Quantitative aspects of phytooestrogen metabolism in sheep fed on subterranean clover (Trifolium subterraneum cultivar clare) or red clover (Trifolium pratense)

1970 ◽  
Vol 21 (5) ◽  
pp. 713 ◽  
Author(s):  
DA Shutt ◽  
RH Weston ◽  
JP Hogan
Keyword(s):  
Residence Time ◽  
Trifolium Pratense ◽  
Daily Intake ◽  
Red Clover ◽  
Subterranean Clover ◽  
Trifolium Subterraneum ◽  
Biochanin A ◽  
Free Form ◽  
The Mean ◽  
High Level

Studies have been made of the digestion and metabolism in Merino wethers of the isoflavones in subterranean clover (Trtfolium subterraneurn cv. Clare) and red clover (T. pratense). The dietary intake of isoflavones with both clovers was about 9 g per day. With the subterranean clover, the isoflavones were predominantly genistein and biochanin A, and slight teat length increases in the wethers ingesting this clover indicated a low level of oestrogenicity. With the red clover formononetin represented 60% of the isoflavone present and the wethers on this diet exhibited maximal teat length increases indicating a high level of oestrogenicity. Less than 1 % of the daily intake of the isoflavones was excreted as such in the faeces and urine; hence most of these compounds were metabolized or retained in the sheep. The dietary isoflavones were found to disappear rapidly from the rumen, and it was estimated by using marker techniques that the removal of these compounds from the stomach (rumen, reticulum, omasum, and abomasum) was virtually complete. Equol (7,4'-dihydroxyisoflavan), a metabolite of formononetin, was the predominant phyto-oestrogen in the digesta and excreta when red clover was given. The excretion of 3.9 g/day of this compound, mainly in urine, was equivalent to 70% of the intake of formononetin. It was calculated that about 86% of the equol produced in the rumen was absorbed from that organ; the mean residence time for equol in the rumen was estimated to be 1.7 hr. The isoflavones were present in blood plasma mainly in conjugated forms. Equol predominated with both clovers. The levels of equol were much lower with the subterranean clover than with the red clover diet; the concentrations of the conjugated form were respectively 13 and 300-440 �g/100 ml. Equol in the free form, although not detectable with the subterranean clover, was present at 4-10 �g/100 ml with red clover. The data were considered to be consistent with the conclusion that equol accounts for most of the phyto-oestrogenic activity in sheep fed on clovers containing high levels of formononetin.

scholarly journals Oestrogenic Isoflavone Content in Natural Strains of Subterranean Clover (Trifolium subterraneum L.) from Sardinia

2006 ◽  
Vol 1 (7) ◽  
pp. 1934578X0600100 ◽  
Author(s):  
Aldo Tava ◽  
Luciano Pecetti ◽  
Alessia Bertoli ◽  
Efisio Piano
Keyword(s):  
Trifolium Pratense ◽  
Red Clover ◽  
Subterranean Clover ◽  
Trifolium Subterraneum ◽  
Biochanin A ◽  
Lower Amount ◽  
Positive Role ◽  
Isoflavone Concentration ◽  
Trifolium Pratense L ◽  
Natural Strains

A collection of subterranean clover (Trifolium subterraneum L.) genotypes from Sardinia was evaluated for the content of oestrogenic isoflavones to assess differences in total and individual compounds. Daidzein, genistein, and their respective 4′-methoxy derivatives biochanin A and formononetin, were detected in leaves and quantified by GC/FID and GC/MS methods. A large variation among genotypes for individual compounds and for the total isoflavone concentration was observed. Total isoflavones were measured in the range 0.8-13.4 mg/g fresh weight (0.3-4.5% dry matter). Daidzein was usually present in lower amount compared to the other isoflavones. Genistein and biochanin A were found in most cases in higher concentration than formononetin. Phyto-oestrogens have been shown to have both oestrogenic and anti-oestrogenic properties, and their effects entail a positive role for the human health. T. subterraneum revealed the presence of higher concentration of isoflavones compared to other sources of these compounds, e.g. soybean (Glycine max (L.) Merrill) and red clover (Trifolium pratense L.). This study provided information on the biodiversity of the natural strains from Sardinia, emphasizing the importance of these genetic resources for their variation in the amount of total and individual isoflavones.

