Browsing by Author "Beelders, Theresa"

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    Comprehensive phenolic profiling of Cyclopia genistoides (L.) Vent. by LC-DAD-MS and -MS/MS reveals novel xanthone and benzophenone constituents
    (MDPI, 2014-08) Beelders, Theresa; De Beer, Dalene; Stander, Maria A.; Joubert, Elizabeth
    A high-performance liquid chromatographic (HPLC) method coupled with diode-array detection (DAD) was optimized for the qualitative analysis of aqueous extracts of Cyclopia genistoides. Comprehensive insight into the phenolic profile of unfermented and fermented sample extracts was achieved with the identification of ten compounds based on comparison with authentic reference standards and the tentative identification of 30 additional compounds by means of electrospray ionization mass spectrometry (ESI-MS) and tandem MS detection. Three iriflophenone-di-O,C-hexoside isomers, three xanthone-dihydrochalcone derivatives and one dihydrochalcone are herein tentatively identified for the first time in C. genistoides. Of special interest is one iriflophenone-di-O,C-hexoside present in large amounts. New compounds (tentatively) identified for the first time in this species, and also in the genus Cyclopia, include two aromatic amino acids, one flavone, an iriflophenone-di-C-hexoside, a maclurin-di-O,C-hexoside, two tetrahydroxyxanthone-C-hexoside isomers, a tetrahydroxyxanthone-di-O,C-hexoside, two symmetric tetrahydroxyxanthone-C-hexoside dimers, nine glycosylated flavanone derivatives and five glycosylated phenolic acid derivatives. The presence of new compound subclasses in Cyclopia, namely aromatic amino acids and glycosylated phenolic acids, was demonstrated. The HPLC-DAD method was successfully validated and applied to the quantitative analysis of the paired sample extracts. In-depth analysis of the chemical composition of C. genistoides hot water extracts gave a better understanding of the chemistry of this species that will guide further research into its medicinal properties and potential uses.
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    HPLC method development for the characterisation of the flavonoid and phenolic acid composition of rooibos (Aspalathus linearis) infusions
    (Stellenbosch : Stellenbosch University, 2011-12) Beelders, Theresa; De Villiers, A. J.; Joubert, E.; De Beer, D.; Sigge, G. O.; Stellenbosch University. Faculty of AgriSciences. Dept. of Food Science.
    ENGLISH ABSTRACT: Rooibos tea, produced from the endemic South African fynbos plant Aspalathus linearis, has various healthpromoting benefits which can largely be attributed to its phenolic composition. In this study, the reversedphase liquid chromatographic (RP-LC) separation of the principal phenolic constituents of aqueous rooibos infusions was optimised on conventional high-performance liquid chromatography (HPLC) instrumentation. The phenolic constituents comprised dihydrochalcones (aspalathin and nothofagin), flavones (orientin, isorientin, vitexin, isovitexin, luteolin, luteolin-7-O-glucoside and chrysoeriol), flavonols (quercetin, isoquercitrin, hyperoside and rutin), a hydroxycinnamic acid (ferulic acid) and a phenylpropanoid (phenylpyruvic acid glucoside, PPAG). A systematic approach towards method development was adopted: resolution was optimised by simultaneous optimisation of gradient conditions and temperature on a sub-2 6m phase to exploit the benefits of this phase for fast routine analyses. The developed HPLC method, utilising photodiode-array (PDA) detection, yielded complete separation of the 15 target analytes on the 1.8 µm C18 column, thermostatted to 37°C, within 37 min (total analysis time: 50 min). The method was successfully validated and demonstrated its suitability for the fast, quantitative analysis of aqueous infusions of unfermented and fermented rooibos. Mass spectrometric (MS) and tandem MS detection confirmed peak purity and permitted the tentative identification of 13 additional phenolic compounds, including a flavonol Odiglycoside (quercetin-3-O-robinobioside), a luteolin-6-C-pentoside-8-C-hexoside and a novel C-8-hexosyl derivative of aspalathin reported here for the first time. The HPLC-PDA method was subsequently applied to a large number of fermented rooibos samples representative of different production seasons (2009, 2010 and 2011) and quality grades (grades A, B, C and D) to capture as much potential variation in the phenolic composition as possible. Production season had no clear effect on the levels of the individual phenolic compounds in ‘cup-of-tea’ rooibos infusions, whilst high quality tea (grades A and B) was associated with higher levels of phenolic compounds and soluble solids than low quality tea (grades C and D). Steam-pasteurisation of the plant material, required to obtain a product of high microbiological quality, induced significant reductions in the mean values of most of the phenolic compounds in rooibos infusions. The major phenolic constituents of steam-pasteurised, fermented rooibos were isoorientin and orientin, whilst quercetin-3-O-robinobioside, PPAG and aspalathin were also present in high concentrations. Representative content values of the major phenolic compounds present in a typical ‘cup-of-tea’ rooibos infusion were thus obtained and the generated data are suitable for inclusion in food composition databases. The application of comprehensive two-dimensional liquid chromatography (LCxLC) was investigated as an alternative approach for the detailed investigation of rooibos phenolics. The combination of hydrophilic interaction chromatography (HILIC) in the first dimension and RP-LC in the second dimension offered different separation selectivities and hence a high degree of orthogonality. HILICxRP-LC provided a significant improvement in resolution, as is evident from practical peak capacities in excess of 2000 and 800 for the off-line and on-line methods, respectively. Further optimisation, particularly of the first dimension separation, is however required to improve the LCxLC separation of complex rooibos phenolic fractions.
