Made available in DSpace on 2014-06-11T19:29:07Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-07-30Bitstream added on 2014-06-13T20:18:55Z : No. of bitstreams: 1 cavassani_tb_me_araiq.pdf: 1899529 bytes, checksum: c90c7f090f98ce6f94ef871d0d70f937 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / É consenso entre cientistas do mundo inteiro que existem moléculas com o poder de alterar o funcionamento do sistema endócrino humano e de diversas espécies de animais, conhecidas atualmente como alteradores endócrinos. Dentro deste grupo de substâncias, merece destaque as isoflavonas presentes em maior abundância nos grãos de soja, principalmente a genisteína (GEN) e a daidzeína (DAID), apontadas como responsáveis por desencadear inúmeros efeitos endócrinos em espécies aquáticas. Portanto, o objetivo do presente trabalho foi otimizar e validar um novo método para determinação de GEN e DAID em efluente de ETE, água subterrânea, potável e de rio; aplicando o método já validado nestas matrizes na cidade de Araraquara e avaliar o comportamento sazonal destes alteradores endócrinos. O método apresentado é baseado na pré-concentração e simplificação de amostra proporcionado pelo uso de SPE aplicado a 2 litros de amostras de água de rio e subterrânea e ao volume de 1,5 litro de efluente de ETE, analisados por HPLC-UV utilizando um sistema MeOH/Água como alternativa ao uso de acetonitrila. A exatidão mostrou-se na faixa de 75 a 110%, com precisão entre 0,5 e 14 %. O LQ alcançado com o referido método foi de 6,3 e 188 ng L-1 para daidzeína e 2,17 e 187 ng L-1 para a genisteína nas matrizes de água subterrânea, superficial e efluente de ETE, respectivamente. A aplicação do método nas amostras de água subterrânea e do Ribeirão das Cruzes demostrou que as concentrações de DAID e GEN situaramse abaixo dos LD. Já para as amostras de efluente de ETE, estas moléculas foram identificados em todas as amostras coletadas, com concentrações variando entre 60-306 ng L-1 para GEN e de 76 a 288 ng L-1 para DAID. Comparando-se os dados de temperatura e pluviosidade com as concentrações destes fitoestrógenos no período amostrado, percebe-se que a temperatura... / It is a common thought among scientists worldwide that there are molecules with the capacity of altering the functioning of the endocrine system of humans and several species of animals, currently known as endocrine modifiers. Inside this group of substances, the highlighted ones are the isoflavones more abundantly present in soybean grains, especially genistein (GEN) and daidzein (DAID), pointed as those responsible for triggering a number of endocrine effects in aquatic species. Therefore, the aim of the present work was optimize and validate a new method for the determination of GEN and DAID in an effluent of a Sewage Treatment Unit (STU), as well as subterranean, drinking and river waters, applying the method already validated in these matrixes in the city of Araraquara, and then evaluate the seasonal behavior of these endocrine modifiers. The method presented is based on the sample pre-concentration and simplification enabled by the use of SPE applied to 2 liters of samples from subterranean and river waters, and to the volume of 1.5 liter of STU effluent, analyzed through HPLC-UV using a system of MeOH/Water as an alternative to the use of acetonitrile. The accuracy was shown to be in the range of 75 to 110%, with a precision between 0.5 and 14 %. The LQ reached with the method referred was of 6.3 and 188 ng L-1 for daidzein, and 2.17 and 187 ng L-1 for genistein in the matrixes of subterranean and superficial waters, as well as STU effluent, respectively. The application of the method in the samples of subterranean water and also in that of the Cruzes stream demonstrated that the DAID and GEN are below the LD. For the samples of STU effluent, such molecules were identified in all samples collected, with concentrations ranging between 60-306 ng L-1 for GEN and 76-288 ng L-1 for DAID. Comparing the temperature and rainfall data with the concentrations of these... (Complete abstract click electronic access below)

