Le principal objectif de cette recherche est l’évaluation de nouveaux adsorbants nommés Metal-Organic Frameworks (MOFs) pour la séparation des isomères de l'hexane afin d'améliorer l'indice d'octane de l’essence. La séparation des isomères de l'hexane est actuellement réalisée par Total Isomerization Processes (TIP) basé sur l’utilisation de la zéolithe 5A qui permet d’isoler les paraffines «non normales». Afin d'améliorer et de tester d’autres adsorbants, des structures MOFs flexibles et rigides ont été synthétisées et évaluées pour cette séparation au travers de séries de courbes de perçage avec des mélanges d’isomères de l’hexane nHEX, 3MP, 22DMB et 23DMB. Cela a permis d'obtenir des isothermes d'adsorption à l'équilibre et une analyse de leurs performances a permis de trouver les structures plus performantes. L’UiO-66(Zr) fonctionnalisé par des groupes –Br, –NO2, et –NH2, les solides mésoporeux MIL-100(Cr) et son analogue fonctionnalisé MIL-100(Cr) greffé avec des alkylamines, le solide microporeux MIL-125(Ti) fonctionnalisé avec un groupe –NH2 et le tétracarboxylate de fer(III) microporeux MIL-127(Fe) sont les solides rigides étudiés. Les structures flexibles sont les dicarboxylates de fer(III) MIL-53(Fe) fonctionnalisés –(CF3)2, –2CH3, le ZIF-8 à base d’imidazolate et un polymorphe MIL-88B(Fe) fonctionnalisé –2CF3. La caractérisation de ces adsorbants cristallins a été réalisée par combinaison de diffraction des rayons X (XRPD), spectroscopie infrarouge (IR), analyse thermogravimétrique (TGA) et la porosimétrie d’adsorption d’azote. Les solides MIL-53(Fe)–(CF3)2 et ZIF-8 démontrent un effect de tamis moléculaires avec un comportement remarquable. / The main goals of this research are the synthesis of new specific adsorbents named Metal-Organic Frameworks (MOFs) for the separation of hexane isomers in order to improve the octane number of the gasoline. The separation of hexane isomers is actually performed using the conventional Total Isomerization Processes (TIP) with zeolite 5A which isolates only «non-normal paraffins». In order to improve and to test other alternatives, flexible and rigid frameworks were synthesized, performing a set of breakthrough curves with hexane isomers nHEX, 3MP, 22DMB and 23DMB with the purpose of obtaining adsorption equilibrium isotherms and further analysis of their performances in order to find new frameworks that offer better results. This concerned first the rigid frameworks UiO-66(Zr) functionalized with the functional groups –Br, –NO2, and –NH2; the mesoporous MIL-100(Cr) and its functionalized analogue MIL-100(Cr) grafted with alkylamines, the microporous Ti MOF MIL-125 functionalized with the functional group –NH2 and the iron tetracarboxylate MIL-127(Fe). The flexible frameworks were the Zn imidazolate ZIF-8, the iron(III) dicarboxylates MIL-53(Fe) functionalized with the functional groups –(CF3)2 and –2CH3 and the MIL-88 functionalized with the functional group –2CF3. The characterization of these crystalline adsorbents was achieved by X-Ray Powder Diffraction (XRPD), Infra-Red spectroscopy (IR), Thermogravimetric Analysis (TGA) and nitrogen surface area measurement. MIL-53(Fe)–(CF3)2 and ZIF-8 demonstrated molecular sieve effects with interesting and promossing behaviour for hexane isomers separation.

