Effect of hydroalcoholic extract of Myracrodruon urundeuva All. and Qualea grandiflora Mart. leaves on the viability and activity of microcosm biofilm and on enamel demineralization

Abstract Objectives: The aim of this study was to assess the effect of Myracrodruon urundeuva All. and Qualea grandiflora Mart. leaves hydroalcoholic extracts on viability and metabolism of a microcosm biofilm and on enamel demineralization prevention. Methodology: Microcosm biofilm was produced on bovine enamel using inoculum from pooled human saliva mixed with McBain saliva, under 0.2% sucrose exposure, for 14 days. The biofilm was daily-treated with the extracts for 1 min. At the end, it was analyzed with respect to viability by fluorescence, CFU counting and extracellular polysaccharides (phenol-sulphuric acid colorimetric assay) and lactic acid (enzymatic assay) production. The demineralization was measured by TMR. The data were compared using ANOVA or Kruskal-Wallis (p<0.05). Results: M. urundeuva All. at 100, 10 and 0.1 μg/mL and Q. grandiflora Mart. at 100 and 0.1 μg/mL reduced biofilm viability similarly to positive control (chlorhexidine) and significantly more than the negative-vehicle control (35% ethanol). M. urundeuva at 1000, 100 and 0.1 μg/mL were able to reduce both lactobacilli and mutans streptococci CFU counting, while Q. grandiflora (1000 and 1.0 μg/mL) significantly reduced mutans streptococci CFU counting. On the other hand, the natural extracts were unable to significantly reduce extracellular polysaccharides and lactic acid productions neither the development of enamel carious lesions. Conclusions: The extracts showed antimicrobial properties on microcosm biofilm, however, they had no effect on biofilm metabolism and caries protection.


Introduction
Dental caries involves dental biofilm rich in acidogenic and aciduric bacteria such as Streptococcus mutans, Streptococcus sobrinus, Lactobacillus sp., Veillonella, Actinomyces, bifidobacteria and fungi, 1 which are metabolically active under frequent sugar exposure, producing acids that induce tooth demineralization. 2 Mechanical disorganization of dental biofilm by brushing and rationing sugar consumption are key strategies to prevent the disease. In addition, conventional antimicrobial oral mouthrinses can be recommended for patients at high-risk level. 3 However, their antimicrobial properties may not reflect into an anti-caries effect and, additionally, may induce some side-effects such as taste alteration, tooth staining and mucosa desquamation. 4,5 Therefore, scientists are directing attention to folk medicine in order to find alternative antimicrobial agents against oral diseases as dental caries. 6 Brazil is the country harboring the highest plant M. urundeuva has antimicrobial action, 8,9 including action against mutans streptococci, 10 as well as analgesic, hepatoprotective, antidiarrheal, colonic anastomotic wound healing and anti-ulcerogenic effects. 11 Q. grandiflora exhibits anti-ulcerogenic action in the ethanolic extract of its bark. 12 Besides, this extract has an antioxidant effect, 13 analgesic and anticonvulsive potential 14 and antibacterial action. 15 Regarding dental caries, a previous study tested the effect of aqueous extracts of M. urundeuva on mutans streptococci counts and on dental enamel micro-hardness of rats submitted to cariogenic challenges. The extract promoted significant reduction of mutans streptococci counts as well as enamel demineralization. 16 Recently, our research group showed that both hydroalcoholic extracts of M. urundeuva and Q. grandiflora leaves (isolated or combined) had antimicrobial action; however, they did not prevent enamel caries formation under the mutans streptococci biofilm model. 17 Therefore, there is no consensus about the anti-caries action of the extracts. Furthermore, there is no information about their mechanism of action under more complex biofilm models (such as multispecies or microcosm biofilm).

Microcosm biofilm formation and treatments
The human saliva was defrosted and mixed with McBain saliva 21 in a proportion of 1:50. The microcosm biofilm was produced as described in previous studies. 5,19 The samples were placed in a 24-well plate and the solution containing human saliva and McBain saliva was added to each well (v=1.5 mL/well), which was incubated at 5% CO 2 and 37°C for the first 8 h.
Thereafter, the samples were washed with PBS and   Rogosa (Kasvi; Curitiba, Paraná, Brazil) supplemented with 0.13% glacial acetic acid to assess the number of lactobacilli. 24 The plates were then incubated at 5% CO 2 and 37°C. After 48 h, the CFU numbers were counted and transformed in log 10 CFU/mL.

