Nosocomial, or hospital acquired, infections, are ubiquitous within the modern clinical setting leading to over $5 billion annually of related healthcare costs in North America. All indwelling devices are highly susceptible to bacterial colonization where physico-chemical interactions between bacteria and biomaterial surfaces have been implicated as determinant factors in the fate of the initial adhesion processes. It has been proposed that by exploiting interference strategies within this critical step of infection the ability to create 'non-infective' biomaterials may be developed. / This thesis demonstrates the effectivity of North American cranberry (Vaccinium macrocarpon) derived proanthocyanidins in preventing the adhesion of pathogenic bacteria to biomaterial surfaces. Specifically, using a model of catheter associated urinary tract infection, significant reductions in initial adhesion of uropathogenic Escherichia coli and Enterococcus faecalis to PVC and PTFE were observed. With the application of colloidal theory, a mechanism of steric interference was determined as responsible for these effects. / The evidence presented implicates PAC as a molecule of interest for the development of novel biomaterials with increased resistance to bacteria colonization.

Phytochemicals found in grains complement those found in fruits and vegetables. These phytochemicals, though minor compounds, contribute to the antioxidant properties which are related to the health benefits associated with the consumption of whole grain. In this thesis project, nine barley genotypes of diverse origin namely CI2230 from Nepal, CI1248 from Israel, 3 Peruvian genotypes; Peru 3, Peru 16 and Peru 35, Hokuto Hadaka from Japan, EX116; a cross between Moroccan and Canadian genotype, EX83; a cross between two Canadian genotypes and EX127; a cross between Canadian and German genotypes were studied. The genotypes were categorized based on appearance into purple, black and yellow grains. Phenolic acids and flavonoids were identified and quantified in these diverse genotypes using HPLC-ESI-MS analysis. The main classes of dietary flavonoids studied in the barleys were anthocyanins and flavan-3-ols. Phenolic acids were identified and quantified (p-coumaric, ferulic, sinapic, caffeic, vanillic). Three ferulic acid dehydrodimers (8-0-4’ DFA, 8-5’ benzofuran form and 5-5’ DFA) were also identified. The most abundant dimeric flavan-3-ols were procyanidins B3 and prodelphinidin B3. The monomeric unit, (+)-catechin, was the most abundant while catechin glucoside (m/z 451) was also identified. Among the Peruvian genotypes, Peru 16 and Peru 35 exhibited relatively high levels of total PA content. Total phenolic content and antioxidant activities of methanolic, acetone and alkali hydrolyzed extracts of the nine barley genotypes was determined by the Folin- Ciocalteau assay, DPPH radical scavenging assay and oxygen radical absorbance capacity (ORAC assay).The acetone extract exhibited the highest antioxidant capacity using all the methods of analysis. Furthermore, dark colored grains were found to exhibit higher contents of phenolic compounds. The phenolic acids, PAs and anthocyanins identified and quantified had significant contribution to the overall antioxidant capacity of the barley whole grain. Four hull-less genotypes namely CI2230, EX127, CI1248 and Peru 35 were further partially sprouted to establish the effects of sprouting on phenolic acid composition. Partial sprouting was observed to significantly increase the soluble conjugated phenolic acids. The barley genotypes studied were found to contain different quantities of phytochemicals and had high proanthocyanidin content thereby rendering them as alternative sources of antioxidants. Barley sprouts present a possible novel food ingredient with improved properties such as phenolic acid composition and other benefits such as easier incorporation into food products under development.

Phytochemicals found in grains complement those found in fruits and vegetables. These phytochemicals, though minor compounds, contribute to the antioxidant properties which are related to the health benefits associated with the consumption of whole grain. In this thesis project, nine barley genotypes of diverse origin namely CI2230 from Nepal, CI1248 from Israel, 3 Peruvian genotypes; Peru 3, Peru 16 and Peru 35, Hokuto Hadaka from Japan, EX116; a cross between Moroccan and Canadian genotype, EX83; a cross between two Canadian genotypes and EX127; a cross between Canadian and German genotypes were studied. The genotypes were categorized based on appearance into purple, black and yellow grains. Phenolic acids and flavonoids were identified and quantified in these diverse genotypes using HPLC-ESI-MS analysis. The main classes of dietary flavonoids studied in the barleys were anthocyanins and flavan-3-ols. Phenolic acids were identified and quantified (p-coumaric, ferulic, sinapic, caffeic, vanillic). Three ferulic acid dehydrodimers (8-0-4’ DFA, 8-5’ benzofuran form and 5-5’ DFA) were also identified. The most abundant dimeric flavan-3-ols were procyanidins B3 and prodelphinidin B3. The monomeric unit, (+)-catechin, was the most abundant while catechin glucoside (m/z 451) was also identified. Among the Peruvian genotypes, Peru 16 and Peru 35 exhibited relatively high levels of total PA content. Total phenolic content and antioxidant activities of methanolic, acetone and alkali hydrolyzed extracts of the nine barley genotypes was determined by the Folin- Ciocalteau assay, DPPH radical scavenging assay and oxygen radical absorbance capacity (ORAC assay).The acetone extract exhibited the highest antioxidant capacity using all the methods of analysis. Furthermore, dark colored grains were found to exhibit higher contents of phenolic compounds. The phenolic acids, PAs and anthocyanins identified and quantified had significant contribution to the overall antioxidant capacity of the barley whole grain. Four hull-less genotypes namely CI2230, EX127, CI1248 and Peru 35 were further partially sprouted to establish the effects of sprouting on phenolic acid composition. Partial sprouting was observed to significantly increase the soluble conjugated phenolic acids. The barley genotypes studied were found to contain different quantities of phytochemicals and had high proanthocyanidin content thereby rendering them as alternative sources of antioxidants. Barley sprouts present a possible novel food ingredient with improved properties such as phenolic acid composition and other benefits such as easier incorporation into food products under development.

