Estudo das propriedades antimicrobianas de Caryocar villosum (Aubl.) Pers. sobre bactérias cariogênicas

Abstract

Dental caries is a public health problem that is associated with an increase in acidogenic Streptococcus mutans and Lactobacillus, involved in the onset and progression of lesions, respectively. The gold standard antimicrobials in combating these bacteria have shown adverse effects that encourage the establishment of studies to identify alternative substances for the prevention of the disease. The objective of this work was to evaluate the antimicrobial potential of Caryocar villosum extracts against bacteria responsible for caries disease, Streptococcus mutans, and Lactobacillus sakei. Pulp, seed and peel extracts of C. villosum, obtained with different solvents (ethanol, hydroalcohol, hexane and methanol) at concentrations ranging from 5 to 0.009 mg/mL, were tested. To determine the Minimum Inhibitory Concentration (MIC), 50 μL of extract solutions or chlorhexidine at different concentrations was added to the 96-well plate along with 50 μL of bacterial culture. As a negative control, sterile Brain and Heart Infusion Broth (BHI) was used instead of the extract solution. After incubation at 37º C for 48 hours, resazurin was added to assess cell viability. The nature of the antimicrobial activity was evaluated through the death time of the microorganisms challenged at concentrations of ½ MIC and MIC of the extracts, incubated at 37º C. Ten μL aliquots were plated, at increasing time intervals up to 24 hours, in Petri dishes with BHI agar, incubated at 37º C for 24 hours to count colony forming units (CFU/mL). The release of intracellular proteins from bacteria was evaluated to assess the possible action of the extracts on the bacterial cell wall, after incubation at 37ºC for 5 hours. To assess the interference of extracts on the ability of bacteria to adhere to the surface, concentrations between 0.312 and 5 mg/mL were used in microtiter plates with cultures of the two bacteria and incubated at 37º C for 24 hours. After this period, the wells were washed with distilled water, and methanol, crystal violet and finally glacial acetic acid were added. The number of microorganisms adhered to the plate was measured by absorbance at 540nm. The extracts showed antimicrobial activity in MIC varying between 1.25 and 2.5 mg/mL, for the two tested bacteria. All extracts tested against L. sakei showed bactericidal action, whereas against S. mutans the activity varied between bacteriostatic and bactericidal. Even though results indicate a bactericidal capacity for some extracts, in the protein release test, there was no indication of cell wall impairment, suggesting that the mechanism of action of the extracts on bacteria is in another cell target. The ability to reduce bacterial adhesion to the surface was observed at all concentrations tested, including sub-inhibitory ones, being an important result, since caries is a biofilm-dependent disease. The results of the present study are relevant for the use of different extracts of C. villosum in combating and preventing caries. New studies must be conducted to identify the active compounds present in the extracts, in addition to the evaluation of toxicity and damage to the oral mucosa for safe use.