115 0 C ( f r o m E t 2 0 ) . C 2 3 H 2 8 0 8 ( f o u n d 430.1622 f o r 430.1628 by HR-MS). UV X ^ ??SH (log * ) : 270 ( 3 . 4 0 ) , 235sh ( 4 . 1 4 ) , 210 ( 4 . 9 2 ) . [ a ] 25 +108 0 i n CHC1 3. 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 ( s e e T a b l e V) . 1 3ONMR ( s e e T a b l e V I ) . MS m/z ( r e l i n t ) : 430 [M +] ( 4 0 ) , 249 ( 6 ) , 233 ( 1 0 ) , 224 ( 1 8 ) , 219 ( 1 5 ) , 209 ( 8 ) , 208 ( 2 4 ) , 207 ( 4 0 ) , 206 ( 1 7 ) , 205 ( 7 ) , 203 ( 9 ) , 197 ( 4 2 ) , 196 ( 1 6 ) , 195 ( 2 3 ) , 194 ( 1 5 ) , 192 ( 1 3 ) , 191 ( 3 4 ) , 182 ( 2 0 ) , 181 ( 6 0 ) , 180 ( 2 8 ) , 179 ( 1 0 0 ) , 178 ( 1 5 ) , 177 ( 8 ) , 176 ( 1 5 ) , 175 (7) , 169 ( 5 5 ) , 168 ( 1 3 ) , 167 ( 1 2 ) , 166 ( 3 6 ) , 165 ( 7 8 ) , 161 ( 1 9 ) , 154 ( 1 4 ) , 153 ( 1 3 ) , 152 ( 2 1 ) , 151 (24) 139 ( 7 ) , 138 ( 1 5 ) , 133 ( 1 3 ) , 125 ( 1 1 ) , 115 ( 1 0 ) , 95 ( 1 5 ) , 93 ( 1 3 ) , 91 ( 1 3 ) , 81 ( 1 5 ) , 79 ( 1 5 ) . S e s a r t e m i n 5. (36 mg). Mp 115-116 °C ( f r o m E t 2 0 ) . C 2 3 H 2 8 0 8 ( f o u n d 430.1627 f o r 430.1628 by HR-MS). UV X M e 0 H ( l o g e ) : max J 270 ( 4 . 1 1 ) , 211 ( 4 . 8 3 ) . [ a ] J 5 + 50 0 i n CHC1 3. 1H-NMR ( s e e T a b l e V). 1 3C~NMR ( s e e T a b l e V I ) . MS m/z ( r e l i n t ) : 430[M +] ( 4 7 ) , 265 ( 5 ) , 249 ( 9 ) , 235 ( 7 ) , 234 ( 8 ) , 233 ( 1 1 ) , 224 ( 1 8 ) , 222 ( 6 ) , 219 ( 1 7 ) , 209 ( 7 ) , 208 ( 2 4 ) , 207 ( 5 1 ) , 206 ( 1 5 ) , 205 (8) , 203 ( 1 1 ) , 197 ( 3 6 ) , 196 ( 2 1 ) , 195 ( 3 8 ) , 194 ( 2 1 ) , 193 (10) , 192 ( 1 4 ) , 191 ( 4 6 ) , 190 ( 9 ) , 189 ( 9 ) , 182 ( 2 3 ) , 181 ( 7 2 ) , 180 ( 3 3 ) , 179 ( 1 0 0 ) , 178 ( 2 2 ) , 177 ( 1 1 ) , 176 ( 1 9 ) , 175 (9) , 169 ( 4 2 ) , 168 ( 1 5 ) , 167 ( 1 2 ) , 166 ( 3 5 ) , 165 ( 8 9 ) , 161 ( 2 6 ) , 154 ( 1 0 ) , 153 ( 1 8 ) , 152 ( 2 6 ) , 151 ( 2 6 ) , 138 ( 1 1 ) , 135 (11) , 133 ( 1 6 ) , 125 ( 1 3 ) , 121 ( 8 ) , 115 ( 1 1 ) , 110 ( 1 0 ) , 105 (10) , 95 ( 1 8 ) , 93 ( 1 5 ) , 91 ( 1 5 ) , 81 ( 1 8 ) , 79 ( 1 5 ) . 78 E p i - y a n q a m b i n 6. (127 mg). Mp 119-120 °C ( f r o m E t 2 0 ) . UV X^eOH ( l o g e ) : 272 ( 3 . 5 1 ) , 238sh ( 4 . 1 1 ) , 210 ( 4 . 8 0 ) . [ a ] 25 +122 0 i n CHC1 3. 1H-NMR ( s e e T a b l e V). 1 3C-NMR ( s e e T a b l e V I ) . MS m/z ( r e l i n t ) : 4 4 6 [ M + ] ( 3 7 ) , 265 ( 6 ) , 250 ( 1 0 ) , 249 ( 9 ) , 235 ( 1 2 ) , 224 ( 2 6 ) , 223 ( 9 ) , 219 ( 1 2 ) , 208 ( 1 3 ) , 207 ( 5 1 ) , 206 ( 10) , 1 97 ( 3 2 ) , 1 96 ( 2 4 ) , 195 (68) , 1 94 (18) , 191 ( 1 0 ) , 189 (8) , 182 (39) , 181 ( 1 0 0 ) , 179 (12) , 177 (12) , 176 ( 2 1 ) , 169 (42) , 1 68 ( 1 6 ) , 1 67 (13) , 166, ( 1 0 ) , 1 65 (21 ), 154 ( 1 1 ) , 1 53 ( 1 4 ) , 151 ( 2 0 ) , 138 ( 1 3 ) , 125 ( 1 4 ) , 1 10 ( 1 0 ) , 95 ( 9 ) , 93 ( 1 3 ) , 91 (11) , 81 ( 1 5 ) , 79 (9) . Yanqambin 7. (115 mg) Mp 119-121 0 ( f r o m E t 2 0 ) UV X MeOH ( l o g e ) : 270 ( 3 . 5 2 ) , 235 ( 4 . 1 4 ) , 210 ( 4 . 9 3 ) . [ a ] J 5 +45.1 0 i n C H C I 3 . 1H-NMR ( s e e T a b l e V). 1 3C-NMR ( s e e T a b l e V I ) . MS m/z ( r e l i n t ) : 4 4 6 [ M + ] ( 2 7 ) , 265 ( 6 ) , 250 ( 8 ) , 249 ( 8 ) , 235 ( 1 0 ) , 224 ( 1 9 ) , 223 ( 8 ) , 219 ( 9 ) , 208 ( 1 2 ) , 207 ( 4 8 ) , 206 ( 8 ) , 197 (20) , 196 ( 2 8 ) , 195 ( 5 1 ) , 194 ( 2 0 ) , 193 ( 1 0 ) , 191 ( 1 1 ) , 190 ( 8 ) , 189 ( 9 ) , 182 ( 2 7 ) , 181 ( 8 2 ) , 179 ( 1 2 ) , 177 ( 1 3 ) , 176 (21) , 169 ( 2 5 ) , 168 ( 1 5 ) , 167 ( 1 2 ) , 166 ( 8 ) , 165 ( 1 7 ) , 163 ( 8 ) , 154 ( 8 ) , 153 ( 1 7 ) , 152 ( 9 ) , 151 ( 1 8 ) , 149 ( 1 3 ) , 145 ( 9 ) , 138 ( 1 1 ) , 137 ( 1 3 ) , 136 ( 8 ) , 135 ( 1 2 ) , 131 ( 1 1 ) , 128 ( 8 ) , 125 ( 2 1 ) , 123 ( 1 4 ) , 121 ( 1 2 ) , 119 ( 1 5 ) , 117 ( 9 ) , 115 ( 1 3 ) , 111 ( 1 3 ) , 110 ( 1 9 ) , 109 ( 1 9 ) , 107 ( 1 1 ) , 105 ( 1 5 ) , 97 ( 2 0 ) , 96 ( 1 3 ) , 95 ( 3 3 ) , 93 ( 2 0 ) , 91 ( 2 4 ) , 85 ( 1 6 ) , 83 ( 2 3 ) , 82 ( 1 7 ) , 81 ( 3 6 ) , 79 ( 1 9 ) . 7 9 D i h y d r o s e s a r t e m i n 8. (21 mg) UV X ( l o g e ) : 282 ( 3 . 2 5 ) , 232sh ( 3 . 8 7 ) , 218 ( 4 . 0 8 ) . [ a ] 25 +11.8 0 i n CHC1 3. 1H-NMR (400 MHz) ( C D C 1 3 ) : 6 1.56 (1H, br s, e x c h a n g e a b l e w i t h D 2 0 , OH), 2.40 (1H, m, J = 5 Hz, H-3), 2.55 (1H, dd, J = 14, 11 Hz, A r C H 2 ) , 2.73 (1H, m, H-4), 2.92 (1H, dd, J = 14, 5 Hz, A r C H 2 ) , 3.73-4.08 (4H, H-5, CH 2OH), 3.83 (3H, s, O C H 3 ) , 3.84'(6H, s, 2 X O C H 3 ) , 3.89 (3H, s, O C H 3 ) , 4.78 (1H, d, J = 6.3 Hz, H-2), 5.95 (2H, s, O C H 2 0 ) , 6.40 (2H, s, H-2', H-6'), 6.53 (1H, s, H-2" o r H-6"), 6.54 (IH, s, H-2" o r H-6"). MS m/z ( r e l i n t ) : 432[M +] ( 2 3 ) , 414 ( 1 . 2 ) , 383 ( 2 . 4 ) , 368 ( 1 . 3 ) , 353 ( 1 . 1 ) , 249 ( 6 ) , 233 ( 1 7 ) , 224 ( 1 4 ) , 219 ( 1 8 ) , 208 ( 2 1 ) , 207 ( 1 2 ) , 206 ( 1 2 ) , 195 ( 2 2 ) , 193 ( 1 4 ) , 183 ( 1 0 ) , 182 (82) , 181 ( 1 0 0 ) , 180 ( 1 8 ) , 179 ( 5 3 ) , 169 ( 1 0 ) , 167 ( 2 2 ) , 166 ( 2 1 ) , 165 ( 4 5 ) , 153 ( 1 8 ) , 152 ( 1 3 ) , 151 ( 3 8 ) , 148 ( 1 8 ) , 137 ( 1 4 ) , 136 ( 1 2 ) , 123 ( 2 3 ) , 105 ( 1 2 ) , 95 ( 2 7 ) , 93 ( 1 2 ) , 91 ( 2 2 ) , 79 ( 1 8 ) . j3-Dihydroyangambin 9. (13 mg) UV X MeOH ( l o g e ) : 280 ( 3 . 7 5 ) , max 227sh ( 4 . 2 5 ) , 213 ( 4 . 5 8 ) . [a] J 5 +15.1 0 i n CHC1 3. 1H-NMR (400 MHz) (CDC1 3) 6 1.78 (1H, br s, e x c h a n g e a b l e w i t h D 2 0 , OH), 2.03 (1H, m, J = 5 Hz, H-3), 2.52 (1H, dd, J = 14, 7 Hz, A r C H 2 ) , 2.8 (1H, dd, J = 14, 5 Hz, A r C H 2 ) , 2.88 (1H, m, H-4), 3.70-4.05 (4H, m, H-5, CH 2OH), 3.84 (6H, s, 2 X O C H 3 ) , 3.89 (6H, s, 2 X O C H 3 ) , 3.94 (6H, s, 2 X O C H 3 ) , 4.70 ( I H , 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 ( r e l i n t ) : 4 4 8 [ M + ] ( 2 9 ) , 399 ( 6 ) , 265 ( 3 ) , 263 ( 5 ) , 249 ( 1 8 ) , 240 ( 2 1 ) , 235 ( 1 6 ) , 233 ( 9 ) , 224 ( 4 2 ) , 223 ( 2 3 ) , 222 ( 4 5 ) , 221 ( 1 2 ) , 219 ( 1 4 ) , 210 ( 2 0 ) , 208 80 (14) , 207 (18) , 205 ( 1 1 ) , 198 ( 1 1 ) , 197 (27) , 196 ( 3 2 ) , 195 (83) , 1 93 ( 1 8 ) , 191 ( 1 3 ) , 189 (10) , 183 (17) , 1 82 (77) , 181 (100) , 179 (18) , 177 ( 1 2 ) , 1 76 (13) , 1 69 (48) , 168 (21 ) , 167 (46) , 165 ( 2 0 ) , 161 ( 1 1 ) , 1 54 (19) , 1 53 (19) , 1 52 ( 2 2 ) , 151 (50) , 1 49 ( 1 2 ) , 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 ( 1 2 ) , 121 ( 1 5 ) , 1 10 ( 1 2 ) , 109 ( 1 8 ) , 1 07 (15) , 1 06 ( 1 1 ) , 105 (17) , 95 (20) , 93 ( 1 7 ) , 92 ( 1 2 ) , 91 (29) , 81 (23) , 79 ( 2 2 ) . 4. RESULTS E l e v e n of t h e major c o n s t i t u e n t s of a d i e t h y l e t h e r e x t r a c t of V. e l o n g a t a b a r k were i s o l a t e d a s d e s c r i b e d i n t h e E x p e r i m e n t a l s e c t i o n - a n d i d e n t i f i e d by s p e c t r o s c o p i c means. 0 -S i t o s t e r o l was t h e o n l y p h y t o s t e r o l i d e n t i f i e d . The s t i l b e n e s , 3, 4', 5 - t r i m e t h o x y - t r a n s - s t i l b e n e (1a) and i t s c i s isomer were f o u n d i n a p p r o x i m a t e l y e q u a l p r o p o r t i o n s . O n l y t h e f o r m e r has been d e s c r i b e d as a n a t u r a l l y o c c u r r i n g c o n s t i t u e n t . E u s i d e r i n (2) and a h i t h e r t o u n d e s c r i b e d compound, v i r o l o n g i n ( 3 ) , c o m p r i s e d t h e n e o l i g n a n s . F o u r b i s - t e t r a h y d r o f .uran l i g n a n s were i d e n t i f i e d : e p i - s e s a r t e m i n ( 4 ) , s e s a r t e m i n ( 5 ) , e p i - y a n g a m b i n (6) and yangambin ( 7 ) . F i n a l l y , two new t e t r a h y d r o f u r a n compounds, d i h y d r o s e s a r t e m i n (8) and 0 -d i h y d r o y a n g a m b i n (9) were i d e n t i f i e d . 81 P r o t o n s E p i -s e s a r t e m i n (4) S e s a r t e m i n (5) E p i -yangambi n (6) Yangambin (7) 1H 2.91(1H),m 3.05(1H),m 2.95(1H),m 3.08(1H),m 2H 4.85(1H),d J = 5.5 4.70(1H),d J=4.0 4.84(1H),d J=5.0 4.75(1H),d J=5. 5 4aH 3.70-3.95 ( 1H) ,m 4. 15-4.50 (1H),m 3.70-4.00 (1H),m 4.20-4.43 (1H),m 4j3H 3.15-3.5 (IH),M 3.70-4.10 (1H),m 3.20-3.55 (IH),m 3.82-3.97 (1H),m 5H 3.15-3.50,m 3.05(1H),m 3.20-3.55 3.08(1H),m 6 H 4.41(1H),d J = 7.0 4.70(1H),d J = 4.0 4.43(1H),d J=7.0 4.75(1H),d J=5. 5 8aH 4.00-4.25 (1H),m 4.15-4.50 (1H),m 4.00-4.25 (1H),m 4.20-4.43 (1H),m 8/3H 3.70-3.95 (1H),m 3.70-4.10 (1H),m 3.70-4.00 (1H),m 3.82-3.97 (1H),m 2 ' , 2 " 6* , 6" 6 . 5 6 ( 2 H ) , s 6.53(2H),s 6.55(4H),s 6.57(4H),s 6.58(4H),s OCH 3 3.90(3H),s 3.87(3H),s 3.85(3H),s 3.91(3H),s 3.87(3H),s 3.82(3H),s 3.88(12H),s 3.83(6H),s 3 . 8 6 (12H),s 3.83(6H),s OCH 2 0 5.95(2H),s 5.95(2H),s - -T a b l e V - 1H-NMR s p e c t r a o f b i s - t e t r a h y d r o f u r a n l i g n a n s i s o l a t e d from V i r o l a e l o n g a t a b a r k . D a t a a r e p r e s e n t e d ( i n o r d e r ) a s c h e m i c a l s h i f t ( 6 , r e l a t i v e t o . TMS); i n t e g r a l v a l u e ( n u m b e r of p r o t o n s ) ; m u l t i p l i c i t y o f p e a k s ; c o u p l i n g c o n s t a n t ( i n H z ) . S p e c t r a were r e c o r d e d i n CDC1 3 a t 80 MHz. 82 C a r b o n Number E p i -S e s a r t e m i n (4) S e s a r t e m i n (5) E p i -yangambin (6) Yangambin (7) 1 54.52 54.41 54.36 54.29 2 87.59 8 5 . 8 5 ( 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 8 6 . 0 2 ( 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 1 5 3 . 2 9 ( f ) 153.32 153.39 3" 153.21 1 5 3 . 4 8 ( 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 OCH 3 56.13,56.66, 60.77 56.24,57.51 60.86 56.07,60.68 56.16,60.73 OCH 2 0 1 00. 1 1 100.09 - -T a b l e VI - 1 3C-NMR s p e c t r a of b i s - t e t r a h y d r o f u r a n l i g n a n s i s o l a t e d f r o m V i r o l a e l o n g a t a b a r k . C h e m i c a l s h i f t s a r e g i v e n i n S(ppm) r e l a t i v e t o TMS; 6(TMS)= 5(CDC1 3) + 77.0 ppm; r e c o r d e d a t 20 MHz i n CDC1 3 . V a l u e s f o l l o w e d by l e t t e r s a r e i n t e r c h a n g e a b l e . 8 3 a. 3 , 4 ' - 5 - t r i m e t h o x y - c i s - s t i l b e n e and 3 , 4 ' , 5 - t r i m e t h o x y - t r a n s - s t i l b e n e Compounds 1a and 1b ( F i g u r e 7) were p r e s e n t i n a p p r o x i m a t e l y e q u a l amounts and beha v e d s i m i l a r l y on TLC u s i n g s e v e r a l s o l v e n t s y s t e m s . B o t h formed a p i n k c o l o u r upon s p r a y i n g w i t h H 2 S O „ . They were s e p a r a t e d u s i n g t h e c h r o m a t o t r o n and c r y s t a l l i z e d f r o m d i e t h y l e t h e r . The UV s p e c t r u m of 1b d i s p l a y e d bands of a p p r o x i m a t e l y e q u a l i n t e n s i t y a t 319 and 305 n a n o m e t e r s , c o r r e s p o n d i n g t o Band I and I I , r e s p e c t i v e l y , t h a t a r e c h a r a c t e r i s t i c o f s t i l b e n e s ( H i l l i s and I s h i k u r a , 1966). The s p e c t r a l d a t a o b t a i n e d f o r 1b a r e i n agreement w i t h t h o s e p r e s e n t e d f o r 3 , 4 ' , 5 - t r i m e t h o x y -t r a n s - s t i l b e n e ( B l a i r e t aJL, 1969). The s i g n a l s o f t h e v i n y l p r o t o n s a r e r e p o r t e d by B l a i r e t a_l (1969) t o be b r o a d s i n g l e t s i n a 60 MHz 1H-NMR s p e c t r u m . The b e t t e r r e s o l u t i o n o f t h e 400 MHz s p e c t r u m o b t a i n e d h e r e showed t h e v i n y l p r o t o n s i g n a l s t o be d o u b l e t s w i t h a c o u p l i n g c o n s t a n t o f 16 Hz. T h i s v a l u e i s c h a r a c t e r i s t i c o f u n s y m m e t r i c a l l y s u b s t i t u t e d t r a n s s t i l b e n e s and compound 1b was t h u s a s s i g n e d . The UV s p e c t r u m of 1a, a s i n g l e band a t 283 nm, i s s u g g e s t i v e o f a c i s - s t i l b e n e d e r i v a t i v e ( H i l l i s and I s h i k u r a , 1966). The mass s p e c t r u m i s v e r y s i m i l a r t o t h a t o f 1b, b o t h d i s p l a y i n g a p a r e n t i o n o f mass 270. In t h e 400 MHz 'H-NMR s p e c t r u m , t h e a r o m a t i c AB p a t t e r n o f C-3',5';C- 2',6', t h e s i g n a l s o f t h e two v i n y l p r o t o n s and t h e C-2, C-4 and C-6 p r o t o n s i g n a l s a r e a l l p r e s e n t b u t s h i f t e d u p f i e l d somewhat from t h o s e o f 1b. The v i n y l p r o t o n s i g n a l s a p p e a r a s d o u b l e t s 84 Figure 7 - Structures from V i r o l a of stilbenes and neolignans isolated elongata bark. 85 F i g u r e 8 - S t r u c t u r e s of b i s - t e t r a h y d r o f u r a n l i g n a n s i s o l a t e d from V i r o l a e l o n g a t a b a r k . 86 Figure 9 - Structures of tetrahydrofuran lignans isolated from V i r o l a elongata bark. 87 w i t h a c o u p l i n g c o n s t a n t of 12 Hz, a v a l u e t h a t i s c h a r a c t e r i s t i c of u n s y m m e t r i c a l l y s u b s t i t u t e d c i s s t i l b e n e s . On t h e b a s i s o f t h e s e d a t a compound 1a i s a s s i g n e d a s t h e c i s is o m e r of 1b. The o c c u r r e n c e of t h i s compound i s n o t e w o r t h y s i n c e s t i l b e n e s a r e n o r m a l l y p r e s e n t n a t u r a l l y i n t h e more s t a b l e t r a n s form (Drewes and F l e t c h e r , 1974). b. E u s i d e r i n and V i r o l o n g i n Compounds 2 and 3 ( F i g u r e 7) d e m o n s t r a t e d s i m i l a r b e h a v i o r by TLC. B o t h d i s p l a y e d a p i n k c o l o u r upon s p r a y i n g w i t h H 2 S O a . Compound 2 was fo u n d t o have a m o l e c u l a r w e i g h t of 386 by MS. P r o t o n and m e t h o x y l c o u n t s by NMR and t h e MS r e v e a l e d t h e f o r m u l a C 1 8 H 1 3 0 2 (OMe)„ and t h e MS f r a g m e n t m/z = 208 s u g g e s t e d t h e p r e s e n c e of t h e s y s t e m ( C H 3 0 ) 3 C 6 H 2 C H C H C H 3 . F u r t h e r a n a l y s i s showed t h e s p e c t r a l d a t a t o be i d e n t i c a l t o t h a t r e p o r t e d f o r t h e b e n z o d i o x a n n e o l i g n a n , e u s i d e r i n ( F e r n a n d e s e t a l , 1980; G o t t l i e b e t a l , 1976; Hobbs and K i n g , 1960). The p r e c i s e s t r u c t u r e of t h a t compound has been e s t a b l i s h e d d e f i n i t i v e l y by l a n t h a n i d e i n d u c e d s h i f t d a t a ( B r a z F i l h o e_t a l , 1976). The c h e m i c a l s h i f t o f Me~3 ( 5 1.26) and t h e v a l u e f o r J (8 Hz) by 'H-NMR a r e e v i d e n c e f o r t h e t r a n s r e l a t i o n s h i p of t h e Ar-2/Me-3 g r o u p s ( F e r n a n d e z e t a l , 1980) and s u p p o r t t h e i d e n t i f i c a t i o n of compound 2 as e u s i d e r i n . E u s i d e r i n has p r e v i o u s l y been r e p o r t e d from V i r o l a  g u g g e n h e i m i i and V i r o l a p a v o n i s ( F e r n a n d e s e_t a_l, 1980) and v a r i o u s g e n e r a of t h e f a m i l y L a u r a c e a e ( G o t t l i e b e t a l , 1976). Compound 3 b e a r s c o n s i d e r a b l e r e s e m b l a n c e t o e u s i d e r i n ( 2 ) , b a s e d upon i t s UV, 1H-NMR s p e c t r u m and mass s p e c t r u m . The 88 presence of the MS fragments wi th m/z = 209 and 208 suggest the presence of the groups (CH 3 0)3C 6 H 2 CH 2 CHCH3 and ( C H 3 0 ) 3 C 6 H 2 C H C H C H 3 , r e s p e c t i v e l y . T h i s , combined w i th the appearance of two s e t s , each of two e q u i v a l e n t aromat ic p ro tons and f i v e methoxy groups in the 1H-NMR spectrum i s i n d i c a t i v e of the presence of an 8 - 0 - 4 ' - n e o l i g n a n . The 1H-NMR spectrum c l o s e l y ressembled tha t r epo r t ed fo r 1 - (3 ,4 ,5- t r ime thoxypheny l ) -2-a l l y l -2 ,6-d ime thoxyphenoxy ) -propane , a compound i s o l a t e d from seeds of M y r i s t i c a f r ag rans ( I soga i §_t §_1, 1973), a l s o of the M y r i s t i c a c e a e . The on ly d i f f e r e n c e s are in the occur rence of the s i g n a l s a t t r i b u t e d to the aromat ic a l l y l i c s u b s t i t u e n t as a doub le t (3H, J = 6 Hz) at 6 1.87 and m u l t i p l e t s between 6 6.05 (1H) and 6.25 and 5 6.2 and 6.5 (1H) in the spectrum of compound 3. These th ree s i g n a l s would, i n s t e a d , be expected to r e s u l t from an a romat i c p ropeny l group (Fernandes et a_l, 1980). Double resonance exper iments f u r t h e r suppor ted the ass ignments . I r r a d i a t i o n at 6 1.9 produced the c o l l a p s e of the s i g n a l at 5 6.05-6.25 and i r r a d i a t i o n at 6 6.2 caused the doub le t at 5 1.87 to appear as a s i n g l e t . c . E p i - s e s a r t e m i n , Sesa r temin , Epi-yanqambin and Yanqambin Compounds 4 and 5 ( F igure 8) behaved s i m i l a r l y in v a r i o u s chromatograph ic sys tems. Two s p o t s , forming a brown c o l o u r upon sp r a y i ng wi th H2SO<,, were d i s t i n g u i s h e d by TLC . Both compounds were found to have formulae C 2 3 H 2 6 0 8 by HR-MS and the 1H-NMR c l e a r l y i n d i c a t e d the presence of four methoxy and one methy lened ioxy group and two p a i r s of e q u i v a l e n t a romat i c 89 p r o t o n s . Fu r the rmore , s i g n a l s between 5 3.0 and 4.5 i n d i c a t e d the presence of a b i s - t e t r a h y d r o f u r a n r i n g . These compounds were conc luded to be 2 , 6 - d i a r y l - 3 , 7 - d i o x a b i c y c l o f 3 , 3 , 0 ] - o c t a n e type l i g n a n s . The C-1, C~5 bond of n a t u r a l l y o c c u r r i n g b i s -t e t r a h y d r o f u r a n l i g n a n s i s c h a r a c t e r i s t i c a l l y in the c i s c o n f i g u r a t i o n and the 1H-NMR and 1 3C-NMR s p e c t r a ob t a i ned (Tab les V and VI) i n d i c a t e tha t t h i s i s a l s o t rue of compounds 4 and 5. The a r y l s u b s t i t u e n t s of C-2 and C-6 can be e i t h e r a x i a l or e q u a t o r i a l , a l l o w i n g fo r th ree types of s t e r e o i s o m e r s . Compound 4 was conc luded to be an a x i a l -e q u a t o r i a l isomer on the b a s i s of the f o l l o w i n g f e a t u r e s of i t s 1H-NMR spectrum ( P e l t e r and Ward, 1978; Russe l and Fenemore, 1973): 1) a d i f f e r e n c e in C-1 and C-5 methine p ro ton chemica l s h i f t s (5 2.91 and 6 3 .15-3 .5 , r e s p e c t i v e l y ) , 2) a d i f f e r e n c e in the chemica l s h i f t s of the b e n z y l i c p ro tons at C-2 and C-6 ( 6 4.85 and 6 4 .41 , r e s p e c t i v e l y ) , 3) the presence of one a x i a l (C-4) methylene p ro ton u p f i e l d ( 6 3.15-6 3.5) from the "no rma l " p o s i t i o n ( 6 3.8 to 5 4.0) due to s h i e l d i n g by the a x i a l a romat ic r i n g and an e q u a t o r i a l methylene p ro ton (C-8) down f i e l d between 6 4.0 and 6 4 .25 , due to d e s h i e l d i n g by the e q u a t o r i a l a romat i c r i n g . Ch iba et a l (1980) used 1 3C-NMR to e s t a b l i s h the p o s i t i o n s of a r y l groups of unsymmet r i ca l l y s u b s t i t u t e d b i s -t e t r a h y d r o f u r a n l i g n a n s and Greger and Ho fe r ( l 980 ) based t h e i r ass ignment of the s t e r e o c h e m i s t r y on data ob t a i ned us i ng the l an than ide induced s h i f t t e c h n i q u e . In the p resen t s tudy , the 90 r e l a t i v e c o n f i g u r a t i o n s were a s s i g n e d on the b a s i s of 1 3C~NMR s p e c t r a . The chemica l s h i f t s of carbon atoms 1' and 1" are p a r t i c u l a r l y u s e f u l in de te rm in ing the s t e r eochem i s t r y of at tachment of the pheny l group to the b i s - t e t r a h y d r o f u r a n ske l e t on ( P e l t e r and Ward, 1978). Compounds 6 and 7 ( F igu re 8) are known, s ymmet r i c a l l y s u b s t i t u t e d b i s - t e t r a h y d r o f u r a n l i g n a n s and were i d e n t i f i e d on the b a s i s of t h e i r me l t i ng p o i n t s , o p t i c a l r o t a t i o n s and UV, IR, MS and 'H-NMR s p e c t r a l data which agreed c l o s e l y wi th p u b l i s h e d i n f o rma t i on (Abe et a l , 1974; B r i ggs et a l , 1968; Chen et a l , 1976; J e f f r i e s et a l , 1961; L a i e_t a l , 1973). The 1 3C-NMR data of compounds 6 and 7 were compared wi th those of the unsymmet r i ca l l y s u b s t i t u t e d compounds 4 and 5 and the ass ignments made on t h i s b a s i s . The 1' and 1" carbons of the d i e q u a t o r i a l l y s u b s t i t u t e d compound, yangambin (7 ) , have chemica l s h i f t s of 136.66 ppm, a va lue tha t compares we l l w i th those p u b l i s h e d by P e l t e r and Ward (1978) fo r s i m i l a r l i g n a n s . The a x i a l 3 ,4 ,5-t r ime thoxypheny l s u b s t i t u e n t of epi-yangambin (6 ) , which has a chemica l s h i f t of 133.89 ppm (Tab le V I ) , i s e a s i l y d i s t i n g u i s h e d from the e q u a t o r i a l one. The s i m i l a r i t y between t h i s va lue (133.89 ppm) and the s i g n a l seen in the spectrum of compound 4 (133.97 ppm) i s taken as ev idence tha t the 3 ,4 ,5-t r imethoxy pheny l s u b s t i t u e n t i s in the a x i a l p o s i t i o n in compound 4. The remain ing C-1 s i g n a l (135.88 ppm) was a s s i g n e d to the 3-methoxy-4,5-methylenedioxyphenyl s u b s t i t u e n t which was a l r e a d y known, from the 1H-NMR spect rum, to be in the e q u a t o r i a l p o s i t i o n , by d e f a u l t . The 1 3C-NMR spectrum of the 91 d i e q u a t o r i a l compound 5 s u p p o r t s t h e s e a s s i g n m e n t s s i n c e i t has s i g n a l s a t 135.86 ppm and 136.77 ppm, v a l u e s w h i c h c o r r e s p o n d c l o s e l y t o t h o s e a s s i g n e d a b o v e f o r e q u a t o r i a l 3-m e t h o x y - 4 , 5 - m e t h y l e n e d i o x y p h e n y l a n d 3 , 4 , 5 - t r i m e t h o x y p h e n y l s u b s t i t u e n t s , r e s p e c t i v e l y . A l l o t h e r 1 3C-NMR s i g n a l s showed c l o s e a g r e e m e n t w i t h t h e s p e c t r a r e p o r t e d f o r s i m i l a r b i s t e t r a h y d r o f u r a n l i g n a n s ( P e l t e r a n d Ward, 1 9 7 8 ) . The mass s p e c t r a o f compounds 4 and 5 a r e a l m o s t i d e n t i c a l a n d b e a r c l o s e r e s e m b l a n c e t o t h o s e o f compounds 6 and 7 w h i c h a r e a l s o a l m o s t i d e n t i c a l . The m o l e c u l a r f o r m u l a e f o r a l l o f t h e f r a g m e n t s d e s c r i b e d f o r compounds 4 and 5 were o b t a i n e d f r o m mass m e a s u r e m e n t s by h i g h r e s o l u t i o n mass s p e c t r o m e t r y . C o r r e s p o n d i n g s t r u c t u r e s a r e drawn i n F i g u r e 10. The b a s i c f r a g m e n t a t i o n p a t h w a y s d e s c r i b e d by P e l t e r ( 1 9 6 7) and D u f f i e l d ( 1 9 6 7) a r e e a s i l y i d e n t i f i a b l e . The f r a g m e n t a t i o n p a t t e r n o f compounds 6 a n d 7 h a s a l s o been i n c l u d e d ( F i g u r e 11) f o r c o m p a r i s o n . As h a s been o b s e r v e d by o t h e r s ( P e l t e r , 1967; T a n i g u c h i , 1 9 7 2 ) , r e l i a b l e d i f f e r e n c e s i n t h e s t r u c t u r e of i s o m e r s a r e n o t i d e n t i f i a b l e , d. D i h y d r o s e s a r t e m i n a n d 0 - D i h y d r o y a n q a m b i n Compounds 8 a n d 9 ( F i g u r e 9) a r e t h e most p o l a r c o n s t i t u e n t s o f t h e e t h e r e x t r a c t . They a r e s e p a r a t e d e a s i l y by TLC, compound 8 g i v i n g a s a l m o n - p i n k c o l o u r e d s p o t w i t h H 2 S O a and compound 9 a g r e y c o l o r a t i o n . The mass s p e c t r a o f t h e s e two compounds were e s p e c i a l l y i n f o r m a t i v e s i n c e t h e y p r o d u c e d p a r e n t i o n s w i t h m a s s e s 432 and 448, r e s p e c t i v e l y . T h e s e v a l u e s a r e j u s t 2 mass u n i t s g r e a t e r t h a n t h e p a r e n t F i gure 10 - Scheme of f r a g m e n t a t i o n of b i s - t e t r a h y d r o f u r a n l i g n a n s , e p i - s e s a r t e m i n and s e s a r t e m i n , by mass s p e c t r o m e t r y F i g u r e 11 - Scheme of f r a g m e n t a t i o n of b i s - t e t r a h y d r o f u r a n l i g n a n s , epi-yangambin and yangambin, by mass s p e c t r o m e t r y 94 i o n s of s e s a r t e m i n and yangambin, r e s p e c t i v e l y . F u r t h e r s i m i l a r i t i e s i n t h e mass s p e c t r a o f a l l f o u r compounds were a p p a r e n t . The mass s p e c t r u m o f d i h y d r o s e s a r t e m i n (8) showed two s e r i e s o f f r a g m e n t s : 181, 169, 168 and 165, 153, 152, i n d i c a t i n g t h e p r e s e n c e of b o t h 3~methoxy-4,5-m e t h y l e n e d i o x y p h e n y l and 3 , 4 , 5 - t r i m e t h o x y p h e n y l s u b s t i t u e n t s . The g r e a t e r abundance of t h e peak a t m/z = 181 t h a n t h a t a t m/z = 165, was t a k e n as e v i d e n c e t h a t t h e t r i m e t h o x y s u b s t i t u t e d a r o m a t i c g r o u p was a b e n z y l , r a t h e r t h a n a p h e n y l , s u b s t i t u e n t . The r e m a i n i n g f e a t u r e s of t h e mass s p e c t r u m r e s e m b l e t h o s e e x p e c t e d from a s u b s t i t u t e d t e t r a h y d r o f u r a n . The f r a g m e n t a t i o n p a t t e r n has been i n t e r p r e t e d a c c o r d i n g t o t h e schemes p r e s e n t e d by P e l t e r e_t a_l (1966) and P e l t e r ( 1 967) ( F i g u r e 12). The 1H-NMR s p e c t r u m s u p p o r t e d t h e p r o p o s a l t h a t t h e compound was a s u b s t i t u t e d t e t r a h y d r o f u r a n and p r o v i d e d i n f o r m a t i o n on i t s r e l a t i v e c o n f i g u r a t i o n . H a l l (1964) and H a l l ejt a l (1972) have d i s c u s s e d t h e a p p l i c a b i l i t y of NMR d a t a i n a s s i g n i n g t h e c o n f o r m a t i o n o f f u r a n o s e s i n s o l u t i o n . A t l e a s t 20 c o n f o r m e r s a r e r e c o g n i z e d . The r e l a t i v e i m p o r t a n c e of e a c h i n t h e d i s t r i b u t i o n o f a l l p o s s i b l e c o n f i g u r a t i o n s w i l l v a r y d e p e n d i n g upon t h e n a t u r e of t h e s u b s t i t u e n t s and t h e s o l v e n t u s e d . U n f o r t u n a t e l y , t h e v a r i e t y of t e t r a h y d r o f u r a n l i g n a n s p r e s e n t l y known i s n o t g r e a t and d e t a i l e d NMR s p e c t r a have been r e p o r t e d i n o n l y a l i m i t e d number o f c a s e s . Few d a t a a r e t h e r e f o r e a v a i l a b l e f o r c o m p a r i s o n . From t h e e x i s t i n g F i g u r e 12 - Scheme of f r a g m e n t a t i o n of t e t r a h y d r o f u r a n l i g n a n . d i h y d r o s e s a r t e r n i n , by mass sp e c t r o m e t r y 97 d a t a , i t appears that the f ea tu re tha t can be most e a s i l y determined i s the r e l a t i v e c o n f i g u r a t i o n at the benzy l c a r b o n . A c i s o r i e n t a t i o n of c o n s t i t u e n t s about the C-2-C-3 bond w i l l r e s u l t in d e s h i e l d i n g of the benzy l p ro ton and a s h i f t down f i e l d in i t s resonance to approx imate l y 6 5.5 ( B i r c h and Smi th , 1964; B i r c h et a l , 1967; Inoue et a l , 1981; Sarkanen and W a l l i s , 1973a and b; Smi th , 1963). A t r ans o r i e n t a t i o n r e s u l t s in the C-2 pro ton hav ing a chemica l s h i f t of approx imate l y 6 4 . 7 . The c o r r e s p o n d i n g s i g n a l observed in the spectrum of compound 8 i s a d o u b l e t , 6 4 .78 . T h i s va lue agrees we l l w i th the assignment of the t r ans c o n f i g u r a t i o n . The c o u p l i n g cons tan t may be expected to vary c o n s i d e r a b l y , depending upon the nature of the s u b s t i t u e n t s and the favoured con fo rmat ion of the furan r i n g . The p u b l i s h e d data are i n s u f f i c i e n t to a l l ow the use of c o u p l i n g cons tan t as a d e f i n i t i v e c r i t e r i o n fo r a s s i g n i n g c o n f i g u r a t i o n . The measured v a l u e , J = 6.3 Hz i s , however, in l i n e wi th s e v e r a l v a lues p r e v i o u s l y r e p o r t e d fo r the t r ans c o n f i g u r a t i o n about the C-2-C-3 bond of a s u b s t i t u t e d t e t r a h y d r o f u r a n (Sarkanen and W a l l i s , 1973a; Smi th , 1963). The r e l a t i v e c o n f i g u r a t i o n of the t e t r a h y d r o f u r a n l i g n a n , ( + ) - l a r i c i r e s i n o l has been e s t a b l i s h e d by chemica l methods. I t s o p t i c a l r o t a t i o n has been r epo r t ed as [a] = + 17.5 0 (Weinges, 1961). The o p t i c a l r o t a t i o n of compound 8 was found to be [a] = + 11.8 ° . These two va lues appear to be s u f f i c i e n t l y s i m i l a r to form the b a s i s f o r a s s i g n i n g the r e l a t i v e c o n f i g u r a t i o n of the remain ing carbon of compound 8. 