Dissertations / Theses on the topic 'Isoflavones'

Orientadores: Pedro Luiz Rosalen, Severino M. Alencar
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba
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Resumo: Entre as própolis brasileiras, urna nova própolis ainda não classificada de acordo com Park et al., 2002 e denominada de própolis vermelha originária do estado de Alagoas (Nordeste do Brasil), tem mostrado resultados interessantes em relação a sua composição química e atividade biológica em estudos preliminares. Assim, o objetivo principal deste trabalho é identificar a origem botânica da própolis vermelha, a composição química e atividade biológica do extrato etanólico da própolis (EEP) e do extrato etanólico da resina da planta (EER), avaliar o efeito sazonal sobre a composição química e atividade biológica do EEP e do EER. Esses objetivos foram atingidos através das seguintes metodologias: 1- observação do comportamento de visita das abelhas à . vegetação próxima à colméia; 2- comparação dos perfis químicos dos vegeÜlis visitados pelas abelhas coletoras de resina e da própolis vermelha, obtidos por cromatografia líquida de alta eficiência em fase reversa e análises complementares, estabelecendo-se assim, as características entre ambos os materiais, visando à identificação do marcador biológico botânico e 3- avaliação da influência do efeito sazonal anual sobre a própolis vermelha e sua vegetação fonte por meio da atividade antimicrobiana e perfil químico, com coletas bimensais das amostras durante o período de 1 ano. A atividade antimicrobiana foi avaliada por meio da Concentração Inibitória Mínima (CIM) e Concentração Bactericida Mínima (CBM) e os microrganismos usados foram: O Streptococcus mutans UA159, Streptococcus sobrinus 6715, Staphylococcus aureus ATCC25923 e Actinomyces naeslundii ATCC 12104. Os resultados demonstraram o mesmo perfil quimico entre o EEP e o EER da planta Dalbergia ecastophyllum (L.) Taub., cuja característica foi a alta concentração relativa das isoflavonas 3-hidroxi-8,9-dimetoxipterocarpin e medicarpina. Os perfis químicos do EEP e do EER, obtidos ao longo do ano, através do testes químicos, apresentaram-se distintos dos perfis dos demais 12 tipos de própolis brasileiras já classificadas e variaram quantitativamente de acordo com a sazonalidade. A CIM variou entre 15,6-125 'mu'g/rnL e a CBM de 31,2 - 500 'mu'g/mL considerando os 4 microrganismos àvaliados. Conclui-se que esta própolis, cuja origem botânica é a Dalbergia ecastophyllum, pode ser classificada corno o 13° tipo de própolis, de acordo Park et al., 2002, e tanto o EEP quanto o EER apresentaram alta atividade antimicrobiana, os quais poderão ser utilizados para pesquisas futuras de novas moléculas no controle da cárie dental
Abstract: Among the Brazilian propolis, new propolis not yet classified, according to Park et al., 2002 and called of red propolis, originary from Alagoas state (Northeast of Brazil), have showed interesting results in preliminary studies in our laboratories. Thus, the main objective of this work is to identify the botanical marker of red propolis, the chemical composition and biological activity of extract ethanolic of propolis (EEP) and the extract ethanolic of resin of plant (EER), verifying the effect of seasonal period on chemical composition and antimicrobial activity of EEP and EER. This objective was reached through the following methodologies: 1- observation of bee behavior in visiting vegetation next beehive; 2- comparison of chemical profile of vegetables visited by bees for collecting resin with red propolis, gotten for liquid chromatography of high efficiency in phase reverse and complementary analyses, for establishing the common characteristics between both materiaIs, aiming to identification of botanical biological marker; 3- evaluation of influence seasonal annual effect on the red propolis and its vegetation source by means of the antimicrobial activity and chemical profile, with bymonthly collections of the samples during the period of 1 year. The antimicrobial activity was evaluated by Minimal Inhibitory Concentration (MIC) and Minimal Bactericidal Concentration (MBC) and microorganism used were Streptococcus mutans UA159, Streptococcus sobrinus 6715, Staphylococcus aureus ATCC25923 e Actinomyces naeslundii ATCC 12104. The results had the same demonstrated to chemical profile between the EEP and the EER of the plant Dalbergia ecastophyllum (L.) Taub. Whose characteristic was the high relative concentration of isoflavonas 3-hidroxi-8,9 dimetoxipterocarpin and medicarpina. The chemical profiles of EEP and EER varied quantitatively according to seasonal effect The CIM varied between 15.6-125 'mu'g/rnL and the CBM of 31.2 - 500 'mu'g/rnL. One concludes that these propolis, whose botanical origin is the Dalbergia ecastophyllum, can be classified as 13° type of propolis, according to Park et al., 2002 and the EEP and EER showed high antibacterial activity which can be used for future molecule research in the control of caries dental
Mestrado
Farmacologia, Anestesiologia e Terapeutica
Mestre em Odontologia

Orientador: Sonia Nair Bao
Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: Este estudo foi proposto para avaliar se a exposição perinatal (gestacional e lactacional) ou crônica a isoflavonas em dieta contendo soja ou na forma de concentrados de isoflavonas pode comprometer a saúde reprodutiva de coelhos machos. No primeiro experimento, fêmeas foram alimentadas com dieta contendo soja ou dieta isenta de soja e alfafa, suplementada com 10 ou 20 mg/kg/dia de isoflavonas ao longo da gestação e lactação. O grupo controle foi mantido somente com a dieta isenta de soja e alfafa. Na desmama, foram avaliados o peso e a morfologia dos órgãos do aparelho reprodutor e os níveis séricos de testosterona de parte dos filhotes machos. O restante deles foi submetido à dieta controle desde a desmama até a fase adulta. Após a puberdade, os animais foram avaliados quanto ao comportamento sexual, qualidade do sêmen e morfologia dos órgãos reprodutivos. No segundo experimento, fêmeas foram alimentadas com as mesmas dietas empregadas no primeiro experimento, porém a suplementação com isoflavonas foi realizada com doses variando de 2,5 a 20 mg/kg/dia. As doses de isoflavonas foram selecionadas com base em estimativas da ingestão de isoflavonas a partir do consumo de alimentos derivados da soja. Após a desmama, os filhotes machos receberam a mesma dieta fornecida para suas respectivas mães até o fim do experimento. Foi avaliado nestes animais a idade à puberdade, qualidade do sêmen e o comportamento sexual, e, na 33ª semana de vida o peso e a morfologia dos órgãos reprodutivos. Os resultados deste estudo foram baseados em dados obtidos da avaliação de 100 machos num período de 3 anos. O número de espermatozóides esteve de acordo com os valores da literatura para coelhos da raça Nova Zelândia e não variou significativamente em relação ao grupo controle, embora o volume de sêmen tenha sido menor em coelhos expostos à alta dose de isoflavonas (20 mg/kg/dia). O peso dos órgãos reprodutivos não diferiu estatisticamente do grupo controle e não houve evidência de malformações genitais, alterações metaplásicas, ou qualquer outra alteração histopatológica correlacionada com os tratamentos. Nos jovens, a análise histológica dos testículos não revelou diferenças no desenvolvimento gonadal. Coelhos suplementados de forma crônica com 20 mg/kg/dia de isoflavonas apresentaram menor ingestão de alimentos e peso corporal na fase adulta. Este achado é economicamente importante na produção animal; todavia os animais alimentados com a dieta contendo soja apresentaram na 33ª semana de idade consumo de alimento e peso corporal maiores em 6 e 4% respectivamente do que os animais do grupo controle (P < 0,05). Apesar dos recentes alertas, os resultados deste estudo não suportam a hipótese de que a exposição à isoflavonas em doses compatíveis com o consumo de alimentos à base de soja possa comprometer a saúde reprodutiva masculina
Abstract: This study was proposed to determine if perinatal (that is gestation and lactation) or chronic exposure to isoflavones trough consumption of soy containing diet or semipurified soy isoflavones may disrupt male reproductive health of rabbits. In the first experiment, groups of dams were fed either soy containing diet or soy and alfafa free diet supplemented with soy isoflavones at levels of 10 and 20 mg/kg/day throughout gestation and lactation. The control group was kept on soy and alfafa free diet only. Reproductive organs weight and morphology and serum levels of testosterone of part of the male offspring were evaluated at weaning. Remaining males were subjected to the control diet from weaning to adulthood. Sexual behavior, semen quality and reproductive organs morphology were evaluated after puberty. In the second experiment, groups of dams were fed same diets employed in the experiment 1, but supplementation with isoflavones were performed with doses ranging from 2,5 to 20 mg/kg/day. Dose levels of isoflavones were selected on the basis of the reported estimative of isoflavones intake from the consumption of soy-based foods. After weaning, male offspring received the same diet, which was given to the respective mother. The age that males reached puberty, semen quality and sexual behavior were evaluated in these animals and at 33 weeks of age reproductive organs weight and morphology were analyzed. Results of this study were sustained by data from the evaluation of 100 males in a period of 3 years. Sperm counts was within literature values for New Zealand rabbits and did not vary significantly in relation to control group, although semen volume has been lesser in rabbits exposed to high levels of isoflavones (20 mg/kg/dia). Reproductive organs weight did not differ statistically from the control, and there was no gross evidence of genital malformations, metaplastic changes, or any histopathologic alteration that was correlated with the treatments. In the young rabbits, histological analysis of the testes did not revel differences in gonadal development. Rabbits chronically supplemented with 20 mg/kg/day of soy isoflavones showed lesser food intake and body weight at adulthood. This find is economically important in animal production; however the animals fed soy containing diet showed food consumption and body weight 6 and 4%, respectively higher than animals of the control group at 33 weeks of age (P <0.05). In conclusion, despite recent alerts, results of this study did not support the hypothesis that isoflavones consumption at dietary levels may impair male reproductive health
Doutorado
Biologia Celular
Doutor em Biologia Celular e Estrutural

Flavonoids are non-nutrient secondary metabolites ubiquitous in plants, associated with protection against various diseases, such as cancer and cardiovascular diseases. Dietary flavonoids are normally found as conjugated glycosides except, notably, in fermented foods where although there may be losses in total flavonoid content, levels of liberated aglycones can be relatively high. There has been considerable interest in the relationship between the form and structure of the ingested flavonoids and the consequences for efficiency of absorption. The research focused firstly on β-D-glucosidases (β-D-glycoside glucohydrolase, EC 3.2.1.21) extracted from different plant sources and characterised. The enzyme was found at the highest levels in almond and apple seeds. The optimum reaction conditions of the enzyme from apple seeds were determined to be pH 5.5 at 65ºC, and the enzyme extract was stable at 4ºC for at least 12 weeks. Kinetic characterisation of the enzyme from selected materials was carried out by using para-nitrophenyl-β-D-glucopyranoside (p-NP-Glc) as substrate. The Km and Vmax of the enzyme from apple seed extract were determined, for the first time, to be 5.48 ± 0.34 mM and 15.60 ± 0.95 U/mg protein (n = 8), respectively, with the protein content of the extract being 0.728 ± 0.019 mg/ml. Secondly, isoflavone contents from different sources were investigated. Soy bean and its products are were found to be good sources of daidzin and genistin; kudzu was the best source of puerarin; red clover and chickpea were good sources of formononetin and biochanin A. Passion fruit was found to be an interesting source of isoflavones outside the legume family. By using selected enzyme sources and isoflavone sources, a novel natural style soy-based food was developed in which isoflavones existed predominately as aglycones. The food, derived using soya and enzymes from waste sources, may have further potential.

