Submitted by Mariana Gabriela Fonseca (marigfonseca@yahoo.com.br) on 2018-09-17T13:01:19Z No. of bitstreams: 1 Fonseca, MG_Dissertacao Mestrado Corrigida-merged (1).pdf: 5024373 bytes, checksum: 7883e82b0d1dc259f5f8c6567a8c452b (MD5) / Approved for entry into archive by Adriana Aparecida Puerta null (dripuerta@rc.unesp.br) on 2018-09-17T14:03:13Z (GMT) No. of bitstreams: 1 fonseca_mg_me_rcla.pdf: 5024373 bytes, checksum: 7883e82b0d1dc259f5f8c6567a8c452b (MD5) / Made available in DSpace on 2018-09-17T14:03:13Z (GMT). No. of bitstreams: 1 fonseca_mg_me_rcla.pdf: 5024373 bytes, checksum: 7883e82b0d1dc259f5f8c6567a8c452b (MD5) Previous issue date: 2018-07-20 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Ainda que consideráveis avanços tecnológicos no setor agrícola tenham proporcionado melhorias no bem-estar humano, milhares de pessoas ainda enfrentam miséria e fome ao redor do mundo. O progresso alcançado no último século, com a Revolução Verde, apesar de ter impulsionado a produção global de alimentos, também trouxe consequências catastróficas para o ambiente natural. Além disso, a população mundial continua a crescer exponencialmente, em especial em regiões emergentes como nos continentes africano e asiático. Todos esses fatores, somados aos inúmeros limitantes das produções agrícolas, representam uma grande ameaça à segurança alimentar do planeta, além de trazer prejuízos econômicos exorbitantes. Dentre os limitantes agrícolas, as fitopatologias causadas por bactérias do gênero Xanthomonas respondem por perdas de produção que podem chegar a 100%, quando em situação epidêmica. As produções de tomate, pimentão e mandioca podem ser reduzidas gravemente quando acometidas por Xanthomonas euvesicatoria, causadora da mancha bacteriana em pimentão e tomateiros, e Xanthomonas axonopodis pv. manihotis, causadora da bacteriose da mandioca. O combate atual para estas doenças baseia-se no uso de compostos cúpricos, notadamente danosos à saúde humana e ambiental, mas há casos, como o da bacteriose da mandioca, em que não há controle químico eficaz, sendo medidas de controle e manejo a única possibilidade de amenizar o problema. Como alternativa, estudos vem apontado a possibilidade de uso de metabólitos secundários de microrganismos, incluindo fungos da Antártica, no combate às Xanthomonas ssp. A presente pesquisa avaliou o potencial bactericida de extratos produzidos por 156 fungos isolados de diversas amostras de solo e sedimentos marinhos antárticos contra X. euvesicatoria e X. ax. pv. manihotis. Vinte e sete extratos foram bioativos para pelo menos uma das espécies de Xanthomonas testadas. Os extratos bioativos foram produzidos por 23 diferentes fungos, sendo que 9 foram isolados de amostras de sedimentos marinhos, 5 de solo abaixo de madeira podre, 6 de solo com biofilme e 3 isolados de solo abaixo de barra de ferro. O gênero de fungos Pseudogymnoascus, bastante comum na Antártica, foi predominante dentre os isolados foram produtores de compostos bioativos contra as espécies testadas. / Yet considerable technological advances in the agricultural sector have provided improvements in human well-being, thousands of people still face misery and hunger around the world. The progress achieved in the last century within the Green Revolution, despite having boosted global food production, has also brought catastrophic consequences to the natural environment. In addition, the world population continues to grow exponentially, especially in emerging regions such as the African and Asian continents. All these factors, coupled with the numerous constraints of agricultural production, pose a great threat to the planet's food security, as well as exorbitant economic losses. Among the agricultural limitations, phytopathologies caused by bacteria of the genus Xanthomonas respond by losses of production that can reach 100%, when in an epidemic situation. The tomato, pepper and cassava productions can be severely reduced when affected by Xanthomonas euvesicatoria, which causes bacterial spot in pepper and tomatoes, and Xanthomonas axonopodis pv. manihotis, which causes cassava bacterial blight. The current fight for these diseases is based on the use of copper compounds, notably harmful to human and environmental health, but there are cases, such as cassava bacterial blight, in which there is no effective chemical control, and management measures are the only possibility of mitigating the problem. As an alternative, studies have indicated the possibility of using secondary metabolites of microorganisms, including Antarctic fungi, in the control of Xanthomonas ssp. The present study evaluated the bactericidal potential of extracts produced by 156 fungi isolated from several soil samples and marine sediments against X. euvesicatoria and X. ax. pv. manihotis. Twenty-seven extracts were bioactive for at least one of the Xanthomonas species tested. Bioactive extracts were produced by 23 different fungi, of which 9 were isolated from marine sediment samples, 5 from soil under rotten wood, 6 from soil with biofilm and 3 soil isolates below iron bar. The genus of fungi Pseudogymnoascus, very common in Antarctica, was predominant among the isolates were producers of bioactive compounds against the species tested.