98 The c o n f i g u r a t i o n i s t h e r e f o r e , 2S, 3R, .4R, i d e n t i c a l to tha t of ( + ) - l a r i c i r e s i n o l . The mass spectrum of compound 9 ( F igure 13) i s ana logous to tha t of d i h yd rosesa r t em in (8) ( F igure 12) sugges t i ng that i t , t o o , i s b i o s y n t h e t i c a l l y r e l a t e d to a b i s - t e t r a h y d r o f u r a n l i g n a n ; d i f f e r i n g on ly in that one furan r i n g i s opened, l e a v i n g a f r ee hyd roxy l g roup . When the 1H-NMR spectrum i s examined, however, i t i s apparent tha t a d i f f e r e n c e e x i s t s in the type of a romat ic s u b s t i t u e n t s p resen t in compound 9 and yangambin or ep i-yangambin . Ins tead of two s i n g l e t s , each a r i s i n g from two e q u i v a l e n t a romat ic p r o t o n s , one s i n g l e t i n t e g r a t i n g fo r 2 p ro tons (6 = 6.66) and two s i n g l e proton s i n g l e t s at 8 6.63 and 6.36 are obse r ved . T h i s pa t t e rn i s s i m i l a r to the s i n g l e t s at 6 6.84 and 6 6.43 which were observed fo r the 3 ,4-methy lenedioxy-6-methoxyphenyl group observed by Russe l and Fenemore (1973). A l though the p o s i t i o n i n g of the a romat i c groups cannot be unambiguously d e f i n e d on the b a s i s of on l y the 1H-NMR spect rum, the s t r u c t u r e has been t e n t a t i v e l y a s s i gned on the b a s i s of a compar ison of the chemica l s h i f t s w i th data from r e l a t e d compounds. In the 'H-NMR spectrum of d i h yd rosesa r t em in (8 ) , the chemica l s h i f t of the C-2 1 and C-6' p ro tons of the methoxy-methy lened ioxybenzy l s u b s t i t u e n t , which are r e s o l v e d i n t o two peaks at 6 6.53' and 6 .54, a re down f i e l d from the s i g n a l of the co r r e spond ing C-2" and C-6" p ro tons of the 3 ,4 ,5- t r ime thoxy-pheny l s u b s t i t u e n t oppos i t e (6 6 .40 ) . Assuming the same r e l a t i o n s h i p to e x i s t in the 1H-NMR spectrum 99 of /3-dihydroyangambin, t h e lo w e r f i e l d s i g n a l (a 2 p r o t o n s i n g l e t , 5 = 6.66) i s a s s i g n e d t o t h e p r o t o n s o f t h e b e n z y l s u b s t i t u e n t . T h i s would c o r r e s p o n d t o t h e two e q u i v a l e n t p r o t o n s o f t h e 3 , 4 , 5 ~ t r i m e t h o x y - b e n z y l g r o u p . The r e m a i n i n g two a r o m a t i c s i n g l e t s ( i n t e g r a t i n g a t 1 p r o t o n e a c h ) , w i t h c h e m i c a l s h i f t s 5 6.63 and 6 6.36 a r e a s s i g n e d t o t h e C-5" and C-2" p r o t o n s , r e s p e c t i v e l y . T h e s e c h e m i c a l s h i f t s a r e i n agreement w i t h 1H-NMR d a t a p u b l i s h e d f o r o t h e r l i g n a n s ( R u s s e l and Fenemore, 1973; T a n i g u c h i and Oshima, 1972a and b ) . The o p t i c a l r o t a t i o n of compound 9, [ a ] = +15.1 0 i s s i m i l a r t o t h a t of (+)- l a r i c i r e s i n o l , [ a ] = +17.5 0 (Weinges, 1961), s u g g e s t i n g s i m i l a r c o n f i g u r a t i o n s a t c a r b o n s 2,3 and 4. A l t h o u g h t h e c h e m i c a l s h i f t , 5 = 4.75, i s i n d i c a t i v e of a t r a n s c o n f i g u r a t i o n a b o u t C2-C3, t h e c o u p l i n g c o n s t a n t m e asured from t h e 400 MHz s p e c t r u m , J = 8.5, i s l a r g e r t h a n t h a t o f compound 8, J = 6.3, and o t h e r v a l u e s p u b l i s h e d f o r t e t r a h y d r o f u r a n l i g n a n s w i t h a n a l o g o u s c o n f i g u r a t i o n s . The 1H-NMR d a t a f o r compounds 8 and 9 a r e s u f f i c i e n t l y d i f f e r e n t t o s u g g e s t t h a t t h e i r r e l a t i v e c o n f i g u r a t i o n s a r e a l s o d i f f e r e n t and no f u r t h e r a t t e m p t was made t o a s s i g n t h e s t e r e o c h e m i s t r y o f compound 9. 100 PART B. EXAMINATION OF THE BIOLOGICAL ACTIVITY OF V i r o l a e longa ta EXTRACTS 1. INTRODUCTION T h i s s e c t i o n d e s c r i b e s the s e r i e s of b i o l o g i c a l exper iments tha t were c a r r i e d out in an e f f o r t to determine i f a t o x i c c o n s t i t u e n t was present in the bark r e s i n and to t r y to e l u c i d a t e the nature of the e f f e c t s observed in mice i n j e c t e d wi th e x t r a c t s of t h i s m a t e r i a l . The methods used to prepare the e x t r a c t s were somewhat d i f f e r e n t from those used to o b t a i n p u r i f i e d compounds fo r s p e c t r o s c o p i c a n a l y s i s . F u l l d e t a i l s have been i n c l u d e d in the f o l l o w i n g s e c t i o n . 2. MATERIALS AND METHODS The c o l l e c t i o n and i d e n t i f i c a t i o n of p l an t m a t e r i a l has been d e s c r i b e d in Part A, 2a. Except where i n d i c a t e d o t h e r w i s e , the bark samples which had been p r e se r ved in methanol immediate ly a f t e r be ing c o l l e c t e d were used in the p r e p a r a t i o n of e x t r a c t s fo r b i o l o g i c a l t e s t i n g . The e x t r a c t s ob ta ined from bark which had been d r i e d and that which had been p r e se r ved in methanol were i n d i s t i n g u i s h a b l e by TLC and HPLC. a . P r e p a r a t i o n of e x t r a c t s A l k a l o i d a l and n o n - a l k a l o i d a l e x t r a c t s V i r o l a e l onga t a bark (28 g dry weight) was m i l l e d and e x t r a c t e d e x h a u s t i v e l y at 20 0 C w i th 100% methano l . The 278 mg of m a t e r i a l e x t r a c t e d was suspended in water , a c i d i f i e d ( to 101 pH 3 wi th H C l ) , and e x t r a c t e d wi th d i e t h y l e t h e r . The aqueous phase was next b a s i f i e d (pH 12 wi th NaOH) and e x t r a c t e d w i th d i ch lo romethane . T h i s formed the a l k a l o i d a l f r a c t i o n (12.5 mg). The remain ing aqueous and d i e t h y l e ther f r a c t i o n s were combined and n e u t r a l i z e d to form the n o n - a l k a l o i d a l f r a c t i o n (260 mg). Aqueous e x t r a c t D r i e d V. e l onga ta bark (30 g) was m i l l e d and e x t r a c t e d w i th d i s t i l l e d water at 20 0 C fo r t h r e e , e i g h t hour p e r i o d s . The mix ture was a g i t a t e d c o n s t a n t l y du r i ng e x t r a c t i o n . A f t e r f i l t r a t i o n of the combined e x t r a c t s , the s o l u t i o n was evapora ted in vacuo and s to r ed at 4 0 C u n t i l use . D i e t h y l e ther e x t r a c t The methanol p r e se r ved bark was d r i e d , m i l l e d and e x t r a c t e d f i v e t imes w i th dry d i e t h y l e t h e r . The e x t r a c t was f i l t e r e d and c o n c e n t r a t e d by r o t a r y e vapo ra t i on in. vacuo and the brown, syrupy r e s i d u e s t o r ed at -30 0 C u n t i l use . b. F r a c t iona t ion of e x t r a c t s  Aqueous e x t r a c t T h i s e x t r a c t was f r a c t i o n a t e d by ion exchange chromatography. A column was p repared from tho rough l y washed Dowex 50WX8 (2-50 mesh, H + form) r e s i n . The sample was d i s s o l v e d in 1N HCl and a p p l i e d to the co lumn. I t was e l u t e d wi th aqueous s o l u t i o n s of pH 6, 8, 10 and I2 ( ad jus ted us ing NaOH). F r a c t i o n s were n e u t r a l i z e d immediate ly a f t e r c o l l e c t i o n . The f r a c t i o n s were moni tored fo r the presence of 1 02 i n d o l e a l k a l o i d s and amino a c i d s by TLC us ing E h r l i c h ' s and n i n h y d r i n r e a g e n t s , r e s p e c t i v e l y . D i e t h y l e ther e x t r a c t The s e p a r a t i o n of t h i s e x t r a c t was s i m i l a r to that d e s c r i b e d in Par t B, 2b of t h i s c h a p t e r , c . Chromatographic a n a l y s i s of e x t r a c t s  HPLC Q u a n t i t a t i v e a n a l y s i s of e x t r a c t s was c a r r i e d out u s i ng a V a r i a n MCH-10 reve rse phase column on a V a r i a n HPLC wi th a V a r i a n v a r i a b l e wavelength d e t e c t o r . The sample was e l u t e d wi th methanol/water by g r a d u a l l y i n c r e a s i n g the methanol c o n c e n t r a t i o n from 50% to 100% over a 20 minute p e r i o d . The UV abso rb ing substances, were de t e c t ed at 250 nm. Q u a n t i f i c a t i o n was performed by comparing peak h e i g h t s of the sample chromatogram wi th the va lues from a s tandard curve prepared us ing known q u a n t i t i e s of pure compounds. TLC Because the four major c o n s t i t u e n t s of the d i e t h y l e ther e x t r a c t were not r e a d i l y r e s o l v e d by HPLC, t h i n l a y e r chromatography systems were used to es t imate the r e l a t i v e amounts of these c o n s t i t u e n t s . Polygram S i l i c a g e l U V 2 5 a TLC p l a t e s were used wi th e i t h e r pe t ro leum e t h e r / d i e t h y l e t h e r / a c e t o n i t r i l e (6/6/1) or hexane/ c h l o r o f o r m / e thano l (25/25/1) as d e v e l o p i n g s o l v e n t s . 1 0 3 d . Assay of spontaneous motor a c t i v i t y The appara tus used to measure spontaneous locomotor a c t i v i t y i s a m o d i f i c a t i o n of the " j i g g l e cage" (K innard and Watzman, 1 9 6 6 ) . The same procedure was used to measure spontaneous motor a c t i v i t y of mice in the p r e v i o u s l y d e s c r i b e d study on the b i o l o g i c a l a c t i v i t y of J . p e c t o r a l i s . Complete d e t a i l s of the appara tus and the method fo r r e c o r d i n g spontaneous motor a c t i v i t y are p r o v i d e d in Chapter I I , s e c t i o n 2a. The spontaneous motor a c t i v i t y was measured in a r b i t r a r y u n i t s . The measur ing dev i ce was s e n s i t i v e not on l y to g ross b e h a v i o r a l a c t i v i t y , but a l s o to more s u b t l e behav io r such as grooming or even b r e a t h i n g . The s i g n a l s produced were rough ly p r o p o r t i o n a l in magnitude to the type of a c t i v i t y obse r ved . Female Swiss mice ( 3 0 - 3 5 g) were used in t h i s study to examine the e f f e c t s of the e x t r a c t s . A l l an imals were of comparable ages and were ma in ta ined under s tandard c o n d i t i o n s of an imal c a r e . E x t r a c t s were i n j e c t e d i n t r a p e r i t o n e a l l y in a l i q u o t s of e i t h e r 5 0 or 1 0 0 M L D i s t i l l e d water was used as a v e h i c l e fo r the aqueous e x t r a c t s . Because of i t s s o l u b i l i t y p r o p e r t i e s and low t o x i c i t y (Budden et a l , 1 9 7 9 ; Worth ley and S c h o t t , 1 9 6 6 ) , Tween 8 0 (Sigma) in water at a c o n c e n t r a t i o n of 1 0 % (v/v) was used as a v e h i c l e fo r a d m i n i s t e r i n g the d i e t h y l e ther e x t r a c t s . Record ing of locomotor a c t i v i t y was begun immediate ly a f t e r i n j e c t i o n and c a r r i e d out f o r 6 0 m inu tes . Because of the e f f e c t of such v a r i a b l e s as l i g h t i n g (Walsh and Cummins, 1 9 7 6 ) , no i s e ( I n g l i s , 1 9 7 5 ) , the presence 1 04 or absence of an observer (Nor ton , 1980), or time in the a n i m a l s ' c i r c a d i a n rhythm (Hughes e_t a_l, 1978), these c o n d i t i o n s were s t a n d a r d i z e d . Locomotor a c t i v i t y was measured under ambient f l u o r e s c e n t l i g h t and in the presence of a cons tan t machine no i se of low i n t e n s i t y . A l l assays were c a r r i e d out between 7 and 10 PM in the presence of a s i n g l e o b s e r v e r . e. Assay of a n t i - a g g r e s s i v e a c t i v i t y L ignans were examined fo r t h e i r a b i l i t y to i n h i b i t i s o l a t i o n induced a g g r e s s i o n . Male Swiss mice were i s o l a t e d by hous ing them s i n g l y in cages fo r a p e r i o d of at l e a s t s i x weeks. T h i s procedure r e s u l t e d in d i s t i n c t b e h a v i o r a l responses in most m ice . Two types of behav io r were d i s t i n g u i s h e d when an i s o l a t e d mouse was p l a ced in a cage wi th a mouse not p r e v i o u s l y i s o l a t e d ( s o c i a l i z e d mouse) . Some of the i s o l a t e d mice l o s t the behav io r i sms that norma l l y accompany s o c i a l i z a t i o n . I n s t e ad , they r e s i s t e d any at tempts at con tac t made by the s o c i a l i z e d mouse, becoming ne rvous , assuming a d e f e n s i v e pos tu re and e m i t t i n g sharp squeaks . Mice which responded in t h i s f a s h i o n were used in a s tudy of the e f f e c t of the l i g n a n , e p i - s e s a r t e m i n , on t h i s a n t i s o c i a l b e h a v i o r . Most of the remainder of the i s o l a t e d mice were openly a g g r e s s i v e when p l a c e d in the same cage as a s o c i a l i z e d mouse. A f t e r a r e l a t i v e l y b r i e f p e r i o d , d u r i n g which the mice e x p l o r e d each o t h e r ' s o r a l and a n o g e n i t a l r e g i o n s , the i s o l a t e d mouse g e n e r a l l y a t t a c k e d and f i g h t i n g ensued . The four b i s - t e t r a h y d r o f u r a n l i g n a n s i s o l a t e d from V. e l onga t a 1 05 bark were examined fo r t h e i r a b i l i t y to reduce the a g g r e s s i v e n e s s observed in i s o l a t e d mice . 3. RESULTS The on l y p h y s i o l o g i c a l l y a c t i v e substances known from the genus V i r o l a are the a n t i - s c h i s t o s o m a l n e o l i g n a n s , sur inamens in and v i r o l i n from V i r o l a sur inamens is (Barata e_t a l , 1978), the a n t i - f u n g a l n e o l i g n a n , (+)-gua iac in from V i r o l a  c a r i n a t a ( G o t t l i e b , Maia and R i b e i r o , 1976) and t r yp tamine and /3-carboline d e r i v a t i v e s from v a r i o u s spec i e s ( A g u r e l l e_t a l , 1969; Holmstedt et a l , 1980). I t has been proposed tha t the potent h a l l u c i n o g e n i c a c t i v i t y of the t r yp t am ines , 5-MeODMT and DMT accounts fo r the use of V i r o l a bark r e s i n as an arrow po i son ( G a l e f f i et a l , 1983; Maia and Rodr igues , 1976). T h i s h ypo thes i s was examined. The a l k a l o i d s of the V. e l onga t a bark used in t h i s study had been ana l y sed and shown to c o n s i s t of a s i n g l e t r yp t am ine , 5-MeODMT, at a c o n c e n t r a t i o n of 0.245 mg/g dry weight (McKenna et a_l, 1984). Five-MeODMT was the on l y t r yp tamine de t e c t ed by G a l e f f i e_t a_l (1983) in t h e i r a u t h e n t i c sample of the arrow p o i s o n . The q u e s t i o n of the importance of t h i s compound to the p h a r m a c o l o g i c a l e f f e c t s of the bark r e s i n was approached by p r e p a r i n g a methano l i c e x t r a c t of the bark and s e p a r a t i n g i t i n t o a l k a l o i d a l and n o n - a l k a l o i d a l f r a c t i o n s ( d e s c r i b e d in M a t e r i a l s and Methods, s e c t i o n 2a ) . The e f f e c t s of the e x t r a c t s were compared w i th r espec t to the g ross b e h a v i o r a l responses observed in mice a f t e r i n t r a p e r i t o n e a l i n j e c t i o n . 106 The r e s u l t s are summarized in Tab le V I I . The r e s u l t s c l e a r l y i n d i c a t e tha t the an ima l s ' g ross behav io r was a l t e r e d to a g r ea t e r extent by the n o n - a l k a l o i d a l e x t r a c t than by the a l k a l o i d a l e x t r a c t . Oppos i t e e f f e c t s were e l i c i t e d by the two e x t r a c t s . Whereas the a l k a l o i d a l e x t r a c t induced a m i l d degree of h y p e r a c t i v i t y at the h i ghes t doses t e s t e d , a comparable dose of the n o n - a l k a l o i d a l f r a c t i o n r e s u l t e d in a marked r e d u c t i o n in a c t i v i t y and a s t a t e of s tupor l a s t i n g s e v e r a l h o u r s . a . Examinat ion of the aqueous f r a c t i o n fo r t o x i c i t y The l ack of t o x i c i t y of the methano l i c e x t r a c t of V. e longa ta r a i s e d the p o s s i b i l i t y that a t o x i c c o n s t i t u e n t not e x t r a c t e d by t h i s so l v en t might be p r e s e n t . Mebs e_t aJL (1982) have r epo r t ed the t o x i c na ture of a p o l y p e p t i d e of an aqueous e x t r a c t of the l a r vae of Diamphida n i g r o r n a t a , a Bushman arrow p o i s o n . To examine whether V. e l onga t a con t a i ned a t o x i n which was e i t h e r i n s o l u b l e i n , or i n a c t i v a t e d by o rgan i c s o l v e n t s , an aqueous e x t r a c t of the bark was examined fo r pha rmaco log i c a l a c t i v i t y . T h i s e x t r a c t was a d m i n i s t e r e d i n t r a p e r i t o n e a l l y to female Swiss mice in doses of 40, 100 and 200 mg/kg. W i th in two minutes of i n j e c t i n g 200 mg/kg f requent w r i t h i n g was obse r ved . The an imals became somewhat uncoord ina ted and, w i t h i n f i v e minutes of i n j e c t i o n , motor a c t i v i t y was s i g n i f i c a n t l y r educed . The an imals l ay prone and an i n c r ea sed r a t e of r e s p i r a t i o n , accompanied by p i l o e r e c t i o n , was obse r ved . A f t e r a p e r i o d of two hours , normal behav io r g r a d u a l l y resumed. 1 07 Dose adm in i s t e r ed Behav iora l . response Amt. bark r ep resen ted by e x t r a c t Amt. 5-MeODMT in a l k a l o i d f r a c t ion Non-a l k a l o i d a l f r a c t i o n A l k a l o i d a l f r a c t i o n in jec ted (mg) m g mg/kg 5 (20)t 0.05 1 .0 s i i g h t reduct ion in a c t i v i t y no e f f e c t 1 0 (40) 0.09 1 .9 r e d u c t i o n in a c t i v i t y no e f f e c t 20 (80) 0.19 3.8 i n a c t i v i t y : 1 durat ion hr no e f f e c t 40 (160) 0.36 7.5 i n a c t i v i t y : 2 durat ion hr s i h y p e r a c t : 10 min 80 (320) 0.74 15.0 i n a c t i v i t y : > 3 h r dura t ion s i h y p e r a c t : 15 min Tab le VII - Gross b e h a v i o r a l responses of Swiss mice to a d m i n i s t r a t i o n of a l k a l o i d a l and n o n - a l k a l o i d a l e x t r a c t s of V i r o l a e l onga ta bark . Three mice per dose were o b s e r v e d : e x t r a c t s . w e r e adm in i s t e r ed by i n t r a p e r i t o n e a l i n j e c t i o n . t numbers in b r a cke t s r e f e r to dose ( in mg/kg) of n o n - a l k a l o i d a l f r a c t i o n 108 Lower doses of t h i s e x t r a c t (100 and 40 mg/kg) produced s i m i l a r b e h a v i o r a l changes , but of l e s s e r i n t e n s i t y and s h o r t e r d u r a t i o n . Three mice were i n j e c t e d w i th each dose and complete recovery a f t e r 3 hours was observed in a l l c a s e s . An attempt was made to concen t r a t e the b i o l o g i c a l a c t i v i t y of t h i s e x t r a c t by f r a c t i o n a t i n g i t u s i n g ion exchange chromatography. A l l of the f r a c t i o n s o b t a i n e d , however, caused w r i t h i n g and reduced a c t i v i t y l e v e l s when i n j e c t e d i n t o mice . Each f r a c t i o n was examined f o r the presence of p o l a r a l k a l o i d s or amino a c i d s by s p r a y i n g t h i n l a y e r chromatograms of the f r a c t i o n s wi th E h r l i c h ' s , i o d o p l a t i n a t e and n i n h y d r i n r e a g e n t s . Only nega t i v e r e s u l t s were o b t a i n e d . The p o s s i b i l i t y tha t the i r r i t a n t a c t i v i t y observed in the aqueous e x t r a c t r e s u l t e d from the presence of a complex p h e n o l i c mix ture was c o n s i d e r e d . Tann ic a c i d (J. L. Baker) was i n j e c t e d at a dose of 20 mg/kg. I t produced a response which was i n d i s t i n g u i s h a b l e from that of the aqueous e x t r a c t of the V i r o l a ba rk . The mice responded by w r i t h i n g w i t h i n two minutes of the i n j e c t i o n . P i l o e r e c t i o n , a prone pos tu re and a r e d u c t i o n in a c t i v i t y l a s t i n g approx imate l y th ree hours were a l s o obse r ved . 109 b. Examinat ion of the d i e t h y l e x t r a c t for d e p r e s s i o n of  spontaneous motor a c t i v i t y Having determined that s t r o n g l y t o x i c c o n s t i t u e n t s were absent from both the aqueous and the methanol f r a c t i o n s and tha t the n o n - a l k a l o i d a l f r a c t i o n of the methanol e x t r a c t possessed the s t ronges t e f f e c t when a d m i n i s t e r e d to mice , t h i s f r a c t i o n was examined f u r t h e r . P r e l i m i n a r y f r a c t i o n of the n o n - a l k a l o i d a l f r a c t i o n by TLC on s i l i c a ge l y i e l d e d non-polar f r a c t i o n s which s t i l l possessed the a b i l i t y to reduce the a c t i v i t y l e v e l s of mice i n j e c t e d p e r i t o n e a l l y . More than one of the f r a c t i o n s demonstrated t h i s a c t i v i t y . Because of t h i s , and the time consuming nature of and v a r i a b i l i t y in the b i o a s s a y , the approach of f r a c t i o n a t i o n , gu ided by bioassay," was abandoned. I n s t ead , the s e p a r a t i o n and p u r i f i c a t i o n of the major c o n s t i t u e n t s of the a c t i v e d i e t h y l e ther e x t r a c t of the bark was under taken . The b i o l o g i c a l a c t i v i t i e s of the major compounds were then examined at c a r e f u l l y measured doses . A m o d i f i e d v e r s i o n of the " j i g g l e cage " was used to r e co rd locomotor a c t i v i t y . T h i s approach a l lowed a q u a n t i t a t i v e es t imate of the compound's e f f e c t i v e n e s s in r educ ing locomotor a c t i v i t y . T h i r t e e n compounds were i s o l a t e d and i d e n t i f i e d from the d i e t h y l e ther e x t r a c t . These were: the u b i q u i t o u s p h y t o s t e r o l , ^ - s i t o s t e r o l ; two s t i l b e n e d e r i v a t i v e s , 3 , 4 ' , 5 - t r i m e t h o x y - c i s -s t i l b e n e (1a) and i t s t r ans isomer (1b ) ; the n e o l i g n a n s , e u s i d e r i n (2) and v i r o l o n g i n (3 ) ; the b i s - t e t r a h y d r o f u r a n 110 l i g n a n s , e p i - s e s a r t e m i n (4 ) , sesa r temin (5 ) , epi-yangambin (6) and yangambin (7 ) ; and the t e t r a h y d r o f u r a n l i g n a n s d i h yd rosesa r t em in (8) and /3-dihydroyangambin (9 ) . In a d d i t i o n , two u n i d e n t i f i e d a romat i c c o n s t i t u e n t s , compounds X and Y, were i s o l a t e d and t e s t e d . The i s o l a t i o n and i d e n t i f i c a t i o n of these compounds by s p e c t r o s c o p i c means has been d e s c r i b e d in Part A of the p resen t c h a p t e r . The a b i l i t y of each of these compounds to suppress spontaneous motor a c t i v i t y in mice was t e s t e d . A pure sample of each compound was i n j e c t e d i n t r a p e r i t o n e a l l y in a dose of 25 mg/kg. Some of the compounds t e s t e d caused marked r e d u c t i o n in spontaneous locomotor a c t i v i t y . T h i s was e s p e c i a l l y obv ious in the case of the b i s - t e t r a h y d r o f u r a n l i g n a n s . The r e d u c t i o n in motor a c t i v i t y caused by ep i - sesa r t em in i s ev iden t in the d i m i n i s h e d e l e c t r i c a l output of the f o r c e t r ansduce r ( F igure 14). T h i s s i g n a l decreased s t e a d i l y between 10 and 60 minutes f o l l o w i n g i n j e c t i o n and g r a d u a l l y began to recover between 80 and 90 minutes p o s t - i n j e c t i o n . T y p i c a l t ime cou rses fo r the r e d u c t i o n in motor a c t i v i t y induced by ep i - se sa r t em in and e p i -yangambin can be observed in F i g u r e 15. A l l of the compounds i s o l a t e d were t e s t e d in t h i s way. The average of the 24 measurements of locomotor a c t i v i t y made (at 5 minute i n t e r v a l s ) d u r i n g the 2 hour p e r i o d f o l l o w i n g i n j e c t i o n was c a l c u l a t e d in each c a s e . The r e s u l t s of t h i s t e s t i n g are p resen ted in Tab le V I I I . Ten of the t h i r t e e n compounds t e s t e d produced a s i g n i f i c a n t r e d u c t i o n i n a c t i v i t y I l l F i g u r e 14 - Example of o u t p u t of " j i g g l e c a g e " f o r c e t r a n s d u c e r u s e d t o measure s p o n t a n e o u s motor a c t i v i t y o f m i c e . a) r e c o r d e d d u r i n g n o r m a l motor a c t i v i t y , b) r e c o r d e d a f t e r i n j e c t i o n o f e p i - s e s a r t e m i n (25 mg/kg): C h a r t 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.) F i g u r e 15 - E f f e c t of b i s - t e t r a h y d r o f u r a n l i g n a n s , e p i -s e s a r t e m i n and e p i - y a n g a m b i n on s p o n t a n e o u s l o c o m o t o r a c t i v i t y of m i c e . V a l u e s a r e means, N= 3: SEMs a r e o m i t t e d from g r a p h but were a l l l e s s t h a n 15%. 1 1 3 Compound Dose(mg/kg) t Reduct ion in a c t i v i t y t-% C o n t r o l l e v e l SEM(n=4) ^ - s i t o s t e r o l 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 c o n t r o l Tween 80 1 00 7 Tab l e VI I I - E f f e c t of p u r i f i e d compounds of V . e longa ta bark on spontaneous locomotor a c t i v i t y of Swiss mice . t Compounds were i n j e c t e d in 50 M1 of 25% aqueous Tween 80. t- Va lues are averages of 4 i n j e c t i o n s per compound: SEM = s tandard e r r o r of mean. 1 1 4 l e v e l . 3 , 4 ' , 5 - t r i m e t h o x y - c i s - s t i l b e n e (1a ) , v i r o l o n g i n (3 ) , e p i - s e s a r t e m i n (4 ) , sesa r temin (5 ) , epi-yangambin (6 ) , yangambin (7) and d i h yd rosesa r t em in (8) a l l produced approx imate l y a 50% r e d u c t i o n in a c t i v i t y at the dose a d m i n i s t e r e d . 3 , 4 ' , 5 - T r i m e t h o x y - t r a n s - s t i l b e n e (1b) , e u s i d e r i n (2) and 0-dihydroyangambin (9) showed l e s s e r , but s t i l l s i g n i f i c a n t a c t i v i t y . Only the unknown compounds (X and Y) and /3-s i tos te ro l were i n a c t i v e . c . Q u a n t i f i c a t i o n of major c o n s t i t u e n t s of d i e t h y l e ther  e x t r a c t Because such a l a rge percentage of the compounds t e s t e d possessed s i g n i f i c a n t i n h i b i t o r y a c t i v i t y on mice , i t became of importance to determine the c o n c e n t r a t i o n s of each of the compounds in the e x t r a c t s . Only then c o u l d the r e l a t i v e importance of each c o n s t i t u e n t to the o v e r a l l pha rmaco log i c a l response of the bark r e s i n be es t ima ted a c c u r a t e l y . H igh performance l i q u i d chromatography was u t i l i z e d to q u a n t i f y the UV abso rb ing compounds of the e x t r a c t . The r e s u l t s of t h i s a n a l y s i s are p resen ted in Tab le IX. It can be seen from Tab le IX that the four b i s -t e t r a h y d r o f u r a n l i g n a n s a r e , q u a n t i t a t i v e l y , the most important c o n s t i t u e n t s . Toge the r , they compr ise approx imate l y 38%, by we ight , of the t h i r t e e n compounds q u a n t i f i e d . More i m p o r t a n t l y , w i th respec t to the p resen t s tudy , i s the f a c t that they r ep resen t approx imate l y 87% of the c o n s t i t u e n t s that were a c t i v e in supp ress ing locomotor a c t i v i t y . 1 1 5 Compound RT(min) Rf C o n c e n t r a t i o n Mg/g d r y wt b a r k 1 2 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 . 1 9 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 - s i t o s t e r o l 0.75 0.33 95 t-T a b l e IX - Q u a n t i t a t i v e a n a l y s i s of t h i r t e e n most common c o n s t i t u e n t s o f d i e t h y l e t h e r e x t r a c t of V. e l o n g a t a b a r k . s o l v e n t 1 = p e t e t h e r / e t h y l e t h e r / a c e t o n i t r i l e ( 6 / 6 / 1 ) . s o l v e n t 2 = h e x a n e / c h l o r o f o r m / e t h a n o l ( 2 5 / 2 5 / 1 ) . t amounts e s t i m a t e d , b a s e d upon t h e r e l a t i v e s i z e of UV a b s o r b i n g s p o t s on TLC. t- amount e s t i m a t e d , b a s e d upon amount i s o l a t e d f r o m 1500 g o f d r i e d b a r k . 