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Theses and dissertations (Applied Sciences)

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    The isolation and characterization of phytoalexin and constitutive agents from plants for mycotoxin control
    (2000) Mohanlall, Viresh; Odhav, Bharti
    Plant medicine is an important area of commercial activity in South Africa. This is a rapidly expanding market, thus we are evaluating natural and stressinduced compounds (phytoalexins) from plants as agents that may be able to control mycotoxins. Natural compounds from Bridelia micrantha, Warburgia salutaris, Lippia javanica and Scenecio serratuloides and stress-induced compounds (phytoalexins) from Citrus sinensis cv Valencia were screened for antitunqal and antimycotoxic activity by bioautography against a test organism (Cladosporium cladosporoides) and mycotoxin producing fungi (Fusarium moniliforme and Aspergillus flavus).
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    The potential of spice oils in the control of mycotoxin producing fungi
    (2000) Juglal, Sarla; Odhav, Bharti
    Spice oils are known to exhibit antifungal activity and therefore have the potential to control mycotoxin production. There is a need in the food industry to find measures to control mycotoxins that are frequently associated with grains that form the staple diet of the majority of the population in South Africa. Clove, cinnamon, oregano, tumeric, eucalyptus, neem, aniseed, mace and nutmeg oils were tested to determine their inhibitory potential against growth of Aspergillus parasiticus and Fusarium moniliforme using the agar overlay technique. Varying concentrations of the spice oils, ranging from 0.1 ppm to 2.0 ppm, were incorporated into broth cultures of A. parasiticus and maize patty cultures ofF. moniliforme. Levels of production of aflatoxins and fumonisin were determined using standard thin layer chromatography and highpressure liquid chromatography methods. In addition, the active component of the spice oils were isolated, characterised and tested. The inhibitory potential of these compounds for field use was tested by incorporating clove oil, whole cloves and ground cloves in samp
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    Concurrent analysis of the mycotoxins, cyclopiazonic acid, moniliformin and ochratoxin A using capillary zone electrophoresis
    (2000) Govender, Urishani; Odhav, Bharti
    Mycotoxins are a group of natural poisons produced by certain strains of fungal species when they grow under favourable conditions on a wide variety of different substrates. These toxins have been implicated in a wide range of acute diseases in man and animals. Their toxic effects include oesophageal cancer and liver diseases in humans, and carcinogenic effects in experimental rats and poultry. Hence, there is a need to monitor toxin levels in food commodities.
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    Antimicrobial activity of ciprofloxacin-coated gold nanoparticles on selected pathogens
    (2014-08-08) Moodley, Nivrithi; Odhav, Bharti
    Antibiotic resistance amongst bacterial pathogens is a crisis that has been worsening over recent decades, resulting in serious and often fatal infections that cannot be treated by conventional means. Diseases caused by these drug resistant agents result in protracted illnesses, greater mortality rates and increases in treatment costs. Improvements to existing therapies and the development of novel treatments are urgently required to deal with this escalating threat to human health. One of the more promising strategies to combat antibiotic resistance is the use of metallic nanoparticles. Research into this area has shown that the binding of antibiotics to nanoparticles enhances their antimicrobial effects, reduces side-effects due to requirement of lower dosages of the drug, concentrates the drug at the interaction site with bacterial cells and in certain cases, has re-introduced susceptibility into bacterial strains that have developed drug resistance. Furthermore, these nanoparticles can be used in cancer treatment in similar drug delivery roles. Based on the promising data that demonstrated the synergistic effects of antimicrobial agents with nanoparticles, the aim of our research is to determine the effect of ciprofloxacin-conjugated gold nanoparticles