Purification and identification of antiviral components from Laggera pterodonta by high-speed counter-current chromatography

Although Laggera pterodonta, a folk medicine has been widely used for several centuries as an antiviral agent, there have been no studies to identify its antiviral components. A bioassay-guided phytochemical examination of L. pterodonta disclosed that its aqueous extract, which was made up of three dicaffeoylquinic acids showed significant inhibitory activity of virus replication. Then a simple and efficient preparative high-speed counter-current chromatography (HSCCC) method was successfully established by using ethyl acetate–n-butanol–water (3:2:5, v/v) as the two-phase solvent system to isolate and purify three bioactive dicaffeoylquinic acids, 3,5-O-dicaffeoylquinic acid, 3,4-O-dicaffeoylquinic acid and 4,5-O-dicaffeoylquinic acid. The flow rate was 1.5 ml/min and revolution speed was 800 rpm. The isolation was done in less than 6 h, and 34.6 mg of 3,5-O-dicaffeoylquinic acid, 29.4 mg of 3,4-O-dicaffeoylquinic acid and 27.1 mg of 4,5-O-dicaffeoylquinic acid was yielded from 600 mg of the crude sample in one-step separation with the purity of 98.3%, 96.7% and 96.2%, respectively, as determined by high-performance liquid chromatography (HPLC). The structures of these three bioactive dicaffeoylquinic acids were identified by ultraviolet (UV), electrospray ionization mass spectrometry (ESI-MS), proton nuclear magnetic resonance (1H NMR) and carbon-13 nuclear magnetic resonance (13C NMR). In the antiviral experiment, three dicaffeoylquinic acids all showed significant inhibitory activity against herpes simplex virus-1 (HSV-1), herpes simplex virus-2 (HSV-2) and influenza viruses A (IVA) in vitro with high selectivity indexes. However, among the three compounds, 3,5-O-dicaffeoylquinic acid and 4,5-O-dicaffeoylquinic acid were the more active than 3,4-O-dicaffeoylquinic acid against HSV-1, HSV-2 and IVA. This study demonstrated a direct link between the antiviral activity of L. pterodonta and the three dicaffeoylquinic acids.