Submitted by Marcos Anselmo (marcos.anselmo@unipampa.edu.br) on 2017-06-05T13:30:10Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) BRUNA PIAIA RAMBORGER.pdf: 1379039 bytes, checksum: 750df4846ea9e6b47021886f60adead4 (MD5) / Approved for entry into archive by Marcos Anselmo (marcos.anselmo@unipampa.edu.br) on 2017-06-05T13:30:25Z (GMT) No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) BRUNA PIAIA RAMBORGER.pdf: 1379039 bytes, checksum: 750df4846ea9e6b47021886f60adead4 (MD5) / Made available in DSpace on 2017-06-05T13:30:25Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) BRUNA PIAIA RAMBORGER.pdf: 1379039 bytes, checksum: 750df4846ea9e6b47021886f60adead4 (MD5) Previous issue date: 2017-03-20 / A utilização de pesticidas visa o aumento da produção e qualidade dos produtos agrícolas. Porém, estes compostos podem ocasionar danos ao meio ambiente e ao ser humano devido a sua vasta utilização e toxicidade. O 2,4-Diclorofenoxiacetico (2,4-D) é um herbicida muito utilizado para diversas culturas no Brasil e no mundo para combater ervas daninha de folha larga. Possui características de alta solubilidade em água, mobilidade e persistência levando a contaminação de água e solo e em sua fórmula comercial, utilizada em lavoura, apresenta a classificação toxicológica I (extremamente tóxico). Desta forma, para a recuperação de áreas contaminadas com o 2,4-D, a utilização de plantas com capacidade de degradar, estabilizar e/ou remover contaminantes, conhecida como fitorremediação, torna-se extremamente importante devido o baixo custo e diversidade vegetal para tal fim. O presente estudo avaliou a possível capacidade de fitorremediação do Plectranthus neochilus (boldo) exposto ao pesticida comercial (Aminol) em solo e água através de extrações consecutivas (intervalo de dias). Após esse período de exposição, foram analisadas as respostas dessa planta em termos da presença do 2,4-D no chá das folhas, capacidade antioxidante total (DPPH), análise de polifenóis totais e flavonoides para as plantas expostas ao composto em solo e água. Nas plantas expostas na água também foi verificado a capacidade antioxidante total pelo método do fosfomolibdênio e a quantificação de compostos fenólicos. Após 15 dias de experimento, o 2,4-D não foi mais detectado nas amostras de solo e a planta não foi necessária na descontaminação desta matriz devido a degradação do composto ocorrer nesse mesmo período. Diferentemente, na água, o 2,4-D permaneceu até 67% em 60 dias de experimento, o que proporcionou a utilização de dois grupos de tratamento com a planta (um grupo de plantas por 30 dias e um novo grupo nos 30 dias restantes no mesmo sistema), e assim, obteve-se uma descontaminação de até 49% do 2,4-D. O composto não foi detectado no chá das folhas da planta e a capacidade antioxidante total, polifenóis e flavonóides apresentaram-se diminuídos viii em solo (todo experimento) e água (primeiros 30 dias). Entretanto para aquelas plantas que estavam na água nos 30 dias restantes, houve um aumento nessas análises próximo ao nível basal (grupo branco). Na quantificação dos compostos fenólicos (ácido caféico, ácido cumárico e ácido ferúlico) presente no chá dessas plantas observou-se que no grupo de plantas dos primeiros 30 dias houve um aumento do ácido cumárico e acido ferúlico, comparado ao grupo de plantas não expostas ao 2,4-D no tratamento 1 e uma diminuição do ácido caféico no tratamento 2. Nos 30 dias restantes com as novas mudas, observou-se uma diminuição do ácido cumárico e aumento dos ácidos cafeico e ferúlico no tratamento 1 e 2. Os resultados indicaram que a planta teve capacidade de fitorremediar o 2,4-D na água e, embora o composto tenha causado danos no sistema antioxidante, obteve aumento dos compostos fenólicos quantificados tornando o chá da planta útil após a fitorremediação. / The use of pesticides is aimed at increasing the production and quality of agricultural products. However, these compounds can cause harm to the environment and to humans due to their wide use and toxicity. 