This diploma thesis is about soybean as functional food. Functional food contain components with positive effect on human health. In soybean there are lot of these components and very valued are phytoestrogens. Phytoestrogens has estrogenic activity with positive effect in prevention of osteoporosis, breast and prostate cancer, cardiovascular disease and menopausal problems. In experimental part was measured level of isoflavones in different kinds of soy food. The method for separation was high performance liquid chromatography with mass spectrometry.

In plant and food research the functional significance of herbs, spices and other plants, including their components is very popular topic. Plant material contains many components that are beneficial to human health by reducing the risk of chronic degenerative diseases. It is necessary to define the individual substances to understand and explain their biological effects. The first part of thesis describes the selected plants (elderberry, nettle, marigold, milk thistle, sea buckthorn, sage, purple coneflower and thyme) and also describes their medicinal properties and uses. The second part of thesis deals with phenolic and flavonoid substances. In the third (experimental) part, of thesis there were qualitatively and quantitatively determined flavonoid compounds in selected plant species. For identification the technique combining liquid chromatography and mass spectrometry - HPLC/MS was used. Extraction was carried out by vortexing and Soxhlet method, using two different concentrations of metanol. These metods were then statistically compared.

Wine contains organic acids, which determine its final quality. Tartaric and malic acids are found in dominant concentrations. Also present are citric, lactic and succinic acids, and some volatile acids such as formic and acetic acid. The process of winemaking is associated with changes in the concentration of wine acids. The composition of acids in wine is also affected by the type, quality and ripeness of grapes, grape manufacture and wine production. The aim of this study was to analyse the concentrations of tartaric, malic, citric, lactic, succinic, formic and acetic acid during the winemaking process in different press fractions of 8 varieties of white wines from the Vineyard centre Mělník - Chloumek and in 5 commercially purchased red wines, using HPLC with DAD detection, and to analyse the trend of changes in different phases of the winemaking process. The white wine press fractions were supplied following the wine press (A), settling of juice (B), in the phase of partially fermented juice (C) and during the maturation of young wine (D-G.). The samples were taken within 2-3 days. There were no red wine press fractions available from the vineyard. 5 bottles of different varieties of red wine were thus purchased from the shop and analysed. The GraphPad Prism programme was used to perform statistical analyses (ANOVA). The hypothesis suggesting that tartaric acid would be the dominant acid was confirmed in 5 varieties of white wine and in all red wines. The hypothesis suggesting a difference between red and white wines was also confirmed. There were significant differences in the concentration and composition of acids between young white wines and red wines. The hypothesis that the concentration of all acids would increase was not confirmed. Tartaric acid showed the most linear trends of decrease, followed by malic acid. The trend of change in the concentrations of succinic, lactic, citric and formic acid increased in some varieties and it decreased in others. The grape variety, weather during summer and autumn and the manufacturing processes all influence the acid composition in wine. These factors can explain the differences seen between different varieties of wine.

The diploma thesis deals with the determination of vitamin E in grains of wheat kernels with unusual coloring. In the theoretical part is mentioned botanical characterization of wheat, barley and tritordeum, vitamin E and its chemical and biological aspects and basics of chromatography with a focus on the high performance liquid chromatography (HPLC). The analysis to quanify vitamin E was performed in the experimental part of the thesis. The analysis was conducted with samples of winter and spring wheat, spring barley and spring tritordeum from harvests of 2014 and 2015. These were varieties with unusual coloring of the grain. The evaluation was conducted by variety (winter wheat, spring wheat, spring barely, spring tritordeum) and by the color of the kernels (blue aleuron, purple pericarp, yellow pericarp). Subsection part of the thesis is dedicated to a quantification of vitamin E during long-term storage. Based on the determination of total vitamin E amount in all analyzed varieties of cereal grains with a different color is not statistically significant difference in the values of the total amount of vitamin E. The difference between the content of vitamin E in the samples of the same varieties of different cereals harvest from a year 2014 and a year 2015 were not statistically significant. All varieties of wheat were quantified four vitamers: alpha-tocopherol, beta-tocopherol, alpha-tocotrienol and beta-tocotrienol. The amount of other vitamers was below the limit of detection. The total content of vitamin E and contents of individual vitamers coincide with the data given in the literature for wheat varieties with standard color of grain. Spring barley contained a slightly higher average levels of total vitamin E amount in comparison with varieties of spring and winter wheat. The total average amount of vitamin E in varieties of spring tritordeum was also slightly higher than in varieties of winter and spring wheat. From the long term storage point of view, it is clear that the total amount of vitamin E in both varieties is relatively stable and shows no clear trend in function of storage time.

