Made available in DSpace on 2015-04-11T13:38:44Z (GMT). No. of bitstreams: 1 Vanessa.pdf: 3684235 bytes, checksum: 9a9ca68627cdbe2a70c583d22130850f (MD5) Previous issue date: 2012-02-27 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / In this work was realized a phytochemical study and evaluations of extracts and phases of Andira retusa (Fabaceae) regarding activities such as antioxidant, cytotoxic and antimicrobial. This is the first chemical and biological study with this species. There was realized two collects of vegetal material, whose extracts were analyzed by TLC. From chromatographic fractionation of dichloromethane phase of barks methanolic extract resulted in the isolation of lupeol and the mixture of β-sitosterol and stigmasterol. There were also fractionated: the flowers dichloromethane extract; the dichloromethane and ethyl acetate phases from the barks dichloromethane extract and the ethyl acetate phase from the barks methanolic extract. The NMR 1H spectrum of these extracts and phases showed signs characteristic of terpenes (possibly steroids), aromatic compounds (likely flavonoids and chalcones) and aldehydes groups were observed, but were not possible to isolate the compounds to realize the structural identification. The barks, leaves and branches methanolic extracts, as well as the ethyl acetate and butanolic phases from the barks dichloromethane and methanolic extracts, showed a major antioxidant potencial against free radical DPPH. The extracts of 1 DCM Flower and 2 MeOH Bark showed high cytotoxic potential against Brine shrimp Lethality and on cells L929. All extracts showed low activity against Aeromonas hydrophila. However, when analyzed for antimicrobial activity against other microorganisms (Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa), there were observed the activity for the extracts: 1 DCM Branches (1000 μg/mL), 1 MeOH Leaves (1000 e 500 μg/mL) and 1 MeOH Flowers (1000 μg/mL) against S. aureus, 1 MeOH Leaves (1000 μg/mL) against all three microorganisms assayes and 1 DCM Flowers (1000 μg/mL) against S. aureus and E. coli. Of all phases tested, only phase 2 CaM 3.1 (1000 μg/mL) showed antimicrobial activity against S. aureus. The results of biological activities showed that many of the phases whose demonstrated biological activities came from inactive or with low activity crude extracts, which indicated the importance of fractionating biogically active extract as well as those chemically intere / Neste trabalho foi realizado o estudo fitoquímico e avaliações dos extratos e fases de Andira retusa (Fabaceae) quanto às atividades: antioxidante, citotóxica e antimicrobiana. Este é o primeiro estudo químico e biológico com esta espécie. Foram realizadas duas coletas de material vegetal, e extratos obtidos dos mesmos foram analisados por CCD. Do fracionamento cromatográfico da fase diclorometânica do extrato metanólico das cascas foi possível isolar o lupeol e a mistura de -sitosterol e estigmasterol. Foram também realizados os fracionamentos: do extrato diclorometânico das flores e das fases diclorometânica e acetato de etila obtida do extrato diclorometânico das cascas e da fase acetato de etila obtida extrato metanólico das cascas. Os espectros de RMN de 1H dos extratos e fases mostraram sinais característicos de terpenos (possivelmente esteroides), compostos aromáticos (possivelmente flavonoides e chalconas) e grupos aldeídos, mas não foi possível isolar as substâncias para realizar a identificação estrutural. Os extratos metanólicos das cascas, das folhas e dos galhos, assim como as fases de polaridade intermediária e mais polares dos extratos diclorometânico e metanólicos das cascas, apresentaram um maior potencial antioxidante frente ao radical livre DPPH. Dos extratos e fases testados para atividade citotóxica frente à Artemia salina e sobre as células L929, os que tiveram resultados significativos foram os extratos diclorometânico das flores da 1ª coleta e metanólico das cascas da 2ª coleta. Todos os extratos apresentaram baixa atividade contra Aeromonas hydrophila. No entanto, frente a outros micro-organismos (Staphylococcus aureus, Escherichia coli e Pseudomonas aeruginosa) foram ativos os extratos: 1 Galho DCM (1000 μg/mL), 1 Folha MeOH (1000 e 500 μg/mL) e 1 Flor MeOH (1000 μg/mL) contra S. aureus, 1 Folha MeOH (1000 μg/mL) contra os três micro-organismos e 1 Flor DCM (1000 μg/mL) contra S. aureus e E. coli. Das fases testadas, somente a fase 2 CaM 3.1 (1000 μg/mL) apresentou atividade sobre S. aureus. Os resultados das atividades biológicas mostraram que várias das fases que apresentaram atividades provinham de extratos brutos inativos ou com baixa atividade, o que indica a importância do fracionamento de extratos biologicamente ativos e daqueles quimicamente interessantes.