2,4-Dichlorophenoxyacetic (2,4-D) is a widely used herbicide for various crops in Brazil and the world to combat broadleaf weeds. It has characteristics of high solubility in water, mobility and persistence leading to contamination of water and soil and in its commercial formula, used in farming, it presents toxicological classification I (extremely toxic). Thus, for the recovery of areas contaminated with 2,4-D, the use of plants capable of degrading, stabilizing and /or removing contaminants, known as phytoremediation, becomes extremely important due to the low cost and plant diversity for this purpose. The present study evaluated the possible phytoremediation capacity of Plectranthus neochilus (boldo) exposed to the commercial pesticide (Aminol) in soil and water through consecutive extractions (interval of days). After this period, the plant's responses were analyzed in terms of the presence of 2,4-D in leaf tea, total antioxidant capacity (DPPH), total polyphenols and flavonoids analysis for plants exposed to soil and water. In the plants exposed in the water it was also verified the total antioxidant capacity by the phosphomolybdenum method and the quantification of phenolic compounds. After 15 days of experiment, 2,4-D was no longer detected in the soil samples and the plant was not necessary in the decontamination of this matrix due to degradation of the compound occurring in the same period. Differently, in water, 2,4-D remained up to 67% in 60 days of experiment, which provided the use of two treatment groups with the plant (a group of plants for 30 days and a new group in the remaining 30 days in the same system), and thus a decontamination of up to 49% of 2,4-D was obtained. The compound was not detected in tea leaves of the plant and total antioxidant capacity, polyphenols and flavonoids were decreased in soil (whole experiment) and water (first 30 days). However, for those plants that were in the water in the remaining 30 days, x there was an increase in these analyzes near the basal level (compared to blanc - plants in contact with water only). In the quantification of the phenolic compounds (caffeic acid, coumaric acid and ferulic acid) present in the tea of these plants, it was observed that in the group of plants of the first 30 days there was an increase of the coumaric acid and ferulic acid, compared to the group of plants not exposed to 2,4-D in treatment 1 and a decreased in caffeic acid in treatment 2. In the remaining 30 days with the new seedlings, there was a decrease of the coumaric acid and increase of the caffeic and ferulic acids (treatment 1 and 2). The results indicated that the plant had the capacity of phytoremediation of the 2,4-D in water and, although the compound caused damages in the antioxidant system, it obtained an increase of the quantified phenolic compounds, making tea of the plant useful after phytoremediation.

This diploma´s thesis deals with the determination of total antioxidant activity of red and white gooseberries and red, white and black currants. There are available information about small fruits, an overview of natural and synthetic antioxidants and a summary of the methods to determine the total antioxidant activity in the theoretical part. Frozen fruit samples were provided by Research and Breeding Institute of Pomology Holovousy Ltd. in the project QI111A141 – Research of new technologies in the cultivation of gooseberries and currants with a focus on quality and use of fetuses (Ministry of Agriculture). The total antioxidant activity of red and white gooseberries and currants was measured by spectrophotometric methods using free radicals ABTS•+ and DPPH and was expressed as a Trolox equivalent. The total antioxidant activity of black currants was measured by electron paramagnetic resonance using free radicals ABTS•+ and DPPH and expressed as a TEACABTS and a TEACDPPH. There were measured 8 species of red gooseberries, 6 species of white gooseberries, 12 species of red currants, 6 species of white currants and 8 species of black currants in this thesis. The highest values of total antioxidant activity showed species of red and white gooseberries Alan, Karmen, Pax and Invicta and species of red and white currants Rovada, Orion and Olin. Among black currants, species Ometa and Ruben had the highest values of total antioxidant activity.