La graine de soja contient de grandes quantités d'isoflavones (génistéine, daidzéine et glycitéine). En raison de leurs propriétés phytoestrogéniques, ces composés peuvent avoir des effets bénéfiques sur la santé humaine, mais ils peuvent aussi être perçus comme perturbateurs endocriniens, en particulier dans les laits pour nourrissons. La teneur et la composition en isoflavones de la graine diffèrent selon la fraction considérée. Les cotylédons contiennent de la génistéine et de la daidzéine, tandis que les germes, avec une teneur quatre à dix fois supérieure, contiennent majoritairement de la daidzéine et de la glycitéine. Le génotype influence fortement la teneur en isoflavones totales. Le déterminisme du pourcentage des isoflavones est essentiellement génétique. Ce travail porte sur l'étude du déterminisme génétique à l'origine des variations de teneurs et de compositions en isoflavones dans les germes et les cotylédons de la graine, en tenant compte également du net décalage de l'accumulation entre ces deux compartiments, au cours du développement de la graine. Dans un premier temps, les gènes des isoflavone synthases (IFS) de variétés très différenciées pour leurs teneurs et profils d'isoflavones ont été séquencés, puis les expressions des gènes clefs de la biosynthèse (neuf chalcone synthases (CHS), une chalcone réductase (CHR), quatre chalcone isomérases (CHI) et les deux isoflavone synthases (IFS) ont été suivies par RT-PCR quantitative dans les cotylédons et dans les germes, à trois stades critiques du développement de la graine (25, 40 et 60 jours après floraison). La seconde partie de ce travail a été consacrée à l'étude de l'expression de différents gènes candidats de la flavonoïde 6-hydroxylase (F6H) catalysant la première étape de la synthèse de la glycitéine. Le polymorphisme des séquences génomiques IFS1 et IFS2 des isoflavone synthases n'a pas montré de lien avec les différences de teneurs en isoflavones entre les variétés. L'activité transcriptionnelle des gènes de biosynthèse des isoflavones souligne l'existence d'une régulation bien distincte de cette synthèse dans ces deux compartiments. Les taux d'expression des gènes cibles ne sont pas toujours reliés avec les différences de teneurs ou de profils dans les germes et les cotylédons, suggérant ainsi l'effet prépondérant des régulations post-traductionnelles, notamment dans la formation du complexe multienzymatique de biosynthèse de ces composés. Nous avons aussi mis en évidence une forte expression du gène CHS9 codant pour la chalcone synthase 9, avec un profil correspondant plus à l'accumulation des isoflavones dans le germe que dans les cotylédons. Les gènes CHS7 et CHS8 codant pour les chalcone synthases 7 et 8, déjà signalés comme fortement corrélés à la synthèse des isoflavones, sont plus liés à l'accumulation dans les cotylédons que dans les germes. Ces travaux montrent aussi que le gène F6H signalé dans la littérature ne s'exprime pas dans les germes. En revanche, deux candidats dont la séquence est similaire à 79% ont été étudiés. Le gène F6H3 est le seul à s'exprimer dans la graine, uniquement dans le germe. Son expression n'a pas été détectée dans les germes d'une lignée mutante qui ne produit pas de glycitéine. Ce gène est donc un candidat potentiel clef pour la synthèse de la glycitéine dans le germe. La structure particulière de l'enzyme correspondante pourrait indiquer une forte implication de l'architecture du complexe enzymatique et des contraintes qui en découlent dans l'utilisation préférentielle d'une voie ou d'une autre dans ce schéma de biosynthèse
The soybean seed contains large amounts of isoflavones (genistein, daidzein, and glycitein). Owing to their phytoestrogenic properties, these compounds can have beneficial effects on human health, but they can also be considered as endocrine disruptors, for example in infant formulas. The isoflavone content and composition in the seed depend on the considered fraction. The cotyledons contain only genistein and daidzein, while the hypocotyls are four to ten times more concentrated and contain three isoflavones, mostly daidzein and glycitein. The genotype has a strong influence on total isoflavone content, and even more on the percentage of individual isoflavones in cotyledons and hypocotyls. The objective of this work is to investigate the genetic determinism that underlies such contrasted contents and compositions between the two seed fractions, and the relation between main biosynthetic steps and genotypic differences. First, the genes of isoflavone synthases (IFS) were sequenced in varieties with highly contrasted content and composition. The expression of different keys genes of the biosynthesis (nine chalcone synthases (CHS), a chalcone reductase (CHR), four chalcone isomerases (CHI) and the two isoflavone synthases (IFS) have then been followed by quantitative RT - PCR in the cotyledons and hypocotyls, at three critical stages of seed development (25, 40 and 60 days after flowering). Second, the expression of different candidate genes for the flavonoid 6-hydroxylase (F6H) which catalyzes the first step in the synthesis of the glycitein has been investigated. The polymorphism of the genomic sequences IFS1 and IFS2 of isoflavone synthases was not correlated with differences in isoflavone contents. The transcriptional activity of key genes of the biosynthesis of isoflavones pointed out the existence of a distinct regulation of isoflavone biosynthesis between the two seed fractions. The expression levels of target genes were not always related to differences in isoflavone content or compositions in the hypocotyls and cotyledons. This suggests the overriding effect of post-translational regulation, especially in the formation of multienzyme complex of biosynthesis of these compounds. The chalcone synthase gene CHS9 was highly expressed, with a profile similar to the accumulation of isoflavones in hypocotyls. The chalcone synthase genes CHS7 and CHS8 expressions, already reported as highly correlated to the biosynthesis of isoflavones were more related to accumulation in the cotyledons than in hypocotyls. This work has also shown that the F6H gene, reported in the literature was not expressed in the hypocotyls. However, two candidates with as highly similar coding sequence (79%) have been studied. The F6H3 gene is the only one expressed in the seed, more precisely in the hypocotyls but it was not expressed in the cotyledons. Moreover, it was not expressed in a mutant line which did not accumulate glycitein. This gene is therefore a key potential candidate for the synthesis of the glycitein in hypocotyls. The particular structure of the corresponding enzyme may indicate a strong involvement of the architecture of the multienzyme complex of isoflavones biosynthesis and the constraints arising in the preferential use of a track or another in this scheme of biosynthesis

Orientador: Gabriela Alves Macedo
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos
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Resumo: A soja atrai considerável atenção no atual cenário econômico mundial devido ao seu elevado potencial nutritivo e efeitos potencialmente benéficos à saúde humana, que são atribuídos principalmente às isoflavonas. Esta classe de fenóis heterocíclicos é conhecida por suas atividades biológicas, tais como as atividades estrogênica, antioxidante e antitumoral, sendo as formas agliconas mais ativas do que as glicosiladas. Contudo, estudos têm mostrado que a eficácia clínica das isoflavonas está relacionada com a capacidade de produção de equol, um metabólito da daidzeína que, segundo a literatura vigente, é produzido exclusivamente pela ação da microbiota intestinal. Assim, há evidências de que nem todos os indivíduos são capazes de metabolizar as isoflavonas a equol, sendo essa variabilidade atribuída às diferenças na composição da microflora intestinal, hábitos alimentares, fatores genéticos, dentre outros. Neste contexto, os produtos à base de soja são uma forma de incluir as isoflavonas na dieta, sendo que o extrato hidrossolúvel de soja (EHS) é um substrato que tem se apresentado com potencial para produção de novos alimentos com apelo saudável. Desse modo, com o propósito de aumentar o conteúdo de isoflavonas bioativas e avaliar a viabilidade de um processo biotecnológico para produção de equol in vitro, neste projeto foi investigada a aplicação de culturas starters e bactérias lácticas probióticas na fermentação do EHS, aliado à ação do extrato bruto de tanase obtido a partir de Paecilomyces variotti. Além disso, também foi avaliada a biotransformação dos compostos fenólicos e atividade antioxidante do produto obtido. O teor de fenóis totais foi avaliado pelo método de Folin-Ciocalteau, a atividade antioxidante pelos métodos in vitro ORAC e de sequestro de radicais DPPH e o perfil químico dos extratos, antes e depois dos processos de biotransformação, foi avaliado por CLAE-DAD com co-injeção de padrões de isoflavonas. Após o processo fermentativo e/ou tratamento enzimático do EHS, houve um significativo aumento no teor de fenólicos totais e capacidade antioxidante, evidenciada por ambos métodos empregados (ORAC e DPPH), quando comparados com o controle do EHS sem reação. Além disso, foi verificada uma modificação no perfil polifenólico das amostras do EHS biotransformadas evidenciado por CLAE-DAD, resultando em um aumento na concentração das formas agliconas em relação às glicosiladas, bem como o aumento da concentração de equol após os processos de biotransformação propostos. Os resultados obtidos por CLAE-DAD confirmaram que o extrato de tanase de P. variotty foi capaz de biotransformar as formas glicosiladas (daidzina e genistina) das isoflavonas em suas respectivas formas agliconas (daidzeína e genisteína), indicando uma atividade diglicosídica do extrato semipurificado de tanase. Pelo que se tem conhecimento, a hidrólise de isoflavonoides glicosilados por tanase, bem como a formação de equol, é um relato inédito na literatura demonstrando que é possível desenvolver um processo in vitro para a obtenção deste composto bioativo, sem a presença de bactérias intestinais, utilizando apenas uma biotransformação enzimática
Abstract: The soy attracts considerable attention in the current global economic scenario due to their high nutritional potential and potentially beneficial effects to human health, which are mainly attributed to isoflavones. This class of heterocyclic phenols is known for its biological activities, such as estrogenic, antioxidant and antitumor activities, and the aglycone forms are more active than the glycosylated. However, studies have shown that the clinical efficacy of isoflavones is related to the capacity to produce equol, a metabolite of daidzein, which according to the current literature it is produced exclusively by the action of the gut microbiota. Thus, there is evidence that not all individuals are able to metabolize isoflavones to equol and this variability is attributed to differences in the composition of gut microflora, diet, genetic factors, among others. In this context, the soy-based products are a way to include the isoflavones in the diet, and the soymilk is a substrate that has shown the potential to produce new foodstuff with healthy appeal. Thus, in order to increase the content of bioactive isoflavones and assess the viability of a biotechnological process for in vitro production of equol, in this project was investigated the application of starters and probiotic lactic bacteria strains in the fermentation of soymilk, combined with the action of the crude extract of tannase obtained from Paecilomyces variotii. Furthermore, it was also evaluated the biotransformation of phenolic compounds and antioxidant activity of the obtained product. The total phenolic content was assessed by Folin-Ciocalteau method, the antioxidant activity by in vitro methods of ORAC and DPPH radical sequestration and the chemical profile of the extracts, before and after the processes of biotransformation, was evaluated by HPLC-DAD with co- injection of standards of isoflavones. After the fermentation process and/or enzymatic treatment of the soymilk, there was a significant increase of the total phenolic content and antioxidant capacity, as evidenced by both methods employed (ORAC and DPPH), compared with the standard soymilk. Furthermore, there was observed a change in the polyphenolic profile of biotransformed samples of soymilk, evidenced by HPLC-DAD, resulting in an increase in the concentration of the aglycones in relation to glucosides forms, as well as there was a significant increase of the concentration of equol after the biotransformation processes proposed. The results obtained by HPLC-DAD confirmed that the extract of tannase from P. variotii was able to biotransform the glucosides forms (daidzin and genistin) of isoflavones in their aglycone forms (daidzein and genistein), indicating a diglycosidase activity of the crude extract of tannase. From what is known, the hydrolysis of glucosides isoflavone by tannase, as well as the formation of equol, is an unpublished report in the literature demonstrating that it is possible to develop an in vitro method for obtaining this bioactive compound, without the presence of gut bacteria process using only an enzymatic biotransformation
Mestrado
Ciência de Alimentos
Mestra em Ciência de Alimentos

