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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.Sc.  (Agr.) University  A THESIS SUBMITTED  o f B r i t i s h C o l u m b i a , 1970  IN PARTIAL FULFILMENT OF  THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE  i n the Department of  ANIMAL SCIENCE  We a c c e p t t h i s t h e s i s as c o n f o r m i n g to  the  r e q u i r e d staiTcfard  THE UNIVERSITY OF BRITISH COLUMBIA June 1974  In presenting  t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f the requirements  for an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r reference and study.  I f u r t h e r agree that permission f o r extensive  copying  of t h i s thesis f o r s c h o l a r l y purposes may be granted by the Head of my Department or by h i s representatives.  I t i s understood that  copying or p u b l i c a t i o n of t h i s thesis f o r f i n a n c i a l gain s h a l l not be allowed without my w r i t t e n permission.  James Stuart Gammie  Department o f ANIMAL SCIENCE The U n i v e r s i t y of B r i t i s h Columbia Vancouver 8, Canada  ii  ABSTRACT  Examination of Vicia amen'cana and Astragalus miser var. serotinus for estrogenic and anti-estrogenic a c t i v i t y demonstrated low potency uterotrophic compounds interfering with synthetic hormones in mammalian reproductive tracts. was affected by stage of growth.  The potency of the extracts  A toxic fraction was present in  Astragalus miser var. serotinus at f u l l bloom stage.  Hormonal a c t i v i t y  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 yields 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 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 l u s t r a t e d that N, P and K applications would s i g n i f i c a n t l y 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 ii  TABLE OF CONTENTS CHAPTER I. II.  PAGE INTRODUCTION  1  LITERATURE REVIEW  4  The Occurrence o f Flavonoids i n Nature  4  The Biosynthesis of Plant Flavonoids  5  Flavonoids i n Animals  7  Phenolic Compounds -- Plant and Animal D i s t r i b u t i o n and Relations  9  Functions of Flavonoids i n Plants . . .  10  Plant Phenols as N u t r i t i v e Sources  10  Phenols and Plant Pathogens  10  Plant Phenols and Plant Growth  11  The Occurrence of Phenolic Compounds i n Animals Distribution  12 12  Phenolic Compounds i n Animals; Estrogens i n Growth and Reproduction  13  Hormonal Control of Reproduction  14  The Bioassay of Estrogens The Occurrence of Plant Hormones A f f e c t i n g Animal Reproduction  15 18  Historical  18  Estrogens Isolated from Plant Forage Species . .  19  H i s t o l o g i c a l Changes of the Mammalian Reproductive Tract from the E f f e c t s of Plant Estrogens  20  iv PAGE  CHAPTER B e n e f i c i a l E f f e c t s of Plant Estrogens i n Animal Growth and F i n i s h i n g  22  Anti-Estrogenic A c t i v i t y of Forages  23  The E f f e c t s of Plant Extracts on P i t u i t a r y Function  27  Metabolism of Plant Estrogens by Animals  ....  28  Factors A f f e c t i n g the Estrogenic A c t i v i t i e s of Plants  31  Location  31  Season and Growth Stage  31  V a r i e t a l V a r i a t i o n i n the Estrogenic A c t i v i t y of Plants The E f f e c t s of D e f o l i a t i o n on Estrogenic Potencies of Legumes The E f f e c t s of Drying on the Estrogenic A c t i v i t y o f Legumes The E f f e c t s of Plant Pathogens on Estrogenic  33  Potency o f Plant Species  36  Mineral Elements and Plant Estrogen A c t i v i t y  32  34  . .  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 f o r Assessment of B i o l o g i c a l Separation  Activity. .  Procedure of Plant Extracts  U l t r a v i o l e t Absorption Spectra o f Plant Extracts  45 47 49  V  CHAPTER  PAGE U l t r a v i o l e t Spectra i n the Presence of Selected Reagents  49  Infrared Spectroscopic Plant Extracts  51  Examination of  Results and Discussion IV.  51  THE EFFECTS OF FERTILIZER TREATMENTS ON THE ESTROGENIC COMPONENTS OF ALSIKE CLOVER (TRIFOLIUM T HYBRIDIUM) WHITE CLOVER (TRIFOLIUM REPENS VAR. LADINO) AND ALFALFA (MEDICAGO SATIVA VAR. VERNAL) . . . Introduction Materials and Methods  69 69  .  70  Seed, P l o t 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 A p p l i c a t i o n -- P l o t Maintenance Procedure  72  Harvesting and Plant Storage  73  Fractionation o f Plant Material  73  Thin Layer Chromatography of Plant Extracts . . .  73  Quantitative Determination o f Estrogenic Constituent of the Plant Extracts Results and Discussion Summary and Conclusions  74 76  . .  82  Experiment I  82  Experiment I I . . . .  83  REFERENCES  85  APPENDIX  97  vi  LIST OF TABLES TABLE 1.  2.  3. 4.  5.  6.  7.  8. 9.  PAGE THE RELATIVE DOSE RATE OF ANIMAL ESTROGENS TO PRODUCE EQUIVALENT RESPONSES IN THE OVARECTOMIZED RAT UTERINE WEIGHT TEST  17  THE POTENCIES AND FORAGE CROP DISTRIBUTION OF THE ESTROGENIC AROMATIC COMPOUNDS AS ASSESSED BY MOUSE BIOASSAY  24  PROXIMATE ANALYSIS OF VICIA AMERICANA AND ASTRAGALUS MISER VAR. SEROTINUS . .  53  ETHER AND CHLOROFORM EXTRACT WEIGHTS (GMS) OF VICIA AMERICANA AND ASTRAGALUS MISER VAR. SEROTINUS (350 GM DRY MATTER SAMPLES)  54  THE EFFECTS OF ETHER AND CHLOROFORM EXTRACTS ON THE UTERUS OF THE LABORATORY RAT (EXPRESSED AS % OF BODY WEIGHT)  55  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  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  EFFECTS OF FERTILIZER TREATMENTS ON ESTROGENIC COMPONENTS OF LEGUME SPECIES . ......  79  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  vii LIST OF FIGURES FIGURE  1. 2.  PAGE  The Major Flavonoid Classes I s o l a t e d from Plant Species The D i s t r i b u t i o n of the Flavonoid Classes i n Nature  3.  Flavonoid Precursors and B i o s y n t h e t i c I n t e r r e l a t i o n ships of the Flavonoid Classes  4.  The Major Estrogenic Compounds I s o l a t e d from Legume Species  5. 6. 7.  ;la 6 8 . 19a  The Major Estrogenic Isoflavones and t h e i r Metabolic Products i n the L i v e r and Rumen  28a  Sampling Transects f o r Plant Species Vegetative Stage  43  —  Sampling Transects f o r Plant Species -Seed;Pod Stage  43  8.  Growth Stages f o r Analysis o f V i c i a americana  44  9.  Growth Stages f o r A n a l y s i s o f Astragalus miser var. serotinus F r a c t i o n a t i o n Procedure f o r the E x t r a c t i o n of Plant Estrogenic/Antiestrogenic Compounds  46  11.  P l o t Layout and F e r t i l i z e r Treatments  70a  12.  T r i f o l i u m repens var. l a d i n o . Fertilizer Trials  71  10.  13.  44  Random Design f o r  Chromatographic M o b i l i t i e s of Isoflavones and Coumestrol i n Domestic Legumes . . . . . . . . . . . . . . . .  77  ACKNOWLEDGEMENTS  The author wishes to thank Dr. W.D. K i t t s , Chairman of the D i v i s i o n o f Animal Science f o r h i s guidance and assistance during the course o f t h i s project. Additional thanks are extended to Miss G. Wilson and Mr. D. Pearce f o r 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 f o r photographic a s s i s t a n c e . The author also wishes to thank Dr. A.B. Beck and Dr. E.M. B i c k o f f f o r supplying pure isoflavone and coumestrol samples f o r 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 o f i n v e s t i g a t i o n i n the past f i f t y years.  The flavonoids are the l a r g e s t group of plant  phenols; they possess c l o s e l y r e l a t e d chemical  and s t r u c t u r a l properties  which r e f l e c t t h e i r unique biosynthetic pathways found only i n the plant kingdom (Geissman 1962; Faust 1965; Margma 1970). The basic nine carbon skeleton (Figure 1) with attachment of acetate units r e s u l t s i n highly s p e c i f i c products, many of which are r e s t r i c t e d to i s o l a t e d f a m i l i e s or genera of plants.  Flavonoids and  t h e i r d e r i v a t i v e s have found wide a p p l i c a t i o n i n the food industry: f l a v o u r i n g , colouration and usage as antioxidants and enzyme i n h i b i t o r s i s well documented.  Medicinal products, i n c l u d i n g potent central  nervous system drugs and hormone precursors, are also included i n the range of flavonoid products i n commerce  (Geissman 1962; Harborne 1967;  Heftmann 1967). In the area of domestic l i v e s t o c k growth and reproduction, producers and researchers have observed f o r over f o r t y 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 (Pieterse 1956; B i c k o f f 1968).  those o f the animal estrogens  Ingestion of these plant materials  at low concentrations has increased both growth and reproductive performance o f ruminants; adverse e f f e c t s , i n c l u d i n g d y s t o c i a , trans-  FLAVONOLS  Figure 1  ISOFLAVONES  The Major Flavonoid Classes Isolated from Plant Species.  2 i e n t and permanent i n f e r t i l i t y , have resulted from high l e v e l s of plant hormones i n the forages.  Bradbury and White (1954) confirmed reproduc-  t i v e problems were caused by the isoflavone and coumestan classes of the flavonoids i s o l a t e d from c u l t i v a t e d legume species. The occurrence of reproductive interference i n 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 u n i d e n t i f i e d  compounds may act as pro-estrogens, being converted by rumen fermentations to a c t i v e metabolites, whose e f f e c t s could be b e n e f i c i a l or deleterious on animal reproductive t r a c t s (Braden 'et aj_. 1967;  B i c k o f f 1968).  The commercial l i v e s t o c k industry has benefited from the rapid development and a p p l i c a t i o n of synthetic growth hormones i n 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 c l o s e r examination i n t o the b e n e f i c i a l properties of range and c u l t i v a t e d legumes i n s t i m u l a t i n g the production of high q u a l i t y meat f o r 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 v a r i a b l e :  environmental factors account f o r  greater than ninety per cent of these a l t e r a t i o n s i n a c t i v i t i e s .  This  two part study examined both the e f f e c t s 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 d i s t r i b u t e d  species of native plants used as forages -- V i c i a americana and Astragalus miser var. s e r o t i n u s , located on a primary beef production range, were assessed f o r t h e i r e f f e c t s on animal reproductive t r a c t s throughout the growing season.  Correlations with n u t r i t i v e values, and examination of  the compounds present i n the plant extracts were also conducted.  3 The second part of the study involved  the e f f e c t s of f e r t i l i z e r  a p p l i c a t i o n s on plant y i e l d s and estrogenic isoflavone important c u l t i v a t e d legume species: clover ( T r i f o l i u m bybridum),  alfalfa  levels.  Three  (Medicago s a t i v a ) ,  alsike  and ladino clover ( T r i f o l i u m repens),  were q u a n t i t a t i v e l y assessed f o r estrogenic isoflavones to c o n t r o l l e d f e r t i l i z e r a p p l i c a t i o n s .  when subjected  This study could present guide-  l i n e s f o r the forage producer i n c o n t r o l l i n g estrogenic a c t i v i t i e s legumes by the j u d i c i o u s use of f e r t i l i z e r treatments.  of  4  CHAPTER II LITERATURE REVIEW  THE OCCURRENCE OF FLAVONOIDS IN NATURE The water soluble flavonoid pigments occur almost u n i v e r s a l l y in higher p l a n t s , but are d i s t i n c t l y absent i n lower orders.  True  flowering plants (angiosperms) synthesize pigments i n stems and  leaves;  b a c t e r i a , algae, and fungi lack biosynthetic routes f o r flavonoids (Geissman 1962).  Liverworts and mosses contain anthocyanin and  flavonoids i n mono and diglycoside forms; the ferns resemble the angiosperms i n t h e i r pigment content, and possess the chalcone class —  a p r i m i t i v e c h a r a c t e r i s t i c 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 f l a v o n o i d s : i . e . flavones, f l a v o n o l s , flavanones and the majority o c c u r as glycosides.  leucoanthocyanins;  Anthocyanin pigments con-  s t i t u t e ; important colouring m a t e r i a l s ; imparting orange, s c a r l e t , crimson, mauve, blue, yellow and i v o r y to plants. These pigments have been located i n almost every plant part: stamen s t y l e and pod, leaves and stems.  c o r o l l a , s e p a l , bract, The anthocyanins show  importance as taxonomic markers at the genus and family l e v e l s . Detailed d e s c r i p t i o n s of the flavonoid c l a s s e s , t h e i r d i s t r i b u t i o n s , and t h e i r r o l e as taxonomic i n d i c a t o r s are presented by Bate-Smith (1962) and Harborne (1962).  5 The Biosynthesis of Plant  Flavonoids  The cytoplasmic route f o r biosynthesis o f plant flavonoids through the shikimic acid pathway to supply the Cg - a n d the g l y c o l y t i c pathway to supply the Cg has been well e s t a b l i s h e d (Faust 1965; Margma 1970).  This process occurs mainly i n green leaves;  l e a f synthesis occurs e a r l y i n the c h l o r o p l a s t s ; l a t e r photo reactions make flavonoid precursors a v a i l a b l e , and may promote Cg - Cg l i n k i n g , (Weinstein et al_. 1961; R o s s i t e r and Beck 1967b). l a t i o n i s i s o l a t e d i n the vacuoles.  Flavonoid accumu-  A pool of carbon substrates i s  u t i l i z e d -- sucrose during e a r l y l e a f growth, and carbon dioxide during l e a f expansion phases.  This carbon pool i s also u t i l i z e d i n  the production of c e l l p r o t e i n , c e l l w a l l , starches, and i n the r e s p i r a t o r y cycle ( R o s s i t e r 1972).  The combination o f the carbon  pool i n t o the flavonoid skeleton i s r e 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 c l a s s o f the flavonoids  serves as an intermediate i n flavonoid biosynthesis Z i l g and Grisebach  (Bate-Smith 1959.;  1968).  The coumestans and isoflavones are b i o g e n e t i c a l l y r e l a t e d ; coumestans belong to a c l a s s o f i s o f l a v o n e s ; the isoflavone daidzein serves as a precursor i n the production of the coumestans; the rearrangement o f the isoflavone molecule r e s u l t s i n the coumestan d e r i v a t i v e s o f the isoflavones (Goodwin 1965; Harborne 1967; Wong 1968).  Figure  3  i l l u s t r a t e s the s t r u c t u r a l r e l a t i o n s h i p s and  precursors o f the flavonoids. The synthesis o f the flavonoids i s under s p e c i f i c gene c o n t r o l , with a basic pigment pattern common to nearly a l l the higher  6 Flavonoid Complement  Phylogenetic Tree  Complete range of flavonoids (biflavonyls rare)  Angiosperms  most f l a v o n o i d s , but u s u a l l y simple types. B i f l a v o n y l s characteristic  Gymnosperms  Spermatophytes s t r u c t u r a l l y simple flavonoids: 3-deoxyanthocyani ns  Ferns.  