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Studies of simple phenols in plants : 1. Phenolic acids in ferns. 2. The uptake of phenolic compounds… Glass, Anthony David Melville 1970

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STUDIES OF SIMPLE PHENOLS IN PLANTS 1. PHENOLIC ACIDS IN FERNS 2 . THE UPTAKE OF PHENOLIC COMPOUNDS BY PITYROGRAMMA CALOMELANOS AND HORDEUM VULGARE 3.  THE PHENOL GLUCOSYLATION REACTION IN HIGHER PLANTS by ANTHONY DAVID MELVILLE GLASS  B.Sc,  U n i v e r s i t y o f Wales, Swansea, 1959  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n t h e Department of Botany We a c c e p t t h i s t h e s i s a s c o n f o r m i n g t o t h e required standard  THE UNIVERSITY OF BRITISH COLUMBIA AUGUST, 1970  In  presenting  this  an a d v a n c e d d e g r e e the I  Library  further  for  agree  in  at  University  the  make  it  partial  freely  that permission for  this  representatives. thesis  for  It  financial  gain  of  The U n i v e r s i t y o f B r i t i s h V a n c o u v e r 8, C a n a d a  of  of  Columbia,  British for  extensive by  the  Columbia  shall  not  the  requirements  reference copying of  Head o f  is understood that  written permission.  Department  fulfilment  available  s c h o l a r l y p u r p o s e s may be g r a n t e d  by h i s of  shall  thesis  I  agree  and this  be a l l o w e d  that  study. thesis  my D e p a r t m e n t  copying or  for  or  publication  w i t h o u t my  - i Abstract I . Systematic  s t u d i e s of the d i s t r i b u t i o n of p h e n o l i c  a c i d s have been l a r g e l y r e s t r i c t e d t o angiosperm f a m i l i e s . T h i s s u r v e y of f o r t y - s i x s p e c i e s of f e r n s extended the work begun by Bohm and Tryon,  and  brought the t o t a l number of s p e c i e s examined t o ninety-two.  p-Coumaric a c i d , c a f f e i c a c i d ,  a c i d , p-hydroxybenzoic a c i d , p r o t o c a t e c h u i c  ferulic acid,  and v a n i l l i c a c i d were found t o be w i d e s p r e a d . A time study of the c o n c e n t r a t i o n s of these compounds i n P t e r l d l u m a q u i l l n u m and A t h y r l u m f e l l x femina suggests t h a t c e r t a i n of these a c i d s undergo c o n s i d e r a b l e t u r n o v e r d u r i n g the g r o w i n g season. I I . K i n e t i c s t u d i e s of the uptake of hydroquinone g l u c o s i d e by r o o t s of Hordeum v u l g a r e and by gametophytes of P i tyrogramma calomelanos showed t h a t t h i s compound i s a c t i v e l y t r a n s p o r t e d , and suggested t h a t c a t e c h o l g l u c o s i d e i s absorbed by a s i m i l a r mechanism. Hydroquinone e x h i b i t e d a d i f f e r e n t p a t t e r n of uptake w h i c h p o s s e s s e d some of the f e a t u r e s of an a c t i v e system. The  l a t t e r compound  i s a b s o r b e d by p a s s i v e d i f f u s i o n . A  concentration  gradient favouring d i f f u s i o n i s maintained  by  c o n v e r s i o n of t h i s phenol t o i t s c o r r e s p o n d i n g glucoside.  the  -iiI I I . A survey of twenty-nine  s p e c i e s of f e r n s showed  t h a t these p l a n t s , l i k e f l o w e r i n g p l a n t s , a r e capable of g l u c o s y l a t i n g a d m i n i s t e r e d  phenols.  C e r t a i n a c i d i c compounds, i d e n t i f i e d by J.B. Pridham a s p h e n o l i c g l u c o s i d e - 6 - s u l p h a t e s , a r e formed when s i m p l e phenols catechol, resorcinol,  such as hydroquinone,  etc., are administered to  g e r m i n a t i n g seeds of V l c l a f a b a . The p r e s e n t s t u d i e s i n d i c a t e t h a t these compounds a r e commonly formed when s i m p l e phenols a r e a d m i n i s t e r e d t o h i g h e r p l a n t s . S i n c e Pridham s i d e n t i f i c a t i o n was based upon t h e i d e n t i f i c a t i o n o f h y d r o l y s i s p r o d u c t s , w i t h o u t r e f e r e n c e t o a u t h e n t i c compounds, and s i n c e s e v e r a l of t h e p r o p e r t i e s o f the supposed s u l p h a t e s were i n c o n s i s t e n t w i t h t h e known p r o p e r t i e s of sugar s u l p h a t e s , a r b u t i n - 6 - s u l p h a t e was c h e m i c a l l y s y n t h e s i z e d . Comparisons of t h e p r o p e r t i e s of t h i s a u t h e n t i c compound w i t h the n a t u r a l product  showed  t h a t t h e two compounds were q u i t e d i s s i m i l a r and t h e r e f o r e suggest was i n c o r r e c t .  t h a t Pridham s i d e n t i f i c a t i o n  -iiiTABLE OF CONTENTS Page ABSTRACT  i  TABLE OF CONTENTS  i i i  COMMON AND SYSTEMATIC NAMES OF COMPOUNDS LIST OF TABLES  ... v i v i i  LIST OF FIGURES  ix  ACKNOWLEDGEMENT  xi  I.  PHENOLIC ACIDS I N FERNS  INTRODUCTION  1  LITERATURE REVIEW  2  MATERIALS AND METHODS Plant material  6  Preparation of plant extracts  6  RESULTS AND DISCUSSION The s u r v e y  •  The q u a n t i t a t i v e s t u d y LITERATURE CITED  9 13 2?  -ivII.  UPTAKE OF PHENOLIC COMPOUNDS BY PITYROGRAMMA AND HORDEUM Page  INTRODUCTION  29  LITERATURE REVIEW  31  MATERIALS AND METHODS Plant material  38  A d m i n i s t r a t i o n o f chemicals and d e t e r m i n a t i o n o f uptake  41  L a b e l l e d compounds and t h e i r preparation  42  RESULTS AND DISCUSSION Free p h e n o l s  45  Phenolic glucosides  60  LITERATURE CITED III.  72  THE PHENOL GLUCOSYLATION REACTION I N HIGHER PLANTS  INTRODUCTION  75  LITERATURE REVIEW  76  MATERIALS AND METHODS Plant material Preparation of arbutin-6-sulphate Preparation of gentiobiosides  80 .... 80 83  MATERIALS AND METHODS ( c o n t i n u e d )  Page  A d m i n i s t r a t i o n o f compounds  84  Preparation of plant extracts and chromatography  85  RESULTS AND DISCUSSION Fern survey  8?  Survey f o r a r b u t i g e n i n  87  Identity of arbutigenin  90  S i g n i f i c a n c e o f the p h e n o l glucosylation reaction  96  LITERATURE CITED  101  -viCOMMON AND SYSTEMATIC NAMES OF COMPOUNDS COMMON NAME  SYSTEMATIC NAME  arbutin  p-hydroxyphenyl beta-D-glucoside  caffeic  acid  3 . 4 - dihydroxycinnamic a c i d  catechol  1 . 2 - dihydroxybenzene  o-coumarlc a c i d  2- hydroxycinnamlc a c i d  p-coumarlc a c i d  4-hydroxycinnamic a c i d  ferulic  3 - methoxy-4-hydroxycinnamic a c i d  gallic  acid acid  3,4,5-trihydroxybenzoic acid  gentiobiose  6-0-(beta-D-glucopyranosyl)-Dglucopyranose  gentislc  2.5- dihydroxybenzoic a c i d  acid  hydroquinone  1,4-dihydroxybenzene  phloroglucinol  1»3»5-trihydroxybenzene  protocatechuic acid  3»4-dlhydroxybenzoic acid  resorcinol  1 . 3 - dihydroxybenzene  salicylic sinapic  acid  acid  2-hydroxybenzoic a c i d 4 - hydroxy-3.$-dimethoxyc±nnamic acid  syringlc  acid  k-hydroxy-3»5-dimethoxybenzoic acid  vanillic  acid  4-hydroxy-3-methoxybenzoic acid  -vii-  LIST OF TABLES TABLE  PAGE Phenolic acids i n ferns  1. 2.  3.  4.  5.  Cinnamic and b e n z o i c a c i d s i n some ferns  11  A c o m p i l a t i o n o f the r e s u l t s o f t h e f i r s t and p r e s e n t f e r n s u r v e y s f o r phenolic acids •  12  V a r i a t i o n i n the c o n c e n t r a t i o n o f c i n n a m i c a c i d and b e n z o i c a c i d d e r i v a t i v e s i n the e t h a n o l - s o l u b l e f r a c t i o n of Pterldlum aqullinum V a r i a t i o n i n the concentration of c i n n a m i c a c i d and b e n z o i c a c i d d e r i v a t i v e s i n the e t h a n o l - i n s o l u b l e f r a c t i o n of Pterldlum aqullinum  ...17  ....17  V a r i a t i o n i n the c o n c e n t r a t i o n o f c i n n a m i c a c i d and b e n z o i c a c i d d e r i v a t i v e s i n the e t h a n o l - s o l u b l e f r a c t i o n of Athyrium f e l i x - f e m i n a  17  V a r i a t i o n I n the c o n c e n t r a t i o n o f cinnamic a c i d and b e n z o i c a c i d d e r i v a t i v e s i n the e t h a n o l - i n s o l u b l e f r a c t i o n of Athyrium f e l i x - f e m i n a  18  7.  C h a r a c t e r i s t i c s o f unknown compounds observed i n P t e r l d l u m and A t h y r i u m  18  8.  Dry w e i g h t e s t i m a t e s f o r P t e r l d l u m and A t h y r i u m d u r i n g the time study  18a  6.  Uptake o f p h e n o l i c compounds by Pltyrogramma and Hordeum 1.  Uptake o f hydroquinone a s a f u n c t i o n o f time  51  2.  Uptake o f hydroquinone as a f u n c t i o n o f t i m e , as i n f l u e n c e d by m e t a b o l i c inhibitors  52  Uptake o f hydroquinone a s a f u n c t i o n of e x t e r n a l concentration  5^  3.  -viii4.  Uptake o f hydroquinone as a f u n c t i o n of e x t e r n a l c o n c e n t r a t i o n ......  .. 5^  5»  Uptake o f hydroquinone as i n f l u e n c e d by p o t e n t i a l t r a n s p o r t c o m p e t i t o r s • • • • • • • 5 6  6.  Uptake o f hydroquinone a s i n f l u e n c e d by pH  7.  Uptake o f Rb as i n f l u e n c e d by t h e presence o f hydroquinone •••  7a, Uptake o f 8. 9. 10. 11. 12* 13,  ,...58  86  ,58  3  59&  H labelled catechol  Uptake o f a r b u t i n a s a f u n c t i o n of time  63  Uptake of a r b u t i n a s a f u n c t i o n of e x t e r n a l c o n c e n t r a t i o n •  ••••••63  Uptake o f a r b u t i n a s a f u n c t i o n of e x t e r n a l c o n c e n t r a t i o n  65  Uptake o f a r b u t i n a s i n f l u e n c e d by m e t a b o l i c i n h i b i t o r s  ..........67  Uptake o f a r b u t i n a s i n f l u e n c e d by p o t e n t i a l t r a n s p o r t c o m p e t i t o r s  ..68  Uptake of a r b u t i n as i n f l u e n c e d by pH  70  The Phenol G l u c o s y l a t i o n R e a c t i o n 1,  Ferns which are capable of g l u c o s y l a t i n g h y d r o q u i n o n e and c a t e c h o l  2*  3.  •••  »,,,88  Results of the survey f o r the a b i l i t y t o produce a r b u t i g e n i n and hydroquinone g e n t i o b i o s i d e  89  Chromatographic c h a r a c t e r i s t i c s of compounds encountered i n t h i s study  92  -ix LIST OF FIGURES FIGURE  PAGE Phenolic acids i n ferns  1.  V a r i a t i o n i n the c o n c e n t r a t i o n s o f c i n n a m i c a c i d s and b e n z o i c a c i d s i n the base h y d r o l y s a t e o f the e t h a n o l soluble f r a c t i o n of Pterldlum  20  2.  V a r i a t i o n i n the c o n c e n t r a t i o n s o f c i n n a m i c a c i d s and b e n z o i c a c i d s i n the a o i d h y d r o l y s a t e o f t h e e t h a n o l s o l u b l e f r a c t i o n o f P t e r l d l u m ...........21  3.  V a r i a t i o n i n the c o n c e n t r a t i o n s o f c i n n a m i c a c i d s and b e n z o i c a c i d s i n the base h y d r o l y s a t e o f t h e e t h a n o l insoluble f r a c t i o n of Pterldlum  .22  V a r i a t i o n i n the concentrations of c i n n a m i c a c i d s and b e n z o i c a c i d s i n the base h y d r o l y s a t e o f the e t h a n o l soluble f r a c t i o n of Athyrium  23  V a r i a t i o n i n the c o n c e n t r a t i o n s o f c i n n a m i c a c i d s and b e n z o i c a c i d s i n the a c i d h y d r o l y s a t e o f the e t h a n o l soluble f r a c t i o n of Athyrium  24  V a r i a t i o n i n the c o n c e n t r a t i o n s o f c i n n a m i c a c i d s and b e n z o i c a c i d s i n the base h y d r o l y s a t e o f the e t h a n o l i n s o l u b l e f r a c t i o n of Athyrium  25  Diagram o f unknown compounds observed i n P t e r l d l u m and A t h y r i u m  26  4.  5.  6.  7.  Uptake o f p h e n o l i c compounds by Pltyrogramma and Hordeum la. lb.  Uptake o f hydroquinone as a f u n c t i o n o f time  53  Uptake o f hydroquinone as a f u n c t i o n o f t i m e , a s i n f l u e n c e d by m e t a b o l i c inhibitors  53  -X-  2a.  Uptake o f hydroquinone a s a f u n c t i o n of e x t e r n a l c o n c e n t r a t i o n , by b a r l e y roots • •••••55  2b.  Uptake o f hydroquinone as a f u n c t i o n of e x t e r n a l c o n c e n t r a t i o n , by Pltyrogramma gametophytes  55  The d a t a o f F i g . 2 a i s shown i n t h e form o f a double r e c i p r o c a l p l o t  57  3a. 3b.  4a.  The e f f e c t o f c a t e c h o l , r e s o r c i n o l , and p h l o r o g l u c i n o l on t h e uptake o f hydroquinone The e f f e c t o f pH upon hydroquinone uptake 86  ....57  .59  4b.  The e f f e c t o f hydroquinone upon uptake  5.  The uptake o f a r b u t i n as a f u n c t i o n o f t i m e , by b a r l e y r o o t s •••<» ......64  6a.  The uptake o f a r b u t i n as a f u n c t i o n o f e x t e r n a l c o n c e n t r a t i o n , by b a r l e y roots .••••••••••••••••••••66  6b,  The uptake o f a r b u t i n as a f u n c t i o n o f e x t e r n a l c o n c e n t r a t i o n , by Pityrogramma .•.•••.........  7a, 7b.  8,  Rb  The d a t a o f F i g , 6a i s shown i n t h e form o f a double r e c i p r o c a l p l o t  59  66 ...69  The e f f e c t o f g l u c o s e , c a t e c h o l g l u c o s i d e , and hydroquinone upon the uptake o f a r b u t i n •  69  The i n f l u e n c e o f pH upon t h e uptake o f a r b u t i n by b a r l e y r o o t s «...  71  -xiAcknowledgement To Dr. B.A.  Bohm, under whose s u p e r v i s i o n t h i s work  was conducted, goes my a p p r e c i a t i o n f o r h i s p a t i e n c e  i  and encouragement  t h r o u g h o u t t h e p e r i o d of my  studies.  He has been most generous i n h i s p r o v i s i o n of r e s e a r c h f a c i l i t i e s , as w e l l as i n p e r m i t t i n g me t o pursue and d e v e l o p my i n t e r e s t s i n t r a n s p o r t phenomena. I am g r a t e f u l t o Dr. G.H.N. Towers f o r v a l u a b l e d i s c u s s i o n s c o n c e r n i n g my own r e s e a r c h and a l s o f o r the group meetings where a w i d e r range of t o p i c s was  discussed. The f i n a n c i a l a s s i s t a n c e of the N a t i o n a l Research  C o u n c i l of Canada and of the Department of Botany i s gratefully  acknowledged.  PHENOLIC ACIDS I N FERNS  -1INTRODUCTION Cinnamic a c i d and b e n z o i c a c i d d e r i v a t i v e s a r e widespread i n n a t u r e , o c c u r r i n g i n t h e form o f c o n j u g a t e s w i t h a host o f d i f f e r e n t organic molecules. Considerable  interest  and r e s e a r c h a c t i v i t y have been d i r e c t e d toward the s t u d y o f t h e i r d i s t r i b u t i o n as w e l l as v a r i o u s a s p e c t s o f t h e i r metabolism  i n t h e anglosperms. 1  s t u d y by Bohm and Tryon  By c o n t r a s t , p r i o r t o t h e  p r a c t i c a l l y no i n f o r m a t i o n was  a v a i l a b l e r e g a r d i n g these compounds i n f e r n s . T h i s s t u d y was performed  t o obtain f u r t h e r information regarding the  d i s t r i b u t i o n o f c i n n a m i c and b e n z o i c a c i d d e r i v a t i v e s i n f e r n s , as w e l l as t o s t u d y t h e q u a n t i t a t i v e changes o c c u r r i n g i n t h e l e v e l s o f these compounds throughout t h e growing  season o f two s p e c i e s o f f e r n s .  -2LITERATURE REVIEW The  r e s u l t s o f e x t e n s i v e s t u d i e s by a l a r g e  number of workers have shown t h a t t h e p h e n o l i c  acids  a r e w i d e s p r e a d t h r o u g h o u t t h e p l a n t kingdom. B a t e 2 Smith r e p o r t s t h a t i n t h e l e a v e s of angiosperms p-coumaric a c i d , c a f f e i c a c i d , f e r u l l c a c i d , and s i n a p i c a c i d were found i n 4 9 , 63, 4 8 , and 32$ of those 3 p l a n t s examined. Tomaszewski f o u n d p - h y d r o x y b e n z o i c a c i d and g e n t i s i c a c i d i n t h e l e a v e s of 9 7 $ o f 122 p l a n t s examined. P r o t o c a t e c h u i c a c i d has been found i n a l m o s t every p l a n t t h a t has been s y s t e m a t i c a l l y 4  examined . Most commonly t h e p h e n o l i c a c i d s o c c u r , n o t a s t h e f r e e a c i d s , b u t as c o n j u g a t e s w i t h a v a r i e t y o f o r g a n i c compounds. F o r example, c h l o r o g e n i c a c i d , a c a f f e i c q u i n l c a c i d d e p s i d e has been shown t o be p r a c t i c a l l y 5,6 u b i q u i t o u s among h i g h e r p l a n t s sides are considered  . Phenolic a c i d glyco-  t o be r a r e amongst h i g h e r p l a n t s ,  whereas by c o n t r a s t t h e p h e n o l i c a c i d e s t e r s enjoy a 7  w i d e s p r e a d d i s t r i b u t i o n . The terms s o l u b l e and 8  i n s o l u b l e - e s t e r s were c o i n e d by Basyouni e t a l . t o d e s c r i b e those p h e n o l i c a c i d e s t e r s w h i c h were s o l u b l e and  i n s o l u b l e , r e s p e c t i v e l y , i n ethanol. Within the  e t h a n o l - s o l u b l e group f a l l compounds such as g l u c o s e 7 5,6 9 e s t e r s , q u i n i c a c i d e s t e r s , a c y l a t e d a n t h o c y a n i n s , and 10 8 2 - 0 - c a f f e o y l a r b u t i n • Basyouni c o n s i d e r e d t h a t c e r t a i n  -3-  o f the e t h a n o l - i n s o l u b l e e s t e r s , namely enzyme-ester conjugates  might be h i g h l y i m p o r t a n t  intermediates i n  l i g n i n b i o s y n t h e s i s . However, i t i s not suggested  that  a l l o f the e t h a n o l - i n s o l u b l e p o o l e x i s t s i n t h i s form. 11 Smith  e.g., demonstrated t h a t a l k a l i n e h y d r o l y s i s o f  l i g n i n preparations yielded a v a r i e t y of phenolic acids presumed t o be p r e s e n t as l i g n i n - b o u n d e s t e r s . 12 The s t u d y by I b r a h i m e t a l . nonvascular  showed t h a t even i n  p l a n t s , such as mosses and l i v e r w o r t s ,  p h e n o l i c a c i d s c o u l d be found. T h i s same s u r v e y i n c l u d e d t h r e e s p e c i e s o f f e r n s which a l l c o n t a i n e d p h e n o l i c a c i d s . The f i r s t d e t a i l e d s u r v e y of the f e r n s f o r the presence o f p h e n o l i c a c i d s was conducted by Bohm and 1 T r y o n . These workers examined f o r t y - s i x s p e c i e s and i d e n t i f i e d p-coumarlc a c i d , c a f f e i c a c i d , f e r u l i c  acid,  s i n a p i c a c i d , p-hydroxybenzoic a c i d , p r o t o c a t e c h u i c a c i d , v a n i l l i c a c i d , and s y r l n g l c a c i d as t h e major p h e n o l i c a c i d s . S t u d i e s o f p h e n o l i c metabolism i n f e r n s 13  are e x t r e m e l y  l i m i t e d . Young e t a l . ,  i n a survey of  p l a n t s f o r the presence o f L - p h e n y l a l a n i n e  ammonia-  l y a s e a c t i v i t y , showed t h a t t h i s enzyme i s p r e s e n t i n Osmunda clnnamomea and P t e r l d l u m a q u l l i n u m . S i n c e  this  enzyme c o n v e r t s p h e n y l a l a n i n e t o cinnamic a c i d , i t s presence r e p r e s e n t s a p r e r e q u i s i t e f o r the f o r m a t i o n o f 1 cinnamic and b e n z o i c a c i d d e r i v a t i v e s . Bohm and T r y o n 14 administered DL-phenylalanine-1 C and D L - p h e n y l a l a n i n e 14 -3C i n s e p a r a t e e x p e r i m e n t s t o l e a v e s o f Cyathea  a r b o r e a and observed r a d i o a c t i v i t y i n p-coumarlc a c i d , c a f f e i c a c i d , and f e r u l i c a c i d . These o b s e r v a t i o n s i n t e r p r e t e d as s u g g e s t i n g a pathway e s s e n t i a l l y  were  similar  t o t h a t found i n angiosperms f o r t h e b i o s y n t h e s i s o f cinnamic  acid derivatives. 8  Apart  from t h e work by B a s y o u n i  there i s l i t t l e  I n f o r m a t i o n a v a i l a b l e on t h e c o n c e n t r a t i o n s o f p h e n o l i c a c i d s i n p l a n t t i s s u e s as a f u n c t i o n o f age. B a s y o u n i s t u d i e d t h e l e v e l s o f p-coumaric a c i d , f e r u l i c a c i d , s i n a p i c a c i d , v a n i l l i c a c i d , and s y r i n g i c a c i d o b t a i n e d by t h e a l k a l i n e h y d r o l y s i s o f t h e e t h a n o l - s o l u b l e  fract-  i o n , and p-coumaric a c i d and f e r u l i c a c i d r e l e a s e d by a l k a l i n e h y d r o l y s i s of the ethanol i n s o l u b l e f r a c t i o n of wheat p l a n t s . L e v e l s o f these compounds were d e t e r m i n e d o v e r a p e r i o d o f f i f t y - s e v e n days f o l l o w i n g g e r m i n a t i o n . 