|Year : 2022 | Volume
| Issue : 4 | Page : 385-389
Comparison of antifungal activity of probiotics, coconut oil and clotrimazole on candida albicans – An In vitro study
V Divyadharsini1, TN UmaMaheswari1, S Rajeshkumar2
1 Department of Oral Medicine and Radiology, Saveetha Dental College, Saveetha Institute of Medical and Technical Sciences (SIMATS), Saveetha University, Chennai, Tamil Nadu, India
2 Department of Pharmacology, Saveetha Dental College, Saveetha Institute of Medical and Technical Sciences (SIMATS), Saveetha University, Chennai, Tamil Nadu, India
|Date of Submission||20-May-2021|
|Date of Decision||17-Aug-2021|
|Date of Acceptance||15-Dec-2021|
|Date of Web Publication||09-Dec-2022|
T N UmaMaheswari
162, Saveetha Dental College, Saveetha Institute of Medical and Technical Sciences (SIMATS), Saveetha University, Poonamallee High Road, Velappanchavadi, Chennai - 600 077, Tamil Nadu
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Background: Oral candidiasis is the most prevalent opportunistic infection of oral cavity. Candida albicans causes oral lesions in the vast majority of cases. Antifungal regimens such as azoles and polyenes are commonly used to control candida infections. Virgin coconut oil has been proven to have antifungal, antimicrobial, and antioxidant properties. Probiotic bacteria have been used to modify microflora ecosystems and also have antifungal activity. Aim: To evaluate the antifungal activity of probiotics, coconut oil, and clotrimazole against oral Candida albicans species. Materials and Methods: Coconut oil and probiotics were tested against clotrimazole, a positive control. Candida albicans were isolated and incubated in Rose Bengal Agar at 30° C for 48 h. The growth inhibitory effect of the test materials was evaluated using the direct contact method by adding 25, 50, and 100 μL of the test material as well as control to candida in a sterile tube and incubated at 37° C aerobically for 24 h. Results: The mean zone of inhibition for clotrimazole was 36 mm, whereas for coconut oil it was 13.6 mm, and for probiotics, it was 11.6 mm. Clotrimazole had the highest inhibitory effect at 100 μL concentration. The difference between the groups was statistically significant (P-value 0.002). Conclusion: The antifungal activity of clotrimazole was found to be higher than that of coconut oil and probiotics against C. albicans. Future studies should concentrate on other herbal alternatives which will be as effective as clotrimazole for the management of oral candidiasis.
Keywords: Antifungal, candid mouth paint, coconut oil, disc diffusion method, lactobacillus, oral candidiasis
|How to cite this article:|
Divyadharsini V, UmaMaheswari T N, Rajeshkumar S. Comparison of antifungal activity of probiotics, coconut oil and clotrimazole on candida albicans – An In vitro study. J Indian Acad Oral Med Radiol 2022;34:385-9
|How to cite this URL:|
Divyadharsini V, UmaMaheswari T N, Rajeshkumar S. Comparison of antifungal activity of probiotics, coconut oil and clotrimazole on candida albicans – An In vitro study. J Indian Acad Oral Med Radiol [serial online] 2022 [cited 2023 Feb 5];34:385-9. Available from: http://www.jiaomr.in/text.asp?2022/34/4/385/363013
| Introduction|| |
The most prevalent opportunistic infection affecting the oral mucosa is the oral candidiasis, and the majority of the lesions are caused by Candida albicans., Other species responsible for candidiasis are Candida glabrata, Candida tropicalis, and Candida krusei. Candida is harmless commensal found in 30%–60% of healthy individuals. It was first described in 1838 by pediatrician Francois Veilleux and isolated in 1844 by J.H. Bennett from the sputum of a tuberculosis patient. Normal oral microflora consists of bacteria, fungi including Candida albicans, mycoplasma, protozoa, and possibly viral flora. Around 30% to 50% people carry Candida albicans as normal oral microflora organism. Candida is a weak pathogen, and the very young, the very old, and the very sick are usually affected with candidiasis. Most candida infections only affect mucosal linings, but rare systemic manifestations may have a fatal course. In elderly patients, candida infections areis common and underdiagnosed, particularly in those who wear dentures. It can be prevented with a good oral care regimen. Candidiasis serves as a marker for systemic diseases, such as diabetes mellitus and is most commonly seen in immunocompromised patients. C albicans is a normal commensal of the mouth and generally causes no problems in healthy people. Overgrowth of candida species, can cause local discomfort, burning sensation, altered taste sensation, dysphagia from oesophageal overgrowth resulting in poor nutrition, slow recovery, and prolonged hospital stay. In immunocompromised patients, infection can spread through the bloodstream or upper gastrointestinal tract leading to severe infection with significant morbidity and mortality.
