ABSTRACT The Genus Casearia is a rich source of cleordane - type diterpenes. To search for practical sources of potential cleordane - type diterpenes, and to study the structure and activity relationship of cleordane - type diterpenes, the twigs and leaves of Casearia membranacea Hance ( Flacourtiaceae ) was collected for phytochemical and anti-tumor investigation. Bioassay-directed fractionation of an ethyl acetate layer of Casearia membranacea has resulted in the isolation of six new clerodane-type diterpenes. The structures of these cleordane-type diterpenes were established and designated as Caseamembrins G¡ãL¡]1¡ã6¡^and their derivatives compounds 7 and 8. The structures of compounds 1¡ã8 were determined by application of NMR techniques included 1H NMR, 13C NMR, DEPT, COSY, HMQC, HMBC, NOESY and another physical methods which include MS, UV, IR and optical rotation, and the published reports about the data of related compounds. The spectral data of 1¡ã8 are in conformity with the basic skeleton of cleordane-type diterpenes previously isolated from Casearia membranacea Hance. The basic structures of 1¡ã8 contain two 6- menbered ring. The structures were identified as rel-(2S,5R,6R,8S,9S,10R, 18S,19R)-2-hydroxy-6-butanoyloxy-18,19-acetyloxy-18,19-epoxy-cleroda-3,13(16),14-triene (Caseamembrin G, 1¡^¡Brel-(2S,5R,6R,8S,9S,10R,18S, 19R)-2-hydroxy-6,18-dibutanoyloxy-19-acetyloxy-18,19-epoxy-cleroda -3,13(16),14-triene (Caseamembrin H, 2¡^¡Brel-(2S,5R,6R,8S,9S,10R,18S ,19R)-2-(2-methylbutanoyloxy)-6-hydroxy-18-methoxy-19-acetyloxy-18,19-epoxy-cleroda-3,13(16),14-triene¡]Caseamembrin I, 3¡^¡Brel-(2R,5R ,6R,8S,9S,10R)-2-(2-methylbutanoyloxy)-6-hydroxy-cleroda-3,13(16),14 -triene-18,19-dicarboxaldehyde ( Caseamembrin J, 4¡^¡Brel-(2S,5R,6R,7R, 8S,9S,10R)-2,7-diacetyloxy-6-hydroxy-cleroda-3,13(16),14-triene-18,19-dicarboxaldehyde¡]Caseamembrin K, 5¡^¡Brel-(2S,5R,6S,7R,8S,9S,10R, 18S,19R)-2-butanoyloxy-6,7-dihydroxy-18-butanoyloxy-19-acetyloxy-18,19-epoxy-cleroda-3,13(16),14-triene¡]Caseamembrin L, 6¡^¡A¤Îrel-(2S,5R ,6R,8S,9S,10R,18S,19R)-2-O-acetyl-6-butanoyloxy-18,19-acetyloxy-18,19-epoxy-cleroda-3,13(16),14-triene ( 7¡^¡Brel-(2S,5R,6R,8S,9S,10R,18S,19 R)-2-O-acetyl-6,18-dibutanoyloxy-19-acetyloxy-18,19-epoxy-cleroda-3,13(16),14-triene¡]8¡^. It is worthy to mention that this is the first report of the isolation of 1¡Ð6 from a natural source and their derivatives compounds 7 and 8. Compound 1 exhibited moderate cytotoxicity against¡]Hepa59T/VGH¡^ ,¡]KB¡^and ¡]Hela¡^cancer cells , but compound 3 and 4¡B5 show no activity against those cancer cell lines.

As part of the continuing work of the International Cooperative Biodiversity Group (ICBG), plant extracts were received from Madagascar. The extracts were screened for cytotoxicity using the A2780 human ovarian cancer cell line bioassay. The crude extract of Casearia nigrescens was fractionated and yielded five known compounds and one new compound that were cytotoxic. Mass spectrometry and 1D and 2D NMR techniques were used to determine the structure of the isolated compounds. The dichloromethane fraction of Casearia nigrescens was weakly active in the A2780 human ovarian cancer cell line bioassay. Further separation of the dichloromethane fraction resulted in the isolation of five known compounds (casearlucin A, caseamenbrol A, rel-18(S),19(R)-diacetoxy-18,19-epoxy-6(R)-hydroxy-2(S)-(2ξ-methylbutanoyloxy)-5(R),8(S),9(S),10(R)-cleroda-3,13(16),14-triene, casearlucin B, and rel-18(S),19(R)-diacetoxy-18,19-epoxy-2(S)-(2ξ-ethylbutanoyloxy)-5(R),8(S),9(S),10(R)-cleroda-3,13(16),14-triene) and one new compound (casearlucin L). Based on the available literature this was the first investigation of the natural products of Casearia nigrescens. The structure of the known compounds was determined by comparison of the NMR, MS, optical rotation, UV, and IR data with the data found in the literature. The structure of casearlucin L was determined by NMR data and comparison with the NMR data for similar known compounds. / Master of Science

SOUSA, Antonio Honório de. Estudo químico de Croton Limae A. P. S. Gomes, M. F. Sales & P. E. Berry (Euphorbiaceae). 2014. 279 f. Tese (Doutorado em química)- Universidade Federal do Ceará, Fortaleza-CE, 2014. / Submitted by Elineudson Ribeiro (elineudsonr@gmail.com) on 2016-10-04T15:43:43Z No. of bitstreams: 1 2014_tese_ahsousa.pdf: 13571451 bytes, checksum: dcde11370b56473403f20f179360abe9 (MD5) / Approved for entry into archive by Jairo Viana (jairo@ufc.br) on 2016-10-10T19:44:57Z (GMT) No. of bitstreams: 1 2014_tese_ahsousa.pdf: 13571451 bytes, checksum: dcde11370b56473403f20f179360abe9 (MD5) / Made available in DSpace on 2016-10-10T19:44:57Z (GMT). No. of bitstreams: 1 2014_tese_ahsousa.pdf: 13571451 bytes, checksum: dcde11370b56473403f20f179360abe9 (MD5) Previous issue date: 2014 / The present work reports the chemical study related to the stem and the roots of Croton limae, collected in Andaraí/BA. The phytochemical investigation of ethanol extract from the stem lead to the isolation of two kaurane-type diterpenes, ent-kaur-16-en-18-oic acid and