scholarly journals Factors Affecting the Separation and Bioactivity of Red Clover (Trifoliumpratense) Extracts Assayed against Clostridium sticklandii, a Ruminal Hyper Ammonia-producing Bacterium

2012 ◽  
Vol 7 (12) ◽  
pp. 1934578X1200701 ◽  
Author(s):  
Isabelle A. Kagan ◽  
Michael D. Flythe
Keyword(s):  
Ethyl Acetate ◽  
Trifolium Pratense ◽  
Red Clover ◽  
Normal Phase ◽  
Biochanin A ◽  
Factors Affecting ◽  
Clostridium Sticklandii ◽  
Tlc Plates ◽  
Phenolic Extract ◽  
Layer Chromatography

Red clover ( Trifolium pratense) is rich in phenolic compounds. Both the crude phenolic extract and biochanin A, an isoflavonoid component of the extract, suppress growth of Clostridium sticklandii, a bovine, Gram-positive, ruminal hyper-ammonia producing bacterium (HAB). The purpose of this study was to determine if other components of red clover extract contributed to its anti-HAB activity. Extracts of the Kenland cultivar of red clover, collected 0 h and 24 h after cutting, were separated by normal-phase thin-layer chromatography (TLC) in either ethyl acetate-hexanes (9:1, v/v) or ethyl acetate-methanol (4:1, v/v). Bands on TLC plates were assayed by either overlaying the plates with agar seeded with C. sticklandii, or setting the excised bands face-down onto plates of bacteria-seeded agar. Biochanin A inhibited C. sticklandii growth on TLC plates when as little as 8 nmol was present in the extract. An antimicrobial band, seen in a previous bioassay, was not found, suggesting that this second compound may be more labile than biochanin A.

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.

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 (< 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 > 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 <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.

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.

In vivo metabolic tracking of 14C-radiolabelled isoflavones in kudzu (Pueraria lobata) and red clover (Trifolium pratense) extracts

2009 ◽  
Vol 102 (10) ◽  
pp. 1523-1530 ◽  
Author(s):  
Jonathan G. Mun ◽  
Michael D. Grannan ◽  
Pamela J. Lachcik ◽  
Adam Reppert ◽  
Gad G. Yousef ◽  
...  
Keyword(s):  
Trifolium Pratense ◽  
Enterohepatic Circulation ◽  
Red Clover ◽  
Root Culture ◽  
Biochanin A ◽  
Blood Collection ◽  
Pueraria Lobata ◽  
Sprague Dawley ◽  
Sprague Dawley Rats

Absorption, distribution and elimination of 14C-labelled isoflavone-containing extracts from kudzu (Pueraria lobata) root culture and red clover (Trifolium pratense) cell culture were investigated in an in vivo rat model. The predominant isoflavones in the kudzu extract were the glycosides puerarin, daidzin and malonyl daidzin, while in the red clover extract, the major isoflavones were formononetin and its derivatives, genistein and biochanin A, with radioactivities of 3·770 and 7·256 MBq/g, respectively. Male Sprague–Dawley rats, implanted with a jugular catheter and a subcutaneous ultrafiltrate probe, were orally administered with 14C-labelled isoflavone extracts from either kudzu or clover cell cultures. Serum, interstitial fluid (ISF), urine and faeces were collected using a Culex® Automated Blood Collection System for 24 h. Analysis of bone tissues revealed that radiolabel accumulated in the femur, tibia and vertebrae at 0·04, 0·03 and 0·01 % of the administered dose, respectively, in both kudzu and red clover treatments. The liver accumulated the greatest concentration of radiolabel among the tissues tested, at 1·99 and 1·54 % of the administered kudzu and red clover extracts, respectively. Serum and ISF analysis showed that both extracts were rapidly absorbed, distributed in various tissues, and largely eliminated in the urine and faeces. Urine and faeces contained 8·53 and 9·06 % of the kudzu dose, respectively, and 3·60 and 5·64 % of the red clover dose, respectively. Serum pharmacokinetics suggest that extracts from kudzu may undergo enterohepatic circulation.

scholarly journals The red clover (Trifolium pratense) isoflavone biochanin A inhibits aromatase activity and expression

2008 ◽  
Vol 99 (2) ◽  
pp. 303-310 ◽  
Author(s):  
Yun Wang ◽  
Wai Man Gho ◽  
Franky L. Chan ◽  
Shiuan Chen ◽  
Lai K. Leung
Keyword(s):  
Breast Cancer ◽  
Enzyme Activity ◽  
Oestrogen Receptor ◽  
Transcriptional Control ◽  
Trifolium Pratense ◽  
Red Clover ◽  
Biochanin A ◽  
Luciferase Reporter ◽  
Aromatase Activity ◽  
Postmenopausal Symptoms

