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Free radicals have been implicated in the progression of various diseases, such as cancers and cardiomyopathies. When the body is overburdened with free radicals and endogenous antioxidants become depleted, oxidative stress ensues with resultant damage to biomolecules. During oxidative stress high levels of reactive oxygen species are generated, cellular viability decreases, and apoptosis and lipid peroxidation are induced. Supplementation with exogenous supplements rich in antioxidants, such as herbal remedies containing polyphenols, could result in increased protection against oxidative stress. The aim of the study was to assess the effect of Burkea africana and Syzygium cordatum in a cellular oxidative stress model for the potential development of an antioxidant supplement. Crude aqueous and methanolic extracts were prepared by solvent maceration, while a polyphenolic-rich extract was created through liquid-liquid extraction. Polyphenolic content and antioxidant activity was assessed in cell-free systems. Polyphenolic content was determined through the Folin-Ciocalteau and aluminium trichloride methods, while antioxidant activity was assessed by the Trolox Equivalence Antioxidant Capacity and 1,1-diphenyl-2-picrylhydrazyl radical assays. Identification of phytochemical classes was done through thin layer chromatography and biochemical reactions. Inherent cytotoxicity of samples was determined in four cell cultures (3T3-L1 pre-adipocytes, C2C12 myoblasts, normal human dermal fibroblasts and U937 macrophage-like cells) using the neutral red uptake assay. The effect on oxidative stress was assessed in 2,2`-azobis-(2-methylpropionamidine) dihydrochloride-exposed U937 macrophage-like cells with regards to reactive oxygen species generation, cytotoxicity, apoptosis, lipid peroxidation and GSH depletion. Both B. africana and S. cordatum showed enrichment of polyphenols from the aqueous extract, to methanolic extract, to polyphenolic-rich extract. Antioxidant activity showed the same trend, which correlated well with the increased concentration of polyphenols, such as catechin, gallic acid and myricetin. Samples indicated toxicity in the 3T3-L1 and C2C12 cell lines, though no toxicity was noted in the U937 cell line and normal human dermal fibroblast cultures. Free radical-induced generation of reactive oxygen species, cytotoxicity, lipid peroxidation and apoptosis was successfully reduced by crude extracts of B. africana and the polyphenolic-rich extracts of both plants between concentrations of 10 and 20 ìg/ml. The crude extracts of S. cordatum were mostly ineffective in reducing these parameters, even though cell viability was increased. B. africana pre-treatment decreased reduced glutathione concentrations significantly in a dose-dependent manner, while the methanolic and polyphenolic-rich extract of S. cordatum increased concentrations moderately. Polyphenolic-rich extracts of B. africana and S. cordatum had the most potent decrease in oxidative stress-related parameters in the present study, which could be attributed to the polyphenolic content and antioxidant activity. Limited cytotoxicity was apparent in two of the four cell lines tested; further isolation and purification needs to be carried out to assess the bioactive constituents which do not elicit a toxic response. Further investigation through the use of quantitative structure–activity relationship modeling could give more insight on conformational and chemical changes that need to be brought about to modify the bioactive phytochemicals for reduced cytotoxicity, but increased antioxidant activity. Copyright / Dissertation (MSc)--University of Pretoria, 2013. / Pharmacology / unrestricted

Please read the abstract in the section, 00front, of this document / Dissertation (MSc)--University of Pretoria, 2008. / Zoology and Entomology / unrestricted

Pentachlorophenol, (PCP) is an organochlorine compound which was first developed in the 1930’s. PCP is said to be the most toxic of the chlorophenols and is classified as a hazardous substance and a probable human carcinogen. PCP has proven to be cytotoxic to a number of cell lines translating to its effect on various organs. The aim of the study was to assess organochlorine-induced hepatotoxicity in a mechanistic manner using an in-house developed procedure. Also, the possible hepatoprotective effect of methanolic extracts of the bark of two medicinal plants, Burkea africana (BA) and Syzygium cordatum (SC), as well as the known hepatoprotective agent, N-acetyl cysteine (NAC), were investigated. In addition to PCP, two of its major metabolites, tetrachloro-1,2-hydroquinone (TCHQ) and tetrachloro-1,4-benzoquinone (TCBQ) were also evaluated. A hepatocarcinoma cell line (HepG2) was used to investigate the effect of these compounds on different parameters of cellular function. Cytotoxicity was assessed using the neutral red uptake assay. Cytochrome P4501A1 (CYP1A1) activity was determined using ethoxy-resorufin-O-deethylation as surrogate. Generation of reactive oxygen species (ROS) was investigated by measuring dichlorofluorescein diacetate cleavage. Effects on mitochondrial membrane potential were determined using JC-1 staining, whilst necrosis was investigated by assessing plasma membrane integrity using propidium iodide (PI)staining. The degree of apoptotic death was determined by quantifying caspase-3 activity. Assays were repeated with an additional 1 h pre-treatment of the cells with either NAC, SC or BA in order to investigate whether these compounds were able to protect against the toxicity induced by PCP and its metabolites. The IC50 values of PCP, TCHQ and TCBQ were 68.0, 144.0 and 129.4 μM, respectively. All three test compounds induced CYP1A1 activity with PCP being the most potent. TCBQ produced extensive ROS generation. TCHQ also induced ROS generation, whilst PCP appeared to have no significant effect on ROS generation. All test compounds caused mitochondrial depolarization. None of the test compounds caused an increase in necrotic cell death. PCP, TCHQ and TCBQ had negligible effects on apoptosis. Both SC and BA alleviated the toxic effects observed in cells treated with PCP. Minor increases in viability occurred in cells pre-treated with plant extracts prior to exposure to both metabolites. NAC, as well as both plant extracts, greatly reduced CYP 1A1 activity induced by PCP. NAC, SC and BA exacerbated CYP1A1 induction in cells exposed to concentrations of TCBQ and TCHQ that initially produced little or no effect on CYP1A1 activity. Contrarily, decreased CYP1A1 activity was observed in cells exposed to concentrations of TCBQ and TCHQ where extensive induction of CYP1A1 occurred. NAC, as well as both plant extracts, suppressed ROS generation in cells exposed to all test compounds. In cells exposed to PCP and TCBQ more extensive mitochondrial depolarization was seen when pre-treated with NAC and plant extracts than when exposed to the compounds alone. Negligible effects were seen in pre-treated cells exposed to TCHQ. BA and SC caused increases in necrotic death in cells exposed to the test compounds. NAC, BA and SC had negligible effects on the changes in caspase-3 activity induced by the test compounds. From the results it is proposed that PCP induces its own metabolism by increasing CYP1A1 activity. It also causes mitochondrial insult which could lead to the opening of the mitochondrial permeability transition pore and subsequent release of cytochrome C, activation of caspases and eventually apoptotic cell death. With regard to TCHQ and TCBQ, results suggest that extensive ROS generation caused damage to various cellular macromolecules and that this could be the main cause of their toxicity. NAC, SC and BA appeared to alleviate toxicity in certain instances. Further investigation is required in order to assess them as possible hepatoprotective agents. Copyright / Dissertation (MSc)--University of Pretoria, 2011. / Pharmacology / unrestricted