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The oral intake of green tea, black tea and some of the tea constituents has been demonstrated to protect against various diseases and show protective effects on the cell. Tea is able to regulate gene expression, regulate enzymes, regulate the cell cycle, cause cell cycle arrest, induce apoptosis, and inhibit the proteasome. The aim of this study was to find proteins by a proteomic approach that may be modulated in mice drinking tea in contrast to control animals that receive water. Three groups; control, low dose, and a high dose tea group were chosen to determine the effect of tea on protein regulation of C57BL/6 male mice. Daily liquid consumption was measured, and even though the high dose group consumed less liquid they still ingested more tea than the low dose group at the end of the study. Weight gain was measured for all the groups but no significant differences were found. Some differences were found in organ weights of the low and high dose groups. There was no dose dependent effect for the liver and small intestine, while the colon showed a positive and the pancreas a negative dose dependent effect. Small intestine and liver proteins were separated by one and two dimensional gel electrophoresis. No significant differences were found for the small intestine and liver when the proteins were separated by tricine SDS PAGE. However some significant differences were found on the glycine SDS PAGE gels of both the small intestine and the liver. The small intestine had three significant bands at 66kDa, 45kDa and 10kDa. The three significant liver bands were at 110kDa, 66kDa and 14kDa. HPLC analysis of the liver 66kDa band showed that the band consisted of only one protein while the 14kDa band consisted of possibly two proteins. MS analysis of the 14kDa band identified the proteins as hypothetical protein XP_358319 (15 190Da) and immunoglobulin Ą chain (13 140Da). Although the identified proteins match the molecular weight of the 14kDa band these results will need to be confirmed by MudPIT. Thirty 2DE spots of the small intestine were regulated by tea. Ten of these spots were analyzed by MALDI TOF MS, but only seven of these proteins were identified. These proteins were S-phase kinase associated protein p19, hypothetical protein XP_903753, unnamed protein product, adenylyl cyclase-associated protein 2, developmental control protein, lysosomal acid phosphatase, and cytochrome P450 (CYP2D13). All seven the small intestinal proteins will need to be confirmed by de novo sequencing, to ensure the positive identification of the proteins. Currently there is no 2DE map in literature of the small intestine. This study will provide the first 2DE map of the murine small intestine proteins. Thirty three 2DE spots of the liver were regulated by tea. Twenty of these were analyzed by MALDI TOF MS, but only fifteen of these proteins were idenitifed. These regulated proteins are: superoxide dismutase, and glutathione peroxidase that are antioxidant enzymes to counteract oxidative stress, detoxification enzymes like glutathione S-transferase mu-1, glutathione peroxidase theta-1 and cytochrome b5. Annexin A4 is able to help stabilize plasma proteins and the cytoskeleton and may induce apoptosis, keratin 8 may help with network formation and reinforcement of cellular membranes, malate dehydrogenase for energy expenditure and ketohexokinase in carbohydrate metabolism, while ubiquitin conjugating enzyme E2 plays a role in protein turn over. Other identified proteins include inosine-triphosphate-pyrophosphatase, triosephosphate-isomerase, and myoglobin. This study provides a novel 2DE map for liver protein regulation by tea. This was the first study that has taken a proteomic approach to the identification of the overall regulation of proteins by tea. The aim of this study was met by identifying the tea regulated proteins and elaborating on the protective effects and possible cancer chemo preventative effects of tea. / Dissertation (MSc (Biochemistry))--University of Pretoria, 2007. / Biochemistry / unrestricted

Please read the abstract in the section 07back.pdf of this document. / Dissertation (MSc (Biochemistry))--University of Pretoria, 2007. / Biochemistry / unrestricted