flavones  Lycopods -Horsetails-  flavonols leucoanthocyanins chalcones flavanones  few flavonoid types mosses  only  3-deoxyanthocyanins flavonols. glycoflavones  'Red algae fungi  Flavonoids absent  bacteria Harborne (1967)  Figure 2  The D i s t r i b u t i o n 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 C j  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; ability to synthesize the flavonoid molecule is absent.  the  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  Cinnamic Acids  Lignin Coumarins Benzoic Acids 3 Malonate  Flavones  Chalcones  +  Stilbenes  Isoflavones  Aurones Anthocyanins Flavones Catechins  (After Goodwin 1965; Wong  Figure 3  1968)  Flavonoid Pre-cursors and Biosynthetic I n t e r r e l a t i o n s h i p s of the Flavonoid Classes. 00  9 for the d i s t i n c t brown-violet colouration i n weevil l a r v a e , and qui nones for pigment patterns i n aphid species.  However, vertebrate species do not  accumulate plant pigments to the same extent as i n s e c t species, and the majority are s u c c e s s f u l l y degraded and eliminated from the animal body.  Phenolic Compounds -- Plant and Animal D i s t r i b u t i o n and Relations The process o f estrogen and androstane synthesis i n animals occurs by the successive degradations of c h o l e s t e r o l .  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 i n plants (Bennett e_t al_. 1966).  Pregnenolone conversion to  progesterone was discovered i n the A f r i c a n shrub Holarrhena floribunda by Z a l k o w e t al_. (1964); estrone and e s t r i o l have been i s o l a t e d from Butea and willow catkins (Butenandt and Jacob 1933; Skarzynski 1933), and c h o l e s t r o l from red algae species (Tsuda et al_. 1958).  Little  knowledge i s a v a i l a b l e on the function of these i s o l a t e d animal s t e r o i d s in p l a n t s .  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 u n l i k e l y f o r the steroids 1967).  (Heftmann  Various theories on the a c t i v i t i e s of animal s t e r o i d s i n plants  have been proposed, and include the e f f e c t of the hormones i n increasing plant c e l l permeability to e l e c t r o l y t e s 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 i n the flowering plants and the fungi (Love and Love 1945; Mirocha et a]_. 1969).  10 FUNCTIONS OF FLAVONOIDS IN PLANTS Plant Phenols as N u t r i t i v e Sources The phenolic flavonoids and t h e i r esters are generally stable end products, although a c t i v e interconversions occur (Grisebach and Bopp 1959).  Normally the products are not translocated from t h e i r  c h l o r o p l a s t i c s i t e s of synthesis.  S o i l bacteria and fungi have evolved  enzymes f o r cleavage and u t i l i z a t i o n of phenols f o r food  resources  (Towers 1964), although i t i s not c l e a r 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 o f phenolic catechin glycosides by tea shoots; the sugar and aglycone was oxidized to y i e l d carbon dioxide. These r e s u l t s imply the use o f flavonoids as r e s p i r a t o r y substrates by plants.  Phenols and Plant PathogensCadman (1960) noted that tannins occurring i n the bark of trees formed i n s o l u b l e complexes with v i r u s e s : When the phenols were applied to roots and leaves a t l e v e l s 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 i n 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 s t r u c t u r e , 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 t h e i r d e r i v a t i v e s ( i . e . chlorogenic acid) occur  11 in Targe concentrations  i n the c e l l sap of h i g h l y r e s i s t a n t species;  i n h i b i t i o n of Venturia species i n apple and pear v a r i e t i e s , and disease resistance i n seeds, due to the presence of anthocyanins contained  i n the seed coats of Pisum arvense, have also been reviewed  by Claus (1961). Plant phenols have been located accumulating i n plant c e l l s and vacuoles adjacent to fungi i n f e c t e d c e l l s ; increased protein synthesis and r e s p i r a t i o n by t i s s u e s t h i s accumulation.  also occur simultaneously  with  Cruickshank (1962) indicated that attack by various  fungal pathogens caused d i f f e r e n t rates of synthesis o f f l a v o n o i d s ; Cruickshank and P e r r i n (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 i n f e c t i n g fungus to stimulate flavonoid formation, or the a b i l i t y of the invading fungus to t o l e r a t e the phenol produced.  The n a t u r a l l y occurring  flavonoids i n disease conditions could serve as precursors to more t o x i c products, i . e . P i s a 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 r o l e o f plant phenolics and growth promotion.  Harborne (1967) indicated that  phenols were concerned i n dormancy of seedlings, also i n root and shoot growth —  i n i n h i b i t i n g and s t i m u l a t i n g the indole a c e t i c acid  oxidase enzyme system.  S t e n l i d (1963) proposed that a l l 4' hydroxy  flavonoids ( i . e . kaempferol) were co-factors f o r the oxidation of  12 indole a c e t i c acid oxidase and therefore growth i n h i b i t o r s ; 3' 4' dihydroxy flavonoids ( i . e . q u e r c i t i n ) i n h i b i t e d the d e s t r u c t i o n of indole a c e t i c a c i d and were growth s t i m u l a t o r s .  S t e n l i d (1962) also reported  that s y n t h e t i c anthocyanidins at concentrations of 3 x 10~ molar to 7  -4 10  molar increased root growth i n wheat seedlings, and reversed the  i n h i b i t i o n i n root growth produced by the a d d i t i o n of indole a c e t i c 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 i n l e t t u c e v a r i e t i e s not normally l i g h t r e q u i r i n g f o r dormancy c o n t r o l .  At concentrations  of 40 to 80 milligrams per l i t e r , naringenin i n h i b i t e d seed germination and competed with g i b b e r e l i n f o r the control of dormancy i n the same system.  These r e s u l t s i n d i c a t e the possible role of flavonoids i n  plant growth.  THE OCCURRENCE OF PHENOLIC COMPOUNDS IN ANIMALS  Distribution In d i r e c t contrast to the plant kinggdom, the animal kingdom contains phenolic compounds i n l i m i t e d amounts; number of s t r u c t u r a l types have been i s o l a t e d .  only a r e s t r i c t e d The e s s e n t i a l amino  acid tryptophan i s the most widely d i s t r i b u t e d phenol i n the animal body; i t s role as a precursor f o r 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 c l a s s o f tryptophan d e r i v a t i v e s 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 l i m i t e d i n t h e i r synthesis to s p e c i f i c t i s s u e systems i n the animal body, many b i o l o g i c a l l y i n a c t i v e compounds occur t r a n s i t o r i l y , being d e r i v a t i v e s of catechols and amines, or intermediate breakdown products of d i e t a r y phenols.  Phenolic Compounds i n Animals; Estrogens' i n Growth and Reproduction The development of secondary sexual c h a r a c t e r i s t i c s and regulation of the reproductive cycle i n the female are both d i r e c t l y affected by the output o f estrogens and progestins produced p r i m a r i l y by the o v a r i e s ; secondary sources include the adrenal cortex and the placenta.  The estrane s e r i e s of s t e r o i d s (C-jg) possess an aromatic  "A" r i n g and hydroxy! grouping at Cg.  Non-steroids possess estrogenic  a c t i v i t y due to a phenolic function a t the para p o s i t i o n or a f l u o r i n e molecule a t 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 r a p i d l y and are l e s s potent than non water soluble s t e r o i d s . The profound changes caused by ovarectomy i n d i c a t e the large influence of estrogens on the female reproductive t r a c t .  Pre-pubertal  ovarectomy r e s u l t s i n i n f a n t i l e genital t i s s u e s ; the female cycles f a i l to appear.  Post-pubertal ovarectomy  r e s u l t s i n the cessation of  the menstrual c y c l e , uterine vaginal mucosal and f a l l o p i a n tube atrophy, with secondary sexual c h a r a c t e r i s t i c s disappearing f o l l o w i n g ovarectomy.  14 The anabolic e f f e c t s o f estrogens are e s p e c i a l l y prevalent during the estrous tissue.  c y c l e , and s p e c i f i c a l l y i n uterine and mammary  Means and O-Malley (1972) i s o l a t e d l i p o p r o t e i n binder f r a c t i o n s  s p e c i f i c f o r the uptake o f estrogens i n these t i s s u e types; the phenolic, the non-polar portion and r i n g s u b s t i t u t i o n a l l contributed to a c t i v e binding, (Korenman 1969) r e s u l t i n g i n highly s p e c i f i c uptake by the t i s s u e of the estrane s e r i e s . The s p e c i f i c anabolic e f f e c t s of estrogens on the reproductive t r a c t 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 a c t i o n r e s u l t i n g i n increased protein s y n t h e s i s ;  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  lining;  3.  Increased c e r v i c a l mucus s e c r e t i o n ;  4.  D i r e c t competition f o r amino acids i n gluconeogenesis f o r protein s y n t h e s i s ;  5.  Increased water and f l u i d uptake by the u t e r i n e 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 d i r e c t influence on estrogen production and gamete formation by the p i t u i t a r y -- f o l l i c l e s t i m u l a t i n g and l e u t i n i z i n g hormones.  gonadotropins  With maturity,  f o l l i c u l a r development, rupture, and progesterone secretion f o r pregnancy  15 maintenance occur r e g u l a r l y as a c h a r a c t e r i s t i c of the estrous c y c l e , 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 c o n t r o l l e d by a feedback mechanism of estrogen and progesterone acting d i r e c t l y on receptor c e l l s i n the basal hypothalamus.  Jensen and DeSombre  (1972) postulated that s p e c i f i c hypothalamic receptor s i t e 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 r e s u l t s i n reduced output  of p i t u i t a r y gonadotropins; the system remains i n constant balance and controls the l e v e l s 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 , i n t r a v a g i n a l l y or by i n j e c t i o n . B i c k o f f (1968) confirmed that f o r estrogen assessments, uterine weight increases i n d i c a t e low concentrations of estrogens. quantitative.  These r e s u l t s are  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 v a r i a t i o n s of the bioassay technique.  Lack of c o r r e l a t i o n  grazing ruminants.  e x i s t s i n laboratory animals and  Techniques i n v o l v i n g sheep as t e s t animals have  been developed (Lamond and Southcott 1962; Francis and M i l l i n g t o n Lindsay 1968).  1965;  Moule et al_. (1963) indicated that vaginal c o r n i f i c a t i o n  could be induced i n ewes by low estrogen l e v e l s when primed with progesterone; a s p e c i f i c method i n which the t e s t animals were not s a c r i f i c e d .  16 Optimal methods and factors a f f e c t i n g t h i s method were examined by Lang and Lamond (1962; 1965). 1964)  Increase i n wether teat length (Braden et a l .  can be used f o r q u a n t i t a t i v e estrogen e f f e c t s over a narrow  range; i t s s i m p l i c i t y makes the method a general i n d i c a t o r of activity.  estrogenic  The uterine weight increase applied d i r e c t l y to sheep has  shown popularity f o r 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 i n t a c t ewes and adult ovarectomized ewes and r e s u l t s can be correlated with wether bioassay r e s u l t s (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 r a t . 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 f r a c t i o n i n the supernatant of r a b b i t u t e r i which showed s t e r o i d s p e c i f i c i t y f o r estrogens was  described  by Korenman (1968).  These  r e s u l t s indicated that a s e n s i t i v e hormone assay method f o r i n v i t r o determination of the competitive was  possible.  binding a b i l i t i e s of uterine s t e r o i d s  Korenman (1969) noted that the r e l a t i v e binding  affini-  t i e s of plasma estrogens p a r a l l e l e d uterotropic a c t i v i t y . Peterson and Common (1972) described  a radio immunoassay  technique f o r e s t r a d i o l u t i l i z i n g a synthetic antibody to estrogens. The competitive  protein binding and radio immunoassay techniques are  q u a n t i t a t i v e indices of e s t r o g e n i c i t y which are independent of whole body e f f e c t s , and w i l l f i n d f u r t h e r a p p l i c a t i o n s f o r the b i o l o g i c a l assessment of uterine s t e r o i d 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 Maximum Uterine Weight Increase in 4.0 hours  Hormone  33% Increase i n Uterine Weight in 6.0 hours  70% Increase i n Uterine Weight in 6.0 hours  17 6-Estradiol  0.025  0.100  0.150  Estriol  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  Diethylstilbestrol  0.078  0.156  (From Dorfmann, 1962).  0.625  18 THE OCCURRENCE OF PLANT HORMONES AFFECTING ANIMAL REPRODUCTION  Historical During the 1940's, breeding abnormalities became widespread in sheep pastured on a European clover c u l t i v a r T r i f o l i u m subterraneaum in Western A u s t r a l i a .  Abnormally high pasture intakes of the c l o v e r  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 a s s o c i a t i o n of the c l o v e r species with reduced reproductive performance; the isoflavone g e n i s t e i n (5,7,4 trihydroxy isoflavone) was  i s o l a t e d from clover leaves; contents  the leaves reached l e v e l s of 0.70% was confirmed (1954).  on a dry matter basis.  of  Genistein  to be e s t r o g e n i c a l l y a c t i v e by mouse bioassay (Curnow  Curnow and R o s s i t e r (1955) reported that more than 120  v a r i e t i e s of T r i f o l i u m subterraneum contained g e n i s t e i n . The c l a s s i c "clover disease" occurs a f t e r animal exposure to plant estrogens f o r s i x months or more.  Pathological conditions i n c l u d i n g  swollen and p r o l i f e r a t e d 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 i n d i c a t e t y p i c a l symptoms  of excess estrogen s t i m u l a t i o n on the reproductive t r a c t , and have been reviewed by Meyers (1951) and Moule et al_.  (1963).  