Topical application of antifungal agent is used to treat most of the infection in a simple and effective manner. Nevertheless, topical agents may not be effective in treating chronic mucocutaneous candidiasis with immunosuppression. In such instances, systemic administration of antifungal agent is required. Antifungal agents, such as azoles (fluconazole, ketoconazole) and polyenes (amphotericin B or nystatin), are commonly used in the treatment of candida infections.
Coconut has proved to have a significant inhibitory action against common oral pathogens. Therefore, it can contribute to oral health to a great extent and is useful for the development of medicines against various diseases. The parts of its fruit like coconut kernel and tender coconut water have antimicrobial and antioxidant properties.
Probiotics are live microorganisms that, when administered in adequate amounts, confer a health benefit on the host. Probiotics consumption leads to improvement of intestinal health, improves the symptoms of lactose intolerance, and reduces the risk of various diseases. A variety of well-characterized strains of Lactobacilli and Bifidobacteria are available for human use. Probiotic bacteria have been used to modify microflora ecosystems and have shown some success as a therapeutic for oral diseases.
This study aims to evaluate and compare the antifungal activity of clotrimazole, coconut oil, and probiotics against Candida.
| Materials and Methods|| |
The study was approved by Institutional Human Ethical Committee (Ref No. IHEC/SDC/OMED-2002/21/54). Checklist for Reporting in-vitro Studies (CRIS) guidelines was followed for conducting this study. [Flowchart 1] shows the items in CRIS guidelines.
Coconut oil and probiotics were tested against clotrimazole, a positive control, at three different concentrations 25 μL, 50 μL, and 100 μL.
Sample preparation and handling
Rose Bengal Agar, a selective medium to detect and enumerate yeasts and moulds, in various samples was chosen to culture Candida albicans. Neutral pH media with antibiotics was selected for this study as it has an advantage for fungal growth compared to acidified media as the later, may inhibit fungal growth or fail to inhibit bacterial growth and may restrict the size of mold colonies. Besides providing better isolation of slow-growing fungi, rose bengal dye is also taken up my fungal isolates, thereby aiding in their recognition. Candida albicans were isolated and incubated in Rose Bengal Agar with chloramphenicol at 37° C for 48 h.
Coconut oil and probiotics as test materials and clotrimazole as a positive control were used in this study. The fresh coconut was grated and pressed to obtain coconut milk. The resultant coconut milk was left to rest for 48 h in a sterile and closed container. After 48 h, the liquid and solid parts were separated, and the oil found on the surface was then extracted and stored in a dark and sterile bottle until use. Probiotics containing Lactobacillus acidophilus strain were used in this study as it has better antifungal activity compared to other Lactobacillus species. Candid mouth paint from Glenmark containing 1% w/v Clotrimazole I.P. in glycerine I.P. and propylene glycol I.P. base was used as control.
