INTRODUCTION:

Dental caries has the high prevalence as oral cavity health related problems in the world. In Indonesia, regarding oral and tooth health have elevated from 23.2% (2007) to 25.5% (2013) of which dental caries is the most the common found (1). Dental plaque consists of various oral bacterial species of which S. sanguinis, S. mutans, and L. acidophillus are reported to be predominant.

Those bacteria are capable of producing acids derived from sugar metabolism which can dissolve the tooth enamel, resulting in caries or cavities. Caries can further spread to the dentine and pulp (2). Noteworthy, caries and poor oral health have been correlated with several cardiovascular diseases cases (3).

Streptococcus sanguinis is a Gram-positive bacterium with no spore formation, lives as a facultative anaerobe, and non-motile(4)^.S. sanguinis plays role as pioneer bacteria that colonizes the surface of the teeth. This pioneering bacteria co-aggregate with other bacteria to form dental plaques (2). In addition, S. sanguinis can produce sortase A enzyme to enter the bloodstream. Bacteria has been reported to cause endocarditis(5). The ability of bacteria adhere to the epithelial cells are considered as virulence factor. Lactobacillus acidophillus is a Gram-positive and rod-shaped bacteria(6), L. acidophillus is reported to attach on to the surface of the teeth following S. sanguinis colonization (7).

Dental caries can potentially be prevented by inhibiting the growth of biofilms on the surface of the teeth. Applying mouthwash containing active substances such as chlorhexidine, cetypirinidium chloride, and fluoride has been reported to be effective against dental plaques. However, the side effects such as irritation of the digestive tract and discoloration of the tooth have been reported following long term uses of those active ingredients (8).

Zerumbone is an essential oil component found in several plants of family Zingiberaceae. Those plants can be found across Southeast Asia such as Indonesia, China Bangladesh, Vietnam, Japan, Burma, Nepal, Sri Lanka, Jamaica, and Nigeria, and has been widely used as spices as well as part of traditional remedy. Zingiber zerumbet (Indonesian: Lempuyang Gajah) in particular, is used as a treatment for a toothache in India (9). Zerumbone is known to have an antibacterial effect on S. mutans (10). Essentially, this compound exhibit no cytotoxicity on healthy mammalian cells (11) which may refer to safety. It is interesting to find out whether this particular compound may serve as inhibitor to oral biofilm producers S. sanguinis and L. acidophilus single species biofilms.

MATERIAL AND METHODS:

Sample Preparation:

Zingiber zerumbet L. Roscoe ex Sm rhizomes, Family Zingiberaceae obtained from Kulonprogo, DIY, Indonesia. The taxonomy identification was confirmed in the Pharmacognosy Laboratory, Department of Pharmaceutical Biology, Faculty of Pharmacy, Universitas Gadjah Mada, Yogyakarta, Indonesia.

Materials:

Zerumbone was obtained from the distillation and recrystallization process. Distilled water (aquades) sterile, crystal violet (Himedia, Germany) (1% in sterile distilled water), Brain Heart Infusion (Oxoid, Germany), ethanol 95% (Merck, Germany), sucrose (Merck, Germany), Listerine® (Indonesia), NA (oxoid, UK), chloramphenicol (Sigma Aldrich, Germany), MHA (Muller hinton agar) (Oxoid, UK), anaerogen gas pack (Oxoid, UK), 96 well flat-bottom polystyrene microplate (Iwaki, Japan), 24 well flat-bottom polystyrene microplate (Iwaki, Japan), Dimethyl sulfoxide (DMSO) (Merck, Germany).

Equipments:

Some equipments used in this research were laminar airflow, incubator (Sakura, Japan), micropipette pipetman (Gilson, France), multichannel micropipette (Socorex, Swiss), microtiter plate reader (Optic Iveymen System 2100-C, Spain), spectrophotometry (Geneys 10 UV Scanning, 335903) (Thermo Scientific Spectronic, USA), autoclave (Sakura, Japan)

GC-MS Analysis:

Gas Chromatography Mass Spectrometry (GC-MS) was conducted to confirm the identity and purity of Zerumbone. GC-MS was performed at the Organic chemistry laboratory in Faculty of Mathematics and Natural Sciences UGM. Zerumbone was obtained in this study that was diluted and injected to a capillary column Restek Rtx-5 MS. Helium gas was used by flow rate of 28.4ml/min with 13.7 kPa pressure and the injection port was heated to 300°C.

Microorganisms and Inoculum Preparation:

This study used S. sanguinis ATCC 10566 and L. acidophilus ATCC 4356. Culture were grown on MHA (Oxoid, UK) at 37°C for 48 hours in anaerobic conditions (anaerobic gas was put in anaerobic chamber and closed tightly). After 48 hours, some of the colonies were formed on BHI broth and incubated at 37°C in anaerobic condition until number of the cells necessary for the experiment was reached.

