INTRODUCTION:

Periodontitis is an inflammation of the periodontal tissue caused by certain microorganisms that cause the damage to the periodontal ligament and alveolar bone characterized by the probing depth, recession, or both^1-3. In 2013, the prevalence of periodontal disease in Indonesia had a percentage of 96.58%⁴. The periodontal disease affects almost all age groups in society⁵. Data on the Basic Health Research in 2018 from the Ministry of Health of the Republic of Indonesia shows that 57.6% of Indonesians have dental and mouth problems and receive services from dental medical personnel^6-7.

Periodontitis can be divided into two types, which are aggressive periodontitis and chronic periodontitis^8-9. Aggressive periodontitis around 90% is caused by Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans) and and the rest is caused by another bacterium such as Fusobacterium nucleatum (F. nucleatum), Porphyromonas gingivalis (P. gingivalis), Tannerella forsythia (T. forsythia), Treponema denticola (T. denticola), Prevotella intermedia (P. intermedia), Prevotella nigrescens (P. nigrescens), Campylobacter rectus (C. rectus), Eikenella corrodens (E. corrodens) and Parvimonas micra (P. micra)¹⁰. Periodontitis treatment is usually done by scaling, root planning, and the administration of materials and drugs¹¹. Materials and drugs used for periodontal therapy are periodontal pack or periodontal dressing, analgesics, and antibiotics^12-14. In periodontal treatments, the local antibiotic products that are commonly used are minocycline, doxycycline, metronidazole, tetracycline, and chlorhexidin. The use of tetracycline, minocycline, and doxycycline show more effective effects, while metronidazole and chlorhexidin show minimal effects^15,16.

These days, innovation of drug formulations is improving and developing. Mucoadhesive is this innovation options, because mucoadhesive provides benefits such as good comfort and easy application of drugs without pain. Various mucoadhesive forms are tablets, films, patches, disks, strips, ointments and gels. Mucoadhesive patches offer greater flexibility and comfort than any other forms¹⁷. Mucoadhesive gingival patches are a form of drug supplies designed to provide an effective amount of medication based on the therapy applied to the gingiva. Mucoadhesive gingival patches have some functions such as periodontal pack or periodontal dressing. Mucoadhesive gingival patches consist of liners, adhesives, pharmaceutical contents, and backings. A liner is a patch which is removed before application and is attached to the mucosa. Adhesives has the function as adhesives and backing’s role is as anchoring patches¹⁸. The mucoadhesive gingival patch is applied to the gingiva after a periodontal surgery. Mucoadhesive gingival patches have the primary function to prevent the post-periodontal surgical infections, protect the surgical wounds from chewing trauma, and prevent the onset of pain due to any contact with food¹⁹. Additional ingredients such as antioxidants and high antimicrobials are needed in the mucoadhesive gingival patch which serves to accelerate the healing of periodontal treatments. This is because the main content of mucoadhesive gingival patch is HPMC and CMC-Na which are not antioxidants and antimicrobial ingredients. HPMC is a gelling agent that works to produce a gel that is clear, easily soluble in water, and has low toxicity. CMC-Na has a function as a good film-forming, biodegradable, and has low toxicity^20,21. The addition of antioxidants made from natural ingredients is also important because it aims to minimize the adverse side effects of synthetic ingredients.

Some natural compounds have the potential to inhibit the bacteria that cause periodontitis, like thymol and carvacrol which are found in Thymus vulgaris essential oil. Thymol and carvacrol are the main compounds contained in T. vulgaris essential oil which have a potential as an antioxidant, antimicrobial, antitussive and antispasmodic. Previous studies have proven the use of T. vulgaris essential oil as an inhibitor of oral bacteria. Thymol is more effective in inhibiting Gram-negative bacteria as the cause of aggressive periodontitis, such as A. actinomycetemcomitans and F. nucleatum. T. vulgaris essential oil can inhibit A. actinomycetemcomitans which is shown by the presence of a 32µg/mL inhibition zone activity measured using the disc diffusion method²².

Furthermore, T. vulgaris essential oil has been investigated and proven its ability to inhibit oral cavity bacteria. However, so far there is only few research on mucoadhesive gingival patches which are formulated with T. vulgaris essential oil in inhibiting A. actinomycetemcomitans and F. nucleatum. The aim of this study is to investigate the ability of mucoadhesive gingival patch contained with Thymus vulgaris essential oil to inhibit the growth of A. actinomycetemcomitans and F. nucleatum bacteria.

