Theses and Dissertations (Biosciences)
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Item The antifungal activity and bioanalysis of fractions from Tulbaghia violacea (leaf and root) acetone extracts.(Vaal University of Technology, 2022-12) Makgati, Mogau Mafise Brian; Mitema, A. Dr.; Takaidza, S. Dr.Infectious diseases represent a critical problem to health and cause morbidity and mortality worldwide. Despite the significant progress in human medicine, infectious diseases caused by microorganisms such as fungi are still a major threat to public health. Tulbaghia violacea is one medicinal plant that has been used traditionally to manage fungal infections. The present study aimed to determine the antifungal activity and perform bioanalysis of acetone fractions from T. violacea extracts. Tulbaghia violacea crude leaf and root acetone extracts and their fractions were tested against seven fungal species for antifungal activity. The plant extracts were prepared using the maceration method, and column chromatography was utilized to obtain fractions. The plant extract’s total phenolic and flavonoid contents were determined using the Folin-Ciocalteu and aluminum chloride colorimetric methods, respectively. The antioxidant activity was evaluated using the free radical scavenging methods 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2′-azino-bis-3-ethylbenzthiazoline-6-sulphonic acid (ABTS•+) assays. The antimicrobial activity of T. violacea crude leaf and root extracts and their fractions was determined using the well diffusion, microtiter, and time-kill assay. The compounds present in the T. violacea leaf and root extracts and their fractions were determined using GC-MS. The results indicated that the total phenolic content (TPC) of all four samples of T. violacea leaf extracts ranged from 0.597-5.003 mg of Gallic acid equivalent/g, whereas total flavonoid content (TFC) varied between 5.324- 15.844 mg of Quercetin/g. The TPC and TFC of all root extracts ranged between 3.19-8.41 mg of GAE/g and 10.28-12.40 844 mg of QE/g, respectively. DPPH leaf fractions ranged from 48-50% and 60-61% for root extracts at 0.5 mg/mL. The ABTS assay also showed a dose-dependent radical scavenging activity. The scavenging activity ranged between 92-95% (crude leaf extract and fractions) and 77-84% (crude root extract and fractions). The investigations using GC-MS revealed a total of 21 metabolites with varied phytochemical activity. For antimicrobial activity, all T. violacea leaf and root acetone extracts and their fractions suppressed the growth of all fungal strains with leaf extracts zones of inhibition ranging between 3-18 mm whereas the root extracts ranged between 3.3-21 mm. The MIC of leaf and root acetone extracts and their fractions ranged between 0.78-12.5 mg/mL and 0.39-12.5 mg/mL, respectively whereas the MFC values of the plant leaf and root acetone extracts and their fractions ranged between 0.78- ˃ 50 mg/mL and 0.39-25 mg/mL, respectively. The time-kill curve assay demonstrated that most of the leaf extract and their fractions at 2 MIC demonstrated fungistatic activity averaging 3-6.23 log10 kill against C. albicans whereas, almost all root extract and their fractions demonstrated fungicidal activity, averaging 0.04-3 log10 kill. The membrane permeability assay indicated membrane damage in a dose-dependent manner for all extracts. In conclusion, T. violacea possesses phytocomponents with good potential as antifungals.Item Antimycobacterial activity of synthetic compounds isolated from South African medicinal plants against mycobacterium tuberculosis(Vaal University of Technology, 2014-11) Ledwaba, Elizabeth Ramadimetsa; Bapela, N. B., Dr.; Van Wyk, ChristaTuberculosis (TB) remains one of the most difficult infectious diseases to control in the world today. The disease spreads easily in overcrowded, badly ventilated places and among people who are undernourished. Trends in the incidence of TB together with the development of multi-drug (MDR-TB) and extensively drug resistant (XDR-TB) strains of TB raises the need to intensify the search for more efficient drugs to combat this disease. Herbal remedies used in traditional medicine provide an interesting and largely unexplored source for the discovery of potentially new drugs for infections such as TB. The aim of the study was to evaluate the in vitro antimycobacterial activity of synthesized compounds from medicinal plants against Mycobacterium tuberculosis (M. tuberculosis). About 40 synthesized compounds isolated from South African medicinal plants were screened against H37RV using microplate alamar blue assay (MABA). Identified active compounds were screened against resistant strains of M. tuberculosis (MDR, XDR and pre-XDR) and sensitive clinical isolates of TB. Cytotoxicity and synergistic drug combination studies were done on active compounds to validate their toxicity and synergy levels. Cytotoxicity was done by sulforhodamine assay (SRB) against the C2C12 cell line. Only six compounds showed activity against M. tuberculosis with minimum inhibitory concentration (MIC) below 10μg/ml. The results obtained indicated that the cytotoxicity effects of the three compounds on C2C12 cells demonstrated marginal toxicity except for MVB 282/61215 which showed a high toxicity at the lowest concentration of 0.156μg/ml with over 100% viable cells at the highest concentration (5μg/ml). MVB 282/61271 had the highest percentage cell viability (65%) at the lowest concentration. Only two compounds had a higher potency evoking a bigger response at low concentrations with treated cells still viable after 3 days of incubation with the compound which was comparable with the treatment of isoniazid (INH). Synergistic activity of the six compounds was less in INH combination as compared to the rifampicin’s (RIF) combination. The results demonstrated that the synergistic interaction between the compounds and RIF could the antituberculosis acitivity. In conclusion the synergistic effects with RIF translate to lower dosing requirements of the compounds and the potential to combat multidrug resistant TB. In deed there is no doubt that natural products, with their range of interesting chemical structures and powerful antimycobacterial effects are certain to remain important participants in the development of new generations of antimycobacterial drugs.Item Assessing the genetic diversity of Alternaria Bataticola in South Africa using molecular markers(2015) Chalwe, Joseph Musonda; Pillay, Michael; Adebola, PatrickSweetpotato (Ipomoea batatas) is an important food crop that is grown in many countries. A number of viral and fungal sweetpotato diseases have been reported worldwide. One of the major and most economic diseases of the sweetpotato is Alternaria blight which is caused by the fungal pathogen Alternaria bataticola. This disease can be managed in a short term using fungicides and cultural practices. However, a long term and inexpensive approach is through the development of resistant cultivars. A prerequisite to this approach is the knowledge of the genetic diversity of this fungal pathogen. This study assessed the genetic diversity of 25 South African isolates of A. bataticola from naturally infected leaves and stems collected from different sweetpotato growing regions in South Africa by (i) characterising the isolates based on their morphology (ii) pathogenicity tests (iii) random amplified polymorphic DNA (RAPD) (iv) variation of the ITS2 sequences and (v) prediction of the ITS2 secondary structures. The isolates revealed some variation in colony colour pigments after culturing but Koch’s postulates were confirmed by their pathogenicity tests. The analysis of RAPD and variation of the ITS2 sequences showed high levels of variation (100%) among the isolates. Dendrograms generated from these analyses had many subclusters and did not cluster the isolates according to their geographic origins. The ITS2 secondary structures were predicted and can be used to identify and distinguish the isolates. This information in addition to the genetic diversity of the A. bataticola isolates will aid plant breeders in the development of resistant sweetpotato cultivars and early management of blight disease in South Africa.Item Assessing the genetic diversity of South African sweetpotato germplasm using DNA and protein markers(2013-06) Selaocoe, Maleshoane EllenSweetpotato is one of the most important food crops in developing countries including South Africa. Currently two major types of cultivars are grown in South Africa: one is the orange-fleshed sweetpotato (OFSP) which has high β-carotene content, a precursor of vitamin A. The second type is the cream-fleshed sweetpotato (CFSP) which has low β-carotene content but is high in dry