Original Article

Antimicrobial Efficacy of Two Mouthrinses Against Candida albicans: An

In Vitro Study
Nagappan Nagappan, Bharath Champakesan1, Nagaland Tirupati, Thomson Maridasan D’cruz2, Palanivel Pandian
Ramasubramanian3, Parvathy Premnath4

Department of Public Background: Mouthrinses have been used for centuries with the objective of

Abstract
Health Dentistry, Chettinad
reducing the amount of microorganisms in the oral cavity. Mouthrinses are
Dental College and
Research Institute, Chennai,
used as adjuncts to mechanical oral hygiene. Therapeutic mouthwashes are
1
Department of Public often recommended as an adjunct to mechanical plaque control for prevention
Health Dentistry, Vinayaka of plaque accumulation and to maintain gingival and periodontal health.
Mission’s Sankarachariyar Mechanical control alone for reducing recalcitrant biofilms in the oral cavity has
Dental College, Salem, been challenged because it is considered to be rather time-consuming and most
Tamil Nadu, 2Aesthetic importantly insufficient for effective oral hygiene. The aim of this study was to
Avenue Multi Speciality evaluate the antimicrobial efficacy of herbal and 0.2% chlorhexidine gluconate
Dental Clinic, Mumbai,
mouthrinse against Candida albicans. Materials and Methods: The minimum
Maharashtra, 3Department
of Public Health Dentistry, inhibitory concentration and antimicrobial effectiveness (zone of inhibition)
Best Dental Science College, of a herbal mouthrinse and 0.2% chlorhexidine mouthrinse were determined
Madurai, 4Department of by broth macro-dilution and agar well diffusion method, respectively. Results:
Public Health Dentistry, The zone of inhibition of C. albicans was 26 mm for the 0.2% chlorhexidine
Asan Memorial Dental mouthrinse, whereas it was 12 mm for the Arowash liquid mouthrinse. Conclusion:
College and Hospital, Chlorhexidine mouthrinse (0.2%) has a better antimicrobial efficacy against the
Chengalpattu, Tamil
C. albicans when compared to herbal mouthrinse (Arowash liquid).
Nadu, India
Keywords: Antimicrobial efficacy, Candida albicans, chlorhexidine, herbal

Introduction effects as an adjunct to the improved purity and efficacy

of pharmaceutical drugs.[3] However, the accompanying
N   
ature always stands as a golden mark to
  
exemplify the outstanding phenomenon of
symbiosis. The biotic and abiotic elements of nature
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]
are all interdependent. Nature has provided a complete
storehouse of remedies to cure all ailments of Indigenous system of medicine such as Siddha,
humankind. The knowledge of drugs has accumulated Ayurveda, and Unani depends on the use of various
over thousands of years as a result of inquisitive nature plants species.[4] Given the drawbacks of modern
of human beings, so that today we possess many medicine today, there is a growing focus on the
effective means of ensuring health care.[1] Natural importance of medicinal plants in the traditional health
products, including plants, animals, and minerals have care system. A look at the research and investments
been the basis of treatment of human diseases. History that are going into medicinal plants highlights the fact
of medicine dates back practically to the existence of that they will play an important role in human health.
human civilization.[2]
Address for correspondence: Dr. N. Nagappan,
Allopathy or modern medicine is the method of treating Department of Public Health Dentistry, Chettinad Dental College
and Research Institute, Kelambakkam,
disease with remedies that produce effects different Chennai 603103, Tamil Nadu.
from those caused by the disease itself.[3] It appears that E-mail: [Link]@[Link]
modern medical practice accepts the presence of side This is an open access journal, and articles are distributed under the terms of the
Creative Commons Attribution-NonCommercial-ShareAlike 4.0 License, which allows
Access this article online others to remix, tweak, and build upon the work non-commercially, as long as
Quick Response Code: ­appropriate credit is given and the new creations are licensed under the identical terms.
Website: [Link] For reprints contact: reprints@[Link]

How to cite this article: Nagappan N, Champakesan B, Tirupati N,

D’cruz TM, Ramasubramanian PP, Premnath P. Antimicrobial efficacy of
DOI: 10.4103/jpbs.JPBS_16_19
two mouthrinses against Candida albicans: An in vitro study. J Pharm
Bioall Sci 2019;11:S293-6.

