Preparative separation and purification of four cis–trans isomers of coumaroylspermidine analogs from safflower by high-speed counter-current chromatography

• An HSCCC method for isolation and purification in this research has been created.
• Four cis–trans isomers of coumaroylspermidine analogs from Safflower have been successfully isolated and purified.
• A two-phase solvent system composed of chloroform, methanol and water (1:1:1, v/v/v) was used in research.
• Four components were obtained in one-step separation within 210 min.
• The purities of these four components are 95.5%, 98.1%, 97.5% and 96.2%, respectively.

High-speed counter-current chromatography (HSCCC) was successfully applied for the first time to isolate and purify four cis–trans isomers of coumaroylspermidine analogs from Safflower. HSCCC separation was achieved with a two-phase solvent system composed of chloroform–methanol–water (1:1:1, v/v/v) with the upper phase as the mobile phase. In a single run, a total of 1.3 mg of N1, N5, N10-(E)-tri-p-coumaroylspermidine (EEE), 4.4 mg of N1(E)-N5-(Z)-N10-(E)-tri-p-coumaroylspermidine (EZE), 7.2 mg of N1(Z)-N5-(Z)-N10-(E)-tri-p-coumaroylspermidine (ZZE), and 11.5 mg of N1,N5,N10-(Z)-tri-p-coumaroylspermidine (ZZZ) were obtained from 100 mg of crude sample. High Performance Liquid Chromatography (HPLC) analysis showed that the purities of these four components are 95.5%, 98.1%, 97.5% and 96.2%, respectively. The chemical structures were identified by ESI-MS, 1H NMR and 13C NMR.