Development of advanced chromatographic techniques for the in-depth phenolic profiling of rooibos

[摘要] ENGLISH ABSTRACT: Rooibos (Aspalathus linearis) is a South African fynbos plant with known health-promotingproperties, consumed mainly as a herbal tea. The health-promoting properties of rooibos areassociated with its phenolic composition. During herbal tea production, the plant material is'fermented, which reduces the phenolic content. This led to development of unfermented(green) rooibos tea with increased phenolic content. Conventionally the phenolic compoundsare quantitatively and qualitatively analysed using reversed phase (RP) high performanceliquid chromatography (HPLC) with diode array detection (DAD) and mass spectrometry(MS).This thesis reports the design of a validated quantitative RP-HPLC-DAD method toenable quantification of four eriodictyol-glucopyranoside isomers that have not beenpreviously quantified in rooibos. External authentic reference standards were used to identifyand quantify phenolic compounds with the help of MS, which also confirmed peak purity forthe quantified compounds. The plant material of 10 rooibos plants were sub-divided beforeprocessing prepare green, semi-fermented and fermented products from each. Mixtures ofacetonitrile and ethanol with water (0, 20, 40, 60, 80 and 100%) were evaluated for maximalextraction of phenolics from the plant material. Extracts prepared with 40% acetonitrilerepresenting maximal extraction from the plant material, as well as water extracts (foodingredient extracts), were analysed. For the first time aspalathin was quantified in rooiboswith its known degradation products, the eriodictyol-glucopyranoside isomers, iso-orientinand orientin. In addition, a phenylpropanoid and eight other phenolic compounds were alsoquantified.Complex natural samples such as rooibos contain a range of phenolic compounds,some of which remain unidentified due to challenges in their separation. This led to thedevelopment of a comprehensive two dimensional (2D) separation technique to gain indepthqualitative information on the phenolic composition of rooibos. Normal phase (NP)high performance countercurrent chromatography (HPCCC) was used to develop the firstdimension (1D) separation. A gradient elution using an ethyl acetate, n-butanol, watersolvent system was used to separate the phenolic compounds followed by an extrusion step(60 min analysis time, 48 fractions collected). The second dimension (2D) separation usedultra (U)HPLC to ensure rapid analysis and maximum efficiency. The 2D separation methodwas developed from the quantitative method with further development aimed at obtaining ahigh practical peak capacity in a reasonable analysis time. The practical peak capacity wasdetermined as a function of the 2D flow-rate and gradient time, as well as the 1D fractioncollection time. The off-line NP-HPCCC×RP-UHPLC method was applied to green andfermented rooibos samples. DAD was used to construct contour plots to elucidate quantitative and qualitative differences, while MS detection was used for tentativeidentification of previously unidentified compounds. A total of 39 compounds, 18 of whichwere not previously identified in rooibos, were identified using MS detection in positiveionisation mode. Most of the newly identified compounds were very polar. The combinationof NP-HPCCC with RP-UHPLC separations was characterised by a high degree oforthogonality (~80%), contributing to a high practical peak capacity (3293) and improvedseparation of especially the novel polar phenolic compounds.

[发布机构] Stellenbosch University