Studies on the Chemical and Biological Properties of Coumestrol and Related Compounds

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Agricultural Research Service UNITED STATES DEPARTMENT OF AGRICULTURE 6

Studies on the Chemical and Biological Properties of Coumestrol and Related Compounds

Technical Bulletin No. 1408

Agricultural Research Service UNITED STATES DEPARTMENT OF AGRICULTURE

Contents Page Page Introduction 1 Synthesis of coumestans—con. Analytical procedures 3 Synthesis from previously Biological procedures 6 formed coumestans 32 Chromatographie procedures 7 Physical properties of coumestans Qualitative procedure 8 Ultraviolet absorption spectral Quantitative procedure 9 measurements 32 Other Chromatographie pro- Proton magnetic resonance cedures 10 spectral measurements 35 Isolation 14 Mass spectral measurements 37 Isolation of coumestrol from Chromatography 41 Ladino clover 15 Coumestrol in plants 43 Large-scale processing of al- Variations as affected by cut- falfa for coumestrol 16 tings and stages of growth __ 43 Isolation of compounds closely- Variations as aifected by loca- related to coumestrol 17 tion and climatic conditions __ 46 Characterization of coumestans __ 18 Variations as aifected by varietal Coumestrol 20 and genetic differences 47 Trifoliol 20 Variations as related to disease. 48 Medicagol 24 Mechanism of biosynthesis 53 4'-0-methylcoumestrol 24 Utilization of coumestrol by the 3'-Methoxycoumestrol 24 animal 55 Lucernol 25 Metabolism 58 Sativol 25 Estrogenic potency 59 11, 12-Dimethoxy-7-hydroxy- Antifertility effects 53 coumestan 26 Hyperestrogenic syndrome 05 4',7-Dihydroxyflavone 26 Hypoestrogenic syndrome 69 3',4',7-Trihydroxyflavone 26 Antigonadotropic factors 72 Synthesis of coumestans 27 Effects on rate of growth 73 Cyclization of 4-hydroxy-3- Other biological effects 77 phenylcoumarin derivatives _ _ 27 Correlation of structure and Oxidation of flavylium salts 27 activity 78 Condensation of catechol with Patents 81 substituted 4-hydroxycou- Summary 82 marins 30 Literature cited 83

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Washington, D.C. Issued November 1969 For sale by the Superintendent of Documents, U.S. Government Printing Office Washington, D.C. 20402—Price 60 cents

Studies on the Chemical and Biological Properties of Coumestrol and Related Compounds

By E. M. BicKOFF, R. R. SPENCEK, S. C. WITT, and B. E. KNUCKLES, chemists, Western Utilization Research and Development Division, Agricultural Research Service

Introduction

Natural estrogens in plants the Western Utilization Research have assumed considerable im- and Development Division portance since their connection (WURDD) as well as reports with infertility in grazing sheep from Cheng and co-workers at was demonstrated in 1946 by Iowa State (66, 67), Engle and Bennetts and co-workers (19)'^ co-workers at Ohio State (98), and in 1948 by Curnow and co- and Pieterse and Andrews at workers (83), These reports were Purdue (197) definitely estab- followed in 1951 by the isolation lished that both alfalfa and La- from subterranean clover of the dino clover frequently display sig- isoflavone, genistein (56)y which nificant estrogenic activity. In was shown to be estrogenically 1955, work was begun at active in mice (i9, 82). Subse- WURDD on the occurrence, bio- quently, the estrogenically active logical properties, and isolation isoflavones, formononetin, bio- of the Ladino clover estrogen(s). chanin A, daidzein, and praten- Surprisingly, the major com- sein, were found in both subter- pound, which was named coumes- ranean and red clovers (39, 68, trol (30), was not an isoflavone 100, 221). At first genistein was as were all previously reported believed to be the factor that in- forage estrogens. Instead, it # terfered with the breeding of »turned out to be a benzofurocou-11 sheep (83). However, more re- marin derivative belonging to anl\ cent work suggests that formo- entirely new class of compounds, nonetin may be more important which were later named coumes- (8Í, 182, 184,). tans (85). These compounds have Until 1953, alfalfa and Ladino subsequently been shown to be clover were considered to be biogenetically related to the iso- among the nonestrogenic forages flavones (118). Coumestrol has a (5i). However, experiments in close structural relationship not ^ Italic numbers in parentheses refer only to the isoflavones, but also to Literature Cited, p. 83. to the natural estrogen, estradiol. 2 TECHNICAL BULLETIN 1408, U.S. DEPT. OF AGRICULTURE

H,C OH /VOH OH 0

Genistein Estradiol

^^X-OH ^ XV.OH

HO-^/N /^O 0 Coumestrol Diethylstilbestrol

FIGURE 1.—Structural formulas of genistein, estradiol, coumestrol, and diethylstilbestrol {22), and the synthetic estrogen, di- 222), white {105, 167), bur ethylstilbestrol (fig. 1). In fact, {167), subterranean {18, 167, Whalley {US) proposed that the 183, 18i), and strawberry (18, estrogenic activity of coumestrol 105,167), as well as meadow flora can be attributed to its stilbene- (179), Chinese milk vetch (217), like structure, which is analo- soybeans (217), soybean sprouts gous to that of diethylstilbestrol. (217), pea silage,^ and commerc- Studies established that cou- ial samples of frozen peas and mestrol was the dominant estro- beans (2). Studies on the varia- gen of alfalfa {S9, 122), Ladino tion in coumestrol content of al- clover {39, 122), and the annual falfa and Ladino clover, as related medics {18, 10U, 183), Although to environmental factors (36, 37, the isoñavones account for most 127), indicated that coumestrol of the estrogenic activity of red content was greatly affected by and subterranean clovers, their disease (í3, 16U) and insect in- contribution to the estrogenicity festation (163), of alfalfa and Ladino clover is small {122), Subsequently, cou- ^ Personal communication from J. H. mestrol was found in a wide Adler, animal physiologist, Hadassah variety of clovers, e.g., red (167, Medical School, Israel, 1967. CHEMICAL AND BIOLOGICAL PROPERTIES OF COUMESTROL

The discovery of coumestrol isolated from other plants. The stimulated a number of studies first compound of this basic related to its physiological effects structure was wedelolactone, on animals. Coumestrol has been which Govindachari and co- shown to be considerably more workers (111, 112) isolated from potent estrogenically for both Wedelia calendtilacea in 1956. mice (39) and sheep (57) than Other naturally occurring cou- has genistein. Reproductive prob- mestans are erosnin isolated lems attributed to alfalfa and from Pachyrrhizus erosus (88), Ladino clover, such as those re- psoralidin from Psoralea coryli- ported by Engle and co-v^orkers folia (65, 86, HI), and norwede- (98), Wright (22i), and Adler lolactone from Eclipta alba and Trainin (6, 7, 8), can be ex- (1Í8). The structural formulas plained bv the presence of cou- of these naturally occurring coumestrol. Estrogenically active al- mestans are given in figure 2. falfa was shown to beneficially Coumestans are also structurally affect the rate of growth of cattle similar to the naturally occurring (176) and sheep (103, 19Í), Al- isoflavonoids inermin (77), trifo- though later results suggested lirhizin (59), pisatin (81, 196), that coumestrol was not the caus- pterocarpin (60, 6Í, 198), and ative agent, coumestrol did im- homopterocarpin (6Jp, 171) (fig. prove the quality of the meat ob- 3). This similarity in structure tained from sheep (131). was further demonstrated by In addition to coumestrol, Bowyer and co-workers (53), seven other coumestans have who converted homopterocarpin been isolated from alfalfa (Medi- into coumestrol dimethylether. cago sativa) (H) and Ladino clo- This technical bulletin summa- ver (TrifoliuTYi repens) (38). rizes and correlates 13 years of They are trifolio! (il, 160), research on coumestrol and re- 4'-0-methylcoumestrol (4-2), me- lated compounds in this labora- dicagol (161), lucernol (205), tory as well as at other research sativol (205), 3'-methoxycoumes- institutions throughout the world. trol (4-5), and 11,12-dimethoxy- It also includes some of the stud- 7-hydroxycoumestan (206). This ies on other forage estrogens as type of compound has also been they relate to coumestrol.

Analytical Procedures

An early method of determin- estrogenic activity has been mea- ing estrogenic activity in plant sured by the increase in uterine material was the Allen-Doisy test weight of ovariectomized or im- (96), which is based on a deter- mature female mice or rats (í9, mination of the onset of estrus in the test animal by the appear- 56, 83, 197). Once the estrogenic ance of cornified epithelial cells compound is identified, Chromato- in vaginal smears. In most of the graphie methods (156) can often work on the isolation of estro- be used for its quantitative deter- genic constituents from forages, mination. TECHNICAL BULLETIN 1408, U.S. DEPT. OF AGRICULTURE

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0> 6 TECHNICAL BULLETIN 1408, U.S. DEPT. OF AGRICULTURE Biological procedures During the isolation of the feeding the samples of whole al- plant estrogen, coumestrol, from falfa or clover, it was necessary Ladino clover, estrogenic activity to prepare extracts for assay. was measured by a mouse-uter- Fresh forages were mascerated ine-weight bioassay (29), This in acetone in a mechanical blend- bioassay was also employed for er. Acetone was the preferred our initial studies on the occur- solvent because it extracted more rence of coumestrol in plants. estrogenic activity and less total Booth and co-workers (51) ob- solids than the other solvents served that a number of com- tested (table 1.). After concen- monly used vegetable oils were tration of the aqueous-acetone themselves capable of producing extract, the estrogenic substances an estrogen-like response in mice. were reextracted into ether. This Therefore, test materials were eliminated the presence of water- administered orally to eliminate soluble factors that sometimes re- any complications that might duce uterine response. Dried for- have arisen from subcutaneous ages having moderate estrogenic injections in oil. This procedure activity, i.e., requiring less than more closely duplicated the nor- 10 grams of plant material for mal route of ingestion by live- assay, were Soxhlet-extracted stock and, also, eliminated the with acetone for 24 hours. Dried need for a laborious purification forages having low estrogenic ac- procedure (199), Sufficient sam- tivity, i.e., requiring more than ple or extract was incorporated 10 g. of plant material for assay, into the test diet to cause at least received a preliminary purifica- a doubling of the uterine weight tion by extracting with chloro- during the test period. form. This removed a large Since a graded uterine re- amount of nonestrogenic mate- sponse could not be obtained from rial. The residual meal was then

TABLE 1.—Relative effectiveness of organic solvents for extracting estrogenic activity from Ladino clover meal

Average uterine weight at dif- ^ , ferent Soxhlet extraction times Solvent 12 hours 16 hours 24 hours Milligrams Milligrams Milligrams Control 9 9 9 Acetone 51 62 78 Alcohol 38 63 49 Benzene-alcohol (2:1)' 38 64 59 Chloroform 9 9 9 Ether __ __ 17 Petroleum ether 9 9 9 ' Proportion by volume. Source: Bickoff, E. M., Booth, A. N., Livingston, A. L., and others (29), CHEMICAL AND BIOLOGICAL PROPERTIES OF COUMESTROL 7

TABLE 2.—Uterine response to graded levels of estrogen

Quantity fed Uterine weight Diet per mouse ± S.E/ Micrograms Milligrams Control 9.73 0.33 Coumestrol 100 13.74 .73 200 18.74 .97 300 25.68 1.05 400 34.39 1.23 Diethylstilbestrol 025 11.64 .41 050 17.61 .83 075 22.86 .95 100 33.83 .99 ^ S.E. = standard error mean. Source: Bickoff, E. M., Booth, A. N., Livingston, A. L., and others (29). treated in the manner described plus diethylstilbestrol, usually 0.1 for the moderately active sam- mg./lO g.-diet). The mice were ples. The various fractions pre- sacrificed just prior to exhaustion pared during the isolation of of the food supply. The freshly coumestrol as well as the pure excised uteri were carefully trim- compounds v^ere dissolved in ace- med and weighed without blot- tone or alcohol and incorporated ting or slitting. Since there was into the mouse diet. a lack of correlation between body Estrogenic activity v^as eval- weight and uterine weight, the uated by feeding the test diets assay results were expressed as (10 g./mouse) to immature fe- the average uterine weight per male mice. The mice, caged in mouse rather than per gram of groups of five, were fed the test body weight. Results could also diet ad libitum until they had be expressed in terms of diethyl- consumed about 50 g. of ration. stilbestrol or coumestrol equiva- Each assay included a negative lency by constructing dosage- control (basic ration only) and response curves from data such a positive control (basic ration as those given in table 2.

Chromatographie procedures Since alfalfa contained at least of coumestrol under ultraviolet five substances which give a re- (UV) light (U) lent itself to sponse in the mouse bioassay Chromatographie techniques. Ini- (122)y a more selective test was tially, a qualitative procedure required for the determination of (167) for detecting small coumestrol. Chromatographie amounts of coumestrol in plant techniques were investigated as they are simple, rapid, and per- material was developed, which mit the screening of a large num- was refined to a rapid, quantita- ber of samples. In addition, the tive paper Chromatographie pro- characteristic blue fluorescence cedure (158, 159). 8 TECHNICAL BULLETIN 1408, U.S. DEPT. OF AGRICULTURE

TABLE 3.—Sequence of Chromatographie solvent systems for the identification of conmestrol

Type of Average Rf values cnromatography Solvents and proportions by volume for coumestrol ^ Filter paper Chloroform-acetic acid-water (2:1:1) 0.51 Acetic acid-water (1:1) .57 Silicic acid Chloroform-ether (1:1) .56 Ethyl acetate-Skellysolve B (3:i) __. [74 Acetone-Skellysolve B (1:3) .24 Ethyl ether-Skellysolve B (7:3) .36 'As measured from the leading edge of the fluorescent spot produced by a reference coumestrol sample. Source: Lyman, R. L., Bickoif, E. M., Booth, A. N., and Livingston, A. L. (167).

Qualitative procedure both fresh and dry plant mate- In our procedure, an acetone ^^^^^ ^^^ ^^ve Rf values for cou- extract of dry plant material was "^estrol m 10 solvent systems chromatographed sequentially in (table 4). He reported that sys- two solvent systems on heavy ^ems 1, 2, 8, and 9 gave the filter paper, then in four systems best results with plant extracts, on silicic acid chromatostrips Where impurities tended to mask (167). The solvent systems in the coumestrol spot, the spot was order of use are summarized in eluted and rechromatographed in table 3. An empirical visual esti- a different solvent system to mate of the amount of coumestrol verify the presence of coumestrol. was made by observing the rela- Although Chromatographie pro- tive intensity of fluorescence cedures are not exact when com- eluted from the fourth chroma- pared to the bioassay, they are tostrip. more specific and give a reason- Recently, Chury (69) dis- ably good estimate of the relative cussed extraction procedures for amount of coumestrol.

TABLE 4.—Chury's solvent systems ^

Solvent R, values for system No. Solvents and proportions by volume coumestrol 1 Acetone-acetic acid-water (3:1:6) 0.088-0.10 2 Acetone-water (3:7) .10 - .16 3 Chloroform-acetic acid-water (2:1:1) __ .33 - .40 4 Benzene-acetic acid-water (2:2:1) .19 - .23 5 Butanol-acetic acid-water (4:1:5) .91 - .93 6 Methanol-benzene (10:40) .77 _ .80 7 Acetic acid-water (30:20) .55 - .60 8 Acetic acid-water-HCl (50:35:15) .55 - .60 9 ^ Benzene-acetone (9:1) 0 10 Methanol-chloroform (lí:89) .91 - .95 ^ Ascending chromatography. Source: Chury, Jiri (69). CHEMICAL AND BIOLOGICAL PROPERTIES OF COUMESTROL

Quantitative procedure Extracts were prepared from the forage samples for assay A sensitive quantitative paper (158), Fresh samples were rapid- Chromatographie procedure, us- ly mascerated with 95 percent ing a fluorometer, was developed. alcohol in a mechanical blender. The design of the instrument During cooperative studies with {1Í) provided for the insertion various laboratories throughout of paper chromatograms between the United States on the factors a UV radiation source-filter com- that influence the coumestrol con- bination and a detector-filter com- tent of alfalfa, this procedure bination. Intensity of ñuorescence was modified. Fresh material was proportional to galvanometer was extracted with methanol by readings. The method for the standing for at least 3 days (127^ quantitative measurement of cou- 2IS), Samples treated in this mestrol on paper chromatograms manner could be stored for at (159), using a fluorometer, in- least 3 months with no appreci- volved spotting known quantities able loss of coumestrol. Dried of pure coumestrol on sheets of samples were first rehydrated, Whatman No. 1 paper; develop- then extracted with either 95 per- ing the chromatograms in 50 percent ethanol or methanol. Recent- cent acetic acid, ascending; and ly, Chury (69) reported that a preparing a standard curve from higher yield could be obtained if the galvanometric readings. Mea- an enzymatic hydrolysis was car- surements from at least four ried out prior to alcohol extrac- spots of the same concentration tion. Extracts of samples low in were averaged to obtain each coumestrol had to be further point on the standard curve. purified to remove the waxes and Typical fluorometric measure- fatty materials that interfered ments are shown in table 5. Using with the paper Chromatographie this technique, quantities of cou- phase of the analysis. In the mestrol in the range of 0.2 to 1.0 earlier work, these impurities micrograms could be estimated were removed by extraction with with a standard deviation of petroleum ether. However, later about 7 percent. we found that chloroform was a more satisfactory solvent for extracting the impurities. In this TABLE 5.—Typical fluorometric procedure, the alcohol had to be measurements for coumestrol removed from the original extract. The aqueous phase was Galvanometer Coumestrol reading ^ then made basic with sodium car- concentration- bonate. After the green color was Operator A Operator B removed with chloroform, the Micrograms Millimeters Millimeters aqueous phase was acidified. Cou- 0.2 62.2 62.0 mestrol and the other phenolic .6 40.7 40.4 compounds were then reextracted 1.0 16.2 15.7 into ether, and a concentrate rep- ^ Average of 10 separate spots. Full- resenting a tenfold increase over scale deflection is 100 millimeters. the original extract was prepared. Source: Livingston, A. L., Bickoff, E. M., Guggolz, Jack, and Thompson, Extracts were analyzed on paper C. R. (159). chromatograms and coumestrol 10 TECHNICAL BULLETIN 1408, U.S. DEPT. OF AGRICULTURE concentration was reported on a the first dimension. This system dry weight basis, in parts per had the advantage of moving con- million. This method is sensitive taminating green pigments and to about 2 p.p.m. of coumestrol, lipoid material near the solvent with a maximum error of about front and gave a very good sepa- ±5 percent. ration of the polyphenols. Aque- Recently the paper Chromato- ous ammonia (2N) was found to graphie procedure was adapted be the most suitable solvent for for the quantitative measurement the second dimension. After the of coumestans and flavones in above separation, the concentra- alfalfa samples using two-dimen- tions of the individual isoflavones sional paper chromatography were determined by spectrophoto- Us), Samples were spotted two metric measurement. The level of to four times heavier than in coumestrol was too low to mea- the coumestrol analysis and de- sure by UV absorption. Its con- veloped in 50 percent acetic acid, centration was estimated visually ascending for the first dimension, based on the intensity of its fluo- followed, after drying, by iso- rescence. As little as 0.1 /xg. of propyl alcohol-concentrated am- coumestrol was detected by this monium hydroxide (2:1), de- technique. scending for the second dimen- While studying plant estrogens sion. Fluorometric measurements in sheep, Lindner {155) developed were made on each compound us- a procedure for determining mi- ing two templates to eliminate crogram quantities of coumestrol ñuorescence from surrounding and the estrogenic isoflavones in spots (fig. 4). The relative con- plasma and depot fat of sheep. centration of each compound was The assay procedure consisted determined from a coumestrol essentially of (1) extraction and calibration curve. After a correc- preliminary fractionation of es- tion had been made for the differ- trogens by solvent partition; (2) ence between the fluorescence in- paper Chromatographie separa- tensity of coumestrol and that of tion of the estrogens; (3) spec- an individual compound, the con- trophotometric determination of centration of the compound was the isoflavones after elution with calculated in the same manner as ethanol; and (4) fluorometric for coumestrol. determination of 5-deoxy-isoflavones and coumestrol, both on Other Chromatographie paper strips and after elution procedures with aqueous methanol. Figures Wong {222) developed a two- 5 and 6 represent flow-sheets for dimensional paper Chromato- the determination of these com- graphie method for the separation pounds in plasma and in body fat, and detection of coumestrol as respectively. Steps (3) and (4) well as the known estrogenic iso- could be satisfactorily replaced flavones in red clover. A purified by vapor phase chromatography alcohol extract of the fresh red of their trimethylsilyl ether de- clover was chromatographed on rivatives. Losses during the ex- Whatman 3MM paper, using the traction and purification proced- descending technique. The single ures were determined by the use phase system, benzene-acetic acid- of radioactive internal standards water (125:72:3), was used for and averaged 35 percent. CHEMICAL AND BIOLOGICAL PROPERTIES OF COUMESTROL 11

