Dihydrochalcones (DHCs) are specialised metabolites with a limited natural distribution, found in significant amounts in Malus x domestica Borkh. (cultivated apple) and wild Malus species. Among them, M. x domestica accumulates significant amounts of phloridzin, whilst trilobatin and sieboldin are abundant in some wild relatives. DHCs have demonstrated a wide range of bioactive properties in biomedical models. Some DHCs have also been reported to act as flavour enhancers. Phloridzin may act as an anti-diabetic compound by blocking sodium-linked glucose transport and renal reabsorption of glucose in kidneys. Despite the protective effects reported in mammal models, little is known about how these metabolites are biosynthesised and what is their function in planta, where it has been hypothesised a role for phloridzin in plant growth. The biosynthetic pathway leading to DHC formation has been proposed in apple, and some steps have been characterised recently. DHC pathway diverts from the main phenylpropanoid pathway most probably from 4-coumaroyl-CoA by the action of a yet unknown reductase that would produce 4-dihydrocoumaroyl-CoA. Then, chalcone synthase (CHS) catalyses its condensation to form phloretin. Phloretin can be directly glycosylated at position 2′- or 4′ by the previously characterised 2′- and 4′-O-UDP-glycosyltransferases PGT1 and PGT2, to produce phloridzin or trilobatin, respectively. However, sieboldin has been postulated to derive from hydroxylation in position 3 of phloretin before been glycosylated, and the key responsible enzyme producing 3-hydroxyphloretin has not been yet discovered. The main aim of this PhD proposal was to provide a better understanding of the physiological functions of DHCs in apple, as well as to contribute to the elucidation of the biosynthetic pathway as the molecular basis for future genetic engineering in apple. Towards this aim, functional characterisation was conducted in MdPGT1 knockdown apple lines by RNAi silencing and CRISPR/Cas9 genome editing to assess the physiological effect of targeting a key biosynthetic gene involved in phloridzin biosynthesis. In addition, molecular, transcriptomic and metabolomic analyses were integrated to evaluate candidate genes accounting for 3-hydroxylase activity involved in DHC biosynthesis in wild Malus species accumulating sieboldin. Moreover, a de novo transcriptome assembly was carried out in an intergeneric hybrid between M. x domestica and Pyrus communis L. known to accumulate intermediate levels of DHCs compared to apple, in order to identify additional genes potentially involved in DHC pathway. We compared the physiological effect of reducing phloridzin through PGT1 knockdown by RNAi silencing and CRISPR/Cas9 genome editing. Knockdown lines exhibited characteristic impairment of plant growth and leaf morphology as reported in literature, whereas genome edited lines exhibited normal growth despite reduced foliar phloridzin. Bioactive brassinosteroids and gibberellins were found to be key players involved in the contrasting effects on growth observed following phloridzin reduction. Moreover, a cytochrome P450 from wild M. toringo (K. Koch) Carriere syn. sieboldii Rehder, and M. micromalus Makino was identified as dihydrochalcone 3-hydroxylase (DHCH), proving to produce 3-hydroxyphloretin and sieboldin in yeast. Different DHCH allele isoforms found in domesticated apple and M. toringo and M. micromalus correlated with sieboldin accumulation in a Malus germplasm collection. Finally, the assembled de novo transcriptome of the intergeneric apple/pear hybrid integrated to functional annotation and metabolomic analysis resulted in the identification of genes potentially involved in DHC biosynthesis, providing the basis for future biochemical characterisation. Altogether these results contribute to get insight into the roles of DHCs in apple and to illustrate how CRISPR/Cas9 genome editing can be applied to dissect the contribution of genes involved in phloridzin biosynthesis in apple. Furthermore, the present PhD thesis contributes to the state-of-the-art by elucidating key missing steps in the biosynthesis of DHCs, which could be relevant for future establishment of genetic engineered lines that contribute to assess physiological effects of altering DHCs content, as well as to establish heterologous expression systems to produce de novo DHCs.