as antimicrobial agents. To achieve this aim our objectives were: (i) to synthesize citrate-capped and ciprofloxacin-conjugated gold nanoparticles; (ii) to determine the physical and chemical characteristics of the ciprofloxacin-nanoparticle hybrid molecule; (iii) to investigate the antimicrobial activity of the conjugated nanoparticles against various species of common pathogens and (iv) to investigate the anti-cancer potential of the citrate-capped nanoparticles against a Caco-2 cell line. In this study, citrate-capped gold nanoparticles were conjugated to the antibiotic, ciprofloxacin, and their antibacterial and anti-cancer activity was evaluated. Initial experiments involved the synthesis and characterization of gold nanoparticles and ciprofloxacin conjugated nanoparticles. The gold nanoparticles were synthesized using the Turkevich citrate reduction technique which has been extensively used in studies thus far. The synthesized nanoparticles were characterized for specific absorbance using a UV-Spectrophotometer. The bond between the nanoparticles and ciprofloxacin was characterized by FTIR. Ultra structural details of the gold nanoparticles were established by TEM. The colloidal stability of the nanoparticles was determined by spectroscopic analysis. The antibacterial activity of the ciprofloxacin-conjugated gold nanoparticles was studied by exposure to pathogenic bacteria (Staphyloccocus aureus, E. coli, Klebsiella pneumoniae, Enterocococcus spp., Enterobacter spp., and Psuedomonas spp.). MIC values were measured to give indication of antimicrobial effect. These bactericidal properties of the conjugate nanoparticles were further investigated by electron microscopy. To evaluate the action of the citrate capped gold nanoparticles on cancer cells, we exposed Caco-2 cells to various concentrations of the nanoparticles and its effect was evaluated by measuring the viability of the cells. The results showed that 0.5 mM trisodium citrate reduced gold chloride to yield gold nanoparticles, which were spherical and 15 to 30 nm (by TEM characterization) and had an absorption maxima of 530 nm. The ciprofloxacin conjugated nanoparticles had an absorption maxima of 667nm. The colloidal stability, which is used to assess whether the synthesized particles will retain their integrity in solution showed that citrate-capped GNPs were most stable at 37°C over a 14 day storage period while ciprofloxacin-conjugated GNPs were found to be most stable at 4°C over a 14 day period. The FTIR results showed that chemical bonding in the conjugated nanoparticles occurs between the pyridone moiety of ciprofloxacin and the nanoparticle surface. The antimicrobial results of ciprofloxacin-conjugated GNPs had a significantly improved killing response compared to ciprofloxacin on both Gram positive and Gram negative bacteria. The citrate-capped GNPs are shown to exert a similar cytotoxic effect to gemcitabine on the Caco-2 cell line at a concentration of 0.5 mM. These results indicate that combining gold nanoparticles and ciprofloxacin enhances the antimicrobial effect of the antibiotic. The conjugate nanoparticles increase the concentration of antibiotics at the site of bacterium-antibiotic interaction, and thus enhance the binding and entry of antibiotics into bacteria. This has great implications for treatment of infection, as these antibiotic-conjugated nanoparticles can be incorporated into wound dressings, be administered intravenously as drug delivery agents, be engineered to possess multiple functionalities in addition to antibacterial activity and act as dual infection tracking and antimicrobial agents. Likewise, in this study, gemcitabine, an anticancer drug and gold nanoparticles were shown to kill cancer cells. In addition to their use in photothermal therapy and as drug delivery agents, the nanoparticles themselves possess anti-cancer activity against the Caco-2 cells. Thus, they have potential to act alone as a form of cancer treatment if functionalized with certain targeting agents that are specific to cancer cells, reducing the side-effects that come with regular chemotherapeutic drugs. It can be concluded that ciprofloxacin-conjugated gold nanoparticles enhance antibacterial effects of the antibiotic ciprofloxacin against bacterial cells and citrate-capped gold nanoparticles have anti-cancer activity against the Caco-2 cell line.
