Les intoxications ayant pour cause la prise de toxiques végétaux sont relativement fréquentes. Ces intoxications font suites à une ingestion accidentelle ou volontaire (aliments contaminés, préparations à but « thérapeutique », acte suicidaire, .). Le nombre de ces intoxications est sous-estimé car les signes cliniques sont souvent non spécifiques et les médecins méconnaissent la dangerosité des composés présents et l'incidence des intoxications par les plantes. La sous-estimation du nombre d'intoxications est également liée au manque de méthodes analytiques développées spécifiquement pour la détermination de ces toxines. Enfin de nombreuses molécules restent, à l'heure actuelle, indétectables avec les outils analytiques disponibles. L'objectif de cette thèse était de mettre en lumière l'implication des plantes en toxicologie médico- légale et de proposer des méthodes analytiques pour des composés qui, jusqu'alors, n'étaient pas disponibles. Nous avons développé un screening en CLHP-SM/SM et en CLHP-HRSM/SM pour l'analyse du plus grand nombre de phytotoxines simultanément dans des prélèvements biologiques. Ces méthodes ont été éprouvées par plusieurs cas réels d'intoxication. Nous avons développé la première méthode d'analyse toxicologique de l'hypoglycine A, de l'atractyloside et du carboxyatractyloside applicable en médecine légale. Nous avons enfin approfondi la connaissance des principes toxiques du faux manguier. L'ensemble de ce travail confirme l'importance de l'analyse toxicologique des phytotoxines en médecine légale. Les travaux réalisés ont permis d'approfondir les connaissances sur les toxiques végétaux et de combler certaines lacunes analytiques / Plant poisonings are relatively common. They are caused by accidental or voluntary ingestion (contaminated food, "therapeutic" preparations, suicide attempts.). The number of such poisonings is underestimated, since the clinical signs are often non- specific and doctors are unaware of the incidence of plant poisonings or of the dangerous nature of the compounds present. The lack of analytical methods developed specifically to determine these toxins is another factor in the underestimation of the number of plant poisonings. Furthermore, many molecules are still undetectable with the analytical tools currently available. The aim of this thesis was to highlight the part played by plants in forensic toxicology, and to propose analytical methods for compounds, which up to now had not been available. We have developed a screening method in HPLC-MS/MS and HPLC-HRMS/MS for analysing the highest number of phytotoxins simultaneously in biological samples. The methods were tested on several real poisoning cases. We developed the first toxicological analysis method applicable in forensic medicine for hypoglycin A, atractyloside and carboxyatractyloside. We then deepened our knowledge of the toxic principles of the sea mango. This work, as a whole, confirms the importance of toxicological analysis of phytotoxins in forensic medicine. The work carried out enabled us to improve our knowledge of plant poisons and to fill some analytical gaps

