Antimicrobial Activity of Essential Oil Against Oral Strain

Original Article Int J Clin Prev Dent 2018;14(4):216-221ㆍhttps://doi.org/10.15236/ijcpd.2018.14.4.216 ISSN (Print) 1738-8546ㆍISSN (Online) 2287-6197

Antimicrobial Activity of Essential Oil against Oral Strain

Chungmu Park1, Hyunseo Yoon2

Departments of 1Clinical Laboratory Science and 2Dental Hygiene, Dong-Eui University, Busan, Korea

Objective: In this study, we tried to investigate the antimicrobial activity of natural herbaceous plant-derived essential oils against oral disease-causing bacterial strains and establish the scientific evidences to apply them to the dentifrice, mouthwash, and other uses. Methods: We used the disk diffusion method with 23 types of 100% natural essential oils to verify the antimicrobial effect against Streptococcus mutans, Porphyromonas gingivalis, and Lactobacillus rhamnosus. After adhering to the surface of the plate culture medium, 20 l of the essential oil was prepared, and the diameter of the transparent ring was measured after incubation for 24 hours. All the experiments were repeated three times. Results: Among 23 types of oils, 13 were effective against all three strains: myrrh, ginger, basil, carrot seed, tea tree, patchouli, ylang ylang, cypress, lemongrass, cinnamon, peppermint, lavender, and eucalryptus. Seventeen oils were effective against S. mutans, and myrrh, basil, and carrot seed showed high antimicrobial activity. Eighteen oils were effective against P. gingivalis, and tea tree, carrot seed, and cinnamons showed high antimicrobial activity. Sixteen oils were effective against L. rhamnosus, and carrot seed and peppermint cinnamon showed high antimicrobial activity. Conclusion: Consequently, 13 essential oils showed the antimicrobial activity against three bacterial strains, which indicates these essential oils might be used as the effective materials to suppress the growth of oral-disease inducing microorganisms. Moreover, essential oils that have been analyzed activities in this study will be mixed up within the constant range and analyzed their antimicrobial effects to examine the synergistic activity among them.

Keywords: anti-microbial agents, essential oil, Lactobacillus rhamnosus, Porphyromonas gingivalis, Streptococcus mutans

Introduction creased, people are paying more attention to natural extracts with fewer side effects than the synthetic additives or extracts As the interest in economic development and health has in- [1]. The natural extracts are obtained from medicinal plants and edible plants by various methods such as compression, Corresponding author Hyunseo Yoon freeze extraction, solvent extraction, and steam distillation, Department of Dental Hygiene, Dong-Eui University, 176 which were used for oral medication or skin treatment. Eongwang-ro, Busanjin-gu, Busan 47340, Korea. Tel: Among them, the essential oils that condense fragrances ex- +82-51-890-2688, Fax: +82-505-182-6878, E-mail: yoonhs@ tracted from herb and other plants are more useful because deu.ac.kr they contain many bioactive components [2,3]. These natural https://orcid.org/0000-0002-7455-5506 extracts or oils are used as the natural preservatives for food Received September 6, 2018, Revised November 2, 2018, and grains [4] or cosmetic functional additives [5]. Accepted December 4, 2018 The essential oil is the common product of extracts of flow, stem, and leave components of plants [6]. The essential oil has

Copyright ⓒ 2018. Korean Academy of Preventive Dentistry. All rights reserved. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

