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Some aspects of the biochemical genetics of coumarins in forage legumes Bradner, Norman Richard 1961

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SOME ASPECTS GF THE BIOCHEMICAL GENETICS OF COUMARINS IN FORAGE LEGUMES by -NORMAN RICHARD BRADBER B.S.A., Unive r s i t y of B r i t i s h Columbia, 1959 A. THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN AGRICULTURE i n the D i v i s i o n of PLANT SCIENCE We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA-APRIL 1961 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree a t the U n i v e r s i t y o f B r i t i s h Columbia, I agree t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and study. I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the Head of my Department o r by h i s r e p r e s e n t a t i v e s . I t i s understood t h a t copying or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be allowed without my w r i t t e n p e r m i s s i o n . _B*pa**»e»t of VLAWT $>£j£fiJC£ The U n i v e r s i t y of B r i t i s h Columbia, Vancouver 8 , Canada. Date Vlg^ <! ; |<H I  i i ABSTRACT Coumarin compounds responsible f o r bitterness, 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 clover (Melilotus alba) do not occur i n the non-bitter "Cumino" varie t y of M. alba or i n the species M. dentata. Neverthe-le s s , i n v e s t i g a t i o n showed that compounds which are very probably coumarins do occur i n the non-bitter s t r a i n s . A search f o r coumarins i n a l f a l f a (Medicare s a t i v a ) , c l o s e l y r e l a t e d to sweet clover taxonomieally, did not lead to the discovery of coumarins but did lead to the characteri-zation of a new flavonoid t e n t a t i v e l y characterized as 3:3 = '+ trihydroxy -5:7 dimethoxy flavone. i i i TABLE OP CONTENTS I INTRODUCTION 1 II REVIEW OF LITERATURE 3 A The natural coumarins 3 B Coumarins i n animal metabolism h C Coumarins i n plant metabolism 5 D Plant breeding f o r coumarin freedom i n sweet clover 7 E Analysis of coumarin 9 F Coumarin biosynthesis 11 II I INVESTIGATION I 13 A Materials 13 B Methods l*f C Observations and r e s u l t s 17 D Discussion of r e s u l t s 28 IV INVESTIGATION II 31 A Materials 32 B Methods 33 C Observations and re s u l t s 39 D Discussion of re s u l t s V SUMMARY AND CONCLUSIONS - 51 VI LITERATURE CITED 53 i v ACKNOWLEDGEMENT S My appreciation and thanks are accorded the following 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 of Plant Science, f o r interest and guidance Dr. J. J. R. Campbell, D i v i s i o n of Animal Science, f o r the use of equipment Dr. H. McLean, Dr. J . A. F. Gardner, Mr. G. M. Barton, a l l of the Forest Products Laboratory, Canada Dept. of Northern A f f a i r s and National Resources, f o r assistance i n chemical characterization Mr. Jan Brussel, Mr. Don Pearce, technicians, D i v i s i o n of Plant Science, f o r help i n laboratory and f i e l d The National Research Council of Canada f o r f i n a n -c i a l support Thanks are accorded to my thesis committee: Dr. V. C. Brink, D i v i s i o n of Plant Science, (Chair-man) . Mr. W. E. P. Davis, Canada Dept. of Agri c u l t u r e , Agassiz, B.C. Dr. C. A. Hornby, D i v i s i o n of Plant Science. Dr. N. A. MacLean, D i v i s i o n of Plant Science. Dr. W. D. K i t t s , D i v i s i o n of Animal Science. Dr. G. Rouse, Dept. of Botany. 1. I IHTRODUCTIOU Coumarin, as the substance primarily responsible f o r the u n p a l a t a b i l i t y of common sweet clover (MelHotus alba and Melilotus o f f i c i n a l i s ) , and fo r the t o x i c i t y , i n d i r e c t l y , of spoiled sweet clover hay, has claimed the interest of forage crop breeders. The absence of coumarin i n Melilotus dentata and i n rela t e d forage legumes such as a l f a l f a , and the develop-ment, through hybridization and se l e c t i o n of the eoumarin-free, highly palatable "Cumino"-variety 1 of sweet clover provides encouragement to breeders interested i n the genetical modifi-cation of the chemical composition of forage crops. Despite the evident progress i n the se l e c t i o n of a coumarin-free sweet clover, several questions remain unanswered about coumarin i n forage legumes v i z . (1) I f coumarin per se i s absent i n coumarin-free sweet clover, are other coumarin r e l a t i v e s present? In other words, what are the bio-genetie implications and (2) Are coumarin derivatives of importance present i n forage legumes other than sweet clover? Coumarins are known to be p h y s i o l o g i c a l l y active i n a va r i e t y of ways when fed to animals. The coumarins may be important i n plants although t h e i r p h y s i o l o g i c a l r o l e i s not known. It i s worthy of record that coumarin i t s e l f i s known to induce epinasty and to prevent germination of seed of ce r t a i n non-coumarin con-tai n i n g plants. 1 A vari e t y developed at the forage laboratory, Canada Department of Agriculture, Saskatoon, Saskatchewan. 2. Coumarin and i t s chemical r e l a t i v e s are heterocyclic organic compounds of the C 5 C 3 "type" and are not "favoured" by very s p e c i f i c tests f o r t h e i r recognition. Perhaps t h i s i s one of the major reasons f o r the lack of knowledge about these compounds i n plants. However, many coumarins fluoresce b r i l -l i a n t l y i n u l t r a - v i o l e t l i g h t and use of t h i s fact may be of value i n t h e i r recognition i n screening t e s t s . To-day, more-over, chromatographic techniques can be used to a s s i s t i n t h e i r recognition. Separation and i d e n t i f i c a t i o n i s d i f f i c u l t but when chromatography i s used i n conjunction with other tech-niques such as, in f r a - r e d spectra, u l t r a - v i o l e t spectra, and colour t e s t s , paper chromatography provides a means of i d e n t i -f y i n g many di f f e r e n t though related compounds. It was decided therefore, that, with the common occurrence of fluorescence, i n u l t r a - v i o l e t l i g h t , of coumarins, and, with the powerful t o o l of chromatography, a study of these substances i n forage legumes was warranted. This study could prove to be usefu l to the forage breeder and to the plant physiologist. 3. II REVIEW OF LITERATURE A The natural coumarins Natural coumarins have been studied f o r many years by chemists and a number of workers have reviewed t h e i r d i s -t r i b u t i o n (29, 9*+, 95). Coumarins are recorded i n legumes, c i t r u s crops, grasses and orchids; and Spath (103) stated that the parent compound, coumarin, has been i s o l a t e d from approxi-mately eighty d i f f e r e n t species of plants. Despite the fact that there i s a widespread interest and considerable l i t e r a t u r e on the occurrence of these compounds i n plants, i n only a r e l a t i v e l y few instances have these compounds i n l e a f , seed, and seedling been p r e c i s e l y characterized. Notable i s the work of Spath (103) with umbelliferous species and Seshadri (9*0 with plants native to India. Scopoletin has been i s o l a t e d i n oats (^ 3), i n tobacco (10, 11), i n potatoes (1, 2), and cou-mestrol i n ladino clover and a l f a l f a (12, 13, Ih, 33, 113). Goodwin and Kavanagh (M-2) demonstrated the presence of com-pounds i n many plants exhibiting a fluorescence s i m i l a r to that of scopoletin; blue, b l u i s h or bluish-white fluorescence was demonstrated i n 109 species out of a t o t a l of 135 species be-longing to 126 genera and 69 f a m i l i e s . From the foregoing discussion i t would seem that cou-marins are found primarily i n plant t i s s u e . However, one occurrence of coumarins i n animal t i s s u e i s on record; Lederer (60) has shown that 3th benzo-coumarins are constitu-ents of the scent gland of the beaver. In plants too, there i s a class of compounds very si m i l a r to the coumarins - the chromones. These d i f f e r from the coumarins i n the po s i t i o n of the carbonyl group i n the hetero-c y c l i c r i n g . = 0 Benzo-eC-pyrone Benzo-V-pyrone coumarin chromone Compounds i n plants of such wide occurrence as flavones, i s o -flavones, flavonols, flavanones and flavanolones may be re-garded as "benzo" derivatives of t h i s basic chromone structure. These and other related compounds, the water-soluble plant pigments, the anthocyanins, chalcones and aurones, are wide-spread. (7, 9, 15, 36, hO, 50, 76, 88, 120). Because t h e i r chemical structure i s similar to that of the coumarins, they can be e a s i l y confused with the coumarins. B Coumarins i n animal metabolism As e a r l i e r stated many coumarins