Submitted by Edileide Reis (leyde-landy@hotmail.com) on 2013-04-22T14:46:07Z No. of bitstreams: 5 André Barreiros 5.pdf: 1310338 bytes, checksum: d59dc4bf426cac1e442be4eb9ee6d894 (MD5) André Barreiros 4.pdf: 1401764 bytes, checksum: 07e4fdbbe9dd39da5506e4d366f674d3 (MD5) André Barreiros 3.pdf: 1612776 bytes, checksum: 615c8ab2914ba19f8eb7af1b4c5d83cf (MD5) André Barreiros 2.pdf: 1211895 bytes, checksum: c2f0cb1c002d0263b0c0a42dbdda2a14 (MD5) André Barreiros 1.pdf: 239109 bytes, checksum: d5ef73879c018bc308a7c7bb697a9ebc (MD5) / Made available in DSpace on 2013-04-22T14:46:07Z (GMT). No. of bitstreams: 5 André Barreiros 5.pdf: 1310338 bytes, checksum: d59dc4bf426cac1e442be4eb9ee6d894 (MD5) André Barreiros 4.pdf: 1401764 bytes, checksum: 07e4fdbbe9dd39da5506e4d366f674d3 (MD5) André Barreiros 3.pdf: 1612776 bytes, checksum: 615c8ab2914ba19f8eb7af1b4c5d83cf (MD5) André Barreiros 2.pdf: 1211895 bytes, checksum: c2f0cb1c002d0263b0c0a42dbdda2a14 (MD5) André Barreiros 1.pdf: 239109 bytes, checksum: d5ef73879c018bc308a7c7bb697a9ebc (MD5) Previous issue date: 2005 / O presente trabalho descreve o estudo fitoquímico de Dioclea violacea Mart. além da avaliação das atividades antioxidante e imunomoduladora das substâncias isoladas. Dioclea violacea é uma trepadeira pertencente à família Leguminosae (Fabaceae), subfamília Faboideae (Papilionoideae), tribo Phaseoleae, que ocorre no litoral desde a Guiana até o estado de São Paulo. O caule de um espécime foi coletado no município de Umburanas, Bahia, em área de vegetação de caatinga e relevo de tabuleiro. Após secagem e moagem o material foi submetido à maceração com metanol. O extrato metanólico obtido foi particionado fornecendo os extratos hexânico, clorofórmico e acetato de etila. Os extratos foram submetidos a purificação separadamente, sendo seus constituintes isolados e purificados através de técnicas de CC e CCDP em gel de sílica 60, 60H, 60PF, Poliamida 6 e 11 e permeação em gel de Sephadex LH20. Deste modo, as substâncias isoladas tiveram suas estruturas elucidadas através da análise de dados de RMN de 1H, 13C incluindo experimentos DEPT, ?nOe diff?, além de técnicas bidimensionais como HETCOR, HOMO/COSY, HMQC, HMBC, NOESY, auxiliadas por EMIE, FAB, UV, IV, [a]D25 e CD. Do extrato hexânico foram isolados e identificados a-tocoferol, estigmast-4-en-3-ona, b-sitosterol, estigmasterol, lupeol e b-amirina. Do extrato clorofórmico foram isolados ácido oleanólico, a nova flavanona 7,4?-diidroxi-6-metoxiflavanona, além das 7-hidroxi-6-metoxiflavanona, 5,7-diidroxiflavanona, 5,7-diidroxi-8-metoxiflavanona, 7-hidroxiflavanona, 4?,7-diidroxiflavanona, 7,3?,4?-triidroxiflavanona já anteriormente isoladas de outras fontes. Além disso, foram também isolados deste extrato o 7-hidroxi-6-metoxiflavanonol, 2?,4? diidroxichalcona, 2?,4,4?-triidroxi-3-metoxichalcona, 2?,3,4,4?-tetraidroxichalcona, além da lactona lasiodiplodina e de um novo biflavonóide a,b?-epoxi,-2,2?,4?-triidroxichalcona-(b®4?)-7,4?-diidroxi-6-metoxiflavanona. Enquanto que, do extrato acetato de etila foram isolados a epicatequina, a proantocianidina do tipo A denominada 3?,4?,7-triidroxiflavana-(2b®7,4b®8)-3-prenil-fustina, e outras duas proantocianidinas do tipo A2; epicatequina-(2b®7,4b®8)-epicatequina e epigalocatequina-(2b®7,4b®8)-epicatequina. As substâncias isoladas foram submetidas a testes de atividade antioxidante pelos métodos de inibição da autooxidação do b-caroteno, e seqüestro do radical livre DPPH, sendo que a epicatequina e as proantocianidinas epicatequina-(2b®7,4b®8)-epicatequina e epigalocatequina-(2b®7,4b®8)-epicatequina foram as mais ativas. Algumas das substâncias foram submetidas a testes de atividade imunomoduladora pelos métodos de inibição da proliferação de linfócitos e inibição da síntese de NO, onde a 7 hidroxiflavanona demonstrou maior atividade. A substância mais ativa 7-hidroxiflavanona foi sintetizada a partir da reação de esterificação do ácido cinâmico com o resorcinol em presença de ácido polifosfórico, seguida de rearranjo de Fries e ciclização, porém com baixo rendimento (1,6%). Os produtos principais da reação foram a 7-hidroxi-3,4-diidro-4-fenilcumarina (68,7%) e a 5-hidroxi-3,4-diidro-4-fenilcumarina (11,2%). / Salvador

Orientador: Alba Regina Monteiro Souza Brito / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-22T00:33:18Z (GMT). No. of bitstreams: 1 Faria_FelipeMeirade_D.pdf: 5634635 bytes, checksum: 78374f3fbaac985834cc384c5652485c (MD5) Previous issue date: 2013 / Resumo: As doenças inflamatórias intestinais (DII) compreendem doença de Crohn e reto-colite ulcerativa inespecífica, ambas caracterizadas por processo inflamatório crônico e descontrolado no intestino mediado por células do sistema imune. DII tem sido objeto de extensas pesquisas nos últimos anos; indefinição sobre etiologia e ausência de tratamento eficaz os justificam. Nesse cenário, produtos naturais surgem como alternativas terapêuticas, uma vez que moléculas com atividade farmacológica pronunciada, especialmente compostos fenólicos, têm merecido destaque. Neste trabalho foram avaliadas três frações orgânicas (aquosa - Aq, butanólica - BuOH, e acetato de EtOAc) obtidas a partir das cascas de Rhizophora mangle em modelos experimentais de colite induzida por dextrana sal sódico (DSS) em camundongos e ácido 2,4,6-tri-nitro-benzeno-sulfônico (TNBS) em ratos. BuOH (0.5 e 1.5 mg.Kg-1) e EtOAc (1.5 mg.Kg-1) foram efetivas no modelo de colite por DSS, enquanto BuOH 0.5 mg.K-1 e EtOAc 1.5 mg.Kg-1 mostraram-se eficazes na colite aguda por TNBS. No modelo sub-crônico de colite os tratamentos BuOH e EtOAc reduziram novamente os