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The nature of the genetic difference in the coumarin metabolism of bitter and non-bitter Melilotus alba… Freyman, Stanislaw 1963

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i THE NATURE OF THE GENETIC DIFFERENCE IN THE COUMARIN METABOLISM OF BITTER AND NON-BITTER Melilotus alba Desr. by STANISLAW FREYMAN B.Sc.(Agric), University of Pretoria, 1959. A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN AGRICULTURE in the Division of PLANT SCIENCE We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA APRIL 1963 In presenting this thesis in p a r t i a l fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make i t freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It i s understood that copying or publication of this thesis for financial gain shall not be alloived without my written permission. Dopartmont of ^ P u f t M "T S C j £ N C f e The University of British Columbia, Vancouver 8, Canada. Date Yvy i i ABSTRACT The phenylpropanoid compound, coumarin, i s r e s p o n s i b l e f o r the b i t t e r n e s s and i n d i r e c t l y , t o x i c i t y i n the forage legume sweet c l o v e r ( M e l i l o t u s a l b a ) . Coumarin, mainly i n the "bound" form as the j3> g l u c o s i d e of o-coumaric a c i d i s abundant i n the b i t t e r p l a n t s , but not i n the shoots o f the low-coumarin "Denta" and "Cumino" v a r i e t i e s , and i n the s p e c i e s M. d e n t a t a . S u b s t a n t i a l q u a n t i t i e s of t h e coumaric a c i d g l u c o s i d e h a v e b e e n found i n t h e seed of t h e s e low-coumarin p l a n t s . T h e search f o r a compound t h a t s u b s t i t u t e s f o r coumarin i n the shoots o f low-coumarin p l a n t s , l e d t o the i s o l a t i o n of a f l a v o n o i d , somewhat resembling q u e r c e t i n . Feeding of o and p-coumaric a c i d s t o both h i g h and low-coumarin p l a n t s l e d to the methoxylation o f these compounds p r i o r t o g l y c o s i d a t i o n . When cinnamic a c i d was f e d , no d i f f e r e n c e i n the metabolic products could be d e t e c t e d from those normally, found i n c o n t r o l p l a n t s . ACKNOWLEDGEMENTS My a p p r e c i a t i o n and thanks are accorded the f o l l o w i n g persons and i n s t i t u t i o n s : Dr. V.C.Brink. D i v i s i o n o f Pla n t Science f o r i n t e r e s t and guidance. Dr. H . McLean, Dr. J.A.F. Gardner, Mr. G.M.Barton, a l l of the F o r e s t Products Laboratory,Department o f F o r e s t r y , f o r a s s i s t a n c e i n chemical c h a r a c t e r i s a t i o n . Mr. Don. Pearce and David Armstrong, f o r h e l p i n the f i e l d and greenhouse. The N a t i o n a l Research C o u n c i l of Canada f o r f i n a n c i a l support. Thanks are accorded to my t h e s i s committee: Dr. V.C.Brink, D i v i s i o n of P l a n t Science (Chairman) Dr. G.A. Hornby, D i v i s i o n o f Plant, Science Dr. A .J.Renney, D i v i s i o n o f Plant Science D r . W . D . K i t t s , D i v i s i o n o f Animal Science D r . K . Beamish, Department o f B i o l o g y .and 3 o t a n y Dr. J . J . R i c h t e r , Department o f A g r i c u l t u r a l Economic i i i TABLE OF CONTENTS I INTRODUCTION 1 I I REVIEW OF LITERATURE 5 A The nature of couinarin and i t s r e l a t i v e s 5 B. Coumarin i n sweet c l o v e r 7 C. Function of Coumarins 10 ( i ) The state of coumarin in the plant 10 ( i i ) Toxicity and disease resistance 12 ( i i i ) Role i n flavonoid synthesis 13 ( i v ) Role in lignin synthesis 13 ( v ) Fate of coumarin i n the plant 14 ( v i ) Intact plant response to coumarin 14 D. Biosynthesis of coumarins 15 I I I INVESTIGATIONS AND RESULTS 21 A. Analysis of seed 21 ( i ) M a t e r i a l s 21 ( i i ) Methods 22 ( i i i ) R e s u l t s 23 B. Analysis of sugar 27 ( i ) I-iateriais . 27 ( i i ) Methods 27 ( i i i ) R e s u l t s 29 C. A comparative study of green shoots 29 ( i ) Soxhlet extraction 30 ( i i ) Steam d i s t i l l a t i o n 30 ( i i i ) Liquid-liquid extration 31 ( i v ) A comparison.of aqueous extracts (a) M a t e r i a l s 32 (b) Methods 32 (c) R e s u l t s 36 D. Feeding Experiments. i v ( i ) M a t e r i a l s 55 ( i i ) Methods 55 (a) Seed 56 (b) Shootso 56 ( i i i ) R e s u l t s 58 (a) Coumarin 55 (b) o-coumaric a c i d 53 (c) p-coumaric a c i d 64 (d) cinnamic a c i d 6 6 IV DISCUSSION AND CONCLUSION 68 V LITERATURE CITED 76 1 I INTRODUCTION Sweet c l o v e r ( M e l i l o t u s a l b a ) , a forage crop important i n western Canada and i n a number o f other areas i n t h e mid and h i g h l a t i t u d e s , i s l i m i t e d i n u s e f u l n e s s by a measure o f u n p a l a t i b i l i t y and t o x i c i t y a t t r i b u t e d to the presence i n i t s t i s s u e s o f the chemical compound coumarin. Some success i n producing low coumarin s t r a i n s o f the crop has attended the e f f o r t s o f p l a n t b r e e d e r s . Nontheless a p r a c t i c a l end has not y e t been achieved and, i t i s apparent t h a t an a i d to progress would be more i n f o r m a t i o n on the b i o c h e m i s t r y and g e n e t i c s o f coumarin and i t s r e l a t i v e s i n sweet c l o v e r . To provide such knowledge was a prime o b j e c t i v e of t h i s study; a d d i t i o n a l l y i t was hoped t h a t p a t t e r n s r e l a t -i n g to coumarin s y n t h e s i s i n p l a n t s , l a r g e l y known from work with r a d i o - t r a c e r s , c o u l d be confirmed by means of g e n e t i c b l o c k s and f e e d i n g t r i a l s . A n o t a b l e f e a t u r e o f h i g h e r p l a n t s , and some micro-organisms, i s t h e i r a b i l i t y to s y n t h e s i z e a great v a r i e t y of p h e n o l i c compounds. T h i s a b i l i t y i s not shared by the animal kingdom. Although coumarin i t s e l f i s not a phenol, i t never-t h e l e s s i s c l o s e l y i n v o l v e d i n the metabolic pathways concerned with the s y n t h e s i s o f p h e n o l i c compounds, and coumarins with p h e n o l i c s u b s t i t u t i o n s are very o f t e n formed. Knowledge o f the pathways i n v o l v e d i n the b i o s y n t h e s i s of p h e n o l i c compounds has l a r g e l y been gained d u r i n g the l a s t f i v e years and they are no l o n g e r regarded as being r a t h e r remote from the main metabolic processes of the t i s s u e s . Modern methods, p a r t i c u l a r l y those u s i n g r a d i o - a c t i v e t r a c e r s , have shown t h a t a c e t i c a c i d and s h i k i m i c a c i d can f u n c t i o n as p r e c u r s o r s of aromatic r i n g s and hence p h e n o l i c compounds would appear to be c l o s e l y l i n k e d to the g e n e r a l carbohydrate metabolism of the p l a n t . On the other hand very l i t t l e i s known of the p h y s i o l o g i c a l f u n c t i o n s o f p h e n o l i c s and coumarin i n the metabolism of the p l a n t , yet t h e i r v a r i e t y and abundance suggests t h a t they are i m p o r t a n t 0 Chemical substances that p l a y a primary r o l e i n normal metabolism are more or l e s s common to most organisms. However, they are o f l e s s e r consequence i n comparative b i o -chemistry than the s o - c a l l e d secondary products, the seeming-l y s t a b l e end products of metabolism, such as coumarins. These compounds r e v e a l the e x i s t e n c e o f metabolic processes p e c u l i a r to the organism i n which they o c c u r c Of a l l such secondary substances the most abundand and d i v e r s e are those c o n t a i n i n g p h e n o l i c h y d r c x l groups. Many coumarins, probably i n the bound g l y c o s i d i c forms, occur i n r e l a t i v e l y l a r g e q u a n t i t i e s i n a number of p l a n t f a m i l i e s such as Leguminosae, Rutaceae, U m b e l l i f e r a e , Solanaceae and L a b i a t a e . 3 . In the f a m i l y Leguminosae where many s p e c i e s used f o r forage occur, the coumarins assume a very c o n s i d e r a b l e p r a c t i c a l importance. F o r example i n the sweet c l o v e r s , e s p e c i a l l y M e l i o t u s a l b a and M_j_ o f f i c i n a l i s , coumarin lowers p a l a t i b i l i t y , and i s i n v o l v e d i n the development o f a hemorrhagic d i s e a s e of l i v e s t o c k , a t t r i b u t e d to the p r o d u c t i o n of dicumarol, i n p o o r l y cured sweet-clover hay. P a l a t a b l e , low-coumarin sweet c l o v e r v a r i e t i e s have been developed from h y b r i d s of the b i t t e r M . a l b a and a n o n - b i t t e r , low coumarin s p e c i e s M. d e n t a t a which i s of l i t t l e economic v a l u e . I n h e r i t a n c e of coumarin p r o d u c t i o n i n the p l a n t i s r e l a t i v e l y simple, and i s governed, most probably, by two p a i r s o f genes. Although much progress has been made i n the develop-ment o f low coumarin v a r i e t i e s o f sweet c l o v e r , they are not f r e e of the compound. Knowledge o f the compounds o r compound which s u b s t i t u t e f o r coumarin i n the low coumarin p l a n t s might be u s e f u l to p l a n t b r e e d e r s 0 C o n f i r m a t i o n and e x t e n s i o n o f b i o c h e m i c a l sequences r e l a t i v e t o coumarin i n p a r t i c u l a r , and p h e n o l i c s i n g e n e r a l might w e l l prove t o be of s i g n i -f i c a n c e . Using then sweet c l o v e r as the o r g a n i c s u b j e c t , answers were sought to the f o l l o w i n g q u e s t i o n . I f coumarin i s absent i n these p a l a t a b l e v a r i e t i e s , what r e l a t e d compound f u n c t i o n s as i t s s u b s t i t u t e ? Where do the genes exert t h e i r e f f e c t along the metabolic pathway, p r i o r to or a f t e r the 4. formation o f coumarin? When coumarin. and i t s chemical r e l a t i v e s are " f e d " to n o n - b i t t e r v a r i t i e s , i n what way are these compounds metabolized? Making use o f chromatography i n c o n j u n c t i o n with o t h e r techniques o f s e p a r a t i o n and i d e n t i f i c a t i o n , an attempt was made t o answer these questions i n the hope o f adding to the knowledge of the b i o c h e m i s t r y and g e n e t i c s o f coumarins i n sweet c l o v e r . 5. 11 REVIEW OF LITERATURE A. The nature of coumarin and i t s relatives. Coumarin is one of the more important compounds belonging to the pherfyipropane group, which has a nine carbon C^ - C^  ^skeleton, and i s the most prominent structural unit among the aromatic substances in higher plants„ This struct-ure i s "employed" in the chemistry of the plant in many ways. c - c - c Coumarin was f i r s t isolated from tonka beans by Vogel in 1820 (90) and was i n i t i a l l y considered to be a benzoic acid derivative. Its synthesis by Perkin (61) in 1868 from salicylaldehyde by means of the classical reaction named after him, established i t s relation to o-hydroxycinnamic acid. This latter compound loses a molecule of water as i t cyclizes to form coumarin and the lactone ring. o-hydroxycinnamic acid Coumarin Coumarin, therefore, i s a heterocyclic compound belonging to a class known as benzopyrones, of which two distinct types are recognized. 6 Benzo °< pyrone BenzoXpyrone coumarin chromone The c o l l e c t i v e term "coumarins", r e f e r s to compounds p o s s e s s i n g the b a s i c benzo pyrone s t r u c t u r e ; v a r i a n t s occur through the d i f f e r e n t hydoxyl, methoxyl or methyl s u b s t i t u t i o n s . The f o l l o w i n g are r e p r e s e n t a t i v e coumarins. OH u m b e l l i f e r o n e S c o p o l e t i n Daphnetin ( 7 hydroxy (6 methoxy, 7 hydroxy (7,8 dihydroxy coumarin) coumarin) coumarin) ; R e l a t e d c l o s e l y i n metabolism to the coumarins are the f l a v a n o i d compounds of wide occurrence i n p l a n t s and which may be regarded as "benzo" d e r i v a t i v e s o f the b a s i c chromone s t r u c t u r e . 