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    Xanthones and benzophenones from Cyclopia genistoides (honeybush) : chemical characterisation and assessment of thermal stability
    (Stellenbosch : Stellenbosch University, 2016-03) Beelders, Theresa; Joubert, E.; De Beer, D.; Sigge, G. O.; Stellenbosch University. Faculty of AgriSciences. Dept. of Food Science.
    ENGLISH ABSTRACT: Numerous health-promoting benefits may be derived from the consumption of honeybush tea, a herbal infusion prepared from the leaves and fine stems of the endemic Cape fynbos genus, Cyclopia. These health-promoting benefits are attributed to its phenolic constituents and therefore insight into the nature, quantities and biological activities of individual compounds are required. Information regarding the thermal stability of these compounds is also crucial, as the plant material is subjected to a high-temperature chemical oxidation process (“fermentation”) to develop the sought-after characteristic sensory attributes of the herbal tea product. In this study, the phenolic composition of Cyclopia genistoides, a commercially important species, was comprehensively characterised by high-performace liquid chromatography (HPLC) coupled with diode-array and mass spectrometric detection. A species-specific HPLC method was developed for the qualitative analysis of aqueous extracts prepared from “unfermented” and “fermented” C. genistoides plant material and was subsequently validated for the quantification of 18 phenolic compounds in these types of extracts. The major phenolic constituents included the C-glucosyl xanthone mangiferin (1) and its regio-isomer isomangiferin (2), and the benzophenone 3-β-D-glucopyranosyliriflophenone (3). The presence of novel benzophenone and xanthone derivatives in C. genistoides was demonstrated for the first time, including an iriflophenone-di-O,C-hexoside derivative, present in large quantities. This compound was isolated and unambiguously identified by nuclear magnetic resonance spectroscopy as 3-β-D-glucopyranosyl-4-β-D-glucopyranosyloxyiriflophenone (4) – a novel benzophenone unique to Cyclopia. 3-β-D-Glucopyranosylmaclurin (5), present in small quantities, was also isolated. The isolated benzophenones (4 and 5) exhibited mammalian α-glucosidase inhibitory activity, while 4 and 3 were also marginally effective in increasing glucose uptake in vitro. Compound 4 was ineffective as antioxidant in the DPPH assay, but the most effective in the ORAC assay, compared to the other compounds tested (1, 2, 3, 5). Degradation of compounds 1-4 in C. genistoides plant material under simulated fermentation conditions (80 °C/24 h and 90 °C/16 h) followed first-order degradation kinetics and their thermal stability decreased in the order 4 > 2 > 3 > 1. An increase in the degree of glucosylation significantly increased the thermal stability of the benzophenones, whereas glucosylation at C-4 of the dibenzo-γ-pyrone structure, as opposed to C-2, increased the stability of the tetrahydroxyxanthones in the plant material matrix. This was also confirmed for individual compounds (1-5) in aqueous model solutions (pH 5). Inclusion of 5 in the model systems provided additional insight into structure-stability relationships. Increased B-ring hydroxylation significantly increased the first-order degradation rate constants of the benzophenones. Oxidative coupling of the polyhydroxybenzophenone 5 with the formation of its corresponding xanthones (1 and 2) led to substantial increases in the thermal stability of 1 and 2 compared to that of 5. Increased temperatures increased the degradation rates of all compounds in both the plant material matrix and model solutions. The thermal stability of 1, tested at pH 3-7, was found to be pH-dependent, with increased degradation rates observed at higher pH. Thermally-induced reactions principally included isomerisation, dimerisation and cleavage of O-linked sugar moieties; conversion of all benzophenones to the xanthones occurred to varying degrees. Of special interest was the rapid and predominant conversion of 5 to 1 and 2.