matter. Most South Africans prefer the CFSP although the OFSP offers more advantages. This presents a challenge to plant breeders to develop new varieties that will combine the desirable qualities of both the cultivars. To achieve this goal, plant breeders need knowledge about the genetic variation of the crop to develop an efficient breeding programme. This study assessed the genetic relationships of 28 orange- and cream-fleshed sweetpotato accessions by (i) examining the variation in leaf proteins, (ii) using random amplified polymorphic DNA (RAPD) and, (iii) using variation of the ITS region. The analysis of proteins, RAPD and variation of the ITS region polymorphism levels were 55.6%, 98% and 16.5%, respectively. Dendrograms generated from all the analyses generally clustered the accession according to their flesh colour and country of origin. Analysis of molecular variance (AMOVA) found a significant difference between OFSP and CFSP and a significant difference between the South African and non-South African germplasm. The high genetic diversity in the South African sweetpotato germplasm is a positive indicator for a breeding programme that has a number of targets such as breeding for nutritional improvement, disease resistance and drought toleranceItem Assessing the morphological variation and characterising the proteins of bambara groundnut (Vigna Subterranea L. Verdc)(Vaal University of Technology, 2016-12) Evangeline, Unigwe Amara; Adebola, Dr. P.; Pillay, Prof. M.Bambara groundnut (Vigna subterranea L. Verdc) is an underutilized crop in the African continent. It is a drought tolerant crop and fixes atmospheric nitrogen. Bambara groundnut is primarily grown for the protein content of its seeds and is mainly produced by small scale farmers at the subsistence level. However, despite its importance as a subsistence crop in many African countries, only local landraces of bambara groundnut are still cultivated. Mass selection of a few local varieties for the main agronomic characteristics has been carried out. All the bambara groundnut germplasm in South Africa has not been morphologically characterized. Although the protein of bambara groundnut is of good quality and is rich in lysine, there is no information on the characterisation of these proteins. The presence of antinutritional factors in the crop has also received little attention. This study focused on three major objectives including: (I) to assess the extent of morphological variations among thirty selected landraces of bambara groundnut, (II) to characterize the major seed proteins in these accessions using one dimensional gel electrophoresis, and (III) to determine the presence of any anti-nutritional factors in the seeds of the selected bambara groundnut landraces. 30 accessions of bambara groundnut were evaluated for their variability in agronomic and morphological traits. The field experiment was conducted at ARC-VOPI in Roodeplaat research farm during the 2014/2015 summer cropping season. The field trial was arranged as a complete randomized block design with 3 replications. 18 quantitative traits were recorded to estimate the level of genetic variability among accessions. 4 different methods were employed to extract seed proteins from 30 bambara groundnut accessions in order to ascertain the best method for protein extraction. These methods included: 10%-80% isopropanol, 10% trichloroacetic acid (TCA) in acetone solution, sonication and 2x Lammeli buffer extraction methods. The quick start Qubit® fluorometer protein kit was used to determine the protein concentration in each sample. The samples were then subjected to one dimensional gel electrophoresis. For antinutritional analysis, 5 factors (condensed tannins, free and phytic acid phosphate, polyphenol and trypsin contents) were used to determine the amount of antinutrient in 30 bambara seeds that were ground to a fine powdery flour. 3 replicates of all the samples were ground for each assay evaluated. The flour was then immediately extracted and used for the different assays. The analysis of variance revealed significant differences only in 10 of the 18 phenotypic traits that were evaluated. The UPGMA cluster analysis based on the quantitative traits produced four distinct groups of genotypes and a singleton. Genotypes SB11-1A, SB19-1A, SB12-3B and Bambara-12 were found to possess good vegetative characters and are recommended for use as suitable parents when breeding cultivars for fodder production. Desirable yield and yield-related traits were identified in B7-1, SB4-4C, SB19-1A, Bambara-12 and SB16-5A and are recommended as suitable parental lines for bambara groundnut grain production improvement. The quantitative characters therefore provided a useful measure of genetic variability among bambara genotypes and will enable the identification of potential parental materials for future breeding programmes in South Africa. Out of the 4 different seed protein extraction methods exploited for this study, the 2x Laemmli buffer extraction method produced the best result with clear protein bands. A unique feature from all extraction methods was the presence of a common protein band at ̴ 75 kDa. All extraction methods except 10 % TCA-Acetone resolved common banding patterns in all the bambara groundnut samples. This data suggests that there is very little or no intraspecific genetic diversity among the seed proteins of bambara groundnut accessions studied. There was wide variation in the content of the five antinutritional compounds among the thirty bambara groundnut accessions. The mean values for condensed tannin content ranged between 0.20 - 6.20 mg/g. Free phosphate recorded an overall mean of 1.71 mg/g while a range of 1.35 - 4.93 mg/g was observed by phytic acid phosphate (PAP). The polyphenol content had an overall mean of 0.39 mg/g and trypsin inhibitor (TIA) was quite variable among the bambara groundnut accessions ranging from 5.30 - 73.40 TIA/mg. Generally, higher levels of antinutrients were observed in this study compared to the other studies. The results obtained in this study led to a conclusion that although variations exits among the accessions studied, further research is required to verify the extent of morphological variations, the efficiency of protein extractions methods evaluated and the effects of these antinutrients in human and animal feeds.Item Assessing the pollutant removal efficiency of a wetland as a polishing treatment for municipal wastewater(Vaal University of Technology, 2021-02-16) Mphuthi, Betty Refilwe; Osifo, P., Prof.; Walmsley, T. A., Dr.Pollution of aquatic systems by wastewater containing pathogens, heavy metals and high concentrations of nutrients is of great concern due the ecological risks they impose. The toxic effects of metals may occur even at low concentrations because of potential bio magnification in the food chain. Excessive nutrients cause algal blooms which depletes oxygen and prevents sunlight from penetrating into the water, thereby killing fish and other aquatic organisms. This study investigated the pollutant removal efficiency of a riparian wetland located in Sebokeng, Emfuleni local municipality, South Africa. The study was carried out to assess the water quality of a wetland located downstream of the Sebokeng wastewater treatment plant by monitoring and analysing the physico-chemical parameters which included pH, temperature, electrical conductivity, nutrient levels (nitrates, phosphates, nitrites) and heavy metals. The water samples were collected from the effluent discharge of the treatment plant, upstream and downstream of the wetland. Plant uptake of heavy metals in a riparian wetland, nitrification as well as denitrification processes have been historically recorded as the main processes that contribute to the high removal of pollutants in a wetland. The contaminant concentrations of the influent and the effluent were used to estimate the wetland efficiency in improving the water quality that passes through it and its potential effects on improving the quality of irrigation waters. The heavy metals of interest included Al, Cd, Cr, Cu, Fe, Pb, Mn and Zn. Most heavy metals within the wetland occurred at low concentrations (lower than detectable limits and within the discharge limits for irrigation purposes). The results indicate that the average removal efficiencies for Electrical Conductivity (EC), Total coliforms (TC), E. coli, BOD5, COD, TSS, carbonate hardness, aluminium, iron, manganese, copper, nitrite, nitrate, sulfate and ortho-phosphate were 43 %, 51%, 85%, 60%, 61%, 61%, 21%, 67%, 52%, 51%, 83%, 56%, 89%, 49% and 54% respectively. The study showed that this wetland can provide up to 89% removal efficiency of pollutants. Of particular significance was the high