Preparative separation of anti-oxidative constituents from Rubia cordifolia by column-switching counter-current chromatography.

ABSTRACT

An effective column-switching counter-current chromatography (CCC) protocol combining stepwise elution mode was successfully developed for simultaneous and preparative separation of anti-oxidative components from ethyl acetate extract of traditional Chinese herbal medicine Rubia cordifolia. The column-switching CCC system was interfaced by a commercial low-pressure six-port switching valve equipped with a sample loop, allowing large volume introduction from the first dimension (1st-D) to the second dimension (2nd-D). Moreover, to extend the polarity window, three biphasic liquid systems composed of n-hexane/ethyl acetate/methanol/water (1212, 2323, 5656 v/v) were employed using stepwise elution mode in the 1st-D. By valve switching technique the whole interested region of 1st-D could be introduced to second dimension for further separation with the solvent system 5546 v/v. Using the present column-switching CCC protocol, 500 mg of crude R. cordifolia extract were separated, producing milligram-amounts of four anti-oxidative components over 90% pure. Structures of purified compounds were identified by (1)H and (13)C NMR.