The phytopathogenic fungus <i>Leptosphaeria maculans</i> can cause blackleg disease on crucifers, which results in significant yield losses. Fungal diseases involve interactions between pathogenic fungi and host plants. One aspect of these interactions is mediated by secondary metabolites produced by both fungi and host plants. Phytotoxins and elicitors as well as phytoanticipins and phytoalexins are metabolites produced by fungi and plants, respectively. This thesis describes and discusses the isolation, structure, biological activity and biosynthesis of the secondary metabolites produced by L. maculans.<p> The elicitor-toxin activity bioassay guided isolation of elicitors and phytotoxins produced by <i>L. maculans</i> in a chemically defined medium lead to the isolation of general elicitors, <i>sirodesmin PL</i> (165) and <i>deacetylsirodesmin PL</i> (166), and specific elicitors, <i>cerebrosides C</i> (14) and D (31) from minimum medium (MM) culture under standard conditions. The known phytotoxins sirodesmin PL (165) and deacetylsirodesmin PL (166) induced the production of <i>phytoalexin spirobrassinin</i> (122) in both resistant plant species (brown mustard, <i>Brassica juncea</i> cv. Cutlass) and susceptible plant species (canola, B. napus cv. Westar). A mixture of cerebrosides C (14) and D (31) induced the production of the phytoalexin rutalexin (127) in resistant plant species (brown mustard, B. juncea cv. Cutlass) but not in susceptible plant species (canola, B. napus cv. Westar). New metabolites leptomaculins A-E (267-269, 272 and 274) and deacetylleptomaculins C-E (270, 273 and 275) were isolated from elicitor-phytotoxin active fractions but did not display detectable elicitor activity or phytotoxicity after purification.<p> New metabolites maculansins A (299) and B (300), which were not detected in cultures of L. maculans incubated in MM, were isolated from cultures of <i>L. maculans</i> incubated in potato dextrose broth (PDB). Maculansins A (299) and B (300) displayed higher phytotoxicity on brown mustard than on canola and white mustard (<i>Sinapis alba cv. Ochre</i>) but did not elicit detectable production of phytoalexins in either brown mustard or canola. Metabolite 2,4-dihydroxy-3,6-dimethylbenzaldehyde (212) was produced in higher amount in cultures of L. maculans incubated in PDB than in MM and displayed strong inhibition effect on the root growth of brown mustard and canola. <i>L. maculans</i> incubated in MM amended with high concentration of NaCl produced a new metabolite, 8-hydroxynaphthalene-1-sulfate (293), and a known metabolite, bulgarein (294), which are likely involved in the self-protection. The potential intermediates involved in the biosynthesis of sirodesmin PL (165) were investigated using deuterium labeled precursors: [3,3-2H2]-L-tyrosine (251a), [3,3-2H2]O-prenyl-L-tyrosine (312a), E-[3,3,5,5,5-2H5]O-prenyl-L-tyrosine (312b), [5,5-2H2]phomamide (171a), [2,3,3-2H3]-L-serine (233d) and [5,5-2H2]cyclo-L-tyr-L-ser (252a). Intact incorporation of [5,5-2H2]phomamide (171a) into sirodesmin PL (165) suggested that leptomaculin D (272) and E (274), and deacetylleptomaculin D (273) and E (275) are not intermediates in the biosynthesis of sirodesmin PL (165). They are more likely the catabolic metabolites of sirodesmin PL (165). Phomamide (171), the intermediate in the biosynthetic pathway of sirodesmin PL (165), is likely biosynthesized by coupling of prenyl tyrosine (312) with serine (233) rather than prenylation of cyclo-L-tyr-L-ser (252). When [3,3-2H2]-L-tyrosine (251a), [3,3-2H2]O-prenyl-L-tyrosine (312a), and E-[3,3,5,5,5-2H5]O-prenyl-L-tyrosine (312b) were fed into cultures of L. maculans, a â proton exchange was detected by 1H NMR through intrinsic steric isotope effect, which occurs before the formation of phomamide (171). The biosynthesis and catabolism of sirodesmin PL (165) were proposed based on the results obtained in this work.