ent-kaur-16-en-15-oxo-18-oic acid, two clerodane-type diterpenes, 3,12-dioxo-15,16-epoxy-4α-hydroxycleroda-13(16),14-diene and 3-oxo-4α-hydroxy-13,14,15,16-tetranorclerodan-12-oic acid, and the flavonoid quercetin 3-O-β-D-glucopyranoside. The investigation of the hexane extract from the roots lead to the isolation of one triterpene, acetyl aleuritolic acid, the new dimer ent-17(α-pinen-10’-yl)-15-oxokauran-18-oic acid, two news clerodane diterpenes, 3-oxo-15,16-epoxy-4α,12-dihydroxycleroda-13(16),14-diene and 15,16-epoxy-3α,4α,12-trihydroxycleroda-13(16),14-diene, one halimane-type diterpene, 15,16-epoxy-3α,12-dihydroxyhalima-5(10),13(16),14-triene and the mixture of steroids β-sitosterol and stigmasterol. From the ethanol extract of the roots, it was possible to isolate the flavonoids kaempferol 3-O-β-glucopyranoside and ombuine 3-O-β-rutinoside and the three new clerodane diterpenes 3α,4α,15,16-tetrahydroxyclerod-13-ene, 6-(β-D-glucopyranosyl)-3,12-dioxo-15,16-epoxi-4α-hydroxycleroda-13(16),14-dieno and 3-oxo-4α,12-dihydroxy-14,15,16-trinorclerodan-13-oic acid. Four aromatic derivatives amides from ent-kaur-16-en-18-oic acid were prepared through nucleophilic substitutive reactions. The corresponding methyl esters from the ent-kaur-16-en-18-oic acid and ent-kaur-16-en-15-oxo-18-oic acid were also obtained. Two new derivatives from 3,12-dioxo-15,16-epoxi-4α-hidroxicleroda-13(16),14-dieno were prepared through reduction reaction and another one by the biotransformation of diterpene, made by the fungus Rhizopus stolonifer. Some isolated compounds and derivatives were submited to cytotoxic activity using ovarian (OVCAR-8), glioblastoma (SF-295) and colon (HCT-116) cell lines, and the compounds ent-kaur-16-en-15-oxo-18-oic acid and ent-17(α-pinen-10’-yl)-15-oxokauran-18-oic acid registered activity during preliminaries assays. The secondary metabolites were isolated through usual chromatography techniques, using thin layer chromatography, column chromatography, size exclusion chromatography and high performance liquid chromatography. The determination of the structure of the isolated compounds was performed through physical (melting point and optical rotation) and spectrometric techniques, such infrared (IR), high resolution mass spectrometry and nuclear magnetic resonance (NMR), including bidimensional experiments, and comparison with literature data. / O presente trabalho relata o estudo químico do caule e das raízes de Croton limae, coletado no município de Andaraí-BA. A investigação fitoquímica do extrato etanólico do caule levou ao isolamento de dois diterpenos do tipo caurano, ácido ent-caur-16-en-18-oico e ácido ent-caur-16-en-15-oxo-18-oico, dois diterpenos do tipo clerodano, 3,12-dioxo-15,16-epoxi-4α-hidroxicleroda-13(16),14-dieno e ácido 3-oxo-4α-hidroxi-13,14,15,16-tetranorclerodan-12-oico, e do flavonoide 3-O-β-D-glicopiranosilquercetina. A investigação do extrato hexânico das raízes levou ao isolamento de um triterpeno, ácido acetilaleuritólico, do dímero inédito ácido ent-17(α-pinen-10’-il)-15-oxocauran-18-oico, de dois novos diterpenos clerodanos, 3-oxo-15,16-epoxi-4α,12-dihidroxicleroda-13(16),14-dieno e 15,16-epoxi-3α,4α,12-trihidroxicleroda-13(16),14-dieno, um diterpeno do tipo halimano, 15,16-epoxi-3α,12-dihidroxihalima-5(10),13(16),14-trieno, e da mistura dos esteroides β-sitosterol e estigmasterol. Do extrato etanólico das raízes foram isolados dois flavonoides, 3-O-β-D-glicopiranosilcanferol e ombuina-3-O-β-rutinosídeo, e três diterpenos clerodanos inéditos, 3α,4α,15,16-tetrahidroxicleroda-13-eno, 6-(β-D-glicopiranosil)-3,12-dioxo-15,16-epoxi-4α-hidroxicleroda-13(16),14-dieno e ácido 3-oxo-4α,12-dihidroxi-14,15,16-trinorclerodan-13-oico. Foram preparadas quatro amidas aromáticas derivadas do ácido ent-caur-16-en-18-oico e os respectivos ésteres metílicos dos ácidos ent-caur-16-en-18-oico e ent-caur-16-en-15-oxo-18-oico. Foram preparados dois derivados reacionais obtidos através de reações de redução do diterpeno clerodano 3,12-dioxo-15,16-epoxi-4α-hidroxicleroda-13(16),14-dieno e outro através da biotransformação deste diterpeno pelo fungo Rhizopus stolonifer. Alguns compostos isolados e derivados foram submetidos a testes de atividade citotóxica, utilizando linhagens de células tumorais de ovário (OVCAR-8), glioblastoma (SF-295) e colón (HCT-116), onde testes preliminares indicaram que os compostos ent-caur-16-en-15-oxo-18-oico e ácido ent-17(α-pinen-10’-il)-15-oxocauran-18-oico apresentaram atividade. Os metabólitos secundários foram isolados através de técnicas cromatográficas usuais, utilizando cromatografia em camada delgada, cromatografia em coluna, cromatografia por exclusão molecular e cromatografia líquida de alta eficiência. A determinação estrutural foi realizada através de métodos físicos (ponto de fusão e rotação óptica) e do uso de técnicas espectroscópicas e espectrométricas como infravermelho (IV), espectrometria de massas de alta resolução e ressonância magnética nuclear de hidrogênio (RMN 1H) e carbono-13 (RMN 13C), incluindo experimentos bidimensionais, além de comparação com dados da literatura.

