Diploma thesis on the theme Concentration of selected phytoestrogens in the diet of dairy cattle and their distribution in to the blood and milk deals about transmittance of phytoestrogens from real fed TMR in the South Moravian conventional breedings of dairy cows into the blood plasma of animals and to milk as a product intended for hu-man consumption. A review of literature is devoted to the occurrence of phytoestrogens in forage and feed, their metabolism and effects on animals and humans. The experimental part of the thesis is devoted to the identification and quantification of selected phytoestrogens in feed and their penetration into blood plasma and milk. Determination of phytoestrogens was performed by HPLC - MS analysis.

Regular soy consumption has been shown to reduce cardiovascular (CV) risk through plasma cholesterol reduction. According to the current health claim, this benefit is attributed to soy protein (SP). Dietary intervention trials indicate that isoflavones (ISO), weak phytoestrogens in soy, may also contribute by offering additional vascular and metabolic protection. Equol, a metabolite of the ISO daidzein (DAZ) with greater estrogenic potency, may be an important mediator of such effects. This thesis examines effects of soy, in particular, ISO consumption on CV risk factors and the potential for ISOs to enhance cognition, possibly through improvements of circulatory function. Two crossover design intervention trials were undertaken: a food-based intervention, investigating differential effects of SP and ISO on plasma lipids and other risk factors for CVD, and an ISO supplementation trial, examining effects on cognition and vascular function. Both addressed whether benefits were dependent on equol production. In the first trial, 91 subjects with untreated mild hypercholesterolemia were randomised to consume each of the following three diets in random order for sequential 6 week periods: (S) soy foods containing 24 g of SP and 75-90 mg ISO per day, (SD) soy/dairy foods containing 12 g SP, 12 g dairy protein (DP) and 75-90 mg ISO per day or (D) dairy foods containing 24 g DP only per day. At the end of each diet period, blood lipids, flow-mediated dilatation (FMD) of the brachial artery, blood pressure, arterial compliance and anthropometric measures were assessed. Compared with the control diet (D), there was a small but significant reduction in total cholesterol on the S diet only (2.8 + 1.1%, P<0.05), which could be accounted for by a decrease in saturated fat intake. FMD was found to be significantly improved when SD and S diet data were nested (P=0.03). Plasma triglycerides (TG) improved on both the SD and S diets compared with D (P<0.01). Other lipid, metabolic and vascular parameters did not differ between diets. There were no differences in outcomes between equol (n=30) and non equol producers (n=61). In a subsequent 12 week double-blind supplementation trial, 34 healthy males were randomised to take 4 capsules providing 120mg ISO per day or a matching placebo for 6 weeks, after which they crossed over to the alternate supplement. FMD and cognitive assessments relating to measures of memory and executive function were performed at the beginning and end of each treatment phase. Spatial working memory, a test in which females consistently perform better than males, was significantly improved by ISO supplementation (P<0.02). However, other measures of cognition and FMD were unaffected and there were no differences between equol (n=8) and non-equol producers (n=26). These interventions indicate that ISOs offer specific health benefits, independent of equol production. ISO supplementation can enhance specific cognitive processes which appear dependent on estrogen activation. Additionally, soy foods containing ISOs improved FMD and TG but were unable to improve LDL cholesterol, even in equol producers. Thus dietary ISOs may reduce CV risk but the validity of the current health claim for SP is questioned. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1345614 / Thesis (Ph.D.) - University of Adelaide, School of Molecular and Biomedical Science, 2008

