Potencial biotecnológico de extratos, frações e diterpenos isolados de Croton blanchetianus Baill. sobre biofilmes orais

Abstract

The mouth has a very diversified microbioma, with favorable conditions for the development of oral biofilms. The presence of these biofilms, associated with other factors such as diet and/or inadequate hygiene, can trigger a carious process. The mechanical removal is the most used in the treatment, however, the use of antimicrobials in hygiene products can help in the prevention of caries. Some microorganisms present resistance to traditional antimicrobials, so the search for new antimicrobial agents is necessary. In the brazilian flora can be found medicinal plants, species of the genus Croton demonstrate proven biological activities, in some cases, diterpenes are responsible for the therapeutic action. In this sense, the objective of this work was to evaluate the antimicrobial and antibiofilm potential of extracts, acid and neutral fractions and diterpenes isolated from three specimens of C. blanchetianus against dental caries-related oral strepococci. The extraction of extracts, acid and neutral fractions and isolation of diterpenes occurred in parallel to the antimicrobial action tests. The solutions with the substances were submitted to serial dilutions and the concentrations obtained ranged from 125 to 1.8 μg/mL. The antimicrobial action was verified by minimum inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) tests using S. parasanguinis ATCC903 and S. mutans ATCC700610/UA159 oral streptococci. For the determination of the antibiofilm action the biomass quantification and cell viability were performed in two different situations: in the inhibition of the formation of biofilms and in the action on pre-formed biofilms of S. parasanguinis and S. mutans, with two diterpenes, in the same concentrations previously mentioned. Electromicrographs were obtained by scanning electron microscopy (SEM), in order to visualize S. mutans biofilms after contact with the diterpenes. From the extraction and isolation process were obtained nine hexane extracts, thirty three acid fractions and fifty four neutral fractions, three diterpenes: 3,4-seco-bieren-3-oic acid (ICB1) unprecedented in the literature, methyl 12-hydroxy-3,4- seco-cleistanta-8,11,13,15(4)-penten-3-oate (ICB4) and ent-3,4-seco-atisan-4(18),16-dien- 3-oic acid (ICB8). The hexane extracts of the roots had the lowest values of MIC and MBC, 15.6 and 62.5 μg/mL. Acid fractions presented MIC and MBC of 7.8 μg/mL on S. mutans and 15.6 μg/mL on S. parasanguinis. The lowest value of MIC and MBC among diterpenes was ICB4, 7.8 μg/mL on S. parasanguinis. The inhibition of biofilm formation of S. parasanguinis, by ICB4, occurred up to the concentration of 3.9 μg/mL, with biomass reduction of up to 94% and the decrease in cell viability was 5.42 Log CFU/mL. ICB8 inhibited S. mutans biomass up to 99.9% and cell viability decreased about 3.92 Log CFU/mL. The action on preformed biofilms was also observed in both diterpenes: ICB4 reduced biofilm biomass by up to 75% and ICB8 reduced biomass by up to 95.8%. Electron micrographs showed structural changes in cell morphology. The study points out that the substances ICB4 and ICB8 have antibiofilm action with the potential to be transformed into anticary products.