Diseases caused by bacteria, fungi and viruses pose a significant threat especially to poor rural communities. Viral infections are frequently complicated by secondary bacterial and fungal infections which remain a major challenge globally and in particular, in sub Sahara Africa amongst humans and animals alike. The main aim of this study was to develop a low toxicity plant extract or isolated compound active against viral, bacteria and fungal pathogens from selected plant species. Seven tree species that were investigated were Acokanthera schimperi, Carissa edulis, Ekebergia capensis, Podocarpus henkellii, Plumbago zeylanica, Annona senegalensis and Schrebera alata traditionally used in the treatments of various ailments were selected and extracted using solvents of varying polarity. Extracts of selected plants were tested for activity against two Gram positive and two Gram negative bacterial namely Enterococcus faecalis and Staphylococcus aureus and two Gram-negative species, Pseudomonas aeruginosa and Escherichia coli respectively, three fungal pathogens: Candida albicans, Cryptococcus neoformans and Aspergillus fumigates and four enveloped animal viruses: feline herpes virus–1 (FHV-1, dsDNA), canine distemper virus (CDV, ssRNA), canine parainfluenza virus-2 (CPIV-2, ssRNA) and lumpy skin disease virus strain V248/93 (LSDV, dsDNA). The presence of antioxidant constituents in the different extracts and cytotoxicity against three cell types CRFK, bovine dermis and Vero cells were determined. Bioautography and the serial microplate dilution methods were used to determine the number of antimicrobial compounds and antimicrobial activity of extracts against bacterial and fungal pathogens. Virucidal and attachments assays were used to determine the activity against viral pathogens. Qualitative antioxidant activities of extracts were tested using the DPPH reagent and cytotoxicity using the MTT assay. Biological activity was observed in all the extracts against one or more organisms on bioautography. The intermediately polar system (CEF) separated more active constituents. Some extracts had compounds with similar Rf values active against one or more organisms. In both the antibacterial and antifungal assays, acetone extracts had the highest activity followed by DCM against one or more pathogens. Hexanes extracts were the least active. P. henkellii extracts had more active compounds against the bacteria and Annona senegalensisagainst the fungi. In the micro-dilution assay, S. aureus was the most susceptible bacterial organism to extracts of the different plant, followed by P. aeruginosa andEscherichia coli, and E. faecalis the least. C. neoformans on the other hand was the most susceptible fungal pathogen. In the antiviral assay, although activity was observed with hexane extracts of some plants in the virucidal assay, the most potent inhibition was observed with the acetone and methanol extracts of Podocarpus henkelii against CDV and LSDV in the virucidal assay and acetone extracts in the attachment assay. In general the hexane was the least toxic while the intermediate polarity extracts were generally the most toxic indicating that highly polar compounds were possibly poorly or highly absorbed through membranes in the former and later respectively. Of the three cell types used CRFK was the most sensitive followed by bovine dermis and Vero cells the least. Cytotoxicity studies of extracts of the different plants revealed A. senegalensis and A. schimperi extracts were the most toxic plants in the cellular assay. These plants are toxic to animals and the cytoxicity is in line with the in vivo toxicity. The protective effects of antioxidant constituents in some extracts varied and appear to be influenced by the metabolism of the type of cell in culture. It also appears to suggest that metabolism in kidney derived cells can be influenced by species variation in the origin of cells. P. henkellii was selected for isolation of bioactive compound. Three compounds were isolated and their structure elucidated using 13C and 1H NMR and mass spectrometric data. The antibacterial, antifungal and antiviral activity of the isolated compounds 7’, 4’, 7’’, 4’’’, tetramethoxy amentoflavone (C1), isoginkgetin (C2) and Podocarpusflavone–A (C3) were determined. Compound C2 was the most active against E. coli and S. aureus (MIC = 60 ìg/mE) and a selectivity index (SI) value of 16.67. The compound was also active against A. fumigatus and C. neoformans (SI = 33.33) suggesting both antibacterial and antifungal activity with relative safety. Compound C3 had a broad spectrum of activity against E. faecalis and P. aeruginosa with SI values of 4. A less potent activity of the compounds was obtained in both the virucidal and attachment assays against test pathogens, indicating the lower activity of the