A pilot study on the antimicrobial efficacy of Coleus Aromaticus on Lactobacillus acidophilus – Comparative In-Vitro analysis of five samples on a single plate

INTRODUCTION

Dental caries is a prevalent chronic condition that gradually damages tooth tissues through the demineralization of enamel and dentin due to bacterial infection and dietary acids in dental plaque. Bacteria such as Streptococcus, Actinomyces, and Lactobacillus contribute to this condition, with Lactobacillus often linked to cavity formation, especially in children.^[1]

Chlorhexidine is a commonly used mouthwash with antimicrobial properties. Its effectiveness varies depending on the concentration, with the ability to either inhibit bacterial growth (bacteriostatic) or directly kill bacteria (bactericidal). It works by penetrating the cell wall, causing cytoplasmic lysis and leading to cell death within 20 s of application.^[2] Despite its advantages, 0.2% chlorhexidine has some drawbacks, including tooth staining, throat irritation, reduced taste sensation, potential gastric irritation, and the promotion of tartar formation. To address these limitations, many researchers have explored natural alternatives that offer safety, cost-effectiveness, and minimal to no side effects. Over the years, studies have focused on natural derivatives such as basil (Tulasi), aloe vera, neem, green tea, garlic solutions, and pomegranate as potential alternatives to 0.2% Chlorhexidine.^[3,4]

The primary objective of this study is to develop a phytochemical-based therapeutic oral rinse designed to inhibit the growth of Lactobacillus, with a comparison to the most commonly used mouthwash, 0.2% chlorhexidine.

MATERIAL AND METHODS

The study was approved by the Institutional Ethics Committee, 27^th April 2023, Ethical approval number (IEC/Approval No.304). All procedures performed in the study were conducted in accordance with the ethical standards given in 1964 Declaration of Helsinki, as revised in 2013.

RESULTS

The inhibitory zone diameter in the control group (chlorhexidine mouthwash IP), A1 at 100 µL, A2 at 200 µL, B1 at 100 µL, and B2 at 200 µL was found to be 23, 24, 27, 17, 22 mm, respectively, as shown in Figure 1. Table 1 indicates that the inhibitory effect on Lactobacillus varies among the treatments.

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Figure 1:

(a) The single plate with five samples (A1, A2, B1, B2, C) at different concentrations with the respective measurement of zones of inhibition (Red arrows), (b) Schematic showing the parts of the plate and measurements taken.

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The varying degrees of antibacterial activity among the different treatments, with treatment with A2 showing the strongest inhibition, followed by the control group (chlorhexidine mouthwash IP). Treatment B2 demonstrated moderate inhibitory activity, while both treatments A1 and B1 exhibited relatively weaker inhibition.

DISCUSSION

Dental caries, a prevalent and chronic ailment, is characterized by the gradual deterioration of enamel, dentin, and cementum, leading to the decalcification of these tissues and the breakdown of organic substances. This infection arises from the demineralization of enamel and dentin due to the action of organic acids produced by bacteria found in dental plaque through anaerobic metabolism, playing a pivotal role in the development of dental caries. Among these bacteria, Lactobacillus is particularly noteworthy as a major contributor to dentinal caries. Lactobacilli are closely linked to the advancement of lesions, primarily observed in rampant caries. Evidence suggests that acidophilus, a microorganism known for its significant acid-producing and acid-tolerant characteristics, plays a pivotal role in the development of dental caries.^[5,6]

Chlorhexidine (0.2%) is commonly employed as a mouthwash for the prevention of bacterial and microbial growth. Its effectiveness varies depending on the concentration used, as it can exhibit both bacteriostatic and bactericidal effects. It permeates the cell wall, leading to cytoplasmic lysis and cell death within a mere 20 s of application. Despite its numerous advantages, chlorhexidine is not without its drawbacks, which include tooth staining, throat irritation, reduced taste sensation, gastric discomfort, and the promotion of tartar formation.^[7]

To address these limitations, researchers have explored natural alternatives that are not only safe and cost-effective but also associated with minimal to no side effects. Over the years, various natural derivatives such as Tulasi, aloe vera, neem, green tea, garlic solutions, and pomegranate have been studied for their potential benefits. Despite the widespread use of chlorhexidine, there is a continuous preference for alternative, natural or organic mouth rinses. In our study, we specifically investigated C. aromaticus due to its multifaceted effects and its minimal impact on side effects. C. aromaticus, a member of the Lamiaceae family, is also known as Mexican mint and is locally referred to as “karpuravalli.” It possesses significant antimicrobial and antibacterial properties, along with bacteriostatic and fungistatic qualities. Previous research has delved into the antibacterial and anticancer potential of extracts derived from its leaves.^[8]

The purpose of this study was to develop a phytochemical therapeutic oral rinse capable of inhibiting the growth of Lactobacillus in comparison to the most commonly used mouthwash, 0.2% chlorhexidine. In the control group, where 0.2% chlorhexidine Mouthwash IP served as the reference or standard treatment, the inhibitory zone diameter was 23 mm, consistent with findings from previous studies involving other microorganisms.