Oxidative processes play important role in cell physiology and pathology as well. Balance of these processes is supplied by cooperating antioxidative systems; function of antioxidant defense systems depens on high levels of antioxidants in organism. Presented work is focused on developement and optimization of methods for analysis of important enzyme and non-enzyme antioxidants as well as total antioxidant capacity of selected types of biological material. Extractions and analyses of vitamin E, carotenoids, superoxide dismutase, catalase, peroxidase and lipoxygenase in barley and malt were optimized. RP-HPLC and HPLC/ESI-MS were used for analysis of vitamin E, phenolic and carotenoid content, spectrophotometry was used for enzymes activity analysis. A new methods for catalase and lipoxygenase activities were developed and compared with direct UV methods. Superoxide dismutase activity was determined by commercial diagnostic kit. A colorimetric method was used for peroxidase activity determination. Some kinetic parameters of enzymes were provided too. Optimized methods were used in the analyses of antioxidants in plant material - in barley and malt - in sets of samples of 6 varieties cultivated in four different locations for two years. Content of individual antioxidants differed depending on the variety, but usually were not found significant differences in the levels, depending on growing location. Perhaps climatic conditions have the greatest influence on levels of low molecular weight and enzymatic antioxidants at the specific location; oxidation processes are influenced both the quantity of moisture, both by sunlight, which induces oxidative processes in cultivated plants. The activity of antioxidants in barley caryopses is rapidly increasing during the malting process; an elevated temperature and moistness first induces activation the enzyme systems including antioxidant. In caryopsis is metabolic activity increased during which we can expect an increased production of radicals; for this purpose can antioxidant systems be activated that protect cells from damage by oxidative stress. In the second part of work optimized methods were applied in two clinical trials focused on study of the influence of exogenous antioxidants intake on metabolic and antioxidant status in human organism. In the first clinical study influence of food supplement containing polyunsaturated fatty acids and vitamin E on metabolism of hyperlipidaemics was evaluated. After 3-month supplemenation a lipid profile was improved and serum antioxidant levels increased. The second experiment was focused on enzyme and non-enzyme antioxidant levels in healthy subjects after temporarily intake of specific foods rich in antioxidants. After two-month intake plasma phenolic substances were slightly increased. Total antioxidant capacity and activities of enzyme antioxidants were not affected. Results of both clinical exeriments showed that supplying of antioxidants in natural form or in the form of food supplements does not markedly affect metabolism of healthy subjects, while in patients with chronic diseases antioxidant supplementation can positively influence metabolic status. Results of this work showed that optimized methods are suitable for analyses of antioxidant status parameters and also for monitoring of exogenous antioxidant intake.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The deficit of estrogen that accompanies menopause may be involved in the metabolic changes and increased oxidative stress during non-reproductive female life. Hormone replacement therapy (HRT) has been used to attenuate the menopausal symptoms. It is prescribed either as the replacement of estrogen alone or the combination of estrogen with progestins. Superoxide dismutase (SOD) is a key enzyme in the control of reactive oxygen species levels and SOD modulators may have potential use as therapeutic agents in oxidative stress-associated disorders. The objectives of this study were to evaluate: i) the effects of natural and synthetic estrogens and progestins on the activity of SOD from human blood in vitro; and ii) the effect of the hormone therapy with estrogen or estrogen plus progestin on the markers of oxidative stress in the blood of postmenopausal women and the relationship among these markers and the