0 F o l l o w i n g the e a r l y pinneer work on coumarin, a c e r t a i n i n t e r e s t was maintained, mainly because o f i t s c h a r a c t e r i s t i c a l l y p l e a s a n t , sweet odor and i t s u s e f u l n e s s t o the perfume and f l a v o r i n g i n d u s t r i e s . Knowledge o f the chemistry o f coumarin developed r a p i d l y i n the l a t t e r h a l f o f the 19th century, due mainly to the synthetic method known as the Pechmann condensation (60), i n which phenols react with B-ketonic esters i n the presence of s u l f u r i c acid. 0. O H CH3COCH2COOC2H5 H 2 S O 4 The reaction proceeds from very simple s t a r t i n g materials and gives a good y i e l d of coumarin. Coumarins i n t h e i r reactions may behave as unsaturated lactones or as substituted benzenes. The reactions whiijh involve a l k a l i are usually the reactions of coumaric aci d . The chemistry of coumarin, therefore, consists mainly of substitution i n the r i n g system through such well known reactions as n i t r a t i o n , halogenation, sulfonation, arsenation and mercuration and have been extensively reviewed by Wawzonek (92), Sethna and Shah (76) and Spath (83). B. Coumarin i n Sweet Clover. The f i r s t person to rel a t e the b i t t e r taste of common sweet clover (Melilotus spp) to i t s coumarin content and to develop a quantitative chemical t e s t f o r coumarin, was Obermayer (5#) i n 1913. In spite of the fa c t that the value of sweet clover as a s o i l improving crop was recognized by 1900, i t s use f o r a g r i c u l t u r a l purposes spread rather slowly 8. because of i t s low p a l a t a b i l i t y . The b i t t e r t r a i t o f the p l a n t and i t s "weedy" h a b i t made i t so u n d e s i r a b l e t h a t c e r t a i n S t a t e s o f America at f i r s t l e g i s l a t e d , a g a i n s t i t . The u n p o p u l a r i t y of sweet c l o v e r grew when i t became known t h a t f e e d i n g s p o i l e d sweet c l o v e r hay may cause a " b l e e d i n g d i s e a s e " i n l i v e s t o c k ( 70, 82) due to the f o r m a t i o n o f a t o x i c substance, dicuraarol, from coumarin ( 2 1 ) . When such hay i s eaten, dicumarol reduces the c l o t t i n g power of the bl o o d , by competing with V i t a m i n K, and animals may b l e e d to death from s l i g h t wounds or from i n t e r n a l hemorrhages (22, 74). I t i s i n t e r e s t i n g t o note t h a t i n s p o i l a g e o f hay o f ot h e r coumarin-containing p l a n t s such as sweet v e r n a l g r a s s , dicumarol i s produced. I n g e s t i o n o f these hays a l s o causes the hemorrhagic d i s e a s e . The d i s t r i b u t i o n o f n a t u r a l coumarins i n p l a n t s , and t h e i r r o l e i n animal metabolism has been reviewed by Bradner ( 9 ) . Attempts t o produce coumarin-free sweet c l o v e r v a r i e t i e s were i n i t i a t e d over 30 years ago and an e x t e n s i v e £ l i t e r a t u r e r e c o r d s p r o g r e s s . A d e t a i l e d review o f the s u b j e c t has been presented r e c e n t l y by Bradner (49). Only a s h o r t review of the p e r t i n e n t m a t e r i a l i s presented here. E a r l y attempts to i s o l a t e coumarin-free p l a n t s o f M e l i l o t u s a l b a or M O f f i c i n a l i s were u n s u c c e s s f u l (86). Kuznetsov (55) and l a t e r B r ink (10, 11) r e p o r t e d t h a t a c e r t a i n s p e c i e s , found i n E a s t e r n Europe and Western A s i a was e n t i r e l y f r e e o f the 9 b i t t e r t r a i t . This plant was i d e n t i f i e d to be M dentata, a species of minor a g r i c u l t u r a l s i g n i f i c a n c e . Through i n t e r s p e c i f i c crosses with the aid of g r a f t i n g of the al b a n i s t i c hybrids to normal plants and by subsequent back crossing to the agronomically desirable M alba (79, 12) low coumarin v a r i e t i e s have been developed, notably "Cumino" (36) and "Denta" (81). The genetics involved i n the inheritance of coumarin i n sweet clover had attracted some i n t e r e s t . Among the e a r l i e r workers, Stevenson and White (85), using a colorimetic procedure, f o r coumarin determination, showed that the low coumarin character resulted from a single recessive gene e This conclusion was l a t e r confirmed by Horner and White (45)j Rinke (67) using a s l i g h t l y modified colorimetric t e s t , concluded that high-coumarin content was inherited as a simple recessive. On the other hand, Smith (80) who success-f u l l y crossed M. dentata with M. alba,, from an analysis of back cross progenies, concluded that two main genes f o r reduced coumarin content had been obtained from M. dentata. One of these genes was recessive to the gene f o r high content and the other displayed no dominance. Webster (93) using an emroyo culture technique successfully crossed high coumarin M. o f f i c i n a l i s with low coumarin M. alba. From the analysis of the F2 population he suggested a single major-recessive gene 10. f o r low-coumarin content was i n v o l v e d i n h i s i n h e r i t a n c e p a t t e r n s . Goplen, G r e e n s h i e l d s and Baenziger (34) have r e p o r t e d t h a t two genes are concerned with the i n h e r i t a n c e o f coumarin i n p l a n t s . The gene des i g n a t e d as "b" determines the type o f coumarin present, whether f r e e or bound, i n coumarin-c o n t a i n i n g p l a n t s , while the gene symbolized a 3 "cu" governs the p r o d u c t i o n o f coumarin. In the absence o f "Cu", the "b" gene has no apparent e f f e c t . Thus, a c c o r d i n g t o Goplen et a l , i n BB CuCu genotypes coumarin i s f r e e , i n bb CuCu coumarin i s bound and i n BB cucu coumarin i s absent. C. F u n c t i o n of Coumarins. In s p i t e of the f a c t t h a t the chemistry o f coumarins and p h e n o l i c s i s w e l l known, the f u n c t i o n s o f these compounds i n the p l a n t are s t i l l obscure (30). A n o t a b l e f a c t i s t h a t t h e r e i s s t i l l some u n c e r t a i n t y concerning the s t a t e i n which coumarin e x i s t s i n the i n t a c t p l a n t t i s s u e . T h i s o n l y emphasizes how l i t t l e i s known of i t s metabolic a c t i v i t i e s , ( i ) The s t a t e o f coumarin i n the p l a n t . In e a r l y work on coumarin i n sweet c l o v e r , the assumption was made t h a t the f r e e form o f the compound predominates i n the p l a n t . Subsequent i n v e s t i g a t i o n s demon-s t r a t e d the presence o f bound, i n a d d i t i o n t o f r e e coumarin, and i t was thought t h a t both forms were nor m a l l y present (75, 40) and t h a t the r e l a t i v e amounts o f f r e e and bound coumarin 11 . are gene c o n t r o l l e d (74, 34, 3 5 ) . S c h a e f f e r et a i (73) i n d i c a t e d t h a t because o f the u n c e r t a i n t y about the degree of h y d r o l y s i s of the g l u c o s i d e d u r i n g i t s i s o l a t i o n , r a t i o s c a l c u l a t e d f o r these two compounds would be v i r t u a l l y meaningless. More r e c e n t l y , a f t e r a r e a p p r a i s a l of the r e l a t i o n s h i p between f r e e and bound coumarin, Haskins and Gorz (41) s u b s t a n t i a t i n g the find3 of Rudorf and Schwarze (71), came to the c o n c l u s i o n t h a t when s u i t a b l e e x t r a c t i o n procedures are used, v i r t u a l l y a l l the coumarin i s o btained i n . t h e bound form. However, Brown, ( 1 9 ) , s t i l l more r e c e n t l y , presented i n d i r e c t evidence t h a t f r e e coumarin can e x i s t , at l e a s t ephemerally, i n H i e r o c h l o a o d e r a t a . "Bound" coumarin i s a term r e f e r r i n g t o the /3 -g l y c o s i d e s of both the c i s and the t r a n s isomers o f o-hydroxy-cinnamic a c i d . A c o n t r o v e r s y arose as t o which isomer y i e l d s coumarin more r e a d i l y on h y d r o l y s i s . The t r a n s form would have to undergo i s o m e r i s a t i o n to the c i s form f o r s u c c e s s f u l l a c t o n e r i n g f o r m a t i o n . Brown et a l . (15) e a r l i e r suggested t h a t coumaric a c i d g l y c o s i d e , i . e . the t r a n s form, i s a l s o an i n t e r m e d i a t e i n the f o r m a t i o n o f coumarin. Kosuge ( 5 2 ) , on the o t h e r hand, contends t h a t o n l y the yft-glucoside o f o-coumarinic a c i d i s i d e n t i c a l with bound coumarin, that, i s the c i s and not the trans isomer. The two g l u c o s i d e s can be d i s t i n g u i s h e d by the p r e f e r e n t i a l h y d r o l y s i s o f the g l u c o -s i d e o f coumarinic a c i d by p- gluco3idase ( 5 2 ) . Haskins and 12 Gorz (42) have a l s o d e s c r i b e d methods of a n a l y s i s which permit the c i s and t r a n s g l u c o s i d e s t o be determined s e p a r a t e l y , (ii.) T o x i c i t y and d i s e a s e r e s i s t a n c e . C e r t a i n suggestions f o r the p h y s i o l o g i c a l f u n c t i o n s of coumarins i n p l a n t s have been put forward. Among the suggestions are t h a t these compounds p l a y an important r o l e i n d i s e a s e r e s i s t a n c e . The form a t i o n o f coumarin i n c e r t a i n p a t h o l o g i c a l c o n d i t i o n s l e d Best (5) to suggest t h a t they may be the end products o f pathogen-altered pathways. Best (4) demonstrated t h a t tobacco i n f e c t e d with tomato s p o t t e d w i l t v i r u s produces l a r g e amounts of s c o p o l e t i n . U r i t a n i and Hoshiya (88) suggest t h a t the accumulation o f s c o p o l e t i n and u m b e l l i f e r o n e i n the sound f l e s h next t o the i n j u r e d p o r t i o n o f the tu b e r i n f e c t e d with C e r a t o s t o m e l l a f i m b r i a t a . may a i d i n r e s i s t i n g the i n f e c t i o n . Furthermore, they found the growth of C. f i m b r i a t a to be i n h i b i t e d by both o f these coumarins i n very low d i l u t i o n s . S i n c e Coumarin per se i s normally harmful t o p l a n t s , the f o r m a t i o n o f a g l u c o s i d e may be a mechanism o f d e t o x i f i c a t i o n (47, 57, 63). However, i t i s d i f f i c u l t to conceive that coumarin, formed so abund-a n t l y i n p l a n t s , i s merely a t o x i c waste product, yet many ' p h e n o l i c s a p p l i e d a r t i f i c i a l l y t o p l a n t s are very o f t e n r a p i d l y g l y c o s y l a t e d (62.) S o l u b i l i z a t i o n f o r t r a n s l o c a t i o n of coumarins may be an important aspect o f g l y c o s i d e formation 13. (62) and phenylpropanoids may e x i s t i n the f r e e form o n l y when t a k i n g part i n some metabolic p r o c e s s , such as the f o r m a t i o n o f f l a v o n o i d s o r l i g n i n s . ( i i i ) Role i n f l a v o n o i d s y n t h e s i s . The d e s i g n a t i o n o f f l a v o n o i d compounds by "C5-.C3-C5" b r i n g s out the c l o s e r e l a t i o n s h i p t h a t e x i s t s between them and the cinnamic a c i d s and n e a r l y r e l a t e d coumarins which are designated by "C^-Cy1. A number o f r a d i o — a c t i v e CV-C3 compounds such as p h e n y l a l a n i n e , cinnamic a c i d and f e r u l i c a c i d , have been shown to be i n c o r p o r a t e d i n t o the G^{B )-C-j p o r t i o n of f l a v o n o i d compounds ( 2 4 , 37, 66) e x a m p l i f i e d by the f a c t t h a t c a f f e i c a c i d y i e l d s q u e r c e t i n i n buckwheat (87.) Civ) Role i n l i g n i n s y n t h e s i s Although the P l a n t can u t i l i z e a number of v a r i o u s l y s u b s t i t u t e d C^-C^ u n i t s f o r s y n t h e s i s of the d i f f e r e n t b u i l d -i n g u n i t s f o r l i g n i n , the h i g h e s t e f f i c i e n c y i s shown wit h those compounds which have the 3ame s u b s t i t u t i o n p a t t e r n s as the u n i t s themselveso A s e r i e s o f d i f f e r e n t l y s u b s t i t u t e d p4hydroxlated cinnamic a c i d s are extremely good p r e c u r s o r s , and p l a y a very important r o l e i n l i g n i f i c a t i o n ( 1 3 , 1 4 , 1 7 , 9 5 ) . L i t t l e i s known about the succeeding s t e p s , although' i t i s assumed t h a t the c a r b o x y l i c a c i d i s reduced to the corresponding a l c o h o l p r i o r t o p o l y m e r i z a t i o n (18). 