Considerando que diversas atividades biológicas dos ácidos clorogênicos já foram descritas na literatura, uma melhor compreensão da biossíntese e do acúmulo desses polifenois nas plantas está diretamente vinculada aos parâmetros de qualidade de fitoterápicos. Este trabalho teve o objetivo de monitorar a variação na concentração de metabólitos secundários do grupo dos ácidos clorogênicos no tecido foliar de diversas espécies diante de diferentes tratamentos pós-coleta. Este monitoramento foi realizado através da quantificação das substâncias por cromatografia líquida de ultra eficiência acoplada à espectrometria de massas em sequência (CLUE-EM/EM). Na primeira fase do estudo, foram coletadas folhas de cada espécie selecionada para o estudo (Caju - Anacardium occidentale L.; Graviola - Annona muricata L.; Pata-de-Vaca - Bauhinia variegata L.; Limão - Citrus limon (L.) Burm. f.; Café - Coffea arabica L.; Pitanga - Eugenia uniflora L.; Alecrim - Rosmarinus officinalis L., e Jambolão - Syzygium cumini (L.) Skeels). As folhas foram submetidas a um processo de extração e os extratos obtidos foram analisados por CLAE-EM/EM. Foram encontradas nove substâncias monossubstituídas nas posições 3, 4 e 5 pertencentes aos subgrupos dos ácidos p-cumaroilquínicos, cafeoilquínicos e feruloilquínicos dos ácidos clorogênicos, além de ácidos dicafeoilquínicos. A identificação dos sinais cromatográficos foi realizada por meio dos padrões de fragmentação de cada composto. Na segunda fase do estudo, foram coletadas quatro folhas dos indivíduos em estudo. As folhas foram congeladas em nitrogênio líquido (para interrupção do metabolismo) em tempos diferentes: imediatamente após a coleta (T0), 30 min após a coleta (T0,5), 1 h após a coleta (T1) e 2 h após a coleta (T2). Este procedimento foi realizado uma vez por mês durante seis meses. Em outro momento foram retiradas seis folhas de três indivíduos de espécies selecionadas (Alecrim, Pitanga, Pata-de-Vaca, Limão e Café) e cada folha foi congelada nos tempos T0 (imediatamente após a coleta), T6 (6 h após a coleta), T12 (12 h após a coleta), T24 (24 h após a coleta), T48 (48 h após a coleta) e Ts (após secagem das folhas - aproximadamente 30 dias após a coleta). O material coletado foi extraído e analisado por CLUE-EM/EM, no modo MRM. O método analítico quantitativo foi validado, considerando os parâmetros de linearidade, exatidão e precisão. As concentrações dos ácidos clorogênicos em estudo não apresentaram um padrão de variação que se relacione com os tempos de tratamento ou um aumento linear nas respostas dentro do intervalo de 2 h após o início do catabolismo do tecido. As amostras com tempos de tratamento mais longo, por sua vez, apresentaram aumento significativo de diversos compostos nas folhas que não tiveram seu metabolismo interrompido 30 dias após a coleta. Este acúmulo de ácidos clorogênicos corrobora a hipótese de que o aumento da concentração destes compostos pode estar relacionado ao catabolismo de polímeros fenólicos de maior massa. / Considering that diverse chlorogenic acids\' biological activities have been described, a better understanding of their biosynthesis and accumulation is closely related to phytotherapics\' quality parameters. This research aimed to monitor the variation on the concentration of secondary metabolites from the chlorogenic acids\' group on the leaf\'s tissue of diverse species, towards different post-harvest treatments. The monitoring was performed through the quantification of the compounds by UPLCMS/ MS. On the first phase of the project, leaves from each selected species (Cashew - Anacardium occidentale L.; Soursop - Annona muricata L.; Pata-de-Vaca - Bauhinia variegata L.; Lemon - Citrus limon (L.) Burm. f.; Coffee - Coffea arabica L.; Cherry - Eugenia uniflora L.; Rosemary - Rosmarinus officinalis L., and Jambolan - Syzygium cumini (L.) Skeels) were harvested. This material was submitted to an extraction process, and the obtained extracts were analyzed by HPLC-MS/MS. Nine substances were found, monossubstituted on positions 3-, 4- and 5-, belonging to the subgroups of p-coummaroylquinic, caffeoylquinic and feruloylquinic acids, in addition to dicaffeoylquinic acids. The chromatographic signals\' identification was preceded observing the fragmentation pattern of each compound. On the second phase of the study, four leaves were collected from each individual. The leaves were frozen with liquid nitrogen (to interrupt the metabolism) on different moments - immediately after the collection (T0), 30 min after de collection (T0,5), 1 h after the collection (T1) and 2 h after the collection (T2). This procedure happened once a month during six months. On August, 2016, a different collection was performed for a broader observation: six leaves were harvested from three individuals from selected species (Rosemary, Cherry, Pata-de-Vaca, Limon and Coffee), and each leave was frozen on T0 (immediately after the collection), T6 (6 h after the collection), T12 (12 h after the collection), T24 (24 h after the collection), T48 (48 h after the collection) and Ts (after leaf\'s drying - approximately 30 days after collection). The harvested material was extracted and analyzed by UPLC-MS/MS, on MRM mode. The analytical method was validated for linearity, accuracy and precision. Chlorogenic acids\' concentrations did not show a pattern of variation that related to the different treatments or a linear increase on response until 2 hours after the beginning of the tissue catabolism. The samples submitted to a broader treatment interval, however, showed a significant rise on the concentration of different compounds, on leaves that did not have their metabolism interrupted until 30 days after the collection. This accumulation of chlorogenic acids agrees with the hypothesis that the raise on the concentration of these substances can be related to the catabolism of polymeric phenols of higher mass.

