Zugl.: Hannover, Univ., Diss., 2009

Magister Scientiae (Medical Bioscience) - MSc(MBS) / The blood brain barrier (BBB) is a signaling interface between the blood and the central nervous system (CNS), which prohibits the entry of harmful blood-borne substances into the brain micro-environment, thus maintaining brain homeostasis. The crucial role of the BBB is protecting the CNS, which may adversely be affected by alcohol. The central component of the BBB, endothelial cells (ECs), regulates BBB transport by regulating the permeability both transcellularly and through their paracellular junctions, by structures called tight junctions (TJs) that are composed of proteins. The aim of this study was to investigate the in vitro effects of ethanol (EtOH) and fermented rooibos (Rf) on a monolayer of bEnd5 mouse brain ECs, by determining the effects of EtOH and Rf on bEnd5 (i) cell viability (ii) cell proliferation (iii) rate of cell division (iv) cell toxicity (v) claudin-5 transcription (vi) permeability across a monolayer of bEnd5 ECs and (vii) morphology, for a selected experimental timeline of 24, 48, 72, and 96hrs. We then investigated if the simultaneous exposure of Rf and EtOH could reverse or alleviate the EtOHinduced effects on the bEnd5 ECs. EtOH metabolism induces oxidative stress and results in a range of adverse physiological effects. Aspathalus linearis (rooibos) contains many phenolic compounds, of which the main antioxidant activity is attributed to aspalathin. Our underlining hypothesis is that the antioxidants in an aqueous rooibos extract may therefore protect against the potential oxidant damaging effects of alcohol on the BBB. Cells were exposed for 24hrs to selected concentrations of EtOH (25mM and 100mM), a concentration of Rf containing equivalent of 1.9nM aspalathin, and the combinations of EtOH and Rf. Cell viability and cell toxicity was determined, while cell proliferation and rate of cell division was estimated using the trypan blue exclusion assay. Real time quantitative PCR was implemented to quantify claudin-5 transcription, normalized against housekeeping genes, GAPDH and HPRT. Transepithelial electrical resistance (TEER) was measured using the Ohm Millicell-electrical resistance system, while bEnd5 monolayer morphology was analysed using the Zeiss scanning electron microscope. Both concentrations of EtOH led to an overall decrease in cell viability, and a decreased number of live cells across 72hrs. Consistent with this, EtOH resulted in increased cell toxicity across the 96hr experimental timeframe and a diminished rate of cell division. The transcription of claudin-5 in bEnd5 ECs exposed to 25mM and 100mM EtOH varied dramatically across the 96hr timeframe. While 25mM EtOH resulted in an overall decrease in TEER, cells exposed to 100mM EtOH only decreased TEER between 48 and 96hrs. Morphologically, both concentrations of EtOH led to compromised paracellular spaces as endorsed by high definition SEM analysis. The administration of Rf on its own resulted in an initial decrease in viability, followed by recovery between 72 and 96hrs. Exposure to Rf diminished live cell numbers at 72 and 96hrs, accompanied by a compromised rate of cell division and an overall increase in cell toxicity. In addition, Rf down-regulated claudin-5 transcription across the course of the experiment, particularly between 24 and 48hrs. In alignment with this, Rf also led to an increase in BBB permeability from 24 to 96hrs. However, SEM studies were not able to discriminate any differences between control and Rf treated cells. Our study showed that the BBB could be protected against the adverse effects of EtOH, and this at the plasma concentration induced by 500ml’s of Rooibos tea. The simultaneous exposure of Rf and EtOH was able to negate the effects of EtOH on cell viability, cell proliferation, and cell toxicity but exacerbated the effects of EtOH on claudin-5 transcription and paracellular permeability. Morphologically, co-exposure with Rf only reversed the effects of 25mM EtOH while exacerbating the effects of 100mM EtOH at 96hrs. In conclusion, EtOH was shown to be detrimental to the integrity of bEnd5 ECs, and the addition of a minuscule quantity of the Rf extract was able to partially alleviate excess ROS-induced effects.

