Antimicrobial Efficacy of Two Mouthrinses Against Candida albicans: An In Vitro Study.

BACKGROUND: Mouthrinses have been used for centuries with the objective of reducing the amount of microorganisms in the oral cavity. Mouthrinses are used as adjuncts to mechanical oral hygiene. Therapeutic mouthwashes are often recommended as an adjunct to mechanical plaque control for prevention of plaque accumulation and to maintain gingival and periodontal health. Mechanical control alone for reducing recalcitrant biofilms in the oral cavity has been challenged because it is considered to be rather time-consuming and most importantly insufficient for effective oral hygiene. The aim of this study was to evaluate the antimicrobial efficacy of herbal and 0.2% chlorhexidine gluconate mouthrinse against Candida albicans.
MATERIALS AND METHODS: The minimum inhibitory concentration and antimicrobial effectiveness (zone of inhibition) of a herbal mouthrinse and 0.2% chlorhexidine mouthrinse were determined by broth macro-dilution and agar well diffusion method, respectively.
RESULTS: The zone of inhibition of C. albicans was 26 mm for the 0.2% chlorhexidine mouthrinse, whereas it was 12 mm for the Arowash liquid mouthrinse.
CONCLUSION: Chlorhexidine mouthrinse (0.2%) has a better antimicrobial efficacy against the C. albicans when compared to herbal mouthrinse (Arowash liquid).

INTRODUCTION

Nature always stands as a golden mark to exemplify the outstanding phenomenon of symbiosis. The biotic and abiotic elements of nature are all interdependent. Nature has provided a complete storehouse of remedies to cure all ailments of humankind. The knowledge of drugs has accumulated over thousands of years as a result of inquisitive nature of human beings, so that today we possess many effective means of ensuring health care.[1] Natural products, including plants, animals, and minerals have been the basis of treatment of human diseases. History of medicine dates back practically to the existence of human civilization.[2]

Allopathy or modern medicine is the method of treating disease with remedies that produce effects different from those caused by the disease itself.[3] It appears that modern medical practice accepts the presence of side effects as an adjunct to the improved purity and efficacy of pharmaceutical drugs.[3] However, the accompanying problem of side effects is gaining increased notice as the public justly criticizes that such pure and potent drugs can cure one ailment while causing another.[3]

Indigenous system of medicine such as Siddha, Ayurveda, and Unani depends on the use of various plants species.[4] Given the drawbacks of modern medicine today, there is a growing focus on the importance of medicinal plants in the traditional health care system. A look at the research and investments that are going into medicinal plants highlights the fact that they will play an important role in human health.

The mouth is considered as the mirror of the body, and the health of the oral cavity has been closely associated with systemic health.[5] Dental caries, gingival disease,[6] and periodontal disease[5] are the most frequently occurring oral diseases in the world.

There has been a vigorous search for many years for chemical agents that could supplement mechanical plaque control and thus reduce or prevent oral disease. Antiseptic agents have received the bulk of attention over the years. Finally, they found use of mouthrinse with antimicrobial activities could be an adjunct for daily use.[6]

The 0.2% chlorhexidine mouthrinse can effectively prevent dental plaque and gingivitis in the absence of other oral hygiene procedures. An experimental gingivitis study model established the potential for antimicrobial mouthrinses in clinical practice. This led to a variety of subsequent studies, which demonstrated the anti-plaque and anti-gingivitis effectiveness of chlorhexidine mouthrinses as adjuncts to usual oral hygiene methods. As a result, 0.2% chlorhexidine mouthrinses became widely used in many European countries.[5]

Currently there are commercialized oral rinses, which contain natural compounds with antimicrobial activity and are known to be safe for human use. Natural extracts such as Eucalyptus globules (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.[7]

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.

To the best of our knowledge, no past researches have been conducted on these combinations of herbs against the Candida albicans. So the aims of this study were to evaluate the minimum inhibitory concentration (MIC) of a herbal mouthrinse (Arowash liquid) and to compare its antimicrobial efficacy with a commercially available mouthrinse (0.2% chlorhexidine gluconate) against C. albicans.

MATERIALS AND METHODS

Study design

This was an in vitro experimental study on microbiological analysis of herbal and 0.2% chlorhexidine gluconate mouthrinse.

Test materials

Control mouthrinse: Hexidine mouthrinse (ICPA Health Products, Andheri (East), Mumbai, India)

Experimental mouthrinse: Arowash liquid mouthrinse (Cadila Pharmaceuticals, Cadila Pharmaceutical Ltd. Ahmedabad, Gujarat, India)

Test organisms: C. albicans (ATCC 10231)

Review board clearance

Prior to the commencement, the study was submitted for approval, and clearance was obtained from Scientific Review Board, Saveetha University, Chennai, Tamil Nadu, India.

Approval from authorities

The study was carried out in Department of Microbiology, Sri Muthukumaran Medical College and Hospital, Chennai, Tamil Nadu, India. Permission to conduct the study was obtained from the Dean and Head of the Department (Microbiology) of the college.

Inoculum preparation procedure for fungal strains was followed as per the method by Wikler.[8]

MIC was found by broth macro-dilution method.[9]

The zone of inhibition was found by agar well diffusion method.[8]

Control solution

Chlorhexidine control: 1 mL 0.2% chlorhexidine gluconate solution + 1 mL inoculum

Positive growth control: 1 mL brain heart infusion broth + 1 mL inoculum

Negative growth control: 1 mL brain heart infusion broth + 1 mL sterile saline

RESULTS

Table 1 describes the estimation of MIC of control and experimental mouthrinses. Among the control mouthrinses, negative control mouthrinse did not inhibit growth, positive control mouthrinse inhibited growth, and 0.2% chlorhexidine mouthrinse inhibited growth at 12.5% concentration.