The aim of this research was to investigate the phytochemistry of two species Sanicula europaea and Teucrium davaeanum which are traditionally used in treatment of wounds. Four compounds were isolated from the 80% methanolic extract of S. europaea; bis-(2-ethylhexyl) phthalate (1), palmitic acid (2), rosmarinic acid (3), saniculoside N (4). Compounds 1 and 2 were isolated for the first time from this species. The structure elucidation of the isolated compounds was on the basis of 1D, 2D NMR spectroscopy and mass spectrometry measurements. Two compounds were isolated from the crude glycosides extract of T.davaeanum; 6 is a phenylethanoid glycoside and 8 is an iridoid glycoside, from the data available these may be new compounds for which the names davaeanuside A and davaeanuside B are proposed respectively." The total polyphenol content of S. europaea L, T. davaeanum leaves-flowers and T. davaeanum stem were found to be 5.0, 1.20 and 0.65 mg per 100 mg dried plant material respectively. A study of the antioxidant activity of the 50 % ethanol extracts of S. europaea and T. davaeanum showed that on a mg/mg basis S. europaea and T. davaeanum have approximately 5%, 8 % antioxidant capacity of Trolox respectively. A study of the cytotoxic activity of davaeanuside A (6), iridoid glycoside (7), davaeanuside B (8) and saponin compound (10) isolated from the crude glycosides extract of T. davaeanum revealed that saponin compound (10) inhibited the growth of Hela cells by 50 % at 50 μg/ml, P< 0.001, but the other compounds did not show activities against the tested cell lines at 100 μg/ml. The results of this work provide some basis for the traditional use of these species in the treatment of wounds. / Ministry of high education in Libya

The aim of this research was to investigate the phytochemistry of two species Sanicula europaea and Teucrium davaeanum which are traditionally used in treatment of wounds. Four compounds were isolated from the 80% methanolic extract of S. europaea; bis-(2-ethylhexyl) phthalate (1), palmitic acid (2), rosmarinic acid (3), saniculoside N (4). Compounds 1 and 2 were isolated for the first time from this species. The structure elucidation of the isolated compounds was on the basis of 1D, 2D NMR spectroscopy and mass spectrometry measurements. Two compounds were isolated from the crude glycosides extract of T.davaeanum; 6 is a phenylethanoid glycoside and 8 is an iridoid glycoside, from the data available these may be new compounds for which the names davaeanuside A and davaeanuside B are proposed respectively." The total polyphenol content of S. europaea L, T. davaeanum leaves-flowers and T. davaeanum stem were found to be 5.0, 1.20 and 0.65 mg per 100 mg dried plant material respectively. A study of the antioxidant activity of the 50 % ethanol extracts of S. europaea and T. davaeanum showed that on a mg/mg basis S. europaea and T. davaeanum have approximately 5%, 8 % antioxidant capacity of Trolox respectively. A study of the cytotoxic activity of davaeanuside A (6), iridoid glycoside (7), davaeanuside B (8) and saponin compound (10) isolated from the crude glycosides extract of T. davaeanum revealed that saponin compound (10) inhibited the growth of Hela cells by 50 % at 50 μg/ml, P< 0.001, but the other compounds did not show activities against the tested cell lines at 100 μg/ml. The results of this work provide some basis for the traditional use of these species in the treatment of wounds.

Orientador: Ione Salgado / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-20T06:41:46Z (GMT). No. of bitstreams: 1 SantosFilho_PlinioRodriguesdos_D.pdf: 1637587 bytes, checksum: 89bdaa896ffa24aac0e2f5b00b8ece3a (MD5) Previous issue date: 2012 / Resumo: A nitrato redutase (NR) corresponde ao primeiro passo na assimilação do nitrato em plantas. Recentemente, essa enzima tem sido também relacionada à síntese de óxido nítrico (NO). Entre várias ações sinalizadoras para as plantas, o NO promove o acúmulo de fenilpropanóides pela ativação da expressão de enzimas iniciais dessa via. Contudo, uma correlação entre metabolismo de nitrogênio, emissão de NO e acúmulo de fenilpropanóides não foi estabelecida. Por isso, neste trabalho foi analisado o efeito do suprimento de nitrato e da deficiência na NR sobre a síntese de aminoácidos, a emissão de NO e o metabolismo de fenilpropanóides em diferentes tecidos de Arabidopis thaliana selvagem e mutante duplo deficiente para a NR (nia1 nia2). Análises cromatográficas mostraram que a mutante é deficiente na síntese de sinapoil malato (SM), fenilpropanóide predominante nas folhas, resultando no acúmulo de seu precursor sinapoil glicose (SG) e derivados de kaempferol. Essa deficiência não foi causada pela baixa assimilação do nitrato, já que a