profoundly modify animal metabolism.. In feeding t r i a l s with animals, i t has been observed that coumarin i t s e l f acts as a narcotic f o r rabbits, frogs and earthworms and many other animals (51*). It i s also a sedative and hypnotic f o r mice (95). Wasicky (122) found that the coumarins, pimpinellin and osthruthin, have a s l i g h t t o x i c e f f e c t on r a t s , mice, and guinea pigs. Spath and Kuff-ner (lOh) demonstrated that many natural coumarins have 5. powerful effects on fresh water f i s h ; the f i s h gradually lose t h e i r balance, and remain steady or swim on t h e i r backs, move-ment i s then suspended, and f i n a l l y they die. Because of the physiological importance of coumarins i n animal metabolism, considerable interest has centered on those coumarin-containing grasses, e.g. sweet vernal grass, and legumes, e.g. sweet clover, ladino clover, used as stock feed. S p e c i f i c a l l y , the sweet clovers (M. alba and M. o f f i c i n a l i s ) . used as forage crops, are characterized by the presence of a " b i t t e r p r i n c i p l e " which a f f e c t s t h e i r p a l a t a b i l -i t y as stock feed, and which imparts to the plant a d i s t i n c t , but not unpleasant, v a n i l l a - l i k e odour. This property has long been associated with the presence of coumarin or i t s derivatives i n the tissue of the plant. In addition to ren-dering plants of t h i s crop r e l a t i v e l y unpalatable to herbi-vorous animals, the coumarin i s the parent material from which the anticoagulant "dicumarol" i s derived i n spoiled sweet clover hay (102, 10?, 123). A coumarin i s o l a t e d by Bickoff et a l (12, 13, Ih, 6k) from ladino clover, a l f a l f a , and other legumes and c a l l e d by him "coumestrol" induces oestrus. Other compounds such as genistein and biochanin A ( 8 l , 82) , formono-netin ( 8 ) , though reacting l i k e coumestrol are not coumarins but isoflavones. C Coumarins i n plant metabolism Hot only are coumarins widely d i s t r i b u t e d i n the plant kingdom but they may be p h y s i o l o g i c a l l y active i n plants. 6. Although the r o l e of coumarins i n plants i s not understood, coumarins applied a r t i f i c i a l l y t o plants have given many i n -t e r e s t i n g e f f e c t s . Coumarin per se favours c e l l enlargement i n such plant tissue as oat co l e o p t i l e and leaf blade at low concentrations but checks c e l l enlargement at higher concen-t r a t i o n s (69, 112, 118). Similar checking effects upon root elongation have been found for coumarin and c e r t a i n derivatives (5, 6^, 7^, 80, 92). Thus coumarins appear to act as " a n t i -auxins" and offset or counteract the usual growth enhancing eff e c t of auxins i n the plant (117). Coumarin also i n h i b i t s -mitosis i n onion, l i l y (28) and oats (*+7). Coumarins, and many other chemically unrelated com-pounds i n h i b i t germination of seeds (55> 56). Evenari (3*0 has. stated that germination i n h i b i t o r s are widespread i n plants and appear i n f r u i t pulp, f r u i t coats, endosperm, seed coats, embryo, leaves, bulbs, and roots. The main known i n h i b i t o r s active i n the aforementioned tissues are the following sub-stances or belong to the following chemical groups: hydrogen cyanide, ammonia, ethylene, mustard o i l s , organic acids, un-saturated lactones (some of which are coumarins), aldehydes, e s s e n t i a l o i l s , and al k a l o i d s . Special attention has been given to i n h i b i t o r s i n seed of i r i s , wild oats, l e t t u c e , beets, and cabbage. Juices of lemon (97) and peach f r u i t s (93) also have marked effects on the germination of the seed contained within t h e i r respective f r u i t s . It i s probable that germination i n h i b i t o r s are present i n the seeds and f r u i t s of most species and that they are involved i n a widespread mechanism of dormancy (3^) . A search f o r special chemical configurations r e l a t i n g to dormancy phenomena has been undertaken and i t has been stated that compounds having the structure of unsaturated lactones possess the property of i n h i b i t i n g seed germination and other growth phenomena ( 3 , 6 6 , 7^, 117) . Coumarin and parasorbic a c i d are compounds i n t h i s category. D Plant breeding f o r coumarin freedom i n sweet clover When one considers the widespread d i s t r i b u t i o n of coumarins and t h e i r p h y s i o l o g i c a l a c t i v i t y i n plants and ani-mals, i t i s understandable that a substantial l i t e r a t u r e i s devoted to coumarin and i t s r e l a t i v e s i n forage grasses and legumes. Most of the l i t e r a t u r e however, r e l a t e s to species containing large quantities of coumarin per se, such as sweet clover and sweet vernal grass, but very l i t t l e to species which may contain l i t t l e coumarin per se but, possibly, sub-s t a n t i a l quantities of other coumarins. The l i t e r a t u r e oh the development of coumarin-free sweet clover i s extensive since so many workers i n various f i e l d s contributed d i r e c t l y , or i n d i r e c t l y , to the successful release of M. alba v a r i e t y "Cumino". Because of the importance of t h i s work a short review of the subject i s presented here. The existence of coumarin per se i n plants has been known for a long time. The f i r s t recorded evidence of the i s o l a t i o n of coumarin from plant t i s s u e occurred i n 1820 when i t was i s o -l a t e d from Tonka beans (119). Since that time coumarin has 8 been used i n a v a r i e t y of ways e.g. as a perfume base, as an adulterant i n v a n i l l a extract. Interest i n coumarin i n sweet clover developed when i t was thought that t h i s crop could be used as an i n d u s t r i a l source of coumarin. Further interest was aroused i n the early 1920's when the u n p a l a t a b i l l t y of sweet clover was att r i b u t e d to coumarin. Thereafter, i t oc-curred to two Canadian workers K i r k (57), Kirk et a l (58), and Stevenson et a l (106), that a sweet clover free of coumarin could be bred i n order that the p a l a t a b i l i t y of the crop might be improved. E a r l y attempts to select low coumarin plants were tedious 0*1, 108) u n t i l Clayton and Larmour (27) i n t r o -duced a rapid method to detect free coumarin. Subsequently v a r i e t i e s low i n free coumarin were selected but, l a t e r i t was found that the selections s t i l l contained coumarin as a glyco-side, i n these strains the bound coumarin was not detected by Clayton and Larmour's t e s t . However, simple autol y s i s of the plant tis s u e before the test was conducted, overcame t h i s ob-st a c l e and t o t a l coumarin could then be measured (86, 87). In the search f o r non-bitter sweet clovers a coumarin-free species of sweet clover, M. dentata, or i g i n a t i n g i n Asia Minor, was found (18, 19) and plants from t h i s species were u t i l i z e d i n future breeding programmes (26, hS, 52, 85, 101, 107, 121). In crossing M. alba and M. dentata another problem arose; i t was found that the hybrid contained a chlorophyll l e t h a l which prevented the plants from growing to maturity. This problem was overcome when Brink (20) and Smith (100) demonstrated the f e a s i b i l i t y of grafting the chlorophyll d e f i c i e n t seedling onto normal M. o f f i c i n a l i s plants. 