216 Chungmu Park and Hyunseo Yoon：Antimicrobial Activitiy of Various Essential Oils

been known to have pharmacological effects such as relieving is the leading strain causing periodontal disease [2]. Among pain and alleviating acne through antimicrobial and anti-in- essential oils, peppermint and lemongrass oils showed anti- flammatory activities against acne-causing bacteria [6-9]. microbial effects against P. gingivalis [20]. Moreover, it is proven to be an effective antioxidant in food First, diverse mint-flavored essential oils were selected spices and utilized in various fields such as seeking psycho- since they would be used for the additives for dentifrice or logical stability [10-12]. mouthwash. Then, their antimicrobial activities were ana- Thus far, chlorohexidine, cetylpyridinium chloride and flu- lyzed through the inhibited growth against three representa- orine have been commonly used for additive agents for tive microorganisms including S. mutans, P. gingivalis, and mouthwashing solution. By the way, they have various side ef- Lactobacillus rhamnosus which can evoke oral disease. fects such as teeth stain, unpleasant taste, and oral pain in spite Thourghout this study, essential oils that contain antimicrobial of their outstanding bacteriostatic activity [13]. Therefore, activities against oral-disease causing strains. many researchers have tried to search natural resources- de- rived materials which can inhibit the proliferation of micro- Materials and Methods organisms that cause oral diseases to overcome these prob- lems [1]. Specifically, the studies verified the antimicrobial 1. Materials activity of the plum extract [14], bamboo salt [15], green tea leaf, mulberry leaf, and matte leaf extract [16] against We used 100% natural essential oils sold by UK-based Streptococcus mutans which is the leading strain causing den- Aqua Oleum. There were 23 types of oils as shown in Table 1. tal caries. Other studies identified the antimicrobial activity They were mixed with Tween 20 (Anatrace Products, LLC, tea tree oil [17] and orange oil [18] among the essential oils. Maumee, OH, USA) and then diluted with triple distilled wa- Moreover, the studies also exhibited the antimicrobial activity ter into 50 % (v/v) to be the test sample. of Alnus firma extract [19], myrrh extract, rhatany extract, and chamomile extract against Porphyromonas gingivalis which

Table 1. Characteristics of materials

Extraction Case No. Oil name Botanical name Cultivated area methoda 1 Myrrh Commiphora myrrha Somalia 1 2Rosemary Rosemarinus officinalis France 1 3 Ginger Zingiber officinale Nigeria 1 4Pine Pinus Sylvestris Siberia 1 5Basil Ocimum basilicum Egypt 1 6 Carrot seed Daucus carota Belgium 1 7Tea tree Melaleuca alternifolia Australia 1 8Neroli Citrus aurantium subsp. amara or Bigaradia Tunisia 1 9 Patchouli Pogostemon patchouli Indonesia 1 10 Palmarosa Cymbopogon martini India 1 11 Ylang Ylang Cananga odorata Masagascar 1 12 Grape Fruit Citrus paradisi California 2 13 Cypress Cupressus sempervirens France 1 14 Lemongrass Cymbopogon citratus Guatemala 1 15 Majoram Origanum majorane Mediterranean Sea 1 16 Lemon Citratus limon Asia 3 17 Cederwood Cedrus atlantica Morocco 1 18 Cinnamon Cinnamomum zeylanicum Sri Lanka 1 19 Mandarin Citrus reticulata Italy 2 20 Peppermint Mentha piperita United Kingdom 1 21 Lavender Lavandula angustifolia France 1 22 Eucalryptus Eucalyptus globulus Spain 1 23 Spearmint Mentha spicata Europe 1 a1: water vapor distillation, 2: cooling extraction method, 3: cooling compression extraction method.