14 15 T h i s s t u d y as w e l l as those by Urban , and G r i f f i t h s i n d i c a t e d t h a t there i s a considerable v a r i a t i o n i n the c o n c e n t r a t i o n s o f p h e n o l i c a c i d s d u r i n g t h e growing season. F u r t h e r m o r e , p h y s i o l o g i c a l f a c t o r s such as t h e a v a i l a b i l i t y o f water and t h e type o f i l l u m i n a t i o n r e c e i v e d have been shown t o I n f l u e n c e t h e l e v e l s o f 16 p h e n o l i c compounds i n p l a n t s . I n B a s y o u n i s s t u d y t h e p h e n o l i c a c i d s r e l e a s e d by a l k a l i n e h y d r o l y s i s o f t h e e t h a n o l s o l u b l e f r a c t i o n reached a maximum a t n i n e days. T h e r e a f t e r a p r o g r e s s i v e d e c r e a s e i n q u a n t i t y was r e c o r ded. V a n i l l i c and s y r i n g i c a c i d s showed a second i n c r e a se, r e a c h i n g a p l a t e a u a t about f o r t y - s e v e n days. The e t h a n o l - i n s o l u b l e f r a c t i o n reached a maximum c o n c e n t r a t i o n a t t w e n t y - f i v e days.  -5T h e r e a f t e r t h e r e was a d e c l i n e t o atpplateau a t about t h i r t y - f i v e days. p-Coumaric a c i d i n c r e a s e s t o a second maximum a t f i f t y - s e v e n days.  -6-  MATERIALS AND METHODS Plant  material P l a n t s used i n these s t u d i e s were o b t a i n e d  v a r i e t y of sources.  from a  B r i t i s h Columbia f e r n s were c o l l e c t e d  by t h e a u t h o r and Dr Bruce Bohm d u r i n g t h e summer o f 1967. The  d e p a r t m e n t a l greenhouses m a i n t a i n a c o l l e c t i o n o f  f e r n s which was drawn upon. F e r n s from H a w a i i were c o l l e c t e d by Dr Bohm i n December 1967 a t t h e H a w a i i N a t i o n a l P a r k . Dr T.M.C.Taylor, f o r m e r l y o f t h i s department, p r o v i d e d samples o f t h e Hymenophyllaceae from H a w a i i and Maui. Mecodium w r l g h t l l , from t h e Queen C h a r l o t t e I s l a n d s , was c o l l e c t e d by Dr W . B . S c h o f i e l d o f t h i s department. P l a n t s from t h e U n i v e r s i t y o f Tokyo B o t a n i c a l Garden and from Kew Gardens were s u p p l i e d by t h e d i r e c t o r s o f t h e s e s institutes. Preparation of plant extracts A. The s u r v e y F r e s h p l a n t m a t e r i a l was c u t i n t o s m a l l p i e c e s and homogenized i n b o i l i n g 80% e t h a n o l i n a Waring B l e n d o r . The  e t h a n o l s o l u b l e m a t e r i a l was t h e n e x t r a c t e d by c o n t -  inuous b o i l i n g f o r f i f t e e n minutes on a h o t p l a t e . The e t h a n o l i c s o l u t i o n was s e p a r a t e d from t h e i n s o l u b l e m a t e r i a l by f i l t r a t i o n and t h e n e v a p o r a t e d t o d r y n e s s under an a i r j e t . The e t h a n o l i c r e s i d u e was e x t r a c t e d w i t h b o i l i n g w a t e r and f i l t e r e d t h r o u g h C e l l t e . The f i l t r a t e was d i v i d e d i n t o two p o r t i o n s . A c i d h y d r o l y s i s was performed upon one p o r t i o n by a d j u s t i n g t h e s o l u t i o n t o  -71N w i t h HC1, ing i t  to  b r i n g i n g the  s o l u t i o n to  c o o l t o room t e m p e r a t u r e .  were e x t r a c t e d  with ether  for  nine  boiling,  Acid  hours i n a  continous  The s e c o n d p o r t i o n was s u b j e c t e d t o  hydrolysis  by a d j u s t i n g  heating  the  s o l u t i o n to b o i l i n g ,  t o room t e m p e r a t u r e . t o pH 2 w i t h HC1 were e v a p o r a t e d amount  of  prior  to ether  and a l l o w i n g  extraction.  paper.  cool  Ether  extracts  small  ethanolic  sol-  chromatog-  Chromatograms were d e v e l o p e d by d e s c e n d i n g  for  formic a c i d for  the the  first  i acetic acid  direction,  second d i r e c t i o n .  were l o c a t e d by e x a m i n i n g t h e light  these  to  acidified  s h e e t s o f Whatman No 1  c h r o m a t o g r a p h y e m p l o y i n g benzene (10i7»3 v / v )  it  to dryness and d i s s o l v e d i n a  95% e t h a n o l , A l i q u o t s o f  basic  IN w i t h NaOH,  B a s i c h y d r o l y s a t e s were  u t i o n s were a p p l i e d t o raphy  s o l u t i o n to  allow-  hydrolysates  extractor.  the  and  followed  i  water  by 2%  Phenolic acids  paper under  ultraviolet  (3660 A ) , t h e n by f u m i n g i t w i t h ammonium h y d r o x i d e  under the  same w a v e l e n g t h ,  and f i n a l l y  by s p r a y i n g  it  23 with diazotized spray of  5% NaOH. Q u a n t i t i e s  were d e t e c t a b l e B.  p-nitroaniline  by t h e s e  The q u a n t i t a t i v e  a s low a s 1-2  over-  /ug. of  acid  methods.  extraction  were e m p l o y e d  s t u d y as t h o s e o u t l i n e d above f o r  f e r n s were c o l l e c t e d f r o m t h e t h e y were g r o w i n g w i l d . their  by a n  study.  The same methods o f this  followed  the  University  At approximately  emergence a b o v e g r o u n d , f o r t y  for  survey.  grounds  where  ten days  fiddleheads  All  of  after each  s p e c i e s were l a b e l l e d ive  tape.  Thereafter  by means o f a l o o s e r i n g samples  (single fronds)  of adhes-  were  analysed  every  t e n d a y s . The e t h a n o l - i n s o l u b l e m a t e r i a l was  dried  f o r t w e n t y - f o u r hours, and i t s weight added t o t h a t  of the d r i e d e t h a n o l - s o l u b l e weight estimate. dissolved applied  residue  The d r i e d e t h e r  t o e a c h o f two s h e e t s  each case  one s h e e t  to obtain a dry  e x t r a c t s were  I n 4 m l . o f 95% e t h a n o l  oven  each  a n d 0.1 m l . a l i q u o t s  o f Whatman No 1 p a p e r . I n  was e m p l o y e d t o l o c a t e t h e p h e n o l i c  a c i d s a s d e s c r i b e d a b o v e . The a c i d s were e l u t e d f r o m t h e unsprayed sheet corresponding  with  95% e t h a n o l .  t o a g i v e n a c i d was times with  In each case the area c u t from the paper,  and  e l u t e d three  10 m l . q u a n t i t i e s o f e t h a n o l .  The  e t h a n o l i c s o l u t i o n s thus obtained  were  concentrated  t o a volume o f 10 m l . a n d t h e a b s o r b a n c e o f e a c h d e t e r m i n e d u s i n g a Beckman DU  spectrophotometer a t the  absorption  maximum o f t h e p a r t i c u l a r  centration  o f e a c h a c i d was  to standard expression plant  calibration  sample  compound.  t h e n d e t e r m i n e d by  curves  o f t h e w e i g h t i n mg.  and e x t r a p o l a t e d  The  con-  reference t o an  p e r g. d r y weight of  tissue. Three g. samples of the e t h a n o l - i n s o l u b l e  were e a c h t r e a t e d w i t h  100 m l . o f IN NaOH f o r two h o u r s  a t room t e m p e r a t u r e . The f i l t r a t e  was a c i d i f i e d  with  f o r nine  HC1 a n d e x t r a c t e d w i t h  Analyses  residues  were t h e n p e r f o r m e d  ether  t o pH 2  hours.  i n t h e same manner a s  d e s c r i b e d above f o r the e t h a n o l - s o l u b l e  fractions.  -9RESULTS AND DISCUSSION A. The s u r v e y The  r e s u l t s o f t h i s s u r v e y a r e shown i n T a b l e  Page 11. Of t h e f o r t y - s i x s p e c i e s o f f e r n s  1,  examined  o n l y f o u r gave c o m p l e t e l y n e g a t i v e r e s u l t s f o r t h e presence o f p h e n o l i c a c i d s . The " b a s i c  complement"  1  r e f e r r e d t o by Bohm and Tryon , namely p-coumaric a c i d , c a f f e i c a c i d , f e r u l i c a c i d , p-hydroxybenzoic a c i d , p r o t o c a t e c h u i c a c i d , and v a n i l l i c a c i d o c c u r r e d i n most p l a n t s . Table 2, Page 12 shows t h e percentage of the p l a n t s  examined w h i c h c o n t a i n e d t h e above a c i d s .  W i t h t h e e x c e p t i o n o f v a n i l l i c and f e r u l i c a c i d s t h e d i s t r i b u t i o n of a c i d s  i n t h i s s u r v e y c l o s e l y resembles 1  t h a t f o u n d i n t h e s u r v e y by Bohm and Tryon . F e r u l i c a c i d can be d e r i v e d  from c a f f e i c a c i d by m e t h y l a t i o n .  V a n i l l i c a c i d can be d e r i v e d by a b e t a - o x i d a t i o n  from e i t h e r f e r u l i c  acid  type of r e a c t i o n o r p r o t o c a t e c h u i c  a c i d by m e t h y l a t i o n . Other t h a n f e r u l i c a c i d , t h e o t h e r substrates  f o r these r e a c t i o n s  i . e . , c a f f e i c a c i d and  protocatechuic a c i d , are equally d i s t r i b u t e d i n both s u r v e y s . As a t r i b e t h e Blechneae i s remarkable f o r the r a r i t y of t h e m e t h o x y l a t e d a c i d s . Of those examined i n t h i s s u r v e y and t h a t o f Bohm 23$ p o s s e s s e d v a n i l l i c a c i d and 38$ p o s s e s s e d f e r u l i c a c i d . Of t h e n i n e t y - t w o s p e c i e s examined i n t h e two s u r v e y s 74$ c o n t a i n e d vani l l i c a c i d and 74$ c o n t a i n e d f e r u l i c a c i d . The d i s t r i b u t i o n of c a f f e i c a c i d w i t h i n t h e t r i b e Blechneae i s  -10-  typical  o f t h a t f o r the whole group (77%  87%)  compared t o  so t h a t the immediate p r e c u r s o r o f f e r u l i c a c i d would appear t o be a v a i l a b l e w i t h i n the t r i b e . B o t h the Hymeno p h y l l a c e a e and the D a v a l l i e a e a r e u n u s u a l o f f e r u l i c a c i d (17%  f o r the  rarity  and 20% r e s p e c t i v e l y ) , whereas van-  i l l i c a c i d i s n o r m a l l y d i s t r i b u t e d . These o b s e r v a t i o n s reflect  may  a h i g h l e v e l o f the f e r u l i c a c i d b e t a - o x i d a t i o n  enzyme. I n making i n t e r p r e t a t i o n s based upon the absence o f a g i v e n c h e m i c a l i t s h o u l d be borne i n mind t h a t we are d e a l i n g here w i t h secondary m e t a b o l i t e s , the f u n c t i o n s o f which a r e not always c l e a r . The v a r i a b i l i t y i n concent r a t i o n s o f p h e n o l i c compounds throughout the growing season has a l r e a d y been r e f e r r e d t o and i n p a r t i c u l a r , i n P t e r l d l u m and A t h y r i u m  the c o n c e n t r a t i o n s o f v a n i l l i c  a c i d and t o a l e s s e r e x t e n t f e r u l i c a c i d were q u i t e  low  a t the b e g i n n i n g o f the season. However, even c o n s i d e r i n g these f a c t s the t a x a d i s c u s s e d would appear t o be a t y p i c a l of the whole group. The  r a r i t y o f s i n a p i c and s y r i n g i c a c i d s f o l l o w s the 12  same t r e n d observed occurrence was  f o r gymnosperms by I b r a h i m  . The  of s y r i n g y l s u b s t i t u t e d a c i d s i n l e a f e x t r a c t s  found t o be s t r o n g l y c o r r e l a t e d w i t h the  occurrence  o f s y r i n g y l r e s i d u e s i n the l i g n i n . A r e a s o n a b l y  clear  c u t l i n e can be drawn between the gymnosperms and  angio-  sperms on t h i s b a s i s . Presumably the appearance o f the s y r i n g y l type o f s u b s t i t u t i o n p a t t e r n r e p r e s e n t s a i v e l y r e c e n t a c q u i s i t i o n amongst v a s c u l a r p l a n t s .  relat-  -11T A B L E 1. C I N N A M I C A N D BENZOIC ACIDS IN SOME FERNS  *  Source  PCA a b  CAF a b  FER SIN a b a b  HBA a b  PRO a b  VAN a b  Ophioglossaceae— Botrychium  multifidum  B. virginianum  J . G . Gmelin  Swartz  BC  + + + + + + — — — — + + + +  + + + +  — — — —  + +  HI  + + + +  --  + +  + +  +  -  _  — —  —  _  BC  Glcicheniaceae— Dicranopteris  emarginata  (Brack.)  Robinson Hymenophyllaceae— Hymcnophyllum  barbatum V. den Bosch  Mecodium recurvum (Gaud.) Copel. M. wrightii (V. den Bosch.) Copel. Vandenboschia cyrtotheca (Hilleb.) Copel. V. davalliodes (Gaud.) Copel. Conocoinmus  minutus (Blume) V .  MI  HI QC MI HI  TOK TOK TOK TOK  Polypodiaceae— Woodsieae Woodsia oregana D . C . Eaton Ctenitis decomposita  (R. Br.) Copel.  Dryopteris auslriaca (Jacq.) Woynar. D. felix-mas (L.) Schott. Cymnocarpium  dryopteris (L.)  Lastrea globulifera Polystichum  Newm.  Brack.  andersonii Hopkins  Thclypteris  UBC  BC BC BC HI  P. lonchitis (L.) Roth  U BC  BC  phegopteris  Slosscn  BC  Asplenieae— unilaterale Lamarck  HI  Blechneae— brasilicnse Dcsv.-var.  crispum  hort.  TOK  B. discolor (Forst.) Keys  TOK  B. orientate L.  TOK  B. spicant (L.) Roth.  BC  Doodia  dives Kunzc hillcbrandii  TOK Robinson  S. cyatheoides Kaulf. Woodwardia fornwsana  HI  HI  orientalis S.W.  var.  Ros.  W. unigemmata  Dennstaedtia wilfordii (Moore) Koidz. Microlepia setosa (Smith) Alston Coniogramma  pilosa (Brack.) Hier  Cryptogramma  crispa (L.) R. Br.  arifolia (Burm.) Moore  rotundifolia  Adiantum  pedatum  UBC HI  Hook L.  UBC BC  UBC UBC BC  Pteridcae— Pteridium  aquilinum  P. aquilinum  K u h n var. aquilinum  K u h n var. decomposita  Pteris longifolia  L.  KEW HI UBC  Davallieae— Nephrolepis  cordifolia (L.) Prcsl  HI  Polypodieae— Pldebotium Pleopeltis Polypodium  + + — + — + + + + + + + + + + +  +  +  -  +  - --  + + + + — + _ _ + + + — + — — + + + + + — —  -  1  + -J1  — — — —  _  -  + +  -  +  _ _  + + — +  —  — +  + +  1  + + — — _ _ + + + + — —  — + + + + + + + + +  - -  + + + _ + + + — + +. + — _L_  T"  + _ + + + +  --  + + _ — + + — —  + —  ,i. "T"  -  +  +  -- -  + + +  + + + + + + + — — + + + + — — — + +  + + +  -- -  + + +  + + + +  + + + +  _  +  -- --  + +  -- --  — + _ + _ _ + + + + +  + + + + +  +  TOK  Gymnogrammeae—  Heinionitis  + + + + + + + + — — + + + + + — — + + + + + — —  + + +  TOK  (Makina) Nakci  Dcnnstacdtciac—  Pellcac  + + + +  BC  Dryopterideae  Sadlerin  —  MI  Cyaihea fauriei (Christ.) Copel. C. hancockii Copel. C. metteniana (Hance) Christ, et Tard. C. podophylla Hook  Blechnum  + + — — _ _ + + — — — — + + + + — —  + + + +  den  Bosch. Cyatheaceae—  Asplenium  + +  —  sp. thunbergiana glycyrrhiza  UBC Kaulf.  HI  D . C . Eaton  BC  + + + + + + + + + + + — + — + — + — —  — +  + + + + + + •+ +  + + + + + + + — "++  + + + + + — + + —  _ _ — — — —  — — — —  + + — +  + + _ — — _ + + + + + + + + + + — — — + —  + —  + + + + + + _ _ + + — _ + + + + + — — + + — — + + ' ++ + + + + + + + +  + + + + + +  + +  + + + +  -  + + + + + — — + + + + — —. — — — + + + + — —  —  +  -  +  -  +  —  —  + +  + + + + + + + + + + — — + + + +  * Source of material: British Columbia (BC); University of British Columbia Greenhouses (UBC); Maui, Hawaiian Islands (MI); Hawaii, Hawaiian Islands (HI); Queen Charlotte Islands, British Columbia (QC); University of Tokyo Botanical Gardens ( T O K ) ; Kew Gardens (KEW). Explanation of symbols: P C A , p-coumaric acid; C A F , caffeic acid; F E R , ferulic acid; SIN, sinapic acid; H B A , p-hydroxybenzoic acid; P R O , protocatechuic acid; V A N , vanillic acid; a, acidic hydrolysis; b, basic hydrolysis; + , present; —, absent.  -12TABLE 2.  1  A COMPILATION OF THE RESULTS OF THE FIRST AND PRESENT FERN SURVEYS FOR PHENOLIC ACIDS  Phenolic acid  F i r s t survey  Second s u r v e y  Percentage  p-Coumaric  40  40  87  Caffeic  43  37  87  Ferulic  39  29  74  Sinapic  2  4  7  o-Coumaric  2  0  2  p-Hydroxybenzoic  34  38  78  Protocatechuic  27  24  56  Vanillic  42  26  74  Gentisic  1 1 1  0  1 1 1  Salicylic Syringic  0 0  * A t o t a l o f 46 s p e c i e s of f e r n s were examined i n each s u r v e y . The numbers shown i n d i c a t e t h e number of s p e c i e s w h i c h c o n t a i n e d a p a r t i c u l a r a c i d . The Percentage column shows combined r e s u l t s i n w h i c h v a l u e s f o r t h e two s u r v e y s a r e p o o l e d . The r e s u l t s of t h i s survey have been p u b l i s h e d i n P h y t o c h e m l s t r y 81629  -13-  B. The q u a n t i t a t i v e  study  The r e s u l t s o f t h e p h e n o l i c a c i d time s t u d y w i t h t h e two f e r n s a r e p r e s e n t e d i n t a b l e s 3 - 6 and f i g u r e s 1 - 6 . The v a l u e s i n t a b l e s 3 and 5 r e p r e s e n t t h e averages o f f o u r r e p l i c a t e s , w h i l e those i n t a b l e s 4 and 6 a r e t h e averages  o f two r e p l i c a t e s . I n d i v i d u a l v a l u e s were  u s u a l l y w i t h i n 10 % o f t h e mean. C o n s i d e r i n g t h e n a t u r e o f t h e e x p e r i m e n t a l m a t e r i a l and t h e purpose o f t h e s t u d y , namely t o observe t h e g e n e r a l t r e n d s throughout  the seas-  on, t h i s v a r i a n c e was n o t c o n s i d e r e d t o d e t r a c t from t h e findings. I t i s c l e a r from these d a t a t h a t t h e r e i s a s i g n i f i c a n t degree o f m e t a b o l i c a c t i v i t y i n these two f e r n s p e c i e s w i t h r e g a r d t o t h e p h e n o l i c a c i d s . I t would seem 2 t h a t Bate-Smith  i s q u i t e wrong when he s t a t e s t h a t " t h e  p h e n o l i c a c i d s found i n t h e l i v i n g t i s s u e s a r e t h e r e f o r e s t a b l e and c h a r a c t e r i s t i c end p r o d u c t s " . I n g e n e r a l t h e c i n n a m i c a c i d d e r i v a t i v e s were p r e s e n t i n h i g h e r concent r a t i o n s than the correspondingly s u b s t i t u t e d benzoic a c i d . S i g n i f i c a n t f l u c t u a t i o n s were observed  i n t h e conc-  e n t r a t i o n s o f t h e c i n n a m i c a c i d s d u r i n g t h e growing season. I n b o t h s p e c i e s t h e o v e r a l l t r e n d was a r a p i d a c c u m u l a t i o n t o a maximum f o l l o w e d by a d e c l i n e t o a p l a t e a u which was m a i n t a i n e d f o r t h e remainder  of the  season. I n P t e r i d i u m t h e major a c i d was p-coumaric and the maximum reached a t f i f t y days. T h i s p a t t e r n i s s i m i 18 l a r t o t h a t observed by Kuc and N e l s o n i n t h e i r study  -14-  o f wheat p l a n t s . T h e i r k i n e t i c s t u d i e s showed t h a t t h e q u a n t i t y o f p-coumaric a c i d p e r u n i t weight o f l i g n i n reached a maximum a t s i x t y days and t h e r e a f t e r d e c l i n e d . 16  S i m i l a r l y , i n Basyouni s study  an e a r l y maximum was  f o l l o w e d by a d e c l i n e t o a p l a t e a u . I n A t h y r i u m t h e majo r p h e n o l i c a c i d was c a f f e i c  and t h e maximum c o n c e n t r a -  t i o n was reached a t a much e a r l i e r d a t e , a t twenty days. Despite considerable f l u c t u a t i o n s the concentration of t h i s a c i d d e c l i n e d a f t e r t h i s t i m e . By c o n t r a s t t h e b e n z o i c a c i d s showed a more d e c i d e d t r e n d towards a g r a d u a l i n c r e a s e i n c o n c e n t r a t i o n t h r o u g h o u t t h e growing season. T h i s i s p a r t i c u l a r l y t r u e o f v a n i l l i c a c i d which was f r e q u e n t l y absent - a l t o g e t h e r i n t h e e a r l y s t a g e s o f the study, b u t r e a c h e d i t s h i g h e s t c o n c e n t r a t i o n a t e i g h t y days. I n both species l a r g e r q u a n t i t i e s o f a c i d s were p r e s e n t as b a s e - s e n s i t i v e e t h a n o l - s o l u b l e  conjugat-  es t h a n a c i d - s e n s i t i v e forms. I n P t e r l d l u m t h e a c i d - l a b i l e forms showed l i t t l e v a r i a t i o n d u r i n g t h e s t u d y . I t would appear u n l i k e l y t h a t these forms r e p r e s e n t  signif-  icant q u a n t i t i e s of active metabolites i n t h i s plant. A t h y r i u m . i n c o n t r a s t , showed as much v a r i a t i o n i n i t s a c i d - l a b i l e conjugates  as i n i t s b a s e l a b l l e forms. =  In Pteridium a l k a l i n e h y d r o l y s i s of the ethanolinsoluble f r a c t i o n released quantities of phenolic acids a t l e a s t e q u a l t o those  r e l e a s e d from t h e e t h a n o l - s o l u b l e  f r a c t i o n . I n A t h y r i u m o n l y s m a l l q u a n t i t i e s o f a c i d s were bound i n t h i s manner.  -15-  The  most s t r i k i n g o b s e r v a t i o n i n t h i s s t u d y i s the  dynamic s t a t u s of the p h e n o l i c a c i d s , p a r t i c u l a r l y  p-  coumaric a c i d i n P t e r l d i u m and c a f f e i c a c i d i n A t h y r i u m . B o t h o f these compounds I n c r e a s e d  rapidly i n i t i a l l y  and  l a t e r d e c l i n e d i n c o n c e n t r a t i o n . I n a s t u d y of l i g n i n 19  b i o s y n t h e s i s i n wheat p l a n t s Stone e t a l .  found t h a t  the g r e a t e s t i n c r e a s e i n the p r o d u c t i o n of l i g n i n r e d f o r t y - f i v e t o seventy days a f t e r s e e d i n g .  occur-  Higuchi  20  and Brown  a l s o observed a s l o w e r r a t e of  lignification  i n younger wheat p l a n t s . T h e i r o b s e r v a t i o n s the s l o w e r r a t e i s due  t o the c u m u l a t i v e  suggest t h a t  e f f e c t of  slow-  e r r e a c t i o n s a t s e v e r a l stages r a t h e r t h a n r e l a t i v e i v i t y o f an enzyme system a t any one h e t i c pathway. I t may  inact-  stage of the b i o s y n t -  w e l l be t h a t d u r i n g the e a r l y  s t a g e s of growth, when p h o t o s y n t h e t i c a c t i v i t y i s u s u a l l y pronounced, l a r g e q u a n t i t i e s of p o t e n t i a l l i g n i n  precurs-  o r s are s e t a s i d e i n the form of e s t e r s , g l y c o s i d e s , e t c . t o e n t e r the main l i g n i n pathway a t a l a t e r t i m e . maximum c o n c e n t r a t i o n o f c a f f e i c a c i d was  The  reached i n  A t h y r i u m a;t t w e n t y days. At t h i s age the l e a v e s were about 30 cm;, t a l l ,  p a l e g r e e n i n c o l o u r , and  still  partly  c i r c i n a t e . These young l e a v e s emerge between f u l l y grown l e a v e s of the same rhizome and are t h u s c o n s i d e r a b l y shaded a t t h i s age. C o n s i d e r i n g the b i o s y n t h e t i c o r i g i n 21  of p h e n y l p r o p a n o i d  m o l e c u l e s from c a r b o h y d r a t e s  i t is  i n t r i g u i n g t h a t c a f f e i c a c i d s h o u l d r e a c h i t s maximum c o n c e n t r a t i o n a t a time when the p h o t o s y n t h e t i c p o t e n t i a l  -16-  i s s t i l l f a r from b e i n g r e a l i s e d . Perhaps  phenylpropan-  o i d molecules synthesized i n the o l d e r leaves are t r a n s p o r t e d t o t h e younger l e a v e s i n p r e p a r a t i o n f o r t h e l i g n i f i c a t i o n process. A l t e r n a t i v e l y , the h y d r o l y s i s of f o o d r e s e r v e s l o c a t e d i n t h e rhizome may p r o v i d e t h e n e c e s s a r y raw m a t e r i a l s . 22  H i l l i s and Swain  a n a l y s e d l e a v e s o f Prunus  domestica  f o r t o t a l p h e n o l s , l e u c o a n t h o c y a n i n s and f l a v o n o l s a t i n t e r v a l s d u r i n g t h e growing season and found t h a t t h e amounts i n c r e a s e d r a p i d l y u n t i l t h e l e a v e s reached maximum s i z e and t h e n d e c r e a s e d . T h e i r r e s u l t s suggest t h a t i n the o l d e r l e a v e s t h e p h e n o l i c compounds a r e e i t h e r metabo l i z e d or translocated t o other parts of the plant. D u r i n g t h e course o f t h e e x a m i n a t i o n s o f these f e r n s s e v e r a l " unknown " compounds were observed. These are i l l u s t r a t e d i n diagram ( F i g 7 ) . The b e h a v i o r o f these compounds under u.v. l i g h t  (366OA);  and t h e i r c o l o u r  r e a c t i o n t o d i a z o t i z e d p - n i t r o a n i l i n e are recorded i n t a b l e 7. One compound I n p a r t i c u l a r ( No 1 i n F i g 7 ) proved t o be i n t e r e s t i n g i n t h a t i t s o c c u r r e n c e was r e s t r i c t e d t o l e a v e s o f P t e r l d l u m between t h e ages o f twenty and t h i r t y days. A t i t s maximum i t appeared i n h i g h c o n c e n t r a t i o n b u t t h e q u a n t i t y dropped  rapidly  t h e r e a f t e r and f a i l e d t o r e a p p e a r t h r o u g h o u t t h e r e m a i n d e r o f t h e p l a n t s l i f e . Attempts t o i s o l a t e t h e compound f o r s t r u c t u r a l a n a l y s i s f a i l e d , due t o i t s instability.  -17-  T A B L E 3. V A R I A T I O N IN T H E C O N C E N T R A T I O N OF CINNAMIC ACID A N D BENZOIC ACID DERIVATIVES IN T H E E T H A N O L SOLUBLE FRACTION O F Pteridium  aquilinum  Concentration (mg/g dry wt) at acid* PCA CAF FER PHBA PROTO VAN  Base Acid Base Acid Base Acid Base Acid Base Acid Base Acid  10 days  20 days  30 days  40 days  50 days  0-440* 0-183 0063 0-200 0091 0-023 0-105 0090 0000 0076 0068 0-000  0-341 0-184 0-087 0-176 0-188 0-120 0-115 0-109 0030 0015 0082 0 000  0-760 0-117 0-293 0-100 0158 0055 0-102 0-046 0035 0030 0061 0025  1-063 0-217 0-272 0-170 0-184 0-119 0-139 0052 0-095 0-101 0066 0030  1-106 0-192 0-222 0-132 0-176 0-104 0-135 0056 0-163 0-025 0-107 0-145  60 days • 0-978 0-155 0-235 0091 0-160 0-067 0-103 0059 0000 0047 0-113 0 055  70 days  80 days  0-913 0-144 0169 0099 0-133 0-077 0-145 0052 0-052 0049 0108 0 045  0-910 0-141 0-280 0-093 0-124 0-073 0095 0-066 0000 0 045 0-134 0-136  * Key: P C A , p-coumaric acid; C A F , caffeic acid; F E R , ferulic acid; P H B A , p-hydroxybenzoic acid; P R O T O , protocatechuic acid; V A N , vanillic acid; Base, alkaline hydrolytic procedure; Acid, acidic hydrolytic procedure. T A B L E 4.; V A R I A T I O N IN T H E C O N C E N T R A T I O N O F CINNAMIC ACID A N D BENZOIC ACID DERIVATIVES I N T H E ETHANOL-INSOLUBLE FRACTION O F Pteridium  aquilinum  Concentration (mg/g dry wt) at Phenolic acid*  10 days  20 days  30 days  40 days  50 days  60 days  70 days  80 days  PCA CAF FER PHBA PROTO VAN  0-078* 0045 0017 0032 0024 0000  0-158 0-064 0054 0019 0-152 0 037  0-460 0085 0-137 0023 0020 0000  0-550 0-192 0-178 0-118 0-269 0-000  1-071 0-331 0-216 0075 0000 0 000  0-467 0-219 0-125 0-159 0-260 0000  0-612 0-217 0-208 0000 0 000 0000  0-634 0-247 0-222 0-107 0-436 0-234  * For Key, see Table-3. T A B L E 5. V A R I A T I O N I N T H E C O N C E N T R A T I O N O F CINNAMIC A C I D A N D BENZOIC ACID DERIVATIVES I N T H E E T H A N O L - S O L U B L E F R A C T I O N O F Athyrium  felix-femina  Concentration (mg/g dry wt) at acid PCA CAF FER PHBA PROTO VAN  Base • Acid Base Acid Base Acid Base Acid Base Acid Base Acid  10 days  20 days  30 days  40 days  50 days  60 days  70 days'  80 days  0-604 0-101 0-703 0197 0 000 0000 0-100 0-056 0-050 0-049 0015 0000  0-388 0-181 1-333 0-854 0051 0016 0060 0057 0-163 0-044 0024 0-000  0-391 0-172 0-490 0 371 0094 0064 0-079 0088 0-169 0063 0-144 0-109  0-368 0070 0-476 0-276 0-184 0-161 0-130 0-000 0-181 0-115 0-234 0-317  0-371 0056 0-867 0-235 0-185 0000 0-161 0-032 0-285 0076 0-250 0-256  0-228 0000 0-149 0-100 0-152 0000 0-118 0000 0051 0-000 0-225 0095  0-253 0169 0-558 0-208 0-153 0-061 0074 0-051 0-118 0-214 0-258 0-277  0-304 0000 0-573 0-144 0-128 0098 0-104 0-000 0156 0-243 0-261 0-393  :  -18-  T A B L E 6. V A R I A T I O N IN T H E C O N C E N T R A T I O N O F CINNAMIC A C I D A N D BENZOIC A C I D DERIVATIVES IN T H E E T H A N O L - I N S O L U B L E F R A C T I O N OF Athyrium felix-femina  Phenolic acid  , 10 days  20 days  Concentration (mg/g dry wt) at " 30 days 40 days 50 days 60 days  PCA CAF FER PHBA PROTO VAN  0-113 0-252 0-000 0-090 0-144 0-000  0096 0-245 0-000 0-044 0-100 oooo  0-117 0-391 0-029 0034 0095 0000  0077 0-188 0-059 0-000 0-123 0 000  0-076 0-108 0000 0-047 0-118 0-107  0-073 0-149 0-061 0-066 0-243 0-135  T A B L E , 7. CHARACTERISTICS O F U N K N O W N COMPOUNDS OBSERVED IN Pteridium  Unknown compound*  Sourcef  Fluorescence at 3660 A  Fluorescence with N H  1 2 3 4 5 6 7 8 9 10 11  P (a, b) P. A (a, b) P, A (a) P,A(b) P, A (b) P, A (a) A (a) P, A (a) P, A (a) A(b) P(a)  Yellow None None None • None None None Blue None None None  Intensified None None None Blue None None Intensified Blue None Blue  3  — 70 days  —, 80 days  0-063 0-111 0-044 . 0063 0-149 0-150  AND  0061 0-122 0-075 0-109 0-370 0-214  Athyrium  Colour reaction with DPNAJ Mauve Green • Mauve Blue to mauve Orange Mauve Grey None Mauve Blue Violet  * Numbers correspond to unknown in diagram (Fig. 7). P, Pteridium; A, Athyrium; a, acid, hydrolysate of ethanol-soluble fraction; b, alkaline hydrolysate of ethanol-soluble fraction. X Diazotizcd p-nitroaniline ovcrsprayed with dilute sodium hydroxide.  t  The r e s u l t s o f t h i s q u a n t i t a t i v e s t u d y have been p u b l i s h e d i n P h y t o c h e m i s t r y 8»371  -18a-  TABLE 8 .  DRY WEIGHT ESTIMATES FOR PTERIDIUM AND ATHYRIUM DURING THE TIME STUDY  AGE OF PLANTS (days)  PTERIDIUM  10  ATHYRIUM  g.  5.3  20  9.5  g.  7.6  30  13.7  g.  6.9  40  14.7  g.  6.9  50  16.8  g.  6.4  60  10.8  g.  6.7  70  12.3  g.  10.3  80  19.8  g.  6.9  LEGEND FOR Fig.  1.  FIGURES  1-6  V a r i a t i o n I n the c o n c e n t r a t i o n s and b e n z o i c  of cinnamic a c i d s  a c i d s i n the base h y d r o l y s a t e  of the  e t h a n o l - s o l u b l e f r a c t i o n of P t e r i d i u m . Fig.  2. V a r i a t i o n i n the c o n c e n t r a t i o n s and b e n z o i c  of cinnamic a c i d s  a c i d s i n the a c i d h y d r o l y s a t e  of the  e t h a n o l - s o l u b l e f r a c t i o n of P t e r l d l u m . Fig.  3 . V a r i a t i o n i n the c o n c e n t r a t i o n s and b e n z o i c  of c i n n a m i c a c i d s  a c i d s i n the base h y d r o l y s a t e  o f the  e t h a n o l - i n s o l u b l e f r a c t i o n of P t e r i d i u m . Fig.  4. V a r i a t i o n i n the  and b e n z o i c  concentrations  of c i n n a m i c a c i d s  a c i d s i n the base h y d r o l y s a t e  of  the  e t h a n o l - s o l u b l e f r a c t i o n of A t h y r i u m . Fig.  5. V a r i a t i o n i n the c o n c e n t r a t i o n s and b e n z o i c  a c i d s i n the a c i d h y d r o l y s a t e  ethanol-soluble Fig.  6. V a r i a t i o n i n the  and b e n z o i c  of c i n n a m i c a c i d s of the  f r a c t i o n of A t h y r i u m . concentrations  of c i n n a m i c a c i d s  a c i d s i n the base h y d r o l y s a t e  o f the  e t h a n o l - i n s o l u b l e f r a c t i o n of A t h y r i u m .  The  symbols used i n the f o l l o w i n g f i g u r e s are  f o l l o w s :-  A, p-coumaric a c i d ;  caffeic acid;  • . f e r u l i c a c i d ; A, p-hydroxybenzoic a c i d ; o, e c h u i c a c i d ; •, v a n i l l i c a c i d .  as  protocat-  -20-  Fig.  10 20 30  1.  40 50  60 70 80  TIME (days)  -21-  Fi«. 2.  -23F i g . 4.  10 20 30 40 50 60 70 80 TIME (daysj  -26-  DIAGRAM OF UNKNOWN COMPOUNDS OBSERVED I N PTERIDIUM AND ATHYRIUM.  1.0  S-^ was t h e o r g a n i c phase o f b e n z e n e : a c e t i c (10:7O,v/v).  S  2  acid:water  was 2% f o r m i c a c i d . The f i g u r e s a l o n g  the axes r e p r e s e n t R^ v a l u e s . The s p o t s l a b e l l e d A,B, and C a r e c a f f e i c a c i d , p-coumarle a c i d , and f e r u l i c a c i d , r e s p e c t i v e l y , and a r e i n c l u d e d as markers. C h a r a c t e r i s t i c s o f t h e above compounds may be found i n Table 7 Page 18.  -27LITERATURE CITED t.  Bohm, B.A. and T r y o n , R.M. I 9 6 7 . P h e n o l i c compounds i n f e r n s 1. A s u r v e y o f some f e r n s f o r c i n n a m i c and b e n z o i c a c i d d e r i v a t i v e s . Can J . Botany. itlt585.  2. B a t e - S m i t h , E.C. 1 9 5 4 . F e r u l i c , s l n a p i c and r e l a t e d a c i d s i n l e a v e s . Chem. and I n d . 1457. 3.  Tomaszenski, M. i 9 6 0 . The o c c u r r e n c e o f p-hydroxyb e n z o i c a c i d and some o t h e r simple p h e n o l s i n v a s c u l a r p l a n t s . B u l l . Acad. P o l o n . S c i . , s e r . S c i . b i o l . 8:61.  4 . Harborne, J.B. and Simmonds, N.W. 1964. Chap. 3. B i o c h e m i s t r y o f P h e n o l i c Compounds. Academic P r e s s , London and New York. 5. P o l i t i s , J . 1949. C h l o r o g e n i c a c i d i n p l a n t s and i t s d i s t r i b u t i o n . Compt. Rend. 228:265. 6. Sondheimer, E. 1962. The c h l o r o g e n i c a c i d s and r e l a t e d compounds. P r o c . Sym. P l a n t . P h e n o l i c s . N o r t h A m e r i c a . Oregon S t a t e U n i v . 7. Harborne, J.B. and C o r n e r , J . I 9 6 I . P l a n t p o l y p h e n o l s 4. Hydroxycinnamic a c i d - s u g a r d e r i v a t i v e s . Biochem. J . 81.: 242. 8. E l - B a s y o u n l , S a i d . Z., N e i s h , 1964. P h e n o l i c a c i d s i n derivatives of phenolic intermediates i n l i g n i n 2:627.  A.C. and Towers, G.H.N. wheat 3. I n s o l u b l e a c i d s as n a t u r a l b i o s y n t h e s i s . Phytochem.  9. Harborne, J.B. 1964. Chap. 4 . B i o c h e m i s t r y o f P h e n o l i c Compounds. Academic P r e s s . London and New York. 10.  Haslam, E., Naumann, M.O. and ( i n p a r t ) B r i t t o n , G. 1964. P h e n o l i c c o n s t i t u e n t s o f V a c c i n i u m V i t l s i d a e a . J . Chem. Soc. 5 6 4 9 .  11.  Smith, D.C.C. 1955.  E s t e r groups i n l i g n i n .  Nature.  176:267. 12.  I b r a h i m , R.K., Towers, G.H.N, and G i b b s , R.D. I 9 6 2 . S y r i n g i c a c i d s and s i n a p i c a c i d s a s i n d i c a t o r s o f d i f f e r e n c e s between major groups o f v a s c u l a r p l a n t s . J . L i n n e a n Soc. London. Botany. 58:223.  -2813. Young, M.R., Towers, G.H.N, and N e i s h , A.C. 1966. Taxonomic d i s t r i b u t i o n o f ammonia-lyases f o r L - p h e n y l a l a n i n e and L - t y r o s l n e i n r e l a t i o n t o l i g n i f i c a t i o n . Can J . Botany. 44:341 11  14. Urban, R. 1959. P h y s i o l o g i s c h e Untgrsuchungen Uber E i n i g e F l a v o n o i d e und O x y z i m t s a u r e n . I I . Untersuchungen u b e r den J a h r e z e i t l i c h e n und Tagesperiodischen V e r l a u f der Stoffbidung. P l a n t a . 52:565. 15. G r i f f i t h s , L.A. 1958. P h e n o l i c a c i d s and f l a v o n o i d s o f Theobroma oacoa., s e p a r a t i o n and i d e n t i f i c a t i o n by paper chromatography. Biochem. J , 70:120. 16. E l - B a s y o u n i , S a i d . Z. 1964. Ph.D. T h e s i s . Dept. Botany. M c G i l l Univ. Montreal. 17.  B a t e - S m i t h , E.C. 1958. P l a n t p h e n o l i c s as taxonomic g u i d e s . P r o c . L i n n . Soc. Lond. 169:198.  18. Kuc, J . and N e l s o n , O.E. 1964. The abnormal l i g n i n s produced by the b r o w n - m i d r i b mutants o f maize. 1. The b r o w n - m i d r i b - 1 mutant. A r c h . Biochem. B i o p h y s . 105:103. 19. Stone, J.E., B l u n d e l l , M.J. and Tanner, K.G. 1951. The f o r m a t i o n o f l i g n i n i n wheat p l a n t s . Can. J . Chem. 2 £ : 7 3 4 . 2 0 . H i g u c h i , T. and Brown, S.A. 1963. S t u d i e s o f l i g n i n b i o s y n t h e s i s using i s o t o p i c carbon. X I . Reactions l e a d i n g t o l i g n i f i c a t i o n i n young wheat p l a n t s . Can. J . Biochem. P h y s i o l . 4 1 : 6 5 . 21.  N e i s h , A.C. i 9 6 0 . B i o s y n t h e t i c pathways o f a r o m a t i c compounds. Ann, Rev. P l a n t P h y s i o l . 11:55.  2 2 . H i l l i s , W.E. and Swain, T. 1959. The p h e n o l i c c o n s t i t u e n t s o f Prunus d o m e s t l c a . 2. The a n a l y s i s o f t i s s u e s o f the V i c t o r i a plum t r e e . J . S c i . Food A g r i c . 1 0 : 1 3 5 . 2 3 . Bray, H.G., Thorpe, W.V. and White, K. 1950. The f a t e of c e r t a i n o r g a n i c a c i d s and amides i n t h e r a b b i t . Biochem. J . 4 6 : 2 7 1 .  THE UPTAKE OF PHENOLIC COMPOUNDS BY PITYROGRAMMA CALOMELANOS AND HORDEUM VULGARE  -29INTRODUCTION  In  order  development  to  a  f u l f i l  living  organism  supply  of  nutrients  bodies  of  multicellular  erent  compounds  tissue.  Thus  thesis  to  ported  from  from  must  their  must  f o r growth  receive  i t s environment. organisms  be  hormones  specific  i t s potential  moved  target sites  from  areas,  of  of  to  site  nutrients  the  diff-  tissue  their  synthesis to  continuous  Within  a variety  t r a n s p o r t e d from  are  a  and  may  of be  storage  syntrans-  organs,  etc. In ing  some  organism  such  as  may  entry of  result  from  that  at  E.g.,  taining  a  Nitella  into  purely physical  they  levels  c o n s i d e r a b l y below  ution.  molecules  It i s characteristic  however,  many m o l e c u l e s  even  the  diffusion.  organisms, of  cases  can  forces  living  i n excess  environmental  translucens i s capable  cytoplasmic potassium  liv-  maintain concentrations  vastly  their  of  the  of,  or  distribof  main-  c o n c e n t r a t i o n which  i s  1 1200  times  Such  phenomena a r e  since  they  that  involve  Nevertheless achieve the  expense  such to  the  of  there uptake of  medium  i n which  thermodynamically a  decrease  exist of a  wide  been  i t from  of  non-spontaneous of  of  the  active  system.  which  compounds  d e r i v e d energy.  by  growing.  mechanisms  variety  called  uptake  i t is  entropy  i n nature  metabolically  mechanisms has  distinguish  the  Uptake  transport  passive  at  so  mechanisms.  by as  Rosenberg  has d e f i n e d a c t i v e t r a n s p o r t a s " t h e movement  o f a substance by o t h e r f o r c e s i n a d d i t i o n t o t h e chemi c a l ( o r a n a l o g o u s ) p o t e n t i a l g r a d i e n t o f t h i s substance A b e t t e r d e f i n i t i o n might i n c l u d e some r e f e r e n c e metabolic  to a  component o f t r a n s p o r t .  The l i t e r a t u r e p e r t a i n i n g t o a c t i v e t r a n s p o r t i n p l a n t s and a n i m a l s i s c o n s i d e r a b l e . N a t u r a l l y , t h e work has p r i m a r i l y been concerned w i t h t h e movement o f n u t r i e n t s u b s t a n c e s such as s u g a r s , amino a c i d s , i n o r g a n i c i o n s e t c . , and f o r t h e most p a r t secondary  metabolites  have r e c e i v e d l i t t l e a t t e n t i o n . The f o l l o w i n g s t u d i e s were performed t o g a i n some u n d e r s t a n d i n g  o f t h e mech-  anisms o f uptake o f p h e n o l i c compounds i n h i g h e r plants.  -31LITERATURE REVIEW S e v e r a l c r i t e r i a a r e now w e l l e s t a b l i s h e d as 2,3 c h a r a c t e r i s t i c of active transport  systems  . Taken  s i n g l y these c r i t e r i a do n o t by t h e m s e l v e s e s t a b l i s h a c t i v e transport,- b u t when s e v e r a l o f t h e c r i t e r i a c a n be shown t o o c c u r s i m u l t a n e o u s l y  the p r o c e s s becomes  highly probable. 2,3 Criteria f o ractive  transport  1. S a t u r a t i o n phenomena. When t h e r a t e o f uptake i s no l o n g e r a l i n e a r f u n c t i o n of the d i f f e r e n c e i n concentration  on b o t h  s i d e s o f t h e membrane, e s p e c i a l l y a t h i g h e r  con-  c e n t r a t i o n s , s a t u r a t i o n phenomena o c c u r . When uptake i s plotted against external concentration ported  of the trans-  compound, t h e form o f t h e p l o t c l o s e l y approx-  imates a r e c t a n g u l a r  h y p e r b o l a . By a n a l o g y w i t h enzyme  k i n e t i c s a M i c h a e l l s c o n s t a n t ( K ) i s employed t o m  c h a r a c t e r i z e t h e a f f i n i t y o f t h e t r a n s p o r t system f o r its  substrate.  