danos causados pelo TNBS, embora BuOH tenha se mostrado mais eficiente. A avaliação de mediadores envolvidos nesses modelos experimentais mostrou que tanto BuOH quanto EtOAc exercem ação sobre a atividade da glutationa peroxidase (GSH-Px) e mieloperoxidase (MPO) bem como sobre os níveis das citocinas pró-inflamatórias fator de necrose tumoral-? (TNF-?), interferon-? (IFN-?), interleucinas 1?, 6 e 12 (IL-1?, IL-6 e IL-12), além de reduzirem a expressão de ciclo-oxigenase-2 (COX-2) no modelo de DSS. No modelo sub-crônico de colite induzida por TNBS, às duas frações atuaram sobre a atividade da GSH-Px e catalase (CAT) e sobre os níveis de glutationa (GSH), TNF-? e IL-12; EtOH também reduziu os níveis de IL-6 e levou ao aumento de citocina anti-inflamatória interleucina-10 (IL-10), BuOH por sua vez mostrou-se eficaz em reduzir o índice de peroxidação lipídica (TBARS) e atividade da MPO, atuando também sobre a expressão de ciclo-oxigenase-1 (COX-1). A análise fitoquímica revelou presença de procianidinas oligoméricas nas duas frações ativas, BuOH e EtOAc, sugerindo que estas substâncias sejam responsáveis pelos efeitos produzidos pelos tratamentos experimentais. As frações BuOH e EtOAc apresentaram efeitos importantes sobre os componentes imune, inflamatório e oxidante em modelos experimentais de colite induzida por TNBS e DSS em doses consideradas baixas para uma mistura de substâncias. As procianidinas de R. mangle surgem então como alternativa terapêutica interessante sobre as DII / Abstract: Inflammatory bowel diseases (IBD) comprises Crohn's disease and ulcerative colitis, both are characterized by an immune-mediated chronic inflammatory process in the gut. IBD have been studied over the past few years; undefined etiology and the absence of effective treatments justify these studies. In this scenario, natural products arises as therapeutic alternative, once various studies in animal models have shown efficient treatments. Nowadays, molecules with pronounced pharmacological activities, especially phenolic compounds, have been highlighted. In this work three organic fractions (aqueous - Aq, butanolic - BuOH and ethyl acetate - EtOAc) obtained from Rhizophora mangle barks were evaluated in experimental models of dextran sodium sulphate (DSS)-induced colitis in mice and 2,4,6-tri-nitro-benzene sulphonic acid (TNBS)-induced colitis in rats. BuOH (0.5 and 1.5 mg.Kg-1) and EtOAc (1.5 mg.Kg-1) were effective in the DSS-induced model, while BuOH (0.5 mg.Kg-1) and EtOAc (1.5 mg.Kg-1) were also effective in acute TNBS-induced model of colitis, which lead to the evaluation of these last ones in sub-chronic TNBS-induced models of colitis. In this model, both treatments reduced the injury provoked by the instillation of TNBS, although BuOH has shown more efficiency. The evaluation of the mediators involved in these experimental models showed that either BuOH as EtOAc act on the glutathione peroxidase (GSH-Px) and myeloperoxidase activities as well as on pro-inflammatory cytokines tumor necrosis factor- (TNF-?), interferon- (IFN-?) and interleukins -1?, 6 and 12 (IL-1, IL-6 and IL-12) levels, and down-regulation of ciclo-oxygenase-2 (COX-2) expression in the DSS-induced model of colitis. Whereas in the sub-chronic TNBS-induced model of colitis, both fractions showed effects on the GSH-Px and catalase (CAT) activities, and glutathione (GSH), TNF-? and IL-12 levels; EtOAc also reduced IL-6 levels and lead to the enhancement of the levels of an anti-inflammatory cytokine, interlukin-10 (IL-10), on the other hand BuOH showed to be efficient in reducing lipid peroxidation index (TBARS) and MPO activity, acting on the upregulation of cyclo-oxygenase-1 (COX-1). Phytochemistry analysis reveals the presence of oligomeric procyanidins in both fractions, BuOH and EtOAc, suggesting that these substances are responsible for the effects produced by the experimental treatments. BuOH and EtOAc fractions presented important effects on the immune, inflammatory and oxidant components in the experimental models of TNBS and DSS-induced colitis using doses, considered, lower for a mixture of substances. The procyanidins of R. mangle became an interesting alternative therapeutic for IBD / Doutorado / Farmacologia / Doutor em Farmacologia

Parmi les métabolites secondaires impliqués dans la qualité du raisin et du vin, les tanins condensés ou proanthocyanidines (PAs) jouent un rôle majeur, en particulier dans l'astringence et la stabilité de la couleur du vin. Ces molécules sont également impliquées dans la défense des plantes contre des stress biotiques et abiotiques. En outre, les effets bénéfiques des PAs pour la santé humaine sont bien documentés. Les PAs de la vigne ont la particularité d’être estérifiées avec de l’acide gallique. Une réaction d’acylation appelée galloylation est responsable de cette modification. Les études montrent que la galloylation influence les propriétés œnologiques et pharmacologiques des PAs. Dans la baie de raisin, les PAs sont synthétisés dans les premiers stades de développement, principalement dans les pellicules et les pépins. Un nombre relativement faible d'étapes enzymatiques sont nécessaires pour la biosynthèse de la structure de base de ces métabolites et les gènes correspondants sont aujourd'hui largement connus chez les plantes modèles, y compris chez la vigne. Cependant, les mécanismes moléculaires impliqués dans les étapes finales, y compris la galloylation, ne sont encore que partiellement connus. Des résultats antérieurs obtenus après la recherche de QTL influençant la composition du raisin, et en particulier le taux de