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.

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 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.

Genetic variability of principal isoflavones in red clover

2006 ◽  
Vol 86 (Special Issue) ◽  
pp. 1345-1347 ◽  
Author(s):  
Yousef A Papadopoulos ◽  
R. Tsao ◽  
K. B. McRae ◽  
A. E. Mellish ◽  
S. A. E. Fillmore
Keyword(s):  
Genetic Variability ◽  
Trifolium Pratense ◽  
Red Clover ◽  
Broad Sense ◽  
Biochanin A ◽  
Growth Stages ◽  
Broad Sense Heritability ◽  
Food Ingredients ◽  
Isoflavone Concentration ◽  
Trifolium Pratense L

Isoflavones, known for their health benefits, are abundant in red clover (Trifolium pratense L.). Total isoflavone concentrations can be 30 times that of soybean, indicating that red clover is a good source of nutraceutical and functional food ingredients. In this study, tissue samples of 13 red clover cultivars were taken at two growth stages (late-bud stage and late-flowering stage) to determine the concentration of individual isoflavones using HPLC. Individual isoflavone concentrations and total isoflavone concentration differed significantly according to red clover cultivar. We found significant genetic variability for total isoflavone concentration and individual isoflavone concentrations; these differences were not related to ploidy level (diploid vs. tetraploid). Broad-sense heritability (H = genetic variance/total variance) ranged from 0 to 83% and was influenced by isoflavone type and sampling date. The results of this study suggest that there is significant genetic variability for isoflavone concentrations among currently available red clover cultivars. Key words: Trifolium pratense L., biochanin A, formononetin, growing stages, broad-sense heritability

scholarly journals Integrated Phytochemical Analysis Based on UPLC-MS and Network Pharmacology Approaches to Explore the Quality Control Markers for the Quality Assessment of Trifolium pratense L.

Molecules ◽  
2020 ◽  
Vol 25 (17) ◽  
pp. 3787
Author(s):  
Liyu Luo ◽  
Wenya Gao ◽  
Yan Zhang ◽  
Chang Liu ◽  
Guopeng Wang ◽  
...  
Keyword(s):  
Quality Control ◽  
Quality Assessment ◽  
Chlorogenic Acid ◽  
Trifolium Pratense ◽  
Red Clover ◽  
Biochanin A ◽  
Leguminous Plant ◽  
Network Pharmacology ◽  
Trifolium Pratense L ◽  
Novel Method

Red clover consists of the overground parts and inflorescence of Trifolium pratense L., a leguminous plant belonging to the genus Trifolium. It is widely distributed worldwide and has long been used in traditional medicine. In this study, a combination approach using UPLC-MS and network pharmacology was applied to explore the quality control markers for the quality assessments of red clover. Firstly, UPLC-MS was used to identify the compounds in different parts of red clover. Twenty-eight compounds were totally identified. According to the traditional clinical efficacy of red clover, a compound-target-function network was constructed by network pharmacology to discover the main active compounds based on the identified compounds. Nine compounds of chlorogenic acid, daidzin, calycosin-7-O-β-d-glucoside, genistin, ononin, daidzein, genistein, formononetin, and biochanin A were filtrated and further confirmed in rat plasma in view of the blood-absorbed components taking effects. Finally, a novel method for simultaneously detecting the nine quality control markers was developed by UPLC-QQQ-MS in an effort to assess the quality of red clover. For all samples, the average contents of the nine compounds measured from high to low consist of formononetin, ononin, biochanin A, genistin, daidzin, calycosin-7-O-β-d-glucoside, genistein, daidzein, and chlorogenic acid. The samples from Gansu province showed the best quality in the three producing areas This study provides new strategies to explore the quality control markers and develops a novel method for the quality assessment of red clover.

Comparisons of Floral Response of Seed Lots of Dollard Red Clover, Trifolium pratense L. 1

Crop Science ◽  
1965 ◽  
Vol 5 (5) ◽  
pp. 425-428 ◽  
Author(s):  
R. J. Bula ◽  
R. G. May ◽  
C. S. Garrison ◽  
C. M. Rincker ◽  
J. G. Dean
Keyword(s):  
Trifolium Pratense ◽  
Red Clover ◽  
Trifolium Pratense L

scholarly journals Volatile Constituents of Trifolium Pratense and T. Repens from N.E. Italian Alpine Pastures

2009 ◽  
Vol 4 (6) ◽  
pp. 1934578X0900400 ◽  
Author(s):  
Aldo Tava ◽  
Daniele Ramella ◽  
Maris Grecchi ◽  
Paolo Aceto ◽  
Renato Paoletti ◽  
...  
Keyword(s):  
Steam Distillation ◽  
Trifolium Pratense ◽  
Red Clover ◽  
Volatile Oil ◽  
Volatile Fraction ◽  
Fresh Weight ◽  
Fresh Weight Basis ◽  
Qualitative And Quantitative ◽  
Aerial Parts ◽  
Trifolium Pratense L

The composition of the volatile fraction of two important forage legumes from Italian sub-alpine N.E. pastureland, namely Trifolium pratense L. subsp. pratense (red clover) and T. repens subsp. repens (white clover) were investigated. The volatile oil was obtained from the fresh aerial parts by steam distillation and analyzed by GC/FID and GC/MS. The oil yield was 0.018 and 0.021% (weight/fresh weight basis) for T. pratense and T. repens, respectively. Several classes of compounds were found in both the oils, including alcohols, aldehydes, ketones, terpenes, esters, hydrocarbons, phenolics and acids. Qualitative and quantitative differences were found.

Polycross‐Progeny Testing of Red Clover (trifolium pratense L. 1

Crop Science ◽  
1968 ◽  
Vol 8 (4) ◽  
pp. 451-454 ◽  
Author(s):  
Norman L. Taylor ◽  
W. A. Kendall ◽  
W. H. Stroube
Keyword(s):  
Trifolium Pratense ◽  
Red Clover ◽  
Progeny Testing ◽  
Trifolium Pratense L
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