compounds against tested viral pathogens. The studies further suggest structural activity relationship in the antimicrobial activity of biflavonoids. The compounds C1 and C2 had no toxic effect on the three cell types and mutagenicity studies indicated no activity of these compounds. Podocarpusflavone-A occurs in every species of Podocarpus so far investigated, except P. latifolius. These studies represent the first isolation of bioactive compounds from P. henkellii. Although a different extractant was used than that used by traditional healers, the presence of antiviral compounds in Podocarpus henkelii against two unrelated viruses may justify on a chemotaxonomic basis the traditional use of related species Podocarpus latifoliusand Podocarpus falcatus in the traditional treatment of canine distemper infection in dogs. / Thesis (PhD)--University of Pretoria, 2011. / Paraclinical Sciences / unrestricted

O verapamil (VER) é um composto quiral comercializado como mistura racêmica dos enantiômeros (+)-(R)-VER e (-)-(S)-VER. O VER é biotransformado em norverapamil (NOR) e em outros metabólitos por vias dependentes do CYP. O tolueno e o n-hexano são solventes orgânicos que podem alterar o metabolismo de medicamentos dependente do CYP. Assim, o estudo investiga a estereosseletividade na farmacocinética do verapamil administrado a ratos na dose de 10 mg kg-1, sob forma racêmica, e do seu metabólito, norverapamil, bem como a influência do n-hexano e do tolueno na disposição cinética dos enantiômeros (+)-(R) e (-)-(S)-VER e (R)- e (S)-norverapamil em animais tratados com os solventes por inalação em câmara de exposição do tipo nose only nas concentrações de 88, 176 e 352 mg/m3 para o n-hexano e 94, 188 e 376 mg/m3 para o tolueno. Os enantiômeros do VER e do NOR foram separados na coluna de fase quiral Chiralpak® AD e analisados por LC-MS/MS (m/z = 441,3->165,5 para os enantiômeros do norverapamil e m/z 455,3->165,5 para os enantiômeros do verapamil). A análise farmacocinética foi realizada com base no modelo monocompartimental. A farmacocinética do verapamil é estereosseletiva em ratos do grupo controle não tratado com os solventes com acúmulo plasmático do eutômero (-)-(S)-VER (AUC0-? = 250,8 vs 120,4 ng mL-1 h; P<=0,05, teste de Wilcoxon). O metabólito (S)-NOR também foi acumulado no plasma dos animais do grupo controle com razão S/R relativa ao parâmetro AUC0-? de 1,5. Os parâmetros farmacocinéticos AUC0-?, Cl/F, Vd/F e t1/2 relativos aos enantiômeros (-)-S e (+)-(R)-VER e aos enantiômeros (S) e (R)-NOR não foram alterados pela exposição em câmara de exposição do tipo nose only ao n-hexano nas concentrações de 88, 176 e 352 mg/m3 e ao tolueno nas concentrações de 94, 188 e 376 mg/m3; teste de Kruskall-Wallis; P<=0,05. No entanto, a exposição ao n-hexano nas concentrações de 176 mg/m3 e 352 mg/m3 e ao tolueno nas concentrações de 94 mg/m3, 188 mg/m3 e 376 mg/m3 resultou em perda da enantiosseletividade observada para o grupo controle. / Verapamil (VER) is a chiral compound which is commercialized as a racemic mixture of the (+)-(R)-VER and (-)-(S)-VER enantiomers. VER is biotransformed into norverapamil (NOR) and other metabolites through CYP-dependent pathways. Toluene and n-hexane are organic solvents that can alter the metabolism of CYP-dependent drugs. The present study investigated the stereoselectivity in the pharmacokinetics of racemic VER administered to rats at a dose of 10 mg kg-1 and of its metabolite NOR. In addition, the influence of n-hexane and toluene on the kinetic disposition of the (+)-(R) and (-)-(S)-VER and (R)- and (S)-NOR enantiomers was analyzed in animals exposed by nose-only inhalation to n-hexane at concentrations of 88, 176 and 352 mg/m3 and to toluene at concentrations of 94, 188 and 376 mg/m3. The VER and NOR enantiomers were separated on a Chiralpak® AD chiral phase column and analyzed by LC-MS/MS (m/z = 441.3->165.5 for the NOR enantiomers and m/z 455.3->165.5 for the VER enantiomers). Pharmacokinetic analysis was performed using a monocompartmental model. The pharmacokinetics of VER was stereoselective in control rats not treated with the solvents, with plasma accumulation of the (-)-(S)-VER eutomer (AUC0-? = 250.8 vs 120.4 ng mL-1 h; P<=0.05, Wilcoxon test). The (S)-NOR metabolite was also found to accumulate in plasma of control animals, with an S/R AUC0-? ratio of 1.5. The pharmacokinetic parameters AUC0-?, Cl/F, Vd/F and t1/2 obtained for the (-)-S-VER, (+)-(R)-VER, (S)-NOR and (R)-NOR enantiomers were not altered by nose-only exposure to n-hexane at concentrations of 88, 176 and 352 mg/m3 or to toluene at concentrations of 94, 188 and 376 mg/m3 (P<=0.05), Kruskal-Wallis test). However, exposure to 176 and 352 mg/m3 n-hexane and to 94, 188 and 376 mg/m3 toluene resulted in the loss of enantioselectivity observed for the control group.

