ORIGINAL RESEARCH

https://doi.org/10.5005/jp-journals-10015-2247
World Journal of Dentistry
Volume 14 | Issue 6 | Year 2023

Effects of Curcumin, Curcumin–Rosemary, and Chlorhexidine Mouthwashes on Streptococcus mutans in Patients Undergoing Orthodontic Treatment: An In Vivo Study

Sujitha Sridharan¹, Poornima R Jnaneshwar², Ravi Kannan³

^1-3Department of Orthodontics and Dentofacial Orthopedics, SRM Dental College, Chennai, Tamil Nadu, India

Corresponding Author: Poornima R Jnaneshwar, Department of Orthodontics and Dentofacial Orthopedics, SRM Dental College, Chennai, Tamil Nadu, India, Phone: +91 9500044266, e-mail: poorni01@gmail.com

Received on: 02 May 2023; Accepted on: 05 June 2023; Published on: 22 August 2023

ABSTRACT

Aim: The aim of the study was to evaluate the effects of curcumin mouthwash and curcumin–rosemary mouthwash against Streptococcus mutans (S. mutans) colonies in patients undergoing orthodontic treatment.

Materials and methods: Around 48 subjects were randomly allocated to three groups, group I (chlorhexidine mouthwash), group II (curcumin mouthwash), and group III (curcumin–rosemary mouthwash). Plaque samples were collected at three stages, T₁—before bonding, T₂—1 month after bonding orthodontic attachments, and T3—after the use of the prescribed mouthwash for a period of 1 month. The collected samples were cultured and the number of S. mutans colonies were counted. The quality of the mouthwash was assessed using a questionnaire. Since the data were normally distributed, paired t-test and one-way analysis of variance (ANOVA) were used to evaluate the difference within and among the groups, respectively. Categorical data were analyzed using the Chi-square test.

Results: The results obtained showed a significant reduction in the S. mutans colonies at T3 after the use of mouthwash for 1 month in all three groups. Patients in groups II and III had temporary discoloration of the tongue, whereas patients in group I had a burning sensation in the oral mucosa on prolonged use.

Conclusion: All three types of mouthwashes were effective in the reduction of S. mutans colonies in orthodontic patients.

Clinical significance: The new herbal mouthwash are cost-effective and had negligible side effects when compared to chlorhexidine. Therefore, they can be used as mouthwashes for all patients regardless of socioeconomic background.

How to cite this article: Sridharan S, Jnaneshwar PR, Kannan R. Effects of Curcumin, Curcumin–rosemary, and Chlorhexidine Mouthwashes on Streptococcus mutans in Patients Undergoing Orthodontic Treatment: An In Vivo Study. World J Dent 2023;14(6):541-546.

Source of support: Nil

Conflict of interest: None

Keywords: Curcumin, Chlorhexidine, Mouthwash, Rosemary, Streptococcus mutans.

INTRODUCTION

Oral health is essential for general well-being which further influences the quality of life to a greater extent. The key factor responsible for the development of periodontitis, dental caries, and other oral infections are microorganisms, which colonize the surface of the tooth and are part of dental plaque along with food debris. Poor oral hygiene not only causes damage to the periodontium, teeth, and other oral structures, it also increases the risk of heart disease, stroke, and adds to diabetic complications.¹^-³

Maintaining optimal periodontal health is of the utmost importance throughout orthodontic treatment as orthodontic brackets and the bonding resins have a greater tendency to accumulate plaque due to their irregular surface. Klukowska et al. suggested that plaque accumulation was 2–3 times more in patients undergoing orthodontic treatment when compared to nonorthodontic patients.⁴

Plaque accumulation in the resin-enamel junction may cause enamel demineralization leading to the development of white spot lesions. Tufekci et al. proved that acidogenic bacteria like Streptococcus mutans (S. mutans) and Lactobacilli increase drastically in the dental plaque around the orthodontic brackets. These bacteria cause the demineralization of enamel in that region leading to an increase in the incidence of white spot lesions.⁵ Tanner et al. proved that S. mutans have a strong association with white spot lesions.⁶ It is necessary to control the growth of these bacteria at the early stages of orthodontic treatment, which can be achieved by the inclusion of mouthwashes in routine oral hygiene practice along with the other mechanical plaque removal techniques.