1 1 6 d . E f f e c t of b i s - t e t r a h y d r o f u r a n 1iqnans on i s o l a t i o n induced  a g g r e s s i o n A f t e r the a d m i n i s t r a t i o n of compounds 4 to 7, i t was observed tha t the t r e a t e d mouse o f t e n behaved i n an unusua l l y p a s s i v e manner. At doses lower than those r e q u i r e d to suppress locomotor a c t i v i t y , d i s t i n c t b e h a v i o r a l changes c o u l d be d i s c e r n e d . Normal e x p l o r a t o r y a c t i v i t y was very much reduced and a gene ra l h e s i t a n c y was appa ren t . The mice were e s p e c i a l l y t i m i d and when handled showed l i t t l e r e s i s t a n c e . They were ext remely r e l u c t a n t to defend themse lves . These o b s e r v a t i o n s l ed to the h ypo thes i s tha t the b i s -t e t r a h y d r o f uran l i g n a n s possessed a n t i - a g g r e s s i v e a c t i v i t y and tha t t h i s may p l ay a r o l e in the use of V. e l onga t a bark as an arrow p o i s o n . T h i s h ypo thes i s was t e s t e d by examining the e f f e c t of these four compounds upon a g g r e s s i o n induced in mice by i s o l a t i o n . The i n d u c t i o n of agg re s s i on in mice by some s o r t of i s o l a t i o n procedure has been used e x t e n s i v e l y as an expe r imen ta l model of a g g r e s s i o n (B ra in and Jones , 1982; Sco t t and F r e d e r i c s o n , 1951). A v a r i e t y of parameters have been measured as i n d i c a t o r s of a g g r e s s i v e n e s s . In the p resen t s t udy , i s o l a t e d mice were s e l e c t e d f o r e x p e r i m e n t a t i o n , based upon e i t h e r ove r t agg re s s i v eness or t h e i r nervousness upon i n t r o d u c t i o n to a group-housed mouse. These two groups of mice d i s p l a y e d d i f f e r e n t b e h a v i o r . Consequen t l y , d i f f e r e n t measurements were made in e i t h e r c a s e . The l e s s a g g r e s s i v e group of i s o l a t e d mice were used to 1 1 7 examine the e f f e c t of d i f f e r e n t doses of the most abundant b i s - t e t r a h y d r o f u r a n l i g n a n , ep i - sesa r t em in (4) on agg re s s i on r e l a t e d b e h a v i o r . These mice were observed to i n t e r a c t w i th newly i n t r oduced group-housed mice in a c h a r a c t e r i s t i c manner. Each i s o l a t e d mouse r e s i s t e d at tempts at s o c i a l i z a t i o n by the group-housed mouse, adop t i ng a d e f e n s i v e pos tu re and e m i t t i n g a s e r i e s of sharp squeaks . When not c o n f r o n t e d by the group-housed mouse, they d i s p l a y e d e x p l o r a t o r y b e h a v i o r , combined w i th p u r s u i t and a n o g e n i t a l i n v e s t i g a t i o n of the group-housed mouse. T h i s l a t t e r a c t i v i t y has been c o r r e l a t e d w i th a g g r e s s i v e behav io r and i s p r e d i c t i v e of i t (Simon et a l , 1983). As i n d i c a t o r s of b e h a v i o r , these three a c t i v i t i e s were measured d u r i n g a 5 minute i n t e r v a l beg inn ing 15 minutes a f t e r i n j e c t i o n of l i g n a n . The r e s u l t s are p resen ted in Tab l e X. The r e s u l t s i n d i c a t e tha t ep i - sesa r t em in had a dose-dependent e f f e c t upon the behav ior observed in p r e v i o u s l y i s o l a t e d m ice . Not on ly was the t ime spent in e x p l o r a t o r y behav io r reduced upon a d m i n i s t r a t i o n of t h i s l i g n a n , but the d e f e n s i v e behav io r observed in response to s o c i a l con t a c t by the group-housed mouse, was a t t enua t ed s i g n i f i c a n t l y . Moreover , the time spent in a c t i v e l y pu r su ing the group housed mouse was markedly r educed , even at the lowest dose t e s t e d (3.1 mg/kg). T h i s i s e s p e c i a l l y sugges t i v e of a r e d u c t i o n in a g g r e s s i v e n e s s . To v e r i f y t h i s i n i t i a l o b s e r v a t i o n , i s o l a t e d mice which demonstrated o v e r t l y a g g r e s s i v e behav io r were used to t e s t the e f f e c t of the four b i s - t e t r a h y d r o f u r a n l i g n a n s . Each was 1 18 Dose of Behav ior observed in 5 minute p e r i o d Ep i - sesa r t em in Number of % time in % t ime in p u r s u i t (mg/kg) squeaks e x p l o r a t ion p l u s a n o g e n i t a l i n ves t i ga t 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 Tab l e X - E f f e c t of e p i - s e s a r t e m i n on th ree b e h a v i o r a l parameters r e l a t e d to agg re s s i v enes s of i s o l a t e d m ice . Va lues are averages of th ree i n j e c t i o n s of each dose . 1 19 i n j e c t e d at doses of 2 5 mg/kg and the e f f e c t on the r e ad ines s of the i s o l a t e d mouse to a t t a c k the group-housed mouse was obse r ved . F i v e minutes a f t e r i n j e c t i o n , the mice were p l a c e d toge ther and the time e l apsed be fo re the f i r s t a t t ack by the i s o l a t e d mouse was measured. The data of Tab le XI i n d i c a t e that each of the l i g n a n s t e s t e d s i g n i f i c a n t l y suppressed the tendency of the p r e v i o u s l y i s o l a t e d mouse to a t t a c k . The mice t r e a t e d in t h i s way were p a r t i c u l a r l y p a s s i v e , o f f e r i n g l i t t l e r e s i s t a n c e to the s o c i a l con tac t i n i t i a t e d by the group housed p a r t n e r . They responded by remain ing q u i e t l y in one co rne r of the cage and hudd l i ng w i th eyes p a r t i a l l y c l o s e d , when c o n t a c t s were made. 4. DISCUSSION The r e s u l t s of t h i s study i n d i c a t e that the arrow po i son prepared from V i r o l a bark may we l l be unique among a b o r i g i n a l arrow p o i s o n s . The p h y s i o l o g i c a l e f f e c t s observed in mice were s u b t l e and r e s t r i c t e d to b e h a v i o r . No s e r i o u s d i s r u p t i o n of normal p h y s i o l o g y c o u l d be d i s t i n g u i s h e d and no m o r t a l i t y was observed in any of the mice t e s t e d , d e s p i t e the a d m i n i s t r a t i o n of r e l a t i v e l y l a r g e doses of bark e x t r a c t . The Yanomamo d a r t s ana l y sed by G a l e f f i et auL (1983) were r e p o r t e d to each c a r r y approx imate l y 150 mg of r e s i n . Labora to ry mice r e a d i l y s u r v i v e d doses of 2 0 0 mg/kg of V . e l onga t a bark e x t r a c t . I t seems u n l i k e l y that w i l d game, which would be expec ted to weigh in excess of 1 kg and would , t h e r e f o r e , r e c e i v e a s i g n i f i c a n t l y l e s s e r dose than tha t 1 20 Compound Latency to a t t a c k : t ime (min . ) ep i - sesa r t em in (4 ) >30 sesa r temin (5 ) >30 epi-yangambin(6) >30 yangambi n(7) >30 c o n t r o l 3.7 Tab le XI - E f f e c t of b i s - t e t r a h y d r o f u r a n l i g n a n s on agg re s s i v enes s of m i ce . La tency to a t t a ck r e f e r s to the t ime between the i n t r o d u c t i o n the i s o l a t e d mouse (to which l i g n a n had been admin i s t e r ed ) to the s o c i a l i z e d mouse and the time of the f i r s t a t t a c k of the i s o l a t e d mouse. 121 adm in i s t e r ed to the mice in t h i s s tudy , would s u f f e r any m o r t a l i t y from a d m i n i s t r a t i o n of the r e s i nous p o r t i o n of the d a r t s . The sugges t i on tha t the t r yp tamine c o n s t i t u e n t s of the bark are r e s p o n s i b l e fo r i n t e r r u p t i n g the normal responses of i n j u r e d game, in t h i s way f a c i l i t i a t i n g i t s c a p t u r e , i s an i n t r i g u i n g one. If t r u e , i t i s an i n t e r e s t i n g e t h n o b i o l o g i c a l phenomenon. There seems to be no p r e p a r a t i o n w i th comparable e f f e c t s amongst the many types of arrow p o i s o n s . The pr imary a l k a l o i d of the ma jo r i t y of the bark samples of V i r o l a  e l onga ta that have been ana l ysed i s 5-MeODMT ( A g u r e l l et a l , 1969; 1980; G a l e f f i et a l , 1983; Holmstedt et a l , 1980; McKenna e_t a l , 1984). When t e s t e d fo r a b i l i t y to b ind to s e r o t o n i n r e cep to r s i t e s (Glennon and Gessne r , 1975), b e h a v i o r a l e f f e c t s in rodents (Gessner and Page, 1962; Glennon et a l , 1980; Ho et a_l, 1970) and psychotomimet ic a c t i v i t y in man ( S h u l g i n , 1978), 5-MeODMT i s c o n s i s t e n t l y shown to be the most potent n a t u r a l l y o c c u r r i n g t r y p t a m i n e . However a p p e a l i n g t h i s h ypo thes i s i s , the data p resen ted here o f f e r no support fo r i t . The n o n - a l k a l o i d a l and a l k a l o i d a l f r a c t i o n s from e q u i v l e n t amounts of V. e longa ta bark were compared and the n o n - a l k a l o i d a l f r a c t i o n was c l e a r l y observed to have a g r ea t e r e f f e c t on the behav io r of the mice to which they were a d m i n i s t e r e d (Tab le V I I ) . The i n j e c t i o n of the n o n - a l k a l o i d a l f r a c t i o n at a dose of 3.8 mg/kg was r e s p o n s i b l e fo r a r e d u c t i o n in a c t i v i t y l a s t i n g s e v e r a l h o u r s , wh i l e the a l k a l o i d a l f r a c t i o n from an e q u i v a l e n t amount of 1 22 bark (0.19 mg/kg) produced no obse rvab le change in b e h a v i o r . B e h a v i o r a l changes in mice in response to 5-MeODMT a d m i n i s t r a t i o n have been de t e c t ed at doses as low as 0.5 mg/kg (Ho et a l , 1970; Chapter I I , t h i s t h e s i s ) . It i s necessa ry to c o n s i d e r the p o s s i b i l i t y tha t the l e v e l of 5-MeODMT in the e l onga ta bark sample used to prepare the e x t r a c t s fo r these exper iments was u n u s u a l l y low. G a l e f f i et a_l (1983) r epo r t that 5-MeODMT compr ised some 8% (by weight) of the r e s i n c o a t i n g the t i p s of the Yanomamo d a r t s ana l y sed by them. A l though the bark r e s i n , i t s e l f , has not been used in t h i s s tudy , i t i s l i k e l y tha t a methano l i c e x t r a c t of the bark bears a c l o s e resemblance to the r e s i n wi th r e spec t to type and amount of chemica l c o n s t i t u e n t s . The a l k a l o i d a l f r a c t i o n (which c o n s i s t e d e n t i r e l y of 5-MeODMT) compr ised 4.5% (by weight) of the methano l i c e x t r a c t . A l though t h i s f i g u r e i s s l i g h t l y l e s s than tha t r epo r t ed by G a l e f f i e_t a l (1983) , the d i f f e r e n c e i s not s u f f i c i e n t to e x p l a i n the r e l a t i v e l a ck of a c t i v i t y observed from the a l k a l o i d a l f r a c t i o n in these expe r imen t s . The r e d u c t i o n in locomotor a c t i v i t y observed by the non-a l k a l o i d a l e x t r a c t i s l i k e l y the r e s u l t of the complex mixture of p h e n o l i c s p r e s e n t . The i d e n t i t y of the red-brown c o n s t i t u e n t which forms upon exposure of the r e s i n to a i r i s not known. I t has been suggested tha t i t i s the r e s u l t of the o x i d a t i o n of f l a v o n o i d c o n s t i t u e n t s and perhaps the fo rmat ion of pigmented dimers ( G o t t l i e b , 1979). The c o n t r i b u t i o n of the non-po lar c o n s t i t u e n t s i d e n t i f i e d 1 23 from the d i e t h y l e ther e x t r a c t to the b i o l o g i c a l a c t i v i t y of the n o n - a l k a l o i d a l f r a c t i o n i s d i f f i c u l t to a s s e s s . The r e s u l t s of the locomotor a c t i v i t y s t u d i e s c a r r i e d out show that compounds 1 to 9 a r e , to v a r y i n g deg rees , e f f e c t i v e in r educ ing spontaneous motor a c t i v i t y at moderate ly h igh doses ( 1 0-25 mg/kg). It i s u n c l e a r , however, whether the c o n c e n t r a t i o n s of these c o n s t i t u e n t s i s h igh enough in the r e s i n to make a s i g n i f i c a n t c o n t r i b u t i o n to i t s o v e r a l l b i o l o g i c a l e f f e c t s . The b i s - t e t r a h y d r o f u r a n l i g n a n s ep i - se sa r t em in (4 ) , sesa r temin (5 ) , epi-yangambin (6) and yangambin (7 ) , because of t h e i r h igh c o n c e n t r a t i o n s compared to the other c o n s t i t u e n t s of the e x t r a c t , would be expected to c o n t r i b u t e p r o p o r t i o n a t e l y more to the t o t a l b i o l o g i c a l a c t i v i t y . The b i s - t e t r ahyd ro f uran l i g n a n , p i n o r e s i n o l |3-D-glucoside has been shown to have hypo tens i ve a c t i v i t y and s y r i n g a r e s i n o l 0-D-g lu cos i de i s c l a imed to i n c r e a s e the performance of an imals under s t r e s s ( reviewed by MacRae and Towers, 1984). The mechanism by which these two l i g n a n s exe r t t h e i r e f f e c t i s not unde r s tood . S i m i l a r l y , the mechanism by which compounds 4 to 7 reduce i s o l a t i o n induced agg re s s i on i s comp le te l y unknown. Many agents are now known which a f f e c t agg re s s i on in i s o l a t e d m ice . Among those which have been shown to reduce i t are a n t i p s y c h o t i c drugs such as ch lo rp romaz ine ( K r e i s k o t t , 1981) or r e s e r p i n e ( La i e_t a l , 1975), the a n x i o l y t i c drugs such as b a r b i t u r a t e s or benzod i azep ines (Haefe ly e_t a_l, 1981), the 1 24 a l k a l o i d s morphine and muscimol ( Posh i v a l o v , 1982), c e r t a i n o p i a t e pep t i des such as met-enkepha l in and neo-endorphin and c e r t a i n h y p o t h a l a m i c - p i t u i t a r y p e p t i d e s such as somatos ta t i n ( Posh i v a l o v , 1982). The .doses r e q u i r e d fo r an e f f e c t to be observed range between 1 and 25 mg/kg fo r a l l but the a n x i o l y t i c d rugs , f o r which much h igher doses are r e q u i r e d . The b i s - t e t r a h y d r o f u r a n l i g n a n s , e p i - s e s a r t e m i n , s e s a r t e m i n , epi-yangambin and yangambin a r e , by compar i son , moderate ly e f f e c t i v e a n t i - a g g r e s s i v e a g e n t s . It i s p o s s i b l e that the n e o l i g n a n s , e u s i d e r i n and v i r o l o n g i n , as we l l as the t e t r a h y d r o f u r a n l i g n a n s d i h yd rosesa r t em in and 0-dihydroyangambin , may a l s o p l a y a minor r o l e in the pha rmaco log i c a l a c t i v i t y of the r e s i n . The e ther e x t r a c t of Magnol ia obovata ba rk , a Ch inese m e d i c i n a l agen t , has been shown to have s eda t i v e e f f e c t s in mice (Watanabe e_t a_l, 1973). The major c o n s t i t u e n t s are neo l i gnans ( F u j i t a e_t a_l, 1972) not u n l i k e e u s i d e r i n and v i r o l o n g i n . The a v a i l a b l e ev idence i n d i c a t e s that V. e l onga ta bark does not c o n t a i n a h i g h l y t o x i c c o n s t i t u e n t which might e x p l a i n i t s use as an arrow p o i s o n . Tox i c compounds have not p r e v i o u s l y been r epo r t ed from the f am i l y M y r i s t i c a c e a e . None was de t e c t ed d u r i n g a thorough s c r een ing of V. e l onga ta ba rk . Moreover , methanol e x t r a c t s of V.. p a v o n i s , V. s e b i f e r a and V. c a l o p h y l l a have a l s o been t e s t e d by a d m i n i s t e r i n g them to mice at a dose of 100 mg/ml and found no ev idence of t o x i c i t y or m o r t a l i t y . I t can be conc luded tha t the use of V i r o l a s p . as an arrow po i son i s e i t h e r based on a more s u b t l e b i o l o g i c a l 1 2 5 a c t i v i t y than t o x i c i t y o r , a l t e r n a t i v e l y , that i t i s wi thout a p h y s i o l o g i c a l b a s i s . It shou ld be po in t ed out tha t s e v e r a l important u n c e r t a i n t i e s remain r ega rd ing the use of V. e l onga ta as an arrow p o i s o n . The f i r s t concerns the exact means of p r e p a r i n g the r e s i n . The present study may be c r i t i c i z e d because r e s i n p repa red in the manner of the Yanomamos has not been used . S ince the method of p r e p a r i n g the r e s i n i s s t r a i g h t f o r w a r d and i n v o l v e s no th ing more than c o l l e c t i n g and warming the bark over a f i r e , i t seems improbable that the da r t t i p r e s i n d i f f e r s s i g n i f i c a n t l y from an e x t r a c t of the ba rk . The p o s s i b i l i t y e x i s t s , however, tha t the a p p l i c a t i o n of heat promotes the fo rmat ion of some a c t i v e c o n s t i t u e n t and t h i s s h o u l d , t h e r e f o r e , be borne in mind. The source of the V i r o l a sample i s a l s o an important c o n s i d e r a t i o n . The bark used in t h i s study was c o l l e c t e d in Pe ru , whi le the m a t e r i a l used in the p r e p a r a t i o n of a u t h e n t i c Yanomamo arrow po i son i s ob t a i ned from t r ee s growing in no r the rn B r a z i l . The p o s s i b i l i t y that geography may c o n t r i b u t e to q u a l i t a t i v e or quant i t i a t i v e v a r i a t i o n in chemica l c o n s t i t u e n t s must be c o n s i d e r e d . F i n a l l y , there i s some doubt as to the r e l a t i o n s h i p between V. e l onga t a and V. t h e i o d o r a , the two sources of Yanomamo arrow p o i s o n . V i r o l a t h e i o d o r a i s c o n s i d e r e d a separa te s p e c i e s by S chu l t e s (1969) and most of the sources of arrow po i son have been a t t r i b u t e d to t h i s s p e c i e s . S p e c i a l i s t s in the taxonomy of t h i s genus have g e n e r a l l y regarded V. 1 26 t h e i o d o r a and V. e longa ta to be synonomous (Rodr igues , 1980; Smith and Wodehouse, 1937) and in the l a t e s t monograph, on ly the l a t t e r name i s r e cogn i zed (Rodr igues , 1980). A l though the s p e c i e s concept i s not yet comp le t e l y r e s o l v e d , the re i s gene ra l agreement that two types can be d i s t i n g u i s h e d in the f i e l d . Both are used as a snuf f and arrow po i son (P rance , G . T . , p e r s . comm.). The present study i s the f i r s t r epo r t of the n o n - a l k a l o i d a l c o n s t i t u e n t s of e i t h e r t r e e . A compara t i ve study of the chemis t r y of these two types i s now in o r d e r . Gaps e x i s t in our unders tand ing of the e t h n o l o g i c a l a spec t s of V i r o l a r e s i n arrow p o i s o n . Of p a r t i c u l a r importance i s the way in which i t i s used fo r h u n t i n g . Sa l a the (1931) has r epo r t ed tha t i t i s used fo r hun t ing monkeys and b i r d s . Becher (1960) , Prance (1970) and S chu l t e s and Holmstedt (1968) have p o i n t e d out tha t t h i s i s a slow a c t i n g arrow po i son and that the wounded an imal must be f o l l owed fo r some time so as to a l l ow the substance to take e f f e c t . I t i s c o n c e i v a b l e tha t a s s a y i n g the r e s i n by i n t r a p e r i t o n e a l i n j e c t i o n in mice i s hot an a p p r o p r i a t e expe r imenta l sys tem. E i t h e r the mode of a d m i n i s t r a t i o n or the response of the p a r t i c u l a r an ima l used may i n t r oduce unsuspected v a r i a b l e s . 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MULTIPLE SCREENING OF AMAZONIAN EUPHORBIACEAE FOR  BIOLOGICAL ACTIVITIES 1. INTRODUCION The f a m i l y , Euphorb iaceae , i s w e l l r ep resen ted among the p l a n t s used by ind igenous South Amer icans (Appendix A ) . It i s the t h i r d most important p l a n t f am i l y in the Amazon r e g i o n , w i th respec t to numbers of spec i e s used as m e d i c i n a l p l a n t s by the l o c a l i n h a b i t a n t s (Chapter I ) . I t compr ises some 6% of the a lmost 800 s p e c i e s of m e d i c i n a l p l a n t s enumerated (Appendix A).- The range of pha rmaco log i c a l a c t i o n s observed i s broad and i n c l u d e s p r e p a r a t i o n s e f f e c t i v e a g a i n s t wounds, s k i n i n f e c t i o n s , m a l i g n a n c i e s , i n f l ammat ions and used as t o n i c s and a p h r o d i s i a c s . Many s p e c i e s are po isonous and t h e i r use i s p robab l y based on t h i s p r o p e r t y . C e r t a i n s p e c i e s are used w ide ly as m e d i c i n a l s by the l a r g e mes t i zo p o p u l a t i o n . They are f r e q u e n t l y p r e s c r i b e d by h e r b a l i s t s and can be purchased in the market p l a c e s . These i n c l u d e A l cho rnea c a s t a n e i f o l i a , P h y l l a n t h u s u r i n a r i a and Cro ton l e c h l e r i . . A l c h o r n e a c a s t a n e i f o l i a i s a sma l l t r ee that grows, p a r t l y submerged d u r i n g the p e r i o d when the r i v e r s are h i g h , a long the banks of the Amazon's major t r i b u t a r i e s . I t s bark i s h i g h l y esteemed f o r the t reatment of rheumat ic 1 35 c o n d i t i o n s and i t s l eaves are taken as a d e c o c t i o n to s t i m u l a t e , or r e j u vena t e , the o l d e r man. P h y l l a n t h u s u r i n a r i a , and perhaps a l s o Phy l l an thus amarus, are used to t r e a t a number of a i lmen t s of the kidney and are b e l i e v e d to be e f f e c t i v e in c aus ing the f r agmenta t ion and/or e x p u l s i o n of k idney and g a l l s t ones . Croton l e c h l e r i i s t r ee of the t i e r r a  f irme whose t h i c k red bark r e s i n i s c o l l e c t e d and used as a wound treatment and fo r i n t e r n a l i n j u r i e s and m a l i g n a n c i e s . From pe r sona l e x p e r i e n c e , the c l a i m s conce rn ing i t s a b i l i t i e s to promote h e a l i n g of s u p e r f i c i a l wounds seem not to be g r e a t l y exaggera ted . Two s p e c i e s , J a t ropha cu r cas and J a t ropha  g o s s y p i i f o l i a , are dooryard c u l t i v a r s . They are both used w ide ly in the t reatment of burns and sk in i n f e c t i o n s and o c c a s i o n a l l y to t r e a t m a l i g n a n c i e s . In Jonathan H a r t w e l l ' s survey of p l a n t s used aga in s t c ance r , the f am i l y Euphorb iaceae i s very we l l r ep resen ted ( H a r t w e l l , 1969). The genera Aca l ypha (9 s p e c i e s ) , Croton (9 s p e c i e s , i n c l u d i n g C. pa l anos t i gma , Chamaesyce (both C. hyssop i f o l i a and C. thymi f o l i a , among o t h e r s ) , J a t ropha (9 s p e c i e s , i n c l u d i n g J . cu r cas and J.. gossyp i i f o l ia ) , Manihot (Manihot e s c u l e n t a ) , and P h y l l a n t h u s (3 s p e c i e s , i n c l u d i n g P. u r i n a r i a ) are a l r e ady r ep resen ted in the l i t e r a t u r e . Only a few of the p l a n t s l i s t e d above have been examined p r e v i o u s l y f o r t h e i r b i o l o g i c a l l y a c t i v e chemica l c o n s t i t u e n t s . Of those tha t have, J a t ropha cu r c a s has been found to c o n t a i n a compound, j a t r ophone , that possesses ant i tumour a c t i v i t y (Kupchan et. a_l, 1976). S i m i l a r l y , 1 36 Phy l l an thus b r a s i l i e n s i s , a p l an t tha t i s c l o s e l y r e l a t e d to P. u r i n a r i a and P. amarus, has been shown to c o n t a i n the ant i tumour subs tance , p h y l l a n t h o c i n (Kupchan, 1977). One of the s p e c i e s , Croton l e c h l e r i , has been found to c o n t a i n an a l k a l o i d , t a s p i n e , tha t i s not on ly a n t i -in f lammatory (Pers inos-Perdue et a_l, 1979), but a l s o possesses the a b i l i t y to i n h i b i t v i r a l r e ve r se t r a n s c r i p t a s e ( S e t h i , 1977). Major c o n s t i t u e n t s of s e v e r a l o ther s p e c i e s have been found to be l i g n a n s : dimers of pheny lp ropano id u n i t s . R e c e n t l y , J a t ropha gossyp i i f o l i a has been shown to c o n t a i n the l i g n a n , 2-p ipe rony l -3-ve r a t r y l -3R-7-bu t y ro l a c tone (Cha t t e r j ee et a l , 1981). Phy l l an thus u r i n a r i a has been s t u d i e d i n t e n s i v e l y from the po in t of view of i t s chemis t r y and f i v e l i g n a n s have been i d e n t i f i e d : p h y l l a n t h i n , h y p o p h y l l a n t h i n , n i r a n t h i n , n i r t e t r a l i n , and p h y l t e t r a l i n (An janeyu lu , 1973). These compounds are s i m i l a r in s t r u c t u r e to the p o d o p h y l l o t o x i n group of l i g n a n s . P o d o p h y l l o t o x i n , and r e l a t e d compounds, have r e c e n t l y been shown to have a n t i v i r a l a c t i v i t y (Bedows and H a t f i e l d , 1982). The v a r i e t y in s t r u c t u r e of secondary p roduc t s from Euphorb iaceous s p e c i e s i s a l r e ady known to be g r e a t . The predominant b i o l o g i c a l a c t i v i t y observed fo r compounds produced by p l a n t s of t h i s f am i l y i s t o x i c i t y (K inghorn , 1979). The p o s s i b i l i t y tha t the presence of t o x i c compounds c o u l d e x p l a i n the widespread uses of Euphorb iaceous s p e c i e s in the Amazon was c o n s i d e r e d . I t i s appa ren t , from what l i t t l e we know of the d i s e a s e s 1 37 of p r i m i t i v e Amazonian s o c i e t i e s , tha t i n f e c t i o n s and i n f e c t i o u s d i s e a s e s have always f i g u r e d p rominen t l y ( B l ack , 1975; B lack et a l , 1978; Buck et a l , 1968; L a r r i c k et a l , 1979). I n f e c t i o n s of the eye , ear and mouth are not uncommon and the tendency fo r i n j u r i e s or i n s e c t b i t e s to become i n f e c t e d i s always great ( L a r r i c k et a_l, 1979). The i n c i dence of exposure to the funga l pathogens H i s top lasma capsu la tum or P a r a c o c c i d i o d e s b r a s i l i e n s i s i s h igh ( L a r r i c k e_t §_1,1979; Mok and N e t t o , 1978). The v i r a l d i s e a s e s , herpes s imp l ex , mononuc leos is ( Eps te in-Ba r r v i r u s ) , ch i cken pox, h e p a t i t i s B and c y tomega lov i rus d i s e a s e s have been shown to be endemic to a l l the B r a z i l i a n t r i b e s examined ( Ba ruzz i et aJL, 1971; B l a ck , 1975; Nee l et a_l, 1968). Amazonians are r e g u l a r l y exposed to a v a r i e t y of p ro tozoan and h e l m i n t h i c p a r a s i t e s ( L a r r i c k et. a l , 1979) . It i s reasonab le to expect tha t a p r o p o r t i o n a t e l y l a rge f r a c t i o n of the m e d i c i n a l p l a n t s used by ind igenous Amazonians would be u t i l i z e d in the t reatment of i n f e c t i o u s d i s e a s e s of t h i s n a t u r e . The t o x i c p r o p e r t i e s of Euphorb iaceous s p e c i e s may be a p p l i e d in such a way. These o b s e r v a t i o n s suggested tha t the s c r e e n i n g of Amazonian Euphorb iaceous s p e c i e s f o r a v a r i e t y of pha rmaco log i c a l a c t i v i t i e s r e l a t e d to t o x i c i t y or growth r e g u l a t i o n , would c o n s t i t u t e an i n t e r e s t i n g s t udy . T h i r t y - f o u r s p e c i e s from t h i s f a m i l y were s e l e c t e d at random and samples of l e a f , bark (or the a e r i a l p o r t i o n s of herbaceous spec i e s ) were c o l l e c t e d . S i x t een of these 34 s p e c i e s had some 1 38 e t h n o b o t a n i c a l use . E x t r a c t s of the p l an t m a t e r i a l were p repared and examined fo r t h e i r a b i l i t y to i n h i b i t the growth of b a c t e r i a , y e a s t s , dermatophyt i c f u n g i , v i r u s e s and po ta to tumours, as we l l as t h e i r t o x i c i t y to b r i ne sh r imp . A n t i b a c t e r i a l a c t i v i t y a g a i n s t the gram nega t i ve o rgan i sm, E. c o l i and the gram p o s i t i v e bac t e r i um , S. aureus was examined. The yeas t s t e s t e d i n c l uded b rewer ' s y e a s t , S. c e r e v i s i a e and the pathogen, C. a l b i c a n s . Four dermatophyt i c fung i were t e s t e d : Microsporum c a n i s , M. fu lvum, M. gypseum and T r i chophy ton g a l l i n a e . Two an imal v i r u s e s fo r which l a b o r a t o r y t echn iques fo r h a n d l i n g are a v a i l a b l e were examined fo r t h e i r s e n s i t i v i t y to the e x t r a c t s . These were S i n d b i s v i r u s , a membrane e n c a p s u l a t e d , s i n g l e s t randed RNA v i r u s and murine c y t o m e g a l o v i r u s , a membrane e n c a p s u l a t e d , double s t r anded DNA v i r u s . The a b i l i t y of the 34 Euphorb iaceous e x t r a c t s to i n h i b i t the growth of tumours e l i c i t e d by Agrobac te r ium tumefac iens in po ta to tuber d i s c s was a l s o s t u d i e d . T h i s b i o l o g i c a l assay has been shown to be p r e d i c t i v e of an e x t r a c t ' s a b i l i t y to i n h i b i t the growth of 3PS leukemia c e l l s i_n v i v o ( F e r r i g n i et a l , 1982) . F i n a l l y , the gene ra l t o x i c i t y of e x t r a c t s was a s sessed us i ng the b r i n e shr imp assay (Meyer et a_l, 1982). T o x i c i t y to b r i n e shr imp (Artemia s a l i n a ) has been used to moni tor the presence of a v a r i e t y of compounds i n c l u d i n g p e s t i c i d e s , p o l l u t a n t s , myco tox ins , a n e s t h e t i c s , d i n o f l a g e l l a t e t o x i n s , morphine a n a l g e s i c s , coca r c i nogens and ca r c i nogens (Meyer et 1 39 a l , 1982). It i s a l s o capab le of d e t e c t i n g pho rbo l e s t e r s , the growth r e g u l a t o r y compounds tha t are widespread in the f am i l y Euphorb iaceae (Kinghorn et a_l, 1977). The p resen t study i s in tended to p rov ide a d e s c r i p t i o n of c e r t a i n of the b i o l o g i c a l a c t i v i t i e s of an e t h n o b o t a n i c a l l y impor tan t , yet c h e m i c a l l y l i t t l e known, group of p l a n t s . I t was hoped tha t t h i s approach would y i e l d gene ra l i n f o rma t i on on the types and po t enc i e s of the pha rmaco log i c a l a c t i v i t i e s among the s p e c i e s t e s t e d which w i l l , pe rhaps , h e l p e x p l a i n t h e i r e t h n o b o t a n i c a l u ses . It may be p o s s i b l e to ga in some a p p r e c i a t i o n of the extent to which the b i o l o g i c a l a c t i v i t i e s of t h i s group of p l a n t s i s known to ind igenous Amazonians (or what has been t r a n s m i t t e d i n t o the e t h n o b o t a n i c a l l i t e r a t u r e ) . T h i s i n f o rma t i on may l ead to the even tua l i s o l a t i o n and i d e n t i f i c a t i o n of n o v e l , b i o l o g i c a l l y a c t i v e c o n s t i t u e n t s . 