Non‐alcoholic fatty liver diseases (NAFLD) is manifested in the absent of alcohol abuse. This disease is the major cause of liver failure and death among adults and children worldwide, including South Africa. Its increasing prevalence urges the need of therapeutic intervention. The main objectives of this study were to investigate the following: (1) The effect of 38.9% high fat diet (HFD)‐induced insulin resistance and fatty liver in male Wistar rats, (2) The efficacy of aqueous extracts from Sutherlandia frutescens leaves and Prunus africana bark and metformin in the treatment of HFDinduced insulin resistance and fatty liver. Male Wistar rats were fed on HFD (the HF group) or normal rat chow (the LF group) for 12 weeks. Even though the HFD‐fed rats had developed insulin resistance by week 12, fatty liver developed by week 16. After week 12, the HF group was divided into four groups of 6‐7 rats each and three of those groups were gavaged with either 0.125 mg P. africana extract/kg bwt/day (the HF+Pa group) or 50 mg S. frutescens extract kg bwt/day (the HF+Sf group) or 16 mg metformin/ kg bwt/day (HF+Met group), while kept on the same diet for an additional of 4 weeks, to investigate whether two medicinal plant extracts and metformin can prevent HFD to induce fatty liver or not. After 16 weeks, the liver histological images revealed that the HF group developed fatty liver in the form of both microsteatosis and macrosteatosis. Fatty liver was confirmed by significant increased liver total lipid (TL) and activities of glucose‐6‐phosphate dehydrogenase (cG6PD) and xanthine oxidase (XO), mitochondrial NADH oxidase (mNOX) and by a decrease (P<0.05) in the activities of the homogenate superoxide dismutase (hSOD) and mitochondrial complex II in the HF group, when compared to the LF group. Since the activities of mCS and cACL enzymes were not changed in the HF group, hence increased cG6PD activity in the HF group indicates that there was increased NADPH demand for lipid accumulation from activated NEFAs taken up by the liver from circulation and for maintenance of the NADPH‐dependent antioxidants and oxidants, respectively. The obtained data also show that mitochondria of the HFD‐fed rats adapted to an increase in energy availability, thereby compensation through decreasing complex II activity, to allow electron flux from β‐oxidation to respiratory chain in the HF group. Liver TL content was significantly decreased in the rats treated with metformin and P. africana extract, but not in the rats treated with S. frutescens when compared to the HF group (P < 0.05). However, the TL content remained >5% per liver weight in all treated groups. The present study demonstrates that these two plant extracts and metformin have different glucogenic and lipogenic effects from that presented by HFD alone when compared to the LFD alone. In conclusion, metformin and P. africana extract can attenuate HFD‐induced fatty liver without changing the dietary habits. Hence S. frutescens extract is less effective in the prevention of HFD‐induced fatty liver. A change in the dietary habits is recommended to be considered during the use of these three remedies in the treatment of HFD‐induced insulin resistance and fatty liver. All three treatments enhanced antioxidant capacity, and may improve insulin resistance and fatty liver mediated by the present HFD through different mechanism of actions in the liver.

In Kenya, the prevalence of diabetes is estimated at 3-10 percent of the population. These figures could be higher because most type 2 diabetics are diagnosed many years after onset. Out of this number, 15 percent are people below 30 years of age who need prompt education to avoid complications that are associated with diabetes (DMI centre, 2004). Due to inadequate or lack of proper information, most patients especially those with type 2 diabetes are diagnosed through complications. Untreated or poorly managed diabetes is now the leading cause of eye disease and kidney failure in the world. Diabetes is the largest cause of kidney failure in the developed world, and is the fourth leading cause of global death by disease in the world (IDF, 2007). At the Kenyatta National Hospital in Nairobi, Kenya, it is the leading cause of all non-accident related amputations. It is with such statistics in mind and the grim reality of poor and inadequate health services that this research is based. The wide use of selected medicinal plants for the treatment and management of diabetes warrants the further study of these plants for potential use and commercialization. The data obtained can also be invaluable for use and reference when using these plants for medicinal purposes. The medicinal plant studied in the research is widely used in Kenya by many communities and was chosen based on ethno-pharmacological references using traditional medicinal practitioners as well as patient’s recommendations. Different in vitro and in vivo assays were studied to try and elucidate the mechanisms of action as well as the organs targeted during treatment using this plant.

