Separation of triterpenoid saponins from the root of Bupleurum falcatum by counter current chromatography: The relationship between the partition coefficients and solvent system composition

Abstract

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 (1) H 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.

Isolation and purification of two antioxidant isomers of resveratrol dimer from the wine grape by counter‐current chromatography

“…To obtain an efficient separation of target compounds, the previous articles on CCC demonstrated that the two‐phase solvent system should meet the following requirements: (i) sufficient sample solubility (<30 s); (ii) short settling time; (iii) appropriate partition coefficient ( K ) values (usually between 0.5 and 2); (iv) no decomposition or denaturation of the sample; and (v) satisfactory retention of the stationary phase. According to the previous reports, two‐phase solvent systems composed of n ‐hexane/ethyl acetate/methanol/water and light petroleum/ethyl acetate/methanol/water were widely used and also very efficient for CCC separation of polyphenol. The two‐phase solvent system was selected according to the K values of each target compound.…”

Caspase-4 is essential for saikosaponin a-induced apoptosis acting upstream of caspase-2 and γ-H2AX in colon cancer cells

“…Previously, our group found that SSa could induce the activation of various caspases and poly (ADP-ribose) polymerase (PARP), decrease Bcl-2 and X-linked inhibitor of apoptosis (XIAP) expression, and induce apoptosis in human colon carcinoma (HCC) cells [ 14 ]. In addition, SSa has been shown to have cytotoxicity against human gastric cancer, breast cancer, and hepatoma [ 15 - 18 ].…”

Application of linear gradient elution in countercurrent chromatography for the separation of triterpenoid saponins from the roots of Pulsatilla koreana Nakai

“…Effective CCC separation depends on the partition coefficient ( K D ) of each solute, and the K D values of the solutes depend on the selection of a suitable biphasic solvent system . Solvent mixtures of chloroform/isopropanol/methanol/water have frequently been used as the solvent system for the separation of saponins . Table shows the K D values and the settling time of each solute for solvent systems 1 to 6 as well as the phase ratio of the solvent system when the composition of the solvent system is varied.…”

“…Table shows the K D values and the settling time of each solute for solvent systems 1 to 6 as well as the phase ratio of the solvent system when the composition of the solvent system is varied. The addition of isopropanol afforded high K D values and prolonged the separation time of the compounds . However, an increase in isopropanol ratio caused a decrease in stationary phase retention, which is indicated by the increased settling time of the bi‐phase.…”