Thesis (PhD)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: The present study employed a two-phased approach to investigate the possible mechanisms involved in the chemopreventive properties of rooibos (Aspalathus linearis) and different honeybush species (Cyclopia spp.) in vitro. In the first phase, the effect of unfermented methanol and aqueous herbal tea extracts against the growth parameters (cell viability, proliferation and apoptosis) of normal (CRL 7761); premalignant (HaCaT); and malignant (CRL 7762) skin cells was evaluated and compared to green tea extracts. The predictive potential of polyphenol content (total polyphenol and flavanol/proanthocyanidins) and antioxidant properties (ABTS; ORAC; FRAP and LPO) in the biological activity of extracts in cells was also assessed. Of the herbal teas, the methanol extract of rooibos was the most active and it inhibited the growth of skin cells presumably by inducing mitochondrial dysfunction via membrane depolarisation. At lower concentrations, this activity was associated with inhibition of cell proliferation that was selective for cancer cells whilst higher concentrations induced apoptosis that was more prominent in premalignant cells. The strong antioxidant properties of the extracts implicated the role of pro-oxidative polyphenol/iron interactions involving monomeric flavonoids and polymeric proanthocyanidins in the cytotoxic effects of rooibos. The strong relationship between total polyphenolic and flavanol/proanthocyanidins content, antioxidant properties and reduction of cell viability indicated that these parameters (polyphenols and antioxidant properties) can serve as predictive tools for the cytotoxic effects of rooibos in vitro. The aqueous extracts of honeybush species, although weaker, displayed similar effects to rooibos extracts in cells with C. genistoides being the most effective at selectively inhibiting the proliferation of cancer cells whilst the pro-apoptotic activity of C. subternata and C. intermedia was more prominent in premalignant cells. The underlying mechanisms are also likely to result from pro-oxidative mechanisms resulting from polyphenol/iron interactions that mainly involve polymeric flavanol-like proanthocyanidin compounds in honeybush. In contrast, the methanol extracts exhibited weaker cytotoxic effects and protected cancer cells from going into apoptosis. The cytoprotective effects of honeybush species are possibly mediated by the major monomeric compounds such as mangiferin and hesperidin through antioxidant mechanisms that result in reduction of oxidative stress. Due to the possible dual role of the monomeric and polymeric compounds in the honeybush extracts, the total polyphenolic content of these herbal teas may not be a good
indicator of biological activity in vitro. However, as aqueous extracts displayed high
flavanol/proanthocyanidins content and exceptional activity in the ABTS assay, these
parameters may be considered as indicators of cytotoxicity. On the other hand,
methanol extracts, particularly from the xanthone-rich species (C. genistoides and C.
longifolia) which exhibited the weakest cytotoxic effects, were more active in the
ORAC thus this assay may be a useful predictor for cytoprotective activity. In the
second phase, an in vitro UVB/HaCaT model which used IL-1α as a biomarker for
early inflammation was developed and validated with known anti-inflammatory
compounds, dexamethasone and ibuprofen. It was used to determine the specific
mechanisms involved in the modulatory effects of the herbal tea extracts against
inflammation. Rooibos extracts and the aqueous extract of honeybush enhanced the
cytotoxic effects of UVB in the model and exhibited indirect anti-inflammatory effects
as they removed icIL-1α containing cells via apoptosis. In contrast, methanol extracts
of honeybush exacerbated icIL-1α by protecting UVB stimulated cells from
undergoing apoptosis. In conclusion, methanol extract of rooibos and aqueous
extracts of honeybush species may be useful in protecting the skin after UVB
exposure. These herbal tea extracts may block initiation and delay the promotion
stage during skin carcinogenesis by removing premalignant cells via apoptosis and
preventing onset of inflammation. In contrast, due to their cytoprotective effects,
methanol extracts of honeybush may be more effective at preventing oxidative stress
in skin before UVB exposure. Future studies should focus on the effects of extracts
and polyphenolic fractions on the oxidative status of the cells and development of
