Thesis (MSc (Biomedical Technology))--Cape Peninsula University of Technology, 2018. / The challenge of antimicrobial resistance has increased drastically over the years as more microorganisms are becoming resistant to the available conventional treatments. The burden of antimicrobial resistant infections is intensified by the increase in immunocompromising conditions such as HIV/AIDS and cancer. Due to this challenge, pharmaceutical companies, health sectors and researches are in search of new antimicrobial agents that can solve the problem at hand. Medicinal plants are a reliable source for drug discovery as it is estimated that 25% of modern medicine originated from plants. They have also been used traditionally as sources of medicine in the treatment of many human ailments. Plants can also be applied in the field of nanotechnology. Nanotechnology is a promising field in medicine as it has the potential to offer improved methods for disease diagnostics and therapeutics. The use of plants in nanotechnology brings about biologically friendly nanomaterials. Cotyledon orbiculata is one of the well-known and common plants of South Africa that is used in traditional medicinal practices. The nanotechnology applications as well as the antimicrobial and immunomodulatory effects of this plant were evaluated. The ability of C. orbiculata to synthesize silver nanoparticles was determined. Optimisation of silver nanoparticle synthesis using water extract of C. orbiculata was done at different conditions. The conditions evaluated include, reaction temperature (25 and 70°C), silver nitrate concentration (1 and 3mM), plant extract concentration (1.5, 3 and 6mg/ml) and reaction time. The synthesis of silver nanoparticles using this plant was successful. The optimal conditions for the synthesis of silver nanoparticles using C. orbiculata were 3mg/ml of the C. orbiculata extract, 3mM silver nitrate at a reaction temperature of 70°C for 2 hours. Under these conditions, spherical, crystalline nanoparticles with sizes of 20-40nm were produced. The antimicrobial and immunomodulatory properties of C. orbiculata extracts and silver nanoparticles were evaluated. Antimicrobial activity was evaluated against Staphylococcus aureus, Staphylococcus epidermidis, Methicillin resistant Staphylococcus aureus (MRSA), Pseudomonas aeruginosa and Candida albicans, using the micro-dilution assay to determine the minimum inhibitory concentration (MIC). The results obtained revealed that all extracts of C. orbiculata have antimicrobial properties against all the microorganisms tested. The MICs of the extracts ranged from 3.13 to 50mg/ml and the MBC/MFC from 6.25 to >100mg/ml. The methanol extract exhibited better antimicrobial activity in comparison to the others extracts whereas the water extract had better antifungal properties. The chloroform extract showed the lowest activity in both antibacterial and antifungal studies. Silver nanoparticles also exhibited antimicrobial activity against all the microorganisms tested. It’s MICs against these microorganisms ranged from 5–80μg/ml and MBC/MFC from 20-160μg/ml. The silver nanoparticles were highly active than the water extract against both the bacteria and the fungi. Immunomodulatory effects of the plant extracts and silver nanoparticles were determined by evaluating cytokine production using the enzyme linked immunoassay (ELISA) assay. All the extracts and silver nanoparticles of C. orbiculata were found to have anti-inflammatory properties. The water extracts showed more anti-inflammatory activity against the cytokines than the other extracts. However the silver nanoparticles were more active than the water extract. The findings from this study confirmed that C. orbiculata have antimicrobial and immunomodulatory effects. This provided scientific evidence of the traditional use of this plant in the treatment of skin infections and inflammatory conditions.