Orientador: Gabriela Alves Macedo / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-25T09:23:41Z (GMT). No. of bitstreams: 1 Queiros_LiviaDias_M.pdf: 1473794 bytes, checksum: 8841b5d38503f939739da3243001d1ed (MD5) Previous issue date: 2014 / Resumo: A soja atrai considerável atenção no atual cenário econômico mundial devido ao seu elevado potencial nutritivo e efeitos potencialmente benéficos à saúde humana, que são atribuídos principalmente às isoflavonas. Esta classe de fenóis heterocíclicos é conhecida por suas atividades biológicas, tais como as atividades estrogênica, antioxidante e antitumoral, sendo as formas agliconas mais ativas do que as glicosiladas. Contudo, estudos têm mostrado que a eficácia clínica das isoflavonas está relacionada com a capacidade de produção de equol, um metabólito da daidzeína que, segundo a literatura vigente, é produzido exclusivamente pela ação da microbiota intestinal. Assim, há evidências de que nem todos os indivíduos são capazes de metabolizar as isoflavonas a equol, sendo essa variabilidade atribuída às diferenças na composição da microflora intestinal, hábitos alimentares, fatores genéticos, dentre outros. Neste contexto, os produtos à base de soja são uma forma de incluir as isoflavonas na dieta, sendo que o extrato hidrossolúvel de soja (EHS) é um substrato que tem se apresentado com potencial para produção de novos alimentos com apelo saudável. Desse modo, com o propósito de aumentar o conteúdo de isoflavonas bioativas e avaliar a viabilidade de um processo biotecnológico para produção de equol in vitro, neste projeto foi investigada a aplicação de culturas starters e bactérias lácticas probióticas na fermentação do EHS, aliado à ação do extrato bruto de tanase obtido a partir de Paecilomyces variotti. Além disso, também foi avaliada a biotransformação dos compostos fenólicos e atividade antioxidante do produto obtido. O teor de fenóis totais foi avaliado pelo método de Folin-Ciocalteau, a atividade antioxidante pelos métodos in vitro ORAC e de sequestro de radicais DPPH e o perfil químico dos extratos, antes e depois dos processos de biotransformação, foi avaliado por CLAE-DAD com co-injeção de padrões de isoflavonas. Após o processo fermentativo e/ou tratamento enzimático do EHS, houve um significativo aumento no teor de fenólicos totais e capacidade antioxidante, evidenciada por ambos métodos empregados (ORAC e DPPH), quando comparados com o controle do EHS sem reação. Além disso, foi verificada uma modificação no perfil polifenólico das amostras do EHS biotransformadas evidenciado por CLAE-DAD, resultando em um aumento na concentração das formas agliconas em relação às glicosiladas, bem como o aumento da concentração de equol após os processos de biotransformação propostos. Os resultados obtidos por CLAE-DAD confirmaram que o extrato de tanase de P. variotty foi capaz de biotransformar as formas glicosiladas (daidzina e genistina) das isoflavonas em suas respectivas formas agliconas (daidzeína e genisteína), indicando uma atividade diglicosídica do extrato semipurificado de tanase. Pelo que se tem conhecimento, a hidrólise de isoflavonoides glicosilados por tanase, bem como a formação de equol, é um relato inédito na literatura demonstrando que é possível desenvolver um processo in vitro para a obtenção deste composto bioativo, sem a presença de bactérias intestinais, utilizando apenas uma biotransformação enzimática / Abstract: The soy attracts considerable attention in the current global economic scenario due to their high nutritional potential and potentially beneficial effects to human health, which are mainly attributed to isoflavones. This class of heterocyclic phenols is known for its biological activities, such as estrogenic, antioxidant and antitumor activities, and the aglycone forms are more active than the glycosylated. However, studies have shown that the clinical efficacy of isoflavones is related to the capacity to produce equol, a metabolite of daidzein, which according to the current literature it is produced exclusively by the action of the gut microbiota. Thus, there is evidence that not all individuals are able to metabolize isoflavones to equol and this variability is attributed to differences in the composition of gut microflora, diet, genetic factors, among others. In this context, the soy-based products are a way to include the isoflavones in the diet, and the soymilk is a substrate that has shown the potential to produce new foodstuff with healthy appeal. Thus, in order to increase the content of bioactive isoflavones and assess the viability of a biotechnological process for in vitro production of equol, in this project was investigated the application of starters and probiotic lactic bacteria strains in the fermentation of soymilk, combined with the action of the crude extract of tannase obtained from Paecilomyces variotii. Furthermore, it was also evaluated the biotransformation of phenolic compounds and antioxidant activity of the obtained product. The total phenolic content was assessed by Folin-Ciocalteau method, the antioxidant activity by in vitro methods of ORAC and DPPH radical sequestration and the chemical profile of the extracts, before and after the processes of biotransformation, was evaluated by HPLC-DAD with co- injection of standards of isoflavones. After the fermentation process and/or enzymatic treatment of the soymilk, there was a significant increase of the total phenolic content and antioxidant capacity, as evidenced by both methods employed (ORAC and DPPH), compared with the standard soymilk. Furthermore, there was observed a change in the polyphenolic profile of biotransformed samples of soymilk, evidenced by HPLC-DAD, resulting in an increase in the concentration of the aglycones in relation to glucosides forms, as well as there was a significant increase of the concentration of equol after the biotransformation processes proposed. The results obtained by HPLC-DAD confirmed that the extract of tannase from P. variotii was able to biotransform the glucosides forms (daidzin and genistin) of isoflavones in their aglycone forms (daidzein and genistein), indicating a diglycosidase activity of the crude extract of tannase. From what is known, the hydrolysis of glucosides isoflavone by tannase, as well as the formation of equol, is an unpublished report in the literature demonstrating that it is possible to develop an in vitro method for obtaining this bioactive compound, without the presence of gut bacteria process using only an enzymatic biotransformation / Mestrado / Ciência de Alimentos / Mestra em Ciência de Alimentos