Made available in DSpace on 2014-06-11T19:29:09Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-03-05Bitstream added on 2014-06-13T20:38:27Z : No. of bitstreams: 1 passareli_f_me_araiq.pdf: 8997721 bytes, checksum: a69e7054a6bba5e85800139250ac1c79 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Tendo em vista o pequeno acervo de padrões de referência disponíveis para uso no controle de qualidade de fitoterápicos preparados a partir de espécies nativas brasileiras, este trabalho tem como objetivos preparar e caracterizar padrões de alta pureza de casearinas a partir de Casearia sylvestris, uma planta nativa da flora brasileira, que há muito tempo vem sendo usada pela população. A estratégia utilizada para o isolamento das casearinas consistiu no fracionamento do extrato etanólico das folhas por extração em fase sólida utilizando sílica e carvão ativo, cromatografia em coluna em fase normal, além de desenvolvimento de método em CLAE preparativo em fase reversa no modo isocrático e reciclante. Para as análises por CLAE preparativo no modo isocrático, inicialmente, alguns parâmetros como resolução, carga e volume de injeção foram otimizados em coluna analítica e posteriormente foi feita a transferência de escala para coluna preparativa utilizando fator de escalonamento de 21,2. A transferência foi adequada, resultando no isolamento de 10,5 mg da casearina J, 30,0 mg da casearina X e 3,0 mg da casearina Z. Em modo reciclante, foram isolados 60,0 mg da caseargrewiina F. As análises de pureza dessas substâncias foram feitas através de CLAE- DAD, calculando-se primeiramente a pureza de pico, que foi de 100% para as três casearinas analisadas e, posteriormente, a pureza cromatográfica, pelo método de normalização de área, sendo de 85,5, 97,2, e 98,6% para as casearinas J, X e caseargrewiina F, respectivamente. As análises por CLAE-DAD-EM foram importantes para confirmar o resultado de pureza de pico, assim como para obter os dados de massas dos contaminantes presentes nessas amostras. O grau de pureza por DSC não pôde ser obtido, devido à ausência do pico referente à fusão das casearinas, inferindo que as substâncias... / Taking into account the few library of reference standards available for the use in quality control of phytotherapic agents from Brazilian native species, the aim of this work was to prepare and characterize high purity standards of casearins from Casearia sylvestris, a native plant of the Brazilian flora that has been used traditionally by population for a long time as remedy. The strategy to isolate the casearins was to fraction at the ethanolic extract from leaves by solid phase extraction using silica and active charcoal, column chromatography (normal phase) and, additionally, the method development in preparative HPLC (reverse phase) using isocratic e recycling mode. For the analyses by preparative HPLC using isocratic mode, firstly of all some parameters like resolution, amount of sample and volume injection were optimized in analytical column and then the scale-up to preparative column was carried out using a scale factor of 21.2. The scale-up was appropriate, leading to the isolation of 10.5 mg of the casearin J, 30.0 mg of the casearin X and 3.0 mg of the casearin Z. In recycling mode, 60.0 mg of the caseargrewiin F were isolated. The purity analyses of these substances were carried out by HPLC-DAD, firstly measuring the peak purity, wich was 100.0% for the three analyzed casearins, and then, the chromatographic purity, by the area normalization method, wich was 85.5, 97.2 and 98.6% for the casearins J, X and caseargrewiin F, respectively. The HPLC-DAD-MS analyses were important to confirm the peak purity results as well as to obtain the mass data of contaminants wich are usually present in these samples. The measurement of the purity degree by DSC method was not possible due to the absence of fusion peak, thus it suggests an amorphous state for these substances. From the TG/DTG/DTA curves, it was possible to suggest the absence of volatile... ((Complete abstract click electronic access below)

Orientador: Alberto José Cavalheiro / Banca: Fernando Batista da Costa / Banca: João Carlos Palazzo de Mello / Resumo: Tendo em vista o pequeno acervo de padrões de referência disponíveis para uso no controle de qualidade de fitoterápicos preparados a partir de espécies nativas brasileiras, este trabalho tem como objetivos preparar e caracterizar padrões de alta pureza de casearinas a partir de Casearia sylvestris, uma planta nativa da flora brasileira, que há muito tempo vem sendo usada pela população. A estratégia utilizada para o isolamento das casearinas consistiu no fracionamento do extrato etanólico das folhas por extração em fase sólida utilizando sílica e carvão ativo, cromatografia em coluna em fase normal, além de desenvolvimento de método em CLAE preparativo em fase reversa no modo isocrático e reciclante. Para as análises por CLAE preparativo no modo isocrático, inicialmente, alguns parâmetros como resolução, carga e volume de injeção foram otimizados em coluna analítica e posteriormente foi feita a transferência de escala para coluna preparativa utilizando fator de escalonamento de 21,2. A transferência foi adequada, resultando no isolamento de 10,5 mg da casearina J, 30,0 mg da casearina X e 3,0 mg da casearina Z. Em modo reciclante, foram isolados 60,0 mg da caseargrewiina F. As análises de pureza dessas substâncias foram feitas através de CLAE- DAD, calculando-se primeiramente a pureza de pico, que foi de 100% para as três casearinas analisadas e, posteriormente, a