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: Cyclopia genistoides (honeybush) has been identified as an excellent resource for the production of a xanthone-enriched extract due to its high mangiferin content and successful cultivation. The predominant xanthone present in C. genistoides is mangiferin, a potent antioxidant proven to exhibit a wide range of bioactivities that contribute greatly to the health-promoting abilities of honeybush extracts. Isomangiferin, the regio-isomer of mangiferin and of comparable antioxidant capacity to mangiferin, is another valuable compound present in substantial quantities in C. genistoides. A xanthone-enriched extract would find possible application in functional food/beverage products that provide health benefits beyond basic nutrition. In the current study, the effect of ethanol (EtOH) concentration (0-100%, v/v), plant material size (milled vs. teabag fraction), extraction time (0-60 min) and elevated extraction temperatures on the extraction of xanthones from unfermented C. genistoides was investigated. Single factor experiments showed the best extraction efficiency, evaluated in terms of extract yield, xanthone yield and xanthone content of the extract, was achieved by extracting milled plant material with 20-60% EtOH (v/v) for 30 min at elevated temperatures (70°C). Response surface methodology (RSM) to evaluate the individual and interaction effects of process variables, namely EtOH concentration (0-100%, v/v) and temperature (0-70°C) was used to further optimise the extraction process. EtOH concentration was found to have the largest effect on extraction efficiency (p < 0.05), whilst temperature had a negligible effect. Optimal levels of EtOH concentration (40%, v/v) and temperature (70°C) for maximum extract and mangiferin yields were successfully achieved within the experimental domain, using 10 mL/g solvent:solid ratio and 30 min extraction time. Ultrafiltration (UF) was subsequently employed to facilitate further xanthone enrichment of the unfermented C. genistoides extract (40% EtOH, v/v). A series of laboratory scale membrane devices (centrifugal membrane units, stirred cell and tangential flow ultrafiltration (TFU) system) were used in an up-scale approach to determine the effect of membrane material (regenerated cellulose (RC) vs. polyethersulphone (PES)), molecular weight cut off (MWCO; 3 kDa, 10 kDa, 30 kDa), feed concentration (1% vs. 3% soluble solids (SS)) and operating parameters (transmembrane pressure (TMP) and feed flow rate) on membrane performance and permeate quality. The best performing membrane in terms of productivity and xanthone enrichment was the 10 kDa RC membrane when using an extract concentration close to that of industrially prepared extracts (3% SS). RSM was used to further optimise UF of unfermented C. genistoides through a 10 kDa RC membrane in the TFU system. The individual and interaction effects of TMP (0.82-2.04 bar) and feed flow rate (200-444 mL/min) on permeate flux, xanthone enrichment and the fouling index were investigated. The individual effects of TMP and feed flow rate had a significant effect on all measured responses, while their interaction only affected average permeate flux and fouling index significantly. Optimal TMP and feed flow rate values of 2.04 bar and 444 mL/min, respectively, were selected within the experimental domain, restricted by equipment constraints. Validation of the combined protocol including ethanol-water extraction and UF using plant material from ten different unfermented C. genistoides batches resulted in enriched extracts containing 10.6-17.8% xanthone content. During UF, average mangiferin and isomangiferin enrichments of 20% and 22%, respectively, were obtained. Whilst no correlation was found between the feed concentration of the extracts, xanthone enrichment and fouling index, a strong linear correlation (R2 = 0.98) was found between feed concentration and permeate yield. / AFRIKAANSE OPSOMMING: Cyclopia genistoides (heuningbos) is geidentifiseer as ‘n uitstekende bron vir die produksie van ‘n xantoon-verrykte ekstrak weens sy hoë mangiferien-inhoud sowel as suksesvolle verbouing daarvan. Die oorheersende xantoon teenwoordig in C. genistoides is mangiferien, ‘n kragtige antioksidant met ‘n bewese wye reeks bioaktiwiteite wat grootliks bydra tot die gesondheidsvoordele van heuningbosekstrakte. Isomangiferien, die regio-isomeer van mangiferien met vergelykbare antioksidant-aktiwiteit as mangiferien, is nog ‘n waardevolle verbinding teenwoordig in aansienlike hoeveelhede in C. genistoides. ‘n Xantoon-verrykte ekstrak kan moontlik toegepas word in funksionele voedsel- of drankie-produkte, wat gesondheidsvoordele bo en behalwe die basiese voedsaamheid inhou. Die effek van etanol (EtOH)-konsentrasie (0-100%, v/v), plantmateriaal grootte (gemaal teenoor teesakkie-fraksie), ekstraksietyd (0-60 min) en ekstraksietemperatuur op die ekstraksie van xantone uit ongefermenteerde C. genistoides is ondersoek. Enkelfaktor eksperimente het getoon dat die beste ekstraksie-effektiwiteit, in terme van ekstrakopbrengs, xantoonopbrengs en xantooninhoud van die ekstrak, bereik is deur gemaalde plantmateriaal met 20-60% EtOH (v/v) vir 30 min by verhoogde temperature (70°C) te ekstraheer. Respons-oppervlak Metodologie (ROM) is aangewend om die individuele en interaktiewe effekte van die veranderlikes, naamlik EtOH-konsentrasie (0-100%, v/v) en temperatuur (0-70°C) te ondersoek asook om die ekstraksieproses verder te optimiseer. EtOH-konsentrasie het die grootste effek op die ekstraksie-effektiwiteit gehad (p < 0.05), terwyl die effek van temperatuur onbeduidend was. Optimale vlakke van EtOH-konsentrasie (40% v/v) en temperatuur (70°C) vir maksimum ekstrak- en mangiferienopbrengs is binne die eksperimentele domein is gevind, met die gebruik van 10 mL/g oplosmiddel:vastestof verhouding en ‘n ekstraksietyd van 30 min. Ultrafiltrasie (UF) is daarna gebruik om verdere xantoon-verryking van die ongefermenteerde C. genistoides ekstrak (40% EtOH, v/v) te fasiliteer. ‘n Reeks labratoriumskaal membraantoestelle (sentrifugale membraaneenhede, ‘n geroerde selsisteem en ‘n kruisvloei-ultrafiltrasie (KVU) sisteem) is gebruik in ‘n opskaleringsbenadering om die effek van die membraanmateriaal (geregenereerde sellulose (RS) vs. polyetersulfoon (PES)), molekulêre gewig afsnit (MWCO; 3 kDa, 10 kDa, 30 kDa), voerkonsentrasie (1% vs. 3% oplosbare vastestowwe (OV)) en operasionele parameters (transmembraandruk (TMD) en voervloeispoed) op membraanprestasie en permeaatkwaliteit te bepaal. Die membraan met die beste prestasie in terme van produktiwiteit en xantoon-verryking was die 10 kDa RS membraan wanneer gebruik met ‘n ekstrakkonsentrasie na aan dié van die industrieel vervaardigde ekstrakte (3% OV). ROM is gebruik om die KVU van ongefermenteerde C. genistoides deur ‘n 10 kDa RS membraan verder te optimiseer. Die indiwiduele en interaktiewe effekte van TMD (0.82-2.04 bar) en voervloeispoed (200-444 mL/min) op permeaatvloei, xantoon-verryking en die blokkeringindeks is ondersoek. Die individuele effekte van TMD en voervloeispoed het ‘n betekenisvolle effek op alle gemete response gehad, terwyl hul interaksie net gemiddelde permeaatvloei en besoedelingsindeks beduidend beïnvloed het. Optimale TMD en voervloeispoed waardes van 2.04 bar en 444 mL/min, onderskeidelik, is geselekteer binne die eksperimentele domein, wat bepaal is deur die beperkings van die toerusting. Die geldigheid van die gesamentlike protocol, insluitende etanol-water ekstraksie en UF, is getoets deur plantmateriaal van tien verskillende ongefermenteerde C. genistoides monsters te gebruik. Dit het gelei tot verrykte ekstrakte wat 10.6-17.8% xantone bevat het. UF het onderskeidelik gemiddelde mangiferien- en isomangiferien-verryking van 20% en 22% gelewer. Geen korrelasie is gevind tussen die voerkonsentrasie van die ekstrakte en die besoedelingsindeks nie, maar ‘n goeie liniêre korrelasie (R2 = 0.98) is tussen voerkonsentrasie en permeaatopbrengs gevind.