2. S p e c i f i c i t y . Whilst the rate of d i f f u s i o n o f chemicals i n t o c e l l s has been shown by t h e c l a s s i c a l s t u d i e s o f C o l l a n d e r and B a r l u n d t o depend upon l i p i d  solubility  and m o l e c u l a r d i m e n s i o n s ( g i v e n e q u i v a l e n t  electro-  chemical p o t e n t i a l gradients),active transport  systems  e x h i b i t a h i g h degree o f s e l e c t i v i t y independent o f these c h a r a c t e r i s t i c s . Thus g l u c o s e ,  b u t n o t mannose  -321s a c t i v e l y absorbed by the i n t e s t i n a l tissue of cer5  t a i n mammals . 3.  Temperature s e n s i t i v i t y . Unusually high temperature c o e f f i c i e n t s , of the  order of enzymatic reactions, are frequently  character-  i s t i c of active transport systems. 4.  Metabolic dependence. This i s probably one of the most important c r i t e r i a .  Several parameters of the experimental tissue can be adjusted to demonstrate t h i s dependence,e.g., anaerobi c or low temperature conditions can be imposed, or s p e c i f i c chemical i n h i b i t o r s of metabolism such as cyanide or 2,4-dinitrophenol  may  be added to the t i s s u e .  5 . " U p h i l l " transport. B y " u p h i l l , i n t h i s context,is meant the movement of a compound against i t s electrochemical p o t e n t i a l gradi e n t . The c r i t e r i o n of u p h i l l transport must be i n t e r preted with care since i t i s well known that specialized secretory c e l l s transport molecules  i n the d i r e c t i o n of  the gradient. Furthermore, the entry of a molecule into the c e l l may be followed by i t s conversion to a d i f f e r ent molecular species, so that a concentration  gradient  favouring d i f f u s i o n i s maintained. Thus the bulk of the 6  glucose absorbed i n transport studies by B i e l e s k i was recovered  i n the form of  sucrose.  U n t i l quite recently the experimental approach to transport has been that of k i n e t i c studies. Clearly,  -33the e x p e r i m e n t e r i s l i m i t e d by t h e f a c t t h a t t r a n s p o r t , by d e f i n i t i o n , c a n be measured o n l y a c r o s s phase bounda r i e s , and hence t h e k i n e t i c t r e a t m e n t o f uptake by i n t a c t t i s s u e s has been t h e major t o o l o f workers i n t h i s f i e l d . The c r i t e r i a d e s c r i b e d above c a n be a p p l i e d , almost without  m o d i f i c a t i o n , t o any enzyme mediated  p r o c e s s , and t h e r e f o r e i t i s h a r d l y s u p r i s l n g t h a t t h e b a s i c model f o r a c t i v e t r a n s p o r t r e p r e s e n t s a s i m p l e e x t e n s i o n o f t h e M i c h a e l l s - M e n t o n model o f enzyme 7  a c t i o n . I f t h e i r equation  E + S ES  \ £2 ^ .  k  3 ^  k4  t-  ES E + P  i s i n t e r p r e t e d so t h a t E r e p r e s e n t s o r s i t e , and S r e p r e s e n t s then the observations  the t r a n s p o r t u n i t  t h e t r a n s p o r t e d compound,  of active transport are w e l l  accounted f o r . The t r a n s p o r t molecule (S) i s b e l i e v e d t o r e a c t w i t h a membrane component, termed a c a r r i e r o r t r a n s p o r t s i t e t o form a complex (ES) w h i c h i s t h e n moved from one s i d e o f t h e membrane t o t h e o t h e r . The complex i s t h e n thought t o b r e a k down r e l e a s i n g t h e t r a n s p o r t e d molecule i n t o t h e c e l l . U n l i k e t h e enzyme model, t h e s u b s t r a t e i s b e l i e v e d t o remain unchanged and t h e c a r r i e r i t s e l f becomes m o d i f i e d as a r e s u l t o f the t r a n s p o r t p r o c e s s . There i s good e v i d e n c e ,  however,  - 43  t h a t the t r a n s p o r t of c e r t a i n compounds i n v o l v e s m o d i f i c a t i o n of t h e s u b s t r a t e as a p r e r e q u i s i t e of a b s o r p t i o n . Thus t h e uptake of n i n e sugars has been 8  demonstrated by Roseman e t a l . t o be a s s o c i a t e d w i t h t h e i r p h o s p h o r y l a t i o n by a h e a t - s t a b l e p r o t e i n  (HPr),  w h i c h i s i t s e l f p h o s p h o r y l a t e d by p h o s p h o e n o l p y r u v a t e . Mutants l a c k i n g the enzymes w h i c h p h o s p h o r y l a t e e i t h e r HPr o r the sugars a r e u n a b l e t o accumulate t h e s e s u g a r s . Whether the s u b s t r a t e o r the t r a n s p o r t s i t e i s m o d i f i e d as a consequence of t r a n s p o r t t h e r e i s a metab o l i c dependence f o r r e g e n e r a t i o n o f t r a n s p o r t capacity. The a c t u a l t r a n s l o c a t i o n mechanism throughfetheyuimiembrane remains a m y s t e r y . S e v e r a l models have been proposed t o a c c o u n t f o r the t r a n s p o r t of s p e c i f i c i o n s by •1,9.10 animal t i s s u e s ,  but as y e t we a r e s t i l l a l o n g way  from a d e t a i l e d u n d e r s t a n d i n g of the t r a n s p o r t mechanism  a t the m o l e c u l a r l e v e l . The t r a n s p o r t s i t e i s g e n e r a l l y c o n s i d e r e d t o be  a p r o t e i n . The c r i t e r i a d i s c u s s e d above a r e i n k e e p i n g w i t h t h i s n o t i o n e.g., s p e c i f i c i t y , temperature sensitivity,  M i c h a e l i s - M e n t e n k i n e t i c s e t c . Furthermore  i n h i b i t i o n of t r a n s p o r t has been a c h i e v e d by r e a g e n t s 11 which r e a c t s p e c i f i c a l l y w i t h p r o t e i n s  . Also,  inhibit-  ors of p r o t e i n s y n t h e s i s such as c h l o r a m p h e n i c o l p r e v e n t t r a n s p o r t systems from b e i n g s y n t h e s i z e d i n 12 bacteria .  -3513  Kennedy e t a l . have i s o l a t e d a b e t a - g a l a c t o s i d e t r a n s p o r t i n g p r o t e i n from E s c h e r i c h i a c o l l a f t e r f i r s t t r e a t i n g a l l o t h e r p r o t e i n s o f two s t r a i n s o f b a c t e r i a (Induced and non-induced f o r t h e t r a n s p o r t s y s t e m ) , w i t h N - e t h y l m a l e i m i d e (NEM) i n t h e presence o f t h e n a t u r a l s u b s t r a t e o f t h e t r a n s p o r t i n g p r o t e i n . The c e l l s were t h e n washed f r e e o f t h e NEM and s u b s t r a t e , and 14 treated with C-NEM. F o l l o w i n g f r a c t i o n a t i o n a r a d i o c h e m i c a l l y pure p r o t e i n was I s o l a t e d and shown t o be a b s e n t from t h e non-induced o r t r a n s p o r t - n e g a t i v e s t r a i n s . Proteins involved i n the binding of transport s u b s t r a t e s have now been i s o l a t e d f r o m a v a r i e t y of 12  sources  . Some s u c c e s s has been a c h i e v e d i n t h e study  of t r a n s p o r t p r o t e i n s by t h e i r r e l e a s e f o l l o w i n g o s m o t i c shock. I n some cases r e s t o r a t i o n o f an o s m o t i c a l l y shocked, and c o n s e q u e n t l y i m p a i r e d p o r t system has been o b t a i n e d  trans-  by t h e a d d i t i o n o f 14  crude and p u r i f i e d p r o t e i n s r e l e a s e d by shock t r e a t m e n t . R e g a r d i n g t h e t r a n s p o r t of p h e n o l i c compounds i n p l a n t s , t h e a u t h o r was a b l e t o f i n d o n l y a v e r y l i m i t e d 15  amount o f i n f o r m a t i o n i n t h e l i t e r a t u r e . Pridham s t u d i e d t h e phloem t r a n s l o c a t i o n o f s e v e r a l phenols and  t h e i r corresponding beta-glucosides  in Vlcia  faba.  The phenols were a p p l i e d t o t h e l e a v e s and t h e i r d i s t r i b u t i o n t h r o u g h o u t t h e p l a n t m o n i t o r e d by d i s s e c t i n g the p l a n t i n t o 1 cm p o r t i o n s , and chromatographing t h e methanolic e x t r a c t s . A l s o , the " a p h i d - s t y l e t " technique  -36-  was  15  employed u s i n g Macroslphum p l s l .  The  p h e n o l s were  r e a d i l y absorbed and t r a n s l o c a t e d , and e v i d e n c e of a more r a p i d t r a n s f e r of the g l u c o s i d e s t h a n the c o r r e s ponding a g l u c o n e s p r e s e n t e d .  Studies of  intestinal  t r a n s p o r t of monosaccharides by A l v a r a d o 16  and Crane  5  as  w e l l as by Landau e t a l . have shown t h a t s e v e r a l p h e n o l i c g l u c o s i d e s are a c t i v e l y t r a n s p o r t e d , s h a r i n g the same t r a n s p o r t s i t e as g l u c o s e . The  I n h i b i t i o n of 17  r e a b s o r b t i o n i n k i d n e y t u b u l e s of dog, 18  and i n t e s t i n e  and  glucose rat  by compounds such as a r b u t i n and  kidney salicin,  suggests t h a t these t i s s u e s too p o s s e s s a common s i t e f o r the t r a n s p o r t o f g l u c o s e and p h e n o l i c Lefevre  19  glucoside.  s u g g e s t s t h a t the human e r y t h r o c y t e membrane  p o s s e s s e s s i t e s which are c a p a b l e of b i n d i n g  phenolic  h y d r o x y l g r o u p s . However, p h e n o l , p h l o r o g l u c i n o l , p - m e t h y l p h e n o l , and m e t h y l p h l o r o g l u c i n o l were a l l found t o be r e l a t i v e l y i n a c t i v e as i n h i b i t o r s o f sugar t r a n s 5  port. Alvarado  found t h a t f r e e p h e n o l s were r e a d i l y  accumulated by i n t e s t i n a l t i s s u e and a c t u a l l y i n h i b i t e d the t r a n s p o r t o f sugars and p h e n y l g l u c o s l d e s . U n l i k e o f the g l u c o s i d e s , the form of the i n h i b i t i o n was  that found  not t o be c o m p e t i t i v e . Furthermore maximal r a t e s of e n t r y were not observed a t c o n c e n t r a t i o n s as h i g h as 50  mM.  The  passage o f s a l i g e n i n through the w a l l of r a t i l e u m  was  observed t o o c c u r a t r a t e s up t o t h r e e times f a s t e r 20  t h a n the c o r r e s p o n d i n g  glucoside, s a l i c i n .  The  mech-  anism of f r e e p h e n o l a c c u m u l a t i o n i n a n i m a l t i s s u e i s  -37not understood  5  but i t i s b e l i e v e d  21  t h a t such  llpid-  s o l u b l e s u b s t a n c e s d i s t r i b u t e themselves a c c o r d i n g t o t h e i r p a r t i t i o n c o e f f i c i e n t s between the aqueous  incub-  a t i o n medium and the l i p i d phases o f the c e l l s . P h e n o l i c g l y c o s i d e s such as a r b u t i n , however, l i k e sugars, are water s o l u b l e , l i p i d I n s o l u b l e s o l u t e s w i t h h i g h m o l e c u l a r w e i g h t s and a r e p o o r l y absorbed, i f a t a l l , by s i m p l e d i f f u s i o n mechanisms.  -38-  MATERIALS AND METHODS Plant material F e r n gametophytes were grown i n l i q u i d  culture  under a s e p t i c c o n d i t i o n s . Spores o f t h e f e r n Pltyrogramma  calomelanos were c o l l e c t e d by p l a c i n g  l e a v e s o f t h i s p l a n t , a d a x i a l s u r f a c e downward, on s h e e t s o f aluminum f o i l . Spores were s e p a r a t e d from e x t r a n e o u s s p o r a n g i a l d e b r i s by suspending 1 g o f t h e s p o r e s i n d i s t i l l e d w a t e r and w i t h d r a w i n g t h e s u s p e n s i o n by p i p e t t e . The d e b r i s f l o a t e d a t t h e s u r f a c e o f t h e w a t e r and was l e f t b e h i n d . The spore s u s p e n s i o n was filtered  and resuspended  i n a. 3% aqueous sodium hypo-  c h l o r i t e s o l u t i o n , w i t h c o n s t a n t s h a k i n g f o r t e n minu t e s . A l l subsequent  o p e r a t i o n s were conducted a t a  Troemner C l e a n A i r S t a t i o n w i t h p r e v i o u s l y a u t o c l a v e d equipment and s o l u t i o n s . The spore s u s p e n s i o n was filtered  i n a Biichner f u n n e l and washed w i t h 500 mlr» o f  d i s t i l l e d w a t e r . F o l l o w i n g t h i s t h e spores were r e s u s pended i n 100 ml,'* o f a n u t r i e n t s o l u t i o n c o n t a i n i n g z500 mg,» Ca(N03) , 250 mgs (NHj^SOij., 125 mgs MgSOj^, 2  125  nigs K HPOi4., 25 g o f s u c r o s e , 500 mg,s o f y e a s t 2  e x t r a c t , p l u s 1 ml o f a t r a c e element m i x t u r e c o n t a i n i n g 3 g MnS0^.4H 0, 500 mg* 2  H3BO3,  500 mgr. ZnSO^. 7 H 0 , 2  25 xngi CuSOip5H 0, 25 mg:* Na MoO^, p e r l i t r e , 2  2  dissolved  i n d i s t i l l e d w a t e r and made up t o a l i t r e . The s u s p e n s i o n was  s t i r r e d by means o f a magnetic  stirring  d e v i c e t o main-  t a i n t h e homogeneity o f m i x t u r e . One ml a l i q u o t s were  -39-  removed and i n t r o d u c e d i n t o 50 ml erlenmeyer f l a s k s c o n t a i n i n g h ml*; o f the same n u t r i e n t medium. The c o t t o n wool p l u g s were covered w i t h a l a y e r o f Saranwrap  t o p r e v e n t d r y i n g out o f the medium. The o  f l a s k s were m a i n t a i n e d a t 25 C i n a S h e r e r growth chamb e r under f l u o r e s c e n t l i g h t i n g (9000 l u x ) , s u b j e c t e d t o a r e p e a t i n g c y c l e o f 12 h r s l i g h t f o l l o w e d by 12 h r s o f d a r k n e s s f o r the p e r i o d o f t h e i r growth. Any contamina t i o n o f the c u l t u r e s became apparent w i t h i n 2 t o 3 days i n the form o f f u n g a l c o l o n i e s o r t u r b i d i t y o f the medium due t o b a c t e r i a l growth. Uncontaminated  cultures  became g r e e n w i t h i n 5 days. Growth proceeded by a s e r i e s of p a r a l l e l d i v i s i o n s to give a 5 or 6 c e l l e d  filament  a t about 10 d a y s . T h i s was f o l l o w e d by the onset o f a t w o - d i m e n s i o n a l growth p a t t e r n w h i c h gave r i s e t o the c h a r a c t e r i s t i c h e a r t shaped gametophyte The gametophytes  w i t h i n 3 weeks.  formed a mat o f t i s s u e on the bottom  o f the f l a s k w h i c h c o u l d be washed o r o t h e r w i s e manipu l a t e d w i t h o u t d i s t u r b i n g i t . P r i o r t o the b e g i n n i n g o f an uptake experiment the gametophytic mats were washed ( i n s i t u , i n the f l a s k s ) w i t h d i s t i l l e d w a t e r . The e x c e s s w a t e r was t h e n removed by i n v e r t i n g the f l a s k s and a l l o w i n g them t o d r a i n f o r 15 m i n u t e s . The use o f gametophytic t i s s u e f o r uptake s t u d i e s was t h o u g h t i i n i t i a l l y ,  t o r e p r e s e n t a good c h o i c e o f  p l a n t m a t e r i a l , s i n c e the organism i s a s i m p l e , p l a n a r body u n c o m p l i c a t e d by v a s c u l a r development  and f u r t h e r -  -40more, o b v i a t e d t h e n e c e s s i t y o f u s i n g e x c i s e d t i s s u e . However t h e r a t e o f growth was slow and more i m p o r t a n t , much v a r i a b i l i t y was found i n t h e e x p e r i m e n t a l  results.  Thus t h e f e r n t i s s u e was r e p l a c e d by b a r l e y r o o t s . B a r l e y seeds, ( v a r i e t y Conquest) were g e r m i n a t e d 22  a c c o r d i n g t o t h e method d e s c r i b e d by E p s t e i n  . Forty  gm.; o f seeds were a l l o w e d t o s t a n d f o r 10 minutes i n a 3% sodium h y p o c h l o r i t e s o l u t i o n . The h y p o c h l o r i t e s o l u t i o n was poured o f f and t h e seeds a e r a t e d f o r 18 h r s i n 200 ml*' o f d i s t i l l e d w a t e r . The seeds were then p l a c e d upon l a y e r s o f c h e e s e - c l o t h s t r e t c h e d a c r o s s t h e t o p s o f p l a s t i c boxes ( o f t h e s o r t used as food c o n t a i n e r s i n r e f r i g e r a t o r s ) . The c o n t a i n e r s were f i l l e d w i t h d i s t i l l e d w a t e r and t h e ends o f t h e c h e e s e - c l o t h  allowed  t o d i p i n t o t h e w a t e r . The seeds were c o v e r e d w i t h a l a y e r o f c h e e s e - c l o t h and a e r a t e d f o r 4 days i n d a r k n e s s . By 4 days t h e r o o t s had grown down i n t o t h e w a t e r t o a l e n g t h o f about 4 t o 5 cm . The r o o t s were c u t o f f a t a l e v e l o f 1 cm below t h e c h e e s e - c l o t h and immersed i n 500 m l , o f d i s t i l l e d w a t e r . T h i s t r e a t m e n t  served t o mix  the r o o t s t h o r o u g h l y . The r o o t s were removed from t h e w a t e r and spun d r y ( b y hand) i n a square o f gauze w h i c h was drawn t o g e t h e r t o form a pouch. The r o o t s were t h e n weighed out i n 1 g p o r t i o n s , a l t h o u g h f o r c e r t a i n  exper-  iments 0 . 5 o r even 2 g q u a n t i t i e s were used. The r o o t samples were p l a c e d on l a y e r s o f gauze a p p r o x i m a t e l y  7 cm,*  square and t h e c o r n e r s o f t h e gauze drawn t o g e t h e r and s t a p l e d t o form a bag. A p i e c e o f c o t t o n was t i e d t o  -41each bag so t h a t i t c o u l d be e a s i l y m a n i p u l a t e d . The bags were p l a c e d i n a e r a t e d h o l d i n g - w a t e r u n t i l  suff-  i c i e n t number o f bags had been p r e p a r e d . The bags were then removed from t h e w a t e r and spun d r y (by hand) i n r e a d i n e s s f o r t h e a c t u a l uptake  period.  A d m i n i s t r a t i o n of c h e m i c a l s . The r a d i o a c t i v e c h e m i c a l s were d i s s o l v e d i n g l a s s d i s t i l l e d w a t e r (pH 6 . 3 - 6 . 5 ) I n a l l experiments except those s p e c i f i c a l l y examining the i n f l u e n c e of pH. I n t h e l a t t e r experiments t h e r a d i o a c t i v e c h e m i c a l s were d i s s o l v e d In c i t r a t e / p h o s p h a t e b u f f e r e d s o l u t i o n s . I n most e x p e r i ments t h e t r e a t m e n t s o l u t i o n (20 ml.) was c o n t a i n e d i n a 30 m l . beaker. However, experiments i n v o l v i n g g a s s i n g w i t h N2 were c o n t a i n e d i n 50 m l . e r l e n m e y e r f l a s k s . I n the  e a r l i e r experiments u s i n g f e r n t i s s u e the c h e m i c a l s  ( i n s o l u t i o n ) were added t o t h e gametophytic mats i n the f l a s k s . I n a l l experiments two r e p l i c a t e t r e a t m e n t s were p r e p a r e d . D u r i n g t h e course of the uptake p e r i o d the r a d i o a c t i v e s o l u t i o n s were a e r a t e d c o n t i n u o u s l y . D e t e r m i n a t i o n of u p t a k e . Uptake was determined by c o u n t i n g an a l i q u o t of an e t h a n o l i c e x t r a c t of the p l a n t m a t e r i a l p r e p a r e d a t the end of t h e uptake p e r i o d . F o r t h e l a t t e r procedure the p l a n t m a t e r i a l was removed from the r a d i o a c t i v e and i m m e d i a t e l y r i n s e d i n 1 l i t r e of d i s t i l l e d  solution water.  The samples were t h e n a l l o w e d t o s t a n d f o r two p e r i o d s  -42of 15 minutes each l n l i t r e s o f d i s t i l l e d w a t e r . T h i s t r e a t m e n t s e r v e d t o remove any r a d i o a c t i v e m a t e r i a l which adhered t o t h e r o o t s u r f a c e o r was p r e s e n t  i n the  c e l l f r e e space. The p l a n t t i s s u e was then e x t r a c t e d w i t h b o i l i n g 80$ e t h a n o l  (100 ml.) f o r 20 m i n u t e s .  The r o o t e x t r a c t s were c o n c e n t r a t e d a r o t a r y evaporator  t o a s m a l l volume on  and t h e n made up t o 10 m l . i n a  v o l u m e t r i c f l a s k . The f e r n e x t r a c t s were e v a p o r a t e d t o d r y n e s s and then e x t r a c t e d w i t h b o i l i n g w a t e r , w h i c h s e r v e d t o remove t h e c h l o r o p h y l l . The aqueous e x t r a c t s ?? were f i l t e r e d t h r o u g h C e l l t e and t h e n c o n c e n t r a t e d  to a  volume o f 10 m l . a s d e s c r i b e d above. One m l . a l i q u o t s , from each 10 m l . s o l u t i o n , were t a k e n and counted i n 10 m l . of a dloxane-based s c i n t i l l a t i o n f l u i d , i n a Nuclear  Chicago s c i n t i l l a t i o n c o u n t e r .  