As pollution becomes an ever-increasing threat to the global environment pressure is being placed upon industry to "clean-up" its act, both in terms of reducing the possibility of new pollution and cleaning up already contaminated areas. It was with this in mind that Amplats embarked on a phytoremediation project to decontaminate nickel-polluted soils at one of their mine sites in Rustenburg, using the nickel hyperaccumulating plant, Berkheya coddii, which is endemic to the serpentine areas near Barberton, Mpumalanga. Besides the applied aspects pertaining to the development of the phytoremediation process we were also interested in more academic aspects concerning the transport and storage of nickel within the plant tissues. In order that the progress of nickel could be followed through the plant, a radio-tracer of ⁶³nickel was placed in the soil and its movement within the plant followed by analysing the plant material, at set intervals, using a liquid scintillation counter. From these studies it was found that the nickel appeared to be transported from the roots to the leaves of the plant via the xylem. It appeared that the nickel was not confined to the leaf to which it was initially transported and so movement of nickel within the phloem also appears to occur in B. coddii. As nickel is generally toxic to most plants, hyperaccumulators contain elements that nullify the toxic effect of nickel. In the case of Berkheya coddii it is thought that the accumulated nickel is bound to malate to form a harmless nickel complex. With this in mind an assay for L-malic acid was developed in order that any effect on L-malic acid, caused by growing Berkheya coddii on soils containing various concentrations of nickel, could be determined. This method also enabled comparisons of L-malic acid concentrations to be made between hyperaccumulators and non-hyperaccumulators of various plant species. From the L-malic acid comparisons it was found that the nickel concentration within soils affected the levels of L-malic acid within B. coddii and that the levels of L-malic acid within B. coddii were greater than that of a closely related non-hyperaccumulator, suggesting that L-malic acid is indeed involved in the hyperaccumulation mechanism within B. coddii. B. coddii was chosen as the tool in nickel phytoremediation at Rustenburg Base Metal Refineries as it was found to accumulate up to 2.5% nickel in the dry biomass, it grows rapidly and has a large above-ground biomass with a well developed root system, and it is perennial and so does not need to be planted each season. Earlier work had shown that the nickel levels in the roots were comparatively low (up to 0.3% nickel in the dry material) and thus, for ease of harvesting and to ensure the continued vegetative growth of the plant on the planted sites, it was decided that the leaves and stems of the plants would be harvested at the end of each growing season. The plant was also found to accumulate low levels (0.006 - 0.3 %) of precious metals, including platinum, palladium and rhodium, within its above ground biomass, making it attractive for the remediation of certain soils that contain low levels of these metals. Before B. coddii could be introduced to the Rustenburg area a comparison of the climatic and soil conditions of Barberton, the area to which B. coddii is endemic, and Rustenburg needed to be made to ensure that the plant would be able to survive the new conditions. These comparisons showed that Rustenburg receives on average, 484 mm less rain per year than Barberton, indicating that irrigation was required when the Rustenburg sites were planted out with B. coddii, in order to reduce water stress. Rustenburg was also found to be, on average, 4.6°C warmer than Barberton, but as B. coddii growth responds to wet/dry seasons, as opposed to hot/cold seasons, it was not felt that this temperature difference would have a negative effect on the growth of the plants. The soil comparisons showed the contaminated Rustenburg sites to be serpentine-like in nature, with respect to Barberton, again giving confidence that the plant would adapt to the conditions occurring at the contaminated sites. However, to ensure optimal growth, nutrient experiments were also performed on B. coddii to ascertain the ideal macronutrient concentrations required, without inhibiting nickel uptake. These trials indicated that the individual addition of 250 mg/l ammonium nitrate, 600 mg/l calcium phosphate, 2 000 mg/l calcium chloride, 600 mg/l potassium chloride and 250 mg/l magnesium sulphate enhanced plant growth and nickel uptake, suggesting that, for phytoremediation purposes, these nutrients should be added to the medium in which the plants are growing. The growth-cycle of naturally occurring B. coddii plants in Barberton was also studied in order that seedlings could be germinated, in greenhouses, at the correct time of year so that the plants could be sown as the naturally occurring plants were germinating. From this information the seeds of the plants could be collected at the correct time of year and the above ground biomass harvested when the nickel concentrations were at their highest. It was found