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 (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 (MSc Food Sc) (Food Science)--University of Stellenbosch, 2005. / Near infrared spectroscopy (NIRS) and UV/Vis spectrophotometric methods were investigated as rapid and/or more economical methods to quantify the major monomeric phenolic compounds, soluble solid content (SSC), total polyphenol content (TPC) and total antioxidant activity (TAA) of green rooibos and Cyclopia genistoides.

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.

Thesis (PhD(FoodSc))--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Potential beneficial and/or adverse modulatory effects of polyphenol-enriched extracts of rooibos (Aspalathus linearis) and honeybush (Cyclopia spp.) on the antioxidant homeostasis in the liver were investigated. Phase II metabolism of aspalathin and mangiferin, the major polyphenols of rooibos and honeybush respectively, was assessed for potential glucuronidation and sulphation. Glucuronidation resulted in a loss of antioxidant activity for aspalathin and mangiferin in post-column HPLC-DAD-DPPH• and HPLC-DAD-ABTS•+ assays and also a decreased activity of iron chelating properties of mangiferin in the FRAP assay. Two independent studies for 28 and 90 days with polyphenol-enriched extracts (PEEs) of unfermented rooibos [Aspalathus linearis (PER)] and honeybush [Cyclopia. subternata (PECsub) and C. genistoides (PECgen)] in male Fischer rats were conducted to assess possible beneficial and/or adverse biological effects. PECgen was only included in the 28 day study. PEEs were characterised by in vitro antioxidant assays and HPLC analysis. The importance of detailed chemical characterization of rooibos and honeybush when investigating biological effects in vivo is clear as distinctive biological effects and major differences in compositions were evident. Biological parameters included were serum chemical parameters, activities of selected antioxidant enzymes, levels of glutathione and the modulation of expression of oxidative stress and antioxidant defense related genes in the liver. Sub-chronic (90 days) exposure of rats to PER and PECsub both adversely affected iron absorption significantly (P<0.05) and significantly (P<0.05) and markedly lowered levels of reduced glutathione (GSH) in the liver. The high levels of polyphenol intake were implicated and interaction with glutathione was postulated to occur via catechol o-quinone conjugations with GSH. This was also implicated in the significantly (P<0.05) increased activity of glutathione reductase (GR) following 28 days. These findings suggest that PEEs from rooibos and honeybush have the potential to alter the glutathione homeostasis, which could contribute to oxidative status in the liver. PECsub resulted in alterations in the liver biliary system which was manifested as significantly (P<0.05) increased serum total bilirubin (Tbili) and alkaline phosphatase (ALP), depending on the age of the rats, level of total polyphenols and duration of exposure. The expression of a number of oxidative stress and antioxidant defense related genes were differentially altered by the PEEs of rooibos and honeybush in rat liver and further indicated potential oxidative stress. Modulatory effects of PEEs on expression of 84 of these genes in rat liver were assessed with a quantitative real-time reverse transcription polymerase chain reaction (RT-PCR) array and provided additional insights into the possible adverse and protective effects of rooibos and honeybush. Further investigation on total polyphenol dose levels and time of exposure in the application of PEEs