Ein hoher Verzehr von Obst und Gemüse scheint das Risiko der Inzidenz verschiedener Erkrankungen zu reduzieren. Es wird vermutet, dass eine Gruppe sekundärer Pflanzeninhaltsstoffe, die Flavonoide, hierfür verantwortlich sind. Mögliche Effekte auf die intestinale Barrierefunktion dieser Substanzklasse sind jedoch weitgehend ungeklärt. Parazelluläre Eigenschaften epithelialer Zellen werden hauptsächlich durch die Zell-Zell-Kontakte der Tight Junction (TJ) insbesondere durch die Proteine Occludin und die Claudine definiert. Ziel dieser Arbeit war es, die Effekte des am häufigsten vorkommenden Flavonoids Quercetin auf die Barrierefunktion der Kolonkarzinom-Zelllinie Caco-2 zu untersuchen. Hierbei zeigte sich, dass Quercetin konzentrationsabhängig (50-200 µM) den transepithelialen Widerstand erhöhte. Die Wirkung von 200 µM Quercetin war bereits nach 4 h Inkubation erkennbar und erreichte nach 48 h maximale Werte. Der Wirkverlust, welcher nach 72 h Inkubation eintrat, konnte durch eine tägliche Gabe des Flavonoids verhindert werden. Weiterhin zeigte sich, dass der Quercetin-induzierte Widerstandsanstieg durch mukosale oder serosale Zugabe gleichermaßen auslösbar war. Western Blot-Analysen der TJ-Proteine Occludin, Claudin-1, -3, -4 und -7 ergaben, dass der durch Quercetin-induzierte Widerstandsanstieg insbesondere mit einer Zunahme der Expression des abdichtenden TJ-Proteins Claudin-4 einherging. Quercetin erhöhte ebenfalls die mRNA-Expression von Claudin-4 (quantitative RT-PCR) und bewirkte eine Aktivierung des Claudin-4-Promotors (Luciferase-Reportergen-Analysen). Mittels Immunfluoreszenz-Färbungen und Laserscanning-Mikroskopie konnte ein vermehrter Einbau von Claudin-4 in die TJ nachgewiesen werden. Funktionelle Untersuchungen mittels radioaktiven Fluxmessungen zeigten, dass das Flavonoid die parazelluläre Permeabilität für Natrium und Chlorid reduzierte, aber die Durchlässigkeit von Mannitol als parazellulärer Marker unverändert blieb. Wir konnten hiermit erstmals nachweisen, dass Quercetin die Expression des abdichtenden TJ-Proteins Claudin-4 in den TJ-Komplex verstärkte, wodurch die Ionen-Durchlässigkeit für Natrium und Chlorid vermindert wurde. Das führte zu einer Abdichtung der intestinalen Barriere. Dieser direkte Effekte von Quercetin könnte eine neue Möglichkeit für die Behandlung oder Prävention von Diarrhöe-bedingten intestinalen Barrieredefekten darstellen. / High dietary intake of fruits and vegetables is associated with a reduced disease risk. A group of secondary plant compounds, the flavonoids, are supposed to be important in this respect, but there is still limited information about their effects on intestinal barrier function. Paracellular properties of epithelial cells are defined for the most part by the tight junctional complex with the corresponding tight junction (TJ) proteins occludin and the claudin gene family. Therefore, the aim of our study was to elucidate the effects of quercetin, a common flavonoid, on the barrier function of the colonic epithelial cell line Caco-2. Addition of quercetin to the Caco-2 monolayer applied to the mucosal and serosal culture medium increased transepithelial resistance in a concentration-dependent manner (50-200 µM). The effect of 200 µM quercetin was already observable after 4 hours and reached maximal levels after 48 hours. The loss of action after 72 hours was blocked by a daily addition of the flavonoid. The effect of quercetin was not different after mucosal or serosal addition. Western blot analysis of occludin, claudin-1, -3, -4, and -7 revealed that the resistance rise was associated specifically with an elevated expression of the barrier-sealing TJ protein claudin-4. The mRNA expression and the promotor activity of claudin-4 were found increased by the flavonoid using quantitative RT-PCR and luciferase reporter gene assays. Immunofluorescent staining analyzed by confocal laser scanning microscopy primarily revealed a strong increase of claudin-4, localized within the TJ as well as in subjunctional regions. Radioactive tracer fluxes revealed a reduced paracellular permeability of sodium and chloride by quercetin, whereas the permeability of the uncharged solute mannitol was not altered. We demonstrated that the flavonoid quercetin increases the expression of claudin-4 within the tight junctional complex, which caused a decrease of the paracellular permeability for sodium and chloride. This leads to a sealing of the intestinal barrier for ions. Thus, this novel direct effect of quercetin may be utilized for the treatment or prevention of diarrhea-causing intestinal barrier defects in inflammatory bowel diseases.

