Open Collections

UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Methods for the isolation and purification of ethanol-insoluble, phenolic esters in mint Majak, Walter 1972

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-UBC_1972_A1 M34.pdf [ 5.52MB ]
Metadata
JSON: 831-1.0101334.json
JSON-LD: 831-1.0101334-ld.json
RDF/XML (Pretty): 831-1.0101334-rdf.xml
RDF/JSON: 831-1.0101334-rdf.json
Turtle: 831-1.0101334-turtle.txt
N-Triples: 831-1.0101334-rdf-ntriples.txt
Original Record: 831-1.0101334-source.json
Full Text
831-1.0101334-fulltext.txt
Citation
831-1.0101334.ris

Full Text

11 2 ^ 3 'IETH0D3 FOR THE ISOLATION AND PURIFICATION 0 ETHANOL-INSOLUBLE, PHENOLIC ESTERS IN HINT by WALTER MAJAK B.Sc, K c G i l l U n i v e r s i t y , Montreal, 1963 M.Sr., Delhousie U n i v e r s i t y , H a l i f a x , 19 \ THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n the Department o f Botany We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA A p r i l 1972 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree a t the U n i v e r s i t y o f B r i t i s h Columbia, I agree t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and study. I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y purposes may be g r a n t e d by the Head of my Department or by h i s r e p r e s e n t a t i v e s . I t i s understood t h a t c o p y i n g or p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be allowed without my w r i t t e n p e r m i s s i o n . Department o f The U n i v e r s i t y o f B r i t i s h Columbia Vancouver 8, Canada i ABSTRACT Previous k i n e t i c , i s o t o p i c studies have suggested that " i n s o l u b l e " phenolic esters are the d i r e c t precursors of l i g n i n . Heretofore, the " i n s o l u b l e " esters have been detected by the chromatographic examination of gross hy d r o l y s i s products of ethanol-insoluble residues and/or acetone powders. In t h i s study, new, d e f i n i t i v e methods were developed f o r the i s o l a t i o n and p u r i f i c a t i o n of the ethanol-insoluble, phenolic esters of Mentha arvensis. "Insoluble" conjugates of c a f f e i c (compound 1), f e r u l i c (compound 2) and para-coumaric (compound 3) acids were p u r i f i e d and were shown to be e l e c t r o p h o r e t i c a l l y and chromatographically homogeneous. A m u l t i p l i c i t y of " i n s o l u b l e " pools of c a f f e i c acid was detected i n Mentha. Two pools of " i n s o l u b l e " c a f f e i c acid were d i s t i n g u i s h e d on the basis of t h e i r anionic m o b i l i t y at pH 1.9. An " i n s o l u b l e " macromolecular c a f f e y l conjugate was p a r t i a l l y p u r i f i e d . S t r u c t u r a l i n v e s t i g a t i o n s indicated that the c a f f e i c acid moiety of compound 1 was covalently associated with amino acids. Radioactive s u l f u r was incorporated i n t o compounds 1, 2 and 3 and the evidence suggested that the 35 S resided i n an anionic, a c i d i c group. U l t r a - v i o l e t spectroscopy demonstrated that the phenolic moieties of i i compounds 1, 2 and 3 were linked v i a t h e i r r e s pective carboxyl groups. Time-course tracer studies were performed, f o r the f i r s t time, on a p u r i f i e d , " i n s o l u b l e " pool of c a f f e i c , 14 14 f e r u l x c and para-coumaric a c i d . When CC^ and C-phenylalanine were administered, a de c l i n e i n the s p e c i f i c a c t i v i t y of compounds 1,2 and 3 was indicated i n two out of three experiments. The s p e c i f i c a c t i v i t y of the c a f f e o y l moiety of compound 1 followed a p a r a l l e l course i n each experiment. Compound 2, the f e r u l i c acid-containing conjugate, c o n s i s t e n t l y possessed the highest s p e c i f i c a c t i v i t y as compared to compounds 1 and 3. The s p e c i f i c a c t i v i t i e s of various pools of c a f f e i c acid were compared at the six-hour metabolic period. The s p e c i f i c a c t i v i t y of " soluble" c a f f e i c acid was 2 - 3 times greater than the s p e c i f i c a c t i v i t y of the c a f f e o y l moiety of compound 1. The c a f f e i c acid moiety of the high-molecular-weight conjugate was not r a d i o a c t i v e . The author suggests that compound 2 i s an "active, i n s o l u b l e e s t e r " v/hich may be d i r e c t l y involved i n l i g n i f i c a t i o n . i i i TABLE OF CONTENTS Page ABSTRACT i TA3LE OF CONTENTS i i i LIST OF TABLES v i i LIST OF FIGURES. v i i i ACKNOWLEDGEMENT . x i i INTRODUCTION 1 LITERATURE REVIEW. . 4 MATERIALS AND METHODS Plant material 14 The preparation of reagents and adsorbents 14 PAW reagent 14 Spray reagents 14 Adsorbents 15 Analysis of phenolic acids and a u t o l y s i s of acetone powders 16 Apparatus f o r electrophoresis 16 Radioactive compounds and t h e i r administration 16 Elemental, amino acid and i s o t o p i c analyses 17 Commercial enzyme preparations 17 RESULTS AMD DISCUSSION I n i t i a l observations of the products of hydrolysis of i n s o l u b l e residues , Some properties of the ethanol-insoluble phenolic esters that were revealed i n the ear l y attempts of i s o l a t i o n Detection of the ethanol-insoluble, phenolic acid conjugates i n M. arvensis  The d e r i v a t i o n of major phenolic acid pool i n M. arvensis  Detailed procedures devised f o r the preparative i s o l a t i o n of fluorescent, ethanol-insoluble, phenolic conjugates ... From acetone powders Preparation of acetone powders . Ex t r a c t i o n of acetone powders ... P r e c i p i t a t i o n of high-molecular-weight components Adsorption by DEAE-Cellulose ... Resolution by TLC Resolution by HVPE, paper chrom-atography and TLC From ethanol-insoluble residues From buffe r e x t r a c t i o n of fr e s h material A p p l i c a t i o n of the methods to the " i n s o l u b l e " ohenolic esters of wheat. P a r t i a l p u r i f i c a t i o n of the high-molecular-weight conjugate of c a f f e i c acid i n M. arvensis C r i t e r i a of n u r i t y f o r compounds 1, 2 and 3 \ RESULTS AND DISCUSSION (continued) Adsorptive properties of compound 1 49 On dextran and polyacrylamide gels .. 49 On DEAE-Cellulose 52 On poly amide 53 P a r t i a l c h a r a c t e r i z a t i o n of " i n s o l u b l e " esters 54 Compound 1 P a r t i a l acid h y d r o l y s i s 54 Enzymatic h y d r o l y s i s „ 56 Mild a l k a l i n e h y d r o l y s i s 56 Estimation of percentage by weight of c a f f e i c acid i n compound 1 58 Linkage of c a f f e i c acid 59 Determination of s u l f u r i n compound 1 59 35 Incorporation of S i n t o compounds 1, 2 and 3 60 Attempts to i d e n t i f y the non-c a f f e o y l moiety(ies) 61 Is the c a f f e o y l moiety of compound 1 a r t e f a c t u a l l y associa-ted with another molecule? ...... 65 Gel f i l t r a t i o n i n the presence of 7M urea 65 Electrophoresis i n PAW .... 66 Compounds 2 and 3 68 Compound 4 71 v i RESULTS AND DISCUSSION ( c o n t i n u e d ) The c a f f e o y l c o n j u g a t e i n the C band . 73 The h i g h - m o l e c u l a r — w e i g h t c a f f e y l c o n j u g a t e 73 Time-course t r a c e r experiments P r e l i m i n a r y o b s e r v a t i o n on the " i n s o l u b l e " c a f f e i c a c i d o f Mentha ... 76 E s t i m a t i o n of r e l a t i v e p o o l s i z e s o f compounds 1, 2 and 3 78 S p e c i f i c a c t i v i t y d e t e r m i n a t i o n 79 Turnover s t u d i e s on compounds 1, 2 and 3 80 R e l a t i v e i n c o r p o r a t i o n of p h e n y l a l a n i n e -14 14 " C and c i n n a m i c a c i d - C i n t o v a r i o u s c a f f e i c a c i d p o o l s 88 SUMMARY AND CONCLUSIONS 93 LITERATURE CITED 101 v i i L i s t of tables Table Page I. Types of Phenolic Acid Conjugates I d e n t i f i e d i n Plants 5 I I . U l t r a - v i o l e t Spectra and Fluorescent Colors of Isolated Compounds 38 I I I . R Values xlOO 48 IV. Concentrations of Amino Acids Derived from Four D i f f e r e n t I s o l a t e s of Compound 1 63 V. Radioactive Compounds Administered and t h e i r Incorporation i n t o Soluble C a f f e i c Acid and the C a f f e i c Acid of Compound 1 82 v i i i L i s t o f f i g u r e s F i g u r e P a g e 1. C i n n a m i c a c i d s a n d r e l a t e d c o m p o u n d s 2 2. B i o g e n e t i c r e l a t i o n s h i p s o f p h e n o l i c a c i d s 6 3. H y p o t h e t i c a l o u t l i n e o f l i g n i n b i o s y n t h e s i s i n w h e a t 9 4 . S e p h a d e x G-25 c o l u m n c h r o m a t o g r a p h y o f a q u e o u s e x t r a c t s o f a c e t o n e p o w d e r s o f M . a r v e n s i s 23 5. S e p h a d e x G-10 c o l u m n c h r o m a t o g r a p h y o f e x t r a c t c o n t a i n i n g f l u o r e s c e n t , c a f f e o y l c o n j u g a t e s . . . . 26 6. D i a g r a m m a t i c r e p r e s e n t a i o n s o f e l e c t r o p h o r e -t o g r a m s 28 7 . T h e d e r i v a t i o n o f m a j o r p h e n o l i c a c i d p o o l s b y m e t h o d I 30 8. T h e d e r i v a t i o n o f m a j o r p h e n o l i c = c i d p o o l s b y m e t h o d I I 31 i x Figure Page 9. P u r i f i c a t i o n of the fluorescent, ethanol-i n s o l u b l e phenolic conjugates by method 13 33 10. Slectrophcretogram developed at pH 2 and photographed i n u.v. l i g h t 41 11a. Sephadex G-100 column chromatography of extract containing high-molecular-weight c a f f e y l conjugate 43 l i b . Diagrammatic representation of anionic bands (actual size) obtained a f t e r HVPE of Sephadex G-100 eluate containing high-molecular-weight c a f f e y l conjugate 43 12. U l t r a - v i o l e t absorption spectra of high-molecular-weight c a f f e y l conjugate, c a f f e i c acid and compound 1 i n d i s t i l l e d water 46 13a. Column chromatography of compound 1 and c a f f e i c acid 50 13b. Sephadex G-25 column chromatography of compound 1 50 X Figure Page 14. Diagrammatic representation of electrophoreto-gran of p a r t i a l acid hydrolyzates and standards 55 15. U l t r a - v i o l e t absorption spectra of compound 3 and para-coumaric acid i n d i s t i l l e d water 69 16. U l t r a - v i o l e t absorption spectra of compound 2 and f e r u l i c acid i n d i s t i l l e d water 70 17. U l t r a - v i o l e t absorption spectrum of compound 4 i n methanol containing 1% water 72 18. U l t r a - v i o l e t absorption spectra of compound 1 and the c a f f e o y l conjugate of the C band i n d i s t i l l e d water 75 19. Changes with time i n the s p e c i f i c a c t i v i t i e s of c a f f e i c acids of M. arvensis a f t e r the 14 administration of NaH CO^ 77 20. Changes with time i n s p e c i f i c a c t i v i t i e s of compound 1 and c a f f e i c acid moiety of compound 1 14 a f t e r administration of NaH CO^ and phenylala-n i n e - ^ 1 ^ 83 x i F i g u r e Page 21. Changes w i t h time i n s p e c i f i c a c t i v i t i e s o f compounds 2 and 3 a f t e r a d m i n i s t r a t i o n o f 14 p h e n y l a l a n i n e - U - C 84 22. Changes w i t h time i n s p e c i f i c a c t i v i t i e s o f compounds 1, 2 and 3 and c a f f e i c a c i d moiety of compound 1 a f t e r a d m i n i s t r a t i o n of c i n n a m i c a c i d - 3 - 1 4 C 86 23 Sephadex G-100 column chromatography of h i g h -m o l e c u l a r - w e i g h t c a f f e y l c o n j u g a t e 91 x i i ACKNOWLEDGEMENTS The author wishes to express his sincere gratitude to Dr. G.H.N. Towers f o r h i s encouragement and advice during the i n v e s t i g a t i o n s and for his valuable c r i t i c i s m s during the preparation of t h i s manuscript. The author also wishes to thank Dr. I.E.P. Taylor f o r the frequent use of h i s high voltage e l e c t r o p h o r e s i s apparatus and f o r h i s supervision of the amino acid analyses. 1 INTRODUCTION The ubiquity of phenolic acids i n higher plants has " 1 2 3 been established i n the l a s t decade x' ' and they have been shown to be d i r e c t l y involved i n the biosynthesis 4 5 of coumarins, flavonoids and l i g n i n s ' . Investigations have centered on the hydroxylated and methoxylated cinnamic acids (Figure 1) which occur, i n combined form as glycosides or esters i n p r a c t i c a l l y 1 every higher plant . C a f f e i c acid esters such as c h l o r -6 7 ogenic acids and/or rosmarinic acids (Figure 1) are often the major phenolic esters encountered i n ethanolic extracts of p l a n t s . An aqueous ethanolic extract of fresh plant material i s the c l a s s i c a l s t a r t i n g point f o r the i s o l a t i o n of low-molecular-weight, phenolic acid d e r i v a t i v e s . However, phenolic acids can also be l i b e r a t e d by h y d r o l y s i s of 8 9 ethanol-insoluble residues ' or a u t o l y s i s of acetone powders 1^. On the basis of k i n e t i c studies, i t has been suggested that these " i n s o l u b l e " esters are natural intermediates i n the biosynthesis of l i g n i n s 1 1 . The biosynthesis o f . l i g n i n s remains a major, unresolved problem i n plant biochemistry. Heretofore, the study of i n s o l u b l e esters has been confined to chromatographic examinations of gross hy d r o l y s i s products derived from crude ethanol-insoluble 2 OH H O ^ O H COOH 31 2i 0 I CO-CH=CH Chlorogenic acid HO, HO OH OH CH=CH-COOH CH = CH-CO I 0 CHCOOH CH 2 OH OH Rosmarinic acid para-Coumaric:( R=R=H) Caffeic:(R=OH.R=H) Ferulic:(R=OCH3.R=H) Sinapic:(R=R=OCH3) Figure 1. Cinnamic acids and r e l a t e d compounds. 3 residues and acetone powders. The purpose of t h i s study-was to examine t h i s ethanol-insoluble f r a c t i o n i n some d e t a i l and to develop p r a c t i c a l and r e l i a b l e methods f o r r e s o l v i n g the various pools of phenolic a c i d s . With the successful development of such methods i t became possible to carry out preliminary studies of the chemical nature of one of these compounds. I t was also p o s s i b l e to conduct, f o r the f i r s t time, k i n e t i c , i s o t o p i c studies on a p u r i f i e d , ethanol-insoluble pool of c a f f e i c a cid, para-coumaric acid and f e r u l i c a c i d . 4 LITERATURE REVIEW Various phenolic acids have been described i n plants. Figure 1 i l l u s t r a t e s the commonly-occurring cinnamic acids which are u s u a l l y found conjugated i n the form of esters and glycosides. An exceptional array of phenolic acid conjugates has been i d e n t i f i e d . The examples selected f o r Table I i l l u s t r a t e a diverse c r o s s - s e c t i o n of phenolic acid conjugates which includes esters of quinic acid, c y c l o a r t e n o l (a t r i t e r p e n o l ) , f a r n e s i f e r o l (a sesquiter-penol), malvidin (an anthocyanidin) and kaempferol (a f l a v o n o l ) . F e r u l i c and c a f f e i c acid were found associated with 52 a C-28 n Y d r o x y - f a t t y acid i n oat kernels . The a n t i -oxidant property of these compounds i s a t t r i b u t e d to the hydroxycinnamoyl residues. In 1968, Synge reviewed the d i s t r i b u t i o n of non-53 protein amino acid conjugates . He concluded that the only well-substantiated report of a phenolic acid acylated to an amino acid was g a l l i c acid (3,4,5-trihydroxy 33 33To benzoic) which occurred as N - g a l l o y l l e u c i n e ' Amides of decarboxylated<products of amino acids such as putrescine and agmatine are more frequent. These include f e r u l o y l p u t r e s c i n e " ^ and para-coumaroylaqmatine^ (Table I ) . Stoessl and c o l l a b o r a t o r s have r e c e n t l y i d e n t i f i e d 5 54 f e r u l i c and para-coumaric amides of 2 - h y d r o x y p u t r e s c i n e These s u b s t a n c e s accumulate i n the l e a v e s o f r u s t - i n f e c t e d , r e s i s t a n t wheat. T a b l e I Types of P h e n o l i c A c i d Conjugates I d e n t i f i e d i n P l a n t s Conjugate Source 3 - C a f f e o y l q u i n a t e ( c h l o r o g e n i c a c i d ) 5 - C a f f e o y l q u i n a t e ( n e o c h l o r o g e n i c a c i d ) 3 , 5 - D i c a f f e o y l q u i n a t e ( i s o c h l o r o g e n i c a c i d ) 1 , 4 - D i c a f f e o y l q u i n a t e ( c y n a r i n ) 3 - F e r u l o y l q u i n a t e 3-para-Coumaroylquinate P o l y g a l l o y l q u i n a t e 3 - C a f f e o y s h i k i m a t e C a f f e o y l m a l a t e C a f f e o y l t a r t a r a t e a l p h a - C a f f e o y l - 3 , 4 - d i h y d r o x y p h e n y l l a c t a t e ( r o s m a r i n i c a c i d ) C a f f e o y l g l u c o s e C a f f e o y l g l u c o s a m i n e C a f f e i c a c i d g l u c o s i d e C y c l o a r t e n o l f e r u l i c a c i d e s t e r F a r n e s i f e r o l B ( I I ) para-coumaric a c i d e s t e r C h o l i n e s i n a p i c a c i d e s t e r C h o l i n e i s o f e r u l i c a c i d e s t e r K a e m p f e r o l - 3 ( f e r u 1 o y 1 s o p h o r o t r i o s i d e ) M a l v i d i n - 3 ( o a r a - c o u m a r o y l r u t i n o s e ) - 5 -g l u c o s i d e N - G a l l o y l l e u c i n e F e r u l o y l p u t r e s c i n e para-Coumaroylaqmatine widespread ' . , .12,15 widespread ' . , * ,,4,13 widespread ' 14 a r t e c h o k e c o f f e e & tomato ,17,18 widespread ' r , . . 