pureza cromatográfica, pelo método de normalização de área, sendo de 85,5, 97,2, e 98,6% para as casearinas J, X e caseargrewiina F, respectivamente. As análises por CLAE-DAD-EM foram importantes para confirmar o resultado de pureza de pico, assim como para obter os dados de massas dos contaminantes presentes nessas amostras. O grau de pureza por DSC não pôde ser obtido, devido à ausência do pico referente à fusão das casearinas, inferindo que as substâncias... (resumo completo, clicar acesso eletrônico abaixo) / Abstract: Taking into account the few library of reference standards available for the use in quality control of phytotherapic agents from Brazilian native species, the aim of this work was to prepare and characterize high purity standards of casearins from Casearia sylvestris, a native plant of the Brazilian flora that has been used traditionally by population for a long time as remedy. The strategy to isolate the casearins was to fraction at the ethanolic extract from leaves by solid phase extraction using silica and active charcoal, column chromatography (normal phase) and, additionally, the method development in preparative HPLC (reverse phase) using isocratic e recycling mode. For the analyses by preparative HPLC using isocratic mode, firstly of all some parameters like resolution, amount of sample and volume injection were optimized in analytical column and then the scale-up to preparative column was carried out using a scale factor of 21.2. The scale-up was appropriate, leading to the isolation of 10.5 mg of the casearin J, 30.0 mg of the casearin X and 3.0 mg of the casearin Z. In recycling mode, 60.0 mg of the caseargrewiin F were isolated. The purity analyses of these substances were carried out by HPLC-DAD, firstly measuring the peak purity, wich was 100.0% for the three analyzed casearins, and then, the chromatographic purity, by the area normalization method, wich was 85.5, 97.2 and 98.6% for the casearins J, X and caseargrewiin F, respectively. The HPLC-DAD-MS analyses were important to confirm the peak purity results as well as to obtain the mass data of contaminants wich are usually present in these samples. The measurement of the purity degree by DSC method was not possible due to the absence of fusion peak, thus it suggests an amorphous state for these substances. From the TG/DTG/DTA curves, it was possible to suggest the absence of volatile... ((Complete abstract click electronic access below) / Mestre

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / The present work reports the chemical study related to the stem and the roots of Croton limae, collected in AndaraÃ/BA. The phytochemical investigation of ethanol extract from the stem lead to the isolation of two kaurane-type diterpenes, ent-kaur-16-en-18-oic acid and ent-kaur-16-en-15-oxo-18-oic acid, two clerodane-type diterpenes, 3,12-dioxo-15,16-epoxy-4α-hydroxycleroda-13(16),14-diene and 3-oxo-4α-hydroxy-13,14,15,16-tetranorclerodan-12-oic acid, and the flavonoid quercetin 3-O-β-D-glucopyranoside. The investigation of the hexane extract from the roots lead to the isolation of one triterpene, acetyl aleuritolic acid, the new dimer ent-17(α-pinen-10â-yl)-15-oxokauran-18-oic acid, two news clerodane diterpenes, 3-oxo-15,16-epoxy-4α,12-dihydroxycleroda-13(16),14-diene and 15,16-epoxy-3α,4α,12-trihydroxycleroda-13(16),14-diene, one halimane-type diterpene, 15,16-epoxy-3α,12-dihydroxyhalima-5(10),13(16),14-triene and the mixture of steroids β-sitosterol and stigmasterol. From the ethanol extract of the roots, it was possible to isolate the flavonoids kaempferol 3-O-β-glucopyranoside and ombuine 3-O-β-rutinoside and the three new clerodane diterpenes 3α,4α,15,16-tetrahydroxyclerod-13-ene, 6-(β-D-glucopyranosyl)-3,12-dioxo-15,16-epoxi-4α-hydroxycleroda-13(16),14-dieno and 3-oxo-4α,12-dihydroxy-14,15,16-trinorclerodan-13-oic acid. Four aromatic derivatives amides from ent-kaur-16-en-18-oic acid were prepared through nucleophilic substitutive reactions. The corresponding methyl esters from the ent-kaur-16-en-18-oic acid and ent-kaur-16-en-15-oxo-18-oic acid were also obtained. Two new derivatives from 3,12-dioxo-15,16-epoxi-4α-hidroxicleroda-13(16),14-dieno were prepared through reduction reaction and another one by the biotransformation of diterpene, made by the fungus Rhizopus stolonifer. Some isolated compounds and derivatives were submited to cytotoxic activity using ovarian (OVCAR-8), glioblastoma (SF-295) and colon (HCT-116) cell lines, and the compounds ent-kaur-16-en-15-oxo-18-oic acid and ent-17(α-pinen-10â-yl)-15-oxokauran-18-oic acid registered activity during preliminaries assays. The secondary metabolites were isolated through usual chromatography techniques, using thin layer chromatography, column chromatography, size exclusion chromatography and high performance liquid chromatography. The determination of the structure of the isolated compounds was performed through physical (melting point and optical rotation) and spectrometric techniques, such infrared (IR), high resolution mass spectrometry and nuclear magnetic resonance (NMR), including bidimensional experiments, and comparison with literature data. / O presente trabalho relata o estudo quÃmico do caule e das raÃzes de Croton limae, coletado no municÃpio de AndaraÃ-BA. A investigaÃÃo fitoquÃmica do extrato etanÃlico do caule levou ao isolamento de dois diterpenos do tipo caurano, Ãcido ent-caur-16-en-18-oico e Ãcido ent-caur-16-en-15-oxo-18-oico, dois diterpenos do tipo clerodano, 3,12-dioxo-15,16-epoxi-4α-hidroxicleroda-13(16),14-dieno e Ãcido 