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

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E T H N O B I O L O G I C A L AND C H E M I C A L OF S E L E C T E D AMAZONIAN  INVESTIGATIONS PLANTS  by  WILLIAM  DONALD  MACRAE  B.Sc, 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 THE  SUBMITTED  IN PARTIAL FULFILMENT  REQUIREMENTS  FOR  DOCTOR OF  THE  DEGREE  OF  PHILOSOPHY in  THE  FACULTY  OF  GRADUATE  Biology  We  STUDIES '  Department  accept t h i s t h e s i s as conforming to the r e q u i r e d standard  THE  UNIVERSITY  OF  March  ©  William  BRITISH  COLUMBIA  1984  Donald  MacRae,  1984  OF  In  presenting  requirements  this for  thesis  an  British  it  freely available  for  that  Columbia,  or  by  for  understood  that  his  a  t  e  reference  and  study.  I  extensive be  her or  shall  ,>1<SH' <  copying  granted  by  the  of  publication  not  be  allowed  Columbia  of  this  It  this  without  make  further  head  representatives.  of  ^  University shall  The U n i v e r s i t y o f B r i t i s h 1956 Main M a l l Vancouver, Canada V6T 1Y3  D  the  Library  permission.  Department  at  the  the  may  or  f u l f i l m e n t of  that  for  copying  f i n a n c i a l gain  degree  agree  purposes  department  for  I  permission  scholarly  partial  advanced  of  agree  in  thesis  of  my  is  thesis my  written  i i  Abstract The has  ethnobotanical  been  allowed plants  surveyed, the  literature  compiled  identification  with  specific  and of  of  Amazonian  organized.  South  This  information  c e r t a i n taxonomic  uses which  seem p r o m i s i n g  America  groups for  and  further  research. The  use  of  hallucinogenic alkaloidal  J u s t i c i a p e c t o r a l i s as Virola  based  compounds c o u l d  pharmacological indicative  e f f e c t s of  snuffs  an  e x t r a c t on  of  strongly  presence  constituents.  Nor  did  inhibitory  synergistic effect  Vi rola  bark.  smooth muscle presence also at  of  this  coumarin  present,  high  E x t r a c t s of  and  was  concentrations. towards  four  explain  the  of  infections. the  use  upon  Coumarin  a r o m a . The  coumarin  may  plant  a  was  role  caused  shown  to  i n i t s use  the  not  activity  wholly  a  from  the was  contraction of  the and  use  of  Vi rola  elongata  as  both  for  e f f e c t s of  snuff  an  may  certain  constituent  considered. The  to  constituent  observed of  mice  responsible  hypnotic as  of  which  smooth muscle  was  any  r e l a x a t i o n of  result  treatment  be  the  the  Betaine,  fungi  i n the  that  responses  inhibitory  shown t o  possibility  play  and  No  psychoactive  psychoactive  plant  to e l i c i t  Strong  was  plant  mice were  the  the  dermatophytic  the  investigated.  i n the  umbelliferone.  observed  extract  the  activity  and  to  J . p e c t o r a l i s e x t r a c t s have  5-methoxy-N,N-dimethyltryptamine, of  additive  been  detected  the  or  has  be  of  the  an  hallucinogenic  is  snuff  and an arrow  were e v a l u a t e d alkaloidal activity  for  their  fraction  plant  alkaloidal activity  of  material  fraction  lignans  stilbenes,  motor a c t i v i t y possibility  for  the  lignans  inhibitory aureus,  viruses,  of  the  S.  activities  the  certain  species  of  towards  infection  least  activity  polyphenolic  were  non-polar two  identified.  reduce  the  The  spontaneous in mice.  responsible  The  for  the  poison is' r a i s e d .  towards four  were  Escherichia  coli,  dermatophytic  in potato d i s c s  p r o p o r t i o n of  the  dermatophytes,  tumours and t h e  observed are  species  biological  bis-tetrahydrofuran  partly  formation  aureus,  same  The non-  Amazonian E u p h o r b i a c e a e  two y e a s t s ,  potato  its  induced aggression  activity  tumour  biological  The a n t i v i r a l  effect.  from t h e  were shown t o  at  of  four  bark  A non-  from the  j3-sitosterol,  lignans,  shrimp. A large  against  viruses,  to  as an arrow  species  to b r i n e  use of  are  elongata  Staphylococcus  were a c t i v e  addition  two n e o l i g n a n s ,  they  Thirty-four  toxicity  fraction  the presence  and i s o l a t i o n  that  use of V i r o l a  In  the  s p o n t a n e o u s motor  had no s i g n i f i c a n t  tetrahydrofuran  bis-tetrahydrofuran  two a n i m a l  in  was examined and some of  extract.  and two  screened  alkaloidal  to  of  on mouse b e h a v i o r .  E l e v e n compounds were i s o l a t e d  this  isomeric  of  the  was a t t r i b u t e d  compounds.  effects  caused a reduction  i n mice w h i l e  amount of  part  p o i s o n was e x a m i n e d . E x t r a c t s  brine  fungi, and  extracts  at  least  one  s h r i m p . The  discussed with respect  to  i n Amazonian e t h n o m e d i c i n e . of  one of  the  plants  Amanoa was e x a m i n e d . The i n h i b i t o r y by m u r i n e c y t o m e g a l o v i r u s  was  screened, a activity found  to  i v  result the  from  lignan,  the presence  a- ( -  hours),  this  already  infected  similar  to that  examined  )-peltatin.  compound  podophyllotoxin.  of a  single At  prevented  cells.  low d o s e s ( l O  Lignans  naturally  ng/ml  but  f o r two  t o be  of compounds  of s t r u c t u r a l  podophyllotoxin  type  not  observed.  other  in  very  lignan,  anti-murine  activity  as  of v i r u s e s  observed  occurring  cytomegalovirus  was  was  of a v a r i e t y  activity  identified  the r e p l i c a t i o n  It'sactivity  of another  for antiviral  compound,  than  the  types  were  V  T a b l e of  Contents  Abstract  i i  List  of  Tables  List  of  Figures  xii  Acknowledgement  xiv  CHAPTER  x  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 LITERATURE  CHAPTER  CITED  25  II  Justicia  pectoralis  : A STUDY OF THE BASIS FOR  USE AS A V i r o l a SNUFF ADMIXTURE 1.  INTRODUCTION  2. MATERIALS  3.  11  ITS 30 30  AND METHODS  a.  Plant  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  32  c.  Behavior  33  d.  Rat  e.  Antimicrobial tests  34  f.  Antiviral  35  RESULTS  material  .31  experiments  stomach s t r i p e x p e r i m e n t s  tests  31  33  35  vi  a.  E x a m i n a t i o n of  Justicia  pectoralis  for  alkaloids 35  b.  Compound 1  c.  Behavioral  d.  Effect  36 effects  of Just ic ia p e c t o r a l i s  of J u s t i c i a p e c t o r a l i s  5-MeODMT i n d u c e d b e h a v i o r a l e.  Effect  of  f.  Gross behavioral  g.  Effect  h.  Effect  on  responses  39  5-MeODMT on mouse a c t i v i t y  of  Justicia  extracts  37  effects  co-injections pectoralis  of  of of  41  5-MeODMT  44  5-MeODMT and  extracts  44  Justic ia pectorali s  extracts  on smooth m u s c l e of  47  i.  Analysis  aromatic  j.  Quantification  of  c o u m a r i n s of  k.  E x a m i n a t i o n of  J.  pectoralis  1.  Screening  for  other  constituents J.  for  51 pectoralis  ....53  lignans  biological activities  55 57  4. DISCUSSION  59  LITERATURE  63  CHAPTER  CITED  III  AN ETHNOPHARMACOLOGICAL EXAMINATION BARK,  OF V i r o l a  elongata  A SOUTH AMERICAN ARROW POISON  INTRODUCTION PART A .  ISOLATION  69 69  AND IDENTIFICATION OF THE MAJOR NON-  POLAR CONSTITUENTS OF V i r o l a  e l o n g a t a BARK  73  1.  INTRODUCTION  73  2.  EXPERIMENTAL  73  vii  a.  Extraction  b.  Chromatography  3. COMPOUNDS 4.  73 74  ISOLATED  74 80  3,4',5-trimethoxy-cis-stilbene  and  3,4',5-  trimethoxy-trans-stilbene  83  b.  Eusiderin  87  c.  Epi-sesartemin, and  d. B.  Virola 1.  material  RESULTS a.  PART  of p l a n t  and v i r o l o n g i n sesartemin,  epi-yangambin  yangambin  88 0-yangambin  D i h y d r o s e s a r t e m i n and EXAMINATION elongata  OF  BARK  THE  91  BIOLOGICAL ACTIVITY  OF  EXTRACTS  100  INTRODUCTION  2. M A T E R I A L S  AND  100 METHODS  100  a.  Preparation  of e x t r a c t s  b.  Fractionation  c.  Chromatographic  d.  Assay  of spontaneous  e.  Assay  of a n t i - a g g r e s s i v e  100  of e x t r a c t s analysis  101 of e x t r a c t s  motor  102  activity  103  activity  104  3. R E S U L T S a.  105  Examination  of the aqueous  fraction for  toxicity b.  106  Examination of the d i e t h y l depression  c.  Q u a n t i f i c a t i o n of major diethyl  d.  of spontaneous  Effect  ether  ether motor  extract for activity  constituents  of  extract  of b i s - t e t r a h y d r o f u r a n  109  114 lignans  on  viii  isolation  induced  aggression  116  4. D I S C U S S I O N  119  LITERATURE  CHAPTER  CITED  127  IV  STUDIES  ON  THE  PHARMACOLOGICAL  ACTIVITY  OF  AMAZONIAN  EUPHORBIACEAE  PART FOR  134  A . M U L T I P L E S C R E E N I N G OF  AMAZONIAN  EUPHORBIACEAE  BIOLOGICAL A C T I V I T I E S  134  1 . INTRODUCTION 2. M A T E R I A L S  3.  134  AND  METHODS  a.  Plant  b.  Preparation  c.  Antimicrobial  d.  Antiviral  e.  Potato  f.  Toxicity  to brine  g.  Analysis  of data  139  material  139  of plant  extracts  140  screening  140  activity  disc  141  tumour  assay  143  shrimp  144 144  RESULTS a.  Antimicrobial  activity  b.  Antiviral  c.  I n h i b i t i o n of potato  d.  Toxicity  e.  Correlation  activity  to brine  150 tumour  between  Antimicrobial  formation  shrimp biological  4. D I S C U S S I O N a.  145  153 156  assays  156 167  activity  167  ix  b.  Antiviral  activity  c.  Antitumour  d.  Toxicity  170  activity  to brine  172  shrimp  172  5. C O N C L U S I O N PART  B.  173  a-( -  CONSTITUENT  )- PELTATIN,  OF  Amanoa  THE A N T I V I R A L  sp  176  1 . INTRODUCTION 2. M A T E R I A L S  AND  METHODS  a.  Plant  b.  Antiviral  c.  Chromatography  176  material  3. R E S U L T S AND PART  176  176  assays  177 177  DISCUSSION  C. T H E A N T I V I R A L A C T I O N  178 OF  LIGNANS  1 . INTRODUCTION 2. M A T E R I A L S  183 183  AND  METHODS  ...184  a.  Chemicals  184  b.  Cells  184  c.  Antiviral  d.  Effect  and v i r u s e s screening  of time  of lignans  of treatment  186 187  3. R E S U L T S  188  4. D I S C U S S I O N  193  LITERATURE  APPENDIX OF  A  CITED  - LIST  ETHNOBOTANICAL BIBLIOGRAPHY:  APPENDIX  OF  201  AMAZONIAN.ANGIOSPERMS  INTEREST  APPENDIX  207  A  B - S P 4 1 0 0 COMPUTER  231 PROGRAM(BASIC)  236  X  List  I.  Phylogenetic  of  Tables  distribution  of  species  of  Amazonian  a n g i o s p e r m s h a v i n g documented e t n o b o t a n i c a l II.  III.  Effect  of  5-MeODMT and  of  co-administration pectoralis  c o u m a r i n and u m b e l l i f e r o n e Just ic ia  'H-NMR s p e c t r a  13  C-NMR  of  extracts Effect bark  in  elongata  elongata  r e s p o n s e s of  of  of V i r o l a  different 56  lignans  bark  81  bis-tetrahydrofuran  from V i r o l a  of  46  bis-tetrahydrofuran  of  Gross b e h a v i o r a l  mice  pectoralis  from V i r o l a spectra  40  spontaneous  Levels  of  of  on  activity  administration  VIII.  extracts  locomotor  isolated VII.  of  alkaloidal elongata  lignans  bark  -82  S w i s s mice and  to  non-alkaloidal  bark  107  p u r i f i e d compounds of V i r o l a  elongata  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  Swiss mice IX.  X.  analysis  constituents  of d i e t h y l  Effect  Effect  of  of  of  thirteen ether  most common  extract  of V i r o l a  bark  parameters XI.  113  Quantitative  elongata  ..14  on  Effect  isolated VI. '  extracts i n mice  s a m p l e s of V.  pectoralis  spontaneous locomotor a c t r v i t y  Justicia  IV.  Just ic ia  use  115  epi-sesartemin related  on t h r e e  behavioral  to aggressiveness  bis-tetrahydrofuran  lignans  of on  mice  ....118  xi  aggressiveness XII.  Antimicrobial  i n mice screening  Euphorbiaceous XIII.  Antiviral  screening  activity  of extracts  148  of 151  of Euphorbiaceous  plants  of Agrobacteriurn induced  formation  154  S c r e e n i n g of e x t r a c t s for  plants for  plants  inhibition  tumour XVI.  fungus  Screening of e x t r a c t s for  of ...146  Screening of Euphorbiaceous  Euphorbiaceous XV.  of e x t r a c t s  plants  anti-dermatophytic XIV.  120  their  toxicity  of Euphorbiaceous  to brine  shrimp,  plants  Artemia  salina •XVII.  157  Summary  of b i o l o g i c a l  Euphorbiaceous XVIII. 2 X  pair  plant XIX.  XX.  tables  f o r agreement used  to screen  t h e 85 164  Values  of Fisher's  tables  of Table  Summary  exact  test  f o r contingency  XVIII  165  of e t h n o b o t a n i c a l information activity  o f t h e 34 s p e c i e s  for their  replication  of Sindbis  virus  inhibition  of 168  of lignans  of time  and  tested  Examination  Effect  between  extracts  Euphorbiaceae  XXII.  of 159  o f t h e 14 a s s a y s  biological  XXI.  of extracts  plants  2 Contingency  each  screening  of lignan  of Sindbis  effect  a n d MCMV  treatment  virus  on 189  upon  infection  192  xii  List  1. D i s t r i b u t i o n  Amazonian  ethnobotanical 2. S t r u c t u r e s  of  (50  documented  ( 1 ) ,coumarin  (2) a n d  (3)  38 locomotor  activity 43  o f a) s e r o t o n i n ( l  of  on s m o o t h  6. H P L C  n g / m l ) a n d b)  48  ( 10 Mg/ml) a n d ( b )  ( 1 0 Mg/ml) o n s m o o t h  chromatogram  betaine  muscle  (a) coumarin  (umbelliferone)  of the organic  muscle  fraction  52  of  leaves  J . pectoralis  Structures Vi rola  from  of s t i l b e n e s  54 and neolignans  isolated  from  elongata  8. S t r u c t u r e s  lignans  isolated  elongata  85  of t e t r a h y d r o f u r a n  Vi rola  elongata  Scheme  of fragmentation  lignans,  84  of b i s - t e t r a h y d r o f u r a n  Vi rola  9. S t r u c t u r e s  10.  with  18  o f 5-MeODMT o n s p o n t a n e o u s  jug/ml)  5. E f f e c t  7.  angiosperms  mice  4. E f f e c t  of  Figures  uses  of betaine  umbelliferone 3. E f f e c t  of  epi-sesartemin  lignans  isolated  from 86  of b i s - t e t r a h y d r o f u r a n and sesartemin  by  mass  spectrometry 11.  Scheme  of fragmentation  lignans,  epi-yangambin  92 of b i s - t e t r a h y d r o f u r a n and yangambin  by mass  spectrometry 12.  Scheme  of fragmentation  93 of t e t r a h y d r o f u r a n  lignan,  xi i i  dihydrosesartemin, 13.  Scheme  Example  Effect and of  16.  motor  epi-yangambin  lignan,  output  used  to  96  record  activity  111  on  lignans,  spontaneous  epi-sesartemin  locomotor  activity  mice  112  Chromatotron  elution  o f Amanoa  Structure from  18.  95  spectrometry  of b i s - t e t r a h y d r o f u r a n  fraction 17.  by mass  of transducer  spontaneous 15.  spectrometry  of fragmentation of t e t r a h y d r o f u r a n  /3-dihydroyangambin 14.  by mass  of a-( -  Amanoa  Structures  profile  of ethyl  acetate  sp. leaves  179  )- p e l t a t i n  isolated  sp  .181  of l i g n a n s  tested  for antiviral  activity 19.  185  Effect  of time  of l i g n a n  treatment  murine  cytomegalovirus infection  on  inhibition  of 191  xi v  Acknowledgement An e t h n o b o t a n i c a l strongly I  contributors  as w e l l  and  of  was  his  the  beneficial Just ic ia  thanks  t o be a b l e  laboratory.  made p l a n t  the  important  I  Our  Botanical Justicia Botanical  of  of  and  work  of  advisors,  who  supplied  and  gave  in cooperation  with  role  he p l a y e d  pectoralis Research,  His  following  information interest  for  Prof.  his  was  in  helpful  and  analysis. R.E.  Schultes  i n many w a y s . name i s  cited  the  This  has is  and  from  u s e s of  both  elongata. personal  and D i r e c t o r ,  the  of  in d e s c r i b i n g  the  and a l s o D r .  concerning  of  study  the  New York  and,  was e s p e c i a l l y  available  and V i r o l a  Professor  work  work.  sp.  times  Museum c o n c e r n i n g  information  the  research  exchange  this  this  to acknowledge  Schultes,  to  Towers,  this  field  fieldwork  number of  pectoralis wish  G.H.N.  for  free  material  course  from the  Justicia  R.E.  the  degree,  t o work  b o t h d u r i n g the  The e t h n o b o t a n i c a l  evident  to P r o f .  t o many a s p e c t s  influenced  a  no e x c e p t i o n  t o many c o n t e m p o r a r y  p e c t o r a l i s and V i r o l a  he k i n d l y  is  is  time.  fortunate  D e n n i s McKenna, in  as  study  large  e n t h u s i a s m and s u p p o r t  generously  that,  This  approach  informants.  owe s p e c i a l  unlimited  one.  t o an e s p e c i a l l y  my p r e d e c e s s o r s  I  ethnobiological  interdisciplinary  am i n d e b t e d ,  I  or  c o m m u n i c a t i o n of Harvard  ethnobotany G.T.  Botanical  ethnobotany  Garden, of  University  and taxonomy  Prance,  both  Prof.  Director for  of  of  helpful  Justicia  XV  pectoralis  and V i r o l a  A great collection  elongata.  many p e o p l e may be c r e d i t e d w i t h a s s i s t i n g  of  plant  material.  Museum,  generously  for  identification  the  collected. kindly  All  of  identified  Museum. D r .  de P e r u ,  assisted  in  plant  the  Franklin  kindly  the  of  much of  species  by D r .  of  M.J.  the  Sr.  Ayala,  of  for  consultation  necessary  Puricaca  of  the  Pucallpa,  for  the  their  cited  source  concerning medicinal W i t o t o and B o r a Nuevo  Cdrdoba,  mestizo  I n d i a n s of  for  Enrique  assistance  plant  their Donez,  in plant  R a m i r o D i a z of  Balsas.  this  Peruana, I  helpful graciously is  communications thanks  and t h e  to  Boras  and M a r i o C o r d o b a p r o v i d e d  the of  Andres  valuable  Among t h e many I n d i a n  and  may be numbered Don J u a n  These are  just  am  Summer  Juan Ruiz Macedo,  Don J u a n P e s o of  for  regard.  N i c o l e Maxwell  owe s p e c i a l  hospitality.  collecting.  Pucallpa,  I  Puco U r q u i l l o  informants contacted,  M o z o m b i t e of  personal  use.  in  and equipment a v a i l a b l e .  and Wes T h i e s s e n of  several  and  Departamento  de A m a z o n i a  i n f o r m a t i o n and h o s p i t a l i t y . of  were  authorization  o f f e r e d both  Brillo  collected  Historia  A d r i a n a L y o a z a and N i c o l e M a x w e l l  the  arranged  t h e Museo  information.  as  and  Field  Universidad Nacional  Linguistics,  Field  the C h i c a g o  of  the  material  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  to Dick R u t t e r  Institute  of  made t i m e a v a i l a b l e  and e x p o r t .  plant  Euphorbiaceae  made h e r b a r i u m f a c i l i t i e s  grateful  the  Huft  of  Chicago  and a s s i s t a n c e  Director  procurement  collection  de A g r i c u l t u r a Dr.  provided advice  Ramon F e r r y r a ,  Naturale  Tim Plowman,  in  Nina Rumi,  a few of  the  and Don many  xvi  Peruvians  who,  interest,  offered  The  in a spirit  information  laboratory  interdisciplinary in  muscle  NMR  advice spectra  obtained U.B.C. on  many  The  M.P.  in  Dept.  of Medical strains  and Pete  h i s lab.  me  University,  data  were  U.B.C, fungi.  tested  a n d D r . G.H.  recorded  Chemistry, facilitated  I am  grateful  and t o Dr.  by to  for  Barbara  the  I would  like  Microbiology,  out the studies  Vancouver,  A l l of  U. o f W a s h i n g t o n , f o r  of Medical  of the l i g n a n s  Gopaul  Department,  was  Felipe Balza.  tumefaciens  Dept.  Sam  spectra  a n d Drummond,  Microbiology,  smooth  r o t a t i o n s were  of spectral with  U.B.C,  and behavior.  of Biochemistry,  to carry  Some  Forintek,  on a n t i v i r a l  were  provided  Sheriha,  to  U.B.C, activity by D r . E .  E l Fateh  Libya.  Finally, British  and o p t i c a l  assistance  Drummond,  activity.  care  of dermatophytic  D r . J.B. Hudson,  also  provided  r e s o l u t i o n mass  of D r s . P i e r c e  discussions  allowing  Swan,  animal  the Aqrobacterium  sporulating  for  spectra  interpretation  Dept  was  the s e r v i c e s of the Chemistry  Gordon,  providing  thank  research  available for recording  and the high  Infrared  common  uses.  Bob K a n t y m i r  Crawford  concerning  through  helpful  Dill,  of t h i s  equipment  the instruments  U.B.C  Dr.  Terry  on p l a n t  c o n t r a c t i o n a n d mouse l o c o m o t o r  provided the  made  phase  i n nature.  the greenhouse.  generously  of f r i e n d s h i p and a  the financial  Columbia,  the  Natural  for  a Postgraduate  support  i n t h e form  Science  of a Graduate  and E n g i n e e r i n g Fellowship  of the U n i v e r s i t y of  Research  i s gratefully  Fellowship, Council  and  o f Canada  acknowledged.  1  I.  1.  ETHNOBIOLOGY:  biotic not  terms  resources  are, are  ethnobotany for  the  by man.  numerous and t h e y  backgrounds,  INTRODUCTION  AN INTERDISCIPLINARY  Ethnobiology, overlapping  GENERAL  and e t h n o p h a r m a c o l o g y  study Its  APPROACH  of  the  students  use of  come from d i f f e r e n t  as many d e f i n i t i o n s  of  earth's  and p r a c t i t i o n e r s  b r i n g i n g w i t h them v a r y i n g  perhaps,  the  are  disciplines  points  of  are  and  view.  "ethnobiology"  as  There there  ethnobiologists. The t e r m s ,  ethnobiology,  ethnopharmacology  have been  of  sanctioned  scientifically  descriptions their  1967; H o l m s t e d t 1976).  used by  a p p l i e d to  observations,  Bruhn and H o l m s t e d t  (1982)  many  or  of  plants,  ( E f r o n e_t  pharmacology products).  involved  and b o t a n y  The o n l y  ethnobiology.  It  is  and al,  1963; S c h u l t e s  have  and  succinctly  as  ethnobiology  disciplines  types  identifications,  investigations  1983; S c h u l t e s ,  the  t h e 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 o b s e r v e d by m a n . "  Ethnobotany basic  and  indigenous peoples  and B r u h n ,  d e f i n e d ethnopharmacology "  loosely  and e x p e r i m e n t a l  principals,  Swain,  ethnobotany  (or  may be s i m i l a r l y are  zoology  term which used in  ethnology  the  in  or  the  defined.  The  anthropology, case  encompasses a l l present  or  work  of  animal  situations  for  this  is  reason.  2  My c o n c e p t i o n of having it  been  general  the  objectives  c o n s i d e r a t i o n of  ethnobiology  societies.  Acceptance  that  of  of  tendency  the  unique,  workers.  Neither  there  In  an  attempt  follows and  does  a  importance  science. in  science  i n d u s t r i a l i z e d and  from our our  scientific  have b r o a d e n e d  or  not common. T h e r e  not  m e t h o d , but  in other  primitive  simple technologies  own i s  society  the  only  t o adopt  ways i s  to accept the  is  the  corollary  necessarily  trivial  false.  beliefs  interface of  that  result  the of  between  primitive  ethnobiology,  is  not  use of  by man has  testing  tradition.  When one q u e r i e s  came t o  appropriate friend,  know of answer;  a relative,  on t h e  other  it  an e s s e n t i a l  the part  emerged s l o w l y ,  by g e n e r a t i o n s  of  of  a plant that  the  an e l d e r  use,  one sometimes g e t s  the  shamanic  varies;  it  was  learned  how  the  i n f o r m a t i o n was o b t a i n e d or  the  oral  an I n d i a n or m e s t i z o a b o u t  from a  from a n e i g h b o r i n g community.  h a n d , one a s k s a n a t i v e  in  as  view  inquisitive  has been s u s t a i n e d by t h e  been t r a i n e d that  is  method and  a l w a y s a smooth o n e . The s c i e n t i f i c  and t h a t  they  scientific  p e o p l e s , which  plants  empirical  the  primitives  If,  is  objectives  t h e v a l u e of  in  not  study,  role,  western  information gained  The  is  the  systems d i f f e r e n t  primacy  study  concensus view.  this  r a p i d advances  gap between  a widespread  of  i n modern  conceptual  belief  of  i n f l u e n c e d by many p r e v i o u s  The r e c e n t  or  field  c o i n c i d e w i t h some i m a g i n a r y  to c l a r i f y  of  this  tradition, from t h e  specialist t h e answer  plant,  itself.  who has seldom According  3  to  the  shaman,  of  the  plant,  explanation. certain  the  process  The  plants  Peruvian  as  mestizo  doctores  or They  teach)  (Luna,  1983).  made  during  trance  like  of  hallucinogenic  amazing  thing  similar  forms,  all  Americas  the  1944).  The of  (Browman  and  The  i f the  remarkable ago,  modern  about an  to  the and  Cape  pain.  alkaloid  with  concept  the  and,  Since from  same  then, the  poppy,  invocations  regulated tobacco.  as  of  Amazon,  (Eliade, the  1964;  cultures  barren  of  land  teachers It  is  until  little been  are  ironic,  fundamental  discovered pain  by  that  in  the  human  neurophysiology  the  normal  availability  poppy  plant.  The  body  Trabucchi,  has  ensued.  peptides, of  the  or  years  importance morphine, to system  which  also  1978).  A  The endorphins,  compound  euphoric  ten  binding  present  and  easily  i t indicates  are  the  of  however,  c e n t r a l nervous  the  but  the  approximately  of  prevents  not  the  of  in  Eskimos  of  in  truly  1976).  figuratively,  (Costa  the  Metraux,  neurons  receptors  by, The  on  upon  course,  or  of  to  (plants  the  thought.  i t has  ensenan  only  the  example,  its  i t i s common,  Chine,  plants  to  spirit  i s that  in  as  knew v e r y  discovery  dependent of  1979;  For  science  rethinking  provocative was  well  que  potent  Eurasia  i s taken  insight.  peptides  major  as  not  the  routinely refers  subjects  induced,  traced  scientific  and  to  of  be  Schwartz,  receptors  bind  can  Horn  specific that  tribes,  the  and  up  listening  shaman  practice  much  and  are  ayahuasca  this  summoning  vegetales  states  i m p l i c a t i o n s of  reconciled that  about  beliefs  Fuegians  of  establishing contact  that  use  i s one  morphine  properties  of  4  Papaver  somniferum were,  Sumerians  some 6000 y e a r s ago  Many  similar  convulsant currently  the  Euphorbiaceae,  et  in  and S t i l l e ,  in experimental a_l,  i n d u c e r s of  1981).  For  esters,  tabacum)  Tubocurarine (Atropa  neuromuscular  neurotransmission from  ( E v a n s and S o p e r , have been  found t o  p h y s i o l o g i s t s have  distinguished  the  (from Amanita Loganiaceae)  t r a n s m i s s i o n , and r o t e n o n e  and in  atropine studies  (Derris  spp.)  autumnale)  by most c y t o g e n e t i c i s t s  repair Merck  (Caffea  s p . and Thea s p . )  p r o c e s s e s . The l i s t Index,  one c i t a t i o n important  it  is  difficult  constituent It  is  i n the  to pick  of  plants  is  (Colchicum and  effects  inhibition  of  o f DNA  a page on w h i c h a t  useful  or  p r o d u c e d by a p l a n t instructive  to  the least  economically or m i c r o o r g a n i s m ,  i m a g i n e what modern  and m e d i c i n e might have been l i k e  constituents  of  c o u l d be c o n t i n u e d . L o o k i n g a t  to a s c i e n t i f i c a l l y  does n o t a p p e a r . biology  Colchicine  h u n d r e d p a p e r s have been w r i t t e n on t h e  caffeine  (from  muscaria).  inhibitor.  several  al,  were e x c i t e d by n i c o t i n e  both used e x t e n s i v e l y  used r o u t i n e l y  e_t  be  (Yip  they  of  they  tools  1978;  commonly u s e d as a m e t a b o l i c is  species  i n mammalian c e l l s  or m u s c a r i n e  are  is  tumour p r o m o t e r s and i m p o r t a n t  animal  (from p l a n t s  belladonna)  Phorbol  Recently,  n e u r o n s b a s e d upon whether  a  (Menispermaceae),  1980).  interferon  many y e a r s ,  Picrotoxin,  of  research  1977).  the  1972).  investigations  are  cancer  potent  Nicotiana  known t o  examples may be c i t e d .  used w i d e l y  Weinstein  (Swain,  from A n a m i r t a c o c c u l u s  (Hoffmeister of  coincidentally,  had t h e  or microorganisms never  organic  been  available  5  to u s . small  Knowledge fraction  naturally or  in  of  these  what  areas  it  is  would p r o b a b l y  now. W i t h o u t  biologically  o c c u r r i n g compounds as m o d e l s , v e r y  chemotherapeutic  agents  would y e t  be r e d u c e d t o a  few  active,  antibiotics  have been s y n t h e s i z e d by  chemi s t s . Even s u c h a r e l a t i v e l y economic  importance  recognized. of  the  of  economically  contained plant  was  i n e x c e s s of wholesale tropical (U.S.  $8.0 b i l l i o n  forests,  from b i o l o g i c a l l y  primitive  is  American  own s o c i e t y ' s  importance  of  the  of  the  respect  general to  to  In  be  and  that,  Morris  between  filled  the  one-half  in the  consumer, 1980,  value  U.S.  was  the  had c l i m b e d 1982).  imported to to  to  The  the U.S.  from  be $25 m i l l i o n  1980). argument  indebted to  societies  plants  t e m p e r e d by t h e  dollar  the  widely  Farnsworth  has been e s t i m a t e d  active plant  The d e g r e e  to  as  approximately  ( F a r n s w o r t h and L o u b ,  space,  deeply  not  indicate  annually.  Task F o r c e ,  sufficient  and m e d i c i n e a r e  our  which  medicinal plants alone,  is  the p r e s c r i p t i o n s  unchanged but  v a l u e of  Given  1981).  p r o d u c t s . The v a l u e ,  Interagency  further.  figures all  that  matter  compounds c o n t i n u e  t o be $ 3 . 0 b i l l i o n ,  percentage  are  (Oldfield,  1973, 25% of  estimated  to medicine  important  have p r e s e n t e d  1959 and  plants  Educated estimates  o b t a i n e d from p l a n t s (1976)  straightforward  the  t h a t modern  information  science  obtained  compounds, c o u l d be e x p a n d e d for  plants  h e l d by  the  has a l r e a d y .been c o n t r a s t e d  lack  of  acknowledgment  knowledge.  observation  that  The i r o n y the  of  of this  "most u s e f u l  with  the situation drugs  6  derived of  the  from h i g h e r scientific  therapeutic  plants  enquiry  effects"  have been  'discovered'  into alleged  as a  folkloric  ( F a r n s w o r t h and L o u b ,  result  claims  1982).  Many m i c r o b i a l p r o d u c t s have been d i s c o v e r e d as a of  a random s c r e e n i n g p r o g r a m . F o r  plants  are  Moreover,  not  nearly  since  they  part  of  rich  history  the  the m a t e r i a l  past,  of  the  has been t h e of  as amenable are  investigation  they  of  out  constituents  plants  out.  the U.S.  The most e x t e n s i v e  National  Cancer  evidence  significantly those  for  follow-up study,  that  the  occurrence  higher  upon.  still  man  Less  lead to  biological  In  development  and r e s e r p i n e .  OBJECTIVES OF  of  higher  these  the  activity  plants  have  been  has been c o n d u c t e d by  ( S u f f n e s s and D o u r o s ,  antitumour  Spjut  in p l a n t s  s e l e c t e d at  of  Institute  P l a n t s were s c r e e n e d  interesting  2.  the  a  1973) .  carried  than  important  t o draw  l e d to  such s t u d i e s  with novel  an  u s e d by p r i m i t i v e  Some random s c r e e n i n g p r o g r a m s of  1982).  are  available  digitalis  today,  higher  man. As a r e s u l t ,  p r e f e r r e d a p p r o a c h and has  commonly c a r r i e d  (Gran,  of  reasons,  result  suchr an a p p r o a c h .  primitive  use by man i s  such drugs as a t r o p i n e ,  discovery  to  macroscopic,  w o r l d of  plant  various  of  activity.  and P e r d u e  antitumour having  In  (1976)  activity  a history  of  an  presented was  folk  use  random.  ETHNOBIOLOGY  The f o r e g o i n g d i s c u s s i o n has d e v e l o p e d t h e  idea  that  one  7  of  the  primary  objectives  of  ethnobiology  is  naturally  occurring constituents  with novel  activity.  A natural  this  describe  and e x p l a i n  constituents the  use of  for  the  in  the  of  the d i s t r i b u t i o n plant  medieval  origin  gradual  e x t e n s i o n of  plants  aim i s of  in medicine  being  responsible  as  various  plant  phytochemistry,  natural  products chemistry  of  synthesizing this  broad p e r s p e c t i v e are  distributed  in  the p l a n t  been a t t e m p t e d  by o n l y a few  Farnsworth,  al,  t y p e of  et  valuable  insights  kingdom i s  the  it  role  of  i n f o r m a t i o n on t h e p r e s e n c e  of  even compounds w i t h s p e c i f i c  in taxa  not  previously  Perdue,  1976).  biologically  chemical  this  in p r e d i c t i n g  sort,  of  the  computer  1982;  1958).  is  in  provide  activity,  (Barclay too, the  is  and considered  occurrence  of  ( S p j u t and  Perdue,  in handling  large  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  i n f o r m a t i o n on p l a n t s ,  This  provide  biological  information,  active constituents  The a p p l i c a b i l i t y  b a s e s of  a c t i v e compounds  s p e c i f i c compounds,  examined c h e m i c a l l y  tool  to gain a  constituents  would be e x p e c t e d t o  Ethnobotanical  of  an immense one and has  or p o s s i b l y  data  so as  various  predictive  by some t o be a u s e f u l  fields  and chemotaxonomy.  1962; K a r r e r ,  Moreover,  1976).  related  ( F a r n s w o r t h and L o u b ,  evolution.  certain  chemical  i n f o r m a t i o n may e v e n t u a l l y  into  to  followed a  biologically  1974; H e g n a u e r ,  descriptive  the  information  on how v a r i o u s  active  has p o i n t e d  of  The t a s k  in  to  (1972)  i n f o r m a t i o n on t h e taxa  a desire  biologically  Swain  of  biological  modern taxonomy. T h e r e has  a c c u m u l a t i o n of  constituents  kingdom.  the d i s c o v e r y  currently  being  and  8  investigated Loub,  1982; F a r n s w o r t h There  that  is  another  this  al,  basic  the aspect  property statement  unlikely  to c o n t r i b u t e  other  between a way  hand,  of  directly  of  plant  the  ethnobotanical  only  This w i l l  complete ecosystems of  ethnobotany,  result  since  in  t o be of  g r o u p s of  the  of  but a l s o  these,  too,  of  the is  information,  on h a b i t a t s  and  inevitably  even  altered  t h e d o c u m e n t a t i o n of  plant  use  vegetal  drastic  One of  is  unrecorded, of  materials.  This  existence  in  approach.  of  biota,  the  context  scientific  loss  are  on  roles  IMPORTANCE OF ETHNOBIOLOGY The q u e s t i o n of  can,  the  i m p o r t a n c e as t h e  world's  the c e r t a i n t y  is  the  It  is  relationship  if  the  the  advantage.  man and p l a n t s  information,  work  criticized.  science.  the  between  cultures,  therefore,  continues  3.  of  economic d e v e l o p m e n t .  identification  face  to b a s i c  of  by man  i n d u s t r i a l i z e d w o r l d expands and  on s p e c i f i c  as a r e s u l t  a plant  a reductive  interactions  e n d u r a n c e of  low.  nature  kingdom i n an h i s t o r i c a l  the  predictably  use of  summarize an a s p e c t  d e c l i n i n g as  the  in e t h n o b i o l o g i c a l  be r e g a r d e d as a u n i q u e  not p o s s i b l e u s i n g o n l y  The e x t e n t  not  objective  c o n c e r n i n g the  man and t h e  and  1981).  e t h n o b i o l o g y most o f t e n  can a l s o  A single  the  et  1977; F a r n s w o r t h  s h o u l d not be o v e r l o o k e d . The s u b j e c t i v e  approach is Yet  ( F a r n s w o r t h and B i n g e l ,  of  i n c l u d i n g many human  and  activity little  known  cultures,  change.  IN THE TROPICS  future  of  tropical  e c o s y s t e m s of  the  9  world  i s an  time out  in  this  and  that  type  of  work.  drugs  tropical  and  factors.  species  this  70%  global  forest  of  1981).  i s agreed  described al,  1971).  information The  Of  these,  most  tropical  moist  forecast  that  of  often  we  et  10  the 10  and  a_l,  16 only  %  for  certainly  important  indicates  that  parallels  studying  this  ethnobiology, uncertain for  1977).  between  million  species  known the  most  40  and  (Meyers,  the  species  (Meyers, the  of  almost  Covering  contain  of  planet's  already  1981;  Raven  rudimentary  1971). p r e d i c t i o n of  i s that  of  of  1970).  least  extent  for  species  potential  (Bruning,  to  possess  cited  forest  5  has  from  forests account  they  carried  the  in  t r o p i c s i s the  i t i s the  between  (Raven  the  for  of  diversity  which  Tropical  estimated  estimates  ranging  in  surface,  that  many  synthesized  reason  be  region  identified  (Raffauf,  this  tropics  fertile  so  at  can  the  enormous  productivity  earth's  It  with  why  presented  compounds  region.  world's  biomes,  been  biology,  the  the  study  i n t e r a c t i o n s are  compelling  facing  50%  and  diversity  future  the  clear  The  of  of  especially  1981).  organisms  equally  7%  an  (Oldfield,  has  biologists  plants,  were  branch  only  be,  using  origin  any  of  are  i s not  in chemical in  for  natural  Evidence  increase  one  ethnobiological  inter-specific  diversity  An  of  to It  of  plants  kingdoms  intra-  people  continues  important  all  pertinent  h i s t o r y . Although  anywhere  been,  or  especially  the  Richards  future  (1952;  of  1973)  the who  et  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 a s 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 o f t h e e a r t h by t h e end o f t h e 2 0 t h century."  Other less  predictions dramatic.  that,  There  by t h e y e a r  tropical and  are generally  moist  i s broad  2000,  forests  very will  less  pessimistic  agreement large  be  among  segments  reduced  1974; G o l l e y  and Medina,1975;  1972;  Lanly  and Clement,  1979; M e y e r s ,  1974,  1975, a n d  1976;  UNESCO, The  for  watershed  approximately  the  1980;  1977).  of t h i s  areas,  a  vulnerable ecosystem  a n d b;  et  a l ,  Persson,  1973;  Sommer,  cultivation  1971).  to modification to tolerate  importance  have  such  been  identified  by t h e e x p a n d i n g  points  towards t h e  change  of the  (Gentry,  seasonally  f r a c t i o n of the  has been  however,  as  (Denevan,  to resist  total  realized  quite  i s t h e most  and the a b i l i t y pressures  (Fearnside,  ranching  evidence  small  This,  forests  above  practiced  of the c o n t r i b u t i o n  relatively  accounts  tropical  cited  and c a t t l e  ecosystem  system  of d e f o r e s t a t i o n are  factors  to the o v e r a l l productivity (Meggers,  moist  forecasts  The emerging  The magnitude  recently  The  rates  harvesting  1980).  ability  inundated area,  disruptive  timber  Shane,  limited  Predicted  of s h i f t i n g  population,  1980a  o f t h e Amazon R i v e r  of the g l o b a l  The major  pattern  (Farnworth  Gomez-Pompa  1976; R i c h a r d s ,  40% o f t h e w o r l d ' s  1980).  representative 1979).  of the world's  1978).  vast  .(Goodland,  specialists  t o remnants  Golley,  1977; P o o r e ,  b u t n o t much  of this  has not been  of t h e r e l a t i o n s h i p s between  complex established.  the trees  of the  11  inundated  regions  has  recently  the  major Any  been  source likely  forests  a n d t h e many documented of p r o t e i n  scenario  i n the next  evolutionary  scale,  the  biota  planet's  seemingly  (Goulding,  a very  i s of a drastic 1981).  situation,  as p o s s i b l e  ethnobotany contribution Tropical  Biology,  indigenous have  It the  OF T H I S  The  objectives  basic  approach  was  general  biota  biological methods  for this  only  in Tropical  These  but  a r e aimed of  allowed.  tropical  Descriptive  valued  Priorities in r e l a t i o n s h i p of the  i s considered  i n s i g h t s which  of the present was  to select  after  years  the plants active  that  efficacy  this  study  likely  may of  to  be research.  were s e v e r a l - f o l d .  instances  of  traditional  by b i o l o g i c a l constituents  and i d e n t i f i c a t i o n  expected  time  The l o n g s t a n d i n g  of b i o l o g i c a l l y  isolation  1980).  1971; R e s e a r c h  their  of  STUDY  u s e , t o examine  presence the  i n many  by s c i e n t i f i c  4. APPROACH  plant  with  i n the extent  Priorities  t o be a m i n o r ,  et_ a l ,  tropical  on an  on t h e b i o l o g y  i n the l i m i t e d  1980).  peoples  resulted  obtained  The  (Raven  (Research  information  i s considered  are also  certain priorities for  1980; T r o p i c a l D e f o r e s t a t i o n ,  organisms  interval  In preparation  Biology,  a s much  fish  population.  reduction  have  accumulating  proposed  Fish  of the moist  short  research  at  1980).  f o r the enlarging  century,  unavoidable  of f r u i t i f e r o u s  f o r the future  (Meyers,  been  species  process  of the plant  lead  and provide  f o rt h e  and t o attempt  of the a c t i v e would  assays  constituents.  to conclusions some  insight  on  1 2  into  its  use by a r e l a t i v e l y  was hoped t h a t structure be n o v e l  either  (preferably and t h a t  the  primitive  culture.  biological activity  both)  of  the  a contribution  Moreover,  or  the  chemical  active constituent(s)  to basic  it  would  s c i e n c e might  be  made. The f i r s t of  plants  criteria plants  of  stage  in t h i s  ethnobotanical  came  into play  interest this  level.  potential plant's  for  to assay  o b t a i n i n g new  in  the  probability  of  assumed t h a t  active  the  clearly  was a l s o  constituents  l i k e l i h o o d of  a plant  in a plant  containing  w i t h many documented e t h n o b o t a n i c a l  b)  w h i c h had not a l r e a d y  been a r r a n g e d  data  of  and monographs of  botanists,  pharmacologists, missionaries  the  of  been s t u d i e d  Pertinent  locations  works  chemical  It  such  uses chemically  information  i n c l u d i n g the  anthropologists explorers,  and h e r b a r i u m l a b e l s .  