The choice of chemical mouthwashes available in the market is numerous; the prominent being chlorhexidine, which has been tested extensively and proven to be very effective in reducing plaque accumulation and limiting the growth of harmful bacteria. Brightman et al. and Anderson et al. showed that the use of 0.12% chlorhexidine mouthwash and routine mechanical plaque removal techniques are effective in controlling plaque accumulation and gingivitis in orthodontic patients.⁷^,⁸ However, chlorhexidine has a few limitations like staining of the teeth and tongue, mucosal irritation, and bad taste.⁹^,¹⁰

Recent researchers have focused on finding an effective alternative to chlorhexidine in the form of herbal agents incorporated in dentifrices, toothpaste, and mouthwashes.¹¹^-¹³

One of the herbal agents that has a wide range of favorable antimicrobial properties and easily available is curcumin. Negi et al. proved that turmeric oil has antimicrobial properties.¹⁴ They are mainly because of the constituents of curcuma longa which are phenolic compounds, essential oils, steroids, and alkaloids.¹⁵ Medical uses of curcumin are highly extensive, they are used as antibacterial, antiviral, and antifungal agents.¹⁵ They are used in certain immunological conditions, treatment of arthritis, and renal dysfunctions. They are used as antihemorrhagic agents, stabilizing blood pressure, and possess neuroprotective, and antioxidant properties. It is commonly used as an antibacterial agent in wound healing.¹⁵ Antibacterial effects of curcumin on S. mutans colonies are by three predominant mechanisms, they are bacterial lysis, inhibition of bacterial activity, and inhibition of production of extracellular polysaccharide and interference with bacterial adhesion.¹⁶

Oliveira et al. proved that R. officinalis L. (Lamiaceae) has both antimicrobial and antiinflammatory properties and hence can be used to reduce oral infections caused by oral microorganisms.¹⁷ Antibacterial effects of rosemary may be due to its constituents like 1,8-cineole, camphor, and α-pinene.¹⁷ Extracts of rosemary have been found to be antibacterial to microbes present in bacterial plaque, possess cytotoxic and genotoxic properties against the microbes, and possess antiinflammatory activity.¹⁸ Antibacterial effects of rosemary are mainly because of the inhibition of bacterial adhesion to the host cell surface or enamel surface.¹⁸

The efficacy of the two herbal agents, curcumin and rosemary has been evaluated individually. It was proven that both agents were effective in reducing dental plaque and microbial colonies.¹⁹^-²² Levine et al. studied the synergistic effects of curcuminoids and rosemary leaf extract on the neoplastic cell lines of canines and concluded that they possessed cytotoxic properties and reduced the proliferation of various types of neoplastic cell effects.²³ However, the cumulative antimicrobial effects of these two herbal agents as mouthwash have not been tested before. In addition, all the previous studies focus on the effects of these mouthwashes on the oral health of nonorthodontic patients.

Hence the primary purpose of this study was to test the antimicrobial efficacy of two types of mouthwash against S. mutans bacterial colonies and compare the same with chlorhexidine mouthwash which has been extensively tested and proven to be effective against S. mutans. The mouthwashes tested were curcumin mouthwash and the novel curcumin–rosemary mouthwash. Waghmare et al. and Mali et al. evaluated the effects of curcumin mouthwash in nonorthodontic patients but its effect on S. mutans colonies was not evaluated.¹⁹^,²⁰ The second aim of the study was to assess patient’s perception of the quality of the mouthwashes used in this study by using a questionnaire.

MATERIALS AND METHODS

This prospective study was approved by the Institutional Review Board and Ethical Committee (SRMDC/IRB/2017/MDS/NO.109), started in the month of June 2018, and was conducted for a period of 6 months. The total sample size calculated (power of 95%) was 48. Subjects who were older than 18 years, were to undergo fixed appliance mechanotherapy with nonextraction protocol were selected based on the following inclusion criteria; a full complement of teeth in permanent dentition (not including the third molars), no carious lesions, restorations, periodontal or systemic diseases. The degree of malocclusion was not considered for the study. Subjects who were active smokers and on antibiotic or probiotic therapy were excluded from the study. Around 48 subjects were randomly allocated by block randomization with a block size of four, to either of the three mouthwash groups—group I (chlorhexidine mouthwash), group II (curcumin mouthwash), and group III (curcumin–rosemary mouthwash) as 16 per group. All the subjects were blinded by giving the mouthwash in opaque containers without labeling.

Composition of the Mouthwash

Group I, 16 subjects received chlorhexidine mouthwash—chlorhexidine gluconate diluted to 0.2% w/v in flavored aqua base.
Group II, 16 subjects received curcumin mouthwash in the following composition—10 mL of the mouthwash contained 0.1% curcumin, 0.001% flavoring agent, and the rest comprised distilled water.²⁴
Group III, 16 subjects received curcumin–rosemary mouthwash and it was prepared to the following dilution—10 mL of the mouthwash contained 0.1% curcumin, 0.1% rosemary, 0.001% flavoring agent, and the rest comprised distilled water.

