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.

Thesis (MSc Food Sc)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: The sensory profiles, phenolic composition and colour of honeybush infusions, prepared from six Cyclopia species (C. sessiliflora, C. longifolia, C. genistoides, C. intermedia, C. subternata and C. maculata), were determined to establish the variation between species. The results of the sensory study were used to create a honeybush sensory wheel and lexicon. The “characteristic” sensory profile of honeybush tea can be described as a combination of floral, sweet, fruity and plantlike flavours with a sweet taste and a slightly astringent mouthfeel. Sensory results indicated that the species could be divided into three distinct groups; group A (C. sessiliflora, C. intermedia and C. genistoides), group B (C. longifolia and C. subternata) and group C (C. maculata). Group A was associated with fynbos floral, fynbos sweet and plantlike attributes, group B with rose geranium and fruity sweet attributes and group C with woody, boiled syrup and cassia/cinnamon attributes. Gas chromatography-olfactometry analysis of the C. maculata aroma fraction indicated that the spicy note of its aroma could possibly be explained by the high concentration of the volatile component eugenol. However, none of the aroma impact volatiles had a specific cassia/cinnamon note. Large variation in the composition of the honeybush infusions was revealed through the quantification of the soluble solids, total polyphenol and individual monomeric polyphenolic compounds, as well as the absorbance (“colour”). Infusions of C. genistoides, C. longifolia and C. sessiliflora had the highest soluble solids and total polyphenol content, as well as the highest absorbance values. Only mangiferin, isomangiferin, hesperidin and compound C (unidentified compound) were detected in all six Cyclopia species. Cyclopia genistoides, C. longifolia and C. sessiliflora, in order of prominence, contained the highest concentration of both mangiferin and isomangiferin whereas C. genistoides and C. maculata contained the highest hesperidin content. The bitter taste present in certain Cyclopia species appeared to be due to a high mangiferin content, however, compounds such as isomangiferin and compound C might also have played a role. The effect of fermentation (oxidation) temperature (80°C and 90°C) and time (8 h, 16 h, 24 h and 32 h) of C. genistoides, C. subternata and C. maculata on the sensory characteristics of their infusions was also investigated. Fermentation for longer than 8 h resulted in an increase in positive sensory attributes and a decrease in negative sensory attributes rather than the formation of new sensory attributes. A fermentation temperature/time combination of 80°C/24 hours or 90°C/16 h was required for C. genistoides, C. subternata and C. maculata. Fermenting C. genistoides at 90°C would result in a honeybush infusion with slightly less rose geranium notes whereas C. subternata can be fermented at either 80°C or 90°C, depending on whether floral or apricot jam notes are desired. Cyclopia maculata should preferably not be fermented at 90°C due to an increase in negative sensory attributes (hay/dried grass and green grass). Fermentation reduced the soluble solids content, total polyphenol content, colour and concentration of individual polyphenolic compounds. Changes in the taste and mouthfeel of honeybush tea could be attributed to changes in the polyphenolic composition caused by the high temperature oxidation. Mangiferin associated with the bitter taste of C. genistoides, while in C. subternata astringency may be partly attributed to the mangiferin and isomangiferin content. The study substantiated the need for further research on the contribution of the major phenolic compounds towards the taste and mouthfeel of Cyclopia species. / AFRIKAANSE OPSOMMING: Die sensoriese profiel, fenoliese samestelling en kleur van heuningbostee, berei van ses Cyclopia spesies (C. sessiliflora, C. longifolia, C. genistoides, C. intermedia, C. subternata en C. maculata), is bepaal ten einde die mate van variasie vas te stel. Die resultate van die sensoriese studie is gebruik om 'n sensoriese wiel en leksikon vir heuningbostee te ontwikkel. Die “karakteristieke” sensoriese profiel van heuningbostee kan beskryf word as 'n kombinasie van blomagtig, soet, vrugtig en plantagtige geure met 'n soet smaak en 'n effense frankheid. Sensoriese resultate het aangedui dat die spesies in drie groepe verdeel kon word; groep A (C. sessiliflora, C. intermedia and C. genistoides), groep B (C. longifolia and C. subternata) en groep C (C. maculata). Groep A is met fynbos blom, fynbos-soet en plantagtige geure geassosieer, groep B met roos geranium en vrugtige-soet geure en group C met houtagtige, gekookte stroop en kassia/kaneel geure. Gaschromatografie-olfaktometrie analises van C. maculata se aroma fraksie het getoon dat die speseryagtige aroma moontlik as gevolg van die hoë konsentrasie van die vlugtige komponent, eugenol, kon wees. Geen van die aroma-impak vlugtige verbindings het egter 'n spesifieke kassia/kaneelagtige noot gehad nie. Groot variasie in die samestelling van heuningbostee ten opsigte van die inhoud van oplosbare vastestowwe, totale polifenole en monomeriese fenoliese verbindings, asook die absorbansie (“kleur”) is aangetoon. Heuningbostee berei van C. genistoides, C. longifolia en C. sessiliflora het die hoogste oplosbare vastestowwe en totale polifenol inhoud, asook die hoogste absorbansie waardes gehad. Slegs mangiferien, isomangiferien, hesperidien en verbinding C (ongeïdentifiseerde verbinding) is in al ses Cyclopia spesies geïdentifiseer. Cyclopia genistoides, C. longifolia en C. sessiliflora, in volgorde van belangrikheid, het die hoogste konsentrasie van beide mangiferien en isomangiferin gehad teenoor C. genistoides en C. maculata wat die hoogste hesperidien konsentrasie gehad het. Die bitter smaak teenwoordig in sekere Cyclopia spesies blyk moontlik as gevolg van die hoë mangiferien inhoud te wees, hoewel komponente soos isomangiferien en komponent C dalk ook 'n rol mag speel. Die effek van die fermentasie temperatuur (80°C en 90°C) en tyd (8 h, 16 h, 24 h en 32 h) van C. genistoides, C. subternata en C. maculata op die sensoriese eienskappe van heuningbostee is ondersoek. Fermentasie vir langer as 8 h het tot 'n toename in positiewe sensoriese eienskappe en afname in negatiewe sensoriese eienskappe gelei eerder as die ontstaan van nuwe sensoriese eienskappe. Om heuningbostee met 'n optimum sensoriese profiel te verkry is 'n fermentasie temperatuur/tyd kombinaise van 80°C/24 h of 90°C/16 h nodig vir C. genistoides, C. subternata en C. maculata. Cyclopia genistoides wat by 90°C gefermenteer word sal minder van die roos geranium note bevat, terwyl C. subternata by 80°C of 90°C gefermenteer kan word, afhangende of 'n blomagtige of 'n appelkooskonfyt noot verlang word. Fermentasie by 90°C word nie aanbeveel C. maculata nie as gevolg van die toename van sekere negatiewe sensoriese eienskappe (hooi/droe gras aroma en -geur en groen gras aroma). Fermentasie het die inhoud van oplosbare vastestowwe, totale polifenole, individuele polifenoliese verbindings, asook kleur verminder. Veranderinge in die smaak en mondgevoel van heuningbostee kon toegeskryf word aan die veranderinge in die polifenoliese inhoud as gevolg van die hoë temperatuur oksidasie. Mangiferien is met die bitter smaak van C. genistoides geassosieer, terwyl mangiferien and isomangiferien moontlik deels frankheid in C. subternata veroorsaak. Die studie het die noodsaaklikheid vir verdere navorsing op die bydrae van die hoof fenoliese verbindings tot die smaak en mondgevoel van Cyclopia spesies gestaaf.

