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The estrogen-like activity and identification of certain isoflavones present in red clover Hogg, Robert William 1962

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THE* ES'.TROGEN-LIKE, ACTIVITI AND! IDENTIFICATIOE OF CERTAIN IS'OFEAVONES' PRESENT IN, RED; GLOVER by ROBERT WILLIAM HOGG B. S'. A., The University of British Columbia, I960 A THESIS; SUBMITTED IN PARTIAL, FULFILMENT OF-THE REQUIREMENTS' FOR THE DEGREE OF Master of Science in Agriculture in the Division of Animal Science We accept this thesis as conforming to the required, standard Members of the Division' THE UNIVERSITI OF BRITISH COLUMBIA April,, 1962. In presenting this thesis in p a r t i a l fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make i t freely-available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It i s understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of {Z,A**22#d&ea*££s The University of British Columbia, Vancouver 8, Canada. ABSTRACT A study was carried out to determine the relative estrogen-like activity attributed to the isoflavone compounds present in red clover plants (Trifolium pratense). The pure compounds were fed as additives to a non-estrogenic basal diet at total dosages ranging from zero to fifteem milligrams per mouse. The biological assay used for the determination of the estrogem-like activity was a slight modification of that outlined, by Kitts et a l . i n 1959 (63). The isoflavone found to possess the greatest estrogen-like activity was Genisteim followed by Daidzein, Biochanin A and Formononetirt. A non-isoflavone compound, Coumestrol, was found to posses-s a greater estrogen^like activity than any of the isoflavones' considered. Mixtures of the pure isoflavones were bio assayed' to observe any inhibitory or synergistic characteristics of one upon another. Mochanin; A appeared, to exert a slight inhibitory action and1 formononetdin a synergistic character. An electrophoretic technique was developed which facilitated, the; detection of the isoflavones i n plant extracts of monthly harvested red. clover. An attempt was made: to associate the biological activity of the red clover samples and the compounds x^ hicfa were observed in' these same samples using electrophoresis* -idoL-ACKMOWLEDQMEHT The author wishes to express his thanks to Dr. ¥• D... Kitts, Associate Professor in the Divisioni of Animal Science for his direction and' criticism.' throughout the course of this study* Thanks is also extended to the staffs of the Division of Plant Science and the Central. Animals Depot for their cooperation in this' investigation. The author is grateful, to Dr. K. Venkataraman of the national Chemical laboratories at Poona, India for supplying samples of synthetically prepared Formononetin and Biochanin A. Thanks i s also extended to Dr. E. M. Bickoff of the Western Utilization Research and Development Division,. Agricultural Research Service, U. S. Dt. A. i n California for supplying samples of naturally isolated isoflavones. Dr» HiT McLean' of the1 Forest Products Research Laboratory, University of British Columbia, i s thanked for the infrared analysis, and aid i n interpretation of the results obtained!, on the iinidentified substance Isolated from red clover* TABLE OF CONTENTS ACKNOWOTGMENT ABSTRACT INTRODUCTION 1 LITERATURE REVIEW k The Isoflavones h The Identification of Isoflavones. 12 The Isolation of Isoflavones 15>,' Coumestrol 1? The Biological Significance 19 The Effect of Estrogens on the Female Reproductive Tract 19 Plant Estrogens 21 Estrogenic Activity and Structure 26 Non-estrogenie Effects of the Isoflavones; 2ff MATERIALS AND' METHODS • 29 I. The Experimental Compounds 2§-II. The Bioassay Procedure 32 HI* The Preparation of Diets Containing the Pure Isoflavones 33 IV. The Preparation of Rations' Containing Two or More Isoflavones 3& V. The Preparation of Redi Clover Extracts for1 BLoassay 36 VI. The Electrophoresis Procedure 38) RESULTS' AND; DISCUSSION X, The Bioassay of Genistein, BLochanin A, Daidzein, Ebrmononetini and Coumestrol hh - i i -I I. The Results of the Bioassay of Isoflavone Mixtures £85 I H . The Results, of the Bioassay of Red Glover 6© IW. The Electrophoresis Procedure 63 V. The Estrogen-like Activity versus Electrophoresis Analysis of Red! Glover f 1 SUMMARY AND! CONCLUSIONS IS BIBLIOGRAPHE 11 INTRODUCTION! Within the large group of compounds referred to as flavonoids, there exists a minor subdivision of naturally occurring and synthetically prepared substances designated as isoflavones. To date fourteen isoflavones are known to occur in plant material, however their functional role i n these biological systems has not been clearly defined. The isoflavones do not take part, to any discernable extent, in the process of photosynthesis and as they are of common occurrence in most higher plants i t i s thought that they may i n some way be associated with the oxidation-reduction systems of plant tissue ( 9 7 ) • Isoflavones occur in plant tissue as aglycones and as glycosides, rhamnose and glucose being the carbohydrates most commonly found. The f i r s t natural isoflavone was isolated from. Prunus bark, charac-terized and called prunetin (56). The work of Baker and Robinson i n 1925 (6), in which 7 methoxyisoflavone was synthesized, was rapidly followed by additional synthesis procedures by which other chemically pure isoflavones were obtained. The fact that these compounds were of biological significance to a system other than that from which they were isolated was realized when they were identified in plant material which was thought to have caused repro-ductive disorders in grazing sheep. The compounds appeared to exert an action similar to that of the natural estrogens in the grazing animals. The presence of such estrogen-like compounds in plants was first reported in 1926 by Loewe ( 7 3 ) . However i t was not until 1 9 U U when extensive re-productive failures occurred in ewes of some Australian flocks grazing on subterranean clover, that serious consideration was given to their presence. Bennetts (1U), at this time, reported such reproductive disturbances as dystocia, uterine prolapse and general female infertility resulting in low -2-lambing rates. Biological assays developed as modifications of the original Allen and Doisey test (1) have classified the isoflavones present in the clover as possessing an estrogen-like effect in laboratory animals as well as in sheep. The extent of this activity varies between the different iso-flavones (UU)• The isoflavones possessing an estrogen-like activity which can be isolated from red clover are Biochanin A, Daidzein, Formononetin, Genistein, Pratensein and the coumarin-like derivative Coumestrol ( 6 l , 81;, 101). Variations in the reported activity levels of these compounds resul-ting from the use of differing bioassay procedures has led the author to investigate as many of the estrogen-like compounds, of plant material as possible using one technique over a range of values such that their rela-tive intensities could be determined. The compounds used in these studies were naturally isolated or obtained by chemical conversion of available synthetic compounds. The substances studied were genistein, daidzein, biochenin A, formononetin and coumestrol. The f i r s t study involved the biological assay of the individual com-pounds at increasing levels to determine their estrogenic intensity relative to one another. Associated with this, a study considering mixtures of the compounds was carried out to determine the possibility of a synergistic or inhibitory effect of one upon the other(s). The second study involved the development of an electrophoretic tech-nique for the identification of these active compounds in plant extracts. Such a technique was advisable owing to inadequate results obtained by the use of chromatography. -3-The third study involved an attempt to associate the estrogenic actl vity of red clover with the compounds identified electrophoretically in the extracts at various stages of the growing period of the plant. LITERATURE REVIEW The Isoflavones The chemical class of the Isoflavones is a minor subdivision of the larger group of compounds referred to as flavonoids- The designation i "flavonoid" encompasses a large group of naturally occurring compounds which consist of two benzene rings joined by a propane bridge of form. C5-C-G-C-C6. Such compounds include: chalcones, aurones, flavanones, flavones and isoflavones, flavonals, flavanonols, anthoeyanidins and catechins. However, as the name indicates isoflavones are natural Isomers of the parent structure and are of the form C5-C-C-C5. Isoflavone compounds are synthesized in greater or lesser amounts by-most higher plants and on the basis of their distribution in the plant kingdom i t i s thought that they may serve some useful function in plant tissue, perhaps in some association with the plants* oxidation-reduction system ( 9 7 ) • No positive role can be assigned to such compounds in photo-synthetic processes other than that they are resulting endproducts. Fourteen isoflavones are known to occur in plant material. This number excludes glycosides which may be present in a number of forms for any one of the given compounds. Table I gives the common name, substitution and natural source of the known isoflavones ( 9 7 ) • It would appear that iso-flavones occur in a l l portions of the plant tissue — roots, rhizomes, bark, heartwood, sapwood, leaves, flowers, seeds, fruits and resin secre-tions. They are present as free entities or glycosides and the occurrence of several differing glycosides of one compound i n any plant is not un-common. An example of two forms of one compound which exist simultaneously would be genistin, the 7 glucoside of genistein, and sophoricoside, the Table I Natural Isoflavones and their Occurrence Parent compound Name Substitution Occurrence Daidzein Formononetin Pseudobaptigenin Maximin Genistein Prunetin Biochanin A (Olmelin) Orobol (Norsantal) Santal Tectorigenin Muningin Tlatlancuayin •Drigenin Pratensein Soyabean as 7-glucoside, daidzin Ononis spinosa as glucoside ononinj subterranean and red clover Baptisia tinctoria as 7-rhamnoglucoside pseudo-bap t i sin. Root of Tephrosia maxima Genista tincteria and soya-7,U*-(OB>2 7-OH-U*-OCH3 7-OH-3,,Ul-CH202 ?-0CR2CH=C (CH3):2-3 jii *GH202 5,T,1*i-(OH>3 5,U'-(OH);2-7-OCH3 5,7-(OH)2-U'-OCH3 ^ ^ S U ' - t O H ^ 5,3SliM0%-7-0CH3 $,T,li*-(OH)'3-c>.OCH3. 6,l+«-(OH>2-5,7-COCH3))2 5,2*-(0CE3}2-6,7-CH202 5,7,3l-(OH))3-6,U,,5,i-(OCH3>3Root of Iris florentina bean as 7 glucoside-genistinj Sophora japonica as h1-glucoside and kl rhamno-glucoside; Trifolium species Prunus bark as li" glucoside, prunitrin Clover, germinated grain of Cicer arietinum Orobus tuberosus as the glucoside, oroboside Santalwood, Barwood Rhizome of Iris tectorum Max as 7 glucoside tectorigin Heartwood of Pterocarpus  angolensis Iresine celosioides L. 5,7,x'-(0H);3,x'0CE3 Red clover (structure proposed) Taken froim Venkataraman, K» "Flavones and Isoflavones" p.56 (97). (Pratensein, 101) -6-k* glucoside of the same compound (100). The sugar residues of the iso-flavones are found most commonly at the 7 or k* position of the structure, glucose and rhamnose being of most common occurrence. The occurrence of isoflavones as the aglycone is more common than the corresponding form for flavones (97). Figure I gives the basic chemical structure for flavones and isoflavones as well as the individual structures for those compounds which will be considered in detail. The majority of the work in the field has been concerned with the study of pure isoflavones and to a lesser extent with the glycosides. It is unfortunate that removal of the sugar residue by mineral acid hydrolysis leaves no indication as to the site of attachment (100). The glycosides are usually water and alcohol soluble and ether insoluble whereas the aglycones are alcohol and ether soluble and unless highly hydroxylated show no water solubility. Methylation of the hydroxyl groups eliminates water solubility. The isoflavones and their glycosides are normally con-sidered as pigments and range from pale cream to rust in intensity. Those compounds which are isolated from red clover and considered i n this study were a l l pale cream or colorless. In 1910 Finneraore (£6) assigned the f i r s t isoflavone structure to a compound of natural origin. This compound was prunetin and had been solvent extracted from prunus bark. Since this time isoflavones have been isolated from a large variety of plant species. The parent compound and the mono-and penta- hydroxy isoflavones have not been shown to occur naturally. Three of the compounds are polyhydroxy and the remaining possess one or more ether groups. No naturally occurring isoflavone i s known in which the B; ring is unsubstituted. The ? position is always occupied, eight compounds having hydroxyl groups and three possessing methoxy groups at FIGURE I The Chemical Structure of Compounds Discussed in the Contexf Basic flavone structure OCH. Biochanin - A 5,7 Dihydroxy-4'methoxy isoflavone C I 6 H I 2 ° 5 Basic isoflavone structure OCH, Formonone t i n 7 Hydroxy-4'methoxy isoflavone C | 6 H | 2 0 4 ( C ^ I 2 ° 5 ) 0 Daid i in C 21 ^  20^ 9 7 Glucone of Daidzein Daidze in C | 5 H | 0 ^ 4 7,4' Dihydroxy isof lavone (C6H | 205) 0 Genistin c 2 l H 2 0 ° I O 7 Glucone of Genistein 0 ^ , 0 Coumestrol C I 5 H 8 ° 5 3,9 Dihydroxy-6H-benzofuro-[3,2-C] [l] benzopyran 6-one Genistein CJ5H1QO5 5,7,4* Trihydroxy isoflavorvje } OH OCH, Pratensein C,g H, 2 0g (proposed) 5,7,3' Trlhydroxy-4'methoxy isoflavone -8-this site (97). The recently isolated compound pratensein (101) has not been included in the above figures as i t s substitution in the B ring is not definite. This compound would appear to be a trihydroxy methoxyiso-flavone. The most significant characteristic regarding the determination of the chemical structure of isoflavones is their susceptibility to alkaline hydrolysis and the resulting products. Five per cent ethanolic potassiumi hydroxide and mild heat hydrolyzes an isoflavone to formic acid and desoxy-benzoin which can be recrystallized for accurate melting point confirmation. More severe conditions cause the breakdown of desoxybenzoin to phenol and phenylacetic acid. This pathway of breakdown is characteristic of a l l isoflavones and was f i r s t observed by Finnemore (56) and considered later by venkataraman (97). Substitution positions in the isoflavone series occur in the A ring at the 5$ 5,7 J or 5,6,7 positions and in the B ring at h'j 3t,Utj or 3l,hl»5>r. The ultraviolet absorption spectra of iso-flavones indicate maxima at wave lengths of 320 and 260 millimicrons. Table H. l i s t s the ultraviolet absorption spectra maxima'- for several naturally occurring isoflavones. The color reactions associated with the isoflavone complex will be discussed under means of identification of these compounds at a later point. The f i r s t synthesis of an isoflavone was carried out in 1925 by Baker and Robinson (6) who at this time synthesized 7' methoxyisoflavone, and later in 1928 synthesized genistein (7). It was not until 193k that the parent compound, isoflavone, was synthesized (6U). Ten known glycosides have been synthesized by reacting the isoflavone in question, for example, - 9 -Table II The Ultraviolet Absorption Spectra of some Naturally Occurring Isoflavones in Ethanol (97) Name ^max™1 log Ei Xmax m u log E Formononetin 300' 2^ 0 Genistein 325* 263 Biochanini A 325* 263 Prunetin 325* 263 Orobol - 28? Santal - 263 Tlatlancuayin. (in chloroform) 320' 3-86 21*5 278* U.312 « Inflection Taken from Venkataraman, K. "-Flavones and Isoflavones" p.58 (97). -10-genistein in acetone with aqueous potassium hydroxide and acetobromoglucose to form genistin (97). It has been previously mentioned that removal of the sugar residue i s facilitated by mineral acid hydrolysis. The inter-conversion of the isoflavones is also possible as demonstrated, by the removal of the kT methyl ether group of formononetin to produce daidzein (31)• Advances in chemical synthesis have reduced much of the ambiguity of certain compounds. An example of this is Bose's synthesis of formono-netin and its identification with biochanin B (29), and also the proof that pratensol was equivalent to biochanin A (97). The interrelations of many of these compounds is self evident from their structures, biocha-nin A being the U r methyl ether of genistein as formonometin is the k1 methyl ether of daidzein. Similarily prunetin is the 7 methyl ether of genistein (8). Other compounds in the series also demonstrate these close similarities and before an accumulation of literature developed many com-pounds bore different names on similar structures which led to some con-fusion. In many cases elimination of impurities i n what were thought to be new compounds resulted in their identification with those already known. A large volume of literature has accumulated on various methods for the synthesis of isoflavones (7,8,9,10,11,29,lib,59,97,100) . The possible means of the biogenesis of these compounds has been considered by Gelssman (57). He states that one must consider that a l l carbon is originally derived from' carbon dioxide and the relationships between amino acids and carbon moieties must be remembered. As very l i t t l e conclusive proof is available which would indicate definite paths of formation much is based on speculation regarding chemical pathways which would be mechanistically feasible. One possibility would be the rearrange--It-merit of a common precursor (not identified) into a number of differing f l a -vonoid moieties. Geissman has also suggested that isoflavones may be formed in the plant from a C^ -C-C-C fragment, the aromatic ring present forming the B ring. Although they are very similar in the positioning of hydroxyl and other substituting groups no biogenic evidence is available to support the transformation of a flavonone to a flavone or isoflavone. In 1959 Grisebach (60) using labelled D>L-phenylalanine found that the carbon skeleton of phenylalanine is incorporated into 7 hydroxy k* methoxyisoflavone i n red clover and a rearrangement involving either the migration of a phenyl or benzoyl group occurs in the biogenesis* This work outlines possible biogenic pathways for flavones, isoflavones and coumestrol in red clover. Additional information regarding the possibi-l i t i e s of the biogenic origin of isoflavones is considered by Geissman (58) and Seshadri (88). Isoflavones with additional fur an or pyran rings are relatively common in plants and form the basis of many large complex compounds in these systems. These forms have not been considered here but have been referred to in the review by Venkataraman (97)• The compounds which were specifically studied in this program are:: Biochanin A, 5,7 dihydroxy k1 methoxyisoflavonej Formononetin, 7 hydroxy U' methoxyisoflavonej Daidzein,, 7,Ur dihydroxyisoflavone, and Genistein, 5,7,l*r' trihydroxyisoflavone. These compounds have a l l been isolated from Red clover. A f i f t h isoflavone has recently been isolated from red. clover by Wong (101); and called Pratensein. It is present at very low levels and preliminary structural determinations would indie at ec'.that i t is 5,7,3" trihydroxy U* methoxy isoflavone. The individual character of the above -12-mentioned compounds will be given when identification is discussed. The Identification of Isoflavones; In general the isoflavones are thought to be pigments ranging in intensity from colorless to a soft rust color. The compounds considered in this study were a l l colorless or very pale yellow in nature* It has been mentioned that the determination of chemical structure can be readily facilitated by hydrolytic fission (alkaline hydrolysis} and by observation of the maxima in the ultraviolet absorption spectra (Table II)). A number of specific color reactions are also characteristic of the isoflavones* Briggs (3U) has shown that the presence of a free hydroxyl group at the 3,$ or 8 position is required for the characteristic rust color in alcoholic or aqueous ferric chloride. Pope et a l * (81*) has reported this color reac-tion for the compound Biochanin A and observed a red color when the iso-flavone was in excess i n methanolic ferric chloride and a green color when the ferric chloride was in excess. Addition of hydrochloric acid resulted in the green color while sodium hydroxide caused the red color* This would indicate that the color observed is dependent on the pH of the solution. Pope also observed varying shades of peach to rust when a solution of the compounds spotted on paper was sprayed with diazotized; p-amino phenyl-2-diethyl aminoethyl sulphone. The color reaction asso-ciated with a diazotized salt i s normally indicative of the presence of an aromatic structure* It has also been observed that certain isoflavones exhibit a weak visible fluorescence in concentrated, sulfuric acid* Iso-flavones are recognized as giving similar color reactions to flavones and thus reduction with sodium, amalgan followed by concentrated hydrochloric acid produces a pink color which is indicative of a flavonoid type com--13-pound (101). Walters (98) has stated that a change in color from red to purple when genistin i s placed in concentrated sulfuric acid indicates the presence of impurities in the sample. A considerable amount of work has been done involving the chromato-graphic separation and identification of these compounds. Bate-Smith et al. have studied the chromatographic characteristics' of formononetin using the solvent system- butanol-acetic acid-water as well as several others. Pope and Elcoate (82) carried out similar studies on genistein. In 19$k Pope and Wright (8U) reported the character of genistein,,. bio-chanin A, and formononetin in a number of different solvent systems with resulting Rf values. Because of variations resulting in Rf values depending on the specific conditions, no mention will be made of them. A number of the more common solvents used are: acetic acidi:. water (l s l ) , butanolracetic acidtwater (U:lr5>), acetonetwater (3:7), acetic acid: waterrhydrochloric acid (3:6:1), five per cent potassium carbonate, benzenetacetic aci&twater (2:2:1), and isopropanol:water (6cU). Guggolz, in 1961 (6l) outlined an elaborate repetitive chromatographic separation and identification of the above' mentioned compounds including daidzein from forages. The procedure involves the elution and rerunning of the compounds through several different solvent systems, the final run being completed on a s i l i c i c acid strip. The resulting Rfs for the compounds in the various solvent systems have been reported (.61). The determination of the position of the isoflavones on a chromato-gram is readily facilitated by the fluorescent character of the compounds in ultraviolet light. This fluorescence occurs at a wave length of 253 nnllimicrons and to a lesser extent at 366 millimicrons. At a wave length -1U-of 2^ 3 inilliiiiicrons forraononetin produces a fluorescent character (pale bright blue)1 as does daidzein. This fluorescence is markedly increased by exposure of the paper to ammonia fumes or in the presence of sodium hydroxide. Genistein and biochanin A can be observed as shadowed or dark spots due to the ultraviolet light absorbing character of these compounds. A number of developing sprays have also been used. These include diazotized salts, ferric chloride, magnesium and hydrochloric acid, and neutral or basic lead acetate (97)• A composite table of the common characteristics of identification has been prepared. Table III. Common Characteristics used in the Identification of Isoflavones ,Fluorescence Ferric Compound Melting Point; Crystals in U.¥. light Spray*, chloride Biochanin A 2l!i.0C. — dark orange rust/green Daidzein 325°C colorless blue cream -— Formononetin 262°G — blue creams Genistein 296 C White dark peach rust/green 6-sided rods * A diazotized spray In 1952 Hashimoto (62) reported briefly on the electromigration of flavonoids and sugars in a buffer solution of two per cent borax. Iden-tification of the spots was carried out by immersion in a five per cent etherial solution of lithium aluminum hydride followed by rapid drying and a dilute hydrochloric acid spray. The spots appeared red to dark green in color. Developments in the use of electrophoresis as an identi--15-f i c a t i o n technique l e d the author to i n v e s t i g a t e i t s p o s s i b i l i t i e s f o r the i s o f l a v o n e s e r i e s * The I s o l a t i o n of Isoflavones The i s o l a t i o n of prunetin by Finnemore i n 1910' (56) was the f i r s t i s o l a t i o n of an isoflavone from a n a t u r a l source. The compound was extracted fromi prunus bark w i t h uhot i n d u s t r i a l s p i r i t s " and p u r i f i e d by ether and ammonium> carbonate extractions followed by r e c r y s t a l l i z a t i o n from a l c o h o l . Other e a r l y extractions and studies were c a r r i e d out by Walz (99) • This procedure i s s t i l l the basis f o r the e x t r a c t i o n of Isoflavones and hormone-like compounds from p l a n t sources. Burnham et a l . i n 1930 (35) and Skarzynski i n 1933 (91) used t h i s basic procedure: f o r the analysis of estrogenic compounds from plant sources, and though no mention i s made to them, these compounds could w e l l have been i s o -flavones. The basic procedure involves the ex t r a c t i o n of the plant m a t e r i a l , i f dry with alcohol, i f wet with acetone. The extract i s then concentrated and the c h l o r o p h y l l and extraneous matter removed by benzene e x t r a c t i o n followed by e x t r a c t i o n of the estrogens with ether and/or a d i l u t e base. Various s l i g h t modifications have been developed and w i l l be mentioned below. In I9I4I Walters (98)1 o u t l i n e d a method f o r i s o l a t i n g g e n i s t i n from soybeans and obtained a 0.1 per cent y i e l d . This method w i l l be discussed i n d e t a i l at a l a t e r point as i t was used as an i s o l a t i o n technique i n t h i s study. Robinson (86) c a r r i e d out extractions on the d i f f e r e n t s t r u c t u r a l portions of the plants and reported that the estrogen-like compounds -16-(possibly isoflavones) appeared in greatest intensities in the leaves followed by the roots and petioles. Bradbury