Informed consent was obtained from all the patients after explaining the purpose of the study and the use of mouthwash. Oral prophylaxis was done and appropriate oral hygiene instructions were given to all the subjects prior to the commencement of orthodontic treatment.

An orthodontic toothbrush and toothpaste were provided to every patient and they were advised to brush their teeth twice daily for a minimum of 3 minutes. Patients were also advised not to undergo any oral prophylactic procedure on the day of sample collection.

The first set (T1) of plaque samples was obtained before the commencement of orthodontic treatment. Patients underwent fixed mechanotherapy with orthodontic metal brackets of McLaughlin Bennett Trevisi prescription (0.022” slot size). The second (T2) plaque sample was collected 1 month after bonding from the buccal surfaces of the maxillary right central incisor, maxillary left first premolar, mandibular left central incisor, and mandibular right first premolar. After collection of the plaque samples (T1 and T2), patients in group I were given chlorhexidine mouthwash and were advised to use 5 mL of the mouthwash diluted with an equal amount of water twice a day for 1 month while patients in group II (curcumin mouthwash) and patients in group III (curcumin–rosemary mouthwash) were advised to use 10 mL of undiluted mouthwash twice a day for 1 month (Flowchart 1). All the patients were advised to rinse their mouths with the respective mouthwash for 60 seconds.²⁰ They were also provided with a chart and were instructed to make an entry after using the mouthwash and were requested to bring the same to ensure patient compliance during subsequent visits. A reminder was sent to all the patients through a text message every day for a period of 1 month.

Flowchart 1: Flowchart depicting the methodology

The third (T3) set of plaque samples was collected from the same tooth surface after 1 month of use of the prescribed mouthwash.

All the samples were collected early in the morning, placed in an appropriate transport medium, and transported immediately to the laboratory. In the laboratory the samples were mixed in a vortex and after agitation about 10 μL of the solution was inoculated in Mutans-Sanguis Agar plates using a cotton swab and incubated at 37° C for 48 hours. The number of S. mutans colonies was measured using a colony counter. The results were expressed as a number of S. mutans colony-forming units per mL (CFU/mL).

The feedback for the mouthwashes was obtained with the help of a questionnaire. The participants were requested to fill out the questionnaire and submit it at the end of the study. This helped in assessing the patient’s acceptance and perception of the prescribed mouthwashes. The questionnaire used in this study had six questions related to the mouthwashes with answers for most of them in yes or no format. The questionnaire used in this study was validated after conducting a pilot study.

Statistical Analysis

Data were analyzed with the nMaster software 2.0 version. The normality of data was studied using the Kolmogorov–Smirnoff test. In order to find the difference in the S. mutans colonies before and after bonding, paired t-test was used and one-way analysis of variance (ANOVA) was used to find the difference in the S. mutans colonies between the groups after the use of the mouthwash. The probability value of 0.05 was considered significant. Categorical data obtained from the questionnaire were analyzed using the Chi-square test.

RESULTS

The mean reduction in S. mutans colonies after the use of mouthwashes for 1 month (T2–T3) was highest for group I followed by groups II and III. Nevertheless, this mean reduction was significant for all the groups (Table 1). The mean differences at T1 and T2 were 2.53 × 10⁴ CFU/mL for group I, 1.69 × 10⁴ CFU/mL for group II, and 1.47 × 10 CFU/mL for group III but there was no significant increase in colony count between T1 and T2 except for group III (Table 1). When the reduction of the S. mutans colony was analyzed before and after the use of mouthwashes (T2-T3), it was highly significant for group I (p = 0.0005), group II (p = 0.0005) and group III (p = 0.005) (Table 1).

