Nous avons porté une attention particulière à 2 marqueurs chimiotaxonomiques de la famille botanique des orchidées, les stilbénoïdes et dérivés glucosyloxybenzyliques d’acides organiques, et les avons recherchés dans deux espèces tropicales. L’investigation phytochimique de Cyrtopodium paniculatum (Ruiz & Pav.) Garay a permis l’isolement de 35 stilbénoïdes dont 12 nouvellement décrits. L’évaluation de leur activité cytotoxique in vitro sur la lignée cellulaire cancéreuse UG-87 montre que seuls les stilbénoïdes dimères semblent aptes à induire une perturbation de l’intégrité cellulaire. Nous avons également exploré la composition chimique d’Arundina graminifolia (D.Don) Hochr. De ses parties aériennes, 9 stilbénoïdes (dont 2 nouvelles structures ont été isolés) ainsi que des 7 nouveaux dérivés hydroxybenzyliques de l’acide (R) 2-benzylmalique, les arundinosides. De ses parties souterraines, plus de 40 arundinosides ont également pu être identifiés. Nous avons finalement évalué le potentiel cytoprotecteur de stilbénoïdes et arundinosides d’ A. graminifolia sur lignée cellulaire PC12, sans qu’aucune activité relevée n’ait été concluante. / The aim of the study was focused on two chemotaxonomic markers of the family Orchidaceae, namely stilbenoids and the glucosyloxybenzyl derivatives of organic acids. We specifically explored these two chemical families in two tropical orchid species. The chemical investigation of Cyrtopodium paniculatum (Ruiz & Pav.) Garay led to the isolation of 35 stilbenoids, including 12 newly described compounds. Their cytotoxic activity on UG-87 cancer cell line was evaluated. The result obtained demonstrated that stilbenoids dimers were the only compounds capable of disturbing the cellular integrity. In parallel, we explored the chemical composition of Arundina graminifolia (D.Don) Hochr. From its aerial parts, 9 stilbenoids (including 2 newly described ones) together with 7 new (R) glucosyloxybenzyl 2-benzylmalate derivatives, the arundinosides, were isolated. From its underground parts, over 40 arundinoside-like compounds have also been identified. The cytoprotective evaluation of stilbenoids and arundinosides against beta-amyloid induce toxicity on PC12 cells was evaluated, however, no significant result was obtained from the biological evaluation.

Submitted by REBECA PREVIATE MEDINA null (rebecapm_2@hotmail.com) on 2016-07-12T01:22:08Z No. of bitstreams: 1 tese completa final.pdf: 17929997 bytes, checksum: 2ca69cdbb8845123f617feff1388a3eb (MD5) / Approved for entry into archive by Ana Paula Grisoto (grisotoana@reitoria.unesp.br) on 2016-07-12T20:30:01Z (GMT) No. of bitstreams: 1 medina_rp_dr_araiq_par.pdf: 1819041 bytes, checksum: 6a9b96f8294269d0ff2fc5f05f6dd74b (MD5) / Made available in DSpace on 2016-07-12T20:30:01Z (GMT). No. of bitstreams: 1 medina_rp_dr_araiq_par.pdf: 1819041 bytes, checksum: 6a9b96f8294269d0ff2fc5f05f6dd74b (MD5) Previous issue date: 2016-06-22 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Os Produtos Naturais desempenham um papel dominante na descoberta e desenvolvimento de novos fármacos para o tratamento de diversas doenças. Com isso, a busca por novas fontes de substâncias bioativas tem se intensificado, com destaque para estudos realizados com organismos e micro-organismos marinhos. As algas vermelhas marinhas (Rhodophyta) são as principais produtoras de metabólitos secundários biologicamente ativos dentre as algas e produzem principalmente substâncias halogenadas. Dentre os micro-organismos, podemos destacar os fungos endofíticos de algas marinhas, pois apresentam alta diversidade de espécies e diversidade estrutural dos metabólitos secundários, os quais têm mostrado potencial antitumoral, antimicrobiano e antifúngico. Sendo assim, este trabalho teve como objetivo explorar o potencial das algas vermelhas Asparagopsis taxiformis (estágio Falkenbergia) e Pyropia spiralis, bem como de seus fungos endofíticos, como fontes de substâncias bioativas. O estudo de A. taxiformis e de P. spiralis levou à identificação de dezesseis substâncias por CG-EM, como os ácidos eicosapentaenóico e araquidônico, além