Elution-extrusion counter-current chromatography separation of five bioactive compounds from Dendrobium chrysototxum Lindl

The elution-extrusion counter-current chromatography (EECCC) method was firstly developed by Berthod in 2003 and has been used in natural products separation in recent years. The advantages of this method have been well documented such as reducing the separation time and solvent consumption. In the EECCC method, the time point of the extrusion step is very important during the whole separation process as it directly affects the resolutions, separation time and solvent consumption. However, how to choose a suitable time point to perform the extrusion step without decreasing the resolution has not been studied yet. In the present study, a strategy for systematically calculating the time point for extrusion was developed in theory and five bioactive compounds from the extract of Dendrobium chrysototxum Lindl. were separated and compared using normal CCC and EECCC method. Our results demonstrated that the accurate time point to perform the extrusion could be calculated and reduced both separation time and solvent consumption without losing separation performance. Using this EECCC method, five bioactive compounds were separated and purified with high purity. The separation time and solvent consumption were decreased from 200 min to 100 min and 5-2.5L during the separation process while the resolutions were still acceptable. Finally, 63 mg, 48 mg, 97 mg, 162 mg and 43 mg of hydroxyl phenanthrenes and bibenzyls with the purity of 98.7%, 98.0%, 98.2%, 99.0% and 98.7%, respectively were isolated from 1.2 g crude extract of D. chrysototxum Lindl. initially purified by column chromatography in one step separation. The purities of compounds were determined by HPLC. Their structures were identified by electrospray ionization-mass spectrometry (ESI-MS) and NMR.