Caracterização fitoquímica, avaliação dos efeitos sinérgicos e antibiofilmes dos extratos de Rosmarinus officinalis L. e Plinia cauliflora sobre microrganismos envolvidos na infecção endodôntica secundária
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2024-02-23
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Universidade Estadual Paulista (Unesp)
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Com o aumento de microrganismos resistentes nos canais radiculares, os estudos de extratos de plantas com ação antibacteriana têm aumentado visando terapias alternativas. O objetivo deste estudo foi avaliar os efeitos sinérgicos sobre Enterococcus faecalis, Enterococcus faecium, Candida albicans, Fusobacterium nucleatum e Porphyromonas gingivalis e a ação antibiofilme dos extratos hidroetanólicos de Rosmarinus officinalis L. (alecrim) e Plinia cauliflora (jabuticaba). Foram determinadas a Concentração Inibitória Mínima (CIM) e Concentração Microbicida Mínima (CMM) por microdiluição em caldo e Índice de Concentração Inibitória Fracionada e Índice de Concentração Microbicida Fracionada por microdiluição checkerboard. Posteriormente, a ação da concentração efetiva dos extratos foi avaliada sobre biofilmes formados em poços de placa de microtitulação. Os biofilmes foram expostos a dois diferentes períodos de tratamento com os extratos, por 5 min e 24 h, sendo n=10 para cada grupo experimental. Como controles foram utilizados solução salina 0,9% (C+) e hipoclorito de sódio 2,5% (C-), totalizando 5 grupos para cada microrganismo e cada tempo experimental. Em seguida, os biofilmes foram submetidos ao teste de MTT. Foram realizadas análises da atividade citotóxica dos extratos sobre as linhagens de queratinócitos humanos (HaCat) pelo teste de MTT. A análise estatística foi feita aplicando método ANOVA e teste de Dunn (significância de 5%). Ambos os extratos demonstraram fitocompostos fenólicos e flavonóides e possuem ação antioxidante. Os extratos apresentam ação antimicrobiana para as cepas estudadas e quando foram associados, apresentaram concentrações aditivas para cepa de E. faecalis. Em relação a propriedade antibiofilme, o extrato de alecrim apresentou boa redução da viabilidade microbiana de E. faecalis, com redução de 52% para o tempo de 5 min e de 40% para 24 h. Para as cepas de microrganismos anaeróbios, para a cepa de F. nucleatum, o extrato de jabuticaba reduziu em até 73% o biofilme. Para a cepa de P. gingivalis houve redução de até 41% para o extrato de jabuticaba e de até 57% para o extrato de alecrim. Quanto a citotoxicidade em HaCat, demonstrou que após tratamento com diferentes concentrações dos extratos, ambos apresentaram citotoxicidade estatisticamente semelhante ao hipoclorito de sódio 2,5%. Diante disso, pode-se concluir que ambos os extratos apresentam ação antioxidante e antimicrobiana em culturas planctônicas, exceto o extrato de jabuticaba para C. albicans. Quanto a associação dos extratos, não houve ação sinérgica, mas obteve-se resultado aditivo para cepa clínica de E. faecalis 2. A ação antibiofilme foi efetiva para as cepas de E. faecalis, F nucleatum e P. gingivalis. A citotoxicidade dos extratos apresentou semelhança estatística com o hipoclorito de sódio 2,5% para ambos.