14 (v) Fate of coumarin i n the plant. In order to i d e n t i f y metabolic products formed , x . 14 from coumarin, Kosuge and Conn (53)}administered coumarin 3 C to excised shoots from matured plants of Melilotus alba, and found that only very small quantities of the isotype .collected i n the plant t i s s u e . Instead the coumarin was rapidl y converted to m e l i l o t y l glucoside, m e l i l o t i c acid and at lea s t two other unidentified compounds0 Under the same conditions radioactive o-coumaric acid was converted primarily to o-coumaryl glucoside and i n small amounts to coumarin, m e l i l o t i c acid, m e l i l o t y l glucoside and at least two other unidentified coumpounds. Brown (19) fed coumarin 2 C ^ to sweet vernal grass and recovered extremely small amounts of the i n coumarin and in the aglycone of the c i s glycoside 0 (vi) Intact plant responses to coumarin. When coumarins are applied a r t i f i c i a l l y to plants they have many i n t e r e s t i n g v i s i b l e e f f e c t s . Coumarin per se favors c e l l enlargement i n some plants at low concentrations, but has an i n h i b i t o r y e f f e c t on growth and seed germination at high concentrations. This f i e l d has been reviewed i n greater d e t a i l (9). Other than an i n d i c a t i o n that coumarin may act as an "anti-auxin" and counteract the usual growth, enhancing e f f e c t of auxins i n the plant (89), these studies 1 5 . throw very l i t t l e l i g h t on the metabolic function of coumarin at a chemical l e v e l . D. Biosynthesis of coumarins. Despite the fact that the widespread d i s t r i b u t i o n of coumarins i n higher plants has attracted attention f o r many years, information concerning the o r i g i n of these com-pounds i s very recent, but su r p r i s i n g l y , very much progress has already been made. 0f-;-E a r l i e r theories have been c r i t i c a l l y reviewed by Reppel ( 6 5 ) . Among the e a r l i e r schemes was one suggested by Haworth (43) i n 1942 f o r the formation of 7 r h y d r o x y c o u m a r i n s through the. oxidation of 4-hydroxycinnamic aci d . I t i s , i n t e r e s t i n g to note that a l l the members of the coumarin family synthesized by plants, except coumarin i t s e l f , have a' phenolic oxygen at position 7 , that i s para to the side chain (28, 44). Since the families i n which coumarins are predominant are also r i c h i n the les s complex C5-C3 structures, suggesting close biogenetic r e l a t i o n s h i p between the two classes of compounds (32), p-coumaric acid should play an important role in these syntheses. Haworth 1s i n t e r e s t i n g suggestion has been supported by Birch ( 7 ) and i t was subsequently more generally assumed that coumarins are derived from phenyl-proparioid acids of the cinnamic acid type ( 6 , 30). Brown ( 1 6 ) , using radio-16 o active isotopes, has shown that p-coumaric acid i s over 70 times les s e f f i c i e n t than cinnamic acid as a precursor of coumarin i n Hierochloa odorata. and t h u 3 p-coumaric acid i s not s i g n i f i c a n t -l y involved i n the biosynthesis of coumarin i t s e l f . I t appears that i n higher plants, cinnamic acid i s a common precursor of a l l coumarins, and that ortho or para hydroxylation of t h i s compound leads subsequently to the formation of coumarin and the 7-hydroxycoumarins respectively. Different enzyme systems may be required f o r the formation of the lactone r i n g and the 7-hydroxycoumarins. The mechanism by which the lactone r i n g of coumarin i s formed i s s t i l l not f u l l y understood, and theories have been published which do not involve orthohydroxylation of a precursor (59). However the formation o-coumaric glucoside before the formation of coumarin supports the b e l i e f that ortho-hydroxylation i s an es s e n t i a l feature of coumarin biosynthesis. In t h i s connection i t i s of in t e r e s t that Buhler and Mason (20) have shown that i n the presence of dihydroxyfumarate, peroxidase can hydroxylate cinnamic acid i n both the ortho and para p o s i t i o n s . Recently there has been considerable i n t e r e s t i n the occurrence and function of the hydroxylated cinnamic acids i n plants (2, 3, 25, 29, 31, 49, 56) since they are involved not only i n coumarin, but. also i n flavonoid synthesis. These compounds f a l l into the phenolic category and two biosynthetic routes have been established as those usually responsible f o r 17. the f o r m a t i o n of aromatic r i n g s which may be ragarded as p h e n o l i c , e n o l i c or oxygen h e t e r o c y c l i c . These two r o u t e s i n v o l v e : a) A poly-J5-keto a c i d i n t e r m e d i a t e produced by h e a d - t o - r a i l condensation of a c e t a t e u n i t s (26, 7> 3, 28). A c e t a t e has been shown to be a very e f f e c t i v e source of the u n i t comprising r i n g A i n f l a v o n i d s (38, 48, 77, 91). b) Formation o f C5-C3 i n t e r m e d i a t e s through the s h i k i m i c - p h r e p h e n i c a c i d pathway (26, 27, 51). The d e t a i l e d study o f the b i o s y n t h e s i s of the coumarins almost c e r t a i n l y f o l l o w s the s h i k i m i c - p r e p h e n i c a c i d pathway, and not by the h e a d - t o - t a i l condensation of o f a c e t a t e u n i t s . The f o l l o w i n g i s the most accepted scheme f o r the b i o s y n t h e s i s o f coumarins: 18, Carbon d i o x i d e COOH 3hikimic a c i d S i n a p i c T F e r u l i c HOOc C M ^ O - C O O H T v J I O a f f e i c 7 hydroxy coumarin t OH coumarin phrephenic a c i d C H t - £ < ? - C o o H OH quinone i n t e r m e d i a t e I C H = C H - C 0 0 H — C H - C O O H ©H o-hydroxy cinnamic OH p-hydroxy cinnamic a c i d p h e nylpyruvic a c i d C H x - C H n H j L - ^ o o H C H = C H - C O O H L - p h e n y l a l a n i n e cinnamic a c i d . 19 -An i n t e r e s t i n g f e a t u r e o f the b i o s y n t h e s i s o f coumarins i s the known p a r t i c i p a t i o n o f g l y c o s i d e s i n 14 the p r o c e s s . When carbon-dioxide-C was a d m i n i s t e r e d to H i e r o c h l o a odorata, Brown (15) found t h a t the t o t a l C"^ i n o-coumaric a c i d , r e c o v e r e d a f t e r the g l u c o s i d e was h y d r o l y z e d , reached a maximum i n about. 4 days. The peak i n the t o t a l C"^ o f coumarin was not reached u n t i l 8 to 17 days a f t e r a c t i v a t i o n . Both showed a d e c l i n e a f t e r r e a c h i n g the peak, i n d i c a t i n g t h a t o-coumaryl g l u c o s i d e , the t r a n s form, and coumarin are both metabolic i n t e r m e d i a t e s r a t h e r than end products, and t h a t the glucoside i s formed f i r s t . The s p e c i f i c a c t i v i t y o f coumarin l i b e r a t e d by emulsin h y d r o l y s i s from the g l u c o s i d e was c o n s i s t e n t l y lower than t h a t o f " f r e e " coumarin from coumarinic a c i d g l u c o s i d e . In a d d i t i o n Brown (19) i n d i c a t e d t h a t there i s c u r r e n t l y no t r a c e r evidence to support the view t h a t coumarinic a c i d g l u c o s i d e i s h y d r o l y z e d t o coumarin i n v i v o and suggests t h a t coumarin i s an i n t e r m e d i a t e i n the f o r m a t i o n of coumarinic a c i d g l u c o s i d e from o-coumaryl g l u c o s i d e or t h a t coumarin and coumarinic a c i d g l u c o s i d e are formed independently from a common p r e c u r s o r , the separate p o o l i d e a . Kosuge and Conn (54) i n s i m i l a r s t u d i e s u s i n g M e l i l o t u s a l b a found that when r a d i o a c t i v e o-coumaryl g l u c o s i d e was f e d to p l a n t s , i t was converted to coumarin. They suggest t h a t .through i s o m e r i z a t i o n , the c i s form acts as an i n t e r m e d i a t e i n the c o n v e r s i o n . However, the t u r n o v e r of the coumaric a c i d 2 0 . g l u c o s i d e s t o coumarin i s very slow, and the same a p p l i e s to m e l i l o t i c a c i d g l u c o s i d e , which may h y d r o l y z e t o m e l i l o t i c a c i d o n l y s l i g h t l y more r e a d i l y . The a c t i v i t y o f ( i - g l u c o s i d a s e i n the b i o s y n t h e s i s o f coumarin i s a l s o o f some s i g n i f i c a n c e . U n l i k e emulsin, i t shows s p e c i f i c i t y i n t h a t i t h y d r o l y z e s the c i s p r e f e r e n -t i a l l y t o the t r a n s isomer. Schaefer et a l . ( 2 6 ) suggest t h a t the f o r m a t i o n o f - g l u c o s i d a s e i n sweet c l o v e r i s g e n e t i c a l l y c o n t r o l l e d . I t i s o f i n t e r e s t t h a t "Cumino", a low coumarin v a r i e t y a p p a r e n t l y d e f i c i e n t i n o-coumaric a c i d g l u c o s i d e s , has ve r y high / 3 - g l u c o s i d a s e a c t i v i t y ( 5 4 . ) 2 1 111 INVESTIGATIONS AND RESULTS A A n a l y s i s o f seed An attempt was made to d e t e c t the e x i s t e n c e o f a compound which may a c t as a s u b s t i t u t e f o r coumarin i n coumarin-f r e e sweet c l o v e r v a r i e t i e s , by a comparative study o f e x t r a c t s . A b a s i s f o r the attempt l a y i n the assumption t h a t the coumarin s u b s t i t u t e was c l o s e l y r e l a t e d t o coumarin and t h a t , l i k e coumarin, i t e x i s t e d i n the coumarin-free v a r i e t i e s i n the g l y c o s i d i c water s o l u b l e s t a t e . Aqueous e x t r a c t s , t h e r e f o r e , o f the seed o f non-b i t t e r M. d e n t a t a f n o n - b i t t e r "Curaino" and "Denta" sweet c l o v e r v a r i t i e s and common, white blossom M. a l b a were chromatogrammed comparatively i n order t o e s t a b l i s h any major d i f f e r e n c e s i n the occurrence o f s p e c i f i c chemical c o n s t i t u e n t s , ( i ) M a t e r i a l s The s p e c i e s and v a r i e t i e s o f sweet c l o s e r used were as f o l l o w s : a) n o n — b i t t e r , coumarin-free M e l i l o t u s d e n t a t a from A.E. H a l l o w e l l , U.S.D.A., B e l t s v i l l e , Md. b) n o n - b i t t e r , coumarin-free white sweet c l o v e r , v a r i e t y "Cumino" from R. G r e e n s h i e l d s , and B.P.Golpen. Forage L a b o r a t o r y , Canada Dept. o f A g r i c u l t u r e , Saskatoon, Saskatchewan. c) n o n - b i t t e r , coumarin-free, white sweet c l o v e r , v a r i e t y "Denta" from W.K.Smith, Forage and Range Research, A.R.S., C.R., U.S.D.A., U n i v e r s i t y o f Wisconsin, Madison, W i s c o n s i n . d) Canadian common, b i t t e r , white sweet c l o v e r from 22 B u c k e r f i e l d ' s L t d . , Vancouver, B.C. e) Medicago s a t i v a a l f a l f a , v a r i e t y "Rhizoma" from the U n i v e r s i t y of B r i t i s h Columbia, Vancouver, B.C. ( i i ) Methods. The e x t r a c t i o n procedure f o l l o w e d somewhat t h a t of Charaux (23) who f i r s t e x t r a c t e d g l u c o s i d e s o f "coumarin" from sweet c l o v e r v i z : a) 30 g. seed was ground i n a W i l e y - M i l l u s i n g a 40 mesh s c r e e n . b) The ground seed was immersed i n 250 ml. b o i l i n g water, and allowed t o b o i l f o r 5 minutes. c) The hot mixture was then f i l t e r e d through cheese-cloth„ d^ The aqueous f i l t r a t e was e x t r a c t e d i n a s e p a r a t o r y f u n n e l with an equal volume of d i e t h y l - e t h e r f o r f u r t h e r p u r i f i -c a t i o n , and, a l s o , t o remove any f r e e coumarin. e) The aqueous l a y e r was f i l t e r e d through Whatman § 1 f i l t e r Paper. f ) The e x t r a c t was a p p l i e d f o r chromatography t o Whatman #3 paper, and the s o l v e n t s used were: chloroform; methanol; water (5:5:1) of Jenson (50\ 10% methanol, and w a t e r 0 g) The chrornatograms were scanned under u.v. l i g h t (both 3660°A and 2537°A max.) The Charaux ( i b i d ) e x t r a c t i o n procedure i n c l u d e d f u r t h e r p u r i f i c a t i o n steps u s i n g n e u t r a l l e a d acetate p r e c i p i t a t i o n , f o l l o w e d by b a s i c l e a d a c e t a t e treatment o f the f i l t r a t e , 23. resusperition o f the formed p r e c i p i t a t e i n water and b u b b l i n g h^S gas through t o form an i n s o l u b l e b l a c k l e a d s u l f i d e . The g l y c o s i d e s remained i n s o l u t i o n a f t e r f i l t r a t i o n o r centrifuga-? t i o r i . S i n ce these p u r i f i c a t i o n steps e l i m i n a t e d compounds which were not normally found i n the r e g i o n o f the chromato-graras occupied by the compounds which were of i n t e r e s t i n t h i s study - and were t h e r e f o r e o f no i n t e r f e r e n c e - the s i m p l i f i e d procedure d e s c r i b e d on the p r e v i o u s page was f a v o r e d . A few c o l o u r t e s t s were used to a s s i s t i n the i d e n t i f i c a t i o n o f the spots on the chromatograms. a) Whitest - the chromatogram was exposed t o NH-j vapours and was immediately observed i n v i s i b l e l i g h t and under u l t r a - v i o l e t l i g h t . b) NaOH t e s t - a drop o f IN NaOH was a p p l i e d t o the spot o f i n t e r e s t o r e l s e the sheet was sprayed with the NaOH s o l u t i o n . c) HCI t e s t - the sheet was sprayed with IN HCI, d) D i a z o t i s e d - p - n i t r o a n i l i n e t e s t - the diazonium s o l u t i o n was prepared as o u t l i n e d by Roberts and Lfnk ( 6 9 ) . 