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DEPOT FAT

EXTRACTED WITH 60 ML. BENZENE-LIGHT PETROLEUM (1:1)

ORGANIC SOLVENT TISSUE RESIDUE EXTRACT

RE-EXTRACTED WITH 0.4N PARTITIONED SEQUENTIALLY N aO H ( 3 X 30 ML.) WITH ALKALI EXTRACTS 1 TO 3

COMBINED EXHAUSTED TISSUE ORGANIC PHASE ALKALI EXTRACTS (DISCARDED) (DISCARDED)

ADJUSTED TO pH 9.9 EXTRACTED WITH ETHYL ACET AT E ( 4 X 2 VOLUM ES)

CRUDE PHENOLIC AQUEOUS PHASE EXTRACT* (DISCARDED)

3|{ FURTHER TREATMENT AS FOR CRUDE ETHYL EXTRACT *'E" FROM PLASMA. SEE FIGURE 5

FIGURE 6.—Schematic flowsheet for the extraction of isoflavones from the body fat of sheep (155).

Adler and co-workers {9) mod- 10), the second. Coumestrol was ified Lindner's method (155) for eluted from the second chromato- determining both free and conju- plate and its concentration esti- gated coumestrol in whole bovine mated fluorometrically by Lind- blood. A crude ethyl acetate extract of the blood was purified by ner's technique (155). Conjugated sequential thin layer chromato- coumestrol was converted to free graphy. Chloroform-methanol- coumestrol by enzymatic hydroly- water (9:10:1) was used as the sis and its concentration estimat- first developer and cyclohexane- ed in the same manner as cou- ethyl acetate-methanol (45:45: mestrol. Isolation Isolation procedures for natu- successive extraction with sol- rally occurring phenolic com- vents of increasing polarity. Once pounds are generally based upon the crude compound is extracted CHEMICAL AND BIOLOGICAL PROPERTIES OF COUMESTROL 15 from the plant tissue, it may be commonly used Chromatographie purified by fractional crystalliza- techniques. Final purification is tion, by solvent partition, or by generally accomplished by recry- separations employing any of the stallization, sublimation, or both. Isolation of coumestrol from Ladino clover Coumestrol was first isolated The materials that did not ex- from Ladino clover in 1956 (26, hibit acidic properties were re- 31 ). The mouse-uterine-weight moved by partitioning between bioassay was employed to guide chloroform and dilute alkali (pH the various steps of the isolation. 10-11). The aqueous layer was Reground Ladino clover meal, acidified (pH 6) and re-extracted high in estrogenic activity, was with ether, which, after evapora- extracted with hot water to re- tion of the other, gave a yellow move the water soluble materials, product. including saponins. This was fol- Coumestrol was separated lowed by a Skellysolve C extrac- from the other phenolic and fla- tion to remove fats, waxes, chlo- vonoid-type compounds by a se- rophylls, and carotenoids. The ries of six countercurrent distri- crude coumestrol was separated butions (CCD). The solvent sys- from the residual meal by ether tems and the order in which they extraction, which gave a dark were employed are presented in tarry material upon evaporation table 6. The number of transfers of the solvent. Coumestrol and and the position of coumestrol at the other phenolic compounds the end of each distribution are were further purified by taking also given. The results of the es- advantage of their weakly acidic trogenic assays of the various properties. They are preferen- fractions obtained during the iso- tially soluble in organic solvents lation procedure are summarized when in their normal or lactone in table 7. For final purification, form. However, in their open coumestrol was repeatedly re- ring form (basic salt), they are crystallized from methanol-chlo- preferentially soluble in water. rof orm and sublimed.

TABLE 6.—Solvent systems employed in purification of coumestrol by countercurrent distribution No. of Coumestrol Solvents and proportions by volume transfers location Tubes ' Acetone-ether-water-Skellysolve B (10:5:5:2) 100 69-90 Chloroform-carbon tetrachloride-methanol-water (2:2:3:2) ^^^ 56-öü Methaiiol-benzene-ether-water (4:4:1:1) 100 30-60 Skellysolve B-ethyl acetate-methanol-carbon tetrachloride-water (1:1:1:1:1) 100 25-58 Acetone-carbon tetrachloride-water (2:1:1) 100 on ¿n Acetone-carbon tetrachloride-water-methanol (10:5:5:1) 280 ^0-6^^ ' Refers to location in 100 tubes of the Craig countercurrent distribution instrument. Source: Bickoif, E. M., Booth, A. N., Lyman, R. L., and others (31), 16 TECHNICAL BULLETIN 1408, U.S. DEPT. OF AGRICULTURE

TABLE 7.—Biological potency of fractions obtained during the isolation of coumestrol from Ladino clover

Amount fed Uterine Material tested per mouse weight Milligrams Milligrams Control (basal ration) 10 Ladino clover meal 3,000 15 Residue after water extraction _ 3,000 27 Residue after Skellysolve extraction 3,000 65 Concentrate from ether extraction 200 36 After extraction with alkali, and transfer back to ether 10 30 Coumestrol .67 95 Diethylstilbestrol .0004 72 Source: Bickoff, E. M., Booth, A. N., Lyman, R. L., and others {31).

Large-scale processing of alfalfa for coumestrol

For large-scale isolation {S2), and a number of other phenolic alfalfa was used as the starting compounds co-crystallized from material because of its commer- the upper phase as greenish-yel- cial availability {H5), The pro- low solids (about 70 percent cou- cedures for extracting coumestrol mestrol ). Recrystallization, first from Ladino clover were em- from isopropyl alcohol and then ployed with several modifications dimethylformamide (DMF), re- in order to facilitate the handling moved most of the green contam- of the large quantity of meal (45 inants and left a light yellow tons) {32), Acetone instead of product containing 85 percent ether was employed to extract coumestrol. This crude product the defatted meal. The acetone was purified by acetylation and extracted 2.8 percent of the sol- recrystallization of its acetate ids and 37 percent (6,200 g.) of from chloroform. Basic hydroly- the coumestrol originally present sis regenerated pure coumestrol. in the meal. The acetone concen- Paper chromatography was em- trate was distributed between ployed to follow this large-scale chloroform and alkali in a man- isolation of coumestrol. ner similar to that described for Portions of this coumestrol as Ladino clover. After acidification, well as some of the cruder frac- a dark gummy mass was col- tions were supplied to several lected instead of a yellow crystal- cooperating agencies to evaluate line solid. This was purified by their potential growth-promoting partitioning between methanol- properties with sheep {193) and water-ether-hexane. Coumestrol cattle {209). CHEMICAL AND BIOLOGICAL PROPERTIES OF COUMESTROL 17 Isolation of compounds closely related to coumestrol The chloroform filtrate from certain solvent systems with ade- the final purification of coumes- quate separations being made. Fi- trol acetate contained several gure 7 shows a schematic chro- hundred grams of crystalline ma- matogram of a typical CCD sepa- terial. This was combined with ration. For preliminary identifi- the crude coumestrol acetate ob- cation, a Roman numeral was as- tained by reworking various signed to an individual compound fractions of the large-scale pro- after it was isolated. Figure 8 cessing. After further recrystal- presents the sequence of these lization of these combined ace- distributions. tates from chloroform, deacetyla- A number of compounds were tion yielded over a kilogram of present in minute amounts and material containing about 60 per- were not detected by paper chro- cent coumestrol. Two-dimen- matography in the original prep- sional paper chromatograms of aration or in the initial fractions. this crude coumestrol prepara- However, as the major compo- tion showed the presence of at nents were removed by CCD, the least 15 other blue or yellow flu- minor ones became visible on the orescing compounds. A number developed chromatograms. A of these were isolated by CCD total of 12 compounds were iso- iil). Fifty-eight distributions lated. These included coumestrol, employing four solvent systems trifoliol, salicylic acid, tricin, and (table 8) were required to sepa- eight compounds which at that rate coumestrol and 11 of the time were of unknown structure. other phenolic compounds. By These have been subsequently using dimethylformamide (DMF) characterized as medicagol (com- as a carrier solvent, over 200 g. pound I ), 4'-0-methylcoumestrol of material were distributed in (compound II), 3'-methoxycou-

TABLE 8.—Solvent systems employed in countercurrent distributions of alfalfa phenolics

Solvent Solvents and proportions Starting Total Solids system by volume material solids per run Grams Grams A Skellysolve B-ether-methanol- Crude coumestrol water-DMF' (2:5:3:1:0.4). preparation. 1,000 40 B Ethyl acetate-Skellysolve B-ether- Fraction 2 860 215 methanol-water-DMF Fraction 11 56 28 (12:12:10:5:4:8). Fraction 12 96 24 C Acetone-Skellysolve B-ether-water Combined fractions (2:1:1:1). 4 and 7. 200 10 D Water-DMF-acetone-carbon Fraction 19 36 12 tetrachloride (2:4:3:5). ^ DMF = dimethylformamide. Source: Bickoif, E. M., Livingston, A. L., Witt, S. C, and others i^l). 18 TECHNICAL BULLETIN 1408, U.S. DEPT. OF AGRICULTURE SOLVENT FRONT

< OH

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FIGURE 7.—Example of a paper chromatogram of a typical countercurrent distribution separation {Jfl). See page 17 for names of numbered compounds.

mestrol (compound III), 4',7-di- and ll,12-dimethoxy-7-hydroxy- hydroxyflavone ( compound IV ), coumestan (compound VIII). Fig- 3', 4', 7-trihydroxyflavone (com- ure 2 presents the structures of pound V), lucernol (compound the coumestans that were iso- VI), sativol (compound VII), lated. Characterization of Coumestans The trivial name coumestan heterocyclic, four ring system was proposed by Mentzer {85) having the systematic name 6H- for the skeletal structure of the benzofuro [3,2,c] [1] benzopyran- CHEMICAL AND BIOLOGICAL PROPERTIES OF COUMESTROL 19

CRUDE CRYSTALLINE PREPARATION

CCD (Countercurrent Distribution) SOLVENT SYSTEM A

CCD, SOLVENT SYSTEM B ACETYLATE and RECRYSTALLIZE

TRIFOLIOL COMPOUNDS I and II ACETATE ACETATE

ACETYLATE and RECRYSTALLIZE TLC, THIN-LAYER CHROMATOGRAPHY

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COUMESTROL COUMESTROL and COMPOUND III ACETATE

TRICIN PRECIPITATES PAPER CHROMATOGRAPHY

COMPOUND IE

SOLVENT SYSTEM C I FRACTION 10 FRACTION 11 FRACTION 12 FRACTION 13 FRACTION 14

COMPOUND IS" SALICYLIC ACID CCD, SOLVENT SYSTEM B

FRACTION 15

I CCD, SOLVENT SYSTEM D COMPOUND Y COMPOUND H COMPOUND m

FRACTION 23

COMPOUND mn

FIGURE 8.—Sequence of countercurrent distributions for the isolation of coumestans and flavones from alfalfa (-4Í).

6-one (44-) and has been accepted lished by the American Chemical by Chemical Abstracts. The Society (160), names coumarino-c oumarone Both physical and chemical (113), c o u m a r i n o-benzofuran means have been employed in the (133) y and benzof ur ocoumarin characterization of the coumes- (132) have also been applied to tans. Derivatives such as acetates this class of compounds, of which and methyl ethers are useful to coumestrol is representative indicate the number of hydroxyl (113), The name coumarino- groups. Fusion studies and step- coumarone was not acceptable wise degradation to known com- according to the Ring Index pub- pounds have been employed to 20 TECHNICAL BULLETIN 1408, U.S. DEPT. OF AGRICULTURE determine the ring structure as tion of functional groups. Syn- well as the location of some of thesis is usually used for final con- the functional groups. Both UV and proton magnetic resonance firmation of structure. Table 9 (p.m.r.) spectral analyses have summarizes the physical data for been used in pinpointing the loca- these compounds.

Coumestrol The presence of two free hy- scribed for wedelolactone (111). droxyl groups was indicated by Methylative ring opening of cou- the formation of diacetoxyl and mestrol gave a trimethylether dimethoxyl derivatives (31), The methyl ester (fig. 10, lia), which empirical formula, CisHgOg, dif- was readily saponified to the fered from that of a typical fla- corresponding 2- ( 2,4-dimethoxy- vonoid by the absence of two hy- phenyl) -6-methoxybenzof uran-3- drogen atoms. Fusion studies carboxylic acid (fig. 10, lib). yielded only resorcinol and /3-re- Heating decarboxylated the acid sorcylic acid, giving an indication to 2- ( 2,4-dimethoxyphenyl ) -6- of the number and position of the benzofuran (fig. 10, III). Ozo- hydroxyl groups on the two nolysis of this product, followed rings. The UV absorption spec- by treatment with peroxide, trum of coumestrol (fig. 9) dif- gave 4-methoxy-2(2,4-dimethoxy- fered markedly from spectra benzoyl)-benzoic acid (fig. 10, of known isoflavones and fla- IV), which was hydrolyzed to vones having the hydroxyl pat- give the 2,4-dimethoxy- and 2- tern indicated by the fusion prod- hydroxy-4-methoxybenzoic acids ucts. (fig. 10, V and VI). These were the breakdown products expected The structure of coumestrol for a compound having the same as 3,9-dihydroxy-6H-benzofuro [3, basic ring structure as wedelolac- 2,c] [l]benzopyran-6-one (fig. 10, tone. The structure of coumestrol la) (44') was established by step- (7,12-dihydroxycoumestan, fig. wise degradation, following a 10, la) was then confirmed by its procedure similar to that de- unequivocal synthesis (P^).

Trifoliol Trifoliol was originally iso- and its 12-methoxyl derivative. lated from Ladino clover (38) This suggested that the methoxyl and later from alfalfa (H). Ele- group was probably in the mental analysis showed that tri- 12-position, and that one of its foliol was a monomethoxyl com- hydroxyl groups was in the pound having the empirical for- 7-position. Trifoliol was de- mula CieHioOe. The presence of graded to its benzofuran in the two phenolic hydroxyl groups same manner as described for was indicated by the formation coumestrol. Comparison of the of diacetoxyl and dimethoxyl de- p.m.r. spectra of trifoliol and its rivatives. UV spectral measure- benzofuran derivative showed ments indicated that trifoliol was that the D-ring (fig. 10) was sub- structurally related to coumestrol stituted at the 10- and 12-posi- CHEMICAL AND BIOLOGICAL PROPERTIES OF COUMESTROL 21

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X X \J II II N O- eu 24 TECHNICAL BULLETIN 1408, U.S, DEPT. OF AGRICULTURE Medicagol Medicagol was isolated from al- formula of CiTHnOr,, indicating falfa as a mixture with a second that the parent compound could compound ( 4'-0-methylcoumes- have been derived from a cou- trol) from which it could not be mestan containing a methylene- separated by standard tech- dioxyl and a hydroxyl group. The niques. Although thin-layer chro- presence of the methylenedioxyl matography (TLC) afforded a group was further indicated by a slight separation, its use was im- positive Hansen test (12^) and practical for isolating sufficient by p.m.r. and infrared (IR) spec- quantities for characterization troscopy. Strong acid hydrolysis studies. UV spectral studies of the mixture suggested that each of the medicagol acetate mixture compound had a hydroxyl group gave 7, 11, 12-trihydroxycoumes- in the 7-position. The two com- tan and 4'-0-methylcoumestrol. pounds were separated by frac- This further indicated that medi- tional crystallization of their cagol must be T-hydroxy-ll, 12- mixed benzofuran derivatives. methylenedioxy coumestan, Elemental analysis of medicagol which was confirmed by its syn- benzofuran gave an empirical thesis (161).