that the plants began to germinate as the first rains fell, which was generally at the beginning of September, and plant maturity was reached at about five months, after which flowers were produced. Seeds were produced from the flowers and these matured and were wind-dispersed one month to six weeks after full bloom, usually during February. The plants then started to die back and dry out and dormancy was reached about nine months after germination, generally in about mid- to late- May. It was found that the nickel concentration was at its highest about one month after the plants had begun to dry out and thus it was decided that the above ground biomass would usually be harvested at the end of April each season, in order to achieve maximum nickel recovery. Finally, in order that the plant's potential for use in phytoremediation could be fully assessed, field trials at the contaminated sites in Rustenburg were performed. Germination procedures were developed for the mass production of B. coddii and it was found that, although fully formed plants could be propagated in tissue culture, it was cheaper and faster to germinate the seeds in speedling trays, containing a zeolite germination mix, in greenhouses. It was also found that the seeds had a low germination rate, due to dehydration of the embryos and thus, in order to obtain the number of plants required, four to five times the amount of seeds needed to be sown. The two-month-old seedlings were transferred to potting bags, containing a mixture of potting soil and RBMR soil, and grown up in the greenhouse for a further three months. This growth period allowed B. coddii to adapt to the RBMR soil and also ensured that the plants were relatively healthy when transplanted into three prepared sites at RBMR. The plants were allowed to grow for the entire season after which the above ground biomass, comprising the leaves and stems, was harvested, dried and then ashed in an ashing vessel designed by the author, with the help of Mr K Ehlers. The ashed material was acid-leached with aqua regia in order that the base metals (mainly nickel) and precious metals could be removed from the silicates and carbonised material. The acid solution was then neutralised, causing the base metals (mainly nickel) and precious metals to be precipitated. This precipitate was then smelted with a flux in order that nickel buttons could be formed. Thus, from all the phytoremediation trials it was found that this process is highly successful in employing B. coddii for the clean-up of nickel-contaminated sites. This constitutes the first time that such a complete phytoremediation process has ever been successfully developed with B. coddii as the phytoremediation tool. It also appears to be the first time that phytoremediation has been performed "commercially" to produce a saleable metal product. The success of this project has stimulated Amplats to continue with, and expand it, to include more studies on phytoremediation as well as in the biomining of certain areas containing very low levels of precious metals which, with conventional techniques, were previously not worth mining. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 1998.

Mycoplasmas conform most closely with the theoretical concept of 'minimum cells', existing as the smallest, free-living organisms capable of self-replication. They survive as parasites of plants, insects, animals or humans, with the most common human colonising species being Mycoplasma hominis. M. hominis has been characterised as a human pathogen responsible for a variety of infections, which pose a significant threat particularly to immunocompromised patients and neonates. However little has been elucidated about the cell physiology and molecular structure of this organism. Of interest to this study were the investigation of the heat shock response of M. hominis and the diagnostic assays used for its detection. The heat shock response is a ubiquitous physiological feature of all organisms and displays unprecedented conservation. This phenomenon is particularly evident in the 70 kDa family of heat shock proteins (hsp70) which exhibits a high degree of homology between different species. The hsp70 gene from M. hominis was cloned and preliminary partial sequencing indicated the similarity with other hsp70 homologs. The regulation of hsp70 expression at the transcriptional and translational levels was investigated. The level of hsp70 mRNA was found to increase correspondingly in response to heat shock, more visibly than the level of hsp70 protein. However imrnunochemical studies of the M. hominis hsp70 translation product demonstrated further the homology with other species. To facilitate rapid diagnosis of M. hominis infections, a PCR ELISA diagnostic assay was developed and optimised. The amplification of a conserved region of the M. hominis 16S rRNA gene was linked to subsequent hybridisation to an appropriate capture probe in a microtiter plate format. The sensitivity of the assay was comparable to other molecular assays although the PCR ELISA produces more rapid results and is less labour intensive. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 1998.

