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 Table of Contents  
ORIGINAL RESEARCH
Year : 2021  |  Volume : 13  |  Issue : 1  |  Page : 45-52

Eugenol and thymol as potential inhibitors for polymicrobial oral biofilms: An in vitro study


1 Program Doctoral Faculty of Pharmacy, Universitas Gadjah Mada, Yogyakarta, Indonesia
2 Department of Pharmaceutical Biology, Faculty of Pharmacy, Universitas Gadjah Mada, Yogyakarta, Indonesia
3 Department of Oral Biology, Faculty of Dentistry, Universitas Gadjah Mada, Yogyakarta, Indonesia

Date of Submission23-Jul-2020
Date of Decision10-Aug-2020
Date of Acceptance23-Oct-2020
Date of Web Publication28-Jan-2021

Correspondence Address:
Prof. Triana Hertiani
Department of Pharmaceutical Biology, Faculty of Pharmacy, Universitas Gadjah Mada, Sekip Utara, Yogyakarta 55281
Indonesia
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/1319-2442.308357

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  Abstract 

Aim: Dysbiosis of polymicrobial biofilms causes dental caries. In a search for a new effective anticaries agent from eugenol and thymol, this study aimed to investigate the efficacy of eugenol and thymol on polymicrobial (Streptococcus sanguinis, Lactobacillus acidophilus, Actinomyces viscosus, and S. mutans) biofilms. Materials and Methods: Antibacterial and antibiofilm activities were tested using the microdilution method. Antibiofilm activities consisted of inhibiting biofilm formation and degradation of polymicrobial biofilms. Tests were conducted using the microdilution method on 96-well microtiter plates. Tests were done at concentration of 1% v/v, 0.5% v/v, 0.25% v/v, and 0.125% v/v. The compound for biofilm staining was 1% v/v crystal violet, and this study used a microplate reader at a wavelength of 595 nm. The minimum biofilm inhibition concentration (MBIC50) and the value of minimum biofilm eradication concentration (MBEC50) were calculated to determine the effectiveness of antibiofilm test compounds against polymicrobial biofilms. The comparative compound used chloramphenicol and Listerine. Scanning electron microscope (SEM) was used to observe the morphological changes in the biofilm after the treatment. Results: Eugenol and thymol showed inhibitory activity against the formation of polymicrobial biofilms. In cells treated with eugenol on polymicrobial biofilms, the matrix of extracellular polymeric substance (EPS) became degraded. Thymol inhibited the biofilms’ growth and damaged the EPS which protect bacterial biofilms. Conclusion: Based on these results, it can be concluded that eugenol and thymol have an inhibiting effect on the formation of polymicrobial biofilms at 24 h and has a great potential in anticaries.

Keywords: Antibiofilm, Anticaries, Eugenol, Polymicrobial, Thymol


How to cite this article:
Utami DT, Pratiwi SU, Haniastuti T, Hertiani T. Eugenol and thymol as potential inhibitors for polymicrobial oral biofilms: An in vitro study. J Int Oral Health 2021;13:45-52

How to cite this URL:
Utami DT, Pratiwi SU, Haniastuti T, Hertiani T. Eugenol and thymol as potential inhibitors for polymicrobial oral biofilms: An in vitro study. J Int Oral Health [serial online] 2021 [cited 2021 Mar 5];13:45-52. Available from: https://www.jioh.org/text.asp?2021/13/1/45/308357


  Introductions Top


Biofilms are related to oral health problems, including caries, periodontitis, halitosis, and gingivitis. Dental caries is a chronic disease which can affect human health in childhood or adolescence as well as in adulthood,[1] due to decay of hard parts of the tooth such as enamel, dentin, and cementum. The tooth surface destruction by acid formation is a result of fermentation of carbohydrates into lactic acid by several oral microorganisms, that is, Streptococcus mutans, Lactobacillus acidophilus, Actinomyces viscosus, Nocardia spp., and Candida albicans.[2],[3],[4] Those microbes form biofilms as a polymicrobial culture on hard tooth surfaces and soft epithelial tissue which protect them from the unsupported environment.[5] Microbial diversity in polymicrobial biofilms can increase the protection capacity.[6],[7] The most recent research of oral biofilm inhibitors has only been focused on monomicrobial culture, even though oral polymicrobial biofilms present a greater threat which can lead to chronic infectious diseases.[8]