2. MATERIALS AND METHODS a . P l an t m a t e r i a l P l an t s were c o l l e c t e d in Peru in 1981. Voucher specimens have been d e p o s i t e d at UNAP ( I q u i t o s , P e ru ) , San Marcos (L ima, P e ru ) , Ch icago F i e l d Museum and the UBC he rba r ium. De te rm ina t i ons were c a r r i e d out by Dr . M.J . H u f t , Ch icago F i e l d Museum. Samples were p rese r ved in methanol soon a f t e r c o l l e c t i o n . An e x t r a c t of p o d o p h y l l i n , the r e s i n of Podophyl lum s p . rhizome was i n c l u d e d in each of the assays as a p o s i t i v e c o n t r o l . I t i s known to c o n t a i n p o d o p h y l l o t o x i n , as we l l as a 1 40 number of o ther l i g n a n s , w i th an t i t umour , a n t i v i r a l and t o x i c a c t i v i t y (MacRae and Towers, 1984). b. P r e p a r a t i o n of p l an t e x t r a c t s L ea f , ba rk , or the a e r i a l pa r t of herbaceous p l a n t s , was homogenized in a Waring b lender and e x t r a c t e d e x h a u s t i v e l y w i th methanol at 20 ° C . The e x t r a c t s were c o n c e n t r a t e d by e vapo ra t i on i_n vacuo and p a r t i t i o n e d between water and e t h y l a c e t a t e . The f r a c t i o n s were s e p a r a t e d , d r i e d by e vapo ra t i on in  vacuo and d i s s o l v e d in e i t h e r 95% or 50% aqueous e thano l f o r the e t h y l a c e t a t e and aqueous f r a c t i o n s , r e s p e c t i v e l y . The f i n a l c o n c e n t r a t i o n of the e x t r a c t s was 100 mg/ml. They were s t o r e d at -30 ° C . c . A n t i m i c r o b i a l s c r een ing B a c t e r i a and yeas ts The e x t r a c t s were examined fo r t h e i r a b i l i t y to i n h i b i t the growth of the b a c t e r i a , E. c o l i and S. aureus and the yeas t s S. c e r e v i s i a e and C. a l b i c a n s . The paper d i s c assay method was used to assess a n t i m i c r o b i a l a c t i v i t y . Overn ight c u l t u r e s of the b a c t e r i a and yeas t s were a p p l i e d to agar c o n t a i n i n g n u t r i e n t b ro th (D i f co ) or Sabouraud 's dex t rose b r o t h ( D i f c o ) , r e s p e c t i v e l y , u s ing a s t e r i l e c o t t o n swab. P l an t e x t r a c t s (100 mg/ml) were a p p l i e d in 10 nl a l i q u o t s (img) to s t e r i l e 6 mm d iameter d i s c s of Whatman No. 1 f i l t e r pape r . These were a i r d r i e d and a p p l i e d to the agar p l a t e s to which b a c t e r i a or yeast had been added. They were incuba ted at 30 °C u n t i l a d i s t i n c t " l awn" was v i s i b l e (24 to 48 h o u r s ) . 141 The zone of i n h i b i t i o n around any of the d i s c s was noted and i t s d iameter r e c o r d e d . Assays were c a r r i e d out in t r i p l i c a t e . Dermatophyt ic fung i S p o r u l a t i n g c u l t u r e s of M. c a n i s , M. gypseum, M. fulvum and T . g a l l i n a e were ob ta ined from the Department of Med i c a l M i c r o b i o l o g y , U .B .C . The method used to assay the e x t r a c t s fo r i n h i b i t i o n of growth was a m o d i f i c a t i o n of tha t r e p o r t e d by Brancato and G o l d i n g (1953) . Spore suspens ions were prepared by add ing 1 ml of s t e r i l e d i s t i l l e d water to a mature c u l t u r e on an agar s l a n t and v o r t e x i n g i t b r i e f l y . Us ing a sharp wire t i p , a sma l l amount of the spore suspens ion was t r a n s f e r r e d to a p l a t e of Sabouraud 's aga r , in which had been i n c l u d e d v a r i o u s amounts of p l an t e x t r a c t and 1% e t h a n o l . The p l a t e s were l e f t at 20 °C fo r 14 days . The d iameter of the c o l o n i e s formed was measured. The assay was c a r r i e d out in t r i p l i c a t e , d . Ant i v i r a l a c t i v i t y A n t i v i r a l a c t i v i t y was assayed by de te rm in ing the e f f e c t s of e x t r a c t s on p laque forming a b i l i t y of S i n d b i s v i r u s and murine c y tomega lov i rus (MCMV). S i n d b i s v i r u s i s a s i n g l e s t r anded RNA v i r u s in the f a m i l y T o g a v i r i d a e wh i l e MCMV i s a double s t randed DNA v i r u s of the herpes v i r u s g roup . In each c a s e , the e f f e c t of the e x t r a c t s on v i r u s p r i o r to i n f e c t i o n and on c e l l s a l r e a d y i n f e c t e d wi th v i r u s , was measured. V i r u s e s and c e l l s S i n d b i s v i r u s (ob ta ined o r i g i n a l l y from Dr . D .E . Vance) had been grown in BHK-21 c e l l s and p u r i f i e d by the techn ique 1 42 d e s c r i b e d p r e v i o u s l y (Mosmann and Hudson, 1973). It was s t o r e d in t i s s u e c u l t u r e medium at -70 °C p r i o r to use . Murine c y tomega lov i rus (Smith s t r a i n ) had been propagated in ME c e l l s , p u r i f i e d and s t o r e d in the same manner. Both v i r u s e s were assayed by t h e i r e f f e c t on mouse (3T3 s t r a i n ) embryo c e l l s (passages 18 to 21 ) . C e l l s were c u l t i v a t e d in D u l b e c c o ' s m o d i f i e d E a g l e ' s medium (Gibco) c o n t a i n i n g 0.37% sodium b i c a r b o n a t e , 10% f e t a l bov ine serum (G ibco ) , 100 Un i t s /m l p e n i c i l l i n G (S igma), 100 Mg/ml' s t r ep tomyc in s u l f a t e (Sigma) and 2 Mg/ml econazo le ( C i l a g -Chemie AG) . They were incubated at 37 °C in an atmosphere of 95% a i r : 5% carbon d i o x i d e . P laque forming assay The assays were c a r r i e d out s i m i l a r l y f o r S.indbis v i r u s and MCMV. They d i f f e r e d on ly in the d i l u t i o n of the v i r u s prepared i n i t i a l l y and in the s i z e of the p e t r i d i s h e s on which the mouse embryo c e l l s were grown. In the case of S i n d b i s v i r u s , the p laques are r e l a t i v e l y l a r g e (4-5 mm) and 60 mm diameter d i s h e s were used ; MCMV produces sma l l e r p l aques (approx 1 mm) and 35 mm diameter d i s h e s were used . The v i r u s e s were exposed to p l a n t e x t r a c t s e i t h e r p r i o r to i n f e c t i o n ( p r e - i n f e c t i o n ) or a f t e r i n f e c t i o n ( pos t -i n f e c t i o n ) of c e l l s . In the t reatment p r e - i n f e c t i o n assay , a p p r o p r i a t e d i l u t i o n s of v i r u s were exposed to the p l an t e x t r a c t in D u l b e c c o ' s m o d i f i e d Eag le medium (MEM) c o n t a i n i n g 1% e t h a n o l . C o n c e n t r a t i o n s of 1, 10 and 100 Mg/ml e x t r a c t were 143 t e s t e d . A f t e r 2 h o u r s i n c u b a t i o n a t 37 °C, t h e v i r u s / p l a n t e x t r a c t m i x t u r e was a d d e d t o a m o n o l a y e r o f mouse embryo c e l l s and i n c u b a t e d f o r a n o t h e r 2 h o u r s a t 37 °C. F i n a l l y , t h e v i r u s / p l a n t e x t r a c t m i x t u r e was removed and t h e m o n o l a y e r o f c e l l s o v e r l a y e d w i t h D u l b e c c o ' s MEM c o n t a i n i n g 5% FBS and 0.5% a g a r o s e . A f t e r t h e p l a t e s had s o l i d i f i e d , t h e y were i n c u b a t e d a t 37 °C u n t i l t h e p l a q u e s were s u f f i c i e n t l y w e l l d e v e l o p e d t o be c o u n t e d . The t r e a t m e n t p o s t - i n f e c t i o n a s s a y was c a r r i e d o ut by f i r s t i n f e c t i n g m o n o l a y e r s of mouse embryo c e l l s w i t h a p p r o p r i a t e amounts of v i r u s i n D u l b e c c o ' s MEM f o r 2 h o u r s . The v i r u s s u s p e n s i o n was t h e n removed and t h e p l a t e s were o v e r l a y e d w i t h D u l b e c c o ' s MEM c o n t a i n i n g 5% FBS, t h e p l a n t e x t r a c t i n e t h a n o l ( f i n a l c o n c e n t r a t i o n 1%), and 0.5% a g a r o s e . A f t e r t h e o v e r l a y had s o l i d i f i e d , t h e p l a t e s were i n c u b a t e d a t 37 °C u n t i l t h e y were r e a d y f o r c o u n t i n g . P l a q u e s were c o u n t e d a g a i n s t a d a r k b a c k g r o u n d u s i n g t h e u n a i d e d e y e . e. P o t a t o d i s c tumour a s s a y The p r o c e d u r e u s e d was t h a t of F e r r i g n i e_t a_l ( 1 9 8 2 ) , w i t h some m i n o r m o d i f i c a t i o n s . P o t a t o e s (Solanum t u b e r o s u m , r e d R u s s e t v a r i e t y ) were s u r f a c e s t e r i l i z e d by i m m e r s i n g i n 20% b l e a c h f o r 15 m i n u t e s . S t e r i l e d i s c s of 15 mm d i a m e t e r and 4 mm t h i c k n e s s were p r e p a r e d from t h e t u b e r s u s i n g a c o r k h o l e b o r e r and a k n i f e c o n t a i n i n g r a z o r b l a d e s . The d i s c s were r i n s e d t h r e e t i m e s w i t h s t e r i l e d i s t i l l e d w a t e r , d r a i n e d and a p p l i e d t o a l a y e r o f 1% a g a r ( D i f c o ) i n p e t r i d i s h e s . 1 44 Twenty M1 of an overnight culture of Agrobacterium  tumefaciens, s t r a i n 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 i t y to br ine shr imp The method used has been described by Meyer e_t a l (1982). Brine shrimp (Artemia salina) eggs (New Technology, Ltd.) were incubated in a r t i f i c i a l seawater (Marine Enterprises, Ltd.) for 48 hours at 20 °C. The larvae were counted into groups of 10 and placed in 5 ml of a r t i f i c i a l seawater, to which had been added the plant extract in DMSO ( f i n a l concentration 1%, v/v). Concentrations of 10, 100 and 1000 Mg/ml extract were tested. Survival was measured afte r 24 hours incubation at 20 °C. g. Analysis of data The screening for i n h i b i t i o n of growth of bacteria and yeasts allowed some degree of qu a n t i f i c a t i o n , based upon the width of the zone of i n h i b i t i o n surrounding the extract saturated f i l t e r paper di s c . In the case of the assays for anti-dermatophytic fungus a c t i v i t y , the diameter of the colony 1 45 was measured a f t e r growth in the presence of d i f f e r e n t c o n c e n t r a t i o n s of p l a n t e x t r a c t . R e s u l t s were expressed as the minimum c o n c e n t r a t i o n r e q u i r e d to reduce the diameter of the co lony to 25% of tha t of un t r ea t ed c o l o n i e s . T h r e e - f o l d d i l u t i o n s of p l a n t e x t r a c t were t e s t e d and the minimum dose r e q u i r e d to s a t i s f y t h i s c r i t e r i o n was r e c o r d e d . The a n t i - v i r a l , a n t i - p o t a t o tumour and b r i n e shr imp t o x i c i t y assays i n v o l v e d the c o l l e c t i o n of a more e a s i l y q u a n t i f i e d set of d a t a . Data e x h i b i t i n g e a s i l y d i s c e r n i b l e dose-response r e l a t i o n s h i p s were sub j e c t ed to l o g i t t r a n s f o r m a t i o n and the L D 5 0 c a l c u l a t e d by l e a s t mean squares a n a l y s i s (Ashton , 1972; Hafner and Noack, 1977). 3. RESULTS a . A n t i m i c r o b i a l a c t i v i t y The r e s u l t s of the a n t i b a c t e r i a l and a n t i f u n g a l s c r een ing are p resen ted in Tab l e s XII and X I I I . The two s p e c i e s of b a c t e r i a t e s t e d d i f f e r e d markedly in t h e i r response to the e x t r a c t s . The growth of E. c o l i was i n h i b i t e d by e x t r a c t s of on l y two of the 34 s p e c i e s of Euphorb iaceae (6%) wh i le S. aureus was i n h i b i t e d by e x t r a c t s of 26 (or 76%) of the s p e c i e s t e s t e d (Table X I I ) . C. a l b i c a n s was comp le t e l y r e s i s t a n t to a l l the e x t r a c t s and S. c e r e v i s i a e was i n h i b i t e d by the e x t r a c t s of on l y two s p e c i e s . The four dermatophyt i c f ung i t e s t e d were more s e n s i t i v e to the presence of the e x t r a c t s (Table X I I I ) . Microsporum  c a n i s was the most s e n s i t i v e of the dermatophytes , be ing T a b l e XII Spec i es P a r t E x t r a c t * Zone of i n h i b i t i on(w i d t h , in mm) E. c o1 i S. a u r e u s C. a 1b i cans S. c e r e v i s i a e A c a l y p h a b e n e n s i s l e a f o - - - -a - 2 - -bark o - 4 - -a - 4 - -A. d i v e r s i f o l i a 1 ea f o - 2 - -a - 2 - -A. m a c r o s t a c h y a bark o - - - -A. s t a c h y u r a bark o - 3 - -a - 2 - -A l c h o r n e a c a s t a n e i f o l i a bark o - - - -A. d i s c o l o r bark a 0 - 1 - -a - 2 - -A . t r i p l i n e r v i a bark o - 1 - -•Amanoa a f f . o b l o n q i f o l i a l e a f a o - 1 - -a - 1 - -bark o - - - -A p a r i s t h m i u m cordatum l e a f a o - 2 - -bark a o - 2 - -a - 2 - -Apodandra l o r e t e n s i s bark o - 2 - -a - 2 - -C a r y o d e n d r o n o r i n o c e n s e bark o - 1 - -Chamaesyce h y s s o p i f o l i a a e r i a 1 a o - 1 - -a - 1 - -C. thym i f o l i a aer i a l o - 1 - -C n i d o s c o l u s p e r u v i a n u s l e a f o - - - -bark a o - 3 - -C o n c e v e i b a strum mart ianum bark a o - 1 - -a - 1 - -C r o t o n c u n e a t u s l e a f o - 1 - 2 a - 2 - -bark o - - - -a C . 1 e c h l e r i bark o - 2 - -C . pa 1anost igma bark a o - 2 - -a - 2 - -C. t r i n i t a t i s aer i a 1 o - 1 - -D i d y m o c i s t u s c h r y s a d e n i u s l e a f a o - - - -bark o - 1 - 4 Hevea b r a s i 1 i e n s i s l e a f a o - - - -a - - - -J a t r o p h a c u r c a s aer i a 1 o - - - -a - - - -J . q o s s y p i i f o l i a aer i a l o - - - -J . w e b e r b a u e r i l e a f o - - - -bark a o - - - -Mabea maynens i s l e a f a o - 3 - -a - 3 - -M . n i t i da bark o 3 1 - -a - - - -Manihot e s c u l e n t a a e r i a 1 o - 1 - -a - - - -Maprouna g u i a n e n s i s bark o - 1 - -a - 1 - -P h y l l a n t h u s amarus aer i a 1 o - - - -a - 3 - -P. o r b i c u l a t u s aer i a 1 o 5 3 - -a - - - -P. pseudo-conami aer i a l o - - -a - - - -P. u r i nar i a aer i a l o - - - -a - 4 - -P o d o c a l y x l o r a n t h o i d e s l e a f o - - - -a - 1 - -bark o - - - -Secur inec ja c o n g e s t a aer i a 1 a o - - -Podophy11i n t r e s i n o - - -a - 2 T a b l e XII - A n t i m i c r o b i a l s c r e e n i n g of e x t r a c t s o f E u p h o r b i a c e o u s p l a n t s . R e s u l t s a r e e x p r e s s e d as a b s e n c e ( - ) o r p r e s e n c e ( w i d t h of zone of i n h i b i t i o n in mm) of a n t i m i c r o b i a l a c t i v i t y . * o = o r g a n i c f r a c t i o n ; a = aqueous f r a c t i o n t P o d o p h y l l i n r e s i n e x t r a c t i s i n c l u d e d as a p o s i t i v e c o n t r o l T a b l e X I I I Spec i es P a r t E x t r a c t * C o n c e n t r a t i o n p r o d u c i n g >75% nh i b i t i on of growth( „g/ml ) M. can i s M. gypseum M. f u l v u m T. ga11i nae A c a l y p h a b e n e n s i s l e a f 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. d i v e r s i f o l i a l e a f o <0.125 0 25 0 25 0 25 a <0.125 - 2 0 0 5 A. m a c r o s t a c h y a bark o <0.125 0 5 0 5 0 25 A. s t a c h y u r a bark o <0.125 0 25 0 5 0 25 a <0.125 1 0 1 0 0 25 A l c h o r n e a c a s t a n e i f o l i a bark o 1 .0 0 25 0 5 0 25 a 1 .0 - - -A. d i s c o l o r bark o <0.125 0 25 1 0 0 25 a 2 .0 - - -A. t r i p1 i n e r v i a bark o 0 . 25 0 25 1 0 0 5 a 1 .0 1 0 1 0 1 0 Amanoa a f f . o b l o n q i f o l i a l e a f 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 l e a f 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 l o r e t e n s i s bark o 0. 25 <0.125 0 25 0 25 a 0 . 25 2 . 0 2 0 0 25 C a r y o d e n d r o n o r i n o c e n s e bark o 1 .0 1 . 0 2 0 2 0 a - 2 . o - 2 0 2 0 Chamaesyce h y s s o p i f o l i a aer i a l o 0 . 5 1 0 0 5 0 5 a <0.125 2 . 0 - 2 0 C. thym i f o l i a a e r i a l o <0.125 <0.125 1 0 0 25 a 0 . 5 - - 1 0 C n i d o s c o l u s p e r u v i a n u s l e a f 0 <0.125 e 1 0 0 5 bark a o <0.125 <0.125 <0.125 <0.125 C o n c e v e i b a s t r u m mart ianum bark a o 0 . 25 0. 5 1 o 0 25 a 0 . 25 - - 2 0 C r o t o n c u n e a t u s l e a f 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. l e c h l e r i bark o <0.125 <0.125 - 0 25 C. pa 1anost iqma bark a o <0.125 <0.125 0 .5 <0.125 a <0.125 - 2 .0 1 0 C. t r i n i t a t i s a e r i a l o <0.125 0 . 5 1 0 0 125 D i d y m o c i s t u s c h r y s a d e n i u s l e a f a o <0.125 - - 2 0 a - - - 1 0 bark o - - - 2 0 a 1 .0 2 .0 2 0 2 0 Hevea b r a s i l i e n s i s l e a f o 0 . 25 2 .0 2 0 2 0 J a t r o p h a c u r c a s a e r i a 1 a o <0.125 - - -a <0.125 1 .0 - -J . cjossyp i i f o l i a a e r i a 1 o 1 .0 - - -a 1 .0 - - -J . w e b e r b a u e r i 1 eaf o 0 . 5 2 .0 2 0 2 0 bark a o <0.125 2 0 2 0 1 0 Mabea maynens is l e a f a o 1 .0 1 0 1 0 1 0 a <1 . 25 2 0 1 0 0 5 M. n i t i d a bark o <0. 125 . 25 0 5 0 25 Man ihot e s c u l e n t a aer i a l a o <0.125 <0.125 <0.125 <0.125 a 1 .0 - - -Maprouna g u i a n e n s i s bark o <0.125 0 25 0 25 0 25 P h y l l a n t h u s amarus aer i a 1 o <0.125 2 0 - 0 25 a 0 . 5 2 0 2 0 1 0 P. o r b i c u 1 a t u s aer i a l o <0.125 0 25 0 5 <0.125 a <0.125 2 0 - 2 0 P. pseudo-conami a e r i a 1 o <0.125 0 5 2 0 1 0 a 0 . 25 - 1 0 -P. u r i n a r i a a e r i a l o o . 2 5 - - 0 25 a <0.125 2 0 2 0 1 0 P o d o c a l y x l o r a n t h o i d e s l e a f 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 S e c u r i n e q a c o n q e s t a aer i a l a o <0.125 0 5 1 0 1 0 Podophy11 i n t r e s i n o 0 . 5 0 5 0 5 0 . 25 T a b l e X I I I - S c r e e n i n g of E u p h o r b i a c e o u s p l a n t s f o r a n t i - d e r m a t o p h y t i c fungus a c t i v i t y . The v a l u e s r e c o r d e d r e f e r to the dose of e x t r a c t r e q u i r e d to i n h i b i t c o l o n y growth by a t l e a s t 75%. * o = o r g a n i c f r a c t i o n ; a = aqueous f r a c t i o n t P o d o p h y l l i n r e s i n e x t r a c t i s i n c l u d e d as a p o s i t i v e c o n t r o l 150 i n h i b i t e d to some degree by at l e a s t one of the e x t r a c t s of each of the 34 s p e c i e s t e s t e d . Microsporum gypseum was i n h i b i t e d by 97% of the s p e c i e s , a l though 6 of the a c t i v e e x t r a c t s were e f f e c t i v e on l y at h igh c o n c e n t r a t i o n s (>2 mg/ml). Microsporum fulvum was i n h i b i t e d by 91% of the Euphorb iaceous s p e c i e s t e s t e d , 21% of these be ing of low a c t i v i t y . F i n a l l y , 94% of the e x t r a c t s i n h i b i t e d T . g a l l i n a e and 12% of these were of low po tency . The e x t r a c t of p o d o p h y l l i n , known to c o n t a i n p o d o p h y l l o t o x i n l i g n a n s wi th a n t i v i r a l a c t i v i t y , was modera te ly e f f e c t i ve in i n h i b i t i n g the growth of each s p e c i e s of dermatophyte , b. A n t i v i r a l a c t i v i t y The percentage r e d u c t i o n in number of v i r a l p laques formed in response to t reatment of S i n d b i s v i r u s and murine c y t o m e g a l o v i r u s , both be fo re and a f t e r i n f e c t i o n , i s p resen ted in Tab le XIV. The e x t r a c t s are p a r t i c u l a r l y e f f e c t i v e in i n a c t i v a t i n g both v i r u s e s when a p p l i e d to them p r i o r to i n f e c t i o n of the c e l l s . With the excep t i on of the bark of D idymoc i s tus c h r y s a d e n i u s , which i n a c t i v a t e d MCMV but not S i n d b i s v i r u s , at l e a s t one of the e x t r a c t s of each of the s p e c i e s t e s t e d was e f f e c t i v e in i n h i b i t i n g i n f e c t i o n by both v i r u s e s . The o rgan i c f r a c t i o n s were g e n e r a l l y more e f f e c t i v e than the aqueous ones . F o r t y of the 42 o rgan i c f r a c t i o n s t e s t e d i n a c t i v a t e d S i n d b i s v i r u s and a l l i nac . t i va ted MCMV. The aqueous f r a c t i o n s were s l i g h t l y l e s s e f f e c t i v e . F o r t y - e i g h t per cent i n a c t i v a t e d S i n d b i s v i r u s wh i le 91% i n a c t i v a t e d MCMV. When the e x t r a c t s were a p p l i e d to c e l l s which had a l r e ady T a b l e XIV I n h i b i t i o n of P l a q u e Forming A b i l i t y S i ndb s v i r u s Mur ine c y t o m e g a l o v i r u s Spec i es P a r t E x t . Treatment Treatment T reatment T r e a t m e n t * P r e - i n f e c t i on Post - i n f e c t i on P r e - i n f e c t i on P o s t - i n f e c t i on 1 10 1 0 0 LCs o 1 10 1 0 0 LC 5 o 1 1 0 1 0 0 LCs o 1 1 0 1 0 0 LCs o 1/9/ uQ/ 1/9/ 1/9/ 1/9/ n9/ c 9 / «/9/ ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml ml A c a l y p h a b e n e n s i s l e a f o 1 0 0 1 0 0 1 0 0 <1 1 2 2 - 1 0 0 1 0 0 1 0 0 <1 0 0 1 -a 0 0 0 - 2 0 1 - 8 9 1 0 0 1 0 0 < 1 0 0 0 -bark o 1 0 0 1 0 0 1 0 0 <1 2 2 2 - 1 0 0 1 0 0 1 0 0 <1 0 0 tox -a 3 3 6 9 8 9 3 . 0 1 2 1 - 1 0 0 1 0 0 1 0 0 < 1 0 0 tox -A. d i v e r s i f o l i a l e a f o 1 0 0 1 0 0 1 0 0 <1 0 2 4 tox 16 9 8 1 0 0 1 0 0 <1 0 0 0 -a 1 0 0 1 0 0 1 0 0 <1 0 0 1 - 1 0 0 1 0 0 1 0 0 < 1 0 0 0 -A. m a c r o s t a c h y a bark o 1 0 0 1 0 0 1 0 0 <1 0 0 6 - 9 9 1 0 0 1 0 0 <1 0 0 0 -a 0 0 o - 0 0 3 - 0 0 9 9 - 0 0 0 -A. s t a c h y u r a bark o 1 0 0 1 0 0 1 0 0 <1 0 0 13 - 9 8 1 0 0 1 0 0 <1 0 0 0 -a 1 0 0 1 0 0 1 0 0 < 1 0 0 0 - 1 0 0 1 0 0 1 0 0 <1 0 0 0 -A l c h o r n e a c a s t a n e i f o l i a bark o 9 6 1 0 0 1 0 0 <1 1 0 6 - 6 5 1 0 0 1 0 0 . 3 0 0 0 0 -a 0 0 0 - O 0 0 - 7 2 1 0 0 1 0 0 . 2 2 0 O 0 -A. d i s c o l o r bark o 14 5 6 1 0 0 3 . 9 17 4 1 tox 2 0 6 0 1 0 0 tox . 8 6 0 0 tox -a 0 0 0 - 0 0 0 - 1 0 0 1 0 0 1 0 0 <1 0 0 0 -A. t r i p l i n e r v i a bark o 1 0 0 1 0 0 1 0 0 <1 5 3 7 6 4 3 5 7 2 1 0 0 1 0 0 . 2 2 0 0 0 -a 1 0 0 1 0 0 1 0 0 < 1 0 21 tox 17 1 0 0 1 0 0 tox < 1 0 0 tox -Amanoa a f f . o b l o n q i f o l i a 1 eaf o 1 0 0 1 0 0 tox < 1 1 0 0 1 0 0 tox < 1 1 0 0 1 0 0 tox <1 1 0 0 1 0 0 tox <1 a 1 0 0 1 0 0 tox <1 2 3 9 5 tox 2 . 0 1 0 0 1 0 0 tox <1 1 0 0 1 0 0 tox <1 bark o 9 2 1 0 0 1 0 0 <1 4 3 6 9 9 5 1 . 9 1 0 0 1 0 0 1 0 0 < 1 1 0 0 1 0 0 1 0 0 <1 a 0 0 0 - 0 0 0 - 0 9 1 1 0 0 7 . 7 0 0 1 0 0 2 7 0 Apar i s thmiurn cordatum l e a f 0 8 7 8 8 9 9 . 0 8 0 0 tox - 2 6 9 9 1 0 0 1 . 3 0 0 tox -a 2 2 5 8 7 9 8 . 0 0 0 0 - 8 2 1 0 0 1 0 0 < 1 0 0 0 <1 bark o 6 3 71 8 4 . 15 9 2 4 1 0 0 3 . 8 9 6 1 0 0 1 0 0 <1 0 0 0 -a 0 0 . 0 - 0 6 2 9 1 4 7 9 2 1 0 0 1 0 0 <1 0 0 0 -Apodandra l o r e t e n s i s bark o 1 0 0 1 0 0 1 0 0 <1 0 6 7 5 5 1 5 7 1 0 0 1 0 0 . 9 1 0 0 0 -a 4 1 6 0 7 9 2 . 9 13 17 tox - 1 0 0 1 0 0 1 0 0 < 1 0 0 0 -C a r y o d e n d r o n o r i n o c e n s e bark o 0 0 0 - 0 0 0 - 6 5 1 0 0 1 0 0 . 3 0 0 o 0 -a 1 0 0 1 0 0 1 0 0 <1 0 0 tox - 1 0 0 1 0 0 tox <1 0 0 tox -Chamaesyce h y s s o p i f o l i a aer i a l o 1 0 0 1 0 0 1 0 0 <1 0 12 18 - 9 7 1 0 0 1 0 0 <1 0 0 0 -a 0 0 0 - 0 0 0 - 6 4 9 9 1 0 0 . 5 3 0 0 0 -C. thym i f o 1 i a aer i a 1 0 1 0 0 1 0 0 1 0 0 <1 7 4 1 tox 15 1 0 0 1 0 0 1 0 0 <1 0 0 tox -a 0 0 0 - 0 0 0 - 0 0 0 - 0 0 0 -C n i d o s c o l u s p e r u v i a n u s l e a f o 1 0 0 1 0 0 1 0 0 <1 1 0 0 1 0 0 tox < 1 8 2 1 0 0 1 0 0 <1 0 0 tox -a 0 0 0 - 0 0 5 1 2 0 8 0 0 9 8 4 1 0 0 0 -bark o 1 1 2 6 6 0 5 3 2 7 3 5 7 9 12 0 0 1 0 0 2 2 0 0 0 -a 12 2 5 6 3 4 7 3 6 3 8 4 0 6 . 1 1 0 0 1 0 0 1 0 0 <1 0 0 0 -C o n c e v e i b a s t r u m mart ianum bark o 9 9 1 0 0 1 0 0 <1 0 0 4 8 2 2 6 1 0 0 1 0 0 1 0 0 <1 0 0 0 -a 1 0 0 1 0 0 1 0 0 <1 0 12 2 5 4 0 1 0 0 1 0 0 1 0 0 < 1 0 0 0 -C r o t o n c u n e a t u s l e a f o 1 0 0 1 0 0 1 0 0 <1 0 0 0 - 5 8 9 9 1 0 0 . 6 3 0 0 0 -a 1 0 0 1 0 0 1 0 0 <1 0 0 0 - 1 0 0 1 0 0 1 0 0 < 1 0 0 0 C. c u n e a t u s 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. l e c h l e r i 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. p a l a n o s t i q m a 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. t r i n i t a t i s a e r i a l 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 -D i d y m o c i s t u s c h r y s a d e n i u s l e a f 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 b r a s i 1 i 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 -J a t r o p h a c u r c a s 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 . g o s s y p i i f o 1 i 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 . w e b e r b a u e r i 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 m a y n e n s i s l e a f 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. n i 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 M a n i h o t e s c u l e n t a 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 p j u i a n e n s i s 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 -P h y l l a n t h u s amarus aer i a l 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. o r b i c u l a t u s 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 i a l 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. u r i n a r 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 -P o d o c a l y x l o r a n t h o i d e s l e a f 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 -S e c u r i n e q a c o n q e s t a aer i a l 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 r e s i n o 98 100 tox <1 0 0 tox - 1 1 12 tox - 100 100 tox <1 T a b l e XIV - A n t i v i r a l s c r e e n i n g of e x t r a c t s of E u p h o r b i a c e o u s p l a n t s . R e s u l t s a r e e x p r e s s e d as per c e n t r e d u c t i o n i n number of p l a q u e s o b s e r v e d in the c o n t r o l s . * o = o r g a n i c e x t r a c t ; a = aqueous e x t r a c t t P o d o p h y l l i n r e s i n e x t r a c t i s i n c l u d e d as a p o s i t i v e c o n t r o l 1 53 been i n f e c t e d w i th v i r u s ( t reatment p o s t - i n f e c t i o n ) , a much sma l l e r percentage of e x t r a c t s produced a r e d u c t i o n in the number of v i r a l p laques formed. S i n d b i s v i r u s was s u s c e p t i b l e to 23 (68%) and MCMV to on l y 8 (24%) of the 34 s p e c i e s t e s t e d . Both l e a f and bark m a t e r i a l demonstrated a n t i v i r a l a c t i v i t y . L i t t l e d i f f e r e n c e between the number of o rgan i c and aqueous f r a c t i o n s that possessed a c t i v i t y (29 and 25 , r e s p e c t i v e l y ) was obse r ved . No c o r r e l a t i o n between a n t i v i r a l a c t i v i t y r e s u l t i n g from p r e - i n f e c t ion and p o s t - i n f e c t i o n a p p l i c a t i o n was e v i d e n t . The s t rong a n t i v i r a l a c t i o n of the Amanoa s p . t e s t e d was of n o t e . The o rgan i c and aqueous f r a c t i o n of the bark p revented i n f e c t i o n by both S i n d b i s v i r u s and MCMV comp le te l y at a c o n c e n t r a t i o n of 1 ag/ml . The e x t r a c t s were e f f e c t i v e when a p p l i e d • a c c o r d i n g to e i t h e r the p r e - i n f e c t i o n or p o s t -i n f e c t i o n p r o t o c o l s . An e x t r a c t of the a n t i v i r a l p o d o p h y l l i n r e s i n was e f f e c t i v e a g a i n s t both v i r u s e s when a p p l i e d p r i o r to i n f e c t i o n but i n h i b i t e d the r e p l i c a t i o n of on l y MCMV when a p p l i e d as fo r the p o s t - i n f e c t i o n p r o t o c o l . I t i n h i b i t e d the i n f e c t i o n comp le t e l y at a c o n c e n t r a t i o n of 1 jug/ml. c . I n h i b i t i o n of pota to tumour fo rmat ion The r e s u l t s of the s c r een ing f o r ant i tumour a c t i v i t y were a l s o c h a r a c t e r i z e d by a very h igh p r o p o r t i o n of a c t i v e e x t r a c t s (Table XV) . Th i r t y -one (91%) of the s p e c i e s t e s t e d were e f f e c t i v e in i n h i b i t i n g the fo rmat ion of tumours. T h i s r ep re sen t ed 53 of the 84 o rgan i c and aqueous e x t r a c t s . T a b l e XV Spec i es P a r t E x t r a c t * Per c e n t i n h i b i t i o r i of tumour f o r m a t i o n ( S E M ) 0 . 