Isoflavonas são compostos fenólicos de ocorrência principalmente na família Leguminosae, utilizados, entre outros, como alternativa para a reposição hormonal em mulheres no período pós-menopausa, devido a sua ação estrogênica seletiva. Em vista disso, foi investigada a presença das isoflavonas daidzeína, genisteína, formononetina e biochanina A em 153 espécies de Leguminosas nativas do Rio Grande do Sul através de cromatografia líquida de alta eficiência. Destas, 42 espécies apresentaram ao menos uma das quatro isoflavonas investigadas. Pertencente à família Leguminosae, o gênero Trifolium é encontrado em quase toda a zona temperada e subtropical do mundo, sendo que a espécie Trifolium riograndense é nativa do estado do Rio Grande do Sul. As isoflavonas daidzeína, genisteína, formononetina e biochanina A foram quantificadas nesta espécie, que apresentou 0,187 mg de daidzeína; 0,223 mg de genisteína; 16,683 mg de formononetina e 1,207 mg de biochanina A (por grama de amostra seca). A parte da planta com maior concentração de isoflavonas foi a folha, com 7,331 mg por grama de amostra seca, e a parte da planta com menor teor destas isoflavonas foi a raiz, com 2,806 mg por grama de amostra seca. Formononetina foi a isoflavona majoritária em todas as partes da planta analisadas. Ainda, foram realizados ensaios de atividade antiinflamatória do extrato das folhas de Trifolium riograndense. Foram utilizados os modelos de quimiotaxia in vitro em câmara de Boyden e indução de edema em pata de ratos, utilizando a carragenina como agente flogístico. Pelo ensaio de quimiotaxia, verificou-se que o extrato das folhas do trevo em estudo apresentou inibição da migração leucocitária de forma significativa em diversas concentrações testadas. Através do ensaio de edema em pata de rato, verificou-se que a administração por via oral de extrato de Trifolium riograndense em ratos, na dose de 100 mg/kg, inibiu a formação do edema provocado pela carragenina, significativamente.
Isoflavones are polyphenolic compounds of Leguminosae Family. They are used as alternative therapy for treating symptoms of the menopause, due their selective action in oestrogenic receptors. So, the presence of isoflavones daidzein, genistein, formononetin and biochanin A was investigated in 153 species of the Leguminosae Family that are natives of South Brazil, by high performance liquid chromatography. Forty two species showed at least one of four investigated isoflavones. The genus Trifolium belongs to Leguminosae Family. It is found in temperate and subtropical regions in the world. Trifolium riograndense is a species native of Rio Grande do Sul – Brazil. The isoflavone content in this species was 0.187 mg g-1 of dry material (DM) to daidzein; 0.223 mg g-1 of DM to genistein; 16.683 mg g-1 of DM to formononetin, and 1.207 mg g-1 of DM to biochanin A. The leaf was the part of the plant with more isoflavone content, 7.331 mg of DM. The root was the part with less isoflavone content, 2.806 mg of DM. The majority isoflavone in all parts was formononetin. The dry extract of leaves of Trifolium riograndense was submitted to biological tests. The anti-inflammatory activity was analyzed by Boyden chamber assay, it allowed observe chemotaxis phenomenon, and by induced rat paw oedema using carrageenan as flogistic agent. Through chemotaxis model, potential activity could be observed to extract of Trifolium riograndense. In the induced rat paw oedema test, a significantly inhibition of oedema was observed in the animals treated with extract of Trifolium riograndense (100 mg/kg).

Epidemiological and clinical studies researching the effects of soy food intake require a high compliance to a soy diet protocol. Measuring isoflavones in the bodily fluids is the most objective method in determining dietary compliance because isoflavones are highly and positively correlated with soy food intake and specific to soy foods. Urine analysis is preferred over plasma due to its noninvasiveness, which aids in compliance. Our study investigated the accuracy of collecting weekly and single day samples from 19 premenopausal women on a daily soy diet protocol in comparison to monthly samples for determining dietary compliance. We compared urinary isoflavone excretion rates (UIER) of weekly samples and single day samples to the UIER of a monthly sample that consisted of urine collected daily. Correlations were high between all samples and the monthly UIER. The correlations of the mean of all weeks UIER and single day UIER with the monthly UIER were 0.96 and 0.89, respectively. No large differences were seen when samples were stratified by ethnicity, 8MI, and equol excretor status. The small degree of increased accuracy in determining dietary compliance in measuring monthly UIER compared to weekly does not justify the extra time and effort required by the subjects and staff. Therefore, we conclude that analyzing weekly UIER is an accurate and feasible method of determining dietary compliance with a soy-based diet
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Soybeans and products thereof are considered as a very contradictory legume across layman and expert opinions. Some see it almost as a “superfood” while others avoid it due to its biologically active content with an unclear effect on the organism. As a lot of research on soybeans was already performed the content varies considerably and it is not easy to grasp the issue correctly. The objective of the research part of the thesis is a structuralized study of already published scientific knowledge to clarify the current state of the art. Experimental part of the thesis focuses on characterisation of soy and its products in terms of active ingredients content and determination of antioxidants and antimicrobial effect. After that, the cytotoxic effect on human heterogeneous colorectal adenocarcinoma epithelial cell line and mouse melanoma cell line is determined by in vitro testing. All obtained information and results of experimental part could possibly serve as a starting point for further study of soy and soy products, both in the dissertation and in the submission of a grant project.

Six isoflavones derived from Millettia griffoniana namely, 4’-methoxy-7-O-[(E)-3-methyl-7hydroxymethyl-2,6 octadienyl]isoflavone (7-O-DHF), Griffonianone C (Griff C), 7-O-geranylformononetin (7-O-GF), 3’,4’-dihydroxy-7-O-[(E)-3,7-dimethyl-2,6-octadienyl]isoflavone (7-O-GISO), Griffonianone E (Griff E), 4’-O-geranylisoliquiritigenin (4-O-GIQ) were tested for potential oestrogenic activities in three different oestrogen receptor alpha (ERα) dependent assays, namely a recombinant yeast assay, a reporter gene assay based on stably transfected MCF-7 cells (MVLN cells) and the induction of alkaline phosphatase in Ishikawa cells. The oestrogenic activities of isoflavones from Millettia griffoniana could be completely suppressed by the pure oestrogen antagonist, fulvestrant. The expression of Ki-67, proliferating cell nuclear antigen (PCNA) and cyclin D1 (CD1) mRNA used as indicator of cell proliferation in MCF-7 cells was assayed. Based on these in vitro results, Griff C was further tested in vivo. The main objective of this part of the work was to study the mechanistic basis of the oestrogenicity Three different doses of Griff C (2, 10, or 20 mg/kg BW) of Griff C in ovariectomised Wistar rats. 17β-oestradiol (E2: 10 µg/kg BW) was used as positive control. They were treated daily for three consecutive days and sacrificed 24 hours after receiving the last dose. The whole uterus was removed and weight. Liver and vena cava fragments were also collected and stored together with uteri in liquid nitrogen for subsequent real-time PCR to evaluate the effects of Griff C on the regulation of some relevant oestrogen–responsive genes in the uterus, the liver and the vena cava. The role of Griff C in apoptosis or in cell survival, through mediation of the phosphatidylinositol 3 kinase-Akt (PI3K-Akt) signaling pathway, was also investigated. Western blot analysis revealed that Griff C slightly increased the phosphorylation of Akt at its serine 473 residue. In this work, oestrogenic properties of the isoflavones derived from Millettia griffoniana are described using reporter gene assays and the oestrogen-inducible alkaline phosphatase Ishikawa model for the first time. These in vitro data were verified in vivo showing the regulation of the expression of various relevant oestrogen-responsive genes by Griff C. The spectrum of its activity was clearly similar to that of 17β-oestradiol on uterine hepatic and vena cava tissues of ovariectomised rats except for the proliferative response. However Griff C remained 100 to 1000 times less effective than oestradiol. These findings confirmed that some of the biological effects attributed to Millettia griffoniana are closely related to oestrogen-mediated action.

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Universidade Estadual Paulista (UNESP)
A osteoporose é uma doença crônica que afeta principalmente mulheres na pós-menopausa, e caracteriza-se por perda de massa óssea e deterioração da arquitetura do tecido ósseo, sendo considerada como um sério problema de saúde pública. A Terapia de Reposição Hormonal (TRH) é talvez o tratamento mais efetivo para reduzir a perda óssea, no entanto vem acompanhada por efeitos adversos como o aumento do risco de câncer de mama e de endométrio. Terapias alternativas, como a ingestão de isoflavonas, vêm sendo propostas a fim de se prevenir a osteoporose. As isoflavonas, um tipo de fitoestrógeno, parecem desenvolver atividade biológica parecida com a dos estrógenos de mamíferos, pois apresentam habilidade para se ligarem aos receptores estrogênicos. Além disso, estudos em animais e em humanos têm mostrado que as isoflavonas vêm se constituindo numa alternativa interessante para o controle do metabolismo lipídico e da obesidade. Além das isoflavonas, uma das possíveis maneiras de se prevenir a perda óssea é por meio da ingestão de quantidades adequadas de cálcio, um dos componentes dietéticos de maior importância para a manutenção da integridade óssea. Desta forma, o objetivo deste trabalho foi estudar o efeito da ingestão do iogurte de soja, fermentado com Enterococcus faecium e Lactobacillus jugurti, suplementado com isoflavonas e cálcio sobre o peso corpóreo e tecido ósseo de ratas maduras ovariectomizadas. Os animais foram divididos em 5 grupos, cada qual com 9 animais: pseudo-ovariectomizado, ovariectomizado; ovariectomizado que ingeriu o produto fermentado suplementado com isoflavonas e cálcio, ovariectomizado que ingeriu o produto fermentado suplementado com cálcio e ovariectomizado que ingeriu placebo suplementado com cálcio. O tratamento durou 3 meses e foram utilizados tíbias e fêmures de cada animal para... .
Osteoporosis is a chronic disease that affects mainly postmenopausal women, and it is characterized by bone loss and deterioration of the bone tissue architecture, and it considered a serious problem of public health. The Hormonal Replacement Therapy Hormonal (HRT) is the most effective treatment to reduce the bone loss, however it may be associated with adverse effects as the increase of the risk of endometrium and breast cancer. Alternative therapies, as intake of isoflavones, have being proposed to prevent the osteoporosis. Isoflavones, a type of phytoestrogens, seem to have biological activity like the mammals estrogens, because they present ability to bind to the estrogens receptors. Moreover, studies in animals and in human have shown that isoflavones may be an interesting alternative for the control of the lipidic metabolism and the obesity. On the other hand, the intake of adequate amounts of calcium, one of the dietary components of bigger importance for the maintenance of the bone integrity, is also considerate to be able to prevent the bone loss. Then, the objective of this research was to study the effect of the intake of the soy yoghurt, fermented with Enterococcus faecium and Lactobacillus jugurti, supplemented with isoflavones and calcium on the corporal weight and bone tissue of ovariectomized mature rats. The animals had been divided in 5 groups, each one with 9 animals: sham-ovariectomized, ovariectomized; ovariectomized treated with soy yoghurt supplemented with isoflavones and calcium, ovariectomized treated with soy yoghurt supplemented with calcium and ovariectomized treated with placebo supplemented with calcium. The treatment lasted 3 months and the tibia and femur of each animal were used for following analysis: bone length; mechanical assay of three points; weight, volume and bone density; bone mineral content; bone... (Complete abstract, click electronic address below).