10% Na^CO^ i s f i r s t sprayed onto the f i l t e r paper i n order t o open any l a c t o n e r i n g p r e s e n t . I t i s f o l l o w e d by the d i a -zonuim s o l u t i o n . The f o r m a t i o n o f c o l o u r s i s noted. ( i i i ) R e s u l t s Of the thr e e g l y c o s i d i c s o l v e n t s used f o r chromatography 24. chloroform: methanol: water (5:5:1) proved to be most satis f a c t o r y and henceforth, unless otherwise stated, i t s use may be presumed. A l l three v a r i t i e s of sweet clover as well as M. dentata yielded a very conspicuous blue non-fluorescent u.v. absorbing spot or band with an Rf.84: t h i s spot or band i s not found i n tlie corresponding region of a l f a l f a . This rather heavy concentration attracted attention and further investiga-t i o n was pursued. There was good reason, from the extraction procedure used, to believe that the blue absorbing region included a glycoside or glycosides. Accordingly bands and spots from a l l sources were separately eluted with 10% Methanol, the volume reduced to ca. 10 ml, and boiled for 5 min. i n 3N HCI. The materials were then extracted with diethyl-ether and portions of the ether solutions were chromatogrammedo Also autolysed seed f l o u r was extracted with ethanol. The extracts should contain both glycosides and many aglycones released by the enzymatic hydrolysis. Portions of alcoholic extracts from the separate sources were chromatogrammed. A summary of the r e s u l t s i s presented i n F i g . 1 as a serni-diagramatic chromatogram. 25 Chloroform 5 Methanol 5t Water 1 O 0 O Q O 1.0 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 F i g . 1. Semi-diagramatic chromatogram showing conspicuous non-fluorescent, u.v. absorbing spots, of s i m i l a r nature i n a l f a l f a and b i t t e r and non-bitter sweet clovers obtained from seed extracts, solvent: chloroform, methanol, water : (5:5:1) 1. ex aqueous, a l f a l f a seed 2, 3, 4, 5» ex aqueous common sweet'clover, "Curaino", "Denta", and M0 dentata respectively. 6. Coumarin 7, 8, 9, 10 acid hydrolysis, ether extraction 11, 12, 13, 14 a u t o l y s i s , ethyl alcohol extraction. Colour reactions, supra vide. f o r the chromatospots 26 depicted in the diagram of Fig. 1 were obtained and are summarized in Table 1. The observations are consistent with the view that water extraction yields glycosides which on hydrolysis or autolysis yield coumarin. Apparently seed of both bitter M. alba and non-bitter "Cumino", "Denta" and M c  dentata contain glycosides of coumaric acid. Table 1. Colour reactions of chromatospots numbered as in F i g . l . Spot Spray reagent p-nitroaniline NaOH HCI 1. - - - -2. - - - white f l 3. - - . - n 4. _ - - tt 5 . - - tt 6 . red f l bright y . f l — UoV. 7 . red . f l . n -a. red f l . -9 . red f l . -10. red f l . — 11. red f l . 12. red f l . -13. red f l . -14. red ' f l . -uv - ultra violet y . f l yelloxv fluorescence w.fl white " 27 B A n a l y s i s o f sugar I t was noted t h a t i n the l i t e r a t u r e reviewed, r e f e r e n c e was made c o n s i s t e n t l y t o " f r e e " and "bound" coumarin. I t was t a c i t l y accepted t h a t bound coumarin e x i s t s i n the g l y c o s i d i c form and no mention was made of the sugar o r sugars a c t u a l l y i n v o l v e d . In very r e c e n t l i t e r a t u r e , authors tend to be somewhat more s p e c i f i c and the - g l u c o s i d e o f o-coumaric a c i d has been s y n t h e s i z e d . An attempt was made to determine whether or not the blue absorvent band, from the chromatogrammed seed e x t r a c t s , i s a g l y c o s i d e ; i f t h i s proved t o be f a c t , as might be supposed, i t became a matter o f i n t e r e s t to know the i d e n t i t y of the a s s o c i a t e d sugar or sugars. ( i ) M a t e r i a l s Common white sweet c l o v e r seed and green t i s s u e was used. ( i i ) Method The seed was e x t r a c t e d i n the same manner as p r e v i o u s l y stated„ The green t i s s u e was e x t r a c t e d as f o l l o w s : a) F r e s h p l a n t m a t e r i a l was p l a c e d i n hot 95% e t h a n o l and allowed t o b o i l f o r 5 min. b) The contents were then t r a n s f e r r e d t o a Waring B l e n d o r where the t i s s u e was ground i n e t h a n o l . 28. c) The extract was f i l t e r e d , and the f i l t r a t e transferred to a f l a s h evaporator where the alcohol was evaporated. d) The remaining residue was then taken up i n water and any insoluble matter, which included most of the chlorophyll, was f i l t e r e d o f f . I f any chlorophyll remained i n the solution, i t precipitated out a f t e r the f i l t r a t e stood over-night i n a freezer. e) Both the seed and the green tiss u e extracts were applied to Whatman #3 f i l t e r paper and developed using the descending method of chromatography with chloroform: methanol: water (5:5:1) solvent. A f t e r elution of the blue band, hydrolysis with 3N HCI and scrubbing with an equal volume of ethyl ether, the aqueous layer was concentrated i n a f l a s h evaporator and applied to Whatman § 3 f i l t e r paper. Alongside the unknown, an aqueous solution of glucose, mannose, arabinose, xylose and galactose was spotted. The chromatogram was developed by l e t t i n g ethyl acetate: pridine: water (8:2:1) solvent move for 48 hours down the sheet which has had i t s lower edge cut with pinking scissors to f a c i l i t a t e solvent run-off„ The sugar spots were detected on the paper by f i r s t spraying 3% p-anisidine hydrochloride i n moist butanol (46), and then by placing the sheet f o r ca. 5 min. i n an oven at 100°C f o r colour development• 29. ( i i i ) R e s u l t s The same b l u e , coumarin y i e l d i n g compound present i n the seed, was found i n the green t i s s u e o f b i t t e r common sweet c l o v e r . F o l l o w i n g h y d r o l y s i s , the aqueous l a y e r y i e l d e d a spot, which, on s p r a y i n g with p - a n i s i d i n e h y d r o c h l o r i d e and subsequent development at 100°C turned a g r e e n i s h brown c o l o u r i n the same way as g l u c o s e 0 T h i s c o l o u r r e a c t i o n i s t y p i c a l o f a l l aldohexoses. The spot a l s o moved e x a c t l y the same d i s t a n c e as glucose and has thus an RQ value o f 1. The blue band, t h e r e f o r e , i s the /3 - g l u c o s i d e o f o-coumaric a c i d , and i s present i n the green t i s s u e and seed bf b i t t e r M. a l b a and i n the seed but not i n the green t i s s u e of the n o n - b i t t e r v a r i e t i e s "Cumino" and "Denta" and i n M. d e n t a t a . C. A comparative study o f green s h o o t s . S i n c e the seed o f both the b i t t e r and n o n - b i t t e r sweet c l o v e r p l a n t s c o n t a i n the J2>-glucoside of o-coumaric a c i d , i t i s not s u i t e d f o r a comparative study; a t t e n t i o n was, t h e r e f o r e , t u r n e d to the green shoots of the p l a n t s i n the search of a compound s e r v i n g as a s u b s t i t u t e o f coumarin i n non-b i t t e r p l a n t s . T h i s study was c a r r i e d out on a comparative b a s i s , with a somewhat s i m i l a r approach t o t h a t o f A l s t o n et a l (1) , who, by chromatographic analyses o f l e a f e x t r a c t s o f 3 0 . h y b r i d s i n the genus B a p t i s i a demonstrated a p a t t e r o f i n h e r i t a n c e i n which components o f parent s p e c i e s were p r e s e n t . Fo r a s t r i c t comparison of the chemical c o n s t i t u e n t s , the c o l l e c t i n g , e x t r a c t i n g and chromatography o f the b i t t e r and non-b i t t e r p l a n t s , was normally performed s i m u l t a n e o u s l y under i d e n t i c a l c o n d i t i o n s . ( i ) Soxhlet e x t r a c t i o n . B i t t e r M. a l b a , n o n - b i t t e r v a r i e t y "Cumino" and M. dentata shoots were c o l l e c t e d , allowed t o w i l t f o r enzymatic h y d r o l y s i s o f the g l y c o s i d e s , a i r d r i e d at 40°C and ground i n a Wiley Mill.;. The ground m a t e r i a l was then e x t r a c t e d f o r 12 hours i n a s o x h l e t e x t r a c o r u s i n g c h l o r o f o r m , acetone and methanol as s o l v e n t s . Since so many p l a n t c o n s t i t u e n t s are s o l u b l e i n these s o l v e n t s , chromatographic s e p a r a t i o n o f the e x t r a c t s was h i g h l y u n s a t i s f a c t o r y and no d i f f e r e n c e c o u l d be d e t e c t e d o ( i i ) Steam d i s t i l l a t i o n Since coumarin per se i s h i g h l y v o l a t i l e , steam d i s t i l l a t i o n was used i n an attempt t o i s o l a t e a r e l a t e d compound with 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 , which might e x i s t i n coumarin-free v a r i e t i e s . Aqueous e x t r a c t s o f common white blossom sweet c l o v e r and "Cumino" v a r i e t y were ob t a i n e d as d e s c r i b e d p r e v i o u s l y , f o r the i s o l a t i o n o f o-coumaric g l u c o s i d e from the green t i s s u e . The aqueous e x t r a c t s were hydrolyzed by adding it) ml. of cone. HCI t o 100 ml. b o i l i n g e x t r a c t , and r e f l u x i n g f o r about 5 minutes. I t was noted t h a t on a d d i t i o n of the a | i d a f l a k y brown p r e c i p i t a t e was formed. The hydro l y z e d e x t r a c t was s t e a m d i s t i l l e d u n t i l about 100 ml. o f the d i s t i l l a t e was c o l l e c t e d ' . In both" cases some i n s o l u b l e m a terialjwas d i s t i l l e d over with the water and a very s t r o n g d i f f e r e n c e i n odor between the f i t t e r and n o n - b i t t e r steam d i s t i l l a t j e s was noted. However, an attempt t o i s o l a t e any o f the v o l a t i l e c o n s t i t u e n t s by e t h y l e t h e r e x t r a c t i o n , f o l l o w e d by chromatography proved u n s u c c e s s f u l , ( i i i ) L i q u i d - l i q u i d e x t r a c t i o n . Hydrolyzed and unhydrolyzed aqueous e x t r a c t s o f b i t t e r and n o n - b i t t e r p l a n t shoots were e x t r a c t e d f o r 12 hours i n a continuous l i q u i d - l i q u i d e x t r a c t o r , u s i n g e t h e r , benzene and petroleum e t h e r as s o l v e n t s . The e x t r a c t s were a p p l i e d t o .'Whatman #3 f i l t e r paper and the s o l v e n t used f o r s e p a r a t i o n was descending chlo r o f o r m : methanol: water (5:5:1) Some d i f f e r e n c e s , i n the appearance o f bands, could be d e t e c t e d ; however, they were mostly i n c o n s i s t e n t and r a t h e r c o n f u s i n g . E t h e r removed, from the h y d r o l y z e d n o n - b i t t e r ;' p