Study of the phytoestrogen content of goat’s rue (Galega orientalis ), alfalfa (Medicago sutiva) and white clover (Trifolium repens) Hannu Saloniemi, Kaarlo Kallela and Ilkka Saastamoinen Saloniemi, H., Kallela, K. & Saastamoinen, 1.1993. Study of the phytoestrogen content of goat’s rue (Galega orientalis), alfalfa (Medicago saliva) and white clover (Trifolium repens). Agric. Sci. Finl. 2: 517-524. (College of Veterinary Medicine, Dept, Basic Veterinary Medicine, Sect. Animal Hygiene, FIN-00581 Helsinki, Finland.) Studies were conducted to determine the phytoestrogen content of goat’s rue (Galega orientalis Lam.), alfalfa (Medicago saliva L.) and white clover (Trifolium repens L.), all belonging to the Fabaceae family subjected to test cultivation at research stations of the Agricultural Research Centre of Finland. Apart from some insignificant quantities, goat’s rue did not contain any known phytoestrogens. Even in biological studies ithad no estrogenic effect. The estrogenic effect of alfalfa was apparently due to coumestrol, which was discovered in the samples in quantities of 34-65 ppm. All white clover varieties contained very small quantities of estrogenic isoflavones and coumestrol, and they did not explain the increased weight of the immature rat uterus observed in the biological studies. Key words: phytoestrogens, goafs rue, alfalfa, white clover Introduction Studies of the effects of phytoestrogens have been conducted since the early 1940 s as a result of widely spread fertility problems observed in Aus- tralian sheep (BENNETTS et al. 1946). In Finland these studies started in the 19605. Their aim was primarily to determine whether phytoestrogens were involved in the fertility disorders in cows which occurred commonly especially in the spring at the beginning of the grazing season (Kallela 1964). Interest in phytoestrogens has generally been aroused by their adverse properties. They may, however, also be beneficial while increasing the growth rate of animals and the milk yield of cows (Refsdal 1976,Petterson et al. 1984). Ac- cording to recent studies, they may also have a prophylactic effect against some hormone-related human malignancies (Adlercreutz et al. 1991, Rose 1992). The known phytoestrogens are either isofla- vonoids or coumarines. Of the isoflavonoids, bio- chanin-A and genistein, which in monogastric an- imals have an estrogenic effect, are broken down in the rumen of ruminants into inactive paraethylphe- nol (Pettersson et al. 1984). Two other phytoes- trogens, daidzein and formononetin, are converted by ruminal microbia into active equol. Coumestrol, which belongs to coumarins, is absorbed and is active as such (Pettersson et al. 1984). Many Finnish fodder and pasture plants contain small amounts of phytoestrogens. The highest con- centrations of phytoestrogens occur in red clover, so that all varieties ofred clovercontain phytoestro- gens. Abundant feeding of a diet based on red clover silage has been shown to causefertility prob- lems in cattle (Kallela et al. 1984). Despite its numerous good properties, red clover also has dietary and feed-technological drawbacks: it is poorly resistant to treading and it is not very 517 Agric. Sei. Finl. 2 (1993) https://www.c-info.fi/en/info/?token=Sbzf40UCSYyTStje.d72SrtFxSLVs1A4TjcpfEQ.YbcqmeArhxPBH4JDO-MjaQ9snRm8m0UTaAjv-rVtfwqhmFZWuVho0w7UzZ-Uaeu2VRZCs4q_mtWGVkJAQeVjnPlzer94FDFnHLY_sIOOQlu3d1pxRngktVny-MiNCgbfa7CL6GmQ2V7Jed3MU9FeHjXrJSEpUYMRibIYkEjp4UFuvywEKqdYD8f29zDpfMSm89elu5w6s9uVX6wJBd6accutZW9SM3id8meZFUBJt4FATN8uYxukATwt22BmQjP0N4pju-ict8WFH7WuNJFKOQa0U-qbVdt1IHO-jyaR546q--avyvOXEC7YLpZT