Atmospheric aldol self-reactions of octanal, heptanal and hexanal in a range of aqueous H2SO4 w/v% concentrations as a catalyst were studied in both bulk liquid-liquid experiments and gas-liquid experiments. Initially, a new practical methodology was developed and enhanced to monitor aldol reactions in aqueous acidic media. The evaluation of a quenching and extracting method were performed, confirming the suitability, reliability and reproducibility of the extraction method. In bulk studies, aldol products of the three aldehydes were separated and identified by preparative HPLC, GC-MS and NMR. The major aldol products observed at high acid concentrations were alpha,β-unsaturated aldehyde (dimer), trialkyl benzene (trimer) and tetraalkylcycloocta-tetraene (tetramer). The trimer of octanal was formed as trioxane in low sulfuric acid concentration and the possible mechanism accretion reaction pathways of high and low acid concentrations are proposed in this study. A systematic kinetic study of octanal, heptanal and hexanal in the bulk experiments at 65, 60 and 55 w/v% H2SO4 at 294 K were monitored using gas chromatographic equipped with a flame ionisation detector (GC-FID). The rate constants were generally estimated using second order kinetics and observed to increase as a function of sulfuric acid concentrations and also as the chain length of aliphatic aldehyde increased. The aldol self-reaction in the bulk experiment was too fast at room temperature to be easily measured using a quenching method therefore attempts were made to follow the reaction at low temperature (0 °C). The result at low temperature indicated that the rate constant of aldehyde was reduced but there was an issue of rapid rise in temperature as a result of mixing concentrated sulfuric acid with aqueous solution of the aldehyde. A gas bubbling system was developed which better simulates atmospheric reality, and which also resolves the issue of temperature rise on mixing. Two different methodologies were used: one in which the aldehyde was continually added, and one where a fixed amount was added from the gas phase and the reaction was then allowed to proceed, monitored at selected time intervals. The precision and accuracy of the fixed method was then further improved by the addition of an internal standard (IS). Using this, the concentrations of aliphatic aldehydes (C6-C8) were calibrated using an experimentally determined response factor and used to follow the loss of the reactant aldehydes. Similar methods were applied to the aldol dimers (C6-C8), which were purified and used to calibrate the chromatographic response. The rate constant for octanal, heptanal and hexanal at 76 wt% and 294 K were 0.0969 M-1 s-1, 0.1497 M-1 s-1 and 0.2622 M-1 s-1 respectively. There are some observations based upon the results presented in this thesis that may be of atmospheric significance: (i) phase separation between organic and aqueous layers in both the bulk experiment and in the bubbling system; (ii) the acid strength dependence and concentration-dependence of the various products; (iii) the faster rates than previously reported, and variation between bulk and bubbling; and (v) the time-dependent colour changes. Further work to explore these observations is proposed.

Made available in DSpace on 2016-06-02T19:56:44Z (GMT). No. of bitstreams: 1 3706.pdf: 2821060 bytes, checksum: 5975ad59eeeedfbd6e16578292306171 (MD5) Previous issue date: 2011-08-01 / Universidade Federal de Sao Carlos / The objective of this study was to verify the influence of the Si/Al ratio (11.5, 15.0, 25.0, 40.0 and 140.0) in zeolite ZSM-5 in the conversion, activity, selectivity and stability as bifunctional catalysts (Pt/H-ZSM-5) applied to the isomerization reaction of n-hexane. To prepare the bifunctional catalysts, initially the zeolites were submitted, successively, to ion exchange with ammonium cations, ion exchange cations with platinum, calcination and activation process. From the results of the isomerization of n-hexane, it was observed that with the increase of the Si/Al ratio, that is, decreasing the amount of aluminum, there was a reduction in activity and conversion. This is due to decrease the number of acid sites present, responsible to isomerize the carbocation generated in these sites. In contrast, the increase of the Si/Al ratio leads to a better selectivity to the formation of isomers. Among the catalysts, the Pt/H-ZSM-5 (15.0) showed the best result of conversion and activity. In order to compare the Pt/H-ZSM-5 catalysts, the reaction was carried out using the isomerization catalyst Pt/H-Beta (9.0). This catalyst had the best result that all the others, this result may possibly be due to increased acidity of the material and its morphological characteristics, such as type and diameter of pores etc. and/or the crystallite size of zeolites used. / O objetivo deste estudo foi verificar a influência da razão Si/Al (11,5, 15,0, 25,0, 40,0 e 140,0) na zeólita ZSM-5 através da conversão, atividade, seletividade e estabilidade de catalisadores bifuncionais (Pt/H-ZSM-5) aplicados a reação de isomerização do n-hexano. Para preparar os catalisadores bifuncionais, inicialmente as zeólitas foram submetidas, sucessivamente, a troca iônica das zeólitas com cátions amônio, troca iônica com cátions platina, processos de calcinação e ativação. A partir dos resultados da isomerização do nhexano observou que com o aumento da razão Si/Al, isto é, diminuição da quantidade de alumínio, obteve-se uma redução na atividade e conversão. Tal fato se deve a diminuição do número de sítios ácidos presentes, sendo que os mesmos são responsáveis por isomerizar os carbocátions gerados nos sítios. Em contrapartida, o aumento da razão Si/Al acarreta em uma melhor seletividade a formação dos isômeros. Dentre os catalisadores Pt/H-ZSM-5, o catalisador Pt/H-ZSM-5 (15,0) foi o que apresentou melhor resultado de atividade e conversão. A fim de comparar os catalisadores Pt/H-ZSM-5, a reação de isomerização do nhexano foi realizada utilizando o catalisador Pt/H-Beta (9,0). O mesmo obteve melhor resultado que todos os outros, podendo tal resultado ser, possivelmente, devido a maior acidez do material e suas características morfológicas, como tipo e diâmetro de poros etc. e/ou pelo tamanho do cristalito das zeólitas utilizadas.