9 Further inte r e s t i n sweet clover was created when i t was r e a l i z e d that not only was coumarin responsible f o r the un-p a l a t a b i l i t y of the crop but i t was also involved i n the formation of "dicumarol" i n spoiled sweet clover hay (22, 23, 63, 67, 83, 89, 102, 105, 123). After many years of sel e c t i o n a few seeds of a eoumarin-free var i e t y were c o l l e c t e d and these seeds were sent to the forage laboratory at Saskatoon, Saskatchewan. A f t e r a number of years of intensive s e l e c t i o n M. alba v a r i e t y "Cumino" was released, a notable achievement made possible by many workers. E Analysis of coumarin Before terminating t h i s t o p i c a short discussion of the chemical methods f o r coumarin measurement i s i n order since the success of the sweet clover breeding programme de-pended on them to a large extent: i t was es s e n t i a l that inex-pensive and rapid t e s t s be developed f o r screening large numbers of plants. E a r l y investigators (30, 31, 32) used the steam d i s t i l l a t i o n method outlined by Obermayer (75) but t h i s method was too cumbersome to screen large populations. The development of rapid colour t e s t s , referred to above, by Clay-ton and LarmouT (27) overcame the d i f f i c u l t y of t h i s problem. However, t h i s method only measured the free coumarin and f a i l e d to detect bound coumarin. Roberts and Link (86, 87) reported that with autolysis of the l e a f material p r i o r to extraction of the coumarin, the colorimetric method measured t o t a l cou-marin content. A few years l a t e r , u t i l i z i n g the methods of 10. Ufer (115, 116) a fluorometric procedure was developed which measured coumarin content q u a n t i t a t i v e l y on a photofluorometer (98), or semiquantitatively (99, 12*+) by v i s u a l assessment of the fluorescence when samples were placed i n u l t r a - v i o l e t l i g h t . For a rapid determination of the presence or absence of t o t a l coumarin, the fluorometric method described by White, Savage, and Johnston (125) and recently modified by Goplin, Greenshields, and White (U-9), i s currently being used. Associated with the fluorometric measurement of cou-marin i s the use of fluorescence of compounds when they are viewed under u l t r a - v i o l e t l i g h t . Fluorescence of coumarins on f i l t e r paper chromatograms can be quite c h a r a c t e r i s t i c (9» 109, 110). A l l heterocyclic substances absorb u l t r a -v i o l e t l i g h t and many fluoresce i n i t ; many authors have com-p i l e d tables of compounds with t h e i r fluorescent colours (7, 25> 35, 36, 50, 51, 77, 110). Because many excellent reviews and texts have been written on the subject of paper chromato-graphy (16, 6l, 65, Ilk) t h i s topic w i l l not be discussed. In addition to the purely physical means to measure or i d e n t i f y coumarins, there are chemical methods. These con-s i s t of two main methods to di s t i n g u i s h the coumarin r i n g ; namely those based on the hydrolysis of the compound by alka-l i n e reagents (59, 62, 78, 127) and those methods based on the preparation of some s p e c i a l derivatives (2k), In order to conduct tests with coumarins, s p e c i a l attention should be paid to the means of i s o l a t i n g the compounds i n a pure form. Many coumarins can be extracted from plant 11 materials by suitable solvents (e.g. ether, benzene, acetone). However, care should be taken that the extractants do not cause changes i n the chemical makeup of the compounds. For example, Duncan and Dustman ( 3 D > proved that destruction of coumarin takes place i n a prolonged ether extraction. If coumarins are present as glycosides, the aglycones may be i s o l a t e d a f t e r preliminary hydrolysis; a l t e r n a t i v e l y , the glycoside may be extracted using alcohol or water. Natural coumarins are either neutral or s l i g h t l y a c i d and appear i n the corresponding f r a c t i o n s of the plant extract (29). In the i s o l a t i o n i t i s usual to take advantage of the f a c t that the coumarin r i n g i s opened by warm d i l u t e a l k a l i with the f o r -mation of the coumarinate s a l t , which allows the removal of neutral material (29). On decomposition of the s a l t with acid, the coumarlnic r i n g c y c l i s e s spontaneously, regenerating the coumarin whieh i s thus e a s i l y separated from acids formed by the hydrolysis of the other constituents; however, the method i s not i d e a l because i t i s d i f f i c u l t to avoid degradation of the coumarins (29). F Coumarin biosynthesis It was e a r l i e r mentioned that l i t t l e i s known of the function of the natural coumarins i n plants or, i n f a c t , of the benzenoid compounds generally (71). One way t o learn more of t h e i r functions or function would be to trace the pathways or pathway by which they are synthesized i n the or-ganism. 12 Several approaches may be taken to the study of the biosynthesis of eoumarins and benzenoid compounds generally. Possibly the most powerful i s through the use of radiotracer compounds. Neish (71), i n a recent review, has given a concise statement on the present status of knowledge obtained through t h i s approach. Another approach not adequately treated, as yet, i s that of " i n j e c t i o n or feeding" t r i a l s i n l i v i n g tissues of possible r e l a t i v e s and precursors of coumarins and r e l a t e d benzenoids followed by analysis of chemical changes as i n d i -cated by disappearance, accumulation etc. Yet another approach i s to i s o l a t e enzymes involved i n the biosynthetic pathways and the study of the enzymatic reactions i n v i t r o . Closely r e l a t e d to the l a t t e r two approaches i s that of the use of genetic blocks and the study of gene-controlled reactions i n r e l a t e d species and v a r i e t i e s . The genetic approach has con-tr i b u t e d s u b s t a n t i a l l y to the knowledge of flavonoid bio-synthesis (U-0) but no contributions have been forthcoming as yet i n the biosynthesis of coumarins: a beginning i s made i n the investigations to follow. 13. I l l INVESTIGATION I A comparative study of extracts from coumarin-free sweet clover, and from coumarin-containing sweet clover. The principal aim of this investigation was to as-certain i f coumarin relatives replaced coumarin, in coumarin-free sweet clover. One means by which differences might be detected between non-bitter M. dentata, non-bitter "Cumino" sweet clover, and bitter common white sweet clover would be by chromatographing suitable extracts. The approach assumes that the solubility characteristics of the coumarins are simi-lar. By this method, clues regarding the pathway of sub-stitution for coumarin might be recorded in differences observed on paper chromatograms. A Materials The species and varieties of sweet clover used were as follows: a) non-bitter, coumarin-free Melilotus dentata from A. E. Hallowell, U.S.D.A., Beltsville, Md. b) non-bitter coumarin-free white sweet clover, variety "Cumino" from R. Greenshields, and B. R. Goplen. Forage laboratory, Canada Dept. of Agriculture, Saskatoon, Saskatchewan. c) Canadian common, bitter white sweet clover from Bucker-field's Ltd., Vancouver. B.C. 1>+. B Methods EXTRACTION Establishment of s p e c i f i c chemical constituents occurring i n non-bitter sweet clovers, which do not occur i n the b i t t e r s t r a i n s , as has been stated, rests i n the expec-t a t i o n that differences may be found by chromatographing comparable tissue extracts. Materials from many extraction procedures were compared; only two, which appeared to be reasonably s a t i s f a c t o r y , are given below, a) Green leaf and stem tissue . 1. S l i c e t i s s u e into small pieces and immerse immediately i n hot 70% ethyl alcohol; then grind material i n a Waring Blendor. 2. Shake for 12 hours; extract twice more with equal volumes of solvent. 3 . F i l t e r the combined extract; cor.centrate i n a f l a s h evaporator to approximately 10$ of the o r i g i n a l volume @ 35°C. h. Extract the solution with d i e t h y l ether i n a l i q u i d -l i q u i d extractor and repeat as necessary. 5. Concentrate the ether extract to approximately % of the volume @> 3 5 ° C 6. Stripe the ether solution on Whatman #3 f i l t e r paper and develop the chromatogram eight hours with g l a c i a l acetic acid: water solvent ( 1 : 1 ) , (A:W) at about room temperature 22°C. 7. Stripe the residue l e f t i n the l i q u i d - l i q u i d extractor and develop i t i n n-Butyl alcohol: g l a c i a l acetic 15. a c i d : water 0+:l:5 organic phase) (B:A:W). 8. A l l ehromat©grams are seanned under u l t r a - v i o l e t l i g h t (3660 A and 2537 A). b) Seed 1. Grind the seed i n a Wiley m i l l using 20 mesh screen. 2. Shake the material with 70$ ethyl alcohol f o r 12 hours; extract twice more with equal volumes of solvent. 3. F i l t e r the combined extract, and concentrate i n a f l a s h evaporator to approximately 10$ of the volume © 35°C Extract the solution with d i e t h y l ether i n a l i q u i d -l i q u i d extractor and repeat as necessary. 5. Reduce the volume of the combined ether extracts and treat as above. 6. Take the residue, or residues l e f t i n the l i q u i d -l i q u i d extractor and treat with basic lead acetate, centrifuge and c o l l e c t the p r e c i p i t a t e . Suspend the p r e c i p i t a t e i n 70$ ethyl alcohol and bubble through H 2S gas; centrifuge and c o l l e c t the s o l u t i o n ; repeat t h i s procedure on the solution from the f i r s t c e n t r i -fugation using neutral lead acetate. 7. The a l c o h o l i c solutions of neutral lead and basic lead acetate "operations" are striped on #3 papers and developed i n B:A:W solvent. 