Made available in DSpace on 2014-07-29T16:11:45Z (GMT). No. of bitstreams: 1 Dissertacao Stela.pdf: 754626 bytes, checksum: 312169c0104b8d6d919af0eb6ee8cf69 (MD5) Previous issue date: 2010-03-05 / Isoflavones are flavonoids found in abundance in grains of soy (Glycine max L.) and its derivatives. These substances have estrogen like structure and weakly estrogenic action. They are used for treatment of menopause symptoms as an alternative to conventional hormone replacement therapies, because they cause fewer side effects. Soy isoflavones are amongst herbal products of great interest to obtain solid dosage forms. High assay variability of isoflavones in commercially available capsules and tablets shows the need of appropriate drug development and quality control for this herbal product. The aim of this work was drug development and quality control of soy isoflavone solid dosage forms, using standardized dry extract of soy. To evaluate the quality of the extract microbiologic and assay analysis of this drug s markers were performed. Results obtained from microbiological test did not show microbiologic contamination of the extract, as Escherichia coli, Staphylococcus aureus, Candida albicans and Aspergillus niger did not grow. To evaluate assay and dissolution percentage of both markers analytical methodology for quantification of daidzein and genistein by high performance liquid chromatography was developed, and also a method to analyze dissolution profile of the obtained tablets. Isocratic method was used for chromatographic analysis of the markers, with mobil phase of methanol with 0.1% of acetic acid, water with 0.1% of acetic acid and acetonitrile in proportion 64:32:4, flow of 0.7 mL/min and temperature of 30°C. At this condition, peaks were symmetric, had resolution lower than 2, with retention times of 3,13 and 3,72 for daidzein and genistein, respectively. Assay evaluation of the markers daidzein and genistein showed that concentrations in the extract were 27,8% and 11,1%, respectively. To accomplish preformulation study, thermal analysis and powder and granules flow evaluation were made. Six formulations of isoflavone tablets were developed, one by direct compression and five by wet granulation. Results of thermal analysis showed no possible extract interactions between tested excipients and extract. Wet granulation was the best method to obatain tablets, because it presented the best compressional properties with flow velocity between 4,5 and 6,7 s/100g and compressibility between 16,85% and 26,06%. GU3 formulation, which presented the best performance according to evaluated parameters, made by wet granulation, showed 92,66% and 92,76% of daidzein and genistein, respectively, disintegration of 15 minutes, and its flow was categorized as excellent. In analytical dissolution method development of isoflavone tablets solubility test was performed to define the best medium, and water with 3% of sodium lauryl sulfate dissolved the greatest amount of daidzein and genistein at tested temperature, 37°C, in comparison with the other medium. Other parameters defined through method development were paddle apparatus, stirring speed of 100 rpm and 900 mL of medium volume. Results showed the possibility to obtain promising solid dosage forms to deliver isoflavone, and optimized quality control methodologies to analyze raw material and finished product. / Isoflavonas são flavonóides encontrados em abundância nos grãos de soja (Glycine max L.). Essas substâncias