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Abstract(s)
A produção de radicais livres é um processo comum e necessário dos organismos vivos, e como consequência disso, os mesmos podem reagir e causar danos oxidativos que são responsáveis pelo envelhecimento e doenças degenerativas. De forma a combater estes efeitos o organismo consegue desenvolver defesas antioxidantes, porém algumas atitudes e hábitos podem ajudar nestes mecanismos de defesa. Sendo assim, uma dieta rica em compostos fenólicos pode ser de suma importância devido às suas propriedades antioxidantes. As frutas, vegetais e seus derivados são as principais fontes destes compostos. Além disso, os polifenóis também influenciam a qualidade dos produtos, pois atuam no processo de oxidação, tendo um papel importante na cor e evolução da sensação gustativa (adstringência). Sendo assim a determinação destes compostos é uma tarefa importante a fim de avaliar a potencial aplicação destes produtos como fonte de antioxidantes. O presente trabalho descreve a monitorização de polifenóis em bebidas comercias disponíveis no mercado. A presença dos polifenóis traduz o potencial de inibição de processos de oxidação característico dessas bebidas. Esta medida foi efetuada por deteção ótica e permite estimar quantitativamente os teores de compostos fenólicos totais medidos pela atividade enzimática da Horseradish peroxidase (HRP) e uma enzima artificial de nanocubos de Azul de Prússia (NcAP). Foi realizado um estudo para avaliar a linearidade e os limites de deteção para ambos os ensaios. Os resultados foram calculados com base em antioxidantes de referência. Neste trabalho utilizaram-se o ácido gálico (AG) e ácido tânico (AT). Em termos gerais, os NcAP mostraram ser eficientes na atividade mimética da peroxidase na reação típica de H2O2 e 3,3',5,5'-tetrametilbenzidina (TMB). O mecanismo catalítico foi confirmado por análise colorimétrica. Para o desenvolvimento da metodologia foram realizadas otimizações de temperatura, concentração, pH e tempo de reação. Com a enzima natural verificou-se que a melhor condição de reação foi 0,4 mM de TMB, 4,0 mM de H2O2, 0,03 µg/mL de HRP, pH 4, temperatura ambiente e um tempo de reação de 2 minutos. Os compostos fenólicos foram detetados até níveis µM, com um LOD = 6,62 µM para ácido gálico (AG) e 0,41 µM para ácido tânico (AT). Os valores de R2 de 0,9856 e 0,9912 para deteção de AG e AT, respetivamente, sugerem uma boa linearidade. A mesma metodologia foi reproduzida com a enzima artificial, neste caso, NcAP. Foram otimizados parâmetros como o pH, temperatura e tempo de reação para um valor de concentração de NcAP de 0,01 µg/mL. Os compostos fenólicos foram também detetados em níveis µM com um LOD = 5,63 µM para AG e 1,51 µM para AT. Os valores de R2 de 0,986 e 0,977 para deteção de AG e AT, respetivamente, sugerem uma boa linearidade. Por fim, ambas as metodologias foram aplicadas na avaliação de compostos fenólicos em bebidas comerciais. Os resultados através da metodologia com HRP sugerem resultados próximos para os diferentes antioxidantes analisados, por outro lado, os resultados com NcAP apresentam algumas discrepâncias. No entanto, apesar dos resultados obtidos serem promissores, podemos concluir que a etapa de análise de amostras reais com a metodologia NcAP ainda carece de otimizações para seguimento em trabalhos futuros.
