
Dental caries is a sucrose-dependent disease caused by an interaction between cariogenic and symbiotic oral microorganisms1). The interaction between sugars and biofilm formation is the primary cause of dental caries2,3). Sucrose affects the formation of extracellular polysaccharides (EPS), as well as affects the growth and adhesion of the biofilm4). EPS, an important dental biofilm component, is produced by glucosyltransferases (GTF) of Streptococcus mutans and is called glucan5). EPS, especially water-insoluble EPS, play an important role in the adhesion and deposition of S. mutans on the tooth surface6). EPS composition and production are affected by various factors such as temperature, carbon, nitrogen, divalent ions, and medium content7,8). The increased solubility of EPS reduced biofilm adhesion9). Particularly, water-insoluble EPS change the structure of the biofilms and increase their porosity10).
Xylitol is a naturally occurring sugar alcohol that cannot be metabolized by oral microorganisms for energy production. When xylitol is absorbed by microorganisms for metabolism, it accumulates as the toxic sugar-phosphate11). The growth of S. mutans exposed to xylitol is inhibited12), which affects biofilm formation and structure13). S. mutans exposed to xylitol produced a thinner biofilm containing less EPS than those exposed to sucrose14). This reduced microbial adhesion15) and increased biofilm-removal efficacy9).
S. mutans, a representative oral bacterium that causes dental caries, forms biofilms and metabolizes various carbohydrates to produce organic acids. Especially, activity and acidogenicity of S. mutans are enhanced by sucrose16,17). S. mutans secretes GTFs enzyme encoded by three gtfB, gtfC and gtfD genes18,19). GTFs produce EPS from sucrose, GtfB (known as GTF-1) and GtfC (known as GTF-SI) produce water-insoluble EPS, and GtfD (known as GTF-S) produces water-soluble EPS20,21). GTFs play a major role in biofilm formation and cause dental caries22). An increasing research interest exists in methods other than the traditional methods of biofilm removal for the prevention and treatment of dental caries23). Recently, research has been conducted at the gene and protein levels on the possibility of GTFs controlling EPS-producing activity or development as an anti-caries vaccine for the therapeutic agent24).
Angelica gigas Nakai Root (AGN) is a medicinal plant having various therapeutic effects. The 70% ethanol extract of AGN (EAGN) has an antibacterial effect against S. mutans and inhibits biofilm formation by S. mutans16). However, there have been no studies on the effects of EAGN on the antibacterial and metabolic activities including biofilm formation and GTF activity of S. mutans according to exposure to xylitol and sucrose.
This study aimed to determine the effect of EAGN on the biofilm formation and metabolic activities of S. mutans according to exposure to xylitol and sucrose, and to confirm the possibility of using EAGN as an effective medicinal plant-derived anti-cariogenic substance.
The 70% EAGN was prepared according to the method reported by Jeong (2024)16). It was prepared as 250 mg/ml stock solution in dimethyl sulfoxide (Sigm-Aldrich Chemical Co., St. Louis, MO, USA), and concentrations of 2.5 mg/ml, 3.75 mg/ml, 5 mg/ml and 6.75 mg/ml were used in the experiment.
S. mutans (KCCM 40105) purchased from Korea Microbiological Conservation Center (KCCM, Seoul, Korea) was cultured in an incubator (Daihan Scientific Co., Daegu, Korea) for 24 hours at 36.5°C using brain-heart infusion (BHI) broth medium (MB cell; KisanBio, Seoul, Korea) and a BHI broth medium containing 1% xylitol and sucrose, respectively. According to the method described by Jeong (2024)16), 5×106 CFU/ml was prepared and used in the experiment. Growth of S. mutans was measured at an optical density (OD) of 600 nm using a UV-Vis spectrophotometer (X-ma 1200; Human Corp., Seoul, Korea).
