Protective properties of rooibos (Aspalathus linearis) flavonoids on the prevention of skin cancer

Gwashu, Asanda (2015-03)

Thesis (MSc)--Stellenbosch University, 2016

Thesis

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.

AFRIKAANSE OPSOMMING : Ultraviolet B (UV-B) blootstelling is een van die vernaamste oorsake van velkanker en veroorsaak ‘n reeks van veranderings op sellulêre vlak wat insluit effekte soos oksidatiewe skade, DNS beskadiging en inflammasie. Die keratinosiete en makrofage in the vel speel ‘n belangrike role gedurende inflammasie en skei verskillende sitokienes af wanneer die selle blootgestel word aan UV-B insluitende toksiese stowwe soos lipopolisakkariedes (LPS). Die ongekontroleerde en kroniese uitskeiding van die sitokienes kan baie nadelig wees en lei tot verskillende velsiektes in sluitende kanker. Rooibos is bekend vir ‘n verskeidenheid van gesondheids voordele wat onder andere anti-inflammatoriese effekte insluit wat gekoppel word aan die antioksidant eienskappe van die flavonoïede. In die huidige studie is die totale polifenole en flavanol inhoud van water en methanol ekstrakte van onderskeidelik groen en geoksideerde rooibos vergelyk, terwyl the antioksidant eienskappe geëvalueerwas met behulp van die FRAP en ABTS toetse. Verder is die anti-inflammasie van rooibos ondersoek in die UV-B/HaCaT en die LPS/makrofaag inflammatoriese sel modelle. Die methanol ekstrak afkomstig van groen rooibos (MUF), het die hoogste antioksidant aktiwiteitg ehad met ‘n ooreenkomtige hoë vlak van totale polifenole, flavanole, die monomeriese flavonole wanneer dit met die water ekstrak (AUF)vergelyk word. Die water en methanol ekstrakte van die ge-oksideerde rooibos het ‘n laer aktiwiteit wanneer bogenoemde parameters en antioksidant aktiwiteit ter sprake kom. Die MUF ekstrak was verder gefraksioneer met behulp van kolom-chromatografie met XAD-2 as matriks en fraksies met verskillende polariteite is verkry. Die belangrikste rooibos flavonoïede was gekonsentreer in die X-3 en X-4 fraksies wat ook die hoogste antioksidant eienskappe gehad het, alhoewel dit ooreensstem met die MUF ekstrak. Die mees polêre fraksies, X-1 en X-2 bevat minder polifenole en was geassossieer met ‘n lae antioksidant aktiwiteit. Die anti-inflammatoriese eienskappe van die rooibos ekstrakte en kolom fraksies was getoets in ‘n UV-B/HaCaT keratinosiet inflammasie model waartydens die opeenhoping van interleukin 1 alfa (IL-1α) en sel lewensvatbaarheid getoets is. In die afwesigheid van UV-B lig het die methanol ekstrakte en die flavonoïed verrykte kolom fraksies X-3 en X-4, IL-1α opeenhoping verhoog. Die verhoogte akkumulasie het gepaard gegaan met ‘n verlaging in sel lewensvatbaarheid en‘n verhoging in apoptose (seldood) wat dui op ‘n pro-inflammatoriese effek. Die mees polêre fraksie, X-1 het die sel se oorlewing en apoptose betekenisvol verlaag terwyl IL-1α akkumulasie verhoog was. Hierdie verhoging in IL- 1α kan moontlik verband hou met ‘n gepaardgaande sel nekrose, IL- 1α uitskeiding en die aktiveering van ‘n outokriene pro-inflammatoriese terugvoer meganismes. ‘n Soortgelyke effek is waargeneem met die mees nie-polêre fraksieX-5, maar sonder ‘n verlies in die lewensvatbaarheid en apoptose wat ‘n pro-inflammatoriese effek aandui. Die water ekstrakte and die polêre fraksie X-2 het egter IL-1α verlaag met minimale effekte op die groei van die selle wat dui op ‘n anti-inflammatoriese reaksie. In die teenwoordigheid van UV-B het al die ekstrakte en kolom fraksies, IL-1α verlaag met die methanol en flavonoïed verrykte ekstrakte die mees effektiefste. Die IL-1α verlaging was verder geassosieer met ‘n verdere daling in beide die sel lewensvatbaarheid en die induksie van apoptose. Dit kom voor as of rooibos IL-1α verlaag deur selle te verwyder met behulp van seldood, alhoewel daar ‘n kritieke balans bestaan wat sal bepaal watter tipe geinduseer word, byvoorbeeld die induksie van apoptose waartydens IL-1α verwyder word of deur nekrose waartydens dit vrygestel word. By hoë konsentrasies en dalende ATP vlakke word induksie van apoptose verhoed en gevolglik vind nekrose plaas wat inflammasie verder kan stimuleer. Hierdie pro-inflammatoriese effekte moet dus verhoed word. Die anti-inflammatoriese effek van rooibos ekstrakte en kolom fraksies was ook geëvalueer in die LPS-geinduseerde THP-1 makrofaag inflammatoriese model metTNF-α uitskeiding as eindpunt. Al die rooibos ekstrakte het TNF-α verlaag sonder om ‘n noemenswaardige effek te hê op sel lewens vatbaarheid en die induksie van apoptose. Die water ekstrak en die mees polêre kolom fraksie (X-1) was opmerklik die aktiefste by die hoogste konsentrasies terwyl fraksie X-2 die kleinste effek gehad het. Die flavonoied verrykte fraksies X3 and X-4 asook die mees nie-polêrefraksie (X-5) het ook die TNF-α uitskeiding verminder wat gepaarde gegaan het met die induksie van apoptose by die hoogste konsentrasies. Omdat daar minimale effekte op die lewensvatbaarheid van die makrofage by lae konsentrasies verkry is, kandie effek van die water ekstrakte en die meer polêre kolom fraksies aan ‘n tipiese anti-inflammatoriese effek toe geskryf word. Die LPS/makrofaag model is baie meer weerstandbiedend teenoor die pro-oksidatiewe effekte van die rooibos flavonoiede en is ‘n ideale model om die anti-inflammatoriese eienskappe van rooibos verder te ondersoek. In teenstelling hiermee het rooibos ‘n indirekte anti-inflammatoriese effek in die UV-B/HaCaT inflammatoriese model gehad vi deur die induksie van apoptose en die verwydering van IL-1α. Afhangende van die konsentrasie vlakke kan dit ook nekrotiese sel dood veroorsaak vanwee die pro-oksidant effekte van die rooibos flavonoiede met moontlike bydra en die rol van yster wat vrygestel word. Dit lei tot ‘n verdere verhoging van die inflammatoriese reaksie in the model vanwee‘n outomatiese terugvoer meganisme. Die anti-inflammatories effekte van die meer polêre rooibos verbindings behoort verder ondersoek te word in die UV-B/HaCaT keratinosiet inflammatoriese model, veral om die moontlike rol van die tannien-agtige proanthosianidiniene en ander nie-flavonoïed verbindings te evalueer. In die geval sal die aandag gevestig moet word op die geoksideerde rooibos omdat die flavonoïede-verrykte methanol en kolom fraksies die anti-inflammatoriese effek verberg as gevolg van hul sitotoksiese effekte. Verder moet die effek van rooibos op die modulering van verskillende sel boodskap stimuli ten opsigte van die induksie van inflammasie beter gedefinieer word ten einde die kankerwerende eienskappe van rooibos verder te ondersoek.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/98536
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