Development of HPLC method for Cyclopia subternata phenolic compound analysis and application to other Cyclopia spp.

Abstract

Analysis of phenolic compounds in Cyclopia spp. (honeybush) is very important because of commercial interest in the plant material as an herbal tea and in extracts for the nutraceutical and cosmeceutical markets. An existing HPLC-DAD method was adapted and proved reproducible as evidenced by good intra- and inter-day precision. The method allows the quantification of several well-known as well as unidentified phenolic compounds. Its suitability for quantification of the major phenolic compounds present in the water extracts of unfermented and fermented Cyclopia subternata, Cyclopia intermedia, Cyclopia genistoides and Cyclopia sessiliflora was evaluated. Known compounds that were identified and quantified include mangiferin (0.1–11.8 g/100 g), isomangiferin (0.1–3.2 g/100 g), eriocitrin (not detected–0.4 g/100 g), hesperidin (0.2–1.2 g/100 g) and scolymoside (not detected–1.8 g/100 g), while narirutin and luteolin were detected in most samples, but could not be quantified due to low concentrations and co-elution. An unidentified compound (traces–4.8 g hesperidin equivalents/100 g), unidentified hydroxycinnamic acid derivative (not detected–0.3 g mangiferin equivalents/100 g), unidentified flavanone (not detected–2.0 g hesperidin equivalents/100 g) and an eriodictyol-glycoside (not detected–0.5 g hesperidin equivalents/100 g) could also be quantified. Large variation was observed within a sample series of the same extract type due to the nature of the plant material. Qualitative and quantitative differences were observed between species, while “fermentation”, a high-temperature process involving non-enzymatic oxidation, decreased the content of most phenolic compounds. Results using DAD as the only detection method should be handled with care, as the tentative identification of compounds 9 and 10, based on retention time and UV–Vis spectrum, could not be confirmed using liquid chromatography–electrospray ionisation-mass spectrometry.