Rozprawy doktorskie na temat „Resveratrol”

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O resveratrol é uma fitoalexina produzida em função de estresses bióticos e abióticos e foi encontrado em um número limitado de espécies de plantas, incluindo o amendoim (Arachis hypogaea) e seus parentes silvestres. Este tem também propriedades antioxidantes e em função disso é um promissor antitumoral, cardioprotetor e neuroprotetor. O amendoim é um alotetraploide de constituição genômica AB sendo A. ipaënsis e A. duranensis os doadores de seus genomas B e A, respectivamente. Estudos prévios demonstraram que algumas espécies silvestres do gênero Arachis produzem resveratrol e que ampla variabilidade genética para diferentes características existe entre acessos das espécies silvestres, incluindo ampla variação nos níveis de resistência a doenças fúngicas e nematoides. Portanto, a avaliação de um maior número do gênero e a investigação da variação da produção de resveratrol também entre acessos é fundamental para uma exploração mais adequada das espécies silvestres do gênero Arachis. O melhor entendimento das causas da variação na produção de resveratrol sem dúvida contribuirá para o uso das mesmas e o primeiro passo para esse entendimento sem duvida passa pela identificação do padrão de expressão da resveratrol sintase (RS). Este trabalho tem como objetivos avaliar a variação na produção de resveratrol entre espécies (A. hypogaea, A. stenosperma, A. valida, A. wiliamsii, A. duranensis, A. ipaënsis e um anfidiploide sintético proveniente do cruzamento artificial entre A. duranensis e A. ipaënsis) e entre acessos de duas espécies(A. hypogaea e A. stenosperma) por meio de HPLC e avaliar a expressão relativa da resveratrol sintase por meio de RT-qPCR em A. hypogaea, A. duranensis, A. ipaënsis e um anfidiploide sintético. Folhas foram coletadas de plantas cultivadas em casa de vegetação para os grupos teste e controle e três repetições biológicas ...
Resveratrol is a phytoalexin produced under biotic and abiotic stresses. It has been found in a restricted number of plant species including peanut (Arachis hypogaea) and its wild relatives. This phytochemical has antioxidant properties, being considered a promising antitumour, cardioprotective and neuroprotective agent. Peanut is an allotetraploid specie with an AABB genomic constitution. Arachis ipaënsis and A. duranensis are the donors of the B genome and the A genome, respectively. Previous studies have shown that wild species of the genus Arachis can produce resveratrol. Therefore, the evaluation of other species of thus genus and the analysis of the variation in the production of resveratrol between accessions is essential for better exploitation of wild species in the genus Arachis. It also important the understanding of the variation in resveratrol concentration between species and accessions and that could be reached through the better understating of the role resveratrol synthase.. . The aims of this work were to evaluate the variation in resveratrol production between species (A. hypogaea, A. stenosperma, A. valida, A. wiliamsii, A. duranensis, A. ipaënsis and in a synthetic amphidiploid derived from these two wild species A. duranensis and A. ipaënsis) and between accessions of the same specie (A. hypogaea and A. stenosperma) through HPLC, and to quantify the expression of resveratrol synthase by RT-qPCR in A. hypogaea, A. duranensis, A. ipaënsis and in a synthetic amphidiploid. Leaves for the test and control groups were collected from plants cultivated in a greenhouse and three biological replicates were evaluated for each specie. The synthesis of resveratrol in leaves was induced by treatment with UV for 2 hours and thirty minutes and then the difference in the concentration of resveratrol between species and between the accessions and cultivars of the same species was analyzed. We found new ...

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Resumo: O resveratrol é uma fitoalexina produzida em função de estresses bióticos e abióticos e foi encontrado em um número limitado de espécies de plantas, incluindo o amendoim (Arachis hypogaea) e seus parentes silvestres. Este tem também propriedades antioxidantes e em função disso é um promissor antitumoral, cardioprotetor e neuroprotetor. O amendoim é um alotetraploide de constituição genômica AB sendo A. ipaënsis e A. duranensis os doadores de seus genomas B e A, respectivamente. Estudos prévios demonstraram que algumas espécies silvestres do gênero Arachis produzem resveratrol e que ampla variabilidade genética para diferentes características existe entre acessos das espécies silvestres, incluindo ampla variação nos níveis de resistência a doenças fúngicas e nematoides. Portanto, a avaliação de um maior número do gênero e a investigação da variação da produção de resveratrol também entre acessos é fundamental para uma exploração mais adequada das espécies silvestres do gênero Arachis. O melhor entendimento das causas da variação na produção de resveratrol sem dúvida contribuirá para o uso das mesmas e o primeiro passo para esse entendimento sem duvida passa pela identificação do padrão de expressão da resveratrol sintase (RS). Este trabalho tem como objetivos avaliar a variação na produção de resveratrol entre espécies (A. hypogaea, A. stenosperma, A. valida, A. wiliamsii, A. duranensis, A. ipaënsis e um anfidiploide sintético proveniente do cruzamento artificial entre A. duranensis e A. ipaënsis) e entre acessos de duas espécies(A. hypogaea e A. stenosperma) por meio de HPLC e avaliar a expressão relativa da resveratrol sintase por meio de RT-qPCR em A. hypogaea, A. duranensis, A. ipaënsis e um anfidiploide sintético. Folhas foram coletadas de plantas cultivadas em casa de vegetação para os grupos teste e controle e três repetições biológicas ...
Abstract: Resveratrol is a phytoalexin produced under biotic and abiotic stresses. It has been found in a restricted number of plant species including peanut (Arachis hypogaea) and its wild relatives. This phytochemical has antioxidant properties, being considered a promising antitumour, cardioprotective and neuroprotective agent. Peanut is an allotetraploid specie with an AABB genomic constitution. Arachis ipaënsis and A. duranensis are the donors of the B genome and the A genome, respectively. Previous studies have shown that wild species of the genus Arachis can produce resveratrol. Therefore, the evaluation of other species of thus genus and the analysis of the variation in the production of resveratrol between accessions is essential for better exploitation of wild species in the genus Arachis. It also important the understanding of the variation in resveratrol concentration between species and accessions and that could be reached through the better understating of the role resveratrol synthase.. . The aims of this work were to evaluate the variation in resveratrol production between species (A. hypogaea, A. stenosperma, A. valida, A. wiliamsii, A. duranensis, A. ipaënsis and in a synthetic amphidiploid derived from these two wild species A. duranensis and A. ipaënsis) and between accessions of the same specie (A. hypogaea and A. stenosperma) through HPLC, and to quantify the expression of resveratrol synthase by RT-qPCR in A. hypogaea, A. duranensis, A. ipaënsis and in a synthetic amphidiploid. Leaves for the test and control groups were collected from plants cultivated in a greenhouse and three biological replicates were evaluated for each specie. The synthesis of resveratrol in leaves was induced by treatment with UV for 2 hours and thirty minutes and then the difference in the concentration of resveratrol between species and between the accessions and cultivars of the same species was analyzed. We found new ...
Mestre

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências da Saúde, Programa de Pós-Graduação em Farmácia, Florianópolis, 2010
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O resveratrol (RV) é um polifenol pertencente à classe dos estilbenos, presente na dieta humana. Esta substância tem demonstrado inúmeras atividades farmacológicas incluindo antitumoral, anti-inflamatória, antioxidante, cardioprotetora, entre outras. Entretanto, a biodisponibilidade oral desta substância é praticamente nula, em decorrência do seu elevado metabolismo colônico e hepático. Por outro lado, estudos relatam os benefícios do RV sobre a pele, fazendo com que a liberação tópica deste composto constitua uma interessante alternativa à administração oral para o tratamento de várias desordens cutâneas. Neste sentido, a nanoencapsulação do RV pode ser considerada uma alternativa promissora para a aplicação tópica cutânea. Neste trabalho, nanopartículas lipídicas sólidas (NLS), nanoemulsões (NE) e microemulsões (ME) foram preparadas pela técnica de difusão do solvente a quente e avaliadas quanto a sua capacidade de incorporar o RV, assim como de liberar este fármaco através da pele. Visando uma forma farmacêutica para uso tópico, nanogéis foram preparados pela adição de hidroxietilcelulose às dispersões coloidais. Um método analítico por CLAE foi desenvolvido e validado, com o intuito de determinar a concentração de RV nas formulações e monitorar a concentração de fármaco permeado ou retido na pele. O tamanho médio das partículas foi aproximadamente de 221, 119 e 22 nm, para as NLSs, NEs e MEs, respectivamente. Todas as preparações apresentaram potencial zeta negativo. Elevados valores de eficiência de encapsulação foram obtidos para todas as formulações testadas. Entretanto, o teor de RV variou significativamente em função do tipo de nanocarreador e da quantidade de fármaco inicialmente adicionada às formulações. Em especial, a ME apresentou uma maior capacidade de incorporar o RV, aumentando a sua concentração cerca de 28 vezes em relação a sua solubilidade aquosa. As formulações em dispersão e em gel foram avaliadas em estudos de permeação cutânea utilizando células de difusão tipo Franz e pele de orelha de porco como modelo de membrana por um período de 8 horas. O fluxo de permeação, o tempo de latência e o coeficiente de permeabilidade foram estimados após a construção do gráfico da quantidade permeada (µg/cm²) versus tempo (h). Estudos de microscopia confocal de varredura laser (MCVL) foram realizados no intuito de observar a difusão do fármaco incorporado nas NLSs, NEs e MEs através da pele, utilizando o vermelho de Nilo (VN) como marcador fluorescente. A permeação e a retenção do RV foram significativamente afetadas pelo tipo de nanocarreador lipídico. O fluxo e o coeficiente de permeabilidade diminuíram na seguinte ordem: MERV < NLSRV < NERV. Por outro lado, a quantidade permeada através da pele aumentou após a aplicação da ME apresentando a mais elevada concentração de fármaco, quando MEs com diferentes teores de RV foram testadas. O tempo de latência não variou para a maioria das formulações, e a quantidade de fármaco retido na pele demonstrou ser significativamente reduzida, somente quando as NLS foram aplicadas. As fotomicrografias obtidas por MCVL evidenciaram a permeação do VN nas camadas profundas da pele quando as dispersões coloidais foram testadas, o que não foi observado com uma solução do mesmo utilizada como controle, em que o marcador fluorescente permaneceu nas camadas superficiais da pele. Além disso, a menor penetração do corante a partir das MEs pode ser visualizada, confirmando os resultados obtidos nos estudos de permeação.

The goal of this research project was to synthesize derivatives of transresveratrol. In order for resveratrol to be activated and used by the body it needs to bind to Human Serum Albumin (HSA), a protein in blood plasma. The derivatives were synthesized to improve the ability of resveratrol to enter cells as well as improve their ability to bind to HSA. The three derivatives that were synthesized have converted one of the hydroxyl groups on resveratrol to an ether with a methylene chain terminated by a carboxylic acid. By varying the lengths of the methylene chain we varied the water solubility of the resveratrol derivative. This brought the research closer to the goal of determining how this would affect the binding ability to HSA. Currently three derivatives have been synthesized and purified once by column chromatography.

Plant polyphenols are a vast family of natural compounds present in many foods and drinks. Many of them have noteworthy biological properties. Resveratrol, for example, can help prevent cardiovascular disease in humans, because it is able to lower blood pressure and lipid peroxidation, to induce vasodilatation and to reduce platelet aggregation. Moreover, resveratrol has antioxidant, anti-inflammatory, and anti-atherosclerotic properties, resulting in protection of the cardiovascular system, improvement of age-related cognitive decline and prevention and therapy of cancer. These potential positive effects are hampered by the low bioavailability of this compound and of polyphenols in general. As a result of a low level of absorption and a rapid metabolism in intestinal and liver cells, only small amounts of polyphenols are found in the bloodstream, and then mostly as metabolites. The major aim of my doctorate work has been the development of “pro-drugs”, resistant to metabolism during absorption and capable of regenerating the natural compound thanks to the action of ubiquitous enzymes. Resveratrol was chosen as model polyphenol. The project is carried out in collaboration with a group of organic chemists who synthesise the compounds and also contribute analytical know-how. Since polyphenols generally have a low solubility in water, and solubility is a key factor contributing to the bioavailability of a compound, a first resveratrol derivative was synthesised in which succinyl linkers connected resveratrol hydroxyls on one side and glucose residues on the other via carboxyester bonds. Pharmacokinetic studies with this compound showed that the levels and the composition of the metabolite mix in the bloodstream were the same as those obtained using resveratrol itself. This suggested that the compound was hydrolysed to resveratrol in the gastro-intestinal tract, before absorption. The carboxyester bond system thus turned out to be too labile in vivo, and therefore of limited usefulness. Other bond systems, such as the ether and sulfonate linkages, proved on the contrary to be too stable We therefore turned to other functionalities. In one approach the acetal bond system was used to link protective/solubilising groups to resveratrol hydroxyls. This type of protecting group is hydrolysed under acid conditions, but turned out to be rather too stable for use in vivo. Ex vivo and in vivo experiments with a series of acetal derivatives bearing short oligo-ethylene glycol chains however allowed us to evaluate and appreciate the positive effect this type of substituents can have on the absorption process. In the search for a protective functionality with ideal stability characteristics, we then turned to yet another, more labile, bond type, which cannot be specified here. To confer water solubility, polyhydroxylated moieties or PEG chains were introduced via this bond system. The work has so far led to satisfactory levels of absorption with two regioisomeric mono-substituted derivatives: uM levels of resveratrol-containing molecules were measured in blood samples, persisting for hours. However, the two free hydroxyl groups in these compounds are targets for glucuronosyltransferases, which largely modify them before the regeneration of resveratrol can be completed. A new approach has now been adopted, incorporating the positive aspects which have emerged from the investigation of acetal derivatives. In these new compounds short oligo-ethyleneglycol chains are linked to all three hydroxyl groups of resveratrol to improve absorption while offering protection. These constructs may be expected to be well absorbed (depending on chain length) and to hydrolyse with suitable kinetics. Only preliminary results with one compound are available at the time this thesis is submitted. During my doctorate I had the opportunity to spend a six-month period in the laboratory of prof. E. Mervaala at the Institute of Biomedicine of the University of Helsinki, Finland. There I performed a preliminary study on the possible positive effects of resveratrol and caloric restriction against sunitinib-induced cardiotoxicity and renal damage in spontaneously hypertensive rats, using normotensive WKY rats as control. Sunitinib is a tyrosine kinase inhibitor (TKI). The results did not reveal statistically significative differences between control and treated rats. Further experiments will be needed before a definite conclusion can be reached. Finally, it should be mentioned that in a side project I participated in studies on the metabolism of polyphenols by cultured cells. Work with monolayers of colonic Caco-2 cells revealed a remarkable heterogeneity in the expression of Phase II metabolism enzymes (sulfo- and glucuronosyl-transferases) within the same cell line. It was however possible to regenerate a uniform activity in the different populations by cultivating the cells with low concentrations of a xenobiotic compound (in our case quercetin).
I polifenoli vegetali sono una vasta famiglia di composti naturali presenti in molti cibi e bevande. Molti di loro possiedono notevoli proprietà biologiche. Il resveratrolo per esempio, può aiutare a prevenire le malattie cardiovascolari, perchè è in grado di abbassare la pressione sanguigna, ridurre la perossidazione lipidica l’aggregazione piastrinica. Inoltre, il resveratrolo ha proprietà antiossidanti, anti infiammatorie e anti aterosclerotiche, risultanti nella protezione del sistema cardiovascolare, nel miglioramento del declino cognitivo legato all’età e nelle prevenzione e terapia del cancro. Questi potenziali effetti terapeutici trovano però un ostacolo nella scarsa biodisponibilità di questo composto e dei polifenoli in generale. Come risultato di un basso livello di assorbimento e di rapido metabolismo nelle cellule intestinali ed epatiche, nel sangue vengono ritrovate solo piccole quantità di polifenoli, e principalmente sotto forma di metaboliti. Lo scopo del mio progetto di dottorato è lo sviluppo di prodrugs di resveratrolo, il nostro polifenolo modello, resistenti al metabolismo durante l’assorbimento intestinale capaci di rigenerare il composto naturale grazie all’azione di enzimi ubiquitari. Il progetto è avvenuto in collaborazione con un gruppo di chimici organici che hanno sintetizzato i composti utilizzati in questa tesi. Dato che i polifenoli generalmente sono poco solubili in acqua, e la solubilità è un fattore determinante per la biodisponibilità di un composto, un primo precursore di resveratrolo è stato ottenuto funzionalizzando gli ossidrili con gruppi glucosio, attraverso un linker succinico (RGS); studi di farmacocinetica con questo composto mostrano che i livelli e la composizione dei metaboliti in circolo sono però del tutto analoghi a quelli ottenuti somministrando resveratrolo; questo suggerisce un’idrolisi del derivato a resveratrolo nel tratto intestinale, prima dell’assorbimento. Quindi, l’utilità del legame carbossiestereo in vivo è limitata. Sono stati successivamente sintetizzati altri precursori del resveratrolo, con un legame acetalico che lega gruppi protettivi/solubilizzanti agli ossidrili. Il legame acetalico è caratterizato da una bassa polarità e da uno scarso ingombro sterico, e si prevede quindi che favorisca il passaggio attraverso le bio-membrane. È suscettibile all’idrolisi in acido ma è comunque ancora troppo stabile per l’uso in vivo. Esperimenti ex vivo ed in vivo, con una serie di derivati acetalici recanti corte catene di oligo-etilene glicole utilizzando segmenti d’intestino, hanno permesso di determinare che la lunghezza della catena del gruppo solubilizzante influenza positivamente l’assorbimento. Alla ricerca di funzionalità protettive con ideali caratteristiche di stabilità, si è quindi passati ad un altro tipo di legame, più labile, che non può essere specificato qui. Tramite questo legame, allo scopo di conferire solubilità, sono state introdotte porzioni poliidrossilate o catene di PEG. Questa strategia ha portato a livelli di assorbimento soddisfacenti con due derivati (regioisomeri) monosostituiti: concentrazioni dell’ordine del uM di derivati del resveratrolo si ritrovano nel sangue, persistenti per ore. Tuttavia, i due idrossili liberi di questi composti sono substrati per le glucuronosiltrasferasi, e questa modificazione avviene prima che la rigenerazione di resveratrolo sia completa. Un nuovo approccio è stato ora adottato, incorporando gli aspetti positivi che sono emersi dagli esperimenti condotti con i derivati acetalici. In questi nuovi composti corte catene di oligo-etileneglicole sono legate ai tre idrossili del resveratrolo per aumentare l’assorbimento e nel contempo fornire protezione. Questi nuovi derivati dovrebbero essere ben assorbiti (in funzione della lunghezza della catena) e con un’adatta cinetica di idrolisi. Al momento della pubblicazione di questa tesi sono disponibili solo risultati preliminari. Durante il mio corso di dottorato ho avuto l’opportunità di trascorrere un periodo di sei mesi presso il gruppo di ricerca del Prof. Mervaala’s all’Università di Helsinki, dove ho condotto uno studio sui possibili effetti positivi del resveratrolo e della restrizione calorica sulla cardiotossicità e il danno renale indotti dal sunitinib in ratti sponteaneamente ipertesi, usando ratti normotesi come controllo. Il sunitinib è un inibitore delle tirosin-chinasi (TKI). I risultati preliminari non hanno permesso di evidenziare differenze statisticamente significative fra i controlli e i ratti trattati. Per trarre conclusioni maggiormente significative, sarà necessario condurre ulteriori esperimenti. Per finire, in durante lo stadio iniziale del progetto ho partecipato ad uno studio sul metabolismo di polifenoli in cellule in cultura. Gli studi di assorbimento/metabolismo con monostrati di cellule intestinali Caco-2 hanno messo in luce l’esistenza di eterogeneità nell’espressione degli enzimi metabolici di Fase II (solfo- e glucuronosil-trasferasi) all’interno della stessa linea cellulare. È comunque possibile uniformare nuovamente popolazioni con diversa attività metabolica coltivando le cellule in presenza di concentrazioni minime di xenobiotico (nel nostro caso quercetina).