Transcription factors (TFs) are ubiquitous gene expression regulators and play essential roles in almost all biological processes. This Ph.D. project is primarily focused on the functional characterisation of MYB112 - a member of the R2R3-MYB TF family from the model plant Arabidopsis thaliana. This gene was selected due to its increased expression during senescence based on previous qRT-PCR expression profiling experiments of 1880 TFs in Arabidopsis leaves at three developmental stages (15 mm leaf, 30 mm leaf and 20% yellowing leaf). MYB112 promoter GUS fusion lines were generated to further investigate the expression pattern of MYB112. Employing transgenic approaches in combination with metabolomics and transcriptomics we demonstrate that MYB112 exerts a major role in regulation of plant flavonoid metabolism. We report enhanced and impaired anthocyanin accumulation in MYB112 overexpressors and MYB112-deficient mutants, respectively. Expression profiling reveals that MYB112 acts as a positive regulator of the transcription factor PAP1 leading to increased anthocyanin biosynthesis, and as a negative regulator of MYB12 and MYB111, which both control flavonol biosynthesis. We also identify MYB112 early responsive genes using a combination of several approaches. These include gene expression profiling (Affymetrix ATH1 micro-arrays and qRT-PCR) and transactivation assays in leaf mesophyll cell protoplasts. We show that MYB112 binds to an 8-bp DNA fragment containing the core sequence (A/T/G)(A/C)CC(A/T)(A/G/T)(A/C)(T/C). By electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation coupled to qPCR (ChIP-qPCR) we demonstrate that MYB112 binds in vitro and in vivo to MYB7 and MYB32 promoters revealing them as direct downstream target genes. MYB TFs were previously reported to play an important role in controlling flavonoid biosynthesis in plants. Many factors acting upstream of the anthocyanin biosynthesis pathway show enhanced expression levels during nitrogen limitation, or elevated sucrose content. In addition to the mentioned conditions, other environmental parameters including salinity or high light stress may trigger anthocyanin accumulation. In contrast to several other MYB TFs affecting anthocyanin biosynthesis pathway genes, MYB112 expression is not controlled by nitrogen limitation, or carbon excess, but rather is stimulated by salinity and high light stress. Thus, MYB112 constitutes a previously uncharacterised regulatory factor that modifies anthocyanin accumulation under conditions of abiotic stress. / Transkriptionsfaktoren (TFs) sind ubiquitäre Regulatoren der Genexpression und spielen eine essentielle Rolle in nahezu allen biologischen Prozessen. Diese Doktorarbeit hat vor allem die funktionelle Charakterisierung von MYB112 zum Thema, einem Mitglied der R2R3-MYB-TF-Familie aus der Modellpflanze Arabidopsis thaliana. Ausgesucht wurde das Gen aufgrund seiner erhöhten Expression in seneszenten Blättern, basierend auf vorangegangenen qRT-PCR Expressions-Profiling Experimenten für 1880 TFs in Arabidopsis Blättern aus drei Entwicklungsstadien (15 mm Blatt, 30 mm Blatt und 20 % vergilbtes Blatt). MYB112-Promotor-GUS-Fusionslinien wurden generiert um das Expressionsmuster von MYB112 detailliert zu untersuchen. Unter Zuhilfenahme transgener Ansätze in Kombination mit Metabolomics und Transcriptomics können wir zeigen, dass MYB112 eine wichtige Rolle in der Regulation des pflanzlichen Flavonoid-Metabolismus spielt. In MYB112 Überexpressoren und MYB112-defizienten Mutanten kommt es zu erhöhter bzw. verminderter Anthocyanin-Akkumulation. Expressions-Profiling zeigt, dass MYB112 einerseits als ein positiver Regulator des Transkriptionsfaktors PAP1 fungiert, was zu einer erhöhten Anthocyanin-Biosynthese führt, andererseits als negativer Regulator von MYB12 und MYB111 auftritt, welche beide die Flavonol-Biosynthese kontrollieren. Wir haben früh auf MYB112 reagierende Gene durch eine Kombination verschiedener Ansätze identifiziert. Diese umfassen Genexpressions-Profiling (Affymetrix ATH1 Microarrays und qRT-PCR) und Transaktivierungs-Experimente in Mesophyll-Protoplasten aus Blättern. Wir zeigen, dass MYB112 an eine 8-bp DNA-Fragment, welches die Kernsequenz (A/T/G)(A/C)CC(A/T)(A/G/T)(A/C)(T/C) aufweist. Mit Hilfe von Electrophoretic Mobility Shift Assay (EMSA) und Chromatin-Immunopräzipitation gekoppelt mit qPCR (ChIP-qPCR) zeigen wir, dass MYB112 in vitro und in vivo an die Promotoren von MYB7 und MYB32 bindet was sie damit als direkte Zielgene von MYB112 identifiziert. Es wurde bereits gezeigt, dass MYB TFs eine wichtige Rolle bei der Kontrolle der Flavonoid-Biosynthese in Pflanzen haben. Viele Faktoren, die oberhalb des Anthocyanin-Biosyntheseweges agieren, werden bei Stickstofflimitierung oder erhöhter Saccharose-Konzentration auch verstärkt exprimiert. Außer den erwähnten Bedingungen können auch andere Umweltparameter, wie z. B. erhöhter Salzgehalt und Starklicht zu erhöhter Expression führen. Im Gegensatz jedoch zu einigen anderen MYB TFs, die einen Einfluss auf Gene des Anthocyanin-Biosyntheseweges ausüben, ist die Expression von MYB112 nicht durch Stickstoff-Limitierung oder Kohlenstoffüberfluss kontrolliert, sondern wird durch erhöhten Salzgehalt sowie Starklicht stimuliert. Somit ist MYB112 ein neuer Regulator, der eine Anthocyanin-Akkumulation unter abiotischen Stressbedingungen kontrolliert.