3 eg/ml 1 . 0 ^g/ml 3 . 0 y g / m l ED 5 o ( j,g/ml ) A c a l y p h a b e n e n s i s l e a f o 70( 6 ) 9 1 ( 2 ) 99 ( 1 ) 1 7 a 87 4 ) 9 4 ( 2 ) 98 ( 1 ) 0 44 bark 0 94 2 ) 8 1 ( 4 ) 1 0 0 ( 1 ) 0 16 a 68 9 ) 8 5 ( 6 ) 1 0 0 ( 0 ) 0 35 A. d i v e r s i f o l i a l e a f o 21 19) 4 5 ( 1 7 ) 5 3 ( 9 ) 2 0 3 a 12 3 ) 1 7 ( 6 ) 1 1 ( 4 ) A. m a c r o s t a c h y a bark o 26 13) 9 1 ( 5 ) 1 0 0 ( 0 ) 0 5 0 a 2 2 ) 0 ( 13) 0(11) A. s t a c h y u r a bark o 85 9 ) 99 ( 1 ) 1 0 0 ( 0 ) 0 17 a 45 6 ) 6 0 ( 5 ) 9 4 ( 1 ) 0 51 A l c h o r n e a c a s t a n e i f o l i a bark o 83 6 ) 9 0 ( 4 ) 9 9 ( 1 ) 0 14 a 36 4 ) 7 4 ( 7 ) 9 4 ( 3 ) 0 52 A. d i s c o l o r bark o 76 7 ) 8 3 ( 5 ) 8 7 ( 6 ) 0 01 a 0( 10) 6 4 ( 7 ) 6 6 ( 5 ) 1 75 A. t r i p 1 i n e r v i a bark o 28( 9 ) 4 3 ( 1 1 ) 1 0 0 ( 0 ) 0 62 a 0( 11) 0 ( 12) 0 ( 14) Amanoa a f f . o b l o n q i f o l i a l e a f 0 84 6 ) 9 3 ( 3 ) 9 6 ( 2 ) 0 0 3 a 15 8 ) 1 3 ( 8 ) 2 6 ( 1 3 ) bark o 1 1 11) 7 4 ( 6 ) 6 9 ( 8 ) 0 0 9 a 0 12) 0 ( 9 ) 7 ( 6 ) A p a r i s t h m i u m cordatum l e a f o 23 12) 5 7 ( 1 0 ) 7 8 ( 7 ) 0 93 a 0 10) 1 7 ( 1 1 ) 0(11) bark o 17 7 ) 6 0 ( 6 ) 8 7 ( 2 ) 0 8 9 a 0 15) 7 0 ( 8 ) 9 3 ( 2 ) 1 28 -Apodandra l o r e t e n s i s bark o 0 12) 0 ( 14) 0(11) a 1 1 ( 6 ) 1 1 ( 3 ) 8 1 ( 1 8 ) 1 76 C a r y o d e n d r o n o r i n o c e n s e bark o 45 8 ) 7 4 ( 8 ) 8 9 ( 3 ) 0 41 a ' 121 6 ) 8 ( 4 ) 1 1 ( 8 ) Chamaesyce h y s s o p i f o l i a aer i a 1 o 53( 5 ) 7 2 ( 5 ) 7 9 ( 4 ) 0 25 a 34( 9 ) ' 4 7 ( 5 ) 8 7 ( 5 ) 0 75 C. thym i f o l i a aer i a l o 38 5 ) 7 2 ( 5 ) 9 5 ( 1 ) 0 51 a 0 10) 0 ( 1 2 ) 9 ( 7 ) C n i d o s c o l u s p e r u v i a n u s l e a f o 3 6 ) 7 ( 7 ) 0 ( 7 ) a 8 7 ) 0 ( 5 ) 4 ( 3 ) bark o 0 5 ) 4 9 ( 8 ) 1 0 0 ( 0 ) 1 0 3 a 19 7 ) 2 6 ( 7 ) 2 8 ( 8 ) C o n c e v e i b a s t r u m mart ianum bark o 601 8 ) 8 8 ( 4 ) 9 7 ( 2 ) 0 57 a 66 10) 7 9 ( 5 ) 8 1 ( 4 ) 0 0 5 C r o t o n c u n e a t u s l e a f o 72( 8 ) 8 5 ( 4 ) 9 1 ( 6 ) 0 . 0 8 a 36( 9 ) 6 2 ( 1 0 ) 8 9 ( 4 ) 0 6 0 bark o 0( 10) 2 ( 4 ) 5 ( 9 ) a 0( 8 ) 0 ( 1 1 ) 0 ( 1 1 ) C. l e c h l e r i bark o 0 11) 3 0 ( 7 ) 8 1 ( 4 ) 1 7 5 a 0 9 ) 0 ( 7 ) 0 ( 1 1 ) C. p a l a n o s t i cjma bark o 7 9 5 ) 8 5 ( 5 ) 9 1 ( 3 ) 0 0 2 a 0 9 ) 6 2 ( 5 ) 8 1 ( 3 ) 1 5 7 C. t r i n i t a t i s aer i a l o 17 9 ) 7 4 ( 1 3 ) 8 7 ( 9 ) 0 7 8 a 0 1 1 ) 0 ( 9 ) 0 ( 1 3 ) D i d y m o c i s t u s c h r y s a d e n i u s l e a f o 7 2 9 ) 7 4 ( 9 ) 9 1 ( 8 ) 0 1 1 a 0 4 ) 0 ( 1 0 ) 0 ( 9 ) bark o 0 1 3 ) 6 4 ( 1 2 ) 7 7 ( 9 ) 1 5 6 a 4 9 ) 0 ( 12 ) 0 1 3 ) Hevea b r a s i l i e n s i s l e a f o 0 1 3 ) 7 ( 8 ) 6 5 ) a 3 5 ) 0 ( 7 ) 1 5 ) J a t r o p h a c u r c a s aer i a 1 o 0 1 5 ) 8 1 ( 7 ) 8 2 8 ) 1 4 3 a 14 1 2 ) 0 ( 1 3 ) 4 9 ) J . q o s s y p i i f o l i a aer i a 1 o 5 1 2 ) 0 ( 1 0 ) 0 9 ) a 2 5 ) 0 ( 1 3 ) 0 1 2 ) J . w e b e r b a u e r i l e a f o 9 1 1 ) 1 2 ( 1 3 ) 5 9 ) a 0 1 1 ) 10(11) 7 9 ) bark 0 0 1 3 ) 6 6 ( 1 3 ) 8 3 9 ) 1 5 2 a 5 ( 1 1 ) 4 ( 1 0 ) 9 1 2 ) Mabea m a y n e n s i s l e a f 0 0 ( 1 5 ) 6 5 ( 7 ) 9 4 7 ) 1 3 3 a 0 ( 1 3 ) 0 ( 9 ) 8 7 6 ) . 2 6 4 M. n i t i da bark o 1 6 ( 1 3 ) 7 0 ( 1 1 ) 7 9 ( 1 0 ) 0 9 1 a 0 ( 1 4 ) 1 7 ( 1 2 ) 8 3 1 1 ) 0 6 0 Manihot e s c u l e n t a aer i a 1 o 7 7 ( 1 1 ) 6 4 ( 1 5 ) 8 7 ( 8 ) 0 0 3 a 5 7 ( 1 3 ) 6 2 ( 1 3 ) 8 7 ( 1 0 ) 0 3 2 Maprouna q u i a n e n s i s bark o 6 2 ( 1 5 ) 7 4 ( 1 3 ) 7 5 ( 1 3 ) 0 0 5 a 0 ( 1 5 ) 0 ( 1 3 ) 1 2 ( 1 1 ) P h y l l a n t h u s amarus aer i a 1 o 8 1 ( 6 ) 7 9 ( 8 ) 9 4 ( 5 ) 0 0 5 a 0 ( 1 0 ) 8 3 ( 7 ) 9 0 ( 5 ) 1 3 1 P. o r b i c u l a t u s aer i a 1 o 7 6 ( 5 ) 9 8 ( 3 ) 1 0 0 ( 0 ) 0 2 4 a 0 ( 1 1 ) 3 8 ( 1 0 ) 8 9 ( 8 ) 1 5 5 P. pseudo-conami aer i a 1 o 8 9 ( G) 9 6 ( 5 ) 1 0 0 ( 1 ) 0 17 a 7 4 ( 9 ) 7 7 ( 8 ) 1 0 0 ( 3 ) 0 3 4 P. ur i nar i a aer i a 1 o 0 ( 1 1 ) o d o ) 8 5 ( 5 ) 2 7 3 a 3 2 ( 8 ) 5 5 ( 7 ) 96( 4 ) 0 6 4 P o d o c a l y x l o r a n t h o i d e s l e a f o 2 3 ( 1 2 ) 3 4 ( 1 0 ) 8 0 ( 8 ) 1 17 a 0 ( 1 0 ) 0 ( 7 ) 0 ( 9 ) bark o 0 ( 1 1 ) 0 ( 8 ) 0 ( 1 2 ) a 0 ( 8 ) 0 ( 1 1 ) 5 ( 7 ) S e c u r i n e q a c o n q e s t a aer i a 1 o 1 9 ( 1 2 ) 4 9 ( 9 ) 9 1 ( 7 ) 0 8 8 a 9 ( 1 2 ) 0 ( 1 1 ) 0 ( 9 ) Podophy1 l i n t r e s i n o 4 ( 1 2 ) 7 2 ( 9 ) 9 3 ( 5 ) 0 9 8 T a b l e XV - S c r e e n i n g of e x t r a c t s of E u p h o r b i a c e o u s p l a n t s f o r i n h i b i t i o n of A g r o b a c t e r i um induced tumour f o r m a t i o n . * o = o r g a n i c f r a c t i o n ; a = aqueous f r a c t i o n t P o d o p h y l l i n r e s i n e x t r a c t i s i n c l u d e d as a p o s i t i v e c o n t r o l 1 56 E x t r a c t s of b o t h l e a f and ba r k showed a n t i t u m o u r a c t i v i t y and no d i f f e r e n c e i n t h e a c t i v i t i e s of t h e aqueous and o r g a n i c f r a c t i o n s was a p p a r e n t . The a n t i t u m o u r a c t i v i t i e s o b s e r v e d were g e n e r a l l y q u i t e s t r o n g : t h e E D 5 0 s r a n g e d between 0.01 and 3.0 Mg/ml. P o d o p h y l l i n was m o d e r a t e l y e f f e c t i v e i n i n h i b i t i n g tumour f o r m a t i o n . d. T o x i c i t y t o br i n e s h r imp A l a r g e p e r c e n t a g e of t h e e x t r a c t s examined were t o x i c t o b r i n e s h r i m p ( T a b l e X V I ) . The ran g e o f t o x i c i t i e s o b s e r v e d was g r e a t and t h e L C 5 0 v a r i e d between l e s s t h a n 1 Mg/ml and 1 mg/ml, t h e h i g h e s t dose t e s t e d . E x t r a c t s w i t h an L C 5 0 l e s s t h a n 1 mg/ml were c l a s s e d as a c t i v e . T w e n t y - s e v e n o f t h e 34 E u p h o r b i a c e o u s s p e c i e s (79%) p r o d u c e d a t o x i c r e s p o n s e , by t h i s c r i t e r i o n . A p p r o x i m a t e l y t h r e e t i m e s as many o r g a n i c f r a c t i o n s (32) as aqueous f r a c t i o n s (10) were t o x i c . P o d o p h y l l i n d e m o n s t r a t e d a h i g h d e g r e e of t o x i c i t y ( L C 5 0 << 1 Mg/ml) t o w a r d s b r i n e s h r i m p . e. C o r r e l a t i on between b i o l o g i c a l a s s a y s The d a t a s e t s of T a b l e s X I I t o XVI a r e d e t a i l e d and c o n t a i n much i n f o r m a t i o n . A l a r g e number o f . e x t r a c t s w i t h a c t i v i t i e s i n e a c h o f t h e b i o l o g i c a l a s s a y s i s t h e p r e d o m i n a n t t r e n d . T h i s i n f o r m a t i o n has been summarized i n T a b l e X V I I . A l t h o u g h e a c h of t h e a s s a y s m e a s u r e s a somewhat d i f f e r e n t a s p e c t o f t h e b i o l o g i c a l a c t i v i t y of t h e e x t r a c t , c e r t a i n a s s a y s a r e more c l o s e l y r e l a t e d , i n t e r m s of t h e n a t u r e o f t h e a c t i v i t y d e t e c t e d , t h a n o t h e r s . The a s s a y s m e a s u r i n g a n t i b a c t e r i a l a c t i v i t y (Nos. 3 and 4 ) , f o r example, would be T a b l e XVI Per c e n t mortc i l i t y of b r i n e shr imp S p e c i e s P a r t E x t r a c t * 10 100 1000 LCs o i/O/ml „g/ml eg/ml »,g/ml A c a l y p h a b e n e n s i s 1 eaf o 0 20 100 1 16 a 0 0 56 -bark o 0 0 100 214 a 0 0 20 -A. d i v e r s i f o l i a l e a f o 0 0 24 -a 0 0 0 -A. m a c r o s t a c h y a bark o 0 40 100 104 a 0 0 0 -A. s t a c h y u r a bark o 0 0 100 214 a 0 0 0 -A l c h o r n e a c a s t a n e i f o l i a bark o 8 72 100 41 a 0 0 4 -A. d i s c o l o r bark o- 0 24 72 399 a 0 0 4 -A . t r i p i i n e r v i a bark o 0 28 100 1 10 a 0 68 100 92 Amanoa a f f . o b l o n q i f o l i a l e a f o 76 92 100 6 . 1 a 52 84 100 14.3 bark o 0 8 32 -a 0 8 44 875 Apar i s thmiura cordatum l e a f o 0 60 100 95 a 0 0 12 -bark o 0 8 96 254 a 0 0 0 -Apodandra l o r e t e n s i s bark o 0 4 100 142 a 0 0 0 -C a r y o d e n d r o n o r i n o c e n s e bark o 72 96 100 5 .8 a 8 56 96 76 Chamaesyce h y s s o p i f o l i a aer i a 1 o 0 0 0 -a 0 0 0 -C. thymi f o l i a aer i a 1 o 76 100 100 1 . 8 a 0 0 0 -C n i d o s c o l u s p e r u v i a n u s l e a f o 0 4 44 -a 0 0 0 -bark o 0 68 100 92 a 0 0 4 -C o n c e v e i b a s t r u m mart ianum bark o 12 88 100 30 a 4 8 12 -C r o t o n c u n e a t u s l e a f o 100 100 100 <<1 a 0 16 100 120 bark o 68 100 100 2.6 a 0 8 100 130 C. l e c h l e r i bark o 40 92 100 15 a 0 0 0 -C. p a l a n o s t i q m a bark o 92 96 100 1 . 3 a 0 8 24 -C. t r i n i t a t i s aer i a 1 o 24 76 100 27 a 0 0 0 -D i d v m o c i s t u s c h r y s a d e n i u s l e a f o 0 20 52 615 a 0 0 0 -bark o 40 68 96 30 a 0 0 100 2 14 Hevea b r a s i l i e n s i s l e a f o 0 0 76 1 106 a 0 0 4 -J a t r o p h a c u r c a s aer i a 1 o 20 28 32 -a 8 4 12 -J . q o s s y p i i f o l i a aer i a 1 o 24 28 36 -a 0 36 32 -J . w e b e r b a u e r i l e a f o 0 4 88 401 a 0 0 0 -bark 0 0 20 84 326 a 0 0 12 -Mabea m a y n e n s i s l e a f 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 M a n i h o t e s c u l e n t a aer i a 1 o 0 8 16 -a 0 0 28 -Maprouna q u i a n e n s i s bark o 24 64 100 30 a 0 0 24 P h y l l a n t h u s amarus aer i a 1 o 1G 24 92 ' 14 a 0 0 0 -P. o r b i c u l a t u s 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 -P o d o c a l y x l o r a n t h o i d e s l e a f o 4 0 4 -a 0 0 0 -bark o 4 36 84 193 a 0 0 8 -S e c u r i n e q a c o n q e s t a aer i a 1 o 96 100 100 0 . 0 9 a 0 0 92 642 Podophy1 l i n t r e s i n o 100 100 100 <<1 T a b l e XVI - S c r e e n i n g of e x t r a c t s o f E u p h o r b i a c e o u s p l a n t s f o r t h e i r t o x i c i t y t o b r i n e s h r i m p , A r t e m i a s a l i n a . E x t r a c t s h a v i n g a L C s o < 1000 ^g/ml were c l a s s i f i e d as a c t i v e . * o = o r g a n i c f r a c t i o n ; a = aqueous f r a c t i o n t P o d o p h y l l i n r e s i n i s i n c l u d e d as a p o s i t i v e c o n t r o l . T a b l e XVII Spec i es P a r t * Ext B i o l o g i c a l Assay t 1 2 3 4 5 6 7 8 9 10 1 1 12 13 14 A c a l y p h a b e n e n s i s l e a f 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. d i v e r s i f o l i a l e a f 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. m a c r o s t a c h y a bark o - - - - <0. 13 0 . 5 0 . 5 0 . 25 <1 .0 - <1 .0 - 0 5 104 A. s t a c h y u r a 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 -A l c h o r n e a c a s t a n e i f o l i a bark o - - - - 1 .0 0 . 25 0 . 5 0 . 25 <1 .0 - 0 . 3 0 - 0 14 4 1 a - - - - 1 .0 - - - - - 0.22 - 0 52 -A. d i s c o l o r 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 . t r i p 1 i n e r v 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 a f f . o b l o n q i f o l i a 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 A p a r i s t h m i u m cordatum l e a f o - + 2 - - <0. 13 0 . 5 1 .0 0 . 5 0 . 0 8 - 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 l o r e t e n s i s 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 -C a r y o d e n d r o n o r i n o c e n s e 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 h y s s o p i f o l i a 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 . 5 3 - 0 75 -C. thym i f o 1 i 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 - - - - - -C n i d o s c o l u s p e r u v i a n u s 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 - - -C o n c e v e i b a s t r u m mart ianum 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 -C r o t o n c u n e a t u s l e a f o - + 1 - + 2 <0. 13 0 . 5 0 . 5 0 . 25 <1 .0 - 0 . 6 3 - 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. l e c h l e r i bark o + 2 _ _ <0. 13 <0. 13 0 . 2 5 <1 .0 - 0 . 28 - 1 8 15.2 a - - - - - - - - - - - 487 - -C. p a l a n o s t i q m a bark o - + 2 - - <0. 13 <0. 13 0 5 <0. 13 14 155 0 . 8 6 - 0 02 1 . 3 a - + 2 - - <0. 13 - 2 0 1 .0 <1 .0 391 <1 .0 15 1 6 -C. t r i n i t a t i s aer . o - + 1 - - <0. 13 0 . 5 1 0 0 .13 0 . 5 12 1 .4 \ 1 ~ 0 78 27 D i dymoc i s t u s c h r y s a d e n i u s l e a f a o - - - - <0. 13 - - 2.0 1 . 3 31 4 . 6 133 0 1 1 615 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 b r a s i l i e n s i s l e a f o - - - - 0 . 25 2 .0 2 0 2 .0 <1 .0 - 0 . 6 <1 .0 - -a - - - - - - - - - - <1 .0 - - -J a t r o p h a c u r c a s aer . o - - - - <0. 13 - - - <1 .0 88 7 . 0 - 1 4 -a - - - - - - - - < 1 .0 32 22 - - -J . q o s s y p i i f o l i a aer . o - - - - <0. 13 - - - <1 .0 37 1 . 5 - - -a - - - - <0. 13 1 .0 - - <1 .0 512 1 . 7 - - -J . w e b e r b a u e r i l e a f 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 Mabea maynens i s l e a f a o - + 3 - - 1 .0 1 .0 1 0 1 .0 <1 .0 724 <1 .0 - 1 3 -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 e s c u l e n t a 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 . 1 8 - 0 32 -Maprouna q u i a n e n s i s bark o - + 1 - - <0. 13 0 . 25 0 25 0. 25 <1 .0 - <1 .0 - 0 05 30 a - + 1 - - - - - - - - 0. 10 - - 5380 P h y l l a n t h u s 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. o r b i c u l a t u s 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 . 6 5 - 0 17 15 a - - - - 0 .25 - 1 0 - - 40 18 - 0 34 214 P. u r i n a r i a aer . o - - - - 0 . 2 5 - - 0 . 2 5 <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 -P o d o c a l y x l o r a n t h o i d e s l e a f 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 - - -S e c u r i n e q a c o n q e s t a aer . o - + 1 - <0. 13 0 . 5 1 0 1 .0 0 .01 0.81 0.81 0 . 33 0 88 0 . 0 9 a - - - - - - - - - 22 1 . 7 22 - 642 Podophy11i n + r e s . o - + 2 - - 0 . 5 0 . 5 0 5 0 . 2 5 <1 .0 - 4 . 3 <1 .0 0 98 <1 .0 T a b l e XVII - Summary of b i o l o g i c a l s c r e e n i n g of e x t r a c t s of E u p h o r b i a c e o u s p l a n t s Legend f o r T a b ! e XVII t B i o a s s a y s to which numbers r e f e r : 1 = = i n h i b i t i o n of growth of E_. c o l i 2 = = i n h i b i t i o n of growth of S . a u r e u s 3 = = i n h i b i t i o n of growth of C. a l b i cans 4 = = i n h i b i t i o n of growth of S . c e r e v i s e a e 5 = = i n h i b i t i o n of growth of M. c a n i s 6 = = i n h i b i t i o n of growth of M . gypseum 7 = = i n h i b i t i o n of growth of M. fu1vum 8 = = i n h i b i t i o n of growth of T . g a 1 1 i n a e 9 = ; a n t i - S i n d b i s v i r u s a c t i v i t y : p r e - i n f e c t i o n 10 = = a n t i - S i n d b i s v i r u s a c t i v i t y : p o s t - i n f e c t i o n 1 1 = = ant i -MCMV a c t i v i t y : p r e - i n f e c t i o n 12 = = ant i-MCMV a c t i v i t y : p o s t - i n f e c t i on 13 = = i n h i b i t i o n of Aqrobacter iurn induced p o t a t o tumours 14 = = t o x i c i t y t o A r t e m i a s a l i n a * P o d o p h y l l i n r e s i n e x t r a c t i s i n c l u d e d as a p o s i t i v e c o n t r o l . * o = o r g a n i c f r a c t i o n ; a = aqueous f r a c t i o n 162 e x p e c t e d t o r e s e m b l e e a c h o t h e r more c l o s e l y , w i t h r e s p e c t t o t h e r e s u l t s o b t a i n e d , t h a n would two a s s a y s u t i l i z i n g d i s t a n t l y r e l a t e d o r g a n i s m s . D a t a has been p r e s e n t e d w h i c h i n d i c a t e s t h a t t h e p o t a t o d i s c tumour a s s a y i s p r e d i c t i v e of i n v i v o a n t i l e u k e m i c a c t i v i t y ( F e r r i g n i e t a_l, 1982) and i t i s b e i n g c o n s i d e r e d as a p r e l i m i n a r y s c r e e n i n g a s s a y f o r a n t i n e o p l a s t i c a g e n t s ( C a s s a d y e_t a l , 1981). T o x i c i t y t o b r i n e s h r i m p , w h i l e not s p e c i f i c f o r a n t i t u m o u r a c t i v i t y , shows some d e g r e e o f c o r r e l a t i o n w i t h r e s u l t s o f a s s a y s f o r c y t o t o x i c i t y ( G a l s k y e t a l , 1980 and 1981; Meyer e t a l , 1982). B o t h a s s a y s have been u s e d o n l y r e l a t i v e l y r e c e n t l y and more d e s c r i p t i v e i n f o r m a t i o n on t h e d e g r e e of a s s o c i a t i o n between t h e a c t i v i t i e s measured i s n e eded. The r e s u l t s of T a b l e s XII t o XVI have been summarized i n t h e form o f 2 X 2 c o n t i n g e n c y t a b l e s i n T a b l e X V I I I . E a c h of t h e s e t s o f f o u r numbers r e p r e s e n t s a c o m p a r i s o n o f t h e r e s u l t s of t h e two b i o a s s a y s i n d i c a t e d . The f o r m a t f o r e a c h of t h e s e i s i l l u s t r a t e d i n t h e f o l l o w i n g t a b l e . 1 63 Test A p o s i t i v e negat ive p o s i t i v e a b Tes t B negat ive c d The l e t t e r (a) r e f e r s to the number of e x t r a c t s in which both Tes t A and Tes t B produced a p o s i t i v e r e s u l t ; (b) i n d i c a t e s the number hav ing a nega t i ve r e s u l t in Test A and a p o s i t i v e r e s u l t in Tes t B, e t c . These data were sub jec ted to s t a t i s t i c a l a n a l y s i s u t i l i z i n g B iomed i ca l Data Packages program P:4F to compute va lues fo r F i s h e r ' s exact t e s t . These s t a t i s t i c s ( f o r a two t a i l e d t e s t ) have been summarized in Tab le XIX. The va lues observed fo r t h i s s t a t i s t i c range from l e s s than 0 .0001, i n d i c a t i n g a c l o s e a s s o c i a t i o n of r e s u l t s , to 1.0,. i n d i c a t i n g no a s s o c i a t i o n of r e s u l t s . S ince no i n h i b i t o r y a c t i v i t y towards C. a l b i c a n s was observed in any of the e x t r a c t s , a s s o c i a t i o n s between b ioassay No. 3 and any of the o ther b i oassays c o u l d not be c a l c u l a t e d . The co r r e spond ing columns of Tab le XIX are b l ank . C e r t a i n i n t e r e s t i n g t rends can be d i s t i n g u i s h e d in these d a t a . The va lues of 0.4941 fo r F i s h e r ' s exact t e s t a p p l i e d 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 T a b l e XVIII - 2 X 2 C o n t i n g e n c y t a b l e s f o r agreement between each p a i r of the 14 a s s a y s used t o s c r e e n the 85 p l a n t e x t r a c t s . Data a r e summarized from T a b l e X V I I . 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 . 0 0 0 0 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 0 0 1 7 8 0 . 3 6 0 0 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 . 0 8 0 0 0 .1774 0 . 0 5 7 0 0 . 0 3 4 7 0.4941 1 .000 - 0.4941 0 .0283 0 .0066 0 .0034 0 .0030 0 .0074 1 .000 0 . 2 0 2 0 0. 3512 0 . 1 1 7 8 1 2 3 4 5 6 7 8 9 10 11 T a b l e XIX - V a l u e s of F i s h e r ' s e x a c t t e s t f o r c o n t i n g e n c y t a b l e s of T a b l e XVIII 12 13 1 66 the r e s u l t s of the s c r een ing of gm nega t i ve and gm p o s i t i v e b a c t e r i a i n d i c a t e s no a p p r e c i a b l e degree of a s s o c i a t i o n between these r e s u l t s . Nor d i d the r e s u l t s of the i n h i b i t i o n of E. c o l i growth s i g n i f i c a n t l y resemble the r e s u l t s of any of the o ther b i o a s s a y s . The r e s u l t s ob ta ined fo r S. au reus , however, were shown to be q u i t e c l o s e l y a s s o c i a t e d wi th the data fo r i n h i b i t i o n of growth of each of the dermatophyt i c fung i t e s t e d . The s i m i l a r i t y in the r e s u l t s ob t a i ned in the s c r e e n i n g fo r ant i-dermatophyte a c t i v i t y i s suppor ted by the o b s e r v a t i o n tha t the co r r e spond ing va lue fo r F i s h e r ' s exact t e s t i s l e s s than 0.0001 in a l l c a s e s . I t i s i n t e r e s t i n g tha t the r e s u l t s of the a n t i - S i n d b i s v i r u s ( p r e - i n f e c t i o n t reatment ) s c r een ing were a l s o c l o s e l y a s s o c i a t e d wi th the r e s u l t s of the an t i -de rma tophy t i c f ung i s c r e e n i n g . T h i s was not the case fo r any of the o ther th ree a n t i - v i r a l t e s t s c a r r i e d o u t . No a s s o c i a t i o n between the pre-i n f e c t i o n and p o s t - i n f e e t i o n t reatment of e i t h e r S i n d b i s v i r u s (p= 0.1429) or MCMV (p= 0.5421) was i n d i c a t e d . The r e s u l t s of the assays of the p r e - i n f e c t i o n t reatment of S i n d b i s v i r u s and MCMV were somewhat s i m i l a r (p= 0.0178) whi le the p o s t -i n f e c t i o n t reatment r e s u l t s showed no s i g n i f i c a n t a s s o c i a t i o n (p= 0.2278) . The r e s u l t s of the po ta to d i s c tumour i n h i b i t i o n assay were a s s o c i a t e d wi th the r e s u l t s of the i n h i b i t i o n of S. aureus (p= 0 .0001 ) , each of the an t i -de rma tophy t i c fung i assays and a n t i - S i n d b i s v i r u s ( p r e - i n f e c t i o n ) , as we l l as anti-MCMV (pre- and p o s t - i n f e c t ion) assays to some extent (p= 167 0 .035-0 .08 ) . The p a t t e r n of a s s o c i a t i o n of the b r i ne shr imp t o x i c i t y r e s u l t s w i th the r e s u l t s of the o ther b i oassays i s s i m i l a r to that of the ant i- tumour r e s u l t s . The s i n g l e excep t i on i s that a n t i - S. aureus a c t i v i t y and b r i n e shrimp t o x i c i t y show no a s s o c i a t i o n . The va lue of F i s h e r ' s s t a t i s t i c f o r the compar ison of ant i tumour a c t i v i t y and b r i ne shr imp t o x i c i t y i s 0. 1 1 78. 4. DISCUSSION a . A n t i m i c r o b i a l a c t i v i t y The percentage of Euphorb iaceous s p e c i e s e x h i b i t i n g a n t i m i c r o b i a l a c t i v i t y i s comparable to tha t of p l a n t s s e l e c t e d from a l l f a m i l i e s . Khan et a^ L (1980) r e p o r t e d tha t 98% and 18% of 60 s p e c i e s of A f r i c a n m e d i c i n a l p l a n t s i n h i b i t e d the growth of S. aureus and E. c o l i , r e s p e c t i v e l y . Ieven et a l (1979) observed 78% and 31% of 100 s p e c i e s of h ighe r p l a n t s wi th m e d i c i n a l uses to i n h i b i t the growth of these two s p e c i e s . It i s a p p a r e n t l y common fo r S. aureus to be more s e n s i t i v e than E. c o l i to a n t i m i c r o b i a l agents from p l a n t s . With the excep t i on of J a t ropha cu r cas and J . g o s s y p i i f o l i a , a l l of the s p e c i e s of Euphorb iaceae whose m e d i c i n a l use i s concerned wi th the t reatment of wounds or sk i n c o n d i t i o n s , were i n h i b i t o r y to S. aureus (Table XX) . Few of the Euphorb iaceous e x t r a c t s e x h i b i t e d any i n h i b i t o r y a c t i v i t y towards y e a s t s . None p reven ted the growth T a b l e XX Inh i b i t o r y A c t i v i t y o r T o x i c i t y Towards: Spec i es Col 1 . F o l k Use gm-ve gm+ve dermato p o t a t o No. b a c t e r i a b a c t e r i a y e a s t phyt i c fungus v i r u s tumour Artern i a A c a l y p h a b e n e n s i s 1 14 - + - + + + + A. d i v e r s i f o l i a 63 - + - + + + -A. m a c r o s t a c h y a 91 - - - + - + + A. s t a c h y u r a 1 13 - + - + - + + A l c h o r n e a c a s t a n e i f o l i a 95 rheumat ism, t o n i c - - - + - + + A. d i s c o l o r 81 - + - + + + + A. t r i p i i n e r v i a 31 - + - + + + + Amanoa a f f . o b l o n q i f o l i a 70 - + - + + + + A p a r i s t h m i u m cordatum 29 - + - + + + + Apodandra l o r e t e n s i s 56 - + - + + + + C a r v o d e n d r o n o r i n o c e n s e 84 s k i n d i s e a s e s - + - + - + + Chamaesyce h y s s o p i f o l i a 65 wounds, c a n c e r - + - + - + -C. thym i f o 1 i a 38 wounds, c a n c e r - + - + + + + C n i d o s c o l u s p e r u v i a n u s 133 a n t i - a p h r o d i s i a c - - - + + + + C o n c e v e i b a s t r u m mart ianum 101 - + - + + + + C r o t o n c u n e a t u s 93 - + + + - + + C. l e c h l e r i 62 wounds, c a n c e r + + + + + C. pa 1 a n o s t i gma 76 wounds, c a n c e r - + - + + + + C. t r i n i t a t i s 48 s o r e t h r o a t , c h e s t a i l m e n t s - + - + + + + D i d y m o c i s t u s c h r y s a d e n i u s 27 - + + + + + + Hevea b r a s i i i e n s i s 54 - - - + + - -J a t r o p h a c u r c a s 67 p u r g a t i v e , c a n c e r , s k i n c o n d . - - - - + + -J . pjossyp i i f o 1 i a 68 p u r g a t i v e , c a n c e r , s k i n c o n d . - - • - - + - -J . weberbauer i 136 aphrod i s i ac - - - - + - + Mabea maynens i s 79 - + - + + + -M. n i t i d a 53 s k i n c o n d i t i o n s + + - + + + + M a n i h o t e s c u l e n t a 61 b u r n s , s k i n i n f e c t i o n s - + - + + + -Maprouna g u i a n e n s i s 73 - + - + - + + P h y l l a n t h u s amarus 37 k i d n e y a i l m e n t s , l i v e r - + - + + + + P. o r b i c u l a t u s 49 + + - + + + + P. pseudo-conami 112 - - + + + + P. ur i nar i a 36 k i d n e y a i l m e n t s , l i v e r - + - + + + + P o d o c a l y x l o r a n t h o i d e s 89 - + - + + + + S e c u r i n e q a c o n q e s t a 30 + + + + + F r e q u e n c y +ve 6 76 6 91 79 91 79 Resu l t (%) T a b l e XX - Summary of e t h n o b o t a n i c a l Data a r e summarized f rom i n f o r m a t i o n and b i o l o g i c a l T a b l e s XII to X V I I I . a c t i v i t y of the 34 s p e c i e s of E u p h o r b i a c e a e t e s t e d . 1 70 of C. a l b i c a n s , wh i le 2 out of 34 s p e c i e s were a c t i v e a g a i n s t S. c e r e v i s i a e . S c reen ing s t u d i e s of a l l f a m i l i e s of h ighe r p l a n t s , by compar i son , have de t e c t ed a much h ighe r percentage (10-42%) of e x t r a c t s i n h i b i t o r y towards yeas t s (Farnsworth e_t a l , 1966; Fong et a l , 1972; Ieven et a l , 1979). The percentage of e x t r a c t s i n h i b i t i n g the growth of dermatophyt i c f ung i (91%) i s h i gh compared to that observed in o ther s t u d i e s : 2.6% (Farnsworth et_ a_l, 1966); 2.1% (Fong et a l , 1972); and 65% (Ieven et a l , 1979). As in the case of a n t i - S . aureus a c t i v i t y , 8 out of the 10 s p e c i e s of Euphorb iaceae tha t were used in the t reatment of s k i n c o n d i t i o n s are i n h i b i t o r y towards the dermatophyt i c fung i t e s t e d . b. A n t i v i r a l a c t i v i t y It i s somewhat s u r p r i s i n g tha t the e x t r a c t s were so un i f o rm l y e f f e c t i v e in i n a c t i v a t i n g both S i n d b i s v i r u s and MCMV when a p p l i e d to them at low c o n c e n t r a t i o n s . The E D 5 0 s of most of the a c t i v e e x t r a c t s was l e s s than 1 jug/ml (Table XV I I ) . The i n a c t i v a t i o n of v i r u s e s by d i r e c t a p p l i c a t i o n of a wide v a r i e t y of p l a n t e x t r a c t s has been r e p o r t e d p r e v i o u s l y (Konowalchuk and S p e i r s , 1976 and 1978). T h i s has been a t t r i b u t e d to the presence of p h e n o l i c compounds in the .aqueous e x t r a c t s a p p l i e d to the v i r u s e s (Cheo and L i n d n e r , 1964; John and Mukundan, 1978; Takech i and Tanaka, 1981). Among the p h e n o l i c compounds w i th a c t i v i t y i s the u b i q u i t o u s compound, t ann i c a c i d (Konowalchuk and S p e i r s , 1978). These 171 s o r t s of compounds would be expected to occur predominant l y in an aqueous e x t r a c t . The v i r u s i n a c t i v a t i n g a c t i v i t y of the aqueous e x t r a c t s t e s t e d in the present study may be the r e s u l t of p h e n o l i c compounds. A l a r g e percentage of the o rgan i c f r a c t i o n s , however, were a l s o h i g h l y e f f e c t i v e in i n a c t i v a t i n g •both S i n d b i s v i r u s and MCMV and i t i s d i f f i c u l t to a t t r i b u t e t h i s a c t i v i t y to p h e n o l i c c o n s t i t u e n t s . It i s known that sapon ins can a l s o i n a c t i v a t e c e r t a i n v i r u s e s ( Rage t l i and We int raub, 1974; R a g e t l i , 1975) and i t i s p o s s i b l e that the presence of t h i s , or o ther c l a s s e s of l i p o p h i l i c compounds w i th the a b i l i t y to d i s r u p t the s t r u c t u r e of biomembranes may be r e s p o n s i b l e . A l though d i r e c t v i r a l i n a c t i v a t i o n i s a p o t e n t i a l l y important b i o l o g i c a l a c t i v i t y , an agent which i n h i b i t s v i r u s r e p l i c a t i o n in a l r e a d y i n f e c t e d c e l l s would seem to be more u s e f u l as a t h e r a p e u t i c agen t . Of the 34 spec i e s of Euphorb iaceae t e s t e d , 23 (68%) i n h i b i t e d i n f e c t i o n by S i n d b i s v i r u s and 8 (24%) were e f f e c t i v e a g a i n s t MCMV. These are very h igh percentages compared w i th what has been observed from the h ighe r p l an t s c r e e n i n g programs c a r r i e d out by Farnsworth e_t a l (1966) ( 3% of s p e c i e s a c t i v e ) , Fong et a l (1972) (8.3% s p e c i e s a c t i v e ) and Van den Berghe e_t a_l (1979) (8% spec i e s a c t i v e ) . I t i s i n t e r e s t i n g to no te , a g a i n , that the r e s u l t s of the a n t i - S i n d b i s v i r u s and anti-MCMV assay were not a s s o c i a t e d , s t a t i s t i c a l l y , i n d i c a t i n g tha t the requ i rements f o r , and compounds i n v o l v e d i n , the i n h i b i t i o n of i n f e c t i o n by each v i r u s are q u i t e d i f f e r e n t . 