The African potato is used as an African traditional medicine for its nutritional and medicinal properties. Most research has been carried out on H. hemerocallidea, with very little or nothing on other Hypoxis spp. The main aim of this project was to provide scientific data on the anticancer, anti-inflammatory and antioxidant properties of H. hemerocallidea, H. stellipilis and H. sobolifera chloroform extracts and their active compounds. The hypoxoside and phytosterol contents of the three Hypoxis spp. were determined using TLC, HPLC and GC. H. hemerocallidea and H. sobolifera chloroform extracts contained the highest amounts of hypoxoside and β-sitosterol, respectively. For the anticancer properties, cytotoxicity of the Hypoxis extracts and its purified compounds were determined against the HeLa, HT-29 and MCF-7 cancer cell lines (using MTT), and PBMCs (using CellTiter-Blue®). H. sobolifera had the best cytotoxicity against the three cancer cell lines, whereas H. stellipilis stimulated HeLa and HT-29 cancer cell growth. IC50 values of hypoxoside and rooperol were determined. DNA cell cycle arrest (using PI staining) occurred in the late G1/early S (confirmed by increased p21Waf1/Cip1 expression) and G2/M phases after 15 and 48 hrs, respectively, when treated with Hypoxis extracts and rooperol. H. sobolifera and rooperol activated caspase-3 and -7 (using fluorescently labelled antibodies) in HeLa and HT-29 cancer cells, and caspase-7 in MCF-7 cancer cells after 48 hrs. Annexin V binding to phosphatidylserines in rooperol treated U937 cells confirmed early apoptosis after 15 hrs. The TUNEL assay showed DNA fragmentation in the three cancer cell lines when treated with H. sobolifera and rooperol for 48 hrs. A shift pass the G2/M phase has led to the investigation of endoreduplication, which was confirmed by cell/nucleus size, and anti-apoptotic proteins (Akt, phospho-Akt, phospho-Bcl-2 and p21Waf1/Cip1). U937 cell differentiation to monocyte-macrophages was optimized using PMA and 1,25(OH)2D3, which was confirmed by morphological and biochemical changes. For the anti-inflammatory properties, Hypoxis extracts and rooperol significantly increased NO production in monocyte-macrophages (pre-loaded with DAF-2 DA) and phagocytosis of pHrodoTM E. coli BioParticles®. The treatments had no effect on COX-2 expression in monocyte-macrophages. The phytosterols significantly increased IL-1β and IL-6 secretion xv (using the FlowCytomix Multiplex human Th1/Th2 10plex Kit I) in the PBMCs of one donor. For the antioxidant properties, Hypoxis extracts and rooperol significantly increased ROS production in undifferentiated and differentiated U937 cells, which were pre-loaded with DCFH-DA. Hypoxis extracts and purified compounds had ferric reducing activities, but only rooperol had ferric reducing activities significantly greater than ascorbic acid. β-sitosterol, campesterol and cholesterol significantly increased SOD activity in Chang liver cells, while H. stellipilis, H. sobolifera and rooperol decreased SOD activity. Anticancer, anti-inflammatory and antioxidant properties of the Hypoxis extracts may be attributed to the β-sitosterol content, because Hypoxis chloroform extracts contained very little or no hypoxoside. Unidentified compounds, and synergistic and additive effects of the compounds may have contributed to the biological effects. This study confirms previous reports that rooperol is the active compound. Results provide scientific data on the medicinal properties of one of the most frequently used medicinal plants in South Africa.

