1.Background

The inhibition of microbial growth and biofilm formation is important to reduce oral infectious diseases¹⁾. Antibiotics have been used to control infectious diseases, and their intensive use has led to the emergence of resistant bacterial strains^2,3). Antibiotics have also been used to effectively control oral infectious diseases, however, they have many side effects⁴⁾. Medicinal plants used in traditional medicine are known to have various biological activities and are safe with minimal or no side effects^5,6). Research and interest in medicinal plants as a resource for finding safe substances to control new pathogenic microorganisms is growing^2,3). The development of safe plant-derived medicinal substances is necessary to effectively control oral infectious microorganisms⁴⁾.

Angelica is classified into three species: the Korean species is Angelica gigas Nakai (AGN), the Chinese species is Angelica sinensis, and the Japanese species is Angelica acutiloba⁷⁾. The Korean name for AGN is Cham-dang-gui, which grows naturally or is cultivated, and the dried roots are used in traditional herbal medicine prescriptions⁸⁾. The AGN root is known to have various pharmacological effects, including anticancer, neuroprotective, anti-inflammatory, anemia and cardiovascular system, antioxidant, and improving immunity⁹⁾. In addition, there have been no reports of the side effects of AGN¹⁰⁾, and its safety has already been confirmed in animal¹¹⁾ and clinical¹²⁾ studies. Despite the various pharmacological effects and safety profiles of AGN, there are few reports on its antimicrobial effects. In particular, there are no reports on the anti-oral microbial effects of AGN.

2.Objectives

The purpose of this study was to reveal the anti-oral microbial effect and the microbial and biochemical changes in oral microorganisms according to the concentration of the ethanol extract of AGN (EAGN) root, and to confirm the possibility of using EAGN as a plant-derived functional substance for controlling oral infectious microorganisms.

1.Study design

1) Preparation of EAGN

Dried AGN roots were purchased from Goodherb Co. (Seoul, Korea) and used for ethanol extract. AGN roots (500 g) were subjected to extraction with 70% ethanol according to a previous reported method¹⁾. The extracted solvent was soaked at room temperature for 48 hours, filtered with filter paper (Whatman No. 2; Whatman Inc., Maidstone, UK), concentrated with a rotary vacuum evaporator (Eyela A-1000; Eyela, Tokyo, Japan) and freeze-dried at −70°C. EAGN was dissolved in dimethyl sulfoxide (DMSO) (Sigma-Aldrich Chemical Co., St. Louis, MO, USA) to make a stock solution (250 mg/ml), and the stock solution of EAGN was diluted and used at concentrations 2.5 mg/ml, 3.75 mg/ml, 5.0 mg/ml, and 6.75 mg/ml.

2) Oral microbial culture

Streptococcus mutans, Lactobacillus casei, and Aggregatibacter actinomycetemcomitans, which are oral pathogenic bacteria, and Candida albicans, which is a fungus, were purchased from the Korea Microbiological Conservation Center and Korean Collection for Type Cultures (Table 1). S. mutans and A. actinomycetemcomitans were cultured with Brain Heart infusion agar and broth (MB cell Ltd., Seoul, Korea), L. casei was cultured with De Man–Rogosa–Sharpe (MRS) agar and broth (MB cell Ltd.), and C. albicans was cultured with Potato dextrose agar (PDA, MB cell Ltd.) and potato dextrose broth (MB cell Ltd.) in an incubator (Daihan Scientific Co., Daegu, Korea) at 36.5°C for 24 hours and were diluted to 5×10⁶ CFU/ml according to a previous reported method¹³⁾.

Oral Microbial Strains for Culture

Class name	Strain	Culture media	Aero condition	Staining
Bacteria	Streptococcus mutans KCCM 40105	Brain heart infusion (BHI) Agar and Broth	Facultative anaerobic	Gram positive
Bacteria	Lactobacillus casei KCCM 12452	De Man–Rogosa–Sharpe (MRS) Agar and Broth	Facultative anaerobic	Gram positive
Bacteria	Aggregatibacter actinomycetemcomitans KCTC 2581	BHI Agar and Broth	Microaerophilic	Gram negative
Fungi	Candida albicans KCCM 11282	Potato dextrose agar (PDA) and potato dextrose broth (PDB)	Aerobic	Gram positive

KCCM: Korea Microbiological Conservation Center, KCTC: Korean Collection for Type Cultures.