A total of 100 μl S. mutans prepared at 5×106 CFU/ml was spread on a BHI agar (MB cell; KisanBio) plate and BHI agar plates containing 1% xylitol and sucrose, respectively. Paper discs (Ф6 mm, Advantec Toyo Kaisha Ltd., Tokyo, Japan) absorbing EAGN of each concentration and penicillin antibiotic discs (10 µg; Oxoid Ltd., Hampshire, United Kingdom) were placed on each agar plate. Agar plates for the experiment were cultured in the incubator for 24 hours at 36.5°C, and the clear zone created around the paper disk was measured by a Vernier caliper (Mitutoyo Co., Kanagawa, Japan).
BHI broth and BHI broth containing 1% xylitol and sucrose inoculated with 5×106 CFU/ml S. mutans were treated with EAGN of each concentration and were cultured using the incubator for 24 hours at 36.5°C in 96-well plates.
For the biofilm formation assay, the medium was removed from the 96-well plates, washed with distilled water, stained with 0.1% crystal violet (MB cell; KisanBio) at room temperature for 15 minutes, treated with 95% ethanol and measured at OD 570 nm using a microplate reader (SunriseTM; Tecan, Grödig, Austria).
To measure GTF activity, a GTF activity kit (R&D Systems Inc., Minneapolis, MN, USA) was used. The prepared reagents were added 96-well plates according to the manufacturer’s instructions, and then measured at OD 620 nm using a microplate reader (SunriseTM; Tecan).
BHI broth and BHI broth containing 1% xylitol and sucrose inoculated with 5×106 CFU/ml S. mutans and treated with EAGN were cultured in 96-well plates for 24 hours at 36.5°C and used for the calcium assay. A calcium assay kit (Abcam, Cambridge, UK) was used to measure free calcium ions in the medium. The Ca2+ concentration was measured at OD 575 nm using a microplate reader (SunriseTM; Tecan) in 96-well plates treated with the reagents according to the manufacturer’s protocol.
S. mutans (5×106 CFU/ml) and each concentration of EAGN were treated with 5 ml BHI broth and BHI broth containing 1% xylitol and sucrose, and cultured for 24 hours. According to the method of Jeong (2024)16), acid production was measured by the pH value using a pH meter (TTBH Pte Ltd., Singapore) and the buffering capacity was measured as the amount of 1 M NaOH required to bring the pH value of the medium to pH 7.
Our results, which were obtained from three independent experiments, are expressed as mean±standard deviation and analyzed using SPSS 25.0 (IBM Corp., Amonk, NY, USA). Significant differences were determined using one-way analysis of variance.
The results of antibacterial effects of EAGN in BHI agar and BHI agar containing 1% xylitol and sucrose inoculated with S. mutans by disk diffusion test are shown in Fig. 1 and Table 1. In BHI containing 1% xylitol, EAGN showed antibacterial activity similar to that of BHI only, however, in BHI containing 1% sucrose, it increased compared to those of BHI only and BHI containing 1% xylitol (Fig. 1).
Resistant Results of 70% Ethanol Extract of Angelica gigas Nakai Root (EAGN) Against Streptococcus mutans in Brain-Heart Infusion (BHI) Agar Containing 1% Xylitol and Sucrose Using Disk Diffusion Test
Strain | Streptococcus mutans (KCCM 40105) | |||
---|---|---|---|---|
Media | BHI agar | BHI+1% xylitol | BHI+1% sucrose | |
DMSO | – | – | – | |
EAGN (mg/ml) | ||||
2.5 | + | + | + | |
3.75 | + | + | ++ | |
5.0 | + | + | ++ | |
6.25 | ++ | ++ | ++ | |
Penicillin G (10 µg) | +++ | +++ | +++ |
–: resistrant (<6 mm), +: susceptible (6∼10 mm), ++: more susceptible (11∼15 mm), +++: most susceptible (>16 mm), BHI: brain-heart infusion, DMSO: dimethyl sulfoxide, KCCM: Korea Microbiological Conservation Center.