Highbush blueberry (Vaccinium corymbosum L.) has one of the highest antioxidant capacities and flavonoid concentrations of any fruit or vegetable, and regular consumption of blueberries has been connected to a wide range of health benefits. A diversity of flavonoids (flavonols, anthocyanins, proanthocyanidins) are likely responsible for many of the health benefits, and these compounds also significantly contribute to the organoleptic properties of ripe blueberries. Despite the potential importance of these flavonoids in diet, there has been little investigation into the molecular genetics of blueberry flavonoid biosynthesis. Therefore, I developed a real-time quantitative PCR protocol to monitor expression of flavonoid genes throughout development and ripening. Following evaluation of five reference genes, expression profiling of biosynthetic genes revealed that flavonoid synthesis is tightly controlled at the transcriptional level in a biphasic developmental pattern. These results are discussed in relation to flavonoid metabolite accumulation profiles, which were produced as part of a collaboration. Finally, in conjunction with a second group of collaborating scientists, some promising preliminary evidence is provided suggesting that the hormone abscisic acid might have a role in regulating ripening initiation in blueberry. / Graduate

Este trabalho descreve o estudo quÃmico de Croton muscicarpa (Euphorbiaceae), planta nativa do Nordeste do Brasil, popularmente conhecida como âvelame-de-cheiroâ. A espÃcie foi coletada na Chapada Diamantina no estado da Bahia, e sua investigaÃÃo quÃmica foi realizada atravÃs do isolamento e caracterizaÃÃo de metabÃlitos secundÃrios presentes nos extratos etanÃlico dos talos e hexÃnico das raÃzes. Foram isolados dos talos os sesquiterpenos, espatulenol e 6-metÃxi-cipereno, este Ãltimo de descriÃÃo inÃdita na literatura. Das raÃzes foram isolados o triterpeno, Ãcido acetil aleuritÃlico (AAA) e uma polimetoxiflavona, a 5-hidroxi-3,6,7,3â,4â-pentametoxiflavona (artemetina). O isolamento dos constituintes quÃmicos foi realizado por mÃtodos cromatogrÃficos convencionais e a determinaÃÃo estrutural das substÃncias isoladas realizada atravÃs do uso de tÃcnicas espectromÃtricas como: Infravermelho (IV), Espectrometria de Massa (EM) e RessonÃncia MagnÃtica Nuclear de HidrogÃnio (RMN 1H) e Carbono-13 (RMN 13C), incluindo tÃcnicas bidimensionais (COSY, HSQC, HMBC e NOESY) e comparaÃÃo com os dados descritos na literatura. / Este trabalho descreve o estudo quÃmico de Croton muscicarpa (Euphorbiaceae), planta nativa do Nordeste do Brasil, popularmente conhecida como âvelame-de-cheiroâ. A espÃcie foi coletada na Chapada Diamantina no estado da Bahia, e sua investigaÃÃo quÃmica foi realizada atravÃs do isolamento e caracterizaÃÃo de metabÃlitos secundÃrios presentes nos extratos etanÃlico dos talos e hexÃnico das raÃzes. Foram isolados dos talos os sesquiterpenos, espatulenol e 6-metÃxi-cipereno, este Ãltimo de descriÃÃo inÃdita na literatura. Das raÃzes foram isolados o triterpeno, Ãcido acetil aleuritÃlico (AAA) e uma polimetoxiflavona, a 5-hidroxi-3,6,7,3â,4â-pentametoxiflavona (artemetina). O isolamento dos constituintes quÃmicos foi realizado por mÃtodos cromatogrÃficos convencionais e a determinaÃÃo estrutural das substÃncias isoladas realizada atravÃs do uso de tÃcnicas espectromÃtricas como: Infravermelho (IV), Espectrometria de Massa (EM) e RessonÃncia MagnÃtica Nuclear de HidrogÃnio (RMN 1H) e Carbono-13 (RMN 13C), incluindo tÃcnicas bidimensionais (COSY, HSQC, HMBC e NOESY) e comparaÃÃo com os dados descritos na literatura.

