Total synthesis of the pyrrolizidine alkaloid NP25302: (+)-NP25302 is an unusual vinylogous urea containing pyrrolizidine alkaloid shown to exhibit cell adhesion inhibition. It was envisaged that this natural product could be accessed by a novel 5-endo-dig cyclisation to construct the pyrrolizidine core, and a Curtius rearrangement to install the vinylogous urea motif. This methodology was first tested on a model system, furnishing nor-NP25302 from L-proline in 12 steps and 9% overall yield. The total synthesis of (±)-NP25302 was completed in 9 steps and 26% overall yield from ethyl 2-nitropropionate using similar methodology. Studies into the stereospecificity of the Au(I)-catalysed cyclisation of monoallylic diols: During the synthesis of (+)-isoaltholactone in the Robertson group, the key Au(I)-catalysed cyclisation was observed to occur with some stereospecificity. Further investigations were therefore conducted into the stereochemical outcome of this reaction using stereodefined allylic alcohols, and from the combined results a mnemonic was proposed to predict the stereochemistry of the products of this reaction. Studies into the total synthesis of ascospiroketals A and B: Investigations were conducted into the total synthesis of the recently isolated natural products ascospiroketals A and B. A second generation synthesis was used to construct advanced intermediates 1 and 2.

O presente estudo visou avaliar os possiveis efeitos imunotóxicos e imunoteratogênicos da integerrimina N-óxido presente no resíduo butanólico (RB) do ,Senecio brasiliensis, uma planta hepatotóxica que contém alcalóides pirrolizidínicos (APs). Tem sido relatado o uso de APs como fitoterápicos em todo o mundo e também como contaminantes de pastagens e alimentos, sendo assim responsáveis por intoxicações em animais de produção e humanos; porém, até o momento, não há relatos de sua ação sobre o sistema imune. Assim, após a extração da planta, quantificou-se no RB de Senecio brasiliensis, por meio de análise elementar e de espectro (1H e 13C), cerca de 70% de integerrimina N-óxido. O RB foi administrado a ratos Wistar machos adultos, nas doses de 3, 6 e 9 mg/kg, por gavagem, durante 28 dias. Foram avaliados o ganho de peso, o consumo de alimento, os órgãos linfóides e a fenotipagem de sua células, a atividade de fagócitos, as respostas imunes humoral e celular e atividade proliferativa de linfócitos. Já ratas gestantes, receberam as mesmas doses do RB do 6º ao 20º dia de gestação. No final da gestação, parte das fêmeas foi destinada à cesariana e as restantes prosseguiram à termo até o nascimento, para a análise das proles aos 60 dias de idade. Os fetos provenientes da cesariana foram avaliados quanto ao tamanho, peso, viabilidade e malformações externas e também submetidos à análise óssea. As mães tiveram seu desempenho reprodutivo avaliado. Naquelas proles avaliadas até 60 dias de idade, procedeu-se a avaliação dos órgãos linfóides e dos parâmetros hematológicos. O presente estudo revelou que integerrimina N-óxido promoveu diminuição no ganho de peso corporal de ratos machos adultos, redução na atividade proliferativa das células T e interfência na contagem de células sanguíneas destes animais. Por outro lado, esses mesmos animais não apresentaram diferenças na atividade de fagócitos, na fenotipagem de linfócitos e nas respostas imunes estudadas. Em ratas gestantes, o RB causou hepatotoxidade, diminuição do ganho de peso materno, diminuição no tamanho das placentas. A análise fetal revelou menor ossificação. Quando da avaliação da prole aos 60 dias de idade, não foram observados efeitos no ganho de peso atribuídos ao RB, nos órgãos linfóides, e também parâmetros hematológicos e bioquímicos. Concluindo, os estudos aqui realizados permitem sugerir que as doses baixas de integerrimina N-óxido ora empregadas, não promovem efeitos imunotóxicos significantes, bem como não apresentaram potencial teratogênico, seja por meio das avaliações clássicas de teratologia quanto naqueles protocolos de