palatable; it disappears from fallows in 2-3 years and the oppressional losses of dried red clover are great. Partly for these reasons attempts have been made to experiment with new legume varieties suit- able for the Finnish climatic conditions which might have a positive effect on the fodder quality and palatability. These plants include white clover {Trifolium repens), alfalfa (Medicago saliva) and goat’s rue ( Galega orientalis). At present, their adaptability to the Finnish conditions is experi- mented at agricultural research stations. In this con- text, it has been considered justified to also exam- ine their content of phytoestrogens and estrogenic effects on rats. Material and methods Experimental Samples ofwhite clover, alfalfa and goat’s rue were collected during the summer of 1991 from five agricultural research stations in different parts of Finland. There were four white clover {Trifolium repens) varieties: ‘Undrom’, ‘Jögeva’, ‘Sandra’ and ‘Tammisto’. Samples of ‘Undrom’ were col- lected from the South Savo, Karelia, Kainuu and North Ostrobothnia Research Stations, while samples of ‘Jögeva’, ‘Sandra’ and ‘Tammisto’ were collected from the South Savo Research Sta- tion. Samples of goat’s rue {Galega orientalis) were collected from the Karelia and Sata-Häme Research Stations and those of variety ‘Jokioinen’ of alfalfa {Medicago sativa) from the Sata-Häme Research Station. Fertilization was very similar in all the research stations. During the growing period samples were col- lected, as a rule, mainly four times: twice from the spring growth (white clover at the pasture and sil- age stage, alfalfa and goat’s rue at the bud and early blossom stages) and twice from the aftermath. As far as possible, the samples were collected at the same stage of development at all research sta- tions. Samples from the second aftermath of white clover were obtained only from the South Savo Research Station, but even there the growth of clo- ver was poor. The samples consisted of the entire above- ground part of the plant. The samples were ground in a meat chopper immediately after cutting. There- after they were allowed to stand for 30 min at +37°C for the conjugated phytoestrogens to hydro- lyse (Francis and Millington 1965a) before mixing in absolute ethanol. The samples were then stored in a refrigerator for closer chemical analyses and biological studies. Studies conducted on subterranean clover have revealed that chrushing leaf tissue releases glu- cosides to free isoflavones (aglucones) through en- zyme hydrolysis (Beck 1964, Francis and Millington 1965b). Accordingly, when different parts of red clover are milled, enough enzyme (p glucosidase) was released from every part of the plant (stems not included) to allow complete hydro- lysis (McMurray et al. 1986). The adequacy of the hydrolysation method (mac- eration and incubation for 30 min at 37°C) was established in tests with red and white clover, in which theresults of this method and acid hydrolysis were compared (Table 1). Chemical analyses The method described earlier (Kallela and Saas- tamoinen 1978) was adapted for this study as follows: The plant samples warmed up at room temperat- ure (50 g in absolute ethanol) were mixed intensely for 5 min. The procedure was repeated the next day whereafter the samples were filtered through a Buchner funnel. The filtrate was evaporated using a vacuum evaporator (+ 40°C) to reach 100 ml. An aliquot was diluted and filtered through an Acro- disc CR filter (Gelman) before high performance liquid chromatography (HPLC). Daidzein, genistein, formononetin and bio- chanin-A were determined using a UV detector, and coumestrol was determined by fluorometry. The liquid chromatograph used was a Hewlett Packard 1050 chromatograph provided with an automatic sampler and a UV detector. The fluorometer was a Perkin ElmerLS-4. A Windows- based Chem Station program of Hewlett Packard in 518 Agric. Sei. Fint. 2 (1993) Table 1. A comparison between the amounts of isoflavones after the present hydrolysis method and hydrochloric acid hydrolysis. Species Date of Treatment Isoflavones % in DM Daidzein Genistein Formo- Bioch.-A. Totalcollection nonetin Red clover Sample 1 1.7.92 A 0.024 0.076 0.214 0.828 1.142 A + B 0.028 0.081 0.252 0.926 1.286 A + C 0.024 0.074 0.245 0.866 1.209 Sample 2 1.7.92 A 0.025 0.084 0.203 0.888 1.199 A+B 0.024 0.083 0.212 0.863 1.182 A + C 0.028 0.076 0.224 0.858 1.206 White clover Sample 1 7.6.93 A 0.001 0.002 0.014 0.006 0.022 A + D 0.000 0.002 0.015 0.004 0.022 A = maceration + 30 min 37°C B = 4.6 gDM, +Bo°, 2 h (reflux) 4-mol HCL 1 ml in 30 ml alcohol C = 4.6 gDM, +Bo°, 2 h (reflux) 4-mol HCL 10 ml in 30 ml alcohol D =4.0 gDM, +7s°, 1 h (reflux) 25% HCL 10 ml in 80 ml alcohol a 486 computer was used forrecording and calcula- tion of the results of samples. The conditions were as follows: column Lichrosorb 100 RP-18 spm 250x4mm Hewlett Packard, flow rate Iml/min, acetonitrile-water solution, initially 40%, after 5 min 70%, after 8.5 min 80% and after 9 min 100% to complete 15 min, was used as mobile phase. The apparatus was stabilised for 7 min with 40% aceto- nitrile before each run. UV-254/nm; fluorometer ex 304 nm, em 454 nm. The following commercial preparations were used as standards: daidzein and formononetin (K & K Laboratories ICN, USA), genistein (Sigma Chemical CO, USA) and bio- chanin-A (Aldrich-Chemie, Germany). Standards were diluted to absolute alcohol concentrations of 1-20 pg/ml. The injection used was 20 pi, while on the fluorometer it was 10 pi or less. Biological studies The studies were conducted using the method de- scribed earlier (Kallela 1975) with the amount of extract fed to the rats equalling 3 g of plant dry matter per day. Uterine weight was used as the indicator of the effect of the treatment. The extract added to the feed of the test rats was prepared based on an earlier study (Kallela 1964) as follows: The plant samples were cooked in etha- nol three times: for 30 min, 1 h and 15 min, and filtered through a Buchner funnel. The ethanol fil- trates were pooled and ethanol was evaporated at +4O°C using a vacuum evaporator. At the end of evaporation, some (approx. 10 ml) distilled water was added and evaporation was continued until the water started to distil over. The water residue was transferred with the distilled water and diethyl ether (ca. 40 ml and 250 ml in small doses, respectively) into a separating funnel which was shaken intensely for 5 min, allowed to stand for 30 min and reshaken for 5 min. After the different layers had separated, the ether extract was collected. The water residue was treated in the same way three more times. The ether extracts were pooled and washed with due care in the separation funnel, shaking with a small amount of water (ca. 50 ml). Finally the ether ex- tract was evaporated to reach 500 ml using a vac- uum evaporator. The extract was added to the feed of the test rats at the calculated doses from which ether was evaporated in a fume hood. HPLC analyses were conducted on the phytoes- trogen