C'est le deficit en strogenes associe a la menopause qui est en grande partie responsable de l'osteoporose post-menopausique. Or, l'abstention therapeutique etant frequente chez les femmes menopausees, il est indispensable d'envisager, chez celles ne pouvant ou ne souhaitant pas suivre une hormonotherapie substitutive, d'autres approches preventives visant a retablir une certaine impregnation oestrogenique. La nutrition pourrait constituer une strategie alternative ou complementaire, moins onereuse, permettant d'eviter les problemes d'observance frequemment associes aux prises medicamenteuses de longue duree. C'est donc a ce titre que l'efficacite preventive de la consommation d'aliments riches en phyto-oestrogenes demande a etre demontree. Dans ce contexte, l'objectif de ce travail de these etait de definir quel pouvait etre l'impact d'une supplementation quotidienne en isoflavones de soja sur l'osteopenie induite par ovariectomie chez la rate agee de 7-15 mois, modele de perte osseuse consecutive au deficit oestrogenique. Le but de la premiere etude etait d'evaluer l'eventuel impact preventif de la genisteine et de la daidzeine (incorporees separement a l'alimentation), et de comparer leur efficacite respective a celle du 17-ethinyloestradiol. Une seconde experimentation a consiste a determiner les effets preventifs potentiels, non plus de l'une ou l'autre des molecules, mais d'un melange d'isoflavones de soja. De facon a definir si ces effets dependaient du niveau de consommation : 3 pourcentages d'isoflavones (incorporees a l'alimentation quotidienne) ont ete testes. Enfin, par opposition aux 2 approches preventives precedentes, une derniere experimentation avait pour but d'evaluer si les isoflavones de soja etaient

In wissenschaftlichen wie auch in öffentlichen Kreisen wird kontrovers diskutiert, ob durch die Aufnahme von hormonell aktiven Substanzen aus Nahrungs‐ oder Nahrungsergänzungsmitteln mit positiven oder negativen Einflüssen auf die menschliche Gesundheit und Lebensqualität zu rechnen ist. Das Interesse an solchen Substanzen ist in den letzten Jahren permanent gewachsen, und der Markt erhielt einen zusätzlichen Zulauf, als Studien zeigten, dass die klassische Hormonersatztherapie für postmenopausale Frauen aus Östrogen‐Progestagen‐Kombinationen mit einem erhöhten Risiko für hormonabhängige Krebsarten wie Brust‐ oder Endometriumskarzinomen verbunden ist. Demgegenüber implizieren epidemiologische Beobachtungen aus ostasiatischen Ländern, dass die Aufnahme sojareicher Nahrung mit einer verringerten Inzidenz für diese hormonabhängigen Krebsarten korreliert zu sein scheint. Stofflich betrachtet stehen in diesem Zusammenhang Isoflavone wie Genistein und Daidzein, die in großen Mengen z. B. in Sojabohnen vorkommen, im Fokus des wissenschaftlichen Interesses. Eine Vielzahl von in vitro und in vivo Studien ergab für Isoflavone ein heterogenes Wirkspektrum. Neben hormonellen Wirkungen wie schwach östrogenen bzw. antiöstrogenen Eigenschaften, sind ebenfalls eine Reihe nicht‐hormoneller Wirkungen, wie beispielsweise antioxidative Eigenschaften oder die Fähigkeit zur Hemmung von Tyrosinkinasen, beschrieben. Trotz der hohen Anzahl an Studien zu Isoflavonen ist immer noch unklar wie valide die Beobachtungen beim Menschen sind, da ihre Langzeitwirkungen experimentell kaum untersucht sind. Daher war es das Ziel dieser Arbeit, zur Aufklärung der Langzeitwirkung von Isoflavonen als Nahrungsbestandteile und damit zum Verständnis ihrer Wirkmechanismen beizutragen, um anschließend mit den erzielten Resultaten die Risikobewertung dieser Substanzklasse zu optimieren. Zu diesem Zweck wurden in einem generationsübergreifenden Fütterungsexperiment die Einflüsse eines sojabasierten Futters mit denen eines, mit isoliertem Genistein supplementierten, Futters verglichen und anschließend denen eines phytoöstrogenfreien Futters gegenüber gestellt. Sowohl in juvenilen als auch adulten Ratten wurden die gewebespezifischen, physiologischen und molekularen Antworten des Uterus evaluiert. In einer zusätzlichen Studie sollte zudem geklärt werden, inwieweit Gestagene die genistein‐ bzw. östrogenregulierten Prozesse im Uterus adulter ovariektomierter Ratten zu modulieren vermögen. Anhand etablierter molekularbiologischer Methoden zur Gen‐ (qPCR) und Proteinexpression (Western Blot), aber auch durch neu zu etablierende immunohistochemische Analysen wurden diverse physiologische (z. B. Körpergewicht, Futteraufnahme, spezifische Organgewichte) und molekulare Marker (z. B. uterine Markergene für Östrogenität oder Proliferation) analysiert und untereinander verglichen. Bei der Betrachtung der Ergebnisse wird deutlich, dass die lebenslange, kontinuierliche Isoflavonexposition der Mütter während fetaler und pränataler Entwicklungsphasen unterschiedliche gewebespezifische Effekte in juvenilen und adulten Weibchen der Tochtergeneration auslöst. Während auf der einen Seite die bereits in utero begonnene Exposition mit Genistein in juvenilen Tieren in einer relativ starken Uterotrophie sowie östrogenen Effekten hinsichtlich der Expressionsmuster einiger Markergene resultiert, zeigt Genistein in den gleichen Tieren andererseits auch Eigenschaften die als antiproliferativ und antiöstrogen zu bewerten sind. Interessanterweise sind diese Effekte im adulten Organismus anschließend nicht mehr nachweisbar. Des Weiteren zeigen unsere Untersuchungen, dass die Östrogenresponsivität des juvenilen Uterus nicht durch diätetische Isoflavone modifiziert wird. Genistein wirkt in diesen Tieren als Östrogenrezeptor β Partialagonist, wobei das antiproliferative sowie antiöstrogene Potenzial erst in hohen Konzentrationen erkennbar wird. Im Gegensatz zum juvenilen zeigt sich im adulten Uterus eine erheblich gesteigerte Östrogenresponsivität infolge der lebenslangen, kontinuierlichen und bereits in utero begonnenen Isoflavon‐ bzw. Genisteinexposition. Diese Tiere reagieren mit dramatisch erhöhten Uterusfeuchtgewichten auf Östrogenrezeptor α vermittelte östrogene Stimuli. Bemerkenswerterweise sind diese stark erhöhten Uterusfeuchtgewichte jedoch nicht auf vermehrte Proliferation, sondern auf eine gesteigerte Einlagerung von Luminalflüssigkeit zurückzuführen. Analysen zur Genexpression von Aquaporinen deuten zwar auf östrogenabhängige Mechanismen hin, jedoch konnte kein direkter Bezug zur chronischen Isoflavon‐ bzw. Genisteinexposition nachgewiesen werden. Wie schon in den Untersuchungen am juvenilen Uterus beobachtet, vermittelt Genistein auch im adulten Uterus seine molekularen Wirkungen als tendenzieller Östrogenrezeptor β‐selektiver Partialagonist. Unsere Analysen der Expression spezifischer uteriner Markergene legen darüber hinaus die Vermutung nahe, dass Genistein seine molekularen Wirkungen präferenziell über den klassischen ERE‐abhängigen Signalweg vermittelt, da durch Genistein besonders Gene reguliert werden, die diese Transkriptionsfaktor Bindungsstellen in der Promotorregion besitzen. Die Rolle von Gestagenen bei genisteinregulierten Prozessen im Uterus ist nicht ganz klar. Während unsere Ergebnisse zeigen, dass Progesteron in einem kurzfristigen dreitägigen experimentellen Design, unabhängig von der Ab‐ bzw. Anwesenheit von 17β‐Östradiol, kaum einen Einfluss auf genisteinregulierte Prozesse hat, legen die Ergebnisse unserer längerfristigen Experimente hingegen die Vermutung nahe, dass Gestagene die chronischen Effekte von Isoflavonen modulieren können. Schlussfolgernd kann anhand unserer Studien festgehalten werden, dass die molekularen Wirkungen von Genistein stark von Faktoren wie der Expositionsdauer, der Expositionskonzentration sowie dem Alter des Organismus abhängen. Unsere Daten zeigen eindeutig, dass eine traditionelle sojabasierte ostasiatische Diät in keiner Weise mit einer, durch isolierte Isoflavone supplementierten, westlichen Diät gleichzusetzen ist. Des Weiteren konnten wir im Rahmen dieser Arbeit zeigen, dass die Exposition mit diätetischen Isoflavonen während der Embryogenese einen lebenslangen Einfluss auf die Nachkommen hat, indem die östrogenresponsivität dieser Individuen massiv beeinflusst wird.

Recent years have witnessed a tremendous growth in the number of gold-catalyzed highly selective chemical transformations. The aim of this study was to develop new gold catalytic methods to mediate carbon-carbon, carbon-oxygen and carbon-nitrogen bond forming reactions.
Chapter 2 describes new annulations catalyzed by gold(I) of simple o-hydroxyaldehydes with alkynes. This annulation efficiently generates isoflavanone-type structures, which have many possible applications in the synthesis of isoflavanone natural products. This chapter details the development and the optimization of the reaction conditions. The annulation was applied to various aldehydes and arylalkynes with good efficiency. Furthermore, it incorporated all atoms of both starting materials into the product and thus has a theoretical atom economy of 100%.
Chapter 3 extends the scope of the annulations developed in Chapter 2. Indeed, 2,3-dihydro-1-tosylquinolin-3(1H)-one derivatives, aza-isoflavanones, have been efficiently synthesized via a gold-catalyzed annulation of 2-tosylaminobenzaldehyde and alkynes. The annulation was applied to various arylalkynes with good efficiency. This annulation can be used for the synthesis of a range of potentially bioactive molecules.
Chapter 4 describes applications in the total synthesis of the annulation developed in chapter 2. (+/-)-Pterocarpan and analogues have been efficiently synthesized via the annulation of salicylaldehydes and o-methoxymethoxylphenylacetylene followed by a one-pot reduction and acidic cyclization of the ketones. In addition, isoflavone derivatives have been synthesized rapidly via the annulation of salicylaldehyde and arylacetylenes followed by IBX/DMSO oxidation of the isoflavanones.
As part of our ongoing studies on metal-catalyzed atom economical reactions, the gold/silver-catalyzed addition of arenes to mono-substituted allene, generate exclusive terminal addition products are described in Chapter 5.
Finally, Appendix A explores the use of commercially available polymer-supported triphenylphosphine as a catalyst for the efficient gamma-addition of methyl 2-butynoate with various pronucleophiles. In addition, optimization studies shows no need of a co-catalyst for the reaction in the presence of a mixture of water:toluene (5:1). The polymer-supported triphenylphosphine is recoverable and can be reused efficiently. This gamma-addition is highly atom-economical and the reaction time was reduced under microwave irradiation.

Soybean [Glycine max (L.) Merr.] seeds contain isoflavones that have positive impacts on human health. Field and greenhouse experiments were conducted in Quebec Canada to determine the effects of management and environmental factors [seeding date (late May and mid June), row spacing (20-, 40- and 60-cm), weeds (presence or absence), irrigation levels (low, moderate, and high) and genotypes (Proteina, Orford, and Golden)] and of foliar applications of elicitor compounds (i.e., LCOs, chitosan, and actinomycetes spores), on the isoflavone concentrations of mature soybean seeds, and other important seed characteristics. Our results indicated that environmental and agronomical factors have a great impact on soybean seed isoflavone concentrations of early maturity soybean cultivars. Year, seeding date, and weeds affected total and individual isoflavone concentrations, row spacing had no effect. Total isoflavone concentration was greater in 2003 than 2004. Seeding in mid June increased isoflavone concentration by 38%, compared to seeding in May. The presence of weeds increased total isoflavone concentrations by 9%. Isoflavone concentrations were significantly affected by cultivars and irrigation levels. In both of two growing seasons, Proteina had significantly greater isoflavone concentrations compared to Orford. Irrigation effects on isoflavone concentrations differed between years and cultivars. However, most responses were observed with the lower of the two irrigation levels, which increased isoflavone concentrations by as much as 60% compared to a non-irrigated control. Our results suggest that under greenhouse conditions most biotic elicitors tested increased the concentration of individual and total isoflavones in soybean seeds when compared to untreated control plants. LCOs proved to be the most effective in studies contrasting various elicitors. Response of field-grown plants was more variable than that of greenhouse-grown plants.

