Dental caries is a bacterial infectious disease accompanied by the demineralization process and a multifactorial disease caused by an interaction among bacteria in the plaque, food and saliva¹⁾. The plaque is composed of bacteria, protein and polysaccharides when streptococci are accumulated in the mouth after bacteria adhere to the initial acquired pellicle. Then this creates acids, forming a cluster solidly and causes demineralization²⁾. Therefore, the reduction of bacteria in the plague can prevent diseases caused by the plague and proper management is extremely important.

For preventing dental caries, several physical methods including toothbrushing are used. In addition to this, a variety of oral rinses are used as auxiliary tools to prevent dental caries. In general, oral rinses are used to feel refreshingness from toothbrushing and remove bad breath³⁾ and known to reduce the numerical value of oral bacteria, especially, Streptococcus mutans^4-6). Among various oral rinses, chlorhexidine is coupled to hydroxyapatite, brown pellicle and the plague and inhibits growth of bacteria by being activated by gradation for 12 to 24 hours when coupled to hydroxyapatite⁷⁾. Weitz et al.⁸⁾ argued that using 0.12% of chlorhexidine for toothbrushing reduced the plague and Brecx et al.⁹⁾ reported that chlorhexidine and meridol were most effective in inhibiting the plague after comparing the inhibitory effects of listerine, a phenolic substance and meridol and chlorhexidine, solutions containing fluorinated amines. However, the long-term use leads to side effects such as tolerance¹⁰⁾. For this reason, there are active studies on natural substances with higher stability and less harmful effects on tissues, recently. Namba et al.¹¹⁾ conducted a growth inhibitory experiment using 60 kinds of extracts from Chinese and Japanese medicinal plants and reported that the minimum growth inhibitory concentration of magnolol and hinokiol, component of Magnoliae cortices, was 6.25 μg/ml and thus they had strong antimicrobial effects on S. mutans. Also, Lee¹²⁾ reported that the Curcuma aromatica extract had a high antimicrobial effect on S. mutans. These studies imply that strong substances for a selective inhibition of dental caries-causing bacteria are required to prevent diseases caused by oral microorganisms.

Glycyrrhiza uralensis is a plant called Gamcho and the part of root and stem with skin or the skinless part is used, mainly. Gafner et al.¹³⁾ said that licoricidin and licorisoflavan A contained in Gamcho inhibited bacteria causing dental caries and gum diseases.

Psoralea corylifolia is a leguminous plant called Bogolji which is spicy and little bitter. Systemically, this plant is reported to be effective in expanding the coronary arteries, increasing blood flow and the number of white blood cells, exciting smooth muscles, increasing the blood and forming an antimicrobial effect¹⁴⁾.

Asa rum siebodii Miquel is a plant called Saeshin that has thin roots and is very spicy. Yu et al.¹⁵⁾ reported that this inhibited adhesion of S. mutans to the tooth surface and insoluble glucan synthesis.

Erythrina variegata is a leguminous plant called Haedongpi and in Korea, this refers to the bark of E. variegata. This is known to be effective for gastritis, arthritis, neuralgia, rhinitis, stroke, cough and sputum and so on¹⁶⁾.

The effects of these natural herbal plants vary depending on the extraction or refinement method. Jang et al.¹⁷⁾ reported that no antimicrobial effect was observed in the hot water and ethanol extracts, but a strong antimicrobial effect was observed in the sugar extract when the antimicrobial effects of Citrus unshiu peel were compared depending on the extraction methods of hot water, ethanol and sugar. Likewise, Jung¹⁸⁾ reported that there was an increase in the antimicrobial effect when the ethanol extract was used rather than the water extract after comparing the antimicrobial effects of Opuntia ficus-indica var. saboten Makino.

Therefore, this research aimed to identify the possibility of natural herbal extracts as alternative preventing agents of dental caries by comparing the antimicrobial effects of natural herbal extracts on S. mutans with the existing oral rinses, based on the minimum growth inhibitory concentration and minimum and minimum bactericidal concentration and analyzing toxicity using human gingival fibroblasts.

1) Selection of the natural herbal

The four natural herbal used in this study were selected based on their low price and accessibility and purchases from kyeongdong market, seoul and stored at refrigiator (Table 1).