The antibacterial potency of the extracts of the seed of Garcinia kola (bitter kola) was investigated in this study against a panel of referenced, environmental and clinical bacterial strains. The killing rates of the active extract as well as their potential for combination antibacterial therapy with standard antibiotics were also elucidated using standard procedures. The aqueous and acetone extracts of the seed were screened for activity against 27 bacterial isolates. The aqueous extract exhibited activity mainly against Gram positive organisms with Minimum inhibitory concentration (MIC) values ranging from 5 mgml-1 – 20 mgml-1, while the acetone extract showed activity against both Gram negative and Gram positive organisms with MIC values ranging from 10 mgml-1 - 0.156 mgml-1. The acetone extract also showed rapid bactericidal activity against Staphylococcus aureus ATCC 6538 with a 3.097 Log10 reduction in counts within 4 hours at 0.3125 mgml-1 and a 1.582 Log10 reduction against Proteus vulgaris CSIR 0030 at 5 mgml-1 after 1 hour. In addition, the aqueous, methanol and acetone extracts of the seeds also exhibited activity against four clinical strains of Staphylococcus isolated from wound sepsis specimens. The MIC values for the aqueous extract were 10 mgml-1 for all the isolates while the acetone and methanol extracts had lower values ranging from 0.3125 - 0.625 mgml-1. The acetone extract was strongly bactericidal against Staphylococcus aureus OKOH3 resulting in a 2.70 Log10 reduction in counts at 1.25 mgml-1 within 4 hours of exposure and a complete elimination of the organism after 8 hours. The bactericidal vi activity of the same extract against Staphylococcus aureus OKOH1 was weak, achieving only a 2.92 Log10 reduction in counts at 1.25 mgml-1 (4× MIC) in 24 hours. In the test for interactions between the acetone extract of the seeds and antibiotics, synergistic interactions were observed largely against Gram positive organisms using the FIC indices, (indices of 0.52 - 0.875) with combinations against Gram negatives yielding largely antagonistic interactions (indices of 2.0 to 5.0). Synergy (≥ 1000 times or ≥ 3 Log10 potentiation of the bactericidal activity) against both Gram negative and Gram positive organisms was detected by time kill assays mainly involving the antibiotics tetracycline, chloramphenicol, amoxycillin and penicillin G. Combinations involving erythromycin and ciprofloxacin consistently gave antagonistic or indifferent interactions. We conclude that the acetone extract of Garcinia kola seeds possess strong bactericidal activities against both Gram positive and Gram negative organisms and can be therapeutically useful in the treatment of bacterial infections including the problematic staphylococcal wound infections. In addition, the acetone extract can be a potential source of broad spectrum resistance modifying compounds that can potentially improve the performance of antibiotics in the treatment of drug resistant infections.

Infectious diarrhea results in 2 to 5 million deaths worldwide per year, and treatments that are safe, effective, and readily available are under investigation. The field of medicinal ethnobotany focuses on plants that are used by different cultural groups for treating various diseases and evaluates these plants for efficacy and cytotoxicity. In the present study, ethnobotanical research was conducted with Central Anatolian villagers in Turkey. Folk concepts and etiologies surrounding diarrhea were analyzed, as were salient plant-based remedies for diarrhea. Reviewing the literature, 91 plant species were described as anti-diarrheal in all of Turkey. In Central Anatolia, villagers described 35 species. For continued research via bactericidal and bacteriostatic bioassays, 15 plants were selected. Methanolic and aqueous extracts of medicinally used plant parts were evaluated for inhibitory properties against 10 diarrhea-causing bacteria in the first bioassay, and later 21 bacteria in a second assay utilizing spectrophotometry. The cytotoxic properties were also evaluated in an Alamar Blue Assay using HepG-2, PC-3, and SkMEL-5 human cell lines. While several extracts showed bactericidal and bacteriostatic properties, the methanolic extract of R. canina galls inhibited the most bacteria at the lowest concentrations. They were not cytotoxic. Thus, R. canina methanolic gall extracts were selected for bio-assay guided fractionation. Antibacterial activity was maintained in the third fraction which was composed of almost pure ellagic acid. The bioassay was repeated with standard ellagic acid, and the polyphenol retained potency in inhibiting multiple bacterial strains. Several other extracts showed promise for safe, effective anti-bacterial remedies for diarrhea.

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Magister Scientiae (Medical Bioscience) - MSc(MBS) / Obesity is a growing public health concern globally, particularly in developed countries such as the United States (US). More than 30% of Americans are considered obese. In the past 20 years in America, incidence of obesity has increased significantly (Mokdad et al., 2003). As a consequence, the Centers for Disease Control (CDC) anticipates that 1 in 3 American adults will be diabetic by 2050 (Boyle et al., 2001; Hedley et al., 2004). The most important cause of obesity is poor nutrition, absence of physical exercise and unfavourable lifestyle changes (James et al., 2001).