Preparative high-speed counter-current chromatography (HSCCC) was used to separate and purify bioactive constituents from the stems and leaves of Lophatherum gracile Brongn. Six flavone C-glycosides each at over 95% purity including two new compounds were obtained in one-step separation by HSCCC with an optimized two-phase solvent system composed of ethyl acetate-n-butanol-ethanol-water at volume ratio of 4:2:1.5:8.5 (v/v/v/v). The experiment yielded 19.9mg of luteolin 6-C--D-galactopyranosiduronic acid (12)--D-glucopyranoside (1), 28.5mg of luteolin 6-C--L-arabinopyranosyl-7-O--D-glucopyranoside (2), 31.5mg of isoorientin (3), 44.8mg of orientin (4), 25.3mg of swertiajaponin (5) and 12.1mg of apigenin 6-C--D-galactopyranosiduronic acid (12)--D-glucopyranoside (6) from 500mg of crude extracts. The purity of these compounds was determined by high-performance liquid chromatography (HPLC). Their chemical structures were identified by electron spray ionization mass spectroscopy (ESI-MS), H-1 and C-13 nuclear magnetic resonance spectroscopy (NMR).