imunoteratogenicidade. / The aim of this study was to evaluate the possible immunotoxic and immunoteratogenic effects of integerrimine N-oxide content in the butanolic residue (BR) of Senecio brasiliensis, a hepatotoxic plant that contained pyrrolizidine alkaloids (PAs). PAs have been reported as a pasture and food contaminant and as herbal medicine used worldwide and are related to promote poisoning events in livestock and human beings. After the plant extraction, BR residue from Senecio brasiliensis was submitted to elemental and spectral analyses (1H and 13C ) and was found to contain approximately 70% of integerrimine N-oxide by, which was administered to adult male Wistar rats at doses of 3, 6 and 9 mg/kg for 28 days, by gavage. Body weight gain, food consumption, lymphoid organs and their cell phenotype, phagocyte analysis, humoural and cellular immune responses, and lymphocyte proliferation were evaluated. In addition, pregnant rats received the same doses of BR from the 6th to the 20th gestational day. In late pregnancy, some of the dams were destined for cesarean section and the other females followed the pregnancy until the birth for offspring analysis at 60 days old. Fetuses from cesarean section were evaluated for size, weight, viability and external malformations and also subjected to bone analysis. The reproductive performance of females were performed. The present study showed that integerrimine N-oxide could promote in adult male Wistar rats, body weight gain impairment, interference with blood cell counts and a reducing T cell proliferative; however, no differences in phagocyte activities, lymphocytes phenotyping and immune responses here evaluated were observed. In pregnant rats, BR caused hepatotoxicity, decreased body weight gain and reduced placental size. Fetus analysis showed lower ossification. When 60-days old offspring were it was not observed any toxic effects resulted from BR exposition in body weight gain, lymphoid organs, haematological and biochemical parameters of these rats. It is concluded that low doses of integerrimine N-oxide here employed did not produce marked immunotoxic effects and seems to be a teratogenic signs by both methods, the classical evaluations or by immunoteratological protocols.

Li, Yanhong. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 192-212). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

O presente estudo visou avaliar os possiveis efeitos imunotóxicos e imunoteratogênicos da integerrimina N-óxido presente no resíduo butanólico (RB) do ,Senecio brasiliensis, uma planta hepatotóxica que contém alcalóides pirrolizidínicos (APs). Tem sido relatado o uso de APs como fitoterápicos em todo o mundo e também como contaminantes de pastagens e alimentos, sendo assim responsáveis por intoxicações em animais de produção e humanos; porém, até o momento, não há relatos de sua ação sobre o sistema imune. Assim, após a extração da planta, quantificou-se no RB de Senecio brasiliensis, por meio de análise elementar e de espectro (1H e 13C), cerca de 70% de integerrimina N-óxido. O RB foi administrado a ratos Wistar machos adultos, nas doses de 3, 6 e 9 mg/kg, por gavagem, durante 28 dias. Foram avaliados o ganho de peso, o consumo de alimento, os órgãos linfóides e a fenotipagem de sua células, a atividade de fagócitos, as respostas imunes humoral e celular e atividade proliferativa de linfócitos. Já ratas gestantes, receberam as mesmas doses do RB do 6º ao 20º dia de gestação. No final da gestação, parte das fêmeas foi destinada à cesariana e as restantes prosseguiram à termo até o nascimento, para a análise das proles aos 60 dias de idade. Os fetos provenientes da cesariana foram avaliados quanto ao tamanho, peso, viabilidade e malformações externas e também submetidos à análise óssea. As mães tiveram seu desempenho reprodutivo avaliado. Naquelas proles avaliadas até 60 dias de idade, procedeu-se a avaliação dos órgãos linfóides e dos parâmetros hematológicos. O presente estudo revelou que integerrimina N-óxido promoveu