ORIGINAL RESEARCH

https://doi.org/10.5005/jp-journals-10005-2795
International Journal of Clinical Pediatric Dentistry
Volume 17 | Issue S-1 | Year 2024

Antimicrobial Efficacy of Mangifera indica, Mentha arvensis, and Chlorhexidine Mouthwashes on Streptococcus mutans and Candida albicans in Children: A Comparative In Vivo Study

Shweta Kajjari¹ https://orcid.org/0000-0001-9016-1067, Vanishree B K² https://orcid.org/0000-0003-4855-681X, Sruthi Janardhanan³, Vidyavathi H Patil⁴ https://orcid.org/0000-0002-3007-9317, Chaitanya Uppin⁵ https://orcid.org/0000-0002-8527-5411, Shivayogi M Hugar⁶

^1,4–6Department of Pediatric and Preventive Dentistry, KLE V. K. Institute of Dental Sciences, KLE Academy of Higher Education and Research, Belagavi, Karnataka, India

²Department of Pediatric and Preventive Dentistry, M. A. Rangoonwala College of Dental Sciences & Research Centre, Pune, Maharashtra, India

³Department of Pediatric and Preventive Dentistry, Unity Speciality Dental Centre, Kunhimangalam, Kerala, India

Corresponding Author: Shweta Kajjari, Department of Pediatric and Preventive Dentistry, KLE V. K. Institute of Dental Sciences, KLE Academy of Higher Education and Research, Belagavi, Karnataka, India, Phone: +91 9964525733, e-mail: drshwetakajjari@gmail.com

ABSTRACT

Background: Herbal medicines are both promotive and preventive in inhibiting dental caries. Most commonly used natural plant extracts like mango (Mangifera indica) and pudina (Mentha arvensis) as an entire or together are scientifically well proven to be safe and effective against various oral health problems, especially in preventing dental caries and periodontal diseases.

Aim: The present study aims to comparatively evaluate the effect of Mangifera indica, Mentha arvensis, and Chlorhexidine (CHX) (0.2%) mouthwashes on Streptococcus mutans (S. mutans) and Candida albicans (C. albicans) in children.

Materials and methods: By random allocation, 45 healthy children were selected between the age-group of 7 and 10 years of both genders from a residential school. They were divided into three study groups of 15 in each; group I—CHX (control group), group II—Mangifera indica mouthwash, and group III—Mentha arvensis mouthwash. Stimulated whole saliva sample collected for the amount of colony forming units and cultured for S. mutans and C. albicans. The data was tabulated, and statistical analysis was done with one-way variance analysis of the variance (ANOVA), Tukey’s post hoc honestly significant difference (HSD) test, and Bonferroni and Kruskal–Wallis test.

Results: Mangifera indica (group II) and Mentha arvensis (group III) showed a significant reduction of salivary counts at baseline, 14th day, and 21st day period and minimal effect on C. albicans for Mangifera indica. Among the three types of mouthwash, the CHX was the most superior; it showed a statistically significant reduction overall. When compared to Mangifera indica with Mentha arvensis, Mentha arvensis showed a statistically significant reduction in S. mutans and C. albicans and had an almost equivocal effect like CHX.

Conclusion: Both herbal mouthwashes had a significant reduction in the S. mutans and C. albicans in children. So, it can be considered an active ingredient in mouthwashes. It may serve as a natural antimicrobial mouthwash with no adverse effects.

How to cite this article: Kajjari S, K VB, Janardhanan S, et al. Antimicrobial Efficacy of Mangifera indica, Mentha arvensis, and Chlorhexidine Mouthwashes on Streptococcus mutans and Candida albicans in Children: A Comparative In Vivo Study. Int J Clin Pediatr Dent 2024;17(S-1):S78–S83.

Source of support: Nil

Conflict of interest: None

Keywords: Candida albicans, Chlorhexidine, Mangifera indica, Mentha arvensis, Salivary Streptococcus mutans.

INTRODUCTION

“Dental caries is a complex, multifactorial and microbial disease which is neither self-limiting nor amenable to short-term pharmacological management.”¹^,³ It is an infectious microbial disease involving an interplay between numerous internal factors, such as tooth surface morphology, general health, nutritional and hormonal factors, and a variety of external factors—like microbial flora, diet, oral hygiene, fluoride, plaque, and saliva.⁴

Dental caries can be prevented by targeting the primary risk factors like microorganisms, diet, plaque, etc. For decades, sucrose has been considered to be a major external contributor to dental caries, among other etiological factors. Plaque control is often achieved by using preventive measures like the combination of chemical and mechanical approaches. The various vehicles for delivery of chemical methods are toothpaste, mouthwashes, gels, varnishes, chewing gums, spray, and irrigators, etc.⁵