20 C a e s a l p i t u a western hemlock 21 Phaseolus 22 c h i c o r y 7 Mentha & o t h e r l a b i a t e s widespread ' 24 15 19 N i c o t i a n a 26 p o t a t o b e r r i e s . .,27,28 r i c e bran o i l ' F e r u l a 29 c r u c i f e r s 31 S i oara 30 pea 32 32 o o t a t o Q u e r ^ u s 33a,33b a e g i l o p s ' g r a p e f r u i t 35 b a r l e y " 6 The cinnamic acids are derived from phenylalanine and tyrosine and they are r e l a t e d b i o g e n e t i c a l l y as shown below^. SHIKIMIC ACID CHORISMIC ACID PHENYLALANINE • TYROSINE CINNAMIC ACID * para-COUMARIC ACID \ CAFFEIC ACID I FERULIC ACID SINAPIC ACID Figure 2. Biogenetic r e l a t i o n s h i p s of phenolic acids. I t has been suggested that some of these hydro-xy l a t i o n s and O-methylations of cinnamic acids occur 3 S through the quinoyl esters . An enzyme preparation from potatoes catalyzes the hydroxylation of para-3 6 coumaroylquinic acid to chlorogenic acid . On the other hand, cinnamic acid 4-hydroxylase and para-coumaric acid hydroxylase have been shown to occur i n 37 plants and have been p a r t i a l l y p u r i f i e d . There i s not enough information to determine the r e l a t i v e importance of hydroxylations of fre e acids versus 7 h y d r o x y l a t i o n s o f e s t e r s or o t h e r d e r i v a t i v e s . There are r e l a t i v e l y few examples o f macromolecular or sub—macromolecular p h e n o l i c a c i d c o n j u g a t e s known. Wheat f l o u r c o n t a i n s a g l y c o p r o t e i n i n which f e r u l i c 38 77 a c i d i s e s t e r i f i e d w i t h a x y l a n c h a i n ' . A f u n g i t o x i c , c h l o r o g e n i c a c i d ( 3 - 0 - c a f f e o y l q u i n i c a c i d ) - amino a c i d 39 a d d i t i o n product has been i s o l a t e d from p o t a t o . However, subsequent e v i d e n c e was p r e s e n t e d which i n d i c a t e d t h a t the amino a c i d p o r t i o n was a contaminant d e r i v e d from 40 chromatography paper . P u r i f i e d , h e a t - s t a b l e f a c t o r s ( a b s o r p t i o n maxima 315-325 nm) i s o l a t e d from s p i n a c h 41 42 c h l o r o p l a s t s were shown t o enhance p h o t o p h o s p h o r y l a t i o n ' , p h o t o r e d u c t i o n ^ a n d the l i g h t - d e p e n d e n t , enzyme a c t i v i t y 43 o f r i b u l o s e d i p h o s p h a t e c a r b o x y l a s e . Krogmann and S t i l l e r proposed t h a t the chromophoric group was a f l a v o n e or a p h e n o l i c compound such as c h l o r o g e n i c or 44 c a f f e i c a c i d I A l i b e r t and co-workers r e p o r t e d the pr e s e n c e o f p h e n o l i c a c i d s i n p a r t i a l l y p u r i f i e d , p r o t e i n f r a c t i o n s 45 from oak l e a v e s . I t i s s u r p r i s i n g t h a t t h e s e f r a c t i o n s were not s u b j e c t e d t o s i m p l e d i a l y s i s or e l e c t r o p h o r e s i s t o t e s t the h y p o t h e s i s t h a t the p h e n o l i c compounds, g a l l i c , e l l a g i c and p r o t o c a t e c h u i c a c i d s (which are p r e s e n t i n these l e a v e s ) were adsorbed to the p r o t e i n s . However, the authors d i d suggest t h a t t h e s e p h e n o l i c 8 a c i d s c o u l d be l i n k e d through a c i d - and/or a l k a l i -45 s e n s i t i v e hydrogen bonding Bound, i n s o l u b l e p h e n o l i c a c i d s were o r i g i n a l l y 46 47 48 49 d e t e c t e d i n l i g n i n p r e p a r a t i o n s ' ' ' When n a t i v e l i g n i n from sugar cane i s b r i e f l y exposed t o m i l d a l k a l i , subsequent e x t r a c t i o n w i t h e t h e r y i e l d s para-coumaric a c i d , f e r u l i c a c i d , p a r a - h y d r o x y b e n z o i c a c i d , v a n i l l i c a c i d (3-methoxy-4-hydroxybenzoic) and 46 s y r i n g i c a c i d (3,5-dimethoxy-4-hydroxybenzoic) . Smith p o s t u l a t e d t h a t t h e s e a c i d s o c c u r as e s t e r r e s i d u e s 46 a t t a c h e d t o pre-formed l i g n i n s A l k a l i - h y d r o l y z a b l e , p h e n o l i c a c i d c o n j u g a t e s were o d e t e c t e d 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 o f wheat shoots . The q u a n t i t a t i v e changes i n the l e v e l s o f t h e s e a c i d s d u r i n g growth and development were compared w i t h l e v e l s o f " s o l u b l e " a c i d s o c c u r r i n g as h y d r o l y z a b l e g l y c o s i d e s and e s t e r s . Dynamic f l u c t u a t i o n s i n c o n c e n t r a t i o n were observed over an eight-week p e r i o d . F o r example, a r a p i d d e c l i n e i n " s o l u b l e " f e r u l i c a c i d was observed w i t h a c o n c o m i t a n t i n c r e a s e i n the c o n c e n t r a t i o n o f " i n s o l u b l e " f e r u l i c a c i d ^ . Subsequent t r a c e r experiments r e v e a l e d t h a t the r a t e 14 Id o f i n c o r p o r a t i o n of C0o " C - p h e n y l a l a n i n e and t y r o s i n e was more r a p i d i n t o the " i n s o l u b l e e s t e r s " of the hydroxy-cinnamic a c i d s than i n t o the s o l u b l e e s t e r s 1 1 . An 9 experiment was conducted i n which wheat seedlings were 14 exposed to CC^ f o r two hours and t h i s was followed by per i o d i c measurements of the s p e c i f i c a c t i v i t i e s of the " i n s o l u b l e " cinnamic acids and l i g n i n aldehydes* 1. These l i g n i n aldehydes are degradation products obtained by the a l k a l i n e nitro-benzene oxidation of ethanol-insoluble residues"^. The k i n e t i c data indicated that " i n s o l u b l e " esters behaved more l i k e precursors of l i g n i n than did the soluble e s t e r s ^ . El-Basyouni, Neish and Towers proposed the following scheme f o r l i g n i n biosynthesis i n wheat (Figure 3): PHENYLALANINE CO, ACTIVE INSOLUBLE ESTERS LIGNIN TYROSINE INSOLUBLE ESTERS SOLUBLE ESTERS t CINNAMIC ACID HYDROXY CINNAMIC ACIDS Figure 3. Hypothetical o u t l i n e of l i g n i n biosynthesis i n wheat. 10 T a y l o r and Zucker, on the o t h e r hand, have emphasized 13 51 the r o l e of the s o l u b l e e s t e r s i n l i g n i f i c a t i o n ' T r a c e r s t u d i e s on p h e n y l p r o p a n o i d metabolism i n Xanthium l e a v e s and p o t a t o t u b e r s i n d i c a t e d t h a t c h l o r o g e n i c and i s o c h l o r o g e n i c a c i d s were a c t i v e l y t u r n i n g over w h i l e " i n s o l u b l e " c a f f e i c a c i d was c o m p a r a t i v e l y i n a c t i v e ^ 1 . However, the t r a c e r s were a d m i n i s t e r e d t o e x c e e d i n g l y s m a l l samples (e.g., 5 l e a f d i s c s weighing 120 mg) and the a c t i v e , i n s o l u b l e p o o l s may not have been d e t e c t e d . F u r t h e r more, the 50% aqueous methanol e x t r a c t t h a t was d i s c a r d e d i n t h e i r f r a c t i o n a t i o n p rocedure may have y i e l d e d y e t another p o o l o f p h e n o l i c a c i d s , (see " R e s u l t s and D i s c u s s i o n F r i t i g and co-workers i n s t u d i e s on coumarin b i o -s y n t h e s i s were unable t o f i n d a c t i v i t y i n the i n s o l u b l e 55 p h e n o l i c d e r i v a t i v e s of t o b a c c o t i s s u e c u l t u r e s . How-ever, the l e v e l s o f i n s o l u b l e p h e n o l i c a c i d s were v e r y 5 6 low as might be expected i n t i s s u e c u l t u r e s which are not undergoing e x t e n s i v e l i g n i f i c a t i o n . On k i n e t i c grounds, they c o n c l u d e d t h a t the f o r m a t i o n of the coumarin, 55 s c o p o l e t i n , was v i a the f r e e hydroxycinnamic a c i d s and not through g l u c o s i d e s or s o l u b l e e s t e r s ^ as p o s t u l a t e d by Brown Fuchs, R o h r i n g e r and Samborski have c a r r i e d out e x t e n s i v e s t u d i e s on the response of p h e n o l i c metabolism i n wheat t o r u s t i n f e c t i o n ^ * ^ ' ^ . They have c o n f i r m e d 11 the p r e v i o u s o b s e r v a t i o n t h a t the " i n s o l u b l e " e s t e r s o f wheat are more r a p i d l y l a b e l l e d than the " s o l u b l e " e s t e r s . When 1 4 C - p h e n y l a l a n i n e was a d m i n i s t e r e d (22-hour m e t a b o l i c p e r i o d ) , the r a t i o s o f the a c t i v i t i e s i n the " i n s o l u b l e " f e r u l i c a c i d t o the a c t i v i t i e s i n the " s o l u b l e " f e r u l i c a c i d were 11:1 ( s u s c e p t i b l e , h e a l t h y l e a v e s ) , 2:1 ( s u s c e p -t i b l e , r u s t i n f e c t e d l e a v e s ) , 9:1 ( r e s i s t a n t , h e a l t h y 5 8 l e a v e s ) and 9:1 ( r e s i s t a n t , r u s t i n f e c t e d l e a v e s ) U s i n g " C - p h e n y l a l a n i n e , E l l i s demonstrated t h a t the i n c o r p o r a t i o n o f l a b e l i n t o the e t h a n o l - i n s o l u b l e , p h e n o l i c e s t e r s o f Mentha was as g r e a t as the t o t a l l a b e l i n the 61 p o o l of r o s m a r i n i c a c i d . E l l i s o b t a i n e d h i s r e s u l t s & 2 by the f o l l o w i n g procedure : The e t h a n o l - i n s o l u b l e r e s i d u e was h y d r o l y z e d i n a l k a l i , a c i d i f i e d , e x t r a c t e d w i t h e t h e r and the t o t a l a c t i v i t y i n the e t h e r phase was, d e s i g n a t e d as the l a b e l i n the " e t h a n o l - i n s o l u b l e " e s t e r p o o l . Whether t h i s i s a d i r e c t measurement o f the a c t i v i t y o f the " i n s o l u b l e " p h e n o l i c a c i d s i s q u e s t i o n a b l e . F u r t h e r -more, s i n c e t h e r e i s a m u l t i p l i c i t y o f " i n s o l u b l e " p h e n o l i c a c i d p o o l s i n Mentha (see " R e s u l t s and D i s c u s s i o n " ) t h i s procedure i s o b v i o u s l y an o v e r s i m p l i f i c a t i o n . On the b a s i s of t h e i r s t u d i e s , E l - B a s y o u n i and N e i s h * ^ 6 3 and l a t e r , Hope proposed t h a t the m e t a b o l i c a l l y - a c t i v e , i n s o l u b l e , p h e n o l i c a c i d s were a s s o c i a t e d w i t h p r o t e i n s . The i n i t i a l c r i t e r i a were based on the presence of l a b e l l e d 12 p h e n o l i c a c i d s i n ammonium s u l f a t e p r e c i p i t a t e s and i n Sephadex G-200 e l u a t e s ^ . On the b a s i s o f Sephadex G-25 chromatography a low-molecular-weight (^SOOO g/M) and a h i g h - m o l e c u l a r - w e i g h t (^5000 a/M) po o l o f bound p h e n o l i c a c i d s were d e s c r i b e d i n wheat s e e d l i n g s * ^ . When "^C-p h e n y l a l a n i n e was a d m i n i s t e r e d , the s p e c i f i c a c t i v i t y o f the s o - c a l l e d " p r o t e i n " p h e n o l i c a c i d s d e c r e a s e d w h i l e t h a t o f the " n o n - p r o t e i n " a c i d s i n c r e a s e d d u r i n g a one-hour m e t a b o l i c p e r i o d . (The f e e d i n g experiment c o n s i s t e d o f t h r e e t i m e - c o u r s e samples a t 1,2 and 4 hours and the p r e c e e d i n g k i n e t i c c o r r e l a t i o n i s o n l y i n agreement w i t h the f i g u r e s f o r the 1 and 2 hour i n t e r v a l s ) . Hope conclu d e d t h a t t h i s d a t a supported the e a r l i e r r e s u l t s o f E l - B a s y o u n i . However, these crude, o v e r l a p p i n g f r a c t i o n s were not p u r i f i e d , nor were they s u b j e c t e d t o d i a l y s i s o r e l e c t r o p h o r e s i s t o t e s t the p o s s i b i l i t y of n o n - c o v a l e n t bonding. Furthermore, the m o l e c u l a r weight l i m i t s mentioned above were e s t a b l i s h e d on the b a s i s o f g e l chromatography i n c o n v e n t i o n a l b u f f e r s . Under these c o n d i t i o n s p h e n o l i c c o n j u g a t e s are s t r o n g l y adsorbed t o the g e l , t r u e g e l f i l t r a t i o n i s not o p e r a t i o n a l and the 73 e s t i m a t i o n of m o l e c u l a r weight l i m i t s can be m i s l e a d i n g On the o t h e r hand, when a crude f r a c t i o n i s ex c l u d e d from a d e x t r a n g e l , the e a r l y e l u t i o n can be the r e s u l t o f 73 complex f o r m a t i o n or a g g r e g a t i o n e s p e c i a l l y when p h e n o l i c a c i d s a re i n v o l v e d (see " R e s u l t s and D i s c u s s i o n " ) . 13 I t i s obvious from the f o r e g o i n g d i s c u s s i o n t h a t new, d e f i n i t i v e methods were r e q u i r e d f o r the i s o l a t i o n o f e t h a n o l - i n s o l u b l e e s t e r s . The work t o be d e s c r i b e d i n t h i s t h e s i s c oncerns the i s o l a t i o n and p u r i f i c a t i o n o f bound p h e n o l i c a c i d s o t h e r w i s e known as e t h a n o l - i n s o l u b l e e s t e r s . A h i g h - m o l e c u l a r - w e i g h t and a l o w - m o l e c u l a r -weight (<10,000 g/M) po o l were r e s o l v e d from acetone powders and/or e t h a n o l - i n s o l u b l e r e s i d u e s o f Mentha a r v e n s i s . With the development of these methods i t v/as then p o s s i b l e t o conduct t i m e - c o u r s e t r a c e r s t u d i e s on " i n s o l u b l e " , p h e n o l i c c o n j u g a t e s t h a t were c h r o m a t o g r a p h i c a l l y and e l e c t r o p h o r e t i c a l l y homogenous. 14 MATERIALS AND METHODS Plant M a t e r i a l . Mentha arvensis L. was grown under greenhouse conditions supplemented with a sixteen-hour photoperiod. Non-flowering shoots that were at l e a s t 3 months old were harvested. A e r i a l shoots of Equisetum arvense L. were c o l l e c t e d on the U.B.C. endowment lands. Two-week-old Thatcher wheat seedling (Triticum aestivum L.) were grown i n f l a t s i n the greenhouse. The Preparation of Reagents and Adsorbents. PAW Reagent L i q u i f i e d phenol, U.S.P., was r e d i s t i l l e d under n i t r o g combined with equal volumes of reagent grade g l a c i a l a c e t i c acid (Baker and Adamson) and stored i n amber glass b o t t l e s at 4°. The PAW reagent (phenol: a c e t i c acid : water, 1 : 1 : 1 ) was f r e s h l y prepared by the additi o n of d i s t i l l e d water. Spray Reagents The cadmium ninhydrin reagent was prepared according 6 5 to the method of Heilmann e_t al_ . Sugars were detected by the para-anisidine-phosphate reagent developed by 15 Bryson e t al_ and Mukherjee e t al_ . D i a z o t i z e d p a r a -n i t r o a n i l i n e , which i s s p e c i f i c f o r phenols, was used 6 8 a c c o r d i n g t o the procedure of Bray e_t a l  Adsorbents The TLC ad s o r b e n t s , C e l l u l o s e powder MN 300 G (Macherey, Nagel & Co.) and A v i c e l were washed b e f o r e s p r e a d i n g . C e l l u l o s e was washed w i t h 60% e t h a n o l , acetone, the chromatographic s o l v e n t t o be used and acetone. A v i c e l was washed w i t h hot e t h a n o l , 10% a c e t i c a c i d , d i s t i l l e d water and acetone. The p r e p a r a t i v e TLC p l a t e s were 1 mm t h i c k and e i t h e r 19 x 55 cm (lon g ) o r 20 .x 20 cm ( s h o r t ) . DEAE C e l l u l o s e was p r e c y c l e d w i t h 0.5 N NaOH and washed w i t h d i s t i l l e d water u n t i l the e l u a t e was n e u t r a l . I t was c o n v e r t e d t o the a c e t a t e form by bat c h w i s e procedure w i t h 10% a c e t i c a c i d and f i n a l l y washed w i t h water t o n e u t r a l pH. For p r e p a r a t i v e i s o l a t i o n s , V/hatman No. 3 MM chroma-tography paper was washed w i t h d i s t i l l e d water f o r 24 ho u r s . The chromatography paper was handled w i t h surgeon's g l o v e s t o av o i d c o n t a m i n a t i o n . E l e c t r o p h o r e t o g r a m s and chromatograms were c r o s s -s t i t c h e d w i t h t r a n s p a r e n t n y l o n t h r e a d . When an e x c i s e d band was sewn t o a s t r i p o f chromatography paper i t was c r o s s - s t i t c h e d on both s i d e s , the blank u n d e r l a y was 16 removed and the f l a p s were c r o s s - s t i t c h e d to the band. A n a l y s i s of P h e n o l i c A c i d s and A u t o l y s i s of Acetone Powders. Methods f o r the a n a l y s i s of p h e n o l i c a c i d s by paper chromatography and u l t r a - v i o l e t a b s o r p t i o n s p e c t r o s c o p y 8 64 have been d e s c r i b e d by o t h e r s ' . A u t o l y s i s of acetone powders was performed a c c o r d i n g t o the method of E l - B a s y o u n i and N e i s h * ^ . Apparatus f o r E l e c t r o p h o r e s i s For low v o l t a g e paper e l e c t r o p h o r e s i s , a Gelman d e l u x e e l e c t r o p h o r e s i s chamber (No. 51170) supported Whatman No. 3 MM chromatography paper (7 x 21 cm) e q u i l -i b r a t e d i n PAW. High v o l t a g e paper e l e c t r o p h o r e s i s (HVPE) was 69 performed i n a m o d i f i e d M i c h l a p p a r a t u s . The b u f f e r 70 s o l u t i o n s employed were f o r m u l a t e d by Ambler R a d i o a c t i v e Compounds and t h e i r A d m i n i s t r a t i o n L - F h e n y l a l a n i n e - U - 1 4 C , N a 2 H 1 4 C 0 3 , and N a 2 3 5 S 0 4 were purchased from New England N u c l e a r , Boston, Mass. 14 Cinnamic a c i d - 3 - C was purchased from Schwartz B i o r e s e a r c h , Orangeburg, New York. The i s o t o p e s were a d m i n i s t e r e d to 17 50 g l o t s o f mint shoots 4-6 i n t e r n o d e s i n l e n g t h . F r e s h l y c u t shoots were r e c u t under water b e f o r e u s e . Each l o t r e c e i v e d the l a b e l l e d a c i d i n 3 ml 0.02 H pH 7 phosphate b u f f e r under c o n s t a n t i l l u m i n a t i o n (9000 l u x ) and temperature ( 1 9 ° ) . The s o l u t i o n s were imbibed w i t h i n 1.5 hours and t h i s was f o l l o w e d w i t h d i s t i l l e d v/ater. E l e m e n t a l , Amino A c i d and I s o t o p i c A n a l y s e s E l e m e n t a l a n a l y s e s were accomplished by Dr. C. D a e s s l e ( O r g a n i c M i c r o a n a l y s e s , M o n t r e a l ) . Compound 1 (1 mg) was h y d r o l y z e d in_ vacuo w i t h 5.7 N HC1 f o r 24 hr a t 110° and the h y d r o l y z a t e was d r i e d i n a vacuum d e s i c c a t o r over KOH p e l l e t s . Amino a c i d a n a l y s e s were performed by Dr. I.E.P. T a y l o r and co-workers a t the Botany Department, U n i v e r s i t y of B r i t i s h Columbia. The 71 method of Spackman e_t al_ was employed u s i n g a Beckman 120 C amino a c i d a n a l y z e r . A l l r a d i o a c t i v i t y measurements were c a r r i e d out i n a N u c l e a r Chicago L i q u i d S c i n t i l l a t i o n Spectrometer (724 System). Aquasol ( N u c l e a r Chigo) was used as the s c i n t i l l a t i o n f l u i d f o r c o u n t i n g the r a d i o a c t i v e samples. Commercial Enzyme P r e p a r a t i o n s T r y p s i n , p e p s i n ( p o r c i n e stomach mucosa), papain and p e c t i n a s e ( f u n g a l ) were purchased from Calbiochem. 18 T r y p s i n was used a c c o r d i n g t o the procedure o f G u i d o t t i e_t a_l. P e p s i n was used a c c o r d i n g t o Ambler's 70 method . The r e a c t i o n s w i t h papain and p e c t i n a s e proceeded f o r 12 hours a t 28° i n 0.01M N - e t h y l m o r p h o l i n e -4 pH 7 b u f f e r c o n t a i n i n g 3 x 10 H c y s t e i n e . The s u s c e p t i b i l i t y o f compound 1 t o the h y d r o l y t i c a c t i v i t y of these- enzymes was assayed by the f o l l o w i n g method: The m i x t u r e was f r e e z e - d r i e d a f t e r the i n c u b a t i o n p e r i o d . The m o b i l i t y and f l u o r e s c e n c e o f the t r e a t e d sample (100/jg compound 1) was then compared t o a s t a n d a r d s o l u t i o n o f compound 1 a f t e r HVPE a t pH 2. 19 RESULTS AND DISCUSSION I n i t i a l O b s e r v a t i o n s of the Products of H y d r o l y s i s o f  I n s o l u b l e Residues P r e l i m i n a r y i n v e s t i g a t i o n s w i t h Equisetum arvense showed t h a t p a r a - h y d r o x y b e n z o i c , f e r u l i c and p a r a -coumaric a c i d s were r e l e a s e d by enzymatic a u t o l y s i s o f acetone powders or by a l k a l i n e h y d r o l y s i s of e t h a n o l " i n s o l u b l e r e s i d u e s . Furthermore, s h o r t term exposures 14 o f a e r i a l shoots (2 hr) t o CO^ i n d i c a t e d t h a t t h e s e p o o l s of p h e n o l i c a c i d s were r a p i d l y l a b e l l e d . However, experiments w i t h Equisetum were abandoned a t t h i s time because of the d i f f i c u l t i e s i n v o l v e d i n growing the p l a n t d u r i n g the w i n t e r months. Hentha  a r v e n s i s was s e l e c t e d because of i t s a v a i l a b i l i t y and c l o n a l u n i f o r m i t y . T h i s p l a n t was a l s o found t o be an e x c e l l e n t source o f " i n s o l u b l e " p h e n o l i c a c i d s . F e r u l i c a c i d (75 /ag) , para-coumaric a c i d (180 p.g) and c a f f e i c a c i d (750 ^ug) were l i b e r a t e d (2N NaOH, 3 hours, 25°) from the b u f f e r e d e x t r a c t o f 1 g of acetone pov/der of Mentha a r v e n s i s . I f the r e m a i n i n g c e l l u l a r d e b r i s was r e - e x t r a c t e d w i t h 7M urea, a second p o o l o f c a f f e i c a c i d (225 jag) was 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 . 