This project was focused on development of extraction and purification protocol for novel cytotoxic compound isolated from soil cyanobacterium Nostoc muscorum which would allow to determine its molecular structure via NMR (nuclear magnetic resonance) and MS (mass spectrometry) techniques. Purified cytotoxin was subjected to several biochemical and microscopical experiments in order to assess its toxicological parameters, determine the mechanism of action and evaluate potential application in biotechnology or pharmacy.

Plants have been utilized as a source of medicines since ancient times. They contain a vast range of secondary metabolites which play important roles in different diseases. The Scope of this study is to define the function of secondary metabolites from Andrographis paniculata (Kalmegh) and Silybum marianum (Milk Thistle) against DNA damage, which initiates many diseases. Sequential extraction of both plant materials was performed with different polarity solvents. Qualitative analysis was performed with Gas (GC) and High Performance Liquid Chromatography (HPLC). Primary extracts screening studies were performed against cytotoxicity, antioxidant and soluble collagen assays (Sircol dye). Further bioactivity was confirmed using the single-cell gel electrophoresis (Comet assay) to estimate levels of DNA damage. Fourier Transform Infrared analysis of bioactive extracts was performed to identify the functional groups present in them . Subsequently, bioactive extracts were further separated into acid, base, phenol and neutral fractions. These fractions and bioactive extracts were screened with the free radical assays to identify the scavenging activity. Chemical mapping of the bioactive fraction was performed with High Performance Liquid Chromatography (HPLC). Preparative-HPLC was performed to separate the compounds which were present in the bioactive fractions. MTT assay, Hydroxyl radical and nitric oxide radical scavenging activity assays were performed to screen the fractions and DNA protective activity of the bioactive fractions was confirmed with single-cell gel electrophoresis. These bioactive fractions were de-replicated with hyphenated techniques like LCMS and LCMS/MS to identify the molecular weight of the compounds and Quadrupole time-of-flight mass spectrometry was performed to identify the accurate mass of the compounds. Sequential extraction separates the non-polar and polar compounds present in the plant material. Qualitative analysis confirmed the presence of fatty acids in the non-polar extracts of both plants using GC and the presence of standard constituents in the polar extracts of both plants using HPLC. It also helps in chemical mapping of the extracts. Acetone, Methanol1 and Methanol2 extracts from either plant are non-cytotoxic. The high antioxidant activity is observed in methanol extracts from Andrographis paniculata and in acetone/methanol2 extracts from Silybum marianum. Extracts that protect against UVA and UVB damage also increased soluble collagen production in Human Dermal Fibroblast (HDF) in culture. Primary Screening helped to select six extracts out of twelve extracts for further analysis. Comet assay confirmed DNA protective activity in Methanol1 extract of Milk thistle and Acetone, Methanol2 extracts from Kalmegh. These three extracts were further fractionated into 38 fractions out of which three fractions that are F1, F13 and F31 fractions confirm the DNA protection activity. De-replication of the bioactive fractions was performed with LC-ESI-MS/MS which confirm twenty one compounds and accurate mass of fifteen compounds was determined using Q-tof mass spectrometry.

