Avaliou-se a segurança de um fitoterápico, constituído de extratos fluidos de Aristolochia cymbifera (“cassaú”), Plantago major L.(“transagem”), Luehea grandiflora Mart.(“açoita-cavalo”), Myrocarpus frondosus Allemão (“cabreúva”), Piptadenia colubrina Benth (“angico”) (Cassaú Composto® ), através de estudos de toxicidade aguda e subcrônica, tendo como base a resolução Nº 90, de 16 de março de 2004 da ANVISA (Agência Nacional de Vigilância Sanitária). Para o teste de toxicidade aguda, ratos Wistar de ambos os sexos foram tratados por via oral com uma única dose de 26 ml/kg, correspondendo a 20 vezes a dose terapêutica indicada pelo fabricante para seres humanos adultos. Os resultados revelaram haver sinais de toxicidade sistêmica com o aparecimento de ataxia, porém de forma transitória e reversível, não causando interferência no desenvolvimento ponderal dos animais, nos consumos de água e ração, nas produções de urina e fezes, bem como alterações macroscópicas nos órgãos dos animais. Avaliou-se também a exposição a doses repetidas do fitoterápico (toxicidade subcrônica). Constituiram-se 4 grupos experimentais (10 animais/sexo/dose), onde administrou-se por via oral a ratos Wistar, durante 30 dias, doses diárias de 1,3 ml/kg, 6,5 ml/kg e 13 ml/kg, respectivamente a dose terapêutica indicada pelo fabricante para seres humanos adultos, 5 vezes, e 10 vezes a dose terapêutica, além de um grupo controle, onde administrou-se o veículo do fitoterápico. Os resultados revelaram ausência de toxicidade sistêmica, fundamentados na ausência de alterações hematológicas e bioquímicas sangüíneas, bem como peso e análises histopatológicas dos órgãos, nos diferentes grupos. As flutuações nos consumos de água e ração, bem como produções de urina e fezes, não influenciaram de maneira negativa o desenvolvimento ponderal dos animais. Concluiu-se portanto, que a utilização do fitoterápico nas doses e períodos referidos pode ser considerado segura.

Avaliou-se a segurança de um fitoterápico, constituído de extratos fluidos de Aristolochia cymbifera (“cassaú”), Plantago major L.(“transagem”), Luehea grandiflora Mart.(“açoita-cavalo”), Myrocarpus frondosus Allemão (“cabreúva”), Piptadenia colubrina Benth (“angico”) (Cassaú Composto® ), através de estudos de toxicidade aguda e subcrônica, tendo como base a resolução Nº 90, de 16 de março de 2004 da ANVISA (Agência Nacional de Vigilância Sanitária). Para o teste de toxicidade aguda, ratos Wistar de ambos os sexos foram tratados por via oral com uma única dose de 26 ml/kg, correspondendo a 20 vezes a dose terapêutica indicada pelo fabricante para seres humanos adultos. Os resultados revelaram haver sinais de toxicidade sistêmica com o aparecimento de ataxia, porém de forma transitória e reversível, não causando interferência no desenvolvimento ponderal dos animais, nos consumos de água e ração, nas produções de urina e fezes, bem como alterações macroscópicas nos órgãos dos animais. Avaliou-se também a exposição a doses repetidas do fitoterápico (toxicidade subcrônica). Constituiram-se 4 grupos experimentais (10 animais/sexo/dose), onde administrou-se por via oral a ratos Wistar, durante 30 dias, doses diárias de 1,3 ml/kg, 6,5 ml/kg e 13 ml/kg, respectivamente a dose terapêutica indicada pelo fabricante para seres humanos adultos, 5 vezes, e 10 vezes a dose terapêutica, além de um grupo controle, onde administrou-se o veículo do fitoterápico. Os resultados revelaram ausência de toxicidade sistêmica, fundamentados na ausência de alterações hematológicas e bioquímicas sangüíneas, bem como peso e análises histopatológicas dos órgãos, nos diferentes grupos. As flutuações nos consumos de água e ração, bem como produções de urina e fezes, não influenciaram de maneira negativa o desenvolvimento ponderal dos animais. Concluiu-se portanto, que a utilização do fitoterápico nas doses e períodos referidos pode ser considerado segura.

Avaliou-se a segurança de um fitoterápico, constituído de extratos fluidos de Aristolochia cymbifera (“cassaú”), Plantago major L.(“transagem”), Luehea grandiflora Mart.(“açoita-cavalo”), Myrocarpus frondosus Allemão (“cabreúva”), Piptadenia colubrina Benth (“angico”) (Cassaú Composto® ), através de estudos de toxicidade aguda e subcrônica, tendo como base a resolução Nº 90, de 16 de março de 2004 da ANVISA (Agência Nacional de Vigilância Sanitária). Para o teste de toxicidade aguda, ratos Wistar de ambos os sexos foram tratados por via oral com uma única dose de 26 ml/kg, correspondendo a 20 vezes a dose terapêutica indicada pelo fabricante para seres humanos adultos. Os resultados revelaram haver sinais de toxicidade sistêmica com o aparecimento de ataxia, porém de forma transitória e reversível, não causando interferência no desenvolvimento ponderal dos animais, nos consumos de água e ração, nas produções de urina e fezes, bem como alterações macroscópicas nos órgãos dos animais. Avaliou-se também a exposição a doses repetidas do fitoterápico (toxicidade subcrônica). Constituiram-se 4 grupos experimentais (10 animais/sexo/dose), onde administrou-se por via oral a ratos Wistar, durante 30 dias, doses diárias de 1,3 ml/kg, 6,5 ml/kg e 13 ml/kg, respectivamente a dose terapêutica indicada pelo fabricante para seres humanos adultos, 5 vezes, e 10 vezes a dose terapêutica, além de um grupo controle, onde administrou-se o veículo do fitoterápico. Os resultados revelaram ausência de toxicidade sistêmica, fundamentados na ausência de alterações hematológicas e bioquímicas sangüíneas, bem como peso e análises histopatológicas dos órgãos, nos diferentes grupos. As flutuações nos consumos de água e ração, bem como produções de urina e fezes, não influenciaram de maneira negativa o desenvolvimento ponderal dos animais. Concluiu-se portanto, que a utilização do fitoterápico nas doses e períodos referidos pode ser considerado segura.

