Seasonal Variation of Red Clover (Trifolium pratense L., Fabaceae) Isoflavones and Estrogenic Activity

Abstract

Red clover (Trifolium pratense L., Fabaceae) dietary supplements are currently used to treat menopausal symptoms because of their high content of the mildly estrogenic isoflavones daidzein, genistein, formononetin and biochanin A. These compounds are estrogenic in vitro and in vivo, but little information exists on the best time to harvest red clover fields to maximize content of the isoflavones and thus make an optimal product. Samples of cultivated red clover aboveground parts and flower heads were collected in parallel over one growing season in northeastern Illinois. Generally, autohydrolytic extracts of aboveground parts contained more isoflavones and had more estrogenic activity in Ishikawa endometrial cells, compared with extracts of flower heads. Daidzein and genistein content peaked around June to July, while formononetin and biochanin A content peaked in early September. Flower head and total aboveground parts extracts exhibited differential estrogenic activity in an Ishikawa (endometrial) cell-based alkaline phosphatase (AP) induction assay, whereas nondifferential activity was observed for most extracts tested in an MCF-7 (breast) cell proliferation assay when tested at the same final concentrations. Ishikawa assay results could be mapped onto the extracts' content of individual isoflavones, but MCF-7 results did not show such a pattern. These results suggest that significant metabolism of isoflavones may occur in MCF-7 cells, but not in Ishikawa cells, and therefore caution is advised in the choice of bioassay used for the biological standardization of botanical dietary supplements.

Phyto-oestrogens in herbage and milk from cows grazing white clover, red clover, lucerne or chicory-rich pastures

“…This is in accordance with previous findings (Antignac et al, 2004;Hoikkala et al, 2007;Steinshamn et al, 2008). The very high concentration of equol in milk from especially cows grazing red clover was expected, since red clover has a high concentration of formononetin (0.8 to 11 mg/g DM) but also biochanin A (0.8 to 5 mg/g DM) depending on the part (flower, stem or leaves) and maturity of the plant, cultivar and environment (Sivesind and Seguin, 2005;Booth et al, 2006). Formononetin is metabolized by microbes in the rumen via daidzein to equol which is the major isoflavone absorbed to the blood circulation after feeding red clover/grass silage, whereas biochanin A is demethylated to genistein to form mainly p-ethyl phenol (Lundh, 1995).…”

Genetic variability of isoflavones in the USDA red clover core collection

“…Sivesind & Seguin (2005), related isoflavone concentrations in ten red clover cultivars in Quebec, ranging from 8 923 to 12 753 µg g -1 of DM. Booth et al (2006) also found similar isoflavones concentrations ranging from 3 070 to 10 210 µg g -1 of DM when analysing only one red clover cultivar.…”

“…It is also important to highlight that is time saving to harvest at vegetative stage once that is known that leaves have a greater concentration of isoflavones than flowers, and the cultivation period is smaller (Sivesind & Seguin, 2005;Booth et al, 2006;Tsao et al, 2006).…”

“…Future breeding activities need to take this into account, exploiting the wide range of genetic diversity present in this species in order to develop new varieties to supply the demands from this new market (Greene et al, 2004). However, total and individual isoflavones concentration are affected by many factors including fertility management, plant maturity, tissue type, genetics, sampling methodology and seasonal variation Booth et al, 2006).…”

Fresh and ensiled forages as a source of estrogenic equol in bovine milk: a review

“…Vetter (1995) reported comparable or higher contents in flowers than in leaves (Table 1). Booth et al (2006) determined formononetin and daidzein contents in flower heads by one order of magnitude lower than in above-ground parts. The most thorough study was carried out by Tsao et al (2006).…”