Use of countercurrent chromatography during isolation of 6-hydroxyluteolin-7-O-β-glucoside, a major antioxidant of Athrixia phylicoides.

Athrixia phylicoides, an indigenous South African herbal tea, has potential as a source of nutraceutical antioxidant extracts. Countercurrent chromatography (CCC) was employed as part of a multi-step process to isolate one of the major antioxidant compounds in A. phylicoides extracts. Antioxidant activity of the extracts was comparable to commercial nutraceutical extracts from Aspalathus linearis and Cyclopia spp. in a range of assays. The extracts were tested for radical scavenging (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) di-ammonium radical cation (ABTS·⁺) scavenging, 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) scavenging and oxygen radical absorbance capacity (ORAC)), ferric reducing antioxidant potential (FRAP) and iron chelating activity, as well as inhibition of microsomal lipid and linoleic acid emulsion oxidation. After extraction optimisation, the antioxidant activity of the major phenolic compounds in an A. phylicoides extract was determined using the on-line HPLC-diode-array-DPPH and -ABTS·⁺ radical scavenging assays. Major compounds reported for the first time included chlorogenic acid, 1,3-dicaffeoylquinic acid, several hydroxycinnamic acid derivatives, including dicaffeoyl quinic acids, and an unidentified flavone-hexose. Finally, CCC was used in conjunction with liquid-liquid partitioning and semi-preparative reversed-phase HPLC to isolate 6-hydroxyluteolin-7-O-β-glucoside (a major antioxidant) and quercetagetin-7-O-β-glucoside (a minor compound present in CCC fraction containing 6-hydroxyluteolin-7-O-β-glucoside) from an A. phylicoides extract. The chemical structures of the isolated compounds were confirmed by LC high-resolution electrospray ionisation MS, as well as ¹H, ¹³C and 2D NMR spectroscopy. This is the first report of the isolation of these compounds from A. phylicoides.