Lam, Hilary. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 182-191). / Abstracts in English and Chinese. / ACKNOWLEDGEMENTS --- p.I / ABSTRACT --- p.III / TABLE OF CONTENTS --- p.V / LIST OF FIGURES --- p.X / LIST OF TABLES --- p.XIX / LIST OF ABBREVIATIONS --- p.XXII / Chapter Chapter 1: --- LITERATURE REVIEW --- p.1 / Chapter 1. --- Aristolochia --- p.1 / Chapter 1.1 --- "Aristolochia, as a plant" --- p.1 / Chapter 1.2 --- The chemicals in Aristolochia --- p.1 / Chapter 1.3 --- "Aristolochia, as herbal remedies" --- p.3 / Chapter 1.4 --- The Aristolochia poisoning cases --- p.4 / Chapter 1.5 --- The mechanism of AAs --- p.6 / Chapter 1.6 --- Renaming CHN to AAN --- p.6 / Chapter 1.7 --- Banning Aristolochia herbs --- p.7 / Chapter 1.8 --- The possible cause of ANN --- p.8 / Chapter 1.8.1 --- Misuse of Chinese Medicine --- p.8 / Chapter 1.8.2 --- Substitution --- p.9 / Chapter 1.8.3 --- The complexities of the herbal nomenclature --- p.9 / Chapter 1.8.4 --- Adulteration --- p.11 / Chapter 1.9 --- Methods for authenication --- p.12 / Chapter 1.9.1 --- Traditional methods for authentication --- p.12 / Chapter 1.9.2 --- The advantage of using molecular methods --- p.13 / Chapter 1.9.2.1 --- DNA fingerprinting --- p.13 / Chapter 1.9.2.2 --- DNA sequencing --- p.15 / Chapter 1.10 --- Method selection rationale --- p.15 / Chapter 1.11 --- The need for molecular authentication of six medicinal herbs --- p.17 / Chapter 1.11.1 --- The herb Mutong --- p.17 / Chapter 1.11.1.1 --- The poisoning cases reported --- p.19 / Chapter 1.11.1.2 --- Other authentication studies of Mutong --- p.19 / Chapter 1.11.2 --- The herb Muxiang --- p.20 / Chapter 1.11.2.1 --- Chemical profile --- p.21 / Chapter 1.11.2.2 --- Other authentication studies of Muxiang --- p.21 / Chapter 1.11.3 --- The herb Baiying --- p.22 / Chapter 1.11.3.1 --- The poisoning cases reported --- p.23 / Chapter 1.11.3.2 --- Other authentication studies of Baiying --- p.24 / Chapter 1.11.4 --- The herb Fangj --- p.i 24 / Chapter 1.11.4.1 --- Chemical profile --- p.25 / Chapter 1.11.4.2 --- The poisoning cases reported --- p.26 / Chapter 1.11.5 --- The herb Madouling --- p.26 / Chapter 1.11.6 --- The herb Zhushalian --- p.27 / Chapter 1.12 --- Aristolochia specific markers --- p.28 / Chapter 1.13 --- Significance of the research --- p.29 / Chapter Chapter 2: --- OBJECTIVE --- p.30 / Chapter Chapter 3: --- MATERIALS AND METHODS --- p.31 / Chapter 3.1 --- Samples source --- p.31 / Chapter 3.2 --- Total DNA extraction --- p.39 / Chapter 3.2.1 --- Cetyltriethylammonium bromide extraction --- p.39 / Chapter 3.2.2 --- Commercial kit extraction --- p.40 / Chapter 3.3 --- DNA amplification --- p.42 / Chapter 3.4 --- DNA fingerprinting --- p.43 / Chapter 3.4.1 --- DNA concentration determination --- p.43 / Chapter 3.4.2 --- ISSR fingerprinting --- p.44 / Chapter 3.5 --- Agarose gel electrophoresis --- p.45 / Chapter 3.6 --- Purification of PCR product --- p.46 / Chapter 3.7 --- Cloning of PCR product --- p.47 / Chapter 3.7.1 --- Ligation --- p.47 / Chapter 3.7.2 --- Transformation --- p.48 / Chapter 3.7.3 --- Cell cultivation --- p.48 / Chapter 3.7.4 --- Plasmid extraction --- p.49 / Chapter 3.7.5 --- Insert confirmation --- p.49 / Chapter 3.8 --- DNA sequencing --- p.50 / Chapter 3.8.1 --- Cycle sequencing --- p.50 / Chapter 3.8.2 --- Purification of cycle sequencing product --- p.51 / Chapter 3.8.3 --- DNA analysis --- p.51 / Chapter 3.9 --- Sequence analysis --- p.52 / Chapter Chapter 4: --- AUTHENICATION OF MUTONG --- p.53 / Chapter 4.1 --- Results --- p.53 / Chapter 4.1.1 --- Sequence alignment --- p.54 / Chapter 4.1.1.1 --- trnL-trnF sequences --- p.54 / Chapter 4.1.1.2 --- psbA-trnH sequences --- p.55 / Chapter 4.1.2 --- Percentage similarity analysis --- p.64 / Chapter 4.1.3 --- Dendrogram analysis --- p.67 / Chapter 4.2 --- Discussion --- p.73 / Chapter 4.2.1 --- Evaluation of chloroplast trnL-trnF region in differentiation of Mutong --- p.73 / Chapter 4.2.2 --- Evaluation of chloroplast psbA-trnH region in differentiation of Mutong --- p.74 / Chapter 4.2.3 --- Evaluation of using DNA sequencing in differentiation of Mutong --- p.75 / Chapter 4.3 --- Conclusion --- p.77 / Chapter Chapter 5: --- AUTHENICATION OF MUXIANG --- p.78 / Chapter 5.1 --- Results --- p.78 / Chapter 5.1.1 --- Sequence alignment --- p.79 / Chapter 5.1.1.1 --- trnL-trnF sequences --- p.79 / Chapter 5.1.1.2 --- psbA-trnH sequences --- p.80 / Chapter 5.1.2 --- Percentage similarity analysis --- p.88 / Chapter 5.1.3 --- Dendrogram study --- p.91 / Chapter 5.2 --- Discussion --- p.97 / Chapter 5.2.1 --- Evaluation of chloroplast trnL-trnF region in differentiation of Muxiang --- p.97 / Chapter 5.2.2 --- Evaluation of chloroplast psbA-trnH region in differentiation of Muxiang --- p.99 / Chapter 5.3 --- Conclusion --- p.100 / Chapter Chapter 6: --- AUTHENICATION OF BAIYING --- p.102 / Chapter 6.1 --- Results --- p.102 / Chapter 6.1.1 --- Sequence alignment --- p.103 / Chapter 6.1.2 --- Percentage similarity analysis --- p.107 / Chapter 6.1.3 --- Dendrogram analysis --- p.107 / Chapter 6.2 --- Discussion --- p.109 / Chapter 6.2.1 --- Evaluation of chloroplast psbA-trnH region in differentiation of Solarium and Aristolochia --- p.109 / Chapter 6.2.2 --- Molecular authentication of Baiying --- p.112 / Chapter 6.3 --- Conclusion --- p.113 / Chapter Chapter 7: --- AUTHENICATION OF FANGJI --- p.114 / Chapter 7.1 --- Results --- p.114 / Chapter 7.1.1 --- Sequence alignment --- p.115 / Chapter 7.1.1.1 --- trnL-trnF sequence --- p.115 / Chapter 7.1.1.2 --- psbA-trnH sequence --- p.116 / Chapter 7.1.2 --- Percentage similarity analysis --- p.123 / Chapter 7.1.3 --- Dendrogram study --- p.126 / Chapter 7.2 --- Discussion --- p.132 / Chapter 7.2.1 --- Evaluation of chloroplast trnL-trnF region in differentiation of Fangji --- p.132 / Chapter 7.2.2 --- Evaluation of chloroplast psbA-trnH region in differentiation of Fangji --- p.133 / Chapter 7.3 --- Conclusion --- p.133 / Chapter Chapter 8: --- AUTHENICATION OF MADOULING --- p.135 / Chapter 8.1 --- Results --- p.135 / Chapter 8.1.1 --- Sequence alignment --- p.136 / Chapter 8.1.1.1 --- trnL-trnF sequence --- p.136 / Chapter 8.1.1.2 --- psbA-trnH sequence --- p.136 / Chapter 8.1.2 --- Percentage similarity analysis --- p.143 / Chapter 8.1.3 --- Dendrogram study --- p.146 / Chapter 8.2 --- Discussion --- p.152 / Chapter 8.2.1 --- Evaluation of chloroplast trnL-trnF region in differentiation of Madouling --- p.152 / Chapter 8.2.2 --- Evaluation of chloroplast psbA-trnH region in differentiation of Madouling --- p.153 / Chapter 8.3 --- Conclusion --- p.153 / Chapter Chapter 9: --- AUTHENICATION OF ZHUSHALIAN --- p.155 / Chapter 9.1 --- Results --- p.155 / Chapter 9.1.1 --- Sequence alignment --- p.156 / Chapter 9.1.1.1 --- trnL-trnF sequence --- p.156 / Chapter 9.1.1.2 --- psbA-trnH sequence --- p.157 / Chapter 9.1.2 --- Percentage similarity analysis --- p.157 / Chapter 9.1.3 --- Dendrogram study --- p.162 / Chapter 9.2 --- Discussion --- p.166 / Chapter 9.2.1 --- Evaluation of chloroplast trnL-trnF region in differentiation of Zhushalian --- p.166 / Chapter 9.2.2 --- Evaluation of chloroplast psbA-trnH region in differentiation of Zhushalian --- p.171 / Chapter 9.3 --- Conclusion --- p.171 / Chapter Chapter 10: --- ARISTOLOCHIA SPECIFIC MARKER --- p.172 / Chapter 10.1 --- ISSR fingerprinting --- p.172 / Chapter 10.2 --- Discussion --- p.178 / Chapter Chapter 11: --- CONCLUSION --- p.180 / BIBLIOGRAPHY --- p.182 / APPENDIX - MATERIALS PREPARATION --- p.192

There is community concern about toxic contaminants in Chinese herbal medicines. The two areas of contamination that attract most attentions are the nephrotoxic chemical, aristolochic acids found to be present in some Chinese herbs and resulting in renal failure of over 200 patients in Belgium, and heavy metals such as lead, arsenic, cadmium, mercury and chromium, which can cause systemic, CNS, neurological and developmental pathologies. Currently there is a lack of systematic information about the aristolochic acid content in Aristolochia species and related genera, nor have there been any studies on metal contamination conducted in Australia which can provide some scientific basis for assessment of potential risk of Chinese herbal medicines posed to consumers in Australia. This research aimed at addressing these concerns by firstly carrying out a systematic measurement of the contents of aristolochic acids in some relevant raw herbal medicines (CHM) and