Casein proteins are important escpecially for cheese making. In addition to technology and other factors, casein concentration in milk and relative representation of individual casein fractions influence cheese yield. Majority fractions are alfa-casein, beta-casein and kappa-casein. For the determination of proteins in the milk can be used electrophoretic techniques or near infra-red spektrometry. High performance liquid chromatography (HPLC) is going to be used. HPLC is widely used analytical method, which is based on differential affinity of the substances to the mobile and the stationary phase. The thesis has introduced the method of determining the casein fractions by HPLC. The method was partially optimized using cow milk and calibrated to the standard solutions, which were purchased casein fractions derived from the bovine milk. Real samples of cow, sheep and goat milk were analyzed. The goat milk was available in several variants: Sanski goat milk and white shorthair goat from domestic or farm breeding. All milk samples were analyzed for casein and casein fractions proportions. Chromatograms show that the casein formula is different in cow, sheep and goat milk. Different concentrations of the casein fraction in the milk of various animal species have been statistically detected in almost all cases. The exception was the concentration of beta-casein in the milk of white shorthair goat from the domestic and the farm breeding and cow milk. In these types of milk there was no statistically significant difference in the concentration of beta-casein. Analysis of individual samples of white shorthair goat milk from the farm breeding shown that casein content in the milk of different animals reared under the same conditions may be different. Ratios of beta-casein to alfa-casein, beta-casein to kappa-casein and alfa-casein to kappa-casein were evaluated in all samples. Interspecific comparisons proved that ratios were statistically significantly different in all milks. In the milk of Sanski goat, there was found the stable ratio of beta-casein to alfa-casein during lactation. For all goat milk there were typical higher values of the ratio of beta-casein to kappa-casein. The ratio of alfa-casein to kappa-casein was highest in the sheep milk. Despite some shortcomings, it is possible to use the HPLC method possible for these purposes, but it is necessary to continue with its optimization.

Objetivos: Simular um ajuste de dose em pacientes com sobrepeso e com obesidade que utilizam a doxorrubicina (DOX), que proporcione curvas de concentração plasmática e concentrações máximas (Cmáx) semelhantes às de pacientes com peso normal. Metodologia: Determinou-se as concentrações plasmáticas de DOX em pacientes com câncer de mama após a administração i.v. de 60 mg/m2, com ajuste de dose pelo BSA e com tempo de infusão de 40 minutos (0,66 h). As pacientes foram divididas em três grupos: a) pacientes com peso normal (IMC até 24,9) (n = 3); b) pacientes com sobrepeso (IMC 25,0 – 29,9) (n = 5); c) pacientes com obesidade (IMC acima 30,0) (n = 2). Os tempos de coleta utilizados foram: 0,66, 1,66, 8,66, e 24,66 h. Os perfis farmacocinéticos da DOX foram avaliados após quantificação da DOX através de metodologia por CLAE desenvolvida e validada. Resultados e Discussão: Os parâmetros farmacocinéticos ASC e Cmáx foram analisados por abordagem não-compartimental e compartimental. Em ambas as análises encontraram-se diferenças significativas (α = 0,05) entre os grupos com sobrepeso e com obesidade comparados com o grupo com peso normal para ASC e diferenças significativas entre o grupo com sobrepeso comparado ao grupo de pacientes com peso normal para o Cmáx. Após simulação de ajuste de dose pelo peso e pelo IMC, para as pacientes com sobrepeso e obesas, obtivemos novos valores de ASC e Cmáx. Houve diferenças significativas (α = 0,05) para o grupo de pacientes com sobrepeso comparado ao grupo com peso normal para a ASC e nenhuma diferença significativa entre os grupos estudados para Cmáx. Os percentuais de diferença da ASC e Cmáx do grupo das pacientes com sobrepeso e obesas comparados com o grupo de pacientes com peso normal foram diminuindo quando a dose era ajustada pelo peso e IMC. Conclusões: O ajuste de dose pelo índice BSA, comumente usado na prática clínica, não produz concentrações plasmáticas iguais em grupos de pacientes com peso normal e com sobrepeso, indicando que outros índices devem ser considerados, como peso e IMC. / Objectives: to simulate a dose adjustment in patients with overweight and obesity using doxorrubicin (DOX), providing plasma concentrations similar to those of patients with normal weight. Methodology: plasma concentrations of DOX were determined in patients with breast cancer after the iv. administration of 60 mg/m2 of DOX, adjusted by the BSA and with an infusion time of 40 minutes (0.66 h). The patients were divided into three groups: (a) patients with normal weight (BMI < 24.9) (n = 3); b) patients with overweight (BMI 25.0 – 29,9) (n = 5); c) patients obese (BMI > 30.0) (n = 2). Samples were collected at 0.66, 1.66, 8.66, and 24,66 h. DOX's pharmacokinetic profiles were evaluated after quantification of DOX using a new HPLC method developed and validated. Results and discussion: pharmacokinetic parameters (AUC and Cmax) were analyzed by non-compartmental and compartmental approaches. Significant differences (α = 0.05) between overweight and obese groups when compared with the normal weight group were found with respect to AUC and significant difference between the group with overweight compared to the patients with normal weight was found for Cmax. After simulating the dose adjustment by weight and by BMI for overweight and obese patients, new values for Cmax ASC were calculated. The new values obtained using both weight and BMI were closer to the normal group than those obtained with the BSA. The percentage of difference of ASC and Cmax of the overweight and obese group compared with the Group of patients with normal weight were lower when the dose was adjusted by weight and IMC. Conclusions: dose adjustment index BSA, commonly used in clinical practice, produced different plasma concentrations in groups of patients with normal weight, overweight, and obese, indicating that other indexes must be considered, such as weight and BMI.

