Evalúa los residuos del plaguicida clorpirifos en semillas de quinua (Chenopodium quinua W.) en el período de cosecha, producidos en el distrito de La Molina de la región Lima. Los objetivos específicos fueron los siguientes; 1) Determinar la concentración del plaguicida clorpirifos en mg/kg (ppm) en las muestras de semillas de quinua y 2) Verificar si la concentración del plaguicida clorpirifos en las semillas de quinua se encuentra dentro del Límite Máximo Permisible según normativa de la Unión Europea y del Perú. Se evaluaron 12 muestras de quinua de un campo del distrito de La Molina y 1 muestra de quinua orgánica (libre de plaguicida) proveniente de la provincia de Yauyos, la cual ayudó para la validación del método analítico. La validación del método cumplió con los criterios establecidos por la Comisión Europea. Los análisis de las 12 muestras de quinua provenientes del distrito de La Molina presentaron residuos del plaguicida clorpirifos, 11 muestras no sobrepasan el LMR (0.05 mg/kg) establecido por las legislaciones de Canadá, Unión Europea y nuestro país, solo 1 muestra sobrepasó dicho LMR, con una concentración de 0.136 mg/kg. Los resultados muestran la exposición del plaguicida clorpirifos en semillas quinua, por ende, su presencia en nuestra dieta diaria y en la exportación. / Tesis

Memoria para optar al título de Ingeniero en Alimentos / La empresa Biofresco, procesadora de hortalizas, genera desechos orgánicos, siendo uno de los principales residuos las hojas de apio (Apium graveolens L. var. dulce). Sabiendo que éstas poseen fitoquímicos con propiedades antioxidantes y pigmentos como la clorofila, el principal objetivo de la siguiente investigación, fue el aprovechamiento de este desecho agrícola, por medio del estudio de la cinética de secado por convección con aire caliente, para el desarrollo de una infusión con aporte de antioxidantes, y por otro lado, con la hoja fresca, el desarrollo de un extracto enriquecido en clorofila para posterior uso como colorante natural. Para el estudio de secado de las hojas se trabajó a tres temperaturas (35, 45 y 55°C). Se realizó un ajuste de los datos experimentales, utilizando 6 modelos matemáticos establecidos en la literatura. Basándose en análisis estadístico, el modelo Page Modificado presentó el mejor ajuste. Los valores de la difusividad efectiva del agua se estimaron en el rango de 6,5 x 10-10 - 2,8 x 10-9 (m2/s) y el valor de la energía de activación fue de 61,83 (kJ/mol). Para el desarrollo de la infusión se trabajó con hoja secada a 35, 45 y 55°C con 4 tiempos de extracción (3, 5, 7 y 10 min). Siendo la infusión obtenida con la hoja secada a 35°C, por un tiempo máximo de contacto de la muestra con el agua caliente de 7 min, la que entregó el mejor valor agregado de acuerdo a las propiedades antioxidantes. Un metabolito secundario de la hoja de apio; apigenina, fue cuantificado por HPLC-DAD, encontrándose una cantidad entre 1,5 – 2 mg AP/100 g hoja de apio ps en las infusiones con hoja seca a 35°C. Finalmente, para el desarrollo integral de la infusión se realizó una evaluación sensorial con consumidores, los cuales en general, mostraron un grado de indiferencia hacia el producto. Por otra parte, el desarrollo del extracto enriquecido en clorofila se obtuvo por medio del método de maceración con etanol 95% como solvente extractante. Este análisis presentó una cantidad de 25,1 g de clorofila a/Kg de hoja pf. Por lo tanto, con el desarrollo de estos alimentos con propiedades funcionales, se logró valorizar los residuos de hoja de apio / Biofresco company, a vegetable processor, generates organic waste like celery leaves (Apium graveolens L. var. dulce), one of the main residues that this industry produces. Knowing that these celery leaves have phytochemicals with antioxidant properties and pigments such as chlorophyll, the main objective of the following research was to take advantage of these organic waste, through the study of hot air convection drying kinetics of celery leaf, by the development of an infusion with antioxidant contribution and, on the other hand, an extract enriched in chlorophyll for later use as a natural colorant using fresh leaves. For the study of the drying process of the leaves it has been worked at three temperatures (35, 45 and 55°C). An adjustment of the experimental data was made using 6 estabilished-in-literature mathematical models. Based on statistical analysis, the Modified Page model presented the best fit. The values of the effective diffusivity of the water were estimated in the range of 6.5 x 10-10 to 2.8 x 10-9 (m2/s) and the value of the activation energy was 61.83 (kJ/mol). For the infusion development it has been worked with leaf dried at 35, 45 and 55° Celsius with 4 extraction times (3, 5, 7 and 10 minutes). The best value added according to antioxidant properties was obtained with the leaf dried at 35° C, with a maximum contact time between leaf and hot water of 7 minutes. A secondary metabolite of the celery leaf, apigenin, was quantified by HPLC-DAD, finding quantities between 1,5 and 2 mg AP/100 g celery leave dw. Finally, a sensory evaluation was performed with consumers for the integral development of the infusion, which in general showed a degree of indifference to the product. On the other hand, the development of the extract enriched in chlorophyll has been obtained through the maceration method with 95% ethanol as extractant solvent. This analysis showed an amount of 25.1 g of chlorophyll a/kg of fresh leaf weight. Therefore, with the development of these foods with functional properties, it was possible to value the residues of celery leaf

Memoria para optar al título de Químico Farmacéutico / Los flavonoides son compuestos polifenólicos que se encuentran distribuidos en el reino vegetal. Las propiedades antioxidantes de estos compuestos se relacionan con su capacidad de atrapar radicales libres (ej. radical hidroxilo) y apagar especies excitadas (ej. oxígeno singlete). Los flavonoides poseen la capacidad de interactuar con distintas proteínas desde que se administran, al estar en la circulación sanguínea, con proteínas estructurales celulares, pudiéndose modificar tanto su biodisponibilidad como sus efectos farmacológicos. Estudios recientes sugieren que la unión con albúmina afecta la absorción, biodisponibilidad y actividad de los flavonoides, dependiendo de la estructura del polifenol. El objetivo general de este trabajo es desarrollar metodologías cromatográficas HPLC para la determinación del efecto antioxidante frente al radical hidroxilo, de polifenoles que están interactuando con albúmina en medio acuoso tampón pH 7,4, siguiendo el consumo del polifenol en función del tiempo. Así, se desarrollaron dos metodologías analíticas por Cromatografía Líquida de Alta Eficiencia (HPLC). Una fue desarrollada con la finalidad de lograr una óptima separación del flavonoide y sus respectivos productos de oxidación. Para esta metodología se utilizó una columna de fase reversa C8 y una gradiente de elución isocrática. Por otra parte, el seguimiento del consumo de cada flavonoide en función del tiempo se realizó en una columna C18, donde estos compuestos eluyen a tiempos menores en comparación con la metodología utilizada para la separación de los posibles productos de oxidación. Ambas metodologías fueron validadas en forma preliminar utilizando concentraciones del rango micromolar, presentando una adecuada relación lineal entre la señal cromatográfica y la concentración. La metodología se aplicó eficientemente para la determinación del efecto antioxidante de morina cuando está interaccionando con albúmina en medio acuoso tampón pH 7,4 y se comparó con el medio homogéneo. Los resultados muestran que morina presenta una mayor reactividad con el radical hidroxilo cuando está interactuando con la proteína, lo que indica que aumenta su eficiencia antioxidante / Flavonoids are polyphenolic compounds that are distributed in the plant kingdom. The antioxidant properties of these compounds are related to their ability to trap free radicals (eg. hydroxyl radical) and quench excited species (eg. singlet oxygen). Flavonoids have the ability to interact with different proteins since they are administered, being in the bloodstream, with cellular structural proteins, being able to modify both their bioavailability and their pharmacological effects. Recent studies suggest that binding with albumin affects the absorption, bioavailability and activity of flavonoids, depending on the structure of the polyphenol. The general objective of this work is to develop HPLC chromatographic methodologies for the determination of the antioxidant effect against the hydroxyl radical of polyphenols that are interacting with albumin in aqueous buffer pH 7.4, following the consumption of polyphenol as a function of time. Thus, two analytical methodologies were developed by High performance Liquid Chromatography (HPLC). One was developed with the purpose of achieving an optimum separation of the flavonoid and its respective oxidation products. For this methodology, a C8 reverse phase column and an isocratic elution gradient were used. On the other hand, the monitoring of the consumption of each flavonoid as a function of time was carried out in a C18 column, where these compounds elute at shorter times in comparison with the methodology used for the separation of the possible oxidation products. Both methodologies were validated in a preliminary way using concentrations of the micromolar range, presenting an adequate linear relationship between the chromatographic signal and the concentration. The methodology was applied efficiently for the determination of the antioxidant effect of morin when interacting with albumin in an aqueous buffer pH 7.4 and was compared with the homogeneous medium. The results show that morin presents a greater reactivity with the hydroxyl radical when it is interacting with the protein, which indicates that it increases its antioxidant efficiency

