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Estrogenic activities of native and cultivated legume species Gammie, James Stuart 1974

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ESTROGENIC ACTIVITIES OF NATIVE AND CULTIVATED LEGUME SPECIES by JAMES STUART GAMMIE B . S c . (Ag r . ) U n i v e r s i t y o f B r i t i s h Co lumbia , 1970 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n the Department o f ANIMAL SCIENCE We accept t h i s t h e s i s as conforming to the r e q u i r e d staiTcfard THE UNIVERSITY OF BRITISH COLUMBIA June 1974 In presenting t h i s thesis in p a r t i a l f u lfilment of the requirements for an advanced degree at the University of B r i t i s h 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. I t i s understood that copying or publication of this thesis for fi n a n c i a l gain shall not be allowed without my written permission. James Stuart Gammie Department of ANIMAL SCIENCE The University of B r i t i s h Columbia Vancouver 8, Canada ABSTRACT i i Examination of Vicia amen'cana and Astragalus miser var.  serotinus for estrogenic and anti-estrogenic act iv i ty demonstrated low potency uterotrophic compounds interfering with synthetic hormones in mammalian reproductive tracts. The potency of the extracts was affected by stage of growth. A toxic fraction was present in Astragalus miser var. serotinus at fu l l bloom stage. Hormonal act iv i ty was not correlated with proximate analysis results for both species. Examination of the extract components revealed the overall structures of phenolic and aromatic compounds, including isoflavones, in a dynamic state throughout the growing season. The effects of topical f e r t i l i z e r application on alsike clover (Trifolium hybridum), ladino clover (Trifolium repens var. ladino) and a l f a l f a (Medicago sativa) indicated that dry matter y ields were s l ight ly but ins igni f icant ly affected by f e r t i l i z e r application. A method for the quantitative analysis of the free estrogenic isoflavones biochanin A and genistein was developed. Estimation of these two isoflavones, in addition to coumestrol and formononetin, i l lust rated that N, P and K applications would s ignif icant ly affect the level of these plant sterols in the legume species. Alsike clover increased in total isoflavone content with phosphate addition; ladino clover increased total isoflavones to phosphate deficiency and complete f e r t i l i z e r s ; a l f a l f a did not respond to f e r t i l i z e r treatment. Total estrogenic compounds on a dry matter basis were less affected in the three species than were individual isoflavone components. i i i TABLE OF CONTENTS CHAPTER PAGE I. INTRODUCTION 1 I I . LITERATURE REVIEW 4 The Occurrence of Flavonoids in Nature 4 The Biosynthesis of Plant Flavonoids 5 Flavonoids in Animals 7 Phenolic Compounds -- Plant and Animal Distribution and Relations 9 Functions of Flavonoids in Plants . . . 10 Plant Phenols as Nutritive Sources 10 Phenols and Plant Pathogens 10 Plant Phenols and Plant Growth 11 The Occurrence of Phenolic Compounds in Animals 12 Distribution 12 Phenolic Compounds in Animals; Estrogens in Growth and Reproduction 13 Hormonal Control of Reproduction 14 The Bioassay of Estrogens 15 The Occurrence of Plant Hormones Affecting Animal Reproduction 18 His t o r i c a l 18 Estrogens Isolated from Plant Forage Species . . 19 Histological Changes of the Mammalian Reproductive Tract from the Effects of Plant Estrogens 20 CHAPTER i v PAGE Beneficial Effects of Plant Estrogens i n Animal Growth and Finishing 22 Anti-Estrogenic A c t i v i t y of Forages 23 The Effects of Plant Extracts on P i t u i t a r y Function 27 Metabolism of Plant Estrogens by Animals . . . . 28 Factors Affecting the Estrogenic A c t i v i t i e s of Plants 31 Location 31 Season and Growth Stage 31 Varietal Variation in the Estrogenic A c t i v i t y of Plants 32 The Effects of Defoliation on Estrogenic Potencies of Legumes 33 The Effects of Drying on the Estrogenic A c t i v i t y of Legumes 34 The Effects of Plant Pathogens on Estrogenic Potency of Plant Species 36 Mineral Elements and Plant Estrogen A c t i v i t y . . 37 I I I . HORMONAL ACTIVITY OF TWO SPECIES OF NATIVE B.C. RANGE LEGUMES 41 Introduction 41 Materials and Methods 42 Plant Material 42 Extraction Procedure 45 Bioassay for Assessment of Biological A c t i v i t y . . 45 Separation Procedure of Plant Extracts 47 Ul t r a v i o l e t Absorption Spectra of Plant Extracts 49 V CHAPTER PAGE Ul t r a v i o l e t Spectra in the Presence of Selected Reagents 49 Infrared Spectroscopic Examination of Plant Extracts 51 Results and Discussion 51 IV. THE EFFECTS OF FERTILIZER TREATMENTS ON THE ESTRO-GENIC COMPONENTS OF ALSIKE CLOVER (TRIFOLIUM T HYBRIDIUM) WHITE CLOVER (TRIFOLIUM REPENS VAR. LADINO) AND ALFALFA (MEDICAGO SATIVA VAR. VERNAL) . . . 69 Introduction 69 Materials and Methods . 70 Seed, Plot Preparation and Layout 70 Seeding Rates and F e r t i l i z e r Treatments 72 Seeding and F e r t i l i z e r Application -- Plot Maintenance Procedure 72 Harvesting and Plant Storage 73 Fractionation of Plant Material 73 Thin Layer Chromatography of Plant Extracts . . . 73 Quantitative Determination of Estrogenic Constituent of the Plant Extracts 74 Results and Discussion 76 Summary and Conclusions . . 82 Experiment I 82 Experiment I I . . . . 83 REFERENCES 85 APPENDIX 97 vi LIST OF TABLES TABLE PAGE 1. THE RELATIVE DOSE RATE OF ANIMAL ESTROGENS TO PRODUCE EQUIVALENT RESPONSES IN THE OVARECTOMIZED RAT UTERINE WEIGHT TEST 17 2. THE POTENCIES AND FORAGE CROP DISTRIBUTION OF THE ESTROGENIC AROMATIC COMPOUNDS AS ASSESSED BY MOUSE BIOASSAY 24 3. PROXIMATE ANALYSIS OF VICIA AMERICANA AND ASTRAGALUS MISER VAR. SEROTINUS . . 53 4. ETHER AND CHLOROFORM EXTRACT WEIGHTS (GMS) OF VICIA AMERICANA AND ASTRAGALUS MISER VAR. SEROTINUS (350 GM DRY MATTER SAMPLES) 54 5. THE EFFECTS OF ETHER AND CHLOROFORM EXTRACTS ON THE UTERUS OF THE LABORATORY RAT (EXPRESSED AS % OF BODY WEIGHT) 55 6. ABSORPTION MAXIMA AND THE EFFECTS OF REAGENTS ON THE ULTRA-VIOLET ABSORPTION SPECTRA OF COMPOUNDS ISOLATED FROM ETHER AND CHLOROFORM EXTRACTS OF VICIA AMERICANA AND ASTRAGALUS MISER VAR. SEROTINUS 58 7. THE EFFECTS OF FERTILIZER TREATMENTS ON THE DRY MATTER YIELDS OF RANDOMLY GROWN 1.8 x 3.1 M PLOTS OF ALSIKE CLOVER, LADINO CLOVER, AND ALFALFA 78 8. EFFECTS OF FERTILIZER TREATMENTS ON ESTROGENIC COMPONENTS OF LEGUME SPECIES . . . . . . . 79 9. ANALYSIS OF VARIANCE FOR THE EFFECTS OF FERTILIZER TREATMENTS ON THE ESTROGENIC CONSTITUENTS OF TRIFOLIUM REPENS, TRIFOLIUM HYBRIDUM, AND MEDICAGO SATIVA 81 APPENDIX TABLE 1. ANALYSIS OF VARIANCE FOR THE EFFECTS OF FERTILIZER TREATMENTS VERSUS CONTROLS (FERTILIZER TREATMENT CROSSED WITH ESTROGENIC .CONSTITUENTS AND REPLICATES NESTED) 97 v i i LIST OF FIGURES FIGURE PAGE 1. The Major Flavonoid Classes Isolated from Plant Species ;la 2. The Distribution of the Flavonoid Classes i n Nature 6 3. Flavonoid Precursors and Biosynthetic Interrelation-ships of the Flavonoid Classes 8 4. The Major Estrogenic Compounds Isolated from Legume Species . 19a 5. The Major Estrogenic Isoflavones and th e i r Metabolic Products i n the Liver and Rumen 28a 6. Sampling Transects for Plant Species — Vegetative Stage 43 7. Sampling Transects for Plant Species --Seed;Pod Stage 43 8. Growth Stages for Analysis of V i c i a americana 44 9. Growth Stages for Analysis of Astragalus miser var. serotinus 44 10. Fractionation Procedure for the Extraction of Plant Estrogenic/Antiestrogenic Compounds 46 11. Plot Layout and F e r t i l i z e r Treatments 70a 12. Trifolium repens var. ladino. Random Design for F e r t i l i z e r T r i a l s 71 13. Chromatographic M o b i l i t i e s of Isoflavones and Coumestrol in Domestic Legumes . . . . . . . . . . . . . . . . 77 ACKNOWLEDGEMENTS The author wishes to thank Dr. W.D. K i t t s , Chairman of the Division of Animal Science for his guidance and assistance during the course of this project. Additional thanks are extended to Miss G. Wilson and Mr. D. Pearce for technical help and f i e l d work, as well as Mr. L. Dunn for s t a t i s t i c a l analyses and Mr. W. Foster for photographic assistance. The author also wishes to thank Dr. A.B. Beck and Dr. E.M. Bickoff for supplying pure isoflavone and coumestrol samples for the experiments. 1 CHAPTER I INTRODUCTION The phenolic compounds produced by a large number of plant species have been the subject of a great amount of investigation in the past f i f t y years. The flavonoids are the largest group of plant phenols; they possess closely related chemical and structural properties which r e f l e c t their unique biosynthetic pathways found only in the plant kingdom (Geissman 1962; Faust 1965; Margma 1970). The basic nine carbon skeleton (Figure 1) with attachment of acetate units results in highly s p e c i f i c products, many of which are res t r i c t e d to isolated families or genera of plants. Flavonoids and the i r derivatives have found wide application in the food industry: flavouring, colouration and usage as antioxidants and enzyme in h i b i t o r s i s well documented. Medicinal products, including potent central nervous system drugs and hormone precursors, are also included in the range of flavonoid products in commerce (Geissman 1962; Harborne 1967; Heftmann 1967). In the area of domestic livestock growth and reproduction, producers and researchers have observed for over forty years that many forage and pasture legume and grass species contain compounds with structures and a c t i v i t i e s resembling those of the animal estrogens (Pieterse 1956; Bickoff 1968). Ingestion of these plant materials at low concentrations has increased both growth and reproductive performance of ruminants; adverse e f f e c t s , including dystocia, trans-FLAVONOLS ISOFLAVONES Figure 1 The Major Flavonoid Classes Isolated from Plant Species. 2 ient and permanent i n f e r t i l i t y , have resulted from high levels of plant hormones in the forages. Bradbury and White (1954) confirmed reproduc-tiv e problems were caused by the isoflavone and coumestan classes of the flavonoids isolated from cultivated legume species. The occurrence of reproductive interference in animals by plant materials i s widespread, and i s not r e s t r i c t e d to s p e c i f i c locations and environmental influences (Samuel 1967). Many as yet unidentified compounds may act as pro-estrogens, being converted by rumen fermentations to active metabolites, whose effects could be beneficial or deleterious on animal reproductive tracts (Braden 'et aj_. 1967; Bickoff 1968). The commercial livestock industry has benefited from the rapid development and application of synthetic growth hormones in the f i n i s h i n g of marketable animals. Recent r e s t r i c t i o n s on the use of these growth promotants warrant.a closer examination into the beneficial properties of range and cultivated legumes in stimulating the production of high quality meat for the consumer. Research to date has indicated that the hormone-like a c t i v i t i e s of plants are extremely variable: environmental factors account for greater than ninety per cent of these alterations in a c t i v i t i e s . This two part study examined both the effects of stage of growth and f e r t i l i z e r n u t r i t i o n on the estrogenic potencies of legumes. Two widely distributed species of native plants used as forages -- V i c i a americana and Astragalus  miser var. serotinus, located on a primary beef production range, were assessed for t h e i r effects on animal reproductive tracts throughout the growing season. Correlations with n u t r i t i v e values, and examination of the compounds present in the plant extracts were also conducted. 3 The second part of the study involved the effects of f e r t i l i z e r applications on plant yields and estrogenic isoflavone l e v e l s . Three important cultivated legume species: a l f a l f a (Medicago s a t i v a ) , alsike clover (Trifolium bybridum), and ladino clover (Trifolium repens), were quantitatively assessed for estrogenic isoflavones when subjected to controlled f e r t i l i z e r applications. This study could present guide-lines for the forage producer in controlling estrogenic a c t i v i t i e s of legumes by the judicious use of f e r t i l i z e r treatments. 4 CHAPTER II LITERATURE REVIEW THE OCCURRENCE OF FLAVONOIDS IN NATURE The water soluble flavonoid pigments occur almost universally in higher plants, but are d i s t i n c t l y absent in lower orders. True flowering plants (angiosperms) synthesize pigments in stems and leaves; bacteria, algae, and fungi lack biosynthetic routes for flavonoids (Geissman 1962). Liverworts and mosses contain anthocyanin and flavonoids in mono and diglycoside forms; the ferns resemble the angiosperms in t h e i r pigment content, and possess the chalcone class — a primitive characteristic which l i n k s the woody (gymnosperms) and flowering plants (angiosperms). (Figure 2) The seven hundred species of gymnosperms contain a wide range of flavonoids: i.e. flavones, flavonols, flavanones and leucoanthocyanins; the majority occur as glycosides. Anthocyanin pigments con-s t i t u t e ; important colouring materials; imparting orange, s c a r l e t , crimson, mauve, blue, yellow and ivory to plants. These pigments have been located in almost every plant part: c o r o l l a , sepal, bract, stamen style and pod, leaves and stems. The anthocyanins show importance as taxonomic markers at the genus and family levels. Detailed descriptions of the flavonoid classes, th e i r d i s t r i b u t i o n s , and t h e i r role as taxonomic indicators are presented by Bate-Smith (1962) and Harborne (1962). 5 The Biosynthesis of Plant Flavonoids The cytoplasmic route for biosynthesis of plant flavonoids through the shikimic acid pathway to supply the Cg - a n d the gl y c o l y t i c pathway to supply the Cg has been well established (Faust 1965; Margma 1970). This process occurs mainly in green leaves; leaf synthesis occurs early in the chloroplasts; l a t e r photo reactions make flavonoid precursors available, and may promote Cg - Cg l i n k i n g , (Weinstein et al_. 1961; Rossiter and Beck 1967b). Flavonoid accumu-la t i o n i s isolated in the vacuoles. A pool of carbon substrates i s u t i l i z e d -- sucrose during early leaf growth, and carbon dioxide during leaf expansion phases. This carbon pool i s also u t i l i z e d in the production of c e l l protein, c e l l w a l l , starches, and in the respiratory cycle (Rossiter 1972). The combination of the carbon pool into the flavonoid skeleton i s re s t r i c t e d almost e n t i r e l y to the flowering and woody plants. The chalcone class of the flavonoids serves as an intermediate in flavonoid biosynthesis (Bate-Smith 1959.; Z i l g and Grisebach 1968). The coumestans and isoflavones are biogenetically related; coumestans belong to a class of isoflavones; the isoflavone daidzein serves as a precursor in the production of the coumestans; the rearrangement of the isoflavone molecule results i n the coumestan derivatives of the isoflavones (Goodwin 1965; Harborne 1967; Wong 1968). Figure 3 i l l u s t r a t e s the structural relationships and precursors of the flavonoids. The synthesis of the flavonoids i s under s p e c i f i c gene control, with a basic pigment pattern common to nearly a l l the higher 6 Phylogenetic Tree Angiosperms Gymnosperms Spermatophytes Ferns. Lycopods -Horsetails-mosses 'Red algae fungi bacteria Flavonoid Complement Complete range of flavonoids (biflavonyls rare) most flavonoids, but usually simple types. Biflavonyls characteristic s t r u c t u r a l l y simple flavonoids: 3-deoxyanthocyani ns flavones flavonols leucoanthocyanins chalcones flavanones few flavonoid types 3-deoxyanthocyanins flavonols. glycoflavones Flavonoids absent only Harborne (1967) Figure 2 The Distribution of the Flavonoid Classes in Nature 7 plants; genes controlling the biosynthesis interact with each other, competing for precursors (Geismann 1962). Gene mutations also block or increase the synthesis along existing alternate pathways, resulting in structural variations in the Cj 5 ring skeleton and in the number and attachment of the sugar residues (Harborne 1967). Flavonoids in Animals The phenolic compounds are rare in animals in comparison to their distribution in the plant kingdom. The animal body is capable of synthesizing phenyl propane units and phenylalanine and tyrosine; the ability to synthesize the flavonoid molecule is absent. With the exception of the isoflavones, degradation of flavonoid molecules by the hepatic system to m-hydroxylphenyl acetic acid and carbon dioxide for urinary and respiratory excretion occurs in the animal body (Williams 1964). Numerous insect species accumulate plant flavonoids through ingestion and failure to degrade to simpler products (Morris and Thompson 1963). Accumulation of flavones and anthocyanins occurs in insects, notably butterflies, where the larvae ingest Poa and Festuca, and accumulate flavonoid glycosides (Ford 1941). Snails, hydra polyps and earthworm ingestion and storage has been documented by Kubista (1950), Roots and Johnston (1966). The utilization of C 2 8 and C 2 g plant steroids by insects ( i .e., blowfly and tobacco hornworm) in the production of ecdysones for moulting, larval growth and metamorphosis has been reviewed by Robbins et al_. (1941). 