Orientador: Silmara Marques Allegretti / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-12T15:42:28Z (GMT). No. of bitstreams: 1 Oliveira_ClaudineideNascimentoFernandesde_M.pdf: 7853084 bytes, checksum: e21dff7dd2d35d9545d8717dc30fdb79 (MD5) Previous issue date: 2008 / Resumo: A esquistossomose mansonica e uma doenca cronica e debilitante. Estima-se que mais de 200 milhoes de pessoas estejam infectadas no mundo pela doenca, e no Brasil estima-se que existe de 8 a 10 milhoes de pessoas infectadas. O tratamento da esquistossomose e baseado na quimioterapia com o praziquantel, principalmente devido a sua atividade contra todas as especies de Schistosoma patogenicas ao homem. Infelizmente, o uso extensivo e inapropriado desse farmaco pelo mundo, culminou com o aparecimento de esquistossomas tolerantes, gerando uma preocupacao sobre a selecao da resistencia a esse medicamento. Entao, para se controlar a esquistossomose, ha necessidade de se desenvolver novas opções de farmacos, como alternativa ao praziquantel. As plantas medicinais vem sendo aplicadas e testadas como novas alternativas medicamentosas para o tratamento de parasitoses. Este estudo teve por objetivo avaliar a atividade esquistossomicida da planta Phyllanthus amarus L. sobre Schsistosoma mansoni, linhagem BH em camundongos Swiss. Foram testados os extratos hexanico e etanolico nas concentracoes 100, 150 e 250mg/Kg, e a fracao de Lignanas 50 e 100mg/Kg, administrados em dose unica por tubagem esofagica. Os animais foram divididos em dois grupos de acordo com o periodo de tratamento (30 ou 45 dias apos a infeccao para avaliar a acao dos extratos nos vermes jovens e adultos, respectivamente). Foram analisados os seguintes parametros: quantidade/ porcentagem de vermes adultos nas veias mesentericas, porta e nas visceras; proporcao entre machos e femeas; reducao no numero de vermes; reducao no numero de ovos eliminados para o ambiente externo e retidos no tecido intestinal; alteracao no oograma; aspecto visual dos granulomas nas visceras; alteracao no tamanho dos granulomas encontrados no figado. De acordo com os resultados obtidos, o tratamento feito com o extrato etanolico 250mg/Kg no 30° dia de infeccao, foi o que apresentou maior reducao do numero de vermes (63%), e o grupo tratado com o extrato hexanico 100mg/Kg nesse mesmo periodo foi o que apresentou maior quantidade de vermes nas visceras (n=3,1). Ja os tratamentos feitos com o extrato etanolico 100mg/Kg e a fracao de Lignanas 50mg/Kg apos 30 dias de infeccao, conseguiram cessar a postura de ovos, o que fez com que os orgaos desses grupos fossem pouco lesados. Ocorreu ausencia de granulomas nos figados observados histologicamente para o grupo tratado com o extrato etanolico 100mg/Kg. No 45° dia apos a infeccao o grupo tratado com o extrato hexanico 150mg/Kg foi o que apresentou resultado mais significativo, uma vez que ele conseguiu reduzir o numero de ovos imaturos e aumentar o de ovos maduros, indicando que este tratamento alterou a oviposicao do verme. Os resultados obtidos para os extratos hexanico e etanolico e fracao de Lignanas demonstraram potencial atividade nos diferentes parametros avaliados, evidenciando que a planta P. amarus possui efeito contra o S. mansoni linhagem BH. / Abstract: The schistosomiasis is a chronic and debilitating disease. It is estimated more than 200 million people worldwide are infected by the disease, and in Brazil is estimated 8 to 10 million people infected. The treatment of schistosomiasis is based on praziquantel chemotherapy, mainly due to its activity against all species of pathogenic Schistosoma to humans. The inappropriate and extensive use of this drug in the world culminated in the appearance of tolerant worms, generating a concern about the selection of resistance this drug. So, to control schistosomiasis, there is need to develop new options for drugs, as an alternative to the praziquantel. Medicinal plants have been implemented and tested as a new alternative drug for the treatment of parasitic. The objective of this study was to assess the presence of antichistosomal activity of the plant Phyllanthus amarus L. using Schistosoma mansoni infected mice of BH strain. The hexane and ethanolic extracts with 100, 150 and 250mg/Kg, concentrations and the Lignans fraction 50 and 100mg/kg, were administered with single oral dose by esophageal intubation. The animals were divided into two groups according to the treatment period (30 or 45 days after infection to