CAPES; Fundação Araucária / A espécie Moringa oleifera (Moringaceae) é uma planta que possui ampla aplicação industrial, alto valor nutricional e que, além disso, também exibe diversas atividades biológicas. Utilizadas na medicina popular, as folhas de M. oleifera já demonstraram possuir grande variedade de moléculas bioativas, inclusive compostos fenólicos, os quais são, possivelmente, os responsáveis pelo potencial antioxidante desta parte da planta. Apesar do crescente interesse sobre a espécie e, especificamente, sobre o seu potencial fitoquímico, são escassos os trabalhos que relatam o isolamento e a identificação dos compostos bioativos presentes nas suas folhas, principalmente em exemplares cultivados no Brasil. Sendo assim, os objetivos deste trabalho foram comparar dois métodos de extração de compostos bioativos e, na sequência, isolar bioguiadamente compostos com atividade antioxidante das folhas de M. oleifera coletadas no município de Itajaí (Santa Catarina, Brasil). O monitoramento bioguiado foi realizado com ensaios in vitro de determinação da atividade antioxidante: capacidade de redução do reagente Folin-Ciocalteau, FRAP, sequestro dos radicais DPPH e ABTS, além do ORAC. A técnica de CLAE-DAD foi utilizada para a caracterização química e acompanhamento das etapas do isolamento. A principal diferença prática entre os métodos de extração avaliados foi o preparo de um extrato hidroalcoólico inicial, no processo de extração 1. A partir dos resultados de determinação da atividade antioxidante, interpretados com o auxílio de ferramentas estatísticas (teste de Tukey e teste t pareado), foi possível perceber que o potencial das folhas de M. oleifera sofreu variações em função da forma de extração e dos solventes utilizados. Em geral, as frações produzidas a partir do processo de extração 1 apresentaram maior atividade antioxidante e perfis cromatográficos com sinais mais intensos. Com base nestes resultados, a fração obtida com acetato de etila, no processo de extração 1, foi selecionada para dar sequência ao isolamento bioguiado. A purificação desta fração em coluna aberta preenchida com sílica gel gerou 61 subfrações, as quais, após análise de CCD, foram agrupadas em 18. A avaliação da atividade antioxidante das subfrações agrupadas mostrou que cinco apresentavam grande potencial. Contudo, em função do rendimento, apenas três puderam dar sequência ao isolamento. Nesta etapa, uma análise adicional foi realizada: a determinação da atividade antioxidante por CLAE on-line com o ABTS•+, que permitiu definir quais dos compostos presentes nas três subfrações possuíam maior potencial e, por isso, seriam isolados. Desta forma, cinco compostos foram isolados pela técnica de CLAE semipreparativa, sendo que dois foram testados frente ao ensaio de sequestro do DPPH•. Os valores de EC50 obtidos, 30,34 e 38,72 μg/mL, estão próximos aos encontrados na literatura para substâncias isoladas de outras matrizes naturais. A técnica de RMN permitiu identificar um flavonol glicosilado. Os resultados deste trabalho mostraram que as folhas de M. oleifera coletadas em Itajaí são fonte de compostos fenólicos com potencial antioxidante e, por isso, promissoras para aplicação nas indústrias de cosméticos, alimentos e farmacêutica. / Moringa oleifera (Moringaceae) is a plant that has wide industrial application, high nutritional value and, also, exhibits several biological activities. Used in folk medicine, M. oleifera leaves have already been shown to possess a wide variety of bioactive molecules, including phenolic componds, which are possibly responsible for the antioxidant potential of this part of the plant. Despite the growing interest in this species and, specifically, in its phytochemical potential, there are few studies about the isolation and identification of bioactive compounds present in M. oleifera leaves, especially in specimens grown in Brazil. Therefore, the aims of this work were to compare two methods for extracting bioactive componds and, than, to isolate compounds with antioxidant activity of M. oleifera leaves collected in Itajaí (Santa Catarina, Brazil) by a bioguided study. The bioguided monitoring was carried out with in vitro assays to determine the antioxidant activity: Folin-Ciocalteau reagent reduction capacity, FRAP assay, DPPH and ABTS radical scavenging methods and, also, the ORAC assay. HPLC-DAD technique was used for chemical characterization and monitoring of the isolation stages. The main practical difference between the evaluated extraction methods was the preparation of an initial hydroalcoholic extract, in the extraction process 1. From the results of the antioxidant activity determination, interpreted with the aid of statistical tools (Tukey’s test and paired t-test), it was possible to see that the potential of M. oleifera leaves varied depending on the extraction form and on the solvents used. In general, the fractions prepared from the extraction process 1 showed higher antioxidant activity and chromatographic profiles with more intense signals. Based on these results, the fraction obtained with ethyl acetate, in the extraction process 1, was selected for the bioguided isolation. The purification of this fraction on an open column of silica gel generated 61 subfractions, which, after TLC analysis, were grouped in 18. The evaluation of the antioxidant activity of grouped subfractions showed that five of them presented great potential. However, depending on the yield, only three could follow the isolation. In this step, an additional analysis was performed: the determination of the antioxidant activity by an on-line HPLC method with the ABTS•+. This technique allowed defining which of the compounds presented in each subfraction had higher potential and, therefore, would be isolated. In this way, five compounds were isolated by semipreparative HPLC, two of them were tested by the DPPH• scavenging assay. The obtained EC50 values, 30.34 and 38.72 μg/mL, are close to those found in literature for substances isolated from other natural matrices. The NMR technique allowed identifying a flavonol glucoside. The results of this work showed that M. oleifera leaves collected in Itajaí are source of phenolic compounds with antioxidant potential and, therefore, are promising for the application in cosmetics, food and pharmaceutical industries.

