The isoflavones in kudzu roots, especially the C-glycosylated isoflavone puerarin, have been linked to many health benefits. Puerarin contains a carbon-carbon glycosidic bond that can withstand hydrolysis. The C-glycosylation reaction in the biosynthesis of puerarin has not been thoroughly investigated, with conflicting reports suggesting that it could take place on daidzein, isoliquiritigenin, or 2,7,4ʹ-trihydroxyisoflavanone. Kudzu species were identified for use in comparative transcriptomics. A non-puerarin producing kudzu was identified as Pueraria phaseoloides and a puerarin producing kudzu was identified as Pueraria montana lobata. Through the use of the plant secondary product glycosyltransferase (PSPG) motif, glycosyltransferases (UGTs) were identified from the transcriptomes. The UGTs that had higher digital expression in P. m. lobata were examined further using additional tools to home in on the UGT that could be responsible for puerarin biosynthesis. One of the UGTs identified, UGT71T5, had previously been characterized from kudzu as a C-glycosyltransferase involved in puerarin biosynthesis through in vitro enzyme activity (with daidzein) and a gain of function approach in soybean hairy roots. Previous studies have not supported the end-product of a pathway such as daidzein as the target for C-glycosylation, and no genetic analysis of UGT function had been conducted in kudzu. The activity of recombinant UGT71T5 with daidzein was confirmed in the present work. Following the development of a kudzu hairy root system, UGT71T5 expression was then knocked down by RNA interference (RNAi). When compared to control hairy roots there was a large reduction in puerarin content in the UGT71T5-RNAi roots, confirming the role of this enzyme in puerarin biosynthesis. Isotopic labeling of kudzu plants revealed that labeled daidzein could be directly incorporated into puerarin; however, the percent incorporation of daidzein was substantially lower than that of L-phenylalanine, a compound at the start of the pathway to isoflavone synthesis. The knockdown of 2-hydroxisoflavanone synthase (2-HIS) in kudzu hairy roots blocked formation of puerarin and daidzin (7-O-glycosyldaidzein), and was accompanied by accumulation of C-glycosylated isoliquiritigenin and C-glycosylated liquiritigenin. These compounds were found in low amounts in control hairy roots, but were virtually absent in UGT71T5 knockdown hairy roots. The knockdown of 2-hydroxyisoflavanone dehydratase (2-HID) in kudzu hairy roots resulted in a slight reduction in puerarin but no change to daidzin levels, suggesting that C-glycosylation might stabilize the substrate for 2-HID which can otherwise spontaneously dehydrate. Taken together these results reveal that UGT71T5 is likely the major C-glycosyltransferase involved in puerarin biosynthesis in kudzu. They also provide evidence for an alternative pathway to puerarin biosynthesis through the C-glycosylation of isoliquiritigenin or its immediate precursor. In one pathway, UGT71T5 acts as an operationally soluble enzyme that can directly C-glycosylate daidzein, and in the other pathway UGT71T5 acts as part of a metabolic channel for conversion of a C-glycosylated earlier precursor to puerarin. Other UGT enzymes identified in this work did not show C-glycosyltransferase activity; however, three enzymes showed activity in vitro that could be useful for introducing novel regiospecificity in biochemical synthesis of flavonoid glycosides.

Kudzu (Pueraria lobata) plant is an edible leguminous vine. This study focused on the utilization of kudzu starch and kudzu root extract in beef patties. We hypothesized that a) physicochemical and sensory properties of beef patties formulated with kudzu starch, is comparable to those of potato starch; b) the kudzu root extract is rich in isoflavones, and isoflavones quantity is not affected during cooking. In Study I, beef patties were formulated using modified commercially available kudzu and potato starch each at 2.0, 4.0 and 6.0% (wt/wt). Starch treated beef patties were compared with respect to change in physical, chemical, color, textural and consumer responses as affected by starch type (kudzu, potato) and starch level (2, 4, 6 %). Additionally starch treated patties were compared to all-beef patties. Kudzu starch treated patties were significantly lower in moisture % (62.7 vs. 64.4), higher in fat % (9.1 vs. 8.3), protein % (26.3 vs. 24.7), hardness (9.3, vs. 6.9 N) and gumminess (3.7 vs. 1.9 N) compared to potato starch treated patties. Starch treated samples were significantly lighter in color and had lower (P <0.05) expressible moisture compared to all-beef patties. Patties with 6% kudzu or potato starch were significantly higher in cooking yield than all-beef patties. No significant difference existed in consumer overall liking scores of kudzu or potato starch treatments and control beef patties with no added starch. The overall liking scores ranged between 5 ‘neither like nor dislike’ and 6 ‘like slightly’ for all samples. In study II, kudzu root extract was prepared, and using HPLC, ten isoflavones were detected with puerarin and daidzein accounting for 95% of the total isoflavones. Beef patties were formulated with kudzu root extract at 0, 1, and 3% (wt/wt), and four isoflavones were detected in uncooked and cooked patties, considering other isoflavones diluted to undetectable levels in patties. Results indicated that cooking did not change the amount of isoflavones in beef patties. This study illustrates the characteristics of kudzu starch compared to conventionally used potato starch in meat model system and verifies the thermal stability of isoflavones in beef patties.