© 2019 Journal of Pharmacy and Bioallied Sciences | Published by Wolters Kluwer - Medknow S293
 Nagappan, et al.: Antimicrobial efficacy of herbal and chlorhexidine mouthrinses

The mouth is considered as the mirror of the body, and Test materials
the health of the oral cavity has been closely associated Control mouthrinse: Hexidine mouthrinse (ICPA
with systemic health.[5] Dental caries, gingival disease,[6] Health Products, Andheri (East), Mumbai, India)
and periodontal disease[5] are the most frequently
occurring oral diseases in the world. Experimental mouthrinse: Arowash liquid mouthrinse
(Cadila Pharmaceuticals, Cadila Pharmaceutical Ltd.
There has been a vigorous search for many years for Ahmedabad, Gujarat, India)
chemical agents that could supplement mechanical
plaque control and thus reduce or prevent oral disease. Test organisms: C. albicans (ATCC 10231)
Antiseptic agents have received the bulk of attention over Review board clearance
the years. Finally, they found use of mouthrinse with Prior to the commencement, the study was submitted
antimicrobial activities could be an adjunct for daily use.[6] for approval, and clearance was obtained from
The 0.2% chlorhexidine mouthrinse can effectively Scientific Review Board, Saveetha University, Chennai,
prevent dental plaque and gingivitis in the absence of Tamil Nadu, India.
other oral hygiene procedures. An experimental gingivitis Approval from authorities
study model established the potential for antimicrobial
The study was carried out in Department of
mouthrinses in clinical practice. This led to a variety of
Microbiology, Sri Muthukumaran Medical College
subsequent studies, which demonstrated the anti-plaque
and anti-gingivitis effectiveness of chlorhexidine and Hospital, Chennai, Tamil Nadu, India. Permission
mouthrinses as adjuncts to usual oral hygiene methods. to conduct the study was obtained from the Dean
As a result, 0.2% chlorhexidine mouthrinses became and Head of the Department (Microbiology) of the
widely used in many European countries.[5] college.

Currently there are commercialized oral rinses, which Inoculum preparation procedure for fungal strains was
contain natural compounds with antimicrobial activity followed as per the method by Wikler.[8]
and are known to be safe for human use. Natural MIC was found by broth macro-dilution method.[9]
extracts such as Eucalyptus globules (eucalyptus),
Eugenia caryophyllus (clove), Sanguinaria canadensis The zone of inhibition was found by agar well diffusion
(sanguinarine), Cinnamomum verum (cinnamon), and method.[8]
Mentha spicata (spearmint) are among the extracts Control solution
added to the formation of many commercialized
mouthrinse antimicrobial agents due to the broad Chlorhexidine control: 1 mL 0.2% chlorhexidine
range effects toward oral microbes and pathogens.[7] gluconate solution + 1 mL inoculum

Arowash liquid is a judicious combination of three Positive growth control: 1 mL brain heart infusion broth
herbs—Acacia catechu extract, Glycyrrhiza glabra + 1 mL inoculum
extract, and Syzygium aromaticum extract—which Negative growth control: 1 mL brain heart infusion
is responsible for antiseptic, anti-inflammatory, broth + 1 mL sterile saline
astringent, local anesthetic, and mouth freshener effect.
To the best of our knowledge, no past researches have Results
been conducted on these combinations of herbs against Table 1 describes the estimation of MIC of control
the Candida albicans. So the aims of this study were and experimental mouthrinses. Among the control
to evaluate the minimum inhibitory concentration mouthrinses, negative control mouthrinse did not
(MIC) of a herbal mouthrinse (Arowash liquid) and to inhibit growth, positive control mouthrinse inhibited
compare its antimicrobial efficacy with a commercially growth, and 0.2% chlorhexidine mouthrinse inhibited
available mouthrinse (0.2% chlorhexidine gluconate) growth at 12.5% concentration.
against C. albicans.
Table 2 describes the minimum fungicidal concentration
Materials and Methods (MFC) for experimental mouthrinse against
Study design
oral bacterial strains. The minimum bactericidal
concentration (MBC) for C. albicans was 50%.
This was an in vitro experimental study on
microbiological analysis of herbal and 0.2% Table 3 describes the zone of inhibition for control and
chlorhexidine gluconate mouthrinse. experimental mouthrinse against C. albicans. The zone

