|Year : 2019 | Volume
| Issue : 2 | Page : 56-60
Effectiveness of Eugenia caryophyllus in toothpaste against oral microbial in the saliva of healthy subjects in Indonesia
Rosalina Tjandrawinata1, Armelia Sari Widyarman2, Dewi Liliany1
1 Department of Dental Materials, Faculty of Dentistry, Trisakti University, Grogol Jakarta Barat, Indonesia
2 Department of Microbiology, Faculty of Dentistry, Trisakti University, Grogol Jakarta Barat, Indonesia
|Date of Web Publication||18-Jun-2019|
Dr Rosalina Tjandrawinata
Department of Dental Materials, Faculty of Dentistry, Trisakti University, Jln. Kyai Tapa 260, Grogol Jakarta Barat 11440
Source of Support: None, Conflict of Interest: None
Background: Toothpaste is essential in the process of oral care. One of the components of toothpaste, Eugenia caryophyllus, might have an anti-inflammatory and antibacterial effect. Objectives: The objective of this study is to analyze the effectiveness of E. caryophyllus in the toothpaste against Streptococcus mutans, Lactobacillus, and total microbial load in the saliva of healthy subjects after toothbrushing using E. caryophyllus toothpaste. Methods: Saliva (n = 10, aged 18–25 years) were collected before and 2 weeks after toothbrushing using toothpaste-containing E. caryophyllus (Antiplaque, Triple-Ace, Indonesia). The total microbial load of saliva was determined by the measurement of colony-forming unit (CFU) number on brain–heart infusion agar, at 37°C, for 24 h, in anaerobic-condition. Real-time polymerase chain reaction technique was used to quantify the S. mutans and Lactobacillus deoxyribonucleic acid using SYBR green and 16S-rRNA gene-specific primers for S. mutans and Lactobacillus. Primers were 5'-ATTCCCGCCGTTGGACCATTCC-3' (fwd); 5'-CCGACAAAGACCATTCCATCTC-3' (rvs) and 5'-CTTGTACACACCGCCC GT CA-3' (fwd); 5'-CTCAAAACTAAACAAAGTTTC-3' (rvs) for S. mutans and Lactobacillus, respectively. Data were analyzed using t-pair test with P < 0.05 set as the level of significance. Results: The result showed that there was a significant reduction of total microbial load, S. mutans, and Lactobacillus number in the saliva after toothbrushing. The total microbial number in the saliva was significantly decreased before (5.84 ± 0.43 log CFU/mL) and 2 weeks after toothbrushing (5.27 ± 0.61 log CFU/mL) (P < 0.05). The number of S. mutans and Lactobacillus was also significantly decreased before (8.27 ± 0.11 and 2.34 ± 0.71 log CFU/mL) and after toothbrushing (8.18 ± 0.11 and 1.91 ± 0.25 log CFU/mL) (P < 0.05). Conclusion: E. caryophyllus toothpaste may reduce the number of total microbial, S. mutans, and Lactobacillus in the saliva of healthy subjects. Further studies are needed to explore this result.
Keywords: Eugenia caryophyllus, Lactobacillus, Streptococcus mutans, toothpaste
|How to cite this article:|
Tjandrawinata R, Widyarman AS, Liliany D. Effectiveness of Eugenia caryophyllus in toothpaste against oral microbial in the saliva of healthy subjects in Indonesia. Sci Dent J 2019;3:56-60
|How to cite this URL:|
Tjandrawinata R, Widyarman AS, Liliany D. Effectiveness of Eugenia caryophyllus in toothpaste against oral microbial in the saliva of healthy subjects in Indonesia. Sci Dent J [serial online] 2019 [cited 2020 Jul 9];3:56-60. Available from: http://www.scidentj.com/text.asp?2019/3/2/56/260558
| Background|| |
According to the Indonesian Ministry of Health (KEMENKES RI, 2013), Indonesians' awareness of dental and oral health is poor. Prevalent dental and oral diseases in Indonesia include tooth cavities, tartar or calculus, periodontitis, halitosis, and dental hypersensitivity. These diseases are caused by bacteria which turn accumulated dental plaque into glucose and acid, which causes caries and periodontal diseases. Specific types of acid-producing bacteria, especially Streptococcus mutans and Lactobacillus sp., colonize the dental surface and cause damage to the hard tooth structure in the presence of fermentable carbohydrates, e.g., sucrose and fructose. S. mutans and Lactobacillus sp. are present in cariogenic biofilms and play a significant role in the carious process. The treatment of dentine surfaces to prevent biofilm formation and reduce bacteria growth may assist in the prevention of caries initiation and progression.
