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Methoxylated flavones from Citrus sp peels as resistance modifying compounds
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A Tuberculose permanece uma das principais causas de morte por doença infeciosa, afetando uma em quarto pessoas globalmente. Mais recentemente, a disseminação de novas formas multirresistentes de Mycobacterium tuberculosis mostrou poder tornar esta doença numa das piores epidemias do nosso tempo, sendo as opções terapêuticas diminutas e ineficientes. Esta resistência, intrínseca ou adquirida, pode envolver bombas de efluxo de antibióticos, mutações genéticas que evitam a atividade antibiótica ou mudanças na parede celular. Este trabalho teve como foco a atividade inibitória de bombas de efluxo por certas polimetoxiflavonas , nomeadamente a 3,5,6,7,8,3′,4′-heptametoxiflavona, extraída da casca de Citrus reticulata Blanco. As cascas de C. reticulata possuem uma das maiores concentrações de polimetoxiflavonas do género Citrus. Usando o aparelho extrator de Soxhlet e diclorometano como solvente, obtiveram-se 6.1g de extrato a partir de 293.9g de casca, originando um total de 0.0137g de 3,5,6,7,8,3′,4′-heptametoxiflavona pura. Depois de purificada através de HPLC semi-prep, em combinação com técnicas de cromatografia em coluna, a estrutura da heptametoxiflavona foi confirmada por NMR. Foram determinados os valores da Concentração Minima Inibitória (MIC) da 3,5,6,7,8,3′,4′-heptametoxiflavona pura, do extrato de diclorometano de casca de C. reticulata e das frações 3 e 6 da primeira coluna cromatográfica realizada, usando-se Mycobacterium smegmatis como modelo para Mycobacterium tuberculosis. Realizaram-se ainda ensaios de modulação com rifampicina e brometo de etídio com a mesma cultura de M. smegmatis. Três das quatro amostras testadas mostraram atividade moduladora significativa, reduzindo o valor de MIC da rifampicina 4 a 8 vezes. O modulador mais potente foi a fração 3, incrementando a suscetibilidade da cultura de M. smegmatis 8 vezes em relação ao brometo de etídio (MF=8). A 3,5,6,7,8,3′,4′-heptametoxiflavona pura foi a segunda amostra mais potente, com um aumento de suscetibilidade de M. smegmatis em relação ao brometo de etídio mais baixo do que em relação à rifampicina (MF= 2 e MF=8, respetivamente). Concluímos que esta polimetoxiflavona representa um potencial muito interessante como ferramenta de otimização da terapêutica anti- tuberculótica com rifampicina.
Tuberculosis remains one of the main causes of death due to an infectious disease, affecting one in four people globally. More recently, the spreading of multi-drug resistant Mycobacterium tuberculosis strains has threatened to become one of the worst epidemics of our time, with therapeutic options getting slimmer and less effective. This resistance, acquired or intrinsic, acts through activation of efflux pumps that expel antibiotics, genetic mutations to avoid their activity or changes in cell wall. This research focused on the efflux pump inhibitory activity of some polimethoxyflavones, mainly 3,5,6,7,8,3′,4′-heptamethoxyflavone, extracted from Citrus reticulata Blanco peel. C. reticulata peels have shown to possess one of the highest counts of olimethoxyflavones of the genus Citrus. 6.1g of dichloromethane extract of mandarin orange peel, obtained by Soxhlet apparatus from 293.9g of peel, yielded 0.0137g of pure 3,5,6,7,8,3′,4′-heptamethoxyflavone. 3,5,6,7,8,3′,4′-heptamethoxyflavone was purified successfully through semi-preparative HPLC combined with column-chromatography techniques. The structure was confirmed through NMR. Minimum inhibitory concentration (MIC) values of the pure 3,5,6,7,8,3′,4′- heptamethoxyflavone, dichloromethane extract and fractions 3 and 6 from the chromatographic fractionation process were determined using Mycobacterium smegmatis as a model of Mycobacterium tuberculosis. Modulation assays with rifampicin and etydium bromide were performed using the same M. smegmatis culture. Three of the four samples tested had significant modulating activities by reducing the MIC value of rifampicin (MFRIF) 4 to 8-fold. The most potent modulator was the fraction 3, as it increased the susceptibility of M. smegmatis towards EtBr and RIF remarkably (MF = 8). Pure 3,5,6,7,8,3′,4′-heptamethoxyflavone was the second most potent sample tested, with an increased susceptibility of M. smegmatis to EtBr lower than that observed towards RIF (MF=2 an MFRIF=8, respectively), showing a very interesting potential to optimize the therapeutic activity of one of the main drugs used in Tuberculosis treatment.
Tuberculosis remains one of the main causes of death due to an infectious disease, affecting one in four people globally. More recently, the spreading of multi-drug resistant Mycobacterium tuberculosis strains has threatened to become one of the worst epidemics of our time, with therapeutic options getting slimmer and less effective. This resistance, acquired or intrinsic, acts through activation of efflux pumps that expel antibiotics, genetic mutations to avoid their activity or changes in cell wall. This research focused on the efflux pump inhibitory activity of some polimethoxyflavones, mainly 3,5,6,7,8,3′,4′-heptamethoxyflavone, extracted from Citrus reticulata Blanco peel. C. reticulata peels have shown to possess one of the highest counts of olimethoxyflavones of the genus Citrus. 6.1g of dichloromethane extract of mandarin orange peel, obtained by Soxhlet apparatus from 293.9g of peel, yielded 0.0137g of pure 3,5,6,7,8,3′,4′-heptamethoxyflavone. 3,5,6,7,8,3′,4′-heptamethoxyflavone was purified successfully through semi-preparative HPLC combined with column-chromatography techniques. The structure was confirmed through NMR. Minimum inhibitory concentration (MIC) values of the pure 3,5,6,7,8,3′,4′- heptamethoxyflavone, dichloromethane extract and fractions 3 and 6 from the chromatographic fractionation process were determined using Mycobacterium smegmatis as a model of Mycobacterium tuberculosis. Modulation assays with rifampicin and etydium bromide were performed using the same M. smegmatis culture. Three of the four samples tested had significant modulating activities by reducing the MIC value of rifampicin (MFRIF) 4 to 8-fold. The most potent modulator was the fraction 3, as it increased the susceptibility of M. smegmatis towards EtBr and RIF remarkably (MF = 8). Pure 3,5,6,7,8,3′,4′-heptamethoxyflavone was the second most potent sample tested, with an increased susceptibility of M. smegmatis to EtBr lower than that observed towards RIF (MF=2 an MFRIF=8, respectively), showing a very interesting potential to optimize the therapeutic activity of one of the main drugs used in Tuberculosis treatment.
Descrição
Trabalho Final de Mestrado Integrado, Ciências Farmacêuticas, Universidade de Lisboa, Faculdade de Farmácia, 2019
Palavras-chave
Citrus reticulata Blanco Polimethoxyflavones Efflux pump inhibitors Tuberculosis Mestrado Integrado - 2019