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Neste trabalho são apresentados os ensaios realizados para a caracterização da cultura de Rauvolfia sellowii Müll. Arg. (Apocynaceae). Foram determinados os parâmetros: pH, peso fresco, peso seco, produção de alcalóides, e consumo de açúcares. A curva de crescimento foi similar às observadas para as culturas de células de outras espécies, estendendo-se até o dia 14. As culturas de R. sellowii não converteram a sacarose em glicose e frutose no meio de cultura. A sacarose foi absorvida diretamente do meio. A absorção completou-se no dia 22. Não houve mudança significativa no pH do meio durante o desenvolvimento celular. A vomilenina foi identificada como o alcalóide majoritário. As alterações na produção deste alcalóide foram avaliadas pela modificações nas condições de cultivo, supressão de auxinas, aumento da concentração de sacarose e mio-inositol, adição de extrato de levedura (eliciador biótico), e adição dos precursores triptofano e triptamina. Em todos os casos houve a supressão da produção de vomilenina. Com exceção da adição de precursores, os tratamentos provocaram o acúmulo de substâncias desconhecidas, possivelmente outros alcalóides. / This work aimed to characterize the growth parameters of a Rauvolfia sellowii Müll. Arg. (Apocynaceae) cell suspension culture. The parameters analyzed were pH variation, biomass accumulation (fresh weight and dry weight), production of alkaloids and sugars uptake. The growth curve was similar to that observed for other plant cells, the culture cycle lasted for 14 days. R. sellowii cultures did not converted sucrose into glucose and fructose in the culture medium. Sucrose was directly taken up from the medium. The complete uptake occurred at day 22. There were no significant changes in the medium pH during the cell development. The major alkaloid accumulated in the cultures was characterized as the vomilenine. The regulation of vomilenine production was also evaluated by changing the culture conditions: depletion of auxins, increase of sucrose and myo-inositol concentration, addition elicitor (yeast extract) and precursor feeding (tryptophan and tryptamine). In all the conditions tested, vomilenine production was suppressed. Moreover, with the exception of precursor feeding, all treatments caused the accumulation of unknown substances, possibly alkaloids.

Neste trabalho são apresentados os ensaios realizados para a caracterização da cultura de Rauvolfia sellowii Müll. Arg. (Apocynaceae). Foram determinados os parâmetros: pH, peso fresco, peso seco, produção de alcalóides, e consumo de açúcares. A curva de crescimento foi similar às observadas para as culturas de células de outras espécies, estendendo-se até o dia 14. As culturas de R. sellowii não converteram a sacarose em glicose e frutose no meio de cultura. A sacarose foi absorvida diretamente do meio. A absorção completou-se no dia 22. Não houve mudança significativa no pH do meio durante o desenvolvimento celular. A vomilenina foi identificada como o alcalóide majoritário. As alterações na produção deste alcalóide foram avaliadas pela modificações nas condições de cultivo, supressão de auxinas, aumento da concentração de sacarose e mio-inositol, adição de extrato de levedura (eliciador biótico), e adição dos precursores triptofano e triptamina. Em todos os casos houve a supressão da produção de vomilenina. Com exceção da adição de precursores, os tratamentos provocaram o acúmulo de substâncias desconhecidas, possivelmente outros alcalóides. / This work aimed to characterize the growth parameters of a Rauvolfia sellowii Müll. Arg. (Apocynaceae) cell suspension culture. The parameters analyzed were pH variation, biomass accumulation (fresh weight and dry weight), production of alkaloids and sugars uptake. The growth curve was similar to that observed for other plant cells, the culture cycle lasted for 14 days. R. sellowii cultures did not converted sucrose into glucose and fructose in the culture medium. Sucrose was directly taken up from the medium. The complete uptake occurred at day 22. There were no significant changes in the medium pH during the cell development. The major alkaloid accumulated in the cultures was characterized as the vomilenine. The regulation of vomilenine production was also evaluated by changing the culture conditions: depletion of auxins, increase of sucrose and myo-inositol concentration, addition elicitor (yeast extract) and precursor feeding (tryptophan and tryptamine). In all the conditions tested, vomilenine production was suppressed. Moreover, with the exception of precursor feeding, all treatments caused the accumulation of unknown substances, possibly alkaloids.