1 72 c . A n t i t u m o u r a c t i v i t y The p e r c e n t a g e of t h e s p e c i e s t e s t e d w h i c h e x h i b i t e d a n t i - p o t a t o tumour a c t i v i t y was v e r y h i g h . The e x t r a c t s of 31 of 34 ( o r 91% o f s p e c i e s ) had an E D 5 0 l e s s t h a n 5 nq/ml. F e r r i g n i e t a_l (1982) o b s e r v e d 27% of t h e i r sample of E u p h o r b i a c e o u s s e e d s t o e l i c i t d e m o n s t r a b l e a n t i t u m o u r a c t i v i t y . T h i s d i f f e r e n c e may be a r e s u l t of t h e m e t h o d o l o g y a p p l i e d o r may r e f l e c t c h e m i c a l d i f f e r e n c e s between t h e v e g e t a t i v e m a t e r i a l u s e d i n t h i s s t u d y and t h e s e e d s u s e d by F e r r i g n i e_t a l ( 1 9 8 2 ) . I t i s r e a s o n a b l e t o e x p e c t t h a t t h e s t r a i n of A_;_ t u m e f a c i e n s and t y p e o f p o t a t o u s e d w o u l d i n t r o d u c e some v a r i a b i l i t y a l t h o u g h t h e r e i s i n s u f f i c i e n t i n f o r m a t i o n a v a i l a b l e t o d i s c u s s t h i s p o s s i b i l i t y f u r t h e r a t t h i s t i m e . d. T o x i c i t y t o b r i n e s h r i m p T h i r t y - o n e of t h e 34 s p e c i e s t e s t e d were t o x i c t o b r i n e s h r i m p . T h i s i n d i c a t e s t h a t t o x i c c o n s t i t u e n t s a r e p r e v a l e n t i n t h e s p e c i e s of t h i s f a m i l y . Meyer e_t a_l (1982) f o u n d t h a t t h e e x t r a c t s of t h e s e e d s of 18 o f 41 (43%) s p e c i e s o f E u p h o r b i a c e a e were t o x i c t o A r t e m i a . In t h e i r s t u d y , t o x i c i t y was d e f i n e d on t h e b a s i s of an L D 5 0 o f l e s s t h a n 1000 Mg/ml. I f a s i m i l a r c r i t e r i o n i s a p p l i e d t o t h e p r e s e n t s t u d y , t h e number of a c t i v e s p e c i e s i s r e d u c e d t o 27 ( 7 9 % ) . The g r e a t e r p r o p o r t i o n o f t o x i c s p e c i e s d e t e c t e d i n t h i s s t u d y s u p p o r t s t h e i d e a t h a t t h e v e g e t a t i v e p l a n t m a t e r i a l u s e d i n t h i s s t u d y i s d i f f e r e n t , w i t h r e s p e c t t o b i o l o g i c a l l y a c t i v e compounds p r e s e n t , f r o m t h e s e e d s u s e d by Meyer e t a_l ( 1 9 8 2 ) . K i n g h o r n 173 et_ a l (1977) examined a s e r i e s of pho rbo l e s t e r s fo r t h e i r t o x i c i t y to b r i n e shr imp. Ar temia were s e n s i t i v e to many of the phorbo l e s t e r s t e s t e d and t h i s assay s h o u l d , t h e r e f o r e , be u s e f u l fo r examining the Euphorb iaceae in which pho rbo l e s t e r s are w idespread . Many of the e x t r a c t s examined in the p resen t s tudy e x h i b i t e d a degree of t o x i c i t y to b r i ne shr imp that was as h igh as some of the pure compounds examined by K inghorn et_ a l (1977) . The recent i d e n t i f i c a t i o n of i r r i t a n t pho rbo l d e r i v a t i v e s from seeds of J a t ropha cu r cas and J . g o s s y p i i f o l i a (Adol f et a_l, 1984) r a i s e the q u e s t i o n of whether t h e s e , or s i m i l a r c o n s t i t u e n t s , are a l s o p resen t in v e g e t a t i v e m a t e r i a l of these p l a n t s . 5. CONCLUSION On the b a s i s of the compar ison w i th i n f o rma t i on in the l i t e r a t u r e , the s p e c i e s of Euphorb iaceae sampled appear to be e s p e c i a l l y r i c h in agents which i n h i b i t the growth of dermatophyt i c f u n g i , c e r t a i n v i r u s e s and tumours on po ta to d i s c s and in d i s p l a y i n g gene ra l t o x i c i t y . Whether these d i v e r s e b i o l o g i c a l a c t i v i t i e s are d i f f e r e n t m a n i f e s t a t i o n s of the same compounds or are produced s e p a r a t e l y by d i f f e r e n t compounds i s not known. Any, or a l l , of these b i o l o g i c a l a c t i v i t i e s c o u l d form the b a s i s f o r the use of these s p e c i e s of Euphorb iaceae in Amazonian e thnomed i c ine . Approx imate l y 80% of the s p e c i e s used in the t reatment of s k i n i n f e c t i o n s were a c t i v e in each of the four above ment ioned b i o a s s a y s . (Table XX) . Ten of the 16 174 s p e c i e s w i th m e d i c i n a l uses are used in the t reatment of c o n d i t i o n s tha t are l i k e l y to be the r e s u l t of e i t h e r i n f e c t i o u s organisms or m a l i g n a n c i e s . The numbers of s p e c i e s a c t i v e a g a i n s t S. au reus , the dermatophyt i c f u n g i , at l e a s t one of the v i r u s e s t e s t e d , po ta to tumours and b r i n e shr imp are 26, 31, 27, 31 and 31, r e s p e c t i v e l y . The ma jo r i t y of the s p e c i e s a c t i v e in these b i oassays have no documented use as a m e d i c i n a l agen t . T h i s c o u l d be a r e s u l t e i t h e r of t h e i r p r o p e r t i e s never hav ing been d i s c o v e r e d by a b o r i g i n a l peop les or the incomple te nature of the e t h n o b o t a n i c a l i n f o rma t i on a v a i l a b l e . Such c o n s i d e r a t i o n s i n vo l v e the assumpt ion that the assays used are p r e d i c t i v e on ly of the s o r t s of b i o l o g i c a l a c t i v i t i e s tha t might , in p r a c t i c e , be u s e f u l t h e r a p e u t i c a l l y . T h i s i s not the c a s e . A c e r t a i n percentage of f a l s e p o s i t i v e r e s u l t s have been observed us ing the po ta to d i s c tumour assay ( F e r r i g n i et. a l , 1982) and l i m i t a t i o n s in p r e d i c t a b i l i t y are inheren t i n any b i o l o g i c a l a s say . In t h i s r e s p e c t , i t i s i n t e r e s t i n g to note some of the q u a n t i t a t i v e a spec t s of the d a t a . The p o t e n c i e s of the b i o l o g i c a l a c t i v i t i e s of m e d i c i n a l l y used s p e c i e s a r e , on the average , no g rea t e r than those of the a c t i v e s p e c i e s wi th no r e c o r d e d ' e t h n o b o t a n i c a l use . Some of the s t ronges t a c t i v i t i e s , f o r example the a n t i v i r a l a c t i v i t y of Amanoa s p . , are p resent in s p e c i e s hav ing no known e t h n o b o t a n i c a l use . If the o b j e c t i v e of t h i s s c r een ing program had been the a t ta inment of p o s i t i v e r e s u l t s in any of the b i o l o g i c a l t e s t s c a r r i e d o u t , the re would, in r e t r o s p e c t , have been no 1 7 5 advantage ob ta ined from s e l e c t i n g p l a n t s wi th uses in e thnomed ic ine . It shou ld be emphasized that t h i s c o n c l u s i o n i s not in tended to app ly to o ther s c r e e n i n g programs fo r b i o l o g i c a l a c t i v i t y . It i s r e s t r i c t e d to the a p p l i c a t i o n of these s p e c i f i c b ioassays to the Amazonian s p e c i e s of Euphorb i aceae . Nor does i t imply tha t e t h n o b o t a n i c a l i n f o r m a t i o n conce rn ing i n d i v i d u a l s p e c i e s of Euphorb iaceae w i l l not be h e l p f u l in d i s c o v e r i n g s i g n i f i c a n t new b i o l o g i c a l l y a c t i v e c o n s t i t u e n t s . I t i s in tended as a summary statement fo r t h i s d e s c r i p t i v e s t udy . 176 PART B. a 2i Z Iz PELTATIN, THE ANTIVIRAL CONSTITUENT OF Amanoa sp . 1 . INTRODUCTION In a s c r een ing p r o j e c t of the Amazonian f l o r a , e x t r a c t s from the bark and l eaves of c e r t a i n s p e c i e s of Euphorb iaceae were observed to i n h i b i t the development of S i n d b i s v i r u s and murine c y tomega lov i rus in t i s s u e c u l t u r e c e l l s . A sample of Amanoa sp . e x h i b i t e d the s t r onges t a n t i v i r a l a c t i v i t y of the 34 s p e c i e s examined. E x t r a c t s of both the l eaves and bark were h i g h l y e f f e c t i v e in i n h i b i t i n g the fo rmat ion of p laques by both v i r u s e s . Both the o rgan i c and aqueous f r a c t i o n s were a c t i v e a g a i n s t the v i r u s e s and the former d i s p l a y e d s i g n i f i c a n t l y g rea te r a c t i v i t y . The l e a f e x t r a c t was s e l e c t e d fo r a p r o j e c t aimed at the i s o l a t i o n of the a c t i v e c o n s t i t u e n t ( s ) . 2. MATERIALS AND METHODS a . P l an t m a t e r i a l The p l a n t m a t e r i a l was c o l l e c t e d near I q u i t o s , Pe ru . Voucher spec imens(D. MacRae No. 70) have been d e p o s i t e d at U N A P ( l q u i t o s ) , San Marcos ( L ima ) , Ch icago F i e l d Museum and UBC Herbar ium. The i d e n t i f i c a t i o n of the p l a n t , Amanoa a f f . o b l o n g i f o l i a M u e l l . A r g . , was c a r r i e d out by D r . M.J. H u f t , Ch icago F i e l d Museum. P l an t m a t e r i a l was p r e se r ved in methanol p r i o r to e x t r a c t i o n . The l e a f (41 g dry weight) sample was homogenized i n 177 methanol and e x t r a c t e d e x h a u s t i v e l y wi th tha t so l v en t at 20 ° C . The combined e x t r a c t s were f i l t e r e d , evapora ted in vacuo and p a r t i t i o n e d between e t h y l a ce t a t e and d i s t i l l e d water . The r e s u l t i n g o rgan i c and aqueous e x t r a c t s were evapora ted in  vacuo and d i s s o l v e d in 95% and 50% e t h a n o l , r e s p e c t i v e l y and s t o r e d at -30 ° C . b. A n t i v i r a l assays The method fo r measur ing a n t i v i r a l a c t i v i t y i s d e s c r i b e d , in d e t a i l in Part C of t h i s c h a p t e r . B r i e f l y , murine c y tomega lov i rus (MCMV) was a p p l i e d to p e t r i d i s h e s c o n t a i n i n g an a lmost c o n f l u e n t monolayer of mouse (3T3) embryo c e l l s . A f t e r a p e r i o d of i n c u b a t i o n , which a l lowed fo r i n f e c t i o n by the v i r u s , the medium was removed and r e p l a c e d wi th a s o l i d medium ( c o n t a i n i n g 0.5% agarose) in which the p l a n t e x t r a c t s were d i s s o l v e d . C e l l s were incuba ted at 37°C u n t i l the v i r a l p laques were s u f f i c i e n t l y we l l deve loped fo r c o u n t i n g . They were counted w i th the unaided eye and per cent i n h i b i t i o n of p laque fo rmat ion was c a l c u l a t e d . c . Chromatography The e x t r a c t was r e s o l v e d us ing a r o t a r y TLC d e v i c e , the Chromatotron (Ha r r i son Research A s s o c i a t e s ) . A 2 mm p l a t e p repared from S i l i c a Ge l P F 2 5 « (Merck) was used e x c l u s i v e l y . E l u t i o n was c a r r i e d out us ing hep tane/ch lo ro fo rm/e thano l (25/25/3) at a f low ra te of 3 ml/min. F r a c t i o n s were c o l l e c t e d at two minute i n t e r v a l s . The e l u a t e was mon i tored by r e c o r d i n g i t s absorbance at 254 nanometers. 1 78 3. RESULTS AND DISCUSSION The i n i t i a l s e p a r a t i o n of the o rgan i c f r a c t i o n of the Amanoa l eaves (350 mg e x t r a c t ) y i e l d e d three major U.V. abso rb ing peaks , each c o n s i s t i n g of s e v e r a l components. The e l u t i o n p r o f i l e i s seen in F i g u r e 16. The p o l a r component of the e x t r a c t , which d i d not move in the so l v en t system hep tane/ch lo ro fo rm/e thano l (25/25/3) , was e l u t e d wi th acetone and c o l l e c t e d as three l a r g e f r a c t i o n s . The f r a c t i o n s c o l l e c t e d were each reduced to 3 ml volume by e vapo ra t i on and 2 M1 d i l u t e d 100 t imes wi th t i s s u e c u l t u r e medium ( D u l b e c c o ' s mod i f i ed minimum e s s e n t i a l medium c o n t a i n i n g 5% f e t a l bovine serum). The sample was d i l u t e d a f u r t h e r 5 t imes wi th minimum e s s e n t i a l medium c o n t a i n i n g 5% f e t a l bov ine . se rum (5%-MEM) and 0.5% agarose which was o ve r l a yed on a monolayer of mouse embryo c e l l s which had j u s t been i n f e c t e d w i th MCMV. The medium was a l l owed to s o l i d i f y and the p l a t e s were incubated at 37 °C u n t i l p laques had deve loped . The assay was c a r r i e d out in d u p l i c a t e . A c t i v e f r a c t i o n s were i d e n t i f i e d by t h e i r a b i l i t y to reduce the number, of MCMV p laques by more that 50%. F r a c t i o n s 19 through 26 were a l l c l a s s i f i e d as a c t i v e ( F igure 16) . I t i s c l e a r from the f i g u r e tha t these f r a c t i o n s co r respond to the e l u t i o n of a s t rong U.V. abso rb ing peak. The f r a c t i o n s were moni tored by TLC, combined and sub j ec t ed to f u r t h e r p u r i f i c a t i o n by i d e n t i c a l chromatograph ic p r o c e d u r e s . The a n t i v i r a l a c t i v i t y was a t t r i b u t a b l e to a 179 TIME(min.) g u r e 16 - C h r o m a t o t r o n e l u t i o n p r o f i l e of e t h y l a c e t a t e f r a c t i o n of Amanoa s p . l e a v e s . F r a c t i o n s w i t h a n t i - v i r a l a c t i v i t y a r e b l o c k e d o u t . 180 s i n g l e compound. The f o l l o w i n g s p e c t r a l data were ob ta ined fo r t h i s a c t i v e compound. a-(- ) - P e l t a t i n . Mp 229-230 ° C . [a] 25 (CHC1 3 ) -118 ° . U.V. X MeOH. 274, 240sh, 214. 1H-NMR. (80 MHz, CDC1 3 ) 5 6.35 (s , 2H, H-2 ' , H-6 ' ) , 6.21 (s , 1H, H-8), 5.93 ( s , 2H, 0 " C H 2 - 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, 2XOCH 3 ) , 3.1 - 3.5 (m, 1H, H-4), 2.6 - 2.75 (3H, H-2, H-3, H-4). MS m/z ( r e l . i n t . ) 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 ( i l ) , 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 s p e c t r a i n d i c a t e d that the compound might be a d i b e n z o b u t y r o l a c t o n e l i g n a n . The c h a r a c t e r i s t i c chemica l s h i f t of the C-8 pro ton and the equ i va l ence of the C-2' and C-6' p ro tons suggested that i t may be a - p e l t a t i n . T h i s was c o r r o b o r a t e d by a compar ison of i t s mass spectrum wi th tha t p u b l i s h e d fo r t h i s compound ( D u f f i e l d , 1967). a - P e l t a t i n e x i s t s as (+) and (-) d i a s t e r e o m e r s , depending upon the r e l a t i v e c o n f i g u r a t i o n at C-1. Based upon the c h a r a c t e r i s t i c chemica l s h i f t (5 4.58) and the o p t i c a l r o t a t i o n [a] (CHC1 3 ) = -118 ° , which were measured fo r the compound i s o l a t e d , i t was determined to be the (-) isomer ( F igu re 17). 181 OH H »-(-)-Peltat in F i g u r e 17 - S t r u c t u r e of a-( - ) - p e l t a t i n i s o l a t e d from Amanoa sp . 1 82 a - P e l t a t i n has r e c e n t l y been r e p o r t e d t o r e d u c e s i g n i f i c a n t l y t h e c y t o p a t h i c e f f e c t r e s u l t i n g f r o m i n f e c t i o n by h e r p e s s i m p l e x v i r u s - 1 ( H S V - l ) (Markkanen e_t a_l, 1981a). The r e l a t e d compound, p o d o p h y l l o t o x i n , a w e l l known c o n s t i t u e n t of t h e m e d i c i n a l r e s i n o f P o d o p h y l l u m s p . , was o b s e r v e d t o p r o d u c e a v e r y s i m i l a r e f f e c t (Markkanen e_t a l , 1981b). Bedows and H a t f i e l d (1982) have o b s e r v e d t h a t p o d o p h y l l o t o x i n i s e f f e c t i v e i n i n h i b i t i n g b o t h t h e c y t o p a t h i c e f f e c t and i n f e c t i v i t y p r o d u c e d by m e a s l e s v i r u s and HSV-1. They o b s e r v e d a - p e l t a t i n , however, t o have o n l y a s l i g h t a n t i v i r a l e f f e c t . By c o m p a r i s o n , F a r n s w o r t h e_t a_l ( 1 966) d e t e c t e d no a n t i v i r a l a c t i v i t y of an e x t r a c t o f P o d o p h y l l u m  p e l t a t u m a g a i n s t v a c c i n i a v i r u s , p o l i o v i r u s t y p e I I I and p s e u d o r a b i e s v i r u s . In c o m p a r i n g t h o s e r e s u l t s w i t h t h e ones o b t a i n e d i n t h i s s t u d y , t h e d i f f e r e n c e s i n t h e a s s a y s used s h o u l d be b o r n i n mind. M e a s l e s v i r u s i s a s i n g l e s t r a n d e d RNA v i r u s ( p a r a m y x o v i r u s g r o u p ) , w h i l e HSV-1 and MCMV a r e d o u b l e s t r a n d e d DNA v i r u s e s of t h e genus h e r p e s v i r u s . M o r e o v e r , i t i s not s t a t e d w h i c h i s o m e r of a - p e l t a t i n was u s e d . The a n t i v i r a l a c t i v i t y we o b s e r v e d from a- ( - ) - p e l t a t i n may be s p e c i f i c t o t h a t i s o m e r . a- ( - ) - P e l t a t i n i s t h e f i r s t l i g n a n t o be r e p o r t e d f r o m t h e genus Amanoa and t h e f i r s t l i g n a n o f i t ' s c l a s s ( c y c l o l i g n a n - 9 ' - 9 - 1 i g n a n o l i d e ) t o be r e p o r t e d f r o m t h e f a m i l y E u p h o r b i a c e a e . 183 PART C. THE ANTIVIRAL ACTIVITY OF LIGNANS 1. INTRODUCTION The c y c l o l i g n a n o l i d e , a-( - ) - p e l t a t i n , was i d e n t i f i e d as the potent a n t i v i r a l agent from an Amazonian s p e c i e s of Amanoa. T h i s o b s e r v a t i o n , combined wi th recent r e p o r t s (Bedows and H a t f i e l d , 1982; Markkanen et a l , 1981a and b) that o ther c y c l o l i g n a n o l i d e s a l s o produce a n t i v i r a l e f f e c t s s t i m u l a t e d t h i s compara t i ve study on the e f f e c t s of a v a r i e t y of l i g n a n s on v i r u s i n f e c t e d c e l l s . C y c l o l i g n a n o l i d e s d i s p l a y a d i v e r s i t y of b i o l o g i c a l e f f e c t s which c o u l d form the b a s i s f o r the mechanism of t h e i r a n t i v i r a l a c t i o n . Some d e r i v a t i v e s of p o d o p h y l l o t o x i n , the best known example of t h i s c l a s s of compounds, can damage DNA d i r e c t l y ( Lo ike and Ho row i t z , 1976a), wh i le o the r s are known to i n t e r f e r e w i th n u c l e o s i d e uptake and n u c l e i c a c i d metabol ism (Lo ike and Horow i tz , 1976b). S t i l l o the r s cause fundamental a l t e r a t i o n s in c e l l u l a r metabol ism (Waravdekar e_t a l , 1953). F i n a l l y , c e r t a i n p o d o p h y l l o t o x i n d e r i v a t i v e s b ind to t u b u l i n (Brewer et a l , 1979) i n t e r f e r i n g w i th i t s p o l y m e r i z a t i o n to form m i c r o t u b u l e s and , in t h i s way, i n h i b i t i n g m i t o s i s . To p r o v i d e f u r t h e r i n f o rma t i on on the mode of a n t i v i r a l a c t i o n of l i g n a n s , the f o l l o w i n g s t u d i e s were c a r r i e d o u t : 1) a wide v a r i e t y of l i g n a n s were t e s t e d f o r t h e i r p o s s i b l e e f f e c t s on v i r u s i n f e c t e d c e l l s ; 2) the a n t i v i r a l a c t i o n s of p o d o p h y l l o t o x i n and a-184 p e l t a t i n a g a i n s t two v i r u s e s , S i n d b i s v i r u s and murine c y tomega lov i rus (MCMV) were compared; 3) d i f f e r e n t t imes du r i ng the p rocess of i n f e c t i o n were examined to determine which stage was s e n s i t i v e to l i g n a n a c t i o n . 2. MATERIALS AND METHODS a . Chemica ls The l i g n a n s used were ob ta ined from a v a r i e t y of s o u r c e s . P o d o p h y l l o t o x i n was p u r i f i e d from a sample purchased from Sigma. a - P e l t a t i n was i s o l a t e d from Amanoa sp . as d e s c r i b e d in Part A. J u s t i c i d i n B and d i p h y l l i n , as we l l as i t s a p i o s i d e and a c e t y l a t e d a p i o s i d e were p rov ided by Dr . G. H. S h e r i h a , Dept . of Chemis t r y , A l Fa teh U n i v e r s i t y , L i b y a . P l i c a t i c a c i d , d i h y d r o f e r u l i c a c i d , m a t a i r e s i n o l , a - c o n i d e n d r i n , and d i m e t h y l r e t r o d e n d r i n were a g i f t of Dr . E r i c Swan, F o r i n t e k , Vancouver . A r c t i i n was i s o l a t e d from Arc t ium lappa and s e s a r t e m i n , e p i s e s a r t e m i n , yangambin, ep iyangambin , d i h y d r o s e s a r t e m i n and 0-dihydroyangambin were i s o l a t e d from V. e l onga t a (Chapter I I , t h i s t h e s i s ) . The s t r u c t u r e s of these compounds are p resen ted in F i g u r e 18. b. C e l l s and v i ruses The p r e p a r a t i o n of v i r u s e s and procedures fo r m a i n t a i n i n g c e l l s has been d e s c r i b e d in Part A, t h i s c h a p t e r . 185 Podophyllotoxin co-(-)-Peltatin DiphyllinR OH oo -Conidendrin CH,0 Dimethyl-OD-ret rodendri n Justicidin B F i g u r e 18 - S t r u c t u r e s of l i g n a n s t e s t e d f o r a n t i v i r a l a c t i v i t y . 186 c . A n t i v i r a l s c r e e n i n g of 1 i g n a n s The s a m p l e s were d i s s o l v e d i n 95% e t h a n o l ( 1 0% e t h a n o l i n t h e c a s e o f p l i c a t i c a c i d ) . T h e s e were d i l u t e d w i t h t i s s u e c u l t u r e medium s u c h as t o a l l o w f o r a f i n a l e t h a n o l c o n c e n t r a t i o n of 1%. The a s s a y s were c a r r i e d o ut by a l l o w i n g t h e compound t o r e m a i n i n c o n t a c t w i t h t h e c e l l s d u r i n g t h e p e r i o d o f e x p o s u r e t o t h e v i r u s and a l s o a f t e r t h e v i r u s had been removed and w h i l e t h e c e l l s were b e i n g i n c u b a t e d f o r s e v e r a l d a y s t o a l l o w f o r t h e d e v e l o p m e n t o f v i r a l p l a q u e s . N e a r l y c o n f l u e n t l a y e r s of mouse (3T3) embryo c e l l s (between p a s s a g e 15 and 22) were b a t h e d i n D u l b e c c o ' s minimum e s s e n t i a l medium (MEM) c o n t a i n i n g e i t h e r Sin,dbis v i r u s o r m u r i n e c y t o m e g a l o v i r u s (MCMV) a t a t i t e r c a l c u l a t e d t o a l l o w f o r a c o u n t a b l e number of p l a q u e s and t h e compound t o be t e s t e d . P o d o p h y l l o t o x i n , a - p e l t a t i n , j u s t i c i d i n B, and d i p h y l l i n and i t s a p i o s i d e s were t e s t e d a t c o n c e n t r a t i o n s o f 0.01, 0.10 and 1.0 Mg/ml, w h i l e t h e r e m a i n i n g compounds were t e s t e d a t 1.0, 10 and 100 Mg/ml. A f t e r 2 h o u r s o f i n c u b a t i o n a t 37 °C, t h e s o l u t i o n c o n t a i n i n g v i r u s and l i g n a n was removed and t h e p l a t e s were o v e r l a y e d w i t h D u l b e c c o ' s MEM c o n t a i n i n g 5% f e t a l b o v i n e serum ( F B S ) , 0.5% a g a r o s e and t h e a p p r o p r i a t e l i g n a n a t t h e same c o n c e n t r a t i o n . A f t e r t h e p l a t e s had s o l i d i f i e d , t h e y were i n c u b a t e d u n t i l t h e p l a q u e s had d e v e l o p e d s u f f i c i e n t l y f o r c o u n t i n g . 187 d . E f f e c t of time of t reatment Three s tages in the p rocess of i n f e c t i o n were examined fo r s e n s i t i v i t y of the v i r u s to the presence of the a n t i v i r a l l i g n a n s , p o d o p h y l l o t o x i n and a - p e l t a t i n : the p r e - i n f e c t i o n p e r i o d ; the p e r i o d du r i ng i n f e c t i o n ; and the p e r i o d a f t e r i n f e c t i o n : P r e - i n f e c t ion t reatment Suspens ions of v i r u s were exposed to the v a r i o u s c o n c e n t r a t i o n s of each compound in D u l b e c c o ' s MEM, c o n t a i n i n g 0.1% e thano l f o r a p e r i o d of 2 hou r s . A l i q u o t s were added to monolayers of mouse embryo c e l l s , d i l u t i n g the v i r u s and l i g n a n mixture 25,000 t imes . A f t e r i n c u b a t i n g the c e l l s w i th v i r u s at 37 °C fo r a f u r t h e r 2 hou r s , the medium was removed and the c e l l s o ve r l a yed wi th MEM c o n t a i n i n g 5% FBS and 0.1% aga rose . The p l a t e s were incuba ted u n t i l p laques had deve loped . Treatment dur ing i n f e c t ion V i r u s was combined wi th e i t h e r p o d o p h y l l o t o x i n or a-p e l t a t i n in D u l b e c c o ' s MEM (1% e thano l f i n a l c o n c e n t r a t i o n ) , which was then a p p l i e d to a monolayer of mouse c e l l s and the p l a t e s were incuba ted fo r 2 hours at 37 ° C . The medium was removed and r e p l a c e d wi th a s o l i d o v e r l a y as d e s c r i b e d above. Treatment p o s t - i n f e c t i o n Mouse embryo c e l l monolayers were exposed to v i r u s suspended in D u l b e c c o ' s MEM fo r 2 hours at 37 ° C . The v i r u s was removed and a s o l i d o v e r l a y was added. T h i s c o n t a i n e d , in 188 a d d i t i o n to D u l b e c c o ' s MEM wi th 5% FBS and 0.5% aga rose , e i t h e r p o d o p h y l l o t o x i n or a - p e l t a t i n and 1% e t h a n o l . The p l a t e s were incuba ted u n t i l p l aques were v i s i b l e . 3. RESULTS P o d o p h y l l o t o x i n and a - p e l t a t i n are h i g h l y e f f e c t i v e in p r e v e n t i n g the development of MCMV p laques in mouse embryo c e l l s (Table XX I ) . Both compounds are approx imate l y equa l in po tency , r educ ing the number of p laques formed by a lmost 50% at a c o n c e n t r a t i o n of 10 ng/ml . Ne i t he r compound, however, had any e f f e c t on the fo rmat ion of S i n d b i s v i r u s p laques in mouse embryo c e l l s , even at a dose as h igh as 1 Mg/ml. The a r y l n a p t h a l e n e compounds, j u s t i c i d i n B and d i p h y l l i n , as w e l l as i t s a p i o s i d e and a c e t y l a t e d a p i o s i d e , were e f f e c t i v e in r educ ing the number of S i n d b i s v i r u s p laques and, to some e x t e n t , a l s o the MCMV p l a q u e s . C l e a r dose-response were not e v i d e n t . None of the o ther l i g n a n s t e s t e d showed any a n t i v i r a l a c t i v i t y , even at doses as h igh as 100 Mg/ml. To attempt to ga in f u r t h e r i n f o rma t i on on the mode of a c t i o n of the a n t i v i r a l l i g n a n s p o d o p h y l l o t o x i n and a-p e l t a t i n , the compounds were a p p l i e d at v a r i o u s t imes du r i ng the p rocess of v i r a l i n f e c t i o n . To determine whether these l i g n a n s had any d i r e c t e f f e c t on the v i r u s e s , v i r u s was i ncuba ted w i th l i g n a n fo r two hours p r i o r to be ing used to i n f e c t the c e l l s ( p r e - i n f e c t i o n t r e a tmen t ) . To t e s t whether e a r l y or l a t e s tages of the i n f e c t i v e p rocess were a f f e c t e d , c e l l s were i n f e c t e d wi th v i r u s in the presence of l i g n a n % I n h i b i t i o n of P l a q u e F o r m a t i o n L i g n a n T e s t e d 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 <//ml j/g/ml »/g/ml „g/ml „g/ml „g/ml ,/g/ml P o d o p h y l l o t o x i n 8 3 4 . nt t nt 35 74 100 nt nt a - p e l t a t i n 0 6 7 nt nt 49 85 100 nt nt J u s t i c i d i n B 42 . 