8. The chromatograms are scanned as described before. SPOT TESTS Many colour tests are a v a i l a b l e to a i d i n the iden-16. t i f i c a t i o n of unknown heterocyclic compounds. A number of the tests that proved usef u l i n t h i s i n v e s t i g a t i o n are presented here. a) HH^ test - the chromatogram containing the p u r i f i e d spot was exposed to N H 3 vapours and immediately observed i n 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 test - a few drops of I N. FaOH solution was applied to the p u r i f i e d spot on the f i l t e r paper chromatogram and the colour of the spot was noted (91). c) HCl test - a few drops of concentrated HCl were applied to the spot on the f i l t e r paper chromatogram and the colour of the spot was noted. d) Diazotized-p-nitro a n i l i n e test - The diazonium solution was prepared as outlined by Roberts and Link (86). The tes t solution was spotted on f i l t e r paper and a few drops of 10% Ia 2 G03 added. The formation of colours, on the addition of a few drops of the diazonium reagent, was noted. The examination of the u l t r a - v i o l e t absorption spectra of p u r i f i e d compounds i s an excellent means of substantiating suspected differences between compounds. e) U l t r a - v i o l e t absorption spectrum - The compound was d i s -solved i n 95% ethyl alcohol (2mg./l.) and the u l t r a - v i o l e t spectra noted using 1 ml. quartz cuvettes i n a Beckman D U spectrophotometer. 17. C\ Observations and resu l t s The p r i n c i p l e fluorescing and absorbing spots, seen, when comparable chromatograms were examined under u l t r a - v i o l e t are given with t h e i r approximate Rf values, i n the tables to follow. Their colours and i n t e n s i t i e s for various reasons are usef u l only i n a very general way, and are recorded i n an abbreviated form. v.l.B.F. - very l i g h t Blue Fluorescence l.B.F. - l i g h t Blue Fluorescence B.F. - Blue Fluorescence b.B.F. - bright Blue Fluorescence B.W.F. - Blui s h White Fluorescence l.W.F. - l i g h t White Fluorescence W.F. - White Fluorescence Y.W.F. - Yellowish White Fluorescence Y.F. - Yellow Fluorescence l.M.F. ;. r l i g h t Mauve Fluorescence M.F. - Mauve Fluorescence 1.0.F. - l i g h t Orange Fluorescence b.B.G.F. - bright Blue Green Fluorescence v.l.B.G.F. - very l i g h t Blue Green Fluorescence v.l.Ab. - very l i g h t Absorbance l.Ab. - l i g h t Absorbance d.Ab. - dark Absorbance 18. Spots, fluorescent and absorbing, seen when non-bitter and b i t t e r sweet clover l e a f extracts were compared under u l t r a -o v i o l e t i l l u m i n a t i o n ( 3 6 6 O A ) . TABLE 1 - A comparison of procedural ether extracts Common "Cumino" Dentata sweet clover sweet clover sweet clover Spot colour Rf (A:W) colour Rf(A:W) colour Rf (A:W), 1 - - - - B.W.F. .19 2 - - - - v.l.B.F. .26 3 l.B.F. .37 - - v.l.B.F. .36 h l.M.F. Y.W.F. .^ 9 v.l.B.F. 5 M.F. .59 - - b.B.F. .57 6 - - - - d.Ab. .62 7 l.W.F. .65 W.B.F. .70 b.B.F. .69 8 - - - - d.Ab. .76 9 b.B.F. .82 - - Y.F. .82 10 W.F. .88 b.B.F. .86 l.B.F. .86 11 — W.F. Y.F. .92 19. TABLE 2 - A comparison of residues l e f t i n the liquid-liquid extractor Common "Cumino" Dentata sweet clover sweet clover sweet clover Spot colour Ef(B:A:W) colour Rf (B:A:W) colour Rf (B:A; 1 B.W.F. .05 B.W.F. .05 B.W.F. .06 2 b.B.F. .07 b.B.F. .09 - -3 l.Ab. .11 l.Ab. .12 Y.F. .11 if b.B.F. .15 b.B.F. .16 l.B.F. .18 5 l.Ab. .19 d.Ab. .21 l.Ab. .22 6 Y.W.F. .2h Y.W.F. .26 v.l.B.F. .25 7 l.M.F. .27 v.l.B.F. .31 M.F. .29 8 - - - - v.l.B.F. .32 9 - - - - v.l.B.F. 10 l.Ab. .52 v.l.Ab. l.Ab. M 11 v.l.B.G.F. .60 v.l.B.F. .57 b.B.G.F. .5^  12 v.l.B.F. .75 v.l.B.F. .75 — — 20. TABLE 3 - A comparison of materials precipitated by neutral lead acetate Common "Cumino" Dentata sweet clover sweet clover sweet clover Spot colour Rf(B:A:W) colour Rf(B:A:W) colour Rf(B:A:W) 1 b.B.F. .Oh b.B.F. .Oh b.B.F. .0^-2 b.B.F. .10 b.B.F. .10 b.B.F. .09 3 l.Ab. .16 l.Ab. .16 l.Ab. .13 h b.B.F. .20 b.B.F. .22 b.B.F. .18 5 v.l.B.F. .31 v.l.B.F. .3^  l.B.F. .29 6 v.l.B.F. M V.l.B.F. M l.B.F. .53 7 V.l.B.F. .77 v.l.B.F. .79 v.l.B.F. .72 TABLE h - A comparison of materials pre c i p i t a t e d by basic lead acetate Common "Cumino" Dentata sweet clover sweet clover sweet clover Spot colour Rf(B:A:W) colour Rf(B:A:W) colour Rf(B:A:W) 1 l.B.F. .01+ v.l.B.F. .Oh M.F. .06 2 l.O.F. .16 1.0.F. .16 Y.F. .1^ (25375) 3 - - - - d.Ab. .16C2537A) h - - - l.B.F. .20 5 v.l.B.F. .28 v.l.B.F. .28 d.Ab. .25C2537A) 6 v.l.B.F. ,h? 7 - v.l.B.F. .60 21. Spots, fluorescent and absorbing, seen when non-bitter and b i t t e r sweet clover seed extracts were compared under o u l t r a - v i o l e t i l l u m i n a t i o n ( 3 6 6 O A ) . TABLE 5 - A comparison of materials pre c i p i t a t e d by neutral lead acetate Common sweet clover "Cumino" sweet clover Spot colour Rf (B:A:¥) colour Rf (B:A:¥) 1 d.Ab. .Ik d.Ab. .13 2 v.l.Ab. .22 l.Ab. .2k 3 d.Ab. d.Ab. .3^  k v.l.B.F. .60 v.l.B.F. .53 5 v.l.B.F. .78 v.l.B.F. .7k TABLE 6 - A comparison of materials p r e c i p i t a t e d by basic lead acetate Common sweet clover "Cumino" sweet clover Spot colour Rf(B:A:W) colour Rf(B:A:W) 1 d.Ab. .2k d.Ab. .25 2 v.l.B.F. .82 v.l.B.F. .8k 22. TABLE 7 - A comparison of ether extracts Common sweet clover "Cumino" sweet clover Spot colour Rf(A:W) colour Rf (A:W) 1 d.Ab. .23 v.l.Ab. .16 2 v.l.B.F. .55 v.l.B.F. .51 3 v.l.B.F. .75 v.l.B.F. .72 h v.l.B.F. .80 l.B.F. . 8 2 5 d.Ab. . 8 8 v.l.Ab. . 8 8 6 l.B.F. .95 B.B.F. .96 General and s p e c i f i c q u a l i t a t i v e t e s t s , physical and chemical, i n d i c a t i v e of flavonoids and coumarins i n more or less pure state have appeared from time to time over a period of nearly a century and are now quite numerous. Swain (109), and Geissman (36), and others have provided summary statements on the usefulness of many of these tests and have recorded the behavior of a long l i s t of coumarins and flavonoids with these t e s t s . Furthermore, they have recorded t h e i r reaction on paper chromatograms. Spots then, on the chromatograms of the sweet clover extracts obtained above, were screened using a number of the qu a l i t a t i v e tests recommended as us e f u l by Swain (109) and Geissman (36). Only u l t r a - v i o l e t fluorescence, i n a c i d and al k a l i n e media, and diazonium tests are recorded below. How-ever, i t should be pointed out that only materials eluted and paper chromatographed several times were tested f o r the following records. 23. TABLE 8 - A comparison of two spots of possible int e r e s t isolated from "Cumino" sweet clover and Common sweet clover Test Untreated(U.V.) N ^ ( V i s u a l ) H H 3 ( U . V . ) 2N. NaOH (U.V.) 2N.HC1.(U.V.) Diazotized-P-nitro-a n i l i n e and Na2G03 "Cumino" sweet clover spot 10 Table 1 colour Blue colourless bright Blue Blue Green bright Blue l i g h t Purple Common sweet clover spot 9 Table 1 colour Blue colourless bright Blue Blue green l i g h t e r Blue l i g h t Purple 2h. TABLE 9 - A comparison of two add i t i o n a l spots of possible interest isolated from "Cumino" sweet clover and Common sweet clover Test "Cumino" sweet clover spot 7 Table 1 colour Untreated(U.V.) Bluish white Fluores-cence N h T 3(Visual) N H3(U.V.) 2N.NaOH(U.V.) 2N.HC1.(U.V.) Diazotized-P-nitro-a n i l i n e and Na2CC>3 Yellow Yellow Green Green bright Blue inconclusive Common sweet clover spot 7 Table 1 colour Bluish White Fluores-cence Yellow l i g h t yellow Green b.yellow(also visual) Blue inconclusive 25. TABLE 10 - A comparison of additional spots isolated from "Cumino" sweet clover and Common sweet clover Test "Cumino" sweet clover spot h Table 1 colour UntreatedCU.V.) light Yellow Fluores-cence If H 3(Visual) N H3(U.V.) 2N.?aOH(U.V.) 2N.HC1.