A Study on the Antibacterial Effect of Chamaecyparis Obtusa Extract on the Causative Agent of Dental Caries
Yu-Ri Choi1, Mi-Sook Choi2, Haeng-Eun Lee3, Seoul-Hee Nam3*
1Department of Dental Hygiene, Hallym polytechnic University, Chuncheon, 24210, Republic of Korea e
2Department of Dental Hygiene, Andong Science College, Andong, 36686, Republic of Korea
3Department of Dental Hygiene, College of Health Sciences, Kangwon National University, Samcheok, 25949, Republic of Korea
ABSTRACT
We conducted to evaluate the antibacterial effect of the Chamaecyparisobtusa extract on Sreptococcusmutans (S. mutans). The strains used in this experiment were investigated for S. mutans. The activity of Chamaecyparisobtusaextract was measured the diameter (mm) of the clear zone by the paper disk (Φ8 mm) diffusion method and colony forming units (CFU). The significant difference among groups in one-way ANOVA and Duncan test.As a result, the ethanol extract of Chamaecyparisobtusa showed the strongest antimicrobial activity against S. mutans. The clear zone of S. mutans was found to be 14 mm, 15.5 mm and 17 mm at 20 mg/mL,30 mg/mLand at 40 mg/mL, respectively. In addition, the Chamaecyparisobtusa extract exhibited concentration-dependent inhibition of CFU over 1.25 mg/mL, with 100% inhibition at a concentration of 40 mg/mL. These results show that Chamaecyparisobtusa extract has anticariogenic effect on S. mutans.
Therefore, Chamaecyparisobtusa extract can be used effectively in the prevention of oral diseases.
Keywords
Antibacterial activity, Dental caries, Plant extract, Chamaecyparisobtusa, Streptococcus mutans
Introduction
Oral health is essential to general health and well-being. Good oral health enables individuals to eat and enjoy a variety of foods, communicate effectively, and have self-esteem, social confidence, and a good overall quality of life [1]. In fact, oral health affects every aspect of people’s lives. To achieve oral health, oral health care is important [2]. With the increasing interest in oral health care, the prevention of oral diseases is now more prevalent than the prevention of other diseases. However, people in developed and developing countries still suffer from oral diseases [3].
According to a recent report of the World Health Organization (WHO), 60% of adults around the world suffer from oral diseases related to dental caries. This indicates that suffering from dental caries persists worldwide [4, 5]. Dental caries is a disease caused by complex actions of bacteria, dietary factors, and host factors. The role of bacteria is especially essential. The bacterium Streptococcus mutans (S. mutans) is known to be a major causative agent of dental caries [6]. It induces dental caries through its adhesion, proliferation, and acid production on the tooth surface [7]. S. mutans secretes an enzyme called glucosyl transferase (GTase) that induces dental caries outside or on the surface of the microbial body and breaks down sucrose in food to form insoluble glucan on the tooth surface. Glucan also attaches to the tooth surface with other microorganisms in the oral cavity to create a biofilm [8]. Therefore, to prevent and reduce dental caries, the growth of S. mutans and the formation of biofilms in the oral cavity should be inhibited.
Antibiotics, which are substances that inhibit the growth of microorganisms, have been widely consumed as therapeutic medicines until now. In recent years, however, due to the emergence of resistant bacteria from long-term administration of antibiotics, various side effects have appeared,
which are considered very serious problems [9]. Accordingly, much attention has been paid to the development of antibiotics with low resistance using microorganisms and through the study of the antibacterial activity of natural products [10]. In recent years, as a part of basic research to prevent dental caries, interest in microbial inhibition using natural extracts has increased, and various studies on it have been conducted [11]. Magnolol and hinokiol [12], gymnemic acid [13], Japanese green tea extract [14], and cardinol [15] have been reported to have an anticaries effect on the oral cavity. Gold [16] and plum extract have been found to have antibacterial effects on cariogenic bacteria, and green tea, mulberry, and mate leaves extracts have been found to have growth inhibition effects on S. mutans [17].
Plant extracts have long been known to have antibacterial and antioxidant activities. In particular, essential oils, which are volatile secondary metabolites, have anti-inflammatory, antibacterial, and anti-allergic functions. Thus, they are being studied as natural pharmaceutical supplements or food preservatives [18]. In addition, the antibacterial effects of phytoncide and aroma essential oil, which are essential oils extracted from plants, are widely known [19].
