Plant Polyphenols and Their Association with Proteins - American

7 Plant Polyphenols and Their Association with Proteins

Downloaded by NORTH CAROLINA STATE UNIV on September 14, 2013 | http://pubs.acs.org Publication Date: January 20, 1983 | doi: 10.1021/bk-1983-0208.ch007

J O H N M c M A N U S , T E R E N C E H. L I L L E Y , and E D W I N H A S L A M University of Sheffield, Department of Chemistry, Sheffield S3 7HF, United Kingdom

Plant polyphenols (syn. vegetable tannins) which complex strongly with proteins are based on two structural types. The first group, the proanthocyanidins, possess an oligomeric flavan-3-ol structure and the second class is composed of esters of gallic acid, (R) and (S)-hexahydroxydiphenic acid, and their derivatives usually with D-glucose. Polyphenols associate with proteins principally by intermolecular hydrogen bonding. A hypothesis is advanced to explain the propensity of polyphenols, as their molecular size increases, to precipitate proteins from solution. An important corollorary of this theory is that simple phenols (M.Wt. < 200) should display similar behaviour if they can be maintained in solution at sufficiently high concentrations. For a c e n t u r y and more t h e e l u c i d a t i o n o f t h e c h e m i s t r y o f a n a t u r a l p r o d u c t (J_, s e c o n d a r y m e t a b o l i t e ) has been a d o m i n a n t theme of organic chemistry. Today t h e e m p h a s i s has c h a n g e d . The q u e s t i o n - "what i s t h e r o l e o f s e c o n d a r y m e t a b o l i s m i n t h e l i f e o f p l a n t s and m i c r o - o r g a n i s m s ? " - i s one t o w h i c h i n v e s t i g a t o r s i n c r e a s i n g l y address themselves. The p r o b l e m r e m a i n s e s s e n t i a l l y u n r e s o l v e d a l t h o u g h numerous i d e a s have been p u t f o r w a r d . Several p r o p o s i t i o n s c e n t r e on t h e s u g g e s t i o n t h a t i t i s t h e p r o c e s s o f secondary metabolism and n o t , i n t h e general c a s e , t h e secondary m e t a b o l i t e s themselves which a r e o f importance t o t h e organism (2). T h e s e s u g g e s t i o n s do n o t e x c l u d e t h e p o s s i b i l i t y , indeed p r o b a b i l i t y , that the d i s t i n c t i v e p r o p e r t i e s o f i n d i v i d u a l seconda r y m e t a b o l i t e s have, over t h e course o f e v o l u t i o n , secured a p a r t i c u l a r n i c h e f o r an o r g a n i s m i n t h e l i v i n g w o r l d . Contrasting w i t h t h i s v i e w p o i n t a r e t h o s e w h i c h s u g g e s t t h a t an o r g a n i s m ' s c a p a c i t y t o d e f e n d i t s e l f a g a i n s t p r e d a t i o n by some o r g a n i s m s and t o a t t r a c t o t h e r s i n c l u d e d t h e e v o l u t i o n o f an a b i l i t y t o s y n t h e s i s e an a r r a y o f s e c o n d a r y m e t a b o l i t e s w h i c h i n a p p r o p r i a t e c i r c u m s t a n c e s m i g h t r e p e l o r a t t r a c t o t h e r o r g a n i s m s (3 > ^ 0 • 0097-6156/83/0208-0123$06.00/0 © 1983 American Chemical Society

In Plant Resistance to Insects; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