Reconhece-se actualmente que o vinho tem propriedades benéficas no organismo humano, sendo constituído por compostos que exercem uma influência positiva no metabolismo dos indivíduos que o consomem com regularidade e moderação. O resveratrol (trans-3,4’,5-trihidroxiestilbeno) é uma fitoalexina que está presente em diversos alimentos e bebidas, em particular no vinho tinto. Das várias classes de polifenóis existentes no vinho, o resveratrol (estilbeno) é um dos compostos mais estudados do ponto de vista de importância biológica, devido aos potenciais efeitos benéficos para a saúde humana. Na primeira parte deste trabalho foi desenvolvido e validado um método analítico sensível, com vista a determinar, quantitativamente, as concentrações de resveratrol em amostras de vinhos tintos portugueses, utilizando a extracção em fase sólida (SPE) e a cromatografia líquida de alta eficiência acoplada a um detector de díodos (HPLC-DAD). Na segunda parte, foram implementadas técnicas, tais como, o método de difusão em disco e o método da microdiluição, que permitiram avaliar a actividade antimicrobiana do resveratrol em diversos microrganismos, nomeadamente bactérias Gram-positivas (Bacillus cereus, Staphylococcus aureus e Enterococcus faecalis) e Gram-negativas (Escherichia coli, Klebsiella pnemoniae, Salmonella typhimurium e Pseudomonas aeruginosa). O efeito do resveratrol em bactérias Gram-positivas foi também analisado, recorrendo-se às curvas de morte, de forma a tentar assim compreender se o resveratrol actua como um agente bactericida ou bacteriostático. Analisou-se, ainda, a acção do resveratrol em Bacillus cereus, recorrendo à microscopia e à citometria de fluxo, de forma a tentar compreender o mecanismo de acção deste composto. Finalmente, foi também avaliada a actividade antimicrobiana do resveratrol em diferentes estirpes de Helicobacter pylori, utilizando o método da difusão em disco e o método da diluição em agar. Para além disso, verificou-se a hipótese do resveratrol e amostras de vinho tinto (devidamente quantificadas na primeira parte deste trabalho) inibirem a actividade da urease de H. pylori, considerada um dos principais factores de virulência deste microrganismo. Complementou-se, ainda, o estudo analisando cineticamente a actividade da urease na presença do resveratrol. O método analítico foi validado de acordo com normas internacionalmente aceites quanto a: especificidade, linearidade, limites de detecção (LOD) e de quantificação (LOQ), precisão, exactidão, estabilidade e eficiência de extracção. Verificou-se ainda a aplicabilidade do método, analisando 186 amostras de vinhos tintos comerciais, provenientes de diferentes regiões, castas e anos. Os resultados obtidos demonstraram que os níveis de trans-resveratrol nos vinhos tintos, variaram entre 0,05 e 10,9 μg/mL, enquanto que as concentração de cis-resveratrol variaram entre 0,04 e 8,71 μg/mL. No que diz respeito à actividade antimicrobiana do resveratrol, verificou-se que este composto possui actividade contra todas as bactérias Gram-positivas em estudo. Os resultados demonstraram que o resveratrol tem um efeito bacteriostático que afecta o crescimento da célula bacteriana em todas as estirpes testadas. Quanto ao mecanismo de acção do resveratrol, os resultados obtidos sugerem que o crescimento celular do Bacillus cereus foi inibido, o que por sua vez terá levado à paragem da divisão celular. Na última parte deste trabalho, confirmou-se a actividade antibacteriana do resveratrol em diferentes estirpes de Helicobacter pylori. Para além disso, quer o resveratrol, quer os vinhos tintos analisados demonstraram um efeito inibitório na actividade da urease de Helicobacter pylori. A análise cinética revelou que o resveratrol é um inibidor da urease não competitivo e reversível. O presente trabalho contribuiu por um lado, para o melhor conhecimento do teor de resveratrol nos vinhos portugueses, e por outro demonstrou o potencial do resveratrol como agente antibacteriano, podendo no futuro ter potenciais aplicações tanto na conservação dos alimentos como na terapia clínica.
It is now recognized that wine has beneficial properties for the human health, since it is constituted by compounds that exert a positive influence on the individuals’ metabolism, provided that the consumption is moderate. The phytoalexin resveratrol (3,4’,5-trihydroxistilbene) is commonly found in foodstuffs and drinks, particularly in red wine. From the various classes of polyphenols presented in wine, resveratrol (stilbene) is one of the most studied from the viewpoint of biological importance, due to the possible beneficial effects on the human health. In the first part of this work it was developed and validated a sensitive analytical method for the quantitative analysis of resveratrol in portuguese red wines, using solid phase extraction (SPE) and liquid chromatography coupled with diode array detector (HPLC-DAD). In the second part the antimicrobial activity of the compound was evaluated by means of different techniques, such as the disc diffusion and microdilution methods, and the studied microorganisms included Gram-positive (Bacillus cereus, Staphylococcus aureus and Enterococcus faecalis) and Gram-negative (Escherichia coli, Klebsiella pneumoniae, Salmonella typhimurium and Pseudomonas aeruginosa) bacteria. We also analyzed the effect of resveratrol on Gram-positive organisms using time-kill assays, in order to understand whether resveratrol acts as a bactericide or bacteriostatic. In order to understand the mechanism of action of this compound, its action against Bacillus cereus was analysed by microscopy and flow cytometry. Finally, the antimicrobial activity of resveratrol against different strains of Helicobacter pylori was evaluated using both the disk diffusion and agar dilution methods. In addition, we verified the ability of resveratrol and red wine samples (properly quantified in the first part of this work) to inhibit the urease activity of H. pylori, since this is considered a major virulence factor of this microorganism. Moreover, we complemented the study analyzing kinetically urease activity in the presence of resveratrol. The analytical method was fully validated according to internationally accepted guidelines, regarding specificity, linearity, limits of detection (LOD) and quantification (LOQ), precision, accuracy, stability and extraction efficiency. Once validated, the methodology was applied to 186 commercial red wine samples from different regions, grape varieties and vintage. The results showed that the content of trans-resveratrol in red wines ranged from 0.05 to 10.9 µg/mL, while the concentration of cis-resveratrol ranged from 0.04 to 8.71 µg/mL. Regarding the antimicrobial activity of resveratrol, we found that this compound has activity against all tested Gram-positive bacteria. The results demonstrated that resveratrol has a bacteriostatic effect which affects bacterial cell growth in all tested strains. As regards the mechanism of action of resveratrol, the results suggest that cell growth of Bacillus cereus was inhibited, which may in turn stop cell division. In the last part of this work, we confirmed the antibacterial activity of resveratrol against different strains of Helicobacter pylori. Furthermore, both resveratrol and red wines showed an inhibitory effect on urease activity of Helicobacter pylori. The kinetic analysis revealed that resveratrol is a non-competitive and reversible inhibitor of urease. The present work contributed to the best knowledge of portuguese wines regarding the levels of resveratrol, and demonstrated the possible use of this compound as an antibacterial agent, and may in future have potential applications for both food preservation and clinical therapy.

Resveratrol (3,4,5-trihidroxiestilbeno), um polifenol pertencente ao grupo de compostos denominados fitoalexinas, é sintetizado por uma ampla variedade de plantas, tais como as videiras, em resposta a infecções fúngicas e a exposição à radiação UV. Nos vinhos este composto está presente em elevadas concentrações, sendo considerado um dos constituintes com maior potencial antioxidante. Esta elevada capacidade de absorver os radicais livres presentes em diversos processos biológicos permite ao resveratrol previnir doenças cardiovasculares e diversos tipos de câncer. O principal objetivo do presente estudo foi determinar in vitro o efeito radioprotetor do resveratrol em cultura celular com auxílio dos testes de citotoxicidade do resveratrol (IC50%) e da dose letal 50% da radiação gama (DL50). Os estudos in vitro do nível de toxicidade do resveratrol, verificado pelo ensaio de citotoxicidade utilizando-se o método de incorporação do vermelho neutro, e da determinação da dose letal 50% (DL50) da radiação gama, oriunda de uma fonte de Cobalto-60 (Co-60), foram realizados em cultura de células da linhagem NCTC Clone 929 da ATCC. O IC50% do resveratrol foi de aproximadamente 50 M/L. A DL50 da radiação gama apresentou um valor de aproximadamente 354 Gy. Baseando-se nestes resultados biológicos foram realizados estudos do efeito radioprotetor do resveratrol nas mesmas condições experimentais, constatando-se que o resveratrol nas concentrações entre 12,5 M/L e 25 M/L apresentou um efeito radioprotetor mais acentuado.
Resveratrol (3,4,5 trihydroxystilbene), a phenolic phytoalexin occurring naturally in a wide variety of plants, such as grapevines, in response to injury as fungal infections and exposure to ultraviolet light. In the wines this compound is present at high levels and is considered one of the higest antioxidant constituents. This high capacity to scavenge the free radicals generated by several biologic processes by resveratrol can provide a prevention of human cardiovascular diseases and several types of cancer. The main objective of this study was to determine the in vitro radioprotective effect of resveratrol in cell culture with the aid of the tests of cytotoxicity of resveratrol (IC50%) and lethal dose 50% of gamma radiation (LD50). Studies of the level of resveratrol toxicity, found by cytotoxicity test performed by neutral red uptake assay, and lethal dose 50% (LD50) of gamma radiation from source of Cobalt-60 (Co-60) was performed in cell culture NCTC Clone 929 from ATCC. The IC50% of resveratrol was about 50M/L. The DL50 of gamma radiation showed a value of about 354Gy. On the basis of these biological results, it was performed studies of radioprotective effect of resveratrol on the same experimental conditions, verifying that the resveratrol in concentrations between 12.5M/L and 25M/L showed a more pronounced radioprotective effect.

Obesity is a major epidemic and an independent risk factor for heart disease. Food derived compounds such as resveratrol has been reported to have strong medicinal properties and shown potential in preventing and reversing heart diseases. This study investigated the cardioprotective properties of resveratrol in an animal model of diet induced obesity and possible cellular mechanisms. Obese prone (OP) and obese resistant (OR) rats were fed with high fat diet while, sprague dawley (SD) rats that served as control were fed with normal lab chow for a total of 17 weeks. During the last 5 weeks of study, treatment group received resveratrol daily by oral gavage at a dosage of 2.5 mg/kg body weight. Treatment with resveratrol significantly improved cardiac isovolumic relaxation time (IVRT), thiobarbituric acid reactive substance (TBARS), tumor necrosis factor α (TNF-α), interleukin 6 (IL-6), triglycerides, glucose, low density lipoprotein and increased insulin in OP rats and TNF-α, glucose and leptin in OR rats. Cardiac calcium handling proteins such as sarcoendoplasmic reticulum Ca2+-ATPase2a (SERCA2a), phospholamban (PLB) and phosphorylated PLBthr17 and PLBser16 were unchanged in all the groups. Excess circulating lipids cause cellular dysfunction in major organs. Adult rat cardiomyocytes were incubated for 24 hours with different doses (10, 100 and 200µM) of palmitic acid (PA). One group cardiomyocytes were pretreated with resveratrol for 45minutes prior to addition of PA. Incubation with 200µM PA significantly increased the number of round shaped cardiomyocytes and apoptosis and resveratrol treatment prevented these changes. Cardiomyocytes contractility measurement showed 200µM PA resulted in reduced rate of relaxation and resveratrol prevented this reduction. Western blot analysis showed that the PA induced a 17% decrease in SERCA2a expression and SERCA2A:PLB ratio was preserved with resveratrol treatment. The change in SERCA2a with PA and resveratrol exposure was statistically not significant. In conclusion, resveratrol treatment reversed cardiac abnormalities in OP rats, but not in OR rats. Resveratrol treatment prevented PA induced contractile abnormality in adult cardiomyocytes. Importantly, this study showed that resveratrol can act directly on cardiomyocytes and protect against damage from exposure to high levels of lipids.
February 2015

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Dissertacao (Mestrado)
IPEN/D
Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP

Recently, the compound resveratrol has had media attention as an anti carcinogen. However, the bioavailability of resveratrol is low in the human system due to its hydrophobic nature. Therefore, it must be administered in high dosages to be effective. A plethora of derivatives have been synthesized that have the potential of resveratrol but sadly share low bioavailability. The first effort of this research was an attempt to produce a more hydrophilic ester of resveratrol. Failing this, the final product was synthesized using a glycine derivative to produce 4-[(1E)-2-(3,5-diacetyloxyphenyl)ethenyl]phenyl N-[(1,1-dimethylethoxy)carbonyl]-glycinate.