of rooibos and honeybush as dietary supplements and functional foods is recommended and will also be of value in anticipated regulatory requirements for future substantiation of safety and efficacy. / AFRIKAANSE OPSOMMING: Die moontlike voordelige en/of nadelige modulerende effekte van polifenol-verrykde ekstrakte van rooibos (Aspalathus linearis) en heuningbos (Cyclopia spp.) op die antioksidant homeostasis in die lewer is ondersoek. Die fase II metaboliete van aspalatien en mangiferin, die hoof verbindings in rooibos en heuningbos onderskeidelik, is ondersoek t.o.v. glukuronidering en sulfonering. Glukuronidasie het gelei tot n verlies in antioksidant aktiwiteit van aspalatien en mangiferin soos bepaal in post-kolom HPLC-DAD-DPPH• en HPLC-DADABTS•+ toetse, asook verminderde interaksie met yster van mangiferin in die FRAP toets. Twee onafhanklike studies van 28 en 90 dae is onderneem met polifenol-verrykde ekstrakte (PVEs) van ongefermenteerde rooibos [Aspalathus linearis (PVR)] en heuningbos [Cyclopia. subternata (PVCsub) and C. genistoides (PVCgen)] in manlike Fisher rotte om die moontlike voordelige en/of nadelige biologiese effekte te ondersoek. PECgen was slegs in die 28 dae studie ingesluit. PVEs is gekarrakteriseer deur in vitro antioksidant en HPLC analises. Die belang van chemiese karaktirisering van rooibos en heuningbos tydens ondersoeke na biologiese aktiwiteit is duidelik aangesien verskeie en variërende biologiese aktiwiteite en verskille in die komposisie in die huidige studie gesien is. Die biologiese parameters wat ondersoek is om die effek van die PVEs te bepaal het serum kliniese parameters, aktiwiteit van geselekteerde ensieme, glutatioon en evaluering van die ekspressie van oksidatiewe en antioksidant verwante gene in die lewer, ingesluit. Sub-kroniese (90 dae) blootstelling van rotte aan PVR en PVCgen het yster absorpsie negatief beïnvloed. Die beduidende (P<0.05) verlaagde vlak van gereduseerde glutatioon in die lewer was toegeskryf aan die hoë vlakke van polifenole ingeneem tydens die studie en word moontlik veroorsaak deur n spesifieke katekol o-konjugasie van GSH. Hierdie interaksie was ook moontlik die oorsaak van n beduidende (P<0.05) toename in die aktiwiteit van glutatioon reduktase. Dié bevindinge het moontlike implikasies t.o.v die glutatioon homeostase en is n moontlike indikase dat PVEs van rooibos kan bydra tot oksidatiewe stres. PVCsub het veranderinge in die lewer gal-sisteem tot gevolg gehad aangesien daar n beduidende (P<0.05) verhoging in die serum totale bilirubin en alkalien fosfaat was. Hierdie veranderinge is beïnvloed deur die ouderdom, vlakke van die totale polifenole en die periode van blootstelling. Die uitdrukking van oksidatiewe en antioksidant verwante gene is op verskillende maniere beïnvloed deur die PVEs van rooibos en heuningbos in rot lewer and dien as n verdere indikasie van onderliggende oksidatiewe stres. Die modulerende effekte van PVEs op geenuitdrukking het gelei tot additionele insig aangaande die moontlike skadelike of beskermende eienskappe van PVEs vir gebruik as kruie produkte of dieet aanvullings. Die indikasies van moontlike oksidatiewe stres was duidelik van biologiese parameters en modulering van geenuitdrukking in die lewer, en vereis verdere ondersoek na die polifenool dosis en periode van toediening voordat PVEs van rooibos en heuningbos as funksionele voedsel produkte gebruik word. Hierdie ondersoek sowel as toekomstige ondersoeke in hierdie verband sal van waarde wees vir regulatoriese vereistes omtrent die veiligheid en effektiwiteit van rooibos en heuningbos kruie produkte.

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.