The plant phenylpropanoid pathway is initiated from deamination of phenylalanine to form cinnamic acid followed by hydroxylation and methylation of the aromatic ring to generate a variety of phenolic compounds including lignin monomers, flavonoid compounds and sinapate esters. The incorporation of phenylpropanoid metabolism served as a key step in the early land-colonization of plants from aqueous environment since phenolic compounds play important roles in plant development and abiotic/biotic stress responses. Lignin is a heteropolymer of hydroxycinnamyl alcohols that are derived from the major branch of plant phenylpropanoid pathway. The main function of lignin is to enhance the strength of plant cell wall and waterproof the vascular system for long-distance transportation of water and solutes. In addition, lignin is also involved in protecting plants against pathogen attack. My Ph.D. research is to investigate how lignin biosynthesis contributes to plant immunity. The results showed that the expression of major lignin biosynthetic genes was induced upon host fungal pathogen infection. Moreover, a mutant disrupted in the lignin gene F5H1 showed enhanced susceptibility when challenged with several fungal pathogens. F5H1 encodes a ferulic acid 5-hydroxylase that is uniquely present in angiosperm plants, leading to the biosynthesis of syringyl lignin monomer, which is not present in gymnosperm plants. Subsequent research demonstrated that f5h1 mutation impaired the penetration (pre-invasion) resistance but did not impact post-invasion resistance. Furthermore, the pathogen-induced expression of lignin genes was independent of well-characterized defensive signaling pathways, and regulated by a novel regulating mechanism. F5H1 contributes to pmr2-mediated resistance but acts independently of other molecular components of penetration resistance including PEN1, PEN2, and PEN3. In contrast to f5h1, a knockout mutant of flavonoid pathway gene chalcone isomerase (CHI/TT5) showed enhanced resistance to host anthracnose pathogen Colletotrichum higginsianum in a salicylic acid (SA)-dependent manner. Taken together, our results for the first time provide genetic evidence demonstrating that lignin biosynthetic gene F5H1 plays critical roles in plant penetration resistance and that an uncharted pathway in flavonoid metabolism confers an SA-dependent resistance pathway in Arabidopsis.

Submitted by Marcos Anselmo (marcos.anselmo@unipampa.edu.br) on 2017-06-05T14:11:26Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) MARCIA ROSULA POETINI SILVA.pdf: 1891478 bytes, checksum: e4874e22f3dbc89ee3033d4701e4a8a2 (MD5) / Approved for entry into archive by Marcos Anselmo (marcos.anselmo@unipampa.edu.br) on 2017-06-05T14:11:58Z (GMT) No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) MARCIA ROSULA POETINI SILVA.pdf: 1891478 bytes, checksum: e4874e22f3dbc89ee3033d4701e4a8a2 (MD5) / Made available in DSpace on 2017-06-05T14:11:58Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) MARCIA ROSULA POETINI SILVA.pdf: 1891478 bytes, checksum: e4874e22f3dbc89ee3033d4701e4a8a2 (MD5) Previous issue date: 2017-02-21 / Os flavonoides são compostos naturais presentes em diversos tipos de plantas, são quimicamente classificados como polifenóis e geralmente encontrados em sua forma livre ou glicosilada. A Hesperidina (Hsd) é um flavonoide cítrico, encontrado principalmente em frutas cítricas como a laranja e o limão, classificado como flavanona glicosídica e possui propriedades antioxidantes, anti-inflamatórias, anticancerígenas, antivirais, hipolipidêmica, entre outras. O ferro (Fe) é um nutriente fundamental para todas as células vivas, mas em excesso pode ser tóxico, por causar danos oxidativos através da formação de radicais livres, como a reação de fenton, os quais resultam em fenômenos de estresse oxidativo. Uma desregulação no metabolismo do Fe está associado com dano celular e também a doenças neurodegenerativas como a doença de Parkinson e Alzheimer. O objetivo deste estudo foi avaliar o efeito neuroprotetor da hesperidina na exposição aguda ao ferro na forma de sulfato ferroso em Drosophila melanogaster adultas macho. As moscas foram divididas em quatro grupos: 1) controle, 2) hesperidina (10 μM), 3) Fe (20 mM) na forma de sulfato ferroso, 4) hesperidina (10 μM) + Fe (20 mM). As moscas foram concomitantemente expostas ao Fe e Hsd na dieta em papel filtro (dissolvidos em sacarose 1 %) por 48 horas, de acordo com seus respectivos grupos. Para as análises in vivo foram avalizadas a sobrevivência e os comportamentos (testes como geotaxia negativa, campo aberto e base/topo) e ex vivo a atividade da acetilcolinesterase (AChE) na cabeça e no corpo, viabilidade celular e mitocondrial e determinação dos níveis de dopamina na cabeça das moscas. A atividade da catalase (CAT), superóxido dismutase (SOD) e glutationa S-transferase (GST), os níveis de peroxidação lipídica (TBARS), níveis de espécies reativas (ER), o teor de tióis não protéicos (NPSH), os tióis totais e os níveis de ferro na cabeça e no corpo de Drosophila melanogaster também foram avaliados. A exposição ao Fe aumentou significativamente a mortalidade das moscas, enquanto que as sobreviventes apresentaram déficit locomotor significativo com atividade aumentada de AChE. No entanto, a suplementação dietética com Hsd causou uma diminuição significativa na mortalidade, melhora da atividade locomotora e restauração da atividade da AChE em moscas expostas ao Fe. O metal também causou decréscimo nos níveis de tióis totais e não proteicos e na atividade das enzimas SOD, CAT e GST, acompanhadas com aumento significativo na geração de ER e TBARS, assim como aumento nos níveis de Fe na cabeça e no corpo e redução nos níveis de dopamina na cabeça das moscas expostas ao elemento. Efeitos esses prevenidos pela hesperidina. A hesperidina apresentou o potencial antioxidante e amenizou o efeito causado pela exposição aguda ao Fe em Drosophila melanogaster. / Flavonoids are natural compounds present in many plant types, are chemically classified as polyphenols and are generally found in their free or glycosylated form. Hesperidin (Hsd) is