Thesis (MScFoodSc)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: The flavanone aglycones, hesperetin and eriodictyol, have been identified as potential taste modulators with reported sweetness-enhancing and bitterness-masking properties, respectively. Reduction of the sugar content of food products has become important in view of the global obesity epidemic. Taste modulators have shown potential to enhance the sweet taste of reduced-sugar foods without unfavourably affecting their flavour profile. On the other hand, bitterness-masking taste modulators are useful to mask the bitter taste of functional phytochemical ingredients. In the current study, Cyclopia maculata (honeybush) was investigated as potential source of hesperetin- and eriodictyol-enriched extracts. Hesperetin and eriodictyol were present mainly below the quantification limit in C. maculata plant material, including unfermented leaf and stem material, unfermented and fermented tea, as well as the fermented by-product (< 40 mesh and > 12 mesh). Conversely, their rutinoside and modulatinginactive derivatives, hesperidin and eriocitrin were present at substantially higher concentrations in the plant material. The stems and by-product were shown to be good sources of hesperidin, but not eriocitrin. The qualitative and quantitative phenolic profile of the by-product was similar to that of the stems. The tea processing by-product was therefore selected to optimise extraction of flavanone glycosides for subsequent de-glycosylation of the flavanone glycosides to aglycones. The by-product was subjected to ultrasound-assisted extraction to investigate its potential as renewable source of the flavanone glycosides. Response surface methodology (RSM) was employed to optimise and study the individual and interactive effects of the process variables, namely ethanol concentration (% v/v), time (min), temperature (°C), and solvent:solid ratio (mL/g), on flavanone glycoside extraction. The hesperidin yield and content (of extract), as well as extract yield, increased with an increase in extraction time, temperature and solvent:solid ratio. Practical process restrictions limited global optimisation and only an optimum of 52.8% (v/v) ethanol for extract and hesperidin yield could be reached. Temperature was the parameter with the most significant effect (p < 0.05) on extraction efficiency among those studied. Practical process parameter values that were feasible for industrial application (52.8% (v/v) ethanol, 20 mL/g solvent:solid ratio, 60°C and 30 min) were selected for the preparation of a flavanone glycoside-enriched extract from the tea processing by-product. The flavanone glycoside-enriched extract was subjected to acid-catalysed hydrolysis to deglycosylate hesperidin and eriocitrin to hesperetin and eriodictyol, respectively. RSM was employed to optimise the acid hydrolysis process and to study the effect of the hydrolysis parameters (temperature (°C) and time (min)) on hydrolysis efficiency. At the maximum temperature (92.1°C) and corresponding optimum time (98.4 min) ca 80% conversion of hesperidin to hesperetin was achieved. Substantially more eriodictyol formed during acid hydrolysis than eriocitrin present in the initial extract owing to the deglycosylation of unidentified glycosides with the same aglycone. Unidentified breakdown products imparting a red colour to the acid-hydrolysed extract were also observed. The total phenolic content of the acid-hydrolysed extract was significantly higher (p < 0.05) than that of the unhydrolysed extract, indicating the formation of unidentified compounds with the ability to reduce the Folin-Ciocalteau reagent, although no significant difference (p ≥ 0.05) between the antioxidant activities of these extracts, as assessed with the DPPH radical scavenging and ORAC assays, was observed. The potential of enzymatic bioconversion as an alternative to acid-catalysed hydrolysis was investigated using commercial hesperidinase. Bioconversion resulted only in de-rhamnosylation with ca 100% conversion of hesperidin to hesperetin-7-O-glucoside in an aqueous C. maculata extract at pH 4.0 and 40°C. / AFRIKAANSE OPSOMMING: Die flavanoon aglikone, hesperetien and eriodiktiol, is geïdentifiseer as potensiële smaakmoduleerders met berigte soetheid-versterkende en bitter-maskerende eienskappe, onderskeidelik. Vermindering van die suikerinhoud van voedselprodukte het belangrik geword in die lig van die wêreldwye vetsugepidemie. Smaakmoduleerders het die potensiaal getoon om die soet smaak van voedsel met verlaagde suikerinhoud te versterk sonder om hul geurprofiel ongunstig te beïnvloed. Andersyds is bittermaskerende smaakmoduleerders nuttig om die bitter smaak van funksionele fitochemiese bestanddele te maskeer. In die huidige studie is Cyclopia maculata (heuningbos) ondersoek as ‘n potensiële bron van hesperetien- and eriodiktiol-verrykte ekstrakte. Hesperetien and eriodiktiol was hoofsaaklik teenwoordig onder die kwantifiseringsperk in C. maculata plantmateriaal, insluitend ongefermenteerde blaar- en stokmateriaal, ongefermenteerde en gefermenteerde tee, asook die gefermenteerde byproduk (< 40 maas en > 12 maas). Hierteenoor was hul rutinosiedes en modulerend-onaktiewe derivate, hesperidien and eriositrien, teenwoordig in aansienlik hoër konsentrasies in die plantmateriaal. Die stokmateriaal en byproduk is getoon om goeie bronne van hesperidien, maar nie eriositrien nie, te wees. Die kwalitatiewe en kwantitatiewe fenoliese profiel van die byproduk was soortgelyk aan dié van die stokke. Die teeprosesseringsbyproduk is dus geselekteer om die ekstraksie van flavanoonglikosiede, voorafgaande hul de-glikosilering na aglikone, te optimeer. Die byproduk is aan ekstraksie met behulp van ultrasoniese klank onderwerp om die potensiaal daarvan as hernubare bron van flavanoonglikosiede te ondersoek. Respons-oppervlak Metodologie (ROM) is gebruik om die individuele en wisselwerking effekte van die proses veranderlikes, naamlik etanolkonsentrasie (% v/v), tyd (min), temperatuur (°C), en oplosmiddel:vastestof verhouding (mL/g), op flavanoonglikosied ekstraksie te optimiseer en te bestudeer. Die hesperidienopbrengs en -inhoud (van ekstrak), sowel as die ekstrakopbrengs, het toegeneem met ‘n toename in die ekstraksietyd, - temperatuur en oplosmiddel:vastestof verhouding. Praktiese prosesbeperkings het die globale optimisering beperk en slegs ‘n optimum van 52.8% (v/v) etanol vir ekstrak- en hesperidienopbrengs kon bereik word. Temperatuur was die parameter met die mees beduidende effek (p < 0.05) op ekstraksie doeltreffendheid van dié wat bestudeer is. Praktiese prosesparameterwaardes wat haalbaar is vir industriële toepassing (52.8% (v/v) etanol, 20 mL/g oplosmiddel:vastestof verhouding, 60°C en 30 min) is geselekteer vir die voorbereiding van 'n flavanoonglikosied-verrykte ekstrak uit die teeprosesseringsbyproduk. Die flavanoonglikosied-verrykte ekstrak is aan suur-gekataliseerde hidrolise onderwerp om hesperidien en eriositrien na hesperetien en eriodiktiol, onderskeidelik, te de-glikosileer. ROM is gebruik om die suurhidrolise proses te optimeer en die effek van die hidrolise parameters (temperatuur (°C) en tyd (min)) op hidrolise doeltreffendheid te bestudeer. Ongeveer 80% omskakeling van hesperidien na hesperetien is behaal teen die maksimum temperatuur (92.1 °C) en ooreenstemmende optimum tyd (98.4 min). Aansienlik meer eriodiktiol is tydens suurhidrolise gevorm as eriositrien wat in die oorspronklike ekstrak teenwoordig was, as gevolg van de-glikosilering van ongeïdentifiseerde glikosiede met dieselfde aglikoon. Ongeïdentifiseerde afbreekprodukte, wat 'n rooi kleur aan die suurgehidroliseerde ekstrak gegee het, is ook waargeneem. Die totale fenoliese inhoud van die suurgehidroliseerde ekstrak was beduidend hoër (p < 0.05) as dié van die ongehidroliseerde ekstrak, wat die vorming van onbekende verbindings met die vermoeë om die Folin-Ciocalteau reagens te reduseer aandui, hoewel daar geen beduidende verskil (p ≥ 0.05) tussen die antioksidant-aktiwiteite van hierdie ekstrakte, soos bepaal met die DPPH radikaal blussings- en ORAC toetse, waargeneem is nie. Die potensiaal van ensiematiese bio-omskakeling as 'n alternatief vir suur-gekataliseerde hidrolise is ondersoek met behulp van kommersiële hesperidinase. Bio-omskakeling het slegs tot de-ramnosilering gelei met ca 100% omskakeling van hesperidien na hesperetien-7-O-glukosied in 'n C. maculata waterekstrak by pH 4.0 en 40°C.