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patchouli, ylang ylang, grape fruit, cypress, lemongrass, lemon, 2. Tested strains and cultivation condition cederwood, cinnamon, peppermint, lavender, and spearmint. To measure the antimicrobial effect of the samples against Sixteen oils showed the antimicrobial effect against L. the strains causing oral diseases, we procured the S. mutans rhamnosus: myrrh, ginger, basil, carrot seed, tea tree, patch- (KCTC 3065) and P. gingivalis (5352) strains from Korean ouli, palmarosa, ylang ylang, cypress, lemongrass, marjoram, Collection for Type Cultures in Daejeon, Korea and the L. cinnamon, peppermint, lavender, eucalryptus, and spearmint. rhamnosus (KCOM 1213) strains from the Korean Collection 2. Comparison of antimicrobial activity of for Oral Microbiology S. mutans and L. rhamnosus strains essential oil against S. mutans were cultured on agar medium of brain heart infusion (Difco Laboratories Inc., Detroit, MI, USA) while P. gingivalis Table 3 shows the measurement results of the diameter of strains were cultured on tryptic soy agar (Difco) medium con- the transparent rings using the disk diffusion method to check taining 10% sheep blood, 0.1% hemin (Sigma, St. Louis, MO, the antimicrobial activity against S. mutans. Among 17 essen- USA), and containing vitamin K1 (Sigma). tial oils that had the antimicrobial activity against S. mutans, myrrh showed the highest antimicrobial activity at 18.34 mm 3. Measurement of antimicrobial activity using and followed by basil at 18.08 mm, and carrot seed at 17.91 disk diffusion method mm (p<0.001). We measured the antimicrobial activity of essential oil 3. Comparison of antimicrobial activity of against S. mutans, P. gingivalis, and L. rhamnosus using the essential oil against P. gingivalis disk diffusion method [21]. All strains were tested in accord- ance with NCCLS Guide Line M11-A6 (National Committee Table 4 shows the measurement results of the diameter of for Clinical Laboratory Standards, 2004). Each strain was in- the transparent rings using the disk diffusion method to check oculated on a plate medium, a sterilized paper disk (Advantec; the antimicrobial activity against P. gingivalis. Among 18 es- Toyo Roshi, Ltd., Tokyo, Japan) with a diameter of 8 mm ad- sential oils that had the antimicrobial activity against P. gingi- hered to the surface of the plate medium, 20 l of the prepared valis, tea tree showed the highest antimicrobial activity at 50 % (v/v) essential oil was dropped, and then it was cultivated in an anaerobic incubator (5% CO2, 10% H2, and 85% N2) at 37°C for 20 hours. The diameter (mm) of the formed trans- Table 2. Antimicrobial activities against Streptococcus mutans, parent rings was measured after the incubation. All tests were Porphyromonas gingivalis, Lactobaillus rhamnosus repeated three times. Case No. Oil name S. mutans P. gingivalis L. rhamnosus 4. Analysis method 1 Myrrh ＋＋＋ 2Rosemary − ＋ − To compare the antimicrobial activities of essential oils, we 3Ginger ＋＋＋ conducted the one-way ANOVA using IBM SPSS ver. 25.0 4Pine ＋ −− (IBM Co., Armonk, NY, USA) and the post-analysis using the 5Basil ＋＋＋ 6 Carrot seed ＋＋＋ Duncan technique. All tests were repeated three times. 7 Tea tree ＋＋＋ 8Neroli −− − Results 9 Patchouli ＋＋＋ 10 Palmarosa ＋ − ＋ 11 Ylang Ylang ＋＋＋ 1. Antimicrobial activity according to the type of 12 Grape Fruit ＋＋ − the essential oil 13 Cypress ＋＋＋ 14 Lemongrass ＋＋＋ Table 2 shows the results of the disk diffusion method to 15 Marjoram −− ＋ check the antimicrobial activities of essential oils. 16 Lemon − ＋ − Seventeen oils showed the antimicrobial effect against S. mu- 17 Cederwood ＋＋ − 18 Cinnamon ＋＋＋ tans: myrrh, ginger, pine, basil, carrot seed, tea tree, patchouli, 19 Mandarin −− − palmarosa, ylang ylang, grape fruit, cypress, lemongrass, ceder 20 Peppermint ＋＋＋ wood, cinnamon, peppermint, lavender, and eucalryptus. 21 Lavender ＋＋＋ Eighteen oils showed the antimicrobial effect against P. gin - 22 Eucalryptus ＋＋＋ givalis: myrrh, rosemary, ginger, basil, carrot seed, tea tree, 23 Spearmint − ＋＋

