Traditional Tibetan medicine is important for discovery of drug precursors. However, information about the chemical composition of traditional Tibetan medicine is very limited due to the lack of appropriate chromatographic purification methods. In the present work, A. kansuensis was taken as an example and a novel two-dimensional reversed-phase/hydrophilic interaction liquid chromatography(HILIC) method based on on-line HPLC-DPPH bioactivity-guided assay was developed for the purification of analogue antioxidant compounds with high purity from the extract of A. kansuensis. Based on the separation results of many different chromatographic stationary phases, the first-dimensional (1D) preparation was carried on a RP-C18HCE prep column, and 2 antioxidant fractions were obtained from the 800mg crude sample with a recovery of 56.7%. A HILIC-XAmide prep column was selected for the second-dimensional (2D) preparation. Finally, a novel antioxidant beta-carboline Alkaloids (Glusodichotomine AK) and 4 known compounds (Tricin, Homoeriodictyol, Luteolin, Glucodichotomine B) were purified from A. kansuensis. The purity of the compounds isolated from the crude extract was >98%, which indicated that the method built in this work was efficient to manufacture single analogue antioxidant compounds of high purity from the extract of A. kansuensis. Additionally, this method showed great potential in the preparation of analogue structure antioxidant compounds and can serve as a good example for the purification of analogue structure antioxidant carboline alkaloids and flavonoids from other plant materials. CI - Copyright (c) 2017 Elsevier B.V. All rights reserved.