Rapid Isolation and Purification of Phorbol Esters from Jatropha curcas by High-Speed Countercurrent Chromatography

In this work, a high-speed countercurrent chromatography (HSCCC) method was established for the preparation of phorbol esters (PEs) from Jatropha curcas. n-Hexane–ethyl acetate–methanol–water (1.5:1.5:1.2:0.5, v/v) was selected as the optimum two-phase solvent system to separate and purify jatropha factor C₁ (JC1) with a purity of 85.2%, as determined by HPLC, and to obtain a mixture containing four or five PEs. Subsequently, continuous semipreparative HPLC was applied to further purify JC1 (99.8% as determined by HPLC). In addition, UPLC–PDA and UPLC–MS were established and successfully used to evaluate the isolated JC1 and PE-rich crude extract. The purity of JC1 was only 87.8% by UPLC–UV. A peak (a compound highly similar to JC1) was indentified as the isomer of JC1 by comparing the characteristic UV absorption and MS spectra. Meanwhile, this strategy was also applied to analyze the PE-rich crude extract from J. curcas. It is interesting that there may be more than 15 PEs according to the same quasi-molecular ion peaks, highly similar sequence-specific fragment ions, and similar UV absorption spectrum.