Studies on the Chemical and Biological Properties of Coumestrol and Related Compounds (
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2 2 5 :; 1111/2.8 1111/2.5 :~ 1/1/1 .8 11111 . 1.0 1.0 32 W /////3.2 I~ 1111/ . ."'3 . 163 IE ~~~ .; 13.6 ~ I.\.ii ...c: I~ .0 :i I~ L. " I ~ u 1.1 ~t.:.u. 1.1 ~L.L.;. -- I 111111.8 111111.25 111111.4 111111.6 111111.25 111111.4 111111.6 MICROCOPY RESOLUTION TEST CHART MICROCOPY RESOLUTION TEST CHART NATIONAL BUREAU Of STANDARDS·1963·A NATIONAL BUREAU Of STANDARDS·1963·A Studies on the Chemical and Biological Properties of Coumestrol and Related Compounds ( Technical Bulletin No. 1408 Agricultural Resear~h Service UNITED STATES DEPARTMENT OF AGRICULTURE !/ Contents Page Page". Introduction ___ ___ ___ ___ __ __ ___ _ 1 Synthesis of coumestans-con. Analytical prol;edures _________ ,,_ 3 Synthesis from previously Biological procedures _. ________ 6 formed. cO'lrnestans __ _ __ __ __ 32 Chromatographic procedures _ __ 7 Physical properties of coumestans Qualitative procedure 8 Ultraviolet· absorption spectral Quantitative procedure _ _ _ _ _ _ 9 measurements ___ ___ __ __ __ __ 32 Other chromatographic pro- Proton magnetic resonance cedures __________________ 10 spectral measurements 35 Isolation _________ . _ • ___ ___ __ __ _ 14 Mass spectral measurements _ __ 37 Isolation of cournestrol from Chromatography - - - -- -- - _ 41 Ladino clover ______________ 15 Coumestrol in plants ____________ 43 Large-srllle processing of al- Variations as affected by cut falfa 'for coumestrol __ __ _ __ _ 1e tings and sta~'E!s of growth __ 43 Isolation of compoundu closely Variations as affected by loca related to coumestral _. 17 tion and climatic conditions _ _ 46 Characterization of coumestans __ 18 Variations as affected by varietal Coumestrol _ _ _ __ .. ___ . .. 20 and genetic differences ___ .._ 47 Trifoliol _ .• _ _ .• _ _ _ 20 Variations as related to disease_ 48 Medicagol ______ • ___ ..24 Mechanism of biosynthesis 53 4'-Q..methylcoumestrol __ . _ _ _ _ :1:4 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 -.--------- 63 coumestan -___ - - - . __ . __ - - . 26 Hyperestrogenic syndrome -.. 65 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 derivath,es 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 Trade names and the names of commercial companies are used in this publication solely to provide specific information. Mention of a trade name or manufacturer does not constitute a guarantee or warranty of the product by the U.S. Department of Agriculture or an endorsement by the Depart ment over other products not mentioned. 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. ~. .BICKOFF, R. R. SPENCER, S. C. WITT, and B. E. KNUCKLES, chemists, Western 'Utilization Researrk and Development Division, Agricultu-ral 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)1 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), which nificant estrogenic activity. In was shown to be estrogenically 1955, work was begun at active in mice (49, 82). Sutse 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, troT (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 sheep (83). However, more re marin derivative belonging to an cent work suggests that formo entirely new class of compounds, nonetin may be more important which were later named coumes (84,182,184). 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 (54). However, experiments in close structural relationship not only to the isoflavones, but also 1 Italic nurnhers in parentheses refer to Literature Cited, p. 83. to the natural estrogen, estradiol, 1 \) 2 TECHNICAL ntJLLIllTIN 140S~ U.S. DEPT.OJl' .AGRICULTURE ,,:.- HO o Genistein Estradiol OH OH Coumestrol Diethylstilbestrol FIGURE I.-Structural formulas of genistein, estradiol, coumestrol, and diethylstilbestrol (.l1.l1). and the synthetic estrogen, di 222), white (105, 167), bur ethylstilbestrol (fig. 1). In fact. (167), subterranean (18, 167, Whalley (48) proposed that the 183, 184), and strawberry U8, 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,2 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 (39, 122), Ladino tion in coumestrol content of al clover (39, 122), and the annual falfa and Ladino clover, as related medics (18, 104, 183). Although to environmental factors (36, 37, the isoflavones account for most 127), indicated that coumestrol of the estrogenic activity of red content was greatly affected by and subterraneiin clovers, their disease (43, 161,.) and insect in contribution to the estrogenicity festation (163). of alfalfa and Ladino clover is small (122). Subsequently, cou 2 Personal communication from J. H. mestrol was found in a wide Adler, animal physiologist, Hadassah varie'ty of clovers, e.g., red (167, Medical School, Israel, 1967. CHEMICAL AND BIOLOGICAL PROPERTIES OF COUrdESTROL 3 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 calendulacea in 1956. mice (39) and shE!ep (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 PsoraZea coryli ported by Engle and co-workers folia (65, 86, 141), and norwede (98), Wright (224), and Adler lolactone from Eclipta alba and Trainin (6, 7, 8), can be ex (148). The structural formulas plained by the presence C)f cou of these naturally occurring cou mestrol. Estrogenically active al mestans are given in figure 2. falfa was shown to beneficially Coumestans are also structuraHy affect the rate of growth of cattle similar to the naturally occurring (176) and sheep (103, 194). Al isoflavonoids inermin (77), trifo though later results suggested lirhizin (59), pisatin (81, 196), that coumestroI was not the caus pterocarpin (60, 64, 198), and ative agent, coumestrol did im homopterocarpin (64, 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 (Med'i into coumeR+;rol dimethylether. cago sativa) (41) and Ladino clo This technical bulletin summa ver (T?'ifoUum ?·epens) (38). rizes and correlates 13 years of They are trifoliol (41, 160), research on coumestrol and re 4'-O-methylcoumestrol (42), 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 (45), and 1l,12-dimethoxy It aiso includes some of the stud 7 -hydroxycoumestan (206). This ies on othe'l' 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 (49, mination of the onset of estrus in 56, 83, 197). Once the estro'genic the test animal by the appear ance of cornified epithelial cells compound is identified, chromato in vaginal smears~ In most of the graphic methods (156) can often work on the isolation of estro be used for its quantitative deter genic constituents from forages, mination.