l a n t e x t r a c t s , a d u l l f l u o r e s c e n t compound, which d i d not appear i n the cor r e s p o n d i n g r e g i o n o f a chromatogram of', s i m i l a r l y t r e a t e d b i t t e r p l a n t e x t r a c t s . Instead l a r g e q u a n t i t i e s o f coumarin were recovered from the " b i t t e r " e x t r a c t s , 32. The d u l l f l u o r e s c e n t compound was l a t e r found t o be i d e n t i c a l w ith one i s o l a t e d and i n v e s t i g a t e d i n a manner d e s c r i b e d i n the f o l l o w i n g s e c t i o n . ( i v ) A comparison o f aqueous e x t r a c t s . (a) M a t e r i a l s F r e s h green f i e l d grown shoots o f common b i t t e r M. a l b a , n o n - b i t t e r s t r a i n s "Cumino" and "Denta", and n o n - b i t t e r s p e c i e s M. d e n t a t a were used. (b) Methods I s o l a t i o n procedures. The e x t r a c t i o n procedure f o l l o w e d was e x a c t l y the same as d e s c r i b e d p r e v i o u s l y , with e x c e p t i o n a l care b e i n g g i v e n t o minimize h y d r o l y s i s of the g l y c o s i d e s , by p l a c i n g the p l a n t s i n b o i l i n g a l c o h o l as soon as p o s s i b l e a f t e r they had been c u t . The two chromatography s o l v e n t s used most e x t e n s i v e l y f o r i s o l a t i o n / w e r e c h l o r o f o r m : methanol: water (5:5:1) and 1% a c e t i c a c i d . Bands of i n t e r e s t were cut out and e l u t e d with 10% e t h a n o l . A n a l y t i c a l procedures A number o f g l y c o s i d i c and p h e n o l i c s o l v e n t s were used f o r chromatography and f o r Rf value measurements the chromatograms were run on Whatman #1 f i l t e r paper i n a s o l v e n t - s a t u r a t e d atmosphere at 20°C i 1°C. 33 Many colour tests are available to help i n the i d e n t i f i c a t i o n of unknown heterocyclic and phenolic compounds. A number of tests which proved useful i n t h i s investigation are as follows. a) FeCl-j/K^FeCCN)^ - 1 gm. of each compound i s dissolved i n 100 ml. H 20. I f phenolic hydroxyl group i s present, a blue colour appears. b) FeCl^ - Production of colour with 1% aqueous FeCl-} solution, i s general with a l l classes of poly-hydroxy flavonoid compounds (30). c) Diazo reactions; A few diazonium s a l t s were used, which under proper conditions . couple with aromatic compounds contain-ing a powerfully electron-releasing group; e.g.-OH. Substitution usually occurs para to the a c t i v a t i n g group. Azo compounds formed by the coupling reaction are usually strongly coloured, (i.) Diazotized benzidine hydrochloride. Solution (1):5 gms. benzidine added to 14 ml. 37$ HCI and the suspension i s dissolved i n 980 ml. water. Solution (2): 10% NaN02 - fre s h l y prepared. Two volumes of solution (2) were mixed with 3 volumes of solution (1) and sprayed a f t e r 10 minutes. 34. ( i i ) D i a z o t i z e d p - n i t r o a n i l i n e - T h i s t e s t was con-ducted as o u t l i n e d i n the a n a l y s i s o f the seed, ( i i i ) D i a z o t i z e d s u l f a n i l i c a c i d - G . l g d i a z o t i z e d s u l f a n i l i c a c i d was d i s s o l v e d i n 20 ml. 10$ N a 2C0 3. Mg-HCl t e s t - T h i s t e s t d e v i s e d by Shinoda (78) i s s p e c i f i c f o r f l a v o n o i d compounds. To a s m a l l amount o f the t e s t m a t e r i a l i n aqueous s o l u t i o n , a s m a l l p i e c e o f Mg. r i b b o n was added and then a few drops of cone. HCI. The f o r m a t i o n of c o l o u r was noted f o r a p e r i o d o f 15 min. T h i s t e s t was a l s o c a r r i e d out by s p r a y i n g the spot on chromatography paper f i r s t w ith an e t h a n o l i c suspension o f f i n e Mg. powder, immediately f o l l o w e d by cone. HCI. NHj fumes - A f t e r the spot was exposed t o the fumes i t was observed under v i s i b l e and u.v. l i g h t . T h i s i s not a very s p e c i f i c t e s t but i s an i n d i c a t i o n o f the degree of methoxylation o f f l a v o n o i d and p h e n o l i c compounds. Cone. HNO^ - The f i l t e r paper was sprayed with the concentrated a c i d . Some p h e n o l i c compounds d i s s o l v e i n c o n c e n t r a t e d n i t r i c a c i d t o g i v e a b r i l l i a n t b l u e c o l o u r . HCI - Spots or bands on the chromatography paper were exposed t o HCI fumes. 1% B o r i c a c i d - 1% o x a l i c a c i d . F l a v o n o l s with a f r e e 5 hydroxy group r e a c t with b o r i c a c i d i n the presence 3 5 . of organic mineral acids to give bright yellow or green-ish yellow fluorescence. Ammoniacal AgNO-j - 14 gms. AgNO^ were dissolved in 100 ml. H20 and 6N NH^ OH was added un t i l the silver oxide formed just dissolved. With this reagent, o-dihydroxy compounds give a colour reaction. Vanillin-p-toluene sulfonic acid - 2 gms. v a n i l l i n and 1 g. p-toluene sulfonic acid was dissolved in 100 ml. absolute ethyl alcohol. After spraying vthe chromato-gram was heated in an oven at 80 - 100°C. for five to ten minutes. A positive reaction indicates the presence of a phloroglucihol or catechol type of compound. Sodium molyhydate - 1 g. was dissolved in 10,0 ml. water. This reagent reacts with o-dihydroxy compounds. KMnO^  - 1% aqueous KMnO^ , when sprayed on paper, leaves a red film except where a redox reaction has taken place, and the area of the spot remains white or turns slightly yellow. Neutral PbAc - 5% aqueous solution of neutral lead acetate. Basic PbAc - 5 $ aqueous solution of basic lead acetate Alcoholic A1C13 - 5 g. of A I C I 3 w a s d i s s o l v e d in 100 ml. 95% ethanol. After spraying, the paper was allowed to dry and was then exposed to ammonia fumes. 36 p) N e u t r a l AgNO^ - The a c e t o n e - s i l v e r n i t r a t e reagent was prepared by adding 50 ml. of s a t u r a t e d aqueous AgNO-j to 1 l i t e r o f acetone and then adding j u s t enough water to d i s s o l v e the p r e c i p i t a t e . A b s o r p t i o n s p e c t r a of compounds from bands o f p o s s i b l e i n t e r e s t i n the v i s i b i l e and u l t r a v i o l e t l i g h t range were obtained u s i n g 3 ml. s i l i c a c u v e t t e s i n a Beckman DK-2 spectrophotometer; the compounds were d i s s o l v e d i n 95$ ethanol o r e t h y l a c e t a t e . F o r i n f r a - r e d a b s o r p t i o n s p e c t r a , 0 .5 mg. o f p r e -c i p i t a t e d m a t e r i a l was mixed with 0 .6 g. anhydrous potas-sium bromide and put i n t o a p e l l e t i n g u n i t . The p e l l e t was prepared u s i n g a p r e s s u r e o f 20,000 l b s . per square i n c h , and i n s e r t e d i n a Beckman JR-3 spectrophotometer and a r e c o r d i n g o b t a i n e d , (c) R e s u l t s . F i r s t attempt. When the aqueous e x t r a c t s o f the b i t t e r and non-b i t t e r p l a n t s were a p p l i e d to Whatman #3 f i l t e r paper and a s e r i e s o f d i f f e r e n t g l y c o s i d i c s o l v e n t s were used f o r s e p a r a t i o n by the descending method, a white f l u o r e s c e n t band appeared c o n s i s t e n t l y on chromatograms o f the non-b i t t e r p l a n t s i n e x a c t l y the same r e g i o n as t h a t occupied by o — c o u m a r i c a c i d g l u c o s i d e on the corresponding chromatograms of the b i t t e r p l a n t s 0 No o-coumaric a c i d 37 glucoside was detected i n the coumarin-free plants, and the white fluorescent compound appeared to be present i n the M. alba v a r i t i e s "Cumino" and "Denta" as well as i n M. dentata. While the /3-glucoside of o-couraaric acid was found to be abundant i n the b i t t e r common white blossom sweet clover, the fluorescent compound appeared to be present i n f a r smaller quantities i n the coumarin-free plants. The solvents with the corresponding Rf values are represented i n Table 11o TABLE 11 - Rf values given by a number of solvents of a f l u o r -escent compound is o l a t e d from aqueous extracts of non-bitter plants. Solvent Rf chloroform: methanol: water 5 5 1 0.83 n-butanol: acetic acid: water 4 1 5 0.63 n—butanol: ethanol: water 40 11 19 0.55 ethyl acetate: acetic acid: water 9 2 2 0.62 15$ acetic acid 0.90 38 The f l u o r e s c e n t band, which had been separated by descending c h l o r o f o r m : methanol: water ( 5 : 5 : 1 ) was e l u t e d from Whatman #3 f i l t e r paper w i t h 10% e t h a n o l . P a r t o f the e l u a t e was h y d r o l y z e d by b o i l i n g under r e f l u x f o r t e n minutes with 3N HCI. On h y d r o l y s i s a s m a l l p r e c i p i t a t e was formed and a very s t r o n g , c h a r a c t e r i s t i c a l l y p l e a s a n t odor was e m i t t e d . When NaOH was added to a p o r t i o n of the h y d r o l y z e d s o l u t i o n , i n o r d e r t o n e u t r a l i s e the a c i d , the c o l o u r changed t o y e l l o w e x a c t l y at the n e u t r a l p o i n t and the odor could no l o n g e r be d e t e c t e d . The compound formed on a c i d treatment, presumably the aglycone, was removed with e t h y l e t h e r . The aqueous l a y e r was run f o r 48 hours w i t h e t h y l a c e t a t e ; p y r i d i n e ; water ( 8 : 2 : 1 ) s o l v e n t and sprayed with a n i s i d i n e h y d r o c h l o r i d e , however, no sugar c o u l d be d e t e c t e d . The e t h y l e t h e r l a y e r was a l s o s p o t t ed and the chromatogram developed u s i n g descending chloroform: methanol: water ( 5 ; 5 : 1 ) . The Rf value o f the compound, which appeared as a d u l l l i g h t f l u o r e s c e n t spot on the paper, i s 0 . 8 3 , and i s i d e n t i c a l t o the compound ex-t r a c t e d i n a continuous l i q u i d - l i q u i d e x t r a c t o r with e t h e r , from the h y d r o l y z e d aqueous e x t r a c t s of n o n - b i t t e r p l a n t s . A number o f spray reagents were used and the r e a c t i o n s are presented i n Table 1 1 1 . 39. TABLE 111 - Colo u r r e a c t i o n s produced by a compound formed on a c i d treatment o f a f l u o r e s c e n t compound i s o l a t e d from n o n - b i t t e r p l a n t s . Reagent Re a c t i o n HCI -HNO3 -KMnO^ -F e C l 3 -A l C l ^ NH^ vapour -Na 2C0 3 -NH vapour -d i a z . - p - i n t r o a n i l i n e -d i a z . b e n z i d i n e h y d r o c h l o r i d e red The volume o f the unhydrolyzed e l u a t e was c o n s i d e r -a b l y reduced i n a f l a s h evaporator,( removing p r a c t i c a l l y a l l the e t h a n o l p r e s e n t ) , and was p l a c e d i n a r e f r i g e r a t o r . A f t e r a few days a f l a k y white p r e c i p i t a t e appeared, which was separated by c e n t r i f u g a t i o n and d r i e d at 40°C. i n a 40. vacuum oven. An i n f r a - r e d a b s o r p t i o n spectrum o f the p r e c i p i t a t e d compound i n d i c a t e d t h a t no aromatic r i n g i s i n v o l v e d , and the spectrum resembled t h a t o f a f a t t y a c i d e s t e r . Since an a l i p h a t i c compound i s i n c o n s i s t e n t with what would be expected to serve as a s u b s t i t u t e f o r coumarin i n n o n - b i t t e r p l a n t s , f u r t h e r c h a r a c t e r i z a t i o n was not attempted. Second attempt The search f o r a c l o s e l y r e l a t e d compound t o coumarin, i n n o n - b i t t e r p l a n t s , was continued on a comparative b a s i s , u s i n g aqueous e x t r a c t s . A l a r g e r v a r i e t y o f chromatography s o l v e n t s were t e s t e d , and a very i n t e r e s t i n g s e p a r a t i o n was obtained,with ~1> a c e t i c a c i d , a s o l v e n t used by Brown (19). Fi g u r e 11 - Chromatographic comparison o f e x t r a c t s from b i t t e r and n o n - b i t t e r p l a n t s . a c e t i c a c i d Common "Cumino" Sweet C l o v e r . "Denta" '0o68 0.64 Rf M.Dentata A b l u e absorbent band common to both the b i t t e r and n o n - b i t t e r p l a n t s , d i f f e r e n t i a t e d i n t o a very dark 41 absorbent region with a s l i g h t l y higher Rf value i n the coumarin-free p l a n t 3 . This dark region was e s p e c i a l l y con-spicuous on chromatograms of extracts from young non-bitter shoots, where l i t t l e interference from yellow pigments was encountered, and the compound was found to be at l e a s t as abundant as o-coumaric acid glucoside i n b i t t e r sweet clover plants. The type of resolution i s best represented diagraraatically ( Fig.11) The dark compound i s highly unstable, even i n the dry form, and w i l l degrade turning yellow on the f i l t e r paper within three to four days, much sooner i n the presence of l i g h t . The darker upper region of the absorbent band was eluted with 10$ ethanol, the volume was reduced i n a f l a s h evaporator and i t was immediately stored i n a freezer. On thawing the aqueous eluate, a white p r e c i p i t a t e was formed„ The pre c i p i t a t e was found to be only s l i g h t l y soluble i n water, sparingly soluble i n ether, p r a c t i c a l l y insoluble i n 0. 