3-oxo-4α-hidroxi-13,14,15,16-tetranorclerodan-12-oico, e do flavonoide 3-O-β-D-glicopiranosilquercetina. A investigaÃÃo do extrato hexÃnico das raÃzes levou ao isolamento de um triterpeno, Ãcido acetilaleuritÃlico, do dÃmero inÃdito Ãcido ent-17(α-pinen-10â-il)-15-oxocauran-18-oico, de dois novos diterpenos clerodanos, 3-oxo-15,16-epoxi-4α,12-dihidroxicleroda-13(16),14-dieno e 15,16-epoxi-3α,4α,12-trihidroxicleroda-13(16),14-dieno, um diterpeno do tipo halimano, 15,16-epoxi-3α,12-dihidroxihalima-5(10),13(16),14-trieno, e da mistura dos esteroides β-sitosterol e estigmasterol. Do extrato etanÃlico das raÃzes foram isolados dois flavonoides, 3-O-β-D-glicopiranosilcanferol e ombuina-3-O-β-rutinosÃdeo, e trÃs diterpenos clerodanos inÃditos, 3α,4α,15,16-tetrahidroxicleroda-13-eno, 6-(β-D-glicopiranosil)-3,12-dioxo-15,16-epoxi-4α-hidroxicleroda-13(16),14-dieno e Ãcido 3-oxo-4α,12-dihidroxi-14,15,16-trinorclerodan-13-oico. Foram preparadas quatro amidas aromÃticas derivadas do Ãcido ent-caur-16-en-18-oico e os respectivos Ãsteres metÃlicos dos Ãcidos ent-caur-16-en-18-oico e ent-caur-16-en-15-oxo-18-oico. Foram preparados dois derivados reacionais obtidos atravÃs de reaÃÃes de reduÃÃo do diterpeno clerodano 3,12-dioxo-15,16-epoxi-4α-hidroxicleroda-13(16),14-dieno e outro atravÃs da biotransformaÃÃo deste diterpeno pelo fungo Rhizopus stolonifer. Alguns compostos isolados e derivados foram submetidos a testes de atividade citotÃxica, utilizando linhagens de cÃlulas tumorais de ovÃrio (OVCAR-8), glioblastoma (SF-295) e colÃn (HCT-116), onde testes preliminares indicaram que os compostos ent-caur-16-en-15-oxo-18-oico e Ãcido ent-17(α-pinen-10â-il)-15-oxocauran-18-oico apresentaram atividade. Os metabÃlitos secundÃrios foram isolados atravÃs de tÃcnicas cromatogrÃficas usuais, utilizando cromatografia em camada delgada, cromatografia em coluna, cromatografia por exclusÃo molecular e cromatografia lÃquida de alta eficiÃncia. A determinaÃÃo estrutural foi realizada atravÃs de mÃtodos fÃsicos (ponto de fusÃo e rotaÃÃo Ãptica) e do uso de tÃcnicas espectroscÃpicas e espectromÃtricas como infravermelho (IV), espectrometria de massas de alta resoluÃÃo e ressonÃncia magnÃtica nuclear de hidrogÃnio (RMN 1H) e carbono-13 (RMN 13C), incluindo experimentos bidimensionais, alÃm de comparaÃÃo com dados da literatura.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / Croton cajucara Benth (Euphorbiaceae) is a popular medicinal plant in the Amazon region of Brazil for the treatment of liver and kidney disorders and also to lower blood cholesterol. The trans-dehydrocrotonin (t-DCTN), is the major clerodane diterpene isolated from the stem bark of Croton cajucara, that showed gastroprotective, hypoglycemic and hypolipidemic effects. Since drugs that possess pharmacological properties are often associated with contradictory cardiovascular and hepatic effects with possible cytoprotection or cytotoxicity properties, the present study aimed to examine the effects of t-DCTN (i) in the tests of lethality to Artemia sp and in primary cultures of mesencephalic and hepatic cells in vitro; (ii) on hepatotoxicity induced in vivo by acetaminophen or galactosamine/LPS in mice and (iii) on arterial blood pressure and heart rate in vivo, and on chronotropism and inotropism on isolated preparations of rat atria and aortic rings. The t-DCTN (0.3 to 300 Micromolar) demonstrated low toxicity to Artemia sp (LC50 of 670  80 Micromolar), and manifested no per se cytotoxicity on primary cultures of mesencephalic cells but could effectively revert the reduced cell viability induced by neurotoxin, 6-OHDA (200 Micromolar). On the other hand, t-DCTN (0.3â300 Micromolar) displayed cytotoxicity similar to ethanol (50-400 Milimolar) in primary cultures of hepatocytes. It, however offered hepatoprotection against acetaminophen (500 mg/kg)-induced hepatotoxicity in mice, evidenced from biochemical parameters of hepatic glutathione, and malonaldehyde, and serum AST and ALT levels. Nevertheless, the histological scores in liver tissues were not significantly altered by t-DCTN pretreatment. t-DCTN pretreatment also offered protection against galactosamine/LPS-induced hepatotxcity through restoration of glutathione and reductions in serum AST and ALT levels. In pentobarbital sodium anesthetized normotensive rats, t-DCTN produced hypotensive and bradycardia responses in a dose-dependent manner. The hypotensive effect of t-DCTN (10 mg/kg) was not affected by atropine, propranolol or hexamethonium but was abolished by L-NAME. In isolated right atria, t-DCTN inhibited the spontaneous beating but it was unable to reduce the isoproterenol-induced increase in heart beat. The inotropism was unchanged in the presence of t-DCTN in isolated left atria. In isolated rat aortic rings, t-DCTN relaxed the tonic contraction induced by phenylephrine (1 Micromolar), which was abolished in endothelium denuded or in L-NAME treated tissues. t-DCTN possessed low toxicity to Artemia sp; is devoid of neurotoxicity to mesencephalic cells; either induced hepatotoxicity or hepatoprotection, depending on the models used, and further suggested a possible inhibitory action on cytocrhrome-P450. The hypotensive action of t-DCTN may possibly involve in part the nitric oxide release from endothelium, and in part a direct relaxant effect on vascular smooth muscle. Taken together the data available in literature with the present observations suggest a caution while extrapolating animal data for a promising therapeutic utility of t-DCTN especially in the treatment of hepatic disorders or diabetes associated