recuperação do conteúdo de aminoácidos na mutante não alterou seu perfil metabólico. Porém, a maior disponibilidade de nitrato aumentou a atividade da NR, a emissão de NO e os níveis de SM e diminuiu os níveis de SG, nos dois genótipos. O cultivo in vitro da mutante na presença de malato afetou a produção de SM de maneira dose-dependente, enquanto substâncias doadoras de NO causaram apenas um pequeno aumento em SG. Porém, a combinação malato/doador de NO promoveu a recuperação de SM ao nível da selvagem. Esse efeito sinergístico do NO com o malato também ocorreu quando as folhas da mutante foram infiltradas com esses compostos. Além disso, a atividade da enzima sinapoil glicose:malato sinapoil transferase (SMT) foi menor na mutante e a adição de NO aumentou a síntese de SM. Ainda, as folhas da mutante foram incapazes de acumular antocianinas sinapoiladas ao nível da selvagem quando submetidas a um estresse luminoso. Nos botões florais apenas derivados de kaempeferol e quercetina foram identificados e não houve diferença entre selvagem e mutante. Nas raízes não foram identificados fenilpropanóides, provavelmente porque esses compostos só são acumulados nesse órgão na presença de luz. Em relação ao acúmulo de aminoácidos, as folhas do mutante apresentaram níveis reduzidos de todos os aminoácidos parecendo atuar como fonte desses compostos para os botões florais, que não apresentaram nenhuma diferença em relação à selvagem. A glutamina recuperou os níveis de aminoácidos nas folhas, mas não causou diferença nos botões florais. Nas raízes, não houve diferença no conteúdo de aminoácidos entre selvagem e mutante, quando cultivadas no solo, mas in vitro, a mutante foi deficiente, provavelmente pela limitação de nutrientes nessa condição. Esses resultados indicam que o metabolismo dos ésteres de ácido sinápico nas folhas, controlado por aciltransferases dependentes de sinapoil glicose, está comprometido no mutante nia1 nia2 e sugere um potencial papel sinalizador para o NO na ativação dessas aciltransferases. Ainda, o efeito da deficiência na NR nos níveis de aminoácidos parece alterar as relações de fonte e dreno na planta e a folha foi o órgão mais afetado / Abstract: The nitrate reductase (NR) is the first step in nitrate assimilation in plants. Recently, this enzyme has also been related to the synthesis of nitric oxide (NO). Among various signaling actions for plants, NO promotes the accumulation of phenylpropanoids by activating the expression of the initial enzymes of this pathway. However, a correlation between nitrogen metabolism, NO emission and accumulation of phenylpropanoids has not been established. Therefore, this work analyzed the effect of nitrate supply and NR deficiency on the synthesis of amino acids, emission of NO and phenylpropanoid metabolism in different tissues of wild type and NR double-deficient (nia1 nia2) Arabidopsis thaliana plants. Chromatographic analysis showed that the mutant is deficient in the synthesis of sinapoylmalate (SM), the major phenylpropanoid in the leaves, resulting in accumulation of its precursor sinapoylglucose (SG) and kaempferol derivatives. This deficiency was not caused by the low nitrate assimilation, since the recovery of the amino acid content in the mutant did not change its metabolic profile. In contrast, an increased supply of nitrate enhanced NR activity and NO production, and increased SM and decreased SG levels in both genotypes. The in vitro cultivation of mutant in the presence of malate affected the production of SM in a dose-dependent manner, whereas NO donors caused only a slight increase in SG. However, the combination of malate/NO donor promoted the recovery of SM at the level of wild type plants. The synergistic effect of NO with malate in the recovery of SM also occurred when the mutant leaves were infiltrated with these compounds. Furthermore, sinapoylglucose:malate sinapoyltransferase (SMT) activity was reduced in the mutant, and the addition of NO increased SM synthesis. Additionally, mutant leaves were unable to accumulate sinapoylated anthocyanins at the level of wild type when exposed to light stress. In the flower buds just kaempeferol and quercetin derivatives were identified and there was no difference between wild type and mutant. In the roots, phenylpropanoids were not identified, probably because these compounds are accumulated in this organ only in the presence of light. Regarding the accumulation of amino acids, the mutant leaves showed reduced levels of all amino acids and appeared to act as a source of these compounds to the flower buds that showed no difference from the wild plant. Glutamine recovered the amino acid levels in leaves, but caused no difference in flower buds. In the roots, there was no difference in the amino acid content between wild type and mutant, when grown in soil, but in vitro, the mutant was deficient, probably due to nutrient limitation in this condition. These results indicate that hydroxycinnamate ester metabolism in leaves, controlled by the sinapoylglucose-dependent sinapoyltransferases, is compromised in nia1 nia2 mutant and suggests a potential signaling role for NO in the activation of these acyltransferases. Additionally, the effect of NR deficiency in the levels of amino acids appears to alter the relationship of source and sink in the plant and the leaf is the most affected organ / Doutorado / Bioquimica / Mestre em Biologia Funcional e Molecular