20 Some P r o p e r t i e s of the E t h a n o l - i n s o l u b l e P h e n o l i c E s t e r s  t h a t were Revealed i n the E a r l y Attempts o f I s o l a t i o n The r e p o r t e d o c c u r r e n c e o f h i g h - m o l e c u l a r - w e i g h t f r a c t i o n s c o n t a i n i n g bound cin n a m i c a c i d s i n w h e a t " ^ ' ^ and b a r l e y s e e d l i n g s ^ and i n oak l e a v e s 4 ^ have been d i s c u s s e d i n the L i t e r a t u r e Review. The a u t h o r s ' methods f o r s t u d y i n g t h e s e f r a c t i o n s were a p p l i e d t o b u f f e r e d e x t r a c t s o f acetone powders of E_. arvense and M. a r v e n s i s i n o r d e r t o determine t h e i r u s e f u l n e s s . A c c o r d i n g l y , two grams o f acetone powder (E_. arvense or M. a r v e n s i s ) were e x t r a c t e d f o r one hour a t 2° w i t h 80 mis 0.1M pH 7 phosphate b u f f e r . The f i l t r a t e was c e n t r i f u g e d at 30,000 x g f o r one minute. The s u p e r n a t a n t was f r e e z e - d r i e d and a p p l i e d t o a Sephadex G-25 column (1 x 30 cm) e q u i l i b r a t e d w i t h the s t a r t i n g b u f f e r . The exc l u d e d volume was c o l l e c t e d and f r e e z e - d r i e d . T h i s f r a c t i o n was r e d i s s o l v e d and a p p l i e d t o a Sephadex G-200 column (1 x 30 cm) e q u i l i b r a t e d w i t h .05H pH 7 phosphate b u f f e r . F i v e m i l l i l i t e r f r a c t i o n s were c o l l e c t e d and the p h e n o l i c a c i d s were l i b e r a t e d w i t h a l k a l i (2N NaOH, 6 hours, 2 5 ° ) . Para-hydroxy b e n z o i c a c i d ( i n E. arvense) and c a f f e i c 21 a c i d ( i n M. a r v e n s i s ) were found t o be the major p h e n o l i c a c i d s a s s o c i a t e d w i t h the s o - c a l l e d h i g h - m o l e c u l a r - w e i g h t f r a c t i o n s . I f thes e f r a c t i o n s were s u b j e c t e d t o d i a l y s i s however, a major p o r t i o n o f the bound p h e n o l i c a c i d s escaped t o the e x t e r n a l medium. There are a t l e a s t two ways of e x p l a i n i n g t h e s e r e s u l t s : These d i a l y s a b l e p h e n o l i c c o n j u g a t e s must have had a m o l e c u l a r weight between 5000 g/K (the i o n - e x c l u s i o n l i m i t o f Sephadex G-25) and 10,000 g/M ( t h e c l a s s i c a l l e v e l above which p r o t e i n s are g e n e r a l l y thought t o be r e t a i n e d i n the d i a l y s i s bag) i n which case they cannot be d e s i g n a t e d as t r u e , h i g h - m o l e c u l a r - w e i g h t f r a c t i o n s . The o t h e r e x p l a n a t i o n i s t h a t the p h e n o l i c m o i e t i e s were weakly a s s o c i a t e d e i t h e r v i a hydrogen bonding o r a d s o r p t i o n . The e a r l y attempts a t s e p a r a t i n g bound phenols on Sephadex were c o m p l i c a t e d by a d s o r p t i o n e f f e c t s . When b u f f e r e d e x t r a c t s were a p p l i e d t o Sephadex columns e q u i l i b r a t e d i n c o n v e n t i o n a l b u f f e r s , the i n i t i a l e l u t i o n p r o f i l e s were not r e p r o d u c i b l e even i f the g e l was p r e -c y c l e d w i t h s o l v e n t s such as 7M urea and/or e t h a n o l . I f a p o r t i o n of the p r e c y c l e d g e l was made a l k a l i n e , a y e l l o w c o l o r was ob s e r v e d . Orth_p_-dihydroxy p h e n o l i c compounds gave a s i m i l a r r e a c t i o n w i t h base. I t was conc l u d e d t h a t g e l f i l t r a t i o n d e p a r t e d from normal b e h a v i o u r not o n l y by l a t e e l u t i o n , the r e s u l t o f a d s o r p t i o n , but a l s o through i r r e v e r s i b l e b i n d i n g . 22 An e l u t i o n p a t t e r n based on normal g e l f i l t r a t i o n must be r e p r o d u c i b l e on the same bed. I f i t cannot be d u p l i c a t e d on the same column one c o n c l u d e s t h a t the pore s t r u c t u r e has been a l t e r e d . These e f f e c t s can be reduced, however, by the use of a n t i - a d s o r p t i v e agents such as 7H ure a or 73 phenol : a c e t i c a c i d : water T r a c e A ( F i g u r e 4) i s an e l u t i o n p r o f i l e o f a b u f f e r e d e x t r a c t from the acetone powder of M. a r v e n s i s . The Sephadex G-25 column was developed w i t h s t a n d a r d phosphate b u f f e r a t 2 ° . Tr a c e B i s an e l u t i o n p r o f i l e o f the same e x t r a c t on an i d e n t i c a l column e q u i l i b r a t e d w i t h 7M u r e a : 0.1% a c e t i c a c i d . I t was c o n c l u d e d , t h e r e f o r e , t h a t i n the presence of c o n v e n t i o n a l b u f f e r s the major p o o l o f bound p h e n o l i c a c i d s i s s t r o n g l y adsorbed t o the d e x t r a n g e l . On prolonged s t a n d i n g , i t becomes i m p o s s i b l e t o e l u t e t h e s e components even w i t h the use of a n t i - a d s o r p t i v e s o l v e n t s . P r e c i p i t a t i o n w i t h (NH^^SO^ has been i n c l u d e d as a p u r i f i c a t i o n s t e p by o t h e r s 1 ^ ' 4 ^ ' ^ . I f an aqueous e x t r a c t (see page 34) of an acetone powder o f M. a r v e n s i s i s p r e c -i p i t a t e d w i t h ( N H 4 ) 2 S 0 4 to a f i n a l c o n c e n t r a t i o n o f 70% the p r e c i p i t a t e appears t o c o n t a i n a p h e n o l i c component(s) as evidenced by absorbance at 325 nm (c i n n a m i c a c i d s : 87 290 - 330 nm) . Two components were r e s o l v e d from t h i s p r e c i p i t a t e by the f o l l o w i n g p r o c e d u r e : 0-8 0-6 i i i Column e q u i l i b r a t e d wi th . -0-1M pH7 phosphate • • 7M urea A A 7M urea o o Absorbance at 280nm at 3 2 5 n m - i 0-4 0-2 0-0 B 6 :f3.-rr6_-r-.8crz8rrra— • — I s 8 10 12 14 F R A C T I O N S (x1-0ml) 16 40 Figure 4. Sephadex G—25 column chromatography of aqueous extracts of acetone powders of M. arVensis (Refer to text f o r A, B, and C). 24 The (NH^^SO^. p r e c i p i t a t e was i n i t i a l l y resuspended and c e n t r i f u g e d a g a i n to-remove r e s i d u a l s u p e r n a t a n t . I t was then d i s s o l v e d i n 0.1M pH 7 phosphate b u f f e r c o n t a i n i n g 0.02M m e r c a p t o e t h a n o l . Acetone (or dioxane) was added t o b r i n g the f i n a l c o n c e n t r a t i o n t o 65% and the p r e c i p i t a t e ( t r a c e C, F i g u r e 4) was c o l l e c t e d as b e f o r e . The super-n a t a n t ( t r a c e B, F i g u r e 4) was f r e e z e - d r i e d . G e l chromatography was performed on Sephadex G-25 M (1 x 21 cm) e q u i l i b r a t e d w i t h 7M u r e a : 0.1% a c e t i c a c i d and the sample volume was 0.1 ml. Thus p r e c i p i t a t e C r e p r e s e n t s a h i g h - m o l e c u l a r - w e i g h t f r a c t i o n and t h i s i s shown by i t s e l u t i o n p r o f i l e on Sephadex G-25 i n F i g u r e 4. Chromatography o f the f r e e z e -d r i e d s u p e r n a t a n t on G-25 shows t h a t i t i s a m i x t u r e of low and h i g h m o l e c u l a r weight components ( t r a c e B, F i g u r e 4 ) . P r e c i p i t a t e C d i d not y i e l d p h e n o l i c a c i d s on h y d r o l y s even when gram q u a n t i t i e s were used. T h e r e f o r e , (NH.J^SO, ' 4 2 4 p r e c i p i t a t i o n had a c h i e v e d n o t h i n g . However, o r g a n i c s o l v e n t p r e c i p i t a t i o n had s e p a r a t e d a major, n o n - p h e n o l i c component from the aqueous e x t r a c t of an acetone powder and t h i s f e a t u r e was s u b s e q u e n t l y a p p l i e d t o the f i n a l methods of p u r i f i c a t i o n . 25 D e t e c t i o n o f the S t h a n o l - I n s o l u b l e P h e n o l i c a c i d Conjugates  i n M. a r v e n s i s . G e l chromatography on Sephadex G-25 e q u i l i b r a t e d w i t h 7M u r e a : 0.1% a c e t i c a c i d r e v e a l e d a major p o o l o f bound p o l y p h e n o l s i n aqueous e x t r a c t s of acetone powders ( F i g u r e 4 ) . The f o l l o w i n g experiments d e s c r i b e how the components of t h i s major p o o l were o r i g i n a l l y r e s o l v e d . The b u f f e r e d e x t r a c t o f 1 g acetone powder was brought t o 40?£ dioxane and, a f t e r c e n t r i f u g a t i o n , the f r e e z e -d r i e d s u p e r n a t a n t was chromatographed on Sephadex G-25 e q u i l i b r a t e d w i t h 7M urea : 0.1% a c e t i c a c i d . The u r e a was removed on Sephadex G-10 e q u i l i b r a t e d i n 50% aqueous acetone and the f r a c t i o n s a b s o r b i n g at 325 nm were pooled and f r e e z e - d r i e d . When the c o n c e n t r a t e d e l u a t e was sub-j e c t e d t o h i g h v o l t a g e paper e l e c t r o p h o r e s i s a t pH 1.9 a group o f f l u o r e s c e n t , a n i o n i c bands was r e s o l v e d . T h e i r f l u o r e s c e n c e was enhanced w i t h ammonia v a p o r s . When the f r e e z e - d r i e d s u p e r n a t a n t from 10 g acetone powder was a p p l i e d t o a Sephadex G-10 column (10 x 60 cm) e q u i l i b r a t e d w i t h 50% acetone : 0.1% a c e t i c a c i d , the e l u t i o n p r o f i l e i n F i g u r e 5 was o b t a i n e d . Three p r i n c i p a l peaks were r e s o l v e d i n the system. The two minor peaks were not examined and whether they r e p r e s e n t analogs of h i g h e r m o l e c u l a r weight i s a matter f o r c o n j e c t u r e . 26 F i g u r e 5 . Sephadex G-10 column chromatography of e x t r a c t c o n t a i n i n g f l u o r e s c e n t , c a f f e o y l c o n j u g a t e s . 27 A t t e n t i o n was d i r e c t e d to the major peak ( F r a c t i o n s No. 18-29). These f r a c t i o n s were pooled and a n a l y z e d by e l e c t r o p h o r e s i s . An a r r a y o f f l u o r e s c e n t bands was observed m i g r a t i n g towards the anode. T y p i c a l e l e c t r o -p h o r e t i c p a t t e r n s are i l l u s t r a t e d d i a g r a m a t i c a l l y i n F i g u r e 6. Band A r e s o l v e s i n t o B and C a t pH 1.9. When B and C were developed a g a i n at pH 5.7, the same a n i o n i c , f l u o r e s c e n t p a t t e r n s were observed (B and C ) . The same p h e n o l i c a c i d s ( c a f f e i c , para-coumaric and f e r u l i c ) were l i b e r a t e d when B and/or C were e l u t e d and h y d r o l y z e d . T h e r e f o r e , two groups o f bound p h e n o l i c a c i d s were d i f f e r e n t i a t e d on the b a s i s of t h e i r m o b i l i t y a t pH 1.9. Furthermore, th e s e two groups were not i n t e r c o n v e r t i b l e as judged by a second e l e c t r o p h o r e t i c run of B which y i e l d e d o n l y B a t pH 1.9. S i m i l a r l y , C y i e l d e d o n l y C. The compounds i n the B band were chosen f o r p r e p a r a t i v e i s o l a t i o n because of t h e i r h i g h e r c o n c e n t r a t i o n and g r e a t e r s t a b i l i t y . The g e n e r a l scheme f o r the p u r i f i c a t i o n o f the B components i s d e s c r i b e d i n F i g u r e 9 i n the f o l o w i n g s e c t i o n . N o t i c e t h a t d u r i n g i s o l a t i o n on a l a r g e s c a l e s i x compounds were r e s o l v e d from the B group and .these were d e s i g n a t e d as compounds 1,2,3,4,5,and 6. 28 F i g u r e 6 . Diagrammatic r e p r e s e n t a t i o n s of e l e c t r o p h o r e t o g r a m s . I 2 | 10 I £ | 3 0 c m + Ul t rav io le t f luorescence B lue Absorbs (Blue/NH 3) fTT^I Orange yZ7A B' 29 The D e r i v a t i o n of Mai or P h e n o l i c A c i d P o o l s i n M. a r v e n s i s The p r e v i o u s o b s e r v a t i o n s and c o n c l u s i o n s l e d the author to d e v e l o p two p a r a l l e l schemes f o r the i s o l a t i o n o f the bound, " i n s o l u b l e " p h e n o l i c c o n j u g a t e s ( F i g u r e s 7 and 8 ) . In t h e s e f l o w diagrams the acetone- and e t h a n o l -s o l u b l e f r a c t i o n s r e p r e s e n t the pool of f r e e p h e n o l i c a c i d s , g l y c o s i d e s and e s t e r s such as r o s m a r i n i c a c i d and c h l o r o g e n i c a c i d ( F r a c t i o n s IA and T I A ) . Aqueous e x t r a c t s of the acetone- or e t h a n o l - i n s o l u b l e r e s i d u e s r e v e a l a second p o o l of p h e n o l i c a c i d c o n j u g a t e s ( F r a c t i o n s IB and I I B ) . The PAW s o l u b l e f r a c t i o n c o n t a i n s a t h i r d s o urce of bound p h e n o l i c a c i d s ( F r a c t i o n s IC and I I C ) . The amount of p h e n o l i c a c i d s t h a t can be l i b e r a t e d from Acetone Powder IC and Residue IIC i s n e g l i g i b l e . These are d e s i g n a t e d as the l i g n i n - c e l l w a l l f r a c t i o n s . They y i e l d e d the p h e n o l i c aldehydes para-hydroxybenzaldehyde, v a n i l l i n and s y r i n g a l d e h y d e on a l k a l i n e n i t r o b e n z e n e o x i d a t i o n performed a c c o r d i n g t o the method of Stone and B l u n d e l l 5 0 . Although acetone powders (Method I) were used as the s t a r t i n g p o i n t f o r l a r g e - s c a l e i s o l a t i o n s , the a l t e r n a t e scheme (Method I I ) was pursued to d i s c o v e r whether F r a c t i o n s IIB and IIC c o n t a i n e d the same p h e n o l i c c o n j u g a t e s as F r a c t i o n IB and IC. 30 F i g u r e 7. The d e r i v a t i o n o f major p h e n o l i c a c i d p o o l s by Method I . Mentha a r v e n s i s e x t r a c t w i t h c o l d acetone I n s o l u b l e (ACETONE POWDER IA) S o l u b l e (FRACTION IA) e x t r a c t w i t h water » * d r y w i t h acetone I n s o l u b l e (ACETONE POWDER IB) S o l u b l e (FRACTION IB) e x t r a c t w i t h PAW dr y w i t h acetone I n s o l u b l e (ACETONE POWDER IC) S o l u b l e (FRACTION IC) Water c o n t a i n i n g 0.1% a c e t i c a c i d and 0.1% mercap t o e t h a n o l The extracted acetone powder i s combined w i t h acetone, p u l v e r i z e d i n a Waring b l e n d e r and c o l l e c t e d on a Buchner funnel• 31 F i g u r e 8. The d e r i v a t i o n o f major p h e n o l i c a c i d p o o l s by Method I I . Mentha a r v e n s i s e x t r a c t w i t h hot e t h a n o l I n s o l u b l e (RESIDUE IIA) e x t r a c t w i t h water d r y w i t h acetone S o l u b l e (FRACTION IIA) I n s o l u b l e (RESIDUE IIB) e x t r a c t w i t h PAW d r y w i t h acetone I n s o l u b l e (RESIDUE IIC) S o l u b l e (FRACTION IIB) S o l u b l e (FRACTION IIC) Water c o n t a i n i n g 0.1% a c e t i c a c i d and 0.1% m e r c a p t o e t h a n o l . The e x t r a c t e d acetone powder i s combined w i t h acetone, p u l v e r i z e d i n a Waring b l e n d e r and c o l l e c t e d on a Buchner f u n n e l . 32 F i g u r e 9 d e s c r i b e s the g e n e r a l scheme (Method IB) f o r p u r i f y i n g the f l u o r e s c e n t , e t h a n o l - i n s o l u b l e c o n j u g a t e s o f Mentha from e i t h e r F r a c t i o n IB or I I B ( F i g u r e s 7 and 8 ) . The d e t a i l e d account o f each s t e p w i l l be d e s c r i b e d i n the next s e c t i o n . A t h i r d method, i n v o l v i n g the b u f f e r e d e x t r a c t i o n o f f r e s h p l a n t m a t e r i a l w i l l a l s o be d e s c r i b e d i n the next s e c t i o n . 33 F i g u r e 9. P u r i f i c a t i o n o f the f l u o r e s c e n t , e t h a n o l -i n s o l u b l e p h e n o l i c c o n j u g a t e s by Method IB, FRACTION IB o r I I B p r e c i p i t a t i o n w i t h acetone and dioxane P r e c i p i t a t e s S upernatant a d s o r p t i o n on D E A E - C e l l u l o s e H 20 and 10% a c e t i c a c i d e l u a t e s PAW e l u a t e ( f r e e z e - d r y ) p r e p a r a t i v e e l e c t r o -p h o r e s i s (pH 1.9) A n i o n i c band a t 6 cm A n i o n i c band a t 3 cm paper chromato-graphy and TLC No. 1 No. 2 No. 3 No. 4 No. 5 No. 6 34 D e t a i l e d procedures Devised f o r the P r e p a r a t i v e I s o l a t i o n  o f F l u o r e s c e n t , E t h a n o l - i n s o l u b l e , P h e n o l i c C o n j u g a t e s . From Acetone Powders. (1) P r e p a r a t i o n of Acetone Powders The shoots were d i c e d i n t o a c h i l l e d , k i n g - s i z e Waring b l e n d e r , c o l d acetone (-20°) was added and the m i x t u r e was comminuted a t h i g h speed f o r 45 seconds. The e x t r a c t was f i l t e r e d immediately through a Buchner f u n n e l and washed s e v e r a l times w i t h c o l d a c e t o n e . The powder was a i r - d r i e d f o r 10 minutes on a sheet of chromatography paper and then t r a n s f e r r e d f o r one hour t o a d e s i c c a t o r connected t o a vacuum pump t o remove the l a s t t r a c e s o f acetone. The d r i e d acetone powders were s t o r e d i n a deep f r e e z e . (2) E x t r a c t i o n o f Acetone Powder In the f o l l o w i n g s t e p s the temperature was m a i n t a i n e d a t 2° u n l e s s o t h e r w i s e i n d i c a t e d . F i f t y grams of acetone powder were combined w i t h 2000 ml d i s t i l l e d water c o n t a i n i n g 0.1% g l a c i a l a c e t i c a c i d and 0.1% m e r c a p t o e t h a n o l . The m i x t u r e was s t i r r e d f o r one hour, squeezed through f i n e n y l o n n e t t i n g and the e x t r a c t was c e n t r i f u g e d f o r ten minutes i n a S o r v a l l GSA r o t o r at 16,000 x g. The s u p e r n a t a n t was decanted, s h e l l - f r o z e n and f r e e z e - d r i e d . 35 (3) P r e c i p i t a t i o n of H i q h - m o l e c u l a r - w e i q h t Components The volume of the f r e e z e - d r i e d e x t r a c t was a d j u s t e d t o 400 ml w i t h d i s t i l l e d water c o n t a i n i n g 0.1% mercapto-e t h a n o l . While s t i r r i n g r a p i d l y , dioxane (15°) was added to g i v e a f i n a l c o n c e n t r a t i o n of 33% and the s t i r r i n g proceeded f o r 15 minutes. The m i x t u r e was c e n t r i f u g e d a t 27,000 x g f o r . 2 minutes and the s u p e r n a t a n t was decanted and f r e e z e - d r i e d . The volume of the f r e e z e - d r i e d s u p e r n a t a n t was a d j u s t e d to 250 ml w i t h 0.0IM pH 6 phosphate b u f f e r . While s t i r r i n g r a p i d l y , acetone was added t o g i v e f i n a l c o n c e n t r a t i o n of 50%. The m i x t u r e was s t i r r e d , c e n t r i f u g e d and f r e e z e - d r i e d by the above method. (4) A d s o r p t i o n by D E A E - C e l l u l o s e ( a c e t a t e c o u n t e r i o n ) The f r e e z e - d r i e d s u p e r n a t a n t was d i s s o l v e d i n a minimum amount of aqueous 2% a c e t i c a c i d and combined w i t h 50 g D E A E - C e l l u l o s e (Whatman, DE-32, m i c r o g r a n u l a r form, suspended i n 500 ml water) p r e v i o u s l y c o n v e r t e d from the f r e e base to the a c e t a t e form. The m i x t u r e was s t i r r e d f o r 40 minutes, the g e l was c o l l e c t e d on Whatman No.l on a Buchner f u n n e l and washed w i t h 3 x 500 ml water and then w i t h 10 x 500 ml 5% a c e t i c a c i d . The washed g e l was suspended t w i c e i n 250 ml PAW, s t i r r e d f o r 40 minutes and f i l t e r e d . The f i l t r a t e s from t h i s b a tchwise e l u t i o n were combined and f r e e z e - d r i e d . To c o m p l e t e l y remove the phenol i t was n e c e s s a r y to add water, melt and then s h e l l -36 f r e e z e the f r a c t i o n s a t l e a s t f i v e times d u r i n g the f r e e z e - d r y i n g p r o c e d u r e . The presence of o r t h o - d i h y d r o x y -phenyl d e r i v a t i v e s i s i n d i c a t e d by the f o r m a t i o n of a y e l l o w c o l o r i n the presence of a l k a l i and so, washing was t e r m i n a t e d when the f i l t r a t e gave o n l y a p a l e y e l l o w c o l o r w i t h NaOH. The p r o g r e s s o f e l u t i o n was i n d i c a t e d by the appearance o f b r i g h t y e l l o w c o l o r when the PAW e l u a t e was made a l k a l i n e . (5) R e s o l u t i o n by TLC The f r e e z e - d r i e d e l u a t e was s t r e a k e d from edge to edge on long C e l l u l o s e MN 300 G p l a t e s and chromatographed at room temperature f o r 48 hours i n BAW. The s p e c i f i c bands (see T a b l e I I ) , were e l u t e d w i t h water and r e -* chromatographed on s h o r t A v i c e l p l a t e s i n BzAWE and on s h o r t A v i c e l p l a t e s i n AnAW. The p u r i f i e d compounds were e l u t e d , f r e e z e - d r i e d and s t o r e d at - 2 0 ° . (6) R e s o l u t i o n by HVPE, Paper Chromatography and TLC T h i s s t e p was used f o r i s o l a t i n g compounds 4,5 and 6. Furthermore, t h i s a c c e l e r a t e d procedure c o n s i s t e n t l y gave h i g h y i e l d s of compounds 1,2 and 3. Hence i t was used f o r i s o l a t i n g the l a b e l l e d compounds i n t r a c e r e x p e r i m e n ts . The f r e e z e - d r i e d PAW e l u a t e from s t e p (4) was d i s s o l v e d i n a minimum amount of water and a p p l i e d as a band 14 i n c h e s from the anode end on Whatman No. 3MM • See T a b l e I I I f o r "Key to Solvents'*. 