Two-dimensional chromatography based on on-line HPLC-DPPH bioactivity-guided assay for the preparative isolation of analogue antioxidant compound from Arenaria kansuensis

- A novel two-dimensional semipreparative HPLC-DPPH-DAD method was established for targeted seperation of antioxidant directly.
- A novel two-dimensional preparative system was established through RP-C18HCE coupling with NP-XAmide and successfully purify the single antioxidant compound from coeluted fraction.
- A novel antioxidant compound from traditional Tibetan medicine Arenaria Kkansuensis was found with the method.
- Two groups of analogical antioxidant compounds were isolated from A. Kkansuensis with the method.

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 800 mg 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 β-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.

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