| Abstract|| |
Context: Epigallocatechin-3-gallate (EGCG) is a flavonoid extracted from green tea that demonstrated antimicrobial activity.
Aims: To evaluate the efficacy of EGCG 0.5%, 1%, and 2% concentrations as an antimicrobial solution in dentin caries-like lesions induced in a bacterial-based in vitro model.
Materials and Methods: Twenty-five human dentin specimens were submitted to a microbial-based caries model by immersion in brain heart infusion (BHI) broth inoculated with Streptococcus mutans UA159, for 5 days. After the demineralization period, the specimens were randomly divided into groups: Group I: 0.9% saline solution; Group II: 2% chlorhexidine digluconate; Group III: 0.5% EGCG; Group IV: 1% EGCG; and Group V: 2% EGCG. After the treatments, carious dentin samples were harvested from dentin specimens and analyzed by colony-forming unit (CFU) counts. Data were analyzed by ANOVA and Tukey's test.
Results: Log reduction values (SD, CFU.mg -1 ) for Groups I-V were: 5.02 (0.16), 3.96 (0.43), 4.74 (0.26), 4.89 (0.56), and 4.91 (0.40), respectively. There was no statistical difference between the EGCG concentrations and saline solution (P > 0.05). Furthermore, there was no statistical difference between EGCG concentrations (P > 0.05). However, there was a statistically significant difference between the chlorhexidine digluconate group and the other groups (P < 0.05).
Conclusion: EGCG at the studied concentrations were not effective in eliminating S. mutans from dentin caries-like lesions.
Keywords: Dentin; catechin; streptococcus mutans
|How to cite this article:|
de Assis JS, Lima RA, Marques Lima JP, Azevedo Rodrigues LK, Santiago SL. Effect of epigallocatechin-3-gallate application for remaining carious dentin disinfection. J Conserv Dent 2015;18:51-5
|How to cite this URL:|
de Assis JS, Lima RA, Marques Lima JP, Azevedo Rodrigues LK, Santiago SL. Effect of epigallocatechin-3-gallate application for remaining carious dentin disinfection. J Conserv Dent [serial online] 2015 [cited 2020 Jan 25];18:51-5. Available from: http://www.jcd.org.in/text.asp?2015/18/1/51/148896
| Introduction|| |
Conventional treatment of dentinal caries is focused on the surgical removal of the damaged dental areas and, subsequently, restoration using a filling material. Not long ago, the main mode used by dentists for treating deep carious lesions was by taking out the diseased tissues. However, the aim of operative dental treatment is to maintain tooth integrity as long as possible. Thus, the modern and desirable management of deep caries lesions should be centered on the principles of minimum intervention dentistry.
In dentin, caries lesions develop at different depths being the superficial layers (infected layer) the most damaged, making it difficult the preservation and remineralization of this substrate. However, in deeper layers (affected layer), although demineralization is also present, the cross-banded ultra-structure of the collagen matrix is preserved. Thus, the elimination of pathogens present in affected dentin can make the preservation of this substrate possible, especially in those areas where collagen is not injured by the carious process.  Besides, the sealing of infected carious dentine below dental restorations makes the phenotypic and genotypic diversity of the surviving microbiota less complex. However, it has also been suggested that bacterial growth occurs beneath the restoration and this growth may be restricted to those genotypes best able to exploit the environment with limited availability of nutrients.  Consequently, bactericide substances can be a viable option for dentin disinfection that decreases the microbial load. Therefore, the use of cavity disinfectant solutions has been recommended. 
Chlorhexidine digluconate is considered a gold standard agent in controlling biofilm formation due to its ability to significantly decrease the levels of oral microorganisms. An ideal cavity disinfectant should also present a low toxicity or, preferably, no toxic effects to the pulp cells, especially to odontoblasts.  However, it has been previously demonstrated that chlorhexidine presents cytotoxic effects on a variety of cell lines,  similar to different chemical agents indicated for use as cavity disinfectants.  This way, it is important to look for natural alternatives for decreasing dentin contamination that lowers the pulp cells. 
In this regard, leaves of Camellia sinensis contain polyphenolic components with antimicrobial activity against a wide spectrum of microbes. The most abundant components in green tea are polyphenols, in particular flavonoids such as the catechins, catechin gallates, and proanthocyanidins.  This polyphenolic component exhibits antimutagenic, anti-inflammatory, antidiabetic, cancer-preventive, and antimicrobial properties. 
Over 50% of green tea is composed of Epigallocatechin-3-gallate (EGCG), and it has been suggested that it is the main flavonoid responsible for most of the potential health benefits attributed to green tea intake.  The effects of green tea polyphenols, more specifically against S. mutans, have been previously demonstrated. EGCG is indicated as the major polyphenol component in tea that interferes with the virulence of cariogenic bacteria. 