pathogen and nutrient removal efficiency. A t-test was performed in order to determine the statistical significance of the wetland pollutant removal efficiencies. All p-values calculated were well below 0.05 and the removal efficiencies are therefore considered statistically significant. For this particular ecosystem the findings show that there is no great concern about metal pollution since most of the metals tested for were below the minimum limit for irrigation stipulated by the South African water regulation department (DWAF 1996a). Therefore, the wetland effluent water qualifies for both agriculture and landscape irrigation. Future considerations in choosing to use wetlands as a polishing facility for wastewater treatment systems are highlighted in the study.Item Biochemical and molecular characterization of heavy metal resistant bacteria isolated from the Klip River, South Africa(Vaal University of Technology, 2014) Chihomvu, Patience; Stegmann, P., Dr.; Pillay, M., Prof.The Klip River has suffered severe anthropogenic effects from industrial, agricultural, mining and domestic activities. As a result harmful contaminants such as heavy metals have accumulated in the river, causing microorganisms inhabiting the environment to develop mechanisms to protect them from the harmful effects of the contaminants. The current study deals with the isolation and characterization of heavy metal resistant bacteria isolated from the Klip River Catchment. Water and sediment samples were collected from 6 sites of the Klip River, and the Vaal Barrage (control). In-situ parameters, such as pH, turbidity, salinity, conductivity, temperature and dissolved oxygen were determined. Lead, iron, cadmium, nickel, zinc and copper concentrations of water were determined by atomic absorption spectroscopy. For bacterial analysis sediment and water samples were collected in sterile glass jars and bottles respectively. Heavy metal resistant bacterial isolates were screened on heavy metal constituted Luria Bertani (LB) agar. Biochemical profiles of the isolates were constructed using the API 20E® strips, antibiotic susceptibility tests were done and growth studies were carried out using spectrophotometric methods. The isolates were identified using 16SrDNA sequencing and alignment. A partial sequence of the copper resistance gene pcoA was amplified from strains Lysinibacillus sp. KR25 [KJ935917], and Escherichia coli KR29 [KJ935918]. The pcoR gene was amplified from E. coli (KR29) and the partial sequence for the chromate resistance gene chrB, was amplified from Pseudomonas sp. KR23 [KJ935916]. The gene fragments were then sequenced and translated into protein sequences. The partial protein sequences were aligned with existing copper and chromate resistance proteins in the Genbank and phylogenetic analysis was carried out. The physico-chemical properties of the translated proteins were predicted using the bioinformatics tool Expasy ProtParam Program. A homology modelling method was used for the prediction of secondary structures using SOPMA software, 3D-protein modelling was carried out using I-TASSER. Validation of the 3D structures produced was performed using Ramachandran plot analysis using MolProbity, C-score and TM-scores. Plasmid isolation was also carried out for both the wild type strains and cured derivatives and their plasmid profiles were analysed using gel electrophoresis to ascertain the presence of plasmids in the isolates. The cured derivatives were also plated on heavy metal constituted media. Antibiotic disc diffusion tests were also carried out to ascertain whether the antibiotic resistance determinants were present on the plasmid or the chromosome. The uppermost part of the Klip River had the lowest pH and thus the highest levels of heavy metal concentrations were recorded in the water samples. Turbidity, salinity and specific conductivity increased measurably at Site 4 (Henley on Klip Weir). Sixteen isolates exhibiting high iron and lead resistance (4 mM) were selected for further studies. Antibiotic susceptibility tests revealed that the isolates exhibited multi-tolerances to drugs such as Ampicillin (10 μg/ml), Amoxcyllin (10 μg/ml), Cephalothin acid (30 μg/ml), Cotrimoxazole (25 μg/ml), Neomycin (30 μg/ml), Streptomycin (10 μg/ml), Tetracycline (30 μg/ml), Tobramycin (10 μg/ml) and Vancomycin (30 μg/ml). Growth studies illustrated the effect of heavy metals on the isolates growth patterns. Cadmium and chromium inhibited the growth of most of the microorganisms. The following strains had high mean specific growth rates; KR01, KR17, and KR25, therefore these isolates have great potential for bioremediative applications. Using 16SrDNA sequencing the isolates were identified as KR01 (Aeromonas hydrophila), KR02 (Bacillus sp.), KR04 (Bacillus megaterium), KR06 (Bacillus subtilis), KR07 (Pseudomonas sp), KR17 (Proteus penneri), KR18 (Shewanella), KR19 (Aeromonas sp.), KR22 (Proteus sp.), KR23 (Pseudomonas sp.), KR25 (Lysinibacillus sp.), KR29 (Escherichia coli), KR44 (Bacillus licheniformis) and KR48 (Arthrobacter sp.). Three heavy metal resistance genes were detected from three isolates. The pcoA gene was amplified from strains Lysinibacillus sp KR25, and Escherichia coli KR29; pcoR gene from E. coli KR29 and the chrB gene, from Pseudomonas sp. KR23. The genes encoding for heavy metal resistance and antibiotic resistance were found to be located on the chromosome for both Pseudomonas sp. (KR23) and E.coli (KR29). For Lysinibacillus (KR25) the heavy metal resistance determinants are suspected to be located on a mobile genetic element which was not detected using gel electrophoresis. The translated protein sequence for pcoA_25 showed 82% homology with the copper resistant protein form Cronobacter turicensis [YP003212800.1]. Sequence comparisons between the pcoR partial protein sequence found in E. coli KR29 showed 100% homology with 36 amino acids (which was 20% of the query cover) from a transcriptional regulatory protein pcoR found in E. coli [WP014641166.1]. For the chrB partial protein sequence detected in Pseudomonas sp. (KR23), 97% of the query sequence showed 99% homology to a vitamin B12 transporter btuB in Stenotrophus sp. RIT309.Item Characterisation of Amaranthus Tricolor mutant plants with increased drought-tolerance(2010-02) Kgang, Itumeleng Eugenia; Laloo, Neelan; Van Emmenis, LynelleAmaranthus tricolor (A. tricolor) is a nutritious vegetable crop that is used as a subsistence and cash crop in the rural areas in Africa. Its yield and production is severely limited by abiotic stresses such as drought. Mutation technology, using gamma irradiation, was previously employed as a tool to create genetic variation in order to select for lines with improved drought-tolerance. During irradiation, 160 Gy (Gray) was selected as the optimal dosimetry that allowed subsequent seed germination. The resulting mutant lines were screened over several generations under field and greenhouse conditions and seven promising drought-tolerant lines were selected. Here we report on physiological and morphological studies of two of these Amaranthus mutant lines (#2 and #5) to confirm the enganced drought-tolerance. Plants were grown in the greenhouse in plastic pots containing germination mix with fertiliser. They were exposed to 21 days of well-watered condition, 19 days of drought-stress conditions and 7 days of re-watering. shoot height, leaf area, protein content and relative water content (RWC) of the fresh and dry material were determined colorimetrically under well-watered and drought-stress conditions, while anthocyanin was only measured during well-watered conditions. Shoot height, leaf area, number of leaves per plant and the protein content were significantly reduced under water-stress conditions. Under well-watered condition mutant #5 grew faster with the shoot length significantly higher than mutant #2 and the wild type. Even though drought adversely affected shoot lenght, mutant#5 still performed better than mutant #2 and the wild type under drought-stress conditions. While under both well-watered and drought-stress conditions, the wild type plants had bigger leaf area compared to the two mutant lines. After 16 days of drought-stress conditions, all the leaves of the wild type plants were dried out, as a result no wild type plants recovered after 8 days re-watering. Meanwhile, both mutant #2 and #5 plants recovered significantly after 8 days of re-watering. The wild type was tolerant compared to the two mutant lines. Protein content for mutant #2 plants was higher under both well-watered and drought-stress conditions but was not significantly different from mutant #5 plants compared to the wild type plants after 19 days of drought-stress conditions. Furthermore, genetic