B a r r e t t et al_. (1965) i n d i c a t e d progressive decreases i n lamb crops occurred when ewes ingested red c l o v e r pasture v a r i e t i e s f o r 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 r e s u l t e d i n 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 a g e n t ;  appreciable losses i n  o f f s p r i n g numbers are apparent. Many reports have appeared on the widespread occurrence of plant hormones a f f e c t i n g animal reproduction. reviewed  Bradbury and White (1954)  the e a r l y 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 a d d i t i o n , review a r t i c l e s  have appeared which i n d i c a t e the d i s t r i b u t i o n o f plant a c t i v i t i e s . These include:  B i c k o f f (1968), Moule e t a l _ . (1963) i n B r i t a i n , Samuel  (1967) i n the U.S.A., Symington (1965) i n Rhodesia, K i t t s (1960) i n Canada, and Bankov (1970) who reported on estrogenic pasture crops i n the U.S.S.R.  Estrogens  Isolated From Plant Forage Species Forage compounds i s o l a t e d and a c t i n g on animal  t r a c t s as u t e r o t r o p i c hormones include the coumestans: 4  reproductive coumestrol and  methoxycoumestrol, and the isoflavones biochanin A, d a i d z e i n ,  formononetin,  g e n i s t e i n and pratensin.  Structures and potencies of  these plant flavonoids are given i n Figure 4 and 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.  Table 2 Coumestrol  was f i r s t i s o l a t e d from ladino c l o v e r by B i c k o f f e_t al_. i n 1957; d i f f e r i n g i n s t r u c t u r e , but b i o g e n e t i c a l l y r e l a t e d to the isoflavones. Mouse bioassay confirmed the potency o f 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  HO  OCH, Biochanin - A  16 12°5 5,7 Dihydroxy - 4' methoxy isbfalvone  (C H ) )0 6  1 2  5  Daidzin  C  H  Formononetin  16 12°4 7 Hydroxy - 4' methoxy isoflavone C  H  Q  ^21^20^9  7 Glucone of Daidzein  Daidzein  ^15 10^4 H  7,4' Dihydroxy isoflavone  0 Pratensein  C  5,7,3' Trihydroxy  Figure 4  16 12°6 H  —4'' methoxy isoflavone  Coumestrol  C  15 8°5 H  3,9 Dihydroxy - "6H-benzofuro[3,2-C][l]benzopyran 6-one  The Major Estrogenic Compounds Isolated from Legume Species  20 a c t i v e i n sheep depending on administration route.  The s t i l b e n e  structure of coumestrol resembling d i e t h y l s t i l b e s t r o l accounts f o r part of t h i s a c t i v i t y according to B i c k o f f (1968), as does the planar r i n g structure and s i m i l a r distances between the hydroxyl groups as noted by Shemesh et a K  (1972).  H i s t o l o g i c a l Changes of the Mammalian Reproductive Tract from the E f f e c t s of Plant Estrogens The vaginal histology of ewes was examined by B e l l and Sanger (1958) on estrogenic pastures.  C e l l 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 e f f e c t s 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 f i g u r e s : 1.  I n h i b i t e d 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.  F a i l u r e 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 a d d i t i o n a l theories regarding the primary cause of i n f e r t i l i t y of animals on legume d i e t s .  Engle (1957)  indicated that ewes on ladino clover pasture experienced delayed estrus with conception occurring l a t e i n the c y c l e ; 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 s e r v i c e .  B a r r e t t et al_. (1965) found a 25% lambing  21 rate of ewes on red c l o v e r d i e t s , and Turnbull et al_. (1966) noted the s e v e r i t y of c y s t i c endometrial on red c l o v e r pastures.  hyperplasia s i g n i f i c a n t l y higher i n ewes  Cyst numbers greater than 10 per c e r v i x or  uterus r e s u l t e d i n a lower proportion of animals pregnant post s e r v i c e . Turnbull et al_. (1966) indicated that embryonic death i n 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 i n animals with c y s t i c - hyperplasia.  endometrial  O'Brien (1971) examined sperm numbers recovered from the  u t e r i and cervixes of ewes grazing on oat grass and red c l o v e r pastures. Sperm numbers of 350 were recovered  from the red c l o v e r groups, while  control animals y i e l d e d 17,160 mature spermatozoa per c e r v i x . author postulated that the major cause of i n f e r t i l i t y was  The  non-fertiliz-  ation due to the absence of sperm numbers reaching the f a l 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 c o r r e l a t e d with the number of uterine cysts present, which are associated with hypertrophy and s w e l l i n g of the c e r v i c a l and uterine mucus membranes.  This  was  conformed by L i g h t f o o t et a l . (1967) and O'Brien (1971).  Moule et al_.  (1963) a t t r i b u t e d e a r l y embryonic death before attachment of the f e r t i l i z e d embryo to the endometrial  wall.  Bauminger and  (1969), using ovarectomized, progesterone treated r a t s and  Lindner increased  dosages of g e n i s t e i n , concluded that two phenol groups at 7 and of g e n i s t e i n were e s s e n t i a l f o r estrogenic a c t i v i t y .  4'  These authors  concluded that g e n i s t e i n mimics many, but not a l l , of the actions of 17-g-Estradiol, i n promoting uterine growth and water i m b i b i t i o n with r e s u l t i n g hyperplasia; but f a i l s to induce the implantation of the delayed b l a s t o c y s t 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 B e n e f i c i a l E f f e c t s of Plant Estrogens i n Animal Growth and F i n i s h i n g The rapid r i s e i n the a p p l i c a t i o n of synthetic estrogens f o r f i n i s h i n g l i v e s t o c k , and the recent removal of d i e t h y s t i l b e s t r o l commercial  f i n i s h i n g operations  from  prompts i n v e s t i g a t i o n into the p o s s i b l e  use of plant e x t r a c t s as s u b s t i t u t e s f o r the potent s t e r o i d  analogues.  Various experiments have been conducted to measure p l a n t 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 p o s i t i v e e f f e c t s with wethers; growth responses, gains, and meat q u a l i t y were a l l improved on d i e t s 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 d i e t s containing 114 ppm  coumestrol.  Varied and inconclusive r e s u l t s 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 r e c e i v i n g s t i l b e s t r o l implants without a l f a l f a d i e t s . Matsushima a l s o indicated that steers on d i e t s of 25, 100 and 250  ppm  of coumestrol showed no improvement i n growth response; 10.0 mg of DES per day resulted i n a p o s i t i v e response of 10% over control animals. George and Turnbull (1966) pastured i n t a c t Merino ram lambs-on control pastures of perennial rye grass (Lolium perenne L.) ( T r i f o l i u m repens L.) u n t i l weaning.  and white c l o v e r  At weaning 38 animals remained  on the grass dominant pasture, and 23 were placed on red c l o v e r ( T r i f o l i u m pratense L.) dominant pasture, composed of 95% red c l o v e r . 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 < greater i n the red c l o v e r pastured animals.  0.05)  23 Commercial e x t r a c t i o n techniques  f o r lucerne separation of  coumestrol have been described by B i c k o f f (1961).  I t i s obvious  more research i n t o the preparation of e x t r a c t s and applied n u t r i t i o n programmes i s necessary to obtain maximum b e n e f i c i a l e f f e c t s of the plant estrogens.  Further work i s required to determine i f the  b e n e f i c i a l e f f e c t s of plant hormones override the deleterious a c t i v i t i e s on l i v e s t o c k . Two  types of forages may  have to be developed;  one containing a low estrogen content f o r breeding stock, and  the  other with high estrogenic a c t i v i t y to promote growth and f a t t e n i n g in s t e e r s , wethers and poultry.  Anti-Estrogenic A c t i v i t y of Forages Many forages contain f a c t o r s which reduce the e f f e c t s of the endogenous animal estrogens, producing a hypo-estrogenic A s i n g l e plant species may  syndrome.  contain high l e v e l s of estrogenic isoflavones  and coumestans, yet t h e i r b i o l o g i c a l e f f e c t s are masked by a n t i estrogens.  B i c k o f f (1968) noted poor c o r r e l a t i o n between b i o l o g i c a l  and chemical  assays, due to the two f r a c t i o n s i n a s i n g l e plant species.  Emmens (1965) e s t a b l i s h e d 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 e s t r a d i o l and estrone a c t i v i t y ; Shutt (1967) suggested the weak estrogen could displace e s t r a d i o l from the uterine receptor s i t e s .  B i c k o f f 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 i n h i b i t e d 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  8,000 yg  1.0  A l f a l f a , Ladino c l o v e r , red c l o v e r , Subterranean clover  Biochanin A  18,000 yg  0.46  A l f a l f a , red c l o v e r , Subterranean clover  Formononetin  32,000 yg  0.26  A l f a l f a , red c l o v e r , Ladino c l o v e r , Subterranean clover  Genistein  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 c l o v e r , Barrel Medic, Peas  1.20 yg  6,900  Estrone  a  C o n t r o l groups uterine weight = 9.6 mg  ^At the dose l e v e l to produce a 25.0 mg uterus (After B i c k o f f 1968).  Date palm  1  ,  25 a c t i v i t y o f coumestrol.  Adler (1962) confirmed that the a l f a l f a a n t i -  estrogens reduced the potency o f coumestrol  to one tenth on female  genital t r a c t s when administered simultaneously to animals, and i n d i c a t e d that the compounds could be r e l a t e d to progesterone or androgens i n s t r u c t u r e . Anti-estrogenic a c t i v i t y has been reported by Adler (1962), B i e l y and K i t t s (1964) and Cook and K i t t s (1964) i n legumes, grasses and needles o f Pinus ponderosa.  Adler (1962) reported on a coumestrol  analogue present i n a l f a l f a which was e f f e c t i v e against both coumestrol and 17 B - e s t r a d i o l ; i n d i c a t i n g i t s v e r s a t i l i t y as an anti-estrogen. Studies on the structures o f compounds and a n t i - e s t r o g e n i c a c t i v i t i e s by Terenius  (1968)demonstrated i n v i t r o i n h i b i t i o n of 17 B-estradibl  by m i r o e s t r o l , a plant estrogen with one f i f t h the a c t i v i t y o f 17 3-estradiol.  The most estrogenic isomer of e s t r a d i o l was the most  e f f e c t i v e uptake i n h i b i t o r ; non-steroid c a r b o x y l i c acids and t h e i r isomers also blocked uterine uptake of 17 B - e s t r a d i o l . Terenius f u r t h e r c l a s s i f i e d anti-estrogens into two types:  (1971)  (a) progesterone/  testosterone structures which do not i n h i b i t or reduce the concentration of estrogens i n the target t i s s u e ; t h e i r mode o f a c t i o n i s unknown, and (b) compounds which reduce the concentration of estrogens at the s i t e of action i n the target t i s s u e s by a f f e c t i n g estrogen metabolism, which would include the rates o f conjugation and rates of e x c r e t i o n . Terenius (1968), Rochefort and Capony (1972) proposed that the mammalian uterus has a l i m i t e d number of receptor/binder s i t e s ; competitive binding occurs, and anti-estrogens could form e i t h e r a durable complex with the s i t e s , or a s h o r t - l i v e d complex when the  26 endogenous estrogen supply i s of short duration.  This s h o r t - l i v e d  complex i s too t r a n s i e n t to exert c e l l and t i s s u e growth e f f e c t s , but i n both cases the receptor s i t e a v a i l a b i l i t y f o r animal i s reduced.  estrogens  The anti-estrogens have high a f f i n i t i e s f o r 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 s i t e s f o l l o w s , and as a consequence reduced p r o t e i n synthesis by the uterus i s the r e s u l t .  These r e s u l t s were confirmed by Folman and  Pope (1967), and Terenius  (1971).  Additional workers added to the s t r u c t u r e s and of the anti-estrogens:  Korenman (1969) noted that the  activities molecular  dimensions of m i r o e s t r o l , anti-estrogens and 17 B - e s t r a d i o l were s i 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 - e s t r a d i o l ; these groups contribute to the binding a f f i n i t y ; the presence of a hydrophobic portion of the skeleton and s i z e were important i n "loose" binding of e s t r a d i o l to the receptor s i t e s . miroestrol molecule.  These structures are part of the  Shemesh et al_. (1972), using a competitive  protein binding technique with r a b b i t uterine c y t o s o l , demonstrated that the non-steroids coumestrol  and g e n i s t e i n a c t i v e l y competed f o r  binding s i t e s with 17 3 - e s t r a d i o l ; the binding a f f i n i t y was r e l a t e d to the potency of the compounds: parts coumestrol  1 part of 17 B - e s t r a d i o l to 70  to 175 parts g e n i s t e i n 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 p o s i t i o n s 7 and 12 of  27 the isoflavones and coumestans were e s s e n t i a l f o r i n t e r a c t i o n with the uterine cytosol receptor.  Proestrogens were designated  by the  p o s i t i o n i n g of 7 and 12 methoxy groups; t h e i r a b i l i t y to bind to the uterus was only a f t e r a c t i v e ruminal/hepatic  The E f f e c t s of Plant Extracts on P i t u i t a r y  demethylation in vivo.  Function  The maintenance of the estrous cycle i s under the d i r e c t control of the a n t e r i o r 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 s t i m u l a t i n g or l e u t i n i z i n g hormones d i r e c t l y a f f e c t s the c y c l i c nature of estrus.  Plant phenols have been shown  to a f f e c t both the ovarian and gonadotropic systems. found anti-estrogenic and anti-gonadotropic  Chury (1965)  activities in alfalfa  e x t r a c t s ; L e a v i t t and Wright (1965) confirmed these r e s u l t s , and noted that the primary e f f e c t 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.  L e a v i t t and Meismer (1967) discovered  that  coumestrol could cause p e r s i s t e n t estrus without o v u l a t i o n . and Wright (1965) reported that coumestrol was 2.9 x 10  Leavitt  as e f f e c t i v e  as e s t r a d i o l on the uterus i n a c t i v i t y , and 8.5 x 10"^ as e f f e c t i v e as e s t r a d i o l i n 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 i n 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 B i c k o f f (1968) confirm various authors findings on the a c t i v i t y of coumestrol both as a s t i m u l a t i n g and retarding compound on p i t u i t a r y / o v a r y a c t i v i t y of mammals.  