galloylation des PAs, et des études transcriptomiques après surexpression de facteurs de transcription régulant la biosynthèse de la voie des PAs, ont permis l'identification de gènes potentiellement impliqués dans ces étapes. Des gènes de shikimate déshydrogénase (SDH) ont été identifiés. Ces gènes interviendraient en amont, pour la biosynthèse de l'acide gallique. Trois glucosyltransférases ainsi identifiées et déjà caractérisées au laboratoire sont impliquées dans la biosynthèse de l'ester de glucose de l'acide gallique (β-glucogalline), qui servirait d'intermédiaire pour la galloylation des PAs. Ces méthodes de criblage ont également permis d’identifier 2 acyltransférases de type sérine carboxypeptidase, nommées glucose acyltransférases (GATs) qui seraient capables de catalyser la dernière étape de galloylation: le transfert de l'acide gallique depuis la β-glucogalline sur les PAs. Le premier objectif de cette thèse a été de déterminer la fonction des SDHs codées par les gènes de vigne. Certaines SDHs recombinantes produites de façon hétérologue chez E.coli ont la capacité à produire de l'acide gallique in vitro. Leur niveau d’expression au cours du développement et dans différents tissus de la baie a également été établi. Les résultats obtenus in vitro sont étayés par le profil métabolique (acide gallique, β-glucogalline et PAs) de hairy-roots de vigne transformées avec un gène de SDH. Le second objectif de cette thèse a été de valider la fonction des GATs par expression transitoire dans des feuilles de tabac et des tests enzymatiques in vitro. La transformation transitoire de feuilles de vigne avec les GATs a permis de moduler la concentration d’esters phénoliques et nomment des flavan-3-ols galloylés in planta. L’étude de ces gènes a été étendue aux plantes vasculaires par des analyses phylogénétiques et a permis d’identifier des motifs peptidiques potentiellement impliqués dans les mécanismes étudiés et reflétant la sub-fonctionnalisation de certains gènes. Ce travail a fourni des informations sur les bases génétiques et les mécanismes moléculaires impliqués dans la biosynthèse de l'acide gallique et son transfert en deux étapes sur les flavan-3-ols (galloylation). De nouvelles hypothèses sur l'intervention de différents transporteurs et la nature des molécules transportées pourront être formulées. / Among the secondary metabolites involved in grape berry and wine quality, condensed tannins or proanthocyanidins (PAs) play a major role, especially in astringency and color stability of wine. These molecules are also involved in plant defence against biotic and abiotic stresses. Furthermore, the beneficial effects of PAs to human health are well documented. In grapevine, PAs have the distinctive feature of being esterified with gallic acid. An acylation reaction called galloylation is responsible for this modification. Studies show that the galloylation influences oenological and pharmacological properties of PAs. In the grape berry, PAs are synthesized in the early stages of development, mainly in skin and seeds. A relatively small number of enzymatic steps are required for the biosynthesis of the basic structure of these metabolites and the corresponding genes are now widely known in model plants, including in grapevine. However, the molecular mechanisms involved in the final steps, including galloylation, are only partially known. Earlier results obtained after the search of QTL influencing the composition of the grape berry, especially the galloylation ratio of PAs, and transcriptomic studies after overexpression of transcription factors that regulate PAs biosynthesis pathway, have allowed the identification of genes potentially involved in these steps. Shikimate dehydrogenase (SDH) genes were identified. These genes would intervene upstream, for the biosynthesis of gallic acid. Three identified glucosyltransferases, already characterized in the laboratory, are involved in the biosynthesis of glucose ester of gallic acid (β-glucogalline), which could serve as an intermediary for PAs galloylation. These screening methods have also helped to identify 2 serine carboxypeptidase-like acyltransferases, called glucose acyltransferases (GATs) which are capable of catalyzing the last step of galloylation: the transfer of gallic acid from β-glucogalline to PAs. The first objective of this thesis was to determine the function of the SDHs encoded by grapevine genes. Recombinant SDHs, produced heterologously in E. coli, have the capacity to generate gallic acid in vitro. Their level of expression during development and in different tissues of the berry was also established. In vitro results are supported by the metabolic profile (gallic acid, β-glucogallin and PAs) of grapevine hairy -roots transformed with a SDH gene. The second objective of this thesis was to validate the function of the GATs by transient expression in tobacco leaves and in vitro enzyme assays. The transient transformation of grapevine leaves with GATs allowed to modulate the concentration of phenolic esters and notably galloylated flavan-3-ols in planta. The study of these genes was extended to vascular plants by phylogenetic analyses which allowed to identify peptide motifs potentially involved in the studied mechanisms and reflecting the sub-functionalization of certain genes. This work has provided informations on the genetic basis and molecular mechanisms involved in the biosynthesis of gallic acid and its two-step transfer on flavan-3-ols (galloylation). New hypotheses on the intervention of different carriers and nature of transported molecules can be proposed.