CAPES; CNPq; Fundação Araucária / A qualidade das águas é atualmente um assunto que tem atraído a atenção de pessoas de todas as áreas, especialmente quanto à contaminação dos ambientes aquáticos pelos chamados poluentes emergentes. Esses contaminantes constituem compostos orgânicos ou inorgânicos, sintéticos ou naturais, contidos nos bens de consumo utilizados por toda população e que tem como destino final os corpos aquáticos. O objetivo deste trabalho foi verificar a presença de produtos farmacêuticos e de higiene pessoal (cafeína, ácido acetilsalicílico, ácido salicílico, cetoprofeno, naproxeno, genfibrozila, fenofibrato, estradiol, etinilestradiol, estrona, 4-metilbenzilideno cânfora, metoxicinamato de octila e octacrileno) no rio Iguaçu, da nascente até sua foz, e em seus principais afluentes na Região Metropolitana de Curitiba. A determinação dos contaminantes emergentes foi realizada utilizando a extração em fase sólida e cromatografia em fase líquida com detector de arranjo de diodos e cromatografia em fase gasosa acoplada a espectrometria de massas. Realizou-se validação da metodologia empregada de acordo com os padrões estabelecidos pela Agência Nacional de Vigilância Sanitária. O método para extração e quantificação dos 13 compostos mostrou-se eficiente para a determinação de 10 desses analitos nas amostras de água. A cafeína foi o composto mais presente nas amostras; o ácido acetilsalicílico foi o produto farmacêutico encontrado em maiores concentrações; o estradiol, o hormônio sexual feminino detectado com maior frequência e o metoxicinamato de octila, o produto de higiene pessoal mais detectado. Os resultados obtidos neste trabalho indicam grande influência antrópica na bacia do Alto Iguaçu, principalmente devido à presença de esgotos domésticos. Observou-se também que as estações de tratamento de esgoto contribuem com a entrada de nutrientes e contaminantes emergentes que não são eficientemente removidos nos tratamentos utilizados. A falta de saneamento básico impacta negativamente na qualidade das águas do rio Iguaçu, que encontra-se altamente degradado devido a influência dos afluentes da Região Metropolitana de Curitiba. / Water quality is a currently subject that has attracted the attention of people from all areas, especially the contamination of aquatic environments by so-called emerging pollutants. These contaminants are organic or inorganic, natural or synthetic compounds, contained in products used throughout the population and that reacht water bodies. With this work, we intended to verify the presence of pharmaceuticals and personal care products (caffeine, acetylsalicylic acid, salicylic acid, ketoprofen, naproxen, gemfibrozil, fenofibrate, estradiol, ethinylestradiol, estrone, 4– methylbenzylidencamphor, octylmethoxycinnamate and octacrilene) in Iguaçu River, from source to mouth, and its major tributaries in the Metropolitan Region of Curitiba. The determination of emerging contaminants was done using solid phase extraction and liquid chromatography with diode array detector and gas chromatography tanden mass spectrometry. Validation of the methodology was conducted in accordance with standards established by the Agência Nacional de Vigilância Sanitária. The method for extraction and quantitation of the 13 compounds proved to be efficient for the determination of 10 of these analytes in water samples. Caffeine was the main compound present in the samples; acetylsalicylic acid was the pharmaceutical product founded in the highest concentractions; estradiol, the female sexual hormone more frequently detected and octylmethoxycinnamate, the personal care product longer detected. The results of this study indicate intense anthropogenic influence in the Upper Iguaçu basin, mainly due to the presence of domestic sewage. It was also observed that the wastewater treatment plants contribute to the entry of nutrients and emerging contaminants that are not efficiently removed in the treatments used. The lack of sanitation has a negative impact on water quality of the Iguaçu river, which is highly degraded due to the influence of the tributaries of the Metropolitan Region of Curitiba.

Este estudo utilizou os princípios da glacioquímica para determinar e analisar as variações nas concentrações aniônicas de um testemunho de neve e firn obtido pela perfuração no manto de gelo da antártica ocidental no verão austral de 2004/05. O testemunho IC-6 (81°03'S, 79°51'W), de 34,65 m de profundidade, obtido a 750 m de altitude, foi subamostrado em sala limpa (CLASE 100), usando um sistema de derretimento contínuo desenvolvido pela equipe do Climate Change Institute (CCI) da Universidade do Maine (EUA). Esse processo gerou 1.368 amostras para análises por cromatografia iônica, cerca de 58 amostras por metro, permitindo detalhamento sazonal da variabilidade das concentrações dos íons majoritários. O testemunho representa 66 ± 3 anos de dados ambientais, segundo a datação baseada na variação sazonal dos íons Cl-, Na+, Mg+2 e SO4-2. O testemunho de 23,61 m em equivalente d’água, corrigido para variações em densidade, representa uma acumulação liquida média anual de 0,36 m (em equivalente d’água). Assim, a camada ao fundo foi formada no ano de 1938 (± 3 anos). As concentrações iônicas médias medidas no IC-6, são: [(Na+= 66,92 ± 2,32 μg L-1), (K+= 3,31 ± 0,18 μg L-1); (Mg+2= 10,07 ± 0,25 μg L-1); (Ca+2 = 16,93 ± 0,38 μg L-1); (Cl- = 155,74 ± 4,40 μg L-1); (NO3- = 56,01 ± 0,80 μg L-1); (SO4 2 = 55,65 ± 1,36 μg L-1); e (CH3SO3 (MS) = 14,11 ± 1,19 μg L-1)]. As maiores concentrações de Na+, Cl-, e Mg+2 foram interpretadas como picos de invernos, associadas diretamente ao aerossol dos mares circundantes em respostas, provavelmente, a advecção mais intensa de massas de ar (marinho) sobre as plataformas de gelo, e portanto são também traçadores marinhos. Já o perfil (série) de sulfato está em antifase, em relação às variações nas espécies Na+, Cl- e Mg+2. De origem predominantemente marinha, o sulfato total apresentou maiores concentrações durante a primavera e verão (períodos de maior atividade biológica nos mares circumpolares), possivelmente marcando a variação sazonal da atividade biológica na região. Embora em alguns intervalos essa ―antifase‖ não fique tão clara, é o que ocorre na maior parte do testemunho IC-6, condição que auxiliou na interpretação da variação sazonal observada principalmente