Thesis (M.Sc. (Biochemistry) -- University of Limpopo, 2018 / Dicerocaryum senecioides is a plant widely used as a nutritional source. It is used also for treatment of measles, wounds and to facilitate birth in domestic animal and humans in many parts of southern Africa (Mampuru et al., 2012). Findings in our laboratory have shown that a dichloromethane fraction of D. senecioides possesses antiinflammatory properties in human t-lymphocytes (Madiga, 2009), while the methanol crude extract possesses anti-proliferative and proapoptotic properties against Jurkat T cancer cells (Mphahlele, 2008). In this study, the probable anti-cancer effect of D. senecioides crude methanol leaf extract was investigated on cervical HeLa cancer cells. Dried powdered leaves of D. senecioides were extracted with absolute methanol to obtain a crude extract. To assess the cytotoxicity effect of the extract, KMST-6 and HeLa cell cultures were exposed to various extract concentrations (0 to 600 µg/ml) for 24 and 48 hours and subjected to the MTT assay. The results showed the extract to have no significant increase in the viability inhibition of HeLa cells at all tested concentrations after 24 hours of treatment. However, treatment with 400, 500 and 600 µg/ml of the extract for 48 hours revealed significantly increased HeLa cell viability inhibition. Furthermore, the extract showed to have no effect on the viability of normal human fibroblast KMST-6 cells at concentrations below 600 µg/ml, after 24 and 48 hours of treatment, thus showing selective cytotoxicity of the extract. To determine the mode of cell death associated with the increase in HeLa cell viability inhibition, the Hoechst 33258 nuclear staining assay and inverted light microscopy were employed. The data proposed apoptosis as the mode of cell death associated with the inhibition of HeLa cell viability. This was evidenced by changes in cell morphology such as the loss of HeLa cell radial extensions, cell shrinkage, as well as nuclear morphological features such as chromatin condensation. Apoptosis induction was further confirmed by the annexin-V/PI and multicaspase assays, using flow cytometry. The results showed an increase in the percentage of cells stained with annexin-V/PI, as well as increased caspase activity in extract-treated HeLa cells. To elucidate proapoptotic mechanisms of the extract, Western blotting analysis as well as the human apoptosis antibody array kit were used. This was to measure the expression profile of a number of apoptosis regulatory proteins. The results demonstrated modulation of some anti- and pro-apoptotic proteins, as well as the release of mitochondrial proteins required xiii for initiation of apoptosis, in the cytoplasm. The D. senecioides extract showed to have no effect on the cell division cycle of HeLa cells as determined by the PI staining assay. In conclusion, D. senecioides crude methanol leaf extract induced some degree of apoptosis in cervical HeLa cancer cells via the intrinsic apoptosis pathway. This was by modulating some of the members of the Bcl-2 family of proteins, which, facilitated the release of cytochrome C and activation of a caspase cascade. / South African Medical Research Council (SAMRC)

As plantas para fins medicinais são amplamente utilizadas no Brasil, porém existem poucos estudos que comprovam a eficácia como também a segurança do uso. Piper aduncum usada por muitas comunidades como planta medicinal para tratar e curar diferentes doenças, dentre estas, desordens gastrointestinais. O objetivo deste estudo foi avaliar o potencial antimicrobiano e toxicológico do extrato bruto aquoso das folhas Piper aduncum e aproximar os efeitos em testes laboratoriais aos possíveis efeitos observados no consumo das infusões. Folhas saudáveis de Piper aduncum foram utilizadas para preparar os extratos brutos aquosos em três concentrações (20 g/L, 10 g/L e 5 g/L). Foram realizados testes de ágar difusão em poço contra as bactérias enteropatogênicas Sthaphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922, Salmonella typhimurium ATCC 1402 e Shigella flexneri ATCC 12022. Testes de fragilidade osmótica eritrocitária humana e o teste citotóxico