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19.50 mm and followed by carrot seed at 17.44 mm, and cin- is used for enhancing refreshing sensation in the oral health namon at 16.82 mm (p<0.001). field [22]. This study is intended to find the essential oils which are 4. Comparison of antimicrobial activity of used in a dentifrice or mouthwash as mint for flavor or taste essential oil against L. rhamnosus and have the antimicrobial activity against bacteria causing Table 5 shows the measurement results of the diameter of tooth decay and periodontal disease. the transparent rings using the disk diffusion method to check the antimicrobial activity against L. rhamnosus. Among 16 es- Table 4. Antibacterial effect of various essential oils against Porphy- sential oils that had the antimicrobial activity against L. rham- romonas gingivalis nosus, carrot seed showed the highest antimicrobial activity at 16.65 mm and followed by peppermint at 16.33 mm, and cin- Oil name Inhibition zone (mm) F p-value namon at 15.84 mm (p<0.001). Myrrh 15.27±0.79ef 40.352 ＜0.001 Rosemary 15.02±0.92ef Ginger 15.74±0.44fg Discussion Basil 15.03±0.12ef Carrot seed 17.44±0.87gh Attention on natural extract has been increasing because of Tea tree 19.50±1.41i the adverse effect of existing drug, and it is no exception in the Patchouli 15.28±0.32ef bc oral-related area. Chlorhexidine is the leading antibiotic sub- Ylang Ylang 12.40±0.47 b stance, but authorities recommend the use for a minimum prod Grape Fruit 11.54±0.47 Cypress 12.55±0.43bc due to various side effects such as mucosal pain and pigmenta- Lemongrass 13.28±0.94cd tion [13]. The functional plants are used in the form of secon- Lemon 9.81±0.49a dary metabolite using various extraction methods, and one of Cederwood 14.37±0.97de gh them is the essential oil. The essential oil has the basic struc- Cinnamon 16.82±0.26 gh ture consisting of oxygen, hydrogen, and carbon. It contains Peppermint 16.72±0.78 Lavender 15.31±1.07ef ketone, oxides, aldehyde, ester, and alcohol components. Eucalryptus 15.49±0.34ef Essential oils are used for a variety of functions such as re- Spearmint 10.36±0.16a lieving pain, sedation, antioxidant, and antimicrobial [7-11], it Values are presented as mean±standard deviation. The same lettrers (a-i) indicate significane difference.

Table 3. Antibacterial effect of various essential oils against Strepto- coccus mutans Table 5. Antibacterial effect of various essential oils against Lacto- bacillus rhamnosus Oil name Inhibition zone (mm) F p-value Oil name Inhibition zone (mm) F p-value Myrrh 18.34±0.26f 74.609 ＜0.001 Ginger 13.08±0.70c Myrrh 11.72±0.85b 46.812 ＜0.001 Pine 11.05±0.18b Ginger 15.62±1.18fg Basil 18.08±0.29f Basil 15.40±0.55f Carrot seed 17.91±0.17f Carrot seed 16.65±0.57g Tea tree 17.89±0.36f Tea tree 12.84±0.39cd Patchouli 14.18±0.47d Patchouli 13.82±0.67de Palmarosa 15.04±0.39d Palmarosa 13.93±0.22e Ylang Ylang 10.87±0.54ab Ylang Ylang 9.84±0.52a Grape Fruit 9.95±0.10a Cypress 9.57±0.35a Cypress 12.79±0.75c Lemongrass 10.48±0.59a Lemongrass 10.65±0.47ab Marjoram 11.69±0.76b Cederwood 12.77±0.10c Cinnamon 15.84±0.36fg Cinnamon 16.00±0.68e Peppermint 16.33±0.42fg Peppermint 16.10±0.44e Lavender 12.69±0.57bc Lavender 16.27±0.98e Eucalryptus 13.26±0.59cde Eucalryptus 14.75±1.25d Spearmint 10.23±0.40a Values are presented as mean±standard deviation. The same lettrers Values are presented as mean±standard deviation. The same lettrers (a-f) indicate significane difference. (a-g) indicate significane difference.