The use of traditional and natural medicines in primary healthcare or alternative therapy is on the increase. However, the safety and efficacy of these medicines have not yet been confirmed. Pharmacognosy, the study of the properties of drugs, potential drugs or drug substances of natural origin and the search for new drugs from natural resources, is therefore of extreme importance in today’s healthcare environment. Cotyledon orbiculata L. var. orbiculata (Haw.) DC., a succulent shrub that is widely distributed over the whole of southern Africa, is an example of a plant used in traditional medicine for its antiepileptic effects. Oxidative stress can either be the cause of, or be secondary to epilepsy pathogenesis. Lipid peroxidation causes the disruption of cell membranes which leads to cell destruction and, in the case of neurological disorders, neurodegeneration. Reactive species have also been found to influence neurotransmission by affecting neurotransmitter metabolism and functions. Reactive species can therefore be responsible for the development of convulsions. Conventional anti-epileptics have shown to exert neuroprotective effects but information or research regarding their ability to prevent epilepsy from becoming chronic does either not exist or is not promising. Antioxidants have potential in the treatment of epileptic seizures as well as the prevention of chronic epilepsy by preventing the effects that oxidative stress has on neurotransmitter metabolism and functions that cause alterations in neuronal excitability and seizure threshold, ultimately leading to epileptic foci. The aim of this study was to evaluate the potential of the bufadienolide orbicusides of C. orbiculata and analogues as anti-epileptic treatment through antioxidant activity. Initially the isolation of novel antioxidants from C. orbiculata leaf juice was attempted. The antioxidant activity of the concentrated juice and fractions resulting thereof were evaluated with two assays. The thiobarbituric acid (TBA) assay was used to measure the extent of lipid peroxidation and nitroblue tetrazolium (NBT) assay was used to measure superoxide scavenging activity in rat brain homogenate. The low concentrations of orbicusides prompted the determination of the activity of two commercial bufadienolides (bufalin and cinobufotalin) and two bufadienolide analogues, synthesised by the esterification of trans-androsterone and androstanolone, respectively, using coumalic acid, producing Compound 1 and Compound 2. The toxicity of the commercial bufadienolides and synthesised analogues were evaluated by using the MTT assay (a cell viability assay). C. orbiculata juice showed significant pro-oxidant activity in both assays. Bufalin showed significant pro-oxidant activity in the TBA assay. Cinobufotalin showed no significant activity. Compound 1 showed pro-oxidant activity in the TBA assay and Compound 2 showed slight antioxidant activity in the NBT assay. The commercial bufadienolides showed low cell viability, indicating significant toxicity. The synthesised analogues showed a significant reduction in toxicity (despite Compound 2 being moderately toxic) when compared to the toxicity of the commercial bufadienolides. The low concentrations of orbicusides in the plant material and the antioxidant assay results of the two commercial bufadienolides suggested that the orbicusides may not be involved in the antioxidant properties of C. orbiculata. However, the antioxidant activity of Compound 2 showed that altering the pyrone moiety of bufadienolides could possibly improve antioxidant activity. The reduced toxicity and slight antioxidant activity of the synthesised bufadienolide analogues motivates further investigation. / MSc (Pharmaceutical Chemistry), North-West University, Potchefstroom Campus, 2014

The use of traditional and natural medicines in primary healthcare or alternative therapy is on the increase. However, the safety and efficacy of these medicines have not yet been confirmed. Pharmacognosy, the study of the properties of drugs, potential drugs or drug substances of natural origin and the search for new drugs from natural resources, is therefore of extreme importance in today’s healthcare environment. Cotyledon orbiculata L. var. orbiculata (Haw.) DC., a succulent shrub that is widely distributed over the whole of southern Africa, is an example of a plant used in traditional medicine for its antiepileptic effects. Oxidative stress can either be the cause of, or be secondary to epilepsy pathogenesis. Lipid peroxidation causes the disruption of cell membranes which leads to cell destruction and, in the case of neurological disorders, neurodegeneration. Reactive species have also been found to influence neurotransmission by affecting neurotransmitter metabolism and functions. Reactive species can therefore be responsible for the development of convulsions. Conventional anti-epileptics have shown to exert neuroprotective effects but information or research regarding their ability to prevent epilepsy from becoming chronic does either not exist or is not promising. Antioxidants have potential in the treatment of epileptic seizures as well as the prevention of chronic epilepsy by preventing the effects that oxidative stress has on neurotransmitter metabolism and functions that cause alterations in neuronal excitability and seizure threshold, ultimately leading to epileptic foci. The aim of this study was to evaluate the potential of the bufadienolide orbicusides of C. orbiculata and analogues as anti-epileptic treatment through antioxidant activity. Initially the isolation of novel antioxidants from C. orbiculata leaf juice was attempted. The antioxidant activity of the concentrated juice and fractions resulting thereof were evaluated