na série do cloro. O perfil de excesso de sulfato apresenta perfil similar ao de sulfatos total, com picos concomitantes. Além da forte correlação com o íon SO4-2, também é observada uma correlação fraca a moderada com o íon nitrato. Picos concomitantes deste íon com o excesso de sulfato representam eventos episódicos como é o caso das erupções vulcânicas de grande magnitude. A variabilidade da concentração de nitrato não esta associada ao aerossol marinho, como aponta a falta de correlação entre esse ânion e o Cl-, Na+ e Mg2+. Porém, série de nitrato apresenta muitos períodos bem marcados e correlacionados com as concentrações de excesso de sulfato, devendo representar a ocorrência de eventos episódicos, como erupções vulcânicas. Entretanto, a análise de íons maiores nesse estudo não possibilitou a identificação de eventos específicos, será necessário o uso de técnicas complementares para determinação de elementos traços. Sugere-se que o nitrato seria transportado e depositado por massas de ar provenientes da estratosfera ou da alta troposfera e que grandes concentrações dessa espécie poderiam estar associadas ao registro de ocorrências de eventos vulcânicos. Essa característica parece ser coerente com os picos correlacionáveis nos perfis (séries) de nitrato e sulfatos. Além da variação sazonal (observada principalmente no perfil de cloro), foram identificados outros padrões recorrentes no tempo (ciclos), principalmente nas séries de dos íons Na+, Cl- e Mg2+ (origem marinha) e NO3-. O principal ciclo identificado, de aproximadamente 17,3 anos, necessita melhor investigação. A secundária, em torno de 10 anos, estaria associada ao ciclo solar (de 10,7 anos). Também são observados ciclos com períodos entre 2 a 5 anos, que poderiam estar associados ao fenômeno ENOS (El Niño - Oscilação Sul). Ao comparar as concentrações médias do IC-6 com de outros sítios no interior da Antártica, observa-se uma abrupta redução ao atravessar as montanhas Transantárticas em direção ao Polo Sul geográfico. Sugere-se que cordilheira esteja barrando o transporte dos aerossóis marinhos para o interior do continente devido a um efeito orográfico sobre a precipitação. / This study employed glaciochemical principles to determine and analyze the variation of anionic concentrations of a firn and snow core obtained from the Western Antarctic Ice Sheet, in the summer of 2004/05. The IC-6 core (81°03'S, 79°51'W), reaching 34.65 m in depth, was extracted at 750 m above sea level. This core was subsampled in a Class 100 clean room, employing a discrete continuous melting system developed by the team at the Climate Change Institute (CCI), University of Maine, USA. This process produced 1,368 samples for ionic chromatographic analyses, approximately 58 samples per meter, permitting a seasonal-scale resolution of the main ion concentrations and variabilities. This core represents 66 ± 3 years of environmental data, according to Cl-, Na+, Mg+2 e SO4-2 ion seasonal variations. The 23.61 m core, in water equivalent, corrected for the density variation, represents an annual net accumulation average rate of 0.36. The deepest layer was deposited in 1938 (± 3 ). Core mean ionic concentrations are: [(Na+= 66,92 ± 2,32 μg L-1); (K+= 3,31 ± 0,18 μg L-1); (Mg+2= 10,07 ± 0,25 μg L-1); (Ca+2 = 16,93 ± 0,38 μg L-1); (Cl- = 155,74 ± 4,40 μg L-1); (NO3- = 56,01 ± 0,80 μg L-1); (SO4 2 = 55,65 ± 1,36 μg L-1); and (CH3SO3 (MS) = 14,11 ± 1,19 μg L-1)]. The largest concentrations of Na+, Cl-, e Mg+2 were interpreted as winter peaks, directly associated with the aerosols from the surrounding seas, probably, in response to the intensification of marine air mass advection on the ice shelves, and, thus, also being marine tracers. The sulphate profile (series) presents an antiphase, with relation to Na+, Cl- e Mg+2 species variations. Predominantly of marine origin, total sulphates presented greater concentrations during Spring and Summer (periods of greater biologic activity in the Southern Ocean), possibly marking the seasonal variation of biologic activity in the region. Although in some intervals of this ―antiphase‖ are not clearly evident, they are consistent throughout most of the IC-6 core, assisting with the interpretation of the observed seasonal variations, particularly when related to chlorine data series. The sulphate excess profile is similar to total sulphate profile, showing concomitant spikes. Besides the strong correlation to tSO4-2 ion, a weak to moderate correlation was observed for nitrate ions. Coinciding peaks for this ion with excess sulphate may represent episodic events, such as presented by volcanic events of great magnitude. The nitrate concentration variability is not associated to marine aerosols, as shown by the lack of correlation between this anion and Cl-, Na+ e Mg2+. The nitrate series presents many well marked periods and seem to be correlated to excess sulphate concentrations, possibly representing the occurrence of episodic events, such as volcanic eruptions. Even so, the major ions analyses proposed by this work did not make the identification of such episodic events clear. Such events need to be addressed with complementary techniques to determine the specific trace elements. These results suggest that nitrate is transported and deposited by stratospheric or high tropospheric air masses, and that great concentrations of this species could be associated to the recorded volcanic events. This characteristic appears to be coherent with the spikes in the nitrate and sulphate profiles. Besides the seasonal variation (observed, principally, in the chlorine profile), other time cycle/patterns were identified, mainly those related to Na+, Cl- e Mg2+ ion series (of marine origin) and NO3-. The main identified cycle, approximately 17.3 years, ensues to be better investigated. A second cycle, presenting a 10 year period, is possibly associated to the solar cycle (10.7 years). Shorter cycles of 2 and 5 year periods could possibly be related to the ENSO phenomenon. On comparing average concentrations of the IC-6 core with other sites, farther within the Antarctic continent, an abrupt reduction was observed, from the Trans-Antarctic mountains to the Geographic South Pole, suggesting that this mountain range could be a barrier for marine aerosol transport to the interior of the continent, due to an orographic effect on the precipitation.

