Reversed-phase liquid chromatographic isolation of lubimin and solavetivone from Hyoscyamus muticus "hairy" root cultures

A high-performance liquid chromatographic reversed-phase procedure was developed to estimate the concentration of sesquiterpene phytoalexins secreted into the media by Agrobacterium transformed root cultures of Hyoscyamus muticus. An isolation procedure based on solid-phase extraction (C₁₈ Sep-pak) results in very rapid analysis and gives full recovery, compared to sequential chloroform extraction. A simple isocratic procedure permits quantification of sesquiterpene production by these root cultures in response to exposure to fungal elicitors. Calibration curves for lubimin and solavetivone content were obtained by fractionating sufficient material to provide a measurable weight. The identity of these compounds was confirmed by TLC, UV, GC-MS, and NMR. This experimental system is being used to examine the regulation of sesquiterpene biosynthesis, and to develop large-scale processing techniques for the production of secondary metabolises from plant roots grown in bioreactor systems.