Online isolation and purification of four phthalide compounds from Chuanxiong rhizoma using high-speed counter-current chromatography coupled with semi-preparative liquid chromatography

The phthalide compounds of Chuanxiong rhizoma including senkyunolide A, levistolide A, Z-ligustilide and 3-butylidenephthalide, have been reported as the biologically active compounds because of their therapeutic effects. In this work, online high-speed counter-current chromatography coupled with semi-preparative liquid chromatography instrument was set up, and online separation of the four compounds has been simultaneously achieved using this instrument. In this study, using all the selected solvent system, Z-ligustilide and 3-butylidenephthalide were eluted in one peak by high-speed counter-current chromatography. Using high-speed counter-current chromatography with a solvent system of n-hexane–ethyl acetate–methanol–water–acetonitrile (8:2:5:5:5, v/v), 3.6 mg of senkyunolide A (94.4%) and 3.0 mg of levistolide A (95.3%) were obtained from 100 mg of the crude extract. Coeluted Z-ligustilide and 3-butylidenephthalide peak fraction (8 mL) from high-speed counter-current chromatography was directly transferred and injected to the semi-preparative liquid chromatography for further separation. Finally, 5.6 mg of Z-ligustilide (97.5%) and 4.8 mg of 3-butylidenephthalide (99.3%) were obtained. The identification of these four compounds was performed by quadrupole time-of-flight mass spectrometer, 1H and 13C nuclear magnetic resonance spectrometer.

► Online separation instrument was set up using a six-way valve for the first time.
► Online separation instrument is counter-current chromatography coupled with HPLC.
► Online separation of the four compounds has been achieved using this instrument.