Oral diseases are significant challenges for healthiness all over the world. Carbohydrate oral diseases fermentation of foods by oral bacteria reduces the pH of plaque and demineralizes enamel that results from the formation of dental caries [1-3]. Dental caries and periodontal diseases are the most common challenges for oral healthiness, following oral and pharyngeal cancers and oral tissue lesions . Oral healthiness is an integral status for general well-being related to the quality of life and increases beyond the functions of the craniofacial complex . The association between and the activities of microbial species present in the oral cavity has been reported . Bacteria species inhabit the oral cavity, and some cause oral diseases . The increase in dental caries increases acidogenic and aciduric Gram-positive bacteria, such as Streptococcus mutans and S. sobrinus, lactobacilli, and actinomycetes that metabolize sucrose to organic acids (mainly lactic acid) that dissolve the calcium phosphate in teeth and cause decalcification and eventual decay . Streptococcus mutans is the principal cariogenic bacterium in humans [7, 8]. It was reported that S. mutans bacteria are colonized in environments rich in sucrose and glucan-mediated on tooth surfaces . The sticky polymer produced by sucrose named glucan produced of S. mutans is a starting factor for dental caries formation due to trapping oral bacteria, food debris, and salivary components . Researchers seek a safe antibacterial compound with anti-cariogenic properties that have minimum side effects. Chemotherapeutic structures prepared from natural products have significant importance for producing novel drugs . Some studies have reported using medicinal plants as a source of chemotherapeutic agents to inhibit oral diseases [12, 13]. Curcumin is a natural component of the rhizome Curcuma longa, also known as turmeric, and it is used for medicinal purposes, mainly to treat inflammatory conditions . Curcumin shows antibacterial activity by disordering the 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) membranes in the bacteria . Studies have reported the antibacterial activity of curcumin against Gram-positive (S. aureus and Enterococcus faecalis) and Gram-negative (Escherichia coli and Pseudomonas aeruginosa) .
Since curcumin is an antibacterial compound, but it has not been used against S. mutans, biofilm formation, and dental caries development. The present study was conducted to evaluate the antibacterial effects of administration of curcumin against S. mutans, biofilm formation, and dental caries development.
Materials and Methods
Curcumin (EC No. 2072805) was prepared from Sigma-Aldrich Chemical Company (St. Louis, MO). Streptococcus mutans (PTCC 1683) was prepared from the Iranian Biological Resources Center (Tehran, Iran). Approval for this study was obtained from International Center for Intelligent Research (ICIR-2020-184163).
The investigation antibacterial activity by minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC)
Antibacterial activity of curcumin MIC and MBC against S. mutans was investigated by microdilution procedure as reported by previous studies . The MIC was considered the lowest concentration that prevents visible bacterial growth and MBC as the highest dilution that produced no bacterial growth on solid medium. All the experiments were replicated in triplicate.
Biofilm formation assay
Biofilm formation for S. mutans was conducted as reported by others  by using 105-106 CFU mL−1 S. mutans, 150 μL of BHI with 5% (w/v) sucrose at different times.
Dental caries Development
The effects of curcumin on oral colonization and cariogenic potential of S. mutans were investigated in 16 rats and two experimental groups (8 rats per group), as grouped as control and curcumin. The animals were fed with erythromycin water (100 μg mL-1) and a regular diet for 7 days to minimize the microbial load. The bacteria were colonized by oral swab, and animals received a 5% sucrose diet in all the trials. To colonize, the rats were inoculated with 1.4 × 1010 CFU of a streptomycin-resistant strain of S. mutans (MT8148R), in molars surfaces once for 7 days. The rats were received curcumin twice/a day (20 mg/kg body weight daily) by topical administration for 28 days. The samples were collected 4 weeks next and scored based on previous studies .
The data were analyzed by SPSS software (version of 23). The data for In vitro sections were analyzed by ANOVA test. The data for In vivo section were analyzed by T-test. Since data were normal, Pearson correlation was also used.
Results and Discussion
The results showed that using curcumin in the concentration of 128 µg/mL could inhibit the growth of S. mutans, while in the concentration of 256 µg/mL could kill S. mutans. The results for killing S. mutans are presented in Figure 1.