proprietary medicines (CPM)- 27 CHM, and 7 CPM, and secondly analysing the contents of five heavy metals in 100 CHM, 50 CPM, and 5 commonly used Chinese medicinal formulae (CMF) in the form of raw herbs, and finally evaluating the potential systemic metal toxicity caused by routine ingestions of Chinese medicines in the common form of encapsulated concentrated powder extracts formulated for the treatment of seasonal allergic rhinitis by means of measuring the metal concentrations in blood collected from 71 patients in a randomised double-blind control clinical trial (RCT). Results showed that four of the 37 CHM and two of the 7 CPM contained the banned toxic aristolochic acids. Some of these contaminated medicines could still be purchased over-the-counter in Victoria. Quantitative screening of metal contamination in CHM found that metal concentrations were much lower in the aqueous solutions than in the acid-digested samples. Almost all CHM, CPM and the 5 CMF contained the five heavy metals. Contrary to popular perception, their metal concentrations in the clinically ingested form were extremely low. Their prescribed ingested contents calculated as percentages of the universally recognised regulatory safety standards, the WHO provisional tolerable weekly intake (PTWI), would produce only small percentages of the PTWI for the metal concerned. This was true even when the metal intakes from any forms of Chinese medicines were added to the normal Australian daily dietary metal exposure. These new approaches of analysing the aqueous extractions, as well as interpretation with reference to the WHO regulatory standards and in combination with the Australian normal daily diet, are more relevant and realistic. The RCT in vivo study demonstrated no significant metal accumulation in the blood of both the real treatment group and the placebo control group, thus, attesting to the encouraging finding of the herbal medicine analysis. In conclusion, there is still much to improve in Australia in terms of enforcing the regulation of banning the sale of Chinese herbal medicines that might contain the highly nephrotoxic aristolochic acids. On the other hand, all forms of Chinese medicines in Victoria are safe, and do not appear to pose significant health concerns in terms of metal contamination.

Orientador: Lucia Maria Xavier Lopes / Banca: Conceição de Fátima Alves Olguim / Banca:Maria de Meneses Pereira / Resumo: O presente trabalho descreve o isolamento e a elucidação estrutural dos constuintes químicos de Aristolochia gigantea Mart. A. gigantea desenvolve um forte sistema subterrâneo de caules e raízes (tubérculos e rizomas). Os extratos etanólicos destas partes da planta foram submetidos a cromatografia (CC, CCDP e CLAE), resultando em doze e oito substâncias, respectivamente. Entre as quais, uma nova aristolactama, aristolactama 9-[O-b-glicopiranosil-O-(1 ׳׳® ׳ 2 )-b-glicopiranosídeo], e duas alcamidas, cis- e trans-N-p-coumaroil-3-O-metildopamina juntamente com as substâncias conhecidas alantoína, b-sitosterol, E-nerolidol, (+)-kobusina, (+)- eudesmina, cis- e trans-N-feruloiltiramina, trans-N-coumaroiltiramina, cis- e trans-Nferuloil- 3-O-metildopamina, aristolactama Ia 8-β-O-glicosídeo, aristolactama Ia N-β- glicosídeo, aristolactama IIIa e magnoflorina. Suas estruturas foram determinadas por análises espectroscópicas. Os efeitos dos extratos, da alantoína, (+)-kobusina e (+)-eudesmina na ação das fosfolipases e proteases dos venenos das serpentes Bothrops jararacussu e Crotalus durissus terrificus foram avaliados. A alantoína foi o inibidor mais eficiente das fosfolipases enquanto a (+)-eudesmina e os extratos apresentaram atividade inibitória moderada. Além disso, os extratos, a alantoína e a (+)-eudesmina mostraram atividade moderada para as proteases de Bothrops jararacussu enquanto a (+)-kobusina apresentou maior potencial inibitório. / Abstract: The present work describes the isolation and structural elucidation of the chemical constituents of Aristolochia gigantea Mart. A. gigantea develops a strong system of subterranean stems and roots (tuberous or rhizomatous roots). Ethanol extracts of these plant parts were subjected to chromatography (CC, TLC, and HPLC) to give twelve and eight