Original Article

Comparison of the Effect of Fluoride 0.2% and a Combined Mouthwash (Flavonoid Compounds and Fluoride 0.2%) Against Streptococcus mutans and Lactobacillus acidophilus: In Silico and In Vitro Study

Please cite this article as follows: Omidi B, SarveAhrabi Y, Nejati Khoei S. Comparison of the effect of fluoride 0.2% And a combined mouthwash (flavonoid compounds and fluoride 0.2%) Against Streptococcus mutans and Lactobacillus acidophilus: in silico and in vitro study. Avicenna J Dent Res. 2023; 15(4):142-149. doi:10.34172/ajdr.1676

Background

Dental caries is one of the most common chronic and preventable diseases in the world (1). In general, caries is considered to be the result of the interaction between caries-causing oral flora and fermentable carbohydrates on tooth surfaces over time (2). According to several epidemiological studies, Streptococcus mutans is related to dental caries, and it seems that this gram-positive bacterium plays the main role in the initiation of caries (3). S. mutans affects tooth enamel by fermenting sucrose and producing lactic acid. In addition, this bacterium uses sucrose to make dental plaque. Dental plaque is made of dextran, which is a type of polysaccharide (4). S. mutansproduces three types of glucosyltransferases (GTF-I, GTF-SI, and GTF-S). The glucosyltransferase gene (gtfB) is encoded by GTF-I, is involved in the initial stages of glucan synthesis from sucrose, and is insoluble in water. This gene is one of the most important factors in the virulence of this bacterium (5-7). Glucan also plays a role in the formation of dental plaque and the firm adhesion of microorganisms to the tooth surface, resulting in the accumulation of acid and the beginning of decalcification on the surface of the enamel (8). Further, using advanced molecular techniques, several studies have shown that lactobacilli are located in the advanced areas of carious lesions, and probably these bacteria are related to dental caries (9). The reason for surface decay is the effect of acidic products resulting from bacterial fermentation, following the digestion of the protein matrix (matrix) by bacteria that cause tooth decay (10). Flavonoids are a group of naturally occurring polyphenolic compounds characterized by the flavan core and are one of the most common classes of compounds in fruits, vegetables, and plant-derived beverages (11). More than 8000 compounds with flavonoid structures have been identified. Together with carotenoids, they are responsible for the vibrant colors of fruits and vegetables. Some of the best-known flavonoids are quercetin and kaempferol. In plants, these compounds protect against UV rays, pathogens, and herbivores (12). Flavonoids are considered health-promoting and disease-preventing food supplements. Epidemiological, clinical, and animal studies show that flavonoids have antibacterial (13), antifungal (14), anticancer (15), and antioxidant (16) effects. According to the contents, this study focused on an in vitro and in silico comparison of the effects of fluoride 0.2% and a combination mouthwash (Flavonoid compounds and fluoride 0.2%) against S. mutans and L. acidophilus for use in mouthwashes and toothpastes.

Methods

In Silico: The crystal structure of glucansucrase from S. mutans (3AIE) and the structure of glucan-1, 6-alpha-glucosidase from L. acidophilus (4AIE) were received from https://www.rcsb.org (Protein Data Bank). The structures of flavonoids as ligands were received from https://pubchem.ncbi.nlm.nih.gov (PubChem).

In Vitro: All flavonoid compounds had been purchased from Sigma-Aldrich Company (Germany). The bacterial strains (S. mutans ATCC 35668 and L. acidophilus ATCC 4356) were organized by the Iranian Industrial Microorganisms Collection Center (Lyophilized). Microbiological assessments had been accomplished with the use of a Memmert- INC153T2T3 incubator.

In Silico

Ligand Preparation

The 2-dimensional (2D) structures of flavonoids (Table 1) as ligands (No. 1-10) were retrieved from ChemDraw Ultra 12.0.2.1076 (Figure 1) and saved in.pdb format. Use ChemBio3D Ultra 12.0 for the optimization of the ligands (17).

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Figure 1.

2D Structures of Flavonoid Compounds (1-10). Note. 2D: 2-dimensional

Figure 1.

2D Structures of Flavonoid Compounds (1-10). Note. 2D: 2-dimensional

Protein Preparation

The 2D structure of glucansucrase from Streptococcus mutans (PDB ID: 3AIE) and glucan-1, 6-alpha-glucosidase from Lactobacillus acidophilus (PDB ID: 4AIE) were obtained from the Protein Data Bank (https://www.rcsb.org/) with a resolution of 3.00 Å (Å: Angstrom) and in.pdb format. Subsequently, using Discovery Studio Client 4.5 software, the ligands and water molecules were separated from the main structures. Finally, the structures were prepared by adding polar hydrogens using AutoDockTools-1.5.6 software and saved in.pdbqt format.