Figure 1: Kinetic of killing curve during 24 h incubation. Higher time and concentrations resulted in killing more bacteria
The results for biofilm production are depicted in Figure 2. The results revealed that the increase in concentration and time decreased biofilm formation. The results approve that curcumin decreases biofilm formation in higher concentrations, but it needs more time. The results showed a positive correlation (r2=0.985, P=0.001) between biofilm formation and antibacterial activity. The decrease in bacterial count results in a decrease in biofilm formation.
Figure 2: The results for biofilm production. Higher time and concentrations resulted in decreased biofilm formation
Dental caries development
The results for dental caries development are illustrated in Figure 3. The results demonstrated that administration of curcumin reduced caries scores in smooth surface caries (Figure 3-A) and sulcal surface caries (Figure 3-B) compared to the control group (P<0.05).
Figure 3: The effects of curcumin on dental caries in rats. The administration of curcumin reduced dental caries development in smooth and sulcal
This study was conducted to evaluate the effects of administration of curcumin against S. mutans, biofilm formation, and dental caries development. The results showed that administration of curcumin showed antibacterial activity and decreased biofilm formation and dental caries development. The results revealed that administration of curcumin in more time and higher concentrations decreased a significant number of bacteria. It means that curcumin needs more time for killing S. mutans. Studies have reported the antibacterial activity of curcumin against Gram-positive (S. aureus and E. faecalis) and Gram-negative (E. coli and P. aeruginosa) . Curcumin shows antibacterial activity by disordering DPPC membranes in the bacteria . Another study showed that curcumin changes bacteria membrane by thinning and disrupting the membrane . Therefore, curcumin shows antibacterial activity by influencing on bacteria membrane. It needs more time for killing and inhibiting bacteria. Seemingly, curcumin slowly penetrates in bacteria membrane and destroys bacteria. Parallel with this claim, and it was reported that natural compounds slowly change the permeability of the cell membrane and penetrate bacteria [19,20]. It shows the highest effect in the highest concentration. It could be argued that curcumin may be degraded while affecting the bacteria and thus higher concentration of curcumin provides enough curcumin for affecting the bacteria. It was observed a positive correlation between antibacterial activity and biofilm formation. Seemingly, curcumin shows antibacterial activity and decreases S. mutans, resulting in decreased biofilm formation. It is well known that glucans mediate biofilm formation. It was reported that anti-biofilm agents decrease biofilm formation by damaging physical integrity and stability and reducing the availability of binding sites for S. mutans . Curcumin may decrease biofilm formation, reducing the availability of binding sites for S. mutans, because it showed antibacterial activity.
Curcumin administration reduced caries scores in smooth surface caries and sulcal surface caries. The results approve the topical administration of curcumin reduces caries development. The efficiency of curcumin on caries scores might be attributed to its effects on the pathogenicity of S. mutans. The S. mutans damages teeth by enzyme GTF activity. Studies have reported that S. mutans have adverse effects on teeth by increasing GTF activity and bacterial glycolytic . In sum, the results approve that the administration of curcumin decreases S. mutans population resulting in caries scores.
In conclusion, curcumin showed antibacterial activity and anti-biofilm formation. It also decreased reduced caries scores. All the findings approve of curcumin as a safe agent that could be used as a compound in toothpaste structure. Curcumin can be considered an appropriate compound for antibacterial activity against S. mutans. We suggest using curcumin for tooth care.
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
All authors made substantial contributions to conception and design, acquisition of data, or analysis and interpretation of data; took part in drafting the article or revising it critically for important intellectual content; gave final approval of the version to be published; and agree to be accountable for all aspects of the work.
Conflict of Interest
The authors declared no conflicts of interest in the present study.
Elham Sadat Afraz:
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Ilia Asadi, Mojtaba Pakravan, Khashayar Bahramsari, Mahsa Rekabi, Nasrollah Moradi-kor, Elham Sadat Afraz Inhibitory Effects of Administration of Curcumin Against on Streptococcus Mutans: In vitro and In vivo Studies, J. Med. Chem. Sci., 2022, 5(4) 436-441