Orientador: Roberto Herminio Moretti / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-04T02:30:32Z (GMT). No. of bitstreams: 1 Freitas_DanielaDeGrandiCastro_D.pdf: 989026 bytes, checksum: c9f4c5d7a467d08716626299b2917043 (MD5) Previous issue date: 2005 / Resumo: Nutrientes são necessários para o desenvolvimento e crescimento dos indivíduos. No entanto, uma nutrição preventiva pode proteger, minimizar ou mesmo protelar riscos a saúde que podem ser causados por pré-disposições genéticas, maus hábitos alimentares e até agressões do meio ambiente. As evidências epidemiológicas estão continuamente providenciando recomendações para que as pessoas consumam mais frutas e vegetais como medida preventiva para reduzir o risco de diversas doenças degenerativas. É notável o aumento do consumo de alimentos saudáveis devido à crescente preocupação com a melhoria da qualidade de vida, porém, o ritmo da vida moderna exige alimentos práticos. Dentro deste contexto, do crescimento visível do desenvolvimento de alimentos para fins especiais, da necessidade de combinar saúde e praticidade, e de reunir alguns ingredientes que vem recebendo grande atenção para a aplicação nestes alimentos funcionais, é que se elaborou o estudo de desenvolvimento e estabilidade de barra de cereais a base de proteína de soja, gérmen de trigo e aveia, incluindo como ingrediente a lecitina de soja, e suplementada com vitaminas C e E. Este trabalho teve por objetivo o desenvolvimento da formulação da barra de cereais e a avaliação das características físico-químicas, vitamínicas, enzimáticas, sensoriais e de textura durante o seu armazenamento. Um estudo preliminar de inativação enzimática no gérmen de trigo foi realizado devido à susceptibilidade a rancidez, fator adverso de qualidade de cereais desidratados. Amostras de aveia laminada integral e gérmen (produto natural inalterado da moagem do trigo) foram caracterizadas e a atividade de lipase e peroxidase medidas. Foi utilizado um delineamento fatorial completo para estudar os efeitos dos fatores ¿temperatura¿ e ¿tempo¿ na inativação das enzimas e para a determinação das condições ótimas de redução de atividade no gérmen de trigo. Foram determinadas como condições ótimas tratamentos térmicos à temperatura na faixa de 180 a 190°C, com duração de 4,8 a 8,0 minutos e obtidas reduções significativas (p£0,5) na atividade residual da lipase e peroxidase. O gérmen de trigo teve seu teor de umidade ajustado para 28% e a torração foi realizada em estufa a 180°C por 8,0 minutos, para que pudesse ser utilizado como ingrediente da barra de cereais. Desenvolveu-se então uma formulação de barra de cereais funcional sabor banana com alto teor protéico, à base de proteína de soja, gérmen de trigo e aveia, e enriquecida de vitaminas C (ácido ascórbico) e E (acetato de a-tocoferol 50%). A barra de cereais e seus principais ingredientes foram submetidos a análises físico-químicas para caracterização e três formulações variando o teor de ácido ascórbico foram avaliadas quanto à aceitação sensorial e intensidade ideal de doçura e acidez. A proteína de soja texturizada utilizada apresentou elevados teores dos oligossacarídeos rafinose e estaquiose (1,92 g/100g e 4,66 g/100g) e de isoflavonas totais (283,49 mg/100g) se comparados ao grão, farinha integral e isolado protéico. A formulação final da barra de cereais apresentou 15,31% de proteína e elevado teor de vitamina E (118,0 mg/100g) e minerais como fósforo (P), cálcio (Ca), cobre (Cu), ferro (Fe), manganês (Mn) e zinco (Zn). A formulação adicionada de 1,1g/100g de ácido ascórbico obteve maior aceitação sensorial diferindo significativamente (p£0,5) das demais amostras. Esta formulação, preferida pela equipe sensorial, foi então submetida ao estudo de estabilidade durante o seu armazenamento. Foram utilizados como embalagens três filmes (A: PET/PEBD; B: PETmet/PEBD; C: PET/PEBD/AL/PEBD) com diferentes propriedades de barreira. As barras de cereais foram armazenadas em condições de temperatura e umidade relativa ambiente por um período de 180 dias. Durante o armazenamento, estudou-se a estabilidade das vitaminas C, vitamina E, e a atividade de lipase e peroxidase. No período de 180 dias, as barras de cereais acondicionadas nos três materiais testados apresentaram uma retenção de vitamina C menor que 50%. No entanto, a embalagem com folha de alumínio proporcionou menor velocidade de reação e maior tempo de meia vida de 146 dias. Os teores de vitamina E (acetato de a-tocoferol) nas barras de cereais apresentaram pequenas variações durante o armazenamento. Houve um aumento significativo (p£0,5) da atividade de peroxidase durante o período de 90 dias de estocagem nas embalagens testadas, provavelmente devido à regeneração dessa enzima, enquanto que a atividade de lipase não demonstrou aumento significativo durante os 180 dias de estocagem. Foram acompanhados também a umidade, atividade de água, pH, acidez total e medidas físicas associadas à textura como resistência ao corte, dureza e coesividade. A avaliação sensorial foi realizada através de teste de aceitação e intenção de compra durante o armazenamento. As barras de cereais formuladas com os ingredientes alternativos apresentaram variações na atividade de água (Aa), umidade e acidez total durante o armazenamento. A umidade das barras de cereais tendeu a um aumento, o que acarretou influência significativa (p£0,5) nas características de textura de resistência ao corte e dureza. As barras de cereais formuladas com proteína texturizada de soja, gérmen de trigo e aveia obtiveram boa aceitação sensorial perante consumidores nos atributos de sabor e aspectos visuais, porém, a aceitação tendeu a um declínio ao longo do tempo, associado à diminuição da intenção de compra do produto. O efeito das diferentes propriedades de barreira dos filmes de embalagens testados foi significativo (p£0,5) na estabilidade das barras de cereais ao longo do período de estocagem / Abstract: Nutrients are required for development and growth of individuals. However a preventative nutrition could be protected, minimized or delayed by way from health risks arising from genetic aggressions, the environment and also eating habits. Epidemiological evidence is constantly providing recommendations that people should consume more fruits and vegetables as a preventative measure to reduce the risk of various degenerative diseases. The increase in consumption of health foods is notable, due to the increasing concern with the improvement of life quality, although the pace of modern life demands practical foods. Within this context of a visible growth in the development of foods for special ends, of the need to combine health and practicality and aiming to bring together some ingredients which have received special attention for application in such functional foods, a study of the development and stabilization of a cereal