ANTIMICROBIAL POTENTIAL OF RESIN MATRICES LOADED WITH COFFEE COMPOUNDS

Abstract

This study evaluated the biological behavior of the coffee compounds Trigonelline (T), chlorogenic acid (C) and nicotinic acid (N), correlating with their release from a resin matrix. Minimum inhibitory concentration (MIC) was evaluated against S. mutans UA159, and cytotoxicity was assessed on OD-21 cells. Resin matrices (BisGMA/TEGDMA 70/30 wt%, camphorquinone/EDMAB 0.5/1 wt%) were doped with coffee compounds in different concentrations (10/20/30/40/50 wt%), performing 15 groups (T10-T50, C10-C50, N10-N50), and a control group with no coffee compound. Degree of conversion (DC%) was analyzed by Fourier Transform Infrared spectroscopy. Antimicrobial properties were evaluated by bioluminescence (Luciferase assay). The release from loaded matrices was analyzed over time (24h, 6, 14, 21 and 28 days), using high-performance liquid chromatography (HPLC). Data were submitted to ANOVA/Tukey’s test (α=0.05). MIC for T and C was 6 mg/mL, and 4 mg/mL for N. None of them were cytotoxic. Only T50 and C50 showed lower DC% than control (α<0.05). Some groups (T30/T40/T50/C40/C50/N50) were strongly antimicrobial, reducing bacterial activity approximately five times compared to control (α<0.05). For T30, T40, T50, C40 and C50, the HPLC showed a release above or closer to MIC values mainly in 24 h, but for N50, up to 28 days. In conclusion, the coffee compounds presented antimicrobial activity, depending on their concentration when added in resin matrices, being found a correlation with their release.

1. Introduction

Natural compounds have been used in the treatment of different diseases and the research in this area continues to increase, stimulating the development of new commercial products with greater pharmacological activity, lower toxicity and improved biocompatibility.¹ In addition, they can be an important source for the discovery of new drugs with antimicrobial activity.² In dentistry, we can apply the use of these compounds in the prevention and treatment of dental caries.

Dental caries is multifactorial and depends on the interaction of several etiological factors: host, diet and microbiota. Streptococcus mutans, a Gram-positive bacterium often found in the dental biofilm, is one of the major caries pathogens.³ This species can ferment a wide variety of sugars and, at the same time, maintain healthy metabolism even at a low pH, causing enhanced tooth demineralization, which can result in more extensive caries lesion formation.⁴

The number of caries lesions has decreased,^5-7 however, a large part of the population still suffers from this disease. Alternatives for the prevention and control of dental caries, an infectious disease, have been continuously investigated and, due to its bacterial origin,⁸ the search for antimicrobials able to interfere directly in its progression has been a consistent strategy for dental researchers.

The use of natural antimicrobials can inhibit the growth of microorganisms or even destroy them,² with less concern over damage to resident cells and tissues. Some studies have verified antimicrobial action by several natural compounds,^9-11 including coffee,^12-15 that is one of the most complex beverages from a chemical point of view. This complexity is due to the great diversity of natural components in its composition, as well as the interaction of compounds formed during its processing.¹⁶ Several beneficial characteristics to health have been attributed to coffee,^17,18 such as its antimicrobial activity. In particular, the coffee compounds trigonelline, chlorogenic acid and nicotinic acid have shown bioactivity, especially against S. mutans,^19,20 and may be helpful for dental caries prevention.

However, there are no studies that have incorporated these compounds into dental resin-based materials for the purpose of making antimicrobial dental materials. Dental sealants, varnishes, cements, adhesive systems and restorative composites would be likely vehicles for these compounds and could act for prevention and treatment of dental caries. The organic matrix of these materials typically consists of methacrylate monomers, diluents and initiators/inhibitors, and may readily incorporate new antimicrobial monomers in order to maximize their performance.^7,21

The purpose of the present study was to evaluate the biological effects of different coffee compounds, their influence on S. mutans when added in dental resin matrices, as well their release to aqueous environment over time.