Thesis (MScFoodSc)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: In light of the limited and inconsistent supply of good quality honeybush tea, a species-specific sensory profile and the physicochemical characteristics of Cyclopia intermedia (honeybush) tea were determined to ultimately establish the optimum fermentation parameters for this herbal tea on laboratory-scale and to validate these findings on commercial-scale. The characteristic sensory profile of C. intermedia can be described as sweet tasting and slightly astringent with a combination of “fynbos-floral”, “fynbos-sweet”, “fruity” (specifically “apricot jam”, “cooked apple”, “raisin” and “lemon/lemon grass”), “woody”, “caramel/ vanilla” and “honey-like” aromas. The flavour can be described as distinctly “fynbos-floral”, “fynbos-sweet” and “woody”, including hints of “lemon/lemon grass” and “hay/dried grass”. The results of the sensory study were used to create a C. intermedia sensory wheel and lexicon, and an elementary grading system that categorised samples into “good”, “average” and “poor” sensory quality was proposed. Physicochemical parameters, i.e. soluble solids (SS) content, absorbance as a measure of colour, and turbidity, were evaluated as possible rapid predictors of sensory quality. High SS content, absorbance and turbidity correlated strongly with “poor” sensory quality. A linear relationship existed between the physicochemical parameters. The effect of fermentation temperature (70, 80 and 90°C) and time (12, 16, 24, 36, 48 and 60 h) on the sensory and physicochemical characteristics of C. intermedia was determined on laboratory-scale. Increasing fermentation time increased the intensity of positive sensory attributes, while decreasing the intensity of negative sensory attributes. The SS content, colour and turbidity of infusions decreased with increasing fermentation time, while the SS content and turbidity of infusions increased with increasing fermentation temperature. Fermentation at 90°C for 36 h on laboratory-scale produced C. intermedia with the best sensory properties, while preserving the SS content and colour of infusions. Fermentation at 70°C and 80°C required longer fermentation times for development of positive sensory attributes. Fermentation at 90°C was subsequently validated on commercial-scale. Laboratory-scale fermentation of the same batches of plant material was also carried out concurrently to allow direct comparison of the scale of fermentation on tea quality. Commercial-scale fermentation, despite increased variability as a result of increased batch volumes and heating difficulties, produced C. intermedia of “good” sensory quality after 24 and 36 h of fermentation. Increasing fermentation time had little effect on the SS content and colour of infusions of tea produced on commercial-scale, but turbidity increased significantly after 36 h. Thus, to produce C. intermedia with consistently good quality on commercial-scale, fermentation at 90°C for 24 to 36 h is recommended. Increasing fermentation time past 48 h should be avoided to prevent turbidity and the development of sensory attributes characteristic of over-fermented tea. However, due to the large variability of commercial-scale honeybush tea production, it is recommended that each batch be monitored between 24 and 36 h to determine when optimum fermentation has been obtained. / AFRIKAANSE OPSOMMING: Beperkte en wisselvallige beskikbaarheid van goeie gehalte heuningbostee noodsaak die optimisering van fermentasie parameters vir Cyclopia intermedia. Optimisering van fermentation parameters is op laboratorium skaal gedoen, gevolg deur validasie van die parameters op kommersiële skaal. Vooraf is die spesie-spesifieke sensoriese profiel en die fisies-chemiese eienskappe van C. intermedia tee bepaal. Die kenmerkende sensoriese profiel van C. intermedia kan beskryf word as soet en effens vrank met 'n kombinasie van "fynbos-blomagtige", "fynbos-soet", "vrugtige" (spesifiek "appelkooskonfyt", "gekookte appel", "rosyntjie” en “suurlemoen/sitroen gras"), "houtagtige", "karamel/vanilla" en "heuningagtige" aromas. Die smaak kan beskryf word as "fynbos-blomagtig", "fynbos-soet" en "houtagtig", met 'n tikkie "suurlemoen/sitroen gras" en "hooi/gedroogde gras". Die resultate van die sensoriese studie is gebruik om 'n C. intermedia sensoriese wiel en leksikon, asook 'n basiese graderingstelsel wat tee monsters in "goeie", "gemiddelde" en "swak" sensoriese kwaliteit klassifiseer, te ontwikkel. Fisies-chemiese parameters: oplosbare vastestof (SS) inhoud; absorbansie as 'n maatstaf van kleur; en troebelheid, is geëvalueer as moontlike indikasies van sensoriese kwaliteit. Hoë SS inhoud, absorbansie en troebelheid waardes het sterk met "swak" sensoriese kwaliteit gekorreleer. 'n Lineêre verwantskap bestaan tussen die fisies-chemiese parameters en kwaliteit. Die effek van fermentasie temperatuur (70, 80 en 90°C) en -tyd (12, 16, 24, 36, 48 en 60 h) op die sensoriese en fisies-chemiese eienskappe van C. intermedia is op laboratorium skaal bepaal. Verlenging van fermentasie tyd het die intensiteit van die positiewe sensoriese eienskappe verhoog, terwyl dit die intensiteit van negatiewe sensoriese eienskappe verminder het. Die SS inhoud, kleur en troebelheid van die tee het met verlengde fermentasie tyd afgeneem, terwyl die SS inhoud en troebelheid met verhoging van fermentasie temperatuur toegeneem het. Fermentasie by 90°C vir 36 h op laboratorium skaal het tee met die beste sensoriese eienskappe geproduseer, met behoud van die SS inhoud en kleur. Fermentasie by 70°C en 80°C het 'n langer fermentasie tyd vir die ontwikkeling van positiewe sensoriese eienskappe vereis. Fermentasie by 90°C is daaropvolgens op kommersiële skaal uitgevoer, met gelyktydige laboratorium skaal fermentasie van dieselfde plantmateriaal lotte om die direkte effek van die skaal van fermentasie op tee kwaliteit te bepaal. Kommersiële fermentasie, ten spyte van verhoogde wisselvalligheid as gevolg van groot volumes tee en probleme met verhitting, het tee van "goeie" sensoriese kwaliteit na fermentasie periodes van 24 en 36 h geproduseer. Verlenging van fermentasie tyd het min uitwerking op die SS inhoud en kleur van kommersiel gefermenteerde tea gehad, maar troebelheid het beduidend na 36 h toegeneem. Fermentasie by 90°C vir 24 - 36 h word gevolglik aanbeveel om tee met goeie gehalte op kommersiële skaal te produseer. Fermentasie vir langer as 48 h moet vermy word om troebelheid te voorkom en die ontwikkeling van sensoriese eienskappe kenmerkend van oor-gefermenteerde tee te vermy. As gevolg van faktore wat groot variasie in kommersiële skaal heuningbostee produksie kan teweegbring, word aanbeveel dat elke produksielot tussen 24 en 36 h gemonitor word om die optimum fermentasie tyd te bepaal.