bar based on soy protein, wheat germ and oat was elaborated, with the addition of soy lecithin and supplemented with vitamins C and E. Thus the objective of this study was the development and formulation of a cereal bar and an evaluation of its physicochemical, vitamin, enzymatic, sensory and texture characteristics during storage. A preliminary study of the enzymatic inactivation of wheat germ was carried out due to its susceptibility to rancidity, a process reducing the quality of dehydrated cereals. Samples of whole rolled oats and wheat germ (natural, unaltered product from wheat milling) were characterized and their lipase and peroxidase activities determined. A complete factorial design was used to study the effects of time and temperature in the inactivation of the enzymes and to determine the optimal conditions to reduce their activity in wheat germ. The optimal conditions were shown to be a temperature in the range from 180 to 190°C for between 4.8 and 8.0 minutes, obtaining significant (p£0,5) reductions in the residual activities of lipase and peroxidase. The moisture content of the wheat germ was adjusted to 28%, which was then roasted at 180°C for 8.0 minutes such that it could be used as an ingredient in cereal bars. A functional, banana flavored cereal bar was formulated with high protein content, based on soy protein, wheat germ and oat, and enriched with vitamins C (ascorbic acid) and E (50% a-tocopherol). The cereal bar and its main ingredients were characterized by way of physicochemical analyses, and three formulations, varying in their ascorbic acid contents, prepared and submitted to a sensory acceptance test and ideal test for the intensity of sweetness and acidity. The textured soy protein used showed high contents of the oligosaccharides raffinose and stachyose (1.92 g/100g and 4.66 g/100g) and of total isoflavones (283.49 mg/100g) as compared to the bean, whole flour and protein isolate. The final formulation of the cereal bar showed 15.31% protein and high contents of vitamin E (118.0 mg/100g) and minerals such as phosphorus (P), calcium (Ca), copper (Cu), iron (Fe), manganese (Mn) and zinc (Zn). The formulation with 1.1 g/100g of ascorbic acid was the most accepted sample, differing significantly (p£0,5) from the other samples. This most accepted formulation was submitted to an evaluation during storage. Three films were used for packaging: (A: PET/PEBD; B: PETmet/PEBD; C: PET/PEBD/AL/PEBD), each with distinctive barrier properties. The cereal bars were studied under environmental conditions of temperature and relative humidity for 180 days. Vitamins C and E stabilities and the activities of lipase and peroxidase were determined during storage. The cereal bars packaged in the three materials tested showed vitamin C retention below 50% after 180 days storage. However the packaging including aluminum foil provided a lower reaction velocity and longer half life of 146 days. The vitamin E (a-tocopherol acetate) contents of the cereal bars presented slight variations during storage. There was a significant increase in peroxidase activity during the first 90 days of storage in all the types of packaging tested, probably due to regeneration, whilst lipase activity showed no significant increase during the 180 days of storage. The moisture content, water activity, pH and total acidity were also determined during storage as well as the textural measurements of cutting resistance, hardness and cohesiveness. The acceptance and intention to buy tests were used for the sensory evaluation during storage. The cereal bars formulated with alternative ingredients showed variations in water activity (Aw), moisture content and total acidity during storage. The moisture contents of the cereal bars tended to increase during storage, with a significant (p£0,5) influence on the textural characteristics of cutting resistance and hardness The cereal bars formulated with textured soy protein, wheat germ and oats were well accepted by the consumers in the sensory evaluation (flavor and visual aspect), although the acceptance tended to decline during storage, associated with a decrease in intent to buy. The effect of the different barrier properties of the packaging films tested was significant (p£0,5) in the stability of the cereal bars during storage / Doutorado / Tecnologia de Alimentos / Doutor em Tecnologia de Alimentos

Os isolados protéicos de soja são utilizados como ingredientes em diversos alimentos e sua utilização vêm aumentando juntamente com o aumento das pesquisas sobre os metabólitos secundários da soja, as isoflavonas. Alguns efeitos benéficos vem sendo associados às isoflavonas, entre estes a sua ação antioxidante, a redução ao risco de câncer, doenças cardiovasculares e osteoporose. O objetivo deste estudo foi o de otimizar as condições de extração das isoflavonas e de suas formas conjugadas a partir da farinha desengordurada de soja, visando o preparo de isolado protéico de soja. Os resultados mostraram que a obtenção de isolados protéicos de soja com teor aumentado de isoflavonas depende da utilização de condições brandas de centrifugação para a separação do precipitado isoelétrico, assim como da utilização de água acidificada na sua lavagem. A presença de isoflavonas no isolado resulta de três fatores, o primeiro referindo-se à associação entre isoflavonas e proteínas através de interações hidrofóbicas, eletrostáticas, e pontes de hidrogênio; o segundo à menor solubilidade das isoflavonas presentes na farinha desengordurada de soja no pH isoelétrico; e o último ao processo de carreamento (físico) das isoflavonas pelas proteínas insolubilizadas. / Soy protein isolates are used as ingredients in several food products and their use is increasing together with the increase of the researches on the secondary metabolites of soy, the isoflavones. Some beneficial effects have been associated to the isoflavones, among these their antioxidant action, prevention of cancer, cardiovascular diseases and osteoporosis. The objective of this study was to optimize the extraction conditions of the isoflavones from the defatted soy flour, seeking the preparation of soy protein isolates. The results showed that the obtention of soy protein isolates with increased content of isoflavones depends on the use of mild conditions of centrifugation for the separation of the isoelectric precipitate, as well as on the use of water acidified in the washing step. The presence of isoflavones in the isolates resulted from three factors, the first refers to the association between isoflavones and proteins through hydrophobic; and electrostatic interactions, and hydrogen bonding; the second to the decreased solubility of the isoflavones extracted from the defatted soy flour in the isoelectric pH; and the last to the carrying process (physical) of isoflavones by the precipitating proteins.