The progression of skin ageing in individuals is multifaceted and provoked by various aspects, including hereditary and a variety of environmental causes, for instance UV (ultra violet) radiation, resulting in the morphological modifications in the dermal layer of the skin (Makrantonaki & Zouboulis, 2007:40) Transformations caused by ageing skin, in which degenerative alterations exceed regenerative alterations are recognised by the thinning and wrinkling of the epidermis in conjunction with the appearance of lines, creases, crevices and furrows, particularly emphasised in lines of facial expressions (Aburjai & Natsheh, 2003:990). For human beings to continue to exist in a terrestrial atmosphere, the loss of water from the skin must be cautiously synchronised by the epidermis, a task dependent on the multifaceted character of the stratum corneum (Rawlings & Harding, 2004:43). The stratum corneum (SC) is responsible for the main resistance to the penetration of most compounds; nevertheless the skin represents as an appropriate target for delivery. The target site for anti-ageing treatment includes the epidermal and dermal layers of the skin. Therefore, the need to apply fatty materials to the skin is practically intuitive and may perhaps be as old as man’s existence itself (Lodén, 2005:672). Natural therapies have been used for several decades for taking care of skin illnesses and a wide variety of dermatological disorders, such as inflammation, phototoxicity, atopic dermatitis and alopecia areata (Aburjai & Natsheh, 2003:988). Using the skin as an alternative route for the administration of honeybush extracts for the treatment of ageing skin may be beneficial. Tea contains more than 500 chemical compounds, including, tannins, flavonoids, amino acids, vitamins, caffeine and polysaccharides. Tea polyphenols (flavonoids) have proven anti-inflammatory, antioxidant, antiallergic, antibacterial and antiviral effects (Aburjai & Natsheh, 2003:990). Unfortunately using the skin as an alternative route for administering drugs (transdermal drug delivery) has numerous limitations. One of these limitations is the barrier function of the skin (Naik et al., 2000:319). Because of the skin’s outstanding ability to protect the body against unwanted substances from its surroundings, it is necessary to use methods to enhance drug penetration through the skin. The aim of this study was to formulate two 2% semisolid formulations containing two different honeybush extracts as the active ingredient, and to determine which of the formulations deliver mangiferin and hesperidin best to the target site (dermis). Cosmetic formulations of a natural origin, is designed to protect the skin against exogenous or endogenous harmful agents, as well as to balance the dermal homeostatis lipids altered by dermatosis and ageing (Aburjai & Natsheh, 2003:988). Stability tests over a three month period were also performed on the different formulations. To determine the stability of the different semi-solid formulations, the formulated products were stored at 25 °C/60% RH (relative humidity), 30 °C/60% RH and 40 °C/75% RH. HPLC analysis was used to determine the concentrations of the ingredients in all the formulated products. Other stability tests included appearance, pH, viscosity, mass loss, zeta potential and particle size determination. Unfortunately a change in colour, viscosity, zeta potential, mass loss, particle size and concentration of the ingredients in both the formulations, indicated that the products were unstable from the first month of stability testing. A 2% Cyclopia maculata cream as well as a 2% Cyclopia genistoides cream was formulated. Franz cell diffusion studies as well as membrane release studies were performed over a 12 h period, followed by tape stripping experiments to determine which semi-solid formulation delivered mangiferin and hesperidin the best to the dermal layer of the skin. The results of the different formulations were compared. Unfortunately there was no significant penetration by any of the honeybush extracts. Results were inconclusive and unquantifiable due to unconvincing penetration results. The antioxidant properties of both the extracts and the active ingredients were calculated. Antioxidant studies by the use of the TBA-assay method were done to determine whether the honeybush extracts, mangiferin and hesperidin as well as their semisolid formulations had any antioxidant activities. Both the honeybush extracts and the semisolid formulations showed promising results. Mangiferin and hesperidin did not show any antioxidant activity on their own, therefore the assumption can be confirmed that plants do function synergistically. A clinical study was also conducted to see whether honeybush extracts have the potential to hydrate the skin, counteracting the symptoms and signs of skin ageing. Clinical efficacy studies were done to determine whether the honeybush formulations had any skin hydrating effects in the treatment against skin ageing. The results were statistically inconclusive and variations between the subjects were very high due to skin variations at different skin sites. There was however a trend that Cyclopia genistoides performed the best. / Thesis (MSc (Pharmaceutics))--North-West University, Potchefstroom Campus, 2013.