and White (31) in isolating, genistein and formononetin from subterranean clover did not extract the plant material in the usual manner but rather expressed the juice from the plants, heated i t and on cooling collected a residue which separated out and was termed the "chloroplast" fraction. The dried residue was then used for solvent extraction. This method was used later by Beck (13)* Pope and Elcoate (82)) using the basic solvent extractions isolated bio-chanin A from red clover. Purification in this case was carried out by fractionation in a Celite column. Bate-Smith et a l . (12) also used this method to isolate formononetin from red clover. Many extractions have been accelerated by using a Waring blender instead of extended1 refluxing and result in very similar yields. Cheng using naturally isolated com-pounds reported that synthetics exerted the same estrogenic activity as did those isolated from the plant source (1*3) * Curnow (U?) and Pope (81t) have both reported on the isolation of various isoflavones from red and subterranean clover. Virtanen( 97a) isolated formononetin readily by the ether soluble fraction being further extracted with petroleum' ether and sublimed the undissolved residue i n vacuo at 260°to 280°C. Thompson (96) observed a one hundred times concentration of the estrogenic components of plants by carrying out a preliminary extraction with dilute alkali, acidifying the aqueous extract and re-extracting the now acid fraction with ether. Extraction procedures used by Kitts et a l . (65) do not in-volve the use of dilute alkali. Guggolz in 196l (6l) outlined a long chromatographic procedure for the identification and isolation of genistein, daidzein, formononetin and biochanin A from red clover using an additional preliminary extraction with Shellysolve to remove fats and extraneous -17-matter. Rehydration of dried clover samples before extraction was reported by Livingston in 1961 (71)• There.would appear to be some controversy as to whether dried samples possess similar or slightly lower levels of estro-gen-like compounds than do fresh extractions (22)'. This loss is attributed to failing respiration during drying, however, in many cases the decrease in potency is negligible. Goumestrol The compound coumestrol is a recently observed factor which appears to be present in certain varieties of alfalfa, ladino and strawberry clover and possibly other forage plants. A discussion of the compound has been separated from those mentioned previously as i t is not of an isoflavone nature. However, as this compound bears some structural similarity , and a definite similarity in biological action i t was considered i n the basic research program. The structure of the compound coumestrol is given in Figure I. In 19$7 Bickoff et al. (17) reported the identification and subsequent isolation of a crystalline substance from alfalfa which possessed an estro-gen-like activity similar to, though more intense than, the reported activity for known isoflavones. The compound was solvent extracted from the alfalfa meal, purified by countercurrent distribution and recrystallization from methanol. The isolate was shown to be. a coumarin derivative and was called, coumestrol. Goumestrol exhibited a brilliant blue fluorescence in acid or neutral solution, a green-yellow fluorescence in strong alkali, and melted with slight decomposition at 385°C. The methanolic ultraviolet spectra showed maxima at wave lengths of 208 , 2^ 3 and 3U3 millimicrons and the emperical formula was determined to be Ci£%0-£. Additional confirmation - 1 8 -of the compound's character was obtained later in 1957• This original isolation of the compound from a natural source was rapidly followed by a chemical synthesis and the assignment of the systematic name, 3 , 9 Dl-hydroxy-6E-benzofuro(3,2-C) (1) benzopyran 6 one (19,52)). Subsequent studies have demonstrated the presence of coumestrol i n other forage crops (18,, 2 1 , ?1)) and also the fact that in alfalfa particu-larily, the concentration of the active compound appears to increase and/ or remain relatively high throughout the growing season (22,23,25)'. Some controversy has developed regarding this point and other plant estrogen-like compounds. Goumestrol may be isolated from both fresh and dried' forages, the biological activity being higher in the fresh clover, the decrease probably resulting from decomposition on drying (22)). As the extraction procedure for wet, fresh forage is similar to that outlined for the isoflavones only the dry extraction will be briefly considered. The dried ground forage was in i t i a l l y extracted with water and Skellysolve C before ether extraction for removal of the estrogen-like substance* These i n i t i a l extractions plus the use of ether as the preferred solvent eliminated many impurities and facilitated subsequent purification using five per cent sodium carbonate, countercurrent distribution and recrystallization from methanol* Bickoff et al. (18)' has reported details regarding the countercurrent solvents and' the purification technique. The identification of coumestrol was readily facilitated when i t was realized that the brilliant fluorescence, visible on chromatographic papers, was associated with the compound. This fact associated with advancing s i l i c i c acid chromatographic techniques led to the development by Bailey (5) of an apparatus which measured the fluorometric intensity quantitatively -19-of the compound on chromatography strips. Livingston et al. (JO) proved this apparatus accurate for the quantitative range necessary and demonstrated that storage of the papers led to a decrease in the fluorescent character of the compound over a one hundred day period. The solvent used in the chro-matographic studies was acetic acid and water ( l r l ) which was found to give an Rf value of approximately 0.5. In i960 Bickoff et a l . (2U) carried out rather extensive studies regarding the effect of altering the coumestrol molecule and the relation-ship between various molecular characteristics and i t s biological activity. Acetylation of both hydroxyl groups did not significantly reduce the estro-genic activity nor did opening of the lactone ring. Decreases in activity were noted when the compound was esterified, the furan ring was opened, one hydroxyl group was removed, and when additional hydroxyl groups were placed on the molecule. Removal of both hydroxyl groups completely eliminated the estrogenic activity. Coumestrol would appear to be thirty times as estrogenically active as genistein. Bickoff et a l . (2U) has stated that the lower activity of the isoflavones may result from the fact that the oxygen at position four i s ketonic and results in a single bond i n the 3,U position while coumestrol possesses a double bond at this site. The Biological Significance The Effect of Estrogens on the Female Reproductive Tract The estrogens of the mammalian body are, and (3 estradiol, estriol and estrone. Hereafter these compounds will be referred to inclusively in the term "estrogens"'. The estrogens control and regulate the female sexual behavior and the development of primary and secondary sex characteristics ( 2 ) . The major source of estrogens in the female is the overy,, particularily the i -20-Graafian f o l l i c l e . A secondary source, of l i t t l e significance in the normal animal is the adrenals. Estrogens are specifically involved i n the control of the female sexual cycle, changes in which can he' observed externally % variations i n the uterine and vaginal cytology of most small laboratory animals. The following events may be observed during the sexual cycle of the normal mature intact animal and also following the injection of estrogen into an immature or ovariectomized mature animal* Reference is made primarily to laboratory rats- and mice in which the injection of estrogens cause the typical changes in the endometrial cytology of the uterus encountered! in the normal intact animal. Estrogens have been found to cause increased vascularity and accelerated mitotic activity in the uterine tissue, resulting in an increased weight of this organ. In castrate mice and rats the injection of estrogens also results in the accumulation of water i n the uterine lumen which contributes to the increased weight. In other species the uterine interstitium becomes very edematous (77) • It has also been shown that the increase i n uterine weight is pro-portional to the amount of estrogen administered. These conditions occur in the normal animal at "estrus1*, identified by the presence of cornified cells and some leucocytes in prepared vaginal smears. In the immature or castrate animal such conditions may be observed1 following administration of an estrogen or estrogen-like compound. Injection of estrogenic compounds into an immature rodent results in an immediate increase in the uterine water content followed by a later increase in the dry weight. The water uptake appears to be related to vasodilation and changes i n permeability of the uterine vessels,, however, ' the water increase appears to be largely of an extracellular nature* The -21-increase in dry weight at the later phase is associated with some protein synthesis ( 7 2 ) . Astwood ( 3 ) developed a six hour bioassay for the deter-mination of estrogenic compounds based on the accumulation of water in the mucosal stroma and the resulting increase in organ weight. He also found that the water increase in noncastrates was maximal when injection occurred during proestrus and minimal in the early stages of diestrus (1;). Later work states that the rapid influx of water within six hours increases the intra and extracellular moisture from 7 9 * 6 to 8 5 * 3 per cent. This high level gradually decreases to 8 2 per cent as cell division occurs ( 3 7 ) ; , The endometrium and musculature appeared to be similarily affected and following a single injection normal water levels return approximately 5 © ' hours post-injection. Prior to the work of Astwood, Allen and Doisy ( 1 ) had developed a technique which gave relatively conclusive results and allowed the analysis of a compound to determine its estrogenicity non-quantitatively. The criterion of estrogenicity i s satisfied when injection of the compound into a mature ovariectomized rat results in vaginal corni-fication similar to that observed during the "estrus"1 phase of the sexual cycle, Ostrovsky ( 7 8 ) has shown that the plant estrogens possess a similar contact character to the natural estrogens* This means that both plant and natural estrogens are contact hormones and that surface appli-cation will result in similar effects as caused by the same compounds being transported via the blood stream to the contact organ. Plant Estrogens Although estrogen-like compounds had previously been observed i n plant material by Loewe in 1 9 2 6 C ? 3 ) l i t t l e significance was attached to their presence un t i l 1 9 ^ when extensive reproductive failures were observed) i n -22-some Australian sheep flocks (14). Such disorders as dystocia and uterine prolapse as were reported were of such significance that extensive research was carried out to ascertain the causal agent. The flocks primarily affected had been grazed on pastures consisting predominantly of a locally developed, strain of subterranean clover var. dwalganup 0S)» Transfer of the affected ewes to other forages did not restore the lost f e r t i l i t y (100). Investi-gation of the forage led to the conclusion that the isoflavone constitutients, particularily genistein were causing the adverse effects* Extracts of willow and alder were among the fi r s t plant materials found to demonstrate an estrogenic effect. At this time workers found that the greatest activity appeared to coincide with the rapid spring growth period and following this stage the green parts of the plant demonstrated l i t t l e or no activity (35). Studies of subterranean clover have shown that the leaves possess the highest concentrations of estrogenic substances followed by the roots and petioles (86). It has been noted that application of estro-gens to plants appears to stimulate the vegetative growth (6?). As the plant estrogens are of highest concentration in the leaves (probable site of production) in the early spring, the possibility of them being growth stimulants in the plant cannot be disregarded. Skarzynski i n 1933 (91) demonstrated the presence in willow of an estrogenic compound, the crystal-line character and melting point of which was identical to estriol but possessing a much greater estrogenic activity. At this same time true estrogens (estrone and estriol) were isolated from plant sources (36). In 19ljl the mouse uterine weight assay was elaborated by Evans (5U) and with minor modifications is the assay s t i l l employed. Immature female mice weighing between six and eight grams were injected twice daily with -23-the compound of question for 3 days. Eighteen hours following the last injection the mice were sacrificed and the uteri dissected and weighed. The increase in weight in excess of the control, i f any, is proportional to the estrogenic potency of the compound. Present day techniques usually use mice