Masters Degrees (Biochemistry)

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Browsing Masters Degrees (Biochemistry) by browse.metadata.advisor "Gelderblom, Wentzel Christoffel Andreas"

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    Protective properties of rooibos (Aspalathus linearis) flavonoids on the prevention of skin cancer
    (Stellenbosch : Stellenbosch University, 2015-03) Gwashu, Asanda; Gelderblom, Wentzel Christoffel Andreas; Louw, Ann; Stellenbosch University. Faculty of Science. Department of Biochemistry
    ENGLISH ABSTRACT : Ultraviolet-B (UV-B) radiation is a major cause of skin cancer resulting in an array of events including oxidative damage, DNA damage and inflammation. The keratinocytes and skin macrophages play a pivotal role in inflammation and are known to release a wide range of cytokines in response to UV-B and/or other toxicants such as lipopolysaccharide (LPS). The chronic release of the cytokines, if not controlled, may be detrimental leading to a variety of skin diseases including cancer. Rooibos is well known for its health benefits which include anti-inflammatory effects that are attributed to the anti-oxidant properties of the flavonoids. In the current study the aqueous and methanol extracts of unfermented and fermented rooibos were compared in terms of their polyphenol and flavanol content, while their antioxidant properties were assessed in the FRAP and ABTS assays. The methanol extract of unfermented (MUF) rooibos, which contained the highest levels of total polyphenols and flavanol content as well as the monomeric flavonoids, exhibited the strongest antioxidant properties when compared to its aqueous counterpart (AUF). The fermented rooibos methanol and aqueous (MF and AF) extracts exhibited similar but weaker responses compared to the unfermented extracts. The MUF extract was further fractionated by column chromatography utilising an XAD-4 resin resulting in five major fractions with different polarity. The major rooibos flavonoids were enriched in fractions X-3 and X-4, which also exhibited the highest antioxidant activity although it was similar to the MUF extract. The most polar fractions, X-1 and X-2, contained less flavonoids and exhibited a weaker antioxidant activity. The anti-inflammatory effects of the rooibos extracts and column fractions were investigated in the UV-B/HaCaT inflammation model monitoring interleukin 1α (IL-1α) production and cell viability indices. In the absence of UV-B exposure the methanol extracts and the flavonoid-enriched fractions, X3 and X-4, increased IL-1α with a decrease in cell viability and increase in apoptosis, suggestive of a pro-inflammatory effect. The most polar fraction X-1 drastically decreased cell viability and apoptosis while IL-1α was increased, which may be attributed to necrotic cell death and a subsequent pro-inflammatory stimulation via an autocrine feedback pathway. A similar effect was noticed with the non-polar fraction X-5, however without adversely affecting the cell growth parameters suggestive of a direct pro-inflammatory effect. The aqueous extracts and the polar fraction X-2 had the opposite effect by decreasing IL-1α with minor effects on cell viability and apoptosis at low concentrations, suggesting an anti-inflammatory effect. In the presence of UV-B all the extracts and most of the column fractions resulted in a decrease in IL-1α accumulation in comparison to the control, with the methanol unfermented extract and flavonoid enriched fractions being the most active. A further decrease in cell viability and apoptosis was also observed at the higher concentrations. Therefore, rooibos may aid the removal of IL-1α indirectly presumably by inducing cell death although a critical balance appear to exist in the type of cell death, e.g. via apoptosis by which IL-1α is removed or via necrosis where the cytokine is released. The anti-inflammatory effects of the rooibos extracts and column fractions were also monitored in LPS-induced THP-1 derived macrophages monitoring the release of TNF-α. All the extracts decreased TNF-α release with minor effects on the cell growth parameters. The aqueous fermented extract and the most polar fraction, X-1 were the most active in decreasing TNF-α and fraction X-2 the exhibited the lowest activity at the highest concentrations. The flavonoid enriched column fractions, X-3 and X-4 as well as the non-polar X-5 column fractions reduced the excretion of TNF-α, although cell viability was decreased and apoptosis was increased at higher concentrations. The LPS/macrophage inflammatory model seems to be more resistant to the pro-oxidant effects of the rooibos flavonoids and therefore provides an ideal model to further characterise the anti-inflammatory properties of rooibos. In the UV-B/HaCaT inflammatory model the rooibos-enriched flavonoid extracts seem to remove cytokines through inducing apoptotic cell death thereby indirectly inhibiting inflammation. However, depending on the concentration levels, it could also stimulate inflammation under certain conditions by exhibiting pro-oxidant effects, presumably via iron interactive mechanisms. The anti-inflammatory effects of the more polar rooibos constituents, presumably the tannin-like proanthocyanidins and/or the non-flavonoid constituents of rooibos, exhibiting a lower antioxidant potency, should be further investigated utilising the UV-B/HaCaT keratinocyte inflammatory model. In this regard, the further characterization of fermented rooibos is of interest as the flavonoid enriched MUF and column fractions seems to mask the anti-inflammatory effects due to adverse effects on cell growth indices. The modulation of different cells signalling pathways associated with inflammation need to be characterized to better define the chemopreventive properties of rooibos.