Table 1

Estimation of minimum inhibitory concentration (MIC) of control and experimental mouthrinses

Strains	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%
Candida albicans	−	+	−	−	−	+	+	+	+	+	+	+	+	+	+

CHX = chlorhexidine, + = positive bacterial growth, − = negative bacterial growth

Table 2 describes the minimum fungicidal concentration (MFC) for experimental mouthrinse against oral bacterial strains. The minimum bactericidal concentration (MBC) for C. albicans was 50%.

Table 2

Minimum fungicidal concentration (MFC) for experimental mouthrinse

Strains	50%	25%	12.5%	MFC value
Candida albicans	−	+	+	50%

+ = positive bacterial growth, − = negative bacterial growth

Table 3 describes the zone of inhibition for control and experimental mouthrinse against C. albicans. The zone of inhibition for control and experimental mouthrinse was 26 and 12 mm, respectively.

Table 3

Zone of inhibition for control and experimental mouthrinse against Candida albicans

Mouthrinse	Candida albicans
Control mouthrinse	26 mm
Experimental mouthrinse	12 mm

DISCUSSION

The most common dental diseases are plaque-related infections. In recent years, there has been a lot of concern about caries prevention, especially in the developing countries and lower socioeconomic societies. Hence, there is always a quest for natural mouthrinse, which is economical, safe, and easy to prepare, in order to achieve better oral health with widespread use as a home remedy.[10]

Mouthrinses have been used for centuries for medicinal and cosmetic purposes, but it is only in recent years that the rationale behind the use of ingredients has been subjected to scientific research and clinical trials. The advent of mouthrinses containing chlorhexidine has been major breakthrough in the research as a chemical means to prevent oral diseases.[11] Since its conception, chlorhexidine has proven its effectiveness beyond dispute, and its different formulations are used routinely in both general dental practice and teaching institutions. Chlorhexidine gluconate has gained considerable interest in the dental community since its introduction as a 0.2% mouthrinse in an experimental gingivitis study.[5]

Arowash liquid is a judicious combination of three Ayurvedic herbs such as A. catechu, G. glabra, and S. aromaticum. A. catechu extract contains “tannins,” which have astringent and antioxidant effects, thereby protecting the mucosal layers. G. glabra extract, containing “glycyrrhizin,” is a reputed drug for gastric and duodenal ulcers, and possesses soothing properties. Glycyrrhizin has also been reported to have anti-inflammatory action. “Clove oil” is a reputed antiseptic, antibacterial, antifungal, antiviral, and a local anesthetic. It is an invaluable remedy in inflammation of the mouth and pharynx, and as a local analgesic and dental antiseptic.

The present in vitro study was conducted to estimate the MIC of a herbal mouthrinse and to compare its antimicrobial efficacy with a commercially available mouthrinse (0.2% chlorhexidine gluconate) against C. albicans. The mouthrinses used in this study were experimental mouthrinse (Arowash liquid [combination of A. catechu extract, G. glabra extract, clove oil]) and control mouthrinse (hexidine [0.2% chlorhexidine mouthrinse]).

The strain used in this study was internationally accepted C. albicans (ATCC 10231). The study was conducted in the Department of Microbiology, Sri Muthukumaran Medical College, for a period of 1 month. To determine the MIC, MBC, and zone of inhibition for mouthrinses, broth macro-dilution method, subculture method, and agar well diffusion method were used, respectively.

The data were tabulated according to the presence (positive) or absence (negative) of growth of fungal strain in the experimental mouthrinse dilutions assessed. The experimental mouthrinse showed antimicrobial activity in dilution method and it 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 fungal strain used. The positive control mouthrinse inhibited the growth in fungal strain used. The 0.2% chlorhexidine gluconate control mouthrinse did not inhibit growth on fungal strain used.

MIC for the experimental mouthrinse was found to be 25% for C. albicans. The current experimental mouthrinse is a combination of clove oil, A. catechu, and G. glabra. So far studies have reported only the MIC for any one of the aforementioned components.

In this study, the zone of inhibition for control mouthrinse (hexidine) for C. albicans was found to be 19, 24, 19, and 26 mm, respectively, which was lower than that in the study conducted by Sedighinia et al.[12] The reason for this difference in values is because of variation in product formulation.

In this study, the experimental mouthrinse inhibited the zones in C. albicans about 12 mm, whereas in the study conducted by Aneja et al.[13] clove oil extract did not inhibit the zones for C. albicans. 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.

In this study, chlorhexidine mouthrinse was found to be more effective against C. albicans. Similar results have been reported in studies conducted by Nakamoto et al.[14] and Bruschi et al.[15] Chlorhexidine is bacteriostatic at low concentrations and bactericidal at high concentrations. At low concentrations, it increases the permeability of the bacterial cell membrane with leakage of intracellular components. At high concentrations, it causes precipitation of bacterial cytoplasm and cell death.[16] In addition, it is actively adsorbed to the negative groups on the bacterial cell surface, thereby preventing them from adhering to the tooth pellicle. The fungicidal activity of chlorhexidine is also due to its penetration in the region of the glycerol moieties with subsequent breakdown of the permeability barrier and leaking of cytoplasmic content.[17] Thus, the lower efficacy against C. albicans can be attributed to the basic differences in outer cell structure between bacteria and fungi, with the latter possessing a rigid outer chitin wall.

CONCLUSION

The MIC of Arowash liquid for C. albicans was 25%. The MFC of Arowash liquid for C. albicans was 50%. Chlorhexidine mouthrinse (0.2%) has a better antimicrobial efficacy against the C. albicans when compared to herbal mouthrinse (Arowash liquid).

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.