S294 Journal of Pharmacy and Bioallied Sciences ¦ Volume 11 ¦ Supplement 2 ¦ May 2019
 Nagappan, et al.: Antimicrobial efficacy of herbal and chlorhexidine mouthrinses

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: Minimum fungicidal concentration (MFC) for properties. Glycyrrhizin has also been reported
experimental mouthrinse to have anti-inflammatory action. “Clove oil” is a
Strains 50% 25% 12.5% MFC value reputed antiseptic, antibacterial, antifungal, antiviral,
Candida albicans − + + 50%
and a local anesthetic. It is an invaluable remedy in
+ = positive bacterial growth, − = negative bacterial growth
inflammation of the mouth and pharynx, and as a local
analgesic and dental antiseptic.
The present in vitro study was conducted to estimate
Table 3: Zone of inhibition for control and experimental
the MIC of a herbal mouthrinse and to compare its
mouthrinse against Candida albicans
Mouthrinse Candida albicans
antimicrobial efficacy with a commercially available
mouthrinse (0.2% chlorhexidine gluconate) against
Control mouthrinse 26 mm
Experimental mouthrinse 12 mm 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
of inhibition for control and experimental mouthrinse mouthrinse (hexidine [0.2% chlorhexidine mouthrinse]).
was 26 and 12 mm, respectively.
The strain used in this study was internationally
Discussion accepted C. albicans (ATCC 10231). The study was
The most common dental diseases are plaque-related conducted in the Department of Microbiology, Sri
infections. In recent years, there has been a lot of concern Muthukumaran Medical College, for a period of
about caries prevention, especially in the developing 1 month. To determine the MIC, MBC, and zone
countries and lower socioeconomic societies. Hence, of inhibition for mouthrinses, broth macro-dilution
there is always a quest for natural mouthrinse, which is method, subculture method, and agar well diffusion
economical, safe, and easy to prepare, in order to achieve method were used, respectively.
better oral health with widespread use as a home remedy.[10] The data were tabulated according to the presence
Mouthrinses have been used for centuries for medicinal (positive) or absence (negative) of growth of fungal
and cosmetic purposes, but it is only in recent years strain in the experimental mouthrinse dilutions assessed.
that the rationale behind the use of ingredients has The experimental mouthrinse showed antimicrobial
been subjected to scientific research and clinical trials. activity in dilution method and it was 50% in the pilot
The advent of mouthrinses containing chlorhexidine experiment, so the highest dilution used for the sensitivity
has been major breakthrough in the research as a test was 50%. The negative control mouthrinse did not
chemical means to prevent oral diseases.[11] Since its inhibit growth of fungal strain used. The positive control
conception, chlorhexidine has proven its effectiveness mouthrinse inhibited the growth in fungal strain used.
beyond dispute, and its different formulations are used The 0.2% chlorhexidine gluconate control mouthrinse
routinely in both general dental practice and teaching did not inhibit growth on fungal strain used.
institutions. Chlorhexidine gluconate has gained MIC for the experimental mouthrinse was found to
considerable interest in the dental community since its be 25% for C. albicans. The current experimental
introduction as a 0.2% mouthrinse in an experimental mouthrinse is a combination of clove oil, A. catechu,
gingivitis study.[5] and G. glabra. So far studies have reported only the
Arowash liquid is a judicious combination of three MIC for any one of the aforementioned components.
Ayurvedic herbs such as A. catechu, G. glabra, and In this study, the zone of inhibition for control
S. aromaticum. A. catechu extract contains “tannins,” mouthrinse (hexidine) for C. albicans was found to be
which have astringent and antioxidant effects, thereby 19, 24, 19, and 26 mm, respectively, which was lower
protecting the mucosal layers. G. glabra extract, than that in the study conducted by Sedighinia et al.[12]
containing “glycyrrhizin,” is a reputed drug for The reason for this difference in values is because of
gastric and duodenal ulcers, and possesses soothing variation in product formulation.