No description available.

No description available.

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

Les travaux de recherche présentés dans ce manuscrit ont pour but de développer des procédés industriels pour la production de quatre ingrédients alimentaires infantiles ayant des propriétés hypoallergéniques et des teneurs réduites en phytoestrogènes. Les propriétés nutritionnelles des protéines de riz et de soja en font des sources intéressantes. Néanmoins, plusieurs problématiques liées aux caractéristiques des produits apparaissent aujourd’hui : la présence de phytoestrogènes (isoflavones) dans les isolats protéiques de soja ; la difficulté à solubiliser et isoler les protéines de riz et la forte allergénicité des protéines dans le cas du soja. Ces travaux présentent l’étude du fractionnement des protéines de soja et de riz pour le développement : d’isolat protéique à teneur réduite en isoflavones ; isolat protéique de riz ayant une teneur supérieure à 90% de protéines ; hydrolysats protéiques de soja et de riz dont le profil de poids moléculaire est maitrisé et potentiellement hypoallergénique. Afin d’y parvenir, la réduction de la taille des protéines par des processus enzymatiques puis le contrôle de leur poids moléculaire ont dû être étudiés. Concernant l’élimination des phytoestrogènes (isoflavones), deux méthodes ont permis d’atteindre de hauts rendements d’extractions. En premier lieu, l’étude de l’extraction par éthanol via une optimisation à petite échelle, suivie d’une mise à l’échelle industrielle ont permis de développer un premier produit à teneur résiduelle en isoflavones inférieure à 50 μg/g de produit sec représentant une réduction de près de 98% de la teneur en isoflavones. Le second procédé étudié a été la rétention des isoflavones sur résine d’adsorption à partir d’un hydrolysat protéique de soja préalablement mis au point, et ceci, par l’utilisation de solution aqueuse sans étape préalable d’extraction. Ce procédé a fait l’objet d’une mise à l’échelle industrielle et d’une étude du comportement chromatographique des isoflavones. L’extraction des isoflavones par eau subcritique et CO2 supercritique est aussi présentée dans cette thèse. Elle a permis de mettre en évidence l’influence de la polarité des différents composés et de la teneur en protéines des produits de soja utilisés. Ces travaux de thèse ont aussi permis de définir un nouveau procédé pour la production d’isolat protéique de riz par l’intermédiaire d’enzymes de types cellulolytiques et amylases, à partir de coproduits issus de l’industrie du sirop de glucose. Des études sur des matières moins transformées telles que le son de riz et la farine ont aussi été étudiées pour la concentration des protéines. L’étude de l’hydrolyse des protéines de soja et de riz a été possible par le suivi de différents indicateurs tels que le pH, la solubilité des protéines, le degré d’hydrolyse, le profil de poids moléculaire par électrophorèse et par chromatographie d’exclusion stérique. Ces procédés ont permis la production de quatre nouveaux ingrédients pouvant être testés pour leurs caractéristiques hypoallergéniques avant une éventuelle production industrielle / The objectives of these works were to develop industrial processes for the production of four infant food ingredients with hypoallergenic properties and reduced levels of phytoestrogens. For this purpose, the nutritional properties of the rice and soy protein are promising. However, due to the presence of phytoestrogens (isoflavones) the consumption of soy protein isolates is a big concern for infant food security because the high exposure to these compounds, known to be endocrine disruptors. Consequently, it was first intended to develop a soy protein isolate with reduced content of isoflavones below 50 μg/g following the recommendations of French and European health authorities. Rice protein isolates are either non-existent on the market, or extremely rare. Therefore, the development of rice protein isolate with a minimum content of 90 % protein was another objective. For the sensitive population, such as infants, the aim of this work was also to develop soy and rice protein hydrolysates conferring hypoallergenic properties. To achieve this goal, the reduction of the size of proteins and the control of their molecular weight was studied. Two methods were used to achieve high extractions yields. A study of ethanol extraction ranging from small-scale optimization to industrial scale was used for a final product with a residual content in isoflavones below 50 μg/g. The second method was to retain isoflavones on adsorption resin from a soy protein hydrolysate. This was possible without preliminary extraction step by solvent. This method was also tested in the industrial scale. The chromatographic behavior of different isoflavones was also studied. The extraction of isoflavones with subcritical water and supercritical CO2 is also presented in this thesis even though these methods were not retained. These pressurized extractions showed the influence of the polarity of isoflavones and the protein content of soy products onto the isoflavone extraction. These works also identified a novel process for the production of rice protein isolate by the hydrolysis of polysaccharides with cellulolytic enzymes and amylases from concentrated protein byproducts from the glucose syrup industry. Studies on less processed materials such as rice bran and flour were also studied for protein isolation. The study of the hydrolysis by proteases of soy and rice proteins were monitored by various indicators such as pH, protein solubility, the degree of hydrolysis, the molecular weight profile by electrophoresis, and size exclusion chromatography. These processes are enabled for the production of four new ingredients that will be tested for their hypoallergenic characteristics before a large scale production.