pathologies. / Croton cajucara Benth. (Euphorbiaceae), planta medicinal da regiÃo AmazÃnica do Brasil, à usada no tratamento de desordens hepÃticas, renais e hipercolesterolemia. A transdesidrocrotonina (t-DCTN), principal diterpeno clerodano isolado da casca do caule do CrÃton cajucara, possui efeito gastroprotetor, hipoglicÃmico e hilpolipidÃmico. Uma vez que substÃncias com esse perfil farmacolÃgico sÃo geralmente associadas a efeitos contraditÃrios sobre o sistema cardiovascular ou hepÃtico com possÃveis propriedades citoprotetora ou citotÃxica, o presente estudo objetivou avaliar os efeitos da t-DCTN (i) no teste de toxicidade para Artemia sp e para cultura de cÃlulas mesencefÃlicas primÃrias e cÃlulas hepÃticas in vitro; (ii) na toxicidade induzida in vivo por acetaminofeno ou galactosamina/LPS em camundongos e (iii) na pressÃo arterial mÃdia e freqÃÃncia cardÃaca in vivo, e no cronotropismo e inotropismo em Ãtrio isolado e anÃis de aorta isolada de rato. A t-DCTN (3-300 Micromolar) possui baixa toxicidade para Artemia sp (CL50 = 670  80 Micromolar) nÃo sendo detectada atividade citotÃxica da t-DTCN sobre cÃlulas mesencefÃlicas primÃrias per si, revertendo a diminuiÃÃo da viabilidade celular induzida por 6-OHDA (200 Micromolar). Por outro lado, a t-DCTN (0,3-300 Micromolar) demonstrou citotoxicidade semelhante ao etanol (50-400 Milimolar) em cultura primÃria de hepatÃcitos de rato de acordo com os teste do MTT, embora tenha apresentado proteÃÃo na hepatotoxicidade induzida por acetaminofeno (500 mg/kg) em camundongos, pelos testes da glutationa, malonaldeÃdo, AST e ALT em contraste, os escores histolÃgicos de tecido hepÃtico de camundongos nÃo foram significativamente alterados pela t-DCTN para cÃlulas tratadas com acetaminofeno, mas mostrou hepatoproteÃÃo na lesÃo induzida por galactosamina/LPS, revertendo os nÃveis de glutationa induzida pela hepatotoxina os nÃveis sÃricos de AST e ALT. Em animais normotensos anestesiados com pentobarbital, a t-DCTN produziu hipotenÃÃo e bradicardia de forma dose dependente. A hipotenÃÃo induzida por t-DCTN (10 mg/kg) nÃo foi alterada pelo prÃ-tratamento com atropina, propranolol e hexametÃnio, sendo reduzida pelo L-NAME. A t-DCTN inibe a freqÃÃncia de contraÃÃo espontÃnea de Ãtrio direito isolado, mas nÃo interfere no aumento dos batimentos atriais induzido pelo isoproterenol. O inotropismo nÃo foi alterado pela t-DCTN em Ãtrio esquerdo isolado. A t-DCTN à capaz de relaxar contraÃÃes submaximais de fenilefrina (1 Micromolar), sendo seu efeito parcialmente inibido pela retirada do endotÃlio ou pela presenÃa de L-NAME. Portanto, a t-DCTN possui baixa toxicidade para Artemia sp, nenhuma neurotoxicidade para cÃlulas mesencefÃlicas, hepatotoxicidade e hepatoproteÃÃo, dependendo da tÃcnica utilizada, sugerindo uma possÃvel aÃÃo inibitÃria no citocromo P450. A hipotenÃÃo induzida pela t-DCTN provavelmente se deve a sua aÃÃo combinada bradicardizante e vasodilatadora sendo esse efeito parcialmente mediado pela liberaÃÃo de NO endotelial e parte por efeitos na musculatura lisa vascular. Em conjunto, os dados disponÃveis na literatura com as presentes observaÃÃes sugerem que a t-DCTN està envolvida com propriedades citotÃxicas bem como citoprotetoras e, portanto, cuidados deve ser tomados quanto a extrapolaÃÃo dos dados em animais para a promissora atividade terapÃutica, especialmente para tratamento de desordens hepÃticas ou patologias associadas à diabetes.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / O presente trabalho descreve o primeiro estudo quÃmico das folhas de Croton pedicellatus Kunth.(Euphorbiaceae), um arbusto, nativo da AmÃrica do Sul, popularmente conhecido como velame, nome dado a vÃrias espÃcies de Croton. O extrato etanÃlico das folhas foi submetido a procedimentos clÃssicos de cromatografia, incluindo Cromatografia LÃquida de Alta EficiÃncia (CLAE), resultando no isolamento, de 15 metabÃlitos secundÃrios: dois sesquiterpenos conhecidos como blumenol A e roseosÃdeo; cinco diterpenos clerodanos furÃnicos: 12,20:15,16-diepoxi-3-hidroxi-7,20-dioxocleroda-13(16),14-dien-18,2-olÃdeo; 12,20:15,16-diepoxi-2,8-dihidroxi-3-acetoxi-7,20-dioxocleroda-13(16),14-dien-18-oato de metila; 12,20:15, 16-diepoxi-2-hidroxi-3-acetoxi-7,20-dioxocleroda-13(16),14-dien-18-oato de metila; 12,20:15,16-diepoxi-3,8-dihidroxi-2-acetoxi-7,20-dioxocleroda-13(16),14-dien-18-oato de metila e 12,20:15,16-diepoxi-3-hidroxi-2-acetoxi-7,20-dioxocleroda-13(16), 14-dien-18-oato de metila; seis flavonoides: alpinumisoflavona; canferol; 3-O-glicopiranosilquercetina; 6ââ-O-p-cumaroil-β-icopiranosilcanferol (tilirosÃdeo), 6ââ-O-p-cumaroil-β-galactopiranosilcanferol e 6ââ-O-p-cumaroil-β-galactopiranosil-3â-metoxi canferol, alÃm dos esterÃides β-sitosterol e seu derivado 3-O-glicosilado. A determinaÃÃo estrutural de todos os metabÃlitos secundÃrios isolados foi realizada atravÃs do emprego de tÃcnicas espectromÃtricas como ressonÃncia magnÃtica nuclear de hidrogÃnio-1 e carbono-13 (RMN de 1H e de 13C uni e bidimensionais), espectroscopia na regiÃo do infravermelho (IV), espectrometria de massas de alta resoluÃÃo, obtida com ionizaÃÃo por electrospray e comparaÃÃo com dados da literatura. / The present work describes for the first time the chemical investigation from leaves of Croton pedicellatus Kunth. (Euphorbiaceae), a shrub, native to South America and popularly known as âvelameâ, name given to several Croton species. The ethanol extract from leaves was subjected to classical chromatographic procedures including high performance liquid chromatography (HPLC), resulting in the