28 Metabolism of Plant Estrogens by Animals 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 p r o t e i n , 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 a c t i v e forms i n 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 g e n i s t e i n , and formononetin to d a i d z e i n , have been e s t a b l i s h e d by Nilsson (1961, 1962).  No evidence of more extensive degradation i n  rumen l i q u o r has been indicated i n vivo by Batterham et al_. (1965). Braden (1967) incubated biochanin A and formononetin i n rumen l i q u o r preparations; rapid demethylation of s i x t y percent of the two isoflavones to g e n i s t e i n and daidzein r e s u l t e d .  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 i n the duodenal-jejunal area w i t h i n two to three hours- a f t e r i n g e s t i o n ; Lindner (1967) indicated that the presence of l i v e r t o x i n s , s t r e s s , or reduced n u t 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 i n formononetin and daidzein. Genistein end products are simple phenols,  28a Figure 5  The Major Estrogenic Isoflavones and Their Metabolic Products i n the L i v e r and Rumen  / m Biochanin A (0.8 x 10-5 A c t i v i t y of Diethylstilbestrol)  (After Braden and Shutt  Genistein (1.1 x IO" A c t i v i t y of Diethylstilbestrol) 5  1970)  Paraethyl Phenols ( E s t r o g e n i c a l l y Inactive)  29 such as P-ethyl phenol; phenol production  resulted i n a loss of  estrogenic a c t i v i t y ; formononetin degradation estrogenic a c t i v i t y  produced equol with  25% that of genistein (Shutt 1969).  Shutt et a]_. (1970), using sheep on red clover dominated pastures, indicated that l e s s than 1% of the ingested (9.0 gms/day) were excreted i n the feces and urine.  isoflavones D a i l y urinary  excretion of 3.9 gms/day of equol was equivalent to 70% percent of the d a i l y intake of formononetin. produced by the rumen was  E i g h t y - s i x percent of the equol  absorbed by the rumen epithelium.  Shutt and Braden (1968) found plasma l e v e l s of 50 ygs/100 ml and uterine t i s s u e l e v e l s of 46 ygs/uterus of equol (7,4* flavan) i n sheep on red clover d i e t s .  These l e v e l s represent more  than 70% of the t o t a l estrogens extracted from the plants. (1967) demonstrated how b i o l o g i c a l response:  dihydroxyiso-  Under  plasma l e v e l s of plant compounds affected  i n sheep, plasma g e n i s t e i n l e v e l s of 1.0  to  5.0 ygs/100 ml of plasma e l i c i t e d graded uterine growth responses, while l e v e l s above 5 ygs/100 ml of plasma r e s u l t e d i n maximum uterine growth response.  Adipose t i s s u e storage of conjugated plant estrogens  occurred i n excess of plasma l e v e l s .  Lindner also pointed out that  these l e v e l s should not a f f e c t consumer health. The a c t i v i t y of plant estrogens i n animals i s affected by previous d i e t .  Lindsay et a]_. (1970) found that the c e r v i c a l mucus  response of ewes to subterranean c l o v e r species high i n genistein was  reduced by a ten-day pre-feeding  period on d i e t s containing  genistein at l e v e l s of 1.1% of the plant material on a dry matter  30 basis.  Formononetin response d i d not depend on previous d i e t .  Deactivation of genistein into non-active phenols by the rumen microorganisms occurred i n animals conditioned to g e n i s t e i n d i e t s ; formononetin was not deactivated. No adaptation by rumen m i c r o f l o r a species or t i s s u e oxidative systems, such as polyphenol oxidase, occurred when formononetin was pre-fed a t 1.2% o f p l a n t dry matter content.  Rapid genistein degradation to P-ethyl phenol l e d 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 i n 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 a l t e r a t i o n s which r e s u l t e d in rapid degradation of the isoflavone.  Cayen and Common (1965)  i n j e c t e d fowl with l a b e l l e d coumestrol; no coumestrol or equol was recovered i n the urine. This suggested d i f f e r e n t metabolic routes f o r the coumestans, and probably contributed to the higher metabolic a c t i v i t y o f the coumestans. Species d i f f e r e n c e s a l s o e x i s t i n the metabolism o f plant estrogens —  more e f f i c i e n t conjugation and e x c r e t i o n of phyto-  estrogens occurs i n c a t t l e ; formononetin i s metabolized at a f a s t e r rate i n sheep than i n c a t t l e .  Braden and Shutt (1971) suggested  from these r e s u l t s 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 e f f e c t s of estrogenic pastures could be p a r t i a l l y a t t r i b u t e d to t h i s 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 i n plant species are extremely v a r i a b l e ; 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 l o c a t i o n have ranged from 10.4 to 125.4 ppm. M i l l i n g t p n (1964) reported on v a r i a t i o n s of barrel medic tribuloides);  (Medicago  coumestrol content ranged from 40 to 180 ppm. A l l  plants were harvested a t f u l l bloom stage, with year l o c a t i o n action being h i g h l y s i g n i f i c a n t .  inter-  Davies and Dudzinsky (1965) noted  that subterranean c l o v e r , between s i t e and between year differences in a c t i v i t i e s were highly c o r r e l a t e d .  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 i n the s p r i n g , c o i n c i d i n g with the dominance of the pasture by the legume.  Estimates on the potencies o f 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 o f growth; coumestrol increased during the second year of c u l t i v a t i o n  levels  with successive growth  stages, and reached a maximum between 10 and 25 days a f t e r f u l l bloom  32 of 270 pptn. B i e l y 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 e f f e c t s have also been  examined on red clover by Flux et al_. (1963), Dedio and Clark (1968), and R o s s i t e r (1972). The r e s u l t s can be summarized i n that the maximum isoflavone/ coumestan biosynthesis occurred e a r l y during the l e a f unfolding  stage;  isoflavones competed f o r carbon substrates ( s o l u b l e 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 i n d i c a t o r f o r determining  isoflavone/coumestan l e v e l s , and  consequently e s t r o g e n i c i t y of the plant species.  V a r i e t a l V a r i a t i o n i n the Estrogenic A c t i v i t y of Plants Large ranges of estrogenic isoflavones e x i s t i n d i f f e r e n t v a r i e t i e s of plants.  Francis, M i l l i n g t o n and B a i l e y (1967) examined  over 100 species of the genus T r i f o l i u m ; t o t a l contents of isoflavones reached values of up to 0.25%  of the t o t a l dry weight.  B a i l e y and  Francis (1971) surveyed 76 l i n e s of T r i f o l i u m subterraneaum, section Calycomorphum; a basic isoflavone pattern and evolutionary  progression  of isoflavone contents was apparent; Francis and M i l l i n g t o n (1965) noted that isoflavone patterns are s i g n i f i c a n t i n r e l a t i o n 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 to 0.94%; biochanin A of 0.60%  to 1.12%  0.35%  of dry weight; high c o r r e l a t i o n s  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. -12  Total leaf isoflavone levels were  -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 f o r c e l l wall and c e l l  protein synthesis.  Mild grazing r e s u l t e d i n 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 r e s u l t e d , did the e f f e c t of d e f o l i a t i o n a f f e c t estrogenic potency of the ingested legumes. Frequency of c u t t i n g 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 r e s u l t e d i n the a c t i v i t i e s of subsequent harvests during the growing season to f o l l o w c l o s e l y that of plants allowed to grow without i n t e r f e r e n c e . When cuttings were made past the vegetative stage of the a l f a l f a , subsequent c u t t i n g s possessed l i t t l e or no estrogenic a c t i v i t y .  Ladino clover at any c u t t i n g  followed a trend of high estrogenic potency during the e a r l y 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 l e v e l s a t f u l l bloom stage, with a detectable increase during e a r l y and l a t e seed stages.  The E f f e c t s of Drying on the Estrogenic A c t i v i t y of Legumes Drying legume forage f o r preservation has not always given consistent r e s u l t s 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  c l o v e r was  maintained a f t e r drying; Francis and Milington (1965) i n d i c a t e d that the estrogenic a c t i v i t y of dried subterranean  c l o v e r was n e g l i g i b l e  35 when compared with green m a t e r i a l .  Nilsson (1959) showed that a r t i f i c -  i a l drying and e n s i l i n g did not change the a c t i v i t y of red c l o v e r , while f i e l d curing lowered the b i o l o g i c a l a c t i v i t y .  Youngman (1963)  demonstrated that the estrogenic a c t i v i t y o f legumes increased with the age of c u t t i n g f o r both hay and s i l a g e ; curing had l i t t l e e f f e c t . Dedio and Clark (1969) working with red clover concluded that isoflavone l e v e l s d i d 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 o f a c t i v i t y due to breakage o f the isoflavone glycoside bonds occurred when samples were r a p i d l y d r i e d ; fresh red clover t o t a l isoflavone content (biochanin A plus formononetin) was 1.28% of dry weight.  Drying the forage at 80°C r e s u l t e d i n t o t a l  isoflavone  content of 1.21%'of t o t a l dry weight; while f r e e z i n g the samples resulted i n t o t a l l e v e l s of 1.20% of the t o t a l dry weight. Two reasons have been proposed to explain the d i f f e r e n t e f f e c t s 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 c l o v e r species during extended storage r e f l e c t e d a greater s t a b i l i t y of the isoflavones over the coumestans.  Leaching or destruction of  the g l y c o s i d i c bonds or the isoflavone molecules could occur, p a r t i c u l a r l y in unfavourable, wet haying periods.  B i c k o f f e t aj_. (1960) proposed  that increased a c t i v i t y a f t e r drying of red and subterranean clovers compared with a l f a l f a and ladino c l o v e r , 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 E f f e c t s of Plant Pathogens on Estrogenic Potency of Plant Species Evidence has accumulated which i n d i c a t e s one of the major sources o f isoflavone/coumestan v a r i a t i o n i s i n the action o f b a c t e r i a , f u n g i , and i n s e c t attack on the i n d i v i d u a l plant species.  Loper and  Hanson (1964) observed a one hundred f o l d increase i n l e a f coumestrol content of a l f a l f a f o l l o w i n g l e a f spot i n f e c t i o n .  Normal coumestrol  values were 2.1 ppm, and rose to 183.7 ppm a f t e r mild i n f e c t i o n . t r o l content was p o s i t i v e l y and d i r e c t l y c o r r e l a t e d with severity.  Coumes-  disease  The same authors (Loper e t a l . 1967) indicated that plant  s e l e c t i o n and breeding f o r disease resistance reduced the coumestrol content of a l f a l f a ; fungicide a p p l i c a t i o n s to reduce f o l i a r  diseases  resulted i n r e l a t i v e l y low and constant l e v e l s of coumestrol; fungicide sprayed stems and leaves averaged 28.6 ppm, t o t a l coumestrol while Unsprayed plants averaged 103.3 ppm. l e a f v i r u s i n f e c t i o n on lucerne.  M i l l i k a n (1971) examined red  The r e s u l t s i n d i c a t e d 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 l e a f v i r u s i n f e c t i o n occurred on the exposed l e a f surfaces.  Leaf rust  i n f e c t i o n (Uromyces s t r i a t y s ) also affected t o t a l coumestan content; Francis and M i l l i n g t o n (1971) found a s i x f o l d increase from 15 to 80 ppm i n the leaves of the burr medic, which was d i r e c t l y r e l a t e d to the degree of rust i n 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 f o l l o w i n g i n f e c t i o n : Hess and Hadwiger (1971) examined the anti-fungal i s o f l a v o n e , phaseolin; under pathogen attack, the D.N.A. template was stimulated  37 to produce increased l e v e l s 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 i n f e c t i o n and was affected by a range of i n f e c t i o n s i n c l u d i n g aphids.  Loper stated that:  "The  increased l e v e l s may  be a general  increase i n flavonoid production, e s p e c i a l l y occurring in damaged areas when protein synthesis i s reduced, protein synthesis being i n v e r s e l y r e l a t e d to flavonoid production." Sherwood et al_. (1970) indicated that a time course of coumestan/isoflavone accumulation p a r a l l e l e d the development of i n f e c t i o n ; the concentration of flavonoids was r e l a t e d to the degree of i n f e c t i o n , t r a n s l o c a t i o n of coumestans from i n f e c t e d plant areas to healthy areas did not occur.  The host plant was demonstrated to  be the p r i n c i p l e c o n t r i b u t o r of flavonoid precursors and enzymes i n flavonoid biosynthesis  —  t h i s process occurred i n the i n f e c t e d  t i s s u e , not i n the surrounding  healthy t i s s u e . The same authors  also determined that the mechanism of induction of flavonoid synthesis was r e l a t e d to the metabolism of the i n f e c t i n g organisms and not to mechanical damage, with coumestan/isoflavone accumulation associated with the c a t a l y s i s of c e l l s leading to c e l l necrosis and t i s s u e degradation.  Mineral Elements and Plant Estrogen A c t i v i t y The f i n d i n g that inorganic n u t r i e n t s e s s e n t i a l f o r plant growth d i r e c t l y a f f e c t e d plant coumestan and isoflavone l e v e l s 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 a p p l i c a t i o n rates of 216 kg/hectane i n d i c a t e d by bioassay techniques that the estrogenic a c t i v i t y increased when no treatment was a p p l i e d ; plants r e c e i v i n g 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 i n potencies from those r e c e i v i n g phosphate only.  Dry matter y i e l d s  increased by a f a c t o r of 2 to 3 with phosphoric a c i d a d d i t i o n . Rossiter and Beck (1966) examined phosphate supply and potencies of the Dwalganup s t r a i n of T. subterraneum.  Decreasing  the phosphate  supply r e s u l t e d i n a doubling of the formononetin/genistein and a decrease i n dry matter productions.  content,  In the Mount Barker  s t r a i n , genistein and biochanin A l e v e l s doubled with increasing phosphate d e f i c i e n c y ; formononetin, increased by a f a c t o r of 4.  the major estrogenic i s o f l a v o n e ,  R o s s i t e r (1970) confirmed the e f f e c t s  of phosphate l e v e l s on formononetin; at the l e a f emergence stage.  