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A deficiência de micronutrientes afeta de maneira substancial o estado nutricional, a saúde e o desenvolvimento de uma parcela significativa da população de países desenvolvidos e em desenvolvimento. Os minerais, entre eles o ferro, desempenham funções essenciais à vida, o que justifica a grande atenção dada as carências deste nutriente. Dentre as estratégias utilizadas para o combate e prevenção das carências nutricionais, o enriquecimento de alimentos de baixo custo ou de grande consumo pelos grupos mais vulneráveis, tem se apresentado como uma alternativa eficaz. Resultados expressivos de redução dos casos de anemia ferropriva vêm sendo alcançados em programas de Política Nacional de Alimentação que prevêem a adoção de medidas essenciais, como o enriquecimento de alimentos amplamente utilizados pela população, tais como, o pão e o leite....
The deficiency of micronutrients affects substantially the nutricional state, the health and the development of a significant part of the population, both in developed and in developing countries. Minerals, among them the iron, play vital roles in life, which accounts for the great attention that has been given to the lack of these nutrients. Among the strategies used for prevention of malnutrition, the enrichment of low cost foods or greatly consumed ones by the most vulnerable groups has been shown as an efficient alternative. Outstanding results of reduction in cases of iron deficiency anemia have been reached in the Food National Politics programs that imply the adoption of essential policies, as for the enrichment of food widely consumed by the population, such as bread and milk. Therefore, the purpose of this paper was to study the sensorial viability of enrichment of low cost soy produts , such as milk, bread, yogurt ice cream and peanut candy like, from several sources of iron, as well as to quantify, through HPLC, the isoflavone contents in these products, aiming, if necessary, a further supplementation of those ones. The products have been enriched with three sources of iron FeSO4.7H2O, iron aminoacid chelate and NaFeEDTA and evaluated on the sensorial properties (difference from control and acceptance test) compared with a non-enriched sample (control). Isoflavones have been quantified through HPLC in a first stage in the products enriched with the three sources of iron and were subsequently analyzed in the following materials: soy grains with rind, peeled and cooked soy, okara soy residues, pasteurized soy milk and soy yogurt...(Complete abstract, click electronic address below).

Orientadores: Carla Beatriz Grespan Bottoli, Carol Hollingworth Collins
Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Quimica
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Resumo: O uso de padrões analíticos em análises cromatográficas é de fundamental importância para obter resultados analíticos confiáveis tanto em processos de validação de métodos quanto em análises rotineiras. Porém, o custo dos padrões de referência e sua disponibilidade para comercialização tornam o processo analítico muito caro e demorado. Neste trabalho foram obtidos padrões analíticos de isoflavonas de soja através da cromatografia líquida preparativa. Inicialmente foi desenvolvida uma método analítica por cromatografia líquida de alta eficiência, para separação e identificação de isoflavonas em extrato seco de soja. A seguir, foi realizada a transposição da escala analítica para a escala preparativa, iniciando pelo método da transposição direta. A caracterização e a pureza das isoflavonas obtidas foram verificadas a partir da pureza cromatográfica e pelos espectros na região do ultra violeta e visível, complementados pela espectrometria de massas e pela ressonância magnética nuclear. Os padrões obtidos por cromatografia preparativa apresentaram um teor de pureza de 93,1 % para daidzina, 99,8 % para daidzeína, 89,5 % para genisteína e 87,7 % para glicitina, permitindo, assim, o seu uso como padrões em análises rotineiras. Para demonstrar a aplicabilidade dos padrões obtidos, foi desenvolvido e validado um método para extração, hidrólise ácida e determinação das isoflavonas agliconas contidas em grãos de soja. Os resultados do conteúdo total de isoflavonas foram 283,5 ± 10,7 mg/100 g para soja não transgênica (BRS133) e 228,2 ± 13,8 mg/100 g para soja transgênica (BRS245RR), demonstrando diferenças significativas no conteúdo destas duas variedades de soja.
Abstract: The use of analytical standards in chromatographic analyses is very important to get trustworthy analytical results for validation of methodologies and for routine analyses. However, the cost of the available reference standards is high, prejudicing the overall analytical process. In this work, a transposition from analytical to preparative scale was carried out to obtain analytical standards of isoflavones from soybeans. An analytical methodology using high performance liquid chromatography (HPLC) was developed for separation and identification of isoflavones in dry soy extract. The transposition of the analytical parameters to the preparative scale was done initially through direct transposition. The characterization and purity of the isoflavones was determined by HPLC with spectra from a DAD detector, complemented by mass (MS+/-) and nuclear magnetic resonance spectrometries. The resulting isoflavone purities, after preparative separation and lyophilization, were 93.1 % for daidzin, 99.8 % for daidzein, 89.5 % for genistein and 87.7 % for glycitin, allowing their use as standards in routine analyses. To demonstrate the applicability of the standards obtained, an approach for extraction, acid hydrolysis and determination of the total amounts of isoflavone aglicones in soybeans was developed and validated. The results indicated total isoflavone contents of 283.5 ± 10.7 mg/100 g for non-transgenic (BRS133) and 228.2 ± 13.8 mg/100 g for transgenic (BRS245RR) soybeans, demonstrating significant differences in the isoflavone content of these two different soybeans.
Doutorado
Quimica Analitica
Doutor em Ciências

Red clover (Trifolium pratense L.) contains isoflavones, which are of interest because of their benefits for human health as well as their adverse effects on the fertility of farm animals. Isoflavone concentration in 10 cultivars ranged from 8923 to 12753 mug g-1 DM averaged across sites, harvests, and years. One cultivar, 'Start', distinguished itself with particularly low isoflavone levels. Concentrations varied according to plant part with leaves having the highest levels followed by stems and inflorescences, when averaged across maturity levels and cultivars (11970, 4896 and 3297 mug g-1 DM, respectively). Greatest content was found in leaves and stems during vegetative stages. Fresh herbage contained higher isoflavone content than either silage or hay (14464, 12200 and 11604 mug g-1 DM, respectively). Foliar application of yeast extract, chitosan, and acetic acid elicitors overall showed modest (12-15%) increases in isoflavone concentration over untreated control plants. Differences were not observed between elicitor concentrations used.

Title from PDF of title page (University of Missouri--Columbia, viewed on Feb 23, 2010). The entire thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file; a non-technical public abstract appears in the public.pdf file. Dissertation advisor: Dr. David A. Sleper; Dr. Henry T. Nguyen. Vita. Includes bibliographical references.

El proyecto pretende valorar una nueva terapia en el tratamiento del trastorno por consumo de cocaína. Recientemente se ha descubierto que un inhibidor de la enzima aldehído deshidrogenasa-2 reduce el consumo de cocaína en animales (ALDH2). Se trata de un derivado de la daidzina-daidzeína, una de las isoflavonas que contiene la soja. Los extractos de soja y la daidzina-daidzeína se han utilizado para el tratamiento del alcoholismo en animales y humanos. Se considera de interés conocer si puede ser eficaz en el trastorno por consumo de cocaína y si interfiere en el metabolismo del acetaldehído. Se desarrollaron tres ensayos clínicos en humanos: uno de farmacocinética a dosis única de diferentes preparados de extracto de soja para elegir uno y ser usado en los otros ensayos; otro mecanístico para evaluar la interacción entre el extracto de soja y el alcohol; y un tercero exploratorio para valorar la eficacia de un inhibidor natural de la ALDH2 (daidzina-daidzeína) en pacientes con trastorno por consumo de cocaína. El objetivo del estudio era evaluar la eficacia de un inhibidor selectivo de la ALDH2 de origen natural (daidzina-daidzeína, contenida en extracto de soja) en el tratamiento del Trastorno por Consumo de Cocaína y conocer si interfiere en el metabolismo del acetaldehído. Con la administración concomitante de extracto de soja y alcohol, no se observó alteración de signos vitales ni efectos subjetivos o adversos; por otro lado, ningún voluntario afecto de Trastorno por consumo de cocaína adquirió abstinencia completa, pero se observó una alta retención al tratamiento, una disminución de la severidad del consumo en la escala Severity Dependence Scale (SDS) y mejoría del estado de salud física en distintas áreas del SF-36 Health Survey. Nuestros resultados demuestran la no interferencia de daidzina-daidzeína con el metabolismo del acetaldehído y sugieren una posible función reduciendo la gravedad del consumo de cocaína. Así, la comorbilidad alcohol-cocaína, no sería criterio de exclusión en el posible uso de daidzina-daidzeína como tratamiento del consumo de cocaína.
The project aims to evaluate a new therapy in treating cocaine use disorder. Recently it was discovered that an inhibitor of the enzyme aldehyde deshidrogenada-2 (ALDH2) reduces cocaine use in animals. It is a derivative of daidzin-daidzein, one of the isoflavones contained in soy. Daidzin-daidzein and soy extracts have been associated with reductions of alcohol use in animals and humans. It is interesting to know whether it can be effective in cocaine use disorder and if it interacts in the metabolism of acetaldehyde. We carried out three clinical trials in humans: a single dose pharmacokinetics of different soy extract prepared to choose one to use in the other clinical trials; another mechanistic to assess the interaction between soy extract and alcohol; and a third exploratory to evaluate the efficacy of an inhibitor of aldehyde deshidrogenase-2 (daidzin-daidzein) in patients with a cocaine use disorder. The objective of the study was to evaluate the efficacy of an inhibitor of aldehyde deshidrogenase-2 (daidzin-daidzein, contained in soy extract) in the treatment of the cocaine use disorder and to know if interacts with the acetaldehyde metabolism. With the administration of soy extract and alcohol, it was not observed disturbance of the vital sings and subjective or adverse effects; on the other hand, no volunteer with cocaine use disorder reached hole abstinence, but a high retention in treatment, a decrease of the Severity Dependence Scale (SDS) and an improvement in the different areas of the SF-36 Health Survey was observed. Our results demonstrate that daidzin-daidzein did not interfere in the acetaldehyde metabolism and suggest a possible function decreasing the severity of cocaine consumption. Cocaine-alcohol comorbidity, would not be an exclusion criteria in the possible use of daidzin-daidzein like a treatment for cocaine consumption.

Thesis (Ph. D.)--Ohio State University, 2003.
Title from first page of PDF file. Document formatted into pages; contains xxiii, 249 p. : ill. Advisor: Robert W. Brueggemeier, College of Pharmacy. Includes bibliographical references (p. 234-249).

Cardiovascular diseases (CVD) are the leading cause of morbidity and mortality in the United States. It is also well recognized that the incidence of CVD is substantially increased in postmenopausal women due to the loss of estrogen. Experimental and clinical data support vascular protective effects of estrogen by various mechanisms. However, administration of estrogen is also associated with an increased incidence of heart disease which limits its therapeutic potential. Given the demonstrated risks of conventional estrogen therapy, a search for novel, cost-effective, alternative vasoactive agents for prevention of CVD is of major importance in the effort to decrease the burden of CVD morbidity. Genistein, a major soy isoflavone, may be one of those alternative agents because of its selective affinity to estrogen receptor-beta and various beneficial effects on CVD. However, the mechanism of the cardioprotective effects of genistein is still unclear. The objectives of this study were (1) to investigate the effect of genistein on the expression of endothelial nitric oxide synthase (eNOS) both in vitro and in vivo; (2) to define the mechanism by which genistein regulates eNOS expression; and, (3) to examine whether genistein protects against tumor necrosis factor-alpha (TNF-α)-induced apoptosis in human aortic endothelial cells (HAECs). The results demonstrated that genistein, at physiologically achievable concentrations (1-10 μM) in individuals consuming soy products, enhanced the expression of eNOS protein and subsequently elevated nitric oxie (NO) synthesis in both HAECs and human umbilical vein endothelial cells, concomitant with the increased eNOS mRNA expression (2.6-fold of control) and eNOS promoter activity, suggesting that genistein activates eNOS transcription. Furthermore, dietary supplementation of genistein to spontaneously hypertensive rats restored aortic eNOS levels, improved aortic wall thickness, and alleviated hypertension, confirming the biological relevance of the in vitro findings. However, the effects of genistein on eNOS and NO were not mediated by activation of estrogen signaling, mitogen-activated protein kinase, phosphatidylinositol 3-kinase/Akt kinase, protein kinase C or inhibition of typrosine kinases, but possibly through activating the cAMP/protein kinase A/cAMP responsive elemant binding protein pathway. These data suggest that genistein has direct genomic effects on the vascular wall that are unrelated to its known actions, leading to increase in eNOS expression and NO synthesis, thereby improving vascular homeostasis. We also found that genistein (5-10 μM) significantly inhibited TNF-α-induced apoptosis in HAECs as determined by caspase-3 activation, apoptotic cell detection and DNA laddering. The anti-apoptotic effect of genistein was associated with an enhanced expression of anti-apoptotic Bcl-2 protein and its promoter activity that was ablated by TNF-α. Moreover, this anti-apoptotic effect of genistein was not mediated by extracellular signal-regulated kinase 1/2, protein kinase A, or estrogen receptor. However, inhibition of p38 mitogen-activated protein kinase (p38) by SB203580 completely abolished the cytoprotective effect of genistein, suggesting that genistein acted through the p38-dependent pathway. Accordingly, stimulation of HAECs with genistein resulted in rapid and dose-dependent activation of p38. Unlike TNF-α which specifically activated p38α, genistein selectively induced phosphorylation of p38β, suggesting that p38β, but not p38α, is essential for the cytoprotective effect of genistein. These findings provide the evidence that genistein acts as a survival factor for vascular ECs to protect cells against apoptosis via activation of p38β. Taken together, the resuls of the present study suggest that genistein can act directly on vascular ECs, improves endothelium homeostasis by promoting eNOS expression and endothelial-derived NO synthesis through activating the cAMP/PKA/CREB cascade, and protects against TNF-α-induced apoptosis via activation of p38 β. These data potentially provide a basic mechanism underlying the physiological effects of genistein in the vasculature.
Ph. D.