Herbal agents are known to have antibacterial effects. Studies have proved that herbal agents decrease the bacterial biofilm formations of S. mutans, Enterococcus faecalis, and Porphyromonas gingivalis., Mechanical action of toothbrushing has been proved to effectively remove oral biofilm. There exist many modern treatments and preventive methods for periodontal disease and caries prevention, such as brushing the teeth with toothpaste or using mouthwash. Toothpaste is commonly used to clean plaque on tooth surfaces; moreover, it reduces tooth hypersensitivity due to abrasion and attrition, remineralizes enamel and dentine, and has antiseptic effect.,,
Eugenia caryophyllus, commonly known as eugenol, is naturally found in clove bud (Syzygium aromaticum) and clove oil. Eugenol is commonly used in dentistry in zinc oxide-eugenol (ZOE) cement form as restorative or cementation material, and ZOE is often used to fill deep cavities since it has a healing effect on dental pulp. Eugenol has also been recommended as a desensitization agent. One study found that clove oil dominates peripheral main mechanisms and that the eugenol in clove oil can suppress the sensitive sensory receptors in the teeth. Eugenol is also known to be an antibacterial agent., Despite these intriguing properties, eugenol is not currently a common ingredient in toothpaste. The present study, therefore, seeks to examine whether eugenol-based toothpaste can be effective at reducing the pathogenic microorganisms known to cause oral cavities. Ultimately, we hope to increase both the effectiveness of toothpaste and people's awareness of the importance of dental and oral care.
| Materials and Methods|| |
The research participants (10 subjects) were patients in Trisakti University Dental Hospital (RSGM FKG USAKTI) who were randomly selected according to the inclusion criteria such as patients without tooth decay, without periodontal disease, with calculus and periodontal index scores being 0, and who do not in orthodontic treatment. All ten patients provided informed consent and were 18–25 years old (young adult). This study received permission from the local ethics committee (019/S1/KEPK/FKG/9/2017).
Collection of saliva samples
Participants were asked to brush their teeth for 1 min, two times daily with 1 cm toothpaste containing E. caryophyllus (Antiplaque, Triple-Ace, Indonesia) for 2 weeks. Saliva samples were collected from the participants before and immediately after 2 weeks. Saliva samples were collected by asking participants to spit into a sterile funnel inserted into a 15 mL macro-centrifugal tube. Each sample was then divided into 2 mL samples which were stored in a cooler and then refrigerated at −20°C until the next step.
Saliva (10 uL) samples were cultured in brain–heart infusion agar at 37°C for 24 h in anaerobic conditions. The total microbial load of each sample was determined by measuring the number of colony-forming unit (CFU) in the agar after 24 h.
Deoxyribonucleic acid extraction
Each sample was centrifuged at 4500 g for 15 min. The supernatant was eliminated and the natant (bacteria cells) was obtained. Then 1 mL phosphate-buffered saline was added to the sample in the centrifuge tube with a pellet for washing, after which the sample was transferred to a 1.5 mL micro-centrifugal tube and homogenized in a vortexer and centrifuged at 10,000 g for 10 min. The supernatant was then again eliminated and 100 μL H2O was added to the natant. The 1.5 mL micro-centrifugal tube was then sealed with Sherlock and incubated in a water bath for 20 min at 100°C. Subsequently, the tube was transferred into ice (0°C) for 10 min. The sample was then homogenized in a vortexer and centrifuged at 10,000 g for 2 min.