isolation of 15 secondary metabolites: two sesquiterpenes known as blumenol A and roseosÃdeo; five clerodane furan diterpenes: 12,20:15,16-diepoxy-3-hydroxy-7,20-dioxocleroda-13(16),14-dien-18,2-olide; methyl-12,20: 15,16-diepoxy-2,8-dihydroxy-3-acetoxy-7,20-dioxocleroda-13(16),14-dien-18-oate; methyl-12.20:15,16-diepoxy-2-hydroxy-3-acetoxy-7,20-dioxocleroda-13 (16),14-dien-18-oate; methyl-12,20:15,16-diepoxy-3,8- dihydroxy-2-acetoxy-7,20 -dioxocleroda-13(16),14-dien-18-oate; methyl-12,20:15,16-diepoxy-3-hydroxy-2-acetoxy-7,20-dioxocleroda-13(16),14-dien-18-oate, and six flavonoids: alpinumisoflavone, kaempferol, quercetin-3-glucoside, Kaempferol-3-β-D-(6-trans-p-coumaroyl)glucopyranoside (tiliroside), kaempferol-3-β-D-(6-trans-pcoumaroyl)galactopyranoside and kaempferol-3â-methoxy-3-β-D-(6-trans-pcoumaroyl)glucopyranoside, besides the steroids β-sitosterol and its derivative 3-glucosylated. The structure determination of all secondary metabolites isolated was performed by spectrometric techniques such as hydrogen-1 and carbon-13 nuclear magnetic resonance (1H NMR and 13C one/two- dimensional), infrared spectrometry (IR), high resolution mass spectrometry and comparison with literature data.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / Sabendo da importÃncia de cÃlulas de mamÃferos em cultura para avaliar a citotoxicidade de novas substÃncias com aÃÃo terapÃutica, o presente trabalho determinou, inicialmente, a atividade citotÃxica por MTT e hemolÃtica de 7 diterpenos clerodanos (Ãcido hardiwickiico, casearinas L, O, U e sua forma degradada, e casearinas X e Y) isolados a partir das folhas de Casearia sylvestris frente a um painel de 9 linhagens de cÃlulas tumorais e a eritrÃcitos de camundongos. Na ausÃncia de hemÃlise de todos os diterpenos, a Casearina U (Cas U) mostrou ser o mais ativo contra cÃlulas tumorais. A atividade citotÃxica da Cas U parece depender do anel formado pelos carbonos C-18 e C-19, uma vez que a hidrÃlise Ãcida e sua abertura levam à diminuiÃÃo ou perda total da bioatividade. Posteriormente, os estudos de mecanismo de aÃÃo com a Cas U (0,4; 0,8 e 1,6 Âg/mL) revelaram reduÃÃo concentraÃÃo-dependente na viabilidade celular por azul de tripan e na sÃntese de DNA por incorporaÃÃo de BrdU, sendo o mecanismo antiproliferativo da Cas U independente de aÃÃo inibitÃria sobre a topoisomerase I. As anÃlises morfolÃgicas feitas por hematoxilina/eosina e por incorporaÃÃo de brometo de etÃdio/laranja de acridina mostraram alteraÃÃo no padrÃo de morte em favorecimento da necrose proporcional à concentraÃÃo, como desintegraÃÃo membranar e picnose nuclear (1,6 Âg/mL), embora tenha ocorrido tambÃm retraÃÃo celular, condensaÃÃo e fragmentaÃÃo da cromatina nucleares (0,4 e 0,8 Âg/mL), sinais condizentes com apoptose. Nos ensaios de fragmentaÃÃo do DNA por citometria de fluxo e de genotoxicidade por cometa, a atividade da Cas U foi concentraÃÃo-dependente e sem diferenciaÃÃo entre cÃlulas normais (linfÃcitos perifÃricos humanos) e cancerosas (HL-60). A avaliaÃÃo antitumoral (10 e 25 mg/kg/dia, intraperitoneal; 25 mg/kg/dia, oral) em camundongos transplantados com Sarcoma 180 revelou atividade apenas na maior dose via intraperitoneal, causando reduÃÃo de 90 % do crescimento tumoral e alteraÃÃes renais incipientes e reversÃveis, enfatizando a potencialidade da Cas U como molÃcula modelo para a sÃntese de novos compostos com propriedades anti-cÃncer. / Knowing the importance of mammalian cell culture in evaluating the cytotoxicity of new substances with therapeutic action, this work initially examined the cytotoxicity (by MTT assay) and hemolytic activity of 7 clerodane diterpenoids (acid hardiwickiico, casearins L, O and U and its degradation product, and casearins X and Y) isolated from leaves of Casearia sylvestris against a panel of 9 tumor cell lines and on mouse erythrocytes. All diterpenes studied showed no hemolytic effect, while casearin U (Cas U) was found to be the most active against tumor cells. Cytotoxic activity of Cas U seems to depend on the ring structure formed by carbons C-18 and C-19, since acid hydrolysis and ring opening led to a decrease or total loss of bioactivity. Subsequently, studies on the mechanism of action of Cas U (0.4, 0.8 and 1.6 Âg/mL) revealed a concentration-dependent decrease in cell viability as determined by trypan blue dye exclusion and in DNA synthesis assayed by BrdU incorporation, where this antiproliferative mechanism of Cas U was not found to be dependent on an inhibitory action on topoisomerase I. Morphological analysis assessed by hematoxylin/eosin and ethidium bromide/acridine orange staining showed alterations in the pattern of cell death toward necrosis according to concentration, as seen by membrane disintegration and pyknotic nuclei (1.6 Âg/mL). However, there also were cell volume reduction, and condensation and fragmentation of nuclear chromatin, consistent signs with apoptosis. DNA fragmentation was examined by flow cytometry and genotoxicity determined with the comet assay, and Cas U activity was found to be dependent on concentration but did not differ between normal (human peripheral lymphocytes) and malignant (HL-60) cells. Antitumor activity of Cas U was tested in mice transplanted with sarcoma 180 (10 and 25 mg/kg/day, intraperitoneally; 25 mg/kg/day, orally), and only the highest intraperitoneal dose was found to effective, leading to 90 % inhibition of tumor growth, with reversible changes in the kidneys. These findings point to the potential of Cas U as a model molecule to synthesize new compounds with anticancer properties.

