Synthetic dyes that are used as color additives in foodstuffs are regulated by the Food and Drug Administration (FDA) under the Food, Drug, and Cosmetic Act (FD&C). The use of synthetic dyes not approved by the FDA, or the addition of dyes approved by the FDA above their maximum concentration limits in foodstuffs necessarily constitutes illegal food adulteration. Recently, rhodamine B (RhB), a bright-pink synthetic dye not approved for use in foodstuffs, has become an adulterant of interest due to its discovery in a large variety of food samples, and its identity as a potential carcinogen. Numerous chromatographic and spectroscopic methods have been developed for the analysis of RhB in food samples, as well as standard methods available for the analysis of synthetic dyes. However, due to the complexity of real food samples and the chemical diversity of synthetic dye concomitants, comprehensive chromatographic methods tend to be time consuming and expensive. Herein, we report a method for the determination of RhB in foodstuffs for the screening of real samples prior to subsequent full-blown chromatographic analysis, saving valuable time and resources. This screening method employs thin-layer chromatography (TLC) as a separation method, and, due to the strong yellow-orange fluorescence exhibited by RhB when excited with ultraviolet or green light, direct fluorescent measurement of RhB on the TLC plate using a fiber optic probe coupled to a commercial spectrofluorometer or to an instrumental set up for laser-induced fluorescence measurements. Qualitative analysis of RhB is based on its retardation factor, excitation and fluorescence spectra, and fluorescence lifetime. Quantitative measurements are made directly from the TLC plate to provide analytical figures of merit comparable to traditional fluorometric methods in liquid solution. The ability of the new method to determine RhB in food samples is then demonstrated with the analysis of chili powder.

Vitamin C is industrially produced by the Reichstein method, which uses gluconobacters to oxidize sorbitol to sorbose then a chemical process to convert sorbose to 2-keto-L-gulonic acid (2-KLG). The establishment of a more extensive microbial process for 2-KLG production translates into a less expensive and more efficient production of vitamin C. I examined pure strains and mixed cultures for their ability to produce 2-KLG using thin layer and high performance liquid chromatography. The DSM 4027 mixed culture produced the highest yield, 25 g/L, of 2-KLG from 100 g/L of sorbose, while the gram-negative rods isolated from DSM 4027 produced 8.8 g/L, and B. megaterium isolated from DSM 4027 produced 1.4 g/L. Thus, the gram-negative rods in the mixed culture were the primary 2-KLG producer, but B. megaterium in the DSM 4027 mixture enhanced this synthesis. Authentic pure cultures of Gluconobacter oxydans IFO strain 3293 and ATCC strain 621 produced 3.4 g/L and 5.7 g/L, respectively. Attempts to co-culture the isolated B. megaterium with the isolated gram-negative rods and authentic Gluconobacter strains did not increase 2-KLG production, nor did growing the cultures on B. megaterium spent media. Bacillus megaterium produced an unidentified keto-compound detected on the TLC chromatograms, which suggested that B. megaterium converted sorbose to an intermediate that may then be converted by the gram-negative rods in DSM 4027 to 2-KLG. Limited phenotypic tests suggested that the gram-negative rods in the DSM 4027 mixture are not gluconobacters. / Master of Science

As caprilinas são acilgliceróis de cadeia média com diversas aplicações nos campos alimentício, farmacêutico e cosmético. A esterificação direta é um processo importante para a síntese de caprilinas, dado que o glicerol é subproduto da produção de biodiesel e que a tricaprilina, material de partida para a glicerólise e hidrólise, não é um composto abundante naturalmente. As reações existentes para a síntese de acilgliceróis podem ser catalisadas por via química ou enzimática, sendo esta última superior em diversos aspectos. Dois dos principais contrapontos à utilização de enzimas são seu alto custo e dificuldade de recuperação. A imobilização de enzimas é uma das maneiras existentes para contornar estas questões. A maioria dos estudos científicos relativos à obtenção destes compostos refere-se aos acilgliceróis de cadeia longa. Portanto, é necessário aprimorar a esterificação direta entre o glicerol e ácido caprílico catalisada por lipase, através da compreensão da cinética reacional e da influência dos parâmetros reacionais. A existência de uma metodologia de separação, identificação e quantificação simples e eficaz também é importante, tornando as pesquisas relacionadas às caprilinas mais rápidas e menos complicadas. Desta maneira, este trabalho consistiu de três etapas principais: (1) a imobilização de lipases em resinas de troca aniônica, visando reduzir os custos em relação à lipase imobilizada comercial; (2) o desenvolvimento de um método de cromatografia em camada delgada capaz de quantificar as caprilinas formadas; (3) o estudo da reação de esterificação para a obtenção de caprilinas em regime descontínuo e descontínuo-alimentado. A resina DOWEX® 1X2-400 foi a que apresentou a melhor eficiência de imobilização (EI) da Palatase®. Sua atividade lipolítica foi apenas 12,7% inferior à versão imobilizada comercial da lipase, a Lipozyme®RM IM, indicando a viabilidade de substituição desta última. O desenvolvimento de método de cromatografia em camada delgada resultou em relações lineares para a detecção e quantificação de ácido caprílico, monocaprilina, dicaprilina e tricaprilina com R2 e R2pred de 99,3%/98,73%; 98,9%/97,6%; 99,8%/99,8% e 99,7%/99,5%, respectivamente, e com valor-p de 0,000. As reações em regime descontínuo foram realizadas seguindo planejamento fatorial completo, no qual foram variadas a temperatura (30 ou 70°C), a proporção molar entre os reagentes (1:1 ou 3:1 - ácido caprílico : glicerol) e o tempo reacional (6 ou 10h). A análise de regressão do rendimento reacional expresso em porcentagem de consumo de ácido caprílico indicou que, nestas condições, os fatores relevantes para o resultado final são a temperatura e a proporção molar, bem como a interação entre eles. As melhores condições para a obtenção de mono e dicaprilinas foram, portanto, de 30ºC e razão molar de 1:1. O estudo da cinética da reação indica que 40,4% do ácido caprílico são consumidos na primeira hora e que o consumo máximo ocorre na terceira hora de reação. Houve uma queda drástica no rendimento reacional, quando a reação foi conduzida em regime descontínuo-alimentado, provavelmente devido à menor eficiência catalítica da lipase devido ao baixo teor de água na primeira hora de reação. / Caprylins are medium chain acylglycerols that find several applications in food, pharmaceutical and cosmetic fields. Direct esterification is an important route for the synthesis of caprylins, since glycerol is a byproduct of biodiesel production and tricaprylin, the starting material of glycerolysis and hydrolysis, is not a naturally abundant compound. The glycerides synthesis can be catalyzed by either chemical or enzymatic routes, the latter being superior in several aspects. The biggest issues regarding the use of enzymes are their high cost and difficult recovery. Using immobilized enzymes can minimize these concerns. Most of scientific studies concerning the obtainment of these compounds address to long chain acyglycerols. Thus, the improvement of lipase catalyzed esterification of glycerol and caprylic acid is necessary, by understanding the reaction kinetics and the role of reaction parameters. A simple and effective method of separation, identification and quantification of caprylins is also a relevant contributing factor for easier and faster researches. Therefore, this work consisted of three main steps: (1) immobilization of lipases on anion exchange resins in order to produce a cheaper alternative to imported commercial immobilized lipases; (2) development of a thin layer chromatographic method capable of quantifying caprylins; (3) studying the obtainment of caprylins by direct esterification in batch and fed batch systems. DOWEX® 1X2-400 resin presented the best immobilization efficiency (IE) results. Additionally, its lipolytic activity was only 12.7% lower than the commercial Lipozyme® RM IM, suggesting that Lipozyme\'s substitution is feasible. Regarding the developing of a thin layer chromatography method, linear correlations were obtained for detection and quantification of caprylic acid, monocaprylin, dicaprylin and tricaprylin, with a R2 and R2pred of 99,3%/98,73%; 98,9%/97,6%; 99,8%/99,8% e 99,7%/99,5%, respectively, with a p-value of 0,000. The batch reactions were carried out according to a complete factorial design, in which the temperature (30 or 70ºC), the molar ratio between the reagents (1:1 or 3:1) and reaction time (6h or 10h) were varied. The regression analysis of reactional yield expressed in terms of percentage of caprylic acid consumption indicated that only the temperature, molar-ratio and their interaction were important for reaction yields, within the studied conditions. The best conditions related to the obtainment of mono and dicaprylins were 30ºC and molar ratio of 1:1. The reaction kinetics indicates that 40.4% of the caprylic acid is consumed within one hour of reaction and that the maximal consumption was achieved in the third hour. A drastic drop in reaction yield was observed when the fed batch mode was adopted, probably due a lower catalytic efficiency presented by lipase as a result of the lower water content in the first hour of reaction.

