Orientador: Alexandra Christine Helena Frankland Sawaya / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-24T09:51:06Z (GMT). No. of bitstreams: 1 Lopez_BegonaGimenez-Cassina_M.pdf: 5145110 bytes, checksum: e91f0a5d069796983ac9064ccc7af78c (MD5) Previous issue date: 2014 / Resumo: A palavra própolis tem origem Grega: pro significa em defensa de, e polis significa comunidade. A própolis é usada pelas abelhas para fortalecer as paredes da colmeia e para cobrir as paredes internas e por devido a sua atividade antimicrobiana. A composição química da própolis é variável segundo a biodiversidade e a origem geográfica. A própolis vermelha, encontrada no nordeste e norte do Brasil, apresenta promissoras atividades biológicas: atividade antimicrobiana, antiparasitária, antioxidante, citotóxica, antiinflamatória, analgésica, efeitos antiobesidade, contra psoríase e hepatoprotetores. A composição química da própolis vermelha, relatada na literatura, parece variar qualitativa e quantitativamente, inclusive entre amostras coletadas na mesma região. Isto pode ser devido a variações sazonais, a flora direitamente ao redor das colméias ou aos métodos de análise. A maioria das classes de substâncias já identificadas pode ser analisada adequadamente por espectrometria de massas com ionização por eletrospray e cromatografia líquida. Os constituintes de média e alta polaridade (como fenólicos, flavonóides e benzofenonas) são responsáveis por muitas das atividades biológicas reportadas à própolis vermelha. A cromatografia líquida de ultra-eficiência acoplada à espectrometria de massas (UHPLC-MS) é um método moderno, rápido e sensível. Permite avaliar os perfis químicos das amostras e determinar sua composição qualitativamente, comparando os espectros de massas (MS/MS) dos componentes das amostras com informações na literatura. Portanto UHPLC-MS é a ferramenta mais adequada para a avaliação da composição de matrizes complexas como a própolis. A comparação do perfil químico de diversas amostras de própolis vermelha brasileira e a avaliação de sua atividade antimicrobiana e antioxidante permitirá identificar substâncias que possam estar contribuindo para sua bioatividade, e levar á identificação de marcadores químicos para o controle de qualidade destas amostras, bem como possibilitará definir se há um ou vários tipos de própolis vermelha / Abstract: The word, propolis is of Greek origin: pro means in defense of and polis means community. Propolis is used by bees to strengthen the hive walls and cover the inner walls for its antimicrobial activity. The chemical composition of propolis varies depending on the biodiversity and geographic origin. Red propolis from the north and northeast of Brazil shows promising biological activity: antimicrobial, antiparasitic, antioxidant, cytotoxic, anti-inflammatory, analgesic, antiobesity, antipsoriasic and hepatoprotective effects. The chemical composition of red propolis reported in literature seems to vary qualitatively and quantitatively, even between samples collected in the same region. This may be due to seasonal variations, the flora directly around the hives or differences in the analytical methods. Most of the compounds already identified can be analyzed by mass spectrometry with electrospray ionization and liquid chromatography. These compounds of medium and high polarity (phenolics, flavonoids and benzophenones) are responsible for most of the biological activities reported. Ultra efficient liquid chromatography coupled with mass spectrometry (UHPLC-MS) is a modern, fast and sensitive method. It allows the characterization of the chemical profile of the samples and determines their qualitative composition, by comparison of the mass spectra (MS/MS) of the compounds with data from literature. Therefore it is the most adequate tool to evaluate the composition of complex matrixes such as propolis. The comparison of the chemical profile of diverse red propolis samples and evaluation of their antimicrobial and antioxidant activities allow us to identify the compounds responsible for these activities, indicate the marker compounds and define if there is one type, or several types, of red propolis / Mestrado / Fármacos, Medicamentos e Insumos para Saúde / Mestra em Ciências