isoflavones were already evident  In phosphate d e f i c i e n t leaves i n  c o n t r a s t , the concentration of isoflavones increased during the l a t e r growth stages. In pot c u l t u r e experiments,  Rossiter (1969) examined  nitrogen d e f i c i e n c i e s on growth and estrogenic potencies of plant parts of T. subterraneum.  Nitrogen d e f i c i e n c y was associated with  increased isoflavone l e v e l s .  In the f i r s t t r i f o l i a t e leaves, the  t o t a l concentrations of formononetin,  g e n i s t e i n and biochanin A were  doubled from 3.7% to 7.1% with low nitrogen supply.  Biochanin A  l e v e l s were l e s s a f f e c t e d than the other i s o f l a v o n e s , and remained unchanged at a concentration of 1.2% of the t o t a l l e a f weight.  39 S u l f u r supply also a f f e c t s the isoflavone l e v e l s . and Barrow (1972) examined s u l f u r supply i n T. subterraneum.  Rossiter A  consistent tendency existed f o r isoflavone l e v e l s to decrease as s u l f u r supply  increased.  Otter (1966) summarized the e f f e c t s 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) f o r  protein synthesis p r e v a i l , a l a r g e r part of endogenous metabolites (soluble sugars and CO^) are channeled i n t o primary compounds -- proteins and c e l l w a l l s .  A reduced number of carbon molecules are a v a i l a b l e  for secondary processes -- anthocyanin and flavonoid pigments.  At  low nitrogen l e v e l s , the protein carbon saved (from the d e f i c i e n c y i n protein synthesis) i s more than adequate to account f o r extra i s o f l a v o n e / coumestan glycoside carbons f o r glycoside formation."  The addition  of f e r t i l i z e r , as indicated by Schoo and Rains (1970), r e s u l t s i n a reduction of isoflavone l e v e l s . "normalized"  This reduction may r e s u l t from a  isoflavone metabolism and/or a d i l u t i o n e f f e c t from increased  dry matter due to increased plant growth. Rossiter (1972) confirmed that low s o i l phosphate l e v e l s decreased protein content per c e l l and that isoflavone l e v e l s increased on a dry matter basis. s t r a t e competition  This f i n d i n g adds evidence to support the sub-  hypothesis  c i t e d by R o s s i t e r (1972), which i n d i c a t e d  that isoflavone/coumestan formation  i s more d i r e c t l y r e l a t e d to sugars  and starches than to plant protein l e v e l s .  The amounts of isoflavones  formed per c e l l depend on the supply o f carbon substrates i n the formation of sugars and starches. At f u l l expansion stage, isoflavone synthesis i n legumes normally ceases.  While the l e a f was expanding under the e f f e c t o f  40 l e v e l s of mineral supplementation, the formation of c e l l protein and c e l l p r o t e i n and c e l l walls takes preference over isoflavone/coumestan formation, due to the substrate competition e f f e c t on the supply of carbon substrates; with the r e s u l t of large v a r i a t i o n s i n isoflavone content and estrogenic a c t i v i t y of the legume species.  41  CHAPTER I I I HORMONAL ACTIVITY OF TWO SPECIES OF NATIVE B.C. RANGE LEGUMES  INTRODUCTION  The widespread occurrence of plant compounds having e f f e c t s on animal reproduction has been well documented i n c u l t i v a t e d legume species (Samuel 1967; B i c k o f f 1968).  Native legumes and grass species  also cause hormonal imbalances a f f e c t i n g the reproductive t r a c t .  Wada  (1963) reported on a c t i v i t y o f Chinese milk vetch Astragalus s i n i c u s , Symington (1965) on high-veld pastures of Central A f r i c a .  Cook and  K i t t s (1964) and Stevenson e t 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 f r a c t i o n s ; s o i l c o n d i t i o n s , f o l i a r pathogens and growth stages are important i n a f f e c t i n g the plant hormone l e v 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 t r a c t .  Experiments to assess the types of compounds present,  and the e f f e c t s of growth stage on potency of the legume species were also conducted.  42 MATERIALS AND METHODS  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 I n t e r i o r dry b e l t of B.C.  as described by Barneby (1964), were harvested during the  grazing season.  The Farwell Creek area, south of Riske Creek,  1971 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 f o r l o c a t i o n s i t e s ; a c t i v e grazing of l i v e s t o c k was conducted on t h i s t y p i c a l Cariboo grassland range during the spring and summer grazing season from May 1 to June 21st 1971.  Animals ranging on t h i s area include  h e i f e r s , and cows with calves.  A t o t a l of 2,900 animals u t i l i z e the  u n i t , with r e s u l t i n g heavy overgrazing of both grass and legume species. Harvesting of the legumes was on randomly located transect l i n e s i n t r i p l i c a t e f o r each growth stage.  Plants were hand c l i p p e d  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  V i c i a americana and Astragalus miser var. serotinus at which samples were c o l l e c t e d f o r a n a l y s i s . Only healthy specimens free of disease or i n s e c t i n f e s t a t i o n of varying heights were sampled to maintain  43  Numerals represent transect l i n e s harvested at s p e c i f i c stages of maturity: I. Ill.  Vegetative stage. Seed pod stage.  44  Figure 9  Astragalus miser var. serotinus - at illustrated growth stages  analyses conducted  45 representative samples o f the areas.  Eight hundred gram samples  (wet weight) were c o l l e c t e d a t each date; following packaging and s e a l i n g i n p l a s t i c bags, quick freezing a t 0°C commenced; t h i s temperature was maintained u n t i l the analyses were conducted. Proximate analysis of the samples a t the s p e c i f i c growth stages was conducted according to the methods of the A.O.A.C. (1960).  E x t r a c t i o n Procedure Figure 10 i l l u s t r a t e s the procedure employed f o r e x t r a c t i o n of phytoestrogens.  This i s a modification of the e x t r a c t i o n methods  of Beck (1964) and B i e l y and K i t t s (1964).  A l l solvents were reagent  grade and d o u b l e - d i s t i l l e d i n glass before use to e l i m i n a t e peroxides and i n t e r f e r i n g aromatic contaminants.  Ultraviolet  spectrophotometer  t e s t i n g f o r increased absorption of the ether and chloroform solvents at 300 my i n a 1.0 cm path length quartz c e l l s confirmed the absence of peroxides.  i Bioassay f o r Assessment of B i o l o g i c a l A c t i v i t y A s i x 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 i n j e c t e d with 0.2 ml of p h y s i o l o g i c a l s a l i n e subcutaneously, or 0.025 micrograms of 17-8-estradiol dissolved in 0.2 ml of s a l i n e . The ether and chloroform f r a c t i o n s were taken to dryness and weighed.  The f r a c t i o n s were dissolved i n a convenient solvent  46 Chopped forage (350 gms dry matter)  I  Macerated i n food blender with 0.1N HCl f o r 3 minutes. pH adjusted to 7.0  I  Incubated 30 minutes at 37°C  I  Boiled 10 minutes i n 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 i n 95% Ethanol /3 l i t e r s v 100 gms Concentrated to approx.'100 ml. Water added to give approx. 70% Ethanol s o l u t i o n (V/V) [  ;  Chlorophyll and l i p i d s extracted with 3 - 500 ml portions of petroleum ether (B.P. 50-70°C) u n t i l no colour remains i n ether f r a c t i o n pH adjusted to 7.2 with 40% Sodium Hydroxide  I  Evaporated i n vacuum to remove a l l Ethanol Washed with 5 - 800 ml portions o f 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 E x t r a c t s (Fraction B) evaporated to dryness (25°C) Extracts'weighed | Extracts dissolved i n 25.0 ml ToluenerEthanol  Figure 10  (50:50)  F r a c t i o n a t i o n Procedure f o r 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. again taken to dryness.  The extracts were  The ether e x t r a c t f r a c t i o n (Fraction A) was  dissolved i n p h y s i o l o g i c a l s a l i n e (0.2 ml/animal).  The  chloroform  f r a c t i o n (Fraction B) was prepared s i m i l a r l y ; 15.0 grams of dry matter equivalent was dissolved i n s a l i n e containing 0.025 micrograms of 17-B-estradiol chloroform  i n order to assess the anti-estrogenic a c t i v i t y of the  extracts.  S a c r i f i c e by ether i n h a l a t i o n of the animals  occurred s i x hours post i n j e c t i o n . The a c t i v i t i e s o f the f r a c t i o n s were assessed by the increase or decrease o f gross uterine weight expressed as a percentage of body weight, when compared with the control groups.  Separation  Procedure f o r Plant Extracts  Thin layer chromatographic plates (20 cm x 20 cm) were coated with s i l i c a gel G (Merck) a t a thickness of 0.25 mm.  P r i o r to use  the plates were a c t i v a t e d by heating to 110°C f o r 30 minutes. samples dissolved i n toluene: ethanol  1.0 ml  (50:50) from the e x t r a c t i o n  procedure were taken to?dryness and redissolved i n 0.5 ml toluene: ethanol plates.  (50:50) to ensure m o b i l i t y and separation on the t h i n layer Samples were streaked on the plates with a Camag "Chromato-  charger" a p p l i c a t o r .  Four l i n e s of 40.0 m i c r o l i t e r s each were applied  per plate at the o r i g i n , r e s u l t i n g i n a t o t a l a p p l i c a t i o n per plate of 160 m i c r o l i t e r s ; drying with forced hot a i r a f t e r each 40.0 microl i t e r a p p l i c a t i o n gave uniform streaks o f minimum width with a continuous surface area being applied. per e x t r a c t .  A t o t a l of ten plates were run  The developing solvent was composed of chloroform:  48 methanol (91:9). The solvents were d i s t i l l e d p r i o r to use, and confirmed to be peroxide f r e e .  Ascending chromatography with a  saturated atmosphere was employed. 25°C.  The temperature was maintained a t  The solvent f r o n t was run to the f u l l  length of the p l a t e s .  The plates were a i r dried f o r one minute following the run, bands were observed and t h e i r positions marked under u l t r a v i o l e t l i g h t (3650 Angstroms), a f t e r 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 d i a z o t i z e d s u l f a n i l i c acid to t e s t f o r any phenols not v i s i b l e i n the u l t r a violet light.  Rf values and colours f o r each of the bands were  recorded. The ten r e p l i c a t e plates were combined, the bands removed from the plates by vacuum, mixed f o r 1.0 minute with 10.0 ml of spectroscopic methanol, and centrifuged f o r 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 o x i d a t i o n 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 i n the f i r s t separation to ensure p u r i t y 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 p l a t e s . Viewing under u l t r a v i o l e t l i g h t and spraying with d i a z o t i z e d s u l f a n i l i c acid ensured the absence of i n t e r f e r i n g materials and eliminated overlapping bands present with the extracts from the f i r s t chromatographic separation.  49 U l t r a v i o l e t Absorption Spectra of Plant Extracts Absorption data of the p u r i f i e d e x t r a c t s was obtained by d i s s o l v i n g the extracts i n d i s t i l l e d spectroscopic grade methanol. Sample concentration was adjusted so that the major absorption peaks had an o p t i c a l 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 l i n e a r recorder was employed f o r absorption spectra determinations. The spectrophotometer was c a l i b r a t e d 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 a t 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 c o n s i s t i n g of an equal area of thin layer s i l i c a gel was subjected to developing i n 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 c a l c u l a t e d f o r absorption peaks.  U l t r a v i o l e t Spectra i n the Presence of Selected Reagents As a guide i n determining s t r u c t u r a l 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 i n methanol.  50 Reagents: 1.  Sodium Methoxide (NaOme):  2.5 gms o f f r e s h l y cut  m e t a l l i c sodium were added to 100 ml o f dry d i s t i l l e d spectroscopic methanol.  Three drops of t h i s s o l u t i o n  was added to the e x t r a c t s i n methanol. the 2.  A f t e r 5.0 minutes,  spectrum was re-run to check f o r decomposition.  Aluminum Choloride ( A l C l ^ ) : grade A l C I  3  5.0 grams of fresh reagent  were dissolved i n 100 ml of d i s t i l l e d  spectroscopic grade methanol. a fresh sample of e x t r a c t .  6.0 drops were added to  The spectra were recorded  a f t e r thorough mixing o f 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 reagent. 3  The spectra were recorded and the s o l u t i o n s were discarded. 4.  Sodium Acetate (NaOAc): NaOAc was used.  Anhydrous powdered reagent grade  Excess NaOAc was added to the cuvette  containing 0.45 ml o f the fresh stock s o l u t i o n of the extract.  A l l spectra were recorded w i t h i n 2.0 minutes  a f t e r the a d d i t i o n of the reagent. 5.  Boric Acid (HQBO.J: H3BO3 was employed.  Anhydrous powdered reagent grade S u f f i c i e n t H^Bo^ was added to the  cuvette containing the e x t r a c t s with NaOAc to give a saturated s o l u t i o n . the  The s o l u t i o n s were discarded a f t e r  spectra were recorded.  51  Infrared Spectroscopic Examination of Plant E x t r a c t s In order to examine the s t r u c t u r e s of the skeletons and  attached groups, i n f r a r e d analysis was conducted on the e x t r a c t s of the two legume species harvested at the vegetative stage.  A Beckman  IR-5A i n f r a r e d spectrophoid meter with double beam scanning was employed.  The machine was c a l i b r a t e d with a polystyrene f i l t e r  (Beckman Instruments Ltd.) p r i o r to sample determinations.  The  samples dissolved i n toluenerethanol (50:50) from the e x t r a c t i o n procedure were evaporated to dryness, and r e - d i s s o l v e d 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 s i z e was kept constant i n order to obtain a uniform sample s i z e on the p l a t e .  A second sodium choloride plate  was positioned over the samples, and the spectra were determined i n 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 c h l o r i d e p l a t e ; the same procedure as was u t i l i z e d with the samples was used f o r the blank p l a t e .  Sodium chloride p l a t e s 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 i n Table 3.  