Effect of forage on the content of phyto-oestrogens in bovine milk

Phyto-oestrogens are believed to have a range of beneficial effects on predominant Western diseases. A few studies on phyto-oestrogens in milk exist and show that the composition can be affected by feeding. Therefore, the aim was to study how feeding of lucerne and grass/clover silages (GCSs) affects the concentration of phyto-oestrogens in bovine milk. Sixteen Danish Holstein cows were assigned to a 4 × 4 latin square design with four cows per treatment per period of 3 weeks. The four treatment diets were lucerne silage (LS), 2/3 lucerne silage and 1/3 maize silage (2/3LS), 1/3 lucerne silage and 2/3 maize silage (1/3LS) and GCS. Milk was collected at the end of each period and feed samples on day 6, 13 and 20 in each experimental period. Milk and pooled feed samples were analysed for the concentration of isoflavones, coumestans and lignans. The content of isoflavones was higher and the content of coumestrol lower in the GCS diet than in LS, 2/3LS and 1/3LS diets. For the LS, 2/3LS and 1/3LS diets, the concentration of coumestrol and secoisolariciresinol increased with the proportion of lucerne while the concentration of isoflavones was similar across the diets. The concentrations of the formononetin, daidzein and equol in the milk were significantly higher for the GCS diet than for the LS, 2/3LS and 1/3LS diets. In particular, the concentration of equol was 62-291 times higher for GCS. The concentration of coumestrol was significantly lower for the GCS diet compared to the LS, 2/3LS and 1/3LS diets. No pattern for the concentration of lignans was observed. In conclusion, a high concentration of isoflavones, particularly equol, and a low concentration of coumestrol were measured in the milk from the GCS diet. In contrast, a low concentration of isoflavones and a high concentration of coumestrol were measured in the milk from the LS, 2/3LS and 1/3LS diets. However, the concentration of phyto-oestrogens in bovine milk is low compared to food sources rich in phyto-oestrogens but the high concentration of equol could possibly be of therapeutic importance.     

Isoflavones in several clover species and in milk from goats fed clovers

Isoflavones that exist in forages such as clover can move in milk, and may have beneficial effects on human health. We measured the isoflavones in subterranean (cv Yarloop, Geraldton, Dinninup and Dwalganup) and red (cv Merviot and Hamua) clovers, grown in a mountain environment, and in the milk of two goats fed a fresh mixture of the four subterranean clovers gathered at flowering stage. Isoflavone glycosides were hydrolysed with an improved beta glycosidase method. Free aglycones (FA) and the hydrolysed glycosides + free aglycones fraction (HF) were analysed by HPLC. In milk, the methanol extracted compounds were deconjugated and similarly analysed. Total HF was highest in Yarloop (19 g kg(-1) dry matter) and was three times lower in Merviot. Formononetin was the major compound (46-64% of total HF). FA varied from 30 to 73% of HF. Milk contained formononetin (50 microg L(-1)), similar amounts of equol and p ethylphenol (about 250 microg L(-1)), and equol like compounds (1120 microg L(-1)). Equol, which has estrogenic activity, and p ethylphenol were produced by biotransformation of formononetin, and biochanin A or genistein, respectively. The modulation of isoflavone amounts in milk and the estimation of their interests for human health warrant further investigation.     

Effect of mycorrhization on the isoflavone content and the phytoestrogen activity of red clover

Red clover, known for its estrogenic activity due to its isoflavones content (biochanin A, genistein, daidzein and formononetin), was inoculated with the arbuscular mycorrhizal fungus Glomus mosseae. Once the symbiotic fungus was well established, plants were harvested and we determined the root and shoot dry weight as well as the P-content. In roots and leaves the levels of biochanin A, genistein, daidzein and formononetin were quantified by reversed-phase HPLC and the estrogenic activity of the leaves was measured by a transactivation assay using a yeast two-plasmid system. Mycorrhization increased the levels of biochanin A in the root and the shoot and reduced the levels of genistein in the shoot of red clover. The levels of the other isoflavones were not affected. The shoot biomass of mycorrhizal plants more than doubled compared with non-mycorrhizal control plants, and this growth-stimulating effect of arbuscular mycorrhiza did not affect the estrogenic activity of red clover. In a control P treatment, the biomass of red clover was greatly enhanced. However, the estrogenic activity was reduced. These results suggest that, in contrast to an enhanced shoot biomass production after P application with a reduced estrogenic activity, with arbuscular mycorrhiza the shoot biomass of red clover can be enhanced without a negative effect on estrogenic activity.     