Mouthwash is known to inhibit dental caries. Fluoride, chlorhexidine, and cetylpyridinium chloride are effective against caries. Research has shown that the bioactive components of these chemical substances can cause irritation of the digestive tract and discoloration of the teeth.[9] The abundance of secondary metabolites in some medicinal plants is attractive for the discovery of natural compounds against dental caries.

Eugenol and thymol are commonly used as flavoring agents of food. They have many pharmacology applications such as their antioxidants,[10] anti-inflammatory,[11],[12] fungicidal,[13],[14] and antimicrobial activities.[15],[16] Eugenol and thymol are both phenolic compounds and responsible for the antibacterial and antibiofilm activities of specific essential oils. Thymol and eugenol have a broad spectrum for antimicrobial activity.[17]

Eugenol and thymol showed inhibition activity against the formation of mono and polymicrobial biofilms of Pseudomonas aeruginosa, Escherichia coli, S. aureus, and C. albicans. The results also showed the effective activity of eugenol and thymol in breaking down mono and polymicrobial biofilms.[18] Eugenol is the principal constituent of the clove oil from Syzygium aromaticum, which showed an antibacterial effect on S. mutans, S. sobrinus, and S. sanguinis.[19],[20] Clove oil constitutes 88.58% of eugenol and is the main component that inhibits A. viscosus and S. mutans.[21] Eugenol is known to have an inhibitory effect against L. acidophilus.[22] Thymol is isolated from the essential oils of Thyme spp., for example, Thyme vulgaris, T. zygis, and T. citriodorus.[23] Thymol is also known to inhibit the growth of L. acidophilus.[24] Thymol can decrease S. sanguinis planktonic bacteria in aerobic conditions.[25] Those reports were conducted in an aerobic environment. Dental health problems are mostly related to anaerobic conditions; therefore, a study to measure the potency toward polymicrobial oral biofilms in an anaerobic state is needed to better understand the potential for natural treatments of oral microbial biofilms. In this study, we aimed to evaluate the effectiveness of eugenol and thymol as an antibiofilm agent in polymicrobial cultures.


  Materials and Methods Top


Setting and design

This in vitro study was conducted by crystal violet assay in 96-well microtiter plates. The total duration of the study was 3 months. This study was conducted in Laboratory Microbiology, Department Pharmaceutical Biology, Faculty of Pharmacy, Universitas Gadjah Mada, Indonesia. Serial concentration of test compounds (1%, 0.5%, 0.25%, and 0.125% v/v) were used. Polymicrobial oral biofilms were various. Among these oral biofilms, S. mutans ATCC 25175, S. Sanguinis ATCC 10566, L. acidophilus ATCC 4356, A. viscosus ATCC 15987 were selected based on the inclusion criteria for our study. The bacterial stock was kept frozen in glycerol at –80°C. After that, it was thawed at room temperature. The organisms were maintained by culturing in Brain Heart Infusion (BHI) and incubating them at 37°C for 18–24 h.

Materials

Materials used in this research were eugenol (Sigma Aldrich, Germany), thymol (Sigma Aldrich, Germany), 96 and 24 well microtiter plates (Iwaki, Japan), Brain Heart Infusion (BHI) (Oxoid, UK), sucrose (Oxoid, UK), crystal violet (Himedia, India), coverslip, 95% ethanol MRS (Merck, Germany, MHA (Muller Hinton agar) (Oxoid, UK), anaerogen gas pack (Oxoid, UK), chloramphenicol (Sigma Aldrich, Germany), and Listerine (Indonesia).