research  s y s t e m of  Cronquist  (1981).  not  present notes  and  travellers  As much of  has  is  this  and information  as p o s s i b l e was c o l l e c t e d and o r g a n i z e d a c c o r d i n g t o classification  the  was  on S o u t h A m e r i c a n p l a n t s  systematically.  i n a wide v a r i e t y  The  belonging to a taxon:  a)  The e t h n o b o t a n i c a l  The  used t o a s s e s s  being present.  to  defined  laboratory.  and  Many  was g i v e n  i n f o r m a t i o n was a s s e s s e d .  plants  compounds would be g r e a t e r  selection  t o work.  Preference  t a x o n o m i c p o s i t i o n and e t h n o b o t a n i c a l  concerning related  the  upon w h i c h  w i t h w e l l e s t a b l i s h e d and r e l a t i v e l y  u s e s w h i c h were amenable  data  at  approach involved  This  is  the  presented  1 3  as A p p e n d i x A . to  the  The  i n f o r m a t i o n has been s u m m a r i z e d , a c c o r d i n g  distribution  documentation at has a l s o (1980),  of  the  species  order  been a r r a n g e d whose  dimensional advantage  of  relationships each g r o u p ,  level,  o r d e r s of  not  only  in Table  1 a c c o r d i n g to  classification  the  allowing,  ethnobotanical  and f a m i l y  in Figure  s y s t e m of  scheme of  with  consists  I.  It  Dahlgren of  a  two  a n g i o s p e r m s . T h i s has  t h e main  the  phylogenetic  between t h e  orders,  but  the  relative  i n numbers of  species  described,  to  size  be  of  easily  perceived. The arises for  information gathered  from t h e  biases  of  degree It  of is  survey  Angiosperms are  is,  known t o  with ethnobotanical important,  Solanaceae It  I  and F i g u r e  much b e t t e r  are  uses.  Apocynaceae,  with 95,  can be seen  ethnobotanical  T,  especially  the  from F i g u r e  species  used,  number of  in each group i s  the  Subclasses  the  species  most  are  Rubiaceae  46 and 45 s p e c i e s , the  Class  i n number of  that are  Euphorbiaceae,  1 that  and  r e p r e s e n t e d than  rich  The f a m i l i e s  53, 47,  interest  this  higher  that  the M a g n o l i o p s i d a , the  a c c o r d i n g t o numbers of  Leguminosae,  and p r o v i d e d a  in  i n d i g e n o u s A m a z o n i a n s . The  however,  and A s t e r i d a e  information  selected  r e p r e s e n t e d among t h e m e d i c i n a l  Class L i l i o p s i d a . Within Rosidae  was  It  observations.  from T a b l e  widely  poisonous p l a n t s Magnoliopsida  of  subjective.  whose d a t a  c o m b i n i n g the  more e x t e n s i v e  verification evident  inherently  the a u t h o r s  c o m p i l a t i o n . However,  way made t h e  is  the  and  respectively. species  generally  a  of  14  Table  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 of s p e c i e s of Amazonian angiosperms having documented e t h n o b o t a n i c a l use.  Taxa are arranged C r o n q u i s t (1981).  according to Table I i s a  Taxon  t h e c l a s s i f i c a t i o n scheme summary o f A p p e n d i x A.  Number o f s p e c i e s w i t h documented use  Division: Maqnoliophyta Class: Maqnoliopsida Subclass I. Magnoliidae Order: Magnoliales F a m i l y : Annonaceae Family: Myristicaceae Order: Laurales Family: Monimiaceae Family: Lauraceae Order: Piperales Family: Piperaceae Order: Aristolochiales Family: Aristolochiaceae Order: Ranunculales Family: Menispermaceae Order: Papaverales Family: Papaveraceae S u b c l a s s I I . Hamamelidae Order: Urticales Family: Moraceae Family: Cecropiaceae Subclass I I I . Caryophyllidae Order: C a r y o p h y l l a l e s Family: Phytolaccaceae Family: Nyctaginaceae Family: Cactaceae Family: Chenopodiaceae Family: Amaranthaceae Family: Portulacaceae Family: Basellaceae Family: Caryophyllaceae Order: Polygonales Family: Polygonaceae  of  793 727 1 04 42 12 30 6 2 4 29 29 8 8 18 18 1 1 17 17 10 7 22 20 3 2 4 1 5 3 1 1 2 2  15  Table I(continued) No. spec ies  Taxon Subclass IV. D i l l e n i i d a e Order: D i l l e n i a l e s Family: Dilleniaceae Order: Theales Family: Caryocaraceae Family: Marcgraviaceae Family: Quiinaceae Family: Clusiaceae Order: Malvales' Family: Tiliaceae Family: Sterculiaceae Family: Bombacaceae Family: Malvaceae Order: V i o l a l e s Family: Flacourtiaceae Family: Bixaceae Family: Violaceae Family: Turneraceae Family: Passifloraceae Family: Cucurbitaceae Family: Begoniaceae Order: S a l i c a l e s Family: Salicaceae Order: E r i c a l e s Family: Ericaceae Order: Primulales Family: Theophrastaceae Family: Myrsinaceae Subclass V. Rosidae Order: Rosales Family: Connaraceae Order: Fabales Family: Leguminosae Order: Myrtales Family: Lythraceae Family: Thymelaeaceae Family: Onagraceae Family: Melastomataceae Family: Combretaceae Order: Santalales Family: Olacaceae Family: Loranthaceae Family: Balanophoraceae  3 4 3 1 10 3 4 2 10 14 3 2 1 1 9 1 1 3 1 4  4 95 1 3 1 3 3 2 5 1  3  80  18  19  31  1 3 5  4 95 1 1  8  230  16  Table  I(continued) Taxon  Order: Celastrales Family: Celastraceae Family: Icacinaceae Family: Dichapetalaceae Order: Euphorbiales Family: Euphorbiaceae Order: Linales Family: Erythroxylaceae Family: Humiriaceae Order: Polygalales Family: Malpighiaceae Family: Vochysiaceae Order: Sapindales Family: Sapindaceae Family: Burseraceae Family: Anacardiaceae Family: Simaroubaceae Family: Meliaceae Family: Rutaceae Order: Geraniales Family: Oxalidaceae Subclass VI. Asteridae Order: Gentianales Family: Loganiaceae Family: Gentianaceae Family: Apocynaceae Family: Asclepiadaceae Order: Solanales Family: Solanaceae Family: Convolvulaceae Family: Hydrophyllaceae Order: Lamiales Family: Boraginaceae Family: Verbenaceae Family: Lamiaceae Order: Scrophulariales Family: Scrophulariaceae Family: Gesneriaceae Family: Acanthaceae Family: Pedaliaceae Family: Bignoniaceae Order: Campanulales F a m i l y : Campanulaceae Order: Rubiales Family: Rubiaceae Order: Asterales Family: Asteraceae  No.  spec i e s  5 2 2 1 47 47  0  1 2 8 22 17 5 27 6 5 6 3 2 5  1 1 247 68 8 4 53 3 49 45 3 1 29 8 8 13 44 4 4 18 1 17 4 4 46 46 17 17  17  Table  I(continued)  Class:  66  Liliopsida  Subclass I: Alismatidae Order: Alismatales Family: Alismataceae Subclass I I : Arecidae Order: Arecales Family: Arecaceae Order: Cyclanthales Family: Cyclanthaceae Order: Arales Family: Araceae Subclass I I I : Commelinidae Order: Commelinales F a m i l y : Commelinaceae Order: Cyperales Family: Cyperaceae F a m i l y : Poaceae S u b c l a s s IV: Z i n g i b e r i d a e Order: Bromeliales Family: Bromeliaceae Order: Zingiberales F a m i l y : Musaceae Family: Zingiberaceae Family: Costaceae F a m i l y : Cannaceae Family: Marantaceae S u b c l a s s V. Liliidae Order: Liliales Family: Pontederiaceae Family: Liliaceae Family: Iridaceae Family: Agavaceae Family: Smilacaceae Family: Dioscoreaceae Order: Orchidales Family: Orchidaceae  2 2 2 29 5 5 1 1 23 23 8 1 1 7 2 5 13 1 1 12 4 • 1 4 1 2 14 8 1 1 2 1 1 2 6 6  Figure  1-  D i s t r i b u t i o n o f A m a z o n i a n a n g i o s p e r m s 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 , a r r a n g e d a c c o r d i n g to t h e c l a s s i f i c a t i o n scheme o f D a h l g r e n ( 1 9 8 0 ) . Numbers r e f e r t o s p e c i e s t h a t have a t l e a s t one 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 . D a t a a r e s u m m a r i z e d f r o m A p p e n d i x A.  19  reflection exception  of  the  i s the  number order,  utilized  in proportion  majority  of  of  species  in  Orchidales, to  i t s species  which  i t s size.  are  rare,  that  This  small  group.  seems may  in  A  not  be size  notable to  be  because  the  and  easily  not  obtained. The list  broad  played  a  perspective significant  ethnobotanical selected bases  were  for  elongata  problems  activities  for  uses a  of  of  the  screening members  of  Just icia pectorali s additive  to  Yanomamo.  its  the  Its  documented  by  In  in  study  might  Acanthaceae, Eighteen (Table very  well  i n my  program the  i s an  never to  other  ability  herb  has  for  a  to  well  produce  an  the  well  alone  for  as  a  scientific  identified indicated  The  that  family,  ethnobotanically.  i n which  Related  Solanaceae  of  as  reasons  i t s use  results.  used  been  The  ethnobotanical  I.  Vi rola  snuffs  considerations  Asteridae,  to  tribes  subjected  being  with  biological  biological  botanists.  important  in Table  Apocynaceae, their  been  finally  Euphorbiaceae.  also  interesting  subclass,  represented  various  Acanthaceous  and  studies  p e c t o r a l i s and  of  and  of  and  hallucinogenic  this  species  ethnobotanical  chemical  unacculturated  snuff,  this  selection  family,  based  problem, yield  the  Justicia  i s moderately  I ) . The  for  have  Amazonian  Asteraceae, known  the  addition  ethnobotanical its  in  by  i n v e s t i g a t i o n . The  anthropologists  however,  scrutiny.  Vi rola  use  inclusion  snuff,  role  i n v e s t i g a t i o n s of  the and  permitted  and  uses  are  i t i s placed,  families  \s  (Rubiaceae,  Bignoniaceae)  complex  known  are  biologically  well  active  20  chemical  constituents.  are  medicinally  used  interesting are  number  of Asian  activities  recently  been  shown  to display  and Banerjee,  1979).  members  of the family  Acanthaceae  reports  have  been  of J u s t i c i a with  isolated.  p r i m a r i l y of the b i s - t e t r a h y d r o f u r a n  (Ghosal  No  species  and a v a r i e t y o f compounds  biological  lignans,  have  A  of the genus,  the South  are l i t t l e  Justicia,  class.  antidepressant  Finally,  from  These They  action  American  known  chemically.  the Americas  have  been  made. The  use of V i r o l a  established dual  uses  arrow The  of V i r o l a  family,  a  (Gottlieb,  biological studied  1979).  activities.  question  could  poison  or  i f some  marked  biological  especially  well  studied  The  only  explain other  to experimental  respect  was  with  analysis.  (30  In a d d i t i o n  /3-carboline are also  interesting  elongata,  has  to i t s indole  been  alkaloids.  of h a l l u c i n o g e n i c resin  toxic,  responsible  o n e . I t was  an  intriguing.  ethnobotanically  the use of t h e bark compound  The  i n the Amazonian  possess  V.  well  as both  and n e o l i g n a n s  the presence  activity,  interesting  species,  with  theiodora  and  of these  a  literature.  chemically.  tryptamine  Some  of whether  alkaloids  utilized,  v a r i e t y of l i g n a n s  chemically  amenable  well  i s also  are especially  i s important  of p s y c h o t r o p i c a wide  and V i r o l a  hallucinogen  and r e l a t i v e l y  alkaloids,  The  elongata  Myristicaceae,  number  known  a n d an  as h a l l u c i n o g e n s  i n the ethnobotanical  I t i s moderately  species), to  phenomenon  poison  flora.  species  assumed  or  a s an  arrow  otherwise  was  considered  t o be  readily  an  21  Although  the subject  hallucinogenic  plant  constituents  of  this  It  i s i n t r i g u i n g that  groups  thesis,  of South  materials This  some  peregrina  indole  of source  (Malpighiaceae).  Many  pharmacologically  actions.  Indole  this  class  group.  classes  terms former  refers  objective effects  are involved:  Virola  spp.  i n many  compounds  comprise  distinguished  i n meaning to a  observation  the agent  a psychosis.  of e i t h e r  term.  Hallucinogenic  impossible  to quantitate.  one o f t h e p o t e n t i a l  indole  compound  may  have.  four  have  been  range of a n d j3-  used  of  two o f t h e s e v e n hallucinogenic Although  synonomously, the  highly  produces  Problems  dependent  Moreover,  The o t h e r  with  i s largely a  upon  dose  hallucinogenic  pharmacological  t o an  behavioral  are associated  activity  or  these  i n t e r p r e t a t i o n , the l a t t e r  mimic  of the subject,  1982).  and a r e o f t e n  that  at least  of the plants  as having  (Hollister,  subjective  a  the tryptamine  which  function  t o have  detected  activity  drugs.  Anadenanthera  constituents  have  indole  using  and B a n i s t e r i o p s i s spp.  and found  been  of  (Myristicaceae),  of both  o f compounds  overlap  that  order.  distinct  the custom  compounds  These  psychotomimetic  adopted  of the indole  examined  carboline  and c u l t u r a l l y  i t i s considered  spp. (Rubiaceae)  is in  a l k a l o i d s as ceremonial  plants  (Leguminosae),  Psychotria  only  Indians  t o t h e theme  of the subject  geographically  American  containing  groups  i s peripheral  clarification  i s e s p e c i a l l y s o when  major  use  of the use and e f f e c t s of  effects  and activity  that  pharmacological  an  actions  is  22  may a l s o r e s u l t  i n b e h a v i o r a l a l t e r a t i o n s . The p r e c i s e nature  of the b i o l o g i c a l e f f e c t s of such compounds i s t h e r e f o r e difficult  to d e f i n e . T h i s i s p a r t i c u l a r l y t r u e when animals  are used as models to q u a n t i f y the pharmacological The  term, p s y c h o t r o p i c ,  r e f e r s to the a b i l i t y  mood or c o g n i t i v e processes.  As d e f i n e d by Lewin  i n c l u d e s agents with hypnotic, and  response. to a l t e r  (1927), i t  inebriant, excitant,  euphoriant  h a l l u c i n o g e n i c a c t i v i t y . Because i t i s a more general  term  for d e s c r i b i n g agents with a c t i v i t y on the c e n t r a l nervous system, i t s use i s a p p r o p r i a t e  when the nature of the  b i o l o g i c a l a c t i v i t y under i n v e s t i g a t i o n i s not defined  (Hoffmeister  and S t i l l e ,  completely  1982). I t i s used i n t h i s  t h e s i s t o d e s c r i b e the e f f e c t s of the a d m i n i s t r a t i o n  of 5-  methoxy-N,N-dimethyltryptamine and e x t r a c t s of J . p e c t o r a l i s and V. elongata The  t o mice f o r t h i s  reason.  t h i r d , and f i n a l , part of t h i s research p r o j e c t was  somewhat d i f f e r e n t  i n approach. While i n Peru, I became aware  of the importance of s p e c i e s of the f a m i l y , Euphorbiaceae i n f o l k medicine t h e r e . T h i s i s the t h i r d ranking of number of e t h n o b o t a n i c a l  f a m i l y i n terms  uses (Table I ) . I t s s p e c i e s are  known to c o n t a i n a great d i v e r s i t y 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 . The most widely probably  diterpenes  d i s t r i b u t e d compounds are  of the phorbol  a v a r i e t y of b i o l o g i c a l a c t i v i t i e s ,  e s t e r c l a s s . These possess foremost among which are  complex r e g u l a t o r y a c t i o n s on mammalian t i s s u e s (Evans and Soper, 1978). The Amazonian s p e c i e s of Euphorbiaceae have been examined with  respect  to t h e i r chemical  c o n s t i t u e n t s or  23  pharmacological A  activity  significant  ethnobotanical  proportion  o f wounds,  ulcers,  conditions. Their  to involve  replication  of microorganisms  some a s p e c t  of c e l l u l a r  to inhibit  dermatophytic toxicity  of  the  species.  some  used  have a documented studies  activity an  some w i t h were  and p o t a t o  that  extract  yeasts,  was  to  activities  the  species activity  and a l s o  ethnobotanical  an o b j e c t i v e . The  identified.  that  with  pursued  and  of  action  d i d , and d i d  i n the case  elongata,  marked  these  relative  u s e . As  constituents  provide  information  the  in plants  and  of  i n s i g h t i n t o the mechanism  o f A m a n o a s p . was  was  for their  tumours  aim of the study  hoped  A  documented  examined  the b i o l o g i c a l  of chemical  also  biological  of b a c t e r i a ,  of J . p e c t o r a l i s and V ir o l a  was  responsible  on  These  t o a n a l y s i s by b i o a s s a y .  viruses  The  action  or v i r u s e s , or the r e g u l a t i o n of  without,  I t was  of d e t e c t i n g  identification  of  shrimp.  the medicinally  not,  of  the r e p l i c a t i o n  would allow  likelihood  others  information  known  obtained  either the i n h i b i t i o n  plants,  fungi, animal  to brine  descriptive little  of  a r e e s p e c i a l l y amenable  uses,  of  mestizo  mechanism  proliferation.  of Euphorbiaceous  cases.  species  i n the treatment  be h y p o t h e s i z e d  ability  of the Euphorbiaceous  of  or  and cancerous  ethnobotanical  number  the Indian  infections  variety  small  by  o.f P e r u  activities  a very  i n t e r e s t are used  population  may  i n only  of  the  novel  antiviral  biological activity  t h e compound  24  These the of the  three  a n a l y s i s of 34  species  subsequent  these,  a  chapters  Vi rola  of  this  i ) the  elongata  Euphorbiaceae  a n a l y s i s of  species in  projects:  of  Amanoa,  thesis.  the are  study  resin, for  of and  J.  iii)  biological  antiviral dealt  pectoralis, the  with  as  screening  activities  constituent  i i )  of  and one  independent  of  25  LITERATURE CITED C h a p t e r I_  B a r c l a y , A.S. a n d P e r d u e , R.E. 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Yip,  Y.K., P a n g , R.H.L., U r b a n , C. a n d V i l c e k , J . ( 1 9 8 1 ) P a r t i a l p u r i f i c a t i o n a n d c h a r a c t e r i z a t i o n o f human a ( i m m u n e ) 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 7 8 , 1 6 0 1 1605.  30  II.  Justicia FOR  1.  I T S U S E AS A V i r o l a  snuffs  widespread  use of t r y p t a m i n e  i n the Orinoco  been  well  Bark  resin  documented  and northern during  of species  one o f t h e s o u r c e s  of these  1909) a n d t h e i r  (Schultes  and Holmstedt,  known  Virola  that  based  there  herbaceous later  established of  as a custom  et a l ,  Barker  described  as  1967). reported  1909  (Koch-  established  constituents (1953),  snuff.  of  and the  the addition  resin  was  o f an  This  plant  pectoralis var. stenophylla  to Vi rola  1968).  snuffs  i s widespread  (Brewer-Carias  1971; P r a n c e ,  was  has  1953, h o w e v e r ,  important  and Holmstedt,  which  well  Not u n t i l  other  (1960),  leaves  t h e Yanomamo g r o u p  Chagnon  1968).  as J u s t i c i a  (Schultes  dry, pulverized  a r e now  areas  (Wassen,  as e a r l y  to the pulverized  identified  drainage  (Myristicaceae)  The m i s s i o n a r y ,  Zerries  additive  (Acanthaceae) its  BASIS  hallucinogenic  century  snuffs  uses  existed  snuffs.  anthropologist,  was  THE  ADMIXTURE  based  Amazon  the past  of V i r o l a  Griinberg,  it  SNUFF  OF  INTRODUCTION  The  as  p e c t o r a l i s : A STUDY  The a d d i t i o n  i s now  well  amongst  the t r i b e s  and 'Steyermark,  1972; S c h u l t e s ,  of  1976;  1978; S e i t z ,  1967). The snuffs  reason  f o r the i n c l u s i o n of J . p e c t o r a l i s  i s not understood.  suggested  that  i t i s added  Several because  Indian  informants  o f i t s aroma  in Virola have  (Prance,  1972;  31  Schultes  and Holmstedt,  however,  that  Holmstedt, the  sole  al,  i t may p o s s e s s  1968).  other  The r e p o r t s  ingredient  intoxication  1968; S e i t z ,  (Brewer-Carias  1971; P r a n c e ,  and that  comm.)  i s evidence,  (Schultes  J. pectoralis  and  constitutes  i tproduces  and Steyermark,  G.T., p e r s .  There  properties  that  of a snuff  1967).  a state of  1 9 7 6 ; C h a g n o n e_t  are especially  interesting. The of  Vi rola  1980).  chemistry snuffs  -  a.  of other  be a v a i l a b l e . T h i s  basis  Plant  The  i s known  no i n f o r m a t i o n American study  (Holmstedt  of the chemistry  a l , of J .  on t h e c h e m i c a l  species  of t h i s  of the possible  plant  used  in this  study  i n 1 9 8 1 . I t was i d e n t i f i e d  deposited  e_t  genus  appears  physiological undertaken.  AND METHODS  by D r . Timothy  Museum. V o u c h e r  specimens  herbarium,  I t has been  pointed  was c o l l e c t e d  i n Pucallpa,  as Just i c i a p e c t o r a l i sv a r .  Plowman  of the Chicago  ( D . M c K e n n a N o . 1) h a v e  a t UBC h e r b a r i u m ,  San Marcos  Peru.  studied  constituents  material  stenophylla  of  well  of the a c t i v e  f o r t h e u s e o f J . p e c t o r a l i s was t h e r e f o r e  MATERIALS  Peru  been  nothing  Moreover,  constituents  2  have  In contrast,  pectoralis.  to  and pharmacology  Chicago  Lima  Field  Museum,  a n d UNAP h e r b a r i u m ,  out (Schultes,  that  Just icia pectoralis var. stenophylla  form  of J . p e c t o r a l i s and i t i s u n l i k e l y  Field been University Iquitos,  R.E., p e r s . i s probably that  comm.) a  growth  the difference  32  in  name  Prof.  used  Schultes  considered  greenhouse mature  ethanol times rotary added  minutes  plants  (1150  and  acetate.  fractions  The  the  original  b.  Chromatography  Polygram  Silica  precoated (HPLC) phase  layer  variable  automated  spectra  g  G  UV  were  hours  ether,  0.478  on  in  stems  boiling four  concentrated  a  (500  steam  by  ml)  was  bath  for  The  aqueous  fraction  with  diethyl  ether,  acetate  and  and  42.3  g,  was  carried  100%  20 was  then  aqueous  respectively,  weight,  UV  2 5  a  (TLC)  (Brinkman)  performed  Varian  obtained  not  and  extracted  water  ethyl  performance  were  Bruker  is  spectroscopy  Model  were  FT-100 using  on  a  Gas  cellulose  Perkin  using (Eastman)  chromatography  Varian with  with  resonance  instrument a  out  MCH-10 Varian  reverse Series  634  chromatography/mass  obtained  magnetic  and  liquid  5000 HPLC  detector.  (GC/MS) d a t a  a  48  0.931,  GC/MS. N u c l e a r on  celite.  chromatography gel  with  under  flowers  was  and  heated  grown  placed  Distilled  fresh  and  wavelength  spectrometry  recorded  for  1150  separations and  combined  was  for  leaves,  homogenate  through  p l a t e s . High  column  The  diethyl  accounted  of  Thin  washed,  were  were  continuously  distinction  v e g e t a t i v e l y and  g)  which  keeping  report.  i_n v a c u o .  filtered  varietal  cut  filtrates  residue  d i f f e r e n c e . In  Freshly  homogenized.  the  the  propagated  conditions.  the  extracted  genetic  subsequent  evaporation to  ethyl  the  were  and  and  a  suggestion,  in  Plants  of  reflects  and  Elmer  a  Finnegan  (NMR)  spectra  infrared 710B  (IR)  Infrared  1020 were  33  Spectrophotometer. c.  Behavior  experiments  Locomotor a c t i v i t y version which  of  a  "jiggle  was measured i n mice u s i n g an  cage"  (Robbins,  were u s e d .  intraperitoneal  The t e s t  injection  and t h e mouse p l a c e d  w i r e mesh cage w h i c h was  force  transducer  amplified  by e l a s t i c  1000 t i m e s  integrator.  voltage  value  of  written.  that  every  This apparatus  of  minute  intervals.  movement of  and t o  fine  It  ability  constituents  to  d.  Rat  a f i v e minute p e r i o d  by  to  a l t h o u g h the All  toxicity  was  input  for  at  five  activity  produced a  and compounds were  injection. and  Because  1979),  u s e d as a v e h i c l e  for  the  Tween  a 25% (v/v)  aqueous  activity  the  case.  experiments  The p r o c e d u r e u s e d f o r  evaluating  the  of  lipophilic  a_l,  of  the  the  (Budden e t  One h u n d r e d nl  SP  was  units,  former  extracts  Tween 80 was u s e d i n e a c h  stomach s t r i p  was  absolute  both locomotor  intraperitoneal  low  value  in a r b i t r a r y  sensitive  signal.  substances administered. of  and summed t h e  s o l u b i l i z e both h y d r o p h i l i c  and i t s  signal  sampled the  p r o d u c e d an a v e r a g e  was  (Eastman Kodak C o . )  solution  signals  t h e mouse,  larger  administered to.mice  18 cm  100 m i l l i s e c o n d s , c a l c u l a t e d  over  body movements,  proportionately  Centre,  isometric  b a n d s . The t r a n s d u c e r  successive  difference  degree  i n an  s u s p e n d e d from an  A computer program which  between  mice  and m o n i t o r e d u s i n g a S p e c t r o p h y s i c s  approximately  difference  80  Swiss  s u b s t a n c e was a d m i n i s t e r e d by  cubic  its  Female  had been b r e d and m a i n t a i n e d by t h e A n i m a l C a r e  U.B.C.  4100  1977).  adapted  of  34  extracts on smooth muscle was based on that described by Vane (1957). B r i e f l y , a male Wistar rat was s a c r i f i c e d 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 s t r i p as described by Vane (1957). The stomach s t r i p was suspended v e r t i c a l l y in a 5 ml capacity bath containing Kreb's solution (composition in g/1: NaCl = 6.9; KC1 = 0.35; CaCl -2H 0 = 0.36; 2  = 0.29; KH PO„ = 0.16; NaHC0 2  3  2  MgSO„-7H 0 2  = 2.1; dextrose = 1.0) through  which was bubbled continuously a mixture of 95% oxygen and 5% carbon dioxide. The lower end of the s t r i p 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 s t r i p . 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 s t r i p 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 a c t i v i t y . 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  d e x t r o s e agar  paper  respectively,  swabs.  Sterile  tested  had been a p p l i e d , were p l a c e d on the  plates  were e x a m i n e d f o r  inhibition  filter  paper  24 and 48 h o u r s ,  f.  filter  plates,  discs after  Antiviral  f o r m a t i o n of  were t e s t e d  (MCMV)  included  agarose  i n the  m o n o l a y e r s and t h e later of  for  RESULTS  a.  Examination  of  The r e p o r t s  their  the  overlay  Sindbis are  Stille,  G.T.,  that  pers.  to  surface.  evidence  but  as  comm.)  agents  be The  the  inhibit  the  and m u r i n e Extracts  were  infected  and MCMV, r e s p e c t i v e l y . in Chapter  pectoralis  the  for  is  sole  cell  are  alkaloids  used,  not  ingredient  that  this  1970). T h i s that  (Schultes,  of  a  a_l,  plant  an  snuff 1971;  alone  may  The most  (Hoffmeister  fact, J.  o n l y as  activity.  alkaloids  Complete  III.  1976; Chagnon e t  suggest  indicating  N,N-dimethyltryptamine that  to  a p p l i e d to v i r u s  J.' pectoralis  1982; S c h u l t e s ,  indicated  virus  or even h a l l u c i n o g e n i c  psychotropic  preliminary  agar  growth a r o u n d  ability  presented  Justicia  to V i r o l a ,  have p s y c h o t r o p i c potent  extract  i n c u l t u r e d mouse c e l l s .  ( B r e w e r - C a r i a s and S t e y e r m a r k , Prance,  of  the  p l a q u e s w h i c h d e v e l o p e d were c o u n t e d 2 and  the assay  3.  admixture  for  p l a q u e s by S i n d b i s  cytomegalovirus  details  t o which  cotton  tests  Extracts  5 days  discs,  using  and  combined w i t h  pectoralis  1970; S c h u l t e s ,  the p h a r m a c o l o g i c a l a c t i v i t y  of  J.  may  contain  1972) pectoralis  36  may of  be a t t r i b u t a b l e  t o the presence  three  J . pectoralis  alkaloids  by T L C . Samples  developed  with  ethyl  extracts  were  and sprayed  1984).  N,N-Dimethyltryptamine,  and  applied  with  Ehrlich's  classes  any  No E h r l i c h ' s  of the three Each  using  precipitation aqueous each  (Martello  (19/1)  layer  observed  after  produced  a distinct  caused  with  were  (upper  eta l ,  included as  compound  and Farnsworth,  could  be d e t e c t e d i n  plates  using  A single  reagents f o r  n-propanol/0.3  spot  with  colour  ammonium  reagent  tt  0.46 w a s  reagent. I t  when  sprayed  with the  reagent  which  ( S t a h l , 1969)  forming  for alkaloids  N NH OH  Rf v a l u e  compounds  the deposition of platinum,  iodoplatinate  with  was c h r o m a t o g r a p h e d o n  modification of Dragendorff's  for quaternary  Only the  a precipitate  Dragendorff's  salmon-pink  of a l k a l o i d s  1962).  yielding  fraction  spraying with  Bregoff-Delwiche  and  (McKenna  and Dragendorff's  This  as a developer.  specific  (5/4/1)  f o rthe presence  was p o s i t i v e ,  of the reagents. thin  N NH OH 4  plates,  5-OH-N,N-dimethyltryptamine,  positive  was t e s t e d  Meyer's  fraction  cellulose  g e lG  extracts.  extract  Valser's,  examined f o r  to Silica  reagent  5-methoxy-N,N-dimethyltryptamine  standards.  were  ether/2-butanone/6  phase)  b.  or other  alkaloids. The  is  of indole  a gray  (Stahl,  spot  1969).  C o m p o u n d J_  The silica  compound  using  ( 1 ) was p u r i f i e d  n-propanol/0.3  by c o l u m n  N. NHi,OH  (3/1)  c h r o m a t o g r a p h y on  as eluant  a n d was  37  crystallized data  were  Compound 3250br, (100  The  from  aqueous  methanol.  J_ ( b e t a i n e ) 2900,  : 2 1 5 nm. I R v max  UV X max  11  IR s p e c t r u m  3.31 ( 9 H ,  was i d e n t i c a l  t o that  sample  T L C a n d by HPLC 2  with  evidence,  for  inner  spectrum  betaine  salt)  matched  that  (Sigma).  1 co-chromatographed  acetonitrile/H 0 this  hydroxide 1  an a u t h e n t i c  s ) .  reported  1 9 8 1 ) a n d t h e 100 MHz H-NMR  cellulose  1  1  2  Compound  cm" :  1 6 5 0 , 1 4 8 0 , 1 4 0 0 , 1 3 3 0 , 9 8 0 , 9 3 0 a n d 8 8 0 . H-NMR  MHz, D 0 ) 5 : 3.9 ( 2 H , s ) ,  (Poucert,  of  spectral  obtained.  (carboxymethyl-trimethylammonium  of  The f o l l o w i n g  with  (retention  a flow  rate  betaine  time  = 4.7 m i n .  of 1 ml/min.).  i t was c o n c l u d e d  that  b o t h on  compound  using  2%  On t h e b a s i s 1 was  betaine(Figure 2). c.  Behavioral The  first  pectoralis account  compound  known to  approach  t o the ethnopharmacology that  the use of t h i s  thepossibility might  behavioral extracts  of Just ic ia p e c t o r a l i s  established  for  eliminate  ef f e c t s  be p r e s e n t .  changes  were  i n mice  observed.  t o accompany  psychotropic plant  that  This  a non-alkaloidal  injected  A variety  and S t i l l e ,  this  with  did not  psychotropic  question,  J . pectoralis  of behavioral  theadministration  rodents(Hoffmeister  alkaloids did not  as a snuff.  To approach  ofJ .  changes a r e  of psychotropic  1982).  agents  Alterations in  38  Figure  2 -  S t r u c t u r e s of b e t a i n e ( 1 ) , coumar i n (2) and u m b e l l i f e r o n e ( 3 ) , compounds i s o l a t e d Just ic ia pectoralis.  from  39  spontaneous  locomotor a c t i v i t y  (Hollister,  1982).  ether  extracts  of  The a q u e o u s , J.  intraperitoneally  are  pectoralis  t o mice at  commonly  ethyl  a c e t a t e and  were  administered  a d o s e of  c h a n g e s were o b s e r v e d and l o c o m o t o r results  of  in Table  the  II.  spontaneous  Each  extract  activity  locomotor  often  provides  estimate  acetate  relatively responses  weak a c t i v i t i e s  Ef f e c t  of  of  of  Just ic ia  the  pectoralis  the  of  alone,  was o b t a i n e d  has p s y c h o t r o p i c  activity.  of  the  None of  presence  the  of  Virola  that  the  hallucinogenic  important  indoles  Holmstedt  et  al,  use of  activity  N,N-Dimethyltryptamine dimethyltryptamine  on 5-MeODMT  (DMT)  (5-MeODMT)  of  and are,  al,  manufacture  of  role  based s n u f f s indole  when  is  is  based  constituents.  quantitatively,  1980; McKenna e t known  its  J.  5-methoxy-N,N-  barks  is  that  was c o n s i d e r e d . T h e r e  their  i n most V i r o l a  two c o n s t i t u e n t s  indicate  activity,  strong evidence  the  extracts.  the  to  snuff  used i n  in  strongest  extracts  used i n c o m b i n a t i o n w i t h V i r o l a  these  and level  reduction  three  Mice  responses  S i n c e no e v i d e n c e  on the  presented  in a c t i v i t y .  activity  The  activity.  induced b e h a v i o r a l  pectoralis,  of  p o s s e s s e d the  o b s e r v e d were i n d i c a t i v e  psychotropic d.  fraction  recorded.  with eyes c l o s e d ,  The r e c o r d i n g  The e t h y l  was  Behavioral  r e c o r d i n g are  displayed piloerection. a quantitative  diethyl  250 mg/kg.  caused a reduction  adopted a huddled p o s t u r e ,  observed  (Agurell 1984).  from a l l  snuffs  of  and t h e i r  At  et  the  most  al,  1969;  least  one  the V i r o l a presence  in  of  species the  40  %  Extract t Injected  normal  activity  leveKmin.  post  injection)  5  10  15  20  25  30  35  40  45  50  55  60  aqueous  93  91  1 04  92  95  86  85  88  77  79  75  77  ethyl acetate  96  1 07  101  64  52  61  58  44  53  57  51  59  diethyl ether  92  94  98  91  94  87  83  88  77  82  75  88  2 5 % T w e e n 80 ( 1 00 / i l )  91  96  1 1 0 1 04  92  94  109  11 2  95  99  11 4  91  Table  II - E f f e c t of Just i c i a p e c t o r a l i s e x t r a c t s spontaneous locomotor a c t i v i t y i n mice.  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 dissolved i n 100 M 1 o f 2 5 % Tween 80.  at doses  on  o f 250  mg/kg  41  snuffs  themselves  i s well  Holmstedt  and Lindgren,  ten  aspotent  It  times would,  chosen  designed  to test  pectoralis  Five-MeODMT  be e x p e c t e d  effects  as a model  1967).  (Agurell  a psychotomimetic  therefore,  pharmacological  documented  compound  to account  (Shulgin, f o r most  This  was r e s p o n s i b l e  ofthe  studies  some c o m p o n e n t  for modulating  1982).  t r y p t a m i n e was  f o rc o - a d m i n i s t r a t i o n  the hypothesis that  1969;  i sapproximately  a s DMT  of the snuffs.  et_ a j ^ ,  of J .  the effects  oft h e  tryptamines. Five-MeODMT  i s known  t o cause  behavioral  changes  i n rats  of  produce  excitation,  1 mg/kg  salivation tremor range  (Ahlborg  produced  levels  Grahame-Smith, 1978)  have  activity  b y 5-MeODMT  quantified  and t o vary  chosen  t o examine  some  behavioral  response  t o 5-MeODMT.  o f 5-MeODMT o n m o u s e  Solutions aqueous Activity  constituent  Tween  o f 5-MeODMT 80  levels  the  in activity  animal  t o be  manner.  This  the p o s s i b i l i t y  that  that J .  alters the  activity  (Sigma)  monitored  in  a n d Hedden,  shown  were  dissolved  (100 M D a n d a d m i n i s t e r e d  were  a n d some  1971). The  for measuring  been  Doses  e t a l , 1968;  (Shah  i n a dose-dependent  contains  Effect  b y DMT  have  pectoralis  e.  Changes  (Ahlborg  i n devices  The responses  was t h e r e f o r e  tremor  i s dose-dependent  e_t a _ l , 1 9 6 8 ) .  1971) a n d i n m i c e  levels.  reproducible method  i n rats  been  intraperitoneally.  hyperactivity,  compound  (Ahlborg  produced  injected  of characteristic  e t a l . , 1968; G r a h a m e - S m i t h ,  by t h i s  1-10 mg/kg  a number  a t 5 minute  i n 2%  intraperitoneally. intervals  for  60  42  minutes show  after  that  injection.  a l l doses  The r e s u l t s p r e s e n t e d  o f 5-MeODMT  tested  produced  an i n i t i a l  period  minutes.  The degree  of h y p e r a c t i v i t y  dependent (2.5  manner.  o r 12.5 mg/kg) d o s e  returned  almost  Administration data  reductions minutes (1971)  post  with  reduction normal level  previously  level  been  by  for  to last  respectively.  1.25 mg/kg  at high  a similar  but that,  doses,  markedly  ( 3 - f o l d a t 12.5 m g / k g ) .  60  and Grahame-Smith i n rats  r e s p e c t i v e l y . The study (50%  and 70% normal  activity  n o t shown) h a v e n o t  however,  has been  decreasing  shown t o  activity  o f 2.5 a n d 25 mg/kg in activity  was  observed  60 a n d 120 m i n u t e s ,  The r e s u l t s p r e s e n t e d  activity  mg/kg;  o f 5-MeODMT  of gradually  approximately  at least  in activity  i n the present  The r e d u c t i o n  o f 5-MeODMT p r o d u c e  until  o f 5-MeODMT,  the administration  injection.  in significant  (1968)  increases  Dimethyltryptamine,  1971).  them  e t a_l  a s an e f f e c t  a similar pattern following  persisted  5-MeODMT; d a t a  reported  (Grahame-Smith,  doses  after  post  high  levels  ( 1 . 2 5 a n d 0.625  resulted  observed  0 . 6 2 5 mg/kg  administration.  levels  similar  dose-  the activity  30 m i n u t e s doses  Ahlborg  3 a n d 1 mg/kg  activity  produce  very  in activity  after  which  injection.  observed  injected  after  in contrast,  in activity  in a  10 t o 20  o f a l o w ( 0 . 3 1 mg/kg) o r a  of intermediate  n o t shown),  lasting  varied  o f 5-MeODMT,  t o normal  3  ( 0 . 3 1 t o 1 2 . 5 mg/kg)  of hyperactivity  In t h e case  i n Figure  here  indicate  reduction  activity  t h a t low  i n motor  levels  increase  43  "1  10  1  20  1  30  1 i.0  1  T  50  60  TIME AFTER INJECTlON(minutes) Figure  3 - E f f e c t o f 5-MeODMT o n s p o n t a n e o u s a c t i v i t y of mice.  locomotor  Mice were injected with 5-MeODMT at time 0 and locomotor 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 minute intervals thereafter. Administration o f v e h i c l e a l o n e ( c o n t r o l ) i s n o t s h o w n : maximum d e v i a t i o n from normal a c t i v i t y l e v e l o b s e r v e d was 22%. Values a r e m e a n s , N=3; SEMs a r e n o t d r a w n b u t w e r e a l l l e s s t h a n 5%.  44  f.  Gross  behavioral  Changes  i n behavior  with  the effects  dose  o f 0.31 mg/kg  hyperactivity period  (1978) the  sudden  noses. fine  body  At s t i l l  an e x t r e m e l y a n d showed This  were  Jerkiness,  1978),  were  also  i n j e c t i o n , a l l of these t o be r e d u c e d  levels  were  Effect  slowly  were  head  a series  i n frequency  by a  a n d Hedden  elicited  when  ( 2 . 5 a n d 12.5 posture  with  multiple  was p e r i o d i c a l l y a n d  increased,  a n d pawed  t o an  r i g i d i t y and  i n mice After  their  b u t t h e number o f compared  twitching,  behavioral  followed  of  crouched  observed.  of  doses  was r e d u c e d  greatly  3. A  the animal l a y  flattened  behavior  the animals  activity  period  As Shah  higher  c h a r a c t e r i s t i c a l l y produced  Hedden,  began  g.  when  locomotor  animal.  trembling,  post  responses  fright  movements.  movements  untreated  and  DMT,  of the hindlegs  Gross  which  posture.  adopted  interrupted  during  well  i n Figure  10 m i n u t e s ,  a flattened  was d i s t u r b e d .  backwards  briefly  i n an i n i t i a l  60 m i n u t e s  for  t h e mice  extension  presented  l a s t i n g approximately  reported  mg/kg),  b y 5-MeODMT c o r r e l a t e  activity  resulted  displaying  animal  o f 5-MeODMT  induced  on motor  of roughly  quietly,  effects  b y DMT  (Shah  15-20 m i n u t e s  effects  and s e v e r i t y .  gradually Normal  activity  regained.  of c o - i n j e c t i o n s  o f 5-MeODMT a n d J u s t i c i a  pectoralis  extracts  A in  dose  activity  extracts  o f 5-MeODMT w h i c h level  was s e l e c t e d  forsynergistic  would  produce  t o examine  effects.  a marked  elevation  the J . pectoralis  Injections  o f 5 mg/kg  5-  45  MeODMT, c o m b i n e d extracts, results  were  each  level,  there  extracts  i n Table  caused  5-MeODMT  slight  From  this  induced  pectoralis  pharmacological tryptamine  doses  monoamine  t o occur  indicate for  that  oxidase  1971),  plays  ability  results  o f 5-MeODMT w e r e 5-MeODMT, e i t h e r Just i ci a  t h e former  there  a role  observed alone,  i t i s concluded  ori n  that the  to-that  obvious  i s evidence  (Ahlborg, either  enzyme  important  system  i n the behavior  1979)  enzyme  metabolites  Plant  activity  inhibit the  the existing  t o suggest  1968).  behavioral  (Brattsten,  Although  to inactive  which  ejb a _ l , 1 9 7 6 ; H o l m s t e d t  oxidases  i s t h e most  a  ofthe  c o n t a i n compounds  t h e most  plants.  the physiological  obtained  with the  extracts.  i s comparable  function  i n higher  to inhibit  potentiate  i n agreement  (Bhattacharya  converting tryptamines  Smith, also  that  The o b s e r v a t i o n s  b y 5-MeODMT. C o n s t i t u e n t s w h i c h  a l , 1980) a n d m i x e d  known  in activity  do n o t c o n t a i n any c o n s t i t u e n t w i t h  responsible f o r modulating  enzyme,  that  of any o f t h e J u s t i c i a  were  h a l l u c i n o g e n s . N o r do t h e y  induced  the fact  hyperactivity.  of experiments  activity  changes  r e c o r d e d . The  reductions  any of t h e three  extracts  Just ic i a  A l l of the p r e v i o u s l y mentioned  of high  series  levels  effect  a d m i n i s t r a t i o n o f 5 mg/kg with  of the three  I I I . Despite  behavioral responses  effects  combination  et  of each  and the a c t i v i t y  measurements.  behavioral  are  2 5 0 mg/kg  was n o s i g n i f i c a n t  upon  locomotor  J.  extract  the gross  after  made  are presented  alone,  of  with  that  are  data system  (Grahame-  the latter  c o n s t i t u e n t s with the could  possibly  o f t r y p t a m i n e s . The  experiments  do n o t s u p p o r t  46  Per c e n t Normal t Spontaneous Locomotor A c t i v i t y (Minutes Post I n j e c t i o n )  Sample  Injected  5  5 mg/kg 5-MeODMT + 2 5 0 mg/kg aqueous e x t r a c t  5 mg/kg 5-MeODMT + 2 5 0 mg/kg e t h y l a c e t a t e e x t .  5 mg/kg 5-MeODMT + 2 5 0 mg/kg e t h y l e t h e r e x t .  5 mg/kg 5-MeODMT  100  Table  M1 2 5 % Tween  8 0 +•  1 0  15  20  25  30  176  204  204  1 28  1 08  105  155  194  204  126  101  95  1 64  198  215  1 34  1 18  1 04  1 70  214  217  121  1 09  1 08  92  104  100  95  107  101  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 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 on s p o n t a n e o u s motor a c t i v i t y of m i c e .  