Dental caries is a representative oral diseases associated with bacteria in the oral cavity [1], and the related bacterium is known to be Streptococcus mutans [2]. The characteristics of S. mutans is a gram-positive facultative anaerobic bacterium and unlike other streptococci species in the oral cavity, it is resistant to bacitracin [3]. The virulence factors of S. mutans are aciduricity, glucosyltransferases (GTF), and glucan binding proteins [4-7], and it is known that bacterial biofilm are more related to dental caries than planktonic bacteria due to the characteristics of these bacteria [8,9]. Therefore, it is recognized that dental caries can be prevented by managing dental biofilm.

The aciduricity of S. mutans is that it can survive in strong acidity by excreting hydrogen inside the bacteria to the outside by membrane-associated proton-translocating adenosine triphosphate synthase locatpresent in the bacterial membrane [4,10]. Another virulence factor is glycosyltransferase, and S. mutans has three types of glycosyltransferase [5], GtfB, GtfC, and GtfD. GtfB and GtfC are proteins of 162 kDa and 149 kDa in size, respectively, that produce insoluble glucans, and GtfD is an enzyme of 155 kDa that forms soluble glucans [11-13]. The glucans formed in this way play an important role in the formation of biofilms by attaching bacteria to the dental surface and serve as a protective barrier against external substances [14,15]. Finally, glucan-attached proteins have been reported to be involved in the initial aggregation of bacteria and dental biofilm formation via soluble glucans [16,17].

Rosmarius officinalis, L., commonly known as rosemary, is a plant belonging to the Lamiaceae family and is widely found in the Mediterranean region [18,19]. Rosemary is mostly used as a spice in cooking, and is also used as a natural preservative and medicinal plant in the food industry [20]. In addition, many plant compounds exhibiting pharmacological activity are isolated and used as oils and by boiling. The compounds of rosemary most frequently reported to have pharmacological actions are caffeic acid, carnosic acid, chlorogenic acid, monomeric acid, oleanolic acid, rosmarinic acid, ursolic acid, carnosol, eucalyptol, rosmadial, rosmanol, rosmaquinone A and B, and secohinochio. These compounds promote various effects due to interactions between organic systems [21,22].

R. officinalis, L. is a rich source of phenolic compounds, the properties of which are contained in the extracts and essential oils, and these phenolic compounds exhibit antibacterial effects [23]. Rosemary extracts have been reported to have antibacterial activity against Gram-positive and Gram-negative bacteria, including Staphylococcus aureus and Escherichia coli [24,25].

A recent study reported that metabolites of Candida albicans promote biofilm formation of S. mutans [26]. When the medium in which C. albicans was cultured was mixed with a fresh medium and the formation of S. mutans biofilms was compared, a larger amount of biofilm was formed than that of S. mutans biofilms cultured in the fresh medium. In addition, it was observed that more biofilms were formed in a mixed culture environment of C. albicans and S. mutans than in a biofilm cultured solely of S. mutans [27]. This phenomenon was reported to be due to the fact that metabolites secreted by C. albicans enhance the production of the glycosyltransferase of S. mutans, thereby increasing biofilm formation. In this study, we investigated the effect of a rosemary extract containing a natural antibacterial effect in a toothpaste on the initial biofilm of S. mutans related to dental caries using this toothpaste.

1. Bacterial and fungal cultures

The bacteria used in this study was Streptococcus mutans ATCC 25175, and the fungus was Candida albicans ATCC 10231. S. mutans was cultured under aerobic conditions at 37℃ using Trypticase soy broth (TSB) (BD biosceince, San Jose, CA, USA). C. albicans was cultured under aerobic conditions at 37℃ using sabouraud dextrose liquid medium (BD biosceince, San Jose, CA, USA). It was subcultured and used until each bacterial and fungal sensitivity test and biofilm test were completed.

2. Preparation of experimental materials

To investigate the effect of the abrasive, samples were prepared by adding various concentrations of rosemary extract powder to a dentifrice product based on 17% dental type silica.