Effects of pH and aluminium on the growth of temperate pasture species. II. Growth and nodulation of legumes

1991 ◽  
Vol 42 (5) ◽  
pp. 893 ◽  
Author(s):  
DC Edmeades ◽  
FPC Blamey ◽  
CJ Asher ◽  
DG Edwards
Keyword(s):  
White Clover ◽  
Trifolium Pratense ◽  
Inorganic Nitrogen ◽  
Red Clover ◽  
Subterranean Clover ◽  
Trifolium Subterraneum ◽  
Lotus Corniculatus ◽  
Culture Solution ◽  
Solution Ph ◽  
Lotus Pedunculatus

Ten temperate pasture legumes inoculated with appropriate rhizobia were grown for 31 days in flowing solution culture. Solution ionic strength was approximately 2700 8M and contained inorganic nitrogen (150 , 8M NO3-) only at the commencement of the experiment. Solution pH was maintained at 4.5, 5.0, 5.5 and 6.0. Also, five aluminium (Al) treatments were imposed, with nominal Al concentrations of 0, 3, 6, 12 and 24 8M (2.5, 7.1, 8.3, 11.2 and 24.7 8M Al measured) at pH 4.5. Solution pH <6 . 0 markedly reduced total dry mass (TDM) in all cultivars of white clover (Trifolium repens) cvv. 'Grasslands Pitau, Huia, G18 and Tahora' and red clover (Trifolium pratense) cvv. 'Grassland Turoa and Pawera', and to a lesser extent in the two subterranean clover (Trifolium subterraneum) cvv. 'Tallarook and Woogenellup'. In contrast, solution pH had no effect on the growth of Lotus corniculatus cv. Maitland, while Lotus pedunculatus cv. Maku grew best at pH 4.5. Lotus pedunculatus cv. Maku grew best in solution where the sum of the activities of the monomeric Al species {Alm} was maintained at 5.9 8M. The growth of all other species was decreased with Al in solution, a 50% reduction in TDM being associated with c. 6 8M {Alm] for white clover and subterranean clover, and c. 3 8M in red clover and Lotus corniculatus cv. Maitland.

Effects of pH and aluminium on the growth of temperate pasture species. I. Temperate grasses and legumes supplied with inorganic nitrogen

1991 ◽  
Vol 42 (3) ◽  
pp. 559 ◽  
Author(s):  
DC Edmeades ◽  
FPC Blamey ◽  
CJ Asher ◽  
DG Edwards
Keyword(s):  
White Clover ◽  
Festuca Arundinacea ◽  
Trifolium Pratense ◽  
Inorganic Nitrogen ◽  
Red Clover ◽  
Subterranean Clover ◽  
Trifolium Subterraneum ◽  
Solution Culture ◽  
Pasture Legumes ◽  
Effects Of Ph

A flowing solution culture experiment was conducted in which four temperature pasture grasses and 11 temperate pasture legumes were grown in solutions of low ionic strength for 29 days at six aluminium (Al) levels with average Al3+ activities {Al3+} of 1.0, 5.1, 5.9, 9.9, 20.8, 41.6 8M at pH 4.5. Growth of these species was also measured at pH 5.5 in the absence of Al. In the absence of added Al, lucerne (Medicago sativa) cv. Hunter River and phalaris (Phalaris aquatica) cv. Grasslands Maru grew better at pH 5.5 than at 4.5. The reverse was so for ryegrass (Lolium perenne) cv. Grasslands Nui and subterranean clover (Trifolium subterraneum) cv. Tallarook. Increasing the pH from 4.5 to 5.5 had no effect on the growth of the other 11 species and cultivars tested. Micromolar activities of Al (<10 8M) markedly reduced the growth of all species. Four groups were tentatively identified in terms of their tolerance to Al. The two cultivars of ryegrass and the two red clover (Trifolium pratense) cultivars, together with Lotus corniculatus cv. Maitland, were relatively sensitive to Al. A 50% reduction in total dry matter (TDM) was associated with {Al3+} of 2-3 8M. The four white clover (Trifolium repens) cultivars, tall fescue (Festuca arundinacea) cv. Grasslands Roa, phalaris cv. Grasslands Maru, and Lucerne cv. Hunter River were intermediate in their tolerance, with {Al3+} of 3-5 8M reducing TDM by 50%. The two subterranean clover cultivars were more tolerant to Al than white clover (50% TDM reduction at 5-6 8M {Al3+}. Lotus pedunculatus CV. Grasslands Maku was exceptional in its tolerance to Al. Its growth was unaffected by {Al3+} up to 5 8M and 50% growth reduction was associated with {Al3+} of 7 8M The results of this study emphasize the need to conduct solution culture experiments at realistic solution concentrations and ionic strengths when examining the effects of Al on plant growth.