Mikania é o maior gênero da tribo Eupatorieae, com cerca de 430 espécies, sendo amplamente distribuído no Brasil, com aproximadamente 200 espécies descritas. Dentro deste gênero, Mikania cordifolia, M. micrantha e M. glomerata são comumente chamadas de guaco e estão entre as espécies mais usadas popularmente, principalmente para o tratamento de febre, reumatismo, doenças do trato respiratório e contra picadas de cobra. No presente trabalho, foram realizados estudos fitoquímicos destas três espécies e foram analisados os efeitos dos tipos de adubação, da radiação solar e do estresse hídrico sobre a produção de metabólitos secundários. Primeiramente, através de estudos fitoquímicos, foram identificados 62 compostos. As frações polares das espécies apresentaram como compostos majoritários derivados fenilpropanóicos, destacando-se o Ácido 3,5-dicafeoilquínico em M. glomerata e M. micrantha e o Ácido Fertárico em M. cordifolia. Na espécie M. glomerata também foram identificados derivados do Ácido glucárico di e tri esterificados por ácidos caféicos, até o momento não descritos na literatura, além de flavonóides sulfatados, os quais também estão presentes em M. micrantha. Nas frações apolares foi verificada a presença de triterpenos, principalmente em M. cordifolia e derivados do Ácido Caurenóico em M. glomerata e M. micrantha. As lactonas sesquiterpênicas foram encontradas em M. cordifolia (esqueleto melampolido) e em M. micrantha (esqueleto micranolido). A análise dos cultivares de M. glomerata e M. cordifolia, revelou uma grande influência das condições de cultivo na composição dos metabólitos secundários. Os experimentos mostraram uma grande variação entre a concentração dos compostos presentes nas duas espécies de Mikania, sendo verificado que as condições de cultivo e principalmente as taxas de luminosidade são de extrema importância na acumulação desses metabólitos. O estresse hídrico não apontou efeitos significativos no acúmulo de substâncias em M. glomerata. As análises histoquímicas se mostraram eficazes na obtenção de informações a respeito da localização de compostos fenólicos nas folhas de M. glomerata. / Mikania is the largest gender of the tribe Eupatorieae, with about 430 species, being distributed thoroughly in Brazil, with approximately 200 described species. In this gender, Mikania cordifolia, M. micrantha and M. glomerata are commonly called \"guaco\" and are among the more popularly used species, mainly for the treatment of fever, rheumatism, diseases of the breathing tract and against snake bites. In the present work, phytochemical studies of these three species were carried out and the effects of the fertilization types, the solar radiation and the hydric stress upon the production of secondary metabolites were analyzed. Firstly, through phytochemical studies, 62 compounds were identified. The polar fractions of the species presented as majority compounds phenylpropanoic derivatives: 3,5-Dicaffeoylquinic acid in M. glomerata and M. micrantha and Fertaric acid in M. cordifolia. In M. glomerata were also identified glucaric acid derivatives di- and tri-esterified with cafeic acids, which are no described in the literature until the moment, besides sulfated flavonoids, which are also present in M. micrantha. In the apolar fractions the triterpenes presence were verified mainly in M. cordifolia and kaurenoic acid derivatives in M. glomerata and M. micrantha. The sesquiterpene lactones were found in M. cordifolia (melampolide skeleton) and in M. micrantha (micranolide skeleton). The analysis of M. glomerata and M. cordifolia cultivars revealed a great influence of the cultivation conditions in the secondary metabolites composition. The experiments showed a great variation among the concentration of the compounds in the two species of Mikania, being verified that the cultivation conditions and mainly the taxes of solar radiation are of extreme importance in the accumulation of these metabolites. The hydric stress didnt have significant effects in the accumulation of substances in M. glomerata. The histochemical analyses were effective in supply information regarding location of phenolic compounds in the leaves of M. glomerata.