(U.V.) Diazotized-P-nitro-aniline and Na 2C03 No change No change bright-yellow Fluorescence quenched inconclusive Common sweet clover spot 5 Table 1 colour Mauve Fluorescence No change Violet Blue Violet inconclusive 26. TABLE 11 - A comparison of d i e t h y l ether extracts of non-bitter and b i t t e r sweet clover l e a f when the developed chromatograms are treated with sodium carbonate and diazotized-p-nitro a n i l i n e Common "Cumino" Dentata sweet clover sweet clover sweet clover Spot colour Rf (A:W) colour Rf (A:W) colour Rf (A:W) 1 - - - - n i l .19 2 - - - - n i l .26 3 n i l .37 - - n i l .36 k n i l n i l n i l A 9 5 n i l .59 - - n i l .57 6 - - - - n i l .62 7 n i l .65 n i l .70 Blue .69 8 - - - - Mauve .76 9 n i l .82 - - Mauve .82 10 n i l .88 l i g h t purple .86 Mauve .86 11 _ _ n i l .9h Mauve .92 27. TABLE 12 - A comparison of u l t r a - v i o l e t absorption spectra of compounds of possible inte r e s t i s o l a t e d from d i e t h y l ether extracts (Table 1) Common sweet clover "Cumino" sweet clover Spot minima maxima Spot minima maxima 9 2h5 270 10 250 270 7 2k$ 270 7 253,303 285,327 k 25^ ,265 258,271 See Figures 1, 2, 3, k. Examination of Tables 1, 2, 3, and k reveals the following: a) that procedural neutral lead acetate precipitates from b i t t e r and non-bitter sweet clovers have a very s i m i l a r composition. b) that procedural basic lead acetate p r e c i p i t a t e s , from b i t t e r and non-bitter sweet clovers, show very small com-po s i t i o n a l differences. c) that procedural d i e t h y l ether extracts from b i t t e r and non-bitter sweet clovers, as indicated by u l t r a - v i o l e t fluorescence and Rf. values showed marked differences. d) that residues from b i t t e r and non-bitter sweet elovers, as indicated by u l t r a - v i o l e t fluorescence and Rf. values, showed marked differences. Substance, roughly i s o l a t e d by paper chromatography, eluted, and further p u r i f i e d by paper chromatography, and exa-mined by comparative spot t e s t s , supported the fact that c e r t a i n differences i n b i t t e r and non-bitter sweet clovers i n Figure 1 - U.V. absorption spectrum of "Cumino" sweet clover spot 10 Table 1 220 240 260 280 300 320 340 360 380 400 WAVELENGTH (Mu) Figure 2 - U.V. absorption spectrum of Common sweet clover spot 9 Table 1 220 240 260 280 300 320 340 360 380 400 WAVELENGTH (Mu) 28. the procedural diethyl ether soluble fraction are to be a t t r i -buted to different, but possibly related, compounds. However the behaviour of compounds or compound represented by spots 7 in Common and "Cumino" sweet clovers (Table 1) suggests caution, for in neutral media differences in Rf. values and fluorescence were very slight; in 2 N.NaOH, though a marked difference in fluorescence occurred. Closer study of spots 9 - 1 0 (Table 1) (Table 8) did not support a possible d i f -ference at this chromatographic locus. On the other hand a difference in the region of spots *f and 5 (Table 1) (Table 10) seemed to be substantiated. Support for a difference in the nature of the compounds in the region of spot 7 (Table 1) was found in the ultra-violet absorption spectra (Table 1 2 ) . Comparisons of chromatograms of diethyl ether extracts from Dentata and Common sweet clover, treated with sodium car-bonate and diazotized-p-nitro aniline, (a general test for coumarins and many other compounds) showed differences in colour in the "Rf region" where many coumarins are found (Table 11) . Comparative chromatograms also showed many fluorescence differences at l o c i which not necessarily showed differences in the diazo reaction (Table 1 ) . D Discussion of results The search for differences in coumarin metabolism in bitter and non-bitter sweet clover is like looking for the proverbial "needle in the haystack." The differences may be Figure 3 - U.V. absorption spectrum of "Cumino" sweet clover spot 7 Table 1 220 240 260 280 300 320 340 360 380 400 WAVELENGTH (Mu) Figure h - U.V. absorption spectrum of Common sweet clover spot 7 Table 1 220 240 260 280 300 320 340 360 380 400 WAVELENGTH (Mu) 29. established early i n a metabolic sequence, i n which case one might expect the b i t t e r coumarin of Common sweet clover to be "replaced" by compounds i n non-bitter sweet clover which d i f f e r quite markedly chemically from coumarin. On the other hand, as has been pointed out, the inheritance of "non-bitterness" i s f a i r l y simple and, i n general, i n other metabolic changes r e l a t e d to simple gene differences, the chemical change i s f a i r l y simple, too, e.g. addition or loss of -OH groups, ad-d i t i o n or loss of -OCH^ groups. Moreover, the "Cumino" "non-b i t t e r character" was transfered to Common sweet clover and then repeated backcrossing to Common sweet clover followed. Thus, "Cumino" should genetically be very similar to "Common" except f o r the "non-bitter" character; i t follows then that the chemical differences revealed on chromatograms etc. are very l i k e l y to be associated with the coumarin metabolism of these legumes. On the other hand many differences i n morphology and physiology exist between non-bitter species M. dentata and b i t t e r species M. alba and M. o f f i c i n a l i s . Nevertheless the non-bitter character i n M. dentata i s the source of the non-b i t t e r character i n "Cumino". Therefore, i t i s reasonable to suppose that the common metabolism i n "Cumino" and "dentata" i s much the same. The coumarin metabolism of Common sweet clover i s f a i r l y complex even though the l i t e r a t u r e and breeders tend to emphasize only coumarin per se. However, i t should be remem-bered that even i n Common sweet clover the c l o s e l y r e l a t e d 30. m e l i l o t i c a c i d occurs with coumarin. Coumarin i s also found as the aglycone and glycoside, and coumaric a c i d or acids are concomitant compounds. Knowledge of the coumarin metabolism of Common sweet clover i s very probably incomplete. However the metabolic changes associated with the "non-bitter gene or genes" are probably revealed i n the production of new cou-marins; some support f o r t h i s view i s the appearance of cou-marins i n other legumes e.g. coumestrol i n a l f a l f a and ladino clover; a n g e l i e i n and p s o r a l i n i n Psoralea c o r y l i f o l i a ; p-Coumaric ac i d i n Dariesia l a t i f o l i a and T r i f o l i u m pratense. In conclusion i t would appear that d i e t h y l ether extracts are well worth studying since differences i n the sweet clovers have been recorded f o r these extracts and f u r -ther, these differences appear to be coumarin compounds. Ad-d i t i o n a l l y , i t would be of interest i n future investigations to examine these differences as i n d i v i d u a l i s o l a t e s . 31. IV INVESTIGATION II A search f o r coumarin metabolism i n a l f a l f a . The genus Medlcago, to which a l f a l f a belongs, and the genus Melilotus. to which sweet clover belongs, are c l o s e l y r e l a t e d taxonomically. The closeness of the r e l a t i o n s h i p would be accepted on almost any basis of scrutiny. Both genera contain species of major economic importance; i n the one genus, Melilotus. as has already been discussed, coumarin and i t s metabolites are of very considerable sig n i f i c a n c e ; on the other hand, as yet, no f i r m evidence of a coumarin metabolism has been recorded f o r a l f a l f a . An i n d i c a t i o n that a coumarin metabolism exists i n the a l f a l f a s (and other legumes), as well as i n the sweet clovers, comes with the discovery of the oestrogenieally active "coumestrol" i n a l f a l f a , by Bickoff and associates, which may involve a coumarin i n i t s biosynthesis. To determine whether or not other coumarins occur i n a l f a l f a became the objective of t h i s second study. Although coumarin per se occurs i n Common sweet clover to the extent of .5 to 1,5% of the dry forage, a coumarin counterpart for a l f a l -fa does not seem to be on record. I f i t does exist i t may not be easy to recognize f o r as has been pointed out, s p e c i f i c chemical and physical tests for coumarins, as d i s t i n c t from a number of other heterocyclic compounds, are not a v a i l a b l e . An entree to a possible coumarin metabolism of a l f a l f a seemed to off e r i t s e l f i n the record of a b r i l l i a n t l y f l u o r e s -cent, but reasonably stable compound, occurring i n a l f a l f a 32. seeds ( 2 1 ) . Qualitative t e s t s , other than fluorescence also gave some support to the b e l i e f that the compound f e l l into the general category of the compounds to be sought f o r . The report to follow i s e s s e n t i a l l y the record of the i s o l a t i o n from a l f a l f a and p a r t i a l characterization of t h i s one compound of probable i n t e r e s t . Adding somewhat to the im-portance to be attached to the i s o l a t i o n and characterization of the fluorescent compound i n a l f a l f a i s the fact that other forage legumes, such as red clover, white clover, etc. also carry b r i l l i a n t l y fluorescent compounds. These d i f f e r , very l i t t l e , probably, from the a l f a l f a compound and i t i s f a i r to surmise that characterization of the one w i l l tend to make easy the characterization of the others. It i s understandable, that some data comparing a l f a l f a i s o l a t e s and red clover and sweet clover i s o l a t e s are presented; p r i n c i p l e i n t e r e s t , nonetheless, focuses on the i s o l a t i o n and characterization