t Normal 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 was e s t a b l i s h e d f o r e a c h mouse by m e a s u r i n g a c t i v i t y f o r 10 m i n u t e s p r i o r t o i n j e c t i o n . +• C o n t r o l .  47  this  hypothesis  life  of  duration  h.  J.  pectoralis.  of  three J . J.  pectoralis  pectoralis  Dimethyltryptamine known t o a n t a g o n i z e (serotonin)  activity, central  the  action  pectoralis  (Glennon  could exert  directly  on the  them w i t h t r y p t a m i n e s , for  serotonin  testing  this  A strip a rat  of  of  these  receptors  or  is  as that  effect  by  J.  either  by c o m p e t i n g stomach  afforded a relatively  by c o n t r a c t i n g l e n g t h of  (Vane,  such a r a t  with s e r o t o n i n ,  (corresponding  The m u s c l e r e l a x e s serotonin. This  from t h e  low c o n c e n t r a t i o n s  treatment  of  agents  was c o n s i d e r e d . The r a t  minutes  removal  This  affinity  behavioral  1 ng/ml 5 - h y d r o x y t r y p t a m i n e  length).  1959).  The p o s s i b i l i t y  smooth m u s c l e p r e p a r e d  the  minimum l e n g t h  are  hallucinogenic  receptor  exposed to of  affected  for  strip  easy  means  hypothesis.  hydroxytryptamine of  with  1980).  some s u b t l e  receptors  the  5-hydroxytryptamine  action  stomach r e s p o n d s t o  a tracing  of  serotonin  the  derivatives  ( B a r l o w and K h a n ,  et_ a l ,  in  c h a n g e s was  tryptamine  serotonin  t h e mechanism of  half  on smooth m u s c l e  correlates  suggesting that  to  extracts  on smooth m u s c l e  hallucinogens  of  the  extracts,  and o t h e r  pharmacological a c t i v i t y  of  in  t h e m a g n i t u d e nor  5-MeODMT i n d u c e d b e h a v i o r a l  the  Effeet  assay  increase  responses observed. Neither  of  of  acting  Any  5-MeODMT would be e x p e c t e d t o be m a n i f e s t e d  behavioral  by any  for  1957).  of  region  5-  Figure  4a (RSS)  (serotonin).  Within  the muscle r e a c h e s  completely is  shows  stomach s t r i p  t o a 2% r e d u c t i o n  response  fundic  within quite  in  two  its  total  two m i n u t e s reproducible  of and  48  Figure  4 - E f f e c t of (50 y g / m l )  (a) s e r o t o n i n (ing/ml) on s m o o t h m u s c l e .  and  (b)  betaine  Increase in 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 muscle. I n t e r v a l of perfusion of compound is delimited a r r o w s : c h a r t s p e e d = 6 mm/min: a m p l i f i c a t i o n = 16X.  49  the degree  of  contraction  Muscle p r e p a r a t i o n s hydroxytryptamine extracts  at  ether  extract  observed.  In  completely  however,  contrast,  for  specific  ethyl the  fractions  RSS  to  the  light  activity  i n an a t t e m p t  to  Aqueous  2a.  It  retained  of  was e x p o s e d t o relaxation alone  the  was  elicited  a concentration  observed  in  antagonism  p r o d u c e d no e f f e c t  the  on t h e  nature  the these  1  and  extracts  impractical.  on t h e  a  of  ether  The  response  of  the  constituents  smooth m u s c l e p r e p a r a t i o n s  ether  isolate  fractions  of  of  J.  responsible may  the  shed  pectoralis.  were f r a c t i o n a t e d  and i d e n t i f y  fraction  only  its  was  fractionated  was o b s e r v e d t h a t  ammonium compound, of  5-  The further  a c t i v e compounds.  fraction  The aqueous  because  pectoralis  5-hydroxytryptamine.  aqueous and d i e t h y l  was  at  on t h e p h a r m a c o l o g i c a l a c t i v i t y  section  the  r e n d e r e d an e x a m i n a t i o n of  fraction  A knowledge for  contraction  5-hydroxytryptamine  acetate  RSS  a similar  The s p o n t a n e o u s a c t i v i t i e s  aqueous  J.  and p r o d u c e d a marked  the aqueous e x t r a c t  s t r o n g and l o n g l a s t i n g mg/ml.  three  eliminated  induced c o n t r a c t i o n  alone,  the  5-  of .1 mg/ml.  t h e m u s c l e . When t h e  extract  1 ng/ml  c o m b i n e d w i t h one of  hydroxytryptamine of  i n a d o s e - d e p e n d e n t manner.  were p e r f u s e d w i t h  a concentration  The e t h e r  relaxation  varies  by t h o s e betaine,  reaction  as d e s c r i b e d  contraction  fractions  containing  w h i c h had a l r e a d y  with reagents  inducing  for  the  in  activity  quaternary  been  identified  alkaloid  detection.  50  A  commercial  assayed  concentrations  lower  at  than  TLC  2.4%.  this,  spot  extract of  column  activity  was  Crystalline  the  to  for  the  was  low  muscle  was  as  4b).  10  at  but  of  present  the  and  not  the  comparison  betaine,  induced  presence  were  levels  in  by  pure  contraction to  at  slight  estimated,  amounts  relatively  Contractions  were  betaine  was  and  Mg/ml,  observed  of  strong  a  observed  (Figure  attributable  the  observed compounds RSS,  The  the 5a  two  fraction.  as  measured  on  to  caused  RSS  b).  Smooth be  The  compounds  using  a  muscle  obtained  compound, a  was  by  of  to  be  the  this  marked  the  relaxing  from at  two  these  to  fractions. fractions.  the  in  the  from  the data  diethyl  When  between  length  i t s resting  spectral  from  (spasmolytic)  concentrations  compounds  isolated  in  increase  returned  following  f r a c t i o n a t e d by chloroform/methanol  concentrated  rapidly of  fraction  silica  were  each  removal  and  ether  eluate.  Mg/ml,  muscle.  (Figures  the  the  100  following  Mg/ml)  pectoralis  smooth  chromatography as  and  effect  induced  was  fraction  gradient  10  J.  a l i q u o t of  applied  It  (Sigma)  betaine.  Ether  An  RSS.  contractions  with  the  hydrochloride  concentration  of  size  A l l of  amount  the  (100  the  The  extract  aqueous  on  concentrations  reproducible. aqueous  betaine  contraction. This  elicited  of  of  for activity  prolonged high  sample  of  length  medium were ether  obtained  51  Compound (3.70), 90  2  (coumarin).  273  (52),  (3.70).  89  (53),  MS  64  Mp  67-69  m/z  °C.  UV  (rel.int):  (12),  63  (41),  X max  nm  146[M ]  (72),  +  62  (16),  (log  51  e):  310  118  (13),  (100),  50  (12).  MeOH Compound e):  3  325  1720,  (umbelliferone).  (4.22),  1620,  (100  MHz,  (1H,  d,  1575,  (sh), 1420,  acetone-D-6) J  =  8 Hz,  int):  162[M ]  (14),  85  (32),  43  +  On  254  (13),  83  223-225  242  1330, 5  H-5),  (67),  Mp  6.82  134  (14),  IR  1240,  1150,  78  (IH,  (2H,  (74),  v ^  (sh).  : 7.86  105  (35),  d,  m,  J  H-6  nm  cm" :  3200  910,  850.  =  Hz,  97  (38),  Xmax 1  x  12  and  (39), 69  UV  °C.  1  H-8).  (18), 57  95  (32),  (log br,  H-  NMR  H-4),  7.52  MS  (rel  m/z (14),  55  91  (40),  51  (100).  the  obtained,  basis  of  Compounds  hydroxycoumarin  melting  2 and  3  points  were  (toluene/acetone,  with 1/1)  (50%  by  aqueous  the as  respectively  authentic and  and  identified  (umbelliferone),  co-chromatographed  chromatography  the  standards  high  on  spectral coumarin  and  (Figure  2).  silica  gel  performance  acetonitrile),  data 7-  They  liquid  confirming  their  identity. i.  Analysis A  in  of  possible  Virola  snuff  aromatic  constituents  explanation i s that  (Prance,  1972;  Schultes  fragrant  nature  is  "Jamaica  garden  balsam"  for  the  i n c l u s i o n of  i t contributes  a  and  1968;  indicated  Holmstedt, by  one  (Schultes  of  and  J.  desirable  aroma  Seitz,  i t s common Holmstedt,  pectoralis  1967).  names, 1968).  To  A  Figure  5 - E f f e c t o f ( a ) c o u m a r i n ( 1 0 Mg/ml) a n d (b) u m b e l 1 i f e r o n e (10 u g / m l ) on s m o o t h  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 s p e e d = 6 mm/min: a m p l i f i c a t i o n = 16X.  muscle. by a r r o w s :  chart  53  establish for J.  its  the  nature  aroma,  pectoralis  hours. each  of  s a m p l e s of  identified, pectoralis  volatile  only by  appears  to  leaves  responsible and stems  steam d i s t i l l a t i o n  was a n a l y z e d by GC/MS.  a single  its  constituents  f r e s h and d r i e d  were s u b j e c t e d  The d i s t i l l a t e  sample,  the  constituent  In  for  of  2  the  case  of  was p r e s e n t .  This  was  mass s p e c t r u m , as c o u m a r i n . The aroma o f to  be a r e s u l t  of  the  single  c o u m a r i n s of  J.  pectoralis  J.  constituent,  coumarin. j.  Quanti f i c a t i o n The e t h e r  of  extract  contained,  as p r i m a r y  c o u m a r i n and u m b e l l i f e r o n e .  To p r o v i d e  i n f o r m a t i o n on t h e  of  metabolites, HPLC.  sample of in  various  Samples  and f l o w e r s  presence  of  s a m p l e s of  young,  green  were e x t r a c t e d  leaves  these J.  constituents,  quantitative  two  important  pectoralis  leaves,  mature,  exhaustively  were a n a l y z e d by pigmented  with methanol. A  collected  from t h e p l a n t  while  P e r u was a l s o a n a l y s e d .  The m e t h a n o l i c  extracts  evaporated  ir\ v a c u o and p a r t i t i o n e d  dichloromethane.  The l a t t e r  i n m e t h a n o l and a n a l y s e d using  by HPLC.  50% aqueous m e t h a n o l  a b s o r b a n c e was m o n i t o r e d a t The l e a f constituents,  extracts  between  fraction  (flow  and u m b e l l i f e r o n e  and dissolved  separated and  the  250 nm.  were q u a n t i f i e d by c o m p a r i s o n w i t h peak  Company)  growing  were  water  = 1ml/min)  c o n t a i n e d a s major  authentic  was  was e v a p o r a t e d ,  c o u m a r i n and u m b e l l i f e r o n e  c h r o m a t o g r a m s of  it  The sample was rate  leaves  s a m p l e s of  UV a b s o r b i n g (Figure  heights  coumarin  6).  Samples  of (J.T.  (K and K L a b o r a t o r i e s ,  Baker  Ltd.).  The  54  Figure  6 - HPLC c h r o m a t o g r a m of (methanolic  extract)  the  of  organic  Just ic ia  fraction  pectorali s .  Flow r a t e I m l / m i n , i s o c r a t i c , 50% MeOH, detection at 254 nm, c h a r t speed= 0.5 c m / m i n . Compound w i t h r e t e n t i o n t i m e of 5.91 i s u m b e l l i f e r o n e ; t h a t w i t h r e t e n t i o n time of 8.62 i s c o u m a r i n .  55  results  are  presented  and u m b e l l i f e r o n e Justicia  is  several  limited  reach higher  the  major  of  only  orders  are of  species  constituents  al,  one of  pectoralis of  chemically,  1980; Munakata  et  the  six  (Ghosal al,  are  21 l i g n a n s  moiety  acid  fraction, spectral 'H-NMR,  spray  reagent  have  the  behavior  of  sample of the  To d e t e r m i n e  chloroform/acetone  in J .  of  the in  It  1980).  gel  and HPLC:  diethyl isolated  to  lignans the (1963)  ether and  those  °C),  obtained  Furthermore,  chromatographic  Retention rate  contain"  (132-135  (Sigma).  developed  flow  but  three J u s t i c ia  the was  as  al,  All  species whether  melting point  /3-sitosterol  e l u t e d w i t h 25% aqueous m e t h a n o l ,  and  1970; Okigawa  pectoralis,  unknown compound on two  (95/5)  plant  lignans  d e s c r i b e d by B e r o z a  reagent.  ( T L C : R f = 0 . 4 5 on S i l i c a  the  appear,  previously  and P o w e l l ,  present  Its  they  the  1979; G h o s a l e_t  UV and mass s p e c t r u m were i d e n t i c a l  an a u t h e n t i c  systems  with t h i s  were o b t a i n e d .  for  in  from J u s t i c i a  were p r e s e n t  one compound,  reacted data  but  than  known t o c o n t a i n  was u s e d t o examine TLC s e p a r a t i o n s Only  that  and B a n e r j e e ,  moiety.  of  at  1965; Ohta and M u n a k a t a ,  the m e t h y l e n e d i o x y p h e n y l  species  lignans  Just ic ia  identified  fractions.  present  extract,  for  the  chromotrophic  this  coumarin  leaves,  1982; O l a n i y i  this  of  of  magnitude h i g h e r  1970; O l a n i y i ,  containing  importance  they  i n more mature  J.  seven  been a n a l y s e d  The  s a m p l i n g , to b e " s e q u e s t e r e d  levels  Examination Of  et  Not  UV a b s o r b i n g compounds of  from t h i s  k.  IV.  secondary metabolites  evident.  concentrations other  as  in Table  with time  = 6.10 min  = 1 ml/min.)  was  56  Concentration  Sample  Per  Coumar i n  Analyzed  flowers  young  in  Cent Dry Weight  Umbelli ferone  N.D.  N . D . t-  leaves  mature  0.42(0.04)  0.30(0.04)  1 . 18(0.08)  0.58(0.05)  0.93(0.05)  0.44(0.04)  leaves  leaves(Peru)  T  Table t  IV  - L e v e l s of c o u m a r i n and u m b e l l i f e r o n e i n d i f f e r e n t s a m p l e s of J u s t i c i a p e c t o r a l i s .  F i g u r e s a r e the a v e r a g e of t h r e e d e v i a t i o n o f mean i n b r a c k e t s .  $ N.D.  = Not  Detectable  determinations;  standard  t  57  identical  to  that  of  an a u t h e n t i c  sample of  0-sitosterol  (S i gma). The d i e t h y l  ether  and e t h y l  pectoralis  were examined f o r  diphyllin,  lignans  Samples  of  known from s e v e r a l  of  Dr.  (1/1)  G.H.  0.57 and 0 . 7 9 ,  as d e v e l o p e r .  Sheriha,  and r e a c t e d  evidence  the p r e s e n c e  for  pectoralis  extracts  The c h e m i s t r y different that  was of  from t h a t  This difference  of  the  Just ic ia  B and  species.  gel G using and j u s t i c i d i n  University)  had Rf  either  compound i n  blue  reagent. the  B  values  Both d i s p l a y e d a s t r o n g  with chromotrophic a c i d of  J.  justicidin  Diphyllin  Fateh  of  No  J.  obtained.  J.  of  of  other  p e c t o r a l i s appears  any of  the  other  have been e x a m i n e d . The a p p a r e n t  note. all  El  respectively.  fluorescence  fractions  the p r e s e n c e  were c h r o m a t o g r a p h e d on S i l i c a  toluene/acetone (gifts  acetate  may r e f l e c t  species previously  t o be  species  absence  s t u d i e d are  in  of  phylogenetic  quite this  genus  lignans  is  distance  native  of  since  to Asia  or  Africa. 1.  Screening  for  Although J . admixture,  there  indicating  that  of  biological  pectoralis is it  and i s  pulmonary also  infections  nasal  is  known as a  uses.  known t o the  in  (Maxwell,N.,  snuff  ethnobotanical In  pers.  states  Puinave Indians a d e c o c t i o n to  (Schultes, the  information  the Colombian  the  f o r m of  and pneumonia  t o be e f f e c t i v e  cavity  activities  best  has m u l t i p l e  u s e d by them i n  been s a i d  the  is  considerable  Vaupes and Amazonas, i t  yakayu  of  other  1978).  treatment  of  It  as  treat has  infections  c o m m . ) . An h e r b a r i u m  58  specimen c o l l e c t e d herbarium l a b e l Colombia rickets  is  stating  (Schultes,  treatment  chest  a variety against the  of  the  of  dermatophytic  the  the  plant  ether  growth  extract,  upon t h e  Caribbean  relieve  with  of  or  J.  wounds  for  bacteria  however,  g r o w t h of  coli,  cerevisiae  upon  against  were  the and  tested  aureus, yeasts,  the  M. g y p s e u m , M. extracts  tested.  The  inhibitory  effect  diethyl  effect  fungi.  separately  was o b s e r v e d a t  fulvum  had any  a concentration  C o u m a r i n and u m b e l l i f e r o n e were t e s t e d  have  the  activity  Staphylococcus  the d e r m a t o p h y t i c  activity  in  dependent  fractions  or y e a s t s  i n h i b i t e d at  to  1977).  inhibitory  the  an  was examined by  exerted a strong  each of  No i n h i b i t o r y  is  Escherishia  None o f  1976),  It  poultice  pectoralis  Microsporum c a n i s ,  the  (Wong,  1977).  been r e p o r t e d  (Morton,  use of  medicinal  (Morton,  coughs  has a l s o  and S a c c h a r o m y c e s  g r o w t h was c o m p l e t e l y  fungi.  Meta,  known  and as a wound  bacterium,  bacterium,  fungi,  well  This hypothesis  and T r i c h o p h y t o n g a l l i n a e . upon t h e  used in  children afflicted  effects  gram p o s i t i v e  albicans  a c c o m p a n i e d by an  widely  m i c r o o r g a n i s m s . The t h r e e  gram n e g a t i v e  Candida  It  infections  activity.  extracts  is  a relatively  1945)  1962).  possible that of  is  treat  to  (Roig y Mesa,  antimicrobial testing  tea  and a p h r o d i s i a c  is  it  A m e r i c a and t h e  ( S t e h l e and S t e h l e , sudorific  Triana  1978).  u s e d as a p e c t o r a l  expectorant  to  pectorali s is  in Central  It  that  as a d e c o c t i o n  Just ic ia plant  by J o s e J .  Their of  on  1 Mg/ml. these  d o s e s as h i g h  as  59  50  Mg/ml,  diethyl major  i n d i c a t i n g that  ether  extract  is  the  activity  not a t t r i b u t a b l e  of  to e i t h e r  their  three  ability  extracts to  of  inhibit  J.  the  c y t o m e g a l o v i r u s and S i n d b i s chapter  III).  extracts  No e f f e c t s  at  pectoralis replication  virus  were a l s o  these  of  tested  were o b s e r v e d from any 10  for  murine  (methods d e s c r i b e d  c o n c e n t r a t i o n s as h i g h as  evidence,  studies,  that  from e i t h e r  the  s a m p l e s of  chemical constituents activity,  was  detectible  of  in  the  Mg/ml.  found.  effects  t h e c h e m i c a l or J.  capable  of  Nor d i d J .  upon t h e  pectoralis eliciting pectoralis  behavioral  the  and i d e n t i f i e d : Betaine (Guggenheim, methyl  betaine,  is  widely  1951).  hallucinogenic extracts  groups for  methionine v i a transferase  the  report  examined the  only  r e s p o n s e s of  remethylation  is  autonomic nervous  of  isolated  and a n i m a l s  of.homocysteine  1 9 6 5 ) . Few  to  methyl  s t u d i e s of  have been p u b l i s h e d c o n c e r n i n g  was t h a t  effects  5-  as a s o u r c e of  enzyme b e t a i n e - h o m o c y s t e i n e  nature  mice t o  were  in both p l a n t s  known f u n c t i o n  ( S h a p i r o and S c h l e n k ,  pharmacological first  the  have any  c o u m a r i n and u m b e l l i f e r o n e .  distributed  Its  biological  analysed possessed  MeODMT. T h r e e compounds w i t h b i o l o g i c a l a c t i v i t y  the  of  DISCUSSION No  The  the  constituents. The  4.  antifungal  of  Hunt and Renshaw  a series  of  derivatives  s y s t e m . Most d e r i v a t i v e s  t o have a m u s c a r i n i c e f f e c t ;  i.e.,  (1929)  to  of  a  betaine. who  betaine  on  were o b s e r v e d  reduce b l o o d p r e s s u r e  60  and h e a r t betaine,  rate. is  brief  "practically A fraction  Their  and r e s t r i c t e d  without  of  consideration  action  an e x t r a c t  of  containing betaine  been r e p o r t e d  to  al,  1965).  induced hypnosis  in  anti-inflammatory  study  on p u r e  the  rats  betaine.  Pluchea  as a major  extract  in  To what e x t e n t  in  other  More r e c e n t l y ,  has been r e p o r t e d  that  doses,  it  electric  is  capable  of  agent  shock as w e l l as c h e m i c a l  of  They a r e  plants  (Karrer,  spasmolytic, (Patyra,  al,  like  in b a c t e r i a ,  as w e l l a s  1980)  and t h i s  spasmolytic  and A d e s i n a ,  action,  not is  betaine, fungi  appears  known.  a  non-  At  are  high  distributed  and a b r o a d  relaxing,  range  or  for  coumarin  (Achterrath-  t o be a f e a t u r e  (Lee  1983a and b ) .  which a f f e c t s  of  i n d u c e d by  umbelliferone  common t o many c o u m a r i n d e r i v a t i v e s  1971; O j e w o l e  is  has been r e p o r t e d p r e v i o u s l y  1963a and b)  in  convulsants.  1 9 5 8 ) . A smooth m u s c l e  action  Tuckermann e t is  present  properties  betaine  blocking convulsions  is  present  ( F r e e d e_t §_1, 1 9 7 9 ) .  C o u m a r i n and u m b e l l i f e r o n e , widely.  The smooth  the  to betaine  anti-convulsive  et  barbiturate  1966).  can be a t t r i b u t e d  specific  smooth m u s c l e  the c r u d e e x t r a c t  the  has  and p o s s e s s e d  the c r u d e e x t r a c t it  system."  1968; P r a s a d  1965)  presented  is  constituent,  potentiated  of  it  lanceolata  isolated  ( P r a s a d e_t a_l,  results  that  autonomic nervous  inducing effect  w i t h the  compound,  statement  ( P r a s a d e_t a_l,  activity  muscle c o n t r a c t i o n  parent  1967; D a s g u p t a et_ a_l,  Furthermore,  c l o s e agreement  the  induce c o n t r a c t i o n s  (Dasgupta,  the  plant,  (Compositae),  preparations  to  on t h e the  of  and  This  Fujiwara,  general  many smooth m u s c l e s ,  that  61  apparently pressure  results  in a lowering  in animals  treated  ( O j e w o l e and A d e s i n a , its  derivatives,  temperature the  toxic  (Ito  et  B o t h of of  these  1951; K i t a g a w a , actions  coumarin  a volatile  1979).  aroma of  J.  is  overall  sedative  small  effect  in  of  animals,  this it  50 mg/kg. Whereas  localized  effects  the  At d o s e s  approaching  and h y p n o t i c  activity  Kreitmair,  be r e l a t e d  to  1949). the  ability  respiration  Coumarin  acting  is  also  effective  on macrophages  to  1979).  and s w e e t l y  compound and  aromatic  as an a r o m a t i c  sole  ingredient  of  these  J.  pectoralis  respect.  produces it  nasal  varied  activity  is  regions  pharmacologically across  1975).  body  (Koh and W i l l o u g h b y ,  Finally,  additive  responsible  of  active  has  (Kirk for  and  the  be  snuff  is  for  activities  not  clear.  coumarin  and a n a l g e s i a  reasonable during  the  to expect  snuff  taking,  at that  it  head may be e x p o s e d  levels  as a r e s u l t analgesia  induced at  or  lower  of  is  to  The  is  Administered parenterally  mucosa. L o c a l  could possibly  biological  observed  sedation  not  h i g h d o s e s may be a c h i e v e d that  scopoletin  pectoralis.  and h y p n o t i c  interesting  1958).  mitochondrial  agent,  the  The c o n t r i b u t i o n the  reduce  1956a and b;  applications It  to  sedative  e_t a l ,  phagocytosis  many c o m m e r c i a l Othmer,  ability  inhibit  1956a; P a i  is  r a t e and b l o o d  coumarin,  could possibly  as an a n t i - i n f l a m m a t o r y stimulate  the  c o u m a r i n has  compounds t o  (Kitagawa,  w i t h the  ( K i t a g a w a and I w a k i ,  these  heart  1983a and 1 9 8 3 b ) . C o u m a r i n , and some of  possess  level,  al,  of  to  doses  of  such possible  to  its  absorption  sedative/hypnotic doses  than  if  62  administered The  parenterally.  ability  (Ojewole  and  of  Adesina,  admininistration in  blood  effect  scopoletin  is often  pressure  is a  1983b)  one  psychological  changes.  umbelliferone  could  one  of  the  stressful  is also  1956).  or  It  whether  The  lower  reduce of  accompanied  (Szara,  direct  to  aspects  Tryptamine  transient  i s not  known  that  the  increases  whether  i t i s mediated  pressure  of  pressure  interest.  by  possibility blood  blood  by  coumarin  and  or  thereby  tryptamines,  this  reduce  is  intriguing. In  conclusion,  pectoralis its  of  effects  to  of  umbelliferone  the  snuff  has  support  plant to  was  as  a  snuff.  contribute been  raised.  the  belief  obtained.  affect  hallucinogenic  i t i s combined  and  to  m„ice a p p e a r  5-meODMT, t h e  which  coumarin  evidence  hallucinogenic  administration  action with  is a  no  The to  Nor  directly  tryptamine  of  possibility the  that  J.  does the  Vi rola, that  pharmacological  63  LITERATURE Chapter  CITED 11  A c h t e r r a t h - T u c k e r m a n n , U., K u n d e , R., F l a s k a m p , E . , I s a a c , 0. a n d T h i e m a r , K. ( 1 9 8 0 ) P h a r m a c o l o g i c i n v e s t i g a t i o n s o f c h a m o m i l e . V . I n v e s t i g a t i o n s on t h e s p a s m o l y t i c e f f e c t s o f c o m p o u n d s o f c h a m o m i l e a n d k a m i l l o s a n on t h e i s o l a t e d g u i n e a p i g i l e u m . P l a n t a Med. 39, 38-50. 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The b i s - i s o q u i n o l i n e a l k a l o i d ,  related  the  discovery  compounds from s p e c i e s  alkaloids,  s u c h as  and c a r d i a c  strychnine,  glycosides  ( L e w i s and E l v i n - L e w i s , diterpene the  alkaloids  of  1982)  the  constituents  while others  have  (batrachotoxins shown t h a t  of  arrow  and o t h e r s ) .  to possess antitumour  the  well  best  known a r e  the  the (Bisset,  alkaloids),  pumilotoxins  histrionicotoxins  et  poison  phorbol  frogs  ester,  1971).  Some of  p o i s o n s have  proven  valuable  and c a r d i a c tools  in  research  and Perdue  poisons are  five  than h i g h e r  glycosides)  (1976)  s p e c i e s as arrow  have  t i m e s more  plants  random. The use of V i r o l a  indole  a_l,  Spjut  activity  and  known e x a m p l e s  used in China  and  active  (Loganiaceae)  of members of  (Sakata  been a p p l i e d as  arrow  tubocurarine,  and t h e  (tubocurarine  s o u r c e s of  pharmacologically  from C o l o m b i a n a r r o w  f r o m Hura c r e p i t a n s  as m e d i c i n a l agents  Less  alkaloids)  huratoxin, active  i n many  from S t r y c h n o s  (triterpene  (Daly,  arrow  the Menispermaceae,  Aconitum s p .  alkaloids)  the D e n d r o b a t i d a e  of  some of  1977).  (cis-decahydroquinoline  of  are  batrachotoxins  (spiropiperidine  potent  from a v a r i e t y  A p o c y n a c e a e and M o r a c e a e ,  1981),  of  lead,  of  poisons  is  likely  selected  at  70  interesting, but  not  only  e t h n o l o g i c a l l y and  pharmacologically.  Reports  Vi rola  theidora  and  by  Yanomamo  (Waika)  accompanied  the  Brazil was  were  applied  to  amongst  cite  describe the  Waika  distinguished  Venezuela  by  observations an  a  bark  This  source  which  preparation  of  dart-tip  arrow  (Bauer,  1965).  Although  i t has  may  a  mixture  preparation 1966), been  the  reported  carefully  an  of  are  by  almost  related the  of  bark  followed  Virola a  well  the  the Rio  heated  identical  to Rio  Vi rola  the  as  as  is  have the  (Biocca, sole  Tototobi. over  a  fire  and  repeatedly  over  the  fire.  procedure  by  living  the to  in  the  more  than  tribes some  tribes  e t al,  Biocca has  also  Holmstedt  (1968)  of  arrow of  arrow  by  flows tips,  has  tribe  tips  Vi rola  which  (1970)  300  the  and  resin  Sanama  in  (Schultes  bark  Prance  poison  the  1965;  bamboo  the  and  ingredient  S t r i p s of  as  used  that  preparation  material  readily  Waika  and  snuff  and  arrow  been  resin  Schultes  of  same  (i960)  an  reported  the  1970).  manner  Waikas),  Becher  by  Vi rola  poison  the  established practice  is applied  heating  and  sp.  Prance,  describe  theiodora the  as  1968;  Waikas  from  of  arrow  poisons  of  northern  Menispermaceae to  been  and  Schultes  plant  the  reported  curare  their  use  Holmstedt,  the  of  of  tree  l i a n a s of also  poison. and  resin  hallucinogenic  that  (1931)  Loganiaceae  use  are  of  arrow  an  bark  southern  use  the  of  of  group.  from  use  as  Salathe  the  the  elongata  a l t e r n a t e l y as  Holmstedt(1971) first  Virola  on  anthropologically,  observed  (closely  kilometers  north  Tototobi. theiodora  and  V.  elongata  have  been  considered  to  71  be  synonomous  (Rodrigues,  by  authorities  1980;  to  d i s t i n g u i s h V.  of  the  ease  (Schultes pointed same  and  that  the  V.  growing  used  in  Vi rola to  which  their  have  variation  been  between  methyltryptamine,  (Agurell  et  al,  1984).  These,  number  of  elicit  at  Gessner  their  of  been  snuff  Both  to  doses et  were  usually  Holmstedt  other  indole  psychotomimetic  Hedden,  are  the  (Gessner 1968;  1978).  the et  It  are  that  al,  was  with  respect V.  considerable N-  present  known  animals,  to  al, have  foremost  their  1956).  et  and  behavioral  Page,  1962;  has  been  active  responsible  suggested  constituents for  responses  Ho  of  e_t a _ l , et  that of  to  N,N-  Gessner 1971;  a  among  ability  5-methoxy-N,N-dimethyltryptamine normal  V. are  and  McKenna  a l k a l o i d s , are  (Szara,  and  they  alkaloids  and  the  5-methoxy-N,N-  1980;  activity  in  has  poison.  alkaloids,  major  in  firme  theiodora  and  basis  comm.)  found  arrow  there  indole  Grahame-Smith,  biologically that  i n man  and  disrupt  al,  V.  the  pers.  and  prefers  field  extensively  Although  the  the  tierra  and  studied  on  generally  N,N-dimethyltryptamine  hallucinations  and  poison  have  1969;  and  (G.T.,  on  group  Schultes  in  watercourses  physiological activities  shown  low  the  samples,  dimethyltryptamine been  to  examined.  dimethyltryptamine  is  Prance  this  species  recognized  occurring  of  1937).  separate  alkaloidal constituents.  elongata  which  a be  preparation  species  taxonomy  v a r i e t i e s are  theiodora close  the  as  1968).  two  the  Wodehouse,  i t can  Holmstedt,  locality,  both  and  theiodora  with  out  elongata  Smith  in  al,  incapacitating  the  rodents 1961; 1970;  indole  Vi rola  have  Shah  alkaloids  arrow wounded  72  animal.  Yanomamo d a r t s  to  contain  in  high  These to  a  single  the In  the  (8%  attribute  presence  of  been  alkaloid,  concentration  workers  have  this  present  by  the  examined  weight)  an  experimental  Vi rola  administered  approach, if  the  observed to  and  tryptamines  severity  to  explain  has  been  quantified.  determine  i f toxic  caused the  (Galeffi  e f f e c t i v e n e s s of  problem  behavior  found  e_t a l , the  1983).  arrow  poison  indole constituent.  study,  were  and  5-methoxy-N,N-dimethyltryptamine  ethnopharmacological elongata  chemically  to  of  taken. mice  I t was  and  the  the  intended,  as  an  this of  effects by  arrow  on  this  present  d i s r u p t i o n of  bark  to  Extracts  c o n s t i t u e n t s were  behavioral  use  approach  or  sufficient poison.  73  PART A .  ISOLATION AND IDENTIFICATION OF THE MAJOR NON-POLAR CONSTITUENTS  1.  OF V i r o l a  elongata  BARK  INTRODUCTION D u r i n g the c o u r s e  of  pharmacological effects was e n c o u n t e r e d biological of  the d i f f i c u l t y  in  large  of  numbers of  section  isolate  the  nature  was  difficulty  potency  of  locomotor  activity  of  the  sample a  being  better  t h e major  compounds  biological activity.  isola'tion  the  function  decided that  and p u r i f y  the  T h i s was a  in q u a n t i f y i n g  it  some  relative  fractions.  t o measure t h e i r  describes  extracts,  the  mice and t h e  As a r e s u l t ,  and t h e n  Vi rola  certain  involved  a p p r o a c h would be t o first  of  studies dealing with  in determining  effects  fractionated.  the  and i d e n t i f i c a t i o n  This of  these  compounds.  2.  EXPERIMENTAL  a.  E x t r a c t i o n of The b a r k  on t h e Voucher  Plant  Material  was c o l l e c t e d  near  Rio Ampiyacu, a Peruvian specimens  herbarium  in  (D.  Iquitos,  the  village  tributary  McKenna N o .  59)  of  of the  out  The d r i e d b a r k  (1.3  room t e m p e r a t u r e  Amazon.  San M a r c o s H e r b a r i u m i n L i m a ,  by D r . kg)  W.A.  Rodrigues,  ether.  UNAP  the  identification  INPA, M a n a u s ,  was m i l l e d and e x t r a c t e d  with d i e t h y l  Nuevo  were d e p o s i t e d a t  C h i c a g o F i e l d Museum and t h e UBC h e r b a r i u m . The was c a r r i e d  Brillo  four  Brazil. times  at  74  b.  Chromatography After  evaporation  chromatography Associates) Silica  i_n v a c u o  the  on a C h r o m a t o t r o n  (Harrison  u t i l i z i n g preparative  gel  PF  2 5  ,  (Merck).  petroleum ether/ethyl 3/3/1)  yielded  purity  for  residue  Repeated  the  spectroscopy.  subjected  They a r e  coated  chromatography  following  to  Research  (4 mm) p l a t e s  ether/acetonitrile  e a c h of  was  with  (12/12/1;  compounds i n  presented  in  with  6/6/1; sufficient  order  of  elut ion.  3.  COMPOUNDS  ISOLATED  0 -sitosterol. data  agree  (425 mg) Mp 133-135 ° C  with data  Chromatography  obtained  on TLC  (silica)  heptane/chloroform/ethanol ether/ethyl of  for  sample  2  (4.97). 3.78 J  UV X c y c l o h e x . ( max 1  H-NMR  (3H,  s,  = 2 H z , H-2,  d,  J  (2H, 239  3  H-6),  = 12 H z , v i n y l ) , d,  J  (12),  l o 3g  e  (12),  sample  (Sigma).  and p e t r o l e u m  (12/12/1)  1a.  ) . 83  was  identical  to  6.43  t,  (1H,  6.77  (2H,  H-6').  212 ( 8 ) ,  235sh ( 4 . 6 4 ) ,  5 3.65  3  (1H,  (23 mg) Mp 73-74 ° C  (4.50),  2  (CDC1 ):  6.32  = 9 Hz, H-2', 224  PMR and MS  that  (Sigma).  (400 MHz)  OCH ),  an a u t h e n t i c  ( 2 5 / 25/1)  3,4',5-trimethoxy-cis-stilbene Et 0).  2  using  ether/acetonitrile  an a u t h e n t i c  (from E t 0 ) ,  J  d, d,  (6H,  s,  = 2 H z , H-4), J  3  6.43  = 9 Hz, H-3',  MS m/z  196 ( 1 1 ) ,  (rel  int)  195 ( 1 3 ) ,  214  2 X OCH ),  = 12 H z , v i n y l ) , J  (from  (2H,  d,  6.52  (1H,  H-5'),  7.21  270[M ] +  181  (100),  (10),  169  75  (10),  165 ( 1 0 ) , 153 ( 1 3 ) , 152 ( 1 9 ) , 149 ( 1 2 ) , 141 ( 9 ) , 135  (9) , 83  127 ( 8 ) , 115 ( 1 5 ) , 104 ( 1 0 ) , 95 ( 1 1 ) , 91  ( 1 1 ) , 81  ( 1 5 ) , 71  ( 1 5 ) , 69 ( 2 2 ) .  3,4',5-trimethoxy-trans-stilbene (from 303  Et 0).  (4.72),  (CDC1 ): 3  4),  UV X ^  2  235sh  149  270[M ] +  x  ( l o g e ) : 335sh  (4.42),  216 ( 4 . 6 6 ) .  1  (4.45),  H-NMR  318  ( 4 0 0 MHz)  6.38 ( I H , d , J = 2 H z , H-  6.90 ( 1 H , d , J = 16 H z , v i n y l ) ,  d, J = 9 Hz,  7.04 ( 1 H , d , J =  7.44 ( 2 H , d , J = 9 H z , H-2', H - 6 ' ) .  (100),  (4.68),  MS m/z  83 ( 9 ) , 76 ( 1 1 ) , 71  3  3.28  1  H-NMR  234sh  0  (4.24),  ( 8 0 MHz) ( C D C 1 ) : 3  C  (from  Et 0). ^  230 ( 4 . 7 8 ) .  2  UV XMeOH ( l o g max  [a]  2  3.9-4.3  4.9-5.25  ( 2 H , m, = C H ) ,  3  r  -10.4 i n 0  5 1.26 ( 3 H , d , J = 6 H z , M e - 3 ) ,  ( 2 H , d , J = 7 H z , C H ) , 3.85 ( 3 H , s , O C H ) ,  X OCH ),  3  3.88 ( 9 H , s , 3  ( I H , m, H - 3 ) , 4 . 5 6 ( 1 H , d , J = 8 H z , 2  2 H z , H - 6 ) , 6.48 ( 1 H , ( r e l int)':  91  ( 2 5 ) , 70 ( 2 3 ) , 69 ( 2 7 ) .  2. ( 2 8 mg) Mp 9 3 - 9 5  271 ( 3 . 2 1 ) ,  CHC1 .  5.7-6.25  H-2),  ( 1 H , m, C H = ) , 6.37 ( 1 H , d , J  d , J = 2 H z , H " 8 ) , 6.58 ( 2 H , s , H-2', H-  6*).  MS m/z  (3),  3 1 2 ( 3 ) , 311 ( 4 ) , 3 0 2 ( 5 ) , 2 1 0 ( 1 0 ) , 2 0 9 ( 7 2 ) , 2 0 8  205  (rel  2 3 9 ( 1 4 ) , 224 ( 1 1 ) , 212 ( 8 ) , 196 ( 9 ) , 195  ( 3 7 ) , 141 ( 7 ) , 135 ( 7 ) , 128 ( 8 ) , 115 ( 1 2 ) , 104 ( 7 ) ,  Eusiderin  =  '  C  0  181 ( 7 ) , 169 ( 7 ) 167 ( 1 3 ) , 165 ( 8 ) , 153 ( 9 ) , 152 ( 1 3 ) ,  (12),  e):  e  d , J = 2 H z , H-2, H - 6 ) , 6.90 ( 2 H ,  16 H z , v i n y l ) ,  (10) ,  h  3  H-5'),  int)  o  1 b . (41 mg) Mp 5 6 - 5 7  6 3.81 ( 9 H , s , 3 X O C H ) ,  6.65 ( 2 H ,  H-3',  l  ( 1 7 ) , 85 ( 1 1 ) ,  386[M ] +  ( 2 2 ) , 372 ( 5 ) , 3 4 4 ( 3 ) ,  343 (100),  ( 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  (7),  137  (14),  ( 1 3 ) , 135  119  ( 1 3 ) , 95  ( 1 3 ) , 91  3.  (3.35),  227  (CDC1 )  6  Me-9'),  2.71  J  J  =  14,  OCH ),  3.83  3  (1H,  m,  6.53  (2H,  (5),  210  (55), 177  5.5  163  dd,  Hz,  (6H,  J J  = =  H-7),  s,  2 X  6.2-6.5  13,  (100),  208  ( 6 ) , 182  ( 1 7 ) , 162 ( 1 4 ) , 148  ( 1 1 ) , 147  (10),  135  ( 2 2 ) , 134  ( 9 ) , 133  (10),  107  ( 1 9 ) , 105  85  ( 1 1 ) , 83  ( 1 2 ) , 81  70  ( 1 1 ) , 69 ( 2 5 ) .  Hz,  3.80  3  ( I H , m,  +  , 195  ( 1 9 ) , 194  ( 5 1 ) , 179  ( 6 ) , 166  ( 7 ) , 153  ( 2 3 ) , 131  ( 7 ) , 165  ( 5 ) , 137 ( 1 1 ) , 125  ( 1 9 ) , 118  =  6Hz,  (1H,  (6H,  dd,  s,  2  X  6.05-6.25 H-6),  ( 2 7 ) , 211 (87),193  ( 3 0 ) , 178  ( 1 1 ) , 151  ( 1 8 ) , 138  J  H-8),  s , H-2,  268  MHz)  3.10  ( r e l i n t ) 402[M ]  (73)  ( 8 ) , 119  H-7),  (2H,  (80  (3H, d,  s, O C H ) ,  6.43  ( l o g e)  H  H-NMR  1.87  4.2-4.5  ( 2 8 ) , 167  149  ( 1 9 ) , 120  (3H,  ( 7 ) , 181  (14) ,  122. ( 7 ) , 121  = 8.0  m/z  ( 1 0 ) , 161  1  ( 1 0 ) , 97  ( 1 4 ) , 79 ( 2 8 ) .  X^|°  in CHC1 .  (1H, H-7'),  ( 4 0 ) , 209  ( 4 ) , 168  o i l . UV  Me-9),  J  OCH3),  MS  ( 1 8 ) , 81  ( 7 ) , 147  ( 1 1 ) , 121  ( 1 1 ) , 103  3  3.78  H-5').  ( 7 ) , 191  0  6Hz,  s , H-3',  ( 1 2 ) , 169  (7),  Colourless  (3H, d,  (1H,  ( 1 7 ) , 83  ( 4 8 ) , 148  ( 8 ) , 123  ( 2 3 ) , 104  [ a ] j p -12.4  (4.04).  H-8'),  192  ( 5 0 ) , 85  ( 1 8 ) , 149  ( 1 8 ) , 132  ( 1 9 ) , 105  (34 mg).  1.22  =  ( 1 0 ) , 150  ( 1 9 ) , 133  ( 1 0 ) , 107  Virolongin  3  ( 6 ) , 151  (39),  (16),  (23),  164  150  ( 1 6 ) , 136 ( 6 ) , 123 ( 9 ) ,  ( 9 ) , 115  ( 9 ) , 109  ( 2 5 ) , 103  ( 1 5 ) , 95  ( 1 2 ) , 93  ( 1 0 ) , 91  (40),  ( 1 3 ) , 79  ( 3 3 ) , 78  ( 1 3 ) , 77  ( 3 4 ) , 71  (18),  77  Epi-sesartemin (found  4. ( 9 7 m g ) . 1 1 4 - 1 1 5  430.1622  (3.40),  235sh  f o r 430.1628  (4.14),  0  C  (from  Et 0).  ^??S  [ a ] 25 +108  2  3  H  (log  H  b y H R - M S ) . UV X  210 ( 4 . 9 2 ) .  C  2  2  I R i>KBr max  3  u  cm 1360,  "  : 2900,  1  2820,  8  * ) : 270  i n CHC1 .  0  0  8  1625, 1585, 1540, 1500, 1450,  1410,  1 3 2 0 , 1 2 3 0 , 1 2 0 0 , 1 1 2 5 , 1 0 8 0 , 1 0 4 0 , 1 0 0 0 , 9 2 5 , 8 3 0 . 'H-  NMR  (see Table  V) .  430  [ M ] ( 4 0 ) , 249 ( 6 ) , 233 ( 1 0 ) , 224 ( 1 8 ) , 219 ( 1 5 ) , 209 ( 8 ) ,  208  ( 2 4 ) , 2 0 7 ( 4 0 ) , 2 0 6 ( 1 7 ) , 2 0 5 ( 7 ) , 203 ( 9 ) , 197 ( 4 2 ) , 196  13  ONMR  (see Table  V I ) . MS m/z  (rel  int):  +  (16),  195 ( 2 3 ) , 194 ( 1 5 ) , 192 ( 1 3 ) , 191 ( 3 4 ) , 182 ( 2 0 ) , 181  (60),  180 ( 2 8 ) , 179 ( 1 0 0 ) ,  (7) ,  178 ( 1 5 ) , 177 ( 8 ) , 176 ( 1 5 ) , 175  169 ( 5 5 ) , 168 ( 1 3 ) , 167 ( 1 2 ) , 166 ( 3 6 ) , 165 ( 7 8 ) ,  161  (19),  154 ( 1 4 ) , 153 ( 1 3 ) , 152 ( 2 1 ) , 151 ( 2 4 ) 139 ( 7 ) , 138  (15),  133 ( 1 3 ) , 125 ( 1 1 ) , 115 ( 1 0 ) , 95 ( 1 5 ) , 93 ( 1 3 ) , 91 ( 1 3 ) ,  81  ( 1 5 ) , 79 ( 1 5 ) .  Sesartemin (found  5. ( 3 6 m g ) . Mp  430.1627  115-116  f o r 430.1628  °C ( f r o m  Et 0). 2  b y H R - M S ) . UV X  M  e  0  H  max 270  (4.11),  211 ( 4 . 8 3 ) .  + 50  0  i n CHC1 . 3  V I ) . MS m/z  1  3  H  2  8  (log  0  8  e):  J  H-NMR ( s e e  V).  (47),  265 ( 5 ) , 249 ( 9 ) , 235 ( 7 ) , 234 ( 8 ) , 233 ( 1 1 ) , 224  222  (see Table  5  2  Table  1 3  C~NMR  [a]J  C  ( r e li n t ) : 430[M ] +  (18),  ( 6 ) , 2 1 9 ( 1 7 ) , 2 0 9 ( 7 ) , 208 ( 2 4 ) , 207 ( 5 1 ) , 2 0 6 ( 1 5 ) ,  205  ( 8 ) , 2 0 3 ( 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  (72),  180 ( 3 3 ) , 179 ( 1 0 0 ) ,  (9) ,  178 ( 2 2 ) , 177 ( 1 1 ) , 176 ( 1 9 ) , 175  169 ( 4 2 ) , 168 ( 1 5 ) , 167 ( 1 2 ) , 166 ( 3 5 ) , 165 ( 8 9 ) , 161  (26),  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  ( 1 0 ) , 95 ( 1 8 ) , 93 ( 1 5 ) , 91 ( 1 5 ) , 81  ( 1 8 ) , 79 ( 1 5 ) .  78  Epi-yanqambin (log +122  e ) : 272 ( 3 . 5 1 ) ,  i n CHC1 .  0  3  MS m/z 235  6. ( 1 2 7 m g ) . Mp  1  H-NMR  119-120  238sh  (4.11),  (see Table  ( r e li n t ) : 446[M ]  °C ( f r o m  V).  1 3  Et 0).  210 ( 4 . 8 0 ) .  C-NMR  UV X^eOH  2  [ a ] 25  (see Table V I ) .  ( 3 7 ) , 265 ( 6 ) , 250 ( 1 0 ) , 249 ( 9 ) ,  +  ( 1 2 ) , 2 2 4 ( 2 6 ) , 2 2 3 ( 9 ) , 2 1 9 ( 1 2 ) , 208 ( 1 3 ) , 2 0 7 ( 5 1 ) ,  ( 10) , 1 97 ( 3 2 ) , 182  (8) ,  (39) ,  1 96 ( 2 4 ) , 181  (100),  195  (68) ,  1 94 ( 1 8 ) ,  179  (12) ,  177  (12)  191 ( 1 0 ) , ,  1 68 ( 1 6 ) ,  1 67 ( 1 3 ) ,  166,  (10),  1 65 (21 ) ,  154 ( 1 1 ) ,  (14),  151  138  125  (14),  1 10 ( 1 0 ) ,  95  91  ( 1 1 ) , 81  Yanqambin e):  (rel 224  7. ( 1 1 5 mg) Mp  H-NMR  119-121  235 ( 4 . 1 4 ) ,  (see Table  i n t ) : 446[M ] +  ( 9 ) , 93  (13),  0  (from  210 ( 4 . 9 3 ) .  V).  1 3  C-NMR  Et 0) 2  UV X MeOH  [ a ] J 5 +45.1  (see Table  0  (  l  o  g  in  V I ) . MS m/z  ( 2 7 ) , 265 ( 6 ) , 250 ( 8 ) , 249 ( 8 ) , 235 ( 1 0 ) ,  ( 1 9 ) , 2 2 3 ( 8 ) , 2 1 9 ( 9 ) , 208 ( 1 2 ) , 207 ( 4 8 ) , 2 0 6 ( 8 ) , 197  (20) , (8),  196 ( 2 8 ) , 195 ( 5 1 ) , 194 ( 2 0 ) , 193 ( 1 0 ) , 191 ( 1 1 ) , 190 189 ( 9 ) , 182 ( 2 7 ) , 181 ( 8 2 ) , 179 ( 1 2 ) , 177 ( 1 3 ) , 176  (21) , (8), 138  1  1 53  ( 1 5 ) , 79 ( 9 ) .  270 ( 3 . 5 2 ) ,  CHCI3.  (13),  189  176 ( 2 1 ) , 169  (42) ,  (20),  206  169 ( 2 5 ) , 168 ( 1 5 ) , 167 ( 1 2 ) , 166 ( 8 ) , 165 ( 1 7 ) , 163 154 ( 8 ) , 153 ( 1 7 ) , 152 ( 9 ) , 151 ( 1 8 ) , 149 ( 1 3 ) , 145  (9),  ( 1 1 ) , 137 ( 1 3 ) , 136 ( 8 ) , 135 ( 1 2 ) , 131 ( 1 1 ) , 128 ( 8 ) , 125  (21),  123 ( 1 4 ) , 121 ( 1 2 ) , 119 ( 1 5 ) , 117 ( 9 ) , 115 ( 1 3 ) , 111  (13),  110 ( 1 9 ) , 109 ( 1 9 ) , 107 ( 1 1 ) , 105 ( 1 5 ) , 97 ( 2 0 ) , 96  (13),  95 ( 3 3 ) , 93 ( 2 0 ) , 91  (36),  79 ( 1 9 ) .  ( 2 4 ) , 85 ( 1 6 ) , 83 ( 2 3 ) , 82 ( 1 7 ) , 81  79  Dihydrosesartemin 232sh  (3.87),  MHz)  (CDC1 ):  2.40  (1H,  3  ArCH ), 2  X OCH ),  m, J = 5 H z , H - 3 ) ,  (6),  i n CHC1 .  0  3  with  H-NMR ( 4 0 0  D 0 ,OH), 2  2 . 5 5 ( 1 H , d d , J = 14, 11 H z ,  3.83 ( 3 H , s , O C H ) ,  3  3.84'(6H, s, 2  3  4.78 ( 1 H ,  d , J = 6.3 H z ,  6.40 ( 2 H , s , H-2', H - 6 ' ) ,  6.54 ( I H , s , H-2" o r H - 6 " ) .  ( 2 3 ) , 414 ( 1 . 2 ) ,  1  2  2  2  +  +11.8  (3.25),  2.92 ( 1 H , d d , J = 14, 5 H z , A r C H ) ,  ( 4 H , H-5, C H O H ) ,  o r H-6"),  ( l o g e ) : 282  6 1.56 ( 1 H , b r s , e x c h a n g e a b l e  (2H, s, OCH 0),  432[M ]  [ a ] 25  3.89 ( 3 H , s , O C H ) ,  3  2"  218 ( 4 . 0 8 ) .  2 . 7 3 ( 1 H , m, H - 4 ) ,  3.73-4.08  5.95  8. (21 mg) UV X  383 ( 2 . 4 ) ,  6.53 ( 1 H , s , H-  MS m/z  368 ( 1 . 3 ) ,  H-2),  (rel  i n t ) :  353 ( 1 . 1 ) , 2 4 9  233 ( 1 7 ) , 2 2 4 ( 1 4 ) , 2 1 9 ( 1 8 ) , 2 0 8 ( 2 1 ) , 2 0 7 ( 1 2 ) ,  206  (12),  195 ( 2 2 ) , 193 ( 1 4 ) , 183 ( 1 0 ) , 182 ( 8 2 ) , 181 ( 1 0 0 ) ,  (18),  179 ( 5 3 ) , 169 ( 1 0 ) , 167 ( 2 2 ) , 166 ( 2 1 ) , 165 ( 4 5 ) , 153  (18),  152 ( 1 3 ) , 151 ( 3 8 ) , 148 ( 1 8 ) , 137 ( 1 4 ) , 136 ( 1 2 ) , 123  (23),  105 ( 1 2 ) , 95 ( 2 7 ) , 93 ( 1 2 ) , 91  j3-Dihydroyangambin  180  ( 2 2 ) , 79 ( 1 8 ) .  