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.

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.