Real-time polymerase chain reaction
We used the following polymerase chain reaction (PCR) mix containing 5 μL SYBR Green reagent (Applied Biosystems, USA), 1 μL for each forward and reverse primers, 3 μL deoxyribonucleic acid (DNA) sample, and nuclease-free water. Each diluted DNA bacterial sample was inserted into a PCR tube along with the PCR mix and sealed and then centrifuged at 1500 rpm for 1 min. The PCR tube was then inserted into PCR machine, and the temperature and design plate were programmed on a computer. The PCR result was then obtained and analyzed. The primers used in this research are shown in [Table 1], and the quantitative PCR program information is given in [Table 2].
We used the Shapiro–Wilk test to assess the data's normality and the Levene test to assess homogeneity. We analyzed the differences in total bacterial load, S. mutans, and Lactobacillus at baseline and 2 weeks after toothbrushing with eugenol group using paired t-tests. Wilcoxon signed-rank test for nonnormal data. P < 0.05 was considered statistically significant. Statistical analysis was performed using IBM SPSS Statistics of Windows, Version 20.0. (IBM Corp., Armonk, NY, USA).
| Results|| |
After 2 weeks of brushing with the eugenol-based toothpaste, participants' saliva showed a significant reduction in total microbial load, including S. mutans and Lactobacillus numbers. As shown in [Figure 1], the total number of microbes significantly decreased from 5.84 (±0.43) log CFU/mL before the experiment to 5.27 (±0.61) log CFU/mL after the 2-week intervention (P < 0.05). [Figure 2] and [Figure 3] show that S. mutans and Lactobacilli also significantly decreased from 8.27 (±0.11) and 2.34 (±0.71) log CFU/mL) before the experiment, respectively, to 8.18 (±0.11) and 1.91 (±0.25) log CFU/mL after the experiment, respectively (P < 0.05).
|Figure 1: Total plate count from saliva samples before and after treatment with toothpaste containing Eugenia caryophyllus|
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|Figure 2: The number of Streptococcus mutans present in saliva samples before and after treatment with toothpaste containing Eugenia caryophyllus|
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|Figure 3: The number of Lactobacillus sp. present in saliva samples before and after treatment with toothpaste containing Eugenia caryophyllus|
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| Discussion|| |
In this study, eight participants who brushed their teeth using eugenol toothpaste for 2 weeks were found to reduce their oral population of S. mutans by >50% compared to before the treatment. However, the population of S. mutans in one participant was only reduced by 30% after treatment, while another one participant had an increase of S. mutans in his oral population, with a value of 120%. This was likely due to eating and cleansing habits of the participant. The presence of sugar promotes the biosynthesis of insoluble glucans, which cause bacteria to firmly adhere to the tooth surface.,, In contrast, Lactobacilli were not as greatly affected by the use of eugenol toothpaste for 2 weeks. Only one participant exhibited a reduction of Lactobacillus bacteria by 50%. However, the majority of participants (70%) still exhibited some reduction of Lactobacilli, ranging from 8% to 50%. Three participants showed a slight increase. Toothbrushing with eugenol toothpaste reduced the total bacterial load in nine participants (90%), including by >50% in eight participants. Overall, the results suggest that eugenol is more effective at reducing S. mutans than Lactobacilli.
Actually, dental caries can be prevented using an antimicrobial agent to suppress the growth of cariogenic microorganisms, such as S. mutans and Lactobacilli. Studies of cavities in children have furthermore identified S. mutans as the most common risk factor of cavity development. S. mutans and Lactobacilli are both anaerobic, facultative bacteria that can survive in any circumstance in oral cavities. These bacteria ferment sugars from food such as glucose, sucrose, lactose, trehalose, mannitol, and sorbitol. Of these, the most cariogenic is sucrose, which is easily fermented by bacteria. It also functions as a substrate in forming extracellular and intracellular polysaccharides via interactions with glucosyltransferase, which is produced by S. mutans. Extracellular polysaccharides, particularly glucan which does not dissolve in water, mediate preliminary attachment by S. mutans and other oral bacteria to tooth surfaces and thus facilitate the formation of dental plaque. In general, S. mutans is the main microbe responsible for forming dental caries although other acidogenic microorganisms are involved to lesser degrees. Lactobacilli account for 1% of microflora in oral cavities, and some Lactobacillus species are naturally present in the saliva (e.g., Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus fermentum, Lactobacillus plantarum, Lactobacillus rhamnosus, and Lactobacillus salivarius) and produce organic acid using a hetero-fermentative method.