Recentemente foram isolados da esponja marinha Axinella cf. corrugata dois compostos derivados da esculetina: o éster metílico do ácido 4-esculetínico e o éster etílico do ácido 4-esculetínico. Este último apresentou importante atividade contra o vírus da SARS. Este projeto teve como meta isolar e cultivar as linhagens de fungos associadas à esponja Axinella cf. corrugata, bem como analisar seus extratos por HPLC-PDA-MS, objetivando a possível detecção desses compostos (ou derivados) nesses extratos. Avaliações preliminares levaram à obtenção de 11 amostras potencialmente relacionadas a esses compostos. Dentre elas, uma apresentou espectros no UV e de massas muito similares aos obtidos para o aduto de sódio do éster metílico do ácido 4-esculetínico. No entanto análises espectroscópicas mais detalhadas por RMN - 1H, RMN - 13C, HSQC, HMBC e COSY da amostra purificada permitiram identificar o composto isolado, a 1,3,6-trihidroxi-8-metil-9H-xanten-9-ona. Simultaneamente às análises químicas, os extratos obtidos a partir das linhagens fúngicas isoladas da esponja Axinella cf. corrugata tiveram suas atividades biológicas avaliadas frente a microrganismos e células tumorais humanas, resultando em mais de 20% dos extratos com alguma atividade biológica. / Recently two compounds derived from esculetin have been isolated from the marine sponge Axinella cf. corrugata: the methyl ester of esculetin-4-carboxylic acid and the ethyl ester of esculetin-4-carboxylic acid. The latter displayed antiviral activity against the SARS virus. This project aimed the isolation and the growth of fungal strains associated to the sponge Axinella cf. corrugata, and the subsequent analysis of the fungal extracts by HPLC-PDA-MS, aiming the possible detection of the esculetin compounds (or derivatives) in those extracts. Preliminary analysis yielded 11 samples potentially related to these compounds. Among these extracts, one presented UV and MS spectra very similar to the spectra obtained for the sodium adduct of the methyl ester of esculetin-4-carboxylic acid. However, a detailed spectroscopic analysis of a pure compound isolated by RMN - 1H, RMN - 13C, HSQC, HMBC e COSY allowed the identification of the compound, which is 1,3,6-trihydroxy-8-methyl-9H-xanthen-9-one. Simultaneously to the chemical analysis of the fungal crude extracts, the biological activities of the obtained extracts were evaluated against microrganisms and human tumoral cell lines. More than 20% of the extracts displayed some biological activity.

Cyanobacteria are notorious for their potential to produce toxins with human health effects, particularly the hepatotoxic microcystins (MCs), but cyanobacteria also produce other bioactive compounds. A wide variety of oligopeptides including aeruginosins, cyanopeptolins and cyanobactins may be as toxic as MCs. To investigate the production of these compounds, an UPLC QTOF-MS/MS method was developed to compare the metabolomic profiles of various strains of a common bloom-forming and toxigenic species, Microcystis aeruginosa, as well as those obtained from lakes with mixed cyanobacterial assemblages. Although many compounds could not be confirmed, MCs were rarely the dominant secondary metabolite in any sample. Since the biological role of MCs remains unknown, I tested the hypothesis that MCs provide protection against oxidative stress as induced through exposure to the herbicide atrazine and UV radiation in pure cultures of toxic vs non-toxic strains. Results were inconclusive and varied between strains suggesting other mechanisms exist to counter oxidative stress.

New methodology has been utilized to provoke or increase targeted metabolic pathways in microbes. The low hanging fruit of natural products has been discovered over the last 50 years. To continue finding new metabolites to be used as possible drug candidates, methodology development such as those proposed herein are necessary. This methodology uses extracts from known pathogenic bacteria to elicit production of latent biosynthetic pathways from environmental bacterial isolates that may be active against the original pathogenic strains. A new compound, MAV-1 (1) of the diketopiperazine family (Figure 1) was isolated and identified utilizing these techniques. The structure of MAV-1 (1) was defined by a combination of mass spectroscopy (MS) and nuclear magnetic resonance (NMR) spectroscopy. Discovery of MAV-1 (1), a possible precursor to other known compounds, demonstrates the continuing utility of microbial sources with new chemodiversity.