Chez les plantes vasculaires, le métabolisme des phénylpropanoïdes conduit à la synthèse de précurseurs de biopolymères structuraux tels que la lignine, ainsi que de nombreux composés antioxydants et anti-UV. Ce métabolisme phénolique est apparu lors de la colonisation des terres par les plantes et a été critique pour leur adaptation à ce nouvel environnement. Physcomitrella patens, une bryophyte phylogénétiquement proche des premières plantes terrestres, est un bon modèle pour l'étude de certains caractères ancestraux. P. patens est dépourvue de lignine. En combinant des approches phylogénomique, génétique et biochimique, ce travail démontre le rôle essentiel de deux BAHD hydroxycinnamoyl tranférases dans le métabolisme phénolique de la mousse et la formation de précurseurs de la cuticule, une couche hydrophobe qui recouvre les parties aériennes de la plante et lui confère une imperméabilité. Il suggère également que deux hydroxycinnamoyl transférases sont requises pour la formation des composés phénoliques solubles accumulés par la mousse. Une exploration préliminaire du métabolisme des flavonoïdes chez ce modèle révèle d’autre part le caractère incomplet et primitif de cette voie métabolique. / In vascular plants, the phenylpropanoid metabolism leads to the synthesis of precursors of structural biopolymers such as lignin and of essential antioxidants and UV screens. The phenolic pathway leading to these compounds appeared upon plant land colonization and is thought critical for their adaptation to this new environment. Physcomitrella patens is a bryophyte, an early-diverging land plant and thus a good model to reveal ancestral traits. P. patens is devoid of lignin. Combining phylogenomic, genetic and biochemical approaches, this work demonstrates the essential role of two BAHD hydroxycinnamoyl transferases in the moss phenolic metabolism and in the formation of precursors of the cuticle, a hydrophobic layer, covering and conferring impermeability to the aerial parts of the plant. It also suggests that two nonredundant hydroxycinnamoyl transferases are required for the formation of the soluble phenolic compounds accumulated in moss. A preliminary exploration of the flavonoid metabolism in this model in addition reveals primitive features of this metabolic route.

<p>The phenylpropanoid pathway gives rise to a wide variety of specialized metabolites, but the majority of carbon flux going through this pathway is directed towards the synthesis of the lignin monomers: <i>p</i>-coumaryl alcohol, coniferyl alcohol and sinapyl alcohol. Lignin is a major impediment in biomass saccharification, which negatively affects animal feed and biofuel production. In an effort to improve biomass for the latter purposes, researchers have altered the polymer through genetic manipulations and generated biomass with lower recalcitrance to saccharification; however, in many cases these efforts have resulted in plant dwarfism. To date, we do not have a full understanding of the extent of lignin modifications a plant is able to tolerate without affecting its growth. More importantly, the mechanism that links dwarfism and modifications in lignin content and composition remains unknown. To contribute to answering these questions, we designed a strategy to incorporate a novel monomer into the lignin of <i>Arabidopsis thaliana</i>. We used mutants in genes that code for enzymes and regulators of the phenylpropanoid pathway to redirect the pathway’s flux towards the synthesis of <i>p</i>-coumaraldehyde and prevent the incorporation of <i>p-</i>coumaryl alcohol. Despite being mutated for the genes typically considered to be required for monolignol biosynthesis, the plants we generated continue to incorporate <i>p-</i>coumaryl alcohol into their lignin. This result suggests that the pathway’s architecture has not been completely elucidated and that there are more enzymes involved in lignification than previously thought. Additionally, we explored the connection between perturbations in phenylpropanoid metabolism and plant growth, by using an inducible