Despite advances in healthcare, cardiovascular disease (CVD) remains the leading cause of death in the United States. Elevated levels of plasma cholesterol are highly predictive of CVD and stroke and are the principal driver of atherosclerosis. Unfortunately, current cholesterol lowering agents, such as statins, are not known to reverse atherosclerotic disease once it has been established. In preclinical models, agonists of nuclear receptor, LXR, have been shown to reduce and reverse atherosclerosis. Phytosterols are bioactive non-cholesterol sterols that act as LXR agonists and regulate cholesterol metabolism and transport. We hypothesize that stigmasterol would act as an LXR agonist and alter intestinal cholesterol secretion to promote cholesterol elimination. Mice were fed a control diet, or a diet supplemented with stigmasterol (0.3% w/w) or T0901317 (0.015% w/w), a known LXR agonist. In this experiment we analyzed the sterol content of bile, intestinal perfusate, plasma, and feces. Additionally, the liver and small intestine were analyzed for relative levels of transcripts known to be regulated by LXR. We observed that T0901317 robustly promoted cholesterol elimination and acted as a strong LXR agonist. Stigmasterol also promoted cholesterol elimination but did not alter LXR-dependent gene expression. Stigmasterol promoted transintestinal cholesterol secretion through an LXR-independent pathway.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniversitet, 2004. / Härtill 4 uppsatser.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The present work describes the synthesis of twenty-two new derivatives (4-25) from stigmasterol (1) and seven new derivatives (26-32) from ursolic acid (2) via esterification and coupling reactions with different amino acids. All the structures of the compounds were confirmed by 1H and 13C NMR. The derivatives were tested for their antimicrobial activity, antitumor activity against cancer cells HT -29 (colorectal) and inhibition of POP, AChE and BChE enzymes. In antimicrobial activity among the compounds tested, ursolic acid (1) showed the greatest potential for inhibition. The results obtained from enzyme inhibition tests were satisfactory, generally the activities of the starting compounds 1 and 2 were increased after the introduction of the amino acids. Among the derivatives evaluated, ursolic-proline-proline-OH acid (32) showed the highest inhibitory capacity of AChE and BChE enzymes and may be considered a dual cholinesterase inhibitor, which can provide greater efficacy in the treatment of Alzheimer's disease. In the evaluation of the antitumor effect, after the first 24 h, most of ursolic acid derivatives showed inhibition effect in cell viability between 33.7 and 70%. After 72 h, the derivative 32 showed the best performance because it did not increase the number of viable cells at any concentration tested, indicating that this compound may be a candidate for antitumor drug. / O presente trabalho descreve a síntese de vinte e dois novos derivados (4 - 25) do estigmasterol (1) e sete novos derivados (26 - 32) do ácido ursólico (2) através de reações de esterificação e de acoplamento com diferentes aminoácidos. Todas as estruturas dos compostos foram confirmadas por RMN 1H e RMN 13C. Os derivados foram testados quanto à sua atividade antimicrobiana, atividade antitumoral frente à célula cancerígena HT-29 (colorretal) e de inibição das enzimas POP, AChE e BChE. Na atividade antimicrobiana, dentre os compostos testados, o ácido ursólico (1) demonstrou o maior potencial de inibição. Os resultados obtidos nos ensaios de inibição enzimática foram satisfatórios, geralmente ocorrendo um aumento da atividade dos compostos de partida 1 e 2 após a introdução dos aminoácidos. Dentre os derivados avaliados, o ácido ursólico-prolina-prolina-OH (32) apresentou a maior capacidade inibitória das enzimas AChE e BChE, podendo ser considerado um inibidor colinesterásico dual, o que pode proporcionar maior eficácia no tratamento da doença de Alzheimer. Na avaliação do efeito antitumoral, após as primeiras 24 h, a maioria dos derivados do ácido ursólico demonstrou efeito de inibição da viabilidade celular entre 33,7 e 70 %. Após 72 h, o derivado 32 foi o que apresentou melhor desempenho, pois não aumentou o número de células viáveis em nenhuma concentração testada, o que indica que este composto pode ser um candidato a fármaco antitumoral.