A balanced diet and an appropriate feeding are the two most important requirements of aquaculture. Research on feed, quality control and biological evaluation are important to formulate correct diets because without correct and suitable feeding, fish are unable to stay healthy and productive. Furthermore, quality and composition of feeds deeply affect the nutritional and organoleptic characteristics of the final product. In the last years, fish meal has been gradually and partially substituted by plant-based products, because of their improved sustainability and lower costs. Soybean meal is the principal vegetable protein source, due to its high protein content, good amino acid profile, high digestibility, low cost, consistency and availability. Soybean meal contains isoflavones which are polyphenolic compounds (genistein, daidzein, glycitein) that, as phytoestrogens, may act as estrogen receptor agonists or antagonists causing biological activity on fish in addiction to possible changes on the fillet quality. The potential effects of isoflavones may affect the soybean meal utilization as an alternative protein source in aquaculture feed. In the meantime, it is important, for consumer safety, to evaluate the potential amount of the isoflavones in edible tissue and their antioxidant effect that could enhance the product shelf life. Our objective was to examine, on the one hand, the effect of dietary isoflavones on growth, reproduction and health in rainbow trout (Oncorhynchus mykiss), and on the other hand, on the quality of fish food. Rainbow trout were fed three experimental diets containing different concentrations of isoflavones (0, 500, 1500 ppm) for 70 days. Growth, estrogenic activity expressed by vitellogenin (VTG) protein levels in plasma and liver, plasma 17β-estradiol (E2) levels, gonadal development, state of stress and histological changes in various tissues were evaluated in a subsample of the animals at the end of feeding trial. Complementary analyses on growth performance, proximate composition, lipid oxidation and isoflavone deposition were carried out on the remaining part of the animals for each treatment, after being stored at 4 °C for 1 and 7 days. Neither growth performance nor the relative mRNA levels of the liver Insulin Growh Factor I (Igf-I) were influenced by different levels of dietary isoflavones. Plasma and liver VTG protein levels and plasma E2 were unaffected by the treatments and the correlation between E2 plasma levels and VTG densitometry values was significant (p < 0.05). The fish gonadosomatic index (GSI) did not significantly differ among the three experimental groups but correlated with plasma VTG densitometry values (p < 0.05). Plasma, muscle and fin cortisol concentrations fell within the normal welfare range and were not correlated to isoflavone levels. Histologically, the distal intestine showed a normal morphology with well-differentiated enterocytes, as well as the liver showed normal hepatocytes. A supranuclear accumulation of lipid droplets in enterocytes and some lipid droplets in hepatocytes were observed in all the tested groups, suggesting an impact of basal dietary lipid on transport/metabolism of fat in the fish. Inclusion and different levels of isoflavones in diets did not affect the performance of trout used for final product analysis either. Indeed, trout showed similar body weight at caught, 337 g on average, and an average gutted body weight of 299 g. Biometric indexes, skin and fillet colour, rheological characteristics, proximate composition and fatty acids profile of fillet were not affected by the different dietary treatments. Otherwise, time of storage reduced fillet yield (56.4 vs. 53.6%; p < 0.01), and skin lightness (59.2 vs. 51.5; p < 0.001); red index moved to more negative values (p < 0.001) and yellow index decreased (6.99 vs. 5.07; p < 0.001). Fillet pH (6.22 vs. 6.34; p < 0.001) and lightness increased (38 vs. 43.6; p < 0.001) while yellow index (6.20 vs. 4.52; p < 0.001) and shear force decreased (0.94 vs. 0.80 g kg-1; p < 0.001). According to the time of storage, trout fillet showed an increase of water, a loss of crude protein and an increase of total volatile basic nitrogen content (19.3 vs. 21.2 mg 100 g-1; p < 0.001). As a result of PUFAs omega-6 decrease (13.1% vs. 12.7%; p < 0.05), omega-3/omega-6 ratio increased (1.05 vs. 1.20; p < 0.05) during storage. The results evidenced an accumulation of isoflavones on trout fillets, even if isoflavone concentration in fillets was not significantly influenced by the content of dietary isoflavones in both storage times. Lipid peroxidation expressed as thiobarbituric acid reactive substances (TBARS) levels in fillet on day 1 of refrigeration was significantly higher than that at day 7 (p < 0.05) but no difference resulted among groups fed different isoflavone levels, evidencing no effect of feed soy isoflavones on fillet lipid oxidation. Overall, these results suggest that the isoflavone tested doses do not compromise rainbow trout reproduction, growth and health; although a moderate transfer of isoflavones from diet to fillet was observed, quality, technological and nutritional characteristics and lipid oxidation, were not affected by dietary treatment. Our data demonstrate that, with regard to isoflavone contents and their potential effects, soybean meal can replace fish meal in rainbow trout diets at a high level with no negative effects on fish performance and final product quality.
Una dieta bilanciata e un’appropriata alimentazione sono le due maggiori richieste da parte dell’acquacoltura. Gli studi scientifici sui mangimi, il controllo qualità e la valutazione dell’aspetto biologico sono importanti per formulare una corretta e idonea dieta per le specie ittiche. Senza un’adeguata alimentazione, il pesce non può crescere nei tempi stabiliti e mantenersi in salute, oltre al fatto che la qualità e la composizione dei mangimi influenzano profondamente le caratteristiche nutrizionali e organolettiche del prodotto finale. Negli ultimi anni, la farina di pesce è stata sostituita gradualmente e parzialmente dai prodotti derivati dalle piante, per il loro basso costo e la loro sostenibilità rispetto alla farina di pesce. La farina di soia è la principale risorsa proteica vegetale per l’alimentazione animale, per il suo elevato contenuto proteico, il buon profilo amminoacidico, l’elevata digeribilità, il costo ridotto, nonché l’ampia disponibilità. La farina di soia contiene isoflavoni (genisteina, daidzeina, gliciteina) che sono composti polifenolici nonché fitoestrogeni ed in quanto tali possono agire come agonisti o antagonisti dei recettori degli estrogeni endogeni, agendo biologicamente sulla specie ittica e modificando le caratteristiche qualitative del prodotto finale. I potenziali effetti degli isoflavoni possono influenzare l’utilizzo della farina di soia come risorsa proteica alternativa nei mangimi per pesci. Risulta importante valutare per la sicurezza del consumatore anche il potenziale ammontare degli isoflavoni nel tessuto edibile della specie alimentata con la soia, oltre che il loro effetto antiossidante che potrebbe aumentare la shelf life del prodotto. Il nostro obiettivo si prefiggeva, da un lato, di esaminare l’effetto degli isoflavoni della soia sulla crescita, la riproduzione e la salute della trota iridea (Oncorhynchus mykiss), dall’altro, di valutare la qualità della carne del pesce. Le trote sono state alimentate con tre diete contenenti differenti concentrazioni di isoflavoni (0, 500, 1000 ppm) per un tempo di 70 giorni. Al termine della prova di alimentazione, una parte delle trote sperimentali sono state utilizzate per valutare la crescita, l’attività estrogenica (espressa in livelli di proteine del tuorlo nel plasma e nel fegato), i livelli plasmatici di estradiolo, lo sviluppo gonadico, lo stato di stress e i cambiamenti istologici nei vari tessuti. Analisi complementari su performance di crescita, composizione prossimale, ossidazione lipidica e deposito degli isoflavoni nel filetto sono state effettuate sulla rimanente parte delle trote, dopo la loro conservazione a 4°C, per 1 e 7 giorni. I diversi dosaggi di isoflavoni non hanno influenzato né la performance di crescita né le concentrazioni di mRNA del Fattore di Crescita Insulino Simile (Igf-I) nel fegato. I livelli della vitellogenina (VTG) in plasma e fegato e di estradiolo plasmatico (E2) non sono stati condizionati dalle diete utilizzate, inoltre la correlazione tra livello plasmatico di E2 e i valori densitometrici della VTG si è rivelata significativa (p < 0.05). L’indice gonadosomatico (GSI) delle trote non ha mostrato differenze tra i tre gruppi sperimentali ed è risultato correlato con i valori densitometrici della VTG plasmatica (p < 0.05). Le concentrazioni di cortisolo rilevate in plasma, muscolo e pinna si sono collocate in un range di valori bassi, compatibili con una condizione di benessere, e non hanno mostrato correlazioni con il livello di isoflavoni nelle diete. Dal punto di vista istologico, l’intestino distale presentava una morfologia normale con enterociti ben differenziati, così come il fegato che, a sua volta, mostrava epatociti normali. In tutti i gruppi testati, si è evidenziato un accumulo sopranucleare di gocce lipidiche che suggerisce un effetto dei lipidi della dieta basale sul trasporto/metabolismo dei grassi nel pesce. L’inclusione di differenti livelli di isoflavoni nelle diete non ha influenzato nemmeno la performance delle trote utilizzate per l’analisi del prodotto finale in momenti diversi durante la conservazione. Infatti, le trote mostravano un simile peso del corpo alla cattura, con una media di 337 g, ed una media del peso del corpo eviscerato di 229 g. Gli indici biometrici, il colore della pelle e del filetto, le caratteristiche reologiche, la composizione chimica e il profilo acidico dei grassi del filetto non sono stati modificati dai diversi trattamenti alimentari. D’altro canto, il tempo di conservazione ha ridotto significativamente la resa dei filetti (56.4 vs. 53.6%; p < 0.01) e la luminosità della pelle (59.2 vs 51.5; p < 0.001), l’indice del rosso ha mostrato valori più negativi (p < 0,001) e l’indice del giallo è diminuito (6.99 vs. 5.07; p < 0.001). Il pH del filetto (6.22 vs. 6.34; p < 0.001) e la sua luminosità sono aumentati (38 vs. 43.6; p < 0.001), mentre l’indice del giallo (6.20 vs. 4.52; p < 0.001) e lo sforzo di taglio sono diminuiti (0.94 vs. 0.80 g kg-1; p < 0.001). Sempre sulla base del tempo di conservazione, il filetto ha mostrato un aumento della percentuale d’acqua, una perdita in proteine ed un aumento del contenuto di azoto basico volatile (19.3 vs. 21.2 mg 100 g-1; p < 0.001). Durante la conservazione, inoltre, è risultata una diminuzione degli acidi grassi polinsaturi (PUFA) omega-6 (13.1% vs. 12.7 %; p < 0.05) e un aumento del rapporto omega-3/omega-6 (1.05 vs. 1.20; p < 0.05). I risultati hanno evidenziato, per entrambi i tempi di conservazione, un accumulo di isoflavoni nei filetti di trota, anche se la concentrazione non è stata influenzata significativamente dal contenuto degli isoflavoni presenti nelle diete. L’ossidazione lipidica, espressa come livelli di TBARS (thiobarbituric acid reactive substances), dei filetti conservati dopo 1 giorno di conservazione a 4 °C, è risultata significativamente più alta che al giorno 7 (p < 0.05), ma nessuna differenza si è riscontrata tra i gruppi alimentati con diversi livelli di isoflavoni, evidenziando nessun effetto da parte degli isoflavoni della soia sull’ossidazione lipidica del filetto. I risultati ottenuti sembrano indicare che gli isoflavoni, alle dosi testate, non compromettano la riproduzione, la crescita e la salute della trota; anche se si è osservato un moderato trasferimento degli isoflavoni dalla dieta al filetto, la qualità, le caratteristiche nutrizionali e l’ossidazione lipidica non risultano influenzati dal trattamento alimentare. In merito al contenuto di isoflavoni nella farina di soia e al loro potenziale effetto, i nostri risultati dimostrano che la farina di pesce può essere sostituita dalla farina di soia nelle diete somministrate alle trote senza aver effetti negativi sulla performance e sulla qualità del prodotto finale.