têm estrutura semelhante ao estrógeno e possuem uma ação fracamente estrogênica. São utilizadas no tratamento dos sintomas da menopausa como alternativa às terapias de reposição hormonal convencionais, por provocarem menor número de efeitos colaterais. As isoflavonas de soja estão entre as matérias-primas vegetais com ações de interesse para obtenção de formas farmacêuticas sólidas. A alta variabilidade de teores de isoflavonas em cápsulas e comprimidos disponíveis comercialmente demonstra a necessidade de desenvolvimento farmacotécnico e controle de qualidade adequados para este fitoterápico. O objetivo deste trabalho foi o desenvolvimento analítico e farmacotécnico de formas farmacêuticas sólidas de isoflavona de soja, utilizando como matéria-prima extrato seco de soja padronizado. Para verificar a qualidade do extrato foi realizada análise microbiológica e análise do teor dos marcadores. Os resultados obtidos através do ensaio microbiológico não demonstrou contaminação microbiológica do extrato, não tendo crescimento de Escherichia coli, Staphylococcus aureus, Candida albicans e Aspergillus niger. Para avaliação do teor e da porcentagem dissolvida de ambos os marcadores foram desenvolvidas e validadas tanto metodologia analítica de quantificação de daidzeína e genisteína por cromatografia líquida de alta eficiência quanto metodologia para avaliar o perfil de dissolução dos comprimidos obtidos. Para análise cromatográfica dos marcadores foi utilizado método isocrático, com fase móvel constituída por metanol com 0,1% de ácido acético, água com 0,1% de ácido acético e acetonitrila na proporção 64:32:4 , fluxo de 0,7 mL/min e temperatura de 30ºC. Nesta condição os picos foram simétricos, tiveram resolução maior que 2, com tempos de retenção de 3,13 e 3,72 da daidzeína e genisteína, respectivamente. A avaliação do teor dos marcadores daidzeína e genisteína, demonstrou que as concentrações no extrato foram 27,8% e 11,1%, respectivamente. Em relação ao estudo de pré-formulação foi feita análise térmica e a avaliação das propriedades dos pós e grânulos. Foram desenvolvidas seis formulações de comprimidos de isoflavona, sendo uma por compressão direta e cinco por granulação úmida. Nos resultados da análise térmica não foram observadas possíveis interações dos excipientes com o extrato. O melhor método de obtenção dos comprimidos foi por granulação úmida, pois apresentou melhores propriedades compressionais com velocidade de escoamento variando entre 4,5 e 6,7 s/100g e compressibilidade entre 16,85% e 26,06%. A formulação por granulação úmida 3 foi a que apresentou melhor desempenho segundo os parâmetros analisados, apresentando teor de 92,66% e 92,76% de daidzeína e genisteína, respectivamente, desintegração de 15 minutos, e seu fluxo foi caracterizado como excelente. No desenvolvimento do método analítico para dissolução dos comprimidos de isoflavona foi feito o teste de solubilidade para definir o melhor meio, sendo o meio água com 3% de lauril sulfato de sódio o que dissolveu maior quantidade de daidzeína e genisteína na temperatura de 37ºC em comparação com os outros meios. Os demais parâmetros definidos através do desenvolvimento do método foram aparato pá, velocidade de rotação de 100 rpm e 900 mL de volume de meio. Os resultados mostraram a possibilidade de obtenção de formas farmacêuticas sólidas promissoras para veiculação de isoflavona, além de metodologias otimizadas para controle de qualidade para análise da matéria-prima e do produto acabado.