74 tox * nt nt 13 14 tox nt nt D i p h y l 1 i n 7 18 84 nt nt 7 1 1 '19 nt nt D i phy11 i n 8 14 100 nt nt 26 10 9 nt nt ap i os i de D i p h y l 1 i n 20- 96 95 nt nt 33 4 25 nt nt ap i os i d e - O A c D i m e t h y l r e t r o d e n d r i n nt nt 3 7 tox nt nt 1 6 tox a - C o n i dendr i n nt nt. 7 0 5 nt nt 2 5 0 P I i c a t i c a c i d nt . nt 1 6 0 nt nt 2 1 3 Mata i r e s i no 1 nt nt 7 4 tox nt nt 4 0 tox A r c t i i n nt nt 4 5 3 nt nt 3 4 3 D i h y d r o f e r u 1 i c nt nt 4 4 0 nt nt 1 3 2 ac i d E p i s e s a r t e m i n nt nt 2 7 2 nt nt 0 3 5 S e s a r t e m i 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 h y d r o v 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 T a b l e XXI - E x a m i n a t i o n of l i g n a n s f o r t h e i r e f f e c t on r e p l i c a t i o n of S i n d b i s V i r u s and MCMV in mouse c e l l s . R e s u l t s a r e the a v e r a g e of an exper iment c a r r i e d out i n d u p l i c a t e . t nt = not t e s t e d . $ tox = t o x i c to c e l l s ; p l a q u e s were not c o u n t e d . 190 ( t reatment d u r i n g i n f e c t i o n ) and c e l l s a l r e a d y i n f e c t e d wi th v i r u s were exposed to l i g n a n fo r the complete d u r a t i o n of the c u l t u r e p e r i o d ( p o s t - i n f e c t i o n t r e a t m e n t ) . The e f f e c t s of l i g n a n s on MCMV i n f e c t i o n are p resen ted in F i g u r e 19 wh i l e th,e data conce rn i ng S i n d b i s v i r u s can be found in Tab le XXI I . F i g u r e 19 suppor ts the i n i t i a l f i n d i n g tha t p o d o p h y l l o t o x i n and a - p e l t a t i n are h i g h l y e f f e c t i v e in i n h i b i t i n g the fo rmat ion of p l aques by MCMV when a p p l i e d c o n t i n u o u s l y to i n f e c t e d c e l l s . As shown in Tab le XXI , a-p e l t a t i n i s s l i g h t l y more potent than p o d o p h y l l o t o x i n . When the c e l l s are i n f e c t e d w i th v i r u s and exposed to p o d o p h y l l o t o x i n or a - p e l t a t i n c o n c u r r e n t l y , the l i g n a n s are on l y s l i g h t l y l e s s e f f e c t i v e in i n h i b i t i n g p laque f o r m a t i o n . T h i s i s somewhat s u r p r i z i n g s i n c e the d u r a t i o n of exposure to l i g n a n i s on l y two hou r s ; compared wi th s e v e r a l days (assuming tha t i t i s not me tabo l i zed by the c e l l s ) in the case of the p o s t - i n f e c t i o n t r ea tment . When the v i r u s i s exposed d i r e c t l y to p o d o p h y l l o t o x i n , no e f f e c t was d e t e c t e d at the h ighes t c o n c e n t r a t i o n t e s t e d (40 Mg/ml). a - P e l t a t i n , on the o ther hand, caused a dose-dependent r e d u c t i o n in the number of p laques formed from t r e a t e d v i r u s e s . T h i s e f f e c t was approx imate l y three o rde r s of magnitude weaker than tha t produced by t reatment d u r i n g or a f t e r i n f e c t i o n by v i r u s . The number of p laques were reduced by a lmost 50% a f t e r two hours p r e - i n f e c t i o n t reatment w i th a-p e l t a t i n . S i n d b i s v i r u s responded q u i t e d i f f e r e n t l y to t reatment 191 LOG CONCENTRATlON(ng/ml) F i gu re 19 - E f f e c t of time of l i g n a n t reatment on i n h i b i t i o n of murine c y tomega lov i rus i n f e c t i o n in mouse embryo c e l l s . S o l i d f i g u r e s r e f e r to a-( - ) - p e l t a t i n ; open p o d o p h y l l o t o x i n . Squares = p r e - i n f e c t i o n t r ea tment ; t reatment d u r i n g i n f e c t i o n ; c i r c l e s = p o s t - i n f e c t i o n ones to t r i a n g l e s = t r ea tment . Compound % R e d u c t i o n in P l a q u e s ' T reatment 0 .005 0.01 0. 1 10 40 Protoco1 „g/ml l/g/ml „g/ml „g/ml eg/ml p o d o p h y l l o t o x i n 0 1 0 1 0 p r i o r t o i n f e c t i o n a - p e l t a t i n 2 1 0 1 0 p o d o p h y l 1 o t o x i n 0 1 0 13 58 d u r i n g i n f e c t i o n a - p e l t a t i n 0 1 2 0 52 p o d o p h y l 1 o t o x i n 1 0 1 0 0 a f t e r i n f e c t i o n a - p e l t a t i n 0 2 0 1 1 T a b l e XXII - E f f e c t of t ime of l i g n a n t rea tment upon i n h i b i t i o n of S i n d b i s V i r u s i n f e c t i o n in mouse embryo c e l l s . R e s u l t s a r e the a v e r a g e of an exper iment c a r r i e d out i n t r i p l i c a t e . 1 93 wi th p o d o p h y l l o t o x i n or a - p e l t a t i n (Table XX I I ) . Ne i the r pre-i n f e c t i o n nor p o s t - i n f e c t i o n t reatment wi th e i t h e r l i g n a n had any e f f e c t , even at c o n c e n t r a t i o n s as h igh as 40 Mg/ml. Expos ing the c e l l s to l i g n a n at the same time as they are be ing i n f e c t e d w i th S i n d b i s v i r u s , however, r e s u l t e d in a dose-dependent r e d u c t i o n in the number of v i r a l p laques u l t i m a t e l y formed. T h i s e f f e c t , t o o , i s much weaker (1/1000 t imes as potent ) as tha t observed fo r l i g n a n t reatment d u r i n g or a f t e r i n f e c t i o n by MCMV. 4. DISCUSSION A l though the number of compounds t e s t e d was s m a l l , the r e s u l t s i n d i c a t e that a n t i v i r a l a c t i v i t y i s s p e c i f i c to c e r t a i n c l a s s e s of l i g n a n s . The t e t r a h y d r o f u r a n l i g n a n s , d i h yd rosesa r t em in and /3-dihydroyangambin, and the b i s -t e t r a h y d r o f uran l i g n a n s , e p i s e s a r t e m i n , s e s a r t e m i n , epiyangambin and yangambin, were a l l w i thout e f f e c t . B i s-t e t r a h y d r o f uran l i g n a n s are known to have a number of b i o l o g i c a l e f f e c t s i n c l u d i n g the a b i l i t y to i n h i b i t c y c l i c AMP phosphod i e s t e r a se (N ika ido et_ a_l, 1981), a n t i - s t r e s s a c t i v i t y (Brekhman and Dardymov, 1969), hypo tens i ve a c t i v i t y (S ih e_t a l , 1976) and b e h a v i o r a l e f f e c t s (Chapter I I I , t h i s t h e s i s ) . Ne i t he r of the b u t a n o l i d e l i g n a n s t e s t e d , m a t a i r e s i n o l or a r c t i i n , d i s p l a y e d any a n t i v i r a l a c t i v i t y . M a t a i r e s i n o l , l i k e c e r t a i n b i s - t e t r a h y d r o f u r a n l i g n a n s , i s an i n h i b i t o r of the enzyme, c y c l i c AMP phosphod i e s t e r a se (N ika ido et, a_l, 1981). P l i c a t i c a c i d i s a r e a c t i v e compound and i s r e s p o n s i b l e f o r 194 p roduc ing asthma and e l i c i t i n g an a l l e r g i c response in man (Chan-Yeung et a l , 1973). I t , t o o , was without e f f e c t on the r e p l i c a t i o n of the two v i r u s e s t e s t e d . D i m e t h y l r e t r o d e n d r i n and a - c o n i d e n d r i n , l i k e p o d o p h y l l o t o x i n and a - p e l t a t i n , are c y c l o l i g n a n o l i d e s . Ne i t he r had any a n t i v i r a l a c t i v i t y . The e f f e c t of the a r y l n a p t h a l e n e l i g n a n s , j u s t i c i d i n B and d i p h y l l i n and i t s a p i o s i d e s in i n h i b i t i n g S i n d b i s v i r u s i n f e c t i o n i s i n t e r e s t i n g . A l though these compounds are not known to have any e f f e c t on m i c ro tubu l e s or upon n u c l e o s i d e t r a n s p o r t , j u s t i c i d i n B i s h i g h l y t o x i c to f i s h (Munakata et a l , 1965). The mechanism of a c t i o n of t h i s p i s c i c i d a l compound i s comp le t e l y unknown and i t i s worthwhi le c o n s i d e r i n g that the a n t i v i r a l and p i s c i c i d a l a c t i v i t i e s of these compounds share some aspec t s r ega rd ing t h e i r mode of a c t i o n . The exper iments in which v i r u s i s exposed to p o d o p h y l l o t o x i n or a - p e l t a t i n b e f o r e , du r i ng or a f t e r i n f e c t i o n y i e l d e d some g e n e r a l , though q u i t e i n t e r e s t i n g c o n c l u s i o n s . The anti-MCMV a c t i v i t y of these compounds cannot be e x p l a i n e d by a d i r e c t e f f e c t upon the v i r u s . P o d o p h y l l o t o x i n had no e f f e c t at the doses t e s t e d , whi le a-p e l t a t i n caused a s l i g h t i n a c t i v a t i o n of MCMV v i r u s which was not of s u f f i c i e n t magnitude to e x p l a i n the anti-MCMV a c t i v i t y . The a b i l i t y of a - p e l t a t i n to damage the v i r u s d i r e c t l y i s c o n s i s t e n t w i th p r e v i ous r e p o r t s tha t t h i s , and o ther p o d o p h y l l o t o x i n type l i g n a n s hav ing a 4 ' - h y d r o x y l group, possess the a b i l i t y to cause DNA f ragmenta t ion ( Lo ike and H o r o w i t z , 1976a). It i s noteworthy tha t a - p e l t a t i n had no 195 co r r e spond ing e f f e c t upon the s i n g l e s t randed RNA c o n t a i n i n g S i n d b i s v i r u s . A s t rong anti-MCMV e f f e c t of p o d o p h y l l o t o x i n and a~ p e l t a t i n i s observed on l y when c e l l s are t r e a t e d d u r i n g or a f t e r i n f e c t i o n . Because t reatment a f t e r i n f e c t i o n was the most e f f e c t i v e reg imen, i t can be conc luded tha t n e i t h e r attachment nor p e n e t r a t i o n of the v i r u s are s tages which are d i s r u p t e d by these l i g n a n s . T h i s statement shou ld be q u a l i f i e d by n o t i n g that i n f e c t i o n by S i n d b i s v i r u s was i n h i b i t e d to some extent by r e l a t i v e l y h igh c o n c e n t r a t i o n s of both p o d o p h y l l o t o x i n and a - p e l t a t i n (Tab le XX I I ) . I t appears tha t these compounds do have a weak, but s i g n i f i c a n t , i n h i b i t o r y e f f e c t on the e a r l y s tages of the S i n d b i s v i r u s i n f e c t i o n . It i s s i g n i f i c a n t tha t the anti-MCMV e f f e c t i s e v iden t whether the c e l l s are t r e a t e d fo r two hours d u r i n g i n f e c t i o n or fo r s e v e r a l days a f t e r i n f e c t i o n . From F i g u r e 19, i t appears tha t the anti-MCMV a c t i v i t y of the pos t- t rea tment p r o t o c o l was approx imate l y three t imes more potent than tha t of the t reatment du r i ng i n f e c t i o n p r o t o c o l . T h i s d i f f e r e n c e in a n t i v i r a l potency i s d i s p r o p o r t i o n a t e l y sma l l r e l a t i v e to the d i f f e r e n c e in the dose of l i g n a n a p p l i e d in each p r o t o c o l . The most obv ious e x p l a n a t i o n s suggested by t h i s r e s u l t are e i t h e r that the e f f e c t of the two l i g n a n s i s i r r e v e r s i b l e and the time of exposure i s unimportant or that some stage of v i r u s development a f f e c t e d by them i s t r a n s i e n t and, i f i n t e r r u p t e d , the p rocess i s not c o n t i n u e d . Such a stage would need to e x i s t both d u r i n g and a f t e r the f i r s t two hours of v i r u s i n f e c t i o n . 1 96 It has been suggested that the a n t i v i r a l a c t i v i t y of p o d o p h y l l o t o x i n type l i g n a n s i s a r e s u l t of t h e i r a f f i n i t y f o r t u b u l i n and t h e i r a b i l i t y to i n t e r r u p t i t s agg rega t i on (Bedows and H a t f i e l d , 1982; Markkanen et a l , 1981b). C e r t a i n drugs which a f f e c t t u b u l i n have been s t u d i e d wi th respec t to t h e i r a n t i v i r a l a c t i v i t i e s and the r e s u l t s a r e , by no means, c o n c l u s i v e . The e a r l y o b s e r v a t i o n s tha t c o l c h i c i n e or the V inca a l k a l o i d s c o u l d suppress v i r a l i n f e c t i o n s were encourag ing from the c l i n i c a l v i e w p o i n t . We ins te in and Chang ( i960) found that c o l c h i c i n e was e f f e c t i v e in supp res s i ng the e a r l y s tages of i n f e c t i o n of mice by i n f l u e n z a and encepha lomyoca rd i t i s . v i r u s e s . The c o l c h i c i n e d e r i v a t i v e , demeco l c i ne , demonstrated a n t i v i r a l a c t i v i t y a g a i n s t v a c c i n i a , p o l i o , ECHO and Coxsack ie B v i r u s e s in t i s s u e c u l t u r e and-Newcast le d i s e a s e v i r u s in the d e v e l o p i n g c h i c k embryo ( K a t s i l a m b r o s , 1962). C o l c h i c i n e and demeco lc ine a l s o suppressed i n f e c t i o n of r a b b i t cornea c e l l s and r a b b i t t i s s u e c u l t u r e c e l l s by herpes s implex v i r u s (Tokumaru and A v i t a b i l e , 1971). The V in ca a l k a l o i d , v i n b l a s t i n e , i n c r e a s e d the s u r v i v a l of mice i n f e c t e d by mengo v i r u s ( Johnston , 1965) and v i n c r i s t i n e was a c t i v e a g a i n s t F r i e n d and Rauscher leukemia v i r u s e s in mice ( C h i r i g o s , 1965). V i n b l a s t i n e and v i n c r i s t i n e were a l s o e f f e c t i v e aga in s t herpes s implex v i r u s i n f e c t i o n in r a b b i t cornea or r a b b i t k idney t i s s u e c u l t u r e c e l l s (Tokumaru and A v i t a b i l e , 1971). In a s i g n i f i c a n t number of s t u d i e s , however, these a n t i m i t o t i c subs tances have been found to have no e f f e c t upon 1 97 v i r a l r e p l i c a t i o n . C o l c h i c i n e was observed to be i n e f f e c t i v e a g a i n s t p o l i o v i r u s (Kovacs, 1962), REO v i r u s (Da les , 1963) and v a c c i n i a v i r u s (Solovyov and Men tkev i ch , 1965) in t i s s u e c u l t u r e . V i n c r i s t i n e had no e f f e c t on m u l t i p l i c a t i o n of mengo v i r u s ( Johnston, 1965) or v a c c i n i a , polyoma, Rous sarcoma or encepha lomyoca rd i t i s v i r u s e s (Freeman e_t a_l, 1965). S eve ra l s t u d i e s have p r o v i d e d ev idence tha t the c e s s a t i o n of RNA s y n t h e s i s tha t norma l l y accompanies the metaphase s t a t e , and a l s o c o l c h i c i n e induced metaphase a r r e s t , i s r e s p o n s i b l e fo r the i n t e r r u p t i o n of v i r a l i n f e c t i o n . In the case of p o x v i r u s , i t was shown that uncoa t ing of the v i r u s was i n h i b i t e d due to the absence of a c e l l u l a r p r o t e i n produced in response to i n f e c t i o n ( J o k l i k , 1964). Newcast le d i s e a s e v i r u s d i d undergo normal a t tachment , p e n e t r a t i o n and e c l i p s e in c o l c h i c i n e t r e a t e d c e l l s but f u r t h e r r e p l i c a t i o n of the v i r u s was p r e v e n t e d , appa r en t l y due to the absence of c e l l u l a r RNA s y n t h e s i s (Marcus and Robb ins , 1963). Ev idence has been p resen ted which i n d i c a t e s tha t m i t o t i c i n h i b i t o r s a l s o i n h i b i t the f i n a l stage of v i r a l assembly . C o l c h i c i n e reduced the e x t r a c e l l u l a r p r o d u c t i o n of S em l i k i F o r e s t v i r u s by 75 to 90% and the e f f e c t was a t t r i b u t e d to the d e p o l y m e r i z a t i o n of m i c r o t u b u l e s (R ichardson and Vance , 1978). In compar ing t h i s r e s u l t w i th the data r e p o r t e d h e r e , i t i s important to note t h a t , i n the study of R i cha rdson and Vance (1978) , the c o n c e n t r a t i o n of c o l c h i c i n e used was 1000 f o l d g r ea t e r than that r e q u i r e d to i n h i b i t m i t o s i s . From the r e s u l t s r epo r t ed he re , i t i s e v iden t that p o d o p h y l l o t o x i n 198 i n h i b i t s MCMV i n f e c t i o n at the minimum dose r e q u i r e d to prevent m i t o s i s . An oppos ing e f f e c t of c o l c h i c i n e on v i r a l i n f e c t i o n s has a l s o been r e p o r t e d . I n f e c t i v i t y of herpes s implex v i r u s type 2 DNA to r a b b i t k idney c e l l s was enhanced 5 to 7 f o l d by expos ing c e l l s to c o l c h i c i n e , c o l c e m i d or v i n b l a s t i n e p r i o r to i n f e c t i o n (Farber and E b e r l e , 1976). A g a i n , the doses used were c o n s i d e r a b l y g rea te r than those r e q u i r e d to i n h i b i t m i t o s i s . The a v a i l a b l e ev idence i n d i c a t e s that m i c r o t u b u l e s p l a y a r o l e in v i r a l i n f e c t i o n s . Judg ing from the v a r i a t i o n in the r e s u l t s , i t appears that the nature of tha t r o l e may vary a g rea t d e a l , depending upon the s p e c i f i c v i r u s , the s p e c i f i c c e l l type and the stage of the p rocess of i n f e c t i o n . The r e s u l t s p resen ted here conce rn i ng the a n t i v i r a l a c t i v i t y of a-p e l t a t i n and p o d o p h y l l o t o x i n ' do not support the hypo thes i s tha t the i n h i b i t i o n of m i c r o t u b u l e s by these two compounds i s r e s p o n s i b l e fo r t h e i r a n t i v i r a l a c t i v i t y . Two p i e c e s of ev idence may be c i t e d : 1) these l i g n a n s exe r t an a n t i v i r a l a c t i o n aga in s t murine c y tomega lov i rus but not S i n d b i s v i r u s . 2) the a n t i v i r a l e f f e c t of the l i g n a n s a g a i n s t MCMV was not observed to be r e a d i l y r e v e r s i b l e . It seems u n l i k e l y that m i c r o t u b u l e s are a requirement fo r the m u l t i p l i c a t i o n of MCMV but not S i n d b i s v i r u s . The i r r e v e r s i b i l i t y of the a n t i v i r a l a c t i v i t y i s not c o n s i s t e n t w i th what i s known of the a n t i m i t o t i c behav io r of these 199 compounds ( D u s t i n , 1978). Another c e l l u l a r a c t i v i t y known to be i n h i b i t e d by p o d o p h y l l o t o x i n i s n u c l e o s i d e t r a n s p o r t . A l though the c o n c e n t r a t i o n r e q u i r e d to e l i c i t a d e t e c t i b l e e f f e c t i s somewhat g r ea t e r than that r e s u l t i n g in i n h i b i t i o n of m i t o s i s , the e f f e c t i s s t rong and q u i t e s p e c i f i c (Lo ike and Horow i t z , 1976b; M i z e l and W i l s o n , 1972). The s t u d i e s of both Lo ike and Horowi tz (1976a and b) and M i z e l and Wi lson (1972) have p r o v i d e d c o n v i n c i n g ev idence that the i n h i b i t i o n of t u b u l i n agg rega t i on and n u c l e o s i d e uptake by p o d o p h y l l o t o x i n , as w e l l as by c o l c h i c i n e , are q u i t e independent phenomena. For these r easons , i t i s worthwhi le c o n s i d e r i n g tha t the mode of the a n t i v i r a l a c t i o n of p o d o p h y l l o t o x i n and a - p e l t a t i n i n v o l v e s an e f f e c t upon n u c l e o s i d e t r a n s p o r t . The same arguments c i t e d above aga in s t the hypo thes i s that the a n t i v i r a l a c t i v i t y i s the r e s u l t of t u b u l i n b i n d i n g may be a p p l i e d h e r e : i . e . , the re i s no a v a i l a b l e e x p l a n a t i o n fo r the s e n s i t i v i t y of on ly MCMV or fo r the l a ck of r e v e r s i b i l i t y of the e f f e c t . I n h i b i t i o n of n u c l e o s i d e uptake by p o d o p h y l l o t o x i n has c l e a r l y been shown to be r e a d i l y r e v e r s i b l e ( Lo ike and Ho row i t z , 1976a). Moreover , the c a p a c i t y for n u c l e o s i d e uptake i s not a b s o l u t e l y r e q u i r e d fo r c e l l growth and i t i s not c l e a r why v i r u s r e p l i c a t i o n shou ld be a f f e c t e d under these c o n d i t i o n s . The s y n t h e s i s of RNA, DNA and p r o t e i n has been shown to be u n a f f e c t e d by t reatment w i th e i t h e r p o d o p h y l l o t o x i n or c o l c h i c i n e (Lo ike and Ho row i t z , 1976a: M i z e l and W i l s o n , 1972). 2 0 0 I t must be conc luded tha t the s i t e of a c t i o n of p o d o p h y l l o t o x i n and a - p e l t a t i n which i s r e s p o n s i b l e fo r t h e i r a n t i v i r a l a c t i v i t y i s unknown. I f i t i n v o l v e s i n h i b i t i o n of e i t h e r t u b u l i n aggrega t ion or n u c l e o s i d e t r a n s p o r t then i t appears tha t the s u s c e p t i b l e s i t e i s a c r i t i c a l s tage which, i f i n t e r r u p t e d du r i ng the f i r s t two hours f o l l o w i n g v i r u s i n f e c t i o n , u l t i m a t e l y p reven t s the normal development of the r e p l i c a t i v e c y c l e . 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A n t i v i r a l a c t i v i t y . L l o y d i a 41, 43-47. W a r a v d e k a r , V.S., P a r a d i s , A.D. and L e i t e r , J . ( 1 9 5 3 ) . Enzyme c h a n g e s i n d u c e d i n n o r m a l and m a l i g n a n t t i s s u e s w i t h c h e m i c a l a g e n t s . I I I . E f f e c t o f a c e t y l p o d o p h y l l o t o x i n - c j -p y r i d i n i u m c h l o r i d e on c y t o c h r o m e o x i d a s e , c y t o c h r o m e c, s u c c i n o x i d a s e , s u c c i n i c d e h y d r o g e n a s e and r e s p i r a t i o n of sarcoma 37. J . N a t l . C a n c e r I n s t . 14, 585-592. W e i n s t e i n , L. and Chang, T.-W. ( 1 9 6 0 ) . The e f f e c t o f c o l c h i c i n e and some c h e m i c a l l y r e l a t e d compounds on e x p e r i m e n t a l v i r a l i n f e c t i o n s . A n t i b i o t . Chemother. ( W a s h i n g t o n , D.C.) 10, 180-187. 207 APPENDIX A L i s t 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 r i p a r i a 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 c a p i t e l l a t a K6 49 C. d e b i l i s A1 A2 49 C. sprucei T 49 Dialyanthera otoba A2 30,31 D. p a r v i f o l i a C6 38 Iryanthera c r a s s i f o l i a A2 49 I. grandis T 49 I . longi f o l i a A2 49 I. macrophylla R1 64 I. polyneura A2 49 I. t r i c o r n i s A2 49 I. u l e i A2 49 Osteophloem platyspermum D1 D2 55 V i r o l a a l b i d i f l o r a 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 2 0 8 V. l o r e t e n s i s T 49 v . m e l i n o n i i A2 49 V. pe ruv i ana K1 46, 49 V. r u f u l a R1 4 V. s c h u l t e s i i A2 49 V. seb i f e r a B2 R1 S 2,49 V. su r inamens i s A2 A8 C8 49 V. t h e i o d o r a 02 R1 49 V i r o l a sp . G1 4 Order : L a u r a l e s F a m i l y : Monimiaceae S iparuna q u i a n e n s i s A2 B2 CI 2 S. sp . B2 C1 G2 2, 36 F a m i l y : Lauraceae Nectandra p i c h i i r i n C5 K2 1 5 N. r a d i a c i P2 1 5 Ocotea venenosa 02 45 Cou roup i t a sp . A2 25 Order : P i p e r a l e s F a m i l y : P ipe raceae Peperomia e m a r q i n e l l a N 2, 51 P. g l a b e l l a JI 51 P. macrostachya F1 P2 55 P. serpens N 51 P ipe r aduncum A1 A8 C5 E3 2 P. a legreanum K1 2 P. a n t i rheumat icum B2 2 P. bogotense A2 2 P. caudatum 02 42 P. d a c t y l o s t igmum K1 51 P. e r y t h r o x y l o i d e s C7 I 1 K1 1 P. f u t u r i T 2 P. gen i cu l a tum K1 42 P. h i sp idum 04 P2 U2 2 P. hostmannianum A5 55 P. i n t e r i t u m K5 55 P. i g u i t o s e n s i s T 2 P. marginatum CI 1 1 P. nigrum D3 1 1 P. nubigenum A1 2 P. ob l iquum 02 2 P. pe l t a tum A2 K1 2 P. s c h u l t e s i i D3 F1 61 P. serpens N 2 | \ s u d i s i l v e s t r e A2 B2 C5 1 1 E3 F1 G9 P. t i n g e n s U 2 P. tube rcu la tum B2 C6 N 04 1 1 P ipe r s p . A1 K1 K2 02 2 Pothomorphe umbe l l a ta F1 02 55 2 0 9 Orde r : A r i s t o l o c h i a l e s F a m i l y : A r i s t o l o c h i a c e a e A r i s t o l o c h i a a n q u i c i d a N 1 5 A. barba ta N 1 5 A. b r a s i l i e n s i s T 1 5 A. g r a n d i f l o r a 12 1 5 A. maxima N 12 1 5 A. m e d i c i n a l i s K6 47 A. p i l o s a K1 1 5 A r i s t o l o c h i a sp . A2 C2 K7 N T 2, 11, 15 O rde r : Ranuncu la les F a m i l y : Menispermaceae Abuta caud i cans 02 42 A. q r a n d i f l o r a 02 P2 2, 1 1 A. imene 02 03 1 1 , 42,53 A. macrocarpa 02 1 1 A. r u f e s cens F3 02 1 1 , 42 A. s p l e n d i d a 02 53 A. vaupesens i s 02 53 A. v e r r u c u l o s a 02 1 1 Andontocarya t r i p e t a l a C6 53 Anomospermum r e t i c u l a t u m 02 53 Chondrodendron tomentosum B2 F3 14 28, 42 * N 02 P2 C. t ox i f e rum 02 2, 42 C issampe los andromorpha 02 1 1 C. p a r e i r a F1 03 P2 1 1 , 42 Coccu lus imene 02 42 Curarea tecunarum G1 02 42, Orthomene schomburgk i i 01 55 T e l i t o x i c u m peruvianum 02 A2 55 O rde r : Papavera les F a m i l y : Papaveraceae Boccoc i a f r u t e s c e n s A2 C3 C6 04 1 1 Subc l ass I I . Hamamelidae O rde r : U r t i c a l e s F a m i l y : Moraceae Brosimium u t i l e D2 D3 1 i Ch lo rophora t i n c t o r i a C6 K1 U3 2, 1 1 F i c u s a n t h e l m i n t h i c a 01 42 F. a t rox 02 42 F. d u l c e r i a C6 1 1 F. q l a b r a t a C6 1 1 H e l i c o s t y l i s scabra N a u c l e o p s i s m e l l o - b a r r e t o i A2 54 03 37 P o u l s e n i a armata T 2 Pseudolmedia l a e v i q a t a 01 54 21 0 F a m i l y : Cec rop iaceae C e c r o p i a t o l i m e n s i s Coussapoa cinnamomea C. m a g n i f o l i a C. o r thoneura C. t r i n e r v i a Pourouma cucura P. s c h u l t e s i i Subc l a ss I I I . C a r y o p h y l l i d a e O r d e r : C a r y o p h y l l a l e s F a m i l y : Phy to l accaceae P e l t e v e r i a a l l i a c e a e Phy to l a c ca bogo tens i s P. r i v i n o i d e s F a m i l y : Nyc tag inaceae Boerhaav ia c o c c i n e a Nea p a r v i f l o r a F a m i l y : Cactaceae Ep iphy l l um sp . Opunt ia s p . T r i c h o c e r e u s pachanoi Neora imondia mac ros t i bas F a m i l y : Chenopodiaceae Chenopodium ambrosiodes F a m i l y : Amaranthaceae •A l t e rnan the ra Lehmanni i Amaranthus hyb r idus Gomphrena sp . I r e s i n e c e l o s i a I r e s i n e sp . F a m i l y : Po r tu l a caceae C a l a n d r i n i a c au l e s cens P o r t u l a c a pe renn i s Ta l inum pan i cu l a tum F a m i l y : B a s e l l a c e a e Andredera d i f fusa F a m i l y : Ca r yophy l l a c eae Drymar ia p a u c i f l o r a O r d e r : Po l ygona l es F a m i l y : Polygonaceae Polygonum punctatum Rumex o b t u s i f o l i a Subc l ass IV. D i l e n i i d a e O r d e r : D i l l e n i a l e s F a m i l y : D i l l e n i a c e a e C u r a t e l l a amer icana D a v i l l a l acunosa T e t r a c e r a r o t u n d i f l o r a P2 03 T J3 J3 B2 A2 A2 C6 M 01 A6 C6 M 03 C2 U4 R2 R1 R1 R2 R2 CI U1 A2 A8 B2 R2 T F3 T T T C2 C5 04 C3 2 54 54 54 54 61 54 1 1 1 1 , 1 1 , 2 2 39 39 39 39 47 2 42 1 1 1 1 2 2 2 2 2 1 1 48 53 53 B2 E4 A2 A8 U2 hi 1 1 2 2 21 1 O r d e r : Thea les F a m i l y : Caryocaraceae Caryocar glabrum C. g r a c i l e C. tessmanni i Ca ryocar sp . F a m i l y : Marcgrav iaceae Souroubea c r a s s i p e t a l a S. g u i a n e n s i s S. p a c h y p h y l l a F a m i l y : Qu i inaceae Qui ina l e p t o c l a d a F a m i l y : C l u s i a c e a e C a r a i p a p a r r i e l l i p t i c a C l u s i a amazonica C_. C. 1 i n e o l a t a r e n g g e r i o i d e s  C l u s i a sp . K ie lmeyera rosea  Symphonia g l o b u l i f e r a  V i sm i a angusta  V. f e r r u g i n e a  V. tomentosa O r d e r : Ma l va l e s F a m i l y : T i l i a c e a e Ape iba t i b o u r o u  Luehea sp . T r i u m f e t t a l a p p u l a F a m i l y : S t e r c u l i a c e a e Guazuma ulmi f o l i a  H e r r o n i a camargoana  S t e r c u l i a a p e t a l a  Theobroma subincanum F a m i l y : Bombaceae Bombax globosum  Pa t i noa i c t h y o t o x i c a F a m i l y : Malvaceae Abelmoschus e s c u l e n t u s A b u t i l o n H i b i scus v i rgatum abelmoschus Ma lachra r u d i s Malva v e r t i c i l l a t a Malvastrum peruvianum Pavonia c a n c e l l a t a P. hooke r i S i da g lomerata S. s e tosa 03 01 03 03 F3 K2 J l E3 A2 J3 A6 T A2 C3 R2 A4 A1 A2 A2 A2 T A6 A1 A2 V F1 R2 K2 A1 03 C l P6 C4 K6 A8 A2 C3 C l T T T 42 47 2 47 53 61 61 53 55 1 1 2 1 1 39 2 55 5 55 3 1 5 2 2 1 1 55, 1 1 55 55 47 2 2 1 1 1 1 2 2 2 1 5 1 1 1 1 61 21 2 O r d e r : V i o l a l e s F a m i l y : F l a c o u r t i a c e a e Banara q u i a n e n s i s 01 61 Ca rpo t roche amazonica 01 2 C a s e a r i a r e s i n i f e r a 01 2 C. s y l v e s t r i s A1 A2 1 1 C a s e a r i a sp . A4 D1 U2 2, 3 Lunan ia p a r v i f l o r a 01 47 Mayna amazonica B2 04. 43, 53 M. l i n q u i f o l i a A2 53 M. l o n g i f o l i a C2 53 M. mur i c i da 01 47 M. t o x i c a 01 55 Ryania a n g u s t i f l o i a 01 02 55 R. p y r i f e r a 01 2 R. spruceana 01 2 F a m i l y : B ixaceae B ixa o r e l l a n a C1 H1 04 1 1 B. purpura C1 H1 04 1 1 B. urucurana C1 H1 04 1 1 F a m i l y : V i o l a c e a e C o r y n o s t y l i s v o l u b i l i s C6 K3 53 Hybanthus l ana tus D1 2 F a m i l y : Turneraceae Turnera u l m i f o l i a D6 F1 13 Q 1 1 F a m i l y : P a s s i f l o r a c e a e P a s s i f l o r a v i t i f o l i a C4 K2 P2 R1 1 1 F a m i l y : Cucu rb i t a ceae Angur i a umbrosa 01 53 Angur i a sp . 