A cultura popular atribui ao gênero Passiflora várias propriedades medicinais e funcionais. Frutas frescas e secas, cascas, infusões e suco da polpa de maracujás silvestres são consumidos e comercializados para controlar ansiedade, insônia, tremores, diabetes e obesidade, entre outras indicações. Dessa forma, os objetivos deste trabalho foram caracterizar e comparar as folhas e frutos (pericarpo, polpa e sementes) das espécies P. edulis Sims variedades BRS Sol do Cerrado, Ouro Vermelho e Gigante Amarelo, P. alata Curtis, P. setacea DC e P. tenuifila Killip em relação ao seu potencial funcional, avaliando-se conteúdos de compostos fenólicos totais, proantocianidinas, vitamina C, alcaloides, tipos e teores de flavonoides, controle potencial de glicemia pós-prandial (inibição de α-amilase e α-glicosidase in vitro) e capacidade antioxidante in vitro. O principal flavonoide encontrado tanto em folhas como em frutos foi a homoorientina e as maiores concentrações foram encontradas em folhas, seguido de pericarpo. Os maiores teores de alcaloides foram detectados nas polpas dos frutos de P. edulis (0,1-0,2 mg/100 g b.s.). As maiores concentrações de compostos fenólicos foram detectadas nas folhas de P. edulis e P. alata, porém as sementes de P. tenuifila e principalmente P. setacea apresentaram os teores mais elevados em comparação com as outras frações. Em relação às proantocianidinas, este é o primeiro estudo que mostra a presenças destes compostos em Passiflora spp, em concentrações significativamente maiores nas sementes. Além disso, o estilbeno piceatanol (3,4,3\',5\'-tetrahidroxi-trans-estilbeno) também foi encontrado nas sementes de P. edulis variedades BRS Ouro Vermelho (2 mg/100 g b.s.) e Gigante Amarelo (3 mg/100 g b.s.) e P. alata (10 mg/100 g b.s.). Os compostos fenólicos encontrados nas sementes de todas as espécies de Passiflora analisadas se mostraram potentes inibidores das enzimas α-amilase e α-glicosidase. Este trabalho mostrou que os frutos e as folhas de Passiflora spp podem ser considerados boas fontes de compostos bioativos, especialmente com atividade antioxidante, apresentando, portanto, potenciais efeitos benéficos sobre a saúde humana. / Folk culture assigns to the genus Passiflora various medicinal and functional properties. Fresh and dried fruits , barks , teas and wild passion fruit pulp juice are consumed and sold to control anxiety, insomnia , tremor , diabetes and obesity , among other indications .Thus, the aim of this study were to characterize the leaves and fruit (pericarp , pulp and seeds) of the species P. edulis Sims varieties BRS Sol do Cerrado, Ouro Vermelho e Gigante Amarelo, P. alata Curtis, P. setacea DC e P. tenuifila Killip in relation to their functional potential , evaluating content of total phenolic compounds , proanthocyanidins , vitamin C , alkaloids , types and levels of flavonoids, potential control of postprandial glycemia (inhibition of α-amylase and α-glucosidase in vitro) and in vitro antioxidant capacity. The main flavonoid found in both leaves and fruits was homoorientina and the highest concentrations were found in leaves, followed by pericarp . The highest levels of alkaloids were detected in the pulp of the fruits of P. edulis ( 0.1-0.2 mg/100 g b.s. ) . The highest concentrations of phenolic compounds were identified in the leaves of P. edulis and P. alata, but the seeds of P. tenuifila and especially P. setacea showed higher levels compared with the other fractions . With respect to proanthocyanidin, this is the first study to show the presence of these compounds in Passiflora spp at concentrations significantly increased in seeds. Furthermore, piceatannol stilbene ( 3,4,3\',5\'- tetrahydroxy -trans- stilbene ) was also found in the seeds of P. edulis varieties BRS Ouro Vermelho (2 mg/100 g d.w.) and Gigante Amarelo (3 mg/100 g d.w.) and P. alata (10 mg/100 g d.w.). Phenolic compounds found in the seeds of all species analyzed are good inhibitors of α-amylase and α-glicosidase. This work showed that the fruit and leaves of Passiflora spp. can be considered good sources of bioactive compounds, especially with antioxidant activity, thus presenting potential beneficial effects on human health.