Varietal reactions of Trifolium subterraneum L. to Phaseolus virus 2 Pierce.

1954 ◽  
Vol 5 (4) ◽  
pp. 598 ◽  
Author(s):  
EM Hutton ◽  
JW Peak
Keyword(s):  
Trypan Blue ◽  
Continuous Distribution ◽  
Systemic Infection ◽  
Subterranean Clover ◽  
Trifolium Subterraneum ◽  
Epidermal Cells ◽  
Severe Strain ◽  
Resistant Varieties ◽  
Lethal Reaction ◽  
The Mean

A number of varieties of subterranean clover (Trifolium subterraneum L.) were tested with a severe strain of Phaseolus virus 2 Pierce. Northam First Early, Dwalganup, and Pink Flowered gave lethal necrotic reactions while the rest developed mottles and chlorosis of different degrees of intensity. Reductions in the mean fresh weights of plants of five subterranean clover varieties ranged from 26.1 to 76.7 per cent. Epidermal cells of mottled leaves contained irregular aggregates of viroplasts stained by treatment with phloxine-trypan blue. Viroplasts in young tip leaves did not stain if the plants were held at temperatures below 44.6°F while those in mature basal leaves were not so affected. The lethal-reactors Northam First Early, Dwalganup, and Pink Flowered were resistant in the field to Phaseolus virus 2. With hand inoculation in the glass-house an average of 10 per cent. of plants of these varieties remained free from systemic infection, whereas all the plants of mottle-reacting varieties became systemically infected. The virus had a discontinous distribution in plants of lethal-reactors, compared with a continuous distribution in those of mottle-reactors such as Mount Barker. The lethal reaction is heritable in a dominant fashion in most crosses, so that the development of desirable new virus- resistant varieties is possible.

scholarly journals Variety, time of harvest and conditions during growing season have impact on red clover isoflavone content

2018 ◽  
Vol 27 (2) ◽  
Author(s):  
Eeva Arja Mustonen ◽  
Mikko Tuori ◽  
Päivi Kurki ◽  
Mika Isolahti ◽  
Juhani Taponen ◽  
...  
Keyword(s):  
High Performance ◽  
Trifolium Pratense ◽  
Red Clover ◽  
Weather Conditions ◽  
Biochanin A ◽  
Growing Seasons ◽  
Crop Varieties ◽  
Isoflavone Content ◽  
Trifolium Pratense L ◽  
Time Of Harvest

Red clover (Trifolium pratense L.) is the predominant legume used in northern European agriculture. Official red clover variety trials are conducted by Natural Resources Institute Finland (Luke) to determine the value of field crop varieties. The trials used for the current analysis were conducted in Luke units in southern Finland (Mikkeli) and northern Finland (Ruukki) in two consecutive years. Plant samples for isoflavone analyses were collected from four varieties grown as four replicates and harvested twice during both growing seasons. The four main isoflavones biochanin A, genistein, daidzein and formononetin were analysed using high performance liquid chromatography. Total phytoestrogen content in the varieties varied in the range of 11.2−14.8 mg g-1 dry matter (DM). The variety and the time of harvest had most effect on the isoflavone, especially formononetin, contents of red clover. A more northern growing area and challenging weather conditions were associated with increased isoflavone concentrations.

scholarly journals Soluble Phenolic Compounds in Different Cultivars of Red Clover and Alfalfa, and their Implication for Protection against Proteolysis and Ammonia Production in Ruminants

2015 ◽  
Vol 10 (7) ◽  
pp. 1934578X1501000 ◽  
Author(s):  
Isabelle A. Kagan ◽  
Ben M. Goff ◽  
Michael D. Flythe
Keyword(s):  
Phenolic Compounds ◽  
Trifolium Pratense ◽  
Phenolic Content ◽  
Colorimetric Assay ◽  
Red Clover ◽  
Biochanin A ◽  
Total Phenolic ◽  
Ammonia Production ◽  
Response Factors ◽  
Soluble Phenolics