4'-0-Methylcoumestrol 4'-0-methylcoumestrol was iso- oxy) phenyl]- 6-methoxylbenzo- lated as a mixture with medica- furan (the benzyloxybenzof uran gol. Thin-layer Chromatographie of 4'-0-methylcoumestrol) con- comparison of the mixture with firmed its identity. A small known compounds suggested that amount of natural 4'-0-methylcou- it was 4'-0-methylcoumestrol. mestrol was subsequently obtain- Definite location of the hydroxyl ed by treatment of the mixture group on the unknown compound with 86 percent sulfuric acid. Un- was established by Dreparation der these conditions, medicagol of the benzyloxybenzof uran deriv- was completely converted to 7, 11, atives of the mixture. The benzo- 12-trihydroxycoumestan, while furans were separated by frac- 4'-0-methylcoumestrol remained tional crystallization. Elemental analysis of the benzyloxybenzo- unchanged. These were separated furans substantiated that one by CCD, and the natural product of the compounds had been a was compared with an authentic monomethoxyl derivative of cou- sample of 4'-0-methylcoumestrol mestrol. Comparison with au- {135), with which it was identi- thentic 2- [2-methoxy-4- ( benzy 1- cal (U2),

3'-Methoxycoumestrol 3'-Methoxycoumestrol was were obtained by paper chroma- isolated as a mixture with cou- tography. Elemental analysis in- mestrol from which it could not dicated that it was a monome- be easily separated. Limited thoxyl compound having the em- quantities of the pure compound pirical formula CieHioOe. Forma- CHEMICAL AND BIOLOGICAL PROPERTIES OF COUMESTROL 25

tion of diacetoxyl and dimethoxyl coumestrol vâs decarboxylated derivatives demonstrated the to the benzof uran. presence of tvô hydroxyl groups. Comparison of the p.m.r. spec- UV spectral studies indicated tra of the methylether and benzo- that the tvô hydroxyls vêre not furan derivatives of 3'-methoxy- ortho to each other {136) and coumestrol indicated that the that one vâs in the 7-position compound vâs substituted in {135), the 7-, 11-, and 12-positions. To establish the ring substitu- This functional group assign- tion pattern of 3'-methoxycou- ment was confirmed by com- mestrol, it vâs desirable to de- parison of the methylated deriva- grade the compound to its benzo- tive of 3'-methoxycoumestrol furan derivative. Since it was with 7, 11, 12-trimethoxy coumes- difficult to obtain sufficient ma- tan {161), with which it was iden- terial for this purpose by paper tical. Comparing the UV spectrum of 3'-methoxycoumestrol chromatography, the mixture of with those of known coumestans coumestrol and 3'-methoxycou- indicated that the methoxyl group mestrol vâs taken to their ortho- was in the 11-position. methöxycinnamic acid derivatives Definite assignment of the via methylative ring opening, fol- methoxyl group to the 11-position lovêd by hydrolysis. These deriv- and confirmation of the structure atives vêre separated by CCD of 3'-methoxycoumestrol were ac- and the or¿/¿o-methoxycinnamic complished by its unequivocal acid derivative of 3'-methoxy- synthesis {i5).

Lucernol The UV spectrum of lucernol of an or¿/¿o-dihydroxyl grouping. was similar to that of the cou- The p.m.r. spectral studies indi- mestans. Analytical data indicated that lucernol was either cated that no methoxyl groups 7,11,12-trihydroxycoumestan or 6, were present. The formation of 7, 12-trihydroxycoumestan. Com- triacetoxyl and trimethoxyl de- parison of the physical properties rivatives confirmed the presence of three free hydroxyl groups. of lucernol with those of 7, 11, Degradation by alkaline fusion 12-trihydroxycoumestan {161) gave resorcinol and hydroxyhy- proved them to be different. droquinone, which suggested the Thus, the structure of lucernol number and possible positions of had to be 6, 7, 12-trihydroxy- the hydroxyl groups on the two coumestan {205), This structure rings. UV spectral studies indi- was later confirmed by its syn- cated a hydroxyl group at the 7- thesis by Kalra and co-workers position as well as the presence {138),

Sativol Analysis of sativol, its acetoxyl two hydroxyl groups. Degrada- and methoxyl derivatives, in- tion by alkaline fusion gave dicated that it was a mono- a mixture of four compounds methoxyl compound containing that were identified as re- 26 TECHNICAL BULLETIN 1408, U.S. DEPT. OF AGRICULTURE sorcinol, ^-resorcylic acid, pyro- Therefore, the lone methoxyl gallol, and pyrogallol carboxylic group had to be at the 7-position acid. As with lucernol, these and the hydroxyl groups at the products suggested the presence 8- and 12-positions. The location of one oxygen-containing func- of the lone methoxyl group was tional group on one ring and two confirmed by p.m.r. spectroscopy on the other. Systematic degrada- through a comparison of the tion using the same procedure as ether-acetate shift of the aro- described for coumestrol indi- matic protons of sativol with cated that sativol had substi- those observed for several cou- tuents at the 7-, 8-, and 12-posi- mestans of known structure. tions. This substitution pattern Thus, the structure of sativol is was confirmed by p.m.r. studies. 7-methoxy-8, 12-dihydroxycou- UV spectral studies indicated mestan (205), Kalra and co- that sativol did not contain a hy- workers (138) confirmed this droxyl group at the 7-position or substitution pattern by the syn- an or¿/¿o-dihydroxyl grouping. thesis of sativol methylether. ll,12-Dimethoxy-7- hydroxycoumestan Elemental analysis of 11, 12-di- thentic 7, 11, 12-trimethoxycou- methoxy-7-hydroxycoumestan and mestan (161), UV spectral studies its acetoxyl and methoxyl deriva- indicated that the hydroxyl group must be located at the 7-position. tives indicated that it was a di- The structure of this compound methoxylcoumestan containing as ll,12-dimethoxy-7-hydroxycou- one hydroxyl group. Its methoxyl mestan was confirmed by synthe- derivative was identical with au- sis (173, 206). 4',7-Dihydroxyflavone 4',7-Dihydroxyflavone was iso- data indicated that the compound lated from both alfalfa and Lad- was a flavone. Alkaline fusion ino clover (iO), The empirical studies suggested that the com- formula, C15H10O4, was typical pound was 4',7-dihydroxyflavone. of a flavonoid compound. The This was confirmed by compari- presence of two hydroxyl groups was demonstrated by the forma- son of the natural compound tion of a diacetoxyl derivative. with an authentic sample of 4', Color reactions and UV spectral 7-dihydroxyflavone. 3, 4',7-Trihydroxyflavone 3',4',7-Trihydroxyflavone, iso- derivative confirmed the presence lated from both alfalfa (i6) and of three hydroxyl groups. The Ladino clover (157), had an em- UV spectral shifts suggested that pirical formula of C15H10O.5, the compound was 3',4',7-trihy- which suggested that it was a droxyflavone. This was confirmed trihydroxyflavone. Color tests by comparison with an authentic and UV spectral studies confirmed the basic flavone struc- sample of 3',4',7-trihydroxyfla- ture. Formation of a triacetoxyl vone. CHEMICAL AND BIOLOGICAL PROPERTIES OF COUMESTROL 27 Synthesis of Coumestans Four approaches have been techol with substituted 4-hydroxy- employed for the synthesis of coumarins, and (4) preparation coumestans. They are: (1) cycli- of a new coumestan from one zation of appropriately substituted 4-hydroxy-3-phenylcoumar- prepared by any of the above ins, (2) oxidation of flavylium procedures. The four methods •salts, (3) condensation of ca- will be discussed separately. Cyclization of 4-hydroxy-3-phenylcoumariii derivatives The first synthesis of coumes- ified through its diacetoxyl deriv- trol was accomplished by Emer- ative. son and BickofF (P^). They con- Kawase (139) shortened the densed 2,4-dimethoxyphenylace- synthesis of coumestrol by start- tonitrile (fig. 11, I) with resor- ing with 2,4-dimethoxybenzoic cinol (fig. 11, II) in a Hoesch- acid methyl ester instead of re- type reaction to give a-(2,4-di- sorcinol (fig. 11, II), which gave methoxyphenyl) -2, 4-dihydroxy- 2,4-dimethoxybenzoyl-(2,4 - dime- acetophenone (fig. 11, III). thoxyphenyl) acetonitrile as the This product was then con- intermediate. This was converted densed with methyl chlorofor- to coumestrol by demethylation mate using the procedure de- and simultaneous ring closure. scribed by Gilbert and co- Other coumestans have also been workers (109) to give a methyl- synthesized using similar proce- formate derivative (fig. 11, IV). dures. They include wedelolac- Hydrolysis with alkali gave 3- tone trimethylether by Govinda- ( 2,4-dimethoxypheny 1 ) -4,7-dihy- chari and co-workers (Hi), lu- droxycoumarin (fig. 11, V), cernol and sativol dimethylether which was converted to coumes- by Kalra and co-workers (138), trol (fig. 11, VI) by demethyla- as well as the basic unsubstituted tion with simultaneous ring clo- parent coumestan by Des- sure. The crude product was pur- champs-Vallet and Mentzer (85),

Oxidation of flavylium salts In connection with a study of was oxidized to a carbomethoxy- anthocyanins and related com- benzofuran (fig. 12, IV), which pounds, Jurd (133, 13i) found rapidly lactonized on acidification that coumestan-type compounds to coumestrol (fig. 12, V). The could be prepared by the oxida- synthetic coumestrol was identi- tion of flavylium salts. For the cal with the natural compound in synthesis of coumestrol (fig. 12, all respects. V), 2,4-dihydroxybenzaldehyde A number of other naturally was condensed with w-methoxy- occurring coumestans have been 2, 4-dibenzyloxyacetophenone synthesized using Jurd's proce- (fig. 12, I) to give the benzyloxy- dure. They include the following: flavylium salt (fig. 12, II). Acid trifoliol by Livingston and co- hydrolysis gave 2',4',7-trihydroxy- workers (160), medicagol by 3-methoxyflavylium chloride (fig. 'Jurd (132), 3'-methoxy cou- 12, III). This flavylium salt mestrol by Bickoff and co- 28 TECHNICAL BULLETIN 1408, U.S. DEPT. OF AGRICULTURE

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TABLE 10.—Physical data for synthetic coumestans

Melting point Figure -— ■ Compound 13, III Parent Acetate Methylether compound derivative derivative ° c. ° c. ° c. 7,10-Dihydroxycoumestan e 278-79 247-48 230-31 7-Methoxy-lO-hydroxycoumestan c 234-35 236 230-31 7-Hydroxy-lO-methoxycoumestan f 333-34 288-89 230-31 7,11-Dihydroxycoumestan g >350 253 211.5 7-Methoxy-ll-hydroxycoumestan d 281-82.5 229-31 211.5 7,13-Dihydroxycoumestan b >325 214 '204 7-Hydroxy-13-methoxycoumestan a '303 214-15 '204 ' Reported melting point is 195° C. (133). ' Reported melting point is 289° C. {133). Source: Spencer, R. R., Knuckles, B. E., and Bickoff, E. M. (207). workers (^5), and 4'-0-methyl- which was debenzylated with acid coumestrol by Jurd (133). to give the desired flavylium salt. Jurd's procedure has been used The flavylium salt was then oxi- to synthesize a number of cou- dized to give the coumestan (fig. mestans that are presently not 13, III). The orientation pattern known to occur in nature. In addi- of the D-ring (fig. 13, I) is gov- tion to 7,10,12-trihydroxy- erned by the aldehyde. An alde- coumestan (the parent of trifo- hyde (fig. 13, II) substituted at liol) (160), a series of isomers of Ri (Ro = R3 = H) will produce sub- coumestrol, which have a differ- stitution at the 13-position in the ent substitution pattern in the D-ring (207)y have been pre- D-ring. Similarly, Rs (Ri = R3 = pared. An appropriately substi- H) substituted aldehydes produce tuted ort/io-hydroxybenzaldehyde the 11-series and Ra (Ri = R2 = H), was condensed with w-methoxy-2, the 10-series. Table 10 gives a 4-dibenzyloxyacetophenone to summary of the coumestans that form the benzyloxyflavylium salt, were prepared in this study. Condensation of catechol with substituted 4-hydroxycoumarins With his synthesis of wedelo- has been found to react with a 4- lactone, Wanzlick (219) devel- hydroxycoumarin in this reac- oped a novel method of synthesiz- tion. Therefore, the only substi- ing the coumestan structure. tution that can be obtained in the Wedelolactone (fig. 14, III) was D-ring is at the 11- and 12-posi- formed by the condensation tions. However, any substitution of 4,5-dihydroxy-7-methoxycou- can be effected in the A-ring de- marin (fig. 14, I) with catechol pending on the 4-hydroxycou- (fig. 14, II) in an aqueous-ace- marin used in the coupling reac- tone mixture of potassium ferro- tion. Employing this reaction, cyanide and sodium acetate. Ca- Livingston and co-workers (161) techol is the only compound that synthesized medicagol by conden- CHEMICAL AND BIOLOGICAL PROPERTIES OF COUMESTROL 31

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o 32 TECHNICAL BULLETIN 1408, U.S. DEPT. OF AGRICULTURE sing 4,7-dihydroxycoumarin with Fukui and co-workers {108), 11, catechol to give 7,11,12-tTÍhy- 12 - dimethoxy-T-hydroxycoumes- droxycoumestan. This was then tan from 7-benzyloxycoumarin by converted to medicagol by direct methylenation with di-iodo-meth- Malleshawar and co-workers ane. Similarly, medicagol dimeth- {173), and erosnin from 5-hy- ylether was synthesized from droxypsoralene by Fukui and 4-hydroxy-7-methoxycoumarin by Nakayama {107), Synthesis from previously formed coumestans Jurd {185) synthesized the 4'- coumestan (fig. 13, Illd) were 0-methyl and 7-0-methyl deriva- prepared from their respective tives of coumestrol by selective diacetates by Spencer and co- alkylation of coumestrol diace- workers {207). They also pre- tate. The 7-0-methyl derivative pared 7,13-dihydroxycoumestan was prepared directly from cou- (fig. 13, Illb) by demethylation mestrol diacetate by selective of 7-hydroxy-13-methoxycoumes- methylation followed by hydro- tan {207), lysis. In order to prepare the 4'- Spencer and co-workers {206) 0-methyl derivative, however, it synthesized 11,12-dimethoxy-7- was necessary to block the 7-po- hydroxycoumestan from 7,11, sition with a base stable group. 12-trihydroxycoumestan (fig. 15, Thus, selective benzylation of la). The 11- and 12-positions coumestrol diacetate under simi- were first blocked by the forma- lar conditions gave 7-0-benzyl- tion of the diphenylme- coumestrol monoacetate. After thylenedioxyl derivative (fig. 15, hydrolysis, this compound was lia). Benzoylation (fig. 15, lib), methylated, forming 7-0-benzyl- followed by acid hydrolysis, rege- 4'-0-methylcoumestrol. Debenzy- nerated the 11,12-dihydroxyl de- lation of the latter compound rivative (fig. 15, lb). Methylation gave 4' - 0 - methylcoumestrol, of the 11- and 12- positions then which was identical with the formed 7-0-benzoyl-ll,12-dimeth- natural 4'-0-methylcoumestr ol. oxycoumestan (fig. 15, Ic), which Using this procedure, 10-hydroxy- upon basic hydrolysis yielded 7-methoxycoumestan (fig. 13, 11, 12-dimethoxy-7-hy dr oxycou- IIIc) and ll-hydroxy-7-methoxy- mestan (fig. 15, Id). Physical Properties of Coumestans The physical properties of cou- These properties also assist in mestans, such as UV and determining the substitution pat- p.m.r. spectroscopy, and chromatography can be used as aids tern and type of ring substi- for structural determination. tuents. Ultraviolet absorption spectral measurements Coumestans generally exhibit micron region (band III) and the high intensity UV absorption in 200 to 215 millimicron region the 340 to 355 millimicron region (band IV) along with low inten- (band I), in the 230 to 250 milli- sity absorption in the 300 to 320 CHEMICAL AND BIOLOGICAL PROPERTIES OF COUMESTROL 33

3 o N o II II Ti

o SI O

0) S I

tí

CO X

o -Û u -^ 34 TECHNICAL BULLETIN 1408, U.S. DEPT. OF AGRICULTURE millimicron region (band II) (fig. acetate (135), indicating that of 9). A shift in band I of a coumes- the two hydroxyl groups only one tan indicates the presence of a hy- (the 7-hydroxyl) is sufficiently droxyl group at the 7-position. acidic to be ionized by the weak- For example, in the spectrum ly basic sodium acetate. of lucernol (6,7,12-trihydroxy- In the presence of sodium ace- coumestan) a shift with sodium tate, boric acid chelates with acetate is observed (fig. 16). phenolic compounds, including However, when the 7-hydroxyl is coumestans, which contain ortho- blocked, as in sativol (fig. 17), dihydroxyl groups to cause a this shift is not observed. This bathochromic shift. The spectra can be explained by the fact that of coumestans that do not con- the hydroxyl group in the 7-posi- tain this grouping are not ap- tion is conjugated with the lac- preciably affected. For example, a tone carbonyl and, therefore, is 5 to 10 m/x shift is observed in the strongly acidic. In confirmation spectrum of lucernol (fig. 16) but of this observation, band I of cou- not in the spectrum of sativol (fig. mestrol (Amax 344 millimicron, 17). A summary of the peak loca- table 11) is shifted to 387 millimicrons in sodium ethylate but tions and shifts of representative only to 362 millimicrons in sodium coumestans is given in table 11.

225 250 275 300 325 350 375 400 425 450 WAVELENGTH, mM

FIGURE 16.—Ultraviolet absorption spectra of lucernol in ethanol: (1) neutral, (2) sodium acetate, (3) boric acid-sodium acetate. CHEMICAL AND BIOLOGICAL PROPERTIES OF COUMESTROL 35

0.8 1 1 1 1 1 K\ A 0.6 -^A i\ — \ - \ 1 L \ 1 \ I 0.4 1 \ O 1 \

FIGURE 17.—Ultraviolet absorption spectra of sat!vol in ethanol: (1) neutral, (2) sodium acetate, (3) boric acid-sodium acetate.