A soja contém alto conteúdo protéico e de lipídeos poliinsaturados e representa a principal fonte de isoflavonas. Por este grão não fazer parte da dieta brasileira, produtos contendo soja surgiram no mercado como fontes de isoflavonas, adaptados ao paladar da nossa população. Os condimentos também têm sido incorporados aos ditos \"alimentos funcionais\" pela sua relação com efeitos benéficos à saúde. Este trabalho objetivou determinar o teor de isoflavonas dos produtos contendo soja e/ou derivados, o teor de flavonóides de diferentes condimentos e marcas, e a capacidade antioxidante destas amostras. Os resultados mostram que o quibe vegetariano destaca-se como fonte de isoflavonas, e o chocolate pela capacidade antioxidante. Entre os condimentos, houve diferença significativa entre tipos e marcas. O orégano destacou-se quanto à capacidade antioxidante e a salsa quanto ao teor de flavonóides. Alimentos contendo soja ou derivados e os condimentos podem representar fontes de compostos bioativos para a população brasileira. / Soy contains high protein and polyunsaturated lipids content and represents the main source of isoflavones. This grain is not part of the Brazilian diet, so some products containing soy emerged as sources of isoflavones, adapted to the taste of our population. Spices have also been incorporated into \"functional foods\" by their relation to health benefits. This study aimed to determine the isoflavone content of products containing soy and/or derivatives, flavonoids content in different brands and condiments, and antioxidant capacity of these samples. The results showed that vegetarian \"quibe\" is a good source of isoflavones, and chocolate had the highest antioxidant capacity. Among spices, there were significant differences between the types and brands. Oregano had the highest antioxidant capacity and parsley had the highest flavonoids content. Foods containing soy or derivatives and condiments may represent sources of bioactive compounds for Brazilian population.

Chan Ho Yee. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (leaves 121-138). / Abstracts in English and Chinese. / ACKNOWLEDGEMENTS --- p.II / ABSTRACT --- p.III / 摘要 --- p.V / ABBREVIATIONS --- p.VI / "TABLE OF CONTENTS, " --- p.VII / LIST OF FIGURES AND TABLES --- p.XI / Chapter CHAPTER 1 --- GENERAL INTRODUCTION --- p.1 / Xenobiotic-metabolizing enzymes --- p.1 / Cytochrome P450 1 family --- p.4 / CYP1A1 --- p.5 / CYP1A2 --- p.5 / CYP1B1 --- p.5 / Transactivation of CYP1 enzymes by Aryl hydrocarbon receptor (AhR) --- p.8 / Implication of PAHs and CYP1 family in breast cancer --- p.10 / Potential role of phytochemicals on cancer prevention --- p.11 / Significance of this project --- p.13 / Chapter CHAPTER 2 --- MATERIALS AND METHODS --- p.14 / Chemicals --- p.14 / Maintenance of cells --- p.14 / Preparation of cell stock --- p.14 / Cell recovery from liquid nitrogen stock --- p.15 / Measurement of cell viability --- p.15 / Preparation of cell lysates (NP-40 cell lysis buffer) --- p.15 / XRE-luciferase gene reporter assay --- p.16 / Manipulation of DNA and RNA --- p.17 / Separation and purification of DNA from agarose gel --- p.17 / Separation of DNA from acrylamide gel --- p.17 / Restriction digestion --- p.18 / Ligation of DNA fragments --- p.18 / Transformation of DH5 a --- p.19 / Small scale plasmid purification from DH5a (mini prep) --- p.19 / Large scale plasmid isolation from DH5a (maxi-prep) --- p.20 / Construction of XRE activated luciferase reporter gene --- p.21 / Measurement of DMBA-DNA adduct formation --- p.21 / Semi-quantitative RT-PCR Assay --- p.22 / ENZYME ACTIVITIES --- p.23 / Isolation of microsomes --- p.23 / EROD activities in intact cells --- p.23 / EROD inhibition assay --- p.24 / Stattstical Analysis --- p.24 / Chapter CHAPTER 3 --- BAICALEIN INHIBITS DMBA-DNA ADDUCT FORMATION BY MODULATING CYP1A1 AND 1B1 ACTIVITIES --- p.26 / Introduction --- p.26 / Results --- p.28 / EROD activities in MCF-7 cells and inhibition assay --- p.28 / Baicalein suppressed DMBA-induced XRE-driven luciferase