The phytopathogenic fungus <i>Leptosphaeria maculans</i> can cause blackleg disease on crucifers, which results in significant yield losses. Fungal diseases involve interactions between pathogenic fungi and host plants. One aspect of these interactions is mediated by secondary metabolites produced by both fungi and host plants. Phytotoxins and elicitors as well as phytoanticipins and phytoalexins are metabolites produced by fungi and plants, respectively. This thesis describes and discusses the isolation, structure, biological activity and biosynthesis of the secondary metabolites produced by L. maculans.<p> The elicitor-toxin activity bioassay guided isolation of elicitors and phytotoxins produced by <i>L. maculans</i> in a chemically defined medium lead to the isolation of general elicitors, <i>sirodesmin PL</i> (165) and <i>deacetylsirodesmin PL</i> (166), and specific elicitors, <i>cerebrosides C</i> (14) and D (31) from minimum medium (MM) culture under standard conditions. The known phytotoxins sirodesmin PL (165) and deacetylsirodesmin PL (166) induced the production of <i>phytoalexin spirobrassinin</i> (122) in both resistant plant species (brown mustard, <i>Brassica juncea</i> cv. Cutlass) and susceptible plant species (canola, B. napus cv. Westar). A mixture of cerebrosides C (14) and D (31) induced the production of the phytoalexin rutalexin (127) in resistant plant species (brown mustard, B. juncea cv. Cutlass) but not in susceptible plant species (canola, B. napus cv. Westar). New metabolites leptomaculins A-E (267-269, 272 and 274) and deacetylleptomaculins C-E (270, 273 and 275) were isolated from elicitor-phytotoxin active fractions but did not display detectable elicitor activity or phytotoxicity after purification.<p> New metabolites maculansins A (299) and B (300), which were not detected in cultures of L. maculans incubated in MM, were isolated from cultures of <i>L. maculans</i> incubated in potato dextrose broth (PDB). Maculansins A (299) and B (300) displayed higher phytotoxicity on brown mustard than on canola and white mustard (<i>Sinapis alba cv. Ochre</i>) but did not elicit detectable production of phytoalexins in either brown mustard or canola. Metabolite 2,4-dihydroxy-3,6-dimethylbenzaldehyde (212) was produced in higher amount in cultures of L. maculans incubated in PDB than in MM and displayed strong inhibition effect on the root growth of brown mustard and canola. <i>L. maculans</i> incubated in MM amended with high concentration of NaCl produced a new metabolite, 8-hydroxynaphthalene-1-sulfate (293), and a known metabolite, bulgarein (294), which are likely involved in the self-protection. The potential intermediates involved in the biosynthesis of sirodesmin PL (165) were investigated using deuterium labeled precursors: [3,3-2H2]-L-tyrosine (251a), [3,3-2H2]O-prenyl-L-tyrosine (312a), E-[3,3,5,5,5-2H5]O-prenyl-L-tyrosine (312b), [5,5-2H2]phomamide (171a), [2,3,3-2H3]-L-serine (233d) and [5,5-2H2]cyclo-L-tyr-L-ser (252a). Intact incorporation of [5,5-2H2]phomamide (171a) into sirodesmin PL (165) suggested that leptomaculin D (272) and E (274), and deacetylleptomaculin D (273) and E (275) are not intermediates in the biosynthesis of sirodesmin PL (165). They are more likely the catabolic metabolites of sirodesmin PL (165). Phomamide (171), the intermediate in the biosynthetic pathway of sirodesmin PL (165), is likely biosynthesized by coupling of prenyl tyrosine (312) with serine (233) rather than prenylation of cyclo-L-tyr-L-ser (252). When [3,3-2H2]-L-tyrosine (251a), [3,3-2H2]O-prenyl-L-tyrosine (312a), and E-[3,3,5,5,5-2H5]O-prenyl-L-tyrosine (312b) were fed into cultures of L. maculans, a â proton exchange was detected by 1H NMR through intrinsic steric isotope effect, which occurs before the formation of phomamide (171). The biosynthesis and catabolism of sirodesmin PL (165) were proposed based on the results obtained in this work.