Of the r a d i o -  a c t i v i t y t a k e n up by t h e p l a n t t i s s u e 90$ was r e c o v e r a b l e i n t h e e t h a n o l - s o l u b l e f r a c t i o n . A l l uptake  values  were c o r r e c t e d a c c o r d i n g l y . Radioactive -T-  chemicals "  C l a b e l l e d hydroquinone was purchased f r o m Schwarz B i o r e s e a r c h  Inc. S o l u t i o n s of the appropriate  m o l a r i t y were p r e p a r e d w i t h u n l a b e l l e d hydroquinone and t h e n a s m a l l a l i q u o t of t h e e t h a n o l i c s o l u t i o n of 14 C hydroquinone was added t o b r i n g t h e s p e c i f i c a c t i v i t y t o approximately and  5jiC/mmole. T r i t i a t e d hydroquinone  c a t e c h o l were p r e p a r e d by t r i t i a t i o n of t h e r i n g  p r o t o n p o s i t i o n s o f hydroquinone and c a t e c h o l by r e f l u x ing  5 mmole of t h e phenol i n 27.5 ml* of t r i t i a t e d  -43t r i f l u o r a e e t i c a c i d ( p r e p a r e d from  3  H2O and t r i f l u o r -  a c e t i c a n h y d r i d e ) w i t h 10 mg. o f Pd on c h a r c o a l f o r 48  29  hours • L a b i l e t r i t i u m was removed by s u c c e s s i v e s o l u t i o n and e v a p o r a t i o n and t h e phenols were f i n a l l y c r y s t a l l i z e d from e t h a n o l . The s p e c i f i c a c t i v i t i e s of the phenols thus o b t a i n e d were 44/iC/mmole f o r hydroquinone and 31 /iC/mmole f o r c a t e c h o l . The s p e c i f i c  activities  of a l l compounds as a d m i n i s t e r e d i n s p e c i f i c e x p e r i m e n t s a r e shown I n the a p p r o p r i a t e t a b l e s l i s t i n g t h e r e s u l t s of those e x p e r i m e n t s .  The p r e p a r a t i o n of the b e t a - D - g l u c o  s i d e s of hydroquinone and c a t e c h o l was a c h i e v e d  by making  use of t h e c a p a c i t y of h i g h e r p l a n t s t o g l u c o s y l a t e a d m i n i s t e r e d p h e n o l s . Thus t h e t r i t i a t e d phenols were s e p a r a t e l y f e d t o shoots of Zea mays ( v i a the c u t e n d s ) . A f t e r 2 days the p l a n t s were e x t r a c t e d i n b o i l i n g e t h a n o l . The e t h a n o l i c e x t r a c t s were evaporated ness, e x t r a c t e d w i t h b o i l i n g water, and f i l t e r e d  80$  t o drythrough  C e l i t e . The c o n c e n t r a t e d aqueous e x t r a c t s were a p p l i e d , i n t h e f o r m o f bands, t o s h e e t s of Whatman No 3 chromatography paper, w h i c h were d e v e l o p e d i n t - b u t a n o l t a c e t i c acidswater  ( 3 « 1 » 1 v / v ) . The g l u c o s i d e s were e l u t e d  from t h e papers w i t h w a t e r and then r e a p p l i e d t o f r e s h sheets of Whatman No 3 paper. By t h i s method t h e g l u c o s i d e s were p u r i f i e d t h r o u g h t h r e e more s o l v e n t systems, namely, b u t a n o l 1 p y r i d i n e : w a t e r ( 6 I 4 I 3 v / v ) , 80$ i s 6 p r o p a n o l , and butanol»ethanol1water  (40illil9 v/v).  -44-  The  p u r i f i e d compounds were found t o have i d e n t i c a l  u l t r a v i o l e t s p e c t r a t o a u t h e n t i c samples. Q u a n t i t i e s of u n l a b e l l e d catechol beta-D-glucoside,  r e q u i r e d f o r the  d i l u t i o n of the t r i t i a t e d g l u c o s i d e , were o b t a i n e d G a u l t h e r i a a d e n o t h r l x . P u r i f i c a t i o n of the was  from  glucoside  a c h i e v e d by paper chromatography as d e s c r i b e d above.  Hydroquinone b e t a - D - g l u c o s i d e  ( a r b u t i n ) a n d a l l o t h e r chem-  i c a l s employed i n t h i s study were c o m m e r c i a l l y  available.  -45-  RESULTS AND  DISCUSSION  The uptake of p h e n o l i c m o l e c u l e s by p l a n t t i s s u e s i s of i n t e r e s t from two p o i n t s of v i e w . F i r s t l y ,  in a  g e n e r a l sense our knowledge of t r a n s p o r t phenomena i s extended t o secondary m e t a b o l i t e s , w h i c h have l a r g e l y been i g n o r e d i n t r a n s p o r t s t u d i e s . S e c o n d l y , the uptake of  these m o l e c u l e s , f a r from b e i n g an a r t i f i c i a l  lab-  o r a t o r y a r t i f a c t , r e p r e s e n t the v e r y r e a l s i t u a t i o n i n which p l a n t s a r e p l a c e d by v i r t u e of the i n t r o d u c t i o n of  p h e n o l i c s I n t o the s o i l by the death and decay of  p l a n t m a t e r i a l . S i n c e many of these compounds a r e known to have p o t e n t p h y t o t o x i c p r o p e r t i e s , the e c o l o g i c a l s i g n i f i c a n c e of t h e i r presence may be c o n s i d e r a b l e . ^ ; Thus some s p e c i e s of G a u l t h e r i a (which produce q u a n t i t i e s of c a t e c h o l g l u c o s i d e ) may measure of advantage  large  d e r i v e some l a r g e  over competing s p e c i e s due t o the  presence of t h i s compound and the more t o x i c a g l u c o n e , c a t e c h o l , i n the  soil.  Experiments on the uptake of f r e e phenols Fig  l a shows the uptake of hydroquinone from the  e x t e r n a l medium as a f u n c t i o n of t i m e . C l e a r l y hydroquinone e n t e r s the r o o t t i s s u e v e r y r e a d i l y ( 0 . 2 7 5 mg./hr/g. f r e s h w e i g h t of r o o t ) . These o b s e r v a t i o n s concur w i t h 2,20  those on a n i m a l t i s s u e s  . In the study by A l v a r a d o and  5  Crane, o r c i n o l a c c u m u l a t i o n i n hamster i n t e s t i n e o c c u r r e d r e a d i l y and e q u i l i b r a t i o n between the t i s s u e and the i n c u b a t i o n medium o c c u r r e d w i t h i n 30 m i n u t e s . I n the p l a n t  -46t i s s u e , however, the f a c i l e c o n v e r s i o n to i t s corresponding  of hydroquinone  g l u c o s i d e might serve t o m a i n t a i n  a concentration gradient, favourable  t o continued  dif-  f u s i o n . Chromatography of t h e e t h a n o l i c e x t r a c t of t h e r o o t t i s s u e showed t h a t 100$ o f the r a d i o a c t i v i t y  present  was I n t h e form o f a r b u t i n . The i n s e t ( P i g l b ) shows the e f f e c t s o f low temperature ( i c e b a t h 1-3°C), N , and 2  2,4-dinitrophenol  on the uptake o f hydroquinone from the  medium. W h i l s t t h e t r e a t m e n t s  can be seen t o reduce the  r a t e o f uptake, t h e r e d u c t i o n i s s m a l l . The i n i t i a l r a t e s were reduced by 30$, 44$, and 21$ r e s p e c t i v e l y . The uptake of a r b u t i n was reduced by ?4#, 59%, and 75%  22 r e s p e c t i v e l y by t h e same t r e a t m e n t s .  Epstein  obtained  a 75% r e d u c t i o n o f Na+ uptake by b a r l e y .roots, when the i n c u b a t i o n medium was bubbled w i t h N  2  i n place of a i r .  Comparisons o f t h e r a d i o a c t i v i t i e s p r e s e n t  In the eth-  a n o l e x t r a c t s of the r o o t t i s s u e , and the r a d i o a c t i v i t i e s l o s t from the e x t e r n a l s o l u t i o n s i n t h e same t i m e ,  showed  t h a t i n t h e c o n t r o l 72% o f t h e uptake was r e c o v e r e d i n the e t h a n o l i c e x t r a c t , w h i l s t 50%, 18%, and 22%, r e s p e c t i v e l y were r e c o v e r e d  from t h e above mentioned t r e a t m e n t  e x t r a c t s . The d i f f e r e n c e s between uptake as determined by l o s s from t h e medium and by e t h a n o l i c e x t r a c t i o n of the r o o t s c a n be a t t r i b u t e d , i n p a r t , t o a f r e e space component which i s l o s t d u r i n g d e s o r p t i o n . However, t h i s component s h o u l d be e q u i v a l e n t r e g a r d l e s s o f t h e t r e a t ment. I f , however, the uptake o c c u r r e d by d i f f u s i o n , then  -47under c o n d i t i o n s of a r r e s t e d metabolism a l a r g e propo r t i o n of the phenol would remain i n the f r e e f o r m . During desorptlon desorption observation  t h i s c o u l d be l o s t by d i f f u s i o n t o  s o l u t i o n . This explanation  the  i s s u p p o r t e d by  t h a t the e t h a n o l i c e x t r a c t s c o n t a i n e d  no  the free  p h e n o l s . F i g 2a summarizes the e f f e c t of e x t e r n a l concent r a t i o n of hydroquinone upon uptake by b a r l e y r o o t s . T h i s form of s a t u r a t i o n can be c h a r a c t e r i s t i c of a c t i v e t r a n s p o r t systems. However, i n the case of p h e n o l s , w h i c h a r e so r e a d i l y g l u c o s y l a t e d , t h e r e i s a n o t h e r r e a s o n a b l e e x p l a n a t i o n . The  v e r y r a p i d r a t e of e n t r y of the  phenol  c o u l d produce an i n t e r n a l c o n c e n t r a t i o n a p p r o a c h i n g t h a t of the e x t e r n a l c o n c e n t r a t i o n as a l i m i t . A t  this  concentration„the g l u c o s y l a t i o n c a p a c i t y of the t i s s u e w e l l be s a t u r a t e d , and  represent  may  the l i m i t i n g f a c t o r  f o r f u r t h e r u p t a k e . I f the d a t a of F i g 2a a r e redrawn i n the form of a double r e c i p r o c a l p l o t , then a K 1.7  x 10~^  M i s obtained.  value  m  of  I n a study of the b i o s y n t h e s i s 24  of p h e n o l i c g l u c o s i d e s , Yamaha and C a r d l n i  isolated,  from wheat germ, an enzyme w h i c h t r a n s f e r r e d the  glucose  moiety of u r i d i n e d i p h o s p h a t e - g l u c o s e t o hydroquinone, f o r m i n g a r b u t i n . The M. The  K  m  v a l u e f o r t h i s enzyme was  i n s e t ( F i g 2b) r e p r e s e n t s  2 x  10-3  the r e s u l t s f r o m the  same type of experiment u s i n g the f e r n gametophyte t i s s u e . The  form of the p l o t i s f a i r l y t y p i c a l of sub-  s t r a t e - i n h i b i t e d enzyme r e a c t i o n s . I t may higher concentrations  be t h a t a t  the p h e n o l i s c a p a b l e of  inhibiting  -48e i t h e r t h e g l u c o s y l a t i o n r e a c t i o n , o r r e a c t i o n s upon which g l u c o s y l a t i o n depends. F i g 3b i l l u s t r a t e s t h e e f f e c t o f t h r e e  simple  p h e n o l s , r e s o r c i n o l , c a t e c h o l , and p h l o r o g l u c i n o l on the uptake o f hydroquinone. The p l o t i s i n t h e form o f a double r e c i p r o c a l p l o t where V r e p r e s e n t s uptake and S represents  t h e c o n c e n t r a t i o n o f t h e s u b s t r a t e , hydro-  quinone. The i n h i b i t i n g p h e n o l s a r e m a i n t a i n e d  a t 2.5mM.  The i n h i b i t i o n I s n o t c o m p e t i t i v e . The e f f e c t o f v a r i o u s p h e n o l s upon t h e t r a n s p o r t o f sugars and p h e n o l i c 5 g l u c o s i d e s was i n v e s t i g a t e d by A l v a r a d o and found t o be o f t h e same form as observed h e r e . C o n s i d e r i n g the 25 a f f i n i t y of phenols f o r binding t o p r o t e i n s , i t i s not s u p r i s i n g t o f i n d t h a t m e t a b o l i c a l l y dependent p r o c e s s es a r e i n h i b i t e d i n t h i s way. Presumably t h e I n h i b i t i o n c o u l d o c c u r by a n o n - s e l e c t i v e b i n d i n g ' t o  transport  p r o t e i n s o r t o enzymes i n v o l v e d i n t h e m e t a b o l i c 26 component o f t r a n s p o r t . S t e n l i d  has shown t h a t t h e  a g l y c o n e s o f c e r t a i n f l a v o n o i d s i n h i b i t the a b s o r b t i o n of sugars by e x c i s e d wheat r o o t s . The  corresponding  g l y c o s i d e s were found t o be l e s s a c t i v e . These same a g l y c o n e s were found t o i n h i b i t o x i d a t i v e  phosphorylation  and hence t h e e f f e c t upon uptake may be mediated v i a t h i s d i r e c t i n h i b i t i o n o f energy p r o v i d i n g r e a c t i o n s . I n o r d e r t o examine t h e q u e s t i o n o f a n o n - s e l e c t i v e 86 i n h i b i t i o n o f t r a n s p o r t t h e uptake o f  Rb was i n v e s t -  i g a t e d i n t h e presence o f v a r i o u s c o n c e n t r a t i o n s o f  -49-  hydroquinone. The a b s o r p t i o n of t h i s i o n by b a r l e y r o o t s has been shown by E p s t e i n  22  t o be a m e t a b o l i c a l l y depend-  ent p r o c e s s . F i g 4b shows the e f f e c t of d i f f e r e n t concen86 t r a t i o n s of hydroquinone upon the uptake of  Rb from 5 mM  s o l u t i o n s . I t would appear t h a t hydroquinone i n t e r f e r e s , i n a n o n - s e l e c t i v e f a s h i o n , w i t h uptake  processes.  The i n f l u e n c e of e x t e r n a l pH upon i n t a c t c e l l  activ-  i t i e s has been used t o l o c a l i z e t h a t a c t i v i t y a t t h e c e l l 27  s u r f a c e . Thus W i l k e s and Parmer  showed t h a t t h e pH  e f f e c t upon i n v e r t a s e a c t i v i t y of the y e a s t c e l l  was  i d e n t i c a l w i t h t h a t upon the i s o l a t e d enzyme. They conc l u d e d t h a t t h e enzyme was l o c a t e d a t the c e l l  surface.  The e f f e c t of pH upon hydroquinone uptake i s shown i n F i g 4a. The e f f e c t i s n o t t y p i c a l of enzyme-mediated p r o c e s s e s . The s l o p e of the l i n e p r o b a b l y  r e f l e c t s the d i m i n i s h i n g  a v a i l a b i l i t y of t h i s compound w i t h i n c r e a s i n g pH. Phenols r e a d i l y o x i d i z e and p o l y m e r i z e  a t a l k a l i n e pH, and i n the  case of hydroquinone t h e e x t e r n a l s o l u t i o n was observed t o become brown d u r i n g the course of t h e e x p e r i m e n t . The e n t r y o f c a t e c h o l i n t o b a r l e y r o o t s was found t o o c c u r r e a d i l y , a l t h o u g h n o t as r a p i d l y as the e n t r y of hydroquinone. Table 7a c o n t a i n s the d a t a f o r the uptake 3  of  l a b e l l e d c a t e c h o l by b a r l e y r o o t s . The r a t e of uptake  was d e t e r m i n e d t o be 0.161  mg/hr/g. f r e s h weight of r o o t s .  Chromatography o f t h e e t h a n o l i c e x t r a c t s of the r o o t t i s s u e a f t e r a b s o r p t i o n showed t h a t a l l the p h e n o l was present  i n t h e f o r m of the  beta-D-glucoside.  -50-  I t would appear h i g h l y l i k e l y t h a t the e n t r y of f r e e p h e n o l s , such as hydroquinone, o c c u r s by a p r o c e s s of p a s s i v e d i f f u s i o n , w h i c h i s m a i n t a i n e d by the t r a p p i n g of absorbed phenol t h r o u g h g l u c o s y l a t i o n . T h i s h y p o t h e s i s i s based upon the o b s e r v a t i o n s t h a t 1.  :-  phenols a r e r e a d i l y g l u c o s y l a t e d by p l a n t t i s s u e s , thus m a i n t a i n i n g c o n c e n t r a t i o n g r a d i e n t s f a v o u r a b l e f o r continued d i f f u s i o n ,  2 . the K  m  f o r uptake of hydroquinone and f o r i t s g l u c o -  s y l a t i o n are extremely close, 3 . 100$ of the r a d i o a c t i v i t y r e c o v e r e d i n the e t h a n o l i c e x t r a c t was i n the form of the g l u c o s i d e of the absorbed phenol, 4 . under c o n d i t i o n s of a r r e s t e d metabolism t h e r e was a f a i r l y l a r g e l o s s of absorbed p h e n o l d u i n g the desorption period, 5 . pH e f f e c t s a r e a t y p i c a l of t r a n s p o r t p r o c e s s e s , and 6.  t h e r e was no i n d i c a t i o n of a c o m p e t i t i v e type of i n h i b i t i o n between r e l a t e d phenols f o r u p t a k e , r a t h e r the i n h i b i t i o n resembled the u n c o m p e t i t i v e type of Inhibition.  -51-  TABLE 1 14 The uptake o f  C l a b e l l e d hydroquinone (3*6 ^iC/mmole)  by b a r l e y r o o t s (2 g. samples) a s a f u n c t i o n o f t i m e . Hydroquinone c o n c e n t r a t i o n was 5 TIME  TOTAL RADIOACTIVITY OF INCUBATION MEDIUM  0 mins  a. 8.136  15 mins  30 mins  d.p.m.  0  d.p.m.  b. 8 . 0 9 0 X 10* d.p.m.  0  d.p.m.  X 10*  120 mins  180 mins  d.p.m.  a. 7 . 1 3 0 X 1 0  5  d.p.m.  1.006  b. 7.180 X 1 0  5  d.p.m.  0 . 9 0 9 X 10*  d.p.m.  1.042 X 1 0  5  d.p.m.  d.p.m.  1.112  X 10  5  d.p.m.  a. 7 . 0 9 4 X 10^ d.p.m. b. 6 . 9 7 8 X  60 mins  UPTAKE OP RADIOACTIVITY FROM MEDIUM  io  5  X 10  5  a. 6 . 9 0 9 X 1 0  5  d.p.m.  1.227  X 10  5  d.p.m.  b. 6.782 X 1 0  5  d.p.m.  1.308  X 10  5  d.p.m.  a. 6 . 4 9 9 X 1 0  5  d.p.m.  1.637 X 1 0  5  d.p.m.  b. 6 . 3 8 2 X 1 0  5  d.p.m.  1.708 X 1 0  5  d.p.m.  a. 6 . 0 8 2 X 1 0  5  d.p.m.  2 . 0 5 4 X 10^  b. 5 . 9 1 6 X 1 0  5  d.p.m.  2.173  X 10  5  d.p.m. d.p.m.  * I n t h i s t a b l e , and a l l o t h e r s t h a t f o l l o w i n t h i s s e c t i o n , a and b r e f e r t o r e p l i c a t e t r e a t m e n t s . In a l l graphs t h e mean o f these two v a l u e s i s p l o t t e d .  -52-  TABLE 2 14 The u p t a k e o f  C l a b e l l e d hydroquinone (3»6 /iC/mmole)  by b a r l e y r o o t s  (2 g. samples) a s a f u n c t i o n o f t i m e , a s  i n f l u e n c e d by low t e m p e r a t u r e , N 2,4-dinitrophenol  1 5 mins  30 mins  60 mins  *  atmosphere, and 10"^ M  (DNP)  CONTROL ( 28°C)  1 C  N  a.  0  0  0  0  b.  0  0  0  0  a.  1.006  0.687  0.507  0.855  b.  0.909  0.646  0.625  0.760  a.  1.042  0.749  0.514  0.976  b.  1.112  0.837  0.702  0.816  a.  1.227  0.866  0.758  0.923  b.  1.308  0.897  0.654  1.087  TIME 0 mins  2  G  A l l v a l u e s must be m u l t i p l i e d by 1 0  5  d.p.m.  2  DNP  -5314 F i g l a t The uptake o f function Fig l b (inset)  C l a b e l l e d hydroquinone as a  o f t i m e , by b a r l e y  roots.  s The e f f e c t o f low temperature  N  and 2 , 4 - d i n i t r o p h e n o l -•- upon t h e uptake o f hydroquinone. ( c o n t r o l -•-)  •  1  2  Time in hours  3  2  -o  -54-  TABLE 3 3  The uptake o f H l a b e l l e d hydroquinone (6 jiC/mmole), over a p e r i o d of t h r e e h o u r s , a s a f u n c t i o n o f e x t e r n a l c o n c e n t r a t i o n , by b a r l e y r o o t s (1 g. samples). . EXTERNAL CONCENTRATION (mM)  UPTAKE x 10"^ d.p.m. a. b.  1.0  7.05  7.15  1-5  8.39  8.59  2.0  9.84  9.50  2.5  10.86  10.73  5.0  14.08  12.62  7.5  16.04  12.29  10.0  16.19  15.49  15.0  13.62  15.35  20.0  13.83  14,59  TABLE 4 3  The uptake of H l a b e l l e d hydroquinone (44 /aC/mmole), over a p e r i o d o f t h r e e h o u r s , a s a f u n c t i o n of e x t e r n a l c o n c e n t r a t i o n , by Pltyrogramma EXTERNAL CONCENTRATION (mM)  UPTAKE x 1Q--5 d.p.m. a. b.  1.0  6.06  5.66  2.5  7.63  9.82  5.0  12.90  19.60  10.0  12.30  16.90  15.0  5.80  9.90  20.0  3.30  4.30  -553 F i g 2a : The uptake o f H l a b e l l e d hydroquinone a s a function of external concentration,  by b a r l e y  roots. 3 F i g 2b ( i n s e t ) s The uptake o f H l a b e l l e d hydroquinone as a f u n c t i o n o f e x t e r n a l c o n c e n t r a t i o n , by Pityrogramma  gametophytes.  E  a •a co  ra a. 3  5 10 15 20 Hydroquinone concentration  10 Hydroquinone  15  concentration  20 (mM)  (mM)  -56-  TABLE 5 14 The uptake o f  C l a b e l l e d hydroquinone ( 1 . 5 uC/mmole)  by b a r l e y r o o t s ( 0 . 5 g. samples),  over a p e r i o d o f t h r e e  hours, a s i n f l u e n c e d by t h e presence of some s i m p l e phenols,  maintained  a t a c o n c e n t r a t i o n o f 2 . 5 mM  HYDROQUINONE CONCENTRATION  1 . 2 5 mM  UPTAKE x 10  d.p.m.  1. CONTROL  2. + CATECHOL  a. 1.197 b. 1.061  a. 0.903 b. 0.872  2.50  mM  a. 1.362 b. 1 . 4 7 5  a. 1.076 b. 1.157  5.00  mM  a. 1.570 b. 1.658  a. 1.116 b. 1.233  3.  + RES0RCINOL 4 . + PHLOROGLUCINOL  1.25 mM  a. 0 . 7 3 3 b. 0 . 7 8 3  a. 1.087 b. 0 . 9 9 9  2.50  mM  a. 0 . 9 3 5 b. 0.891  a. 1.144 b. 1.173  5.00  mM  a. 1 . 4 5 0 b. 1.316  a. 1.391 b. 1.339  -57-  F i g 3a  i The d a t a of F i g 2a a r e shown i n the form of a double r e c i p r o c a l  m  plot.  o  .5  1.  5.  .2  F i g 3b  i The e f f e c t of c a t e c h o l  .4  .8  r e s o r c i n o l - o - , and  p h l o r o g l u c i n o l - A - on the uptake of hydroquinone  -58-  TABLE 6 14 C l a b e l l e d hydroquinone ( 5 « 0 /aC/mmole) by  The uptake o f  b a r l e y r o o t s (1 g. s a m p l e s ) , a s i n f l u e n c e d by t h e pH of the medium. Hydroquinone c o n c e n t r a t i o n was 5 mM. pH OF MEDIUM  UPTAKE OF HYDROQUINONE x 10*"^ d.p.m. b.  1.782  a. 1.397  b.  1.639  5  a. 1.243  b.  1.309  6  a.  1.386  b.  1.463  7  a.  1.053  b.  1.190  8  a.  0.888  b.  0.660  3  a.  4  1.628  TABLE 7 86  The uptake o f  Rb ( 0 . 