Objective: To develop a method for separation and purification of alkaloids from the crude extract of Corydalis yanhusuo by combination of semi-preparative HPLC and high-speed counter-current chromatography(HSCCC). Method: First

high speed counter-current chromatography was successfully performed with a two-phase solvent system

petroleum ether-ethyl acetate-Methanol-water(2: 3: 3: 2).From 300 mg of crude extract

a mixture and two compounds were obtained.And then

the mixture was separated by semi-preparative HPLC. The structures of the target compounds were identified by ESI-MS and NMR. Result: 4 compounds with glaucine (125 mg

1)

tetrahydropalmatine (40.5 mg

2)

tetrahydroberberine (23.5 mg

3) and d-corydaline (10 mg

4) were obtained with purities of 96.5%

97.54%

98.3% and 97.3%

respectively. Conclusion: Combination of HSCCC and semi-preparative HPLC is an efficient method for separation of alkaloids from the crude of C.yanhusuo.