Effects of mixtures of red clover and maize silages on the partitioning of dietary nitrogen between milk and urine by dairy cows

Eight multiparous lactating Holstein-Friesian cows were used to evaluate the partitioning of dietary nitrogen (N) from diets based on mixtures of red clover and maize silages in comparison with diets based on ryegrass silage. All cows received 4 kg/day of a standard dairy concentrate with one of four forage treatments in an incomplete changeover design with three 4-week periods. Three treatments were based on mixtures of red clover and maize silage. N intake was altered both by varying the ratio of these silages (40/60 and 25/75 on a dry matter (DM) basis) and by an additional treatment for which the DM intake of the 40/60 mixture was restricted to the level achieved with grass silage. Rumen passage rates were estimated from faecal excretion curves following a pulse oral dose of Dysprosium-labeled silage and urinary excretion of purine derivatives (PD) was used as an index of rumen microbial protein synthesis. Red clover silage mixtures led to significantly increased feed intake (21.5, 20.7 and 15.2 kg DM/day for 40/60 and 25/75 red clover/maize silage mixtures and grass silage, respectively), milk production (25.8, 27.8 and 20.0 kg/day for the same treatments, respectively) and milk component yields, but were without effect on milk fat and protein concentrations. The large increase in the yield of milk (24.5 kg/day) and milk components for the restricted red clover/maize silage treatment, in comparison with the grass silage treatment, was proportionately greater than the increase in DM intake (16.6 kg DM/day). There were no significant treatment effects on diet digestibility, while the higher intakes of red clover silage mixtures were associated with higher rumen passage rates (5.82%, 6.24% and 4.55%/h, respectively). There were significant effects of both N intake and forage source on the partitioning of dietary N between milk and urine. When dietary protein was diluted by the inclusion of maize silage, red clover silage led to increased milk N and reduced urinary N in comparison with grass silage. Improvements in N utilisation may be related to increased dietary starch and/or rumen passage rates leading to increased microbial protein synthesis for these treatments. Urinary excretion of PD was significantly higher for all diets based on mixtures of red clover and maize silages, in comparison with grass silage. Urinary N output was close to literature predictions based on N intake for the diet based on ryegrass silage, but 40 to 80 g/day (25% to 30%) less than predicted for the diets based on the mixtures of red clover and maize silages.     

Quantification of isoflavones in red clover by high-performance liquid chromatography

An RP-HPLC method for the determination of daidzein, genistein, formononetin and biochanin A in red clover (Trifolium pratense L.) was developed and validated. The compounds are quantified after hydrolytic extraction using an internal standard. On a base-deactivated C(18) column good separation of the analytes, also from accompanying substances, and excellent peak shape are achieved by gradient elution with aqueous sulfuric acid and acetonitrile. The method was applied to the analysis of different red clover cultivars.     

Fertility and growth of nulliparous ewes after feeding red clover silage with high phyto-oestrogen concentrations

The study aimed to determine the effects of red clover (Trifolium pratense) silage with high phyto-oestrogen content on ewe performance during their first breeding season. Red clover silage containing formononetin, biochanin A, genistein, and daidzein was fed to 10 nulliparous ewes of the prolific Finnish Landrace breed before, during and after the breeding season, for a total of 5 months. A control group of 10 ewes was fed with grass silage. The mean numbers of foetuses per pregnancy were 2.1±0.7 and 2.2±0.8 for the red clover and control groups, respectively. The total mass of the uterus with its contents was significantly greater in ewes of the red clover group compared with those of the control group. This difference was mainly explained by the greater volume of foetal fluids. Serum progesterone concentration in the red clover group was significantly lower over the entire period analysed than in the control group. In conclusion, the fecundity of the ewes was not reduced by red clover feed with high phyto-oestrogen concentrations. The volume of foetal fluids increased that could increase the risk for vaginal prolapse before the term.     

