Development of an efficient fractionation method for the preparative separation of sesquiterpenoids from Tussilago farfara by counter-current chromatography

A novel application of counter-current chromatography (CCC) to enrich plant extracts using direct and continuous injection (CCC-DCI) was developed to fractionate sesquiterpenoids from the buds of Tussilago farfara L. In this study, an n-hexane-acetonitrile-water (HAcW) solvent system was separately pumped into the CCC column, and an extraction solution (45% acetonitrile) was directly and continuously injected into the CCC column. Since the extraction solution was used as a mobile phase in this method, solvent consumption could be greatly reduced. To enrich the extraction solution (315.9 g/5.4 L), only 4.2 L water, 4.6 L acetonitrile, and 1.2 L n-hexane were used, including the extraction step. Finally, 6.8 g of a sesquiterpenoid-enriched (STE) fraction was obtained from the crude extract (315.9 g) of Tussilago farfara (1 kg) in a single CCC run with a separation time of 8.5 h. The sample injection capacity of CCC-DCI was greater than 300 g; this amount of sample could not be handled in conventional CCC or other fractionation methods with the same column volume. Moreover, three major sesquiterpenoids (1: tussilagone, 2: 14acetoxy-7 beta-(3'-ethyl cis-crotonoyloxy)-1 alpha-(2'-methylburyryloxy)-notonipetranone, and 3: 7 beta-(3'-ethyl cis-crotonoyloxy)-1 alpha-(2'-rnethylburyryloxy)-3,14-dehydro-Z-notonipetranone) were purified from the STE fraction by CCC, and their chemical structures were elucidated by H-1 NMR and C-13 NMR. A quantification study was conducted, and the contents of compounds 1-3 in the CCC-DCI fraction were higher than those of conventional multi-step fractionations performed in series: solvent partitioning and open column chromatography. Furthermore, the average CCC-DCI recoveries were 96.1% (1), 96.9% (2), and 94.6% (3), whereas the open column chromatography recoveries were 77.7% (1), 66.5% (2), and 58.4% (3). The developed method demonstrates that CCC is a useful technique for enriching target components from natural products. (C) 2017 Elsevier B.V. All rights reserved.