Allocation sequence, randomization and blinding
Antifungal activity of clotrimazole, coconut oil, and probiotics was determined using the Disc Diffusion method. A total of 25 μL, 50 μL, and 100 μL of clotrimazole, coconut oil, and probiotics was randomly dispensed into the wells and placed on its surface at equal distance. The plates were then incubated at 37° C aerobically for 24 h. The investigator and statistician were blinded and analyzed the results. Growth inhibitory effect was evaluated using the direct contact method of the test materials as well as control to Candida in a sterile tube. The reading was performed after 24 h of incubation for Candida albicans, the diameter of the zone of inhibition around each disc was measured and recorded as the mean diameter (mm).
The normality was checked based on the Shapiro–Wilk Test (α = 0.05). The zone of inhibitions of clotrimazole, coconut oil, and probiotics were compared against one another by the parametric One-way ANOVA and Turkey Kramer test for multiple independent groups and independent t-test for two independent groups. The significance (P-value) was set at 0.05.
| Results|| |
The antifungal susceptibility test showed that C. albicans was susceptible to clotrimazole, coconut oil, and probiotics by having a clear zone of inhibition.
[Figure 1] shows the growth inhibitory effect of 1% clotrimazole, coconut oil, and probiotics at 25 μL, 50 μL, and 100 μL concentrations.
|Figure 1: Showing growth inhibitory effect of clotrimazole, coconut oil and probiotics|
Click here to view
[Table 1] shows the comparison of the zone of inhibition diameter between different concentrations of teat materials and control. It was found that the mean zone of inhibition for clotrimazole was 30 mm at 25 μL, 33 mm at 50 μL, and 45 mm at 100 μL, whereas for coconut oil it was 15 mm at 25 μL, 10 mm at 50 μL, and 16 mm at 100 μl; and for probiotics it was 15 mm at 25 μL, 10 mm at 50 μL, and 10 mm at 100 μL.
|Table 1: Showing diameter of the zone of inhibition of 1% clotrimazole, coconut oil, and probiotics at different concentrations|
Click here to view
Percentage of inhibitory effect at 25 μL concentration was 50% for clotrimazole, 25% for coconut oil, and 25% for probiotics. At 50 μL concentration, the percentage of inhibitory effect was 62% for clotrimazole, 19% for coconut oil, and 19% for probiotics. At 100 μL concentration, the percentage of inhibitory effect was 63% for clotrimazole, 23% for coconut oil, and 14% for probiotics [Figure 2].
|Figure 2: Showing percentage of inhibitory effect of clotrimazole, coconut oil and probiotics at 25, 50 and 100 micro litre concentrations|
Click here to view
The normality was assessed using Shapiro–Wilk test, and the P value was found to be 0.73, and all the groups had been distributed normally. The difference between all the groups was analyzed using the ANOVA test, and the results were statistically significant (P-value 0.002). Turkey Kramer test was applied to compare the zone of inhibition between two groups. On comparing the zone of inhibition of clotrimazole and coconut oil, clotrimazole had a greater antifungal effect, and the results were statistically significant (P-value 0.004). On comparison of inhibitory zone between clotrimazole and probiotics, clotrimazole was found to have a greater inhibitory effect, and the results were statistically significant (P-value 0.003). On assessing the antifungal activity of coconut oil and probiotics, coconut oil had more inhibitory effect, and the difference between the groups was not statistically significant (P- value 0.88).