Resveratrol (3, 4, 5-trihidroxiestilbeno) é um polifenol produzido por uma grande variedade de plantas em resposta ao estresse e encontrado predominantemente em cascas de uvas. Este princípio ativo apresenta vários benefícios à saúde, como a capacidade antioxidante, relacionada à prevenção de diversos tipos de câncer e do envelhecimento precoce da pele. No entanto, apresenta baixa biodisponibilidade quando administrado por via oral, o que torna interessante sua aplicação tópica. O principal objetivo deste trabalho foi a incorporação de resveratrol em hidrogéis poliméricos para obtenção de um sistema de liberação utilizado topicamente contra o desenvolvimento de desordens cutâneas, como o envelhecimento cutâneo e o câncer de pele. As matrizes poliméricas compostas por poli(N-vinil-2-pirrolidona) (PVP), poli(etileno glicol) (PEG) e ágar ou PVP e propano-1,2,3-triol (glicerina) e irradiadas a 20 kGy foram caracterizadas pelos ensaios de fração gel e intumescimento; sua biocompatibilidade preliminar foi avaliada in vitro por meio do ensaio de citotoxicidade utilizando o método de incorporação do vermelho neutro. Devido à baixa solubilidade do resveratrol em água, verificou-se o efeito da adição de 2% de etanol às matrizes. Todas as matrizes estudadas, contendo ou não álcool, apresentaram alto grau de reticulação, capacidade de intumescimento e não apresentaram toxicidade em ensaio preliminar de biocompatibilidade. Os dispositivos foram obtidos pela incorporação de resveratrol nas matrizes poliméricas, realizada de forma direta e indireta, ou seja, antes e após irradiação, respectivamente. Os dispositivos obtidos pelo método direto foram submetidos aos ensaios de fração gel, intumescimento e citotoxicidade e apresentaram-se semelhantes às respectivas matrizes. Os dispositivos contendo 0,05% de resveratrol obtidos pelo método direto e os dispositivos contendo 0,1% de resveratrol obtidos pelo método indireto foram submetidos ao ensaio de cinética de liberação durante 24 h. A quantificação do resveratrol liberado foi realizada por cromatografia líquida de alta eficiência (HPLC). Apenas os dispositivos obtidos pelo método indireto apresentaram capacidade de liberar o resveratrol incorporado, que apresentou capacidade antioxidante após liberação.
Resveratrol (3, 4, 5-trihydroxystilbene) is a polyphenolic produced by a wide variety of plants in response to injury and found predominantly in grape skins. This active ingredient has been shown to possess benefits for the health, such as the antioxidant capacity which is related to the prevention of several types of cancer and skin aging. However, the oral bioavailability of resveratrol is poor and makes its topical application interesting. The purpose of this study was to immobilize resveratrol in polymeric hydrogels to obtain a release device for topical use. The polymeric matrices composed of poli(N-vinyl-2-pyrrolidone) (PVP), poly(ethyleneglycol) (PEG) and agar or PVP and glycerol irradiated at 20 kGy dose were physical-chemically characterized by gel fraction and swelling tests and its preliminary biocompatibility by in vitro test of cytotoxicity using the technique of neutral red uptake. Due to low solubility of resveratrol in water, the addition of 2% ethanol to the matrices was verified. All matrices showed a high crosslinking degree, capacity of swelling and the preliminary cytotoxicity test showed nontoxicity effect. The devices were obtained by resveratrol immobilizaton in polymeric matrices, carried out in a one-or-two-steps process, that is, before or after irradiation, respectively. The one step resveratrol devices were characterized by gel fraction, swelling tests and preliminary biocompatibility, and their properties were maintained even after the resveratrol incorporation. The devices containing 0,05% of resveratrol obtained by one-step process and 0,1% of resveratrol obtained by two-steps process were submitted to the release test during 24 h. Resveratrol quantification was done by high performance liquid chromatography (HPLC). The results obtained in the kinetics of release showed that only the devices obtained by two-step process release the resveratrol, which demonstrate antioxidant capacity after the release.

O resveratrol (Res), um polifenol presente no vinho tinto, é conhecido por possuir potente atividade antioxidante. O efeito do resveratrol (Res) frente à nefrotoxicidade do antineoplásico cisplatina (cDDP) foi avaliado em ratos neste estudo. Os animais foram tratados com Res (25 mg/Kg de peso copóreo, ip., dose única) 30 minutos antes da administração de cisplatina (5 mg/Kg de peso copóreo, ip., dose única) e foram sacrificados depois de 2 ou 5 dias do tratamento. Após 5 dias, o aumento da creatinina sérica, volume urinário e proteinúria, que são marcadores de alterações renais, apresentaram significativa redução (p < 0,05) com a administração de resveratrol. Os ratos tratados com cisplatina apresentaram necrose tubular aguda e maior marcação imuno-histoquímica para células ED1 e linfócitos T no córtex e medula externa renal. Estas alterações foram menos intensas nos animais tratados com resveratrol. Após 2 dias, a administração de cisplatina aos ratos induziu aumento na concentração de malonaldeído (MDA) e reduziu nos níveis de glutationa (GSH) no tecido renal, que não foram amenizadas pelo resveratrol. Os resultados desse estudo indicam que o tratamento com resveratrol atenuou as alterações renais funcionais, histológicas e imuno-histoquímicas induzidas pela cisplatina. O efeito protetor provavelmente está relacionado à diminuição de infiltrado de células inflamatórias no tecido renal
Resveratrol (Res), a polyphenolic present in red wine, is known to possess potent antioxidant properties. The ability of resveratrol to protect against the nephrotoxicity of the antineoplastic agent cisplatin (cDDP) was evaluated in rats. The animals were treated with Res (25 mg/Kg body weight, ip., single dose) 30 minutes before administration of cDDP (5 mg/Kg body weight, ip., single dose) and then, sacrificed in 2 or 5 days followed by the treatment. After 5 days with resveratrol administration, the enhanced serum creatinine levels, urinary volume and urinary protein, which are indicative of renal injury, shown a significant reduction (p < 0.05). The cisplatintreated rats presented a tubular cell necrosis and increase immunostaining for ED1 and T-lymphocytes in the renal cortex and outer medulla. Those alterations were less intense in animals treated with resveratrol. After 2 days, administration of cisplatin to rats induced a higher malondialdehyde levels (MDA), and reduction in glutathione (GSH) concentrations in kidney tissue that were not prevented by resveratrol. In this study, the results indicate that resveratrol treatment attenuated the functional, histological and immunohistochemical renal alterations induced by cisplatin. The protect effect is relatated to the decrease of cells infiltrated at kidney tissue.

SirT1 is a class III histone deacetylase that has beneficial roles in various diseases related to aging such as cancer, diabetes and neurodegenerative disease. Resveratrol is a natural compound that mimics most of the beneficial effects attributed to SirT1. Resveratrol has toxicity towards cancer cells and has been reported to be a direct activator of SirT1. Interestingly, SirT1 over-expression has also been reported to be toxic. We set out to determine if resveratrol toxicity is mediated through activation of SirT1. We have assessed resveratrol toxicity in embryonic stem cells and mouse embryonic fibroblast (MEFs) across different SirT1 genotypes. Our data indicates that SirT1 is not implicated in resveratrol toxicity in either normal or transformed MEFs. Thus, resveratrol toxicity does not appear to be mediated by SirT1.

Resveratrol (trans-3,5,4′-trihydroxystilbene), a naturally occurring polyphenol present in grapes and red wine has undergone investigation as a potential cancer chemopreventive agent. Resveratrol is rapidly and extensively metabolised to its major phase II metabolites, including resveratrol sulfates and glucuronides. It is not yet known whether resveratrol metabolites contribute to the beneficial effects attributed to resveratrol, or whether resveratrol regeneration can occur from the metabolites. Resveratrol and metabolite pharmacokinetics were investigated in the plasma of healthy human volunteers receiving 0.5, 1.0, 2.5 and 5.0 g resveratrol daily. Concentrations were also quantified in malignant and non-malignant colon tissue removed from colorectal cancer patients, who received 0.5 or 1.0 g resveratrol daily. A mixture of resveratrol monosulfates and individual monoglucuronide isomers were synthesised. Resveratrol monosulfates were administered to mice to determine their pharmacokinetics. The effects of the metabolites on proliferation in HCA-7, HT-29 and HCEC colon cell lines were assessed. In healthy volunteers, resveratrol metabolites were shown to be the major species recovered from plasma. Average volunteer plasma AUClast for resveratrol-4′-O-glucuronide and resveratrol-3-O-sulfate were approximately 36- and 81-fold greater respectively than for resveratrol, following 0.5 g resveratrol dosing. In colon tissues from cancer patients resveratrol generally predominated, with resveratrol sulfate glucuronide being the most prominent metabolite. In mice administered monosulfates, resveratrol formation was found to occur, with measurable concentrations in plasma, mucosa, liver, lung and pancreas. The conversion of resveratrol sulfates to resveratrol was also observed in cells in vitro. Uptake of the metabolites and intracellular resveratrol correlated with effects on proliferation. Whilst resveratrol monoglucuronides had a limited inhibitory effect on cell proliferation, monosulfates caused a more pronounced reduction, with the greatest effect in HT-29 followed by HCA-7 cells, and little or no effect in HCEC cells. Further investigations will improve our understanding of the role of resveratrol metabolites in chemoprevention.

Thesis (MSc)--Stellenbosch University, 2001.
ENGLISH ABSTRACT: Grapevine is constantly under attack from a wide variety of pathogens including viruses, bacteria and fungi. In order to ensure survival, the grapevine has developed a vast array of defense mechanisms to combat invading organisms. A key element of this disease resistance is the production of phytoalexins, of which resveratrol is the primary component. The synthesis of resveratrol, together with other structural and biochemical defense mechanisms equips the plant to combat a number of pathogens resulting in the production of healthy grapes for the vinification of top quality wine. As part of the active disease response resveratrol is synthesised de novo in the berry skin at the site of infection, on recognition of the pathogen. Here it is able to limit the damage caused by the pathogen as well as preventing it from spreading. This gives the plant the opportunity to initiate its systemic acquired resistance thereby protecting the rest of the plant and preventing secondary infections. The fermentation of red wine on the grape skins allows for the extraction of resveratrol from the skin into the wine. Red wines therefore have a significantly higher concentration of resveratrol than white varieties, which contain little or no resveratrol at all. It is for this reason that the moderate consumption of wine, in particular red wine, is synonymous with a healthy lifestyle. The antioxidant and anti-inflammatory activities of resveratrol are important contributors to the cardiovascular benefits derived from the consumption of red wine. It now seems, however, that significant cardiovascular protection is derived from the synergistic action of resveratrol, the polyphenols and the alcohol in wine. With the wholesomeness of any food or beverage being of extreme importance, the aim of this project was to manipulate wine yeast to produce resveratrol during fermentation. This required the introduction of an entire metabolic pathway, by integrating plant genes into the yeast. Resveratrol synthase utilises three malonyl-CoA and one pcoumaroyl- CoA molecules to produce one molecule of resveratrol, Saccharomyces cerevisiae produces malonyl-CoA but no p-coumaroyl-CoA. Therefore, the following genes were obtained to enable yeast to produce p-coumaroyl-CoA: PAL, encoding phenylalanine ammonia-lyase to convert phenylalanine into cinnamic acid; C4H, encoding cinnamate-4- hydroxlyase to convert cinnamic acid into p-coumaric acid; and 4CL9 or 4CL216 encoding CoA-ligases to convert the p-coumaric acid into p-coumaroyl-CoA. To attain high-level expression, the genes were subcloned under the control of the phosphoglycerate kinase gene (PGK1) promoter and terminator. Due to integration problems with these expression cassettes and the fact that the yeast was able to consume p-coumaric acid, the 4CL9, 4CL216 and Vst1 (encoding resveratrol synthase) genes were subcloned under the control of the alcohol dehydrogenase (ADH2) and PGK1 promoters into episomal plasmids, respectively. A laboratory yeast strain containing both the Vst1 and 4CL9, or the Vst1 and 4CL216 genes was evaluated for its ability to utilise p-coumaric acid and produce resveratrol. Northem analysis confirmed that the Vst1, 4CL9 and 4CL216 genes were transcribed and over-expressed compared to the control strain. The transformants expressing the CoA-ligase genes utilised the p-coumaric acid faster than the control, although it was not possible to determine whether p-coumaroyl-CoA was produced. No resveratrol was produced under the assay conditions used. The results indicated that the yeast is unable to produce active resveratrol synthase, which is required to catalyse the final reaction in the production of resveratrol. Posttranslational modification, such as overglycosylation and disulphide formation, of the heterologous protein in yeast has been indicated as the possible reason for the lack of enzyme activity. This introduces an exciting area of research for the development of biotechnological tools with the ability to increase the production of active heterologous proteins in yeast.
AFRIKAANSE OPSOMMING: Wingerde word voortdurend deur 'n groot verskeidenheid patogene, insluitende virusse, bakteriee en swamme, aangeval. Ten einde oorlewing te verseker, het die wingerdstok In wye reeks verdedigingsmeganismes ontwikkel om weerstand te bied teen indringerorganismes. 'n Belangrike faktor in hierdie weerstand teen siektes is die produksie van fitoaleksiene, waarvan resveratrol die hoofkomponent is. Oeur die sintese van resveratrol, asook ander strukturele en biochemiese verdedigingsmeganismes, word die plant toegerus om weerstand te kan bied teen In hele aantal patogene ten einde gesonde druiwe te produseer wat gebruik kan word vir die vinifikasie van topgehalte wyn. As deel van die aktiewe reaksie teen siektes, word resveratrol de novo in die dop van die korrel by die plek van infeksie gesintetiseer sodra 'n patogeen herken word. Hier kan dit die skade deur die patogeen veroorsaak, beperk en verhoed dat dit versprei. Oit gee aan die plant die geleentheid om sy sistemies-verworwe weerstand te inisieer, en daardeur die res van die plant te beskerm, sowel as sekondere infeksies te verhoed. Die fermentasie van rooiwyn op die druifdoppe maak voorsiening vir die ekstraksie van resveratrol uit die dop na die wyn. Die konsentrasie van resveratrol in rooiwyn is dus beduidend hoer as in die wit varietelte, wat geen of baie min resveratrol bevat. Oit is dan juis die rede waarom die matige inname van wyn, veral rooi wyn, gesien word as In integrale deel van 'n gesonde leefwyse. Resveratrol se aktiwiteit as antioksidant en antiinflammatoriese middel lewer In belangrike bydrae tot die kardiovaskulere voordele wat verkry word uit die inname van rooiwyn. Oit blyk egter nou dat die beduidende kardiovaskulere beskerming gesetel is in die sinergistiese werking van resve ratro I, die polifenole en die alkohol in wyn. Aangesien die heilsaamheid van enige voedsel of drank van die uiterste belang is, was dit die doel van hierdie projek om wyngis te manipuleer ten einde tydens die fermentasieproses resveratrol te produseer. Hiervoor moes 'n volledige metaboliese pad daargestel word deur plantgene in die gis te inkorporeer. Resveratrol-sintase maak gebruik van drie maloniel-KoA-molekules en een p-kumarotel-Kos-molekule om een molekule resveratrol te produseer. Saccharomyces cerevisiae produseer maloniel-KoA, maar nie p-kumaroiel-Kcs, nie. Oie volgende gene is dus aangewend om die gis in staat te stel om p-kumarolel-Koe, te produseer: PAL, wat fenielalanien-ammoniak-liase enkodeer om fenielalanien om te sit na kaneelsuur; C4H, wat sinnamaat-4-hidroksliase enkodeer om kaneelsuur om te sit na p-kumaarsuur; en 4CL9 of 4CL216 wat KoA-ligases enkodeer om p-kumaarsuur om te sit na p-kumarolel-Kos, Om hoevlak-uitdrukking te verkry, is die gene gesubkloneer onder beheer van die fosfogliseraat-kinase-geen(PGK1)- promotor en -terminator. As gevolg van integrasieprobleme met hierdie uitdrukkingskassette en die feit dat die gis die p-kumaarsuur kon verteer, is die 4CL9-, 4CL216- en Vst1- (wat resveratrol-sintase enkodeer) gene na episomale plasmiede gesubkloneer onder beheer van die alkohol-dehidrogenase(ADH2)- en PGK1-promotors onderskeidelik. 'n Laboratorium-gisstam wat 6f beide die Vst1-geen en die 4CL9-geen, 6f die Vst1-geen en die 4CL216-geen bevat het, is geevalueer vir die verrnoe om pkumaarsuur te benut en resveratrol te produseer. Noordelike klad analises het bevestig dat die Vst1-, 4CL9- en 4CL216-gene getranskribeer en ooruitgedruk was in vergelyking met die kontrole-stam. Die transformante wat die KoA-ligases uitgedruk het, het die pkumaarsuur vinniger benut as wat die kontrole dit gedoen het, alhoewel dit nie moontlik was om vas te stel of o-kurnarotel-Kos, geproduseer is nie. Met die essai-kondisies wat gebruik is, is geen resveratroI geproduseer nie. Die resultate het daarop gedui dat die gis nie daartoe in staat is om aktiewe resveratrol-sintase, wat nodig is vir die katalise van die finale reaksie in die produksie van resveratrol, te produseer nie. Naomsettingsmodifikasies van die heteroloe protelen in die gis, soos oor-glikosilasie en disulfiedvorming, is aangewys as die moontlike rede vir die gebrek aan ensiemaktiwiteit. Dit stel In opwindende veld vir verdere navorsing voor, naamlik die ontwikkeling van biotegnologiese middele met die vermoe om die produksie van aktiewe heteroloe protelene in gis te verhoog.