serum levels of estradiol and progesterone. The in vitro effect of steroid hormones (17 β-estradiol 17-acetate, progesterone, β-estradiol 3-benzoate and medroxyprogesterone 17-acetate) was evaluated in the enzyme purified from human erythrocytes (CuZnSOD) (Sigma) and in samples of erythrocytes (cytosolic CuZnSOD) and platelets-rich plasma (PRP) (MnSOD and cytosolic and extracellular CuZnSOD) obtained from healthy men and women. Hormones caused a dose-dependent stimulation of erythrocyte CuZnSOD activity at low concentrations (physiological), but this effect was abolished at higher concentrations. The combination of an estrogen with a progestin had a synergic effect on the erythrocyte CuZnSOD activity. In the PRP the activity of MnSOD was not affected by hormones, whereas the CuZnSOD activity was modulated only by the natural, but not by the synthetic hormone derivatives. Four groups of women were selected to evaluate blood markers of oxidative stress: premenopausal women (n=24), postmenopausal women without hormone therapy (HT) (n=31), postmenopausal women with estrogen-only HT (ET) (n=12) and estrogen plus progestin HT (EPT) (n=16). The levels of protein carbonyl, lipid peroxidation and the activity of catalase and glutathione peroxidase did not differ among groups. However, the activities of SOD isoforms (CuZn and MnSOD) and total plasma antioxidant power (FRAP) were significantly higher in postmenopausal women under EPT compared with postmenopausal women without HT, whereas ET increased only the activity of CuZnSOD in postmenopausal women. The duration of HT and serum E2 levels were positively correlated with the activity of CuZnSOD and the total antioxidant power of plasma (FRAP levels), whereas progesterone levels were positively correlated with the activity of CuZnSOD and negatively correlated with protein carbonyl levels. The total antioxidant power of plasma was positively correlated to the CuZnSOD activity and to the GPx activity. The present study demonstrated for the first time, that the natural and synthetic steroid hormones have a direct biphasic effect on CuZnSOD activity of human erythrocytes in vitro. We also observed that the hormone replacement therapy increase the antioxidant status of postmenopausal women due to an increase of the enzymatic antioxidant defenses and this effect is more remarkable with the combined hormone therapy (estrogen plus progestin). / O déficit de estrogênio, que acompanha a menopausa pode ser relacionado às alterações metabólicas e ao aumento do estresse oxidativo, observados na fase não reprodutiva feminina. A terapia de reposição hormonal é utilizada para atenuar os sintomas da menopausa. Ela é prescrita como reposição de estrogênio ou uma combinação de estrogênio com progestina. A superóxido dismutase (SOD) é uma enzima chave no controle dos níveis de espécies reativas de oxigênio e, moduladores da SOD podem ser úteis como agentes terapêuticos em desordens associadas ao estresse oxidativo. Os objetivos deste estudo foram avaliar: i) os efeitos, in vitro, de estrógenos e progesterona, naturais e sintéticos, sobre a atividade da SOD presente em sangue humano, e ii) o efeito da terapia hormonal com estrogênio ou estrogênio mais progestinas sobre os marcadores de estresse oxidativo no sangue de mulheres na pós-menopausa e a relação entre esses marcadores e os níveis séricos de estradiol e progesterona. O efeito, in vitro, de hormônios esteroides (acetato de 17β-estradiol (E2), progesterona, 3-benzoato de 17β-estradiol e 17-acetato de medroxiprogesterona) foi avaliado na enzima purificada a partir de eritrócitos humanos (CuZnSOD) (Sigma), e em amostras de eritrócitos (CuZnSOD citosólica) e de plasma rico em plaquetas (PRP) (CuZnSOD, citosólica e extracelular, e MnSOD, mitocondrial), obtidas a partir de homens e mulheres saudáveis. Os hormônios, em concentrações baixas (fisiológica), causaram uma estimulação, dose dependente, da atividade da CuZnSOD eritrocitária, embora, este efeito tenha sido suprimido em concentrações mais elevadas. Ademais, a combinação de um estrogênio com uma progestina apresentou um efeito sinérgico sobre a atividade da CuZnSOD eritrocitária. No PRP a atividade da MnSOD não foi afetada por hormônios, enquanto que a atividade da CuZnSOD foi modulada apenas pelos esteroides naturais. Quatro grupos de