Proton magnetic resonance spectral measurements The p.m.r. spectra of coumes- preciable effect on the shielding tans, like their UV spectra, have of the H-5 proton. In spectrum 2 several characteristic features. of figure 18, one of the low field Although most of the proton re- or¿/¿o-doublets has remained un- sonances occur in the range of changed while the other has T = 2.50 to 3.50 of the aromatic shifted upfield. The unshifted region, the signals from two of doublet is assigned to H-5 and the protons (H-5 and H-10) are the shifted resonance to H-10. A located significantly dov^nfield jö-furanal proton is also produced from the other aromatic protons. as a result of decarboxylation. Its Figure 18, spectrum 1, shoves signal is found in the region these peaks in sativol dimethyl- T = 2.8 to 2.9. Since the only pro- ether. Decarboxylation of a cou- ton that is known to couple to mestan has a major effect on the H-3 is H-13, a 1.0 Hz splitting of position of H-10, due to the remo- the H-3 signal provides direct ev- val of the paramagnetic shift idence for the absence of substitu- caused by the nearby carbonyl tion at H-13. This doublet is group, but does not have an ap- clearly evident in spectrum 2 of 36 TECHNICAL BULLETIN 1408, U.S. DEPT. OF AGRICULTURE

•Si^ + 'C" lOO n3^+ O ' lO O O ü ^ ' U2 03

2 os :i 00 Oí 00 ooo O 00 00 'o

o ^^ 00(N00 oooo o 00 ï> S OTHO OIHTH rH O O i (M(M

3-S .ST3 lOO ICO

CO .3 ^ Oi CO o o o O C/2 CÔ

3. '^ OOO (NCO t> IH) l> CO 00 0 ^ TjíCOCO ■^^ 'íCp "T^ T^ TÍ^ T^ ^^

ce O o t COOI U2 a 1^ :i.COC

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ce i00(N i^ iCOCO co

Ji (M Oq t-C C

a "^ 0(M io(M c 00 (M oa ii^ Tj< lO t-lO Ti<-rji LO r}< TJH Ti< lO i^COCOCOCOCOCOCOCO CO CO CO

PQ < I OS 2 •

§ o st s «.'S § o -Pr-H o o o CQ o (M s go -5-lg o'u •■SOS'S-1^ OH IXl-rH 00

Mass spectral measurements Although not a new technique, coumarin carbonyl. Coumarins mass spectrometry has only re- that are unsubstituted on the py- cently been applied to the study rone ring show this fragmentation of the naturally occurring oxy- to a more marked extent.^ gen heterocyclics. Barnes and Oc- Coumestrol shows an intense colowitz {15) reported the mass molecular ion that is so stable spectra of a number of com- that the loss of CO is the only pounds containing the coumarin significant fragmentation (fig. structure to serve as a guide to 19). The monomethyl derivatives more complex systems. The char- of coumestrol show the loss of acteristic fragmentation under one CH3 and two CO functions electron impact is the ready loss of CO from the pyrone ring to that results in the formation of a form an ion having the benzo- benzofuran in which the D-ring furan structure followed by a has also been converted to a 5- further loss of the remaining member ring (fig. 20). oxygen atoms, again as CO. Only preliminary studies have ^ Personal communications from C. S. been carried out on coumestans.^'^ Barnes and J. L. Occolowitz, research chemists, C. S. R. Research Laborator- Apart from the breakdown of ies, Australia, 1965. methoxyl functions, the only * Personal communication from Chris major fragmentation results Falshaw, chemist, Massachusetts Insti- from the loss of CO from the tute of Technology, Boston, 1964. 38 TECHNICAL BULLETIN 1408, U.S. DEPT. OF AGRICULTURE

fi O o o CO 1

ir» un CNI o CO ^ <

«lo O >- r-) O :r. r>4 1— CO ^ LU II < __ o > o _J '^ ■•^ 2

^ S3 TH »O

w to

o O u ta 3

un tí Oí

tíc- o w

I od CHEMICAL AND BIOLOGICAL PROPERTIES OF COUMESTROL 39

o c4

00 co*^

CO ñ o o 5à o oo O o ñ ft

1 CO w O CÔ ü os

■gco ■ga. O 2 ■

it! o ÍL^Tf ^ O -§ 00 có CO HO áoo § Ö •ta o ^^ ^ Cs¡(M* Co> (N loO ft »^ "^ 5^ «5 -^ CO 00

(M2 > coco 'S'" w G 'Stí §o So çj- o

CO ^ ft ^ H3 ^ (M* (M* W r-iíO g ô Oí >»^ . -ü «O W 'oq >» tíoo ^ t be tí

CO •w o o >^ iH t- 00 '«^ o '<* oa TH ^(N IH IH 00 10 (N(M00LOCO00LOrH00TÍ

TH 1—1 Ö tí ^ TH .1-4 o TH O a wI £00 O OiCOOi00T-JO50qr-H tí ^ tí o I I I CO ^ Co e tj» o O) Ö § o ^ S TH 'S o !ooTí ;OCÍT—lOOiT-H'^^CO ^ w o ., ^ O •^* 10 .V 1^ >» o" X «i " o 09 05 o « TH O TH CO <5 '^ 1 r> 00 ^^ O CO '^^ <^ "^ CO N g ^ T3 >» CO C<Í ' CO (M W C« PQ Xi I bC •p-ttí Ö -M c3 Oí so í3 tí -M ^« tí t» O '^ t~ t^ " " IC (M CO C3 O) í^ ^ O 00 (M •§ w 00 Tl< o 05 O'^^'*^ 'o ta tí §1 CÖ «Ho tí tí o ^ tí u^.2 03 s S S d S tí Ö -^ (N (N 00 10 CO íO 00 00 ^ .• tí O tí 4J lili u o »> g^ I n3 O) CO oN tí tí o o iS CO S tí O) >> ü 0 is| X >> tí !rt«H ^ Qi +3 tí J ^ tí o X tí tJ o cu CÔ «* y-^ g ü S tí T 2 bÓ QJ tí O tí (N i tí TH ü (M ^í g S S o '^ cQ tí rtí as tí w ï ?^ r-t TH 4J «r^ tí tí tí J-" Ji tí ÍH C g OÍS'3 tí-e ^ .s H H «I SdJS s -•^ tí -M O) «H (M «H o o S)5 o '^ o tí tí Ulm

6 ^ rtíc « :z; CD l> OOOï o ^ CHEMICAL AND BIOLOGICAL PROPERTIES OF COUMESTROL 41

OH OH

268 240

FIGURE 19.—The mass spectral fragmentation pattern of coumestrol, (See text footnote 3, page 37.)

►=o

239

FIGURE 20.—The mass spectral fragmentation pattern of 4'-0-methylcoumestrol. (See text footnote 3, page 37.)

Trifoliol behaves more like -CH3 -CO -CO coumestrol and shows the loss of (312 —^297 —»^269 —^241). one CH3 and one CO function Although these measurements do -CH3 -CO not give as much information as (298 p.m.r. spectroscopy about the mo- 283 255). lecular structure of these com- Its monomethyl derivatives lose pounds, they are useful in deter- one CH3 and two CO functions, mining the molecular weights of similar to the monomethyl cou- the compounds and the presence mestrol derivatives of the pyrone ring. Chromatography Coumestans characteristically oped chromatograms to ammonia fluoresce when exposed to UV vapor or spraying them with di- light. This property makes them lute base usually intensifies the very adaptable to Chromato- fluorescence and, in some cases, graphie studies. It also offers a changes the color of the spots tool to assist in their separation (table 14). In general, coumes- and purification. Exposing devel- tans containing only hydroxyl- 42 TECHNICAL BULLETIN 1408, U.S. DEPT. OF AGRICULTURE

» Oc C3

■v-^, CO r—I eu CO ■+-> . ^ Pi. o ^ 0) ^ 3 52 KH o 1^1 où ce CD öc -p o^ 2 .2 í3 ^ si 0 fi 0 ^ o g

■^^ o

-»-* bD ^ '-rj S ¿ ••tí-^ ^5 äo'ö ¿^ I C3 0) 0^ X ftr-H 0) o CO ^ S2S 5L( -M -M S3^ o; oS cC ;¿ Ö CMPPQ pq S Soi> rfi Ö ^ S;§ . 0 fi 'fi S «.> ^ ± Ö Ö (Mt>0 fi "*"* ^ S Sa>

t?2 O)fi fiCD 00 ff ^ 5-( iT) t- CO. rH (>3 >

•S 2 2 o o p w 3 o ?3 fH ^ '^ ' ci «H «H -^^'^ . 5 Q) 0) O^^ W 2 Ci CÖ CÖ Ci ^ >» <3; o -M -♦-> -+-> < Ci Ci Ci •^ OTHi fi SS QPQ o T^ OH CO 1-^021-1 CHEMICAL AND BIOLOGICAL PROPERTIES OF COUMESTROL 43 functional groups exhibit white free hydroxyl group have Rf to gray fluorescence. The intro- values in the 0.6 to 0.8 region, duction of a single methoxyl and those containing three or group at the 7-position changes more hydroxyl groups have Rf the fluorescence to violet or pink. values below 0.3 in these solvent A blue fluorescence is generally systems. Thus, by employing the exhibited by coumestans with combination of Rf values and other substitution patterns. The color under UV light, paper chro- relative fluorescence intensity of matography can be an aid in de- the coumestans, as compared to termining the structure of a cou- coumestrol, is given in table 14. mestan. At this laboratory, the solvent Although, the best resolution systems that gave the best reso- of these compounds has been ob- lution of these compounds were tained with paper chromatogra- 50 percent acetic acid, ascending, phy, thin-layer chromatography and isopropyl alcohol-concen- (TLC) gave better resolution of trated ammonium hydroxide their methylated and acetylated (2:1), descending. These systems derivatives. Silicic acid chroma- have been used both for one- and tostrips have also been used for two-dimensional chromatography- separating their degradation The Rf values for the cou- products (169). Ether-Skelly- mestans in these solvents are solve B (7:3) gave the best sepa- given in table 14. These values ration of methylether deriva- represent a ratio between the dis- tives, while chloroform was the tance the compound travels and best for acetylated derivatives. the distance the solvent travels. Other workers have applied both In general, coumestans contain- paper Chromatographie and TLC ing two free hydroxyl groups, the techniques to the separation of remaining substituted with meth- coumestans. The solvent systems oxyl or methylenedioxyl groups, they used are discussed under have Rf values in 0.4 to 0.6 re- '^Analytical Procedures^' and gion. Those coumestans with one ^'Coumestrol in Plants." Coumestrol in Plants Estrogenic activity of forages grees of success. However, be- is extremely variable (36, 1Í2, cause of great environmental US, 197, 211) and has been variations occurring under nor- shown to be related to such fac- mal field conditions, it has been tors as cuttings, stages of difficult t o correlate estrogenic growth, diseases, and genetic and activity or coumestrol content environmental diflFerences. Since with plant growth and develop- 1950, many studies have at- ment. Therefore, recent work on tempted to correlate estrogenic coumestrol content of alfalfa has activity with one or more of been carried out under controlled these variables, with varying de- environmental conditions. Variations as affected by cuttings and stages of growth Cheng and co-workers (66, hays, obtained evidence that es- 67), studying estrogenic sub- trogenic activity increased with stances in alfalfa and clover number of cuttings. In a more ex- 44 TECHNICAL BULLETIN 1408, U.S. DEPT. OF AGRICULTURE tensive study on alfalfa, Pieterse uced alfalfa meals contain less and Andrews (197) found that than 100 p.p.m. coumestrol estrogenic activity increased with (lJf5). Loper (162), studying al- maturation, although the estro- falfa grown under controlled en- genie pattern varied from cutting vironmental conditions, found ev- to cutting. In later studies, Bick- idence that senescence may cause off and co-workers (23 ^ 28) the accumulation of coumestrol. found that the estrogenic activity Francis and Millington (10Jf) of alfalfa increased as the plant found that the coumestrol con- matured and usually reached its tent of annual medic species in- highest level at full bloom or creased with maturation and sub- seedhead stage (table 15). Simi- sequent senescence. Although cou- lar results were also obtained mestrol levels were compara- with white clover clones, includ- tively low at all growth stages up ing the Ladino variety (37), In to flowering, they reached consid- cooperative studies by WURDD erable proportions in the natu- with commercial dehydrators rally dried standing plants, espe- (IJfJfy li5)y the coumestrol con- cially Medicago littoralis Harbin- tent of alfalfa during the grow- ger (335 p.p.m.) (table 16). In ing season was shown to vary the flowering plants, coumestrol greatly with number of cuttings was concentrated in the mature as well as with stage of plant de- leaves. However, in the dried velopment. By allowing the standing plants, the stems and plants to mature to the seed pod pods contained the majority of stage, far beyond the normal har- the coumestrol. Loper (162) was vest age, dehydrated alfalfa unable to find any coumestrol in meals containing from 340 to 560 either the immature or mature p.p.m. coumestrol were obtained. seed pods of barrel medic plants Generally, commercially prod- (M e die a go littoralis Rhode).

TABLE 15.—Variation in estrogenic activity with stage of growth of alfalfa, 1958

Amount of coumestroP as determined from the following cuttings Stage of maturity — ZTTTTl, First Second Third Fourth Fifth Sixth ^ P.p.m. P.p.m. P.p.m. P.p.m. P.p.m. P.p.m. Vegetative . 13 0 6 11 7 12 Early bud _. 0 0 1 20 11 Full bud 0 0 111 17 10 1/10 bloom 7 0 92 49 23 1/4 bloom 9 20 47 27 1/2 bloom 33 63 93 43 17 Full bloom 77 262 98 103 44 Dough 120 94 181 93 41 Seedhead 123 187 210 198 ^ As determined by mouse-uterine-weight bioassay and reported on a dry- weight basis. " First cutting of 1959 crop. Source: Bickoff, E. M., Booth, A. N., Livingston, A. L., and Hendrickson, A. P. (28). CHEMICAL AND BIOLOGICAL PROPERTIES OF COUMESTROL 45

§5 ILOOOOCMçOOS ö* CO TH O (N LO Tj< *r CO

<> tOLOOOTÎ o, lO o o CO c

CO o • OOIOOOOOOOOÏ

snoiAqo ^. OOLOO(MTi<00 gi^ OOOi 00 lO THTH o CO

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5>

Acor>co o .í: O

*^(X)C01OO5THXOC0

si puooag JO ^SJTj[

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feo

o ^ 13 •43 Oí 03 o o O O Si CÖ .Êf > O c« s?H s s ^ 05 T3 o 3^ rr^z |§* WüoSoQpqW -ÖCO

TH «5 . gtí Ö^ O)^

< s-S g^ 03 .. Ö 0) ft iJ w '^ CO f^ CO Ä CO

^^ I*^ P^ R^ ^^ P^ F^ 46 TECHNICAL BULLETIN 1408, U.S. DEPT. OF AGRICULTURE

Furthermore, he found that se- medic plants exhibiting severe nescence by itself did not lead to physiogenic leaf spotting. Work- large amounts of coumestrol. ing with soybeans, Wada and Ya- However, he found coumestrol hara {217) reported that coumes- concentrations as high as 2,362 trol was higher in the sprouts p.p.m. in the mature leaves of than in the bean itself.

Variations as affected by location and climatic conditions In cooperative studies with Moapa variety of alfalfa grown Crops Research Division of ARS, under different climatic condi- Bickoff and co-workers {37) tions (table 18). The alfalfa found considerable differences grown at 5,000 feet was under al- between the estrogenic activity of most continuous moisture condi- Ladino clover grown in South tions from rain and fog. Conse- Carolina and that grown in Cali- quently, these plants had much fornia. Similarly, Ochi and co- less sunshine than those grown workers {189) in Japan attrib- at sea level or 2,000 feet. The uted the variation in estrogenic plants that had the least sun- potency among Ladino clover shine and the most moisture con- samples to differences in site of tained three times the level of sample collection. Millington and coumestrol. co-workers {183) found that the Loper and Hanson {164^) stud- coumestrol content of Cyprus and ied the coumestrol content of commercial barrel medic vari- Ranger alfalfa grown under con- eties grown in adjacent plots in a trolled environmental conditions number of Australian localities and found essentially no differ- varied considerably (table 17). ences between plants grown at Cooperative studies on alfalfa by different temperature regimes. WURDD with commercial dehy- Hanson and co-workers {127) drators in California, South Da- were unable to find any correla- kota, Pennsylvania, and Kansas tion between light intensity and further confirmed that geo- coumestrol content in field tests. graphic location can influence TT. T coumestrol-r> .,1 . content . TT {1^5). •• i. j« j ^PersonalT"! 7 communication • x- from4. E. J. Britten,' m Hawaii, studied Britten, agronomist, University of the coumestrol content of the Hawaii, 1961.

TABLE 17.—Variation in coumestrol content of barrel medics groivn at different localities in Australia [Mean of 10 estimates; measured in parts per million of coumestrol ± standard error]

Location Variety Mendel Perth Cunderdin Merredin Toodyay Cyprus 50 ± 3.8 55 ± 2.6 40 ± 4.6 180 ± 14.2 160 ± 12.6 Commercial 45 ± 3.4 45 ± 2.6 100 ± 6.9 150 ± 14.8 210 ± 13.8 Source: Millington, A. J., Francis, C. M., and McKeown, N. R. (183). CHEMICAL AND BIOLOGICAL PROPERTIES OF COUMESTROL 47

TABLE 18.—Variation in coumestrol content of alfalfa grown at different experiment stations in Hawaii

Station elevation Rainfall Average Source of Coumestrol (feet) temperature moisture Inches/year ° F. P.p.m. Sea level 12 67-85 Irrigation 39.5 2,000 75 61-73 Rain 31.5 5,000 100 49-63 Rain and fog 99.0 Source: See text footnote 5, page 46.

Variations as affected by varietal and genetic differences Francis and Millington (lOi) ranged from 6 to 25 p.p.m., while found wide differences in the cou- estrogenic activity ranged from mestrol content of a number of 106 to 182 percent of the control. medic species and varieties The alfalfa clone with the lowest grown under similar conditions. estrogenic activity contained the Wide variations in the estrogenic least coumestrol, while the clone content of 56 strains of alfalfa, with the greatest estrogenic ac- all harvested in the bud stage, tivity contained the most coumes- have been reported by Stob and trol. However, the coumestrol co-workers {211). Ladino clover concentration d i d not coincide has been shown to contain a mod- with the estrogenic activity for erate amount of coumestrol {S7y the intermediate clones. With six 122), while strawberry (105), clones, including the above four, red (222) white (105), and sub- they conducted crossbreeding ex- terranean clovers (183) and Tri- periments (21i)' Using estro- foliiiTn nigrescens (105) contain genic activity as a guide, they only small amounts. concluded (on the basis of these Hanson and co-workers (127) six clones) that it should be pos- found that nearly all of the var- sible to develop alfalfa of either iation in coumestrol content of high or low estrogenic activity. five alfalfa varieties in the one- An extensive study on the tenth bloom stage was nongenetic magnitude and nature of the var- and therefore concluded that iability of coumestrol content of breeding only for high or low alfalfa (125, 126, 127) confirmed coumestrol content did not ap- the earlier observations that cou- pear promising. However, the ex- mestrol content is influenced by tent to which coumestrol content can be changed by selection de- stage of growth, variety, and lo- pends also on variation among in- cation. For this study, five vari- dividual plants, which was not eties, Buffalo, Du Puits, Lahon- measured. Stuthman and co- tan. Ranger, and Vernal, repre- workers (213) studied the cou- sentative of the germ plasm mestrol content of four alfalfa found in principal alfalfa vari- clones that previously had been eties grown in the United States, reported to differ in estrogenic were grown in replicated plots in activity. Coumestrol content California, Iowa, Kansas, Ne- 48 TECHNICAL BULLETIN 1408, U.S. DEPT. OF AGRICULTURE braska, North Carolina, Pennsyl- The most significant correlation vania, and Utah. More than 99 between variables and coumestrol percent of the plot variation was was between defoliation scores estimated t o be environmental. and coumestrol content.

