Thesis (MSc)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: Chronic inflammation is suggested to contribute to cancer development and therefore a potential target for chemoprevention. In the skin, keratinocytes and macrophages play an integral part in acute and chronic inflammation, with interleukin 1-α (IL-1α) and tumor necrosis factor α (TNF-α) as key cytokines governing this process. Green tea (Camellia sinensis) and the South African herbal teas, rooibos (Aspalathus linearis) and honeybush (Cyclopia spp.) displayed antiinflammatory effects in mouse and human skin. To further investigate the antiinflammatory properties of green tea and the herbal teas, rooibos and honeybush (C. subternata and C. maculata) herbal teas, suitable cell culture models were developed and validated utilising human keratinocytes (HaCaT) and monocyte (THP- 1) derived macrophages. Aqueous extracts of the green tea and unfermented herbal teas were prepared and their chemical composition determined by high performance liquid chromatography (HPLC) and the antioxidant activity characterised utilising different antioxidant assays. Green tea and rooibos exhibited similar antioxidant activities while C. maculata displayed the lowest overall antioxidant activity of all the extracts, despite possessing the highest mangiferin level, the major polyphenol in honeybush. The modulation of cytokine release was studied in (i) an UVB-induced pre-exposure HaCaT model monitoring the accumulation of IL-1α and (ii) a LPS stimulated THP-1 macrophage model monitoring the TNF-α release, utilising both a pre-exposure and co-exposure extract regimens. In the pre-exposure HaCaT inflammatory model the UVB-induced IL-1α was decreased by the green tea extract while a far weaker response was obtained with the rooibos extract. Both the honeybush extracts displayed a significant effect in the reduction of IL-1α with C. subternata exhibiting a slight increased protection at a lower extract concentration. In the pre-exposure THP-1 derived macrophage model, green tea and the herbal tea extracts inhibited TNF-α release in a dose dependent manner in the absence of an overt loss in cell viability and apoptosis at lower extract concentrations, suggesting a typical anti-inflammatory effect. In the co-exposure model, the different extracts also exhibited an anti-inflammatory effect at the lowest concentrations in the absence of apoptosis while at higher extract concentrations the effect was masked by a decrease in cell viability and increased apoptosis. C. maculata exhibit differential effects when considering the inhibition of cytokine production and, depending on the cell model, either exhibited a weaker or stronger effect when compared to C. subternata and rooibos. Phenolic diversity of the different teas is likely to explain the differential effects in the antioxidant assays and cell culture models with respect to the regulation of the production of the inflammatory markers. Proposed mechanism for the anti-inflammatory effects include the modulation of oxidative stress via various pathways and the subsequent down regulation of nuclear factor kappa β (NFκB) and activated protein-1 (AP-1) which are key regulators of cytokine production governing the inflammatory response. / AFRIKAANSE OPSOMMING: Kroniese inflammasie van die vel kan bydra tot die ontwikkeling van kanker en is dus ’n potensiële area om te teiken in die voorkoming van velkanker. Keratinosiete en makrofage speel ’n integrale rol in akute en chroniese inflammasie van die vel en TNF-α en IL-1α is die belangrikste sitokiene wat hierdie proses inisieer. Dit is bekend dat ekstrakte van groen tee (Camellia sinensis) en die Suid-Afrikaanse kruietees, rooibos (Aspalathus linearis) en heuningbos (Cyclopia spp.) ‘n anti-inflammatoriese effek op die vel van muise en mense het. Om die anti-inflammatoriese aktiwitieit van groen tee, rooibos en 2 heuningbos kruietees (C. subternata en C. maculata) verder te ondersoek en te definieer is geskikte selkultuurmodelle ontwikkel en gevalideer deur gebruik te maak van menslike keratinosiete (HaCaT) en monosiet (THP-1) afgeleide makrofage. Water ekstrakte van groen tee en ongefermenteerde kruietees is voorberei en die chemiese samestelling deur hoë druk vloeistof chromatografie (HDLC) bepaal. ‘n Verskeidenheid van antioksidant bepalingstoetse is gebruik om die antioksidant aktiwiteit van die ekstrakte te meet. Groen tee en rooibos het