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.