evaluate the effect of the extracts in young and adult worms respectively). Analyzes performed consists in a set of parameters: quantity / percentage of adult worms in the veins mesenteric, port and the viscera; proportion of male and female worms, worms reduction; eggs reduction eliminated environment and retained in the intestinal tissue; change in oograma; visual aspect of granulomas in the viscera; change in the size of granulomas found in the liver. According to the results, the treatment with ethanolic extract 250mg/Kg in 30th days of infection, presented the greatest reduction in the number of worms (63%), and the group treated with the hexane extract 100mg/kg in same period presented the highest number of worms in the viscera (n=3.1). The treatments made with ethanolic extract 100mg/kg and the Lignans fraction 50mg/kg after 30 days of infection managed to stop the egg laying. So that the organs of these groups were slightly injured. There was absence of granulomas in the liver observed histological for the group treated with ethanolic extract 100mg/kg. In the 45th days after infection the group treated with the hexane extract 150mg/Kg presented the most significant result, as to reduced the number of immature eggs, and increased of mature eggs, indicating that this treatment has changed the worm oviposition. The results for the ethanol, hexane extracts and fraction of Lignans demonstrated potential activity in the various parameters measured, suggesting that the plant P. amarus has effect against S.mansoni strain BH. / Mestrado / Mestre em Parasitologia

O verapamil (VER) é um composto quiral comercializado como mistura racêmica dos enantiômeros (+)-(R)-VER e (-)-(S)-VER. O VER é biotransformado em norverapamil (NOR) e em outros metabólitos por vias dependentes do CYP. O tolueno e o n-hexano são solventes orgânicos que podem alterar o metabolismo de medicamentos dependente do CYP. Assim, o estudo investiga a estereosseletividade na farmacocinética do verapamil administrado a ratos na dose de 10 mg kg-1, sob forma racêmica, e do seu metabólito, norverapamil, bem como a influência do n-hexano e do tolueno na disposição cinética dos enantiômeros (+)-(R) e (-)-(S)-VER e (R)- e (S)-norverapamil em animais tratados com os solventes por inalação em câmara de exposição do tipo nose only nas concentrações de 88, 176 e 352 mg/m3 para o n-hexano e 94, 188 e 376 mg/m3 para o tolueno. Os enantiômeros do VER e do NOR foram separados na coluna de fase quiral Chiralpak® AD e analisados por LC-MS/MS (m/z = 441,3->165,5 para os enantiômeros do norverapamil e m/z 455,3->165,5 para os enantiômeros do verapamil). A análise farmacocinética foi realizada com base no modelo monocompartimental. A farmacocinética do verapamil é estereosseletiva em ratos do grupo controle não tratado com os solventes com acúmulo plasmático do eutômero (-)-(S)-VER (AUC0-? = 250,8 vs 120,4 ng mL-1 h; P<=0,05, teste de Wilcoxon). O metabólito (S)-NOR também foi acumulado no plasma dos animais do grupo controle com razão S/R relativa ao parâmetro AUC0-? de 1,5. Os parâmetros farmacocinéticos AUC0-?, Cl/F, Vd/F e t1/2 relativos aos enantiômeros (-)-S e (+)-(R)-VER e aos enantiômeros (S) e (R)-NOR não foram alterados pela exposição em câmara de exposição do tipo nose only ao n-hexano nas concentrações de 88, 176 e 352 mg/m3 e ao tolueno nas concentrações de 94, 188 e 376 mg/m3; teste de Kruskall-Wallis; P<=0,05. No entanto, a exposição ao n-hexano nas concentrações de 176 mg/m3 e 352 mg/m3 e ao tolueno nas concentrações de 94 mg/m3, 188 mg/m3 e 376 mg/m3 resultou em perda da enantiosseletividade observada para o grupo controle. / Verapamil (VER) is a chiral compound which is commercialized as a racemic mixture of the (+)-(R)-VER and (-)-(S)-VER enantiomers. VER is biotransformed into norverapamil (NOR) and other metabolites through CYP-dependent pathways. Toluene and n-hexane are organic solvents that can alter the metabolism of CYP-dependent drugs. The present study investigated the stereoselectivity in the pharmacokinetics of racemic VER administered to rats at a dose of 10 mg kg-1 and of its metabolite NOR. In addition, the influence of n-hexane and toluene on the kinetic disposition of the (+)-(R) and (-)-(S)-VER and (R)- and (S)-NOR enantiomers was analyzed in animals exposed by nose-only inhalation to n-hexane at concentrations of 88, 176 and 352 mg/m3 and to toluene at concentrations of 94, 188 and 376 mg/m3. The VER and NOR enantiomers were separated on a