2. Materials and methods

Minimal Inhibitory Concentration (MIC)

The coffee compounds investigated in this study were trigonelline, chlorogenic acid and nicotinic acid, purchased from Sigma Aldrich (St. Louis, MO, USA). The figure 1 shows their molecular structure and respective molecular weight. In order to evaluate the antibacterial properties of these compounds, the MIC (minimum inhibitory concentration) was determined using S. mutans UA159 planktonic cultures. The concentration of starting inoculum used was 6 x 10⁵ CFU/ml, according to the recommendations of the Clinical and Laboratory Standards Institute (CLSI). The microdilution of the compounds was done in a 96-well plate, being assigned wells for bacterial suspension and for culture medium without any compound to serve as positive and negative control, respectively. Then, the plate was incubated in anaerobic conditions (90% N₂, 5% CO₂, and 5% H₂) overnight at 37°C. Experiment was conducted in triplicate. The MIC of trigonelline, chlorogenic acid, and nicotinic acid was determined as the lowest concentration at which UA159 growth was completely inhibited after exposure for 24h.

Figure 1 -.

A: Trigonelline molecular structure, B: Chlorogenic acid molecular structure, C: Nicotinic acid molecular structure.

Cytotoxicity

To evaluate the effect of trigonelline, chlorogenic acid and nicotinic acid on cell viability, undifferentiated dental pulp cells (OD-21) were used. Some in vivo and in vitro studies have shown that resin-based materials, as composites or adhesives, can damage the pulp tissue (Chen et al., 2019; Costa et al., 2002) or pulp cells (Salehi et al., 2015; Caldas et al, 2019). Thus, undifferentiated dental pulp cells have relevance to restorative dentistry, since they can differentiate into odontoblast-like cells, and consequently, replace damaged pulp cells by restorative procedures or caries.^22,23

OD21 cells were cultured in 96-well plate (10⁴ cells/well) (n = 5) in Dulbecco’s modified Eagle’s medium (Lonza Walkersville, MD, USA), containing 10% fetal bovine serum and supplemented with an antibiotic solution (1% Penn-Strep) at 37°C in a humidified atmosphere of 5% carbon dioxide (CO₂) in air for 24 hours to allow cells to adhere and spread. Cells were then treated with serial dilutions of each coffee compound (6, 0.6 and 0.06 mg/mL for trigonelline and chlorogenic acid, and 4, 0.4 and 0.04 mg/mL for nicotinic acid). The highest concentrations were determined based on the results obtained from the MIC test, performed previously. Cell cultures in a medium without any bioactive compound served as control. After incubation for 24h, metabolic activity of the cells was measured using a water-soluble methyl tetrazolium salt, MTS (3-[4,5,dimethylthiazol-2-yl]-5-[3-carboxymethoxy-phenyl]-2-[4-sulfophenyl]-2H-tetrazolium) assay (n=5). Then, 10μl (1:10 dilution) of MTS was added to each well and incubated for 3 hours at 37°C, and the concentration of metabolic byproducts as a function of cell number was colorimetrically (absorbance) measured at 490 nm using a plate reader (Epoch, BioTek Instruments, VT, USA).

Resin Synthesis

A model resin matrix was prepared with a mixture of 70 wt% BisGMA (bisphenol A-glycidyl-dimethacrylate and 30 wt% TEGDMA (triethylene glycol-dimethacrylate), both from Esstech Inc. (Essington, PA, USA). To this mixture, 0.5 wt% of Camphorquinone and 1.0 wt% of EDMAB (Ethyl 4-dimethyl aminobenzoate), both purchased from Sigma Aldrich (Milwaukee, WI, USA), were added as photoinitiator and accelerator, respectively. These components were mixed with a centrifuge (SpeedMixer DAC150 FVZ, Flacktek, SC, USA), for two periods of 1 minute, each at 1300 rpm. Afterwards, the coffee compounds were added to the model resin matrix in the specific percentages relative to the total mass, in accordance with the Table 1. For Luciferase assay, a composition without any coffee compound was used to act as control group.

Table 1 -.

Experimental groups

Groups	Resin Matrix (wt %)	Compound	Concentration (wt %)
T10	90	Trigonelline	10
T20	80		20
T30	70		30
T40	60		40
T50	50		50
C10	90	Chlorogenic acid	10
C20	80		20
C30	70		30
C40	60		40
C50	50		50
N10	90	Nicotinic acid	10
N20	80		20
N30	70		30
N40	60		40
N50	50		50

Degree of conversion (DC%)