soortgelyke antioksidant aktiwiteite getoon, terwyl C. maculata die swakste algehele aktiwiteit getooon het, ten spyte van die teenwoordigheid van hoёr vlakke van mangiferin, die belangrikste polifenoliese verbinding in heuningbos. Modulasie van sitokiene is verder bestudeer in (i) ’n UVB-geïnduseerde vooraf-blootstelling HaCaT model, waartydens akkumulering van IL-1α gemonitor is en (ii) ‘n lipopolisakkaried (LPS)-gestimuleerde THP-1 makrofaag model, waar die vrystelling van TNF-α gemonitor is. Vir die THP-1 model is beide die voor en gelyktydige blootstelling benaderings vir die ekstrakte met LPS gebruik. In die keratinosiet model, waar die selle aan ekstrakte blootgestel is voor UVB bestraing, is IL-1α beduidend verlaag deur die groen tee ekstrak, terwyl ’n swakker reaksie gesien is met rooibos. Beide heuningbos ekstrakte het ’n beduidende invloed in die vermindering van IL-1α getoon, waar C. subternata ’n effense verhoogde beskerming teen selsterfte by ‘n laer ekstrakkonsentrasie toon. Blootstelling van die makrofage aan al vier ekstrakte voor LPS stimulasie (vooraf-blootstelling), het gelei tot inhibisie van TNF-α vrystelling op ’n dosis afhanklike wyse en die afwesigheid van apoptose en selsterftes by lae ekstrak konsentrasievlakke. Hierdie waarnemings dui op ’n tipiese antiinflammatoriese effek. In die gelyktydige-blootstelling model verlaag al die ekstrakte TNF-α vrystelling teen die laagste ekstrak konsentrasievlakke, in die afwesigheid van apoptose en met geen effek op seldood nie. Hoёr ekstrak konsentrasievlakke het sitotoksisiteit en verhoogde apoptose getoon, dus was die anti-inflammatoriese effek gemaskeer. C. maculata toon ‘n variërende effek met betrekking tot antioksidant aktiwiteit en die bekamping van sitokien produksie, afhangend van die model wat bestudeer is. Die verskeidenheid fenoliese verbindings teenwoordig in die verskillende tee ekstrakte is waarskynlik die rede vir die effekte wat waargeneem is tydens antioksidant toetsing en selkultuurmodelle. Die anti-inflammatoriese meganismes wat deur hierdie studie voorgestel word sluit die modulasie van oksidatiewe stres via verskeie metaboliese paaie in. Modulasie van oksidatiewe stres lei tot af-regulering van kernfaktor-kappaB (NF-κB) en aktiveerderproteïen- 1(AP-1), wat sleutel reguleerders van sitokien produksie tydens inflammatoriese respons is.

Thesis (PhD)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Phytoestrogen containing extracts of Cyclopia, an indigenous South African fynbos plant used to prepare honeybush tea, may serve as a source of new estrogen analogues. It would be of great benefit if these new analogues would not only prevent the development and progression of breast cancer which, globally, is responsible for the highest number of cancer associated deaths among females, but also have a reduced side-effect profile when compared to current treatments and, in addition, also alleviate menopause associated symptoms. In this study three extracts, P104, SM6Met, and cup-of-tea, from two species of Cyclopia, C. genistoides and C. subternata, were evaluated for their potential to modulate molecular targets involved in prevention and treatment of breast cancer. We show that the phytoestrogenic extracts of Cyclopia antagonise estrogen-induced cell proliferation both in vitro as well as in vivo. Furthermore, our study presents various molecular mechanisms whereby the Cyclopia extracts may be eliciting this effect. Importantly, we show, for the first time, that the Cyclopia extracts behave as ERα antagonists and ERβ agonists which, with respect to the known role of the ER subtypes in breast cancer, where the ERα subtype is associated with the stimulation of cell proliferation and the occurrence of breast cancer, while ERβ ameliorates the action of ERα in breast cancer and could act as an inhibitor of breast cancer development, may be beneficial for the prevention or treatment of breast cancer. In addition, we also show that the extracts of Cyclopia behave as selective estrogen receptor degraders by down-regulating ERα protein levels while stabilising ERβ protein levels, which not only provides a possible molecular explanation for the observed ERα antagonism and ERβ agonism, but, in addition, may be beneficial as higher ERα levels are associated with malignant breast cancer tumours, while higher ERβ levels are associated with benign tumours. Furthermore, we show that the Cyclopia extracts