9. ( 1 3 mg) UV X MeOH  ( l o g e ) : 280 ( 3 . 7 5 ) ,  max 227sh  (4.25),  [a]  213 ( 4 . 5 8 ) .  MHz)  (CDC1 )  2.03  ( 1 H , m, J = 5 H z , H - 3 ) ,  3  ArCH ), 2  8.5  265  +15.1  i n CHC1 .  0  3  6 1.78 ( 1 H , b r s , e x c h a n g e a b l e  ( 4 H , m, H-5, C H O H ) , 2  3  Hz, H-2),  D 0 , OH), 2  2.88 ( 1 H , m,  H-4),  3  3  6.36 ( 1 H , s , H - 2 " ) , . 6 . 6 3 MS m/z  H-NMR ( 4 0 0  3.84 ( 6 H , s , 2 X O C H ) , 3 . 8 9  3.94 ( 6 H , s , 2 X O C H ) ,  s , H-2', H - 6 ' ) .  with  1  2 . 5 2 ( 1 H , d d , J = 14, 7 H z , 2  s, 2 X OCH ),  (2H,  5  2.8 ( 1 H , d d , J = 14, 5 H z , A r C H ) ,  3.70-4.05 (6H,  J  4.70 ( I H , d , J =  (1H, s, H-5"),  ( r e li n t ) :448[M ] +  6.66  ( 2 9 ) , 399 ( 6 ) ,  ( 3 ) , 263 ( 5 ) , 249 ( 1 8 ) , 240 ( 2 1 ) , 235 ( 1 6 ) , 233 ( 9 ) ,  (42),  224  2 2 3 ( 2 3 ) , 2 2 2 ( 4 5 ) , 221 ( 1 2 ) , 2 1 9 ( 1 4 ) , 2 1 0 ( 2 0 ) , 2 0 8  80  (14) ,  207  (18)  (83) ,  1 93  (18),  (100) ,  179  ,  198  205 ( 1 1 ) , 191  (13),  189  177 ( 1 2 ) ,  ( 1 8 ),  197  (11), (10) ,  1 76  183  (13) ,  (27) , (17) ,  1 69  196 ( 3 2 ) , 1 82  195  (77) ,  181  168 (21 ) , 167  (48) ,  (46) ,  165  (20),  161  (11),  1 54  (19) ,  1 53  (19) ,  1 52  (22),  151  (50) ,  1 49  (12),  1 48  (22) ,  1 39  (20) ,  1 38  (28) ,  1 37  (27) ,  1 36  (16) ,  1 35  (12) ,  1 33  (10) ,  1 25  (15) ,  124  (12) ,  1 23  (13) ,  1 22  (12),  121  (15),  1 10  (12),  109  (18),  1 07  (15) ,  1 06  (11),  105  (17) ,  95  4.  ( 1 7 ) , 92  (12),  91  (29) ,  (23) ,  81  79 ( 2 2 ) .  RESULTS Eleven  extract  of the major c o n s t i t u e n t s  o f V.  Experimental Sitosterol 3,  93  (20) ,  4',  were has  found  section-and was  the only  were  isolated  identified phytosterol  i n approximately  been d e s c r i b e d  equal  by  were  epi-yangambin  identified:  identified. (1a) and  dihydroyangambin  compounds, (9) were  Four  The  i n the  means.  stilbenes,  Only  the  former  constituent.  compound,  virolongin  b i s - t e t r a h y d r o f .uran (4), sesartemin  (7). Finally,  dihydrosesartemin identified.  0-  i t s c i s isomer  proportions.  epi-sesartemin  (6) and yangambin  tetrahydrofuran  as 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  the neolignans.  ether  spectroscopic  (2) and a h i t h e r t o u n d e s c r i b e d  comprised  lignans  bark  5-trimethoxy-trans-stilbene  Eusiderin (3),  elongata  of a d i e t h y l  two  new  (8) and  0-  (5),  81  Epiyangambi n (6)  Protons  Episesartemin (4)  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  4j3H  Sesartemin (5)  Yangambin (7)  3.70-3.95 ( 1H) ,m  4. 1 5 - 4 . 5 0 (1H),m  3.70-4.00 (1H),m  4.20-4.43  3.15-3.5 (IH),M  3.70-4.10 (1H),m  3.20-3.55 (IH),m  3.82-3.97 (1H),m  (1H),m  5H  3.15-3.50,m  3.05(1H),m  3.20-3.55  3.08(1H),m  6H  4.41(1H),d J = 7.0  4.70(1H),d J = 4.0  4.43(1H),d J=7.0  4.75(1H),d J=5. 5  4.15-4.50 (1H),m  4.00-4.25 (1H),m  4.20-4.43 (1H),m  3.70-4.10 (1H),m  3.70-4.00 (1H),m  3.82-3.97 (1H),m  6.56(2H),s 6.53(2H),s  6.55(4H),s  6.57(4H),s  6.58(4H),s  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  3.86(12H),s 3.83(6H),s  5.95(2H),s  5.95(2H),s  -  -  8aH  4.00-4.25 (1H),m 3.70-3.95  8/3H  (1H),m 2' , 2" 6* , 6" OCH  OCH  Table  2  3  0  V - H-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 i s o l a t e d from V ir o l a e l o n g a t a bark. 1  lignans  Data a r e p r e s e n t e d ( i n order) as chemical shift(6, relative to. TMS); i n t e g r a l v a l u e ( n u m b e r o f 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 a t 80 MHz. 3  82  Carbon Number  EpiSesartemin (4)  Sesartemin  Yangambin  (5)  Epiyangambin (6)  (7)  1  54.52  54.41  54.36  54.29  2  87.59  85.85(c)  87.66  85.88  4  71 .03  71.87(d)  71 .00  71 .91  5  49.96  54.41  49.90  54.29  6  82.11  86.02(c)  82.06  85.88  8  69.99  72.00(d)  69.77  71.91  1 '  1 35.88  135.86  136.69  136.66  1 "  133.97  136.77  133.89  136.66  2'  105.73(a)  105.73(e)  103.00  102.98  2"  102.73  1 02.94(e)  102.69  1 02.98  3'  153.21  153.29(f)  153.32  153.39  3"  153.21  153.48(f)  153. 1 3  153.39  4'  149.06(b)  149.20(g)  137.60  137.64  4"  137.09  136.77  137.00  137.64  5'  143.62(b)  143.72(g)  153.32  153.39  5"  153.21  153.48  153. 1 3  153.39  6'  101.39(a)  101.52(e)  103.00  102.98  6"  102.73  102.94(e)  102.69  102.98  OCH  3  OCH  2  56.13,56.66, 60.77 0  1 00. 1 1  56.24,57.51 60.86 100.09  56.07,60.68  -  -  T a b l e VI - C-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 i s o l a t e d from V i r o l a elongata bark. 13  Chemical 5(CDC1 ) followed 3  56.16,60.73  lignans  s h i f t s a r e g i v e n i n S ( p p m ) r e l a t i v e t o TMS; 6 ( T M S ) = + 7 7 . 0 ppm; r e c o r d e d a t 20 MHz i n C D C 1 . Values 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 . 3  83  a.  3,4'-5-trimethoxy-cis-stilbene and  3,4',5-trimethoxy-trans-  stilbene Compounds approximately several  with  chromatotron spectrum  H SO„.  that  those  (Blair  from  signals  with  i scharacteristic  stilbenes The  UV s p e c t r u m  suggestive  displaying spectrum,  ion  the aromatic  o f mass  signals  those  area l l present  o f 1b. T h e v i n y l  broad resolution of  thevinyl  proton  o f 16 H z . T h i s  substituted  trans  assigned. band  a t 2 8 3 nm, i s  (Hillis  and Ishikura,  to that  o f 1b, b o t h  2 7 0 . I n t h e 4 0 0 MHz  protons  f o r 1b  of the vinyl  t o be  constant  similar  AB p a t t e r n  o f t h e two v i n y l  t o Band I  obtained  The b e t t e r  derivative  i svery  equal  3,4',5-trimethoxy-  showed  o f 1a, a s i n g l e  spectrum  a parent  for  a coupling  1b was t h u s  of a cis-stilbene  T h e mass  here  T h e UV  of stilbenes  data  of unsymmetrically  a n d compound  ether.  The s i g n a l s  spectrum.  upon  corresponding  e t a_l ( 1 9 6 9 )  obtained  t o be d o u b l e t s  colour  of approximately  1969).  on T L C u s i n g  using the  diethyl  presented  by B l a i r 1  4 0 0 MHz s p e c t r u m  proton  separated  The s p e c t r a l  e t aJL,  i n a 60 MHz H-NMR  similarly  arecharacteristic  1966).  with  are reported  singlets  signals  were  present i n  a pink  a t 319 a n d 305 n a n o m e t e r s ,  and I s h i k u r a ,  protons  1966).  formed  and c r y s t a l l i z e d  i n agreement  value  and behaved  Both  They  2  trans-stilbene  from  systems.  II, respectively,  (Hillis  the  amounts  o f 1b d i s p l a y e d b a n d s  intensity  are  equal  solvent  spraying  and  1a a n d 1b ( F i g u r e 7) w e r e  o f C-3',5';C-  'H-NMR  2',6', t h e  a n d t h e C - 2 , C-4 a n d C-6  but shifted  proton  signals  upfield appear  somewhat as doublets  84  F i g u r e 7 - S t r u c t u r e s of s t i l b e n e s and from V i r o l a elongata bark.  neolignans  isolated  85  Figure  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 lignans i s o l a t e d from V i r o l a e l o n g a t a bark.  86  Figure 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 from V i r o l a elongata bark.  lignans  isolated  87  with  a coupling  characteristic On  the basis  isomer since  o f 12 H z , a v a l u e  of unsymmetrically  of these  data  stilbenes trans  a r enormally  form  (Drewes  that i s  substituted  compound  o f 1b. T h e o c c u r r e n c e  stable b.  constant  c i s stilbenes.  1a i s a s s i g n e d  of t h i s  compound  present  naturally  and F l e t c h e r ,  as the c i s  i s noteworthy i n t h e more  1974).  E u s i d e r i n and Virolongin Compounds  behavior with 386  a  Compound  b y MS. P r o t o n  revealed 208  by T L C . B o t h  H SO . 2  2 a n d 3 ( F i g u r e 7) d e m o n s t r a t e d  Further that  and methoxyl  (Fernandes  C  1  8  H  0  2  counts (OMe)„  e t a l , 1980; G o t t l i e b  e_t a l , 1 9 7 6 ) .  Filho  thevalue  for J  trans  relationship  1980)  and support  eusiderin.  various  Compound based  The c h e m i c a l  6  pavonis  3 bears  i t s UV, H-NMR 1  3  eusiderin  induced shift  Hobbs a n d K i n g ,  shift  for  (Fernandez  the et a l ,  2 as  reported  from  Virola  e_t a _ l , 1 9 8 0 )  (Gottlieb  resemblance  spectrum  data  o f Me~3 ( 5 1.26)  o f compound  (Fernandes  considerable  2  t o be i d e n t i c a l t o  groups  of the family Lauraceae  upon  3  compound h a s been  E u s i d e r i n h a s p r e v i o u s l y been  genera  3  (8 H z ) b y 'H-NMR a r e e v i d e n c e  guggenheimi i and V i r o l a  m/z =  (CH 0) C H CHCHCH .  data  by l a n t h a n i d e  the identification  weight of  a n d t h e MS f r a g m e n t  e t a l , 1976;  o f t h e Ar-2/Me-3  spraying  b y NMR a n d t h e MS  neolignan,  The p r e c i s e s t r u c t u r e of that  (Braz  upon  a molecular  of t h e system  f o r the benzodioxan  definitively  (2),  3  colour  t o have  showed t h e s p e c t r a l  established  and  1  the presence  analysis  reported  1960).  2 was f o u n d  the formula  suggested  d i s p l a y e d a pink  similar  et a l ,  and 1976).  to eusiderin  a n d mass  spectrum. The  88  presence the  of  t h e MS f r a g m e n t s w i t h m/z = 209 and 208 s u g g e s t  presence  of  the  groups  (CH 0) C H CHCHCH , 3  3  6  2  of  two  3  6  respectively.  3  appearance  (CH 0)3C H CH CHCH3  sets,  e a c h of  The  1  of  the  presence  of  and  2  This,  combined w i t h  two e q u i v a l e n t  p r o t o n s and f i v e methoxy g r o u p s indicative  2  in the  1  the  aromatic  H-NMR s p e c t r u m  is  an 8 - 0 - 4 ' - n e o l i g n a n .  H-NMR s p e c t r u m c l o s e l y  ressembled that  reported  for  1 -(3,4,5-trimethoxyphenyl)-2-allyl-2,6-dimethoxyphenoxy)propane,  a compound i s o l a t e d  ( I s o g a i §_t §_1, 1 9 7 3 ) , differences the at  aromatic 6 1.87  6.2  allylic  (1H)  would,  propenyl group experiments 6 1.9  in  of  substituent  the  instead,  at  of  the  6 6.05  s p e c t r u m of  result  6 6.2  caused the  signal  at  doublet at  = 6 Hz)  These  three  from an  1980). Double  the  J  to  and 6.25 and 5  s u p p o r t e d the a s s i g n m e n t s . of  only  attributed  (3H,  compound 3.  be e x p e c t e d t o a_l,  (1H)  fragrans  The  signals  as a d o u b l e t  between  (Fernandes et  further  the M y r i s t i c a c e a e .  occurrence  produced the c o l l a p s e  irradiation a  i n the  and m u l t i p l e t s  and 6.5  signals  are  also  from s e e d s of M y r i s t i c a  aromatic  resonance Irradiation  at  5 6 . 0 5 - 6 . 2 5 and  5 1.87  to appear  as  singlet.  c.  Epi-sesartemin,  Sesartemin,  Compounds 4 and 5 ( F i g u r e chromatographic  systems.  E p i - y a n q a m b i n and Yanqambin 8)  behaved  Two s p o t s ,  similarly  f o r m i n g a brown  upon s p r a y i n g w i t h H SO<,, were d i s t i n g u i s h e d by T L C . 2  compounds were f o u n d t o have f o r m u l a e C the  1  H-NMR c l e a r l y  one m e t h y l e n e d i o x y  indicated  2 3  the p r e s e n c e  g r o u p and two p a i r s  of  H  2 6  of  0  8  in  various  colour Both  by HR-MS and  f o u r methoxy  equivalent  and  aromatic  89  protons. the  Furthermore,  presence  of  between  5 3.0  and 4 . 5  a bis-tetrahydrofuran  ring.  T h e s e compounds  were c o n c l u d e d t o be type  lignans.  configuration  4 and 5. axial  lignans  and the  V and VI)  equatorial its  1  1) shifts  i s o m e r on t h e  2.91  5 4.0)  this of  the  spectra  is  also  cis  obtained  true  of compounds  C-2 and C-6 c a n be three types  of  the  either  of  following  and Ward,  i n C-1  axialfeatures  1978; R u s s e l  (  in (  presence of  6 3.15-6  due t o  and C-5 m e t h i n e  and 6 3 . 1 5 - 3 . 5 ,  C-2 and C-6  the  equatorial 6 4.25,  C-NMR  for  basis  (Pelter  a difference  p r o t o n s at  upfield  that  13  in  bis-  of  and  1973):  (5  3)  H-NMR and  allowing  a difference  2)  occurring  Compound 4 was c o n c l u d e d t o be an  H-NMR s p e c t r u m  Fenemore,  naturally  characteristically  substituents  or e q u a t o r i a l ,  stereoisomers.  1  is  indicate  The a r y l  indicated  2,6-diaryl-3,7-dioxabicyclof3,3,0]-octane  The C - 1 , C~5 bond of  tetrahydrofuran  (Tables  signals  3.5)  the  methylene  one a x i a l from the  positions  et of  al  (1980)  aryl  tetrahydrofuran  g r o u p s of  lignans  the  used  assignment  of  lanthanide  induced s h i f t  (C-4)  axial  (C-8)  13  of  benzylic  respectively),  methylene  aromatic  proton (  6 3.8  equatorial  and G r e g e r  aromatic  to e s t a b l i s h  unsymmetrically  technique.  In  the bis-  based  obtained using  the p r e s e n t  and  ring.  substituted  and H o f e r ( l 9 8 0 ) on d a t a  to  r i n g and an  d o w n f i e l d between 6 4 . 0  C-NMR  stereochemistry  the  "normal" p o s i t i o n  due t o d e s h i e l d i n g by t h e  Chiba  shifts  6 4 . 8 5 and 6 4 . 4 1 ,  proton  chemical  respectively),  chemical  s h i e l d i n g by t h e  proton  study,  their the the  90  relative  configurations  spectra.  The c h e m i c a l  particularly attachment skeleton are  useful  of  the  (Pelter  known,  were a s s i g n e d on t h e  shifts  phenyl group to and Ward,  symmetrically  optical  rotations  which agreed c l o s e l y 1974; B r i g g s  al,  1961; L a i  et  al,  e_t a l ,  on t h e  and UV,  The  1 3  compound, value  1'  and  yangambin  that  Ward (1978)  b a s i s of IR,  for  shift  distinguished this  value  (7),  similar  MS and  al,  the  compound 4. to  the  of  from t h e  ( 1 3 3 . 8 9 ppm) and t h e  phenyl  is  shifts  equatorial  spectral  substituent  is  one.  signal  signal  from t h e  1  The  et  is  substituted  136.66 ppm, a and  (6),  w h i c h has a  easily  The s i m i l a r i t y seen  in  the a x i a l  the  that  s p e c t r u m of the  was  substituent  C-NMR  between  3,4,5-  position  ( 1 3 5 . 8 8 ppm)  13  this  3,4,5-  H-NMR s p e c t r u m ,  p o s i t i o n , by d e f a u l t .  et  compounds 6  of  epi-yangambin  in  (Abe  p u b l i s h e d by P e l t e r  taken as e v i d e n c e  The r e m a i n i n g C-1  known,  of  diequatorially  3-methoxy-4,5-methylenedioxyphenyl  was a l r e a d y  'H-NMR  1976; J e f f r i e s  The a x i a l  equatorial  8)  melting  a s s i g n m e n t s made on  133.89 ppm ( T a b l e V I ) ,  compound 4 ( 1 3 3 . 9 7 ppm) trimethoxy  the  lignans.  of  unsymmetrically  have c h e m i c a l  substituent  of  their  C-NMR d a t a  compares w e l l w i t h t h o s e  trimethoxyphenyl chemical  1" c a r b o n s of  are  bis-tetrahydrofuran  1968; Chen e t  1973).  C~NMR  bis-tetrahydrofuran  s u b s t i t u t e d compounds 4 and 5 and t h e The  1"  with published information  and 7 were compared w i t h t h o s e of  basis.  and  13  Compounds 6 and 7 ( F i g u r e  substituted  points,  1'  of  stereochemistry  the  1978).  and were i d e n t i f i e d  al,  c a r b o n atoms  in d e t e r m i n i n g the  lignans  data  of  basis  t o be i n  in assigned which the  s p e c t r u m of  the  91  diequatorial has  compound  5 supports  these  s i g n a l s a t 1 3 5 . 8 6 ppm a n d 1 3 6 . 7 7 ppm, v a l u e s  correspond  c l o s e l y t o those assigned  methoxy-4,5-methylenedioxyphenyl substituents, close  agreement with  The mass identical  the spectra  lignans  spectra  and bear  for  a l l of the fragments described  and  fragmentation  o f compounds  11) f o r c o m p a r i s o n . 1967;  Taniguchi,  structures  6 and 7 has a l s o  of  isomers  d.  Dihydrosesartemin  constituents  and 0  of the ether  T L C , compound  H SO  a  these  4 and 5 were mass  i n Figure  fragmentation  by o t h e r s  differences  10.  (1967)  been i n c l u d e d  (Figure (Pelter,  i n the structure  -Dihydroyanqambin 9) a r e t h e most  9 a grey  coloured  c o l o r a t i o n . The mass  two compounds were e s p e c i a l l y i n f o r m a t i v e parent  These values  ions  are just  with  polar  e x t r a c t . They a r e s e p a r a t e d  8 g i v i n g a salmon-pink  a n d compound  produced  formulae  by P e l t e r The  6  are not i d e n t i f i a b l e ,  Compounds 8 a n d 9 ( F i g u r e  2  a r e drawn  As h a s been o b s e r v e d reliable  o f compounds  resolution  pathways described  1972),  1978).  f o r compounds  D u f f i e l d (1967) a r e e a s i l y i d e n t i f i a b l e .  pattern  showed  f o rs i m i l a r  The m o l e c u l a r  f r o m mass m e a s u r e m e n t s by h i g h  The b a s i c  phenyl  signals  t o those  identical.  Corresponding  3-  4 and 5 a r e almost  resemblance  7 which a r e a l s o  spectrometry.  C-NMR  reported  and  obtained  13  ( P e l t e r and Ward,  almost  which  and 3,4,5-trimethoxy  o f compounds  close  since i t  above f o re q u a t o r i a l  r e s p e c t i v e l y . A l l other  bistetrahydrofuran  by  assignments  spot  easily with  spectra of since  they  masses 432 a n d 4 4 8 , r e s p e c t i v e l y .  2 mass  units  greater  than  the parent  F i gure  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  spectrometry  Figure  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  lignans,  epi-yangambin  and yangambin,  by mass  spectrometry  94  ions  of  sesartemin  similarities  and  yangambin,  i n t h e mass  spectra  respectively. of a l l four  Further  compounds  were  apparent. The series  mass  of  spectrum  fragments:  indicating  181,  the presence  methylenedioxyphenyl The  greater  m/z  =  165,  was  taken  substituent.  The  those  fragmentation  the  schemes  (Figure The  was  remaining  165,  pattern  a t m/z that  a  a  has  the  of  152,  been  substituents. than  that  at  trimethoxy rather  than  t h e mass  a  phenyl,  spectrum  tetrahydrofuran.  interpreted  e_t a_l ( 1 9 6 6 )  was  substituted  a on  20  supported  according  and  tetrahydrofuran  i t s relative  (1972)  have  to  Pelter  discussed of  ( 1 967)  furanoses  are recognized.  The  vary  depending  solvent  used.  lignans  presently  been  Unfortunately,  reported  therefore  the nature  known  available  the  a  (1964)  great  and  relative  o f NMR  of  and  At of  will the  tetrahydrofuran  d e t a i l e d NMR  From  data  importance  configurations  l i m i t e d number  f o r comparison.  and  in solution.  substituents  the v a r i e t y  i s not  in only  of  Hall  the  provided  the a p p l i c a b i l i t y  i n the d i s t r i b u t i o n of a l l p o s s i b l e upon  that  and  configuration.  the conformation  conformers  the proposal  each  are  181  substituted  Pelter  spectrum  assigning  have  =  benzyl,  features  from  by  and  3~methoxy-4,5-  t h e peak  H-NMR  1  ejt a l  least  both  group  presented  information  in  153,  168  two  12).  compound  Hall  169,  evidence  expected  The  (8) showed  3,4,5-trimethoxyphenyl  of as  aromatic  dihydrosesartemin  of  and  abundance  substituted  resemble  of  of cases.  spectra Few  the e x i s t i n g  data  Figure  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  lignan. dihydrosesarternin,  by mass  spectrometry  97  data,  it  appears  determined A cis  is  the  of  Smith,  in  its  results  in  the  s p e c t r u m of  resonance  of  insufficient  to allow  previously  for  rotation  1961).  to  = + 11.8  of  the  ring.  al,  1981;  A trans  Sarkanen  of  observed This  in  value  configuration.  and t h e  favoured  coupling constant  as a  trans  agrees  considerably,  are  line  the  The  The p u b l i s h e d d a t a  in  and  orientation  shift  6 4.78.  will  (Birch  assigning configuration.  the  carbon.  shift  6 5.5  substituents  use of  The  with several  configuration  tetrahydrofuran  of  the  been e s t a b l i s h e d has  been  The o p t i c a l °.  similar  benzyl  C-2-C-3 bond  a chemical  trans  easily  measured  values  about  (Sarkanen  the  C-2-  and  1963).  has  (Weinges,  relative  the  configuration  (+)-lariciresinol  sufficiently  1963).  a doublet,  however,  Smith,  The r e l a t i v e  [a]  for  a substituted  1973a;  optical  the  Hz i s ,  reported  C-3 bond of Wallis,  furan  criterion  = 6.3  1967; Inoue e t  of  nature  conformation  J  the  may be e x p e c t e d t o v a r y  the  the  The c o r r e s p o n d i n g s i g n a l  assignment  d e p e n d i n g upon t h e  definitive  about  at  to a p p r o x i m a t e l y  Smith,  compound 8 i s  coupling constant  value,  al,  can be most  b e n z y l p r o t o n and a  C-2 p r o t o n h a v i n g 6 4.7.  w i t h the  that  configuration  the  1973a and b;  approximately  be  of  1964; B i r c h e t  and W a l l i s ,  feature  constituents  in d e s h i e l d i n g  downfield  Its  the  relative  orientation  result  well  that  These to  configuration  rotation two v a l u e s  the  by c h e m i c a l  r e p o r t e d as  form t h e of  tetrahydrofuran  basis  of  [a]  lignan,  methods.  = + 17.5  0  compound 8 was  appear for  to  be  assigning  r e m a i n i n g c a r b o n of  found  the  compound 8.  98  The c o n f i g u r a t i o n of  is  therefore,  3R,.4R,  identical  to  that  (+)-lariciresinol. The mass s p e c t r u m of  to  2S,  that  it,  of  too,  dihydrosesartemin  is  biosynthetically  lignan;  differing  leaving  a free  examined, the  type  compound 9 ( F i g u r e  in  (Figure  related  that  one  it  aromatic  is  apparent  furan  that  substituents  I n s t e a d of  arising  aromatic  integrating  for  singlets  8 6.63 and 6.36 a r e  at  This  2 protons  pattern  is  similar  6.43 w h i c h were o b s e r v e d  for  p o s i t i o n i n g of  spectrum, basis  of  related (8),  the  structure  has  a c o m p a r i s o n of compounds. In  the  chemical  shift  of  two  two p e a k s a t  signal  of  exists  to  the  the  singlets, one  each  singlet  single  proton  singlets  at  6 6.84 and 6  3,4-methylenedioxy-6-  basis  of  and Fenemore  only  the  shifts  'H-NMR s p e c t r u m of the C-2  1  and C - 6 '  are  (1973).  groups cannot  been t e n t a t i v e l y  6 6.53' and 6 . 5 4 ,  1  Assuming the  substituent  a s s i g n e d on with data  same r e l a t i o n s h i p  to e x i s t  the  from  dihydrosesartemin p r o t o n s of  which are  downfield  opposite  be  H-NMR  (6  in the  the  resolved  from  t h e c o r r e s p o n d i n g C-2" and C - 6 " p r o t o n s of  3,4,5-trimethoxy-phenyl  in  observed.  methoxy-methylenedioxybenzyl s u b s t i t u e n t , into  is  i n compound 9 and  and two  the c h e m i c a l  the  H-NMR s p e c t r u m  the a r o m a t i c  u n a m b i g u o u s l y d e f i n e d on t h e  that  opened,  a difference  m e t h o x y p h e n y l g r o u p o b s e r v e d by R u s s e l A l t h o u g h the  is  protons,  (6 = 6.66)  analogous  suggesting  ring 1  is  bis-tetrahydrofuran  present  yangambin or e p i - y a n g a m b i n . from two e q u i v a l e n t  12)  to a  h y d r o x y l g r o u p . When t h e  however, of  only  (8)  13)  the the  6.40). 1  H-NMR  spectrum  99  of  /3-dihydroyangambin,  singlet,  5 =  6.66)  substituent. protons two  This  chemical  protons,  The similar  with  optical  of  of  from  similar  no  8,  J  further  compound  9.  and Oshima,  of compound  each),  8 and  J  with  t o t h e C-5"  shifts  are in  lignans  (Russel  [ a ] = +15.1  [ a ] = +17.5  0  is indicative  = 8.5,  is  0  (Weinges, 2,3  and  constant i s larger  than  values  analogous  c o n f i g u r a t i o n s . The  published for  9 are s u f f i c i e n t l y  different  configurations are also to assign  the  4.  of a  and o t h e r  made  and  1972a a n d b ) .  the coupling  spectrum,  = 6.3,  was  5 = 4.75,  C2-C3,  relative  attempt  9,  remaining  c o n f i g u r a t i o n s at carbons  lignans with  their  The  1 proton  f o r other  benzyl  equivalent  group.  chemical  proton  of the  are assigned  published  shift,  t h e 400 MHz  f o r compounds that  These  at  (+)- l a r i c i r e s i n o l ,  the chemical  o f compound  suggest and  rotation  c o n f i g u r a t i o n about  data  6 6.36  1973; T a n i g u c h i  tetrahydrofuran NMR  and  (a 2  t o t h e two  (integrating  H-NMR d a t a  suggesting  measured that  1  to that  Although trans  5 6.63  signal  to the protons  correspond  respectively.  Fenemore,  1961),  would  singlets  shifts  agreement and  i s assigned  field  of the 3,4,5~trimethoxy-benzyl  aromatic  C-2"  the lower  1  H-  to  different  stereochemistry  100  PART B.  EXAMINATION  OF THE BIOLOGICAL  Virola  1.  elongata  OF  EXTRACTS  INTRODUCTION This  section  experiments a toxic  that  injected prepare  describes  the  series  out  was p r e s e n t  nature  with extracts the  the  were c a r r i e d  constituent  to e l u c i d a t e  extracts  of of  the this  details  have  MATERIALS  been  effects  in Part  otherwise,  bark  obtained  to determine  resin  observed  and t o  in  mice  from t h o s e  to  used  spectroscopic analysis. following  if  try  The methods u s e d  different  and i d e n t i f i c a t i o n  been d e s c r i b e d the  immediately  preparation  bark  material.  the  biological  Full  section.  AND METHODS  The c o l l e c t i o n  methanol  the  were somewhat  included in  of  i n an e f f o r t  in  t o o b t a i n p u r i f i e d compounds f o r  2.  ACTIVITY  of  A,  2a. Except  of  plant  where  material  indicated  s a m p l e s w h i c h had been p r e s e r v e d after  extracts  biological  testing.  The  from b a r k w h i c h had been d r i e d and t h a t  been p r e s e r v e d  in  b e i n g c o l l e c t e d were u s e d i n  for  has  i n m e t h a n o l were i n d i s t i n g u i s h a b l e  the  extracts  which  had  by TLC and  HPLC. a.  Preparation  of  extracts  A l k a l o i d a l and n o n - a l k a l o i d a l Virola extracted  elongata  bark  exhaustively  mg of m a t e r i a l  at  extracts  (28 g d r y 20  e x t r a c t e d was  0  weight)  C with  was m i l l e d  and  100% m e t h a n o l . The 278  suspended in water,  acidified  (to  101  pH 3 w i t h H C l ) , phase was  next  and e x t r a c t e d basified  dichloromethane. mg).  This  (pH  with d i e t h y l  12 w i t h NaOH)  c o m b i n e d and n e u t r a l i z e d  to  form t h e  ether  (12.5  fractions  non-alkaloidal  were  fraction  extract  D r i e d V.  elongata  w i t h d i s t i l l e d water  bark  at  20  The m i x t u r e was a g i t a t e d  (30  g)  evaporated  in v a c u o and s t o r e d a t ether  filtered  five  times  syrupy  chromatography. 50WX8  dissolved  in  was  bark  C until  was d r i e d ,  was  use.  milled  ether.  evaporation  s t o r e d at  -30  0  and  The e x t r a c t  by r o t a r y  fractionated  by  A c o l u m n was p r e p a r e d  (2-50 mesh, H  +  form)  pH 6,  8,  were n e u t r a l i z e d  The f r a c t i o n s  ion  in. v a c u o  C until  was and  use.  the  exchange  from t h o r o u g h l y  resin.  1N H C l and a p p l i e d t o  Fractions  collection.  0  solution  After  extracts  w i t h aqueous s o l u t i o n s of NaOH).  periods.  extract  This extract  Dowex  the  with dry d i e t h y l  residue  F r a c t i o n a t i o n of Aqueous  hour  extract  and c o n c e n t r a t e d  brown,  4  eight  extracted  during e x t r a c t i o n .  the combined e x t r a c t s ,  The m e t h a n o l p r e s e r v e d extracted  three,  constantly  of  Diethyl  was m i l l e d and  C for  0  filtration  b.  fraction  with  mg). Aqueous  the  The aqueous  and e x t r a c t e d  formed t h e a l k a l o i d a l  The r e m a i n i n g a q u e o u s and d i e t h y l  (260  ether.  The sample column. It  was was  10 and I 2 ( a d j u s t e d immediately  were m o n i t o r e d f o r  washed  eluted using  after the presence  of  1 02  indole alkaloids ninhydrin  and amino a c i d s  reagents,  Diethyl  ether  c.  in Part  and  respectively.  extract  The s e p a r a t i o n of described  by TLC u s i n g E h r l i c h ' s  B,  this  2b of  extract this  Chromatographic a n a l y s i s  of  was  similar  to  that  chapter, extracts  HPLC Quantitative  analysis  V a r i a n MCH-10 r e v e r s e Varian  variable  absorbing  extracts  was c a r r i e d  detector.  by g r a d u a l l y  from 50% t o  The sample was  i n c r e a s i n g the  100% o v e r  s u b s t a n c e s , were d e t e c t e d a t  chromatogram w i t h the v a l u e s known q u a n t i t i e s  of  methanol The UV  250 nm. Q u a n t i f i c a t i o n the  sample  from a s t a n d a r d c u r v e  pure  eluted  a 20 m i n u t e p e r i o d .  was p e r f o r m e d by c o m p a r i n g peak h e i g h t s of  using  out u s i n g a  p h a s e column on a V a r i a n HPLC w i t h a  wavelength  with methanol/water concentration  of  prepared  compounds.  TLC Because extract  the  were not  chromatography amounts of plates  f o u r major readily  r e s o l v e d by HPLC,  constituents.  were u s e d w i t h e i t h e r  as d e v e l o p i n g  of  the  (6/6/1)  diethyl  thin  s y s t e m s were u s e d t o e s t i m a t e  these  acetonitrile  constituents  layer  the  Polygram S i l i c a  relative  gel U V  petroleum ether/ d i e t h y l  or h e x a n e / c h l o r o f o r m / e t h a n o l  solvents.  ether  2 5 a  TLC  ether/ (25/25/1)  1 03  d.  A s s a y of  s p o n t a n e o u s motor  The a p p a r a t u s activity  is  Watzman,  1966).  u s e d t o measure  a m o d i f i c a t i o n of  on t h e  details  of  biological activity  the  apparatus  in  of  are  The m e a s u r i n g d e v i c e  behavioral  activity,  g r o o m i n g or  even  proportional Female examine t h e  J.  to  pectoralis.  (30-35  effects  the  in Chapter  not  t o more s u b t l e  g)  and were m a i n t a i n e d  Complete  II,  in  section  arbitrary  only  to  gross  behavior  s u c h as  p r o d u c e d were  roughly  of  activity  were u s e d i n  extracts.  described  recording  was measured  type  and  measure  previously  sensitive  the  (Kinnard  All  this  animals  under  observed. study  to  were of  standard  conditions  care.  5 0 or  were  injected  aqueous e x t r a c t s .  low  toxicity  Because  (Budden e t  Tween 8 0 (Sigma)  al,  i n water  u s e d as a v e h i c l e  for  R e c o r d i n g of  immediately  intraperitoneally  100 M L D i s t i l l e d  the  extracts.  the  The s i g n a l s  S w i s s mice  Extracts either  breathing.  in magnitude  c o m p a r a b l e ages animal  but a l s o  of  used t o  provided  was  locomotor  cage"  and t h e method f o r  The s p o n t a n e o u s motor a c t i v i t y  units.  of  "jiggle  of m i c e  s p o n t a n e o u s motor a c t i v i t y 2a.  the  spontaneous  The same p r o c e d u r e was  s p o n t a n e o u s motor a c t i v i t y study  activity  water of  1979;  at  solubility  Worthley  administering  of  the  effect  of  (Walsh and Cummins,  1976),  noise  of  and c a r r i e d  was  out  such v a r i a b l e s (Inglis,  for  properties  and S c h o t t ,  the d i e t h y l  locomotor a c t i v i t y  injection  of  u s e d as a v e h i c l e  a concentration  after  Because  its  was  in a l i q u o t s  and  1966),  1 0 % (v/v)  was  ether  begun  for as  1975),  6 0 minutes. lighting the  presence  1 04  or  absence  animals'  of  an o b s e r v e r  circadian  (Norton,  rhythm  1980),  (Hughes  e_t a_l,  or  time  1978),  constant carried  fluorescent  light  m a c h i n e n o i s e of out  between  low  7 and  and i n  10 PM i n  was  the p r e s e n c e  intensity. the  All  the  these  c o n d i t i o n s were s t a n d a r d i z e d . L o c o m o t o r a c t i v i t y under a m b i e n t  in  of  assays  presence  of  measured a  were a  single  observer. e.  Assay  of  anti-aggressive  Lignans isolation  were examined f o r  in cages  This procedure  responses  their  i n d u c e d a g g r e s s i o n . Male  h o u s i n g them s i n g l y weeks.  activity  for  resulted  i n most m i c e .  d i s t i n g u i s h e d when an  the  lost  accompany s o c i a l i z a t i o n . made by t h e  assuming a d e f e n s i v e which  of  behavior.  the  lignan,  Most of  openly  aggressive  mouse.  After  the  isolated  least  they  six  were i n a cage  ( s o c i a l i z e d mouse).  Instead,  by  behavioral  behavior  behaviorisms  that  with  Some of  normally  resisted  p o s t u r e and e m i t t i n g  any  attempts  epi-sesartemin, remainder  a relatively  of  in  brief  oral  on t h i s  the  the  i s o l a t e d mice  attacked  four  lignans  regions,  were  the  from V .  mice  the  and f i g h t i n g e n s u e d . isolated  the  socialized  d u r i n g which  and a n o g e n i t a l  of  Mice  antisocial  same cage as a  period,  i s o l a t e d mouse g e n e r a l l y bis-tetrahydrofuran  sharp squeaks.  f a s h i o n were u s e d i n a s t u d y  when p l a c e d  e x p l o r e d each o t h e r ' s  inhibit  s o c i a l i z e d mouse, becoming n e r v o u s ,  responded in t h i s  effect  of  at  i s o l a t e d mouse was p l a c e d  the  contact  a p e r i o d of  Two t y p e s  isolated  at  to  S w i s s m i c e were  in d i s t i n c t  a mouse not p r e v i o u s l y i s o l a t e d mice  ability  The  elongata  1 05  bark  were e x a m i n e d f o r  aggressiveness  3.  their  observed  in  to  isolated  genus V i r o l a  physiologically are  the  mice.  1978),  carinata  the  potent  al,  hallucinogenic  and DMT a c c o u n t s poison  et  (Galeffi  hypothesis  for et  neolignan,  from v a r i o u s 1980).  It  activity the  al,  neolignans,  (+)-guaiacin 1976)  the  5-MeODMT, a t 1984).  bark  e_t  and  al, the  5-MeODMT  resin  1983; M a i a and R o d r i g u e s ,  of  the V.  elongata  a concentration  bark  of  of  as an arrow 1976).  the  (1983)  authentic  e_t a_l  in  poison.  The q u e s t i o n of  pharmacological effects preparing a methanolic into alkaloidal  their the of  only  the bark of  This  section  this  tryptamine,  2a).  were compared w i t h r e s p e c t  r e s p o n s e s o b s e r v e d i n mice a f t e r  (McKenna  detected  sample of this  study  the  by  arrow  compound t o  the  r e s i n was a p p r o a c h e d by  the  and n o n - a l k a l o i d a l  and M e t h o d s ,  a single  tryptamine  i m p o r t a n c e of  extract  used i n  0.245 mg/g d r y w e i g h t  Five-MeODMT was  Galeffi  extracts  from V i r o l a  (Agurell  tryptamines,  use of V i r o l a  e_t  and t r y p t a m i n e  species  had been a n a l y s e d and shown t o c o n s i s t  Materials  (Barata  has been p r o p o s e d t h a t  of  the  was e x a m i n e d .  The a l k a l o i d s  a_l,  known from  surinamensis  M a i a and R i b e i r o ,  derivatives  1969; H o l m s t e d t  substances  from V i r o l a  anti-fungal  (Gottlieb,  /3-carboline  active  the a n t i - s c h i s t o s o m a l  s u r i n a m e n s i n and v i r o l i n  et  reduce  RESULTS The o n l y  al,  ability  bark and s e p a r a t i n g  fractions  (described  The e f f e c t s t o the  of  it  in  the  gross  behavioral  intraperitoneal  injection.  106  The r e s u l t s  are  summarized i n T a b l e  The r e s u l t s behavior extract  clearly  was a l t e r e d than  elicited  by the  by t h e  alkaloidal  two e x t r a c t s .  stupor a.  of  several  E x a m i n a t i o n of The l a c k  elongata  of  raised  extracted  the  have r e p o r t e d  of  solvent toxic  larvae  To examine whether  an aqueous e x t r a c t pharmacological This extract  of  in,  of  injecting  reduced.  for  that  of  toxic ity  a toxic  of  extract  or  elongata  of  V.  constituent Mebs e_t aJL  a polypeptide  of  an  not (1982)  aqueous  a Bushman  arrow  contained a toxin  which  inactivated  by o r g a n i c  b a r k was e x a m i n e d  solvents,  for  40,  intraperitoneally  100 and 200 mg/kg. W i t h i n  200 mg/kg f r e q u e n t  injection,  The a n i m a l s  a p e r i o d of  V.  became somewhat  respiration,  fraction  and a s t a t e  the m e t h a n o l i c  was a d m i n i s t e r e d  m i n u t e s of  of  doses  activity.  i n d o s e s of  minutes  in a c t i v i t y  were  extract  highest  Diamphida n i g r o r n a t a ,  the  S w i s s mice  The a n i m a l s  the  non-alkaloidal  fraction  nature  poison.  insoluble  at  alkaloidal  m i g h t be p r e s e n t .  of  either  the  aqueous  extract  was  the  non-alkaloidal  Opposite e f f e c t s  Whereas t h e  the p o s s i b i l i t y  the  by t h e  gross  hours.  toxicity  by t h i s  extent  extract.  i n a marked r e d u c t i o n  lasting  the a n i m a l s '  hyperactivity  a c o m p a r a b l e d o s e of  resulted  that  to a greater  induced a m i l d degree tested,  indicate  VII.  uncoordinated and,  motor a c t i v i t y lay  writhing  was  p r o n e and an  two h o u r s ,  normal b e h a v i o r  female  two observed.  within  five  significantly  increased  a c c o m p a n i e d by p i l o e r e c t i o n ,  was  to  rate  of  was o b s e r v e d .  gradually  After  resumed.  1 07  Dose  administered  Amt. b a r k represented by e x t r a c t injected(mg)  5 (20)t  Behavioral.  Amt. 5-MeODMT in a l k a l o i d f r a c t ion mg  response  Nonalkaloidal fraction  Alkaloidal fraction  mg/kg  siight reduct ion in a c t i v i t y  0.05  1 .0  1 0(40)  0.09  1 .9  r e d u c t i o n in activity  20  (80)  0.19  3.8  inactivity:1 durat ion  hr  40  (160)  0.36  7.5  inactivity:2 durat ion  hr  si  hyperact: 10 min  80  (320)  0.74  15.0  inactivity:>3hr durat ion  si  hyperact: 15 min  T a b l e VII  effect  no  effect  no  effect  - G r o s s b e h a v i o r a l r e s p o n s e s of S w i s s m i c e t o 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 o n g a t a b a r k .  T h r e e m i c e p e r dose were o b s e r v e d : e x t r a c t s . w e r e intraperitoneal injection. t numbers fraction  no  in brackets  refer  to dose  (in  mg/kg)  of  administered  by  non-alkaloidal  108  Lower  d o s e s of  this  similar  behavioral  shorter  duration.  complete  recovery  An a t t e m p t activity  of  extract  (100 and 40 mg/kg)  changes,  but  of  T h r e e mice were after  lesser  extract  the  by f r a c t i o n a t i n g  it  using  however,  caused w r i t h i n g  and r e d u c e d a c t i v i t y  injected  into mice.  fraction  presence  of  layer  alkaloids  chromatograms of  iodoplatinate  the  fractions  was  fractions  and n i n h y d r i n  ion  obtained, levels  examined f o r  or amino a c i d s with  reagents.  cases.  biological  All  polar  the  in a l l  exchange chromatography.  Each  of  and  w i t h e a c h dose and  observed  was made t o c o n c e n t r a t e  this  intensity  injected  3 h o u r s was  produced  when  the  by s p r a y i n g  thin  Ehrlich's,  Only n e g a t i v e  results  were o b t a i n e d . The p o s s i b i l i t y the  aqueous e x t r a c t  that  the  resulted  irritant from t h e  activity presence  phenolic  m i x t u r e was c o n s i d e r e d . T a n n i c  injected  at  was  of  (J.  in  a complex L.  Baker)  was  a d o s e of  20 mg/kg.  It  produced a response  which  indistinguishable  from t h a t  of  t h e aqueous e x t r a c t  of  Virola  bark.  of  injection.  the  reduction also  acid  observed  The mice  Piloerection,  in a c t i v i t y  observed.  r e s p o n d e d by w r i t h i n g  lasting  within  two  the  minutes  a p r o n e p o s t u r e and a  approximately  three  h o u r s were  109  b.  Examination  of  the d i e t h y l  s p o n t a n e o u s motor  that  non-alkaloidal  p o s s e s s e d the fraction  was examined  gel  p o s s e s s e d the  ability  demonstrated t h i s consuming n a t u r e  of  yielded to  toxic  constituents  the of  undertaken.  the  methanol  the methanol  the  fractions  one of  and v a r i a b i l i t y  in  the  and p u r i f i c a t i o n ether  cage"  the  of  of  the  mice  time the  the of  major the  bark  major  measured d o s e s . A  was u s e d t o  T h i s approach allowed  compound's e f f e c t i v e n e s s  by  was a b a n d o n e d .  extract  The b i o l o g i c a l a c t i v i t i e s  "jiggle  of  bioassay,  g u i d e d by b i o a s s a y , "  modified version  of  levels  and t h e  this,  carefully  estimate  this  still  fractions  of  the  fraction  the  Because  the a c t i v e d i e t h y l  activity.  and  extract  which  activity  compounds were t h e n examined a t  locomotor  were  fractions  non-alkaloidal  More t h a n  separation  of  constituents  when a d m i n i s t e r e d t o m i c e ,  reduce  activity. of  of  non-polar  fractionation,  Instead,  d e p r e s s i o n of  further.  peritoneally.  a p p r o a c h of  effect  fraction  TLC on s i l i c a  injected  fraction  strongest  Preliminary  was  strongly  from b o t h t h e aqueous and t h e  the  for  activity  Having determined that absent  extract  a in  record  quantitative reducing  locomotor  activity. 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 diethyl  ether  extract.  ^-sitosterol;  two  stilbene  and i t s  eusiderin  (1a) (2)  These w e r e :  stilbene  the  ubiquitous  derivatives,  trans  and v i r o l o n g i n  isomer (3);  the  phytosterol,  3,4',5-trimethoxy-cis-  (1b); the  from  the  neolignans,  bis-tetrahydrofuran  110  lignans,  epi-sesartemin  and yangambin  (7);  (4),  and t h e  dihydrosesartemin  (8)  sesartemin  (5),  tetrahydrofuran  epi-yangambin lignans  and /3-dihydroyangambin  (9).  In  addition,  two u n i d e n t i f i e d a r o m a t i c  constituents,  compounds X and  were i s o l a t e d and t e s t e d .  The  and i d e n t i f i c a t i o n  these  compounds by s p e c t r o s c o p i c means has  P a r t A of  the p r e s e n t  The a b i l i t y  of  e a c h of  e a c h compound was  Y,  been d e s c r i b e d  of in  chapter.  