Thesis (Master of Environmental Health)--Cape Peninsula University of Technology, 2020 / Background: The development of metabolic disease is accompanied by changes in gut microbiota phenotype, including a decrease of beneficial bacteria and increase of pernicious bacteria of the gastrointestinal tract. A Western (high-fat and high-sugar) diet, sedentary lifestyle and altered gut microbiota diversity have been associated with an increased risk of developing metabolic diseases such as type 2 diabetes and its associated risk factor, obesity. Many researchers have studied the link between the gut microbiota and diet. Hence our in vitro study is aimed at investigating the potential prebiotic effect of an aspalathin-rich unfermented rooibos extract, Afriplex GRT™ and aspalathin on the faecal bacterial diversity of vervet monkeys fed Western diet. Methodology: A total of six vervet monkeys (Chlorocebus pygerythrus) were selected from monkeys fed either a maize based normal diet (standard diet group; n=3) or a high fat diet (Western diet group; n=3) for more than 5-years. Faecal samples were collected from the animals in both groups at the Primate Unit and Delft Animal Centre (PUDAC) between 7 – 9 AM. Faecal samples from the two groups were divided into culture-independent baseline samples (before culture) and culture-dependent samples (after anaerobic culture). The culture-dependent samples were cultured under anaerobic conditions at 37°C for 10 hours, with or without Afriplex GRT™ extract or aspalathin. Bacterial genomic DNA (gDNA) was extracted from all samples using the NucleoSpin® DNA Stool extraction kit. Purified gDNA was sent for metagenomic sequencing for 16S rRNA gene analysis of microbial diversity using an Ion Torrent Next-generation Sequencing platform. Results: Results indicated that the Western diet affects the abundance of several bacterial species. Afriplex GRT™ and aspalathin significantly enhanced the relative abundance of health promoting butyrate-producing bacteria such as Faecalibacterium prausnitzii in both standard and Western diet groups (p= 0.02 and p=0.04, respectively). A similar trend was observed in other beneficial bacteria such as Eubacterium spp., Sutterella spp., and Dorea longicatena. Conclusion: Based on the data observed, it can be suggested that Afriplex GRT™ has a beneficial prebiotic effect on gut microbiota diversity and gut health.

Thesis (MTech (Food Technology))--Cape Peninsula University of Technology, 2015. / Rooibos iced tea (RIT), as one of the products of Rooibos is fast becoming very popular as a beverage in society due to the benefits of the phenolic compounds that are associated with this herbal tea. Some of the commercially available products have been found to contain, if any, lower contents of the major phenolic compounds, namely aspalathin and its oxidation products, iso-orientin and orientin. Their presence is considered as indicators of a good quality product. The purpose of this study was to investigate the effect of ultraviolet-C (UV-C) light as an alternative treatment to heat treatment on the shelf life, pH, phenolic composition, colour and microorganisms associated with Rooibos. Two formulations of RIT were used in order to determine the efficacy of the UV-C on the shelf life whilst three formulations were used for the physicochemical analysis. Only one formulation was used for inoculation with three spoilage bacteria, yeast and mould spoilage microorganisms namely; Escherichia coli K12, Staphylococcus aureus, Salmonella sp., Saccharomyces cerevisiae and Cladosporium sp. The UV-C dosages of 0, 918, 1 836, 2 754 and 3 672 J.l -1 were used to treat the RIT using a pilot-scale UV-C system with a turbulent flow at a constant flow rate of 4000 l.hr-1 . A log count of 4 log10 was considered the limit for the spoilage growth since it is the average log10 afternormal pasteurisation. The use of UV-C treatment was found to have significantly (p1) effect on the overall colour difference of the RIT in formulations A, B, and C. All the spoilage microorganisms were significantly reduced by UV-C dosage to less than 4 log10 except the Cladosporium sp. The S. cerevisiae was the most sensitive microorganism whilst Cladosporium sp. was the most resistant. The effect of UV-C on the spoilage microorganism followed the sequence: S. cerevisiae>Salmonella sp.>S. aureus>E. coli K12>Cladosporium sp. This study indicated that microbiological reduction was achieved as a function of increasing UV-C dosage. In order to achieve the highest log10 reduction, the highest UV-C dosage of 3 672 J.l-1 may be used. However, the dosage may need to be increased in order to achieve the desired results in the treatment of Cladosporium sp. It can thus be concluded from the above investigations that UV-C dosage treatment of 3 672 J.l-1 is optimum in the non-thermal treatment of RIT / South African Association for Food Science & Technology Cape Peninsula University of Technology Bursary

