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      Source of variation of isoflavone concentrations in perennial clover species

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          Abstract

          Introduction:

          Clover has attracted considerable interest not only as a valuable livestock forage plant, but also as an important source of isoflavones. The current study was aimed to assess the variation of concentration of three isoflavones in clover species grown under a cool temperate climate environment in Lithuania.

          Materials and Methods:

          Isoflavone contents were quantified in the plant parts of 21 accessions belonging to five perennial species of genus Trifolium ( T. pratense, T. repens, T. medium, T. rubens, and T. pannonicum). Daidzein, formononetin, and genistein concentrations were quantified in separate plant parts by reversed-phase high-performance liquid chromatography. The analyte extraction was performed from dried and ground leaves, stems, flowers, and roots. The procedure included acid hydrolysis of isoflavone glycosides to aglycones.

          Results:

          According to the averaged sum of the three isoflavones quantified in leaves-stems-flowers, the five clover species ranked as follows: T. medium (7.54-3.62-2.31 mg/g) > T. pratense> T. rubens> T. pannonicum> T. repens (0.191-0.204-0.171 mg/g). The contribution of individual compound to the total isoflavone content depended on the species, accession, and plant part. The major part of the isoflavones is concentrated in leaves or stems; however, there is a great variation also.

          Conclusion:

          There exists a large variation in the total as well as in individual concentration of isoflavones among the clover species and plant parts and within species. With regard to isoflavone concentration and variability within species, some accessions of T. medium and T. pratense can be considered a highly promising source of phytoestrogens.

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          Most cited references42

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          Phytoestrogens: a review of the present state of research.

          Phytoestrogens are a diverse group of plant-derived compounds that structurally or functionally mimic mammalian estrogens and show potential benefits for human health. The number of articles published on phytoestrogens has risen dramatically in the past couple decades. Further research continues to demonstrate the biological complexity of phytoestrogens, which belong to several different chemical classes and act through diverse mechanisms. This paper discusses the classification of phytoestrogens, methods of identification, their proposed mechanisms of action and botanical sources for phytoestrogens. The effects of phytoestrogens on breast and prostate cancers, cardiovascular disease, menopausal symptoms and osteoporosis will also be examined including research on benefits and risks. Copyright 2003 John Wiley & Sons, Ltd.
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            Evaluation of estrogenic activity of plant extracts for the potential treatment of menopausal symptoms.

            Eight botanical preparations that are commonly used for the treatment of menopausal symptoms were tested for estrogenic activity. Methanol extracts of red clover (Trifolium pratense L.), chasteberry (Vitex agnus-castus L.), and hops (Humulus lupulus L.) showed significant competitive binding to estrogen receptors alpha (ER alpha) and beta (ER beta). With cultured Ishikawa (endometrial) cells, red clover and hops exhibited estrogenic activity as indicated by induction of alkaline phosphatase (AP) activity and up-regulation of progesterone receptor (PR) mRNA. Chasteberry also stimulated PR expression, but no induction of AP activity was observed. In S30 breast cancer cells, pS2 (presenelin-2), another estrogen-inducible gene, was up-regulated in the presence of red clover, hops, and chasteberry. Interestingly, extracts of Asian ginseng (Panax ginseng C.A. Meyer) and North American ginseng (Panax quinquefolius L.) induced pS2 mRNA expression in S30 cells, but no significant ER binding affinity, AP induction, or PR expression was noted in Ishikawa cells. Dong quai [Angelica sinensis (Oliv.) Diels] and licorice (Glycyrrhiza glabra L.) showed only weak ER binding and PR and pS2 mRNA induction. Black cohosh [Cimicifuga racemosa (L.) Nutt.] showed no activity in any of the above in vitro assays. Bioassay-guided isolation utilizing ER competitive binding as a monitor and screening using ultrafiltration LC-MS revealed that genistein was the most active component of red clover. Consistent with this observation, genistein was found to be the most effective of four red clover isoflavones tested in the above in vitro assays. Therefore, estrogenic components of plant extracts can be identified using assays for estrogenic activity along with screening and identification of the active components using ultrafiltration LC-MS. These data suggest a potential use for some dietary supplements, ingested by human beings, in the treatment of menopausal symptoms.
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              Determination of isoflavones in red clover and related species by high-performance liquid chromatography combined with ultraviolet and mass spectrometric detection.

              High-performance liquid chromatography-UV-electrospray ionization-mass spectrometric detector (HPLC-UV-ESI-MSD) method for determination of isoflavones in red clover (Trifolium pratense L.) and related species has been developed. The separated isoflavones including aglycones, glycosides and glycoside malonates, were individually analyzed and identified by their molecular ions and characteristic fragment ion peaks using LC-MSD under MS and MS-MS mode, and in comparison with the standard isoflavones. A total of 31 isoflavones were detected in red clover. Several isoflavones were also identified for the first time in related species, T. repense L. (white clover), T. hybridum L. (alsike clover) and T. campestre Schreber (hop trefoil). Based on reversed phase HPLC, all 10 isoflavone aglycones, daidzein, formononetin, genistein, pseudobaptigenin, glycitein, calycosin, prunetin, biochanin A, irilone and pratensein in acidic hydrolyzed extracts were successfully separated within 40 min and quantified individually by UV and MS detectors. For the 10 target compounds, the investigated concentrations ranged from approximately 24 to approximately 12500 ng/ml for UV detection and approximately 6 to approximately 3125 ng/ml for MS detection, and good linearities (r2 > 0.999 for UV and r2 > 0.99 for MS) for standard curves were achieved for each isoflavone. The accuracy and repeatability (n = 10) were within 15% for these 10 compounds. This is the first method reported that enables the simultaneous quantitation of all 10 isoflavone aglycones in red clover and related species.
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                Author and article information

                Journal
                Pharmacogn Mag
                Pharmacogn Mag
                PM
                Pharmacognosy Magazine
                Medknow Publications & Media Pvt Ltd (India )
                0973-1296
                0976-4062
                Jan-Feb 2014
                : 10
                : Suppl 1
                : S181-S188
                Affiliations
                [1] Chemical Research Laboratory, Institute of Agriculture, Lithuanian Research Centre for Agriculture and Forestry, Kėdainiai, Lithuania
                [1 ] Department of Grass Breeding, Institute of Agriculture, Lithuanian Research Centre for Agriculture and Forestry, Kėdainiai, Lithuania
                [2 ] Laboratory of Genetics and Physiology, Institute of Agriculture, Lithuanian Research Centre for Agriculture and Forestry, Kėdainiai, Lithuania
                [3 ] Department of Pharmacognosy of Lithuanian University of Health Sciences, Kaunas, Lithuania
                Author notes
                Address for correspondence: Dr. Bronislava Butkutė, Chemical Research Laboratory, Institute of Agriculture, Lithuanian Research Centre for Agriculture and Forestry, Akademija, Kėdainiai district, LT-58344, Lithuania. E-mail: brone@ 123456lzi.lt
                Article
                PM-10-181
                10.4103/0973-1296.127373
                4047584
                e2b051a4-4c02-4c43-ac66-3f518ae52e0a
                Copyright: © Pharmacognosy Magazine

                This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-Share Alike 3.0 Unported, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                : 01 October 2012
                : 11 November 2012
                : 21 February 2014
                Categories
                Original Article

                Pharmacology & Pharmaceutical medicine
                concentration variation,daidzein,formononetin,genistein,plant part,trifolium species and accessions

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