system to track the changes in gene expression and metabolism that occur when phenylpropanoid metabolism is restored in a lignin biosynthetic mutant. The use of an inducible system allowed us to not only determine the metabolic processes affected in this mutant, but the proximal sequence of events that lead to restored growth when a functional copy of the mutant gene is induced. Finally, we redirected the flux through the pathway to assess the effects of simultaneously modulating lignin content and composition. Through this project we discovered that redirecting phenylpropanoid flux towards the synthesis of sinapyl alcohol in lignin-deficient mutant backgrounds, results in plant dwarfism. The growth impairment of these mutants can be overcome by providing exogenous coniferyl alcohol, suggesting that dwarfism in these mutants is caused by deficiency in coniferyl alcohol and/or derivatives thereof and not lignin alone. Altogether these projects allowed us to define the cellular processes affected by perturbations in phenylpropanoid homeostasis and the role of other phenylpropanoids besides lignin in this process.</p>

Submitted by Maike Costa (maiksebas@gmail.com) on 2017-02-24T14:21:28Z No. of bitstreams: 1 arquivo total.pdf: 7731405 bytes, checksum: 68a495488b791e03507898e6666f5361 (MD5) / Made available in DSpace on 2017-02-24T14:21:28Z (GMT). No. of bitstreams: 1 arquivo total.pdf: 7731405 bytes, checksum: 68a495488b791e03507898e6666f5361 (MD5) Previous issue date: 2015-08-31 / The genus Piper is the largest the family Piperaceae, its species are distributed especially in neotropical region of the globe. The phytochemical research of Piper species originated numerous scientific papers in many parts of the world, which led to the isolation of many bioactive compounds such as kavalactones, lignoids, chromones, flavonoids and chalcones, terpenes, steroids, prenylated benzoic acids, aristolactams, phenylpropanoids and amides. Piper arboreum is an erect shrub with about 4.0 meters high. It is popularly known as “fruto de morcego”, “alecrim-de-Angola”, “pau-de-Angola” or “beto-preto” and used in Brazil, in the form of decoction for the treatment of rheumatism, bronchitis, flu and colds and used against venereal and urinary tract diseases. This work reports the phytochemical study of the leaves and stem of P. arboreum Aublet. Using usual chromatographic methods and spectroscopic techniques of IR, MS, 1H and 13C NMR and comparison with the literature data it was possible to isolate and identify from the leaves and stem of Piper arboreum, a mixture of steroids: β-sitosterol and stigmasterol, four aristolactams: Piperolactam A, Piperolactam B, Piperolactam C and Cefaronone B, three phenylpropanoids: 3,4,5-trimethoxyphenyl-propanoic acid, 3,4-dimethoxyphenyl-propanoic acid and 3,5-dimethoxy-4-hydroxyphenyl-propanoic acid and three amides: Arboreumine, N-(12,14-dimethoxy-13-hydroxydihydrocinamoil)-3-pyridin-2-one and N-[12-(15,16-methylenedioxyphenyl)-8( E),10 (E) -heptadienoil-pyrrolidine. The last two compounds are being reported for the first time in the literature. / O gênero Piper é o maior da família Piperaceae, suas espécies estão distribuídas especialmente na região neotropical do globo terrestre. A investigação fitoquímica de espécies de Piper originou inúmeros trabalhos científicos em várias partes do mundo, o que levou ao isolamento de diversos compostos bioativos tais como cavalactonas, lignoides, cromonas, flavonoides e chalconas, terpenos, esteroides, ácidos benzoicos prenilados, aristolactamas, fenilpropanoides e amidas. Piper arboreum é um arbusto ereto de aproximadamente 4,0 metros de altura. Popularmente é conhecida como fruto de morcego, alecrim-de-Angola, pau-de-Angola ou beto-preto e utilizada no Brasil na forma de decocto para o tratamento de reumatismo, bronquite, gripe e resfriado e empregada contra doenças venéreas e do trato urinário. Este trabalho reporta o estudo fitoquímico das folhas e caule de P. arboreum Aublet. Utilizando-se métodos cromatográficos usuais e técnicas espectroscópicas de IV, EM e RMN de 1H e 13C uni e bidimensionais e a comparação dos dados com a literatura foi possível isolar e identificar das folhas o e do caule de Piper arboreum, uma mistura de esteroides: β-sitosterol e estigmasterol, quatro aristolactamas: Piperolactama A, Piperolactama B, Piperolactama C e Cefaronona B, três fenilpropanoides: Ácido 3,4,5-trimetoxifenil-propanóico, Ácido 3,4,-dimetoxifenil-propanóico e Ácido 3,5-dimetoxi-4-hidroxifenil-propanóico e três amidas: Arboreumina, N-(12,14-dimetoxi-13-hidroxidihidrocinamoil)-3-piridin-2-ona e N-[12-(15,16-metilenedioxifenil)-8(E),10(E)-heptadienoil-pirrolidina. Os dois últimos compostos estão sendo relatados pela primeira vez na literatura.