Orientador: Leonardo Susumu Takahashi / Resumo: Os aminoácidos e seus metabólitos são importantes reguladores das principais vias metabólicas dos peixes, essenciais para manutenção, crescimento, reprodução, comportamento, imunidade e resistência a diversos agentes estressores. Nesse sentido, os objetivos dos trabalhos foram avaliar a influência que uma dieta deficiente em aminoácidos essenciais exerce na capacidade imune e oxidativa de tilápias do Nilo (O. niloticus) em fase de terminação, e averiguar a reposta imunológica e oxidativa de juvenis de tilápia alimentados com dieta contendo níveis crescentes de inclusão do aminoácido histidina desafiados com lipopolissacarídeo. Ao todo foram utilizadas 774 tilápias (264 em fase de terminação e 510 juvenis) em dois experimentos distintos com delineamentos inteiramente ao acaso. No primeiro ensaio foram avaliadas 11 dietas experimentais (controle e dietas deficientes em cada um dos dez aminoácidos essenciais) em variáveis do sistema imune não especifico e atividade hepática de enzimas do sistema antioxidante de tilápias, e o segundo, os níveis de inclusão de histidina na dieta sobre as mesmas variáveis, em juvenis de tilápias desafiados com lipopolissacarídeo. Os resultados mostraram que dietas deficientes em aminoácidos essenciais influenciam na capacidade imunomodulatória e oxidativa de tilápias em terminação, e o nível ideal de inclusão de histidina na dieta pode potencializar essas capacidades frente a um desafio. Investigar o efeito do metabolismo de aminoácidos no potencia... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Amino acids and their metabolites are important regulators of the main metabolic pathways of fish, essential for maintenance, growth, reproduction, behavior, immunity and resistance to various stressors. In this sense, the objectives of the works were to evaluate the influence that a diet deficient in essential amino acids has on the immune and oxidative capacity of Nile tilapia (O. niloticus) in the process of termination, and also to investigate the immune and oxidative response of juvenile tilapia fed a diet containing increasing levels of inclusion of the amino acid histidine challenged with lipopolysaccharide. In total, 774 tilapia (264 in the finishing phase and 510 juveniles) were used in two different experiments with completely randomized designs. In the first trial, 11 test diets (control and diets deficient in each of the ten essential amino acids) were evaluated on non-specific immune system variables and liver activity of tilapia antioxidant system enzymes, and the second, the levels of inclusion of histidine in the diet on the same variables, in tilapia juveniles challenged with lipopolysaccharide. The results show that diets deficient in essential amino acids influence the immunomodulatory and oxidative capacity of finishing tilapia, and the ideal level of histidine inclusion in the diet can enhance these capacities in the face of a challenge. Investigating the effect of amino acid metabolism on the immune and oxidative potential of tilapia can be a tool in hel... (Complete abstract click electronic access below) / Doutor

Se determinó los niveles de las enzimas antioxidantes: Glutation Peroxidasa (GPx), Superóxido Dismutasa (SOD) y Catalasa (CAT); y la concentración de Malondialdehído (MDA) como un indicador de la peroxidación lipídica, en eritrocitos de 60 personas aparentemente sanas: 30 residentes de la altura (Cerro de Pasco, 4340 m.s.n.m.) y 30 residentes de nivel del mar (Lima, 150 m.s.n.m.). Se encontró diferencia estadísticamente significativa entre los niveles eritrocitarios de Glutation peroxidasa de los nativos de altura (66,86 ± 8,97 U GPx / g Hb) y los de nivel del mar (37,78 ± 8,87 U GPx / g Hb), (p < 0,001). No se encontró diferencia estadísticamente significativa entre los valores obtenidos para la Superóxido dismutasa de la altura y de nivel del mar: 4140,56 ± 1147,69 U SOD / g Hb vs. 4104,35 ± 1917,9 U SOD / g Hb, respectivamente. La actividad de la enzima Catalasa fue menor en los sujetos de altura (275,27 ± 44,21 k CAT / g Hb) en relación con los de nivel del mar (414,29 ± 81,07 k CAT / g Hb), siendo esta diferencia estadísticamente significativa (p < 0,001). Los niveles de Malondialdehído en los nativos de altura también fueron menores (8,60 ± 1,91 nMol MDA / g Hb) que los obtenidos a nivel del mar (11,16 ± 1,55 nMol MDA / g Hb), (p < 0,001). / It was determined the levels of the antioxidants enzymes: Glutathione Peroxidase (GPx), Superoxide Dismutase (SOD) and Catalase (CAT); and the concentration of Malondialdehyde (MDA) like an indicator of the lipid peroxidation in erythrocytes of 60 seemingly healthy people: 30 residents at the high altitude (Cerro de Pasco, 4340 m above sea level) and 30 residents at sea level (Lima, 150 m above sea level). It was found significant difference stadistic between erythrocyte levels of Glutathione peroxidase higher in the native of high altitude (66,86 ± 8,97 U GPx / g Hb) and people living at sea level (37,78 ± 8,87 U GPx / g Hb), (p < 0,001). It was not found significant difference stadistic between the values obtained of Superoxide dismutase at high altitude and sea level: 4140,56 ± 1147,69 U SOD / g Hb vs. 4104,35 ± 1917,9 U SOD / g Hb, respectively. The Catalase enzyme activity was smaller in subjects of high altitude (275,27 ± 44,21 k CAT / g Hb) that in subjects at sea level (414,29 ± 81,07 k CAT / g Hb), this difference was significant (p < 0,001). The levels of Malondialdehyde in the native of high altitude were also smaller (8,60 ± 1,91 nMol MDA / g Hb) that in those obtained at sea level (11,16 ± 1,55 nMol MDA / g Hb), (p < 0,001). / Tesis

Los hongos son fuente de alimento y medicina desde tiempos remotos. Por su valor nutracéutico y como fuente de nutricéuticos se han utilizado para mantener y mejorar la salud, preservar la juventud y promover la longevidad. También son fuente de compuestos químicos bioactivos útiles para la preparación de productos farmacéuticos. De las 700 especies de hongos comestibles investigadas, sólo unos 50 hongos tienen valor medicinal y entre ellos Grifola frondosa es uno de los más notables e investigados, especialmente como estimulador del sistema inmune y por sus propiedades antitumorales en la lucha contra el cáncer. En los bosques andino patagónicos de Argentina y Chile existen dos representantes del género Grifola: G. gargal Singer y G. sordulenta (Mont.) Singer, para los cuales no se ha investigado suficientemente sobre su biología y por lo tanto no se ha desarrollado aún una tecnología apropiada para su cultivo, como tampoco se han hecho estudios sobre sus posibles propiedades medicinales y aplicaciones no solo terapéuticas sino también biotecnológicas. Por ello se investigó la factibilidad de cultivarlos en condiciones controladas y a su vez se profundizó en el conocimiento de distintos aspectos biológicos de interés. Por otra parte, la actividad medicinal hallada en políporos y en especial en Grifola frondosa apoya la formulación de la hipótesis que plantea también la presencia de actividad antioxidante y/o antigenotóxica en estas especies. La confirmación de tales propiedades es necesaria para sostener más investigación sobre su uso medicinal y resulta de importancia en la propuesta de posibles variedades de productos a partir de ellos, lo cual otorgaría un plus como valor agregado de estos hongos. Para comprender aún más las condiciones de crecimiento de estas especies se realizaron cuatro campañas recorriendo diferentes bosques de robles del Parque Nacional Lanín. Allí, con la ayuda de residentes del lugar y micólogos, se pudieron recolectar fructificaciones sólo en determinados lugares, así como también se tomaron datos de las colonias en crecimiento y muestras de G. gargal causando podredumbre. Asimismo, se aislaron dos cepas (cepa B y G9) de G. gargal, una proveniente de un árbol en pie y otra tomada de un roble caído que llevaba más de 20 años produciendo fructificaciones. La historia de los bosques de roble, informada por otros autores, revela que durante la última glaciación fueron fragmentados y en consecuencia se desarrollaron procesos de variabilidad genética entre los mismos. Algunas de estas variables son el contenido y calidad de ciertos compuestos polifenólicos los cuales se sabe que son importantes en la biología de los hongos degradadores