A atividade antiviral de compostos flavonoídicos tem sido amplamente investigada nos últimos anos. Nesse contexto, a primeira etapa da presente tese teve por objetivo avaliar a atividade anti-herpética in vitro dos principais isoflavonóides de Glycine max (soja): genisteína, daidzeína, gliciteína e cumestrol. Dentre os isoflavonóides investigados, a genisteína e o cumestrol mostraram uma interessante atividade frente aos vírus HSV-1 (cepa KOS e 29R, sensível e resistente ao aciclovir, respectivamente) e HSV-2 (cepa 333), interferindo em diferentes etapas do ciclo de replicação dos vírus. Devido à reduzida hidrossolubilidade desses isoflavonóides, foi proposta a sua incorporação em nanoemulsões de uso tópico. As formulações foram otimizadas através de um experimento fatorial completo do tipo 23 . O efeito dos fatores tipo de óleo (óleo de rícino ou miristato de isopropila), co-tensoativo iônico (oleilamina ou ácido olêico) e fosfolipídeo (DSPC ou DOPC) sobre as propriedades das nanoemulsões e a retenção da genisteína na pele de orelha suína foi investigado. Nanoemulsões compostas de miristato de isopropila/ oleilamina/DOPC apresentaram um menor diâmetro de gotícula e uma maior retenção de genisteína na pele de orelha suína enquanto que a combinação miristato de isopropila/oleilamina/DSPC mostrou o menor índice de polidispersão. A viscosidade das formulações selecionadas foi ajustada através do uso de hidroxietilcelulose visando à obtenção de produtos de uso tópico, obtendo-se hidrogéis de comportamento pseudoplástico. Na sequência, um conjunto de resultados obtidos demonstrou que a composição das formulações (especialmente o fosfolipídeo DOPC e o agente espessante hidroxietil celulose) pode influenciar a liberação e a retenção dos isoflavonóides em mucosa esofágica suína. A integridade da mucosa também desempenha um papel no aumento da permeação/retenção do cumestrol, conforme ilustrado nas imagens de microscopia confocal, utilizando vermelho do Nilo como marcador fluorescente. De maneira geral, a incorporação dos isoflavonóides nas nanoemulsões aumenta a atividade anti-herpética desses compostos in vitro. O conjunto dos resultados demonstra que as formulações desenvolvidas são potenciais carreadores de uso tópico para genisteína e cumestrol no tratamento das infecções herpéticas. / The antiviral activity of flavonoid compounds has been extensively investigated in recent years. In this context, the first step of this study was to evaluate the antiherpes activity in vitro of the main isoflavonoids of Glycine max (soybean): genistein, daidzein, glycitein and coumestrol. Among the investigated isoflavonoids, genistein and coumestrol showed interesting activity against HSV-1 (KOS and 29R strains, which are acyclovir-sensitive and acyclovir–resistant strains, respectively) and HSV-2 (333 strain), interfering at different stages of the virus replication cycle. Due to the low hydrosolubility of these isoflavonoids, their incorporation into topical nanoemulsions was proposed in this study. The formulations were optimized through a 23 full factorial design. The factors effect of oil type (castor oil or isopropyl myristate), ionic co-surfactant (oleylamine or oleic acid) and phospholipid type (DSPC or DOPC) on physicochemical properties and genistein retention into porcine ear skin was investigated. Nanoemulsions composed of isopropyl myristate/ DOPC/oleylamine showed the smaller particle size and higher genistein retention into skin, whereas the formulation composed of isopropyl myristate/DSPC/oleylamine exhibited the lower polydispersity index. The viscosity of selected formulations was adjusted with hydroxyethyl cellulose to obtain topical products, which showed nonNewtonian behavior. In sequence, a set of results showed that formulations composition (especially the phospholipid DOPC and the thickening agent hydroxyethyl cellulose) could influence the release and retention of isoflavonoids in porcine esophagus mucosa. The integrity of mucosa plays a role on the increase of coumestrol permeation/retention, according to confocal microscopy images, using Red Nile as fluorescent label. In general terms, the incorporation of isoflavonoids into nanoemulsions increases the antiherpes activity of these compounds in vitro. The overall results show that the formulations developed in this study are potential topical carriers for genistein and coumestrol in the treatment of herpes infections.

