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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.

Phytochemical analysis of both HEA1 and the crude plant extract showed the presence of phenolics, tannins and saponins. Saponins were the predominant secondary metabolites and were mostly abundant in the plant extract and to a lesser extent in the active compound. Steroidal saponins, tannins and phenolics were also detected in the plant extract, but only the phenolics were detected in the active compound. The results of the phytochemical analysis showed that those compounds that were not present in the active compound could be removed from the crude extract during the TLC purification process. Investigation on the mechanism of action of the crude plant extract on the sterol production by A. flavus showed that the plant extract affected ergosterol biosynthesis by causing an accumulation of oxidosqualene in the ergosterol biosynthetic pathway resulting in a decline in ergosterol production. An oscillatory response in lanosterol production was observed in the presence of the plant extract, which may be an adaptation mechanism of A. flavus to unfavourable conditions and compensation for the loss of enzyme activity which may have occurred as a result of the accumulation of oxidosqualene. The antifungal activity of the plant extract on ergosterol production by A. flavus may also be due to saponins which target the cell membrane and ergosterol production in fungi. The effect of the plant extract on the fungal cell wall of A. flavus also showed that the plant extract caused a decline in β-(1, 3) glucan production by inhibiting β-glucan synthase. The plant extract also affected the chitin synthesis pathway of A. flavus, by causing a decline in chitin production, which was due to the inhibition of chitin synthase. Investigation of chitinase production using 4MU substrates showed that the plant extract caused an accumulation of chitobioses, by activating chitobiosidases and endochitinases. A decline in N-acetylglucosaminidase activity in the presence of the plant extract was observed and this prevented the formation of N-acetylglucosamine. The accumulation of chitobiosidase and endochitinase may be as a result of autolysis that may be triggered by A. flavus as a survival mechanism in the presence of the plant extract and as a compensatory mechanism for the loss of β-glucans and chitin. The antifungal effect of the plant extract on various components of the cell wall of A. flavus, makes T. violacea aqueous plant extract an ideal chemotherapeutic agent against both human and plant pathogens of Aspergillus. The broad spectrum of antifungal activity of T. violacea against A. flavus also eliminates any chances of the fungus developing resistance towards it and would make it a candidate for use as a potential antifungal agent. Further identification and possible chemical synthesis is needed to shed light on the safety and efficacy of the active compound for further development as a chemotherapeutic agent.

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.