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
Avaliar o efeito da isoflavona da soja sobre o tecido mamário em mulheres na pós-menopausa. Trata-se de estudo clínico, prospectivo, randomizado, duplo-cego, placebo controlado, envolvendo 80 mulheres na pós-menopausa com sintomas vasomotores, idade entre 45 a 70 anos, acompanhadas no Ambulatório de Climatério e Menopausa da Faculdade de Medicina de Botucatu-Unesp, de janeiro de 2005 a dezembro de 2006. Na randomização, 40 pacientes receberam 100 mg isoflavona da soja/dia (duas cápsulas de 125 mg de extrato seco de glicine Max) e 40 pacientes placebo (duas cápsulas de lactose) durante 10 meses. A densidade mamária (DM) foi avaliada pela mamografia e o parênquima mamário pela ultrassonografia de mamas no início e após dez meses de seguimento. Para análise estatística foram utilizados o teste t-Student, ANOVA, teste de Mann-Whitney e teste do Qui-Quadrado. Na comparação das características clínicas iniciais entre os grupos de usuárias de isoflavona e placebo, não houve diferenças significantes, com valores médios de idade de 55,1±6,0 e 56,2±7,7 anos, tempo de menopausa de 6,6±4,8 e 7,1±4,2 anos e IMC de 29,7±5,0 e 28,5±4,9 kg/m2, respectivamente (p>0,05). Concluíram o estudo 32 pacientes sob isoflavona e 34 sob placebo. Na comparação da densidade mamográfica entre os momentos inicial e final, não houve diferença estatisticamente significativa. Na avaliação do parênquima mamário pela ultrassonografia, não houve diferença entre os grupos. Na comparação entre os momentos dentro de cada grupo, não foram constatadas diferenças significativas nos parâmetros da mamografia e ultrassonografia. No período de 10 meses, o uso de isoflavona de soja não modificou o tecido mamário, avaliado pela mamografia e ultrassonografia, em mulheres na pós-menopausa
To evaluate the effect of soy isoflavone on breast tissue in postmenopausal women. This study is a prospective, randomized, double-blind, placebo-controlled trial involving 80 postmenopausal women with vasomotor symptoms, aged 45-70 years, followed in Climacteric and Menopause Clinic of the Botucatu Medical School-UNESP, from January 2005 to December 2006. At randomization, 40 patients received 100 mg of soy isoflavone/day (two capsules of 125 mg standardized soy extract, Glicine max) and 40 patients, placebo (two capsules of lactose) for 10 months. The breast density was evaluated by mammography and breast parenchyma by ultrasound, at baseline and after ten months of follow-up. The Student t-test, ANOVA, Mann-Whitney and Chi-Square were used in the statistical analysis. In comparison of baseline clinical characteristics between the isoflavone and placebo groups, there were no significant differences, with mean age of 55.1 ± 6.0 and 56.2 ± 7.7 years, duration of menopause 6.6 ± 4.8 and 7.1 ± 4.2 years and BMI 29.7 ± 5.0 and 28.5 ± 4.9 kg/m2, respectively (p> 0.05). Concluded the study, 32 patients on isoflavone and 34 in the placebo group. In comparison in mammographic density (MD) between moments, baseline and final, there was no difference statistically significant. In the evaluation of breast parenchyma by ultrasound, there was no difference between groups. In comparing the moments within each group, there were no significant differences in the parameters of mammography and ultrasound. In 10 months, the use of soy isoflavone did not affect breast tissue, as assessed by mammography and ultrasound, in postmenopausal women

Orientador: Hélia Harumi Sato
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos
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Resumo: O presente trabalho visou o estudo da produção da ?-glicosidase pela linhagem de Aspergillus sp. utilizando-se resíduos agrícolas como o farelo de trigo, casca de maracujá e bagaço de cana-de-açúcar, a imobilização da enzima em diferentes suportes como lentes PVA - Lentikats®, sol-gel, Eupergit, Amberlite, gelatina e alginato de cálcio, e a aplicação da enzima livre e imobilizada na conversão de isoflavonas glicosiladas de soja em isoflavonas agliconas. O fungo foi identificado como Aspergillus niger LBA 02. O extrato enzimático bruto obtido de A. niger LBA 02 apresentou atividade de ?-glicosidase, CM -celulase, amilase, poligalacturonase e pectinase. Foi obtida maior atividade de ?-glicosidase (44,81 U/g) na fermentação da linhagem A. niger LBA 02 em meio semissólido composto por 25 g de farelo de trigo e 5,7 mL de água destilada, após 240 h de fermentação a 30°C. Os efeitos da adição do extrato de levedura e dos sais KH2PO4, NH4NO3, MgSO4.7H2O, no meio de farelo de trigo, para a produção de ?-glicosidase por A. niger LBA 02 não foram significativos nos níveis estudados. Os resíduos casca de maracujá e bagaço de cana-de-açúcar adicionados no meio de farelo de trigo não atuaram como indutores da produção de ?-glicosidase, nos níveis estudados. Dentre os 7 métodos de imobilização testados, as técnicas de sol-gel e lentes PVA - Lentikats® apresentaram melhores resultados para a imobilização da enzima ?-glicosidase. A enzima livre apresentou atividade ótima a 65°C e pH 4,5, enquanto a enzima imobilizada em sol-gel mostrou atividade ótima na faixa de 60 - 65°C. A temperatura de 50°C foi fixada como temperatura ótima de trabalho para a enzima imobilizada em lentes PVA - Lentikats®, pois acima da temperatura de 60°C ocorreu a fusão das lentes. A imobilização da ?-glicosidase não alterou o pH ótimo de atividade da enzima, permanecendo em 4,5. A imobilização resultou em um pequeno aumento da estabilidade térmica da ?-glicosidase. A ?-glicosidase imobilizada em lentes PVA - Lentikats® apresentou-se mais estável do que a enzima livre, após 3 h de tratamento na faixa de 45 a 55°C. O tempo de meia vida da ?-glicosidase imobilizada em sol-gel a 70°C foi 0,88 h. A ?-glicosidase imobilizada em sol-gel apresentou cerca de 10% de atividade residual após 3 h a 70°C, enquanto que a enzima livre foi inativada após 1 hora a 70°C. A ?-glicosidase imobilizada em sol-gel apresentou valores de KM na faixa de 1,0 a 1,25 mM de celobiose, a 60°C, estimados pelos métodos de Lineweaver - Burk, Hanes - Woolf e Solver, enquanto que a enzima livre apresentou valores de 0,92 a 1,69 mM de celobiose, sugerindo que não houve alteração da afinidade da enzima pelo substrato celobiose com a imobilização. A imobilização da enzima em lentes PVA - Lentikats® resultou em um aumento dos valores de KM estimados em 3,61; 2,7 e 3,03 mM de celobiose, a 50°C, respectivamente, pelos métodos de Lineweaver - Burk, Hanes - Woolf e Solver indicando que a imobilização resultou em diminuição da afinidade da enzima imobilizada pelo substrato. A taxa de conversão relativa da solução 1,5 mM de celobiose em tampão acetato 0,5 M pH 5,0 a 50°C, utilizando-se ?-glicosidase imobilizada em lentes PVA - Lentikats® em processo contínuo foi de 100% após 5 h, entretanto a porcentagem de conversão diminuiu para 40% após 148 h. A ?-glicosidase livre e imobilizada produzida pelo micro-organismo A. niger LBA 02 foi capaz de hidrolisar as isoflavonas glicosiladas de soja em suas formas agliconas. O teor da isoflavonas agliconas aumentou no extrato de isoflavonas de soja tratadas com ?-glicosidase livre e imobilizada em lentes PVA - Lentikats® e sol-gel, sendo que a daidzeína aumentou aproximadamente 2,6; 10,8 e 12,2 vezes e o teor de genisteína aumentou 11,7; 11,4 e 11,4 vezes quando aplicada a enzima ?-glicosidase livre e imobilizada em lentes PVA - Lentikats® e sol-gel, respectivamente, ao final de 24 h
Abstract: This work aimed to study the production of ?-glucosidase by Aspergillus sp. strain using agricultural residues such as wheat bran, passion fruit peel and sugarcane bagasse, the immobilization of the enzyme in different support such as lens - shaped PVA - Lentikats®, sol-gel, Eupergit, Amberlite, gelatin and calcium alginate and the application of free and immobilized enzyme in the conversion of isoflavone glucosides in soy isoflavone aglycones. The fungus was identified as Aspergillus niger LBA 02. The crude enzyme extract obtained from A. niger LBA 02 showed activity of ?-glucosidase, CM-cellulase, amylase, polygalacturonase and pectinase. It was obtained higher ?-glucosidase activity (44.81 U / g) in the fermentation of strain A. niger LBA 02 in semisolid media composed of 25 g of wheat bran and 5.7 mL of distilled water, after 240 h of fermentation at 30 °C. The effects of the addition of yeast extract and salts KH2PO4, NH4NO3, MgSO4.7H2O in wheat bran medium for production of ?-glucosidase by A. niger LBA 02 were not significant in the levels studied. The passion fruit peel waste and sugarcane bagasse added in the culture media of wheat bran did not act as inducers of the production of ?-glucosidase levels studied. Among the methods of immobilization tested, the sol-gel technique and lens - shaped PVA - Lentikats® showed better results for ?-glucosidase immobilization. The free enzyme showed optimum activity at 65 °C and pH 4.5, while the enzyme immobilized in sol-gel showed optimum activity in the range of 60 - 65 °C. A temperature of 50 °C was set as the working optimum temperature for the enzyme immobilized in lens - shaped PVA - Lentikats® because above 60 °C occurred melting of the lenses. Immobilization of ?-glucosidase did not alter the optimum pH of the enzyme activity, remaining at pH 4.5. Immobilization resulted in a small increase in the thermal stability of ?-glucosidase. The ?-glucosidase immobilized in lens - shaped PVA - Lentikats® showed to be more stable than the free enzyme after 3 h of treatment in the range of 45 to 55 °C. The half-life of the ?-glucosidase immobilized in sol-gel at 70 °C was 0.88 h. The ?-glucosidase immobilized in sol-gel showed about 10% residual activity after 3 h at 70 °C while free enzyme was inactivated after 1 h at 70 °C. The ?-glucosidase immobilized in sol-gel showed KM in the range of 1.0 to 1.25 mM of cellobiose at 60 °C, estimated by the method of Lineweaver - Burk, Hanes - Woolf and Solver while free enzyme showed KM values in the range of 0.92 to 1.69 mM of cellobiose, suggesting no change in the affinity of the enzyme for the substrate cellobiose after immobilization. The immobilized enzyme in lens - shaped PVA - Lentikats® resulted in an increase in the KM values estimated 3.61, 2.7 and 3.03 mM of cellobiose at 50 °C, respectively, by methods Lineweaver - Burk, Hanes - Woolf and Solver indicating that immobilization resulted in decreasing affinity of the immobilized enzyme to the substrate. Relative conversion rate of 1.5 mM cellobiose solution in acetate buffer 0.5 M pH 5.0 at 50 °C, using ?-glucosidase immobilized in lens - shaped PVA - Lentikats® in continuous process was 100% after 5 h, but the conversion decreased to 40% after 148 h. The ?-glucosidase produced by the micro-organism A. niger LBA 02, free and immobilized was able to hydrolyze isoflavone glycosides from soy in their aglycone forms. The content of isoflavone aglycones increased in soy isoflavones extract treated with ?-glucosidase free and immobilized in lens - shaped PVA - Lentikats® and sol-gel, and the daidzein increased approximately 2.6, 10.8 and 12.2 times, and genistein increased content of 11.7, 11.4 and 11.4 fold when applied to ?-glucosidase enzyme free and immobilized in lens - shaped PVA - Lentikats ® and sol-gel, respectively, at the end of 24 h
Doutorado
Ciência de Alimentos
Doutora em Ciência de Alimentos