Phytoncide is a volatile aromatic component of plants that is obtained by distilling plants with water vapor. This and other volatile components are composed of compounds such as phenolics, terpenoid, alkaloid, phenylpropane, acetogenin, and steroid, which have dozens to 200 species [20, 21]. Phytoncide essential oil has been reported to have antibacterial and antifungal effects [22].
A large amount of phytoncide with a peculiar scent is produced on the stems of Chamaecyparisobtusa, an evergreen coniferous tree of the genus Charmaecyparis of the Cupressaceae family, generally known as “hinoki cypress,” which grows wild in Japan and Taiwan and has been successfully cultivated in the southern part of Korea. [23]. From ancient times, such tree has been believed to have a natural healing effect. Recently, the essential oil extracted from the phytoncide on the stems of the tree has been found to have antioxidant and antibacterial effects. Thus, Chamaecyparisobtusa is used as a medicinal plant and for forest bathing [24].
This study was conducted to find out if a natural product with well-known antibacterial and antifungal effects can be used for therapeutic purposes against resident oral flora. It investigated the antibacterial activity of Chamaecyparisobtusa extract against dental caries and evaluated the potential of such extract for the prevention and treatment of dental caries.
Materialsand Methods
Bacterial culture
To measure the antibacterial activity, S. mutans (KCTC 3065/ATCC 25175) was incubated in Brain Heart Infusion (BHI; BD Bioscience, Franklin Lakes, NJ) at 37°C under microaerophilic conditions for 24 hours. The S. mutans was diluted to a concentration of 1×105 colony forming units (CFU/mL).
Sample extraction
Chamaecyparisobtusa used in this study was grown in Gyeongsan, Gyeongsangbuk, South Korea, was purchased from Cheongmyeong Co., Ltd. After adding 70% ethanol to 100 g of crushed Chamaecyparisobtusa, extraction was done in a heated mantle at 60°Ϲ for 12 hours. The extract was filtered by using filter paper (Advantec No. 2, Toyo, Japan), and the Chamaecyparisobtusa extract was concentrated and lyophilized by using a rotary vacuum evaporator (N-1300E.V.S. EYELA Co.,Japan). The concentrated Chamaecyparisobtusa was
lyophilized using a freeze dryer (Ilshin Lab Co., South Korea) to obtain the Chamaecyparisobtusa powder. The final samples were stored at -20°C until use.
Disc diffusion method
A paper disc was placed on top of BHI agar medium, and 100 μL of each experimental group (1.25, 2.5, 5, 10, 20, 30 and 40 mg/mL) was dropped onto an 8 mm filter paper disk (Advantec Toyo Kaisha, Ltd.). After keeping it at 37°C for 24 hours, the diameter of the clear zone was measured.
Antibacterial test
Mixed extracts were prepared at the 1.25 mg/mL, 2.5 mg/mL, 5 mg/ml, 10 mg/mL, 20 mg/mL, 30 mg/mL and 40 mg/mL concentrations, and S. mutans-containing solution of 100 μL was inoculated into the medium. The microplate was incubated anaerobically for 24 hours at 37°C, and then the mixture in each well was uniformly smeared in an BHI agar medium. After incubation for 24 hours, the number of CFU present in the agar plates was checked.
Statistical analysis
The software (IBM SPSS Statistics 24.0, SPSS Inc., Chicago, IL, USA) was used to evaluate significant differences in the antibacterial activity with one-way ANOVA. In post hoc analysis used the Duncan test at a 0.05 significance level
Results
This study investigated the antibacterial effect of Chamaecyparisobtusa extract on S. mutans, the causative agent of dental caries. Chamaecyparisobtusa ethanol extract was isolated and concentrated to measure its antibacterial effect and properties against S. mutans using the paper disk method. The results are as follows (Figure 1). When Chamaecyparisobtusa extract was applied to S. mutans at concentrations of 20, 30, and 40 mg/mL, it inhibited the growth of the bacteria by 14, 15.5, and 17 mm, respectively. When Chamaecyparisobtusa extract was applied at concentrations of 20, 30, and 40 mg/mL, bacterial growth inhibition zones were observed in proportion to the increase in the concentration of the extract. The strongest antibacterial effect was observed in the 40mg/mL group, which showed a 17mm bacterial growth inhibition zone.
Figure 1. Antibacterial activity of the ethanol extract of Chamaecyparisobtusa on the growth of S.mutansvia paper disk method.
