Antibacterial and antioxidant effect of ethanol extracts of Terminalia chebula on Streptococcus mutans

Abstract Objective Dental caries is a high prevalent chronic bacterial infectious disease caused by plaque, a bacterial colony deposited on tooth surfaces and gum tissues. Streptococcus mutans is a primary cariogenic bacterium commonly found in the human oral cavity. Oral hygiene products containing antibacterial ingredients can be helpful in caries management. In this study, we investigated the anticaries mechanism of the ethanol extract of Terminalia chebula (EETC) on S. mutans and suggest its possible application as a functional ingredients for oral hygiene products. Materials and methods The EETC was prepared from the Terminalia chebula fruit. Disk diffusion, minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and colony forming unit (CFU) were analyzed to observe the antibacterial activity of EETC. The glucan formation was measured using the filtrate of bacterial culture medium and sucrose. Gene expression was analyzed using RT‐PCR. Cytotoxicity was analyzed using the MTT assay. The radical‐scavenging activities of DPPH and ABTS were also tested to verify the antioxidant activity of EETC. Results The antibacterial activity of the EETC was explored through a disc diffusion analysis and CFU measurement. EETC treatment decreased insoluble glucan formation and gene expression of glycosyltransferase B (gtf B), glycosyltransferase C (gtf C), glycosyltransferase D (gtf D), and fructosyltransferase (ftf). The MIC and MBC of EETC on S. mutans were not cytotoxic to gingival fibroblasts. In addition, we observed DPPH and ABTS‐radical scavenging activities of EETC. Conclusions These results indicate that the antibacterial and antioxidant effects of EETC may contribute to oral hygiene products for dental caries management.

resistance is growing. In order to overcome these shortcomings, attempts to discover natural antibacterial ingredients for oral care are continuing.
The oral cavity is a suitable environment for bacterial growth and propagation. The presence of bacteria in the mouth readily stimulates the formation of dental plaque, which accumulates on both hard and soft tissues as dental calculus. The cause of tooth decay is the production of insoluble saccharide polymers and organic acids by oral bacteria. Glycosyltransferase (GTase) and fructosyltransferase (FTase) convert saccharide into insoluble polymers such as glucan and fructan (Wexler et al., 1993). These insoluble polymers adhere to the enamel surface and facilitates stable bacteria growth and incorporation of many types of bacteria. Taken together, locally attached bacteria produce persistent organic acids through glycolysis, which change the acidic environment around the tooth surface, thereby leading to demineralization. Streptococcus spp. and Lactobacillus spp. have been reported as being causative bacteria of tooth decay. Streptococcus mutans is one of the main causes of bacterial tooth decay. S. mutans is also an acid-resistant bacteria and therefore contributes to sustained demineralization in acidic environments.
The antibacterial effect of Terminalia chebula fruit extracts have been reported on Salmonella typhi, Staphylococcus epidermidis, Staphylococcus aureus, Bacillus subtilis, and Pseudomonas aeruginosa in accordance with the extraction methods (Kannan et al., 2009).
The antibacterial effect of T. chebula fruit extracts on Streptococcus mutans has been also reported (Nayak et al., 2014). However, our present understanding of the genetic mechanism in anticaries activity of the extract of T. chebula is insufficient. In addition to considering the antibacterial function of the extract of T. chebula on S. mutant in the oral cavity, we also need to investigate its safety on oral cells.
In the present study, we determined the antibacterial effects of an ethanol extract of T. chebula (EETC) on S. mutans and elucidated the biological mechanisms that support its anticaries effect. This result verifies that EETC is safe for gingival epithelial cells and effective in treating anticaries through antibacterial activity.

| Plant material
The ethanol extract of the T. chebula fruit was provided by COSMAX  (Daejeon, Korea) and was cultured in brain-heart infusion (BHI) broth (Becton, Dickinson and Company, Baltimore, MD, USA) or BHI agar at 37 C.

| Drug susceptibility test
Drug susceptibility was assessed using the disc diffusion method. Briefly, a bacterial suspension in agarose solution was inoculated on BHI agar plates and the gel was allowed to solidify completely at room temperature. Whatman filter discs with infused drug were placed on the plates and cultured for 24 h. Drug susceptibility was assessed using a linear fitting of the squared radius (diameter in mm) of the inhibition zones.