8-sitosterol and fucosterol are converted to cholesterol which acts as a precursor for the ecdysones. Anthocyanin pigments may also be responsible Phenyl al an i n e Y Tyrosine S Flavones Aurones Cinnamic Acids 3 Malonate Chalcones + Stilbenes Anthocyanins Flavones Catechins Lignin Coumarins Benzoic Acids Isoflavones (After Goodwin 1965; Wong 1968) Figure 3 Flavonoid Pre-cursors and Biosynthetic Interrelationships of the Flavonoid Classes. 00 9 for the d i s t i n c t brown-violet colouration in weevil larvae, and qui nones for pigment patterns in aphid species. However, vertebrate species do not accumulate plant pigments to the same extent as insect species, and the majority are successfully degraded and eliminated from the animal body. Phenolic Compounds -- Plant and Animal Distribution and Relations The process of estrogen and androstane synthesis in animals occurs by the successive degradations of cholesterol. Side chain cleavages and hydroxyl group attachment produce pregnenolone (White et al. 1968). Both of these s p e c i f i c animal hormones have been located in plants (Bennett e_t al_. 1966). Pregnenolone conversion to progesterone was discovered i n the African shrub Holarrhena floribunda by Zalkowet al_. (1964); estrone and e s t r i o l have been isolated from Butea and willow catkins (Butenandt and Jacob 1933; Skarzynski 1933), and cholestrol from red algae species (Tsuda et al_. 1958). L i t t l e knowledge i s available on the function of these isolated animal steroids in plants. Plant and animal reproductive processes d i f f e r so widely that analagous a c t i v i t i e s seem unlikely for the steroids (Heftmann 1967). Various theories on the a c t i v i t i e s of animal steroids i n plants have been proposed, and include the effect of the hormones in increasing plant c e l l permeability to electrolytes and water (Hechter and Lester 1960), the stimulation of c e l l d i f f e r e n t i a t i o n (Karlson 1963), the control of the flowering process (Bonner e_t al_. 1963), and sexual development and d i f f e r e n t i a t i o n in the flowering plants and the fungi (Love and Love 1945; Mirocha et a]_. 1969). 10 FUNCTIONS OF FLAVONOIDS IN PLANTS  Plant Phenols as Nutritive Sources The phenolic flavonoids and their esters are generally stable end products, although active interconversions occur (Grisebach and Bopp 1959). Normally the products are not translocated from t h e i r chloroplastic sites of synthesis. Soil bacteria and fungi have evolved enzymes for cleavage and u t i l i z a t i o n of phenols for food resources (Towers 1964), although i t i s not clear i f higher plants can u t i l i z e these compounds as sources of n u t r i t i o n . Harborne (1967) presented evidence of the rapid oxidation of phenolic catechin glycosides by tea shoots; the sugar and aglycone was oxidized to y i e l d carbon dioxide. These results imply the use of flavonoids as respiratory substrates by plants. Phenols and Plant Pathogens-Cadman (1960) noted that tannins occurring in the bark of trees formed insoluble complexes with viruses: When the phenols were applied to roots and leaves at levels of 0.1 to 0.6%, fungus growth was retarded. The same author postulated that enzyme or hormone secretion by the fungi i n i t i a t e d and conditioned the action of the plant phenols, which in turn would i n h i b i t the fungal growth. The ultimate t o x i c i t y of the phenol would depend on i t s structure, concentration and d i s t r i b u t i o n when a fungus was present on the host plant. Steroids and their derivatives ( i . e . chlorogenic acid) occur 11 in Targe concentrations i n the c e l l sap of highly resistant species; i n h i b i t i o n of Venturia species in apple and pear v a r i e t i e s , and disease resistance in seeds, due to the presence of anthocyanins contained in the seed coats of Pisum arvense, have also been reviewed by Claus (1961). Plant phenols have been located accumulating in plant c e l l s and vacuoles adjacent to fungi infected c e l l s ; increased protein synthesis and respiration by tissues also occur simultaneously with this accumulation. Cruickshank (1962) indicated that attack by various fungal pathogens caused di f f e r e n t rates of synthesis of flavonoids; Cruickshank and Perrin (1963) theorized that the s u s c e p t i b i l i t y of plants to pathogens may be due to the i n a b i l i t y of the infecting fungus to stimulate flavonoid formation, or the a b i l i t y of the invading fungus to tolerate the phenol produced. The naturally occurring flavonoids in disease conditions could serve as precursors to more toxic products, i.e. Pisa t i n i s produced from flavonoid precursors only when disease conditions are present. Plant Phenols and Plant Growth Much speculation has been advanced regarding the role of plant phenolics and growth promotion. Harborne (1967) indicated that phenols were concerned in dormancy of seedlings, also in root and shoot growth — in i n h i b i t i n g and stimulating the indole acetic acid oxidase enzyme system. Stenlid (1963) proposed that a l l 4' hydroxy flavonoids ( i . e . kaempferol) were co-factors for the oxidation of 12 indole acetic acid oxidase and therefore growth i n h i b i t o r s ; 3' 4' dihy-droxy flavonoids ( i . e . quercitin) inhibited the destruction of indole acetic acid and were growth stimulators. Stenlid (1962) also reported that synthetic anthocyanidins at concentrations of 3 x 10~ 7 molar to -4 10 molar increased root growth i n wheat seedlings, and reversed the i n h i b i t i o n in root growth produced by the addition of indole acetic acid. P h i l i p s (1961) examined dormancy control by naringenin and found that naringenin could induce a l i g h t requirement in lettuce va r i e t i e s not normally l i g h t requiring for dormancy control. At concentrations of 40 to 80 milligrams per l i t e r , naringenin inhibited seed germination and competed with gibberelin for the control of dormancy in the same system. These results indicate the possible role of flavonoids in plant growth. THE OCCURRENCE OF PHENOLIC COMPOUNDS IN ANIMALS  Distribution In direct contrast to the plant kinggdom, the animal kingdom contains phenolic compounds in limited amounts; only a re s t r i c t e d number of structural types have been isolated. The essential amino acid tryptophan i s the most widely distributed phenol in the animal body; i t s role as a precursor for melanin, adrenalin and noradrenalin has been established. Tyrosine functions as the basis of the iodinated phenol thyroxine, the peptide hormone i n s u l i n , and the neurohypophyseal hormones oxytocin and vasopressin. The t h i r d 13 major class of tryptophan derivatives are the hydroxylated indole amines -- serotonin precursors, and catechol amines associated with the central nervous system, sympathetic nerve endings, and the adrenal medulla. Although the phenols are limited in th e i r synthesis to s p e c i f i c tissue systems in the animal body, many b i o l o g i c a l l y inactive compounds occur t r a n s i t o r i l y , being derivatives of catechols and amines, or intermediate breakdown products of dietary phenols. Phenolic Compounds in Animals; Estrogens' in Growth and Reproduction The development of secondary sexual characteristics and regulation of the reproductive cycle in the female are both d i r e c t l y affected by the output of estrogens and progestins produced primarily by the ovaries; secondary sources include the adrenal cortex and the placenta. The estrane series of steroids (C-jg) possess an aromatic "A" ring and hydroxy! grouping at Cg. Non-steroids possess estrogenic a c t i v i t y due to a phenolic function at the para position or a fluorine molecule at Cg, (Rosenberg and Dorfman 1958). Estrogenic potency also depends on water s o l u b i l i t y ; more soluble compounds are excreted rapidly and are less potent than non water soluble steroids. The profound changes caused by ovarectomy indicate the large influence of estrogens on the female reproductive tract. Pre-pubertal ovarectomy results in i n f a n t i l e genital tissues; the female cycles f a i l to appear. Post-pubertal ovarectomy results in the cessation of the menstrual cycle, uterine vaginal mucosal and fa l l o p i a n tube atrophy, with secondary sexual characteristics disappearing following ovarectomy. 14 The anabolic effects of estrogens are especially prevalent during the estrous cycle, and s p e c i f i c a l l y i n uterine and mammary tissue. Means and O-Malley (1972) isolated lipoprotein binder fractions s p e c i f i c for the uptake of estrogens in these tissue types; the phenolic, the non-polar portion and ring substitution a l l contributed to active binding, (Korenman 1969) resulting in highly s p e c i f i c uptake by the tissue of the estrane series. The s p e c i f i c anabolic effects of estrogens on the reproductive tract can be summarized: 1. Increased R.N.A. polymerase a c t i v i t y , R.N.A. synthesis and complementary protein formation which feeds back on the c e l l n u c l e i , amplifying gene action resulting i n increased protein synthesis; 2. P r o l i f e r a t i o n of vaginal e p i t h e l i a l c e l l s and the uterine endometrial l i n i n g ; 3. Increased cervical mucus secretion; 4. Direct competition for amino acids in gluconeogenesis for protein synthesis; 5. Increased water and f l u i d uptake by the uterine c e l l s . Hormonal Control of Reproduction Extensive review a r t i c l e s , (Hansel and Snook 1970; Cupps 1972; Jensen and DeSombre 1972), have confirmed the direct influence on estrogen production and gamete formation by the p i t u i t a r y gonadotropins -- f o l l i c l e stimulating and l e u t i n i z i n g hormones. With maturity, f o l l i c u l a r development, rupture, and progesterone secretion for pregnancy 15 maintenance occur regularly as a characteristic of the estrous cycle, in conjunction with the r i s e and f a l l of F.S.H./L.H. output. Anterior p i t u i t a r y output of the gonadotropins has been shown to be controlled by a feedback mechanism of estrogen and progesterone acting d i r e c t l y on receptor c e l l s in the basal hypothalamus. Jensen and DeSombre (1972) postulated that s p e c i f i c hypothalamic receptor site s bind with c i r c u l a t i n g estrogens, i n h i b i t i n g the secretion of hypothalamic releasing factors. This feedback mechanism results in reduced output of p i t u i t a r y gonadotropins; the system remains in constant balance and controls the levels of c i r c u l a t i n g plasma estrogens. The Bioassay of Estrogens The assessment of estrogenic a c t i v i t y has been based on the degree of vaginal c o r n i f i c a t i o n or increase i n uterine weight occurring when estrogens are administered o r a l l y , intravaginally or by i n j e c t i o n . Bickoff (1968) confirmed that for estrogen assessments, uterine weight increases indicate low concentrations of estrogens. These results are quantitative. Progesterone and androgens also react p o s i t i v e l y on uterine weight increases, as was noted by Biggers (1954). Moule et al.(1963) reviewed sixteen methods and variations of the bioassay technique. Lack of correlation exists in laboratory animals and grazing ruminants. Techniques involving sheep as test animals have been developed (Lamond and Southcott 1962; Francis and Millington 1965; Lindsay 1968). Moule et al_. (1963) indicated that vaginal c o r n i f i c a t i o n could be induced in ewes by low estrogen levels when primed with proges-terone; a s p e c i f i c method in which the test animals were not s a c r i f i c e d . 16 Optimal methods and factors affecting this method were examined by Lang and Lamond (1962; 1965). Increase in wether teat length (Braden et a l . 1964) can be used for quantitative estrogen effects over a narrow range; i t s s i m p l i c i t y makes the method a general indicator of estrogenic a c t i v i t y . The uterine weight increase applied d i r e c t l y to sheep has shown popularity for s t i l b e s t r o l and estrogen assay; t h i s method r e l i e s on the use of immature intact ewes and adult ovarectomized ewes and results can be correlated with wether bioassay results (Lamond and Lang 1965). Table 1 i l l u s t r a t e s the dose response of animal estrogens on the uterus of the ovarectomized rat. The i d e n t i f i c a t i o n of a s p e c i f i c estrogen binding 8S macromol-ecule fraction in the supernatant of rabbit uteri which showed steroid s p e c i f i c i t y for estrogens was described by Korenman (1968). These results indicated that a sensitive hormone assay method for i n v i t r o determination of the competitive binding a b i l i t i e s of uterine steroids was possible. Korenman (1969) noted that the r e l a t i v e binding a f f i n i -t i e s of plasma estrogens paralleled uterotropic a c t i v i t y . Peterson and Common (1972) described a radio immunoassay technique for estradiol u t i l i z i n g a synthetic antibody to estrogens. The competitive protein binding and radio immunoassay techniques are quantitative indices of estrogenicity which are independent of whole body e f f e c t s , and w i l l find further applications for the biological assessment of uterine steroid competitors. 17 TABLE 1 THE RELATIVE DOSE RATE OF ANIMAL ESTROGENS TO PRODUCE EQUIVALENT RESPONSES IN THE OVARECTOMIZED RAT UTERINE WEIGHT TEST Minimal Dose in Micrograms to Produce Hormone 33% Increase in Uterine Weight in 6.0 hours 70% Increase in Uterine Weight in 6.0 hours Maximum Uterine Weight Increase in 4.0 hours 17 6-Estradiol 0.025 0.100 0.150 E s t r i o l 0.029 0.078 0.039 Estrone 0.450 1.250 50.0 Equilin 0.312 1.250 5.0 Equilenin 0.546 0.625 50.0 D i e t h y l s t i l b e s t r o l 0.078 0.156 0.625 (From Dorfmann, 1962). 18 THE OCCURRENCE OF PLANT HORMONES AFFECTING ANIMAL REPRODUCTION His t o r i c a l During the 1940's, breeding abnormalities became widespread in sheep pastured on a European clover c u l t i v a r Trifolium subterraneaum in Western Aust r a l i a . Abnormally high pasture intakes of the clover due to low r a i n f a l l and f e r t i l i z e r shortages stimulated the outbreaks of i n f e r t i l i t y , with lambing percentages dropping to eight per cent. Bennetts ejt al_. (1946) suggested the association of the clover species with reduced reproductive performance; the isoflavone genistein (5,7,4 -trihydroxy isoflavone) was isolated from clover leaves; contents of the leaves reached levels of 0.70% on a dry matter basis. Genistein was confirmed to be estrogenically active by mouse bioassay (Curnow (1954). Curnow and Rossiter (1955) reported that more than 120 v a r i e t i e s of Trifolium subterraneum contained genistein. The c l a s s i c "clover disease" occurs after animal exposure to plant estrogens for six months or more. Pathological conditions including swollen and proliferated uterine and vaginal epithelium, hyperplasia of the uterine glands, c y s t i c u t e r i , enlarged and abnormal udder development are present. These conditions a l l indicate typical symptoms of excess estrogen stimulation on the reproductive t r a c t , and have been reviewed by Meyers (1951) and Moule et al_. (1963). Barrett et al_. (1965) indicated progressive decreases in lamb crops occurred when ewes ingested red clover pasture varieties for f i v e -year periods. Other researchers, (Morley et a]_. 