v anglickom jazyku Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biophysics and Physical Chemistry Student: František Lach Supervisor: Mgr. Pavel Bárta, PhD Consultant: Mgr. Lucie Hyršová Title of diploma thesis: In vitro saturation study of 99m Tc-HYNIC-ramucirumab on PC-3 cell line The number of malignant tumours in the population has increased in recent years. Due to the frequent serious sides effects of chemotherapeutic drugs on the whole organism, targeted antitumor therapy is at the forefront. Due to its specific effect on the regulatory and signal pathways of protein structures, monoclonal antibodies are used for the target anti-tumour therapy. The basic properties of the growing tumour include vasculogenesis (the ability to build new blood vessels from the endothelial precursors) and angiogenesis (the process of self-inducing formation of blood vessels). Endothelial tumour progenitors include vascular endothelial growth factor (VEGF). VEGF activates its biological activity by binding to its transmembrane tyrosine-kinase receptors VEGFR. Indeed, the inhibition of the vascular endothelial factor receptors is the target of some monoclonal antibodies. Ramucirumab is a monoclonal antibody that selectively inhibits VEGF receptor type 2 (VEGFR-2) and thereby...

Objetivo: Avaliar a farmacocinética da vildagliptina em animais sadios e diabéticos, através da análise dos níveis plasmáticos totais e livres teciduais, empregando-se a técnica de microdiálise. Metodologia: A doença foi induzida nos animais através da administração de 42mg/kg de aloxano através da via intravenosa (i.v.). A vildagliptina foi administrada nas doses de 50 mg/kg (n = 6) e 75 mg/kg (n = 6) via i.v. nos animais diabéticos e na dose de 50 mg/kg (n = 6) nos animais sadios. As concentrações plasmáticas foram quantificadas por CLAE-EM-EM em método desenvolvido e validado. A ligação às proteínas plasmáticas foi determinada por microdiálise, assim como a avaliação tecidual. As sondas de microdiálise foram calibradas in vitro através de diálise e retrodiálise e in vivo utilizando retrodiálise. Para determinação das concentrações teciduais, uma segunda metodologia foi desenvolvida e validada em CLAE-EM-EM. Avaliações compartimentais (software Scientist ®) e não compartimentais (software Excel ®) foram realizadas. Resultados e Discussão: A ligação as proteínas plasmáticas apresentou um valor médio de 9,44 % ± 3,23, condizente com valores encontrados na literatura. Os valores de Ke, clearance, tempo de meia vida, MRT e VDss não apresentaram diferença estatística significativa entre as diferentes doses administradas nos animais diabéticos e entre os animais sadios. As calibrações in vitro por diálise e retrodiálise apresentaram uma recuperação média de 30%, sem diferença estatística entre as duas metodologias empregadas (α = 0,05). A recuperação in vivo também apresentou o mesmo valor médio de recuperação. A penetração tecidual do fármaco em animais diabéticos para as diferentes doses estudadas apresentou mesmo valor nos tecidos estudados, uma média de 0,20. A penetração tecidual semelhante no animal diabético pode ser devido ao dano similar entre os órgãos sofrido durante a indução da doença. Já os animais sadios apresentaram penetração tecidual similar no músculo sem diferença estatística significativa em relação aos diabéticos, entretanto no fígado foi observada uma penetração quarenta e quatro vezes inferior a observada no músculo. Essa disparidade pode ser atribuída a diferença de expressão de proteínas transportadoras no fígado do animal diabetico quando comparado ao sadio. O perfil farmacocinético plasmático foi semelhante entre os dois grupos avaliados, sendo que os parâmetros não diferiram estatisticamente (α = 0,05). Foi empregado o modelo de dois compartimentos para prever as concentrações teciduais. A previsão supõe concentrações superiores as encontradas experimentalmente, contradizendo dados de literatura. Esses dados são inéditos na literatura e demostram a importância da determinação do fármaco em tecidos alvo, uma vez que nem sempre modelos matemáticos conseguem prever a realidade fisiológica. Conclusões: As metodologias analíticas para quantificação da vildagliptina em microdialisado e plasma foram desenvolvidas e validadas, seguindo os requisitos do FDA. O perfil farmacocinético plasmático foi adequadamente descrito pelo modelo de 2 compartimentos. Os perfis teciduais obtidos nesse trabalho podem contribuir para o melhor entendimento dos mecanismos farmacológicos envolvidos e contribuir para futura otimização de terapias. / Objective: To evaluate the pharmacokinetics of vildagliptin in healthy and diabetic animals using a microdialysis