mulheres foram selecionados para avaliar marcadores sanguíneos de estresse oxidativo: mulheres na pré-menopausa (n = 24), mulheres na pós-menopausa sem terapia hormonal (TH) (n = 31), mulheres na pós-pausa utilizando TH, composta apenas de estrogênio (ET) (n = 12), ou de uma combinação de estrogênio mais progestinas (EPT) (n = 16). Os níveis de proteína carbonilada, peroxidação lipídica e da atividade de catalase e glutationa peroxidase (GPx) não diferiram entre os grupos. No entanto, as atividades das isoformas da SOD (CuZn e MnSOD) e o poder antioxidante total do plasma (FRAP) foram significativamente maiores em mulheres na pós-menopausa sob EPT em comparação com mulheres na pós-menopausa sem TH, enquanto que a ET aumentou apenas a atividade da CuZnSOD em mulheres na pós-menopausa. A duração da TH e os níveis séricos de E2 foram positivamente correlacionados com a atividade da CuZnSOD e com o poder antioxidante total do plasma (FRAP), enquanto que os níveis de progesterona foram positivamente correlacionados com a atividade da CuZnSOD e negativamente correlacionados com os níveis de proteína carbonilada. O poder antioxidante total do plasma foi positivamente correlacionada com a atividade da CuZnSOD e da GPx. O presente estudo demonstrou, pela primeira vez, que os hormônios esteroides, naturais e sintéticos, têm um efeito direto e bifásico na atividade da CuZnSOD eritrocitária humana in vitro. Também foi observado que a terapia de reposição hormonal aumenta a capacidade antioxidante de mulheres na pós-menopausa devido a um aumento das defesas antioxidantes enzimáticas (SODs) e que este efeito é ainda mais pronunciado com o uso de terapia hormonal combinada (estrogênio e progestinas).

This Diploma thesis deals with the production of the concentrate from the Elderberry marc extract (Sambucus nigra L.). The basic chemical characteristics in this concentrate were determined. The theoretical part of the thesis deal with the botanic characteristic of the Elderberry and its berries. The characteristic of the phenolic compounds, mainly anhocyanins was summarized. The use of the anthocyanin pigments in the food industry, the possibilities of their extraction and the concentrate production was discussed. Thereafter the methods of the anthocyanin content and the phenolic content determination were described. In the experimental part of study, the production of the anthocyanin rich extract from Elderberry marc was optimized. The extraction of the marc by the 1:1 ethanol/water (V/V) solvent with the ratio marc/solvent 1:2 (w/V) showed the most convenient. The production of the concentrate followed. During the concentration on the rotary evaporator the pigment degradation was investigated. The degradation decreased with the decrease of the temperature. The final concentrate was prepared from the extract on the rotary evaporator by 45 °C for 15 minutes. This product had the 7times lower volume compared to the initial extract. In the final concentrate come of the chemical characteristic were determined. By liquid chromatography (HPLC) the major pigments cyanidin-3-sambubiosid and cyanidin-3-glucosid were determined. Their total content was determined to 34,9 gl-1. The total anthocyanin content was determined by pH differential method to 7,1 gl-1 (expressed as cyanidin-3-glucoside equivalent). The total phenolic content was determined to 27,1 gl-1 (expressed as gallic acid equivalent) by Folin-Ciocalteu method. By electron paramagnetic resonance the increase in antioxidant activity with the increase in concentration was found. Up to 42 aromatic compounds was determined in Elderberry marc, its extract and the final concentrate.

This thesis deals with the total antioxidant activity (TAA) evaluation of various sorts of tea (green, black, half-fermented, white, mate, rooibos and herbal) depending on the process and time of their treatment. The main emphasis is put on testing a new method of measuring the total antioxidant activity by flow coulometry. The results acquired by this method are compared to the results of the chemiluminescent Trolox equivalent antioxidant capacity (TEAC) method which is a standard for measuring the total antioxidant capacity in various materials and raw materials. The work discusses the reasons for the differences in the values found by both methods.