1963; Clark 1965), 19 have shown that short term exposures to estrogenic pastures during breeding periods have resulted in reproductive f a i l u r e s which are d i f f i c u l t to diagnose as to causative agent; appreciable losses in offspring numbers are apparent. Many reports have appeared on the widespread occurrence of plant hormones affecting animal reproduction. Bradbury and White (1954) reviewed the early l i t e r a t u r e , and noted that greater than f i f t y species of plants contained estrogenic substances. In addition, review a r t i c l e s have appeared which indicate the d i s t r i b u t i o n of plant a c t i v i t i e s . These include: Bickoff (1968), Moule e t a l _ . (1963) in B r i t a i n , Samuel (1967) in the U.S.A., Symington (1965) in Rhodesia, K i t t s (1960) in Canada, and Bankov (1970) who reported on estrogenic pasture crops in the U.S.S.R. Estrogens Isolated From Plant Forage Species Forage compounds isolated and acting on animal reproductive tracts as uterotropic hormones include the coumestans: coumestrol and 4 methoxycoumestrol, and the isoflavones biochanin A, daidzein, formononetin, genistein and pratensin. Structures and potencies of these plant flavonoids are given in Figure 4 and Table 2. Table 2 i l l u s t r a t e s major d i s t r i b u t i o n of the forage estrogens. Coumestrol was f i r s t isolated from ladino clover by Bickoff e_t al_. in 1957; d i f f e r i n g in structure, but biogenetically related to the isoflavones. Mouse bioassay confirmed the potency of coumestrol as t h i r t y times the potency of genistein (Table 2), and f i f t e e n to one hundred times as 19a HO Biochanin - A 5,7 Dihydroxy HO OCH, C16H12°5 - 4' methoxy isbfalvone C16H12°4 Formononetin 7 Hydroxy - 4' methoxy isoflavone ( C 6 H 1 2 ) 5 ) 0 Q Daidzin ^21^20^9 7 Glucone of Daidzein Daidzein ^15H10^4 7,4' Dihydroxy isoflavone 0 Pratensein C16H12°6 Coumestrol C15H8°5 5,7,3' Trihydroxy —4'' methoxy isoflavone 3,9 Dihydroxy - "6H-benzofuro-[3,2-C][l]benzopyran 6-one Figure 4 The Major Estrogenic Compounds Isolated from Legume Species 20 active in sheep depending on administration route. The stilbene structure of coumestrol resembling d i e t h y l s t i l b e s t r o l accounts for part of this a c t i v i t y according to Bickoff (1968), as does the planar ring structure and similar distances between the hydroxyl groups as noted by Shemesh et aK (1972). Histological Changes of the Mammalian Reproductive Tract from the  Effects of Plant Estrogens The vaginal histology of ewes was examined by Bell and Sanger (1958) on estrogenic pastures. Cell d i s t o r t i o n , degeneration and clumping were common; these authors suggested.lowered f e r t i l i t y was due to the prevention of normal implantation of the f e r t i l i z e d ova. Sanger and Bell (1961) compared ladino clover effects on f e r t i l i z a t i o n of sheep ova; 59% of ova cleavage occurred with sheep on ladino clover pasture, 75% of ova cleavage occurred on blue grass pastures. Three explanations were advanced to explain the lowered f e r t i l i t y figures: 1. Inhibited f o l l i c l e formation; 2. Interference with sperm a c t i v i t y and t r a v e l ; 3. Failure of f o l l i c u l a r rupture (a remote p o s s i b i l i t y ) . Several researchers have advanced additional theories regarding the primary cause of i n f e r t i l i t y of animals on legume diet s . Engle (1957) indicated that ewes on ladino clover pasture experienced delayed estrus with conception occurring late in the cycle; 41% lambed to f i r s t service on ladino pastures; 66% of the controls on grass pastures lambed to f i r s t service. Barrett et al_. (1965) found a 25% lambing 21 rate of ewes on red clover d i e t s , and Turnbull et al_. (1966) noted the severity of c y s t i c endometrial hyperplasia s i g n i f i c a n t l y higher i n ewes on red clover pastures. Cyst numbers greater than 10 per cervix or uterus resulted in a lower proportion of animals pregnant post service. Turnbull et al_. (1966) indicated that embryonic death in the f i r s t s i x t y days contributed to i n f e r t i l i t y in animals with c y s t i c endometrial - hyperplasia. O'Brien (1971) examined sperm numbers recovered from the uteri and cervixes of ewes grazing on oat grass and red clover pastures. Sperm numbers of 350 were recovered from the red clover groups, while control animals yielded 17,160 mature spermatozoa per cervix. The author postulated that the major cause of i n f e r t i l i t y was n o n - f e r t i l i z -ation due to the absence of sperm numbers reaching the fal l o p i a n tubules. Reproductive f a i l u r e i s also p o s i t i v e l y correlated with the number of uterine cysts present, which are associated with hypertrophy and swelling of the cervical and uterine mucus membranes. This was conformed by Lightfoot et a l . (1967) and O'Brien (1971). Moule et al_. (1963) attributed early embryonic death before attachment of the f e r t i l i z e d embryo to the endometrial w a l l . Bauminger and Lindner (1969), using ovarectomized, progesterone treated rats and increased dosages of genistein, concluded that two phenol groups at 7 and 4' of genistein were essential for estrogenic a c t i v i t y . These authors concluded that genistein mimics many, but not a l l , of the actions of 17-g-Estradiol, in promoting uterine growth and water imbibition with resulting hyperplasia; but f a i l s to induce the implantation of the delayed blastocyst on the endometrial wall of the uterus. Thus, many hypotheses have been advanced to explain the actions of the plant phenolics on lowered f e r t i l i t y rates of grazing ruminants. 22 Beneficial Effects of Plant Estrogens in Animal Growth and Finishing The rapid r i s e in the application of synthetic estrogens for f i n i s h i n g livestock, and the recent removal of d i e t h y s t i l b e s t r o l from commercial f i n i s h i n g operations prompts investigation into the possible use of plant extracts as substitutes for the potent steroid analogues. Various experiments have been conducted to measure plant estrogen extracts on weight gains and carcass q u a l i t i e s . O l d f i e l d et al_. (1966) obtained positive effects with wethers; growth responses, gains, and meat quality were a l l improved on diets of crude a l f a l f a meals, acetone extracts of a l f a l f a , and coumestrol. George and Turnbull (1966) reported improved p a l a t a b i l i t y of lamb carcasses when animals received diets containing 114 ppm coumestrol. Varied and inconclusive results have been obtained with steers. Matsushima (1961) measured growth rates of steers on high coumestrol a l f a l f a d i e t s ; the gains obtained (1.09 kg/day) were equal to those animals receiving s t i l b e s t r o l implants without a l f a l f a diets. Matsushima also indicated that steers on diets of 25, 100 and 250 ppm of coumestrol showed no improvement in growth response; 10.0 mg of DES per day resulted in a positive response of 10% over control animals. George and Turnbull (1966) pastured intact Merino ram lambs-on control pastures of perennial rye grass (Lolium perenne L.) and white clover (Trifolium repens L.) u n t i l weaning. At weaning 38 animals remained on the grass dominant pasture, and 23 were placed on red clover (Trifolium pratense L.) dominant pasture, composed of 95% red clover. At twelve months of age, body.Weight-, t e s t i c u l a r weight, epididymal sizes and mature sperm numbers were a l l s i g n i f i c a n t l y (P < 0.05) greater in the red clover pastured animals. 23 Commercial extraction techniques for lucerne separation of coumestrol have been described by Bickoff (1961). I t i s obvious more research into the preparation of extracts and applied n u t r i t i o n programmes i s necessary to obtain maximum beneficial effects of the plant estrogens. Further work i s required to determine i f the beneficial effects of plant hormones override the deleterious a c t i v i t i e s on livestock. Two types of forages may have to be developed; one containing a low estrogen content for breeding stock, and the other with high estrogenic a c t i v i t y to promote growth and fattening in steers, wethers and poultry. Anti-Estrogenic A c t i v i t y of Forages Many forages contain factors which reduce the effects of the endogenous animal estrogens, producing a hypo-estrogenic syndrome. A single plant species may contain high levels of estrogenic isoflavones and coumestans, yet the i r biological effects are masked by a n t i -estrogens. Bickoff (1968) noted poor correlation between biological and chemical assays, due to the two fractions in a single plant species. Emmens (1965) established that a compound could act as an estrogen or an estrogen i n h i b i t o r , depending on dosage, while Folman and Pope (1966) and Shutt (1970) demonstrated that coumestrol and genistein could block estradiol and estrone a c t i v i t y ; Shutt (1967) suggested the weak estrogen could displace estradiol from the uterine receptor s i t e s . Bickoff et al_. (1960), studying lucerne, found that some samples which e n t i r e l y lacked estrogenic a c t i v i t y on uterine growth response contained compounds which inhibited the estrogenic 24 TABLE 2 THE POTENCIES AND FORAGE CROP DISTRIBUTION OF THE ESTROGENIC AROMATIC COMPOUNDS AS ASSESSED BY MOUSE BIOASSAY Compound Amount (Micrograms) to produce a 25.0 mg uterus (a) Relative Potency (b). Distribution Genistein 8,000 yg 1.0 A l f a l f a , Ladino clover, red 1 clover, Subterran-ean clover Biochanin A 18,000 yg 0.46 A l f a l f a , red clover, Subterran-ean clover Formononetin 32,000 yg 0.26 A l f a l f a , red clover, Ladino clover, Subterran-ean clover Coumestrol Diacetate 340 yg 24.0 A l f a l f a , Ladino clover Coumestrol 240 yg 35.0 A l f a l f a , Ladino clover, Barrel , Medic, Peas Estrone 1.20 yg 6,900 Date palm aControl groups uterine weight = 9.6 mg ^At the dose level to produce a 25.0 mg uterus (After Bickoff 1968). 25 a c t i v i t y of coumestrol. Adler (1962) confirmed that the a l f a l f a a n t i -estrogens reduced the potency of coumestrol to one tenth on female genital tracts when administered simultaneously to animals, and indicated that the compounds could be related to progesterone or androgens in structure. Anti-estrogenic a c t i v i t y has been reported by Adler (1962), Biely and Ki t t s (1964) and Cook and K i t t s (1964) in legumes, grasses and needles of Pinus ponderosa. Adler (1962) reported on a coumestrol analogue present in a l f a l f a which was effective against both coumestrol and 17 B-estradiol; indicating i t s v e r s a t i l i t y as an anti-estrogen. Studies on the structures of compounds and anti-estrogenic a c t i v i t i e s by Terenius (1968)demonstrated in v i t r o i n h i b i t i o n of 17 B-estradibl by miroestrol, a plant estrogen with one f i f t h the a c t i v i t y of 17 3-estradiol. The most estrogenic isomer of estradiol was the most effective uptake i n h i b i t o r ; non-steroid carboxylic acids and thei r isomers also blocked uterine uptake of 17 B-estradiol. Terenius (1971) further c l a s s i f i e d anti-estrogens into two types: (a) progesterone/ testosterone structures which do not i n h i b i t or reduce the concentration of estrogens in the target tissue; t h e i r mode of action i s unknown, and (b) compounds which reduce the concentration of estrogens at the s i t e of action in the target tissues by affecting estrogen metabolism, which would include the rates of conjugation and rates of excretion. Terenius (1968), Rochefort and Capony (1972) proposed that the mammalian uterus has a limited number of receptor/binder s i t e s ; competitive binding occurs, and anti-estrogens could form either a durable complex with the s i t e s , or a short-lived complex when the 26 endogenous estrogen supply i s of short duration. This short-lived complex i s too transient to exert c e l l and tissue growth e f f e c t s , but in both cases the receptor s i t e a v a i l a b i l i t y for animal estrogens i s reduced. The anti-estrogens have high a f f i n i t i e s for the s p e c i f i c cytoplasmic binder s i t e s , but do not enter the nuclear s i t e s . Expression of the a c t i v i t y of the compound retained at the binding sites follows, and as a consequence reduced protein synthesis by the uterus i s the result. These results were confirmed by Folman and Pope (1967), and Terenius (1971). Additional workers added to the structures and a c t i v i t i e s of the anti-estrogens: Korenman (1969) noted that the molecular dimensions of miroestrol, anti-estrogens and 17 B-estradiol were si m i l a r ; the distances between the number three and seventeen, three and eighteen hydroxyl groups resembled the distances between the three and seventeen hydroxyl groups of 17 6-estradiol; these groups con-tribute to the binding a f f i n i t y ; the presence of a hydrophobic portion of the skeleton and size were important in "loose" binding of estradiol to the receptor s i t e s . These structures are part of the miroestrol molecule. Shemesh et al_. (1972), using a competitive protein binding technique with rabbit uterine cytosol, demonstrated that the non-steroids coumestrol and genistein ac t i v e l y competed for binding s i t e s with 17 3-estradiol; the binding a f f i n i t y was related to the potency of the compounds: 1 part of 17 B-estradiol to 70 parts coumestrol to 175 parts genistein produced equivalent i n h i b i t i o n of the uptake of t r i t i a t e d 17 B - e s t r a d i o l . These authors suggested that the presence of free hydroxyl groups at positions 7 and 12 of 27 the isoflavones and coumestans were essential for interaction with the uterine cytosol receptor. Proestrogens were designated by the positioning of 7 and 12 methoxy groups; thei r a b i l i t y to bind to the uterus was only after active ruminal/hepatic demethylation in vivo. The Effects of Plant Extracts on P i t u i t a r y Function The maintenance of the estrous cycle i s under the d i r e c t control of the anterior p i t u i t a r y . Any interference with the production or release of f o l l i c l e stimulating or l e u t i n i z i n g hormones d i r e c t l y affects the c y c l i c nature of estrus. Plant phenols have been shown to affect both the ovarian and gonadotropic systems. Chury (1965) found anti-estrogenic and anti-gonadotropic a c t i v i t i e s in a l f a l f a extracts; Leavitt and Wright (1965) confirmed these r e s u l t s , and noted that the primary effect of the extracts was to i n h i b i t the release of p i t u i t a r y hormones. Leavitt and Meismer (1967) discovered that coumestrol could cause persistent estrus without ovulation. Leavitt and Wright (1965) reported that coumestrol was 2.9 x 10 as e f f e c t i v e as estradiol on the uterus i n a c t i v i t y , and 8.5 x 10"^ as effective as estradiol in blocking p i t u i t a r y gonadotropin release. The primary a c t i v i t y of coumestrol, unlike e s t r a d i o l , i s to cause uterine growth in a reduced form before blocking p i t u i t a r y gonadotropin release. Extensive review a r t i c l e s by Samuel (1967) and Bickoff (1968) confirm various authors findings on the a c t i