| MICs and MBCs
MICs for EETC was determined in triplicate via the broth dilution method after incubation at 37 C for 24 h. Briefly, EETC were added to 10 ml volumes of liquid medium resulting in 0, 0.1, 0.5, 1, 5, 10, 15, 20, 25, 30 μg/ml concentration and the cultured strain was inoculated such that the absorbance was 0.1 at 600 nm. After 24 h, the absorbance of the culture medium was measured at 600 nm. The concentration of EETC with an absorbance of 0.1 ± 001 or less was taken as the MIC of EETC. To determine the MBC for the EETC, bacteria were cultured for 24 h in a liquid medium containing a 10-10 À7 serial dilution of EETC. 100 μl of the diluted bacterial solutions was smeared on agar plate and incubate for 3 days before counting the number of colonies. Then the number of bacteria per 100 μl was estimated by inverting the dilution factor and we defined the minimum concentration of the extract required to kill 99.99% of the bacterial cells compared to the control group to be the MBC.

| Colony forming unit (CFUs)
The number of viable cells was measured using CFU. S. mutans was cultured until the absorbance of 600 nm was 0.4. 100 μl of the cultured medium was added to 900 μl liquid medium in which the EETC was added (0, 0.1, 0.5, 1, 5, 10, 15, 20 μg/ml) to make a 10-fold diluted bacterial solution. Dilution was repeated to 10 À5 . 100 μl of the diluted bacterial solution was smeared on agar plates, incubated for 3 days, and colonies were counted.

| Glucan formation
S. mutans was cultured in liquid media. The media was centrifuged at 10,000 rpm for 30 min and supernatant was filtered using 0.22 μm filter. After then, a filtrate was prepared using an Amicon Ultra Centrifugal Filter (MWCO 30 kDa,Cat. No. UFC903008,Millipore) and used as a bacterial enzyme solution containing GTase. 200 μl of enzyme solution and/or EETC was added to 800 μl of sucrosecontaining substrate solution [sucrose 12.5 mg, NaN 3 0.25 mg/ml of 50 mM potassium phosphate buffer (pH 6.5)] to make 1 ml reaction solution which were reacted at 37 C for 36 h. Then reaction solution was centrifuged then and supernatant was discarded. 4 ml of 50 mM potassium phosphate buffer (pH 6.5) was added and sonicated for 5 min. The absorbance of this solution was measured at 550 nm.

| RT-PCR
Total RNA was isolated using the TRIzol reagent according to the manufacturer's instructions (Invitrogen, Carlsbad, CA, USA). Firststrand cDNA synthesis was performed using 2 μg of the total RNA and Promega's reverse transcription system (Madison, WI, USA). The primer sequence for RT-PCR was shown in Table 1. PCR amplification was carried out in a reaction mixture containing 0.5 μg first-strand cDNA and 10 pmol primers and consisted of 30 cycles. The amplified PCR product was electrophoresed on a 2% agarose gel in 1 Â Tris-Borate-EDTA buffer containing ethidium bromide and visualized using the Gel Doc 2000 system (BioRad Laboratories, CA, USA). Images were analyzed using the Image J program in the same pixel area (National Institutes of Health, Bethesda, MD, USA).

| MTT assay
Immortalized gingival fibroblasts were grown in DMEM/F12 (3:1) supplemented with 10% FBS, 1 Â 10 À10 M cholera toxin, 0.4 mg/ml hydrocortisone, 5 μg/ml insulin, 5 μg/ml transferrin and 2 Â 10 À11 M triiodothyronine at 37 C in a humidified atmosphere of 5% CO 2 . Cells (5 Â 10 3 cells/well) were seeded into 96-well culture plates and left overnight to adhere. These cells were treated with various concentrations of EETC for 24 and 48 h, respectively. Viable cells were detected by incubating with 5 mg/ml MTT solution for an additional 4 h at 37 C, followed by the dissolution of the produced formazan product in the cells with 100 μl of DMSO. Absorbance was measured at 570 nm with a microplate reader (Synergy™ HTX Multi-Mode Microplate Reader, BioTek).

| Statistical analysis
Statistical analyzes were conducted using InStat GraphPad Prism ver.

| Antibacterial effect of EEDC on S. mutans
The disk diffusion method was used to verify the growth inhibition effect of the EETC on S. mutans. 1 Â 10 4 CFU of S. mutans were T A B L E 1 Primer sequence and annealing temperature for RT-PCR