de la madera. En consecuencia esto indicaría una variabilidad entre cepas de los diferentes bosques de robles para G. gargal. Dos cepas, correspondientes a G. gargal (cepa A) y G. sordulenta, fueron obtenidas del Centro de Investigación y Extensión Forestal Andino Patagónico (CIEFAP). Hallar las condiciones óptimas en la producción de micelio y cuerpos fructíferos es un paso fundamental para la producción optimizada de los compuestos con valor nutricional, y como fuente de nutricéuticos y fármacos hipotéticamente presentes en estos hongos. El análisis del crecimiento micelial de G. gargal y G. sordulenta en agar nutritivo reveló que para ambas especies, las mejores condiciones de cultivo fueron pH 4, 18ºC y medio de cultivo MYPA 16 suplementado con 0,4% de cáscara de girasol en polvo. Así, fue posible disminuir el tiempo para la obtención de inóculo de excelente calidad y el cultivo de las cepas en este medio fue posteriormente utilizado en forma rutinaria para su mantenimiento y para su uso como inóculo. Los resultados presentados fueron analizados por una posible pérdida de vigor (luego de tres años de subcultivos) y se halló que ambas cepas no perdieron vigor y que además hubo una mejora en la velocidad de colonización por G. sordulenta y ello se puede explicar por haber desarrollado una adaptación a los ingredientes del medio, concluyendo que las condiciones de rutina empleadas fueron adecuadas para conservar su vigor por más de 12 subcultivos. El aumento en contenido de polvo de cáscara de girasol no modificó la velocidad de colonización de la cepa A de G. gargal por lo que no se justifica su aumento en el medio de cultivo por encima del 0,4 % para esta especie; no se encontraron diferencias en la velocidad de colonización entre las cepa A, B y G9. Usualmente se observa en el stock de cepas de los laboratorios de micología que algunas cepas producen primordios y/o verdaderos cuerpos fructíferos un tiempo después que son almacenadas. Esto llevó a utilizar este método de cultivo in vitro en condiciones controladas para dilucidar los procesos que subyacen en la morfogénesis fúngica, i.e.: la diferenciación del micelio vegetativo a micelio reproductivo. Las observaciones realizadas con lupa y microscopio a G. frondosa, G. gargal y G. sordulenta, fueron comparadas con las observaciones realizadas por otros autores en la bibliografía hallándose algunas diferencias de importancia taxonómica en cuanto a velocidad de crecimiento, morfología de las colonias, cambios de coloración en el medio de cultivo y tipo de degradación de compuestos fenólicos, estudiada in vitro. El aspecto al microscopio de las hifas generativas, gloeopleuras, esqueletales, presencia de clamidosporas y estructuras cristaloides, también en comparación con las observaciones de otras investigaciones, indicó algunas diferencias que permiten conocer mejor la variabilidad entre cepas de estas especies. El estudio de las formaciones morfogénicas in vitro de G. gargal y G. sordulenta fue útil para detectar cambios producidos en estos cultivos luego de aplicar diferentes condiciones lumínicas. La irradiación con luz blanca durante el crecimiento vegetativo de G. gargal previno una demora significativa del crecimiento de micelio en cajas de Petri causadas por temperaturas desfavorables (c.a. 21ºC). Las diferentes condiciones de luz produjeron cambios en el metabolismo secundario y en la diferenciación morfogénica de los cultivos. La respuesta a estas condiciones lumínicas fue mayor en G. gargal que en G. sordulenta. Los resultados indicaron que ambas especies de Grifola fueron sensibles a la irradiación lumínica, con respuestas morfogénicas de distinto tipo e intensidad, si bien la luz blanca fue más efectiva. En ausencia del estímulo luminoso, ambas especies del género fueron capaces de mostrar eventos morfogénicos. En el caso de G. gargal, en presencia del estímulo luminoso, la cepa A fue más sensible que la B en la presentación de respuestas fotomorfogénicas. Estos resultados registrados para los distintos anchos de banda de irradiación lumínica son sugerentes de la participación de más de una molécula fotorreceptora. Para estimar la capacidad ligninolítica in vitro, se cultivó G. gargal y G. sordulenta en medios diferenciales conteniendo diferentes compuestos fenólicos. Para fines comparativos y para poder extrapolar los resultados al cultivo, resultó útil realizar este estudio en forma simultánea con otras dos especies poliporales que tienen una buena performance en fermentaciones en estado sólido empleando cáscara de girasol como sustrato: G. frondosa y Ganoderma lucidum (dos cepas). El estudio reveló la expresión de enzimas polifenol oxidasas en diferentes momentos del crecimiento y con diferentes patrones. Se sugiere que una de estas sería la enzima lignina peroxidasa (LiP). En otro estudio se verificó la actividad de lacasas. Asimismo, la presencia de la enzima con actividad de Mn-peroxidasa (MnP), fue también detectada, ya que en presencia de 20 ppm de Mn(II) en sustratos a base de cáscara de girasol la velocidad de crecimiento micelial fue mayor; asimismo en el caso de G. sordulenta causó un aumento en la densidad micelial aparente. En otro análisis similar se estudió el crecimiento de estas especies en medios diferenciales conteniendo diferentes fuentes de carbohidratos para determinar la actividad de la enzima celobiosa deshidrogenasa. El crecimiento fue en todos los casos moderado a bajo. Se determinó que G. gargal crece mejor en xilulosa y pectina, lo cual indicaría una preferencia por la fracción hemicelulósica de los sustratos. Grifola sordulenta por su parte, crece bien en celulosa y xilulosa y presentó actividad de la enzima celulosa deshidrogenasa. La velocidad de crecimiento y la densidad de la colonización en los medios de carbohidratos fue mayor en las colonias de G. frondosa y Ganoderma lucidum superando a G. gargal y G. sordulenta que si bien crecieron en todos los medios, mostraron menor habilidad para colonizarlos. Por lo cual ambos estudios en medios diferenciales mostraron una menor performance in vitro, lo cual permitió prever un desempeño regular a bajo al extrapolar estos resultados al cultivo en sustratos basados en cáscara de girasol. En algunas especies el cultivo de micelio en medios líquidos ofrece ciertas ventajas como reducción de las pérdidas por contaminación, utilización de espacios más reducidos, y fácil recolección de la biomasa y de los metabolitos disueltos en el medio. Se realizaron cultivos de G. gargal y G. sordulenta en medio líquido agitado empleando Erlenmeyers de 250 ml y 500 ml, y frascos de 3 l, así como también en medio líquido estacionario empleando fuentes de vidrio de 4 l. Se compararon los diferentes sistemas considerando el trabajo que requiere cada técnica y la cantidad de biomasa que produce. Para G. gargal se encontró favorable el cultivo en frascos de 3 l, y para G. sordulenta en Erlenmeyers de 250 ml, obteniéndose una biomasa de 4 y 18 g/l en 20 días de cultivo, respectivamente. En ambas especies la temperatura óptima para este cultivo fue de c.a. 18ºC. En este trabajo se determinó que la suplementación con diferentes reguladores del crecimiento vegetal y/o vitaminas o aminoácidos, o sólo con bencilaminopurina (0,1-10 mg/l) a los medios de ambas especies no incrementa significativamente la biomasa. Por otra parte, mediante el empleo de inóculo homogeneizado se consiguió disminuir la variabilidad dentro de los tratamientos en comparación con el uso de discos de micelio cultivado en agar como inóculo. Los diferentes protocolos suministraron material micelial con diferentes cualidades para ensayar posteriormente las propiedades antioxidantes de estas especies. El spawn en granos es el material que se emplea para inocular grandes masas de sustrato en el cultivo de hongos. La evaluación del crecimiento de micelio de G. gargal y G. sordulenta mediante el bioensayo de crecimiento lineal de Duncan (1997) reveló que ambas especies pueden ser cultivadas en granos de trigo, girasol, maíz y combinaciones de trigo con mijo y maíz con girasol; se determinó que el mejor crecimiento se logra para ambas especies con el cultivo en granos de trigo en el rango de pH de 5,3 a 6,4. La colonización completa de los granos de trigo, según la técnica de producción habitual en botellas de un litro, se produce más rápidamente a 24ºC, en comparación con el crecimiento del micelio en medios semisólidos y líquidos (c.a. 18ºC). En el cultivo en granos no se hallaron diferencias en el número de botellas que completaron la colonización a los días 25 y 30. Para ambas especies y para todos los tipos de granos, la mayor proporción de botellas colonizadas se observó a los 30 días. Considerando la cantidad de granos por gramo de spawn, el empleo de granos de trigo es el más indicado. Se empleó el test de crecimiento lineal de Duncan para evaluar la velocidad de colonización, la densidad aparente, el incremento del contenido de proteínas y de actividad de lacasas, y la degradación de fibras de G. gargal y de G. sordulenta en 20 formulaciones de sustrato a base de cáscara de girasol. Seguidamente se realizó otro ensayo para estudiar el efecto de ciertos suplementos