To observe the antibacterial activity of Chamaecyparisobtusaextract against S. mutans, Chamaecyparisobtusa extract was added to S. mutans at the concentrations of 1.25, 2.5, 5, 10, 20, 30, and 40 mg/mLon a BHI agar medium, respectively. After 24 hours, the number of CFU of the S. mutans and the growth inhibition rate compared to the control were measured. The results are as follows (Figure 2).
Figure 2. Inhibitory effect of Chamaecyparisobtusa on biofilm formation by S. mutans, as observed by the number of CFU.
In the control, which was not treated with Chamaecyparisobtusa extract, a high number of S.
mutans bacteria was detected: 3.7±0.2 × 1011mg/mL. In the medium to which 1.25 mg/ml of Chamaecyparisobtusa extract was added, the number of S. mutans CFU was 1.8±0.2 × 108 CFU;
in the medium to which 2.5 mg/mLof the extract was added, 2.8±0.1 × 107 CFU; in the medium to which 5 mg/mLof the extract was added, 1.5±0.1 × 105CFU; in the medium to which 10 mg/mLof the extract was added, 5.8±0.2 × 103 CFU/ml; in the medium to which 20 mg/mLof the extract was added, 2.1±0.1 × 102CFU; in the medium to which 30 mg/mLof the extract was added, 1.5±0.1 × 102CFU; and in the medium to which 40 mg/mLof the extract was added, 0.0 CFU/ml. As shown in Figure 2, compared with the control, even in the medium to which only 1.25 mg/mLof the extract was added, the S. mutans colony formation was significantly reduced;
and in the medium to which 40 mg/mLof the extract was added, no S. mutans colony formation was observed at all (Figure 3).
Figure 3. Inhibitory effect of Chamaecyparisobtusa on the number of CFUsecreted by S.
mutans.
The statistical analyses showed that the antibacterial effect of Chamaecyparisobtusaextract on S.
mutans significantly differed according to the concentration of the extract that was applied (Table 1). The CFU of the control was 3.7±0.2 × 1011a, and the number of CFUs decreased significantly as the concentration of the Chamaecyparisobtusaextract increased (p < 0.05). However, there was no statistically significant difference in the number of CFU when 20-40 mg/mLof the extract was applied (p > 0.05). At the 40mg/mLconcentration of the extract, no CFU appeared, so 40 mg/mLcan be considered the optimal concentration in terms of antibacterial power.
Table 1. Antibacterial activity of ethanol extract of Chamaecyparisobtusa via number of CFU.
Group S. mutans 1.25 mg/mL
2.5 mg/mL
5 mg/mL
10 mg/mL
20 mg/mL
30 mg/mL
40 mg/
mL P- Value
24h 3.7±0.2 1011a
1.8±0.2 108b
2.8±0.1 107c
1.5±0.1 105d
5.8±0.2 103e
2.1±0.1 102f
1.5±0.1
102f 0.0f 0.000
*The significant difference among groups in one-way ANOVA. Different letters (a, b, c, d, e and f) by the presented significant result of the post hoc Duncan test (p<0.05).
Discussions
S. mutans, a resident oral flora, causes dental caries, which leads to tooth loss. Therefore, to prevent oral disease, it is important to manage disease-causing bacteria among resident oral flora [25]. However, the use of antibiotics may discolor the teeth or dry the mouth. Therefore, studies have been conducted to find out if plant extracts with antibacterial effects can be used as antibacterial agents without the side effects of antibiotics [26]. In particular, studies have been conducted on the prevention or suppression of oral diseases through bacteriostasis or sterilization of oral bacteria using natural extracts [27]. Many of these studies used the Chamaecyparisobtusa leaf extract, as its biological effects are known. In particular, its antibacterial and anti- inflammatory activities have been reported [25]. As Chamaecyparisobtusa essential oil has been found to have high effects on physiological activities, such as anti-inflammatory and antioxidant effects as well as antibacterial and insect repellent effects that alleviate atopy and prevent mold and mites, research and other efforts to find more functional substances are continuing [28].
According to a paper on the environmental change and inhibition of adhesion of S. mutans to the natural extract, gold, as an antibacterial activity medium, when S. mutanswas administered to the culture medium with a concentration of 50 mg/mL, the polysaccharide formation was reduced, which showed antibacterial power [29]. However, the Chamaecyparisobtusa extract that was used in this study showed a bacteria killing effect at the lower concentration of 40 mg/mL, which confirmed its stronger antibacterial activity compared to that of gold.