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P o l y p h e n o l s are a d i s t i n c t i v e group o f h i g h e r p l a n t secondary m e t a b o l i t e s ( 5 ) . Two i m p o r t a n t c l a s s e s - t h e p r o a n t h o c y a n i d i n s ( 1 , F i g u r e 1) and e s t e r s o f g a l l i c a c i d and (R) and ( S ) - h e x a d y d r o x y d i p h e n i c a c i d ( F i g u r e 5) a r e u n i q u e and t h e i r mode o f b i o s y n t h e s i s i n the p l a n t e n a b l e s m o l e c u l e s o f m o l e c u l a r w e i g h t > 500 and i n many c a s e s o f a t l e a s t 2-3,000 t o be formed. These m o l e c u l e s , i n r e l a t i v e l y low c o n c e n t r a t i o n s , m o r e o v e r p o s s e s s the impo r t a n t p r o p e r t y o f p r e c i p i t a t i n g p r o t e i n s , p o l y s a c c h a r i d e s and some a l k a l o i d s f r o m s o l u t i o n . This association with proteins i s the b a s i s o f t h e use o f p o l y p h e n o l s ( s y n . t a n n i n s ) as a g e n t s f o r the c o n v e r s i o n o f raw a n i m a l s k i n s t o d u r a b l e , impermeable l e a t h e r (6). I t has a l s o been r e c o g n i s e d as u n d e r l y i n g many o t h e r phenomena a s s o c i a t e d w i t h p l a n t t i s s u e s . Thus i t i s h e l d t o be r e s p o n s i b l e f o r the a s t r i n g e n c y o f u n r i p e f r u i t , b e v e r a g e s s u c h as t e a and w i n e , f o r t h e i m p a i r e d n u t r i t i o n a l c h a r a c t e r i s t i c s o f some f o o d s and f o r the i n a c t i v a t i o n o f m i c r o b i a l enzymes and p l a n t viruses. The c a p a c i t y o f p l a n t s t o m e t a b o l i s e t h e s e two c l a s s e s o f p o l y p h e n o l i s a p r i m i t i v e c h a r a c t e r i s t i c t h a t has t e n d e d t o become l o s t w i t h i n c r e a s i n g p h y l o g e n e t i c s p e c i a l i s a t i o n . Accordi n g t o B a t e - S m i t h (7) t h e i r i m p o r t a n c e t o t h e p l a n t l i e s i n t h e i r e f f e c t i v e n e s s as r e p e l 1 a n t s t o p r e d a t o r s b r o u g h t a b o u t by t h e i r a b i l i t y t o p r e c i p i t a t e p r o t e i n s w h e t h e r t h i s be an e x t r a c e l l u l a r m i c r o b i a l enzyme o r the s a l i v a r y p r o t e i n o f a b r o w s i n g a n i m a l . P r o a n t h o c y a n i d i n s and P r o c y a n i d i n s - In a c l a s s i c a l s t u d y B a t e S m i t h (8) used t h e p a t t e r n s o f d i s t r i b u t i o n o f the t h r e e p r i n c i pal c l a s s e s o f p h e n o l i c m e t a b o l i t e s , which are found i n the l e a v e s o f p l a n t s , as a b a s i s f o r c l a s s i f i c a t i o n . The b i o s y n t h e s i s of these phenols - ( i ) p r o a n t h o c y a n i d i n s ; ( i i ) g l y c o s y l a t e d f l a v o n o l s and ( i i i ) h y d r o x y c i n n a m o y l e s t e r s - i s b e l i e v e d t o be a s s o c i a t e d w i t h the d e v e l o p m e n t i n p l a n t s o f t h e c a p a c i t y t o s y n t h e s f s e t h e s t r u c t u r a l p o l y m e r l i g n i n by the d i v e r s i o n f r o m p r o t e i n s y n t h e s i s o f the a m i n o - a c i d s L - p h e n y l a l a n i n e and L - t y r o sine. V a s c u l a r p l a n t s t h u s e m p l o y one o r more o f t h e p - h y d r o x y cinnamyl a l c o h o l s ( 2 , 3 , and 4 ) , w h i c h a r e d e r i v e d by e n z y m i c r e d u c t i o n (NADH) o f the coenzyme A e s t e r s o f the c o r r e s p o n d i n g h y d r o x y c i n n a m i c a c i d s , as p r e c u r s o r s t o l i g n i n . The same coenzyme A e s t e r s a l s o f o r m the p o i n t s o f b i o s y n t h e t i c d e p a r t u r e f o r the t h r e e g r o u p s o f p h e n o l i c m e t a b o l i t e s ( ? , i i , i i i ) , F i g u r e 1. The two p r i n c i p a l c l a s s e s o f p r o a n t h o c y a n i d i n s f o u n d (10) i n p l a n t t i s s u e s a r e t h e p r o c y a n i d i n s ( 1 , R = H) and the p r o d e l p h i n i d i n s ( 1 , R = OH). P r o a n t h o c y a n i d i n s o f mixed a n t h o c y a n i d i n c h a r a c t e r ( 1 , R = H o r OH) have been n o t e d . In any t i s s u e where p r o a n t h o c y a n i d i n s y n t h e s i s o c c u r s t h e r e i s i n v a r i a b l y found a range o f m o l e c u l a r s p e c i e s - f r o m the monomeric f l a v a n - 3 - o l s ( c a t e c h i n s , g a l l o c a t e c h i n s ) t o t h e p o l y m e r i c f o r m s (1) and b i o s y n t h e t i c work (11) s u g g e s t s a v e r y c l o s e r e l a t i o n s h i p between t h e m e t a b o l i s m o f t h e p a r e n t f l a v a n - 3 " o l and the s y n t h e s i s o f p r o a n t h o c y a n i d i n s , F i g u r e 4.