de nucleosídeos, como a uridina e a timidina, obtidos de A. taxiformis, e a uridina e a inosina, obtidas de P. spiralis. Os extratos brutos e frações das algas estudadas foram avaliados frente ao fungo fitopatogênico Cladosporium sphaerospermum, frente à enzima acetilcolinesterase e frente a linhagens tumorais de carcinoma de ovário (OVCAR-8), adenocarcinoma de cólon humano (HCT-116) e glioblastoma - humano (SF-295), sendo que os mesmos não apresentaram atividades antifúngica e anticolinesterásica significativas e apenas as frações hexânicas e butanólicas de ambas as algas apresentaram atividade citotóxica moderada frente à linhagem SF-295. Em relação ao estudo dos micro-organismos associados às algas, foi possível isolar sete linhagens de fungos endofíticos da alga A. taxiformis e três linhagens de P. spiralis, que foram cultivadas em meio de batata e dextrose (MBD) preparado com água do mar ou água ultra pura. Após o período de incubação, foram obtidos os extratos brutos em acetato de etila, que foram avaliados quanto ao perfil químico por CLAE e RMN, e perfil biológico por meio dos bioensaios in vitro. Após a prospecção inicial, Nemania bipapillata (AT-05), Sarocladium strictum (PS-01), Annulohypoxylon stygium (AT-03) e Hypoxylon sp. (AT-06) foram selecionados para investigação. O estudo de N. bipapillata, cultivado em água do mar e MBD, levou ao isolamento da dicetopiperazina ciclo(Pro-Tir), tirosol e das isocumarinas, (3R)-5-hidroximetil-meleína e (3R)-5-carbóxi-meleína. Ao cultivar essa linhagem fúngica em água ultra pura e MDB, foram isolados dois norsesquiterpenos e três sesquiterpenos do tipo botriano inéditos na literatura e um conhecido, 4β-acetoxi-9β,10β,15α-triidroxiprobotridial. A análise do extrato bruto de S. strictum, cultivado em água ultra pura e MDB, levou à identificação em mistura de duas dicetopiperazinas e ao isolamento da isoflavona daidzeína e de seis ftalatos naturais, dos quais dois ainda não foram descritos na literatura e nem sintetizados. A. stygium e Hypoxylon sp. foram cultivados em água do mar e MDB, sendo que A. stygium forneceu o pirogalol, que apresentou atividade contra Escherichia coli e Staphylococcus aureus resistente a meticilina, além do tirosol e (˗)-(3R,4R)-3,4,5-triidróxi-tetralona, enquanto Hypoxylon sp. forneceu (3R)-scytalona, (3R,4R)-4-hidróxi-scytalona e daidzeína. Portanto, esses resultados ressaltam a importância da investigação dos organismos marinhos, bem como de seus micro-organismos associados, e contribuem para o conhecimento da quimiodiversidade de organismos de origem marinha da costa brasileira, ainda sub-explorada do ponto de vista químico. / Natural Products have a major role in the discovery and development of new drugs used for diseases treatments. Thus, the search for new sources of bioactive compounds has been intensified and studies have increasingly focused on marine organisms and microorganisms. Marine red algae (Rhodophyta) are the major producers of biologically active secondary metabolites among algae, and biosynthesize mainly halogenated compounds. Among microorganisms, we highlight endophytic fungi from marine algae, as they present marked species diversity as well as structural diversity of secondary metabolites. Such metabolites have shown interesting potential as antitumor, antimicrobial and antifungal agents. Therefore, this project aimed to explore the potential of red algae Asparagopsis taxiformis (Falkenbergia stage) and Pyropia spiralis and their endophytic fungi, as novel sources of bioactive compounds. The study of A. taxiformis and P. spiralis led to identification of sixteen compounds by GC-MS, as eicosapentaenoic and arachidonic acids, in addition to nucleosides uridine and thymidine, obtained from A. taxiformis, and uridine and inosine, obtained from P. spiralis. The crude extracts and fractions from the studied algae were evaluated against phytopathogenic fungus Cladosporium sphaerospermum, against acetylcholinesterase enzyme and against tumor cell lines of ovarian carcinoma (OVCAR-8), human colon adenocarcinoma (HCT-116) and human glioblastoma (SF-295). No