[Table 1.] The List of Medicinal Plants Used for This Study

The List of Medicinal Plants Used for This Study

2) Extraction and concentration of the natural herbal extract

The four natural herbal extract were extracted using distilled water and ethanol as solvent. The distilled water extraction were performed as follows: 50 g of the four herbs were allowed to settle and deposit in 500 ml of distilled water for four days, filtered, pressure concentrated and frozen at −70℃ deep freezer (DF8510; IlShin BioBase Co. Ltd., Namyangju, Korea) for 12 hours after which dried using freeze dryer (FD8508; IlShin BioBase Co. Ltd.). The freeze dried extracts were weighted, and dissolved in distilled water and dimehylsulfoxide (DMSO; Sigma, San Jose, CA, USA) at 10 mg/ml, sterilized using 0.22 μm polyvinylidene difluoride (PVDF) filter (BIOFIL, Ashland, VA, USA).

3) Selection of mouth rinsing solutions

The mouth rinsing solutions used in this study were selected based in their diversity of ingredients and market share, and sterilized using 0.22 μm PVDF. Description of each medicine are in Table 2.

[Table 2.] The List of Mouth Rinsing Solution Used for This Study

The List of Mouth Rinsing Solution Used for This Study

The bacteria used in this study is S. mutans ATCC 25175 provided by Department of Oral Microbiology, College of Dentistry, Dankook University. The bacteria was inoculated in liquid trypticase soy broth (TSB; BD Biosciences, San Jose, CA, USA) and cultured for 12 hours in 37℃ incubator (IB-11E; Jeiotech, Daejeon, Korea).

The cultured bacteria were counted using Petroff Hausser counting chamber (Hausser Scientific, Horsham, PA, USA) and diluted to 1×10⁶ cells/ml at TSB. 180 μl of liquid TSB was dispensed to each well of 96-micro well plate and 180 μl of the four herbs and mouth rinsing solutions were dispensed to each well serially diluted with 20 μl bacterial culture and incubated for 12 hours. Cell growth were measured using spectrophotometer (precision microplate reader, Emax precision; Molecular Device Corp., San Diego, CA, USA) by measuring absorbance at 660 nm.

To study minimal bactericidal concentration, live cell were counted at lower and higher concentration than MIC. Bacteria cultured at ethanol extract of the natural herbal extract were 1/10 diluted in phosphate buffered saline (PBS). The diluted cell were serially diluted to 10⁶ factor. 100 μl of diluted bacteria culture were inoculated at brain heart infusion (BD Biosciences) agar plate using triangle spreader (SPL Life Science, Pocheon, Korea) and cultured for 36 hours. The plate with 200 colonies were selected and the total cell in 100 μl were calculated based on their dilution factor.

To measure antibacterial activity against S. mutans using disk diffusion method, trypticase soy agar (TSA) agar plate were used. Sterilized cotton tips were used to equally spread bacteria culture in the TSA agar plate. Sterilized paper disk with 0.4, 0.8, 5 mg/disk of each herb extract and the mg/disk paper disk were placed on the agar plate after drying for 10 minutes. After incubating for 48 hours in 37℃ incubator, clear zone around the disk were analyzed to determine antibacterial activity.

To observe the presence of cytotoxicity in the ethanol extract of the natural herbal extract, the activity of normal gingival fibroblast were measured using MTT (Thiazoly Blue Tetrazolium Bromide 98%; Sigma). Bacteria were inoculated at 1×10⁵ cells/ml in 96-micro well plate with different concentration of the herb extract and cultured for 24 hours. Then the cultured supernatant were eliminated and 100 μl of 4 mg/ml MTT solution in PBS (Gibco BRL, Grand Island, NY, USA), sterilized with 0.22 μm PVDF filter, were carefully dispensed and mixed gently while setting the plate in the ground. Then the plates were cultured in CO₂ incubator (MCO-18AC; Sanyo Electric Co. Ltd., Gunma-ken, Japan) for 4 hours, removed the reaction solution, eluted formazan using 200 μl of DMSO, and shook 30 minutes using shaker (SH 30; Finepcr, Seoul, Korea) after covering with aluminum foil. After 30 minutes of shaking, 100 μl of the elution were transferred to new 96-micro well plate and measured the absorbance at 570 nm using spectrophotometer. All experiments were triplicated.

1) Minimum growth inhibition concentration of natural herbal distilled-water extracts against S. mutans

As a result of measuring the minimum growth inhibition concentration against S. mutans, it was found that all natural herbal distilled-water extracts did not have antimicrobial effects similarly to the control group (Fig. 1).