v i t y of coumestrol both as a stimulating and retarding compound on pituitary/ovary a c t i v i t y of mammals. Metabolism of Plant Estrogens by Animals 28 A large amount of l i t e r a t u r e has accumulated regarding the metabolic routes and fate of plant flavonoids by the ruminant. The a b i l i t i e s of ingested coumestrol and genistein to stimulate the incorporation of precursors into protein, phopholipids and uterine R.N.A., were confirmed by Noteboom and Gorski (1963). Biggers and Curnow (1954) concluded the forage estrogens were pro-estrogens, being converted to more active forms in the rumen and l i v e r . Active rumen/liver demethylation of biochanin A, formononetin, and methoxylated isoflavones into formaldehyde and phenols, interconversions of biochanin A to genistein, and formononetin to daidzein, have been established by Nilsson (1961, 1962). No evidence of more extensive degradation i n rumen liquor has been indicated in vivo by Batterham et al_. (1965). Braden (1967) incubated biochanin A and formononetin in rumen liquor preparations; rapid demethylation of s i x t y percent of the two isoflavones to genistein and daidzein resulted. Figure 5 i l l u s t r a t e s the major metabolic products of rumen action on plant estrogens. Rapid and complete absorption occurs in the duodenal-jejunal area within two to three hours- after ingestion; Lindner (1967) indicated that the presence of l i v e r toxins, stress, or reduced nut r i t i o n a l status could impair hepatic function and a l t e r the demethylation process. Shutt and Braden (1968) reported that animals consuming estrogenic forages excreted more urinary breakdown products. This resulted from a reduction of a 2-3 double bond and a 4-oxygen group present in formononetin and daidzein. Genistein end products are simple phenols, Figure 5 The Major Estrogenic Isoflavones and Their Metabolic Products in the Liver and Rumen 28a / m Biochanin A (0.8 x 10-5 A c t i v i t y D i e t h y l s t i l b e s t r o l ) of Genistein (1.1 x IO" 5 A c t i v i t y of D i e t h y l s t i l b e s t r o l ) (After Braden and Shutt 1970) Paraethyl Phenols (Estrogenically Inactive) 29 such as P-ethyl phenol; phenol production resulted in a loss of estrogenic a c t i v i t y ; formononetin degradation produced equol with estrogenic a c t i v i t y 25% that of genistein (Shutt 1969). Shutt et a]_. (1970), using sheep on red clover dominated pastures, indicated that less than 1% of the ingested isoflavones (9.0 gms/day) were excreted in the feces and urine. Daily urinary excretion of 3.9 gms/day of equol was equivalent to 70% percent of the daily intake of formononetin. Eighty-six percent of the equol produced by the rumen was absorbed by the rumen epithelium. Shutt and Braden (1968) found plasma levels of 50 ygs/100 ml and uterine tissue levels of 46 ygs/uterus of equol (7,4* dihydroxyiso-flavan) in sheep on red clover diets. These levels represent more than 70% of the total estrogens extracted from the plants. Under (1967) demonstrated how plasma levels of plant compounds affected biological response: in sheep, plasma genistein levels of 1.0 to 5.0 ygs/100 ml of plasma e l i c i t e d graded uterine growth responses, while levels above 5 ygs/100 ml of plasma resulted i n maximum uterine growth response. Adipose tissue storage of conjugated plant estrogens occurred in excess of plasma levels. Lindner also pointed out that these levels should not affect consumer health. The a c t i v i t y of plant estrogens in animals i s affected by previous diet. Lindsay et a]_. (1970) found that the cervical mucus response of ewes to subterranean clover species high i n genistein was reduced by a ten-day pre-feeding period on diets containing genistein at levels of 1.1% of the plant material on a dry matter 30 basis. Formononetin response did not depend on previous diet. Deactivation of genistein into non-active phenols by the rumen micro-organisms occurred in animals conditioned to genistein di e t s ; formononetin was not deactivated. No adaptation by rumen microflora species or tissue oxidative systems, such as polyphenol oxidase, occurred when formononetin was pre-fed at 1.2% of plant dry matter content. Rapid genistein degradation to P-ethyl phenol led to genistein's estrogenic a c t i v i t y being reduced over long ingestion periods. Formononetin underwent l i t t l e loss in a c t i v i t y ; equol conversion and production of O-desmethylangolensin s t i l l occurred or increased, suggesting no ruminal or enzyme alterations which resulted in rapid degradation of the isoflavone. Cayen and Common (1965) injected fowl with labelled coumestrol; no coumestrol or equol was recovered in the urine. This suggested different metabolic routes for the coumestans, and probably contributed to the higher metabolic a c t i v i t y of the coumestans. Species differences also e x i s t in the metabolism of plant estrogens — more e f f i c i e n t conjugation and excretion of phyto-estrogens occurs in c a t t l e ; formononetin i s metabolized at a faster rate in sheep than in c a t t l e . Braden and Shutt (1971) suggested from these results that the lower s u s c e p t i b i l i t y of c a t t l e to the effects of estrogenic pastures could be p a r t i a l l y attributed to this metabolic rate function. 31 FACTORS AFFECTING THE ESTROGENIC ACTIVITIES OF PLANTS Estrogenic and anti-estrogenic a c t i v i t i e s in plant species are extremely variable; both environment and inheritance patterns govern these levels. Location In studies by the U.S.D.A. (1965) average coumestrol values in a l f a l f a due to location have ranged from 10.4 to 125.4 ppm. Millingtpn (1964) reported on variations of barrel medic (Medicago  t r i b u l o i d e s ) ; coumestrol content ranged from 40 to 180 ppm. A l l plants were harvested at f u l l bloom stage, with year location i n t e r -action being highly s i g n i f i c a n t . Davies and Dudzinsky (1965) noted that subterranean clover, between s i t e and between year differences in a c t i v i t i e s were highly correlated. Season and Growth Stage Squires (1966) bioassayed the estrogenic a c t i v i t y of ladino clover pastures using sheep teat length; the pasture a c t i v i t y was maximum in the spring, coinciding with the dominance of the pasture by the legume. Estimates on the potencies of a l f a l f a by Legg (1950); K i t t s et al_. (1959); Kohler (1962); and U.S.D.A. (1965) indicated s l i g h t a c t i v i t y throughout the f i r s t year of growth; coumestrol levels increased during the second year of c u l t i v a t i o n with successive growth stages, and reached a maximum between 10 and 25 days after f u l l bloom 32 of 270 pptn. Biely and K i t t s (1964) postulated that an inverse r e l a t i o n -ship existed between estrogenic and anti-estrogenic a c t i v i t y of a l f a l f a throughout the growing season. Growth stage effects have also been examined on red clover by Flux et al_. (1963), Dedio and Clark (1968), and Rossiter (1972). The results can be summarized in that the maximum isoflavone/ coumestan biosynthesis occurred early during the leaf unfolding stage; isoflavones competed for carbon substrates (soluble sugars) destined for c e l l protein and c e l l wall synthesis. Rossiter (1972) indicated that the soluble sugar content of leaves at f u l l expansion stage could serve as an indicator for determining isoflavone/coumestan l e v e l s , and consequently estrogenicity of the plant species. Varietal Variation in the Estrogenic A c t i v i t y of Plants Large ranges of estrogenic isoflavones e x i s t in different var i e t i e s of plants. Francis, Millington and Bailey (1967) examined over 100 species of the genus Trifolium; tot a l contents of isoflavones reached values of up to 0.25% of the total dry weight. Bailey and Francis (1971) surveyed 76 l i n e s of Trifolium subterraneaum, section Calycomorphum; a basic isoflavone pattern and evolutionary progression of isoflavone contents was apparent; Francis and Millington (1965) noted that isoflavone patterns are s i g n i f i c a n t in relation to the centres of o r i g i n of the plant species. Dedio and Clark (1968) examined Canadian red clover v a r i e t i e s and noted formononetin ranges of 0.35% to 0.94%; biochanin A of 0.60% to 1.12% of dry weight; high correlations existed between biochanin A and formononetin estimates. They suggested 33 this could serve as a basis for selectively breeding plants for low and high isoflavone varieties. The selection of plants for isoflavone levels has been further clarified by Francis and Millington (1965) who indicated that single genes control varietal differences, the release of bound isoflavones, plant methylation of daidzein and genistein to formononetin and biochanin A, and the quantities of isoflavones present. The production of a mutant strain of T. subterraneum with low isoflavone levels, having negligible estrogenic activity, has been confirmed by Millington et al_. (1966); i f proven disease resistant, the application as a pasture species will no doubt benefit the Australian livestock producer. The Effects of Defoliation on Estrogenic Potencies of Legumes Rossiter (1969) examined grazing and defoliation effects on the estrogenic potency of T. subterraneum. Total leaf isoflavone levels were -12 -12 reduced from 84 x 10 to 43 x 10 grams per gram of dry matter as a result of defoliation; genistein levels were mostly affected, being reduced from 1.2% to 0.6% of leaf dry matter. Repeated defoliations were more effective than single clippings, giving lowered leaf genistein levels of 0.4% of the total leaf dry matter content. Pro-tection of the plants from grazing had negligible effects on the concentrations of formononetin, biochanin A, and genistein in fully expanded leaves. Rossiter (1969) also studied the effects of severe defoliation on isoflavone levels; a reduction in isoflavones occurred; this suggested decreased levels of soluble carbohydrates available, 34 which reduced isoflavone synthesis and increased catabolism of the anthocyanins to supply carbon substrates for c e l l wall and c e l l protein synthesis. Mild grazing resulted in no s i g n i f i c a n t reductions in isoflavone l e v e l s ; only i f pasture stocking i n rates exceeding 3.7 ewes per acre, when increased intake of pasture plants resulted, did the effect of def o l i a t i o n affect estrogenic potency of the ingested legumes. Frequency of cutting on the estrogenic potencies of a l f a l f a and ladino clover was examined by K i t t s (1959). Cutting of a l f a l f a at the vegetative stage resulted in the a c t i v i t i e s of subsequent harvests during the growing season to follow closely that of plants allowed to grow without interference. When cuttings were made past the vegetative stage of the a l f a l f a , subsequent cuttings possessed l i t t l e or no estrogenic a c t i v i t y . Ladino clover at any cutting followed a trend of high estrogenic potency during the early phases of vegetative growth; a c t i v i t y declined to i n s i g n i f i c a n t levels at f u l l bloom stage, with a detectable increase during early and late seed stages. The Effects of Drying on the Estrogenic A c t i v i t y of Legumes Drying legume forage for preservation has not always given consistent results on estrogenic a c t i v i t i e s . Davies and Dudzinski (1965) found that the potency of f i e l d cured subterranean clover was maintained after drying; Francis and Milington (1965) indicated that the estrogenic a c t i v i t y of dried subterranean clover was negligible 35 when compared with green material. Nilsson (1959) showed that a r t i f i c -i a l drying and en s i l i n g did not change the a c t i v i t y of red clover, while f i e l d curing lowered the biological a c t i v i t y . Youngman (1963) demonstrated that the estrogenic a c t i v i t y of legumes increased with the age of cutting for both hay and sil a g e ; curing had l i t t l e e ffect. Dedio and Clark (1969) working with red clover concluded that isoflavone levels did not d i f f e r from fresh clover samples when oven dried at temperatures below 80°C, or when the samples were frozen; l i t t l e or no loss of a c t i v i t y due to breakage of the isoflavone glycoside bonds occurred when samples were rapidly dried; fresh red clover total isoflavone content (biochanin A plus formononetin) was 1.28% of dry weight. Drying the forage at 80°C resulted in total isoflavone content of 1.21%'of total dry weight; while freezing the samples resulted in total levels of 1.20% of the total dry weight. Two reasons have been proposed to explain the di f f e r e n t effects of drying on the estrogenic a c t i v i t y of clovers and a l f a l f a . Swierstra (1958) suggested that the greater retention of a c t i v i t y of clover species during extended storage reflected a greater s t a b i l i t y of the isoflavones over the coumestans. Leaching or destruction of the glycosidic bonds or the isoflavone molecules could occur, p a r t i c u l a r l y in unfavourable, wet haying periods. Bickoff et aj_. (1960) proposed that increased a c t i v i t y after drying of red and subterranean clovers compared with a l f a l f a and ladino clover, could be due to a less stable estrogen i n h i b i t o r or breakdown enzyme, or due to a more stable estrogen precursor or intermediate product. 36 The Effects of Plant Pathogens on Estrogenic Potency of Plant Species Evidence has accumulated which indicates one of the major sources of isoflavone/coumestan variation i s in the action of bacteria, fungi, and insect attack on the individual plant species. Loper and Hanson (1964) observed a one hundred fold increase in leaf coumestrol content of a l f a l f a following leaf spot infection. Normal coumestrol values were 2.1 ppm, and rose to 183.7 ppm after mild in f e c t i o n . Coumes-t r o l content was po s i t i v e l y and d i r e c t l y correlated with disease severity. The same authors (Loper et a l . 1967) indicated that plant selection and breeding for disease resistance reduced the coumestrol content of a l f a l f a ; fungicide applications to reduce f o l i a r diseases resulted in r e l a t i v e l y low and constant levels of coumestrol; fungicide sprayed stems and leaves averaged 28.6 ppm, total coumestrol while Unsprayed plants averaged 103.3 ppm. M i l l i k a n (1971) examined red leaf virus infection on lucerne. The results indicated that the range of coumestrol i n disease free plants was 6.0 to 25.0 ppm, and the estrogenic a c t i v i t y when measured by bioassay increased when red leaf virus infection occurred on the exposed leaf surfaces. Leaf rust infection (Uromyces s t r i a t y s ) also affected total coumestan content; Francis and Millington (1971) found a six f o l d increase from 15 to 80 ppm in the leaves of the burr medic, which was d i r e c t l y related to the degree of rust in f e c t i o n . Explanations have been advanced to explain the rapid r i s e and accumulation of coumestans and isoflavones following infection: Hess and Hadwiger (1971) examined the anti-fungal isoflavone, phaseolin; under pathogen attack, the D.N.A. template was stimulated 37 to produce increased levels of phaseolin. I t was concluded that increased isoflavone concentrations represented a plant defence mechanism versus the pathogen. Loper (1968) noted that coumestrol could increase independently of infection and was affected by a range of infections including aphids. Loper stated that: "The increased levels may be a general increase in flavonoid production, especially occurring in damaged areas when protein synthesis i s reduced, protein synthesis being inversely related to flavonoid production." Sherwood et al_. (1970) indicated that a time course of coumestan/isoflavone accumulation paralleled the development of i n f e c t i o n ; the concentration of flavonoids was related to the degree of i n f e c t i o n , translocation of coumestans from infected plant areas to healthy areas did not occur. The host plant was demonstrated to be the pr i n c i p l e contributor of flavonoid precursors and enzymes in flavonoid biosynthesis — this process occurred in the infected tissue, not in the surrounding healthy tissue. The same authors also determined that the mechanism of induction of flavonoid synthesis was related to the metabolism of the infecting organisms and not to mechanical damage, with coumestan/isoflavone accumulation associated with the cata l y s i s of c e l l s leading to c e l l necrosis and tissue degradation. Mineral Elements and Plant Estrogen A c t i v i t y The finding that inorganic nutrients essential for plant growth d i r e c t l y affected plant coumestan and isoflavone levels and estrogenic a c t i v i t y was f i r s t examined by Alexander and Rossiter (1952). 