diversity was examined in all the Amaranthus lines using random amplified polymorphic DNA (RAPD) analysis. Nineteen arbitrary RAPD markers were used of which two detected polymorphisms (OPA) 07 and OPA 16).Item Characterization and identification of microbial communities in pigeon droppings using Culture-Independent techniques(2015-08) Leareng, Samuel Keeng; Pillay, M.; Feto, Naser AliyePigeon droppings, found in abundance in most cities and towns where pigeons are found, are a source of potential yeast and molds into the environment. Invasive fungal infections are a cause of morbidity and often mortality in immunocompromised individuals. The objective of this study was to the identification of bacterial and mold agents from pigeon droppings. Pigeon droppings samples were collected from three locations during the winter and summer months and studied for the occurrence of bacteria, yeast and molds by utilising culture-independent techniques. Amplification of the 16S rDNA gene and the internal transcribed spacer (ITS) region, cloning and ARDRA and DGGE were used for the characterisation of the microbial populations followed by sequencing. Several mold and yeasts, as well as bacteria were found to be present in pigeon droppings, which can spread into the environment and be transmitted to immunocompromised individuals and children. DGGE analysis of the bacterial communities revealed banding patterns that clustered all but one winter samples and all summer samples, showing a high similarity among the microbial members in both seasons and sample locations. Fungal DGGE analysis revealed clusters that grouped summer and winter samples from Johannesburg and Pretoria while VUT samples were clustered on their own. From the identification of fungal and bacterial DNA, Cryptococcus species was the majority of fungi isolated from the dropping samples. Geotrichum, Kazachstania and Fusarium species were isolated from phylotypes obtained from ITS amplicons analysed by ARDRA. Lactobacillus and Enteroccoccus species, organisms usually found in the gastrointestinal tract were the common bacterial members identified. The results showed no difference in microbial communities across all sample locations, while seasonal changes also had no impact in microbial community patterns.Item Co-production of inulinase by Kluyveromyces marxianus and Saccharomyces cerevisiae in solid state fermentation(2014-02) Molefe, Nnana Mantsopa; Padayachee, T.; Reddy, P.Solid-state fermentation (SFF) has emerged as a good method for the production of microbial enzymes such as inulinases. The use of low-cost agricultural plants and agro-industrial residues as substrates in SSF processes provides a value adding alternative to these otherwise under/or un-utilised vegetation. Production of inulinases, using various inulin-containing plant materials as carbon sources was studied using pure and mixed cultures of yeast strains. All substrates resulted in different levels of enzyme activity. A mixed culture of Kluyveromyces marxianus and Saccharomyces cerevisiae produced an extracellular exoinulinase when grown on different types of inulin-containing plant materials. Initial inulinase production was achieved as follows: 10 IU/gds (garlic cloves), 15 IU/gds (parsnips), 10 IU/gds (wheat bran) and 7 IU/gds (amadumbe) by K. marxianus and S. cerevisiae in a mixed culture. The production of inulinases by a mixed culture of K. marxianus and S. cerevisiae under SSF was further optimized by investigating initial moisture content, temperature, carbon source, nitrogen source, inoculum volume and inoculum ratio. The highest inulinase activity attained was in garlic cloves (85 IU/gds), followed by parsnips (65 IU/gds), wheat bran (37 IU/gds) and amadumbe (25 U/gds). The activities yielded 5.6 fold higher inulinase than in preliminary studies. The optimum pH and temperature of the crude enzyme were 5.0 and 50 oC, respectively. The pH and temperature stability of the enzyme was steady for 1 hour retaining about 64% activity. The average inulinase/invertase activity (I/S) ratio of 1.0 by crude inulinases was also observed after 48 hours. The crude extracellular enzyme extracts from the garlic cloves, parsnips, amadumbe and wheat bran were partially purified by ammonium sulphate precipitation and showed a specific activity of 9.03 U/mg, 0.08 U/mg, 4.12 U/mg and 0.133 U/mg respectively. The Km and Vmax values of the inulinase were 21.95 mM and 2.09 μM/min; 19.79 mM and 1.38 μM/min; 31.59 mM and 0.51 μM/min; and 25.74 mM and 0.23 μM/min, respectively. All extracts demonstrated potential for large-.scale production of inulinase and fructose syrup.Item A Comparative study between the prevalence of MTHFR A1298C SNP and homocysteine metabolism in an elderly black South African population(Vaal University of Technology, 2018-08) Dippenaar, Luzanne; Lebea, P. J., Dr.; Grobler, C. J., Dr.Background: Cardiovascular diseases are one of the most common causes of death worldwide. This is not only a problem in developed countries, it is of major concern for public health in developing countries as well. Increased homocysteine is an independent risk factor for cardiovascular diseases. Nutritional deficiencies of folate, vitamin B6 and vitamin B12 are associated with hyperhomocysteinemia. MTHFR A1298C, a single nucleotide polymorphism, is similarly linked with higher concentrations of homocysteine. The aim of this study was to determine the prevalence of MTHFR A1298C in a black elderly population, along with folate, vitamin B6 and vitamin B12 and to evaluate the effect on homocysteine levels. Methodology: The research design was an observational cross-sectional study and was ethically approved. A total of 84 elderly who attend a day-care centre (also met inclusion criteria) were purposively selected. DNA was extracted and frozen on the day of blood collection. The MTHFR A1298C genotype was determined with real time PCR. Homocysteine, folate, vitamin B6 and vitamin B12 serum levels were detected with commercial assay kits. Results: Homocysteine was found to be elevated with a median of 17.78 µmol/L (interquartile range 13.98-21.03 µmol/L). Serum folate, vitamin B6 and vitamin B12 medians were in the normal range. Although, 5.95% and 22.62% of the population were deficient and possibly deficient for vitamin B12, respectively. MTHFR A1298C frequency was as follow: 89.29% (AA), 9.52% (AC) and 1.19% (CC), with no significant correlation (p>0.05) with homocysteine. Vitamin B12 correlated significantly with homocysteine levels. Conclusion: Vitamin B12 deficiency had an effect on homocysteine levels. Overall, nutritional deficiencies are not responsible for the hyperhomocysteinemia in this population. In conclusion from this study showed MTHFR A1298C frequency in black South Africans does not contribute to homocysteine as a risk factor for cardiovascular disease. Keywords: Cardiovascular disease, elderly, folate, homocysteine, MTHFR A1298C, vitamin B6, vitamin B12.Item The construction and evaluation of a novel tubular photobioreactor at a small pilot plant scale(2012-07) Kutama, Makonde; Stegmann, P.; Van Wyk, C.The mass production of algae for commercial purposes has predominately been carried out in open ponds systems. However, open ponds systems have a number of disadvantages such as poor light utilization, requirement for large areas of land and high risks of contamination. On the other hand, photobioreactors have attracted much interest because they allow a better control of the cultivation conditions than open systems. With photobioreactors, higher biomass productivities are obtained and contamination can be easily prevented. Photobioreactors can also be engineered to manipulate the light and dark photosynthetic reactions thus enhancing biomass productivity. The main objective of this study was to construct a novel tubular photobioreactor which had the ability to expose the cultured alga to light and dark phases with the aim of optimizing the algal biomass production. A novel tubular photobioreactor with the ability to manipulate the cultured alga’s light and dark photosynthetic reactions was constructed in this study. The alga Spirulina platensis was chosen as the test organism in this novel tubular photobioreactor due to a number of reasons such as its globally socioeconomic importance, its tolerance of higher pH and temperature values which makes it almost impossible to contaminate. The cultivation process of Spirulina in the photobioreactor was investigated through alternating light and dark cycles in an attempt to increase the photosynthetic efficiency of the culture. The effect of different light intensities on the growth of Spirulina in the novel tubular photobioreactor was investigated and it was found that the best light