Production of secondary metabolites in plant tissue cultures The goal of this study is to determine the influence of sodium molybdate and sodium tungstate as elicitors on production of scopoletin in cell suspension culture of Angelica archangelica L. The culture was grown in a liquid culture medium Murashige and Skoog on a roller apparatus in the dark and light. The content of scopoletin was in cells and in the culture medium determined by high performance liquid chromatography with fluorometric detection. The results show that sodium molybdate served as an elicitor to the production of scopoletin positively, application of sodium tungstate did not increase scopoletin production in any case. The highest production of scopoletin after application of sodium molybdate versus the control cells was reached in the suspension culture of Angelica archangelica L. cultured in the dark at a concentration of 25,50 mg/l. Scopoletin content increased by 166,7 %. After application of sodium tungstate the production always decreased, in the medium of a suspension culture of Angelica archangelica L. cultivated in the light at a concentration of 66.00 mg/l, the production decrease was by up 62,5 %.

Physalis angulata L. (Solanaceae), popularmente conhecida como camapu ou balãozinho, é uma espécie que vem ganhando importância econômica em decorrência de suas atividades biológicas, como citotóxica, antibacteriana e anti-inflamatória. Dentre os diversos metabólitos secundários conhecidos para esta espécie, destacam-se os flavonoides e alcaloides. É amplamente difundido que o metabolismo secundário das plantas pode ser alterado por fatores bióticos e abióticos. Nesse sentido, esse trabalho objetivou destacar a influência do Potato virus Y, estirpe O (PVYO), isolado de camapu, no metabolismo secundário, avaliando as quantidades e os perfis de flavonoides e alcaloides também em plantas de camapu infectadas. A infecção viral foi induzida em plantas obtidas por sementes, os metabólitos foram extraídos a partir de folhas, foram analisados em CLAE e CG-EM e comparados com aqueles provenientes de plantas sadias. Não foram observadas diferenças qualitativas nos perfis tanto dos flavonoides quanto dos alcaloides. Por outro lado, houve diminuição no teor de flavonoides totais nas plantas infectadas na infecção sistêmica. Além disso, houve aumento significativo do derivado de atropina nas plantas infectadas em comparação com os demais tratamentos. Dessa forma, sugerimos que a infecção induzida pelo PVYO em plantas de camapu teve maior impacto na via das substâncias nitrogenadas em relação às fenólicas / Physalis angulata L. (Solanaceae), popularly known as \"camapu\" or \"balãozinho\", has become economically important due to its biological activities, such as cytotoxic, antibacterial and anti-inflammatory. Among the secondary metabolites, flavonoids and alkaloids have already been described for this species. Secondary metabolism can be influenced by both biotic and environmental agents. The main goal of this work was to analyze the influence of Potato virus Y (PVYO) on the secondary metabolism of infected \"camapu\" plants, evaluating total amount and profiles of flavonoids and alkaloids. Viral infection was induced in plants obtained from seeds. The compounds were extracted from leaves, analyzed in HPLC and GC/MS and compared with those from healthy plants. There were no qualitative differences between healthy and infected plants concerning to alkaloid and flavonoid profiles. On the other hand, it was noticed a lower flavonoid concentration in the systemic infection of infected plants. An increase of an atropine derivative was observed in infected plants. In conclusion, according to the results we propose that the PVYO induced infection in plants of \"camapu\" had stronger impact on the nitrogenous compounds than on phenolics