To investigate potential sources of novel grain protectors, this study evaluated, firstly, the bioactivity of ethanolic extracts (66) prepared from 29 species belonging to 11 different genera of Neotropical Annonaceae against the maize weevil Sitophilus zeamais (Coleoptera: Curculionidae). A screening assay demonstrated that the most pronounced effects (acute and chronic) on S. zeamais were caused by extracts from the Annona montana, A. mucosa, A. muricata and A. sylvatica seeds, and, to a lesser extent, by extracts prepared from leaves of A. montana, A. mucosa, A. muricata, and Duguetia lanceolata. However, the most active extracts (from seeds) did not affect fungal growth and aflatoxin production of Aspergillus flavus (Ascomycota: Trichocomaceae). Using the maize weevil as bioindicator, bioguided fractionations were then conducted in order to isolate, purify and characterize the possible active compound(s) from the most interesting extracts. By means of different chromatographic procedures, nine compounds (five acetogenins, three steroids, and one aromatic compound) were isolated. The acetogenins rolliniastatin-1 and ACG4 (structural determination in progress) and the aromatic compound 2,4,5- trimethoxystyrene as well as the steroids campesterol, stigmasterol, and sitosterol [tested in mixture (8.44 + 12.37 + 79.19%, respectively)] showed promising grain protective properties. Furthermore, the obtained results indicate that compounds from different chemical natures have a synergistic effect on the overall biological activity of the crude extracts. In a second study, the acute and chronic toxicity of selected ethanolic seed extracts from Annona species (A. montana, A. mucosa, A. muricata, and A. sylvatica) and an acetogenin-based commercial bioinsecticide (Anosom® 1EC) were investigated against the cabbage looper Trichoplusia ni (Lepidoptera: Noctuidae) and the green peach aphid Myzus persicae (Hemiptera: Aphididae). In the laboratory, extracts of A. mucosa and A. sylvatica as well as Anosom® were especially active through oral and topical administration. A greenhouse trial showed that a formulated A. mucosa extract and Anosom® were highly effective (>98% mortality) against third instar of T. ni larvae, and comparable to a pyrethrin-based commercial insecticide (Insect Spray®) used as a positive control. Similar to results with T. ni, A. mucosa extract showed the greatest aphicidal either in laboratory or greenhouse bioassays. In a third study, the acaricidal activity [against the citrus red mite Panonychus citri (Acari: Tetranychidae)] of the ethanolic extract from A. mucosa seeds (most active) was investigated. In laboratory tests, it exhibited levels of activity superior to commercial acaricides/insecticides of natural origin [Anosom® 1EC (annonin), Derisom® 2EC (karanjin), and Azamax® 1.2EC (azadirachtin + 3- tigloylazadirachtol)] and similar to a synthetic acaricide [Envidor® 24 SC (spirodiclofen)]. Finally, the compatibility of A. mucosa seed extract with three entomopathogenic fungi species (Beauveria bassiana, Isaria fumosorosea and Metarhizium anisopliae) was assessed. In overall, it was compatible with the three entomopathogenic fungi species when tested at recommended concentrations for target pest species control. Therefore, this study argues for the use of derivatives from Neotropical Annonaceae as a useful component in the framework of integrated pest management (IPM) programs. / Visando investigar potenciais fontes de novos protetores de grãos, este estudo avaliou, primeiramente, a bioatividade de extratos etanólicos (66) obtidos de 29 espécies pertencentes a 11 diferentes gêneros de anonáceas neotropicais sobre o gorgulho-do-milho Sitophilus zeamais (Coleoptera: Curculionidae). A triagem inicial demonstrou que os efeitos (agudos e crônicos) mais pronunciados sobre S. zeamais foram causados pelos extratos de sementes de Annona montana, de A. mucosa, de A. muricata e de A. sylvatica, seguidos pelos extratos de folhas de A. montana, de A. mucosa, de A. muricata e de Duguetia lanceolata. No entanto, os extratos mais ativos (sementes) não afetaram o crescimento vegetativo e a produção de aflatoxinas de um isolado de Aspergillus flavus (Ascomycota: Trichocomaceae). Fracionamentos biomonitorados foram então realizados a fim de isolar, purificar e caracterizar o(s) composto(s) ativo(s) majoritário(s) dos extratos mais promissores, utilizando-se, para isso, o gorgulho-do-milho como bioindicador. Por meio de diferentes procedimentos cromatográficos, foram isolados nove compostos: cinco acetogeninas, três esteroides e um composto aromático. As acetogeninas roliniastatina-1 e ACG4 (determinação estrutural em andamento), o composto aromático 2,4,5-trimetoxiestireno e os esteroides campesterol, estigmasterol e sitosterol [testados em mistura (8,44 + 12,37 + 79,19%, respectivamente)] mostraram promissoras propriedades protetoras de grãos. Em geral, os resultados obtidos indicaram que compostos de diferentes naturezas químicas têm efeito sinérgico sobre a atividade biológica dos extratos brutos. No segundo estudo, foi avaliada a toxicidade aguda e crônica dos extratos selecionados de sementes de Annona (A. montana, A. mucosa, A. muricata e A. sylvatica) e de um bioinseticida comercial à base de acetogeninas (Anosom® 1EC) sobre a lagarta-mede-palmo Trichoplusia ni (Lepidoptera: Noctuidae) e sobre o pulgão-verde Myzus persicae (Hemiptera: Aphididae). Em laboratório, os extratos de A. mucosa e de A. sylvatica e o bioinseticida Anosom® foram especialmente ativos através da administração oral e tópica. Em casa de vegetação, um extrato formulado de A. mucosa e Anosom® foram altamente eficazes contra larvas de terceiro ínstar de T. ni, com eficácia comparável ao de um inseticida comercial à base de piretrinas (Insect Spray®) utilizado como controle positivo. Similar aos resultados com T. ni, o extrato de A. mucosa apresentou a maior atividade aficida, tanto