en la velocidad de colonización y densidad aparente en 10 formulaciones más. Con la información recolectada se puede afirmar que ambas especies pueden crecer en estos sustratos, que la cáscara de girasol no necesita suplementos, como salvado o sustrato gastado de Pleurotus ostreatus, para sostener un buen crecimiento del micelio. Para G. gargal la colonización mejoró con un tratamiento ácido del sustrato, o con el agregado de cofactores enzimáticos (Mn(II) y Zn(II)), o de otras fuentes lignocelulósicas como roble, álamo o paja de trigo; para G. sordulenta se encontró que la colonización mejora sólo en cuanto a la densidad con un tratamiento ácido del sustrato, o con el agregado de cofactores enzimáticos (NH4(I), Mn(II) y Zn(II)). Con el establecimiento de un cultivo axénico de G. gargal y G. sordulenta sobre troncos sintéticos artificiales empleando cáscara de girasol como sustrato, se encontró que ambas especies pueden colonizar bien el sustrato, si bien con un ciclo productivo más extenso y mayor riesgo de contaminación a posteriori que para otras especies cultivadas con la misma técnica. La inducción con choque térmico a 5ºC produjo una sensible inducción de primordios, y se lograron algunas fructificaciones. El intercambio gaseoso es crucial para el desarrollo de los basidiomas, como era esperable según los antecedentes registrados en la literatura sobre la producción de G. frondosa. La secuencia de producción de ambas especies en las diferentes etapas es similar a la de G. frondosa: primordios granulares grises, que luego se diferencian en fructificaciones que se suceden en formas conocidas como cerebro, coliflor y racimo. Las propiedades antioxidantes de extractos metanólicos de cuerpos fructíferos, micelio de cultivo líquido y/o de cultivo de granos de trigo fueron analizadas en cuanto a sus propiedades de extinción de radicales (radical DPPH), y poder reductor, asimismo también se comparó el contenido de compuestos fenólicos y se caracterizaron los extractos metanólicos con la técnica de cromatografía de capa delgada. Los resultados hallados permitieron conocer las propiedades antioxidantes de G. gargal y G. sordulenta. Estas especies resultaron poseer muy buenos atributos antioxidantes, especialmente poder reductor. Las diferentes bioformas de micelio y también las formas de cultivo modifican cuali y cuantitativamente el contenido de antioxidantes causando variaciones en la actividad secuestrante de radicales y en el poder reductor. También se encontró que el micelio puede ser inducido a modificar su contenido en antioxidantes usando reguladores de crecimiento vegetal, y que la variación en el contenido de una propiedad antioxidante es independiente de la producida en otra propiedad antioxidante. La actividad antioxidante se debió en parte a la presencia de compuestos fenólicos pero no fueron los únicos compuestos activos. El análisis de cromatografía de capa delgada reveló además que la mayoría de los compuestos antioxidantes eran de naturaleza polar, en éstos siempre se hallaron las características de revelado correspondientes a compuestos fenólicos, y además en algunas bandas también se reveló la presencia de flavonoides. Por su parte los metabolitos apolares se observaron en todos los cromatogramas de los extractos. En algunos casos podría asociarse a compuestos fenólicos, mientras que en otros podría deberse a metabolitos no fenólicos. En G. sordulenta se halló que la actividad antioxidante estuvo parcialmente asociada a compuestos flavonoides. Las propiedades antigenotóxicas de cuerpos fructíferos y micelio de cultivo líquido de G. gargal; así como las harinas de granos de trigo fermentados con G. gargal, G. sordulenta y/o G. frondosa, fueron estudiadas con el test de mutación y recombinación somática en Drosophila melanogaster. El agente químico utilizando para causar las mutaciones (promutágeno) fue el DMBA (7-12-dimetilbenzo(α)antraceno). Con el estudio de la toxicidad de DMBA en distintos tratamientos pudo observarse un incremento en la mortalidad de las larvas de 9 a 45%, sin embargo cuando se agregaron los extractos fúngicos esta mortalidad disminuyó. La mutación y recombinación se evaluó como el número de spots blancos por cada cien ojos, mostrando un aumento en la frecuencia en aquellos tratamientos que contenían DMBA, y una disminución en los co-tratamientos conteniendo ambos: DMBA, y extractos fúngicos en el siguiente orden: fructificación de G. gargal, las tres harinas de granos colonizados y micelio de cultivo líquido de G. gargal. Se concluyó que el material evaluado resultó no tóxico per se y en combinación con el promutágeno y procancerígeno DMBA pudieron disminuir su mortalidad y la genotoxicidad. La respuesta protectora de los materiales fúngicos activó mecanismos de detoxificación en la larva de D. melanogaster que pueden ser desmutagénicos o bioantimutagénicos, y que fueron causados por ciertos compuestos bioactivos presentes en estos hongos superiores con actividad antigenotóxica como p.e. fenólicos, ácido linoleico, polisacáridos y polipéptidos. Luego de demostrar la ausencia de genotoxicidad, las propiedades antioxidantes y las propiedades antigenotóxicas de G. gargal y G. sordulenta, aun es necesario realizar estudios adicionales sobre la producción de cuerpos fructíferos por medio de la fermentación en estado sólido, y así facilitar su disponibilidad para su consumo como alimento. Hasta entonces una alternativa es la producción de micelio y metabolitos por medio del cultivo líquido. Finalmente, en un apartado final de la tesis se muestra información del contenido mineral y de diferentes nutrientes analizados en muestras de micelio, fructificaciones y granos de trigo colonizados, y como una novedad con potencial biotecnológico se sugiere la obtención de una harina de trigo colonizado por estos hongos para ser empleada como un alimento funcional. / As of ancient times, fungi have been and still are a source of food and medicine. Due to their nutraceutical value, they have been used to maintain and improve health, preserve youth and promote longevity. They are also a source of bioactive chemical compounds, which are, in turn, useful for the preparation of nutriceuttical and pharmaceutical products. Of the 700 edible mushroom species investigated to date, only 50 have medicinal value. Among these 50 is Grifola frondosa which is one of the most thoroughly investigated, particularly as a stimulator of the immune system and for its antitumoral properties. Grifola gargal Singer and G. sordulenta (Mont.) Singer, two representatives of the genus Grifola, in the andean patagonian forest in Argentina and Chile, have not been fully studied to date. Nor have their medicinal properties with their corresponding therapeutic applications been investigated. The purpose of this Ph. D. thesis was therefore to study the possibilities of cultivating G. frondosa under control conditions. On the other hand, the medicinal activity observed in polypores, particularly in G. frondosa, gives support to the hypothesis on the presence of antioxidant and /or antigenotoxic activity in these species. Confirming such properties is absolutely necessary to conduct further research in favor of its medicinal properties and to promote the proposal of possible varieties of products derived from them, which would, in turn increase the value of these fungi. To further learn about the growth conditions of these species four campaigns were done in the oak forests located within Lanín National Park. With the help of mycologists and people living in the area, fruiting bodies were collected. Growth data and samples of rotting G. gargal were also collected. Two new strains (strain B and G9) of G. gargal were isolated, one from a standing tree and another from a fallen oak which had been producing fruits for a period longer than 20 years. Previous research on the history of oak forests reveals that during the last ice age they were fragmented, thus inducing the development of processes of genetic variability. Some of these sources of variability variables are the content and quality of certain polyphenolic compounds which are known to be important in the biology of fungal decomposers of wood. This could therefore be indicative of variability among different strains of oak woodlands to G. gargal. Two strains, corresponding to G. gargal (strain A) and G. sordulenta, were obtained from the Centro de Investigación y Extensión Forestal Andino Patagónico (CIEFAP). Reaching to the optimal conditions for the production of mycelium and fruiting bodies is key to the optimized production of compounds with nutritional, nutraceutical and also as a source for nutriceuticals and pharmaceuticals hypothetically present in these fungi. Analysis of mycelial growth of G. gargal and G. sordulenta on nutrient agar medium revealed that for both species, the best culture conditions were pH 4, 18 ° C and culture medium supplemented with 0.4% MYPA of sunflower husk powder. It thus took shorter time to obtain high quality seed and cultivation of strains in this medium was subsequently used routinely both for its maintenance and use as inoculum. Results were analyzed by a possible