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 (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.

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

Thesis (PhD)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Insights into the role of oxidative stress and pancreatic β-cell dysfunction in the pathogenesis of type 2 diabetes (T2D) reveals an opportunity for the development of novel therapeutics that directly protect and preserve β-cells. The protective role of dietary antioxidants, such as plant polyphenols, against oxidative stress induced diseases, including T2D, is increasingly under scrutiny. Polyphenol-rich extracts of Cyclopia spp, containing mangiferin, may provide novel therapeutics. An aqueous extract of unfermented Cyclopia maculata, containing more than 6 % mangiferin, was assessed for its protective effect in pancreatic β-cells in vitro, ex vivo and in vivo under conditions characteristic of T2D. The effect of mangiferin was also evaluated in vitro and ex vivo, with N-acetyl cysteine (NAC) as an antioxidant control. In this study, we established in vitro toxicity models in RIN-5F insulinoma cells based on conditions β-cells are exposed to in T2D; i.e. lipotoxicity, inflammation and oxidative stress conditions. To achieve this, cells were exposed to the following stressors: palmitic acid (PA), a pro-inflammatory cytokine combination and streptozotocin (STZ), respectively. Thereafter, the ability of the C. maculata extract, mangiferin and NAC to protect RIN-5F cells from the effects of these stressors was assessed by measuring β-cell viability, function and oxidative stress. Cell viability was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, adenosine triphosphate and annexin-V and propidium iodide assays. Cell function was evaluated by measuring glucose stimulated insulin secretion, cell proliferation and cellular calcium. To assess oxidative stress in the RIN-5F cells, diaminofluorescein-FM and dihydroethidium fluorescence, and superoxide dismutase enzyme activity were measured. The in vitro findings were then verified in isolated pancreatic rat islets using methods and models established in the RIN-5F experiments. The protective effect of the extract, NAC and metformin was assessed in STZ induced diabetic Wistar rats, using two treatment regimes, i.e. by treating rats with established diabetes and by pretreating rats prior to induction of diabetes by STZ. Glucose metabolism, oxidative stress and pancreatic morphology were assessed by performing an oral glucose tolerance test, measuring serum insulin, triglycerides, nitrites, catalase and glutathione. Hepatic thiobarbituric acid reactive substances and nitrotyrosine were also assessed. Immunohistochemical labelling of pancreata with insulin, glucagon and MIB-5 was used for morphological assessment. The extract improved β-cell viability, function and attenuated oxidative stress, most apparently in STZ and PA induced toxicity models comparable with NAC both in vitro and in isolated islets. Mangiferin was not as effective, showing only marginal improvement in RIN-5F cell and islet function, and oxidative stress. Pretreatment of STZ induced diabetic Wistar rats with extract was as effective as, if not better than, metformin in improving glucose tolerance, hypertriglyceridaemia and pancreatic islet morphology related to improved β-cell function. This study demonstrated that the aqueous extract of unfermented C. maculata was able to protect pancreatic β-cells from STZ and PA induced toxicity in vitro and ex vivo. In vivo, pretreatment with the extract improved glucose metabolism and pancreatic islet morphology in STZ induced diabetic Wistar rats. / AFRIKAANSE OPSOMMING: Insigte oor die rol wat oksidatiewe stres en pankreas β-sel disfunksie in die patogenese van tipe 2-diabetes (T2D) speel, bied 'n geleentheid vir die ontwikkeling van nuwe terapeutiese middels wat β-selle direk daarteen beskerm. Die beskermende rol van antioksidante in die dieët soos plantaardige polifenole teen oksidatiewe stres geinduseerde siektes soos T2D, is toenemend onder die soeklig. Polifenolryk ekstrakte van Cyclopia spp wat mangiferin bevat mag nuwe terapeutiese middels lewer. ‘n Waterekstrak van ongefermenteerde Cyclopia maculata wat meer as 6% mangiferin bevat, is ondersoek vir sy beskermende effek op pankreas ß-selle in vitro, ex vivo en in vivo teen kondisies kenmerkend aan T2D. Die effek van mangiferin is ook in vitro en ex vivo geëvalueer, met N-asetielsistien (NAC) as 'n antioksidant kontrole. In hierdie studie is in vitro toksisiteitsmodelle in RIN-5F insulinoomselle gevestig. Die modelle is gebaseer op toestande waaraan β-selle blootgestel word tydens T2D; d.w.s. lipotoksisiteit, inflammasie en oksidatiewe stres. Hiervoor is die selle aan die volgende stressors blootgestel: palmitiensuur (PA), ‘n pro-inflammatoriese sitokien mengsel en streptozotosien (STZ). Vervolgens is die vermoë van die C. maculata ekstrak, mangiferin en NAC om die RIN-5Fselle teen hierdie stressors te beskerm, beoordeel deur die meting van β-sellewensvatbaarheid, funksie en oksidatiewe stres. Sellewensvatbaarheid is bepaal met 3-(4,5-dimetielthiazol-2-yl)-2,5-difenieltetrazolium bromied, adenosientrifosfaat en anneksien-V and propidium jodied toetse. Selfunksie is geëvalueer d.m.v. glukose gestimuleerde insuliensekresie, selproliferasie en sellulêre kalsium bepaling. Oksidatiewe stres in die RIN-5Fselle is geëvalueer d.m.v. diaminofluorescein-FM en dihidroethidium fluoressensie bepalings, asook meting van superoksied dismutase ensiemaktiwiteit. Die in vitro bevindings is daarna in geїsoleerde rot pankreaseilande bevestig deur die metodes en modelle wat in die RIN-5F eksperimente gebruik is. Die antidiabetiese effekte van die ekstrak, NAC en metformien in STZ-geїnduseerde diabetiese Wistar rotte is bepaal d.m.v. twee behandlingsregimes, d.w.s. die behandeling van rotte met gevestigde diabetes of deur die behandeling voor die induksie van diabetes te begin. Glukose metabolisme, oksidatiewe stres en veranderinge in die pankreasmorfologie is ondersoek d.m.v. orale glukose toleransie toetse en die bepaling van serum insulien, trigliseriedes, nitriete, katalase en glutationien. Hepatiese tiobarbituursuur reaktiewe stowwe en nitrotirosien is ook geëvalueer. Immunohistochemiese kleuring van pankreas snitte is gebruik vir morfologiese assessering van insulien, glukagon en MIB-5. Die ekstrak het mees opvallend β-sel lewensvatbaarheid en funksie verbeter, terwyl oksidatiewe stres verminder is in die STZ- en PA-geїnduseerde toksisiteitmodelle. Bogenoemde effekte van die ekstrak in vitro en in die geїsoleerde eilande was vergelykbaar met die van NAC. Mangiferin was minder effektief, met slegs ‘n marginale verbetering in die funksie van RIN-5Fselle en eilande, asook t.o.v. oksidatiewe stres. Behandeling van die Wistar rotte met die ekstrak voor induksie van diabetes met STZ was net so effektief, of selfs beter as metformien in terme van verbeterde glukosetoleransie, trigliseriedvlakke en die morfologie van pankreas eilande wat verband gehou het met β-sel funksie. Hierdie studie het getoon dat die waterekstrak van ongefermenteerde C. maculata pankreas β-selle teen veral STZ- en PA-geїnduseerde toksisiteit in vitro en ex vivo beskerm het. In vivo het behandeling met die ekstrak voor en na induksie van diabetes, glukosemetabolisme en die morfologie van pankreas eilande in STZ-geїnduseerde diabetiese Wistar rotte verbeter.