Phytosterols are steroid chemicals produced by plants for the purposes of membrane function and hormone production. Phytosterols can cause endocrine disruption in aquatic species at very low concentrations and are suspected of contributing to endocrine disruption linked to pulp and paper effluent. Wastewater from the pulp and paper industry is often treated biologically in aerated stabilization basins (ASBs) that expose phytosterols to a range of redox zones. Phytosterol removal in ASBs varies and stigmasterol has even been shown to increase across the treatment system. Little is known about the microbial processes that occur within ASBs and their effect on phytosterol removal. The objective of this research was to assess the biotransformation potential of phytosterols in a simulated ASB treatment system and to improve understanding of the processes that occur within the various redox zones and their impact on the removal of phytosterols. To assess the biotransformation of phytosterols under aerobic conditions, three assays were conducted using a stock aerobic culture fed with pulp and paper wastewater. The assays tested three conditions: phytosterols present as a sole added carbon source, phytosterols with dextrin as an added carbon source, and phytosterols with ethanol as a solubilizing agent and added carbon source. Phytosterol biotransformation was found to be limited by low phytosterol solubility. When solubilized, phytosterol removal occurred in two phases: an initial near-linear removal, followed by accelerated removal during the culture's stationary stage, possibly due to the release of extracellular cholesterol oxidase. The anoxic and anaerobic biotransformation of phytosterols was examined through a series of three semi-batch cultures maintained under nitrate-reducing, sulfate-reducing and fermentative/methanogenic conditions, all developed from stock cultures fed with pulp and paper wastewater. Phytosterol removal was significant in the nitrate-reducing culture, although microbial activity and phytosterol removal declined in later stages. Phytosterol removal was also observed in the sulfate-reducing culture, although there was a significant lag period before removal occurred. No phytosterol removal was observed in the fermentative/methanogenic culture. Phytosterol biotransformation was also examined in the context of a lab-scale ASB fed continuously with pulp and paper wastewater. The steady-state ASB effluent and sediment characteristics were examined over three hydraulic retention times (HRTs). Effluent quality was not significantly affected by a change in HRT but sediment characteristics were significantly affected and, at shorter HRTs, phytosterols accumulated in the sediment. Wastewater bioassays demonstrated the release of phytosterols during the breakdown of solids. This research improves the understanding of biological processes within ASBs and their effect on phytosterol removal.