affect the nuclear localization and distribution of both ER subtypes in a manner that provides an additional molecular explanation for the observed ERα antagonism and ERβ agonism. Investigation of the molecular processes involved in the promotion and progression of breast cancer, such as the distribution of cells between the phases of the cell cycle, cancer cell invasion, and the regulation of genes governing these processes provides evidence that the Cyclopia extracts are not as proliferative as estrogen. In addition, Cyclopia extracts display anti-inflammatory properties, which may be beneficial as inflammation is an enabling characteristic in cancer development and progression. Furthermore, this study, for the first time, shows that the phytoestrogenic extracts of Cyclopia are absorbed, are not toxic, and display biological ERα antagonist activity in vivo by retarding uterine growth. Thus, we propose that the Cyclopia extracts act as selective estrogen receptor subtype modulators with potential to be developed as a nutraceutical for the treatment or prevention of breast cancer. / AFRIKAANSE OPSOMMING: Fitoëstrogeen-bevattende ekstrakte van Cyclopia, ‘n inheemse Suid Afrikaanse fynbosplant wat gebruik word vir die voorbereiding van heuningbostee, mag as ‘n bron van nuwe estrogeen-analoë dien. Dit sal baie voordelig wees indien hierdie nuwe analoë nie net die ontwikkeling en progressie van borskanker sal voorkom nie, aangesien borskanker wêreldwyd verantwoordelik is vir die grootste getal kankerverwante sterftes onder vroue, maar ook ‘n verminderde newe-effek profiel vertoon in vergelyking met huidige behandelings en ook, boonop, simptome wat met menopouse geassosieer word, sal verlig. In hierdie studie is drie ekstrakte, P104, SM6Met, en cup-of-tea, vanaf twee spesies van Cyclopia, C. genistoides en C. subternata, geëvalueer vir hul potensiaal om die molekulêre teikens betrokke by die voorkoming en behandeling van borskanker te moduleer. Ons wys dat die fitoëstrogeniese ekstrakte van Cyclopia antagoniseer estrogeen-geïnduseerde selproliferasie beide in vitro as ook in vivo. Verder bied ons studie ook verkskeie molekulêre meganismes aan oor hoe die Cyclopia ekstrakte hierdie effek mag ontlok. ‘n Belangrike bevinding is dat ons vir die eerste keer wys dat die Cyclopia ekstrakte hulself as ERα -antagoniste en ERβ-agoniste gedra wat, met betrekking tot die erkende rol van die ER-subtipes in borskanker, waar die ERα-subtipe geassosieer word met die stimulasie van selproliferasie en die gebeurtenis van borskanker, terwyl ERβ die aksie van ERα onderdruk en as ‘n inhibeerder van borskankerontwikkeling kan dien, voordelig mag wees vir die voorkoming of behandeling van borskanker. Ons wys boonop ook dat die ekstrakte van Cyclopia hulself soos selektiewe estrogeen- reseptor-degradeerders gedra deurdat hul ERα-proteïnvlakke verlaag terwyl hul ERβ-proteïnvlakke stabiliseer. Dit verksaf nie net ‘n moontlike molekulêre verduideliking vir die waargeneemde ERα-antagonisme en ERβ-agonisme nie, maar mag ook voordelig wees in borskanker aangesien hoër ERα-vlakke geasosieer word met kwaadaardige borskankertumors en hoër ERβ-vlakke met nie-kwaadaardige tumors. Verder wys ons dat die Cyclopia ekstrakte die lokalisering en verspreiding van beide ER-subtipes in die selkern op so ‘n wyse beïnvloed dat dit ‘n addisionele molekulêre verduideliking bied vir die ERα-antagonisme en ERβ-agonisme wat waargeneem is. Verdere ondersoek van die molekulêre prosesse betrokke by die promosie en progressie van borskanker, soos die verspreiding van selle tussen die fases van die selsiklus, die beweging van kankerselle na omliggende weefsels, en die regulering van gene wat hierdie prosesse beheer, verskaf bewyse dat die Cyclopia-ekstrakte nie so proliferatief is soos estrogeen nie. Die ekstrakte van Cyclopia vertoon boonop ook anti-inflamatoriese eienskappe, wat voordelig mag wees aangesien inflammasie ‘n bydraende eienskap in kankerontwikkeling en -progressie is. Verder wys hierdie studie vir die eerste keer dat die fitoëstrogeniese ekstrakte van Cyclopia geabsorbeer word, nie toksies is nie, en dat hulle biologiese ERα-antagonis aktiwiteit vertoon deurdat hulle uterus-groei vertraag in vivo. Dus stel ons voor dat die Cyclopia-ekstrakte optree soos selektiewe-estrogeen-reseptor-subtipe-moduleerders met die potensiaal om ontwikkel te word as ‘n nutraseutiese middel vir die behandeling of voorkoming van borskanker.

