
Oral biofilm, commonly called dental plaque, adheres to tooth surfaces and constitute a critical etiological factor in the development of various oral diseases, including dental caries and periodontal diseases1). The formation of a dental biofilm begins with the adhesion of resident oral microorganisms to the acquired pellicle, a proteinaceous layer that coats the tooth surface through salivary interactions. Among these microorganisms, Streptococcus mutans, one of the most prevalent bacterial species in the oral cavity, plays a pivotal role in biofilm development2,3). S. mutans synthesizes adhesive, water-insoluble extracellular polysaccharides (EPS), particularly glucans, through the metabolism of dietary sugars, thereby facilitating the aggregation and colonization of diverse microbial communities within biofilms2,3). Furthermore, S. mutans metabolizes fermentable carbohydrates to produce organic acids, lowering the environmental pH to below 5.5, a critical threshold for initiating tooth demineralization4). Consequently, inhibition of S. mutans growth is a promising strategy for suppressing biofilm formation, thereby mitigating plaque-induced oral diseases and maintaining oral health.
Recently, natural compounds with antimicrobial activity have gained significant attention, with increasing reports of their potential in addressing systemic diseases and combating oral pathogens5-10). Among these natural resources, Artemisia princeps (AP), commonly called mugwort, has been widely used as a traditional medicinal herb in East Asian countries, such as Korea and Japan, because of its diverse bioactive compounds. In particular, AP Pampanini from Ganghwa, Korea, is known to contain various polyphenolic compounds and has been reported to exhibit various biological activities, such as antibacterial activity against oral bacteria and analgesic, antibacterial, and anti-inflammatory effects on various systemic diseases11-15). However, no studies have evaluated the effects of AP extract on the formation of oral biofilms caused by S. mutans.
This study aimed to evaluate the effects of an AP extract from Ganghwa, Korea, on the growth and biofilm formation of S. mutans. Furthermore, we propose that natural products have potential as alternative antimicrobial agents by comparing their efficacy with chlorhexidine, a widely used oral disinfectant in dentistry.
The S. mutans (KCTC 3065) strain was purchased from the Korean Collection for Type Cultures (KCTC, Daejeon, Korea). The strain was activated by inoculating it into brain heart infusion broth (BHI) (Difco BD; Becton, Dickinson and Company, Franklin Lakes, NJ, USA) and incubating it at 37°C for 24 hours prior to use in the experiments. The AP used in this study was a dried Ganghwa mugwort product grown in Korea and was purchased online (Eommaaeson Co., Gimcheon, Korea). The dried AP was extracted three times with 10 times (w/v) of 99.5% methanol solution and then centrifuged to obtain a methanol AP extract at a concentration of 10,000 ppm. The solution was diluted with methanol for use in subsequent experiments. MeOH, the solvent used for extraction, served as the negative control (NC), and a 0.12% chlorhexidine solution (CHX) (Hexamedine; Bukwang Pharm Co., Ltd., Seoul, Korea) was used as the positive control (PC).
After treating the AP extract diluted to various concentrations (ranging from 1 to 2,000 ppm) in a culture medium of S. mutans, it was incubated at 37°C for 24 hours. From each group, 0.1 ml of the cultured medium was spread onto the BHI agar plates and incubated for 48 hours. The bacterial colonies were counted and expressed as colony-forming units per milliliter (CFU/ml). A minimum bactericidal concentration (MBC) assay was conducted to evaluate the effects of AP extract on the growth of S. mutans. Measurements were performed independently with four replicates.
Biofilm formation assays were performed as described previously16). Briefly, saliva samples were collected from healthy adults, following a protocol approved by the Institutional Review Board of Kyungpook National University (Approval No. 2023-0246). All participants provided informed consent prior to sample collection. The saliva samples were centrifuged at 4°C and 4,000 rpm for 10 minutes (Centrifuge 1580R; Labogene Inc., Daejeon, Korea), and the supernatant was filtered through a 0.22 μm membrane filter (Merck Millipore, Burlington, MA, USA). To form an acquired pellicle, the filtered saliva samples were added to 12-well plates (SPL Life Sciences Co., Ltd., Pocheon, Korea) and incubated at 37°C for 6 hours. After removing the initial saliva medium, the AP extract or control was added to 1.8 ml of fresh saliva medium containing 1% sucrose as the dietary sugar for EPS formation, followed by the addition of 0.2 ml of bacterial suspension at 1×108 CFU/ml, and the plates were incubated at 37°C for 48 hours. The biofilm was stained by removing the medium, adding a 0.1% crystal violet solution, and incubating at room temperature for 15 minutes. Stained biofilms were washed twice with sterile distilled water. Images of the stained biofilm were captured, and the biofilm was dissolved in 20% acetic acid for quantification. A 0.2 ml aliquot of the solution was transferred to a 96-well plate, and the absorbance was measured at 590 nm using a microplate reader (Thermo Fisher Scientific, Waltham, MA, USA). Measurements were performed independently with four replicates.
All statistical analyses were performed using IBM SPSS Statistics (version 29.0; IBM Corp., Armonk, NY, USA), with the significance level set at p<0.05. The Shapiro–Wilk test was used to assess the normality of the data. Differences in means between groups were analyzed using one-way analysis of variance, followed by Tukey’s post-hoc test.
The results demonstrated a significant reduction in the CFU of S. mutans with increasing extract concentrations. The MBC was determined to be 1,250 ppm. AP showed an inhibitory effect on colony formation starting from 100 ppm, and only a few colonies were observed at 1,000 ppm (data not shown). Based on these findings, subsequent experiments were conducted using AP extract at 100 and 1,000 ppm concentrations.