technique. Methodology: Diabetes was induced in animals by administration of 42 mg/kg of alloxan intravenously (iv). Vildagliptin was administered intravenously as 50 mg/kg (n = 6) and 75 mg/kg doses (n = 6) in the diabetic animals and as a 50 mg/kg dose (n = 6) in healthy animals. Plasma concentrations were quantified by a HPLC-MS-MS method developed and validated. The plasma protein binding was determined by microdialysis and tissue evaluation. Microdialysis probes were calibrated in vitro using dialysis and retrodialysis and in vivo using retrodialysis. A second method was developed and validated using HPLC-MS-MS to determine tissue concentrations. Results and Discussion: Mean plasma protein was 9.44% ± 3.23, consistent with values reported in the literature. The values of Ke, clearance, half-life, MRT and Vdss showed no statistical difference between the evaluated doses in diabetic animals and between healthy animals (α = 0.05). Calibrations in vitro by dialysis and retrodialysis showed mean recovery of 30%, with no statistical difference between the two methodologies. Mean recovery in vivo also showed the same value. The tissue penetration of the drug in diabetic animals for the different doses studied showed the same value in both tissues studied, an mean of 0.20. The tissue penetration similar in diabetic animals could be due to the similar damage suffered between organs during induction of the disease. The healthy animals showed similar muscle penetration, compared with diabetics animals, although the liver showed a penetration forty four times lower than muscle. This discrepancy can be attributed to differential expression of transporter proteins in the liver of diabetic animals, when compared to the healthy group. The plasma pharmacokinetic profile was similar between the investigated groups, and the parameters did not differ. The two-compartment model was employed to describe the data and used to predict the concentration in the tissues. This is the first study to present these tissue profiles, which presented concentrations below the estimated by the model. These data demonstrate the importance of determining the drug inside the target tissue, as the mathematical models sometimes cannot predict physiology. Conclusions: The analytical methods for the quantification of vildagliptin in microdialysate and plasma were developed and validated by following the requirements of the FDA. The plasma pharmacokinetic profile was correctly described by the model of two compartmental models. The novel tissue profiles obtained in this study may contribute to a better understanding of the pharmacological mechanisms involved and contribute to optimization of future therapies.

O tomate (Solanum lycopersicum L.) é considerado um alimento funcional por sua propriedade antioxidante. O fruto contém dos carotenoides, ácido L-ascórbico, vitamina E e substâncias fenólicas. Estudos indicam que o consumo do tomate pode retardar o envelhecimento e favorecer o funcionamento do sistema imunológico em humanos, embora não seja possível afirmar uma relação direta entre a prevenção ou cura de qualquer carcinoma com o consumo de produtos à base licopeno. Na década de 1990, a Empresa Brasileira de Pesquisa Agropecuária (EMBRAPA) criou o projeto TOMATEC, o tomate ecologicamente cultivado. A produção é baseada em um manejo diferenciado, que objetiva a otimização do uso do solo, menor consumo de pesticidas, herbicidas e incentivo à produção familiar dos agricultores. O estudo objetiva observar a influencia dos diferentes manejos: tradicional e TOMATEC na produção de (15E)-licopeno nos frutos do tomate, em diferentes cultivares do Brasil. Assim, foi utilizada a técnica de cromatografia líquida de alta eficiência acoplada a detector por arranjo de diodos (CLAE-DAD), em fase estacionária polimérica composta de 30 carbonos (C30). Esta fase estacionária é capaz de separar carotenoides, incluvise, isômeros geométricos do licopeno. As amostras foram coletadas, tratadas, extraídas e analisadas sob as mesmas condições para avaliação das concentrações de (15E)-licopeno nos frutos. Ensaios realizados foram capazes de demonstrar, de maneira objetiva, que a metodologia proposta conduziu a resultados confiáveis à qualidade desejada, de acordo com o guia de validação proposto pelo Instituto Nacional de Metrologia, Normalização e Qualidade Industrial (INMETRO). Foram utilizadas ferramentas estatísticas para comparar os teores de (15E)-licopeno, em diferentes Estados do Brasil, sob diferentes manejos. Os resultados indicam os maiores teores da substância em cultivares convencionais e mostram que o ensacamento do fruto é um fator decisivo para a biossíntese do (15E)-licopeno