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The m a j o r i t y o f t h e s t r u c t u r a l and b i o s y n t h e t i c work i n t h i s f i e l d has been d i r e c t e d t o w a r d s a n u n d e r s t a n d i n g o f t h e p r o c y a n i d i n s ( 1 , R = H) - t h e m a j o r g r o u p o f n a t u r a l p r o a n t h o c y a n i d i n s , ( 1 1 ) F r u i t b e a r i n g p l a n t s have p r o v e d t o be p a r t i c u l a r l y r i c h s o u r c e s o f o l i g o m e r i c condensed p r o c y a n i d i n s ( 1 1 , 12, 13) and s t u d i e s have c o n c e n t r a t e d upon t h e f o u r m a j o r n a t u r a l l y o c c u r r i n g d i m e r i c p r o c y a n i d i n s ( B - 1 , B-2, B-3 and B-4, F i g u r e 2) w h i c h have been i s o l a t e d f r o m f r u i t , f r u i t p o d s , seeds a n d seed s h e l l s , l e a v e s and o t h e r t i s s u e s o f a w i d e r a n g e o f p l a n t s . The p r o c y a n i d i n s o c c u r f r e e and u n g l y c o s y l a t e d and a l m o s t i n v a r i a b l y t h e y a r e found w i t h one o r b o t h o f t h e p a r e n t f 1 a v a n - 3 - o l s , ( + ) - c a t e c h i n o r ( - ) - e p i c a t e c h i n , F i g u r e 2. The p a t t e r n s o f o c c u r r e n c e o f t h e p r o c y a n i d i n s a r e most e f f e c t i v e l y r e v e a l e d by h p l c o r by two d i m e n s i o n a l paper c h r o m a t o g r a p h y and t h e y may be used a s a t a x o n o m i c g u i d e ( 1 1 ) . The a b s o l u t e s t e r e o c h e m i s t r y i n p r o c y a n i d i n s B-1 and B-2 was d e t e r m i n e d a s kR and t h e C-C i n t e r f l a v a n bond t h u s o c c u p i e s a q u a s i - a x i a l bond a t C-4 on t h e " u p p e r " f l a v a n - 3 - o l h e t e r o c y c l i c r i n g (]k). Conversely the absolute stereochemistry i n p r o c y a n i d i n s B-3 and B-4 was shown t o be and h e r e t h e i n t e r f l a v a n bond i s t h u s i n a q u a s i - e q u a t o r i a l p o s i t i o n on t h e h e t e r o c y c l i c r i n g ( 1 4 ) . Thus t h e p r i n c i p a l p r o c y a n i d i n d i m e r s ( F i g u r e 2) a l l p o s s e s s a t r a n s o r i e n t a t i o n o f t h e C~3 h y d r o x y l group and t h e f l a v a n s u b s t i t u e n t a t C-4. D e s p i t e t h e s u c c e s s f u l a p p l i c a t i o n o f s p e c t r o s c o p i c methods t o the s t r u c t u r e d e t e r m i n a t i o n o f t h e d i m e r i c p r o c y a n i d i n s anomalous f e a t u r e s o f t h e s p e c t r a l e d t o s p e c u l a t i o n (_V2, J 5 , J 6 ) t h a t r e s t r i c t e d r o t a t i o n e x i s t e d a b o u t t h e i n t e r f l a v a n bond. A d e t a i l e d s t u d y (14) c o n f i r m e d t h i s h y p o t h e s i s and d e f i n e d two d i f f e r e n t forms o f h i n d e r e d r o t a t i o n a s s o c i a t e d w i t h t h e two g r o u p s o f p r o c y a n i d i n d i m e r s (B-1 and B-2, kR - a b s o l u t e c o n f i g u r a t i o n ) and (B-3 and B-4, 4 S - a b s o l u t e c o n f i g u r a t i o n ) . Examina t i o n o f m o l e c u l a r models shows t h a t , a l t h o u g h s e v e r a l c o n f o r m a t i o n s a r e a c c e s s i b l e by i n t e r f l a v a n bond r o t a t i o n , t h e r e e x i s t s one p r e f e r r e d conformation f o r each o f the p a i r s o f n a t u r a l p r o c y a n i d ins. I f t h e s e a r e v i e w e d f r o m d i f f e r e n t a n g l e s t h e y bear an a l m o s t o b j e c t t o m i r r o r image r e l a t i o n s h i p ; the structures are q u a s i - e n a n t i o m e r i c ( F i g u r e 3). Elaboration of the oligomeric p r o c y a n i d i n s (J^, R = H) by t h e a d d i t i o n o f f u r t h e r i d e n t i c a l f l a v a n - 3 - o l u n i t s , b e a r i n g i n mind t h e c o n f o r m a t i o n a l r e s t r a i n t s a b o u t t h e i n t e r f l a v a n bond, l e a d s t o two g e n e r a l s t r u c t u r e s w h i c h may a d o p t h e l i c a l c o n f o r m a t i o n s w i t h o p p o s i t e h e l i c i t i e s ( 1 1 ) . The c e n t r a l c o r e o f t h e s e p o l y m e r s i s compcse d o f r i n g s A a n d C o f t h e f l a v a n r e p e a t u n i t and r i n g B - t h e o r t h o - d i h y d r o x y p h e n y l r i n g - p r o j e c t s l a t e r a l l y from t h i s c o r e . E a r l i e r p r o p o s a l s (_12^ J_3, 17) and t h e r e s u l t s o f b i o s y n t h e t i c e x p e r i m e n t s (18) have been adumbrated i n t o a scheme o f b i o s y n t h e s i s f o r t h e p r o c y a n i d i n s ( F i g u r e k) i n w h i c h i t i s s u g g e s t e d t h a t t h e y a r e formed a s b y p r o d u c t s d u r i n g t h e f i n a l s t a g e o f t h e s y n t h e s i s o f t h e p a r e n t f 1 a v a n - 3 ~ o l s t r u c t u r e s , ( + ) - c a t e c h i n and ( - ) e p i c a t e c h i n (_[_[, _18) . A two s t e p r e d u c t i o n o f t h e f 1 av-3~en-3-ol