Using a leaf sample of C. aromaticus at a concentration of 100 µL, the inhibitory zone diameter for this treatment measured 24 mm, indicating a slightly larger inhibition zone compared to the control. However, when the C. aromaticus leaf sample was applied at a higher concentration of 200 µL, it produced a significantly larger inhibitory zone with a diameter of 27 mm, surpassing both the control and the lower concentration treatment. This suggests that the higher concentration of the C. aromaticus leaf sample possesses a more potent antibacterial effect against Lactobacillus. This could be probably due to the presence of phytochemicals present in the leaves. The antibacterial effect of C. aromaticus was probably due to the presence of phytochemicals, volatile oil compounds, phenolic compounds, etc.

C. aromaticus contains numerous volatile oils and cells that secrete essential oils, each with a diverse range of chemical compositions. It also includes phenolic substances, primarily made up of eugenol, methyl eugenol, and thymol, which make up a significant portion of its phytochemical composition. It is important to note that these specific constituents are particularly prevalent in C. aromaticus found in the Indian region, specifically the variety containing Wild Oregon, as highlighted by authors Baslas and Kumar in 1981. Furthermore, despite the extensive array of phytochemicals and volatile oils present in C. aromaticus, there are noteworthy concentrations of y-terpinene and p-cymene. These components are of particular interest because they serve as precursors for thymol and carvacrol, as explained by Prudent.^[9,10]

The preference for using extracts lies in the ability to produce higher concentrations, thus potentially increasing efficacy and obtaining a more purified form that minimizes unintended side effects resulting from impurities. When C. aromaticus extracts were employed at a concentration of 100 µL, the inhibitory zone diameter was 17 mm, notably smaller than that observed with 0.2% chlorhexidine mouthwash IP. This indicates that the homeopathic extract at this concentration exerts a weaker antibacterial effect compared to 0.2% chlorhexidine mouthwash IP. Similarly, when the extract was used at a higher concentration of 200 µL, the inhibitory zone diameter measured 22 mm, reflecting a larger inhibition zone. This implies that the higher concentration of the homeopathic extract has a relatively stronger antibacterial effect than the lower concentration and underscores the variation in inhibitory effects on Lactobacillus based on the raw material and concentration of the extracts.

As dental caries continue to increase among both children and adults, ongoing research aims to develop a vaccine capable of preventing or reducing caries incidence. Such a vaccine would offer significant benefits, especially for individuals with intellectual disabilities and those suffering from systemic diseases that compromise their immunity, such as type 2 diabetes. However, it is important to acknowledge that developing such a vaccine is a complex and lengthy process. In this context, natural or organically derived mouth rinses emerge as an alternative to synthetic rinses. They hold the potential to address concerns related to antimicrobial resistance, which can be exacerbated by the use of synthetic products. In addition, these natural rinses may help maintain a healthy balance of normal oral commensals within the oral cavity while providing effective oral care.^[11-13]

In this study, a cost-effective culture method was employed by consolidating five samples onto a single plate to assess the inhibitory growth of chlorhexidine, guided by the literature review indicating an inhibition zone of 23 mm. In summary, the study suggests that C. aromaticus extract could be a valuable and accessible resource for oral health in rural India, where microbial control is vital in preventing dental caries, and it offers potential benefits with fewer adverse effects compared to chlorhexidine. This finding may lead to the development of cost-effective and sustainable oral health interventions in these communities.

CONCLUSION

The prevention of dental caries primarily hinges on the management of microbial growth. Within the constraints of this study, it can be inferred that C. aromaticus, extracted from dried leaves in the form of an aqueous extract, demonstrated superior antimicrobial efficacy against Lactobacillus when compared to 0.2% chlorhexidine mouthwash and oral rinse. The main implication of this research is its potential utility in school-based preventive oral health programs in rural India, where C. aromaticus leaves are readily available and cultivated naturally and are associated with minimal to no adverse effects on the oral cavity.