37 chromatography paper, 10 i n c h e s wide. The paper was wetted w i t h b u f f e r and b l o t t e d by sta n d a r d p r o c e d u r e s . The c o n d i t i o n s f o r e l e c t r o p h o r e s i s were as f o l l o w s : pH 1.9 ( a c e t a t e - f o r m a t e b u f f e r ) ; 3000 v o l t s ; i n i t i a l c u r r e n t of 100 mAmp; 60 minutes; i n i t i a l temperature o o 16 i n c r e a s i n g t o 21 . Two p r i n c i p l e f l u o r e s c e n t bands were r e s o l v e d m i g r a t i n g 3 cm and 6 cm i n the d i r e c t i o n o f the anode. Each band was e x c i s e d and s t i t c h e d to a s t r i p o f chromatograph paper and developed i n BzAWE. The e l e c t r o p h o r e t i c band m i g r a t i n g 3 cm y i e l d e d pure compound 4 and a mi x t u r e o f compounds 5 and 6 a f t e r chromatography. The l a t t e r two were r e s o l v e d by s t i t c h i n g the mixed band to chromatography paper and d e v e l o p i n g i t i n 2% HCOOH. The band m i g r a t i n g 6 cm y i e l d e d pure compound 1 and a mi x t u r e o f compounds 2 and 3. The mixture was e l u t e d , f r e e z e - d r i e d and se p a r a t e d by m u l t i p l e development on s h o r t A v i c e l p l a t e s i n AnAW. Thus a c l a s s of n a t u r a l l y - o c c u r i n g compounds had been r e s o l v e d , and-they a re c h a r a c t e r i z e d by t h e i r e l e c t r o n e g a t i v i t y and f l u o r e s c e n c e . T h e i r wavelength of maximum absorbance, bathochromic s h i f t s and c o l o r under u l t r a - v i o l e t l i g h t are l i s t e d i n T a b l e I I . Compound 1, o c c u r r i n g as .003% of the f r e s h weight, was the major c o n s t i t u e n t and a t l e a s t 10 times more c o n c e n t r a t e d than No. 3 or 2. A t t e n t i o n was d i r e c t e d t o No. 1, 2 and 3 i n which the r e s p e c t i v e p h e n o l i c groups 38 were i d e n t i f i e d as c a f f e i c , f e r u l i c and para-coumaric a c i d s by co-chromatography wi t h a u t h e n t i c s t a n d a r d s i n f i v e s o l v e n t svstems. T a b l e I I U l t r a - v i o l e t S p e c t r a and F l u o r e s c e n t C o l o r s of I s o l a t e d comoounds Compound X max(nm) Water Bathochromic s h i f t (nm)  + NaOH + H3B0 3/ NaOAc C o l o r s u.v. u.v./NH, 1 2 325 322 312 59 49 4 6 19" 345,285, 47 250* 322 320 0 24 + + sky-b l u e b l u e b l u e b l u e -w hite b l u e -green absorbs dark-b l u e p a l e - b r i g h t -y e l l o w y e l l o w b r i g h t -y e l l o w absorbs b r i g h t -y e l l o w I n f l e c t i o n • * In excess NaBH^ + In 10% water i n e t h a n o l * + In methanol c o n t a i n i n g 1% water.. 3 9 From E t h a n o l - i n s o l u b l e Residues Mentha, 100 g f r e s h - w e i g h t , was homogenized w i t h 2 l i t e r s 95% e t h a n o l i n a b l e n d e r and t h o r o u g h l y e x t r a c t e d v/ith h o t 80% e t h a n o l . The e t h a n o l i n s o l u b l e r e s i d u e was c o l l e c t e d and e x t r a c t e d w i t h 500 ml water c o n t a i n i n g 0.1% a c e t i c a c i d and 0.1% m e r c a p t o e t h a n o l . The f i l t r a t e was f r e e z e - d r i e d and t r e a t e d a c c o r d i n g t o s t e p s (4) and (6) above. From B u f f e r E x t r a c t i o n o f F r e s h M a t e r i a l Mentha, 300 g f r e s h - w e i g h t , was blended w i t h 1 l i t e r pH 5.3 p y r i d i n i u m a c e t a t e b u f f e r c o n t a i n i n g 0.02M merca p t o e t h a n o l The c e l l u l a r d e b r i s was s e p a r a t e d by f i l t r a t i o n through n y l o n n e t t i n g f o l l o w e d by c e n t r i f u g a t i o n . The c l e a r , b u f f e r e d e x t r a c t was f r e e z e - d r i e d and r e d i s s o l v e d i n 250 ml 0.01M pH 6 phosphate b u f f e r c o n t a i n i n g 0.0 2^ m e r c a p t o e t h a n o l . High-m o l e c u l a r - w e i g h t components were p r e c i p i t a t e d by the method d e s c r i b e d i n p a r t ( 3 ) . The f r e e z e - d r i e d s u p e r n a t a n t was d i s s o l v e d i n 100 ml d i s t i l l e d water and t h i s was a p p l i e d t o a 4 x 40 cm. polyamide (Woelm) column p r e v i o u s l y e q u i l i b r a t e d v/ith d i s t i l l e d v/ater. The column bed was e l u t e d w i t h 500 ml d i s t i l l e d water and the e l u a t e was f r e e z e - d r i e d . The f r e e z e - d r i e d polyamide e l u a t e was developed c o n s e c u t -i v e l y on Whatman No. 3 MM chromatography paper i n the f o l l o w i n s o l v e n t s : BAW (48 h o u r s ) ; BzAW : water : e t h a n o l (10 : 5 : 40 20, 48 h o u r s ) ; Butanol : e t h a n o l : water ( 4 : 1 : 2:2, 24 h o u r s ) ; * BzAWE and 2% HCOOH . Compounds 1, 2 and 3 were i d e n t i f i e d by u.v. s p e c t r o p h o t o m e t r y a f t e r e l u t i o n . A p p l i c a t i o n of the Methods to the " I n s o l u b l e " P h e n o l i c E s t e r s of Wheat Method IB was a p p l i e d to 14-day-old T h a t c h e r wheat shoots to see whether s i m i l a r compounds c o u l d be d e t e c t e d . A c c o r d i n g l y , the e x t r a c t o f two grams of acetone powder of T r i t i c u m aestivum was p r e p a r e d by s t e p s 2 and 3 (see p r e v i o u s s u b s e c t i o n s ) and s u b j e c t e d t o KVPE (300 v o l t s , 90 minutes, pH 2, i n i t i a l c u r r e n t 100 mA, i n i t i a l temperature 14° i n c r e a s i n g to 2 4 ° ) . There were two prominent, a n i o n i c f l u o r e s c e n t , bands (see i n s e t , F i g u r e 10). The f a s t e r - m o v i n g band was e l u t e d , h y d r o l y z e d i n a l k a l i (N KaOH, 50°, one hour, under n i t r o g e n ) , and the e t h e r e x t r a c t of the h y d r o l y z a t e was chromatographed i n two dimensions (BzAW x 2% HCOOH). F e r u l i c a c i d was d e t e c t e d by i t s f l u o r e s c e n c e under u.v. l i g h t s , R f's, r e a c t i o n w i t h d i a z o t i z e d p a r a - n i t r o a n i l i n e and u.v. a b s o r p t i o n spectrum. T h e r e f o r e , wheat shoots c o n t a i n a compound s i m i l a r i n some r e s p e c t s t o compounds 1, 2 and 3. A l t h o u g h No. 1 m i g r a t e s f a s t e r than the f e r u l i c a c i d - c o n t a i n i n g band of wheat, bot h compounds e x h i b i t a n i o n i c b e h a v i o u r a t pH 2 ( F i g u r e 10). The author recommends these methods f o r the i s o l a t i o n and p u r i f i c a t i o n of bound, " i n s o l u b l e " f e r u l i c a c i d from wheat. See T a b l e I I I f o r "Key t o S o l v e n t s . 41 F i g u r e 10. E l e c t r o p h o r e t o g r a m developed at pH 2 and photographed i n u.v. l i g h t . C a f f e i c a c i d ( C F ) , c h l o r o g e n i c a c i d (CH), u m b e l l i f e r o n e (U>') , compound 1 and aqueous e x t r a c t s of acetone powders of wheat and mint are i l l u s t r a t e d . 42 P a r t i a l p u r i f i c a t i o n o f the H i q h - M o l e c u l a r - W e i q h t  Conjugate of C a f f e i c a c i d i n M. a r v e n s i s . S o l u b l e c o n j u g a t e s of p h e n o l i c a c i d s i n c l u d i n g r o s m a r i n i c a c i d are e l i m i n a t e d by e x t r a c t i o n w i t h e t h a n o l (or acetone) f o l l o w e d by e x t r a c t i o n v/ith water ( F i g u r e s 7 and 8 ) . The r e m a i n i n g c e l l u l a r d e b r i s i s r e - e x t r a c t e d w i t h c o l d acetone and s u b s e q u e n t l y d r i e d . T h i s second acetone powder (Acetone Powder IB or Residue IIB) i s the s t a r t i n g p o i n t f o r the i s o l a t i o n o f the h i g h - m o l e c u l a r -weight c o n j u g a t e of c a f f e i c a c i d . The second acetone powder (lOg) was e x t r a c t e d f o r one hour wi t h 150 ml PAW, f i l t e r e d through M i r a c l o t h and the f i l t r a t e was f r e e z e - d r i e d . The f r e e z e - d r i e d e x t r a c t was d i s s o l v e d i n a minimum volume of 7M urea : 0.1% a c e t i c a c i d , f i l t e r e d and the f i l t r a t e was a p p l i e d to a Sephadex G-100 column (2.5 x 15 cm) e q u i l i b r a t e d w i t h the same s o l v e n t . The e l u t i o n p a t t e r n r e v e a l s a s i n g l e major peak a b s o r b i n g a t 325nm ( F i g u r e 11a). F r a c t i o n s No. 6-9 were combined and d i a l y s e d a g a i n s t water c o n t a i n i n g 0.01% m e r c a p t o e t h a n o l . The d i a l y s i s proceeded f o r 48 hours w i t h two - 4 l i t e r changes o f water and the bag c o n t e n t s were f r e e z e - d r i e d . A p a l e y e l l o w powder (lOmg) was o b t a i n e d . 43 F i g u r e 11a. Sephadex G-100 column chromatography of e x t r a c t c o n t a i n i n g h i g h - m o l e c u l a r - w e i g h t c a f f e y l c o n j u g a t e . F i g u r e l i b . Diagrammatic r e p r e s e n t a t i o n of a n i o n i c bands ( a c t u a l s i z e ) o b t a i n e d a f t e r HVPE of Sephadex G-100 e l u a t e c o n t a i n i n g h i g h - m o l e c u l a r - w e i g h t c a f f e y l c o n j u g a t e . 4 4 45 C a f f e i c a c i d was r e l e a s e d on t r e a t i n g the powder w i t h 2N HC.1 (90°, 30 m i n u t e s ) . The f r e e z e - d r i e d hydro-l y z a t e was chromatographed i n two dimensions (BzAW x 2% HCOOH) and the c a f f e i c a c i d was i d e n t i f i e d by i t s f l u o r e s c e n c e , c o l o r r e a c t i o n w i t h d i a z o t i z e d p a r a - n i t r o -a n i l i n e , chromatographic R f v a l u e s and u.v. spectrum. The h i g h m o l e c u l a r weight, deduced from chromatography on G-100 and d i a l y s i s , and the l a c k of f l u o r e s c e n c e are two f e a t u r e s which d i s t i n g u i s h t h i s form o f bound c a f f e i c a c i d from compound No. 1 which i s d e r i v e d from the water-s o l u b l e p o o l . The u.v. a b s o r p t i o n spectrum of the macro-m o l e c u l a r form e x h i b i t s a maximum at 320 nm and 287 nm. ( C a f f e i c a c i d has a minimum at 315 nm and a s h o u l d e r around 285 nm a t n e u t r a l pH, F i g u r e 12). The macro-m o l e c u l a r c a f f e y l c o n j u g a t e i s s t r o n g l y adsorbed on paper e l e c t r o p h o r e t o g r a m s , DEAE c e l l u l o s e ( a c e t a t e c ounter i o n ) and Sephadex G-10 and G-15. I t i s s t a b l e i n d e f i n i t e l y when s t o r e d as a f r e e z e - d r i e d powder at 0° and under n i t r o g e n . In n e u t r a l s o l u t i o n i t i s g r a d u a l l y o x i d i z e d by the a i r t o a brown s u s p e n s i o n . E l e c t r o p h o r e s i s (3000 v o l t s , pH 2, 45 minutes) of the i s o l a t e d compound(s) r e v e a l e d a s e r i e s of a n i o n i c bands m i g r a t i n g 0 - 6 cm ( F i g u r e l i b ) . These bands are d e t e c t e d under u.v. l i g h t by t h e i r y e l l o w c o l o r a t i o n i n the presence of ammonia. Figure 12. U l t r a - v i o l e t absorption spectra of high-molecular-weight c a f f e y l conjugate (X), c a f f e i c acid (Y) and compound 1 (Z) i n d i s t i l l e d water 47 C r i t e r i a o f P u r i t y f o r Compound No. 1, 2 and 3 The i s o l a t e d compounds No. 1, 2 and 3 m i g r a t e d as s i n g l e d i s c r e t e bands toward the anode under the f o l l o w i n g c o n d i t i o n s o f h i g h - v o l t a g e paper e l e c t r o p h o r e s i s : pH 2.0 ( a c e t a t e - f o r m a t e b u f f e r ) ; 3000 v o l t s ; i n i t i a l c u r r e n t of 100mA; 40 minutes; i n i t i a l temperature 16° i n c r e a s i n g t o 21°. Each compound mi g r a t e d 3.2 cm as compared t o ze r o f o r u m b e l l i f e r o n e and c a f f e i c a c i d and 10.8 cm f o r c y s t e i c a c i d . The p u r i f i e d compounds migrated as s i n g l e s p o t s on paper chromatograms and on c e l l u l o s e TLC p l a t e s i n a v a r i e t y o f s o l v e n t systems. T a b l e I I I l i s t s the r e s p e c t i v e R f v a l u e s o f No. 1, 2 and 3 as w e l l as the r e l a t i v e p o s i t i o n o f c a f f e i c a c i d . The compounds showed s p e c i f i c c o l o r s under u l t r a -v i o l e t l i g h t and they possessed c h a r a c t e r i s t i c u.v. a b s o r p t i o n s p e c t r a and s h i f t s (see T a b l e I I ) . F i n a l l y , w i t h r e s p e c t t o the i s o l a t i o n p r o c e d u r e s , the s p e c i f i c e l u t i o n from DEAE C e l l u l o s e and the e l e c t r o -p h o r e t i c b e h a v i o u r a t pH 2 e l i m i n a t e s v i r t u a l l y a l l the n e u t r a l and c a t i o n i c p l a n t c o n s t i t u e n t s . 48 T a b l e I I I R-. Values xlOO * S o l v e n t s Compound BAW BzAW BzAWE T o l HCOOH+ + + • Pyr C a f f e i c A c i d 91 2 60 86 20 No. 1 73 * • 74 0 17 39 + 26 5 + 68 No. 2 80 • * 75 0 34 58 + 34 10 + 68 No. 3 80 * * 77 0 34 5 8 + 39 22 + 68 Key t o S o l v e n t s : BAW, b u t a n o l : a c e t i c - a c i d : water (4 : I : 5~, upper phase) ; BzAw, benzene : a c e t i c a c i d water (10 : 7 : 3, upper phase); BzAWE, BzAW : water e t h a n o l (50 : 5 : 20); T o l , t o l u e n e : b u t a n o l : p y r i d i n e ( c o n t a i n i n g 5% a c e t i c a c i d ) : water ( 1 : 4 : 2 : 1 ) ; HCOOH, 2% f o r m i c a c i d ; Pyr:, b u t a n o l : p y r i d i n benzene : water ( 5 : 3 : 1 : 3 ) . AnAW, a n i s o l e : a c e t i c a c i d : water (70 : 30 : 2 ) . 1 s were determined on Whatman No. 3 MM chromatography paper u n l e s s o t h e r -wise i n d i c a t e d . On MN C e l l u l o s e 300G. + On A v i c e l . + + R e f e r s t o t r a n s - c a f f e i c a c i d . + + + Rj. c h l o r o g e n i c a c i d = 58. R_ R f c i s - c a f f e i c a c i d = r o s m a r i n i c a c i d = 34. 49 A d s o r p t i v e P r o p e r t i e s of Comnound 1 On Dextran and F o l y a c r y l a m i d e G e l s The s t r o n g i n t e r a c t i o n between Sephadex g e l s and p h e n o l i c c o n j u g a t e s i n mint was d e s c r i b e d p r e v i o u s l y ( F i g u r e 4 ) . Compound 1 was observed to d e v i a t e s i m i l a r l y from the normal p a t t e r n of g e l f i l t r a t i o n on Sephadex G-25 (Medium) and G-10. The anomalous b e h a v i o u r was m a n i f e s t e d even i n the s o - c a l l e d a n t i - a d s o r p t i v e systems. In F i g u r e 13b, the t h r e e e l u t i o n p r o f i l e s of compound 1 can be d i r e c t l y compared s i n c e the sample s i z e (0.1ml) and colum dimensions (0.9 x 12.5 cm) were c o n s t a n t . N o t i c e the e x t e n t o f zone b r o a d e n i n g as compared to the e l u t i o n volume (0.7 ml) of the i o n e x c l u s i o n s t a n d a r d s (B l u e Dextran and h o r s e h e a r t cytochrome c ) . Thus No. 1 i s s t r o n g l y r e t a r d e d i n both the PAW and s t a n d a r d b u f f e r systems. The e l u t i o n p r o f i l e i n 7M urea i s s h a r p e r but the zone broadening i s s t i l l l a r g e s i n c e the sample volume was d i l u t e d f i f t y t i m e s . A t a i l i n g e f f e c t i s observed on G-10 i n 7M urea ( F i g u r e 13a). The e l u t i o n p r o f i l e on B i o - g e l P-2 i n 7M u r e a i s the s h a r p e s t but s t r o n g r e t e n t i o n was observed i n two runs out of f o u r . T h e r e f o r e , r e t a r d a t i o n through a d s o r p t i o n cannot be e x c l u d e d . F i n a l l y i t can be p o i n t e d out t h a t r e a l g e l 50 F i g u r e 13a. Column chromatography of compound 1 and c a f f e i c a c i d (Columns were e q u i l i b r a t e d w i t h 7M ur e a : 0.1% a c e t i c a c i d ) Mo. 1 on 3 i o - g e l P-2 • • • (100-200 mesh) No. 1 on Sephadex G-10' A A A C a f f e i c a c i d on Sephadex G-10 o o o F i g u r e 13b. Sephadex G-25 column chromatography of compound 1. Column was e q u i l i b r a t e d w i t h : 0.1% pH 7 phosphate b u f f e r o o o 7M ur e a : 0.1% a c e t i c a c i d • • • PAW A i * 52 f i l t r a t i o n was observed i n t h i n - l a y e r g e l chromatography (TLG). No. 1 m i grated as a d i s c r e t e , f l u o r e s c e n t spot on G-25 and G-5C ( S u p e r f i n e ) e q u i l i b r a t e d w i t h 7M urea : 0.1% a c e t i c a c i d . The r e l a t i v e m i g r a t i o n d i s t a n c e o f No. 1 was 0.64 as compared to cytochrome c on Sephadex G-25 and 0.47 as compared to Blue Dextran on Sephadex G-50. The a n a l y t i c a l a p p l i c a t i o n of TLG i n 7H u r e a : 0.1% a c e t i c a c i d s h o u l d prove to be u s e f u l i n f u t u r e s t u d i e s on compounds 1, 2 and 3. However, column chroma-tography, u s i n g the v a r i o u s g e l c o n d i t i o n s d e s c r i b e d above, should be a voided s i n c e the a d s o r p t i v e p r o p e r t i e s o f these compounds produce e x t e n s i v e zone b r o a d e n i n g . On DSAE C e l l u l o s e ( a c e t a t e c o u n t e r i o n ) E l u t i o n o f No. 1 by the PAW s o l v e n t i s not e f f e c t e d e x c l u s i v e l y by the change i n i o n i c f o r c e s . When a sample of No. 1 (200 pg) i s a p p l i e d to a DEAE C e l l u l o s e column (1.5 x 5 cm) and the column i s washed w i t h 75 ml 10% a c e t i c a c i d , an a d d i t i o n a l 100 ml PAW (7 bed volumes) i s r e q u i r e d to c o m p l e t e l y e l u t e the compound. The zone-b r o a d e n i n g i n d i c a t e s t h a t d e s o r p t i o n i s o c c u r r i n g as w e l l as e l u t i o n through ion-exchange. T h e r e f o r e , as a p r e p a r a t i v e , batchwise, procedure t h i s system has a d i s a d v a n t a g e s i n c e e x c e s s i v e volumes o f PAW would be r e q u i r e d f o r complete e l u t i o n and the PAW r eagent i s not e a s i l y l y o p h i l i z e d . The advantage 53 of the system i s the s p e c i f i c e l u t i o n which i s e f f e c t e d w i t h PAW. Thus, a n i o n i c , p h e n o l i c compounds (such as compounds 1, 2 and 3) are s t r o n g l y adsorbed a t a c i d i c pH and then s p e c i f i c a l l y desorbed w i t h PAW. On Polyamide The s e c t i o n d e s c r i b i n g the i s o l a t i o n procedure u s i n g polyamide (see page 3 9 ) i n d i c a t e d t h a t compound 1 moved w i t h the i n i t i a l e l u a t e , water. However, the f r e e z e -d r i e d b u f f e r e x t r a c t t h a t was a p p l i e d t o the polyamide c o n t a i n e d the r e s i d u a l b u f f e r s a l t s . When pure No. 1 was a p p l i e d t o a s i m i l a r column, e q u i l i b r a t e d i n water, i t was s t r o n g l y adsorbed. E x t e n s i v e washing w i t h water, aqueous e t h a n o l or acetone would not e l u t e the compound. High c o n c e n t r a t i o n s of s a l t , however, c o u l d e f f e c t the e l u t i o n . The compound was c o m p l e t e l y r e c o v e r e d by e l u t i n g w i t h acetone : a c e t i c a c i d : water (1 : 1 : 1) and f r e e z e - d r y i n g . T h i s a l t e r n a t e p r o c e d u r e of e l u t i o n can be a p p l i e d t o aqueous e x t r a c t s i n which the i o n i c s t r e n g t h has been a d j u s t e d c l o s e to z e r o . S i n c e the e l u t i o n i s complete, (as compared to the batchwise procedure w i t h DEAE C e l l u l o s e ) t h i s polyamide system i s recommended f o r the study of a b s o l u t e p o o l s i z e s o f compounds 1, 2 and 3. 