Preparative separation of seven polyphenols from Perillae Folium via pH-zone-refining counter-current chromatography combined with high-speed counter-current chromatography

Abstract

Perillae Folium is a well-known traditional Chinese medicine, and it possesses anti-inflammatory, anti-oxidant, and hypolipidemic effects. The pharmacological properties of Perillae Folium are based on its main functional compositions, such as phenolic acids, flavonoids, and volatile oils. In this study, seven polyphenols, including three phenolic acids and four flavonoid glycosides, were successfully isolated from Perillae Folium by pH-zone-refining counter-current chromatography (pH-ZRCCC) combined with traditional high-speed counter-current chromatography (HSCCC). First, the crude sample was separated by pH-ZRCCC using a biphasic solvent system composed of pet ether–ethyl acetate–acetonitrile–water (1 : 3 : 1 : 5, v/v). The upper phase of the biphasic solvent system added trifluoroacetic acid (10 mM) as the stationary phase, and the lower phase added ammonia water (30 mM) as the mobile phase. In this separation, one compound, rosemary acid (I), with high purity and a mixture were obtained. The mixture was further separated using HSCCC with a ethyl acetate–n-butanol–water (4.8 : 0.2 : 5, v/v) solvent system to obtain apigenin-7-O-[β-D-glucuronopyranosyl (1→2)-O-β-D-glucuronopyranoside] (II), luteolin-7-O-β-D-glucuronide (III), 4-hydroxycinnamic acid (IV), scutellarin (V), caffeic acid (VI), and apigenin-7-O-β-D-glucuronide (VII). The purities of the obtained compounds were above 92.7%. The study demonstrated that the combination of pH-ZRCCC and HSCCC is an effective method for the preparation and separation of polyphenols, particularly the complex mixture of phenolic acids and flavonoids from natural products.