In this study the ability of resveratrol to inhibit the activation of the monocyte respiratory burst was investigated.;Differentiated U937 (dU937) cells were pre-treated with resveratrol before stimulation with f-met-leu-phe (fMLP), phorbol 12-myristate-13-acetate (PMA) or arachidonic acid (AA). The extra-cellular and total production of reactive oxygen species (ROS) were measured by isoluminol and luminol chemiluminescence (CL). Superoxide production was measured using lucigenin. Resveratrol was found to inhibit ROS production induced by all three stimuli. Measurement of ROS production was also confirmed using with two used, 2',7'-dichlorofluorescein (DCF) and dihydrorhodamine (DHR). Again resveratrol inhibited both responses. There were significant between the inhibitory effects of resveratrol on peroxidase-dependent (isoluminol, luminol, DCF and DHR) and independent (lucigenin- ROS measuring principles. Moreover, resveratrol was found to be oxidised by the horseradish peroxidase/hydrogen peroxide system.;The cell signal transduction pathways activated by fMLP, PMA and AA were investigated. Only fMLP was found to activate phosphatidylinositol-3-kinase (PI3K) and Akt, using specific inhibitors of both kinases. Resveratrol inhibited PI3K activity with little direct effect on other kinases shown to regulate the respiratory burst, including Akt, extra-cellular regulated protein kinase (ERK); and protein kinase C (PKC). Akt and ERK were found to be activated by fMLP.;In conclusion, resveratrol was found to be a potent inhibitor of ROS production, particularly if a peroxidase-dependent measuring principle was used. Resveratrol can be oxidised by peroxidases, which inhibit the oxidation of the redox probe. Use of a detection method that did not require peroxidase revealed that resveratrol was still a potent inhibitor of fMLP-induced ROS. Moreover, this inhibitory dose of resveratrol correlated with its ability to inhibit the PI3K-Akt pathway, one of the major regulatory pathways of fMLP-induced ROS production. Modulation of these cell signalling intermediates by resveratrol might represent an important anti-inflammatory pathway and further add to its potential cardioprotective properties.

A espécie redox ativa resveratrol (3,5,4’-trihidroxi-trans-estilbeno), uma fitoalexina encontrada nas uvas e no vinho tinto, apresenta importantes efeitos biológicos como atividade antioxidante, anti-inflamatória, antitumoral e cardioprotetora. O sistema nervoso central (SNC) também é alvo do resveratrol que por sua vez pode atravessar a barreira hematoencefálica e exercer efeito neuroprotetor, modulando importantes funções neurogliais. Fisiologicamente os astrócitos controlam importantes funções cerebrais, principalmente, relacionadas ao metabolismo glutamatérgico, à plasticidade sináptica e neuroproteção; e em situações neuropatológicas, relacionadas ao estresse oxidativo. Assim, neste estudo nós investigamos o efeito do resveratrol sobre importantes parâmetros gliais em células C6 frente a dois modelos de insulto oxidativo: (I) 1 mM de H2O2/0,5 h e (II) 0,1 mM de H2O2/6 h. Avaliamos, nessas condições, o perfil das principais defesas antioxidantes enzimáticas celulares, a genotoxicidade celular e possíveis vias de sinalização que possam contribuir para o melhor entendimento do mecanismo de ação do resveratrol no SNC. Nós demonstramos que o resveratrol modula a captação de glutamato; a atividade da enzima glutamina sintetase; os níveis intra e extracelulares de glutationa; a secreção da proteína S100B; as enzimas SOD, CAT e GPx; a peroxidação lipídica; a frequência de micronúcleos; as enzimas heme oxigenase 1 e iNOS e o fator de transcrição NFκB, de maneira dependente das condições oxidativas do meio e do ambiente redox celular. Dessa forma, nossos resultados mostram que o resveratrol apresenta comportamentos anti e pró-oxidante e que sua ação neuroprotetora pode estar relacionada a modulação da atividade glial e da heme oxigenase 1. Enfim, o resveratrol pode representar um potencial agente terapêutico em patologias que envolvam déficits no sistema glutamatérgico associado ao estresse oxidativo.
The redox active compound, resveratrol (3,5,4 '-trihydroxy-trans-stilbene), a phytoalexin, found in grapes and red wine, has a wide range of biological effects, such as, antioxidant, antiinflammatory, anticancer and cardioprotective. The central nervous system (CNS) is also the target of resveratrol which is able to trespass the blood-brain barrier and exerts neuroprotective effects by modulating important glial parameters. Physiologically, astrocytes control important brain functions, mainly related to glutamatergic metabolism, synaptic plasticity and neuroprotection; and in neurophatology events related to oxidative stress. In this study, we investigated the effect of resveratrol on important parameters in C6 astoglial cells against oxidative insult in two models: (I) 1 mM H2O2/0.5 h and (II) 0.1 mM H2O2/6 h. We evaluated under these conditions, the activity of the major cellular enzymatic antioxidant defenses, the possible genotoxicity and cell signaling pathways that may contribute to better understanding the mechanism of resveratrol in the CNS. Resveratrol modulates glutamate uptake; glutamine synthetase activity; intracellular and extracellular levels of glutathione; S100B secretion; the enzymes SOD, CAT and GPx; lipid peroxidation; the frequency of micronuclei; the enzymes heme oxygenase 1 and iNOS and the transcription factor NFκB depending upon the oxidant conditions of the milieu and the cellular redox environment. Thus, our results show that resveratrol presents anti and pro-oxidant effects and their neuroprotective action may be related to modulation of glial function and heme oxygenase 1. Finally, resveratrol can represent a potential therapeutic agent against pathologies associated to glutamatergic system and oxidative stress.

Resveratrol (RV) is a naturally occurring phytoalexin of the stilbenoid family produced by some plant species, and present in grape skin, peanuts, and red wine. It has been found to exhibit anti-cancer, anti-inflammatory, anti-viral, anti-aging, cardio protective, and anti-oxidant properties. Bioavailability is a huge setback that limits the potentials of RV. As a result, efforts have been made to design and synthesize RV esters and aliphatic acids in an attempt to increase its bioavailability, solubility in water, and possibly improving its biological activities. Resveratrol esters, 3,5,4'-triacetyloxystilbene (2) and Methyl 1,1',1''- (3,4',5-stilbenyl)-1,6-hexanedioate (3) have been synthesized. Compound 3 is a new compound, synthetic yield is 88%, and purity is above 95% based on NMR integration. Both 2 and 3 are good candidates for biological evaluation. 3 was used as a precursor in the synthesis of resveratrol aliphatic acid, 8-(3',5'-dihydroxylstilbene-4''-oxy)-3,6-dioxocotanoic acid (9). First, 2 was hydrolyzed to resveratrol diester, 3,5-diacetyloxystilbene (4). Mitsunobu reaction of 4 and methyl 8-hydroxy-3,6-dioxooctanoate (7) was then carried out to afford methyl 8-(3',5'-diacetyoxystilben-4''-oxy)-3.6-dioxooctanoate (/5), which was then hydrolyzed to afford 9 in total 43.6 % yield. Structures of all newly synthesized compounds were confirmed by 1H and 13C NMR spectroscopy.

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
O gênero Arachis possui 81, espécies distribuídas em nove seções taxonômicas, sendo o amendoim (A. hypogaea L.) a principal espécie, devido ao seu alto valor nutricional. O gênero possui também outras espécies de interesse econômico, que têm sido utilizadas em programas de melhoramento do amendoim (espécies da seção Arachis) e espécies forrageiras de outras seções do gênero. Com relação às demais espécies, o germoplasma da grande maioria está disponível para a caracterização e pode, sem dúvida, contribuir para seu uso futuro. O gênero Arachis é um dos poucos que produzem o resveratrol, uma fitoalexina que, além de ser uma resposta da planta contra estresses, também tem ação antioxidante, sendo indicado para prevenção de cânceres e cardiopatias. A avaliação do potencial de produção de resveratrol foi realizada somente em algumas espécies da seção Arachis, faltando avaliação em outras espécies do gênero e nos tetraploides sintéticos que estão sendo utilizados para facilitar a transferência de alelos de espécies silvestres para a cultivada. Assim, o objetivo desse trabalho foi ampliar o conhecimento sobre resveratrol no gênero Arachis. Para tanto foram analisados 21 acessos de espécies silvestres de cinco diferentes seções do gênero Arachis, oito poliploides sintéticos e três cultivares de amendoim. A produção de resveratrol em um conjunto menor de cinco espécies foi acompanhada também de avaliação da expressão do gene que codifica a enzima resveratrol sintase, RS, para entender a variação observada no teor de resveratrol entre as espécies. Tendo em vista que cada experimento foi realizado em uma época diferente do ano e de modo separado, não foi possível reunir todos os dados e fazer uma comparação entre eles. Em todos os experimentos foram avaliadas folhas submetidas a estresse abiótico por raios ultravioleta (UV), que foi o melhor indutor de síntese em dados de literatura. Os resultados mostraram que 21 acessos de 17 espécies das cinco seções são capazes de produzir resveratrol, sendo o maior teor detectado em A. lignosa, da seção Procumbentes. Em relação ao potencial de produção dos híbridos, foi verificado que a concentração de resveratrol na maioria dos anfidiploides sintéticos é significativamente maior em relação aos parentais silvestres diploides. Além disso, a concentração de resveratrol nos híbridos resultantes do cruzamento entre anfidiploides e A. hypogaea foi significativamente maior que nos anfidiploides, sugerindo que a combinação entre alelos de silvestres e do cultivado tem um efeito positivo no aumento do resveratrol. Em relação às análises moleculares, notou-se que a expressão do gene é imediata ao término da indução, seguindo de forma decrescente. Por outro lado, as concentrações de resveratrol são crescentes em relação ao passar do tempo de coleta após a indução por UV. Os resultados demonstram o potencial do gênero como fonte de resveratrol, seja via amendoim cultivado, por meio de introgressão de alelos silvestres utilizando-se anfidiploides, ou pelo uso per se dos acessos de espécies silvestres, incluindo de outras seções distantes do amendoim, como é a Procumbentes, da qual faz parte A. lignosa, que apresentou o maior teor de resveratrol no estudo.
The Arachis genus comprises 81 species divided into nine taxonomic sections, being peanut (A. hypogaea L.) the main species due to its high nutritional value. The genus also has other species of economic interest, which have been used in breeding programs for peanut (species of Arachis section) and to forage production. Regarding the other species of Arachis, germplasm of most of them is available and their characterization could undoubtedly contribute to their future use. Arachis genus is one of the few that produces resveratrol, a phytoalexin that is synthesized by the plant due to a stress and it is also a antioxidant that has been used to prevention of cancers and heart diseases. The resveratrol evaluation in Arachis was carried out mainly in Arachis hypogaea and in some species of the section Arachis, lacking the evaluation in other species of the genus and in the synthetic tetraploids that have been used to facilitate the transfer of alleles from wild species to the cultivated one. Therefore, the objective of this study was to increase the knowledge about resveratrol in the genus Arachis. Twenty one accessions of wild species from five different sections of the genus Arachis, eight synthetic polyploids and three peanut cultivars were analyzed. The production of resveratrol in a smaller set of five species was also accompanied of evaluation of resveratrol synthase to try to understand the variation observed in the resveratrol content among the species. Since each experiment was performed at a different time of year and separately, it was not possible to gather all the data and make a comparison between them. In all experiments, leaves treated with ultraviolet radiation (UV) were evaluated, which was the best inducer of synthesis in literature data. The results showed that 21 accessions of 17 species of the 5 sections are able to produce resveratrol, being the highest content detected in A. lignosa from Procumbentes section. It was verified that the concentration of resveratrol in the majority of the synthetic amphidiploids is significantly higher in relation to the diploid wild parents. In addition, the concentration of resveratrol in the hybrids resulting from the crossing between amphidiploids and A. hypogaea was significantly higher than in amphidiploids, suggesting that the combination of wild and cultivated alleles has a positive effect on the increase of resveratrol. It was noticed that the expression of the gene is immediate at the end of the induction, following in a decreasing way. On the other hand, the concentrations of resveratrol are increasing in relation to the time of collection after the induction of the material. The results demonstrate the potential of wild species of genus Arachis as a 10 source of resveratrol, either via cultivated peanuts, through introgression of wild alleles using amphidiploids, or through the use of wild species accesses, including from other sections distant from peanut, such as A. lignosa.

Orientador: Marcos Aparecido Gimenes
Resumo: O gênero Arachis possui 81, espécies distribuídas em nove seções taxonômicas, sendo o amendoim (A. hypogaea L.) a principal espécie, devido ao seu alto valor nutricional. O gênero possui também outras espécies de interesse econômico, que têm sido utilizadas em programas de melhoramento do amendoim (espécies da seção Arachis) e espécies forrageiras de outras seções do gênero. Com relação às demais espécies, o germoplasma da grande maioria está disponível para a caracterização e pode, sem dúvida, contribuir para seu uso futuro. O gênero Arachis é um dos poucos que produzem o resveratrol, uma fitoalexina que, além de ser uma resposta da planta contra estresses, também tem ação antioxidante, sendo indicado para prevenção de cânceres e cardiopatias. A avaliação do potencial de produção de resveratrol foi realizada somente em algumas espécies da seção Arachis, faltando avaliação em outras espécies do gênero e nos tetraploides sintéticos que estão sendo utilizados para facilitar a transferência de alelos de espécies silvestres para a cultivada. Assim, o objetivo desse trabalho foi ampliar o conhecimento sobre resveratrol no gênero Arachis. Para tanto foram analisados 21 acessos de espécies silvestres de cinco diferentes seções do gênero Arachis, oito poliploides sintéticos e três cultivares de amendoim. A produção de resveratrol em um conjunto menor de cinco espécies foi acompanhada também de avaliação da expressão do gene que codifica a enzima resveratrol sintase, RS, para entende... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: The Arachis genus comprises 81 species divided into nine taxonomic sections, being peanut (A. hypogaea L.) the main species due to its high nutritional value. The genus also has other species of economic interest, which have been used in breeding programs for peanut (species of Arachis section) and to forage production. Regarding the other species of Arachis, germplasm of most of them is available and their characterization could undoubtedly contribute to their future use. Arachis genus is one of the few that produces resveratrol, a phytoalexin that is synthesized by the plant due to a stress and it is also a antioxidant that has been used to prevention of cancers and heart diseases. The resveratrol evaluation in Arachis was carried out mainly in Arachis hypogaea and in some species of the section Arachis, lacking the evaluation in other species of the genus and in the synthetic tetraploids that have been used to facilitate the transfer of alleles from wild species to the cultivated one. Therefore, the objective of this study was to increase the knowledge about resveratrol in the genus Arachis. Twenty one accessions of wild species from five different sections of the genus Arachis, eight synthetic polyploids and three peanut cultivars were analyzed. The production of resveratrol in a smaller set of five species was also accompanied of evaluation of resveratrol synthase to try to understand the variation observed in the resveratrol content among the species. Since each experim... (Complete abstract click electronic access below)
Doutor

Background: Cardiac hypertrophy is a compensatory enlargement of the heart in response to stress such as hypertension. It is beneficial in reducing stress placed on the heart. However, when the stress is of a chronic nature, it becomes pathological and leads to cardiac dysfunction and heart failure. Current treatments for hypertension and heart failure have proven beneficial but are not highly specific and associated with side effects. Accordingly, there is an important need for alternative strategies to provide safe and effective treatment. Methods: Ten-week-old male spontaneously hypertensive rats (SHRs) and Wistar–Kyoto (WKY) rats were treated with resveratrol (2.5mg/kg/day) for a period of 10 weeks. Systolic blood pressure, and cardiac structure and function were measured in all groups at different time points of resveratrol treatment. Oxidative stress was also determined in all groups after 10 weeks of resveratrol treatment. Results: SHRs were characterized with high blood pressure and concentric hypertrophy from 15 weeks of age. Cardiac functional abnormalities were also evident in SHR from 15 weeks onwards. Resveratrol treatment significantly prevented the development of concentric hypertrophy, and systolic and diastolic dysfunction in SHR without lowering blood pressure. Resveratrol also significantly reduced the oxidative stress levels of cardiac tissue in SHR. Conclusions: Resveratrol treatment was beneficial in preventing the development of concentric hypertrophy and cardiac dysfunction in SHR. The cardioprotective effect of resveratrol in SHR may be partially mediated by a reduction in oxidative stress. Thus, resveratrol may have potential in preventing cardiac impairment in patients with essential hypertension.

