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Plants have been utilized as a source of medicines since ancient times. They contain a vast range of secondary metabolites which play important roles in different diseases. The Scope of this study is to define the function of secondary metabolites from Andrographis paniculata (Kalmegh) and Silybum marianum (Milk Thistle) against DNA damage, which initiates many diseases. Sequential extraction of both plant materials was performed with different polarity solvents. Qualitative analysis was performed with Gas (GC) and High Performance Liquid Chromatography (HPLC). Primary extracts screening studies were performed against cytotoxicity, antioxidant and soluble collagen assays (Sircol dye). Further bioactivity was confirmed using the single-cell gel electrophoresis (Comet assay) to estimate levels of DNA damage. Fourier Transform Infrared analysis of bioactive extracts was performed to identify the functional groups present in them . Subsequently, bioactive extracts were further separated into acid, base, phenol and neutral fractions. These fractions and bioactive extracts were screened with the free radical assays to identify the scavenging activity. Chemical mapping of the bioactive fraction was performed with High Performance Liquid Chromatography (HPLC). Preparative-HPLC was performed to separate the compounds which were present in the bioactive fractions. MTT assay, Hydroxyl radical and nitric oxide radical scavenging activity assays were performed to screen the fractions and DNA protective activity of the bioactive fractions was confirmed with single-cell gel electrophoresis. These bioactive fractions were de-replicated with hyphenated techniques like LCMS and LCMS/MS to identify the molecular weight of the compounds and Quadrupole time-of-flight mass spectrometry was performed to identify the accurate mass of the compounds. Sequential extraction separates the non-polar and polar compounds present in the plant material. Qualitative analysis confirmed the presence of fatty acids in the non-polar extracts of both plants using GC and the presence of standard constituents in the polar extracts of both plants using HPLC. It also helps in chemical mapping of the extracts. Acetone, Methanol1 and Methanol2 extracts from either plant are non-cytotoxic. The high antioxidant activity is observed in methanol extracts from Andrographis paniculata and in acetone/methanol2 extracts from Silybum marianum. Extracts that protect against UVA and UVB damage also increased soluble collagen production in Human Dermal Fibroblast (HDF) in culture. Primary Screening helped to select six extracts out of twelve extracts for further analysis. Comet assay confirmed DNA protective activity in Methanol1 extract of Milk thistle and Acetone, Methanol2 extracts from Kalmegh. These three extracts were further fractionated into 38 fractions out of which three fractions that are F1, F13 and F31 fractions confirm the DNA protection activity. De-replication of the bioactive fractions was performed with LC-ESI-MS/MS which confirm twenty one compounds and accurate mass of fifteen compounds was determined using Q-tof mass spectrometry.

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Made available in DSpace on 2014-10-09T12:34:24Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T14:09:44Z (GMT). No. of bitstreams: 0 / O uso acentuado de fármacos e de produtos de higiene e cuidados pessoais por grande parcela da população e a conseqüente e contínua entrada dos mesmos no ambiente gera uma necessidade cada vez maior de se investigar a presença, o comportamento e os efeitos causados à biota aquática, bem como novas formas de tratamento para efluentes contendo tais substâncias. O cloridrato de fluoxetina é um princípio ativo utilizado em tratamentos de distúrbios depressivos e de ansiedade. Enquanto o surfactante dodecilsulfato de sódio está presente em diversos produtos de limpeza e de cuidados pessoais. O presente estudo teve como enfoque avaliar a toxicidade aguda do cloridrato de fluoxetina, dodecil sulfato de sódio bem como a mistura de ambos frente aos organismos aquáticos Hyalella azteca, Daphnia similis e Vibrio ficheri. A redução da toxicidade do cloridrato de fluoxetina e da mistura após tratamento com radiação ionizante, proveniente de acelerador industrial de elétrons, também foi objeto de estudo. Para Daphnia similis os valores médios de CE5048h encontrados para o fármaco, o surfactante e a mistura não irradiados foram de 14,4 %, 9,62% e 13,8%, respectivamente. Após irradiação das substâncias, a dose de 5 kGy se mostrou a mais efetiva, resultando em valores médios de CE5048h de 84,60% e >90 %, para o fármaco e para a mistura respectivamente. Para Hyalella azteca foram realizados ensaios de toxicidade aguda para coluna d\'água com duração de 96 horas, cujas médias dos valores de CE5096h encontradas para o fármaco, o surfactante e a mistura não irradiados foram de 5,63 %, 19,29 %, 6,27 %, respectivamente. Para o fármaco e mistura irradiados com a dose de 5 kGy, foram de 69,57% e 77,7 %, respectivamente. Para Vibrio ficheri os ensaios de toxicidade aguda para o fármaco não tratado e para o fármaco irradiado com 5 kGy geraram valores de CE5015min de 6,9 % e 32,88 %, respectivamente. Tais resultados evidenciaram a redução da toxicidade das subtâncias - teste após irradiação. / Dissertação (Mestrado) / IPEN/D / Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP

Muitas espécies de animais vivenciam situações nas quais há uma profunda depressão metabólica, como na anóxia, na hipóxia e na hibernação. Durante a reoxigenação ou o despertar, ocorre aumento da produção de espécies reativas de oxigênio (ROS), que tendem a causar danos oxidativos. Diferentes enzimas antioxidantes protegem o organismo contra as ROS, porém não se sabe qual a real importância de cada uma delas durante a reoxigenação. A hibernação é uma das formas de hipometabolismo menos estudadas, fazendo com que haja questionamentos sobre como os hibernantes se protegem das ROS durante o despertar. A análise dos dados existentes é complexa devido à existência de variáveis não controladas, como o efeito do jejum associado à hibernação. Nesta tese, foram desenvolvidos dois projetos. Em um, investigou-se a importância da catalase num ciclo de anóxia e reoxigenação em caramujos pulmonados. No segundo, investigou-se o efeito da hibernação e da privação alimentar no intestino de lagartos teiús. Com base nos resultados, foi possível concluir que a catalase exerce um papel complementar contra os danos oxidativos causados pelas ROS e em conjunto com os demais componentes do sistema antioxidante. Porém, sua função não parece ser essencial, sendo em grande parte compensada pela atividade de glutationa peroxidase. Também foi possível concluir que a hibernação, estudada sem a interferência de drásticas quedas da temperatura, causa nítidas alterações no metabolismo de radicais livres no intestino de lagartos, com queda de atividades enzimáticas e de concentração de glutationa. A ausência de grandes danos oxidativos durante o despertar dos animais mostra que eles têm um sistema antioxidante eficiente. A privação alimentar resultou em respostas semelhantes as da hibernação, mas parece ter causado um certo grau de estresse oxidativo. Os resultados apresentados nesta tese respondem dois questionamentos no estudo do metabolismo de radicais livres em situações que envolvem flutuações na disponibilidade e no consumo de oxigênio. / Many species of animals experience situations in which occurs a profound metabolic depression, like anoxia, hypoxia and hibernation. During reoxygenation or arousal, there is an increase of the production of reactive oxygen species (ROS), which tend to cause oxidative damage. Different antioxidant enzymes protect the organims against the ROS, however the real importance of each one of them during reoxygenation is not known. Hibernation is one of the types of hypometabolism less studied, and questions about how the hibernators protect themselves from ROS during the arousal have not yet been answered. The analysis of the existent data is complex due to the existence of uncontrolled variables. In this thesis were carried out two studies in which were investigated: the importance of catalase in a cycle of anoxia and reoxygenation in pulmonate snails, and the effect of hibernation and of food deprivation in the intestine of tegu lizards. Considering the results, it was possible to conclude that catalase plays an complementary role against the damages caused by ROS and in association with the other components of the antioxidant system. However, its function seems to be non-essential, being greatly compensated by the glutathione peroxidase activity. It was also possible to conclude that hibernation, studied without the interference of drastic falls in temperature, causes clear alterations in free radicals metabolism in the lizards intestine, with a reduction in enzymes activities and in glutathione concentration. The absence of big oxidative damage during the arousal of the animals shows that they have an efficient antioxidant system. Food deprivation resulted in similar responses of those from hibernation, but seemed to cause some degree of oxidative stress. The results presented in this thesis answer two questions in the study of the free radical metabolism in situations that involve fluctuations in oxygen availability and consumption.

Little is known of tissue carbon monoxide (CO) changes after acute exposure because tissue levels are in the order of picomoles per milligram and the technology to measure such low concentrations has only become available relatively recently. This study tested three hypotheses: That tissue CO levels (1) vary among tissues after acute poisoning; (2) change over time; but (3) cannot be predicted by measuring carboxyhaemoglobin (COHb) levels. Twenty four healthy male Sprague-Dawley rats were exposed to 2500 ppm CO in air for 45 min. This non-lethal exposure achieved reproducible COHb values of 66 to 72%. Animals were allowed to recover breathing air and were sacrificed at 30 minute intervals for 150 minutes. An additional nine male animals served as unexposed controls. Accurate measurements of tissue CO levels were made in blood, brain, heart, and skeletal muscle samples. All samples were prepared using the validated technique described by Vreman et al., and Reduction Gas Analysis was used to determine the pmol CO per wet weight tissue. Co-oximetry and gas chromatography were performed on all blood samples. Predictably, blood CO content dropped following exposure, but tissue CO content did not follow the same trend in all tissues. This study supports the hypothesis of (1) tissue and (2) time-related variability of CO concentration in three body tissues after exposure and (3) documents lack of utility of COHb for predicting tissue CO tissue values. / Dissertation (MSc)--University of Pretoria, 2007. / Nuclear Medicine / Unrestricted