condition that favored higher biomass formation was at 600 μ mol m-2 s-1. Five different light/ dark ratios were evaluated at a light intensity of 600 μ mol m-2 s-1 during a batch mode of operation of the novel tubular photobioreactor. The light/ dark ratio of 1:0.25 was found to be the best ratio because it gave the highest biomass in the shortest period of time when compared to the other ratios used. These results seem to suggest that longer light cycle relative to dark cycle results in higher biomass production. The ratio of 1:0.25 was then used to operate the novel tubular photobioreactor in a continuous mode. A maximum biomass productivity of 25 g/m2/day was achieved which corresponded to a net photosynthetic efficiency of 5.7 %. This result was found to be higher than what most photobioreactors could achieve but it was 2.8 g/m2/day lower than the highest ever reported productivity in a photobioreactor when Spirulina is cultivated. The 2.8 g/m2/day lower was attributed to the different materials used in the construction of these two photobioreactors. The photobioreactor which achieved 27.8 g/m2/day was made up of a clear glass whereas the novel tubular photobioreactor was made up of a PVC tubing. PVC tubes tend to change from clear to a milky colour after a certain period when it is used at higher temperature and pH values hence blocks a certain amount of light. Therefore the main recommendation in this study is to use a PVC tubing with a longer life span when used at a higher temperature and pH values.Item Correlating the prevalence of C174G polymorphism with IL-6, TNF-α and Hs-CRP in an elderly black South African population.(Vaal University of Technology, 2019-03) Valentine, Jessica; Lebea, J., Dr.; Grobler, C. J., Dr.Cardiovascular diseases (CVDs) are the leading cause of death worldwide, and the prevalence thereof is on the rise in developing countries due to the demographic transition and urbanization. The inflammatory process, atherosclerosis, is at the root of the majority of CVDs and is caused by unresolved inflammation. Various cardiovascular risk factors such as hyperglycaemia, dyslipidaemia, hypertension, smoking and aging stimulate the development of atherosclerosis through triggering inflammation. Being in a state of chronic low-grade inflammation therefor places an individual at higher risk of developing CVD, with inflammation playing a cause and effect role. The aim of this study was to investigate the inflammatory status of an elderly black South African population by analysis of inflammatory markers HS-CRP, TNF-α and IL-6, as well as the genetic polymorphism C174G associated with increased serum levels of IL-6 in some populations. The research was conducted in the field of Biomedical Sciences as a quantitative, cross-sectional, analytical observational design. The study was ethically approved and involved collection of 84 blood samples from volunteers in a purposively selected population as part of a larger collaborative study. Serum was used to analyse HS-CRP, TNF-α and IL-6 and DNA was extracted from whole blood for analysis of the C174G polymorphism. The median serum HS-CRP of 6.44mg/L (IQR = 2.82 - 9.86mg/L) fell within the highest risk (>5mg/L) of CVD and 75% of participants were at high (3.01-5mg/L) or very high (>5mg/L) risk. The median TNF-α of 0.00pg/mL was within the normal range and only 2.6% of participants had high serum TNF-α levels. The median serum IL-6 level was 1.92pg/mL and was also within the normal range with only 2.6% of participants who had high serum IL-6 levels. For the C174G polymorphism analysis, 98.6% had the GG, 1.4% the GC genotype and no participants had the CC genotype. The median serum IL-6 level of the homozygous GG group was 6.51mg/L, higher than the 4.13mg/L serum IL-6 of the heterozygous GC group. The difference in IL-6 should be considered with caution as only one participant had the C allele. A highly significant (p=0.001) correlation was found between HS-CRP and IL-6, as well as between IL-6 and TNF-α (p = 0.048). The elderly black Sharpeville community is in an increased inflammatory state which puts them at risk of CVD. The prevalence of the C allele in the C174G polymorphism is low in this population. Further research could be conducted as intervention studies to decrease the inflammatory state of the population and influence health policy changes to improve prevention of CVD.Item Cytotoxic and genotoxic studies of crude extracts from the leaves, stems and roots of Tulbaghia Violacea(Vaal University of Technology, 2017-11) Nellvecia, Madike LeratoTulbaghia violacea Harv. (wild garlic) has been used in traditional medicine in Southern Africa for the treatment of various ailments. Despite the widespread use and popularity of this medicinal plant as a herbal medicine, there is contradictory evidence regarding the safety and toxicity of the plant. The phytochemical profiling of the plant has also been neglected in research. The determination of chemical constituents present in plant material as well as the potential toxicity found in plants are preliminary steps necessary for the discovery and development of novel therapeutic agents with improved efficacy. The aim of this study was to evaluate the cytotoxic and genotoxic potential of crude extracts from the leaves, stems and roots of T. violacea. This was performed in vitro using aqueous and ethanol extracts of the leaves, stems and roots. The aim of the study was achieved by three major objectives; (1) to identify the active phytocompounds present in the leaves, stems and roots, (2) to assess the cytotoxicity using the MTT (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) cell proliferation assay, and (3) to evaluate the genotoxic potential of the leaf, stem and root water extracts using the Allium cepa assay. A total of 14 phytochemicals were each extracted separately with distilled water and 70% ethanol by maceration from the leaves, stem and roots of T. violacea. The results of the qualitative phytochemical analysis showed that pharmacologically active compounds such as tannins, terpenoids, flavonoids, saponins, proteins, steroids, cardiac glycosides, phenols and coumarins were present in some organs of T. violacea. However, phlobatannins, leucoanthocyanins, alkaloids, carbohydrates and anthocyanins were absent in all plant parts. Overall, the leaves of the plant contained more active compounds than those present in the stems and roots when both water and 70% ethanol were used as the extractants. The quantitative phytochemical analysis for the Total Flavonoids Content (TFC) and Total Phenolic Contents (TPC) was also assessed. The water (0.027 mg/g) and 70% ethanol (0.053 mg/g) were most effective in extracting flavonoids from the leaves while the least amounts were obtained from the stems and roots. This observation was similar to the TFC were the water extracts of the leaves were the most effective in extracting phenols followed by the stems and roots. The MTT assay was conducted using two cell lines RAW 264.7 and C2C12. The experiment was conducted in triplicates for the leaf, stem and root extracts (water and ethanol) of T. violacea. The experimental design employed a 23 factorial design where three independent variables (concentration, incubation time and type of extracts) were selected using two levels for each variable (high (+) and low (-)). The results illustrated that both the water and ethanol vi extracts only showed a significant reduction in the number of viable cells at the concentration higher than 250 μg/ml treatment for both RAW 264.7 and C2C12 cells. The ethanol extracts from the leaves, stems and roots were found to be toxic towards the RAW 264.7 cells even at lower concentrations at both 24 and 48 h incubation periods (% cell viability < 50%). The water extracts were non-toxic to RAW 264.7 cells except for the water stem extract which showed toxicity after 48 h incubation (IC50 = 9.475 (4.061 to 23.39)). For the C2C12 cells, the lowest potent toxic concentration was 250 μg/ml for the ethanol extract of the stem after 48 h incubation. Overall, the T. violacea plant extracts were non-toxic as percentage cell viability greater than 50% was noted for both extraction solvents in all the plant parts of T. violacea. No cytotoxic activity was observed in all T. violacea plant parts with the C2C12 cell line (IC50 > 30 μg/ml). For the Allium cepa assay, only the water crude extracts of the leaves, stems and roots of T. violacea were used. A similar trend of potent genotoxic activity in the water stem extracts compared to the leaf and root extracts at the concentration ranges studied. Similar to the MTT assay, it is clear from the study that at higher concentrations, the water crude extracts from the leaves, stems and roots of T. violacea is toxic. From this study, it can be concluded that the extraction of compounds using water is more efficient than using ethanol. Overall, the T. violacea leaf extracts extracted the most phytocompounds and showed the highest percentage of viable cells as well as desirable IC50 values. However, preparation of herbal remedies using T. violacea plant extracts should be done with caution due to their possible genotoxic and cytotoxic potential at higher concentrations. This study raises a need to further conduct in vivo cytogenetic studies to ascertain the possible toxic effects of T. violacea crude extracts.Item Determining the effectiveness of water treatment process barriers for the removal of viruses in drinking water.