Estudos recentes têm demonstrado o efeito das isoflavonas da soja, em especial da genisteína (GEN), aplicada topicamente, na prevenção do envelhecimento cutâneo. Esse efeito tem sido relacionado com as suas atividades inibidora de tirosina quinase, antioxidante e estrogênica. Neste contexto, o objetivo do presente trabalho foi desenvolver nanoemulsões de uso tópico contendo GEN. Em uma primeira etapa, foi validada metodologia para a quantificação da GEN por CLAE, utilizando um sistema isocrático com detecção no UV em 327 ou 270 nm. Na segunda etapa, nanoemulsões constituídas de GEN, lecitina de gema de ovo, triglicerídeos de cadeia média (TCMGEN) ou octildodecanol (ODDGEN) e água foram preparadas por emulsificação espontânea. Esse procedimento conduziu à obtenção de nanoemulsões monodispersas com diâmetro de gotícula de 263 e 282 nm, viscosidade de 1,5 e 1,8 cP e potencial zeta de -44 e -42 mV, para TCMGEN e ODDGEN, respectivamente. A quantidade de GEN associada com ambas as formulações foi próxima de 100 % (para 1 mg/mL). A reduzida solubilidade da GEN no TCM e ODD (230 e 138 μg/g, respectivamente) sugere o efeito da lecitina na sua associação com as nanoemulsões. Considerando que os estudos de DSC demonstraram a interação da GEN com TCM, ODD e lecitina, a GEN parece estar localizada tanto no núcleo oleoso como na interface das nanoemulsões. Em uma última etapa, foi realizado o estudo de permeação cutânea da GEN a partir das formulações utilizando células de difusão de Franz. Foi demonstrada a reduzida permeação da GEN (~ 7,5 μg.cm-2.h-1). Existe uma redução significativa da permeabilidade da GEN a partir dos núcleos óleos (~ 3,5 – 5 μg.cm-2.h-1) ou nanoemulsões (~ 3 – 3,5 μg.cm-2.h-1), indicando a afinidade da GEN pelos veículos utilizados. O conjunto dos resultados obtidos demonstra a influência dos componentes das formulações sobre as propriedades físico-químicas das nanoemulsões, bem como no perfil de permeação cutâneo da GEN. / Recent studies have shown the effect of soy isoflavones, especially genistein (GEN), topically administrated, in preventing skin aging. This effect has been related to tyrosine kinase inhibition, antioxidant and estrogenic activities. In this context, the aim of the present work was the development of topic nanoemulsions containing GEN. First, it has been validated an isocratic method to quantify GEN by HPLC with UV detection at 327 and 270 nm. In a second step, nanoemulsions composed by GEN, egg lecithin, medium chain triglycerides (TCMGEN) or octyldodecanol (ODDGEN) and water were prepared by spontaneous emulsification. This procedure yielded monodisperse emulsions with a typical mean droplet size of 263 and 282 nm, viscosity of 1.5 and 1.8 cP and ζ-potential -44 and -42 mV, for TCMGEN and ODDGEN, respectively. The amount of GEN associated with both formulations was close to 100 % (to 1 mg/mL). The low solubility of GEN in TCM and ODD (230 and 138 μg/g, respectively) suggests the role of lecithin on their association with nanoemulsions. Since DSC experiments have demonstrated GEN interactions with TCM, ODD and lecithin, GEN molecules seem to be located in to the oil core and at the interface of nanoemulsions. In a last step, the permeation of GEN from formulations using ear pigskin mounted in Franz diffusion cells was performed. It was shown that GEN permeation was low (~ 7.5 μg.cm-2.h-1). There was a significant reduction of the GEN permeability from oils (~ 3.5 - 5 μg.cm-2.h-1) or nanoemulsions (~ 3 – 3.5 μg.cm-2.h-1), showing the affinity of GEN to the vehicles. In conclusion, the overall results show the effect of the nanoemulsion components on both physicochemical properties of the nanoemulsions and GEN skin permeation profile.

