Cariostatic Efficacy of Cinnamon Water Extract on Streptococcus mutans: An In vitro Study.

BACKGROUND: Dental caries is a chronic, widespread, and infectious disease affecting the large population. Anticariogenic effects of different herbs and natural products are observed, cinnamon among these, showed the ability to inhibit Streptococcus mutans biofilm.
OBJECTIVES: The aim of this study was to assess the cinnamon water extract use on nicotine exposed S. mutans biolfilms.
MATERIALS AND METHODS: Microtiter plates having S. mutans culture were treated using varying nicotine concentration range (0-32 mg/ml). These were then either subjected to cinnamon water (2.5 mg/ml) extract or no treatment and analyzed using a spectrophotometer to see planktonic growth, total growth absorbance, and biofilm formation.
RESULTS: About 2.5 mg/ml of cinnamon water inhibits the S. mutans biofilm (nicotine-induced) considerably at various nicotine levels (0-32 mg/ml).
CONCLUSION: Cinnamon water extract has a powerful inhibitory effect for S. mutans growth. Nicotine-induced S. mutans growth is markedly reduced by treating with cinnamon water extract. Copyright:

INTRODUCTION

Dental caries is one of the most common chronic infectious diseases caused by bacterial colonization. Caries begin with the decalcification of nonorganic part followed by the organic matrix.[1] The polysaccharide-rich biofilm formation and low pH (acidification) are major factors that control caries pathogenesis.[2] Etiology of caries is related to sugar intake, diet, oral bacteria, oral hygiene practices, and protective procedures alterations. Dental caries prevalence is approximately 45.8%, according to NHANES.[3] There are various therapeutics for prevention and treating dental caries. Among those, fluoride constitutes the mainstay for caries prevention. However, additional approaches are required to improve treatment effectiveness. Despite advances in treatment measures, affected individuals experience tooth loss.[1]

Various antiplaque approaches for caries treatment are based on using broad-spectrum microbicidal such as chlorhexidine.[2] Along with mechanical plaque control agents, mouth rinses helps in reducing bacterial count including Streptococcus mutans and hence dental caries.[4]

Natural products contain substances having different biological activities such as anticaries. However, it is quite challenging because of difficult isolation of natural products.[2] Among natural anticaries agents, rosemary (Rosmarinus officinalis), and ginger rhizome (Zingiber officinale) showed antimicrobial activities and no toxicity by USFDA.[56] No study to date has reported caries remineralization using natural agents. Most studies focused on the effect of herbals on bacterial growth and not on biofilms. Thus, understanding the mechanism of natural herbs as anticaries agents remain unknown. Still many natural substances have promising anticaries activity.[2]

Spices and herbs are widely used as an anticaries agent. Cinnamon is obtained from Cinnamomum species, has antimicrobial activity against pathogens such as Streptococcus faecalis, Enterococcus faecalis, S. aureus, and Enterobacter cloacae. These are the main factors responsible for pulpal and periapical diseases.[789] Essential oil, from cinnamomum zeylanicum leaves, is effective against Lactobacillus acidophilus and S. mutans causing caries and plaque.[1011]

The aim of this study was to assess the cinnamon water extract use on nicotine exposed biofilms of S. mutans. S. mutans was considered as it is the most common microorganism associated with dental caries.[12]

MATERIALS AND METHODS

A pilot experiment was conducted before main study to obtain an minimum biofilm inhibitory concentration (MBIC), and minimum inhibitory concentration (MIC) of the cinnamon water on S. mutans growth. Growth inhibition was assessed on TSBS. MBIC and MIC were established as 5 mg/ml in other study.[13] In this pilot study, the MIC was determined as 2.5 mg/ml.

To evaluate cinnamon water effect on S. mutans treated with nicotine, TSBS serial dilutions were made to achieve 0, 0.25, 0.5, 1, 2, 4, 8, 16, and 32 mg/ml nicotine without cinnamon water extract. The same series dilutions were prepared with cinnamon water extract MIC dilution (2.5 mg/ml as evaluated by the pilot experiment). All 96 wells were aliquoted with 190 μl TSBS followed by incubation in 5% CO2 at 37°C for 24 h. After 24 h, planktonic growth, and biofilm was measured using a spectrophotometer at 490 nm. S. mutans biofilms without nicotine and with/without cinnamon water extract (2.5 mg/ml) served as controls.