3) Disk diffusion test

To confirm the anti-oral microbial activity according to the concentration of EAGN, oral microorganisms were cultured, diluted to 5×10⁶ CFU/ml and smeared on agar plates. EAGN treated paper discs (Ф6 mm; Advantec Toyo Kaisha Ltd., Tokyo, Japan) and Penicillin G (10 mcg; Oxoid Ltd., Hampshire, UK) antibiotic discs were place on the prepared agar plates. After culturing for 24 hours, the diameter of the clear zone formed around the discs was measured using a vernier caliper (Mitutoyo Co., Kamagawa, Japan).

4) Measurement of growth and biofilm formation assay

The selective medium inoculated with diluted oral microorganisms was treated with EAGN at concentrations of 2.5 mg/ml, 3.75 mg/ml, 5 mg/ml, and 6.75 mg/ml and then cultured for 24 hours. The growth of oral microorganisms was measured at a wavelength of 600 nm using a UV-Vis spectrophotometer (X-ma 1200; Human Corp., Seoul, Korea). The biofilm formation of oral microorganisms was measured at 570 nm using microplate reader (Sunrise TM; Tecan, Grödig, Austria) after removing the medium, washing with DW, staining with 0.1% crystal violet (MB cell Ltd.) for 15 minutes, and treatment with 95% ethanol, according to a previously reported method¹⁴⁾.

5) Measurement of acid production and buffering capacity

Oral microorganisms were diluted to 5×10⁶ CF/ml and each concentration of EAGN was added to 5 ml selective medium and cultured for 24 hours. Acid production and buffering capacity were measured by the pH value using a pH meter (TTBH Pte Ltd., Petro Centre, Singapore), as previous reported¹⁴⁾. Buffering capacity was measured as the amount of 1M NaOH required to bring the pH of the medium to 7, according to a previously reported method¹⁵⁾.

2.Statistical analysis

The experiments were conducted three times and the results were presented as mean±standard deviation. The results were analyzed using IBM SPSS (version 25.0; IBM Corp., Amonk, NY, USA) and significant differences were determined using one-way analysis of variance.

1.Anti-oral microbial activity of EAGN

The results of a disk diffusion test to confirm the anti-oral microbial activity of EAGN against S. mutans, L. casei, and A. actinomycetemcomitans belonging to bacteria and C. albicans belonging to fungi are shown in Fig. 1. EAGN showed anibacterial effects against S. mutans and A. actinomycetemcomitans at all concentration, and the antibacterial effects of S. mutans and A. actinomycetemcomitans increased with increasing EAGN concentrations. Especially, a more susceptible effect of EAGN concentration was seen above 5.0 mg/ml for S. mutans and above 3.75 mg/ml for A. actinomycetemcomitans (Table 2). However, EAGN had no effect on L. casei and C. albicans.

Fig. 1. Anti-oral Microbial Activity of 70% ethanol extract of Angelica gigas Nakai (scare bar=6 mm). DMSO: dimethyl sulfoxide.

Comparison of Anti-oral Microbial Activity of 70% EAGN by Disk Diffusion Test

Strains	DMSO	EAGN (mg/ml)				Penicillin G (10 units)
		2.5	3.75	5.0	6.75
Streptococcus mutans	−	＋	＋	＋＋	＋＋	＋＋＋
Lactobacillus casei	−	−	−	−	−	＋＋＋
Aggregatibacter actinomycetemcomitans	−	＋	＋＋	＋＋	＋＋	＋＋＋
Candida albicans	−	−	−	−	−	−

−: resistrant (<6 mm), ＋: susceptible (6∼10 mm), ＋＋: more susceptible (11∼15 mm), ＋＋＋: most susceptible (>16 mm).