A wide  range of crude protein and crude f i b e r values are evident throughout the grazing season.  These r e s u l t s are c o n s i s t e n t f o r range legume  52 growth patterns, with decreasing p r o t e i n and increasing crude f i b e r content of the plants found during advancing maturity. Table 4 i n d i c a t e s the ether and chloroform e x t r a c t weights from 350 grams (D.M.) of both species during the grazing season.  The  ether e x t r a c t f r a c t i o n reached maximum at f u l l bloom stage f o r both species and increased again a t seed disseminated stage.  The chloroform  e x t r a c t was maximum a t f u l l bloom i n V i c i a americana, and at seed disseminated stage i n Astragalus miser var. s e r o t i n u s .  These  r e s u l t s i n d i c a t e no c o r r e l a t i o n between e x t r a c t weights and proximate a n a l y s i s data. Bioassay r e s u l t s are tabulated i n Table 5.  V i c i a americana  possessed maximum estrogenic a c t i v i t y at seed disseminated stage, and maximum i n h i b i t o r y e f f e c t s by the chloroform e x t r a c t on e s t r a d i o l a c t i v i t y a t vegetative stage.  In c o n t r a s t , Astragalus miser var.  serotinus r e f l e c t e d maximum uterine growth promoting e f f e c t s a t seed pod stage; maximum i n h i b i t o r y action occurred a t the vegetative stage, as d i d V i c i a americana.  These r e s u l t s agree with K i t t s e t aj_.  (1959),  who noted large v a r i a t i o n s i n plant a c t i v i t y due to plant growth stages, and with B i e l y and K i t t s (1964) who i n d i c a t e d that an inverse r e l a t i o n s h i p e x i s t e d between estrogenic and a n t i - e s t r o g e n i c a c t i v i t i e s during the growing season.  Results from t h i s experiment  demonstrate r e l a t i o n s h i p e x i s t i n g between plant potencies and e x t r a c t weights.  Factors i n f l u e n c i n g t h i s lack of c o r r e l a t i o n  between the weights o f the e x t r a c t s and the potencies of the growth stages include:  53 TABLE 3 PROXIMATE ANALYSIS OF VICIA AMERICANA AND ASTRAGALUS MISER VAR. SEROTINUS  V i c i a americana Harvest Date  Growth Stage  % Dry Matter  % Crude Protein  19/6/71  Vegetative  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 Disseminated  77.7  5.6  18.7  2.6  7.1  % Crude Fiber (ADF)  % Crude Fat.  % Ash  Astragalus miser var. serotinus Harvest Date  Growth Stage  % Dry Matter  % Crude Protein  % Crude Fiber (ADF)  % Crude Fat  % Ash  19/6/71  Vegetative  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 Disseminated  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)  V i c i a americana  Growth Stage  Ether E x t r a c t (Fraction A) gms  Chloroform Extract (Fraction B) gms  Vegetative  0.513  0.405  F u l l Bloom  0.746  0.588  Seed Pod  0.313  0.113  Seed Disseminated  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  F u l l Bloom  0.927  0.187  Seed Pod  0.130  0.089  Seed Disseminated  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 s a l i n e )  Vegetative F u l l Bloom Seed Pod Seed Disseminated (b)  Astragalus  2 (0.025 yg 17-3estradiol)  0.0424 0.0384 0.0387 0.0387  0.0489 0.0543 0.0592 0.0592  3 (15.0 gm D.M. Fraction A ether extract)  4 (15.0 gm D.M. Fraction B chloroform e x t r a c t + 0.025 yg 17-B-estradiol)  0.0408 0.0445 0.0396 0.0453  0.0405 (4, 3, 1 ) * * * 0.0406 (1 . 4 U 4 . 3) 0.0449 ( 1 , 3, 4) 0.0421 •(:!, 4) (4, 3)  miser var. serotinus ( P l o t 2)  Treatment  Vegetative F u l l Bloom Seed Pod Seed Disseminated  1 (0.2 ml s a l i n e )  0.0424 0.0384 0.0438 0.0438  2 (0.025 ug 17-3estradiol) 0.0489 0.0543 0.0628 0.0628  3 (15.0 gm D.M. Fraction A ether extract)  4 (15.0 gm D.M. Fraction B chloroform e x t r a c t + 0.025 yg 17-3-estradiol)  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 d i d not d i f f e r s i g n i f i c a n t l y under Duncan's M u l t i p l e Range Test at the P < .05 l e v e l  ** Animals died p o s t - i n j e c t i o n of the f r a c t i o n * 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 c e l l growth and maturity taking preference over isoflavones for carbon substrates;  3.  The presence of interfering materials, i . e . ,  chlorophyll  artifacts and l i p i d fractions from the extraction procedure. The most pronounced effects of the extracts occurred with the chloroform f r a c t i o n ; 15.0 grams dry matter equivalent of chloroform extract of Vicia americana decreased the potency of 0.025 ugs estradiol 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 l u i d  uptake and c e l 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 significant 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 f u 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 f u 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 p l a n t ; l e v e l s attained a maximum o f 2.7 - 3.2% of plant dry matter at f u l l bloom and e a r l y pod stages, confined mainly to the leaves and p e t i o l e s . Only small amounts were found i n the pods, roots and flowers.  Williams e t al.(1969)  concluded that ingestion of timber milk vetch containing 3% miserot o x i n a t a l e v e l of 4.8 gms/kg body weight was a l e t h a l dose f o r ruminants. The symptoms e x h i b i t e d by the animals two hours post i n j e c t i o n included arching of the back, l o s s o f e q u i l i b r i u m , and rapid drop o f body temperature. i n t e s t i n a l mucosa.  Minute hemorrhagic l e s i o n s were present i n the These e f f e c t s resemble those found by Mosher  (1970), whose ethanolic e x t r a c t i o n procedure f o r miserotoxin  determin-  ation p a r a l l e l e d that f o r the e x t r a c t i o n of plant estrogens. The r e s u l t s of the t h i n l a y e r 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  i n Table 6.  Also included i n t h i s table are the r e s u l t s  of i n f r a r e d spectroscopic examination of the vegetative stage i s o l a t e s from the two legume species.  These t e s t s do not confirm the p o s i t i v e  i d e n t i f i c a t i o n of the e x t r a c t s , but do i n d i c a t e groups of flavonoids present, t h e i r basic s t r u c t u r e s , and the appearance o f new compounds during the growth stages.  Of p a r t 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 i n d i v i d u a l falvonoids would useful to determine the c o n t r i b u t i o n to uterine reactions and competitive binding with the animal The composition  estrogens.  of the e x t r a c t s throughout the growing season i s i n  a dynamic s t a t e , 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.  V i c i a americana -- P l o t 1, Cut 1 (Vegetative Stage) Ether Extract ( F r a c t i o n A) Test Indicates  Line 1 Colour (UV) Colour (NHL)' Rf ***] MeOH 2. NaOMe 3. A1CU  Fluorescent blue Fluorescent dark blue 0.34** 245* 299 SH 233* 281 232* 269  J  <  4. 5. 6.  239* 264 SH 243* 267 SH 232* 266 SH  A1C1, + HCl NaOAC NaOAC + Ho  Infrared  Isoflavone. Flavone or Flavanone. "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  Functional Groups C - H Aromatic, C - c, M u l t i p l e bonds, c y c l i c , Hydroxyl Groups. Phenolic s t r u c t u r e .  Data:  Test Indicates  Line 2 Colour (WV) Colour ( N H J Rf 1. MeOH 2. NaOMe 3. A1C1, 4. AlCIo + HCl  Fluorescent blue Fluorescent blue 0.43 233* 267 SH 232* 286 SH 232* 284 238* 314 SH  6  5. 6.  NaOAC NaOAC + H Bo Q  Infrared  Data:  Q  232* 325 SH 231* 324  Catechin or Xanthone skeleton. "A" ring hydroxyl Groups. Free 5 - hydroxyl Group. Ortho dihydroxyl Groups at 6, 7 or 7, 8. 7 - hydroxyl Group. Ortho-dihydroxyl Groups on Ring "A".  Aromatic, free adjacent OH Groups. C-H Bonds, Phenolic s t r u c t u r e .  Main Absorption Peak •kic  Rf values i n Chloroform: Methanol Solvent 91:9 S H  I n f l e c t i o n Point Values 1-6 expressed i n m i l l i m i c r o n s  59  Table 6 (continued) Line 3 Colour (U.V.) Colour (NH ) Rf** 1. MeOH 2. NaOMe 3. A1C1  Yellow/Green L i g h t Yellow 0.50 268* 319 SH 273* 323 SH 266* 299 SH  4. A1C1 + HCl 5. NaOAC 6. NaOAC + H,Bo,  269* 301 SH 272* 311 SH 268* 322 SH  3  3  3  0  0  Infrared Data:  Test Indicates Flavanone, Aurone, Flavonol. Flavanone s t r u c t u r e . Lacks 5, 7 dihydroxyl Groups. Lacks 6, 7 or 7, 8 dihydroxyl Groups. 5 - hydroxyl Group absent. Lacks 5, 7 dihydroxyl Groups. Lacks 6, 7 dihydroxyl Groups on Ring "A".  C-H, Free OH, Ketone present, Phenolic s t r u c t u r e . Test Indicates  Line 4 Colour (U.V.) Colour ( N H J  Fluorescent Blue Fluorescent Blue  Rf** 1. MeOH 2. NaOMe 3. A i d ,  0.65 2.68* 330 SH 271* 334 SH 274* 297  4. A1C1, + HCl 5. NaOAC  277* 295 SH 277* 319 SH  Infrared Data:  "A" Ring hydroxyl Groups. Lacks 6, 7 or 7, 8 orthodihydroxyl Groups. Lacks 5 - hydroxyl Group. Lacks 6, 7 dihydroxyl Groups on Ring "A".  Free hydroxyl Group. C-H C=0 c-c m u l t i p l e bonds Phenolic s t r u c t u r e . Test Indicates  Line 5 Colour (U.V.) Colour ( N r L ) Rf** 1. MeOH 2. NaOMe  Red Red- i n k 0.84 254* 402 256* 402  3. A1C10  256* 421  4. A1CU + HCl 5. NaOAC 6. NaOAC + HQBOQ  257* 421 256* 405 273* 407  Chalcone, carotenoid or anthocyanin d e r i v a t i v e .  3  J  Infrared Data:  Isoflavone l a c k i n g a 5hydroxyl Group.  -  Lacks "A" r i n g hydroxyl Groups. Ortho-dihydroxyl Groups present. Adjacent hydroxyl Groups. on the nucleus. Ortho-dihydroxyl Groups present on the skeleton.  .C-C Bonds, aromatic r i n g nucleus, free OH groups.  60  Table 6 (continued) 2.  V i c i a americana -- P l o t 1, Cut 1 (Vegetative Stage) Chloroform Extract ( F r a c t i o n B -- Maximum A n t i - e s t r o g e n i c Activity) Line 1  Colour (U.V.) Colour (NH,) 1. 2. 3. 4. 5. 6.  MeOH NaOMe A1C1, A1C1, + HCl NaOAC NaOAC + H Bo 3  Infrared Data:  3  Dull Dull 0.12 235* 237* 235* -  Yellow Yellow  Test Indicates Flavonol, a r t i f a c t , or coumaranone d e r i v a t i v e .  280 SH 279 SH 283 SH 277 SH 280 SH 280 SH  C-H, C-C M u l t i p l e Bond, Phenolic s t r u c t u r e . Line 2  Colour (U.V.) Colour (NH,) Rf** 1. MeOH 2. NaOMe 3. Al Cl  Fluorescent Blue Fluorescent Blue 0.65 277* 322 SH 279* 281* 228 SH  4. A1CU + HCl 5. NaOAC  279* 324 SH 278* 325 SH  J  3  6.  NaOAC + H Bo 3  Infrared Data:  3  281* 325 SH  Test Indicates Flavanone l a c k i n g a 5hydroxyl Group. Lacks a 5-hydroxyl Group. Ortho-dihydroxyl Groups a t 6,7 o r 7, 8. Lacks a 5-hydroxyl Group. Lacks a 5, 7 dihydroxyl structure. 6, 7 dihydroxyl Groups absent from Ring "A".  Free OH groups. C-H, aromatic, CH groups. 2  Line 3 Colour (U.V.) Colour (NH ) Rf** 1. MeOH 2. NaOME  Fluorescent Blue Fluorescent Dark Blue 0.82 257* 282 SH 255* 279  3. ALCLo 4. A1C1- + HCl 5. NaOAC 6. NaOAC + H Bo  257* 233* 234* 221*  3  3  Infrared Data:  3  273 SH 273 SH 272 SH  Aromatic s t r u c t u r e . Hydroxyl groups. Adjacent hydroxyl groups.  Test Indicates Flavene or benzofurane nucleus. C  methylated or glycoside attachment. Ortho-dihydroxyl Groups. 3 adjacent hydroxyl Groups. Free hydroxyl Groups. Ortho dihydroxyl Groups present. 3  61  Table 6 (continued) Line 4 Colour (U.V.) Colour (NHo) Rf** 1. MeOH 2. NaOME  Dull Yellow Green Fluorescent Yellow 0.85 272* 342 SH 272* 340 SH  3. A1CU 4. Al CI ^ + HCl 5. NaOAc 6. NaOAC + H Bo  273SH 272SH 272SH 271SH  J  3  Infrared Data: 3.  3  340SH 340SH 340SH 294SH  Flavanone l a c k i n g a 5hydroxyl 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.  C-H, Phenolic, OH groups, Ring S t r u c t u r e , Aromatic, Adjacent OH groups  V i c i a americana —  P l o t 1, Cut 4 (Seed disseminated staqe). Ether Extract (Fraction A) — maximum estrogenic activity) Line 1  Colour (U.V.) Colour (NH.J Rf** 1. MeOH 2. NaOME 3. A1C1  Fluorescent Blue Intense Fluorescent Blue 0.30 283* 308 295* 336 282* 312  4. Al CI + HCl  282* 312  5.  NaOAC  271*  6.  NaOAC + H Bo  J  3  3  3  3  -  284* 312  Line 2 Colour (U.V.) Colour (NH~) Rf** 1. MeOH 2. NaOME 3. A1CU 4. A1CH + HCl 5. NaOAC 6. NaOAC + H Bo 6  3  Test Indicates  3  Dull Yellow Dull Yellow 0.47 279* 312 286* 334 278* 304 280* 302 279* 336 280 - I n t e n s i t y Decreased  Test Indicates 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. Test Indicates Dihydroflavonol lacks a 5-hydroxyl Group, or a flavonol with a 3-hydroxyl Group with or without a 5-hydroxyl group. 3, 3' o r 4' hydroxyl Groups Ortho dihydroxyl Groups present. Ketone present. Free 7-hydroxyl Group. Ortho di hydroxy! Groups . < present.  62  Table 6 (continued) Line 3 Colour (U.V.) Colour (NH,) Rf 1. MeOH 2. NaOMe 3. A1C1, 4. A1C1, + HCl  Fluorescent Blue Intense Fluorescent Blue 0.55 280* 310 289* 333 281* 305 280* 304  5. NaOAC 6. NaOAC + H,Bo,  277* 333 279* 307 Line 4  Colour (U.V.) Colour (NH,) Rf** 1. MeOH 2. NaOME 3. A1C1, 4. A1C1, + HCl  Fluorescent Blue Intense Fluorescent Blue 0.64 283* 307 294* 335 280* 308 280* 309  5.  NaOAC  283* 335  6.  NaOAC + H Bo 3  3  288* 309  Test Indicates Flavone lacks a 5-hydroxyl Group, or flavonol lacks a 5-hydroxyl Group. 3, 3' or 4' hydroxyl Groups. Lacks ortho-dihydroxyl Groups. Acid stable -- lacks d i hydroxyl Groups or are substituted. 4' hydroxyl Group present. Lacks ortho-dihydroxyl Groups. Test Indicates Flavone or flavonol Lacking a free 5-hydroxyl Group. Free 4' and 3-hydroxyl Groups. Lacks ortho-dihydroxyl Groups. Ketone present or hydroxyl Groups s u b s t i t u t e d . 7 and 4' - hydroxyl Groups present. Lacks ortho-dihydroxyl Groups.  Line 5 Colour (U.V.) Colour (NH,) Rf** 1. MeOH 2. NaOME 3. A1C1, 4.  A1C1, + HCl 0  5. NaOAC 6. NaOAC + H,Bo,  Flavone or flavonol l a c k i n g a Fluorescent Blue Intense Fluorescent free 5-hydroxyl with 3Blue hydroxyl s u b s t i t u t e d . 0.74 283* 311 291* 334 6, 3, 3' or 4' hydroxyl Groups. 275* 308 Ortho-dihydroxyl Groups present. 280* 307 Ketone present or 3 and 5 hydroxyl Groups s u b s t i tuted. 282* 333 7-hydroxyl Group free. 286* B-ring ortho-dihydroxyl Groups present.  Table 6 (continued)  63 Line 6  Colour (U.V.) Colour (NH,) Rf** 1. MeOH 2. NaOME 3. A1C1  Fluorescent Blue Fluorescent Blue 0.84 268* 310 268* 335 268* 310  4.  272* 310  6  3  A1C1 + HCl 3  5. NaOAC 6. NaOAC + H B o 3  3  268* 336 265* 308  Line 7 Colour (U.V.) Colour (NH,)  Fluorescent Blue Fluorescent Dark Blue 0.86 282* 304 288* 340 278* 307 SH  Rf** 1. MeOH 2. NaOMe 3. A1CU 4. A1C1 + HCl  276* 309 SH  5. NaOAC 6. NaOAC + H Bo  279* 335 280* 310 SH  3  3  3  Test Indicates Flavone or flavonol lacks a free 5-hydroxyl Group or flavonol with 3hydroxyl s u b s t i t u t e d . 3,3' or 4' hydroxyl Groups. Lacks ortho-dihydroxyl Groups. 3 and 5 Ketone hydroxyl Groups absent or s u b s t i tuted. 4' and 7 hydroxyl Groups. Lacks ortho-dihydroxyl Groups. Test Indicates Flavone or f l a v o n o l . Lacks a 5-hydroxyl Group with 3-hydroxyl s u b s t i t u t e d . 