compounds, respectively. Among which, a new aristolactam, aristolactam 9-O-b-D-glucopyranosyl-(1 ׳ 2→׳׳ )-b-D-glucoside, and two alkamides, cisand trans-N-p-coumaroyl-3-O-methyldopamine together with the known compounds allantoin, E-nerolidol, b-sitosterol, (+)-kobusin, (+)-eudesmin, cis- and trans-Nferuloyltyramine, trans-N-coumaroyltyramine, cis- and trans-N-feruloyl-3-Omethyldopamine, aristolactam Ia 8-b-O-glucoside, aristolactam Ia N-b-D-glucoside, aristolactam IIIa, and magnoflorine. Their structures were determined by spectroscopic analyses. Effects of extracts, allantoin, (+)-kobusin and (+)-eudesmin on action of phospholipases and proteases from venoms of the snakes Bothrops jararacussu and Crotalus durissus terrificus were evaluated. Allantoin was the most efficient inhibitor of phospholipases whereas (+)-eudesmin and the extracts showed moderate inhibitory activity. In addition, the extracts, allantoin, and (+)-eudesmin showed moderate activity on proteases from Bothrops jararacussu whereas (+)- kobusin showed the highest inhibitory potential. / Mestre

Made available in DSpace on 2014-06-11T19:29:11Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-02-21Bitstream added on 2014-06-13T19:58:53Z : No. of bitstreams: 1 holzbach_jc_me_araiq.pdf: 5449837 bytes, checksum: 65991506015b0b72166c7da9e520f8a7 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O presente trabalho descreve o isolamento e a elucidação estrutural dos constuintes químicos de Aristolochia gigantea Mart. A. gigantea desenvolve um forte sistema subterrâneo de caules e raízes (tubérculos e rizomas). Os extratos etanólicos destas partes da planta foram submetidos a cromatografia (CC, CCDP e CLAE), resultando em doze e oito substâncias, respectivamente. Entre as quais, uma nova aristolactama, aristolactama 9-[O-b-glicopiranosil-O-(1 ׳׳® ׳ 2 )-b-glicopiranosídeo], e duas alcamidas, cis- e trans-N-p-coumaroil-3-O-metildopamina juntamente com as substâncias conhecidas alantoína, b-sitosterol, E-nerolidol, (+)-kobusina, (+)- eudesmina, cis- e trans-N-feruloiltiramina, trans-N-coumaroiltiramina, cis- e trans-Nferuloil- 3-O-metildopamina, aristolactama Ia 8-β-O-glicosídeo, aristolactama Ia N-β- glicosídeo, aristolactama IIIa e magnoflorina. Suas estruturas foram determinadas por análises espectroscópicas. Os efeitos dos extratos, da alantoína, (+)-kobusina e (+)-eudesmina na ação das fosfolipases e proteases dos venenos das serpentes Bothrops jararacussu e Crotalus durissus terrificus foram avaliados. A alantoína foi o inibidor mais eficiente das fosfolipases enquanto a (+)-eudesmina e os extratos apresentaram atividade inibitória moderada. Além disso, os extratos, a alantoína e a (+)-eudesmina mostraram atividade moderada para as proteases de Bothrops jararacussu enquanto a (+)-kobusina apresentou maior potencial inibitório. / The present work describes the isolation and structural elucidation of the chemical constituents of Aristolochia gigantea Mart. A. gigantea develops a strong system of subterranean stems and roots (tuberous or rhizomatous roots). Ethanol extracts of these plant parts were subjected to chromatography (CC, TLC, and HPLC) to give twelve and eight compounds, respectively. Among which, a new aristolactam, aristolactam 9-O-b-D-glucopyranosyl-(1 ׳ 2→׳׳ )-b-D-glucoside, and two alkamides, cisand trans-N-p-coumaroyl-3-O-methyldopamine together with the known compounds allantoin, E-nerolidol, b-sitosterol, (+)-kobusin, (+)-eudesmin, cis- and trans-Nferuloyltyramine, trans-N-coumaroyltyramine, cis- and trans-N-feruloyl-3-Omethyldopamine, aristolactam Ia 8-b-O-glucoside, aristolactam Ia N-b-D-glucoside, aristolactam IIIa, and magnoflorine. Their structures were determined by spectroscopic analyses. Effects of extracts, allantoin, (+)-kobusin and (+)-eudesmin on action of phospholipases and proteases from venoms of the snakes Bothrops jararacussu and Crotalus durissus terrificus were evaluated. Allantoin was the most efficient inhibitor of phospholipases whereas (+)-eudesmin and the extracts showed moderate inhibitory activity. In addition, the extracts, allantoin, and (+)-eudesmin showed moderate activity on proteases from Bothrops jararacussu whereas (+)- kobusin showed the highest inhibitory potential.