Among the herbal drugs currently studied flavonoids occupy a prominent position due to its broad spectrum of therapeutic activities already demonstrated. This work was possible to obtain a fraction rich in flavonoids species Camptosema coriaceum Benth, a Leguminosae. Through the technique of HPLC-DAD-MS was possible to isolate and identify six flavonoid glycosides. Rutin, a glycoside well known was elected as a marker of flavonoid-enriched fraction, this was quantified and its validation method was established. The results showed that the analytical methodology developed for the quantification of rutin is accurate, reproducible and linear over the concentration range 8-48 mg / ml of rutin (R = 0.99943). In order to make this flavonoid-enriched fraction as a technological product, was performed the encapsulation of molecules in β-cyclodextrin rutin and antioxidant tests were made of the material encapsulated and unencapsulated, to evaluate the efficiency of the method pharmaceutics. It was observed that when encapsulated rutin has reduced its antioxidant activity. Additionally, biological tests lymphoproliferation on various carcinoma cell lines were conducted with the crude extract of the species C. coriaceum, with the fraction enriched in flavonoids and rutin, being the most active crude extract. / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Entre os fitoterápicos atualmente estudados, os flavonóides ocupam posição de destaque em virtude de seu amplo espectro de atividades terapêuticas já demonstradas. Neste trabalho foi possível a obtenção de uma fração rica em flavonóides da espécie Camptosema coriaceum Benth, uma Leguminosae. Através da técnica de CLAE-DAD-EM foi possível o isolamento e identificação de 6 flavonóides glicosilados. A rutina, um glicosídeo bem conhecido foi eleita como marcadora da fração enriquecida em flavonóides, esta foi quantificada e seu método de validação foi estabelecido. Os resultados obtidos mostraram que a metodologia analítica desenvolvida para a quantificação da rutina é exata, reprodutível e linear no intervalo de concentração 8 - 48 μg/mL de rutina (R= 0,99943). No intuito de tornar esta fração enriquecida em flavonóides como um produto tecnológico, foi realizado a encapsulação das moléculas de rutina em β-ciclodextrina e foram realizados testes antioxidantes do material encapsulado e do não encapsulado, visando avaliar a eficiência do método farmacotécnico. Foi observado que a rutina quando encapsulada tem sua atividade antioxidante reduzida. Adicionalmente, testes biológicos de linfoproliferação sobre diversas linhagens de carcinoma, foram realizados com o extrato bruto da espécie C. coriaceum, com a fração enriquecida em flavonóides e com a rutina, sendo o extrato bruto o mais ativo.

Due to high global energy demand and a concern for environmental preservation, a renewable energy source is becoming a very important strategy. In this sense, a cellulose is one of the most abundant and promising renewable resources, as its treatment by hydrolysis reaction are obtained biofuels and inputs of great industrial interest, such as: hydroxymethylfurfural (HMF), dihydroxyacetone (DHA), pyruvatedehyde, glyceraldehyde Acetic, formic, lactic and levulic acids. Therefore, the present work proposes an optimization and validation of an analytical methodology in the technique of high performance liquid chromatography (HPLC) for the determination of cellulose conversion products. The optimization of the proposed method was evaluated in a univariate and multivariate fashion. In the HPLC-optimized method, a cation exchange column, a solution of H 2 SO 4, was used as mobile phase with pH = 1.41, column temperature and detector of 42 ° C and 30 ° C respectively, flow of 0.9 mL / Min and total analysis time of 25.40 minutes. 10 analytical standards (glucose, fructose, glyceraldehyde, pyruvate, lactic acid, dihydroxyacetone, formic acid, acetic acid, levulinic acid and HMF) were used for this study. The HPLC method was validated by parameter selection, selectivity, linearity, limit of detection and limit of quantification, precision (repeatability, intermediate precision, instrumental accuracy), accuracy and robustness. These have been taken into consideration both the area and the height of the peaks. Analytical curves showed correlation coefficient ≥ 0.9972. The proposed methodology proved to be sensitive and accurate for a range of 5 to 500 μg / mL. In the instrumental precision, intermediate accuracy and repeatability values of CV ≤ 6,33% were obtained. Accuracy offers assessment methods from 90.58 to 106.87%, the proposed method is robust only for small variations of pH (± 1) and temperature (± 5) for dihydroxyacetone, glucose and pyruvate, which can be explained by the high temperature and pH values. / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Devido à grande demanda energética global e a preocupação com a preservação ambiental, a utilização de fontes de energia renovável está se tornando uma estratégia muito importante. Neste sentido, a celulose é atualmente um dos recursos renováveis mais abundantes e promissores, pois através do seu processamento pela reação de hidrólise são obtidos biocombustíves e insumos de grande interesse industrial, como: hidroximetilfurfural (HMF), dihidroxiacetona (DHA), piruvaldeído, gliceraldeído e os ácidos acético, fórmico, lático e levulínico. Diante disso, o presente trabalho propõe a otimização e a validação de uma metodologia analítica baseada na técnica de cromatografia líquida de alta eficiência (HPLC) para determinação dos produtos da conversão da celulose. A otimização do método proposto foi avaliada de forma univariada e de forma multivariada. No método otimizado por HPLC utiliza uma coluna de troca catiônica, uma solução de H2SO4, como fase móvel com pH=1,41, temperatura da coluna e do detector de 42˚C e 30˚C, respectivamente, fluxo de 0,9 mL/min e tempo total de análise de 25,40 minutos. Para este estudo foram utilizados 10 padrões analíticos (glicose, frutose, gliceraldeído, piruvaldeído, ácido lático, dihidroxiacetona, ácido fórmico, ácido acético, ácido levulínico e HMF). O método de HPLC foi validado através da verificação de parâmetros como, seletividade, linearidade, limite de detecção e limite de quantificação, precisão (repetitividade, precisão intermediária, precisão instrumental), exatidão e robustez. Estes parâmetros foram avaliados levando-se em consideração tanto a área quanto à altura dos picos. As curvas analíticas apresentaram coeficiente de correlação ≥ 0,9972. A metodologia proposta mostrou-se sensível e precisa para a faixa de concentração adotada de 5 a 500 μg/mL. Na precisão instrumental, precisão intermediária e repetitividade foram obtidos valores de CV ≤ 6,33%. A exatidão do método apresentou valores de recuperação de 90,58 a 106,87%, o método proposto é robusto apenas para pequenas variações de pH (±1) e temperatura (±5) para dihidroxiacetona, glicose e piruvaldeído, fato que pode ser explicado pelo alto valor de temperatura e pH, ficando esta variação para estudos futuros.