Fitoestrógenos são compostos naturais de origem vegetal com atividade estrogênica. Estão sendo amplamente investigados para a prevenção de doenças crônicas, coronarianas, câncer de próstata e mama, na redução de riscos de osteoporose e alívio nos sintomas da menopausa. Entre os fitoestrógenos já utilizados encontram-se a daidzeína, genisteína e gliciteína em matrizes complexas como drogas e extratos vegetais, cápsulas e comprimidos, requerendo desenvolvimento e validação de metodologias para a determinação das isoflavonas. As metodologias propostas foram a cromatografia em camada delgada de alta eficiência (CCDAE) e a cromatografia líquida de alta eficiência (CLAE). A fase móvel acetato de etila:hexano (8:2 v/v) foi utilizada para determinação do perfil cromatográfico das isoflavonas agliconas daidzeína, gliciteína e genisteína e a fase móvel acetato de etila:tolueno:ácido fórmico (8: 1: 1 v/v/v) para isoflavonas glicosiladas e não glicosiladas por CCDAE. Para a determinação quantitativa das isoflavonas glicosiladas por CLAE foi proposta uma hidrólise ácida com HCl 3M e aquecimento em banho-maria durante uma hora, como tratamento prévio. A determinação analítica das isoflavonas daidzeína, genisteína, formononetina e biochanina A por CLAE, em modo isocrático foi realizada utilizando coluna cromatográfica monolítica Chromolith® RP-18, 100-4,6mm, fase móvel constituída por água:acetonitrila (6:4 v/v), vazão 0,6mL/min e detecção a 260nm. Os resultados obtidos mostraram linearidade com coeficiente de correlação de 0,9995 para daidzeína, 0,9996 para genisteína, 0,9997 para formononetina e 0.9999 para biochanina A. A precisão e a exatidão apresentaram resultados satisfatórios. Boa resolução e rápida separação dos fármacos em formulações farmacêuticas também foram obtidas. Portanto, pode ser usado nas análises de rotina nos laboratórios de controle de qualidade de fitoterápicos.
Phytoestrogens are natural compounds of plants with estrogenic activity. They are being widely investigated in the prevention of chronic coronary diseases, in prostate and breast cancer, in the reduction of osteoporosis risk and relief of menopause symptoms. Among phytoestrogens already in use are daidzein, genistein and glycitein and they are present in complex matrix such as phytopharmaceuticals, plant extracts, capsules and tablets. These preparations require development and validation of methodologies for quantitative determination of isoflavones. The proposed methodologies include high performance thin layer chromatography (HPTLC) and high performance liquid chromatography (HPLC). The HPTLC coupled with densitometry can be used for quantitative analysis. The mobile phase constituted of ethyl acetate:hexane (8:2 v/v) was used to determine chromatographic profiles of isoflavone aglycones, daidzein, glycitein and genistein. The mobile phase constituted of ethyl acetate:toluene:formic acid (8:1:1 v/v/v) was used for determination of isoflavones glycosides and non-glycosides. For the quantitative determination of isoflavone glycosides with HPLC, an acid hydrolysis with 3M HCl and heating in water-bath for an hour was proposed as sample pretreatment step. The analytic determination of isoflavones daidzein, genistein, formononetin and biochanin A using HPLC was accomplished. The chromatography was carried out in isocratic mode with Chromolith®, a monolithic RP-18 column, (100x4.6mm) with mobile phase constituted of water:acetonitrile (6:4 v/v) operated at a flow rate of 0.6mL/min and detection was made at 260nm. The results showed method linearity with correlation coefficient of 0.9995 to daidzein, 0.9996 for genistein, 0.9997 for formononetin and 0.9999 for biochanin A. The precision and accuracy data presented satisfactory results. Good resolution and faster separation of compounds in pharmaceutical formulations were also obtained. The proposed method can be used in the routine analyses of phytopharmaceuticals in quality control laboratories.

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
The objective of this work was to compare the chemical composition of the soy in conversion for organic agriculture - 1° year in conversion (C1), 2° year in conversion (C2) and 3° year in organic conversion or (C3) - being known the data meteorological of the culture place. To cultivate used it was the CD-216, proceeding from harvest 2005/2006, the City of Santa Helena/PR. The studied variables had been the text of humidity and the centesimal composition (protein, oil, ashes and carbohydrates), the mineral text (P, K, Ca, Mg, Zn, Cu, Fe and Mn) and the text of isoflavones of the grains. Considering the three years in conversion, some factors had differed significantly. The text of humidity in C1 (8,43 %) differed significantly from C2 (9,04 %). For leached ashes and oil texts significant difference between the three years was observed. The raised texts more of oil (21,1 %) and leached ashes (4,79%) had been found in C3. It did not have significant differences between the treatments for the text of protein and carbohydrates, however the biggest texts had been found in C1. In relation to minerals, the studied components had occurred significant differences between all, to the exception of the N. The concentrations of P, K and Cu had been significantly bigger in C1. The concentrations of Mg in C1 and C2 had been similar, differing significantly in C3. The concentrations of Ca, Zn and Mn had been significantly bigger in C3. The concentration of Fe was significantly bigger in C2. The text of total isoflavones differed significantly between C1, C2 and C3, being superior in C3. Isoflavones Daidzina, Genistina and Malonil-Genistina had demonstrated significant differences between the years in conversion. These isoflavones had also presented greaters texts in C3. Isoflavones Malonil-Daidzina, Daidzeína, Genisteína had also presented expressives values in C3, however they had not differed significantly
O objetivo deste trabalho foi comparar a composição química da soja em conversão para agricultura orgânica - 1° ano em conversão (C1), 2° ano em conversão (C2) e 3° ano em conversão ou orgânica (C3) conhecendo os dados meteorológicos do local de cultivo. A cultivar utilizada foi a CD-216, proveniente da safra 2005/2006, do Município de Santa Helena/PR. As variáveis estudadas foram o teor de umidade, a composição centesimal (proteína, óleo, cinzas e carboidratos), o teor de minerais (P, K, Ca, Mg, Zn, Cu, Fe e Mn) e o teor de isoflavonas dos grãos. Considerando os três anos em conversão, alguns fatores diferiram significativamente. O teor de umidade em C1 (8,43 %) diferiu significativamente de C2 (9,04 %). Para os teores de óleo e de cinzas observou-se diferença significativa entre os três anos. Os teores mais elevados de óleo (21,1%) e de cinzas (4,79%) foram encontrados em C3. Não houve diferenças significativas entre os tratamentos para o teor de proteína e de carboidratos, porém os maiores teores foram encontrados em C1. Em relação aos minerais, ocorreram diferenças significativas entre todos os componentes estudados, à exceção do N. As concentrações de P, K e Cu foram significativamente maiores em C1. As concentrações de Mg em C1 e C2 foram semelhantes, diferindo significativamente em C3. As concentrações de Ca, Zn e Mn foram significativamente maiores em C3. A concentração de Fe foi significativamente maior em C2. O teor de isoflavonas totais diferiu significativamente entre C1, C2 e C3, sendo superior em C3. As isoflavonas Daidzina, Genistina e Malonil-Genistina demonstraram diferenças significativas entre os anos em conversão. Essas isoflavonas também apresentaram maiores teores em C3. As isoflavonas Malonil-Daidzina, Daidzeína, Genisteína também apresentaram valores mais expressivos em C3, porém não diferiram significativamente

Orientador: Jorge Nahás Neto
Coorientador: Eliana Aguiar Petri Nahás
Banca: Paulo Traiman
Banca: Eduardo Carvalho Pessoa
Resumo: Avaliar o efeito da isoflavona da soja sobre o tecido mamário em mulheres na pós-menopausa. Trata-se de estudo clínico, prospectivo, randomizado, duplo-cego, placebo controlado, envolvendo 80 mulheres na pós-menopausa com sintomas vasomotores, idade entre 45 a 70 anos, acompanhadas no Ambulatório de Climatério e Menopausa da Faculdade de Medicina de Botucatu-Unesp, de janeiro de 2005 a dezembro de 2006. Na randomização, 40 pacientes receberam 100 mg isoflavona da soja/dia (duas cápsulas de 125 mg de extrato seco de glicine Max) e 40 pacientes placebo (duas cápsulas de lactose) durante 10 meses. A densidade mamária (DM) foi avaliada pela mamografia e o parênquima mamário pela ultrassonografia de mamas no início e após dez meses de seguimento. Para análise estatística foram utilizados o teste t-Student, ANOVA, teste de Mann-Whitney e teste do Qui-Quadrado. Na comparação das características clínicas iniciais entre os grupos de usuárias de isoflavona e placebo, não houve diferenças significantes, com valores médios de idade de 55,1±6,0 e 56,2±7,7 anos, tempo de menopausa de 6,6±4,8 e 7,1±4,2 anos e IMC de 29,7±5,0 e 28,5±4,9 kg/m2, respectivamente (p>0,05). Concluíram o estudo 32 pacientes sob isoflavona e 34 sob placebo. Na comparação da densidade mamográfica entre os momentos inicial e final, não houve diferença estatisticamente significativa. Na avaliação do parênquima mamário pela ultrassonografia, não houve diferença entre os grupos. Na comparação entre os momentos dentro de cada grupo, não foram constatadas diferenças significativas nos parâmetros da mamografia e ultrassonografia. No período de 10 meses, o uso de isoflavona de soja não modificou o tecido mamário, avaliado pela mamografia e ultrassonografia, em mulheres na pós-menopausa
Abstract: To evaluate the effect of soy isoflavone on breast tissue in postmenopausal women. This study is a prospective, randomized, double-blind, placebo-controlled trial involving 80 postmenopausal women with vasomotor symptoms, aged 45-70 years, followed in Climacteric and Menopause Clinic of the Botucatu Medical School-UNESP, from January 2005 to December 2006. At randomization, 40 patients received 100 mg of soy isoflavone/day (two capsules of 125 mg standardized soy extract, Glicine max) and 40 patients, placebo (two capsules of lactose) for 10 months. The breast density was evaluated by mammography and breast parenchyma by ultrasound, at baseline and after ten months of follow-up. The Student t-test, ANOVA, Mann-Whitney and Chi-Square were used in the statistical analysis. In comparison of baseline clinical characteristics between the isoflavone and placebo groups, there were no significant differences, with mean age of 55.1 ± 6.0 and 56.2 ± 7.7 years, duration of menopause 6.6 ± 4.8 and 7.1 ± 4.2 years and BMI 29.7 ± 5.0 and 28.5 ± 4.9 kg/m2, respectively (p> 0.05). Concluded the study, 32 patients on isoflavone and 34 in the placebo group. In comparison in mammographic density (MD) between moments, baseline and final, there was no difference statistically significant. In the evaluation of breast parenchyma by ultrasound, there was no difference between groups. In comparing the moments within each group, there were no significant differences in the parameters of mammography and ultrasound. In 10 months, the use of soy isoflavone did not affect breast tissue, as assessed by mammography and ultrasound, in postmenopausal women
Doutor

Thesis (Ph. D.)--Ohio State University, 2004.
Document formatted into pages; contains 290 p. Includes bibliographical references. Abstract available online via OhioLINK's ETD Center; full text release delayed at author's request until 2005 Nov. 24.