[ES] El cultivo del caqui ha crecido enormemente en la región Valenciana en los últimos 20 años al presentarse como una alternativa interesante al cultivo de cítricos. Sin embargo, su cultivo se enfrenta a tres problemas importantes. En primer lugar, su producción es monovarietal y se centra en la variedad Rojo Brillante, lo que conlleva un riesgo sanitario y de mercado importante que pone de manifiesto la necesidad de aumentar el número de variedades disponibles. En segundo lugar, el cambio climático pone en peligro la producción de caqui en zonas mediterráneas debido a un aumento en la salinidad del agua y suelo. La salinidad afecta especialmente al caqui debido a sus efectos sobre la producción, calidad y postcosecha de la fruta. La falta de patrones tolerantes a la salinidad compatibles con las condiciones de cultivo además agrava este problema, por lo que es necesario el desarrollo de patrones clonales tolerantes a la salinidad. En tercer lugar, la industria del caqui está basada en la aplicación de tratamientos postcosecha de desastringencia. Aunque existen variedades naturalmente no astringentes, su uso en el Mediterráneo es complicado debido a la incompatibilidad con los patrones existentes. Además, la salinidad afecta gravemente la eficacia del tratamiento de desastringencia. Por ello, cabe destacar que se necesita más información acerca de los mecanismos moleculares que controlan el desarrollo de la astringencia para poder desarrollar nuevas variedades y/o unos tratamientos de desastringencia más efectivos. En el contexto de la mejora genética y la biología molecular, pueden distinguirse tres objetivos para mejorar las condiciones de cultivo en esta región: el desarrollo de nuevas variedades de caqui con una época de madurez distinta a Rojo Brillante, el desarrollo de patrones clonales tolerantes a la salinidad y compatibles con variedades no astringentes y la obtención de nuevos conocimientos acerca de las bases moleculares del mecanismo de regulación de la astringencia para optimizar los tratamientos postcosecha. La presente tesis ha contribuido a estos tres objetivos. Respecto a la obtención de nuevas variedades, se ha desarrollado un análisis de diversidad genética del banco de germoplasma del IVIA, lo que es un recurso importante para la planificación de cruces. Para asegurar una mejora genética eficiente y evitar redundancias en la colección, se ha efectuado un análisis de diversidad usando secuencias microsatélites. El análisis ha arrojado datos de gran utilidad, como el descubrimiento de alelos únicos, que permiten la identificación rápida de variedades, o la división de la colección de acuerdo con el tipo de astringencia del fruto, confirmando el fondo genético común de las accesiones que comparten este carácter. Además, el mapa de las relaciones filogenéticas generado será de gran utilidad para la planificación futura de cruces dentro del programa de mejora de caqui. Para abordar el problema de la salinidad, se han realizado distintos ensayos de estrés salino en invernadero con distintas poblaciones de especies utilizadas como patrón de caqui: Diospyros virginiana (tolerante a salinidad), Diospyros kaki (sensible a salinidad y compatible) y Diospyros lotus (sensible a salinidad). Además, debido a los caracteres de gran interés presentes en estas dos especies, se incluyó en los ensayos una población proveniente de un retrocruce entre D. kaki y D. virginiana. Como resultado, varios individuos de estas poblaciones se han seleccionado debido a su tolerancia a la salinidad, ya que podrían tener las características adecuadas para ser patrones clonales. Complementariamente, se han usado aproximaciones fisiológicas, transcriptómicas y de secuenciación masiva de transcriptoma para hacer una primera descripción de los mecanismos de tolerancia a la salinidad presentes en estas especies. Como resultado, se han descrito los principales caracteres relacionados con la tolerancia a la salinidad en el género Diospyros, como la compartimentalización de iones, la expresión en raíz de un gen similar a HKT-1, la abundancia de canales de cloruro o la arquitectura de raíz. Finalmente, para obtener más información sobre la biosíntesis de proantocianidinas, la molécula responsable de la astringencia, se formuló la hipótesis de la existencia de un complejo proteico del tipo MBW (MYB-bHLH-WD40) que podía regular la ruta molecular de forma análoga a la regulación existente en otras especies estudiadas. En primer lugar se secuenció un gen tipo WD40, posible ortólogo de TTG1 de Arabidopsis thaliana. El papel de este gen y otros genes del hipotético complejo (MYB2, MYB4 y MYC1) han sido estudiados mediante un análisis transcriptómico en distintos estadios de madurez de frutos astringentes y no astringentes. Además, se ha observado mediante microscopía la localización celular de estos genes mediante la expresión transitoria con fusión con fluorescencia en hojas de Nicotiana benthamiana y hemos analizado su interacción mediante ensayos de doble híbrido en levadura y BiFC (complementación bimolecular de fluorescencia) mediante expresión transitoria en hojas de Nicotiana benthamiana. Finalmente, hemos analizado los efectos de estos genes y de sus interacciones en la ruta de biosíntesis de proantocianidinas usando el promotor del gen ANR (antocianidin reductasa) y promotores de los genes reguladores MYC1 y MYB4, fusionándolos con una luciferasa como gen delator y coexpresándolos con distintas combinaciones de los genes del complejo MBW mediante expresión transitoria en hojas de Nicotiana benthamiana. Los resultados de estos ensayos confirman la interacción nuclear de estos genes y su papel en la regulación de la producción de la astringencia, dando lugar a un modelo básico con algunas diferencias a los propuestos en otras especies. / [EN] Persimmon culture has vastly grown in the last 20 years as an alternative to citrus production in the Valencian region. However, its production faces three important problems. First, monovarietal production based on Rojo Brillante is a sanitary and market risk that requires to increase the number of varieties available. Second, climate change has threatened persimmon production in the Mediterranean areas because the salinity increase in water and soil. Salinity severely affects production, quality and postharvest life of the fruit. The lack of salt-tolerant rootstocks compatible with the cultural conditions aggravates the problem; hence clonal salt-tolerant rootstocks are needed. Third, persimmon industry relies on the postharvest deastringency treatment of the varieties. Even though there are varieties that are naturally non-astringent, its