Glioblastomas (GBMs) são os tumores primários (gliomas) mais comuns e agressivos do Sistema Nervosos Central, classificados pelos oncologistas como um dos maiores desafios da oncoterapia. O prognóstico dos pacientes é ruim mesmo após terapia cirúrgica seguida de radio e quimioterapia com Temozolomida (TMZ). Células de GBM apresentam resistência intrínseca à apoptose, porém, se mostram mais sensíveis à indução de senescência, autofagia e catástrofe mitótica. Resveratrol (Rsv) é um polifenol com ação neuroprotetora ao tecido neural sadio e, por outro lado, ação citotóxica em células de GBM. Porém, o mecanismo de ação do Rsv nestas células ainda não está esclarecido. O objetivo do presente trabalho é avaliar o mecanismo de ação do Rsv, bem como o efeito do co-tratamento de Rsv e TMZ, em células de GBM. Rsv reduziu o número de células em 3 linhagens de GBM humano, induzindo autofagia e acúmulo de células nas fases S-G2/M do ciclo celular, com aumento de pCdc2(Y15), das ciclinas E, A e B e redução de ciclina D1. A inibição da autofagia induzida pelo Rsv aumentou a toxicidade do composto, ativou a apoptose (acompanhado do aumento de Bax e de clivagem de caspase 3) e inibiu a parada no ciclo celular induzida pelo Rsv, reduzindo os níveis das ciclinas e de pCdc2 (Y15). Estes dados sugerem um papel citoprotetor da autofagia no efeito do Rsv. Por outro lado, Rsv potencializou o efeito citotóxico da TMZ através da inibição da parada em G2/M induzido pela TMZ, sem reduzir o dano ao DNA induzido pela TMZ ou a ativação da proteína de reconhecimento de dano ATM. Núcleos das células expostas ao co-tratamento apresentaram características típicas de catástrofe mitótica, acompanhado pelo acúmulo de ciclina B e redução de pCdc2(Y15). Através de uma ferramenta de análise morfométrica nuclear desenvolvida por nós, mostramos que o cotratamento de Rsv e TMZ induziu um aumento no número de núcleos em catástrofe mitótica. Em conclusão, Rsv apresentou potencial aplicação em GBM a ser testada em modelos in vivo deste tumor, tanto como terapia primária (especialmente se combinado a inibidores de autofagia) ou como adjuvante à TMZ. Uma vez que as células cancerosas são resistentes à morte celular e sabendo que um único composto geralmente modula mais de um mecanismo celular, é importante considerar estas interações para o desenvolvimento de alternativas para sensibilizar as células cancerosas e aumentar a eficácia terapêutica.
Glioblastomas (GBM) are the most common and aggressive primary tumors (gliomas) of the Central Nervous System, classified by oncologists as one of the biggest challenges of oncotherapy. The prognosis for patients is poor, even after surgical therapy followed by radiotherapy and chemotherapy with Temozolomide (TMZ). GBM cells are intrinsically resistance to apoptosis, but more sensitive to the induction of senescence, autophagy and mitotic catastrophe. Resveratrol (Rsv) is a polyphenol with a neuroprotective effect on healthy neural tissue but it has a a cytotoxic activity in GBM cells. Unfortunately, the mechanism of action of Rsv in these cells is still unclear. The aim of this study is to assess the mechanism of action of Rsv, as well as the effect of co-treatment of Rsv and TMZ in GBM cells. Rsv reduced the number of cells in three cell lines of human GBM, inducing autophagy and accumulation of cells in S-G2/M phase of cell cycle, increasing pCdc2 (Y15), pRb (S807/811), cyclins E, A and B, and reducing cyclin D1. Inhibition of Rsvinduced autophagy slightly increased the toxicity of the Rsv , accompained by triggering apoptosis (accompanied by an increase of Bax and cleavage of caspase 3) and inhibition of cell cycle arrest induced by Rsv, reducing the levels of cyclins and pCdc2 (Y15) in relation to Rsv alone, suggesting a cytoprotective role of autophagy in the effect of Rsv. Moreover, Rsv enhanced the cytotoxic effect of TMZ by abrogate the arrest in G2/M induced by TMZ, without reducing the DNA damage induced by TMZ or the activation of protein damage recognition ATM. Nuclei of cells exposed to co-treatment showed typical features of mitotic catastrophe, accompanied by the accumulation of cyclin B and reduced pCdc2 (Y15). Through a tool developed by us, we demonstrate that in fact the cotreatment induced an increase of nuclei with irregularities typical of mitotic catastrophe. In conclusion, Rsv has potential application in GBM, both as primary therapy (especially if combined with inhibitors of autophagy) or as adjuvant to TMZ. Since cancer cells are resistant to cell death and understanding that a single compound usually modulates more than one cellular mechanism, it is important to consider these interactions to the development of alternatives to sensitize cancer cells and increase therapeutic efficacy.

Thesis (M.S.) -- University of Maryland, College Park, 2006.
Thesis research directed by: Dept. of Nutrition and Food Science. Title from t.p. of PDF. Includes bibliographical references. Published by UMI Dissertation Services, Ann Arbor, Mich. Also available in paper.

Resveratrol or trans-3,4’,5-trihydroxystilbene is a naturally occurring phytochemical contained in red grapes skin, nuts and berries. It has been shown over the years to have different biological properties particularly in the chemoprevention of cancer. However, it is metabolised in vivo to sulfates and glucuronides within 1h and is active against different targets in a dose dependant manner. A library of new analogues of resveratrol has been synthesised with the aim of stopping or at least slowing down the metabolism whilst keeping its activity on the inhibition of cancer cell proliferation. Seven new analogues of resveratrol were synthesised in which the phenol substituents were systematically replaced by benzylic alcohols and/or methoxy groups. The library was then assessed in in vitro enzymatic metabolism with mouse and human liver fractions in the presence of a cofactor. The compounds were also evaluated as inhibitors of HCA-7 colorectal cancer cell proliferation.

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Resveratrol is a potent antioxidant, anti-inflammatory, anticancer, and chemoprotective agent. However, it is sensitive to some external agents such as air, light and oxidative enzymes that can reduce its viability and bioavailability for clinical use. In order to provide a controlled release, the aim of this study was to obtain resveratrol-loaded polyester microparticles and to evaluate their physicochemical properties, antioxidant potential and effect on hemolysis of human erythrocytes. Microparticles of poly(3-hydroxybutyrate-co -3-hydroxyvalerate) (PHBV) and poly(-caprolactone) (PCL) containing resveratrol were successfully prepared by simple emulsion/solvent evaporation. All formulations showed suitable encapsulation efficiency values higher than 80%. PHBV microparticles revealed spherical shape with rough surface and presence of pores. PCL microparticles were spherically shaped with smooth surface. Fourier-transformed infrared spectra demonstrated no chemical bond between resveratrol and polymers. X-ray powder diffraction patterns and differential scanning calorimetry analyses indicated that microencapsulation led to a drug amorphization. These PHBV/PCL microparticles delayed the dissolution profile of resveratrol. Release profiles were better fitted to biexponential equation. The hypochlorous acid scavenging activity and 2,2-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) radical cation decolorization assay confirmed that the antioxidant activity of resveratrolloaded into PHBV/PCL microparticles was kept, but was dependent on the microparticle morphology and dissolution profile. Resveratrol-loaded PHBV/PCL microparticles showed no cytotoxic effect on red blood cells. These results support an experimental basis for the use of resveratrol-loaded PHBV/PCL microparticles as a feasible oral drug delivery carrier for controlled release of resveratrol, being an attractive alternative in chronic diseases prevention.
O resveratrol é um potente agente antioxidante, anti-inflamatório, anticancerígeno e quimiopreventivo. No entanto, é sensível a algumas condições externas e ambientais, tais como: ar, luz e enzimas oxidativas, que podem reduzir a sua viabilidade e a sua biodisponibilidade para o uso clínico. Com o propósito de se elaborar sistemas de liberação modificada, o objetivo deste estudo foi a obtenção de micropartículas baseadas em poliésteres, contendo resveratrol, e a avaliação das características físico-químicas, do potencial antioxidante e do efeito sobre a hemólise de eritrócitos humanos desses materiais. As micropartículas de poli(3-hidroxibutirato-co-3 hidroxivalerato) (PHBV) e poli(-caprolactona) (PCL) contendo resveratrol foram preparadas com sucesso pelo método de emulsão simples/evaporação do solvente. Todas as formulações mostraram valores de eficiência de encapsulação adequados, superiores a 80%. As micropartículas de PHBV revelaram formato esférico com superfície rugosa e presença de poros. As micropartículas de PCL apresentaram formato esférico com superfície lisa. Os espectros de infravermelho com transformada de Fourier não demonstraram nenhuma ligação química entre o resveratrol e polímeros após a microencapsulação. As análises de difração de raio-X e de calorimetria exploratória diferencial indicaram que a microencapsulação conduziu a uma amorfização do fármaco. As micropartículas de PHBV e de PCL foram efetivas no controle da liberação do resveratrol. Os perfis de dissolução apresentaram o melhor ajuste para a equação biexponencial. Os ensaios de inibição do ácido hipocloroso e de descolaração do radical catiônico 2,2-azinobis(3-etilbenzotiazolina-6-ácido sulfônico) confirmaram a manutenção da atividade antioxidante do resveratrol presente nas micropartículas de PHBV e de PCL, mas dependente da morfologia das micropartículas e do perfil de dissolução. As micropartículas de PHBV e de PCL contendo resveratrol não apresentaram efeitos citotóxicos sobre os eritrócitos humanos. Esses resultados sugerem que as micropartículas poliméricas elaboradas a partir do PHBV e da PCL, contendo o fármaco, têm viabilidade como sistemas de liberação controlada por via oral do resveratrol, sendo uma alternativa interessante na prevenção de doenças crônicas.

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CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico
FAPEMIG - Fundação de Amparo à Pesquisa do Estado de Minas Gerais
Apesar da grande aplicação do sêmen congelado na bovinocultura, a criopreservação ainda resulta em considerável diminuição da viabilidade e fertilidade espermática. Diversos fatores são responsáveis por essa perda, sendo importante ressaltar os danos causados ao espermatozoide pelo estresse oxidativo. O objetivo do trabalho foi avaliar o efeito antioxidante resveratrol na criopreservação de sêmen bovino. Foram utilizados oito touros holandeses selecionados previamente por exame andrológico. O sêmen foi diluído em meio Tris-gema-glicerol e divididos nos grupos: R0 (controle- sem antioxidante), R1 (50 μM de resveratrol), R2 (100 μM de resveratrol) e R3 (1000 μM de resveratrol). Os espermatozoides após o descongelamento foram avaliados através da análise de motilidade e vigor espermáticos por microscopia de contraste de fase, parâmetros cinéticos por sistema automatizado (CASA), integridade de membrana plasmática e acrossomal, função mitocondrial e fertilidade in vitro. Os dados foram analisados por ANOVA e as médias foram comparadas pelo teste de Tukey-Krammer e Student-Newman-Keuls ao nível de significância de 5% através dos programas Statistical Analysis System (SAS Institute, Cay, NC, USA) e Graph Pad Prism versão 5 (Graph Pad Software, California, Estados Unidos). Na análise de motilidade do sêmen por microscopia óptica observou-se maior média (P<0,05) do tratamento R1 em relação a R3, não sendo estes diferentes (P>0,05) do controle e de R2. Entretanto, os resultados da análise de motilidade progressiva pelo CASA mostraram que o tratamento com resveratrol 50 μM apresentou melhor (P<0,05) resultado em relação ao controle e outros tratamentos. Os dados dos parâmetros cinéticos do CASA também revelaram um número maior de espermatozoides com trajetória linear no grupo R1. Há também mais células com membrana plasmática integra no grupo tratado (R1) em relação ao controle. Na análise de função mitocondrial e na produção in vitro de embriões não houve diferença entre controle e tratamento (P>0,05). Conclui-se que o resveratrol na concentração de 50 μM protege a membrana plasmática de células espermáticas e melhora parâmetros cinéticos do sêmen sem alterar a capacidade fecundante do espermatozoide.
Despite the wide application of frozen semen in livestock, cryopreservation yet results in decrease in viability and fertility of sperm. Several factors are responsible for this loss as injuries caused on sperm by oxidative stress. The aim of the study was to evaluate the antioxidant effect of resveratrol on cryopreservation of bovine sperm. We used eight Holstein bulls selected previously by andrological exam. Semen was diluted in Tris-yolk-glycerol extender and distributed in groups: R0 (control - no antioxidant), R1 (50 μM resveratrol), R2 (100 μM resveratrol) and R3 (1000 μM resveratrol). Motility and vigorous thawed bull sperm were evaluated by phase contrast microscopy and kinetics parameters by an automated system (CASA). Plasmatic and acrosomal membrane integrity, mitochondrial function, and in vitro fertilization were also evaluated in thawed sperm. The data were analyzed by ANOVA and averages were compared by Tukey-Krammer and Student-Newman-Keuls tests by the Statistical Analysis System (SAS Institute, Cay, NC, USA) and Graph Pad Prism versão 5 (Graph Pad Software, California, Estados Unidos). The level of 5% was considered significant. With optical microscopy was observed higher sperm motility in treatment R1 than R3, and they were not different (P>0.05) to control and R2. However, the results of progressive motility analysis by CASA showed that treatment with 50 μM resveratrol had better (P <0.05) results than control and other treatments. The data of the kinetic parameters of CASA also revealed greater (P <0.05) number of spermatozoa with linear trajectory in R1 treatment. There is also more (P <0.05) cells with plasma membrane intact in R1 compared to the control. In mitochondrial function analysis and in vitro embryo production no difference was found between treatment and control (P>0.05). We conclude that the resveratrol 50 μM concentration protects the sperm cell plasmatic membrane and improves kinetic parameters without affect fertilization potential of spermatozoa.

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Dissertacao (Mestrado)
IPEN/D
Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP

As células estreladas hepáticas (HSC) são pericitos específicos do fígado, são células armazenadoras de gordura e produzem componentes de matriz extracelular. As HSCs ao serem ativadas, proliferam, perdem as gotas lipídicas e aumentam a secreção de matriz extracelular. O controle da modulação fenotípica e da proliferação das HSCs é de suma importância para entendermos a fibrose hepática. Esta pesquisa foi realizada com a linhagem celular GRX (representativa das HSCs murinas) obtida a partir de reações fibrogranulomatosas inflamatórias. Estudos anteriores, em nosso laboratório, pesquisaram a ação de diferentes drogas sobre a modulação destas células, tais como: retinol, pentoxifilina, indometacina e insulina. Tem sido relatado que o resveratrol (3,4,5 trihidroxiestilbeno) (RSV) tem ação antioxidante, antiinflamatória, antiproliferativa bem como tem capacidade de ativar ou inibir a transcrição de enzimas envolvidas na regulação de vários eventos celulares, por exemplo metabolismo de lipídeos. Por isso, procuramos utilizar o RSV, um polifenol natural presente na casca das uvas e conseqüentemente em grande quantidade de vinhos tintos, neste modelo celular. As células GRX foram cultivadas (12, 24 e 120 h) em meio DMEM com 5% SFB acrescido ou não de RSV (100 nM, 1µM ou 100 µM) e retinol (RHO) (5µM). Nossos estudos demonstraram que tratamento por 120 h com RSV (100nM e 1µM) inibiu a proliferação das células GRX diminuindo em 35% o número de células. Analisando o efeito de 1µM de RSV sobre o ciclo celular e a apoptose, verificamos que após 120 h de tratamento houve um aumento das células na fase S e Sub-G1. Constatou-se condensação e fragmentação nuclear, sugerindo um possível efeito pró-apoptótico do RSV sobre a célula GRX. Observou-se que o tratamento com RSV (100 nM) por 5 dias não induziu a formação de gotas lipídicas nas células GRX. Porém ao acrescentar RSV+RHO, constatou-se que o RSV não interferiu na síntese e acúmulo de lipídios, provocado pelo RHO (5µM). Propondo que a lipogênese neste modelo de tratamento possa estar associada com a ação da sirtuina (deacetilase NAD-dependente) e do PPARy (receptores ativados por proliferadores de peroxissomos), determinou-se a expressão do mRNA destas duas proteínas. Observou-se que o RSV (100 nM) aumenta a expressão do mRNA da sirtuina 1 (SIRT1) e diminui a expressão de PPARy. Porém, o tratamento com RSV+RHO provocou uma redução na expressão da SIRT1, alterando a relação PPARy/SIRT1, promovendo a síntese de lipídios. Nossos dados mostraram que após 24h de tratamento, o RSV não alterou a incorporação de acetato [C14] em triacilglicerídios (TG), enquanto que, o RHO ou RSV+RHO aumentaram a incorporação do marcador nestes lipídios. O depósito de TG permaneceu constante, porém a síntese diminuiu com o tempo de tratamento. Acredita-se que estes compostos atuam por mecanismos moleculares diferentes modulando o metabolismo de lipídios. Sugerimos, de acordo dados da literatura, que o RHO atua via receptor nuclear LXR formando um heterodímero com PPARy, favorecendo a lipogênese. Por outro lado, o RSV ativa a SIRT1, que reprime o PGC1α (coativador do PPARy). Logo, a inibição do PPARy pelo RSV inibe consequentemente a lipogênese. Concluímos que o resveratrol apresenta um efeito anti-proliferativo, pró-apoptótico sem induzir o fenótipo lipocítico nas células GRX. Desta forma, se mantém em equilíbrio, no espaço de Disse, as células mesenquimais quiescentes e ativadas, contribuindo para restabelecer a homeostase hepática.
The hepatic stellate cells (HSC) are liver-specific pericytes, as well as fat-storing cells and they are also responsible for the extracellular matrix components production. When activated, HSCs lose lipid droplets and increase extracellular matrix secretion. The phenotypical modulation and the HSCs proliferation control have paramount importance to understand the hepatic fibrosis. This research has been carried out with the GRX cell line (HSCs murine representative) which was obtained from inflammatory fibrogranulomatous reactions. Previous studies in our laboratory investigated the action of different drugs on the modulation of these cells, such as: retinol, pentoxifilin, indomethacin and insulin. Resveratrol (RSV) has been referred to as having antioxidant, antiinflammatory and antiproliferative action (3,4,5 trihidroxiestilbeno), as well as the capacity to activate or inhibit the transcription of enzymes involved in the regulation of several cell events, as, for example, lipid metabolism Therefore, we used RSV, which is a natural polyphenol found in the grapes skin and, consequently, in large amounts of red wine, in this cell model. The GRX cells were cultivated (12, 24 and 120 h) in medium DMEM with 5% SFB added RSV or not (100 nM, 1µM or 100 µM) and retinol (RHO) (5µM). Our studies demonstrated that a 120 h treatment with RSV (beginning with 100 nM) inhibited GRX cells proliferation, thus decreasing the cell number in 35%. Assessing the effect of 1µM RSV on the cell cycle and the apoptosis, we found that, after this treatment period, there was an increase of cells in phases S and Sub-G1. Nuclear condensation and fragmentation were observed, which suggested a probable pro-apoptotic RSV effect on the GRX cell. The RSV (100 nM) 120 h treatment did not induce lipid droplets formation in the GRX cells. However, RSV did not interfere in the synthesis or accumulation of lipids caused by RHO (5µM), when RSV+RHO were added. Considering that the lipogenesis in this treatment model might be associated with sirtuin action (deacetilase NAD-dependent) and PPARy (receptors activated by peroxisome proliferators), the mRNA expression of these two proteins was determined. RSV (100 nM) proved to increase sirtuin mRNA expression 1 (SIRT1) and decrease PPARy.expression. However, the treatment with the RSV+RHO caused a reduction in the SIRT1 expression thus alterating the relation PPARy/SIRT1, which promoted the lypid synthesis. Our data showed that after the 24-hour treatment RSV did not change acetate incorporation [C14] in triacylglicerides (TG), whilst RHO or RSV+RHO increased the marker incorporation in these lipids. The TG deposit remained constant, but the synthesis diminished during the treatment period. It appears that these compounds act through different molecular mechanisms when modulating lipids metabolism. According to literature data, we suggest that RHO acts via LXR nuclear receptor, thus forming an heterodímer with PPARy, which favors lypogenesis. On the other hand, RSV activates SIRT1, which inhibits PGC1α (PPARy coactivator). Therefore, the PPARy inhibition by RSV consequently inhibits lypogenesis. We concluded that RSV presents pro-apoptotic, anti-proliferative effect, without inducing lypocytic phenotype in the GRX cells. By this means the mesenquimal activated quiescent cells keep their balance at the Disse space, thus contributing with the hepatic homeostasis restoration.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior
O resveratrol à um polifenol antioxidante natural encontrado especialmente na epicarpo da uva, em nozes e na romà o qual pode inibir a ativaÃÃo de mediadores prÃ-inflamatÃrio e citocinas na fase precoce de expressÃo do gene. Jà à bem conhecido que as lectinas sÃo proteÃnas de ligaÃÃo de aÃÃcares que atuam tanto como molÃculas prÃ- e anti-inflamatÃrias. Assim, o objetivo do presente trabalho foi o de verificar a ligaÃÃo de um composto de polifenol com uma lectina de Canavalia maritima (ConM) com base na sua capacidade para inibir processos prÃ-inflamatÃrios. Para alcanÃar este objetivo, a ConM foi purificada e cristalizada, uma soluÃÃo de 5 mM de resveratrol 5 mM foi utilizada para soaking durante 2 horas de incubaÃÃo. Os cristais obtidos pertencem ao grupo espacial monoclÃnico C2, o refinamento final resultou em um Rfactor de 16,0% e uma Rfree de 25,5%. Resveratrol se liga na rÃgida atravÃs de pontes de hidrogÃnio e interaÃÃo hidrofÃbica com aminoÃcidos que compÃem a quinta e sexta fita-β da folha-β rÃgida da ConM. A ConM complexada com o resveratrol inibiu a oxidaÃÃo do DPPH, mostrando atividade sinÃrgica entre a proteÃna e o ligante com a relaÃÃo mais eficaz de 2:3. Foi verificado ainda que o sÃtio de ligaÃÃo a carboidratos nÃo està diretamente relacionado à atividade antioxidante. à a interaÃÃo entre ConM e resveratrol, que indica o sinergismo destas duas molÃculas em agir como agentes sequestrantes de radicais livres que podem estarem relacionados a capacidade de reduÃÃo do processo inflamatÃrio atravÃs da inibiÃÃo de muitos mediadores prÃ-inflamatÃrios por lecitinas.
Resveratrol is a natural antioxidant polyphenol found especially in grape epicarp, walnuts, and pomegranates, which can inhibit the activation of proinflammatory mediators and cytokines at the early gene expression stage. It is well known that lectins are sugar-binding proteins that act as both pro- and anti-inflammatory molecules. Thus, the objective of this work was to verify the binding of a polyphenol compound with a lectin of Canavalia maritima (ConM) based on their ability to inhibit pro-inflammatory processes. To accomplish this, ConM was purified and crystallized, and resveratrol was soaked at 5 mM for 2 hours of incubation. The crystal belongs to the monoclinic space group C2, the final refinement resulted in an Rfactor of 16.0% and an Rfree of 25.5%. Resveratrol binds in the rigid β-sheet through H-bonds and hydrophobic interaction with amino acids that compose the fifth and sixth β-strands of the rigid β-sheet of ConM. The ConM and resveratrol inhibited DPPH oxidation, showing synergic activity with the most effective ratio of 2:3 and carbohydrate binding site is not directly related to antioxidant activity. It is the interaction between ConM and resveratrol that indicates the synergism of these two molecules in acting as free radicals scavengers and in reducing the inflammatory process through the inhibition of many pro-inflammatory events.

Background and Aims: In the Western countries, liver cirrhosis is the main cause of portal hypertension, which is defined as a clinical syndrome characterized by an increased hydrostatic pressure within the portal venous system. The importance of this syndrome is defined by the frequency and severity of its complications: gastrointestinal bleeding from ruptured gastroesophageal varices, ascites, hepatorenal syndrome and hepatic encephalopathy. In cirrhosis, the initial factor determining the onset of portal hypertension is the increase in intrahepatic vascular resistance, which is due to the morphological changes of the liver, but also to endothelial dysfunction in the porto-hepatic vascular bed, characterized by an exaggerated response to vasoconstrictors and a deficient response to vasodilators. Resveratrol, a polyphenol found in a variety of fruits, possesses a wide range of beneficial properties, e.g. anti-oxidant and anti-inflammatory activity. In the endothelium resveratrol regulates several vasoactive substances and decreases oxidative stress, both factors involved in the pathophysiology of endothelial dysfunction. This study aimed at evaluating the effects of resveratrol on hepatic and systemic hemodynamics, hepatic endothelial dysfunction and hepatic fibrosis in CCl4-cirrhotic (CH) rats. Methods: Resveratrol (10 or 20mg/kg/day) or its vehicle were administered to CH rats orally for two weeks; then the following measurements were done: a) in vivo: mean arterial pressure (MAP), portal pressure (PP), portal blood flow (PBF) and superior mesenteric artery blood flow (SMABF); b) isolated perfused livers: endothelial function assessed by dose-relaxation curves to acetylcholine; c) assessment of fibrosis: Sirius Red staining of liver sections, collagen-1 mRNA expression and α-smooth muscle actin (α-SMA) protein expression, as a surrogate of hepatic stellate cell activation. Results: Resveratrol administration significantly decreased PP without significant changes in MAP, PBF and SMABF. Reduction in PP was associated with an improved vasodilatory response to acetylcholine and with a significant reduction in liver fibrosis. The decrease in hepatic fibrosis was associated with a reduced collagen-1 mRNA expression and α-SMA protein expression. Conclusions: In cirrhotic rats, resveratrol administration reduces portal pressure by improving liver fibrosis and endothelial dysfunction, suggesting that it may be an ideal supplement in the treatment of portal hypertension in patients with cirrhosis.
Introduzione e scopo dello studio: L'ipertensione portale è una condizione fisiopatologica caratterizzata dall'aumento della pressione nel sistema portale che può causare lo sviluppo di manifestazioni cliniche severe quali le emorragie da rottura di varici esofagee e/o gastriche e da gastropatia congestizia, l'ascite, l'encefalopatia epatica e la sindrome epatorenale. L'ipertensione portale può manifestarsi in corso di diverse patologie, fra le quali la più comune nel mondo occidentale è la cirrosi epatica. Nella cirrosi il fattore iniziale alla base dello sviluppo dell'ipertensione portale è l'aumento delle resistenze vascolari intraepatiche, dovuto sia alle alterazioni morfologiche del fegato, sia alla disfunzione endoteliale nel letto vascolare porto-epatico, caratterizzata da un'esagerata risposta a sostanze vasocostrittrici e una risposta ridotta a sostanze vasodilatatrici. Il resveratrolo è un polifenolo di origine vegetale presente nell'uva e nei suoi derivati, negli arachidi, e in diverse altre piante. Tra le numerose proprietà benefiche di questa sostanza sono state studiate soprattutto quella antiossidante ed antinfiammatoria. Il resveratrolo possiede effetti benefici anche a livello endoteliale: è coinvolto nella regolazione di numerose sostanze vasoattive e riduce lo stress ossidativo, fattori entrambi coinvolti nella patogenesi della disfunzione endoteliale. Lo scopo di questo studio era quello di valutare gli effetti del resveratrolo sull'emodinamica sistemica ed epatica, la disfunzione endoteliale epatica e la fibrosi epatica nel modello animale di cirrosi epatica indotta mediante CCl4. Metodi: Il resveratrolo (10 o 20 mg/kg/die) o il suo veicolo sono stati somministrati oralmente per due settimane in ratti cirrotici; successivamente sono state eseguite le seguenti misurazioni: a) in vivo: pressione arteriosa media (PAM), pressione portale (PP), flusso portale (FP) e flusso arterioso mesenterico superiore (FAMS); b) fegati isolati e perfusi: funzione endoteliale valutata attraverso curve dose-risposta all'acetilcolina; c) valutazione della fibrosi epatica: colorazione Sirius Red di sezioni di fegato, espressione dell'mRNA per il collagene-1 ed espressione proteica di α-smooth muscle actin (α-SMA), indice dell'attivazione delle cellule stellate epatiche. Risultati: La somministrazione di resveratrolo ha determinato una riduzione della PP, senza modificazioni significative della PAM, del FP e del FAMS. La riduzione della PP era associata ad un miglioramento della risposta vasodilatatrice all'acetilcolina e della fibrosi a livello epatico. Oltre al miglioramento della fibrosi valutato mediante colorazione Sirius Red, è stata osservata una riduzione dell'espressione dell'mRNA per il collagene-1 e dell' α-smooth muscle actin (α-SMA). Conclusioni: Nei ratti cirrotici il resveratrolo riduce la PP attraverso un miglioramento della fibrosi e della disfunzione endoteliale epatica. Il resveratrolo, dunque, potrebbe essere un supplemento ideale nel trattamento dell'ipertensione portale in pazienti con cirrosi epatica.

A hidrocefalia é uma condição neurológica complexa, em que ocorre o desequilíbrio na dinâmica do líquido cefalorraquidiano (LCR) provocando um distúrbio na produção, circulação e reabsorção do mesmo, o que pode levar a uma redução e/ou bloqueio do fluxo sanguíneo. Apesar do tratamento com derivação liquórica ser eficiente na redução da ventriculomegalia, muitos danos neurológicos não são revertidos com a cirurgia. Estudos demonstram que o estresse oxidativo está envolvido na gênese das lesões da hidrocefalia. O objetivo deste trabalho foi avaliar a resposta neuroprotetora do Resveratrol, reconhecido como um potente antioxidante, na hidrocefalia experimental induzida em ratos Wistar jovens. Foram utilizados ratos machos, com sete dias de vida, que receberam uma injeção de caulim a 15% na cisterna magna para indução da hidrocefalia. Esses animais foram divididos em grupo hidrocefálico sem tratamento (n=20), grupo hidrocefálico tratado com Resveratrol através de aplicação de injeção intraperitoneal (20mg/kg/dia) (n=20) e um grupo de animais que não receberam a injeção de caulim para ser usado como controle (n=10). Foram realizadas avaliações comportamentais e estudos de ressonância magnética. Duas semanas após, os animais foram eutanasiados para coleta dos encéfalos, e realizados testes histológicos, imunoistoquímicos e bioquímicos. Os resultados obtidos neste trabalho mostraram que os animais hidrocefálicos, que receberam diariamente injeção de Resveratrol, apresentaram efeito benéfico nos testes comportamentais mostrando mais agilidade e melhor exploração do ambiente, melhor desenvolvimento sensoriomotor, aprendizagem e capacidade de memorização, apresentaram também discreta diminuição da atividade astrocitária evidenciada pela imunomarcação do GFAP no corpo caloso e exibiram aumento significativo na quantidade de antioxidantes totais no plasma. Conclui-se que o uso do Resveratrol diminuiu a atividade astrocitária, aumentou a quantidade de antioxidantes e melhorou a memória e o desenvolvimento motor dos ratos
Hydrocephalus is a neurological condition complex, wherein the imbalance occurs in the dynamics of cerebrospinal fluid (CSF) causing a disturbance in the production, circulation and absorption thereof, which can lead to a reduction and / or blockage of blood flow. Despite treatment with CSF shunt be effective in reducing ventriculomegaly, many neurological damage is not reversed with surgery. Studies have shown that oxidative stress is involved in the genesis of hydrocephalus injuries. The objective of this study was to evaluate the neuroprotective response Resveratrol, recognized as a potent antioxidant in experimentally induced hydrocephalus in young Wistar rats. male rats were used with seven days of life, which received a 15% kaolin injection into the cisterna magna induction of hydrocephalus. These animals were divided into hidrocefálico untreated group (n = 20), hidrocefálico group treated with resveratrol by intraperitoneal injection application (20 mg / kg / day) (n = 20) and a group of animals that received the kaolin injection to be used as control (n = 10). behavioral assessments and magnetic resonance studies were performed. Two weeks later, the animals were euthanized to collect the brains and performed histological, immunohistochemical and biochemical tests. The results of this study showed that hidrocefálicos animals that received daily injection of Resveratrol showed beneficial effect on behavioral tests showing more agility and better exploitation of the environment, better sensorimotor development, learning and memory capacity, also showed a slight decrease of astrocyte activity evidenced by immunostaining of GFAP in the corpus callosum and exhibited a significant increase in the total amount of antioxidants in plasma. We conclude that the use of Resveratrol decreased the astrocyte activity, increased the amount of antioxidants and improved memory and motor development of mice

Pharmaceutical Sciences
Ph.D.
Resveratrol (RES) has been associated with numerous pharmacological effects. Yet its pharmacokinetics is not clearly understood. It is known to get extensively metabolized into its sulfated and glucuronidated metabolites and has very low circulating RES concentrations in plasma. Although the concentrations of conjugated metabolites of RES have been reported to be much greater than that of RES, they have not been evaluated. This also becomes important in light of positive biological activities reported for sulfated metabolites of RES. Conjugation is a complex process which can sometimes be a reversible process and needs comprehensive evaluation to better understand RES and its metabolites' disposition. There has been a debate among the researchers regarding the differences in kinetics of preformed versus in vivo formed metabolites in the light of guidelines issued by regulatory bodies regarding metabolites in safety testing (MIST). We have addressed the above questions in this work, in addition to evaluating brain permeability of a potent RES analog, trimethoxy-trans-stilbene (TMS). Chapter 1 presents a detailed introduction, hypothesis and significance of my work. Chapter 2 includes the development and validation of a bioanalytical method for quantitation of RES and its metabolites on LC/MS/MS. We were able to develop and validate a robust bioanalytical method to quantitate RES and its four major metabolites resveratrol-4'-glucuronide (R4'G), resveratrol-3-glucuronide (R3G), resveratrol-4'-sulfate (R4'S) and resveratrol-3-sulfate (R3S). In Chapter 3, lung as a possible metabolizing organ for RES was evaluated. This study was performed in vivo in mouse model using multiple site of administration and single site of sampling approach. In vitro studies were also performed to confirm the in vivo results. Inter species differences in RES pulmonary metabolism were also studied. We observed lungs to be the major metabolizing organs for RES with inter species differences in its metabolism. Chapter 4 provides detailed pharmacokinetics of sulfated metabolites of RES, i.e. resveratrol-3-sulfate (R3S) and resveratrol-4'-sulfate (R4'S) in mouse model by both systemic and oral routes. In vitro studies were also conducted to test the desulfation in liver. Although we did not observe any significant RES in plasma, we observed from our in vitro studies that sulfated metabolites were desulfated in liver. Chapter 5 explains the detailed pharmacokinetics of glucuronidated metabolites of RES i.e. resveratrol-3-glucuornide (R3G) after both systemic and oral route. R3G was observed to undergo enterohepatic circulation. Explanation of R3G disposition in hepatocytes and enterocytes were proposed based on our own and reported results. In Chapter 6 we compared the differences in the kinetics of preformed versus in vivo formed metabolites using modeling and simulation approach. Individual models for disposition of RES, R3S and R3G were developed. These models were combined to give a global model for RES metabolism into R3S and R3G. Simulations were performed under two assumptions; preformed versus in vivo formed metabolite kinetics a) are same and b) they are not same. Our results reported that preformed and in vivo formed metabolite kinetics are not same at least for hydrophilic phase II metabolites. Chapter 7 includes method development and validation for quantitation of TMS in plasma and brain of mouse. Chapter 8 includes a steady state study to characterize the pharmacokinetic parameters of TMS, which was used to evaluate brain permeability of TMS. In summary we developed a robust bioanalytical method for direct quantitation of RES and its metabolites, found the lung to be a major metabolizing organ for RES, delineated complex kinetics of conjugated metabolites of RES, and showed differences in preformed versus in vivo formed metabolite kinetics and better brain permeability of TMS.
Temple University--Theses