Biofilm formation induced by S. mutans inoculation was confirmed on plates coated with acquired pellicles from human saliva (Con). In the NC group, where methanol was used as the extraction solvent, the absorbance value for biofilm formation was significantly suppressed compared to that in the untreated Con group; however, the stained biofilm did not show a significant difference (Fig. 1). However, treatment with the AP extract significantly inhibited biofilm formation in a concentration-dependent manner, exhibiting greater inhibitory effects than the PC group treated with CHX (Fig. 1A). The absorbance of the biofilm formed was significantly inhibited in the AP extract-treated groups. Notably, the 1,000 ppm AP extract group showed a 70% reduction in biofilm formation compared to the NC group (Fig. 1B).
CHX mouthwash is a widely used antiseptic in dentistry for managing inflammation, stomatitis, and periodontitis and for pre- and post-surgical disinfection. Although its bactericidal efficacy is excellent, long-term use can lead to adverse effects, such as disruption of the normal oral microbiota, mucosal irritation, and discoloration of dental prostheses. Therefore, research has increasingly focused on developing antiseptic agents derived from natural products that are relatively safe and less toxic.
This study evaluated the effects of AP extract on the growth and biofilm formation of S. mutans, one of the most common and significant bacteria involved in oral biofilm formation. AP, a traditional herbal medicine widely used in East Asia, contains various bioactive polyphenolic compounds, including flavonoids, phenolic acids, lignans, tannins, and artemisinin, which contribute to its physiological effects11,13,17,18).
Mishra et al.19) highlighted the potential of natural antimicrobial agents containing polyphenols as alternatives to chemical antimicrobials for inhibiting biofilm formation. Previous studies have extensively investigated the antimicrobial activities of natural formulations enriched with5-7,11,13,17-22).
In this study, the AP extract at a concentration of 1,000 ppm exhibited greater biofilm inhibition than CHX. This finding highlights the significance of this research in demonstrating the extract’s superior biofilm inhibitory effects over chemical antimicrobials. Furthermore, given its long history of use as a traditional herbal medicine, the AP extract is considered to have notable advantages over conventional chemical antimicrobials, particularly in terms of safety and reduced side effects.
The MBC of the AP extract was 1,250 ppm, confirming its inhibitory effect on the growth of S. mutans. According to Mohammed et al.23), the MBC of the Artemisia herba-alba (Shih) plant extract against S. mutans is 4,000 ppm. Additionally, Kim et al.24) reported that a methanolic extract of Artemisia annua L. exhibited an MBC of 14,000 ppm against Fusobacterium nucleatum subsp. polymorphum and 7,000 ppm against Prevotella intermedia. Although the species of the Artemisia genus and the types of bacteria used in these studies vary, making direct comparisons difficult, there is a consensus across multiple studies that plants belonging to the Artemisia genus possess antimicrobial properties20,25,26).
Furthermore, in the present study, increasing concentrations of AP extract progressively inhibited biofilm formation. Similarly, Park and Oh14) reported that ethanol and methanol extracts of AP exhibited significantly increased inhibitory effects against Cutibacterium acnes and Staphylococcus aureus as the concentration increased from 5,000 ppm to 20,000 ppm. However, differences in the bacterial strains and extract concentrations evaluated in this study and previous studies could contribute to variations in the findings20,24). Additionally, factors such as the solvent used for extraction and the geographical location of cultivation may influence the ratio and chemical composition of bioactive compounds, thereby affecting their biological activity21). Nonetheless, the results of this study align with those of previous studies demonstrating the antimicrobial properties of the polyphenols contained in AP extracts11,13,14,17).
This study confirmed that AP extract strongly inhibited oral biofilm formation in a concentration-dependent manner, indicating its potential as a natural antimicrobial agent for preventing and treating oral diseases. These findings have significant implications for the development of plant-derived solutions for oral health management. However, although both MBC and minimum inhibitory concentration (MIC) assays were conducted to evaluate the antimicrobial activity of the AP extract against S. mutans, accurate absorbance measurements could not be obtained because of the inherent color of the extract, resulting in the inability to acquire MIC data. Moreover, because the experiments were conducted under laboratory conditions, further research is necessary to evaluate the applicability of the extract in oral environments. In addition, it is necessary to evaluate the stability of methanol, the extraction solvent for AP, and to investigate the potential of high-content polyphenol components in AP to inhibit biofilm formation by confirming the polyphenol content in AP through high-performance liquid chromatography analysis. In particular, studies addressing the AP extract’s long-term safety and interactions with the complex oral microbiota are essential for its potential clinical application.
This study demonstrated the antimicrobial and antibiofilm effects of the AP extract against S. mutans, contributing to the expansion and reinforcement of existing research on oral antimicrobial activity using natural products. These findings suggest the potential for developing oral healthcare products utilizing naturally derived compounds and provide valuable foundational data for exploring alternatives to conventional chemical antimicrobials.
None.
Conflict of interest
Ji-Hyun Min has been journal manager of the Journal of Dental Hygiene Science since January 2023. Ji-Hyun Min was not involved in the review process of this editorial. Otherwise, no potential conflict of interest relevant to this article was reported. Ki-Rim Kim declare that she has no conflicts of interest.
Ethical approval
This study was approved by the Institutional Review Board Committee of Kyungpook National University (Approval No. 2023-0246), and written informed consent was obtained from all subjects.
Author contributions
Conceptualization: Ki-Rim Kim. Data acquisition: Ki-Rim Kim. Formal analysis: Ji-Hyun Min and Ki-Rim Kim. Writing-original draft: Ji-Hyun Min. Writing-review & editing: Ji-Hyun Min and Ki-Rim Kim.
Funding
None.
Data availability
Raw data is provided at the request of the corresponding author for reasonable reason.
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