According to a previous study, the measurement of the clear zone of Chamaecyparisobtusa extract against Gram-positive and Gram-negative bacteria that cause food poisoning showed antibacterial effects on all the bacteria [30]. Meanwhile, the results of a study that showed the antibacterial activity of curcuma longa, ginger, and finger root against S. mutans were similar to the results of this study [31].
Therefore, the results of this study confirmed the antibacterial activity of Chamaecyparisobtusa extract against cariogenic bacteria. Particularly, Chamaecyparisobtusa extract has antibacterial activity against S. mutans, which is a typical bacterium that induces dental caries. It can be said that the antibacterial activity of an active ingredient can be exhibited by its penetration into cells or its inhibition of the synthesis of the bacteria with cells [32]. Based on the above results, Chamaecyparisobtusa extract is expected to be used as an antibacterial agent against S. mutans.
Conclusion
This study evaluated the antibacterial activity of Chamaecyparisobtusa extract, which has been used as medicine for a long time, against S. mutans, a cariogenic bacterium. The antibacterial power of Chamaecyparisobtusa extract was found to be higher at higher concentrations. Its optimal concentration in terms of its antibacterial activity against S. mutanswas 40 mg/mL, at which concentration the bacteria were killed completely. As Chamaecyparisobtusa extract exhibited an antibacterial effect against S. mutans, the causative agent of dental caries, we suggest its applicability as a natural antibacterial agent against oral cavities.
Acknowledgement
This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea Government (MSIT) (2020R1C1C1005306).
References
[1] Locker, D. (1988). Measuring oral health: a conceptual framework. Community Dental Health Journal (5), 3-18.
[2] Watt, R. G. (2005). Strategies and approaches in oral disease prevention and health promotion. Bull World Health Organization, 83(9), 711-18.
[3] Ismail, A. I., Tellez, M., Pitts, N. B., Ekstrand, K. R., Ricketts, D., &Longbottom, C et al.
(2013). Caries management pathways preserve dental tissues and promoteoral health.
Community Dent Oral Epidemiology, 41(e),12–40.
[4] Petersen, P. E. (2003). The World Oral Health Report: continuous improvement of oral health in the 21st century–the approach of the WHO Global Oral Health Programme.
Community Dent Oral Epidemiology, (1), 3-23.
[5] Petersen, P. E. (2004). Challenges to improvement of oral health in the 21st century-the approach of the WHO Global Oral Health programme. International Dental Journal, (54), 329-43.
[6] Keyes, P. H., & Jordan, H. V. (1963). Factors influencing the initiation, transmission and inhibition of dental caries. American AssociationAdvancement Science, (75), 261-83.
[7] Gibbons, R. J., & van Houte, J. (1975). Bacterial adherence in oral microbialecology.
Annual Review of Microbiology, 29, 19-44.
[8] McGhee, J. R., &Michalek, S. M. (1981). Immunobiology of dental caries Microbial aspects and local immunity. Annual Review of Microbiology, (35), 595-635.
[9] Stuart, B. L. (1992). The antibiotic paradox: How miracle drugs are destroying the miracle., Perspectives in Biology and Medicine, 114-29.
[10] Brumfitt, W., & Hamilton-Miller, J. (1989). Methicillin-resistant Staphylococcus aureus. The New England Journal of Medicine, (320), 1188-93.
[11] Otaka, S., Makimura, M., Kuroki, T., Nishihara, Y., &Hirasawa, M. (1991).
Anticaries effects of polyphenolic compounds from Japanese green tea. Caries Research, 25(6), 438-43.
[12] Namba T., Tsunezuka M., Hattor M., Kadota S., & Kikuchi T. (1982). Studies on dental caries prevention by traditional chinese medicines Screening of crude drugs for inhibitory action on plaque formation.Journal of physiology & pathology in Korean Medicine, (15), 179-86.
[13] Miyoshi M., Imoto T., &Kasagi T. (1987). Antieurodontic effect of various fraction extracted from the leaves of Gymnemasylveser. Yonsei Medical Journal, (38), 127-37.
[14] Sakanaka, S., Kim, M., Taniguchi, M., & Yamamoto, T. (1989). Antibacterial substances in Japanese green tea extract against Streptoccusmutans-A catiogenicbacteruium. Agricultural and Biological Chemistry, 53(9), 2307-11.
