Counter-current chromatography (CCC) is extremely useful for the separation, purification, and isolation
of natural products. Recently, Berthod et al. [4,5] established an elution–extrusion CCC method
in metabolic analysis by combining regular chromatographic elution with stationary-phase extrusion,
which extends the hydrophobicity window of a counter-current separation. In this study, a novel
overlapping elution–extrusion CCC method was developed and applied to the preparation of natural
cytotoxic andrographolides from the aerial parts of Andrographis paniculata, a well-known Traditional
Chinese Medicine (TCM) with potent anti-inflammatory effect and anti-cancer activity. Its theory
was first developed, and then a series of CCC experiments were performed to investigate the effi-
ciency of the method in the separation of the ethanol extracts from A. paniculata. Results show that
overlapping elution–extrusion CCC is an efficient method to prepare a cytotoxic natural diterpenoid combination
of 14-deoxy-andrographolide and 14-deoxy-11,12-didehydroandrographolide with the molar
ratio of 1:2 as well as andrographolide using an optimized solvent system composed of hexane–ethyl
acetate–ethanol–water (5:5:4:6, v/v) with an on-demand solvent preparation mode. All components
obtained showed potent cytotoxic activity against human hepatocellular liver carcinoma cells HepG2 and
doxorubicin-resistant R-HepG2 cells. Molecular structures have been identified by electrospray ionizationmass
spectrometry (ESI-MS), electrospray ionizationtime-of-flightmass spectrometry (ESI-TOF-MS),
one- and two-dimensional nuclear magnetic resonance (1D- and 2D-NMR). The method appears to be
very useful for the high-throughput purification of natural products