38 Top dressing of ]\_ subterraneum with phosphate and other f e r t i l i z e r combinations at application rates of 216 kg/hectane indicated by bioassay techniques that the estrogenic a c t i v i t y increased when no treatment was applied; plants receiving phosphate supplementation together with other f e r t i l i z e r s , did not d i f f e r s i g n i f i c a n t l y in potencies from those receiving phosphate only. Dry matter yields increased by a factor of 2 to 3 with phosphoric acid addition. Rossiter and Beck (1966) examined phosphate supply and potencies of the Dwalganup st r a i n of T. subterraneum. Decreasing the phosphate supply resulted in a doubling of the formononetin/genistein content, and a decrease in dry matter productions. In the Mount Barker s t r a i n , genistein and biochanin A levels doubled with increasing phosphate deficiency; formononetin, the major estrogenic isoflavone, increased by a factor of 4. Rossiter (1970) confirmed the effects of phosphate levels on formononetin; isoflavones were already evident at the leaf emergence stage. In phosphate deficient leaves in contrast, the concentration of isoflavones increased during the l a t e r growth stages. In pot culture experiments, Rossiter (1969) examined nitrogen deficiencies on growth and estrogenic potencies of plant parts of T. subterraneum. Nitrogen deficiency was associated with increased isoflavone levels. In the f i r s t t r i f o l i a t e leaves, the total concentrations of formononetin, genistein and biochanin A were doubled from 3.7% to 7.1% with low nitrogen supply. Biochanin A levels were less affected than the other isoflavones, and remained unchanged at a concentration of 1.2% of the total leaf weight. 39 Sulfur supply also affects the isoflavone l e v e l s . Rossiter and Barrow (1972) examined su l f u r supply in T. subterraneum. A consistent tendency existed for isoflavone levels to decrease as sulfur supply increased. Otter (1966) summarized the effects of plant n u t r i t i o n and flavonoid l e v e l s : " I f conditions ( i . e . adequate nitrogen supply) for protein synthesis p r e v a i l , a larger part of endogenous metabolites (soluble sugars and CO^) are channeled into primary compounds -- proteins and c e l l walls. A reduced number of carbon molecules are available for secondary processes -- anthocyanin and flavonoid pigments. At low nitrogen l e v e l s , the protein carbon saved (from the deficiency in protein synthesis) i s more than adequate to account for extra isoflavone/ coumestan glycoside carbons for glycoside formation." The addition of f e r t i l i z e r , as indicated by Schoo and Rains (1970), results in a reduction of isoflavone l e v e l s . This reduction may result from a "normalized" isoflavone metabolism and/or a d i l u t i o n effect from increased dry matter due to increased plant growth. Rossiter (1972) confirmed that low s o i l phosphate levels decreased protein content per c e l l and that isoflavone levels increased on a dry matter basis. This finding adds evidence to support the sub-strate competition hypothesis cited by Rossiter (1972), which indicated that isoflavone/coumestan formation i s more d i r e c t l y related to sugars and starches than to plant protein levels. The amounts of isoflavones formed per c e l l depend on the supply of carbon substrates in the formation of sugars and starches. At f u l l expansion stage, isoflavone synthesis in legumes normally ceases. While the leaf was expanding under the effect of 40 levels of mineral supplementation, the formation of c e l l protein and c e l l protein and c e l l walls takes preference over isoflavone/coumestan formation, due to the substrate competition effect on the supply of carbon substrates; with the result of large variations in isoflavone content and estrogenic a c t i v i t y of the legume species. 41 CHAPTER III HORMONAL ACTIVITY OF TWO SPECIES OF NATIVE B.C. RANGE LEGUMES INTRODUCTION The widespread occurrence of plant compounds having effects on animal reproduction has been well documented i n cultivated legume species (Samuel 1967; Bickoff 1968). Native legumes and grass species also cause hormonal imbalances affecting the reproductive t r a c t . Wada (1963) reported on a c t i v i t y of Chinese milk vetch Astragalus sinicus , Symington (1965) on high-veld pastures of Central A f r i c a . Cook and Ki t t s (1964) and Stevenson et a]_. (1972) studied estrogenic a c t i v i t y and range c a t t l e abortions due to needles of Pinus ponderosa. Both estrogenic and anti-estrogenic a c t i v i t i e s have been found simultaneously in plant fractions; s o i l conditions, f o l i a r pathogens and growth stages are important i n affecting the plant hormone lev e l s . The study described below was designed to assess the hormonal a c t i v i t y of two widespread native forage species on the mammalian uterine tract. Experiments to assess the types of compounds present, and the effects of growth stage on potency of the legume species were also conducted. MATERIALS AND METHODS 42 Plant Material V i c i a americana subsp. oregana (Nutt.) (American vetch), a perennial ranging on grassy slopes from B r i t i s h Columbia to southern C a l i f o r n i a as described by U.S.D.A. (1937); and Astragalus miser var.  serotinus (timber milk vetch) -- occurring on the Interior dry belt of B.C. as described by Barneby (1964), were harvested during the 1971 grazing season. The Farwell Creek area, south of Riske Creek, B.C., (Section 20, T.P. 53, L i l l o o e t Land D i s t r i c t ) was chosen for location s i t e s ; active grazing of livestock was conducted on this typical Cariboo grassland range during the spring and summer grazing season from May 1 to June 21st 1971. Animals ranging on this area include heifers, and cows with calves. A total of 2,900 animals u t i l i z e the unit, with resulting heavy overgrazing of both grass and legume species. Harvesting of the legumes was on randomly located transect li n e s in t r i p l i c a t e for each growth stage. Plants were hand clipped at 12 cm on both sides of the l i n e s , and at 2 cm above ground l e v e l . This method eliminated s o i l contamination and simulated c a t t l e grazing conditions. Figures 6 and 7 i l l u s t r a t e sampling methods on l i n e transects. Figures 8 and 9 i l l u s t r a t e the four growth stages of Vi c i a americana and Astragalus miser var. serotinus at which samples were collected for analysis. Only healthy specimens free of disease or insect infestation of varying heights were sampled to maintain 43 Numerals represent transect lines harvested at sp e c i f i c stages of maturity: I. Vegetative stage. I l l . Seed pod stage. 44 Figure 9 Astragalus miser var. serotinus - - analyses conducted at i l lustrated growth stages 45 representative samples of the areas. Eight hundred gram samples (wet weight) were collected at each date; following packaging and sealing in p l a s t i c bags, quick freezing at 0°C commenced; this temperature was maintained u n t i l the analyses were conducted. Proximate analysis of the samples at the s p e c i f i c growth stages was conducted according to the methods of the A.O.A.C. (1960). Extraction Procedure Figure 10 i l l u s t r a t e s the procedure employed for extraction of phytoestrogens. This i s a modification of the extraction methods of Beck (1964) and Biely and K i t t s (1964). A l l solvents were reagent grade and double-distilled in glass before use to eliminate peroxides and i n t e r f e r i n g aromatic contaminants. U l t r a v i o l e t spectrophotometer testing for increased absorption of the ether and chloroform solvents at 300 my in a 1.0 cm path length quartz c e l l s confirmed the absence of peroxides. i Bioassay for Assessment of Biological A c t i v i t y A six hour bioassay using randomly selected pre-pubertal 40.0 ± 5 gram ovarectomized wistar female rats was employed as described by Astwood (1938) and modified by A l l i s o n and K i t t s (1964). The two groups of control animals were injected with 0.2 ml of physiological saline subcutaneously, or 0.025 micrograms of 17-8-estradiol dissolved in 0.2 ml of saline. The ether and chloroform fractions were taken to dryness and weighed. The fractions were dissolved in a convenient solvent 46 Chopped forage (350 gms dry matter) I Macerated in food blender with 0.1N HCl for 3 minutes. pH adjusted to 7.0 I Incubated 30 minutes at 37°C I Boiled 10 minutes in 95% Ethanol /3 1 i t e r s x [ 100 gms; Cooled and f i l t e r e d I Re-extracted 10 minutes in 95% Ethanol /3 l i t e r s v [ 100 gms; Concentrated to approx.'100 ml. Water added to give approx. 70% Ethanol solution (V/V) Chlorophyll and l i p i d s extracted with 3 - 500 ml portions of petroleum ether (B.P. 50-70°C) un t i l no colour remains in ether fraction pH adjusted to 7.2 with 40% Sodium Hydroxide I Evaporated in vacuum to remove a l l Ethanol Washed with 5 - 800 ml portions of Anhydrous Ether (30 minutes contact time each portion) Washed with 5 - 800 ml portions of Chloroform (30 minutes contact time each portion) Ether (Fraction A) and Chloroform Extracts (Fraction B) evaporated to dryness (25°C) Extracts'weighed | Extracts dissolved in 25.0 ml ToluenerEthanol (50:50) Figure 10 Fractionation Procedure for the.Extraction of Estrogenic/ Antiestrogenic Compounds 47 (toluenerethanol 50:50); concentrations were adjusted to contain 15.0 grams of plant material (dry matter) per animal. The extracts were again taken to dryness. The ether extract fraction (Fraction A) was dissolved in physiological saline (0.2 ml/animal). The chloroform fraction (Fraction B) was prepared s i m i l a r l y ; 15.0 grams of dry matter equivalent was dissolved in saline containing 0.025 micrograms of 17-B-estradiol in order to assess the anti-estrogenic a c t i v i t y of the chloroform extracts. S a c r i f i c e by ether inhalation of the animals occurred six hours post i n j e c t i o n . The a c t i v i t i e s of the fractions were assessed by the increase or decrease of gross uterine weight expressed as a percentage of body weight, when compared with the control groups. Separation Procedure for Plant Extracts Thin layer chromatographic plates (20 cm x 20 cm) were coated with s i l i c a gel G (Merck) at a thickness of 0.25 mm. Prior to use the plates were activated by heating to 110°C for 30 minutes. 1.0 ml samples dissolved i n toluene: ethanol (50:50) from the extraction procedure were taken to?dryness and redissolved in 0.5 ml toluene: ethanol (50:50) to ensure mobility and separation on the thin layer plates. Samples were streaked on the plates with a Camag "Chromato-charger" applicator. Four l i n e s of 40.0 microliters each were applied per plate at the o r i g i n , resulting in a total application per plate of 160 m i c r o l i t e r s ; drying with forced hot a i r after each 40.0 micro-l i t e r application gave uniform streaks of minimum width with a continuous surface area being applied. A total of ten plates were run per extract. The developing solvent was composed of chloroform: 48 methanol (91:9). The solvents were d i s t i l l e d prior to use, and confirmed to be peroxide free. Ascending chromatography with a saturated atmosphere was employed. The temperature was maintained at 25°C. The solvent front was run to the f u l l length of the plates. The plates were a i r dried for one minute following the run, bands were observed and thei r positions marked under u l t r a v i o l e t l i g h t (3650 Angst-roms), after a ten second exposure to ammonia vapour. A 1.0 cm wide s t r i p was sprayed along the edge of the solvent run with diazotized s u l f a n i l i c acid to test for any phenols not v i s i b l e i n the u l t r a -v i o l e t l i g h t . Rf values and colours for each of the bands were recorded. The ten replicate plates were combined, the bands removed from the plates by vacuum, mixed for 1.0 minute with 10.0 ml of spectroscopic methanol, and centrifuged for 5.0 minutes at 2,000 rpm. The supernatant was decanted and evaporated three times to 1.0 ml volume under vacuum at 25°C. Excess temperatures were avoided to prevent denaturation and oxidation of the compounds. The extracts from the f i r s t chromatographic separation were again re-dissolved i n 0.5 ml of toluene:ethanol:(50:50) and re-applied to chromatographic plates as in the f i r s t separation to ensure purity of the samples. The second dimension solvent consisted of 18.0 volumes of methanol made to 1.0 normal with ammonia gas, plus 82.0 volumes of chloroform. The solvent system was allowed to go the f u l l distance on the plates. Viewing under u l t r a v i o l e t l i g h t and spraying with diazotized s u l f a n i l i c acid ensured the absence of interfering materials and eliminated overlapping bands present with the extracts from the f i r s t chromatographic separation. U l t r a v i o l e t Absorption Spectra of Plant Extracts 49 Absorption data of the pu r i f i e d extracts was obtained by dissolving the extracts in d i s t i l l e d spectroscopic grade methanol. Sample concentration was adjusted so that the major absorption peaks had an optical density between 0.6 and 0.8. A Unicam S.P. 800 B u l t r a -v i o l e t recording spectrophotometer with attached Unicam S.P. 20 lin e a r recorder was employed for absorption spectra determinations. The spectrophotometer was calibrated with a hoi mi urn oxide f i l t e r (Beckman Instruments Ltd.). Scanning range was between 200 and 450 m i l l i -microns; s l i t width was set at 0.002 mm; matched s i l i c a cuvettes having 0.45 ml capacity and 10.0 mm path length contained the sample. A blank consisting of an equal area of thin layer s i l i c a gel was subjected to developing in the solvent systems; the s i l i c a gel from the area received the same preparation as the sample, and the spectroscopic methanol supernatant was used as a reference blank during a l l determinations. A l l determinations were duplicated and average values calculated for absorption peaks. U l t r a v i o l e t Spectra in the Presence of Selected Reagents As a guide in determining structural types of the flavonoids present, u l t r a v i o l e t spectral s h i f t s with s p e c i f i c reagents were undertaken following determination of the spectra in methanol. 50 Reagents: 1. Sodium Methoxide (NaOme): 2.5 gms of freshly cut metallic sodium were added to 100 ml of dry d i s t i l l e d spectroscopic methanol. Three drops of this solution was added to the extracts in methanol. After 5.0 minutes, the spectrum was re-run to check for decomposition. 2. Aluminum Choloride ( A l C l ^ ) : 5.0 grams of fresh reagent grade A l C I 3 were dissolved in 100 ml of d i s t i l l e d spectroscopic grade methanol. 6.0 drops were added to a fresh sample of extract. The spectra were recorded after thorough mixing of the reagent with the methanol solution. 3. Hydrocholoric Acid (HCl): Concentrated reagent Grade HCl (50 ml) was mixed with demineralized water (100 ml). 3.0 drops were added to the cuvette containing the AT C I 3 reagent. The spectra were recorded and the solutions were discarded. 