Made available in DSpace on 2016-06-02T19:29:31Z (GMT). No. of bitstreams: 1 3411.pdf: 2299382 bytes, checksum: 44ff227a0e9cd4657b1aca56caa5179c (MD5) Previous issue date: 2010-12-20 / Universidade Federal de Sao Carlos / The plants release primary and secondary metabolites in the environment that may influence the development of adjacent vegetation, this interference phenomenon is called allelopathy. It is recognized as an important ecological process because it interferes with the structure, distribution, composition and dynamics of plant communities. Studies on allelopathic interactions may be useful in the search for natural phytotoxins are produced by plants or microorganisms to be used as natural herbicides, more specific and less harmful to the environment. According to floristic survey conducted by IBGE, Myrtaceae family is one of the major in the cerrado ecosystem. This study attempted to clarify the following issues: species of Myrtaceae family present in the cerrado (Brazilian savanna) exhibit allelopathic potential? There is a similar allelopathic response within genera? Among the active species, which is most promising for starting a bioprospecting? Some of these species has potential as a herbicide for use in agroforestry or organic production of food? To answer these questions were conducted experiments of seed germination and seedling growth of target species cultivated and weedy, and chemical extraction of active compounds in leaves of Myrcia tomentosa. The leaves of Blepharocalyx salicifolius, Campomanesia pubescens, Eugenia bimarginata, Eugenia klotzschiana, Eugenia myrcianthes, Eugenia punicifolia, Myrcia bella, Myrcia lingua, Myrcia multiflora, Myrcia splendens, Myrcia tomentosa, Psidium australe, Psidium cinereum, Psidium laruotteanum and Psidium rufum were collected , cleaned, crushed and stored in plastic bags until use. In the first stage, were carried out the germination and growth bioassays using aqueous extracts of leaf in a concentration of 10% (w / v). We selected three target species, Lactuca sativa and Solanum lycopersicum, both eudicotyledonous and a monocotyledonous Allium cepa. The extracts were applied to all Myrtaceae species compared with the control (distilled water). In the second phase, bioassays were carried out using aqueous extracts of leaves at concentrations of 5 and 10% (w / v). We selected as target species three weeds Euphorbia heterophylla, Echinochloa crus-galli and Ipomoea grandifolia. The leaf extracts of B. salicifolius, M. multiflora, M. splendens and M. tomentosa were compared to negative control (distilled water) and positive (herbicide Oxyfluorfen). In the third stage were carried out biotests germination, growth, and coleoptile of wheat (Triticum aestivum) to direct chemical extraction of active compounds of M. tomentosa. The extractions were carried out using dried leaves powdered and organic solvents of different polarity, the extracts were fractionated using chromatographic column and purified by HPLC. The isolated and purified compounds were identified in RMN13C and RMN1H, by comparison of spectra. The results showed that aqueous extracts of twelve out of fifteen donor species evaluated showed allelopathic activity and each species showed a distinct behavior regarding the allelopathic activity, no grouping by taxonomic proximity. Evaluating the four species most active on weeds species, was detected the potential and efficiency of the extracts, since these were more toxic to weeds species than the herbicide. The leaf extracts of M. tomentosa stood out for demonstrating high activity even at low concentrations, so was chemical extraction from powdered of leaves of M. tomentosa were isolated two compounds from the ethyl acetate extract: juglanin and avicularin, the difference between ix both is the presence of one hydroxyl attached to carbon 3' in avicularin molecule, this difference has an phytotoxic effect more pronounced in the juglanin. This paper is the first report of the presence of the flavonoids kaempferol (juglanin) and quercetin (avicularin) in species of Myrtaceae of cerrado (Brazilian savanna) and gave foundation for future studies on bioprospecting of the M. tomentosa, which did not find any reports of allelopathic studies, fractionation and identification of chemical compounds. / Os vegetais liberam metabolitos primarios e secundarios no