heavier (up to eleven grams) and the unknown compound is fed in a non-estrogenic basal diet at various levels. Sacrifice of the animals occurs from six to twelve hours after removal of or last consumption of the experimental feed. Injection procedures are s t i l l used, the unknown compound being suspended i n such media as aqueous egg albumin (26). and peanut o i l (38, 95) • The guinea pig has been found to be impractical as an experimental animal because of i t s susceptibility to uterine cysts (U6). However this anmal is s t i l l used i n some cases as its digestive tract approximates that of the grazing animals* Drasher (U8) has observed strain differences i n the response of mice to estrogens and consequently work in several laboratories using similar assay procedures cannot really be compared. The use of the mouse uterine assay procedure feeding the dry clover as an additive, i.e. not extracting the substances, can be erroneous in that feeding high levels of dried clover tends to lower the feed intake, results in lower body weights and subsequently inaccurate percentages of uterine weight to body weight. Other assay procedures have been based on the time interval required for vaginal opening in immature animals (53,55)). Opening of the vaginal tract is indicative of reproductive maturity and is hastened by the presence of estrogens (87). Carter (39) observed vaginal opening six days following the addition of genistein to the diets of immature female mice. This worker also reported that inclusion of genistein in the diet resulted -2k-i a decreases in the number of young born, the percentage of females dropping litters, the average number of young per l i t t e r , and the average weight of the lit t e r s . Such results are a l l indicative of reproductive interruptions. Genistin fed to male mice resulted in a linear decrease in growth with increasing levels of the compound and at the higher levels caused a loss of weight. Similarily, groups fed at the higher levels demonstrated greater than normal death rates. Increases in the level of genistin fed resulted in linear decreasing testicular weights in which workers were unable to observe spermatozoa (75) • East (50) working with guinea pigs fed subterranean clover observed extended estrus periods and up to 70 per cent failures in conception as well as epithelial changes in the accessory reproductive organs. In this case withdrawal of the clover resulted in immediate recovery. Using mice this same worker reported, sterility or imparement of reproductive function in males and a lack of mating in females. When matings occurred high percentages of stillbirths, resulted. Withdrawal i n this case resulted in recovery of only some of the animals (50). Injection of androgen into male castrate sheep appears to provide some protection against the adverse affects of ingested sub-terranean clover (1»9). Pope (83,8j?) has reviewed the importance of pasture plant estrogens in the reproduction and lactation of grazing animals. Suomalainen (95) noted increases in the non-fat solids in milk,, produced by animals on spring pasture, which he felt resulted from stimu-lation by estrogenic compounds i n the forage. It appears that certain silages possess an even greater estrogenic activity than the fresh plant material (81). This along with the fact that moldy corn possesses estrogenic activity (80) would suggest that microbiological fermentation increases -25-concentration of, or transforms to a more active form, the plant estrogen-like compounds. Seasonal variations in the concentrations of such com-pounds in certain legumes and grasses as well as the effects of harvesting frequencies have been studied by Kitts et a l . i n 1959 (65,66). The estrogenicity of a wide variety of American forages, from varying geo-graphical locations have been determined by Bickoff et al.(2l), as well as a number of associated studies regarding isoflavones, coumestrol and their estrogenic significance. A review of the biologically active compounds by Kohler (67) states that the estrogenic potency of red clover results primarily from the presence of the compounds genistein and formononetin, and that of subter-ranean clover from the compounds genistein and biochanin A. Using the chloroplast isolation developed by Bradbury and White (31), Gurncw (kl} isolated two mgm of genistein per 100 grams of fresh clover and twelve mgnt per 100 grams of dry clover. Cheng et a l . (ijl) have stated that one pound of mixed clover is equivalent to two micrograms of diethylstilbestrol. Both Cheng (U3) and Biggers (2?) have found that the synthetically pre-pared isoflavones possess similar estrogenic activities to those which have been naturally isolated. Increases i n uterine weights have been obtained with genistein fed at a two and one-half mgm level and based on extrapolation of this value genistein is said to be 1/50,000 as estrogen-ically potent as diethylstilbestrol (.£&)• The order of estrogenic intensity for the isoflavones has been stated to bet daidzein (greatest), genistein equal to biochanin A, and formononetin (least), when fed at the two and one-half mgm level (U3). Contrary to the work of Cheng,, Bradbury and White (32,33) have reported that genistein exerts an estrogenic response at the one mgm level. Several values for the estrogenic potency of bio--26-chanin A have been reported (28,30). Comparisons of genistein and diethylstilbestrol show that dose response curves of the two give similar slopes between the range of three to six mgm for genistein and 0.015 to 0.03 ugm for diethylstilbestrol. The activity of genistein can be increased four fold by heating with one and one-half per cent potassium hydroxide (20). This increase probably results from opening of the heterocyclic ring in the molecule and results in a more favorable orientation of the hydroxyl groups. The compound coumestrol has been found to be thirty times as estrogenically active as genistein and the other isoflavones (2b). The results obtained i n this research program may clarify the rela-tive estrogenic intensities of these compounds particularily those observed! at higher levels of intake than have been previously studied. Estrogenic Activity and Structure Improved synthesis techniques and a greater chemical understanding of such compounds, with particular reference to synthetics (for example diethylstilbestrol) has led to some attempts of interpretating the rela-tionship between chemical structure and estrogenic activity. low values of estrogenic potency is a relatively common characteristic for a large number of simple phenols and phenolic stilbenes (92). It was originally thought that estrogenic activity depended on a structural similarity between the tested! compound and natural estrogens. This i s i n part true, however the primary requirement is the presence and spatial arrangement of two free hydroxyl groups (92). The studies of Bickoff et a l . regarding the compound coumestrol and its activity to structure relationship have been previously discussed in this review (2b). -27-Spatial manipulation of the isoflavone molecule results in an arrange-ment similar to that of the natural estrogens* Bradbury and White (32) have demonstrated that the presence of a 2 alkyl substituent reduces the activity of the isoflavone probably resulting from distortion of the coplanarity of the 3 phenyl ring with the chromone ring. Proof of this distortion is indicated by the ultraviolet absorption shift when a 2 alkyl group is added. Isoflavinol has been shown to possess a relatively high activity which drops markedly after introduction of a methoxyl group (68). Warburton (100) states that the presence of a J>. hydroxyl group is required before significant activity will be observed. It i s possible that certain compounds undergo hydroxylation or some other conversion in the organism from an inactive to an active form'. Such compounds are referred to as "pro-estrogens". Non-estrogenic Effects of the Isoflavones Several additional biological effects have been associated' with a few of the isoflavone compounds considered here. Formononetin isolated from red clover has been found to possess certain, antifungal properties, particularily against the strain Sclerotinia trifoliorum (9lfa).This information may lead, to some further understanding of the role of such compounds in the plant system. Japanese workers have found that daidzein, found in many Japanese and Chinese crude drugs possesses an anti-spasmodic effect but does not possess an atropine-like character (89,90). A possible relationship may exist between such an effect and the uterine musculature producing the observed increase in size and weight. While studying the effect of genistin on growth i t was found that moderate levels of the compound caused increases in the weight of the -28-adrenal gland while high levels caused a decrease {hQ>,l&)}» Because of the close association between the ovary and the adrenal (a secondary source of estrogens) this effect may be considered, as resulting from stimulation and finally replacement of the organ as source of estrogen. Very l i t t l e work has been carried out on the isoflavones other than that associated with pure chemistry and the estrogen-like character of such compounds. Other studies are just beginning to take form. MATERIALS AMD METHODS' I. The Experimental Compounds A. Genistein (5>,7,Ur trihydroxyisoflavone) The method used f o r the i s o l a t i o n of g e n i s t e i n was that o u t l i n e d by-Walters i n 19^1 (98) with s l i g h t m o d i f i c a t i o n . Three kilograms of com-mercial, solvent extracted soybean meal were placed i n a cheese c l o t h sack w i t h i n a s i x l i t e r round bottom f l a s k to which was added f i v e l i t e r s of methanol. The mixture was r e f l u x e d at the lowest p o s s i b l e temperature f o r twelve hours, the methanol decanted and an a d d i t i o n a l four l i t e r s of methanol added, a f t e r which r e f l u x i n g was continued f o r an a d d i t i o n a l twelve hours. The combined extracts were concentrated to approximately 300 m i l l i l i t e r s i n a f l a s h evaporator. Approximately two l i t e r s of hot acetone were added to the concentrate u n t i l the r e s u l t i n g p r e c i p i t a t i o n of acetone i n s o l u b l e m aterial ceased. The acetone p r e c i p i t a t i o n removed many impurities such as saponins and carbohydrates. The r e s u l t i n g acetone s o l u t i o n was then f i l t e r e d . The c l e a r acetone s o l u t i o n or f i l t r a t e was then concentrated to a thin, syrup, a f i n a l volume of approximately I4O1 m i l l i l i t e r s . Two volumes of water added t o the concentrate r e s u l t e d i n the p r e c i p i t a t i o n of g e n i s t i n . The small amount of o i l that had accumulated on the surface of the l i q u i d was removed with hexane. The crude p r e c i p i t a t e of g e n i s t i n , the glucone of ge n i s t e i n , was c o l l e c t e d by c e n t r i f u g a t i o n , dissolved i n hot 80 per cent ethanol, treated w i t h charcoal, and f i l t e r e d . The pure compound was c r y s t a l l i z e d at room temperature, and r e c r y s t a l l i z e d three times or u n t i l no purple c o l o r was obtained when seve r a l c r y s t a l s were p l a c e d i n con--30-centrated sulfuric acid, as described by Walters (98). The final crystals were air dried and found to have a melting point of 255>°C, similar to that reported for genistin by Walters (98). Four grams of genistin were placed in a round bottom flask with 90) milliliters of methanol and 20- milliliters of concentrated, hydrochloric acid. The mixture was refluxed for six hours or for one hour after a l l solid material had disappeared from the solution. The solution was cooled and one volume of water added. Upon sitting at room temperature overnight genistein precipitated. This precipitate was collected by ether extraction in a separatory funnel and. subsequent evaporation of the solvent. The crude genistein precipitate was recrystallized from; 6© per cent ethanol three times to ensure purity and the final product air dried. The crystals appeared to be white six-sided rods as reported by Walters (98). The determined melting point using a Kofler Micro Hot Stage was. found to be 287°G. The isolated genistein was found to contain a slight impurity of daidzein, also present in soybeans. Using this procedure a number of extractions totaling seventeen pounds of soybean meal yielded two grams of relatively pure genistein. The genistein used for the biological assays (500; milligrams)) was continually recrystallized from 60 per cent ethanol until no daidzein could be detected. At this time the melting point was found to be 292°C. (Reported 296°C) (98);. The crude and purified products were retained for future use. B. Biochanin A. (5,7 dihydroxy hf methoxyisoflavone) Two grams of synthetically prepared biochanin A were obtained from Br. Venkataraman of the National Chemical Laboratory at Poona, India. -31-The sample used for the bioassay was recrystallized twice from 80^  per cent ethanol at which time a melting point of 212°G was observed* (Reported Zlh°G)) (82). Nb impurities could be detected in the sample* G. Formononetin (7 hydroxy k* methoxyisoflavone) Formononetin was obtained from the same source as Biochanin A. The bioassay sample was recrystallized and the melting point determined to be 263°G. (Reported. 