4. Sodium Acetate (NaOAc): Anhydrous powdered reagent grade NaOAc was used. Excess NaOAc was added to the cuvette containing 0.45 ml of the fresh stock solution of the extract. A l l spectra were recorded within 2.0 minutes af t e r the addition of the reagent. 5. Boric Acid (HQBO.J: Anhydrous powdered reagent grade H3BO3 was employed. S u f f i c i e n t H^Bo^ was added to the cuvette containing the extracts with NaOAc to give a saturated solution. The solutions were discarded after the spectra were recorded. Infrared Spectroscopic Examination of Plant Extracts 51 In order to examine the structures of the skeletons and attached groups, infrared analysis was conducted on the extracts of the two legume species harvested at the vegetative stage. A Beckman IR-5A infrared spectrophoid meter with double beam scanning was employed. The machine was calibrated with a polystyrene f i l t e r (Beckman Instruments Ltd.) prior to sample determinations. The samples dissolved in toluenerethanol (50:50) from the extraction procedure were evaporated to dryness, and re-dissolved completely in 2.0 ml of d i s t i l l e d anhydrous ether. The samples were spotted dropwise (with a i r drying) on a sodium chloride plate 2.5 cm x 2.5 cm x 6.5 mm thickness; drop size was kept constant in order to obtain a uniform sample size on the plate. A second sodium choloride plate was positioned over the samples, and the spectra were determined in the wave number range 650 to 5,000 cnf^. A reference blank spectrum was run by adding 2.0 ml anhydrous ether dropwise to the sodium chloride plate; the same procedure as was u t i l i z e d with the samples was used for the blank plate. Sodium chloride plates were cleaned with d i s t i l l e d acetone, oven dried and dessicated between determinations to prevent moisture uptake on the plate surfaces. RESULTS AND DISCUSSION Proximate analysis data are presented in Table 3. A wide range of crude protein and crude f i b e r values are evident throughout the grazing season. These results are consistent for range legume 52 growth patterns, with decreasing protein and increasing crude f i b e r content of the plants found during advancing maturity. Table 4 indicates the ether and chloroform extract weights from 350 grams (D.M.) of both species during the grazing season. The ether extract fraction reached maximum at f u l l bloom stage for both species and increased again at seed disseminated stage. The chloroform extract was maximum at f u l l bloom in Vic i a americana, and at seed disseminated stage in Astragalus miser var. serotinus. These results indicate no correlation between extract weights and proximate analysis data. Bioassay results are tabulated in Table 5. Vic i a americana possessed maximum estrogenic a c t i v i t y at seed disseminated stage, and maximum in h i b i t o r y effects by the chloroform extract on estradiol a c t i v i t y at vegetative stage. In contrast, Astragalus miser var.  serotinus reflected maximum uterine growth promoting effects at seed pod stage; maximum inh i b i t o r y action occurred at the vegetative stage, as did V i c i a americana. These results agree with K i t t s et aj_. (1959), who noted large variations in plant a c t i v i t y due to plant growth stages, and with Biely and K i t t s (1964) who indicated that an inverse relationship existed between estrogenic and anti-estrogenic a c t i v i t i e s during the growing season. Results from t h i s experiment demonstrate relationship existing between plant potencies and extract weights. Factors influencing this lack of correlation between the weights of the extracts and the potencies of the growth stages include: 53 TABLE 3 PROXIMATE ANALYSIS OF VICIA AMERICANA AND ASTRAGALUS MISER VAR. SEROTINUS Vic i a americana Harvest Date Growth Stage % Dry Matter % Crude Protein % Crude Fiber (ADF) % Crude Fat. % Ash 19/6/71 Vegeta-tive 60.5 9.9 11.4 15.6 2.5 22/7/71 Full Bloom 61.9 10.5 16.9 14.5 2.7 22/8/71 Seed Pod 73.6 9,8 21.7 5.9 6.6 11/9/71 Seed Dissem-inated 77.7 5.6 18.7 2.6 7.1 Astragalus miser var. serotinus Harvest Date Growth Stage % Dry Matter % Crude Protein % Crude Fiber (ADF) % Crude Fat % Ash 19/6/71 Vegeta-tive 48.2 8.4 8.1 15.5 3.0 5/7/71 Full Bloom 49.6 9.0 14.8 3.6 2.9 6/8/71 Seed Pod 73.4 8.7 15.0 2.2 5.5 11/9/71 Seed Dissem-inated 77.7 6.5 15.7 2.8 7.1 54 TABLE 4 ETHER AND CHLOROFORM EXTRACT WEIGHTS (GMS) OF VICIA AMERICANA AND ASTRAGALUS MISER VAR. SEROTINUS (350 GM DRY MATTER SAMPLES) Vici a americana Growth Stage Ether Extract (Fraction A) gms Chloroform Extract (Fraction B) gms Vegetative 0.513 0.405 Full Bloom 0.746 0.588 Seed Pod 0.313 0.113 Seed Dissemin-ated 0.723 0.301 Astragal us miser var. serotinus Growth Stage Ether Extract (Fraction A) gms Chloroform Extract (Fraction B) gms Vegetative 0.724 0.354 Full Bloom 0.927 0.187 Seed Pod 0.130 0.089 Seed Dissemin-ated 0.428 1.161 TABLE 5 THE EFFECTS OF ETHER AND CHLOROFORM EXTRACTS ON THE UTERUS OF THE LABORATORY RAT (EXPRESSED AS % OF BODY WEIGHT) (a) V i c i a americana (Plot 1) Treatment 1 (0.2 ml saline) 2 (0.025 yg 17-3-estradiol) 3 (15.0 gm D.M. Fraction A ether extract) 4 (15.0 gm D.M. Fraction B chloroform extract + 0.025 yg 17-B-estradiol) Vegetative Full Bloom Seed Pod Seed Disseminated 0.0424 0.0384 0.0387 0.0387 0.0489 0.0543 0.0592 0.0592 0.0408 0.0445 0.0396 0.0453 0.0405 (4, 3, 1)*** 0.0406 (1 . 4U4. 3) 0.0449 (1, 3, 4) 0.0421 •(:!, 4) (4, 3) (b) Astragalus miser var. serotinus (Plot 2) Treatment 1 (0.2 ml saline) 2 (0.025 ug 17-3-estradiol) 3 (15.0 gm D.M. Fraction A ether extract) 4 (15.0 gm D.M. Fraction B chloroform extract + 0.025 yg 17 - 3-estradiol) Vegetative Full Bloom Seed Pod Seed Disseminated 0.0424 0.0384 0.0438 0.0438 0.0489 0.0543 0.0628 0.0628 0.0371 ** 0.0448 0.0441 0.0381 (4, 3, 1)*** 0.0411 (1, 4) 0.0420 (1, 3) 0.0449 (4, 3, 1) *** Treatments underlined did not d i f f e r s i g n i f i c a n t l y under Duncan's Multiple Range Test at the P < .05 level ** Animals died post-injection of the fraction * Mean uterine weight as % body weight (N = 8 per group) ^ 56 1. The breakdown or synthesis of isoflavones as affected by available soluble sugar content (Rossiter 1972); 2. A dilution effect of the estrogenic/anti-estrogenic fractions caused by increased plant cel l growth and maturity taking preference over isoflavones for carbon substrates; 3. The presence of interfering materials, i . e . , chlorophyll art i facts and l i p i d fractions from the extraction procedure. The most pronounced effects of the extracts occurred with the chloroform fract ion; 15.0 grams dry matter equivalent of chloroform extract of Vicia americana decreased the potency of 0.025 ugs estra-diol 17-8-estradiol by an average of 24% during the growing season, and Astragal us miser var. serotinus decreased the potency of estradiol by 27.5% throughout the growing season. The increase in uterine f lu id uptake and cel l hyperplasia were affected by the ether extracts, but to a lesser extent than the effects of the chloroform extracts. Vicia  americana showed 7.0% average increase in growth over saline controls; Astragal us miser var. serotinus demonstrated no signif icant estrogenic stimulation in uterine response over the control groups during the growing season. The death of the test group of animals (Fraction A of the fu l l bloom stage of Astragalus miser var. serotinus) indicates the possible presence of miserotoxin (8-glucoside of three nitro-l -propanol) present in the plant at fu l l bloom stage, and at toxic dosage levels. Williams et al_. (1969) established that the concentration of miserotoxin 57 was associated with the maturity of the plant; levels attained a maximum of 2.7 - 3.2% of plant dry matter at f u l l bloom and early pod stages, confined mainly to the leaves and petioles. Only small amounts were found in the pods, roots and flowers. Williams et al.(1969) concluded that ingestion of timber milk vetch containing 3% misero-toxin at a level of 4.8 gms/kg body weight was a lethal dose for ruminants. The symptoms exhibited by the animals two hours post injection included arching of the back, loss of equilibrium, and rapid drop of body temperature. Minute hemorrhagic lesions were present in the int e s t i n a l mucosa. These effects resemble those found by Mosher (1970), whose ethanolic extraction procedure for miserotoxin determin-ation paralleled that for the extraction of plant estrogens. The results of the thin layer chromatographic separation, u l t r a v i o l e t absorption data, and reactions with s p e c i f i c reagents are presented in Table 6. Also included in this table are the results of infrared spectroscopic examination of the vegetative stage isolates from the two legume species. These tests do not confirm the positive i d e n t i f i c a t i o n of the extracts, but do indicate groups of flavonoids present, th e i r basic structures, and the appearance of new compounds during the growth stages. Of part i c u l a r importance i s the presence of isoflavones and phenolic/aromatic structures. Further i s o l a t i o n of the individual falvonoids would useful to determine the contribution to uterine reactions and competitive binding with the animal estrogens. The composition of the extracts throughout the growing season i s i n a dynamic state, with the appearance of new flavonoids at maximum 58 TABLE 6 ABSORPTION MAXIMA AND THE EFFECTS OF REAGENTS ON THE ULTRA-VIOLET ABSORPTION SPECTRA OF COMPOUNDS ISOLATED FROM ETHER AND CHLOROFORM EXTRACTS OF VICIA AMERICANA AND ASTRAGALUS MISER VAR. SEROTINUS 1. Vic i a americana -- Plot 1, Cut 1 (Vegetative Stage) Ether Extract (Fraction A) Line 1 Test Indicates Colour (UV) Colour (NHL)' Rf J ***] < 2. 3. 4. 5. 6. MeOH NaOMe A1CU A1C1, + HCl NaOAC NaOAC + Ho Fluorescent blue Fluorescent dark blue 0.34** 245* 299 SH 233* 281 232* 269 239* 264 SH 243* 267 SH 232* 266 SH Isoflavone. Flavanone. Flavone or "A" Ring hydroxyl Groups. Free 5 - hydroxyl Group. Ortho-dihydroxyl Group at 6, 7 or 7, 8. 5 - hydroxyl Group present. 7 - hydroxyl Group. 6, 7 - dihydroxyl Group on Infrared Data: Functional Groups C - H Aromatic, C - c, Multiple bonds, c y c l i c , Hydroxyl Groups. Phenolic structure. Line 2 Test Indicates Colour (WV) Fluorescent blue Catechin or Xanthone Colour (NHJ Fluorescent blue skeleton. Rf 6 0.43 1. MeOH 233* 267 SH 2. NaOMe 232* 286 SH "A" ring hydroxyl Groups. 3. A1C1, 232* 284 Free 5 - hydroxyl Group. 4. AlCIo + HCl 238* 314 SH Ortho dihydroxyl Groups at 6, 7 or 7, 8. 5. NaOAC 232* 325 SH 7 - hydroxyl Group. 6. NaOAC + H QBo Q 231* 324 Ortho-dihydroxyl Groups on Ring "A". Infrared Data: Aromatic, free adjacent OH Groups. C-H Bonds, Phenolic structure. Main Absorption Peak •kic Rf values in Chloroform: Methanol Solvent 91:9 S H I n f l e c t i o n Point Values 1-6 expressed in millimicrons Table 6 (continued) 59 Line 3 Test Indicates Colour (U.V.) Yellow/Green Flavanone, Aurone, Flavonol. Colour (NH3) Light Yellow Rf** 0.50 1. MeOH 268* 319 SH Flavanone structure. 2. NaOMe 273* 323 SH Lacks 5, 7 dihydroxyl Groups. 3. A1C1 3 266* 299 SH Lacks 6, 7 or 7, 8 dihydroxyl Groups. 4. A1C1 3 + HCl 269* 301 SH 5 - hydroxyl Group absent. 5. NaOAC 272* 311 SH Lacks 5, 7 dihydroxyl Groups. 6. NaOAC + H,Bo, 268* 322 SH Lacks 6, 7 dihydroxyl Groups 0 0 on Ring "A". Infrared Data: C-H, Free OH, Ketone present, Phenolic structure. Line 4 Test Indicates Colour (U.V.) Fluorescent Blue Colour (NHJ Fluorescent Blue Isoflavone lacking a 5-hydroxyl Group. Rf** 0.65 1. MeOH 2.68* 330 SH 2. NaOMe 271* 334 SH "A" Ring hydroxyl Groups. 3. A i d , 274* 297 Lacks 6, 7 or 7, 8 ortho-dihydroxyl Groups. 4. A1C1, + HCl 5. NaOAC 277* 295 SH Lacks 5 - hydroxyl Group. 277* 319 SH Lacks 6, 7 dihydroxyl Groups on Ring "A". Infrared Data: Free hydroxyl Group. C-H C=0 c-c multiple bonds Phenolic structure. Line 5 Test Indicates Colour (U.V.) Red Chalcone, carotenoid or Colour ( N r L ) Red- 3ink anthocyanin derivative. Rf** J 0.84 1. MeOH 254* 402 2. NaOMe 256* 402 Lacks "A" ring hydroxyl Groups. 3. A1C10 256* 421 Ortho-dihydroxyl Groups present. 4. A1CU + HCl 257* 421 Adjacent hydroxyl Groups. 5. NaOAC 256* 405 on the nucleus. 6. NaOAC + HQBOQ 273* 407 Ortho-dihydroxyl Groups - present on the skeleton. Infrared Data: .C-C Bonds, aromatic ring nucleus, free OH groups. Table 6 (continued) 60 2. Vi c i a americana -- Plot 1, Cut 1 (Vegetative Stage) Chloroform Extract (Fraction B -- Maximum Anti-estrogenic A c t i v i t y ) Line 1 Test Indicates Colour (U.V.) Colour (NH,) 1. MeOH 2. NaOMe 3. A1C1, 4. A1C1, + HCl 5. NaOAC 6. NaOAC + H 3Bo 3 Dull Yellow Dull Yellow 0.12 235* 280 SH - 279 SH 237* 283 SH 235* 277 SH - 280 SH - 280 SH Flavonol, a r t i f a c t , or coumaranone derivative. Infrared Data: C-H, C-C Multiple Bond, Phenolic structure. Line 2 Test Indicates Colour (U.V.) Colour (NH,) Rf** J 1. MeOH 2. NaOMe 3. Al Cl 3 4. A1CU + HCl 5. NaOAC 6. NaOAC + H 3Bo 3 Fluorescent Blue Fluorescent Blue 0.65 277* 322 SH 279* -281* 228 SH 279* 324 SH 278* 325 SH 281* 325 SH Flavanone lacking a 5-hydroxyl Group. Lacks a 5-hydroxyl Group. Ortho-dihydroxyl Groups at 6,7 or 7, 8. Lacks a 5-hydroxyl Group. Lacks a 5, 7 dihydroxyl structure. 6, 7 dihydroxyl Groups absent from Ring "A". Infrared Data: Free OH groups. C-H, aromatic, CH2 groups. Line 3 Test Indicates Colour (U.V.) Colour (NH3) Rf** 1. MeOH 2. NaOME 3. ALCLo 4. A1C1- + HCl 5. NaOAC 6. NaOAC + H 3Bo 3 Fluorescent Blue Fluorescent Dark Blue 0.82 257* 282 SH 255* 279 257* -233* 273 SH 234* 273 SH 221* 272 SH Flavene or benzofurane nucleus. C 3 methylated or glycoside attachment. Ortho-dihydroxyl Groups. 3 adjacent hydroxyl Groups. Free hydroxyl Groups. Ortho dihydroxyl Groups present. Infrared Data: Aromatic structure. Hydroxyl groups. Adjacent hydroxyl groups. Table 6 (continued) 61 Line 4 Test Indicates Colour (U.V.) Colour (NHo) Rf** J 1. MeOH 2. NaOME 3. A1CU 4. Al CI ^  + HCl 5. NaOAc 6. NaOAC + H 3Bo 3 Dull Yellow Green Fluorescent Yellow 0.85 272* 342 SH 272* 340 SH 273SH 340SH 272SH 340SH 272SH 340SH 271SH 294SH Flavanone lacking a 5-hydroxyl Group, Flavone or Flavan. Lacks a 5, 7 dihydroxyl structure. Lacks a free 5-hydroxyl Group. Lacks a free 5-hydroxyl Group. Not a 5, 6 dihydroxyl Structure. Dihydroxyl Groups present on the skeleton. Infrared Data: C-H, Phenolic, OH groups, Ring Structure, Aromatic, Adjacent OH groups 3. Vicia americana — Plot 1, Cut 4 (Seed disseminated staqe). Ether Extract (Fraction A) — maximum estrogenic a c t i v i t y ) Line 1 Test Indicates Colour (U.V.) Colour (NH.J Rf** J 1. MeOH 2. NaOME 3. A1C1 3 4. Al CI 3 + HCl 5. NaOAC 6. NaOAC + H 3Bo 3 Fluorescent Blue Intense Fluorescent Blue 0.30 283* 308 295* 336 282* 312 282* 312 271* -284* 312 Flavone Lacking a free 5-hydroxyl Group. 3, 3' or 4' hydroxyl Groups. Ortho dihydroxyl Groups present. Keto group s t a b i l i z e s dihydroxyl Groups. 3, 7 and 4' hydroxyl Groups present. Ortho-dihydroxyl Groups present. Line 2 Test Indicates Colour (U.V.) Colour (NH~) Rf** 6 1. MeOH 2. NaOME 3. A1CU 4. A1CH + HCl 5. NaOAC 6. NaOAC + H 3Bo 3 Dull Yellow Dull Yellow 0.47 279* 312 286* 334 278* 304 280* 302 279* 336 280 - Intensity Decreased Dihydroflavonol lacks a 5-hydroxyl Group, or a flavonol with a 3-hydroxyl Group with or without a 5-hydroxyl group. 3, 3' or 4' hydroxyl Groups Ortho dihydroxyl Groups present. Ketone present. Free 7-hydroxyl Group. Ortho di hydroxy! Groups .< present. Table 6 (continued) 62 Line 3 Test Indicates Colour (U.V.) Fluorescent Blue Flavone lacks a 5-hydroxyl Colour (NH,) Intense Fluorescent Group, or flavonol lacks Blue a 5-hydroxyl Group. Rf 0.55 1. MeOH 280* 310 2. NaOMe 289* 333 3, 3' or 4' hydroxyl Groups. 3. A1C1, 281* 305 Lacks ortho-dihydroxyl Groups. 4. A1C1, + HCl 280* 304 Acid stable -- lacks d i -hydroxyl Groups or are substituted. 5. NaOAC 277* 333 4' hydroxyl Group present. 6. NaOAC + H,Bo, 279* 307 Lacks ortho-dihydroxyl Groups. Line 4 Test Indicates Colour (U.V.) Fluorescent Blue Flavone or flavonol Colour (NH,) Intense Fluores- Lacking a free 5-hydroxyl cent Blue Group. Rf** 0.64 1. MeOH 283* 307 2. NaOME 294* 335 Free 4' and 3-hydroxyl Groups. 3. A1C1, 280* 308 Lacks ortho-dihydroxyl Groups. 4. A1C1, + HCl 280* 309 Ketone present or hydroxyl Groups substituted. 