The production of free radicals is a common and necessary process of living organisms, and consequently, they can react and cause oxidative damage that is responsible for aging and degenerative diseases. To combat these effects, the body can develop antioxidant defenses, but some attitudes and habits can help in these defense mechanisms. Thus, a diet rich in phenolic compounds can be of paramount importance due to their antioxidant properties, fruits, vegetables, and their derivatives are the main sources of these compounds. In addition, polyphenols also influence the quality of the products, as they act in the oxidation process, playing an important role in the color and evolution of the taste (astringency). Therefore, the determination of these compounds is an important task to evaluate the potential application of these products as a source of antioxidants. The goal of this work was to develop a colorimetric enzymatic methodology for the determination of phenolic compounds present in commercial beverages and to reproduce this methodology with the use of an artificial enzyme that mimics the activity of peroxidase since the mimetic enzymes are of scientific and practical significance as they present greater stability and low cost. Here it was reported that Prussian blue nanocubes (NcAP) exhibited interesting peroxidase-like activity in the typical reaction of H2O2 and 3,3',5,5'-tetramethylbenzidine (TMB). For the development of the methodology, optimizations of temperature, concentration, pH, and reaction time were performed. With the natural enzyme, it was found that the best reaction condition was 0,4 mM of TMB, 4,0 mM of H2O2, 0,03 µg/mL of HRP, pH 4, room temperature, and a reaction time of 2 minutes. Phenolic compounds were detected up to µM levels, with a LOD = 6,62 µM for gallic acid (AG) and 0,41 µM for tannic acid (AT). R2 values of 0,9856 and 0,9912 for detection of AG and AT, respectively, suggest good linearity. The same methodology was reproduced with artificial enzyme, in this case, NcAP, where optimizations of the conditions were also performed, but the only change was in the concentration of the nanoenzyme, which in this case was 0,01 µg/mL. Phenolic compounds were also detected at µM levels with a LOD = 5,63 µM for AG and 1,51 µM for AT. R2 values of 0,9861 and 0,9769 for detection of AG and AT, respectively, suggest good linearity. Finally, both systems were used in the evaluation of phenolic compounds in commercial beverages. The results through the methodology with HRP suggest similar results for the different antioxidants analyzed, whereas the results with NcAP show discrepancies. Therefore, despite promising results, the analysis of real samples with the NcAP methodology still needs optimizations for follow-up in future works.
The production of free radicals is a common and necessary process of living organisms, and consequently, they can react and cause oxidative damage that is responsible for aging and degenerative diseases. To combat these effects, the body can develop antioxidant defenses, but some attitudes and habits can help in these defense mechanisms. Thus, a diet rich in phenolic compounds can be of paramount importance due to their antioxidant properties, fruits, vegetables, and their derivatives are the main sources of these compounds. In addition, polyphenols also influence the quality of the products, as they act in the oxidation process, playing an important role in the color and evolution of the taste (astringency). Therefore, the determination of these compounds is an important task to evaluate the potential application of these products as a source of antioxidants. The goal of this work was to develop a colorimetric enzymatic methodology for the determination of phenolic compounds present in commercial beverages and to reproduce this methodology with the use of an artificial enzyme that mimics the activity of peroxidase since the mimetic enzymes are of scientific and practical significance as they present greater stability and low cost. Here it was reported that Prussian blue nanocubes (NcAP) exhibited interesting peroxidase-like activity in the typical reaction of H2O2 and 3,3',5,5'-tetramethylbenzidine (TMB). For the development of the methodology, optimizations of temperature, concentration, pH, and reaction time were performed. With the natural enzyme, it was found that the best reaction condition was 0,4 mM of TMB, 4,0 mM of H2O2, 0,03 µg/mL of HRP, pH 4, room temperature, and a reaction time of 2 minutes. Phenolic compounds were detected up to µM levels, with a LOD = 6,62 µM for gallic acid (AG) and 0,41 µM for tannic acid (AT). R2 values of 0,9856 and 0,9912 for detection of AG and AT, respectively, suggest good linearity. The same methodology was reproduced with artificial enzyme, in this case, NcAP, where optimizations of the conditions were also performed, but the only change was in the concentration of the nanoenzyme, which in this case was 0,01 µg/mL. Phenolic compounds were also detected at µM levels with a LOD = 5,63 µM for AG and 1,51 µM for AT. R2 values of 0,9861 and 0,9769 for detection of AG and AT, respectively, suggest good linearity. Finally, both systems were used in the evaluation of phenolic compounds in commercial beverages. The results through the methodology with HRP suggest similar results for the different antioxidants analyzed, whereas the results with NcAP show discrepancies. Therefore, despite promising results, the analysis of real samples with the NcAP methodology still needs optimizations for follow-up in future works.
Description
Keywords
Deteção colorimétrica Compostos fenólicos Peroxidase Nanoenzimas Colorimetric detection Phenolic compounds Peroxidase Nanoenzymes