Screening bioactive components of Glycyrrhiza uralensis Fisch. with isolated perfused lung extraction and HPLC-ESI-MSn analysis

Abstract

The isolated perfused rat lung (IPL), coupled with high performance liquid chromatography\ andem mass spectrometry analysis (HPLC–ESI-MSn), has been developed as a tool for screening bioactive components in Glycyrrhiza uralensis Fisch. (GU). First, IPL was perfused with the water extract of GU (EGU), the bioactive components in the EGU would selectively combine to the receptors or channels of lung. By changing the pH of perfused solution, the combined components were eluated and then detected by HPLC-ESI-MSn. Four compounds were detected in the desorption eluate of IPL, among these compounds, liquiritin (1), ononin (2) and glycyrrhizic acid (4) were identified by comparing with the chromatography of the standards, while licorice-saponin G2 (3) were determined by analysis of the structure clearage characterization of mass spectrometry. Then, due to the lack of compound 3 sample, compounds 1, 2 and 4 with respective concentrations of 50 μM, 5 μM, 500 nM, 50 nM and 5 nM were applied to evaluate the protective effect of pulmonary epithelial cells (PEC, A549 cell) injury induced by lipopolysaccharide (LPS) for anti-inflammatory activity assessment. The results showed that except the 5 nM group of compound 1, 5 nM and 50 nM groups of compound 2, all other groups could remarkably inhibit the PEC injury (vs LPS group, 2-500 nM groups: p < 0.05; other groups: p < 0.01), all compound showed the dose-dependent effect. In conclusion, IPL coupled with HPLC-ESI-MSn was successfully used to screen the anti-inflammatory components of GU for the first time. The application of IPL coupled with HPLC-ESI-MSn for screening bioactive components of TCMs is rapid, convenient and reliable, and the isolated perfused technology could be extended to isolated heart, liver, kidney, and so on.