Inter- and intra-accession variation of isoflavones in red clover plants

The literature contains a surprising diversity of methods for extracting and analysing isoflavones in red clover. The objective of this study was to measure the extent of variation between red clover varieties and within varieties in terms of isoflavone composition and concentrations. However, few reports exist on how to store samples after harvesting and how to achieve quantitative extraction efficiency. Extraction methods were compared using freeze-dried plant samples with those that were homogenised in methanol. This information is needed for first preserving and then comparing large numbers red clover leaflets from pot and field experiments.

Isoflavones were determined as aglycones after acid hydrolysis, as aglycones are bioavailable. Two accessions of wild red clover plants were used for this experiment. HPLC profiles (without acid hydrolysis) were run to determine any changes during the preservation processes. Homogenisation in methanol generated higher isoflavone contents and lower standard errors than freeze-drying of plant samples. Some of the major isoflavones were quantified; genistein ranged from 57 to 151 µg per gram of fresh leaflets; formononetin from 115 to 1221 µg/g and biochanin A from 0 to 5625.0 µg/g. Interestingly, in the accession with undetectable levels of biochanin A the major isoflavone was tentatively identified as prunetin. Thus there were significant differences in isoflavone concentrations and composition between and within varieties.