Off-line multidimensional countercurrent chromatography in the isolation and purification of chemical constituents from the ethanol extract of Brazilian green propolis

Abstract

Multidimensional countercurrent chromatography (MDCCC) is a versatile technique that is increasingly being used for the purification of compounds in natural products complex matrices. Off-line MDCCC, even if more time-consuming, can be quite useful for exploratory phytochemical work. The ethanol extract of green propolis (EEGP), bee product produced by Apis mellifera L. bees, was initially screened by liquid chromatography coupled to time-of-flight mass spectrometry (LC/Q-TOF-MS) for its dereplication aiming at better understanding its chemical composition, as well as assisting further solvent system selection for its countercurrent chromatography (CCC) fractionation. EEGP was first fractionated (CCC1) using an aqueous biphasic solvent system composed of hexane - ethyl acetate - ethanol -water (1:0.8:1:1, v/v) in normal isocratic elution mode, affording artepillin C and p-coumaric acid as pure compounds and the isomers capillartemisin A/B and aromadendrin - 4'-O-methyl ether/dihydrokaempferide. Off-line second dimension 2D-CCC (CCC2) fractionation of a non-polar fraction with a non-aqueous solvent system (hexane - chloroform - acetonitrile, 1:0.1:1, v/v, reversed phase elution mode), combined with size exclusion chromatography on Sephadex LH-20, afforded anacardic acid 19:1 as well as other phenolic lipid derivatives and cycloartenol triterpene compounds. Further second-dimension CCC x Sephadex LH-20 on a CCC1 fraction afforded the flavonoids betuletol, kaempferide, and isosakuranetin, as well as the prenylated cinnamic acid derivative drupanin. This workflow enabled the annotation of 92 compounds in EEGP by LC/Q-TOF-MS out of which 20 were isolated by MDCCC.

Keywords: Anacardic acids; Flavonoids; Metabolic fingerprinting; Multidimensional countercurrent chromatography; Prenylated phenolic acids.