Effects of the Environment, Cultivar, Maturity, and Preservation Method on Red Clover Isoflavone Concentration

Abstract

Red clover (Trifolium pratense L.) contains isoflavones that are of interest because of their benefits for human health as well as their adverse effects on the fertility of farm animals. A series of field experiments was conducted in Sainte-Anne-de-Bellevue, QC, Canada, to determine the effects of the environment, cultivar, plant maturity, plant part, and preservation method on the concentration of the two predominant isoflavones in red clover, formononetin and biochanin A. In a multi-year, multisite trial, the total isoflavone concentration in 10 cultivars ranged between 8,923 and 12,753 microg g(-1) of DM averaged across sites, harvests, and years. Despite strong environmental effects, the cultivar "Start" consistently had the lowest isoflavone concentrations, with few differences observed among other cultivars. Across stages of maturity, leaves were found to have the highest isoflavone concentration followed by stems and inflorescences (11,970, 4,896, and 3,297 microg g(-1) of DM, respectively). Changes in isoflavone concentrations with increasing maturity varied depending on the plant part. Overall, highest isoflavone concentrations were found in leaves and stems during the vegetative stages, with the formononetin concentration declining until plants initiated flowering, especially in stems, with concentrations then stabilized in both parts. Upon initiation, inflorescences contained similar isoflavone concentrations than leaves, but concentrations decreased rapidly during flower development to fall even below those observed in stems. Inflorescences then had isoflavone concentrations that were as much as 11 times lower than leaves. Fresh herbage contained higher formononetin and total isoflavone concentrations than did silage and hay (14,464, 12,200, and 11,604 microg g(-1) of DM, respectively). The isoflavone concentration in field-grown red clover is thus high but can be affected by a range of agronomic factors.

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).…”

“…This is likely to reflect a higher concentration of phyto-oestrogens in fresh grass than in silage. Supporting this, Sivesind and Seguin (2005) found that the concentration of phytooestrogen was more or less similar among cultivars of red Phyto-oestrogens in herbage and milk clover, but that the concentration of total isoflavones was 22% higher in fresh herbage compared to silage and hay. However, it is important to note that ensiling is a dynamic process dependent on many factors (pH, microbial population, temperature, initial herbage composition) that fluctuate, and therefore these factors potentially influence the concentration of isoflavones in silage.…”

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

“…Similar results were reported from Canada (Sivesind and Seguin, 2005). Average contents and range of the sum of formononetin and biochanin A in ten red clover varieties were 8.84 and 1.53-16.76 mg/g DM, respectively, with overall 55% formononetin, depending on variety, site, stand age, and harvest.…”

“…The highest overall contents were in leaves, followed by stems, petioles, and flowers. Similar data were reported by Sivesind and Seguin (2005). Across stages of maturity, leaves were found to have the highest total isoflavone (formononetin + biochanin A) content followed by stems and inflorescences (11.97, 4.90, and 3.30 mg/g DM, respectively).…”

“…Fresh birdsfoot trefoil and its silages contained only traces of isoflavones. Sivesind and Seguin (2005) observed partly different effects of red clover preservation. They determined formononetin contents 9.02 ± 0.37, 7.22 ± 0.55, and 6.47 ± 0.16 mg/g DM in fresh, ensiled, and field-dried red clover, respectively.…”