of the major fluorescent compound of a l f a l f a . A Materials The species and v a r i e t i e s of forage used were as follows: a) Medlcago satlva v a r i e t y "Rhizoma" from the University of B.C. b) T r i f o l i u m pratense va r i e t y "LaSalle" from Macdonald College, P.Q. e) Melilotus alba. Canadian Common from Buckerfields Ltd., Vancouver, B.C. ' 33. B Methods Method I Approximately one hundred seeds of a l f a l f a , red clover, and sweet clover were placed on f i l t e r paper i n p e t r i plates. The paper was moistened with d i s t i l l e d water and the p e t r i plates incubated at 26°C. for a period of M-8 hours. The colour of the fluorescent material i f any, was then noted o o under long and short wave u l t r a - v i o l e t l i g h t (366GA and 2537A resp e c t i v e l y ) . Method II In order to compare the fluorescent compounds present i n several forage legumes, p a r a l l e l extractions were conducted on germinated and ungerminated seed of Rhlzoma a l -f a l f a , LaSalle. red clover and Common sweet clover. In the extracts i t was hoped that the main compounds responsible f o r the difference i n fluorescence could be crudely separated. One hundred grams of the seed of each species were germinated f o r h8 hours at 26°C, then ground i n a Waring blendor, autolyzed b r i e f l y , and extracted three times with 200 ml. of hot 70$ ethyl alcohol. The solutions were then f i l -tered and concentrated i n a f l a s h evaporator at 35°C to ap-proximately % of the t o t a l volume. The concentrate was then s t r i p e d on No. 3 Whatman f i l t e r paper and developed i n B:A:W solvent. Colours and Rf values were then noted under u l t r a -v i o l e t lamps. •3k. Methods I I I and IV Two methods are l i s t e d below since procedures f o r handling small quantities of plant material d i f f e r rather markedly from those f o r handling large quantities of plant material. I l l A micro i s o l a t i o n procedure 1. One hundred grams of a l f a l f a seed was ground i n a Wiley m i l l using a 20 mesh screen and shaken with 200 ml. of hot 70% ethyl alcohol f o r 12 hours. The extraction was repeated with two portions of alcohol. 2. The crude extract was then concentrated i n a f l a s h evaporator to approximately 10% of the t o t a l volume and then extracted with 200 ml. of d i e t h y l ether i n a l i q u i d - l i q u i d extractor for .12 hours. The ether ex-t r a c t was then reduced to approximately 5% of the t o t a l volume i n a f l a s h evaporator at 30°c« 3. The ether extract was then striped on Whatman Uo. 3 f i l t e r paper and developed i n B:A:W solvent. The papers were dried and observed under u l t r a - v i o l e t lamps to locate the blue-fluorescence. h. The blue-fluorescent bands were then cut out and eluted with 70% ethyl alcohol. The eluate was rechromatogramed and eluted three times to p u r i f y . 5. After the f i n a l elution the alcohol solution was dried and the material taken up i n a minimum quantity of hot water. The water solution was then treated with con-centrated hydrochloric acid, concentrated, cooled, and the f i n e yellowish c r y s t a l l i n e p r e c i p i t a t e c o l l e c t e d . 35. (Diagrammatic presentation of procedure, Figure 5 . ) IV A macro i s o l a t i o n procedure. 1. Two kilograms of a l f a l f a seed were ground i n a Wiley m i l l using a 20 mesh screen and immediately extracted with approximately 16 l i t e r s of hot water f o r three hours. The crude water extract was reduced i n volume to approximately h l i t e r s . 2. The concentrated extract was then extracted with a t o t a l of 800 ml. of d i e t h y l ether i n a large l i q u i d - l i q u i d extraction u n i t . 3 . The crude c r y s t a l l i n e product formed during the extrac-t i o n procedure was c o l l e c t e d . h. The volume of the ether extract was reduced to approxi-mately 10$ of the t o t a l volume and l e f t i n a freezer f o r several weeks. At the end of t h i s time f a i r l y pure cry s t a l s were obtained. (Diagrammatic presentation of procedure, Figure 6 . ) Method V Colour tests and reactions. Many colour tests are avai l a b l e to aid i n the iden-t i f i c a t i o n of unknown heterocyclic compounds. A number of the t e s t s that proved u s e f u l i n t h i s investigation are presented here. a) F e r r i c chloride t e s t - 1.0 mg. of test material was d i s -solved i n 0 . 5 ml. of 70% ethyl alcohol and an alcohol solution of f e r r i c chloride was added and colours noted ( 1 7 ) . Figure 5 - Micro method for seed Grind - (20 mesh) Extract - (70% EtOH) Reduce volume (in vacuo @ 35°C.) Continuous Liquid-Liquid extraction (H 20-Et 20) H^O phase E t 2 0 phase - reduce volume discard Chromatograph (Bu:HAc:H20 solvent) Elute (EtOH 70%) Remove EtOH Dissolve residue i n minimal hot H 20 Add cone. HCl Reduce volume, cool Collect c r y s t a l s of the hydrochloride Figure 6 - Macro method for seed Grind - (20 mesh) Extract - (Hot water) Reduce volume ( i n vacuo @ 35°C.) Continuous Liquid-Liquid extraction (^CMirt^O) H 20 phase Et2© phase Volume reduction i n vacuo Collect crystals C o l l e c t c r y s t a l s i n freezer R e c r y s t a l l l z a t ion 36. b) Neutral and basic lead acetate test - a saturated aqueous solution of both reagents was added to a dilute alcoholic solution of the test material and colours of the precipi-tate noted (35). c) NaOH test - a few drops of 1.0 N. NaOH solution were added to an alcoholic solution of the test material and the colour of the resulting solution noted (91). d) Na^CO^ test - a few drops of 10% Na2C03 solution were added to the alcoholic test solution and the colour noted. e) HCl test - a few drops of concentrated HCl were added to the alcoholic test solution and the colour noted. f) HgSO^ . test - a few drops of concentrated HgSO^ were added to the alcoholic test solution and the colour noted (53, 70). g) HNO^  test - a few drops of concentrated HNO^  were added to the alcoholic test solution and the colour noted (8*+).. h) Mg - HCl test - 1.0 mg. of the test material was dissolved in 1.0 ml. of 70% ethyl alcohol; a small piece of mag-nesium ribbon was added and then a few drops of concen-trated HCl. The formation of colours was noted for a period of fifteen minutes (79, 90, 96). i) Zn - HAc - HCl test - 0.50 grams of screened, graded zinc was added to 1.0 ml. of a water solution of the test material (0.5 mg. per ml.). To this mixture 2.0 ml. of acetone was added, followed by 1.0 ml. of glacial Acetic acid. The mixture was then stirred at 65°C. for ho minutes. 1.0 ml. of concentrated HCl was added slowly and the 37. colour formed i n 15 to 20 minutes noted (6, 37, 38, 39, 79). J) Diazotized - p - n i t r o a n i l i n e t e s t - t h i s test was con-ducted as outlined i n Investigation I (Methods). k) B o r i c - c i t r i c acids test - the boric a c i d and c i t r i c a c i d solutions were prepared as outlined by Wilson (126) and Taubbck (111). 0.5 mg. of the t e s t material was dissolved i n 1.0 ml. of dry acetone and h a l f put i n the b o r i c - c i t r i c a c i d solution and h a l f put i n the c i t r i c a cid s o l u t i o n . The colour i n the b o r i c - c i t r i c a c i d solution was compared with that i n the c i t r i c a c i d solution. 1) Choline-tetraphenyldiboroxide t e s t - some aqueous choline s o l u t i o n was placed on a watch glass and a few drops of tetraphenyldiboroxide (as prepared by Neu (72) ) was added. Then a few drops of the test s o l u t i o n (0.25 mg. i n 1 ml. of methanol) was added and the colour i n daylight and i n u l t r a - v i o l e t l i g h t noted (73). m) HH^ test - the compound on a chromatogram was exposed to ammonia fumes and the colour i n daylight and i n u l t r a -v i o l e t l i g h t noted. Method VI pH fluorescence A rapid method to assess the reaction of a compound to changes i n the hydrogen ion concentration i s presented below. An array of solutions of d i f f e r e n t hydrogen ion con-centrations as outlined by Goodwin and Eavanagh k5) was 38. prepared and a few mis. of each solu t i o n was placed i n p e t r i plates. Pieces of No. 3 Whatman f i l t e r paper containing the test compound were cut up and fl o a t e d on the pH solutions. The reaction as observed under u l t r a - v i o l e t l i g h t was noted im-mediately. Method VII A n a l y t i c a l data derived from the p u r i f i e d c r y s t a l l i n e substance. a) Melting point determination - a few micrograms of the c r y s t a l l i n e test material were spread on a s l i d e , covered and observed on a "Kofler Micro Hot Stage". b) U l t r