[Fig. 1.] Antimicrobial effect of natural herbal extract by distilled water solvent on Streptococcus mutans. GU: Glycyrrhiza uralensis, PC: Psoralea corylifolia, AS: Asa rum siebodii Miquel, EV: Erythrina variegata.

2) Minimum growth inhibition concentration of natural herbal ethanol extracts against S. mutans

As a result of measuring the minimum growth inhibition concentration of natural herbal ethanol extracts, the bacterial growth did not happen at 20 μg/ml for P. corylifolia, at 313 μg/ml for G. uralensis, and at 1,250 μg/ml for E. variegata and A. rum siebodii Miquel (Fig. 2).

[Fig. 2.] Antimicrobial effect of natural herbal extract by ethanol solvent on Streptococcus mutans. GU: Glycyrrhiza uralensis, PC: Psoralea corylifolia, AS: Asa rum sibodii Miquel, EV: Erythrina variegata.

To determine if the results of the minimum growth inhibition concentration of natural herbal ethanol extracts stem from S. mutans, the basic optical density was measured without adding S. mutans (Fig. 3). As a result, the natural herbal extracts except P. corylifolia and G. uralensis obtained an optical density value which showed a similar pattern to the control group.

[Fig. 3.] Absorbance of natural herbal extract by ethanol solvent. GU: Glycyrrhiza uralensis, PC: Psoralea corylifolia, AS: Asa rum sibodii Miquel, EV: Erythrina variegata.

As a result of measuring the minimum growth inhibition concentration against S. mutans, the bacterial growth of natural herbal ethanol extracts was suppressed at 20 μg/ml for P. corylifolia, 313 μg/ml for G. uralensis, and 1,250 μg/ml for E. variegata and A. rum siebodii Miquel. In addition, the bacterial growth of mouth rinsing solutions was suppressed at the concentration of 0.39% for hexamedine, at the concentration of 0.78% for palate cleanser, and at the concentration of 50% for listerine (Fig. 4).

[Fig. 4.] Minimum inhibitory concentration of natural herbal extracts by ethanol and three mouth rinsing solutions against Streptococcus mutans. He: Hexamedine, Ga: Garglin, Li: Listerine, GU: Glycyrrhiza uralensis, PC: Psoralea corylifolia, AS: Asa rum sibodii Miquel, EV: Erythrina variegata.

As a result of measuring the minimal bactericidal concentration against S. mutans, the bacterial death of natural herbal ethanol extracts occurred at 20 μg/ml for P. corylifolia, at 1,250 μg/ml for G. uralensis and E. variegata, and at 2,500 μg/ml for A. rum siebodii Miquel. In addition, the bacterial death of mouth rinsing solutions occurred at the concentration of 0.78% for hexamedine and palate cleanser and at the concentration of 50% for listerine (Table 3).

[Table 3.] Effect of Various Concentration of Several Natural Herbal Extracts by Ethanol and Three Mouth Rinsing Solutions on Streptococcus mutans

Effect of Various Concentration of Several Natural Herbal Extracts by Ethanol and Three Mouth Rinsing Solutions on Streptococcus mutans

As a result of measuring the bacterial growth inhibition to compare the antimicrobial activity against S. mutans, the bacterial growth inhibition zone of both natural herbal extracts and mouth rinsing solutions was increased when the concentration got higher (Fig. 5A, B), and at 5 mg/disk, the inhibition zone was overlapped and the bacterial growth was suppressed in the entire medium (Fig. 5C).

[Fig. 5.] Antimicrobial activity of the ethanol extract of natural herbal and mouth rinsing solutions against the growth of Streptococcus mutans by agar diffusion assay. (A) 0.4 mg/disk, (B) 0.8 mg/disk, (C) 5 mg/disk. He: Hexamedine, Ga: Garglin, Li: Listerine, GU: Glycyrrhiza uralensis, PC: Psoralea corylifolia, AS: Asa rum sibodii Miquel, EV: Erythrina variegata.

As a result of treatment at 10, 39, and 78 μg/ml minimum growth inhibition concentration, to determine if the natural herbal ethanol extracts are toxic to the normal gingival fibroblasts, the optical density value showed a similar pattern to the control group that has no extracts added (Fig. 6). In addition, as a result of treatment by differing the concentration of each extract based on the measurement values of the minimal bactericidal concentration, the other extracts except P. corylifolia were cytotoxic when the concentration got higher (Fig. 7).

[Fig. 6.] The cytotoxicity of natural herbal extracts by ethanol in accordance with concentration. GU: Glycyrrhiza uralensis, PC: Psoralea corylifolia, AS: Asa rum sibodii Miquel, EV: Erythrina variegata.