s p o n t a n e o u s motor a c t i v i t y of  isolation  (6)  these  compounds t o  i n mice was  injected  tested.  suppress A pure  intraperitoneally  sample  i n a dose of  25 mg/kg. Some of  the  compounds t e s t e d  spontaneous  locomotor a c t i v i t y .  the  the  case  of  motor a c t i v i t y  This  following  signal  output  of  injection  and g r a d u a l l y  i n motor a c t i v i t y  of  The a v e r a g e (at  5 minute  injection testing  is  force  evident  began t o  Typical  of  the  presented  in  in Figure  d u r i n g the  in in  the (Figure  10 and 60 m i n u t e s recover  between  time c o u r s e s  for  i n d u c e d by e p i - s e s a r t e m i n  80  the  and e p i -  15.  24 measurements of  intervals)  obvious  transducer  between  in  The r e d u c t i o n  t h e compounds i s o l a t e d were t e s t e d  was c a l c u l a t e d  are  the  decreased s t e a d i l y  yangambin can be o b s e r v e d All  lignans.  c a u s e d by e p i - s e s a r t e m i n  and 90 m i n u t e s p o s t - i n j e c t i o n . reduction  T h i s was e s p e c i a l l y  bis-tetrahydrofuran  diminished e l e c t r i c a l 14).  c a u s e d marked r e d u c t i o n  in  way.  locomotor a c t i v i t y  2 hour p e r i o d  in each c a s e .  in Table V I I I .  this  compounds t e s t e d p r o d u c e d a s i g n i f i c a n t  following  The r e s u l t s  Ten of  the  made  of  this  thirteen  reduction  in  activity  Ill  Figure  14  - Example of output of " j i g g l e cage" f o r c e t r a n s d u c e r used t o measure spontaneous motor a c t i v i t y of mice.  a) r e c o r d e d d u r i n g n o r m a l motor activity, b) recorded after 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 c m / h r .  112  0  Epi-sesartemin (25 mg/kg)  n—i—i—i—i—i—i—i—i—i—r* 20  AO  60  80  100  120  TIME AFTER INJECTION(min.) Figure  15  - 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 s e s a r t e m i n a n d e p i - y a n g a m b i n on locomotor a c t i v i t y of mice.  V a l u e s a r e means, l e s s t h a n 15%.  N=  3:  SEMs a r e o m i t t e d  lignans, e p i spontaneous  from  graph  b u t were a l l  11 3  Dose(mg/kg)  Compound  Reduction % Control  t  ^-sitosterol 1a 1b 2 3 4 5 6 7 8 9 X Y control  Table VIII  t  25 25 25 25 25 25 12.5 6.3 25 25 25 25 25 25 25 Tween 80  in a c t i v i t y level  98 46 62 86 46 41 54 94 51 43 54 48 83 101 104 1 00  t-  SEM(n=4)  8 7 6 7 8 10 9 8 8 9 9 8 9 8 9 7  - E f f e c t of p u r i f i e d compounds of V . e l o n g a t a bark 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 S w i s s m i c e .  Compounds were i n j e c t e d  t- V a l u e s a r e a v e r a g e s of s t a n d a r d e r r o r of mean.  in  50 M1 of  4 injections  25% aqueous Tween per  80.  compound: SEM =  11 4  level.  3,4',5-trimethoxy-cis-stilbene  epi-sesartemin yangambin  (7)  (4),  sesartemin  (5),  (1a),  virolongin  epi-yangambin  and d i h y d r o s e s a r t e m i n  (8)  all  a 50% r e d u c t i o n  administered.  3,4',5-Trimethoxy-trans-stilbene  and 0 - d i h y d r o y a n g a m b i n  significant  activity.  / 3 - s i t o s t e r o l were c.  Only  (6),  produced  approximately  (2)  in a c t i v i t y  (3),  at  the  dose  (1b), but  eusiderin  (9)  showed l e s s e r ,  still  the  unknown compounds (X and Y)  and  inactive.  Quantification  of major  constituents  of  diethyl  ether  extract Because  such a l a r g e  possessed s i g n i f i c a n t of  importance  compounds  in  importance  inhibitory  to determine the  of  percentage  extracts.  the  resin  t h e compounds  activity  on m i c e ,  concentrations  Only  each c o n s t i t u e n t bark  of  of  then c o u l d the to  the  overall  it  tested became  e a c h of  the  relative pharmacological  r e s p o n s e of  the  performance  l i q u i d c h r o m a t o g r a p h y was u t i l i z e d t o q u a n t i f y  UV a b s o r b i n g compounds of analysis It  are  presented  c a n be seen  tetrahydrofuran important 38%,  importantly, that  they  lignans  of  were a c t i v e  in  extract.  in Table  the  are,  IX  that  the  four  quantitatively,  thirteen to  The r e s u l t s  High  of  the  this  IX.  Together,  with respect  represent  the  from T a b l e  constituents.  by w e i g h t ,  be e s t i m a t e d a c c u r a t e l y .  they  the  comprise  bismost approximately  compounds q u a n t i f i e d .  the  approximately  present 87% of  suppressing locomotor  study, the  is  the  More fact  constituents  activity.  that  11 5  Compound  RT(min)  1a 1b 2 3 4 5 6 7 8 9 X Y /3-sitosterol  Table  solvent solvent t  IX  18.80 18.45 15.94 15.22 13.33 13.33 14.47 14.47 1 1 .99 11.22 24.00 22.71  Rf  Concentration  1  2  0.75 0.68 0.63 0.59 0.37 0.31 0.26 0.21 0.15 0.09 0.56 0.53 0.75  0.89 0.77 0.53 0.46  Mg/g  dry bark  0.19 0.18 0.14 0.11 0.05 0.Q3 0.10 0.06 0.33  wt  1 .5 3.0 3.2 1 .6 50 t 50 f 26 f 26 f 1 .2 1 .3 1 1 8 95 t-  - Q u a n t i t a t i v e a n a l y s i s o f t h i r t e e n m o s t common c o n s t i t u e n t s of 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 .  1 = pet ether/ethyl ether/acetonitrile(6/6/1). 2 = hexane/chloroform/ethanol(25/25/1).  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 o f a b s o r b i n g s p o t s on T L C . t- a m o u n t e s t i m a t e d , b a s e d u p o n a m o u n t 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 .  UV  11 6  d.  Effect  of  bis-tetrahydrofuran  1 i q n a n s on i s o l a t i o n  induced  compounds 4 t o  was  aggression After  the  observed that passive  administration the  manner.  locomotor  treated  mouse o f t e n  At d o s e s l o w e r  activity,  discerned.  of  distinct  hesitancy  those  behavioral  Normal e x p l o r a t o r y  and a g e n e r a l  than  activity  was a p p a r e n t .  t i m i d and when h a n d l e d showed l i t t l e extremely  reluctant  to defend  These o b s e r v a t i o n s t e t r a h y d r o f uran that  this  arrow  poison.  effect by  of  in  This hypothesis  these  i n an  required  unusually to  suppress  c h a n g e s c o u l d be was v e r y  much r e d u c e d  The mice were resistance.  especially  They  the  hypothesis  that  possessed anti-aggressive  a role  it  were  themselves.  led to  lignans  may p l a y  behaved  7,  the  use of V .  was  tested  the  activity  elongata  and  b a r k as  by e x a m i n i n g  f o u r compounds upon a g g r e s s i o n  bis-  an  the  induced in  mice  isolation. The i n d u c t i o n of  isolation  i n mice by some s o r t  p r o c e d u r e has been u s e d e x t e n s i v e l y  e x p e r i m e n t a l model of and F r e d e r i c s o n , m e a s u r e d as study,  aggression  aggression  1951).  indicators  of  overt  introduction  aggressiveness  for  or  In  behavior.  their  aggressive  g r o u p of  have  Scott  been  present  nervousness  Consequently,  measurements were made i n e i t h e r  the  1982;  experimentation,  t o a g r o u p - h o u s e d mouse. T h e s e  displayed different  The l e s s  parameters  aggressiveness.  i s o l a t e d mice were s e l e c t e d  upon e i t h e r  an  ( B r a i n and J o n e s ,  A variety of  as  of  based  upon  two g r o u p s of  mice  different  case. i s o l a t e d mice were u s e d  to  11 7  examine t h e bis-  effect  of  tetrahydrofuran  related behavior. newly Each  different lignan,  d o s e s of  t h e most  epi-sesartemin  These m i c e were o b s e r v e d t o  i n t r o d u c e d g r o u p - h o u s e d mice  i s o l a t e d mouse r e s i s t e d a t t e m p t s  series  of  s h a r p s q u e a k s . When not  h o u s e d mouse, t h e y with pursuit  at  mouse. T h i s aggressive 1983).  latter  activity  behavior  and i s  As i n d i c a t o r s  of  injection  of  lignan.  interval  The r e s u l t s  dependent e f f e c t  upon t h e  behavior  behavior defensive the  the  observed  in  the  time  spent  mouse was m a r k e d l y mg/kg).  This  three  spent of  the  emitting group-  combined  group-housed with al,  activities  were  15 m i n u t e s  after  in Table  X.  had a d o s e -  in in  this  response to  in a c t i v e l y  especially  manner.  (Simon e t  observed  time  r e d u c e d , even a t is  it  epi-sesartemin  r e d u c e d upon a d m i n i s t r a t i o n behavior  the  presented  g r o u p - h o u s e d mouse, was a t t e n u a t e d  Moreover,  (3.1  was  of  beginning  are  that  only  of  these  indicate  Not  behavior,  has been c o r r e l a t e d  The r e s u l t s  i s o l a t e d mice.  p o s t u r e and  investigation  behavior,  with  c o n f r o n t e d by t h e  predictive  measured d u r i n g a 5 m i n u t e  interact  s o c i a l i z a t i o n by  displayed exploratory  and a n o g e n i t a l  on a g g r e s s i o n  in a c h a r a c t e r i s t i c  g r o u p - h o u s e d mouse, a d o p t i n g a d e f e n s i v e a  (4)  abundant  previously exploratory  lignan,  social  but  the  contact  by  significantly.  p u r s u i n g the the  lowest  suggestive  of  group housed  dose  tested  a reduction  in  aggressiveness. To v e r i f y  this  initial  demonstrated o v e r t l y effect  of  the  four  observation,  aggressive  behavior  bis-tetrahydrofuran  i s o l a t e d mice were u s e d t o  lignans.  Each  which test  was  the  1 18  Dose of  Epi-sesartemin (mg/kg)  Behavior  Number of squeaks  observed  % time  in 5 minute  in  e x p l o r a t ion  % time  period  in  plus  pursuit  anogenital  i n v e s t i g a t ion  25  4  1 1  0  12.5  3  9  0  6.3  6  21  1  3. 1  6  28  1 2  53  27  24  0  T a b l e X - E f f e c t of e p i - s e s a r t e m i n on t h r e e b e h a v i o r a l p a r a m e t e r s r e l a t e d t o a g g r e s s i v e n e s s of i s o l a t e d mice. Values are  averages  of  three  injections  of  each d o s e .  1 19  injected of  the  at  d o s e s of  2 5 mg/kg and t h e  i s o l a t e d mouse t o a t t a c k  observed. together  F i v e minutes a f t e r and t h e  time  the  effect  on t h e  readiness  g r o u p - h o u s e d mouse  injection,  elapsed before  t h e mice were  the  was placed  first  attack  by  the  each of  the  lignans  the  previously  i s o l a t e d mouse was m e a s u r e d . The d a t a tested  of  T a b l e XI  significantly  indicate  s u p p r e s s e d the  i s o l a t e d mouse t o a t t a c k . particularly contact  passive,  initiated  by r e m a i n i n g q u i e t l y  4.  The mice  offering  by t h e  w i t h eyes p a r t i a l l y  that  tendency treated  little  of  in  this  resistance  group housed p a r t n e r .  i n one c o r n e r closed,  of  the  way  to  They  were  the  social  responded  cage and h u d d l i n g  when c o n t a c t s  were made.  DISCUSSION The r e s u l t s  of  this  p r e p a r e d from V i r o l a arrow  study  indicate  b a r k may w e l l  that  and r e s t r i c t e d  arrow  poison  be u n i q u e among a b o r i g i n a l  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  subtle  the  to behavior.  observed in mice  No s e r i o u s d i s r u p t i o n  n o r m a l 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 observed of  i n any  relatively  of  large  the mice d o s e s of  The Yanomamo d a r t s reported to Laboratory elongata  each c a r r y mice  bark  readily  extract.  receive  bark  analysed  despite  survived It  the  of was  administration  extract. by G a l e f f i  approximately  would be e x p e c t e d t o weigh therefore,  tested,  were  1 5 0 mg of  d o s e s of  seems u n l i k e l y i n e x c e s s of  a significantly  et  auL (1983) resin.  2 0 0 mg/kg of that  V.  w i l d game,  1 kg and w o u l d ,  lesser  were  dose t h a n  that  which  1 20  Compound  Latency  to  attack:  time(min.)  epi-sesartemin(4)  >30  sesartemin(5)  >30  epi-yangambin(6)  >30  yangambi n(7)  >30  control  T a b l e 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 a g g r e s s i v e n e s s of m i c e .  3.7  lignans  on  L a t e n c y t o a t t a c k r e f e r s t o t h e t i m e between t h e i n t r o d u c t i o n t h e i s o l a t e d mouse ( t o w h i c h l i g n a n had been a d m i n i s t e r e d ) t o t h e s o c i a l i z e d mouse and t h e t i m e of t h e f i r s t a t t a c k of t h e i s o l a t e d mouse.  121  administered to mortality  the mice  in  this  from a d m i n i s t r a t i o n  study,  of  the  would s u f f e r  any  r e s i n o u s p o r t i o n of  the  darts.  bark  The s u g g e s t i o n t h a t  the  are  interrupting  injured  responsible game,  in  i n t r i g u i n g one. phenomenon. effects  elongata  that  true,  behavioral al,  it  is  an  effects  1980; Ho e t  man ( S h u l g i n , most p o t e n t  offer  alkaloidal  et  al,  sites  the  its  bark is  (Glennon  rodents  a_l,  1970)  1978),  fractions  responsible while  a reduction  the a l k a l o i d a l  et  al,  1980; bind  to  1975),  and P a g e ,  1962; G l e n n o n  at  is,  the  data  the  on t h e  (Table  a d o s e of  elongata  fraction behavior  VII).  The  presented  and  amounts of V .  in a c t i v i t y  fraction  in  shown t o be  The n o n - a l k a l o i d a l  were a d m i n i s t e r e d  for  primary  tryptamine.  from e q u i v l e n t  fraction  an  Virola  to  consistently  hypothesis  it.  of  comparable  al,  and G e s s n e r ,  effect  non-alkaloidal  et  ability  o b s e r v e d t o have a g r e a t e r  the  with  p o i s o n s . The  non-alkaloidal  they  is  and p s y c h o t o m i m e t i c a c t i v i t y  occurring  for  responses  5-MeODMT ( A g u r e l l  for  the  ethnobiological  s a m p l e s of  (Gessner  5-MeODMT i s  appealing this  no s u p p o r t  arrow  of  capture,  interesting  b a r k were compared and t h e  to which  normal  1983; H o l m s t e d t  in  naturally  However  of  of  1 9 8 4 ) . When t e s t e d  receptor  the  facilitiating  have been a n a l y s e d  1980; G a l e f f i  serotonin  constituents  seems t o be no p r e p a r a t i o n  the m a j o r i t y  McKenna e_t a l ,  here  If  way  amongst t h e many t y p e s of  et  this  There  alkaloid  1969;  for  tryptamine  was of  clearly  the  mice  injection  of  3.8 mg/kg was lasting  from an e q u i v a l e n t  several amount  hours, of  1 22  bark  (0.19  mg/kg)  p r o d u c e d no o b s e r v a b l e  B e h a v i o r a l changes administration (Ho e t  al,  It level  necessary  the  Galeffi  et  (by  weight)  darts not  the  analysed  of  the  with respect alkaloidal  report  bark  study,  bears  of  to low.  5-MeODMT c o m p r i s e d some 8%  it  is  a close  of  which  of  less  difference activity  likely  the  resemblance  to  is  not  the  has  resin  chemical c o n s t i t u e n t s .  the m e t h a n o l i c than  a  Yanomamo  that  of  The  5-MeODMT)  extract.  Although  r e p o r t e d by G a l e f f i  sufficient  to e x p l a i n  o b s e r v e d from t h e  alkaloidal  e_t  the  experiments.  extract  of  the  sample u s e d  (which c o n s i s t e d e n t i r e l y  in  locomotor a c t i v i t y  is  likely  phenolics present.  oxidation  that  coating  weight)  The r e d u c t i o n  It  bark  that  e x p e r i m e n t s was u n u s u a l l y  t y p e and amount of  in these  known.  possibility  methanolic  fraction  not  the  that  this  lack  constituent  thesis).  itself,  relative  of  0.5 mg/kg  resin,  slightly  alkaloidal  low as  bark  this  the  d o s e s as  by them. A l t h o u g h t h e  (by  (1983),  5-MeODMT  of  c o m p r i s e d 4.5%  al  behavior.  tips  to  is  these  resin  fraction  figure  this  in  the  been u s e d i n  extract  at  elongata  for  (1983)  of  II,  the  extracts a_l  response to  to c o n s i d e r  5-MeODMT i n  prepare  in  have been d e t e c t e d  1970; C h a p t e r  is  of  i n mice  change  The  the  result  identity  of  flavonoid constituents  pigmented dimers  (Gottlieb,  The c o n t r i b u t i o n  of  the  of  the  forms upon e x p o s u r e of  has been s u g g e s t e d t h a t  o b s e r v e d by t h e non-  it  the  complex  mixture  red-brown the is  resin the  and p e r h a p s  to a i r  result the  of  is the  formation  1979).  non-polar c o n s t i t u e n t s  identified  1 23  from t h e the  diethyl  ether  non-alkaloidal  results that  of  the  fraction  to  is  the  9 are,  biological activity  difficult  locomotor a c t i v i t y  compounds 1 t o  reducing  extract  to a s s e s s .  studies  to v a r y i n g  s p o n t a n e o u s motor a c t i v i t y  carried  degrees,  at  of  The  out  show  effective  moderately  high  in  doses  ( 1 0-25 m g / k g ) . It these  is  unclear,  constituents  significant  however, is  contribution  epi-yangambin  (6)  high concentrations extract, to  the  the c o n c e n t r a t i o n s  h i g h enough i n t h e  The b i s - t e t r a h y d r o f u r a n (5),  whether  to  its  lignans  overall  the  (7),  total  biological  lignan,  has been shown t o have h y p o t e n s i v e is  animals  stress  under  claimed to  of  their  constituents  of  proportionately  pinoresinol  activity  increase  the more  these  two  and  |3-D-glucoside syringaresinol  the performance  ( r e v i e w e d by MacRae  The mechanism by w h i c h  sesartemin  activity.  The b i s - t e t r a h y d r o f u r a n  0-D-glucoside  effects.  (4),  because  other  w o u l d be e x p e c t e d t o c o n t r i b u t e  t o make a  biological  epi-sesartemin  and yangambin  compared t o  resin  of  lignans  of  and T o w e r s ,  1984).  exert  effect  their  is  not u n d e r s t o o d . Similarly, isolation agents  induced aggression  are  is  completely  now known w h i c h a f f e c t  Among t h o s e  reserpine  barbiturates  drugs (Lai  reduce  s u c h as c h l o r p r o m a z i n e  e_t a l ,  1975),  the  in it  isolated  e_t a_l,  mice.  are  (Kreiskott,  anxiolytic  or b e n z o d i a z e p i n e s ( H a e f e l y  7 reduce  unknown. Many  aggression  w h i c h have been shown t o  antipsychotic or  t h e mechanism by w h i c h compounds 4 t o  1981)  drugs such 1981),  the  as  1 24  alkaloids opiate  m o r p h i n e and m u s c i m o l  peptides  certain  1982).  o b s e r v e d range anxiolytic  between  drugs,  peptides  The.doses required  for  lignans,  anti-aggressive  is  possible  virolongin,  that  as w e l l  for  w h i c h much h i g h e r  certain  as  somatostatin  an e f f e c t all  but  by c o m p a r i s o n ,  to  be  the  doses are  epi-sesartemin,  e p i - y a n g a m b i n and yangambin a r e ,  It  s u c h as  1 and 25 mg/kg f o r  The b i s - t e t r a h y d r o f u r a n  effective  1982),  s u c h as m e t - e n k e p h a l i n and n e o - e n d o r p h i n and  hypothalamic-pituitary  (Poshivalov,  (Poshivalov,  required. sesartemin,  moderately  agents. the  the  neolignans,  eusiderin  tetrahydrofuran  and  lignans  d i h y d r o s e s a r t e m i n and 0 - d i h y d r o y a n g a m b i n , may a l s o p l a y minor  role  ether  extract  agent,  in  the  pharmacological a c t i v i t y  of M a g n o l i a o b o v a t a  bark,  has been shown t o have s e d a t i v e  (Watanabe e_t a_l, (Fujita  e_t a_l,  1973).  1972)  The a v a i l a b l e does not c o n t a i n explain  its  previously  The major  not  unlike  evidence  a highly  use as an arrow been r e p o r t e d  effects  methanol e x t r a c t s  calophylla  that V.  at  a d o s e of  mortality. an arrow  It  are  bark  might  family Myristicaceae.  V.  sebifera  the  of  use of  not None  elongata  by a d m i n i s t e r i n g  c a n be c o n c l u d e d t h a t either  neolignans  elongata  which  100 mg/ml and f o u n d no e v i d e n c e  poison is  mice  T o x i c compounds have  of V.. p a v o n i s ,  have a l s o been t e s t e d  medicinal  in  was d e t e c t e d d u r i n g a t h o r o u g h s c r e e n i n g of V . Moreover,  The  and v i r o l o n g i n .  constituent  poison.  resin.  a Chinese  eusiderin  from t h e  the  constituents  indicates  toxic  of  a  bark. and V .  them t o  toxicity Vi rola  b a s e d on a more s u b t l e  sp.  mice or as  biological  1 25  activity  than  toxicity  physiological It  or,  s h o u l d be p o i n t e d out remain  arrow  The f i r s t  poison.  resin.  prepared Since  involves over  it  should,  therefore,  consideration. while  constituents  arrow in  the  elongata  means of  Yanomamos has n o t  a  as  the  resin  that  be b o r n e  in  the V i r o l a  of  used.  straightforward  and warming the  resin  dart the  the  tip  bark  resin  bark.  the a p p l i c a t i o n  and  The of  heat  and  this  mind. sample  used in  poison is  is  an  preparing  been  some a c t i v e c o n s t i t u e n t  The p o s s i b i l i t y  also  an  important  s t u d y was c o l l e c t e d  the  obtained  or q u a n t i t i a t i v e  is  this  used i n  there  is  preparation  from t r e e s that  of  in  in  authentic  growing  g e o g r a p h y may  variation  some d o u b t as  e l o n g a t a and V .  Yanomamo arrow separate  without  in contribute  chemical  must be c o n s i d e r e d .  Finally, V.  however,  The b a r k  Brazil.  to q u a l i t a t i v e  between  exact  from an e x t r a c t  the m a t e r i a l  Yanomamo arrow northern  the  f o r m a t i o n of  The s o u r c e of  Peru,  the  seems i m p r o b a b l e t h a t  exists,  promotes the  is  s t u d y may be c r i t i c i z e d b e c a u s e  preparing  significantly  possibility  it  important  use of V .  n o t h i n g more than c o l l e c t i n g  a fire,  differs  several  the  concerns  t h e manner of  t h e method of  that  regarding  The p r e s e n t  in  that  basis.  uncertainties  the  alternatively,  poison.  species  theiodora,  Virola  by S c h u l t e s  (1969)  this  to  the  the  theiodora  p o i s o n have been a t t r i b u t e d taxonomy of  to  is  relationship  two  sources  considered a  and most of this  of  the  species.  genus have g e n e r a l l y  sources  of  Specialists  regarded  V.  1 26  theiodora  and V .  elongata  S m i t h and Wodehouse, the  latter  name i s  species  concept  general  agreement  field. G.T., the  pers.  of  aspects the  (1960),  p o i n t e d out the  of  these  r e s i n arrow  is  it  this  the  assaying  substance the  resin  to by  experimental  administration  or  may i n t r o d u c e from t h i s this  and arrow study of  arrow  the  these  the  of  poison.  Clearly,  is  two  is  Primate  5-MeODMT t h a n  q u e s t i o n s are  of  ever  is  A  now i n  order.  ethnological importance (1931)  h u n t i n g monkeys and b i r d s . and H o l m s t e d t  system.  the  the  type  of  (1968)  is  so as  conceivable  injection  Becher  that to  that  in mice  is  hot  t h e mode of  particular  animal  Of p a r t i c u l a r of  has  have  p o i s o n and  some t i m e  Either  behavior that  arrow  It  of  comparative  hunting. Salathe  effect.  the  report  Of p a r t i c u l a r  for  the  (Prance,  first  tree.  the  used  importance  animals  hunted  may be more  easily  with  rodents.  more c o m p l e t e d e s c r i p t i o n s  use and e f f e c t s  the  types  poison.  r e s p o n s e of  view,  poison  either  unsuspected v a r i a b l e s .  point  d i s r u p t e d by  of  in  intraperitoneal  an a p p r o p r i a t e  1980). A l t h o u g h  resolved,  a slow a c t i n g  take  only  c a n be d i s t i n g u i s h e d  wounded a n i m a l must be f o l l o w e d  allow  monograph,  is  and S c h u l t e s is  1980;  there  used f o r  used f o r  (1970)  that  is  (Rodrigues,  latest  i n our u n d e r s t a n d i n g of  it  Prance  types  constituents  i n which  reported that  two  The p r e s e n t  chemistry  of V i r o l a way  the  completely  u s e d as a s n u f f  Gaps e x i s t  is  that  comm.).  the  and i n  not y e t  non-alkaloidal  study  1937)  be synonomous  recognized (Rodrigues,  is  Both are  to  of  field  the arrow  poison w i l l  t o be f u l l y  answered.  observations  be n e e d e d  if  127  LITERATURE Chapter Abe,  CITED III  F . , Y a k a r a , S . , K u b o , K., N o n a k a , G . , O k a b e , H. and N i s h i o k a , I. (1974) S t u d i e s on X a n t h o x y l u m s p p . II. C o n s t i t u e n t s of t h e b a r k of X a n t h o x y l u m p i p e r iturn D C . Chem. 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ACTIVITIES  INTRODUCION The f a m i l y ,  plants the  u s e d by  the  local  almost  important  to  800 s p e c i e s of  probably  large  I).  effective  and c a n be p u r c h a s e d i n  lechleri..Alchornea  grows,  partly  is  highly  is  t h e Amazon r e g i o n ,  enumerated  plants  observed wounds,  by the  (Appendix is  b r o a d and  skin  and u s e d a s  p o i s o n o u s and t h e i r  used w i d e l y  Croton  bark  It  tonics use  and  is  property.  species are  banks of  as m e d i c i n a l s  the  places.  market  castaneifolia  the  the  p r e s c r i b e d by  is  urinaria a small  p e r i o d when t h e  t h e A m a z o n ' s major  esteemed f o r  by  frequently  Phyllanthus  submerged d u r i n g t h e  along the  the  c o m p r i s e s some 6% of  inflammations  Many s p e c i e s a r e  (Appendix A ) .  used as m e d i c i n a l  against  include Alchornea c a s t a n e i f o l i a ,  high,  in  It  m e s t i z o p o p u l a t i o n . They a r e  herbalists  r e p r e s e n t e d among  pharmacological actions  b a s e d on t h i s  Certain  family  species  (Chapter  malignancies,  aphrodisiacs.  well  of m e d i c i n a l p l a n t s  includes preparations infections,  plant  numbers of  inhabitants  A).- The range  is  i n d i g e n o u s South Americans  t h i r d most  with respect  Euphorbiaceae,  treatment  of  These and  tree rivers  tributaries. rheumatic  that are Its  1 35  c o n d i t i o n s and i t s stimulate,  or  leaves  are  taken  as a d e c o c t i o n  rejuvenate,  the  older  man. P h y l l a n t h u s  and p e r h a p s a l s o  Phyllanthus  number of  ailments  effective  i n c a u s i n g the  kidney  and g a l l  f irme whose  wound t r e a t m e n t  and f o r  of  exaggerated.  in  the  occasionally In cancer,  are  resin  internal  (Hartwell, species,  family 1969).  (Manihot  esculenta),  urinaria) Only previously  are  Jatropha  and  survey  for  constituents.  of  of  is  Of  They a r e  both  Jatropha used  infections  and  very  (Kupchan  well  against  represented  (9 s p e c i e s ) ,  Croton  Jatropha  in  listed  biologically  a compound,  used  among o t h e r s ) ,  represented  that  plants  (both C.  and P h y l l a n t h u s  those  activity  curcas  be  Chamaesyce  and J..  the p l a n t s  their  found to c o n t a i n antitumour  curcas  already  a few  abilities  malignancies.  thymi f o l i a ,  including J .  its to  The g e n e r a A c a l y p h a  species,  tierra  and m a l i g n a n c i e s .  b u r n s and s k i n  Euphorbiaceae  and C .  the  wounds seem not  i n c l u d i n g C. p a l a n o s t i g m a ,  hyssopi f o l i a  of  concerning  dooryard c u l t i v a r s .  treat  be  e x p u l s i o n of  tree  injuries  Two s p e c i e s ,  to  a  c o l l e c t e d and u s e d as a  the c l a i m s  Jonathan H a r t w e l l ' s the  is  is  urinaria,  treat  believed  and/or  Croton l e c h l e r i  of  used to  and a r e  superficial  treatment to  are  fragmentation  experience,  gossypiifolia, widely  kidney  red bark  t o promote h e a l i n g greatly  the  stones.  thick  From p e r s o n a l  of  amarus,  to  have,  gossypi i f o l ia) , (3  species,  the  et. a_l,  including  P.  literature. examined  chemical  Jatropha  jatrophone,  (9  Manihot  above have been  active  (9  curcas  that  1976).  has  been  possesses  Similarly,  1 36  Phyllanthus  brasiliensis,  P.  and P.  urinaria  antitumour  contain  the  ability  1 9 7 7 ) . Major found to  lignan, et  al,  of  dimers  related  shown t o c o n t a i n (Kupchan,  is  a_l,  has  not  been  only  1979),  found  but a l s o  several  other  1981).  Phyllanthus  from the  have been  compounds,  point  similar  have r e c e n t l y  (Bedows and H a t f i e l d ,  Euphorbiaceous predominant  in  of  t h e Amazon was is  structure  lignans.  units. contain  the  (Chatterjee  studied  chemistry  and  five  hypophyllanthin,  (Anjaneyulu, to  1973).  the  Podophyllotoxin,  and  been shown t o have a n t i v i r a l  is  of  secondary  already  activity this  The p o s s i b i l i t y the  its  phyllanthin,  in  structure  biological  could explain  view of  been  been  related activity  1982).  species  p r o d u c e d by p l a n t s  has  possesses  have  been shown t o  and p h y l t e t r a l i n  g r o u p of  The v a r i e t y  of  identified:  nirtetralin,  podophyllotoxin  urinaria  to  (Sethi,  species  phenylpropanoid has  the  anti-  transcriptase  of  to  1977).  reverse  gossypi i f o l i a  T h e s e compounds a r e  It  closely  2-piperonyl-3-veratryl-3R-7-butyrolactone  niranthin,  1979).  that et  viral  constituents  Jatropha  intensively lignans  inhibit  is  Croton l e c h l e r i ,  taspine,  be l i g n a n s :  Recently,  has been  (Persinos-Perdue to  that  phyllanthocin  species,  an a l k a l o i d ,  inflammatory the  amarus,  substance,  One of  a plant  that  widespread  the  known t o be g r e a t .  observed  family  is  of  for  of  from The  compounds  toxicity  presence  uses  products  (Kinghorn,  toxic  compounds  Euphorbiaceous  species  in  considered.  apparent,  from what l i t t l e  we know of  the  diseases  1 37  of  primitive  infectious 1975;  et  al,  have  of  tendency  for  infected  is  the  Paracoccidiodes  injuries  et  al,  and  ear or  (Black,  1968; L a r r i c k  and mouth a r e insect et  bites  a_l,  is  high  et  al,  not uncommon  to  1979).  (Larrick  diseases,  (Epstein-Barr  a_l,  infections  figured prominently  (Larrick  brasiliensis  Brazilian  that  become The  incidence  f u n g a l p a t h o g e n s H i s t o p l a s m a c a p s u l a t u m or  and c y t o m e g a l o v i r u s the  eye,  1 9 7 8 ) . The v i r a l  mononucleosis  all  the  always great  exposure to  and N e t t o ,  always  1978; Buck  Infections  and t h e  of  diseases  Black  1979).  Amazonian s o c i e t i e s ,  virus),  diseases  have  e_t §_1,1979; Mok  herpes  simplex,  chicken pox, h e p a t i t i s  been shown t o  tribes  examined  (Baruzzi  1968).  Amazonians are  1975; N e e l  et  variety  of  p r o t o z o a n and h e l m i n t h i c  is  reasonable  et  be endemic  aJL,  1971;  regularly  parasites  B to  Black,  exposed to a  (Larrick  et.  al,  1979) . It fraction  of  expect  the m e d i c i n a l  would be u t i l i z e d this  to  nature.  in  the  The t o x i c  may be a p p l i e d  plants  treatment  in such a  Amazonian E u p h o r b i a c e o u s  of  leaf,  bark  (or  were c o l l e c t e d .  family the  species  for  related  diseases  Euphorbiaceous  an  to  the  of  species  of  of  toxicity  or  interesting  p o r t i o n s of  these  screening  a variety  were s e l e c t e d at  aerial  Sixteen  of  i n d i g e n o u s Amazonians  infectious  suggested that  would c o n s t i t u t e  from t h i s  of  large  way.  pharmacological a c t i v i t i e s  species  a proportionately  u s e d by  properties  These o b s e r v a t i o n s  regulation,  that  of  growth  study.  Thirty-four  random and herbaceous  34 s p e c i e s had some  samples species)  1 38  ethnobotanical  use.  Extracts  p r e p a r e d and examined f o r of  bacteria,  tumours,  as  yeasts, w e l l as  Antibacterial organism,  E.  coli  fungi  their  their  and t h e  their  virus,  sensitivity  for to  the  and  extracts. single  growth potato  negative  bacterium,  Four  S.  aureus  yeast,  S.  dermatophytic  f u l v u m , M. for  available  gypseum  which were examined  These were  Sindbis  s t r a n d e d RNA v i r u s  a membrane e n c a p s u l a t e d ,  and  double  virus.  The a b i l i t y g r o w t h of  potato  gram  M.  h a n d l i n g are  the  shrimp.  Two a n i m a l v i r u s e s  a membrane e n c a p s u l a t e d ,  murine c y t o m e g a l o v i r u s ,  the  viruses  included brewer's  Microsporum c a n i s ,  techniques  s t r a n d e d DNA  the  were  inhibit  to brine  gram p o s i t i v e  and T r i c h o p h y t o n g a l l i n a e .  for  to  fungi,  against  tested  material  pathogen, C. a l b i c a n s .  were t e s t e d :  laboratory  plant  ability  toxicity  activity  and t h e  the  dermatophytic  was e x a m i n e d . The y e a s t s cerevisiae  of  tuber  of  the  34 E u p h o r b i a c e o u s e x t r a c t s  tumours e l i c i t e d d i s c s was a l s o  inhibit  by A g r o b a c t e r i u m t u m e f a c i e n s  s t u d i e d . This b i o l o g i c a l assay  been shown t o  be p r e d i c t i v e  the  3PS  leukemia c e l l s  the  general  g r o w t h of  to  of  an e x t r a c t ' s i_n v i v o  ability  (Ferrigni  to et  in has  inhibit al,  1982) . Finally, using  the  brine  shrimp  presence  brine  of  pollutants,  toxicity  shrimp assay  (Artemia a variety  salina) of  of  (Meyer  et  a_l,  was  assessed  1982). T o x i c i t y  has been u s e d t o m o n i t o r  compounds i n c l u d i n g  mycotoxins, a n e s t h e t i c s ,  morphine a n a l g e s i c s ,  extracts  to  the  pesticides,  dinoflagellate  c o c a r c i n o g e n s and c a r c i n o g e n s  toxins, (Meyer  et  1 39  al,  1982).  growth  It  is  a l s o capable  regulatory  Euphorbiaceae  certain  detecting  compounds t h a t  (Kinghorn  The p r e s e n t  of  study  et is  a_l,  are  intended to provide  the  biological activities  important,  yet  chemically  on t h e  types  among t h e their  of  tested  the  g r o u p of  of  uses. extent  plants  of  is  novel,  2.  MATERIALS AND METHODS  a.  Plant  i n the  family  an  a description  g r o u p of  plants.  general  perhaps,  It  information  pharmacological  activities  help  explain  the  known t o  i n d i g e n o u s Amazonians  the  biological  some  to which  the  activities  ethnobotanical  eventual  biologically  of  ethnobotanically  may be p o s s i b l e t o g a i n  into  literature).  isolation  active  (or  and  constituents.  material  P l a n t s were c o l l e c t e d have been d e p o s i t e d a t Peru),  the  It  i n f o r m a t i o n may l e a d t o  identification  known,  which w i l l ,  what has been t r a n s m i t t e d This  of  a p p r o a c h would y i e l d  ethnobotanical  this  little  and p o t e n c i e s  species  appreciation of  this  widespread  the  1977).  of  was hoped t h a t  phorbol esters,  UNAP  in  Peru in  (Iquitos,  1981. V o u c h e r Peru),  specimens  San M a r c o s  (Lima,  C h i c a g o F i e l d Museum and t h e UBC h e r b a r i u m .  Determinations  were c a r r i e d  F i e l d Museum. Samples  out  by D r . M . J .  were p r e s e r v e d  Huft,  in methanol  Chicago soon  after  collection. An e x t r a c t  of  p o d o p h y l l i n , the  resin  of  Podophyllum s p .  r h i z o m e was  i n c l u d e d i n e a c h of  the a s s a y s  control.  is  p o d o p h y l l o t o x i n , as w e l l a s a  It  known t o c o n t a i n  as a  positive  1 40  number of activity b.  other  lignans,  with antitumour,  (MacRae and T o w e r s ,  Preparation Leaf,  of  bark,  plant or  evaporation acetate.  aerial  part  the  ethyl  final  c.  Antimicrobial  g r o w t h of S.  of  the  and  the  the  and C .  bacteria  and y e a s t s  broth  (Difco)  extracts  (100 mg/ml)  (img)  to  or  in  for  The  They  were  a distinct  Sabouraud's  in  was v i s i b l e  (24  to  agar  swab.  aliquots  agar  They were  assay  dextrose  of Whatman N o .  had been a d d e d . "lawn"  10 nl  the  Overnight  cotton  the  inhibit  and  were a p p l i e d t o  were a p p l i e d discs  to  disc  activity.  d r i e d and a p p l i e d t o  yeast  aureus  using a s t e r i l e  6 mm d i a m e t e r  T h e s e were a i r  or  ability  The p a p e r  antimicrobial  Plant  sterile  coli  albicans.  respectively,  30 ° C u n t i l  100 mg/ml.  and S.  E.  (Difco),  bacteria  was  respectively.  bacteria,  broth  which  50% a q u e o u s e t h a n o l  fractions,  extracts  w a t e r and  d r i e d by e v a p o r a t i o n  their  containing nutrient  paper.  ethyl  were e x a m i n e d f o r  cerevisiae  of  between  yeasts  method was u s e d t o a s s e s s cultures  by  screening  The e x t r a c t s  yeasts  95% or  exhaustively  °C.  Bacteria  the  in e i t h e r  was  were c o n c e n t r a t e d  were s e p a r a t e d ,  a c e t a t e and a q u e o u s  -30  herbaceous p l a n t s ,  and e x t r a c t e d  i_n v a c u o and p a r t i t i o n e d  concentration  s t o r e d at  of  20 ° C . The e x t r a c t s  v a c u o and d i s s o l v e d  toxic  extracts  the  The f r a c t i o n s  and  1984).  homogenized i n a Waring b l e n d e r w i t h methanol at  antiviral  1  filter  plates  incubated 48 h o u r s ) .  to at  141  The zone of its  i n h i b i t i o n a r o u n d any  diameter  r e c o r d e d . Assays  Dermatophytic Sporulating and T .  gallinae  of  t h e d i s c s was  were c a r r i e d  cultures  of M. c a n i s ,  were o b t a i n e d  from t h e D e p a r t m e n t  inhibition  g r o w t h was a m o d i f i c a t i o n of  1 ml of  on an agar  slant  tip,  The method u s e d t o a s s a y  and G o l d i n g  by a d d i n g  (1953).  sterile  a s m a l l amount of of  various  amounts of  were l e f t  20 ° C  spore  agar,  plant for  Ant i v i r a l  extracts  extract  14 d a y s .  activity  on p l a q u e  s t r a n d e d RNA v i r u s  case,  Medical  extracts  reported  t o a mature  briefly.  and  was c a r r i e d  wire  transferred  to  included  1% e t h a n o l .  The d i a m e t e r  by  culture  Using a sharp  s u s p e n s i o n was  for  prepared  of  out  The the  in  plates colonies  triplicate,  in  was a s s a y e d by d e t e r m i n i n g t h e  (MCMV). the  s t r a n d e d DNA v i r u s the e f f e c t  and on c e l l s  of  the  already  Viruses Sindbis  water  forming a b i l i t y  murine c y t o m e g a l o v i r u s  double  that  fulvum  activity  Antiviral of  the  i n w h i c h had been  formed was m e a s u r e d . The a s s a y d.  it  of  s u s p e n s i o n s were  distilled  the  Sabouraud's  at  Spore  and v o r t e x i n g  a plate  triplicate.  M. gypseum, M.  U.B.C.  Brancato  in  fungi  Microbiology, of  out  n o t e d and  and virus  had been grown  of  Sindbis  Sindbis  virus  family  Togaviridae  of  herpes v i r u s  the  extracts  infected  on v i r u s  with v i r u s ,  is  effects  virus a  and  single  w h i l e MCMV i s g r o u p . In  prior  to  a  each  infection  was m e a s u r e d .  cells (obtained  originally  i n BHK-21 c e l l s  from D r .  D.E.  and p u r i f i e d by t h e  Vance)  technique  1 42  described previously in  tissue  culture  cytomegalovirus cells,  (Mosmann and H u d s o n ,  medium a t  (Smith s t r a i n )  p u r i f i e d and s t o r e d  Both v i r u s e s strain)  -70 ° C p r i o r  to use.  in the  (passages  18 t o  effect  21).  in D u l b e c c o ' s modified E a g l e ' s  containing  0.37% sodium b i c a r b o n a t e ,  100 U n i t s / m l  streptomycin Chemie A G ) . 95% a i r :  sulfate  penicillin (Sigma)  forming  were c a r r i e d  initially  virus,  60 mm d i a m e t e r  out  only  and i n  in  the  (Gibco)  bovine  serum  100 Mg/ml' (Cilag-  i n an a t m o s p h e r e  infection) appropriate  dilutions  In of  the  treatment  of  C o n c e n t r a t i o n s of  1,  the  extracts  virus  d i s h e s on case  of  (4-5 mm)  either  infection  and  plaques  prior  (post-  pre-infection the  medium (MEM)  10 and  virus  used.  were e x p o s e d t o  in Dulbecco's modified Eagle  1% e t h a n o l .  the  large  d i s h e s were  or a f t e r  virus  In  S.indbis  MCMV p r o d u c e s s m a l l e r  were e x p o s e d t o p l a n t  cells.  of  the p e t r i  relatively  d i s h e s were u s e d ;  for  dilution  were g r o w n .  (pre-infection)  of  similarly the  1 mm) and 35 mm d i a m e t e r  infection  extract  37 ° C  s i z e of  the p l a q u e s are  The v i r u s e s to  medium  10% f e t a l  (3T3  were  and 2 Mg/ml e c o n a z o l e  t h e mouse embryo c e l l s  (approx  i n ME  assay  and MCMV. They d i f f e r e d  Sindbis  Murine  5% c a r b o n d i o x i d e .  The a s s a y s  which  stored  on mouse  Cells  G (Sigma),  They were i n c u b a t e d a t  Plaque  prepared  was  same manner.  cultivated  (Gibco),  It  had been p r o p a g a t e d  were a s s a y e d by t h e i r  embryo c e l l s  1973).  assay, plant  containing  100 Mg/ml e x t r a c t  were  143  tested.  After  extract  mixture  and  incubated  virus/ cells  plant  37  be  counted.  first  virus  extract After  with  against  they  a dark  Potato  The  disc  of v i r u s  used  was  variety)  20%  bleach  f o r 15 m i n u t e s .  and a  rinsed applied  knife  incubated  developed  carried cells MEM  to  o u t by  with  for 2  hours. were  5% F B S , t h e p l a n t  the plates  were  agarose.  incubated at  f o r counting. Plaques  were  counted  the unaided eye.  that  modifications.  t h i c k n e s s were  well  was  embryo  were  assay  Russet  borer  assay  containing  using  red  4 mm  they  of  5% F B S a n d 0 . 