Comparison of hormonal activity (estrogen,androgen and progestin) of standardized plant extracts for large scale use in hormone replacement therapy

Extracts from red clover (Trifolium pratense), soybean (Glycine max.) and black cohosh (Cimicifuga racemosa) are frequently used as alternative compounds for hormone replacement therapy (HRT) to treat menopausal disorders. Fifteen commercially available products made either from red clover, soybean or black cohosh were tested in in vitro assays in this study. The main polycyclic phenolic compounds of soy and red clover products were biochanin A, genistein, daidzein, formononetin, and glycitein. In red clover products glycitein was not abundant. All the compounds showed clear estrogenic activity through estrogen receptor alpha (ERalpha) and estrogen receptor beta (ERbeta) and affinity to progesterone receptor (PR) and androgen receptor (AR), whereas the compounds from black cohosh did not. This was corroborated by synthetic isoflavones such as biochanin A, daidzein, genistein and formononetin. They exerted affinity to PR and AR in the range of 0.39-110 mM. Statistical analysis applying principal component analysis (PCA) revealed that all red clover and soy products are grouped in different clusters. Red clover products showed a higher affinity to AR and PR than soy products, which is explained by the higher amount of isoflavones present. In vitro assays and chemical analysis showed that theoretical estrogenic activity expressed as equivalent E2 concentration is in the same range as recommended for synthetic estrogens. Broader spectrum of action and hypothesized lower side effects by action through ERbeta make them suitable for alternative hormone replacement therapy.     

Interaction between Sitona lepidus and red clover lines selected for formononetin content

Adult clover root weevil Sitona lepidus show a feeding preference for white clover Trifolium repens over red clover Trifolium pratense. The effects on S. lepidus of three red clover T. pratense lines, selected for high, medium, or low levels of the isoflavone formononetin in foliage, were compared in three experiments using white clover as a control. In a no‐choice slant board experiment, weevil larval weights were greater for larvae feeding on white clover roots than those feeding on roots of the red clovers. The effect of larval root herbivory on plant growth was similar for all four clovers. Following root herbivory, a large increase in root and shoot formononetin levels was observed in the high‐formononetin selection of red clover but little change in the low‐formononetin red clover. In a no‐choice experiment with sexually mature female adult weevils feeding on foliage of the four clovers, all the red clovers had increased weevil mortality. Female weevils eating the high‐formononetin red clover laid fewer eggs than weevils eating white clover. The red clover diet caused a large accumulation of abdominal fat and/or oil in the weevils, whereas weevils feeding on white clover did not accumulate fat/oil. When sexually immature adult weevils were given a choice of foliage from all four clovers, white clover was eaten preferentially, and the low‐formononetin red clover was preferred to the high‐formononetin red clover. The results suggest that formononetin and associated metabolites in red clover may act as chemical defences against adult S. lepidus and that distribution in forage legumes can be manipulated by plant breeding to improve root health.     

Isoflavone Content in Subterranean Clover Germplasm from Sardinia

Subterranean clover (Trifolium subterraneum) is an important pasture legume, and Sardinia is known as a major centre of diversification of this species. As other legumes, this clover produces biologically active flavonoids including the subclass of isoflavones that are natural phytoestrogens with positive health effects. Present sources of isoflavones for medical/nutraceutical treatments are red clover (Trifolium pratense) and soybean (Glycine max). This study assessed the content and composition of flavonoids in 14 subterranean clover genotypes from Sardinia, grown ex‐situ in comparison with two red clover ecotypes, to acquire information on the potential of the species as an alternative source of isoflavones for possible exploitation. Twenty compounds were tentatively identified across the two clovers after HPLC and LC/ESI‐MS analyses, including clovamide, four flavonols, and 15 isoflavones. Most compounds were present as glucosides or glucosyl malonates. Subterranean clover extracts mainly comprised of derivatives of the isoflavones genistein, biochanin A, and formononetin. Compared to red clover, subterranean clover had higher content of total isoflavones and lower concentration of total flavonols. The isoflavone concentration in subterranean clover was higher than literature data for soybean or red clover. The existing genotypic variation warrants the possibility of selecting varieties with high isoflavone concentration for nutraceutical or pharmaceutical purposes.     