Thesis (MSc Food Sc)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: Application of steam treatment to green Cyclopia maculata and C. longifolia to modulate their aroma and flavour profile, without detrimental effects on colour and individual phenolic content, was investigated. Steam treatment (96 °C, atmospheric pressure) of different time periods was applied to the shredded, fresh plant material before drying (STBD; 0, 30, 60, 90 and 120 s) or the herbal tea product after drying (STAD; 0, 1, 2, 3, and 4 min). Steam treatment of 60 s or longer resulted in a significant (p < 0.05) decrease in vegetative (‘green grass’ and ‘hay/dried grass’) and cereal (‘oats/porridge/grains’) aroma and flavour intensities, with an increase in some fruity (‘tropical fruit’ and ‘guava’) and ‘fruity-sweet' aroma intensities. These changes manifested to a greater degree for plant material subjected to STBD, while they were less prominent for C. longifolia compared to C. maculata. Additionally, 19 aroma compounds were instrumentally identified in the volatile fraction of C. maculata infusions prepared from STBD plant material. Of these, seven correlated well with green honeybush vegetative and cereal aroma attributes associated with unsteamed samples. Steaming thus resulted in the rapid loss of some of these highly volatile ‘green’-associated aroma compounds. Steam treatment of more than 60 s, regardless of STBD or STAD, was not detrimental to green colour, and individual phenolic content was not greatly affected. The 30 s STBD treatment of C. maculata, however, led to the rapid loss of green colour and oxidation of phenolic compounds, without considerable aroma improvement. This highlights the need for process control during green honeybush production. Storage stability of steamed (60 s, STBD) and unsteamed green C. maculata was investigated under low (0 °C; moisture impermeable packaging) and normal (25 °C at 60% relative humidity (RH); semi-moisture-permeable sachets) temperature storage conditions (LTS and NTS, respectively) over 6 months and high temperature storage (HTS) conditions (40 °C at 75% RH; semimoisture- permeable sachets) for 1 month. HTS conditions, after 1 month, seemed to emulate changes occurring over 6 months at NTS conditions, with little change detected at LTS conditions. Despite noticeable green colour loss over the respective storage periods at NTS and HTS conditions, individual phenolic compounds were not severely affected. NTS and HTS led to the progressive development of sought-after fermented honeybush sensory attributes, especially in unsteamed samples. These include prominent fruity (‘stewed fruit’, ‘apricot jam’ and ‘marmalade’) aromas and flavours, ‘general sweet’ and ‘fruity-sweet’ aromas and a sweeter taste. The collated descriptive sensory data were used to generate a preliminary sensory wheel for green honeybush aroma, and another for flavour, taste and mouthfeel. The sensory profile of green honeybush was finally described as a dominant vegetative aroma and flavour, prominent sweetassociated and slightly fruity aroma, with sweet and notably bitter tastes and an astringent mouthfeel. By combining the sensory data, it was clear that the major effects of storage outweighed those of steam treatments, suggesting that storage of 3 to 6 months may improve sensory quality, although colour may be slightly compromised. Immediate sensory manipulation and thus improvement, however, may be achieved to a lesser degree in the short term by STBD. / AFRIKAANSE OPSOMMING: Die toepassing van stoombehandeling van groen Cyclopia maculata en C. longifolia vir die modulasie van hul aroma en geur profiel, sonder gepaardgaande nadelige gevolge op kleur en individuele fenoliese inhoud, is ondersoek. Stoombehandeling (96 °C, atmosferiese druk) van verskeie tydstippe is toegepas op die gekerfde, vars plantmateriaal voor droging (SBVD; 0, 30, 60, 90 en 120 s) of die groen teeproduk ná droging (SBND; 0, 1, 2, 3 en 4 min). Stoombehandeling van 60 s of langer het tot 'n betekenisvolle (p < 0.05) afname in vegetatiewe (‘groen gras’ en ‘hooi/gedroogde gras’) en graan (‘hawer/pap/graan’) aroma en geur intensiteite gelei, met 'n toename in sommige vrugtige (‘tropiese vrugte’ en ‘koejawel’) en ‘vrugtige-soet’ aroma intensiteite. Hierdie veranderinge was meer merkbaar in die SBVD behandeling, terwyl C. longifolia minder vatbaar vir hierdie veranderinge was as C. maculata. Daarbenewens is 19 aromaverbindings in die vlugtige fraksie van C. maculata aftreksel voorberei van SBVD plantmateriaal instrumenteel geïdentifiseer. Sewe hiervan het goed gekorreleer met heuningbos vegetatiewe en graan aromas wat verband hou met ongestoomde monsters. Stoombehandeling het dus gelei tot die vinnige verlies van sommige van hierdie baie vlugtige ‘groen’-geassosieërde aromaverbindings. Stoombehandeling vir langer as 60 s, ongeag van SBVD of SBND, het nie die groen kleur en individuele fenoliese inhoud grootliks nadelig beïnvloed nie. ‘n SBVD behandeling van 30 s het egter tot die vinnige verlies van groen kleur en oksidasie van fenoliese verbindings, sonder aansienlike aroma verbetering van C. maculata gelei. Dít beklemtoon die noodsaaklikheid vir prosesbeheer tydens groen heuningbosproduksie. Stabiliteit van gestoomde (60 s, SBVD) en ongestoomde groen C. maculata tydens opberging onder lae (0 °C; vog-ondeurlaatbare verpakking) en normale (25 °C by 60% relatiewe humiditeit (RH); semi-vogdeurlaatbare sakkies) temperatuur opbergingtoestande (LTO en NTO, onderskeidelik) oor 6 maande en hoë temperatuur opbergingtoestande (40 °C by 75% RH; semivogdeurlaatbare sakkies; HTO) vir 1 maand, is ondersoek. HTO het na 1 maand soortgelyke veranderinge teweeggebring as NTO na 6 maande, terwyl min verandering by LTO bespeur is. Ten spyte van merkbare groen kleur verlies oor die onderskeie tydperke by NTO en HTO is individuele fenoliese verbindings min beinvloed. NTO en HTO het gelei tot die progresiewe ontwikkeling van gesogte sensoriese eienskappe van gefermenteerde heuningbos, veral in ongestoomde monsters. Dit sluit in prominente vrugtige (‘gestoofde vrugte’, ‘appelkooskonfyt’ en ‘marmelade’) aromas en geure , ‘algemene soet’ en ‘vrugtige-soet’ aromas en 'n soeter smaak. Al die beskrywende sensoriese data is saamgestel om 'n voorlopige sensoriese wiel vir groen heuningbos aroma op te stel, asook een vir geur, smaak en mondgevoel. Die sensoriese profiel van groen heuningbos kan beskryf word as 'n oorheersende vegetatiewe aroma en geur, prominente soet-verwante en effens vrugtige aroma, met soet en veral bitter smaak en ‘n vrank mondgevoel. Deur die sensoriese data te kombineer kon duidelik uitgewys word dat die belangrikste gevolge van opberging dié van stoombehandeling oortref. Dit dui daarop dat opberging van 3 tot 6 maande sensoriese kwaliteit kan verbeter, hoewel kleur effens benadeel kan word. In die kort termyn kan onmiddellike sensoriese manipulasie en dus verbetering egter in 'n mindere mate deur SBVD bereik word.