5. NaOAC 283* 335 7 and 4' - hydroxyl Groups present. 6. NaOAC + H 3Bo 3 288* 309 Lacks ortho-dihydroxyl Groups. Line 5 Colour (U.V.) Fluorescent Blue Flavone or flavonol lacking a Colour (NH,) Intense Fluorescent free 5-hydroxyl with 3-Blue hydroxyl substituted. Rf** 0.74 1. MeOH 283* 311 2. NaOME 291* 334 6, 3, 3' or 4' hydroxyl Groups. 3. A1C1, 275* 308 Ortho-dihydroxyl Groups present. 4. A1C1, + HCl 280* 307 Ketone present or 3 and 5 0 hydroxyl Groups substi-tuted. 5. NaOAC 282* 333 7-hydroxyl Group free. 6. NaOAC + H,Bo, 286* - B-ring ortho-dihydroxyl Groups present. Table 6 (continued) 63 Line 6 Test Indicates Colour (U.V.) Colour (NH,) Rf** 6 1. MeOH 2. NaOME 3. A1C13 4. A1C1 3 + HCl 5. NaOAC 6. NaOAC + H 3Bo 3 Fluorescent Fluorescent 0.84 310 335 310 Blue Blue 268* 268* 268* 272* 310 268* 336 265* 308 Flavone or flavonol lacks a free 5-hydroxyl Group or flavonol with 3-hydroxyl substituted. 3,3' or 4' hydroxyl Groups. Lacks ortho-dihydroxyl Groups. 3 and 5 Ketone hydroxyl Groups absent or substi-tuted. 4' and 7 hydroxyl Groups. Lacks ortho-dihydroxyl Groups. Line 7 Test Indicates Colour (U.V.) Colour (NH,) Rf** 1. MeOH 2. NaOMe 3. A1CU 4. A1C13 + HCl 5. NaOAC 6. NaOAC + H 3Bo 3 Fluorescent Blue Fluorescent Dark Blue 0.86 282* 304 288* 340 278* 307 SH 276* 309 SH 279* 335 280* 310 SH Flavone or flavonol. Lacks a 5-hydroxyl Group with 3-hydroxyl substituted. 3, 3' or 4' hydroxyl Groups. Lacks ortho-dihydroxyl Groups. Ketone present or substituted hydroxyl Groups. Free 7-hydroxyl Groups. Lacks ortho-dihydroxyl Groups. 4: Astragalus miser var. serotinus -- Plot 2, Cut 1 (Vegetative Stage) Ether Extract (Fraction A) Line 1 Test Indicates Colour (U.V.) Dull Yellow Chalcone, carotenoid or Colour (NH,) Dull Yellow Green anthocyanin derivative Rf** J 0.34 or hydrolysis product. 1. MeOH 277* 406 2. NaOMe 273* 405 3. A1C1, 271* 418 Ortho-dihydroxyl Groups. 4. Aicr+ HCl 273* 416 5. NaOAC 271* -6. NaOAC + H 3Bo 3 272* -Infrared Data: OH, C-H, Ketone Phenolic structure. Adjacent OH groups. Aromatic structure. Table 6 (continued) 64 Line 2 Test Indicates Colour (U.V.) Fluorescent Blue Isoflavone lacks a free 5 -Colour (NH,) Dark Purple hydroxyl Group. Rf** J 0.58 1. MeOH 275* 312 SH 2. NaOMe 284* 325 SH "A" Ring hydroxyl Groups. 3. A1C1, 278* 311 SH Lacks free 5-hydroxyl Group. 4. A l C l o + HCl 277* 310 SH Lacks free 5-hydroxyl Group. 5. NnOAC 278* - Lacks 7 hydroxyl Group or 0 2 at posi t ion # 6 . . . . LacKs 6, 7 dihydroxyl Groups on Ring "A". 6. NaOAC + H 3 Bo 3 276* — Infrared Data: Aromatic nucleus Free OH groups C-H Bonds. Line 3 Test Indicates Colour (U.V.) Fluorescent Br ight Flavonol with free 3-hydroxyl Colour (NH,) Yellow with or without free 5 -Fluorescent Bright hydroxyl. Yellow Rf** 0.95 1. MeOH 269* 324 SH 2. NaOMe 279* 324 SH 7-hydroxyl Group. 3. A1C1 268* 322 SH No ortho-dihydroxyl Groups. 4. Al CI 3 + HCl 269* 322 SH 3 and 5-hydroxyl Groups absent or subst i tuted. 5. AnOAC 275* 327 SH 6 and 8 oxygen subst i tuents . 6. NaOAC + H 3 Bo 3 270* 324 SH No B-Ring ortho-dihydroxyl Groups. Infrared Data: Aromatic nucleus groups. OH groups, C-H Bonding, OH free groups. Line 4 Test Indicates Colour (U.V.) Br ight Red (Green Xanthone, aurone or chalcone Colour (NH,) . in v i s i b l e hydrolysis product. spectrum ) Bright Red Rf** 0.97 1. MeOH 262* 324 SH 2. NaOMe 249* 348 Free hydroxyl Groups. 3. A1C1, 265* 323 4. A1C1- + HCl 267* 323 Ketone present. 5. NaOAC 259* 286 SH Free hydroxyl Group. 6. NaOAC + H,Bo, 264* 327 SH Ortho-dihydroxyl Groups O 0 absent. Infrared Data: Aromatic, free OH groups C-0 present, phenolic present Table 6 (continued) 65 6. Astragalus miser var. serotinus -- Plot 2, Cut 1 (Vegetative Stage) Chloroform Extract (Fraction B — Maximum A n t i e s t r o -genic A c t i v i t y ) Line 1 Test Indicates Colour (U.V.) Fluorescent Light Flavanone lacks a free 5-Colour (NH,) Blue hydroxyl Group. Fluorescent Light Blue Rf** 0.07 1. MeOH 278* 320 SH 2. NaOME 280* 324 SH Lacks "A" Ring hydroxyl Groups. 3. A i d , 276* - Lacks 5, 6, 7, 8 hydroxyl o Groups. 4. A l C l o + HCl 5. NaOAC 276* -280* 326 SH Lacks 5, 7 dihydroxyl Groups. 6. NaOAC + H,Bo, 280* - Lacks 6, 7 dihydroxyl Groups o 0 on Ring "A". Infrared Data: Aromatic, free hydroxyl groups, c-c bonds phenolic present Line 2 Test Indicates Colour (U.V.) Dark Blue Flavanone or dihydro-flavanone Colour (NH,) Purple with 5' or 4' hydroxyl Rf** 0.37 Group. 1. MeOH 273* 323 SH 2. NaOME 238* 292 SH 5-hydroxyl present. 3. A1CU 228* 292 Free 5-hydroxyl Group. 4. A1C1, + HCl 5. NaOAC 227* 273 SH Free 5-hydroxyl Group. 278* - Lacks 5-7 di hydroxyl Group. 6. NaOAC + H,BoQ 276* - Lacks 6, 7 dihydroxyl Groups on Ring "A". Infrared Data: Aromatic, phenolic, C-C multiple bonds C-H, OH groups. Line 3 Test Indicates Colour (U.V.) Dark Blue Flavanone with 5-hydroxyl Group. Colour (NHJ Purple Rf** ^ 0.69 1. MeOH 2 7 7 * 327 SH 2. NaOME 278* - "A" Ring hydroxyl Group. 3. A l C l o 275* 317 SH Lacks 6, 7, Or 7, 8 ortho-4. A1CH + HCl 5. NaOAC 275* 318 SH di hydroxyl Groups. 276* - Lacks a 7 hydroxyl Groups. 6. NaOAC + H,Bo, 273* - Lacks 6, 7 dihydroxyl Groups on Rinq "A". Infrared Data: Aromatic structure, C-c multiple bonds, OH group, Phenolic present. Table 6 (continued) 66 6. Astragalus miser var. serotinus -- Plot 2, Cut 3 (Seed Pod Stage) Ether Extract (Fraction A) Maximum Estrogenic A c t i v i t y Line 1 Test Indicates Colour (U.V.) Colour (NH,) Rf** J 1. 2. MeOH NaOMe 3. A1C1 4. A1C1, + HCl 5. NaOAC NaOAC + H 3Bo 3 Red (Green i n v i s i b l e Red spectrum) 0.07 399* 304 SH 399* 306 SH 408* 305 SH 408* 308 SH 397* 304 SH 399* 311 SH Aurone • chlorophyll fragment. Lacks a free 4' hydroxyl Group. Lacks "B" ring ortho-di hydroxyl Groups. I I I I n H Lacks a free 4' and/or 6 hydroxyl Group. Lacks "A" Ring ortho-di hydroxyl Groups. Line 2 Test Indicates Colour (U.V.) Fluorescent Blue Coumaranone or flavonoid Colour (NH,) Fluorescent Blue hydrolysis product. Rf** J 0.20 1. MeOH 266* 263 SH 2. NaOMe 266* 263 SH 3. A1C1, 311* 266 SH Ortho-dihydroxyl Groups. 4. A1C1, + HCl 311 265 SH Ketone or substituted hydroxyl Groups. 5. NaOAC - 265 SH 6. NaOAC + H 3Bo 3 - 266 SH Line 3 Test Indicates Colour (U.V.) Fluorescent Blue Flavan or p a r t i a l flavonoid skeleton. Colour (NH,) Fluorescent Blue Rf** J 0.28 1. MeOH 271* 265 SH 2. NaOMe 270* 267 SH No reactions indicated flavonoid nucleus and attached Groups not present. 3. A1C1, 271* 265 SH 4. A1C1X + HCl 271* 266 SH 5. AnOAC 272* 269 SH 6. NaOAC + H 3Bo 3 271 268 SH Table 6 (continued) 67 Line 4 Test Indicates Colour (U.V.) Colour (NH,) Rf** J 1. MeOH 2. NaOMe 3. A1C1, 4. A1C1, + HCl 5. NaOAC 6. NaOAC + H 3Bo 3 Fluorescent Blue Fluorescent Blue 0.45 271* 266 SH 272* 336 SH 271* 265 SH 271* 265 SH 271* 263 SH 271* 263 SH Coumaranone flavan or hydro-l y s i s by-products. Free hydroxyl Groups. Line 5 Test Indicates Colour (U.V.) Colour (NH,) Rf** J 1. MeOH 2. NaOMe 3. A1C1, 4. A1C1 3 + HCl 5. NaOAC 6. NaOAC + H 3Bo 3 Fluorescent Blue Fluorescent Bl ue 0.62 271* 327 SH 269* 329 SH 271* -271* -271* 328 SH 271* -Flavanone lacking a 5-hydroxyl Groups. Isoflavone or flavonol lacking a 5-hydroxyl Group. Ortho-dihydroxyl Groups. Keto Group of substituted hydroxyl Groups. "B" Ring ortho dihydroxyl Groups. Line 6 Test Indicates Colour (U.V.) • " • • { . . Colour (NH,) Rf** J 1. MeOH 2. NaOMe 3. A1C1, 4. A1C1, + HCl 5. NaOAC 6. NaOAC + H 3Bo 3 Intense Fluorescent Blue Fluorescent Blue 0.73 271* 328 SH 271* 329 SH 274* -.278* -271 328* 280 -Flavanone, isoflavone or ' flavonol lacking a 5-hydroxyl Group. No free 4' hydroxyl Group. Ortho dihydroxyl Groups present. Lacks a 7-hydroxyl Group. Ortho-dihydroxyl Groups present. Line 7 Test Indicates Colour (U.V.) Intense Fluorescent Flavone, flavonol lacks free 5-Colour (NH3) Blue hydroxyl Groupl with 3-Intense Fluorescent substituted. Blue Rf** 0.84 1. MeOH 276* 330 2. NaOMe 270* 333 C-3 hydroxyl group methylated or glycoside attachment. 3. A1C1, 275* 409 Ortho-dihydroxyl groups present. 4. A1C1, 276* 406 Keto group adds s t a b i l i t y to A l C l ^ complex. 5. NaOAC 280* 331 Free 7-hydroxyl Group. 6. NaOAC + H 3Bo 3 276* Ortho dihydroxyl Groups present. 68 estrogenic a c t i v i t y in V i c i a americana. (Lines 5 and 7 of the seed disseminated stage, and Astragalus miser var. s e p o t i n u s — l i n e s 1 and 3 of the seed pod stage.) The results obtained from these experiments add to the observations by previous authors (Towers et al_. 1964), that plant phenolics contained in V i c i a americana and Astragalus miser var.  serotinus are evident, and c h a r a c t e r i s t i c of s p e c i f i c growth stages of these legumes. From these examinations, i t appears that V i c i a americana and Astragalus miser var. serotinus both contain low potency estrogenic/ anti-estrogenic a c t i v i t y . Consumption rates by domestic livestock from bioassay experiment extrapolation would be in the magnitude of one t h i r d of body weight for noticeable effects. However, as noted by Braden and Shutt (1970), pasture estrogens undergo rapid ruminal demethylation into structures with increased estrogenic a c t i v i t y ( i . e . biochanin A conversion to genistein). Previous research by Turnbull et aj_. (1966), K a l l e l a (1972) has indicated that prolonged grazing on estrogenic pastures is responsible for n o n - f e r t i l i z a t i o n due to the lack of sperm migration through the fal l o p i a n tubes of sheep, and de-creased uterine f l u i d content preventing conception in rats. The results presented indicate that mammalian species do respond to the low potency u t e r o t r o p i c compounds present in the legumes examined; further investigations into rumen a c t i v i t i e s of the extracts would give a clearer picture of the effects of the plant products on domestic livestock. 69 CHAPTER IV THE EFFECTS OF FERTILIZER TREATMENTS ON THE ESTROGENIC COMPONENTS OF ALSIKE CLOVER (TRIFOLIUM HYBRIDIUM) WHITE CLOVER (TRIFOLIUM REPENS VAR. LADINO) AND ALFALFA (MEDICAGO SATIVA VAR. VERNAL) INTRODUCTION One of the major factors influencing the levels of estrogenic compounds in legume species i s mineral n u t r i t i o n . The e a r l i e s t occurrence of clover disease during the 1940 era in Australia was due to a lack of phosphate f e r t i l i z e r on pasture swards, coupled with heavy grazing by ruminants. Early research (Alexander and Rossiter 1952) showed that the a c t i v i t y of strains of Trifolium subterraneum was inversely related to the amount of superphosphate applied to the s o i l . The addition of potassium, magnesium, copper, n i t r a t e or lime to the superphosphate did not s i g n i f i c a n t l y affect the estrogenic a c t i v i t y of the plants. Bickoff (1968) has reviewed several reports by various authors, notably concerning Trifolium subterraneum. Evidence i s lacking in North America on the effects of f e r t i l i z e r elements on the estrogenic constituents of standard forage legume species grown for animal feeds. This study involved an assessment of the estrogenic isoflavones biochanin A, formononetin, and genistein, and coumestan, coumestrol, found in three domestic legumes: Trifolium hybridium, Trifolium repens, and Medicago sativa, as affected by topical f e r t i l i z e r applications. 70 MATERIALS AND METHODS Seed, Plot Preparation and Layout Selected Canada #1 grade seeds of als i k e clover (Trifolium  hybridium), white clover (Trifolium repens var. ladino) and a l f a l f a (Medicago sativa),were inoculated with rhizobium bacteria (Nitragin Co. Ltd. "AB" culture) d i r e c t l y before planting to ensure adequate nitrogen f i x i n g bacteria on the root systems. The plot s i t e on the University of B r i t i s h Columbia Plant Science Department experimental area was disced, l e v e l l e d and 648 kgs/ hectare of agricultural lime were applied and thoroughly mixed to neutralize the acid s o i l conditions of the zone. The plot area had remained i n fallow for two growing seasons. Discing and l e v e l l i n g assured a homogenous seed bed. Plot layout for each species i s i l l u s t r a t e d i n Figure 1.1. A random design was chosen for the treatments. Each treatment received a minimum of four replicates. Control plots for each treatment were also replicated by a factor of four. Figure 12 indicates actual ground lay-out of Trifolium repens var. ladino. (Medicago sativa l i e s adjacent to this block and i s replicated in the same manner.) 1.8 m «fl 8» CI Nl 0.9 m CO P2 N2 CO PI CO CO PI K2 CO Kl P2 C2 Nl PI CO C2 CI CL P2 CL K2 CO K2 CO Kl P2 CO CI Nl C2 N2 CO PI Figure 11 Plot Layout and F e r t i l i z e r Treatments 71 Figure 12 Trifolium Repens Var. Ladino Random Design for F e r t i l i z e r T r i a l s . Seeding Rates and F e r t i l i z e r Treatments 72 Seeding Rates Alsike Clover = 5.3 kg/hectare Ladino Clover = 5.3 kg/hectare A l f a l f a =15.9 kg/hectare F e r t i l i z e r treatments Descri ption kg/hectare CO Control -- no f e r t i l i z e r 0 CI 4-10-10 flower and vegetable food 432 C2 4-10-10 648 NO Control -- no f e r t i l i z e r 0 Nl 16-0-0 ni t r a t e of soda 162 N2 16-0-0 324 PO Control — no f e r t i l i z e r 0 PI 0-45-0 treble super phosphate 108 P2 0-45-0 216 KO Control - no f e r t i l i z e r 0 Kl 0-0-60 Muriate of potash 108 K2 0-0-60 216 Seeding and F e r t i l i z e r Application -- Plot Maintenance Procedure Each 1.8 x 3.1 M plot was raked by hand, and f e r t i l i z e r applied in two directions. Seed was applied following f e r t i l i z e r applications, and spread uniformly on each plot. Plots were again evenly raked in two directions and r o l l e d to ensure seed contact with the s o i l surface. The s o i l surface was moistened to a depth of 2.2 cm following seeding to stimulate germination. This procedure was continued intermittently un t i l germination occurred and seedlings became established. During the t r i a l period, s o i l moisture was maintained with sprinkler i r r i g a t i o n application at the f i r s t sign of moisture deficiency. Water was judiciously applied and s o i l puddling was prevented by 73 controlled application rates. A l l weeding and thinning of plots were done by hand; no herbicides were applied to the plot areas. Border areas surrounding and between the plots were continuously mowed to a 2.2 cm height to prevent plot contamination by foreign plant species. Harvesting and Plant Storage A l l blocks were harvested at f u l l bloom stage. Water was with-drawn 72 hours prior to cutting; the plots were mowed at a height of 2.2 cm above ground surface (to eliminate s o i l contamination); each plot y i e l d was recorded; the four plot replicate cuttings were bulked, thoroughly mixed, and four random 1 kg samples taken from the bulked plots. These samples were sealed in p l a s t i c bags and quick frozen at 0°C for storage. Control plots received the same treatment. Samples were analysed for dry matter content according to the methods of the A.O.A.C. (1960). Fractionation of Plant Material Twenty-five gram samples (D.M.) were extracted as in Part 1 (Figure 5) for each treatment. The ether extracts obtained were weighted and adjusted to a concentration of 15.0 mg/ml in toluene: ethanol (50:50) prior to chromatography. Thin Layer Chromatography of Plant Extracts Thin layer chromatographic plates layered with S i l i c a Gel G (Merck) at 0.25 mm thickness were activated by heating at 110°C 74 for t h i r t y minutes. Fifteen m i c r o l i t e r s were applied with constant cold a i r drying at the o r i g i n , spot size was kept constant at 2 30 mm . Each sample was spotted on duplicate plates. Standards consisting of: Biochanin "A" - 2.0, 4.0, 6.0 micrograms Coumestrol - 0.5, 1.0, 2.0 micrograms Formononetin - 0.5, 1.0, 2.0 micrograms Genistein - 2.0, 4.0, 6.0 micrograms were run concurrently on separate plates in the same solvent system. Standards were run with each duplicate sample chromatographed. Two dimensional ascending chromatography was employed; Solvent I consisted of chloroform:methanol (91:9), and Solvent II