em bioensaios em laboratório quanto em casa de vegetação. No terceiro estudo, a atividade acaricida [sobre o ácaro-purpúreo-dos-citros Panonychus citri (Acari: Tetranychidae)] do extrato de sementes de A. mucosa (mais ativo) foi avaliada em bioensaios laboratoriais. O extrato de A. mucosa apresentou eficácia superior aos acaricidas/inseticidas comerciais de origem natural [Anosom® 1EC (anonina), Derisom® 2EC (karanjina) e Azamax® 1.2EC (azadiractina + 3-tigloilazadiractol)] e similar a um acaricida sintético [Envidor® 24SC (espirodiclofeno)]. Finalmente, foi avaliada a compatibilidade do extrato de sementes de A. mucosa com três espécies de fungos entomopatogênicos (Beauveria bassiana, Isaria fumosorosea e Metarhizium anisopliae). De modo geral, o extrato de A. mucosa foi compatível com as três espécies quando testado nas concentrações preconizadas para o controle das espécies-praga alvo. Assim, este estudo fornece importantes subsídios para o uso de derivados de anonáceas neotropicais como um componente útil para os programas de manejo integrado de pragas (MIP).

Tradicionalmente plantas medicinais são utilizadas pela população para a prevenção e tratamento da úlcera gástrica. A úlcera gástrica é uma patologia heterogênea de etiologia multifatorial, que acomete aproximadamente de 8 a 10% da população mundial. Entretanto, os tratamentos terapêuticos disponíveis são muito dispendiosos, possuem limitada eficácia e com vários efeitos colaterais. Extrato de folhas de Phyllanthus tenellus Roxb. é utilizado pela população para o tratamento de problemas gástricos. Esta espécie está incluída na Relação Nacional de Plantas com Interesse ao SUS (ANVISA). Com a finalidade de investigar a atividade antiúlcera foram ensaiados dois modelos de lesões gástricas em animais. No modelo de indução por etanol acidificado, após administração do extrato bruto (extrato hidroetanólico 70% liofilizado) de P. tenellus, verificou-se redução de 3.44% e 4.25% da área de lesão gástrica nas doses 200mg/kg e 400mg/kg, porém não houve diferença significativa entre os resultados. Já no ensaio, utilizando extrato hidroetanólico, fração acetato de etila e fração aquosa, nas doses de 200mg/kg, assim como no ensaio anterior, verificou-se redução da área de lesão gástrica de 0.7%, 1.1% e 0.7% respectivamente, porém não houve diferença significativa entre controle e tratado. No teste, utilizando a fração acetato de etila das folhas de P. tenellus (Phyllanthaceae), houve redução de 9,46 e 22,80% das lesões gástricas nas doses de 100mg/kg e 400mg/kg, porém não houve diferença significativa. No modelo de úlcera gástrica induzida por etanol acidificado em animais pré-tratados com etoricoxibe, indometacina e L-NAME, verificou-se uma tendência de redução da lesão gástrica, porém não houve diferença significativa. Já no modelo de úlcera gástrica induzida por etanol acidificado em animais pré- tratados com NEM, houve diminuição das lesões gástricas em 90% no grupo Salina+FA (p<0,05), comparando-o com o grupo NEM+FA. Em modelo de úlcera subaguda induzida por ácido acético, após 7 dias de tratamento com o extrato hidroetanólico de P. tenellus nas doses de 100, 200 e 400 mg/kg, houve redução significativa nas lesões gástricas em 60% na dose de 100 mg/kg (p<0.01), 47% na dose de 200 mg/kg (p<0,01) e 69% na dose de 400 mg/kg (p<0,001). Realizando avaliação histológica, foi verificado aumento regenerativo do epitélio da mucosa e reparação com cicatrização da lamina própria da mucosa com proliferação fibroblástica e neoformação de vasos sanguíneos, e grande deposição de colágeno, evidenciando que o processo de cicatrização e regeneração está ocorrendo após o tratamento com o extrato hidroetanólico a 70% de P. tenellus. Paralelamente aos estudos farmacológicos, foi determinado o perfil cromatográfico por Cromatografia em Camada Delgada (CCD) do extrato hidroetanólico e frações (clorofórmica, acetato de etila e aquosa). No ensaio de quantificação de flavonoides e de compostos fenólicos foi observado que a fração acetato de etila possui maior concentração de substâncias fenólicas, incluindo flavonoides, seguida do extrato bruto e por último, pela fração aquosa. No experimento de determinação de capacidade antiradicalar observou-se que a fração acetato de etila (CE50=13,09 &#181;g/ml) apresentou uma melhor capacidade de redução do radical DPPH, seguida do extrato hidroetanólico (CE50=19,86 &#181;g/ml) e fração aquosa (CE50=45,76 &#181;g/ml). Estes resultados estão em consonância com a gastroproteção observada no experimento de atividade antiulcera, sugerindo uma possível relação do flavonoide, di-hidromiricetina presente no extrato, com a atividade gastroprotetora e com a facilitação da cicatrização das úlceras gástricas. / Traditionally medicinal plants have been used to prevent and to treat gastric ulcer. Gastric ulcer is a heterogeneous disease of multifactorial etiology, which affects about 8-10% people in the world. However, these pharmaceutical products are not completely effective and produce many adverse effects. Extracts of leaves of Phyllanthus tenellus Roxb. is often used for treating gastric disease by the population. This species is included in RENISUS. In order to investigate antiulcer activity, for this were realized two distinct ulcer models in rats. In acidified ethanol model, the crude extract (hydroethanol extract 70% lyophilized) from leaves of P. tenellus showed a reduction for 3.44% and 4.25% from gastric lesion area at the dose 200mg/kg and 400mg/kg, however there was not diference significant between the results. The assay using hydroethanol extract, ethyl acetate fraction and aqueous fraction at the dose 200mg/kg as in the previous test showed a reduction from gastric lesion area respectively in 0.7%, 1.1% and 0.7% however there was not significant difference between control and treated. The assay using ethyl acetate fraction from the leaves P. tenellus (Phyllanthaceae), showed a