with two assays. The thiobarbituric acid (TBA) assay was used to measure the extent of lipid peroxidation and nitroblue tetrazolium (NBT) assay was used to measure superoxide scavenging activity in rat brain homogenate. The low concentrations of orbicusides prompted the determination of the activity of two commercial bufadienolides (bufalin and cinobufotalin) and two bufadienolide analogues, synthesised by the esterification of trans-androsterone and androstanolone, respectively, using coumalic acid, producing Compound 1 and Compound 2. The toxicity of the commercial bufadienolides and synthesised analogues were evaluated by using the MTT assay (a cell viability assay). C. orbiculata juice showed significant pro-oxidant activity in both assays. Bufalin showed significant pro-oxidant activity in the TBA assay. Cinobufotalin showed no significant activity. Compound 1 showed pro-oxidant activity in the TBA assay and Compound 2 showed slight antioxidant activity in the NBT assay. The commercial bufadienolides showed low cell viability, indicating significant toxicity. The synthesised analogues showed a significant reduction in toxicity (despite Compound 2 being moderately toxic) when compared to the toxicity of the commercial bufadienolides. The low concentrations of orbicusides in the plant material and the antioxidant assay results of the two commercial bufadienolides suggested that the orbicusides may not be involved in the antioxidant properties of C. orbiculata. However, the antioxidant activity of Compound 2 showed that altering the pyrone moiety of bufadienolides could possibly improve antioxidant activity. The reduced toxicity and slight antioxidant activity of the synthesised bufadienolide analogues motivates further investigation. / MSc (Pharmaceutical Chemistry), North-West University, Potchefstroom Campus, 2014

South Africa is a country of great diversity. Different climate zones and a host of different habitats make South Africa the perfect platform for rich floral diversity. This floral diversity lends itself to the study of natural products by discovering new natural drugs that can be used in the treatment of many illnesses. Studies into the antioxidant properties of plants that are used in traditional medicine are an important aspect of research to determine the rationale of the use of plants by traditional healers. Many neurodegenerative diseases, like epilepsy, Parkinson s and Alzheimer s diseases, are linked to oxidative stress. Antioxidants could play a major role as neuroprotective agents and could alter the progression of these diseases. Epilepsy is one of the world s most prevalent central nervous system disorders and affects more than seventy per one thousand children in South Africa. Most of these cases are people in rural areas of South Africa where communities rely on the use of traditional medicine. Cotyledon orbiculata L. var orbiculata (Haw.) DC. is widely used in traditional medicine to treat epilepsy and other central nervous system disorders. The need to screen these plants for activity and toxicity is very important to understand the complex mechanism of action in the treatment of patients. In this study the methanol extract and three different fractions of the methanol extract of Cotyledon orbiculata were used to test for antioxidant activity and toxicity towards neuroblastoma cells. The freeze dried leaves of Cotyledon orbiculata were extracted with methanol using a Soxhlet apparatus. The concentrated extracts were analysed using HPLC (high pressure liquid chromatography) and three major peaks were selected for isolation. Three assays were performed to assess the antioxidant activity and toxicity of the isolated compounds. The thiobarbituric acid assay (TBA) quantifies the extent of the inhibition of lipid peroxidation in rat brain homogenates by the isolated fractions. All of the samples were able to attenuate lipid peroxidation as seen from the results obtained from the TBA assay. The methanol extract showed the best attenuation of lipid peroxidation in the rat brain homogenate with fraction 1 and 2 showing greater attenuation of lipid peroxidation than fraction 3. The nitroblue tetrazolium assay (NBT) quantifies the ability of the fractions to scavenge superoxide radicals in a rat brain homogenate. All samples were able to scavenge superoxide radicals as indicated by the NBT assay. The methanol extract showed the best superoxide scavenging abilities in the assay whereas fraction 1 showed better scavenging abilities than fraction 2 and 3. The 3–(4,5–dimethylthiazol–2–yl)–2,5–diphenyltetrazolium bromide assay (MTT) indicates the toxicity of the fractions towards neuroblastoma cells. The methanol extract and fraction 2 in the highest concentration of 10 mg/ml were the only samples that showed toxicity towards neuroblastoma cells. The molecular structure of a compound from fraction 2 was determined by using nuclear magnetic resonance spectroscopy (NMR), infrared spectroscopy (IR), and mass spectroscopy (MS). This compound was identified as diethyl malate. Diethyl malate is an artefact that is generated in HPLC procedures in the presence of malic acid (which naturally occurs in the leaves of Cotyledon orbiculata) and ethanol. The methanol extract of Cotyledon orbiculata has high antioxidant activity and could be due to the presence of malic acid in the leaves of the plant. The rationale in the use of Cotyledon orbiculata in the treatment of epilepsy could not be determined due to the isolation of an artefact, diethyl malate, obtained from the fraction. Further research should include methods to prevent artefact formation and purification of the samples that are obtained. / Thesis (MSc (Pharmaceutical Chemistry))--North-West University, Potchefstroom Campus, 2012.