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lignin

(1 Figure 1.

R = H or

OH )

Phenol biosynthesis in higher plants.



Figure 2.

Principal naturally occurring dimeric procyanidins.


to



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B-4 Figure 4.

5

6

Biosynthesis of procyanidins.


5


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Is p o s t u l a t e d . I f t h e s u p p l y o f NADPH ( s a y ) i s r a t e l i m i t i n g t h e i n t e r m e d i a t e C-4 c a r b o c a t i o n s (3S o r 3R s t e r e o c h e m i s t r y ) m i g h t e s c a p e f r o m t h e enzyme a c t i v e s i t e and r e a c t w i t h t h e r e d u c t i o n p r o d u c t , t h e f l a v a n - 3 - o l , t o p r o d u c e d i m e r s , t r i m e r s ... and e v e n t u a l l y polymers. The h y p o t h e s i s h a s been e l a b o r a t e d t o a c c o u n t f o r t h e v a r i o u s s t e r e o c h e m i c a l f e a t u r e s (11) and has been s u p p o r t e d by i n v i t r o b i o m i m e t i c e x p e r i m e n t s . The b a l a n c e between t h e m e t a b o l i c f l u x t o t h e f l a v - 3 - e n - 3 - o l and t h e r a t e o f s u p p l y o f t h e b i o l o g i c a l r e d u c t a n t (NADPH) p r o b a b l y d e t e r m i n e s t h e b a l a n c e between t h e l o w e r and h i g h e r o l i g o m e r i c forms o f t h e p r o c y a n i d i n s i n each p l a n t t i s s u e . Thus t i s s u e s w i t h a h i g h f l u x and p o o r s u p p l y o f NADPH w i l l c o n t a i n p r e d o m i n a n t l y t h e h i g h e r o l i g o m e r s - f o r example t h e seed c o a t o f sorghum ( 1 9 ) . T h i s f a c e t o f p r o a n t h o c y a n i d i n m e t a b o l i s m was n o t e d by e a r l y w o r k e r s (20) who r e c o g n i s e d t h e p r e s e n c e i n p l a n t s o f l e u c o a n t h o cyanins (proanthocyanidins) o f q u i t e d i f f e r e n t s o l u b i l i t i e s . With i n c r e a s i n g degrees o f p o l y m e r i s a t i o n the p r o a n t h o c y a n i d i n s a r e more d i f f i c u l t t o s o l u b i l i s e i n aqueous and a l c o h o l i c media and t h o s e w h i c h may be c o a x e d i n t o s o l u t i o n may have m o l e c u l a r w e i g h t s up t o 7,000 - c o r r e s p o n d i n g t o s t r u c t u r e s s u c h a s ( 1 ) where n s 18. R e c e n t work has c o n c e n t r a t e d upon t h e s t r u c t u r a l e x a m i n a t i o n o f these polymers. A l t h o u g h B a t e - S m i t h (8) g e n e r a l l y d i s c o u n t e d a c o n n e c t i o n w i t h l i g n i n , an e x a m i n a t i o n o f t h e p r o a n t h o c y a n i d i n p o l y m e r s , t o a s c e r t a i n i f t h e y do p l a y a s t r u c t u r a l r o l e w o u l d be v e r y v a l u a b l e . E s t e r s o f G a l l i c and H e x a h y d r o x y d i p h e n i c A c i d s - An a c u t e o b s e r v a t i o n made by B a t e - S m i t h ( 8 ) i n h i s t a x o n o m i c work was t h e complete absence from p l a n t t i s s u e s o f 3 , ^ , 5 " t r i h y d r o x y c i n n a m i c acid. In s i t u a t i o n s where i t s o c c u r r e n c e m i g h t w e l l have been p r e d i c t e d , he f o u n d h e x a h y d o r x y d i p h e n i c a c i d and i t s d e r i v a t i v e s ( F i g u r e 5 ) . R e c e n t work has d e m o n s t r a t e d t h e u b i q u i t y i n p l a n t s o f e s t e r s o f R and S h e x a h y d r o x y d i p h e n i c a c i d w i t h D - g l u c o s e and o f t h e i r presumed b i o g e n e t i c p r e c u r s o r s t h e g a l l o y l - D - g l u c o s e s (22, 2 3 ) . G a l l i c a c i d ( F i g u r e 5) t h u s o c c u p i e s a d i s t i n c t i v e p o s i t i o n i n p h e n o l i c metabolism i n p l a n t s . Almost always i t o c c u r s i n e s t e r form but i n c o n t r a s t t o o t h e r n a t u r a l p h e n o l i c a c i d s , w h i c h a r e i n v a r i a b l y f o u n d a s mono- and o c c a s i o n a l l y b i s e s t e r s w i t h p o l y o l s , g a l l i c a c i d i s e n c o u n t e r e d i n a range o f e s t e r i f i e d forms f r o m t h e s i m p l e m o n o e s t e r s t o t h e c o m p l e x p o l y e s t e r s w i t h D - g l u c o s e whose m o l e c u l a r w e r g h t s e x t e n d t o a t l e a s t 2,000. The s t r u c t u r a l p a t t e r n s d i s c e r n e d amongst g a l l i c a c i d m e t a b o l i t e s a r e n e v e r t h e l e s s h i g h l y r e m i n i s c e n t o f t h o s e found i n o t h e r v a r i a n t s o f secondary metabolism i n o t h e r organisms - e.g. p o l y k e t i d e s i n moulds and f u n g i . G a l l i c a c i d i s most commonly a s s o c i a t e d w i t h D - g l u c o s e . Many p l a n t s m e t a b o l i s e s i m p l e e s t e r s and t h e r e a p p e a r s t o be a p r e d i s p o s i t i o n f o r c e r t a i n p o s i t i o n s o f e s t e r i f i c a t i o n on t h e D - g l u c o s e p y r a n o s e c o r e (1 > 6 > 2 > 3 , * 0 . The m e t a b o l i s m o f 3 - p e n t a k i s g a l l o y l - D - g l u c o s e (Figure5) r e p r e s e n t s something o f a m e t a b o l i c