As plantas medicinais são conhecidas há muitos séculos pelos homens, e ainda hoje são amplamente utilizadas no tratamento de enfermidades. A população acredita que é uma alternativa segura e de baixo custo em relação aos produtos farmacêuticos industrializados, e faz seu uso indiscriminado e sem acompanhamento médico. A automedicação e a falta de controle de qualidade das drogas vegetais (DVs) representam um sério risco à saúde de seus usuários. A diversidade de espécies conhecidas como plantas medicinais, e o uso de nomes populares regionais podem ocasionar erros de identificação taxonômica ou mesmo adulteração do produto. Outro fator que pode alterar a qualidade das DVs é a contaminação de origem química ou biológica, proveniente das diversas fases de seu preparo. Com o uso disseminado de DVs e fitoterápicos, cresceu a preocupação com os efeitos adversos e a interação com medicamentos convencionais. Esse panorama evidencia a interdisciplinaridade na área de plantas medicinais. Assim, o projeto, do qual derivou este de mestrado, é interdisciplinar, com a participação de pesquisadores da etnofarmacologia, microbiologia, farmacovigilância e farmacognosia. O objetivo específico desta dissertação de mestrado é avaliar a qualidade das DVs com possíveis ações psicoativas, adquiridas em barracas de rua na cidade de Diadema. As amostras selecionadas e adquiridas pelo grupo da etnofarmacologia foram confrontadas a monografias farmacopêicas, literatura especializada e/ou amostra autêntica, avaliando-se os caracteres macroscópicos, microscópicos, perfis cromatográficos e pureza das DVs. Cortes histológicos foram preparados conforme as técnicas usuais, cromatografias em camada delgada foram realizadas de acordo com literatura e fotografias documentam as análises. De um total de 35 lotes analisados, 88,6% confirmaram sua autenticidade, e apenas 57,1% estavam em conformidade com os valores máximos permitidos para materiais estranhos. Também ficou evidente a baixa qualidade de armazenamento do material e problemas com embalagens e rótulos, em total desacordo com a legislação vigente. Concluiu-se então que as DVs adquiridas demandam atenção e melhorias quanto à sua qualidade, fato que demonstra a necessidade de maior orientação e conscientização dos vendedores quanto aos cuidados para sua comercialização à população. / Medicinal plants have been known for centuries by humanity and are still widely used for the treatment of illnesses. People believe that it is a safer and low cost alternative in relation to manufactured pharmaceutical products and makes indiscriminate use of them, without medical supervision. Self-medication and the lack of quality control of herbal drugs (HDs) represent a serious health risk to users. The diversity of species known as medicinal plants and the use of popular regional names can cause errors in taxonomic identification or even product adulteration. Another factor that may alter the quality of HDs is contamination from biological or chemical origin, from various stages of their preparation. With the widespread use of herbal remedies and HDs, the concern about adverse effects and interactions with conventional medicines has increased. This scenario highlights the interdisciplinarity of the medicinal plants field. Thus, the project, which this master`s program was derived from, is interdisciplinary, involving researchers from ethnopharmacology, microbiology, pharmacovigilance and pharmacognosy. The specific aim of this master\'s project is to evaluate the quality of HDs with possible psychoactive action, acquired in street stalls in the city of Diadema. The samples selected and acquired by ethnopharmacology were confronted with pharmacopoeial monographs, literature and/or authentic samples and the macroscopic and microscopic characters, chromatographic profiles and purity of HDs were evaluated. Histological sections were prepared according to the usual techniques, thin layer chromatographys were performed according to the literature, and photographs document the analysis. From a total of 35 lots, 88.6% confirmed their authenticity, and only 57.1% were in compliance with the maximum limit for the amount of foreign material. The poor quality of material storage and problems with packaging and labels, in total disagreement with the law, was also made evident. We concluded that the acquired HDs require caution and improvement of their quality, which demonstrates the need for more guidance and awareness of vendors about the care involved in the commercialization of HDs to the population.

In many developing countries, farmers and pastoralists still rely on their indigenous knowledge, practices and locally available plants to control nematode parasitic infections, both in livestock and humans. The overall aim of my thesis was to undertake bioassay-guided phyto-chemical study of extracts and their constituents from Ethiopian anti-parasitic plants used by healers to control gastrointestinal nematode parasites in livestock to validate their ethno-medicinal use and to characterise and identify their active ingredients. As a first experiment (Chapter Three), four types of crude extracts (water, 70% methyl-alcohol, absolute methanol and acetone) of four indigenous Ethiopian medicinal plants (Adenia species, Cissus ruspolii, Ipomoea eriocarpa and Euphorbia thymifolia) were screened against Teladorsagia circumcincta egg hatching in vitro, not only as a first step to validate the traditional healers claim but also to choose the