Molecular Docking

Using the AutoDockTools (1.5.7) program, all polar hydrogen atoms have been introduced to the protein structure, and the partial masses of the ligands have been calculated and introduced with the Discovery Studio 4.5 Client program. The grid box for 3AIE was 118 × 118 × 118 dimensional, and dimensions have been additionally taken into consideration as X-center = 62.833, Y-center = 55.306, and Z-center = 3.139. Furthermore, the grid box for 4AIE was 94 × 94 × 94 dimensional, and dimensions have been additionally taken into account as X-center = 9.002, Y-center = 26.444, and Z-center = 27.248. The overall findings obtained through molecular docking were examined using AutoDockTools-1.5.6 software (18). To analyze the results in greater detail, the ligand-receptor complex corresponding to the conformation with the highest binding energy was prepared and assessed for each compounds using Discovery Studio Client 4.5 software (2D and 3D).

In Vitro

The bacteria that were prepared as lyophilized were inoculated in the broth medium. Brain heart infusion (BHI) agar/broth and Man–Rogosa–Sharpe (MRS) agar/broth were used for S. mutans and L. acidophilus., respectively. Next, the bacterial samples were kept for 48 hours in an anaerobic jar at 37 °C. Then, the bacteria suspension was prepared according to McFarland’s 0.5 standard.

Agar Well Diffusion Method

To perform this experiment, wells of 5 mm in diameter were created by a sterile pipette in culture media containing cultured bacterial suspension. Then, the wells were filled with samples (S. mutans wells: Fluoride 0.2% (control), fluoride 0.2% + eriocitrin (a), Fluoride 0.2% + galangin (b). L. acidophilus wells: Fluoride 0.2% (control), fluoride 0.2% + catechin (c), Fluoride 0.2% + eriocitrin (d), Fluoride 0.2% + isorhamnetin (e)). Next, the plates were put inside the incubator for 24 hours at 37 °C. It is worth noting that all the steps were performed near the flame and in a sterile environment (19). This experiment was repeated three times, and their mean was reported in the results.

Broth Dilution Method

To determine the minimum inhibitory concentration (MIC), a series of 9 tubes were used to test the different dilutions of each compound. It is noteworthy that the control sample was diluted in 9 separate tubes. The initial concentration of each compound was 2 mg/mL (2000 µg/mL), which was obtained by inserting 1 mL of the compounds into the first tube containing 1 mL of culture medium at a concentration of 1 mg/mL (1000 µg/mL). Different dilutions were obtained from tubes No. 1 (2 mg/mL) to No. 9 (0.007 mg/mL). To this end, 1 mL of the compounds in the first tube with a concentration of 2 mg/mL was diluted with 1 mL of culture medium in the second tube (BHI broth and MRS broth were utilized for S. mutans and L. acidophilus, respectively). In this way, 1 mL was removed from the first tube and added to the second tube, containing 1 mL. This was continued up to tube No. 9, then 1 mL was removed from the last tube and ejected, which eventually resulted in the half-dilution of the previous tube. Then, 50 μL of microbial suspension containing 1.5 × 108 bacteria were transferred to the tubes. All the test tubes were incubated (for 24 hours at 37 ̊C). After incubation, the tubes were tested for turbidity because of the bacterial growth. All tubes with no bacterial growth were sampled and cultured to determine the minimum bactericidal concentration (MBC) of the compounds. For this purpose, the tubes showing no bacterial growth were cultured on the culture medium (BHI agar and MRS agar were used for S. mutansand L. acidophilus, respectively). After incubation for 24 hours, the cultured plates were controlled for microbial growth. The lowest concentration of compounds in the relevant plates, exhibiting bacterial growth failure, was considered the MBC of those compounds (20).

Results

In Silico

All affinities of flavonoid compounds (1-10) against bacterial strains were calculated, the results of which are provided in Table 2. The affinity of all compounds was reported between -5.2 and -9.2 kcal/mol. Among these, compounds 7 (eriocitrin) and 9 (galangin) demonstrated the highest amount of H-bonds with amino acids against 3AIE; further, these compounds created the highest amount of variety and number of hydrogen bonds with amino acids such as asparagine, glutamine, aspartic acid, and glutamic acid with compound 7 (eriocitrin) and amino acids serine, tryptophan, and threonine with compound 9 (galangin). Compounds 4 (catechin), 7 (eriocitrin), and 10 (isorhamnetin) showed the highest amount of H-bonds with amino acids against 4AIE; moreover, these compounds created the highest amount of variety and number of hydrogen bonds with amino acids such as serine, glutamic acid, aspartic acid, lysine, and histidine with compound 4 (catechin), as well as amino acids aspartic acid, asparagine, leucine, tyrosine, proline, and serine with compound 7 (eriocitrin), along with amino acids arginine, asparagine, and aspartic acid with compound 10 (isorhamnetin). According to Figure 2, compound 7 (eriocitrin) has the ability to bind and inhibit both structures with an affinity of -8.6 for 3AIE and -9.2 for 4AIE. This compound against S. mutans (3AIE) can inhibit the structure by creating hydrogen bonds with amino acids glutamine (864 H), asparagine (625 H), aspartic acid (666 H), and glutamic acid (622 H). Additionally, against L. acidophilus (4AIE), it can inhibit the structure by creating hydrogen bonds with amino acids such as aspartic acid (327 H), asparagine (361 H and 326 H), leucine (285 H), tyrosine (530 H), proline (284 H), and serine (322 H).