54 P a r t i a l C h a r a c t e r i z a t i o n of " I n s o l u b l e " E s t e r s Comoound 1 P a r t i a l A c i d H y d r o l y s i s Compound 1 was i s o l a t e d a c c o r d i n g t o Method IB ( F i g u r e 9) and s u b j e c t e d t o the a d d i t i o n a l , e l e c t r o -p h o r e t i c treatment i n PAW (see page 66). One m i l l i g r a m o f compound 1 was h y d r o l y z e d i n 214 HCl (90°, 60 minutes) and the f r e e z e - d r i e d h y d r o l y z a t e was r e s o l v e d by HVPE (pH 2; 3000 v o l t s ; 100 mA; 30 minutes; temperature i n c r e a s e d from 12° t o 1 6 ° ) . Under these c o n d i t i o n s o f a c i d h y d r o l y s i s seven n i n h y d r i n - p o s i t i v e , c a t i o n i c bands and a f l u o r e s c e n t n e u t r a l band were r e l e a s e d . The c o n v e r s i o n o f 1 t o n e u t r a l and c a t i o n i c compounds was observed f o r d i f f e r e n t time i n t e r v a l s o f a c i d h y d r o l y s i s . F i g u r e 14 i l l u s t r a t e s the r e s u l t s o f the experiment i n which e q u a l q u a n t i t i e s of No. 1 were sub-j e c t e d t o h y d r o l y s e s f o r 10, 20, 40 and 60 minutes. Thus one observes the g r a d u a l d i s a p p e a r a n c e o f No. 1 and the appearance and p r o p o r t i o n a t e i n c r e a s e i n the n e u t r a l and c a t i o n i c bands. The f l u o r e s c e n t n e u t r a l band ( L i n F i g u r e 14) was e x c i s e d and s t i t c h e d t o a s t r i p o f chromatography paper and i t was i d e n t i f i e d as c a f f e i c a c i d by co-chromatography w i t h a u t h e n t i c s t a n d a r d s i n f i v e s o l v e n t systems. The c a t i o n i c bands (M,N,0,P,Q,R,S) appeared w i t h i n 5 5 F i g u r e 14. Diagrammatic r e p r e s e n t a t i o n (h a c t u a l s i z e ) of e l e c t r o p h o r e t o g r a m o f p a r t i a l a c i d h y d r o l y z a t e s and s t a n d a r d s . T S R CZD Q C Z D P ( Z Z > K 0 CZD N t 1 (5555® M C Z D L t ) G7777K 87777% (777777) ( ) + Lysi ne Aspart ic ac id C a f f e i c ac id No. 1 10min 20mln 40min 60min Standards R e l a t i v e i n t e n s i t y o f bands i n d i c a t e d by: t r a c e = I 1 + 777m 56 one hour a t room temperature a f t e r d i p p i n g the e l e c t r o -phoretograms i n the cadmium-ninhydrin r e a g e n t . A f t e r s t a n d i n g f o r s i x months i n the dark, the K band was found t o d e v e l o p a n i n h y d r i n - p o s i t i v e , v i o l e t c o l o r (an i n t e n s e , n i n h y d r i n - p o s i t i v e band, T, a l s o appeared a f t e r s i x months). The v i o l e t c o l o r of the K band was c o m p l e t e l y superimposed on the f l u o r e s c e n t p o s i t i o n o f compound 1. Furthermore, the n i n h y d r i n - p o s i t i v e , v i o l e t c o l o r o f K de c r e a s e d i n the same way ( i . e . from the 10 minute t o the 60 minute samples) as the o r i g i n a l d e c r e a s e i n f l u o r e s c e n c e i n t e n s i t y . T h e r e f o r e i t appeared t h a t t h e c a f f e i c a c i d of compound 1 was c o v a l e n t l y a s s o c i a t e d w i t h a p e p t i d e ( s ) . Enzymatic H y d r o l y s i s H y d r o l y s i s o f compound 1 by p r o t e o l y t i c ( t r y p s i n , p e p s i n , papain) and g l y c o h y d r o l y t i c enzymes ( p e c t i n a s e ) was attempted but the compound was not s u s c e p t i b l e . However, i n one experiment u s i n g papain, e l e c t r o p h o r e s i s of the f r e e z e - d r i e d h y d r o l y z a t e r e v e a l e d a minor, f l u o r e s c e n t , a n i o n i c band m i g r a t i n g f o u r times as f a s t as No. 1 a t pH 2. On the b a s i s of i t s extended m i g r a t i o n and f l u o r e s c e n c e t h i s fragment may r e p r e s e n t a d e r i v e d s u b u n i t c o n t a i n i n g c a f f e i c a c i d . M i l d A l k a l i n e H y d r o l y s i s When c a f f e i c a c i d was s u b j e c t e d t o hot, a c i d i c c o n d i t i o n s (2N HCl, 9 0 ° ) , 60% of the p h e n o l i c a c i d was 57 d e s t r o y e d i n 20 minutes. T h i s was determined s p e c t r o -p h o t o m e t r i c a l l y by m o n i t o r i n g the absorbance a t 325 nm f o r v a r i o u s time i n t e r v a l s . Cinnamic a c i d i s decarboxy-74 l a t e d t o s t y r e n e i n the presence of hot a c i d and i t was expected t h a t c a f f e i c a c i d behaved s i m i l a r l y . Compound No. 1 was not s i g n i f i c a n t l y a f f e c t e d by c o n d i t i o n s o f c o l d a c i d h y d r o l y s i s (25°, 24 h o u r s ) . T h e r e f o r e a l k a l i n e h y d r o l y s i s was attempted. Excess MaBH^ s t a b i l i z e s c a f f e y l compounds i n a l k a l i n e 75 media . NaBH^ (0.5 g) was added to an aqueous s o l u t i o n of No. 1 (0.5 mg i n 5 ml), t h i s was heated t o 50° under n i t r o g e n whereupon 1 ml 2N NaOH was i n t r o d u c e d and the temperature was m a i n t a i n e d f o r one hour. The volume was i n c r e a s e d to 100 ml w i t h water, the s o l u t i o n was a d j u s t e d t o pH 7 w i t h 2N HCl and a p p l i e d t o a DEAE C e l l u l o s e column ( a c e t a t e c o u n t e r i o n ; 3.5 x 3 cm Buchner f r i t t e d g l a s s f u n n e l ) supported on a s i d e arm f l a s k . The column was washed w i t h an a d d i t i o n a l 50 ml water and the f i l t r a t e was d i s c a r d e d . The c a f f e i c a c i d was e l u t e d w i t h 100 ml 10% a c e t i c a c i d and the e l u a t e was f r e e z e - d r i e d . The f r e e z e - d r i e d e l u a t e was d i s s o l v e d i n e t h a n o l and the 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 determined s p e c t r o -p h o t o m e t r i c a l l y . The i d e n t i t y o f the c a f f e i c a c i d was 9 c o n f i r m e d by the u.v. s p e c t r a l s h i f t s w i t h sodium a c e t a t e 76 (hypsochromic) and sodium acetate/H^BO^ (bathochrornic) as w e l l as by co-chromatography w i t h a u t h e n t i c c a f f e i c 58 a c i d i n f i v e s o l v e n t systems. The y i e l d o f c a f f e i c a c i d a f t e r m i l d a l k a l i n e h y d r o l y s i s was 22% (- 2%) based on the average of f o u r s p e c t r o p h o t o m e t r i c d e t e r m i n a t i o n s . I n t e r f e r e n c e by e s c u l e t i n , ( t he coumarin d e r i v e d by the l i g h t - a c t i v a t e d c y c l i z a t i o n o f c i s - c a f f e i c a c i d 7 8 ) was n e g l i g i b l e as judged by i t s absorbance a t 380 nm. No. 1 was s u b j e c t e d t o c o l d a l k a l i n e h y d r o l y s i s (N NaOH, 4 ° , 6 hours, under n i t r o g e n ) i n the absence of NaBH^ and the N a + i o n s were c o l l e c t e d on CM C e l l u l o s e ( H + ) . The g e l was e l u t e d w i t h water and the f r e e z e - d r i e d e l u a t e was s u b j e c t e d t o HVPE at pH 2. A p a r t i a l r e l e a s e of c a f f e i c a c i d was observed and o n l y two n i n h y d r i n - p o s i t i v e bands were r e v e a l e d . One band m i g r a t e d 7 cm from t h e o r i g i n w h i l e the second, l e s s a c i d i c band moved t o the p o s i t i o n o f M (see F i g u r e 14). When t h i s p r o c e d u r e was a p p l i e d t o a one-hour h y d r o l y z a t e a t 50° a s e r i e s o f n i n h y d r i n - p o s i t i v e bands analogous t o M,N,0,P,Q,R and S (see F i g u r e 14) was o b t a i n e d . No. 1 was exposed t o 5N NH^OH (4°, 6 hours under n i t r o g e n ) , the m i x t u r e was f r e e z e - d r i e d and HVFE (pH 2) i n d i c a t e d t h a t No. 1 was s t a b l e under t h e s e c o n d i t i o n s . E s t i m a t i o n of Percentage b y Weight of C a f f e i c a c i d  i n Compound 1 When compound 1 was s u b j e c t e d t o m i l d a l k a l i n e h y d r o l y s i s (see page 57 ) the y i e l d o f c a f f e i c a c i d was found t o be 22% (- 2 % ) . 59 The p e r c e n t a g e by.weight o f c a f f e i c a c i d i n compound 1 was a l s o e s t i m a t e d by comparing the s t a n d a r d c u r v e s f o r compound 1 and a u t h e n t i c c a f f e i c a c i d . Thus an aqueous s o l u t i o n o f compound 1, 13.5 /ag/ml (O.D. = .51), had the same absorbance as a 6.6 /jg/ml s o l u t i o n o f c a f f e i c a c i d . T h i s c o u l d imply t h a t compound 1 i s 50% c a f f e i c a c i d . However, t h i s was p r o b a b l y a maximum e s t i m a t e s i n c e the molar e x t i n c t i o n c o e f f i c i e n t s o f e s t e r s such as 75 e t h y l c a f f e a t e (€ = 36,170) and c h l o r o g e n i c a c i d (€ = 79 — 43 j140) are a p p r o x i m a t e l y t w i c e as g r e a t as the c f o r 75 c a f f e i c a c i d ( € = 20,410) i n n e u t r a l s o l u t i o n . There-f o r e , u s i n g t h i s f a c t o r the e s t i m a t e of the p e r c e n t by weight o f c a f f e i c a c i d i n compound 1 was found t o be a p p r o x i m a t e l y 25?4 and t h i s agrees w i t h the p r e v i o u s e s t i m a t e based on h y d r o l y s i s . L i n k a g e of c a f f e i c a c i d The f l u o r e s c e n c e under u.v. l i g h t and the s h i f t s i n the u.v. s p e c t r a ( T a b l e I I ) i n d i c a t e d t h a t the p h e n o l i c a c i d v/as l i n k e d through the c a r b o x y l group. T h i s was c o n f i r m e d by the absence of a hypsochromic s h i f t i n the pres e n c e of sodium a c e t a t e . D e t e r m i n a t i o n of S u l f u r i n Compound 1 The c h a r a c t e r i s t i c e l e c t r o p h o r e t i c p a t t e r n s a t pH 2 i n d i c a t e d the presence of an i n o r g a n i c a c i d i c group such as phosphate or s u l f a t e . The a n a l y s e s f o r phosphate 94 a c c o r d i n g t o the method o f Fiske-SubbaRow, proved t o 60 be n e g a t i v e when a p p l i e d t o a c i d d i g e s t s o f 0.5 mg of No. 1. However, when compound No. 1 was a n a l y z e d f o r the presence of s u l f u r the t e s t was p o s i t i v e (0.53% s u l f u r based on the a n a l y s i s of 0.665 mg No. 1 ) . One can argue, however, t h a t a s u l f u r c o n t e n t o f l e s s than one per c e n t c o u l d be a t t r i b u t e d t o e x p e r i m e n t a l e r r o r , s i n c e the a n a l y s i s v/as performed on an e x c e e d i n g l y s m a l l sample. 35 I n c o r p o r a t i o n o f S i n t o Compounds 1, 2 and 3. In an attempt t o determine whether t h e s e " i n s o l u b l e " c a f f e i c e s t e r s c o n t a i n e d s u l f u r , an experiment was 35 c a r r i e d out i n which N a 2 SO^ was a d m i n i s t e r e d t o shoots o f M. a r v e n s i s . F i f t y grams o f young shoots were a l l o w e d t o take 35 up a s o l u t i o n o f 244 yuc N a 2 S 0 4 (328 mc/mM) and t o m e t a b o l i z e f o r 43 hours i n l i g h t . Compounds 1, 2 and 3 were i s o l a t e d by Method IB and d i g e s t e d a c c o r d i n g t o the f o l l o w i n g p r o c e d u r e f o r e f f e c t i v e c o u n t i n g : The i s o l a t e d compound was d i g e s t e d i n HNO^ • HCIO^ (3:2) f o r one hour a t 120°. The s o l u t i o n was c o o l e d and a d j u s t e d f o pH 6 w i t h KOH p e l l e t s . The p r e c i p i t a t e d KCIO^ was removed by c e n t r i f u g a t i o n and the s u p e r n a t a n t was counted i n A q u a s o l . The s p e c i f i c a c t i v i t y o f compound No. 1 was found t o be 0.16 yuc/mg. Assuming a m o l e c u l a r weight i n the range 500-5000 g/M f o r compound No. 1, t h i s would g i v e 61 a s p e c i f i c a c t i v i t y o f 80 -800 uc/ml-l. The d i l u t i o n v a l u e s would then be 4000-400. The a c t i v i t y i n No. 2 was 3.7 x 1 0 4 dpm f o r a 5.0 ml s o l u t i o n (O.D.=0.46). 3 The a c t i v i t y i n No. 3 was 2.8 x 10 dpm f o r a 5.0 ml s o l u t i o n (O.D.=1.32). Subsequent amino a c i d a n a l y s i s d i d not r e v e a l the presence of s u l f u r - c o n t a i n i n g amino a c i d s . T h e r e f o r e , 35 i t was c o n c l u d e d t h a t the S r e s i d e d i n an a n i o n i c , a c i d i c group. Attempts t o I d e n t i f y the N o n - c a f f e o y l m o i e t y ( i e s ) 35 The S i n c o r p o r a t i o n and the a n i o n i c b e h a v i o u r of No. 1 prompted a s e a r c h f o r a s u b u n i t t h a t would s e r v e as the s u l f a t e c a r r i e r . One m i l l i g r a m samples o f compound 1 were used i n each o f the f o l l o w i n g e x p e r i m e n t s . Standard a c i d h y d r o l y s e s and chromatographic a n a l y s e s d i d not r e v e a l the presence o f s u g a r s . To a v o i d the p o s s i b i l i t y o f p h e n o l - f u r f u r a l f o r m a t i o n , which i s commonly encountered d u r i n g h y d r o l y s i s , No. 1 was sub-j e c t e d t o m e t h a n o l y s i s f o l l o w e d by h y d r o l y s i s v/ith A m b e r l i t e IR 120 ( H + ) . No sugars were d e t e c t e d . C a f f e i c a c i d i s f r e q u e n t l y e s t e r i f i e d t o q u i n i c a c i d (see T a b l e I ) . A s e a r c h f o r q u i n i c a c i d a c c o r d i n g t o the methods of Br a g t " ^ and C a r t w r i g h t ^ d i d not r e v e a l the presence of t h i s compound. Rosmarinic a c i d ( t he c a f f e o y l e s t e r o f 3,4-dihydroxy-p h e n y l l a c t i c a c i d ) i s a major p h e n o l i c c o n s t i t u e n t o f 62 Mentha ~. P a r t i a l base h y d r o l y s i s d i d not r e v e a l the presence of t h i s moiety. The p o s s i b i l i t y remains t h a t the s u l f a t e i s bound t o the h y d r o x y l o f an amino a c i d such as s e r i n e or to an u n s u b s t i t u t e d p h e n o l i c h y d r o x y l group. Compound 1 d i d not g i v e an immediate, p o s i t i v e t e s t w i t h n i n h y d r i n but a v i o l e t c o l o r appeared s i x months l a t e r . T h i s d e l a y e d n i n h y d r i n r e a c t i o n suggests t h a t an amino g r o u p ( s ) of No. 1 undergoes g r a d u a l un-masking. The d e l a y e d n i n h y d r i n r e a c t i o n i s c o m p l e t e l y superimposable w i t h the f l u o r e s c e n t p o s i t i o n o f No. 1, which i s a n i o n i c on pH 2 e l e c t r o p h o r e t o g r a m s . E v i d e n c e f o r the p e p t i d i c component(s) of compound No. 1 was r e v e a l e d by the r e s u l t s o f p a r t i a l a c i d h y d r o l y s i s ( F i g u r e 14). E l e m e n t a l a n a l y s i s performed on a 2 mg sample of No. 1 r e v e a l e d a n i t r o g e n c o n t e n t of 2.37%. As a r e s u l t o f t h e s e o b s e r v a t i o n s s t u d i e s were i n i t i a t e d on the amino a c i d c o m p o s i t i o n o f compound 1. P r i o r to amino a c i d a n a l y s i s , compound 1 ( i s o l a t e d by Method IB) was s u b j e c t e d to e l e c t r o p h o r e s i s i n PAW f o l l o w e d by column chromatography on D E A E - C e l l u l o s e , Thus, the a n t i - a d s o r p t i v e treatment and the s p e c i f i c e l u t i o n were r e p e a t e d t o ensure maximum p u r i t y . T a b l e IV l i s t s the c o n c e n t r a t i o n s of amino a c i d s (nanomoles amino a c i d per mg No. 1) d e r i v e d from the h y d r o l y s e s of f o u r d i f f e r e n t i s o l a t e s o f compound 1. 63 T a b l e IV C o n c e n t r a t i o n s of Amino A c i d s D e r i v e d from Four D i f f e r e n t I s o l a t e s o f Compound 1. L y s i n e — — 12.9 9.3 A s p a r t i c 8.4 10.2 9.3 16.5 Thr e o n i n e 4.8 5.4 7.8 6.9 S e r i n e 9.3 12.9 13.5 20.4 Glu t a m i c A c i d 24.9 18.0 16.5 20.7 G l y c i n e 45.3 46.8 38.1 49.8 A l a n i n e 14.4 9.6 22.2 15.6 V a l i n e - 7.8 11.4 -I s o l e u c i n e 3.3 4.2 6.0 4.8 L e u c i n e 4.2 7.2 9.0 7.8 * Amino a c i d v a l u e s a r e g i v e n as nanomoles amino a c i d per mg compound 1. 64 The y i e l d s are low ( a p p r o x i m a t e l y 1% by weight o f compound 1) when compared t o the n i t r o g e n c o n t e n t o f 2.37% o b t a i n e d by e l e m e n t a l a n a l y s i s of No. 1. Alt h o u g h a g e n e r a l p a t t e r n o f amino a c i d c o m p o s i t i o n i s r e v e a l e d , the r e l -a t i v e y i e l d s a re i n c o n s i s t e n t . T h i s i s not s u r p r i s i n g i n view of the f a c t t h a t c a f f e i c a c i d accounts f o r a t l e a s t 22% by weight of No. 1 and presence of t h i s p h e n o l i c component may i n t e r f e r e w i t h the h y d r o l y s i s p r o c e s s o f l i b e r a t i n g f r e e amino a c i d s . T h i s c o n c l u s i o n i s supported by the f o l l o w i n g , r e p l i c a t e d experiment performed on the same i s o l a t e o f compound 1. One m i l l i g r a m o f No. 1 was t r e a t e d w i t h f r e s h l y 95 o d i s t i l l e d diazomethane (48 hours, 25 ). The absence of a u.v. s h i f t i n the presence o f NaOH i n d i c a t e d t h a t the p h e n o l i c h y d r o x y l s of the pr o d u c t were m e t h y l a t e d . When t h i s d e r i v a t i v e was s u b j e c t e d t o a c i d h y d r o l y s i s (24 hours, 6N HCl, 110°) the y i e l d s o f g l y c i n e , l y s i n e and a l a n i n e were t r i p l e d , and two a d d i t i o n a l amino a c i d s ( a r g i n i n e and h i s t i d i n e ) appeared i n the same c o n c e n t r a t i o n as g l y c i n e , and the y i e l d o f amino a c i d s i n c r e a s e d t o ap p r o x i m a t e l y 5%. T h i s v a l u e i s s t i l l t o o low t o accomodate a n i t r o g e n c o n t e n t o f 2.37% even i f i t i s assumed t h a t the amide forms o f g l u t a m i c and a s p a r t i c a c i d predominate i n t h i s p e p t i d e . T h e r e f o r e , f o r f u t u r e s t u d i e s on the amino a c i d c o m p o s i t i o n o f compound 1, the author suggests t h a t the 65 a c i d h y d r o l y s i s be performed on the n o n - p h e n o l i c p o r t i o n of the m o l e c u l e . Thus a method w i l l have to be developed whereby c a f f e i c a c i d i s q u a n t i t a t i v e l y s e p a r a t e d v/ithout i n t e r a c t i o n w i t h the r e s i d u e of compound 1. Is the C a f f e o y l Moiety of Compound 1 A r t e f a c t u a l l y  A s s o c i a t e d w i t h Another M o l e c u l e ? The s i g n i f i c a n t degree of n o n - c o v a l e n t complexes 81 82 i n p l a n t e x t r a c t s has been examined by Loomis ' These a s s o c i a t i o n s can be formed as a r e s u l t of r e v e r s i b l e hydrogen bonding between phenols and p r o t e i n s . The p a r t i a l a c i d h y d r o l y z a t e of compound No. 1 y i e l d s seven n i n h y d r i n - p o s i t i v e bands on h i g h - v o l t a g e paper e l e c t r o -p h o r e s i s (see p r e v i o u s s e c t i o n ) . Two complementary experiments were performed t o t e s t whether compound No. 1 was the p r o d u c t o f r e v e r s i b l e hydrogen bonding f o r c e s between c a f f e i c a c i d and a p e p t i d e ( s ) . Gel F i l t r a t i o n i n the Presence of_7M_Urea 1 The e l u t i o n p a t t e r n s f o r compound No. 1 and c a f f e i c a c i d on a Sephadex G-10 column e q u i l i b r a t e d w i t h 7M u r e a : 0.1% a c e t i c a c i d i s i l l u s t r a t e d i n F i g u r e 13a. No. 1 was observed to move near the f r o n t whereas c a f f e i c a c i d was s t r o n g l y r e t a r d e d on the column. T h e r e f o r e , i t may be c o n c l u d e d t h a t the phenol i n No. 1 i s not a f r e e p h e n o l i c a c i d l i n k e d through r e v e r s i b l e hydrogen bonding t o the p e p t i d e s ( s ) . 66 F,l<5Ct_ro_phoresis i n PAW (Phenol : A c e t i c a c i d :_ Water _ 1 _ : _!_)_ To t e s t whether the p e p t i d i c component was r e v e r s i b l y adsorbed to the p h e n o l i c group, No. 1 was s u b j e c t e d t o low v o l t a g e paper e l e c t r o p h o r e s i s i n PAW, the a n t i -83 84 a d s o r p t i v e system developed by Synge and c o l l a b o r a t o r s ' The e l e c t r o p h o r e t i c c o n d i t i o n s were as f o l l o w s : 9 hours; 400 v o l t s : 4 mA; 3° ( c o l d r o o m ) ; pH 1.5 (PAW). Compound 1, c y s t e i c a c i d and u m b e l l i f e r o n e m i g r a t e d 0.3 cm, 1.5 cm and z e r o towards the anode r e s p e c t i v e l y . No. 1 i s e v i d e n t l y near i t s i s o e l e c t r i c p o i n t i n the PAW system. The c y a n i d i n c a t i o n m i g r a t e d 5.0 cm towards the cathode. Compound 1, which i s a n i o n i c and f l u o r e s c n e t , was e l u t e d , f r e e z e - d r i e d , s u b j e c t e d t o p a r t i a l a c i d h y d r o l y s i s and the h y d r o l y s a t e was r e s o l v e d by h i g h v o l t a g e paper e l e c t r o p h o r e s i s (see F i g u r e 14). Seven, c a t i o n i c , n i n h y d r i n - p o s i t i v e bands were o b t a i n e d i n d i c a t i n g t h a t the p e p t i d i c p o r t i o n was c o v a l e n t l y l i n k e d t o the c a f f e i c a c i d . P h e n o l i c a c i d s are r e a d i l y o x i d i z e d t o t h e i r quinones and, i n p l a n t e x t r a c t s , t h i s l e a d s to c o v a l e n t c o n d e n s a t i o n s Ql 82 o f the quinone w i t h the r e a c t i v e groups o f p r o t e i n s ~' Quinone f o r m a t i o n was minimized i n t h e s e p r o c e d u r es by m a i n t a i n i n g reduced c o n d i t i o n s (0.1% m ercaptoethanol) and c o n t r o l l i n g the hydrogen i o n c o n c e n t r a t i o n so t h a t the pH d i d not exceed 6. 