most promising plant extract(s) for further phyto-chemical studies. The egg hatching inhibition (EHI) test results revealed that the anti-parasitic properties of these plants depended on plant species, dose, and solvent polarity. The water extracts of both C. ruspolii and Adenia sp. exhibited largest, up to 100% EHI but also larval migration inhibition activities, and were selected for further studies. The second experiment (Chapter Four) assessed the nature of active constituents in these extracts by physico-chemical methods. It was observed that the major constituents of both plant extracts responsible for the EHI activities are likely highly polar, water-soluble, small and moderately heat-labile molecules. The third and fourth experiments (Chapters Five and Six) consisted of separating Cissus ruspolii and Adenia sp. water extracts into discrete fractions by gel-permeation chromatography, EHI tests of Bio-Gel P-2 fractions followed by thin layer chromatography (TLC) profiling of these fractions to detect separated spots (in day light, under UV-light or after staining with various staining reagents) and also to see how elution patterns of separated spots affected by column parameters. The EHI tests on the fractions obtained revealed that the active constituents of C. ruspolii and Adenia sp. water crude extracts were eluted into few fractions based on their molecular sizes. The TLC profilings of these fractions identified spot patterns of active and inactive fractions, which allowed pooling of active constituents based on their EHI and TLC profiling into three pools for each plant. The fifth experiment (Chapter Seven) was to isolate and purify compounds from these pools using various preparative planar and column chromatographic methods. Sequential applications of column chromatography followed by preparative thin layer chromatography isolated and purified five active compounds from C. ruspolii and two active compounds from Adenia sp. The sixth experiment (Chapter Eight) was to characterize and propose/elucidate structures of compounds from the active fractions using chromatographic, analytical and spectroscopic methods. In this regard, the structures of two oleanane type triterpenoid saponins isolated from one of active fractions of Adenia sp. were proposed based on their mass spectrometry (MS) and nuclear magnetic resonance (NMR) data with support of compounds property, TLC and literature. Similar outcomes for C. ruspolii were not achieved due to lack of sufficient sample to run 13C-nuclear magnetic resonance spectroscopy and distortionless enhancement by polarization transfer (DEPT), contamination of some purified compounds with ill-characterised substance from the preparative TLC matrix and in some cases mass spectrometry (MS) and nuclear magnetic resonance (NMR) data did not support each other. The last experiment (Chapter Nine) was to assess anthelmintic efficacy and safety of C. ruspolii and Adenia sp. crude water extracts in Heligmosomoides bakeri infected mice. This in vivo test revealed that both plant extracts exhibited significant reduction in worm burdens and worm egg excretion, with moderate effects on haematology and organ weights at tolerated dosages. In conclusion, both in vitro and in vivo data revealed that Adenia sp. and C. ruspolii have anthelmintic properties, thus validating traditional healer claims and supporting ethno-medicinal use. The bioassay-guided phytochemical study resulted in the isolation of a number of active compounds from these plants, for some of which a structure has been proposed.

As caprilinas são acilgliceróis de cadeia média com diversas aplicações nos campos alimentício, farmacêutico e cosmético. A esterificação direta é um processo importante para a síntese de caprilinas, dado que o glicerol é subproduto da produção de biodiesel e que a tricaprilina, material de partida para a glicerólise e hidrólise, não é um composto abundante naturalmente. As reações existentes para a síntese de acilgliceróis podem ser catalisadas por via química ou enzimática, sendo esta última superior em diversos aspectos. Dois dos principais contrapontos à utilização de enzimas são seu alto custo e dificuldade de recuperação. A imobilização de enzimas é uma das maneiras existentes para contornar estas questões. A maioria dos estudos científicos relativos à obtenção destes compostos refere-se aos acilgliceróis de cadeia longa. Portanto, é necessário aprimorar a esterificação direta entre o glicerol e ácido caprílico catalisada por lipase, através da compreensão da cinética reacional e da influência dos parâmetros reacionais. A existência de uma metodologia de separação, identificação e quantificação simples e eficaz também é importante, tornando as pesquisas relacionadas às caprilinas mais rápidas e menos complicadas. Desta maneira, este trabalho consistiu de três etapas principais: (1) a imobilização de lipases em resinas de troca aniônica, visando reduzir os custos em relação à lipase imobilizada comercial; (2) o desenvolvimento de um método de cromatografia em camada delgada capaz de quantificar as caprilinas formadas; (3) o estudo da reação de esterificação para a obtenção de caprilinas em regime descontínuo e descontínuo-alimentado. A resina DOWEX® 1X2-400 foi a que apresentou a melhor eficiência de imobilização (EI) da Palatase®. Sua atividade lipolítica foi apenas 12,7% inferior à versão imobilizada comercial da lipase, a Lipozyme®RM IM, indicando a viabilidade de substituição desta última. O desenvolvimento