Dissertation (PhD)--University of Stellenbosch, 2004. / ENGLISH ABSTRACT: This thesis provides the first scientific evidence on the cancer modulating properties of two unique South African herbal teas, rooibos (Aspalathus Iinearis) and honeybush (Cyclopia intermedia) utilizing in vitro as well as in vivo carcinogenesis assays by: • Demonstrating the in vitro antimutagenic activity of aqueous extracts of the herbal teas against the metabolic activated mutagens, 2-acetylaminofluorene (2- AAF) and the mycotoxin, aflatoxin B1 (AFB,) as well as, to a certain extent, against the direct acting mutagen, hydrogen peroxide, utilizing the Salmonella typhimurium mutagenicity assay. • Increasing the activity of hepatic drug metabolizing enzymes, glutathione Stransferase alpha and UPD-glucuronosyl transferase, and reduced the oxidative stress by stabilizing the level of reduced glutathione (GSH) resulting in an increased hepatic reduced to oxidized glutathione ratio (GSG:GSSG). No toxic effects were noticed in rats consuming the herbal teas for 10 weeks as their sole source of drinking fluid. • Demonstrating the ex vivo modulation of 2-AAF- and AFB1-induced mutagenesis by sub- cellular hepatic fractions of rats consuming the herbal teas in the Salmonella mutagenicity assay. Hepatic cytosolic fractions protected against mutagenesis of both mutagens, while the microsomal fractions exhibited a reduced capacity to metabolize AFB1 to its active mutagenic metabolite. • Providing evidence for the in vivo modulation of tumour promotion using the liver as well as the two-stage skin carcinogenesis animal models. The unprocessed herbal teas arrested proliferation of the placental form of glutathione-Stransferase (GSTP+) altered cells as well as reduced the total number of enzyme altered foci in the liver of rats. Topical application of polyphenolic fractions of the various herbal teas prior to 12-0-tetra-decanoylphorbol-13-acetate (TPA) tumour promotion, reduced tumour formation in mouse skin initiated with 7,12-dimethylbenz[ ajanthracene (DMBA). The protective effect was illustrated by a decreased tumour incidence, a reduction in tumour volume as well as a delayed onset of tumour development. The f1avanol/proanthocyanidin content of the fractions could playa major role in the protection against skin tumour promotion. • Proposing possible mechanisms whereby rooibos and honeybush herbal teas could exert their cancer modulating properties with respect to in vitro and ex vivo antimutagenicity, in vivo oxidative status and reduced tumour promotion. • Providing evidence that the herbal teas mimic the cancer modulating properties of green and black teas although differences exist, presumably due to differences in the polyphenolic constituents. • Suggesting that rooibos and honeybush herbal teas may play an important role as chemopreventive agents in the modulation of cancer. / AFRIKAANSE OPSOMMING: Hierdie tesis bevat die eerste ondersoek na die effek van waterige en polifenoliese ekstrakte van rooibos (Aspalathus Iinearis) en heuningbos (Cyclopia intermedia) op verskeie aspekte van kankerontwikkeling. Die twee kruietees is uniek aan Suid-Afrika en kan 'n belangrike rol speel in die voorkoming van kanker. Verskillende in vitro so wei as in vivo studies het die volgende getoon: • Antimutageniese aktiwiteite teen die metabolies-geaktiveerde mutagene, 2- asetielaminofluoreen (2-AAF) en die mikotoksien, aflatoksien B1 (AFB1) in die Salmonella fyphimurium mutagenisiteitstoets. 'n Beperkte mate van beskerming is ook verleen teen die oksidatiewe mutageen, waterstofperoksied, sonder metaboliese aktivering. • Verhoogde aktiwiteite van die fase II ensieme, glutatioon S-tranferase alfa en UDP-glukuronidase, wat liggaamsvreemde verbindings metaboliseer. Die kruietees verlaag die oksidasietoestand soos weerspieel word deur 'n toename van gereduseerde glutatioon tot die geoksideerde vorm in die lewer van rotte wat 10 weke hierdie kruietees gedrink he!. Die kruietees het geen toksiese uitwerking op die rotte gehad nie. • Antimutageniese aktiwiteite van subselluiE~re fraksies van die lewer teenoor 2- AAF en AFB1 in die Salmonella toets. Die sitosolfraksie van die rotlewer bied beskerming teen die ge"induseerde mutagenese van beide mutagene, terwyl die mikrosomale fraksie ook die metaboliese aktivering van AFB1 na die aktiewe mutageniese metaboliet verminder. • In vivo modulering van kankerpromosie met behulp van bekende rotlewer en muisvel kankerontwikkelingsmodelle. In die lewermodel het die ongeprosesseerde kruietees beide die ontwikkeling en getal van GSTP+ fokusse onderskeidelik vertraag en verminder. In die geval van die velkankermodel het aanwending van polifenoliese fraksies van die kruietees beskerming gebied teen die ontwikkeling van velkankers by muise. Die aantal en grootte van die tumors het afgeneem terwyl die verskyning daarvan ook vertraag is. • Verskeie meganismes waardeur rooibos- en heuningboslee moonllik kanker kan moduleer word voorgeslel. Verskille in die polifenoliese sameslelling asook hul onderskeie konsenlrasies kan 'n belangrike rol speel in die kankerveranderende effekle van die lees. • Oal gereelde inname van rooibos- en/of heuningboslee moonllik 'n belangrike rol kan speel in die voorkoming van dieel- en omgewings-geYnduseerde kankers.