A soja é a principal fonte de isoflavonas, tendo sido associada a efeitos benéficos à saúde humana, incluindo a redução do risco de câncer, de doenças cardiovasculares, osteoporose e melhora dos sintomas da menopausa. A população brasileira não consome soja tão habitualmente quanto os países asiáticos e, nesse contexto, as bebidas de soja poderiam ser uma forma de incluir essas substâncias bioativas na dieta ocidental. Contudo, trabalhos que avaliem o teor de isoflavonas desses produtos são escassos na literatura, o que justifica o presente trabalho. Amostras de doze marcas comercialmente disponíveis de bebidas de soja (n = 65), incluindo os produtos elaborados com isolado protéico de soja ou extrato de soja e acrescidos de sucos de fruta e/ou ingredientes aromatizantes, foram analisadas quanto ao teor e perfil de isoflavonas, utilizando-se a cromatografia líquida de alta eficiência. A capacidade antioxidante foi mensurada pelo método de sequestro de radicais livres do DPPH e o teor de fenólicos totais pelo método do Folin-Ciocalteau. Os valores da concentração total de isoflavonas e o teor de fenólicos totais mostraram uma grande variação entre as diversas marcas de bebidas de soja, variando de 0,7 a 4,9 mg isoflavonas/200 mL e de 6 a 146 mg equivalentes de catequina/200 mL para as bebidas contendo sucos de fruta. Bebidas de soja de sabor original ou chocolate apresentaram variação do teor de isoflavona de 4 a 13 mg/200 mL e fenólicos totais variando de 38 a 155 mg equivalentes de catequina/200 mL. O teor protéico variou de 0,8 a 6 g/200 mL e mostrou-se positivamente correlacionado ao teor de isoflavonas das amostras. As formas predominantes de isoflavonas, nos produtos analisados, foram os β- glicosídeos. Os resultados mostraram que o teor e perfil de isoflavonas, assim como os valores de fenólicos totais das bebidas de soja dependem das condições de processamento e que a atividade antioxidante variou significantemente entre os produtos. Os resultados obtidos mostram que o consumo de uma porção de 300 mL de bebida de soja sabor natural poderia resultar em uma ingestão de 20 mg de isoflavonas, o equivalente a ingestão diária Koreana, indicando que as bebidas de soja poderiam representar fontes importantes de isoflavonas para a nossa dieta. Além disso, o teor de isoflavonas solúveis das bebidas de soja diminui com o decorrer do armazenamento associado à redução da solubilidade da proteína de soja. O perfil de isoflavonas e a capacidade antioxidante das bebidas também são alterados. / Soybean is the most important source of isoflavones, which have been associated with beneficial effects in humans, including prevention of cancer, cardiovascular diseases, osteoporosis and relief of menopausal symptoms. Soybean consumption in Brazil is not as significant as in Asian countries and in this context, soy beverages could be a way to include these bioactive substances in ocidental diet. However, studies about isoflavone content in these products are scarce, which accounts for the present work. Samples of 12 different brands of commercially available soy milk drinks (n = 65), including products made from isolated soy protein and soymilk mixed with fruit juices and/or containing flavoring ingredients were analysed for their isoflavone content and profile using highperformance liquid chromatography. The antioxidant activity was measured using 2.2- diphenil-1-picryl-hydrazil (DPPH) free radical method and the total phenolics was determined by Folin-Ciocalteau method. There was a large variation in total isoflavone concentration and total phenolics content among the different brands of soy milks ranging from 0.7 to 4.9 mg isoflavones/200 mL and 6 to 146 mg equivalents of catechin/200 mL of soymilk mixed with fruit juice. For natural or chocolate soy beverages, the isoflavone content varied from 4 to13 mg/200 mL and the total phenolics ranged from 38 to155 mg equivalent of catequina/200 mL. Levels of protein ranged from 0.8 to 6.0 g/200 mL, these values were associated positively with isoflavone content from samples. The β-glycosides were the predominant form of the isoflavones in the products analyzed. The results showed that total isoflavone, phenolic contents and isoflavone profile of soy beverages depends on processing conditions. Also the antioxidant activity varied significantly among products. The results obtained show that the consumption of 300 mL of soy beverage natural flavor would result in an intake of around 20 mg isoflavone, very similar to the Korean daily intake, indicating that soy beverages could represent important sources of isoflavones in our diet. Furthermore, the soluble isoflavone content of soy beverage decreases with the storage time associated with a reduction of the solubility of soy protein. The profile of isoflavones and antioxidant capacity of beverages are also altered.

No description available.

No description available.

No description available.

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.

Isoflavone synthase (IFS; CYP93C) plays a key role in the biosynthesis of the plant secondary metabolites, isoflavonoids. These phenolic compounds, which are well-known for their multiple biological effects, are produced mostly in leguminous plants (family Fabaceae). However, at least 225 of them have also been described in 59 other families, without any knowledge of orthologues to hitherto known IFS genes from legumes (with the single exception of sugar beet - Beta vulgaris, from the family Chenopodiaceae). In view of these facts, this masters thesis has focused on two main objectives: (1) to identify isoflavone synthase genes in selected leguminous and non-leguminous plants exploiting the PCR strategy with degenerate and non-degenerate primers, and (2) to find a system for the verification of the correct function of these genes. Our methodology for the identification of IFS orthologues was successfully demonstrated in the case of two examined legumes - Phaseolus vulgaris L. and Pachyrhizus tuberosus (Lam.) Spreng, in the genomic DNA of which the complete IFS sequences have been newly identified. To design a procedure for ascertaining the correct function of these genes and others once they have been completely described, a pilot study with IFS from Pisum sativum L. (CYP93C18; GenBank number...