Submitted by Renata Lopes (renatasil82@gmail.com) on 2016-04-06T20:09:47Z No. of bitstreams: 1 tatianedeoliveirasouzasenra.pdf: 539017 bytes, checksum: 647e15774caf85c47318364c3b4c57e3 (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2016-04-24T04:11:36Z (GMT) No. of bitstreams: 1 tatianedeoliveirasouzasenra.pdf: 539017 bytes, checksum: 647e15774caf85c47318364c3b4c57e3 (MD5) / Made available in DSpace on 2016-04-24T04:11:36Z (GMT). No. of bitstreams: 1 tatianedeoliveirasouzasenra.pdf: 539017 bytes, checksum: 647e15774caf85c47318364c3b4c57e3 (MD5) Previous issue date: 2013-02-07 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / O presente estudo teve como objetivo avaliar a atividade carrapaticida de formulações hidroetanólicas do carvacrol, (E)-cinamaldeído, trans-anetol e linalol sobre larvas de Rhipicephalus microplus e Dermacentor nitens. Também foram realizados testes avaliando a atividade acaricida do carvacrol, timol, eugenol e (E)-cinamaldeído em diferentes concentrações sobre larvas e ninfas de Rhipicephalus sanguineus e Amblyomma cajennense. Todas as substâncias foram testadas nas concentrações de 2,5; 5,0; 10,0; 15,0 e 20,0 μl/ml, enquanto o timol foi testado nas mesmas concentrações em mg/ml; foram feitas 10 repetições para cada tratamento. Também foram feitos grupos controle com etanol na concentração necessária para solubilizar cada substância. Os grupos experimentais foram mantidos em câmara climatizada (27±1°C e UR>80±10%) por 24 horas e após este período, a mortalidade foi avaliada. Nos testes envolvendo larvas de R. microplus e D. nitens, o carvacrol e (E)-cinamaldeído causaram mortalidade superior a 98% em todas as concentrações testadas, o trans-anetol a partir da concentração de 5,0 μl/ml também causou a morte de 100% das larvas; entretanto, o linalol mesmo na maior concentração não foi eficiente, matando apenas 14,5 e 8,4% de R. microplus e D. nitens, respectivamente. Nos testes com larvas de A. cajennense a concentração de 5,0 μl/ml do carvacrol, timol e (E)-cinamaldeído causou a morte de 100% dos ixodídeos; para o eugenol este percentual só foi observado a partir da concentração de 15,0 μl/ml. Nos testes realizados com larvas de R. sanguineus, os grupos tratados com a menor concentração (2,5 μl/ml) de carvacrol e (E)-cinamaldeído apresentaram mortalidade de 100%; entretanto, para o eugenol, esse percentual só foi observado a partir da concentração de 10,0 μl/ml. Para ninfas desse mesmo ixodídeo, os monoterpenos carvacrol e timol a partir da menor concentração (2,5 μl/ml ou mg/ml) causaram letalidade de 100% em todas as concentrações, diferindo dos resultados obtidos para o eugenol e (E)-cinamaldeído, uma vez que mortalidade de 100% com a utilização dessas substâncias só foram observadas a partir da concentração de 10,0 μl/ml. Nos testes com A. cajennense a mortalidade das larvas tratadas com carvacrol, timol, eugenol e (E)-cinamaldeído na concentração de 2,5 μl/ml foi de 45, 62,7, 10,2 e 81,6%, chegando a 100% na concentração de 5,0 μl/ml para o carvacrol, timol e (E)-cinamaldeído. Para o eugenol esta taxa de mortalidade foi observada a partir da concentração de 15,0 μl/ml. Para ninfas os testes com carvacrol e timol causaram a morte de 100% dos indivíduos a partir das concentrações de 5,0 μl/ml e 10,0 mg/ml, respectivamente; o eugenol só atingiu mortalidade de 100% na concentração de 20,0 μl/ml, enquanto a xii mortalidade máxima no (E)-cinamaldeído não ultrapassou 64%. O conjunto de resultados obtidos neste estudo indica que os monoterpenos carvacrol e timol e os fenilpropanóides eugenol e (E)-cinamaldeído possuem atividade deletéria sobre estes ectoparasitos. Assim, existe a necessidade de novas pesquisas envolvendo outros estágios de desenvolvimento desses carrapatos, a investigação do sinergismo entre as substâncias, além de testes em condições naturais. Quanto a observação preliminar sobre a presença de ectoparasitos em aves silvestres, foram examinadas 72 aves de 17 espécies com prevalência de 29,2% de ectoparasitos. / The present study aimed to evaluate the efficiency of hydroethanolic formulations of carvacrol, (E)-cinnamaldehyde, trans-anetol and linalool against larval stages of Rhipicephalus microplus and Dermacentor nitens. It was also realized assays to evaluate the acaricide effect of carvacrol, thymol, eugenol and (E)-cinnamaldehyde in different concentrations in larvae and nymph of Rhipicephalus sanguineus and Amblyomma cajennense. All substances were tested in concentrations of 2.5, 5.0, 10.0, 15.0 and 20.0 μl/ml, thymol, tested in mg/ml; 10 repetitions were made for each treatment. It was also made with ethanol in a concentration necessary to solubilize each substance. The experimental groups were maintained in BOD incubator (27±1°C e UR>80±10%) during 24 hours and after this period the mortality was available. In the assays with R. microplus and D. nitens larvae, carvacrol and (E)-cinnamaldehyde caused mortality higher than 98% in all the tested concentrations, the trans-anetol caused a mortality of 100% in larvae from a concentration of 5.0 μl/ml; however, linalool was not efficien even at the higher concentration causing a mortality of only 14.5 and 8.4% in R. microplus and D. nitens, respectively. In assays realized with A. cajennense larvae, the concentration of 5.0 μl/ml of carvacrol, thymol and (E)-cinnamaldehyde cased a mortality of 100%; for eugenol this percentage was obtained from the concentration of 15.0 μl/ml. The test realized with R. sanguineus larvae, groups treated with the lower concentration (2.5 μl/ml) of carvacrol and (E)-cinnamaldehyde presented a mortality of 100%; nevertheless, for eugenol this percentage was observed only from the concentration of 10.0 μl/ml. For nymphs of the same species, the monoterpenes carvacrol and thymol from the lower concentration (2.5 μl/ml or mg/ml) caused a lethality of 100% in all concentrations, different from results obtained for eugenol and (E)-cinnamaldehyde, once mortality of 100% with these substances were observed only from the concentration of 10.0 μl/ml. In assays with A. cajennense, the mortality of larvae treated with carvacrol, thymol, eugenol and (E)-cinnamaldehyde in concentrations of 2.5 μl/ml of 45, 62.7, 10.2 and 81.6%, reaching 100% in the concentration of 5.0 μl/ml for carvacrol, thymol and (E)-cinnamaldehyde. For eugenol mortality rate was observed from the concentration of 15.0 μl/ml. For nymphs, tests with carvacrol and thymol caused a mortality of 100% of individuals from concentrations of 5.0 μl/ml and 10.0 mg/ml, respectively; the eugenol only reached this mortality rate at 20.0 μl/ml concentration, while the maximum mortality with (E)-cinnamaldehyde did not exceed 64%. The results indicate that monoterpenes and xiv phenylpropanoids, eugenol and (E)-cinnamaldehyde have deleterious activity in this ectoparasites. Thus, new researches are needed with other developmental stages of these tick species, the investigation of synergism between the substances, aside from tests in natural conditions. As regarding the preliminary observations of the presence of ectoparasites in wild birds, were examined 72 birds from 17 species different with prevalence of 29.2% ectoparasites.