da avaliação do crescimento da raiz de Allium cepa foram realizados para avaliar a toxicidade dos extratos. A triagem fitoquímica foi realizada para determinar os compostos presentes nas infusões. Os resultados obtidos demonstraram que as infusões de P. aduncum não apresentaram efeitos antimicrobianos e não tiveram efeitos tóxicos sobre células sanguíneas, mas apresentaram potencial tóxico no desenvolvimento celular. / Plants for medicinal purposes are widely used in Brazil, but there are few studies that demonstrate the efficacy and the safety use. Piper aduncum is used for many communities as medicinal plant to treat and cure different diseases, including gastrointestinal disorders. The aim of this study was to evaluate the anti-microbial and toxic potential of aqueous crude extract from leaves of Piper aduncum, and approximate the effect in laboratory tests to what is possibly observed with the tea use. Healthy leaves of Piper aduncum were used to prepare the aqueous crude extract in three concentrations (20g/L, 10g/L and 5g/L). The antimicrobial activity was evaluated by the agar wells diffusion against entropathogenic bacteria Sthaphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922, Salmonella typhimurium ATCC 1402 e Shigella flexneri ATCC 12022. Human erythrocyte osmotic fragility and cytotoxic evaluation of Allium cepa roots growth were performed to evaluate the toxicity of the extracts. The phytochemical screening determined the present compound in infusions. The obtained results showed that P. aduncum infusions has no effect as antimicrobial, and it did not have any toxic effect against blood cells, but potential toxic effect on cell development.

MSCAGR / Department of Plant Production

We evaluated the effect of cyanobacterial crude extracts containing microcystin (CCEMC+) from a natural bloom on seed germination and initial development of three economically important legume species: green mung bean Vigna radiata, cowpea Vigna cylindrical and red mung bean Vigna angularis and compared it to crude extracts of cyanobacteria without the toxin (CCEMC–). Results showed that CCEMC+ and CCEMC– caused different effects on seed germination and initial development of the three species. There was a clear inhibition on germination and root growth of the green mung bean exposed to the CCEMC+ (20, 200 and 500 μg/L), indicating that the green mung bean being more sensitive to CCEMC+ when compared to the cowpea and red mung bean. CCEMC+ induced a greater occurrence of abnormal seedlings in the green mung bean, duce to inhibition the germination as well as reduction of fresh weight and root length. The CCEMC– extract caused no harmful effects to germination and seedlings growth of the green mung bean and red mung bean. However, it reduced shoot and root length in cowpea bean, suggesting that the cowpea being more sensitive to both extracts. Our results indicated that the sensitivity in germination and root growth of the green mung bean V. radiata could be used as an indicator to evaluate the toxic effect and monitor the toxin concentration of water contaminated with microcystins. / Nghiên cứu này khảo sát và so sánh các tác động bất lợi của ly trích vi khuẩn lam có chứa và không chứa độc tố lên sự nẩy mầm và sự phát triển ở giai đoạn đầu của ba loại cây họ đậu gồm đậu xanh Vigna radiata, đậu đỏ Vigna angularis và đậu đen Vigna cylindrical. Kết quả cho thấy hai loại ly trích gây ra các tác động khác nhau lên ba loại cây đậu thí nghiệm. Ly trích vi khuẩn lam có chứa độc tố ở nồng độ 20, 200 và 500 μg/L ngăn chặn đáng kể sự nẩy mầm và sự phát triển rể ở đậu xanh. Cả hai loại ly trích có chứa và không chứa độc tố đều ngăn chặn sự nẩy mầm và sự phát triển rể ở đậu đen. Ngược lại ly trích vi khuẩn lam có chứa độc tố ở nồng độ 500 μg/L lại kích thích chiều dài rể, thân mầm và trọng lượng tươi ở đậu đỏ. Kết quả cho thấy đậu đen khá nhạy cảm với cả hai loại ly trích có chứa và không chứa độc tố, trong khi đó đậu xanh nhạy cảm hơn với ly trích có chứa độc tố. Tính nhạy cảm của các loại cây họ đậu khi phơi nhiễm với ly trích vi khuẩn lam có thể được sử dụng để chỉ thị cho sự ô nhiễm và quan trắc độc tố vi khuẩn lam trong môi trường.