| Discussions|| |
Oral candidiasis is the most prevalent opportunistic infection of the oral cavity. Candida albicans causes oral lesions in the vast majority of cases. Treatment will not be successful unless the underlying predisposing factors are addressed that can lead to recurrence. In general, antifungal agents such as azoles and polyenes are the recommended treatment. These causes alterations in RNA or DNA metabolism or leads to intracellular accumulation of peroxide in the Candida cells. Treatment is decided based on the clinical presentation of the disease. In case of mild presentation, a topical antifungal agent is recommended, and 1% clotrimazole mouth paint, nystatin oral suspension, and miconazole gels are prescribed. Moderate and severe infections are preferably treated with systemic antifungal agents such as fluconazole. Clotrimazole is an imidazole compound that has significant activity against a broad range of superficial and systemic infections caused by pathogenic yeasts, dermatophytes, and filamentous fungi, including C. albicans. The primary site of action of clotrimazole is shown as the inhibition of C. albicans respiration by inhibiting the activity of nicotinamide adenine dinucleotide phosphate oxidase (NADH) oxidase at the mitochondrial level. Clotrimazole damages the permeability barrier in the fungal cytoplasmic membrane, thereby inhibiting the biosynthesis of ergosterol in a concentration-dependent manner through the inhibition of demethylation of 14 alpha lanosterol. When ergosterol synthesis becomes inhibited, the cell is no longer able to construct an intact and functional cell membrane. Hence, in the present study, clotrimazole was taken as the standard antifungal agent, against which other antimicrobial agents were tested.
In the present study, clotrimazole showed increased antifungal property when compared to coconut oil and probiotics. Clotrimazole had the highest inhibitory effect at 100 μL. The results are in accordance with studies performed by Khan et al., comparing the antifungal sensitivity of fluconazole, clotrimazole, and nystatin where clotrimazole was most sensitive to Candida albicans compared to the other two agents. Topical clotrimazole delivery is intended to treat candidiasis by effective penetration of drugs into the stratum corneum. Poor dermal bioavailability, lesser penetration effect, and variable drug concentrations limit the efficiency of topical formulation. So, recently many studies are aimed to improve the bioavailability of clotrimazole and to increase muco-retentive property. Bolla et. al., loaded ufosomes into clotrimazole, and the study concluded that it improved the bioavailability. Harish et al., formulated and evaluated the in-situ gel containing clotrimazole for oral candidiasis, and the optimized formulations were able to release the drug for up to 6 h.
Coconut oil is known to exhibit antimicrobial activity against Streptococcus mutans and Candida albicans. It contains 92% saturated fatty acids, approximately 50% of which is lauric acid. Monolaurin and other medium-chain monoglycerides are shown to have the capacity to alter microbial cell walls, penetrate and disrupt cell membranes, and inhibit enzymes involved in energy production and nutrient transfer, leading to the death of the bacteria. Bergsson et al. showed the susceptibility of Candida albicans to several fatty acids and their 1-monoglycerides. In the present study, coconut oil has shown antifungal activity that is lesser that of clotrimazole which was different from results obtained Shino et al. and Ogbolu et al. Shino et al., have concluded that coconut oil and chlorhexidine showed significant antifungal activity which was comparable with ketaconazole. Kannan et al., evaluated the antifungal activity of virgin coconut oil and clotrimazole against Candida albicans, and concluded that coconut oil has strong potential therapeutic value. Coconut oil had the highest inhibitory effect at 100 μL.
According to a World Health Organization (WHO)/Food and Agricultural Organization (FAO) report (2002), probiotics are “live micro-organisms which, when administered in adequate amounts, confer a health benefit on the host.” Hatakka et al. showed a reduced prevalence of Candida albicans after taking probiotics in cheese. Radi et al., compared the antifungal activity of 10 lactobacillus strains and found that lactobacillus acidophilus had the most effective antifungal activity. Therefore, in our study, we chose Lactobacillus acidophilus to test against test materials and control. Results obtained from this study showed that probiotics has lesser anti-fungal activity compared to clotrimazole which is in accordance to the study conducted by Kõll et al. and Shino et al. Probiotics had the highest inhibitory effect at 25 μL.