biomarkers of chemoprevention that can be utilised in vivo and in human skin. / AFRIKAANSE OPSOMMING: In hierdie studie word moontlike velkankerwerende eienskappe van rooibos (Aspalathus linearis) en ‘n aantal heuningbos (Cyclopia spp.) spesies deur twee afsonderlike benaderings bestudeer. Die eerste benadering ondersoek die effek van die kruietee op groeiparameters van velselle [lewensvatbaarheid, groei en dood van normale selle (CRL 7761), vroeë kankerselle (HaCaT) en kankerselle (CRL 7762)]. Tydens eksperimente is die moontlikheid om polifenoolinhoud (totale polifenole, en flavanol/proantosianidiene verhouding) en antioksidant-eienskappe te gebruik om die biologiese funksies van die ekstrakte in die selle te voorspel, geevalueer. Die metanolekstrak van rooibos het die groei van selle die effektiefste gestop, moontlik deur depolarisasie van die mitokondriale membraan. By lae konsentrasies van die ekstrak is die groei van kankerselle selektief gestop, terwyl vroeë kankerselle die sensitiefste by hoër konsentrasies was. Die hoë antioksidant-aktiwiteit van die rooibosekstrak kan moontlik ‘n rol speel in die indusering van sitotoksiese effekte in die selle en kan toegeskryf word aan die pro-antioksidant aktiwiteit van die polifenole weens hul interaksie met yster. ‘n Spesifieke funksie word vir die monomeriese flavonoïede en die polimeriese proantosianidiene geïmpliseer. Die sterk verwantskap tussen die totale polifenoolinhoud, flavanol/proantosianidien inhoud en antioksidant aktiwiteit met die verlaging in selgroei, maak hul relevante parameters van die voorspellingsmodel. Die waterekstrakte van heuningbos induseer ook soortgelyke maar swakker effekte met die induksie van kankersel dood, met C. genistoides die selektiefste en C. subternata en C. intermedia die aktiefste spesies wat die groei van die vroeë kanker selle inhibeer. Die onderliggende meganismes betrokke blyk ook aan ‘n pro-oksidant effek toe geskryf te wees, waartydens spesifieke polifenool/yster interaksies betrokke is. In teenstelling met rooibos, beskerm die metanolekstrak van heuningbos kankerselle teen seldood, wat moontlik verband hou met die antioksidant-eienskappe van die hoof monomeriese polifenole, mangiferien/isomangiferien en hesperidien. Vanweë die dubbele rol van die monomeriese polifenole en polimeriese verbindings in heuninghbosekstrakte is die totale polifenol inhoud nie ‘n goeie indikator van die biologiese aktiwiteit in vitro nie. Daarenteen is die flavanol/proantosianien inhoud en die hoë aktiwiteit in die ABTS antioksidanttoets goeie indikators om seldood te voorspel. In teenstelling hiermee het die metanolekstrakte van die xantoon-ryke spesies (C. genistoides en C. longifolia) ‘n baie lae effek op seldood, maar ‘n hoë aktiwitiet in die ORAC toets
getoon, wat ‘n goeie rigtingwyser is om die beskermende effek in selle te voorspel.
Met die tweede benadering is die anti-inflammatoriese eienskappe en die
onderliggende meganismes van die kruietee ondersoek in ‘n UVB/HaCaT selmodel.
Intrasellulêre interleukin 1α (IL-1α) is as merker gebruik en die model is geëvalueer
deur bekende anti-inflammatoriese verbindings soos dexamethasone en ibuprofin te
gebruik. Die metanolekstrak van rooibos en die waterekstrak van heuningbos het die
toksiese effek van UVB in die model verhoog deur selle met verhoogde vlakke,van
icIL-1α te verwyder deur middel van die induksie van seldood. Die metanolekstrak
beskerm die selle teen die oksidatiewe skade wat deur UVB geïnduseer word en
verwyder nie selle met hoë IL-1α vlakke nie. Ter opsomming blyk dit dat die
metanolekstrak van rooibos en die waterekstrak van heuningbos moontlik gebuik
kan word om die vel te beskerm teen die induksie van icIL-1α en sodoende die
inisiëring van kanker te blokkeer en ook die promosie van kanker te vertraag. Die
beskermende effek van die metanolekstrak kan moontlik aangewend word om die
oksidatiewe skade wat deur UVB veroorsaak word teen te werk deur dit aan te wend
voordat blootstelling plaasvind. Toekomstige studies behoort verdere karakterisering
van die polifenoolsamestelling van die ekstrakte in te sluit en hul effek op die
oksidatiewe status en anti-inflammoriese effekte van selle te bepaal ten einde sekere
merkers te identifiseer vir vel studies in vivo.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/85632 |
| Date | 12 1900 |
| Creators | Magcwebeba, Tandeka Unathi |
| Contributors | Gelderblom, W. C. A., Joubert, E., Swart, P., Stellenbosch University. Faculty of Science. Dept. of Biochemistry . |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | English |
| Type | Thesis |
| Format | xvii, 236 p. : col. ill. |
| Rights | Stellenbosch University |
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