Biochanin A is an isoflavone isolated from red clover (Trifolium pratense), and is a commercially available nutraceutical for women suffering from postmenopausal symptoms. Isoflavones resemble the structure of oestrogen, and display agonistic and antagonistic interactions with the oestrogen receptor. Overexposure of oestrogen is a major contributing factor in the development of breast cancer, and cytochrome P450 (CYP) 19 enzyme, or aromatase, catalyses the reaction converting androgen to oestrogen. In the present study the effect of biochanin A on the gene regulation and enzyme activity of aromatase was investigated. By assaying MCF-7 cells stably transfected with CYP19, biochanin A inhibited aromatase activity and hampered cell growth attributing to the enzyme activity. In addition, 25 μm-biochanin A significantly reduced CYP19 mRNA abundance in the oestrogen receptor-negative breast cancer cells SK-BR-3. The transcriptional control of the CYP19 gene is exon-specific, and promoter regions I.3 and II have been shown to be responsible for CYP19 expression in SK-BR-3 cells. Luciferase reporter gene assays also revealed that biochanin A could repress the transcriptional control dictated by the promoter regulation. Interestingly, genistein did not inhibit aromatase but it might down regulate promoter I.3 and II transactivation. Since genistein is a major metabolite of biochanin A, it might contribute to biochanin A's suppressive effect on CYP19 expression. The present study illustrated that biochanin A inhibited CYP19 activity and gene expression.

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 The red clover (Trifolium pratense) isoflavone biochanin A modulates the biotransformation pathways of 7, 12-dimethylbenz[a]anthracene

2003 ◽  
Vol 90 (1) ◽  
pp. 87-92 ◽  
Author(s):  
Ho Yee Chan ◽  
Huan Wang ◽  
Lai K. Leung
Keyword(s):  
Dna Damage ◽  
Trifolium Pratense ◽  
Red Clover ◽  
Mutagenic Effect ◽  
Cell System ◽  
Dna Lesions ◽  
Biochanin A ◽  
Luciferase Reporter ◽  
Bacterial Cells ◽  
Mcf 7

Several flavonoids have shown their anti-carcinogenic effects in various models. The soyabean isoflavone genistein was demonstrated earlier in our laboratory to be an effective inhibitor of dimethylbenz[a]anthracene (DMBA)-induced DNA damage in MCF-7 cells by curbing cytochrome P450 (CYP) 1 enzymes. The red clover (Trifolium pratense) isoflavone biochanin A is a methylated derivative of genistein, and its anti-mutagenic effect in bacterial cells has been shown previously. Because of its protection against chemical carcinogenesis in an animal model, biochanin A was selected for testing in our established MCF-7 cell system. From the results obtained in the semi-quantitative reverse transcription–polymerase chain reaction and xenobiotic response element (XRE)–luciferase reporter assays, biochanin A could reduce xenobiotic-induced CYP1A1 and -1B1 mRNA abundances through the interference of XRE-dependent transactivation. Enzyme kinetic studies also indicated that biochanin A inhibited both CYP1A1 and -1B1 enzymes with inhibition constant (Ki) values 4·00 and 0·59μM respectively. Since the biotransformation of DMBA was dependent on CYP1 enzyme activities, biochanin A was able to decrease the DMBA–DNA lesions. The present study illustrated that the red clover isoflavone could protect against polycylic aromatic hydrocarbon-induced DNA damage.

scholarly journals Triterpenoids from the wood of Willughbeia cochinchinensis

2018 ◽  
Vol 1 (T5) ◽  
pp. 137-142
Author(s):  
Linh Thi My Lam ◽  
Phu Hoang Dang ◽  
Hai Xuan Nguyen ◽  
Nhan Trung Nguyen ◽  
Mai Thi Thanh Nguyen
Keyword(s):  
Thin Layer ◽  
Ethyl Acetate ◽  
Thin Layer Chromatography ◽  
Silica Gel ◽  
Column Chromatography ◽  
Normal Phase ◽  
Reverse Phase ◽  
Chemical Structures ◽  
First Time ◽  
Layer Chromatography

From the wood of n-hexane extract of Willughbeia cochinchinensis, perform column chromatography on a normal phase silica gel with n-hexane: ethyl acetate obtained in 8 fractions. From GD-D and GD-F fractions, multiple column chromatography on silica gel was performed in combination with normal and reverse phase thin-layer chromatography, four triterpene compounds as epifriedelanol (1), taraxeryl acetate (2), ambolic acid (3) and α- amyrin (4) were isolated. Their chemical structures were determined by spectrocopic methods as well as comparing with data in the literature. These compounds were isolated for the first time from Willughbeia cochinchinensis.

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