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.

Orientador: WellersonRodrigo Scarano / Coorientador: Flávia Karina Dellela / Banca: Luis Antonio Justilin Junior / Banca: Silvana Gisele Pegorin de Campos / Resumo: A Iniciação e Progressão das neoplasias prostáticas apresentam base hormonal, onde se destacam as alterações envolvendo estrógenos e andrógenos inerentes ao processo de envelhecimento, com incremento dos níveis estrogênicos em relação aos androgênicos. O Bisfenol A (BPA) é o xenoestrógeno mais estudado na atualidade e sua atividade estrogênica tem despertado interesse devido à ampla dispersão do composto no meio ambiente. Assim sendo, o BPA poderia contribuir para o aumento da incidência, agressividade e capacidade metastática dos tumores prostáticos. Por outro lado, a quimioproteção com os fitoquímicos dietéticos está associada à redução na incidência e na progressão de diferentes neoplasias, diminuição de processos inflamatórios e estresse oxidativo ocasionados por substâncias potencialmente nocivas. Desta forma, este estudo objetivou avaliar os efeitos dos fitoquímicos 3,3'-Di-indolil-metano (DIM) e Genisteína (Gen) isolados e/ou associados em células tumorais prostáticas humanas expostas ao BPA. Para tanto, as linhagens LNCaP e PC-3 foram cultivadas e submetidas, por 96h, aos tratamentos C (controle), B (BPA 10nM/L), BD (BPA 10nM/L + DIM 25μM/L), BG (BPA 10nM/L + Gen 25μM/L) e BDG (BPA 10nM/L + DIM 25μM/L + Gen 25μM/L); cujas doses foram estabelecidas pelo ensaio de viabilidade celular. Posteriormente, foi realizado Western Blotting para estudo das proteínas envolvidas com processos de sobrevivência, proliferação, morte celular, modulação hormonal e estresse oxidativo. A análise dos resultados mostrou que os tratamentos BD, BG e BDG, na linhagem LNCaP, apresentaram capacidade de sub-regular a expressão de AR. Acerca das proteínas ERK1/2 em PC-3, B provocou um aumento de expressão, enquanto BDG ocasionou uma diminuição. Já na linhagem LNCaP, a redução da expressão desta MAPK ficou por conta do tratamento BG. Para as proteínas JNK1/2, verificou-se a capacidade de B sub-regular a... / Abstract: The prostate cancer initiation and progression present hormonal basis and this is mainly related to changes involving estrogens and androgens inherent aging process, where there is an increment of estrogens levels relative to androgens. Nowadays, Bisphenol A (BPA) is the most studied xenoestrogen and its estrogenic activity has attracted attention for its wide dispersion in the environment. Thus, this compound could contribute to the increased incidence, aggressiveness and metastatic ability of prostate tumors. Moreover, the chemoprotection with dietary phytochemicals is associated with a reduction in the incidence and progression of different cancers, decrease inflammatory processes and oxidative stress caused by potentially harmful substances. Therefore, this study aimed to evaluate the effects of phytochemicals 3,3 '-Di-indolyl-methane (DIM) and Genistein (Gen) isolated and/or associated to prostatic tumor cells exposed to BPA. For this purpose, LNCaP and PC-3 cells were cultured and exposed, for 96 hours, to the treatments C (control), B (BPA10nM/L), BD (BPA 10nM/L + DIM 25μM/L), BG (BPA 10nM/L + Gen 25μM/L) e BDG (BPA 10nM/L + DIM 25μM/L + Gen 25μM/L); which doses were established by cell viability assay. Subsequently, the cells were subjected to protein extraction for Western Blotting in order to analyze proteins involved in survival, proliferation, cell death, hormonal modulation and oxidative stress processes. The results showed that BD, BG and BDG treatments, in LNCaP cells, were able to down-regulate AR expression. About the ERK1/2 proteins in PC-3 lineage, B caused an increase of expression, while BDG was responsible for a decrease. However, in LNCaP, reducing the expression of MAPK was due to BG treatment. For the JNK1/2 protein, B had ability to down-regulate its expression and, on the other hand, phytochemicals from 3 treatments (BD, BG and BDG), increasing its expression in LNCaP. For the same cell lineage, the ERα ... / Mestre