activities --- p.31 / Baicalein inhibited DMBA-induced CYP1A1 and CYP1B1 mRNA expression --- p.31 / The cytotoxic effect of DMBA was reduced by baicalein --- p.35 / Inhibition of DMBA-DNA adduct formation after baicalein treatment --- p.35 / Discussion --- p.39 / Chapter CHAPTER 4 --- INHIBITION OF DMBA-DNA ADDUCT FORMATION BY (-)-EPIGALLOCATECHIN GALLATE AND THEAFLAVINS --- p.41 / Introduction --- p.41 / Results --- p.45 / Persistence of DMBA-induced DNA adducts --- p.45 / Inhibition of theaflavins and EGCG on human recombinant CYP1A1 and CYP1B1 enzyme activities --- p.48 / EGCG suppressed DMBA-induced EROD activity while thealfavin had no significant effect on this --- p.48 / Kinetic analysis of EGCG on CYP1A1 and CYP1B1 activities --- p.53 / Modulation of DMBA-induced XRE-driven luciferase activities by theaflavins and EGCG --- p.56 / The influence of theaflavins and EGCG on CYP1A1 and CYP1B1 abundance --- p.56 / Discussion --- p.65 / Chapter CHAPTER 5 --- ISOFLAVONES PREVENT DMBA-INDUCED CARCINOGENESIS BY INHIBITING CYP1A1 AND CYP1B1 ACTIVITIES --- p.67 / Introduction --- p.67 / Results --- p.70 / Isoflavones inhibited DMBA-induced EROD activity in MCF-7 cells --- p.70 / Inhibition of MCF-7 microsomal EROD activities by isoflavones --- p.70 / Kinetic analysis of the inhibition of human recombinant CYP1 enzymes by isoflavones --- p.74 / XRE-driven Luciferase activities --- p.83 / Both biochanin A and genistein suppressed DMBA-induced CYP1 mRNA expression --- p.83 / Cytotoxicity of DMBA and isoflavones co-treatment --- p.88 / Isoflavones reduced the binding of activated DMBA to DNA --- p.89 / Discussion --- p.93 / Chapter CHAPTER 6 --- IN VITRO EFFECTS OF BAICALEIN AND THEAFLAVINS ON RAT HEPATIC P450 ACTIVITIES --- p.96 / Introduction --- p.96 / Results --- p.98 / Inhibition of EROD and MROD activities in rat liver microsomes by baicalein --- p.98 / Effects of theaflavins on EROD and MROD activities in rat liver microsomes --- p.102 / Kinetic studies for EROD and MROD activities of theaflavins --- p.104 / DISCUSSION --- p.114 / Chapter CHAPTER 7 --- CONCLUSION --- p.116 / APPENDIX 1 PRIMER LISTS --- p.118 / APPENDIX 2 REAGENTS --- p.119 / BIBLIOGRAPHY --- p.121

Yuen Yee Man. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 108-125). / Abstracts in English and Chinese. / TITLE PAGE --- p.1 / ACKNOWLEGDEMENTS --- p.2 / ABSTRACT --- p.3 / 摘要 --- p.5 / table of contents --- p.7 / list of figures and tables --- p.13 / CHAPTER 1 GENERAL INTRODUCTION --- p.16 / Chapter 1.1 --- role of PHYTOESTROGENS in soy and red WINE the PREVENTION OF CARDIOVASCULAR DISEASES (CVD) --- p.17 / Chapter 1.1.1 --- INTRoduction and Classification of Phytoestrogens --- p.17 / Chapter 1.1.2 --- estrogenic1ty of phytoestrogens and theIr abundancesin Plasma --- p.18 / Chapter 1.1.3 --- phytoestrogens as one of the active components In cvd Protection --- p.21 / Chapter 1.1.4 --- effects of Phytoestrogens on LDL Receptor and Apolipoprotein A-1 --- p.22 / Chapter 1.2 --- role of estrogen receptors (ers) in gene regulation --- p.24 / Chapter 1.2.1 --- "structure, Classification and tissue distribution of ERS" --- p.24 / Chapter 1.2.2 --- ligands for ERS --- p.25 / Chapter 1.2.3 --- mechaniSMS OF LIgands-ERS complex in GENE regulation --- p.27 / Chapter 1.2.4 --- ligand-independent ER activation --- p.28 / Chapter 1.3 --- aims and scopes of investigation --- p.29 / Chapter CHAPTER 2 --- MATERIALS AND METHODS --- p.30 / Chapter 2.1 --- chemicals and materials --- p.30 / Chapter 2.1.1 --- Chemicals --- p.30 / Chapter 2.1.2 --- Plasmids --- p.30 / Chapter 2.2 --- mammalian cell culture maintainence --- p.30 / Chapter 2.2.1 --- Maintenance of Cells --- p.31 / Chapter 2.2.2 --- Preparation of Cell Stock --- p.31 / Chapter 2.2.3 --- Cell Recovery from Liquid Nitrogen Stock --- p.31 / Chapter 2.3 --- manipulation of dna --- p.31 / Chapter 2.3.1 --- isolation of HEPG2 cells genonmic DNA --- p.31 / Chapter 2.3.2 --- separation and purification of dna from agarose gel --- p.31 / Chapter 2.3.3 --- Restriction digestionof DNA --- p.32 / Chapter 2.3.4 --- Ligation of DNA Fragments --- p.32 / Chapter 2.3.5 --- Transformation of --- p.32 / Chapter 2.3.6 --- Small Scale Plasmids Purification from DH5a --- p.32 / Chapter 2.4 --- construction of expression and reporter plasmids --- p.33 / Chapter 2.4.1 --- Construction of Estrogen Receptorα (Erα) Expression Vectors --- p.33 / Chapter 2.4.2 --- construction of reporter vectors of LDLR promoter and the Respective Mutants --- p.33 / Chapter 2.4.3 --- Construction of Reporter Vectors of APOAI Promoter and the Respective Mutants --- p.33 / Chapter 2.5 --- determination of promoter transcrtiption activities --- p.34 / Chapter 2.5.1 --- Transient Transfection of Cell with ERa Expression Vector and Promoter Reporter using Lipofectamine PLUS Reagent --- p.34 / Chapter 2.5.2 --- Dual Luciferase Assay --- p.34 / Chapter 2.6 --- semi-quantitative and quantitative rt-pcr assay --- p.34 / Chapter 2.6.1 --- Transient transfection of Cell with ERa Expression Vector Using Lipofectamine PLUS Reagent --- p.34 / Chapter 2.6.2 --- "Isolation of RNA using TRIzol® Reagent (Life Technology, USA)" --- p.35 / Chapter 2.6.3 --- Quantitation of RNA --- p.35 / Chapter 2.6.4 --- First Strand cDNA Synthesis --- p.35 / Chapter 2.6.5 --- Sem卜Quantitative PCR Reactions --- p.35 / Chapter 2.6.6 --- Quantitative PCR Reactions --- p.36 / Chapter 2.7 --- western blotting analysis --- p.36 / Chapter 2.8 --- statistical methods --- p.36 / Chapter CHAPTER 3 --- REGULATION BY PHYSIOLOGICAL LEVEL OF 17B-ESTRADIOL ON APOLIPOPROTEIN A-I AND LOW-DENSITY- LIPOPROTEIN RECEPTOR IN HEPG2 CELLS --- p.37 / Chapter 3.1 --- introduction --- p.37 / Chapter 3.2 --- results --- p.39 / Chapter 3.2.1 --- Determination of transient transfection functionality of estrogen receptors in hepg2 cells --- p.39 / Chapter 3.2.2 --- Effect of 17β-Estradiolon LDLR promoter transcription activity --- p.39 / Chapter 3.2.3 --- Effect of 17β-Estradiol on apoai promoter transcription activity --- p.40 / Chapter 3.2 --- discussion --- p.47 / Chapter CHAPTER 4 --- SOY ISOFLAVONES AND RESVERATROL DISPLAY DIFFERENT MECHANISM IN THE UP-REGULATION OF LOVV-DENSITY-LIPOPROTEIN RECEPTOR IN HEPG2 CELLS --- p.49 / Chapter 4.1 --- introduction --- p.49 / Chapter 4.2 --- results --- p.54 / Chapter 4.2.1 --- Association of ERα and isoflavones or resveratrol on LDLR promoter transcription activity --- p.54 / Chapter 4.2.2 --- Association of ERβ and isoflavones or resveratrol on LDLR promoter transcription activity --- p.54 / Chapter 4.2.3 --- "Role of MAP Kinase, PKA and PKC in isoflavones and resveratrol induced LDLR promoter transcription" --- p.55 / Chapter 4.2.4 --- Identification of promoter regions responsible for induction of LDLR transcription by isoflavones in the presence OF ERα --- p.55 / Chapter 4.2.5 --- Identification of promoter regions responsible for induction of LDLR TRANSCRIPTION BY resveratrol IN THE ABSENCE OF ERα --- p.56 / Chapter 4.3 --- DISCUSSION --- p.75 / Chapter CHAPTER 5 --- SOY ISOFLAVONES AND RESVERATROL UP-REGULATE APOLIPOPROTEIN A-I SIMILAR TO 17B-ESTRADIOL IN HEPG2 CELLS --- p.80 / Chapter 5.1 --- INTRODUCTION --- p.80 / Chapter 5.2 --- RESULTS --- p.84 / Chapter 5.2.1 --- Association of ERα phytoestrogens on APCAI gene expression --- p.84 / Chapter 5.2.2 --- Association of ERβ and isoflavones or resveratrol on APOAI promoter transcription activity --- p.85 / Chapter 5.2.3 --- "Role of MAP Kinase, PKA and PKC in isoflavones and resveratrol in APOAI promoter transcription in the presence of ERα" --- p.85 / Chapter 5.2.4 --- Identification of promoter regions responsible for induction of APOAI transcription by isoflavones and resveratrol in the presence of ERα --- p.85 / Chapter 5.3 --- DISCUSSION --- p.100 / Chapter CHAPTER 6 --- GENERAL DISCUSSION --- p.103 / Chapter CHAPTER 7 --- SUMMARY --- p.106 / BIBLIOGRAPHY --- p.108 / APPENDIX 1 ABBREVIATIONS --- p.126 / APPENDIX 2 MATERIALS AND METHODS --- p.129 / APPENDIX 3 PRIMER LISTS --- p.145 / APPENDIX 4 REAGENTS AND BUFFERS --- p.147

No description available.

No description available.

