A strategy for high-speed countercurrent chromatography purification of specific antioxidants from natural products based on on-line HPLC method with radical scavenging assay

► Novel approach for purification of antioxidants from natural materials was developed. ► HSCCC purification with on-line HPLC/DPPH radical assay was developed. ► Specific antioxidants in red cabbage, perilla and elderberry colors were purified. ► Antioxidants were confirmed to cyanidin-based anthocyanin and luteolin-based flavones. We have proposed a novel and first strategy of high-speed countercurrent chromatography (HSCCC) purification for the efficient and effective discovery of antioxidant from natural product based on on-line HPLC method with radical scavenging assay. To achieve a strategy for HSCCC purification, the antioxidants in materials are identified by on-line HPLC with DPPH radical scavenging assay. Then, the optimal condition of target peaks would be investigated for the two-phase solvent system, and purified by HSCCC. In this study, the specific antioxidants in red cabbage, perilla and elderberry pigments were evaluated by on-line HPLC with DPPH radical scavenging assay, and purified by HSCCC technique. Specific antioxidants could be rapidly pinpointed in complex mixtures by on-line HPLC with DPPH radical scavenging assay. Then, the optimal two-phase solvent systems were investigated using these HPLC peaks. Finally, the purification of these nine antioxidants form three mixtures were performed by HSCCC. Using mass spectrometric analysis, these antioxidants were confirmed to cyanidin-based anthocyanin from red cabbage and elderberry pigments, and luteolin-based flavones from perrilla pigment. Due to the advantages derived from on-line HPLC with DPPH radical scavenging assay and HSCCC technique, a rapid, efficient and effective strategy has been developed for the discovery of antioxidants from natural products.