Journal of Pharmacy and Bioallied Sciences ¦ Volume 11 ¦ Supplement 2 ¦ May 2019 S295
 Nagappan, et al.: Antimicrobial efficacy of herbal and chlorhexidine mouthrinses

In this study, the experimental mouthrinse inhibited the 2. Patwardhan B, Vaidya ADB, Chorghade M. Ayurveda
zones in C. albicans about 12 mm, whereas in the study and natural products drug discovery. Current Science
2004;86:789-99.
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inhibit the zones for C. albicans. Again the difference assignee. Herbal and pharmaceutical drugs enhanced with
could be attributed to the difference in product probiotics. European patent EP1133306A1. 2000.
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better when compared to a single ingredient. Eastern Pharmacist 1998; 42:21-24.
5. Charles CH, Mostler KM, Bartels LL, Mankodi SM.
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more effective against C. albicans. Similar results have chlorhexidine and an essential oil mouthrinse: 6-month clinical
trial. J Clin Periodontol 2004;31:878-84.
been reported in studies conducted by Nakamoto et al. [14]
6. Zimmer S, Kolbe C, Kaiser G, Krage T, Ommerborn M, Barthel
and Bruschi et al.[15] Chlorhexidine is bacteriostatic at low C. Clinical efficacy of flossing versus use of antimicrobial rinses.
concentrations and bactericidal at high concentrations. J Periodontol 2006;77:1380-5.
At low concentrations, it increases the permeability of 7. Kochhar SK. Economic botany in the tropics. 3rd ed. New
the bacterial cell membrane with leakage of intracellular Delhi: MacMillan Publishers India Limited; 1998.
8. Wikler MA Methods for dilution antimicrobial susceptibility
components. At high concentrations, it causes
tests for bacteria that grow aerobically: approved standard.
precipitation of bacterial cytoplasm and cell death.[16] In USA: Clinical and Laboratory Standard Institute; 7th ed. 2006;
addition, it is actively adsorbed to the negative groups 26:M7-A7.
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from adhering to the tooth pellicle. The fungicidal methods to determine the minimal inhibitory concentration
activity of chlorhexidine is also due to its penetration (MIC) of antimicrobial substances. Nat Protoc 2008;3:
163-75.
in the region of the glycerol moieties with subsequent 10. Thomas A, Thakur S, Mhambrey S. Comparison of the
breakdown of the permeability barrier and leaking of antimicrobial efficacy of chlorhexidine, sodium fluoride,
cytoplasmic content.[17] Thus, the lower efficacy against fluoride with essential oils, alum, green tea, and garlic with lime
C. albicans can be attributed to the basic differences in mouth rinses on cariogenic microbes. J Int Soc Prev Community
outer cell structure between bacteria and fungi, with the Dent 2015;5:302-8.
11. Overholser CD. Longitudinal clinical studies with antimicrobial
latter possessing a rigid outer chitin wall. mouthrinses. J Clin Periodontol 1988;15:517-19.
12. Sedighinia F, Safipour Afshar A, Soleimanpour S, Zarif R,
Conclusion Asili J, Ghazvini K. Antibacterial activity of Glycyrrhiza glabra
The MIC of Arowash liquid for C. albicans was 25%. against oral pathogens: An in vitro study. Avicenna J Phytomed
2012;2:118-24.
The MFC of Arowash liquid for C. albicans was 13. Aneja KR, Joshi R. Antimicrobial activity of Syzygium
50%. Chlorhexidine mouthrinse (0.2%) has a better aromaticum and its bud oil against dental caries causing
antimicrobial efficacy against the C. albicans when microorganisms. Ethnobotanical Leaflets 2010;14:960-75.
compared to herbal mouthrinse (Arowash liquid). 14. Nakamoto K, Tamamoto M, Hamada T. In vitro effectiveness
of mouthrinses against Candida albicans. Int J Prosthodont
Financial support and sponsorship 1995;8:486-9.
Nil. 15. Bruschi ML, Lara EH, Martins CH, Vinholis AH, Casemiro
LA, Panzeri H, et al. Preparation and antimicrobial activity
Conflicts of interest of gelatin microparticles containing propolis against oral
There are no conflicts of interest. pathogens. Drug Dev Ind Pharm 2006;32:229-38.
16. Seymour RA. Anti-plaque and anti-calculus agents. ln: Seymour
RA, Heasman PA, editors. Drugs, diseases and the periodontium.
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