5 %  c o n c e n t r a t i o n 1 % ) , a n d 0.5%  ready  cells  the  removed and t h e p l a t e s  MEM  background tumour  containing  sufficiently  had s o l i d i f i e d , were  °C. F i n a l l y ,  in Dulbecco's  then  (final  procedure  some m i n o r  MEM  were  embryo  removed and t h e monolayer  o f mouse  Dulbecco's  in ethanol  o f mouse  had s o l i d i f i e d ,  monolayers  the overlay  was  Dulbecco's  was  °C, t h e v i r u s / p l a n t  a t 37  post-infection  amounts  °C u n t i l  with  mixture  suspension  overlayed  2 hours  the plaques  infecting  a t 37  t o a monolayer  the plates  treatment  appropriate  e.  added  extract  °C u n t i l  The  incubation  f o r another  After  at  The  was  overlayed with  agarose.  37  2 hours  were  with  to a  layer  of  from razor  sterile  1% a g a r  e_t a_l ( 1 9 8 2 ) ,  (Solanum  sterilized  Sterile  containing  times  Potatoes  surface  prepared  three  of F e r r i g n i  discs  of  the tubers  by  tuberosum,  immersing  15 mm using  diameter  and  a cork  hole  b l a d e s . The d i s c s  distilled  (Difco)  water,  in petri  in  were  d r a i n e d and  dishes.  1 44  Twenty M1 of an overnight c u l t u r e of Agrobacterium tumefaciens, s t r a i n B-6 (obtained from Dr. M.P. U n i v e r s i t y of Washington) i n n u t r i e n t a p p l i e d evenly t o each d i s c . A f t e r extracts, dissolved (DMSO) (Eastman  broth  Gordon,  ( D i f c o ) was  2 hours, the p l a n t  i n 10% (v/v) aqueous d i m e t h y l s u l f o x i d e  Kodak Co.), were added  t o the d i s c s i n  a l i q u o t s of 20 M1 and spread e v e n l y . The d i s c s were incubated at  25 °C i n the dark f o r 12 days. The tumours,  which were  between 0.25 and 1.5 mm i n diameter by t h i s time, were illuminated  from the s i d e and counted with the a i d of a  d i s s e c t i n g microscope. f.  Toxic i t y  t o br ine  shr imp  The method used has been d e s c r i b e d by Meyer e_t a l (1982). Brine shrimp incubated for  in a r t i f i c i a l  seawater  (Marine E n t e r p r i s e s , L t d . )  48 hours a t 20 °C. The l a r v a e were counted i n t o groups of  10 and p l a c e d been added v/v).  (Artemia s a l i n a ) eggs (New Technology, Ltd.) were  i n 5 ml of a r t i f i c i a l  the p l a n t e x t r a c t  seawater, t o which had  i n DMSO ( f i n a l c o n c e n t r a t i o n 1%,  C o n c e n t r a t i o n s of 10, 100 and 1000 Mg/ml e x t r a c t were  t e s t e d . S u r v i v a l was measured a f t e r  24 hours i n c u b a t i o n a t 20  °C. g. A n a l y s i s of data The  screening  for inhibition  of growth of b a c t e r i a and  y e a s t s a l l o w e d some degree of q u 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 saturated f i l t e r  surrounding the e x t r a c t  paper d i s c . In the case of the assays f o r  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 concentrations  growth  of p l a n t  in the presence  extract.  Results  minimum c o n c e n t r a t i o n  required  colony  of u n t r e a t e d  t o 25% o f t h a t  dilutions  of p l a n t  extract  required to s a t i s f y  this  The a n t i - v i r a l , toxicity  assays  quantified  involved  and t h e L D  (Ashton,  3.  RESULTS  a.  Antimicrobial The r e s u l t s  are  presented  bacteria extracts. only  Data  1972; H a f n e r  of the a n t i b a c t e r i a l and X I I I .  d i f f e r e d markedly o f E. c o l i  two o f t h e 34 s p e c i e s  tested  (Table  XII).  of o n l y  C. albicans  the presence  canis  mean  squares  1977).  screening  The two s p e c i e s o f  in their  response to the  was i n h i b i t e d  by e x t r a c t s o f  (6%) w h i l e S.  o f 26 ( o r 76%) o f t h e s p e c i e s was c o m p l e t e l y  a n d S. c e r e v i s i a e  resistant  to  was i n h i b i t e d by t h e  two s p e c i e s .  The f o u r d e r m a t o p h y t i c to  to logit  and a n t i f u n g a l  of Euphorbiaceae  was i n h i b i t e d by e x t r a c t s  extracts  easily  discernible  by l e a s t  and Noack,  shrimp  activity  The g r o w t h  the e x t r a c t s  o f a more  exhibiting easily  aureus  all  Three-fold  tumour a n d b r i n e  calculated  5 0  of the  was r e c o r d e d .  were s u b j e c t e d  i n T a b l e s XII  tested  the diameter  a n d t h e minimum d o s e  the c o l l e c t i o n  relationships  transformation analysis  criterion  were e x p r e s s e d a s t h e  colonies.  were t e s t e d  anti-potato  s e t of d a t a .  dose-response  t o reduce  of d i f f e r e n t  fungi  of the e x t r a c t s  was t h e most  sensitive  tested  (Table  were more  XIII).  sensitive  Microsporum  of the dermatophytes,  being  Table  XII Spec i es  Part  Extract*  Zone o f E.  Acalypha  benensis  leaf bark  A.  diversifolia  1 eaf  A.  macrostachya  bark  A.  stachyura  bark  Alchornea A. A.  castaneifolia  di s c o l o r  bark bark  triplinervia  •Amanoa a f f .  bark  oblonqifolia  leaf bark  Aparisthmium  cordatum  leaf bark  Apodandra  loretensis  Caryodendron Chamaesyce C.  orinocense  hyssopifolia  thym i f o l i a  Cnidoscolus  peruvianus  bark bark  Croton  cuneatus  martianum  o a o a o a o  -  S.  aureus  C.  a 1b i c a n s  i n mm) S.  cerevisiae  -  2 4 4 2 2 -  -  -  o a o  -  3 2 -  -  -  a 0 a o  -  1 2 1  a o a o  -  a o a o a o a o  -  -  -  -  -  1 1 -  -  -  -  2  -  -  -  2 2 2 2 1  -  -  1 1 1  -  -  -  -  -  -  aer i al  a o a o  leaf  o  -  -  -  -  -  3  -  -  -  1 1 1 2 -  -  -  --  -  aer i a 1  bark Conceveibastrum  co1i  i n h i b i t i on(w i d t h ,  bark leaf bark  a o a o a o a o a  -  -  2 -  C .  1echler i  bark  -  2  a o a o  -  2 2 1  -  leaf  a o  -  -  -  -  bark  o  -  1  -  4  -  -  -  -  aer i al  a o a o a o  -  -  -  leaf  o  -  -  -  -  -  -  -  -  3 -  -  -  -  3 3 1 1 1 1 3 3 4 1 -  a o  -  -  o a  -  -  C . p a 1 a n o s t igma  bark  C.  aer i a 1  trinitat is  Didymocistus  Hevea  chrysadenius  brasi1iensis  Jatropha  curcas  J.  qossypi  ifolia  J.  weberbaueri  leaf aer i a 1  bark Mabea maynens i s  leaf  M . n i t i da  bark  Manihot  aer i a 1  esculenta  Maprouna  guianensis  Phyllanthus  amarus  bark aer i a 1  P.  orbiculatus  aer i a 1  P.  pseudo-conami  aer i a l  P.  ur i nar i a  aer ial  Podocalyx  loranthoides  leaf bark  Securinecja  congesta  Podophy11i n t  Table  XII  aer i a 1 res i n  - Antimicrobial screening o r p r e s e n c e ( w i d t h of zone * o = organic  -  o  fraction;  of of  a o a o a o a o a o a o a o a o a o a o a o  -  -  --  5 -  -  fraction  -  -  --  --  -  -  -  -  -  -  -  2  e x t r a c t s of Euphorbiaceous p l a n t s . i n h i b i t i o n i n mm) o f a n t i m i c r o b i a l  a = aqueous  -  t  Podophyllin  Results are activity. resin  expressed  extract  is  as  absence(-)  included  as  a positive  control  Table  XIII Concentration Part  Spec i es  Acalypha  benensis  leaf bark  A.  diversifolia  leaf  A.  macrostachya  bark  A.  stachyura  bark  Alchornea  castaneifolia  bark  A.  discolor  bark  A.  t r i p1i nerv i a  bark  Amanoa a f f .  oblonqifolia  leaf bark  A p a r i sthm i urn c o r d a t u m  leaf bark  Apodandra  loretensis  Caryodendron Chamaesyce C.  orinocense  hyssopifolia  bark aer i a l aer ial  thym i f o l i a  Cnidoscolus  bark  peruvianus  leaf bark  Conceveibastrum Croton  cuneatus  martianum  bark leaf bark  Extract* M.  can is  p r o d u c i n g >75% M.  gypseum  M.  of growth(  fulvum  T.  „g/ml ) g a 1 1 i nae <0.125  0 2 1 1 0 2 0  25 0 0 0 25 0 5 5 0 5  0 25 0 25 1 0 2 .0 2 0 <0.125 2 0 0. 5 2 .0 0. 5 2 0 <0.125 2 .0 1 . 0 2 .o 1 0 2 .0 <0.125 e  0 1 0 1 1 1 2 2 2 2 1 2 0 2 0 2 2 2 0 1 1  <0.125  <0.125  <0.125  <0.125  0 . 25 0 . 25 <0.125 2.0 <0.125 2.0  0. 0. 2 0 2  1 o 0 5 0 5 2 0  0 2 0 0 2  5  o a o a o a o  < 0.125 0 . 25 <0.125 <0.125 <0.125 <0.125 <0.125  0 1 2 0 0  o a o a o a o a o a o a o a o a o a o a o a o a 0  <0.125 <0.125 1 .0 1 .0 <0.125 2.0 0 . 25 1 .0 0 . 25 0.5 <0.125 2.0 <0.125 1 .0 <0.125 0 . 25 0 . 25 1 .0 0.5 <0.125 <0.125 0.5 <0.125  0 25 1 0 0 25  a o a o a o a o a  nh i b i t i on  0 0 25 5  -  5 5 5 0  0 0 0 0 0 0 0 0 0 5 0 25 0 0 0 5 0 0  0 0 0 0 0 0  5 25 5 25 5 25  0 0 0 0 0 1 2 1 0 2 0 1 0 1 0 0 2 2 0 2 0 1 0  25 25 25 25 5 0 0 0 5 0 5 0 25 0 25 25 0 0 5 0 25 0 5  25 0 25 5 0  C. C. C.  lechleri  bark  pa 1anostiqma  bark  trinitatis  Didymocistus  aer i al chrysadenius  leaf bark  Hevea  brasiliensis  Jatropha  curcas  leaf aer i a 1  J . cjossyp i i f o l i a  aer i a 1  J.  1 eaf  weberbaueri  M.  maynensis  ni t ida  Manihot Maprouna  guianensis  Phyllanthus  amarus  Table  XIII  <0.125 -  -  -  --  --  2 1 2 2 2  a o a o a o  1 .0 0 . 25  2 .0 2 .0  2 0 2 0  a o a o a o  <0.125 <0.125 1 .0 1 .0 0.5  -  -  1 .0 2 .0  --  --  2 0  2 0  -  <0.125 1 .0 <0.125  <0.125  <0.125  <0.125  -  -  o a o a o a o a o a o  <0.125 0.5 <0.125 <0.125 <0.125 0 . 25 o.25 <0.125 0.5 <0.125 <0.125  2 0 2 1  aer i al  a o  <0.125  0 5  1 0  1 0  res i n  o  0.5  0 5  0 5  0 . 25  bark aer i a 1  leaf  - 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 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 * o = organic t Podophyllin  -  0 0 0 0 0  a o a o  aer i al  aerial  t  <0.125 1 0 0 125  1 0 0 5 0 25  bark  Podophy11i n  0 .5 2 .0 1 0  1 0 1 0 0 5  aer i a 1  conqesta  <0.125 0. 5  1 0 2 0 . 25  pseudo-conami  Securineqa  <0.125 <0.125 <0.125  1 .0 <1 . 25 <0. 125  leaf  P.  loranthoides  a o a o  1 0  aer i al  Podocalyx  0 25  2 0  orbicu1atus  urinaria  -  2 0  P.  P.  <0.125  <0.125  bark  esculenta  <0.125  a o a o a o  bark Mabea  o  0 25  0 25  2 2 0 2 0  2 0 2 1 2 0 2 1  0 25 1 0 <0.125 2 0 1 0 0 25 1 0 0 25 2 0 0 5  0 0 25 0 5  -0 5 0 0  0 5 0 0 0 25 0 0  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 . The v a l u e s i n h i b i t c o l o n y g r o w t h by a t l e a s t 75%.  f r a c t i o n ; a = aqueous f r a c t i o 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  control  -  0 25  recorded  150  i n h i b i t e d to e a c h of  the  some d e g r e e 34 s p e c i e s  i n h i b i t e d by 9 7 % of extracts mg/ml).  the  species, only  species  Finally,  1 2 % of  these  podophyllin,  were of  activity,  g r o w t h of  each s p e c i e s  Antiviral  cytomegalovirus, in T a b l e XIV.  of  The e x t r a c t s  cells.  these  active (>2 the  b e i n g of  low  inhibited T. The e x t r a c t  e f f e c t ive  least  gallinae  of  in  with  inhibiting  species  tested  was e f f e c t i v e  than  aqueous o n e s .  fractions  one of  Forty  Sindbis  inactivated  extracts  and m u r i n e  the  extracts  the  less  virus  of  to  bark  of not  e a c h of  infection more  the  by  both  effective  fractions  inac.tivated  effective.  while  in  MCMV but  42 o r g a n i c  and a l l  presented  them p r i o r  inactivated  the  is  effective  were g e n e r a l l y  virus  Sindbis  virus  infection,  inhibiting  of  were s l i g h t l y  plaques  e x c e p t i o n of  the  in  fractions  viral  particularly  which  at  The o r g a n i c  Sindbis  when a p p l i e d t o  virus,  viruses.  of  and a f t e r  are  Sindbis  inactivated  i n number of  With the  Didymocistus chrysadenius,  When t h e  of  dermatophyte,  both before  the  cent  2 1 % of  treatment  infection  per  the  i n h i b i t e d by 91% of  extracts  reduction  both v i r u s e s  aqueous  6 of  high concentrations  low p o t e n c y .  inactivating  the  extracts  activity  response to  of  although  was m o d e r a t e l y  The p e r c e n t a g e formed i n  the  the  known t o 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  antiviral  tested  at  tested,  9 4 % of  one of  M i c r o s p o r u m gypseum was  M i c r o s p o r u m f u l v u m was  activity.  b.  least  tested.  were e f f e c t i v e  Euphorbiaceous  and  by a t  MCMV. The  Forty-eight  91% inactivated  were a p p l i e d t o c e l l s  w h i c h had  MCMV.  already  Table  XIV Inhibition S i ndb s Part  Spec i es  * 1  Acalypha  benensis  leaf bark  A.  d i vers i f o l i a  leaf  A.  macrostachya  bark  A.  Alchornea A. A.  bark  stachyura castaneifolia  bark bark  discolor  bark  tr i p l i nervi a  Amanoa a f f .  oblonqifolia  1 eaf  bark Aparisthmiurn  cordatum  leaf bark  Apodandra  loretensis  Caryodendron Chamaesyce C.  orinocense  hyssopifolia  bark aer i al aer i a 1  thym i f o 1 i a  Cnidoscolus  bark  peruvianus  leaf bark  Conceveibastrum Croton  cuneatus  martianum  bark leaf  o a o a o a o a o a o a o a o a o a o a 0  a o a o a o a o a 0  a o a o a o a o a  10  100  1/9/ ml  ml  uQ/ ml  100 0 100 33  100 0 100 69  100 0  100 100  100 100  100 100  100 0  100 0 100 100  100 o  100 89  100 0 56  100 100 100 0 100  0 100 100 100  0 100 100  0 100 100  100  100 92  100 100  tox tox  0 87  0 88 58 71  0 99 79 84  0 100  . 0 100 79  100 100 96  0 14  22 63  0 100 41 0 100 100 0 100  0 100 0 1 1 12 99 100 100 100  60 0 100  100 0 100 0 100 0 26 25 100 100 100 100  100  LCs o  1  10  ml  <1  1 2 2 1  <1  3.0 <1 <1 <1  0 0  -  0 0  <1 < 1 <1  0 0 1  -  O  3.9  17  -  0 5  <1 < 1 < 1 <1 <1  0 100 23 43  -  .08  0 0  8.0 . 15  0 9  -  0  <1  2.9  0 13  -  0  100 100  <1 <1  0  0 100 0  -  0  Ability  100  ml  ml  2  2 1  0 2 2 24 0 0 0 0 0 0 0 41 0 37 21 100 95 69  0 0 0 24 6 6 17 0 0 12  2 1  tox 1 6 3 13 0 6 0  tox 0 64  tox tox tox 95  0  tox 0 100 29 75  tox 0  tox 18  LC o 5  1/9/ ml  -  -  16  -  -  -  20  35 17 < 1  0 0 0 0 0 0  0 0 0 0 0 0  100 0  100 99  100 100  100 100  0 0 0 0  100 100  100 100  0 0 0 0 0  100 100 100 100 100  tox  100 100 98 100 99  0 98 100 65 72 60  100 72 100 100  100 100  100 100  tox tox tox  100  36  0 0  <1 < 1  100 0  208 12 6 .1 226  0 0  100 100 100 100  15  51  0 0  100 100 100 100 100 100  tox  < 1  <1 <1  100 89  100 100 99  tox  0 12  ml  100 97 64  100 0 35 38  0 0  ml  100 100  100 0 27  <1 <1  ml  100 100  0  100 100 100 100  LCs o «/9/ ml  100 65  tox  79 40 48 25  100  c9/ ml  -  3.8 147 51  0  53 47  10  n9/ ml  100 100 100 100  0  60 63  LCs o 1/9/ ml  100 100 100 100 100 100  -  <1  100  1/9/ ml  100 100 91 99  <1  100 0  -  40  -  Treatment P o s t - i n f e c t i on  10  1  100 100 0 26 82 96 92 57  2.0 1 . 9  0 41  0  cytomegalovirus  Treatment P r e - i n f e c t i on  0 0 7  -  Forming  Murine  Treatment P o s t - i n f e c t i on  ml  -  Plaque  virus  Treatment P r e - i n f e c t i on  Ext.  of  82 0 0 100 100 100 58 100  100  100 100 100  <1 < 1 <1 < 1 <1  <1 <1  .30 . 22 .86 <1 . 22 < 1  0 0 0  O  1 0  tox tox 0 0 0 0 0 0 0 0  0  tox  0 0  0 0  0 0  <1  0 100  0 100  <1 < 1  100 100  7 . 7  0 0 0 0 0 0 0  100 100 0 0  tox tox tox  1 . 3 < 1 <1 <1 .91 < 1 . 30 <1 <1 . 53 <1  0 100 0 0 100  0 100 98  <1  100 100  100 100 99  100 100  22 <1 <1 < 1  100 100  .63 < 1  100  1  41  100 100  tox  0 0 0 0 0 o  0 0 0 0  0 0 0  tox  0 0  tox  0 0 0  0 0  tox  0 0 0 0 0 0  0 0 0 0 0 0  0 0 0 0 0 0  0 0 0 0 0  0 0 0 0 0 0  -  -  -  --  -  -  <1 <1 <1 270  <1  -  --  -  -  C.  cuneatus  bark  C.  lechleri  bark  C.  palanostiqma  bark  C.  tr i nitat i s  aerial  Didymocistus  chrysadenius  leaf bark  Hevea b r a s i 1 i ens i s  1 eaf  Jatropha  aer i a 1  curcas  J.  gossyp i i fo1i a  aer i a 1  J.  weberbaueri  1 eaf bark  Mabea M.  maynensis  bark  n i t i da  Manihot  esculenta  Maprouna  leaf  pjuianensis  Phyllanthus  amarus  aer i a 1 bark aer ial  P.  orbiculatus  aer i a 1  P.  pseudo-conami  aer i a l  P.  ur inar i a  aer i a 1  Podocalyx  loranthoides  leaf bark  Securineqa Podophy11i n Table  XIV  -  conqesta t  aer i a l res i n  A n t i v i r a l screening i n number of p l a q u e s * o = organic  o a o a o a 0 a o a o a o a o a o a o a o a o a o a o a o a o a o a o a o a o a o a o a o  94 100 100 0 34 100 61 0 53 0 0 0 100 0 100 100 100 100 100 0 100 0 100 96 99 0 37 20 100 0 100 0 100 0 100 0 100 0 100 100 100 94 91 0 98  99 100 100 0 41 100 78 0 88 0 0 0 100 0 100 100 100 100 100 0 100 0 100 99 99 0 51 32 100 0 100 0 100 0 100 0 100 0 100 100 100 96 95 0 100  100 100 100 0 67 100 94 0 100 0 0 0 100 0 100 100 100 100 100 0 100 0 100 100 98 0 78 69 100 0 100 0 100 0 100 0 100 0 100 100 100 99 99 0 tox  <1 <1 <1 14 <1 .50 1 . 3 -  0 0 0 0 20 0 0 0 15 0 0 0 0 0 0 7 0 0 13 0 14 0 6 0 0 15 41 39 0 0 8 4 0 6 100 0 0 0 0 0 0 0 28 0 0  -  <1 <1 <1 <1 <1 <1 <1 <1 <1 < 1 5.2 26 <1 <1 <1 <1 <1 <1 <1 <1 <1 .01 <1  of e x t r a c t s of Euphorbiaceous observed in the c o n t r o l s .  extract;  a = aqueous  extract  t  tox 0 tox 5 tox 69 tox 0 72 0 0 100 13 0 84 78 75 25 91 0 97 0 31 0 tox 77 63 99 0 0 tox 33' tox 48 tox 60 tox 31 18 0 0 0 100 100 tox  0 0 0 0 32 0 36 0 23 0 0 23 0 0 0 17 25 0 73 0 43 0 13 0 0 43 53 43 0 0 56 6 0 34 100 5 46 1 1 0 0 0 0 100 0 0  plants.  155 391 12 31  -1 1 88 32 37 512 6.0 6. 7 724 15 6 . 1 3. 2  -  8. 1 614 74 <1 40 1 1 1 16 782 -  Results  Podophyllin  -  .81 22 -  resin  are  100 100 66 0 52 100 23 0 0 6 1 0 100 60 98 0 0 2 1 22 87 0 58 0 100 100 98 100 81 58 99 91 0 98 97 58 57 0 95 98 100 100 91 100 55 22 1 1  100 100 100 0 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 ' 99 100 0 100 100 100 100 100 100 100 100 99 100 100 100 100 99 100 100 100 100 100 100 100 100 tox  100 100 100 0 98 100 99 34 100 100 73 100 99 100 96 0 99 98 100 0 990 100 100 100 100 100 94 100 99 98 100 100 100 99 12 100 100 100 100 99 100 98 98 12  expressed  extract  is  as  <1 <1 . 28 . 86 <1 1 .4 1 1 4.6 . 34 8.9 <1 .60 <1 7.0 22 1 . 5 1 . 7 .08 34 .64 <1 <1 <1 <1 . 14 . 18 <1 . 10 8. 1 <1 <1 . 37 .65 18 <1 <1 <1 <1 . 10 <1 .81 1 . 7 per  included  0  o 0 0 0 0 0 0 0 0 0 0 100 0 0 0 0 0 0 0 0  o 0 0 0 100 0 0  o 0 0 0 0 0 0 0  o  0 0 0 0 0 62 0 100  cent as  0 0 0 0 0 25 0 0 0 0 0 0 100 0 0 0 0 0 0 0 0 0 0 0 0 100 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 100 0 100  tox 0 tox 26 tox tox tox 0 69 0 0 0 100 0 0 0 0 tox 0 0 tox 0 0 0 0 100 0 0 0 0 tox 0 0 0 tox 0 tox 0 0 0 0 0 100 100 tox  487 15 133 -  --  <1 -  -  <1 -  -. 33 22 <1  reduction  a positive  control  1 53  been  infected with virus  smaller  percentage  number of to  viral  (68%)  23  Both l e a f Little  that  material between  number of  the  was  between  in  the  susceptible  34 s p e c i e s  organic  ( 2 9 and 2 5 ,  tested.  activity.  and aqueous respectively)  antiviral  activity  from p r e - i n f e c t i o n and p o s t - i n f e c t i o n  application  evident.  note.  The o r g a n i c infection  a concentration  action  of  and a q u e o u s  by b o t h S i n d b i s of  infection  of  virus  the  tested  was  bark  and MCMV c o m p l e t e l y  1 a g / m l . The e x t r a c t s  were  effective  the p r e - i n f e c t i o n  or  post-  podophyllin resin  was  protocols.  An e x t r a c t effective  of  against  the a n t i v i r a l both v i r u s e s  but  inhibited  the  post-infection protocol.  completely  at  Inhibition  the  replication  of  were e f f e c t i v e represented  It  53 of  of  screening  by a v e r y  the  the  for  infection  formation for  antitumour  h i g h p r o p o r t i o n of  Thirty-one  inhibiting  infection  1 jug/ml.  of  XV). in  inhibited  tumour  the  to  o n l y MCMV when a p p l i e d as  potato  also characterized (Table  when a p p l i e d p r i o r of  a concentration  The r e s u l t s  extracts  t h e Amanoa s p .  fraction  when a p p l i e d • a c c o r d i n g t o e i t h e r  c.  of  virus  demonstrated a n t i v i r a l  o b s e r v e d . No c o r r e l a t i o n  prevented at  8 (24%)  the  a much  produced a r e d u c t i o n  possessed a c t i v i t y  The s t r o n g a n t i v i r a l of  post-infection),  formed. S i n d b i s  and MCMV t o o n l y  difference  resulting was  extracts  plaques  and b a r k  fractions was  of  (treatment  the  84 o r g a n i c  (91%)  of  the  formation  of  and a q u e o u s  activity  were  active  species  tested  tumours. extracts.  This  T a b l e XV Per Spec i es Acalypha  Part  benensis  leaf bark  A.  divers i f o l i a  leaf  A.  macrostachya  bark  A.  bark  stachyura  Alchornea  castaneifolia  bark  A.  discolor  bark  A.  t r i p 1 inerv i a  bark  Amanoa a f f .  oblonqifolia  leaf bark  Aparisthmium  cordatum  leaf bark  Apodandra  loretensis  Caryodendron Chamaesyce C.  orinocense  hyssopifolia  bark aer i a 1 aer i a l  thym i f o l i a  Cnidoscolus  bark  peruvianus  leaf bark  Conceveibastrum Croton  cuneatus  martianum  bark leaf bark  Extract* o a 0  a o a o a o a o a o a o a 0  a o a o a o a o a o a o a o a o a o a o a o a o a  0.3  eg/ml  cent  i n h i b i t i o ri o f  1 . 0 ^g/ml  70( 6 ) 87 4 ) 94 2 ) 68 9 ) 21 1 9 ) 12 3 ) 26 13) 2 2) 85 9) 45 6 ) 83 6) 36 4 ) 76 7) 0( 10) 28( 9 )  91(2) 94(2) 81(4)  0( 1 1 ) 84 6 ) 15 8 ) 1 1 11) 0 12) 23 12)  0( 12) 93(3)  0 17 0 0  12)  1 1 6( ) 45 8 ) ' 121 6 ) 53( 5 ) 34( 9 )  yg/  17(6) 91(5)  11(4) 100(0)  0( 13)  0(11)  99( 1 ) 60(5) 90(4 ) 74(7) 83(5) 64(7)  100(0) 94( 1 )  4 3(11)  11(3) 74(8)  0  50 17  0  03  0  09  0  93  51 14 52  0(11) 87(2) 93(2)  0 89 1 28-  0(11) 8 1(18)  1 76 41  89(3) 11(8) 79(4)  0 0  25  87(5) 95( 1 ) 9(7) 0(7)  0 0  75 51  26(7) 88(4)  66 10) 72( 8 ) 36( 9 ) 0( 10)  79(5) 85(4)  28(8) 97(2) 81(4) 91(6) 89(4)  0(11)  16 35 03  96(2 ) 26(13) 69(8) 7(6) 78(7 )  7) 601 8 )  0( 8 )  0 2  62  4(3) 100(0)  62(10) 2(4 )  1 7 44  0 0  0  0(5) 49(8)  8 0 19  ( j,g/ml )  o  100(0) 0( 14)  7) 5)  0 3  5  01 75  99( 1 ) 94(3) 87(6) 66(5)  5) 10) 6)  38  ED  0 0 0 0 0 1  8(4) 72(5) 47(5) 72(5) 0(12) 7(7)  '  m l  99( 1 ) 98( 1 ) 100(1) 100(0) 53(9)  0(9) 57(10) 17(11) 60(6) 70(8) 0( 14)  7) 15)  3.0  formation(SEM)  85(6) 45(17 )  13(8) 74(6)  10)  tumour  5(9) 0( 1 1 )  1 03 0  57  0 05 0. 08 0 60  C. C. C.  lechleri  bark  p a l a n o s t i cjma  bark aer i a l  t r i ni t a t i s  Didymocistus  chrysadenius  leaf bark  Hevea b r a s i l i e n s i s  leaf  Jatropha  aer i a 1  J. J.  curcas  aer i a 1  qossyp i i f o l i a weberbaueri  leaf bark  o a o a o a o a o a o a o a o a o a  0 0 79  0  0 5( 0( 0( 16(  a Mabea M.  maynensis  leaf  n i t i da  Manihot  bark  esculenta  Maprouna  0  aer i a 1 bark  quianensis  Phyllanthus  amarus  aer i a 1  P.  orbiculatus  aer i a 1  P.  pseudo-conami  aer i a 1  P.  ur i nar i a  aer i a 1  Podocalyx  loranthoides  leaf bark  Securineqa Podophy1  Table  XV -  conqesta  aer i a 1  l i n t  Screening  res i n  of  * o = organic t Podophyllin  extracts  of  a o a o a o a o a o a o a o a o a o a o a o  Euphorbiaceous  11)  30(7) 0(7)  9) 5)  0 17  85(5)  62(5)  9) 9) 11) 9) 4) 13)  0 72 0 0 4  74(13)  9) 13)  0 3  5) 15) 12) 12)  0 14 5 2 9 0  82 4  10) 13)  0(9) 70(11) 17(12) 64( 62( 74( 0(  0( 15) 81 ( 6 ) 0( 10) 76( 5) 0( 11) 8 9 ( G) 74( 9)  15) 13) 13) 13)  79(8) 83(7) 98(3) 38(10) 96(5) 77(8) o d o ) 55(7)  0( 11) 32( 8) 23( 12)  34(10) 0(7)  0( 10)  0(8)  0( 11) 0( 8) 19( 12) 9( 12)  0(11) 49(9) 0(11) 72(9)  4( 12)  for  81(7) 0( 13)  4(10) 65(7)  62( 15)  plants  0 (9) 77 (9) 0 13) 6 5) 1 5)  66(13)  13) 0( 14) 77( 11) 57( 13)  inhibition  f r a c t i o n ; a = aqueous f r a c t i o 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  control  of  0 02 1 57  0( 10) 64(12) 0 ( 12 ) 7(8) 0(7)  10(11)  11) 15) 13)  1  (11) (3) (3)  (9) 0 (13) 91 ( 8 )  12(13)  11) 11) 13)  (4)  0(9) 74(9)  0( 0(  5)  81 0 91 8 1 87  0 0 5 7 83 9 94 87  8)  0  78  0  1 1  1  56  1  43  1  52  9) 9) 12) 9) 9) 9) 12)  1  7)  6) 79( 10) 83 11) 87( 8) 87( 10) 75( 13) 12( 1 1 ) 94 ( 5) 90 ( 5) 100( 0 ) 89( 8) 100( 1 ) 100( 3) 85( 5)  96( 4 ) 80( 8) 0( 9) 0( 12) 5( 7) 91 ( 7 ) 0( 9) 93( 5)  A g r o b a c t e r i um i n d u c e d  75  tumour  .  33 2 64 0 91 0 60 0 03 0 32 0  05  0 05 1 31 0 24 1 0 0 2  55 17 34  0 1  73 64 17  0  88  0  98  formation.  1 56  Extracts no  of both  difference  fractions were 3.0  showed  was a p p a r e n t . quite  antitumour  of t h e aqueous  The a n t i t u m o u r  strong: the E D  t o br i n e  large shrimp  great  and the L C  mg/ml,  observed  between  effective  5 0  varied  t h e h i g h e s t dose were  Euphorbiaceous  fractions  in  brine  0.01 a n d  inhibiting  contain  data much  activities This  sets  i n each  biological  of Tables  of the assays  of the b i o l o g i c a l  assays  a r e more  closely  d e t e c t e d , than activity  a toxic  times  (10) were  less  5 0  o f t h e 34  response,  a s many  was  by  organic  toxic.  of t o x i c i t y  (LC  << 1  5 0  assays  number  of the b i o l o g i c a l  aspect  antibacterial  an L C  to  and 1  X I I t o XVI a r e d e t a i l e d  i n f o r m a t i o n h a s been each  with  toxic  observed  1 Mg/ml  Twenty-seven  degree  information. A large  Although  activity  fractions a high  than  Extracts  three  were  shrimp.  C o r r e l a t i on b e t w e e n The  less  as a c t i v e .  Approximately  demonstrated  towards  between  examined  of t o x i c i t i e s  (79%) produced  (32) as aqueous  Podophyllin  The range  tested.  classed  species  criterion.  Mg/ml)  of the e x t r a c t s  (Table XVI).  1 mg/ml  trend.  and organic  s h r imp  percentage  brine  this  ranged  and  formation.  Toxic ity  than  activity  activities  s  5 0  M g / m l . P o d o p h y l l i n was m o d e r a t e l y  A  e.  and bark  i n the a c t i v i t i e s  generally  tumour d.  leaf  activity related,  and  of.extracts  assays  summarized measures  i s the  o t h e r s . The a s s a y s  predominant  i n Table  a somewhat  of the e x t r a c t , i n terms  with  XVII. different  certain  of t h e nature  of the  measuring  (Nos. 3 and 4 ) , f o r example,  would  be  Table  XVI Per Species  Acalypha  Part  benensis  1 eaf bark  A.  divers i foli a  leaf  A.  macrostachya  bark  A.  stachyura  bark  Alchornea  castaneifolia  bark  A.  discolor  bark  A.  tr i p i i nerv i a  bark  Amanoa a f f .  oblonqifolia  leaf bark  Aparisthmiura  cordatum  leaf bark  Apodandra  loretensis  Caryodendron Chamaesyce C.  orinocense  hyssopifolia  bark aer i a 1 aer i a 1  thymi f o l i a  Cnidoscolus  bark  peruvianus  leaf bark  Conceveibastrum Croton  cuneatus  martianum  bark leaf bark  Extract*  o a o a o a o a o a o a oa o a o a o a o a o a o a o a o a o a o a o a o a o a o a  10 i/O/ml 0 0 0 0 0 0 0 0 0 0 8 0 0 0 0 0 76 52 0 0 0 0 0 0 0 0 72 8 0 0 76 0 0 0 0 0 12 4 100 0 68 0  cent  mortci l i t y  of  brine  100 „g/ml  1000 eg/ml  20 0 0 0 0 0 40 0 0 0 72 0 24 0 28 68 92 84 8 8 60 0 8 0 4 0 96 56 0 0 100 0 4 0 68 0 88 8 100 16 100 8  100 56 100 20 24 0 100 0 100 0 100 4 72 4 100 100 100 100 32 44 100 12 96 0 100 0 100 96 0 0 100 0 44 0 100 4 100 12 100 100 100 100  shrimp LCs o »,g/ml 1 16 214 104 214 41 399 1 10 92 6 . 1 14.3 875 95 254 142 5.8 76 1. 8 -  -  92 30 <<1 120 2.6 130  C.  lechleri  bark  C.  palanostiqma  bark  C.  tr i n i tat i s  aer i a 1  Didvmocistus  chrysadenius  leaf bark  Hevea b r a s i l i e n s i s  leaf  Jatropha  aer i a 1  curcas  J.  qossypi i f o l i a  aer i a 1  J.  weberbaueri  leaf bark  Mabea M.  maynensis  bark  n i t i da  Manihot  esculenta  Maprouna  leaf  quianensis  Phyllanthus  amarus  aer i a 1 bark aer i a 1  P.  orbiculatus  aer i a 1  P.  pseudo-conami  aer i a 1  P.  ur i nar i a  aer i a 1  Podocalyx  loranthoides  leaf bark  Securineqa Podophy1  Table  XVI  -  conqesta  l i n t  aer i a 1 res i n  S c r e e n i n g of e x t r a c t s of Euphorbiaceous p l a n t s L C s o < 1000 ^g/ml were c l a s s i f i e d a s a c t i v e . * o = organic  fraction;  a = aqueous  fraction  for  t  their  toxicity  Podophyllin  15 1 . 3  100 0 100 24 100 0 52 0 96 100 76 4 32 12 36 32 88 0 84 12 24 0 100 96 16 28 100 24 92 0 100 28 100 100 100 4 4 0 84 8 100 92 100  92 0 96 8 76 0 20 0 68 0 0 0 28 4 28 36 4 0 20 0 0 0 16 8 8 0 64 0 24 0 48 0 84 0 40 0 0 0 36 0 100 0 100  40 0 92 0 24 0 0 0 40 0 0 0 20 8 24 0 0 0 0 0 0 0 0 0 0 0 24 0 1G 0 4 0 48 0 0 0 4 0 4 0 96 0 100  o a o a o a o a o a o a o a o a o a 0 a o a o a o a o a o a o a o a o a o a o a o a o  to  resin  brine  is  shrimp,  included  as  Artemia  27 615 30 2 14 1 106 401 326 1 20 254 30 ' 14 57 15 214 104 193 0.09 642 <<1  s a l i na.  a positive  Extracts  control.  having  a  Table  XVII * Spec i es  Acalypha  benensis  Part  Ext  1  2  3  4  5  leaf  o a o a o a o  +2 +4 +4 +2 +2 -  -  -  <0. 13 0 . 25 <0. 13 <0. 13 <0. 13 <0. 13 <0. 13  0.5  -  +3 +2 -  -  -  <0. 13 <0. 13 1 .0 1 .0 <0. 13 2.0 0 . 25 1 .0 0.25 0.5 <0. 13 2.0 <0. 13 1 .0 <0. 13 0 . 25 0 . 25 1 .0 0.5 <0. 13 <0 . 13 0.5 <0. 13  0 . 25 1 .0 0 . 25 0 . 25 0 . 25 1 .0 2.0 2.0 <0. 13 2.0 0.5 2.0 0.5 2.0 <0. 13 2.0 1 .0 2.0 1 .0 2.0 <0. 13 0.5 <0. 13 0.5 0.5  bark A.  di vers i f o l i a  leaf  A.  macrostachya  bark  A.  stachyura  bark  Alchornea A. 'A.  castaneifolia  bark  discolor  bark  tr i p 1 i nerv i a  bark  Amanoa a f f .  oblonqifolia  1 eaf bark  Aparisthmium  cordatum  leaf bark  Apodandra  loretensis  Chamaesyce C.  bark  hyssopifolia  aer . aer .  thym i f o 1 i a  Cnidoscolus  bark  orinocense  Caryodendron  peruvianus  1 eaf bark  Conceveibastrum Croton  cuneatus  Biological  martianum 1 eaf leaf bark  o a o a 0 a o a o a o a o a o a o a o a o a o a o a o a o a o a o a  --  -  -  --  -  -  --  -  +1  --  +2  -  -  -  +1 +1 +1 +2 +2 +2 +2 +2  +1 +1 +1 +1 +3 +1  +1 +1 +2 -  -  --  -  -  -  -  -  -  -  --  -  -  -  -  --  --  -  +2 -  -  -  -  -  <0. 13 0 . 25 0 . 25 <0. 13 2.0 <0. 13 2.0  1 .0 2.0 0 . 25 0.5  -  0.5 0.2  t 8  9  0 . 25 2.0 1 .0 1 .0 0 . 25 2.0 0.5  <0. 13 0.5 0 . 25 0.5 0 . 25 0.5 0 . 25  <1 . 0 <1 . 0 3.0 <1 . 0 <1 . 0 <1 . 0  0.5 1 .0 0.5 1 .0 1 .0 1 .0 2.0 2.0 2.0 2.0 1 .0 2.0 0.5 2.0 0 . 25 2.0 2.0 2.0 0.5 1 .0 1 .0 <0. 13 1 .0  0 . 25 0 . 25 0 . 25 0 . 25 0.5 1 .0 2.0 1 .0 0.5 2.0 0.5 1 .0 0 . 25 1 .0 0 . 25 0 . 25 2.0 2.0 0.5 2.0 0 . 25 1 .0 0.5 <0. 13 0 . 25 2.0 0 . 25 0.5 2.0  <1 . 0 <1 . 0 <1 . 0 3.9 <1 . 0 <1 . 0 <1 . 0 <1 . 0 <1 . 0 <1 . 0 0.08 8.0 0 . 15 <1 . 0 2.9  7  6  -  Assay  -  0.5  -  0.5 2.0  -  <1 . 0 <1 . 0 <1 . 0  -  <1 5. 47 <1 <1 <1 <1 <1 <1  .0 3 .0 .0 .0 .0 .0 .0  -  -  16  --  -20 35 17 <1 . 0 2.0 1 .9 -  -  3. 8 147 51 -  -  15  -  <1 . 0 208 12.1 6. 1 23 40 -  12  1 1  10  .0 .0 .0 .0 .0 .0 .0  _  <1 . 0 <1 . 0 0.30 0.22 0.86 <1 . 0 0 . 22 <1 . 0 <1 . 0 <1 . 0 <1 . 0 7. 7 1. 3 <1 . 0  -  <1 <1 <1 <1 <1 <1 <1  -  <1 . 0 0.91 <1 . 0 0 . 30 <1 . 0 <1 . 0 0.53 <1 . 0 <1 . 0 41 22 <1 . 0 <1 . 0 <1 . 0 0.63 <1 . 0 <1 . 0 <1 . 0  -  1 0 0 0 2  --  0 5  -  -  --  1 0 0 0 0 -  --  -  <1 . 0 <1 . 0 <1 . 0 270 -  --  7 44 16 35 0  -  0 0 0 0 0 1 0 0 0 0 0 1 -  --  14  13  1 0 0 0 0 —  17 51 14 52 01 75 62 03 09 93 89 28 76 41 25 75 51  0 57 05 08 6  1 16 214 -  -  104  214 4 1 399 1 10 92 6 . 1 14.3 875 95  -  254 142 5. 8 76  -  1.8 92 30 <1 . 0 120 2 . 2 130  C.  lechleri  bark  C.  palanostiqma  bark  C.  t r i ni t a t i s  aer .  D i dymoc i s t u s  chrysadenius  leaf bark  Hevea  leaf  brasiliensis  Jatropha  aer .  curcas  J.  qossypi i f o l i a  aer .  J.  weberbaueri  leaf bark  Mabea maynens i s  leaf  M.  bark  n i t i da  Manihot  aer .  esculenta  Maprouna  quianensis  Phyllanthus  bark aer .  amarus  P.  orbiculatus  aer .  P.  pseudo-conami  aer .  P.  urinaria  aer .  Podocalyx  loranthoides  leaf bark  Securineqa  conqesta  aer .  Podophy11i n +  res.  Table  XVII  o a o a o a o a o a o a o a o a o a o a o a o a o a o a o a o a o a o a o a o a o a o  -  -  +3 +5 -  +2 +2 +2 +1  _  _  -  -  +1 -  -  -  -  -  -  -  -  +4 -  +3 +3 +1 +1 +1 + 1 +3 +3  -  -  -  -  -  +4 +1 +1 +2  Summary o f  -  -  -  -  -  -  -  <0. <0. <0. <0.  13 13 13 13  <0. 13 <0. 13 0.5  <0. 13 -  -  -  2.0 2.0 -  1 .0 0 . 25 <0. 13 <0. 13 <0. 13 1 .0 1 .0 <0. 13 1 .0 <0. 13 <0. 13 <0. 13 1 .0 <0. 13 <0. 13 0.5 <0. 13 <0. 13 <0. 13 0.25 0.25 <0. 13 0.5 <0. 13 <0. 13 <0. 13 0.5  bi o l o g i c a l  -  -  1 .0  -  2.0 1 .0 2.0 0 . 25 <0. 13 0 . 25 2.0 2.0 0 . 25 2.0 0.5  -  2.0 0.5 2.0 1 .0 0.5 0.5  screening  0.25 <0. 13 1 .0 0.13  <1 . 0 14 <1 . 0 0.5  155 391 12  0 . 28 0.86 <1 . 0 1 .4 \ 1  1 . 3 <1 . 0 <1 . 0 < 1 .0 <1 . 0 <1 . 0 <1 . 0 <1 . 0  31 1 1  2 0  2.0 1 .0 2.0 2.0 2.0 1 .0  4. 6 0 . 34 8.9 <1 . 0 0.6 <1 . 0 7.0 22 1. 5 1 . 7 0.08 34 0.64  1 0 1 0 0 5 <0. 13 0 25 2 0 0 5 2 0 1 0 2 0 0 25 2 0 1 0 1 0 0 5  1 .0 0.5 0 . 25 <0. 13 0 . 25 0 . 25 1 .0 <0. 13 2.0 1 .0 0.25 1 .0 0 . 25 2.0 0.5 1 .0 0.25  <1 <1 <1 5. 26 <1  -  0 5 2 0 1 0 2 0 2 0  -  of  extracts  -  .0 .0 .0 2 .0  <1 . 0 1 .0 <1 . 0 <1 . 0 <1 . 0 <1 . 0 <1 . 0 <1 . 0 <1 . 0 0.01 <1 . 0  of  -  88 32 37 512 6.0 6 . 7 724  -  15 6 . 1 3. 2 8. 1 614 74 <1 . 0 40 1 1 1 16 782 -  -  0.81 22  -  <1 . 0 <1 . 0 <1 . 0 <1 . 0 0.14 0.18 <1 . 0 0 . 10 8. 1 <1 . 0 <1 . 0 0 . 37 0.65 18 <1 . 0 <1 . 0 <1 . 0 <1 . 0 0 . 10 <1 . 0 0.81 1. 7 4. 3  Euphorbiaceous  487 15 ~  133 -  -  <1 . 0 -  -  <1 . 0 -  -  -  -  -  -  0 . 33 22 <1 . 0  plants  1 0 1 0 0 1 1 1  02 6 78  15.2 1. 3 27  1 1  615  8  6  4  5  1 3 2 6 o 91 0 60 0 03 0 32 0 05 0 05 1 3 0 24 1 6 0 17 0 34 2 7 0 64 1 2 0 88 0 98  -  30 214 401 326 120 254 2390 30 5380 1 14  -  57 4430 15 214 104 193 0.09 642 <1 . 0  Legend f o r  t  Bioassays  to  w h i c h numbers  Tab!e  XVII  refer:  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.  aureus  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.  cereviseae  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  ga11inae  9 = ;  anti-Sindbis  1 1 = = anti-MCMV 13 == i n h i b i t i o n  virus  activity :  activity: of  12 == a n t i - M C M V  pre-infection  Aqrobacteriurn induced potato  * Podophyllin  resin  * o  fraction;  = organic  10 == a n t i - S i n d b i s  pre-infection  extract  is  included  a = aqueous  as  tumours  a positive  fraction  14 == t o x i c i t y  control.  virus  activity: to  Artemia  T.  activity:  post-infection  p o s t - i n f e c t i on salina  162  expected the  to  results  resemble obtained,  distantly  related  indicates  that  in  vivo  being  shrimp,  while of  (Galsky have  et  not  on  the  results  the  form  of  2 X  the  sets  of  four  of  the  1981;  degree is  of  presented  et  a _ l , 1982)  numbers  association  XII  to  XVI  tables  represents a  bioassays in  activity,  and  and  i t is  to  brine  Toxicity  for  more  between  of  for  shows  some  cytotoxicity  a l , 1982).  recently  which  is predictive  assays  et  to  utilizing  assay  of  respect  Both  assays  descriptive the  needed.  Tables  is illustrated  with  e_t a l , 1 9 8 1 ) .  Meyer  2 contingency  two  been  tumour  results  relatively  of  has  for antitumour  with  and  measured  assays  (Ferrigni  (Cassady  specific  only  two  Data  disc  closely,  p r e l i m i n a r y screening assay  agents  used  activities  these  a  a l , 1980  information  results  as  more  would  activity  correlation  been  The  than  potato  antileukemic  antineoplastic  other  organisms.  the  considered  degree  each  the  have  in Table  The  table.  summarized  XVIII.  comparison  indicated. following  been  of  format  Each  in of  the f o r each  of  1 63  Test A  positive  Test  The l e t t e r Test the  positive  a  b  negat i v e  c  d  B  (a)  refers  A and T e s t  to  the  in Test  number of  B produced a p o s i t i v e  number h a v i n g a n e g a t i v e  result  B,  result  Biomedical  values  for  tailed  test)  observed  Fisher's  for  this  no a s s o c i a t i o n C.  bioassays of  Certain data.  test.  statistic  (b)  of  results.  between  are  positive  analysis  p r o g r a m P:4F  These  to  statistics  from l e s s  of  results,  to  i n any of  b i o a s s a y No. be c a l c u l a t e d .  the  3 and any of  compute  (for The  than  S i n c e no i n h i b i t o r y  was o b s e r v e d  a  two  values  0.0001,  1.0,.  indicating  activity extracts, the  other  The c o r r e s p o n d i n g c o l u m n s  blank.  interesting  The v a l u e s  both  indicates  i n T e s t A and a  statistical  range  association  c o u l d not  T a b l e XIX  to  Data Packages exact  albicans  associations  result;  i n which  have been summarized i n T a b l e XIX.  indicating a close  towards  extracts  etc.  T h e s e d a t a were s u b j e c t e d utilizing  negat i v e  of  trends  0.4941 f o r  c a n be d i s t i n g u i s h e d i n Fisher's  exact  test  these  applied  to  2  2 41 0 42  3  0 0 2 83  0 0 43 42  4  0 2 2 81  2 0 41 42  0 2 0 83  5  2 67 0 16  40 29 3 13  0 69 0 16  1 68 1 15  6  2 53 0 30  22 20 8 35  0 55 0 30  1 54 1 29  53 16  2 14  7  2 52 0 31  36 18 7 24  0 54 0 31  1 53 1 30  52 15  2 14  4 50 5 26  8  2 61 0 22  40 23 3 19  0 63 0 22  2 61 0 22  4 59 10 12  54 9 1 21  53 10 1 21  9  2 60 0 23  35 27 8 15  0 62 0 23  1 61 1 22  4 58 1 1 12  49 6  13 17  48 6  14 17  52 10  9 13  10  0 43 2 40  20 22 23 20  0 42 0 43  0 42 2 41  36 33  6 10  28 27  14 16  27 27  15 16  31 32  1 1 1 1  1 1  2 78 5 0  42 38 1 4  0 80 0 5  2 78 0 5  67 2  13 3  54 26 1 4  53 27 1 4  6 1 19 2 3  61 19 1 4  4 1 39 1 4  12  0 12 2 71  5 7 38 35  0 12 0 73  0 12 2 71  9 60  3 13  7 5 48 25  8 3 47 27  9 54  3 19  9 3 53 20  4 8 34 39  1 1 69  1 4  13  2 52 0 31  38 16 5 26  0 54 0 31  2 52 . 0 31  51 18  3 13  41 14  40 14  14 17  47 16  7 15  43 19  1 1 12  30 24 12 19  53 27  1 4  6 47 6 26  14  2 41 0 42  24 19 19 23  0 43 0 42  2 41 0 42  20 30  4 12  34 9 2 1 21  34 4 20 22  38 35  5 17  37 25  6 17  23 20 19 23  42 38  1 4  8 35 4 38  13 17  34 28  8 15  10  Table  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 t h e 85 p l a n t e x t r a c t s . D a t a a r e s u m m a r i z e d f r o m T a b l e X V I I .  the  1 1 14 a s s a y s  12 used  31 23  12 19  13 to  screen  0.4941  -  -  1 .00  0.4941  -  1 .00  0.0056  -  0.3429  0.5378  0.0015  -  1 .00  0.0000  0.531 1  0.0001  - •  1 .00  0.0000  0.0000  1 .00  0.0001  -  1 .00  0.0000  0.0000  0.0000  1 .000  0.0913  -  0.4703  0.0000  0.0000  0.0000  0.0002  0.2412  1 .000  -  0.4941  0.4065  0 . 8 2 13  1 .00  1 .00  0.1429  1 .00  0.2020  -  1 .00  0.0440  0.0503  0.0570  0.1065  00178  0.3600  1 .00  0.5486  -  1 .00  0.6899  0.7464  0.7393  1 .00  1 .00  0.2278  0.5421  0.531 1  0.0000  -  0.5310  0.0001  0.0088  0.0104  0.0006  0.0800  0.1774  0.0570  0.0347  0.4941  1 .000  -  0.4941  0.0283  0.0066  0.0034  0.0030  0.0074  1 .000  0.2020  0 . 3512  1  2  Table  3  XIX  4  - Values  5  of  Fisher's  6  exact  7  test  8  for  9  contingency  10  tables  of  11  Table  XVIII  12  0.1178  13  1 66  the  results  bacteria between of  E.  the  of  the  s c r e e n i n g of  indicates these  coli  other  no a p p r e c i a b l e  results.  growth  however,  were shown t o be q u i t e  data  inhibition  fungi  tested.  screening  for  observation test  less  It  is  virus  the  than  a s s o c i a t e d with the  anti-viral  tests  assays  carried  the  treatment  similar results  S.  the  of  any  aureus,  obtained  is  of  the  dermatophytic in  the  s u p p o r t e d by  for  Fisher's  of  the  the  exact  anti-Sindbis  for  any  treatment was  closely  anti-dermatophytic of  the  No a s s o c i a t i o n  pre-infection  MCMV were somewhat infection  the  or MCMV (p= 0.5421) of  results  for  results  results  the case  and p o s t - i n f e e t i o n  inhibition  s c r e e n i n g were a l s o  of  out.  the  cases.  the  results  T h i s was not  (p= 0 . 1 4 2 9 )  e a c h of  activity  treatment)  screening.  the  that  of  a s s o c i a t e d with  corresponding value  interesting  association  the  obtained  the  0.0001 i n a l l  (pre-infection  infection  in  anti-dermatophyte  that  is  g r o w t h of  The s i m i l a r i t y  results  closely  and gm p o s i t i v e  of  resemble  The r e s u l t s  of  degree  Nor d i d t h e  significantly  bioassays.  for  gm n e g a t i v e  of  treatment  (p= 0 . 0 1 7 8 )  other  between  either  indicated. of  while  fungi three the  Sindbis  previrus  The r e s u l t s  Sindbis the  of  virus  and  post-  showed no s i g n i f i c a n t  association  (p= 0 . 2 2 7 8 ) . The r e s u l t s  of  the  were a s s o c i a t e d w i t h t h e aureus  (p= 0 . 0 0 0 1 ) ,  assays  and a n t i - S i n d b i s  anti-MCMV  (pre-  potato disc results  e a c h of  the  virus  of  tumour the  inhibition  inhibition  anti-dermatophytic  (pre-infection),  and p o s t - i n f e c t i o n )  assays  to  as  of  assay S.  fungi well  as  some e x t e n t  (p=  167  0.035-0.08). The p a t t e r n results  w i t h the  that  the  of  anti-  S.  of  association  results  anti-tumour  aureus  association.  of  activity  the  other  results.  antitumour  shrimp  bioassays  is  toxicity similar  exception  shrimp t o x i c i t y  Fisher's activity  brine  The s i n g l e  and b r i n e  The v a l u e of  c o m p a r i s o n of  the  of  statistic  for  and b r i n e  is  to  that  show no  the  shrimp t o x i c i t y  is  0. 1 1 7 8 .  4.  DISCUSSION  a.  Antimicrobial  activity  The p e r c e n t a g e antimicrobial selected 98% and  inhibited Ieven et higher these  the al  two  plants.  growth of  gossypiifolia, medicinal  use  inhibitory  of  S.  It  aureus  and E.  uses  of  the  to  apparently  coli  species  were i n h i b i t o r y  of  respectively.  treatment  S.  aureus  of  aureus  agents and  of  of  growth  to  be  from  J.  Euphorbiaceae  t o S.  yeasts.  curcas  that  plants  the  common f o r  the E u p h o r b i a c e o u s e x t r a c t s towards  reported  100 s p e c i e s  inhibit  Jatropha  plants  coli,  to a n t i m i c r o b i a l  concerned w i t h the  activity  of  African medicinal  e x c e p t i o n of  all is  is  exhibiting  a^L (1980)  o b s e r v e d 78% and 31% of  t h a n E.  skin conditions, Few of  Khan e t  with medicinal  With the  species  comparable to that  60 s p e c i e s  species.  more s e n s i t i v e  is  families.  (1979)  plants  Euphorbiaceous  activity  from a l l 18% of  of  whose  wounds  (Table  exhibited  None p r e v e n t e d  or  XX).  any the  growth  Table  XX  Inh i b i t o r y A c t i v i t y  Spec i es  Col 1 .  Folk  gm-ve  Use  Toxicity  Towards:  potato  dermato  gm+ve  bacter i a bacter i a  No.  or  yeast  phyt i c  v i rus  tumour  Artern i a  fungus  Acalypha  benensis  1 14  -  +  -  +  +  +  +  A.  d ivers i f o l i a  63  -  +  -  +  +  +  -  A.  macrostachya  91  -  -  -  +  -  +  +  A.  stachyura  1 13  -  +  -  +  -  +  +  -  -  -  +  -  +  +  Alchornea  castaneifolia  95  rheumatism,  tonic  A.  discolor  81  -  +  -  +  +  +  +  A.  tr i p i i nerv i a  31  -  +  -  +  +  +  +  70  -  +  -  +  +  +  +  29  -  +  -  +  +  +  +  56  -  +  -  +  +  +  +  diseases  -  +  -  +  -  +  +  Amanoa a f f .  