The purpose of this study was to evaluate the efficacy of this catechin at concentrations of 0.5%, 1%, and 2% as an antimicrobial solution in a microbiological model of caries-like dentinal lesions. The null hypothesis tested was that different solutions have similar effectiveness in reducing S. mutans.
| Materials and Methods|| |
This study was randomized, comprising five groups: Group I: 0.9% saline solution (negative control); Group II: 2% chlorhexidine digluconate (positive control); Group III: 0.5% EGCG; Group IV: 1% EGCG; and Group V: 2% EGCG. The teeth were randomly allocated to the groups, with five experimental units per group, according to a computer generated randomization list using a table created in the Excel system (Microsoft, Richmond, CA, USA).  The experimental design was performed in triplicate in order to reduce the inherent bias related to microbiological procedures.
Fifty caries-free third molars were collected, after the patients' informed consent had been obtained, under a protocol reviewed and approved by the local Research and Ethics Committee (Protocol # 329.632). The teeth were stored in a 0.01% (w/v) thymol solution for a month or less and remained refrigerated until use.
Each tooth was fixed in an acrylic device and cut using a water-cooled diamond saw mounted in a cutting machine (IsoMet Low Speed Saw; Buehler, Ilinois USA) in order to obtain the dentin specimen (4 × 4 × 2 mm), a fragment of the dentin central portion of the third molar. The occlusal plane surfaces obtained were assessed by examination under a microscope, at 40× magnification, to ensure complete removal of enamel. Only the occlusal dentin surface was used and the other surfaces were protected with resistant acid varnish (Risqué, São Paulo, SP, Brazil), resulting in an area of 16 mm2 that served as a microbial surface on which caries lesions were produced. The specimens were mounted on metal appliances, which contained two claws to fix them and a handle to allow carriage on the 24-well polystyrene plates, and immersed in sterile distilled water. They were then sterilized by autoclaving to 121°C for 15 min  and stored in 100% humidity.
Microbiological model of caries-like dentinal lesions ,
After sterilization, the specimen and appliance were removed from the distilled water and immersed in sterile brain-heart infusion broth (BHI CM0225; Oxoid LTD, SP, Brazil) containing 5% (w/v) sucrose. All BHI-containing 24-well polystyrene plates, except those that served as contamination controls, were inoculated with 0.1 ml [2 × 10 8 colony-forming unit (CFU) ml-1] of an overnight culture of S. mutans UA159. After 18 hr, Gram test was used to verify the existence of S. mutans only. A specific optical density was determined using a spectrophotometer (Ultrospec 1100 pro; Amershanm Biosciences) and was used for all samples to adjust the inoculum to the same cell number. Inoculation of each BHI-containing 24-well polystyrene plates was performed only on the first day. The culture medium was changed daily during five consecutive days. The 24-well plates were incubated at 37°C in 5% CO 2 during the entire experimental period. At each transfer time, samples of the cultures were streaked onto BHI agar plates and incubated at 37°C in an atmosphere of 5% CO 2 in order to check for purity.
Exposure of the specimens to EGCG and controls
A 0.9% saline solution (NaCl) was prepared at a ratio of 0.9 g/100 ml and previously autoclaved (121°C, 15 min). Chlorhexidine digluconate at 20% (v/v) was diluted to give 2% chlorhexidine digluconate (v/v) and, because it is an organic substance, was sterilized in a filter. EGCG from green tea (EGCG, 95%, wt/wt) (Sigma-Aldrich Corp. St. Louis, MO) was dissolved into distillated water at concentration of 20 mg/ml. Then the EGCG/distillated water was diluted at concentrations of 10 mg/ml and 5 mg/ml, in this way obtaining the three different concentrations used in this study.
On the fifth experimental day, the biofilm formed over the specimens was removed and the caries dentin was exposed. The specimens were randomly allocated to five different treatment groups (five per group).
All dentin specimens were treated for 60 sec with 15 μl of each solution and dried with an absorbent paper. 
Afterwards, carious dentin was collected from the specimens using a # 15 scalpel blade. The dentin samples were weighed in pre-weighed microcentrifuge tubes and 0.9% saline solution (NaCl) was added at the rate of 0.1 ml of solution per 1 mg of dentin. Subsequently, the suspension was serially diluted (1:10-1:100,000) with 0.9% saline solution (NaCl). Samples were plated in triplicate on a BHI agar, and incubated for 48 hr at 37°C in a 5% CO 2 atmosphere. Representative colonies with typical morphology of S. mutans were counted after 48 hr using a colony counter, and the results were expressed in CFU/mg of dentin. 
All experiments were performed in triplicate and the log reduction results were calculated by subtraction of the initial values from the final values of CFU.mg -1 of dentin after being transformed by Log10. The differences between the experimental groups and control groups were analyzed by BioEstat 5.0. One-way analysis of variance (ANOVA) was performed and followed by a post hoc Tukey's test to compare the antimicrobial efficacy between the groups. The level of significance was set at 5%.
| Results|| |
No significant difference between mean values were found for EGCG treatment in different concentrations when compared to Group I (P > 0.05), and there was no statistical difference between the EGCG concentrations (P > 0.05). Only Group II presented statistically different results from the others, as seen in [Figure 1] (P < 0.05).
|Figure 1: Log and standard deviation obtained according to the treatments|
Click here to view
| Discussion|| |
The observed results showed that all EGCG concentrations were not effective in reducing S. mutans levels. This leads the authors to reject the null hypothesis.