Conventionally, eugenol has been used to kill parasites, and studies have shown that it has antimicrobial and antifungal properties. It is also used to treat diarrhea and other digestive ailments; it is thought to kill intestinal parasites and worms. It is therefore already known to be nontoxic and is considered safe as an oral medication. In dentistry, eugenol is commonly used as a component of temporary fillings for dental cavities. It is also used before denture application and to eliminate pulpitis pain or dental hypersensitivity., It is also commonly used as a basic material in toothpaste.,,
Eugenol interacts with bacteria's cellular membranes and functions as a bactericide by causing holes in the bacteria envelope or by causing bacteria cell deformities, depending on the bacterial strain. For instance, it makes the membrane of L. rhamnosus highly permeable, forming nonspecific pores on plasma membranes, which in turn direct the release of absorbing materials. Another study showed the release of alkaline phosphatase which is located between cell wall and cell membrane and normally could not leak outside bacteria indicating that the essential oil can destroy the cell walls of Staphylococcus aureus s trains, resulting in the increase of the permeability of cell wall and the destruction of the cell structure. It has also been reported that eugenol's hydrophobicity enables a partition of the lipids in the bacterial cell membrane and mitochondria, thus disturbing the cell structure and rendering them more permeable. Eugenol has also been shown to affect both Gram-positive and Gram-negative bacteria, despite their different structures. However, the antibacterial effect of this essential oil from clove buds depended on its dose. While the minimal inhibitory concentration is still in debate,, another ingredients in toothpaste and experimental conditions such as the viscosity of saliva, inoculation of bacteria, incubation time, and the sources of essential oil might influence the antibacterial effects of toothpaste against S. mutans and Lactobacillus sp. However, this should be confirmed in further research.
| Conclusion|| |
Our results indicate that toothbrushing using E. caryophyllus toothpaste may reduce the total microbial load, S. mutans, and Lactobacilli number in the saliva of healthy subjects. Therefore, we suggest using E. caryophyllus as an ingredient in toothpaste composition, to reduce oral bacteria. This industrial step, hopefully, will reduce caries and periodontal disease prevalent in the world, especially in Indonesia.
We want to appreciate Faculty of Dentistry, Trisakti University, for funding this study.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Forssten SD, Björklund M, Ouwehand AC. Streptococcus mutans
, caries and simulation models. Nutrients 2010;2:290-8.
Knight GM, McIntyre JM, Craig GG, Mulyani, Zilm PS. The inability of Streptococcus mutans
and Lactobacillus acidophilus
to form a biofilm in vitro
on dentine pretreated with ozone. Aust Dent J 2008;53:349-53.
Mishra S, Routray S, Kumar Sahu S, Bhusan Nanda S, Charan Sahu K. The role and efficacy of herbal antimicrobial agents in orthodontic treatment. J Clin Diagn Res 2014;8:ZC12-4.
Widyarman AS, Widjaja SB, Idrus E. Strawberry extract's effects on Enterococcus faecalis
and Porphyromonas gingivalis
biofilms in vitro
. Sci Dent J 2017;1:1-5. [Full text]
Oliveira MS, Borges AH, Mattos FZ, Semenoff TA, Segundo AS, Tonetto MR, et al.
Evaluation of different methods for removing oral biofilm in patients admitted to the intensive care unit. J Int Oral Health 2014;6:61-4.
Birang R, Naghsh N, Yaghini J, Mosavi F. Desensitizing efficacy of foam containing potassium nitrate 5% and toothpaste containing strontium acetate in dentin hypersensitivity: An eight-week clinical study. J Periodontol Implant Dent 2013;5:18-22.
Pandey R, Koppolu P, Kalakonda B, Lakshmi BV, Mishra A, Reddy PK, et al.
Treatment of dentinal hypersensitivity using low-level laser therapy and 5% potassium nitrate: A randomized, controlled, three arm parallel clinical study. Int J Appl Basic Med Res 2017;7:63-6.
Markowitz K. A new treatment alternative for sensitive teeth: A desensitizing oral rinse. J Dent 2013;41 Suppl 1:S1-11.
Miglani S, Aggarwal V, Ahuja B. Dentin hypersensitivity: Recent trends in management. J Conserv Dent 2010;13:218-24.