Equipment

Types of equipment used in this research were spectrophotometry UV–Vis (Geneys 10 UV Scanning, 335903) (Thermo Scientific, USA), autoclave (Sakura, Japan), incubator, multichannel micropipette (Socorex, Swiss), micropipette pipetman (Gilson, France), micropipette 2–10 μL; 20–200 μL; 100–1000 μL (Socorex, Swiss), 96-well microtiter plates (Iwaki, Japan), microtiter plate reader (Optic Ivymen System 2100-C, Spain), scanning electron microscopy (JEOL JSM-6400, Japan), and transmission electron microscopy (JEOL JSM-6400, Japan).

Bacterial strains

Streptococcus mutans ATCC 25175, S. Sanguinis ATCC 10566, L. acidophilus ATCC 4356, and A. viscosus ATCC 15987 were used in this study. The number of bacterial cells was measured with spectrophotometry (OD600); 0.1 for S. sanguinis; 0.5 for A. viscosus; 0.2 for S. mutans; and 0.44 for L. acidophilus. This absorbance was equivalent to 1.3 x 108 CFU/mL, 1.5 x 108 CFU/mL, 2 x 108 CFU/mL, and 3.3 x 108 CFU/mL, respectively.[26],[27],[28],[29]

Antibacterial activity

A serial concentration was made from stock solution by diluting 40mg test compound in 2mL solution. The solvent was 1% dimethyl sulfoxide (DMSO). The serial concentration was made in four concentrations such as 1%; 0.5%; 0.25%; and 0.125% v/v. The microdilution method was used in 96-well microtiter plates. After this step, BHI media, the suspension of bacteria, and the test compound were added in the plates and incubated at 37°C for 24 h.

Biofilm formation inhibition in vitro

The inhibition effect of eugenol and thymol against biofilm formation were performed using 96-well microtiter plates[30] with a slight modification using anaerobic condition. Thymol and eugenol, with 2% v/v of each compound were diluted in DMSO as the stock solution. Serial concentrations (1%, 0.5%, 0.25%, 0.125% v/v) were used in aquadest sterile. A commercial mouthwash, Listerine was used as positive control which contains Camelia sinensis (Green tea) leave extract, sodium fluoride, eucalyptol, menthol, methyl salicylate, and thymol. Plates were incubated at 37°C in anaerobic conditions for 24 h for intermediate phase biofilms and 48 h for mature phase biofilms. Anaerobic condition was made by anaerobic gas pack that was placed in an anaerobic jar. A 100 μL solution containing BHI, 2% sucrose, bacterial suspension, and essential oil were added to each well. The amount of bacterial suspension was 10% v/v from total solution. Then, microplates were incubated at 37°C for 24 h and 48 h under anaerobic conditions. After the incubation period, the culture medium was removed and rinsed with sterile distilled water. Next, 125 µL crystal violet 1% v/v was used as the staining reagent. After incubated at room temperature 15 minutes and rinsed with water, 200 µL ethanol 96% was added into each well. The optical density was measured at 595 nm. The result was shown as percentage of inhibition. Percentage of minimum biofilm inhibition concentration (MBIC50) was calculated as follows:

[INLINE 1]

The value of OD approaching the cut of point was the value of MBIC50.[30] At least three replicate experiments were conducted for each concentration of eugenol and thymol.

Biofilm degradation assay in vitro

Biofilm degradation assay was similar to biofilm inhibition assay although test compounds were applied to 24 h and 48 h preformed biofilm. Biofilms were incubated for 24 h and 48 h. After the incubation period, the microbial suspension was discarded, and a test compound was added. Listerine 1%v/v was used as a positive control. Suspension of bacteria, media and test compound were incubated for 24 h and 48 h. After the incubation period, the microplate was washed with sterile aquadest. Next, 125 µL crystal violet was added to each well. After incubated at room temperature 15 minutes and rinsed with water, 200 µL ethanol 96% was added into each well. The optical density was measured at 595 nm. The result was shown as percentage of inhibition. Percentage of minimum biofilm Eradication concentration (MBEC50) was calculated as follows:

[INLINE 2]

The value of OD approaching the cut of point was the value of MBEC50.[30] At least three replicate experiments were conducted for each concentration of eugenol and thymol.