Antimicrobial mouthwashes show their mechanisms using three methods, which exhibit apoptosis, inhibition of bacterial growth, and/or cell metabolic inhibition; and depending upon their concentration, their bactericidal and/or bacteriostatic properties vary.⁶

Chlorhexidine (CHX) (0.2%) is the most commonly used mouthwash, which has been shown to effectively reduce the level of S. mutans and has been proven to be the most effective anti-plaque agent. However, this mouthwash has been reported to possess various side effects in its long-term use.⁷

Taking into consideration the side effects of CHX, and therefore, the liking or faith of individuals towards herbal/natural products. Herbal medicine exhibits both promotive and preventive approaches. Natural plant extract like Mangifera indica has been scientifically proven as a remedy against various oral health problems, especially in preventing tooth decay, and is found to be safe and effective.

The mango, commonly grown worldwide and has the botanical name “Mangifera indica L,” belongs to the family “Anacardiaceae.” Mango (Mangifera indica), a plant widely used since the ancient traditional medicinal systems of India, has been reported to possess antibacterial, antiviral, and anti-inflammatory activities. Mango contains tannins, bitter gum, and resins that give it antimicrobial activity against a few microorganisms. Mango twigs have more antibacterial activity when compared to other parts.⁸

Pudina (Mentha arvensis), a perennial aromatic herb belonging to the family Labiatae and genus Mentha, is a crucial culinary plant with immense medicinal use. The entire part of the plant is considered to be antibacterial antifebrile. Pudina contains menthol as an antioxidants as a sort of biologically active component. Pudina (peppermint) leaves contain about 0.5–4% essential oil that is composed of 50–78% free menthol, monoterpene, menthofuran, and traces of jasmine (0.15%).⁹ Sunitha et al. found a significant antifungal activity by aqueous and ethanolic mint leave extracts activity on C. albicans.¹⁰

Literature review revealed very few in vivo studies assessing the effects of Mentha arvensis and Mangifera indica twigs extracts on Streptococcus mutans (S. mutans) and Candida albicans (C. albicans).⁹^,¹¹

The aim of the study was to comparatively evaluate the clinical efficacy of those medicinal plant extracts in effectively reducing caries forming pathogens, thereby incorporating them in mouthwashes as caries preventive adjunct in children.

MATERIALS AND METHODS

Ethical clearance from the Institutional Research and Ethics Committee, KLE V. K. Institute of Dental Sciences, KLE Academy of Higher Education and Research, Belagavi, Karnataka, India, was taken before the commencement of study. A total of 45 healthy children between the age-group of 7 and 10 years from the schools of Belagavi city were divided randomly into three groups of 15 each based on the mouthwashes prescribed:

Group I: The CHX mouthwash 0.2% as a control.
Group II: 50% concentration of Mangifera indica extract.
Group III: 50% concentration of mouthwash from Mentha arvensis extract.

Systemically healthy children who had four decayed, missing, filled permanent teeth (DMFT) and decayed, missing/extracted, filled primary teeth (deft) and those whose parents gave consent for the examination were included in the study.

Children who cannot clear out the saliva completely or who could not brush their teeth without supervision or with a history of antibiotics therapy intake 3 months prior to and during the study period, or children undergoing orthodontic therapy or with intraoral appliances or the presence of any oral pathology or systemically compromised children were excluded from the study.

Preparation of Mouthwashes

Chlorhexidine mouthwash used in the study which was commercially available (Clohex, Dr Reddy’s).
Mangifera indica mouthwash (Figs 1A to C): The Mangifera indica twigs were dried and ground separately into a coarse powder. The powder was weighed approximately 50 gm and transferred into the bottle, for which was then added 10 mL of sterile, deionized distilled water. The added mixture was then shaken well and allowed for 48 hours at 4°C.⁹ It was then filtered to obtain a 50% concentration of Mangifera indica mouthwash with the following ingredients (Table 1).
Mentha arvensis mouthwash (Figs 2A to C): Mentha leaves were obtained from the local market and sun-dried. The dried leaves were finely powdered and weighed 100 gm and then it was macerated with 500 mL of 100% ethanol. It was then subjected to “Soxhlet filtration” using Whatman filter paper to produce a clear filtrate. The filtrate obtained was reduced at a low-temperature of <60°C to get a solid residue of Mentha arvensis extract. Around 50 gm of extract was dissolved in 10 mL of dimethyl formamide to obtain an extract of 50% concentration was made.¹²