significant antifungal or acetylcholinesterase activities were observed, whereas the hexane and butanol fractions from both algae showed moderate cytotoxicity against SF-295 cell line. As for the study on the microorganisms associated to algae, seven endophytic fungi strains were isolated from A. taxiformis and three strains from P. spiralis, which were cultivated in potato dextrose broth (PDB) prepared with either sea water or ultra pure water. After incubation, the ethyl acetate crude extracts were obtained and evaluated for their chemical and biological profiles, by HPLC, NMR and in vitro bioassays, respectively. After the preliminary prospection, Nemania bipapillata (AT-05), Sarocladium strictum (PS-01), Annulohypoxylon stygium (AT-03) and Hypoxylon sp. (AT-06) were selected for further investigation. The study of N. bipapillata, cultivated in sea water and PDB, led to isolation of diketopiperazine cyclo(Pro-Tyr), tyrosol and isocoumarins, (3R)-5-hydroxymethylmellein and (3R)-5-carboxymellein. Cultivation of this fungal strain in ultra pure water and PDB afforded two botryane-type norsesquiterpenes and three sesquiterpenes, unprecedent compounds in the literature and one known, 4β-acetoxy-9β,10β,15α-trihydroxyprobotrydial. Analysis of the crude extract from S. strictum, cultivated in ultra pure water and PDB, afforded a mixture of two diketopiperazines, in addition to isoflavone daidzein and six natural phthalates, including two novel ones which had been neither described in the literature nor synthesized. A. stygium and Hypoxylon sp. were cultivated in sea water and PDB, and A. stygium afforded pyrogallol, which was active against Escherichia coli and methicilin-resistant Staphylococcus aureus (MRSA), in addition to tyrosol and (˗)-(3R,4R)-3,4,5-trihydroxy-1-tetralone. Hypoxylon sp. afforded (3R)-scytalone, (3R,4R)-4-hydroxy-scytalone and daidzein. Such results highlight the importance of research on marine organisms and their associated microorganisms, and contribute to the knowledge of chemodiversity of marine organisms from the still chemically underexplored Brazilian coast.

En raison de la nature symbiotique des micro-organismes endophytes, cette étude visait à étudier l'activité antibactérienne, antifongique et cytotoxique chez les métabolites secondaires des extraits endophytes foliaires de plantes de l'Amazonie brésilienne et du Cerrado. Dans ce travail de thèse 147 micro-organismes cultivables ont été isolés (130 champignons, 3 bactéries e 14 champignons non-identifiés ou inconnus) à partir de 28 plantes (4 espèces collectées au Brésil et 24 en Guyane Française). Tous les micro-organismes furent identifiés par analyse moléculaire des régions spécifiques de l'ADNr en utilisant des techniques de séquençage génomique. Des champignons endophytes de l’ordre des Xylariales furent ceux de plus importante fréquence d’isolation dans cette étude, représentés par 25 isolats. Des extraits bruts à l’AcOEt furent produits à partir de cultures de chaque micro-organisme isolé. Une proportion relative signifiante (23,1%) des extraits démontra une activité sur Candida albicans ATCC 10213, pendant que 4% furent actifs sur Staphylococcus aureus ATCC 29213. Le potentiel cytotoxique des extraits fut évalué pour des lignées cellulaires humaines KB (carcinome cervical utérin), MDA-MB-435 (mélanome), et MRC-5 (fibroblastes de poumon normal), résultant en proportion signifiante avec activité d’inhibition de la prolifération cellulaire (24,4%, 23,1% e 16,3%, respectivement).Dix-huit métabolites secondaires furent isolés et identifiés à partir du fractionnement des extraits bruts de huit endophytes. Dix-sept de ces substances furent déjà décrites précédemment dans la littérature: l’acide piliformique (24) et la griséofulvine (25) isolés à partir de Xylaria cubensis SNB-GCI02; La phomopirone (26), la pyrenocine (27), l’alterpérilenol (28) et la novae-zelandine (29), isolés de Lewia infectoria SNB-GTC2402; 5-métilmelleina (31) et la dihidrosporothrioride (32) isolées à partir de Xylaria sp. SNB-GTC2501; La mycoleptodiscine (34) et la mycoleptodiscine B (35) isolées à