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Carbohydrate OH

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Glucose

3

HO^^OH

OH

Group 1

G

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its

bis tris

1,2,6 1,2,3,6

Gallic acid

1,2^,6 tetrakis

G -Rrntagalloy I-D-glucose .OG Group II OGi -2H

depside \

1

~

Hp

A X

GO^^h^OG 0

3

o

G

A-Gallotannins'

X

0

GO

OG

/?Y

6 GO

i OG V

/ ™ I •2H

-1

OG G -2H

° ^ ^ o c

1.6 -2,4

GO |

I

I

M . GO

0 0 G^GX

0'

OG

G-G

Dimers *>

\ G-G

Trimers ?

Hexahydroxydiphenic acid Figure 5.

Dimers,Trimers ? Ellagitannins - — C

B

Metabolism of gallic acid and hexahydroxydiphenic acid.


132

PLANT RESISTANCE TO

w a t e r s h e d and f r o m t h i s p o i n t a t l e a s t t h r e e d i f f e r e n t b i o s y n t h e t i c pathways d i v e r g e . From a t a x o n o m i c p o i n t o f v i e w t h e s e pathways a r e q u i t e d i s t i n c t (2k). One pathway p r o c e e d s t o f o r m d e p s i d e m e t a b o l i t e s ( g a l l o t a n n i n s , g r o u p M A ) . Here f u r t h e r g a l l i c a c i d m o l e c u l e s a r e e s t e r i f i e d as m-depsides t o 3 - p e n t a k i s g a l l o y l - D - g l u c o s e , p r e f e r e n t i a l l y a t p o s i t i o n s 3, and 6 . This s u p p o r t s t h e v i e w o r i g i n a l l y e x p r e s s e d by Emil F i s c h e r (25) t h a t these e s t e r s a r e not o n l y m i x t u r e s o f isomers but a l s o o f substances of d i f f e r i n g e m p i r i c a l formulae. A s e c o n d and w i d e l y o b s e r v e d p a t t e r n o f m e t a b o l i s m o f $p e n t a k i s - g a l l o y l - D - g l u c o s e i s t h a t ( M B , F i g u r e 5) i n w h i c h t h e s u b s t r a t e i s f u r t h e r t r a n s f o r m e d by o x i d a t i v e c o u p l i n g o f p a i r s o f a d j a c e n t g a l l o y l e s t e r g r o u p s ( 2 , 3 and 4 , 6) on t h e D - g l u c o pyranose r i n g t o g i v e hexahydroxydiphenoyl e s t e r s . Further m e t a b o l i s m o f t h e s e i n t e r m e d i a t e s t h e n a l s o o c c u r s by i n t e r m o l e c u l a r C-0 o x i d a t i v e c o u p l i n g t o f o r m ' d i m e r s ' - s u c h a s (£) i s o l a t e d f r o m Rubus s p e c i e s - and p o s s i b l y ' t r i m e r s ' . Oxidative c o u p l i n g o f g a l l o y l e s t e r groups takes p l a c e w i t h t h e p r e c u r s o r adopting the e n e r g e t i c a l l y preferred ( Ci) conformation o f the sugar r i n g . Coupled w i t h t h e o b s e r v a t i o n that t h i s i s t h e p r e d o m i n a n t mode o f f u r t h e r o x i d a t i v e m e t a b o l i s m o f g a l l o y l e s t e r s i n p l a n t s , i t a l s o seems r e a s o n a b l e t o c o n c l u d e t h a t i t i s t h e e n e r g e t i c a l l y p r e f e r r e d pathway. A t h i r d and r e l a t i v e l y m i n o r pathway o f m e t a b o l i s m o f 3 p e n t a k i s - g a l l o y l - D - g l u c o s e ( I I C , F i g u r e 5) o c c u r s i n some p l a n t s . Here o x i d a t i v e m e t a b o l i s m t a k e s p l a c e v i a t h e e n e r g e t i c a l l y u n f a v o u r a b l e Ci+ c o n f o r m a t i o n o f t h e s u b s t r a t e and c o u p l i n g o f t h e g a l l o y l e s t e r g r o u p s o c c u r s 1, 6 o r 3 , 6 t o g i v e h e x a h y d r o x y diphenoyl e s t e r s o r 2, k t o give the dehydrohexahydroxydiphenoyl ester. The compound g e r a n i i n i s a key f i g u r e i n t h i s pathway o f m e t a b o l i sm ( 2 6 ) . 1