Scanning electron microscopy analysis

SEM was used to observe biofilm morphological changes following the treatments of test compounds. Coverslips were placed on 24-well microtiter plates with 100 µL total volume containing BHI, sucrose, microbial suspension, and test compound. The plate was incubated at 37°C for 24 h in an anaerobic condition.[31] Afterward, coverslips were fixed with glutaraldehyde and examined under SEM at magnifications ranging from 1000x to 5000x.


  Results Top


The antibacterial activities of eugenol and thymol against the oral microorganism were quantitatively measured by the MIC values. The results were obtained with microdilution methods, with a range of MIC values for eugenol and thymol against oral bacteria [Table 1] and [Table 2]. The difference between the groups in thymol (P < 0.05) and eugenol (P < 0.05) was found to be statistically significant. The data showed that antibacterial effect, eugenol 1% v/v against S. sanguinis, S. mutans, and L. acidophilus were 87.6 ± 1.42; 93 ± 0.55; and 87.9 ± 3.98, respectively. Thymol had the highest antibacterial effect on S. sanguinis 62.4 ± 7.9.
Table 1: MIC50, MBIC50, and MBEC50 of eugenol

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Table 2: MIC50, MBIC50, and MBEC50 of thymol

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Oral bacteria such as S. sanguinis, S. mutans, and L. acidophilus are capable of forming the oral biofilm which is known as dental plaque.[32] In this study, eugenol and thymol showed inhibiting oral biofilms. The results showed that eugenol of S. mutans gave the highest activity of 93 ± 0.5 and was better than the inhibition of S. sanguinis 87.6 ± 1.0, L. acidophilus 87.9 ± 3.0, A. viscosus 73.9 ± 0.5. MBIC50 value from eugenol was 0.5% v/v and it was most effective in inhibiting polymicrobial biofilms in the middle phase [Table 1]. Listerine 1% v/v (57.2 ± 0.1) had lower biofilm inhibition than thymol 1% v/v (52.9 ± 0.6). The data showed that increasing the concentration of eugenol and thymol caused a decrease in biofilm growth. Eugenol caused 50% damaging of monospecies S. mutans in the maturation phase. The inhibition effect of thymol 1% v/v (52.9 ± 0.6) was a higher inhibition effect than eugenol 1% v/v (51.6 ± 0.3) on polymicrobial biofilms. Biofilm degradation in vitro at 24 h in monospecies of positive control (Listerine) (53.9 ± 1.0) showed a lower biofilm degradation compared to eugenol against A. viscosus (57.7 ± 4.0). Listerine 1% v/v (83.6 ± 2.0) showed a lower biofilm degradation compared to thymol 1% v/v against A. viscosus (87.2 ± 0.3). Interestingly, Listerine 1% v/v (67.7 ± 0.6) showed a higher biofilm degradation compared to against L. acidophilus (53.8 ± 2.0).

Listerine 1% v/v (65.3 ± 0.7) showed a lower biofilm degradation compared to eugenol 1% v/v against S. sanguinis (68.5 ± 1.0). Listerine 1% v/v (83.5 ± 0.5) showed lower biofilm degradation compared to thymol 1% v/v against A. viscosus (87.2 ± 0.7) in the mature phase. Listerine 1% v/v showed biofilm degradation against S. sanguinis (67.1 ± 1.0), S. mutans (35.1 ± 0.7), and L. acidophilus (51.5 ± 5.6).