Echinometra lucunter, o ouriço-do-mar responsável por 50% dos acidentes por animais marinhos, causa inflamação e dor quando os espinhos entram na pele, efeitos atribuídos ao trauma mecânico, além de acidentes por ingestão de ovas. O líquido celômico e o extrato aquoso de espinhos foram fracionados e purificados até a obtenção de moléculas puras, que foram testadas em modelos de inflamação. Foram feitas análises histológicas do espinho e de atividade enzimática do extrato de espinho. Foi isolada uma molécula do espinho e um peptídeo do líquido celômico, que causaram inflamação e dor. Foi verificada atividade enzimática de catepsina B/X. Foi observada uma estrutura histológica organizada no espinho, com células entre a porção calcificada, algumas contendo grânulos eletrodensos com conteúdo protéico, típicas secretoras. Conclui-se que o espinho e o líquido celômico de E. lucunter possuem toxinas inflamatórias, que participam do envenenamento e o espinho tem células secretoras de toxinas. A catepsina pode auxiliar no mecanismo de reparação do espinho, quando quebrado. / Echinometra lucunter, the sea urchin responsible for 50% of marine animals accidents, cause inflammation and pain by the spine penetration, effects attributed to the mechanical trauma. Accidents were reported after the ingestion of raw. The celomic fluid and spines were fractionated and purified, procedure repeated until pure molecules were obtained, tested for inflammation models. Histological analyses and enzymatic assays were performed. A molecule from spines and a peptide from the celomic fluid caused inflammatory effects. Moreover, a cathepsin B/X activity could be identified in the spines. An organized histological structure in the spine was observed, with cells embedded in a calcified matrix, as well as granulous cells displaying proteic contents, typical of secretory cells. It was possible to conclude that the spine and the celomic fluid of E. lucunter do contain inflammatory toxins that prolong the spine puncturing event itself, and the spine possesses a toxin secretory structure. The cathepsin would be present in a mechanism of tissue remodeling.

The aim of this diploma thesis is the determination of selected organic pollutants with a focus on artificial sweeteners in waste water from wastewater treatment plant (WWTP) Modřice and the subsequent degradation of the thus obtained concentrations in model water using advanced oxidation processes (AOP). Two sweeteners were determined, namely sucralose and saccharin found in waste water. Synthetic sweeteners are not sufficiently cleaned in the WWTP and penetrate the environment. Effective processes for removing organic substances, including alternative sweeteners, are AOP processes. These processes are based on reactions of highly reactive hydroxyl radical (OH·), which is produced by many chemical reactions. Generally, they use ozone (O3) and hydrogen peroxide (H2O2). On sampling, wastewater samples were treated with Solid Phase Extraction (SPE) and then evaluated by HPLC/MS (High Performance Liquid Chromatography). In observing the degradation of selected sweeteners, three AOP methods were used: O3/UV, H2O2/UV and O3/H2O2. For each of these methods, the efficiency of degradation of sucralose and saccharin was investigated. From the evaluated data it was found that the most efficient AOP process for removing artificial sweeteners from water is the O3/H2O2 method.

