Evaluation of different solvent systems for the isolation of Sparattosperma leucanthum flavonoids by counter-current chromatography

Three flavonoids – 2′,4′,6′-trihydroxy-4′-O-β-d-glucopiranosyl dihydrochalcone, 1, pinocembrin-7-O-(-neohesperidoside, 2 and pinocembrin-7-O-(-(6″-O-acetyl) neohesperidoside, 3 – were successfully isolated from the EtOAc extract of leaves of Sparattosperma leucanthum (Vell.) K. Schum (Bignoniaceae) using a two-step counter-current chromatography (CCC). Two different CCC machines were used, with different column axes (P.C. Inc., vertical orientation axis and AECS Quattro HTPrep, horizontal orientation axis). Detailed studies of flavonoids behaviour in several solvent systems made possible the use of the best system for their isolation. HEMWat and its modifications – exchange of alcohol and addition of a fifth solvent – were tested for isolation of the three compounds in a single run, but good K and α values were not achieved. So, HEMWat 4:10:4:10, with upper phase as mobile, was used to isolate compound 3. A mixture of compounds 1 and 2 was recovered and submitted to a new CCC fractionation using a more polar solvent system: EBuWat 8:2:10, upper phase as mobile. Butironitrile–acetonitrile–water (BuCN–ACN–H2O) 5:10:10, upper phase as mobile, was also used for the isolation of the mixture containing compounds 1 and 2, in order to increase the solubility of the compounds in the CCC solvent system. It is the first time that the system BuCN–ACN–H2O is described in literature.