Plantas parasitas são aquelas que retiram os recursos necessários para a sua sobrevivência de outras plantas (hospedeiras) por meio de uma estrutura denominada haustório, que permite que a parasita se conecte ao sistema vascular da hospedeira. As plantas podem responder ao ataque parasita ativando alguns mecanismos de defesa, dentre eles, a produção de substâncias fenólicas. Os viscos constituem um grupo de plantas parasitas aéreas pertencentes a Santalales que podem ser divididos em viscos holoparasitas ou hemiparasitas. Alguns gêneros de viscos são de importância econômica, dentre eles, Phoradendron, um gênero de visco hemiparasita. Esse trabalho teve como objetivos determinar os conteúdos de taninos, flavonoides, fenilpropanoides, carboidratos solúveis, amido, lipídeos, a razão carbono/nitrogênio e avaliar como a relação parasítica afeta o metabolismo das espécies envolvidas. Para tanto, coletou−se no município de Campanha (MG) folhas e ramos de seis indivíduos não parasitados de Tapirira guianensis (FT e RT) e, a partir de nove indivíduos parasitados de T. guianensis, foram coletados ramos e folhas de Phoradendron crassifolium (FP e RP), ramos não infestados da hospedeira e suas folhas (RNI e FRNI) e ramos infestados de T. guianensis e suas folhas (RI e FRI), sendo que ramos infestados foram divididos em região proximal, galha e distal (RPRI, RGRI e RDRI). Como principais resultados estão o aumento no teor de proantocianidinas e de carboidratos solúveis em FRNI e FRI em relação a FT. Ramos infestados sofreram redução nos conteúdos de carboidratos solúveis, amido, lipídeos, nitrogênio e substâncias fenólicas, exceto na região da galha (RGRI) em que aumentos de amido e flavonoides foram verificados. Observou-se ainda que RNI parece ter sofrido um acúmulo de substâncias fenólicas. O conjunto dos resultados obtidos mostra que P. crassifolium afeta o metabolismo primário e o secundário de T. guianensis, com influência mais pronunciada nos ramos do que nas folhas da hospedeira. Pode−se sugerir que a parasita obtêm nitrogênio e carboidratos solúveis de sua hospedeira e que pode ocorrer uma possível realocação de carbono para crescimento, desenvolvimento, manutenção e, ainda, defesa de ramos ainda não infestados. Além disso, sugere−se que o acúmulo de fenólicos em ramos não infestados (RNI) possa ser um mecanismo de defesa usado pela hospedeira com o objetivo de evitar, ou mesmo diminuir, novas infestações em ramos ainda não parasitados. / Parasitic plants are those that withdraw necessary resources for its survival from other plants (hosts) through a structure so-called haustorium which enables the parasite to connect to the host\'s vascular system. Plants can respond to the parasite attack activating some defense mechanisms, including the production of phenolic compounds. Mistletoes are a group of stem parasitic plants belonging to Santalales that can be grouped into holoparasite or hemiparasite mistletoes. Some mistletoes genera are of economic importance among them Phoradendron, a hemiparasite mistletoe. This study aimed to determine the contents of tannins, flavonoids, phenylpropanoids, soluble carbohydrates, starch, lipids, the carbon⁄nitrogen ratio and evaluate how the parasitic relationship affects the metabolism of the involved species. Were collected in Camapanha (MG) leaves and branches from six non−parasitized individuals of Tapirira guianensis, host plant (LT and BT). From nine parasitized individuals of T. guianensis were collected branches and leaves of Phoradendon crassifolium, the parasitic plant (LP and BP), non-infested host branches and their leaves (NIHB and NIHB-L) and infested branches and their leaves (IHB and IHB-L). Infested branches of T. guianensis were divided into proximal, gall, and distal regions (IHB-P, IHB-G and IHB-D). Main results were an increase in the proanthocyanidin and soluble carbohydrates content in NIHB−L and IHB−L in relation to LT. There was a reduction of soluble carbohydrate, starch, lipids, nitrogen, and phenolic compounds in infested branches, except in the gall region (IHB-G), which showed an increase of flavonoids and starch contents. It was also observed that NIHB appears to accumulate phenolic compounds. Results showed that P. crassifolium affects the primary and secondary metabolism of T. guianensis, and branches were affected more intense than the leaves. It can be suggested that the parasite withdraws nitrogen and soluble carbohydrates from its host and this could result in possible carbon relocation for growth, development, maintenance, and also defense of branches not yet infested. In addition, it could be suggested the accumulation of phenolic compounds in non−infested branches (NIHB) as a defense mechanism used by the host in order to prevent or even reduce new infestations of an individual already parasitized.