A separation method using counter current chromatography coupled with an evaporative light-scattering detection system was developed to purify five triterpenoid saponins from the roots of Bupleurum falcatum. The methanol extract was loaded onto a Diaion ® HP20 column and fractionated by a methanol and water gradient elution. The saikosaponin-enriched fraction was obtained by elution with 100% methanol. The two-phase solvent systems used for separation were composed of chloroform/methanol/isopropanol/water at a volume ratio of 60:60:1:60 and 6:6:1:6. The relationship between the isopropanol ratio of each phase and the partition coefficients of the target compounds was investigated by calculating partition coefficient by high-performance liquid chromatography and measuring the accurate composition of each phase by 1H NMR spectroscopy. Each fraction obtained was collected and dried, which yielded the following five saikosaponins from 700 mg of injected sample: saikosaponin B1 (8.7 mg), saikosaponin A (86 mg), saikosaponin B3 (17 mg), saikosaponin B2 (41 mg), and saikosaponin C (33 mg). Saikosaponin A showed the most potent cytotoxicity against human cancer cells (gastric cancer, AGS cells; breast cancer, MCF-7 cells; and hepatoma, HepG2 cells) after 24 h. The IC50 values for the above three cell types were 34.6, 33.3, and 23.4 μmol/L, respectively.
- Bupleurum falcatum
- Counter current chromatography
- Solvent compositions