3, 3' or 4' hydroxyl Groups. Lacks ortho-dihydroxyl Groups. Ketone present or s u b s t i t u t e d hydroxyl Groups. Free 7-hydroxyl Groups. Lacks ortho-dihydroxyl Groups.  4: Astragalus miser var. serotinus -- P l o t 2, Cut 1 (Vegetative Stage) Ether E x t r a c t (Fraction A) Line 1 Colour (U.V.) Colour (NH,) Rf** 1. MeOH 2. NaOMe 3. A1C1, 4. A i c r + HCl 5. NaOAC 6. NaOAC + H Bo J  3  Infrared Data:  3  Dull Dull 0.34 277* 273* 271* 273* 271* 272*  Yellow Yellow Green 406 405 418 416  Test Indicates Chalcone, carotenoid or anthocyanin d e r i v a t i v e or h y d r o l y s i s product. Ortho-dihydroxyl Groups.  -  OH, C-H, Ketone Phenolic s t r u c t u r e . Adjacent OH groups. Aromatic s t r u c t u r e .  Table 6 (continued)  64 Line 2  Colour (U.V.) Colour (NH,) Rf** 1. MeOH 2. NaOMe 3. A1C1,  Fluorescent Blue Dark Purple 0.58 275* 312 SH 284* 325 SH 278* 311 SH 277* 310 SH 278* -  J  4.  AlClo  6.  NaOAC + H Bo  5.  NnOAC  +  HCl  3  276*  3  Infrared Data:  Test Indicates Isoflavone lacks a free 5 hydroxyl Group. "A" Ring hydroxyl Groups. Lacks free 5-hydroxyl Group. Lacks free 5-hydroxyl Group. Lacks 7 hydroxyl Group or 0 at position # 6 . . . . LacKs 6 , 7 dihydroxyl Groups on Ring " A " . 2  —  Aromatic nucleus Free OH groups C-H Bonds. Line 3  Colour (U.V.)  Fluorescent Bright Yellow Fluorescent Bright Yellow 0.95 269* 324 SH 279* 324 SH 268* 322 SH 269* 322 SH  Colour (NH,) Rf** 1. MeOH 2. NaOMe 3. A1C1 4. Al CI 3 + HCl 5. 6.  AnOAC NaOAC + H Bo 3  275* 327 SH 270* 324 SH  3  Infrared Data:  Test Indicates Flavonol with free 3-hydroxyl with or without free 5 hydroxyl.  7-hydroxyl Group. No ortho-dihydroxyl Groups. 3 and 5-hydroxyl Groups absent or s u b s t i t u t e d . 6 and 8 oxygen s u b s t i t u e n t s . No B-Ring ortho-dihydroxyl Groups.  Aromatic nucleus groups. OH groups, C-H Bonding, OH free groups. Line 4  Colour (U.V.) Colour (NH,) . Rf** 1. MeOH 2. NaOMe 3. A1C1, 4. A1C1- + HCl 5. NaOAC 6. NaOAC + H,Bo, O  Infrared Data:  0  Bright Red (Green in v i s i b l e spectrum ) Bright Red 0.97 262* 324 SH 249* 348 265* 323 267* 323 259* 286 SH 264* 327 SH  Test Indicates Xanthone, aurone or chalcone hydrolysis product.  Free hydroxyl Groups. Ketone present. Free hydroxyl Group. Ortho-dihydroxyl Groups absent.  Aromatic, free OH groups C-0 present, phenolic present  65  Table 6 (continued) 6.  Astragalus miser var. serotinus -- P l o t 2, Cut 1 (Vegetative Stage) Chloroform E x t r a c t (Fraction B — Maximum A n t i e s t r o genic A c t i v i t y ) Line 1  Colour (U.V.)  Fluorescent Light Blue Fluorescent Light Blue 0.07 278* 320 SH 280* 324 SH 276* -  Colour (NH,) Rf** 1. MeOH 2. NaOME 3. A i d , o  4.  A l C l o + HCl  5. 6.  NaOAC NaOAC + H,Bo, o  276* 280* 326 SH 280* -  0  Infrared Data:  Infrared Data:  Q  Dark Blue Purple 0.37 273* 323 SH 238* 292 SH 228* 292 227* 273 SH 278* 276* -  Lacks "A" Ring hydroxyl Groups. Lacks 5, 6, 7, 8 hydroxyl Groups. Lacks 5, 7 dihydroxyl Groups. Lacks 6, 7 dihydroxyl Groups on Ring "A".  Test Indicates Flavanone or dihydro-flavanone with 5' or 4' hydroxyl Group. 5-hydroxyl present. Free 5-hydroxyl Group. Free 5-hydroxyl Group. Lacks 5-7 di hydroxyl Group. Lacks 6, 7 dihydroxyl Groups on Ring "A".  Aromatic, phenolic, C-C m u l t i p l e bonds C-H, OH groups. Line 3  Colour (U.V.) Colour (NHJ Rf** ^ 1. MeOH 2. NaOME  Flavanone lacks a free 5hydroxyl Group.  Aromatic, free hydroxyl groups, c-c bonds phenolic present Line 2  Colour (U.V.) Colour (NH,) Rf** 1. MeOH 2. NaOME 3. A1CU 4. A1C1, + HCl 5. NaOAC 6. NaOAC + H,Bo  Test Indicates  Dark Blue Purple 0.69 2 7 7 * 327 SH 278* 275* 317 SH 275* 318 SH 276* 273* -  Test Indicates Flavanone with 5-hydroxyl Group.  "A" Ring hydroxyl Group. Lacks 6, 7, Or 7, 8 ortho4. A1CH + HCl di hydroxyl Groups. Lacks a 7 hydroxyl Groups. 5. NaOAC 6. NaOAC + H,Bo, Lacks 6, 7 dihydroxyl Groups on Rinq "A". Infrared Data: Aromatic s t r u c t u r e , C-c m u l t i p l e bonds, OH group, Phenolic present. 3.  A l C l o  Table 6 (continued) 6.  66  Astragalus miser var. serotinus -- P l o t 2, Cut 3 (Seed Pod Stage) Ether E x t r a c t (Fraction A) Maximum Estrogenic A c t i v i t y Test Indicates  Line 1 Colour (U.V.) Colour (NH,) Rf** 1. MeOH 2. NaOMe  Red (Green i n v i s i b l e Red spectrum) 0.07 399* 304 SH 399* 306 SH  3. A1C1  408* 305 SH  4. A1C1, + HCl 5. NaOAC  408* 308 SH 397* 304 SH  J  NaOAC + H Bo 3  3  399* 311 SH  Line 2 Colour (U.V.) Colour (NH,) Rf** 1. MeOH 2. NaOMe 3. A1C1, 4. A1C1, + HCl  Fluorescent Blue Fluorescent Blue 0.20 266* 263 SH 266* 263 SH 311* 266 SH 311 265 SH  J  5. NaOAC 6. NaOAC + H B o 3  3  -  Colour (U.V.)  Fluorescent Blue  Colour (NH,) Rf** 1. MeOH 2. NaOMe  Fluorescent Blue 0.28 271* 265 SH 270* 267 SH  3. A1C1, 4. A1C1X + HCl 5. AnOAC 6. NaOAC + H Bo  271* 271* 272* 271  3  3  Lacks a free 4' hydroxyl Group. Lacks "B" r i n g orthodi hydroxyl Groups. II  II  n  H  Lacks a free 4' and/or 6 hydroxyl Group. Lacks "A" Ring orthodi hydroxyl Groups. Test Indicates Coumaranone or f l a v o n o i d hydrolysis product.  Ortho-dihydroxyl Groups. Ketone o r s u b s t i t u t e d hydroxyl Groups.  265 SH 266 SH  Line 3  J  Aurone • c h l o r o p h y l l fragment.  265 SH 266 SH 269 SH 268 SH  Test Indicates Flavan or p a r t i a l f l a v o n o i d skeleton.  No reactions i n d i c a t e d flavonoid nucleus and attached Groups not present.  Table 6 (continued)  67 Line 4  Colour (U.V.) Colour (NH,) Rf** 1. MeOH 2. NaOMe 3. A1C1, 4. A1C1, + HCl 5. NaOAC 6. NaOAC + H B o J  3  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 Line 5  Colour (U.V.) Colour (NH,) Rf** 1. MeOH 2. NaOMe 3. A1C1, 4. A1C1 + HCl  Fluorescent Blue Fluorescent Bl ue 0.62 271* 327 SH 269* 329 SH 271* 271* -  J  3  5. NaOAC 6. NaOAC + H B o 3  3  271* 328 SH 271* -  Line 6 Colour (U.V.) •  "  •  •  {  .  .  Colour (NH,) Rf** 1. MeOH 2. NaOMe 3. A1C1, 4. A1C1, + HCl 5. NaOAC 6. NaOAC + H Bo J  3  3  Intense Fluorescent Blue Fluorescent Blue 0.73 271* 328 SH 271* 329 SH 274* .278* 271 328* 280 Line 7  Colour (U.V.) Colour (NH ) 3  Rf** 1. MeOH 2. NaOMe 3. A1C1, 4. A1C1, 5. NaOAC 6. NaOAC + H Bo 3  3  Test Indicates Coumaranone flavan or hydrol y s i s by-products. Free hydroxyl Groups.  Test Indicates Flavanone l a c k i n g a 5-hydroxyl Groups. Isoflavone or flavonol l a c k i n g a 5-hydroxyl Group. Ortho-dihydroxyl Groups. Keto Group of s u b s t i t u t e d hydroxyl Groups. "B" Ring ortho dihydroxyl Groups. Test Indicates Flavanone, isoflavone or ' flavonol l a c k i n g a 5hydroxyl Group. No free 4' hydroxyl Group. Ortho dihydroxyl Groups present. Lacks a 7-hydroxyl Group. Ortho-dihydroxyl Groups present. Test Indicates  Intense Fluorescent Flavone, flavonol lacks free 5Blue hydroxyl Groupl with 3Intense Fluorescent substituted. Blue 0.84 276* 330 270* 333 C-3 hydroxyl group methylated or glycoside attachment. 275* 409 Ortho-dihydroxyl groups present. 276* 406 Keto group adds s t a b i l i t y to A l C l ^ complex. 280* 331 Free 7-hydroxyl Group. 276* Ortho dihydroxyl Groups present.  68 estrogenic a c t i v i t y i n V i c i a americana. disseminated  (Lines 5 and 7 of the seed  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 r e s u l t s obtained from these experiments add to the observations by previous authors (Towers et al_. 1964), that plant phenolics contained i n 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 l i v e s t o c k  from bioassay experiment e x t r a p o l a t i o n would be i n the magnitude of one t h i r d of body weight f o r noticeable e f f e c t s .  However, as noted  by Braden and Shutt (1970), pasture estrogens undergo rapid ruminal demethylation  i n t o structures with increased estrogenic a c t i v i t y ( i . e .  biochanin A conversion to g e n i s t e i n ) . Previous research by  Turnbull  et aj_. (1966), K a l l e l a (1972) has i n d i c a t e d that prolonged grazing on estrogenic pastures i s responsible f o r 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 f a l l o p i a n tubes of sheep, and  de-  creased uterine f l u i d content preventing conception i n r a t s . The r e s u l t s presented  i n d i c a t e that mammalian species do  respond to the low potency u t e r o t r o p i c compounds present i n the legumes examined; f u r t h e r i n v e s t i g a t i o n s into rumen a c t i v i t i e s of the e x t r a c t s would give a c l e a r e r p i c t u r e of the e f f e c t s of the plant products on domestic l i v e s t o c k .  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 o f the major factors i n f l u e n c i n g the l e v e l s of estrogenic compounds i n legume species i s mineral n u t r i t i o n .  The e a r l i e s t  occurrence o f clover disease during the 1940 era i n A u s t r a l i a 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 s t r a i n s of T r i f o l i u m subterraneum was i n v e r s e l y 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 d i d not s i g n i f i c a n t l y a f f e c t the estrogenic of the p l a n t s .  B i c k o f f (1968) has reviewed several  authors, notably concerning T r i f o l i u m subterraneum.  activity  reports by various Evidence i s  lacking i n North America on the e f f e c t s of f e r t i l i z e r elements on the estrogenic  constituents  of standard forage legume species grown f o r  animal feeds. This study involved an assessment of the estrogenic  isoflavones  biochanin A, formononetin, and g e n i s t e i n , and coumestan, coumestrol, found i n three domestic  legumes:  T r i f o l i u m hybridium, T r i f o l i u m repens,  and Medicago s a t i v a , as affected by t o p i c a l f e r t i l i z e r  applications.  70 MATERIALS AND METHODS  Seed, P l o t Preparation and Layout Selected Canada #1 grade seeds o f a l s i k e c l o v e r ( T r i f o l i u m hybridium), white c l o v e r ( T r i f o l i u m repens var. ladino) and a l f a l f a (Medicago sativa),were inoculated with rhizobium b a c t e r i a ( N i t r a g i n Co. L t d . "AB" c u l t u r e ) d i r e c t l y before planting to ensure adequate nitrogen f i x i n g b a c t e r i a on the root systems. The p l o t s i t e on the U n i v e r s i t y 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 a g r i c u l t u r a l lime were applied and thoroughly mixed to n e u t r a l i z e the acid s o i l conditions of the zone. remained i n f a l l o w f o r two growing seasons.  The p l o t area had  Discing and l e v e l l i n g  assured a homogenous seed bed. P l o t layout f o r 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 f o r the treatments.  Each treatment received  a minimum o f four r e p l i c a t e s . Control p l o t s f o r each treatment were also r e p l i c a t e d by a f a c t o r of four.  Figure 12 i n d i c a t e s actual  ground lay-out of T r i f o l i u m repens var. ladino.  (Medicago s a t i v a  l i e s adjacent to t h i s block and i s r e p l i c a t e d i n the same manner.)  1.8 m «fl 8» CI  Nl  N2  PI  CO  K2  Kl  P2  CO  P2  CO  CO  PI  CO  C2  Nl  PI  CO  C2  CI  CL  P2  CL  K2  CO  K2  CO  CI  Nl  N2  CO  0.9 m  CO  Kl  P2  Figure 11  C2  Plot Layout and F e r t i l i z e r Treatments  PI  71  Figure 12  T r i f o l i u m Repens Var. Ladino Random Design f o r F e r t i l i z e r Trials.  72 Seeding Rates and F e r t i l i z e r Seeding Rates  Treatments A l s i k e Clover Ladino Clover Alfalfa  Fertilizer treatments  = 5.3 kg/hectare = 5.3 kg/hectare =15.9 kg/hectare  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  Nl  16-0-0 n i t r a t e of soda  162  N2  16-0-0  324  PO  Control —  PI  0-45-0 t r e b l e 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  0  no f e r t i l i z e r  Seeding and F e r t i l i z e r A p p l i c a t i o n -- P l o t Maintenance  0  Procedure  Each 1.8 x 3.1 M p l o t was raked by hand, and f e r t i l i z e r applied in two d i r e c t i o n s .  Seed was applied  and spread uniformly on each p l o t .  following f e r t i l i z e r a p p l i c a t i o n s , Plots were again evenly raked i n  two d i r e c t i o n s 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 f o l l o w i n g seeding to stimulate germination.  This procedure was continued i n t e r m i t t e n t l y  u n t i l germination occurred and seedlings became e s t a b l i s h e d .  During  the t r i a l period, s o i l moisture was maintained with s p r i n k l e r i r r i g a t i o n a p p l i c a t i o n at the f i r s t sign of moisture d e f i c i e n c y . Water was j u d i c i o u s l y applied and s o i l puddling was prevented by  73 c o n t r o l l e d a p p l i c a t i o n rates.  A l l weeding and thinning o f p l o t s were  done by hand; no herbicides were applied to the p l o t areas.  Border  areas surrounding and between the p l o t s were continuously mowed to a 2.2 cm height to prevent p l o t contamination by foreign plant species.  Harvesting and Plant Storage A l l blocks were harvested a t f u l l bloom stage.  Water was with-  drawn 72 hours p r i o r to c u t t i n g ; the p l o t s were mowed a t a height o f 2.2 cm above ground surface ( t o eliminate s o i l contamination); each p l o t y i e l d was recorded; the four p l o t r e p l i c a t e cuttings were bulked, thoroughly mixed, and four random 1 kg samples taken from the bulked plots.  These samples were sealed i n p l a s t i c bags and quick frozen a t  0°C f o r storage.  Control p l o t s received the same treatment.  Samples  were analysed f o r dry matter content according to the methods o f the A.O.A.C. (1960).  Fractionation o f Plant Material Twenty-five gram samples (D.M.) were extracted as i n Part 1 (Figure 5) f o r each treatment. weighted  The ether e x t r a c t s obtained were  and adjusted to a concentration o f 15.0 mg/ml i n toluene:  ethanol (50:50) p r i o r to chromatography.  Thin Layer Chromatography o f Plant Extracts Thin l a y e r chromatographic plates layered with S i l i c a Gel G (Merck) a t 0.25 mm thickness were a c t i v a t e d by heating a t 110°C  74 f o r 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 s i z e was kept constant at 2 30 mm .  Each sample was spotted on duplicate plates.  Standards  c o n s i s t i n g 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 i n 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 t e s t e d , 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 i n unsaturated atmospheres. Intermediate drying of 3.0 minutes between runs ensured uniform spot sizes i n the second dimensional separation.  Quantitative Determination of Estrogenic Constitutent of the Plant Extracts Quantitative i n 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  d e f l e c t i o n was obtained by attaching a Unicam SP20 Recorder programmed f o r l i n e a r recording.  A #110-850 lamp (Turner) with peak emission at 360  75 m i l l i m i c r o n s , and primary f i l t e r #7-54 ( Transmits 420 my) were u t i l i z e d .  