Variations as related to disease While studying- estrogenic ac- of the differences in coumestrol t i V i t y in white clover, Bickoff content among varieties can be and co-workers (37) found that a explained by varietal differences virus-infected sample of the Lou- in resistance to foliar pathogens. isiana 6 strain contained consid- Lahontan, for example, had the erably more estrogenic activity highest coumestrol content, the than the comparable virus-free greatest leaf loss, and was the sample (table 19). The high level most susceptible to foliar disease. of activity in the diseased sample DuPuits and Vernal, the most re- suggested a possible correlation sistant of the varieties, had the between estrogenic activity and lowest coumestrol content. Simi- virus infection. Later, based on larly, coumestrol was lowest in the results of their extensive forages grown in California and studies, Hanson and co-workers Utah. Incidence of foliar disease (127) concluded that most of the in these two States is generally differences attributed to the lower than in the other States other variables could result, for studied. Bennett and co-workers the most part, from their rela- (18) in Australia have consist- tionship with disease. Thus, the ently found very low levels of trend toward increased coumes- coumestrol (usually less than 5 trol content with successive p.p.m.) in alfalfa free of obvious stages of growth is consistent signs of pathogenic attack. with the common observation The effect of fungicidal control that disease incidence increases of foliar diseases on the accumu- with advancing maturity. Some 1 a t i o n of coumestrol was also

TABLE 19.—Varietal distribution of estrogenic activity in white clover harvested at Clemson, S. C, 1957

Date Clover line of harvest Flov^er density CoumestroP No. per Parts per square foot million 269 May 6 10 0 2682 May 6 15 0 4304 May 7 12 0 4306 May 8 10 7 Louisiana 21 May 9 9 3 Louisiana 6 (virus-free) May 8 6 13 Louisiana 6 (with virus) May 9 2 105 ^ Determined by mouse-uterine-weight bioassay. Source: Bickoff, E. M., Livingston, A. L., Booth, A. N., and others (37). CHEMICAL AND BIOLOGICAL PROPERTIES OF COUMESTROL 49

120 Leaves Stems 100 o Fungicide applied • Fungicide not applied

I 80 ^

\ 60 > .-* 40 O

EARLY BUD ONE-TENTH FULL BLOOM 10 DAYS AFTER BLOOM FULL BLOOM STAGE OF GROWTH

FIGURE 21.—Coumestrol content of leaves and stems of alfalfa at four stages of growth, with and without fungicide application to control foliar diseases {127). studied {127), The coumestrol increase in coumestrol content of content of alfalfa grown in the plants (table 20). sprayed plots was found to be In studies with alfalfa grown relatively low and constant over under controlled environmental all stages of growth (fig. 21). In conditions, the coumestrol con- contrast, coumestrol levels in- tent was found to increase creased with successive stages of sharply after the foliage was in- growth in alfalfa grown in un- fected with either Pseudopeziza sprayed plots. On the last sam- medicaginis {^3, 164) or Leptos- p 1 i n g date, average coumestrol phaerulina briosiana {164), two content of leaves and stems from leaf spotting organisms ( t a b le sprayed plots was 29 p.p.m. as 21). Significant amounts of cou- compared with 103 p.p.m. for un- mestrol were present only in in- sprayed plots, indicating that the fected tissue, and the level of increased coumestrol content was coumestrol was positively corre- primarily due to increased infec- lated with the number of lesions tion by foliar pathogens. Simul- and the severity of i n f e c t i o n. taneous controlled inoculation ex- Further studies {165) confirmed periments {127), in which green- that coumestrol accumulated in house and field-grown alfalfa infected leaflets and was very plants were infected with each of high in the necrotic lesions. In le- four fungi and one virus, sub- sions with a radius of 2.5 milli- stantiated these results. In gen- meters or less, 85 percent of the eral, infection caused a marked coumestrol was located within a 50 TECHNICAL BULLETIN 1408, U.S. DEPT. OF AGRICULTURE

GO 5Ío 05 o Or-i 00 00 c4 00 000 00 ci 03

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w o 5Û ÖS O Ö í3 ^i^ U ^ 0 »o0 v 0 g '5 o ö § (M ■^ S '< Ä ^ PQ g ^ < ^ 'Q3 EH ^ PH CHEMICAL AND BIOLOGICAL PROPERTIES OF COUMESTROL 51

TABLE 21.—Coumestrol content of the leaves and stems of Ranger alfalfa infected with two foliar pathogens

Pathogen Description of tissue analyzed Coumestrol Parts per million Pseudopeziza medicaginis -_ Leaves with two or more lesions per leaflet 183.7 Leaves with one lesion per leaflet 40.4 Stems (no lesions apparent) 5.7 Healthy check . Leaves 1.1 Stems .6 Leptosphaerulina briosiana .Leaves heavily infected* 71.7 Leaves lightly infected^ 28.5 Stems with occasional lesions 7.2 Healthy check . Leaves 1.0 Stems .5 * Lesions necrotic or yellow. ^ Leaves with either few large or many small lesions that were not yellow. Source: Loper, G. M., and Hanson, C. H. (164-)' radius of 1.0 mm. from the center Francis (10^) reported that of the lesion. The portion of the leaf rust could produce a three- leafspot within a 0.5-mm. radius fold to fourfold increase in the contained as high as 2,600 p.p.m. coumestrol content in the leaves coumestrol, while only traces of common burr medic (Medicago (less than 3 p.p.m.) were found polymorpha). Smart and Sher- in the healthy-appearing tissue wood ^ showed that the coumes- between lesions. trol content of rust-infected alfalfa leaflets was inversely pro- Common leafspot (Pseudope- portional to level of resistance ziza medicaginis) infection has and that the urediospores of the also been shown to cause the ac- rust pathogen contained up to cumulation of other coumestans 868 p.p.m. coumestrol. Studies on and of flavones in alfalfa (iS). alfalfa (165) infected with Uro- The concentrations of these com- myces striatus, the alfalfa rust pounds increased several-fold as fungus, showed that in all but the stage of infection increased the most susceptible plants cou- (table 22). The coumestan, sati- mestrol content was positively vol, increased the most with in- associated with lesion size and creased disease development. number. This relationship could These phenolics reached higher not be shown for the most sus- concentrations in the Atlantic va- c e p t i b 1 e plants because of the riety, which is more susceptible massive discharge of uredio- to common leafspot than the Ca- spores that contained over 400 yuga variety. This is in agree- p.p.m. coumestrol. ment with the findings of Loper and co-workers (165) that recur- The interrelationship between rent selection of alfalfa for in- coumestrol levels and disease is creased resistance to common leafspot resulted in fewer leaf- *^ Listed as a private communication spots and lower coumestrol con- of W. W. G. Smart and R. T. Sherwood tent. in (165) of Literature Cited. 52 TECHNICAL BULLETIN 1408, U.S. DEPT. OF AGRICULTURE

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< -^ÜIHTH r^ C^ « M H <î o CHEMICAL AND BIOLOGICAL PROPERTIES OF COUMESTROL 53 not limited to bacterial and fun- demonstrated that coumestrol gal diseases. Infestation by pea concentrations increased in the aphids and spotted alfalfa aphids leaves of barrel medic {Medicago has been found to cause the littoralis Rhode) with the inci- buildup of coumestrol {127, 163). dence of physiogenic leaf spot- The aphid damaged portions of ting. The spots vêre considered the plants contained elevated lev- to be physiogenic because they els of coumestrol, v^hile unda- vêre free of pathogenic fungi maged plant tissue and unin- and bacteria. High levels of cou- fected plants contained less than mestrol vêre found in the affected 1 p.p.m. In addition, the more leaves (up to 2,362 p.p.m.), vîth aphid-resistant alfalfa varieties the major amount concentrated accumulated less coumestrol than in the spotted areas, i.e., the dam- the susceptible ones. Loper {162) aged tissue. Mechanism of biosynthesis

Disease resistance in plants and isoflavones. These com- has been correlated v^ith the me- pounds are synthesized in plants tabolism of phenolic compounds through a combination of two {128), whose occurrence at infec- pathways responsible for aro- tion and wound sites suggests matic ring formation {121), One that they are associated with the route involves a poly-/?-keto-acid plant's resistance mechanism. intermediate produced by head to The similar effects of foliar path- tail condensation of acetate units. ogens, aphid infestation, and The other route, which is respon- physiogenic leaf spotting on the sible for coumarin biosynthesis accumulation of coumestrol sug- {61, 1Í6, 220), involves the inter- gest that disease triggers some vention of Cg-intermediates asso- nonspecific mechanism in the ciated with the shikimic-pre- plant that causes the rapid accu- phenic acid pathway. mulation of this type of com- Detailed studies on the biosyn- pound. thesis of flavones, isoflavones, Although the biosynthetic and coumestrol have been made pathway of coumestrol in the al- by Grisebach and co-workers falfa-pathogen relationship is (116, 117, 118, 121), Acetic acid unknown, it has been shown that was shown to be a very effective its biosynthesis in healthy plants source of the Ce-units compris- is related to that of the flavones ing the A-ring of flavones {129,

HO2C HO

OCH, ^

• Labelled carbon FIGURE 22.—Incorporation of labeled phenylalanine into the B-ring of formononetin {121). 54 TECHNICAL BULLETIN 1408, U.S. DEPT. OF AGRICULTURE

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O CHEMICAL AND BIOLOGICAL PROPERTIES OF COUMESTROL 55

202), The remaining Cg-units, the course of the biosynthesis. which are responsible for the Preliminary feeding experiments formation of the B-ring (fig. 22), showed that acetic acid-(l-^*C) are formed from precursors such and D,L-phenylalanine-l-^^C were as shikimic acid, phenylalanine, incorporated into coumestrol and cinnamic acid. {117), Experiments with cinna- The isoflavones are synthesized mic acid-(3-^^C) provided unin the plant in a manner similar equivocal proof for the occurrence to the flavones, except that an of an aryl migration {118), The aryl migration occurs. Grisebach labeled carbon was located by and Doerr {115, 119, 120) have degradation of coumestrol to its studied this migration in the bio- benzofuran derivative (fig. 23) synthesis of formononetin by red in the manner described under clover. Using labeled phenylala- ''Characterization of Coumes- nine-2-^*C as the starting mate- tans.'' Decarboxylation of the acid rial for the biosynthesis, they (fig. 23, III) to the benzofuran found that the formononetin pro- (fig. 23, IV) revealed that the duced by the plant was labeled total radioactivity had been lo- at the 3-position instead of the cated in the carbonyl group of 2-position (fig. 22). This could the lactone ring, i.e., the 2-posi- only be explained by the migration. If a migration had not oc- tion of the B-ring from the 2- curred, the labeled carbon would carbon to the 3-carbon at some have been located in the 3-posi- stage during the biosynthesis of tion. Therefore, coumestrol must formononetin. be biogenetically related to the According to Grisebach and isoflavones rather than the cou- Barz {117, 118), if coumestrol marins. were biosynthesized in a manner Grisebach {115) proposed a se- similar to the coumarins, aro- ries of reactions to show how the matic ring A and the three car- isoflavones and coumestrol could bon atoms of the lactone ring be synthesized in the plant (fig. would arise from an intact phen- 24). This relationship was proved ylpropane unit and the B-ring by feeding 2',4,4'-trihydroxychal- from either acetate or glucose cone-4'-glucoside {ß-^^C) to al- (fig. 23). If, on the other hand, falfa plants {116), After 72 the biosynthesis were analogous hours, labeled coumestrol, daid- to that of the isoflavones, the A- zein, and formononetin were iso- ring would arise from the con- lated from an ether extract of the densation of three acetate units, dried roots following the scheme while the B- and lactone rings shown in figure 25. Thus, the would arise from a phenylpro- chalcone shown in figure 24 is a pane unit. Also, a rearrangement common intermediate for the bio- involving an aryl migration synthesis of these compounds in would occur at some point during alfalfa. Utilization of Coumestrol by the Animal Interest in naturally occurring animals. These compounds may plant estrogens stems from their cause an estrogenic response, an effects on reproduction in farm infertility syndrome, or effect the 56 TECHNICAL BULLETIN 1408, U.S. DEPT. OF AGRICULTURE

3 Malonyl-CoA + H02C-CH=CH HQHOH

HO, .OH HO. ^ ^OH C-CH=CH -^OH :-ÍH":¿HiyyoH

HO, OH

^ OH OH

-H2O

HO

OH

FIGURE 24.—Proposed biosynthetic pathway for the isoflavones and coumestrol in plants (115).

growth rate of animals. In an at- concerned with studies on plants tempt to understand these effects, in which coumestrol may be the the metabolic role of these com- active constitutent as well as on pounds has been studied. This coumestrol itself, present discussion is primarily CHEMICAL AND BIOLOGICAL PROPERTIES OF COUMESTROL 57

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tí 58 TECHNICAL BULLETIN 1408, U.S. DEPT. OF AGRICULTURE Metabolism Although a considerable num- amounts of coumestrol. Adler ber of reports have been pub- and co-workers (9) found both lished in recent years on the me- free and conjugated coumestrol tabolism of forage estrogens, in in the blood of cows that were an attempt to clarify their role in fed a ration containing estrogenic the infertility syndrome, most of alfalfa meal. Two groups of ani- these studies have been con- mals received 2.56 and 4.8 mg. ducted with the estrogenic isofla- coumestrol per day, respectively. vones. Metabolic studies on the The concentration of free cou- isoflavones have been recently re- mestrol was higher in the blood viev^ed by Bickoif (20), of the cows on the higher cou- It has been suggested (22, 99) mestrol ration, while the conju- that plant estrogens are proestro- gated coumestrol level was simi- genic in character, i.e., they are lar in both groups (table 23). changed into more active forms Estrogenic isoflavones are ex- in the animal body. For example, tensively degraded by the animal methylated coumestans or isofla- to simple acids and phenols. vones might be demethylated to Equol is one of the major detect- more "potent'' compounds.^ If able metabolites. Taken together, this is true, this raises the ques- all of these metabolites account tion of the nature of the com- for only a small percentage of pounds formed, as v^ell as the site the original quantity of isofla- at which this change occurs. It vone consumed (20). Adler ^ was is assumed that the main process unable to find any coumestrol in of activation and inactivation of the urine of mice fed coumestrol. the plant estrogens takes place in Braden ^ was unable to find any the liver (137, 168). This creates obvious metabolic products in the difficulties in investigation, and urine or blood plasma of sheep the processes involved may differ given coumestrol intraruminally. considerably between laboratory Cayen and Common (63) injected and farm animals. At the same tritiated coumestrol intramuscu- time, additional problems arise larly into nonlaying hens and re- from the presence of substances ported that the radioactivity in in plants, as yet largely unknown, the phenolic extracts of the urine which may increase or diminish was distributed between coumes- the potency of forage estrogens trol and unidentified conversion (27, 36). or breakdown products. As they Lindner (155) studied the were unable to detect any ra- presence of the forage estrogens dioactive equol in these extracts, in the plasma and depot fat of they concluded that coumestrol sheep and was able to detect must be degraded in the fowl trace amounts of coumestrol in along pathways quite different the plasma of ovariectomized from those along which the iso- ewes grazing on estrogenic clover flavones are degraded. Their find- pastures that contained trace ^ See footnote 2, page 2. "^ Personal communication from David " Personal communication from A. W. Bennett, Commonwealth Scientific and H. Braden, Commonwealth Scientific Industrial Research Organization, Aus- and Industrial Research Organization, tralia, 1967. Australia, 1967. CHEMICAL AND BIOLOGICAL PROPERTIES OF COUMESTROL 59

TABLE 23.—Fluorometric determination of free and conjugated coumestrol in whole bovine blood

Coumestrol concentration Form High coumestrol Low coumestrol ration ration Micro grams/liter Micro grams/liter Free coumestrol 1.21 0.35 Conjugated coumestrol _ _ _ .50 .55 Total 1.71 .90 Source: Adler, J. H., Weitzkin, G., and Marinov, U. (9), ings are very interesting when acid in uterine cells and also in- considered in the light of evi- creased the acid soluble uridine dence that the biosynthetic path- triphosphate in the uterus. The v^ay of coumestrol in alfalfa re- pattern of metabolic stimulation sembles that of the isoflavones was similar to that of estradiol, (118). This difference in meta- differing only in the degree of bolic routes for coumestrol and stimulation. Since the metabolic the isoflavones may account for responses studied were indicative the much higher relative estro- of typical estrogen activity, Note- genic potency of coumestrol (39, boom and Gorski considered 57). them to be sufficient to establish Lyman and Krueger (168) that coumestrol was similar to were unable to show any effects estradiol in its action on meta- due to coumestrol on plasma lipid bolic pathways in the uterus. distribution or on the glyceride Furthermore, their observation fraction of the cholestrol-induced that a combination of estradiol fatty liver of either normal or and coumestrol gave no additive castrated male rats. Noteboom effects supported the hypothesis and Gorski (187) found in ovar- that forage estrogens perform iectomized rats that coumestrol the same function as natural es- stimulated the incorporation of trogens in triggering anabolic labeled precursors into protein, responses. phospholipid, and ribonucleic

Estrogenic potency

Until recently, the most com- under field conditions with farm monly used method for quantita- animals (184). Consequently, a tive bioassay has been the mea- number of workers, particularly surement of increase in uterine in Australia, (17, 18, 57, 104) weight of immature female mice. have gone directly to sheep as the However, difficulties have often test animal. Criteria for estro- been encountered when attempt- genic response in sheep include ing to correlate results obtained milk secretion (76, 172), teat with mice and other laboratory length (58, 182), and vaginal and animals with the results obtained uterine increases (17, 57, 149). 60 TECHNICAL BULLETIN 1408, U.S. DEPT. OF AGRICULTURE

TABLE 24.—Dose-response data and relative potency of forage estrogens vs. diethylstilbestrol and estrone

Quantity Uterine Quantity to Compound fed per weight produce 25- Relative mouse ± S.E. * mg. uterus potency "^