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Figure 2.

AutoDock Vina Results of Catechin, Eriocitrin, and Isorhamnetin in the Binding Site of Glucan-1,6-alpha-glucosidase From L. acidophilus, as Well as Eriocitrin and Galangin in the Binding Site of Glucansucrase From S. mutans. Note. Bonds are shown by means of Discovery Studio software (2D and 3D). 2D: 2-dimensional

Figure 2.

AutoDock Vina Results of Catechin, Eriocitrin, and Isorhamnetin in the Binding Site of Glucan-1,6-alpha-glucosidase From L. acidophilus, as Well as Eriocitrin and Galangin in the Binding Site of Glucansucrase From S. mutans. Note. Bonds are shown by means of Discovery Studio software (2D and 3D). 2D: 2-dimensional

In Vitro: Herein, we have evaluated and compared the antibacterial activity of flavonoid compounds that performed best in the previous section (In silico). According to Figure 3, which deals with the inhibition zone of compounds against target bacteria, eriocitrin + fluoride 0.2% represented the best performance compared to all compounds. Based on the data in Table 3, it seems that the compound eriocitrin has a wide range of antimicrobial activities. Groups a (Fluoride 0.2% + Eriocitrin against S. mutans) and d (Fluoride 0.2% + Eriocitrin against L. acidophilus) were most effective between all compounds, respectively, (IZ = 26 ± 0.5 mm, MIC = 250 µg/mL, MBC = 500 µg/mL and IZ = 31 ± 0.5 mm, MIC = 125 µg/mL, MBC = 250 µg/mL).

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Figure 3.

Antibacterial Results of Flavonoid Compounds: [Streptococcus mutans: A: Control (Fluoride), B: Eriocitrin + Fluoride - Lactobacillus acidophilus: A: Control (Fluoride), B: Eriocitrin + Fluoride]

Figure 3.

Antibacterial Results of Flavonoid Compounds: [Streptococcus mutans: A: Control (Fluoride), B: Eriocitrin + Fluoride - Lactobacillus acidophilus: A: Control (Fluoride), B: Eriocitrin + Fluoride]