a citrus flavonoid, found mainly in citrus fruits such as orange and lemon, classified as flavanone glycosidic and has antioxidant, anti-inflammatory, anticancer, antiviral, hypolipidemic properties, among others. Iron (Fe) is a fundamental nutrient for all living cells, but in excess it can be toxic because it causes oxidative damage through the formation of free radicals, such as fenton reaction, which result in phenomena of oxidative stress. Deregulation in the metabolism of Fe is associated with cellular damage and also neurodegenerative diseases such as Parkinson's disease and Alzheimer's. The objective of this study was to evaluate the neuroprotective effect of hesperidin on acute exposure to iron in the form of ferrous sulfate in adult male Drosophila melanogaster. The flies were divided into four groups: 1) control, 2) hesperidin (10 μM), 3) Fe (20 mM) as ferrous sulfate, 4) hesperidin (10 μM) + Fe (20 mM). Flies were concomitantly exposed to Fe and Hsd in the diet on filter paper (dissolved in 1% sucrose) for 48 hours, according to their respective groups. Survival and behaviors (tests such as negative geotag, open field and base / top) and ex vivo acetylcholinesterase (AChE) activity in head and body, cell and mitochondrial viability and determination of dopamine on the head of flies. The activity of catalase (CAT), superoxide dismutase (SOD) and glutathione Stransferase (GST), levels of lipid peroxidation (TBARS), reactive species levels (RS), nonprotein thiol content (NPSH), total thiols and iron levels in the head and body of Drosophila melanogaster were also evaluated. Exposure to Fe significantly increased fly mortality, while survivors had significant locomotor deficits with increased AChE activity. However, dietary supplementation with Hsd caused a significant decrease in mortality, improvement of locomotor activity and restoration of AChE activity in Fe-exposed flies. The metal also caused a decrease in the levels of total and non-protein thiols and in the activity of SOD enzymes , CAT and GST, accompanied with a significant increase in RS and TBARS generation, as well as increase in Fe levels in the head and body and reduction in dopamine levels in the head of the flies exposed to the element. These effects prevented by hesperidin. Hesperidin presented the antioxidant potential and attenuated the effect caused by the acute exposure to Fe in Drosophila melanogaster.

A exposição crônica à radiação solar pode contribuir para o aparecimento do câncer de pele, sendo o uso de fotoprotetores um fator primordial na prevenção desses efeitos deletérios. Atualmente, substâncias bioativas tais como a rutina têm sido foco de interesse da comunidade científica graças às suas propriedades fotoprotetoras e antioxidantes, que podem promover aumento dos valores de FPS, além de conferir características multifuncionais às formulações. Achados in vitro recentes indicam que a rutina, quando incorporada em emulsões fotoprotetoras óleo em água, promove aumento da atividade antioxidante e aumento do FPS. No entanto, a realização de estudos clínicos é fundamental para confirmar e quantificar esses resultados, já que a metodologia in vitro possui baixa repetibilidade e ausência de correlação com ensaios in vivo, principalmente quando as formulações analisadas apresentam substâncias antioxidantes em sua composição. O objetivo deste estudo foi avaliar pela primeira vez a atividade da rutina frente ao FPS e sua segurança clínica através da comparação de formulações fotoprotetoras contendo rutina 0.1% (w/w), avobenzona 3.0% (w/w) e octil dimetil PABA 8.0% (w/w) com uma preparação similar sem o composto bioativo. Adicionalmente, hidratação cutânea, FPS in vitro e atividade antioxidante da rutina em associação com outros filtros foram investigados. O perfil de segurança das formulações qualificou as fórmulas para os testes de eficácia clínica. O teste de DPPH confirmou a capacidade antioxidante da rutina, demonstrando cerca de 40% de aumento na capacidade de sequestro de radicais livres na presença do composto bioativo. A rutina em combinação com os filtros UV aumentou o FPS clínico de 7.30 ± 0.60 para 12.37 ± 1.13, o que representa cerca de 70% de aumento. Os resultados encontrados provam que a rutina em combinação com outros filtros pode aumentar significativamente o valor do FPS e que a mesma é segura para uso clínico. / Unprotected chronic exposure to solar radiation can contribute to premature skin cancer and sunscreens are a key factor to avoid those detrimental effects. Currently, there is a growing interest in the photoprotector and antioxidant potential of bioactive substances, such as rutin, that could help to increase the SPF value and add multifunctional characteristics to the formulations. Recent in vitro findings indicated that rutin, when incorporated in oil-in-water photoprotective emulsions can provide antioxidant activity and SPF increase. However, clinical studies are fundamental to determine this activity duo to in vitro methodology lack of repeatability and correlation between the in vivo data, especially when the analyzed formulas contain antioxidant substances. The aim of this study was to evaluate for the first time to date the rutin in vivo SPF and clinical safety by comparing sunscreens formulations containing rutin 0.1% (w/w), butyl methoxydibenzoylmethane 3.0% (w/w) and octyl dimethylPABA 8.0% (w/w) with a similar bioactive-free preparation. Additionally, skin hydration, in vitro SPF and in vitro antioxidant activity of rutin, in association with the UV filters were investigated. The safety profile of the formulations under sun-exposed skin conditions qualified the formulas for clinical efficacy assays. DPPH test confirmed rutin antioxidant properties, demonstrating about 40% increase in radical scavenging potential when the bioactive compound was present. Rutin in combination with the UV filters increased the clinical SPF from 7.30 ± 0.60 to 12.37 ± 1.13, representing about 70% growth in the SPF value. The results obtained proved that rutin in combination with UV filters can improve the SPF value significantly and is safe for clinical use.