Thesis (PhD (Chemistry and Polymer Science))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: Honeybush tea, also known as “South Africa’s sweetest tea”, is a herbal tea made from the leaves and twigs of Cyclopia spp., indigenous to the fynbos biome in the Western and Eastern Cape provinces of South Africa. The pleasant sweet aroma and taste of fermented honeybush, its low tannin content and the absence of caffeine have led to widespread interest in the commercial cultivation and processing of honeybush tea since the mid-1990s. Although more than 20 species of honeybush grow in the wild, only a few species are commercially exploited for the manufacture of tea. Currently the more prominent species are C. intermedia, C. subternata, C. genistoides, and C. sessiliflora. The present research contributes to a comprehensive honeybush research programme being conducted at the Agricultural Research Council (ARC) Infruitec-Nietvoorbij in South Africa. The first phase of the present study, using C. genistoides as representative species, was aimed at developing the necessary methodology for the analysis of extremely low concentrations of honeybush volatiles. A high-capacity headspace sample enrichment probe was applied successfully in conjunction with gas chromatography-mass spectrometry (GC-MS) to analyse the volatile organic compounds present in dry or infused unfermented and fermented honeybush. A total number of 255 volatile compounds were identified in unfermented and fermented honeybush, the majority of which are terpenoids (138; 54%) comprising mostly terpenes, terpene ketones, terpene alcohols and terpene ethers. Of the other compound classes, the aldehydes are the largest group, followed by esters, hydrocarbons and ketones. The stereochemistry of the identified compounds was determined whenever possible. This is the most comprehensive chemical characterisation of the volatile compounds in a South African herbal plant reported to date. A comparative study of green and fermented honeybush showed that the same compounds are, to a large extent, present in both, albeit in different relative concentrations. Not all of the identified honeybush volatiles are necessarily odour-active compounds contributing to the overall typical honeybush aroma. An important aspect of this research was thus the identification of the 46 odour-active compounds in fermented honeybush by means of gas chromatography-olfactometry (GC-O), using detection frequency and aroma extract dilution analysis methods. Fifteen of these compounds, mainly terpenoids, were singled out as the most intense individual contributors to the honeybush aroma based on consideration of all the relevant GC-O data. The odours of certain compounds, i.e. (6E,8Z)-megastigma-4,6,8-trien-3-one, (6E,8E)-megastigma- 4,6,8-trien-3-one, (7E)-megastigma-5,7,9-trien-4-one, 10-epi- -eudesmol, epi- -muurolol and epi- - cadinol, were perceived by GC-O assessors as typically honeybush-like. The quantitative GC-MS data of seven different Cyclopia samples (including four different species and variants thereof) were compared with respect to all the volatile components and particularly with respect to the odour-active compounds. Interesting variations were found in the concentrations of certain odour-active compounds in the various samples. The quantitative data obtained for the odour-active honeybush volatiles and data obtained from the sensory analysis of eight Cyclopia samples (including four different species and variants thereof) were subjected to statistical analysis and interesting associations between compounds with certain sensory aroma attributes were established. The present study has made a major contribution to the scientific knowledge regarding one of South Africa’s most popular indigenous herbal teas. / AFRIKAANSE OPSOMMING: Heuningbostee, wat ook bekend staan as “Suid-Afrika se soetste tee”, word gemaak van die blare en takkies van Cyclopia spp. wat inheems is en voorkom in die fynbosbioom van die Wes- en Oos-Kaapprovinsies van Suid-Afrika. Die aangename soet smaak en aroma van gefermenteerde heuningbos, die lae tannnien-inhoud en die feit dat die tee kafeïenvry is, het gelei tot belangstelling in die kommersiële verbouing en prosessering van heuningbostee gedurende die 1990s. Meer as 20 heuningbosspesies kom in die natuur voor, maar slegs ‘n paar spesies word kommersieel verbou vir die vervaardiging van heuningbostee waarvan C. intermedia, C. subternata, C. genistoides en C. sessiliflora tans die belangrikste spesies is. Die navorsing maak deel uit van ‘n omvattende heuningbos navorsingsprogram wat onder leiding staan van die Landbounavorsingsraad Infruitec- Nietvoorbij in Suid-Afrika. In die eerste fase van die huidige studie is die nodige analitiese metodologie ontwikkel vir die monsterneming en analise van die vlugtige organiese verbindings wat in uiters lae konsentrasies in heuningbos voorkom, deur van ‘n verteenwoordigende spesie, C. genistoides, gebruik te maak. ‘n Sogenaamde “sample enrichment probe” (SEP) is ontwikkel en suksesvol in kombinasie met gaschromatografie-massaspektrometrie (GC-MS) aangewend vir die analise van die vlugtige verbindings aanwesig in die bodamp van sowel droë plantmateriaal as infusies van ongefermenteerde en gefermenteerde heuningbos. ‘n Totaal van 255 vlugtige verbindings is geïdentifiseer, waarvan die meeste hoofsaaklik terpenoïede is (138, 54%) en terpene, terpeenketone, terpeenalkohole en terpeeneters insluit. Die ander verbindingsgroepe, waarvan die aldehiede die grootste groep is, sluit in esters, koolwaterstowwe en ketone. Indien haalbaar, is die stereochemie van die geïdentifiseerde verbindings ook bepaal. Hierdie studie is die mees omvattende chemiese karakterisering van die vlugtige verbindings in ‘n Suid-Afrikaanse kruieplant wat tot dusver onderneem is. ‘n Vergelykende studie het getoon dat ongefermenteerde en gefermenteerde heuningbos tot ‘n groot mate dieselfde verbindings, hoewel in verskillende relatiewe konsentrasies, bevat. Nie al die geïdentifiseerde vlugtige verbindings in heuningbos is noodwendig aroma-aktiewe verbindings wat ‘n bydrae tot die algehele tipiese heuningbosaroma lewer