Chiralpak® AD chiral phase column and analyzed by LC-MS/MS (m/z = 441.3->165.5 for the NOR enantiomers and m/z 455.3->165.5 for the VER enantiomers). Pharmacokinetic analysis was performed using a monocompartmental model. The pharmacokinetics of VER was stereoselective in control rats not treated with the solvents, with plasma accumulation of the (-)-(S)-VER eutomer (AUC0-? = 250.8 vs 120.4 ng mL-1 h; P<=0.05, Wilcoxon test). The (S)-NOR metabolite was also found to accumulate in plasma of control animals, with an S/R AUC0-? ratio of 1.5. The pharmacokinetic parameters AUC0-?, Cl/F, Vd/F and t1/2 obtained for the (-)-S-VER, (+)-(R)-VER, (S)-NOR and (R)-NOR enantiomers were not altered by nose-only exposure to n-hexane at concentrations of 88, 176 and 352 mg/m3 or to toluene at concentrations of 94, 188 and 376 mg/m3 (P<=0.05), Kruskal-Wallis test). However, exposure to 176 and 352 mg/m3 n-hexane and to 94, 188 and 376 mg/m3 toluene resulted in the loss of enantioselectivity observed for the control group.

Submitted in complete fulfillment for the Degree of Doctorate of Philosophy in Biotechnology, Durban University of Technology, Durban, South Africa, 2016. / Plants have provided a source of medicine from the beginning of human history and are the core of modern medicine. Moreover, plant based drug discovery has led to the development of various anticancer drugs (such as vincristine, vinblastine, etoposide, paclitaxel, camptothecin, topotecan and irinotecan). The use of botanical, phytochemical, biological and molecular techniques have facilitated the discovery of anthraquinones from Ceratotheca triloba that can inhibit the human topoisomerase II enzyme (target for anticancer drugs) and kill cancer cells. However, the C. triloba plant has not been extensively studied for its anticancer activity. Therefore, the aim of this study was to further investigate the anticancer activity of C. triloba and determine the classes of compounds that contributed towards its activity. In this study the leaf and root extracts were prepared by using hexane, DCM, hexane: DCM (1:1), methanol and/or water. These extracts were examined for their growth inhibitory potential on three cancer cell lines (A375 [melanoma], MDA-MB-231[breast] and WHCO1 [esophageal]) by using the MTT assay. Then, different mobile phases were prepared for optimizing the separation of the compounds of the active extract by TLC. Column chromatography was performed with the active extract by using five mobile phases (hexane : DCM [60 : 40, 40 : 60], DCM, DCM : ethyl acetate [90 : 10, 70 : 30, 60 : 40, 50 : 50, 50 : 60, 30: 60, 20 : 80], ethyl acetate and ethyl acetate: methanol [80 : 20, 70 : 30, 50 : 50]). The fractions collected from the column were examined for their growth inhibitory potential on two melanoma cell lines (A375 and UACC-62). The IC50 and TGI (total growth inhibition) values of the active fractions were determined. Also, the apoptosis inducing effects of the active fractions and standards (camptothecin and doxorubicin) were determined by using flow cytometer based assays (FITC annexin assay, PE active caspase 3 assay and BD MitoScreen assay). Subsequently, the chemical structures of the compounds that contributed towards the activity of these fractions were obtained by EI-LC-MS analysis. The results demonstrated that the hexane root extract exhibited the best percentage of growth inhibition (%GI) on all three cancer cell lines. The separation of the compounds of the hexane root extract was optimized on TLC plates by using different ratios of hexane and DCM. Column chromatography allowed for fractionation of this extract. Purified compounds were not obtained due to co-elution. Further research would have to be conducted to obtain purified compounds. This may involve the use of mini-column chromatography and PTLC. Overall a total of ten combined fractions were collected from the column. Four of these fractions (F2, F4, F5 and F8) displayed a high %GI on the A375 and UACC-62 cell lines. Moreover, fraction F4 was the most active fraction as it had the lowest IC50 (0.70 µg.ml-1 [A375] and 0.39 µg.ml-1 [UACC-62]) and TGI (12.50 µg.ml-1[A375] and 25 µg.ml-1 [UACC-62]) values in comparison to the other fractions. All four fractions induced depolarization of the mitochondria membrane potential (ΔΨ), caspase 3 activation, early apoptosis (phospholipid phosphatidylserine exposure) and/or late apoptosis in the melanoma cells. The results also revealed that fraction