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ORIGINAL ARTICLE
Year : 2017  |  Volume : 15  |  Issue : 1  |  Page : 89-93

Comparative Evaluation of Antifungal Activity of Piper Betel Leaf Oil, Origanum vulgare Essential Oil and Fluconazole Suspension on Candida albicans − An In Vitro Study


1 Department of Public Health Dentistry, Bapuji Dental College and Hospital, Davangere, India
2 Reader, Department of Public Health Dentistry, Bapuji Dental College and Hospital, Davangere, India

Date of Web Publication14-Mar-2017

Correspondence Address:
Nisha Makkar
Department of Public Health Dentistry, Bapuji Dental College and Hospital, Room No. 08, MCC ‘B’ Block, Davangere 577004, Karnataka
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jiaphd.jiaphd_134_16

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  Abstract 

Introduction: Oropharyngeal candidiasis is an opportunistic mucosal infection caused by Candida albicans. It usually responds to topical treatments such as clotrimazole troches, topical fluconazole, chlorhexidine mouthwash and nystatin suspension. Piper betel leaf oil and Origanum vulgare essential oil have shown some topical antifungal activity. Aim: To determine and compare the antifungal efficacy of piper betel leaf oil, O. vulgare essential oil and fluconazole suspension against C. albicans. Materials and Methods: The zone of inhibition was measured by the cup–plate diffusion method using 100 μl volume of piper betel leaf oil, O. vulgare essential oil and fluconazole suspension, which were pipetted into the wells of the inoculated Sabouraud’s dextrose agar plates. The zone of inhibition was measured in millimetres using Vernier calliper. Minimum inhibitory concentration (MIC) was determined by the broth macro-dilution test by pouring 1 ml of the respective concentrations of the test material to the individual test tubes along with 10 μl of the diluted test organism inoculum. Finally, MIC was calculated using a reflective viewer. Results: The zone of inhibition for O. vulgare essential oil (>40 mm) was more than fluconazole suspension (>35 mm). MIC of O. vulgare essential oil, piper betel leaf oil and fluconazole suspension was 1.6%, 0.4% and 0.8%, respectively. Conclusion: O. vulgare essential oil was found to be a more effective antifungal agent than piper betel leaf oil and fluconazole suspension.

Keywords: Candida albicans, fluconazole, in vitro study, origanum vulgare essential oil, piper betel leaf oil


How to cite this article:
Makkar N, Prasanna SB, Singla H. Comparative Evaluation of Antifungal Activity of Piper Betel Leaf Oil, Origanum vulgare Essential Oil and Fluconazole Suspension on Candida albicans − An In Vitro Study. J Indian Assoc Public Health Dent 2017;15:89-93

How to cite this URL:
Makkar N, Prasanna SB, Singla H. Comparative Evaluation of Antifungal Activity of Piper Betel Leaf Oil, Origanum vulgare Essential Oil and Fluconazole Suspension on Candida albicans − An In Vitro Study. J Indian Assoc Public Health Dent [serial online] 2017 [cited 2019 Dec 15];15:89-93. Available from: http://www.jiaphd.org/text.asp?2017/15/1/89/201931


  Introduction Top


Oropharyngeal candidiasis (OPC) is a frequent opportunistic infection among the immunocompromised and the elderly populations. It is caused by an overgrowth or infection by a yeast-like fungus, primarily, Candida albicans.[1] The incidence of C. albicans isolated from the oral cavity has been reported to be 45% in neonates, 45–65% in healthy children, 30–45% in healthy adults, 50–65% in people who wear removable dentures, 65–88% in those residing in acute and long-term care facilities, 90% in patients with acute leukaemia undergoing chemotherapy and 95% in patients with human immunodeficiency virus.[2] The clinical manifestations include pseudo-membranous and erythematous forms, causing symptoms such as pain, burning sensation and altered taste, which subsequently lead to nutritional compromise.[1] OPC is a complex mixture of yeasts and hyphae surrounded by extra-polymeric matrix material, which encases the cells within to form an impermeable barrier to host defences and antimicrobial therapy.[3]

Various antifungal agents have been used for the treatment of OPC, with the commonly used ones being topical fluconazole, chlorhexidine mouthwash and nystatin suspension.[4] Because many of the available antifungal drugs have undesirable side effects or are very toxic (amphotericin B), produce recurrence, show drug–drug interactions (azoles) or lead to the development of resistance (fluconazole, 5-flucytosine), some of these drugs are ineffective and have become less successful in therapeutic strategies.[5] Therefore, it is necessary to search for effective and less toxic alternative antifungal agents that would overcome these disadvantages.