(2018) Setlhare, Khomotso Charity; Leat, N, Dr.; Pillay, M, Prof.; Ssemakalu, C. C., Dr.The presence of enteric viruses in drinking water poses a health risk to consumers. It is therefore very important for drinking water suppliers to provide water that is pathogen free and fit for human consumption. This can be achieved by an effective water treatment system that ensures the safety of water from the treatment plant until the water reaches the consumer. This study assessed the ability of a conventional water treatment system to remove viruses. The system consisted of three unit processes, namely, clarification, sand filtration and disinfection. These processes were simulated on a bench-scale to determine the effectiveness of each one at removing viruses. Clarification was conducted using a Phipps and Bird jar testing system and three different chemical treatments: (i) Polyelectrolyte (SUDFLOC 3835), (ii) a combination of lime and activated silica and (iii) a combination of lime, activated silica and ferric chloride. Sand filtration was simulated using a Phipps and Bird column filtration system. Disinfection was conducted using free chlorine. The findings from this study showed that the removal or inactivation of viruses increased with an increase in the concentration of chemicals added. For clarification, the combination of lime, activated silica and ferric chloride was the most effective treatment for the removal or inactivation of viruses. Sand filtration was found to be ineffective for the removal of viruses. Disinfection was shown to be the most effective process for the removal or inactivation of viruses. While clarification, sand filtration and disinfection did not remove or inactivate viruses equally, the entire treatment chain is still essential. This is because even if a barrier does not directly remove viruses it ensures that subsequent processes can function effectively. Overall the treatment processes should not be considered as discrete barriers but rather an integrated system that must function throughout to avoid a risk to customers.Item Development of a HPLC method for the detection of Levetiracetam in blood of patients with epilepsy(2016-05) Engelbrecht, Lynette; Grobler, C. J.; Rheeders, M.Approximately 1% of the world’s population has epilepsy, the second most common neurological disorder after stroke. In South Africa almost 1 in every 100 people has epilepsy, affecting all ages. Levetiracetam (LEV), marketed as Keppra® is an anticonvulsant drug used in the treatment of epilepsy. The daily dosage is 500 mg twice daily with a maximum of 3000 mg. The therapeutic range of LEV is between 12-46 μg/ml. Therapeutic drug monitoring (TDM) should be considered for LEV in patients with poor seizure control or long term treatment. TDM depends on accurate drug concentration measurements. In order to provide an accurate measurement, the High performance liquid chromatography (HPLC) method was developed, compared with a commercially available kit, and the stability of the samples was investigated. Ethical approval was obtained from the Human Research Ethics Committee (Medical), VUT (Ethics reference number: 2015024.4). The study was conducted from January to October 2015. This study involved three groups of volunteers who gave written consent. The first group were fifteen healthy MTech students in the Biomedical Technology Department at the Vaal University of Technology (VUT). Their blood samples were used for the analytical validation of the method and for the stability studies over a 4 weeks period. The second group were six patients from Pathcare Laboratories in Potchefstroom, Klerksdorp and Vereeniging who used Levetiracetam. Their blood samples were used to investigate the influence of different collection tubes as well as the handling and storage of samples on the LEV concentration. The third group were forty four patients from Pathcare Laboratories, Cape Town. Their blood samples were transported to Clinical Pharmacokinetic Laboratory (CPL) for routine therapeutic drug monitoring analysis of LEV and used to compare the newly developed HPLC method and the Commercial kit. The HPLC method was successfully developed and validated to determine LEV in human plasma/serum samples. The calibration curves showed good linearity (r2 = 0,999) over the concentration range of 1 – 60 μg/ml. Accuracy, mean extraction recovery, lower limit of detection (LLOD) and lower limit of quantification (LLOQ) were 98-112%, 97,15% (±1,57), 0,5 and 1,0 μg/ml respectively, in plasma standards. The method was shown to be simple and fast, reproducible and effective for routine laboratory analyses in the future. The agreement between the newly developed method and the ClinRep® HPLC complete commercial kit was the same and there was a statistical significant correlation between the two methods (average r=0.999; p-value < 0.0001, F-test with a true value =0). The method was much cheaper than the commercial kit, used less sample (100 μl) and had a longer running time (15 minutes) to ensure no endogenous interference. The costs of the developed method was 71-82% lower than the three commercial kits available in South Africa. Stability experiments were performed to evaluate the stability of LEV in human plasma/serum, simulating the same conditions which occurred during study samples’ analyses. The % RSD was lower than 5% under all the conditions: freeze, fridge, room temperature and auto sampler over the 4 week period. The results showed that both LEV and the I.S (internal standard) were stable in human serum/plasma under all these conditions. The influence of five different collection tubes, Gold (SST Gel), Red, Purple (EDTA)Green (Heparin) and Blue (Sodium Citrate) was investigated. In two patients, decreased levels were observed in tubes containing blue (sodium citrate) and Green (Heparin). The decrease was not statistically significant. This is an important observation and is an indication that anticoagulants may cause some problems due to drug-protein binding and interference in the matrix effect. A cost effective and reliable HPLC-method with minimal sample preparation time for the routine determination of LEV in plasma/serum samples was developed. It was also shown that the plasma/serum samples were stable at different temperatures over a time period. The only collection tubes that may interfere with the concentrations were the Green (Heparin) and Blue (Sodium Citrate) tubes.Item Development of an "A" genome-specific sequence characterised amplified region (SCAR) marker in Musa L. (bananas and plantains)(2014-09) Mabonga, Lloyd; Pillay, M.Most cultivated bananas and plantains (Musa spp. sect. Eumusa), originated from two wild diploid species, Musa acuminata Colla (AA) and Musa balbisiana Colla (BB), which contributed the A and B genomes, respectively. The two genomes confer different traits to a banana plant. Intra- and interspecific hybridization between the wild diploid species, somatic mutations and selection over many thousands of years has given rise to considerable genetic variability in cultivated bananas. Bananas are classified according to its genome composition and a number of morphological traits are used to identify the genomes of a plant. Morphological classification can be misleading since the morphology of plants can be affected by environmental factors. Molecular techniques to identify the genomes of banana have many advantages. The objective of this study was to develop a SCAR (sequence characterized amplified region) marker from a previously reported A genome-specific RAPD fragment that distinguish the A genome of banana from the B genome. This fragment designated OPA17600 was cloned, sequenced and used to design longer 20-mer SCAR primers. Verification of the SCAR primers for its fidelity to the A genome was carried out on a sample of 22 homo-and heterogenomic accessions representing landraces and hybrids of different ploidy and genome combinations. Out of six primers sets that were tested one set (SC3) produced a unique 