Target gene
Primer sequence cultured on an agar plate with a paper disk containing different concentrations of EETC. As shown in Figure 1, 5 $ 20 μg/ml EETC significantly inhibits S. mutans growth. The diameter of the clear zone where the S. mutans growth was suppressed is shown in Table 2, according to the concentration of EETC. We observed that a clear zone of 2.6 ± 0.23 mm at 5 μg/ml EETC, 6.03 ± 0.17 mm at 10 μg/ml, 10.21 ± 0.19 mm at 15 μg/ml, and 14.30 ± 0.11 mm at 20 μg/ml.

| Inhibitory effect of EETC on S. mutans growth
To observe the growth inhibition of EETC, S. mutans cultured in the 0 $ 20 μg/ml EETC was diluted 10 times and then cultured on an agar plate to measure the number of colonies. As shown in Table 3

| MIC and MBC of S. mutans by EETC
In order to measure the MIC required for the EETC to inhibit the S. mutans growth, the absorbance was measured after culturing the S. mutans in a medium containing 0 $ 30 μg/ml of EETC. In addition, S. mutans cultured in a medium containing EETC was diluted 10 times and cultured on an agar plate to count the number of colonies and measured MBC. As a result of this experiment, the MIC of S. mutans was 10 μg/ml and MBC was 20 μg/ml.

| Inhibitory effect of EETC on glucan formation by S. mutans
The glucan formation was analyzed in the reaction mixture with a filtrate of the S. mutans culture medium and sucrose as substrates. As shown in This result indicates that lower concentration than MIC of S. mutans by EETC inhibits glucan formation and related gene expression.

| Effect of EETC on gingival fibroblasts
The effect of EETC antibacterial concentration on gingival fibroblasts was analyzed by MTT assay. Cells were treated with 0 $ 30 μg/ml of EETC for 24 and 48 h, and live cells were analyzed. As shown in Figure 3, we did not observe cytotoxicity below 25 μg/ml of EETC. However, a decrease of viable cells was observed at 30 μg/ml EETC. This result indicates that using MBC (20 μg/ml) of the EETC on S. mutans is safe for gingival fibroblast cells.