Rostliny rodu Echinacea jsou léčivé byliny stimulující imunitní systém. Používají se zvláště v boji proti chřipce a infekcím horních cest dýchacích. V této práci bylo analyzováno 7 komerčních výrobků obsahujících výtažky z rostliny Echinacea vysokoúčinnou kapalinovou chromatografií. Bylo provedeno kvantitativní stanovení derivátů kyseliny kávové, jež jsou látky zodpovědné za léčivé účinky rostliny. Zjištěné obsahy kyseliny chlorogenové, kávové, cichorové a echinakosidu byly porovnány v jednotlivých výrobcích. Dále byly připraveny extrakty z kořene, listů a květů třapatky nachové pomocí extrakce za zvýšeného tlaku extrakčního rozpouštědla při různých podmínkách, aby se z obsahu účinných látek v nich přítomných zjistilo, které extrakční podmínky jsou nejlepší. Z naměřených dat bylo zjištěno, že největší celková koncentrace obsahových látek je přítomna ve výrobku Imunit Echinaceové kapky firmy Simply You. Jako nejlepší extrakční podmínky se ukázaly teplota 80 °C v kombinaci s rozpouštědlem ve složení EtOH/voda = 40/60, a to jak při extrakci kořene, tak i listů a květů.

This diploma thesis is concerned with determination anthocyanins dyes in elderberry (Sambucus nigra L.). In theoretical part there is described general characterization of the plant, its chemical composition, short view of elderberry breeding and possibilities of usage in food and medical industry. Bigger attention is paid to anthocyanins dyes. There is described their characterization, occurrence, factors that have an impact on their stability. Biosynhesis of the pigments and metabolism, benefit effects and their usage in food chemistry are then shortly sumarized. Methods of determination of anthocyanins are discused too. Next chapter deals with high performance liquid chromatography. At the end of theoretical part, there is sumarized findings of anthocyan dyes analysis by expert scientists in various matrix. In experimental part, total of 16 species of elderberry were analyzed. These species are from research and breeding institute of pomology in Holovousy. The dyes were identification and quantification by high performace liquid chromatography coupled with a diode array detector. Detection wavelenght was 520 nm. Gradient of mobile phase was optimilized because of shortening analyses time and resolution improvement. Four dyes were identified by HPLC: cyanidin-3-sambubioside, cyanidin-3-glukoside, cyanidin-3,5-diglukoside a cyanidin-3-sambubioside-5-glukoside. Total amount of anthocyanins ranged from 33627167,0 to 5525284,0 mAU•min-1. The most anthocyanins dyes were determined in Mammut species (33627167,0 mAU•min-1), the lowest amount was measured in Aurea species (5525284,0 mAU•min-1). The most abundant anthocyanin in all species of eldeberry fruit except Haschebrg and Aurea species was cyanidin-3-sambubioside. The concentrations of cynidin-3-glucoside in all of the berries were quantified using regression equation of calibration curve. Mammut species had the most amount of this anthocyan (0,486 mg•ml-1) and the lowest amount had Aurea species (0,033 mg•ml-1).

This thesis deals with the content of adaptogens in vegetable Schizandra chinensis. The theoretical part deals with the plant Schisandra chinensis, its occurrence, characteristics, uses and cultivation. Location of plants Schisandra chinensis are classified according to Köppen climate classification and compared with the climate in the Czech Republic, due to possible prediction of growing plants in the country. Further are described adaptogens (bioactive substances) contained in this plant and to methods for their extraction and analysis. The experimental part of the thesis deals with the identification of schisandrin in the plant Schisandra chinensis grown in our conditions. The assessment was performed by ultra-performance liquid chromatography (UPLC), high-performance liquid chromatography (HPLC) and direct injection into tha mass spectrometry (MS). Schisandrin was successfully identified in all the samples.

The aim of the diploma thesis is the determination of macrolide antibiotics in drinking water by using of high performance liquid chromatography. Erythromycin and clarithromycin were selected such as representative macrolides due to frequently prescribed pharmaceuticals in this time. Solid phase extraction (SPE) by using of Oasis HLB cartridges was applied for pre concentration and purification of chosen analytes in real samples of drinking water. Optimalization of method and analysis were performed by using of high performance liquid chromatography with mass spectrometry detection (HPLC-MS). The suitable method was selected for determination of macrolides in real samples taken from two sources of drinking water, the interception of water in Litovel and Černovír Olomouc.

Diploma thesis is focused on determination of macrolide antibiotics in wastewater. Environmental contamination with drugs currently represents a major problem. To determine the level of contamination is necessary to develop the appropriate analytical techniques. From the group of macrolide antibiotics were selected four representatives: erythromycin, clarithromycin, azithromycin and roxithromycin due to their frequent use in Czech Republic. For their isolation from wastewater was selected solid phase extraction and for analysis high performance liquid chromatography with mass spectrometric detection was chosen. Under optimized conditions, the ten days analysis of wastewater from the WWTP Brno-Modřice, two days analysis of wastewater from the WWTP Mikulov and one day analysis of wastewater from WWTP of University of Veterinary and Pharmaceutical Sciences Brno was performed.