Influence of Harvest Date, Cultivar, and Sample Storage Method on Concentration of Isoflavones in Subterranean Clover

The isoflavones formononetin (7-hydroxy-4′-methoxyisoflavone; FM), biochanin A (5,7dihydroxy-4′-methoxyisoflavone; BA), genistein (5,7,4′-trihydroxyisoflavone; GE), and trace amounts of diadzein (7,4′-dihydroxyisoflavone; DI) are present in subterranean clover (Trifolium subterraneum L.) in concentrations highly dependent on cultivar or germplasm source. The effects of harvest date and sample storage methods on isoflavone concentration of four subterranean clover cultivars were investigated using high performance liquid chromatography techniques. Leaves and petioles were harvested from cultivars ‘Tallarook’, ‘Nangeela’, and ‘Woogenellup’ and PI 209927 in March, April, and May, 1983 at Overton, TX. Samples were methanol extracted (7 days at 5°C) fresh, and after 24 weeks storage either frozen (−l2°C) or dried (48 h at 40°C). Free isoflavones were measured directly from the methanol extract while total (free + glycoside form) was measured in acid hydrolyzed (4 M HCI at 70°C, 2 h) extracts. Formononetin, BA, and GE were detected in all samples. Harvest date, sample storage, and clover cultivar had significant effects on the concentration of FM, BA, and GE. First and second order interactions were also significant for isoflavone concentrations. Isoflavone concentration decreased over harvest date for all cultivars except Woogenellup. Concentration of total FM in Woogenellup increased from 1.1 to 3.4 g kg⁻¹ from March to May. Total isoflavone concentration was unaffected by frozen storage but was decreased by 30 to 50% in dried samples. We conclude that accurate classification of potential isoflavone concentration in subterranean clover should be derived from measurements at several harvest dates over the growing season. Total isoflavone concentrations in subterranean clover samples frozen for 24 weeks was closely related to concentrations in fresh samples.