Animal trypanosomosis is a major constraint in livestock production in Sub-Saharan Africa. With the emergence of resistance against trypanocidal drugs, the cost and environmental concerns raised by vector control, and the challenge of antigenic variation in vaccine development, alternative control measures are being sought. An anti-disease strategy, whereby the immune response or chemotherapy is aimed towards pathogenic factors rather than the parasite itself, constitutes such a novel approach. Congopain is the major cysteine protease in Trypanosoma congolense, and upon release in the bloodstream of infected cattle, acts as a pathogenic factor. It is therefore an attractive candidate for an anti-disease vaccine. It was hence deemed necessary to investigate the variability of congopain-like cysteine proteases before attempting to design drugs and vaccines based on the inhibition of congopain. Most congopain-like cysteine protease genes of T. congolense exist in a single locus of 12-14 copies organised as tandem repeats of 2 kb gene units. A gene unit library of 120 clones was constructed out of several cosmid clones selected in a previous study that contained various lengths of the congopain locus. Some 24 gene unit clones were sequenced, and it was found that congopain genes cluster in three sub-families, named CP1 (8 clones), CP2 (12 clones) and CP3 (4 clones). The latter most characteristically shows a substitution of the active site cysteine by a serine. Isoform specific primers were designed and used to verify the proportions of the three isoforms (one third CP1, half CP2 and a sixth CP3) in the remaining clones of the library. Since this first study was conducted in one isolate, IL 3000, the results were subsequently validated in a large array of isolates, of T. congolense, as well as T. vivax and T. brucei subspecies, by a PCR approach. Finally, to gain access to copies of congopain genes that are not present in the locus, but rather scattered in the genome, an attempt was made to construct a 2 kb size-restricted genomic library. Only 206 clones could be produced, of which a mere 8 coded for congopain-like proteases. The fact that 7 out of 8 of these clones belong to CP3 (thought to be inactive) suggested a cloning artefact, possibly related to the activity of the cloned proteases. Overall, all congopain genes appear very conserved in a given species, with 87-99% identity at protein level. The pre- and pro-region were the most conserved, while the catalytic domain was the most variable, especially around the active site cysteine, with frequent replacement by a serine residue, and in one instance by phenylalanine. The histidine residue of the catalytic triad was also substituted by either a serine or a tyrosine in some instances. The proenzyme cleavage site sequence was also variable, with APEA being the predominant N-terminal sequence. RT-PCR analyses indicated that CP1, CP2 and CP3 mRNA are all present in the bloodstream forms of T. congolense, showing that these variants are likely to be expressed. The conclusion of this study is that, given the high overall conservation of congopain genes in the genome, for the purpose of anti-disease vaccine, it is likely that a single immunogen will suffice to raise antibody able to inhibit all circulating congopain-like cysteine proteases. For chemotherapy however, a more in-depth enzymatic characterisation of the mutants, involving functional recombinant expression, will have to be undertaken. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2008.

African trypanosomiasis is caused by protozoan parasites known as trypanosomes, which are transmitted by the tsetse fly, affecting both humans and animals. Trypanosoma congolense is one of the main trypanosome species affecting cattle and causes the disease known as nagana. Control of animal African trypanosomiasis currently relies on chemotherapy and vector control methods, neither of which has proven satisfactory. An effective vaccine against trypanosomiasis would be the most cost effective solution to control the disease; however, due to the phenomenon of antigenic variation, intrinsic to the parasite’s outer coat of variable surface glycoprotein, this has not yet been achieved. Recent vaccine efforts have been centred on identification of invariant parasite antigens for use as vaccine candidates. Trypanosome cytoskeleton components have in recent years been shown to be capable of providing a protective immune response against trypanosome infection. These include tubulin proteins, which form the main components of the cytoskeleton, as well as microtubule associated proteins (MAPs) and paraflagellar rod proteins. In the present study α- and β-tubulin from T. congolense were recombinantly expressed and their immuno-protective potential in mice assessed. Amplification of both α- and β-tubulin ORFs from T. congolense genomic DNA was followed by cloning of the amplicons into the T-vector pTZ57R/T, and thereafter sub-cloning into the bacterial expression vector, pET238a and the yeast expression vector pPICZαA28. Only the α-tubulin amplicon was successfully sub-cloned into pICZAαA28; however, no protein expression was achieved upon transfection of the methylotrophic yeast, Pichia pastoris, with this construct. Subcloning of both α- and β-tubulin inserts into pET28a was successful. Expression of recombinant α- and β-tubulin as fusion proteins with a histidine tag, both at a size of 55 kDa, was achieved in Escherichia coli host BL21 (DE3). Recombinant proteins were successfully purified using nickel chelate chromatography under denaturing conditions. Refolding was first attempted by dilution of purified denatured proteins in a refolding buffer followed by reconcentration, but was largely unsuccessful. A second, more successful refolding method was performed wherein denatured proteins were refolded by application of a decreasing gradient of urea, while bound to a nickel chelate column. Native tubulin from cultured T.congolense procyclics was successfully purified and renatured using a polymerisation/depolymerisation method for use as a control for immunisation. Mice were immunised separately with refolded recombinant α- and β-tubulin, native tubulin or an irrelevant protein VP4AA expressed in the same way as the tubulins. ELISA analysis confirmed the production of antibodies against each protein. Parasitaemia developed in all mice following challenge with T. congolense. Only the group immunised with β-tubulin recorded no deaths during the monitoring period despite the presence of parasitaemia, with 60% of mice immunised with α-tubulin or VP4AA and the no antigen control and no mice from the native tubulin immunised group surviving. The results showed that partial protection against trypanosomiasis caused by T. congolense infection was achieved in the group immunised with β-tubulin and suggest that β-tubulin may have vaccine potential. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2010.