0 5 /iC/mmole), by b a r l e y r o o t s ' (1 g.  samples) a s i n f l u e n c e d by t h e presence of h y d r o q u i n o n e . Rubidium c o n c e n t r a t i o n was 5mM. CONCENTRATION OF HYDROQUINONE (mM)  UPTAKE OF R b x 1 0 ~ 3 d.p.m. 8 o  1.25  a.  5.535  b.  6.269  2.50  a.  4.338  b.  4.981  5.00  a.  3.773  b.  3.669  10.00  a.  3.380  b.  3.730  15.00  a.  3.607  b.  3.327  20.00  a.  3.357  b.  3.732  -59-  F i g 4a J The e f f e c t o f pH upon hydroquinone uptake  3  4  5  6  7  8  PH  5  10  Hydroquinone  20 concentration  (mM)  F i g 4b i The e f f e c t o f hydroquinone upon  86  Rb uptake  -59a-  TABLE 7a 3  The uptake of H l a b e l l e d c a t e c h o l (31 /iC/mmole) byb a r l e y r o o t s (2 g. samples) over a t h r e e hour p e r i o d . C a t e c h o l c o n c e n t r a t i o n was 5 mM. TIME  UPTAKE  3 hours  a . 5»07 x 10  U  d.p.m.  b. 5 . 7 3 x 10^ d.p.m.  -60-  E x p e r i m e n t s on t h e uptake o f p h e n o l i c  fflucosld.es  P i g 5 shows t h e uptake o f a r b u t i n a s a f u n c t i o n o f t i m e . The r a t e o f uptake ( 0 . 1 6 0 mg./hr/g. r o o t s ) was found t o be a p p r o x i m a t e l y one f o u r t h t h a t o f hydroquinone 20  on a molar b a s i s . A d a m k i e v i c z  found t h a t s a l i g e n i n  e n t e r e d t h e r a t i l e u m a t a r a t e w h i c h was t h r e e times f a s t e r than the glucoside  salicin.  F i g s 6a and 6b i l l u s t r a t e t h e e f f e c t of e x t e r n a l c o n c e n t r a t i o n on t h e uptake o f a r b u t i n by b a r l e y r o o t s and f e r n gametophytes, r e s p e c t i v e l y . The K v a l u e s d e t e r m i n e d from t h e double r e c i p r o c a l p l o t s were 5mM. m  5  Alvarado  o b t a i n e d v a l u e s r a n g i n g from 2 - 3 . 6 mM f o r t h e  t r a n s p o r t o f t h i s compound by i n t e s t i n a l t i s s u e . I t i s q u i t e remarkable t o f i n d such s i m i l a r i t i e s i n t h e p r o p e r t i e s of such d i f f e r e n t  tissues.  Table 11 r e c o r d s t h e e f f e c t o f some m e t a b o l i c i n h i b i t o r s on t h e uptake o f a r b u t i n by b a r l e y r o o t s . DNP i n c o n c e n t r a t i o n s r a n g i n g from 10"*^ M t o 10"^ M caused i n h i b i t i o n s r a n g i n g from 4 t o 7 8 $ . A n a e r o b i c c o n d i t i o n s (a N  2  atmosphere) reduced t h e uptake by  59$ o f t h e c o n t r o l . Low temperature t r e a t m e n t , by means of an i c e - w a t e r b a t h ( 1 - 3 ° C ) , reduced t h e uptake by 7 4 $ . Such l a r g e r e d u c t i o n s c l e a r l y i n d i c a t e t h e e x t e n t of t h e m e t a b o l i c dependence of u p t a k e . U n l i k e t h e e n t r y o f hydroquinone, t h e uptake of a r b u t i n was i n h i b i t e d i n a c o m p e t i t i v e f a s h i o n by the presence o f a p o t e n t i a l t r a n s p o r t a n a l o g u e . Thus  -61-  o a t e c h o l g l u c o s i d e would appear t o share t h e same t r a n s p o r t s i t e as a r b u t i n . Hydroquinone, i n c h a r a c t e r i s t i c fashion,reduces  t h e uptake o f a r b u t i n i n an uncompet-  i t i v e manner. T h i s i s i d e n t i c a l t o t h e e f f e c t o f f r e e p h e n o l s upon t h e t r a n s p o r t o f g l u c o s i d e s by i n t e s t i n a l 5  t i s s u e . I n a n i m a l t i s s u e g l u c o s e t r a n s p o r t has been shown t o be c o m p e t i t i v e l y i n h i b i t e d by s e v e r a l  phenolic  5  glucosides  . The same a u t h o r s have shown t h a t a r b u t i n  t r a n s p o r t i s c o m p e t i t i v e l y i n h i b i t e d by g l u c o s e . The p r e s e n t s t u d i e s i n d i c a t e t h a t i n p l a n t t i s s u e s t h e p h e n o l i c g l u c o s i d e and g l u c o s e do n o t share a common t r a n s p o r t s i t e . I n F i g 7b, i t c a n be seen t h a t t h e uptake o f a r b u t i n i s n o t i n f l u e n c e d a t a l l by t h e p r e s 26  ence o f g l u c o s e .  Stenlid  showed t h a t p h l o r l d z i n ,  w h i c h i s renowned f o r i t s i n h i b i t i o n o f i n t e s t i n a l sugar t r a n s p o r t , has no such e f f e c t i n p l a n t t i s s u e s . F i g 8 i l l u s t r a t e s t h e e f f e c t o f pH upon t h e uptake o f a r b u t i n . The form o f t h e curve i s f a i r l y t y p i c a l o f t h a t t o be observed f o r many k i n d s o f e n z y m a t i c r e a c t i o n . The pH optimum f o r uptake i s 5«0« I t c a n be seen t h a t t h e c h a r a c t e r i s t i c s o f t h e p h e n o l i c g l u c o s i d e t r a n s p o r t systems i n t h e p l a n t s s t u d i e d , p o s s e s s s e v e r a l f e a t u r e s i n common w i t h t h e c o r r e s p o n d i n g systems i n a n i m a l t i s s u e . T r a n s p o r t i s a phenomenon o f v i t a l importance t o l i v i n g organisms, and i f one c o n s i d e r s  t h e s o r t o f environment i n w h i c h t h e  f i r s t organisms a r e thought t o have e v o l v e d ,  i . e . , the  -62primaeval  soup r e f e r r e d t o by O p a r i n  28  , then, c l e a r l y ,  those organisms w h i c h possessed the a b i l i t y t o concent r a t e compounds of v i t a l importance t o t h e i r m e t a b o l i s m would be a t a d i s t i n c t advantage over those l a c k i n g the a b i l i t y . The may  o r i g i n a l t r a n s p o r t s i t e f o r sugars  have been s u f f i c i e n t l y u n s p e c i f i c t o a c c e p t sugar  d e r i v a t i v e s such as a r b u t i n . Thus the s i t u a t i o n i n a n i m a l t i s s u e s appears t o be of t h i s t y p e .  Duplication  f o l l o w e d by m o d i f i c a t i o n c o u l d have g i v e n r i s e t o a t r a n s p o r t s i t e which, w h i l s t s t i l l phenolic  transporting  g l u c o s i d e s , c o u l d no l o n g e r a c c e p t the  free  s u g a r . Thus the s i t u a t i o n observed i n b a r l e y r o o t s i s realized. I t would seem c l e a r t h a t the a c c u m u l a t i o n of arbutin,and  possibly, s t r u c t u r a l l y related glucosides,  i s an a c t i v e p r o c e s s depending upon the u t i l i z a t i o n  of  m e t a b o l i c a l l y d e r i v e d energy. T h i s c o n c l u s i o n i s based upon the o b s e r v a t i o n s 1.  that  there i s a considerable  tr e d u c t i o n of uptake by  t r e a t m e n t s w h i c h reduce o r i n h i b i t metabolism, 2. t h e r e i s c o m p e t i t i o n  f o r uptake by a s t r u c t u r a l l y  r e l a t e d compound ( c a t e c h o l g l u c o s i d e ) , 3»  t h e r e i s a pH e f f e c t t y p i c a l of enzymatic  4.  there i s a s a t u r a t i o n e f f e c t w i t h a K  ffi  t o t h a t observed f o r a n i m a l t i s s u e s .  processes,  value  close  -63-  TABLE 8 14  C l a b e l l e d a r b u t i n ( 1 . 5 9 J*C/mmole) a s  The uptake of  a f u n c t i o n o f t i m e , by b a r l e y r o o t s ( 0 . 5 g. samples) UPTAKE OF ARBUTIN x 10*3 d.p.m.  TIME ( m i n u t e s ) 15  a . 0.121  b. 0.451  30  a. 0.396  b.  60  a.  b. 1.386  1.149  0.528  120  a. 2.953  b.  2.431  180  a. 3.366  b.  3.231  TABLE 9 3  The uptake of  H l a b e l l e d a r b u t i n (10/iC/mmble) o v e r a  p e r i o d of t h r e e h o u r s , as a f u n c t i o n of e x t e r n a l concentration,  by b a r l e y r o o t s (1 g. samples)  CONCENTRATION OF ARBUTIN (mM)  UPTAKE OF ARBUTIN x 10"^ d.p.m.  2.5  a. 3.1357  b.  2.9954  5.0  a. 5.9805  b.  4.5293  7.5  a. 6.5184  b.  6.4589  10.0  a. 6.5626  b.  7.7286  15.0  a. 6 . 9 0 1 4  b. 6 . 8 0 4 6  20.0  a.  6.5747  b.  6.4191  -64F i g 5 t The u p t a k e  o f a r b u t i n as a f u n c t i o n of  "by b a r l e y r o o t s *  time,  -65-  TABLE 10 The uptake o f  3 H l a b e l l e d a r b u t i n (44 /iC/mmole) a s a  f u n c t i o n of e x t e r n a l concentration  by Pltyrogramma.  over a p e r i o d of t h r e e h o u r s . CONCENTRATION OF ARBUTIN (mM)  UPTAKE OF ARBUTIN x 1 0  - 3  0.5  a . 0.613  b. 0.509  1.0  a . 1.313  b. 1.056  5.0  a . 8.694  b. 8.401  10.0  a.11.030  b.12.689  15.0  a.12.280  b. 9.646  20.0  a.11.644  b.11.412  d.p.m.  -663  F i g 6a i The uptake o f H l a b e l l e d a r b u t i n as a f u n c t i o n of e x t e r n a l concentration,  by b a r l e y  roots.  3  F i g 6b ( i n s e t ) : The uptake o f H l a b e l l e d  arbutin  as a f u n c t i o n o f e x t e r n a l c o n c e n t r a t i o n , Pityrogramma.  Arbutin  concentration  (mM)  by  -67-  TABLE 11 The e f f e c t of v a r i o u s m e t a b o l i c  i n h i b i t o r s upon t h e uptake  3  H l a b e l l e d a r b u t i n (10 uC/mmole,.maintained a t a  of  c o n c e n t r a t i o n of 5 mM) o v e r a p e r i o d of t h r e e h o u r s , by b a r l e y r o o t s (1 g. samples) -4  TREATMENT  UPTAKE x 10  C o n t r o l (25°C)  a. 5.434 b. 5.428  0  M DNP  a . 4.923 b. 5 . 5 5 8  4  10*^ M DNP  a. 4.620 b. 3 . 7 8 4  23  10~^ M DNP  a . 1.238 b. 1.199  75  M DNP  a . 1.089 b. 1.260  78  a . 2.192 b. 2.431  59  a . 1.375 b. 1.370  74  10"  6  10"  3  N  atmosphere  2  Low temperature (1-3°C)  d.p.m.  % INHIBITION  -68The uptake o f  3 H l a b e l l e d a r b u t i n (5 uC/mmole) over a  p e r i o d of t h r e e hours, by b a r l e y r o o t s (1 g. s a m p l e s ) , as i n f l u e n c e d by t h e presence of some p o t e n t i a l t r a n s port competitors  maintained  a t a c o n c e n t r a t i o n of 2 . 5 mM  UPTAKE x 10  ARBUTIN CONCENTRATION 1.  CONTROL  2.  d.p.m.  + CATECHOL GLUCOSIDE  mM  a. 0.843 b. 0 . 8 0 0  a. 0 . 3 4 3 b. 0 . 4 4 4  2 . 5 0 mM  a. 1.478 b. 1 . 4 0 3  a. 0 . 8 7 2 b. 0 . 7 3 3  5.00i;mM  a. 2.156 b. 1 . 8 9 6  a. 1.336 b. 1.162  1.25  3.  + GLUCOSE  4.  + HYDROQUINONE  1.25  mM  a. 0 . 7 4 5 b. 0 . 8 9 4  a. 0 . 3 2 6 b. 0.317  2.50  mM  a. 1 . 2 9 0 b. 1.816  a. 0.5^03 b. 0.440  5.00  mM  a. 1.952 b. 2 . 2 7 0  a. 0.726 b. 0 . 5 7 3  -69-  F i g 7a t The d a t a of F i g 6a shown I n the form o f a double r e c i p r o c a l p l o t .  6  -70-  TABLE 13 3  The I n f l u e n c e o f pH upon t h e uptake o f H l a b e l l e d a r b u t i n ( 2 . 5 uC/mmole, m a i n t a i n e d a t 5 mM) by b a r l e y r o o t s (1 g samples) pH  UPTAKE x I O " d.p.m.  2  a . 2.045 b. 2.475  3  a. 4.592 b. 2 . 5 8 5  4  a. 9.735 b. 9 . 8 7 9  5  a.10.473 b.12.740  6  a.11.206 b.10.786  7  a. 6.635 b. 5 . 9 9 8  8  a . 3.657 b. 4 . 0 7 5  3  -71F i g 8 s The  i n f l u e n c e of pH upon the uptake of a r b u t i n  by b a r l e y  roots.  -72LITERATURE  CITED  1. D a v s o n , H. 1 9 6 4 . A T e x t b o o k o f G e n e r a l L i t t l e , Brown and C o . B o s t o n  Physiology.  2. R o s e n b e r g , T . 1 9 5 4 . S . E . B . Symposium v o l . V l l l . A c t i v e T r a n s p o r t and S e c r e t i o n . O x f o r d U n i v . Press. 3.  W l l b r a n d t , W. 1 9 6 4 . S . E . B . Symposium v o l . V l l l . A c t i v e T r a n s p o r t and S e c r e t i o n . O x f o r d U n i v . Press.  4. C o l l a n d e r , R. a n d B a r l u n d , H . 1 9 3 3 . P e r m e a b i l i t a t s s t u d i e n an Chara C e r a t o p h v l l a . A c t a . b o t . fenn. 1 1 t l . 5.  A l v a r a d o , F . a n d C r a n e , R. 1 9 6 4 . S t u d i e s on t h e mechanism o f i n t e s t i n a l a b s o r b t i o n o f s u g a r s . Biochim. et. Biophys. A c t a . ^x\l6.  6. B i e l e s k i , R . L . I 9 6 2 . The p h y s i o l o g y o f s u g a r A u s t r a l i a n . J . B i o l . S c i . Ij£t429. 7. 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Membrane S c i e n c e . 1621632.  transport  proteins.  -7313. Fox,  C.F. and Kennedy, E.P. I965, S p e c i f i c l a b e l l i n g and p a r t i a l p u r i f i c a t i o n o f t h e M p r o t e i n , a component o f t h e b e t a g a l a c t o s i d e t r a n s p o r t system o f E s c h e r i c h i a c o l l . P r o c . Nat. Acad. S c i . U.S. £4:891.  14. Kabach, H.R. and Stadtman, E.R. I 9 6 5 . P r o l i n e uptake by a n i s o l a t e d c y t o p l a s m i c membrane p r e p a r a t i o n o f E s c h e r i c h i a c o l l . P r o c . Nat. Acad. S c i . U.S. £1*920.  15.  Pridham, J.B. 1 9 6 5 * O b s e r v a t i o n s on the t r a n s l o c a t i o n o f p h e n o l i c compounds. Phytochem. £;777.  16.  Landau, B.R., B e r n s t e i n , L. and W i l s o n , T.H. I 9 6 2 . Hexose t r a n s p o r t by hamster I n t e s t i n e i n v i t r o . Amer. J . P h y s i o l . 203:237.  1 7 . M i c h e l , F.Y. 1 9 3 6 . A r b u t i n d i a b e t e s . P r o c . Soc. B i o l . 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Based upon a technique developed by Dr. J.P.Kutney Dept. Chem. Univ. B r i t i s h Columbia, Vancouver.  THE PHENOL GLUCOSYLATION REACTION I N HIGHER PLANTS  -75-  INTRODUCTION When simple p h e n o l s such as hydroquinone o r c a t e c h o l a r e introduced  i n t o the t i s s u e s o f higher p l a n t s they are  g l u c o s y l a t e d t o form t h e c o r r e s p o n d i n g When t h e p h e n o l i s a d m i n i s t e r e d  beta-D-glucosides.  over a p e r i o d o f s e v e r a l  weeks, t h e f i r s t formed g l u c o s i d e  may become  glucosylated  t o form t h e g e n t i o b i o s l d e . The p a u c i t y o f I n f o r m a t i o n  regarding  the p h e n o l  g l u c o s y l a t l o n r e a c t i o n l n f e r n s l e d t h e a u t h o r t o undertake a s u r v e y o f these p l a n t s w i t h a v i e w t o c l a r i f y i n g t h e i r p o s i t i o n with regard  to t h i s r e a c t i o n . During t h i s  s t u d y i t was observed t h a t t h e b e t a - D - g l u c o s i d e was somet i m e s accompanied by s m a l l amounts o f a compound which behaved as a simple d e r i v a t i v e o f t h e g l u c o s i d e . A second s u r v e y was conducted t o determine whether o r n o t the formation  o f t h i s d e r i v a t i v e was common amongst h i g h e r  p l a n t s . S i n c e V l c l a faba produced r e l a t i v e l y l a r g e q u a n t i t i e s o f t h i s compound, and was e a s i l y grown, i t was employed i n an attempt t o i s o l a t e and c h a r a c t e r i z e the compound.  -76-  LITERATURE REVIEW N a t u r a l l y o c c u r r i n g simple According  phenols.  t o Harborne, t h e simple p h e n o l s a r e r a r e  i n t h e p l a n t kingdom. Hydroquinone i s c o n s i d e r e d  t o be  the most w i d e l y d i s t r i b u t e d , h a v i n g been found as i t s 2  3  b e t a - D - g l u c o s i d e , a r b u t i n , i n t h e E r i c a c e a e , Rosaceae, 4 1 S a x i f r a g a c e a e , as w e l l as i n t h e P r o t e a c e a e and 1 Compositae . I n r e c e n t y e a r s s e v e r a l r a t h e r i n t e r e s t i n g d e r i v a t i v e s o f a r b u t i n have been d i s c o v e r e d  i n plants  5  w h i c h c o n t a i n a r b u t i n . Thus F r i e d r i c h  isolated a cryst-  a l l i n e mono-O-acetyl d e r i v a t i v e from t h e l e a v e s o f P y r u s communis and from V a c c i n i u m V l t l s i d a e a . I n f r a - r e d 6* 7 s t u d i e s , as w e l l as t h e d e f i n i t i v e s y n t h e s i s by Haslam »  have shown t h a t t h i s m o l e c u l e i s t h e 6 - a c e t a t e  of arbutin.  Isopyroside, the 2 - 0 - a c e t y l d e r i v a t i v e of a r b u t i n has been i s o l a t e d from t h e l e a v e s o f P y r u s communis by 8  E n t l i c h e r and Kocourek , who c l a i m t o have f a i l e d , a f t e r many a t t e m p t s , t o i s o l a t e t h e 6 - 0 - a c e t y l d e r i v a t i v e , 5  pyroside*  r e p o r t e d by F r i e d r i c h . The former a u t h o r s  suggest t h a t i s o p y r o s i d e i s t h e n a t u r a l l y o c c u r r i n g compound, w h i c h g i v e s r i s e , d u r i n g e x t r a c t i o n , t o p y r o s i d e by t h e m i g r a t i o n o f t h e a c e t y l group from p o s i t i o n 2 o f the g l u c o s e  moiety t o p o s i t i o n 6. I n s i m i l a r f a s h i o n , 9  P e a r l and D a r l i n g  found t h a t e x t r a c t s o f P o p u l u s  s p e c i e s gave e i t h e r p o p u l i n ( t h e 6-monobenzoate o f s a l i c i n ) o r t r e m u l o i d l n (the 2-monobenzoate) depending upon t h e c o n d i t i o n s o f e x t r a c t i o n . Under s l i g h t l y  -77»  a l k a l i n e c o n d i t i o n s t h e 6 b e n z o a t e was o b t a i n e d a s a r e s u l t o f m i g r a t i o n from t h e 2 p o s i t i o n . They q u e s t i o n the v a l i d i t y o f a l l r e p o r t s o f p o p u l i n s i n c e i t s o r i g i n a l 10 i s o l a t i o n by Braconnot i n 1830. 7 Haslam r e p o r t s t h a t , i n a d d i t i o n t o a r b u t i n and pyroside,  extracts of Vaccinium V i t l s  Idaea c o n t a i n  a  compound w h i c h h a s b e e n i d e n t i f i e d a s  2-0-caffeoyl 11 s t u d y b y t h e same a u t h o r showed  a r b u t i n , A more r e c e n t  that extracts of Arctostaphylos Bergenia three  u v a - u r s i as w e l l as  c r a s s l f o l l a and B e r g e n i a  oordifolla.  g a l l o y l e s t e r s o f a r b u t i n . T h e s e were  contained identified,  by t h e i r h y d r o l y s i s p r o d u c t s and by c h e m i c a l  syntheses,  as 2 g a l l o y l a r b u t i n , 6 g a l l o y l a r b u t i n , and  p-galloyl-  phenyl-beta-D-glucoside, Catechol species  has been found as the f r e e p h e n o l i n s e v e r a l 12,13,14, of the Salicaceae. I t has a l s o been r e p o r t -  15 ed t o o c c u r i n A l l i u m c e p a  2 and Psorospermum  . The  micro-  b i a l d e g r a d a t i o n o f p h e n o l i c compounds c a n g i v e r i s e t o c a t e c h o l and i t s d e r i v a t i v e s as s h o r t - l i v e d i n t e r m e d i a t e s 16 p r i o r t o r i n g cleavage . As i t s b e t a - D - g l u c o s i d e , c a t e c h o l has been r e p o r t e d t o o c c u r i n l a r g e q u a n t i t i e s i n three 17 species of Gaultheria . Other simple phenols such as phenol i t s e l f , are  r e s o r c i n o l , p h l o r o g l u c i n o l , and p y r o g a l l o l 1 extremely rare i n nature .  Formation of phenolic  g l y c o s i d e s by p l a n t t i s s u e s 18  C i a m i c i a n and Ravenna  are reported  t o have been t h e  f i r s t w o r k e r s t o show t h a t h i g h e r p l a n t s a r e c a p a b l e o f  -78g l u c o s y l a t i n g a d m i n i s t e r e d p h e n o l s . When maize and bean p l a n t s were t r e a t e d w i t h d i l u t e s o l u t i o n s o f s a l i g e n i n (o-hydroxybenzyl  a l c o h o l ) o r hydroquinone t h e y  recovered  s a l i c i n (o-hydroxymethylphenyl beta-D-glucoside)  or  arbutin, respectively. 19  I n t h e p e r i o d from 1938 t o 1957,  Miller  studied  the f o r m a t i o n o f p h e n o l i c and a l c o h o l i c g l y c o s i d e s produced when f o r e i g n p h e n o l s o r a l c o h o l s a r e i n t r o d u c e d i n t o p l a n t t i s s u e s . The r e s u l t i n g g l y c o s i d e s were i s o l a t e d and i d e n t i f i e d by c o n v e r s i o n t o t h e i r a c e t y l d e r i v a t i v e s . M i l l e r prepared  t h e same a c e t y l d e r i v a t i v e s by c h e m i c a l  s y n t h e s i s , so t h a t comparisons w i t h t h e a u t h e n t i c compounds were p o s s i b l e . M i l l e r s r e s u l t s i n d i c a t e t h a t t h e nature  o f t h e g l y c o s i d i c d e r i v a t i v e may v a r y , n o t o n l y  w i t h t h e p l a n t t o which t h e a g l y c o n e i s a d m i n i s t e r e d , but a l s o w i t h t h e n a t u r e  o f the aglycone  F o r example, t h e s i m u l t a n e o u s h y d r i n and 2 - c h l o r o p h e n o l ,  administered.  feeding of ethylene  chloro-  t o g l a d i o l u s corms, r e s u l t e d  i n t h e f o r m a t i o n o f t h e g l u c o s i d e o f t h e former and t h e g e n t l o b i o s i d e o f the l a t t e r . 