Preparative separation of eight phenolic acids from L. Moench using pH-zone-refining counter-current chromatography and evaluation of their immunomodulatory effects and synergistic potential.

Phenolic acids headed by cichoric acid in L. Moench show remarkable immunomodulatory effects. In this study, pH-zone-refining counter-current chromatography with a two-phase solvent system composed of EtOAc-ACN-HO (4 : 1 : 5, v/v/v) (TFA (10 mM) in the upper phase and NH·HO (30 mM) in the lower phase) was applied to the pre-segmentation enrichment of 3.5 g of a crude sample of L. Moench. Then two fractions of highly concentrated samples were further separated using EtOAc-HO (1 : 1, v/v) and EtOAc--BuOH-ACN-HO (3 : 1 : 1 : 5, v/v/v/v) solvent systems (TFA (10 mM) in the upper phase and NH·HO (10 mM) in the lower phase), separately. As a result, eight phenolic acids including cichoric acid were isolated successfully. Moreover, the immunomodulatory effects of the isolated compounds and the synergy of the binary and ternary compound combinations were first studied using CompuSyn software. And the ternary combination (caffeoyl tartaric acid + feruloyl tartaric acid + cichoric acid) showed a 94.91% synergistic inhibition rate of NO production released by LPS-induced RAW 264.7 cells. This study developed a strategy for the rapid preparative separation and purification of phenolic acids with complex components and large differences in content and provided a theoretical basis for the synergistic use of monomeric compounds in L. Moench.