loss of vigour (after three years of subcultures) and showed that both strains did not lose vigour and that there was a higher rate of colonization in Grifola sordulenta which could be due to the fact that they adapted to the ingredients of the medium, thus indicating that the routine mycelium growing conditions followed were adequate to maintain its force for more than 12 subcultures. On the other hand, the increase in the content of sunflower husk powder (0.4 %) did not alter the rate of colonization of strain A to G. gargal. This indicated that there is no need in increasing the amount of this supplement in this species. It was also observed that there were no differences in the rate of colonization between strain A, B, and G9. It has been observed that in stock strains of mycology laboratories some strains produce primordia and/or real fruitbodies after they are stored. This led us to use the method of in vitro culture under controlled conditions to deep on the processes underlying fungal morphogenesis, i.e the differentiation of vegetative mycelium to reproductive mycelium in these species. Observations of G. frondosa, G. gargal and G. sordulenta under a magnifying glass and microscope were compared with observations by other researchers. These comparisons revealed some differences in taxonomic significance in terms of growth speed, colony morphology, colour changes in the culture medium and type of degradation studied in vitro. The comparison of the microscopic appearance of generative hyphae, gloeopleuras, skeletal, presence of chlamydospores and crystalloid structures with observations from other researchers showed differences which will contribute to better understanding of the variability among strains of these species. The study of the in vitro morphogenic differentiation of G. gargal and G. sordulenta was useful as it helped detect changes in these crops after applying different light conditions. Irradiation with white light during the vegetative growth of G. gargal prevented a significant delay in growth of mycelium in Petri dishes, originated from unfavourble temperatures (c.a. 21 º C). The different light conditions produced, in fact, changes in secondary metabolism and morphogenic differentiation of cultured mycelia. Compared to findings from a study conducted in colonies grown in Petri dishes which had completed their vegetative growth in darkness and had received a thermal shock, in our study the effect of these environmental conditions was greater on G. gargal and similar on G. sordulenta. This indicates that both species of Grifola are sensitive to light irradiation with different morphogenic response type and intensity while white light is more effective. In the absence of light stimulus, both species of the genus were found to have the ability to show morphogenic events. In the case of G. gargal, strain A was observed to be more sensitive than B in the presentation of photomorphogenic responses. These results reported for different bandwidths of light irradiation are indicative of the involvement of more than one photoreceptor molecule. Grifola gargal and G. sordulenta were cultured in differential media containing different phenolic compounds in order to estimate ligninolytic ability in vitro. For comparative purposes and in order to extrapolate results to their cultivation, this study was carried out simultaneously with two other species of poliporales which exhibit a good performance behave naturally in solid state fermentation when using sunflower husks as main substrate. The study revealed polyphenol oxidase enzyme expression at different times and with different growth patterns. It is suggested that one of these could be the enzyme lignin peroxidase (LiP). It was further verified the activity of laccases. Mn-peroxidase (MnP) activity was presumably present because it was also detected in other strains of G. gargal and because addition of Mn (II) increased the rate of colonization with G. gargal cultivation on sunflower husk as substrate and aspect was better in G. sordulenta. Growth of these species in differential media containing different carbohydrate sources was also studied in order to determine the activity of the enzyme cellobiose dehydrogenase. In all cases, growth ranged from moderate to low. It was found that G. gargal grows best in xylulose and pectin, which could be indicaive of a preference for the hemicellulosic fraction of the substrates. In contrast, G. sordulenta was observed to grow well on cellulose and xylulose and evidenced cellulose dehydrogenase activity. Growth rate and density of colonization on media containing carbohydrates was higher in colonies of G. frondosa and Ganoderma lucidum, being even higher than those of G. gargal and G. sordulenta which, although they grew in all media, evidenced lower ability to colonize them. Both studies in differential media showed a lower in vitro performance, thus preannouncing a regular to low growth performance when these results were extrapolated to crop-based substrate sunflower husk. In some species the mycelium growing in liquid media have the advantage of reducing losses due to culture, the use of smaller spaces, and allow for an easy harvesting of the biomass and recovery of the metabolites dissolved in the medium. Cultures of G. gargal and G. sordulenta were carried out in agitated liquid medium using 250 and 500 ml Erlenmeyers flasks, and 3-liter bottles, as well as in liquid medium sources using 4-liter glass containers. Different systems were compared considering the work involved in each technique and the amount of biomass produced. Grifola gargal culture was carried out in 3-liter glass flasks and G. sordulenta was carried out in 250 ml Erlenmeyers flasks, yielding a biomass of 4 and 18 g/l after 20 days of culture, respectively. In both species, optimum temperature for this crop was c.a. 18 °C. In this work it was also determined that supplementation with different plant growth regulators and/or vitamins or aminoacids, or benzylaminopurine alone (0.1-10 mg/l) to the media of both species does not significantly increase biomass. Moreover, the use of homogenous innoculum reduced variability within treatments with respect to the use of disks of mycelium grown on agar as inoculum. The fungal material obtained from this work provided mycelial with different qualities to further test the antioxidant properties of these species. Grain spawn is the material used to inoculate large number of substrates in mushroom cultivation. The evaluation of mycelial growth of G. gargal and G. sordulenta by linear growth bioassay of Duncan (1997) revealed that both species can be cultivated in grains of wheat, sunflower, corn and wheat combinations with millet and corn with sunflower. It was also observed that optimum growth is achieved for both species when cultivation of wheat grains is in the pH range of 5.3 to 6.4. The full colonization of grains of wheat, following the production technique in traditional 1 liter bottles, occurs more rapidly at 24 °C, compared with the growth of mycelium in semisolid media and liquid (c.a. 18 ° C). In grain cultivation there were no differences in the number of bottles that completed the colonization by days 25 and 30. For both species and for all types of grains, the largest proportion of substrates in bottles was colonized after 30 days. Considering the number of grains per gram of spawn grains, using wheat is best recommended. Duncan linear growth test was used to assess the substrate mycelia colonization rate, bulk density, increased protein content and laccase activity, and fiber degradation produced G. gargal and G. sordulenta in 20 formulations based substrate sunflower husk. Another test was further conducted to study the effect of certain supplements on the colonization rate and apparent mycelial density in 10 formulations. Taken together, findings from our study support the conclusion that both species grow in these substrates, that sunflower seed husks needs no supplements such as bran or Pleurotus ostreatus spent substrate to sustain regular growth of the mycelium. For G. gargal colonization was found to improve with an acid treatment of the substrate or with the addition of enzyme cofactors (Mn (II) and Zn (II)), or other lignocellulosic sources such as oak, poplar, wheat straw. For G. sordulenta colonization was found to improve only in terms of mycelial density with an acid treatment of the substrate, or with the addition of enzyme cofactors (NH4 (I), Mn (II) and Zn (II)). The axenic culture of G. gargal and G. sordulenta on artificial synthetic logs using sunflower husks as substrate showed that both species can colonize the substrate, but with a longer production cycle and increased risk of contamination in relation to other mushroom species with the same technique. The thermal shock induction of 5 °C produced a significant induction of primordia and produced some fructifications. Gas exchange is crucial to the development of basidiomas, as expected based on previous research on the production of G. frondosa. The production sequence of both species at different stages is similar to that of G. frondosa: gray granular