In vitro production of radiolabeled red clover (Trifolium pratense) isoflavones

Red clover isoflavones are increasingly used in dietary supplements for their purported estrogenic effects. However, little is known about their metabolism in animals due to a lack of commercially available isotopically labeled tracers. The goal of this research was to establish red clover cell culturing methodology for ¹⁴C-biolabeling of isoflavones. When root, leaf, and petiole-derived suspension cultures were grown in darkness or light, dark-grown, petiole-derived solution cultures produced the highest concentrations of the two major red clover isoflavones, formononetin (0.67 mg/g FM inoculum) and biochanin A (0.13 mg/g FM inoculum). Varying levels and timing of copper chloride elicitor did not significantly affect isoflavone accumulation. Approximately 38% of the ¹⁴C-sucrose dose accumulated in the cells. Eighteen percent of the initial labeled dose was detected in the isoflavone-rich methanolic extract and of that, 22% accumulated in isoflavones.