In recent times, the use of herbal alternatives for the treatment of disease is becoming popular as it has fewer side effects. Modern pharmacology derives approximately 30% of the drugs from plants and synthetic analogues, which were built on prototype compounds derived from plants. Jain et at. assessed antifungal property of Aloevera gel and Triphala and the results showed at higher concentrations it exhibited significant antifungal property and can be used as a promising adjunct for antifungal agents. However, it is important to compare the effectiveness of these drugs with the gold standard. In our study, antifungal activities of coconut oil and probiotics were not as effective as clotrimazole. The decreased antifungal activity of probiotics could be due to differences in cell concentrations. The variation in results across studies could be due to different lactobacilli strains used, the method used for evaluating antifungal effects, Candida species used in the study, and the duration of incubation. The limitations of this study would be sample size calculation was not performed. Future studies should concentrate on other herbal alternatives which will be as effective as clotrimazole for the management of oral candidiasis and the synergistic effect of adding coconut oil and probiotics to clotrimazole.
| Conclusion|| |
This study scientifically proves the antifungal activity of clotrimazole, coconut oil, and probiotics. The antifungal activity of clotrimazole was found to be higher than that of coconut oil and probiotics against Candida albicans. The disadvantage of a topical drug delivery system is poor drug availability, so newer targeted drug delivery systems could be devised for increasing the bioavailability. Future studies on other herbal alternatives which will be as effective as clotrimazole for the management of oral candidiasis or synergistic effect of coconut oil or probiotics in clotrimazole can be tried.
Financial support and sponsorship
Study funded by Saveetha Dental College, Saveetha Institute of Medical and Technical Sciences (SIMATS), Saveetha University.
Conflicts of interest
There are no conflicts of interest.
| References|| |
Jayapriya R, Maheswari TU, Chaithanya VM. Multiple variants of oral candidiasis as a predictor for HIV/AIDS diagnosis-A case report. Biomedicine. 2019;39 (2):372-6.
Sitaraman P, Muthukrishnan A. Considerations for Management of Candida--Associated Leukoplakia. Indian Journal of Public Health Research & Development. 2019 Nov 1;10 (11).
Shino B, Peedikayil FC, Jaiprakash SR, Ahmed Bijapur G, Kottayi S, Jose D. Comparison of antimicrobial activity of chlorhexidine, coconut oil, probiotics, and ketoconazole on Candida albicans isolated in children with early childhood caries: An in vitro
study. Scientifica. 2016 Jan 1;2016.
Iinuma T, Arai Y, Abe Y, Takayama M, Fukumoto M, Fukui Y, et al
. Denture wearing during sleep doubles the risk of pneumonia in the very elderly. J Dent Res 2015;94:28S-36S.
Rodrigues CF, Rodrigues ME, Henriques M. Candida sp. infections in patients with diabetes mellitus. J Clin Med 2019;8:76. doi: 10.3390/jcm8010076.
AL-Ruaby KJ, Kadhum RW. Conventional and molecular diagnosis of oral candidiasis in Iraqi patients. Indian J Forensic Med Toxicol 2019;13:1301-6.
Akpan A, Morgan R. Oral candidiasis. Postgraduate medical journal. 2002 Aug 1;78 (922):455-9.
Haller I. Mode of action of clotrimazole: Implications for therapy. Am J Obstet Gynecol 1985;152:939-44.
Hitchcock CA, Dickinson K, Brown SB, Evans EG, Adams DJ. Interaction of azole antifungal antibiotics with cytochrome P-450-dependent 14 alpha-sterol demethylase purified from Candida albicans. Biochem J 1990;266:475-80.
Ghannoum MA, Rice LB. Antifungal agents: Mode of action, mechanisms of resistance, and correlation of these mechanisms with bacterial resistance. Clin Microbiol Rev 1999;12:501-17.
Jose M, Cyriac MB, Pai V, Varghese I, Shantaram M. Antimicrobial properties of cocos nucifera (coconut) husk: An extrapolation to oral health. J Nat Sci Biol Med 2014;5:359-64.
Williams NT. Probiotics. Am J Health-Syst Pharm 2010;67:449-58.