reduction in 9,46% and 22,80% of gastric lesion area at the dose 100mg/kg and 400mg/kg however there was not significant difference. In gastric ulcer model induced by acidified ethanol in pre-treated animals in ethoricoxib, indomethacin and L-NAME, showed a tendency of reduction from gastric lesion area however there was not significant difference. In gastric ulcer model induced by acidified ethanol in pre-treated animals with NEM showed a reduction from gastric lesion in 90% in the group Salina+FA (p<0,05), comparing with the group NEM+FA. In subacute ulcer model induced by acid acetic , after 7 days of treatment with hydroethanol extract of P. tenellus at the dose 100, 200 and 400 mg/kg showed significant reduction from gastric lesion in 60% at the dose 100 mg/kg (p<0.01), 47% at the dose 200 mg/kg (p<0,01) and 69% at the dose 400 mg/kg (p<0,001). With histological evaluation it was possible to see regenerative increased of the mucosal epithelium and reparation with healing mucosal lamina propria with fibroblast proliferation and blood vessels neoformation, and large collagen deposition showing healing process and regeneration occurring after treatment hydroethanol extract 70% of P. tenellus. At the same time the pharmacological studies it was made the chromatographic profile by Thin Layer Chromatography (TLC), hydroethanol extract and fractions (chloroform, ethyl acetato and aqueous). The assay of quantitation of flavonoids and phenolic compounds it was observed that the ethyl acetate fraction has a higher concentration of phenolic substances including flavonoides, then the crude extract and lastly the aqueous fraction . In the experiment to determine antiradicalar capacity it was noted that the ethyl acetate fraction (CE50=13,09 &#181;g/ml) showed a better radical DPPH reduction capacity, followed by hydroethanol extract (CE50=19,86 &#181;g/ml) and aqueous fraction (CE50=45,76 &#181;g/ml). These results are in line with the gastroprotection observed in gastric ulcer experiment, suggesting a possible relation of the flavonoid , di - hidromiricetina present in the extract, with the gastroprotective activity and facilitating healing of gastric ulcers.

Este trabalho descreve o desenvolvimento de novos métodos cromatográficos em conjunto com técnicas de microextração para determinação de parabenos em produtos cosméticos e leite humano e rifampicina em amostras de plasma. Destaca-se também o desenvolvimento da fase extratora molecularmente impressa para extração em fase sólida (SPE) de parabenos em amostras de leite humano. O capítulo I descreve a avaliação da extração sortiva em barra de agitação (SBSE) em conjunto com a cromatografia líquida (LC) com detecção espectrofotométrica (LC-UV) para análises de parabenos em produtos cosméticos para fins de controle de qualidade. As variáveis do processo de extração SBSE, tais como tempo e temperatura de extração, pH e força iônica da amostra e condições de dessorção foram otimizadas para aumentar a sensibilidade analítica do método e diminuir o tempo de análise. O método SBSE/LC-UV desenvolvido apresentou faixa linear correspondente ao limite de quantificação (LQ) a 2,50 µg. mg-1 e coeficientes de determinação maiores que 0,993. Os valores de coeficiente de variação resultantes das análises de precisão interensaios foram 5,0% para todos os parabenos analisados e a exatidão variou de 90 a 100%. O método desenvolvido foi aplicado para análises de amostras de produtos cosméticos comerciais (cremes hidratantes, antitranspirantes em creme e filtros solares). O método SBSE/LC-UV padronizado e validado resultou nas seguintes vantagens em relação aos métodos convencionais: simplicidade no preparo de amostra, redução do volume de solvente orgânico e da quantidade de amostra utilizada. O capítulo II descreve o desenvolvimento da fase extratora molecularmente impressa para SPE de parabenos em amostras de leite humano e análises por cromatografia em fase líquida com detecção espectrofotométrica (SPE/LC-UV). Os polímeros de impressão molecular, materiais sintéticos baseados em sistemas biomiméticos, foram sintetizados através do processo sol-gel tendo como molde, o benzilparabeno. Após a remoção do molde, cavidades seletivas (sítios de reconhecimento molecular) ao benzilparabeno e análogos estruturais foram reveladas. O método SPE/LC desenvolvido apresentou faixa linear correspondente ao limite de quantificação a 150 ng. mL-1 e coeficientes de determinação 0,992. Os valores de coeficiente de variação resultantes das análises de precisão interensaios foram 13% e a exatidão variou de 86 a 117%. A aplicabilidade do método SPE/LC foi demonstrada através das análises de amostras de leite humano de lactantes. Segundo os parâmetros de validação analítica avaliados, o método padronizado SPE/LC-UV é adequado para análises de parabenos em amostras de leite humano. O capítulo III descreve a avaliação da microextraçao em fase sólida no capilar (in-tube SPME) acoplada à cromatografia líquida (LC) com detecção espectrofotométrica (LC-UV) para análises de rifampicina em amostras de plasma para fins de monitorização terapêutica. As variáveis in-tube SPME, tais como fase estacionária da coluna capilar, volume de amostra, ciclos aspirar/dispensar e vazão foram otimizadas visando obter maior eficiência do processo. O método in-tube SPME/LC desenvolvido apresentou faixa linear correspondente ao limite de quantificação (0,1 µg. mL-1) a 100 µg. mL-1 e coeficientes de determinação igual a 0,998. Os valores de coeficiente de variação resultantes das análises de precisão interensaios foram 2% e a exatidão variou de 83 a 92%. A aplicabilidade do método in-tube SPME/LC, padronizado e validado foi comprovada através das análises de amostras de plasma de pacientes em tratamento com rifampicina. O método in-tube SPME/LC resultou na automação do processo de análise, maior precisão analítica, menor tempo de análise, menor volume de solvente orgânico e de