chloroform:methanol 82:18 — methanol made to 1.0 N with ammonia gas). Various solvent systems were tested, but the chloroform:methanol systems gave the best R.p values and separation of the spots. Both systems were run to approximately 16.0 cm solvent length in unsaturated atmospheres. Intermediate drying of 3.0 minutes between runs ensured uniform spot sizes in the second dimensional separation. Quantitative Determination of Estrogenic Constitutent of the  Plant Extracts Quantitative in s i t u estimation of isoflavones/coumestans on T.L.C. plates was conducted with a Turner model 111 fluorometer with Camag E.L.C. plate scanner door attachment. Greater s e n s i t i v i t y of deflection was obtained by attaching a Unicam SP20 Recorder programmed for l i n e a r recording. A #110-850 lamp (Turner) with peak emission at 360 75 millimicrons, and primary f i l t e r #7-54 ( Transmits below 254 my to 420 my) were u t i l i z e d . A secondary f i l t e r of 2A-15 (passes wave lengths longer than 520 my) for biochanin A, formononetin, and genistein estimations was employed, and a Kodak Wratten #8 Secondary F i l t e r (passes wave lengths longer than 485 my) was found to give maximum readings for coumestrol determinations. Fluorometer s l i t widths were adjusted to give maximum deflections for each isoflavone; and consisted of: 1° S l i t 2° S l i t Biochanin A 10.0 mm 3.0 mm Formononetin 10.0 mm 3.0 mm Coumestrol 3.0 mm 1.0 mm Genistein 10.0 mm 3.0 mm Following development of the plates, coumestrol and formononetin spots were located by t h e i r values and colour appearances under o 3650 A l i g h t . For preliminary i d e n t i f i c a t i o n , the spots were c i r c l e d , eluted from the plates, dissolved in spectroscopic methanol, and u l t r a -v i o l e t absorption spectra recorded and compared to authentic samples of coumestrol and formononetin. For quantitative estimation, spots of coumestrol and formononetin were c i r c l e d on the plates, exposed to ammonia fumes for 10 seconds to enhance th e i r fluorescence, and scanned within 15 seconds of exposure to the ammonia fumes. Biochanin A and genistein spots were located by spraying with diazotized s u l f a n i l i c acid, and corresponding areas of thin layer plates eluted with methanol; comparison of u l t r a v i o l e t absorption spectra, colours with diazotized s u l f a n i l i c acid, and R^  values when 76 compared to r e - c r y s t a l l i z e d samples of biochanin A and genistein confirmed the i d e n t i f i c a t i o n of the isoflavones. Figure 13 i l l u s -trates chromatographic mobilities of the compounds. Quantitative estimation of the two isoflavones was accomplished by spraying with a 5% solution of AlCl3 in 95% d i s t i l l e d ethanol, followed by heating for 3 minutes at 75°C to inte n s i f y the fluorescence. A l l scanning was done within 2 minutes following heating; t r i p l i c a t e readings for each scan were recorded. Standard curves of concentration ys_ fluorescence were calcu-lated for each chromotographic plate. Isoflavone/coumestrol concentrations were determined.on a dry weight basis for each plant species and f e r t i l i z e r treatment. The results are tabulated i n Tables 7 and 8. RESULTS AND DISCUSSION Dry matter yields (Table 7) were s l i g h t l y but i n s i g n i f i c a n t l y (P > .05) affected by f e r t i l i z e r applications, due to large standard errors from the mean for replicate plots for each f e r t i l i z e r treatment. Ladino clover treatments were least affected by f e r t i l i z e r s , probably due to the forage's indeterminate growth pattern. Alsike clover and a l f a l f a responded to phosphate f e r t i l i z e r s , with the trend being to increase dry matter yields s l i g h t l y but i n s i g n i f i c a n t l y over control plots. Solvents: I II Figure 13 Chroma tographi c M o b i l i t i e s of Isoflavones and Coumestrol in Domestic Legumes 78 TABLE 7 THE EFFECTS OF FERTILIZER TREATMENTS ON THE DRY MATTER YIELDS OF RANDOMLY GROWN 1.8 x 3.1 M PLOTS OF ALSIKE CLOVER, LADINO CLOVER, AND ALFALFA (Average of four replicates) Treatment Alsike Ladino Al f a l f a (kgs) (kgs) (kgs) CO 4.7 3.1 3.0 CI 4.3 2.8 3.2 C2 5.0 2.9 2.8 Nl 3.7 2.7 2.6 N2 4.5 2.8 2.3 PI 4.9 2.6 3.6 P2 5.5 2.9 2.9 Kl 5.9 2.8 3.1 K2 4.9 2.7 2.5 TABLE 8 79 EFFECTS OF FERTILIZER TREATMENTS ON ESTROGENIC COMPONENTS OF LEGUME SPECIES Treatment Biochanin A Dry Matter Formononetin Genistein (% D.M.) Total Estrogenic Isoflavones 1. Alsike CO Cl C2 NI N2 Pl P2 Kl K2 Clover 0.011 0.012 0.002 0.002 0.006 0.001 0.019 0.004 0.005 0.002 0.003 0.001 0.001 0.001 0.002 0.009 0.001 0.001 0.004 0.005 0.005 0.005 0.005 0.004 0.030 0.008 0.003 0.018 % 0.020* 0.008* 0.008* 0.010* 0.007* 0.055* 0.013 0.008* 2. Ladino CO Cl C2 NI N2 Pl P2 Kl K2 Clover - Coumesti 0.0001 0.0001 0.0002 0.0001 0.0004 0.0001 0.0001 0.0001 0.0001 -ol 0.0018 0.0016 0.0012 0.0001 0.0009 0.0015 0.0020 0.0005 0.0005 0.007 0.007 0.006 0.001 0.001 0.001 0.001 0.002 0.004 0.009 % 0.008 0.008* 0.001* 0.002 0.003 0.003 0.003* 0.004* 3. A l f a l f a CO Cl C2 NI N2 Pl P2 Kl K2 - Coumesti 0.0001 0.0001 0.002 0.0002 0.0001 0.0004 0.0001 0.0001 0.0002 "Ol 0.0009 0.002 0.001 0.0001 0.0009 0.002 0.002 0.0005 0.0005 0.007 0.007 0.006 0.0004 0.0002 0.001 0.0006 0.002 0.004 0.008 % 0.009* 0.009* 0.0007 0.0012* 0.0034* 0.0027* 0.0026 ,0.0047 Indicates s i g n i f i c a n t difference between control and treatment at P < .05 when crossed with the three estrogenic constituents. (Anova with treatment and constituent crossed and replicate nested in constituent). 80 Estrogenic isoflavones and coumestrol levels were s i g n i f i -cantly (P < .05) affected by f e r t i l i z e r applications, when compared to control plots. Table 8 indicates that a l s i k e clover responded to treatment P2, increasing total isoflavone concentration by a factor of two; ladino clover followed observations by Rossiter (1970) that in low phosphate supplied plants total isoflavone levels increased during l a t e r growth stages, ( i . e . f u l l bloom stage). Highest tota l concentrations of isoflavones are present in control plots and plots receiving complete f e r t i l i z e r s . A l f a l f a plants responded s i g n i f i c a n t l y to complete f e r t i l i z e r applications, and no mineral applications, but total isoflavone content did not increase under individual N, P, or K added. The results are in agreement with Alexander and Rossiter (1951) who examined subterranean clover. These authors noted that T.  subterraneum contained more estrogenic potency when no f e r t i l i z e r was applied; a l f a l f a plants responded in this t r i a l i n the same manner, by increasing total isoflavone content. Schoo and Rains (1971) also confirmed the results with subterranean clover and formononetin content. An analysis of variance for the individual isoflavones s i g n i f i c a n t l y increased by f e r t i l i z e r treatment was conducted for each species. Treatment v£ control for the three estrogenic con-stituents i s presented in Table 9. 81 TABLE 9 ANALYSIS OF VARIANCE FOR THE EFFECTS OF FERTILIZER TREATMENTS ON THE ESTROGENIC CONSTITUENTS OF TRIFOLIUM REPENS, TRIFOLIUM  HYBRIDUM, AND MEDICAGO SATIVA Species Biochanin A Coumestrol Formononetin Genistein Alsike clover P2 Absent P2 P2 Ladino clover Absent N2 C0;P2* CO; CI* A l f a l f a Absent C2, PI CI, PI, P2 CI; C2* Not s i g n i f i c a n t over control P < .05). Alsike clover was most s i g n i f i c a n t l y increased, both in total and in individual free estrogenic isoflavone content, by the addition of 216 kgs/hectare of 0-45-0. Ladino clover constituents responded to nitrogen, (324 kg/hectare) and to control treatments. A l f a l f a i s o -flavones were s i g n i f i c a n t l y (P < .05) increased by the addition of complete f e r t i l i z e r (432 and 648 kgs/hectare of 4-10-10) and super phosphate (108 and 216 kg/hectare of 0-45-0). These results indicate that char a c t e r i s t i c ratios e x i s t in the three legume species of free estrogenic isoflavones; each species containing a genetically determined pattern of the three i s o -flavones as noted by Bailey and Francis (1971). Individual i s o -flavone levels were affected to a greater degree than total isoflavone content under mineral supplements. The importance of high levels of coumestrol and formononetin to the forage producer should be emphasized. Metabolic demethylation by ruminants of these two flavonoids results in the production of equol (7, 4' - dehydroxy isoflavan) as indicated by Shutt and Braden (1968). Further estimates of the potencies of these two compounds are presented in Table 2, by Bickoff (1968), and by Francis and 82 Millington (1971). The levels of estrogenic isoflavones found in these results and affected by macro-element supplements, do not compare with total estrogenic compounds levels in subterranean clover (app. 1% D.M.). However, as was indicated by Lindner (1967), sheep uterine responses at plasma levels of free genistein above 5 mcgm/100 ml of plasma were associated with maximal uterine growth response; plasma levels of 0.5 mcgm/100 ml plasma of formononetin also resulted in detectable uterotrophic a c t i v i t y . S i g n i f i c a n t isoflavone level increases and forage y i e l d s have resulted from these t r i a l s . F e r t i l i z e r applications and the effects on estrogenic constituents of the cultivated legumes have been demonstrated and can serve as guidelines for the forage producer in con t r o l l i n g plant hormonal a c t i v i t i e s , for increased growth and performance of domestic ruminants. SUMMARY AND CONCLUSIONS From the preceding experiments, the can be drawn: Experiment I 1. Vi c i a americana and Astragalus mi ser var. low levels of uterotrophic components in a dynamic state throughout the growing season; flavonoid and aromatic com-pounds prevail in ether and chloroform extracts of the legumes. following conclusions serotinus contain 2. Vi c i a americana demonstrated maximum uterine growth promoting effects in mammals at seed disseminated stage, and maximum growth i n h i b i t o r y a c t i v i t y at vegetative stage. Astragalus  miser var. serotinus possessed maximum uterine growth promot-ing effects at seed pod stage, with maximum inh i b i t o r y a c t i v i t y on the uterus at vegetative stage. No correlation existed between proximate analysis date, extract weights, and estrogenic/anti-estrogenic a c t i v i t i e s for both species. 3. Interference with the a c t i v i t y of animal estrogens by chloro-form extracts of the two legume species was most pronounced; uterine weight decreases of greater than 20% were obtained during the growing season for both legumes, due to injections of 15 gms dry matter equivalent of each species to immature female rats. 4. The experiments demonstrated a toxic fraction present in Astragalus miser var. serotinus at f u l l bloom stage. This component was ether extractable; the symptoms and effects on animals resembled the effects of miserotoxin. Experiment II 1. F e r t i l i z e r treatments of 4-10-10, 16,-0-0, 0-45-0, and 0-0-60 did not s i g n i f i c a n t l y affect the dry matter yields of als i k e clover, (Trifolium repens) ladino clover (Trifolium hybridum), and a l f a l f a (Medicago sativa) when applied at rates between 0 and 648 kgs/hectare, due t o large standard errors from the mean y i e l d s . A technique for the fluorometric determination for quanti-tative measurement of biochanin A and genistein was developed, u t i l i z i n g fluorescent properties of the two isoflavones when spayed with 5% Al Cl 3 in 95% ethanol. This allowed rapid and reproducible quantitations of the isoflavones i n s i t u on s i l i c a gel thin layer chromatographic plates. Estrogenic isoflavone and coumestrol levels were s i g n i f i c a n t l y affected (P < .05) by f e r t i l i z e r treatments; alsike clover increased total isoflavone concentrations by a factor of two. 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F b i l i t y Treatment 1 2.04 2.04 28.0 0.0008* Estrogenic Constituents 2 2.54 1.27 1743.3 Constituent Replicates 6 2.25 3.76 0.51 Error 8 5.82 7.28 - -Total 17 2.56 - -Control vs. C2 Treatment Source of Variance D.F. S.S. M.S. F Proba-b i l i t y Treatment 1 5.63 5.63 5.55 0.04* Esmogenic Constituents 2 7.17 3.58 3.53 Constituent Replicates 6 9.36 1.55 0.00 Error 8 8.11 1.01 -Total 17 2.09 - -Control vs. NI Treatment Source of Variance D.F. S.S. M.S. F Proba-b i l i t y Treatment 1 5.55 5.55 5.33 0.04* Estrogenic Constituents 2 7.66 3.83 3.68 Constituent Replicates 6 9.36 1.55 0.00 Error 8 8.32 1.04 -Total 17 2.15 • - - • * S i g n i f i c a n t at (P < 0.05). Appendix Table 1 (continued) 98 Control vs. N2 Source of Variance Treatment D.F. S .S. M. S. F Proba-bil i ty Treatment 1 1 .52 1. 52 6.00 0.03* Estrogenic Constituents 2 1 .58 7. 93 31.30 Constituent Replicates 6 1. .46 2. 44 0.00 Error 8 2 .02 2. 53 -Total 17 1 .94 -Control vs. PI Source of Variance Treatment D.F. S. S. M. S. F Proba-bil ity Treatment 1 6. 06 6. 06 5.56 0.04* Estrogenic Constituents 2 5. 58 2. 79 2.56 Constituent Replicates 6 7. 89 1. 31 0.00 Error 8 8. 72 1. 09 -Total 17 2. 03 - • Control vs. P2 Source of Variance Treatment D.F S.S M.S F Proba-bil ity Treatment Estrogenic Constituents Constituent Replicates Error Total . 1 2 6 8 17 6.66 3.72 7.14 2.32 1.27 6.66 1.86 1.19 2.90 22.95 6.42 0.00 0.0015* Control vs. K2 Source of Variance Treatment D.F. S.S. M.S. F Proba-bil ity Treatment Estrogenic Constituents Constituent Replicates Error Total 1 2 6 8 17 5.38 1.43 1.12 1.88 2.16 5.38 7.18 1.86 2.35 22.84 30.48 0.00 0.0015* Significant at (P < 0.05). Appendix Table 1 (continued) 99 B. A l f a l f a : Control vs. Cl Treatment Proba-Source of Variance D.F. S.S. M.S. F b i l i t y Treatment 1 5.20 5.20 11.46 0.009* Estrogenic Constituents 2 1.67 8.38 1846.87 Constituent Replicates 6 6.80 1.13 0.24 Error 8 3.63 4.53 -Total 17 1.68 - -Control vs. N2 Treatment Proba-Source of Variance D.F. S.S. M .S. F b i l i t y Treatment 1 1.06 1 .06 7. 83 0.02* Estrogenic Constituents 2 9.29 4 .64 34. 12 Constituent Replicates 6 3.65 6 .08 0. 004 Error 8 1.08 1 .36 Total 17 1.14 Control vs. Pl Treatment Proba-Source of Variance D.F. S.S. M.S. F. bi1i ty Treatment 1 3.72 3.72 6.06 0.03* Estrogenic Constituents 2 1.17 5.88 95.74 Constituent Replicates 6 4.89 8.15 0.01 Error 8 4.91 6.14 -Total 17 1.76 - -Control vs. P2 Treatment Proba-Source of Variance D.F. S.S. M .S. F. b i l i t y Treatment 1 1.39 1 .39 6. 81 0.03* Estrogenic Constituents 2 7.88 3 .94 19. 21 Constituent Replicates 6 3.28 5 .46 0. 00 Error 8 1.64 2 .05 Total 17 1.09 Sign i f i c a n t at (P < 0,05). Appendix Table 1 (continued) 100 C. Ladino Clover: Control vs. CI Treatment Proba-Source of Variance D.F. S .S. M .S. F. b i l i t y Treatment 1 1 .01 .1 .01 75.0 0.01* Estrogenic Constituents •2 1 .51 7 .59 56255.81 Constituent Replicates 6 7 .06 1 .17 8.72 Error 8 1 .08 1 .35 -Total 17 1 .52 -Control vs. C2 Treatment Proba-Source of Variance D.F. S.S. M .S. F. b i l i t y Treatment 1 1.36 1 .36 11.16 0.01* Estrogenic Constituents 2 1.28 6 .43 527.37 Constituent Replicates 6 6.13 1 .02 0.08 Error 8 9.75 1 .21 -Total 17 1.31 -Control vs.Nl Treatment Proba-Source of Variance D.F. S.S. M.S. F. bi 1 i ty Treatment 1 3.53 3.53 8.35 0.01* Estrogenic Constituents 2 4.18 2.09 4.94 Constituent Replicates 6 1.98 3.31 0.00 Error 8 3.38 4.23 -Total 17 1.11 - -Control vs. Kl Treatment Proba-Source of Variance D.F. S.S. M. S. F. bi 1 i ty Treatment 1 1.92 1. 92 7. 80 0.02* Estrogenic Constituents 2 6.14 3. 07 12. 45 Constituent Replicates 6 3.18 5. 30 0. 00 Error 8 1.97 2. 46 Total 17 1.00 Signif i c a n t at (P < 0.05) 101 ) • _ . Control vs. K2 Treatment • Proba-Source of Variance D.F. S.S. M.S. F. b i l i t y Treatment 1 1.05 1.05 10.80 0.01* Estrogenic Constituents 2 9.06 4.53 46.41 Constituent Replicates 6 4.25 7.08 0.00 Error 8 7.81 9.76 -Total 17 1.09 - -S i g n i f i c a n t at (P < 0.05). 

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