3. Establishing conditions for initial biofilm formation in S. mutans

Based on the finding that the initial biofilm of S. mutans is promoted by C. albicans [27], in order to establish the conditions for initial biofilm formation, a mixed culture of S. mutans and C. albicans and a medium (CCM) in which C. albicans was cultured were used. S. mutans initial biofilm was formed using . First, the ratio of S. mutans (1×10⁷ cell) and C. albicans was mixed in TSB containing 2% sucrose at a ratio of 200:1, 100:1, and 50:1 using suspended fluids measuring bacterial and fungal counts in mixed culture experiments. The 8-well class chamber (SPL life science) was inoculated with 400 μl, incubated at 37℃ for 18 hours, stained with a live/dead bacterial storage kit (Invitrogen, Eugene, OR, USA), and Z-stack scan was performed from 0 to 30 μm using a confocal laser scanning microscope (LSM 700, Carl-Zeiss, Oberkochen, Germany). In addition, in the experiment using the C. albicans culture medium, sucrose was mixed with clean TSB and CCM at a ratio of 1:1, 1:2, and 1:4, inoculated into S. mutans (1×10⁷ cell), 400 μl dispensed in the 8-well class chamber (SPL life science), incubated at 37°C for 18 hours, stained with a live/dead bacterial storage kit (Invitrogen, Eugene, OR, USA), and Z-stack scans were performed from 0 to 30 μm using a confocal laser scanning microscope (LSM 700, Carl-Zeiss, Oberkochen, Germany).

Table 1 . Rosemary extract powder content of experimental group and control group

	Main ingredient
Control group	Dental type silica 17%
Experimental group 1	Dental type silica 17%, rosemary extract powder 0.2%
Experimental group 2	Dental type silica 17%, rosemary extract powder 0.4%

4. Effect of dentifrice on the formation of S. mutans biofilm

Clean TSB and C. albicans culture medium were mixed at a ratio of 1:2, and sucrose was added to 2%. Then, S. mutans was inoculated at 1×10⁷ cells/ml, and 400 μl were dispensed into each well of 8-well glass chamber. In addition, S. mutans (1×10⁷ cells) and C. albicans (1×10⁵ cells) were mixed and inoculated into TSB containing 2% sucrose in the mixed culture, and 400 μl were dispensed into each well of 8-well glass chamber. Next, various concentrations of the control group and the experimental group dentifrice were treated, placed in a 37℃ incubator, and cultured for 18 to 20 hours. The initial biofilm produced in the 8-well glass chamber was stained with a live/dead battery storage kit (Invitrogen, Eugene, OR, USA) and subjected to a Z-stack scan from 0 to 30 μm using a confocal laser scanning microscope (LSM 700, Carl-Zeiss, Oberkochen, Germany). For image analysis, three-dimensional images and data were analyzed using the ZEN program (Carl-Zeiss, Oberkochen, Germany).

5. Data analysis and statistical methods

Statistical analysis was performed using IBM SPSS statistics ver 28.0 software (IBM Co., Amonk, NY, USA). Data analysis was analyzed for normal distribution using komogorov-smirnove analysis, and differences between groups were analyzed through Kruskal-Wallis analysis, a nonparametric analysis. Statistical significance between groups was defined as having a P value of less than 0.05. Post-analysis to compare differences between individual groups was performed by the Mann-Whitney U test, which was performed by multiple comparisons by the Bonferroni method.

1. Susceptibility of rosemary extract to S. mutans

When a susceptibility test was conducted using a 96-well plate to investigate the antibacterial activity of rosemary extract against S. mutans, rosemary extract significantly reduced the growth of S. mutans at a concentration of 0.08 mg/ml (p<0.05), completely inhibited the growth of S. mutans at a concentration of 0.16 mg/ml (Figure 1).

Figure 1. Susceptibility of S. mutans to rosemary extract. S. mutans was cultured with and without the rosemary extract at various concentrations in a 96-well polystyrene plate. The growth of S. mutans was measured using a spectrophotometer at wavelength of 660 nm. The experiments were performed three times in duplicate, and the data are expressed as the means±standard deviation. *Asterix indicates that statistically significant differences compared with control (p<0.05).

2. Susceptibility of dentifrice containing rosemary extract to S. mutans

In order to dentifrice whether the antibacterial activity of rosemary extract was confirmed and maintained even when mixed with dentifrice, rosemary extract was included in various concentrations and an antibacterial activity test against S. mutans was performed. As a result, it was found that the dentifrice concentration of 50 mg/ml significantly reduced the growth of S. mutans in all experimental groups including dental silica, the basic ingredient of the dentifrice (p<0.05) (Figure 2).