African animal trypanosomosis or nagana is a tsetse fly-transmitted disease, caused by Trypanosoma congolense, T. vivax and to a lesser extent T. brucei brucei. The disease causes major losses in revenue in many livestock-producing African countries. The available control methods, including chemotherapeutic drugs and insecticidal spraying, have become environmentally unacceptable. Antigenic variation displayed by the parasites has hindered vaccine development efforts. In this context, rather than focusing solely on the parasite itself, efforts in vaccine development have shifted towards targeting pathogenic factors released by the parasites during infection. Congopain, the major cysteine protease of T. congolense, has been shown to act as a pathogenic factor in the disease process. Analysis of the immune response of trypano-tolerant cattle revealed that these animals have the ability to control congopain activity in vivo. Therefore, congopain is an attractive vaccine candidate. To test the protective potential of congopain, immunisation studies had been conducted in cattle using the baculovirus-expressed catalytic domain of congopain (C2) in RWL, a saponin-based proprietary adjuvant from SmithKline-Beecham. Immunised animals were partially protected against a disease caused by an infection with T.congolense. Unfortunately, subsequent attempts to reproduce these results were disappointing. It was hypothesised that this failure could be due to the different expression system (P. pastoris) used to produce the antigen (C2), or the different adjuvant, ISA206 (Seppic), used, thus hinting towards an epitope presentation problem. Congopain had been shown to dimerise at physiological pH in vitro. Sera from trypano-tolerant cattle preferentially recognised the dimer conformation, advocating for protective epitopes to be dimer associated. For that reason, the present study aimed at improving the antigenicity of congopain through firstly, the elucidation of the protective epitopes associated with the dimer, secondly, the determination of the 3-D structure of the protease in order to map protective epitopes to later design mimotopes, and thirdly improve the delivery of congopain to the immune cells while maintaining the conformation of the protease by using a molecular adjuvant, BiP. A dimerisation model was proposed, identifying the amino acid residues forming the dimerisation motif of congopain. In the present study, particular amino acid residues located in the dimerisation motif were mutated by PCR-based site-directed mutagenesis to generate mutants with different dimerisation capabilities. The congopain mutants were expressed in yeast and their dimerisation capability was assessed by PhastGel® SDS-PAGE. The mutations altered both the electrophoretic mobility of the mutants and their enzymatic characteristics compared to wild-type congopain. This advocated for the involvement of these amino acid residues in the dimerisation process, although they seem not to be the only partakers. Wild-type C2 and mutant forms of C2 were heterologously expressed in P. pastoris and purified to crystallisation purity levels. Crystallisation of these proteins is currently underway, but the results are still unknown. While awaiting the crystallisation results, in silico homology modelling was employed to gain insight into the 3-D structure, using cruzipain crystal structure as a template. The modelled 3-D structure of congopain followed the common framework of cathepsin L-like cysteine proteases. Due to time constraints and awaiting the crystal-derived 3-D structure, the 3-D model of congopain was not exploited to design mimotopes with the potential to provide protection against the disease. As it was shown that protective epitopes are likely to be dimer-specific, maintaining the native conformation of congopain is essential for stimulating a protective immune response in animals. Chemically formulated adjuvants usually contain high salt concentration, at acidic or basic pH, thus might change the conformation of the protease. Adjuvants capable of efficiently delivering the antigen to immune cells while maintaining the conformation of the protease were sought. Proteins belonging to the HSP70 family are natural adjuvants in higher eukaryotes. A protein belonging to the HSP70 family was previously identified in T. congolense lysates and is homologous to mammalian BiP. Congopain was genetically fused with T. congolense BiP in order to improve antigen delivery and production of congopain activity-inhibiting antibodies. The chimeric proteins were successfully expressed in both bacteria and yeasts. The low yields of recombinantly expressed chimeras in yeast and problems associated with renaturation and purification of bacteria-expressed chimeras prevented immunisation studies in mice. However, the groundwork was laid for producing BiP-congopain chimeras for use in an anti-disease vaccine for African trypanosomosis. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2009.

