An In vitro Study to Determine the Antibacterial Activity of Chlorhexidine and Herbal Mouthrinses against Enterococcus faecalis.

Background: Oral diseases have a strong history of treating by natural remedies. Chemical plaque reduction techniques, including dental mouthwashes, are intriguing because they can help patients who are unable to maintain appropriate mechanical plaque management. They are a less technically difficult alternative to mechanical control. Aim: This study aimed to compare the antibacterial efficacy of an herbal and 0.2% chlorhexidine gluconate mouthrinse against Enterococcus faecalis. Methodology: The antimicrobial effectiveness (zone of inhibition) of an herbal mouthrinse and chlorhexidine mouthrinse was determined by agar well diffusion method.
Results: The zone of inhibition for control mouthrinse was 19 mm, and there was no zone of inhibition observed for the experimental mouthrinse.
Conclusion: The result of this investigation suggests 0.2% chlorhexidine gluconate has a better antimicrobial activity than herbal mouthrinse. Copyright:

INTRODUCTION

Oral microbes consist of hundreds of different species of bacteria, fungi, protozoa, and archaea in relationship to oral health. Differently, bacteria are responsible for other pathologies, such as dental caries, gingivitis, and periodontitis, which affect even immunocompetent individuals.[1] Some oral bacteria have a key role in the pathogenesis of infectious disorders of the mouth, jaw, and face, as well as other regions of the body.[2] The reduction of germs in the mouth prior to any dental treatments that are conducted through the mouth can help to reduce the incidence of infections in the postoperative period.[3]

Enterococci are transient constituents of the oral microbiome that can cause a wide range of oral and systemic illnesses.[4] Enterococci are known to cause a variety of diseases, some of which can be fatal, such as urinary tract, neonatal, and wound infections, as well as endocarditis and meningitis. Enterococci have low colonization density.[567]

Enterococci is an elegant study that looked into the virulence characteristics of Enterococcus faecalis isolates from the oral cavity, food, and clinical specimens, finding that oral isolates contained the highest percentages of virulence genes, extracellular enzymes, and the ability to build biofilms. As a result, they hypothesized that the oral cavity could serve as a crucial reservoir for virulent E. faecalis strains resistant to antibiotics.[8]

The efficiency of oral microbe removal, among other factors, is determined by the people's dexterity and thoroughness, as well as their compliance. Clinical investigations have shown that a combination of diligent self-performed plaque control and professional prophylaxis procedures three to six times per year can effectively prevent periodontitis advancement. Such significant and time-consuming efforts to get the best outcomes from mechanical cleaning have laid the groundwork for implementing preventative principles, but they also point to the need for creating chemical plaque management adjuncts.[9]

Antiseptic mouthwashes, frequently coupled with other oral hygiene guidelines, are one of the most efficient strategies for reducing the number of germs in the oral cavity.[10] Due to the widespread usage of mouthwashes in several sectors of dentistry, a wide range of mouthwashes are now available. Chlorhexidine, for example, is regarded as the gold standard mouthwash in the dental sector because of its numerous good effects and minimal negative effects.[1112] Plaque inhibition is reduced when chlorhexidine is chemically modified to lessen its staining.[212]

There are currently sold oral rinses that contain natural antimicrobial components that are recognized to be safe for human usage. Natural extracts such as Eucalyptus globulus (Eucalyptus), Eugenia caryophyllus (clove), Sanguinaria canadensis (sanguinarine), Cinnamomum verum (cinnamon), and Mentha spicata (spearmint) are among the extracts added to the formation of many commercialized mouthrinse antimicrobial agents due to the broad range effects toward oral microbes and pathogens.[13]

Arowash liquid is a judicious combination of three herbs – Acacia catechu extract, Glycyrrhiza glabra extract, and Syzygium aromaticum extract – which is responsible for antiseptic, anti-inflammatory, astringent, local anesthetic, and mouth freshener effect. Surprisingly, little or no data on the impact of mouthwashes on E. faecalis is known.

Hence, the current study aimed to compare the antibacterial efficacy of an herbal and 0.2% chlorhexidine gluconate mouthrinse against E. faecalis.

METHODOLOGY

It is an in vitro experimental study on antibacterial activity of herbal and 0.2% chlorhexidine gluconate mouthrinse. This study comprised two mouthrinses. Control mouthrinse: Hexidine (ICPA Health Products, Andheri [East], Mumbai, India). Experimental mouthrinse: Arowash liquid mouthrinse (Cadila Pharmaceuticals Ltd., Gujarat, India). E. faecalis (ATCC 29212) internationally accepted strain was used to compare the antibacterial activity between control and experimental mouthrinses. The review board clearance was obtained from the Scientific Review Board, Saveetha University, India. As per Winkler's method of inoculum preparation, procedure for E. faecalis strain was done.[14] According to Irith Wiegand,[15] broth macrodilution method was used to find a minimum inhibitory concentration (MIC). The zone of inhibition was found by agar well diffusion method.[14]

RESULTS

Table 1 and Figure 1 describes the estimation of MIC of control and experimental mouthrinses against E. faecalis. Among the control mouthrinses, negative control mouthrinse did not inhibit growth, positive control mouthrinse did inhibit growth, and 0.2% chlorhexidine mouthrinse did not inhibit growth of E. faecalis strain used. At 50% concentration, the experimental mouthrinse inhibits the growth of E. faecalis strain used.

