Browsing by Author "Walters, Nico Albertus"

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    Comprehensive characterisation of the phenolic composition of Cyclopia pubescens (honeybush)
    (Stellenbosch : Stellenbosch University, 2019-12) Walters, Nico Albertus; De Beer, Dalene; Joubert, Elizabeth; Williams, Paul James; Stellenbosch University. Faculty of Agrisciences. Dept. of Food Science.
    ENGLISH ABSTRACT: Cyclopia pubescens Eckl. & Zeyh, endemic to South Africa, is under threat of extinction. This threat can be negated through commercial cultivation if utilised as honeybush tea. Lack of knowledge of the phenolic composition of this Cyclopia species is a hurdle in the development of value-added products such as nutraceuticals, providing the incentive for a comprehensive investigation of the phenolic profile of C. pubescens. A reversed-phase (RP) high-performance liquid chromatography-diode-array detection (HPLC-DAD) method using a biphenyl column was developed and validated. Eight phenolic compounds, representing major compounds (3-β-D-glucopyranosyl-4-O-β-D-glucopyranosyliriflophenone, 3-β-D- glucopyranosylmaclurin, 3-β-D-glucopyranosyliriflophenone, mangiferin, isomangiferin, vicenin-2, narirutin and hesperidin), were identified and a further six compounds tentatively identified by comparison of their retention times, UV–Vis spectra and high-resolution mass spectrometric (HR-MS) characteristics with those of authentic reference standards and literature, respectively. The predominant accumulation of the compounds in the leaves or stems of C. pubescens was determined for a small number of available genotypes (n = 17), affording some indication of variation between plants in the field genebank of the Agricultural Research Council. For further elucidation of the phenolic profile, the focus fell on minor phenolic compounds. Increased separation power was required and therefore an analytical comprehensive two-dimensional (2D) separation method utilising normal-phase (NP) high- performance countercurrent chromatography-DAD (HPCCC) as the first dimension and RP ultra(U)HPLC-DAD as the second dimension. High loading capacity and selectivity of HPCCC and high selectivity and resolving power of UHPLC resulted in ca 81% orthogonality. A total of 32 compounds were (tentatively) identified by comparing their UV– Vis and HR-MS characteristics to those of reference standards and literature. The HPCCC method was subsequently upscaled to semi-preparative mode and eight phenolic compounds were isolated in adequate quantities for unambiguous structural elucidation using 2D nuclear magnetic resonance, gas chromatography-MS analysis after acid- catalysed hydrolysis of the glycoside, derivatisation and determination of optical activity. (2R)-5-O-[α-L-rhamnopyranosyl-(1→2)-β-D-glucopyranosyl]eriodictyol, (2S)-5-O-[α-L- rhamnopyranosyl-(1→2)-β-D-glucopyranosyl]eriodictyol and (2S)-5-O-[α-L-rhamnopyranosyl- (1→2)-β-D-glucopyranosyl]-5,7,3ʹ,4ʹ-tetrahydroxyflavan have not been previously reported in literature. The five other compounds include the two flavanones, (2S)-5-O-[α-L- rhamnopyranosyl-(1→2)-β-D-glucopyranosyl]naringenin and R-neo-eriocitrin, the two phenolic acids, 3-O-α-L-arabinopyranosyl-3,4-dihydroxybenzoic acid and 4-O-β-D-glucopyranosyl-cis-4-hydroxycinnamic acid, and an anisole, 4-(4ʹ-O-β-D-glucopyranosyl-3ʹ- methoxyphenyl)-2-butanone. Natural deep eutectic solvents (NADES) (n = 7) were evaluated to improve extraction efficiency and obtain a more representative phenolic profile of C. pubescens. Hot water and 40% EtOH, commonly used for extraction of Cyclopia plant material, represented the benchmark solvents. Stereoisomers were selectively extracted by the different NADES solvents. Application of an on-line HPLC-ABTS assay showed that these stereoisomers also differed in Trolox equivalent antioxidant capacity. Glutamic acid based NADES (n = 2) provided superior extraction efficiency, but low eutectic stability made these solvents unsuitable for high-throughput extraction; therefore, the most suitable NADES for extracting the phenolic compounds of C. pubescens was choline chloride:proline (CcP) (1:3, molar ratio). The optimal extraction temperature for NADES, tested over the range 40 to 90 °C, was 70 °C instead of 93 °C for water when using a 20 min extraction time. The best extraction solvent for phenolic compounds remains 40% EtOH, but NADES can be used to selectively extract phenolic compounds.
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    Development of advanced chromatographic techniques for the in-depth phenolic profiling of rooibos
    (Stellenbosch : Stellenbosch University, 2016-12) Walters, Nico Albertus; De Beer, D.; De Villiers, A. J.; Williams, Paul James; Stellenbosch University. Faculty of AgriSciences. Dept. of Food Science
    ENGLISH ABSTRACT: Rooibos (Aspalathus linearis) is a South African fynbos plant with known health-promoting properties, consumed mainly as a herbal tea. The health-promoting properties of rooibos are associated 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 compounds are quantitatively and qualitatively analysed using reversed phase (RP) high performance liquid chromatography (HPLC) with diode array detection (DAD) and mass spectrometry (MS). This thesis reports the design of a validated quantitative RP-HPLC-DAD method to enable quantification of four eriodictyol-glucopyranoside isomers that have not been previously quantified in rooibos. External authentic reference standards were used to identify and quantify phenolic compounds with the help of MS, which also confirmed peak purity for the quantified compounds. The plant material of 10 rooibos plants were sub-divided before processing prepare green, semi-fermented and fermented products from each. Mixtures of acetonitrile and ethanol with water (0, 20, 40, 60, 80 and 100%) were evaluated for maximal extraction of phenolics from the plant material. Extracts prepared with 40% acetonitrile representing maximal extraction from the plant material, as well as water extracts (food ingredient extracts), were analysed. For the first time aspalathin was quantified in rooibos with its known degradation products, the eriodictyol-glucopyranoside isomers, iso-orientin and orientin. In addition, a phenylpropanoid and eight other phenolic compounds were also quantified. 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 the development of a comprehensive two dimensional (2D) separation technique to gain indepth qualitative information on the phenolic composition of rooibos. Normal phase (NP) high performance countercurrent chromatography (HPCCC) was used to develop the first dimension (1D) separation. A gradient elution using an ethyl acetate, n-butanol, water solvent 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 used ultra (U)HPLC to ensure rapid analysis and maximum efficiency. The 2D separation method was developed from the quantitative method with further development aimed at obtaining a high practical peak capacity in a reasonable analysis time. The practical peak capacity was determined as a function of the 2D flow-rate and gradient time, as well as the 1D fraction collection time. The off-line NP-HPCCC×RP-UHPLC method was applied to green and fermented rooibos samples. DAD was used to construct contour plots to elucidate quantitative and qualitative differences, while MS detection was used for tentative identification of previously unidentified compounds. A total of 39 compounds, 18 of which were not previously identified in rooibos, were identified using MS detection in positive ionisation mode. Most of the newly identified compounds were very polar. The combination of NP-HPCCC with RP-UHPLC separations was characterised by a high degree of orthogonality (~80%), contributing to a high practical peak capacity (3293) and improved separation of especially the novel polar phenolic compounds.