A busca pelo homem por uma vida saudável tem impulsionado pesquisas por novas substâncias capazes de atender tal desejo. O composto fenólico resveratrol (3, 4\', 5- trihidroxiestilbeno) é uma dessas substâncias que apresenta uma variedade de ações farmacológicas, como potencial antioxidante, capacidade antiinflamatória, proteção contra doenças cardíacas e câncer. Apesar dos inúmeros estudos sobre os benefícios do resveratrol à saúde, há poucos dados na literatura sobre sua toxicidade em organismos aquáticos, e principalmente sua concentração no ambiente, tornando o presente estudo fundamental para a contribuição de informações sobre a ecotoxicidade do resveratrol no ambiente aquático. O presente estudo avaliou a toxicidade do resveratrol em embriões e larvas de Danio rerio (zebrafish). Para isso foi realizado o ensaio in vitro de citotoxicidade do resveratrol, ensaios de ecotoxicidade e ensaio de biomarcadores enzimáticos. A avaliação do resveratrol por cromatografia líquida de alta pressão (HPLC) também foi realizada. De acordo com os resultados obtidos, o índice de citotoxicidade (IC50), concentração do resveratrol que causou a morte de 50% das células da linhagem NCTC-L929 foi de 39 mg L-1. A concentração de resveratrol que causa mortalidade em 50% dos organismos expostos (CL50), nos ensaios de ecotoxicidade crônica de curta duração com larvas do peixe Danio rerio foi de 51,37 mg L-1. A CL50 obtida no ensaio de ecotoxicidade aguda no estágio embriolarval do peixe Danio rerio com 96 h de duração foi de 75,33 mg L-1 e a CL50 obtida no ensaio de ecotoxicidade aguda no estágio embriolarval do peixe Danio rerio com 168 h de duração foi de 50,87 mg L-1. Nas concentrações mais elevadas de resveratrol foram observadas deformidades em embriões e larvas. O resveratrol alterou as atividades das enzimas LDH e ChE no estágio embriolarval de Danio rerio. Na análise do resveratrol por HPLC não foi observado degradação do composto.
The concern about human being healthy life has driven researchers to study new compounds capable of reaching that desire. Resveratrol (3, 4\', 5 trihydroxystilbene) a phenolic compound, is one of these substances which presents a variety of pharmacological actions, as antioxidant potential, antiinflammatory capacity, protection against heart and cancer diseases. Despite the numerous studies on the benefits of resveratrol to health, there is little evidence in the literature of its toxicity to aquatic organisms, and especially its concentration in the environment, making the present study fundamental for the contribution of information on the ecotoxicity of resveratrol in the aquatic environment. The present study evaluated the toxicity of resveratrol in embryos and larvae of Danio rerio (zebrafish). For this purpose the in vitro cytotoxicity of resveratrol assay, ecotoxicity assays and enzyme biomarker assay were performed. The evaluation of resveratrol by high pressure liquid chromatography (HPLC) was also performed. According to the results, the cytotoxicity index (IC50), concentration of resveratrol that caused the death of 50% of the cells of the NCTC-L929 lineage was 39 mg L-1. The concentration of resveratrol that causes mortality in 50% of exposed organisms (LC50) in the short-lived chronic ecotoxicity assays with larvae of the Danio rerio fish was 51.37 mg L-1. The LC50 obtained in the embryo-active acute ecotoxicity test of the Danio rerio fish with 96 h duration was 75.33 mg L-1 and the LC50 obtained in the embryo-active acute ecotoxicity assay of the Danio rerio fish with 168 h duration was 50.87 mg L-1. At higher concentrations of resveratrol deformities were observed in embryos and larvae. Resveratrol altered the activities of LDH and ChE enzymes in the embryonal stage of Danio rerio. No degradation of resveratrol was observed in the HPLC analysis of compound.

Resveratrol (RESV), um polifenol presente em diversos compostos naturais, incluindo o vinho, tem sido associado a um efeito quimiopreventivo em diversos tipos de câncer, inclusive no câncer colorretal (CCR). Esse efeito benéfico observado em diversos estudos vem sendo atribuído, sobretudo, à sua ação anti-oxidante; muito embora o RESV também esteja envolvido em diversos outros mecanismos celulares, incluindo a apoptose. Não obstante, uma análise mais acurada sobre a potencial ação antineoplásica do RESV na promoção do CCR ainda se faz necessária. O CCR é um dos tipos de câncer mais frequentes no mundo ocidental, cuja incidência vem aumentando nos países em desenvolvimento e apesar dos esforços dos cientistas e do surgimento de novas drogas, esse tipo de câncer ainda possui altas taxas de morbidade e mortalidade. O presente estudo avaliou a potencial ação anti-neoplásica do RESV, puro e associado ao etanol (EtOH), na carcinogênese do câncer de cólon induzido por Nmetil-N\'-nitro-N-nitrosoguanidina (MNNG) em ratos através da análise de estresse oxidativo com malondialdeído (MDA) e imunoexpressão de Caspase-3 (Casp-3), gamma-H2AX (H2AX), iPCNA (PCNA) e Caveolina-1 (CAV-1). O experimento foi realizado com 48 ratos wistar, os quais foram submetidos à indução da carcinogênese (0.5 ml de solução de MNNG (5 mg/ml) e tratados com placebo, RESV (1mg/kg/dia) e RESV (1mg/kg/dia) associado com EtOH (0,377g/kg/dia). Os ratos foram subdivididos em 6 grupos: controle (G1), MNNG (G2), RESV (G3), RESV+MNNG (G4), RESV + EtOH (G5) e RESV + EtOH + MNNG (G6). Como resultado, o RESV diminuiu a produção de MDA em todos os grupos tratados, evidenciando seu efeito antioxidante sistêmico. RESV aumentou o número de corpúsculos apoptóticos e da apoptose nas criptas displásicas, levando ao aparecimento de espaços criptais acelulares (ECA) e, consequentemente, a uma redução estatisticamente significativa no número de criptas displásicas nos grupos tratados. Através da diminuição da expressão de H2AX, PCNA e CAV1, observamos que o RESV apresentou um efeito protetor no desenvolvimento de lesões pré-neoplásicas na carcinogênese induzida por MNNG. Atribuímos esse efeito à sua ação pró-apoptótica que demonstrou ser um mecanismo efetivo na carcinogênese colônica em ratos, diminuindo o número de criptas displásicas e levando a formação de ECA\'s, um fenômeno ainda não descrito na literatura. Diferente da hipótese inicial, o EtOH mitigou os efeitos pró-apoptóticos do RESV, sem, contudo, diminuir sua ação antioxidante. Assim, concluímos que a ação anti-neoplásica e quimiopreventiva do RESV é efetiva na fase de promoção da carcinogênese colônica induzida por MNNG e que a associação entre RESV + EtOh, a mesma encontrada nos vinhos, não demonstrou ser tão efetiva quanto ao RESV isolado
Resveratrol (RESV), a polyphenol present in several natural compounds, including wine, has been associated with a chemopreventive effect in different types of cancer, including colorectal cancer (CRC). This beneficial effect observed in several studies has been attributed especially due to its anti-oxidant action; more than that, RESV also is involved in several other cellular mechanisms, including apoptosis. Despite of it, a more accurate analysis of a potential anti-neoplastic action of RESV in the promotion phase of CRC is still ongoing. CRC is one of the most frequent types of cancer in the Western world, with incidence increasing in developing countries and despite the efforts of scientists and the development of new drugs, this type of cancer still has high rates of morbidity and mortality. The present study evaluated a potential anti-neoplastic action of RESV, pure and associated with ethanol, on the carcinogenesis of colorectal cancer induced by Nmethyl-N\'-nitro-N-nitrosoguanidine (MNNG) in rats through the analysis of oxidative stress with malondialdehyde (MDA) and immunoexpression of Caspase-3 (Casp-3), gamma-H2AX (H2AX), iPCNA (PCNA) and Caveolina-1 (CAV-1). Our experiments were performed with 48 wistar rats, which were subjected to carcinogenesis (0.5 ml of MNNG solution (5 mg / ml) and treated with placebo, RESV (1 mg / kg / day) and RESV (1 mg / kg / (G1), RESV + EtOH (G5), RESV + MNNG (G4), RESV + EtOH (G5) and RESV + EtOH + MNNG (G6). As a result, RESV decreases the production of MDA in all treated groups, evidencing its systemic antioxidant effect. RESV increased the number of apoptotic corpuscles and apoptosis in dysplastic crypts, leading to the appearance of acellular cryptic spaces (ACE) and, consequently, to a statistically significant reduction in the number of dysplastic crypts in the treated groups. By decreasing expression of H2AX, PCNA and CAV1, we observed that RESV had a protective effect on the development of pre-neoplastic lesions in MNNG-induced carcinogenesis. We attributed this effect to its pro-apoptotic action, which has been shown to be an effective mechanism in colonic carcinogenesis in rats, decreasing the number of dysplastic crypts and leading to the formation of ACE\'s, a phenomenon not yet described in the literature. Unlike the initial hypothesis, EtOH mitigated the pro-apoptotic effects of RESV, but did not diminish its antioxidant action. Thus, we conclude that the anti-neoplastic and chemopreventive action of RESV is effective in the promotion phase of MNNG-induced colonic carcinogenesis and that the association between RESV + EtOh, the same as that found in wines, has not been shown to be as effective as isolated RESV.

El resveratrol és un component del vi, el qual té uns potencials efectes beneficiosos en malalties relacionades amb l'envelliment com poden ser el càncer, la malaltia cardiovascular, la diabetis i les malalties neurodegeneratives, augmentant l'esperança de vida d'organismes simples i mamífers actuant com a mimetic de la restricció calòrica. Els biomarcadors nutricionals poden ser una millor mesura de l'exposició dietètica que les dades estimades mitjançant enquestes alimentàries, perquè tenen tres grans avantatges envers les enquestes : a) els biomarcadors nutricionals són més precisos; b) les dades dietètiques són normalment més inadequades degut a les limitacions en les dades de composició dels aliments; c) l'anàlisi del biomarcador proporciona una mesura més pròxima del nutrient específic que les dades dietètiques perquè és una mesura integrada del metabolisme del component. Un biomarcador ideal ha de ser quantitativament robust, específic, sensible als canvis en la ingesta del component dietètic, i un període de vida mig adequat.
La hipòtesis d'aquesta tesi doctoral ha estat la de determinar els metabòlits urinaris del resveratrol (URM) com a biomarcadors útils de la consumició del vi.
Es van realitzar dos estudis clínics aleatoritzats, creuats, prospectius. En el primer estudi 10 homes sans van consumir 30g d'etanol/dia en forma de cava o ginebra durant 28 dies. En el segon assaig, 10 dones sanes van consumir 20g d'etanol/dia en forma de vi blanc o negre durant 28 dies. A més a més es van analitzar 1000 participants free-living en un estudi per avaluar efectes d'una dieta mediterrània en la prevenció primària de la malaltia cardiovascular (estudi de PREDIMED). Les anàlisis de URM foren realitzades mitjançant LC-MS/MS després d'una extracció en fase sòlida. Es van utilitzar els valors predictius i la corba ROC per determinar l'exactitud de la prova diagnòstica.
S'utilitza una metodologia robusta LC-MS/MS per determinar el URM en humans. La dosis diària de consumició de vi va ser correlacionada positivament amb els URM (r = 0.895, p< 0.001). En l'anàlisi estadístic mitjançant corba ROC es van obtenir els valors òptims de sensibilitat, la especificitat i els valors predictius.
També es van estimar ingesta dietètica del resveratrol i del piceid (R&P) en la població adulta espanyola. Per aquest propòsit es va compilar una completa base de dades de composició d'aliments de R&P en aliments d'origen espanyol. L'estudi on es va aplicar va ser la cohort espanyola de l'estudi European Prospective Investigation into Cancer and nutrition (EPIC), on participen 40685 voluntaris. La mediana estimada i la mitjana de R&P van ser 100 i 933 microgr./d respectivament. La font més important de R&P van ser els vins (98.4%).
Per tant, els URM se poden utilitzar com a una mesura objectiva i útil del consum de vi en estudios clínics i garns estudis epidemiològics free-living.
Resveratrol, a constituent of wine, has been shown to have beneficial effects on diseases of ageing including cancer, cardiovascular disease, diabetes and neurodegeneration, as well as increase stress resistance and extent the lifespan of organisms and mammals as caloric restriction mimetic. Nutritional biomarkers may be a better measure of dietary exposure than self-reported dietary data, because they have three distinct advantages over dietary data: a) biochemical markers of some nutrient intake are more precise than dietary assessment; b) dietary data are often inadequate because of limitation in food composition data; c) biomarker analysis provides a more proximal measure of specific nutrient intake than dietary data because it is useful as an integrated measure of the metabolism of the component. An ideal biomarker should be quantitatively robust, specific, sensitive to changes in intake of the dietary component, and have an adequate half-life.
The hypothesis of this thesis was to assess urinary resveratrol metabolites (URM) as useful biomarkers of wine consumption.
We performed two randomized, crossover trials and a cohort study. In the first study 10 healthy men consumed 30 g ethanol/day as sparkling wine or gin for 28 days. In the second trial, 10 healthy women consumed 20 g ethanol/day as white or red wine for 28 days. Furthermore 1000 free living participants in a study on the effects of a Mediterranean diet on primary prevention of cardiovascular disease (PREDIMED study) were also evaluated. Analyses of URM were carried out by LC-MS/MS after a solid phase extraction. We used predictive values and ROC curve analyses to assess the diagnostic accuracy.
A robust methodology using LC-MS/MS analysis was applied to determinate the URM in humans. The reported daily dose of wine consumption was correlated positively with URM (r = 0.895, p<0.001). With the ROC curve analysis, optimal values of sensitivity, specifity and predictive values were obtained.
We also estimated the dietary intake of resveratrol and piceid (R&P) in the Spanish adult population. For this purpose, a food composition data base (FCDB) of R&P in Spanish foods was compiled. The study included 40685 subjects a who were included in the European Prospective Investigation into Cancer and nutrition (EPIC)-Spain cohort. The estimated median and mean of R&P intake were 100 and 933 microgr./d respectively. The most important source of R&P were wines (98·4%).
URM can be used as an objective an useful measure of wine consumption in clinical and large free-living epidemiologic studies.