primordia that grow into fruitbodies of brain shape, then cauliflower shape and finally cluster flower. The antioxidant properties of methanolic extracts of fruiting bodies, mycelium from liquid culture and/or wheat grains were analyzed in terms of their radical scavenger properties (DPPH radical) and reducing power. The content of phenolic compounds was compared and methanol extracts were characterized using thin layer chromatography. It was thus possible to learn more about the properties of G. gargal and to confirm them for the first time in the case of G. sordulenta. These species have antioxidant reducing power properties. Different bioforms of mycelium and also different culture systems modify forms of qualitative and quantitative antioxidant content causing variations in radical scavenger activity and reducing power. In other words, the mycelium can be induced to change their antioxidant content using plant growth regulators and variability in the content of antioxidant property is independent of that produced in other antioxidant property. Antioxidant activity was found to be due in part to the presence of phenolic compounds but it was not the only active compound. Thin layer chromatography also helped to show that the majority of antioxidant compounds were polar, in these were always revealed the presence of phenolic compounds, and in some bands, also the presense of flavonoids. Non-polar metabolites were observed in all chromatograms of extracts. In some cases they could be associated with phenolic compounds while in others could be associated to non-phenolic metabolites. In G. sordulenta it was found that the antioxidant activity was preferentially associated to flavonoid compounds. Antigenotoxic properties of fruiting bodies and mycelia from liquid cultures of G. gargal, as well as grains of wheat flour fermented with G. gargal, G. sordulenta and/or G. frondosa were also studied to test somatic mutation and recombination in Drosophila melanogaster. The chemical agent used to cause mutations (promutagen) was DMBA (7-12-dimethylbenz (α) anthracene). Research on toxicity revealed that treatment with DMBA increased larval mortality from 9 to 45%. Still, when fungal extracts were added larval mortality rate decreased. Both mutation and recombination were assessed as the number of white spots per hundred eyes, showing an increased frequency in the treatments with DMBA, and a decrease in co-treatments with both: DMBA and fungal extracts in the following order: G. gargal fruiting body, three grain meals colonized and G. gargal liquid culture mycelium. It could be concluded that the material evaluated was not toxic and that in combination with procarcinogenic promutagenic DMBA both mortality and genotoxicity decreased. The protective response observed with fungal materials triggered detoxification mechanisms in D. melanogaster larvae which could be either desmutagenic or bioantimutagenic and which could be produced due to some bioactive compounds present in higher fungi with antigenotoxic activity, such as phenolics, linoleic acid, polysaccharides and polypeptides. Summing up, findings on the absence of genotoxicity and antioxidant and antigenotoxic properties, in the fungal species studied in this Ph.D. thesis, will be greatly benefited from further research on the optimization of both species for the production of fruiting bodies through fermentation in the solid state. In the meantime, the production of mycelia and metabolites in liquid culture medium is the alternative for such optimitization. A final section of this Ph.D. thesis includes information on the mineral content of different nutrients which were analyzed in samples of mycelium and fruiting bodies and colonized wheat grains. A novelty with biotechnological potential derived from this section concerns the obtention of wheat flour colonized by these fungi, which evidenced factibility of being used as functional nutrient.

Os radicais livres são espécies químicas que reagem rapidamente com diversos compostos e alvos celulares, por possuírem tempo de meia vida muito curto e serem espécies altamente instáveis. A formação destes compostos constitui uma ação contínua e fisiológica, cumprindo funções biológicas essenciais as quais ocorrem pela perda ou adição de um único elétron a um composto não radicalar. Estas reações podem ocorrer em processos bioquímicos do sistema imune ou químicos, causando prejuízo às células através da destruição de componentes, como proteínas, lipídios, açúcares e nucleotídeos. Sabe-se que existem compostos que são efetivos contra tais espécies, prevenindo os danos provocados pelo estresse oxidativo. O objetivo deste trabalho foi estudar compostos heterocíclicos que possuam nitrogênio em sua estrutura (azóis), que figuram na literatura como moléculas exemplares de compostos de aplicação farmacológica de amplo espectro. Dentre estes compostos foram analisados os derivados de pirazóis (26 compostos), tiazóis (7 compostos) e 1 isoxazol (3,5-dimetilisoxazol). Estes 34 compostos foram avaliados pela metodologia ORAC (Capacidade de Absorção de Radicais Oxigênio) a fim de determinar e/ou de avaliar seu potencial antioxidante. A escolha do método ORAC se deu pelo fato das moléculas estudadas apresentarem características hidrofílicas e lipofílicas, além de ser um método validado pela literatura, disponível e de ampla aplicação. O método ORAC avalia a capacidade antioxidante da amostra, medindo sua habilidade de proteger a fluoresceína (FL) da oxidação pelo AAPH no meio reacional. O AAPH é um gerador de radicais livres que a 37°C retira hidrogênio do meio, promovendo a redução da fluorescência da fluoresceína em &#955; medido pelo tempo. Seis compostos apresentaram atividade antioxidante de boa à moderada: 3,5-dimetil-1H-pirazol (2.382 &#181;mol eq.Trolox/g); 3-fenil-5-(4-fluorfenil)-1-tiocarbamoil-4,5-diidro-1H-pirazol (6.354 &#181;mol eq.Trolox/g); 3-fenil-5-(2-metoxifenil)-1-tiocarbamoil-4,5-diidro-1H-pirazol (8.739 &#181;mol eq.Trolox/g); 5-(2,4-diclorofenil)-3-fenil-1-tiocarbamoil-4,5-diidro-1H-pirazol (6.022,226 &#181;mol eq.Trolox/g); 2-[5-(4-metoxifenil)-3-fenil-4,5-diidro-1H-pirazol-1-il]-4-feniltiazol (3.135 &#181;mol eq.Trolox/g); e finalmente 2-[5-(3-nitrofenil)-3-fenil-4,5-diidro-1H-pirazol-1-il]-4-feniltiazol (2.700 &#181;mol eq.Trolox/g). Os experimentos com o método ORAC para os azóis estudados apresentaram reprodutibilidade na execução experimental e demonstraram ser uma alternativa viável para estudos de moléculas sintéticas de potencial antioxidante. / Free radicals are chemical species that react rapidly with various compounds and target cells, as they have a very short half life and are highly unstable. The formation of these compounds consists of a continuous, physiological action, which includes essential biological functions and occurs through the loss or addition of a single electron to a non-radical compound. These reactions may occur in biochemical processes of the immune system, or by chemical reactions, causing damage to the cells through the destruction of components such as proteins, lipids, sugars and nucleotides. It is known that compounds exist which are effective against these species, preventing damage caused by oxidative stress. The object of this work was to study heterocyclic compounds that have nitrogen in their structure (azoles), which appear in the literature as exemplary molecules of compounds with a wide spectrum of pharmacological applications. Of these compounds, derivatives of pyrazoles (26 compounds), thiazoles (7 compounds) and 1 isoxazole (3,5-dimethylisoxazole) were analyzed. These 34 compounds were evaluated by the ORAC (Oxygen Radicals Absorption Capacity) in order to determine and/or evaluate its antioxidant potential. The choice of ORAC method is based on the fact that the molecules studied have hydrophilic and lipophilic characteristics, as well as a method validated by the literature, which is available and widely used. The ORAC method evaluates the antioxidant capacity of the sample, measuring its ability to protect the fluorescence (FL) of the oxidation by the AAPH in the reaction medium. AAPH is a generator of free radicals which, at 37°C, removes hydrogen from the medium, promoting the reduction of fluorescence from fluorescein in &#955; measured by time. Six compounds present good to moderate antioxidant activity: 3,5-dimethyl-1H-pyrazole (2.382 &#181;mol eq.Trolox/g); 3-phenyl-5-(4-fluorphenyl)-1-thiocarbamoyl-4,5-dihydro-1H-pyrazole (6.354 &#181;mol eq.Trolox/g); 3-phenyl-5-(2-methoxyphenyl)-1-thiocarbamoyl-4,5-dihydro-1H-pyrazole (8.739 &#181;mol eq.Trolox/g); 5-(2,4-diclorophenyl)-3-phenyl-1-thiocarbamoyl-4,5-dihydro-1H-pyrazole (6.022,226 &#181;mol eq.Trolox/g); 2-[5-(4-methoxyphenyl)-3-phenyl-4,5-dihydro-1H-pyrazole-1-il]-4-phenylthiazole (3.135 &#181;mol eq.Trolox/g); and finally, 2-[5-(3-nitrophenyl)-3-phenyl-4,5-dihydro-1H-pyrazole-1-il]- 4-phenylthiazole (2.700 &#181;mol eq.Trolox/g). Experiments with the ORAC method for the azoles studied present reproducibility in the experimental execution, and have proven to be a viable alternative for studies of synthetic molecules with antioxidant potential.