INSECTS

1

4

l

Comparative Aspects o f Polyphenol Metabolism Proanthocyanidins and t h e c o m p l e x e s t e r s o f g a l l i c and h e x a h y d r o x y d i p h e n i c a c i d show many s t r u c t u r a l s i m i l a r i t i e s a s p l a n t m e t a b o l i t e s . The shape and s i z e o f t h e e s t e r (5) i s t h u s v e r y s i m i l a r t o t h a t o f a p r o a n t h o c y a n i d i n hexamer ( 1 , n = 4 ) . The most s t r i k i n g f e a t u r e o f b o t h s t r u c t u r e s h o w e v e r ^ i s t h e manner i n w h i c h f r e e p h e n o l i c groups a r e d i s t r i b u t e d over t h e s u r f a c e o f t h e m o l e c u l e p r o v i d i n g a s t r u c t u r e w i t h the i n b u i l t c a p a c i t y f o r m u l t i d e n t a t e attachment t o o t h e r s p e c i e s by h y d r o g e n b o n d i n g . A c u r i o u s but p e r h a p s s i g n i f i c a n t o b s e r v a t i o n i s t h a t , a l t h o u g h several plant f a m i l i e s retain the a b i l i t y to biosynthesise d i f f e r e n t t y p e s o f complex p o l y p h e n o l , r a r e l y a r e both b i o s y n t h e t i c c a p a b i l i t i e s d i s p l a y e d m a x i m a l l y . More o f t e n one p a r t i c u l a r s p e c i e s s p e c i a l i s e s i n one p a r t i c u l a r mode. Thus i n t h e E r i c a c e a e many p l a n t s a r e r i c h s o u r c e s o f p r o a n t h o c y a n i d i n s b u t A r c t o s t a p h y l o s u v a - u r s i has o n l y a m i n i m a l p r o a n t h o c y a n i d i n b i o s y n t h e t i c c a p a c i t y but c o m b i n e s w i t h t h i s a v e r y h i g h l e v e l o f g a l l i c a c i d metabolism.