A exposição crônica à radiação solar pode contribuir para o aparecimento do câncer de pele, sendo o uso de fotoprotetores um fator primordial na prevenção desses efeitos deletérios. Atualmente, substâncias bioativas tais como a rutina têm sido foco de interesse da comunidade científica graças às suas propriedades fotoprotetoras e antioxidantes, que podem promover aumento dos valores de FPS, além de conferir características multifuncionais às formulações. Achados in vitro recentes indicam que a rutina, quando incorporada em emulsões fotoprotetoras óleo em água, promove aumento da atividade antioxidante e aumento do FPS. No entanto, a realização de estudos clínicos é fundamental para confirmar e quantificar esses resultados, já que a metodologia in vitro possui baixa repetibilidade e ausência de correlação com ensaios in vivo, principalmente quando as formulações analisadas apresentam substâncias antioxidantes em sua composição. O objetivo deste estudo foi avaliar pela primeira vez a atividade da rutina frente ao FPS e sua segurança clínica através da comparação de formulações fotoprotetoras contendo rutina 0.1% (w/w), avobenzona 3.0% (w/w) e octil dimetil PABA 8.0% (w/w) com uma preparação similar sem o composto bioativo. Adicionalmente, hidratação cutânea, FPS in vitro e atividade antioxidante da rutina em associação com outros filtros foram investigados. O perfil de segurança das formulações qualificou as fórmulas para os testes de eficácia clínica. O teste de DPPH confirmou a capacidade antioxidante da rutina, demonstrando cerca de 40% de aumento na capacidade de sequestro de radicais livres na presença do composto bioativo. A rutina em combinação com os filtros UV aumentou o FPS clínico de 7.30 ± 0.60 para 12.37 ± 1.13, o que representa cerca de 70% de aumento. Os resultados encontrados provam que a rutina em combinação com outros filtros pode aumentar significativamente o valor do FPS e que a mesma é segura para uso clínico. / Unprotected chronic exposure to solar radiation can contribute to premature skin cancer and sunscreens are a key factor to avoid those detrimental effects. Currently, there is a growing interest in the photoprotector and antioxidant potential of bioactive substances, such as rutin, that could help to increase the SPF value and add multifunctional characteristics to the formulations. Recent in vitro findings indicated that rutin, when incorporated in oil-in-water photoprotective emulsions can provide antioxidant activity and SPF increase. However, clinical studies are fundamental to determine this activity duo to in vitro methodology lack of repeatability and correlation between the in vivo data, especially when the analyzed formulas contain antioxidant substances. The aim of this study was to evaluate for the first time to date the rutin in vivo SPF and clinical safety by comparing sunscreens formulations containing rutin 0.1% (w/w), butyl methoxydibenzoylmethane 3.0% (w/w) and octyl dimethylPABA 8.0% (w/w) with a similar bioactive-free preparation. Additionally, skin hydration, in vitro SPF and in vitro antioxidant activity of rutin, in association with the UV filters were investigated. The safety profile of the formulations under sun-exposed skin conditions qualified the formulas for clinical efficacy assays. DPPH test confirmed rutin antioxidant properties, demonstrating about 40% increase in radical scavenging potential when the bioactive compound was present. Rutin in combination with the UV filters increased the clinical SPF from 7.30 ± 0.60 to 12.37 ± 1.13, representing about 70% growth in the SPF value. The results obtained proved that rutin in combination with UV filters can improve the SPF value significantly and is safe for clinical use.

