Application of high-speed counter-current chromatography for the isolation of antiviral eremophilenolides from Ligularia atroviolacea.

Ligularia is mainly distributed in the western regions of China. Most of the species have been traditionally used in folk medicine for the treatment of hepatitis B, asthma, hemoptysis and pulmonary tuberculosis. In our continuation of research on antiviral components from traditional Chinese medicine, Ligularia atroviolacea was tested for inhibitory effects on hepatitis B virus (HBV). A bioassay-guided phytochemical examination of L. atroviolacea disclosed that its ethyl acetate extract, which was made up of two eremophilenolides, showed suppressive activity on the expression of HBV surface antigen (HBsAg) in the HepG2.2.15 cell line. Then a simple and effective preparative high-speed counter-current chromatography method was successfully developed for the isolation and purification of two main active metabolites, 8beta-hydroxyeremophil-3,7(11)-dien-12,8alpha;15,6alpha-diolide and 8beta-methoxyeremophil-3,7(11)-dien-12,8alpha;15,6alpha-diolide from the ethyl acetate extract of L. atroviolacea by a one-step separation using a two-phase solvent system composed of light petroleum (60-90 degrees C)-ethyl acetate-methanol-water (9:1:8:2, v/v/v). The chemical structures of the two eremophilenolides were identified by ESI-MS, (1)H-NMR and (13)C-NMR analysis. The anti-HBV activity of the two purified compounds was measured; both of them showed suppressive activity on the expression of HBsAg in the HepG2.2.15 cell line. The results support the continued and expanded exploitation and utilization of L. atroviolacea.