A growing body of evidence has demonstrated that soy isoflavone consumption may protect against the development of various chronic diseases. This protection could be linked to isoflavone-induced alterations in immune function. However, recent in vitro and animal studies suggest that soy isoflavones may either enhance or suppress immunocompetence, depending upon the isoflavone concentration, target tissue, and a number of other factors. To date, no study has investigated the effect of dietary soy isoflavone supplementation on immune parameters in humans. Therefore, the purpose of this double-blind, placebo-controlled, 4 wk intervention trial was to investigate whether supplementation with soy isoflavones alters indices of immune function in postmenopausal women. Twenty healthy women (50-69 yr), who were not on hormone replacement therapy, were randomly divided into 2 treatment groups. The supplemented group (n=10) consumed soy isoflavone tablets (100 mg/d) for 4 wk, while the control group (n=10) received placebo tablets. Fasting blood samples were drawn at baseline and on d 28 to assess specific immune parameters. In addition, plasma concentrations of genistein and daidzein were quantified at baseline and at the end of the intervention period. Despite high individual variability among subjects, there was a significant increase (p<0.005) in plasma isoflavone concentration in the supplemented group. However, all assessed immune parameters remained unchanged after supplementation and did not differ between the 2 treatment groups. In conclusion, this study suggests that short-term soy isoflavone supplementation at physiologically attainable concentrations does not alter the aforementioned immune parameters in healthy postmenopausal women. Due to the conflicting data concerning the effect of dietary soy isoflavones on immune function, further research in this area is warranted. / Master of Science

A growing body of evidence has demonstrated that soy isoflavone consumption may protect against the development of various chronic diseases. This defense could be linked to isoflavone-induced alterations in immune function. However, to date, no study has examined the effect of soy isoflavone supplementation on human immunity in vivo. Establishing whether isoflavones affect immunity in aging adults is particularly relevant since compromised immune function has been observed in this population. Therefore, the purpose of this double-blind, placebo-controlled, 4-wk intervention trial was to investigate whether supplementation with soy isoflavones influenced the distribution and/or function of specific lymphocytes in postmenopausal women. Healthy postmenopausal women (50-69 y), who were not using hormone replacement therapy, were randomly divided into 2 treatment groups. The experimental group (n=9) consumed two-50 mg soy isoflavone tablets/d for 4 wk, while the control group (n=9) received placebo tablets. Fasting blood samples were drawn at baseline and on d 28 to assess distribution of T-helper cells (CD3+CD4+), T-cytotoxic cells (CD3+CD8+), total T lymphocytes (CD3+), B lymphocytes (CD19+) and natural killer (NK) cells (CD16+CD56+) via flow cytometry. Cytotoxicity of NK cells was quantified based on lactate dehydrogenase release of lysed K562 cancer cells following co-culture with NK cells from subjects. Analysis of plasma isoflavone concentrations by HPLC demonstrated a significant increase (p<0.005) in plasma genistein concentration in the experimental group after 4 wk of supplementation. However, there was no alteration in lymphocyte distribution or NK cell activity in response to isoflavone supplementation, suggesting that short-term soy isoflavone supplementation does not alter these parameters of immunity in healthy postmenopausal women. / Master of Science

Diabetes is growing public health problem in the United States. Both in Type 1 and Type 2 diabetes, the deterioration of glycemic control over time is largely due to insulin secretory dysfunction and significant loss of functional β-cells. As such, the search for novel agents that promote β-cell survival and preserve functional β-cell mass are one of the essential strategies to prevent and treat the onset of diabetes. Genistein, a flavonoid in legumes and some herbal medicines, has various biological actions. It was recently shown that dietary intake of foods containing genistein improves diabetes in both experimental animals and humans. However, the potential anti-diabetic mechanisms of genistein are unclear. In the present study, we first investigated the effect of genistein on β-cell insulin secretion and proliferation and cellular signaling related to these effects in vitro and in vivo. We then determined its anti-diabetic potential