Submitted by Rosangela Silva (rosangela.silva3@unioeste.br) on 2017-08-30T17:39:39Z No. of bitstreams: 2 Isabella Calvo Bramatti.pdf: 1175071 bytes, checksum: b4b43f726550d831d384392724e6c260 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-08-30T17:39:39Z (GMT). No. of bitstreams: 2 Isabella Calvo Bramatti.pdf: 1175071 bytes, checksum: b4b43f726550d831d384392724e6c260 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2017-02-06 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Tamoxifen is classified as a selective estrogen receptor modulator, with much use in the treatment of breast cancer. Quercetin has aroused interest, mainly for its antioxidant action, which may minimize the damage caused by tamoxifen. Due to the lack of studies about the coadministration of quercetin with tamoxifen, the objective of this study was to elucidate the effect upon the cholinergic activity and antioxidante defense. At the study, ovariectomized rats were separated into groups and treated with tamoxifen (5 mg.Kg-1), quercetin (22,5 mg.Kg-1) and the coadministration of tamoxifen (5 mg.Kg-1) plus quercetin (22,5 mg.Kg-1). The hippocampus was isolated and utilized for the determination of acetylcholinesterase and butyrylcholinesterase activity, superoxide dismutase and catalase activity, and the oxidative damage markers (lipid peroxidation and dosage of thiols groups). It was possible observe the oxidant effect of tamoxifen at the hippocampus, where acted like an antagonist at the estrogen receptors, increasing the lipid peroxidation, levels of thiol groups and attenuating catalase activity; in addition to a cholinergic effect, with the decrease of acetylcholinesterase and butyrylcholinesterase activity. When coadministered the quercetin with tamoxifen, it can be seen that at a situation of oxidative stress, the quercetin acts like pro oxidant in the studied. / Tamoxifeno é classificado como um modulador seletivo dos receptores de estrogênio (SERM), sendo muito utilizado no tratamento contra o câncer de mama. A quercetina tem particularmente despertado interesse, principalmente por sua ação antioxidante a qual supostamente poderia vir a minimizar os danos causados pelo tamoxifeno. Devido à falta de estudos sobre a coadministração da quercetina com o tamoxifeno e a ambiguidade sobre a ação de ambos no cérebro, o objetivo deste estudo foi elucidar o efeito sobre a atividade colinérgica e defesa antioxidante. No estudo, ratas ovariectomizadas foram separadas em grupos e tratadas com tamoxifeno (5 mg.Kg-1), quercetina (22,5 mg.Kg-1) e a coadministração de tamoxifeno (5 mg.Kg-1) mais quercetina (22,5 mg.Kg-1). O hipocampo foi isolado e utilizado para a determinação da atividade da acetilcolinesterase, butirilcolinesterase da superóxido dismutase e catalase e dos marcadores de dano oxidativo (lipoperoxidação e dosagem de grupamentos tióis). Foi possível observar a ação oxidante do tamoxifeno no hipocampo, onde agiu como antagonista nos receptores de estrogênio, aumentando a lipoperoxidação, os níveis de tióis e diminuindo a atividade da catalase; além de um efeito colinérgico com a diminuição da atividade da acetilcolinesterase e butirilcolinesterase. Quando coadministrada a quercetina com o tamoxifeno, pode-se observar que em uma situação de estresse oxidativo, a quercetina age como pró-oxidante na dose estudada.

Submitted by Marcos Anselmo (marcos.anselmo@unipampa.edu.br) on 2016-04-04T17:21:32Z No. of bitstreams: 1 Lucian Del Fabbro.pdf: 1272289 bytes, checksum: 12f1723b46015bc56a5601039de65844 (MD5) / Made available in DSpace on 2016-04-04T17:21:32Z (GMT). No. of bitstreams: 1 Lucian Del Fabbro.pdf: 1272289 bytes, checksum: 12f1723b46015bc56a5601039de65844 (MD5) Previous issue date: 2014-03-02 / As micotoxicoses ocorrem através do consumo de alimentos contaminados, sendo uma questão de saúde pública em todo o mundo. A contaminação dos alimentos depende de crescimento fúngico, além de condições climáticas ideais para o desenvolvimento das micotoxinas. A ingestão de micotoxinas por seres humanos ocorre principalmente pela ingestão de produtos vegetais contaminados, bem como pelo consumo de produtos derivados destes alimentos. A Zearalenona (ZEA) é uma lactona do ácido fenólico resorcílico e pode ser produzida por várias espécies de Fusarium, sendo que Fusarium graminearum, proliferatum e culmorum são os principais produtores. A ZEA é um composto estrogênico, cujo os efeitos tóxicos e seus metabólitos têm sido atribuídos principalmente à sua estrutura química semelhante a dos estrógenos naturais. O sistema reprodutivo é um dos principais alvos de toxicidade da ZEA, sendo os órgãos reprodutores, como útero, ovários e testículos os principais órgãos afetados. Frente a esta problemática, o estudo de substâncias naturais capazes de prevenir tal micotoxicose se faz necessária. A crisina é um flavonoide da classe das flavonas. Possui hidroxila livre nos carbonos 5 e 7 do anel, está presente em níveis elevados no maracujá do mato, mel e no própolis. Possui conhecidos efeitos benéficos como atividades antioxidante e anti-inflamatória, além de aumentar níveis de testosterona. Este estudo teve por objetivo avaliar o efeito protetor da crisina frente aos efeitos tóxicos da micotoxina ZEA nos testículos de camundongos Swiss. Para isto, os camundongos foram tratados durante 10 dias consecutivos com crisina (5 e 