Bioassay‐guided separation and identification of the anticancer composition from Curcuma longa L. by the combination strategy of methanol gradient countercurrent chromatography and ultra‐high‐performance liquid chromatography coupled with high‐resolution mass spectrometry

In this study, a combined strategy with methanol gradient countercurrent chromatography and ultra-high-performance liquid chromatography coupled with high-resolution mass spectrometry was introduced for the fractionation and identification of active constituents from Curcuma longa L. The gradient countercurrent chromatography separation was performed using the heptane-ethyl acetate-methanol-water (5:5:2:8, v/v) solvent system, in which the lower phase and methanol were used as the mobile phases. Constituents of turmeric with large partition coefficients were well resolved. Subsequent cytotoxicity analysis showed that the fractions 10, 11, 12, and 15 expressed significantly higher cytotoxicity against B16 mouse melanoma than the other fractions. Four compounds with potent activity, curcumin, demethoxycurcumin, and bisdemethoxycurcumin from fraction 11 and galanal A from fraction 15, were purified, and the half-maximal inhibitory concentrations were 18.5 ± 1.3, 7.8 ± 0.4, 20.4 ± 1.3, and 14.1 ± 0.8 μM, respectively. Ultra-high-performance liquid chromatography coupled with high-resolution mass spectrometry was then applied for compound identification from fraction 10 with constituents in very low content. A total of 14 diarylheptanoids were identified, which are supposed to be cytotoxic constituents. It proved that the strategy based on the combination of bioassay-guided methanol gradient countercurrent chromatography separation and ultra-high-performance liquid chromatography coupled with tandem mass spectrometry-assisted peak identification could be an efficient method for natural product screening and new drug discovery.