Orientador: Vera Nisaka Solferini, Jose Roberto Trigo / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-04T04:07:32Z (GMT). No. of bitstreams: 1 Silva-Brandao_KarinaLucas_D.pdf: 3484343 bytes, checksum: 217e21d76131f077485dd6273837a84f (MD5) Previous issue date: 2005 / Resumo: Uma filogenia dos membros Neotropicais da tribo Troidini (Lepidoptera: Papilionidae) foi obtida a partir da seqüência de três genes codificadores de proteínas: dois mitocondriais (COI e COII) e um nuclear (EF-1?). Análises de Parcimônia e Bayesiana de 33 taxa resultaram em árvores bastante similares, independentemente do método utilizado, com os 27 Troidini sempre formando um ramo monofilético. O gênero Battus é grupo irmão dos demais Troidini, seguido pelo ramo formado pelos taxa Paleotropicais (aqui representados por três espécimes). O gênero Euryades é o próximo ramo, e grupo irmão dos Parides. O gênero Parides é monofilético, e está dividido em quatro grupos principais pela análise de Máxima Parcimônia, com o grupo mais basal composto das espécies com cauda do SE do Brasil. Otimizações de Caráter de dados ecológicos e morfológicos sobre a filogenia proposta para os troidines indicaram que o uso de várias espécies de Aristolochia é o caráter ancestral, ao invés do uso de poucas ou de uma única planta hospedeira. Para os outros três caracteres, os estados ancestrais foram ausência de uma cauda longa, floresta como habitat primário e oviposição de ovos solitários ou em grupos dispersos de vários ovos. Uma filogenia baseada no gene cloroplástico matK e na região não-codificadora entre os genes trnL-trnF, e uma relação química baseada no seu padrão de sesquiterpenos, foram propostas para as plantas do gênero Aristolochia do SE do Brasil. Aristolochia é um gênero monofilético, cujo estado ancestral é a presença de ácidos aristolóquicos (AAs) nas suas folhas. Espécies consideradas derivadas mostram apenas ácidos labdanóicos (LAs) nas folhas. A relação fenética recuperada com os sesquiterpenos não concorda com a relação filogenética para as Aristolochia, e três grupos principais podem ser reconhecidos: germacreno-D, germacreno-C e Z-cariofileno. A distribuição de AAs e LAs sobre a filogenia de Aristolochia pode ser vista como um resultado da evolução de defesas contra herbivoria de insetos fitófagos através da história evolutiva destas plantas. Por outro lado, a diferenciação das estruturas dos sesquiterpenos em espécies filogeneticamente próximas pode ser hipotetizada como resultado de adaptações relacionadas à atração de polinizadores. Análises filogenéticas foram conduzidas para se determinar relações e para investigar a evolução de caracteres na evolução da interação entre Troidini e Aristolochia, tentando responder as seguintes questões: 1) qual o padrão de utilização de Aristolochia por estas borboletas? 2) o padrão visto atualmente está relacionado à filogenia das plantas ou à sua composição química? 3) a distribuição geográfica das Aristolochia pode explicar a utilização de plantas hospedeiras observada atualmente? e 4) como a interação entre Troidini e Aristolochia evoluiu? Foi encontrada uma congruência significativa entre as filogenias de Troidini e Aristolochia e entre a filogenia dos Troidini e o quimiograma de Aristolochia quando apenas as associações com as plantas hospedeiras preferenciais de Troidini foram consideradas. No entanto, o padrão atual do uso de plantas hospedeiras não parece ser limitado pela filogenia das mesmas, nem pelos químicos secundários encontrados nestas plantas nem pela sua similaridade geográfica. O uso atual de plantas hospedeiras nestas borboletas parece ser simplesmente oportunístico, com espécies com uma ampla distribuição geográfica usando mais espécies de plantas hospedeiras do que aquelas com distribuição mais restrita / Abstract: A phylogeny of the Neotropical members of the tribe Troidini (Lepidoptera: Papilionidae) was obtained with sequences of three protein-coding genes: two mitochondrial (COI and COII) and one nuclear (EF-1?). Parsimony and Bayesian analyses of 33 taxa resulted in very similar trees regardless of method used, with the 27 troidines always forming a monophyletic clade. The genus Battus is sister group to the remaining troidines, followed by a clade formed by the Paleotropical taxa (here represented by three specimens). The genus Euryades is the next branch, and sister group of Parides. The genus Parides is monophyletic, and is divided into four main groups by Maximum Parsimony analysis, with the most basal group composed of tailed species restricted to SE Brazil. Character optimization of ecological and morphological traits over the phylogeny proposed for troidines indicated that the use of several species of Aristolochia is ancestral over the use of few or a single host-plant. For the other three characters, the ancestral states were the absence of long tails, forest as the primary habitat and oviposition solitary or in loose group of several eggs. A molecular phylogeny based both on the plastid gene matK and on the non-coding region between the genes trnL-trnF, and a chemical relationship based on their sesquiterpenes pattern, were proposed for plants in the genus Aristolochia from SE Brazil. Aristolochia is a monophyletic genus, whose ancestral state is the presence of aristolochic acids (AAs) in the leaves. Species considered derived show only labdanoic acids (LAs) in leaves. The phenetic relationship recovered with sesquiterpenes does not agree with the phylogenetic relationships for Aristolochia, and three main clusters can be recognized, germacrene-D, germacrene-C and Z-caryophyllene groups. The distribution of AAs and LAs over the phylogeny of Aristolochia can be viewed as a result of the evolution of defenses against herbivory of phytophagous insects through the evolutionary history of these plants. On the other hand, the differentiation of sesquiterpene structures in species phylogenetically close can be suggested to be adaptations related to attraction of pollinators. Molecular phylogenetic analyses were conducted to determine relationships and to investigate character evolution in the Troidini/Aristolochia interaction, trying to answer the following questions: 1) what is the present pattern of use of Aristolochia by these butterflies? 