Biochemistry and Microbiology
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Browsing Biochemistry and Microbiology by Author "Basson, A.K"
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- ItemAssessing the microbial community structure’s diversity associated with nutrients concentration in the natural wetland systems(University of Zululand, 2018) Buthelezi, Nondumiso, Petunia; Mthembu, M.S; Basson, A.KMicrobial communities play a major role in natural wetlands systems biogeochemical cycles. Therefore, understanding the association between their composition, diversity and environmental parameters is significant in order to understand soil microbial ecology associated with nutrients concentration. Such knowledge may enhance the improvement in the management and protection of wetland systems, as nutrients are essential for wetland plant growth. Despite their usefulness, they promote toxin-producing cyanobacteria and facilitates algal biomass growth that cause undesirable eutrophication and oxygen depletion when present in excess. Nutrient pollution therefore is one of the most widespread and challenging environmental problems in the water that usually result to serious environmental and health challenges. For this study, water and sediment samples were collected over a period of twelve months from Lake Icubhu, which is a fresh water wetland, and Nhlabane mouth Estuary. The physicochemical properties such as temperature, potential hydrogen (pH), chemical oxygen demand (COD), total suspended solids (TSS) and nutrients were analyzed in water samples. The InoLab_IDS multi 9310 was used for the analysis of temperature and pH, while the filter technique was used for the analysis of TSS concentrations, and spectrophotometric methods were used for the analysis of nutrients and COD concentrations, using respective kits.The pH ranged between 6.5 -7 in Lake Icubhu, and 6.9 – 7.3 in Nhlabane Estuary. Nitrate and nitrite were highest in the autumn (3.2 mg/l – 0.5 mg/l),and least in the summer (0.1 mg/l – 0.01 mg/l) for both wetlands, phosphorus and ammonium were also examined. The results of nutrients obtained in this study demonstrated a significant threat to the studied wetlands when compared to the South African standard for Water Quality Guidelines of Aquatic ecosystem throughout the study period. It has been observed and concluded from the values obtained in this study that seasonal variations contributed to nutrient cycling. Correlation coefficient of each wetland system were also analyzed in order to understand the pollution load, as well as the effect of physicochemical parameters on the nutrients. The correlation results revealed that all these parameters are interrelated with each other and should be considered together. Specifically, the Illumina Miseq Sequencing analysis method was used in this study to identify microbial community structures. After sequencing, the most represented bacterial community was Proteobacteria in both wetlands. Therefore, the core composition of these bacterial communities consisted of: Proteobacteria, Bacteroides, Actinobacteria, Firmicutes among others. While members of Actinobacteria, bacteroidetes and Acidobacteria were observed in Lake Icubhu, members of Firmicutes, Chloroflexi and Bacteroidetes were also dominant at Nhlabane Estuary. Finally, some representatives of Thermomicrobia, Fusobacteria, Verrucomicrobia and Fusobacteria were also retrieved from some samples. Based on the results of nutrients, microbial diversity and community structuresobtained in this study, it was concluded that seasonal variations and nutrient pollution loads were related to microbial community structures.
- ItemAssessment of chemical markers as surrogates for efficacy and safety of Rooibos extracts(University of Zululand, 2017) Viraragavan, Amsha; Muller, C.J,F; Basson, A.KResearch 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.
- ItemEvaluation of excipients for enhanced intestinal absorption of Rooibos flavonoids(University of Zululand, 2017) Hlengwa, Nokulunga; Muller, C.J,F; Basson, A.K; Bowles, S.L; Joubert, E.Aspalathin, a dihydrochalcone C-glucoside, and a major flavonoid unique to Rooibos, has been shown to ameliorate insulin resistance, improve glucose uptake in vitro and improve glycaemia in animal models. However, the poor bioavailability of aspalathin limits its potential as a nutraceutical. This study investigated the intestinal absorption of aspalathin, its flavone derivatives, orientin and isoorientin, and nothofagin, its 3-deoxy-derivative, from different green Rooibos extracts in an in vitro intestinal epithelial model, the Caco-2 cell model. Addition of excipients and a nanosome preparation of green Rooibos extract to improve absorption of these flavonoid C-glucosides was also investigated. Methods : Seven green Rooibos extracts, prepared using different solvents (60% ethanol, 80% ethanol and an aqueous extracts), were assessed for the intestinal absorption of their major flavonoids, i.e. aspalathin and its flavone derivatives, orientin and isoorientin, as well as nothofagin, the 3-deoxy-derivative of aspalathin. For testing, two samples per extract type were selected from a large set, based on their bioactivity and chemical dissimilarity. These samples were prepared from different batches of plant material. Caco-2 cells were used as an in vitro intestinal epithelial model to assess absorption of the compounds. Intestinal permeability of the compounds was quantified by analysis of the apical and basolateral samples by HPLC-DAD. The relative transport rate (Papp values) was calculated from the detected concentrations of the compounds crossing the Caco-2 monolayer from the apical to the basolateral compartment. The effect of different extract types, addition of excipients (β-cyclodextrin and inulin) and encapsulation of green Rooibos extract in a nanosome formulation on the absorption of the flavonoids was also explored. Results: At the highest soluble and non-toxic concentration of 1 mg/mL, the transport of aspalathin, nothofagin, orientin and isoorientin across the Caco-2 cell monolayer was confirmed, as these compounds were detected in the basolateral compartment by HPLC-DAD analysis. Standardization of the different extracts (n = 7) to their aspalathin equivalent concentration of 150 μM demonstrated that the transport rate of aspalathin (±1.72 x 10-06 cm/s) was not markedly altered by extract composition as determined by the extraction solvent and plant material. The rate transport was not increased by the addition of β-cyclodextrin (6.99 x 10-07 cm/s) and inulin (1.21977 x 10-06 cm/s), nor by using a nanosome (3.020 X 10-07 cm/s), implying that the strategies used in this study did not enhance intestinal absorption of aspalathin. Conclusion: This study has provided novel information about the intestinal absorption of aspalathin which was not significantly altered by extract composition as determined by extract type and plant material variation. Neither the addition of excipients nor nanosome formulation of the extract could enhance the absorption of aspalathin.