F4 (25 µg.ml-1) induced depolarization of the ΔΨ in a higher percentage of A375 (78.11%) and UACC-62 (87.4%) cells than the other fractions and standards. This fraction also induced caspase 3 activation in a high percentage of A375 (90.56%) and UACC-62 (96.78%) cells. Therefore fraction F4 was also the most active fraction in terms of apoptosis activity. Based on our results and literature findings we can deduce that the active fractions induced the intrinsic or extrinsic (type II) apoptosis pathway in the melanoma cells. Six classes of compounds were identified from the four active fractions. These were: benzothiophenones, benzopyranones, naphthoquinones, anthraquinones, androstanes and quinazolines. In conclusion, this is the first study that evaluated the growth inhibition potential of the leaf and root extracts of C. triloba on a panel of cancer cells. This research indicated that the hexane root extract displayed the best levels of cell growth inhibition. The active constituents of this extract were isolated into four fractions which elicited apoptosis inducing effects that promoted the extrinsic (type II) or intrinsic apoptosis pathway in the melanoma cells. Furthermore, fraction F4 contained the most active compounds from C. triloba as it had the lowest IC50 and TGI values (in comparison to the other fractions) and induced depolarization of the ΔΨ in the highest percentage of melanoma cells. It was confirmed that six classes of compounds were accountable for the anticancer activity of these fractions. Thus, the C. triloba plant is a rich source of anticancer compounds. / D

O n-hexano é um solvente usado industrialmente, e pode causar neuropatia periférica. O indicador biológico da exposição ocupacional ao n-hexano é a 2,5-hexanodiona (2,5HD). O objetivo deste estudo foi validar um método para determinação da 2,5HD urinária por cromatografia em fase gasosa, com detetor de ionização de chama. A validação do método levou em consideração dois tipos de tratamento da amostra, com hidrólise ácida e sem hidrólise ácida. Foi utilizada coluna HP1 (15m x 530&#181;m e espessura do filme 1,5&#181;m), gás de arraste He:4,2mL/min. e ciclohexanona como padrão interno. O método apresentou limites de detecção e quantificação, respectivamente 0,05 e 0,1 mg/L, linearidade de 0,1-20mg/mL, r = 0,999, recuperação entre 98-102%, com precisão e exatidão. Para verificar a aplicação do método foram analisadas 87 amostras de trabalhadores de indústrias de calçados (52 do grupo exposto e 35 do grupo controle). As amostras com hidrólise apresentaram níveis de 2,5HD cerca de dez vezes maiores quando comparados com as amostras sem hidrólise, esta diferença foi estatisticamente significante (p<0,0001), mas os níveis de 2,5HD não ultrapassaram o índice Biológico Máximo Permitido (IBMP) de 5mg/g de creatinina, em nenhuma das amostras analisadas. Os resultados indicam que o método validado mostrou aplicação prática para monitorização biológica, e as amostras que passaram pelo tratamento de hidrólise ácida apresentaram maiores níveis de detecção. / N-hexane is a solvent used in industries, and can cause peripheral neuropathy. The bioindicator in the biological monitoring of workers exposed to n-hexane is the 2,5-hexanedione (2,5HD). This study was developed in order to validate a method for 2,5HD analysis in urine by gas chromatography with flame ionization detector considering two types of sample preparing with and without acid hydrolysis. The chosen technique was performed with HP1 column measured 15m by 530&#181;m inner bore. Helio was used as the carrier gas at a flow rate of 4,2mL/min. The detection and quantification limits were respectively 0,05 and 0,1 mg/L. A linear relationship (r = 0,999) was observed in the range from 0,1-20mg/mL. The average recovery was 98-102%, with precision and accuracy. To apply this method, 87 samples from shoe factory workers were analyzed (52 exposed group and 35 control group). The samples with acid hydrolysis show levels ten times higher than samples without acid hydrolysis. This difference was statistically significant (p<0,0001), but this levels weren\'t higher than the Biological Exposure Index (BEI) were recommended value is 5mg/g creatinine, in any of the analyzed samples. These results reveal that the validated method has application for biological monitoring, and the samples with acid hydrolysis showed rates higher than the samples without hydrolysis.

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