Artemisia afra is one of the oldest, most well known and widely used traditional medicinal plants in South Africa. It is used to treat many different medical conditions, particularly respiratory and inflammatory ailments. There is no reported evidence of its use for the treatment of cancer but due to its reported cytotoxicity, an investigation of the mode of cell death induced by an ethanol A. afra extract using two cancer cell lines was done. IC50 values of 18.21 and 31.88 μg/mL of ethanol extracts were determined against U937 and HeLa cancer cells, respectively. An IC50 value of the aqueous extract was greater than 250 μg/mL. The ethanol extract was not cytotoxic against confluent control cell lines, Chang Liver and Vero cells. The effect of the cytotoxic ethanol A. afra extract on U937 and HeLa cells and their progression through the cell cycle, apoptosis and mitochondrial membrane potential was investigated. After 12 hours of treatment with A. afra a delay in G2/M phase of the cell cycle was evident. Apoptosis was confirmed using the TUNEL assay for DNA fragmentation, as well as fluorescent staining with annexin V-FITC. Apoptosis was evident with the positive control and A. afra treatment at 24 and 48 hours. JC-1 staining showed a decrease in mitochondrial membrane potential at 24 hours. It was deduced that A. afra ethanol extract induces caspase-dependent apoptosis in a mitochondrial dependent manner. Plants harbour many compounds that are not only useful to the plants but also to mankind. Many metabolites have been isolated from A. afra and their biological activity characterised. Due to observed apoptosis induction, isolation of cytotoxic compounds was done and a new sesquiterpene lactone from A. afra was isolated. Structural elucidation of the compound was done by IR, 1D and 2D NMR, CD and mass spectrometry and it was identified as isoalantolactone. HeLa cancer cells were treated with isoalantolactone and cytotoxicity was exhibited in a dose-dependent manner. A low IC50 value of 8.15 ± 1.16 μM was achieved. This study showed that isoalantolactone is partly responsible for the observed A. afra cytotoxicity. Due to the evidence of G2/M arrest, the anti-mitotic potential and the possible onset of mitotic catastrophe by A. afra and isoalantolactone was investigated. It was evident from various flow cytometric analysis of cyclin B1 and phospho-H3 and confocal microscopy that A. afra does possess anti-mitotic activity by causing hyperpolymerisation of tubulin and cells progress into the mitotic phase where M arrest is experienced. The anti-inflammatory activity of sesquiterpene lactones is well documented; however, the anti-inflammatory activity of A. afra is not. Here, it is reported that the production of NO and COX-2 protein levels in RAW 264.7 cells decrease in the presence of A. afra and isoalantolactone after stimulation with LPS. The activated NF-κB subunit, p65 was also investigated. The results suggest that A. afra and isoalantolactone inhibit p65 activation as a decrease in the activated subunit was evident. Thus, the results indicate that exposure to A. afra and isoalantolactone induces an anti-inflammatory response. In conclusion, this study shows, for the first time, the mechanism of induced apoptosis, the anti-mitotic and anti-inflammatory activity of A. afra and its isolated compound, isoalantolactone. It also proves that although extensive research may have been done on a particular plant, as with A. afra, more can be discovered leading to the identification of new compounds and integration of signalling pathways that can be exploited for the treatment of various diseases and ailments.

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.

Resistance of human pathogenic bacterial strains results in selective pressure against known antibiotic. However, plant derived compounds that possess antibacterial potential are currently being investigated for treatment of wound infections in diabetic patients as they are inexpensive and non-toxic. Hence, this dissertation was designed to evaluate two medicinal plants (Brachylaena elliptica and Brachylaena ilicifolia) traditionally used in the treatment of various diseases such as diabetes, and its secondary complications in diabetic patients. The in vitro antioxidant activity of both plants were evaluated using DPPH (1, 1-diphenylhydrazl), ferric reducing power, ABTS (2, 2’-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid), NO (nitric oxide) and H2O2 (hydrogen peroxide) techniques. The antibacterial test and Minimum inhibitory concentration (MIC) was determined by agar dilution method against 5 bacteria strains (Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus pyogene, Proteus vulgaris and Proteus mirabilis) infecting wounds in diabetic patients using amoxicillin and ciprofloxacin as positive control. The phytochemical analyses were assessed using standard published methods. Identification of bioactive components in essential oils of both plants were assessed using GCMS. The aqueous and ethanol extracts of both plants were also evaluated to identify bioactive components using LC-MS. The results of the phytochemical analysis revealed the presence of phenols, tannins, flavanoids, flavanols, proanthocyanidins, saponins and alkaloids in both plants. Both plants indicated strong antioxidant activities which might be due to the presence of bioactive compounds. The aqueous and ethanol leaf extracts of both plants demonstrated appreciable broad spectrum activities against these wound pathogens with MIC ranging between 5 and 0.3 mg/ml. The GC-MS analysis of the essential oils of both plants revealed the presence of monoterpenes, oxygenated sesquiterpenes, phenolics and esters. The LC-MS analysis of the aqueous and ethanol leaf extracts of both plants showed that both plants are rich in alkaloids, terpenes, terpenoids, monoterpernoids, and flavanoids. Conclusively, this study has partially justified the ethnomedicinal use of B. elliptica and B.licifolia leaves for the treatment of various diseases, including diabetes and wound infections caused by bacteria in diabetic patients. These may be attributed to the presence of antioxidant compound such as phenols, flavanoids, saponins, tannins, alkaloids and other phytochemical compounds.