A combined chromatographic method using high-speed counter-current chromatography (HSCCC) and Sephadex LH-20 chromatography was established for bioassay-guided separation and purification of water-soluble antioxidants from Carthamus tinctorius L florets. The crude sample II obtained by an initial cleanup step on the AB-8 macroporous resin exhibited a potential ABTS radical cation scavenging activity with the SC50 value of 49.28 mu g/mL. The HSCCC separation was performed with a two-phase solvent system composed of n-butanol-0.1 mol/L HCl (1:1, v/v) at a flow rate of 1.2 mL/min. After a single run, 3 mg hydroxysafflor yellow A (HSYA) with 98% purity was separated as a major component from 20 mg of crude sample II. In order to increase the yield and investigate the minor components, the sample size of HSCCC separation was enlarged to 1 g. Then, HSCCC fractions were subjected to Sephadex LH-20 chromatography and eluted with distilled water at a flow rate of 1.5 mL/min to remove the acid and further separate the components. This separation yielded 184.0 mg of HSYA (1), 10.0 mg of 5,4'-dihydroxyflavone-3,6-di-O-beta-D-glucoside-7-O-beta-D-glucuronide (2), 3.0 mg of 3-O-caffeoylquinic acid (3), and 3.2 mg of 4-O-beta-D-glucosyl-trans-p-coumaric acid (4) from 1 g crude sample II of C tinctorius L. florets. The purity of the separated compounds were over 95% by HPLC analysis, and their chemical structures were confirmed by MS, H-1 NMR and C-13 NMR. Compounds 3 and 4 were found from this plant for the first time. Antioxidant activities assayed in vitro by ABTS radical cation scavenging showed that the SC50 values of the above four compounds were 44.39 +/- 1.62 mu g/mL, 78.13 +/- 1.00 mu g/mL, 21.85 +/- 1.96 mu g/mL, and 31.44 +/- 2.06 mu g/mL, respectively. (C) 2012 Elsevier B.V. All rights reserved.