**Table 1:** Mouthwash preparation
Ingredients	Concentration
Mangifera indica twigs extract	50%
Peppermint oil (sweetening agent)	1 gm/L
Sodium saccharine (sweetening agent)	1 gm/L
Methyl paraben (preservative)	0.05%
Distilled water (volume)	1000 mL

Figs 1A to C: (A) Preparation of Mangifera indica extract by filtration; (B) Solid Mangifera indica extract; (C) 50% concentration of Mangifera indica extract

Figs 2A to C: (A) Soxhlet method of filtration; (B) Solid Mentha arvensis extract; (C) 50% concentration of Mentha arvensis extract

The extract obtained was stored in a refrigerator for preparation of the mouthwash solution. Authorized sweetening agent peppermint oil (1 gm/L), sodium saccharine (1 gm/L), and preservative methylparaben (0.05%) were added to it. The final concentrate was then diluted with 1000 mL of sterile distilled water to make a 50% concentration of mouthwash, and it was dispensed in sterile airtight containers.

Methodology

It was a double-blind, randomized three-parallel arm study wherein the study participants and microbiologists were unaware of which group the subjects belonged to, and each number was allocated for each group and each participant. The study design and treatment protocols were explained in detail to the parents/caretakers as well as to the children who had participated in the study and informed consent was obtained for the same. A sample size of 42 was calculated using the standard sample size formula, which was rounded to 45. A total of 45 healthy children in the age-group of 7–10 years from the schools of Belagavi city were selected and divided into three groups. The study was conducted for about 21 days, and the children were instructed to brush twice daily. A baseline nonstimulated saliva sample was collected only after 1 week of following the same.

Before salivary sample collection, subjects were informed not to eat or drink (except water) 1 hour before just to minimize the possible accumulation of food debris and stimulation of saliva. A baseline salivary sample of 2 mL was collected in the morning by asking the patient to drool the saliva passively into a sterile plastic bottle for 5 minutes. The samples collected in sterile bottles were carried out in a box containing ice (transport media) to maintain the temperature. The samples were tested for the microbiological analysis for the colony-forming units (CFU) of S. mutans and C. albicans by using mitis salivarius, bacitracin agar, and CHROMagar, respectively.

On the next visit to the school, these children were randomly divided into three groups of 15 each, and they were advised CHX (0.2%), Mangifera indica, and Mentha arvensis extract mouthwashes. They were advised to rinse their mouth for 1 minute using 5 mL of the mouthwash daily after breakfast. The second sample was taken only after 14 days after using mouthwashes and counted for colonies. The same procedure was advised to continue for 1 more week. On the 21st day, the children’s salivary sample was taken using the same procedure, and colonies were counted for S. mutans and C. albicans.

Microbiological Assessments

Streptococcus mutans: Salivary samples of 0.5 mL were spread on mitis salivarius agar, and CFU was identified and counted using a digital colony counter.
Candida albicans: A salivary sample of 0.5 mL was spread on CHROMagar, and CFU was identified and counted using a digital colony counter.

Statistical Analysis

The obtained data was tabulated and statistically analyzed by using mean, standard deviation, one-way variance analysis of the variance (ANOVA), Tukey’s post hoc honestly significant difference (HSD) test, Bonferroni, and Kruskal–Wallis test in Statistical Package for the Social Sciences software 17.0. The mean and standard deviations were used for age distribution, decayed, missing, filled permanent teeth (DMFT), and colony count observations for all three mouthwash groups. The results were considered to be statistically significant at a “0.05 probability level.”

RESULTS

Our study group mouthwashes showed a statistically significant reduction (p < 0.001) in the S. mutans group, whereas for C. albicans, only Mentha arvensis showed a statistically significant reduction, but there was a numerical fall found in the Mangifera indica group, which was not statistically significant.

The mean difference of S. mutans counts at different time intervals of baseline, 14th day, and 21st day reveals for CHX, there was a statistically significant reduction of S. mutans count, and for Mentha arvensis significant reduction at the 21st day only and for Mangifera indica it was not statistically significant (Table 2).

**Table 2:** Mean difference of *S. mutans* counts at different time intervals (baseline, 14th day, and 21st day)—Bonferroni statistical analysis
Groups	(I) duration	(J) duration	Mean difference (I–J)	Significance
CHX	Baseline	14th day	1.89817	<0.001^*
	Baseline	21st day	3.05663	<0.001^*
	14th day	21st day	1.15846	<0.001^*
Mangifera indica	Baseline	14th day	0.34262	0.054
	Baseline	21st day	0.34499	0.056
	14th day	21st day	0.00237	1.000
Mentha arvensis	Baseline	14th day	0.18821	0.012
	Baseline	21st day	0.26829	<0.001^*
	14th day	21st day	0.08009	0.600

*Statistically significant

Intergroup comparison of S. mutans counts at baseline, 14th day, and 21st day of the three groups showed statistically significant results (Fig. 3).