DMSO: dimethyl sulfoxide, EAGN: ethanol extract of Angelica gigas Nakai.

2.Inhibitory effect on growth and biofilm formation of oral microorganisms of EAGN

The effects of EAGN on the growth of oral microorganisms and biofilm formation were shown in Fig. 2. Regarding microbial growth (Fig. 2A), EAGN treatment reduced the growth of S. mutans, L. casei, and A. actinomycetemcomitans, in particular, the growth of A. actinomycetemcomitans and L. casei was significantly reduced. C. albicans increased with EAGN treatment. In the case of biofilm formation (Fig. 2B), S. mutans, L. casei and A. actinomycetemcomitans significantly decreased depending on EAGN concentration. A significant reduction in biofilm formation was seen at EAGN concentration above 3.75 mg/ml for S. mutans, at 6.75 mg/ml for L. casei, and above 2.5 mg/ml for A. actinomycetemcomitans (Table 2). The biofilm formation of C. albicans was significantly lower than that of other oral bacteria and was not affected by EAGN.

Fig. 2. Changes in growth (A) and biofilm formation (B) of oral microorganisms according to 70% ethanol extract of Angelica gigas Nakai treatment. OD: optical density, DMSO: dimethyl sulfoxide. **p<0.01, ***p<0.001 compared with DMSO of bacteria. ^###p<0.001 compared with DMSO group.

3.Effect on acid production and buffering capacity of oral microorganisms of EAGN

The effects of EAGN on the acid production and buffering capacity of oral microorganisms are shown in Fig. 3. The pH was significantly reduced by treatment with EAGN in S. mutans, A. actinomycetemcomitans, and CC (Fig. 3A). In the case of S. mutans and A. actinomycetemcomitans, the pH increased slightly with 6.75 mg/ml EAGN. The pH of C. albicans decreased with EAGN treatment, whereas that of L. casei was unaffected by EAGN treatment. The value of 1M NaOH for S. mutans and A. actinomycetemcomitans decreased at an EAGN concentration of 3.75 mg/ml and above, and their buffering capacity increased. However, the buffering capacities of L. casei and C. albicans were not affected by EAGN treatment (Fig. 3B).

Fig. 3. Changes in acid production (A) and buffering capacity (B) according to 70% ethanol extract of Angelica gigas Nakai treatment. DMSO: dimethyl sulfoxide. *p<0.05, **p<0.01, ***p<0.001 compared with DMSO of bacteria. ^##p<0.01 compared with DMSO group.

1.Key results and interpretation

This study was conducted to reveal the anti-oral microbial effect and the microbial and biochemical changes in oral microorganisms according to the concentration of EAGN root, and to confirm the possibility of using EAGN as a functional substance derived from medicinal plants to control oral infectious microorganisms. EAGN showed anti-oral bacterial effects on S. mutans and A. actinomycetemcomitans at all concentration, and S. mutans showed a more susceptible effect at concentrations above 5.0 mg/ml and A. actinomycetemcomitans at 3.75 mg/ml (Table 2). However, EAGN had no effect on L. casei or C. albicans. These results indicate that EAGN has anti-oral bacterial effects against S. mutans, a dental caries-causing bacterium, and A. actinomycetemcomitans, a periodontal disease-causing bacterium, and has the potential to be used as a functional substance to control of these oral bacteria. EAGN treatment reduced the growth of S. mutans, L. casei, and A. actinomycetemcomitans. In particular, A. actinomycetemcomitans growth was significantly reduced at all concentrations tested. Biofilm formation was significantly reduced at concentrations greater than 3.75 mg/ml for S. mutans and 2.5 mg/ml for A. actinomycetemcomitans (Fig. 2). The acid production was significantly increased by treatment with EAGN in S. mutans, A. actinomycetemcomitans, and C. albicans, and the buffering capacity of S. mutans and A. actinomycetemcomitans increased from an EAGN concentration of 3.75 mg/ml and above (Fig. 3). These results indicate that EAGN induced changes were more sensitive to A. actinomycetemcomitans than to S. mutans, especially in terms of growth and biofilm formation.