600 bp in all the A genome containing banana accessions. However, these primers also amplified an 800 bp fragment in all the BB genotypes and some accessions containing the A and B genomes. While previous reports suggested that there was considerable differentiation between the A and B genomes, recent evidence points to the contrary. The presence of the A genome fragment in the B genome genotypes and accessions may be due to recombination between the two genomes, translocations and substitutions. The study concluded that the 600-bp SCAR sequence is conserved across the A genome in Musa and can be used to identify the A genome in banana classification and Musa breeding programmes.Item The effect of a sugar sweetened beverage diet on DNA methylation in a CACO-2 cell line in vitro(Vaal University of Technology, 2020-12) Ndhlovu, Lesego; Pillay, M., Prof.; Ssemakalu, C., Dr.Obesity has steadily increased and represents a major public health problem worldwide, reducing quality of life and causing a range of health problems. Obesity has emerged as the fifth leading risk of global deaths. Annually, 2.8 million adults die as a result of being overweight or obese. The increase of obesity remains inexplicable in terms of genetic susceptibility to obesity. The genetic loci identified by genome-wide association studies (GWASs) explains about 2% of the heritability for obesity. Perhaps other factors such as epigenetics may be involved in the increase of obesity and may offer solutions for the management of obesity. Epigenetics is defined as a heritable change in gene expression without altering the genome sequences. It may help in providing a logical explanation between the genome and environment which shapes obesity risk and may help to explain the "missing heritability". Epigenetics may affect two mechanisms, namely: i) DNA methylation,and ii) histone modifications. DNA methylation might give scientists a link to the rise in obesity.The study aimed to investigate the effect of sugars used as sweeteners in sugar-sweetened beverages (SSB) on DNA methylation in a Caco-2 cell line in vitro. Four major objectives were pursued in the study which were to:(1) stimulate the Caco-2 cells with varying concentrations of sugar sweeteners and assess the morphological changes of the cells; (2) evaluate the cytotoxicity of different concentrations of the sugar sweetener on the Caco-2 cell line using the Alamar blue and LDH assay; (3) obtain genomic DNA from the treated Caco-2 cell line and perform bisulfite conversion and rest; and (4) amplify the WT1, MEG3, TNFRSF9, ATP10A, and CD44 obesity-associated genes and ascertain their degree of methylation. Caco-2 cells were stimulated with sugar sweeteners at varying concentrations (low, medium and high) for an incubation period of 62 days,and images of the cells were captured for morphological characterisation. The incubation condition entailed cells plated in a 12 or 96 well plate, incubated in a humidified 5% CO2 incubator at 37 °C and there is nutrient renewal every three days.Alamar blue, a cell proliferation colourimetric assay and lactate dehydrogenase assays (LDH), a homogenous membrane fluorimetric assay were used for the cytotoxicity studies. The results of the characterisation showed that different concentrations of sugar sweeteners affected the morphology of the cells as the incubation period progressed. The cytotoxicity results of both LDH and Alamar blue depicted low concentration of sweeteners that had low-to-moderate toxicity and the medium and high concentration of the sweeteners had a moderate to high toxicity on the Caco-2 cells. DNA from the Caco-2 cells was extracted. Techniques used to study DNA methylation such as bisulfite conversion, PCR amplification and restriction enzymes that have differential sensitivity to 5-methyl-cytosine were performed. The quality of DNA extracted was good. The bisulfite conversion was conducted andno amplification was observed, as a contingency plan Normal PCR was performed to amplify the CpG islands, and there was amplification. In conclusion, the study showed that a low concentration of a sugar sweetener (fructose: glucose) used in beverages had low toxicity to the Caco-2 cell line and prolonged exposure of the low concentration might have an adverse effect on the cells' morphology. At medium concentrations, the sugar sweetener used in beverages had medium toxicity to Caco-2 cells; prolonged exposure may lead to morphological changes. These findings indicated that control of dietary glucose intake is an important strategy in combating the development of obesity and type-2 diabetes. DNA methylation could not be established.Item The effect of crude water extracts of Tulbaghia violacea Harv. on scaffolds with cardiovascular applications(Vaal University of Technology, 2020-02) Madike, Lerato Nellvecia; Popat, K. C., Prof.; Pillay, M., Prof.Tulbaghia violacea Harv. has found extensive uses in traditional medicine for the treatment of numerous ailments among which are tuberculosis, oesophageal cancer, diabetes and cardiovascular diseases. Current reports show that cardiovascular diseases are now the primary cause of mortality worldwide. Thus, the potential of T. violacea plant extracts against cardiovascular diseases should be explored. The objectives of this study were, (i) to conduct qualitative and quantitative preliminary phytochemical screening of T. violacea aqueous leaf extracts, (ii) to conduct Gas chromatography–mass spectrometry (GC-MS) analysis for screening of compounds present in the plant extract, (iii) to evaluate the antioxidant activity of the T. violacea crude extracts using the DPPH:1.1-diphenyl-2-picrylhydrazyl and ABTS: 2,2-azino-bis 3-ethylebenzthiazoline-6-sulfonic acid assays, (iv) to evaluate the antimicrobial activity of the T. violacea crude extracts using disk diffusion and Minimum inhibitory concentration/Minimum bactericidal concentration (MIC/MBC), (v) to evaluate the antithrombogenic properties of T. violacea crude extracts on polystyrene, (vi) to fabricate polycaprolactone (PCL) and PCL-T. violacea incorporated scaffolds, (vii) to evaluate the antithrombogenic properties of T. violacea crude extracts on the fabricated PCL and PCL-T. violacea fabricated scaffolds and, (viii) to evaluate the growth and differentiation of adipose derived stem cells (ADSCs) on the fabricated scaffolds. The qualitative and quantitative phytochemical screening was conducted using standard procedures. Folin-Ciocalteu method was used to evaluate both total phenolic content (TPC) and total tannin content (TTC), the Aluminium chloride method was used for total flavonoid content (TFC) and GC-MS was used to screen for compounds present in the plant extract. The antioxidant activity was evaluated using DPPH and ABTS and the antimicrobial activity was evaluated using disc diffusion and MIC/MBC assays. The antithrombogenic properties of the T. violacea aqueous leaf extracts was then evaluated using platelet activation and whole blood clotting kinetics on polystyrene discs which have been reported to induce platelet activation. The experiment was performed in the absence and presence of 100 and 1000 μg/ml T. violacea plant extracts for both the platelet activation study which used blood plasma and the whole blood clotting kinetics assay which used fresh whole blood. Platelet adhesion was evaluated using fluorescence microscopy and a scanning electron microscope (SEM) was used to evaluate their morphology. Three scaffolds designated as PCL, 10% Tvio and 15% Tvio were fabricated which consisted of a 10% PCL powder and 10% as well as 15% T. violacea aqueous plant extract with respect to the PCL powder weight. The scaffolds were then characterized using Fourier-transform infrared spectroscopy (FTIR) and Energy-dispersive x-ray spectroscopy (EDS). The scaffolds were then evaluated for their antithrombogenic properties in the presence and absence of 100 and 1000 μg/ml T. violacea plant extracts. Platelet adhesion was evaluated using a fluorescent microscope and the morphology was evaluated using SEM. For the cell study, adipose derived stem cells (ADSCs) were cultured on the designed scaffolds and evaluated for their toxicity, viability, adhesion, proliferation, morphology and differentiation into osteoblasts over a period of 3 weeks. Lactate dehydrogenase (LDH) assay was used for toxicity studies, alamar blue assay was used for viability, fluorescence microscopy was used to evaluate cellular adhesion and proliferation while the alkaline phosphate (ALP) assay was used to evaluate differentiation of the cells into osteoblasts. Cell morphology was evaluated using SEM. Phytochemical screening of the prepared T. violacea aqueous extract revealed the presence of terpenoids, flavonoids, cardiac