2) is the pattern we see today related to the phylogeny of plants or to their chemical composition? 3) can the geographical distribution of Aristolochia explain the host-plant use observed today? and 4) how did the interaction between Troidini and Aristolochia evolve? We found a significant congruence between the phylogenies of Troidini and Aristolochia and between the phylogeny of Troidini and the chemogram of Aristolochia when only the preferred host-plant associations were considered. However, the current pattern of host-plant use of these butterflies does not seem to be constrained by the phylogeny of their food plants, neither by the secondary chemicals in these plants nor by their geographical similarity. The current host-plant use in these butterflies seems to be simply opportunistic, with species with a wide geographical range using more species of host-plants than those with a more restricted distribution / Doutorado / Ecologia / Doutor em Ecologia

Submitted by Renata Lopes (renatasil82@gmail.com) on 2017-04-11T12:35:08Z No. of bitstreams: 1 brunaesteves.pdf: 2439325 bytes, checksum: 09d8c043ba1c10b373f855d1b13f52d1 (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2017-04-17T20:03:33Z (GMT) No. of bitstreams: 1 brunaesteves.pdf: 2439325 bytes, checksum: 09d8c043ba1c10b373f855d1b13f52d1 (MD5) / Made available in DSpace on 2017-04-17T20:03:33Z (GMT). No. of bitstreams: 1 brunaesteves.pdf: 2439325 bytes, checksum: 09d8c043ba1c10b373f855d1b13f52d1 (MD5) Previous issue date: 2017-02-10 / As plantas medicinais são uma grande fonte para o desenvolvimento de novos medicamentos. Neste contexto, o objetivo do presente estudo foi avaliar a atividade anti-inflamatória e antitumoral de extratos de Solidago chilensis (G10, G11, G12), Aristolochia cymbifera (G13) e Piper umbellatum (G14). Estas espécies vegetais foram selecionadas com base em seu extenso uso na medicina popular para o tratamento de queimaduras, doenças reumáticas e inflamatórias, como sedativas, antipiréticas, entre outros. Como metodologia, primeiramente os extratos foram testados para sua citotoxicidade em células RAW264.7 (método de MTT). Em seguida, a atividade antiinflamatória dos extratos foi determinada através da produçao de óxido nítrico (método de GRIESS) e das citocinas TNF-α e MCP-1 (método de ELISA). Uma vez determinado o extrato mais promissor, sua ação foi avaliada sobre a produção de outras citocinas pro-inflamatórias (IL-1β, IL-6 e IL-12), bem como sobre a expressão de moleculas co-estimuladoras, proliferação celular e produção de IFN-. A influência do extrato no processo inflamatório também foi determinada in vivo, utilizando o modelo de edema de orelha. A atividade antitumoral foi determinada em linhagens murinas de câncer de mama (4T1) e de melanoma (B16) pelo método de MTT. Como resultados, nas concentrações não toxicas, o extrato G14 foi o único capaz de reduzir a produção de NO, TNF-α e MCP-1, sendo assim selecionado para os testes posteriores. Este extrato também inibiu a produçao de IL-1β, IL-6 e IL-12, a expressão dos coestimuladores CD40 e CD80, além de reduzir a proliferação de esplenócitos e a produção de IFN- . No ensaio in vivo, o extrato G14 foi efetivo em reduzir o edema, o infiltrado inflamatório, a produção de IL-1β, IL-6 e MCP-1 assim como da enzima mieloperoxidase. Resultados semelhantes foram encontrados nos testes in vitro realizados como o 4-nerolidilcatecol, composto majoritário presente no extrato G14. Em relação a atividade antitumoral, o extrato G12 foi o que mais se destacou, reduzindo a sobrevivência das duas linhagens estudadas em até 50%. Os resultados apresentados nos permitem concluir que tanto o extrato G14 quanto o composto 4NC foram efetivos na redução do processo inflamatório enquanto o extrato G12 se destacou por reduzir a sobrevivência de células tumorais. / Medicinal plants are a great source for the development of new medicines. In this context, the objective of the present study was to evaluate the anti-inflammatory and antitumor activity of Solidago chilensis (G10, G11, G12), Aristolochia cymbifera (G13) and Piper umbellatum (G14) extracts. These plant species were selected based on their extensive use in folk medicine for the treatment of burns, rheumatic and inflammatory diseases, such as sedatives, antipyretics, among others. As a methodology, the extracts were first tested for their cytotoxicity in RAW264.7 cells (MTT method). The anti-inflammatory activity of the extracts was determined by the production of nitric oxide (GRIESS method) and the cytokines TNF-α and MCP-1 (ELISA method). Once the most promising extract was determined, its action was evaluated on the production of other proinflammatory cytokines (IL-1β, IL-6 and IL-12), as well as on the expression of costimulatory molecules, cell proliferation and production of IFN-. The influence of the extract on the inflammatory process was also determined in vivo using the ear edema model. Antitumor activity was determined in murine (4T1) and melanoma (B16) murine cell lines by the MTT method. As a result, in the non-toxic concentrations, the G14 extract was the only one able to reduce the production of NO, TNF-α and MCP-1, being thus selected for the subsequent tests. This extract also inhibited the production of IL-1β, IL-6 and IL-12, the expression of CD40 and CD80 costimulators, as well as reducing splenocyte proliferation and IFN-γ production. In the in vivo assay, G14 extract was effective in reducing edema, inflammatory infiltrate, IL-1β, IL-6 and MCP-1 production as well as the myeloperoxidase enzyme. Similar results were found in the in vitro tests performed as 4-nerolidylcaltechol, the major compound present in the G14 extract. In relation to the antitumor activity, G12 extract was the most outstanding, reducing the survival of the two strains studied by up to 50%. The results show that both the G14 extract and the 4-NC compound were effective in reducing the inflammatory process while the G12 extract was highlighted by reducing the survival of tumor cells.