Phytoestrogens and Their Metabolites in Bulk-Tank Milk: Effects of Farm Management and Season

Phytoestrogens have structures similar to endogenous steroids and may induce or inhibit the response of hormone receptors. The objectives of the present study were to compare the effects of long-term vs. short-term grassland management in organic and conventional dairy production systems, compare organic and conventional production systems and assess seasonal variation on phytoestrogen concentrations in bulk-tank milk. The concentrations of phytoestrogens were analyzed in bulk-tank milk sampled three times in two subsequent years from 28 dairy farms: Fourteen organic (ORG) dairy farms with either short-term or long-term grassland management were paired with 14 conventional (CON) farms with respect to grassland management. Grassland management varied in terms of time since establishment. Short-term grassland management (SG) was defined as establishment or reseeding every fourth year or more often, and long-term grassland management (LG) was defined as less frequent establishment or reseeding. The proportion of red clover (Trifolium pretense L.) in the herbage was positively correlated with milk concentrations of the mammalian isoflavone equol. Therefore, organically produced bulk-tank milk contained more equol than conventionally produced milk, and milk from ORG-SG farms had more equol than milk from ORG-LG farms. Milk produced during the indoor-feeding periods had more equol than milk produced during the outdoor feeding period, because pastures contained less red clover than fields intended for silage production. Organically produced milk had also higher concentrations of the mammalian lignan enterolactone, but in contrast to equol, concentrations increased in the outdoor-feeding periods compared to the indoor-feeding periods. There were no indications of fertility problems on ORG-SG farms who had the highest red clover proportions in the herbage. This study shows that production system, grassland management, and season affect milk concentrations of phytoestrogens. However, compared to soy products, milk concentrations of phytoestrogens are low and future studies are required to investigate if the intake of phytoestrogens from dairy products has physiological effects in humans.     

Dairy cow responses to graded levels of rapeseed and soya bean expeller supplementation on a red clover/grass silage-based diet

The effects of rapeseed and soya bean expeller (SBE) supplementation on digestion and milk production responses in dairy cows were investigated in an incomplete Latin square design using five cows and four 3-week periods. The experimental diets consisted of five concentrate treatments fed at a rate of 9 kg/day: a mixture of barley and oats, which was replaced with rapeseed or SBE at two levels (CP concentration (g/kg dry matter (DM)) of 130 for the control concentrate and 180 and 230 for the two protein supplemented levels). A mixture of grass and red clover silage (1:1) was fed ad libitum and it had a CP concentration of 157 g/kg DM. Supply of nutrients to the lower tract was measured using the omasal canal sampling technique, and total digestion from total faecal collection. Protein supplementation increased omasal canal amino acid (AA) flows and plasma concentrations of AA, and was also reflected as increased milk production. However, N use efficiency (NUE) decreased with increased protein supplementation. Rapeseed expeller (RSE) tended to increase silage DM intake and elicited higher milk production responses compared with SBE and also resulted in a higher NUE. The differences between the protein supplements in nitrogen metabolism were relatively small, for example, there were no differences in the efficiency of microbial protein synthesis or omasal canal flows of nitrogenous components between them, but plasma methionine concentration was lower for soya bean-fed cows at the high CP level in particular. The lower milk protein production responses to SBE than to RSE supplementation were at least partly caused by increased silage DM and by the lower methionine supply, which may further have been amplified by the use of red clover in the basal diet. Although feed intake, diet digestion, AA supply and milk production were all consistently improved by protein supplementation, there was a simultaneous decrease in NUE. In the current study, the milk protein production increased only 9% and energy-corrected milk production by 7% when high level of protein supplementation (on average 2.9 kg DM/day) was compared with the control diet without protein supplementation showing that dairy production could be sustained at a high level even without external protein supplements, at least in the short term. The economic and environmental aspects need to be carefully evaluated when decisions about protein supplementation for dairy cows are taken.     