Siparuna apiosyce (Mart.) A. DC, espécie vegetal aromática da família Monimiaceae Jussieu, encontrada no sudeste brasileiro, conhecida vulgarmente como \"limão-bravo\", é planta medicinal de uso popular. As folhas são empregadas em problemas gástricos e respiratórios. A espécie consta na primeira edição da Farmacopéia Brasileira, sob a denominação \"limoeiro-bravo\", sendo-considerada a folha, como parte usada. Efetuou-se análise macro e miscroscópica de folha e de caule para fins de identificação da droga inteira, em análises de rotina. O estudo microscópico incluiu folha, na forma pulverizada. Empregaram-se as técnicas usuais de análise. Elaboraram-se desenhos esquemáticos e em detalhe das estruturas microscópicas. As descrições são, igualmente, acompanhadas de fotografias e de fotomicrografias. Efetuou-se análise comparativa de folha e desta em mistura com caule, na proporção naturalmente encontrada no vegetal, tendo-se realizado a determinação de parâmetros físico e químico, abordagem fitoquímica e estudo cromatográfico em camada delgada de ambos. Realizou-se avaliação da atividade antimicrobiana dos extratos fluidos frente a Escherichia coli, Staphylococcus aureus e Candida albicans. Foi determinado o teor de óleo essencial presente em folha e em fruto da espécie. Considerando a comercialização da droga, na forma de extrato fluido, estes foram preparados, pelo processo de percolação fracionada (processo \"C\"), segundo a Farmacopéia Brasileira II, a partir das partes aéreas, anteriormente referidas. Efetuou-se a determinação comparativa, para ambos os extratos fluidos, de parâmetros físicos e químico e análise cromatográfica em camada delgada. Para o estudo químico foram elaborados os extratos hexânico, diclorometânico e etanólico, com vistas ao isolamento de componentes de folha e caule. Com o resíduo etanólico efetuou-se processo de extração de alcalóides. O resíduo hexânico e o resíduo bruto de alcalóides foram submetidos à separação por coluna cromatográfica. As frações resultantes foram cromatografadas em camada delgada, tendo sido reunidas aquelas de comportamento cromatográfico semelhante. Posteriormente, efetuaram-se procedimentos específicos para o isolamento de compostos. Entre os vários componentes obtidos, dois foram identificados, por via espectral, tendo sido o estigmasterol e o alcalóide noraporfínico asimilobina. Espectros das substâncias (I.V., RMN 1H, RMN 13C, RMN 13C - DEPT 135º e HETCOR) acompanham o trabalho. / Siparuna apiosyce (Mart.) A. DC., an aromatic medicinal specie of southest Brazil, commonly known as \"limão - bravo\", is traditionally used to treat gastric and pulmonary diseases. The leaves are included in the first edition of the Brazilian Pharmacopeia and officially named \"limoeiro-bravo\". Macro and microscopical descriptions and analysis were made, concerning leaves and twigs. It was performed by usual techniques. Photographies (macro and microscopical analysis) and drawings follow the descriptions. Comparative assays were made considering leaves and twigs with leaves. The author determined physical and chemical parameters, followed by phytochemical screening and thin layer chromatography profile. The respective fluid extrats were prepared by fractionated percolation, as directed under second edition of the Brazilian Pharmacopeia. Physical and chemical parameters and antimicrobial activity of fluid extract. were determined. Essential oil content of leaf and fruit were obtained. Twigs and leaves were extracted by percolation and successively with n-hexane, dichlorometane and ethanol. Extraction procedures for alkaloidal content was performed, with the ethanolic residue, by traditional methods. The hexanic residue and the concentrated alkaloidal phase were submitted to chromatographic column techniques. Specific procedures were applyed for the component isolation. Two compounds were identified by spectrometric determination: stigmasterol and the noraporphine asimilobine alkaloid. This work include RMN 1H, RMN 13C,DEPT 1350and HETCOR spectra.