Changes in the growth of S. mutans in BHI broth containing 1% xylitol and 1% sucrose according to the EAGN concentration are shown in Fig. 2. EAGN reduced the growth of S. mutans in all media, and the inhibitory effect of bacterial growth in BHI containing 1% sucrose and at 3.75 mg/ml EAGN above was greater than those of BHI only and 1% xylitol BHI medium (Fig. 2).
The changes in GTF activity according to EAGN in S. mutans-inoculated BHI medium containing 1% xylitol and sucrose are shown in Fig. 3. GTF activity was most increased in the BHI medium containing 1% sucrose but was significantly decreased to similar values regardless of EAGN concentration according to treatment with EAGN (Fig. 3). GTF activity in the BHI containing 1% xylitol was reduced, but the value was similar to those of in BHI only (Fig. 3).
The changes in cariogenic biofilm formation according to EAGN in the S. mutans-inoculated BHI medium containing 1% xylitol and sucrose are shown in Fig. 4. Biofilm formation was highest in BHI medium containing 1% sucrose but was significantly reduced according to the EAGN concentration, with greatest decrease at 3.75 mg/ml EAGN (Fig. 4). Biofilm formation slightly increased in BHI containing 1% xylitol but gradually decreased according to EAGN treatment, and was similar to those in BHI only at above 3.75 mg/ml EAGN (Fig. 4).
The changes in Ca2+ in S. mutans-inoculated BHI medium containing 1% xylitol and sucrose according to EAGN are shown in Fig. 5. Ca2+ most increased in BHI containing 1% sucrose, but was gradually reduced according to EAGN concentration, and above 3.75 mg/ml EAGN, the degree of reduction was greater than those of other media (Fig. 5). In BHI only and BHI containing 1% xylitol, Ca2+ slightly decreased according to EAGN treatment and the value were constant regardless of the EAGN concentration (Fig. 5).
The changes in acidogenicity and buffering capacity in S. mutans-inoculated medium containing 1% xylitol and sucrose according to EAGN are shown in Fig. 6. The pH value greatly decreased in BHI containing 1% sucrose and acidogenicity increased, but the pH value increased according to EAGN treatment and the values were similar regardless of EAGN concentration (Fig. 6A). The pH value decreased in BHI only and BHI containing 1% xylitol according to EAGN treatment and the value were similar regardless of the EAGN concentration (Fig. 6A).
The measurements of 1 M NaOH greatly increased in BHI containing 1% sucrose, and the buffering capacity was decreased, but it decreased compared to those of BHI only and BHI containing 1% xylitol according to the treatment with EAGN and the measurements were similar regardless of EAGN concentration, and the buffering capacity was increased (Fig. 6B). The measurements of 1 M NaOH slightly increased in BHI only and BHI containing 1% xylitol, the measurements were similar regardless of the EAGN concentration, and the buffering capacity slightly decreased (Fig. 6B).
Changes in GTFs directly affect biofilm formation18,19). GTFs produce EPS from sucrose, GtfB and GtfC produce water-insoluble EPS, and GtfD produces water-soluble EPS20,21). Water-insoluble EPS help the aggregation and adhesion of bacteria, and water-soluble EPS supplies carbohydrates to metabolize or induce the production of water-insoluble EPS25). In this study, EAGN significantly reduced the GTF activity and biofilm formation of S. mutans according to exposure to 1% sucrose, regardless of EAGN concentration. This indicated that EAGN had a direct effect on the GTF activity of S. mutans activated by sucrose, and as a result, biofilm formation was reduced. Substances derived from Terminalia chebula, Psidium guajava, Pongamia pinnata, Azadirachta indica, Syzygium aromaticum, and Mentha piperita inhibited GTF activity, mouth rinses containing substances derived from these six plants demonstrated a 95.9% inhibitory effect on GTF activity, whereas chlorohexidine mouthwash showed a 54% inhibitory effect26). Research on the anti-caries effects of traditional methods is increasing, in particular, research on GTF at the gene and protein levels is increasing24). This study confirmed that EAGN inhibited biofilm formation by directly inhibiting GTF activity.
