Overlapping elution–extrusion counter-current chromatography: A novel method for efficient purification of natural cytotoxic andrographolides from Andrographis paniculata

• Published in: Journal of Chromatography A 2012-02, Vol.1223, p.53-63
• Main Authors: Wu, Dingfang, Cao, Xiaoji, Wu, Shihua
• Format: Article
• Language: English
• Subjects: aerial parts; Andrographis paniculata; Andrographolides; anti-inflammatory activity; anticarcinogenic activity; carcinoma; chemical structure; countercurrent chromatography; cytotoxicity; diterpenoids; electrospray ionization mass spectrometry; ethanol; extrusion; human cell lines; humans; hydrophobicity; ionization; liver; Natural products; nuclear magnetic resonance spectroscopy; Oriental traditional medicine; Overlapping elution–extrusion counter-current chromatography; solvents; Sweep-elution; Traditional Chinese Medicine
• ISSN: 0021-9673
• DOI: 10.1016/j.chroma.2011.12.036

► We have developed a novel overlapping elution–extrusion CCC method. ► Its theory was established and used for predictable isolation. ► Andrographolides from Andrographis paniculata were successfully isolated by the method. ► The overlapping elution–extrusion CCC is a predictable and economic process. ► A general protocol for natural product isolation was proposed. 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 efficiency 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 ionization mass spectrometry (ESI-MS), electrospray ionization time-of-flight mass 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.