South Africa is a country of great diversity. Different climate zones and a host of different habitats make South Africa the perfect platform for rich floral diversity. This floral diversity lends itself to the study of natural products by discovering new natural drugs that can be used in the treatment of many illnesses. Studies into the antioxidant properties of plants that are used in traditional medicine are an important aspect of research to determine the rationale of the use of plants by traditional healers. Many neurodegenerative diseases, like epilepsy, Parkinson s and Alzheimer s diseases, are linked to oxidative stress. Antioxidants could play a major role as neuroprotective agents and could alter the progression of these diseases. Epilepsy is one of the world s most prevalent central nervous system disorders and affects more than seventy per one thousand children in South Africa. Most of these cases are people in rural areas of South Africa where communities rely on the use of traditional medicine. Cotyledon orbiculata L. var orbiculata (Haw.) DC. is widely used in traditional medicine to treat epilepsy and other central nervous system disorders. The need to screen these plants for activity and toxicity is very important to understand the complex mechanism of action in the treatment of patients. In this study the methanol extract and three different fractions of the methanol extract of Cotyledon orbiculata were used to test for antioxidant activity and toxicity towards neuroblastoma cells. The freeze dried leaves of Cotyledon orbiculata were extracted with methanol using a Soxhlet apparatus. The concentrated extracts were analysed using HPLC (high pressure liquid chromatography) and three major peaks were selected for isolation. Three assays were performed to assess the antioxidant activity and toxicity of the isolated compounds. The thiobarbituric acid assay (TBA) quantifies the extent of the inhibition of lipid peroxidation in rat brain homogenates by the isolated fractions. All of the samples were able to attenuate lipid peroxidation as seen from the results obtained from the TBA assay. The methanol extract showed the best attenuation of lipid peroxidation in the rat brain homogenate with fraction 1 and 2 showing greater attenuation of lipid peroxidation than fraction 3. The nitroblue tetrazolium assay (NBT) quantifies the ability of the fractions to scavenge superoxide radicals in a rat brain homogenate. All samples were able to scavenge superoxide radicals as indicated by the NBT assay. The methanol extract showed the best superoxide scavenging abilities in the assay whereas fraction 1 showed better scavenging abilities than fraction 2 and 3. The 3–(4,5–dimethylthiazol–2–yl)–2,5–diphenyltetrazolium bromide assay (MTT) indicates the toxicity of the fractions towards neuroblastoma cells. The methanol extract and fraction 2 in the highest concentration of 10 mg/ml were the only samples that showed toxicity towards neuroblastoma cells. The molecular structure of a compound from fraction 2 was determined by using nuclear magnetic resonance spectroscopy (NMR), infrared spectroscopy (IR), and mass spectroscopy (MS). This compound was identified as diethyl malate. Diethyl malate is an artefact that is generated in HPLC procedures in the presence of malic acid (which naturally occurs in the leaves of Cotyledon orbiculata) and ethanol. The methanol extract of Cotyledon orbiculata has high antioxidant activity and could be due to the presence of malic acid in the leaves of the plant. The rationale in the use of Cotyledon orbiculata in the treatment of epilepsy could not be determined due to the isolation of an artefact, diethyl malate, obtained from the fraction. Further research should include methods to prevent artefact formation and purification of the samples that are obtained. / Thesis (MSc (Pharmaceutical Chemistry))--North-West University, Potchefstroom Campus, 2012.