Siparuna apiosyce (Mart.) A. DC, espécie vegetal aromática da família Monimiaceae Jussieu, encontrada no sudeste brasileiro, conhecida vulgarmente como \"limão-bravo\", é planta medicinal de uso popular. As folhas são empregadas em problemas gástricos e respiratórios. A espécie consta na primeira edição da Farmacopéia Brasileira, sob a denominação \"limoeiro-bravo\", sendo-considerada a folha, como parte usada. Efetuou-se análise macro e miscroscópica de folha e de caule para fins de identificação da droga inteira, em análises de rotina. O estudo microscópico incluiu folha, na forma pulverizada. Empregaram-se as técnicas usuais de análise. Elaboraram-se desenhos esquemáticos e em detalhe das estruturas microscópicas. As descrições são, igualmente, acompanhadas de fotografias e de fotomicrografias. Efetuou-se análise comparativa de folha e desta em mistura com caule, na proporção naturalmente encontrada no vegetal, tendo-se realizado a determinação de parâmetros físico e químico, abordagem fitoquímica e estudo cromatográfico em camada delgada de ambos. Realizou-se avaliação da atividade antimicrobiana dos extratos fluidos frente a Escherichia coli, Staphylococcus aureus e Candida albicans. Foi determinado o teor de óleo essencial presente em folha e em fruto da espécie. Considerando a comercialização da droga, na forma de extrato fluido, estes foram preparados, pelo processo de percolação fracionada (processo \"C\"), segundo a Farmacopéia Brasileira II, a partir das partes aéreas, anteriormente referidas. Efetuou-se a determinação comparativa, para ambos os extratos fluidos, de parâmetros físicos e químico e análise cromatográfica em camada delgada. Para o estudo químico foram elaborados os extratos hexânico, diclorometânico e etanólico, com vistas ao isolamento de componentes de folha e caule. Com o resíduo etanólico efetuou-se processo de extração de alcalóides. O resíduo hexânico e o resíduo bruto de alcalóides foram submetidos à separação por coluna cromatográfica. As frações resultantes foram cromatografadas em camada delgada, tendo sido reunidas aquelas de comportamento cromatográfico semelhante. Posteriormente, efetuaram-se procedimentos específicos para o isolamento de compostos. Entre os vários componentes obtidos, dois foram identificados, por via espectral, tendo sido o estigmasterol e o alcalóide noraporfínico asimilobina. Espectros das substâncias (I.V., RMN 1H, RMN 13C, RMN 13C - DEPT 135º e HETCOR) acompanham o trabalho. / Siparuna apiosyce (Mart.) A. DC., an aromatic medicinal specie of southest Brazil, commonly known as \"limão - bravo\", is traditionally used to treat gastric and pulmonary diseases. The leaves are included in the first edition of the Brazilian Pharmacopeia and officially named \"limoeiro-bravo\". Macro and microscopical descriptions and analysis were made, concerning leaves and twigs. It was performed by usual techniques. Photographies (macro and microscopical analysis) and drawings follow the descriptions. Comparative assays were made considering leaves and twigs with leaves. The author determined physical and chemical parameters, followed by phytochemical screening and thin layer chromatography profile. The respective fluid extrats were prepared by fractionated percolation, as directed under second edition of the Brazilian Pharmacopeia. Physical and chemical parameters and antimicrobial activity of fluid extract. were determined. Essential oil content of leaf and fruit were obtained. Twigs and leaves were extracted by percolation and successively with n-hexane, dichlorometane and ethanol. Extraction procedures for alkaloidal content was performed, with the ethanolic residue, by traditional methods. The hexanic residue and the concentrated alkaloidal phase were submitted to chromatographic column techniques. Specific procedures were applyed for the component isolation. Two compounds were identified by spectrometric determination: stigmasterol and the noraporphine asimilobine alkaloid. This work include RMN 1H, RMN 13C,DEPT 1350and HETCOR spectra.