culture in the Mediterranean area is limited due to rootstock incompatibility. Additionally, the costly postharvest treatment is also affected by salinity. Consequently, more information is needed about the molecular mechanism of astringency biosynthesis in persimmon fruit in order to develop new varieties and/or more effective treatments for removing astringency of the fruit. In the context of breeding and molecular biology, three goals need to be approached in order to improve the cultural conditions: development of new persimmon varieties with maturity date different from Rojo Brillante, development of salt-tolerant clonal rootstocks compatible with non-astringent varieties and research on the molecular bases of the mechanism of astringency production to optimize the postharvest treatments. This research contributes and provides knowledge to address these goals. Concerning breeding of new varieties, the IVIA persimmon germplasm collection is an important resource for breeding of new varieties. To ensure efficient breeding and avoid redundancy in the collection, a SSR analysis was carried out to solve this problem. This analysis has provided useful data, such as the discovery of unique alleles, that allows rapid identification of varieties or the division of the population according to its astringency type, confirming the common genetic background of the accessions concerning to this trait. The generated genetic diversity map will be useful for designing crosses in the breeding program. To address the salinity problem, several salt-stress assays were done in greenhouse using different persimmon rootstocks populations: Diospyros virginiana (salt-tolerant), Diospyros kaki (salt-sensitive fully compatible) and Diospyros lotus (salt-sensitive). Furthermore, due to the desirable characters present in these species, a backcross progeny between D. kaki and D. virginiana was obtained and included in the salt-stress assays. Thus, several individuals from all the populations have been selected, which are tolerant to salinity and could meet the desired characteristics for appropriate persimmon clonal rootstocks. Furthermore we have used physiological, transcriptomic and mass transcriptome sequencing approaches in order to discover the physiological and genetic bases of salinity tolerance in these species. As a result, we described the main facts related to salinity tolerance mechanisms in Diospyros species, such as ion compartmentalization, HKT-1-like root expression, abundancy of chloride channels and root architecture. Finally, in order to get a deeper insight on the biosynthesis of proanthocyanidins, the responsible molecule of astringency, we hypothesized the presence of a MBW protein complex (MYB-bHLHWD40) in persimmon fruit, involved in the regulation of the proanthocyanidins synthesis pathway in a similar way to other species. We firstly sequenced the putative persimmon WD40 orthologue of TTG1 gene from Arabidopsis thaliana. The role of this gene and other genes of the complex (MYB2, MYB4 and MYC1) was studied through transcriptomic analysis in different stages of nonastringent and astringent fruit. Furthermore, we have observed through microscopy the subcellular localization of these genes using fluorescent protein fusions and transient expression in Nicotiana benthamiana leaves, and analyzed its interaction through yeast twohybrid assays and BiFC (bimolecular fluorescence complementation) experiments in Nicotiana benthamiana leaves. In addition, we have analyzed the effects of these interactions in the pathway of proanthocyanidin biosynthesis using the ANR (anthocyanidin reductase) gene promoter and the promoters of some of these genes (MYB4 and MYC1) fused with a luciferase reporter, together with the coexpression of the MBW genes using transient expression in Nicotiana benthamiana leaves. The results of these assays have confirmed the nuclear interaction of these genes and its role in the regulation of astringency biosynthesis, providing a basic model with some differences with respect to the models proposed in other species. / [CA] El cultiu del caqui ha crescut enormement en la regió Valenciana en els últims 20 anys al presentar-se com una alternativa interessant al cultiu de cítrics. No obstant, el seu cultiu s’enfronta a tres problemes importants. En primer lloc, la seua producció es monovarietal i està centrada en la varietat Rojo Brillante, el que suposa un risc sanitari i de mercat important que posa de manifest la necessitat de augmentar el nombre de varietats disponibles. En segon lloc, el canvi climàtic posa en perill la producció de caqui a la zona mediterrània degut a un augment en la salinitat del aigua i del sòl. La salinitat afecta de forma especialment al caqui degut al seus efectes sobre la producció, qualitat i vida post collita de la fruita. La falta de patrons tolerants a salinitat compatibles amb les condicions de cultiu a més agreuja aquest problema, pel que es necessari el desenvolupament de patrons clonals tolerants a salinitat. En tercer lloc, la industria del caqui esta basada en la aplicació de tractaments post collita de deastringència. Encara que existixen varietats naturalment no astringents, el seu us al mediterrani es complicat degut a la incompatibilitat en els patrons existents. A més, la salinitat afecta gravement a la eficàcia del tractament post collita de deastringència. Per això, cal destacar que es necessita conèixer millor els mecanismes moleculars que controlen el desenvolupament de la astringència per a la obtenció de noves varietats i/o uns tractaments post collita més efectius. En aquest context de millora genètica i biologia molecular, es poden distingir tres objectius per a millorar les condicions del cultiu en aquesta regió: el desenvolupament de noves varietats de caqui en una època de maduresa distinta a la de Rojo Brillante, el desenvolupament de patrons clonals tolerants a salinitat i compatibles amb varietats no astringents i la obtenció de nous coneixements sobre les bases moleculars del mecanisme de producció de astringència per optimitzar els tractaments post collita. La present tesi ha contribuït a aquests tres objectius. En el que respecta a la obtenció de noves varietats, s’ha desenvolupat un anàlisi de diversitat genètica del banc de germoplasma de l’IVIA, el qual es un recurs important per a la planificació de creuaments. Per a assegurar una millora genètica eficient i evitar redundàncies a la col·lecció, s’ha efectuat un anàlisi en microsatèl·lits. El anàlisi ha resultat en dades de gran utilitat, com el descobriment de al·lels únics, que permiteixen la identificació rapida de varietats, o