The analysis of the degree of conversion followed the method described in the study of Almeida et al. (2017). Increments of the resin matrices were inserted into a Teflon mold (0.785 mm³), positioned onto an ATR crystal of the FT-IR spectrometer (Alpha-P/Platinum ATR Module, Bruker Optics GmbH, Ettlingen, Germany), and the spectra between 1500 and 1800 cm⁻¹ were recorded, using 40 scans at a resolution of 4 cm⁻¹. Thus, the resin was photocured for 20 s at 1000 mW/cm² irradiance (Demi LED Curing Light, Demetron Kerr, WI, USA), and the spectra were recorded exactly as performed for the unpolymerized increments. The DC% was calculated from the ratio between the integrated area of absorption bands of the aliphatic C=C bond (1638 cm⁻¹) to that of the C=O bond (1609 cm⁻¹), used as an internal standard, which were obtained from the cured and uncured increments, using the following equation bellow:

$$\text{DC}\%=100\times [1-(\text{Rcured/Runcured})],$$

where R is integrated area at 1638 cm⁻¹/integrated area at 1609 cm⁻¹.

Luciferase Assay

Six disks (6x2 mm) were made for each of the experimental resins, including the control group. A silicon matrix with standardized measurements was placed between two slide cover glasses, the resin was inserted in a single increment and the photoactivation was performed for 40 seconds on both sides, at 1000 mW/cm² irradiance (Demi LED Curing Light, Demetron Kerr, WI, USA). After polymerization, the surfaces of both sides of the disk were polished with 600 grit silicon carbide sandpaper. An analysis of surface roughness was performed using a rugosimeter (Mytutoyo, Tokyo, JP), in order to ensure a mean surface roughness of Ra ≅ 0.3μm.

The disks were then immersed in isopropyl alcohol for 20 minutes for sterilization, dried with sterile gauze and loaded into the wells of a 24-well plate. Each well received 1 ml of bacterial suspension of S. mutans UA159 with Todd-Hewitt (TH) media (Thermo Fisher Scientific, MA, USA) supplemented with 1% (wt/vol) sucrose and the plate was incubated with gentle agitation on a microplate shaker (VWR, PA, USA) at 40 rpm in a 5% CO₂ incubator at 37°C for 24 hours.

After the incubation period, the disks were carefully transferred to a black 24-well plate and incubated with fresh media for 1h at 37ºC in 5% CO₂. Then, 10μl of a substrate solution (2 mM d-luciferin in 0.1 M, pH 6, sodium citrate buffer) was added to each well and the bioluminescence activity was measured in a microplate luminometer (GloMax, Promega, WI, USA). Each plate also contained a well with medium and no bacteria as a sterility control, in addition to a positive control well containing bacteria and no inhibitor.

High performance liquid chromatography (HPLC)

In this study, an Agilent chromatograph (1200 Infinity Series, CA, USA) was used under the following chromatographic conditions: C18 column (150 x 4.6 mm, 3.5 μm particle size), 20 μL injection volume (loop), spectrophotometric detector set at 220nm and the mobile phase consisted of two solvents (100% methanol and 0.1% phosphoric acid at a ratio of 20:80) in a flow rate of 1.0 mL / min.

Calibration curves of trigonelline, chlorogenic acid and nicotinic acid were constructed diluting standard solutions at five different concentrations (0.05, 0.1, 0.2, 0.4 e 0.8 mg/ml) in deionized water. All the calibration curves showed a correlation coefficient greater than 0.98.

Then, three discs were made for each experimental resin by placing the resin in a silicon matrix (10x1mm) and performing the photoactivation from one side of the disk, as described before. The specimens were stored in 2 ml deionized water for 24 hours and the compounds released into the water were analyzed by HPLC, with three independent runs, one for each specimen. The same disks were immersed in water again and new chromatographic analyzes were performed in the periods of 7, 14, 21 and 28 days.

Statistics

Statistical analysis was performed using Statgraphics software 5.1 (Manugistics, Rockville, MD, USA). The obtained data were analyzed by one-way analysis of variance (ANOVA). For cytotoxicity, ANOVA was followed by Student-Newman-Keuls multiple comparison test. For degree of conversion and Luciferase assay, the contrast test applied was Tukey HSD. All analyses were performed at a significance level of α=0.05

3. Results

The MIC values for each coffee compound are presented in Table 2.

Table 2 -.

In vitro susceptibilities of planktonic S. mutans UA159

Antimicrobial agent	MIC (mg/ml)
Trigonelline	6
Chlorogenic acid	6
Nicotinic acid	4

Figure 2 shows the result of the cytotoxicity assay. The absorbance values represent a quantitation of the living cells, and values significantly lower than the control group are considered cytotoxic to some degree. According to the statistical analysis, trigonelline, chlorogenic acid and nicotinic acid were not cytotoxic at the concentrations tested, as the MIC values were not lower than that for the control group.