Kechagia M, Basoulis D, Konstantopoulou S, Dimitriadi D, Gyftopoulou K, Skarmoutsou N, et al
. Health benefits of probiotics: A review. ISRN Nutr 2013;2013. doi: 14.5402/2013/481651.
Krithikadatta J, Gopikrishna V, Datta M. CRIS guidelines (checklist for reporting In-vitro studies): A concept note on the need for standardized guidelines for improving quality and transparency in reporting in-vitro studies in experimental dental research. J Conserv Dent 2014;17:301-4.
] [Full text]
Bartolommei G, Tadini-Buoninsegni F, Hua S, Moncelli MR, Inesi G, Guidelli R. Clotrimazole inhibits the Ca2+-ATPase (SERCA) by interfering with Ca2+ binding and favoring the E2 conformation. J Biol Chem 2006;281:9547-51.
Jan CR, Tseng CJ, Chou KJ, Chiang HT. Novel effects of clotrimazole on Ca2+ signaling in Madin Darby canine kidney cells. Life Sci 2000;66:2289-96.
Khan F, Baqai R. In vitro
antifungal sensitivity of fluconazole, clotrimazole and nystatin against vaginal candidiasis in females of childbearing age. J Ayub Med College Abbottabad 2010;22:197-200.
Bolla PK, Meraz CA, Rodriguez VA, Deaguero I, Singh M, Yellepeddi VK, et al
. Clotrimazole loaded ufosomes for topical delivery: Formulation development and in-vitro studies. Molecules 2019;24:3139. doi: 10.3390/molecules24173139.
Harish NM, Prabhu P, Charyulu RN, Gulzar MA, Subrahmanyam EV. Formulation and evaluation of in situ
gels containing clotrimazole for oral candidiasis. Indian J Pharm Sci 2009;71:421-7.
] [Full text]
Quindós G, Gil-Alonso S, Marcos-Arias C, Sevillano E, Mateo E, Jauregizar N, et al
. Therapeutic tools for oral candidiasis: Current and new antifungal drugs. Med Oral Patol Oral Cir Bucal 2019;24:e172-80.
Bergsson G, Arnfinnsson J, Steingrímsson O, Thormar H. In vitro
killing of Candida albicans by fatty acids and monoglycerides. Antimicrobial Agents and Chemotherapy 2001;45:3209-12.
Ogbolu DO, Oni AA, Daini OA, Oloko AP. In vitro antimicrobial properties of coconut oil on candida species in Ibadan, Nigeria. J Med Food 2007;10:384-7.
Kannan N, Mohammed A. Comparative evaluation of antifungal activity of Cocos nucifera oil against candida albicans. Int J Phytother Res 2014;4:27-31.
Hatakka K, Ahola AJ, Yli-Knuuttila H, Richardson M, Poussa T, Meurman JH, Korpela R. Probiotics reduce the prevalence of oral Candida in the elderly—a randomized controlled trial. J Dent Res 2007;86:125-30.
Radi NA, Abdelmonem AA. A study on the antifungal effects of lactobacillus spp. on candida species. Egypt J Med Microbiol (EJMM) 2017;26:122-5.
Kõll P., Mändar R., Marcotte H., Leibur E., Mikelsaar M., Hammarström L. Characterization of oral lactobacilli as potential probiotics for oral health. Oral Microbiol Immunol
Panjwani S, Rai S, Misra D, Misra A. Herbinaturals: A new paradigm in dentistry. J Indian Acad Oral Med Radiol 2016;28:150-4
Jain S, Mujoo S, Daga M, Kalra S, Nagi R, Laheji A. Comparison of antifungal effect of Aloevera gel and Triphala: An in vitro
study. Journal of Indian Academy of Oral Medicine and Radiology 2017;29:90.
Dheepika B, Maheswari U. Aloe vera in oral diseases-A review. Int J Pharm Pharm Sci 2014;6:64-6.
[Figure 1], [Figure 2]