Assessment of chemical markers as surrogates for efficacy and safety of Rooibos extracts

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dc.contributor.advisorMuller, C.J,F
dc.contributor.advisorBasson, A.K
dc.contributor.authorViraragavan, Amsha
dc.date.accessioned2017-07-10T10:41:01Z
dc.date.available2017-07-10T10:41:01Z
dc.date.issued2017
dc.descriptionA thesis submitted to the Faculty of Science and Agriculture in fulfilment of the requirements for the Degree of Master of Science in Biochemistry in the Department of Biochemistry and Microbiology at the University of Zululand, 2017en_US
dc.description.abstractResearch interest in the bioactive polyphenols of rooibos. to which its health-promoting properties are attributed, has escalated. Defining the quality attributes for assessing the efficacy of rooibos health products forms part of a quality control system. The aim of this study was to identify chemical markers in green rooibos extract (GRE) that could predict bioactivity in cell-based assays, with particular focus drawn to the dihydrochalcone C-glucoside, aspalathin, which has displayed antidiabetic effects in vitro and in vivo. METHODS Two ethanol-based (80% and 60% ethanol) and aqueous extracts were prepared from ten randomly selected plant batches of green rooibos. HPLC-DAD analysis was performed to quantify aspalathin and other major flavonoids present in the extracts. A radioimmunoassay was used to measure 2-deoxy-[3H]-D-glucose absorption in C2C12 murine skeletal muscle and C3A human liver cells, exposed to 10 μg/mL of the extracts. The effect of the green rooibos extracts (GRE’s) and reference extracts ARC 2 and GRT on glucose uptake and lipid accumulation was also tested in 3T3-L1 adipocytes. GRE’s were also tested at concentrations ranging from 1 and 100 μg/mL for inhibitory activity against the protein tyrosine phosphatase 1B (PTP1B) enzyme. To investigate the possible mechanism of action of glucose uptake in C2C12 cells, protein expression studies were conducted. Multivariate statistical analysis was performed using principle component analysis (PCA), to elucidate the relationship of extract type, plant batch variation and bioactivity. RESULTS HPLC-DAD analysis of the different GRE’s demonstrated that extraction with 80% ethanol yielded the highest average phenolic compound enrichment (17.09 ± 2.66% aspalathin), compared to the 60% ethanol and aqueous GRE (12.54 ± 2.51% and 9.52 ± 1.85% aspalathin, respectively). In C2C12 cells glucose uptake was related to the phenolic content, as activity increased in the 80 and 60% ethanol extracts (by up to 182% and 142%, respectively. The glucose uptake was comparable to ARC 2 the reference extract. While in C3A cells the aqueous extract appeared to be more effective. Lipid accumulation was greatly enhanced in 3T3-L1 adipocytes by the ethanolic GRE’s. Furthermore, all extracts showed potent inhibitory activity on PTP1B with IC50 values ≤ 20 μg/mL. Principle component analysis demonstrated clustering between the 80% ethanol-based extracts and the reference extracts. For C2C12 glucose uptake and 3T3-L1 lipid accumulation, a positive correlation was demonstrated with aspalathin and nothofagin, isoorientin and orientin as well as isovitexin and vitexin. CONCLUSION The chemical complexity of these different extracts make it difficult to identify single active pharmaceutical ingredients, however activity was associated with aspalathin and the 3-deoxy-dihydrochalcone, nothofagin, and their flavone derivatives.en_US
dc.description.sponsorshipNational Research Foundation, South African Medical Research Council;Agricultural Research Councilen_US
dc.identifier.urihttps://hdl.handle.net/10530/1599
dc.publisherUniversity of Zululanden_US
dc.subjectrooibos --polyphenols --healthen_US
dc.titleAssessment of chemical markers as surrogates for efficacy and safety of Rooibos extractsen_US
dc.typeThesisen_US
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