Figure 2 -.

Graph of absorbance (representing cell viability) of each substance at 3 different concentrations. Groups: Control (no substance + cells); T6 (Trigonelline 6%); T0.6 (Trigonelline 0.6%); T0.06 (Trigonelline 0.06%); C6 (Chlorogenic acid 6%); C0.6 (Chlorogenic acid 0.6%); C0.06 (Chlorogenic acid 0.06%); N4 (4% nicotinic acid); N0.4 (0.4% nicotinic acid); N0.04 (Nicotinic acid 0.04%).

In accordance with Table 3, only for T50 and C50, the degree of conversion was lower than the control group (α<0.05). For the remaining groups, the values of degree of conversion were statistically similar to the control.

Table 3 -.

Means and SD of DC (%)

Experimental groups	DC (%) ± SD*
Control	69.13 ± 1.65 b,c
T10	67.58 ± 2.01 c,d
T20	66.18 ± 2.62 c,d
T30	65.83 ± 1.45 c,d
T40	65.17 ± 1.55 c,d
T50	64.17 ± 2.97 d
C10	65.90 ± 1.49 c,d
C20	65.34 ± 1.54 c,d
C30	64.40 ± 2.00 c,d
C40	64.57 ± 0.99 c,d
C50	63.26 ± 1.25 d
N10	72.07 ± 2.68 a,b
N20	72.87 ± 2.98 a,b
N30	66.25 ± 1.80 c,d
N40	69.13 ± 2.50 b,c
N50	66.71 ± 2.11 c,d

^*Different superscript letters indicate statistically significant differences.

The results of the Luciferase assay are shown in Figure 3. The luminescence values represent a quantitation of the active bacteria, so those values lower than the control group suggest more effective antimicrobial activity. It can be observed that T30, T40, T50, C40, C50 and N50 groups presented significantly lower values than the control group (α<0.05). Therefore, the components that showed effective antimicrobial action, without statistical difference between them, were trigonelline at 30, 40 and 50%, chlorogenic acid at 40 and 50% and nicotinic acid at 50%.

Figure 3 -.

Luciferase assay (luminescence) for the different concentrations of the three compounds.

The figure 5 shows a graph that correlates the obtained Luciferase assay data with the 24-hour elution results (mg/ml). It can be observed that the higher the release for the three coffee compounds the higher the bacterial inhibition, reaching a total inhibition for two of them (T and C). The release for trigonelline reached values very much higher than MIC values, while chlorogenic and nicotinic acids presented a release closer to the MIC values. Another important aspect to mention is that the release above MIC values were highly cytotoxic for S. mutans for all compounds.

Figure 5 -.

Luciferase assay X 24h Elution (mg/ml)

4. Discussion

Trigonelline, chlorogenic acid and nicotinic acid are natural coffee compounds with antimicrobial potential against S. mutans,^19,20 so they could act to prevent and treat dental caries. In the present study, all the studied compounds showed antimicrobial action against S. mutans and were not cytotoxic.

As a biological characterization, the microdilution technique used to determine minimum inhibitory concentration has the advantage of determining a quantitative result. In this study, the value found for trigonelline and chlorogenic acid was 6 mg/mL, and 4 mg/mL for nicotinic acid. These values differ from those in a previous study,¹⁹ which showed that chlorogenic acid and trigonelline were able to inhibit the growth of S. mutans at a lower concentration of 0.8 mg/mL. In that study, the bacterial strain used was S. mutans ATCC 25175. The bacteria were maintained in broth medium of tryptic soy broth with 20% glycerol and incubated at 37°C under anaerobic conditions for 48 h in a candle jar. The inoculum was prepared with a concentration of 4x10⁶ CFU/mL. This methodology differs from that used in the current research and may be the reason for the divergence of the values found.

