The concentration of isoflavones in red clover (Trifolium pratense L.) at flowering stage

The objective of this study was to determine the concentrations of isoflavones in the cultivars and ecotypes of red clover (Trifolium pratense L.) in a whole above ground part of a plant and separately in stems, leaves and flowers at flowering stage. Isoflavones were extracted using acidified aqueous methanol and subsequent analyses of the extracts were carried out by ultra-performance liquid chromatography coupled with a photodiode array detector. Red clover accumulated the highest concentrations of formononetin (51%) and biochanin A (40%) at flowering stage. Concentrations of formononetin, biochanin A, daidzein and genistein at flowering stage ranged between 2.61-4.40, 1.79-3.32, 0.06-0.14 and 0.36-0.59 mg g(-1) dry matter (DM), respectively. The average concentration of all four isoflavones at flowering stage during two years of investigations was 6.66 mg g(-1) DM and ranged from 5.4 to 8.09 mg g(-1) DM. The analysed genotypes formed the following sequence according to total isoflavone concentration mg g(-1) DM: 'Radviliai' (8.09) > 'Vyliai' (7.29) > No. 2739 (7.15) > No. 2331 (6.92) > 'Kirsinai' (6.90) > No. 2177 (6.47) > 'Kamaniai' (6.41) > 'Arimaiciai' (6.28) > 'Sadunai' (6.17) > 'Vyciai' (6.11) > 'Liepsna' (5.44). The highest concentration of isoflavones at flowering stage was accumulated in leaves. The average total concentration of all four isoflavones was as follows: 12.29 mg g(-1) DM in leaves, 2.93 mg g(-1) DM in stems and 1.42 mg g(-1) DM in flowers. Plant material of all the tested cultivars and wild ecotypes, especially leaves of commercial diploid cv. 'Radviliai', tetraploid cv. 'Vyliai' and semi-natural ecotype No. 2739 containing the highest concentration of isoflavones, could be used for dietary supplement production in the pharmaceutical industry as a source of isoflavones.