To the best of the authors' knowledge, no study has previously investigated the effect of catechins on infected dentin. Thus, the discussion of the current results was performed using researches that investigated the antimicrobial effects of EGCG on the growth of S. mutans. According to Xu et al.,  discrepant results of antimicrobial activity of EGCG can be found, depending on the type of bacterial organization. In planktonic cells, MIC (minimum inhibitory concentration) and MBC (minimum bactericidal concentration) values in a BHI medium were 625 μg/ml and >1,250 μg/ml, respectively. Moreover, EGCG inhibited the formation of S. mutans biofilm in a BHI medium with a MBIC 50 (the lowest flavonoid concentration that showed at least 50% inhibition of the formation of biofilms compared with control) of 312.5 μg/ml. In the data presented by Mankovskaia et al.,  the MIC values for Epigallocatechin-3-gallate against S. mutans were within reference ranges of 31.25 μg/ml to 625 μg/ml, depending on the bacterial strain and culture medium. Du et al.,  concluded that antibacterial activity of the dental adhesives was increased after a 200 μg/ml or higher concentration of EGCG incorporated. Hirasawa et al.  demonstrated that more than 2000 μg/ml of EGCG is required in a mouth-rinsing to be effective against S. mutans present in dental plaque. These researches suggest that EGCG may represent a natural alternative for decreasing dentin contamination with a lower possibility of damaging the pulp cells. 
The concentrations in which catechins are effective as an antimicrobial agent are wide, and one of the causes is the application of different methods of determining bacterial susceptibility. Usually, lower levels of EGCG are necessary to inhibit bacterial culture in planktonic cultures than in oral biofilm. This is due to the fact that in planktonic cultures, bacteria find themselves free in suspension, whereas in the biofilms there is a structured and dynamic ecosystem, which acts coordinately. These types of microorganism organizations present distinct properties. , Biofilms are composed of a variety of species' mechanisms that establish communication between them, including transfer of genetic material, either as virulence traits or as bacterial resistance faster than that occurring in culture planktonic. , Such characteristics favor these structures and develop resistance to many therapeutic agents. 
EGCG at concentrations of 0.5%, 1%, and 2% was not effective as a cavity disinfectant on carious dentin in eliminating the pathogen. In a pilot study, these concentrations presented satisfactory results in MIC and MBC tests. Most likely the ineffectiveness in reducing or eliminating S. mutans can be attributed to interactions formed between the tea catechins and the collagen fibrils. Proteins present on the surface dentin that remained from the nutrient-rich BHI medium may bind or even precipitate catechins and that binding may jeopardize their antimicrobial activity. , Therefore, it can be suggested that there were interactions between the catechin and specific sites in the molecular structure of the collagen.
Unsurprisingly, in the present study, chlorhexidine digluconate was able to significantly decrease the levels of S. mutans inside dentinal tubules. These results are in agreement with a previous study that tested this agent in vitro and in situ against cariogenic bacteria. 
This study aimed to evaluate the antimicrobial activity of the flavonoid applied directly on carious dentin, which has not yet been investigated in the literature. In order to perform the cariogenic challenge, the authors employed the microbiological method, in which the dentinal slabs remained immersed for five days in the nutrient medium inoculated with S. mutans. The production of in vitro caries-like lesions may be achieved in different ways, but the microbiological method presents a pattern of collagen degradation morphologically more similar to natural lesions, with an evident infected layer in the dentin caries lesion simulated by that method.  Dentin submitted to a cariogenic challenge seems to be more resistant to antimicrobial therapy. In the development of the dental biofilms, salivary proteins and glycoproteins on oral surfaces and other organisms play a decisive role in bacterial adhesion.  Collagen Type I, the main organic component of dentin, is recognized by streptococci that bind to collagen and may facilitate the bacterial adhesion and tissue penetration.  Therefore, EGCG concentrations used in this study are shown to be greater than those used in studies of culture planktonic and biofilm.
The absence, until this date, of specific answers about the mechanism of EGCG anti-cariogenic points to the need for further research in this direction. There are numerous studies on the anti-cariogenic potential of the flavonoid. However, there is a great variability in the conditions and methods employed, which makes it difficult to attribute this potential anti-cariogenic to a specific factor or a set of elements.
| Conclusions|| |
EGCG concentrations used as a cavity disinfectant was not efficient in reducing S. mutans levels compared to chlorhexidine digluconate.
| Acknowledgement|| |
This project has received financial support (Grant # 131437/2011-9) from CNPq. The authors thank David Queiroz for his help during experimental procedures. The first author received a scholarship during this study from CNPq. This paper was based on a thesis submitted by the first author to the Faculty of Pharmacy, Dentistry and Nursing of the Federal University of Ceará, in partial fulfillment of the requirements for an MS degree in Dentistry.
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Prof. Sérgio Lima Santiago
Rua Monsenhor Furtado, s/n Rodolfo Teófilo, 60.430-355 Fortaleza-CE
Source of Support: None, Conflict of Interest: None