African animal trypanosomosis is a devastating disease affecting livestock mainly found in sub-Saharan Africa. This disease is known as nagana and is transmitted by the trypanosome parasite from the tsetse fly vector to a mammalian host. There are three African trypanosomes namely Trypanosoma vivax, T. congolense and T. brucei brucei that are the causative agents responsible for this disease in African cattle. This disease is serious since it not only affects livestock but also has a negative impact on the sub-Saharan African economy. There is, therefore, a great demand for better control methods of the disease and suitable diagnostic methods. Current control measures such as the use of trypanocidal drugs, tsetse fly eradication methods and trypanotolerant cattle have become inadequate. The defence mechanism of the trypanosome to continuously change its surface coat by a process of antigenic variation has made it impossible to produce a suitable vaccine. Therefore, chemotherapy is still one of the key approaches for control of this wasting disease. The long existence of the current trypanocidal drugs has allowed the development of drug resistance. The development of new chemotherapeutic drugs is focused on targeting the pathogenic factors such as parasite cysteine peptidases that contribute to the disease. Vivapain is the main cysteine peptidase of T. vivax and shares high sequence identity with congopain, the main cysteine peptidase of T. congolense, which was previously shown to be a pathogenic factor contributing to trypanosomosis. Vivapain, thus, has potential as a target for chemotherapeutic drug design. Hence, the first part of this study involved the recombinant expression and enzymatic characterisation of vivapain for future production of new synthetic inhibitors for the use in new trypanocidal drugs. The catalytic domain of vivapain (Vp) was recombinantly expressed in the Pichia pastoris yeast expression system and enzymatically characterised. The main finding from this study was that Vp was only able to hydrolyse a substrate if the P2 position was occupied by either a hydrophobic Phe or Leu residue. Vp was also found to be active close to physiological pH and was inhibited by the reversible cysteine peptidases, leupeptin, antipain and chymostatin and the irreversible cysteine peptidases L-trans-epoxysuccinyl-leucylamido (4-guanidino) butane (E-64), iodoacetic acid (IAA) and iodoacetamide (IAN). A further important aspect of controlling trypanosomosis is the diagnosis of the disease. Clinical, parasitological, molecular and serological techniques have been applied and used to diagnose trypanosomosis. One of the most promising serological techniques has proven to be the enzyme-linked immunosorbent assay (ELISA), more specifically the antibody and antigen detection ELISAs. The main requirement for this technique is a readily available and reproducible antigen such as that produced by recombinant expression. While there are recombinant antigens that are available to be used to detect T. congolense, T. brucei brucei and even T. evansi infections, there are none available to detect T. vivax infections. In the second part of this study, a mutant inactive full length form of vivapain (FLVp) was expressed in a bacterial expression system for the detection of T. vivax infections. Antibodies against this antigen were produced in both chickens and mice. Both the chicken IgY and mice sera were able to detect the recombinant FLVp in western blots. The mice sera were also able to detect native vivapain in a T. vivax lysate, which is very promising for future use of the FLVp antigen and the corresponding antibodies in diagnosis of T. vivax infections in sera of infected animals. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2010.