La diversité moléculaire des huiles essentielles obtenues à partir des racines d'A. longa est répartie essentiellement en sesquiterpènes oxygénés (50,2 - 81,1%) suivie de monoterpènes oxygénés (5,9 - 28,0%), sesquiterpènes hydrocarbonés (0,7 - 18,4%) et monoterpènes hydrocarbonés (0,0 - 0,8%). Mais, l'huile essentielle des parties aériennes est caractérisé par le pin-2-en-8-ol (29,0 - 17,7%) et le 8-acetoxy-pin-2-ene (15,0 - 25,6%). L'ensemble des acides gras identifiés dans les différents organes d'A. longa et de Bryonia dioïca ont été classés en acide gras polyinsaturés (A.G.P.I) variant de 20,1 à 45,4%, en acide gras mono insaturés variant de 10,1 à 41,2%, et en acide gras saturés (A.G.S) variant de 26,7 à 52,2%. Les valeurs pour les deux paramètres (A.G.P.I)/(A.G.S) et (ω-6)/(ω-3) dans les parties aériennes des deux espèces sont de même ordre de grandeur que d'autres espèces comestibles. Par conséquent, les parties aériennes des deux espèces peuvent être considérées comme une source importante et saine d' (ω-6) et (ω-3). L'activité antibactérienne et antifongique des huiles essentielles a été évaluée qualitativement par la méthode de diffusion de disque sur les différentes souches Gram(+), Gram(-) et un champignon (C. albicans). On constate que les souches de Gram(+) sont plus sensibles aux différentes huiles que les souches de Gram(-) et le champignon. Le pouvoir antibactérien des fractions lipidiques d'A. longa et de B. dioïca a été évalué qualitativement quantitativement par la détermination des concentrations minimales inhibitrices (CMI) et des concentrations minimales bactéricides (CMB). Dans la présente étude les résultats des CMI et des CMB montrent que les souches de bactéries testées présentent une sensibilité distincte aux différentes fractions lipidiques. De plus, l'activité antioxydante des extraits a été estimée en appliquant un plan d'expériences de type Plackett-Burman. Les résultats trouvés ont conduit au classement des antioxydants testés par ordre décroissant d'activité, indépendamment de la concentration initiale des radicaux libres en milieu tamponné : l'acide férulique est plus actif que l'extrait de B. dioica, ce dernier est plus puissant que l'extrait d'A. longa / Composition of essential oil is known to depend on intrinsic and extrinsic factors. Experiments were conducted to study the variation in the essential oil composition of Aristolochia longa (Aristolacheae) harvested at different period (August, 2009; September, 2011; March, 2012; and April, 2013). The essential oil composition was assessed by GC–FID and GC–MS analyses. Hence, oxygenated sesquiterpenes (50.2–81.1%) and oxygenated monoterpenes (5.9–28.0%) are the major chemical groups. The antimicrobial properties of essential oils were evaluated against six microorganisms by the disc diffusion method. Increasingly, the essential oil isolated from roots collected in September (2011), exhibited the highest antimicrobial activity against Streptococcus agalactiae (G+) and Enterococcus faecium (G+). The composition of the fatty acids of the roots and aerial parts of A. longa and Bryonia dioïca (Cucurbutaceae) was analyzed by GC-FID and GC-MS. The oils extracted from the aerial parts of both species were rich in polyunsaturated fatty acids with the essential linolenic and linoleic acids being the most prominent compounds. Oleic and linoleic acids were the majors fatty acids in the roots of both species. The antibacterial activity, minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the lipid extracts were determined against a panel of five bacterial strains. The antibacterial activity of the root lipid extracts was particularly important against Enterococcus feacium (G+) (CMI value of 125 μg/mL; CMB values > 250 μg/mL) and Streptococcus agalactiae (G+) (CMI value of 125 μg/mL; CMB values 250 μg/mL for A. longa roots). These results indicate that A. longa and B. dioïca could be considered as good sources of essential fatty acids which can act as natural antibacterial agents. An experimental design is an experimental set-up to simultaneously evaluate several factors at given numbers of levels in a predefined number of experiments, and also to provide maximum chemical information by analysing chemical data. So, Plackett–Burman (PB) experimental was used to appreciate the stability of DPPH and to estimate antioxidant activity of ferulic acid and the plant extracts. Hence, to increase DPPH absorbance at 190μM the buffer type, pH medium, solvent type and reaction time should be maintained at its lower level while buffer concentration, temperature, buffer ratio should be kept at its high level. While, to increase DPPH absorbance at 50 μM is obtained with buffer acetate, lower temperature and short reaction time. Further, the radical scavenging activity of the plant extracts and ferulic acid as a pure compound is examined in the selected conditions by PB design