de método de cromatografia em camada delgada resultou em relações lineares para a detecção e quantificação de ácido caprílico, monocaprilina, dicaprilina e tricaprilina com R2 e R2pred de 99,3%/98,73%; 98,9%/97,6%; 99,8%/99,8% e 99,7%/99,5%, respectivamente, e com valor-p de 0,000. As reações em regime descontínuo foram realizadas seguindo planejamento fatorial completo, no qual foram variadas a temperatura (30 ou 70°C), a proporção molar entre os reagentes (1:1 ou 3:1 - ácido caprílico : glicerol) e o tempo reacional (6 ou 10h). A análise de regressão do rendimento reacional expresso em porcentagem de consumo de ácido caprílico indicou que, nestas condições, os fatores relevantes para o resultado final são a temperatura e a proporção molar, bem como a interação entre eles. As melhores condições para a obtenção de mono e dicaprilinas foram, portanto, de 30ºC e razão molar de 1:1. O estudo da cinética da reação indica que 40,4% do ácido caprílico são consumidos na primeira hora e que o consumo máximo ocorre na terceira hora de reação. Houve uma queda drástica no rendimento reacional, quando a reação foi conduzida em regime descontínuo-alimentado, provavelmente devido à menor eficiência catalítica da lipase devido ao baixo teor de água na primeira hora de reação. / Caprylins are medium chain acylglycerols that find several applications in food, pharmaceutical and cosmetic fields. Direct esterification is an important route for the synthesis of caprylins, since glycerol is a byproduct of biodiesel production and tricaprylin, the starting material of glycerolysis and hydrolysis, is not a naturally abundant compound. The glycerides synthesis can be catalyzed by either chemical or enzymatic routes, the latter being superior in several aspects. The biggest issues regarding the use of enzymes are their high cost and difficult recovery. Using immobilized enzymes can minimize these concerns. Most of scientific studies concerning the obtainment of these compounds address to long chain acyglycerols. Thus, the improvement of lipase catalyzed esterification of glycerol and caprylic acid is necessary, by understanding the reaction kinetics and the role of reaction parameters. A simple and effective method of separation, identification and quantification of caprylins is also a relevant contributing factor for easier and faster researches. Therefore, this work consisted of three main steps: (1) immobilization of lipases on anion exchange resins in order to produce a cheaper alternative to imported commercial immobilized lipases; (2) development of a thin layer chromatographic method capable of quantifying caprylins; (3) studying the obtainment of caprylins by direct esterification in batch and fed batch systems. DOWEX® 1X2-400 resin presented the best immobilization efficiency (IE) results. Additionally, its lipolytic activity was only 12.7% lower than the commercial Lipozyme® RM IM, suggesting that Lipozyme\'s substitution is feasible. Regarding the developing of a thin layer chromatography method, linear correlations were obtained for detection and quantification of caprylic acid, monocaprylin, dicaprylin and tricaprylin, with a R2 and R2pred of 99,3%/98,73%; 98,9%/97,6%; 99,8%/99,8% e 99,7%/99,5%, respectively, with a p-value of 0,000. The batch reactions were carried out according to a complete factorial design, in which the temperature (30 or 70ºC), the molar ratio between the reagents (1:1 or 3:1) and reaction time (6h or 10h) were varied. The regression analysis of reactional yield expressed in terms of percentage of caprylic acid consumption indicated that only the temperature, molar-ratio and their interaction were important for reaction yields, within the studied conditions. The best conditions related to the obtainment of mono and dicaprylins were 30ºC and molar ratio of 1:1. The reaction kinetics indicates that 40.4% of the caprylic acid is consumed within one hour of reaction and that the maximal consumption was achieved in the third hour. A drastic drop in reaction yield was observed when the fed batch mode was adopted, probably due a lower catalytic efficiency presented by lipase as a result of the lower water content in the first hour of reaction.

The direct sampling of analytes from surfaces under atmospheric conditions followed by mass spectrometric analysis is an ever expanding area of scientific research. Atmospheric pressure surface sampling and ionization techniques for mass spectrometry (MS) offer the ability to interrogate samples that could not be studied under vacuum conditions required of more traditional MS surface analysis techniques. The geometry and nature of materials or surfaces that can be analyzed has been greatly expanded as a result. This dissertation characterizes and shows applications of liquid microjunction surface sampling probe (LMJ-SSP) electrospray ionization systems. The presented work compares traditional analytical work flows with novel analytical workflows utilizing LMJ-SSP-MS technology. The increase of throughput and/or chemical information without the sacrifice of analytical figures of merit is shown and discussed. The readout of analytical surfaces; surfaces where analyte has ended up on a surface in a traditional work flow and not just placed there, constitutes the focus of what is presented in the preceding work. Finally the prospects for spatial liquid chromatography-mass spectrometry (LC-MS) as a powerful analytical technology „in wait‟ is discussed and supported by the presented data.