20,21,22,23,24 More r e c e n t l y , the formation of p h e n o l i c g l y c o s i d e s has been re-examined w i t h p a r t i c u l a r  reference  t o t h e mechanism o f p h e n o l i c g l y c o s y l a t i o n . The b i o s y n t h e s i s o f t h e g l u c o s i d e s and g e n t i o b i o s i d e s i s b e l i e v e d t o o c c u r by a t r a n s f e r o f t h e g l u c o s e m o i e t y o f a sugar n u c l e o t i d e t o t h e p h e n o l . A r b u t i n and o t h e r p h e n o l i c g l u c o s i d e s have been s y n t h e s i z e d from UDPGlucose and t h e  -79-  corresponding  p h e n o l i n t h e presence o f enzyme 25,26,27  from a v a r i e t y o f p l a n t s .  preparations  Yamaha and C a r d i n i  t h a t t h e f o r m a t i o n o f hydroquinone  28  showed  beta-D-gentiobioside  o c c u r r e d i n two s t a g e s . U s i n g enzyme p r e p a r a t i o n s from wheat germ, t h e g l u c o s e m o i e t y o f UDPG was t r a n s f e r r e d t o hydroquinone t o g i v e a r b u t i n . T h i s compound was t h e n g l u c o s y l a t e d a t p o s i t i o n 6 (UDPG a g a i n s e r v i n g as t h e g l u c o s e donor) t o g i v e t h e g e n t i o b i o s i d e . T h e i r s t u d i e s i n d i c a t e t h a t s e p a r a t e enzymes mediate t h e two g l u c o s e transfer reactions. The p h e n o l g l u c o s y l a t i o n r e a c t i o n appears t o be w i d e s p r e a d amongst those h i g h e r p l a n t s examined. The s t u d y 29  by Pridham  showed t h a t t h e a b i l i t y t o p e r f o r m  this reaction  was l a r g e l y r e s t r i c t e d t o angiosperms and gymnosperms. Ten a l g a e and two f u n g i r e a c t e d n e g a t i v e l y . Of t h e twelve b r y o p h y t e s examined, most r e a c t e d n e g a t i v e l y o r gave o n l y t r a c e amounts o f t h e a p p r o p r i a t e g l u c o s i d e s . The two species of ferns reacted p o s i t i v e l y . I n 1962 Pridham and S a l t m a r s h  r e p o r t e d t h a t when  seeds o f V l c i a f a b a were t r e a t e d w i t h s i m p l e  phenols,  s m a l l q u a n t i t i e s o f a compound i d e n t i f i e d as t h e p h e n o l g l u c o s i d e - 6 - s u l p h a t e , accompanied t h e b e t a - D - g l u c o s i d e . T h i s compound was observed t o be e x t r e m e l y  labile,  g i v i n g r i s e t o the parent g l u c o s i d e spontaneously, i n aqueous o r m e t h a n o l i c  s o l u t i o n . IN NaOH a t 100°C, r a p i d l y  e f f e c t e d complete h y d r o l y s i s t o t h e p a r e n t g l u c o s i d e . A g l y c o s u l p h a t a s e p r e p a r a t i o n showed n e g l i g e a b l e a c t i v i t y towards t h e compound.  -80MATERIALS AND METHODS Plant material The f e r n s employed i n t h e s e s t u d i e s were  obtained  from the U n i v e r s i t y greenhouses. O t h e r p l a n t s used i n t h e s u r v e y work were c o l l e c t e d on t h e U n i v e r s i t y grounds where t h e y were growing w i l d . F l a t s o f V l c l a faba were grown i n s o i l , i n t h e greenhouse. Chemicals Preparation o f a r b u t i n 6-sulphate a. By d i r e c t s u l p h a t i o n r — " J O H o i b e r g and Mumma  reported that sulphation of a  v a r i e t y o f a l c o h o l i c and p h e n o l i c compounds c a n be achieved diimide  b y means o f s u l p h u r i c a c i d and d i c y c l o h e x y l c a r b o (DCC) i n dimethylformamide (DMF). I t i s suggested  t h a t under t h e s e c o n d i t i o n s a D C C - s u l p h u r l c a c i d complex i s formed w h i c h r a p i d l y b r i n g s about s u l p h a t i o n , w i t h t h e c o n c o m i t a n t p r e c i p i t a t i o n o f d i c y c l o h e x y l u r e a . Thus :ROH  +  HgSO^  0  II  ROS-OH  II  0 The d i r e c t s u l p h a t i o n o f g l u c o s e  or unsubstituted  gluco-  s i d e s under t h e c o n d i t i o n s d e s c r i b e d by H o i b e r g and Mumma as d i l u t e  (  0.024 mM  H2SO4)  g i v e s r i s e t o a mixture o f the  -81monosulphated d e r i v a t i v e s . There i s , however, no s u l p h a t ion  of phenolic hydroxyls. According t o the greater r e a c t -  i v i t y o f the p r i m a r y h y d r o x y l group l o c a t e d a t p o s i t i o n 6 of t h e g l u c o s e moiety, be e x p e c t e d  t h e s u l p h a t i o n of a r b u t i n s h o u l d  t o g i v e the 6 - s u l p h a t e as the major p r o d u c t .  The r e a c t i o n m i x t u r e was prepared by m i x i n g 0 . 2 4 mmole of a r b u t i n and 1.15 mmole o f DCC, d i s s o l v e d i n 7 ml of DMF, w i t h 0 . 2 4 mmole of H S 0 ^ c o n t a i n e d i n 3 ml o f DMF. P r i o r 2  t o m i x i n g , t h e r e a c t a n t s were c o n t a i n e d i n t e s t  tubes  h e l d i n an i c e b a t h . M i x i n g of t h e r e a c t a n t s produced, a f t e r about 15 sees, a p r e c i p i t a t e of d i c y c l o h e x y l u r e a , w h i c h was removed by f i l t r a t i o n . The f i l t r a t e was banded upon s h e e t s o f Whatman No 3 chromatography paper and developed v/v).  l n a m i x t u r e of butanoltpyridine»water  ( 6 » 4 $ 3  B e s i d e s u n r e a c t e d a r b u t i n , a major band, presumably  the 6 - s u l p h a t e , and t h r e e minor bands c o r r e s p o n d i n g t o 2-,  3 - > and 4-monosulphated a r b u t i n s were o b t a i n e d . The  6 - s u l p h a t e was e l u t e d from t h e paper w i t h water, t r a n s f e r r e d t o p y r i d i n e s o l u t i o n , and a c e t y l a t e d , o v e r n i g h t , w i t h 5 nil of a c e t i c a n h y d r i d e . Chromatography of t h e r e a c t i o n mixture i n b u t a n o l i p y r i d i n e t w a t e r gave a s i n g l e a r b u t i n d e r i v a t i v e a t »  ing  »  #  to 2,3i4,4-tetra-0-acetyl  ( 6 J 4 » 3  V / V )  0 . 7 4 , correspond»  arbutin 6-sulphate.  b. D e f i n i t i v e s y n t h e s i s . The  p r e p a r a t i o n of a r b u t i n 6 - s u l p h a t e by a d e f i n -  i t i v e r o u t e i n v o l v e s t h e temporary b l o c k i n g o f a l l p o t e n t i a l l y r e a c t i v e s i t e s except p o s i t i o n 6 , p r i o r t o  -82-  s u l p h a t i o n . F o l l o w i n g s u l p h a t i o n the b l o c k i n g groups are removed. A m i x t u r e  o f 1 0 g o f a r b u t i n and  1 0 g of  t r l p h e n y l m e t h y l c h l o r i d e were d i s s o l v e d i n 4 0 mli. o f p y r i d i n e . A f t e r s t a n d i n g f o r 5 mins, 30 ml;* o f a c e t i c a n h y d r i d e were s l o w l y added, and  the mixture  stand overnight. T r l p h e n y l m e t h y l c h l o r i d e  allowed  reacts  a l l y w i t h the h y d r o x y l group a t p o s i t i o n 6,  to  specific-  thus e f f e c t -  ively blocking i t . Acetylation i s indiscriminate, c o n v e r t i n g a l l f r e e h y d r o x y l groups t o t h e i r The  mixture  was  acetates.  t h e n poured, w i t h s t i r r i n g , i n t o 1  o f i c e w a t e r . S t i r r i n g was  litre  f o r 2 hrs, a f t e r  continued  w h i c h t i m e , the i n i t i a l l y gummy p r e c i p i t a t e had become g r a n u l a r . The and  p r e c i p i t a t e was  r e c r y s t a l l i z e d to constant  from e t h a n o l , t o g i v e 11 (Lit.  f i l t e r e d from the w a t e r  value. 1 9 7 - 1 9 8 ° C ) ,  melting p o i n t (4  times)  g o f f i n e n e e d l e s m.p. of  197°C  6lo-triphenylmethyl,2!3l4|4-  tetra-O-acetyl arbutin ( I ) . To 11  g o f I d i s s o l v e d i n 50 ml. o f g l a c i a l a c e t i c  were added 15 ml.: o f a s a t u r a t e d s o l u t i o n o f HBr g l a c i a l a c e t i c a c i d . The bromide was  precipitated triphenylmethyl-  i m m e d i a t e l y f i l t e r e d o f f , and the  poured i n t o 500 ml;, o f i c e w a t e r . The ed w i t h 500 ml;.: of c h l o r o f o r m and t o g i v e a gum,  w h i c h was  l i z a t i o n to constant 1.5  in  mixture  filtrate was  extract-  e v a p o r a t e d under vacuum  dissolved i n ethanol. Recrystal-  m e l t i n g p o i n t , from e t h a n o l , gave  g of 0 - a c e t y l q u i n o l 2 , * 3 | 4 ! - t r i - 0 - a c e t y l beta-D7  g l u c o s i d e as n e e d l e s m.p.  147-148°C  ( L i t . value  147-148°C).  -83-  The  above compound was  d i s s o l v e d i n DMF  and  sulphated 30  a c c o r d i n g t o the method of H o i b e r g and Mumma . An a l i q u o t of the r e a c t i o n m i x t u r e was  chromatographed on Whatman  No 1 chromatography paper, u s i n g (6i4«3  v/v) as the i r r i g a n t . The  namely,-v the u n r e a c t e d  butanoltpyridinejwater two a r b u t i n d e r i v a t i v e s ,  s t a r t i n g m a t e r i a l and. the  d e r i v a t i v e were r e s o l v e d a t Rf 0 . 9 5 The  s u l p h a t e d d e r i v a t i v e and  sulphated  0.?4 respectively.  and  the a c e t y l a t e d  6-sulphate  produced by d i r e c t s u l p h a t i o n were I d e n t i c a l i n t h e i r c h r o m a t o g r a p h i c b e h a v i o r . The r e a c t i o n m i x t u r e was  r e m a i n d e r of the above  t r a n s f e r r e d to methanolic  solution  A f t e r b u b b l i n g w i t h NH3 the s o l u t i o n  and  t r e a t e d w i t h NH3.  was  a l l o w e d t o s t a n d o v e r n i g h t . Chromatography of  methanolic  s o l u t i o n showed t h a t i t now  contained  this about  e q u a l q u a n t i t i e s of a r b u t i n and a r b u t i n 6 - s u l p h a t e .  The  chromatographic behavior  found  of the l a t t e r compound was  t o be i d e n t i c a l w i t h t h a t of the a r b u t i n  6-sulphate  produced by d i r e c t s u l p h a t i o n . P r e p a r a t i o n of the b e t a - D - g e n t l o b l o s l d e s and  of hydroquinone  catechol The  p r e p a r a t i o n of the above compounds was  by a m o d i f i c a t i o n of the method of H e l f e r i c h and 32  achieved Schmitz-  H i l l e b r e c h t • S e v e r a l a t t e m p t s t o prepare these compounds by t h e i r method r e s u l t e d i n f a i l u r e . I n s t e a d , mixture  of 50 mg  o c t a a c e t a t e and d i s s o l v e d i n 100  of hydroquinone, 250 mg  of  5 mg of p - t o l u e n e s u l p h o n i c  a  gentiobiose a c i d were  ml of a c e t o n e and r e f l u x e d f o r 2 4 h r s .  -84The acetone s o l u t i o n was  e v a p o r a t e d t o d r y n e s s and  r e d i s s o l v e d i n methanol. D e a c e t y l a t i o n was the use  of N H 3 .  then  achieved  Chromatography of the m e t h a n o l i c  by  solution  on s h e e t s of Whatman No 3 chromatography paper, u s i n g b u t a n o l 1 pyridine«water ( 6 i 4 t 3 v/v) as the s o l v e n t , gave a s i n g l e a r b u t i n - c o l o u r e d band a t Rf 0.51*  Mild hydrol-  y s i s , u s i n g 10$ a c e t i c a c i d a t 100°C f o r 10 mins gave hydroquinone and g e n t i o b i o s e as the p r o d u c t s . The responding  c a t e c h o l d e r i v a t i v e was  cor-  prepared by the same  method ( s u b s t i t u t i n g c a t e c h o l f o r h y d r o q u i n o n e ) . A d m i n i s t r a t i o n of compounds a. F o r m a t i o n of g l u c o s i d e s . Leaves of the p l a n t s were c u t and  then r e c u t under  w a t e r . They were t h e n t r a n s f e r r e d t o 5ml of a 5 mM s o l u t i o n of the a p p r o p r i a t e phenol, v i a l , and a l l o w e d t o m e t a b o l i z e  contained  aqueous  i n a small  f o r a p e r i o d of 24  hrs.  b. Survey f o r a r b u t i g e n i n and c a t e c h o l e q u i v a l e n t . Leaves of the p l a n t s were c u t and  then r e c u t under  w a t e r . They were then t r a n s f e r r e d t o 10 ml of a 5 aqueous s o l u t i o n of the a p p r o p r i a t e p h e n o l , and to metabolize  mM  allowed  f o r a p e r i o d of f o u r d a y s .  P r e p a r a t i o n of p l a n t e x t r a c t s I n a l l c a s e s the p l a n t m a t e r i a l was b o i l i n g 80$  e t h a n o l . The  extracted with  ethanol-soluble f r a c t i o n  e v a p o r a t e d t o d r y n e s s under vacuum a t 40°C. The was  was  residue  e x t r a c t e d w i t h w a t e r and f i l t e r e d t h r o u g h C e l i t e .  The aqueous e x t r a c t was  concentrated  under vacuum a t 4o°C.  -85Chromatography F o r the s u r v e y work, t w o - d i m e n s i o n a l  chromatography  was employed. A l i q u o t s of the aqueous e x t r a c t s were a p p l i e d t o s h e e t s o f Whatman No 1 paper, which were d e v e l o p e d by t - b u t a n o l i a c e t i c a c i d : w a t e r (3»1»1 v/v) for  the f i r s t d i m e n s i o n , and butanol»pyridineiwater  (6:4:3 v/v) f o r the second d i m e n s i o n . I n the i s o l a t i o n of  the unknown a r b u t i n d e r i v a t i v e from V l c l a f a b a  s h o o t s , t o which hydroquinone had been a d m i n i s t e r e d for  4 days, the aqueous e x t r a c t s were a p p l i e d i n the  form o f bands t o s h e e t s o f Whatman No 3  paper,which  were d e v e l o p e d i n t - b u t a n o l : a c e t i c a c i d t w a t e r (3*1»1 v/v).  The a p p r o p r i a t e band was  c u t from the  chromato-  grams, e l u t e d w i t h d i s t i l l e d w a t e r , and t h e n r e a p p l i e d to  f r e s h s h e e t s o f No 3 paper. By t h i s method the compound  was p u r i f i e d t h r o u g h t h r e e more s o l v e n t s , namely,  80$  i s o p r o p a n o l , b u t a n o l : p y r i d i n e : w a t e r (6:4:3 v / v ) , and b u t a n o l : e t h a n o l : w a t e r (40:11:19 v / v ) . F i n a l l y the compound was a p p l i e d , i n aqueous s o l u t i o n , t o a s m a l l column o f Woelm P o l y a m i d e , from which i t was with d i s t i l l e d  eluted  water.  V i s u a l i z a t i o n o f compounds by s p r a y r e a g e n t s When d e a l i n g w i t h compounds p o s s e s s i n g f r e e p h e n o l i c h y d r o x y l groups, d i a z o t i z e d p - n i t r o a n l l i n e was  sprayed  d i r e c t l y onto chromatograms, and t h e n o v e r s p r a y e d w i t h 5$ NaOH. I n those cases where p h e n o l i c h y d r o x y l s were b l o c k e d , as f o r example i n the case of a c e t y l d e r i v a t -  -86i v e s , t h e chromatograms were f i r s t sprayed w i t h IN NaOH and a l l o w e d t o s t a n d f o r 10 mins. T h i s  treatment  served t o h y d r o l y z e t h e p h e n o l i c e s t e r s so t h a t  over-  spraying w i t h d i a z o t i z e d p - n i t r o a n i l i n e then v i s u a l i z e d p h e n o l i c s . Sugars were d e t e c t e d by t h e use o f t h e 35 35 p e r i o d a t e / b e n z l d i n e method , n a p h t h o r e s o r c i n o l , o r 35 the p h l o r o g l u c i n o l method .  -87RESULTS AND The  DISCUSSION  f e r n survey Table 1 shows the r e s u l t s of the s u r v e y of twenty-  n i n e s p e c i e s of f e r n s f o r the a b i l i t y t o g l u c o s y l a t e administered  p h e n o l s . Samples of P s i l o t u m nudum were  a v a i l a b l e i n the U n i v e r s i t y greenhouse, so t h i s p l a n t was  i n c l u d e d i n the s t u d y . With the two s p e c i e s of f e r n s  33 examined by Pridham , and  34  Roy  the s i n g l e s p e c i e s examined by  , t h i s b r i n g s the t o t a l t o t h i r t y - t w o s p e c i e s of f e r n s .  A l l f e r n s and  P s i l o t u m r e a c t e d p o s i t i v e l y t o the presence  of hydroquinone and c a t e c h o l , by the f o r m a t i o n corresponding  the  beta-D-glucosides. H  The  of  II  survey f o r a r b u t i g e n i n Por convenience i n r e f e r r i n g t o the unknown compound  d e s c r i b e d i n the i n t r o d u c t i o n , the term  arbutigenin  c o i n e d , s i n c e t h i s compound gave r i s e , most r e a d i l y ,  was to  a r b u t i n , i n aqueous or e t h a n o l i c s o l u t i o n . T a b l e 2 shows the r e s u l t s of a survey f o r the a b i l i t y t o form a r b u t i g e n i n and  i t s c a t e c h o l g l u c o s i d e e q u i v a l e n t . The  plants  were exposed t o 5 mM aqueous s o l u t i o n s of hydroquinone or c a t e c h o l f o r 4 days. The  g e n t i o b l o s i d e s of h y d r o q u i n -  one and c a t e c h o l were i d e n t i f i e d as minor p r o d u c t s i n  one  of the f i r s t p l a n t s examined. S u b s e q u e n t l y a l l e x t r a c t s were examined f o r the presence of these compounds.  -88-  TABLE 1 F e r n s p e c i e s c a p a b l e of g l y c o s y l a t i n g hydroquinone and catechol. Adiantum c a p i 1 1 1 s - v e n e r i s L. A. caudatum L. Mant. A. h i s p l d u l u m Sw. S c h r a d . A s p l e n l u m v i v i p a r u m (L F i i . ) P r . Tent. A t h y r i u m f e l l x - f e m l n a ( L . ) Roth. Campy1oneuron spp. C i b o t l u m splendens (Gaudichaud) K r a j i n a C t e n l t i s decomposita (R.Br.) C o p e l . Cyathea d e a l b a t a t F o r s t ) Sw. Schrand. Cyrtomium spp. D a v a l l i a s o l l d a ( F o r s t ) Sw. Schrad D e n n s t a e d t i a w l l d f o r d i i (Moore) K o i d z Elaphoglossum r e t l c u l a t u m ( K a u l f u s s ) Gaudichaud Elaphoglossum spp. H e m i o n i t l s a r i f o l l a (Burn) Moore Microsorum punctatum Fee Nephrolepis e x a l t a t a (L.) Schott N. h l r s u t u l a ( F o r s t e r ) P r e s 1 P e l l e a e a n d r o m e d l f o l i a ( K l f . ) Fee P. r o t u n d i f o l i a Hook P. v i r l d i s ( F o r s k . ) P r a n t l , E n g l . Phlebodium aureum J . S m i t h Pltyrogramma calomelanos (L.) L i n k ( l e a v e s ) P. calomelanos ( L . ) L i n k (10-day o l d gametophytes) P l a t y c e r l u m b i f u r c a t u m (Cav.) C. Chr. Polystichum aculeatum (L;) Sehott P t e r l d l u m a q u l l i n u m Kuhn v a r . a q u i l i n u m P t e r i s o r e t i c a L. P. l o n g l f o l l a L.  P s i l o t u m nudum ( L . ) B e a v o i s  was a v a i l a b l e f o r e x a m i n a t i o n  and was thus i n c l u d e d i n t h i s s u r v e y . L i k e the above f e r n s i t was c a p a b l e of g l u c o s y l a t i n g hydroquinone and c a t e c h o l .  -89TABLE 2 R e s u l t s o f the s u r v e y f o r the a b i l i t y t o produce a r b u t i g e n i n and g e n t i o b l o s i d e . PLANT  ARBUTIGENIN  P s i l o t u r n nudum ( L . ) B e a v o i s Adlantum caudatum L. Mant. Camp.vloneuron spp. C t e n l t i s decomposita (R.Br.) C o p e l . D e n n s t a e d t l a w i l d f o r d i i (Moore) K o i d z Nephrolenis e x a l t a t a (L.) Schott Phlebodium aureum J . Smith PityroflT'ftTriTrifl. calomelanos (L. ) L i n k P t e r i s c r e t l c a L. A c e r macrophyllum P u r s h Cytisus scoparius (L.) Link E p i l o b i u m a n g u s t i f o l i u m L. G a u l t h e r i a a d e n o t h r i x (Mig.) Maxim. Lypopersicum e s c u l e n t u m M i l l . P e l a r g o n i u m var. Peperomia maculosa Hook P h a s e o l u s v u l g a r i s L. P l a n t a g o major L. Rubus p a r v i f l o r u s N u t t . Sorbus a u c u p a r i a L. T r a d e s c a n t i a spp. T r l f o l i u m p r a t e n s e L. Trlticum vulgare V i l l . V i c i a f a b a L. Zea mays L.  GENTIOBIOSIDE  + + + + + + + + + + trace + + + + + trace  + trace  * The above t a b l e l i s t s the r e s u l t s f o r the hydroquinone d e r i v a t i v e s o n l y . The c o r r e s p o n d i n g c a t e c h o l compounds were formed a c c o r d i n g t o the same p a t t e r n .  -90Of t h e t w e n t y - f i v e  s p e c i e s o f p l a n t s examined, f i f t e e n  were found t o produce q u a n t i t i e s o f a r b u t i g e n i n . I n some c a s e s o n l y t r a c e amounts were observed, w h i l s t i n Pltyrogramma and V i c l a e.g.,  q u a n t i t i e s equal t o  the simple g l u c o s i d e were formed. The f o r m a t i o n o f p h e n o l i c g e n t i o b i o s i d e s i n response t o t h e a d m i n i s t r a t i o n o f f r e e p h e n o l s appears t o be r a r e , s i n c e  only  t h r e e s p e c i e s produced t h e s e compounds. Identity of arbutigenin I n t h e f o l l o w i n g d i s c u s s i o n , r e f e r e n c e w i l l be made t o a r b u t i g e n i n , o n l y . However, i d e n t i c a l  studies  were performed u s i n g t h e c a t e c h o l b e t a - D - g l u c o s i d e d e r i v a t i v e . Therefore,  a l l that i s reported  concerning  a r b u t i g e n i n can apply e q u a l l y w e l l t o the catechol derivative. In i t s colour reaction to diazotized p - n i t r o a n i l i n e , o v e r - s p r a y e d w i t h base, and i t s U . V . s p e c t r u m , a r b u t i g e n i n was  found t o be I d e n t i c a l w i t h a r b u t i n . H y d r o l y s i s  with  IN HC1 a t 100° f o r 1 minute, gave hydroquinone and glucose as the only recognizable  products.  