Submitted by Fernando Bombarda Oda null (fer_oda@fcfar.unesp.br) on 2017-07-20T17:23:30Z No. of bitstreams: 1 Dissertação_Fernando_Oda_Final.pdf: 7648101 bytes, checksum: f78680181e6caaa8a443897d877a8eae (MD5) / Approved for entry into archive by LUIZA DE MENEZES ROMANETTO (luizamenezes@reitoria.unesp.br) on 2017-07-21T18:30:59Z (GMT) No. of bitstreams: 1 oda_fb_me_arafcf_parc.pdf: 1138705 bytes, checksum: a9ec30f0d79f53facd0d1b055e2a1bc4 (MD5) / Made available in DSpace on 2017-07-21T18:30:59Z (GMT). No. of bitstreams: 1 oda_fb_me_arafcf_parc.pdf: 1138705 bytes, checksum: a9ec30f0d79f53facd0d1b055e2a1bc4 (MD5) Previous issue date: 2017-06-19 / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Casearia sylvestris Swartz, conhecida como guaçatonga, está distribuída em todo Brasil e é muito utilizada na medicina popular, com registro etnofarmacológico desde 1877. Muitos desses usos, como o antiofídico, antiulcerogênico e antiinflamatório já foram comprovados em estudos farmacológicos, os quais mostraram também a baixa toxicidade aguda por via oral de seus derivados vegetais. Estudos in vivo revelaram que os diterpenos clerodânicos são os responsáveis pelas atividades antiulcerogênica e anti-inflamatória de C. sylvestris, o que valoriza ainda mais o uso medicinal desta planta. Entretanto, a constatação de que os diterpenos clerodânicos podem sofrer degradação em meio ácido levantaram dúvidas sobre quais substâncias realmente chegam aos receptores e têm atividade: diterpenos ou seus produtos de degradação? Para elucidar essa questão, os diterpenos clerodânicos isolados casearina J (cas J) e O (cas O) foram submetidos isoladamente à degradação ácida em fluido gástrico simulado sem enzimas (PDA) e, posteriormente, metabolizados com enzimas hepáticas da fração S9 (PDA-ME). A metabolização direta dos diterpenos com a fração S9 também foi realizada (PME). As respectivas casearinas remanescentes foram quantificadas em todos os produtos de metabolização. Posteriormente, foram conduzidos ensaios de síntese e/ou liberação de óxido nítrico em macrófagos RAW 267.7 estimulados por LPS in vitro. Para a determinação estrutural dos derivados formados foram utilizadas técnicas cromatográficas (CLAEDAD) e espectrométricas (CLUE-EM, RMN de 1H e 13C). Foram identificadas dez substâncias formadas após degradação ácida da cas J (PDA-J), além da casearina remanescente (5,05%) e seu epímero (PDA-J8). As estruturas propostas desses produtos resultaram da hidrólise do grupamento éster em C-18 (PDA-J1), hidrólise do grupamento éster em C-19 (PDA-J3), formação de dialdeído após quebra do anel diacetálico C (PDA-J2, majoritário), e adições nos grupos aldeídos do PDA-J2 de uma molécula de água (PDJ-7), uma (PDA-J4 e PDA-J4.1) ou duas de etanol (PDJ-5) e hidrólise do éster em C-18 do PDA-J2 (PDJ-6). Foram identificadas nove substâncias formadas após degradação ácida da cas O, além da casearina remanescente (13,1%). Suas estruturas resultaram da hidrólise dos grupos ésteres em C-18 (PDA-O1) e C-19 (PDA-O3), formação do dialdeído após quebra do anel C (PDA-O2, majoritário), adições nos grupos aldeídos do PDA-O2 de uma (PDA-O4 e PDA-O 4.1) ou duas moléculas de etanol (PDA-O5) e hidrólise do éster em C-7 do PDA-O2 (PDA-O5), além da hidrólise do grupamento esterificado em C-6 do PDA-O5 (PDA-O6 ou PDA-O6.1). A metabolização da cas J pela fração S9 gerou duas substâncias correspondentes à quebra do butanoato em C-18 (PME-J1) e formação do seu epímero (PME-J2), permanecendo 36,8% íntegra. Em relação à metabolização da cas O, 36,5% permaneceu íntegra, e ocorreu a formação de uma molécula após quebra dos substituintes esterificados em C-18 e C-19 (PME-O1) e de outra após a formação do dialdeído no anel C (PME-O2). As análises indicaram que as metabolizações enzimáticas dos PDA resultaram nas mesmas substâncias. Os ensaios em macrófagos estimulados por LPS in vitro mostraram que os produtos de degradação ácida e/ou metabolização enzimática das casearinas não têm atividade antiinflamatória in vitro na inibição da liberação e/ou produção de NO, igualmente às casearinas J e O íntegras. / Casearia sylvestris Swartz known as guaçatonga is distributed throughout Brazil and has been widely used in folk medicine since 1877. Many of these uses, such as anti-fungal, antiulcerogenic and anti-inflammatory, have already been proven in pharmacological studies, also demonstrating low acute oral toxicity. In vivo studies have shown that clerodane diterpenes are responsible for the antiulcerogenic and antiinflammatory activities of C. sylvestris, which further enhances the medicinal use of this plant or its derivatives. However, the finding that clerodane diterpenes may suffer degradation in acid medium have raised doubts about the actual substances reached the receptors and carry out activity: diterpenes or their degradation products? In order to elucidate this issue, purified clerodane diterpenes casearin J (cas J) and O (cas O) were subjected to acid degradation in simulated gastric fluid without enzymes (PDA) alone and subsequently metabolized with S9 fraction (PDAME) liver enzymes. A direct metabolization of diterpenes with a S9 fraction (PME) was also performed. Respective remaining casearins were quantified in all the metabolizing products. In the end, macrophage stimulated by LPS in vitro assays of nitric oxide synthesis were conducted using as sample the purified cas J and O, PDA, PME and PDAME. Chromatographic (HPLC-PDA) and spectrometric techniques (UPLC-PDA-MS, 1H and and 13C NMR) were used for the structural determination of the generated derivatives. Ten substances were identified after the acid degradation of cas J (PDA-J), in addition to the remaining casearin (5.05%) and its epimer. Proposed structures of these compounds result of ester group hydrolysis in C-18 (PDA-J1), and in C-19 (PDA-J7), dialdehyde formation after diacetal C ring breaking (PDA-J2, majority), and additions in the aldehyde groups of the PDA-J2 of a water molecule (PDJ-3), one (PDA-J4 and PDA-J4.1) or two ethanol molecules (PDJ-5) and ester hydrolysis in C-7 of PDA-J2 (PDJ-6). Besides the remaining cas O (13.1%), nine compounds were identified after acid degradation. Their structures result of hydrolysis of the esterified groups in C-18 and C-19 (PDA-O1), dialdehyde formation after C-ring rupture (PDA-O2, majority), additions in the PDA-O2 aldehyde groups of one (PDA-O3 and PDA -O3.1) or two ethanol molecules (PDA-O4) and ester hydrolysis at C-6 (PDA-O5). Metabolization of casearin J by the S9 fraction generated two substances corresponding to the breakdown of n-butanoate in C-18 (PME-J1) and formation of cas J epimer (PME-J2), remaining 36.8% intact. In relation to the metabolization of casearin O, 36.5% remained intact and one molecule was formed after breaking the esterified substituents in C-6 and C-7 (PME-O1) and the other after the formation of dialdehyde at C ring (PME-O2). The analyzes indicated that the enzymatic metabolism of the PDA resulted in the same substances. In vitro LPS stimulated macrophage assays have shown that PDA and PME do not have in vitro anti-inflammatory activity of NO inhibition, as well as intact J and O casearins. / CNPq: 130329/2015-0