5N HCI, and, turning yellow, was highly soluble i n a l k a l i . For measurement of the absorption spectrum i n the 1. R. l i g h t range, the p r e c i p i t a t e was washed a few times with 0.5N HCI, dried i n a vacuum oven at 40°C., mixed with anhydrous KBr and p e l l e t e d . For a u.v. absorption spectrum the washed compound was dissolved i n 95/» ethanol. The absorption spectra are presented i n Figures 111 and IV. 42. For chromatography, the ethanolic solution was spotted on Whatman if 1 f i l t e r paper and the compound was run i n a series of solvents. (See Table IV) The spots were then sprayed with a number of colour test reagents.(See Table V) TABLE IV - Rf values of dark absorbent compound i s o l a t e d from non-bitter p l a n t s 0 Rf values Solvent ascending descending 15% acetic acid 0.61 0.74 2 - b u t a n o l sat. water 0.64 -n-butanol: acetic acid: water 4 1 5 0.47 0.46 water - 0.60 butanol: pyridine: water 10 3 3 ' - 0.47 n-butanol: ethanol: formic acid:water 0 . 2 6 5 1 1 1 chloroform, 2 propanol: acetic acid; water 1 2 1 . 1 0 . 5 2 1% acetic acid - 0.68 43. TABLE V - C o l o u r r e a c t i o n s w i t h d i f f e r e n t spray reagent g i v e n by a dark absorbent compound i s o l a t e d from n o n - b i t t e r sweet c l o v e r p l a n t s . Reagent Reaction F e C L 3 / K 3 F e ( C M ) 6 blue 1% K M n 0 4 s l i g h t l y yellow-no red i n r e g i o n of spot 1% F e C l 3 l i g h t brown p - n i t r o a n i l i n e orange k - 1 hour a f t e r s p r a y i n g d i a z o t i z e d s u l f a n i l i c a c i d y e l l o w immediately d i a z o t i z e d b e n z i d i n e orange immediately NH 3 fumes y e l l o w immediately A g N 0 3 acetone A g N 0 3 ammoniacal y e l l o w v a n i l l i n p-toluene s u l f o n i c a c i d l i g h t y e l l o w Na-molybdate NaOH y e l l o w immediately* * On s p r a y i n g the y e l l o w spot with HCI, the c o l o u r disappeared and the spot again appeared dark bl u e absorbent under u.v. l i g h t . I.R. a b s o r p t i o n spectrum o f dark absorbent compound i s o l a t e d from n o n - b i t t e r sweet c l o v e r p l a n t s . PERCENT TP.ANSMITTANCE N O T E C H A N G E O F S C A L E A T 2000 C M 1 Figure I? - U.V. absorption spectrum of dark absorbent compound i s o l a t e d from non-bitter sweet clover plants. The increase i n density range from 0 - l t o 0.5 - 1.5 was necessary because of the high concentration of the s o l u t i o n i n t'r 3 u.v. . region• OPTICAL DENSITY 46 An attempt was made to h y d r o l y z e the dark b l u e absorbing compound with emulsin and HCI. Emulsin was added t o an aqueous s o l u t i o n o f the compound, and allowed to stand f o r a few hours at 37°C., a f t e r which i t was s t o r e d i n a r e f r i g e r a t o r f o r one week. A white f l u o r e s c e n t compound, i d e n t i f i e d as o-coumaric a c i d , was l i b e r a t e d i n sm a l l q u a n t i t i e s . On h y d r o l y s i s by b o i l i n g under r e f l u x f o r ten minutes i n 3N HCI, a brown p r e c i p i t a t e was formed. The p r e c i p i t a t e was separated by c e n t r i f u g a t i o n , d r i e d f o r an IR a b s o r p t i o n spectrum, and d i s s o l v e d i n e t h y l a c e t a t e f o r a u.v. and v i s i b l e l i g h t range a b s o r p t i o n spectrum, and f o r chromatographic a p p l i c a t i o n . The a b s o r p t i o n s p e c t r a of the brown p r e c i p i t a t e and q u e r c e t i n , f o r comparison, are presented i n F i g u r e s V, VI, VII and V l l l . F o l l o w i n g h y d r o l y s i s , no sugar could be d e t e c t e d i n the aqueous m o t h e r - l i q u o r , the dark compound, t h e r e -f o r e , does not seem to be g l y c o s i d a t e d o The brown p r e c i p i t a t e g i v e s an i n f r a - r e d and v i s i b l e l i g h t range a b s o r p t i o n spectrum very s i m i l a r t o t h a t of q u e r c e t i n , and i n a d d i t i o n somewhat resembles q u e r c e t i n i n i t s appearance and p h y s i c a l p r o p e r t i e s such as s o l u b i l i t i e s . Both q u e r c e t i n and the brown compound are s o l u b l e i n a l k a l i n e aqueous s o l u t i o n s with a s t r o n g y e l l o w c o l o u r . When a p p l i e d to chromatography paper, the brown 4 7 . compound appeared as a yellow spot, which, when run i n a series of flavonoid-solvents, gave Rf values d i s t i n c l y d i f f e r e n t to quercetin,( See Table VI), A series of spray reagents were used, and i n addition, the Shinoda colour test carried out on an aqueous solution of the compound, produced a l i g h t orange colour within ten minutes. (See Table V I I ) . TABLE VI - Rf values of yellow spot obtained on acid treatment of a dark absorbent compound isolated from non-bitter t "Cumino" plants compared to quercetin Rf values(descending.) Solvent Yellow spot Quercetin butanol: 2.1'jo acetic acid 1 1 0.94 m.cresol:Acetic acid:water 50 2 48 0.70 .28 phenol: 73 0.76 butanol; acetic acid: water 4 1 5 C 9 9 .80 water 0. 0 Acetic acid:hydrochloric acid 30 3 water 10 • 0 . 6 9 1% acetic acid 0.10 • 0 5 • 48 . TABLE VII - Colours produced by d i f f e r e n t spray reagents on a y e l l o w spot obtained on a c i d treatment of a dark absorbent compound i s o l a t e d from n o n - b i t t e r "Cumino" p l a n t s . Reagent Reaction NH^fumes y e l l o w i n t e n s i f i e d A I C I 3 g r e e n i s h y e l l o w d i a z o t i z e d b e n z i d i n e s l i g h t l y brownish r e d F e C l 3 / K 3 F e ( C N ) 6 blue F e C l 3 -p - n i t r o a n i l i n e g r e e n i s h y e l l o w d i a z o t i z e d s u l f a n i l i c a c i d y e l l o w i s h brown AgNO^ acetone -Mg-HCl very s l i g h t l y orange PbAc. b a s i c y e l l o w i n t e n s i f i e d PbAc. n e u t r a l y e l l o w i n t e n s i f i e d HNO^ -b o r i c a c i d -HCI fumes i n c r e a s e d f l u o r e s c e n t under U.V. v a n i l l i n p-toluene s u l f o n i c a c i d -I.R. absorption spectrum of yellow compound obtained on a c i d treatment of a dark absorbent compound i s o l a t e d from non-bitter "Cumino" plants PERCENT TRANSMITTANCE S PERCENT TR ANSMITT A N O N O T E C H A N G E O f S C A L E A T 2000 C M 6v F i g u r e VI - I.R. a b s o r p t i o n spectrum o f quercet m PERCENT TRANSMITTANCE PERCENT TRANSMITTANCE NOTE CHANGE OF SCALE AT 2000 CM 1 Figure VII U.V. absorption spectrum of yellow compound obtained on acid treatment of a dark absorbent compound isolated from non-bitter "Cumino" plants The increase i n density range from 0 - 1 to .5 -1.5 was necessary because of the high concentration of the solution in the u.v.region. OPTICAL DENSITY F i g u r e V I I I - A b s o r p t i o n spectrum.. i n the v i s i b l e l i g h t range o f a y e l l o w compound ob t a i n e d on a c i d treatment o f a dark absorbent compound i s o l a t e d from n o n - b i t t e r "Cumino" p l a n t s . 53 I n v e s t i g a t i o n s c a r r i e d out so f a r suggest t h a t both the u.v. absorbent compound found i n n o n - b i t t e r p l a n t s and a l s o the brown p r e c i p i t a t e formed on a c i d treatment, are f l a v o n o i d s . An attempt was aiade t o break the •acylated* l i n k a g e and s p l i t the A r i n g from the B r i n g * The B r i n g , t h e o r e t i c a l l y should be c l o s e l y i n v o l v e d i n the metabolic pathways respon-s i b l e f o r phenylpropane compounds, and thus i s c l o s e l y r e l a t e d to coumarin i n b i t t e r p l a n t s . An equal volume of. NaOH wa3 added t o a s o l u t i o n o f the absorbent compound, and a constant stream o f n i t r o g e n was bubbled through, while the s o l u t i o n was kept i n the dark f o r two hours. The a l k a l i v/as n e u t r a l i z e d with 4N HCI, and the whole aqueous mixture e x t r a c t e d with e t h y l a c e t a t e and chromatogrammed. Due to u n s a t i s f a c t o r y r e s u l t s , the procedure was repeated with a s l i g h t m o d i f i c a t i o n i n t h a t the a l k a l i s o l u t i o n was heated f o r one hour. The e t h y l a c e t a t e e x t r a c t and the water l a y e r was a p p l i e d to Whatman § 1 f i l t e r paper and developed i n descending 15$ a c e t i c a c i d , IN NaOH and IH HCI s o l v e n t s . Rather c o n f u s i n g and i n c o n s i s t e n t r e s u l t s were a t t r i b u t e d to i m p u r i t i e s t h a t may have been i n c l u d e d on e l u t i o n o f the o r i g i n a l band. Due to the i n s t a b i l i t y o f the dark compound, i t could not be f u r t h e r p u r i f i e d by repeated chromatography and e l u t i o n . As p r e v i o u s l y d e s c r i b e d , p u r i f i c a t i o n o f the sample f o r a b s o r p t i o n s p e c t r a was achieved by p r e c i p i t a t i o n , f o l l o w e d by repeated washing 54. of the p r e c i p i t a t e i n a weak a c i d with some l o s s o f m a t e r i a l . The problem posed by t h i s method, i s th a t r a t h e r l a r g e q u a n t i t i e s are r e q u i r e d f o r p r e c i p i t a t i o n ; these are not e a s i l y o b tained by o r d i n a r y paper chromato-graphy o S i n c e the s o l u b i l i t y o f the absorbent compound i n d i f f e r e n t s o l v e n t s had been determined, an attempt was made t o e x t r a c t and p r e c i p i t a t e the compound from non-b i t t e r p l a n t s , without the use o f chromatography. The a l c o h o l i c e x t r a c t o f the shoots was evaporated t o dryness i n a f l a s h evaporator and the r e s i d u e was taken up i n IN NaOH, s o a s to d i s s o l v e a l l o f the dark absorbent compound. T h e s o l u t i o n w a s then n e u t r a l i z e d with an e q u i - e q u i v a l e n t amount . o f a c i d . The f i n a l s o l u t i o n was made s l i g h t l y a c i d i c and s t o r e d i n the r e f r i g e r a t o r , i n the hope t h a t the compound would p r e c i p i t a t e out. A f t e r two weeks a p r e c i p i t a t e formed; however, the dark compound was, h e a v i l y contaminated with o t h e r p l a n t c o n s t i t u e n t s . D Feeding Experiments. The i n v e s t i g a t i o n pursued so f a r i n v o l v e d a comparative study of the normal products o f metabolism i n b i t t e r and n o n - b i t t e r p l a n t s . In order t o throw more l i g h t on the s y n t h e s i s o f phenylpropane compounds i n coumarin-free p l a n t s , an attempt was made to determine i n what way coumarin 55. and i t s immediate p r e c u r s o r s , o-coumaric a c i d and cinnamic a c i d , are metabolized when a p p l i e d to normally coumarin-free sweet c l o v e r p l a n t s . ( i ) M a t e r i a l s Both seed and f i e l d grown p l a n t s o f b i t t e r M. a l b a and n o n - b i t t e r v a r i e t i e s "Cumino" and "Denta" and n o n - b i t t e r s p e c i e s M. d e n t a t a were used. For seed a s a t u r a t e d s o l u t i o n of coumarin, o-coumaric a c i d and cinnamic a c i d i n 1% e t h a n o l was used. On f e e d i n g the shoots a s a t u r a t e d s o l u t i o n . o f coumarin, o- and p-couinaric a c i d s and cinnamic a c i d i n 2% e t h a n o l wa3 used. ( i i ) Method (a) Seed The seeds were s c a r i f i e d with sand paper, soaked f o r one minute i n 6/1 K 2 O 2 , then germinated and p e r m i t t e d to grow f o r s i x days on f i l t e r paper s a t u r a t e d with the d i f f e r e n t s o l u t i o n s . ( F i g . IX, (b) Shoots The method used was b a s i c a l l y t h at of Roach and Roberts ( 6 8 ) . Leaves were removed from shoot t i p s and these were cut back about a h a l f - i n c h . The ends were then i n s e r t e d f o r f o u r days i n v i a l s f i l l e d with the s o l u t i o n . S i n c e the t i p o f the shoot turned c h l o r o t i c and w i l t e d w i t h i n about s i x t e e n hours a f t e r i n s e r t i o n i n o- and p-coumaric a c i d , i t was cut back about a h a l f - i n c h every day. ( F i g . X) . 