67 Furthermore, the browning r e a c t i o n r e s u l t i n g from 81 o x i d a t i o n o f phenols was never observed d u r i n g the e x t r a c t i o n p r o c e d u r e s . Fure No. 1 was i s o l a t e d as a white powder a f t e r the f i n a l f r e e z e - d r y i n g s t e p . On pr o l o n g e d exposure t o a i r i t was g r a d u a l l y c o n v e r t e d t o a p a l e green c o l o r and t h i s o x i d i z e d component was removed by TLC chromatography ( A v i c e l , 2% HCOOH) i n a n i t r o g e n atmosphere. 85 P i e r p o i n t has d e s c r i b e d the r e a c t i o n s which o c c u r r e d when t h i o l s and amino a c i d s were i n c u b a t e d w i t h the e n z y m a t i c a l l y d e r i v e d quinone of c h l o r o g e n i c a c i d . The p r o d u c t s o f t h e s e r e a c t i o n s were 6- and 2,6- s u b s t i t u t e d d e r i v a t i v e s of the c a f f e o y l moiety. Amino a c i d s were l i n k e d v i a the n i t r o g e n o f t h e i r amino groups and t h i o l compounds were l i n k e d through the s u l f u r o f t h e i r s u l f - ' h y d r y l . I f t h e s e p r o d u c t s were degraded, they would y i e l d the 6-amino or 6 - t h i o l c a f f e o y l d e r i v a t i v e analogous t o the 6 - a m i n o - c a f f e i c a c i d d e s c r i b e d by B r i e s k o r n and 86 Mosandl . With r e s p e c t t o compound 1 the o n l y f l u o r e s c e n t p r o d u c t o f h y d r o l y s i s was c a f f e i c a c i d as evide n c e d by v a r i o u s s o l v e n t systems and e l e c t r o p h o r e t o g r a m s ( T a b l e I I I ) . The same a p p l i e s f o r No. 2 and 3 which y i e l d e d f e r u l i c and para-coumaric a c i d , r e s p e c t i v e l y . The h y p o t h e s i s t h a t t h e s e compounds may be formed d u r i n g the e x t r a c t i o n p r e c e d u r e i s thus r e j e c t e d . I t i s h i g h l y u n l i k e l y t h a t the same a r t i f a c t c o u l d be formed by 68 t h r e e d i f f e r e n t methods of i s o l a t i o n . Comoounds 2 and 3 The f l u o r e s c e n c e under u.v. l i g h t and the bathochromic s h i f t s i n the u.v. s p e c t r a ( T a b l e I I ) of compounds 2 and 3 i n d i c a t e d t h a t f e r u l i c a c i d ( i n compound 2) and p a r a -coumaric a c i d ( i n compound 3) were l i n k e d v i a t h e i r r e s p e c t i v e c a r b o x y l groups. The u.v. s p e c t r a o f compound 3 and a u t h e n t i c f e r u l i c a c i d are i l l u s t r a t e d i n F i g u r e 15. F i g u r e 16 shows the u.v. s p e c t r a of compound 2 and a u t h e n t i c nara-coumaric a c i d ( t he u.v.•spectrum of compound 1 was p r e v i o u s l y d e s c r i b e d i n F i g u r e 12). The s h o u l d e r t h a t i s a s s o c i a t e d w i t h the major peak i n the u.v. s p e c t r a of- c i n n a m i c acids, i s a t t r i b u t e d t o the f a c t t h a t these compounds e x i s t i n s o l u t i o n as a m i x t u r e 87 o f c i s and t r a n s isomers . For example, c i s - c i n n a m i c a c i d absorbs a t 264 nm whereas t r a n s - c i n n a m i c a c i d absorbs a t 273 nm ( i . e . the c i s isomer absorbs a t a lower wave-87 l e n g t h ) . With r e s p e c t t o compounds 1, 2 and 3, n o t i c e t h a t i n the presumable c i s r e g i o n , the absorbance i s s i g n i f i c a n t l y lower than i n the c o r r e s p o n d i n g f r e e a c i d . C i s and t r a n s isomerism of 1, 2 and/or 3 was never d e t e c t e d c h r o m a t o g r a p h i c a l l y . Hov/ever, whether the s e c o n j u g a t e s are e x c l u s i v e l y t r a n s i s a matter f o r c o n j e c t u r e . U r Wavelength (nm) F i g u r e 15. U l t r a - v i o l e t a b s o r p t i o n s p e c t r a o f compound 3 and ^ a r a - c o u m a r i c ^ a c i d (COU) i n d i s t i l l e d water. ° 225 250 275 300 325 Wavelength (nm) F i g u r e 16. U l t r a - v i o l e t a b s o r p t i o n s p e c t r a o f compound 2 and f e r u l i c a c i d (FER) i n d i s t i l l e d water. 71 Compound 3, ( i s o l a t e d by Method IB) was rechromato-graphed on TLC ( A v i c e l , 2% HCOOH) p r i o r t o amino a c i d a n a l y s i s . The a n a l y s i s was performed on a 10.0 ml s o l u t i o n (O.D. = 1.02). Assuming t h a t the e x t i n c t i o n c o e f f i c i e n t s o f compounds 1 and 3 are the same (see page 79) the f o l l o w i n g y i e l d s of amino a c i d s (nM amino a c i d / mg compound 3) were o b t a i n e d : a s p a r t i c a c i d , 7.2; t h r e o n i n e , 2.4; s e r i n e , 6.0; g l u t a m i c a c i d , 6.0; g l y c i n e , 15.6; a l a n i n e , 9.6; i s o l e u c i n e , 1.04; l e u c i n e , 2.72. Thus the amino a c i d a n a l y s i s of compound 3 resembles t h a t o f compound 1 ( T a b l e I V ) . Amino a c i d a n a l y s i s o f compound 2 v/as n o t attempted because of the ext r e m e l y low c o n c e n t r a t i o n s o f t h i s c o n j u g a t e . 35 The i n c o r p o r a t i o n o f S i n t o compounds 1, 2 and 3 has a l r e a d y been d e s c r i b e d . These p r e l i m i n a r y o b s e r v a t i o n s may i n d i c a t e t h a t the n o n - p h e n o l i c component i s the same i n compounds 1, 2 and 3 and t h i s i s suppo r t e d , i n p a r t , by t h e i r s i m i l a r p h y s i c a l p r o p e r t i e s ( i . e . a d s o r p t i o n , e l e c t r o — p h o r e t i c m o b i l i t y , c hromatographic R f v a l u e s ) . Comoound 4 The u. v. spectrum o f compound 4 i s i l l u s t r a t e d i n F i g u r e 17. The bathochromic s h i f t o f 24 nm (H-BO_/NaOAc) 1.0 Wavelength (nm) F i g u r e 17. U l t r a - v i o l e t a b s o r p t i o n spectrum of compound 4 i n methanol c o n t a i n i n g 1% water. 73 i n d i c a t e d the p r e s e n c e of an o r t h o - d i h y d r o x y group ( T a b l e IX). No u.v. s h i f t was observed upon the a d d i t i o n o f A l C l ^ or sodium a c e t a t e . The p h e n o l i c moiety t h a t was l i b e r a t e d from compound 4 was e x t r e m e l y l a b i l e i n a i r and a l k a l i . The C a f f e o y l Conjugate i n the C Band T h i s c o n j u g a t e was i s o l a t e d by r e p e a t e d HVPE (see F i g u r e 6) and the c a f f e i c a c i d moiety was d e t e c t e d c h r o m a t o g r a p h i c a l l y a f t e r a l k a l i n e h y d r o l y s i s . The s i m i l a r i t y between the u.v. s p e c t r a of compound 1 (the c a f f e o y l c o n j u g a t e of the B band, F i g u r e 6) and the c a f f e o y l c o n j u g a t e of the C band i s i l l u s t r a t e d i n F i g u r e 18. The C band c o n j u g a t e e x h i b i t s the same f l u o r e s c e n c e under u. v. l i g h t as compound 1. These two compounds are r e a d i l y d i s t i n g u i s h e d , however, on the b a s i s o f t h e i r e l e c t r o p h o r e t i c m o b i l i t i e s a t pH 2 (see F i g u r e 6) and whether they d i f f e r on the b a s i s o f charge alone remains t o be seen. The H i g h - m o l e c u l a r - w e i q h t C a f f e y l Conjugate The i d e n t i f i c a t i o n o f the c a f f e i c a c i d component of t h i s c o n j u g a t e was d e s c r i b e d on page 45 . The h i g h -m o l e c u l a r - w e i g h t c a f f e y l c o n j u g a t e was not f l u o r e s c e n t 74 and t h i s i n d i c a t e d t h a t the c a f f e y l moiety was s u b s t i t u t e d a t the o r t h o - d i h y d r o x y p o s i t i o n s . T h i s was s u p p o r t e d by the s m a l l b o r a t e s h i f t (3 nm as compared to 19 nm f o r compound 1 ) . However, s i n c e a bathochromic s h i f t (57 nm) was observed i n a l k a l i t h i s would suggest t h a t o n l y the para p o s i t i o n was o c c u p i e d . The u.v. spectrum of t h i s p a r t i a l l y p u r i f i e d c o n j u g a t e was i l l u s t r a t e d i n F i g u r e 12. The h i g h absorbance a t 280 nm may r e f l e c t a p r o t e i n - t y p e a s s o c i a t i o n w i t h c a f f e i c a c i d . The r i g o r o u s method of i s o l a t i o n ( i . e . chromatography i n 7M urea : 0.1% a c e t i c a c i d , F i g u r e 11a) argues a g a i n s t the p o s s i b i l i t y o f n o n - c o v a l e n t bonding between the c a f f e y l moiety and the macromolecular complement. Wavelength (nm) F i g u r e 18. U l t r a - v i o l e t a b s o r p t i o n s p e c t r a o f compound 1 and the c a f f e o y l c o njugate of the C band (C band cpd.) i n d i s t i l l e d water. 7 6 Time-course T r a c e r Experiments P r e l i m i n a r y O b s e r v a t i o n s on the ''Insoluble" C a f f e i c A c i d of Mentha Time c o u r s e t r a c e r s t u d i e s w i t h C 0 2 were i n i t i a t e d t o examine the t u r n o v e r of i n s o l u b l e c a f f e i c a c i d and the e x p e r i m e n t a l c o n d i t i o n s are d e s c r i b e d below. The two-hour p h o t o s y n t h e t i c p e r i o d was i n i t i a t e d 14 / by r e l e a s i n g 100 / J C CO,> i n t o a c l o s e d chamber (Jencons No. 4 d e s i c c a t o r ) at 18° c o n t a i n i n g f i v e l o t s of c u t shoots o f Mentha a r v e n s i s a t l e a s t f o u r i n t e r n o d e s i n l e n g t h . Shoots were h e l d i n a beaker wi t h t h e i r c u t ends immersed i n water. A f t e r the two-hour f e e d i n g , the shoots were a l l o w e d t o m e t a b o l i z e under atmospheric c o n d i t i o n s . The acetone powders h a r v e s t e d a t the g i v e n i n t e r v a l s were h y d r o l y z e d w i t h 2N HCl f o r 30 minutes on a steambath and e x t r a c t e d c o n t i n u o u s l y w i t h e t h e r ( i n s o l u b l e } a c i d - s e n s i t i v e p o o l ) . The aqueous phase was a d j u s t e d t o 2N NaOH, h y d r o l y z e d f o r 5 hours at room temperature and e x t r a c t e d c o n t i n u o u s l y w i t h e t h e r ( i n s o l u b l e , a l k a l i - s e n s i t i v e p o o l ) . The s o l u b l e c a f f e i c a c i d p o o l was p r e c i p i t a t e d w i t h l e a d a c e t a t e ^ 1 , c o l l e c t e d and h y d r o l y z e d w i t h 5N NaOH f o r one hour a t room temper-a t u r e . 77 F i g u r e 19. Changes v/ith time i n the s p e c i f i c a c t i v i t i e s of c a f f e i c a c i d s of M. a r v e n s i s a f t e r the a d m i n i s t r a t i o n o f NaH^CO,,. 78 Thus, when shoots of M. a r v e n s i s v/ere exposed t o ^CO^ and the changes w i t h time i n the s p e c i f i c a c t i v i t i e s o f the v a r i o u s c a f f e i c a c i d p o o l s were determined, i t was c l e a r t h a t a t l e a s t two d i f f e r e n t p o o l s o f bound, i n s o l u b l e c a f f e i c a c i d e x i s t e d ( F i g u r e 19). Furthermore, the d e c l i n e i n a c t i v i t y f o l l o w i n g a r a p i d i n c r e a s e i n d i c a t e d a t u r n o v e r o f the " i n s o l u b l e " , a l k a l i - s e n s i t i v e p o o l of c a f f e i c a c i d . E s t i m a t i o n of R e l a t i v e F o o l s i z e s of compounds 1, 2 and 3 As a r e s u l t o f b atchwise procedure o f i s o l a t i o n (Method IB) and the a d s o r p t i v e p r o p e r t i e s of t h e s e compounds on DEAE C e l l u l o s e , d e t e r m i n a t i o n o f a b s o l u t e p o o l s i z e s was u n f e a s i b l e . T a y l o r and Zucker r e p o r t e d t h a t i n Xanthium pennsylvanicum, the c o n t e n t of c h l o r o -g e n i c a c i d , i s o c h l o r o g e n i c a c i d and i n s o l u b l e c a f f e i c a c i d remained c o n s t a n t over a 24-hour p e r i o d ^ 1 . Sub-s e q u e n t l y , T a y l o r showed t h a t the c o n c e n t r a t i o n s o f t h e s e p o o l s v a r i e d between d i f f e r e n t organs o f the same p l a n t but no s i g n i f i c a n t net s y n t h e s i s o c c u r r e d i n any of the 13 t i s s u e s over a 21-hour p e r i o d . On t h i s b a s i s the assumption has been made t h a t i n mint shoots t h e r e was no s i g n i f i c a n t net s y n t h e s i s of compounds 1, 2 and 3 d u r i n g the p e r i o d of a s s i m i l a t i o n and metabolism of t r a c e r s . 79 Weighable q u a n t i t i e s (10 - 30 mg) o f compound 1 were i s o l a t a b l e but the amounts of compounds 2 and 3 were too low f o r a c c u r a t e weight d e t e r m i n a t i o n . The r e l a t i v e pool s i z e s o f compounds 1, 2 and 3 were con-s i s t e n t , however, on the b a s i s of s p e c t r o p h o t o m e t r i c d e t e r m i n a t i o n s of 36 samples from t h r e e , t i m e - c o u r s e , t r a c e r s t u d i e s . the e s t i m a t i o n of the r e l a t i v e p o o l s i z e of compound 1, 2 and 3 i s based on the f o l l o w i n g c r i t e r i a . The c a l c u l a t e d molar e x t i n c t i o n c o e f f i c i e n t s o f c a f f e i c , f e r u l i c and para-coumaric a c i d d i f f e r by - 10% about the mean. The p h e n o l i c m o i e t i e s on No. 1, 2 and 3 were i d e n t i f i e d r e s p e c t i v e l y as c a f f e i c , f e r u l i c and para-coumaric a c i d . The p h y s i c a l p r o p e r t i e s ( a d s o r p t i o n , e l e c t r o p h o r e t i c m o b i l i t y , and R f's) of the s e compounds are s i m i l a r and t h e r e f o r e , f o r comparisons t o be made i n k i n e t i c experiments, i t was assumed t h a t the non-p h e n o l i c component.was the same i n each compound. Thus, the r e l a t i v e p o o l s i z e s v/ere e s t i m a t e d by d i r e c t l y comparing the o p t i c a l d e n s i t i e s o f No. 1, 2 and 3 i n a c o n s t a n t volume. U s i n g t h i s method, the r a t i o o f the c o n c e n t r a t i o n s of No. 1 : No. 2 : No. 3 was found t o be 50 : 1 : 5. S p e c i f i c A c t i v i t y D e t e r m i n a t i o n The r a d i o a c t i v e samples o f compounds 1, 2 and 3 80 were i s o l a t e d by Method IB. The c o n c e n t r a t i o n o f each c o n j u g a t e was determined s p e c t r o p h o t o m e t r i c a l l y a t the wavelength of maximum absorbance i n d i s t i l l e d water (see T a b l e I I ) . An a l i q u o t o f the same s o l u t i o n was used f o r l i q u i d s c i n t i l l a t i o n c o u n t i n g . For example the s p e c i f i c a c t i v i t y o f a p a r t i c u l a r sample o f No. 1 was 3 found t o be (0.95'x 10 dpm/mg). The s p e c i f i c a c t i v i t y was a l s o determined a f t e r polyamide chromatography (see page 5 3 ) and i t remained c o n s t a n t a f t e r t h i s t r e a t m e n t . The c a f f e i c a c i d moiety was d e r i v e d by the method o f m i l d a l k a l i n e h y d r o l y s i s i n the presence o f excess NaBH^ f o l l o w e d by ion-exchange (see page 57). The s p e c i f i c 3 a c t i v i t y was found to be 2.09 x 10 dpm/mg. T h i s was a l s o determined a f t e r e t h e r e a l e x t r a c t i o n o f the DEAE C e l l u l o s e e l u a t e and i t was found to remain c o n s t a n t . An a l k a l i n e h y d r o l y z a t e of another sample o f compound 1 was combined w i t h a u t h e n t i c c a f f e i c a c i d (which served as a c a r r i e r ) , a c i d i f i e d , e x t r a c t e d c o n t i n u o u s l y w i t h e t h e r and chromatographed on TLC (3zAW, A v i c e l ) . The a c t i v i t y i n the e t h e r e a l e x t r a c t was found t o be c o m p l e t e l y a s s o c i a t e d w i t h the c a f f e i c a c i d band. Turnover S t u d i e s on Compounds 1, 2 and 3 The i n i t i a l s t u d i e s on the p r o d u c t s o f h y d r o l y s i s 81 i n d i c a t e d t h a t bound, i n s o l u b l e p o o l s of c a f f e i c a c i d were t u r n i n g over ( F i g u r e 19). These r e s u l t s resembled the s i t u a t i o n i n wheat shoots i n which the e t h a n o l -i n s o l u b l e p o o ls of f e r u l i c and para-coumaric a c i d e x h i b i t e d a r a p i d turnover*"''. T h e r e f o r e , s i m i l a r t r a c e r experiments were developed t o see whether compounds 1, 2 and 3 were t u r n i n g over and T a b l e V d e s c r i b e s the v a r i o u s i s o t o p i c t r e a t m e n t s . Turnover experiments u s i n g 14CC>2 i n l i g h t (Experiment I, F i g u r e 20) were c a r r i e d out t o demonstrate t h a t t h e s e compounds are l a b e l l e d through "the normal o b l i g a t o r y 88 pathway(s) from'elementary carbon s o u r c e s " and the r e s u l t s o f Experiment I sup p o r t e d t h i s h y p o t h e s i s . 14 Furthermore, when COg was a d m i n i s t e r e d a d e c l i n e i n r a d i o a c t i v i t y f o l l o w i n g a r a p i d i n c r e a s e was o b t a i n e d and t h i s i n d i c a t e d a t u r n o v e r of compound 1. S i m i l a r l y , 14 when C - p h e n y l a l a n i n e was a d m i n i s t e r e d i n Experiment I I , compounds 1, 2 and 3 e x h i b i t e d a t u r n o v e r ( F i g u r e s 20 and 21). Basyouni et_ a l demonstrated t h a t i n wheat, " i n s o l u b l e " f e r u l i c and para-coumaric a c i d s a t t a i n t h e i r h i g h e s t 14 s p e c i f i c a c t i v i t y w i t h i n two hours of C 0 ? i n t r o d u c t i o n a f t e r v/hich t h e r e i s a r a p i c d e c l i n e 1 * . Experiments I and I I d i f f e r i n t h i s r e s p e c t s i n c e compound 1 reaches a peak a f t e r a twelve-hour i n t e r v a l . Compounds 2 and 3 on the o t h e r hand, e x h i b i t a maximum s p e c i f i c a c t i v i t y Table V R a d i o a c t i v e Compounds Administered and t h e i r I n c o r p o r a t i o n i n t o S o l u b l e C a f f e i c A c i d and the Ca f f e i c . A c i d of Compound 1. Exp. No. Compound Fed C a f f e i c A c i d S p e c i f i c A c t i v i t y S o l u b l e C a f f e i c A c i d o f No. 1 A c t i v i t y mc/mM Fed S p e c i f i c D i l u t i o n A c t i v i t y jiic/mM S p e c i f i c D i l u t i o n A c t i v i t y /Jc/mM I N a H 1 4 C 0 3 + + 4.9 14 I I P h e n y l a l a n i n e - U - C 375 I I I P h e n y l a l a n i n e - U - 1 4 C 375 IV Cinnamic a c i d - 3 - 1 4 C 9.0 150 12.5 + 2.08 1.3 x 10 ! 