diminuição no ganho de peso corporal de ratos machos adultos, redução na atividade proliferativa das células T e interfência na contagem de células sanguíneas destes animais. Por outro lado, esses mesmos animais não apresentaram diferenças na atividade de fagócitos, na fenotipagem de linfócitos e nas respostas imunes estudadas. Em ratas gestantes, o RB causou hepatotoxidade, diminuição do ganho de peso materno, diminuição no tamanho das placentas. A análise fetal revelou menor ossificação. Quando da avaliação da prole aos 60 dias de idade, não foram observados efeitos no ganho de peso atribuídos ao RB, nos órgãos linfóides, e também parâmetros hematológicos e bioquímicos. Concluindo, os estudos aqui realizados permitem sugerir que as doses baixas de integerrimina N-óxido ora empregadas, não promovem efeitos imunotóxicos significantes, bem como não apresentaram potencial teratogênico, seja por meio das avaliações clássicas de teratologia quanto naqueles protocolos de imunoteratogenicidade. / The aim of this study was to evaluate the possible immunotoxic and immunoteratogenic effects of integerrimine N-oxide content in the butanolic residue (BR) of Senecio brasiliensis, a hepatotoxic plant that contained pyrrolizidine alkaloids (PAs). PAs have been reported as a pasture and food contaminant and as herbal medicine used worldwide and are related to promote poisoning events in livestock and human beings. After the plant extraction, BR residue from Senecio brasiliensis was submitted to elemental and spectral analyses (1H and 13C ) and was found to contain approximately 70% of integerrimine N-oxide by, which was administered to adult male Wistar rats at doses of 3, 6 and 9 mg/kg for 28 days, by gavage. Body weight gain, food consumption, lymphoid organs and their cell phenotype, phagocyte analysis, humoural and cellular immune responses, and lymphocyte proliferation were evaluated. In addition, pregnant rats received the same doses of BR from the 6th to the 20th gestational day. In late pregnancy, some of the dams were destined for cesarean section and the other females followed the pregnancy until the birth for offspring analysis at 60 days old. Fetuses from cesarean section were evaluated for size, weight, viability and external malformations and also subjected to bone analysis. The reproductive performance of females were performed. The present study showed that integerrimine N-oxide could promote in adult male Wistar rats, body weight gain impairment, interference with blood cell counts and a reducing T cell proliferative; however, no differences in phagocyte activities, lymphocytes phenotyping and immune responses here evaluated were observed. In pregnant rats, BR caused hepatotoxicity, decreased body weight gain and reduced placental size. Fetus analysis showed lower ossification. When 60-days old offspring were it was not observed any toxic effects resulted from BR exposition in body weight gain, lymphoid organs, haematological and biochemical parameters of these rats. It is concluded that low doses of integerrimine N-oxide here employed did not produce marked immunotoxic effects and seems to be a teratogenic signs by both methods, the classical evaluations or by immunoteratological protocols.

Li Mi. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 125-136). / Abstracts in English and Chinese. / Abstract --- p.i / 論文摘要 --- p.iv / Publications --- p.vi / Acknowledgement --- p.vii / Abbreviations --- p.viii / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Pyrrolizidine alkaloids --- p.1 / Chapter 1.1.1 --- Distribution and plant sources --- p.1 / Chapter 1.1.2 --- Structures and nomenclature --- p.3 / Chapter 1.2 --- PA-containing Chinese medicinal herbs --- p.6 / Chapter 1.3 --- PA-induced toxicity。 --- p.7 / Chapter 1.3.1 --- Acute toxicity and chronic toxicity --- p.7 / Chapter 1.3.2 --- Genotoxicity --- p.8 / Chapter 1.3.3 --- Tumorigenicity --- p.8 / Chapter 1.3.4 --- Hepatotoxicity --- p.8 / Chapter 1.3.5 --- Mechanism of toxic effects --- p.9 / Chapter 1.3.5.1 --- Metabolic pathways --- p.10 / Chapter 1.3.5.2 --- Liver tissue-bound pyrroles --- p.16 / Chapter 1.3.5.3 --- Metabolizing enzymes --- p.17 / Chapter 1.3.5.3.1 --- Phase I metabolizing enzymes --- p.17 / Chapter 1.3.5.3.2 --- Phase II metabolizing enzymes --- p.18 / Chapter 1.3.5.4 --- Species and gender specificity toward toxicity --- p.19 / Chapter 1.3.5.5 --- Structure-activity relationships --- p.20 / Chapter 1.4 --- Prevention of PAs-induced toxicity --- p.23 / Chapter 1.4.1 --- Significance of prevention in humans --- p.23 / Chapter 1.4.2 --- Regulations toward preventing toxicity induced by PAs --- p.24 / Chapter 1.5 --- Aim of the present study --- p.25 / Chapter Chapter 2 --- Qualitative and Quantitative Analysis of PA-containing Chinese Medicinal Herbs --- p.26 / Chapter 2.1 --- Materials and equipments --- p.27 / Chapter 2.1.1 --- Herbal materials --- p.27 / Chapter 2.1.2 --- Chemicals and solvents --- p.27 / Chapter 2.1.3 --- Equipment and instrumentation --- p.27 / Chapter 2.2 --- Preparation of herbal extracts。 --- p.28 / Chapter 2.2.1 --- Crude herbal extract --- p.28 / Chapter 2.2.2 --- Total pyrrolizidine alkaloid extract --- p.28 / Chapter 2.3 --- Qualitative and quantitative analysis of Ligularia hodgsonii --- p.29 / Chapter 2.3.1 --- Methods --- p.29 / Chapter 2.3.1.1 --- HPLC-UV condition --- p.29 / Chapter 2.3.1.2 --- HPLC-MS condition --- p.29 / Chapter 2.3.1.3 --- Calibration curve for clivorine --- p.29 / Chapter 2.3.1.4 --- Recovery test --- p.30 / Chapter 2.3.1.5 --- Sample test --- p.30 / Chapter 2.3.2 --- Results and discussions --- p.30 / Chapter 2.3.2.1 --- Qualitative analysis of PAs in Ligularia hodgsonii --- p.30 / Chapter 2.3.2.2 --- Calibration curve for clivorine --- p.35 / Chapter 2.3.2.3 --- Result of the recovery test --- p.37 / Chapter 2.3.2.4 --- Quantification of PAs in Ligularia hodgsonii --- p.37 / Chapter 2.4 --- Qualitative and quantitative analysis of Tussilago farfara --- p.37 / Chapter 2.4.1 --- Methods --- p.38 / Chapter 2.4.1.1 --- HPLC-UV condition --- p.38 / Chapter 2.4.1.2 --- HPLC-MS condition --- p.39 / Chapter 2.4.1.3 --- Calibration curve for senkirkine --- p.39 / Chapter 2.4.1.4 --- Recovery test --- p.39 / Chapter 2.4.1.5 --- Sample test --- p.39 / Chapter 2.4.2 --- Results and discussions --- p.40 / Chapter 2.4.2.1 --- Qualitative analysis of senkirkine in Tussilago farfara --- p.40 / Chapter 2.4.2.2 --- Calibration curve for senkirkine --- p.42 / Chapter 2.4.2.3 --- Result of the recovery test --- p.42 / Chapter 2.4.2.4 --- Quantification of senkirkine in Tussilago farfara --- p.43 / Chapter 2.5 --- Qualitative and quantitative analysis of Gynura segetum --- p.43 / Chapter 2.5.1 --- Methods --- p.43 / Chapter 2.5.1.1 --- HPLC-UV condition --- p.43 / Chapter 2.5.1.2 --- HPLC-MS condition --- p.44 / Chapter 2.5.1.3 --- Calibration curves for senecionine and seneciphylline --- p.44 / Chapter 2.5.1.4 --- Recovery test… --- p.44 / Chapter 2.5.1.5 --- Sample test --- p.44 / Chapter 2.5.2 --- Results and discussions。 --- p.45 / Chapter 2.5.2.1 --- Qualitative analysis of PAs in Gynura segetum --- p.45 / Chapter 2.5.2.2 --- Calibration curves for senecionine and seneciphylline --- p.49 / Chapter 2.5.2.3 --- Result of the recovery test --- p.49 / Chapter 2.5.2.4 --- Quantification of PAs in Gynura segetum --- p.49 / Chapter 2.6 --- Qualitative and quantitative analysis of Crotalaria sessiliflora --- p.50 / Chapter 2.6.1 --- Methods --- p.50 / Chapter 2.6.1.1 --- HPLC-UV condition --- p.50 / Chapter 2.6.1.2 --- HPLC-MS condition --- p.50 / Chapter 2.6.1.3 --- Calibration curve --- p.51 / Chapter 2.6.1.4 --- Recovery test --- p.51 / Chapter 2.6.1.5 --- Sample test --- p.51 / Chapter 2.6.2 --- Results and discussions --- p.52 / Chapter 2.6.2.1 --- Qualitative analysis of monocrotaline in Crotalaria sessiliflora --- p.52 / Chapter 2.6.2.2 --- Calibration curve for monocrotaline --- p.54 / Chapter 2.6.2.3 --- Result of the recovery test --- p.54 / Chapter 2.6.2.4 --- Quantification of PAs in Crotalaria sessiliflora --- p.55 / Chapter 2.7 --- Qualitative and quantitative analysis of Senecio scandens --- p.55 / Chapter 2.7.1 --- Methods --- p.55 / Chapter 2.7.1.1 --- HPLC-UV condition --- p.55 / Chapter 2.7.1.2 --- HPLC-MS condition --- p.56 / Chapter 2.7.1.3 --- Sample test --- p.56 / Chapter 2.7.2 --- Results and discussions --- p.56 / Chapter 2.7.2.1 --- Qualitative analysis of PAs in Senecio scandens --- p.56 / Chapter 2.7.2.2 --- Quantification of PAs in Senecio scandens --- p.59 / Chapter Chapter 3 --- Hepatotoxicity Induced by PA-containing Chinese Medicinal Herbs --- p.60 / Chapter 3.1 --- Materials and methods --- p.62 / Chapter 3.1.1 --- Reagents --- p.62 / Chapter 3.1.2 --- Animal models --- p.62 / Chapter 3.1.3 --- Determination of the serum ALT activity --- p.64 / Chapter 3.1.4 --- Determination of hepatic GSH level --- p.68 / Chapter 3.1.5 --- Quantitation of liver tissue-bound pyrroles --- p.69 / Chapter 3.1.6 --- Histological assessment of liver morphological changes --- p.70 / Chapter 3.1.7 --- Assessment of hepatocytes apoptosis --- p.71 / Chapter 3.1.8 --- Statistical analysis --- p.72 / Chapter 3.2 --- Results and discussion --- p.72 / Chapter 3.2.1 --- Calibration curves --- p.72 / Chapter 3.2.1.1 --- Calibration curve for the determination of serum ALT activity --- p.72 / Chapter 3.2.1.2 --- Calibration curve of determination of hepatic GSH level --- p.73 / Chapter 3.2.2 --- Hepatotoxicity Study of Crotalaria sessiliflora --- p.74 / Chapter 3.2.2.1 --- Hepatotoxicity at 24 hrs after treatment --- p.74 / Chapter 3.2.2.1.1 --- Correlation between dosage of monocrotaline in Crotalaria sessiliflora and amount of liver tissue-bound pyrroles --- p.74 / Chapter 3.2.2.1.2 --- Effects of Crotalaria sessiliflora on the serum ALT activity --- p.79 / Chapter 3.2.2.1.3 --- The correlation between the elevated level of ALT activity and apoptosis of liver cells --- p.85 / Chapter 3.2.2.1.4 --- Effects of Crotalaria sessiliflora on the hepatic GSH level --- p.86 / Chapter 3.2.2.1.5 --- Histological changes of liver sections --- p.89 / Chapter 3.2.2.2 --- Hepatotoxicity within 4 days after administration --- p.92 / Chapter 3.2.2.3 --- Sub-acute hepatotoxicity within 14 days after administration --- p.93 / Chapter 3.2.2.4 --- Conclusion in hepatotoxicity study of Crotalaria sessiliflora --- p.99 / Chapter 3.2.3 --- Hepatotoxicity Study of Gynura segetum --- p.102 / Chapter 3.2.3.1 --- Correlation between the dosage of PAs present in Gynura segetum and the amount of liver tissue-bound pyrroles --- p.102 / Chapter 3.2.3.2 --- "Effects of Gynura segetum on serum ALT activity, hepatic GSH level and morphological changes of liver" --- p.104 / Chapter 3.2.4 --- Hepatotoxicity Study of Ligularia hodgsonii --- p.108 / Chapter 3.2.4.1 --- Correlation between the dosage of PAs present in Ligularia hodgsonii and the formation of liver tissue-bound pyrroles --- p.108 / Chapter 3.2.4.2 --- Effects of Ligularia hodgsonii on serum ALT activity and hepatic GSH level --- p.111 / Chapter 3.2.5 --- Hepatotoxicity Study of Tussilago farfara --- p.113 / Chapter 3.2.6 --- Hepatotoxicity Study of PA-containing medicinal herbs --- p.115 / Chapter 3.2.6.1 --- Correlation between formation of liver tissue-bound pyrroles and elevated serum ALT level --- p.115 / Chapter 3.2.6.2 --- Correlation between dosage of PAs and amount of liver tissue-bound pyrroles --- p.117 / Chapter 3.2.7 --- Test of Liver Tissue-bound Pyrroles as a biomarker using Senecionis scandentis --- p.118 / Chapter 3.2.8 --- Conclusions --- p.120 / Chapter Chapter 4 --- General Conclusions --- p.121 / Chapter 4.1 --- Qualitative and quantitative analysis of five PA-containing medicinal herbs --- p.121 / Chapter 4.2 --- Hepatotoxicity induced by PA-containing medicinal herbs in rats --- p.122 / Chapter 4.3 --- The correlation between hepatotoxicity induced by PA-containing medicinal herbs and the formation of liver tissue-bound pyrroles --- p.123 / Chapter 4.4 --- Threshold of the amount of liver tissue-bound pyrroles related to the hepatotoxicity induced by PA-containing medicinal herbs --- p.123 / References --- p.125