**Table 1:** Comparison of the difference in number of *S.mutans* colonies before bonding (T1), 1 month after bonding of the orthodontic attachment (T2), and after the use of mouthwash (T3) in three groups evaluated using paired t-test (×10⁴ CFU/mL)
Groups	Stages	Paired differences					T	Degree of freedom	Significance (two-tailed)
		Mean × 10⁴ CFU/mL	Standard deviation	Standard error mean	95% confidence interval of the difference
		Mean × 10⁴ CFU/mL	Standard deviation	Standard error mean	Lower	Upper
Group I n = 16	T1–T2	−0.87500	1.31022	0.32755	−1.57316	−0.17684	−2.671	15	0.017
Group I n = 16	T2–T3	2.53125	1.64792	0.41198	1.65314	3.40936	6.144	15	0.0005
Group II n = 16	T1–T2	−0.32500	1.69017	0.42254	−1.22563	0.57563	−0.769	15	0.454
Group II n = 16	T2–T3	1.68750	1.13835	0.28459	1.08092	2.29408	5.930	15	0.0005
Group III n = 16	T1–T2	−2.15625	1.85938	0.46484	−3.14704	−1.16546	−4.639	15	0.0005
Group III n = 16	T2–T3	1.46875	1.79322	0.44830	0.51321	2.42429	3.276	15	0.005

One-way ANOVA done to evaluate the change in S. mutans colonies before and after the use of the mouthwash (T2-T3) between the three groups suggested that there was no statistically significant difference (p = 0.135) (Table 2).

**Table 2:** One-way ANOVA to compare the *S.mutans* colonies between the three groups at T1, T2, and T3 (× 10⁴ CFU/mL)
	Sum of squares	Degree of freedom	Mean square	F	Significance
T1	49.333	2	24.666	4.022	0.025
T2	3.198	2	1.599	0.557	0.577
T3	5.375	2	2.688	0.957	0.392
T1–T2	28.254	2	14.127	5.277	0.009
T2–T3	10.073	2	5.036	2.091	0.135

The results obtained from the questionnaire suggested that 43.8% of subjects in group II and 18.8% of subjects in group III had discoloration of the tongue, 50% of subjects in group I perceived burning sensation, while 18.3% experienced allergic reactions, and 25% of the subjects had dryness of the mouth (Tables 3 and 4). There was no significant difference in the rating or acceptance of the mouthwashes (Table 5).

**Table 3:** Burning sensation-percentage and Chi-square analysis
				Groups n = 48					Total
				Group I		Group II	Group III		Total
Burning sensation	No	Count		8		16	16		40
	No	%		50.0%		100.0%	100.0%		83.3%
	Yes	Count		8		0	0		8
	Yes	%		50.0%		0.0%	0.0%		16.7%
			Value		Degree of freedom			Asymptotic significance (two-sided)
Pearson Chi-square			19.200		2			0.0005
Likelihood ratio			21.073		2			0.000

**Table 4:** Allergic reaction-percentage and Chi-square test for assessment of the mouthwash ratings between the three groups
				Groups n = 48					Total
				Group I	Group II		Group III		Total
Allergic reaction	No	Count		13	16		16		45
	No	%		81.3%	100.0%		100.0%		93.8%
	Yes	Count		3	0		0		3
	Yes	%		18.8%	0.0%		0.0%		6.3%
			Value			Degree of freedom		Asymptotic significance (two-sided)
Pearson Chi-square			6.400			2		0.041
Likelihood ratio			7.002			2		0.030

**Table 5:** Chi-square test for assessment of the mouthwash ratings between the three groups
				Groups n = 48					Total
				Group I		Group II	Group III		Total
Rating the mouthwash	2.0	Count		2		1	1		4
	2.0	%		12.5%		6.3%	6.3%		8.3%
	3.0	Count		5		3	2		10
	3.0	%		31.3%		18.8%	12.5%		20.8%
	4.0	Count		4		4	5		13
	4.0	%		25.0%		25.0%	31.3%		27.1%
	5.0	Count		5		8	8		21
	5.0	%		31.3%		50.0%	50.0%		43.8%
			Value		Degree of freedom			Asymptotic significance (two-sided)
Pearson Chi-square			2.911		6			0.820
Likelihood ratio			2.909		6			0.820
Linear-by-linear association			2.008		1			0.156

DISCUSSION

The present study was designed to evaluate the combined effects of a mouthwash containing both curcumin and rosemary and it was compared with curcumin and chlorhexidine. Orthodontic fixed mechanotherapy comprises brackets bonded to the patient’s enamel, archwires engaged to brackets, and elastic modules holding the archwire. There are innumerable sites for plaque accumulation with a propensity to increase the bacterial count. Waghmare et al. compared the efficacy of curcumin with chlorhexidine and proved that the herbal mouthwash was as effective in controlling plaque. Chatterjee et al. and Bomdyal et al. evaluated the effects of curcumin extract on periodontal pathogens and proved that curcumin extract had antibacterial activity against these organisms.²¹^,²² Paula et al. evaluated the antimicrobial property of rosemary extract against S. mutans and Candida albicans (C. albicans) and concluded that rosemary extracts showed a significant reduction in S. mutans colonies which correspond to the results demonstrated in the present study.²⁵ Levine et al. studied the combined effects of curcumin and rosemary extract in neoplastic cell lines of canines and concluded that there was an increase in susceptibility of cancer cells due to a higher accumulation of curcumin when a combination treatment was given. In addition, they concluded that there was sustained activation and signaling of pathways that were critical to promote apoptosis of neoplastic cells.²⁶ Numerous types of research have been conducted to analyze the effects of curcumin and rosemary extracts in mouthwashes individually but not together. Hence, studying their synergistic effects on the plaque microflora was imperative.