Submitted by Maria de Lourdes Mariano (lmariano@ufscar.br) on 2017-01-09T12:40:50Z No. of bitstreams: 1 VIDALJR_JoãodeDeus_2014.pdf: 5289373 bytes, checksum: 071875c19c5148dc61481da7f8ee7095 (MD5) / Approved for entry into archive by Maria de Lourdes Mariano (lmariano@ufscar.br) on 2017-01-09T12:40:58Z (GMT) No. of bitstreams: 1 VIDALJR_JoãodeDeus_2014.pdf: 5289373 bytes, checksum: 071875c19c5148dc61481da7f8ee7095 (MD5) / Approved for entry into archive by Maria de Lourdes Mariano (lmariano@ufscar.br) on 2017-01-09T12:41:06Z (GMT) No. of bitstreams: 1 VIDALJR_JoãodeDeus_2014.pdf: 5289373 bytes, checksum: 071875c19c5148dc61481da7f8ee7095 (MD5) / Made available in DSpace on 2017-01-09T12:41:13Z (GMT). No. of bitstreams: 1 VIDALJR_JoãodeDeus_2014.pdf: 5289373 bytes, checksum: 071875c19c5148dc61481da7f8ee7095 (MD5) Previous issue date: 2014-02-26 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Rauvolfia L. is a pantropical genus with 70 species distributed throughout America, Africa and Asia, also being present in several remote islands like Hawaii and French Polynesia. Although having an estimated age of 34 million years, the genus has a wide distribution as a result of its remarkable dispersal capacity, given its distribution associated with water bodies and the fact that its fruits are a food source for many bird species, placing Rauvolfia as a highly interesting group for biogeographical studies. Based on this, we produced distribution maps for the 70 species of the genus from occurrence data obtained from herbarium sheets and revision articles, which were digitalized and georeferenced. Species with similar distribution patterns were grouped and their maps were produced and compared with maps of topography and hydrography. We analyzed this distribution together with the most recent phylogenetic hypothesis for the group to infer the distribution of ancestral nodes and to identify barriers in the distribution among sister groups. The phylogeny used was based on chloroplast markers, while divergence timing was estimated by molecular clock, calculated for the whole tribe Vinceae Duby in a parallel study. Softwares S-DIVA and VIP were used for biogeographical reconstructions, which were interpreted within the context of current biogeographical hypotheses. The genus Rauvolfia is mainly limited by climatic and geographic factors. Moreover, while some vicariance episodes were identified, much of the patterns observed for Rauvolfia present distribution were attributed to long-distance dispersal events, reinforcing the role of dispersal in current discussions about angiosperm biogeography. / Rauvolfia L. é um gênero pantropical, composto por 70 espécies distribuídas ao longo de América, África e Ásia, além de estar presente em diversas ilhas remotas como Havaí e Polinésia Francesa. Apesar de possuir uma idade estimada em 34 milhões de anos, o gênero apresenta uma distribuição ampla resultante de uma notável capacidade de dispersão, atribuída a sua distribuição associada a corpos d’água e ao fato de seus frutos serem uma fonte de alimentos para muitas espécies de pássaros, situando Rauvolfia como um grupo altamente interessante para estudos biogeográficos. Partindo deste princípio, nós produzimos mapas de distribuição para as 70 espécies do gênero a partir de dados de ocorrência obtidos de etiquetas de pranchas de herbários e de trabalhos de revisão, que foram digitalizados e georreferenciados. As espécies com padrões similares de distribuição foram agrupadas e seus mapas foram produzidos e comparados com mapas de relevo e hidrografia. Analisou-se então esta distribuição em conjunto com a mais recente hipótese filogenética para o grupo para se inferir a distribuição dos ramos ancestrais e localizar barreiras na distribuição de táxons irmãos. A filogenia utilizada foi baseada em marcadores cloroplastidiais, enquanto a datação foi estimada através de relógio molecular, inferida para toda a tribo Vinceae Duby em um estudo paralelo. Foram utilizados os softwares S-DIVA e VIP para as reconstruções biogeográficas, que foram interpretadas dentro do contexto de hipóteses biogeográficas atuais. O gênero Rauvolfia se apresentou delimitado principalmente por fatores climáticos e geográficos. Além disso, apesar da identificação de alguns episódios de vicariância, grande parte dos padrões observados para a distribuição atual de Rauvolfia foi atribuído a eventos de dispersão a longas distâncias, reforçando o papel desses eventos nas atuais discussões sobre biogeografia de Angiospermas.