Dietary restriction (DR) increases lifespan robustly in diverse species. Effects of the dietary polyphenol resveratrol consistent with delayed ageing and/or extension of lifespan have been reported. The involvement in the longevity response to DR of the protein Sirt1, which may be activated by resveratrol and deacetylates a range of cellular substrates that includes histone proteins, identifies epigenetic processes as a pathway that may mediate effects of both DR and dietary resveratrol in delaying ageing and/or extending lifespan. Based on a preliminary observation, the hypothesis underlying the study is that some of the beneficial effects of resveratrol on lifespan/aging are mediated through effects on histone expression that oppose changes observed in ageing. A secondary hypothesis, based on a degree of structural similarity between resveratrol and 17β-oestradiol, was that epigenetic effects of resveratrol are mediated through the estrogen receptor (ER). The effect of resveratrol on histone protein expression was investigated in human intestinal Caco-2 cells and human MCF-7 breast cancer cells. Histone H2a, H2b, H3 and H4 expression was decreased in response to resveratrol treatment in both cell lines. In support of our hypothesis that resveratrol affects ageing through reversing ageing-associated changes in histone proteins, higher levels of H2A, H2B, and H4 expression were detected by western blotting in the small intestine of old (38 months) mice than in younger (12 months) mice. To investigate possible consequences of effects of resveratrol, including effects resulting from altered histone expression, we studied the effect of resveratrol on global gene expression in Caco-2 and MCF-7 cells to address several objectives including: (1) investigating if resveratrol has an effect similar to that of DR at the level of gene expression; (2) identifying if genes or pathways affected by resveratrol were also affected by manipulation of the expression level of Sirt1. For both cell types, the number of genes in the intersection between those affected by resveratrol and a compiled list of genes reported in other studies to respond to DR was greater than expected by chance, supporting the view that responses to resveratrol and to dietary restriction have some commonality and that resveratrol may mimic some effects of dietary restriction. We also found that there was very little overlap between genes affected by resveratrol treatment and by knockdown of Sirt1 expression in Caco-2 cells, which adds to accumulating evidence that resveratrol does not act through effects on Sirt1. To investigate if effects of resveratrol - in particular the reduction in histone protein expression - are mediated through the estrogen receptor (ER), Caco-2 and MCF-7 cells were treated with resveratrol in the presence or absence of the ER antagonist fulvestrant, then total cell lysate was analysed by western blotting. The reduction in histone protein (H2a, H2b, H3 and H4) expression was attenuated by fulvestrant, indicating that resveratrol reduced histone expression via an ER-dependent mechanism. For further investigation of effects of resveratrol on histone expression, Caco-2 cells were transfected with a promoter reporter construct comprising the histone H3 promoter upstream of the β- galactosidase reporter gene, and the effect on reporter gene expression of treatment with resveratrol in the presence and absence of the fulvestrant was measured. Resveratrol reduced reporter gene expression and this effect was attenuated by fulvestrant, demonstrating that resveratrol acts to reduce histone H3 expression at the level of transcription through an ER-mediated mechanism. To investigate if the response to resveratrol treatment is through interaction with estrogen response elements (EREs) in the histone H3 promoter we replaced three potential EREs within the histone H3 promoter region included in the promoter-reporter construct with random sequence. Caco-2 cells were then transfected with either original or mutated promoter-reporter construct and treated with resveratrol or the endogenous ER ligand 17-β estradiol in the presence and absence of fulvestrant. Resveratrol and 17-β estradiol both reduced reporter gene expression from both promoter reporter constructs and in all cases responses were attenuated by fulvestrant, indicating that effects of neither compound, although mediated through the ER, are on the specific sequences region we identified and replaced. In conclusion, these data indicate that resveratrol reduces histone expression in both intestinal and breast cancer cells through an ER-mediated mechanism acting at the level of transcription and that this effect may oppose an accumulation of histone proteins (observed in mouse small intestine) that accompanies ageing. With respect to effects on gene expression, resveratrol was found to mimic some effects of dietary restriction but appeared to act through a mechanism independent of Sirt1.

Resveratrol is a natural polyphenol present in many plant species and derived foods including grapes and red wine. It has been shown to possess chemotherapeutic properties in animal cancer models as well as many biological effects in vitro. In this study, the effects of three resveratrol metabolites including resveratrol-3-0-D-glucuronide, resveratrol-4'-O-D-glucuronide and resveratrol-3-0-D-sulphate on the growth of colon cancer cell lines have been investigated. The growth inhibitory effects of resveratrol, piceatannol, pterostilbene and the glucuronide and sulphate metabolites on Caco-2, CCL-228 and HCT-116 cells were assessed using the neutral red and MTT assays. Resveratrol and its metabolites inhibited the growth of cells with ICso values in the range of 9.8-31 f.lM whilst piceatannol and pterostilbene in the range of 21.7-29.6f.lM and 5-34.5f.lM respectively. Apoptotic assessment by DAPI staining, Z-VAD-FMK pre-treatment and percentage of cells in sub-Gl by FACS all revealed the absence of apoptosis. Treatment of differentiated Caco-2 mono layers showed that resveratrol was capable of inhibiting the growth of cells following treatment on the apical but not basolateral membrane and the effects were less profound with the metabolites. Only high concentrations (500f.lM) of metabolites (not used in any ofthe growth studies) appeared to be toxic to normal cells as measured by a haemolysis assay. Resveratrol was capable of causing S phase arrest in all 3 cell lines at 30f.lM whilst the two glucuronides caused GO/GI arrest in Caco-2 and CCL-228 cells only. Resveratrol-3-0-D-sulphate had no effect on the cell cycle. Growth inhibition caused by resveratrol and its two glucuronides was reversed by the addition of an AMP kinase inhibitor (compound C) or an adenosine A3 receptor antagonist (MRS-119l). Treatment with the highly selective A3 receptor agonist, 2CI-m-MECA caused growth inhibition and the A3 receptor was 'detected in all 3 cell lines. Levels of eyelin D 1 as measured by western blot were significantly reduced at higher concentrations (lOOf.lM) but p-AMPK was not reliably increased in all cases. Resveratrol glucuronides were shown to inhibit the growth of three colon cancer cell lines by GO/G 1 arrest and depletion of eyelin D 1. These findings strongly suggest a role of the adenosine A3 receptor in the observed inhibition therefore, providing a novel target for resveratrol and its metabolites.

Sarcopenia, the loss of muscle mass and function as we age, affects all individuals from approximately the 4th decade of life and results in a poor quality of life. The mechanisms responsible for sarcopenia are unclear and it is likely that it is a multifactorial disease. However, it is hypothesised that an increase in systemic and/or muscle pro-inflammatory cytokine levels play a major role. Interferon induced protein 10 (IP10) is a chemokine that has been shown to be increased in serum as we age. Polyphenols are extracts from plants and have been shown to have anti-inflammatory effects with benefits in multiple diseases. Therefore, the main aim of this thesis was to establish an in vitro model to study the effect of increased levels of IP10 on muscle atrophy and inflammation and to examine whether resveratrol treatment was able to protect against IP10 induced effects on skeletal muscle atrophy or inflammation. Primary myoblasts were isolated from rat muscles and treated with 0.1µM, 1µM and 10µM of resveratrol to identify a functional concentration. Treatment of cells with 1µM of resveratrol increased myoblast growth, increased myotube diameter and decreased production of hydrogen peroxide of the cell media. Furthermore, treatment of myoblasts with 1µM resveratrol led to increases in the protein content of known resveratrol targets; MnSOD and catalase and caused transient increases in Sirt1 protein content. Treatment of cells with 1µM resveratrol also increased MnSOD and catalase content of myotubes and resulted in a decrease in the content of Sirt1, coupled with an increase in the acetylation status of proteins compared with untreated myotubes. As these data suggested that 1µM of resveratrol was functional in myotubes, this concentration was used to pre-treat myotubes before IP10 treatment. Treatment of myotubes with IP10 at levels found in older people (200pg/ml) led to decreases in myotube diameter and increases in the atrophy marker, Atrogin1. Pre-treatment of myotubes with resveratrol provided protection against both of these effects. Treatment of myotubes with a concentration of IP10 found in the young (150pg/ml) had no effect on markers of atrophy. Both concentrations of IP10 were found to have similar effects on cytokine release by myotubes and resveratrol treatment had only marginal effects on this. The second aim of this study was to identify an effective concentration of resveratrol in vivo and examine the effect of this on skeletal muscle force generation in adult and old mice. Treatment of mice with 125mg resveratrol/kg/day resulted in an increase in MnSOD and Sirt1 contents of skeletal muscle from young mice but had no effect on skeletal muscle force generation by EDL muscles of either adult or old mice. Overall data suggest that the increase in IP10 levels seen in ageing contributes to sarcopenia; however it is unlikely this acts through changes to the cytokine profile of muscle. Resveratrol prevented some atrophic effects induced by IP10, suggesting that, the mechanism through which resveratrol may protect against sarcopenia may be through the prevention of increases in atrophic pathways.

Mestrado em Biologia Aplicada
O cancro é uma doença com elevada incidência em todo o mundo. O processo de carcinogénese é bastante complexo e envolve uma série de transformações que culminam no desenvolvimento tumoral. É por isso necessário encontrar novas terapias que sejam menos agressivas ou que aumentem a eficiência da quimioterapia no combate ao cancro. Os polifenóis, nomeadamente o resveratrol têm sido apontados como compostos com efeitos quimioprotetores e citotóxicos em vários tipos de cancro, prevenindo e atrasando o desenvolvimento tumoral nos vários estágios do processo carcinogénico. Apesar de ainda não serem totalmente conhecidos os mecanismos pelos quais este composto atua nas células, a sua atividade parece estimular a atividade de sirtuinas. Uma vez que estas desacetilases desempenham um papel fundamental na regulação de vários mecanismos na resposta ao stresse, parecem estar relacionadas com a regulação do desenvolvimento tumoral. O objetivo deste trabalho foi determinar se a diferente ação citotóxica do resveratrol em várias linhas celulares tumorais está relacionada com uma diferente quantidade basal e posterior alteração do conteúdo de sirtuina 1 e sirtuina 3. Os resultados obtidos através do ensaio de avaliação da proliferação celular mostraram que uma concentração de 50 μM de resveratrol provocou uma diminuição do número de células em todas as linhas celulares. Através de microscopia de epifluorescência foi possível observar que este composto provoca alterações morfológicas celulares e na rede mitocondrial de linhas celulares tumorais, sugerindo a morte celular por apoptose. A análise do conteúdo proteico em sirtuina 1 e sirtuina 3 nas células após tratamento com resveratrol mostrou que de uma forma geral a quantidade de sirtuina 1 é aumentada em todas as linhas celulares, embora sem significância estatística. E que apesar de também sem significância estatística, o resveratrol parece aumentar a quantidade da sirtuina 3 em algumas das linhas estudadas, nomeadamente nas MCF-7. Desta forma, sugere-se que existe alguma correlação entre a expressão das sirtuinas e o efeito do resveratrol na inibição do crescimento de linhas celulares tumorais. Se trabalhos futuros completarem e confirmarem os resultados aqui obtidos, o resveratrol poderá assim ser considerado um bom aliado no desenvolvimento de novas terapêuticas no combate a vários tipos de cancro.
Cancer is a disease with high prevalence in the world. The process of carcinogenesis is complex and involves a series of transformations that culminate in tumor development. It is therefore necessary to find new therapies that are less aggressive or which increase the efficacy of chemotherapy in fighting cancer. Polyphenols, namely in particular resveratrol, have been identified as chemopreventive or cytotoxic agents against various types of cancer, preventing and delaying the tumor growth at various stages of the carcinogenic process. Although the mechanisms by which resveratrol acts on cells are not fully known, its activity seems to stimulate the activity of sirtuins. Since these deacetylases play a major role in the regulation of various mechanisms in response to stress, their activity may be related with the regulation of tumor development. The objective of this study was to determine whether different cytotoxic actions of resveratrol on several tumor cell lines is related to a different basal content and subsequent modulation of sirtuin 1 and sirtuin 3. The results obtained from cell proliferation assays showed that 50 μM of resveratrol resulted in a decrease in cell number in all cell lines tested. By means of epifluorescence microscopy it was observed that resveratrol leads to morphological changes in cellular and mitochondrial network of tumor cell lines, which is suggestive of apoptotic cell death. The semi-quantification of sirtuin 1 and sirtuin 3 protein content after resveratrol treatment showed that in general the amount of sirtuin 1 is increased in all cell lines, although not statistically significant. Although not statistically significant, resveratrol seems to increase the amount of sirtuin 3 in some of the cell lines studied, particularly in MCF-7. Although preliminary, the results suggest that there is some correlation between the expression of sirtuins and the effect of resveratrol on growth inhibition of tumor cell lines. If future results complement and confirm this data, resveratrol may thus be considered a good ally in the development of new therapeutics to combat various types of cancer.

The entire thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file; a non-technical public abstract appears in the public.pdf file. Title from PDF of title page (University of Missouri--Columbia, viewed on December 29, 2009). Thesis advisor: Dr. Ingolf Gruen. Includes bibliographical references.

The objective of this thesis was to help improve the safety of paracetamol for patients of all ages by adding evidence to the body of knowledge on the changing risks of paracetamol toxicity in ageing, frailty and non-acute exposures, and the potential for lifespan and healthspan increasing interventions to provide novel mechanisms of paracetamol hepatotoxicity protection. Furthermore, this thesis aimed to investigate the potential of both paracetamol, and lifespan and healthspan interventions in delaying or preventing frailty. Overall the work presented in this thesis used a series of mouse models to contribute to three main findings with clinical significance. Firstly, they demonstrated that there is no increase in susceptibility to paracetamol toxicity in old age or frailty, with either acute, chronic or sub-acute paracetamol exposure. Secondly, they showed that the currently used paracetamol hepatotoxicity therapy, NAC, does not protect against toxicity induced by sub-acute paracetamol exposure. Further studies showed that the mechanism of SIRT1 activation does not provide an alternative protective mechanism. Finally, this thesis found that the lifespan and healthspan increasing interventions of resveratrol and calorie restriction, but not therapeutic paracetamol, were able to delay frailty in aged mice.

Resveratrol, a naturally derived stilbene, is an interesting compound mostly talked about recently because for its anti-cancer properties. Unfortunately it has some shortcomings due to its low bioavailability and low solubility in water. For this reason, my research is to overcome resveratrol's drawbacks by improving its bioavailability and hydrophilicity. My research is focused on syntheses of novel derivatives of resveratrol such as 3, 5-di-O-isobutyroyl resveratrol and 3, 5-di-O-hexanoyl resveratrol using lipase catalyzed hydrolysis. Therefore, the tri-acylated resveratrols 3, 5, 4'-tri-O-isobutyroyl resveratrol and 3, 5,4'-tri-O-hexanoyl resveratrol were first synthesized. 3,5,4'-tri-O-isobutyroyl resveratrol and 3,5,4'-tri-O-hexanoyl resveratrol were then hydrolyzed using lipase (C. Antarctica) to obtain the products 3,5-di-O-isobutyroyl resveratrol and 3,5-di-O-hexanoyl resveratrol. The four compounds 3,5-di-O-isobutyroyl resveratrol, 3,5-di-O-hexanoyl resveratrol, 3,5,4'-tri-O-hexanoyl resveratrol, and 3,5-di-O-hexanoyl resveratrol were characterized by 1H NMR and 13C NMR.

A preservação do sêmen suíno refrigerado para fins de inseminação artificial é a forma mais utilizada no mundo. Para tal, os espermatozoides são diluídos em meios que forneçam substratos para nutrir e proteger estas células. Porém, a faixa de temperatura de armazenamento do sêmen suíno refrigerado não é capaz de cessar completamente os mecanismos metabólicos dos espermatozoides, que em ambiente aeróbico da dose inseminante, produzem e liberam continuamente produtos do metabolismo do oxigênio. A ação das espécies reativas de oxigênio sobre o espermatozoide é uma das razões do declínio na população de células espermaticas estrutural e funcionalmente aptas à fertilizarem os gametas femininos. Portanto, a adição de um composto antioxidante ao meio diluidor poderia melhorar ou manter constante o número de espermatozoides viáveis ao longo do período de conservação da dose inseminante. Neste contexto, este trabalho objetivou verificar se a adição do antioxidante resveratrol geraria modificações positivas em parâmetros celulares relacionados à qualidade do sêmen suíno refrigerado à 15-17°C avaliados por sistema computadorizado da motilidade espermática e citometria de fluxo (experimento in vitro), e se este antioxidante melhoraria os índices de fertilidade na inseminação artificial intrauterina (IAIU) através da recuperação, contagem e identificação dos embriões (experimento in vivo). As concentrações testadas no experimento in vitro não superaram os resultados do tratamento controle (p<0,05), sendo a concentração de 1,0 mM prejudicial ao espermatozoide suíno (p<0,05). A utilização da concentração de 0,01 mM de resveratrol para inseminação das fêmeas suínas no experimento in vivo não apresentou efeito positivo nos índices de taxa de prenhez e fertilidade ajustada total (p>0,05), além de ter resultado em uma baixa taxa de embriões viáveis (p<0,05), quando comparados ao tratamento controle. Deste modo, pode concluir que não é indicado a adição do antioxidante resveratrol ao meio diluidor para inseminação artificial, uma vez que compromete a qualidade das células espermáticas do reprodutor suíno e impacta negativamente na fertilidade das fêmeas.
The preservation of chilled boar semen for artificial insemination is the most performed worldwide. For this, the sperm are extended in medium that provides substrates to nourish and protect these cells. However, the storage temperature range of chilled semen is not able to completely stop the metabolic mechanisms of sperm, which keeps producting and releasing oxygen reactive products in aerobic environment of insemination doses. The action of reactive oxygen species on the sperm is one of the reasons for decreasing sperm population with structural and functional capacity to fertilize the female gamete. Therefore, the addition of antioxidant compound to the extender medium could improve or maintain the number of viable spermatozoa throughout the conservation time of insemination doses. In this context, this work aimed to determine whether addition of antioxidant resveratrol would generate positive changes in cell parameters related to the quality of boar semen cooled to 15-17 °C assessed by computered analysis of sperm motility and flow citometry (in vitro experiment) and whether this antioxidant would improve fertility rates using intrauterine insemination (IUI) by the recovery, counting and embryos identification (in vivo experiment). The concentrations tested in vitro experiment have not exceed control treatment results (p <0.05), with the concentration of 1.0 mM being detrimental to the boar spermatozoa (p <0.05). The concentration of 0.01 mM of resveratrol used for gilts insemination in experiment in vivo has not shown positive effect on pregnancy rate and the total adjusted fertility (p> 0.05) and have resulted in a low rate viable embryos (p <0.05) compared to control. Thus, we conclued that the addition of resveratrol antioxidant in boar extender medium is not suitable for artificial insemination, once it compromises the quality of boar sperm cells and impairs on female fertility.