Micrograms Milligrams Micrograms Control 0 9.6 ± 0.3 Diethylstilbestrol .025 11.8 .2 .050 18.1 .7 .075 23.7 ,8 0.083 100,000 .100 29.2 .4 .200 78.1 6.2 Estrone .50 14.7 .9 .75 16.0 1.3 1.00 23.8 2.5 1.20 6,900 1.50 36.1 2.3 2.00 45.3 6.7 J Coumestrol 100 13.8 .2 200 24.2 .2 300 29.2 1.3 240 35 400 40.7 1.9 500 76.0 6.0 Coumestroldiacetate 250 19.0 1.2 300 22.3 1.6 400 26.7 1.4 340 24 500 42.4 3.3 1,000 87.0 6.2 Genistein 5,000 19.4 .9 7,500 28.0 5.0 8,000 27.0 1.6 12,000 32.4 1.9 8,000 1.00' 15,000 36.6 .6 20,000 52.7 1.9 Daidzein 5,000 17.3 ± 1.3 7,500 18.5 ± 2.6 10,000 24.8 ± 1.3 11,000 .75 15,000 31.2 ± 1.4 Biochanin A 10,000 20.3 ± 2.5 20,000 27.9 ± 2.1 30,000 27.9 ± 2.6 18,000 .46 40,000 45.5 ± 3.8 Formononetin 15,000 16.8 .8 20,000 17.9 1.4 25,000 23.2 1.0 32,000 .26 30,000 27.5 2.3 40,000 26.1 2.0

^ S.E. = standard error. - Measured at the dosage required to produce a 25-mg. uterus. ^ Genistein was arbitrarily assigned a value of 1.00. Source: Bickoff, E. M., Livingston, A. L., Hendrickson, A. P., and Booth, A. N. 39). CHEMICAL AND BIOLOGICAL PROPERTIES OP COUMESTROL 61

Bickoff and co-workers (39) Booth and Bickoff ^^ observed compared the estrogenic poten- that the potency of coumestrol cies of orally fed coumestrol, relative to diethylstilbestrol was genistein, daidzein, biochanin A, about the same, whether both formononetin, diethylstilbestrol, were administered orally or sub- and estrone by the mouse-uter- cutaneously. However, estrone ine-weight bioassay method was more potent subcutaneously, (table 24). For coumestrol, es- being about 1,000 times more po- trone, and diethylstilbestrol, the tent than coumestrol (table 25). logarithms of the uterine weights Pincus and Merrill ^^ also com- were linear with dosage. With pared coumestrol with estrone the isoflavones, however, a linear administered subcutaneously and relationship existed between the obtained results similar to the mean uterine weight and dosage. above. To obtain a uniform basis of Leavitt (151) reported that, by comparison, relative potencies slope-ratio analysis, estradiol ad- were based on the dosage re- ministered subcutaneously was quired to produce a uterine 33,000 times more active than weight of 25 mg. These were de- coumestrol administered orally termined from dosage-response on the uterus of ovariectomized curves, such as those that can be mice and 120,000 times more ac- plotted from the data given in tive in blocking pituitary gonado- table 24. Coumestrol was found tropic function. to be from 30 to 100 times more potent than the isoflavones under the conditions of the assay. Es- ''^ Booth, A. N., and Bickoff, E. M. trone and diethylstilbestrol were Unpublished data. ^^ Personal communication from Gre- respectively about 200 and 3,000 gory Pincus and A. P. Merrill, The times more active than coumes- Worchester Foundation for Experimen- trol. tal Biology, Massachusetts, 1968.

TABLE 25.—Dose-response data for coumestrol, estrone, and diethylstilbestrol injected subcutaneously in com oil over a i-day period

Uterine Compound Total dose weight ± S.E.^

Micrograms Milligrams Control (basal ration plus corn oil) . ^ -. 15.10 ± 1.25 Coumestrol ^ ^ 200 22.80 + 2.12 300 40.04 ± 5.69 Estrone .1 19.32 ■+- 2.11 .2 23.00 ± 2.78 Diethylstilbestrol .05 34.48 ± 4.54 .1 39.84 ± 2.54

^ S.E. = standard error. Source : Booth, A. N., and Bickoff, E. M. Unpublished data. 62 TECHNICAL BULLETIN 1408, U.S. DEPT. OF AGRICULTURE

As early as 1959, Jennings and by mouse-uterine weight bioassay Dow (130) suggested employing (S9). sheep as the test animals since they are highly sensitive to clo- Braden and co-workers (57) ver estrogens. They reported that used the increase in uterine uterine and vaginal morphology weight of ovariectomized ewes to of sheep on estrogenic pastures compare estrogenic potencies of differed from that of sheep main- coumestrol, genistein, biochanin tained on other pastures. Oldfield A, and formononetin. The activ- and co-V7orkers (19í) reported ity of these compounds in rela- that after 3 vêeks on test diets, tionship to diethylstilbestrol var- grovîng lambs fed a high cou- ied depending on the mode of ad- mestrol alfalfa meal (119 p.p.m.) ministration (table 27). Their re- shovêd increased udder and vul- sults indicated that, when given val development over lambs fed a parenterally, synthetic genistein, lov^ coumestrol meal (22 p.p.m.). biochanin A, and coumestrol ex- It vâs even possible to drav^ milk hibited estrogenic activities in from some of the evê lambs on sheep comparable with those ob- the high coumestrol diet. Using served in mice (39, 4-9, 223), arbitrarily assigned values for When administered intramuscu- progressive udder development, larly, however, diethylstilbestrol three independent scorers evalu- was about 100,000 times more ac- ated the evê lambs at 1.25 on the tive than genistein and biochanin high meal and 0.52 on the lov^ A, while coumestrol was about meal. Similar evaluation for 100 times more active than these vulva development gave values of two isoflavones. The negligible activity of formononetin either 2.04 and 1.07 for the high and intraperitoneally or intramuscu- low meals, respectively. larly was thought to be due pos- In measuring the estrogenic sibly to its low solubility, al- activity of annual medic pas- though when injected intraruminally, it had estrogenic activ- tures, Francis and Millington ity of the same order of magni- (lOi) found increases in teat tude as genistein and biochanin length of wethers to correlate A. Coumestrol was still some 15 with coumestrol content. The times more active intraruminally most active varieties produced than the isoflavones. These re- teat lengths equivalent to those sults are summarized in table 27. resulting from injections of 8 Despite lower potencies of the micrograms of diethylstilbestrol forage estrogens when compared daily (table 26). If coumestrol with diethylstilbestrol, they are alone were responsible for the present in certain forages in suf- activity of the pastures, then the ficient quantities to be influential potency of injected diethylstilbes- in animal nutrition and physiol- trol for wether sheep was about ogy. Furthermore, the less potent 8,500 times as great as that of isoflavones are present in Austra- orally administered coumestrol. lian subterranean clover (56) in This value may be compared to sufficient quantities to cause re- the 3,000-fold increase obtained productive problems in sheep. CHEMICAL AND BIOLOGICAL PROPERTIES OF COUMESTROL 63

TABLE 26.—Response in wether teat length to intake of green medic and injection of diethylstilbestrol

Mean coumestrol Coumestrol Mean content of consumed teat Diethyl- Medic variety medic ± during length stilbestrol standard 9-day increase error ^ test

Parts per Milligrams Micrograms million per sheep Millimeters per day M, littoralis 132.0 ± 25 714 2.90 M. truncatula (Cyprus) ___ 104.0 ± 24 617 2.73 M. truncatula (commercial) _ 101.0 ± 24 519 1.94 M. truncatula (Mtr 173) ___ 25.8 ± 5.4 172 .89 M. sativa 15.8 ± 5.0 90 .29 M. polymorpha 11.4 ± 1.1 56 - .13 M. scutellata 9.2 ± 1.9 50 - .25 Control, oats - .54 2.12 2.65

^ Dry-weight basis. Source: Francis, C. M., and Millington, A. J. (104).

Antifertility effects

The estrus cycle of female ani- cular cells) produce estrogen, mals is initiated when the brain, ovulation occurs, i.e., the follicles via the hypothalamus, stimulates rupture and eggs are released, the pituitary gland to secrete the and the follicular cells become gonadotropic hormones. Each of the corpus luteum. The corpus lu- these hormones regulates specific teum secretes progesterone, functions in the female animal. which prepares the uterine lining The follicle-stimulating hormone for implantation. The processes (FSH) influences the develop- of the uterus are regulated by es- ment of the ovaries during pub- trogens. The naturally occurring erty and later acts directly on estrogens are estradiol, estrone, them in promoting the develop- and estriol. They are primarily ment and maturation of the folli- responsible for the regular cles. The interstitial cell-stimu- course of the estrus cycle. Upon lating or luteinizing hormone fertilization and implantation, (LH) depresses the first part of the estrogens and progesterone the ovarian cycle and encourages inhibit the secretion of FSH and the second or luteal phase, LH, thereby preventing ovula- thereby causing the uterine tion. changes associated v^ith pseudo- The estrus cycle is regulated pregnancy or v^ith gestation. by the alternating stimulation of Under its influence, the follicles the ovary by the pituitary gland (ova surrounded by a sac of folli- hormones and the stimulation of 64 TECHNICAL BULLETIN 1408, U.S. DEPT. OF AGRICULTURE

TABLE 27.—Estrogenic activity of isoflavones and coumestrol in ovariectomized ewes [i/r = intraruminally; i/m = intramuscularly; i/p = intraperitoneally]

Total dose Mean uterine Treatment in 4 days Route weight Grams Control 11.7 Genistein grams 9 i/r '13.3 1 i/m '21.4 3 i/m '21.6 Diethylstilbestrol _ _ micrograms 15 i/m '22.3 45 i/m '20.7 Control 12.2 Formononetin grams 1 i/m 12.9 6 i/r 12.6 Genistein do 0.5 i/m '16.4 Control 14.7 Biochanin A grams 20 i/r 15.3 1 i/m '18.4 Control 5.9 Diethylstilbestrol __ micrograms 10 i/m 7.1 20 i/m '14.3 Biochanin A grams 24 i/r '13.4 Genistein do 20 i/r '16.4 Control 10.4 Formononetin grams 1 i/p 10.9 24 i/r '17.4 Diethylstilbestrol ^^ micrograms 15 i/m '14.5 30 i/m ' 22.6 Control 7.8 Diethylstilbestrol _„ micrograms _ _ 20 i/m '15.2 Coumestrol ^ grams 1.4 i/r '14.0 0.03 i/m '18.6 0.10 i/m '19.3 Control 6.2 Coumestrol _ ^ grams .012 i/m '13.9 Biochanin A do 1 i/m '14.0 Diethylstilbestrol _ ^ micrograms 15 i/m '13.9 30 i/m '17.6 ' From Fisher's one-sided test, these values are found significantly greater (P < 0.05) than the control values. Source: Braden, A. W. H., Hart, N. K., and Lamberton, J. A. (57).

the pituitary gland by the They may block ovulation and ovarian hormones. Factors that stimulate pseudopregnancy or contribute to either higher or cause anovulatory estrus, as in lower than normal hormone lev- the case of birth control pills. els can interfere with this cycle. They may also effect the secre- These antifertility factors may tion mechanism in the Fallopian function by inhibiting the secre- tubes, thereby effecting egg tion of, or response to, the endo- movement; change the uterine genous animal estrogens or other lining in such a manner as to hormones effecting reproduction. prevent implantation; render the CHEMICAL AND BIOLOGICAL PROPERTIES OF COUMESTROL 65

mucus composition in the cervix drome in the cattle, which was unfavorable to sperm transport; characterized by irregular estrus or cause a degenerate effect on cycles, cystic ovaries, and de- the reproductive organs. creased fertility. Other hyperestrogenic effects included preco- Hyperestrogenic syndrome cious mammary and genital development in heifer cows. A Excessive estrogen stimulation study of these disturbances, in caused by long periods of grazing relation to the feeding program, estrogenic pastures is well estab- revealed that their appearance lished. Hov^ever, most of these roughly coincided with the avail- studies have dealt v^ith red and ability of fresh alfalfa. In some subterranean clovers, wherein cases, the alfalfa was found to the isoflavones are the predomi- contain as much as 52 micro- nant estrogens and coumestrol is grams equivalents of estradiol either absent or present in very per kg. (on a dry-weight basis). small quantities. Reviews (20, After alfalfa was excluded from 185) covering this work are the rations of two different dairy available. Relatively few cases of herds, there was an increase in reproductive disorders attributa- conception rates and a decrease ble to estrogenic alfalfa and La- in the frequency of cystic ovaries. dino clover have been reported. In Later studies by Lotan and Adler these cases, coumestrol is un- {166) confirmed that increased doubtedly the causative agent. feeding of estrogenic alfalfa in- As early as 1952, Foltin {102), creased the percentage of cows in Israel, suspected that a rela- with irregular estrus cycles and tionship existed between heavy decreased conception rate. alfalfa feeding and seasonal ste- Recently Ayalan,^^ also in Is- rility in cattle. Similar reports rael, isolated coumestrol from al- began to appear in this country. falfa fed to a herd of dairy cattle For example, in one small dairy with complaints of infertility. herd in New England,^- 19 out of Adler ^ ^ has since found coumes- 32 cows were treated for cystic trol as well as 4'-0-methylcoumes- ovaries one or more times during trol, daidzein, and genistein in a single year. Six of the cows had pea silage fed to dairy cattle ex- to be treated from four to six periencing cystic ovaries and ir- times. Since most of the cystic regular heat cycles. cows were not in ''heat,'' it was suggested that the alfalfa hay Dairy cattle fed Ladino clo- fed the animals might contain ver hay have sometimes been enough estrogens to interfere found to require more services with the estrus cycle. Adler and per conception than comparable Trainin {6, 7, 8, 216), in Israel, animals fed timothy hay.^"^ Ochi were the first to clearly associate and co-workers {188) reported a reproductive problems in dairy cattle with the feeding of large ^^ Personal communication from Na- quantities of alfalfa. They re- tán Ayalan, Kimron Veterinary Insti- ported a hyperestrogenic syn- tute, Israel, 1967. ^' See footnote 2, page 2. ^' Personal communication from H. A. ^- Personal communication from H. P. Keener, Professor of Animal Husban- Adams, Eastern States Farmer's Ex- dry, University of New Hampshire, change, 1959. 1957. 66 TECHNICAL BULLETIN 1408, U.S. DEPT. OF AGRICULTURE study on reproductive conditions ovulation occurred normally, but of dairy cows fed Ladino clover. that fertilization did not follow In the animals on clover, estrus as consistently. Of the total eggs seemed to recur earlier after par- recovered from ewes on Ladino turition. More cows showed dis- clover, only 59 percent were fer- tinct signs of estrus and a proper tile, as compared to 75 percent duration of the estrus period for the ewes on bluegrass. There than animals not fed clover. is probably a connection between Even though no particular this observation and that of Coop changes were observed in their and Clark {79), m Australia, who estrus cycle, the occurrence of re- reported that ewes mated on al- productive disorders was a little falfa exhibited a 10 to 12 percent more frequent and the rate of reduction in lambing percentage conception a little lower in cows that was due mainly to reduction fed clover. These trends were in the number of twins. especially pronounced in animals Infertility problems have also fed more than 30 kg. Ladino per been reported in nonruminant day. animals. Bornstein and Adler Engle and co-workers (98) {52) found that an alfalfa ex- studied the reproductive per- tract that supplied the equiva- formance of ewes during three lence of 0.21 />tg. of estradiol per grazing, breeding, and lambing kg. feed significantly increased seasons. Ewes grazed on Ladino oviduct weights and caused clover came into heat and con- ovarian hyperemia in female ceived an average of 21.7 days chicks. Chury and Panek {73) later than ewes grazed on blue- found follicular cysts and cystic grass all season. They also re- hyperplasia of the uterine lining quired more services per concep- in guinea pigs fed large amounts tion and underwent longer lamb- of alfalfa. These effects were as- ing seasons. Based on mouse- sociated with the estrogenic uterine-weight bioassay, the La- substances in the plants. El- dino clover was found to be es- ghamry {90) has shown that estrogenically active while the trogenic extracts of Ladino clo- bluegrass was inactive. Sänger ver exerted two types of influence and co-workers {201), using vir- on the uterus of growing female gin ewes, confirmed the presence rats. Initially, during the first 2 of estrogen-like substance in the weeks of administration, there above Ladino clover pastures. was a stimulatory effect, as shown Extensive cornification and des- by an increase in uterine weight. quamation of the vaginal cells When the estrogenic feeding was was observed in anestrus smears continued for 3 or 4 weeks, an in- from the ewes on Ladino. They hibitory effect occurred. The uteri were similar in appearance to lost weight as compared to the smears from anestrus controls controls and began to degenerate. treated with estradiol, as well as Elghamry concluded that his lat- to the metestrus and diestrus ter effect offered a logical expla- smears of controls undergoing nation for breeding disturbances their normal estrus cycle. In in domestic animals as well as studies on the nature and cause clinical manifestations of infertil- of the delay in conception, Sän- ity resulting from long continued ger and Bell {200) found that ingestion of plant estrogens. CHEMICAL AND BIOLOGICAL PROPERTIES OF COUMESTROL 67

Grouse (80) reported contin- Bornstein and Adler (52) re- uous heat cycles as well as uter- ported that cockerels fed a diet ine and vaginal diseases in chin- containing 10.3 percent alfalfa, chillas. He attributed these prob- equivalent to 1.65 microgram of lems to the alfalfa in the feed estradiol per kg., had significant- and suggested that they might be ly retarded testicular develop- due to estrogenic substances pre- ment as shown by reduced testes sent in the alfalfa. Once alfalfa weight and by inhibition of sperm was eliminated from the feed, the production. Comb growth was conditions corrected themselves. only slightly inhibited. Recently Negus and co-workers (186) Chury (70) reported the isola- studied the population size of col- tion of a crystalline antigonadal onies of the rice rat on an isolated substance from alfalfa that de- island. They suggested that a creased the weight of the seminal sharp change in the quality of vesicles in male rats. However, the green plants, including their this substance had no effect on estrogen contents, could have im- male mice. mediate effects on the reproduc- Pincus and Merrill ^^ compared tive potential of and eventual in- coumestrol with testosterone and fluence on the population size of found that coumestrol also had the rodent colonies in the ñeld. androgen-like activity in cas- Leavitt and Meismer (152) trated male rats. When adminis- found that injecting coumestrol tered alone, coumestrol caused subcutaneously at 10 mg./kg. or the weight of the seminal vesi- at 100 mg./kg. into 5-day-old cles of the rats to increase, but female rats caused persistent an- had no effect on the weights of ovulatory estrus. At 100 days of the ventral prostrate gland or the age, ovaries from animals treated levator ani muscle. When coumes- with coumestrol were entirely trol was combined with testos- follicular but only weighed half terone, an additive effect was as much as the controls. On the obtained on all three tissues other hand, Bierwagen ^^ found (table 28). Lyman and Krueger that when coumestrol was ad- (168) reported that doses of cou- ministered orally at 10 mg./kg. to mestrol diacetate ranging from adult female mice, it did not 20 to 300 mg. per animal given prevent pregnancy. Deanesly ^^ orally over a 21-day feeding per- reported that coumestrol acetate iod did not inhibit testicular de- given to female guinea pigs sub- velopment or adrenal size in cutaneously at levels of 2 to 6 either normal or castrated male mg. did not influence ovulation, rats. They suggested that the ab- the duration of the corpora lutea, sence of any physiological effects or their normal cycle. might be due to the rapid de- There have been only a very gradation of coumestrol diacetate few reports of substances in for- by the liver in the adult male rat, ages that affect the male animal. similar to certain other estrogens (203). ^^ Personal communication from M. E. Oldfield and co-workers (193) Bierwagen, Director of Pharmacologie Research, Bristol Laboratories, 1968. found that coumestrol stimulated ^^ Personal communication from Ruth Deanesly, University of Cambridge, England, 1968. ' See footnote 11, page 61. 68 TECHNICAL BULLETIN 1408, U.S. DEPT. OP AGRICULTURE

