Isolation of Pyrrolizidine Alkaloid Isomers from Symphytum Species by Semi-Preparative Chiral Chromatography

Abstract

Comfrey is a common name given to plants belonging to the genus Symphytum (family Boraginaceae) [1]. The comfrey root and leaf contain varying levels of the hepatotoxic pyrrolizidine alkaloids (PAs) that have been reported to cause veno-occlusive disease in humans [2]. However, the exact alkaloid profile of different species has not been clearly established, in part because comfrey PAs are not commercially available and the isolation of the individual isomers is difficult. Milligram quantities of PA components from Symphytum are needed for use as analytical standards in quantitating these components in dietary supplements containing these botanicals. Results will be presented on the isolation of PAs from the roots of S. uplandicum. Briefly, a 1.0 kg quantity of plant material was extracted with methanol and the PAs were reduced with zinc dust to convert the N-oxides to free bases. The PAs were enriched on a Chem Elut cartridge (Varian Inc.) and then fractionated on a silica gel chromatographic column, followed by further isolation by semi-preparative HPLC. For the compounds occurring as racemic mixtures, their separation was achieved by semi-preparative chiral HPL C. These chiral stationary phases consist of cellulose immobilized on silica with carbamates as chiral selectors (Chiralpak IA and IC, Chiral technologies Inc. West Chester PA). The compositions of individual fractions were monitored by GC-MS or LC-MS and the purified components were characterized by mass and NMR spectroscopic studies [3–5]. The isolated PAs will be used for the development of analytical methods that can be applied to develop chemical profiles of different species of Symphytum and to determine alkaloid contents of comfrey preparations sold in the market.