5 6 . F i g u r e X - Technique f o r f e e d i n g shoots. On the fourth day the shoots were cut eight to ten inches from the t i p and extracted i n the manner described previously. The following i s a s l i g h t l y d i f f e r e n t extraction procedure used with the seedlings: a) The harvested seedlings were placed i n a beaker of d i s t i l l e d water so as to remove any compound from the surface. b) The complete seedlings were then ground i n a mortar with ca. 15 ml. 95$ EtOH. c) The extract was f i l t e r e d and the mortar rinsed with a further 5 ml. 95$ EtOH. d) The residue on the f i l t e r paper was washed with 10 ' %. ml. 80$ EtOH. e) The combined f i l t r a t e was concentrated to a volume of ca. 5 ml. i n a f l a s h evaporator, so as to pre-c i p i t a t e the chlorophyll and other f a t — s o l u b l e material. f) The concentrated extract was passed over a c e l i t e column and washed down; with a 80$ EtOH. g) The volume was once again reduced to ca. 5 ml. Plants used as controls received exactly the same treatment, with the only difference that no compound was dissolved i n the 1$ ethanol, i n the case of the seed; and i n the 2$ ethanol i n the case of the shoots. The e x t r a c t s , both t r e a t e d and c o n t r o l , were a p p l i e d to Whatman tfl f i l t e r paper. Both ascending and descending chromatography, and p h e n o l i c and p h e n o l i c g l y c o s i d e s o l v e n t s were employed, ( i i i ) R e s u l t s (a) Coumarin Coumarin f i r s t of s e v e r a l compounds used i n the f e e d -i n g t r i a l s , proved to be an extremely d i f f i c u l t compound to work w i t h . I t i n h i b i t e d germination to such an exten t , even when used i n very d i l u t e s o l u t i o n s , t h a t the e x t r a c t s o f the few s e e d l ings which were recovered, were i n s u f f i c i e n t f o r any c o n c l u s i v e r e s u l t s . S i m i l a r l y , when f e e d i n g the shoots with coumarin i t was found that w i t h i n a day the stem became c h l o r o t i c and s h r i v e l l e d as f a r as e i g h t inches from the i n s e r t e d t i p . "Free" coumarin seems to be e q u a l l y t o x i c t o the h i g h -coumarin and to the low-coumarin p l a n t s . (b) o-Coumaric A c i d A t t e n t i o n was next given t o the metabolism of the a p p l i e d o-coumaric a c i d . A l l shoot and s e e d l i n g e x t r a c t s f o l l o w i n g o-coumaric 1 II a c i d f e e d i n g s , when a p p l i e d to Whatman jfl f i l t e r paper, y i e l d -ed a d i s t i n c t white f l u o r e s c e n t band at an Rf value o f 0.61, when the chromatogram was developed with descending 1% a c e t i c a c i d . E x t r a c t s o f c o n t r o l p l a n t s d i d not give t h i s band i n the corresponding r e g i o n of the chromatogram„ The d i f f e r e n c e was very conspicuous. 5 9 . In a d d i t i o n , s m a l l q u a n t i t i e s of the ^ - g l u c o s i d e of o-coumaric a c i d were det e c t e d i n the n o n - b i t t e r v a r i e t i e s which had been f e d ; of cource i n the b i t t e r v a r i e t i e s t h e r e was a great d e a l of the o-coumaric a c i d g l y c o s i d e p r e s e n t . The d i f f e r e n c e i s p a r t i c u l a r l y conspicuous i n shoot e x t r a c t s , s i n c e a b s o l u t e l y no t r a c e of the o-coumaric g l u c o s i d e was found to be present i n the c o n t r o l p l a n t s . The f a c t t h a t shoots of non-b i t t e r p l a n t s car., be induced to form very s m a l l q u a n t i t i e s of the o-coumaric a c i d g l u c o s i d e i s r a t h e r i n t e r e s t i n g . The f l u o r e s c e n t band supra vide was e l u t e d u s i n g 10$ e t h a n o l , and the e l u a t e was concentrated i n a f l a s h e vaporator. The f l u o r e s c e n t compound was then hydrolysed by adding 0 . 5 mg. emulsin to 5 ml. of the e l u a t e and l e t t i n g i t stand at 3 7°C. f o r ten minutes. In a d d i t i o n , a c i d h y d r o l y s i s as d e s c r i b e d p r e v i o u s l y was a l s o c a r r i e d out, and i n both cases the same aglycone was l i b e r a t e d . The sugar attached was i d e n t i f i e d as g l u c o s e . R f v a l u e s and c o l o u r r e a c t i o n s f o r both the g l u c o s i d e and the aglycone are presented i n Tables V l l l , IX and X 0 60 TABLE V l l l - Rf values f o r the aglycone o f a white u c v 0 f l u o r e s c e n t g l u c o s i d e , obtained on f e e d i n g o f o-coumaric a c i d to b i t t e r and n o n - b i t t e r sweet c l o v e r p l a n t s . A l l chromatograms run at 2 0°C. + l ° c 0 Solvent ascending Rf v a l u e s descending... n - b u t a n o l : " a c e t i c a c i d : water 0 . 8 6 4 1 5 0 . 8 7 15$ a c e t i c a c i d 0 . 4 6 0 . 5 1 n-butanol: e t h a n o l : f o r m i c acid:water 0 . 8 9 5 1 1 1 chloroform: 2-propanol: a c e t i c a c i d : 1 2 1 water 0 . 8 5 1 2-butanol s a t . water 0 . 9 0 water 0 . 3 5 n-butanol: 27$ a c e t i c a c i d 1 1 0 . 8 9 e t h y l a c e t a t e : a c e t i c a c i d : water 0 . 8 8 a c e t i c a c i d : h y d r o c h l o r i c a c i d : 30 3 water 10 0 . 8 6 i 61. TABLE IX - Rf values f o r a u.v. fluorescent glucoside obtained on feeding of o-coumaric acid to b i t t e r and non-bitter sweet clover plants. A l l chromatograms prepared by descending method at 20°c, £ 1°C. S o l v e n t Rf value n- butanol: pyridine: water 6 4 3 0.58 n—butanol: ether: w a t e r 40 11 19 0.58 e t h v l a c e t a t e : a c e t i c a c i d :w a t e r "9 . 2 2 0.47 a c e t i c a c i d 0.61 chloroform: methanol: water 5 5 1 . 0.70 n- butanol: acetic acid: water 4 1 5 0.50 15% acetic acid 0.68 nr 1.butanol: p y r i d i n e : w a t e r 10 3 3 0.35 chloroform: 2-propanol: a c e t i c a c i d : 1 2 1 water. 1 0.54 6 2 . TABLE X - Colour reactions produced with d i f f e r e n t spray-reagents by aglycone of the fluorescent glucoside obtained on feeding of o-coumaric acid to b i t t e r and non-bitter sweet clover plants. Reagent Colour reaction FeCl 3/K 3Fe(CN) 6 AgN03 Acetone reagent AgN03 Ammoniacal diazotized s u l f a n i l i c acid p - n i t r o a n i l i n e 1% F e C l 3 1% KMnO, blue l i g h t brown 2 - 3 hrs, reddish brown f a i n t yellow .The reactions indicate that the compound has a phenolic hydroxyl group and that the position para to the hydroxyl group i s substituted since no coupling with the diazonium reagent takes place. Many phenolic compounds have Rf values s i m i l a r to those indicated i n Table V l l l . A number of controls were spotted alongside the unknown on the chromatogram and run i n a series of solvents. Some compounds with extremely s i m i l a r Rf values, but i n no case i d e n t i c a l are f e r u l i c , i s o f e r u l i c , c a f f e i c and sinapic acids, o-, m- and p-coumaric 6 3 . 7 . acids, 34 dimethory, 2 hydroxy, 3 methoxy, 2 hydroxy, 5 methoxy cinnamic acids, herniarin, umbelliferone and scopoletin. Using pH fluorescence curves, Goodwin and Kavanagh characterized many coumarins and r e l a t i v e s (33) In order to obtain an ind i c a t i o n of the i n t e n s i t y of fluorescence at d i f f e r e n t pH values, the aglycone was sprayed on chromatography paper with a series of buffe r s . Since no fluoremetric instrument was av a i l a b l e , the fluorescence was observed r i v u a l l y under u.v. l i g h t and numbers were a l l o t t e d according to the i n t e n s i t y . The approximate pH fluorescence curve of the unknown compound i s given i n F i g . I l l Figure XI - Relative i n t e n s i t i e s of fluorescence at d i f f e r e n t pH values of an unknown compound obtained on feeding t r i a l s . 10 9 Ocular scale 8 of f l u o r - 7 escence . inten- ° s i t y 5 4 3 2 1 e 0 2.2 3 . 4 4 . 6 5 . 8 7 . 0 8.2 9 . 4 1 0 . 6 pH 6 4 . In addition an absorption spectrum i n the u.v. l i g h t range was obtained using a Beckman D.U. spectrophotometer. F i g . III). (c) p-Coumaric acid p-Coumaric acid was fed to both b i t t e r and non-b i t t e r plant shoots i n a further attempt to determine the type of hydroxylation or methoxylation that takes place i n the plant when cinnamic acid r e l a t i v e s are fed. A few synthetic p-hydroxylated cinnamic acid r e l a t i v e s were available f o r comparative purposes. I t was hoped that by feeding of p-coumaric acid more l i g h t might be shed on the nature of the phenolic aglycone i s o l a t e d from plants fed with o-coumaric a c i d . The feeding, extracting and chromatography procedures were exactly the same as followed previously. Extracts from both b i t t e r and non-bitter plants yielded a very f a i n t l y v i s i b l e band on chromatographic separation with 1% a c e t i c acid solvent. This band fluoresced a bright blue under u.v. l i g h t following sparying with IN NaOH. To f a c i l i t a t e i n the marking of the band f o r elution, the edges of the sheets were sprayed with NaOH. The s t r i p s were cut out, eluted with 10$ ethanol and acid hydrolyzed. The ethyl ether layer, following extraction, was applied to Whatman #1 f i l t e r paper alongside a series of substituted cinnamic acids. A number of bands were 65. l i b e r a t e d on hydrolysis, probably due to impurities which may have been included with the rather complicated manner of marking out the band f o r e l u t i o n . However, the Rf values and the colour reactions of one rather prominent spot gave every i n d i c a t i o n that the compound i s sinapic a c i d . (Table TABLE XI - Rf values and colour reactions f o r sinapic acid and unknown aglycone i s o l a t e d from plants fed with p-coumaric aci d . Chromatograms prepared by ascending method at 20°c t 1°C. Rf values Solvent Aglycone Sinapic acid n-butanol: acetic acid: water 4 1 5 0.87 0.87 n-butanol: ethanol:formic acid 5 1 1 water 1 * 0.92 0.92 15$ acetic acid 0.50 0.50 Reagent Colour Reaction FeCl 3/K 3Fe(CN) 6 blue blue p - n i t r o a n i l i n e -neutral AgNC>3 i n acetone l i g h t brown l i g h t brown 2 hrs. 2 hrs. (d) Cinnamic acid Cinnamic acid was also applied to both seeds and shoots of b i t t e r and non-bitter v a r i e t i e s and species. How-ever, on chromatographic examination of extracts from treated and control plants, no difference could be detected 0 Figure XII - U.V. absorption spectrum of aglycone of the fluorescent glucoside obtained on feeding o-coumaric acid to b i t t e r and non-bitter sweet clover plants. The increase i n density range from o - 1 to .5 - 1 .5 was necessary because of the high concentration of the solution i n the u.v. region 68 IV. DISCUSSION & CONCLUSION Discussion As early as 1896, Wilson, (94) i n his book "The C e l l i n Development and Inheritance" stated that inheritance i s the recurrence, i n successive generations, of l i k e forms of metabolism. Genetics, as a f i e l d of study, came to existence at the turn of the century , and f o r a number of decades, geneticists overlooked t h i s metabolic aspect. They dealt to a great extent with genes and characters, but gave very l i t t l e attention to the problems of how genes act i n the metabolism and development of organisms. Geneticists were concerned primarily with how genes are inherited rather than with how they act, or the e f f e c t s of t h e i r action. More recently, a s l i g h t l