Amongst the polar organic compounds occurring in unrefined and refined crude oils and the associated polluted production waters, complex mixtures of acids, known historically as naphthenic acids (NAs), have achieved prominence. This is particularly because NAs have been designated a toxicant class of concern in the oil sands process-affected water (OSPW) that has accumulated in vast quantities following exploitation of the oil sands of Northern Alberta, Canada in recent years. However, though there have been calls for NAs to be added to pollutant inventories, at the initiation of the current study, little knowledge existed of the exact composition of refined or unrefined NAs. The overall aim of the current study was therefore to identify individual NAs in refined (commercial) and unrefined (e.g. oil sands process-derived) complex mixtures of acids and then to assess the toxicity of any identified NAs. Individual NAs were tentatively identified by interpretation of the electron ionisation mass spectra of methyl ester derivatives, following comprehensive multidimensional gas chromatography-mass spectrometry (GCxGC-MS). Reference acids were then either purchased, or more commonly, where they were not commercially-available, synthesised, mainly by micro-hydrogenation methods, for co-chromatography and comparison of mass spectra of methyl esters with those of unknowns. The synthetic NAs, purified to >97% were then subjected to toxicological assessments using the Microtox™ assay. In all, 34 compounds were obtained pure enough for testing. Microtox results revealed that the toxicity endpoint (50% Inhibition Concentration, IC50) was between 0.004 and 0.7 mM. Exponential and other correlations were noted between carbon number and toxicity in several of the structural groups of acids assayed, which may be beneficial for predictions of toxicity of non-synthesised acids. Although n-hexanoic acid (IC50 0.7 mM) had the lowest toxicity, adamantane-type acids were the least toxic as a group overall. Conversely, the decahydronaphthalene (decalin)-type acids had the largest range of toxicities (IC50 0.004 to 0.3 mM) and the most toxic acid assayed was 3-decalin-1-yl-propanoic acid. According to USEPA guidelines many individual acids can be said to show low to medium toxicity. Since the acids in commercial and unrefined NAs occur in complex mixtures, an attempt was also made to assess mixture toxicity. Mixtures of individual structural groups of acids (e.g. acyclic isoprenoid acids, n-acids) and a mixture of all 34 acids were assessed. Apart from the adamantane sub-group of acids, all of the mixtures showed toxicities lower than the sum of the parts when calculated using equations for Concentration Addition and Model Deviation Ratios (simply the predicted IC50/Observed IC50). A hypothesis that achievement of a critical micelle concentration is required to produce toxicity was proposed to explain the lower than expected results. Some of the mass spectra of NA present in the commercial and unrefined mixtures were inconsistent with those of any of the alicyclic acids synthesised or purchased. These were hypothesised to be aromatic acids. Fractionation experiments of the NA mixtures using silver ion thin layer chromatography and solid phase extraction (Ag+TLC and Ag+SPE) were carried out in order to provide further evidence for aromatic acids. Ag+TLC allowed separation of a methylated NA mixture from OSPW into three distinct fractions; Ag+SPE resulted in eleven fractions, through the use of a wider range of solvents and differential solvent ratios. Analysis of the fractions by GC-MS revealed that each fraction was largely still made up of unresolved acids (as esters), although one or two fractions revealed some resolved acids. Use of averaged mass spectra and mass chromatography on each fraction revealed further resolved chromatographic peaks and associated interpretable mass spectra. Each of eight of the eleven sub-fractions were examined by GC-MS, in some cases by GCxGC-MS, and all by infrared spectroscopy, ultraviolet visible spectrophotometry and elemental analysis. A number of structures were proposed for the aromatic acids, including those with sulphur-containing moieties. It was noted that far from being minor components, aromatic acids comprised ca.25-40% of the OSPW acid extracts.

Made available in DSpace on 2015-03-26T13:47:12Z (GMT). No. of bitstreams: 1 texto completo.pdf: 765049 bytes, checksum: dcb0cc188aa67726f24e856817503e00 (MD5) Previous issue date: 2013-02-22 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The aim of this study was to evaluate the fertilizing capacity, acrosomal integrity, and qualify and quantify by chromatographic techniques the incorporation of cholesterol to the sperm membrane by cyclodextrin in different diluents on cryopreservation of goat sperm. Four males, Saanen (2) and Parda Alpine (2) breeds were used. It was performed a completely randomized design, with each semen sample divided into the following treatments: TG - Tris-glycerol diluent; TGCCC - Cyclodextrin-cholesterol complex (CCC) + Tris-glycerol diluent; TG15CCC - CCC diluted in isosmotic solution (saline) to semen with 15 minutes of incubation before addition of the Tris-glycerol diluent; EE - egg yolk + ethylene glycol diluent; EECCC - CCC + egg yolk + ethylene glycol diluent; EE15CCC - CCC diluted in isosmotic solution (saline) to semen with 15 minutes of incubation before addition of the egg yolk + ethylene glycol diluent. Fresh semen, after its examination, was submitted to the cryopreservation process and stored in a cryogenic cylinder for 10 days. After thawing, the following analysis were performed: acrosomal integrity, sperm fertilizing capacity through perivitelline membrane of hen egg yolk binding test (MPEY) and analysis of sperm motility and vigor. Besides, techniques of gas and thin layer chromatography were conducted to evaluate the incorporation of cholesterol to the sperm membrane. Data were submitted to Lilliefors and Cochran and Bartlett tests to verify normality and homogeneity of variances, respectively. The characteristics that met the assumptions of these