In vitro production of radiolabeled red clover (Trifolium pratense) isoflavones
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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.
KeywordsRed clover Isoflavones Metabolic tracers Plant cell culture Copper chloride Radiolabeling Trifolium pratense
This research was supported by the National Center for Complementary and Alternative Medicine sponsored Purdue-Universtiy of Alabama-Birmingham Botanicals Center for Dietary Supplement Research (NIH, 2 P50 AT000477-06). The authors would like to thank the University of Illinois James Scholar Program (College of Liberal Arts and Sciences). Nicola Lancki and Aaron McKerracher assisted with tissue culture maintenance and cell extractions. Contributions were also made by William Helferich with TLC analysis assistance, Tristan Kraft with helpful discussions regarding experimental design, and Jeevan Prasain with preliminary isoflavone analyses.
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