Fig. 3: Intergroup comparison of S. mutans counts at baseline, 14th day, and 21st day

Intergroup comparison of S. mutans counts between each group at different intervals showed statistically significant results for CHX-Mangifera indica and CHX-Mentha arvensis at baseline, 14th day, and 21st day (Table 3).

**Table 3:** Intergroup comparison of *S. mutans* counts—Tukey’s *post hoc* HSD analysis
Duration	(I) group	(J) group	Mean difference (I–J)	Significance
Baseline	CHX	Mangifera indica	−0.81642	<0.001^*
	CHX	Mentha arvensis	−0.86130	<0.001^*
	Mangifera indica	Mentha arvensis	−0.04489	1.000
14th day	CHX	Mangifera indica	−2.37196	<0.001^*
	CHX	Mentha arvensis	−2.57126	<0.001^*
	Mangifera indica	Mentha arvensis	−0.19930	0.256
21st day	CHX	Mangifera indica	−3.52805	<0.001^*
	CHX	Mentha arvensis	−3.64964	<0.001^*
	Mangifera indica	Mentha arvensis	−0.12158	0.743

*Statistically significant

Intergroup comparison of C. albicans counts of the three groups was statistically significant only on 14th and 21st days (Table 4 and Fig. 4).

**Table 4:** Intergroup comparison of *C. albicans* counts at baseline, 14th day, and 21st day of the three groups—ANOVA statistical analysis
Duration groups		N	Mean	Standard deviation	p
Baseline	CHX	15	4.723	0.270	0.76
	Mangifera indica	15	4.767	0.160
	Mentha arvensis	15	4.853	0.094
14th day	CHX	15	3.358	0.308	0.001^*
	Mangifera indica	15	4.526	0.163
	Mentha arvensis	15	4.500	0.160
21st day	CHX	15	3.075	0.361	0.001^*
	Mangifera indica	15	4.500	0.160
	Mentha arvensis	15	3.246	0.112

*Statistically significant

Fig. 4: Intergroup comparison of C. albicans counts at baseline, 14th day, and 21st day

Mean and standard deviation values of C. albicans counts at baseline, 14th day, and 21st day reveal a highly statistically significant reduction in CHX and a statistical significant reduction for Mentha arvensis (Table 5).

**Table 5:** Mean and standard deviation values of *C. albicans* counts at baseline, 14th day, and 21st day—Kruskal–Wallis statistical analysis
Groups		N	Mean	Standard deviation	H	P
CHX	Baseline	15	4.723	0.270	25.24	<0.001^*
	14th day	15	3.358	0.308
	21st day	15	3.075	0.361
Mangifera indica	Baseline	15	4.767	0.160	7.67	0.26
	14th day	15	4.526	0.163
	21st day	15	4.500	0.160
Mentha arvensis	Baseline	15	4.853	0.094	19.62	0.001^*
	14th day	15	3.300	0.102
	21st day	15	3.246	0.112

*Statistically significant

DISCUSSION

In the present study, two widely available and most commonly used herbal products in Indian homes, namely, Mangifera indica and Mentha arvensis leaves as 50% concentrated mouthwashes, were studied by comparing their antimicrobial efficacy to CHX, the golden standard antibacterial and antiplaque mouthwashes in children. The antimicrobial efficacy was evaluated against the primary etiologic bacteria, namely, S. mutans and fungus C. albicans.

Age is the critical factor in subject selection, of which the most important is the number of tooth surfaces at risk. Subjects in the age-group 7–10 years were chosen because of the high-risk activity during the mixed dentition stage and also with an increase in adhesion of S. mutans counts.¹³^,¹⁴

The study was carried out in a residential school, as all the subjects would be following the same routine, and the dietary food would be similar throughout the study period, which might eliminate the bias as well. Accessibility and convenience to the investigator for daily supervision were also an important factor. Dental caries is an infectious microbial disease often due to an increased intake of dietary carbohydrates and an increase in the frequency of a cariogenic diet, thus inhibiting the remineralizing cycle as well as in the occurrence of carious lesions. Hence, diet acts as a major causative factor, which was controlled in our study as all the children were taking the same type of diet.¹³^,¹⁴

In the present study, stimulated saliva was collected because unstimulated saliva is less reliable. Chewing helps to wipe out all the bacteria from the tooth surfaces and mix them with salivary secretion. The stimulation of the salivary flow rate also decreases the intraindividual variation of secretion flow, thus increasing the shedding of bacteria from the teeth.¹³

The present study consisted of 57.8% males and 42.2% females. The mean age of the population was 9.40 years in the CHX group, 9.5 years in Mangifera indica group, and 9.93 years in Mentha arvensis group.