MATERIAL AND METHOD:

This study was approved by the Ethics Commission of the Faculty of Dental Medicine, Airlangga University, Surabaya Indonesia (Reference number 464/ HRECC. FODM/VII/2019). The study was an experimental laboratory in vitro. This study design used is in the form of the post-test-only control group design. This study used samples of the bacterial stock of A. actinomycetemcomitans ATCC 43718 and F. nucleatum ATCC 25586 obtained from the Research Center of the Faculty of Dentistry, Airlangga University. The samples were grouped into T. vulgaris essential oil group as the positive control extract, mucoadhesive gingival patch group as the negative control group, T. vulgaris essential oil gingival patch group as the treatment group and doxycycline gingival patch group as the positive control group. The minimum sample size was determined using Lemeshow formula. Blind simple random sampling was done to select the sample.

The making of mucoadhesive gingival patch was carried out using the solvent casting technique. Each ingredient is weighted, they were 1.5gram CMC-Na sprinkled in 30 mg for one night, then crushed until it becomes a gel. Next, it was added with 60.3 grams of hot water gradually to the base of the gel. Ethanol was included by propylene glycol and then stirred until dissolved. The formulas in stages II and III were mixed and stirred until homogeneous. Moreover, the formula is wighted into 70 grams and put it into a Petri dish the n dried in the oven at 45⁰C.

A. actinomycetemcomitans and F. nucleatum bacteria were cultured in BHI media for 24 hours at 37⁰C under anaerobic conditions. Then, they were planted on agar nutrient media, followed by adding T. vulgaris essential oil, mucoadhesive gingival patch, and T. vulgaris gingival patch essential oil to each bacterium and then incubated for 24 hours at 37⁰C. Furthermore, the inhibition zone diameter results were measured using calipers and tested using the statistical analysis. The data then could be analyzed using the normality test using the Kolmogrov-Smirnov test, followed by the Levene homogeneity test (p>0.05). One-way ANOVA was applied to analyze the significance of all treatment groups and continued with the post hoc test (Tukey HSD) (p<0.05).

The analysis results of A. actinomycetemcomitans bacteria in the T. vulgaris essential oil group show that it has the highest inhibition zone compared to doxycycline mucoadhesive patch group which shows the lowest inhibition zone (see Table 1 and Figure 1). While, one-way ANOVA test results expresses a significant difference (p = 0.021; p<0.05), the results of the Tukey HSD test reveals that between T. vulgaris essential oil group and group with doxycycline mucoadhesive gingival patch, there are significant differences (p = 0.023; p<0.05). Furthermore, there is no any significant difference between groups of doxycycline gingival mucoadhesive patches and mucoadhesive patches with T. vulgaris essential oil (p= 0.061; p>0.05). Similarly, the groups of T. vulgaris essential oil and mucoadhesive gingival patch with Thymus vulgaris essential oil, there is no any significant difference (p = 0.816; <0.05).

The analysis of F. nucleatum in the T. vulgaris essential oil group shows the highest inhibitory zone, while the group of mucoadhesive gingival patch with T. vulgaris essential oil expressed the lowest inhibitory zone (see Table 1 and Figure 2). As One-way ANOVA test results reveals a significant difference (p = 0.000; p<0.05), the results of the Tukey HSD test showed that the groups of T. vulgaris essential oil and doxycycline mucoadhesive gingival patch have significant differences (p = 0.017; p<0.05) Meanwhile, the groups of doxycycline mucoadhesive gingival patch and mucoadhesive gingival patch with T. vulgaris essential oil also carry significant differences (p = 0.000; p<0.05). Furthermore, the groups of T. vulgaris essential oil and mucoadhesive gingival patch with T. vulgaris essential oil possess the significant differences as well (p = 0.000; p<0.05).

An independent t-test sample results presents that Thymus vulgaris essential oil group has no any significant differences in A. actinomycetemcomitans and F. Nucleatum bacteria (p = 0.055; p>0.05). While, the group of mucoadhesive gingival patch with Thymus vulgaris essential oil possess a significance difference in A. actinomycetemcomitans and F. nucleatum bacteria (p = 0.000; p<0.05). Moreover, for doxycycline mucoadhesive gingival patch group, there were no any significant differences in A. actinomycetemcomitans and F. nucleatum bacteria (p = 0.161; p>0.05).

Table 1: The calculation results of the bacterial inhibition zones of A. actinomycetemcomitans and F. nucleatum in each group.