25" 25" 0.69 1.45 5.4 x 10-6.2 x 10 -0.75 5.0 x 10 J 0.38 9.8 x 10 S 0.41 2.2 x 10 A f t e r p u l s e - f e e d i n g f o r 1.5 hours f o l l o w e d by a m e t a b o l i c p e r i o d of 4.5 hours. * * o S o l u b l e c a f f e i c a c i d o b t a i n e d by a l k a l i n e h y d r o l y s i s (2N HaOH, 50 , 2 h o u r s ) . + A d m i n i s t e r e d t o each 50 g l o t of M. a r v e n s i s . + + S p e c i f i c a c t i v i t y determined o n l y f o r compound 1. £ £10 a. Cr>T3 O TY ' 8 > 6 o < o 4 L L O LL) a. 2 ui Experiments I & II J L Experiment III J L •oCaffeic acid moiety • Compound No. 1 Phenylalanine-U-l4C NaH KC0 3 3 6 12 24 _^  J 6 12 *" HOURS 24 48 F i g u r e 20. Changes w i t h time i n s p e c i f i c a c t i v i t i e s o f compound 1 and c a f f e i c a c i d moiety of compound 1 a f t e r a d m i n i s t r a t i o n of N a H 1 4 C 0 3 and p h e n y l a l a n i n e -u - 1 4 c . CO QJ •Q C 0) o c cn o M c r t H-O 3 w 2 o a a 3 H-3 H> W r t ^ CU r t H -O 3 O H i LO C n n> ro O 3* QJ 3 iQ CD cn s: r t 3* r t H* 3 0) 3 •a 3" fl) 3 cn H K t ) M QJ o O 3 \ o o Q) 3 H« 3 ft) i a o p-Hi H-O 0) 0 r t n r t -—> ft) O cn O Hi VO n o ai o M 3 c o c 3 a cn QJ r t a> a Hi O r o o o ro QJ 3 a QJ Hi r t fD t l 7 8 SPECIFIC ACTIVITY No.3 (103dpm/O.D.unit) I" cn Fo o a CO ND 4N | CO CD ND ND -IN •is CO C D CO r - i m X / / / 3* / / >—* p CO * 4 No. 2 (IQdpm/QQunit) m X CD 3 fD 3 O ND CO 85 a f t e r a s i x - h o u r i n t e r v a l . The r e s u l t s of Experiment I I 14 a l s o i n d i c a t e t h a t the r a t e of C - i n c o r p o r a t i o n i n t o compound 2 ^ compound 3 ^ compound 1. Q u a n t i t a t i v e h y d r o l y s e s t o determine the s p e c i f i c a c t i v i t y o f f e r u l i c a c i d i n compound 2 and para-coumaric a c i d i n compound 3 were not attempted because o f the extr e m e l y low c o n c e n t r a t i o n s o f the s e c o n j u g a t e s . However, the s p e c i f i c a c t i v i t y o f compound 1 and the s p e c i f i c a c t i v i t y of the c a f f e i c a c i d o f compound 1 f o l l o w a p a r a l l e l c o u r s e on t h r e e experiments ( I I , I I I and IV) and one can p r e d i c t t h a t the same p a t t e r n would a p p l y t o the p h e n o l i c m o i e t i e s o f compounds 2 and 3. The g e n e r a l p i c t u r e i n Experiment I I I ( F i g u r e s 20 and 21) i s one of r a p i d l a b e l l i n g from p h e n y l a l a n i n e -14 C f o l l o w e d by a r e l a t i v e s t a b i l i t y i n the amount o f l a b e l i n t h i s p o o l . Compounds 1, 2 and 3 do not e x h i b i t a s i g n i f i c a n t t u r n o v e r i n Experiment I I I and t h i s c o u l d imply t h a t the r a t e of s y n t h e s i s of the s e compounds i s eq u a l t o t h e i r r a t e o f d e g r a d a t i o n d u r i n g the 48-hour p e r i o d . The s i t u a t i o n i n Experiment I I I resembles the 14 k i n e t i c s o f C 0 2 i n c o r p o r a t i o n i n t o the p o o l of r o s m a r i n i c a c i d i n Mentha^ 1. 14 When ci n n a m i c a c i d - C was a d m i n i s t e r e d , the i n i t i a l f l u c t u a t i o n i n s p e c i f i c a c t i v i t y was f o l l o w e d by a g r a d u a l i n c r e a s e i n the amount of l a b e l i n t h i s p o o l (Experiment IV, F i g u r e 22), and, i n t h i s r e s p e c t , t h e s e EXPERIMENT IV J i i i _ i i ' i i _ 3 6 12 24 _^ J3_ 6 12 24 HOURS Figure 22. Changes with time i n s p e c i f i c a c t i v i t i e s of compounds 1, 2 and 3 and c a f f e i c acid moiety (C.A.M.) of compound 1 afte r administration of cinnamic acid -3- 1 4C (O.D. cal c u l a t e d f o r 200 ml aqueous solutions cn No. 1, 1.00 mg/200 ml, O.D. = 0.19). 87 r e s u l t s resemble those o f Basyouni ejb aJL when they 14 11 a d m i n i s t e r e d c i n n a m i c a c i d - 2 - C to wheat shoots Compound 2, the f e r u l i c a c i d - c o n t a i n i n g c o n j u g a t e , c o n s i s t e n t l y possessed the h i g h e s t s p e c i f i c a c t i v i t y i n a g i v e n experiment as compared to compounds 1 and 3. T h i s i s i n t e r e s t i n g because i t i s more c l o s e l y r e l a t e d t o l i g n i n p r e c u r s o r than e i t h e r 1 or 3. Thus, a t the s i x - h o u r i n t e r v a l i n Experiments I I and IV the s p e c i f i c a c t i v i t y o f compound 2 i s a t l e a s t f o u r times as g r e a t as compound 1 and s i x times as g r e a t as compound 3. In experiment I I I ( a t the s i x - h o u r i n t e r v a l ) i t i s t w i c e as g r e a t as No. 1 or 3. I f we assume a random d i s t r i b u t i o n of l a b e l among the t h r e e c o n j u g a t e s , the r e l a t i v e , s m a l l p o o l o f compound 2 can account f o r i t s h i g h s p e c i f i c a c t i v i t y . T h i s c o r r e l a t i o n i s not apparent w i t h compound 3 whose s p e c i f i c a c t i v i t y i s a p p r o x i m a t e l y the same as t h a t of compound 1 and y e t i t has a po o l s i z e one t e n t h as g r e a t . Thus, a d e c l i n e i n the s p e c i f i c a c t i v i t y o f compounds 1, 2 and 3 was i n d i c a t e d i n two out o f t h r e e experiments 14 14 u s i n g C 0 2 and C - p h e n y l a l a n i n e . I t i s i n t e r e s t i n g t o s p e c u l a t e on the r o l e of compounds 1, 2 and 3, and e s p e c i a l l y compound 2, i n the l i g h t o f i t s h i g h s p e c i f i c a c t i v i t y and the t u r n o v e r d a t a of Experiment I I . Are t h e s e compounds concerned with l i g n i n b i o s y n -88 t h e s i s and i s compound 3 an " a c t i v e , i n s o l u b l e e s t e r " d i r e c t l y i n v o l v e d i n the t r a n s f e r of p h e n y l p r o p a n o i d u n i t s t o l i g n i n ? I f t h e s e compounds are i n v o l v e d i n l i g n i n b i o s y n t h e s i s t h i s i s hard t o r e c o n c i l e w i t h e v i d e n c e i n d i c a t i n g t h a t cinnamyl a l c o h o l s such as c o n i f e r y l a l c o h o l can be p e r o x i d a t i v e l y i n c o r p o r a t e d i n t o model 89 90 l i g n i n s ' . However, experiments w i t h p o t a t o parenchyma have demonstrated l i g n i n f o r m a t i o n i n v i t r o from 91 p r e c u r s o r s such as f e r u l i c and nara-coumaric a c i d s Nozu has emphasized t h a t the e s s e n t i a l r e q u i r e m e n t s f o r a l i g n i n p r e c u r s o r are (a) a f r e e p h e n o l i c h y d r o x y l group (b) a v i n y l l i n k a g e i n the s i d e c h a i n o f the p h e n o l i c 9 2 moiety and (c) a f r e e 5 p o s i t i o n i n the benzene n u c l e u s In o t h e r words, the a l c o h o l i c f u n c t i o n i n the s i d e c h a i n i n not e s s e n t i a l and i t i s c o n c e i v a b l e t h a t e s t e r i f i e d forms of p h e n o l i c a c i d s c o u l d p a r t i c i p a t e i n model l i g n i n f o r m a t i o n . R e l a t i v e I n c o r p o r a t i o n of P h e n y l a l a n i n e - 1 4 C and Cinnamic 14 a c i d - C i n t o V a r i o u s C a f f e i c A c i d Pools T a b l e V compares the s p e c i f i c a c t i v i t y of the c a f f e i c a c i d of No. 1 t o s o l u b l e c a f f e i c a c i d a f t e r p u l s e f e e d i n g f o r 1.5 hours f o l l o w e d by a m e t a b o l i c p e r i o d of 4.5 h o u r s . 14 The r e s u l t s i n d i c a t e t h a t cinnamic a c i d - 3 - C i s 89 a b e t t e r p r e c u r s o r than p h e n y l a l a n i n e f o r both s o l u b l e c a f f e i c a c i d and the c a f f e i c a c i d of No. 1. T h i s d a t a i s i n agreement w i t h E l l i s ' o b s e r v a t i o n s on the i n c o r p -o r a t i o n o f t h e s e p r e c u r s o r s i n t o the c a f f e o y l moiety of r o s m a r i n i c a c i d , the major, s o l u b l e , c a f f e o y l e s t e r o f Mentha^ 1. T a y l o r and Zucker r e p o r t e d t h a t Xanthium l e a v e s c o n t a i n e d a s i g n i f i c a n t q u a n t i t y o f an a l c o h o l - i n s o l u b l e , c a f f e o y l e s t e r . However, a f t e r p u l s e f e e d i n g p h e n y l a l -14 a n i n e - C f o r one hour f o l l o w e d by a m e t a b o l i c p e r i o d o f 3 hours the s p e c i f i c a c t i v i t y o f the s o l u b l e , c h l o r o -g e n i c a c i d i n Xanthium was twelve times as g r e a t as the i n s o l u b l e c a f f e i c a c i d which showed l i t t l e i f any t u r n o v e r ^ . Mentha, on the o t h e r hand, e x h i b i t s a s i g n i f i c a n t t u r n o v e r o f the " i n s o l u b l e " c a f f e i c a c i d i n some experiments and the s p e c i f i c a c t i v i t y of the s o l u b l e c a f f e i c a c i d i s o n l y 2 - 3 times as g r e a t as the " i n s o l u b l e " c a f f e i c a c i d d e r i v e d from compound 1. The magnitude of t h e s e d i l u t i o n v a l u e s ( T a b l e V) s h o u l d be a p p r e c i a t e d i n the l i g h t o f an i m p o r t a n t f e a t u r e which d i s t i n g u i s h e s t h e s e experiments from those o f E l l i s ^ ^ j of E l - B a s y o u n i 1 1 and of T a y l o r and Z u c k e r ^ 1 . The t r a c e r s v/ere a d m i n i s t e r e d to l a r g e batches o f mint shoots (50 g ) . T h i s would i n e v i t a b l y g i v e h i g h e r d i l u t i o n v a l u e s . 90 The h i g h - m o l e c u l a r - w e i g h t c a f f e y l c o n j u g a t e was i s o l a t e d at the s i x - h o u r i n t e r v a l i n Experiment I I t o compare i t w i t h the s o l u b l e c a f f e i c a c i d and the c a f f e i c a c i d on No. 1. The PAW e x t r a c t was d i a l y s e d p r i o r t o chromatography on Sephadex G-100. F i g u r e 23 shows t h a t the c a f f e y l c o n j u g a t e , o c c u r r i n g i n F r a c t i o n s No. 6 - 8 c o i n c i d e d w i t h the p r o f i l e o f r a d i o a c t i v i t y . However, when t h e s e f r a c t i o n s were s u b j e c t e d t o a c i d o r base h y d r o l y s i s f o l l o w e d by c o n t i n u o u s e x t r a c t i o n w i t h e t h e r , 95% of the a c t i v i t y remained i n the aqueous phase and the i s o l a t e d c a f f e i c a c i d was found t o have no r a d i o a c t i v i t y . P r e l i m i n a r y e s t i m a t e s i n d i c a t e t h a t the amount o f c a f f e i c a c i d a s s o c i a t e d w i t h compound 1 i s a p p r o x i m a t e l y equal t o the p o o l of c a f f e i c a c i d r e l a t e d t o the h i g h -m o l e c u l a r - w e i g h t c o n j u g a t e . However, the d a t a shows t h a t the c a f f e i c a c i d moiety i n the h i g h - m o l e c u l a r -weight c o n j u g a t e i s r e l a t i v e l y i n a c t i v e as compared t o the c a f f e o y l moiety of compound 1. The r e s u l t s emphasize, t h e r e f o r e , the importance of s e p a r a t i n g and p u r i f y i n g the v a r i o u s , " i n s o l u b l e " p o o l s of p h e n o l i c a c i d s when k i n e t i c , i s o t o p i c experiments are performed. Mentha c o n t a i n s a m u l t i p l i c i t y o f " i n s o l u b l e " c a f f e i c a c i d p o o l s ( e . g . compound 1, the c a f f e o y l c o n j u g a t e i n the C band and the h i g h - m o l e c u l a r - w e i g h t c a f f e y l c o n j u g a t e ) and the examination of the g r o s s h y d r o l y s i s p r o d u c t of e t h a n o l -i n s o l u b l e r e s i d u e s i n an obvious o v e r s i m p l i f i c a t i o n . 91 F i g u r e 23. Sephadex G-100 column chromatography of h i g h - m o l e c u l a r - w e i g h t c a f f e y l c o n j u g a t e . Absorbance R a d i o a c t i v i t y The column (1 x 11 cm) was d e v e l o p e d i n 7M u r e a : 0.1% a c e t i c a c i d . A c e t i c a c i d , 0.5 ml, was added t o each f r a c t i o n p r i o r t o r a d i o a c t i v i t y measurements. T h i s e l i m i n a t e d the e x c e s s i v e background caused by u r e a . ABSORBANCE. 325nm 93 SUMMARY AND CONCLUSIONS The s o - c a l l e d e t h a n o l - i n s o l u b l e , p h e n o l i c e s t e r s o f Mentha a r v e n s i s were examined i n some d e t a i l . The s a l i e n t f e a t u r e s o f t h i s i n v e s t i g a t i o n a r e summarized as f o l l o w s : 1. Three, major, p h e n o l i c a c i d p o o l s were d e r i v e d from Mentha by the use o f v a r i o u s s o l v e n t s and a n t i - a d s o r p t i v e r e a g e n t s such as phenol : a c e t i c a c i d : water (PAW): (a) " S o l u b l e " p h e n o l i c a c i d c o n j u g a t e s were o b t a i n e d by the c l a s s i c a l method of e x t r a c t i o n w i t h o r g a n i c s o l v e n t s such as e t h a n o l or acetone. (b) When the r e s i d u e ( s ) i n (a) was r e - e x t r a c t e d w i t h water c o n t a i n i n g 0.1% mercaptoethanol and 0.1% a c e t i c a c i d an " i n s o l u b l e " p o o l o f bound, p h e n o l i c a c i d s was s o l u b i l i z e d . (c) When the r e s i d u e s ( s ) i n (b) was r e - e x t r a c t e d w i t h PAW a second p o o l of " i n s o l u b l e " c a f f e i c a c i d was o b t a i n e d and the l i g n i n -c e l l w a l l f r a c t i o n which remained v/as d e v o i d of bound, p h e n o l i c a c i d s . 94 When aqueous e x t r a c t s of acetone powders were chromatographed on Sephadex g e l s e q u i l i b r a t e d i n c o n v e n t i o n a l b u f f e r s , a major p o o l o f bound, p h e n o l i c a c i d was s t r o n g l y adsorbed t o the g e l . E l u t i o n was e f f e c t e d by chromatography of the same e x t r a c t i n 7M ure a : 0.1% a c e t i c a c i d . Ammonium s u l f a t e f r a c t i o n a t i o n o f t h e s e aqueous e x t r a c t s r e s u l t e d i n the c o - p r e c i p i t a t i o n of h i g h - m o l e c u l a r - w e i g h t f r a c t i o n s w i t h low-m o l e c u l a r - w e i g h t , p h e n o l i c c o n s t i t u e n t s . O r g a n i c s o l v e n t p r e c i p i t a t i o n s e p a r a t e d t h e s e f r a c t i o n s e f f e c t i v e l y . The p h e n o l i c components remained i n the s u p e r n a t a n t a t 50% acetone or d i o x a n e . The p h e n o l i c c o n j u g a t e s i n K b ) were r e s o l v e d by h i g h - v o l t a g e paper e l e c t r o p h o r e s i s (HVPE) and two groups (B and C) of bound, p h e n o l i c a c i d s were d i f f e r e n t i a t e d on the b a s i s of t h e i r a n i o n i c m o b i l i t y a t pK 1.9. Both the 3 r e g i o n and the f a s t e r - m o v i n g C r e g i o n y i e l d e d c a f f e i c , o ara-coumaric and f e r u l i c a c i d s on a l k a l i n e h y d r o l y s i s . These n o v e l , p h e n o l i c c o n j u g a t e s are unique, t h e r e f o r e , on the b a s i s o f t h e i r a n i o n i c b e h a v i o u r a t pH 1.9. 95 5. New, d e f i n i t i v e methods were developed f o r the i s o l a t i o n and p u r i f i c a t i o n o f the p h e n o l i c a c i d c o n j u g a t e s of the 3 r e g i o n . The p r e p a r a t i v e i s o l a t i o n o f compounds 1, 2 and 3 ( c o n t a i n i n g c a f f e i c , f e r u l i c and p a r a - c c u m a r i c a c i d s r e s p e c t i v e l y ) i s d e s c r i b e d from acetone powders, e t h a n o l - i n s o l u b l e r e s i d u e s and f r e s h p l a n t m a t e r i a l . 6. " I n s o l u b l e " f e r u l i c a c i d was i s o l a t e d by the a p p l i c a t i o n o f t h e s e methods t o aqueous e x t r a c t s of acetone powders of wheat. 7. Methods are d e s c r i b e d f o r the p a r t i a l p u r i f i c a t i o n of the h i g h - m o l e c u l a r - w e i g h t c a f f e y l c o n j u g a t e which i s d e r i v e d from the PAW f r a c t i o n ( s ) i n 1(c) above. 8. Evidence i s p r e s e n t e d which i n d i c a t e s t h a t compounds i . 2 and 3 are e l e c t r o p h o r e t i c a l l y and c h r o m a t o g r a p h i c a l l y homogeneous. 9. The a d s o r p t i v e p r o p e r t i e s of compound 1 were i n v e s t i g a t e d i n some d e t a i l . Compound 1 e x h i b i t e d e x t e n s i v e zone br o a d e n i n g on B i o g e l P-2 and Sephadex G-10 and G-25 columns e q u i l i -96 b r a t e d i n a v a r i e t y o f s o l v e n t s . S i m i l a r e f f e c t s were m a n i f e s t e d on DEAE C e l l u l o s e and, i n t h i s system, compounds 1, 2 and 3 were s p e c i f i c a l l y desorbed w i t h PAW. The compounds were s t r o n g l y adsorbed t o polyamide but they c o u l d be c o m p l e t e l y r e c o v e r e d by e l u t i o n w i t h acetone i a c e t i c a c i d : water. 10. A method was developed f o r the m i l d , a l k a l i n e h y d r o l y s i s o f compound 1 which y i e l d s 22% (-2%) c a f f e i c a c i d . 11. When compound 1 was s u b j e c t e d to p a r t i a l a c i d h y d r o l y s i s seven, c a t i o n i c , n i n h y d r i n - p o s i t i v e bands and a n e u t r a l f l u o r e s c e n t band v/ere d e t e c t e d a f t e r HVPE a t pH 1.9. The n e u t r a l , f l u o r e s c e n t band was i d e n t i f i e d as c a f f e i c a c i d (compound 1 i s a n i o n i c and f l u o r e s c e n t under th e s e e l e c t r o p h o r e t i c c o n d i t i o n s ) . I t i s proposed t h a t compound 1 c o n t a i n s c a f f e i c a c i d c o v a l e n t l y a s s o c i a t e d w i t h a p e p t i d e ( s ) . 12. Amino a c i d a n a l y s e s of compound 1 were performed on the b a s i s o f : (a) the e v i d e n c e from p a r t i a l a c i d h y d r o l y s i s , (b) e l e m e n t a l a n a l y s i s o f compound 1 which r e v e a l e d a n i t r o g e n c o n t e n t o f 97 2.37% and (c) the d e l a y e d , n i n h y d r i n - p o s i t i v e r e a c t i o n . On a c i d h y d r o l y s i s compound 1 y i e l d e d l y s i n e , a s p a r t i c a c i d , t h r e o n i n e , s e r i n e , g l u t a m i c a c i d , g l y c i n e , a l a n i n e , v a l i n e , l e u c i n e and i s o l e u c i n e . When compound 1 was s u b j e c t e d t o treatment w i t h diazomethane p r i o r t o a c i d h y d r o l y s i s the y i e l d o f amino a c i d s was i n c r e a s e d f i v e f o l d and two a d d i t i o n a l amino a c i d s ( a r g i n i n e and h i s t i d i n e ) appeared. However, the y i e l d of amino a c i d s ( a p p r o x i m a t e l y 5% by weight o f compound 1) was s t i l l low and the r e l a t i v e y i e l d of each amino a c i d was i n c o n s i s t e n t . The p h e n o l i c p o r t i o n o f compound 1 appears t o i n t e r f e r e w i t h the h y d r o l y s i s p r o c e s s of r e l e a s i n g f r e e amino a c i d s and f u t u r e s t u d i e s on amino a c i d c o m p o s i t i o n s h o u l d be d i r e c t e d towards the a n a l y s i s o f the s e p a r a t e d , n o n - p h e n o l i c p o r t i o n o f compound 1. Compound 3 ( c o n t a i n i n g para-coumaric a c i d ) y i e l d e d a s i m i l a r a r r a y o f amino a c i d s on a c i d h y d r o l y s i s . Compound 1 was s u b j e c t e d t o g e l f i l t r a t i o n i n 1]A u r e a : 0.1% a c e t i c a c i d and e l e c t r o p h o r e s i s i n PAW. The r e s u l t s o f these t e s t s s u p p ort the p r e v i o u s c o n c l u s i o n s t h a t compound 1 c o n t a i n s 98 c a f f e i c a c i d c o v a l e n t l y a s s o c i a t e d w i t h a peptideC s ) . R a d i o a c t i v e s u l f u r v/as i n c o r p o r a t e d i n t o compounds 1, 2 and 3 and the e v i d e n c e suggests 35 t h a t the S r e s i d e s i n an a n i o n i c , a c i d i c group. The u.v. s p e c t r a o f compounds 1, 2 and 3 as w e l l as the c a f f e o y l c o n j u g a t e o f the C band and the p a r t i a l l y p u r i f i e d h i g h - m o l e c u l a r -weight c a f f e y l c o n j u g a t e are d e s c r i b e d f o r the f i r s t t i m e . The s p e c t r a l d a t a i n d i c a t e s t h a t the p h e n o l i c a c i d s i n compounds 1, 2 and 3 are l i n k e d v i a t h e i r r e s p e c t i v e c a r b o x y l groups. The c a f f e y l moiety o f the n o n - f l u o r e s c e n t , h i g h - m o l e c u l a r -weight c o n j u g a t e i s p r o b a b l y s u b s t i t u t e d a t the o r t h o - d i h y d r o x y p o s i t i o n ( s ) . On the b a s i s of the p r e v i o u s o b s e r v a t i o n s i t i s proposed t h a t compounds 1, 2 and 3 c o n t a i n the same n o n - p h e n o l i c complement. The r a t i o o f the c o n c e n t r a t i o n s of compound 1 : compound 2 : compound 3 i s e s t i m a t e d t o be 50 : 1 : 5. 99 19. Time-course t r a c e r s t u d i e s on v a r i o u s p o o l s o f c a f f e i c a c i d o f Mentha i n d i c a t e t h a t an " i n s o l u b l e " , a l k a l i - s e n s i t i v e p o o l o f c a f f e i c a c i d i s t u r n i n g o v e r . 14 Id 14 20. " C - p h e n y l a l a n i n e and C-cinnamic a c i d were a d m i n i s t e r e d t o Mentha shoots and the s p e c i f i c a c t i v i t y of compounds 1, 2 and 3 were determined a t v a r i o u s m e t a b o l i c p e r i o d s . Thus, f o r the f i r s t time, t i m e - c o u r s e t r a c e r experiments were performed on a p u r i f i e d p o o l o f " i n s o l u b l e " c a f f e i c , f e r u l i c and para-coumaric a c i d . 