Les phénylpropanoïdes (PPs) constituent l'une des familles les plus abondantes des métabolites secondaires dans le règne végétal. Ils protègent les plantes contre les stress biotiques et abiotiques. De nos jours, les études portent sur l'utilisation des PPs comme alternatifs aux agents antimicrobiens, antioxydants et anti-inflammatoires de synthèse pour leur incorporation dans la formulation des produits alimentaires et pharmaceutiques. Cependant, l'utilisation de PPs est généralement limitée en raison de leur faible solubilité, stabilité et volatilité. L'objectif de notre travail a été d'encapsuler sept PPs dans des molécules cages, les cyclodextrines (CDs), en vue de développer des systèmes naturels et éco-compatibles ayant des applications potentielles dans les domaines alimentaire et pharmaceutique. Trois axes ont été abordés. Le premier axe a porté sur la préparation et la caractérisation des complexes d'inclusion CD/PP en solution et à l'état solide. Les techniques d'"headspace" couplé à la chromatographie en phase gazeuse (HS-CG), spectroscopie UV-visible, ¹H RMN, (2D) ROESY RMN, FTIR, DSC et de la modélisation moléculaire ont été utilisées comme outils pour la caractérisation des complexes obtenus. Des études de phase de solubilité ont été également réalisées. Le deuxième axe a porté sur l'évaluation de l'effet des CDs sur la photostabilité et la vitesse de libéralisation des PPs. Le dernier axe a été orienté vers l'étude des activités anti-radicalaire, antibactérienne et antifongique des complexes d'inclusion CD/PP. Les résultats montrent que les CDs sont capables d'encapsuler les PPs étudiés, réduire leur volatilité, augmenter leur solubilité et photostabilité ainsi que de générer des systèmes de libération prolongée. De plus, l'encapsulation conserve les propriétés antioxydante, antibactérienne et antifongique des PPS. Les résultats de cette étude suggèrent que les complexes d'inclusion des PPs avec les CDs peuvent être considérés comme outils prometteurs pour l'optimisation des formulations alimentaires et pharmaceutiques. / Phenylpropanoids (PPs) are one of the largest families of plants secondary metabolites. They protect plants against biotic and abiotic stresses. Nowadays, extensive research has been dedicated to PPs aiming their use as natural alternatives to synthetic antimicrobial, antioxidant and anti-inflammatory agents in food and pharmaceutical formulations. However, PPs suffer from a low water solubility, high volatility, high light and thermal sensitivity that limit their further use. This current study aimed to encapsulate seven PPs in host cage molecules, cyclodextrins (CDs), in order to develop natural and biocompatible formulation that may find applications in food and pharmaceutical fields. It focused on three main research axes. The first part dealt with the preparation and the characterization of CD/PP inclusion complexes both in solution and in solid state. Characterizations were performed with Static-Headspace-Gas Chromatography (SH-GC), UV-Visible, ¹H NMR, (2D) ROESY NMR, FTIR, DSC and molecular modeling. These investigations were complemented with phase solubility studies. The second part was devoted to the evaluation of the effect of CDs on the PPs photostability and controlled release. The last part aimed to evaluate the CD/PP inclusion complexes as radical scavengers, antibacterial and antifungal agents. Results showed that CDs could successfully encapsulate PPs, reduce their volatility, enhance their solubility and photostability and generate controlled release system. In addition, encapsulation maintained the antioxydant, antibacterial and antifungal properties of PPs. Thus, the CD/PP inclusion complexes could be considered as a promising tool for formulation optimization.