Table 1

Estimation of minimum inhibitory concentration of control and experimental mouthrinses

Strain	Control mouthrinse			Experimental mouthrinse
	Negative	Positive	CHX	50%	25%	12.5%	6.25%	3.12%	1.56%	0.78%	0.39%	0.19%	0.09%	0.04%	0.02%
Enterococcus faecalis	−	+	−	−	+	+	+	+	+	+	+	+	+	+	+

+: Positive bacterial growth, −: Negative bacterial growth, CHX: Chlorhexidine

Figure 1

Minimum inhibitory concentration of an herbal mouthrinse against Enterococcus faecalis

Table 2 and Figure 2 describes the minimum bactericidal concentration (MBC) for experimental mouthrinse against E. faecalis strain. The MBC for E. faecalis was >50%.

Table 2

Minimum bactericidal concentration for experimental mouthrinse

Strains	50%	25%	MBC value (%)
Enterococcus faecalis	+	+	>50

+: Positive bacterial growth, MBC: Minimum bactericidal concentration

Figure 2

Minimum bactericidal concentration of an herbal mouthrinse against Enterococcus faecalis at 25% and 50%

Table 3 and Figure 3 describes the zone of inhibition for control and experimental mouthrinses against E. faecalis. The zone of inhibition for control mouthrinse was 19 mm, and there was no zone of inhibition observed for the experimental mouthrinse.

Table 3

Zone of inhibition for control and experimental mouthrinses against Enterococcus faecalis

Mouthrinse	Enterococcus faecalis (mm)
Control mouthrinse	19
Experimental mouthrinse	00

Figure 3

Antimicrobial efficacy (zone of inhibition) of an herbal and 0.2% chlorhexidine mouthrinse

DISCUSSION

In the Realm of Pharmaceuticals, herbal medications hold a unique position. The effectiveness of plant-derived medications, as well as a growing interest in natural products, has piqued scientists' interest in medicinal plants. When you look at the amount of study and money being put into medicinal plants, it is clear that they will play a big role in human health. Herbal formulations are widely used in India's rural and urban communities. It is apparent that with the collaboration of drug regulatory authorities, scientists, and industry in India, the herbal business may achieve significant progress.

Because the oral cavity is a living ecosystem, eliminating all aspects of the oral microbiota in order to treat dental plaque-related infections would be counterproductive. Rather, it may be preferable to remove only the most cariogenic and periodontopathic parts of the tooth plaque microbiota, leaving the less harmful elements alone.[16]

The goal of this in vitro investigation was to determine the MIC of an herbal mouthrinse and compare it to a commercially available mouthrinse (0.2% chlorhexidine gluconate) in terms of antimicrobial activity against four oral bacterial strains. The mouthrinses used in the present study were experimental mouthrinse (Arowash liquid [combination of A. catechu extract, G. glabra extract, and clove oil]) and control mouthrinse (Hexidine [0.2% chlorhexidine mouthrinse]).

The data were tabulated based on whether each bacterial strain grew (positive) or did not grow (negative) in the experimental mouthrinse dilutions. The experimental mouthrinse showed that antimicrobial activity in dilution method was 50% in the pilot experiment, so the highest dilution used for the sensitivity test was 50%. The negative control mouthrinse did not inhibit growth of any bacterial strain used. The positive control mouthrinse inhibits the growth in all bacterial strains used. The 0.2% chlorhexidine gluconate control mouthrinse did not inhibit growth of any bacterial strain used.

MIC for the experimental mouthrinse was found to be 50% for E. faecalis. The current experimental mouthrinse is a combination of clove oil, A. catechu, and G. glabra. So far, studies have only reported the MIC for any one of the abovementioned components.

The MIC of A. catechu for E. faecalis was 0.2%. This was very low when compared to 50% in the experimental mouthrinse. Similarly, a study by Sedighinia et al.[17] reported that the MIC of G. glabra extract for E. faecalis was 1.25%. Again this was very low and negligible when compared to experimental mouthrinse. The better MIC of the experimental mouthrinse could be due to the synergistic effect of all three components against E. faecalis.

In the current study, the MBC value for experimental mouthrinse was found to be >50% for E. faecalis. The study by Geetha et al.[18] and Sedighinia et al.[17] had also reported similar results.

In the current study, the zone of inhibition for control mouthrinse (Hexidine) for E. faecalis was found to be 19 mm, which was lower than the study conducted by Sedighinia et al.[17] The reason for this difference in values is because of variation in product formulation.

In the current study, the experimental mouthrinse does not inhibit the zones in E. faecalis, whereas the study conducted by Aneja et al.[19] shows that clove oil extract does not inhibit the zones. Similarly, a study by Geetha et al.[18] reported that the zone of inhibition of A. catechu extract for E. faecalis was found to be 8 mm, and Dhanyakumar et al.[20] reported that the zone of inhibition of G. glabra for E. faecalis was found to be 14 mm. Again, the difference could be attributed to the difference in product formulation, thus concluding that a combination is better when compared to a single ingredient.

CONCLUSION

Considering in vitro data obtained in this study, there is no synergistic effect of antimicrobial activity from the combination of A. catechu extract, G. glabra extract, and S. aromaticum extract. The result of this investigation suggests that 0.2% chlorhexidine gluconate has a better antimicrobial activity than herbal mouthrinse. Further studies on clinical significance are recommended.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.