Antibacterial assay:

A microdilution method was used to perform the planktonic growth of inhibition assay. Different concentrations of zerumbone were tested againts S. sanguinis and L. acidophillus. Aliquots of 100µL (media BHI, suspension of the microbia and the test compound) were dispensed into 96 well microplates. Whereas, a suspension of the bacteria S. sanguinis has an OD λ 600 of 0.1 was correlated to a cell concentration of 1.3x10⁸CFU/mL(12)and L. acidophilus has an OD₆₀₀ of 0,4 was correlated to a cell concentration of 3.3x10⁸CFU/mL (13). Chloramphenicol 1% v/v was used as a positive control while the negative control contained only bacterial suspension. Suspension of the treatment incubated with aerob conditions for 18-24 h at 37°C. The OD values were measured by a microplate reader at λ 595nm. Antibacterial assay was conducted in triplicate.

Biofilm inhibition assay:

BHI media containing sucrose 2%, bacterial suspensions and test compounds were transferred to a 96-well polystyrene microtiter plate and incubated for 48 h in an anaerobic condition with 5% CO₂ at 37°C (anaerobic chamber, anaerobic gas, Oxoid, UK). 10μL Zerumbone was diluted in DMSO 2 % to obtain 1% v/v, 0.5% v/v, 0.25% v/v, 0.125% v/v samples; the positive control was mouthwash Listerine® 1%v/v. The negative control consisted of a microbial suspension and solvent used (DMSO). After discarding the media, the plate was rinsed with sterile distilled water to remove planktonic bacteria. 125µL crystal violet solution of 1% was added to each well. The microplate was incubated at room temperature for 15 minutes. The microplate was rinsed with sterile distilled water, followed by an addition of 200µL absolute ethanol. After incubated for 15 minutes at room temperature, OD of samples were measured using a microplate reader at λ 595nm. The whole experiments were performed in triplicate.

Biofilm degradation assay:

This assay similar to biofilm inhibition assay however the test compound was applied to 48 h preformed biofilm. Biofilm formed after 48 h of incubation in media BHI containing sucrose 2%. After the incubation period, microbial suspension was discarded and added with the test compound. Positive control used were mouthwash Listerine® 1%v/v. Suspension of the assay will incubate for 48 h. After the incubation period, microplate washed with sterile aquades and the further process same with biofilm inhibition assay.

Statistical analysis:

The result was shown of mean±SD. The value of OD approaching the cut-off point was the value of MBEC₅₀ (2). Data were analyzed by one way ANOVA followed by post-hoc Benferroni test. P values <0.05 were considered significant.

SEM observation on biofilm morphology:

BHI media and bacteria cell were grown in coverslip and incubated 37°C for 48 h with anaerobic condition (5%CO₂). After the 48 h incubation, coverslip was rinsed with sterile water, added that with the test compound and incubated with anaerobic condition (5% CO2). After 48 h of incubation, 1% glutaraldehid was added. Sampels were coated with carbon by sputtering with auto fine coater and SEM analysis was conducted.

RESULTS:

GC MS Analysis:

GC-MS result showed the percentage of zerumbone detected in as 91.07% (Figure 1). The MS confirmed its identity (Figure 2). Data used was NIST/WILEY library.

Fig 1. Gas chromatogram of of Z. zerumbet rhizomes essential oil

Figure 2. Zerumbone structure

Antibacterial assay:

Zerumbone showed weak growth inhibition on the planktonic bacteria of S. sanguinis and L. acidophillus. Zerumbone 1% can more reduce on S. sanguinis than L. acidophillus respectively (Fig 3.).

Figure 3. Antibacterial activity of zerumbone againts S. sanguinis and L. acidophillus

Biofilm inhibition assay:

Zerumbone 1% v/v revealed that an inhibition effect more on S. sanguinis than L. acidophilus biofilms (Fig.4)

Fig. 4. Inhibition biofilm of zerumbone againts S. sanguinis and L. acidophillus

Degradation biofilm assay:

Zerumbone 1% v/v showed an more degradation effect on S. sanguinis than L. acidophilus biofilms (Fig. 5)

Fig. 5. Biofilm degradation of zerumbone againts S. sanguinis and L. acidophillus

SEM observation on biofilm morphology:

Zerumbone 1% v/v showed an degradation effect membran matrix extraseluler of S. sanguinis and L. acidophillus (Fig 6b,7b)

Fig. 6. Biofilm degradation of S. sanguinis ATCC 10566 on coverslips was monitored by SEM cultured for 48 h; a: control (untreated) cells; b: cells treated with zerumbone 0,5%; 1: magnification 1000x; 2: magnification 5000x

Fig. 7. Biofilm degradation of L. acidophillus ATCC 4356 on coverslips was monitored by SEM cultured for 48 h; a: control (untreated) cells; b: cell treated zerumbon 1%v/v; 1: magnification 1000x; 2: magnification 5000x

DISCUSSION:

Zerumbone is a sesquiterpene compound found in Zingiber zerumbet (14). Zerumbone has a strong bioactive potential to be an anti-cariogenic agent which means a formulation containing this substance as a primary substrate can be used against tooth decay (15). Besides, plants are high terpenoid which have antimicrobial activity (16) (17).