In vitro production of radiolabeled red clover (<Emphasis Type="Italic">Trifolium pratense</Emphasis>) isoflavones

Red clover isoflavones are increasingly used in dietary supplements for their purported estrogenic effects. However, little is known about their metabolism in animals due to a lack of commercially available isotopically labeled tracers. The goal of this research was to establish red clover cell culturing methodology for ¹⁴C-biolabeling of isoflavones. When root, leaf, and petiole-derived suspension cultures were grown in darkness or light, dark-grown, petiole-derived solution cultures produced the highest concentrations of the two major red clover isoflavones, formononetin (0.67 mg/g FM inoculum) and biochanin A (0.13 mg/g FM inoculum). Varying levels and timing of copper chloride elicitor did not significantly affect isoflavone accumulation. Approximately 38% of the ¹⁴C-sucrose dose accumulated in the cells. Eighteen percent of the initial labeled dose was detected in the isoflavone-rich methanolic extract and of that, 22% accumulated in isoflavones.     

The effect of N-fertilisation rate or inclusion of red clover to timothy leys on fatty acid composition in milk of dairy cows fed a commercial silage : concentrate ratio

The aim of this experiment was to, under typical Swedish production conditions, evaluate the effects of grass silages subjected to different N-fertilisation regimes fed to dairy cows on the fatty acid (FA) composition of their milk, and to compare the grass silages in this respect to red clover-dominated silage. Grass silages made from first year Phleum pratense L. leys subjected to three N-fertilisation regimes (30, 90 and 120 kg N/ha, designated G-30, G-90 and G-120, respectively) and a mixed red clover–grass silage (Trifolium pratense L. and P. pratense L.; 60/40 on dry matter (DM) basis, designated RC–G) were produced. The experiment was conducted as a change-over design, including 24 primiparous and multiparous dairy cows of the Swedish Red breed, each of which was allocated to three of the four diets. The cows were offered 11 kg DM of silage and 7 kg concentrates. The silages had similar DM and energy concentrations. The CP concentration increased with increase in N-fertilisation level. There was a linear increase in DM intake of the different silages with increased N fertilisation. There were also differences in concentrations of both individual and total FAs amongst silages. The daily milk production (kg/day) did not significantly differ between treatments, but G-30 silage resulted in higher concentrations of 18:2n-6 in the milk compared with the other two grass silages. The highest concentrations of 18:3n-3 and cis-9, trans-11 18:2 were found in milk from cows offered the RC–G silage. The G-30 diet resulted in higher concentration of 18:2n-6 and the same concentration of 18:3n-3 in the milk as the other grass silages, despite lower intake levels of these FAs. The apparent recoveries of 18:3n-3 from feed to milk were 5.74%, 4.27%, 4.10% and 5.31% for G-30, G-90, G-120 and RC–G, respectively. A higher recovery when red clover is included in the diet confirms previous reports. The higher apparent recovery of 18:3n-3 on the G-30 treatment may be related to the lower silage DM intake, which led to a higher relative proportion of ingested FAs originating from concentrates compared with the G-90 and G-120 diets. With the rates and types of concentrates used in this study, the achieved differences in FA composition among the silages were not enough to influence the concentrations of unsaturated FAs in milk.     

