Stepwise Elution of a Three-phase Solvent System in Centrifugal Partition Extraction: A New Strategy for the Fractionation and Phytochemical Screening of a Crude Bark Extract

摘要

Introduction - Tree bark represents an interesting source of bioactive molecules for the discovery of new pharmaceutical agents. However, the detailed screening of secondary metabolites in crude bark extracts is often hampered by the presence of tannins, which are difficult to separate from other plant constituents. Objective - In the present study, a new centrifugal partition extraction (CPE) method was developed in order to fractionate a crude bark extract of Anogeissus leiocarpus Guill. & Perr. (Combretaceae). Methods - A three-phase solvent system composed of n-heptane, methyl tert-butyl ether, acetonitrile and water was optimised for the stepwise elution at 20 mL/min of different phytochemical classes according to their hydrophobicity. Onedimensional and two-dimensional NMR analyses of the simplified fractions were then performed in order to characterise potentially interesting metabolites. Results - In one step, 5 g of the initial crude extract were efficiently fractionated to yield highly simplified fractions that contained triterpenes, ellagic acid derivatives, flavonoids and phenolic compounds. All undesired compounds, that is, the highly abundant water-soluble tannins (78.8%), were totally removed and each run was rapidly achieved in 90min on a the multi-gram scale and with low solvent volumes. Conclusion - Centrifugal partition extraction in the elution mode using a three-phase solvent system can thus be proposed as an efficient and cost-effective alternative for a rapid fractionationof crude bark extracts and for an effective screening of potentially active secondary metabolites.