partir de Mycoleptodiscus sp. SNB-GTC2304; le mycoepoxidiène (36) et la altiloxina A (37) isolés à partir de Diaporthe pseudomangiferae SNB-GSS10; L’acremonisol (40), le semicochliodinol (41) et le cochliodinol (42) isolés à partir de Chaetomium globosum SNB-GTC2114; la colletofragarone A2 (43) isolée à partir de Colletotrichum sp. SNB-GTC0201; la flavoglaucine (44) isolée à partir de Eurotium rubrum BBS01. Parmi ces substances, la flavoglaucine (44) isolée à partir de E. rubrum BBS01 démontra une activité comparable au contrôle fluconazol en C. albicans (CIM de 4 µg.mL-1). La substance (44) présenta IC50 >10 µM sur cellules normales MRC-5, ce qui en fait un candidat pour des études ultérieures. Dans ce travail, fut identifié pour la première fois l’activité de la colletofragarone A2 (43) isolée à partir de Colletotrichum sp. SNB-GTC0201. La substance inédite nommée pyrrocidine C (30) fut isolée à partir de L. infectoria SNB-GTC2402 et identifiée par des analyses spectroscopiques (Casella et al., 2013). La pyrrocidine C (30) fut active en S. aureus ATCC 10213 (CIM de 2 µg.mL-1) et ne fut pas considérées cytotoxique pour les cellules normales MRC-5 (IC50 >10 µM), démontrant une sélectivité dans l’action antimicrobienne. Ces résultats démontrent la grande diversité des endophytes fongiques chez les plantes de l'Amazonie brésilienne et du Cerrado et la grande chimiodiversité associée aux métabolites secondaires de ces micro-organismes. Des champignons endophytes tropicaux, comme ceux présentés dans cette étude, peuvent apparaître comme une nouvelle source de substances antimicrobiennes et cytotoxiques. / Because of the symbiotic nature of endophytes, this survey aims to investigate the probality of discovering antibacterial, antifungal and cytotoxic activities in secondary metabolites of leaf endophytes isolated from plants of Amazon and Cerrado biomes. In this study, 147 cultivable microorganisms were isolated (130 fungi, 3 bacteria and 14 unidentified or unknown microbes) from 28 plant species (4 species collected in Brazil and 24 in French Guyana). All endophytes were identified by molecular analyses of specific rDNA regions, with genomic sequencing techniques. Fungal endophytes belonging to Xylariales order were the most frequently isolated in this study, represented by 25 isolates. Crude AcOEt extracts were produced from cultures of each isolated endophyte. A significant relative proportion (23,1%) of extracts showed activity in Candida albicans ATCC 10213, while 4% were active in Staphylococcus aureus ATCC 29213. The cytotoxic potencial of the extracts was evaluated for human cell lines KB (uterin cervical carcinome), MDA-MB-435 (melanoma) and MRC-5 (normal lung fibroblasts), and a significant proportion of them showed cellular proliferation inhibition (24,4%, 23,1% e 16,3%, respectively).Eighteen secondary metabolites were isolated by the fractionation of eight endophytic extracts. Seventeen of these substances had already been previously described in the literature: piliformic acid (24) and griseofulvin (25) isolated from Xylaria cubensis SNB-GCI02; phomopiron A (26), pyrenocin A (27), alterperilenol (28) and novae-zelandin A (29) isolated from Lewia infectoria SNB-GTC2402; 5-metilmellein (31) and dihidrosporothriorid (32) isolated from Xylaria sp. SNB-GTC2501; mycoleptodiscin A (34) and mycoleptodiscin B (35) isolated from Mycoleptodiscus sp. SNB-GTC2304; mycoepoxidien (36) and altiloxin A (37) isolated from Diaporthe pseudomangiferae SNB-GSS10; acremonisol A (40), semicochliodinol A (41) and cochliodinol (42) isolated from Chaetomium globosum SNB-GTC2114; colletofragaron A2 (43) isolated from Colletotrichum sp. SNB-GTC0201; flavoglaucin (44) isolated from Eurotium rubrum BBS01. Among these substances, flavoglaucin (44), isolated from E. rubrum BBS01, showed comparable antifungal activity with the positive control fluconazol in C. albicans (MIC of 4 µg.mL-1). Flavoglaucin (44) also showed IC50 >10 µM in normal MRC-5 cells, becoming a good candidate for further studies. In this work, the cytotoxic activity of colletofragaron A2 (43), isolated from