Submitted by Marco Antônio de Ramos Chagas (mchagas@ufv.br) on 2019-03-01T14:42:06Z No. of bitstreams: 1 texto completo.pdf: 2538890 bytes, checksum: ab3ba217b76cea084c264a925be837ef (MD5) / Made available in DSpace on 2019-03-01T14:42:06Z (GMT). No. of bitstreams: 1 texto completo.pdf: 2538890 bytes, checksum: ab3ba217b76cea084c264a925be837ef (MD5) Previous issue date: 2016-07-15 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Grande parte dos pacientes com doença renal crônica (DRC), em hemodiálise (HD), ingere nutrientes em quantidades abaixo das recomendações ao longo do tratamento. Estudos clínicos têm demonstrado associações entre a ingestão de nutrientes antioxidantes e menor formação de radicais livres, bem como os aspectos relacionados à patogênese da doença, mostrando que os antioxidantes apresentam efeito protetor sobre o desenvolvimento de doenças inflamatórias. O objetivo do presente estudo foi avaliar a capacidade antioxidante total da dieta e sua relação com indicadores antropométricos e marcadores metabólicos e do estresse oxidativo em indivíduos em hemodiálise. Tratou-se de um estudo transversal, de caráter descritivo e analítico, do qual participaram 85 indivíduos (56 homens e 29 mulheres, com média de 62,05+ 13,71 anos) com DRC em HD atendidos em um centro de Nefrologia da Zona da Mata de Minas Gerais. Os dados sócio demográficos (idade, sexo, escolaridade, atividade física, tabagismo, etilismo e adesão à dieta); antropométricos (peso, estatura, índice de massa corporal (IMC), perímetros da cintura (PC) e do quadril (PQ), RCQ, RCE, gordura corporal e visceral e massa muscular); metabólicos (glicose, triglicérides, albumina, ferritina, ureia pré e pós diálise, ferro, creatinina, Kt/V, saturação de transferrina e hemoglobina) foram coletados mediante prontuário. O consumo alimentar foi avaliado por meio do questionário de frequência alimentar, referente ao último ano, a partir do qual foram estimadas as calorias e nutrientes com ênfase naqueles com capacidade antioxidante, bem como foi calculada a capacidade antioxidante total da dieta (CATd) em mmol/g pelo ensaio teórico de FRAP dos alimentos, a partir de bases de dados previamente publicadas. Os nutrientes e compostos com propriedade antioxidantes avaliados foram: ácidos graxos: linoleico e α- linolênico; vitaminas A, C, D e E; minerais selênio, cobre, manganês e zinco, e compostos bioativos: Carotenoides: α-caroteno, β-caroteno, β-criptoxantina, luteína + zeaxantina, licopeno e carotenoides totais; flavonóis: quercitina e quempferol; flavanona hesperedina e antocianidinas: definidina e cianidina). O ajuste energético do consumo de tais nutrientes foi feito pelo método residual. A CATd foi fornecida pelo cálculo do FRAP dos alimentos, enquanto a do soro (CATs) e a enzima superóxido dismutase (SOD) foram avaliadas como biomarcadores séricos de estresse oxidativo por meio de protocolos específicos. Resultados: Foram 85 indivíduos com mediana de idade de 61, variando de 20 a 86 anos, sendo 56 (65,9%) do sexo masculino dos quais 41,2% eram idosos. 65,9% tinham ensino fundamental, 48,2% estavam em tratamento entre 3 e 48 meses e 72,9% seguiam a dieta prescrita pelo serviço, 84,7% dos participantes não fumavam e 88,2% não bebiam. Dos marcadores bioquímicos viu-se que a creatinina e o ferro estavam abaixo dos valores de adequação em todos e a ferritina estava adequada em 78,8%. A glicemia estava em níveis elevados em 66,7% dos participantes e os níveis de triglicérides estavam aceitáveis em 63,5% deles. As concentrações de ureia pré-diálise estavam elevadas em 80% dos participantes e a ureia pós diálise abaixo das recomendações em todos eles. Observou-se 87,1% (n=74) de anêmicos segundo a hemoglobina; e 54,7% estavam com o Kt/V. O grupo de cereais e tubérculos foi o que mais contribuiu para o consumo energético, seguido das bebidas não alcoólicas e frutas. A mediana do consumo energético foi de 46,49 Kcal/kg estando acima da adequação preconizada. Quanto ao estado nutricional, as médias de consumo foram diferentes apenas entre vitamina D, ômega 3, definidina e cianidina. O consumo de vitamina C foi excessivo para todos os participantes e a E foi abaixo assim como dos minerais. Houve associações entre o consumo de vitaminas e indicadores antropométrico de adiposidade central. A CATd não se associou com nenhum dos biomarcadores de estresse oxidativo, porém apresentou correlação positiva com o consumo de ácidos graxos ômega 3 e 6 e vitamina E. Não houve correlação entre a CATs e a CATd, mas ambas se correlacionaram positivamente com o consumo de ácidos graxos ômega 3 e 6. Conclusão: De modo geral, a maioria dos pacientes apresentava-se em bom estado nutricional de acordo com índice de massa corporal, destacando-se um número maior de mulheres com sobrepeso quando comparadas com os homens. Os marcadores bioquímicos não se relacionaram de forma expressiva com as demais variáveis, o consumo de antioxidantes estava baixo, sobretudo, o de carotenoides e fenólicos. Dos biomarcadores de estresse oxidativo, destacou- se relação entre o MDA e a SOD, o percentual de gordura corporal que era maior entre os pacientes com menor MDA e o consumo dos ácidos graxos ter sido positivamente correlacionado à CATd e CATs. Sendo estes últimos, os resultados que exigem maiores esclarecimentos frente às vias metabólicas ainda desconhecidas, já que uma limitação de nosso estudo é seu desenho transversal, que não nos permite tirar conclusões em termos de causalidade. / Most patients with chronic kidney disease (CKD) on hemodialysis (HD), ingests nutrients in amounts below the recommended throughout the treatment. Clinical studies have demonstrated associations between the intake of nutrients and antioxidants decreased formation of free radicals, as well as aspects related to the pathogenesis of the disease, showing that antioxidants have a protective effect on the development of inflammatory diseases. The aim of this study was to evaluate the total antioxidant capacity of the diet and its relationship with anthropometric indicators and metabolic markers and oxidative stress in hemodialysis subjects. This was a cross-sectional, descriptive and analytical character, which was attended by 85 individuals (56 men and 29 women, mean 62,05+ 13.71 years) with CKD on HD treated in a center zone of Nephrology da Mata of Minas Gerais. Demographic partner data (age, gender, education, physical activity, smoking, alcohol consumption and adherence to diet); anthropometric (weight, height, body mass index (BMI), waist perimeters (PC) and hip (PQ), WHR, RCE, body and visceral fat and muscle mass); metabolic (glucose, triglyceride, albumin, ferritin, pre and post dialysis urea, iron, creatinine, Kt/V, transferrin saturation and hemoglobin) were collected through medical records. Dietary intake was assessed by food frequency questionnaire, for the last year, from which were estimated calories and nutrients with emphasis on those with antioxidant capacity, and was calculated to total dietary antioxidant capacity (CATd) in mmol / g the theoretical FRAP test food from previously published databases. The nutrients and compounds with antioxidant property evaluated were: fatty acid: linoleic and α-linolenic acid; vitamins A, C, D and E; minerals selenium, copper, manganese and zinc, and bioactive compounds: Carotenoids: α-carotene, β-carotene, β-cryptoxanthin, lutein + zeaxanthin, lycopene, and carotenoids; flavonols: quercetin and kaempferol; flavanone hesperedina and anthocyanidins: definidina and cyanidin). The energy adjustment of consumption of these nutrients was done by the residual method. The CATd was provided by calculating the FRAP food, while serum (CAT) and superoxide dismutase (SOD) were evaluated as serum biomarkers of oxidative stress through specific protocols. Results: There were 85 patients with a median age of 61, ranging from 20 to 86 years, 56 (65.9%) males of which 41.2% were elderly. 65.9% had primary education, 48.2% were treated between 3 and 48 months, and 72.9% followed the prescribed diet for the service, 84.7% of participants did not smoke and 88.2% did not drink. Of biochemical markers was seen that creatinine and iron were below the suitability values in all and ferritin was adequate in 78.8%. Blood glucose was elevated in 66.7% of participants and triglyceride levels were acceptable in 63.5% of them. Pre dialysis urea concentrations were elevated in 80% of participants and the post dialysis urea below the recommendations in all. 87.1% was observed (n = 74) according to anemic hemoglobin; and 54.7% were with Kt/V. The group of cereals and tubers was the largest contributor to energy consumption, followed by soft drinks and fruit. The median energy consumption was 46.49 Kcal / kg which is above the recommended adjustment. Regarding nutritional status, average consumption showed significant differences between vitamin D, omega 3, definidina and cyanidin. The intake of vitamin C was excessive for all participants and E was below as well as minerals. There were associations between vitamin intake and anthropometric indicators of central adiposity. The CATd not associated with any of the biomarkers of oxidative stress, but was positively correlated with the consumption of omega 3 fatty acids and 6 and vitamin E. There was no correlation between the CATs and CATd, but both were positively correlated with the consumption of omega 3 and 6. Conclusion: in general, most of the patients were in good nutritional status according to body mass index, highlighting a greater number of overweight women compared with men. Biochemical markers were not related in a significant way with the other variables, consumption of antioxidants was low, particularly the carotenoids and phenolics. Of oxidative stress biomarkers, stood out relationship between MDA and SOD, the percentage of body fat was higher among patients with lower MDA and consumption of fatty acids have been positively correlated with CATd and CATs. The latter being the results that require further clarification front of the metabolic pathways are still unknown, as a limitation of our study is its cross-sectional design, which does not allow us to draw conclusions in terms of causality.

