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Comprehensive off-line CCC x LC-DAD-MS separation of Cyclopia pubescens Eckl. & Zeyh. phenolic compounds and structural elucidation of isolated compounds
JournalArticle (Originalarbeit in einer wissenschaftlichen Zeitschrift)
ID
4613335
Author(s)
Walters, Nico A.; de Beer, Dalene; de Villiers, André; Danton, Ombeline; Hamburger, Matthias; Joubert, Elizabeth
Comprehensive off-line CCC x LC-DAD-MS separation of Cyclopia pubescens Eckl. & Zeyh. phenolic compounds and structural elucidation of isolated compounds
Chromatography, Liquid; Chromatography, Reverse-Phase; Countercurrent Distribution; Gas Chromatography-Mass Spectrometry; Holoprosencephaly
Abstract
Introduction The minor phenolic constituents ofCyclopia pubescensEckl. & Zeyh. are unknown and one dimensional (1D) liquid chromatography (LC) is unable to provide sufficient separation. Methodology A two-dimensional (2D) LC method incorporating normal-phasehigh performance countercurrent chromatography (NP-HPCCC) in the first dimension (D-1) and reversed-phase ultra-high-performance liquid chromatography (RP-UHPLC) as the second dimension (D-2) was developed. The analytical HPCCC method was subsequently scaled up to semi-preparative mode and fractions pooled based on phenolic sub-groups. The phenolic compounds in selected fractions were subsequently isolated using RP-HPLC on a C(18)column. Isolated compounds were identified by nuclear magnetic resonance (NMR) spectroscopy. The absolute configurations of compounds were determined by optical rotation and electronic circular dichroism spectra. Sugars were identified by gas chromatography-mass spectrometry (GC-MS) analysis. Results The comprehensive off-line 2D CCC x LC method gave a good spread of the phenolic compounds. Orthogonality calculated using both the convex hull and conditional entropy methods were 81%. High-resolution mass spectrometric fragmentation spectra obtained from a quadrupole-time-of-flight instrument and ultraviolet-visible (UV-vis) spectral data were used to (tentatively) identify 32 phenolic compounds from the analytical CCC fractions. Of the seven isolated compounds, (2S)-5-O-[alpha-l-rhamnopyranosyl-(1 -> 2)-beta-d-glucopyranosyl]eriodictyol (3) and (2S)-5-O-[alpha-l-rhamnopyranosyl-(1 -> 2)-beta-d-glucopyranosyl]-5,7,3 ',4 '-tetrahydroxyflavan (4) were newly identified in all plants. The other isolated compounds were identified as (2S)-5-O-[alpha-l-rhamnopyranosyl-(1 -> 2)-beta-d-glucopyranosyl]naringenin (1),R-neo-eriocitrin (2), 3-O-alpha-l-arabinopyranosyl-3,4-dihydroxybenzoic acid (5), 4-O-beta-d-glucopyranosyl-Z-4-hydroxycinnamic acid (6) and 4-(4 '-O-beta-d-glucopyranosyl-4 '-hydroxy-3 '-methoxyphenyl)-2-butanone (7). Conclusions Among the 32 compounds (tentatively) identified, only six were previously identified inCyclopia pubescensusing 1D LC. Most of the isolated compounds were also identified for the first time inCyclopiaspp., improving the knowledge of the minor phenolic compounds of this genus.
