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Berberidis radix, as a traditional Chinese herbal medicine, is well known as the main source of berberine. This herbal medicine has been used for the treatment of many diseases and alkaloids including jatrorrhizine, palmatine, and berberine are major bioactive constituents. Few literatures report the preparative separation of these alkaloids from Berberidis radix. In the present study, the combinative application of ultrasonic extraction and pH-zone-refining countercurrent chromatography was used to extract and purify these alkaloids. The PZRCCC separation was performed in a two-phase solvent system composed of chloroform– methanol–water (4:3:3 volume ratio), where 40 mM HCl was added in the upper aqueous stationary phase as a retainer and 10 mM triethylamine was added in the lower organic mobile phase as an eluter. From a crude sample of 1.5 g, palmatine (251.6 mg), berberine (392.5 mg), and jatrorrhizine (412.6 mg) were obtained in a single run with purities of all being over 93.0%, analyzed by HPLC. The chemical structures of the separated alkaloids were identified by electrospray ionization–mass spectrometry (ESI-MS), 1H-NMR and 13C-NMR.