glycosides, saponins, protein, phenols, tannins, carbohydrates and amino acids. This is the first study that has identified the presence of carbohydrates and amino acids in T. violacea aqueous leaf extracts. Different concentrations of 0.1, 1.0 and 10 mg/ml of plant extract were used to conduct the quantitative phytochemical screening assays. There was a concentration dependent increase in the amount of phenols, tannins and flavonoids as the concentration of the plant extracts increased. This was the first study that evaluated the total tannic content of T. violacea plant extracts. The amount of total phenols was higher than that of flavonoids and tannins at every concentration range studied followed by the total flavonoids and lastly total tannins. The GC-MS analysis showed the presence of 33 compounds among which were 2,4 – Dithiapentate - 2,2-dioxide, Cannabidiol, 2,4,5,7 –Tetrathiaoctane and 2,4,5,7 - Tetrathiaoctane 2-dioxide. The presence of sulphur compounds support the characteristic garlic-like smell as well as some of the biological activities of T. violacea plant extracts. The antioxidant activities based on DPPH (0.49 mg/ml) and ABTS (0.24 mg/ml) suggest that T. violacea can be used as potential antioxidant agents. For the antimicrobial activity using disc diffusion, the extracts exhibited appreciable antibacterial activities against Bacillus subtilis, Serratia marcescens, Staphylococcus aureus and S. epidermidis. The highest zone of inhibition was observed for S. epidermidis at 19.50 ± 0.87 mm. The MIC results revealed that the plant extract of T. violacea was moderately active against B. subtilis, S. aureus, S. epidermidis, E. coli, and S. marcescens with MIC value of 2.5 mg/ml. However, the antimicrobial effect of the extract on S. epidermidis was bactericidal when compared to the bacteriostatic effect on the other active microorganisms. The antithrombogenic results on the polystyrene discs showed a significant reduction in the number of platelets that adhered on the polystyrene surfaces treated with plasma mixed with 100 μg/ml of plant extract when compared to the untreated control and the 1000 μg/ml treatment. For the 1000 μg/ml treatment, there was a significant increase in the number of platelets that adhered to polystyrene surfaces. These results were confirmed by the fluorescence and SEM results which showed a higher platelet count for the 1000 μg/ml treatment when compared to the other groups. The whole blood clotting kinetics study showed delayed blood clotting with the 100 μg/ml treatment over a period of 60 min when compared to the untreated control and the 1000 μg/ml treatment. These results correspond with the lower platelet adhesion observation and thus confirm the anticlotting properties of T. violacea aqueous leaf extracts at lower concentrations. The mean diameter of the scaffolds was recorded on the SEM as 275.60 ± 60.65 nm, 193 ± 30 nm and 537 ± 138 nm for the PCL, 10% Tvio and 15% Tvio scaffolds, respectively. The FTIR spectrum revealed the presence of amide groups as well hydroxyl O–H stretching groups which were the characteristic groups for the presence of T. violacea plant extracts in the polycaprolactone. The EDS results showed the presence of potassium, chlorine and sulphur compounds which were only present in the T. violacea scaffolds in addition to the carbon, oxygen and silicon observed in the PCL scaffold. The fabricated scaffolds were then used to evaluate platelet adhesion and activation on blood plasma in the absence and presence of 100 and 1000 μg/ml T. violacea aqueous leaf extracts. The results showed that the 10% Tvio scaffold was more effective in inhibiting platelet adhesion and activation at every treatment group especially when plasma was used in the absence of T. violacea plant extracts. A similar observation to the polystyrene study was observed were addition of 1000 μg/ml of plant extract resulted in the highest number of activated platelets. The study suggests the potential of the 10% Tvio scaffold in the prevention of platelet adhesion and aggregation. The in vitro cell adhesion, proliferation and differentiation of adipose derived stem cells (ADSCs) on the fabricated T. violacea loaded PCL nanofibers was then evaluated. The LDH assay illustrated less activity on the 10% Tvio scaffold when compared to PCL and 15% Tvio scaffolds however, none of the scaffolds were considered as toxic. The alamar blue assay was used for viability after 4 and 7 days of culture. The results showed a significant increase in cell viability for all scaffolds from day 4 to day 7 with the 10% Tvio scaffold having the highest overall cell viability for both day 4 and day 7 of cell cultures. Immunofluorescence staining was then used to count the number of cells using DAPI (4′,6-diamidino-2-phenylindole) stained images and illustrated that the T. violacea incorporated scaffolds supported better cell growth compared to the PCL scaffold. Cell morphology on the T. violacea scaffolds was denser and spread out into cellular extensions when compared to the PCL scaffold after 7 days of cell culture, supporting the higher number of adhered cells from the fluorescence results. For the long term cell study after week 1 and 3, the ALP results showed a significant difference in ALP activity between week 1 and week 3 for all scaffolds. The highest ALP activity was observed for the 15% Tvio scaffolds which is a marker for initial phase of bone matrix deposition. The designed T. violacea scaffolds supported better cell growth compared to the PCL scaffold and their morphology was more spread out and covered the entire surface of the scaffolds after week 3. Lastly, the cell count and osteocalcin differentiation was more prominent on 10% Tvio scaffold indicating higher levels of the protein marker for bone formation. Thus, supporting the use of the 10% Tvio scaffold for long-term cell studies. In conclusion, the results of this study indicated that the aqueous extract of T. violacea is rich is phytochemicals and also possess a broad range of pharmaceutically important compounds which may be attributed to the high antioxidant and antimicrobial activities identified. The results from this study suggest that T. violacea aqueous extracts have antithrombogenic properties at lower concentrations. Scaffolds fabricated with the incorporation of T. violacea plant extract also confirm the potential antiplatelet activity of the fabricated 10% Tvio scaffold. The results also suggest the potential of the fabricated 10% Tvio scaffold to enhance cell adhesion, proliferation and differentiation over long-term cell studies. It can thus be recommended that T. violacea may be useful for tissue engineering applications and bone repair with prospects of preventing cardiovascular diseases associated with bone defects. This research study has provided the foundation for clinical evaluation and outlined the potential effects of T. violacea aqueous leaf extracts as a clinical drug.Item The effects of biofouling on a reverse osmosis membrane purification system at Sasol, Sasolburg(Vaal University of Technology, 2011-06) Takaidza, Samkeliso; Van Wyk, C. S.; Stegmann, P., Dr.Reverse osmosis (RO) membranes are widely used in water purification. The presence of biofilms in water and industrial water purification systems is prevalent. As a result, biofouling which is a biofilm problem causes adverse effects on reverse osmosis process, which include flux decline, shorter membrane lifetime and an increase in energy consumption The effect of biofouling on RO membranes was investigated at a water treatment facility at Sasol, Sasolburg by investigating the quality of water purified by the RO system and the extent of fouling that is attributed to biofouling. Chemical and microbiological data was averaged based on the results obtained from water analysis and samples from a fouled membrane. Bacteriological plate counts ranged between log 1.5 to 4 cfu/ml in water samples and log 3.9 to 4.5 cfu/cm2 on biofilm from the membrane surface. Water analysis indicated a high conductivity of 121 µS/cm in the feed and 81 ppm of the TDS, whereas in the permeate conductivity was found to be around 6 µS/cm and 3.8 ppm of the TDS. This indicated that components present in the feed were retained by the membrane. This was supported by membrane autopsy which showed that the bacteria and elements found in the feedwater were also present on the membrane surface, hence contributing to fouling. An average of 33% of cellular ATP was measured on the biofilm from membrane sample, showing that the fouling bacteria are metabolically active in situ. The results clearly indicated that an important biological activity occurred at the membrane surface.
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