Les milieux biologiques bruts, provenant des opérations de transformation de biomasse sont souvent caractérisés à la fois par leur caractère polluant et par un potentiel de valorisation important. Le développement de procédés adaptés au traitement de tels milieux complexes présente ainsi beaucoup d’intérêt. Les jus verts générés par la déshydratation de la luzerne (Medicago Sativa) sont caractérisés par une forte teneur en protéines. Outre leur valeur nutritionnelle importante, ces protéines ont des applications potentielles dans plusieurs domaines, notamment environnemental de part leur teneur élevée en Ribulose 1,5 Bisphosphate Carboxylase Oxydase (Rubisco), enzyme responsable de la fixation du CO2 chez les plantes. Dans ce travail la séparation sélective de la Rubisco à partir du jus de luzerne industriel centrifugé par chromatographie d’échange d’ions a été étudiée. Dans un premier temps une méthode d’analyse qualitative et quantitative a été mise au point pour la détection et la quantification de la Rubisco en solution et ainsi le suivi du procédé de séparation. Dans un deuxième temps, le procédé de séparation a été étudié en colonne, en lit fixe et en lit expansé, en utilisant le support échangeur d’anions Q Hyper Z et l’effet de la dilution du milieu sur la capacité dynamique du procédé a été analysé dans les deux cas. Les résultats obtenus ont montré que les deux modes de contact permettent d’avoir des capacités dynamiques de rétention du même ordre de grandeur que celles de la littérature. Après élution, la Rubisco a été concentrée jusqu’à 21 fois et les fractions produites étaient caractérisées par un grand degré de pureté. Par ailleurs, des études d’équilibre et cinétique d’échange ont été initiées dans ce travail et ont démontré que malgré la taille importante de la protéine d’intérêt (560 kDa) les limitations stériques à son transfert ne sont pas plus importantes que dans le cas de protéines plus simples et plus petites et que le support Q Hyper Z présente effectivement une grande affinité pour la protéine. Enfin une première approche théorique a été conduite pour la compréhension des interactions entre la protéine et l’échangeur dans ce milieu complexe. Elle a permis de confirmer l’importance de la prise en compte de la présence d’autres biomolécules dans le milieu sur la rétention de la Rubisco, peut être même plus que celle des sels / Biological raw material derived from bio-refinery processes, is often considered a source of pollution but it seems also to be a promising alternative to potential material recovery. The development of suitable processes for handling such complex biological material has so many concerns. Green juice produced from mechanical dehydration of Alfalfa (Medicago sativa) is an excellent source of protein with high nutritional quality. Ribulose-1,5-bisphosphate carboxylase oxygenase (Rubisco) is the most abundant protein in the green juice, with potential applications in many fields, such as human nutrition, pharmaceuticals, environmental… The aim of this study is to isolate and recover Rubisco produced from an industrial alfalfa green juice, by ion exchange chromatography process. First of all, a qualitative and quantitative analytical method was developed to provide reliable information about Rubisco content monitoring in the separation process. In a second step, the separation process was performed in fixed and expanded bed, using the anion exchanger Q Hyper Z. In both cases, the effect of the dilution of the green juice on the dynamic capacity of the columns was studied. The results showed that the dynamic capacity retention was similar in both columns to those reported in literature. After elution step, Rubisco was concentrated 21 times and produced with high level of purity. Furthermore, kinetic of ion exchange study was initiated. Despite the large size of the protein (560 kDa), steric limitations to mass transfer were not very significant when compared to those of conventional small proteins. The support Q Hyper Z showed an excellent affinity for the protein recovery. Finally, a first theoretical investigation has been conducted for understanding the retention mechanism between the protein and the separation column. This study shows the importance of taking into account the presence of other bio-molecules in order to perform the retention of Rubisco, perhaps even more than that of salts