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TABLE 29.—Teat lengths and glandular weights

Treatment Trial 1 Trial 2 Item Control Coumestrol Control Coumestrol ration ration * ration ration ^ Teat length (centimeters) 3 weeks 1.56 2.21 5 weeks 1.46 2.49 7 weeks 1.48 2.53 Pituitary weight grams __ __ 0.940 1.258 .835 .933 Pituitary- final body weight - mg./kg _. ___ 15.460 19.710 15.75 17.81 Seminal vesicle weight _ _ _ grams _. .80 7.05 Seminal vesicle final body weight . mg./kg _ _ _ 15.09 134.54 ^ Coumestrol added to ration = 112 p.p.m. ^ Coumestrol added to ration = 145 p.p.m. Source: Oldfield, J. E., Fox, C. W., Bahn, A. V., and others (193). mammary growth and increased (50) found that coumestrol did the weights of the seminal vesi- not inhibit reproduction in rats. cles and the pituitary gland in The reasons for the differences in wether lambs (table 29). More- the effects of coumestrol on vari- over, one of the wethers suffered ous male animals are not known. a rectal prolapse, which was con- Differences among species are sidered suggestive of an estro- very likely contributing factors. genic response. Based on a report (17i) that genistin depressed Hypoestrogenic syndrome testicular development and sperm production in male mice, Booth ^^ The concejpt of a hyperestro- investigated the possibility that genic syndrome caused by long coumestrol would cause similar periods of grazing estrogenic effects. High levels of coumestrol pasture is well established. How- diacetate (1.5 g./kg. diet) com- ever, the concept that forages pletely inhibited reproduction in may contain factors that inter- mice when both sexes were fed fere with the plant estrogens or coumestrol. When attempts were with the endogenous animal es- made to ascertain whether one or trogens and produce a hypoestro- both sexes were involved, the re- genic syndrome is not as widely sults were inconclusive. However, accepted. But, evidence for such the data suggested that the re- an antiestrogenic effect is grow- productive failure was due to ing. It is conceivable that forage sterility in the male and that the with a high level of detectable es- effect was reversible. These ef- trogen by chemical assay might fects were similar to those caused have no apparent estrogenic ef- by feeding the males diethylstil- fect on the animal due to the bestrol (1 mg./kg. diet). Booth presence of a sufficiently high level of an antiestrogenic substance. The situation in which * See footnote 10, page 61. antiestrogens would mask or coun- 70 TECHNICAL BULLETIN 1408, U.S. DEPT. OF AGRICULTURE

teract the effect of the plant es- and guinea pigs, Chury and trogens would help to explain Panek (7Í) reported detrimental the frequent lack of correlation effects of alfalfa on ovarian and between chemical and biological uterine functions, resulting in de- assays for forage estrogens. In pression of fertility, which were seeking an explanation for the not associated with the estrogen actions of antiestrogenic sub- in the plant and presumably due stances, Cook and Kitts (78) sug- to antiestrogens. In later studies, gested that they may function by they decided that the weight inactivating the natural estro- changes observed in the pituitary gens in the blood, or by making glands, uteri, and ovaries of the uterus nonresponsive to es- guinea pigs fed only alfalfa were trogen stimulation. probably the result of the mutual It is well established that the effect of estrogenic and antiestro- prolonged administration of mas- genic factors (73). sive doses of estrogens inhibit Chury (71) and Ershoff (99) ovarian development in the imma- both observed that different ture rat (225), In these cases, batches of alfalfa varied marked- Ershoff and co-workers (99) ly in their antiestrogenic activity have shown that the concurrent (table 30). Biely and Kitts (47), feeding of alfalfa, presumably who assayed 13 legumes and rich in antiestrogenic substances, grasses, including alfalfa and could largely counteract this ef- Ladino clover, showed that anti- fect when the estrogen was estra- estrogenic activity varied wide- diol. Similarly, Chury (71) dem- ly depending on species, stage of onstrated that feeding of alfalfa growth, and date of harvest. minimized the effects of small Bickoff (36) found that some doses of estradiol benzoate on the samples of alfalfa, which entirely ovaries and pituitary glands of lacked estrogenic activity on the rats. Working with rats, rabbits, basis of effect on uterine weight

TABLE 30.—Comparative effects of dried alfalfa on the ovarian weight of rats fed massive doses of alpha-estradiol

Alpha- Body weight estradiol Average Supplements fed with basal ration per ovarian kilogram Initial Final weight of ration ± S.E." Milligrams Grams Grams Milligrams Basal 10 41.4 170 17.3 1.1 20 percent alfalfa meal: 1 10 41.2 185 19.4 ± 1.8 2 10 41.0 179 28.1 ± 1.7 3 10 41.2 184 34.2 ± 3.3 4 10 41.4 184 38.3 ± 3.9 5 10 41.4 185 38.1 ± 4.0 6 10 41.1 189 43.3 ± 2.1 Basal ration without alpha-estradiol . _ 0 41.9 209 53.4 ± 3.0

^ S.E. = standard error. Source: Ershoff, B. H., Hernandez, H. J., and Matthews, J. H. (99). CHEMICAL AND BIOLOGICAL PROPERTIES OF COUMESTROL 71

TABLE 31.—Demonstration of presence of an estrogen inhibitor in alfalfa meal

Average uterine response Equivalent amount of alfalfa to supplemental coumestrol meal in control diet (grams/mouse) None 0.3 mg./mouse Milligrams Milligrams 0 9.0 31.1 5 10.4 13.4 10 9.2 9.3 Source: Bickoff, E. M., Livingston, A. L., Booth, A. N., and others (36), of treated mice, contained a sub- the uterine weight of immature stance that was capable of inhib- mice and antagonized the effects iting* the estrogenic response of of both diethylstilbestrol and es- coumestrol. When an extract of trone (78). Ostrovsky and Kitts one of these meals was incorpo- (195) have suggested that birds- rated into a control diet, the nor- foot trefoil contains an antiestro- mal response to coumestrol was gen similar to that found in al- depressed (table 31). Using the falfa. The triterpene, glycyrrhe- Astwood test with rats, Adler (4, tinic acid, which is a constituent 5) confirmed this observation and of crude licorice extract, has reported additional experiments been shown to have antiestro- in which the antiestrogen re- genic activity, but no estrogenic duced the effectiveness of cou- activity {H7), It is interesting to mestrol to approximately one- note the structural similarity of tenth of its actual potency. He this compound to some of the sa- further showed that the anties- ponin aglycones known to be trogenic substance in alfalfa also present in alfalfa and clover inhibited the uterine response to (218). estradiol and diethylstilbestrol (4). This inhibition was quite Effects such as those produced persistent and lasted 5 days after by the antiestrogens in plants the antiestrogenic treatment may also be induced by estro- under conditions of daily estra- genic, androgenic, and proges- diol treatment. ^^ tatic substances (87). Working with synthetic estrogens, Em- Antiestrogenic activity has mens {95) proved that, depend- been reported in other forages, ing on the level administered, a such as fahli hay and oat hay (3). given compound may act either MacDonald (170) reported that as an estrogen or as an estrogen beef cows tend to abort or give inhibitor. In accordance with this birth to weak calves when they observation, Folman and Pope consume yellow pine needles. (101) found that at certain dose Kitts and co-workers (JO, 11) levels both coumestrol and geni- fractionated yellow pine needles stein could markedly inhibit the and found a factor that depressed uterovaginotropic action of estradiol, estrone, and diethylstil- ^See footnote 2, page 2. bestrol. Similar results on the in- 72 TECHNICAL BULLETIN 1408, U.S. DEPT. OF AGRICULTURE teraction between coumestrol and ies employing mice as the test an- estrone have been reported by imal, Leavitt and Wright {15^) the U.S. Cancer Chemotherapy showed that Ladino clover sam- National Service Center, ples, having no significant estro- (CCNSC) 2^ confirming that plant genic activity by bioassay, caused estrogens can also exhibit anties- reproductive anomolies and al- trogenic properties. most completely stopped conception in mice. No single aqueous- Antigonadotropic factors alcohol, ether-alcohol, chloroform, or acetone-chloroform extract of Some evidence exists that the Ladino had any of the proper- plant estrogens, at levels nor- ties of the unfractionated plant. mally encountered in the field, However, all of the extracts in- may sometimes not be as signifi- creased the incidence of nonviable cant in reproductive problems as embryos in the pregnant mice. are certain presently unknov^n es- Chury and Crha {72) investi- trus and estrogen inhibitors. gated the effects of alfalfa and Wright {221^) reported that feed- alfalfa extracts on induced ovu- ing Ladino clover to female rab- lation in rabbits. An unbalanced bits caused infertility. Although alfalfa diet caused a considerable the clover was estrogenically ac- decrease in ovulation and in the tive by rat-assay, it did not cause number of fertilized eggs. The al- any premature stimulation of the falfa extracts completely inhib- reproductive tract of newly- ited ovulation for varying weaned rabbits even though they lengths of time, usually at least ingested the equivalent of ap- 10 days and, in a few cases, over proximately 30 micrograms of a month. They felt that egg de- diethylstilbestrol daily. However generation was mainly caused by when female rabbits, 5 to 12 the changed physiochemical con- months old, were fed clover conditions in the fallopian tubes. taining the equivalent of 60 Since the maximum suppression micrograms of diethylstilbestrol of ovulation occurred when the daily both prior to and during estrogenic content of the alfalfa gestation, it interfered with ovu- was lowest, they concluded that lation and implantation. Since the these phenomena were probably clover did not produce any estro- caused by an unknown antigon- genic effects in the immature adotropic factor, rather than an rabbits, but did cause infertility estrogen. problems in the mature rabbits, it It is interesting that coumes- was suggested that the observed trol itself also exhibits antigona- infertility might be entirely un- dotropic activity. Leavitt and related to the estrogenic content Wright {153) compared the role of the Ladino. of coumestrol with that of estra- In a continuation of these stud- diol in the feedback mechanism of the anterior pituitary gland of ■^ Personal communication from mice. Histologically, the ovaries Joseph Leiter, Assistant Chief for Lab- of the mice receiving coumestrol oratory Activities, Cancer Chemother- or estradiol had become lutein- apy National Service Center of the ized, whereas those of the con- Public Health Service, U.S. Department of Health, Education, and Welfare, trols were still in the various Bethesda, Md., 1958. stages of follicular development. CHEMICAL AND BIOLOGICAL PROPERTIES OP COUMESTROL 73

The g-onadotropes (hormone- pressed and they concluded that producing cells) in the anterior no significant inhibition of hor- lobe of the pituitary gland of the mone release occurred at these controls were abundant. Few feeding levels. In general, their gonadotropes were left after es- studies indicated that coumestrol tradiol treatment, showing that has physiological effects on pitui- the dose used was capable of in- tary function with relatively hibiting production of gonado- large doses being required for tropic hormones. Coumestrol was antigonadotropic effects. In con- shown to block release of the gon- tinued studies, Leavitt (151) and adotropic hormones rather than Leavitt and Meismer (152) con- their production since the gona- firmed that coumestrol influenced dotropes were plentiful in the the release of the gonadotropic coumestrol-fed mice. Dietary cou- hormones but, unlike estradiol, mestrol at several lower levels caused uterine growth before it produced normal estrogenic effects in the mice. However, affected pituitary gonadotropic ovarian growth was not de- function.

Effects on rate of growth During the 1950's it became (212) in 1957 that genistein and clear that the growth rate, the a crude estrogenic extract of clo- efficiency of feed utilization, and ver hay caused increased rate of the chemical composition of meat growth in fattening lambs. in animals could be altered by Booth found that coumestrol hormone administration (12, IS, caused measurable increases in 123, 180, 215). In 1955, Bur- the growth of male rats (50). roughs and co-workers (62) re- O'Dell and co-workers at the ported that feeding 5 to 10 mg. of University of Missouri,-- study- diethylstilbestrol to beef cattle ing growth factors for guinea daily resulted in an increase of pigs, found that an alcohol extract about 20 percent in rate of gain of alfalfa was active, particu- and also increased feed efficiency. larly in males. As this suggested The practice of using synthetic that the factor might be a plant hormones to stimulate rate and estrogen, coumestrol was fed to efficiency of gain in animals has male guinea pigs at a level of 2 since become widely accepted. mg./lOO g. of diet. In the first Synthetic estrogens are now trial, they obtained a remarkable being fed to or implanted in more response, e.g., a gain of 8.7 g./day than 80 percent of fattening during a 4-week period compared steers in this country (22). with 7.5 g./day for the controls. The fact that forage estrogens In two subsequent trials, how- could cause infertility problems ever, the gains were less than the in breeding animals suggested controls, and they concluded that that they might be also capable coumestrol was not the active of producing the same beneficial factor. effects that are obtained with synthetic estrogens. This possi- " Personal communication from B. L. bility was stimulated by a report O'Dell, Professor of Agricultural Chem- from Iowa State University istry, University of Missouri, 1968. 74 TECHNICAL BULLETIN 1408, U.S. DEPT. OF AGRICULTURE

From 1959 through 1964, Old- test consistently demonstrated field and co-workers (i, 103, 190, improved tenderness and juici- 191, 192, 193, 19Í) conducted a ness scores for lamb roasts from series of feeding trials with animals fed high-coumestrol diets lambs to investigate the possible {193, 19Jf). growth-promoting effects of cou- Johnston and co-workers {131) mestrol. The trials progressed studied the effects of five levels from feeding alfalfa meals to of dietary coumestrol ranging feeding coumestrol concentrates from 18 to 151 p.p.m. on the from alfalfa and, finally, to feed- organoleptic quality of lamb. The ing isolated coumestrol. In gen- tests were conducted both in the eral, a trend toward a positive presence and absence of diethyl- growth response was obtained stilbestrol implantation. They with wether lambs, but not with found that tenderness and juici- ewes. Weight differences were ness values of lambs were en- significant for wethers fed crude hanced by increased levels of cou- alfalfa and the coumestrol conmestrol in the ration (fig. 26). centrates, but not for wethers fed Based on taste tests and shear- isolated coumestrol. Organoleptic force measurements, meat

5.8 3.5 A Shear force 5.6 o Tenderness 4.0 □ Juiciness uo 5.4 4.5 S 5.2 5.0 > -n O ¿ 5.0 5.5 ^3 i 4.8 6.0

< 4.6 6.5 1— 4.4 7.0 4.2 7.5 4.0 8.0

DIETARY COUMESTROL, p.p.m.

FIGURE 26.—Effects of five levels of dietary coumestrol on the meat quality of lamb. (Reproduced from a graph by Johnston and co-workers {131).) CHEMICAL AND BIOLOGICAL PROPERTIES OF COUMESTROL 75

TABLE 32.—Average daily gains of steers in two feeding tests

Daily rate of gain Increase in Protein supplement With diethyl- Without diethyl- gain by adding stilbestrol stilbestrol diethyl- in ration in ration stilbestrol Pounds Pounds Percent 1 lb. soybean oil meal 2.07 2.47 19.3 0.5 lb. soybean oil meal plus 1.25 lb. dehydrated alfalfa. 2.30 2.59 12.6 2.5 lb. dehydrated alfalfa 2.41 2.64 9.5 5.0 lb. dehydrated alfalfa 2.46 2.64 7.3

Source: Matsushima, J. K., Clanton, D. C, and Arthaud, V. H. {1T7). from diethylstilbestrol-implanted (245 p.p.m.) coumestrol content lambs that received a diet con- in their daily ration. They attrib- taining 114 p.p.m. coumestrol uted these results to either or all was rated juicy and the most ten- of the following reasons: (1) der. Furthermore, these values that coumestrol was not the fac- for tenderness and juiciness were tor in dehydrated alfalfa that only slightly better than those contributed to greater gains in obtained from lambs that re- cattle; or (2) levels of coumes- ceived 132 p.p.m. coumestrol trol ranging from 122 to 245 without diethylstilbestrol. p.p.m. were too high; or, (3) the quantity of the major nutrients Matsushima and co-workers at in the three samples of dehy- Nebraska (175, 176, 177) have drated alfalfa was so variable reported positive evidence of that the estrogenic effects were weight gain stimulation in steers masked. Further tests {175) fed alfalfa naturally high in es- with alfalfa containing 25, 100, trogenic activity (table 32). They and 250 p.p.m. coumestrol also found that 5 pounds of this al- showed no significant differences falfa in the daily ration produced in gain or feed efficiency among the same daily weight gain in the cattle. pounds (2.46) as did the addition of diethylstilbestrol to the pro- Elam {89) compared the di- tein-equivalent basal diet (2.47). gestibility and nitrogen utiliza- Lower amounts of alfalfa were tion of high (209 p.p.m.) and low also effective but to a lesser ex- (23 p.p.m.) coumestrol alfalfa. tent. When diethylstilbestrol and There were no significant differ- dehydrated alfalfa were com- ences between the two meals in bined, rates of gain were only nitrogen retention. In later stud- slightly better than when either ies {21) employing pure crystal- was administered alone. In later line coumestrol, Elam and Put- experiments, Matsushima and nam again obtained negative co-workers {178) were unable to results for increased nitrogen find any significant differences in retention. gains among steers receiving 4 Beeson at Purdue {16) con- pounds of dehydrated alfalfa pel- ducted feeding experiments with lets having either low (0 p.p.m.), steers in which they found that medium (122 p.p.m.), or high a daily intake of 0.5 pounds of 76 TECHNICAL BULLETIN 1408, U.S. DEPT. OF AGRICULTURE dehydrated alfalfa meal per steer ment of only 4 percent. The dif- increased daily gain 8 percent. ferences in daily gain and feed They also found that it improved efficiencies resulting from these feed efficiency 7 percent and in- treatments were not statistically creased the concentration of vita- significant. Neither treatment mins A and E, carotene, and had any effect on carcass grade, cholesterol in the blood and vita- dressing percentage, or transit min A and carotene in the liver shrink of the steers when com- (table 33). Hov^ever, Stob and pared to the controls. In further co-workers {210), in experiments experiments,^^ three sets of iden- in which high (560 p.p.m.) and tical twin calves were fed a low (50 p.p.m.) coumestrol dehy- coumestrol concentrate equivalent drated alfalfa pellets were fed, to 1.36 g. coumestrol per head were unable to find a growth- daily. No improvement in daily promoting eifect due to coumes- gain or feed efficiency was found. trol. In fact, daily gains of steers In fact in most instances, a slight fed the low coumestrol alfalfa depression occurred. Their latest were significantly greater than studies -^ were conducted with those of steers fed the high cou- pure crystalline coumestrol. Four mestrol ration. sets of identical twin heifer Stob and co-workers {209) also calves were fed differing levels of compared the growth-promoting coumestrol. Fortification of the effects of a coumestrol concen- high energy ration with 50 and trate with that of diethylstilbestrol on steers. Daily feeding ^^ Personal communication from W. of diethylstilbestrol (10 mg.) im- M. Beeson, Lynn Professor of Animal proved the daily gain and feed Nutrition, Purdue University, 1963. efficiency 12 percent. However, ''W. M. Beeson, Martin Stob, and M. T. Mohler. EFFECT OF CRYSTALLINE feeding the coumestrol concen- COUMESTROL ON THE PERFORMANCE OF trate (equivalent to 840 mg. cou- IDENTICAL TWIN HEIFERS. Amer. Dehy. mestrol) resulted in an improve- Res. Council, final rpt., 3 pp. 1966.