Thesis (MSc Food Sc) (Food Science)--Stellenbosch University, 2008. / The change in aspalathin, iso-orientin, orientin and total polyphenol (TP) content of a commercially produced fermented rooibos (FR) extract was monitored throughout production. Particular attention was paid to the effect of spray-drying on FR and unfermented rooibos (UR) extracts. The quality of commercial, South African rooibos iced teas made with FR extract was also investigated with respect to the aforementioned parameters. Subsequently, the effect of heating and storage on the phenolic composition and colour of experimental iced teas containing respectively FR, UR and nano emulsified unfermented rooibos (NEUR) extracts was investigated. The combined effect of pH (pH 3-7) and storage (5, 30 and 40ºC), as well as high (660 mg/L, 0-7 days at 30ºC; UR only) and low (0.5 mg/L) concentrations of H2O2, was determined on reconstituted FR, UR and NEUR extracts. Finally, eight rooibos iced teas (four variants; unflavoured and lemon-flavoured) were analysed for plant-like, hay-like, rooibos and lemon flavour, as well as astringency, using descriptive sensory analysis. The degree of consumer preference of the flavoured variants was determined using the nine point hedonic scale. In all cases, changes in individual flavonoid content were quantified using HPLC. The TP content of the iced teas and commercial extracts was determined using the Folin-Ciocalteu assay. Browning of the iced teas and reconstituted extracts was monitored spectrophotometrically (420 nm). Aspalathin, iso-orientin and orientin were found to be present after all stages of the FR extract production process. Spray-drying, specifically, also did not reduce the content of these flavonoids, or the TP content, in FR and UR extracts. Despite the relatively good retention during the heating and storage of experimental rooibos iced teas, these flavonoids were either absent or present at extremely low levels in commercial iced teas. The latter suggested that either extremely low quantities of extract, no extract at all or extracts of poor quality, were used for the production of the analysed iced teas. Increased degradation was generally observed for sterilisation treatments compared to pasteurisation whilst losses during storage increased with time. The presence of citric acid, due to its pH-lowering effect, and ascorbic acid, due to its antioxidant activity, was integral to the retention of aspalathin, iso-orientin and orientin during heating, but less so during storage. The UR iced teas generally performed better than their FR counterparts, however, NEUR iced teas exhibited the greatest retention of the aforementioned flavonoids. Heating and storage resulted in browning of most iced teas, whilst the TP content increased slightly or remained unchanged. Phenolic retention in FR and UR extracts decreased with increasing pH and temperature, with concomitant browning. However, between pH 5 and 7, the stability of aspalathin was superior in the NEUR extract formulation. The latter also greatly resisted absorbance changes at pH 3 and 4, despite a loss of aspalathin. The phenolic content of UR extract was immediately reduced by high a concentration of H2O2, however, no significant (P≥0.05) changes in absorbance were detected, suggesting the formation of intermediate, colourless oxidation products. Formulations containing ascorbic acid experienced the greatest reductions. This was attributed to the iron reducing ability of this compound, as reduced iron accelerates the rate of the Fenton reaction. At low levels of H2O2, only the FR extract exhibited a loss of phenolic compounds. The level of iron in this extract was the highest. Despite having the greatest aspalathin and total flavonoid content, lemon flavoured unfermented rooibos iced tea (UF/LEMON) was disliked by consumers. Preference was directed away from the plant-like characteristic of this tea and towards rooibos flavour, characteristic of fermented rooibos iced tea. Iced tea comprising both FR and NEUR extract produced a product that 77% of consumers rated positively. Its slight hay-like flavour did not significantly (P≥0.05) reduce the liking of this product compared to fermented rooibos iced tea.