Treatment of diabetes mellitus is mainly focused on glycaemic control regulated by insulin and takes place in insulin sensitive tissues like skeletal muscle which accounts for 75% of glucose metabolism. Plant derived compounds that have anti-diabetic potential are currently being investigated for diabetes treatment as they are cheap and non-toxic. Oleanolic acid (OA), a triterpene found in a wide variety of plants has been shown to have anti-diabetic effects but its mechanism of action, especially on the insulin signalling cascade has not been fully elucidated. The aim of the present study was to investigate the effects of OA on the PI3K/Akt insulin signalling cascade in skeletal muscle and skin of streptozotocin induced diabetic male Sprague-Dawley rats. Male Sprague-Dawley rats (non-diabetic and diabetic) were treated with insulin (4IU/ kg bw), OA (80 mg/kg bw) and a combination of OA + insulin in an acute and sub-chronic study. The study showed that OA does not reduce blood glucose levels in type 1 diabetic rats but enhances insulin stimulated hypoglycaemic effects. In the acute study OA was shown to activate Akt and dephosphorylate GS in skeletal muscle of streptozotocin induced diabetic rats. In the sub-chronic study OA and OA + insulin increased expression of GS in skeletal muscle of diabetic rats. GP expression was decreased by OA and OA + insulin treatments in skeletal muscle whilst in skin it was increased by both treatments. OA increased both GS and GP in skeletal muscle whilst in skin they were decreased. OA + insulin treatment increased GS and decreased GP activities in skeletal muscle and increased activity of both enzymes in skin of diabetic rats. OA increased the amount of glycogen in both muscle and skin whilst OA + insulin reduced the amount of glycogen. OA and OA + insulin treatment showed some protective effects against liver and muscle damage as there were reductions in serum LDH, ALT and AST levels. In conclusion, oleanolic acid in synergy with insulin can enhance activation of the insulin signalling pathway and there was evidence of OA activation of insulin signaling enzymes independent of insulin. / Thesis (M.Sc.)-University of KwaZulu-Natal, Durban, 2013.

According to the 2012 UNAIDS global report, sub-Saharan Africa hosts 69% of the world’s total population living with HIV, South Africa being the most affected with a reported 24% incidence rate. To date, extensive research is being conducted globally, particularly involving anti-HIV treatment that targets the retroviral enzymes: reverse transcriptase, integrase and protease. The discovery of inhibitors to HIV protease which disrupts virion protein assembly has made this enzyme a prime target of anti-retroviral therapies, thus there exists a concerted research initiative to identify compounds with HIV protease inactivation potential. This study employs HIV protease that is isolated and purified from a genetically modified HIV protease overexpressing Escherichia coli strain to monitor the inhibitory capacity of new lead compounds. Optimized growth conditions for HIV protease production displayed that the use of chemically defined media resulted in higher yields of the enzyme. Recent research studies have shown that peptide-based cage and glycosylated compounds displayed HIV protease inhibitor activity in cell free enzymatic reactions that are comparable to commercially available HIV protease inhibitors. However, in contrast it has also been reported that these inhibitors are inactive in whole T-cell assays, when employing HIV infected CD4 cells. It is a well-known fact that potential new chemical entities that do not possess oral bioavailability, in terms of their absorption properties, are not successful candidates within the drug discovery industry. Following this, the current study was designed to determine if inefficient membrane permeability of these promising anti-HIV protease lead compounds could result in their inactivity in whole T-cell assays. Two different methods were considered, a cell-based method using the Madin Darby Canine Kidney strain I (MDCKI) cell line and a non-cell based method, the parallel artificial membrane permeability assay (PAMPA). MDCKI cells have been extensively used to form monolayers that mimic human intestinal membranes whilst the PAMPA utilizes an artificial lipid membrane composition on a filter support. Data from permeability assays using the novel chemically synthesized inhibitors have been compared to commercially available drugs, antipyrine, metoprolol and caffeine, which displayed efficient membrane permeability characteristics, thereby validating the assay. The results indicated that novel cage-derived and glycosylated peptide inhibitors do not possess sufficient passive diffusion properties which may explain their inactivity in whole T-cell assays. / M.Sc. University of KwaZulu-Natal, Durban 2014.