262°C.) (31). No impurities were detected in the sample. D. Daidzein (7,b* dihydroxyisoflavone) Daidzein was obtained from formononetin by means of a chemical con-version outlined by Bradbury and White i n 1951 (31). One gram of formo-nonetin was placed in a small round bottom flask with 2Q> milliliters of hydriodic acid (d. 1.7) and the mixture refluxed for two hours. Extreme care was used to avoid bumping. The mixture was cooled and poured into 100 milliliters of water. Sulfur dioxide gas was then passed through the solution to decolorize i t by removing residual iodine. This reaction involves the removal of the methyl group from, the h l position of formono-netin and i t s replacement with a hydroxyl group to yield daidzein. Approximately 1+00 milligrams of daidzein were obtained, however i t was contaminated with formononetin. The reaction was repeated and the resulting product showed no formononetin after crystallization from 80' per cent ethanol. The crystals formed were colorless needles melting at 321°G. (Reported 325°C.) (31). By this method 270) milligrams of daidzein was obtained and subsequently used for the biological assay* -32-E. Coumestrol (3,9 dihydroxy-6B-benzofuro (3>2-G')}(l) benzopyran 6 one) A sample of coumestrol (.as coumestrol acetate) was obtained from Dr. E. M. Bickoff of the Western Regional laboratory in California. The sample was recrystallized once from a mixture of 80) per cent ethanol and> acetone after which no impurities could be observed. The use of the synthetic isoflavones and coumestrol i n this program is justified because there exists no difference in the estrogenic activity of natural versus synthetic compounds (1*3). II. The Bioassay Procedure The biological assay used to estimate the estrogenic activity of the compounds considered was that outlined by Kitts et a l . i n 1959 (65). In this assay the compound in question is fed at a predetermined level as an additive to a non-estrogenic control ration (Table IV)1. The experimental Table Hf The Composition of the Control Ration (G-56) Rolled oats 5-2.50 pounds Ground wheat 26.25 pounds Fishmeal (70$) 8.7/5 pounds, Meat Scraps 3.75 pounds Skimi Milk powder T.501 pounds-Steamed bone meal 1.00' pounds Iodized salt 0'. 25 pounds 10Q..00' diet (control plus compound, or extract)' is then fed to immature female Swiss albino mice weighing between 8 and 11 grams and 20! to 21 days of -33-age. The ration is fed at a level of 1.6? grams per mouse per day for a three day period giving a total consumption of five grams of feed: per mouse. At the end of the 1(2 hour period the feed i s removed and any re-maining weighed. Eight hours following the removal of the experimental diet the mice are sacrificed, the body weights recorded, the uteri dis-sected, blotted gently between two pieces of f i l t e r paper and finally weighed on a precision balance. The presence of estrogen-like stimulation is indicated by an increase in the weight of the uteri and consequently represents a higher percentage of the body weight* Several slight modifications of this procedure were carried out. The range i n i n i t i a l body weights of the female mice was restricted to 8.5 to 10 grams to increase uniformity. It was found that at the end of 72 hours, and very often sometime prior to this a l l feed had been consumed and animals housed in groups of six often exhibited cannibalistic tendencies!, particularily during the eight hour fasting period. To alleviate this: situation the feed consumption per mouse was increased to 5*5 grams for the three day period and the fasting time reduced from eight to six hours. Such steps eliminate the occurrence of cannibalism and produced similar results to those obtained with less feed and the longer fast period. Thus the groups of experimental animals remained complete and a certain degree of uniformity retained. IH. The Preparation of Diets Containing the Pure Isoflavones Stock solutions of the pure compounds to be added, at varying amounts to the control ration were prepared as follows.. a. Biochanin A, Formononetin and Genistein Five hundred milligrams of the individual compounds, were dissolved -3U-in a mixture of 801 per cent ethanol and acetone (3:1). Because of the expansive sensitivity to temperature changes of the solutions they were not brought to exact volumes until use and were held at a contant tem-perature throughout measurement and transfer to the diet. b» Daidzein Because of the limited amount of the compound available a lesser volume of this solution was prepared. The same solvent was employed. Two hundred f i f t y milligrams- were dissolved i n 125 milliliters of solvent and 20 milligrams in 10'; milliliters for a total of 135 milliliters con-taining 27fO'' milligrams of compound'. The concentrations of the stock solutions was thus two milligrams per m i l l i l i t e r or 2000' micrograms per m i l l i l i t e r , c. Coumestrol Due to the low solubility of this compound i t was necessary to us& 1000 milliliters of the solvent to dissolve 60J.3 milligrams of coumestrol acetate (equivalent to 500 milligrams' of coumestrol). This solution therefore contained only £ 0 0 micrograms of coumestrol per m i l l i l i t e r * Preparation of the experimental diets and the concentration of the tested compounds ranging from' 0.5 to l5.Q; milligrams per mouse per three day period are summarized in Table V. The volume of the stock solution reported was added to 30' grams of the experimental diet. In cases where the amount of stock solution to be added to the diet was less than 10 milliliters additional solvent was added to bring the volume to between 10) and 20' milliliters which ensured adequate dispersion of the compound in the ration. The solvent was removed by evaporation and the dry ration brought to 33 grams with control diet. The rations, were well mixed and stored i n a cool dark place until used. Table V, Values Used f o r the Preparation of the Experimental D i e t s . mcgm mgmi mgmi ml stock comp rd mcgm comp Bd comprd s o l n n / mcgm consumed. comp"d consumed consumed r a t i o n comp "ds, comp*d at 1 . 6 ? consumed / 3 days /3 days at 2000; prepared gm/day /3 days /mouse /6 mice mcgm/ml* CD) 100 1 6 7 . 0 0 5 0 1 . 0 0 ' O . 5 0 1 3.006 1 . 5 0 3 B,C,,D,,Gi 150' 2 5 0 . 5 0 751 . 5 0 0).?5l k . 5 0 9 2.251* B,,C,D„G: 200 33ii.OO 1 , 0 0 2 . 0 0 1 . 0 0 2 6.012 3.006 B , . C , D , , G 250 l a 7 . 5 0 ) 1 , 2 5 2 . 5 0 ) 1 .252' 7 .515 3 .?57 B , C , D , G , „ 500 &35.0O 2 , 5 0 5 . 0 0 ' 2 . 5 0 5 15.030 7 .515 B , C , D„G 750 1 , 2 5 2 . 5 0 3 , 757.50 3.757 22.51+5 11 . 2 7 2 B , C , D , G 1 , 0 0 0 1 , 6 7 0 . 0 0 5 , 0 1 0 . 0 0 5.01O 30.060 15.030. B , C , D , G 1 , 2 5 0 2 , 0 8 7 . 5 0 6 , 2 6 2 . 5 0 6 . 2 6 2 3? . 5 ? 5 18.78? B , C , & i,5oo 2 , 5 0 5 . 0 0 * 7 , 5 1 5 . 0 0 7 .515 45.090 2 2 . 5 4 5 B „ C , D , ; G : 1 ,750' 2,922.50 8,7/67.50 a.76? 5 2 . 6 0 5 26.302 B , G , G 2 , 0 0 0 3,3&>.00 1 0 , 0 2 0 . 0 0 6 0 . 1 2 0 30'. 060= B , C , D , G 2 , 2 5 0 3 , 7 5 7 . 5 0 11,27/2.50 1 1 . 2 7 2 6 ? . 6 3 5 33.817 F 2 , 5 0 0 U,175.00 1 2 , 5 2 5 . 0 0 1 2 . 5 2 5 7&.150 3 7 . 5 7 5 B , C , D , F , ( 2 , 7 5 0 k,592.50 13,771.50 13.777 82 .665 lia. 332 F 3 ,000 5,010.00^ 15,030.00 15.030^  90.180 45.Q9Q' B * C , F , G * As the stock s o l u t i o n of coumestrol contained' only 500 mcgm/ml i t i s necessary to m u l t i p l y the reported values f o r t h i s compound times four. (1) B-biochanin A, C-coumestrol,, D^daidzein, F-formononetin, GVgenistein. NiB. Omission of seve r a l l e v e l s of daidzein xiras necessary because of the l i m i t e d amount a v a i l a b l e . Two a d d i t i o n a l l e v e l s of formononetin were added to substantiate the graph at intermediate p o i n t s . -36-The Preparation of Rations Containing Two or More Isoflavones Due to the insufficient quantities of daidzein i t was necessary to exclude this compound from the study of the effect of two or more of the Isoflavones fed simultaneously. The following combinations were added to the control diet as before, each at a level of 2,500- mcgm per gram of ration or 37Z.57 milliliters of each of the stock solutions. This high level was chosen because a l l compounds at this point had displayed some evidence of an estrogen-like activity. a. Genistein (2500' mcgm/gm) plus; Biochanin A (2500' mcgm/gm) b. Biochanin A (2500 mcgm/gm) plus Formononetin (2500 mcgm/gm) c. Formononetin (2500' mcgm/gm) plus Genistein (2500 mcgm/gm) d. Biochanin A (2500 mcgm/gm) plus Formononetin (2500 mcgm/gm) plus G§nistein (2500 mcgm/gm) All bioassays involved groups of six immature female mice. The animals were housed in circular aluminum pans and water provided ad libitum. The experimental diet was fed in three equal lots once daily for three days. V. The Preparation of Red Clover Extracts for Bioassay Throughout the 1961 growing season a plot consisting- of seven rows of red clover in the second year of growth was maintained in the Agronomy fields of the Division of Plant Science at the University of British Columbia. The plot was irrigated regularity but did not receive any fertilizer during the experimental period. The rows were 20s feet long and a foot andi a half apart. Each row contained approximately UOJ plants. At monthly intervals a row was harvested with hand shears and a represen-tative sample of the fresh forage taken. A small sample of the fresh -37-clover was removed and a dry weight determination carried out. The sample size listed in Table VI was immediately chopped into short lengths and macerated for three minutes with a total of five liters of acetone in a Waring Blender. It was found that this step i s best carried out in three lots each utilizing 1.5> liters of acetone and a portion of the red clover sample. The progressively decreasing sample size takes into account the increase i n dry weight of the plant. The acetone solution was removed by suction filtration and the f i l t e r pack discarded. The extract was then concentrated under vacuum! to approximately 200 to 2^ 3' mil l i l i t e r s . The concentrated solution was transferred to a separatory funnel and repeatedly extracted with 200 milliliters portions of benzene. Table VI. Harvesting Bates and Sample Size of Red Clover Sample size Date Row number (wet weight) May 10, 1961 $ 1,000) grams June 12, 1961 2 £00 grams July 11, 1961 1 li00 grams August 9, 1961 3 3^ 0 grams September 11, 1961 k 300 grams This step removed the chlorophyll fraction and other extraneous material leaving a clear brown solution. The benzene layer has been shown to contain no estrogenic material and consequently may be disgarded (19} • The benzene extracted solution was further concentrated to a volume of approximately 5>0 milliliters and stored i n the refrigerator prior to use. -38-Based on the percentage dry weight of the fresh sample the concen-trated extract was added, to the control diet such that one gram, of feed contains the extract of one gram of the dried clover. The diet was dried, mixed, brought to 33 grams and stored in- a cool dark place prior to use. The remainder of the concentrated extract was retained for studies invol-ving the electrophoretic identification of isoflavones in plant extracts. VX. The Electrophoresis Technique Advances in the use of electrophoresis as a means of identification led the author to investigate the possibilities of such ia the isoflavone series. A. The Power Supply and Electrophoresis Cabinet The power supply used is a commercially produced unit obtained from the Research Specialties limited Company of Richmond;, California, model 1911, BElectrophoresis Power Supply"1. The unit is operated from, a 115 volt A.C. outlet and has an output range of OJ to 750- volts D'.C. with 0 to 200 milliamperes. The electrophoresis cabinet i s constructed entirely of l/k an(i 3/l6 inch plexiglass. The water cooled bed (13 x 1& inches)) is quarter-roundied at the ends such that the paper drapes into a multicompartment buffer trough (13 x 3 l/h x 2 1/2 inches)' in which rests a l/k inch carbon plate electrode. The design of the cabinet i s given in Figure II. The outer protective cover is fitted onto the base at a limit switch connection such that removal of the cover immediately disconnects the power flow irregardless of the state in the power source. B . The Electrophoresis Paper Several weights of paper were considered in this study in an attempt FIGURE II The Electrophoresis Cabinet Buffer Trough Water cooled bed c Water inlet Removable cover Buffer Trough 7 Water outlet ] Base Horizontal view E lectr ica l outlet to power supply © Limit switch o o c i - -1" v Water cooled bed .3-\ \ \ \ \ v \ V y 0 0 © Vert ica l view Scale I inch: 4 inches 4$u to determine which gave optimum compound movement,, wet strength, ease of handling and minimal resistance. The size of sheet used was 9 x 21 inches, the weights considered, Whatman Number 1, 3 and. k* C. The Buffer Systems A number of buffers were prepared and preliminary experiments were designed to determine which produced the optimum movement and separation of the isoflavone compounds. Two liters of each of the following solu-tions were prepared* a. Acetate buffer (U5) 8.8' ml of 0.2M acetic acid plus 1+1.2 ml of 0.2M sodium acetate diluted to 100) ml. Final pH 5.3. b. Tris (hydroxymethyl)aminomethane raaleate (Tris-maleate) buffer (1+5) 50 ml of 0V2M Tris acidi maleate (21..2 gm Tris plus 23.2 gm maleic acid per 1000 ml) plus Sh ml of 0.214 sodiumi hydroxide diluted to 200> ml. Final pH iJh. c. Ammonium' carbonate 15.? gm per l i t e r . Final pH 8 . 