Made available in DSpace on 2016-12-08T16:24:20Z (GMT). No. of bitstreams: 1 PGCA15MA166.pdf: 955554 bytes, checksum: a7513a7a99db2af80031aee98a3385bf (MD5) Previous issue date: 2015-02-20 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / Senecio oleosus is a plant of Astereacea family found in the South and Southeast of Brazil. Species of this genus are recognized to produce liver damage in different animal species, including in human. This study aimed to evaluate the toxicity of S. oleosus in poultry (Gallus gallus domesticus). Green leaves of S. oleosus were collected in the city of Ponte Alta/SC, dried in the shadow, crushed, mixed with feed and fed to four groups of 10 broilers. Group 1 and Group 2 received single doses of 5g/kg and 20g/kg, respectively. Group 3 were given daily doses of 1g/kg for 20 days (amounts corresponding to green plant) and Group 4 (Control) received feed free of S. oleosus. Five broilers from each group were necropsied 30 days after the beginning of the experiment and the five remaining broilers were necropsied 60 days after the start of supply with the plant. Group 1 showed no gross and microscopic changes. In Groups 2 and 3 were observed yellowish color and increase in the volume of the liver, ascites and, microscopically, there was hepatotocyte vacuolation and megalocytosis, fibrosis and biliary epithelium hyperplasia / Senecio oleosus é uma planta da Família Asteraceae encontrada nas regiões Sul e Sudeste do Brasil. Plantas desse gênero são conhecidas por produzirem lesões hepáticas em diferentes espécies de animais, inclusive no homem. Para avaliar a toxicidade do S. oleosus foram conduzidos experimentos em aves (Gallus gallus domesticus). Folhas verdes de S. oleosus foram coletadas no município de Ponte Alta/SC, secadas a sombra, trituradas, misturadas a ração e fornecidas para quatro grupos de 10 frangos de corte. O Grupo 1 e o Grupo 2 receberam doses únicas de 5g/kg e 20g/kg, respectivamente. Ao Grupo 3 foram fornecidas doses diárias de 1g/kg por 20 dias (valores correspondentes a planta verde) e o Grupo Controle recebeu ração livre de S. oleosus. Cincos frangos de cada grupo foram submetidos à necropsia aos 30 dias do início do experimento e os cinco restantes foram submetidos a necropsia aos 60 dias após o início do fornecimento da planta. As aves do Grupo 1 não apresentaram alterações macro e microscópicas. Na necropsia das aves dos Grupos 2 e 3 foi encontrado principalmente aumento de volume e coloração amarela do fígado e ascite. À microscopia foi observado megalocitose, vacuolização de hepatócitos, fibrose e hiperplasia de epitélio biliar

Orientador: Ronaldo Aloise Pilli / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Quimica / Made available in DSpace on 2018-08-04T15:31:59Z (GMT). No. of bitstreams: 1 Pereira_Elaine_M.pdf: 9380147 bytes, checksum: a608a978d005928c3df671eb93be3ed9 (MD5) Previous issue date: 2005 / Mestrado / Quimica Organica / Mestre em Química

Beinwell enthält neben erwünschten Substanzen wie Allantion und Rosmarinsäure auch toxische Pyrrolizidinalkaloide (PA), die die Anwendung limitieren. Ziel der vorliegenden Arbeit war es, die Expression des Gens für Homospermidinsynthase, ein zentrales Enzym der Biosynthese von PA, stark zu verringern. Dafür wurden künstliche microRNA verwendet. Dabei wurden Haarwurzelkulturen mit entsprechenden Transgenen erzeugt. Es wurden Daten zur mRNA-Menge, PA-Gehalt und Allantoingehalt sowie Wachstumsparameter erhoben und zueinander in Beziehung gesetzt.:1. Einleitung 1.1. Gattung Symphytum (Beinwell) 1.2. Pyrrolizidinalkaloide 1.3. Haarwurzeln als Modellsystem und Sekundärstoffproduzenten 1.3.1. Agrobacterium rhizogenes 1.3.2. Haarwurzelkulturen in der Biotechnologie 1.4. Zielstellung 2. Material und Methoden 2.1. Materialien 2.2. Erzeugung und Kultivierung der pflanzlichen Zell- und Organkulturen 2.3. Erzeugung der gentechnisch modifizierten Agrobacterium-Stämme 2.4. Qualitative