[15] Takasago, K. (1990). Inhibitor of cariogenic bacteria. Japan Patent Office.25890.
[16] Paek, J. Y., Kim, Y. H., Kwon, H. J., Kim, E. N., Kim, W. J., & Han, M. D.
(2008). Effects of antibacteria and adhesive inhibition of Scutellariabaicalensis extract on Streptococcus mutans. Journal of Dental Hygiene Science, 8(4), 367-37.
[17] Kim, S. S., Won, J. H., Lee, G. E., Lee, R. R., Lee, J. H., & Kang, K. H. (2017).
Anti-bacteria effect of green tea, mulberry, and mate leaves extracts on S. mutans.
Journal of Digital Convergence, (15), 347-53.
[18] Kim, D. W., Lee, D. I., Im, S. U., Choi, Y. H., & Song, K. B. (2019). Effects of Diospyros kaki peel, Momordicacharantia, and Canavaliagladiata extracts on the cariogenic traits of Streptococcus mutans. Journal of Korean Academy of Oral Health, 43(3),131-35.
[19] Lis-Balchin, M. (1977). Essential oils and aromatherapy: their modern role in healing. Journal of the Royal Society of Health, (117), 324-29.
[20] Kang, H. Y., Lee, S. S., & Choi, I. Y. (1993). The antifungal activity of coniferous ceedle oil. The Korean Society of Wood Science & Technology,13(2), 71-7.
[21] Whittaker, R. H., &Feeny, P. P. (1971). Alleochemics, chemicalinteractions between species. Science, (171), 757-70.
[22] Lee, H. O., Baek, S. H., & Han, D.M. (2001). Antimicrobial Effects of Chamaecyparisobtusa Essential Oil. Korean Journal of Applied Microbiology andBiotechnology, (29), 253-57.
[23] Ahn, J. Y., Lee, S. S., & Kang, H. Y. (2004). Biological activities of essential oil from Chamaecyparis obtuse. Journal of Society of cosmetic scientists of Korea, 30(4), 503-07.
[24] Kim E. H. (2015). Anticariogenic effect of Chamaecyparisobtusa essential oil on Streptococcus mutans. Master's thesis. Wonkwang University. Iksan.
[25] Li, B., Li, X., Lin, H., & Zhou, Y. (2018). Curcumin as a promising antibacterial agent: Effects on metabolism and biofilm formation in S. mutans. BioMed Research International. 28, 4508709.
[26] Capurso, L. (2021). Side effects of antibiotic: diarrhea and Clostridium difficile infection (2021). Recentiprogressi in medicina, 112(1), 4-26.
[27] Paek, J. Y., Kim, Y. H., Kwon, H. J., Kim, E. N., Kim, W. J., & Han M. D.
(2008). Effects of antibateria and adhesive inhibtion of scutellariabaicalensis extract on streptococcus mutans. Journal of Dental Hygiene Science, 8(4), 637-373.
[28] Kim, J. H., Lee, S.O., Do, K.B., Ji, W. D., Kim, S.G., & Back, Y.D. et al. (2018).
Analysis of the component and immunological efficacy of Chamaecyparisobtusa leaf extract. Korean Journal of Clinical Laboratory Science, 50(1), 37-43.
[29] Park, M. J., Choi, W. S., Min, B. C., Kim, H. Y., Kang, H. Y., & Choi, I. G.
(2008). Antioxidant activities of essential oils from ChamaecyparisobtusaThe Journal of the Korean Wood Science and Technology, 36(6), 159-167.
[30] Kim, B. K., Kang, J. H., Oh, G. H., Hwang, J. Y., Jang, S. O., & Kim, M. H.
(2019). Antibacterial and antioxidant activity of Chamaecyparisobtusa extracts. Journal of Life Science, 29(7), 785-791.
[31] Hwang, H. R., Kang, S. M., Lee, E. S., & Kim, B. I. (2019). Antibacterial effect of Zingiberaceae extracts mediated photodynamic therapy on Streptococcus mutans. Journal of the Korean Dental Association, 257(10), 560-68.
[32] Kang, S. K., Shin, M. K., Auh, Q. S., Chun, Y. H., & Hong, J. P. (2007).
Antibacterial effect on oral pathogenic bacteria of phytoncide from ChamaecyparisObtusa. Journal of Korean Academy Oral Medicine. 32(1), 45-55.