oblonqifolia  Aparisthmium Apodandra  cordatum  loretensis orinocense  84  hyssopifolia  65  wounds,  cancer  -  +  -  +  -  +  -  38  wounds,  cancer  -  +  -  +  +  +  +  -  -  -  +  +  +  +  101  -  +  -  +  +  +  +  93  -  +  +  +  -  +  +  +  +  +  +  Carvodendron Chamaesyce C.  thym i f o 1 i a  Cnidoscolus  peruvianus  Conceveibastrum Croton C.  cuneatus  lechleri  martianum  133  62  skin  anti-aphrodisiac  wounds,  cancer  +  C.  p a 1 a n o s t i gma  76  C.  tr i n i tat i s  48  Didymocistus Hevea  chrysadenius  b r a s i i i e n s is  Jatropha  curcas  J . pjossyp i i f o 1 i a J.  weberbauer i  -  +  -  +  +  +  +  -  +  -  +  +  +  +  27  -  +  +  +  +  +  +  54  -  -  -  +  +  -  -  -  -  +  +  -  -  -  +  -  -  -  68  purgative,cancer,skin  cond.  -  -  -  -  -  -  +  -  +  -  +  -  +  +  +  -  +  +  -  +  +  +  +  -  +  -  +  +  +  -  -  +  -  +  -  +  +  -  +  -  +  +  +  +  49  +  +  -  +  +  +  +  112  -  -  +  +  +  +  53 61  esculenta guianensis  orbiculatus  P.  pseudo-conami  P.  ur i nar i a  37  36  loranthoides  Securineqa  conqesta  Frequency  a p h r o d i s i ac  skin burns,  conditions  skin  infections  73  amarus  P.  ailments  -  136  ni t ida  Podocalyx  chest  cond.  M.  Phyllanthus  throat,  purgative,cancer,skin  79  Maprouna  cancer  67  Mabea maynens i s  Manihot  sore  wounds,  kidney  kidney  ailments,  ailments,  liver  liver  89  •  -  +  -  +  +  +  +  -  +  -  +  +  +  +  +  +  +  +  91  79  91  79  Euphorbiaceae  tested.  +  30  +ve  76  6  6  Result(%)  Table  XX  -  Summary o f Data  are  ethnobotanical  summarized  information  from Tables  XII  to  and  biological  XVIII.  activity  of  the  34 s p e c i e s  of  1 70  of  C. a l b i c a n s ,  S.  cerevisiae.  plants,  Screening  of  extracts  1966; Fong e t  dermatophytic  al,  studies:  1972);  anti-S.  studies  is  Euphorbiaceae conditions  are  yeasts  al,  et_ a_l,  were u s e d i n  the  the  towards  higher percentage  (Farnsworth  the  As i n  growth  e_t  treatment  of  that  observed  2.1%  (Fong  the case  10 s p e c i e s  the  against  1979).  1966);  1979).  of  of  a much h i g h e r  inhibiting  al,  8 out  inhibitory  families  towards  (Farnsworth  activity, that  all  h i g h compared t o  and 65% ( I e v e n e t  aureus  of  detected  extracts  (91%)  2.6%  34 s p e c i e s were a c t i v e  1972; I e v e n e t  of  fungi  of  inhibitory  al,  The p e r c e n t a g e  other  2 out  by c o m p a r i s o n , have  (10-42%) al,  while  of  in  et  of  of skin  dermatophytic  fungi  tested. b.  Antiviral It  is  uniformly  activity  somewhat effective  s u r p r i s i n g that  the  in  both S i n d b i s  MCMV when a p p l i e d t o most of  the  active  inactivating  them a t  extracts  extracts  low c o n c e n t r a t i o n s . was  less  than  were so virus  and  The E D  1 jug/ml  5 0  s  of  (Table  XVII).  wide  The i n a c t i v a t i o n  of  variety  extracts  (Konowalchuk attributed  to  of  plant  and S p e i r s , the  .aqueous e x t r a c t s  viruses  a p p l i e d to  Among t h e  phenolic  compound,  tannic  1978).  phenolic  the v i r u s e s  (Cheo and  1978; T a k e c h i and T a n a k a ,  (Konowalchuk  and S p e i r s ,  the  a  been  compounds i n  is  of  previously  T h i s has  compounds w i t h a c t i v i t y  acid  application  has been r e p o r t e d  1976 and  p r e s e n c e of  1964; J o h n and Mukundan,  by d i r e c t  the  Lindner, 1981). ubiquitous  1978).  These  171  sorts  of  compounds would be e x p e c t e d t o o c c u r  an aqueous e x t r a c t . aqueous e x t r a c t s of  tested  however,  •both S i n d b i s this  virus  activity  the  present  Weintraub,  were a l s o  percentage highly  and MCMV and i t  inactivate  1974; R a g e t l i ,  presence  of  this,  is  w i t h the  ability  to d i s r u p t  the  the  It  in  is  viruses  of  of  is  in  the result  organic  difficult  and i t  classes  of  effective  certain  1975)  or o t h e r  activity  s t u d y may be t h e  to p h e n o l i c c o n s t i t u e n t s .  s a p o n i n s can a l s o  inactivating  to  attribute  known  that  (Ragetli  and  p o s s i b l e that  the  l i p o p h i l i c compounds  structure  of  biomembranes may  responsible. Although d i r e c t  important  useful  in already  tested,  and 8 (24%)  high percentages higher  plant  (1966)  species active).  active)  associated,  agent.  23  (68%)  the  against  is  species a c t i v e ) ,  and Van den Berghe interesting virus  are  quite  note,  potentially  which  inhibits  infection  e_t a_l  by  been o b s e r v e d out  al  (1972)  (1979)  not  different.  inhibition  e_t  (8.3%  results  were the  the  species  and anti-MCMV a s s a y  the  from  (8% the  that  very  by F a r n s w o r t h  that  in,  Sindbis  MCMV. T h e s e a r e  Fong e t  indicating  virus  of  again,  statistically,  to  a  34 s p e c i e s  inhibited  were e f f e c t i v e  and compounds i n v o l v e d  each v i r u s  Of  is  would seem t o be more  compared w i t h what has  ( 3% o f  It  an agent  s c r e e n i n g programs c a r r i e d  the a n t i - S i n d b i s  for,  inactivation  infected cells  as a t h e r a p e u t i c  Euphorbiaceae virus  viral  biological activity,  replication  al  in  inactivating  p h e n o l i c compounds. A l a r g e  fractions,  be  The v i r u s  predominantly  of  requirements of  infection  by  1 72  c.  Antitumour The  percentage  anti-potato of  34  activity  (or  Ferrigni  tumour  91% et  of  a_l  of  the  activity  species)  (1982)  activity.  difference  or  may  vegetative Ferrigni strain  e_t a l A_;_  introduce  d.  to  the  This  brine  defined  of  of  of  the  on  this  the  type  of  although  discuss  this  31  nq/ml. of  methodology  between  the  seeds  used  expect  that  used  would  potato there  the  the  to  of  antitumour  of  and  reasonable  5  sample  differences study  extracts  than  their  result  idea  of  that  from  species  is  by  the  insufficient  possibility  that  toxic  family.  seeds  tested  of  Meyer  18  of  Artemia.  the  basis  of  an  further  species  toxic the  the  is  seeds  to  used  plant  less  to in  at  this  material  Meyer  et  than  found  a_l  active  toxicity Mg/ml.  study, The  that  of  1000  study used  brine  prevalent  study,  (79%).  biologically by  are  present  27  to  species  their  the  detected  vegetative respect  In  to  reduced  species  toxic  (1982)  (43%)  of  5 0  is applied  e_t a_l  41  to  LD  were  constituents  toxic  d i f f e r e n t , with  present,  34  were  active  proportion  is  less  5 0  of  a  this  is  The  shrimp  similar criterion  number  the  of  species  extracts  a  be  and  to  indicates  Euphorbiaceae  If  It  high.  exhibited  time.  shrimp.  was  in  which  demonstrable  may  variability  available  Toxicity  the  used  ED  27%  elicit  tumefaciens  Thirty-one  in  an  chemical  (1982).  some  information this  reflect  material  of  to  tested  very  observed  seeds  applied  was  had  Euphorbiaceous This  species  the  greater supports  in  this  study  compounds  (1982).  Kinghorn  173  et_ a l  (1977)  toxicity the  examined a s e r i e s  to brine  of  phorbol esters  for  shrimp. Artemia  were s e n s i t i v e  t o many of  phorbol esters  useful are  for  and t h i s  Many of  examined i n  e x h i b i t e d a d e g r e e of  toxicity  to b r i n e  al  The r e c e n t  (1977).  derivatives (Adolf  et  similar  the  1984)  identification  of  these  5.  CONCLUSION  Jatropha  raise  constituents,  basis  literature, especially  the rich  dermatophytic  are  of  of  species  of  in agents  fungi,  was  curcas  phorbol  and J .  gossypiifolia  of  also  in v e g e t a t i v e  present  whether  these,  or  material  is  not  Any,  or  all,  basis  for  the  which  certain  inhibit  viruses  the  toxicity.  are  in  sampled appear growth  Whether  different  the to  be  of  and tumours on  potato  these  manifestations  produced s e p a r a t e l y  by  of  different  known. of  these  use of  biological activities  these  species  Amazonian e t h n o m e d i c i n e . A p p r o x i m a t e l y treatment  information  Euphorbiaceae  biological activities  compounds  four  irritant  the comparison w i t h  same compounds or a r e  the  present  shrimp that  the q u e s t i o n  d i s c s and i n d i s p l a y i n g g e n e r a l  in  esters  plants.  On t h e  diverse  the  be  p u r e compounds examined by K i n g h o r n et_  from s e e d s of  a_l,  therefore,  in which p h o r b o l  extracts  some of  the  should,  the  a s h i g h as  the  assay  examining the E u p h o r b i a c e a e  widespread.  study  tested  their  of  skin  infections  above m e n t i o n e d b i o a s s a y s .  of  Euphorbiaceae  80% of  the  were a c t i v e  (Table  could  XX).  form in  species  i n e a c h of  Ten of  the  16  used the  174  species  with medicinal that  infectious  o r g a n i s m s or m a l i g n a n c i e s .  one of 26,  the  31,  S.  agent.  properties or  the  incomplete  assays  activities is  inherent  et. a l , i n any  interesting  to  1982)  t h o s e of  note  the  active  antiviral  If  A certain  the  the  of  out,  positive there  least  shrimp  of  of  ethnobotanical  of  of  their  the  of  of  therapeutically. false  w i t h no  tumour  respect,  average,  it  aspects  assay are  is of  the  of  no g r e a t e r  than  recorded'ethnobotanical  activities,  in  positive  in p r e d i c t a b i l i t y  this  the  biological  potato disc  on t h e  are  for  example  present  in  the  species  use. s c r e e n i n g p r o g r a m had been  results  would,  sorts  be u s e f u l  In  peoples  information  biological activities  this  are  the  the assumption t h a t  percentage  of Amanoa s p . ,  objective  either  the q u a n t i t a t i v e  species  strongest  activity  at  The m a j o r i t y  and l i m i t a t i o n s  no known e t h n o b o t a n i c a l  attainment carried  the  species  have no documented use as a  in p r a c t i c e ,  some o f of  fungi,  tumours and b r i n e  involve  only  used s p e c i e s a r e ,  Some of  having  the  b i o l o g i c a l assay.  The p o t e n c i e s  medicinally  use.  case.  either  The numbers of  have been o b s e r v e d u s i n g t h e  (Ferrigni  data.  of  predictive  the  of  been d i s c o v e r e d by a b o r i g i n a l  nature  that might,  not  results  potato  Such c o n s i d e r a t i o n s  used are  of  the d e r m a t o p h y t i c  bioassays  having  treatment  result  respectively.  these  the  T h i s c o u l d be a r e s u l t  never  available.  This  in  t o be t h e  tested,  31 and 3 1 ,  species active medicinal  aureus,  viruses  27,  likely  used in  conditions  active against  are  uses are  i n any  of  retrospect,  the  biological  have been no  the tests  175  advantage  obtained  ethnomedicine. not  intended  biological these  It  from s e l e c t i n g  activity.  Euphorbiaceae.  to other It  not  Nor does  be h e l p f u l  biologically statement  for  is  bioassays  information concerning will  active this  with uses  s h o u l d be e m p h a s i z e d t h a t  to apply  specific  plants  to it  conclusion  s c r e e n i n g programs  restricted the  to the  that  individual  in d i s c o v e r i n g constituents.  of  of  Euphorbiaceae  significant  study.  of  ethnobotanical  species  It  is  for  application  Amazonian s p e c i e s  imply  descriptive  this  in  is  new  i n t e n d e d as a summary  176  a 2i Z Iz  PART B.  PELTATIN,  THE ANTIVIRAL CONSTITUENT OF  Amanoa  1.  sp.  INTRODUCTION In  a screening project  from t h e  bark  and l e a v e s  were o b s e r v e d t o  inhibit  murine c y t o m e g a l o v i r u s  of  of  certain  the  in t i s s u e  e x h i b i t e d the  34 s p e c i e s  examined. E x t r a c t s  effective  both v i r u s e s . active  in  Both  against  the  greater  for  aimed a t  and t h e  activity. the  Sindbis  antiviral  f o r m a t i o n of  former  The l e a f  isolation  of  virus  A sample  activity  leaves  and aqueous  extracts  Euphorbiaceae  cells.  both the  the  organic  viruses  significantly a project  of  of  of  culture  strongest  inhibiting the  species  development  Amanoa s p .  highly  t h e Amazonian f l o r a ,  and of  of  the  and b a r k plaques  fractions  were  by  were  displayed extract  the  was  selected  active  constituent(s).  2. MATERIALS a.  Plant  AND METHODS  material  The p l a n t Voucher  material  specimens(D.  was c o l l e c t e d  MacRae  No.  70)  near  have  Iquitos,  Peru.  been d e p o s i t e d  at  UNAP(lquitos),  San M a r c o s ( L i m a ) ,  C h i c a g o F i e l d Museum and UBC  Herbarium.  identification  the  The  oblongifolia Chicago F i e l d prior  to  Muell.  of  A r g . , was c a r r i e d  Museum. P l a n t m a t e r i a l  plant, out  Amanoa  by D r .  aff.  M.J.  was p r e s e r v e d  in  Huft, methanol  extraction.  The l e a f  (41  g dry  weight)  sample was h o m o g e n i z e d i n  177  m e t h a n o l and e x t r a c t e d °C.  The c o m b i n e d e x t r a c t s  and p a r t i t i o n e d resulting  between  organic  s t o r e d at  -30  Antiviral  in  cytomegalovirus  After the  t h e medium was  of  The  in  respectively  f o r m a t i o n was  activity  Briefly,  mouse  is  and  described,  murine  dishes  (3T3)  which a l l o w e d  containing  embryo for  cells.  infection  removed and r e p l a c e d w i t h a  0.5% a g a r o s e ) were  were s u f f i c i e n t l y  i n which  i n c u b a t e d at well  the  plant  37°C u n t i l  developed for  u n a i d e d eye and per  solid  extracts the  counting.  cent  by  viral They  inhibition  of  calculated.  Chromatography The e x t r a c t  Chromatotron prepared Elution  was  was c a r r i e d at  two m i n u t e  resolved  (Harrison  from S i l i c a  (25/25/3)  its  water.  were e v a p o r a t e d  was a p p l i e d t o p e t r i  incubation,  were c o u n t e d w i t h t h e  at  chapter.  monolayer  were d i s s o l v e d . C e l l s  c.  this  (MCMV)  medium ( c o n t a i n i n g  plaque  20  in v a c u o  and d i s t i l l e d  95% and 50% e t h a n o l ,  C of  a p e r i o d of  plaques  acetate  measuring a n t i v i r a l  confluent  virus,  ethyl  evaporated  at  assays  in Part  an a l m o s t  were f i l t e r e d ,  solvent  °C.  The method f o r in d e t a i l  with that  and aqueous e x t r a c t s  v a c u o and d i s s o l v e d  b.  exhaustively  a flow  Research A s s o c i a t e s ) .  Gel out  PF 5« 2  using  r a t e of  intervals.  absorbance at  using a rotary  (Merck)  was  TLC d e v i c e ,  the  A 2 mm p l a t e  used  exclusively.  heptane/chloroform/ethanol  3 ml/min. F r a c t i o n s  The e l u a t e  254 n a n o m e t e r s .  were  collected  was m o n i t o r e d by  recording  1 78  3.  RESULTS AND DISCUSSION The i n i t i a l  Amanoa l e a v e s  separation  (350 mg e x t r a c t )  absorbing peaks, elution the  profile  extract,  is  seen  three  The f r a c t i o n s by e v a p o r a t i o n  in F i g u r e  fetal  been  the  major  system  reduced to  100 t i m e s  serum).  plates  volume culture  The sample was d i l u t e d a medium c o n t a i n i n g which  mouse embryo c e l l s  were i n c u b a t e d a t  3 ml  medium  37 ° C u n t i l  5%  was  w h i c h had  i n f e c t e d w i t h MCMV. The medium was a l l o w e d  and t h e  acetone  with t i s s u e  (5%-MEM) and 0.5% a g a r o s e of  component of  fractions.  w i t h minimum e s s e n t i a l  on a m o n o l a y e r  the  U.V.  was e l u t e d w i t h  m o d i f i e d minimum e s s e n t i a l bovine  of  c o m p o n e n t s . The  solvent  c o l l e c t e d were e a c h  5% f e t a l  bovine.serum  overlayed  three  several  (25/25/3),  large  fraction  16. The p o l a r  in  and 2 M1 d i l u t e d  medium ( D u l b e c c o ' s  5 times  organic  yielded  w h i c h d i d not move  and c o l l e c t e d a s  further  the  e a c h c o n s i s t i n g of  heptane/chloroform/ethanol  containing  of  to  just  solidify  plaques  had  developed. The a s s a y were i d e n t i f i e d plaques  was c a r r i e d by t h e i r  by more t h a t  out  in d u p l i c a t e .  ability  to  50%. F r a c t i o n s  classified  as a c t i v e  (Figure  that  these  fractions  correspond to  U.V.  absorbing  to  procedures.  16).  the  fractions  number, of MCMV  19 t h r o u g h 26 were  It  is  the  clear elution  from the of  a  all figure  strong  peak.  The f r a c t i o n s subjected  reduce  Active  were m o n i t o r e d by T L C , c o m b i n e d and  further  purification  The a n t i v i r a l  activity  by  identical  chromatographic  was a t t r i b u t a b l e  to a  179  TIME(min.)  gure  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 fraction o f 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 are blocked out.  180  single this  compound. The f o l l o w i n g  data  were o b t a i n e d  for  a c t i v e compound.  )-Peltatin.  a-(-  MeOH. 2 7 4 , H-2', 4.6  spectral  240sh,  H-6'),  (1H,  3.78  (s,  H-2,  H-3,  Mp 229-230 ° C . 214.  6.21  H-1), 6H,  (s,  4.4  H-NMR.  - 4.6 3  MS m/z  (CHC1 )  -118 ° .  3  5 6.35  3  (1H,  3.1  25  (80 MHz, C D C 1 )  1H, H-8),  2XOCH ),  H-4).  1  [a]  (rel.  (s,  5.93  (s,  2H, 0 " C H - 0 ) ,  H-11),  3.8  - 3.9  - 3.5  (m,  int.)  U.V.  1H, H-4), 400[M ]  (1H, 2.6  291  -  H-11),  - 2.75  (100),  +  2H,  4.4  2  355  (3H,  (12),  341  (8),  340  (7),  323  (6),  315  (5),  309 ( 6 ) ,  283  (5),  279  (4),  255 ( 8 ) ,  253  (5),  247  (13),  246  (69),  234  (4),  285  (6),  (23),  228 ( 2 6 ) ,  202  (23),  201  (73),  200  (12),  190 ( 2 3 ) ,  189  (66),  188 ( 6 5 ) ,  184 ( 1 2 ) ,  181  (13),  172 ( 1 0 ) ,  168 ( i l ) ,  167  (51),  165 ( 1 4 ) ,  155 ( 1 7 ) ,  154  (39),  153 ( 1 3 ) ,  152 ( 1 2 ) ,  151  (19),  139 ( 1 7 ) ,  131  115 ( 2 2 ) ,  103 ( 1 0 ) ,  91  The U . V . might  1  H-NMR s p e c t r a  be a d i b e n z o b u t y r o l a c t o n e  chemical 2'  and  (13),  shift  and C - 6 '  of  the  protons  indicated lignan.  C-8 p r o t o n and t h e  suggested that  it  was c o r r o b o r a t e d by a c o m p a r i s o n of that  published for  exists  as  (+)  and  this (-)  relative  configuration  chemical  shift  (CHC1 ) it  The  C - 1 . Based  and the which  was d e t e r m i n e d  compound  characteristic of  mass s p e c t r u m 1967).  upon the  (-)  for  This  with  the  characteristic  rotation  were m e a s u r e d  the C-  a-Peltatin  d e p e n d i n g upon  optical  t o be t h e  the  equivalence  compound ( D u f f i e l d ,  at  (14).  may be a - p e l t a t i n .  its  diastereomers,  4.58)  = -118 ° ,  3  isolated,  (5  that  X  [a]  the  isomer  compound  (Figure  17).  181  OH  H  »-(-)-Peltatin  Figure  17 - S t r u c t u r e Amanoa s p .  of  a-(  - ) -peltatin  isolated  from  1 82  a-Peltatin significantly by  herpes  The  to produce Bedows  They  and  i s effective  effect.  against  pseudorabies  Measles  DNA  stated  activity to  that a-  from  vaccinia  we  effect have  infection  e_t a _ l ,  1981a).  both  the  only  e_t a l ,  that  virus  t o have  o f an  s p . , was  observed  Farnsworth  virus,  known  (Markkanen  by m e a s l e s  however,  activity  those  the d i f f e r e n c e s virus  (paramyxovirus  not  from  cytopathic  a n d HSV-1. a  slight  e_t a_l ( 1 9 6 6 )  extract  poliovirus  of  Podophyllum  type  I I I and  virus.  comparing  stranded  resulting  in inhibiting  By c o m p a r i s o n ,  peltatum  mind.  reduce  of Podophyllum  (1982)  produced  a-peltatin,  no a n t i v i r a l  study,  to  (Markkanen  similar  and H a t f i e l d  detected  In  effect  (HSV-l)  a very  infectivity  observed  antiviral  reported  of the m e d i c i n a l r e s i n  podophyllotoxin effect  virus-1  been  compound, p o d o p h y l l o t o x i n , a w e l l  constituent  1981b).  the cytopathic  simplex  related  observed  has r e c e n t l y  with  i s a single  viruses  while  used  HSV-1  a-  ( -  in  this  be b o r n i n  virus  a n d MCMV a r e d o u b l e herpesvirus.  of a - p e l t a t i n  from  obtained  should  s t r a n d e d RNA  of the genus  isomer  observed  the ones  in the assays  group),  which  results  was  Moreover,  used.  ) -peltatin  The  may  be  i ti s  antiviral specific  isomer. ( -  )- P e l t a t i n  the genus  Amanoa  i s the f i r s t  and t h e f i r s t  (cyclolignan-9'-9-1ignanolide) Euphorbiaceae.  t o be  lignan lignan  t o be of  reported  reported  i t ' s class from  the  family  183  PART C .  1.  THE ANTIVIRAL ACTIVITY  INTRODUCTION The c y c l o l i g n a n o l i d e ,  as  OF LIGNANS  the p o t e n t  antiviral  agent  Amanoa. T h i s o b s e r v a t i o n , and H a t f i e l d ,  - ) -peltatin,  a-(  was  from an Amazonian s p e c i e s  combined w i t h r e c e n t  1982; Markkanen e t  al,  reports  1981a and b)  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  effects  this  a variety  comparative  on v i r u s  study  infected  on t h e  best  action.  metabolism  to  1953).  alterations  polymerization  to  of  possible 2)  (Bedows  that  other  stimulated of  lignans  of  of  al,  biological  t h e mechanism of podophyllotoxin,  the  while others  and n u c l e i c  1976b).  Still  metabolism  are  known  acid  others  cause  (Waravdekar  podophyllotoxin derivatives  1979)  their  compounds, c a n damage DNA  1976a),  in c e l l u l a r  of  interfering  form m i c r o t u b u l e s a n d ,  with  in t h i s  e_t  bind  its way,  mitosis.  To p r o v i d e  1)  class  certain  (Brewer e t  inhibiting  for  w i t h n u c l e o s i d e uptake  Finally,  tubulin  action  this  ( L o i k e and H o r o w i t z ,  fundamental al,  basis  ( L o i k e and H o r o w i t z ,  interfere  of  a diversity  Some d e r i v a t i v e s  known example of  directly to  display  w h i c h c o u l d form t h e  antiviral  effects  of  cells.  Cyclolignanolides effects  identified  further  lignans,  a wide  variety  effects the  the  i n f o r m a t i o n on t h e mode of following of  on v i r u s  antiviral  studies  lignans  actions  of  were c a r r i e d  were t e s t e d  infected  antiviral  for  out:  their  cells;  p o d o p h y l l o t o x i n and a-  184  peltatin  against  cytomegalovirus 3)  two v i r u s e s , (MCMV)  different  virus  and m u r i n e  were c o m p a r e d ;  times  examined t o d e t e r m i n e  Sindbis  d u r i n g the  which  stage  p r o c e s s of was  infection  sensitive  to  were  lignan  action.  2.  MATERIALS AND METHODS  a.  Chemicals The l i g n a n s  u s e d were o b t a i n e d  P o d o p h y l l o t o x i n was p u r i f i e d Sigma. Part  a-Peltatin  A.  Justicidin  and a c e t y l a t e d Dept.  was  of  dihydroferulic  Al  acid,  dimethylretrodendrin Vancouver.  Arctiin  sesartemin,  from Amanoa s p .  B and d i p h y l l i n ,  Chemistry,  University,  matairesinol, were a g i f t  was  as w e l l as  were p r o v i d e d by D r . Fateh  isolated  episesartemin,  of  from a sample p u r c h a s e d  isolated  apioside  from a v a r i e t y  G.  Libya.  Dr.  Eric  its  apioside  H.  Sheriha,  Plicatic  Swan,  (Chapter  compounds a r e b.  Cells  II,  presented  and  epiyangambin,  has  thesis).  in Figure  The s t r u c t u r e s  of  from V . these  18.  and v i r u s e s  The p r e p a r a t i o n cells  this  acid,  Forintek,  d i h y d r o s e s a r t e m i n and 0 - d i h y d r o y a n g a m b i n were i s o l a t e d elongata  in  and  from A r c t i u m l a p p a  yangambin,  from  as d e s c r i b e d  a-conidendrin,  of  sources.  of  been d e s c r i b e d  viruses in Part  and p r o c e d u r e s A,  this  for  chapter.  maintaining  185  Podophyllotoxin  co-(-)-Peltatin  DiphyllinR  CH,0  OH oo -Conidendrin  Figure  18  Dimethyl-OD-ret rodendri n  - Structures activity.  of  lignans  tested  Justicidin B  for  antiviral  186  c.  Antiviral The  the  samples  case  culture  screen ing  of p l i c a t i c medium  concentration The remain to  for  murine  tested  5%  37  and  0.10 at °C,  1.0,  fetal  (MEM)  number  1.0  the s o l u t i o n  developed  they  during  incubated  tissue  22)  were  of plaques  sufficiently  been  of  to  exposure  removed  f o r s e v e r a l days  were  while  (3T3) embryo  and to  allow  and  minimum  Sin,dbis v i r u s  or  calculated  allow  t h e compound justicidin  B,  to  t o be and  tested at concentrations the remaining  Mg/ml. A f t e r  containing with 0.5%  incubated  cells  in Dulbecco's  titer  a-peltatin,  concentration.  were  had  either  (MCMV) a t a  (FBS),  the p e r i o d  bathed  containing  100  t h e compound  plaques.  overlayed  serum  a t t h e same  solidified,  with  in  ethanol  allowing  l a y e r s o f mouse  Mg/ml,  10 a n d  bovine  diluted final  the v i r u s  i t sapiosides  and  o u t by  of v i r a l  15 a n d  t h e p l a t e s were  lignan  being  Podophyllotoxin,  diphyllin  were  for a  the c e l l s after  cytomegalovirus  tested.  and  with  confluent  medium  to allow  carried  were  a countable  0.01,  as  These  (10% ethanol  1%.  the development  essential  d i s s o l v e d i n 95% e t h a n o l  and a l s o  passage  1ignans  acid).  were  the c e l l s  (between  at  of  in contact  Nearly  for  such  assays  the v i r u s  while  were  of  2 hours  virus  and  After  until  for counting.  of  and  were  incubation  l i g n a n was  Dulbecco's agarose  compounds  of  removed  MEM  containing  the  appropriate  the p l a t e s  the plaques  had had  187  d.  Effect  of  Three for  time  of  treatment  stages  in  the  sensitivity  lignans, period;  of  p r o c e s s of  the v i r u s  to  infection  the  presence  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  period during  infection;  were examined of  the  the  antiviral  pre-infection  and the  period  after  infect ion: P r e - i n f e c t ion S u s p e n s i o n s of concentrations 0.1% e t h a n o l monolayers  of  for  of  treatment virus  were e x p o s e d t o  a p e r i o d of  diluting  25,000 t i m e s .  After  virus  for  2 hours,  and t h e  cells  agarose.  a further  overlayed  The p l a t e s  containing  2 h o u r s . A l i q u o t s were added  mouse embryo c e l l s ,  37 ° C  various  e a c h compound i n D u l b e c c o ' s MEM,  lignan mixture at  the  the  incubating  virus  w i t h MEM c o n t a i n i n g  were i n c u b a t e d u n t i l  and  the c e l l s  t h e medium was 5% FBS  plaques  to  with  removed  and 0 . 1 %  had  developed. Treatment Virus peltatin w h i c h was plates  dur ing  i n f e c t ion  was c o m b i n e d w i t h e i t h e r  i n D u l b e c c o ' s MEM (1%  ethanol  then a p p l i e d to a monolayer  were i n c u b a t e d  for  2 hours at  final of  concentration),  mouse c e l l s  and  the  37 ° C . The medium was  removed and r e p l a c e d w i t h a s o l i d o v e r l a y Treatment  a-  p o d o p h y l l o t o x i n or  as d e s c r i b e d  above.  post-infection  Mouse embryo c e l l  monolayers  were e x p o s e d t o  virus  s u s p e n d e d i n D u l b e c c o ' s MEM f o r  2 hours at  37 ° C . The  was  was a d d e d .  This contained,  removed and a s o l i d o v e r l a y  virus in  188  addition  t o D u l b e c c o ' s MEM w i t h 5% FBS  either  p o d o p h y l l o t o x i n or  plates  were i n c u b a t e d u n t i l  3.  a-peltatin  cells  the development  (Table  potency,  XXI).  a concentration effect  as  effective  of  on t h e  The  visible.  its in  antiviral  To a t t e m p t action  compound,  Sindbis  virus  a d o s e as h i g h as  number of  the  even a t  to gain  other  Sindbis  lignans  compounds were a p p l i e d a t  infect  the  cells  late  infection. effect for  of  dose-response tested  showed any  100 Mg/ml.  l i g n a n s p o d o p h y l l o t o x i n and avarious  To d e t e r m i n e  on t h e  early  or  the  cells  were i n f e c t e d w i t h v i r u s  in  virus  during these  was  to b e i n g used  treatment).  infective  times  whether  viruses,  two h o u r s p r i o r  (pre-infection  stages  plaques and,  i n f o r m a t i o n on t h e mode of  the  incubated with lignan  were  virus  Clear  peltatin,  direct  i n mouse  B and d i p h y l l i n ,  apioside,  antiviral  had any  had  1 Mg/ml.  d o s e s as h i g h as  further  however,  plaques  compounds, j u s t i c i d i n  the  lignans  in  Neither  t h e MCMV p l a q u e s .  viral  equal  10 n g / m l .  of  p r o c e s s of  i n mouse embryo  formed by a l m o s t 50%  None of  activity,  in  plaques  r e d u c i n g the  evident.  effective  approximately  a p i o s i d e and a c e t y l a t e d  also  highly  of MCMV p l a q u e s  f o r m a t i o n of  even a t  some e x t e n t ,  were not  the  1% e t h a n o l .  number of  The a r y l n a p t h a l e n e as w e l l  are  B o t h compounds a r e  r e d u c i n g the  embryo c e l l s ,  to  agarose,  RESULTS  preventing  any  and  p l a q u e s were  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  at  and 0.5%  To t e s t  p r o c e s s were  the p r e s e n c e  of  to  whether affected,  lignan  % Inhibition Lignan  Tested  of  Plaque  Formation MCMV  S i ndb i s v i r u s 0.01 „g/ml  0 . 10 „/ml  1 .00 ///ml  10 <//ml  100 j/g/ml nt  35  0.01 »/g/ml  0 . 10 „g/ml  1 .00 „g/ml  10 „g/ml  100 ,/g/ml  74  100  nt  nt  Podophyllotoxin  8  3  4 .  nt  a-peltat i n  0  6  7  nt  nt  49  85  100  nt  nt  42 .  74  tox  nt  nt  13  14  tox  nt  nt  D iphyl1i n  7  18  84  nt  nt  7  1 1  '19  nt  nt  D i phy11i n a p i o s i de D iphyl1i n ap i os i d e - O A c D i methylretrodendr i n  8  14  100  nt  nt  26  10  9  nt  nt  20-  96  95  nt  nt  33  4  25  nt  nt  nt  nt  3  7  tox  nt  nt  1  6  tox  a-Coni dendr i n  nt  nt.  7  0  5  nt  nt  2  5  0  PIicatic  nt .  nt  1  6  0  nt  nt  2  1  3  M a t a i r e s i no 1  nt  nt  7  4  tox  nt  nt  4  0  tox  Arct i i n  nt  nt  4  5  3  nt  nt  3  4  3  D i hydroferu1i c ac i d Episesartemin  nt  nt  4  4  0  nt  nt  1  3  2  nt  nt  2  7  2  nt  nt  0  3  5  Sesartemi n  nt  nt  0  6  4  nt  nt  4  7  6  Ep i yangamb i n  nt  nt  2  0  6  nt  nt  3  1  6  Yangamb i n  nt  nt  3  6  2  nt  nt  7  0  6  D i hydrov i ro1ong i n  nt  nt  7  4  3  nt  nt  1  5  5  0-d ihydroyangamb i n  nt  nt  2  6  2  nt  nt  5  2  4  Justicidin  Table  XXI  B  acid  -  E x a m i n a t i o n of l i g n a n s R e s u l t s a r e the average t  nt  = not  tested.  for their effect of an e x p e r i m e n t  *  t  on r e p l i c a t i o n of S i n d b i s c a r r i e d out i n d u p l i c a t e . $ tox  = toxic  to c e l l s ;  Virus  plaques  a n d MCMV  were  not  i n mouse  counted.  cells.  190  (treatment during virus  infection)  were e x p o s e d t o  lignan  culture  period  lignans  on MCMV i n f e c t i o n  data  and c e l l s for  (post-infection are  concerning Sindbis  virus  Figure  the  19 s u p p o r t s  the  continuously peltatin the  is  cells  formation  presented  to  infected  slightly  are  podophyllotoxin only  slightly  This  is  lignan that  or  less  somewhat is  it  only  is  effect  the  cells)  the  a-Peltatin,  magnitude  the  on t h e  number of  effect  weaker  infection  by a l m o s t  a-  the  lignans  plaque of  in  formation. exposure  days  the  are  highest  (assuming  case  of  the  other  that  by v i r u s .  t o p o d o p h y l l o t o x i n , no  concentration  hand, caused a  plaques  three  tested  treated  orders  p r o d u c e d by t r e a t m e n t The number of  plaques  two h o u r s p r e - i n f e c t i o n  (40  dose-dependent  formed from  was a p p r o x i m a t e l y  than  50% a f t e r  of  during were  treatment  or  reduced with  peltatin. Sindbis  to  treatment.  at  This  applied  to  compared w i t h s e v e r a l  was d e t e c t e d  in  in  in Table XXI,  duration  exposed d i r e c t l y  viruses.  after  surprizing since  th,e  XXII.  effective  inhibiting  is  reduction  19 w h i l e  that  concurrently, in  the  p o d o p h y l l o t o x i n . When  virus  Mg/ml).  of  and e x p o s e d  m e t a b o l i z e d by the  post-infection When t h e  effective  The e f f e c t s  by MCMV when  than  with virus  a-peltatin  of  finding  As shown  with  duration  in F i g u r e  highly  plaques  cells.  two h o u r s ;  not  are  more p o t e n t  infected  infected  c a n be f o u n d i n T a b l e  initial  of  complete  treatment).  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 inhibiting  the  already  virus  responded q u i t e  differently  to  treatment  a-  191  LOG Figure  CONCENTRATlON(ng/ml)  19 - E f f e c t of time 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 i n mouse embryo c e l l s .  Solid figures refer to a-( - ) - p e l t a t i n ; open ones to podophyllotoxin. Squares = p r e - i n f e c t i o n t r e a t m e n t ; t r i a n g l e s = treatment during 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 treatment.  Compound  % Reduction  in  Plaques'  0.01  0. 1  10  „g/ml  l/g/ml  „g/ml  podophyllotoxin  0  1  0  1  0  a-peltat i n  2  1  0  1  0  podophyl1otox i n  0  1  0  13  58  a-peltat i n  0  1  2  0  52  podophyl1otox i n  1  0  1  0  0  a-peltat i n  0  2  0  1  1  0.005  Table  XXII  „g/ml  - E f f e c t o f t i m e of l i g n a n i n mouse embryo c e l l s .  treatment  Results  an e x p e r i m e n t  are  the average  of  upon  Treatment  eg/ml  inhibition  carried  Protoco1  40  out  of  in  prior  during  after  Sindbis  Virus  triplicate.  to  infection  infection  infection  infection  1 93  w i t h p o d o p h y l l o t o x i n or  a-peltatin  (Table  i n f e c t i o n nor p o s t - i n f e c t i o n t r e a t m e n t any e f f e c t ,  even a t  Exposing the being  cells  to  l i g n a n at  ultimately  reduction  in  virus, the  formed. T h i s e f f e c t ,  t i m e s as p o t e n t ) or  after  4.  DISCUSSION  as t h a t  infection  A l t h o u g h the results  indicate  certain  classes  however,  number of too,  they  viral  i s much weaker  that  antiviral  lignans.  lignans  effects  phosphodiesterase  activity  is  episesartemin,  are  (Nikaido  arctiin,  of  the  butanolide  et_ a_l,  effects lignans  certain  bis-tetrahydrofuran  enzyme,  cyclic  Plicatic  acid  to  lignans, bis-  sesartemin,  without  effect.  to  inhibit  1981), a n t i - s t r e s s  (Chapter tested,  III,  Bis-  activity.  lignans,  is  this  cyclic  an  (Sih  compound and i s  et, a_l,  e_t  thesis). or  Matairesinol, inhibitor  AMP  activity  matairesinol  AMP p h o s p h o d i e s t e r a s e ( N i k a i d o a reactive  the  known t o have a number of  d i s p l a y e d any a n t i v i r a l  is  during  small,  1969), h y p o t e n s i v e a c t i v i t y  1976) and b e h a v i o r a l  Neither  (1/1000  specific  The t e t r a h y d r o f u r a n  i n c l u d i n g the a b i l i t y  (Brekhman and Dardymov, al,  in a  treatment  compounds t e s t e d was  e p i y a n g a m b i n and y a n g a m b i n , were a l l  biological  are  by MCMV.  lignans,  t e t r a h y d r o f uran  had  plaques  d i h y d r o s e s a r t e m i n and / 3 - d i h y d r o y a n g a m b i n , and t h e t e t r a h y d r o f uran  lignan  resulted  lignan  pre-  40 Mg/ml.  same t i m e as  observed for  number of  of  the  Neither  with e i t h e r  c o n c e n t r a t i o n s as h i g h as  infected with Sindbis  dose-dependent  XXII).  of  like the  1981).  responsible  for  194  p r o d u c i n g asthma and e l i c i t i n g (Chan-Yeung e t  al,  1973).  replication  the  two v i r u s e s  of  It,  and a - c o n i d e n d r i n ,  like  cyclolignanolides.  Neither  The e f f e c t and d i p h y l l i n infection  is  of  the  and i t s  apiosides  effect  B is  highly  toxic  action  of  antiviral  and p i s c i c i d a l  activities  infection  or  yielded  conclusions.  a-peltatin  be e x p l a i n e d by a d i r e c t  effect  P o d o p h y l l o t o x i n had no e f f e c t peltatin of  caused a s l i g h t  sufficient  The a b i l i t y consistent  of  possess  the  Horowitz,  a-peltatin  type  ability  1976a).  It  lignans  that  compounds  after  compounds c a n n o t  virus.  doses t e s t e d ,  while  of MCMV v i r u s  which  t h e anti-MCMV  this,  directly  and  that  is  group,  (Loike  a-peltatin  awas  activity.  other  having a 4'-hydroxyl  noteworthy  that  interesting  t o c a u s e DNA f r a g m e n t a t i o n is  et  action.  t o damage t h e v i r u s reports  not  (Munakata  these  these  upon t h e  inactivation  virus  p i s c i c i d a l compound  d u r i n g or  of  the  B  exposed to  magnitude to e x p l a i n  with previous  podophyllotoxin  at  fish  though q u i t e  The anti-MCMV a c t i v i t y  Sindbis  considering  of  is  before,  some g e n e r a l ,  to  mode of  in which v i r u s  are  or upon n u c l e o s i d e  worthwhile  regarding their  the  justicidin  compounds a r e  this  is  podophyllotoxin  on  activity.  inhibiting  unknown and i t  The e x p e r i m e n t s  not  in  completely  some a s p e c t s  effect  lignans,  on m i c r o t u b u l e s  The mechanism of  share  i n man  Dimethylretrodendrin  Although these  al,  the  tested.  arylnapthalene  justicidin  is  was w i t h o u t  had any a n t i v i r a l  transport, 1965).  too,  response  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 ,  interesting.  known t o have any  an a l l e r g i c  and  had no  195  corresponding effect Sindbis  upon t h e  single  virus.  A s t r o n g anti-MCMV e f f e c t peltatin after  is  observed only  infection.  most e f f e c t i v e attachment  Because  regimen,  it  infection  some e x t e n t  relatively  of  the  This  on t h e  It  is  whether or  for  several that  protocol the  cells  are  potency in  is  the  time  of  effect  of  exposure  the  is  development  affected  the  is  process  two  three  of  the  the  or  that  inhibitory infection. is  evident infection  19,  it  post-treatment  t i m e s more p o t e n t  lignans  not c o n t i n u e d .  b o t h d u r i n g and a f t e r  virus  than  that  protocol. This difference small  is  relative  to  The  result  are  either  irreversible  and  the  that  some s t a g e  transient  and,  if  of  virus  interrupted,  Such a s t a g e would need t o  first  in  the  in each p r o t o c o l .  s u g g e s t e d by t h i s  by them i s  appears  From F i g u r e  lignan applied  unimportant  It  to  both  two h o u r s d u r i n g  disproportionately  t h e d o s e of  of  are  qualified  inhibited  significant,  infection.  the  which  s h o u l d be  XXII).  or  neither  anti-MCMV e f f e c t  for  was  stages  was  Sindbis  infection  most o b v i o u s e x p l a n a t i o n s that  virus  but  the  the  treated  during  are  statement  t h e anti-MCMV a c t i v i t y  treatment  difference  of  that  was a p p r o x i m a t e l y  antiviral  virus  (Table  a weak,  days a f t e r  during  infection  high concentrations  stages  significant  the  appears  of  early  treated  after  by S i n d b i 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  effect  are  can be c o n c l u d e d t h a t  lignans.  compounds do have  p o d o p h y l l o t o x i n and a~  treatment  by n o t i n g t h a t by  of  when c e l l s  nor p e n e t r a t i o n  d i s r u p t e d by t h e s e  these  s t r a n d e d RNA c o n t a i n i n g  two h o u r s of  virus  exist  infection.  1 96  It  has been s u g g e s t e d t h a t  podophyllotoxin tubulin  type  and t h e i r  and H a t f i e l d , which a f f e c t antiviral  lignans  ability  the a n t i v i r a l  is  to  a result  interrupt  1982; Markkanen e t tubulin  al,  of  its  activity  their  affinity  aggregation  1981b). C e r t a i n  have been s t u d i e d w i t h r e s p e c t  activities  and t h e  results  are,  of for  (Bedows  drugs to  their  by no means,  conclusive. The e a r l y alkaloids  observations  could suppress v i r a l  from t h e c l i n i c a l that of  colchicine  infection  .viruses.  that  viewpoint.  mice  by  activity  B viruses  in  demecolcine a l s o  suppressed i n f e c t i o n  (Tokumaru and A v i t a b i l e , vinblastine, virus  1965)  F r i e n d and R a u s c h e r Vinblastine  tissue In  culture  cells  a significant  antimitotic  1962). of  virus  rabbit  cornea  alkaloid,  of  in  were a l s o rabbit  mice  i n mice  effective  studies,  and  cells  virus  against  (Chirigos, against  rabbit  (Tokumaru and A v i t a b i l e , number of  the  i n f e c t e d by mengo  was a c t i v e  c o r n e a or  in  Colchicine  The V i n c a  and v i n c r i s t i n e  infection  disease  1971).  leukemia v i r u s e s  stages  ECHO and C o x s a c k i e  simplex  survival  found  demonstrated  by h e r p e s  and v i n c r i s t i n e  simplex v i r u s  early  cells  i n c r e a s e d the  (Johnston,  encouraging  encephalomyocarditis  (Katsilambros,  culture  Vinca  and Chang ( i 9 6 0 )  and-Newcastle  embryo  tissue  were  polio,  developing chick  and r a b b i t  the  demecolcine,  vaccinia,  culture  or  s u p p r e s s i n g the  derivative,  against  tissue  in  i n f l u e n z a and  The c o l c h i c i n e  antiviral  infections  Weinstein  was e f f e c t i v e  of  colchicine  1965). herpes  kidney  1971).  however,  these  s u b s t a n c e s have been f o u n d t o have no e f f e c t  upon  1 97  viral  replication.  