No description available.

Objetivo: avaliar os efeitos do tratamento com o Trifolium pratense nos sintomas menopausais em mulheres climatéricas utilizando o Índice Menopausal de Kupperman (IMK) e pelo Inventário de Satisfação Sexual Golombok e Rust versão feminina (GRISS) l. Metodologia: Este estudo foi prospectivo, randomizado, duplo cego e controlado com placebo, e realizado no setor de Ginecologia Endócrina e Climatério da Disciplina de Ginecologia do Departamento de Obstetrícia e Ginecologia do Hospital das Clínicas, da Faculdade de Medicina da Universidade de São Paulo. Foram selecionadas 100 mulheres na faixa etária de 45 a 65 anos com sintomas menopausais, com amenorréia superior há 1 ano e sem tratamento nos últimos 6 meses. Após a seleção, as mulheres foram divididas em dois grupos: Grupo I (n = 50) receberam Trifolium pratense na dose de 40 mg, 1 capsula/dia, por via oral; Grupo II (n = 50) receberam placebo (controle), contendo lactose, 1 cápsula/dia por via oral. A duração do tratamento foi de 12 meses e as mulheres foram avaliadas antes do tratamento com quatro, oito e 12 meses de tratamento, por exames clínico e laboratorial. Aplicaram-se o teste de t Student e o ANOVA para avaliar as diferenças entre os grupos. Resultados: Houve melhora significante dos sintomas após quatro meses de tratamento no IMK, principalmente, as ondas de calores, em relação aos dados antes do tratamento nos dois grupos. Não observamos melhora na avaliação da sexualidade (GRISS). Conclusão: Nossos dados sugerem que o efeito da ministração de 40mg ao dia de Trifolium pratense pode não ser superior ao do placebo, na redução dos sintomas da pós-menopausa durante um ano de estudo / Objective: to evaluate the effects of Trifolium pratense treatment on the climacteric symptoms and sexualiy in postmenopausal women through Kuppermann Menopausal Index (KMI) and the Golombok Rust Inventory of Sexual Satisfaction (GRISS), respectively. Methods: This study was prospective, randomized, double-blind and placebo-controlled study. Hundred women were selected, aged between 45 and 65 years with climacteric symptoms, with menstruation absence (amenorrhea) that was more then one year and without any treatment for last six months. After selection, women were divided into two groups: GI (n = 50) received 40 mg Trifolium pratense (one capsule per day); GII (n = 50) received placebo (control, one capsule of lactose per day). The length of treatment was 12 months and women were evaluated before and after four, eight and twelve months of treatment through clinical and laboratorial exams. The t Student test and ANOVA were applied for analyzing the differences between groups. Results: There was significant ameliorate in the symptoms after four months of treatment through the KMI, mainly in hot flashes, compared to baseline data of both groups. The sexuality evaluation did not found any difference before and after treatment in both groups. Conclusion: Our data suggested that the 40 mg Trifolium pratense effect may be not superior than placebo in relation to decrease the postmenopausal symptoms during one year of study

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.