8 . di. Boric acid-borax (1+5) • 50 ml of 0).2M boric acid plus 115 ml of 0).5M borax (0..2M in terms, of sodium borate)' diluted to 200' ml. Final pB 9.2. e. Borax-sodium hydroxide (1*5)) 50 ml of 0W05M. borax (0w2M i n terms of sodium borate) plus 1+6 mi of 0.2M sodium' hydroxide diluted to 200 ml. Final pH 10.1. f. Carbonate-bicarbonate buffer (1|5) 1*5.' ml of 0.2M anhydrous sodium carbonate plus 5 ml of 0'.2M sodium1 bicarbonate diluted to 200 ml. Final pH 10v7. g. Borax-sodium hydroxide 50 ml of 0»05M borax plus SO ml of 0.2M sodium, hydroxide diluted to 200 ml. Final pH 11.6. D. The Biological Material The biological material used in this study was that prepared for the biological assay of red clover. The extract remaining after the biolo-gical assay wil l hereafter be referred to as the crude extract. The crude extract was further concentrated under vacuum to a volume such that one m i l l i l i t e r contained the extract of four grams, of dried clover. This preparation was then refrigerated. Upon extended r e f r i -geration, or temporary placement in a deep freeze, extraneous matter of a wax-like consistency settled out and accumulated on the sides and bottom of the vessel. A cold one m i l l i l i t e r sample of the extract was carefully removed from the center of the preparation by pipette, transferred to a testtube and extracted, by shaking with one milliliter of diethyl ether. The ether layer was removed and the procedure repeated. The combined ether layers were concentrated! to 0.5 m i l l i l i t e r containing the extract from four grams of clover. This preparation constituted a. "pure, extract1* and was used as such in the electrophoresis studies. Extracts for samples of May, June, July, August and September forages were prepared. It was felt that electrophoretic comparisons would best be made with samples of isoflavones isolated from red clover and not with those of largely synthetic origin used i n the bioassay studies. It was possible to obtain such a set of samples and the following standards: were prepared. The solvent i n a l l cases was 80 per cent ethanol-acetone {ktl)• (a) Genistein, ragm/10) ml; (b) Biochanin A, iu5 mgm/lO) ml; (<*)) Daidzein, -k2-U«0 mgm/iO1 mlj (d) Formononetin, 10,0! mgm/lO' milj (e) Coumestrol.,, It.® mgm /lO ml, A mixed standard was prepared by ccmfoiiaing one mil l i l i t e r of each of tee above, E, The Electrophoresis Procedure The standard solutions x^ ere applied onto the fi l t e r paper using capillary tubes, with a 0,1+ millimeter inside diameter, at a line marked at one-half the length of the sheet. At a later point,, once direction of movement had been determined, the origin was moved to a line h l/h inches from the end of the page to allow a greater distance for spot migration and separation. The applied standard, solutions were dried by means of a forced hot air drier, A five millimeter diameter spot of the applied solutions was used as nearly as possible throughout the study. The paper was then dipped i n a dish containing the buffer solution in such a manner that the applied spots were moistened by capillary movement of the buffer i n the paper. The moistened sheet was then placed on the water cooled bed of the electrophoresis' cabinet. Papers with the origin h l/U inches from the end were placedi such that the applied compounds were proximal to the negative pole, spot migration, being towards the positive electrode. Contact with the electrolytes in the buffer troughs was made by dipping the overhanging ends of the paper directly into the solution. The paper was then blotted, using a second sheet of f i l t e r paper and a tissue pad, to remove excess moisture and dissipate air pockets which alter the pattern of flow. It is important to maintain a relatively constant degree of wetness over the paper and consequently the blotting pressure must be evenly applied. The cabinet cover was placed i n position and the current applied, A number of varying voltages and running periods were tried to obtain the optimum! of separation between the standards. -U3-Upom completion of the run the power was turned off and the ends of the sheet i n contact with the buffer cut away fromi the rest of the paper* Removal of the wet ends eliminated! dripping and possible spot distortion during drying* The paper was hung in a forced air drying oven and dried, at 1009C for 2$ minutes* F, Detection of the Position of the Compounds Trials were carried out with a number of chromogenic sprays which have been reported to give distinctive color reactions with flavonoid %pe compounds* The sprays considered were as followss a. Aqueous sodium hydroxide and heat b* Ethanolic aluminum: chloride c. Perchloric acidi and ferric chloride di. Ferric chloride i n sulfuric acid e. Ethanolic one per cent sodium nitrite f. Five per cent perchloric acidi and sodium nitrite g. Five per cent methanolic ferric chloride h. Neutral lead acetate I. Basic lead! acetate j * Five per cent etherial lithium aluminumi hydride and dilute hydrochloric acid k* Diazotized sulfanilic acid in ten per cent sodium carbonate The sprays were applied to the dried electrophoresis papers using a "Universal Aerosol Spray Kit" prepared by the Nutritional Biochemicals Corporation of Cleveland, Ohio* As well as possible chromogenic developers, the fluorescent character of the compounds in ultraviolet light was observed i n a chromatoview cabinet at wavelengths of 253 366 millimicrons* RESULTS AND DISCUSSION The Bioassay of Genistein, Biochanin A, Daidzein, Eormononetin and  Coumestrol The fact that different workers, interested in the estrogen-like substances in plants, have in many cases developed and advocated the use of a particular assay procedure, has led to some lack of uniformity in the reported estrogen-like potencies- for different compounds. There i s also an evident lack in the literature of work relating to a number of com-pounds, as studied by one procedure, over any extended range of dosages. In many cases a particular compound, for example genistein, has been reported in the literature as simply being active or inactive at a given level (for example active at the one milligram level) (32), It would appear that no one has considered any one compound over an extended range of values to determine i f the response of the uterus to the estrogen-like stimulation increases as concentration increases or i f such an effect bears some relation to that produced by other isoflavones over the same dosage range. In this study the estrogen-like activity of genistein, biochanin A, daidzein, formononetin and coumestrol was determined at total dosages of 0.5 to 15,0 milligrams per mouse. The Control Diet Immature female mice fed the control ration were found to have an average uterine weight of ®+Hk expressed as a per cent of body weight* The standard error of estimate was - 0 , * Q 1 0 . This value is similar to that observed by Ostrovsky in I 9 6 0 ( ? 8 > and Kitts et a l , in 1 9 5 9 ( 6 5 } -45-both of whom used the same strain of mice and the same control ration. As this value has been shown to vary slightly by other workers at this laboratory the author felt that the average uterine weight would best be expressed in terms of a range existing between 0 ) * l 6 and 0.19 per cent (Figures I, II and I H ) ) . Daring the present study animals of this strain (Swiss Albino), age and weight did not exhibit values above or below this range. Genistein It has been reported that genistein elicits an estrogen-like re-sponse when administered per orum at a level of one milligram to the immature female mouse (32). This study would confirm this fact in that the fir s t signs of uterine stimulation, as measured by an increase i n weight, was observed at the one milligram level and thereafter increased as the dosage was increased to the 1 5 milligram level. However when a dose response equation i s derived for the present data the f i r s t definite stimulation appears to occur at the 1 * 7 5 milli,grant level. The response has been expressed in terms of uterine weight as a per cent of body weight because this affords an opportunity to correct for the slight fluctuations arising from differences i n body weights among the assay animals* For example, the dose response line is construc-ted on the basis of dose per mouse rather than dose per unit mouse, hence some of the variation in the assay values is probably due to the fact that mice above the mean weight were receiving relatively less of the effective agent and those below relatively morej this is assuming feed consumption was equal within the group. Data obtained from the bioassay of genistein i s presented In Table VII. The calculated equation was found to be Y=0.0209x + 0 . 1 6 5 1 between 0 and 1 5 milligram total dosages:, -be-when x equals the milligrams per mouse and I the uterine weight as a per cent of body weight. It i s realized that It may be somewhat erroneous to force the observed increases to the conformity of an' arithmetic re^-lationsMp. The response may in fact be other than arithmetic. The correlation coefficient, r - 0.913, is highly significant (P < Q).01) which may tend to indicate that the arithmetic relationship i s valid, however, the possibility of the response not being arithmetic can not be disregarded!. The equation and points from which i t was calculated have been expressed graphically in Figure III. Biochanin A Biochanin A has been reported as possessing an estrogen-like potency similar to that of genistein (bb). The results of this study would indicate that the estrogen-like effect of biochanini A begins to exert itsel f at the five milligram level and continually increases as the dosage increases to the 15 milligram1 level, though to a much lesser extent than, the in-crease observed for genistein. Consequently this study would indicate that biochanin A and genistein do not exert a quantitatively similar estrogenic response and i s contrary to the results reported! by Cheng et a l . in 1955 (kk)>* The data obtained from the bioassay i s presented in' Table- ¥111, the calculated equation being I-0'.0093x + 0!.lbbl, between 3.76 and 15.0 milligram! total dosages, when x equals the milligrams per mouse and J the uterine weight as a percentage of body weight. Here again the imposition of an arithmetic relationship on the data may be a misrepresentation. The correlation coefficient, r= 0.?3&, (0..01< B< 0.05) however would s t i l l indicate that an ari.thm.etic relationship may be valid. The equation and points from which i t was calculated has been graphically presented in Figure III. -47-Table VII. Results of the Bioassay of Genistein Level mcgm /gm; Mgm/ mouse /3 day assay Rb. of mice/ group Av body wt. (gm);» Av. uterine wt. (mgm). Uterine wt, as % B.W. Standardl error i IOO O.50' 6 10.70) 17.38 0.163 0).O10) 150' 0.75 6 lOJtf 17.08 0-.l6i* 0i.0I7 200' 1.00 6 11.10 21.2? 0).191 0.009 250 1.25 5 10.24 16.94 Qi.166 O.015 500 2.53L 6, 10.75 24.52 0.228 01.013 ?5o 3.76 6 101.67 24.1tO 0*228; 01.021 1,000 5.01 6 10.26 30.12 Oi.293 O.0IL4 1,250 6.26 5 lo.?5 32.53 0.302 O.Q2a 1,500 7.52 6 11.13 36.65 0.329 0).oU* 1,7#>' 8.7? 6 10.56 1*2.53 O.li|0'3 Ov0l6 2,000) 10 . 0 2 6 I0i .?6 39.86 0'.371 O.017' 2,5bo 12.53 6 11.3$ ii4.98; 0W397 0i.oi8; 3,000 15.03 6 11.21* 51.99 QJ162 0)^ )15 Calculated equation Y= 0.0>209x + 0.1651 (9k}. (x =mgm/mouse/3 days T = uterine wt. as % B>.W.) r-0.973 (F<0.01) -US-Table ¥1X1 Results of the Bioassay of Biochanin A. Level mcgm /gm Mgm/ mouse /3 diay assay No. of mice/ group. Av. body wt. (gm)). Av. uterine wt. (mgm)). Uterine wt. as %' B.W. Standard! error i 3.76 6 11.06 17.77 0.161 0.0101 1,000- 5*01 6 10».ii2 21.25 G'.20b G.Q12! 1,250 6.26 5 10^85 17.73 0.163- 0.013 1,500 7.52 6 10.93 25.69 0.236 Q..0LI18'> 1,750 8.77 6 1L.30' 26.18» 0.231 Oi.013 2,000! 10.02 6 11.1b 28.26 OJ.253 Q;.Q33 2,50O> 12.53 6 10.93 32.87/ 0.301 QwQZL 3,000' 15.03 6 11.09 26.17 0.235 Qw0l5 Calculated equation T« 0>.0093x + 0>.lliUl (9b:>. (x= mgra/mouse/3 days, T= uterine wt. as %• B.W.)) r = 0.738 (0.01 < P< 0.05)) F I G U R E III 06, The "Uterine Response to Increasing Levels of Genistein and Biochanin A in the Laboratory Mouse Genistein Control range 10 3.0 50 7.0 90 110 mg ms /m ouse/3dav assay pe r iod 13.0 15.0 Coumestrol In the case of coumestrol the uterine response to the estrogen-like stimulation was not arithmetic,, this response being the greatest of a l l substances considered. The data obtained has- been reported in. Table IX and graphically presented in Figure Hf. The calculated, equation was found to be Y = 0.133& iog x + 0.1jl37, between 0' and! 1E>>,0> milligrams total dosage, when x equals the milligrams- per mouse and Y the uterine weight as a per cent of body weight. The correlation coefficient, r = 0.96, P<0.01, would tend to verify this relationship. Coumestrol has been previously reported as being 30 times more estrogenically potent than genistein. The results of this study would indicate that such a relation-ship does in fact exist but only at the lower dosage levels.. As the concentration of the compounds increases the uterine response to genistein increases linearly to a uterine weight as a per cent body weight of Q»46, while that to coumestrol tends to plateau1 at Q>.