Listerine (31.4 ± 1.0) caused a lower degradation than eugenol 1% v/v (41.3 ± 0.7) against polymicrobial biofilms in the mature phase. Listerine (69,2 ± 0,7) was higher than thymol 1% v/v (64,2 ± 3,1) against polymicrobial biofilms in mature phase. In this phase, oral biofilms had already been adhering to the surface of the teeth. Based on the previous study, the antimicrobial was more challenging to penetrate the biofilms in the maturation phase, and eugenol can be added as a potential compound to be a more effective antibiofilm agent.[18] Even though an effective ways to control dental plaque to prevent inhibition of biofilm growth or degrade existing biofilms by antibiofilm.[33]

Scanning electron microscope

SEM was used to show changes of polymicrobial biofilms morphology after cells were treated with eugenol and thymol on polymicrobial biofilms cultured for 24 h biofilms on a coverslip. Bacteria communities on negative control were thick and dense from EPS [Figure 1]. Based on these results, it can be shown that the biofilm was wrapped by an EPS matrix, which can protect it from antimicrobial compounds. The matrix contains protein, nucleic acid and polysaccharides and has an essential role in biofilm resistance. Bacteria could form biofilms with other species, and in the process the biofilm becomes stronger and thicker.[34] After the cells were treated with eugenol in the polymicrobial biofilms, the matrix of EPS can be degraded. Thymol inhibits biofilms growth and damages the EPS which protects these bacterial biofilms. Thymol and eugenol treatment can change the morphology of the polymicrobial biofilm structure.
Figure 1: Inhibition of biofilm formation of polymicrobial on coverslips monitored by SEM cultured for 24 h; cells treated with (A) 0.5% v/v eugenol, (B) 1% v/v thymol, and (C) control (untreated) cells; 1: magnification ×1000; 2: magnification ×5000

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  Discussion Top


Oral biofilms occur when community of bacteria attach to any surface of the teeth. Oral biofilms cause caries and periodontal disease. Oral infection can be correlated with the systematic disease such as cardiovascular diseases, rheumatoid disease, and neurodegenerative diseases.[35]

Toothpastes usually contain stannous fluoride (SnF2) which is still controversial because of its negative effect. SnF2 causes recalcification and erosion of the tooth enamel, gingival inflammation, and biofilm formation. Despite these negative side effects, it can remove halitosis and tooth stains.[36]

One of the pioneer bacteria in oral biofilm surface attachment is S. sanguinis. The pioneer bacteria have several interactions with other bacteria.[37] These interactions among other microbials make the polymicrobial interaction develop as a bacterial community, now known as polymicrobial biofilm. The dysbiosis in polymicrobial biofilms can cause oral diseases such as dental caries, halitosis and gingivitis.[38] In this study, we evaluated eugenol and thymol as potential treatment in the inhibition of polymicrobial oral biofilms.

Based on the results, eugenol had antimicrobial activity against oral planktonic bacteria that was higher than thymol. This study indicated that eugenol has a greater potential than thymol. In previous studies, it was known that eugenol had an antibacterial effect on S. mutans in vitro.[39] The mechanism of eugenol involves the suppression of quorum sensing of S. mutans.[40]

Antiplanktonic mechanism and antibiofilm mechanism are different. Pathogenic bacteria live together to form biofilms and have a strong defense system. In the planktonic phase, bacteria live freely so antimicrobials can enter the target cells and the cell is broken.[34] Bacteria are known to live dormant when they are in an unfavorable habitat.[41] Most microbial biofilm environments are more resistant to antibiofilm agents compared to planktonic bacteria.[42]

Eugenol and thymol had the most inhibition effects against polymicrobial biofilms in the intermediate phase. Thymol also had degradation effects against polymicrobial biofilms in the intermediate and mature phase. Based on the previous study, thymol and eugenol showed inhibition effect against the formation of monomicrobial and polymicrobial oral biofilms. In polymicrobial biofilms, EPS of biofilms is formed from various microbes which help the cells of the pathogenic bacteria to live longer.[6] EPS has resistance against antimicrobial compounds, drought, nutrient deficiency, and conditions non-conducive for the growth of the microbes. EPS helps them live longer. The results showed that thymol and eugenol were able to break down the EPS of monomicrobial and polymicrobial oral biofilms. In this study, 1% v/v Listerine was used as positive control. Listerine contains Camelia sinensis (Green tea) leave extract, menthol, methyl salicylate, thymol and sodium fluoride.[31]