tests were submitted to ANOVA and means were compared by Duncan s test at 5% of probability. When data did not meet the assumptions of normality and homogeneity of variances, the means were compared by Kruskal-Wallis test. Pearson s correlation coefficient was calculated in all features.Addition of cyclodextrin-cholesterol complex did not increase binding of sperm to MPEY (P > 0.05). The EE15CCC treatment was superior to EECCC treatment in maintaining the acrosomal integrity but did not differ from control (EE; P > 0.05). In Tris-glycerol diluent, the values observed in the control treatment were higher (P < 0.05) than values of the other treatments (TGCCC and TG15CCC) in maintaining the acrosomal integrity. There was a negative correlation between the binding assay and acrosomal integrity (r = -0.25) and positive correlation between the binding assay and sperm motility (r = 0.20). The sperm motility and acrosomal integrity were negatively correlated (r = -0.26). In quantitative and qualitative evaluation of cholesterol by chromatographic techniques (gas and thin layer) there was no difference between the samples of semen in different treatments (P > 0.05). Both techniques showed no incorporation of cholesterol to spermatozoa. It was concluding that addition of cholesterol-cyclodextrin complex to the medium did not improve the physical aspects of goat semen, or the sperm binding capacity. Pre-incubation of semen with CCC for 15 minutes before addition of ethylene + egg yolk diluent (EE15CCC) did not enhance the integrity of the acrosome in relation to control (EE). The gas and thin layer chromatography showed, respectively, an efficient method for quantitatively and qualitatively determining the cholesterol present in the cryopreserved goat sperm. The concentration of 1 mg of cholesterol-cyclodextrin complex added to the goat semen was not effective for increasing the concentration of cholesterol in sperm. / O objetivo do presente estudo foi avaliar a capacidade fecundante, integridade acrossomal, além de qualificar e quantificar por técnicas cromatográficas a incorporação do colesterol à membrana plasmática do espermatozoide pela ciclodextrina em diferentes diluentes na criopreservação de espermatozoides caprinos. Foram utilizados quatro machos caprinos das raças Saanen (2) e Parda Alpina (2) seguindo um delineamento inteiramente casualizado, dividido nos seguintes tratamentos: TGcontrole negativo para o diluente a base de Tris-glicerol; TGCCC- Complexo ciclodextrina-colesterol (CCC) + diluente Tris glicerol; TG15CCC- CCC diluído em solução isosmótica ao sêmen (soro fisiológico) com 15 minutos de incubação antes da adição do diluente Tris glicerol; EE- controle negativo para o diluente a base de Gema de ovo + etilenoglicol; EECCC- CCC + diluente Gema de ovo + etilenoglicol; EE15CCC- CCC diluído em solução isosmótica ao sêmen (soro fisiológico) com 15 minutos de incubação antes da adição do diluente Gema de ovo + etilenoglicol. O sêmen fresco, após realização de sua análise física foi submetido ao processo de criopreservação e estocado em botijão de nitrogênio por 10 dias. Após o descongelamento realizou-se análise da integridade acrossomal, capacidade fecundante do espermatozoide por meio do teste de ligação à membrana perivitelina da gema do ovo de galinha (MPGV) e as análises de motilidade progressiva e vigor espermático. Além destes testes foi realizada a avaliação da incorporação do colesterol à membrana plasmática dos espermatozoides pelas técnicas de cromatografia gasosa e de camada delgada. Para verificação da normalidade e homogeneidade dos dados foi empregado, respectivamente, o teste de Lilliefors e Cochran e Bartlett. As características que atenderam as premissas destes testes foram submetidas à ANOVA e as médias foram comparadas pelo teste de Duncan com 5% de probabilidade de erro. Quando as distribuições não atenderam as premissas de normalidade e homogeneidade, as médias foram comparadas pelo teste de Kruskal-Wallis. Realizou-se a correlação simples de Pearson entre todas as características. A adição do complexo ciclodextrina-colesterol não aumentou a ligação dos espermatozoides a MPGV (P>0,05). O tratamento empregando o complexo ciclodextrina-colesterol (CCC) diluído em solução isosmótica ao sêmen com 15 minutos de incubação antes da adição do diluente a base gema de ovo + etilenoglicol (EE15CCC) foi superior aos valores médios do tratamento EECCC na manutenção da integridade acrossomal, porém não diferiu dos valores do tratamento controle para este diluente (EE; P >0,05). No diluente Tris-Glicerol, os valores observados no tratamento controle foi superior (P<0,05) aos valores médios dos demais tratamentos (TGCCC e TG15CCC) na manutenção da integridade acrossomal. Houve correlação negativa entre o teste de ligação e a integridade do acrossoma (r= -0,25) e positiva entre o teste de ligação e a motilidade espermática progressiva (r= 0,20). A motilidade espermática progressiva e a integridade do acrossoma apresentaram correlação negativa (r= -0,26). Na avaliação quantitativa e qualitativa do colesterol pelas técnicas cromatográficas (gasosa e camada delgada) não se verificou diferença entre as amostras do sêmen nos diferentes tratamentos (P>0,05). Ambas as técnicas demonstraram que não houve incorporação do colesterol aos espermatozoides. Conclui-se que o complexo ciclodextrina-colesterol no meio diluidor não melhorou os aspectos físicos do sêmen caprino pós-descongelamento e a capacidade de ligação à membrana perivitelina da gema do ovo de galinha. A pré-incubação do sêmen com o CCC por 15 minutos antes da adição do diluente a base de Etilenoglicol+ gema de ovo (EE15CCC) não proporcionou um aumento na integridade do acrossoma a ponto de diferir dos valores do tratamento controle (EE). A cromatografia gasosa e de camada delgada demonstraram, respectivamente, um eficiente método quantitativo e qualitativo para determinar o colesterol presente no espermatozoide caprino criopreservados. A concentração de 1 mg do complexo ciclodextrina-colesterol adicionada ao sêmen caprino não foi eficaz em aumentar a concentração de colesterol presente no espermatozoide.