20 mg/Kg, via gavagem), no 11° dia os animais receberam uma dose de ZEA (40 mg/Kg, via gavagem). Após 48 horas, os animais receberam uma dose de pentobarbital (180 mg/Kg, intraperitoneal, i.p.) e o sangue foi coletado por punção cardíaca e os testículos removidos, pesados e homogeneizados em tampão para as determinações dos níveis de testosterona, contagem e avaliação da motilidade dos espermatozoides, determinação de indicadores de estresse oxidativo por métodos colorimétricos: catalase (CAT), superóxido dismutase (SOD), glutationa peroxidase (GPx), glutationa redutase (GR), glutationa-S-transferase (GST) e não enzimáticos glutationa reduzida, TRAP (capacidade de reatividade antioxidante total) e TAR (capacidade antioxidante total) e 4-Hydroxynonenal (4-HNE). Foram determinados interleucinas (IL-1β, IL6 e IL10), o fator de necrose tumoral alfa (TNF-α) e a enzima mieloperocidase (MPO). Foram determinados parâmetros apoptóticos, as atividades das Caspases 3 e 6 além de dos níveis de 8-hydroxy-2'-deoxyguanosina (8-OHdG). O tratamento com crisina reverteu a redução dos níveis de testorenona, da contagem e da motilidade dos espermatozoides. O tratamento com crisina reverteu a inibição das enzimas antioxidantes GPx, GR, e GST, além de normalizar os níveis de GSH e 4-HNE. A alteração nos parâmetros inflamatórios causados pela administração de ZEA foi prevenida pela administração da crisina restaurando os níveis xi de IL-1β e IL-6 e TNF-α e reduzindo os níveis de IL-10 e a atividade da MPO. O tratamento com crisina atenuou os danos apoptóticos causados pela ZEA, inibindo a atividade das caspases 3 e 6 e reduzindo os níveis de 8-OHdG. Desta forma o tratamento com crisina preveniu os danos causados pela micotoxina ZEA em camundongos swiss através da modulação do estresse oxidativo, acarretando em uma redução do processo inflamatório e consequentemente apoptótico. Desta forma espera-se que a crisina possa vir a ser um possível tratamento auxiliar no combate a micotoxicoses seja em humanos ou animais. / Ace mycotoxicoses occur through the consumption of contaminated food being a public health issue worldwide. Contamination of food depends on fungal growth beyond the ideal climatic conditions for the development of mycotoxins. The ingestion of mycotoxins by humans occurs primarily by ingestion of contaminated plant products, as well as the consumption of food products derived therefrom. Zearalenone (ZEA) is a phenolic resorcylic acid lactones and can be produced by various Fusarium species, and Fusarium graminearum, culmorum and proliferatum are the main producers. ZEA is an estrogenic compound, whose the toxic effects and its metabolites have been mainly attributed to their similar chemical structure of natural estrogens. The reproductive system is one of the main targets of toxicity of ZEA and the reproductive organs such as the uterus, ovaries and testes those most affected organs. Faced with this problem, the study of natural substances capable of preventing such mycotoxicosis is required. Chrysin is a flavonoid of the flavones class. It has free hydroxyl at carbons 5 and 7 ring, is present at high levels in passion fruit bush honey and propolis. Chrysin has known beneficial effects as an antioxidant activities and anti-inflammatory and it has increased testosterone levels. This study aimed to evaluate the protective effect of chrysin forward to the toxic effects of the mycotoxin ZEA in the testes of Swiss mice. For this, the mice were treated for 10 consecutive days with chrysin (5 and 20 mg / kg by gavage) on day 11, the animals received ZEA, a dose (40 mg / kg by gavage). After 48 hours, the animals received an overdose of pentobarbital (180 mg / kg, intraperitoneal, i.p.) and blood was collected by heart puncture and removed, weighed and homogenized testis for determination of testosterone, count and assessment of sperm motility, determination of oxidative stress indicators by colorimetric methods: catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione reductase (GR), glutathione-S-transferase (GST) and non-enzymatic reduced glutathione, TRAP (reactivity total antioxidant capacity) and TAR (total antioxidant capacity) and 4-hydroxynonenal (4-HNE). Were determined, interleukins (IL-1β, IL-6 and IL-10), tumor necrosis factor (TNF-α) and mieloperoxidase enzyme (MPO). Apoptotic parameters were determined, Caspases 3 and 6 beyond the levels of 8-hydroxy-2'-deoxyguanosine (8-OHdG). The treatment with chrysin reversed the reduction of testorenone levels, count and motility of sperm. Treatment with chrysin reversed the inhibition of antioxidant enzymes GPx, GR, and GST, as well as normalizing levels of GSH and 4-HNE. The alteration in inflammatory parameters caused by the administration of ZEA was xiii prevented by administration of chrysin, restoring IL-1β and IL-6 and reducing IL-10 levels, and reduce TNF-α levels and MPO activity. Treatment with chrysin attenuated apoptotic damage ZEA, inhibiting the activity of caspases3 and 6, reducing levels and 8-OHdG. Thus, the treatment with chrysin prevented the damage caused by mycotoxins ZEA in Swiss mice probably through modulation of the oxidative stress, resulting in a reduction of the inflammatory process and hence apoptosis. Thus it is expected that chrysin may be a possible treatment support combat in mycotoxicosis both in humans and animals.