Preparative Separation and Purification of Four Glycosides from Gentianae radix by High-Speed Counter-Current Chromatography and Comparison of Their Anti-NO Production Effects.

Secoiridoid and iridoid glycosides are the main active components of Gentianaeradix. In this work, one iridoid and three secoiridoid glycosides from Gentianaeradix have been purified by high-speed counter-current chromatography in two runs using different solvent systems. Ethyl acetate-n-butanol-water (2:1:3, v/v/v) was the optimum solvent system to purify ca. 4.36 mg of loganic acid, 3.05 mg of swertiamarin, and 35.66 mg of gentiopicroside with 98.1%, 97.2% and 98.6% purities, respectively, while 31.15 mg of trifloroside with 98.9% purity was separated using hexane-ethyl acetate-methanol-water (1:3:1:3, v/v/v/v). The structures of the glycosides were identified by mass spectrometry and NMR. After separation, the anti-nitric oxide production effects of the compounds on lipopolysaccharide-induced BV-2 murine microglial cells were also evaluated. All of the compounds inhibited the production of nitric oxide in lipopolysaccharide-induced BV-2 cells with high cell viabilities in a concentration-dependent manner, which demonstrated that were able to be used as a nitric oxide inhibitor.