[Fig. 7.] The cytotoxicity of natural herbal extracts by ethanol in the concentration which have antimicrobial activity against Streptococcus Mutans. GU: Glycyrrhiza uralensis, PC: Psoralea corylifolia, AS: Asa rum sibodii Miquel, EV: Erythrina variegata.

Dental caries is a disease caused by the interactions between bacteria, foods, and saliva within the dental plaque and its major causative germ is known as S. mutans, among the germs present in the dental plaques¹⁹⁾. S. mutans, as gram-positive and facultative anaerobic bacteria, generates acid and acidifies the environment within the dental plaque since it is acid-resistant²⁰⁾. Moreover, S. mutans produces glucan, extracellular polysaccharide²¹⁾ to reinforce the formation of the dental plaques within the oral cavity and makes them grow as mature ones²²⁾. So managing the S. mutans present within the dental plaques is paramount.

To manage the dental plaques, many people are using various preventive auxiliary devices. Among which, mouth rinsing solutions is being used for the purpose of preventing gum disease, removing dental plaque and mouth odor, and preventing dental caries. The mouth rinsing solutions includes various ingredients, which have different effects. Therefore, ongoing studies on the various ingredients of the mouth rinsing solutions have been made. Shin and Lee²³⁾ reported that the mouth rinsing solutions containing NaF, cetylpyridinium chloride (CPC), and ursodeoxycholic acid suppresses the generation of dental plaques. Choi et al.²⁴⁾ demonstrated that the mouth rinsing solutions containing CPC and triclosan has gingivitis index- reducing effect, dental plaque-suppressing effect, and mouth odour-suppressing effect. But such synthesized chemical materials are resistant to antibiotics although they suppress the growth of microorganisms residing in the oral cavity¹⁰⁾. For this reason, researchers are putting more interest in natural materials like medicinal herbs, as more effective and stable alternate products.

Accordingly, this study selected G. uralensis, P. corylifolia, A. rum siebodii Miquel, and E. variegata, which are less expensive and easily available, as new materials that can be used as mouth rinsing solutions.

Natural herbal plants were extracted with distilled water and ethanol, respectively, to measure the minimum growth inhibitory concentration of S. mutans depending on concentration, and among which, solvents showing high antimicrobial activity were selected to compare their antibiotic effects with those of mouth rinsing solutions.

Also, to determine the concentration of natural medicinal herbs that can be used safely in the oral cavity, the extracts were treated to the normal gingival fibroblast cells depending on concentration in order to determine its cytotoxicity using MTT.

As a result, the natural herbal plants extracted with ethanol solvent showed that the higher the concentration, the better its growth-suppressing effects against S. mutans and the natural herbal plants extracted with distilled water showed a similar tendency in the changes by concentration to the control group. This was consistent with the findings that every solvent shows its different effects depending on the extracted effective ingredients¹⁷⁾. Jung¹⁸⁾ reported that ethyl acetate is the most effective, in his study on the antimicrobial activity of opuntia ficus-indica var. saboten Makino in oral cavity depending on solvent. Park et al.²⁵⁾ extracted sophora root with three kinds of solvent (water, methanol, ethanol) and compared its antimicrobial effects in oral cavity. As a result, they reported that the extraction with methanol or ethanol showed antimicrobial effect and among which, ethanol was more effective, which was consisted with this study.

As a result of comparing the minimum growth inhibition concentration against S. mutans, it was found that the bacterial growth inhibition of natural herbal ethanol extracts was at 20 μg/ml for P. corylifolia, at 313 μg/ml for G. uralensis, and at 1,250 μg/ml for A. rum siebodii Miquel and E. variegata and that of mouth rinsing solutions was at the concentration of 0.39% for hexamedine, at the concentration of 0.78% for palate cleanser, and at the concentration of 50% for listerine. Lee²⁶⁾, who compared antimicrobial effects against S. mutans, mouth rinsing solutions in nutrient-rich environment and biofilm environment, reported that palate cleanser and hexamedine are the most effective and listerine is the least effective, which appears consistent with the findings of this study. On the other hand, P. corylifolia showed that the higher the concentration is, the higher the optical density is in the minimum growth inhibition concentration. This is attributable to the fact that P. corylifolia has at least 35 ingredients and among which, organic materials are stigmasterol, essential oil, fatty oil, and resin²⁷⁾ and thus they are not mixed with the medium. To clarify this, the minimal bactericidal concentration was measured. As a result, the bacterial death of natural herbal ethanol extracts occurred at 20 μg/ml for P. corylifolia, 1,250 μg/ml for G. uralensis and E. variegata, and 2,500 μg/ml for A. rum siebodii Miquel, and that of mouth rinsing solutions was at the concentration of 0.78% for hexamedine and palate cleanser and at the concentration of 50% for listerine. Thus, it is found that the higher the concentration is, the higher the optical density is due to the presence of organic matters in P. corylifolia. To identify whether they are antimicrobial, disc diffusion method was used and the bacterial growth inhibition was measured at 0.4, 0.8, or 5 mg/disk. As a result, it is found that the higher the concentration is, the higher the antimicrobial activity is. In particular, at 0.8 mg/disk or higher, the growth inhibition began to be overlapped between natural herbal extracts and mouth rinsing solutions. At 5 mg/disk, the growth inhibition was overlapped and the bacteria did not proliferate in the entire medium.