With the increase in resistant microorganisms in root canals, studies of plant extracts with antibacterial action have increased aiming at alternative therapies. The objective of this study was to evaluate the synergistic effects on Enterococcus faecalis, Enterococcus faecium, Candida albicans, Fusobacterium nucleatum and Porphyromonas gingivalis and the antibiofilm action of hydroethanolic extracts of Rosmarinus officinalis L. (rosemary) and Plinia cauliflora (jabuticaba). The Minimum Inhibitory Concentration (MIC) and Minimum Microbicide Concentration (MCC) were determined by broth microdilution and the Fractional Inhibitory Concentration Index and Fractional Microbicide Concentration Index by checkerboard microdilution. Subsequently, the effect of the effective concentration of the extracts was evaluated on biofilms formed in microtiter plate wells. The biofilms were exposed to two different periods of treatment with the extracts, for 5 min and 24 h, with n=10 for each experimental group. As controls, 0.9% saline solution (C+) and 2.5% sodium hypochlorite (C-) were used, totaling 5 groups for each microorganism and each experimental time. Then, the biofilms were subjected to the MTT test. Analysis of the cytotoxic activity of the extracts on human keratinocyte lines (HaCat) was carried out using the MTT test. Statistical analysis was performed using the ANOVA method and Dunn's test (5% significance level). The results that both extracts demonstrated phenolic and flavonoid phytocompounds and have antioxidant action. The extracts have antimicrobial action for the strains studied and when they were combined, they presented additive concentrations only for the E. faecalis strain. Regarding the antibiofilm property, the rosemary extract showed a good reduction in the microbial viability of E. faecalis, with a reduction of 52% for 5 min and 40% for 24 h. For strains of anaerobic microorganisms, in contact with the F. nucleatum strain, the jabuticaba extract reduced biofilm by up to 73%. For the P. gingivalis strain, there was a reduction of up to 41% for the jabuticaba extract and up to 57% for the rosemary extract. As for cytotoxicity in HaCat, it was demonstrated that after treatment with different concentrations of extracts, both showed cytotoxicity statistically similar to 2.5% sodium hypochlorite. Given this, it can be concluded that both extracts have antioxidant and antimicrobial action in planktonic cultures, except the jabuticaba extract for C. albicans. Regarding the association of extracts, there was no synergistic action, but an additive result was obtained for the clinical strain of E. faecalis 2. The antibiofilm action was effective for the strains of E. faecalis, F nucleatum and P. gingivalis. The cytotoxicity of the extracts showed statistical similarity with 2.5% sodium hypochlorite for both.
With the increase in resistant microorganisms in root canals, studies of plant extracts with antibacterial action have increased aiming at alternative therapies. The objective of this study was to evaluate the synergistic effects on Enterococcus faecalis, Enterococcus faecium, Candida albicans, Fusobacterium nucleatum and Porphyromonas gingivalis and the antibiofilm action of hydroethanolic extracts of Rosmarinus officinalis L. (rosemary) and Plinia cauliflora (jabuticaba). The Minimum Inhibitory Concentration (MIC) and Minimum Microbicide Concentration (MCC) were determined by broth microdilution and the Fractional Inhibitory Concentration Index and Fractional Microbicide Concentration Index by checkerboard microdilution. Subsequently, the effect of the effective concentration of the extracts was evaluated on biofilms formed in microtiter plate wells. The biofilms were exposed to two different periods of treatment with the extracts, for 5 min and 24 h, with n=10 for each experimental group. As controls, 0.9% saline solution (C+) and 2.5% sodium hypochlorite (C-) were used, totaling 5 groups for each microorganism and each experimental time. Then, the biofilms were subjected to the MTT test. Analysis of the cytotoxic activity of the extracts on human keratinocyte lines (HaCat) was carried out using the MTT test. Statistical analysis was performed using the ANOVA method and Dunn's test (5% significance level). The results that both extracts demonstrated phenolic and flavonoid phytocompounds and have antioxidant action. The extracts have antimicrobial action for the strains studied and when they were combined, they presented additive concentrations only for the E. faecalis strain. Regarding the antibiofilm property, the rosemary extract showed a good reduction in the microbial viability of E. faecalis, with a reduction of 52% for 5 min and 40% for 24 h. For strains of anaerobic microorganisms, in contact with the F. nucleatum strain, the jabuticaba extract reduced biofilm by up to 73%. For the P. gingivalis strain, there was a reduction of up to 41% for the jabuticaba extract and up to 57% for the rosemary extract. As for cytotoxicity in HaCat, it was demonstrated that after treatment with different concentrations of extracts, both showed cytotoxicity statistically similar to 2.5% sodium hypochlorite. Given this, it can be concluded that both extracts have antioxidant and antimicrobial action in planktonic cultures, except the jabuticaba extract for C. albicans. Regarding the association of extracts, there was no synergistic action, but an additive result was obtained for the clinical strain of E. faecalis 2. The antibiofilm action was effective for the strains of E. faecalis, F nucleatum and P. gingivalis. The cytotoxicity of the extracts showed statistical similarity with 2.5% sodium hypochlorite for both.
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Palavras-chave
Extratos vegetais, Sinergismo farmacológico, Endodontia, Plant extracts, Drug synergism, Endodontics