21. A d e c l i n e i n the s p e c i f i c a c t i v i t i e s o f compounds 1, 2 and 3 f o l l o w i n g a r a p i d i n c r e a s e was i n d i c a t e d i n two out of t h r e e experiments u s i n g 14 Id and ~ C - p h e n y l a l a n i n e . The r e s u l t s s uggest t h a t t h e s e " i n s o l u b l e " p h e n o l i c c o n j u g a t e s were t u r n i n g o v e r . 22. The s p e c i f i c a c t i v i t y o f the c a f f e o y l moiety of compound 1 v/as determined a t the v a r i o u s time i n t e r v a l s . The r e s u l t s i n d i c a t e t h a t the s p e c i f i c a c t i v i t y o f the c a f f e o y l moiety f o l l o w e d a c o u r s e p a r a l l e l t o the s p e c i f i c a c t i v i t y o f compound 1. 100 Compound 2, the f e r u l i c a c i d - c o n t a i n i n g c o n j u g a t e , c o n s i s t e n t l y e x h i b i t e d the h i g h e s t s p e c i f i c a c t i v i t y i n a g i v e n experiment as compared t o compounds 1 and 3. The obvious q u e s t i o n a r i s e s as t o whether compound 2 i s a l i g n i n p r e c u r s o r . The s p e c i f i c a c t i v i t i e s o f o t h e r p o o l s of c a f f e i c a c i d were determined a t the s i x - h o u r m e t a b o l i c i n t e r v a l . The s p e c i f i c a c t i v i t y o f " s o l u b l e " c a f f e i c a c i d i s 2-3 times as g r e a t as the s p e c i f i c a c t i v i t y of the " i n s o l u b l e " c a f f e i c a c i d o f compound 1. The c a f f e i c a c i d moiety of the h i g h - m o l e c u l a r - w e i g h t c o n j u g a t e was not r a d i o a c t i v e . Mentha c o n t a i n s a m u l t i p l i c i t y of " i n s o l u b l e * c a f f e i c a c i d p o o l s (compound 1, the C band compound and the h i g h - m o l e c u l a r - w e i g h t c o n j u g a t e ) and k i n e t i c s t u d i e s c o u l d be meaningless when performed on the g r o s s h y d r o l y s i s p r o d u c t s o f crude, e t h a n o l - i n s o l u b l e r e s i d u e s . 101 LITERATURE CITED Bate - Smith, E.C. 1962. The p h e n o l i c c o n s t i t u e n t s of p l a n t s and t h e i r taxonomic s i g n i f i c a n c e , J . L i n n . Soc (Bot.) 58_: 95-174. Ibrahim, R.K., Towers, G.H.N., and Gibbs, R.D. 1962. S y r i n g i c and s i n a p i c a c i d s as 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 . Soc. (Bot.) 58.: 223-230. Tomaszewski, M. 1960. Occurrence of para-hydroxy-b e n z o i c a c i d and some o t h e r phenols 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 . _8: 61. Harborne, J.B., ed. 1964. 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. M c C a l l a , D.R. and Ne i s h , A.C. 1959. Metabolism of phenyIpropanoid compounds i n S a l v i a . I . B i o s y n t h e s i s of p h e n y l a l a n i n e and t y r o s i n e , Can. J . Biochem. P h y s i o l . 3_7: 531. Sondheimer, E. 1958. On the d i s t r i b u t i o n o f c a f f e i c a c i d and the c h l o r o g e n i c a c i d isomers i n p l a n t s , A r c h . Biochem. 3iophy. 74_: 131. Harborne, J.B. 1966. C a f f e i c a c i d e s t e r d i s t r i b -u t i o n i n h i g h e r p l a n t s , Z. N a t u r f o r s c h . 21b: 604 S l - B a s y o u n i , S. and Towers, G.H.N. 1964. The p h e n o l i c a c i d s i n wheat. I . Changes d u r i n g orowth and development^ Can. J . Biochem 42: 203-210. G l a s s , A.D.M. and Bohm, B.A. 1969. The accumula-t i o n o f ci 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 P t e r i d i u m a q u i l i n u m and Athyrium f e l i x - f e m i n a Phytochem. 3: 371. E l - B a s y o u n i , S.Z. and Nei s h , A.C. 1966. Occurrence of m e t a b o l i c a l l y - a c t i v e bound forms of cin n a m i c a c i d and i t s p h e n o l i c d e r i v a t i v e s i n acetone powders of wheat and b a r l e y p l a n t s , Phytochem. 5_: 683. E l - B a s y o u n i , S.Z., N e i s h , A.C. and Towers, G.H.N. 1964. The p h e n o l i c a c i d s i n wheat. I I I . I n s o l u b l e d e r i v a t i v e s o f p h e n o l i c c i n n a m i c a c i d s as n a t u r a l i n t e r m e d i a t e s i n l i g n i n b i o s y n t h e s i s , Phytochem. _3: 627. 102 12. Corse, J.W. 1S53. A new isomer o f c h l o r o g e n i c a c i d from peaches, Nature 172: 771. 13. T a y l o r , A.O. 1968. The d i s t r i b u t i o n and t u r n o v e r r a t e of s o l u b l e and i n s o l u b l e c a f f e o y l e s t e r s i n Xanthium, Phytochem. 7_: 63 14. P a n i z z i , L. and S c a r p a t i , M.L. 1954. Isolamento e c o n s t i t u z i o n e d e l p r i n c i p i o n a t t i v o d e l c a r c i o f o , Gazz. chim. i t a l . 84: 792 15. van B r a g t , . J . , Rohrbaugh, L.M. and Wender, S.H. 1965. I d e n t i f i c a t i o n of n e o c h l o r o g e n i c and 3 - 0 - f e r u l o y l q u i n i c a c i d s i n tomato p l a n t s , Phytochem. 4: 977. 16. Corse, J.W., Sondheimer, E. and Lundin, R. 1962. 3 - F e r u l o y l q u i n i c a c i d . A 3-methyl-ether o f c h l o r o g e n i c a c i d , T e t r a h e d r o n 1_8: 1207. 17. W i l l i a m s , A.H. 1958. p a r a - C o u m a r y l - q u i n i c a c i d from apple f r u i t , Chem. and Ind. 1200. 18. Harbourne, J.B. and Corner, J . 1961. P l a n t p o l y -p h e n o l s . 4. Hydroxycinnamic a c i d - sugar d e r i v a t i v e s , Biochem. J . 81_: 242-250. 19. Goldschmid, 0. and H e r g e r t , H.L. 1961. Examination of western hemlock f o r l i g n i n p r e c u r s o r s , T a p p i 44: 858. 20. Haslam, E., Haworth, R.D. and Keen, P.C. 1962. G a l l o t a n n i n s . P a r t V I I . T a r a g a l l o t a n n i n s , J . Chem. Soc. 1962: 3814. 21. S c a r p a t i , H.L. and O r i e n t e , G. 1960. I s o l a t i o n from, k i d n e y beans (Phaseolus v u l g a r i s ) o f p h a s e o l i c a c i d ; i t s c o n s t i t u t i o n and s y n t h e s i s , Gazz. chim. i t a l . 90_: 212. 22. S c a r p a t i , M.L. and O r i e n t e , G. 1958. C h i c o r i c a c i d ( d i c a f f e y l t a r t a r i c a c i d ) : i t s i s o l a t i o n from c h i c o r y ( C h i c o r i u m i n t y b u s ) and s y n t h e s i s , T e t r a h e d r o n . 4_: 43 23. B i r k o f e r , L., K a i s e r , C , Nouvertue, W. and Thomas, V. 1961. N a t u r a l l y - o c c u r i n g sugar e s t e r s o f p h e n o l i c a c i d s , Z. N a t u r f o r s c h . 16b: 249 103 24. Bergmann, L., T h i e s , W. and E r d - l s k y , L. 1965. Sas Vorkornmen von G l u c o s a m i n e s t e r n der Hydroxyzimtsauren i n Gewebekulturen von N i c o t i a n a tabacum, Z. N a t u r f o r s c h . 206: 1297. 25. Bohm, B.A. 1968. P h e n o l i c compounds i n f e r n s . I I I . An examination o f some f e r n s f o r c a f f e i c a c i d d e r i v a t i v e s , Phytochem. 1825. 26. Corner, J . J . and Harbourne, J.B. 1960. Cinnamic a c i d d e r i v a t i v e s of p o t a t o b e r r i e s , Chem. and Ind. 76. 27. Ohta, G. and Shimizu, M. 1958. A new t r i t e r -phenoid a l c o h o l , 2 4 - m e t h y l e n e c y c l o a r e t e n o l , as i t s f e r u l a t e , from r i c e bran o i l , Chem. Pharm. B u l l . Tokyo 6_: 325. 28. Ohta, G. and Shimizu, M. 1957. S t u d i e s on the c o n s t i t u e n t s of r i c e bran o i l . I I . S t r u c t u r e of O r y z a n o l - A, Pharm. B u l l . Tokyo 5_: 40 29. Pridham, J.B. 1967. Terphenoids i n P l a n t s . Academic P r e s s , New York and London. 30. S c h u l t z , O.E. and Gmelin, R. 1952. P a p i e r -chromato-graphie der s e n f o l g l u c o s i d - drogen, Z. N a t u r f orschung. 7b*- 5 0 0 • 31. Gmelin, R. and K j a e r , A. 1970. I s o f e r u l i c a c i d c h o l i n e e s t e r : a new p l a n t c o n s t i t u e n t , Phytochem. _9: 667. 32. Harbourne, J.B. 1967. Comparative B i o c h e m i s t r y of the F l a v o n o i d s . Academic P r e s s , London and New York. 33a. N i e r e n s t e i n , M. 1914. Zur k e n n t n i s der s t i c k -s t o f f h a l t i g e n b e s t a n d t e i l l e der p f l a n z e n -g a l l e n , Z e i t s c h . P h y s i o l . Chem. 9_2_: 53 33b. N i e r e n s t e i n , M. 1915. XX The f o r m a t i o n o f e l l a g i c a c i d from g a l l o y l - g l y c i n e by F e n i c i l l i u m , Biochem. J . 9_: 240. 34. Wheaton, T.A. and Stewart, I . 1965. F e r u l o y l -p u t r e s c i n e : i s o l a t i o n and i d e n t i f i c a t i o n from C i t r u s l e a v e s and f r u i t , Nature 206: 620. 1 0 4 S t o e s s l , A.. 1 9 6 7 . The a n t i f u n g a l f a c t o r s i n b a r l e y . IV. I s o l a t i o n , s t r u c t u r e , and s y n t h e s i s o f the h o r d a t i n e s , Can. J . Chem. 4 5 : 1 7 4 5 . Levy, C.C. and Zucker, M. 1 9 6 0 . Cinnamyl and p-coumaroyl e s t e r s as i n t e r m e d i a t e s i n the b i o - s y n t h e s i s o f c h l o r o g e n i c a c i d , J . B i o l . Chem. 2 3 5 : 2 4 1 8 . R u s s e l l , D.W. and Conn, E.E. 1 9 6 7 . The c i n n a m i c a c i d 4 - h y d r o x y l a s e o f pea s e e d l i n g s , A r c h . Biochem. Bio p h y s . 1 2 2 :* 2 5 6 - 2 5 8 . Neukom, H., P r o v i d o l i , L., G r e m l i , H. and Hui, P.A 1 9 6 7 . Recent i n v e s t i g a t i o n s on wheat f l o u r p entosans, C e r e a l Chem. 4_4: 2 3 8 . C l a r k , R.S., Kuc, J . , Henze, R.E. and Quackenbush, F.W. 1 9 5 9 . The n a t u r e and f u n g i t o x i c i t y o f an amino a c i d a d d i t i o n p r o d u c t o f c h l o r o g e n i c a c i d , P h y t o p a t h o l o g y 4J9: 5 9 4 . A l l e n , E.H. and Kuc, J . 1 9 6 8 . a l p h a - S o l a n i n e and a l p h a - c h a c o n i n e as f u n g i t o x i c compounds i n e x t r a c t s of I r i s h p o t a t o t u b e r s , Phyto-p a t h o l o g y 5J3: 7 7 6 . Gee, R., K y l i n , A. and Saltman, P. 1 9 7 0 . Enhancement o f p h o t o p h o s p h o r y l a t i o n and p h o t o r e d u c t i o n by a c h l o r o p l a s t f a c t o r from s p i n a c h l e a v e s , Biochem. Bio p h y s . Res. Commun. 4 0 : 6 4 2 . Black, C . C , San P i e t r o , A., Limbach, D. and G. M o r r i s . 1 9 6 3 . P h o t o s y n t h e t i c p h o s p h o r y l a t i o n c a t a l y z e d by f a c t o r s i s o l a t e d from photo-s y n t h e t i c organisms, P r o c . N a t l . Acad. S c i . U.S. 50_: 3 7 . Wildner, G.F. and C r i d d l e , R.S. 1 9 6 9 . R i b u l o s e d i p h o s p h a t e c a r b o x y l a s e I . A f a c t o r i n v o l v e d i n l i g h t a c t i v a t i o n o f the enzyme. Biochem. Bio p h y s . Res. Commun. 3_7: 9 5 2 . Krogmann, D.W. and S t i l l e r , M.L. 1 9 6 2 . A n a t u r a l l y - o c c u r r i n g c o f a c t o r f o r p h o t o s y n t h e t p h o s p h o r y l a t i o n , Biochem. Bio p h y s . Res. Commun. 7 : 4 6 . 105 A l i b e r t , G., Marigo, G. and 3oudet, A. 1968. Recherches s ur l a b i o s y n t h e s e des composes aromatiques chez l e s vegetaux s u p e r i e u r s : sur l a p r e s e n c e ' d • a c i d e s p h e n o l i q u e s dans une f r a c t i o n p r o t e i q u e i s o l e e des f e u i l l e s Quercus o o d u n c u l a t a , Comptes rendus Acad. S c i . P a r i s . 267: 2144. Smith, D.C.C. 1955. E s t e r groups i n l i g n i n , Nature 176: 267. P e a r l , I.A., Beyer, D.L., Johnson, B. and S. W i l k i n s o n . 1957. A l k a l i n e h y d r o l y s i s o f r e p r e s e n t a t i v e hardwoods, T a p p i 40_: 374. P e a r l , I.A., Beyer, D.L. and Laskowski, D. 1959. A l k a l i n e h y d r o l y s i s o f r e p r e s e n t a t i v e palms, T a p p i 42.: 779. S t a f f o r d , H.A. 1960. Comparison of l i g n i n - l i k e polymers produced p e r o x i d a t i v e l y by ci n n a m i c a c i d d e r i v a t i v e s i n l e a f s e c t i o n s o f Phleum, P l a n t P h y s i o l . _35: 612. Stone, J . E . and B l u n d e l l , H.J. 1951. Rapid m i c r o method f o r a l k a l i n e n i t r o b e n z e n e o x i d a t i o n of l i g n i n and d e t e r m i n a t i o n o f ald e h y d e s , A n a l . Chem. 2J3: 771. T a y l o r , A.O. and Zucker, H. 1966. Turnover and metabolism of c h l o r o g e n i c a c i d i n Xanthium l e a v e s and p o t a t o t u b e r s , P l a n t P h y s i o l . 41: 1350. D a n i e l s , D.G.H., Ki n g , H.G.C., and M a r t i n , H.F. 1963. A n t i o x i d a n t s i n o a t s : e s t e r s o f p h e n o l i c a c i d s . J . S c i . Fd. A g r i c . 14_: 385. Synge, R.L.M. 1968. Occurrence i n p l a n t s o f amino a c i d r e s i d u e s c h e m i c a l l y bound o t h e r w i s e than i n p r o t e i n s , Ann. Rev. P l a n t P h y s i o l . 19: 113. S t o e s s l , A., R o h r i n g e r , R. and Samborski, D.J. 1969. 2 - H y d r o x y p u t r e s c i n e amides as abnormal m e t a b o l i t e s of wheat, T e t r a h e d r o n _33_: 2807 F r i t i g , 3., H i r t h , L. and O u r i s s o n , G. 1970. B i o s y n t h e s i s of the coumarins: s c o p o l e t i n f o r m a t i o n i n tobacco t i s s u e c u l t u r e s , Phytochem. _9_: 1963. 106 56. F r i t i g , B. P e r s o n a l communication. 57. Brown, S.S., Towers, G.H.N, and Chen, D. 1964. B i o s y n t h e s i s of the coumarins - V. Pathways of u m b e l l i f e r o n e f o r m a t i o n i n Hydrangea  m a c r o p h y l l a , Phytochem. _3: 469. 53. Fuchs, A., Ro h r i n g e r , R. and Samborski, D.J. 1967. Metabolism o f aromatic compounds i n h e a l t h y and r u s t - i n f e c t e d l e a v e s o f w h e a t ^ I I . S t u d i e s w i t h ^ L - p h e n y l a l a n i n e - C, L— t y r o s i n e - U - C, and f e r u l a t e -U- "*C, Can. J . Botany 4_5: 2137. 59. R o h r i n g e r , R. and Samborski, D.J. 1967. Aromatic compounds i n the h o s t - p a r a s i t e i n t e r a c t i o n , Ann. Rev. Phytopath. _5_: 77. 60. Samborski, D.J. and Roh r i n g e r , R. 1970. Abnormal m e t a b o l i t e s o f wheat: o c c u r r e n c e , i s o l a t i o n and b i o g e n e s i s o f 2 - h y d r o x y p u t r e s c i n e amides, Phytochem. 9_: 1939. 61. E l l i s , B.E. and Towers, G.H.N. 1970. B i o g e n e s i s o f r o s m a r i n i c a c i d i n Mentha. Biochem. J . 113: 291. 62. E l l i s , B.E. P e r s o n a l communication. 63. Hope, H. 1969. Ph.D. T h e s i s , D a l h o u s i e U n i v e r s i t y , H a l i f a x , Canada. 64. Ibrahim, R.K. and Towers, G.H.N. 1960. The i d e n t i f i c a t i o n , by chromatography o f p l a n t p h e n o l i c a c i d s , A r c h . Biochem, Biophy. 87: 125, 65. Heilmann, J . , B a r r o l l i e r , J . and Watzke, E. 1957. B e i t r a g zur aminosaurebestimmung auf p a p i e r -chromatogrammen, Z. P h y s i o l . Chem. 309: 219. 66. Bryson, J . L . and M i t c h e l l , T . J . 1951. Improved s p r a y i n g r e a g e n t s f o r the d e t e c t i o n o f sugars on paper chromatograms, Nature 167: 864. 67. Mukherjee, S. and S r i v a s t a v a , H.C. 1952. Improved spray r e a g e n t f o r the d e t e c t i o n o f sugars Nature 169: 330. 68. Bray, H.G., Thorpe, W.V. and White, K. 19 50. 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 the r a b b i t . 10. The a p p l i c a t i o n o f paperchromatography to m e t a b o l i c s t u d i e s o f h y d r o x y a c i d s and amides, Biochem. J . 46_: 271. 107 M i c h l , H. .1951. Paper e l e c t r o p h o r e s i s a t p o t e n t i a l d i f f e r e n c e s o f 50 v o l t s per c e n t i -meter, Hschr. Chem. 82_: 489. Ambler, R.P. 1963. The amino a c i d sequence o f Pseudomonas cytochrome c-551, Biochem. J . 89: 349. Spackman, D.H., S t e i n , W.H and S. M o i r e . 1958. Automatic r e c o r d i n g apparatus f o r use i n the chromatography of amino a c i d s , A n a l . Chem. 30_: 1190. G u i d o t t i , G., H i l l , R .J., and K o n i g s b e r g , W. 1962. The s t r u c t u r e o f human haemoglobin. I I . The s e p a r a t i o n and amino a c i d c o m p o s i t i o n of the t r y p t i c p e p t i d e s from the a l o h a and b e t a c h a i n s , J . B i o l . Chem. 237: 2184 Janson, J a n - C h r i s t e r . 1967. A d s o r p t i o n phenomena on Sephadex, J . Chromatog. _2_8: 12. N o l l e r , C R . 1965. Chemistry o f Or g a n i c Compounds. W.B. Saunders and Co. P h i l a d e l p h i a and New York. Schroeder, H.A. 1967. S t a b i l i z a t i o n o f c a f f e y l compounds i n a l k a l i n e media, Phytochem. _6: 1589 J u r d , L. 1956. A s p e c t r o p h o t o m e t r i c method f o r the d e t e r m i n a t i o n of or_thp_-dihydroxy groups i n f l a v o n o i d comoounds, A r c h . Biochem. B i o o h y s . 63: 376. Fausch, H., Kundig, W. and Neukom, H. 1963. F e r u l i c a c i d as a component of a g l y c o p r o t e i n from wheat f l o u r , Nature 199: 287. Kagan, J . 1966. The pho t o c h e m i c a l c o n v e r s i o n of c a f f e i c a c i d t o e s c u l e t i n . A model f o r the s y n t h e s i s o f coumarins in_ v i v o , J . Am. Chem. Soc. 8_8: 2617. 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, Proc. Sym. P l a n t P h e n o l i c s North Amer. Oregon S t a t e U n i v . C a r t w r i g h t , R.A. and Roberts, E.A.H. 1955. T h e o g a l l i n as a g a l l o y l e s t e r o f q u i n i c a c i d , Chem. and Ind. 230. 108 Loomis, W.D. 1969. Removal of p h e n o l i c compounds d u r i n g the i s o l a t i o n of p l a n t enzymes. Methods i n Enzymoloqy V o l . X I I I . Lowenstein, M., Colowick, S.P. and Kaplan, K.A. ed. Academic P r e s s , London. Loomis, W.D. and B a t t a i l e , J . 1966. P l a n t p h e n o l i c compounds and the i s o l a t i o n o f p l a n t enzymes, Phytochem. _5_: 423. B a g d a s a r i a n , M., Matheson, N.A., Synge, R.L.M and Youngson, M.A. 1964. New p r o c e d u r e s f o r i s o l a t i n g p o l y p e p t i d e s and p r o t e i n s from t i s s u e s , Biochem. J . 91.: 91. Synge, R.L.M. and M. Youngson. 1961. M o l e c u l a r -s i e v e methods f o r s e p a r a t i o n s of p o l y p e p t i d e s a c c o r d i n g t o m o l e c u l a r weight, Biochem. J . 78: 31p. P i e r p o i n t , W.S. 1966. The enzymic o x i d a t i o n of c h l o r o g e n i c a c i d and some r e a c t i o n s of the quinone produced, Biochem. J . 9_8: 567. B r i e s k o r n , C.H. and Mosandl, A. 1970. E i n k a f f e e s a v r e e n t h a l t e n d e s p r o t e i d aus u m b e l l i f e r e n f r u c h t e n , T e t . L e t t . 1_: 109. Pridham, J.B. ed. 1963. Methods i n P o l y p h e n o l  C h e m i s t r y . Pergamon P r e s s , O x f o r d . Pridham, J.B. and Swain, T. ed. 1964. B i o s y n t h e t i c  Pathways i n Higher P l a n t s , page 9. Academic P r e s s , New York. H i g u c h i , T. and I t o , Y. 1958. Dehydrogenation p r o d u c t s of c o n i f e r y l a l c o h o l formed by the a c t i o n of mushroom p h e n o l o x i d a s e , r h u s -l a c c a s e and r a d i s h p e r o x i d a s e , J . Biochem. 45: 575. Nakamura, W. 1967. S t u d i e s on the b i o s y n t h e s i s of l i g n i n . I . D i s p r o o f a g a i n s t the c a t a l y t i c a c t i v i t y of l a c c a s e i n the o x i d a t i o n of c o n i f e r y l a l c o h o l , Biochem. J . _62_: 54. Bland, D.E. and Logan, A.F. 1965. The p r o p e r t i e s of s y r i n g y l , g u a i c y l and para-hydroxyphenyl a r t i f i c i a l l i g n i n s , Biochem. J . 95: 515. 109 Nozu, Y. 1967. S t u d i e s on the b i o s y n t h e s i s o f l i g n i n . I I I . Dehydrogenative p o l y m e r i z a t i o n of c o n i f e r y l a l c o h o l by p e r o x i d a s e , J . Biochem. 62: 519. Krupnikova, T.A., Dranik, L . I . and S h k o l ' n i k , M. 1968. Phenol c a r b o x y l i c a c i d s of c o r n (Zea mays L . ) , Doklady Akademii Nauk SSSR. 180: 1497. F i s k e , C.H., and SubbaRow, Y. 1925. The c o l o r i m e t r i c d e t e r m i n a t i o n of phosphorus, J . B i o l . Chem. _66: 375. V o g e l , A . I . 1955. P r a c t i c a l O r g a n i c Chemistry, t h i r d e d i t i o n . Longmans, London. 

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.831.1-0101334/manifest

Comment

Related Items