O trevo-vermelho (Trifolium pratense L.), uma das leguminosas forrageiras mais utilizadas na agricultura mundial, contém as isoflavonas formononetina e biochanina A, e em menores concentrações daidzeína e genisteína. Estes compostos têm ganhado muito interesse devido aos relatos de seus benefícios à saúde humana. Recentemente, no ano de 2009, esta espécie foi incluida na lista da Relação Nacional de Plantas Medicinais de Interesse ao SUS (RENISUS) que contém plantas medicinais com potencial de gerar produtos para serem produzidos e distribuídos pelo SUS. Mas é importante destacar que não existem estudos de quantificação de isoflavonas em plantas de trevo-vermelho cultivados no Brasil. Os objetivos deste trabalho foram validar método de análise de amostras de trevo-vermelho por Cromatografia Líquida de Alta Eficiência (CLAE); quantificar quatro isoflavonas em 78 acessos de trevo-vermelho; verificar as modificações sazonais na concentração de três acessos de trevo-vermelho; aumentar os níveis de isoflavonas através de melhoramento genético; e determinar a atividade anti-inflamatória in vivo e in vitro e de atividade de inibição da acetilcolinesterase de um extrato seco de trevo-vermelho. Para analisar as plantas por CLAE, estas foram hidrolisadas, separadas por diclorometano, e ressuspendidas em metanol. O método de CLAE usado foi validado. As isoflavonas (daidzeína, genisteína, formononetina e biochanina A) foram quantificadas em 78 acessos de trevo-vermelho, os conteúdos das agliconas (expressos em μg/g de planta seca, n=3) destes compostos variaram entre 0,00 a 137,91 para daidzeína; 14,70 a 516,51 para genisteína; 452,97 a 28548,65 para formononetina; e 1967,64 a 20145,27 para biochanina A, e a concentração total de isoflavonas variou entre 9,81 e 36,36 mg/g. O estudo sazonal mostrou que a concentração total das isoflavonas não difere entre as estações, mas verifica-se que na primavera (quando o trevo-vermelho está no estágio reprodutivo) se observam concentrações inferiores, e no inverno (quando a planta está no estágio vegetativo) são encontradas concentrações mais elevadas. No estudo de melhoramento genético foi possível observar um aumento na concentração total de isoflavonas. A atividade anti-inflamatória in vitro foi medida através da mobilização de leucócitos, pelo ensaio de quimiotaxia na câmara de Boyden. A atividade anti-inflamatória in vivo foi avaliada pelo teste de edema de pata de rato induzido por carragenina. Os resultados do teste anti-inflamatório in vitro mostram que houve significante inibição da migração dos leucócitos nas concentrações de 100,0 (94,73% de inibição), 50,0 (95,39% de inibição), 25,0 (94,73% de inibição), 10,0 (84,68% de inibição) e 5,0 (78,75% de inibição) μg/mL de extrato seco de trevo-vermelho. O teste anti-inflamatório in vivo demonstrou significante atividade nas doses testadas, 100 e 50 mg/kg de extrato seco de trevo-vermelho. O percentual médio de inibição do edema foi 63,37%. O teste da inibição da acetilcolinesterase não demonstrou atividade. Os resultados deste estudo sugerem que se pode produzir plantas com concentrações mais elevadas de isoflavonas para a produção de fitomedicamentos com maior qualidade, e o extrato de trevo-vermelho pode ser adequado para o tratamento de doenças inflamatórias. / Red clover (Trifolium pratense L.) is one of the most utilized forage legume in the world agriculture and contains the isoflavones formononetin and biochanin A, and in smaller concentration daidzein and genistein. These compounds have gained a high interest due to their human health benefits. Recently this species was included in the list of Relação Nacional de Plantas Medicinais de Interesse ao SUS (RENISUS) which contains medicinal plants that have potential to generate products to be produced and distributed by SUS. But it is important to highlight that does not exist any study of isoflavones quantification in red clover plants cultivated in Brazil. The aims of this work were to validate a method to analize red clover samples by High Performance Liquid Cromatography (HPLC); to quantify four isoflavones compounds in 78 red clover accessions; to verify seasonal modifications in the isoflavone concentration of three red clover accessions; to increase isoflavones levels through plant breeding; and to determine in vivo and in vitro anti-inflammatory activity, and inhibitory activity of acetylcholinesterase of a red clover dry extract. To analyze the plants by HPLC, they were hydrolyzed, separated by dichloromethane, and resuspended with methanol. The HPLC method used was validated. Isoflavones (daidzein, genistein, formononetin and biochanin A) were quantified in 78 red clover accessions, the aglycone contents (expressed in μg/g of dry material, n=3) of these compounds varied from 0.00 to 137.91 to daidzein; 14.70 to 516.51 to genistein; 452.97 to 28548.65 to formononetin; and 1967.64 to 20145.27 to biochanin A, and the total isoflavone concentration ranged between 9.81 and 36.36 mg/g. The seasonal study showed that the total concentration of isoflavones is not statistically different at different seasons, but is possible to verify that at spring (when red clover is at reproductive stage) we could observe the lowest concentration, and at winter (when red clover is at vegetative stage) we found the highest concentration. In the breeding study we could observe an increase in the total concentration of isoflavones. The in vitro anti-inflammatory activity was measured towards by leucocytes mobilization, by chemotaxis assay in Boyden´s chamber. The in vivo anti-inflammatory activity was tested by a carrageenan-induced rat paw edema test. The results of anti-inflammatory in vitro test showed that there was a significant inhibition of leukocyte migration at concentrations of 100.0 (94.73% of inhibition), 50.0 (95.39% of inhibition), 25.0 (94.73% of inhibition), 10.0 (84.68% of inhibition) and 5.0 (78.75% of inhibition) μg/mL of red clover dry extract. The in vivo anti-inflammatory test demonstrated significant activity in the tested doses, 100 and 50 mg/kg of red clover dry extract. The average of edema inhibition percentage was 63.37%. The inhibition of acetylcholinesterase test did not show activity. The results of this study suggest that we can select or produce plants with higher concentrations of isoflavones to produce phytomedicines with higher quality, and the red clover extract might be suitable for the treatment of inflammatory diseases.