The species Vernonia tweedieana Baker is an herbaceous plant widely distributed in the plains of Paraguai, Argentina and south of Brazil and popularly known as assa-peixe . This plant is used in traditional medicine as an expectorant medicament. So far, this plant was not studied on the phytochemical and biological point of view. This work is a contribution to the phytochemical study of the Asteraceae. The leaves of V. tweedieana Baker were collected in march of 2004, in Ijuí Rio Grande do Sul, Brazil, and identified by Dr. Geraldo C. Coelho (UNIJUÍ). The respective voucher specimen was deposited in the herbarium of the Federal University of Santa Maria (UFSM)-RS (code SMDB 9536). The leaves (1.900 g) were dried in an air circulating stove at 40 ºC, pulverized in mill and extracted by maceration with 65% EtOH at room temperature for seven days. The ethanolic extract was filtered and the ethanol was removed. Finally, the extract was retake in water and partitioned using organics solvents with increased polarity: CH2Cl2, AcOEt and n-BuOH. We report the isolation and identification of six constituents of the CH2Cl2 fraction: the triterpenes a-amyrin, b-amyrin and lupeol and the steroids b- sitosterol, stigmasterol and spinasterol. The flavanone eriodictyoI was isolated from the AcOEt fraction. The constituents were identified through spectral data of the infra-red, GC-MS, 1H-NMR, 13C-NMR and DEPT. No deaths and other signs of toxicity and adverse effect were observed in the evaluation of acute toxicity with doses up to 5.000 mg/kg, that is the maximum dose for acute oral toxicity test for extract of plants. Also, through DPPH method, AcOEt and n-BuOH fractions of the plant showed good antioxidant activity with IC50 22.52 and 17.44 mg/mL, respectively. / O uso de plantas medicinais sempre teve uma importância vital no cotidiano da humanidade. No entanto, apenas uma pequena parte destas já foram estudadas e tiveram suas ações farmacológicas comprovadas cientificamente. Um exemplo de planta ainda desconhecida quimicamente é Vernonia tweedieana Baker (Asteraceae), vulgarmente conhecida como assa-peixe. É uma planta característica da região Sul do Brasil que é usada popularmente para o tratamento de doenças respiratórias, principalmente pelas suas propriedades expectorantes. Este trabalho descreve o isolamento e identificação de seis constituintes químicos presentes no extrato CH2Cl2 e de um no extrato AcOEt das folhas de Vernonia Tweedieana Baker. As folhas foram coletadas em março de 2004, no município de Ijuí RS. A espécie foi localizada e identificada pelo Prof. Dr. Geraldo C. Coelho (DeBQ-UNIJUÍ). Material testemunha encontra-se depositado no Herbário do Departamento de Biologia da UFSM sob o registro n° SMDB 9536. O material vegetal seco e moído (1.900 g) foi macerado utilizando como solvente etanol:água (65:35, v/v). Após sete dias o extrato foi filtrado e concentrado sob pressão reduzida para remover o etanol. Fez-se fracionamento desse extrato bruto com solventes orgânicos de polaridades crescentes (CH2Cl2, AcOEt, n- BuOH). Da fração CH2Cl2 caracterizou-se os triterpenos a e b-amirinas e lupeol, ainda os esteróides b-sitosterol, estigmasterol e espinasterol; e da fração AcOEt o flavonóide eriodictiol. Os compostos isolados foram analisados por CG-EM-IE, IV, RMN de 1H e RMN de 13C e seus dados espectroscópicos foram comparados com os obtidos da literatura. Ainda, na avaliação da toxicidade aguda foi verificado que nenhum dos extratos apresentou toxicidade em doses até 5.000 mg/mL. Também, pelo método do DPPH, foi constatada atividade antioxidante para as frações AcOEt e n-BuOH da planta, apresentando IC50 de 22,52 e 17, 44 mg/mL, respectivamente.

Koffein (1,3,7-Trimethylxanthin) und Coprostanol (5beta-cholestan-3beta-ol) wurden im Berliner Oberflächenwasser nachgewiesen. Ihre Konzentrationen korrelierten mit dem Verunreinigungsgrad der Proben, was nahelegt, dass sie sich als Marker für menschliche Aktivität eignen. Bemerkenswerterweise wurde Koffein in jeder einzelnen Oberflächenwasserprobe oberhalb der Bestimmungsgrenze von 0,025 µg/L gefunden. Um Oberflächenwasserproben in größeren Serien zu untersuchen, war die Entwicklung zweier neuer Methoden erforderlich: ein Immunoassay, basierend auf einem monoklonalen Antikörper für Koffein und eine dispersive flüssig-flüssig Mikroextraktionsmethode (DLLME), gefolgt von Flüssigkeitschromatographie gekoppelt mit Tandem-Massenspektrometrie (LC-MS/MS) für Coprostanol. Der entwickelte Koffein-Immunoassay zeigt die beste je erhaltene Nachweisgrenze für Koffein (0,001 µg/L), erlaubt Hochdurchsatz-Analysen und erfordert keine Probenvorbereitung. Der Assay wurde auch erfolgreich für die Messung von Koffein in Getränken, Haarwaschmitteln, Koffeintabletten und menschlichem Speichel angewendet. Antikörper gegen Coprostanol sind nicht