a - v i o l e t absorption spectrum - the compound was d i s -solved i n spectral grade isopropyl alcohol (3 mg./l.) and the u l t r a - v i o l e t spectrum measured using 3 ml. s i l i c a cuvettes i n a Beckman DK-2 spectrophotometer. c) Infra-red absorption spectrum - 1.0 mg. of c r y s t a l l i n e material and 1.0 gram of potassium bromide (anhydrous) were thoroughly mixed and put into a p e l l e t i n g u n i t . The p e l l e t , prepared using a pressure of 20,000 l b . per square inch, was a c i r c u l a r translucent sheet of K Br and the c r y s t a l l i n e material. The p e l l e t was then inserted i n a Beckman I.R.3 spectrophotometer and the recording made. d) Rf data - the Rf of the i n d i v i d u a l spots was measured using a chromatogrid d i s t r i b u t e d by the C a l i f o r n i a Foun-dation f o r Biochemical Research, Los Angeles 6 3 , C a l i -f o r n i a , U.S.A. e) Carbon-hydrogen analysis - eight mg. of the p u r i f i e d 39. c r y s t a l l i n e product was sent to Clark Micr©analytical Laboratories, 10k£ West Main St., P.O. Box 17, Urbana, I l l i n o i s , U.S.A., f o r t h i s analysis, f ) Nitrogen, Sulphur and Halogens - these tests were conducted according to McGookin (68). C Observations and r e s u l t s The r e s u l t s are tabled (Tables 13 - 19) and need l i t t l e supplementation or complementation. The occurrence of a d i s t i n c t i v e b r i l l i a n t l y -f l u o r e s c i n g compound or compounds associated with germinating a l f a l f a seed i s substantiated i n Table 13: germinating red clover and sweet clover seed, by contrast, did not fluoresce. Nonetheless, i t i s notable that, through the method II (Table l 1 * ) , the presence of alcohol-soluble fluorescent com-pounds i n the l i v i n g or autolyzed tissues of red clover and sweet clover i s established. Table 1*+, furthermore, estab-l i s h e s the occurrence of fluorescent compounds i n hydrolysate and autolysate of germinated seed which do not occur i n the non-germinated seed. The recognition of the major fluorescent compounds d i f f u s i n g to f i l t e r paper or other media from germinating a l -f a l f a seed presents no sp e c i a l d i f f i c u l t y ; they appear to be two i n number and one of these i s the most b r i l l i a n t and probably the most abundant. The p r i n c i p l e fluorescent and absorbing spots seen, ho. when comparable ehromatograms were examined under u l t r a - v i o l e t l i g h t are given with t h e i r approximate Rf. values i n Tables 13 - 16. TABLE 13 - Colour of diffusates from germinating legume seed under v i s i b l e and U.V. l i g h t Seed a l f a l f a sweet clover red clover colour i n V i s i b l e l i g h t yellow exudate yellow exudate yellow exudate colour i n U l t r a - v i o l e t l i g h t bright bluish-fluorescence no fluorescence no fluorescence TABLE l*f - A comparison of fluorescent spots i n germinated and ungerminated a l f a l f a seed extracts from method II Germinated Not Germinated Spot colour Rf (B:A:W) colour R f ( B J A 1 d.Ab. .08 l . B . F . .07 2 W.F. .13 Y.W.F. .12 3 d.Ab. .20 - -if l . B . F . .25 - -5 d.Ab. .37 d.Ab. .35 6 b . B.F. .60 • b . B.F. .6h 7 d.Ab. .79 d.Ab. .83 hi. TABLE 15 - A comparison of fluorescent spots i n germinated and ungerminated Common sweet clover seed extracts from method II Spot 1 2 3 h 5 6 7 8 9 10 11 Germinated colour Rf(B:A:W) Not Germinated colour Rf(B:A:W) v.l.B.F, Y.F. d.Ab. l.B.F. l.Y.F. l.Ab. l.B.F. l.B.F. l.B.F. d.Ab. .05 ,2h .26 .35 .ho .h2 .h8 .61 .82 .90 Y.W.F. d.Ab. v.l.Y.F. d.Ab. l.B.F. l.B.F. l.B.F. d.Ab., .07 .lh .18 .25 .37 .69 .90 k2. TABLE 16 - A comparison of fluorescent spots i n germinated and ungerminated red clover seed extracts Germinated l o t Germinated Spot colour Rf(B:A:W) colour Rf (B:A:W) 1 d.Ab. .09 d.Ab. .08 2 - - l.B.F. .18 3 l.Ab. .21 - -h l.Y.F. .28 l.Y.F. . .25 5 l.B.F. • 32 l.B.F. .37 6 l.B.F. A8 - -7 - l.B.F. 8 l.B.F. .91 l.B.F. .85 For the most b r i l l i a n t compound, which was i s o l a t e d and c r y s t a l l i z e d by several methods, aforementioned, the pH-fluorescence data are given i n Table 17. The wide range of pH over which the compound fluoresces i n the u l t r a - v i o l e t and the attendant colour changes are notable. TABLE 17 - pH - u l t r a - v i o l e t fluoresce response of a l f a l f a i s o l a t e PH Materials Fluorescence -1.6 8.0 N. H 2S G l + Y.G.F. -0.7 *+.0 N. H 2S 0 l + l.Y.G.F. 0.3 1.5 N. E 2 S \ 1.Y.G.F. 1.3 0.1 N. H^O^. B.F. 2.2 98 ml. 0.1 M. c i t r i c acid and 2 ml. 3.0 h.o 5.0 6.0 7.0 8.0 10. h 11.0 11.7 12.6 0.2 N, IfegHPO^ 79A5 ml. 0.1 M. c i t r i c a c i d and 20.55 ml. 0.2 N. Na2HP0^ 61.1+5 ml. 0.1 M. c i t r i c a c i d and 38.55 ml. 0,2 I, Na^PO^ 1+8.5 ml. 0.1 M. c i t r i c a c i d and 51.5 ml. 0.2 5. N a ^ O ^ 36.85 ml. 0.1 M. c i t r i c a c i d and 63.15 ml. 0.2 N. Na^PO^. 17.65 ml. 0.1 M. c i t r i c a c i d and 82.35 ml. 0.2 N. lagHPO^ 2.75 ml. 0.1 M. c i t r i c a c i d and 97.25 ml. 0.2 N. Efa2HP0^ 0.02 M. Na^PO^ 0.2 N. NagHPO^ and 2 M. Na^O^ 0.2 M. Na 2C0 3 0.01 N. NaOH 0.1 N. NaOH B.F. - Blue Fluorescence l.Y.G.F. - l i g h t Yellow Green Fluorescence Y.G.F. - Yellow Green Fluorescence b.Y.G.F. - bright Yellow Green Fluorescence B.F. B.F. B.F. B.F. B.F. l.Y.G.F. Y.G.F. Y.G.F. b.Y.G.F. b.Y.G.F. Y.G.(quenched) Y.G.(quenched) Characterization of the c r y s t a l l i n e product was materially aided by a series of spot t e s t s , the record of which i s given i n Table 18. The flavonoid nature of the com-pound i s indisputably established; furthermore the compound y i e l d s tests very c h a r a c t e r i s t i c of polyhydroxy flavones, v i z . 1) Mg -HCl test - t h i s test i s one of the most u s e f u l q u a l i -t a t i v e tests f o r the study of flavonoid compounds. The appearance of a pink to magenta colour indicates the pre-sence of flavonols, flavanones and flavanolones. Flavones which lack the 3 - hydroxy! group do not usually respond to t h i s t e s t . In addition, the shade of the colour that develops i s f a i r l y c h a r a c t e r i s t i c and three general cate-gories are noted flavones - Orange - red flavonols - red - crimson flavanones - crimson - Magenta 2) HH^ t e s t - the development of a bright yellow spot when a compound i s exposed to NH^ i s a f a i r l y r e l i a b l e test f o r c e r t a i n polyphenolic carbonyl compounds represented by the flavones, flavanones, chalcones and xanthones. This test i s very useful as a rapid means to determine the structures mentioned. 3) F ed3 test - the production of colours with f e r r i c chloride Is a general property of a l l classes of polyhydroxy flavonoid compounds. Ortho-dihydroxyl groups often give h5. green colours but t h i s generalization Is of l i m i t e d value because green colours are also given by many compounds which do not contain the ortho-dihydroxyl grouping. How-ever, the presence of a 3, 5 trihydroxy grouping i n the B ri n g can be detected because deep blue to black colours are produced with t h i s reagent. TABLE 18 - Spot test results given by the c r y s t a l l i n e i s o l a t e from a l f a l f a Name F e C l 3 neut. PMc basic PbAc NaOH ®& 2CG3 HCl HgSO^. HNO^ Mg-HCl Zn.-HAc-HCl Diazo B o r i c - c i t r i c Choline-t.P.b. NH_ Test dark green s o l . yellow ppte. yellow ppte. yellow on paper yellow on paper yellow s o l . bright yellow s o l . yellowish-brown sol, yellow immediately, red i n 10 sees. red i n 30 mins. no reaction bright yellow s o l . reddish brown s o l . yellow on paper U.V. Test greenish yellow greenish yellow d u l l yellow bright reddish orange bright yellowish green Remarks ++(polyhydroxy flavonoids) +(flavones) •(flavones) +(flavones and flavonols) +•(flavones and flavonols) +(flavones and flavonols) ++(flavonols, flavanones, flavanolones) 4 ( s l i g h t flavanone - contaminant?) -No structure l i k e simple coumarins •possible 5-OH gp. •possible 3-OH gp. +(flavones, flavanones, chalcones and xanthones) ±7. TABLE 19 - Physical and chemical data for the c r y s t a l l i n e i s o l a t e from a l f a l f a 19:1 Physical data 1. Colour - pale yellow 2. C r y s t a l form - t h i n needles 3. Melting point - 26l° - 262°C. 19:2 Microanalysis (organic) Carbon - 55.9$ Sulphur - n i l Hydrogen - .