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    Molecular characterization of aflatoxigenic and non-aflatoxigenic aspergillus isolates
    (2007) Mngadi, Phakamile Truth
    For decades the genus Aspergillus (of fungi) has been classified based on morphological and growth criteria. Members of the Aspergillus section Flavi are economically valuable and methods of differentiating them are thus very important. Several molecular methods have been developed to distinguish these strains. Also, a number of biochemical and genetic studies have been used in order to provide a better means of classification (Lee et al., 2004). Aflatoxins, the most frequently studied mycotoxins, are produced by certain Aspergillus species/strains/isolates of fungi. The aflatoxin biosynthetic pathway studies have led to a number of discoveries. Several structural and regulatory genes (and their enzymes) involved in the biosynthesis of aflatoxins have been discovered and purified (Trail et al., 1995). Aflatoxin production and contamination of agricultural crops are major causes of economic losses in agriculture. Thus, better methods of characterization/differentiation are required for both aflatoxigenic and non-aflatoxigenic isolates. Molecular biology is one of the current tools used to differentiate between these isolates. Polymerase Chain Reaction (PCR)-based randomly amplified polymorphic DNA (RAPD) analysis has been used successfully in the analysis of DNA relatedness of species of fungi, bacteria, plants and animals. Dendograms which evaluate/assess the likeness between different isolates has also been used (Martinez et al., 2001). Restriction fragment length polymorphism (RFLP) analysis has been applied to a number of studies to detect differences between fungi and to establish relationships between them. Therefore, the scope of this study was to investigate RAPD analysis (with dendograms) and detection of RFLPs by hybridization as molecular methods that can distinctly differentiate or characterize the aflatoxigenic and non-aflatoxigenic Aspergillus isolates.
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    Chemoprotective action of natural products on cultured human epithelial cells exposed to aflatoxin B1
    (2005) Reddy, Lalini; Odhav, Bharti
    Previous studies indicate that a mutation in the non-oncogenic p53 gene is epidemiologically linked to human HCC (Ozturk, 1991; Chan et al., 2003). Hsu et al. (1991) found this link in Chinese, South African and Asian patients and Hollstein et al. (1993) found the same gene mutation in Taiwanese patients. The incidence of these aberrations is reported to be about 20- 50% in HCC’s (Kishimoto et al., 1997). There is sufficient evidence to indicate that carotenoids in addition to their well known antioxidant properties (Paiva and Russel, 1999), also affect intercellular communication, immune responses, neoplastic transformations and growth control, and cellular levels of enzymes that detoxify carcinogens (Zhang et al., 1991; Brockman et al., 1992; Pryor et al., 2000). To date studies carried out have used the rat (Foote et al., 1970; Gradelet et al., 1998) and the mule duckling model (Cheng et al., 2001) to show the protective effect of these carotenoids against AFB1 exposure. Of the well known carotenoids, lycopene and beta- carotene occur in abundance in fruits and vegetables and are safe for human consumption. Aflatoxin B1 frequently induces mutations of the p53 gene which is linked to HCC. Although there is much evidence from epidemiological studies linking the beneficial aspects of carotenoids to the prevention of cancer, the cellular and molecular mechanisms need to be understood in order to implement large scale intervention strategies to prevent AFB1 induced carcinoma. The use of chemical or dietary interventions to alter the susceptibility of humans to the actions of carcinogens and to block, retard or reverse carcinogenesis is an emerging chemoprotective strategy for disease prevention (Abdulla and Gruber, 2000; Kensler et al., 2003; Bingham and Riboli, 2004). Chemoprotection by natural products involves maintaining cellular integrity, preventing DNA alterations, activation of p53 suppressor protein and apoptosis. The aim of this study was thus to investigate the cellular and molecular mechanisms by which beta-carotene and lycopene may prevent the AFB1-induced toxic changes in human hepatocytes. In order to achieve this aim, the following objectives were set out: i. To optimise an in vitro system for the evaluation of AFB1 damage to cultured hepatocytes. ii. To determine the biochemical protection offered by beta-carotene and lycopene to AFB1-exposed hepatocytes, by measuring the mitochondrial activity, cell viability and ROS levels using appropriate enzyme assays and flow cytometry. iii. To determine the cellular protection offered by beta-carotene and lycopene to AFB1-exposed hepatocytes, by studying the morphological changes at the structural and ultrastructural levels using phase contrast light and electron microscopy respectively. iv. To determine the molecular protection offered by beta-carotene and lycopene to AFB1-exposed hepatocytes, by detecting apoptotic bodies as genomic markers and measuring the levels of p53 protein and AFB1-N7-guanine adducts produced.