fluido biológico. / This work describes the development of chromatographic methods in combination with microextraction techniques for the determination of parabens in cosmetic and human milk samples and of rifampicin in plasma samples. The design of a molecularly imprinted extraction phase for the solid-phase extraction (SPE) analysis of parabens in human milk samples is also reported. Chapter I describes the evaluation of the stir bar sorptive extraction (SBSE) in combination with liquid chromatography (LC) with UV detection (LC-UV) for the analysis of parabens in cosmetics aiming at quality control. The SBSE variables such as extraction time, temperature, pH of the matrix, ionic strength, and desorption conditions have been optimized, in order to establish the parabens partition equilibrium in a short analysis time. The linear range of the SBSE/LC method lay from LOQ to 2.5 µg. mg-1, with a coefficient of determination higher than 0.993. The interday precision of the SBSE/LC method presented a coefficient of variation lower than 5%, and the accuracy ranged from 90 to 99%. The effectiveness of the proposed method for the analysis of commercial cosmetic products such as body creams, antiperspirant creams, and sunscreens has been proven. Therefore, the combination of SBSE with liquid desorption, followed by LC analysis, provides a simple, and selective tool for the analysis of parabens in cosmetic products using minimized volume of organic solvent as well as very small amount of the target sample. Chapter II presents the development of a molecularly imprinted extraction phase for the SPE analysis of parabens in human milk samples. The molecularly imprinted polymers, which are synthetic materials based on biomimetic systems, were synthesized by the sol-gel technology in the presence of the template (benzilparaben). After template removal, selective cavities (benzilparaben) were revealed for parabens selective extraction. The linear range of the SPE/LC method lay from LOQ to 150 ng. mL-1, with a coefficient of determination higher than 0.992. The interday precision of the SPE/LC method presented a coefficient of variation lower than 13%, and the accuracy ranged from 86 to 117%. The effectiveness of the proposed method for the analysis of human milk samples has been demonstrated. According to the evaluated analytical validation parameters, the SPE/LC-UV method is suitable for the analysis of parabens in human milk samples. Chapter III depicts the evaluation of solid-phase microextraction in the capillary (in-tube SPME) in combination with liquid chromatography (LC) with UV detection (LC-UV) for the analysis of rifampicin in plasma samples for therapeutic drug monitoring. The in-tube SPME/LC method was linear over the LOQ (0.1) to 100 g. mL-1 range, with a linear coefficient value (r2) of 0.998. The inter-assay precision presented coefficient of variation 2%, and the accuracy ranged from 83 to 92%. The effectiveness and practicability of the proposed method have been by analysis of plasma samples from ageing patients undergoing therapy with rifampicin. The in-tube SPME/LC method allowed for automated continuous sample preparation (extraction, concentration, desorption, and injection of analytes) and minimized the analysis time as well as the volumes of organic solvent and biological fluid.

Improvements to the properties of recycled rubber, by developing a more selective breakdown process, is an important issue and a global challenge. Devulcanization is the most promising way to achieve this. It is a process which aims to totally or partially cleave monosulfidic (C-S-C), disulfidic (C-S-S-C) and polysulfidic (C-Sx-C) crosslinks of vulcanized rubber. In this study, the devulcanization of sulfur-vulcanized natural rubber with a variety of diphenyldisulfides has been studied using 2,3-dimethyl-2-butene and squalene as model compounds, vulcanized by sulfur and 2-bisbenzothiazole-2,2’-disulfide. 2-aminodiphenyldisulfide; 4-amino-diphenyldisulfide; bis(2-benzamido-)diphenyldisulfide and 2,2’-bithiosalicylic acid were used as aromatic disulfides devulcanization agents. Thermal analysis was used to investigate interactions between various combinations of curatives in the vulcanization systems in the absence of model compounds. Thermogravimetric analysis was used to detect mass changes during vulcanization and whether the samples decompose/evaporate so as not to contaminate differential scanning calorimetry instrumentation. Differential scanning calorimetry was used to investigate reactions occurring between curatives. Comparison of reversing and non-reversing signals in modulated experiments are suggestive of reactions between devulcanization agents and sulfur, and accelerators Vulcanization of the model compounds were performed in 10m evacuated sealed glass ampoules placed in an oil bath at 160 °C and agitated for the duration of vulcanization. After 60 min the ampoules were removed and quenched. These were then devulcanized by exposure to a devulcanization agent in a sealed ampoule at 180 for 60 min. Crosslinked 2,3-dimethyl-2-butene species were isolated and analysed by reversed phase high performance liquid chromatography. Particular attention was paid to changes in the number of sulfur atoms in the crosslinks. Results indicate that of the devulcanization agents tested 4-amino-diphenyldisulfide was the most effective. No detrimental effect on devulcanization efficiency was caused by the substitution of a basic amino group with an acidic carboxylic acid group Gel permeation chromatography was performed on devulcanized squalene crosslinks using a tetrahydrofuran eluent. GPC investigations with squalene were less effective than HPLC experiments with 2,3-dimethyl-2-butene at evaluating the relative efficiency of individual devulcanization agents. Results were, however, consistent with 4-amino-diphenyldisulfide again being the most efficient devulcanization agent.