Collected data were statistically analyzed to compare the effect of cinnamon water on all nicotine concentrations of 0–32 mg/ml. Furthermore, their interactions of total, planktonic, and biofilm, growth were compared.

RESULTS

Cinnamon water extract, nicotine, and interaction in biofilm, total growth, and planktonic showed a significant effect and caused inhibition on 0–1 and 8 mg/ml nicotine concentration. Biofilm growth increase (significant) was noticed at nicotine concentration of 2 and 4 mg/ml.

A significant decrease at 8, 16, and 32 mg/ml nicotine was seen with cinnamon water. The modest decrease at 0.25 mg/ml nicotine, in biofilm growth, and increase (non-significant) at 0.5 mg/ml was seen. Concerning total S. mutans growth, increase at 0.5, 1, 2, and 4 mg/ml of nicotine were significant with cinnamon water. Conversely, at 8, 16, and 32 mg/ml of nicotine concentration, cinnamon extract liquid showed significant growth reduction compared to the zero-nicotine concentration. A very little total growth increase at a nicotine concentration of 0.25 mg/ml was also observed additionally.

Concerning biofilm, a decrease at nicotine at 16 and 32 mg/ml without cinnamon water compared to zero-nicotine concentration without cinnamon liquid was noticed. Furthermore, a nonsignificant biofilm increase at 4 and 8 mg/ml and slight decrease at 1 and 2 mg/ml was seen. At 0.25 and 0.5 mg/ml, no statistically significant growth difference was reported.

These results are depicted in Tables 1–3.

Table 1

Streptococcus mutans biofilm, with cinnamon water at various nicotine concentrations

Groups	Nicotine concentration	n	Mean	SD	Maximum	Minimum
With 2.5 mg/ml cinnamon water extract	0	12	0.22	0.072	0.128	0.386
	0.25	12	0.221	0.076	0.108	0.3
	0.5	12	0.244	0.03	0.180	0.289
	1	12	0.486	0.243	0.12	0.9
	2	12	0.988	0.111	0.586	1.123
	4	12	1.024	0.176	0.788	1.462
	8	12	0.048	0.003	0.038	0.076
	16	12	0.056	0.02	0.052	0.086
	32	12	0.054	0.028	0.038	0.088
Without 2.5 mg/ml cinnamon water extract	0	12	0.731	0.092	0.48	0.924
	0.25	12	0.724	0.112	0.566	0.886
	0.5	12	0.717	0.074	0.498	0.798
	1	12	0.722	0.164	0.386	0.924
	2	12	0.606	0.12	0.488	0.988
	4	12	0.887	0.152	0.653	1.047
	8	12	0.892	0.221	0.566	1.275
	16	12	0.066	0.002	0.037	0.086
	32	12	0.051	0.004	0.048	0.058

SD: Standard deviation

Table 3

Streptococcus mutans (total), with cinnamon water at various nicotine concentrations

Groups	Nicotine concentration	n	Mean	SD	Maximum	Minimum
With 2.5 mg/ml cinnamon water extract	0	12	0.482	0.028	0.442	0.532
	0.25	12	0.498	0.027	0.501	0.612
	0.5	12	0.555	0.032	0.48	0.620
	1	12	0.62	0.06	0.498	0.594
	2	12	0.612	0.042	0.568	0.696
	4	12	0.710	0.069	0.639	0.855
	8	12	0.248	0.007	0.30	0.302
	16	12	0.22	0.019	0.254	0.315
	32	12	0.156	0.010	0.236	0.288
Without 2.5 mg/ml cinnamon water extract	0	12	0.616	0.030	0.534	0.675
	0.25	12	0.602	0.038	0.502	0.69
	0.5	12	0.63	0.053	0.532	0.702
	1	12	0.586	0.098	0.402	0.6648
	2	12	0.622	0.052	0.522	0.699
	4	12	0.734	0.062	0.633	0.875
	8	12	0.902	0.036	0.768	0.93
	16	12	0.303	0.096	0.137	0.403
	32	12	0.302	0.061	0.98	0.306