below 254 my to  A secondary f i l t e r of 2A-15 (passes wave  lengths longer than 520 my) f o r 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 f o r coumestrol  determinations.  Fluorometer  s l i t widths were adjusted to give maximum d e f l e c t i o n s f o r each i s o f l a v o n e ; 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 p l a t e s , coumestrol spots were located by t h e i r  and formononetin  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 p l a t e s , dissolved i n 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 q u a n t i t a t i v e estimation, spots of  coumestrol  and formononetin were c i r c l e d on the p l a t e s , exposed to  ammonia fumes f o r 10 seconds to enhance t h e i r fluorescence, and scanned w i t h i n 15 seconds o f exposure to the ammonia fumes. Biochanin A and g e n i s t e i n spots were located by spraying with d i a z o t i z e d s u l f a n i l i c a c i d , and corresponding  areas of t h i n l a y e r  plates eluted with methanol; comparison of u l t r a v i o l e t absorption spectra, colours with d i a z o t i z e d s u l f a n i l i c a c i d , and R^ values when  76 compared to r e - c r y s t a l l i z e d samples o f biochanin A and g e n i s t e i n confirmed the i d e n t i f i c a t i o n of the i s o f l a v o n e s .  Figure 13 i l l u s -  t r a t e s chromatographic m o b i l i t i e s of the compounds. estimation o f the two isoflavones was accomplished a 5% s o l u t i o n o f A l C l 3  Quantitative by spraying with  i n 95% d i s t i l l e d ethanol, followed by  heating f o r 3 minutes a t 75°C to i n t e n s i f y the fluorescence.  All  scanning was done w i t h i n 2 minutes f o l l o w i n g heating; t r i p l i c a t e readings f o r each scan were recorded. Standard curves o f concentration ys_ fluorescence were c a l c u l a t e d f o r each chromotographic p l a t e .  Isoflavone/coumestrol  concentrations were determined.on a dry weight basis f o r each plant species and f e r t i l i z e r treatment.  The r e s u l t s are tabulated i n  Tables 7 and 8.  RESULTS AND DISCUSSION  Dry matter y i e l d s (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) a f f e c t e d by f e r t i l i z e r a p p l i c a t i o n s , due to large standard errors from the mean f o r r e p l i c a t e p l o t s f o r each f e r t i l i z e r  treatment.  Ladino c l o v e r treatments were l e a s t a f f e c t e d by f e r t i l i z e r s ,  probably  due to the forage's indeterminate growth pattern. alfalfa  A l s i k e c l o v e r and  responded to phosphate f e r t i l i z e r s , with the trend being to  increase dry matter y i e l d s 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:  Figure 13  I II  Chroma tographi c M o b i l i t i e s of Isoflavones and Coumestrol i n 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 r e p l i c a t e s )  Al f a l f a (kgs)  Treatment  Alsike (kgs)  Ladino (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  79  TABLE 8 EFFECTS OF FERTILIZER TREATMENTS ON ESTROGENIC COMPONENTS OF LEGUME SPECIES Dry Treatment 1.  Genistein  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*  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*  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  Ladino Clover - Coumesti-ol CO Cl C2 NI N2 Pl P2 Kl K2  3.  Formononetin  (% D.M.) Total Estrogenic Isoflavones  A l s i k e Clover CO Cl C2 NI N2 Pl P2 Kl K2  2.  Biochanin A  Matter  Alfalfa CO Cl C2 NI N2 Pl P2 Kl K2  0.0001 0.0001 0.0002 0.0001 0.0004 0.0001 0.0001 0.0001 0.0001 - Coumesti"Ol 0.0001 0.0001 0.002 0.0002 0.0001 0.0004 0.0001 0.0001 0.0002  Indicates s i g n i f i c a n t d i f f e r e n c e between control and treatment a t P < .05 when crossed with the three estrogenic c o n s t i t u e n t s . (Anova with treatment and constituent crossed and r e p l i c a t e nested in c o n s t i t u e n t ) .  80 Estrogenic isoflavones and coumestrol l e v e l s were 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 a p p l i c a t i o n s , when compared to control p l o t s .  Table 8 i n d i c a t e s that a l s i k e c l o v e r responded to  treatment P2, increasing t o t a l isoflavone concentration by a f a c t o r of two; ladino clover followed observations  by R o s s i t e r (1970) that  in low phosphate supplied plants t o t a l isoflavone l e v e l s increased during l a t e r growth stages, ( i . e . f u l l bloom stage). concentrations  Highest t o t a l  of isoflavones are present i n control p l o t s and p l o t s  r e c e i v i n g 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 a p p l i c a t i o n s , and no mineral a p p l i c a t i o n s , but t o t a l isoflavone content d i d not increase under i n d i v i d u a l N, P, or K added.  The r e s u l t s are i n agreement with Alexander and Rossiter (1951)  who examined subterranean c l o v e r .  These authors noted that T.  subterraneum contained more estrogenic potency when no f e r t i l i z e r was a p p l i e d ; a l f a l f a plants responded i n t h i s t r i a l i n the same manner, by increasing t o t a l isoflavone content.  Schoo and Rains (1971) also  confirmed the r e s u l t s with subterranean clover and formononetin content.  An analysis of variance f o r the i n d i v i d u a l 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 f o r each species.  Treatment v£ control f o r the three estrogenic con-  s t i t u e n t s i s presented i n 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  A l s i k e clover  P2  Absent  P2  Ladino clover  Absent  N2  Alfalfa  Absent  C2, PI  Genistein P2  C0;P2*  CO; CI*  CI, P I , P2  CI; C2*  Not s i g n i f i c a n t over control P < .05). A l s i k e clover was most s i g n i f i c a n t l y increased, both i n t o t a l and i n i n d i v i d u a l free estrogenic isoflavone content, by the addition of 216 kgs/hectare of 0-45-0.  Ladino clover constituents responded to  n i t r o g e n , (324 kg/hectare) and to control treatments.  Alfalfa iso-  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 r e s u l t s i n d i c a t e that c h a r a c t e r i s t i c r a t i o s e x i s t in the three legume species of free estrogenic i s o f l a v o n e s ; each species containing a g e n e t i c a l l y determined pattern of the three i s o flavones as noted by B a i l e y and Francis (1971).  Individual i s o -  flavone l e v e l s were affected to a greater degree than t o t a l isoflavone content under mineral  supplements.  The importance of high l e v e l s of coumestrol and formononetin to the forage producer should be emphasized.  Metabolic demethylation  by ruminants o f these two flavonoids r e s u l t s i n the production of equol (7, 4' - dehydroxy i s o f l a v a n ) as indicated by Shutt and Braden (1968).  Further estimates of the potencies of these two compounds  are presented i n Table 2, by B i c k o f f (1968), and by Francis and  82  M i l l i n g t o n (1971). The l e v e l s of estrogenic isoflavones found i n these r e s u l t s and affected by macro-element supplements, do not compare with t o t a l estrogenic compounds l e v e l s i n subterranean clover (app. 1% D.M.). However, as was i n d i c a t e d by Lindner  (1967), sheep uterine responses  at plasma l e v e l s of free g e n i s t e i n above 5 mcgm/100 ml of plasma were associated with maximal uterine growth response; plasma l e v e l s of 0.5 mcgm/100 ml plasma of formononetin also r e s u l t e d i n 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 e f f e c t s on estrogenic constituents of the c u l t i v a t e d legumes have been demonstrated and can serve as guidelines f o r the forage producer i n c o n t r o l l i n g plant hormonal a c t i v i t i e s , f o r increased growth and performance of domestic ruminants.  SUMMARY AND CONCLUSIONS  From the preceding experiments, the following  conclusions  can be drawn: Experiment I 1.  V i c i a americana and Astragalus mi ser var. serotinus contain low l e v e l s of uterotrophic components i n a dynamic state throughout the growing season; flavonoid and aromatic compounds p r e v a i l i n ether and chloroform e x t r a c t s of the legumes.  2.  V i c i a americana demonstrated maximum uterine growth promoting e f f e c t s i n mammals a t seed disseminated stage, and maximum growth i n h i b i t o r y a c t i v i t y a t vegetative stage.  Astragalus  miser var. serotinus possessed maximum uterine growth promoting e f f e c t s at seed pod stage, with maximum i n h i b i t o r y a c t i v i t y on the uterus a t vegetative stage.  No c o r r e l a t i o n  existed between proximate a n a l y s i s date, e x t r a c t weights, and estrogenic/anti-estrogenic a c t i v i t i e s f o r both species. 3.  Interference with the a c t i v i t y of animal estrogens by chloroform e x t r a c t s of the two legume species was most pronounced; uterine weight decreases of greater than 20% were obtained during the growing season f o r both legumes, due to i n j e c t i o n s of 15 gms dry matter equivalent of each species to immature female r a t s .  4.  The experiments demonstrated a t o x i c f r a c t i o n present i n Astragalus miser var. serotinus at f u l l bloom stage. This component was ether e x t r a c t a b l e ; the symptoms and e f f e c t s on animals resembled the e f f e c t s 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 a f f e c t the dry matter y i e l d s of a l s i k e c l o v e r , ( T r i f o l i u m repens) ladino clover ( T r i f o l i u m hybridum), and a l f a l f a (Medicago s a t i v a ) 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 f o r the fluorometric determination  f o r quanti-  t a t i v e measurement of biochanin A and g e n i s t e i n was developed, utilizing  fluorescent properties of the two isoflavones when  spayed with 5% Al Cl  3  i n 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 t h i n layer chromatographic p l a t e s . Estrogenic isoflavone and coumestrol l e v e l s 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; increased t o t a l isoflavone concentrations  a l s i k e clover  by a f a c t o r o f two.  Ladino clover and a l f a l f a produced maximum isoflavone l e v e l s even without the a d d i t i o n o f f e r t i l i z e r , and when p l o t s received complete (4-10-10) a p p l i c a t i o n s . 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A l s i k e Clover: Control vs. Cl Treatment  Source of Variance Treatment Estrogenic Constituents Constituent Replicates Error Total  D.F.  S.S.  1 2 6 8  2.04 2.54 2.25 5.82  17  2.56  M.S.  F  bility  2.04 28.0 0.0008* 1.27 1743.3 3.76 0.51 7.28 -  -  -  Control vs. C2 Treatment Source of Variance Treatment Esmogenic Constituents Constituent Replicates Error Total  D.F.  S.S.  1 2 6 8  5.63 7.17 9.36 8.11  17  2.09  M.S.  F  5.63 3.58 1.55 1.01  5.55 3.53 0.00  -  Probability 0.04*  -  -  Control vs. NI Treatment Source of Variance Treatment Estrogenic Constituents Constituent Replicates Error Total  * S i g n i f i c a n t a t (P < 0.05).  D.F. 1 2 6 8 17  S.S. 5.55 7.66 9.36 8.32 2.15 •  M.S.  F  5.55 3.83 1.55 1.04  5.33 3.68 0.00  -  -  - •  Probability 0.04*  98  Appendix Table 1 (continued) Control vs. N2 Treatment Source of Variance Treatment Estrogenic Constituents Constituent Replicates Error Total  Probability  D.F.  S .S.  M.S.  F  1 2 6 8 17  1.52 1.58 1..46 2.02 1.94  1. 52 7. 93 2. 44 2. 53  6.00 31.30 0.00  D.F.  S. S.  M.S.  F  1 2 6 8 17  6. 06 5. 58 7. 89 8. 72 2. 03  6. 06 2. 79 1. 31 1. 09  5.56 2.56 0.00  S.S  M.S  F  1 2 6 8  6.66 3.72 7.14 2.32  6.66 1.86 1.19 2.90  22.95 6.42 0.00  17  1.27  S.S.  M.S.  F  1 2 6 8  5.38 1.43 1.12 1.88  5.38 7.18 1.86 2.35  22.84 30.48 0.00  17  2.16  -  -  Control vs. PI Treatment Source of Variance Treatment Estrogenic Constituents Constituent Replicates Error Total  Probability  D.F  Treatment Estrogenic Constituents Constituent Replicates Error Total  .  -  •  Control vs. K2 Treatment Source of Variance Treatment Estrogenic Constituents Constituent Replicates Error Total  Significant at (P < 0.05).  D.F.  0.04*  -  Control vs. P2 Treatment Source of Variance  0.03*  Probability 0.0015*  Probability 0.0015*  Appendix Table 1 (continued) B.  99  Alfalfa: Control vs. Cl Treatment  Source of Variance Treatment Estrogenic Constituents Constituent Replicates Error Total  S.S.  M.S.  1 2 6 8  5.20 1.67 6.80 3.63  5.20 8.38 1.13 4.53  17  1.68  -  S.S.  M .S.  1 2 6 8  1.06 9.29 3.65 1.08  1 .06 4 .64 6 .08 1 .36  17  1.14  D.F.  F 11.46 1846.87 0.24  Treatment Estrogenic Constituents Constituent Replicates Error Total  D.F.  Probability  F 7.83 34. 12 0. 004  Control vs. Pl Treatment Source of Variance Treatment Estrogenic Constituents Constituent Replicates Error Total  S.S.  M.S.  F.  1 2 6 8  3.72 1.17 4.89 4.91  3.72 5.88 8.15 6.14  6.06 95.74 0.01  17  1.76  -  D.F.  Treatment Estrogenic Constituents Constituent Replicates Error Total  S i g n i f i c a n t a t (P < 0,05).  S.S.  M .S.  F.  1 2 6 8  1.39 7.88 3.28 1.64  1 .39 3 .94 5 .46 2 .05  6. 81 19. 21 0. 00  17  1.09  D.F.  0.02*  Probabi1i ty 0.03*  -  Control vs. P2 Treatment Source of Variance  0.009*  -  Control vs. N2 Treatment Source o f Variance  Probability  Probability 0.03*  Appendix Table 1 (continued) C.  100  Ladino Clover: Control vs. CI Treatment  Source of Variance Treatment Estrogenic Constituents Constituent Replicates Error Total  F.  D.F.  S .S.  M .S.  1 •2 6 8  1 .01 1 .51 7 .06 1 .08  .1.01 7 .59 1 .17 1 .35  75.0 56255.81 8.72  17  1 .52  S.S.  M .S.  F.  Treatment Estrogenic Constituents Constituent Replicates Error  1 2 6 8  1.36 1.28 6.13 9.75  1 .36 6 .43 1 .02 1 .21  17  1.31  Total  D.F.  11.16 527.37 0.08  Source of Variance Treatment Estrogenic Constituents Constituent Replicates Error Total  S.S.  M.S.  F.  1 2 6 8  3.53 4.18 1.98 3.38  3.53 2.09 3.31 4.23  8.35 4.94 0.00  17  1.11  -  Treatment Estrogenic Constituents Constituent Replicates Error Total S i g n i f i c a n t a t (P < 0.05)  S.S.  M. S.  F.  1 2 6 8  1.92 6.14 3.18 1.97  1.92 3.07 5. 30 2.46  7.80 12. 45 0. 00  17  1.00  D.F.  0.01*  Probabi 1 i ty 0.01*  -  Control vs. Kl Treatment Source of Variance  Probability  -  Control vs.Nl Treatment D.F.  0.01*  -  Control vs. C2 Treatment Source of Variance  Probability  Probabi 1 i ty 0.02*  101 )  •  _ .  Control vs. K2 Treatment •  Source of Variance Treatment Estrogenic Constituents Constituent Replicates Error Total  S i g n i f i c a n t at (P < 0.05).  D.F.  S.S.  M.S.  1 2 6 8  1.05 9.06 4.25 7.81  1.05 4.53 7.08 9.76  17  1.09  -  F. 10.80 46.41 0.00 -  -  Probability 0.01*  

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