Figure 2. Susceptibility of S. mutans to toothpaste including rosemary extract. S. mutans was cultured with and without various toothpastes including rosemary extract at various concentrations in a 96-well polystyrene plate. The growth of S. mutans was measured using a spectrophotometer at wavelength of 660 nm. The experiments were performed three times in duplicate, and the data are expressed as the means±standard deviation. *Asterix indicates that statistically significant differences compared with control (p<0.05).

3. S. mutans initial biofilm formation by C. albicans

To meet the conditions of the initial biofilm of S. mutans, it was mixed cultured with various concentrations of C. albicans, stained using a live/dead bacterial staining kit, and observed with a confocal laser microscope. Compared to the control group S. mutans (1×10⁷ cells) (Figure 3A), S. mutans and C. albicans (0.5×10⁵ cells) did not show significant differences in initial biofilm (Figure 3B). In addition, the amount of biofilm increased in the initial biofilm of S. mutans when mixed cultured with C. albicans at 1.0×10⁵ cells and 2.0×10⁵ cells (Figure 3C and 3D). However, as the concentration of C. albicans increased, the amount of dead S. mutans in the biofilm increased.

Figure 3. Effect of C. albicans on formation of S. mutans biofilm. Early biofilm of S. mutans was formed in the presence or the absence of C. albicans at various concentrations. The biofilm was stained with live/dead bacterial staining kit, and then analyzed with CLSM. The image was analyzed with ZEN software. Green and red color indicate live and dead bacteria in the biofilm, respectively. SM: S. muans, BM: base medium, CM: Candida conditioned medium.

4. Effect of a dentifrice containing rosemary extract on the formation of S. mutans early biofilm by C. albicans cultured medium

When forming an initial biofilm of S. mutans using a mixture of the medium in which C. albicans was cultured and TSB, dentifrice containing rosemary extract was treated at different concentrations and the biofilm was examined. Considering the concentration of rosemary extract contained in the dentifrice, it was treated and observed at a low concentration starting from 250 mg/ml. Compared to the test-control group at a concentration of 250 mg/ml, all dentifrice inhibited the initial biofilm formation of S. mutans (Figure 4).

Figure 4. Effect of toothpaste including rosemary extract on early biofilm of S. mutans. Early biofilm of S. mutans using the mixture of CCM and TSB was formed in the presence or the absence of toothpaste including rosemary extract at concentration of 250 mg/ml. The biofilm was stained with live/dead bacterial staining kit, and then analyzed with CLSM. The image was analyzed with ZEN software. Green and red color indicate live and dead bacteria in the biofilm, respectively.

When a dentifrice at a concentration of 125 mg/ml was treated at the time of bifilm formation, the initial biofilm formation of S. mutans was suppressed in all dentifrice, including the test-control group (Figure 5), and when compared to the test-control group, all dentifrice suppressed the formation of biofilm. A decrease was found in the dentifrice of the experimental group.

Figure 5. Effect of toothpaste including rosemary extract on early biofilm of S. mutans. Early biofilm of S. mutans using the mixture of CCM and TSB was formed in the presence or the absence of toothpaste including rosemary extract at concentration of 125 mg/ml. The biofilm was stained with live/dead bacterial staining kit, and then analyzed with CLSM. The image was analyzed with ZEN software. Green and red color indicate live and dead bacteria in the biofilm, respectively.

When a dentifrice at a concentration of 50 mg/ml was treated, there was no difference in initial biofilm formation between the control group and the test-control group, and S. mutans initial biofilm formation was reduced at a concentration of 0.4% in rosemary extract compared to the test-control group (Figure 6).

Figure 6. Effect of toothpaste including rosemary extract on early biofilm of S. mutans. Early biofilm of S. mutans using the mixture of CCM and TSB was formed in the presence or the absence of toothpaste including rosemary extract at concentration of 50 mg/ml. The biofilm was stained with live/dead bacterial staining kit, and then analyzed with CLSM. The image was analyzed with ZEN software. Green and red color indicate live and dead bacteria in the biofilm, respectively.

In order to confirm the exact value, the effect of the dentifrice was investigated by measuring the biofilm area (mass) using the Image J program. Above 125 mg/ml concentration, the initial biofilm formation of S. mutans was significantly reduced in all experimental group dentifrice compared to the control group (p<0.05) (Figure 7A and 7B). At 50 mg/ml, there was a significant decrease compared to the test-control group at a concentration of 0.4% rosemary extract (p<0.05) (Figure 7C).