nie en daarom was die identifisering van die 46 aroma-aktiewe verbindings in geferementeerde heuningbos deur gebruik te maak van gaschromatografie-olfaktometrie (GC-O) deur middel van deteksiefrekwensie en aroma ekstrak verdunningsanalise, ‘n belangrike aspek van die navorsing. Na oorweging van al die tersaaklike GC-O data is 15 van hierdie verbindings, hoofsaaklik terpenoïede, uitgesonder as die verbindings wat die belangrikste bydrae tot die heuningbosaroma lewer. Die reuke van sekere van die verbindings, nl. (6E,8Z)-megastigma-4,6,8-triën-3-oon, (6E,8E)-megastigma-4,6,8-triën-3-oon, (7E)-megastigma-5,7,9-triën-4-oon, 10-epi- -eudesmol, epi- -muurolol, en epi- -cadinol, is deur sommige van die GC-O paneellede as tipies heuningbosagtig beskryf. Die kwantitatiewe GC-MS data van sewe verskillende Cyclopia monsters (insluitende vier verskillende spesies en variante daarvan) is vergelyk met betrekking tot al die vlugtige verbindings, asook veral met betrekking tot die aroma-aktiewe verbindings. Interessante variasies in die konsentrasies van sekere aroma-aktiewe verbindings is in die verskillende monsters waargeneem. Die kwantitatiewe data van die aroma-aktiewe heuningbosverbindings en data verkry uit die sensoriese analise van agt Cyclopia monsters (insluitende vier verskillende spesies en variante daarvan), is onderwerp aan statistiese analises waaruit interessante assosiasies tussen verbindings met sekere sensoriese aroma-eienskappe waargeneem is. Hierdie studie lewer ‘n groot bydrae tot die wetenskaplike kennis aangaande een van Suid- Afrika se mees populêre inheemse kruietees.

Thesis (MScFoodSc)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: The phenolic composition of Cyclopia species is believed to be partially responsible for the numerous health promoting properties associated with their extracts. Current quality control measures do not accommodate variation in phenolic profiles of Cyclopia species. In this study, comprehensive high performance liquid chromatography (HPLC) methods were developed for the improved characterisation of the phenolic composition of aqueous extracts of two Cyclopia species (C. subternata and C. maculata). The methods were developed to be suitable for both routine quantitative analysis on conventional HPLC instrumentation, and the construction of chromatographic fingerprints for further data analysis. The latter entailed similarity analysis and prediction of total antioxidant capacity (TAC). Using a methodical approach, two separate HPLC methods, using diode array detection (DAD), were developed and validated for the analysis of aqueous extracts prepared from unfermented (green) and fermented plant material of C. subternata and C. maculata. Separation was achieved using the same method parameters (column, temperature, mobile phases), except for differing mobile phase gradients. Hyphenation of the developed HPLC methods with mass spectrometry (MS) and tandem MS allowed the confirmation of phenolic compounds previously identified in Cyclopia, and the tentative identification of several additional compounds in Cyclopia species, which are reported here for the first time. These included apigenin-6,8-di- C-glucoside, 3-hydroxyphloretin-30,50-di-C-hexoside, eriodictyol-di-C-glucoside, iriflophenone-di-O,C-hexoside, hydroxymangiferin and hydroxyisomangiferin. Subsequently, a large number of aqueous extracts of randomly selected green C. subternata (n = 64) and C. maculata (n = 50) plant material samples were analysed. Large quantitative variations were observed on intra- and inter-species levels. Cyclopia maculata extracts contained almost six times more mangiferin than extracts from C. subternata. HPLC-DAD analysis produced duplicate fingerprints for each extract which were consequently used for further analysis. The chromatographic fingerprint of a bioactive extract of each species was included in the respective data sets. Similarity analysis was conducted between the fingerprints from the randomly selected extracts and the corresponding active extract. For each species several extracts were determined to have similar “activity” as that of the active extract (n = 15 for C. subternata and n = 45 for C. maculata). Compounds potentially responsible for the activity were tentatively identified with the aid of principal component analysis (PCA) in combination with similarity analysis. PCA was more effective in identifying small differences between fingerprints than similarity analysis based on the correlation coefficients (r) alone. Furthermore, multivariate data analysis was used to construct partial least squares (PLS) regression models for the prediction of TAC from fingerprint data of each species, and available data from two microplate TAC assays. The construction of the models was successful with reasonable errors (< 10%), and permitted the determination of compounds of interest for future research. These included compounds of known identity that had large positive contributions toward the predictions of TAC, or unknown compounds that had small UV signals, but relatively large positive contributions to the models. / AFRIKAANSE OPSOMMING: Die talle gesondheidbevorderingseienskappe van ekstrakte van Cyclopia spesies word gedeeltelik geassosieer met hul fenoliese samestelling. Huidige kwaliteitskontrolemaatreëls is nie in staat om die variasie wat in die fenoliese profiele van die spesies voorkom, te akkommodeer nie. Omvattende hoë druk vloeistof chromatografiese (HPLC) metodes is vir twee Cyclopia spesies, naamlik C. subternata en C. maculata, in hierdie studie ontwikkel vir beter karakterisering van die fenoliese samestelling van waterekstrakte van dié spesies. Die metodes moes ook geskik wees vir roetine analise van C. subternata en C. maculata ekstrakte op konvensionele HPLC instrumentasie, en vir die opstel van chromatografiese vingerafdrukke (fenoliese samestellingsprofiele) vir verdere data analise, soos gelykvormigheidsanalise en die voorspelling van die totale antioksidantkapasiteit (TAC). Twee HPLC metodes, wat van ’n ultraviolet-diode detektor (DAD) gebruik maak, is ontwikkel deur ’n sistematiese benadering te volg. Die onderskeie metodes is vir die ontleding van waterekstrakte van groen (ongefermenteerde) en gefermenteerde plantmateriaal van C. subternata en C. maculata gevalideer. Ongeag die spesie is optimale skeiding met dieselfde kolom, mobiele fase en kolom-temperatuur bereik, maar met verskillende mobiele fase gradiënte. Analise met massaspektrometrie (MS) en tandem MS het die teenwoordigheid van fenoliese verbindings, wat