und quantitative Untersuchungen an Nukleinsäuren 2.4.1. Nukleinsäurepräparation 2.4.2. Gelelektrophorese 2.4.3. Protokolle für die Polymerasekettenreaktion 2.5. Sekundärstoffanalytik 2.5.1. Allantoinbestimmung 2.5.2. Quantifizierung der Pyrrolizidinalkaloide 3. Ergebnisse 3.1. Erzeugung und Untersuchung der Haarwurzelkulturen 3.1.1. Herstellung der Kulturen 3.1.2. Wachstum und Biomasseakkumulation 3.2. Bestimmung der Expression von HSS und DHS mittels qPCR 3.2.1. Methodenetablierung 3.2.2. Expression von HSS und DHS in Haarwurzelkulturen 3.3. Analytik von Sekundärmetaboliten 3.3.1. Bestimmung des Allantoingehalts 3.3.2. Bestimmung des Pyrrolizidinalkaloidgehalts 3.4. Gibt es einen Zusammenhang zwischen HSS-Expression und Alkaloidgehalt? 4. Diskussion 4.1. Erzeugung und Wachstum von Symphytum-Haarwurzelkulturen mit und ohne zusätzliche Transgene 4.2. Produktion von Allantoin in Haarwurzelkulturen 4.3. Beeinflussen die miRNA-Konstrukte die Expression von HSS und DHS? 4.4. Beeinflußt die Regulation der HSS-Expression den PA-Gehalt? 4.5. Beurteilung der PA-Gehalte in Haarwurzelkulturen 4.6. Ausblick 5. Zusammenfassung 6. Verzeichnisse 7. Anhang

This thesis is concerned with the development of transannular iodoamination methodology for the synthesis of pyrrolizidine, indolizidine and tropane alkaloids. Chapter 1 introduces the concept of a ‘transannular cyclisation’ and outlines the utility of such cyclisations in the synthesis of a range of [x.y.z]-azabicyclic alkaloids. Chapter 2 describes the development of a three step lithium amide conjugate addition, ring-closing metathesis and transannular iodoamination protocol for the preparation of the pyrrolizidine scaffold ([3.3.0]-azabicycle). Cyclisation of a hexahydroazocine occurs with concomitant N-debenzylation to give a single diastereoisomer of the corresponding C(7)-iodopyrrolizidine product, which is then elaborated to the known pyrrolizidine, (−)-7a-epi-hyacinthacine A1. Chapter 3 delineates an extension of the methodology described in Chapter 2, and an investigation into accessing alternate diastereoisomeric pyrrolizidine scaffolds via the transannular iodoamination process. These studies culminate in the synthesis of two pyrrolizidine alkaloids, (−)-hyacinthacine A1 and (−)-hyacinthacine A2. Chapter 4 details investigations into the further elaboration of the C(7)-iodopyrrolizidine scaffold synthesised in Chapter 2. A nucleophilic displacement reaction with azide leads to the synthesis of novel 7-deoxy-7-aminoalexine analogues, whilst radical-mediated substitution of the iodide by oxygen allows the synthesis and isolation of the pyrrolizidine alkaloid (−)-1-epi-alexine. Chapter 5 outlines the development of the transannular iodoamination reaction to facilitate the synthesis of the tropane architecture ([3.2.1]-azabicycle). A tandem lithium amide conjugate addition and aldol reaction sequence is followed by ring-closing metathesis to give the required aminocycloheptene. Subsequent treatment with iodine results in transannular cyclisation to give a single iodotropane product which, following elaboration culminates in the synthesis of (+)-pseudococaine. Chapter 6 contains full experimental procedures and characterisation data for all compounds synthesised in Chapters 2, 3, 4 and 5.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / During 36 months a study was conducted in beef cattle farms in Rio Grande do Sul, Brazil to survey outbreaks of Senecio spp. poisoning. The first part of this dissertation reports an outbreak of Senecio brasiliensis poisoning in cows where the main clinical sign was photosensitization. Adult cows that were placed during six months in a 205-hectare pasture heavily infested by Senecio brasiliensis were affected in September, 2013. Photosensitization was seen in 83 out 162 cows. Liver biopsy was performed in all cows under risk and three cows were necropsied. Histopathological findings in the liver related to pyrrolizidine alkaloids toxicosis consisted of fibrosis, megalocytosis and biliary