^18$ 1 Halogens - n i l Nitrogen - n i l 19:3 Rf data (Whatman No. 1 paper) (20°C.) Solvent Spot centre leading edge B:A:W (^-:1:5) -57 .69 organic phase HAc:H20 (1:3) AO A8 Isopropanol:H 20 (1:*+) .06 .11 1 9 I n f r a - r e d spectrum f o r the a l f a l f a i s o l a t e Please see Figure 7 Graph A. Interpretation of the spectrum indicates that the compound contains a carbonyl at the h position and several hy-droxyl groups, s i m i l a r i n many respects to dihydroquercetin (Figure 7 Graph B). A d d i t i o n a l l y , there i s some i n d i c a t i o n that the carbonyl i s chelated with a group at the 3 posi t i o n on the pryone r i n g . Figure 7 Graph A - a l f a l f a i s o l a t e . absorption spectra Graph B - dihydroquercetin PERCENT TRANSMITTANCE PERCENT TRANSMITTANCE »+8. 19:5 U l t r a - v i o l e t spectrum for the a l f a l f a i s o l a t e Please see Figure 8. Maxima Minima 21+1.5 232.5 26h.O 259.0 339.5 287.0 Numerous attempts to c r y s t a l l i z e the unknown com-pound i n such solvents as benzene, chloroform, and ethyl alcohol proved unsuccessful since only a brown s t i c k y residue was formed. After several attempts, approximately 3 . 5 mg. of a dark yellow material (a chloride s a l t ) was precipitated from a water solution treated with concentrated HCl. Because such small yields were obtained from t h i s method, a large scale extraction was attempted using d i e t h y l ether since i t was noted that the compound was sparingly soluble i n t h i s solvent. A crude c r y s t a l l i n e product (approximately 30 mg.) was collected during the extraction procedure, and a pure cry-s t a l l i n e product (approximately 15 mg.) was collected from the d i e t h y l ether extract when i t was reduced i n volume and l e f t i n a freezer f or several weeks. D Discussion An attempt to f i n d a coumarin metabolism i n a l f a l f a led to the study of two b r i l l i a n t l y fluorescing compounds which diffuse from germinating a l f a l f a seed. J u s t i f i c a t i o n Figure 8 - U.V. absorption spectrum of a l f a l f a i s o l a t e 0.9 L U o 0.8 0.7 0.6 £0.5 cn 0.4 C D < 0 .3 02 0.1 0 220 240 260 280 300 320 340 360 380 400 WAVELENGTH (Mu) ±9. for the attempt lay i n the somewhat li m i t e d evidence of spot t e s t s , fluorescence type, and the fact that a coumarin meta-bolism seemed to be widespread i n the legumes. The two compounds are undoubtedly related chemically for i n various procedures they appear together; t h e i r solu-b i l i t i e s , fluorescence, Rf values, response to spot tests are s i m i l a r . If one compound i s characterized very probably characterization of the other would not be d i f f i c u l t . Unquestionably the one compound chosen f o r study i s fla v o n o i d a l . Furthermore i t i s well hydroxylated, but not much more can be stated with certainty. The occurrence of a coumarin-like structure i n the molecule cannot be ruled out e n t i r e l y , but i t i s d e f i n i t e l y not a simple, more or l e s s , symmetrical coumarin. The u l t r a -v i o l e t spectral maxima f a l l within the ranges given by Geiss-man (36) f o r flavones; on the other hand i t i s to be noted that the U.V. spectral maxima f o r coumestrol also f a l l within t h i s range and caused i t s discoverers much concern. The in f r a - r e d spectrum i s most int e r e s t i n g and as was pointed out by H. McLean and G. M. Barton of the Forest Products laboratory, very c l o s e l y resembles that f o r dihydro-q u e r c i t i n , a flavanone. Some evidence of value i n characterization may be gained from the fac t that the a l f a l f a i s o l a t e i s b r i l l i a n t l y blue fluorescent; the fluorescence i s very s i m i l a r to that of the coumarins, umbelliferone, coumestrol and the flavone, 3:*+ - dihydroxy flavone. Dihydroquercitin and q u e r c i t i n are 5 0 . not fluorescent and appear brown i n u l t r a - v i o l e t . Examination i n the laboratory and i n publications, of physical and chemical properties of large numbers of coumarins and flavonoids does not y i e l d any whose properties are closely sim i l a r to those of the a l f a l f a i s o l a t e . The sum of a l l available evidence, some firm and some speculative, leads to the suggestion that the a l f a l f a i s o -l a t e i s a polyhydroxy flavone with the following approximate structure and formula. c±7Elh°7 51. V SUMMARY AND CONCLUSIONS The breeding of forage crops for modifications, losses or gains, of s p e c i f i c chemical constituents i s a comparatively new development i n forage crop breeding. One undertaking of t h i s kind, that of breeding the forage legume, sweet clover, free from the b i t t e r , and i n d i r e c t l y t o x i c , coumarin has met with some success. Success, the l i t e r a t u r e reveals, has attended the undertaking, only a f t e r many errors were made. Some of the mistakes could, very probably, have been avoided i f a better understanding of coumarin metabolism had been known. An understanding of how genes for bitterness and non-bitterness i n sweet clover act i n biochemical terms would provide valuable information for the plant breeder. L i t t l e i s known of the metabolism of coumarin and i t s r e l a t i v e s even i n sweet clover and i n related forage species such as a l f a l f a . From a survey of the l i t e r a t u r e i t was noted that a great deal i s known of the chemistry of coumarins, that a great deal i s known of coumarin pharmacology i n animals, that a l i t t l e i s known of coumarin as i t i s applied to non-coumarin containing plants, but that very l i t t l e i s known of the function and metabolism of coumarin i n the plant. In an attempt to determine the nature of the d i f -ferences i n the actions of genes for non-bitterness and 52. bitterness ( v i z . coumarin free and coumarin present), "Cumino" non-bitter sweet clover and, the genotypieally very s i m i l a r b i t t e r Common sweet clover were compared chromatographically. Compounds not well characterized, but which were very l i k e l y coumarin r e l a t i v e s appeared i n "Cumino" sweet clover. It i s suggested that genes for non-bitterness in,"Cumino" sweet clover, and very probably i n Melilotus dentata, may be responsible f o r methylation or hydroxylation of the coumarin per se and i t s close r e l a t i v e s which occur i n Common sweet clover. The coumarins, thus elaborated i n "Cumino" and M. dentata are non-bitter and are formed by simple additions to the coumarin r i n g or to i t s precursor. A search f o r a metabolic system involving coumarins i n a l f a l f a , a close r e l a t i v e , taxonomically, of sweet clover, l e d to the spe c i a l study of two b r i l l i a n t l y f l u -orescing compounds occurring i n seed and l e a f , which i t was thought, from preliminary examination, were coumarins. Detailed study however showed that the compounds were not l i k e l y coumarins as such, but polyhydroxy flavones, new to the l i t e r a t u r e and new to forage chemistry. The pro-posed formula f o r one of the two cl o s e l y related compounds i s as follows; 3:3 A trihydroxy - 5:7 dimethoxy flavone. 53. YI LITERATURE CITED 1. Andreae, W. A. 19*+8 The Is o l a t i o n of a Blue Fluorescent Compound Scopoletin from Green Mountain Potato Tubers, Infected with Leaf R o l l Virus. Can. J . Res. C. 26 : 31-3^ . 2. Andreae, S. R. and Andreae, W.A. 19*4-9 The Metabolism of Scopoletin by Healthy and Virus Infected Potato Tubers. Can. J . Res. C. 27 : 15-22. 3. Audus, L. J . 19^ 8 Studies on the Phytostatic Action of 2,^-dichlo-rophenoxyacetic A c i d and Coumarin. The Reversi-b i l i t y of Root Growth Inhibitions. New Phytol. M-7 : 196-219. h. Audus, L. J . and Quastel, J. H. 19*4-7 Coumarin as a Selective Phytocidal Agent. Nature. 159 : 320-32*+. 5. Avers, C. J . and Goodwin, R. H. 1956 Studies on Roots IV. E f f e c t s of Coumarin and Scopoletin on the Standard Root Growth Pattern on Phleum pratense. Amer. J . of Bot. *+3 (8) : .612-6207" ~ 6. Barton, G. M. and Gardner, J . A. F. 1958 Determination of Dihydroquercetin In Douglas F i r and Western Larch Wood. Anal. Chem. 30 (2) : 279-281. 7. Bate-Smith, E. C. 1956 The Commoner Phenolic Constituents of Plants and t h e i r Systematic D i s t r i b u t i o n . Scient. Proc. Roy. Dublin Soc. 27 : 165-176. 8. " ' ' , Swain, T. and Pope, G. S. 1953 The Is o l a t i o n of 7 - hydroxy - h methoxy Iso-flavone (Formononetin) from Red Clover ( T r i f o l -ium pratense) and a Note on the Identity of Pr a t o l . Chem. and Ind. 1127. 9. , and Westall, R. G. 1950 Chromatographic Behavior and Chemical Structure 1. Some Naturally Occurring Phenolic Substances. Biochim. Biophys. Acta, k: 5»K 10. Best, R. J. 19M+ A Fluorescent Substance Present i n Plants. 2. Isol a t i o n of the Substance i n a Pure State and i t s I d e n t i f i c a t i o n as 6 - methoxy-7 hydroxy -1:2 benzpyrone. A u s t r a l . J. Exp. B i o l . Med. 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