As plantas medicinais são conhecidas há muitos séculos pelos homens, e ainda hoje são amplamente utilizadas no tratamento de enfermidades. A população acredita que é uma alternativa segura e de baixo custo em relação aos produtos farmacêuticos industrializados, e faz seu uso indiscriminado e sem acompanhamento médico. A automedicação e a falta de controle de qualidade das drogas vegetais (DVs) representam um sério risco à saúde de seus usuários. A diversidade de espécies conhecidas como plantas medicinais, e o uso de nomes populares regionais podem ocasionar erros de identificação taxonômica ou mesmo adulteração do produto. Outro fator que pode alterar a qualidade das DVs é a contaminação de origem química ou biológica, proveniente das diversas fases de seu preparo. Com o uso disseminado de DVs e fitoterápicos, cresceu a preocupação com os efeitos adversos e a interação com medicamentos convencionais. Esse panorama evidencia a interdisciplinaridade na área de plantas medicinais. Assim, o projeto, do qual derivou este de mestrado, é interdisciplinar, com a participação de pesquisadores da etnofarmacologia, microbiologia, farmacovigilância e farmacognosia. O objetivo específico desta dissertação de mestrado é avaliar a qualidade das DVs com possíveis ações psicoativas, adquiridas em barracas de rua na cidade de Diadema. As amostras selecionadas e adquiridas pelo grupo da etnofarmacologia foram confrontadas a monografias farmacopêicas, literatura especializada e/ou amostra autêntica, avaliando-se os caracteres macroscópicos, microscópicos, perfis cromatográficos e pureza das DVs. Cortes histológicos foram preparados conforme as técnicas usuais, cromatografias em camada delgada foram realizadas de acordo com literatura e fotografias documentam as análises. De um total de 35 lotes analisados, 88,6% confirmaram sua autenticidade, e apenas 57,1% estavam em conformidade com os valores máximos permitidos para materiais estranhos. Também ficou evidente a baixa qualidade de armazenamento do material e problemas com embalagens e rótulos, em total desacordo com a legislação vigente. Concluiu-se então que as DVs adquiridas demandam atenção e melhorias quanto à sua qualidade, fato que demonstra a necessidade de maior orientação e conscientização dos vendedores quanto aos cuidados para sua comercialização à população. / Medicinal plants have been known for centuries by humanity and are still widely used for the treatment of illnesses. People believe that it is a safer and low cost alternative in relation to manufactured pharmaceutical products and makes indiscriminate use of them, without medical supervision. Self-medication and the lack of quality control of herbal drugs (HDs) represent a serious health risk to users. The diversity of species known as medicinal plants and the use of popular regional names can cause errors in taxonomic identification or even product adulteration. Another factor that may alter the quality of HDs is contamination from biological or chemical origin, from various stages of their preparation. With the widespread use of herbal remedies and HDs, the concern about adverse effects and interactions with conventional medicines has increased. This scenario highlights the interdisciplinarity of the medicinal plants field. Thus, the project, which this master`s program was derived from, is interdisciplinary, involving researchers from ethnopharmacology, microbiology, pharmacovigilance and pharmacognosy. The specific aim of this master\'s project is to evaluate the quality of HDs with possible psychoactive action, acquired in street stalls in the city of Diadema. The samples selected and acquired by ethnopharmacology were confronted with pharmacopoeial monographs, literature and/or authentic samples and the macroscopic and microscopic characters, chromatographic profiles and purity of HDs were evaluated. Histological sections were prepared according to the usual techniques, thin layer chromatographys were performed according to the literature, and photographs document the analysis. From a total of 35 lots, 88.6% confirmed their authenticity, and only 57.1% were in compliance with the maximum limit for the amount of foreign material. The poor quality of material storage and problems with packaging and labels, in total disagreement with the law, was also made evident. We concluded that the acquired HDs require caution and improvement of their quality, which demonstrates the need for more guidance and awareness of vendors about the care involved in the commercialization of HDs to the population.