Bacteria live in biofilm when their unfavorable habitat and the bacteria are dormant and not active in metabolize. The development of biofilms begins from planktonic stages, adhesion to the surface, the formation of microcolony and secretion extracellular polymeric substance (EPS)(18). The EPS consists of polysaccharide, protein, and DNA and these formed of EPS are amorphous(19). These exopolysaccharides help the bacteria to prevent the diffusion antibiotic and make the biofilm are stronger than planktonic phase because they can aggregate to other bacteria (20)(21). The benefit of EPS can make biofilms that are resistant to the antimicrobial agents, defense system from chemical and infection (22). The life form biofilms are longer than the planktonic bacteria because of the mechanism of resistance to the microbial agents (23). The biofilm can communicate to other bacteria by quorum sensing. Quorum sensing is the chemometic factor or the pheromones in bacteria that form biofilm (24)(25). Furthermore, the function of quorum sensing to control swarming and biofilm formation (26)(27). Bacterial can bind the surfaces of living and nonliving material and consist of single or multispecies microbial (28)(29). Bacteria can live in the human mouth because in the human mouth has high nutrients (30). The bacteria in oral also cause fluctuations in pH so the fluctuations can cause a loss of mineral from the tooth and the maybe result demineralization process (31).

Determining the effect of zerumbone on bacteria, in this study, antibacterial, biofilm inhibition and degradation assay were used. The bacterial phase is known as the floating bacteria (32). The result of this study showed the reduction of planktonic of S. sanguinis and L. acidophillus were fewer than biofilms bacteria. This was due to the mechanism of zerumbone compounds which play a role in the mechanism of degrading biofilms when compared with the results on antibacterial. Based on this study, after treatment with zerumbone for 48 h, the OD values decreased significantly when compared with that the untreated controls (p< 0.05) (Fig. 5). The data showed that zerumbone had the same activity with essential alcoholic mouthwashes. In the previous study, essential alcoholic mouthwashes showed a better antibiofilm than antiplanktonic activity (33). Zerumbone could be a good choice in preventing plaque.

Biofilm inhibition and degradation assay were used cristal violet in this study. The charged surface molecules and polysaccharides in the extracellular matrix can be bound with crystal violet (34). In this study, zerumbone had more biofilm degradation effect than inhibition biofilm growth because zerumbone killed embedded bacteria or the detachment of living bacteria (35).

Streptococcus sanguinis is one of the pioneer bacteria on the surface of teeth. The bacteria co-aggregate or co-adhesive with the other bacteria. After the co-aggregation, the other bacteria colonize and biofilms maturate (2). S. sanguinis biofilms was degraded by zerumbone (Fig. 5) and thus colonization and coaggregation with the other bacteria were reduced.

The early development of caries, L. acidophillus has not been discovered because it can attach on the surface of the tooth after the colonizer bacteria attached (11). Lactobacillus acidophilus can cause caries in dentine and it can be inhibited by zerumbone, so dental caries can be inhibited. Biofilms are closely related to human infection which have high resistance to antibiotics. This is caused by the presence of extracellular matrix membranes produced by biofilms to protect bacteria. There are not many antibiotics available today that can fight infection due to the presence of biofilms. Biofilms formed by microbial communities that aim to protect bacteria from the environment include antimicrobial agents, changes in pH, UV radiation and changes in osmotic pressure (36).

SEM analysis showed that in the untreated of S. sanguinis (Fig. 6a) and L. acidophilus (Fig. 7a) biofilms on coverslip surfaces (Fig 6a,7a) and the presence of 0.5% v/v zerumbone (Fig. 6b) and 1% v/v zerumbone (Fig. 7b) (MBEC₅₀ value). Bacterial biofilms were reduced because of zerumbone. Biofilms matrix of S. sanguinis and L. acidophilus had disruption when treated with zerumbone (Fig. 6b, 7b), so the biofilms no longer had strong defenses that an indicating that zerumbone killed bacteria. Based on the previous study, long term plaque and gingivitis have been reduced by essential oil (37).

Dental plaque is a form of biofilm. Dental plaque is a mature biofilm in the oral cavity (4). Biofilm in the mature phase has more complex structure (38). The result of this study showed that zerumbone acted as a potent biofilm agent that had degradation actions of the existing plaque. Dental plaque can be prevented using an antimicrobial or chemical agent, but it can cause tooth stain in long term use. The natural compounds such as zerumbone is solution for this problems. It has greater potential effect on degradation the plaque than listerine since it contains phenolic compound. The previous studies showed listerine has a burning sensation in the mouth (20).

CONCLUSION:

Zerumbone exhibited antibiofilm effect on S. sanguinis and L. acidophilus and had a great potential to be developed as anti caries. Further research on the compound effect on a polyculture biofilms is worth exploring

ACKNOWLEDGMENT:

Authors extended their gratitude to The Ministry of Research, Technology and Higher Education (Indonesia) for the funding through the PMDSU (Program Magister Menuju Doktor Untuk Sarjana Unggul) research project No. 2940/UN1.DITLIT/DIT-LIT/LT/2019.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

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Received on 04.09.2019 Modified on 07.11.2019

Research J. Pharm. and Tech. 2020; 13(8):3559-3564.

DOI: 10.5958/0974-360X.2020.00629.0