Based on these results, EAGN showed anti-oral bacterial effects on S. mutans and A. actinomycetemcomitans, which are thought to be related to the inhibition of their growth and biofilm formation. The concentration of EAGN for effective antibacterial effects against both S. mutans and A. actinomycetemcomitans was confirmed to be 3.75 mg/ml and above. Therefore, EAGN can be used as a safe functional substance derived from medicinal plants owing to its antibacterial effects against S. mutans and A. actinomycetemcomitans.

2.Comparison with previous studies

There are abundant studies on the various pharmacological effects of AGN⁹⁾, however, studies on its antibacterial effects are rare, and these studies have reported that EAGN has antibacterial effects against Bacillus subtilis, Bacillus cereus, Staphylococcus aureus, Escherichia coli, and Pseudomonas fluorescens¹⁶⁾. This is the first study to investigate the anti-oral microbial effects of EAGN. In this study, S. mutans, L. casei, and A. actinomycetemcomitans, which are pathogenic oral bacteria and C. albicans, which is fungus were used in the experiments. S. mutans causes dental caries and forms biofilms on the tooth surface¹⁷⁾. L. casei causes advanced dental caries¹⁴⁾, and colonizes oral tissues to form biofilm¹⁸⁾. A. actinomycetemcomitans causes aggressive periodontal disease and colonizes dental plaque^19,20). C. albicans is a normal flora in the non-pathogenic state, but attaches to oral epithelial cells or dentures of patients with weakened immune systems and causes opportunistic infections and candidiasis²¹⁾. The main components of AGN are coumarin, a lipid-soluble substance, and polysaccharide, a water-soluble substance²²⁾. Cumarin includes pyranocoumarins (decursin and decursinol algelate), umbelliferone, nodakenin, peucedanone, marmesin, demethylsuberosin and isoimperatorin²³⁾. Decursin and decursinol algelate are the main active components of the biological effects of AGN^23,24). AGN root contains more than 6% of the active components, decursin, decursinol angelate and nodakenin, in particular, decursin and decursinol angelate are not found in Chinese and Japanese Angelica species²⁵⁾. AGN roots extracted with water did not show an antibacterial effect, but only EAGN roots showed an effect²⁶⁾. In addition, among lipid-soluble cumarins, decursin and decursinol angelate have six-membered rings and senecioylic acid-type side chain that show antibacterial properties in structure, and decursin and decursinol angelate showed antibacterial activity against Bacillus subtilis¹⁶⁾. Therefore, the antibacterial effect and inhibition of growth and biofilm formation of EAGN against S. mutans and A. actinomycetemcomitans observed in this study were also thought to be caused by decursin and decursinol angelate. There was a study in which decursin and decursinol angelate showed antifungal effects on the pathogenic fungi Magonaporthe oryzae²⁷⁾, but in another study using EAGN, it did not show an effect on Sacchromyces cerevisiae²⁶⁾, and this study also had no antifungal effect on C. albicans.

3.Limitations

A limitation of this study is that we could not identify substances with antibacterial effects against S. mutans and A. actinomycetemcomitans, because used crude extracts of EAGN. Future studies are needed to identify the components and mechanisms of EAGN root showing antibacterial effects against S. mutans and A. actinomycetemcomitans, and to confirm the effectiveness of EAGN root through clinical application.

4.Conclusion

EAGN showed anti-oral bacterial effects against both S. mutans and A. actinomycetemcomitans at concentrations above 3.75 mg/ml, and these effects are thought to be related to the inhibition of their growth and biofilm formation. Therefore, EAGN can be used as a safe functional substance derived from medicinal plants owing to its antibacterial effects against S. mutans and A. actinomycetemcomitans.

None.

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.

Not applicable.

This work was supported by Youngsan University Research Fund of 2023.

The data supporting the findings of this study are available from the corresponding author upon reasonable request.