against  C o l c h i c i n e was  poliovirus  (Kovacs,  vaccinia  virus  culture.  Vincristine  virus  (Solovyov  (Johnston,  of  1965)  state,  case  of  have  that  for  the  poxvirus,  was  i n h i b i t e d due t o t h e response to  treated  was p r e v e n t e d , synthesis  Evidence  cells  Colchicine  reduced the  inhibit  by 75 t o  d e p o l y m e r i z a t i o n of In  comparing t h i s  important  a cellular  penetration  to note  the  microtubules w i t h the in  (1978),  the c o n c e n t r a t i o n  greater  than  results  reported here,  that  final  90% and t h e  that,  the of  required to it  is  of mengo  sarcoma  or  the  cessation  is  infection.  In  the  the v i r u s  protein  produced  disease  and e c l i p s e  replication of  was  of  in  virus in  the  virus  cellular  RNA  1963). indicates  stage  extracellular  result  tissue  arrest,  the absence  and  metaphase  1964). Newcastle  due t o  1963)  1965).  u n c o a t i n g of  has been p r e s e n t e d w h i c h  also  virus  viral  further  and R o b b i n s ,  inhibitors  Forest  but  apparently  (Marcus  of  shown t h a t  (Joklik,  in  that  i n d u c e d metaphase  d i d undergo normal a t t a c h m e n t , colchicine  e_t a_l,  accompanies the  a b s e n c e of  infection  1965)  provided evidence  interruption  it  (Dales,  p o l y o m a , Rous  (Freeman  normally  ineffective  on m u l t i p l i c a t i o n  or v a c c i n i a ,  and a l s o c o l c h i c i n e  responsible  REO v i r u s  and M e n t k e v i c h ,  viruses  studies  RNA s y n t h e s i s  1962),  had no e f f e c t  encephalomyocarditis Several  o b s e r v e d t o be  of  viral  evident  and V a n c e ,  reported here,  of  colchicine inhibit  Semliki  was a t t r i b u t e d  (Richardson data  mitotic  assembly.  p r o d u c t i o n of  effect  study  that  Richardson u s e d was  mitosis. that  the  1978).  it  is  and V a n c e  1000  From  to  fold  the  podophyllotoxin  198  inhibits prevent  MCMV i n f e c t i o n  been r e p o r t e d .  DNA t o  the  minimum d o s e r e q u i r e d  of  colchicine  to  mitosis.  An o p p o s i n g e f f e c t also  at  rabbit  kidney c e l l s  exposing c e l l s infection  Infectivity was  to c o l c h i c i n e ,  (Farber  were c o n s i d e r a b l y  and E b e r l e , greater  of  on v i r a l  herpes  infections  simplex v i r u s  enhanced 5 to  has  type  7 f o l d by  c o l c e m i d or v i n b l a s t i n e  prior  1976). A g a i n ,  used  than  those  2  the  doses  r e q u i r e d to  to  inhibit  mitosis. The a v a i l a b l e role  in v i r a l  results, great cell  it  deal, type  results  evidence  infections.  appears  that  indicates  Judging the  and t h e  stage  of  the  that  inhibition  responsible evidence  for  may be these  2) not It  the  their  antiviral  but  exert  not  seems u n l i k e l y  w i t h what  is  that  effect  The of  a-  hypothesis  two compounds  Two p i e c e s  is  of  of  the  against  murine  lignans  against  MCMV was  reversible.  not  Sindbis  the a n t i v i r a l  known of  action  virus.  microtubules are  of MCMV but of  an a n t i v i r a l  Sindbis  o b s e r v e d t o be r e a d i l y  irreversibility  specific  activity  the  a  cited:  antiviral  multiplication  support  a  the  may v a r y  the  antiviral  activity.  in  infection.  of m i c r o t u b u l e s by t h e s e  lignans  cytomegalovirus  role  virus,  p r o c e s s of  and p o d o p h y l l o t o x i n ' do n o t  variation  that  specific  peltatin  1)  of  p r e s e n t e d here c o n c e r n i n g the  the  microtubules play  from t h e  nature  d e p e n d i n g upon t h e  that  a requirement virus.  activity  the a n t i m i t o t i c  is  for  the  The not  behavior  of  consistent these  199  compounds  (Dustin,  Another  cellular  podophyllotoxin concentration somewhat the  is  is  activity  nucleoside transport.  than  that  (1976a and b)  reasons,  it  antiviral effect above the  is  are  of  of  the  the  transport. that  the  for  lack  of  reversibility  Inhibition  of  n u c l e o s i d e uptake  not  for  the of  been shown t o be r e a d i l y 1976a). Moreover,  absolutely  why v i r u s  required  replication  conditions.  the  sensitivity the  of  1972).  tubulin  of  RNA,  well  these the  involves cited  activity  of  an  is  i.e.,  there  o n l y MCMV  effect.  by p o d o p h y l l o t o x i n has  capacity  for c e l l  or c o l c h i c i n e  and W i l s o n ,  antiviral  and  have  t h e mode of  reversible for  (Loike  g r o w t h and i t  with  and  nucleoside  under  is  uptake  not  clear  these  DNA and p r o t e i n  shown t o be u n a f f e c t e d by t r e a t m e n t  Mizel  (1972)  inhibition  s h o u l d be a f f e c t e d  The s y n t h e s i s  podophyllotoxin  both Loike  b i n d i n g may be a p p l i e d h e r e :  or  is  of  The same a r g u m e n t s  explanation  Horowitz,  mitosis,  ( L o i k e and H o r o w i t z ,  considering that  no a v a i l a b l e  clearly  of  i n d e p e n d e n t phenomena. F o r  hypothesis  tubulin  is  by p o d o p h y l l o t o x i n , as  is  the  effect  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  upon n u c l e o s i d e against  quite  worthwhile  action  result  that  by  the  inhibition  The s t u d i e s  and n u c l e o s i d e u p t a k e  as by c o l c h i c i n e ,  Although  and M i z e l and W i l s o n  provided convincing evidence aggregation  in  specific  1972).  inhibited  a detectible  resulting  s t r o n g and q u i t e  1976b; M i z e l and W i l s o n , Horowitz  known t o be  r e q u i r e d to e l i c i t  greater  effect  1978).  has  been  either  ( L o i k e and H o r o w i t z ,  1976a:  200  It  must be c o n c l u d e d t h a t  the  site  of  action  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  is  antiviral  If  involves  either  tubulin  appears if  activity  that  infection,  unknown.  aggregation  the  interrupted  is  or  susceptible  d u r i n g the  ultimately  site  first  prevents  cycle.  More s t u d i e s  further  nature  of  which  is  certain to  responsible viruses.  be u s e f u l  p r o c e s s of  It  tools  the for  is for  infection.  is  the  for  then  stage  following  of it  which,  virus  normal development  are  their  inhibition  a critical  two h o u r s  specific the  responsible  nucleoside transport  replicative the  it  of  of  the  needed t o  characterize  biochemical  alteration  inhibition  p o s s i b l e that d i s s e c t i n g the  of  proliferation  of  these  compounds may  prove  molecular  basis  of  the  201  LITERATURE Chapter  CITED IV  A d o l f , W., O p f e r k u c h , H . J . and H e c k e r , E. (1984) Irritant p h o r b o l d e r i v a t i v e s from f o u r J a t r o p h a s p e c i e s . P h y t o c h e m i s t r y 129-132. A n j a n e y u l u , A . S . R . , Rao, K., Row, L . R . and Subrahmanyam, C . 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(Washington, D.C.) 10, 180-187.  207  APPENDIX A List  of Amazonian Angiosperms of E t h n o b o t a n i c a l I n t e r e s t  Taxon  Use Code  References  C l a s s : Magnoliopsida Subclass I. Magnoliidae Order:  Magnoliales  Family: Annonaceae Anaxagorea sp. Annona ambotay A. spinescens A. tessmannii Duguetia r i p a r i a Guatteria calva G. duckeana G. dura Unonopsis v e n e f i c i o r u m X y l o p i a amazonica X. aromatica X. benthamii Family: M y r i s t i c a c e a e Compsoneura c a p i t e l l a t a C. d e b i l i s C. s p r u c e i D i a l y a n t h e r a otoba D. p a r v i f o l i a Iryanthera c r a s s i f o l i a I. grandis I . longi f o l i a I. macrophylla I. polyneura I. t r i c o r n i s I. u l e i Osteophloem platyspermum Virola albidiflora V. bicuhyba V. c a l o p h y l l a V. c a l o p h y l l o i d e s V. c a r i n a t a V. c u s p i d a t a V. elongata V. f l e x u o s a  02 02 03 T 02 02 T 02 G1 K2 G5 K2  02 P5  K6 A1 A2 T A2 C6 A2 T A2 R1 A2 A2 A2 D1 D2 A2 A2 B2 D2 K3 M R1 R1 A4 R1 A2 02 R1 A2  55 2 42 2 42 53 55 61 42,49,51,57,61 55 61 55 49 49 49 30,31 38 49 49 49 64 49 49 49 55 54 49 49 49,2 49 49 49 49  208  V. l o r e t e n s i s v. melinonii V. p e r u v i a n a V. r u f u l a V. s c h u l t e s i i V. s e b i f e r a V. s u r i n a m e n s i s V. t h e i o d o r a V i r o l a sp. Order : L a u r a l e s F a m i l y : Monimiaceae Siparuna quianensis S. s p . Family: Lauraceae Nectandra p i c h i i r i n N. r a d i a c i Ocotea venenosa Couroupita sp. Order : P i p e r a l e s Family: Piperaceae Peperomia e m a r q i n e l l a P. g l a b e l l a P. m a c r o s t a c h y a P. s e r p e n s P i p e r aduncum P. a l e g r e a n u m P. a n t i rheumat icum P. b o g o t e n s e P. caudatum P. d a c t y l o s t igmum P. e r y t h r o x y l o i d e s P. f u t u r i P. gen i c u l a t u m P. h i s p i d u m P. hostmannianum P. i n t e r i t u m P. i g u i t o s e n s i s P. m a r g i n a t u m P. n i g r u m P. nubigenum P. o b l i q u u m P. p e l t a t u m P. s c h u l t e s i i P. s e r p e n s |\ sudisilvestre P. t i n g e n s P. t u b e r c u l a t u m Piper sp. Pothomorphe u m b e l l a t a  T A2 K1 R1 A2 B2 R1 S A2 A8 C8 02 R1 G1  49 49 4 6 , 49 4 49 2,49 49 49 4  A2 B2 CI B2 C1 G2  2 2,  C5 K2 P2 02 A2  1 5 1 5 45 25  N JI F1 N A1 K1 B2 A2 02 K1 C7 T K1 04 A5 K5 T CI D3 A1 02 A2 D3 N A2 E3 U B2 A1 F1  2, 51 51 55 51 2 2 2 2 42 51 1 2 42 2 55 55 2 1 1 1 1 2 2 2 61 2 1 1  P2 A8 C5 E3  I 1 K1 P2 U2  K1 F1 B2 C5 F1 G9 C6 N 04 K1 K2 02 02  2 1 1 2 55  36  209  Order: A r i s t o l o c h i a l e s Family: Aristolochiaceae Aristolochia anquicida A. b a r b a t a A. b r a s i l i e n s i s A. g r a n d i f l o r a A . maxima A. m e d i c i n a l i s A. p i l o s a A r i s t o l o c h i a sp. Order: Ranunculales Family: Menispermaceae Abuta c a u d i c a n s A. q r a n d i f l o r a A . imene A. macrocarpa A. r u f e s c e n s A. s p l e n d i d a A. v a u p e s e n s i s A. v e r r u c u l o s a Andontocarya t r i p e t a l a Anomospermum r e t i c u l a t u m C h o n d r o d e n d r o n tomentosum  N N T 12 N 12 K6 K1 A2 C2 K7 N T  1 1 1 1 1 47 1 2,  02 02 P2 02 03 02 F3 02 02 02 02 C6 02 B2 F3 14 N 02 P2 02 02 F1 03 P2 02 G1 02 01 02 A2  42 2, 1 1 1 14,2 , 5 3 1 1 1 1 ,42 53 53 1 1 53 53 2 8 , 42  Order: Papaverales F a m i l y : Papaveraceae Boccocia frutescens  A2 C3 C6 04  1 1  S u b c l a s s II. Hamamelidae Order: Urticales F a m i l y : Moraceae Brosimium u t i l e Chlorophora t i n c t o r i a Ficus anthelminthica F. a t r o x F. d u l c e r i a F. q l a b r a t a H e l i c o s t y l i s scabra Naucleopsis mello-barretoi P o u l s e n i a armata Pseudolmedia l a e v i q a t a  D2 D3 C6 K1 U3 01 02 C6 C6 A2 03 T 01  1i 2, 1 1 42 42 1 1 1 1 54 37 2 54  *  C. t o x i f e r u m C i s s a m p e l o s andromorpha C. p a r e i r a C o c c u l u s imene Curarea tecunarum Orthomene s c h o m b u r g k i i T e l i t o x i c u m peruvianum  5 5 5 5 5 5 11,  2, 42 1 1 1 1 ,42 42 42, 55 55  15  21 0  Family: Cecropiaceae Cecropia tolimensis C o u s s a p o a cinnamomea C. m a g n i f o l i a C. orthoneura C. t r i n e r v i a Pourouma c u c u r a P. s c h u l t e s i i Subclass III. Caryophyllidae Order: Caryophyllales Family: Phytolaccaceae Pelteveria alliaceae Phytolacca bogotensis P. rivinoides Family: Nyctaginaceae Boerhaavia coccinea Nea p a r v i f l o r a Family: Cactaceae Epiphyllum sp. Opuntia sp. T r i c h o c e r e u s pachanoi Neoraimondia macrostibas F a m i l y : Chenopodiaceae Chenopodium a m b r o s i o d e s F a m i l y : Amaranthaceae • A l t e r n a n t h e r a Lehmanni i Amaranthus h y b r i d u s Gomphrena s p . Iresine celosia Iresine sp. Family: Portulacaceae Calandrinia caulescens Portulaca perennis Talinum paniculatum Family: Basellaceae Andredera d i f fusa Family: Caryophyllaceae Drymaria p a u c i f l o r a Order: Polygonales F a m i l y : Polygonaceae Polygonum p u n c t a t u m Rumex o b t u s i f o l i a S u b c l a s s IV. D i l e n i i d a e Order: D i l l e n i a l e s Family: Dilleniaceae C u r a t e l l a americana D a v i l l a lacunosa Tetracera rotundiflora  P2 03 T J3 J3 B2 A2  2 54 54 54 54 61 54  A2 C6 M 01 A6 C6 M 03  1 1 1 1 ,53 1 1 ,53  C2 U4  2 2  R2 R1 R1 R2  39 39 39 39  R2 CI U1 A2 A8 B2 R2  47 2 42 1 1 1 1  T F3 T  2 2 2  T  2  T  2  C2 C5 04 C3  1 1 48  B2 E4 A2 A8 U2  1 1 2 2  hi  21 1  Order: Theales Family: Caryocaraceae Caryocar glabrum C. g r a c i l e C. tessmanni i Caryocar sp. Family: Marcgraviaceae 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 Family: Quiinaceae Qui ina l e p t o c l a d a Family: Clusiaceae Caraipa p a r r i e l l i p t i c a C l u s i a amazonica C_. 1 i n e o l a t a C. r e n g g e r i o i d e s Clusia sp. Kielmeyera rosea Symphonia g l o b u l i f e r a Vismia angusta V. f e r r u g i n e a V. tomentosa Order: Malvales Family: Tiliaceae Apeiba tibourou Luehea s p . Triumfetta lappula Family: Sterculiaceae Guazuma u l m i f o l i a H e r r o n i a camargoana Sterculia apetala Theobroma s u b i n c a n u m Family: Bombaceae Bombax g l o b o s u m Patinoa icthyotoxica 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 v i rgatum H i b i scus abelmoschus Malachra rudis Malva verticillata Malvastrum peruvianum Pavonia c a n c e l l a t a P. h o o k e r i Sida glomerata S. s e t o s a  03 01 03 03  42 47 2 47  F3 K2  Jl  53 61 61  E3  53  A2 J3 A6 T A2 C3 R2 A4 A1 A2 A2 A2  55 1 1 2 1 1 39 2 55 5 55 3  T A6 A1  1 5 2 2  A2 V F1 K2 R2  1 1 55, 61 1 1 55  A1 03  55 47  Cl  P6 C4 K6 A8 A2 C3 Cl  T T T  2 2 1 1 1 1 2 2 2 1 5 1 1 1 1  21 2  Order: V i o l a l e s Family: Flacourtiaceae Banara q u i a n e n s i s C a r p o t r o c h e amazonica Casearia resinifera C. sylvestris Casearia sp. Lunania p a r v i f l o r a Mayna a m a z o n i c a M. l i n q u i f o l i a M. l o n g i f o l i a M. m u r i c i d a M. t o x i c a Ryania angustifloia R. pyrifera R. s p r u c e a n a Family: Bixaceae Bixa o r e l l a n a B. p u r p u r a B. u r u c u r a n a Family: Violaceae Corynostylis volubilis Hybanthus lanatus Family: Turneraceae Turnera u l m i f o l i a Family: Passifloraceae Passiflora vitifolia Family: Cucurbitaceae A n g u r i a umbrosa Anguria sp. Apodanthera herverae Anisosperma p a s s i f l o r a Cayaponia opthalmica C . racemosa Fevillea cordifolia Gurania r u f i p i l a Luffa operculata Family: Begoniaceae Begonia rossmanniae  01 01 01 A1 A4 01 B2 A2 C2 01 01 01 01 01  A2 D1 U2 04.  02  61 2 2 1 1 2, 3 47 4 3 , 53 53 53 47 55 55 2 2  C1 H1 04 C1 H1 04 C1 H1 04  1 1 1 1 1 1  C6 K3 D1  53 2  D6 F1  13 Q  1 1  C4 K2 P2 R1  1 1  01 03 C3 C1 J1 01 N 01 C3 P5  53 60 1 5 1 5 47 1 1 1 53 1 1  J1  2  Order: S a l i c a l e s Family: Salicaceae S a l i x humboldtiana  C1 C6 P2  11,  Order: Ericales Family: Ericaceae B e f a r i a congesta B. r e s i n o s a Pernettya prostrata  D6 D6 01  53 11, 2  53  21 3  Order: Primulales Family: Theophrastaceae Clavija poeppiqii Family: Myrsinaceae Anthodiscus obovatus A. peruanas Conomorpha c i t r i f o l i a C. l i t h o p h y t a S u b c l a s s V. Rosidae Order: Rosales F a m i l y : Connaraceae Connarus opacus C. s p r u c e i Rourea c u s p i d a t a R. q l a b r a Order: Fabales F a m i l y : Leguminosae Abrus p r e c a t o r i u s Acosmium n i t e n s Alexa imperatracis Anadenanthera p e r e g r i n a Andira araroba A. inermis A. retusa Apurimacia incarum A. m i c h e l l i Bauhinia guianensis B. inermis B. s p l e n d e n s B. tarapotensis Bowdichia v i r g i l i o d e s B. a r i z a Caesalpinia coriaria C. pulcherima Cajanus cajan Campsiandra a n q u s t i f o l i a C. l a u r i f o l i a Cassia a f f i n i s C. a l a t a C. f r u t i c o s a C. h i r s u t a C. macrophylla C. moschata C. o c c i d e n t a l i s C. r u i z i a n a C. t o r a Centrosema p l u m e r i a Chaetocalyx l a t i s i l i g u a Clathropis brachypetala  02  2  02 03 03 P2 V 03  53, 53, 48 47  03 03 01 D4 03  46 47 54 10,  C3 02 03 R1 A2 C6 T 03 03 03 K2 14 B2 B2 E3 C5 03 A8 P2 A2 A2 A2 K1 03 03 C3 C3 T C3 03 A4 03  D6 J l  04  K5 01  G1 P2 C5 03  R2 C3 C3  D2 P2 K2 M P2  60 60  47  1 1 55 42 47 1 5 1 1 2 42 42 15, 42 1 5 1 1 1 1 1 1 ,42 1 1 1 5 42 2 61 3 5 , 61 1 5 1 1 61 42 42 1 1 1 1 ,15 61 1 5 42 2 42  21 4  C l i t o r i a ternatea Copaifera guayanensis C. hymenaefolia Coumarouma o d o r a t a Crotolaria pilosa C r u d i a amazonica Desmodium s p . Dipteryx tetraphylla Entada p o l y p h y l l a E. s c a n d e n s Erythrina corallodendron Heterostemon mimosoides Hoffmanseggia g r a c i l i s Hymenaea c o u r b a r i l Indigofera suffruticosa Inga s p e c t a b i l i s Leguminosa s p . Lonchocarpus l a t i f o l i u s L. n i c o u L. sericeus L. u r u c u Lonchocarpus sp. Lupinus m u t a b i l i s Macrolabium a c a c i a e f o l i u m M. m u l t i j u g u m Mimosa h o s t i l i s M. i n u i s a M. p u d i c a M. v e r r u c o s a Monopteryx a n g u s t i f o l i a M. uaucu Mucuna r o s t r a t a M y r o x y l o n balsamum Ocimum m i c r a n t h u m Ormosia c o c c i n e a 0. lignivalvis 0. macrophylla P i p t a d e n i a macrocarpa P i t h e c e l l o b i u m laetum Prosopis juliflora P s o r a l e a pubescens Pterocarpus rohrii Swartzia auriculata S. brachyrhachis S. cabrerae S. c o n f e r t a S. g i g a n t e a S. longistipitata S. m i c r o c a r p a S. p e n d u l a S. racemosa •  C3 D6 K1 A2 A1 C2 I 1 E2 D1 N 02 V D4 C6 14 B2 A6 C3 03 C3 03 03 04 A1 01 P1 C2 Q K5 C6 C6 A7 A1 R2 01 A1 02 D3 R1 D3 F3 P2 03 C6 C6 C5 01 G5 C5 03 C5  F1 K5 03 D3 V  F3 K6 C5 01 03 02 04  01 Q  R2  1 1 1 5 1 5 1 1 1 1 61 1 5 2 53 1 5 42 55 2 1 1 1 1 1 1 2 1 1 1 1 1 1 2 60 42 61 61 1 5 1 5 1 1 50 53 53 42 1 1 47 47 47 47 1 1 35 1 1 2 61 48 48 48 48 48 61 48 48 48  21 5  S. recurva S. s c h o m b u r g k i i S. schultesii S. sericea S. s i m p l e x Tachiqalia cavipes T . myrmecophila T. paniculata T. ptychophysea Tephrosia sinapou Tephrosia sp. Zornia leptophylla Order: Myrtales Family: Lythraceae Cuphea racemosa Family: Thymelaeaceae Schoenobiblus peruvianus Styrax tessmannii S. y a p o b o d e n s i s F a m i l y : Onagraceae Epilobium denticulatum Family: Melastomataceae Arthrostemma g r a n d i f l o r u m A. v o l u b i l e Graffenrieda rupestris F a m i l y : Combretaceae Combretum a l t e r n i f o l i u m C. c a c o u c i a T e r m i n a l i a catappa Order: Santalales Family: Olacaceae Heisteria pallida H e i s t e r i a sp. Family: Loranthaceae Gaiadendron punctatum Oryctanthus botryostachys Phoradendron p i p e r o i d e s P h r y g i l a n t h u s eugeniodes Psittacanthus collum-cygni Family: Balanophoraceae Corynaea c r a s s a Order: Celastrales Family: Celastraceae Maytenus l a e v i s M. p s e u d o c a s e a r i a Family: Icacinaceae C a l a t o l a columbiana H u m i r i a n t h e r a ampla Family: Dichapetalaceae Stephanopodium peruvianum  Q C6 03 03 T A2 A2 C2 I1 A8 03 04  C2 G1 04 K1 K1 03 04  61 48 48 48 48 54 61 54 61 1 1 60 61  F1  2,  60  A2 02 03 A2 01 A5  52, 49, 54  T  2  F1 J3 P2 A2  2 1 1 61  JI 01 C1 C5 D2  1 5 2,53,55 1 1  B2 R2 BI  35 2  D1 A8 E4 G5 R2 E3  2 1 1 1 1 39 2  G8  22  B2 C5  1 1 2  A9 U4 01  32 42  P2  35  54 54  21 6  Order: Euphorbiales Family: Euphorbiaceae Alchornea c a s t a n e i f o l i a A. cordatum Caryodendron orinocense C n i d o s c o l u s urens Codiaeum v a r i e q a t u m Croton cajucara C. f e r r u g i n e u s C. q l a b e l l u s C. g o s s i p i f o l i u s C. l e c h l e f i C. maqdalensis C. palanostiqma C. p o l y c a r p u s C. r h a m n i f o l i u s C. scaber C. t r i n i t a t i s Croton sp. Euphorbia c o t i n i f o l i a E. c o t i n o i d e s E. p e p l u s E. p i l u l i f e r a E. t h y m i f o l i a Euphorbia sp. Guatteria maqalophylla G. v e n e f i c i o r u m Hippomane m a n c i n e l l a Hura c r e p i t a n s Jatropha angusti J. ciliata J . curcas J. gossypifolia J . urens Mabea n i t i d a Manihot e s c u l e n t a Micrandra spruceana Nealchornia yapurensis Pedilanthus tithymaloides P h y l l a n t h u s acuminatus P. brasiliensis P. c l a d o t r i c h u s P. conami P. niruri P. p i s c a t o r u m P. p s e u d o - c o n a m i P. rosellus P. salviaefolius Sebastiana pachyphylla  B2 T A4 C3 01 P2 T A2 A1 A1 T A2 T T T C2 04 02 A6 01 D2 P4 A1 02 02 01 A6 I 1 11 C3 02 A6 A8 A4 A2 E3 03 C2 14 03 03 03 03 F3 03 03 F3 F1 C3  A2 M  D3 D4 P2 02  02 03 04 03 M 01 03 C3 M F1 12 J3 C5 D6 01  P2 04 P3  22 2 1 1 2 2 2 2 55 2 2, 22 2 2, 55 2 2 2 3 1 5 42 2, 42 2 1 5 1 5 3, 58 42 42 42 2,1 1 ,42 2 15, 42 2,15,42 1 5 2, 1 5 55 1 1 54 53 1 11 , 5,42 2 2 42 42 2 2, 60 2 2 1 5 2  *  21 7  Order: Linales Family: Erythroxylaceae Erythroxylum coca E. novagranatensis Family: Humiriaceae Humiria balsamifera H. c r a s s i f o l i a Humiriastrum piraparanense H. v i l l o s u m Sacoglottis ceratocarpa Schiekia orinocensis Schistostemon macrophyllum Vantanea p a r v i f l o r a Order: Polygalales Family: Malpighiaceae Banisteria leiocarpa B a n i s t e r i o p s i s caapi B. inebriens B. m a r t i n i a n a B. r u s b y a n a Byrsonima c r a s s i f o l i a B. l a n c i f o l i a H e t e r o p s i s macrostachya H. r i p a r i a Hirea apaporiensis H. s c h u l t e s i i Mascagnia g l a n d u l i f e r a Mezia i n c l u d e n s T e t r a p t e r i s methystica T. mucronata T. s i l v a t i c a T. s t y l o p t e r a Family: Vochysiaceae Qualea acuminata Vochysia columbiensis V. f e r r u q i n e a V. l a x i f l o r a V. l o m a t o p h y l l a Order: Sapindales Family: Sapindaceae Cardiospermum q r a n d i f o l i u m Paullinia alata P. e m e t i c a P. y o c o Serjania sp. Toulicia bullata  Cl  K1 K3 C1 K1 K3  35 35  A1 A2 A2 C2 K1 C3 D3 K7 D3 D5 G2 T  49 49 49 49 49 54 49 49  T R1 R1 R1 R1 C5 03 P2 B2 A2 C5 14 01 A2 C2 C3 F1 R1 02 R2 A2 A2 P2  2 2, 47 2, 47 1 1 2 1 1 ,42 2 52 4 7 , 52 52 52 52 52 47 2, 52 52 52  C6 02 A2 J3 02 R2 A2 D3 F3 G1 G4  5 5 , 61 53 5 3 , 60 53 2, 53  03 03 C2 K3 Q P2 J3 P2  42 2 53 2, 1 1 61 2  Jl Jl  218  Family: Burseraceae Bursera gumifera Hedwigia b a l s a m i f e r a Protium heptaphyllum P. n e q l e c t u m Protium sp. Family: Anacardiaceae Anacardium o c c i d e n t a l e L o x o p t e r y g i u m huasango Mauria aurantiodora -M. h e t e r o p h y l l a Rhus s p . S p o n d i a s mombin Family: Simaroubaceae Quassia cedron Simarouba cedron S. versicolor Family: Meliaceae Carapa g u i a n e n s i s T r i c h i l i a oblonga F a m i l y : Rutaceae Angostura t r i f o l i a t a Hortia sp. Rauia r e s i n o s a Ruta q r a v e o l e n s Zanthoxylum s p . Order: Geraniales Family: Oxalidaceae Oxalis lotoides Subclass VI. Asteridae Order: Gentianales Family: Loganiaceae Antonia ovata Buddleia americana P o t a l i a amara S p i g e l i a humboldtiana Strychnos guianensis S. p e c k i i S. s o l i m o e s a n a S. t o x i f e r a Family: Gentianaceae Chelonanthus a l a t u s Chelonanthus chelonoides G e n t i a n a chamuchni Halenia weddelliana  C3 D6 14 K1  1  5  1  5  P1  A1  2 2 2  K1  T 02 A4 A6 C3 13 0 1 Q A7 A7 A7 A7 A2 G5 14  1 1  42 42 2 42  11,25  42 2 42  U1  P2  U1  B2 D3  -  1  5  1  5  2  P2 Q P2 P2  2 2  02  42  D4  55  03 T C3 14 C6 02 02 02 02  2, 42 2 54  1 1  K1  04 04 T 14  J1  N  1 1  42 2 42 42 3 2 2 2  21 9  F a m i l y : Apocynaceae Allamandra ambetti A. cuneata A. l o p e z i i A. markqrafiana Aspidosperma d i s c o l o r A. megalocarpa A. n i t i d u m A. s c h u l t e s i i Aspidosperma sp. Couma m a c r o c a r p a Hancornia speciosa Himatanthus bracteatus H. p h a g e d o e n i c u s H. s u c u u b a Himatanthus s p . Lacmella sp. Macoubea g u i a n e n s i s Macrosiphonia l o n g i f l o r a M. velame Malouetia duckei M. n i t i d a M. t a m a q u a r i n a M a n d e v i l l a anceps M. a n n u l a r i i f o l i a M. c u n e i f o l i a M. n e r i o d e s M. s c a b r a M. s t e y e r m a r k i i M. s t e p h a n o t i d i f o l i a M. s u b c a r n o s a M. t h e v e t i o i d e s M. t r i a n a e M. v a n h e u r e k i i Mandevilla sp. Mesechites t r i f i d a Odontadenia cognata 0. funigera 0. neglecta 0. s y l v e s t r i s Parahancornia krukovii Plumeria tarapotensis  C2 03 A2 P2 P2 A7 A6 A2 P2 A9 A5 N N D3 03 K5 D3 14 03 A2 01 A5 A5 01 A2 A9 A2 N 01 03 A2 A2 03 K2 04 V. D5 K1 A1 B2  C3 02  P2 04 K5 P2 P2 01  01 R2  C5 I 1  01  01 04  15 57 57 57 2 57 2, 57 5 7 , 61 2 3, 57 57 5 7 , 60 57 2, 57 58 57 2, 57 2 2 2 11, 53 47 57 57 57 57 57 57 57 57 57 57 57 47 57 57 57 57 57 57 2  220  Tabernaemontana amygdalaefolia T. grandiflora T. heterophylla T. muricata T. rimulosa T . sananho  .  T. stenoloba T. tetrastachya T. undulata Tabernaemontana s p . Thevetia peruviana Family: Asclepiadaceae Asclepias curassavica Sarcostemma S. c l a u c u m  andinum  Order: Solanales Family: Solanaceae Brunfelsia chiricaspi B. guianensis B. g r a n d i f l o r a B. B. B. B. B.  latifolia maritima mire tastevinii uniflora  Brunfelsia sp. C a p s i c u m pendulum Cestrum laeviqatum C. l o r e t e n s e C. ochraceum C. reflexum Cyphomandra c r a s s i f o l i a C. d o l i c h o r a c h i s C . endopoqon Datura arborea D. s t r a m o n i u m D. s u a v e o l e n s Iochroma f u c h s i o d e s J a l t o m e t a procumbens  A5 A2 Q K3 K2 C2 01 01 B2 C6 03 01  01 M B2  F1 K2 P2 Q K3  1 1 1 1 57 57 57 57 61 57 57 2 42  C2 C3 C6 E3 14 01 N JI  2,11,15  K5 B2 B2 01 B2 B2 C6 R1 A8 G1 01 N, 02 03 01 B2 01 C6 C6 T A8 02 K5 R2 F1  35 21 35  14 01 D3 K1 K5 P2 01 B2 C3 F1 14 K1 K5 PI B2  P1  M  P2  2 1 1  2 2 3 5 , 41 41 23,29,66 1,6,7,8,33 42 42 61 61 61 54 54 54 1 5 42 47 50 61  221  Juanulloa 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 asperolanatum S . a s p e r r imum S. l y c o c a r p u m S. phyllanthum S . procumbens S. s p e c t a b i l e S. s w a r t z i a n u m Solanum a l b i d u m S . apaporum S . campani forme S. c r i n i t i p e s S. j a m a i c e n s e S. l e p i d o t u m S mammosum S n 1 g r urn S. s c a b r i d u m S. s u b i n e r m e S. t o p i r o S. v e r b a s c i f o l i u m Family: Convolvulaceae Ipomea c r a s s i f o l i a Ipomea s p . Merremia a l a t a Family: Hydrophyllaceae Wigandia caracasana Order: Lamiales Family: Boraginaceae Cordia a l l i d o r a C. dentata 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 Tournefortia brevilobata F a m i l y : Verbenaceae C a l l i c a r p a odorata Lantana a f f i n i s L. camara L. f u c a t a Stachtarpheta cayennensis S. s t r a m i n e a Verbena l i t o r a l i s Verbenacea s p .  Al C6 R2 T F3 01 D1 A1 F1 01 K1 A2 04 04 01 04 B2 04 A2 J2 T J3 A7  R2 Jl  P2 Q  C4 01 M  54 54 47 2 2 2 2 2 55 2 2 54 54 54 61 54 61 61 1 1 61 54 54 54  Q C3 C3  2 2 2  K3  2  A2 B4 D3 Q T T K6 D3 Q  1 1 1 1 2 2 2 2 2 2  B2 C5 C1 P1 D1 A2 A2 C3 C1  2 2 1 1  C4 D6 P2 C5 C5  2 2 2 2 2  222  Family: Lamiaceae Hyptis brachiata H. c a p i t a t a H. c a r p i n i f o l i a H. m u t a b i l i s H. s i n u a t a L e p e c h i n i a meyeni Ocimum m i c r a n t h u m Rosmarinus o f f i c i n a l i s S a l v i a haenkei S. m a c r o p h y l l a S. p a l a e f o l i a S. p i c h i n c h e n s i s S a t u r e i a tomentosa Order: Scrophlariales Family: Scrophulariaceae Alonsoa c u a d r i a l a t a A n t i r r h i n u m majus C a l c e o l a r i a inamoena C a s t i l l e j a communis Family: Gesneriaceae B e s l e r i a drymophila Besleria sp. Columnea s p . Nauticalyx sp. Family: Acanthaceae Acantha v i r i d i s Aphelandra aurantiaca A. p i l o s a F i t t o n i a argyroneura F. verschaffeltii J u s t i c i a blackei J. cabrerae J. chlorastachya J . comata J. ideogenes J. pectoralis J. schultesii Mendoncia aspera Ruellia colorata R. h u m b o l d t i a n a Sanchezia t h i n o p h i l a Teliostachya lanceolata Trichanthera gigantea Family: Pedaliaceae Proboscidea peruviana  A2 A2 B2 P2 D2 C1 R2 B2 T F3 04 K2 D1  E3 A4 B4 CI D3  1 1 1 1 2 1 1 1 1 2 50 2 2 2 2 2 2  D2 D3 B3 T  1 5 1 5 2 2  N N T N  1 1 2 2 47  T J2 D4 K1 D4 D7 A2 A2 04 K7 A2 A2 03 C2 C3 A9 R2 C6  1 5 • 56 56 56 56 56 56 56 56 56 2 5 , 56 56 47 56 1 1 56 56 1 1  T  F3 P2  R2 C6  E4 F1  2  223  Family: Bignoniaceae Arrabidaea chica A. x a n t h o p h y l l a Bignonia opthalmica Cremastus sceptrum D i s t i c t e l l a racemosa Jacaranda glabra Macfadyena u n g u i s - c a t i M a r t i n e l l a obovata M u s s a t 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. serratifolia Tabebuia sp. Tanaec ium n o c t u r n u m  A2 J1 JI 14 01 A2 14 02 V C2 D3 01 A2 M A7 R1  A9  02 14 N P2 P2 B2 01  1 1 47 1 5 2 48 1 1 1 1 47, 48 43 1 1 2, 55 2 1 1 43 42 1 1  O r d e r : Campanulales 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 Lobelia decurrens Siphocampylus corymbi f e r u s  I 1 R2 01 01  2  Order: Rubi'ales F a m i l y : Rubiaceae C a l y c o p h y l l u m spruceanum Cephaelis barcellana Chiococca brachiata C . racemosa Coussarea p i l o s i f l o r a Diodia hyssopi f o l i a Duroia h i r s u t a D. k o t c h u b a e o i d e s D. p e t i o l a r i s D. s a c c i f e r a D. s p r u c e i Exostemma p e r u v i a n a Genipa americana Isert ia alba haenkeana hypoleuca rosea Ladenbergia magnifolia Manettia divaricata Pagamea c o r i a c e a P. m a c r o p h y l l a Palicourea elongata P. q a r d n e r i a n a P. r i q i d a Palicourea sp.  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  53 61 42 1 5 2 2 2, 47 46, 47 47 47 46 2 25 1 1 1 1 53 54 1 1 2 54 54 2 2 2 2  50  2, 2  42  224  Psychotria barbiflora P. capitata P. carthagenensis P. involucrata P. n u d i c e p s P. pinularis P. psychotriaefolia P. rufescens Randia formosa Remijia pedunculata Retiniphyllum concolor R. p i l o s a R. s c h o m b u r g k i i R. s p e c i o s u m R. truncatum Retiniphyllum sp. Rubiacea s p . Rudgea s u b s e s s i l i s R. v i b u r n o i d e s Sabicea amazonensis Order: Asterales Family: Asteraceae Acanthospermum a u s t r a l e Ageratum c o n y z o i d e s Ambrosia e l a t i o r Andromachya i g n i a r i a Baccharis g e n i s t e l l o i d e s B. salicifolia B. t r i n e r v i s Bidens a n d i c o l a Clibadium sylvestre Clibadium sp. Eupatorium ayapanoides E. o d o r a t u m E. scabrum E. sternbergianum M i k a n i a guaco Neurolaena lobata S p i l a n t h e s americana  01 01 01 R 01 01 N R M P2 M T D5 C6 C6 C6 A8 S 01 01 Q R  2 2 46 46 46 2 47 1 1 1 1 1 1 54 54 54 54 54 47 2 2 2 47  M C5 P2 P2 E3 E3 G6 A8 D3 14 T B2 A2 03  1 1 1 1 1 5 1 5 1 5 1 5 1 5 1 5 1 1 60 42 1 1 1 1 1 5 42 1 1 1 1  Ul  A8 N P2 Q 14 M CI U1 M N J3  225  Class: Liliopsida S u b c l a s s I: Alismatidae Order: Alismatales Family: Alismataceae Echinodorus g r a n d i f l o r u s Echinodorus sp. S u b c l a s s II: Arecidae Order: Arecales Family: Arecaceae Chamaedorea f r a g r a n s Elaeis quineensis Geonoma s p . Jessenia polycarpa K u e t h i a montana  Q C8 ,  2 2  A7 C6 K1 D3 N  42 1 1 1 5 1 1 1 5  Order: Cyclanthales Family: Cyclanthaceae C a r d u l o v i c i a palmata  D3  61  Order: Arales F a m i l y : Araceae Anthurium c r a s s i n e r v i u m A . eminens A. jenmanii A. scopendrinum A. t e s s m a n n i i A. tikunorum Aracea sp. Caladium b i c o l o r Dieffenbachia obliqua D. p i c t a c D. s e q u i n e D r a c o n t i u m asperum D. l o n g i p e s D. t r i a n a e Heteropsis sp. P h i l o d e n d r o n craspedodromum P. d y s c a r p i u m P. haematinium P. remifolium Urospatha a n t i s y l l e p t i c a U. somndenta Xanthosoma c o n s p u r c a t u m  J2 T B3 D4 G1 J3 J3 U2 01 01 02 U1 N C5 N T 03 G1 S V G1 A1 A2 01  54 61 61 61 46 47 2 61 61 42 2,42 42 61 61 2 47 47 47 47 47 1 5,47 61  Subclass III: Commelinidae O r d e r : Commelinales F a m i l y : Commelinaceae Tradescantia multiflora  226  Order: Cyperales Family: Cyperaceae Cyperus esculentus Cyperus sp. F a m i l y : Poaceae Axonopus micay A. s c o p a r i u s C a l a m a q r o s t i s cuminens Chloris distichophylla Hierochloe redolens S u b c l a s s IV: Z i n g i b e r i d a e Order: Bromeliales Family: Bromeliaceae Ananas anannasoides Order: Zingiberales F a m i l y : Musaceae Heliconia acuminata Musa balbisiana Heliconia brasiliensis H. cannoidea Family: Zingiberaceae Hedychium coronarium Family: Costaceae Costus amazonicus C. cylindricus C. erythrocoryne C. villosissimus Family: Cannaceae Canna sp. Family: Marantaceae Calathea veitcheana Calathea sp.  F3 R2  15 39  F1 F3 F3 F1 R  1 1 15 2 15 2, 53  C1  C6  F1  1 1  B3 D5 B3 B3  K7  N  K7 K7  N  1 1 1 1  K1  1  1 1 1  54  T  E3 K1  54 1 1 54. 1 1  A2  2  R2  50 2  Cl  P3  3  227  S u b c l a s s V. L i l i i d a e Order: Liliales Family: Pontederiaceae Pontederia cordata Family: Liliaceae E u c h a r i s amazonica Family: Iridaceae Eleutherine piicata S i s y r i n c h i u m alatum F a m i l y : Agavaceae Agave a m e r i c a n a Family: Smilacaceae Smilax sp. Family: Dioscoreaceae Dioscorea pozucoensis D. t r i f i d a  scheme of  is  Caesalpiniaceae u s e d by the  C2  54  G8  2 1 1  (1981)  2  T  42  02 J1 J3 A1 C1 T E1 K2  54 53 54 54 54 1 1  the  classification  w i t h one e x c e p t i o n . The  and F a b a c e a e . cited.  61  2  12  used in p r e f e r e n c e  author(s)  50,  42  03  have been a r r a n g e d a c c o r d i n g t o  Cronquist  Leguminosae  2,  C3 D1  Order: Orchidales Family: Orchidaceae Dichaea muricata E r i o p s i s sceptrum One i d i u m p u s i l l u m Phragmipedium e c u a d o r e n s i s Psymorchis p u s i l l a V a n i l l a odorata  Species  K7 R2  to  the  The s p e c i e s  family  Mimosaceae, name l i s t e d  is  that  228  List LETTER  of  SYSTEM  Ethnobotanical No.  Codes Used  Biological  Action  Used  or  For  Condition  ******************************************************* A  skin  2 3 4 5 6 7 8 9  wounds, c u t s absesses, b o i l s u l c e r s , infections burns d e r m a t i t i s , eczema, p s o r i a s i s , e t c . warts leprosy c a u s e s d e r m a t i t i s or a l l e r g y swelling, inflammation s k i n c o l o r a n t or d e p i l a t o r y  B  skeletomuscular  1 2 3 4  b r o k e n bones rheumatism, arthritis muscle s t i f f n e s s bruises  C  digest ive  1 2 3 4 5 6 7 8  carminative, digestive emet i c cathartic, purgative, laxative ant i spasmodic ant i d i a r r h e a l amebicide, antihelminthic t o o t h decay colic  D  respi ratory  1 2 3 4 5 6 7  colds, influenza, asthma b r o n c h i t i s , chest sore throat tuberculosis expectorant sinusitis  1  .  E  circulatory  1 2 3 4  anemia heart ailments hemostat hypotensive  F  excretory  1 2 3  diuretic calcifications kidney a i l m e n t s  coughs, infections  229  G  reproduct ive, female  1 2 3 4 5 6 7 8 9  contraceptive f e r t i l i t y promoter ant i - a b o r t ive abort i fac ient menstrual pain emmenogogue galactogogue u t e r i n e hemmorage aphrodisiac  H  reproduct ive, male  1  aphrodisiac  I  reproduct ive, both sexes  1 2 3  aphrodisiac venereal disease anaphrodi siac  J  sensory  1 2 3  eye a i l m e n t s ear problems gum and mouth  1  analgesic, anesthetic, toothache, headache sedative st imulant h a l l u c inogen narcot ic convulsions, f i t s , epilepsy palsy, paralysis  K  nervous  2 3 4 5 6 7 L  antibiotic  M  ant i t u m o u r , ant i c a n c e r  N  a n t i d o t e s f o r b i t e s and s t i n g s ( p r i m a r i l y :s n a k e s )  0  poison  P  systematic  1  of  diseases  animals  2 3 4  general arrow p o i s o n f i s h poison r e p e l l e n t or t o x i c  1 2 3 4 5 6  sudorific, diaphoretic febrifuge, malaria diabetes swollen glands cholera shock  to  arthropods  230  Q  tonic  R  admixture  S  magic  T  undefined  U  miscellaneous  V  flavouring, aromat i c  to  The c a t e g o r i e s for  which  the  established it  is  available.  the  illnesses  1 2 3 4  herbicide v e t e r i n a r y use tooth extraction tooth c o l o r a t i o n  describing medicinal are  u s e d as  t o accomodate  systematically that  plants  hallucinogen  the  Although i t  as p o s s i b l e ,  Morton(1977).  treatments  information  have  in the  or  conditions  been form i n  which  has been o r g a n i z e d as this  i n f o r m a n t s c o n c e p t s of d i f f e r e d widely.  activity  approach is  limited  by t h e  d i a g n o s i s and t r e a t m e n t  The scheme  is  b a s e d on t h a t  of of  fact  231  BIBLIOGRAPHY Appendix A de A l m e i d a C o s t a , 0 . 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( 1 9 8 0 ) De P l a n t i s t o x i c a r i i s e Mundo Novo t r o p i c a l e Commentationes XXVI. E t h n o p h a r m a c o l o g i c a l notes on t h e f l o r a of N o r t h w e s t e r n S o u t h A m e r i c a . B o t . Mus. L e a f l . H a r v . U n i v . 2 8 , 1-26.  62.  S o u k u p , J . ( 1 9 7 0 ) V o c a b u l a r i o de l o s Nombres V u l q a r e s F l o r a Peruana . C o l e g i o S a l e s i a n o , Lima.  63.  Spix, J.B. Munchen.  64.  T o v a r , 0 . (1952) R e v i s i o n de l a s e s p e c i a s G e n e r o C h u q u i r a g a . P u b l . Museo H i s t . N a t . S e r i e B, B o t a n i c a , N o . 5, L i m a .  65.  Webb, L . J . ( 1 9 4 8 ) G u i d e t o M e d i c i n a l and P o i s o n o u s P l a n t s of Q u e e n s l a n d . 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 Research, Melbourne.  66.  Wren, R . C . ( 1 9 5 6 ) 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 D r u g s and P r e p a r a t i o n s , P i t m a n and S o n s , L o n d o n .  and von M a r t i u s ,  C.F.P.(1831)  Reise  in  de  la  Brasien  Peruanas d e l J a v i e r Prado,  236  APPENDIX  1 - S P 4 1 0 0 COMPUTER  PROGRAM(BASIC)  The p r o g r a m t a k e s i n c o m i n g signals, after amplification 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 b e t w e e n two s u c c e s s i v e s i g n a l s , a n d p r i n t s t h e average v a l u e every 5 minutes over a 2 hour p e r i o d . A=1: I F TB<3 THEN A=100 ALWAYS I F TB=0 THEN POKE #C394, T B POKE $ # 0 1 C 1 0 7 , 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 I F 2 P E E K # C 2 3 4 / 5 0 0 > T THEN GO TO 90 E L S E 65 14:28:18;M/C I F T=24 THEN GO TO 140 E L S E 120 T=T+1 GO TO 50 ACQUIRE 2 END  A=1000  Publications  MacRae, W. D., W h i t i n g , R.F. and S t i c h , H.F. S i s t e r chromatid exchanges induced i n c u l t u r e d mammalian c e l l s by chromate. Chem.-Biol. I n t e r a c t . 26: 281-286(1979). MacRae, W.D., MacKinnon, E.A. and S t i c h , H.F. The f a t e o f U.V.-induced l e s i o n s a f f e c t i n g SCEs, chromosome a b e r r a t i o n s and s u r v i v a l of CHO c e l l s a r r e s t e d by d e p r i v a t i o n o f a r g i n i n e . 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 o f a r g i n i n e d e p r i v a t i o n upon chromosome a b e r r a t i o n s , SCEs and s u r v i v a l of CHO c e l l s t r e a t e d w i t h mutagenic a g e n t s . Mutat. Res. 62: 495-504(1979). MacRae, W.D., MacKinnon, E.A. and S t i c h , H.F. I n d u c t i o n o f s i s t e r chromatid exchanges and chromosome a b e r r a t i o n s i n CHO c e l l s a r r e s t e d i n the c e l l c y c l e by a r g i n i n e d e p r i v a t i o n . I n V i t r o 15: 555-564(1979). MacRae, W.D. and S t i c h , H.F. I n d u c t i o n o f s i s t e r chromatid exchanges i n Chinese hamster ovary c e l l s by t h e r e d u c i n g agents b i s u l f i t e and a s c o r b i c a c i d . T o x i c o l o g y 13: 167-174(1979). MacRae, W.D. and S t i c h , H.F. I n d u c t i o n o f s i s t e r chromatid exchanges i n Chinese hamster o v a r y c e l l s by t h i o l and h y d r a z i n e compounds. Mutat. Res. 68: 351-365(1979) . Yamamoto, E., Wat, C.-K., MacRae, W.D., Towers, G.H.N, and Chan, G.F.Q. P h o t o i n a c t i v a t i o n o f human e r y t h r o c y t e enzymes by <=<-terthienyl and p h e n y l h e p t a t r i y n e , n a t u r a l l y o c c u r r i n g compounds i n the A s t e r a c e a e . FEBS L e t t e r s 107: 134-136(1979). Wat, C.-K., MacRae, W.D., Yamamoto, E., Towers, G.H.N, and Lam, J . P h o t o t o x i c e f f e c t s o f n a t u r a l l y o c c u r r i n g p o l y a c e t y l e n e s and < * - t e r t h i e n y l on human e r y t h r o c y t e s . Photochem. P h o t o b i o l . 32: 167-172(1980). MacRae, W.D., Chan. G.F.Q., Wat, C.-K., Towers, G.H.N, and Lam, J . E x a m i n a t i o n of n a t u r a l l y o c c u r r i n g p o l y a c e t y l e n e s and c < - t e r t h i e n y l f o r t h e i r a b i l i t y t o induce c y t o g e n e t i c damage. E x p e r i e n t i a 36: 1096-1097(1980). MacRae, W.D., I r w i n , D.A.J., B i s a l p u t r a , T. and Towers, G.H.N. Membrane l e s i o n s i n human e r y t h r o c y t e s induced by the n a t u r a l l y o c c u r r i n g compounds <*> - t e r t h i e n y l and p h e n y l h e p t a t r i y n e . Photobiochem. P h o t o b i o p h y s . 1: 309-318 (1980) . MacRae, W.D. and Towers, G.H.N. L e t t e r t o the e d i t o r . J . Ethnopharm. 348(1983).  7: 343-  Yamamoto, E., Wat, C.-K., MacRae, W.D., G a r c i a , F . J . and Towers, G.H.N. Photodynamic h e m o l y s i s caused by ° N - t e r t h i e n y l . P l a n t a Med.(in p r e s s , Nov., 1983).  MacRae, W.D. and Towers, G.H.N. Review: The b i o l o g i c a l a c t i v i t i e s of lignans. Phytochem.(In press, Nov., 1983). MacRae, W.D. and Towers, G.H.N. J u s t i c i a p e c t o r a l i s : A study of the basis f o r i t s use as V i r o l a snuff admixture(in preparation). MacRae, W.D. and Towers, G.H.N. Non-alkalidal constituents of V i r o l a Phytochem.(to be submitted).  elongata.  MacRae, W.D., McKenna, D.J. and Towers, G.H.N. An ethnopharacological examination of V i r o l a 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, ( i n preparation). MacRae, W.D., Hudson, J.B. and Towers, G.H.N. c< - P e l t a t i n : the a n t i v i r a l constituent of Amanoa sp. ( i n preparation). MacRae, W.D., Hudson, J.B. and Towers, G.H.N. A n t i v i r a l a c t i v i t i e s of lignans. (in preparation).  

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