Magister Scientiae (Medical Bioscience) - MSc(MBS) / The blood brain barrier (BBB) is a signaling interface between the blood and the central nervous system (CNS), which prohibits the entry of harmful blood-borne substances into the brain micro-environment, thus maintaining brain homeostasis. The crucial role of the BBB is protecting the CNS, which may adversely be affected by alcohol. The central component of the BBB, endothelial cells (ECs), regulates BBB transport by regulating the permeability both transcellularly and through their paracellular junctions, by structures called tight junctions (TJs) that are composed of proteins. The aim of this study was to investigate the in vitro effects of ethanol (EtOH) and fermented rooibos (Rf) on a monolayer of bEnd5 mouse brain ECs, by determining the effects of EtOH and Rf on bEnd5 (i) cell viability (ii) cell proliferation (iii) rate of cell division (iv) cell toxicity (v) claudin-5 transcription (vi) permeability across a monolayer of bEnd5 ECs and (vii) morphology, for a selected experimental timeline of 24, 48, 72, and 96hrs. We then investigated if the simultaneous exposure of Rf and EtOH could reverse or alleviate the EtOHinduced effects on the bEnd5 ECs. EtOH metabolism induces oxidative stress and results in a range of adverse physiological effects. Aspathalus linearis (rooibos) contains many phenolic compounds, of which the main antioxidant activity is attributed to aspalathin. Our underlining hypothesis is that the antioxidants in an aqueous rooibos extract may therefore protect against the potential oxidant damaging effects of alcohol on the BBB. Cells were exposed for 24hrs to selected concentrations of EtOH (25mM and 100mM), a concentration of Rf containing equivalent of 1.9nM aspalathin, and the combinations of EtOH and Rf. Cell viability and cell toxicity was determined, while cell proliferation and rate of cell division was estimated using the trypan blue exclusion assay. Real time quantitative PCR was implemented to quantify claudin-5 transcription, normalized against housekeeping genes, GAPDH and HPRT. Transepithelial electrical resistance (TEER) was measured using the Ohm Millicell-electrical resistance system, while bEnd5 monolayer morphology was analysed using the Zeiss scanning electron microscope. Both concentrations of EtOH led to an overall decrease in cell viability, and a decreased number of live cells across 72hrs. Consistent with this, EtOH resulted in increased cell toxicity across the 96hr experimental timeframe and a diminished rate of cell division. The transcription of claudin-5 in bEnd5 ECs exposed to 25mM and 100mM EtOH varied dramatically across the 96hr timeframe. While 25mM EtOH resulted in an overall decrease in TEER, cells exposed to 100mM EtOH only decreased TEER between 48 and 96hrs. Morphologically, both concentrations of EtOH led to compromised paracellular spaces as endorsed by high definition SEM analysis. The administration of Rf on its own resulted in an initial decrease in viability, followed by recovery between 72 and 96hrs. Exposure to Rf diminished live cell numbers at 72 and 96hrs, accompanied by a compromised rate of cell division and an overall increase in cell toxicity. In addition, Rf down-regulated claudin-5 transcription across the course of the experiment, particularly between 24 and 48hrs. In alignment with this, Rf also led to an increase in BBB permeability from 24 to 96hrs. However, SEM studies were not able to discriminate any differences between control and Rf treated cells. Our study showed that the BBB could be protected against the adverse effects of EtOH, and this at the plasma concentration induced by 500ml’s of Rooibos tea. The simultaneous exposure of Rf and EtOH was able to negate the effects of EtOH on cell viability, cell proliferation, and cell toxicity but exacerbated the effects of EtOH on claudin-5 transcription and paracellular permeability. Morphologically, co-exposure with Rf only reversed the effects of 25mM EtOH while exacerbating the effects of 100mM EtOH at 96hrs. In conclusion, EtOH was shown to be detrimental to the integrity of bEnd5 ECs, and the addition of a minuscule quantity of t h e Rf extract was able to partially alleviate excess ROS-induced effects. / National Research Foundation (NRF)