y d i f f e r e n t approach led. to a reconsideration of the basic tenets of c l a s s i c a l genetics and also the e a r l i e r notion or concept of the gene. Genetics, today, i s approaching a time when i t can specify what a given gene or a set of cl o s e l y linked genes i s doing, as accurately as biochemistry can specify what an enzyme such as pepsin i s doing, even i f the precise d e t a i l s of what i s happening i n e i t h e r case i s not known. Biochemical genetics may be approached from two d i r e c t i o n s . One i s the study of the chemical structure of the genetic material i t s e l f , namely D.N.A. The other approach, from an opposite d i r e c t i o n , i n -volves the study of the metabolic pathways controlled by 69. g e n e s . I n h i g h e r p l a n t s , t h e r e a r e f e w e x a m p l e s o f t h e l a t t e r a p p r o a c h a n d r e l a t i v e l y l i t t l e i s k n o w n o f g e n e c o n t r o l l e d p r o c e s s e s . S t u d i e s o f g e n e - c o n t r o l l e d r e a c t i o n s i n t h e p l a n t m a y b e u s e f u l i n o r d i n a r y g e n e t i c s a n d b i o c h e m i s t r y , a n d a l s o m a y b e o f c o n s i d e r a b l e p r a c t i c a l s i g n i f i c a n c e , e s p e c i a l l y i n b r e e d i n g p r o g r a m s . T h e p r o d u c t i o n o f l o w - c o u m a r i n v a r i e t i e s o f M , a l b a , i n v o l v e d b a c k - c r o s s i n g f o r f i v e t o s e v e n g e n e r a t i o n s , t o t h e b i t t e r c o m m o n s w e e t c l o v e r p a r e n t . W i t h e a c h g e n e r a t i o n l o w -c o u m a r i n p l a n t s w e r e s e l e c t e d . T h u s , v a r i t i e s s u c h a s " C u m i n o " a n d " D e n t a " h a v e , v e r y p r o b a b l y , a g e n i e c o m p l e m e n t t h e s a m e a s b i t t e r M . a l b a , e x c e p t i n g f o r a o n e o r t w o g e n e d i f f e r e n c e i n v o l v e d i n t h e p r o d u c t i o n o f c o u m a r i c a c i d g l u c o s i d e ( 34). S i n c e t h e g e n e t i c d i f f e r e n c e i s r a t h e r s i m p l e , i t w a s a s s u m e d t h a t t h e p a t h w a y , n o r m a l l y l e a d i n g t o t h e p r o d u c t i o n o f g l u c o s i d e o f o - c o u m a r i c a c i d i n t h e b i t t e r p l a n t s , m i g h t b e o n l y s l i g h t l y a l t e r e d i n t h e n o n - b i t t e r p l a n t s , a n d t h a t t h e e n d p r o d u c t m i g h t b e c l o s e l y r e l a t e d t o o - c o u m a r i c a c i d g l u c o s i d e . T h e a p p l i c a t i o n o f p a p e r c h r o m a t o g r a p h y t o g e n e t i c r e s e a r c h e s r e l a t i v e l y r e c e n t . B e c a u s e o f i t s s i m p l i c i t y , v e r s a t i l i t y a n d c h e a p n e s s , t h e t e c h n i q u e h a s b e a n e f f e c t i v e l y u s e d i n m a n y k i n d s o f b i o l o g i c a l i n v e s t i g a t i o n s , a n d a n e x t e n s i v e b i o c h e m i c a l b a c k g r o u n d i s n o t e s s e n t i a l f o r i t s s u c c e s s f u l a p p l i c a t i o n . F o r a n e x a m p l e , H a d o r n a n d M i t c h e l l (39) w o r k i n g w i t h t h e s e p i a m u t a n t o f D r o s o p h i l a m e l a n o g a s t e r 7 0 . c h r o m a t o g r a p h i c a l l y i s o l a t e d a y e l l o w pigment from the mutant, and showed t h a t i t s chemical s t r u c t u r e was extremely c l o s e l y r e l a t e d to t h a t of the red pigment, normally found i n w i l d type f l i e s . The technique o f f e e d i n g , i n c o n j u n c t i o n with chromato-graphy, of chemical components, e s p e c i a l l y when "marked" with r a d i o - a c t i v e t r a c e r s , i s u s e f u l i n the study o f the metabolism of these components i n a p l a n t . With t h i s background an attempt was made to determine what compound s u b s t i t u t e s f o r coumarin i n the n o n - b i t t e r p l a n t s , and a l s o where the gene f o r low-coumarin e x e r t s i t s e f f e c t s along the s h i k i m i c - p r e p h e n i c a c i d pathway. A comparative study o f the seed o f sweet c l o v e r r e v e a l e d t h a t the /3> - g l u c o s i d e o f o-coumaric a c i d i s present i n both the b i t t e r and n o n - b i t t e r p l a n t s . T h i s would suggest that a development aspect i s a l s o i n v o l v e d i n the pathways concerned with the s y n t h e s i s of phenylpropanoid compounds. I t appears t h a t the normally " l e a k y " genes f o r low-coumarin l o s e " c o n t r o l " i n the seed, which r e s u l t s i n the appearance i n the t i s s u e of o-coumaric a c i d g l u c o s i d e . The | i - g l u c o s i d e of o-coumaryl and o-ccumaric a c i d s could not be d i f f e r e n t i a t e d ; however, on h y d r o l y s i s the g l u c o s i d i c band could not be detected on the chromatograms, but o n l y coumarin which appeared i n q u a n t i t y . A search f o r a compound that s u b s t i t u t e s f o r coumarin i n the shoots of n o n - b i t t e r p l a n t s , was based on the 71 s u p p o s i t i o n that the compound i s c l o s e l y r e l a t e d to coumarin i t s e l f , with the d i f f e r e n c e based on l y on p o s s i b l e s u : s t i t u t i o n s i n the benzene r i n g , and thus has very s i m i l a r p r o p e r t i e s such as s o l u b i l i t i e s and r e a c t i o n to s t r a y reagents. Since coumarin e x i s t s i n the bound g l y c o s i d i c form, i t was assumed t h a t i t s s u b s t i t u t e i n the Eton-bitter p l a n t s would a l s o be g l y c o s i d a t e d , and thus water s o l u b l e . T h i s search proved to be an extremely l e n g t h y task, and was o n l y complicated by the f a c t t h a t there are no coumarin s p e c i f i c reagents a v a i l a b l e , i n s p i t e o f the e x t e n s i v e knowledge of coumarin chemistry. Mo c l o s e r e l a t i v e t o coumarin could be found i n aoundance i n the green shoots of non-b i t t e r p l a n t s , but i n s t e a d a compound was i s o l a t e d from the shoots of a l l the n o n - b i t t e r p l a n t s , which appears to be a flavonoid', with p r o p e r t i e s somewhat s i m i l a r to q u e r c e t i n . Many workers have a l r e a d y demonstrated t h a t phenylpropanoid compounds, of which coumaric a c i d i s a r e p r e s e n t a t i v e , act as f l a v o n o i d p r e c u r s o r s (91). Cinnamic a c i d when fed to sweet c l o v e r , i s a p p a r e n t l y taken up i n the metabolism and " d i s a p p e a r s " i n both the b i t t e r and n o n - b i t t e r p l a n t s . Together with the i s o l a t i o n of a f l a v o n -o i d type compound from n o n - b i t t e r p l a n t s , the r e s u l t s from cinnaiir c a c i d f e e d i n g , would suggest that the compound i s a common p r e c u r s o r i n both the b i t t e r and n o n - b i t t e r p l a n t s . In the b i t t e r p l a n t s , the cinnamic a c i d would undergo or t a o h y d r o x y l a t i o n and then g l y c o a i d a t i o n to form the J3 - g l u c o s i d e of o-coumaric a c i d . In the n o n - b i t t e r p l a n t s , the cinnamic 7 2 . a c i d might be i n c o r p o r a t e d to f o r - r i n g B of a f l a v o n o i d compound. The r i n g A of the f l a v o n o i d compound would be formed by h e a d - t o - t a i l condensation of a c e t a t e u n i t s . The genes f o r low and h i g h coumarin content might exert t h e i r e f f e c t j u s t a f t e r the formation of cinnamic a c i d , or even e a r l i e r . I t appears t h a t the genes, i n s t e a d of b r i n g i n g about the s m a l l change by a h y d r o x y l a t i o n or meth-o x y l a t i o n i n the benzene r i n g of coumaric a c i d , c a r r y the s y n t h e s i s with a s l i g h t a l t e r a t i o n " f a r t h e r " on the pathway, with the f o r m a t i o n of a f l a v o n o i d ; a l s o • g l y c o s i d a t i o n i s h i n d e r e d . Feeding of o-coumaric a c i d l e d not to the p r o d u c t i o n of the ^ 5 - g l u c o s i d e of o-coumaric a c i d , as r e p o r t e d by Kosuge and Conn ( 5 4 ) , but i n s t e a d s i g n i f i c a n t q u a n t i t i e s of a g l y c o -s i d e of an u n i d e n t i f i e d p h e n o l i c compound, resembling i s o f e r u l i c a c i d . In a d d i t i o n Kosuge and Conn, i b i d , d i d not r e p o r t any t o x i c e f f e c t s which were experienced i n t h i s study on f e e d i n g o- and p-coumaric a c i d and coumarin to both b i t t e r and non-b i t t e r p l a n t s . The formation of an u n i d e n t i f i e d p h e n o l i c g l u c o s i d e f o l l o w i n g o-coumaric a c i d f e e d i n g i n a l l the p l a n t s f e d , i r r e s p e c t i v e of v a r i e t y or s p e c i e s , o n l y f u r t h e r supports the s u p p o s i t i o n t h a t the g e n e t i c d i f f e r e n c e between low- and high-coumarin s t r a i n s i s i n i t i a t e d " e a r l i e r " i n the s h i k i m i c a c i d -phenylpropanoid pathway. However, si n c e nc r a d i o - a c t i v e t r a c e r s were used, i t cannot be s t a t e d with c e r t a i n t y t h a t the p h e n o l i c 73 o glucoside produced on feeding of o-coumaric acid i s due to a disruption of a normal metabolic pathway, or, as a r e s u l t of the metabolism of the fed compound. Feeding of p-coumaric acid led to the production, of a glycoside of sinapic acid i n a l l the plants to which i t was applied. The formation of sinapic acid involved a 3,5 methoxylation of the p-coumaric acid . A s i m i l a r methoxylation of the o-coumaric acid would produce 2 hydroxy, 3,5 methoxy cinnamic aci d . Unfortunately no synthetic sample of t h i s compound was available f o r comparison with the unidentified aglycone from o-coumaric acid feeding„ 7 4 . C o n c l u s i o n s 1 . T h e - g l y c o s i d e o f o - c o u m a r i c a c i d , w h i c h o n h y d r o l y s i s y i e l d s c o u m a r i n , w a s f o u n d t o b e p r e s e n t i n t h e s e e d o f b o t h t h e n o n - b i t t e r v a r i e t i e s " C u m i n o " a n d " D e n t a " , i n t h e s e e d o f n o n - - b i t t e r M . d e n b a t a , a n d i n t h e b i t t e r c o m m o n M. a l b a . 2. T h e s e a r c h f o r a c o m p o u n d t h a t s u b s t i t u t e s f o r c o u m a r i n i n t h e s h o o t s o f n o n - b i t t e r p l a n t s , l e d t o t h e i s o l a t i o n o f a c o m p o u n d f r o m n o n - b i t t e r p l a n t s , w h i c h a p j e a r s t o b e a f l a v o n o i d , a n d r e s e m b l e s q u e r c e t i n i n m a n y r e s p e c t s . T h i s c o m p o u n d d i d n o t a p p e a r i n t h e g r e e n t i s s u e s o f b i t t e r * s w e e t c l o v e r . 3 . F e e d i n g o f o - c o u m a r i c a c i d t o s h o o t s o f b i t t e r a n d n o n -b i t t e r p l a n t s , l e d t o t h e f o r m a t i o n o f a p h e n o l i c g l u c o s i d e i n a l l t h e p l a n t s , i r r e s p e c t i v e o f v a r i e t i e s o r s p e c i e s . H y d r o l y s i s y i e l d e d a n a g l y c o n e , s i m i l a r t c i s o f e i u l i c a c i d . T h e a g l y c o n e m i g h t b e 2 h y d r o x y , 3.5 m e t h o x y c i n n a m i c a c i d . 4 . F e e d i n g o f p - c o u m a r i c a c i d t o - s h o o t s o f b i t t e r a n d n o n -b i t t e r p l a n t s , l e d t o t h e f o r m a t i o n o f s i n a p i c a c i d g l y c o s i d e . 5. W h e n c i n n a m i c a c i d w a s f e d . t o p l a n t s , n o d i f f e r e n c e i n t h e m e t a b o l i c p r o d u c t s c o u l d b e d e t e c t e d f r o m t h o s e n o r m a l l y f o u n d i n c o n t r o l p l a n t s . 6. T h e g e n e o r g e n e s f o r l o w c o u m a r i n , a p p a r e n t l y e x e r t t h e i r e f f e c t b e f o r e t h e f o r m a t i o n o f c o u m a r i r i , a n d o - c o u m a r i c 75. a c i d m o s t p r o b a b l y i s n o t a p r e c u r s o r t o a f l a v o n o i d c o m p o u n d i s o l a t e d f r o m n o n - b i t t e r s w e e t c l o v e r p l a n t s . 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