Preparative isolation of antioxidative compounds from Dracocephalum heterophyllum using off-line two-dimensional reversed-phase liquid chromatography/hydrophilic interaction chromatography guided by on-line HPLC-DPPH assay

Traditional Tibetan medicine (TTM) has been valuable for the identification of new therapeutic leads. Nevertheless, reports about the chemical constituents of TTM are meager owing to the lack of suitable purification techniques. In this study, an off-line two-dimensional reversed-phase/hydrophilic interaction liquid chromatography (2D RP/HILIC) technique guided by on-line HPLC-DPPH has been established for the isolation of pure antioxidants from the extract of Dracocephalum heterophyllum. According to the chromatographic recognition outcome of the HPLC-DPPH system, the first-dimensional (1D) separation on the Megress C18 preparative column yielded 6 antioxidative fractions (61.4% recovery) from the ethyl acetate fraction (6.1 g). In the second-dimensional (2D) separation, a HILIC XAmide preparative column was employed. In total, 8 antioxidants were isolated from D. heterophyllum with a purity of >95%, which indicated the efficiency of the developed method to prepare antioxidative compounds with high purity from plant extracts. In addition, this method was highly efficient for the preparation of structural analogues of the antioxidative polyphenols and could be applied for the purification of structural analogues from other resources.