la divisió de la col·lecció d’acord en el tipus d’astringència del fruit, confirmant el fons genètic comú de les accessions que comparteixen aquest caràcter. A més, el mapa de les relacions filogenètiques elaborat serà de gran utilitat per a la planificació futura de creuaments dins del programa de millora de caqui. Per a abordar el problema de la salinitat, s’han realitzat distints assajos d’estrés salí en hivernacle amb distintes poblacions de patró de caqui: Diospyros virginiana (tolerant a salinitat), Diospyros kaki (sensible a salinitat i amb compatibilitat) i Diospyros lotus (sensible a salinitat). A més, degut als caràcters de gran interès presents en aquestes dos espècies, es va incloure en els assajos una població provenient de un retrocreuament entre D. kaki i D. virginiana. Com a resultat, diversos individus d aquestes poblacions s’han seleccionat degut a la seua tolerància a salinitat, ja que podrien tindre les característiques adequades per a ser patrons clonals. Complementàriament, s’han utilitzat aproximacions fisiològiques, transcriptòmiques i de seqüenciació massiva de transcriptoma per a fer una primera descripció dels mecanismes de tolerància a salinitat presents en aquestes espècies. Com a resultat s’han descrit els principals caràcters relacionats amb la tolerància a la salinitat en el gènere Diospyros, com la compartimentalització de ions, la expressió en arrel de un gen similar a HKT-1, la abundància de canals de clorur o la arquitectura de la arrel. Finalment, per a obtenir mes informació sobre la biosíntesi de proantocianidines, la molècula responsable de la astringència, es va formular la hipòtesi de la existència de un complex proteic del tipus MBW (MYB-bHLH-WD40) que podria regular la ruta molecular de forma anàloga a la regulació existent en altres espècies estudiades. En primer lloc, es va seqüenciar el gen ortòlog putatiu del tipus WD40 de TTG1 de Arabidopsis thaliana. El paper de aquest gen i altres gens del hipotètic complex (MYB2, MYB4 i MYC1) han sigut estudiats mitjançant un anàlisi transcripcional en diferents estats de maduresa de fruits astringents i no astringents. A més, s’ha observat amb microscòpia la localització cel·lular de aquestos gens mitjançant la expressió transitòria amb fusió a fluorescència en fulles de Nicotiana benthamiana i hem analitzat la seua interacció amb un assaig de doble híbrid en llevat i BiFC (complementació bimolecular de fluorescència) mitjançant expressió transitòria en fulles de Nicotiana benthamiana. Finalment hem analitzat els efectes de aquests gens i de les seues interaccions en la ruta de biosíntesi de proantocianidines utilitzant el promotor del gen ANR (antocianidin reductasa) i promotors dels gens reguladors MYC1 i MYB4 fusionant-los amb una luciferasa com a gen delator i coexpressant-los amb diferents combinacions dels gens del complex MBW mitjançant expressió transitòria en fulles de Nicotiana benthamiana. Els resultats de aquests assajos confirmen la interacció nuclear de aquestos gens i el seu paper en la regulació de la producció de la astringència, donant lloc a un model bàsic amb algunes diferències als proposats en altres espècies. / Gil Muñoz, F. (2020). Biotechnological tools applied to the improvement of persimmon (Diospyros kaki Thunb) culture [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/153155 / TESIS

No description available.

Thesis (MSc)--University of Stellenbosch, 2011. / ENGLISH ABSTRACT: High molar mass tannin extracts are complex mixtures which are distributed in both molar mass and chemical composition. Condensed tannins from quebracho and mimosa and hydrolysable tannins of tara, chestnut wood and turkey gall were studied. Application of a single analytical technique is not sufficient to elucidate the complete structures present in the extracts. 13C Nuclear Magnetic Resonance (NMR) spectroscopy and Matrix Assisted Laser Desorption/Ionisation Time-of-Flight (MALDI-TOF) mass spectrometry were applied in order to determine the chemical composition and molar mass, respectively. A new mass spectrometric method that can uniquely determine the oligomer microstructure was developed using Collision Induced Dissociation (CID) experiments. Bulk analysis only showed the average composition of the extracts, in order to obtain specific information on the molar mass and chemical composition distributions. Hydrophilic Interaction Liquid Chromatography (HILIC) was used for analysis of the condensed tannins and for the hydrolysable tannins Normal Phase Liquid Chromatography (NP-LC) was utilised. The HILIC separation was up-scaled and the fractions were collected and analysed by MALDI-TOF, and this coupling revealed that separation occurs by molar and chemical composition. For separation of the molecules only by size, Size Exclusion Chromatography (SEC) analyses were carried out; this allowed for relative comparison of the tannin molecules. In conclusion, for characterisation of high molar mass tannins a multi-dimensional approach was necessary since the various distributions present in these extracts are superimposed. / AFRIKAANSE OPSOMMING: Hoë molekulêre massa tannienekstrakte is komplekse mengsels, in terme van beide molekulêre massa en chemiese samestelling. Gekondenseerde tanniene vanaf quebracho en mimosa, en hidroliseerbare tanniene vanaf tara, kastaaiinghout en Turksegal is bestudeer. Die gebruik van ‘n enkele analitiese tegniek is nie voldoende om die volledige struktuur van komponente teenwoordig in die ekstrakte te analiseer nie. 13C KMR-spektroskopie en MALDI-TOF-massaspektroskopie is gebruik om die chemiese samestelling en molekulêre massa, onderskeidelik, te bepaal. ‘n Nuwe metode is ontwikkel vir die bepaling van die oligomeer-mikrostruktuur deur gebruik te maak van botsings-geïnduseerde dissosiasie eksperimente. Grootmaat analise het net die gemiddelde samestelling van die ekstrak bepaal. Hidrofiliese-interaksie-vloeistofchromatografie (HILIC) is gebruik vir die analise van gekondenseerde tanniene en gewone fase-vloeistofchromatografie is gebruik vir die hidroliseerbare tanniene. Die HILIC-skeiding is op groter skaal uitgevoer en die fraksies is versamel en gebruik vir MALDI-TOF analise. Hierdie koppeling het getoon dat skeiding plaasvind op grond van molekulêre massa en chemiese samestelling. Grootte-uitsluitingschromatografie is gebruik vir die skeiding van molekules alleenlik op grootte. Hierdeur kon ‘n relatiewe vergelyking van die tannienmolekules gemaak word. Vir die karakterisering van hoë molekulêre massa tanniene is ‘n multi-dimensionele benadering nodig aangesien die verskeie verspreidings teenwoording in hierdie ekstrakte supergeponeerd is.