Results of our previous studies indicate that the ether fraction of
High-speed countercurrent chromatography (HSCCC) is a liquid-liquid partition chromatographic technique that has recently gained increased interest. This technique relies on the use of centrifugal force for the retention of the stationary phase. HSCCC is more advantageous than other liquid-liquid techniques because of the shorter separation time, wider range of possible solvent systems, and quantitative material recovery (Marston and Hostettmann, 1994, 2006; Ito, 2005; Friesen
and Pauli, 2007). These technical advantages have resulted in extensive use of HSCCC for the preparative separation of various natural products (Tsao and Deng, 2004; Wang et al., 2007).
In this study, we report for the first time one-step isolation and purification of fucosterol from a crude extract of
Methylene chloride,
for the preparation of crude extract and HSCCC separation. The sample was stored at -18℃ until required for experiments.
Preparative HSCCC was performed using a Model CCC- 1000 multilayer coil of polytetrafluoroethylene (PTFE) tubing with an inner diameter of 1.6 mm and a total capacity of 325 mL (Pharma-Tech Research, Baltimore, MD, USA). Considering a distance of
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Preparation of crude extract
Whole
Selection of two-phase solvent systems
The composition of a two-phase solvent system was selected according to the partition coefficient (
Preparation of two-phase solvent system and sample solution
The selected solvent system consisted of
HSCCC separation procedure
The multilayer coil column was first entirely filled with the upper organic phase (stationary phase) at a flow rate of 4.0 mL/min. The lower phase was then pumped into the head end of the inlet column at a flow rate of 1.2 mL/min, while the apparatus was run at 800 rpm. After hydrodynamic equilibrium was established, as indicated by elution of a clear mobile phase at the tail outlet, the sample solution (50 mg in 3 mL of each phase) was injected into the separation column through the injection valve. The effluent from the tail end of the column was monitored continuously by connection to the tail outlet of the coiled column with the evaporative light-scattering detection (ELSD) system through a split valve. The ELSD system was set to a probe temperature of 90℃ and a gain of 1.0, and the nebulizer gas nitrogen adjusted to 1.7 bars. Each peak fraction was collected according to the elution profile and assessed by HPLC. After separation was complete, retention of the stationary phase was measured by collecting the column contents; these were forced out of the column using pressurized nitrogen gas.
HPLC analyses and identification of fractioned peak
The crude extract and each peak fraction from HSCCC were analyzed by HPLC. The analyses were performed with an Agilent Eclipse XDB C18 column (150×4.6 mm I.D.) at a column temperature of 30℃. The mobile phase, consisting of acetonitrile-methanol (70:30, v/v) was isocratically eluted at a flow rate of 0.8 mL/min using the ELSD detector.
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Structural identification of fucosterol
IR (KBr): 3,439, 2,936, 1,626, 823 cm-1. Mass (EI, 70 eV):
1H NMR (400 MHz, CDCl3): 5.36 (1H, br. d,
13C-NMR (100 MHz, CDCl3): 146.9 (C-24), 140.7 (C-5), 121.6 (C-6), 115.5 (C-28), 71.7 (C?3), 56.7 (C-14), 55.7 (C- 17), 50.1 (C-9), 42.3 (C-13), 42.2 (C-4), 39.7 (C-12), 37.2 (C-1), 36.5 (C-10), 36.4 (C-20), 35.2 (C-22), 34.7 (C-25), 31.8 (C-7,8), 31.5 (C-2), 28.2 (C-16), 25.6 (C-23), 24.3 (C- 15), 22.2 (C-26), 22.1 (C-27), 21.0 (C-11), 19.4 (C-19), 18.7 (C-21), 131.1 (C-29), 11.8 (C-18).
Successful separation by HSCCC depends largely on the selection of a suitable two-phase solvent system. This requires consideration of the following:the satisfactory retention of the stationary phase (Yoon, 1995), the settling time of the solvent system should be shorter than 20 s (Oh et al., 1990), the partition coefficient (
To achieve efficient resolution of the target compound, the following two-phase solvent systems were examined:
In the present study, the retention of the stationary phase was 60.1%. The retention of the stationary phase relative to the total column capacity was measured from the volume of the stationary phase collected after complete separation. Our
[Table 1.] The partition coefficient values of fucosterol in different solvents(x) systems
The partition coefficient values of fucosterol in different solvents(x) systems
results indicate that
Fig. 3A shows the results of fucosterol separation by HSCCC from the crude
Thus, we developed an effective method for the preparative separation of fucosterol from