The progression of skin ageing in individuals is multifaceted and provoked by various aspects, including hereditary and a variety of environmental causes, for instance UV (ultra violet) radiation, resulting in the morphological modifications in the dermal layer of the skin (Makrantonaki & Zouboulis, 2007:40) Transformations caused by ageing skin, in which degenerative alterations exceed regenerative alterations are recognised by the thinning and wrinkling of the epidermis in conjunction with the appearance of lines, creases, crevices and furrows, particularly emphasised in lines of facial expressions (Aburjai & Natsheh, 2003:990). For human beings to continue to exist in a terrestrial atmosphere, the loss of water from the skin must be cautiously synchronised by the epidermis, a task dependent on the multifaceted character of the stratum corneum (Rawlings & Harding, 2004:43). The stratum corneum (SC) is responsible for the main resistance to the penetration of most compounds; nevertheless the skin represents as an appropriate target for delivery. The target site for anti-ageing treatment includes the epidermal and dermal layers of the skin. Therefore, the need to apply fatty materials to the skin is practically intuitive and may perhaps be as old as man’s existence itself (Lodén, 2005:672). Natural therapies have been used for several decades for taking care of skin illnesses and a wide variety of dermatological disorders, such as inflammation, phototoxicity, atopic dermatitis and alopecia areata (Aburjai & Natsheh, 2003:988). Using the skin as an alternative route for the administration of honeybush extracts for the treatment of ageing skin may be beneficial. Tea contains more than 500 chemical compounds, including, tannins, flavonoids, amino acids, vitamins, caffeine and polysaccharides. Tea polyphenols (flavonoids) have proven anti-inflammatory, antioxidant, antiallergic, antibacterial and antiviral effects (Aburjai & Natsheh, 2003:990). Unfortunately using the skin as an alternative route for administering drugs (transdermal drug delivery) has numerous limitations. One of these limitations is the barrier function of the skin (Naik et al., 2000:319). Because of the skin’s outstanding ability to protect the body against unwanted substances from its surroundings, it is necessary to use methods to enhance drug penetration through the skin. The aim of this study was to formulate two 2% semisolid formulations containing two different honeybush extracts as the active ingredient, and to determine which of the formulations deliver mangiferin and hesperidin best to the target site (dermis). Cosmetic formulations of a natural origin, is designed to protect the skin against exogenous or endogenous harmful agents, as well as to balance the dermal homeostatis lipids altered by dermatosis and ageing (Aburjai & Natsheh, 2003:988). Stability tests over a three month period were also performed on the different formulations. To determine the stability of the different semi-solid formulations, the formulated products were stored at 25 °C/60% RH (relative humidity), 30 °C/60% RH and 40 °C/75% RH. HPLC analysis was used to determine the concentrations of the ingredients in all the formulated products. Other stability tests included appearance, pH, viscosity, mass loss, zeta potential and particle size determination. Unfortunately a change in colour, viscosity, zeta potential, mass loss, particle size and concentration of the ingredients in both the formulations, indicated that the products were unstable from the first month of stability testing. A 2% Cyclopia maculata cream as well as a 2% Cyclopia genistoides cream was formulated. Franz cell diffusion studies as well as membrane release studies were performed over a 12 h period, followed by tape stripping experiments to determine which semi-solid formulation delivered mangiferin and hesperidin the best to the dermal layer of the skin. The results of the different formulations were compared. Unfortunately there was no significant penetration by any of the honeybush extracts. Results were inconclusive and unquantifiable due to unconvincing penetration results. The antioxidant properties of both the extracts and the active ingredients were calculated. Antioxidant studies by the use of the TBA-assay method were done to determine whether the honeybush extracts, mangiferin and hesperidin as well as their semisolid formulations had any antioxidant activities. Both the honeybush extracts and the semisolid formulations showed promising results. Mangiferin and hesperidin did not show any antioxidant activity on their own, therefore the assumption can be confirmed that plants do function synergistically. A clinical study was also conducted to see whether honeybush extracts have the potential to hydrate the skin, counteracting the symptoms and signs of skin ageing. Clinical efficacy studies were done to determine whether the honeybush formulations had any skin hydrating effects in the treatment against skin ageing. The results were statistically inconclusive and variations between the subjects were very high due to skin variations at different skin sites. There was however a trend that Cyclopia genistoides performed the best. / Thesis (MSc (Pharmaceutics))--North-West University, Potchefstroom Campus, 2013.