EPS has functional regions with negative charged binding to heavy metal ions, including Ca2+, and divalent ions, such as Ca2+ are removed from the environment through EPS binding27,28). Ca2+ in the biofilm composed of EPS binds to the cell wall of bacteria29), and Ca2+ can be released into the biofilm fluid6). Water-insoluble EPS produced by S. mutans exposed to sucrose changed the structure of the biofilm and increased its porosity10). Water-insoluble EPS also forms a thick biofilm but reduces biofilm Ca-binding sites, which decreases the density of bacteria adhering to the biofilm30). This eventually leads to environmental changes and affects biofilm cariogenicity30). Mineral ions including Ca2+ in the medium are closely related to pH, and Ca2+ is released when the pH drop31). In this study, Ca2+ concentration and acidogenicity increased significantly in the S. mutans-inoculated medium containing 1% sucrose compared to the other media. This is the result of a decrease in the number of ca-binding sites that bind the bacteria cell wall to the water-insoluble EPS produced by S. mutans using sucrose. The Ca2+ concentration in the S. mutans-inoculated medium containing 1% sucrose gradually decreased significantly as the concentration of EAGN increased and was also different from those in other media. This result also differed from the change in biofilm formation in the S. mutans-inoculated medium containing 1% sucrose. In the case of 2.5 mg/ml EAGN, Ca2+ was similarly reduced in all types of media, which may be due to a decrease in biofilm formation mainly composed of water-insoluble EPS by EAGN. At concentrations over 3.75 mg/ml EAGN, biofilm formation was constant, however, Ca2+ in the medium decreased significantly as EAGN treatment increased. EAGN treatment decreased acidogenicity and increased buffering capacity regardless of EAGN concentration in the S. mutans-inoculated medium containing 1% sucrose. This suggests the possibility of another way to induce Ca2+ reduction by EAGN. Galla Chinesensis induces remineralization by binding to calcium and acting as a transporter of Ca2+ that supplies calcium to the carious area32,33). Although there were no direct results, the decrease in Ca2+ by EAGN in S. mutans-inoculated medium containing 1% sucrose may be due to the binding of Ca2+ and EAGN.
Therefore, EAGN is a safe anti-cariogenic natural substance to inhibit biofilm formation through the directly inhibiting GTF activity and adjusts the microenvironment for tooth remineralization through reducing Ca2+ and acidogenicity and increasing the buffering capacity according to exposure to sucrose in S. mutans.
This in vitro study dose not reflect changes in the influence of various microenvironments in the oral cavity and their relationship with other oral microorganisms. Further studies are needed to provide direct evidence of the relationship between Ca2+ and EAGN in the S. mutans-inoculated BHI medium containing 1% sucrose.
None.
Conflict of interest
Soon-Jeong Jeong has been serving as an editor-in-chief of the Journal of Dental Hygiene Science since January 2023. She was not involved in the review process of this editorial. So, there was no conflict of interest.
Ethical approval
This article dose not require IRB screening, because commercially available bacterial strain was used.
Author contributions
Conceptualization: Moon-Jin Jeong and Soon-Jeong Jeong. Data acquisition: Sung Ok Kim, Do-Seon Lim, and Kyungwon Heo. Formal analysis: Sung Ok Kim, Do-Seon Lim, and Kyungwon Heo. Funding: Moon-Jin Jeong. Supervision: Soon-Jeong Jeong. Writing-original draft: Soon-Jeong Jeong. Writing-review & editing: Moon-Jin Jeong and Soon-Jeong Jeong.
Funding
This study was supported by research fund from Chosun University, 2023.
Data availability
Raw data is provided at the request of the corresponding author for reasonable reason.
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