Rapid Detection of Antioxidant Flavonoids in Azalea (Rhododendron mucronulatum) Flowers using On-line HPLC-ABTS+ System and Preparative Isolation of Three Flavonoids by Centrifugal Partition Chromatography

Abstract

High-performance liquid chromatography coupled to an on-line ABTS+-based assay (on-line HPLC-ABTS+) system was used to determine the principle antioxidants in azalea flowers. Three flavonoids, myricetin, quercetin, and kaempferol, recovered in ethyl acetate extracts of azalea flowers were determined to have antioxidant activities. These three flavonoids were isolated and purified by successive centrifugal partition chromatography (CPC) using two different biphasic solvent systems, consisting of n-hexane-ethyl acetate-methanol-water (3:5:3:5 or 4:5:4:5, v/v). A total of 46.2 mg of myricetin, 28.9 mg of quercetin, and 10.6 mg of kaempferol with purities of 97.0%, 95.4%, and 93.9%, respectively, were purified from 500 mg of ethyl acetate soluble material from azalea flowers. The structures were identified by their retention time, UV spectra, and ESI-MS in the positive ion mode and were confirmed by NMR experiments.