] [Full text]
Pavithra B. Eugenol – A review. J Pharm Sci Res 2014;6:153-4.
Aishwarya J, Harini N, Karthikeyan M. Clove oil and its role in oral health – A review. Int J Pharm Sci Health Care 2014;3:155-68.
Towaha J. The benefits of cloves eugenol in various industries in Indonesia. Perspektif 2012;11:79-90.
Ajeng R, Haniastuti T, Handajani K. Inflammatory cells infiltration after eugenol administration in Sprague Dawley rat's molars. Majalah Kedokteran Gigi Indonesia 2016;2:66-73.
Newbrun E. Extracellular polysaccharides synthesized by glucosyltransferases of oral streptococci. Composition and susceptibility to hydrolysis. Caries Res 1972;6:132-47.
Bowen WH. Do we need to be concerned about dental caries in the coming millennium? Crit Rev Oral Biol Med 2002;13:126-31.
Shumi W, Lim J, Nam SW, Lee K, Kim SH, Kim MH, et al
. Environmental factors that affect Streptococcus mutans
biofilm formation in a microfluidic device mimicking teeth. Bio Chip J 2010;4:257-63.
Smullen J, Koutsou GA, Foster HA, Zumbé A, Storey DM. The antibacterial activity of plant extracts containing polyphenols against Streptococcus mutans
. Caries Res 2007;41:342-9.
Gross EL, Beall CJ, Kutsch SR, Firestone ND, Leys EJ, Griffen AL. Beyond Streptococcus mutans
: Dental caries onset linked to multiple species by 16S rRNA community analysis. PLoS One 2012;7:e47722.
Islam B, Khan SN, Khan AU. Dental caries: From infection to prevention. Med Sci Monit 2007;13:RA196-203.
Paes Leme AF, Koo H, Bellato CM, Bedi G, Cury JA. The role of sucrose in cariogenic dental biofilm formation – New insight. J Dent Res 2006;85:878-87.
Xu X, Zhou XD, Wu CD. Tea catechin epigallocatechin gallate inhibits Streptococcus mutans
biofilm formation by suppressing gtf genes. Arch Oral Biol 2012;57:678-83.
Duarte S, Gregoire S, Singh AP, Vorsa N, Schaich K, Bowen WH, et al.
Inhibitory effects of cranberry polyphenols on formation and acidogenicity of Streptococcus mutans
biofilms. FEMS Microbiol Lett 2006;257:50-6.
Sharma C, Singh BP, Thakur N, Gulati S, Gupta S, Mishra SK, et al.
Antibacterial effects of Lactobacillus
isolates of curd and human milk origin against food-borne and human pathogens 3. Biotech 2017;7:31.
Tammannavar P, Pushpalatha C, Jain S, Sowmya SV. An unexpected positive hypersensitive reaction to eugenol. BMJ Case Rep 2013;2013. pii: bcr2013009464.
Safrudin I, Maimulyanti A, Pribadi AR. Effect of crushing bud (Syzygium aromaticum
) and distillation rate on main constituents of the essential oil. Am J Essential Oils Natl Prod 2015;2:12-5.
Devi KP, Sakthivel R, Nisha SA, Suganthy N, Pandian SK. Eugenol alters the integrity of cell membrane and acts against the nosocomial pathogen Proteus mirabilis
. Arch Pharm Res 2013;36:282-92.
Oyedemi SO, Okoh AI, Mabinya LV, Pirochenva G, Afolayan AJ. The proposed mechanism of bactericidal action of eugenol, α-terpineol and g-terpinene against Listeria monocytogenes, Streptococcus pyogenes, Proteus vulgaris
and Escherichia coli
. Afr J Biotechnol 2009;8:1280-6.
Xu JG, Liu T, Hu QP, Cao XM. Chemical composition, antibacterial properties and mechanism of action of essential oil from clove buds against Staphylococcus aureus
. Molecules 2016;21. pii: E1194.
Bennis S, Chami F, Chami K, Rhayour A, Elaraki T, Remmal A. Eugenol induces damage of bacterial and fungal envelope. Moroccan J Biol 2004;1:30-9.
[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2]