Effect on Salivary Streptococcus mutans Count

In this study, we found that salivary S. mutans counts were less among the Mentha arvensis group compared to Mangifera indica group among both herbal mouthwashes. In the present study, at different intervals of baseline, 14th day, and 21st day, statistically significant reduction in salivary S. mutans counts was found in all the groups (p = 0.001) in which CHX was the most followed by Mentha arvensis and Mangifera indica.

In a study conducted by Chaudhary et al., the effect of pudina extract was compared with other herbal products like neem, mango, and tulsi. Pudina extract exhibited a wider zone of inhibition, which was obtained at the 50% concentration.¹² Sreevidhya and Geetha conducted an in vitro study in which peppermint oil showed a zone diameter of 16 mm against S. mutans. Mint oil a very good antibacterial activity against S. mutans.¹⁵

Another study conducted by Prashant et al. reported that 50% extract of mango chewing stick showed the maximum zone of inhibition for Streptococcus mitis and proved that it has antibacterial property.⁹ Similar study conducted by Sahni et al., at 48 hours, higher antimicrobial activity was seen for Mangifera indica (mango).¹⁶ Mango sticks contain tannins and resins that have an astringent effect on both the mucous membrane and enamel, forming a layer on it, thus preventing the formation of dental caries.¹⁶ In contrast to our study, Gauniyal and Singh Teotia showed that a 5% ethanolic extract of Mangifera indica (mango) was ineffective against S. mutans and failed to show the zone of inhibition.¹⁷

Vijayaalakshmi and Geetha, in their in vitro study it was noted that herbal mouthwash was checked for its antibacterial activity in reducing S. mutans in comparison with CHX mouthwash, and results showed that herbal mouthwash was equivalent to conventional mouthwash.¹⁸

According to many studies that have been reported on the effects of herbal mouthwashes against specific oral microorganisms, the CHX mouthwash was the most promising and showed superior results.¹⁹^,²⁰

Effect on Candida albicans

On comparing the antifungal activity of CHX, Mangifera indica, and Mentha arvensis mouthwash was found to be as potent as CHX mouthwash. A statistically significant reduction was seen in both CHX and Mentha arvensis between baseline and 21st-day intervals (p < 0.001*). There was a numerical fall in the count, which is seen in the Mangifera indica group, and it was not statistically significant (p = 0.26).

A study by Gauniyal and Singh Teotia reported ethanolic extract of Mentha piperita showed a zone of inhibition against C. albicans and no zone of inhibition for Mangifera indica (mango) extract.¹⁷

A similar study reported by Sunitha et al. noted alcoholic mint leaves inhibited the growth of C. albicans, whereas no zone of inhibition was seen in aqueous mint leaf extract.²¹ According to a study conducted by Machado et al. and Suci and Tyler, the CHX solutions were able to reduce the CFU of Candida biofilm.²²^,²³

The result of this study showed that the Mangifera indica and Mentha arvensis mouthwashes showed a significant reduction in S. mutans counts and C. albicans when baseline values were compared with the 14th and 21st days under adjunctive oral hygiene measures.

No adverse effects on the oral hard and soft tissues or abnormal symptoms/sensations were detected after using the Mangifera indica and Menthaarvensis mouthwashes and were accepted by the children for a period of 21 days. Mangifera indica and Mentha arvensis mouthwashes were evaluated in our study for their potential as novel antimicrobial agents. However, the study was of short duration and had a small sample size. So, more in vivo studies on larger samples need to be conducted to prove its efficacy as an effective antimicrobial and antifungal component in mouthwashes that can be prescribed for children.

CONCLUSION

Both the herbal mouthwashes had a significant reduction in salivary S. mutans levels and minimal effect on C. albicans by Mangifera indica. Both herbal mouthwashes could be economically safer alternatives to commercial products, especially in low socioeconomic groups and remote areas where modern healthcare facilities are not accessible. Further long-term study with a large population group is recommended to determine the efficacy of these herbal mouthwashes in improving oral health status and also on their side effects when long-term uses are recommended.

ORCID

Shweta Kajjari https://orcid.org/0000-0001-9016-1067

Vanishree B K https://orcid.org/0000-0003-4855-681X

Vidyavathi H Patil https://orcid.org/0000-0002-3007-9317

Chaitanya Uppin https://orcid.org/0000-0002-8527-5411

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