Preference of dairy cows for ryegrass, white clover and red clover, and its effects on nutrient supply and milk quality

Two experiments were conducted with 30 dairy cows each, to study the preference for fresh (Experiment 1) and ensiled (Experiment 2) ryegrass, white and red clover. Both experiments consisted of three choice diets with white or red clover or both, offered with ryegrass, and two diets with ryegrass mixed with white or red clover (40% clover). Cows consumed diets with 37.7% fresh white and 45.9% red clover, and no preference was observed when the cows were offered all three forages. By contrast, cows preferred white and red clover silage (73.0 and 69.2%, respectively) over ryegrass silage (of lower nutritive quality). When offered three forages, cows preferred white (59.8%) over red clover (17.5%) and ryegrass (22.7%). Choice diets resulted in diets similar (fresh forages) or higher in nutrient content and digestibility (silages). Treatments did not affect feed intake and performance. Choices compared to mixed diets with red clover silage were preferable regarding the fatty acid composition of the milk fat. Obviously, only large differences in nutrient and energy concentration facilitate preferences for clovers over ryegrass, which could, depending on clover type, be beneficial in terms of the milk's fatty acid composition.     

Effect of fish oil on ruminal biohydrogenation of C18 unsaturated fatty acids in steers fed grass or red clover silages

Red clover and fish oil (FO) are known to alter ruminal lipid biohydrogenation leading to an increase in the polyunsaturated fatty acid (PUFA) and conjugated linoleic acid (CLA) content of ruminant-derived foods, respectively. The potential to exploit these beneficial effects were examined using eight Hereford × Friesian steers fitted with rumen and duodenal cannulae. Treatments consisted of grass silage or red clover silage fed at 90% of ad libitum intake and FO supplementation at 0, 10, 20 or 30 g/kg diet dry matter (DM). The experiment was conducted with two animals per FO level and treatments formed extra-period Latin squares. Flows of fatty acids at the duodenum were assessed using ytterbium acetate and chromium ethylene diamine tetra-acetic acid as indigestible markers. Intakes of DM were higher (P < 0.001) for red clover silage than grass silage (5.98 v. 5.09 kg/day). There was a linear interaction effect (P = 0.004) to FO with a reduction in DM intake in steers fed red clover silage supplemented with 30 g FO/kg diet DM. Apparent ruminal biohydrogenation of C18:2n-6 and C18:3n-3 were lower (P < 0.001) for red clover silage than grass silage (0.83 and 0.79 v. 0.87 and 0.87, respectively), whilst FO increased the extent of biohydrogenation on both diets. Ruminal biohydrogenation of C20:5n-3 and C22:6n-3 was extensive on both silage diets, averaging 0.94 and 0.97, respectively. Inclusion of FO in the diet enhanced the flow of total CLA leaving the rumen with an average across silages of 0.22, 0.31, 0.41 and 0.44 g/day for 0, 10, 20 or 30 g FO/kg, respectively, with a linear interaction effect between the two silages (P = 0.03). FO also showed a dose-dependent increase in the flow of trans-C18:1 intermediates at the duodenum from 4.6 to 15.0 g/day on grass silage and from 9.4 to 22.5 g/day for red clover silage. Concentrations of trans-C18:1 with double bonds from Δ4-16 in duodenal digesta were all elevated in response to FO in both diets, with trans-11 being the predominant isomer. FO inhibited the complete biohydrogenation of dietary PUFA on both diets, whilst red clover increased the flow of C18:2n-6 and C18:3n-3 compared with grass silage. In conclusion, supplementing red clover silage-based diets with FO represents a novel nutritional strategy for enhancing the concentrations of beneficial fatty acids in ruminant milk and meat.     

Receptor binding and transactivation activities of red clover isoflavones and their metabolites

Red clover extracts contain a variety of isoflavones, which have affinity toward estrogen receptor alpha (ERalpha), estrogen receptor beta (ERbeta), androgen receptor (AR), and progesterone receptor (PR). Upon ingestion, they undergo various metabolic transformations. For a complete evaluation of red clover extracts and possible health benefits, the resulting metabolites should also be investigated. Biochanin A, formononetin, genistein, daidzein, dihydrobiochanin A, dihydroformononetin, dihydrogenistein, dihydrodaidzein, 3'-hydroxygenistein, 6-hydroxydaidzein, 6-hydroxydesmethylangolensin, equol, O-desmethylangolensin, angolensin, and p-ethylphenol were tested for their transactivation potential toward ERalpha, AR, and PR in yeast. Competitive binding assays with radiolabeled 17beta-estradiol, 17alpha-methyltrienolone or progesterone assessed binding to the respective ERalpha and ERbeta, AR, and PR. The compounds showed only weak binding affinity to AR and PR, with IC(50) values being greater (i.e., lesser affinity) than 10(-5)M for the respective receptor. So far, beneficial health effects have been attributed to the production of equol. We propose that other metabolites can also contribute to these effects. However, more detailed information for the formation of these metabolites in humans and for bioavailability data are required to confirm our assumptions.     