03 60 Apodanthera herverae C3 1 5 Anisosperma p a s s i f l o r a C1 1 5 Cayaponia op tha lm ica J1 47 C. racemosa 01 1 F e v i l l e a c o r d i f o l i a N 1 1 Guran ia r u f i p i l a 01 53 L u f f a o p e r c u l a t a C3 P5 1 1 F a m i l y : Begoniaceae Begonia rossmanniae J1 2 O r d e r : S a l i c a l e s F a m i l y : S a l i c a c e a e S a l i x humboldt iana C1 C6 P2 11, O r d e r : E r i c a l e s F a m i l y : E r i c a c e a e B e f a r i a conges ta D6 53 B. r e s i n o s a D6 11, 53 Pe rne t t ya p r o s t r a t a 01 2 21 3 O r d e r : P r i m u l a l e s F a m i l y : Theophrastaceae C l a v i j a p o e p p i q i i 02 2 F a m i l y : Myrs inaceae An thod i s cus obovatus 02 03 53, 60 A. peruanas 03 53, 60 Conomorpha c i t r i f o l i a P2 V 48 C. l i t h o p h y t a 03 47 Subc l a ss V. Ros idae O r d e r : Rosa les F a m i l y : Connaraceae Connarus opacus 03 46 C. sp ruce i 03 47 Rourea c u s p i d a t a 01 54 R. q l a b r a D4 03 10, 47 O r d e r : Faba les F a m i l y : Leguminosae Abrus p r e c a t o r i u s C3 D6 J l 04 1 1 Acosmium n i t e n s 02 55 A l exa i m p e r a t r a c i s 03 42 Anadenanthera p e r e g r i n a R1 47 And i r a a ra roba A2 1 5 A. ine rmis C6 K5 01 1 1 A. r e tusa T 2 Apur imac ia incarum 03 42 A. m i c h e l l i 03 42 Bauh in ia g u i a n e n s i s 03 G1 15, 42 B. ine rmis K2 1 5 B. sp lendens 14 P2 1 1 B. t a r a p o t e n s i s B2 C5 1 1 Bowdichia v i r g i l i o d e s B2 03 1 1 , 42 B. a r i z a E3 1 1 C a e s a l p i n i a c o r i a r i a C5 1 5 C. pu l che r ima 03 42 Cajanus ca jan A8 2 Campsiandra a n q u s t i f o l i a P2 61 C. l a u r i f o l i a A2 R2 35, 61 C a s s i a a f f i n i s A2 C3 1 5 C. a l a t a A2 C3 1 1 C. f r u t i c o s a K1 61 C. h i r s u t a 03 42 C. mac rophy l l a 03 42 C. moschata C3 1 1 C. o c c i d e n t a l i s C3 D2 P2 1 1 , 15 C. r u i z i a n a T 61 C. t o r a C3 K2 M P2 1 5 Centrosema p lumer i a 03 42 Chae toca l yx l a t i s i l i g u a A4 2 C l a t h r o p i s b r a c h y p e t a l a 03 42 21 4 C l i t o r i a t e rna t ea C3 F1 1 1 C o p a i f e r a guayanens is D6 1 5 C. hymenaefo l i a K1 K5 03 1 5 Coumarouma odora ta A2 D3 V 1 1 C r o t o l a r i a p i l o s a A1 1 1 C r u d i a amazonica C2 61 Desmodium s p . I 1 1 5 D ip t e r yx t e t r a p h y l l a E2 2 Entada p o l y p h y l l a D1 53 E. scandens N 1 5 E r y t h r i n a c o r a l l o d e n d r o n 02 42 Heterostemon mimosoides V 55 Hof fmanseggia g r a c i l i s D4 2 Hymenaea c o u r b a r i l C6 F3 1 1 I n d i g o f e r a s u f f r u t i c o s a 14 K6 1 1 Inga s p e c t a b i l i s B2 C5 1 1 Leguminosa sp . A6 01 2 Lonchocarpus l a t i f o l i u s C3 03 1 1 L. n i cou 03 1 1 L. s e r i c e u s C3 02 04 1 1 L. urucu 03 2 Lonchocarpus sp . 03 60 Lup inus m u t a b i l i s 04 42 Macro lab ium a c a c i a e f o l i u m A1 61 M. mul t i jugum 01 61 Mimosa h o s t i l i s P1 1 5 M. i n u i s a C2 01 Q 1 5 M. pud ica Q 1 1 M. v e r rucosa K5 50 Monopteryx a n g u s t i f o l i a C6 53 M. uaucu C6 53 Mucuna r o s t r a t a A7 42 Myroxy lon balsamum A1 1 1 Ocimum micranthum R2 47 Ormosia c o c c i n e a 01 47 0 . l i g n i v a l v i s A1 47 0 . mac rophy l l a 02 47 P i p t a d e n i a macrocarpa D3 R2 1 1 P i t h e c e l l o b i u m laetum R1 35 P ro sop i s j u l i f l o r a D3 1 1 P so r a l e a pubescens F3 2 P t e roca rpus r o h r i i P2 61 Swar tz i a a u r i c u l a t a 03 48 S. b r a c h y r h a c h i s C6 48 S. cab re rae C6 48 S. c o n f e r t a C5 48 S. g igan tea 01 48 S. l o n g i s t i p i t a t a G5 61 S. m i c roca rpa C5 48 S. pendula 03 48 S. racemosa C5 48 • 21 5 S. r ecurva Q 61 S. schomburgk i i C6 48 S. s c h u l t e s i i 03 48 S. s e r i c e a 03 48 S. s implex T 48 T a c h i q a l i a c a v i p e s A2 C2 G1 04 54 T . myrmecophi la A2 K1 61 T . p a n i c u l a t a C2 54 T . ptychophysea I 1 K1 61 T e p h r o s i a s inapou A8 03 04 1 1 T e p h r o s i a sp . 03 60 Zo rn i a l e p t o p h y l l a 04 61 O r d e r : M y r t a l e s F a m i l y : Ly th raceae Cuphea racemosa F1 2, 60 F a m i l y : Thymelaeaceae Schoenob ib lus pe ruv ianus A2 02 03 52, 54 S ty rax tessmann i i A2 01 49, 54 S. yapobodens is A5 54 F a m i l y : Onagraceae E p i l o b i u m d e n t i c u l a t u m T 2 F a m i l y : Melastomataceae Arthrostemma g r a n d i f l o r u m F1 2 A. v o l u b i l e J3 P2 1 1 G r a f f e n r i e d a r u p e s t r i s A2 61 F a m i l y : Combretaceae Combretum a l t e r n i f o l i u m JI 1 5 C. c a c o u c i a 01 2 ,53 ,55 T e r m i n a l i a ca tappa C1 C5 D2 1 1 O r d e r : S a n t a l a l e s F a m i l y : O lacaceae H e i s t e r i a p a l l i d a B2 R2 35 H e i s t e r i a sp . BI 2 F a m i l y : Loranthaceae Gaiadendron punctatum D1 2 Oryc tan thus bo t r yos t a chys A8 E4 1 1 Phoradendron p i p e r o i d e s G5 1 1 P h r y g i l a n t h u s eugeniodes R2 39 P s i t t a c a n t h u s co l l um-cygn i E3 2 F a m i l y : Ba lanophoraceae Corynaea c r a s s a G8 22 O r d e r : C e l a s t r a l e s F a m i l y : C e l a s t r a c e a e Maytenus l a e v i s B2 1 1 M. p seudocasea r i a C5 2 F a m i l y : I c ac inaceae C a l a t o l a co lumbiana A9 U4 32 Humi r i an the ra ampla 01 42 F a m i l y : D i chape t a l a ceae Stephanopodium peruvianum P2 35 21 6 O r d e r : Eupho rb i a l e s F a m i l y : Euphorb iaceae A l cho rnea c a s t a n e i f o l i a B2 22 A. cordatum T 2 Caryodendron o r i nocense A4 1 1 C n i d o s c o l u s urens C3 2 Codiaeum var ieqatum 01 2 Croton ca ju ca r a P2 2 C. f e r r u g i n e u s T 2 C. q l a b e l l u s A2 55 C. g o s s i p i f o l i u s A1 2 C. l e c h l e f i A1 A2 M 2, 22 C. maqda lens is T 2 C. pa lanos t iqma A2 2, 55 C. po l y ca rpus T 2 C. r h a m n i f o l i u s T 2 C. scaber T 2 C. t r i n i t a t i s C2 D3 D4 3 Croton sp . 04 P2 1 5 Euphorb ia c o t i n i f o l i a 02 42 E. c o t i n o i d e s A6 02 2, 42 E. pep lus 01 2 E. p i l u l i f e r a D2 1 5 E. t h y m i f o l i a P4 1 5 Euphorb ia sp . A1 3, 58 G u a t t e r i a maqa lophy l l a 02 42 * G. v e n e f i c i o r u m 02 42 Hippomane m a n c i n e l l a 01 02 42 Hura c r e p i t a n s A6 03 04 2,1 1 ,42 J a t ropha angus t i I 1 2 J . c i l i a t a 11 03 15, 42 J . cu r cas C3 M 01 2,15,42 02 03 J . g o s s y p i f o l i a A6 C3 M 1 5 J . urens A8 F1 12 2, 1 5 Mabea n i t i d a A4 55 Manihot e s c u l e n t a A2 1 1 M i c r and ra spruceana E3 J3 54 N e a l c h o r n i a y apu rens i s 03 53 P e d i l a n t h u s t i t h y m a l o i d e s C2 C5 D6 1 1 , 1 5,42 14 01 P h y l l a n t h u s acuminatus 03 2 P. b r a s i l i e n s i s 03 2 P. c l a d o t r i c h u s 03 42 P. conami 03 42 P. n i r u r i F3 P2 2 P. p i s ca to rum 03 04 2, 60 P. pseudo-conami 03 2 P. r o s e l l u s F3 2 P. s a l v i a e f o l i u s F1 P3 1 5 Sebas t i ana p a c h y p h y l l a C3 2 21 7 O r d e r : L i n a l e s F a m i l y : E r y t h r o x y l a c e a e E r y th roxy lum coca C l K1 K3 35 E. novagrana tens i s C1 K1 K3 35 F a m i l y : Humir iaceae Humir ia b a l s a m i f e r a A1 A2 49 H. c r a s s i f o l i a A2 49 Humir ias t rum p i r apa ranense C2 K1 49 H. v i l l o s u m C3 49 S a c o g l o t t i s c e r a t o c a r p a D3 49 S c h i e k i a o r i n o c e n s i s K7 54 Sch is tos temon macrophyl lum D3 D5 G2 49 Vantanea p a r v i f l o r a T 49 O r d e r : P o l y g a l a l e s F a m i l y : Ma lp igh i a ceae B a n i s t e r i a l e i o c a r p a T 2 B a n i s t e r i o p s i s caap i R1 2, 47 B. i n e b r i e n s R1 2, 47 B. m a r t i n i a n a R1 1 1 B. rusbyana R1 2 Byrsonima c r a s s i f o l i a C5 03 P2 1 1 , 42 B. l a n c i f o l i a B2 2 H e t e r o p s i s macrostachya A2 C5 52 H. r i p a r i a 14 01 47, 52 H i r e a a p a p o r i e n s i s J l 52 H. s c h u l t e s i i J l 52 Mascagnia g l a n d u l i f e r a A2 52 Mezia i n c l udens C2 C3 F1 52 T e t r a p t e r i s me thys t i c a R1 47 T . mucronata 02 R2 2, 52 T . s i l v a t i c a A2 52 T . s t y l o p t e r a A2 P2 52 F a m i l y : Vochys iaceae Qualea acuminata C6 55, 61 Vochys i a c o l u m b i e n s i s 02 53 V. f e r r u q i n e a A2 J3 02 R2 53, 60 V. l a x i f l o r a A2 D3 F3 53 V. l oma tophy l l a G1 G4 2, 53 O r d e r : S ap inda l e s F a m i l y : Sapindaceae Cardiospermum q r a n d i f o l i u m 03 42 P a u l l i n i a a l a t a 03 2 P. emet ica C2 53 P. yoco K3 Q P2 2, 1 1 S e r j a n i a sp . J3 61 T o u l i c i a b u l l a t a P2 2 218 F a m i l y : Burseraceae Burse ra gumi fera C3 D6 14 1 5 K 1 P 1 Hedwigia b a l s a m i f e r a A 1 1 5 P rot ium heptaphy l lum K 1 2 P. neqlectum T 2 P ro t ium sp . 02 2 F a m i l y : Anacard iaceae Anacardium o c c i d e n t a l e A4 A6 C3 1 1 13 0 1 Q Loxopteryg ium huasango A7 42 Maur ia a u r a n t i o d o r a A7 42 -M. h e t e r o p h y l l a A7 2 Rhus s p . A7 42 Spondias mombin A2 G5 14 1 1 , 2 5 F a m i l y : Simaroubaceae Quass i a cedron U 1 42 Simarouba cedron P2 2 S. v e r s i c o l o r U 1 42 F a m i l y : Me l i a ceae Carapa g u i a n e n s i s B2 - 1 5 T r i c h i l i a oblonga D3 2 F a m i l y : Rutaceae Angostura t r i f o l i a t a P2 Q 1 5 H o r t i a sp . P2 2 Rauia r e s i n o s a P2 2 Ruta q raveo l ens K 1 1 1 Zanthoxylum sp . 02 42 O r d e r : G e r a n i a l e s F a m i l y : Oxa l i daceae O x a l i s l o t o i d e s D4 55 Subc l a ss V I . A s t e r i d a e O rde r : G e n t i a n a l e s F a m i l y : Loganiaceae An ton i a ovata 03 2, 42 Budd l e i a americana T 2 P o t a l i a amara C3 14 J 1 N 54 S p i g e l i a humboldt iana C6 1 1 S t rychnos g u i a n e n s i s 02 42 S. p e c k i i 02 2 S. so l imoesana 02 42 S. t o x i f e r a 02 42 F a m i l y : Gent ianaceae Che lonanthus a l a t u s 04 3 Che lonanthus c h e l o n o i d e s 04 2 Gent i ana chamuchni T 2 H a l e n i a wedde l l i ana 14 2 21 9 F a m i l y : Apocynaceae A l l amandra ambet t i C2 C3 15 A. cuneata 03 57 A. l o p e z i i A2 02 57 A. markqra f i ana P2 57 Aspidosperma d i s c o l o r P2 2 A. megalocarpa A7 57 A. n i t i dum A6 P2 2, 57 A. s c h u l t e s i i A2 04 57, 61 Aspidosperma sp . P2 2 Couma macrocarpa A9 K5 3, 57 Hancorn ia spec i o sa A5 57 Himatanthus b r a c t ea tu s N P2 57, 60 H. phagedoenicus N P2 57 H. sucuuba D3 01 2, 57 Himatanthus sp . 03 58 Lacme l l a sp . K5 57 Macoubea g u i a n e n s i s D3 2, 57 Mac ros iphon i a l o n g i f l o r a 2 M. velame 14 2 M a l o u e t i a ducke i 03 2 M. n i t i d a A2 01 11, 53 M. tamaquar ina 01 R2 47 M a n d e v i l l a anceps A5 57 M. a n n u l a r i i f o l i a A5 57 M. c u n e i f o l i a 01 57 M. ne r i odes A2 57 M. scabra A9 57 M. s t e y e r m a r k i i A2 C5 I 1 57 M. s t e p h a n o t i d i f o l i a N 57 M. subcarnosa 01 57 M. t h e v e t i o i d e s 03 57 M. t r i a n a e A2 57 M. v a n h e u r e k i i A2 01 57 M a n d e v i l l a s p . 03 47 Mesech i t es t r i f i d a K2 57 Odontadenia cognata 04 57 0 . f u n i g e r a V. 57 0 . n e g l e c t a D5 01 57 0. s y l v e s t r i s K1 04 57 Pa rahancorn ia k r u k o v i i A1 57 P lumer ia t a r a p o t e n s i s B2 2 220 Tabernaemontana a m y g d a l a e f o l i a A5 01 M 1 1 T . g r a n d i f l o r a A2 B2 1 1 T . h e t e r o p h y l l a . Q 57 T . mur i ca ta K3 57 T . r imu losa K2 57 T . sananho C2 F1 K2 57 01 P2 Q T . s t eno loba 01 61 T . t e t r a s t a c h y a B2 K3 57 T . undu la ta C6 57 Tabernaemontana sp . 03 2 T h e v e t i a pe ruv i ana 01 42 F a m i l y : As c l ep i adaceae A s c l e p i a s c u r a s s a v i c a C2 C3 C6 2 ,11,15 E3 14 01 Sarcostemma andinum N 2 S. c laucum JI 1 1 O r d e r : S o l a n a l e s F a m i l y : So lanaceae B r u n f e l s i a c h i r i c a s p i K5 35 B. g u i a n e n s i s B2 14 01 21 B. g r a n d i f l o r a B2 D3 K1 K5 35 01 P2 B. l a t i f o l i a B2 2 B. mar i t ima B2 2 B. mire C6 01 35, 41 B. t a s t e v i n i i R1 41 B. u n i f l o r a A8 B2 C3 F1 23 ,29 ,66 G1 14 K1 K5 01 PI B r u n f e l s i a sp . N, B2 1 ,6 ,7 ,8 ,33 Capsicum pendulum 02 42 Cestrum laev iqa tum 03 42 C. l o r e t e n s e 01 61 C. ochraceum B2 P1 61 C. re f l exum 01 61 Cyphomandra c r a s s i f o l i a C6 54 C. d o l i c h o r a c h i s C6 54 C. endopoqon T 54 Datura a rborea A8 M 1 5 D. stramonium 02 42 D. suaveo lens K5 47 Iochroma f u c h s i o d e s R2 50 J a l tometa procumbens F1 P2 61 221 J u a n u l l o a ochraceae  Markea c o c c i n e a  N i c o t i a n a tabacum  P h y s a l i s pubescens  Saracha aspero lanatum  S . a spe r r imum  S. l ycocarpum  S . phy l l anthum  S . procumbens  S. s p e c t a b i l e  S. swartzianum  Solanum a lb idum  S . apaporum  S . campani forme  S. c r i n i t i p e s  S. jamaicense  S. l ep ido tum S S mammosum n 1 g r urn S. scabr idum  S. subinerme  S. t o p i ro  S. v e r b a s c i f o l i u m F a m i l y : Convo l vu l aceae Ipomea c r a s s i f o l i a  Ipomea sp . Merremia a l a t a F a m i l y : Hyd rophy l l a ceae Wigandia ca racasana O rde r : Lamia les F a m i l y : Borag inaceae C o r d i a a l l i d o r a C. den ta ta C. e c a l y c u l a t a C . l u t e a C. verbenacea H e l i o t r o p i u m argentatum H. t i a r i d i o i d e s T o u r n e f o r t i a b r e v i l o b a t a F a m i l y : Verbenaceae C a l l i c a r p a odora ta Lantana a f f i n i s L. camara L. f u ca t a S t a ch t a rphe t a cayennens i s  S. s t raminea  Verbena l i t o r a l i s  Verbenacea s p . A l R2 54 C6 J l 54 R2 47 T 2 F3 2 01 2 D1 2 A1 2 F1 P2 55 01 2 K1 Q 2 A2 54 04 54 04 54 01 61 04 54 B2 61 04 61 A2 C4 01 M 1 1 J2 61 T 54 J3 54 A7 54 Q 2 C3 2 C3 2 K3 2 A2 B4 1 1 D3 1 1 Q 2 T 2 T 2 K6 2 D3 2 Q 2 B2 2 C5 2 C1 C4 D6 1 1 P1 P2 D1 2 A2 C5 2 A2 C5 2 C3 2 C1 2 222 F a m i l y : Lamiaceae H y p t i s b r a c h i a t a A2 E3 1 1 H. c a p i t a t a A2 A4 B4 1 1 H. c a r p i n i f o l i a B2 CI 2 H. m u t a b i l i s P2 1 1 H. s i n u a t a D2 D3 1 1 L e p e c h i n i a meyeni C1 2 Ocimum micranthum R2 50 Rosmarinus o f f i c i n a l i s B2 2 S a l v i a haenkei T 2 S. mac rophy l l a F3 2 S. p a l a e f o l i a 04 2 S. p i c h i n c h e n s i s K2 2 S a t u r e i a tomentosa D1 2 O r d e r : S c r o p h l a r i a l e s F a m i l y : S c r o p h u l a r i a c e a e A lonsoa c u a d r i a l a t a D2 1 5 A n t i r r h i n u m majus D3 1 5 C a l c e o l a r i a inamoena B3 2 C a s t i l l e j a communis T 2 F a m i l y : Gesner iaceae B e s l e r i a drymophi la N 1 1 B e s l e r i a sp . N 2 Columnea sp . T 2 N a u t i c a l y x sp . N 47 F a m i l y : Acanthaceae Acantha v i r i d i s T 1 5 • Aphe landra a u r a n t i a c a J2 56 A. p i l o s a D4 56 F i t t o n i a a rgy roneura K1 56 F. v e r s c h a f f e l t i i D4 F3 P2 56 J u s t i c i a b l a c k e i D7 56 J . cabre rae A2 56 J . c h l o r a s t a c h y a A2 56 J . comata 04 56 J . ideogenes K7 56 J . p e c t o r a l i s A2 R2 25, 56 J . s c h u l t e s i i A2 56 Mendoncia aspera 03 47 R u e l l i a c o l o r a t a C2 C6 56 R. humboldt iana C3 1 1 Sanchez ia t h i n o p h i l a A9 56 T e l i o s t a c h y a l a n c e o l a t a R2 56 T r i c h a n t h e r a g igan tea C6 E4 F1 1 1 F a m i l y : Peda l i a ceae P robosc idea pe ruv i ana T 2 223 F a m i l y : B ignon iaceae A r r a b i d a e a c h i c a  A. x a n t h o p h y l l a  B ignon ia op tha lmica  Cremastus sceptrum  D i s t i c t e l l a racemosa  Jacaranda g l a b r a  Macfadyena u n g u i s - c a t i  M a r t i n e l l a obovata  Mussat i a hyac i n t h i n a  Pleonotoma j a s m i n i f o l i u m  Pseudocalymma a l l i a c e u m  P y r o s t e g i a venusta  Tabebuia barbata  T . s e r r a t i f o l i a  Tabebuia s p . Tanaec ium nocturnum Orde r : Campanula les F a m i l y : Campanulaceae Centropogon c a l y c i n u s  Isotoma l o n g i f l o r a  L o b e l i a decur rens  S iphocampylus corymbi f e rus O rde r : Rubi 'a les F a m i l y : Rubiaceae Ca l y cophy l l um spruceanum  C e p h a e l i s b a r c e l l a n a  Ch iococca b r a c h i a t a  C. racemosa  Coussarea p i l o s i f l o r a  D i o d i a hyssop i f o l i a Duro ia h i r s u t a D. D. D. D. kotchubaeo ides  pet i o l a r i s sacc i f e r a s p r u c e i  Exostemma pe ruv i ana  Genipa amer icana I s e r t i a a l b a haenkeana  hypo leuca rosea Ladenberg ia m a g n i f o l i a  Mane t t i a d i v a r i c a t a  Pagamea c o r i a c e a P. mac rophy l l a  P a l i c o u r e a e longa ta  P. qa rdne r i ana  P. r i q i d a  P a l i c o u r e a sp . A2 A9 J1 JI 14 01 02 A2 14 1 4 N P2 02 P2 V C2 D3 B2 01 A2 01 M A7 R1 I 1 R2 01 01 A2 04 T U1 C2 T T 01 01 01 01 01 P2 D3 A2 M B2 M G5 P2 P2 T K7 S 01 01 Q 03 48 55 1 1 47 1 5 2 48 1 1 1 1 47, 43 1 1 2, 2 1 1 43 42 1 1 2 50 2, 42 2 53 61 42 1 5 2 2 2, 46, 47 47 46 2 25 1 1 1 1 53 54 1 1 2 54 54 2 2 2 2 47 47 224 P s y c h o t r i a b a r b i f l o r a 01 2 P. c a p i t a t a 01 2 P. c a r t hagenens i s 01 R 46 P. i n v o l u c r a t a 01 46 P. nud iceps 01 46 P. p i n u l a r i s N 2 P. p s y c h o t r i a e f o l i a R 47 P. r u f e s cens M P2 1 1 Randia formosa M 1 1 Remi j i a peduncu la ta T 1 1 R e t i n i p h y l l u m conco lo r D5 54 R. p i l o s a C6 54 R. schomburgk i i C6 54 R. speciosum C6 54 R. t runcatum A8 54 R e t i n i p h y l l u m sp . S 47 Rubiacea s p . 01 2 Rudgea s u b s e s s i l i s 01 2 R. v i b u r n o i d e s Q 2 Sab icea amazonensis R 47 O rde r : A s t e r a l e s F a m i l y : As te raceae Acanthospermum a u s t r a l e M 1 1 Ageratum conyzo ides C5 P2 1 1 Ambrosia e l a t i o r P2 1 5 Andromachya i g n i a r i a E3 1 5 Baccha r i s g e n i s t e l l o i d e s E3 G6 1 5 B. s a l i c i f o l i a A8 D3 14 1 5 B. t r i n e r v i s T 1 5 Bidens a n d i c o l a B2 1 5 C l i bad ium s y l v e s t r e A2 1 1 C l i bad ium s p . 03 60 Eupator ium ayapanoides U l 42 E. odoratum A8 N P2 Q 1 1 E. scabrum 14 M 1 1 E. s te rnberg ianum CI 1 5 M ikan ia guaco U1 42 Neurolaena l o b a t a M N 1 1 S p i l a n t h e s amer icana J3 1 1 225 C l a s s : L i l i o p s i d a Subc l a s s I: A l i s m a t i d a e O rde r : A l i s m a t a l e s F a m i l y : A l i smataceae Ech inodorus g r a n d i f l o r u s Q 2 Ech inodorus sp . C8 , 2 Subc l a s s I I : A r e c i dae O r d e r : A r e c a l e s F a m i l y : Arecaceae Chamaedorea f r ag r ans A7 42 E l a e i s q u i n e e n s i s C6 1 1 Geonoma sp . K1 1 5 J e s s e n i a po l y c a rpa D3 1 1 Kue th i a montana N 1 5 O r d e r : C y c l a n t h a l e s F a m i l y : Cyc lan thaceae C a r d u l o v i c i a palmata D3 61 O r d e r : A r a l e s F a m i l y : Araceae Anthur ium c r a s s i n e r v i u m J2 54 A. eminens T 61 A. jenmani i B3 61 A. scopendrinum D4 61 A. t essmann i i G1 46 A. t ikunorum J3 47 Aracea sp . J3 2 Ca lad ium b i c o l o r U2 61 D i e f f e n b a c h i a o b l i q u a 01 61 D. p i c t a c 01 42 D. sequ ine 02 2,42 Dracont ium asperum U1 42 D. l o n g i p e s N 61 D. t r i a n a e C5 N 61 H e t e r o p s i s sp . T 2 Ph i l odendron craspedodromum 03 47 P. dyscarp ium G1 47 P. haematinium S 47 P. r e m i f o l i u m V 47 Urospatha a n t i s y l l e p t i c a G1 47 U. somndenta A1 A2 1 5,47 Xanthosoma conspurcatum 01 61 Subc l a ss I I I : Commelinidae O rde r : Commel inales F a m i l y : Commelinaceae T r a d e s c a n t i a m u l t i f l o r a 226 O r d e r : C y p e r a l e s F a m i l y : C y p e r a c e a e C y p e r u s e s c u l e n t u s F3 1 5 C y p e r u s s p . R2 39 F a m i l y : Poaceae Axonopus m i c a y F1 1 1 A. s c o p a r i u s F3 1 5 C a l a m a q r o s t i s cuminens F3 2 C h l o r i s d i s t i c h o p h y l l a F1 1 5 H i e r o c h l o e r e d o l e n s R 2, 53 S u b c l a s s IV: Z i n g i b e r i d a e O r d e r : B r o m e l i a l e s F a m i l y : B r o m e l i a c e a e Ananas a n a n n a s o i d e s C1 C6 F1 1 1 O r d e r : Z i n g i b e r a l e s F a m i l y : Musaceae H e l i c o n i a a c u m i n a t a B3 K7 N 1 1 Musa b a l b i s i a n a D5 1 1 H e l i c o n i a b r a s i l i e n s i s B3 K7 N 1 1 H. c a n n o i d e a B3 K7 1 1 F a m i l y : Z i n g i b e r a c e a e Hedychium c o r o n a r i u m K1 54 F a m i l y : C o s t a c e a e C o s t u s a m a z o n i c u s T 54 C. c y l i n d r i c u s Cl P3 1 1 C. e r y t h r o c o r y n e E3 54. C. v i l l o s i s s i m u s K1 1 1 F a m i l y : C a n n a c e a e Canna s p . A2 2 F a m i l y : M a r a n t a c e a e C a l a t h e a v e i t c h e a n a R2 50 C a l a t h e a s p . 2 3 227 Subc l ass V. L i l i i d a e O r d e r : L i l i a l e s F a m i l y : Pon teder i aceae Pon tede r i a co rda t a F a m i l y : L i l i a c e a e E u c h a r i s amazonica F a m i l y : I r i daceae E l e u t h e r i n e p i i c a t a S i s y r i n c h i u m a la tum F a m i l y : Agavaceae Agave amer icana F a m i l y : Smi lacaceae Smilax sp . F a m i l y : D ioscoreaceae D i o s c o r e a pozucoens i s D. t r i f i d a O r d e r : O r c h i d a l e s F a m i l y : Orch idaceae D ichaea mur i ca t a  E r i o p s i s sceptrum  One id ium p u s i l l u m  Phragmipedium ecuadorens i s  Psymorchis p u s i l l a V a n i l l a odora ta K7 R2 C2 G8 C3 D1 03 12 T 02 J1 J3 A1 C1 T E1 K2 2, 50, 61 54 2 1 1 42 2 2 42 54 53 54 54 54 1 1 Spec ies have been ar ranged a c c o r d i n g to the c l a s s i f i c a t i o n scheme of C ronqu i s t (1981) wi th one e x c e p t i o n . The f am i l y Leguminosae i s used in p r e f e r ence to the Mimosaceae, C a e s a l p i n i a c e a e and Fabaceae. The s p e c i e s name l i s t e d i s that used by the au tho r ( s ) c i t e d . 228 L i s t of E t h n o b o t a n i c a l Codes Used LETTER SYSTEM No. B i o l o g i c a l A c t i o n or C o n d i t i o n Used For ******************************************************* A sk in 1 wounds, cu t s 2 abses se s , b o i l s u l c e r s , i n f e c t i o n s 3 burns 4 d e r m a t i t i s , eczema, p s o r i a s i s , e t c . 5 warts 6 l ep rosy 7 causes d e r m a t i t i s or a l l e r g y 8 s w e l l i n g , in f l ammat ion 9 s k i n c o l o r a n t or d e p i l a t o r y B s k e l e t o - 1 broken bones muscular . 2 rheumatism, a r t h r i t i s 3 muscle s t i f f n e s s 4 b r u i s e s C d i g e s t i ve 1 c a r m i n a t i v e , d i g e s t i v e 2 emet i c 3 c a t h a r t i c , p u r g a t i v e , l a x a t i v e 4 ant i spasmodic 5 ant i d i a r r h e a l 6 ameb i c ide , a n t i h e l m i n t h i c 7 t oo th decay 8 c o l i c D r e s p i r a t o r y 1 c o l d s , i n f l u e n z a , coughs , 2 asthma 3 b r o n c h i t i s , ches t i n f e c t i o n s 4 sore th roa t 5 t u b e r c u l o s i s 6 expec to ran t 7 s i n u s i t i s E c i r c u l a t o r y 1 anemia 2 hear t a i lmen t s 3 hemostat 4 hypo tens i ve F e x c r e t o r y 1 d i u r e t i c 2 c a l c i f i c a t i o n s 3 kidney a i lmen t s 229 G rep roduc t i v e , 1 c o n t r a c e p t i v e female 2 f e r t i l i t y promoter 3 ant i-abort ive 4 abor t i fac i en t 5 menst rua l pa in 6 emmenogogue 7 ga lac togogue 8 u t e r i n e hemmorage 9 a p h r o d i s i a c H reproduc t i v e , 1 a p h r o d i s i a c male I r eproduc t i v e , 1 a p h r o d i s i a c both sexes 2 v ene r ea l d i s ease 3 anaphrod i s i a c J sensory 1 eye a i lmen t s 2 ear problems 3 gum and mouth d i s e a s e s K nervous 1 a n a l g e s i c , a n e s t h e t i c , t oo thache , headache 2 s e d a t i v e 3 st imulant 4 h a l l u c inogen 5 narco t i c 6 c o n v u l s i o n s , f i t s , e p i l e p s y 7 p a l s y , p a r a l y s i s L a n t i b i o t i c M ant i tumour , ant i c ance r N a n t i d o t e s fo r b i t e s and s t i n g s of an imals ( p r i m a r i l y : snakes) 0 po i son 1 gene ra l 2 arrow po i son 3 f i s h po i son 4 r e p e l l e n t or t o x i c to a r th ropods P s y s t ema t i c 1 s u d o r i f i c , d i a p h o r e t i c 2 f e b r i f u g e , m a l a r i a 3 d i a b e t e s 4 swo l len g lands 5 c h o l e r a 6 shock 230 Q t on i c R admixture to h a l l u c i n o g e n S magic T unde f ined U m i s c e l l a n e o u s V f l a v o u r i n g , aromat i c 1 h e r b i c i d e 2 v e t e r i n a r y use 3 too th e x t r a c t i o n 4 too th c o l o r a t i o n The c a t e g o r i e s d e s c r i b i n g m e d i c i n a l a c t i v i t y or c o n d i t i o n s f o r which the p l a n t s are used as t rea tments have been e s t a b l i s h e d to accomodate the i n f o r m a t i o n in the form in which i t i s a v a i l a b l e . A l though i t has been o rgan i zed as s y s t e m a t i c a l l y as p o s s i b l e , t h i s approach i s l i m i t e d by the f a c t tha t the in fo rmants concepts of d i a g n o s i s and t reatment of i l l n e s s e s d i f f e r e d w i d e l y . The scheme i s based on that of Mor ton (1977) . 231 BIBLIOGRAPHY  Appendix A de Almeida C o s t a , 0 . (1935) E s t u d i o Farmacognost i co de Manaca. Rev i s t a de F l o r a M e d i c i n a l 1, 345-360. A l t s c h u l , S. von Re i s ( l 973 ) Drugs and Foods from L i t t l e  Known P lants-Notes in Harvard U n i v e r s i t y Herbar ium , Harvard U n i v e r s i t y P r e s s , Cambridge, Mass. B a l l i c k , M. J . , unpub l i shed notes on herbar ium l a b e l s , UNAP Herbar ium, I q u i t o s , Pe ru . B i o c c a , E.(1966) V i a g g i t r a g l i i n d i - A l t o R io Negro-A l to  O r i noco , D i s c h i , Rome. Bod ley , J . H . (1978) P r e l i m i n a r y ethnobotany of the Peruv ian Amazon. 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C o u n c i l f o r S c i e n t i f i c and I n d u s t r i a l Resea r ch , Me lbourne . 66. Wren, R.C. (1956) P o t t e r ' s New C y c l o p e d i a of B o t a n i c a l  Drugs and P r e p a r a t i o n s , Pitman and Sons, London. 236 APPENDIX 1 - SP4100 COMPUTER PROGRAM(BASIC) The p r o g r a m t a k e s i n c o m i n g s i g n a l s , a f t e r a m p l i f i c a t i o n X1000, from t h e f o r c e t r a n s d u c e r , computes t h e a b s o l u t e v a l u e of t h e d i f f e r e n c e between two s u c c e s s i v e s i g n a l s , and p r i n t s t h e a v e r a g e v a l u e e v e r y 5 m i n u t e s o v e r a 2 hour p e r i o d . A=1: I F 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 St i c h , H.F. S i s t e r chromatid exchanges induced i n cultured mammalian c e l l s by chromate. Chem.-Biol. Interact. 26: 281-286(1979). MacRae, W.D., MacKinnon, E.A. and S t i c h , H.F. The fate of U.V.-induced lesions a f f e c t i n g SCEs, chromosome aberrations and s u r v i v a l of CHO c e l l s arrested by deprivation of arginine. Chromosoma 72: 15-22(1979). MacRae, W.D.,MacKinnon, E.A. and S t i c h , H.F. E f f e c t s of arginine deprivation upon chromosome aberrations, SCEs and s u r v i v a l of CHO c e l l s treated with mutagenic agents. Mutat. Res. 62: 495-504(1979). MacRae, W.D., MacKinnon, E.A. and St i c h , H.F. Induction of s i s t e r chromatid exchanges and chromosome aberrations i n CHO c e l l s arrested i n the c e l l cycle by arginine deprivation. In V i t r o 15: 555-564(1979). MacRae, W.D. and S t i c h , H.F. Induction of s i s t e r chromatid exchanges i n Chinese hamster ovary c e l l s by the reducing agents b i s u l f i t e and ascorbic acid. Toxicology 13: 167-174(1979). MacRae, W.D. and S t i c h , H.F. Induction of s i s t e r chromatid exchanges i n Chinese hamster ovary c e l l s by t h i o l 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, n a t u r a l l y occurring compounds i n the Asteraceae. FEBS Letters 107: 134-136(1979). Wat, C.-K., MacRae, W.D., Yamamoto, E., Towers, G.H.N, and Lam, J . Phototoxic e f f e c t s of n a t u r a l l y 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 n a t u r a l l y occurring polyacetylenes and c<-terthienyl f o r t h e i r a b i l i t y to induce cytogenetic damage. Experientia 36: 1096-1097(1980). MacRae, W.D., Irwin, D.A.J., B i s a l p u t r a , T. and Towers, G.H.N. Membrane lesions i n human erythrocytes induced by the n a t u r a l l y occurring compounds <*> - t e r t h i e n y l and phenylheptatriyne. Photobiochem. Photobiophys. 1: 309-318 (1980) . MacRae, W.D. and Towers, G.H.N. L e t t e r to the edi t o r . 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 - t e r t h i e n y l . 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 it s 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). 

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