kommerziell erhältlich. Eine neue Strategie Anti-Coprostanol-Antikörper zu generieren wurde erarbeitet, die eine analoge Verbindung – Isolithocholsäure (ILA) – als Hapten verwendet, mit der eine Gruppe von Mäusen immunisiert wurde. Ein polyklonales Anti-ILA-Serum wurde produziert, welches Coprostanol bindet, aber die niedrige Affinität erlaubte nicht den Aufbau eines Immunoassays, der die Messung von Umweltkonzentrationen des Anayten (im Bereich ng/L) zulässt. Spezifische Anti-ILA-Immunglobuline G wurden auch in den Faeces der Mäuse gefunden. Coprostanol wurde in den Wasserproben durch die Verwendung einer neuentwickelten LC-MS/MS-Methode unter APCI-Ionisation (atmospheric pressure chemical ionisation) gemessen. Konzentrationen oberhalb von 0,1 µg/L wurden nach Voranreicherung der Probe mittels DLLME bestimmt. / Caffeine (1,3,7-trimethylxanthine) and coprostanol (5beta-cholestan-3beta-ol) were detected in samples of Berlin’s surface water. Their concentrations correlated with the contamination status of the samples, suggesting their usefulness as markers of human activity. Remarkably, caffeine concentrations were always well above the limit of quantitation of 0.025 µg/L. In order to screen surface water samples in larger series, the development of two novel methods was required: a monoclonal antibody-based immunoassay for caffeine and a dispersive liquid-liquid microextraction (DLLME) method, followed by liquid chromatography tandem mass spectrometry (LC-MS/MS) for coprostanol. The caffeine immunoassay developed shows the best analytical limit of detection (LOD) obtained so far for caffeine (0.001 µg/L), allows high-throughput analysis, and does not require sample pre-treatment. The assay was also successfully employed to measure caffeine in beverages, shampoos, caffeine tab-lets, and human saliva. Antibodies to coprostanol are not commercially available. A new strategy to generate anti-coprostanol antibodies was elaborated using an analogous com-pound as hapten – isolithocholic acid (ILA) – and immunizing a group of mice. A polyclonal anti-ILA serum was produced, which binds coprostanol but the low affinity did not permit setting up an immunoassay to measure environmental concentrations of the analyte (in the range of ng/L). Specific anti-ILA immunoglobulin G were also found in the faeces of the immunized mice. Coprostanol was quantified in the water samples using a newly developed LC-MS/MS method using atmospheric pressure chemical ionisation (APCI). Concentrations above 0.1 µg/L were determined after sample preconcentration using DLLME. This extraction method also proved to be successful for enrichment of coprostanol-related compounds such as cholesterol, cholestanol, cholestanone, ergosterol, and stigmasterol.

This diploma thesis deals with the optimization of processes for extraction of bioactive lipophilic compounds from fruits of sea buckthorn (Hippophae Rhamnoides) into various types of plant oil bases using simple maceration. The theoretical part of this thesis deals with the characterization of this herb, its botanical classification, traditional use, chemical composition and medicinal effects. Increased attention is also paid to the characterization of individual types of plant oils used, namely camellia, camellia organic, passionflower, kukui and kiwi oil. The experimental part of the thesis deals with application of theoretical knowledge. The profile of total and free fatty acids for individual plant oil bases was determined by GC/FID, furthet the basic fat numbers were also determined. When optimizing the extraction, emphasis was placed not only on the effect of the extraction agent used, but also on the extraction time (1, 3, 5, 7, 10, 14, 21 and 66 days). The macerates were continuously subjected to the determination of selected parameters (total amount of carotenoids, total amount of phytosterols, lutein, neoxanthin, astaxanthin, stigmasterol, -sitosterol and vitamin E) using UV-VIS spectroscopy and HPLC/PDA. Likewise, the peroxide number was monitored during maceration to assess the degree of oxidative degradation of macerates. The recovery of selected total parameters in individual oils did not differ significantly in most cases. On the contrary, the yield of individual monitored parameters differed significantly. At the same time, static maceration under our conditions was not very suitable for the extraction of vitamin E, stigmasterol and total phytosterols.