The cytotoxicity was evaluated based on MIC values, as it would not be necessary higher concentrations to inhibit the bacterial growth. The highest concentrations tested were 6 mg/ml for trigonelline and chlorogenic acid, and 4 mg/ml for nicotinic acid, but none of them exhibited cytotoxicity. This finding is supported by a study²⁴ that also presented a lack of the cytotoxicity of some alkaloids, flavonoids and phenolic acids. However, resin matrices highly doped, specifically with trigonelline (40 and 50%) and nicotinic acid (50%), released higher concentrations than those tested for cytotoxicity. It can be emphasized that this release was also much higher than the value found for MIC and, consequently, unnecessary. Indeed, the luciferase assay shows that the addition of trigonelline at 30% in resin matrices was enough to inhibit the S. mutans growth, with a release closer to the concentration tested for cytotoxicity in this study. For nicotinic acid, concentrations between 40 and 50% should be more reasonable, as the addition of 50% in resin matrices led to a release much higher than MIC, promoting an effective inhibition of bacterial growth (Luciferase assay), but exceeding the concentration tested for cytotoxicity in this study. On contrary, the addition of 40% was not enough to inhibit the bacterial growth. It is convenient to make clear that higher concentrations than 6mg/mL, for trigonelline and chlorogenic acid, and 4mg/mL, for nicotinic acid, were not tested for cytotoxicity in this studies, so it does not mean that they could present cytotoxicity. Further studies could be conducted to extend this investigation

As trigonelline, chlorogenic acid and nicotinic acid did not cause observable damage to pulp cells, the study aimed to evaluate the antibacterial potential of these compounds when incorporated to resins, which could be the basis for the development of new restorative dental materials, such as composites, lining, adhesives or cements capable of avoiding recurrent caries.

In the Luciferase assay, the detection of adenosine triphosphate (ATP), which is present in all living cells, is a quantitative indicator of the presence and number of viable microorganisms. The assay consists of extracting ATP from the microbial cells using the luciferin / luciferase enzyme system and measuring the light generated by means of a luminometer. The light emitted is directly proportional to the amount of ATP present in the sample and depends on the number of microorganism present.

In this study the three compounds tested showed effectiveness against S. mutans when added to a resin matrix, but as a function of concentration. At concentrations of 10 and 20% there was no antimicrobial activity for any of the compounds. Nicotinic acid had effective antibacterial potential only with 50%, which was the highest concentration tested. Chlorogenic acid was able to interfere with bacterial growth when incorporated at 40% and 50%, while trigonelline was efficient at concentrations of 30, 40 and 50%.

The higher efficacy for trigonelline has already been evidenced in a previous study¹⁹, that investigated the antibacterial activity against S. mutans of natural coffee compounds (chlorogenic acid, caffeic acid, ferulic acid, trigonelline, nicotinic acid and caffeine) solutions at 1 mg/mL. For the analysis of the CFU (Colony Forming Units), the study found that the number of CFU was significantly lower for trigonelline (p <0.01) when compared to the chlorogenic and caffeic acids, indicating a stronger antibacterial activity of trigonelline against S. mutans. Also, Daglia et al²⁰ evaluated the inhibition of S. mutans’ adsorption to hydroxyapatite from different coffee compounds such as chlorogenic acid, trigonelline, caffeine and nicotinic acid. These compounds were tested alone or associated, and the results showed that all evaluated compounds were active in this adhesion inhibition, indicating another possible action mechanism of them. The data also indicated that trigonelline was a remarkably effective inhibitor of S. mutans and suggested that this compound is primarily responsible for the antimicrobial activity of coffee.

Antonio et al¹⁹ also evaluated the activity of coffee against Streptococcus mutans and identified the natural compounds that contribute to this action. The authors found that trigonelin and chlorogenic acid showed bacteriostatic, but not bactericidal activity. However, biofilm inhibition results were correlated with the concentration of these compounds in coffee extracts. Nicotinic acid had no bacteriostatic action against S. mutans, so it was suggested that higher concentrations of this acid should be tested.

By means of HPLC it was possible to detect the amount of the compounds released into an aqueous environment. This test had great relevance to evaluate how much of each compound is leached from the resin matrix and if that amount is sufficient to demonstrate antimicrobial behavior. Based on the time release profiles, it is obvious that most of the release occurred in the first 24 hours, mainly for trigonelline.

Trigonelline has a short chain with polar character (Figure 1), which could favor its rapid release (Figure 4). Chlorogenic acid, despite having a longer chain (Figure 1) which would lead to a lower release over time, is no longer released end of 28 days. Nicotinic acid, on the other hand, with a shorter and nonpolar chain (Figure 1), presented a more uniform and gradual release.

Figure 4 -.