ambiente que podem influenciar no desenvolvimento da vegetacao adjacente, este fenomeno de interferencia e denominado alelopatia. E reconhecida como um processo ecologico importante, pois interfere na estrutura, distribuicao, composicao e dinamica de comunidades vegetais. Estudos sobre interacoes alelopaticas podem ser uteis na busca por fitotoxinas naturais, produzidas por plantas ou microrganismos a serem empregados como herbicidas naturais, mais especificos e menos prejudiciais ao ambiente. De acordo com inventario floristico realizado pelo IBGE a familia Myrtaceae e uma das principais em riqueza e diversidade no ecossistema de cerrado. Assim, este trabalho tentou esclarecer as seguintes questoes: especies da familia Myrtaceae presentes no cerrado apresentam potencial alelopatico? Existe semelhanca na resposta alelopatica dentro dos generos? Dentre as especies ativas, qual a mais promissora para se iniciar uma bioprospeccao? Alguma dessas especies apresenta potencial para utilizacao como herbicida em sistemas agroflorestais ou de producao organica de alimentos? Para responder essas perguntas foram realizados bioensaios de germinacao de diasporos e crescimento de plantulas, de especies alvo cultivadas e infestantes de cultura, e extracao quimica de compostos ativos em folhas de Myrcia tomentosa. As folhas maduras de Blepharocalyx salicifolius, Campomanesia pubescens, Eugenia bimarginata, Eugenia klotzschiana, Eugenia myrcianthes, Eugenia punicifolia, Myrcia bella, Myrcia lingua, Myrcia multiflora, Myrcia splendens, Myrcia tomentosa, Psidium australe, Psidium cinereum, Psidium laruotteanum e Psidium rufum foram coletadas, higienizadas, trituradas e armazenadas em embalagens plasticas ate a utilizacao. Na primeira etapa, os bioensaios de germinacao e crescimento, foram efetuados utilizando-se extratos aquosos de po de folhas em concentracao de 10% (p/v). Como especies receptoras foram selecionadas duas eudicotiledoneas Lactuca sativa e Solanum lycopersicum e, uma monocotiledonea Allium cepa, nas quais foram aplicados os extratos de todas as especies de Myrtaceae comparadas ao controle (agua destilada). Na segunda etapa, os bioensaios foram efetuados utilizando-se extratos aquosos de po de folhas em concentracao de 5 e 10% (p/v). Como especies receptoras foram selecionadas as infestantes de cultura Euphorbia heterophylla, Echinochloa crus-galli e Ipomoea grandifolia, nas quais foram aplicados os extratos de folhas de B. salicifolius, M. multiflora, M. splendens e M. tomentosa, comparadas ao controle negativo (agua destilada) e ao positivo (herbicida Oxifluorfem). Na terceira etapa, foram efetuados biotestes de germinacao, crescimento e de coleoptilo de trigo para direcionar a extracao quimica de compostos ativos de M. tomentosa. Para tanto as extracoes foram feitas empregando-se o po de folhas e os solventes organicos de diferentes polaridades. Os extratos foram fracionados em coluna cromatografica e purificados em CLAE. Os compostos isolados e purificados foram identificados em RMN13C e RMN1H, por comparacao de espectros. Os resultados mostraram que extratos aquosos de doze das quinze especies doadoras avaliadas apresentaram atividade alelopatica e cada especie demonstrou comportamento distinto em relacao a atividade alelopatica, nao houve agrupamento por proximidade taxonomica. Avaliando as quatro especies mais ativas sobre especies invasoras, constatou-se o potencial e a eficiencia dos extratos, uma vez que estes foram mais fitotoxicos as especies invasoras que o herbicida. O extrato foliar de M. tomentosa destacou-se por demonstrar elevada atividade mesmo em baixa concentracao, assim foi realizada a extracao quimica do po folhas desta especie. Foram isolados dois compostos do extrato acetato de etila: juglanina e avicularina, o que as diferencia e a hidroxila ligada ao carbono 3 na molecula de avicularina, esta diferenca produziu efeito fitotoxico mais acentuado da molecula de vii juglanina. O presente trabalho relatou pela primeira vez a presenca dos flavonoides kaempferol (juglanina) e quercetina (avicularina) em uma especie de Myrtaceae do cerrado e deu embasamento para futuros estudos relativos a bioprospeccao da especie Myrcia tomentosa, da qual nao se encontrou nenhum relato de estudos alelopaticos, fracionamento e identificacao de compostos quimicos.

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