O trevo-vermelho (Trifolium pratense L.), uma das leguminosas forrageiras mais utilizadas na agricultura mundial, contém as isoflavonas formononetina e biochanina A, e em menores concentrações daidzeína e genisteína. Estes compostos têm ganhado muito interesse devido aos relatos de seus benefícios à saúde humana. Recentemente, no ano de 2009, esta espécie foi incluida na lista da Relação Nacional de Plantas Medicinais de Interesse ao SUS (RENISUS) que contém plantas medicinais com potencial de gerar produtos para serem produzidos e distribuídos pelo SUS. Mas é importante destacar que não existem estudos de quantificação de isoflavonas em plantas de trevo-vermelho cultivados no Brasil. Os objetivos deste trabalho foram validar método de análise de amostras de trevo-vermelho por Cromatografia Líquida de Alta Eficiência (CLAE); quantificar quatro isoflavonas em 78 acessos de trevo-vermelho; verificar as modificações sazonais na concentração de três acessos de trevo-vermelho; aumentar os níveis de isoflavonas através de melhoramento genético; e determinar a atividade anti-inflamatória in vivo e in vitro e de atividade de inibição da acetilcolinesterase de um extrato seco de trevo-vermelho. Para analisar as plantas por CLAE, estas foram hidrolisadas, separadas por diclorometano, e ressuspendidas em metanol. O método de CLAE usado foi validado. As isoflavonas (daidzeína, genisteína, formononetina e biochanina A) foram quantificadas em 78 acessos de trevo-vermelho, os conteúdos das agliconas (expressos em μg/g de planta seca, n=3) destes compostos variaram entre 0,00 a 137,91 para daidzeína; 14,70 a 516,51 para genisteína; 452,97 a 28548,65 para formononetina; e 1967,64 a 20145,27 para biochanina A, e a concentração total de isoflavonas variou entre 9,81 e 36,36 mg/g. O estudo sazonal mostrou que a concentração total das isoflavonas não difere entre as estações, mas verifica-se que na primavera (quando o trevo-vermelho está no estágio reprodutivo) se observam concentrações inferiores, e no inverno (quando a planta está no estágio vegetativo) são encontradas concentrações mais elevadas. No estudo de melhoramento genético foi possível observar um aumento na concentração total de isoflavonas. A atividade anti-inflamatória in vitro foi medida através da mobilização de leucócitos, pelo ensaio de quimiotaxia na câmara de Boyden. A atividade anti-inflamatória in vivo foi avaliada pelo teste de edema de pata de rato induzido por carragenina. Os resultados do teste anti-inflamatório in vitro mostram que houve significante inibição da migração dos leucócitos nas concentrações de 100,0 (94,73% de inibição), 50,0 (95,39% de inibição), 25,0 (94,73% de inibição), 10,0 (84,68% de inibição) e 5,0 (78,75% de inibição) μg/mL de extrato seco de trevo-vermelho. O teste anti-inflamatório in vivo demonstrou significante atividade nas doses testadas, 100 e 50 mg/kg de extrato seco de trevo-vermelho. O percentual médio de inibição do edema foi 63,37%. O teste da inibição da acetilcolinesterase não demonstrou atividade. Os resultados deste estudo sugerem que se pode produzir plantas com concentrações mais elevadas de isoflavonas para a produção de fitomedicamentos com maior qualidade, e o extrato de trevo-vermelho pode ser adequado para o tratamento de doenças inflamatórias. / Red clover (Trifolium pratense L.) is one of the most utilized forage legume in the world agriculture and contains the isoflavones formononetin and biochanin A, and in smaller concentration daidzein and genistein. These compounds have gained a high interest due to their human health benefits. Recently this species was included in the list of Relação Nacional de Plantas Medicinais de Interesse ao SUS (RENISUS) which contains medicinal plants that have potential to generate products to be produced and distributed by SUS. But it is important to highlight that does not exist any study of isoflavones quantification in red clover plants cultivated in Brazil. The aims of this work were to validate a method to analize red clover samples by High Performance Liquid Cromatography (HPLC); to quantify four isoflavones compounds in 78 red clover accessions; to verify seasonal modifications in the isoflavone concentration of three red clover accessions; to increase isoflavones levels through plant breeding; and to determine in vivo and in vitro anti-inflammatory activity, and inhibitory activity of acetylcholinesterase of a red clover dry extract. To analyze the plants by HPLC, they were hydrolyzed, separated by dichloromethane, and resuspended with methanol. The HPLC method used was validated. Isoflavones (daidzein, genistein, formononetin and biochanin A) were quantified in 78 red clover accessions, the aglycone contents (expressed in μg/g of dry material, n=3) of these compounds varied from 0.00 to 137.91 to daidzein; 14.70 to 516.51 to genistein; 452.97 to 28548.65 to formononetin; and 1967.64 to 20145.27 to biochanin A, and the total isoflavone concentration ranged between 9.81 and 36.36 mg/g. The seasonal study showed that the total concentration of isoflavones is not statistically different at different seasons, but is possible to verify that at spring (when red clover is at reproductive stage) we could observe the lowest concentration, and at winter (when red clover is at vegetative stage) we found the highest concentration. In the breeding study we could observe an increase in the total concentration of isoflavones. The in vitro anti-inflammatory activity was measured towards by leucocytes mobilization, by chemotaxis assay in Boyden´s chamber. The in vivo anti-inflammatory activity was tested by a carrageenan-induced rat paw edema test. The results of anti-inflammatory in vitro test showed that there was a significant inhibition of leukocyte migration at concentrations of 100.0 (94.73% of inhibition), 50.0 (95.39% of inhibition), 25.0 (94.73% of inhibition), 10.0 (84.68% of inhibition) and 5.0 (78.75% of inhibition) μg/mL of red clover dry extract. The in vivo anti-inflammatory test demonstrated significant activity in the tested doses, 100 and 50 mg/kg of red clover dry extract. The average of edema inhibition percentage was 63.37%. The inhibition of acetylcholinesterase test did not show activity. The results of this study suggest that we can select or produce plants with higher concentrations of isoflavones to produce phytomedicines with higher quality, and the red clover extract might be suitable for the treatment of inflammatory diseases.