A comparison of the number of S. mutans colonies before and after the use of the prescribed mouthwash (T2-T3) suggested a highly significant reduction in the S. mutans colonies in all three groups (Table 1). This proved that chlorhexidine, curcumin, and curcumin–rosemary mouthwashes were highly effective in reducing the number of S. mutans colonies in orthodontic patients. These observations correspond with the results of Mali et al. who proved that curcumin mouthwash was as effective as chlorhexidine mouthwash in reducing periodontal pathogens.²⁰ Comparison of the number of S. mutans colonies between the three groups using one-way ANOVA suggested that there was no statistically significant difference in the reduction of colony count between the three groups after the use of the prescribed mouthwashes (Table 2). Waghmare et al. compared the effects of chlorhexidine and curcumin and concluded that chlorhexidine had better antiplaque properties than curcumin but both had inhibitory effects on plaque formation and Chatterjee et al. concluded that curcumin mouthwash was as effective as chlorhexidine in reduction of plaque index scores.¹⁹^,²¹ Results of the above-mentioned studies correlate with the present study. Paula et al. observed an inhibition halo when rosemary mouthwash was used against S. mutans and C. albicans, which ascertains the findings of the present study.²⁵

There was however a linear reduction of S. mutans colonies in all three groups after the use of the mouthwashes, thereby proving that both curcumin and curcumin–rosemary mouthwashes are as effective as chlorhexidine in the reduction of S. mutans colonies in orthodontic patients. Curcumin is a powerful antioxidant that is responsible for its antibacterial properties. Curcumin prevents the adhesion of bacteria to host cells, prevents biofilm formation, and protects host cells from bacterial toxins.²⁷ Rosemary is hepatoprotective and has antifungal and antibacterial properties mainly because of the presence of phenolic compounds which are potent antioxidants.²⁸

Anderson et al. studied the effects of chlorhexidine mouthwash on orthodontic patients and concluded that chlorhexidine along with regular oral hygiene practices was effective in reducing plaque levels in orthodontic patients. Similar results were observed in this study but when a questionnaire survey was conducted, patients who used chlorhexidine mouthwash reported burning sensation and allergic reactions in the form of ulcerations on the buccal mucosa. In contrast, the patients who used curcumin and curcumin–rosemary mouthwashes suggested that they noticed staining of the tongue after their use for 1 month (Tables 3 to 5). Discoloration of the tongue was found to be temporary with the use of curcumin mouthwash.¹⁷^,¹⁹

Results of the present study suggest that the two experimental types of mouthwash were as effective individually as chlorhexidine in the reduction of S. mutans colonies in orthodontic patients. Although, chlorhexidine mouthwash was effective in controlling the growth of S. mutans colonies in orthodontic patients, side effects like burning sensation, allergic reactions, and dryness of mouth were reported. In contrast, the chief complaint associated with the use of curcumin and curcumin–rosemary mouthwash was temporary staining of the tongue. Though S. mutans was the only organism studied, the herbal mouthwashes which were nonsynthetic organic in nature proved to be as efficacious as chlorhexidine.

Clinical Significance

New mouthwashes prepared for the present study are cost-effective and can be used by patients from low socioeconomic backgrounds. Therefore, with numerous advantages over chlorhexidine mouthwash and with their minimal and temporary side effects, curcumin or curcumin–rosemary mouthwashes can be effectively used by orthodontic patients. Future research can focus on analyzing the effect of the novel mouthwash (curcumin–rosemary) on other periodontal pathogens.

CONCLUSION

Curcumin and curcumin–rosemary mouthwash were as effective individually as chlorhexidine mouthwash in controlling the growth of S. mutans in orthodontic patients.

Curcumin or curcumin–rosemary mouthwash with its superior properties, cost-effectiveness, and negligible subjective side effects can be definitely prescribed to patients undergoing orthodontic treatment.

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