>Magister Scientiae - MSc / Extracts from the root bark, stem bark, and leaves of R. natalensis were screened for antibacterial activity against standard bacterial strains; Staphylococcus aureas, Escherichia coli and Pseudomonas aureginosa, and fungi; Candida albicans, Trichophyton mentagrophytes or Microsporum gyseum. Chromatographic techniques were utilized to isolate pure compounds. This study validates and documents, in a systematic way, the antimicrobial properties of the R.natalensis used for many years by many people of the world. It also provides valuable information for further phytochemical isolation and characterization studies of active compounds, necessary for the development of new drugs. The extractions were carried out using broad spectrum of solvents (hexane, dichloromethane, ethyl acetate, and methanol).Fractionation was done using standard chromatographic techniques. A total of seven (7)compounds were isolated from R. natalensis. Three of the isolates were characterized and their structures were unambiguously established by detailed spectroscopic analysis that involved high resolution mass spectrometry, 1D and 2D-NMR spectral data experiments 1H, 13C, DEPT,COSY, HMBC, and NOESY. These compounds are: 3-(1-(2,4-dihydroxyphenyl)-3,3-bis(4- hydroxyphenyl)-1-oxopropan-2-yl)-7-methoxy-4H-chromone-4-one (39), Rhuschromone, a novel compound isolated for the first time, 2’,4’ dihydroxychalcone-(4-O-5’’’)-4’’,2’’’,4’’’-trihydroxychalcone (40) and 3-((Z)-heptadec-13-enyl) benzene-1,2-diol (41), an urushiol.Compound 39 recorded the highest activity zone of inhibition (21mm) against S. aureas, which was found to be 50% as active the chloramphenicol standard used. The traditional use of the extracts in infections and inflammatory conditions is rationalized based on the content of the isolated compounds, and it has been proposed that the total crude extract, with its contents of so many bioactive compounds, could be formulated for use in many infections, microbial or fungal.Furthermore, not all of the species studied to date have been fully characterized for potential bioactivities. Thus, there remains a significant research gap spanning the range from lead chemical discovery through process development and optimization in order to better understand the full bioactive potential of many of these plants.

Introduction: The macroporous Amberlite XAD resins were coated with LIX-64N and examined for the rate of uptake of copper . XAD-7 was by far the best support and gave a satisfactory rate of uptake up to loadings of 60% (w/w). The specific surface area of XAD-7 was measured by the adsorption of methylene blue from aqueous solution. The area of the wetted resin was five times less than that of the dry resin. LIX-65N was purified and the anti isomer characterised using spectroscopic techniques . The rate of uptake of copper was not improved by use of purified LIX-65N or by addition of LIX-63. XAD-7 coated with LIX-65N was used in columns. Elution curves for copper showed negligible tailing, and rapid separations of copper from iron (111), nickel, cobalt and magnesium by selective absorption were achieved. Copper was concentrated from very dilute solution at a flow rate of 50 ml min -1 ,and a 99% recovery was obtained. The method was applied to the rapid determination of copper in brass and bronze. A series of long-chain hydroxamic acids were synthesised and tested for suitability as stationary phase on XAD-7. Oleohydroxamic acid and naphthenohydroxamic acid were the most promising. The r ate of uptake of copper was reduced by the use of nonylphenol or amyl alcohol as a diluent. The capacities for copper of the hydroxamic acids were less when supported on XAD-7 than when used as liquid ionexchangers. The distribution coefficients of cobalt, nickel, zinc, lead, vanadium, uranium, iron (111) and copper were measured as a function of pH. XAD-7 coated with oleohydroxamic acid was used in columns for the rapid separation of iron (111) from copper and of copper from nickel, cobalt, lead and zinc. Copper was concentrated from very dilute solution at a flow rate of 45 ml min -1 and a 100,8% recovery was obtained. Copper was successfully separated from nickel by selective elution. The elution curves obtained show negligible tailing. The resin loaded with oleohydroxamic acid lost capacity due to chemical instability. Naphthenohydroxamic acid supported on XAD-7 was not suitable for use in columns, because it was physically unstable.

M.Sc. / The behaviour i.e. the distribution, bioavailability and toxicity of an element depends on the particular species in which it is present. That being said the prediction of the behaviour of species can therefore not be predicted by determination of the total concentration alone. Speciation analysis is thus an important process, which seeks to identify and quantify the species that occur in a sample. By identifying the species one is better equipped to predict the behaviour of the element if it were released into the ecosystem as well as if it were used in industrial processes. The speciation analysis of the dominant chromium species, Cr(III) and Cr(VI), has received much attention as these contradictory species are involved in many industrial processes such as plating and tanning for example. The species have opposing behaviour as Cr(III) is found to be a necessary micronutrient for mammals where as Cr(VI) is known to be toxic. Of the numerous methods developed for chromium speciation analysis, the on-line process of chromatographic preconcentration followed by simultaneous detection with a sensitive detector produces a synergistic effect for the successful speciation analysis of chromium. It was thus decided to study ion chromatography hyphenated with an ICP-OES as a means of studying chromium species. Anion chromatography coupled with ICP-OES was the basis for the development of the first method. Complexation of Cr(III) with EDTA to form a monovalent anion preceded its separation from Cr(VI) as the chromate anion. A carbonate-bicarbonate eluent was used to elute the species from a Dionex AG 9 guard column coupled with an AS 9 separation column. This method proved to be effective and fast, with each analysis taking less than two minutes. The second method was based on cation chromatography in the form of a Dionex CG12A guard column being coupled with an ICP-OES spectrometer as the detector. This method made use of a gradient elution program in which hexavalent chromium was eluted first with ammonium nitrate followed by trivalent chromium with nitric acid. This method was successfully applied to synthetic samples and was found to be effective.