Group	Mean (mm) ± SD
Group	A. *actinomycetemcomitans*	F. *nucleatum*
Essential oil T. vulgaris	13.83 ± 0.54	13.09 ± 0.30
Mucoadhesive gingival patch	0 ± 0	0 ± 0
Mucoadhesive gingival patch T. vulgaris essential oil	13.58 ± 0.77	10.22 ± 0.34
Doxycycline mucoadhesive gingival patch	12.53 ± 0.20	12.05 ± 0.57

The analysis result of A. actinomycetemcomitans bacterial inhibitory shows that T. vulgaris essential oil with mucoadhesive gingival patch from essential oil does not have any significant difference. A. actinomycetemcomitans have the virulence of leukotoxins, adhesin, and bacteriocin. The virulence of bacteria will be disrupted by the activity of T. vulgaris essential oil²³. T. vulgaris essential oil activity is determined by several factors which are composition, additional components contained in the active component, and synergistic interactions between T. vulgaris essential oil and bacteria²⁴. The main composition of T. vulgaris essential oil is thymol and carvacrol. Thymol and Carvacrol interact with bacterial cytoplasmic membranes that cause changes in cytoplasmic permeability and affect lipid composition and bilayer stability. Changes in permeability will cause an increase in the passive flux of protons and across the membrane which then leads to the disruption of cytoplasmic membrane and cell leakage. T. vulgaris essential oil in sufficient concentration has a bactericidal effect²⁵. An additional component in the mucoadhesive gingival patch of T. vulgaris essential oil is CMC-Na and propylene glycol where the role of CMC-Na is as a gel-forming, while propylene glycol has the function as a penetration enhancer, such as the ability to increase the solubility of the material thereby it increases the diffusion through bacterial cell membranes²⁶. The presence of CMC-Na and propylene glycol does not inhibit the mechanism of T. vulgaris essential oil, this is consistent with the previous studies which show that there is no difference in inhibition in both mucoadhesive gingival patch of T. vulgaris and T. vulgaris essential oils. This proves that the content of thymol and carvacrol in the mucoadhesive gingival patches of T. vulgaris essential oil remains effective.

Meanwhile, both T. vulgaris essential oil and mucoadhesive gingival patch from T. vulgaris essential oil have significant differences with doxycycline gingival patch. Doxycycline has bacteriostatic properties; antimicrobial ability is able to inhibit matrix metalloproteinase and to inhibit collagenase. Doxycycline inhibits protein synthesis by reversible binding of the bacterial ribosome subunit, thus, preventing the binding of RNA’s aminoacyl transfer²⁷. Doxycycline is effective against certain amounts of A. actinomycetemcomitans, as it has been reported that MIC doxycycline 0.25-2.0mg/L uses the agar-based test. Doxycycline mucoadhesive gingival patch has lower bacterial inhibitory value than Thymus vulgaris essential oil and mucoadhesive gingival patch of Thymus vulgaris essential oil. This might be due to the presence of doxycycline resistance in previous studies which stated that A. actinomycetemcomitans had 8.9% resistance to doxycycline²⁸.

The results to F. nucleatum bacterial inhibitory analysis shows that between Thymus vulgaris essential oil, mucoadhesive gingival patch contained with Thymus vulgaris essential oil, and doxycycline mucoadhesive gingival patch have the significant inhibitory differences. F. nucleatum has LPS and adhesin virulence factors. Adhesin plays a role as an adhesion and colonization facilities. On the wall of F. nucleatum bacteria, there is LPS which has the role as a protective bacterium from complement activity²⁹. In the results of this study, it is found that the T. vulgaris essential oil and mucoadhesive gingival patch contained with T. vulgaris essential oil are able to inhibit F. nucleatum bacteria. Gram-negative bacteria are less sensitive to Thymus vulgaris essential oil because the Gram-negative outer membrane is more rigid and rich in LPS and more complex which can limit the diffusion of hydrophobic compounds.

The results of T. vulgaris essential oil’s inhibitory influence on A. actinomycetemcomitans and F. nucleatum do not have any significant difference. This proves that the content of thymol and carvacrol can inhibit A. actinomycetemcomitans and F. nucleatum bacteria effectively. The results of this study indicate that the inhibitory ability of mucoadhesive gingival patch contained with T. vulgaris essential oil against F. nucleatum and A. actinomycetemcomitans possesses significant differences. In F. nucleatum, there is FadA, a cell surface protein that regulates adhesion and bacterial invasion. F. nucleatum is able to create a very strong lipopolysaccharide environment and produce butyric acid in the final product of metabolism, which causes F. nucleatum bacteria to be less sensitive compared to A. actinomycetemcomitans. This is consistent with this study that mucoadhesive gingival patch of T. vulgaris essential oil is more sensitive to A. actinomycetemcomitans than F. nucleatum³⁰. From this study, we can conclude that mucoadhesive gingival patch contained with T. vulgaris essential oil can inhibit the growth of A. actinomycetemcomitans and F. nucleatum. It is more sensitive to A. actinomycetemcomitans than F. nucleatum in vitro

ACKNOWLEDGEMENT:

The authors would like to thank Universitas Airlangga for the funding support and research Center Faculty of Dental Medicine Universitas Airlangga Surabaya, Indonesia for laboratory work support.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

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Received on 04.03.2020 Modified on 02.05.2020

Research J. Pharm. and Tech. 2021; 14(2):645-649.

DOI: 10.5958/0974-360X.2021.00115.3