$2. At the 10 milligram' level coumestrol exerts twice the estrogenic potency of genistein while at the l£.0 milligram level the potency is; only 1.25- times that of genistein. Therefore as the amount of genistein administered increases, the uterine response approaches that observed for coumestrol at the same level. It would appear that the limiting response observed' with dosages greater than eight milligrams (a uterine weight as- per cent of body weight of 0.5U) i s probably the result of the limited expansive character of the uterine epithelium and not resulting from a decreased sensitivity to coumestrol. Daidzein Daidzein has been previously reported as being the isoflavone exhibiting Table IX, Results of the Bioassay of Coumestrol Level mcgm1 Mgm/ mouse /3 day assay Eo. of mice / group. Av. hody wt. (gm)< Av. uterine wt (mgm). Uterine' wt. as $> B.¥. Standard, error ^  100 0 . 5 0 6 1 0 . 0 4 3U.90 0 . 3 4 7 0 , 0 3 1 150 0 . 7 5 6 1 0 . 2 9 U3.52 Q).ij22 0 . 0 2 3 2^0 1 . 2 5 6 1 0 .09 4L . 5 0 0-.1O1 0 . 0 2 6 500' 2.5L 6 10). 36 4 7 . 5 6 0 . l i 5 £ 0'.019 750 3.76' 6 10.17 52.90) 0 . 5 2 0 0>.Q2? 1,000: 5 .01 6 1 0 . 3 1 5 1 . 4 8 0.499 0 . 0 3 0 1 , 2 5 0 6 . 2 6 6 11.014 5 8 . 2 5 ; 0 . 5 2 ? 0 . 0 2 7 l , 5 0 a 7 . 5 2 6 9 . 8 4 5 2 . 0 3 Qt.528: 0 . 0 4 3 1 , 7 5 0 85.77/ 6 1 0 , 1 3 5 4 . 6 5 ; 0 . 5 3 9 0 .020; 2,000' 1 0 . 0 2 6 9 , 8 2 5 4 ,95 0 , 5 5 9 0,0.3li 2 , 5 0 0 1 2 . 5 3 . 6 ll.Iit 5 9 . 5 5 Q',534 0 . 0 2 1 3,000 1 5 . 0 3 : 6 11.30) 6 5 . 0 4 0 . 5 7 5 O.0;30 Calculated equation Y> 0 . 1 3 3 4 Log x + 0 . 4 L 3 7 ( 9 l i ) . (x = mgm/mouse/3 days 1 = uterine wt. as %> B.W.) r = 0 . 9 6 (P < 0.01)) -52-the greatest estrogen-like potency (ItU)). The results obtained in-dicate that daidzein does i n fact e l i c i t an estrogen-like response at concentrations lower than that of the other isoflavones, however as the dosage administered increases, the uterine response does not increase to the extent of that obtained with genistein* The slope of the calculated1 arithmetic equation for daidzein i s less; than that of genistein* Therefore daidzein demonstrates a greater estro-gen-like activity than genistein, which decreases i n extent to the 3.5 milligram level and thereafter shows progressively less potency than does genistein* The data obtained from the bioassay of daid-zein are presented i n Table X and have been graphically presented in Figure I¥. The calculated equation was found to be Is0'*Cl5lx + 0*187/2, between 0 and1 12*5 milligram, total dosages, when x equals the milligrams- per mouse and X the uterine weight as a per cent of body weight* The correlation coefficient, r=0>.9?ii., B'<0>*0!,, would indicate an arithmetic relationship exists but here again such an interpretation may be a misrepresentation of the true response* Formononetin This compound has been reported in the literature as possessing l i t t l e or no estrogen-like character (Wi). In this study i t was found that no estrogen-like stimulation occurred until the compound was administered at the 10w68 milligram- level and thereafter this response increased gradually as the dosage increased to the 15 m i l l i -gram level. Consequently the compound does possess an- estrogen-like character which is of very low potency and' observed only when the dosage is relatively high. The data from this assay are presented -5> Table X Results of the Bioassay of Daidzein Level /gift Mgm/ mouse /3 day assay Ho. of mice/ group. Av. body wt. (gm). Av. uterine wt. (nigm))* Uterine wt. as % B.W. Standard error i. 100; 0.50 6 10.2b 18 .b2 0.179 0.01.3 1^ 0 0i.7$ 6 10.76 21.25 0.197/ 0*016 200' 1.00) 6 10.62 22.89 0*216 0*009 250> 1.25 6 io.b5 23.09 0;*221 O.02O 500) 2.51 6 10.32 21.63 C.209 0.010) 150 3.76 6 10.6b 2S-.6Q' 0.268, 0*019 1,000) 5.01 6 ia.o? 25.62 0.25b G*01& 1,50a T.52 6 29*6? 0)*282' o;.oiai 2,000) 101.02 6 9.89 3b.37 0.3b? 0w026 2,500) 12.53 6 10.39 39.6? 0*381 0V018' Calculated equation Y=0.0l5lx + Q)*l8?2 (L9b>» (x = mgm/mouse/3 days ¥= uterine wt* as %> B .¥:.)) r=0.9?b (E<0*01)) Table XI Results of the Bioassay of Formononetin Eevel mcgm /m Mgm/ mouse /3 day assay No. of mice/ group Aw. body wt. (gm)). AT. uterine wt. (.mgm). Uterine wt. as % B.W. Standard! error i 2,2?0 11,27 5 10.68 0.190. Q'.Ollii 2,500) 12.53 6 11.15 21.55 0.193 0,012 2,750 13.78 6 9.71 20.1$ 0-.209 O'.OUlli 3,00® 15.0'3 5 11.26 26.m QW23S& 0v0i5 Calculated! equation I * 0.O119x + 0.0502 (,9b). (x= mgm/mouse/3 days 1-uterine wt. as % B.W.) r= 0..95 P=0.05 F I G U R E IV Mgm/mouse/3 day assoy per iod -56-i n Table XI and have been graphically presented i n Figure Mu The calculated^ equation was found to be Y = 0.0119x0.0502, between 11,27 and 15.03 milligram1 total dosages, when x equals the milligrams, per mouse and' I -the uterine weight as a per cent of body weight. The correlation coefficient, r = 0.°5, P= 0.05, would indicate an arithmetic relationship, however this may again not be a true representation of the response. Discussion-general The response of the five compounds considered is illustrated i n Figure V for relative comparisons. The results of this study appear to contradict several statements which exist in the literature pertaining to the estrogenic!ty of the isoflavones. It i s evident that a l l of the compounds, considered possess an estrogen-like activity to greater or lesser extents over the range of the dosages considered, i n this study. Contrary to the work of Cheng et al. in. 1955 Oik)) genistein; possesses, a markedly greater estrogenic potency than does biochanin A. These workers stated that the two compounds exhibited highly similar estrogen-like potencies, and that daidzein possessed the greatest estrogenic potency within the isoflavones considered. The present study indicates that the conclusions made by Cheng and coworkers are only valid at low dosages (X 3»5 milligrams)1 and that at higher levels genistein is by far the more potent of the four isoflavones. It would have been desirable to assay each individual compound out to a dose level at which the dose line had- reached i t s approximate asymptote i n order to establish the upper limit of the response. Unfor-tunately the quantities' of these relatively rare compounds were not adequate to permit such detailed studies of the dose response relationships. Uterine body weight o O O o c 3 « <• o - - -1 o •o o 3" o a — a. no m o o w o o \ o Q- © no tr » f - 1 3 \ e r « \ < a> J T M \ I (0 -1 \ o. O \ o o CO -< 1 a 3 il \ o <D O 1 c W no — j o 3 ot -n O. 9 ~~' 1 a. r 1 to o <Q c X o O on -£ CM «Q < " — o — 3" a> XI co u> T3 O 3 in 3" CD o c «> O C 3) m 3 O » o (0 3 r < (t> -58-II* The Results of the Bioassay of Isoflavone Mixtures1 Since the estrogen-like activity of forage plants consumed, by grazing animals may possibly be due- to the presence of one or more isoflavones In the forage i t i s of great importance to determine if. inhibitory or synergistic effects, exist between the various compounds* As mentioned earlier the supply of compounds available precluded the conduct of the extensive bioassays necessary to precisely determine the nature and extent of such effects i f they exist. As a preliminary to future work a screening assay was conducted with combinations of geni-stein, biochanin A and formononetin. An arbitrary selection of dosage level had to be made as no existing literature permitted a reasoned selection of dosage levels. Stock solutions were mixed together i n the experimental diets; such that each compound considered i n the mixture was present at a dosage of 12*5 milligrams per mouse per three day assay. This level was. chosen as a l l compounds had' demonstrated a definitive estrogen-like activity at this concentration i n the individual assay studies. The results of this study are presented in Table XIX. The mixture of genistein and biochanin A, at the amounts- used,, produced an estrogen-like response which was approximately midway between the responses observed for the individual compounds.. This would Indicate either, that biochanin A exerts; an inhibitory effect on the extent of the uterine response normally observed after administration of genistein, or that the combined dosage was so far i n excess that the response was partially reversed!. This latter possibility seems unlikely. Biochanin A and formononetin fed simultaneously produced a uterine response greater -59-Table XII Results of the Mo as say of Pure Isoflavone Mixtures Comprds Level mcgm /gm Mgm/ mouse /3 day assay No. of mice/ group Av. body wt. (gm). Av. uterine wt. (mgm)). Uterine wt* as % B.W. Standard error i G 2,500 12.53 6 10.28; 33*93 0*330 O.01U B 2,500 12.53 J B 2,500 12.53 ) 6 10.70- 33*95 0.317 O.032 F 2,500 12.53 ) F' 2,500 12.53 ) 6 10.56 46.68 0.U23 0.019 G 2,500 12.53 J G 2,500' 12.53 B 2,500 12.53 ' 6 10.75 Wi*32 0.1*12 0.022 F 2,500 12.53 J Indi-v idual Res] Donse G 2,500 12.53 6 11*33 0.397 0;.0l8 B 2,^ 30 12.53- 6 10.93 32*87 0.301 0*021 F 2,^ 30; 12.53 6 11.15 21*55 0-.193 0.012 G - genistein, B - Biochanin A, F - formononetin. -60-than that observed when either of the compounds, were fed. individually* Such a result suggests that formononetin possesses a synergistic character. Supporting evidence to this, would be that formononetin'- (of individual low potency)) when fed with genistein produced a response which was slightly greater than that observed when genistein was fed alone. The mixture of genistein, formononetin and biochanin A produced a response slightly below that of pure genistein but well above that observed for pure for-mononetin; pure biochanin A;, the mixture of formononetin and biochanin A; and the mixture of genistein and formononetin. This result would appear to indicate that formononetin exerts a synergistic effect x-rhich i s great enough to overcome or counteract the inhibitory effect of biochanin A and. produce a result approximating -fiiat observed for pure genistein. These preliminary results must be interpreted with a great deal of reservation and should be supported! by much more extensive assays' than' have been possible in the present work. III. The Results of the Bioassay of Red Clover The results of the bioassay of the red clover, harvested monthly, were similar to those previously observed by workers at this laboratory (66). The estrogen-like activity was greatest in the early summer and remained relatively high until July, after which a decrease was observed until September at which time insignificant estrogen-like potency existed i n the sample material. The results of this study have been presented in Table XIIX and graphically presented in Figure VI. The association of electrophoretic studies to this, study have been considered at a later point. - 6 1 -Table A H Restilts of the Bioassay of Red Clover and- the Compounds Identified in the Monthly Extract* Date Ho. of mice/ group-Av. body wt. (gm) Av. uterine wt. (mgm) Uterine wt. as % B . W . Standard error ± Comp *ds in extract May 10: 6 1 0 . 9 3 5 1 . 6 b Q.U72 O.017 F,D,G June 12 6 1 0 . 8 5 3 9 . 6 ? 0 . 3 6 5 0 . 0 1 3 P*D,G July 11 6 1 0 . 2 2 U 8 . 5 2 o.b?b 0 . 0 2 5 F,D,Q Aug. 9 b 1 1 . 2 1 2 6 . 3 0 0 . 2 3 b 0 . 0 1 3 F,H Sept. 11 6 1 0 . 5 3 2 1 . 3 2 0 . 2 0 2 0 . 0 1 8 F,D,G * Extracts assayed such that one gram of feed contained the contents of one gram of dry clover. F - formononetin, D, - daidzein, G - genistein. FIGURE VI 0.5 The Uterine Response of the Lo.boro.tory Mouse to Monthly Horvested Red Clover Samples 0.4 s TJ o 0.3 o »> a t: 0 21 Control range c 5 o i l o oL May 10 June 12 July 11 August 9 September 11 Date of Harvest -63-IV. The Electrophoresis Studies-The use of paper electrophoresis as a method for the identification of isoflavones- as an alternative to the use of chromatography was investi-gated- and the following observations made. The standard' solutions of naturally isolated isoflavones, described under the section Materials and Methods, were used to develop the optimum conditions for the procedure. The standard solutions were genistein, daidzein, biochanin A, formononetin, coumestrol and a composite mixture of these five compounds. Buffer solutions ranging i n pH from 5*3 to 11.6 (Materials and Methods) were tested to determine which would produce the optimum separation and rate of movement* The compounds migrated towards the positive electrode, the migratiott rate and separation improving as the alkalinity of the buffer systems increased. Migration was; extremely restricted at pH values below nine. The optimum' migration was observed using a Borax-sodium; hydroxide buffer of pH 11.6 containing 0.1M sodium borate and' O.IM sodium hydroxide. In the course of a number of trials i t was found that further dilution of the buffer to 0.05M sodium borate and O.O^ M sodium' hydroxide produced a slower and more even migration of the compounds.