7. MCMANUS ET AL. Plant Polyphenols


133

134



Polyphenol

Interactions with Proteins

S t u d i e s o f t h e a s s o c i a t i o n o f p o l y p h e n o l s w i t h p r o t e i n s have a long h i s t o r y (27). Loomis (28) h a s s u c c i n c t l y summarised t h e c o n c l u s i o n s o f t h i s e a r l i e r work. The p r i n c i p a l means whereby p r o t e i n s and p o l y p h e n o l s a r e t h o u g h t t o r e v e r s i b l y c o m p l e x w i t h one a n o t h e r a r e ( i ) h y d r o g e n b o n d i n g , ( i i ) i o n i c i n t e r a c t i o n s and ( i i i ) hydrophobic i n t e r a c t i o n s . W h i l s t t h e major t h r u s t i n e a r l i e r work was t o e m p h a s i z e t h e p a r t p l a y e d by i n t e r m o l e c u l a r h y d r o g e n b o n d i n g i n t h e c o m p l e x a t i o n , H o f f (29) has drawn a t t e n t i o n t o t h e p o s s i b i l i t y t h a t h y d r o p h o b i c e f f e c t s may d o m i n a t e t h e a s s o c i a t i o n between t h e two s p e c i e s . R e c e n t o b s e r v a t i o n s however now p e r m i t a h y p o t h e s i s t o be advanced t o e x p l a i n t h e p r o p e n s i t y o f c o m p l e x p o l y p h e n o l s t o p r e c i p i t a t e p r o t e i n s from aqueous s o l u t i o n ( 3 0 ) . Two s i t u a t i o n s may be e n v i s a g e d . At low p r o t e i n c o n c e n t r a t i o n s t h e p o l y p h e n o l a s s o c i a t e s - p r i n c i p a l l y by h y d r o g e n b o n d i n g v i a t h e o r t h o d i h y d r o x y p h e n y l g r o u p s - a t one o r more s i t e s on t h e p r o t e i n molecule forming a r e l a t i v e l y hydrophobic surface layer (Figure 6a). A g g r e g a t i o n and p r e c i p i t a t i o n t h e n e n s u e . Where t h e i n i t i a l p r o t e i n c o n c e n t r a t i o n i s high the hydrophobic s u r f a c e layer i s formed by c r o s s - l i n k i n g o f d i f f e r e n t p r o t e i n m o l e c u l e s by t h e multidentate polyphenols (Figure 6b). P r e c i p i t a t i o n then f o l l o w s as o u t l i n e d , v i d e s u p r a . T h i s t e n d e n c y t o c r o s s - l i n k p r o t e i n molecules e x p l a i n s the changing s t o i c h i o m e t r y o f the aggregates i n r e l a t i o n t o t h e i n i t i a l p r o t e i n c o n c e n t r a t i o n . An i n t e r e s t i n g c o r o l l o r a r y o f t h i s h y p o t h e s i s i s t h a t s i m p l e phenols such a s p y r o g a l l o l and r e s o r c i n o l s h o u l d a l s o be c a p a b l e o f p r e c i p i t a t i n g p r o t e i n s from s o l u t i o n i f t h e y c a n be m a i n t a i n e d i n s o l u t i o n a t c o n c e n t r a t i o n s s u f f i c i e n t t o push t h e e q u i l i b r i u m i n f a v o u r o f t h e p r o t e i n - p h e n o l c o m p l e x and t h u s e s t a b l i s h a h y d r o p h o b i c l a y e r o f s i m p l e phenol m o l e c u l e s on t h e p r o t e i n s u r f a c e ( F i g u r e 6 c ) . F o r many s i m p l e p h e n o l s t h e l i m i t i s d e t e r m i n e d by t h e i r own _ s o l u b i l i t y but i t c a n be a c h i e v e d i n w a t e r w i t h BSA (3 x 10 m o l a l ) and p y r o g a l l o l (1 m o l a l ) and r e s o r c i n o l (2 m o l a l ) . 5

P o s t s c r i pt Complex p l a n t p o l y p h e n o l s r e a d i l y and r e v e r s i b l y a s s o c i a t e w i t h p r o t e i n s and t h e y c a n p r e c i p i t a t e them f r o m d i l u t e s o l u t i o n . T h i s p r o p e r t y i s however a d i r e c t e x t r a p o l a t i o n o f t h e c h a r a c t e r i s t i c s o f simple phenols themselves. The s t r u c t u r a l d e v i c e r e p r e s e n t e d by t h e p l a n t p o l y p h e n o l s t o a g r e a t e x t e n t o b v i a t e s t h e need f o r a h i g h m o l a l c o n c e n t r a t i o n o f phenol and i t e m b o d i e s t h e added f e a t u r e t h a t c r o s s - l i n k i n g between d i f f e r e n t m o l e c u l a r a g g r e g a t e s may be r e a d i l y a c h i e v e d . The q u e s t i o n s posed i n i t i a l l y r e m a i n . What p u r p o s e do t h e s e molecules serve i n p l a n t metabolism? Does t h e i r p r e s e n c e p o i n t f o r e x a m p l e t o t h e need by p l a n t s e i t h e r now o r d u r i n g t h e c o u r s e of e v o l u t i o n f o r s u c h t a i l o r e d m o l e c u l e s t o r e v e r s i b l y c o a t t h e



(a) Figure 6.

Protein-polyphenol

(b)

association.


(c)

136



s u r f a c e o f p r o t e i n s and hence m o d i f y t h e i r a c t i v i t y a t p a r t i c u l a r p h a s e s o f d e v e l o p m e n t ? Answers t o t h e s e p r o b l e m s w i l l o n l y emerge as and when a g r e a t d e a l more i s known c o n c e r n i n g t h e e n z y m o l o g y o f p r o c e s s e s g o v e r n i n g t h e f o r m a t i o n o f t h e s e m e t a b o l i t e s , how t h e s e p r o c e s s e s a r e r e g u l a t e d and c o n t r o l l e d , and t h e i r r e l a t i o n s h i p t o the network o f p r i m a r y metabolism from which they d e v o l v e . A t r u l y f o r m i d a b l e t a s k b u t a s R o b e r t L o u i s S t e v e n s o n once w r o t e :

and

"To t r a v e l h o p e f u l l y i s a b e t t e r t h i n g than t o a r r i v e , the true success i s t o labour". E l Dorado.