TABLE 33.—Effect of dehydrated alfalfa meal on steers

One-half No pound Item dehydrated dehydrated Increase alfalfa alfalfa meal meal Percent Daily gain pounds 2.05 2.21 8 Feed per hundred weight of _ _ do — 910 842 7 gain. Blood analysis: Vitamin A „ micrograms per 100 milliliters 22 34 55 Carotene do 72 163 126 Vitamin E _ ^ do 38 48 26 Cholesterol do 110 136 24 Liver values : Vitamin A micrograms per gram _ 4.6 11.0 139 Carotene do 10.4 12.0 15

Source: Beeson, W. M. (16). CHEMICAL AND BIOLOGICAL PROPERTIES OF COUMESTROL 77

100 p.p.m. coumestrol did not mestrol is not a growth-stimulant cause any significant change in for cattle. Thus, although cou- the daily gain, feed comsumption, mestrol is similar to diethylstil- or feed efficiency. Higher levels bestrol in its estrogenic effects in of coumestrol (150 and 200 animals, it must have a different p.p.m.) depressed daily gain. metabolic function, since it has From their results, Beeson and little effect on their rate of co-workers concluded that cou- growth or feed efficiency.

Other biological effects Coumestrol and several of the compared them with Captan as other coumestans have been a spore germination inhibitor for screened for a variety of physio- Venturia ineaqualis and Phytoph- logical effects other than those thora infestans and with PCNB already discussed. and Dixon, which are soil fungi- Coumestrol was tested by the cides, as a mycelial growth in- CCNSC ^" in their routine anti- hibitor for Rhizoctonia solani and cancer screening tests for activ- Pythium ultimumr^ Although the ity against three mouse tumors. reference compounds were active As it did not cause any statisti- at concentrations as low as 10 cally significant reduction in p.p.m., the coumestans were inac- tumor weight in the Sarcoma 180 tive at even 1,000 p.p.m. Cruick- or Adenocarcinoma 755 tumor shank ^^ compared these same systems, nor any significant in- four coumestans with pisatin for crease in lifespan in the Leuke- toxicity against mycelial growth mia L-1210 system, it was consid- of Monilinia fructicola and found ered inactive. In addition, cou- them to have no fungitoxic activ- mestrol was tested by Parke, ity. The relationship of coumes- Davis & Co., for its effect on the trol to antifungal activity in red normal serum cholestrol levels in clover was studied by Berken- rats. It was found to have no ef- kamp of Canada Department of fect on either atherosclerotic Agriculture.-^^ His results showed plaques or the serum lipids of hy- that coumestrol was not inhibi- perchloresteremic rats.-^ Jacobs tory to fungi attacking Canadian also found that coumestrol had red clover. The Pesticide Chemi- little effect on the deformability cals Research Branch of ARS ^^ of erythrocytes (red blood cor- tested medicagol as a residue for puscles).^' insecticidal properties. Medicagol Antifungal screening tests have been performed on coumes- '' Personal communication from E. G. Jaworski, Monsanto Chemical Co., trol, 4'-0-methylcoumestrol, medi- 1964. cagol, and trifoliol at several '^ Personal communication from I. A. different laboratories. Monsanto M. Cruikshank, Principal Research Scientist, Commonwealth Scientific and Industrial Research Organization, Aus- '^'' See footnote 21, page 72. tralia, 1965. -" Personal communication from M. L. ^^ Personal communication from Bill Black, G. Rodney, and F. Armstrong, Berkenkamp, plant pathologist, Canada Parke, Davis & Co., 1959. Department of Agriculture, 1968. "^ Personal communication from H. R. ^^ Personal communication from S. A. Jacobs, M.D., Evanston Hospital As- Hall, J. H. Fales, and O. F. Bodenstein, sociation, 1968. USDA, 1965. 78 TECHNICAL BULLETIN 1408, U.S. DEPT. OF AGRICULTURE was not found to be toxic to adult trogen content was low. How- face flies, to DDT-resistant house- ever, they were unable to estab- flies, or to adult male chlordane lish any definite correlations be- resistant German cockroaches. tween mastitis incidence and es- Frank and co-workers (106) trogenic activity of the feed as found a definite relationship be- determined by mouse bioassay. tween the consumption of alfalfa The milk production and breed- and the incidence of clinical ing efficiency did not appear to be mastitis in small herd of dairy effected by the different feeding cows. Over a 2-year period more programs. Kesler and co-workers than twice the number of at- at Pennsylvania State Univer- tacks were recorded for animals sity ^" conducted preliminary receiving alfalfa and bromograss studies on the relationship be- as silage than for those fed al- tween plant estrogens and the most entirely nonlegumes. Ani- solids-not-fat (SNF) content of mals fed alfalfa-bromograss sil- milk. The percent SNF in the age, made when the estrogen con- milk dropped when the cows tent of the plants were relatively went from winter feeding to pas- high, exhibited 3.6 attacks of ture. However, no coumestrol mastitis per cow as compared to was found in the pasture and the 0.8 attacks per cow when the es- study was discontinued. Correlation of Structure and Activity Robertson and Whalley (198) peared to be important for activ- suggested that the estrogenic ac- ity, the introduction of an alkyl tivity of a molecule is dependent group at the 2-position greatly upon certain molecular charac- reduced the activity of genistein. teristics. These include the pres- In contrast, Bradbury and White ence of oxygen-contaning func- (5i, 55) found the isoflav-3-enes tional groups capable of forming to be active only when they had a hydrogen bonds and positioned at 2- or 4-alkyl substituent, and a given distance from each other they were then more active than (140) as well as the presence of genistein. The isoflavenes appear at least one functional phenolic to be more comparable to the stil- group of high hydrogen-bond- bene estrogens than to the isofla- forming capacity (75). Both cou- vone estrogens, since the effects mestrol and genistein contain of substituent groups on their ac- phenolic groups that may satisfy tivity is similar to the effects of the configurational and electros- similar groups in the stilbene se- tatic requirements necessary for ries (20Jf^). The isoflavenes have an estrogenic molecule. stilbene-like structures, e.g., 4', Bradbury and White (55) 7 - dihydroxy-4- e t h y 1-2-methyliso- studied the estrogenic activity of flav-3-ene (fig. 27, Ha), which a series of isoflavones, isoflava- has the same carbon skeleton as nones, and isoflavenes related to diethylstilbestrol (fig. 27, la) and genistein and formononetin. The is the closest approach to it that removal of the 5-hydroxyl group from genistein (forming daid- ^" Personal communication from E. M. zein) decreased its activity (39). Kesler, Professor of Dairy Science, The While the 5-hydroxyl group ap- Pennsylvania State University, 1968. CHEMICAL AND BIOLOGICAL PROPERTIES OF COUMESTROL 79

li il

o -Û ^ ^

o

c

bo g

bo Ö Co

ai

U I II li ö'-O 0 80 TECHNICAL BULLETIN 1408, U.S. DEPT. OF AGRICULTURE still retains the oxygen in the (fig. 27, Illb) only reduces the ac- heterocyclic ring (5^). Lawson tivity about a hundredfold (181). (150) prepared the dimethyl- The introduction of a carbonyl ether of this compound (fig. 27, group at the 2-position appears to lib) and found it to have estro- have little effect on the resultant genic activity of the same order activity, since the activity of the of magnitude as diethylstil- 3-phenylcoumarin (fig. 27, IVa) bestrol dimethylether (fig. 27, is of the same order of magnitude lb). Micheli and co-workers as the 2-dihydro derivative (fig. (181) prepared the diacetates of 27, Ilia). When the 4-ethyl group la and lia (fig. 27) and found on the phenylcoumarin (fig. 27, them to compare closely in activ- IVa) is replaced with either a ity, each being about three times hydrogen (fig. 27, IVb) or a hy- as active as their methylethers. droxyl group (fig. 27, V), the entire activity is lost. Lack of ac- In a review article by Biggers tivity in 4-hydroxycoumarins is (i8), a suggestion was made by general and has been attributed Whalley that coumestrol also de- to destabilization of the 3,4-double rives its estrogenic activity by bond and, therefore, of the stil- virtue of its stilbene-like struc- bene system, which is now free ture. This possibility was investi- to exist in tautomeric keto-enol gated by Bickoff and co-workers forms (208). Formation of the (35) and Micheli and co-workers furan ring by introduction of a (181), who studied the activity 4,6'-oxygen bridge to give cou- of a series of compounds related mestrol dimethylether (fig. 27, to coumestrol, including the Via), stabilizes the 3,4-double above-type compounds. By com- bond and maintains the stilbene- paring the variations in estro- like structure. This restores a genic activity caused by gradual considerable amount of the lost structural changes starting with activity as coumestrol dimethyl- 4',7 - dimethoxy - 4 - ethyl-2-methyl - ether is about one-twentieth as isoflav-3-ene (fig. 27, lib) and active as 4-ethyl-7-methoxy-3-(4- ending with coumestrol dimethyl- methoxyphenyl) coumarin (fig. ether (fig. 27, Via), the impor- 27, IVa) and about one-two thou- tance of alkyl substituents and sandth as active as diethylstil- the 3,4-double bond, i.e., the stilbestrol dimethylether. Coumes- bene-like structure, for estro- trol (fig. 27. VIb) itself is about genic activity can be demon- three times as active as its dime- strated. thylether (fig. 27, Via). Replacement of both the 2- Modification in the structure of methyl and 4-ethyl groups on the coumestrol, such as the addition isoflaven (fig. 27, lib) with hy- or removal of nuclear substi- drogen to from 4',7-dimethoxy- tuents and blocking of the hy- isoflav-3-ene (fig. 27, IIIc) de- droxyl groups, reduces the activ- creases the estrogenic activity of ity of coumestrol (35). The effect the isoflavene (fig. 27, lib) more than 10,000-fold (55), However of substituent groups on the ac- replacing only the methyl group tivity of coumestrol is similar to at the 2-position with hydrogen the effects of similar groups in (fig. 27, Ilia) or the ethyl group the stilbene series (20U). The at the 4-position with hydrogen fact that coumestrol is from 30 to CHEMICAL AND BIOLOGICAL PROPERTIES OF COUMESTROL 81

100 times more active than the exist in keto-enol forms. This re- closely related estrogenic isofla- sults in a single bond in the 3,4- vones (110) is probably due to position of the isoflavone and, the carbonyl group at the 4-posi- consequently, the partial destruction of the isoflavones, which is tion of the estrogenic stilbene primarily ketonic, but which can system.

Patents

Eight patents were issued as a details of methods for isolating result of studies on coumestrol coumestrol from plants and de- performed at Western Regional scribed the preparation and utili- Research Laboratory. These de- zation of feeds containing cou- scribed the isolation of coumes- mestrol. In addition, the physical trol and its estrogenic activity characteristics and estrogenic (26), its purification (92), its properties of coumestrol were synthesis (91, 93), and the prep- discussed. Purification of cou- aration and estrogenic activity of mestrol via its acetate derivative several of its derivatives (2Í, 25, and regeneration of coumestrol 33, 3J,). from its acetate by alkaline hy- The first patent by Emerson drolysis were the subject of the (91) described the synthesis of fourth patent (92), dialky loxyphenyl-2-1 h i o p y r u v i c Patents were obtained by Bick- acids from dialkyloxybenzal rho- off and Booth on the preparation danines using aqueous alkali in and uses of ester (25) and ether the presence of an alkali metal (24) derivatives of coumestrol. Sulfide. This method afforded a These compounds were shown to means of preparing these com- exhibit estrogenic activity simi- pounds in greater yields than pre- lar to that of coumestrol and to viously possible. The use of (2,4- be more stable and more readily dimethoxyphenyl ) thiopyruvic produced in pure, crystalline acid to prepare intermediates for form. The preparation of cou- the synthesis of coumestrol was marone derivatives, i.e., salts also described. The second patent of 2-(2,4-dihydroxyphenyl)-6-hy- was by Emerson and Bickoff droxycoumar one-3-car boxy lie (93) and was on the synthesis acid, from coumestrol by mild of coumestrol from 3-(2,4- alkaline hydrolysis (3i) and use dimethoxyphenyl ) -4,7-dihydroxy- of these salts as estrogenic agents coumarin by heating in the pres- (33) were the subjects of the last ence of a hydrohalic salt of an two patents. aromatic amine, such as aniline The authors assigned a non-ex- hydrochloride. The reaction se- clusive, irrevocable, royalty-free quence was described earlier license on these patents to the under ''Synthesis of Coumestans'' Government of the United States (fig. 11). The preparation of cou- of America for their use mestrol diacetate was also de- throughout the world. Power to scribed. grant sublicenses on these patents A patent on estrogenic factors, was also given to the United by Bickoff and Booth (26) gave States Government. 82 TECHNICAL BULLETIN 1408, U.S. DEPT, OF AGRICULTURE Summary The result of extensive stud- plant that causes the rapid accu- ies on the occurrence and the mulation of this type of com- chemical, physical, and biological pound. Although the biosynthetic properties of the plant estrogen, pathway for coumestrol in dis- coumestrol, and the seven other eased plants has not been stu- alfalfa and Ladino clover coumes- died, it has been shown that tans are described in this bulle- healthy alfalfa biosynthesizes tin. Methods for their isolation coumestrol in a manner analo- from and determination in these gous to the isoflavones. Radioac- plants are described in detail. tive tracer studies proved that an Coumestrol and the other cou- aryl migration occurs at some mestans vêre separated from point during the biosynthesis of other plant constituents by sol- coumestrol, and that coumestrol, vent extraction and countercur- daidzein, and formononetin can rent distribution. Both physical arise from the same intermediate and chemical means vêre em- chalcone. ployed for their structural eluci- Although there is a biosyn- dations, vîth synthesis generally thetic interrelationship between being used for confirmation. coumestrol and the isoflavones, The occurrence of coumestrol they do not appear to be metabol- in nature is widespread. It is the ized along the same pathways. dominant estrogen of alfalfa, The differences in metabolic Ladino clover, and the annual routes may account for the much medics. Both genetic and envi- higher relative estrogenic po- ronmental factors have been tency of coumestrol. Equally im- shown to inñuence the buildup of portant may be structural differ- coumestrol in these forages. Pre- ences. There are certain configu- sent evidence indicates that cou- rational and electrostatic require- mestan levels, including coumes- ments for a molecule to be estro- trol, are quite low in alfalfa free genic. The most important ones, of disease, but that they rapidly as far as diethylstilbestrol is increase to very high levels concerned, are the alkyl substi- shortly after pathogen attack or tuents and the 3,4-double bond, aphid infestation. In barrel i.e., the stilbene structure. The medic, physiogenic leaf spotting furan ring in coumestrol stabil- has been shown to cause the ac- izes this 3,4-double bond. How- cumulation of coumestrol. In all ever, the carbonyl at the 4-posi- cases, coumestrol is concentrated tion of the isoflavones can exist in the immediate vicinity of the in tautomeric forms, which re- damaged tissue. The pathogen- sults in a single bond between the host relationship has not been es- 3,4-positions and, consequently, tablished for the other coumes- the partial destruction of the estrol-containing forages. trogenic stilbene system. The similar effects of foliar The problem of infertility in pathogens, aphid infestation and farm animals caused by the physiogenic leaf spotting on the ingestion of estrogenic alfalfa accumulation of coumestrol sug- and Ladino clover is probably gest that disease "triggers" some more severe than is generally nonspecific mechanism in the recognized. Moderate cases of in- CHEMICAL AND BIOLOGICAL PROPERTIES OF COUMESTROL 83 fertility frequently go undetected lambs. Additional work is neces- or are only transient and, in most sary to confirm this observation. cases, are not associated with the The role of coumestrol in feed being ingested by the ani- the pathogen-host relationship mal. It is becoming increasingly should be elucidated. It is possi- evident that forages can also con- ble that coumestrol is either elab- tain antiestrogenic and antigona- orated by the plant in response to dotropic substances that may act attack or by the pathogen itself. to partly nullify the expected res- Detailed studies of the coumes- ponse to the ingested coumestrol trol-plant-pathogen interrela- or even to that of the normal ani- tionship will provide a better un- mal estrogens. Moreover, it has derstanding of the mechanism of been shov^n that under certain disease resistance. Genetic studies conditions, coumestrol itself can have shown that, by recurrent se- interfere with the endogenous es- lection for resistance to disease, trogens and pituitary hormones. alfalfa very low in coumestrol can be produced. The reduction Most growth studies have in- of coumestrol content by develop- dicated that coumestrol has no ment of new varieties or by con- effect on the rate of growth of trol of pathogens should elimi- either cattle or sheep. However, nate reproductive problems in evidence exists which suggests farm animals caused by the that coumestrol may have benefi- ingestion of high estrogen for- cial effects on carcass quality in ages.

Literature Cited

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