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Abstract(s)
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.
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.
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Keywords
Resveratrol Resveratrol - Vinho tinto Resveratrol - Propriedades antimicrobianas Urease Helicobacter pylori
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Publisher
Universidade da Beira Interior