DPPH-HPLC-DAD analysis combined HSCCC for screening and identification of radical scavengers in Cynomorium songaricum Rupr.

Abstract

A combinative method using the 2,2-diphenyl-1-picrylhydrazyl (DPPH)-high performance liquid chromatography (HPLC)-diode array detector (DAD) and high-speed counter-current chromatography (HSCCC) has been developed to screen and separate radical scavengers from the water extract of Cynomorium songaricum Rupr. Under the target guidance of the DPPH-HPLC-DAD experiment, 3 compounds were found to possess potential antioxidant activities. In order to identify the chemical structures of those compounds, the HSCCC method with a two-phase solvent system composed of ethyl acetate–water (1 : 1; v/v) was developed to isolate and purify the active compounds. Three compounds, 14.3 ± 0.7 mg of (+)-catechin (1), 15.2 ± 0.6 mg of (−)-epicatechin (2) and 11.3 ± 0.4 mg oleuropein (3) were obtained from 600 mg of crude sample with purities of 97.8%, 98.3% and 95.6%, respectively, as determined by HPLC. Their structures were elucidated by electrospray ionization-mass spectrometry (ESI-MS), ¹H nuclear magnetic resonance (NMR) and ¹³C NMR. Among them, oleuropein was obtained from C. songaricum for the first time. The peak order of the three compounds in the HSCCC chromatogram was completely different from that in the HPLC chromatogram. Even both HSCCC and HPLC methods that are employed in this paper were in reversed phase mode. The elution order of HSCCC was determined by the hydrophobicity of the compounds, while the elution order of HPLC may be affected by some other interactions between the stationary phase and the compounds such as hydrogen bonding and steric effects. In addition, antioxidant activities of the three compounds were evaluated by the methods of DPPH radical scavenging assay. All of them showed high radical scavenging activities with the EC₅₀ values being 16.97 ± 0.02, 11.15 ± 0.09 and 41.32 ± 0.20 μg mL⁻¹ for (+)-catechin, (−)-epicatechin and oleuropein, respectively.