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The results of the disk diffusion method to investigate the This study tried to examine the antimicrobial activities against antimicrobial effect against S. mutans showed that 17 essential only three strains despite there are diverse oral-disease caus- oils had the antimicrobial activities. Specifically, myrrh ing microorganisms. In future study, other bacterial strains re- showed the highest antimicrobial activity at 18.34 mm and lated with oral disease should be analyzed and essential oils followed by basil at 18.08 mm and carrot seed at 17.91 mm. confirmed their antimicrobial activity in this study will ana- Other essential oils showing the antimicrobial activity were lyze their synergistic activity when they mixed with other es- ginger, pine, tea tree, patchouli, palmarosa, ylang ylang, grape sential oils. fruit, cypress, lemongrass, cederwood, cinnamon, peppermint, lavender, and eucalryptus. The study by Lee et al. [23] Conclusion reported that the rosemary, lemongrass, orange, and Neem oils showed the antimicrobial activity against S. mutans while the Although essential oils have been widely used in cosmetics, study by Choi and Kang [17] reported that the tea tree oil also foods, and sedatives, there have been few studies of them in showed the antimicrobial activity against S. mutans. However, the oral area. Therefore, additional studies are necessary to the study by Kim et al. [24] showed the results different from test the difference in antimicrobial activity and growth in- this study, and it found no antimicrobial effect of lavender and hibition effect according to concentration based on essential rosemary essential oils against S. mutans. It indicates the need oils that have been proven to have antimicrobial activity. for additional studies to analyze the differences according to Furthermore, additional in vivo tests based on the proved ef- the concentration, surfactant, and other factors. fects may find the potential use of them in oral care products. Eighteen oils had the antimicrobial activities against P. gingivalis. Tea tree showed the highest antimicrobial activity at 19.50 mm and followed by carrot seed at 17.44 mm and cin- References namon at 16.82 mm. Other essential oils showing the anti- 1. Park HR, Hong SJ. Research on natural medicine for wellness microbial activity against P. gingivalis were myrrh, rosemary, and oral health. J Digit Converg 2015;13:357-63. ginger, basil, patchouli, ylang ylang, grape fruit, cypress, lem- 2. Baek HS, Kang SK, Auh QS, Chun YH, Hong JP. Effect of anti- on grass, lemon, cederwood, peppermint, lavender, eucalryp- bacterial effects of myrrh, rhatany, chamomomilla against to oral tus, and spearmint. This study showed the similar results as the microorganisms. J Oral Med Pain 2013;38:299-312. study by Lee et al. [20] that reported the antimicrobial effect of 3. de Rapper S, Kamatou G, Viljoen A, van Vuuren S. The in vitro the peppermint and lemongrass oils against P. gingivalis. The antimicrobial activity of lavandula angustifolia essential oil in study reported that the chamomile also had the antimicrobial combination with other aroma-therapeutic oils. Evid Based Complement Alternat Med 2013;2013:852049. activity against P. gingivalis [2]. The results indicate that addi- 4. Yu MH, Chae IG, Jung YT, Jeong YS, Kim HI, Lee IS. tional studies of more types of oils and extracts are necessary. Antioxidative and antimicrobial activities of methanol extract Sixteen oils had the antimicrobial activities against L. from Rosmarinus officinalis L. and their fractions. J Life Sci rhamnosus. Carrot seed showed the highest antimicrobial ac- 2011;21:375-84. tivity at 16.65 mm and followed by peppermint at 16.33 mm, 5. Bhagava K, Conti DS, da Rocha SR, Zhang Y. Application of and and cinnamon at 15.84 mm. Other essential oils showing the oregano oil nanoemulsion to the control of foodborne bacteria antimicrobial activity against L. rhamnosus were myrrh, gin- on fresh lettuce. Food Microbiol 2015;47:69-73. ger, basil, tea tree, patchouli, palmarosa, ylang ylang, cypress, 6. Oh JY, Kim YC, Lee BS. Hair growth promotion effect and anti-bacterial activity against pityrosporum ovale of peppermint lemongrass, marjoram, lavender, eucalryptus, and spearmint. dil. J Investig Cosmetol 2014;10:261-69. This study showed the similar results as the study by Feniman 7. Campos CA, Gerschenson LN, Flores SK. Development of edi- et al. [25] that reported the antimicrobial effect of cinnamon ble films and coatings with antimicrobial activity. Food oil against L. rhamnosus. Twenty one essential oils except for Bioprocess Technol 2011;4:849-75. neroli and mandarin of 23 essential oils tested for anti- 8. Lodhia MH, Bhatt KR, Thaker VS. Antibacterial activity of es- microbial effect showed to have the antimicrobial activity sential oils from palmarosa, evening primrose, lavender and against the strains causing oral diseases. tuberose. Indian J Pharm Sci 2009;71:134-6. 9. Chae IG, Kim HJ, Yu MH, Kim HI, Lee IS. Antioxidant and anti- In this study, we tested various sorts of essentials were test- bacterial activity of commercially available herbs in Korean ed against three representative oral disease causing micro- markets. J Korean Soc Food Sci Nutr 2010;39:1411-17. oorganisms. As result, 13 essential oils exhibited significant 10. Kazemzadeh R, Nikjou R, Rostamnegad M, Norouzi H. Effect antimicrobial activity, which indicates they could be promis- of lavender aromatherapy on menopause hot flushing: a cross- ing strategy for attenuating the proliferation of three strains. over randomized clinical trial. J Chin Med Assoc 2016;79:

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489-92. hibitory effect of some vegetable oils on Streptococcus mutans 11. Sadeghzadeh J, Vakili A, Bandegi AR, Sameni HR, Zahedi and Lactobacillus casei. J Korean Acad Oral Health 2016;40: Khorasani M, Darabian M. Lavandula reduces heart injury via 24-30. attenuating tumor necrosis factor-alpha and oxidative stress in 19. Choi HJ, Heo NS, Choi TW, Lee YG, Jeong YK, Joo WH. a rat model of infarct-like myocardial injury. Cell J 2017;19:84- Antimicrobial and anti-halitosis effects of alnus firma extracts. 93. J Life Sci 2012;22:1071-6. 12. Lee JY, Ko MY, Kim JS, Park CI. Inhibitory effect of lavender 20. Lee YS, Kim SK, Yang TC, Kim GS, Jeon JG, Chang KW. The essential oil on formalin-induced ultrasonic vocalizations in antibacterial and growth inhibitory effect of some essential oils rats. Korean Soc Cosmet Cometol 2015;5:203-8. against the oral micro-organisms. J Korean Acad Oral Health 13. Scheie AA. Modes of action currently known chemical anti- 2006;30:490-6. plaque agents other than chlorhexdine. J Dent Res 1989;68(spec 21. Balouiri M, Sadiki M, Ibnsouda SK. Methods for in vitro evaluat- iss):1609-16. ing antimicrobial activity: a review. J Pharm Anal 2016;6:71-9. 14. Lee JS, Chung KH. Antimicrobial effect of Prunus mume ex- 22. Wilson R. Aromatherapy: essential oils for vibrant health and tracts against cariogenic bacteria. J Korean Acad Oral Health beauty. New York: Penguin Putnam Inc.; 2002:107-9. 2017;41:65-70. 23. Lee SY, Kim JG, Baik BJ, Yang YM, Lee KY, Lee YH, et al. 15. Oh HN, Choi CH. Effect of bamboo salt on the caries activity Antimicrobial effect of essential oils on oral bacteria. J Korean of Streptococcus mutans. J Korean Acad Oral Health 2016;40: Acad Pediatr Dent 2009;36:1-11. 238-43. 24. Kim MS, Lee KW, Park EJ. Antimicrobial activity of lavender 16. Kim SS, Won JH, Lee GE, Lee RR, Lee SM, Lee JH, et al. and rosemary essential oil nanoemulsions. Korean J Food Cook Anti-bacteria effect of green tea, mulberry, and mate leaves ex- Sci 2017;33:256-63. tracts on S. mutans. J Digit Converg 2017;15:347-53. 25. Feniman CM, Rall VL, Doyama JT, Júnior AF. Cell enumeration 17. Choi TR, Kang MK. Antibacterial effect of tea tree on and visualisation by transmission electron microscopy of Streptococcus mutans. J Korean Soc Dent Hyg 2017;17:613-20. Lactobacillus rhamnosus treated with cinnamon (Cinnamomum 18. Kim SY, Kim HN, Jun EJ, Kim JB, Jeong SH. The growth in- zeylanicum B.) essential oil. Nat Prod Res 2012;26:1721-3.