Concentration of leachates determined by HPLC for Trigonelline, Chlorogenic acid and Nicotinic acid, respectively.

It is also worth to mention that the degree of conversion for resins doped with trigonelline and chlorogenic acid at 50% was statistically lower than the control, and some concerns can be done in this respect. Some studies have shown an inverse correlation between solubility and degree of conversion for dental resins and composites (Gonçalves el al, 2008; Boaro et al., 2013; Brandao et al., 2018). So, as the solubility is related to the leaching of compounds from the material, it can be speculated that the lower conversion could have favored the higher coffee compound release from these resins. The polar chain of trigonelline, associated to the lower conversion of the resin at 50%, could also have contributed to the excessive release in 24 h, which was drastically reduced to a zero rate in the following periods. The longer chain of chlorogenic acid could made the release slower, remaining up to 21 days, even with lower degree of conversion. Despite the degree of conversion of the resins doped with nicotinic at 50% acid be statistically similar to the control, it could be observed a trend of reduction of the DC% values (Table 3), increasing its release, but remaining above its MIC value up to 28 days, which could be associated to their nonpolar chain. Still regarding to the DC%, during the manipulation, the resins loaded with higher concentration of coffee compounds presented an opaque aspect. So, one can assume that this high opacity may have negatively influenced light transmission, thereby decreasing the DC%. This is speculation and needs further investigation (Jardim et al. 2020).

Correlating the MIC, Luciferase and HPLC tests, we can observe that trigonelline was effective against S. mutans at the concentrations of 30, 40 and 50%, which corresponded to 24-hour release values of 6.42; 28.25 and 34.90 mg/ml, respectively. Considering that the MIC of trigonelline was 6 mg/ml, there is a strong correlation between the expected results from the MIC and luciferase assay, and the actual amounts released in the first 24 hours. The same is true for nicotinic acid which had an effective antimicrobial action when incorporated at 50% concentration, because at this level its release at 24 hours was 11.28 mg/ml, which was above its MIC of 4 mg/ml. Note that at the 40% concentration the release was 3.39 mg/ml, which was close to the MIC, but slightly lower and thus did not show a positive effect. For chlorogenic acid, its antimicrobial potential occurred at concentrations of 40 and 50% and the release values were 2.45 and 6.09 mg/ml, respectively. Considering that its MIC is 6 mg/ml, the results are reasonable, but it is a bit surprising that the compound was effective against S. mutans, even without quite reaching the MIC.

According to figure 5, that correlates the obtained data, we can say that the relationship between chemical release and the luciferase assay worked very well, especially for trigonelline and nicotinic acid, in which the concentrations released above the MIC were toxic to bacteria. It is also possible to observe in this figure, that trigonelline was released in much higher concentrations, above MIC values, than the nicotinic and chlorogenic acids, which were released in concentrations closer to their MIC values. This could be related with the polarity of their chains, as explained before.

Taking into account the abovementioned considerations, it seems like that nicotinic acid is a promising compound for a long-lasting effect, as this release from resins loaded at 50% remained above MIC value up to 28 days (4.67 mg/mL), which would ensure antimicrobial potential even at the end of the period tested. Also, their incorporation at this high concentration (50%), did not affect the degree of conversion of the resins.

Although all the compounds demonstrated antimicrobial action at certain concentrations, this effect was studied within the first 24 hours, and the HPLC analysis showed a decrease of the release over time. One option to be considered for future studies is the drug delivery system with release and reabsorption of drugs, ions and molecules.^25,26 Controlled release polymer systems, composed of either capsules or nanoparticles, represent a useful strategy for incorporating active substances, ensuring greater control over their release.^5,8

Moreover, it can be noted in the present study, that only very high concentrations for two of the coffee compounds promoted an antimicrobial effect, which could compromise the mechanical properties of polymeric materials, in the same way as detected for the degree of conversion of the resins doped with trigonelline and chlorogenic acid, both at 50% , and, consequently, make them not suitable for clinical application. Thus, in order to potentiate the antimicrobial action and reduce the amount of compound capable of producing a satisfactory effect, one more suggestion for future investigations would be to incorporate more than one compound into the same dental material.

As conclusion, this study showed that the three compounds tested had effective antimicrobial potential against S. mutans and were not cytotoxic. When incorporated into a resin matrix, it was demonstrated a bactericidal potential that was consistent with their concentration and their release from the resin. Therefore, these materials may be promising adjuncts for the prevention of dental caries disease.