Red clover ( Trifolium pratense) contains soluble phenolic compounds with roles in inhibiting proteolysis and ammonia production. Alfalfa ( Medicago sativa) has been found to have a low phenolic content, but few alfalfa and red clover cultivars have been compared for phenolic content. Total soluble phenolics were quantified by a Folin-Ciocalteu colorimetric assay in nine red clover and 27 alfalfa cultivars. Mean total phenolic contents of red clover and alfalfa were 36.5 ± 4.3 mg/gdw and 15.8 ± 1.4 mg/gdw, respectively, with the greater standard deviation of red clover possibly indicating more diversity in phenolic content. Because different phenolic standards had different response factors in the colorimetric assay, the red clover and 11 alfalfa cultivars were analyzed by HPLC to determine if the differences in total soluble phenolics between genera reflected differences in the amounts of phenolics or in the classes of phenolics responding to the colorimetric assay. Two red clover cultivars differed in total phenolics and phaselic acid. Alfalfa produced different phenolic compounds from red clover, at lower concentrations. Extracts of two red clover cultivars were separated by thin-layer chromatography (TLC), and the bands were assayed for activity against Clostridium sticklandii, a bovine ruminal hyper ammonia-producing bacterium (HAB). Only biochanin A had anti-HAB activity. Inhibitory amounts indicated that five red clover cultivars could be suitable sources of anti-HAB activity.

Urinary sediments in sheep feeding on oestrogenic clover. I. Preliminary observations on changes in certain urinary constituents

1970 ◽  
Vol 21 (6) ◽  
pp. 933 ◽  
Author(s):  
WJ Parr ◽  
P Steele ◽  
B Gabbedy ◽  
MC Nottle
Keyword(s):  
Direct Relationship ◽  
Daily Intake ◽  
Subterranean Clover ◽  
Trifolium Subterraneum ◽  
Urinary Calcium ◽  
Direct Result ◽  
Calcium Excretion ◽  
Total Phenols ◽  
Isoflavone Content ◽  
Daily Excretion

In recent years a series of widespread outbreaks of acute urinary obstruction has occurred in spring in Merino wethers. Animals involved have been grazing oestrogenic strains of subterranean clover at a time when isoflavone concentrations can be expected to be high. Studies have been made on one property on the relationships between these outbreaks, the pasture isoflavone content, and the urinary excretion of phenolic and other constituents by wethers. A trial group of animals was grazed on a dominant sward of the highly oestrogenic Trifolium subterraneum cv. Dinninup during winter and spring. Isoflavone concentrations in the clover reached a peak in early August. However, since the pasture became highly clover-dominant in September and early October the maximum daily intake of isoflavones probably occurred during this period, which also coincided with the occurrence of maximum concentration and maximum daily excretion of total phenols and acid-precipitable material (APM) in the urine. It is suggested that the sediments causing clinical obstructions at this time of the year may be a direct result of increased excretion of phenols. APM showed a highly significant direct relationship with total phenols but its excretion increased at a greater rate than did that of phenols. Urinary calcium excretion also increased at the same time.

scholarly journals Single-Laboratory Validation of UHPLC-MS/MS Assays for Red Clover Isoflavones in Human Serum and Dietary Supplements

2020 ◽  
Vol 103 (4) ◽  
pp. 1160-1166 ◽  
Author(s):  
Ruth N Muchiri ◽  
Richard B van Breemen
Keyword(s):  
Quantitative Analysis ◽  
Human Serum ◽  
Dietary Supplements ◽  
Trifolium Pratense ◽  
Red Clover ◽  
Biochanin A ◽  
Internal Standards ◽  
Selective Reaction ◽  
Coefficients Of Variation ◽  
Trifolium Pratense L