voorheen in Cyclopia spesies geidentifiseer is, bevestig. Verder is ook ’n aantal verbindings vir die eerste keer in Cyclopia tentatief geidentifiseer. Dit sluit apigenien-6,8-di-C-glukosied, 3- hidroksiefloretien-30,50-di-C-heksosied, eriodiktiol-di-C-glukosied, iriflofenoon-di-O,C-heksosied, hidroksiemangiferien en hidroksie-isomangiferien in. Vervolgens is ’n groot aantal ewekansig gekose waterekstrakte van beide groen C. subternata (n = 64) en C. maculata (n = 50) plantmateriaal geanaliseer, en groot kwantitatiewe variasie op intra- en inter-spesievlak waargeneem. Cyclopia maculata ekstrakte het byvoorbeeld byna ses maal die mangiferieninhoud van C. subternata ekstrakte gehad. HPLC-DAD analise van die ekstrakte het duplikaat vingerafdrukke van elke ekstrak geproduseer, wat vir verdere data analise gebruik is. Die chromatografiese vingerafdruk van ’n bioaktiewe ekstrak van elke spesie was by die onderskeie datastelle ingesluit. Gelykvormigheidsanalise is tussen vingerafdrukke van die ewekansig gekose ekstrakte en die ooreenstemmende aktiewe ekstrak uitgevoer. Vir elke spesie is ’n aantal “aktiewe” ekstrakte aangewys (n = 15 vir C. subternata en n = 45 vir C. maculata). Die verbindings wat potensieel verantwoordelik kan wees vir die aktiwiteite is met behulp van hoofkomponentontleding (PCA) in kombinasie met gelykvormigheidsanalise, tentatief aangewys. PCA was egter meer effektief om klein verskille tussen vingerafdrukke aan te dui, in vergelyking met gelykvormigheidsanalise wat slegs op die korrelasie koëffisiënt (r) gebaseer is. Meerveranderlike data analiese is gebruik om “gedeeltelike kleinste kwadrate” (PLS) regressiemodelle, vir die voorspelling van die TAC van beide spesies te bou. Die voorspelling is gebaseer op hul vingerafdruk data en TAC data van twee TAC mikroplaat metodes. Die model-konstruksie was suksesvol met aanvaarbare voorspellingsfoute (< 10%). Verbindings van belang kon ook bepaal word. Dit sluit bekende verbindings in wat groot positiewe bydraes ten opsigte van die voorspelling van TAC getoon het, asook ongeidentifiseerde verbindings wat klein UV-seine getoon het, maar relatiewe groot bydraes tot die modelle gehad het.

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.

Thesis (MScMedSc)--Stellenbosch University, 2012. / Includes bibliography / ENGLISH ABSTRACT: Obesity is a major source of morbidity and mortality worldwide. More than 1.5 billion individuals over the age of 20 years are overweight, with more than 500 million of these individuals being obese. Obesity increases the risk of developing cardiovascular disease, type 2 diabetes and certain types of cancer. Recently, a number of plant extracts have been shown to possess anti-obesity properties in vitro and in various animal models of obesity. The aim of this study was to investigate the effect of a hot water fermented extract of Cyclopia maculata, a South African herbal tea more commonly referred to as honeybush, on lipogenesis and lipolysis in 3T3-L1 pre-adipocytes and adipocytes. To investigate the effect of C. maculata extract on adipogenesis, 3T3-L1 preadipocytes were differentiated in adipogenesis inducing media containing various concentrations. The optimal concentration was determined by screening concentrations ranging from 0 to 1,600 μg/ml. 3T3-L1 pre-adipocytes were differentiated with TNFα or unsupplemented adipogenesis inducing media as positive and negative controls, respectively. Intracellular lipid accumulation was measured by using the Oil O Red stain and a commercial triglyceride assay kit. Cell viability was measured using the 3-(4,5-Dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) and adenosine tri-phosphate (ATP) assays. The expression of PPARγ, C/EBPα, SREBP-1 and PPARα was assessed by Western blot analysis, while the expression of the secreted proteins leptin and adiponectin was assessed by ELISA. The effect of C. maculata extract on lipolysis was investigated by differentiating 3T3-L1 pre-adipocytes in adipogenesis inducing and adipogenesis maintenance media for 8 days until they were mature adipocytes, and thereafter treating with C. maculata extract for 24 hours. The optimal concentration was determined by screening concentrations ranging from 0 to 1,600 μg/ml. Isoproteronol or unsupplemented adipogenesis maintenance media was used as positive and negative controls, respectively. Intracellular lipid break down was measured by using the Oil O Red stain, while glycerol release, a marker of lipolysis, was measured using a commercial kit. Cell viability was measured using the MTT and ATP assays. The expression of HSL and perilipin was assessed by Western blot analysis, while the expression of secreted proteins leptin and adiponectin was assessed by ELISA. Treatment with the C. maculata extract, at most of the concentrations tested, decreased intracellular lipid accumulation in pre-adipocytes. The Oil O Red and the intracellular triglyceride assay, in combination with the cell viability assays, showed that 80 μg/ml optimally reduced intracellular lipid without affecting cell viability. Western blot analysis showed that differentiation of 3T3-L1 adipocytes in the presence of 80 μg/ml of the C. maculata extract decreased the expression of PPARγ2, a key adipogenenic transcription factor, 1.8-fold (p=0.006). PPARγ2 was observed at a smaller size than expected and further studies are needed. The results of the C/EBPα, SREBP-1 and PPARα Western blots were not included in this study and are recommended to be further optimized to reduce non-specific binding. ELISA results showed a significant increase in the secretion of the adipokines, adiponectin (>10-fold, p<0.001) and leptin (1.5-fold, p=0.002). The C. maculata extract was better than the positive control, TNFα, at inhibiting adipogenesis. A concentration of 80 μg/ml of the C. maculata extract maximally induced lipolysis, without affecting cell viability. Western blot analysis showed non-specific binding, and are recommended to be further optimized to reduce non-specific binding. Western blot analysis also showed that acute treatment (24 hours) of mature 3T3-L1 adipocytes with 80 μg/ml increased the expression of the lipolytic protein, HSL (1.6-fold, p=0.025). Perilipin Western blot was not included due to non-specific binding. ELISA results showed an increase in adiponectin (1.5-fold, p=0.015) and leptin (1.2-fold, p=0.067) secretion. Similar results were obtained after treatment with the C. maculata extract or the positive control, isoproteronol. This study shows that treatment of 3T3-L1 pre-adipocytes and adipocytes