O trevo-vermelho (Trifolium pratense L.), uma das leguminosas forrageiras mais utilizadas na agricultura mundial, contém as isoflavonas formononetina e biochanina A, e em menores concentrações daidzeína e genisteína. Estes compostos têm ganhado muito interesse devido aos relatos de seus benefícios à saúde humana. Recentemente, no ano de 2009, esta espécie foi incluida na lista da Relação Nacional de Plantas Medicinais de Interesse ao SUS (RENISUS) que contém plantas medicinais com potencial de gerar produtos para serem produzidos e distribuídos pelo SUS. Mas é importante destacar que não existem estudos de quantificação de isoflavonas em plantas de trevo-vermelho cultivados no Brasil. Os objetivos deste trabalho foram validar método de análise de amostras de trevo-vermelho por Cromatografia Líquida de Alta Eficiência (CLAE); quantificar quatro isoflavonas em 78 acessos de trevo-vermelho; verificar as modificações sazonais na concentração de três acessos de trevo-vermelho; aumentar os níveis de isoflavonas através de melhoramento genético; e determinar a atividade anti-inflamatória in vivo e in vitro e de atividade de inibição da acetilcolinesterase de um extrato seco de trevo-vermelho. Para analisar as plantas por CLAE, estas foram hidrolisadas, separadas por diclorometano, e ressuspendidas em metanol. O método de CLAE usado foi validado. As isoflavonas (daidzeína, genisteína, formononetina e biochanina A) foram quantificadas em 78 acessos de trevo-vermelho, os conteúdos das agliconas (expressos em μg/g de planta seca, n=3) destes compostos variaram entre 0,00 a 137,91 para daidzeína; 14,70 a 516,51 para genisteína; 452,97 a 28548,65 para formononetina; e 1967,64 a 20145,27 para biochanina A, e a concentração total de isoflavonas variou entre 9,81 e 36,36 mg/g. O estudo sazonal mostrou que a concentração total das isoflavonas não difere entre as estações, mas verifica-se que na primavera (quando o trevo-vermelho está no estágio reprodutivo) se observam concentrações inferiores, e no inverno (quando a planta está no estágio vegetativo) são encontradas concentrações mais elevadas. No estudo de melhoramento genético foi possível observar um aumento na concentração total de isoflavonas. A atividade anti-inflamatória in vitro foi medida através da mobilização de leucócitos, pelo ensaio de quimiotaxia na câmara de Boyden. A atividade anti-inflamatória in vivo foi avaliada pelo teste de edema de pata de rato induzido por carragenina. Os resultados do teste anti-inflamatório in vitro mostram que houve significante inibição da migração dos leucócitos nas concentrações de 100,0 (94,73% de inibição), 50,0 (95,39% de inibição), 25,0 (94,73% de inibição), 10,0 (84,68% de inibição) e 5,0 (78,75% de inibição) μg/mL de extrato seco de trevo-vermelho. O teste anti-inflamatório in vivo demonstrou significante atividade nas doses testadas, 100 e 50 mg/kg de extrato seco de trevo-vermelho. O percentual médio de inibição do edema foi 63,37%. O teste da inibição da acetilcolinesterase não demonstrou atividade. Os resultados deste estudo sugerem que se pode produzir plantas com concentrações mais elevadas de isoflavonas para a produção de fitomedicamentos com maior qualidade, e o extrato de trevo-vermelho pode ser adequado para o tratamento de doenças inflamatórias. / Red clover (Trifolium pratense L.) is one of the most utilized forage legume in the world agriculture and contains the isoflavones formononetin and biochanin A, and in smaller concentration daidzein and genistein. These compounds have gained a high interest due to their human health benefits. Recently this species was included in the list of Relação Nacional de Plantas Medicinais de Interesse ao SUS (RENISUS) which contains medicinal plants that have potential to generate products to be produced and distributed by SUS. But it is important to highlight that does not exist any study of isoflavones quantification in red clover plants cultivated in Brazil. The aims of this work were to validate a method to analize red clover samples by High Performance Liquid Cromatography (HPLC); to quantify four isoflavones compounds in 78 red clover accessions; to verify seasonal modifications in the isoflavone concentration of three red clover accessions; to increase isoflavones levels through plant breeding; and to determine in vivo and in vitro anti-inflammatory activity, and inhibitory activity of acetylcholinesterase of a red clover dry extract. To analyze the plants by HPLC, they were hydrolyzed, separated by dichloromethane, and resuspended with methanol. The HPLC method used was validated. Isoflavones (daidzein, genistein, formononetin and biochanin A) were quantified in 78 red clover accessions, the aglycone contents (expressed in μg/g of dry material, n=3) of these compounds varied from 0.00 to 137.91 to daidzein; 14.70 to 516.51 to genistein; 452.97 to 28548.65 to formononetin; and 1967.64 to 20145.27 to biochanin A, and the total isoflavone concentration ranged between 9.81 and 36.36 mg/g. The seasonal study showed that the total concentration of isoflavones is not statistically different at different seasons, but is possible to verify that at spring (when red clover is at reproductive stage) we could observe the lowest concentration, and at winter (when red clover is at vegetative stage) we found the highest concentration. In the breeding study we could observe an increase in the total concentration of isoflavones. The in vitro anti-inflammatory activity was measured towards by leucocytes mobilization, by chemotaxis assay in Boyden´s chamber. The in vivo anti-inflammatory activity was tested by a carrageenan-induced rat paw edema test. The results of anti-inflammatory in vitro test showed that there was a significant inhibition of leukocyte migration at concentrations of 100.0 (94.73% of inhibition), 50.0 (95.39% of inhibition), 25.0 (94.73% of inhibition), 10.0 (84.68% of inhibition) and 5.0 (78.75% of inhibition) μg/mL of red clover dry extract. The in vivo anti-inflammatory test demonstrated significant activity in the tested doses, 100 and 50 mg/kg of red clover dry extract. The average of edema inhibition percentage was 63.37%. The inhibition of acetylcholinesterase test did not show activity. The results of this study suggest that we can select or produce plants with higher concentrations of isoflavones to produce phytomedicines with higher quality, and the red clover extract might be suitable for the treatment of inflammatory diseases.