Terminalia glabrata et Rauvolfia nukuhivensis sont deux espèces végétales polynésiennes endémiques et menacées d’extinction. T. glabrata cohabite avec T. catappa, commune du Pacifique, ce qui contribuerait à sa vulnérabilité par interfertilité. R. nukuhivensis souffre de problèmes de régénération en raison de stress climatiques, de prédation et d’exploitation anthropique. Des approches de génétique et de chimie ont été developpées afin d’étudier ces deux espèces. Pour T. glabrata, les résultats de « barcoding » ont permis d’établir une très forte proximité génétique avec T. catappa. L’analyse métabolomique a révélé une forte variabilité intraspécifique. Les études génétiques de R. nukuhivensis ont permis de relier des espèces distinctes et d’origines géographiques différentes. Elles ont aussi démontré la présence d’un seul groupe de Rauvolfia dont les individus proviennent des îles de Nuku Hiva et de Ua Huka. Ces travaux ont permis d’établir des relations entre les diversités génétiques et chimiques. L’étude des métabolites secondaires de l’écorce de R. nukuhivensis et de la préparation médicinale a permis d’identifier 13 composés, appartenant à 4 familles chimiques (ajmalanes, sarpaganes, macrolines et  carbolines). 8 de ces composés sont nouvellement identifiés dans la nature et une hypothèse de biosynthèse inédite permettant d’expliquer leur co occurrence chez R. nukuhivensis a été établie. Enfin, ces produits ont été soumis à des tests d’activité pharmacologique. La préparation médicinale a stimulé la prolifération cellulaire et la cicatrisation (tissus FHN). Ces tests ont aussi montré la forte inhibition des canaux ioniques Kv11.1 par les nukuhivensiums. / Terminalia glabrata and Rauvolfia nukuhivensis are endangered Polynesian plant species and endemic. T. glabrata co-exists with a common species from the Pacific T. catappa, thus contributing to its vulnerability by interfertility. R nukuhivensis endures regeneration issues due to climate stress, predation and overexploitation. Hence, these species have been classified as protected species by the authorities and are subjected to conservation plans. Because of their heritage value and their traditional uses, they represent species of cultural importance for the country. Genetics and chemistry approaches were conducted for this study. Concerning T. glabrata, barcoding assays established great similarity with T. catappa. Metabolomics data showed infraspecific variability. Phylogenetic data of Rauvolfia species are consistent with their biogeography, and revealed the existence of an only group of individuals from Nuku Hiva and Ua Huka. Genetic diversity is linked to phytochemical occurence. Investigation of R. nukuhivensis bark metabolites and the traditional remedy led to identification of 13 isolated compounds within 8 new ones, belonging to the ajmalan, sarpagan, macroline and β-carboline skeleton. The co-occurrence of these alkaloid skeletons led to establish an unprecedented biosynthesis route. Finally, isolated compounds and the traditional remedy were submitted to bioassays. The traditional remedy induced cell proliferation and wound healing activities on FHN cells and ion channels Kv11.1 were strongly inhibited by

MSc (Chemistry) / Department of Chemistry / Rauvolfia caffra Sond, a species of evergreen trees and shrubs in the dogbane family, (Apocynaceae), is used as a medicinal plant among traditional communities in many countries for the treatment of malaria, diabetes, coughs, gastrointestinal disturbances, skin infections, impotence, insomnia, diarrhoea, dysentery, scabies, worm infections, and both parasitic and microbial infections. Phytochemical studies have revealed that indole alkaloids are the major constituents of the stem bark. However, there are limited studies linking the compounds with the ethnomedicinal uses. The aim of this study is to isolate and characterize bioactive compounds from Rauvolfia caffra Sond. The highest phenolic content found in a fraction was 16.06±0.125 mg GAE/g, while the highest flavonoid content measured was 9.453±0.081 mg QE/g. In the DPPH free radical scavenging activity and reducing power tests, a lowest IC50 value of 0.022±0.003 μg/mL and IC0.5 value 0.518±0.044 μg/mL, respectively, was found. Six compounds were isolated from the stem bark, including lupeol, a pentacyclic tri-terpenoid isolated for the first time from the genus Rauvolfia; raucaffricine, a rare glycoalkaloid of the monoterpenoid indole class; N-methylsarpagine, an indole alkaloid isolated for the time from R. caffra and spegatrine, an indole alkaloid isolated for the first time from R. caffra, respectively. Concerning antimicrobial activity, the highest activity of a fraction was against B. cereus with MIC values as low as 12.5 mg/mL. One fraction at the tested concentration (250 μg/mL) decreased the viability of Plasmodium falciparum (4.149±6.979 %) with an IC50 value of 6.533 μg/mL. The crude extract and some fractions affected the viability of the Trypanosomes at the tested concentration (250 μg/mL), giving -0.133 ± 0.206 %, 11.334 ± 2.692 %, 1.026 ± 0.143 % and 20.769 ± 9.054 % with IC50 values of 18.50 μg/mL, 14.15 μg/mL, 15.58 μg/mL and 34.71 μg/mL, respectively. Furthermore, the fractions did not show significant cytotoxic effects at a concentration of 50 μg/mL. / NRF