SD: Standard deviation

DISCUSSION

The pilot experiment before study was carried out to determine the MBIC, MIC, and antimicrobial activity of cinnamon water extract. Aldehyde in cinnamon is responsible for biological activities and fragrance.[1415] Antimicrobial activity against S. mutans was established in the pilot experiment conducted. As S. mutans inhabit normal oral flora, establishing MIC and MBIC was important to not affect normal oral environment. 16 h S. mutans culture was kept in 96 wells flourished with TSBS and nicotine was added in control and experimental groups to enhance the biofilm growth.[6] Results showed that cinnamon water extracts significantly reduce S. mutans biofilm growth both at low (0.25 mg/ml, 0.5 mg/ml, and 1 mg/ml) and high nicotine concentration (8 mg/ml, 16 mg/ml, and 32 mg/ml). Reduction in colony counts was significant in ethanolic liquorice group establishing its cariostatic potential.[16]

Reduction of S. mutans growth was seen in planktonic and biofilm phase. However, more restriction was seen concerning biofilm. In nicotine group with no cinnamon water extract addition, biofilm formation increase was proportional to increase in nicotine concentration. At 16 and 32 mg/ml of nicotine concentration, significant inhibition of biofilm growth and microbial viability was observed. Similar results were interpreted by study of Huang et al. in 2012, where different S. mutans strains were treated at various nicotine concentrations.[17] Authors concluded that the anticariogenic effect of fluoride-chlorhexidine was highest among the groups. Green tea showed higher cariostatic effects than normal saline.

With cinnamon water extract of nicotine at 2 mg/ml and 4 mg/ml leads to significant bacterial growth increase. These can be attributed to the inhibitory effect of cinnamon water at these concentrations of nicotine and collaborative consequence of nicotine with cinnamon water extract at specific nicotine levels. One study compared caries inhibiting the effect of cinnamon oil and clove oil. Another study in 2011 assessed the efficacy of oral microbiota causing dental caries. This is contrary to popular belief that clove oil is effective in tooth decay and dental plaque. This study justified the present study.[18]

A study by Li et al.[19] evaluated effectiveness of ginger and rosemary, on initial enamel caries remineralization and significant differences between treatments, were observed in terms of microhardness. Compared with the positive control group (NaF dentifrice), significantly greater remineralization was observed with ginger-honey-chocolate treatments. Authors concluded that enhanced remineralization was observed with all treatment systems, with ginger-honey-chocolate being most effective, and this study was consistent with the present study.

CONCLUSION

The cinnamon water addition to products for maintaining oral hygiene could be advantageous for smokers. As no study to date studied nicotine amount in smoker's biofilm, hence, more in-vivo studies to determine this are needed. A test to determine the cytotoxicity of cinnamon water extracts to be determined for safe use. The limitations include only single S. mutans strain consideration. No positive control such as chlorhexidine was used. Furthermore, salivary samples were not considered. In addition, single cinnamon species was used.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

Table 2

Streptococcus mutans planktons, with cinnamon water at various nicotine concentrations

Groups	Nicotine concentration	n	Mean	SD	Maximum	Minimum
With 2.5 mg/ml cinnamon water extract	0	12	0.048	0.007	0.042	0.080
	0.25	12	0.054	0.004	0.036	0.072
	0.5	12	0.052	0.002	0.046	0.067
	1	12	0.052	0.008	0.047	0.069
	2	12	0.08	0.004	0.039	0.069
	4	12	0.073	0.007	0.050	0.089
	8	12	0.080	0.013	0.03	0.097
	16	12	0.090	0.020	0.047	0.121
	32	12	0070	0.017	0.060	0.122
Without 2.5 mg/ml cinnamon water extract	0	12	0.117	0.027	0.090	0.182
	0.25	12	0.90	0.017	0.060	0.120
	0.5	12	0.088	0.022	0.060	0.143
	1	12	0.087	0.04	0.08	0.121
	2	12	0.093	0.035	0.072	0.208
	4	12	0.093	0.037	0.059	0.187
	8	12	0.093	0.024	0.056	0.138
	16	12	0.109	0.057	0.047	0.230
	32	12	0.095	0.03	0.039	0.078

SD: Standard deviation