with 80 μg/ml of C. maculata plant extract inhibits adipogenesis and induces adipolysis, without causing cytotoxicity. A major limitation of the current study is that it was conducted in an in vitro model and does not represent the complexity of obesity as it occurs in humans. However, despite this, we believe that these results are promising and provide support for future in vivo studies to substantiate these preliminary findings. The results of this study is aligned with the Department of Science and Technology’s Ten Year Innovation Plan and the “Farmer to Pharma” value chain that aims to improve our bio-economy by developing our indigenous resources. Moreover, this type of initiative will be able to stimulate job creation, while being able to utilize the very rich South African indigenous knowledge. / AFRIKAANSE OPSOMMING: Vetsug is 'n groot oorsaak van morbiditeit en mortaliteit wêreldwyd. Tans is meer as 1,5 miljard mense oor die ouderdom van 20 jaar oorgewig, met meer as 500 miljoen van hierdie individue wat vetsugtig is. Vetsug verhoog die risiko vir die ontwikkeling van kardiovaskulêre siekte, tipe 2 diabetes en sekere soorte kanker. Onlangs het 'n aantal plantekstrakte anti-vetsug eienskappe in vitro en in verskeie dier modelle van vetsug getoon. Die doel van hierdie studie was om die effek van die Cyclopia maculata, 'n Suid-Afrikaanse kruie-tee, meer algemeen bekend as heuningbos, op lipogenese en lipolise in 3T3-L1 pre-adiposiete en adiposiete te ondersoek. Vir die ondersoek, is 3T3-L1 pre-adiposiete gedifferensieer in ‘n adipogeneseinduserende media met verskillende konsentrasies van ‘n warm water ekstrak van gefermenteerde C. maculata. Die optimale konsentrasie van C. maculata ekstrak is bepaal deur die selle met verskeie konsentrasies te behandel wat gewissel het van 0 tot 1600 mg / mL. 3T3-L1 pre-adiposiete is met adipogenese-induserende media gedifferensieer met of sonder TNFα supplementasie wat as positiewe en negatiewe kontrole, onderskeidelik gedien het. Intrasellulêre lipied-versameling is gemeet deur middel van Oil O Red kleuring en trigliseried-inhoud is bepaal deur 'n kommersiële kit. Sel-lewensvatbaarheid is bepaal deur 3-(4,5-Dimetielthiazol-2- yl)-2,5-difenieltetrazolium bromied (MTT) en adenosien tri-fosfaat (ATP) assays. Die PPARγ, C/EBPα, SREBP-1 and PPARα proteïen uitdrukking is deur middel van Western-blot analise bepaal, terwyl die gesekreteerde proteïene, leptien en adiponektien, deur ELISA bepaal is. Die effek van C. maculata ekstrak op lipolise is ondersoek deur 3T3-L1 preadiposiete in adipogenese-induserende media te differensieer waarna die selle vir ‘n verdere 8 dae in adipogenese-onderhoud media gekultuur is totdat hulle volwasse adiposiete bereik het, voordat die adiposiete behandel is met C. maculata ekstrak vir 24 uur. Die optimale konsentrasie C. maculata ekstrak is bepaal deur die selle met verskeie konsentrasies te behandel wat gewissel het van 0 tot 1600 mg/ml. Adipogenese-onderhoud media met of sonder isoproterenol is onderskeidelik gebruik as die positiewe en negatiewe kontroles. Intrasellulêre lipied afbraak is deur middel van Oil O Red gemeet, terwyl vry gliserol, 'n merker van lipolise, deur ‘n kommersiële kit bepaal is. Sel-lewensvatbaarheid is bepaal deur MTT en ATP assays. Die uitdrukking van HSL is deur middel van Western-blot analise bepaal, terwyl die uitdrukking van die gesekreteerde proteïene, leptien en adiponektien, deur ELISA gemeet is. Ek stel voor dat die perilipin Western blots verder geoptimaliseer word om sodoende nie-spesifieke binding te verminder. Behandeling met C. maculata ekstrak het intrasellulêre lipied-akkumulasie in die pre-adiposiete verminder, by die meeste van die konsentrasies wat getoets is. Die Oil O Red en die intrasellulêre trigliseried toetse, in kombinasie met die sellewensvatbaarheid assays, het getoon dat 80 mg/ml C. maculata ekstrak intrasellulêre lipied optimaal verminder sonder om die sel-lewensvatbaarheid te affekteer. Western blot analise het getoon dat die differensiasie van 3T3-L1 adiposiete in die teenwoordigheid van 80 mg/ml C. maculata ekstrak die uitdrukking van PPARγ2, 'n sleutel adipogenetiese transkripsie faktor, 1.8-voudig (p=0.006) verlaag. PPARy2 is waargeneem by a kleiner grootte as verwag en verdere ondersoek word benodig. Ek stel voor dat die C/EBPα, PPARα en SREBP- 1 Western blots verder geoptimaliseer word om sodoende nie-spesifieke binding te verminder. ELISA resultate het 'n beduidende toename in die sekresie van die adipokines, adiponektien (>10-voudig, p <0.001) en leptien (1.5-voudig, p= 0.002) getoon. Cyclopia maculata ekstrak was beter as die positiewe kontrole, TNFα, om adipogenese te inhibeer. Teen ‘n konsentrasie van 80 mg/ml het C. Maculata ekstrak lipolise maksimaal geïnduseer, sonder om sel-lewensvatbaarheid te beinvloed. ELISA resultate het 'n toename in adiponektien (1.5-voudig, p = 0.015) en leptien (1.2-voudig, p = 0,067) sekresie getoon. Soortgelyke resultate is verkry met die positiewe kontrole, isoproteronol, as met C. maculata ekstrak behandeling. Hierdie studie het getoon dat die behandeling van 3T3-L1 pre-adiposiete en adiposiete met 80 mg/ml C. maculata ekstrak adipogenese inhibeer en adipolise induseer, sonder enige sitotoksisiteit. 'n Beperking van die huidige studie is dat dit in 'n in vitro model gedoen is wat nie die kompleksiteit van vetsug in die mens weerspieël nie. Ten spyte daarvan is resultate belowend en ondersteun dit toekomstige in vivo studies om hierdie voorlopige bevindinge te staaf. Bewys dat ‘n water ekstrak van gefermenteerde C. maculata anti-vetsug eienskappe het kan groot ekonomiese gevolge vir die heuningbos industrie inhou. Die resultate van hierdie studie is in lyn met die Departement van Wetenskap en Tegnologie se tien jaar Innovasie Plan en die "Farm Pharma" waardeketting wat daarop gemik is om ons bio-ekonomie te verbeter deur die ontwikkeling van ons inheemse hulpbronne. Daarbenewens sal hierdie tipe inisiatief potensieel werkskepping stimuleer, terwyl dit die ryk Suid-Afrikaans inheemse kennis aanwend.

Masters of Science / The aim of this study was therefore to evaluate the antioxidant and anti-diabesity potential of a hot water extract of C. maculata in non-cell based assays and correlate the activities with phenolic composition. Total antioxidant capacity (TAC) was assessed in terms of free radical scavenging and iron reducing ability. The DPPH, ABTS, ORAC and FRAP assays were employed. Anti-diabesity potential was assessed in terms of the inhibition of the digestive enzymes, α-glucosidase and pancreatic lipase