Streptococcu mutans initiates pellicle formation. The formation of acellular films consists of proteins in the saliva. After 4 to 24 h, these bacteria become cariogenic biofilms.[43] However, the genus Lactobacilli are often found in caries of parts of the dentine and rarely found in the teeth in the early part of the development of caries. When the growth of S. mutans and L. acidophilus are inhibited, the formation of the pellicles as the etiological origin of dental caries can be prevented and dental caries is inhibited. Eugenol had inhibition biofilm effect in intermediate phase on S. mutans. Thymol had inhibition effect against microbial biofilms in the intermediate phase on S. mutans and L. acidophilus Eugenol had inhibition effect against microbial biofilms in the mature phase on S. mutans.

Eugenol inhibited S. sanguinis biofilms. Based on the previous study, eugenol had an antibacterial effect on S. mutans. Eugenol has monoterpene hydrocarbons. The mechanism of eugenol acts on the cell membrane activity by inactivating enzyme and disrupting genetic material functionality, interfering with the formation of energy production, which disrupts the synthesis of structural components and causes cell death. Eugenol also causes damage to the cytoplasmic membrane. The damage to the plasma membrane can cause disruption of proton motive force (PMF), electron flow, active transport and coagulation of the inside of microbial cells.[44] The mechanism of thymol can penetrate the cells through the cell wall, disrupt the cell membrane and this results in the lysis of the cells.[45]

Eugenol had degradation effects in the intermediate phase on S. sanguinis, S. mutans, L. acidophilus, and A. viscosus. Thymol also had degradation effects in the intermediate phase on S. sanguinis, L. acidophilus, and A. viscosus. Additionally, eugenol had degradation effects in the mature phase on S. sanguinis, S. mutans, L. acidophilus, and A. viscosus. Thymol also had degradation effects in the mature phase on S. sanguinis, L. acidophilus, and A. viscosus. The data indicated that polymicrobial biofilms were more resistant to degradation than single species. Based on the previous study, polymicrobial biofilms were more resistant to antibiotics than single-species biofilms.[33] Therefore, eugenol and thymol can be potentially developed as a therapeutic alternative for polymicrobial biofilms treatment.


  Conclusion Top


Eugenol showed prominent antibacterial effects against S. sanguinis, S. mutans, L. acidophilus, and A. viscosus. Thymol showed antibacterial activity toward A. viscosus. Higher concentration of eugenol was needed to inhibit the polymicrobial culture than thymol. It can be concluded that eugenol and thymol had the most inhibiting effects on the polymicrobial biofilms formation at 24 h.

Future scope

Dental plaque can be prevented using antibiofilm agent. Eugenol and thymol had potential inhibition biofilm growth. In the future, these compounds can formulate as pharmaceutical product such nanoemulsion.

Acknowledgement

We express our gratitude to Faculty of Pharmacy, Universitas Gadjah Mada, Indonesia, and Deputy of Research Reinforcement and Innovation, The Ministry of Research and Technology/National Agency for Research and Innovation (Indonesia) with PMDSU (Master’s Education toward Doctorate) for their support for the research.

Financial support and sponsorship

This research was fully funded by the Deputy of Research Reinforcement and Innovation, The Ministry of Research and Technology/National Agency for Research and Innovation (Indonesia) with PMDSU (Master’s Education toward Doctorate) research project 2020 No. 3166/UN1.DITLIT/DIT-LIT/PT/2020.

Conflicts of interest

There are no conflicts of interest.

Author’s contributions

Diyah Tri Utami designed and performed experiment, analyzed the data, and wrote the paper. Sylvia Utami Tunjung Pratiwi, Tetiana Haniastuti, and Triana Hertiani contributed to the design of the study and co-writing of the paper.

Ethical policy and institutional review board statement

Not applicable.

Patient declaration of consent

Not applicable.

Data availability statement

The data are included within the article.

 
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