Objetivos: Simular um ajuste de dose em pacientes com sobrepeso e com obesidade que utilizam a doxorrubicina (DOX), que proporcione curvas de concentração plasmática e concentrações máximas (Cmáx) semelhantes às de pacientes com peso normal. Metodologia: Determinou-se as concentrações plasmáticas de DOX em pacientes com câncer de mama após a administração i.v. de 60 mg/m2, com ajuste de dose pelo BSA e com tempo de infusão de 40 minutos (0,66 h). As pacientes foram divididas em três grupos: a) pacientes com peso normal (IMC até 24,9) (n = 3); b) pacientes com sobrepeso (IMC 25,0 – 29,9) (n = 5); c) pacientes com obesidade (IMC acima 30,0) (n = 2). Os tempos de coleta utilizados foram: 0,66, 1,66, 8,66, e 24,66 h. Os perfis farmacocinéticos da DOX foram avaliados após quantificação da DOX através de metodologia por CLAE desenvolvida e validada. Resultados e Discussão: Os parâmetros farmacocinéticos ASC e Cmáx foram analisados por abordagem não-compartimental e compartimental. Em ambas as análises encontraram-se diferenças significativas (α = 0,05) entre os grupos com sobrepeso e com obesidade comparados com o grupo com peso normal para ASC e diferenças significativas entre o grupo com sobrepeso comparado ao grupo de pacientes com peso normal para o Cmáx. Após simulação de ajuste de dose pelo peso e pelo IMC, para as pacientes com sobrepeso e obesas, obtivemos novos valores de ASC e Cmáx. Houve diferenças significativas (α = 0,05) para o grupo de pacientes com sobrepeso comparado ao grupo com peso normal para a ASC e nenhuma diferença significativa entre os grupos estudados para Cmáx. Os percentuais de diferença da ASC e Cmáx do grupo das pacientes com sobrepeso e obesas comparados com o grupo de pacientes com peso normal foram diminuindo quando a dose era ajustada pelo peso e IMC. Conclusões: O ajuste de dose pelo índice BSA, comumente usado na prática clínica, não produz concentrações plasmáticas iguais em grupos de pacientes com peso normal e com sobrepeso, indicando que outros índices devem ser considerados, como peso e IMC. / Objectives: to simulate a dose adjustment in patients with overweight and obesity using doxorrubicin (DOX), providing plasma concentrations similar to those of patients with normal weight. Methodology: plasma concentrations of DOX were determined in patients with breast cancer after the iv. administration of 60 mg/m2 of DOX, adjusted by the BSA and with an infusion time of 40 minutes (0.66 h). The patients were divided into three groups: (a) patients with normal weight (BMI < 24.9) (n = 3); b) patients with overweight (BMI 25.0 – 29,9) (n = 5); c) patients obese (BMI > 30.0) (n = 2). Samples were collected at 0.66, 1.66, 8.66, and 24,66 h. DOX's pharmacokinetic profiles were evaluated after quantification of DOX using a new HPLC method developed and validated. Results and discussion: pharmacokinetic parameters (AUC and Cmax) were analyzed by non-compartmental and compartmental approaches. Significant differences (α = 0.05) between overweight and obese groups when compared with the normal weight group were found with respect to AUC and significant difference between the group with overweight compared to the patients with normal weight was found for Cmax. After simulating the dose adjustment by weight and by BMI for overweight and obese patients, new values for Cmax ASC were calculated. The new values obtained using both weight and BMI were closer to the normal group than those obtained with the BSA. The percentage of difference of ASC and Cmax of the overweight and obese group compared with the Group of patients with normal weight were lower when the dose was adjusted by weight and IMC. Conclusions: dose adjustment index BSA, commonly used in clinical practice, produced different plasma concentrations in groups of patients with normal weight, overweight, and obese, indicating that other indexes must be considered, such as weight and BMI.