Equol is known to be a selective androgen modulator and has the ability to bind and inhibit 5-alpha dihydrotestosterone (5α-DHT). Equol is also a selective estrogen receptor modulator and is able to bind beta estrogen receptors with high affinity. As estrogen receptors are found in the hypothalamus, pituitary, and gonads, prenatally administered equol could affect the morphological and reproductive development of offspring. To test this hypothesis, during gestational days 14 to 20, forty-two pregnant Long-Evans rats were given one of six treatments: 1) no treatment, 2) injection with dimethyl sulfoxide (DMSO), 3) injection with 10 mg/kg equol, 4) injection with 21.0 mg/kg equol, 5) injection with 63.0 mg/kg equol, or 6) injection with 90.0 mg/kg flutamide. At birth the pups were weighed, anogenital distance measured, and sex was determined. Some of the animals were sacrificed and trunk blood collected from both the mothers and pups. Serum levels of phytoestrogens, estradiol, testosterone, and 5α-DHT levels were determined. Some pups were allowed to grow up to day 29 and were tested on the forced-swim test with the parameters of time mobile, time immobile, swim distance, and average speed measured. The flutamide treated pups had the lowest anogenital distance. The low equol dose animals had the largest anogenital distance. There were no significant differences in 5α-DHT serum levels in the male offspring among the treatments. However in non-injected control female offspring displayed significantly lower 5α-DHT levels than all the other groups. Mothers treated prenatally with equol displayed significantly higher circulating equol levels compared to controls values. Rats injected with 63.0 mg/kg of equol gained the least weight during pregnancy. Their offspring also had the lowest body weights at birth. Male and female offspring displayed similar behaviors in the Porsolt forced-swim test among the treatment groups. The low and high equol groups displayed the least depressive-like behaviors. The offspring from mothers treated with the medium and high equol doses both gained the most weight from birth to day 29. Treating pregnant rats with equol during the last week of gestation does not appear to have any affect on morphological genital development of the offspring.

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Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmaceutical Botany Candidate: Hana Němcová Supervisor: PharmDr. Jana Karlíčková, Ph.D. Title of Thesis: Comparison of interactions of equol and desmethylangolensin with iron and copper Iron and copper are essential trace elements, which are important for our body. Both elements have a significant effect on the correct function of organs and make part of many enzymes. They are able to accept or donate electrons - conversion between oxidized (Fe3+, Cu2+) and reduced (Fe2+, Cu+) forms. If these metals are excess in organism, they are accumulated in the cells and mediate the creation of free radicals, that destroy cell structures. This deficiency is treated with chelators, which facilitate the excretion of metals from the body. Isoflavonoids are polyfenolic substances, which can have antioxidant effects and they are involved in the scavening of free radicals. Isoflavonoids can have also a pro-oxidative effect, because they are able to reduce metal ions. In this study were tested interactions (chelation and reduction) between the metabolites of isoflavonoids (equol and desmethylangolensin) and ions of iron and copper. Both metabolites only weakly chelate metal ions, but they significantly reduce cupric ions. KEYWORDS: Iron,...

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Phytoestrogens are very intensively studied in recent years because they show different pharmacological effects on human health. For example, it was demonstrated that phytoestrogens reduce the risk of certain cancers (breast, prostate or colon), act as antioxidants in the body and affect the activity of certain enzymes. In addition, also it was demonstrated number of positive effects of soybean phytoestrogens on the health status of women in the menopause, such as reducing the risk of atherosclerotic cardiovascular disease or osteoporosis. Various technologically processed soy beans are important part of the feed rations mainly for high-lactating dairy cows, because they are a valuable source of the energy and high-quality protein, despite their negative effect on reproductive performance. Phytoestrogens consumed from the feed rations are hydrolyzed in the rumen of dairy cow and together with their metabolites are absorbed in the small intestine into the blood. They can be excreted from the body in urine or milk. From this perspective, cow's milk may become a source of phytoestrogens substances, especially equol in the human nutrition. On the other hand, some research results suggest possible potential risks associated with phytoestrogens intake in the diet on human health. At the some age (infants and young children) and group (vegetarians) categories may be dietary intake of phytoestrogens undiserable. According to most experts, a beneficial effect on human health prevail the potential risks. However, the risks associated with phytoestrogens intake in food can not be ignored and therefore phytoestrogens are still actual problem in the field of scientific research.

No / Reactive oxygen species (ROS) are produced by a wide variety of chemicals and physiological processes in which enzymes catalyse the transfer of electrons from a substrate to molecular oxygen. The immediate products of such reactions, superoxide anion radicals and hydrogen peroxide can be metabolised by enzymes such as superoxide dismutase (SOD) and catalase (CAT), respectively, and depending on its concentration by Vitamin C (Vit C). Under certain circumstances the ROS form highly reactive hydroxyl radicals. We examined human sperm and lymphocytes after treatment with six oestrogenic compounds in the Comet assay, which measures DNA damage, and observed that all caused damage in both cell types. The damage was diminished in nearly all cases by catalase, and in some instances by SOD and Vit C. This response pattern was also seen with hydrogen peroxide. This similarity suggests that the oestrogen-mediated effects could be acting via the production of hydrogen peroxide since catalase always markedly reduced the response. The variable responses with SOD indicate a lesser involvement of superoxide anion radicals due to SOD-mediated conversion of superoxide to hydrogen peroxide generally causing a lower level of DNA damage than other ROS. The variable Vit C responses are explained by a reduction of hydrogen peroxide at low Vit C concentrations and a pro-oxidant activity at higher concentrations. Together these data provide evidence that inappropriate exposure to oestrogenic compounds could lead to free-radical mediated damage. It is believed that the observed activities were not generated by cell free cell culture conditions because increased responses were observed over and above control values when the compounds were added, and also increasing dose¿response relationships have been found after treatment with such oestrogenic compounds in previously reported studies.