To identify the cytotoxicity of natural herbal ethanol extracts, extracts were treated to the normal gingival fibroblast cells by concentration. Based on the minimum growth inhibition concentration, extracts were treated at 10, 39, or 78 μg/ml as the concentration when antimicrobial effects began to be shown. As a result, the group treated with extracts showed a similar pattern to the control group, which suggested that the group treated with extracts is nontoxic. Further, based on the minimal bactericidal concentration, extracts were treated depending on concentration. As a result, the extracts other than P. corylifolia showed that the higher the concentration was, the higher the cytotoxicity was. Cho et al.²⁸⁾ reported that G. uralensis extracts were not influential to the cell survival rate at 10 μg/ml, 50 μg/ml, and 100 μg/ml, as a result of the experiment that used CCD-986sk cell strain, fibrinoblast of human skin, using G. uralensis extracts. Chung and Seo²⁹⁾ reported that the cell survival rate by concentration was reduced, as a result of measuring the cytotoxicity of B16 melanoma cell using P. corylifolia extracts through crystal violet. Jung et al.³⁰⁾ reported that there was little cytotoxicity in macrophage, as a result of treating A. rum siebodii Miquel extracts to peritoneal macrophage activated with lipopolysaccharide, and Hwang et al.³¹⁾ reported that the higher the concentration was, the survival rate went down, as a result of examining the survival rate of E. variegata extracts using AGS cell. Similarly, this study showed that there was no cytotoxicity at 10, 39, or 78 μg/ml which was based on the minimum growth inhibition concentration, but the findings from the experiment that treated extracts depending on concentration based on the minimal bactericidal concentration showed that the higher the concentration was, the higher the cytotoxicity was. This suggested that the natural herbal ethanol extracts would be used safely at the concentration of 78 μg/ml or less.

Taken together, 4 kinds of natural herbal extract showed a similar antimicrobial effect when compared with the mouth rinsing solutions, synthesized chemical substance. Therefore, the natural herbal extracts are expected to be used as an alternative preventive agent for dental caries if using them at an appropriate concentration.

In this study, the antibacterial effects of mouth rinsing solutions and natural herbal extracts on S. mutans were measured to evaluate the possibility of using natural herbal extracts as dental caries preventive agents, and the toxicity to normal human gingival fibroblast was observed. The study findings are as follows: Natural herbal extracts showed a more excellent growth inhibitory effect on S. mutans in the ethanol extract than in distilled water. When comparing the minimal growth inhibitory concentration of each extract in S. mutans, Psoralea corylifolia of natural herbal ethanol extracts and Hexamedine of mouth rinsing solutions had a growth inhibitory effect at the lowest concentration, and when comparing the minimal bactericidal concentration of each extract, Psoralea corylifolia of natural herbal ethanol extracts and Hexamedine and Garglin of mouth rinsing solutions had a killing effect at a low concentration. Also, the toxicity of natural herbal ethanol extracts to normal human gingival fibroblast was measured at a minimal growth inhibitory concentration of 10, 39, and 78 μg/ml, no cytotoxicity was found. However, cytotoxicity was found in other extracts excepting Psoralea corylifolia at a high concentration, when the concentration of each extract was changed, based on the minimal bactericidal concentration values.

These results demonstrate that the antibacterial effect of the four kinds of natural herbal extracts was similar to the effect of mouth rinsing solutions, which are synthetic chemicals. Therefore, it is thought that natural herbal extracts can be commercialized as alternative dental caries preventive agents by adding proper concentration.