Literature 1.

2. 3.

4.

5.

6. 7. 8. 9.

10. 11. 12. 13. 14. 15. 16. 17.

Cited

Mothes, K. in "Secondary P l a n t Products" Encylopaedia of P l a n t Physiology New S e r i e s , V o l . 8; Ed. Bell, E . A . and Charlwood B.V.; S p r i n g e r - V e r l a g , Berlin, 1980, p . 1 Bu'Lock, J . D . in "The B i o s y n t h e s i s of Mycotoxins", Ed. Steyn, P.S., Academic P r e s s , New York - London, 1980, p. 1 Janzen, K . H . in "Herbivores - Their I n t e r a c t i o n s w i t h Secondary M e t a b o l i t e s " ; Ed. Rosenthal, G.A. and Janzen, D . H . ; Academic P r e s s , New York - London, 1979, p . 331 Rhoades, D.F. in "Herbivores - Their I n t e r a c t i o n s w i t h Secondary M e t a b o l i t e s " ; Ed. Rosenthal, G.A. and Janzen, D . H . ; Academic P r e s s , Nre York - London, 1979, p. 3. Harborne, J . B . in "Secondary P l a n t Products" Encylopaedia of P l a n t P h y s i o l o g y , New S e r i e s , V o l . 8; Ed. Bell, E . A . and Charlwood, B.V.; S p r i n g e r - V e r l a g , Berlin, 1980, p. 329. Howes, F.N., "Vegetable Tanning M a t e r i a l s " ; Butterworths, London, 1939. Bate-Smith, E.C., Food, 1954, 2 3 , 124. Bate-Smith, E.C., J. Linnaen Soc. ( B o t . ) , 1962, 58, 95. Zenk, M.H. in "Recent Advances in Phytochemistry" V o l . 12; Ed. Swain, T . , Harborne, J . B . and van Sumere, C . , Plenum P r e s s , London and New York, 1978, p. 139. Bate-Smith, E . C . and L e r n e r , N.H., Biochem. J., 1954, 58,126. Haslam, E., Phytochemistry, 1977, 16, 1625. Thompson, R.S., Jacques, D . , Tanner, R . J . N . and Haslam, E., J. Chem. Soc. (Perkin 1 ) , 1972, 1387. Weinges, K., Kaltenhauser, W., Marx, H.-D., Nader, E., Nader, F., Perner, J. and S e i d e r , D . , Annalen, 1968, 711, 184. F l e t c h e r , A.C., Gupta, R.k., P o r t e r , L.J. and Haslam, E . J. Chem. Soc. (Perkin 1 ) , 1977, 1628. Weinges, K., Marx, H . - D . and Perner, J., Chem. B e r . , 1970 103, 2344. J u r d , L. and Lundin, R . , Tetrahedron, 1968, 24, 2652. Geissmann, T . A . and Yoshimura, N.N., Tetrahedron L e t t e r s , 1966, 2669.



7.

MCMANUS ET AL.

Plant

Polyphenols

137

18. Opie, C.T., Porter, L.J., Jacques, D. and Haslam, E . , J . Chem. Soc. (Perkin 1), 1977, 1637. 19. Gupta, R.K. and Haslam, E . , J . Chem. Soc. (Perkin 1), 1978, 892. 20. Robinson, R. and Robinson, G.M., J. Chem. Soc., 1935, 744. 21. Porter, L.J., Czochanska, Z . , Foo, L . Y . , Newman, R . H . , Thomas, W.A. and Jones, W.T., J . Chem. Soc. Chem. Commun., 1979, 375. 22. Okuda, T., Yoshida, T., Mori, K. and Hatano, T., Heterocycles, 1981, 15, 1323. 23. Haslam, E . , Fortschr. chem. org. Naturst., 1982. 24. Magnolato, D., Gupta, R . K . , Haddock, E . A . , Layden, K. and Haslam, E . , Phytochemistry, 1982. 25. Fischer, E . , Ber., 1919, 52, 809. 26. Okuda, T., Nayeshiro, H. and Yoshida, T., Chem. Pharm. Bull. (Japan) 1977, 25, 1862. 27. Van Buren, J . P . and Robinson, W.B., J. Agric. Food Chem., 1969, 17, 772. 28. Loomis, W.D., Methods. Enzymol., 1974, 31, 528. 29. Oh, H . I . , Hoff, J.E., Armstrong, G.S. and Haff, L . A . , J. Agric. Food Chem., 1980, 28, 394. 30. McManus, J., Lilley, T.H. and Haslam, E . , J . Chem. Soc., Chem. Commun., 1981, 309. RECEIVED September 10, 1982


Plant Polyphenols and Their Association with Proteins - American

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