in insulin-deficient and obese diabetic mouse models. The results in our study showed that exposure of clonal insulin secreting (INS1E) cells or isolated pancreatic islets to genistein at physiologically relevant concentrations (1-10 μM) enhanced glucose-stimulated insulin secretion (GSIS), whereas insulin content was not altered, suggesting that genistein-enhanced GSIS is not due to a modulation of insulin synthesis. This genistein's effect is protein tyrosine kinase- and KATP channel-independent. In addition, genistein had no effect on glucose transporter-2 expression or cellular ATP production, but similarly augmented pyruvate-stimulated insulin secretion in INS1E cells, indicating that genistein improvement of insulin secretion in β-cells is not related to an alternation in glucose uptake or the glycolytic pathway. Further, genistein (1-10 μM) induced both INS1 and human islet β-cell proliferation following 24 h of incubation, with 5 μM genistein inducing a maximal 27% increase. The effect of genistein on β-cell proliferation was neither dependent on estrogen receptors, nor shared by 17β-estradiol or a host of structurally related flavonoid compounds. Pharmacological or molecular intervention of PKA or ERK1/2 completely abolished genistein-stimulated β-cell proliferation, suggesting that both molecules are essential for genistein action. Consistent with its effect on cell proliferation, genistein induced cAMP/PKA signaling and subsequent phosphorylation of ERK1/2 in both INS1 cells and human islets. Furthermore, genistein induced protein expression of cyclin D1, a major cell-cycle regulator essential for β-cell growth. Dietary intake of genistein significantly improved hyperglycemia, glucose tolerance, and blood insulin levels in both insulin deficient type 1 and obese type 2 diabetic mice, concomitant with improved islet β-cell proliferation, survival, and mass. These changes were not due to alternations in animal body weight gain, food intake, fat deposit, plasma lipid profile, or peripheral insulin sensitivity. Collectively, these findings provide better understanding of the mechanism underlying the anti-diabetic effects of genistein. Loss of functional β-cell mass through apoptosis is central to the development of both T1D and T2D and islet β-cell preservation and regeneration are very important components of β-cell adaptation to increased apoptosis and insulin resistance and therefore holds promise as a treatment for this disease. In this context, these findings may potentially lead to the development of novel low-cost natural agents for prevention and treatment of diabetes. / Ph. D.

Octylphenol and genistein are two of the growing list of endocrine disrupting chemicals found in the environment that mimic estrogen in reproductive tissue both in vitro and in vivo. It is well established that endogenous estrogens modulate not only the reproductive system, but also the immune system. However, the effects of many endocrine disrupting chemicals, such as octylphenol and genistein, on the immune system have yet to be determined. Preliminary studies on short-term treatment with genistein (0.6 mg) and octylphenol (10 mg) showed that the thymus of orchiectomized (NZBxNZW)F1 males is sensitive to these agents. Further studies focused on the effects of short-term treatment of octylphenol on the morphology and function of the thymus in adult, reproductively intact non-autoimmune C57BL/6 and pre-autoimmune (NZBxNZW)F1 males. Oral dosing of 0.1 mg, 1 mg, or 10 mg of octylphenol 3 times a week for 3 weeks did not affect the morphology or function of the thymus as assessed by its weight, thymocyte cellularity, proportion of immature and mature thymocytes, level of apoptosis, apoptotic rates of stimulated thymocytes, and proportion of mature T cells in the spleen. Furthermore, oral dosing of 0.1 mg, 1 mg, or 10 mg of octylphenol did not result in estrogenic changes in the reproductive tract in our model. Subcutaneous injection of 10 mg of octylphenol resulted in skin lesions that confounded the assessment of its affects on the thymus. Further studies are needed to definitively determine the effects of octylphenol on the immune system of both males and females of various ages and to determine the effect of long-term exposure. / Master of Science

Huang, Hui. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 141-154). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.