Abstract Background Extracts of red clover (Trifolium pratense L.) containing estrogenic and pro-estrogenic isoflavones are used in dietary supplements primarily for the management of menopausal symptoms in women. Objective A UHPLC-MS/MS assay was developed and validated for the quantitative analysis of the six major red clover isoflavones in dietary supplements and in human serum in support of clinical trials. Methods Enzymatic deconjugation of isoflavone glucuronides and sulfate conjugates in human serum specimens was carried out followed by protein precipitation. Isoflavones in red clover dietary supplements were acid hydrolyzed to release aglycons from glycosides. UHPLC separations (&lt; 4 min) were combined with MS/MS using collision-induced dissociation, selective reaction monitoring and deuterated internal standards to measure biochanin A, formononetin, daidzein, genistein, irilone, and prunetin. Results The method was validated with respect to selectivity, specificity, accuracy, linearity, precision, LOD, and LOQ. The calibration curves for all analytes were linear (R2 &gt; 0.998). The mean recovery for low-, medium- and high-quality control standards ranged between 80% and 108%. The precision of the method was assessed using coefficients of variation, which were &lt;15%. Conclusions The UHPLC-MS/MS method is fast, precise, sensitive, selective, accurate, and applicable to the quantitative analysis of red clover isoflavones in different matrices. Highlights This validated UHPLC-MS/MS assay is applicable to the rapid quantitative analysis of red clover isoflavones in human serum and in dietary supplements.

The effects of red clover (Trifolium pratense var. redhead), white clover (Trifolium repens var. S 100) or perennial ryegrass (Lolium perenne var. S 23) on the reproductive performance of sheep

1973 ◽  
Vol 80 (2) ◽  
pp. 323-327 ◽  
Author(s):  
J. E. Newton ◽  
J. E. Betts
Keyword(s):  
Litter Size ◽  
Lolium Perenne ◽  
Perennial Ryegrass ◽  
Reproductive Performance ◽  
White Clover ◽  
Trifolium Pratense ◽  
Red Clover ◽  
The Third ◽  
S 100 ◽  
The Mean

SummaryDuring 3 years, three groups of 20 Scottish half-bred ewes were grazed on perennial ryegrass, white clover or red clover. During this period the two legumes were oestrogenic, in terms of teat-length extension of wethers, but the perennial ryegrass was not. The white clover was only markedly oestrogenic when there was a high proportion of diseased leaves. The mean litter size and lambing percentage of the ewes grazed on red clover was significantly reduced. White clover, although it was oestrogenic, had no effect on mean litter size or lambing percentage. The mean litter size of the ewes on the perennial ryegrass was 2·35, 2·30 and 2·24, on the white clover was 2·20, 2·33 and 2·23 and on the red clover was 1middot;60, 1·67 and 1·78 for the three lambings. Lambing percentage of the ewes on the perennial ryegrass was 175, 225 and 218, on the white clover was 210, 222 and 189, and on the red clover was 160, 128 and 118 for the three lambings. The lambing date of the ewes grazing on the red clover was significantly later at the third lambing.

Biological estimation of oestrogenic activity in red clover (Trifolium pratense): relative potencies of parts of plant and changes with storage

1963 ◽  
Vol 30 (2) ◽  
pp. 243-249 ◽  
Author(s):  
D. S. Flux ◽  
R. E. Munford ◽  
G. F. Wilson
Keyword(s):  
Room Temperature ◽  
Trifolium Pratense ◽  
Red Clover ◽  
Biochanin A ◽  
Reduced Pressure ◽  
Ovariectomized Mice ◽  
Fresh Material ◽  
Freeze Dried ◽  
Relative Potencies ◽  
Different Parts

SummaryImmature ovariectomized mice were used to compare the oestrogenic activities of different parts of the same clover plants; to estimate the relative potencies of biochanin A, genistein and diethylstilboestrol; and to compare the effects of different methods of storage on the oestrogenic potency of red clover leaf and petiole. Test materials were incorporated in the diet fed to the mice and the uterine weight response was used to measure oestrogenic activity.With material harvested at four different times, the leaf or leaf and petiole had the highest activity; small and large stem fractions of the plant were both relatively less active, and blossom and seed head were inactive.Comparison of successive estimates of the relative potency of isoflavones and diethylstilboestrol indicated that the relative responsiveness to the two types of oestrogen did not remain constant. Thus comparisons of estimates of oestrogenic activity of plant material, obtained in terms of diethylstilboestrol in different experiments, could be invalid.The potencies of alcohol, acetone and freeze-dried preparations were compared after storage for 18, 115, 212 and 230 days. Of the procedures tested, the most satisfactory was that in which fresh material was placed in 95% alcohol and the liquor and residue dried and stored at room temperature over concentrated sulphuric acid at reduced pressure.

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