Discussion

Tooth decay is still considered one of the most serious public health problems, and every year, it imposes a great financial burden on healthcare services all over the world, especially in developing countries (21). One of the most important ways to reduce costs is to prevent rotting. Currently, the most effective way to prevent tooth decay, in addition to reducing the intake of sugary substances, is to use mechanical methods such as brushing and flossing (22). However, other methods and chemical antimicrobial agents such as mouthwashes have been used due to the insufficiency of mechanical methods alone to control plaque and prevent periodontal diseases and tooth decay, on the one hand, and the complex bacterial etiology of periodontal diseases and tooth decay, on the other hand (23). Mouthwashes contain effective preventive substances and play a significant role in improving oral health. From a dental point of view, the ideal mouthwash should have properties such as non-discoloration of teeth and mucous irritation, no toxic effects, and a good taste. However, a mouthwash that has all the mentioned properties has not yet entered the market, and researchers are still attempting to find a mouthwash that has the maximum of these properties (24). Oral streptococci are an important part of the collection of dental plaques, and one of the most important members of this collection is S. mutans, which has been associated with caries in several epidemiological studies and is believed to play the main role in the initiation of caries (25). These bacteria make up to 60% of the natural flora of the surfaces inside the mouth, which can cause the formation of biofilm on the surface of the tooth enamel by producing non-soluble viscous polymer materials such as glucan and levan. The primary mechanism of binding S. mutans is the formation of glucan homopolymers from sucrose by glucosyltransferase. Glucosyltransferase catalyzes two main reactions, which include the breakdown of sucrose into glucose and fructose (sucrazy activity) and the transfer of glucose units at the 6-C-3/C position to produce glucan (transferase activity) (26). The present study was conducted with the aim of investigating the effect of combined mouthwash (Flavonoid compounds and fluoride 0.2%) and fluoride 0.2% on the growth of S. mutans and L. acidophilus. Examining the in silico results showed that most of the flavonoid compounds, which were selected in this research, had the ability to inhibit the bacterial samples in a range of -5.2 to -9.2 kcal/mol. The binding energy determines the degree of binding between the compounds and the active sites of the 3AIE and 4AIE. Whatever this number is more negative, binding between the compound and the 3AIE and 4AIE is stronger. In this research, the flavonoid compound eriocitrin was reported to have an effective and stronger interaction than others. It created the highest hydrogen bonds with amino acids such as asparagine, glutamine, aspartic acid, and glutamic acid by glucansucrase from S. mutans, as well as hydrogen bonds with amino acids such as aspartic acid, asparagine, leucine, tyrosine, proline, and serine by glucan-1, 6-alpha-glucosidase from L. acidophilus. Many studies have been reported on the effects of various flavonoid classes in in silico and in vitro conditions. In this regard, Bao et al sought to identify potential inhibitors targeting S. mutans sortase A. They reported that several similar compounds composed of benzofuran, thiadiazole, and pyrrole, which exhibited good affinities and appropriate pharmacokinetic parameters, were potential inhibitors to impede the catalysis of SrtA (27). Likewise, Babaeekhou and Ghane in silico studied the antimicrobial activity of ginger on cariogenic bacteria and stated that some ginger compounds with high affinity to investigated enzymes can be considered candidate compounds for anti-caries drug development studies (28). Eriocitrin is a flavanone-7-O-glycoside between the flavanone eriodictyol and the disaccharide rutinose. It is commonly found in lemons and other citrus fruits. Similarly, Nisar et al evaluated the pharmacological effects of eriocitrin and concluded that eriocitrin has potent biological actions due to its strong antioxidant, antitumor, anti-allergic, antidiabetic, and anti-inflammatory activities. Eriocitrin is more potent in suppressing oxidative stress in diabetes mellitus and other chronic diseases incurred by excessive oxidative stress. During metabolism, eriocitrin is metabolized by the gut microbiota and a chain of molecules such as eriodictyol, methy-eriodictyol, 3,4-dihydroxyhydrocinnamic acid (DHCA), and much more conjugated molecules (29). In vitro results showed that eriocitrin + fluoride 0.2% has the best performance in comparison to all compounds (IZ = 26 ± 0.5 mm, MIC = 250 µg/mL, MBC = 500 µg/mL against S. mutans and IZ = 31 ± 0.5 mm, MIC = 125 µg/mL, MBC = 250 µg/mL against L. acidophilus). In addition, Miyake and Hiramitsu examined antimicrobial substances against oral bacteria from lemon peel and found that 8-geranyloxypsolaren, 5-geranyloxypsolaren, and 5-geranyloxy-7-methoxycoumarin exhibited high antibacterial activities (30). Moreover, Zajkani et al compared the effect of fluoride 0.2% and a combination mouthwash (xylitol and fluoride) on S. mutans and L. acidophilus growths and reported that the fluoride mouthrinse at different concentrations, because of having a good inhibitory effect on S. mutans and L. acidophilus in both methods, was more effective compared with Fuchs mouthrinse (31). Adamczak et al investigated the antibacterial activities of some flavonoids and organic acids, as well as 13 common flavonoids (flavones, flavonols, and flavanones) and 6 organic acids (aliphatic and aromatic acids). They reported that all tested compounds showed antimicrobial properties, but their biological activity was moderate or relatively low. Bacterial growth was most strongly inhibited by salicylic acid (MIC = 250–500 μg/mL). These compounds were generally more active against gram-negative bacteria, such as Escherichia coli and Pseudomonas aeruginosa, than gram-positive ones, including Enterococcus faecalis and Staphylococcus aureus (32).

Conclusion

According to the results of this study, eriocitrin can be used as a new mouthwash, along with fluoride 0.2%.

Acknowledgements

We appreciate all the professors who participated voluntarily in this investigation.

Authors’ Contribution

Conceptualization: Yasin SarveAhrabi, Behin Omidi.

Funding acquisition: Yasin SarveAhrabi, Behin Omidi.

Investigation: Yasin SarveAhrabi, Behin Omidi, Sarina Nejati Khoei.

Methodology: Yasin SarveAhrabi, Behin Omidi, Sarina Nejati Khoei.

Resources: Yasin SarveAhrabi, Behin Omidi.

Supervision: Yasin SarveAhrabi, Behin Omidi.

Writing–original draft: Yasin SarveAhrabi.

Writing–review & editing: Yasin SarveAhrabi, Behin Omidi.

Competing Interests

The authors declare that they have no conflict of interests.

Funding

This project was supported by the Islamic Azad University, Central Tehran Branch, Tehran, Iran.

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