Figure 7. Antibiofilm effect of toothpaste including rosemary extract on early biofilm of S. mutans. The biofilm mass from the biofilm image was analyzed with image J. The experiments were performed three times in duplicate, and the data are expressed as the means±standard deviation. Asterix (*) and sharp (#) indicates that statistically significant differences compared with control and test-control, respectively (p<0.05).

Dental caries is a bacterial disease closely related to S. mutans and is caused by demineralization of the tooth surface. Moreover, with regard to S. mutans, it is known that bacteria in the biofilm state on the tooth surface are more closely related to dental caries than planktonic bacteria in saliva [6]. This is also supported by recent hypotheses and theories related to dental caries. If S. mutans is present at a high rate in the oral streptococci biofilm, it is called a cariogenic biofilm, and it is suggested that dental caries is highly related to the biofilm [28,29]. Accordingly, it was reported that metabolites of C. albicans stimulate the expression of the gtf gene in relation to the initial biofilm formation of S. mutans, leading to better biofilm formation [27]. Accordingly, prior to testing the efficacy of a dentifrice containing rosemary, the conditions for initial biofilm formation in S. mutans were investigated.

When C. albicans and S. mutans were mixed and cultured, S. mutans biofilm formation was observed to increase. However, it was also observed that S. mutans died at the same time as biofilm formation increased at a high rate of C. albicans. Therefore, when the initial biofilm was formed using the medium in which C. albicans was cultured, it was observed that the initial biofilm formation increased as the ratio of C. albicans culture medium increased. This phenomenon occurs because C. albicans and S. mutans produce antibacterial and antifungal substances for each other, and C. albicans is weak to acids, so the growth of C. albicans is inhibited in an acidic environment due to the lactic acid produced by S. mutans. can be suppressed or kill this fungus [30,31]. In addition, the antibacterial and antifungal effects of rosemary on S. mutans and C. albicans were investigated, a dentifrice was mixed using the corresponding minimum concentration, and the effect on biofilm was investigated. The antibacterial effect of rosemary in this study showed similar results to previous studies [32], and the next experiment was conducted with confidence in the method and results of this study.

When the effect of a dentifrice containing rosemary extract on S. mutans early biofilm was investigated using the medium cultured with C. albicans, the initial biofilm formation was significantly suppressed in all groups than in the control group at 125 mg/ml or higher, and the biofilm formation was significantly reduced only at a concentration of 0.4% at 50 mg/ml.

Biofilms have been reported in previous studies to be more resistant to antibacterial substances than planktonic bacteria [33,34], and it has been reported that the extracellular sugar compound secreted by S. mutans acts as a shield to prevent antibacterial substances from penetrating [35], and for this reason, more studies are being conducted on biofilm bacteria that are more related to diseases than planktonic bacteria. The anti-biofilm effect of rosemary extract was also found to have an inhibitory effect on the formation of Candida species, and the anti-biofilm effect of S. aureus and E. coli has also been reported to have an anti-biofilm effect [36].

In conclusion, based on the results of this study, the dentifrice containing rosemary extract shows an inhibitory effect on the initial biofilm formation of S. mutans. This is because the rosemary extract remains in the oral cavity after tooth brushing and suppresses the formation of biofilm on the tooth surface. It is believed that caries can be prevented.

To confirm the antibacterial and anti-biofilm efficacy of the dentifrice containing rosemary extract, we studied the antibacterial and inhibitory effects on S. mutans and C. albicans by using experimental dentifrice containing 0.2% or 0.4% rosemary extract powder and control dentifrice containing dental type silica. As a result of studying the antibacterial activity and inhibitory effect on biofilm formation in C. albicans, the following conclusions were obtained.

1) Rosemary extract has an antibacterial effect on S. mutans, and the effect is maintained even when included in the dentifrice.

2) Rosemary extract showed no antifungal effect on C. albicans.

3) A dentifrice containing rosemary extract inhibits S. mutans early biofilm formation.

4) After tooth brushing with a dentifrice containing rosemary extract, the remaining rosemary extract would inhibit the formation of dental biofilms.

As a result of the above study, the antimicrobial activity and anti-biofilm efficacy of the dentifrice containing rosemary extract could be confirmed. Referring to this, it is thought that it can be used as an evidence when developing a dentifrice in the future.

No potential conflict of interest relevant to this article was reported.