| Antioxidant effect of EETC
To investigate the antioxidant effect of EETC, we tested its radicalscavenging activity by using DPPH and ABTS free radicals. Ascorbic F I G U R E 1 Antibacterial effect of EETC on S. mutans growth. S. mutans (1 Â 10 4 CFU) were cultured on an BHI agar plate with a paper disk containing the EETC. The diameter of the bacterial growth inhibition zone was calculated in millimeters (mm) acid was used as a reductant for the radical-scavenging molecule.
As shown in Figure 4a,b, the radical-scavenging activity of EETC was relatively lower than that of ascorbic acid, but it showed significant activity. The DPPH or ABTS-radical-scavenging activity of EETC was increased in a concentration-dependent manner.  (Struzycka, 2014). Particularly, markedly elevated levels of S. mutans is detected in the palque from the caries lesions and is F I G U R E 2 Inhibitory effect of EETC on glucan formation and gene expression related to insoluble saccharide polymer in S. mutans.
(a) The filtrate of S. mutans culture medium and sucrose were mixed and kept for 36 h at 37 C. After centrifugation, the precipitate was sonicated and the absorbance was measured at 550 nm. *p < 0.001 versus reaction with the filtrate from S. mutant culture without EETC.
(b) RT-PCR was performed with primers for glycosyltransferase B ( gtf B), glycosyltransferase C (gtf C), glycosyltransferase D ( gtf D), and fructosyltransferase (ftf ). Gene expression was observed in 2% agarose gel electrophoresis. The result shown is the representative images from several experiments. Images were captured and density was measured using ImageJ program. Relative density was plotted in fold changes as a graph.
The values of the individual experiments are expressed as the mean ± standard error of three independent experiments. *p < 0.05, **p < 0.001 versus control considered to be the bacteria most closely contributes to the coronal decay (Duchin & van Houte, 1978.). S. mutans can produce organic acids through glycolysis, grows even in environments with a pH of 5.0 or less around the tooth surface biofilm, and maintains a significant glycolysis activity, thus exhibiting strong caries activity (Lemos & Burne, 2008). Facultative anaerobic bacteria Lactobacillus casei, Lactobacillus viscosus, Lactobacillus acidophillus are also acid-resistant and produce lactic acid, and are particularly involved in caries progression such as dentine caries (Duchin & van Houte, 1978). A. viscosus and A. odontolyticus are implicated as the pathogen of root surface caries (Dame-Teixeira et al., 2016).
Biofilm has a thick out layer formed by the insoluble polymer that outside molecules have difficulty penetrating. Bacterial interactions in biofilm easily induce genetic mutations, thereby increasing their resistance to antibiotics and antibacterial substances. Since the degree of deposition and maturity of dental plaques is related to the severity of oral diseases including dental caries, continuous management of dental plaques and maintenance of proper oral hygiene are important for the prevention of oral diseases. Long-term use of synthetic drugs, such as antibiotics, for treatment of dental diseases causes antibioticresistant bacteria. Therefore, for the purpose of preventing and treating oral diseases, there are active studies to observe the bacteriostatic or bactericidal effect of various plant extracts, including herbal medicines. In this study, we found that an ethanol extract of the T. chebula fruit (EETC) is effective in antibacterial use for dental caries that cause the bacteria S. mutans. The antibacterial activity of EETC was confirmed through disc diffusion analysis and CFU measurement.
Terminalia chebula (myrobalan) is widely used in the traditional medicine of India and Iran to treat diseases that include dementia, constipation, and diabetes (Jokar et al., 2016). Many of these beneficial effects of T. chebula fruit are related to the presence of various phytochemicals, including steroids/sapogenins, saponins, anthraquinone derivatives, flavonoids, and tannins (Lee et al., 2007;Rathinamoorthy & Thilagavathi, 2014). The most important component in the fruit is tannin. T. chebula has a tannin content of 32%-45% that includes gallic acid, ellagic acid, chebulic acid, chebulinic acid, punicalagin, and tannic acid. The flavonoids quercetin, catechin, and kaempferol have been detected (Jokar et al., 2016). T. chebula fruit is effective in the treatment of bacterial infections (Kim et al., 2006;Rai & Radhika, 2009). Clinical trials of T. chebula fruit extract as a mouthwash preparation have been reported to reduce plaque accumulation and gingival inflammation (Gupta et al., 2015;Naiktari et al., 2014). It will be necessary to isolation the pure compound from the extract to identify the components, according to their medical value.
We also provide the mechanistic details for anticaries effect of EEDC in this study. Since many studies on the development of natural antibacterial agents have focused only on bacteriostatic and bactericidal analysis, biochemical and genetic analysis are very poor. This limits the data for understanding the antibacterial mechanism. The three characteristics that closely relate S. mutans to dental caries are (1) they form extracellular polymers such as glucan using sugars, Even at the 10 μg/ml EETC for MIC of S. mutans, we observed the remarkable inhibition of the enzymes producing insoluble polymers. In addition, ≤25 μg/ml of EETC was verified to be noncytotoxic in human gingival epithelial cells.
In the present study, we elucidated the antioxidant effect of EETC with DPPH and ABTS-radical scavenging assays. Free radicals are highly reactive with other cellular structures because they contain unpaired electrons. Therefore, free radicals can damage the tissues and cells by stealing their electrons through oxidative reaction.
Various external stimuli and inflammation conditions, such as gum disease, increase oxidative stress in the oral mucosa. High concentrations of reactive oxygen species (ROS) are produced at the plasma membrane in the vicinity of such pathogen as Streptococcus sanguinis and Streptococcus gordonii (Touati, 2000). S. mutans can produce ROS in vitro (Fujishima et al., 2013;Wang et al., 2001).
Therefore, it is very important to find the antioxidants for scavenging these free radicals. From the standpoint of in vitro methods for assessing antioxidative activities, several have been proposed for evaluating them by using 1,1-diphenyl-2-pierylhydrazyl (DPPH) and 2,2 0 -azino-bis-(3-ethylbenzo thiazolin-6-sulfonic acid) diammonium salt (ABTS). DPPH or ABTS-radical-scavenging activity of EETC was increased in a concentration-dependent manner. Particularly, antioxidant activity was observed at lower concentration (≥10 μg/ml) than MIC of the EETC on S. mutans. This result indicates that the EETC can be applied as an antibacterial and antioxidant regardless of cytotoxicity.
In conclusion, this study elucidated that the EETC has effective anticaries activity through the inhibition of glucan formation and related gene expression, as well as antibacterial effects under low cytotoxic concentrations of EETC. Therefore, EETC could be applied to oral hygiene products, with its antibacterial properties, for dental caries management. Further studies are required to isolate which pure compounds are related to antibacterial activity. Clinical or experimental animals studies will be needed to verify the anticariogenic effect of EETC.