ductal proliferation and were present in 119 of the biopsied cows. Six tested cows had increased serum activity of gamma glutamyl transferase. In the second part of this dissertation, a study of an outbreak of seneciosis in sheep in 2014 is reported. Morphological aspects considered important for the establishment of diagnosis in this species were stressed. Ten out of 860 adult sheep with clinical signs of seneciosis died spontaneously and eight sick ewes were euthanized and necropsied. Clinical signs included weight loss, apathy and photosensitization. Four out of seven tested sheep presented increased serum levels of gamma glutamil transferase and two had elevated alkaline phosphatase serum activity. At necropsy, three out of eight ewes presented slightly irregular toughened livers with multifocal nodules, two out of eight ewes had a whitish liver with thickened fibrotic Glisson s capsule partially adhered to the diaphragm, and three out of eight ewes had smooth and grossly normal livers. Histologically, the main hepatic findings that allowed for the establishment of the diagnosis were megalocytosis, proliferation of bile ducts and fibrosis. Spongy degeneration was observed in the brains of all eight necropsied sheep and was more severe at the cerebellar peduncles, mesencephalon, thalamus, and pons. These are suggested as the portions of election to investigate microscopic lesions of hepatic encephalopathy in sheep with chronic seneciosis. / Durante um período de 24 meses foram realizadas visitas periódicas a propriedades de bovinocultura de corte no Rio Grande do Sul, onde havia a suspeita de intoxicação por Senecio spp. Na primeira parte desta dissertação, relata-se a ocorrência de um surto de intoxicação natural por Senecio brasiliensis em bovinos em que o principal sinal clínico observado foi fotossensibilização. O surto ocorreu em setembro de 2013, acometendo vacas adultas que permaneceram durante os seis meses anteriores em um campo de 205 hectares com abundante infestação por Senecio brasiliensis. Fotossensibilização foi observada em 83 vacas de um total de 162 (51,3%). Realizou-se biópsia hepática em todas as vacas do rebanho e três vacas doentes foram necropsiadas. Os principais achados histopatológicos decorrentes de intoxicação por Senecio brasiliensis nos bovinos biopsiados foram fibrose hepática, megalocitose e proliferação de ductos biliares, observados em 119 animais. Seis vacas testadas apresentaram aumento da atividade sérica da gama glutamil transferase. Na segunda parte da dissertação, foi realizado um estudo de um surto de intoxicação por Senecio spp. em ovinos em 2014, dando-se ênfase aos aspectos morfológicos para o estabelecimento do diagnóstico da intoxicação nessa espécie. Dez ovelhas adultas de um rebanho de 860 ovinos morreram com sinais da doença e oito que estavam doentes foram eutanasiadas e necropsiadas. Os sinais clínicos incluíam emagrecimento, apatia e fotossensibilização. Quatro ovelhas, de um total de sete que foram testadas, apresentaram aumento da atividade sérica da gama glutamil transferase e duas apresentaram fosfatase alcalina sérica elevada. Na necropsia, em três dos oito ovinos necropsiados, o fígado estava levemente mais firme, com superfície capsular ligeiramente irregular e com nódulos pálidos na superfície de corte. Em outros três ovinos o fígado era macroscopicamente normal. Em dois dos oito ovinos necropsiados a cápsula de Glisson era brancacenta devido à fibrose, e parcialmente aderida ao diafragma. Histologicamente, as principais alterações observadas, que contribuíram para o estabelecimento do diagnóstico de intoxicação crônica por alcaloides pirrolizidínicos nas ovelhas deste surto foram hepatomegalocitose, proliferação de ductos biliares e fibrose. Todas as ovelhas apresentavam degeneração esponjosa grave no cerebelo e pedúnculos cerebelares, ponte, mesencéfalo e tálamo. Sugere-se que esses sejam os sítios anatômicos de eleição no encéfalo para a observação dessa lesão em ovinos com intoxicação por Senecio spp.