Colletotrichum sp. SNB-GTC0201 was described for the first time. The unpublished substance named pyrrocidin C (30) isolated from L. infectoria SNB-GTC2402 was identified by spectroscopic analyses (Casella et al., 2013). The pyrrocidin C (30) was active in S. aureus ATCC 10213 (MIC of 2 µg.mL-1), and was not considered cytotoxic for normal MRC-5 cells (IC50 >10 µM), showing selectivity in antimicrobial activity. These results demonstrate the great endophytic fungal diversity in plants of Amazon and Cerrado biomes, along with the chemodiversity associated to the secondary metabolites of these endophytes. Tropical fungal endophytes, like those seen in this work, may emerge as a new source of antimicrobial and cytotoxic substances. / Devido à natureza simbiótica dos micro-organismos endofíticos, este estudo teve como objetivo investigar a atividade antibacteriana, antifúngica e citotóxica em metabólitos secundários de extratos de fungos endofíticos foliares de plantas do bioma Amazônia e Cerrado. Neste trabalho de tese foram isolados 147 micro-organismos cultiváveis (130 fungos, 3 bactérias e 14 fungos não-identificados ou desconhecidos) a partir de 28 plantas (4 espécies coletadas no Brasil e 24 na Guiana Francesa). Todos os micro-organismos foram identificados por análise molecular de regiões específicas de DNAr, com uso de técnicas de sequenciamento genômico. Fungos endofíticos da ordem Xylariales foram os de maior frequência de isolamento neste estudo, representados por 25 isolados. Extratos brutos em AcOEt foram produzidos a partir de culturas de cada micro-organismo isolado. Uma proporção relativa significante (23,1%) dos extratos demonstrou atividade em Candida albicans ATCC 10213, enquanto 4% foram ativos em Staphylococcus aureus ATCC 29213. O potencial citotóxico dos extratos foi avaliado para as linhagens celulares humanas KB (carcinoma cervical uterino), MDA-MB-435 (melanoma), e MRC-5 (fibroblastos de pulmão normal), resultando em proporção significante com atividade de inibição da proliferação celular (24,4%, 23,1% e 16,3%, respectivamente). Dezoito metabólitos secundários foram isolados a partir do fracionamento de oito extratos brutos endofíticos. Dezessete destas substâncias já tinham sido descritas anteriormente na literatura: ácido pilifórmico (24) e griseofulvina (25) isoladas de Xylaria cubensis SNB-GCI02; phomopirona A (26), pyrenocina A (27), alterperilenol (28) e novae-zelandina A (29) isoladas de Lewia infectoria SNB-GTC2402; 5-metilmelleina (31) e Dihidrosporothriolida (32) isoladas de Xylaria sp. SNB-GTC2501; mycoleptodiscina A (34) e mycoleptodiscina B (35) isoladas de Mycoleptodiscus sp. SNB-GTC2304; mycoepoxidieno (36) e altiloxina A (37) isoladas de Diaporthe pseudomangiferae SNB-GSS10; acremonisol A (40), semicochliodinol A (41) e cochliodinol (42) isoladas de Chaetomium globosum SNB-GTC2114; colletofragarona A2 (43) isolada de Colletotrichum sp. SNB-GTC0201; flavoglaucina (44) isolada de Eurotium rubrum BBS01. Dentre estas substâncias, a flavoglaucina (44) isolada de E. rubrum BBS01, demonstrou atividade comparável ao controle fluconazol em C. albicans (CIM de 4 µg.mL-1). Esta substância (44) apresentou IC50 >10 µM em células normais MRC-5, tornando-se candidata para estudos posteriores. Neste trabalho foi identificado pela primeira vez a atividade citotóxica da colletofragarona A2 (43), isolada de Colletotrichum sp. SNB-GTC0201. A substância inédita nomeada pyrrocidina C (30) foi isolada a partir de L. infectoria SNB-GTC2402 e identificada através de análises espectroscópicas (Casella et al., 2013). A pyrrocidina C (30) foi ativa em S. aureus ATCC 10213 (CIM de 2 µg.mL-1), e não foi considerada citotóxica para as células normais MRC-5 (IC50 >10 µM), demonstrando seletividade na ação antimicrobiana. Estes resultados demonstram a grande diversidade fúngica endofítica em plantas do bioma Amazônia e Cerrado, e a quimiodiversidade associada aos metabólitos secundários destes micro-organismos. Fungos endofíticos tropicais, como os vistos neste trabalho, podem emergir como uma nova fonte de substâncias antimicrobianas e citotóxicas.