concentration of the extracts. An aliquot of 519 Agric. Sei. Fint. 2 (1993) Table 2. The comparison between HPLC results of phytoestrogens in alcohol and ether extracts. Research station Species and variety Date of sample Isoflavones %in DM collection , Z~Alchol extract Ether extract South-Savo White clover, Jögeva 17.6 0.018 0.021 4.7 0.019 0.020 6.8 0.020 0.024 2.9 0.023 0.034 White clover, Sandra 17.6 0.020 0.024 4.7 0.019 0.020 6.8 0.018 0.022 White clover, Tammisto 17.6 0.021 0.025 4.7 0.019 0.021 6.8 0.022 0.028 2.9 0.034 0.038 White clover, Undrom 17.6 0.020 0.026 4.7 0.023 0.027 6.8 0.024 0.024 2.9 0.029 0.036 Coumestrol ppm in DM Sata Häme Alfalfa, Tammisto 17.6 33.7 34.5 27.6 64.8 71.6 1.8 25.3 18.9 12.8 * 63.0 * analysis unsuccesful etherextract was evaporated in a stream ofnitrogen (+4O°C) to dryness and theresidue was dissolved in an equal volume ofethanol.The sample was diluted and filtered before HPLC analysis. The concentra- tions parallelled those observed in chemical studies (Table 2). Results The study of the phytoestrogen content and estro- genic effect was negative for goafs rue. Insignific- ant amounts of formononetin and biochanin-A were observed only in the early spring samples collected at the Karelia Research Station, and signs ofcoumestrol were observed in the early spring and late autumn samples of the Sata-Häme Research Station. They had no effect on the weight of therat uterus. Alfalfa showed estrogenic activity which was attributable only to coumestrol (Table 3). The quantities of estrogenic isoflavones, formononetin (90-95%) and genistein (5-10%) discovered in the Table 3. Phytoestrogen content of alfalfa (Medicago saliva) and the effects on the weight of immature rat uterus. Variety ”Jokioinen”, Sata-Häme Research Station. Biological study Duration of the experiment 5 days Experimental extract 3 g DM/day Rats/test group 5 rats Rats/control group 8 rats, uterine weight 21.0 ± 2.0 mg Growth stage Date of Coumestrol Increase sample ppm in DM in weight collection of uterus mg Budstage 1 17.6 33.7 +21.2*" Early blossom 27.6 64.8 +s7.9*** First aftermath 1.8 25.3 +l9.3*** Second aftermath 12.8 63.0 2 +46.2*** 1 small amount of formononetin and signs of biochanin-A in samples of bud stage 2 H PLC result of ether extract *** = P< 0.001 white clover varieties were small. In the main part of the material the amount of daidzein and bio- 520 Agric. Sei. Finl. 2 (1993) Table 4, Phytoestrogen content of white clover and the effects on the weight of immature rat uterus Duration of the experiment 5 days Experimental extract 3 g DM/day Rats/test groups 5 rats Rats/control group 8 rats; uterine weight 21.0 ± 2.0 mg Variety Research Growth Date of Isoflavones Coumestrol Increase in station stage sample %in DM ppm in DM weight of collection uterus mg Jögeva South Savo 1 17.6 0.02 0* +18.8'» 2 4.7 0.02 0* +2l.o*** 3 6.8 0.02 0' + 8.6" 4 2.9 0.02 0* +31.1"* Sandra South Savo 1 17.6 0.02 0* +ls.B** 2 4.7 0.02 0* +ls.o*** 3 6.8 0.02 6.8 +l7.s*** 4 2.9 0.03 0* n.a. Tammisto South Savo 1 17.6 0.02 0* +lB.9*** 2 4.7 0.02 0* +l7.s*** 3 6.8 0.02 0* +ls.6*** 4 2.9 0.03 8.9 +3l.7*** Undrom South Savo 1 17.6 0.02 0* + B.6*** 2 4.7 0.02 0* +l7.3*** 3 6.8 0.02 0* +2o.6*** 4 2.9 0.03 5.9 +4s.o*** Undrom Karelia 1 19.6 0.06 5.8 n.a. 2 4.7 0.03 2.2 n.a. 3 20.8 0.04 1.8 n.a. Undrom Kainuu 1 24.6 0.04 2.8 n.a. 2 9.7 0.03 1.6 n.a. 3 9.8 0.04 0* n.a. Undrom N. Ostrabothnia 1 25.6 0.01 0* n.a. 2 9.7 0.02 4.2 n.a. 3 20.8 0.03 8.7 n.a. 1 = pasture stage 2 = silage stage 3 = first aftermath 4 = second aftermath ** = P