Metabolism analysis a) Lactic acid production
For this assay, only the highest and lowest concentrations of each extract were tested. Lactate concentrations were evidenced by means of the enzymatic method (lactic dehydrogenase method, Boehringer; Mannheim, Baden-Württemberg, Germany) according to the manufacturer's instruction. 25 Absorbance was measured at 340 nm using a microplate reader (Fluorstar Optima-BMG Labtech; Ortenberg, Baden-Württemberg, Germany). The values were expressed as mmol lactate/L. 5

b) Extracellular polysaccharides -EPS quantification
The insoluble and soluble EPS were quantified as previously performed. 5 Total carbohydrates were measured using the phenol-sulphuric acid colorimetric assay under absorbance of 490 nm using a microplate reader (Fluorstar Optima-BMG Labtech; Ortenberg, Baden-Württemberg, Germany). 26 The values for both EPS were expressed as μg EPS/mg (biofilm). 5

Transverse microradiography (TMR)
Enamel slices with 80-100 µm of thickness were fixed in a sample-holder together with an aluminum calibration step wedge with 14 steps. Microradiographs were taken using an x-ray generator (Softex; Tokyo, Honshu, Japan) on the glass plates. 17 The glass plates were developed and analyzed using a transmitted light microscope fitted with a 20x objective (Zeiss; Oberkochen, Baden-Württemberg, Germany), a CCD camera (Canon; Tokyo, Honshu, Japan), and a computer containing software from the Inspektor Research System bv (Amsterdam, North Holland, The Netherlands). The cavitation depth (CD, µm) was calculated as previously described. 17

Bacterial viability
Hydroalcoholic extracts of M. urundeuva at 100 μg/mL (62.14%), 10 μg/mL (74.59%) and 0.1 μg/mL (59.81%) and Q. grandiflora at 100 μg/mL (67.19%) and 1 μg/mL (64.50%) presented mean percentage of dead cells similar to the positive control (chlorhexidine, 48.21%), and significantly higher than the negative control group (35% ethanol, 33.79%). The other experimental groups did not differ between themselves and positive and negative controls (p>0.05, Figures   1 and 2). Figure 1 shows the percentage of viable microorganisms from each treatment's group. Figure 2 shows CLSM pictures of a representative biofilm sample from the most effective antimicrobial concentrations of the tested extracts.

Metabolism analysis a) Lactic acid production
None of the extracts was able to significantly reduce lactic acid production compared to negative control; however, chlorhexidine significantly differed from negative control (Figure 3).       17 (2018) only found antimicrobial effect of Q. grandiflora against mutans streptococci at 5 mg/mL, which might be due to differences in the biofilm model between both studies (monospecies biofilm vs. microcosm biofilm) as discussed above.

b) EPS quantification
Despite the extracts being able to reduce bacteria viability as well as the number of lactobacilli and mutans streptococci, they did not interfere in biofilm metabolism, and, therefore, they were unable to reduce caries lesions development, which corroborates A-E) M. urundeuva from 1000 to 0.1 μg/mL, respectively; F-J) Q. grandiflora from 1000 to 0.1 μg/mL, respectively; K) Positive control (chlorhexidine, PerioGard ® ); L) Vehicle (negative) control with a previous study. 17 Despite the treatments having reduced the number of viable bacteria, the microorganisms were still able to produce acid and EPS, which in turn induced enamel demineralization similar to the negative control. Our work provided support for the statement that not all antimicrobial agents have anti-caries potential. 5  In disagreement, a previous study has shown that an aqueous solution of M. urundeuva protected against enamel surface cross-sectional hardness loss in Wistars rats inoculated with mutans streptococci, after 7 weeks of cariogenic challenges. 16 The different result found in the cited study might be due to the greater concentration of the extract (7.5 mg/mL) as well as the type of extract (aqueous) applied by a previous study and to the low velocity of caries development in vivo.
It is also important to consider that a hardness assay is unable to show if the cariogenic challenges induced tooth cavitation, 36 which is considered a limitation of the method.

Conclusions
The extracts showed antimicrobial effects (especially M. urundeuva) on the microcosm biofilm; however, no effect was observed on the biofilm metabolism and neither anti-caries effect under this biofilm model.