Orientador: Iguatemy Lourenço Brunetti / Banca: Cleverton Roberto de Andrade / Banca: Rosemeire Cristina Linhari Rodrigues Pietro / Resumo: As infecções do trato urinário (ITU) são afecções comuns, que podem resultar em níveis significantes de morbidade. Os agentes etiológicos associados a ITU são bactérias Gram-negativas e Gram-positivas, além do fungo comensal Candida albicans. Além dos parâmetros clássicos associados à infecção, as ITU proporcionam o aumento na formação de espécies reativas de oxigênio e nitrogênio, o que também acarreta injúria tecidual, bem como, também ocorre um alto índice de micro-organismos resistentes aos fármacos disponíveis na clínica médica, o que demanda na prospecção por novas abordagens terapêuticas complementares. No Brasil, a espécie Eugenia brasiliensis Lam. é conhecida como "berry brasileira" ou grumixama, e existem poucos relatos na literatura demonstrando sua atividade biológica. Este fator associados à escassez de estudos sobre a espécie brasileira E. brasiliensis, despertou o interesse em novos estudos relacionados à sua ação sobre microrganismos relacionados a ITU e atividade antioxidante. Os extratos utilizados neste estudo foram obtidos a partir da polpa do fruto liofilizado de E. brasiliensis com diferentes solventes, com objetivo de obter dados de sua composição fitoquímica, avaliar suas atividades antioxidantes, antimicrobianas e citotóxicas. Foram utilizados como padrões para os ensaios de atividade antioxidante o ácido elágico, quercetina, ácido gálico, ácido cafeico e trolox. Na caracterização química dos extratos foram determinados, os teores de flavonoides totai... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The urinary tract infections (UTI) are common disorders, which may result in significant levels of morbidity. The etiological agents associated with UTI are Gram-negative and Gram-positive, plus commensal Candida albicans fungus. In addition to the classics associated with infection parameters, the ITU provide increased formation of reactive oxygen and nitrogen species, which also causes tissue damage, as well as a high rate of resistant microorganisms to drugs available in clinical practice, which requires the prospecting for new medicinal approaches. In Brazil, the specie Eugenia brasiliensis Lam. are known as "brazilian berry" or grumixama, and there are few reports in the literature demonstrating your biologic activity. This factor associated with the lack of studies on the brazilian specie E .brasiliensis stir up interest in new research related to the activities of microorganisms related to ITU and antioxidant activity. The extracts used in this study were obtained from the fruit pulp lyophilized of E. brasiliensis with different solvents in order to get data from their phytochemical composition, evaluate their antioxidant activity, antimicrobial and cytotoxicity. For the evaluation of antioxidant activity it was used like standards acid elagic, quercetina, acid gallic, acid cafeico and trolox. In the chemical characterization of the extracts were determined, the contents of total flavonoids... (Complete abstract click electronic access below) / Mestre

Made available in DSpace on 2014-06-11T19:31:03Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-04-09Bitstream added on 2014-06-13T18:41:34Z : No. of bitstreams: 1 delre_pv_dr_sjrp.pdf: 530812 bytes, checksum: 6365c47fdc6445a097bf40dcb11e8b0a (MD5) / O presente trabalho teve como objetivo avaliar a atividade antioxidante de extratos de especiarias, na forma de oleoresinas, aplicados em óleo de soja e sua repercussão sobre o perfil lipídico in vivo. O trabalho experimental foi dividido em quatro ensaios. No Ensaio I, a atividade antioxidante das oleoresinas de orégano, manjericão e tomilho, em diferentes concentrações, foi avaliada no óleo de soja por meio da estabilidade oxidativa. As duas oleoresinas mais efetivas, orégano e tomilho, foram selecionadas na concentração de 3.000 mg.kg-1 para serem submetidas aos ensaios posteriores. Nos Ensaios II e III, o óleo de soja adicionado das oleoresinas de orégano e tomilho ou da mistura das mesmas foi submetido à termoxidação (180ºC/30 horas) e ao teste acelerado em estufa (60ºC/10 dias), cujas amostras foram tomadas nos intervalos de tempo de 0, 5, 10, 15, 20, 25 e 30 horas e 0, 2, 4, 6, 8 e 10 dias, respectivamente. As amostras termoxidadas foram analisadas quanto à estabilidade oxidativa, índice de peróxidos, dienos conjugados, compostos polares totais e perfil de ácidos graxos, enquanto que as amostras do teste acelerado em estufa foram analisadas quanto ao índice de peróxidos e dienos conjugados. No Ensaio IV a atividade antioxidante e hipolipemiante das oleoresinas foram avaliadas in vivo, utilizando ratos Wistar alimentados com dieta hipercolesterolêmica e suplementação das oleoresinas por gavagem (16 mg.kg-1). No sangue coletado após o experimento foram realizadas análises de perfil lipídico (colesterol total e frações, triglicérides) e oxidação da LDL. A partir dos resultados obtidos, verificou-se que em elevada temperatura, termoxidação, as oleoresinas aplicadas de forma isolada apresentaram um poder antioxidante maior que o antioxidante TBHQ, entretanto, a mistura das concentrações, promoveu maior estabilidade ao óleo e menor formação... / This study aims at evaluating the antioxidant activity of spice extracts, in form of oleoresins, applied in soybean oil and its repercussion on the lipid profile in vivo. The experiment was divided into four tests. Test I evaluated the antioxidant activity of oleoresins oregano, basil and thyme, in different concentrations, in soybean oil by means of oxidative stability determination. The most effective oleoresins, oregano and thyme, were selected in a concentration of 3.000 mg.kg-1 and subjected to the subsequent tests. In the Tests II and III, the soybean oil containing either oregano and thyme oleoresins or the mixture of both, was subjected to thermal oxidation (180ºC/30 hours) and to the Schaal oven test (60°C/10 days). The samples were taken in time intervals of 0, 5, 10, 15, 20, 25 and 30 hours and 0, 2, 4, 6, 8 and 10 days, respectively. The thermal oxidated samples were analyzed concerning their oxidative stability, peroxide value, conjugated dienes, total polar compounds and fatty acids profiles, while the samples from the Schaal oven test were analyzed concerning their peroxide value and conjugated dienes. Test IV evaluated the antioxidant and hypolipemiant activities of oleoresins in vivo using Wistar rats that were fed in a hypercholesterolemic diet and oleoresin supplementation by gavage (16 mg.kg-1). The blood collected after the experiment had its lipid profile (total cholesterol and fractions, triglycerides) and LDL oxidation analyzed. Considering the results obtained, it was possible to verify that the oleoresins applied isolatedly presented an antioxidant power greater than the TBHQ antioxidant in high temperatures, thermal oxidation. However, the mixture of concentrations gave more stability to the oil and reduced the formation of polar compounds. It also reduced the formation of saturated fatty acids in detriment of mono and polyunsaturated fatty acids... (Complete abstract click electronic access below)