- This- dilution was not associated1 with any changes of the pH'* Voltages ranging from' 625 to ?50 D.G. were utilized* This variation did1 not alter the relative separation of the Isoflavones but i t was found that at lower voltages the streaking often observed i n mixed plant extracts was reduced and more definitive spots observed following migration* During the course of a run the milliamperage increased front 15 to 20 m.a. The fluorescent character of the compounds, which was used as a means of detection,, was enhanced by the presence of sodium hydroxide -6V in the buffer solution* Whatman, number three f i l t e r paper was found to be most suitable as this grade allowed good migration of the compounds and possessed superior wet strength which facilitated handling of the moistened sheets. Detection of the Spot Position The electrophoresis sheets were dried and placed in a "chromatoview"1 ultraviolet viewing apparatus for observation. At a wavelength of 253 millimicrons daidzein and' formononetin produced a white fluorescence which was' enhanced by the presence of sodium- hydroxide from the buffer solution. Coumestrol similarily produced a brilliant blue fluorescence. Genistein and biochanin A possessing an ultraviolet light absorbing character were not clearly visible on the sheets* Indistinct shadows were present at their positions. The three fluorescent compounds were marked and the paper sprayed with a number of possible developing solutions for the detection of genistein and1 biochanin A and the possibility of chromogenic reactions occurring with the compounds which could be observed with ultraviolet light* The developing sprays' which have, been mentioned, i n the literature were found to be inadequate as the faint color reactions produced were hidden i n the total discoloration of the paper or produced no visible color reaction. Diazotized sulfanilic acid however did produce a signi-ficant color reaction with all. of the compounds and was particularily good with genistein and biochanin A. This substance was later reported in the literature by other workers (61) and had been developed during the same period at both laboratories. The diazotized1 sulfanilic acid was dissolved in- 1© per cent sodium -de-carbonate and sprayed onto the electrophoresis sheets. Genistein and biochanin A produced a deep rust colored spot, formononetin, daidzein and coumestrol appearing as very pale yellow-orange spots. Observation of the papers under ultraviolet light now resulted in deeply shadowed areas at the positions of genistein and biochanin A, however these compounds were now clearly visible without the use of the ultraviolet light. Consequently the combination of ultraviolet light and the diazotized sulf anilic acid spray permitted1 the accurate determination of spot loca-tions- following migration. The use of an ultraviolet hand- lamp allowed the spot migration to be observed in a darkened room during the period of migration. Periods of current application from six to eight hours produced excellent separation of the pure isoflavones and coumestrol* The separation, observed between the pure standards and a composite mix-ture i s illustrated in Figure VII. It can be seen that daidzein migrated' most rapidly and was followed by genistein, formononetin,. biochanin A and coumestrol i n that order. Diffusion of the spots was minimized by making the i n i t i a l applications at the origin as compact as possible. The Electrophoresis of Red Clover Following the development of the electrophoresis procedure,; the extracts of the red. clover samples were analyzed for the presence of genistein,. formononetin, daidzein, biochanin A and coumestrol The extracts were prepared as outlined i n "Materials and Methods" and the. five monthly samples and standards applied to the electrophoresis sheet. These results have been presented i n Figure VIII. Coumestrol and' biochanin A were not observed in any of the monthly samples considered, which may have resulted fromi an inadequate extraction or alteration of these substan--66-ces during extraction. This would appear unlikely as the remaining iso-flavones were observed and the extraction involved only mild! solvents, Daidzein and formononetin' were observed i n a l l samples' and the absence of genistein from; the August sample probably resulted from a slight inadequacy of extraction in this case. The samples appeared to contain a number of substances as can be seen from the additional spots marked on the paper and the occurrence of streaking i n the extracts. The compounds existing at the positions' of coumestrol and- biochanin A i n the extracts were not considered as being these two, a decision based on differences- i n fluore-scent and chromogenic character of these compounds as compared to the standards. The movement of genistein in the plant extracts appeared to be retarded by the presence of extraneous material. The absence of coumestrol from the samples i s in keeping with the results of chromatogra-phic studies previously carried out at this laboratory. Coumestrol has; not been identified in samples of red clover grown in this local,, to date. The extracted samples were found to contain a number of unidentified compounds. Figure IX demonstrates the existence of five such substances (numbered- one to five)). In every case these substances demonstrated' either a fluorescent or chromogenic character differing from- the known compounds which occurred at or near these locations on the electrophoresis paper. Unknowns- "four" and nfive" preceded the migrating isoflavones. The substance at position "five® displayed a distinct pink coloration follxwing the application of diazotized sulfanilic acid. From- the results obtained i t would, appear that electrophoresis can be considered as a highly suitable technique for the identification and possible separation of isoflavones in plant extracts. FIGURE' VII. Separation of Standard Solutions of Isoflavones; by Paper Electrophoresis. {1/31/61} FIGURE VIII* Observations of Isoflavones i n Monthly Harvestedi Red Clover Samples. (9/28/61). FIGURE IX. The Presence of Unidentified Compounds (No* l,.2,,3,b,£} i n Red Clover Samples. (9/20/61). J_, i , SL, " i " I ORIGIN MIXTURE ELECTROPHORESIS OF STANOAR DS Tim* 7 1/2 hrs. NaOH-BORAX pH 11.6 7SOv 20m.a. Spray-Diazotized -R-W.H. 7/31/61 Sulfanilic acid STANDARDS MAY JUNE JULY AUGUST SEPTEMBER STANDARDS ELECTROPHORESIS OF SAMPLES Tim* 8 hrs. NoOH- BORAX pH 11.6 62 5v 20m.a. Spray-Diazotized RWH. 3/28/61 Sulfanilic add JULY J U N E MAY STANDARDS 0 COUMESTROL I BIOCHANIN A FORMONONETIN 9 Q 0 GENISTEIN ELECTROPHORESIS OF SAMPLES Tim* 7 l/a hr». 62 5v 2 0m.a. 9/20/61 NoOH-BORAX pHIL6 Spray- Diozotii ad Sulfanilic acid R.W.H. -71-V. The Estrogen-like Activity versus Electrophoresis Analysis of Red! Glover The electrophoresis analysis demonstrated that daidzein,, formononetin and genistein were present throughout the summer period in the red clover plant. This procedure was not quantitative but as approximately similar amounts of the extract were applied to the paper i t would appear that these compounds are present at a relatively constant amount throughout the season. This may i n fact not be true and varying proportions may exist at different periods-. However the estrogen-like activity of the samples as determined by the bioassay changed considerably over the period considered. The extract of the September sample demonstrated l i t t l e or no estrogen-like activity yet was shown to contain daidzein,, formononetin and genistein. Daidzein and genistein are both relatively active sub-stances as- shown earlier, and formononetin possibly possessing a synergis-tic action as measured! by the mouse uterine response. Figure X depicts the demonstrated activity and1, the compounds present in, both red clover and the pure mixtures. It i s possible that daidzein, not considered in the mixture studies, also possesses a synergistic character. Consequently one is forced to consider the possibility that daid-zein, formononetin and genistein appear to be present throughout the season at insignificant levels to cause a biological effect. The estro-gen-like response of red clover extracts may in fact be due, at least i n part, to some other substance present in the plant or some substance which i s capable of activating the existing isoflavones. On this surmise several samples were run and the compound which appeared at position "five" (Figure IX)) was cut from the sheet and eluted with warm, acetone;. A very small amount of the substance was obtained and a preliminary FIGURE X The Uterine Response of the Laboratory Mouse to Mixed Standards and Red Clover Associated with the Compounds Present 0.5 ', ^-July lH Formononetin, Genistein ^ MaylOjDaidzein 0.4 vGenistein Formononetin "^•Genistein Formononetin Biochanin A "^Genistein Biochanin A ^ June II Formononetin, Genistein, Daidzein 0.3 ^Biochanin A Formononetin . *— Aug 9 Formononetin, Daidzein 0 .2 . Sept II Formononetin, Genistein, Control range Daidzein 0 1 -0.0 Mixed samples ot 12 5 mgm level Red clover response and compounds observed in the samples -73-examination indicated, that the compound may possess an estrogen-like character. It was' also noted that this substance could be identified in the forage samples of May, June and July, associated with a relatively high estrogen-like activity, but did not appear i n the extracts, of August and September, both of which possessed a low es'trogen-like action. A large number of samples containing the compounds at locations "four1* and "five" were run, the spots cut from; the sheets and eluted with acetone. A brown oil-like substance was obtained which would not crystallize from the common solvents or solvent mixtures. The two compounds ("four'1 and "five"); were subjected to infrared analysis and found, to be the same substance. However the distinctiveness of the recording indicated some degree of purity in the samples. The fact that both were the same probably resulted from the excessively heavy spots and the predominance of heavy streaking on the papers, plus possibly, the elution of a third overall spot present but not readily visible. The infrared absorption spectra indicated that the substance possessed no aromatic character, no hydroxyl groups, at least one carbonyl group,, appeared to be of a chain structure and was possibly an aliphatic fatty acid ester. The substance was added to the diet at the 12.5 milligram level and produced, no estrogen-like activity. Inclusion in the diet at the 30 milligram level produced a uterine weight of 0.2580:, as a per cent of body weight, and. definitely indicated: an estrogen-like response. low-ever, this, is considered as a very high level and the response normally observed i n the active red clover samples i s considerably higher. There is a possibility that this substance i n association with the isoflavones present could result i n a synergistic activation to produce the observed -7U-activity. More extensive- studies than were carried out are necessary before definite conclusions can be drawn. SUMMARY AMD CONCLUSIONS. The study indicated, fairly conclusively a number of points regarding, several of the estrogen-like substances present i n red clover and other forage plants. Coumestrol possesses the greatest estrogen-like activity of the substances considered, the response of the mouse uterus to this substance being of a logarithmic nature (Figures IV/, V). The order of the estrogen-like potency of the isoflavones considered, was found to be genistein, greatest, followed, by daidzein,, biochanin A and formononetin, in that order. Daidzein produced a uterine response at the lowest dosage but was surpassed by the response to genistein which produced, an i n i t i a l activity at a slightly greater dosage (Figure V)). Biochanin A appeared to exert a slight inhibitory effect on the response of genistein while formononetin appeared to cause a synergistic action (Figure X). These results; should be interpreted with some reser-vation as the experiment was of a preliminary nature. The estrogen-like potency of monthly harvested..red clover samples; decreased as the growing season progressed until by September only a slight estrogen-like activity was observed (Figure VI)). An electrophoresis technique using a Borax-Sodium hydroxide buffer system (pff 11.6) was developed for the identification of isoflavones in. red clover samples. The monthly harvested red clover samples were found to contain genistein,, daidzein and formononetin. Biochanin A and coumestrol were not observed i n any of the samples. The estrogen-like activity of red! clover was greater in the early season than would be expected from, the compounds found to be present and less than the expected in the late season. This would indicate the presence of a substance in the plant i n the early season which produced a great estrogen-like effect or was' capable of activating the isoflavones present to produce the observed response. 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