This thesis examined the in vitro and in vivo anti-oxidative and anti-diabetic activities of five African medicinal plants which are traditionally used for the treatment of diabetes mellitus viz; Ziziphus mucronata, Cassia singueana, Parkia biglobosa, Khaya senegalensis and Vitex doniana. Ethanol, ethyl acetate and aqueous crude extracts of the stem bark, root and leaf samples of each of the plants (a total of 45 crude extracts) were investigated for detailed anti-oxidative activity and the most active crude extract from each plant was selected for further fractionation with solvents of increasing polarity. Subsequently, the solvent fractions derived from these crude extracts (a total of 21 fractions) were also subjected to the anti-oxidative assays as well as α-glucosidase and α-amylase inhibitory activities assays. Results from these assays revealed that the butanol fractions from crude extracts of Z. mucronata, P. biglobosa, K. senegalensis and V. doniana and the acetone fraction from the crude extract of C. singueana were the most bioactive. Kinetic delineation of the types of enzyme inhibitions exerted by these most active fractions as well as measurement of relevant kinetic parameters (KM, Vmax and Kᵢ) were done using Lineweaver-Burke’s plot. Furthermore, the most active fractions were also subjected to GC-MS analysis and in vivo intervention trial in a type 2 diabetes (T2D) model of rats (except fraction from V. doniana). The in vivo studies revealed that all the fractions possessed potent in vivo anti-T2D activity (to varying extent) and the possible mechanisms of actions were proposed. Furthermore, most of the fractions were able to ameliorate the T2D-associated complications. Analysis of in vivo oxidative stress markers such as glutathione, thiobarbituric acid reactive substances, superoxide dismutase and catalase in the serum, liver, kidney, heart and pancreas of the animals also gave a clue in to the possible mechanism of action. Bioassay guided isolation was used to track the bioactive anti-diabetic compounds from these fractions via column chromatography. The isolated compounds were characterized by ¹H NMR, ¹³C NMR, 2D NMR (in two cases) and mass spectroscopy (in one case). From this study, 2,7-dihydroxy-4H-1-benzopyran-4-one, 3β-O-acetyl betulinic acid, lupeol and bicyclo[2.2.0]hexane-2,3,5triol were identified as the possible bioactive compounds from Z. mucronata, C. singueana, P. biglobosa, K. senegalensis solvent fractions respectively. The findings of this work are important for the relevant government agencies, pharmaceutical industries, scientific community and poor diabetic patients because it might open an avenue for the development of viable and cost effective anti-diabetic herbal products and/or novel plant-derived anti-diabetic drugs. / Ph.D. University of KwaZulu-Natal, Durban 2013.

Diabetes mellitus (DM) is a metabolic disease characterized by chronic hyperglycaemia; this condition is caused by lack of insulin secretion (Type 1) and/or insulin resistance (Type 2). In diabetic patients; carbohydrate, protein and lipid metabolism is disturbed due to the lack of the body’s ability to utilise glucose efficiently. Management of type 1 diabetes involves insulin therapy which may be inconvenient for patients. Therefore alternative methods for management of type 1 diabetes involving medicinal products are being investigated. This study is aimed at investigating the effect of OA on markers of lipid metabolism and on proteins of the insulin signalling pathway in Type 1 diabetic rats as this plant product has anti-hyperglycaemic effects. Male Sprague-Dawley rats were divided into two groups (diabetic and normal). In both groups the rats were further divided into four groups and assigned to treatment as follows: vehicle, insulin, OA and OA plus insulin. Oral glucose tolerance test was performed in fasted and non-fasted diabetic rats for 2 hours. In acute studies the effect OA following treatment of rats was evaluated at 15, 30 and 60 minutes. In sub-chronic studies rats were treated daily for 14 days. OA did not improve glucose tolerance in diabetic rats after 2 hours of administration. However, it enhanced blood glucose lowering effect of insulin and this was statistically significant in fasted rats. In acute studies OA enhanced the effect of insulin in normal and diabetic animals as AKT phosphorylation was increased when insulin was used in combination with OA. OA reduced the expression and activity of HSL in liver tissue after 14 days of treatment in both normal and diabetic rats. In adipose tissue, OA reduced the expression of HSL in diabetic rats. However, OA alone did not reduce the activity of HSL but when it was combined with insulin, a reduction of HSL activity was observed. OA administration had no significant effect on TGA and HDL-c levels but significantly (p < 0.05) reduced total cholesterol and LDL-c in diabetic rats. It had no significant effect on total cholesterol, and increased LDL-c levels in normal rats. Serum AST and ALT levels in diabetic rats were reduced by OA administration but this reduction was not statistically significant. The results of this study suggest that OA enhances the hypoglycaemic effect of insulin, improves lipid profile and possesses hepatoprotective effects. Lastly, OA independently increases AKT phosphorylation and decreases HSL expression and activity. / Thesis (M.Sc.)-University of KwaZulu-Natal, Durban, 2013.