反相高效液相层析法测定新鲜植物材料中的植物雌激素含量

通过适当的样品处理方法,游离的和结合的植物雌激素［大豆素,雌马酚,染料木素,芒柄花素,香豆雌酚和美皂异黄酮］被从新鲜植物材料的提取物中分离出来,并在不同的紫外光波长下,可被ＨＰＬＣ法定量测定,根据滞留时间和标准品的添加,而鉴别出植物雌激素的层析波峰。本方法的测定灵敏度为２ｐｐｍ。白三叶草样品的加样回收率在８０％－１００％之间（平均回收率变异系数为５．４％）。通过比较游离植物雌激素的含量测定,本方法     

The first genetic maps for subterranean clover (Trifolium subterraneum L.) and comparative genomics with T. pratense L. and Medicago truncatula Gaertn. to identify new molecular markers for breeding

This study reports on the construction of the first genetic maps of subterranean clover (Trifolium subterraneum L.), a diploid, inbreeding annual pasture legume, and alignment of its linkage groups with those of red clover (T. pratense L.) and Medicago truncatula Gaertn. Transferability of red and white clover (T. repens L.) simple sequence repeat (SSR) markers to subterranean clover was observed. A total of 343 SSR loci were mapped into eight subterranean clover linkage groups, with 6?C31 loci per linkage group and 27 loci with similar locations between two distinct F ₂ mapping populations. Phenotypic data obtained for flowering time, content of three isoflavonoids (formononetin, genistein and biochanin A), hardseededness, leaf markings, calyx pigmentation and hairiness of stems were analyzed, together with genotypic data. Genomic intervals influencing each trait were assigned to one to three chromosome regions, accounting for 5.5?C59.8% of the phenotypic variance. Syntenic relationships were observed among subterranean clover, red clover and Medicago truncatula genomes. Comparisons of loci shared between the three species indicated that at least two chromosomal regions have undergone duplications in the subterranean clover genome. Candidate genes for isoflavone content were identified using M. truncatula as a reference genome. Synteny-based segmentation observed in Brassicaceae chromosomes helped to account for the apparent segmental-based relationship between the clover genomes, particularly within the subterranean clover lines. The proposed segmental nature of clover genome could account for the extensive variation observed between the parental genotypes, while not preventing production of fertile intercrosses.     

Red clover isoflavones biochanin A and formononetin are potent ligands of the human aryl hydrocarbon receptor

Aryl hydrocarbon receptor (AhR) activation affects the cell cycle and drives cells to apoptosis. Thus, selective AhR modulators (SAhRMs) have previously been implicated in cancer therapy and prevention, particularly for hormone-dependent cancers. In the present study, isoflavones a remedy used to ameliorate menopausal complaints were tested for their potential in transactivating AhR in order to investigate the biological function of red clover isoflavones. The results were compared to the transactivation potentials of other flavonoids and plant-derived indole compounds. We found that the isoflavones biochanin A and formononetin were potent AhR agonists in vitro, with EC(50) values of 2.5 x 10(-7) and 1.3 x 10(-7)mol/l, respectively. These isoflavones are 10 times more potent compared to the indole compounds indole-3-carbinol and diindolylmethane, publicised as powerful AhR agonists with EC(50) values of 5.8 x 10(-6) and 1.1 x 10(-6)mol/l, respectively. Because activated AhR crosstalks with estrogen receptor alpha, future risk-benefit assessments of isoflavones should take into consideration their AhR transactivating potential.