Mild hydrol-  y s i s w i t h 10% a c e t i c a c i d a t 100° f o r 1 minute gave a m i x t u r e o f a r b u t i n and a r b u t i g e n i n . I t was soon found t h a t a r b u t i g e n i n was e x t r e m e l y l a b i l e , s i n c e chromatography i n t h e m i l d e s t o f s o l v e n t s u n f a i l i n g l y gave r i s e t o f r e s h q u a n t i t i e s o f a r b u t i n . A l k a l i n e h y d r o l y s i s by 0.1N NaOH a t 100° c o n v e r t e d a l l t h e a r b u t i g e n i n t o a r b u t i n . S i m i l a r l y , i n e t h a n o l i c s o l u t i o n , the a d d i t i o n  -91of one drop o f I N NaOH was e f f e c t i v e i n c a u s i n g h y d r o l y s i s , a t room t e m p e r a t u r e , t o a r b u t i n . the q u i t e l a r g e number o f a r b u t i n d e r i v a t i v e s i n r e c e n t y e a r s i t seemed l i k e l y t h a t might r e p r e s e n t phenolic  complete  Considering discovered  arbutigenin  such a compound. I t s r e a c t i o n t o t h e  s p r a y r e a g e n t suggested t h a t t h e p h e n o l i c  h y d r o x y l was f r e e , and thus t h e s u b s t i t u t i o n must be upon t h e g l u c o s e m o i e t y . A t about t h i s t i m e , t h e many s i m i l a r i t i e s between t h i s compound and t h e p h e n o l i c  31 g l u c o s i d e 6 - s u l p h a t e r e p o r t e d b y Pridham were n o t e d . Seeds o f V l c i a f a b a were g e r m i n a t e d i n d i l u t e s o l u t i o n s  31 of hydroquinone o r c a t e c h o l , a s d e s c r i b e d by Pridham , and  t h e e t h a n o l i c e x t r a c t s chromatographed. Two compounds  were o b s e r v e d on t h e chromatograms, namely a r b u t i n and a r b u t i g e n i n , Pridham s i d e n t i f i c a t i o n had been made on the b a s i s o f t h e h y d r o l y s i s p r o d u c t s w i t h no r e f e r e n c e t o t h e a u t h e n t i c compound. S e v e r a l o f t h e p r o p e r t i e s o f the supposed s u l p h a t e were i n c o n s i s t e n t w i t h the g e n e r a l p r o p e r t i e s o f sugar s u l p h a t e s , and so i t was d e c i d e d t o p r e p a r e a r b u t i n 6 - s u l p h a t e by c h e m i c a l s y n t h e s i s . The  s y n t h e t i c a r b u t i n 6- s u l p h a t e and a r b u t i g e n i n  were found t o d i f f e r i n b o t h t h e i r c h r o m a t o g r a p h i c and e l e c t r o p h o r e t i c p r o p e r t i e s a s shown i n T a b l e 3»  39 According sugar s u l p h a t e s propylidene  t o Turvey  the a l k a l i n e h y d r o l y s i s of  i s a v e r y slow p r o c e s s .  -D- g a l a c t o s e i 6 - s u l p h a t e  NaOH a t 100°. S t u d i e s o f s u l p h a t e s  Thus d i - O - i s o -  i s s t a b l e t o 2N of various  sugar  -92-  TABLE 3 Chromatographic c h a r a c t e r i s t i c s o f compounds encountered I n t h i s  study  COMPOUND  SOLVENTS A  B  C  D  E  90  66  80  85  83  93  49  61  67  31  88  58  64  78  32  Hydroquinone g l u c o s i d e  89  52  78  66  63  Hydroquinone g e n t i o b i o s i d e  92  35  51  46  23  8?  24  54  60  24  Catechol  glucoside  Catechol gentiobioside Catechol  glucoside-6-sulphate  Hydroquinone  glucoside-6-sulphate  Arbutigenin  90  45  63  Electrophoretic studies COMPOUND  MIGRATION  Arbutigenin  1 cm  Hydroquinone g l u c o s i d e  1 cm  Hydroquinone g l u c o s i d e - 6 - s u l p h a t e  11 cms  * Chromatographic d a t a f o r t h e above s u l p h a t e d compounds and a r b u t i g e n i n were d e t e r m i n e d on Whatman No. 1 chromatography paper. A l l o t h e r d a t a were d e t e r m i n e d on 0 . 5 mm A v i c e l m i c r o c r y s t a l l i n e c e l l u l o s e p l a t e s . A l l R f v a l u e s have been m u l t i p l i e d by 1 0 0 . Key t o s o l v e n t s t A, 2% f o r m i c a c i d 5 B, n - b u t a n o l : e t h a n o l j w a t e r (40:11 * 1 9 ) ; C, n - b u t a n o l : p y i d i n e : w a t e r ( 6 : 4 : 3 ) j D, 80$ i s o p r o p a n o l j E, t - b u t a n o l : a c e t i c a c i d : water ( 3 : 1 : 1 ) . E l e c t r o p h o r e t o g r a m s were d e v e l o p e d a t pH 2, w i t h a n a p p l i e d v o l t a g e o f 3000 v o l t s , and a n i n i t i a l c u r r e n t o f 200 mamps* E l e c t r o p h o r e t o g r a m s were d e v e l o p e d f o r 30 mins u s i n g Whatman No. 3 chromatography paper.  -93d e r i v a t i v e s , i n which carbon 1 i s protected a l k a l i n e degradation,  have e s t a b l i s h e d t h a t  from sulphate  l i b e r a t i o n i s not a h y d r o l y t i c p r o c e s s but the r e s u l t of a b a s e - c a t a l y z e d  e l i m i n a t i o n of sulphate w i t h 36  concomitant p r o d u c t i o n  o f an anhydro r i n g  the  , This  e l i m i n a t i o n r e a c t i o n i s accompanied by i n v e r s i o n a t c a r b o n atom b e a r i n g  the s u l p h a t e  group. Complete  the  de-  s u l p h a t i o n with a l k a l i , of methyl beta-D-galactopyrano s l d e 6 - s u l p h a t e gave the 3,6-anhydro-D-galactopyrano s i d e i n a y i e l d o f about78$, the remainder b e i n g 37 methyl beta-D-galaetopyranoside s t a t e d t h a t a r b u t i g e n i n was  , I t has a l r e a d y been  r e a d i l y hydrolyzed  under  b a s i c c o n d i t i o n s . By c o n t r a s t , c o l d e t h a n o l i c s o l u t i o n s of a r b u t i n 6 - s u l p h a t e were u n a f f e c t e d  by IN NaOH.  When an aqueous s o l u t i o n o f a r b u t i n 6 - s u l p h a t e  was  b o i l e d f o r 10 mins under a l k a l i n e c o n d i t i o n s (0.1N the m i x t u r e was  NaOH)  shown, by paper chromatography, t o  c o n t a i n m a i n l y the u n a f f e c t e d  sulphate  plus small  traces  of a compound w h i c h gave a c o l o u r r e a c t i o n i d e n t i c a l t o a r b u t i n w i t h d i a z o t i z e d p - n i t r o a n i l i n e but f a i l e d  to  cochromatograph w i t h a r b u t i n . T h i s molecule i s most l i k e l y the anhydro d e r i v a t i v e of a r b u t i n . Triphenylmethylchloride rate chemistry  i s w e l l known i n c a r b o h y d -  f o r i t s v a l u e as a b l o c k i n g group w h i c h  s p e c i f i c a l l y r e a c t s w i t h the p r i m a r y h y d r o x y l a t o f g l u c o s e o r i t s d e r i v a t i v e s . The  a d d i t i o n o f a mg  triphenylmethylchloride to a p y r i d i n e s o l u t i o n of  C-6 of  -94a r b u t i g e n i n gave r i s e t o a new  a r b u t i n d e r i v a t i v e which  f a i l e d t o cochromatograph w i t h the t r i p h e n y l m e t h y l d e r i v 6-  a t i v e o f a r b u t i n . Under i d e n t i c a l c o n d i t i o n s a r b u t i n s u l p h a t e gave r i s e t o no new t h a t t h i s treatment  compound. I t i s u n l i k e l y  d i s p l a c e d a s u b s t i t u e n t from  of a r b u t i g e n i n , o t h e r w i s e  the new  C-6  d e r i v a t i v e should  t h e n have cochromatographed w i t h the t r i p h e n y l m e t h y l d e r i v a t i v e o f a r b u t i n . The  only other a l t e r n a t i v e i s  t h a t the s u b s t i t u e n t i s l o c a t e d a t some o t h e r p o s i t i o n . T h i s p o i n t l e a d s t o a c l o s e r s c r u t i n y o f Pridham s methods o f i d e n t i f i c a t i o n . The g l u c o s i d e was methylated,  first  then subjected to a l k a l i n e h y d r o l y s i s to  remove the s u b s t i t u e n t a t C-6,  and f i n a l l y  subjected  t o a c i d h y d r o l y s i s t o c l e a v e the g l u c o s i d i c bond. The  method used f o r m e t h y l a t i o n i n v o l v e d the use  of  methyl i o d i d e i n p y r i d i n e , w i t h s i l v e r oxide as a weak base. The  i n t e r p r e t a t i o n of methylation products  by  this  method has l o n g been open t o q u e s t i o n because o f the m i g r a t i o n o f s u b s t i t u e n t s towards C-6 38 c o n d i t i o n s . Thus Haworth glucose  under a l k a l i n e  o b t a i n e d 2-methyl beta-D-  from 2 , 3 , 4 , - t r i a c e t y l  beta-D-glucose, a f t e r  m e t h y l a t l n g by the above method, f o l l o w e d by h y d r o l y s i s of the a c e t y l g r o u p s . These r e s u l t s a r e most e a s i l y e x p l a i n e d by assuming t h a t d u r i n g the process  the a c e t y l r e s i d u e had m i g r a t e d  2 t o p o s i t i o n 6,  methylation from p o s i t i o n  thus l e a v i n g p o s i t i o n 2 a v a i l a b l e f o r  m e t h y l a t i o n . S i m i l a r l y , the a t t e m p t e d d e a c e t y l a t i o n of  - 9 5 -  the  hexacetate  resulted  i n a mixture  derivatives. identified the  2-,  o f the  3 - , and  6-caffeoyl 31  2 , 3 » 4 - t r i - 0 - m e t h y l g l u c o s e and c o n c l u d e d t h a t p o s i t i o n 6 of the g l u c o s e  The i d e n t i f i c a t i o n  of  sulphate  i o n s l e d him  c o n c l u s i o n t h a t t h e unknown was i n f a c t  sulphate  of  the  above,  there  t h a t P r i d h a m was m i s t a k e n i n h i s  doubt  identification.  The  s e n s i t i v i t y of arbutigenin to a l k a l i n e  probably substituted at moiety.  of t h i s  of  the  p o s i t i o n 2,  of  hydro-  arbutin,  3# o r 4 o f  The v e r y s m a l l amount o f p u r e  obtained by p r e p a r a t i v e lability  the material  c h r o m a t o g r a p h y , due t o  m o l e c u l e , made t h e  more  instructive  a n a l y t i c a l procedures such as I . R . s p e c t r o s c o p y , were n o t a v a i l a b l e  a n d t h e a u t h o r was u n a b l e  t h e compound.  the  crystallization  compound i m p o s s i b l e . T h u s t h e  characterize  6-  the  c a n be l i t t l e  l y s i s would suggest an e s t e r d e r i v a t i v e  glucose  to  phenolic glucoside.  C o n s i d e r i n g the  extreme  7  F o l l o w i n g a c i d h y d r o l y s i s , Pridham  s u b s t i t u t i o n was a t  moiety. the  of 2 - 0 - c a f f e o y l a r b u t i n by Haslam  to  etc  -96-  S l g n l f I c a n c e of the phenol g l u e o s y l a t i o n Phenolic  compounds a r e u b i q u i t o u s  reaction  i n n a t u r e . They  occur as a r e s u l t of b i o s y n t h e t i c a c t i v i t i e s , i n  the  t i s s u e s of a l l p l a n t s . They are found i n s o i l s , as a r e s u l t of the m i c r o b i a l d e g r a d a t i o n of l i g n i n s , t a n n i n s , 40 and a wide v a r i e t y of o t h e r p h e n o l i c  compounds  .  p-Hydroxybenzoic a c i d , v a n i l l i c a c i d , p-coumaric a c i d , and  f e r u l i c a c i d have been i s o l a t e d as the f r e e a c i d s ,  i n concentration  equivalent  t o 4 x 10  M s o i l solution .  A r b u t i n has been e s t i m a t e d t o o c c u r i n c o n c e n t r a t i o n s up t o 6% of dry w e i g h t i n the l e a v e s of V a c c i n i u m V l t l s 42 idaea . C a t e c h o l beta-D-glucoside,.has been o b t a i n e d i n y i e l d s of 0.2$  of the f r e s h w e i g h t i n G a u l t h e r i a  17  a d e n o t h r i x . The  r e l e a s e of these compounds i n t o the  s o i l as a consequence of the d e a t h and m a t e r i a l must be c o n s i d e r a b l e .  decay of  Increasingly,  plant  natural  w a t e r s a r e becoming contaminated by the l a c k of p r o p e r t r e a t m e n t of I n d u s t r i a l e f f l u e n t , i n d i s c r i m i n a t e use  of p e s t i c i d e s e t c , . Among the contaminants can  be  l i s t e d s i m p l e p h e n o l s . Thus R u s s i a n workers have i d e n t i f i e d hydroquinone, r e s o r c i n o l , p y r o c a t e c h o l  and  a 43  v a r i e t y of o t h e r s i m p l e phenols i n p o l l u t e d w a t e r s . Free phenols are e x t r e m e l y p h y t o t o x i c . Exposure t o q u i t e low c o n c e n t r a t i o n s  of hydroquinone or  catechol  can r a p i d l y b r i n g about d a r k e n i n g of p l a n t t i s s u e soon a f t e r w a r d s ,  the d e a t h of the p l a n t . The  appears t o c o n t a i n no s i n g l e e x p l a n a t i o n  and,  literature  f o r the  toxicity  -97of these compounds, but s e v e r a l i s o l a t e d s t u d i e s i m p l i c a t e phenols as i n t e r f e r i n g w i t h o x i d a t i v e phos44 45 phorylation • w i t h the p r o c e s s of m i t o s i s and w i t h 46 membrane f u n c t i o n due  to t h e i r surface a c t i v e nature  Hydroquinone, c a t e c h o l , and a v a r i e t y of simple have been found t o reduce the p e r m e a b i l i t y of  .  phenols erythro-  c y t e s , t o i n o r g a n i c phosphate, t h r o u g h p h y s i c o c h e m l c a l 47 a l t e r a t i o n s i n the c e l l membrane . The a c t i v e t r a n s p o r t 86 of a r b u t i n , as w e l l as Rb has been shown t o be 48 i n h i b i t e d by the presence of hydroquinone . F i n a l l y the r e l a t i v e ease w i t h w h i c h phenols may  become i n v o l v e d  i n hydrogen bonding, or o x i d a t i o n t o quinones has recognized  been  i n r e c e n t y e a r s as a major f a c t o r i n the 49  i n a c t i v a t i o n of enzyme p r e p a r a t i o n s Considering  .  the above i n f o r m a t i o n ,  s u r p r i s i n g t h a t phenols r a r e l y o c c u r , f r e e molecules. Instead  i t i s hardly  i n v i v o , as  the  t h e y are c o n j u g a t e d w i t h a v a r i e t y  of o r g a n i c compounds. The a d m i n i s t r a t i o n of f r e e phenols to higher plants r e s u l t s In t h e i r r a p i d conversion the c o r r e s p o n d i n g g l u c o s i d e s a u t h o r has  or g l u c o s e e s t e r s .  to  The  obsereved t h a t the a d m i n i s t r a t i o n of 2mg  of  hydroquinone t o a s i n g l e shoot of V l c i a f a b a r e s u l t e d i n a 98$ c o n v e r s i o n  to a r b u t i n w i t h i n 4 hrs. S i m i l a r l y  the uptake of hydroquinone by b a r l e y r o o t s , from 2.5  to  20.0  of 48  mM  s o l u t i o n s , r e s u l t e d i n a complete c o n v e r s i o n  the p h e n o l t o a r b u t i n a t a r a t e of 0.275 mg/hr/g r o o t s . The  most r e a s o n a b l e e x p l a n a t i o n f o r the  conjugation  -98-  of a d m i n i s t e r e d phenols i s t h e r e d u c t i o n of t h e i r t o x i c i t y . I n analogous f a s h i o n , phenols a d m i n i s t e r e d t o a n i m a l s a r e d e t o x i f i e d by c o n v e r s i o n t o t h e 50  c o r r e s p o n d i n g g l u c u r o n i d e s o r phenol s u l p h a t e s  .  The a u t h o r has observed t h a t when s o l u t i o n s o f h y d r o quinone o r c a t e c h o l a r e absorbed by V l c i a f a b a and Pltyrogramma calomelanos t h e e f f e c t s a r e f a r more damaging than when e q u i m o l a r s o l u t i o n s of t h e c o r r e s p o n d 51  ing g l u c o s i d e s are absorbed. S i m i l a r l y , the  Pridham  compared  g e r m i n a t i o n of seeds of V l c i a f a b a i n e q u i m o l a r s o l -  u t i o n s o f hydroquinone and a r b u t i n . G e r m i n a t i o n proceeded i n a r b u t i n , b u t i n hydroquinone t h e seeds b l a c k e n e d and died. 29  The survey by Pridham  as w e l l as t h a t by G l a s s and  52  Bohm  showed t h a t t h e phenol g l u c o s y l a t i o n r e a c t i o n  was r e s t r i c t e d t o h i g h e r p l a n t s . I n g e n e r a l t h e a l g a e , f u n g i , and b r y o p h y t e s a r e i n c a p a b l e o f g l u c o s y l a t i n g a d m i n i s t e r e d p h e n o l s . Many f u n g i have been shown t o be c a p a b l e of d e g r a d i n g phenols and m e t a b o l i z i n g t h e p r o d u c t s 53  as a s o l e source o f carbon  .  There i s some e v i d e n c e t o suggest t h a t a l a c k o f t h e a b i l i t y t o g l u c o s y l a t e a d m i n i s t e r e d phenols may be c o r r e l a t e d w i t h an a q u a t i c h a b i t a t . Thus a l l t h e a l g a e 29  examined by Pridham  r e a c t e d n e g a t i v e l y . The o n l y  f l o w e r i n g p l a n t s w h i c h r e a c t e d n e g a t i v e l y were a l s o a q u a t i c . Perhaps such p l a n t s a r e n o t s u b j e c t e d t o the same h i g h c o n c e n t r a t i o n s of phenols a s t e r r e s t r i a l p l a n t s .  -99-  51 Pridham has suggested t h a t a n o t h e r r e a s o n f o r g l u c o s y l a t i o n may be the s o l u b i l i z a t i o n of i n s o l u b l e phenolics.  Many n a t u r a l l y o c c u r r i n g phenols a r e i n s o l u b l e  i n aqueous s o l u t i o n s . G l y c o s y l a t i o n may r e p r e s e n t a p r e r e q u i s i t e f o r t r a n s l o c a t i o n or f u r t h e r metabolism.  54 The same a u t h o r  has r e p o r t e d  t h a t t h e phloem t r a n s -  l o c a t i o n of p h e n o l i c g l u c o s i d e s o c c u r s more r e a d i l y than t h e t r a n s l o c a t i o n of t h e c o r r e s p o n d i n g a g l u c o n e s .  51  Pridham  has a l s o brought a t t e n t i o n t o the g r e a t e r  s t a b i l i t y of g l y c o s i d e s  than the a g l y c o n e s of s i m p l e  p h e n o l s . Thus a t a l k a l i n e pH o r under the i n f l u e n c e of p o l y p h e n o l o x i d a s e s , s i m p l e phenols may be r a p i d l y o x i d i z e d t o quinones w h i c h can b i n d t o p r o t e i n s o r p o l y m e r i z e . The c o r r e s p o n d i n g g l y c o s i d e s s t a b l e under these  are quite  conditions.  L a s t l y , the importance of g l y c o s i d e s  as p o t e n t i a l  sources o f a n t i m i c r o b i a l compounds has been w e l l e s t a b l i s h e d . The d i s r u p t i o n of normal c o m p a r t m e n t a l i z a t i o n i s thought t o b r i n g t o g e t h e r b e t a - g l u c o s i d a s e s and t h e i r phenolic  substrates,  f o l l o w i n g i n v a s i o n of the h o s t  t i s s u e . The r e s i s t a n c e of c e r t a i n v a r i e t i e s of Pyrus communis t o f i r e b l l g h t has been a t t r i b u t e d t o the r e l e a s e of hydroquinone f o l l o w i n g i n v a s i o n by t h e  55  parasite  .  The above d i s c u s s i o n p r e s e n t s s e v e r a l  explanations  f o r t h e phenol g l u c o s y l a t i o n r e a c t i o n i n h i g h e r p l a n t s . I t i s e x t r e m e l y d i f f i c u l t , however, t o r a t i o n a l i z e the  -100-  f u r t h e r m o d i f i c a t i o n s t h a t have been observed i n t h i s s t u d y . The  t o x i c i t y of p h e n o l i c g l u c o s i d e s can  hardly  be reduced by subsequent g l u c o s y l a t i o n (as i n the forma t i o n of g e n t i o b i o s i d e s ) or o t h e r m o d i f i c a t i o n ( i n the case of a r b u t i g e n i n f o r m a t i o n ) . However, s i n c e the  very  presence of these f o r e i g n phenols i n the t i s s u e s represents  a d e p a r t u r e f r o m normal c o n d i t i o n s , these  m o d i f i c a t i o n s may no l o n g e r m a i n t a i n organization.  be I n d i c a t i v e of an organism which i t s p h y s i o l o g i c a l and  biochemical  can  101  LITERATURE CITED 1.  Harborne, J.B. and Simmonds, N.W. 1 9 6 4 . Chap. 3 B i o c h e m i s t r y o f P h e n o l i c Compounds. Academic P r e s s . London and New York.  2.  K a r r e r , W, 1958. K o n s t i t u t i o n und Vorkommen d e r organischen P f l a n z e n s t o f f e . Birkhauser-Verlag, B a s e l and S t u t t g a r t .  3. 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