Since antiquity, plants and their extracts have been used in traditional or folk medicine. The World Health Organization, in 2005, adopted the policy to practice more of traditional medicine in most of the developing nations for primary health care.[6] Piper betel and Origanum vulgare have invaluable medicinal properties such as carminative, diuretic, antiviral, anticancer, immunomodulation, anti-inflammatory, antimicrobial and antioxidant. They have shown some topical antifungal activity, with recent clinical data indicating its efficacy in the treatment of vaginal candidiasis.[7],[8]

The review of the literature showed that none of the studies had compared the antifungal efficacy of piper betel leaf oil, O. vulgare essential oil and fluconazole suspension on C. albicans. Hence, the aim of this study was to compare the antifungal activities of piper betel leaf oil, O. vulgare essential oil and fluconazole suspension on C. albicans to evaluate its use as a therapeutic agent with a research hypothesis that there is a difference in antifungal activities of piper betel leaf oil, O. vulgare essential oil and fluconazole suspension on C. albicans.


  Materials and Methods Top


The protocol for this in vitro study was approved by the Institutional Review Board. Commercially available piper betel leaf oil and O. vulgare essential oil were procured from Kanta Manufacturers, New Delhi. The antimicrobial activity of essential oils was analysed using the strains procured as microbial type culture collections (MTCC) from the Institute of Microbial Technology, Chandigarh. Standard strain of C. albicans (MTCC No. 2091) was grown and maintained on Sabouraud’s dextrose agar slants.

Nutrient broth was prepared according to the manufacturer’s instructions. This was used to get the viable growth of microbes. After autoclaving the broth, it was cooled to 37°C, and 7 ml of this broth was poured into each test tube. Thereafter, freeze-dried form of the microbes was added to it under strict sterile conditions. The test tubes were incubated at 37°C for 48 h. Turbidity in the test tube confirmed the growth of the microbes. Comparison of this turbidity was made with McFarland 0.5 turbidity standard.[8]

Determination of zone of inhibition by agar well diffusion method

The antifungal potential of the testing essential oils was evaluated by Agar well diffusion method.[9]  Petri dish More Detailses containing Sabouraud’s dextrose agar for C. albicans were inoculated with approximately 100 μl of microbial strain using swab technique.

The agar plates were allowed to dry, and five wells or cups of 8 mm diameter were prepared with a sterile cork borer. With a micropipette, 5, 10, 25, 50 and 75 μl volume of each essential oil was propelled directly into the wells of the inoculated, specific media agar plates. The plates were allowed to stand for 10 min for diffusion of the essential oils to take place and incubated at 37°C for 48 h. After incubation for 48 h at 37°C, the plates were examined for the presence of clear zones of growth inhibition surrounding the wells containing the essential oils, which pointed to their efficacy against C. albicans. The zone of inhibition was measured in one plane using Vernier calliper including the well diameter and expressed in millimeters.

Determination of minimum inhibitory concentration

The minimum inhibitory concentration (MIC) is reported as the lowest concentration of the extracts capable of inhibiting the growth of the organism tested.[9] In this study, macro-broth dilution technique was employed to determine the susceptibility of the strains of C. albicans to the oil of O. vulgare and piper betel leaf oil. Susceptibility was expressed as MIC. Fluconazole was used as a positive control against C. albicans. One ml of Sabouraud’s broth for C. albicans was poured into each of the 10 test tubes. The test tubes were plugged with cotton and sterilized in an autoclave. After cooling, 1 ml of the test material was added to test tubes 1 and 2, and the test tubes were capped. The test material was vortexed for 5 s. One ml was withdrawn from the contents of test tube 2 and transferred to test tube 3. This process was continued till the ninth test tube. One ml of properly diluted test organism inoculum was added to each of the test tubes and was incubated at 37°C for 48 h.[9] Following incubation, MIC was calculated as the lowest concentration of the products inhibiting the visible growth of Candida strain using a reflective viewer. All investigations were repeated thrice, and the mean observation was calculated. No statistical tests were employed, and the data obtained were appraised observationally.


  Results Top


In this study, we had examined that the piper betel leaf oil and O. vulgare essential oil exhibited antifungal efficacies against C. albicans at different concentrations. The zone of inhibition of C. albicans was found to increase with increasing volume of piper betel leaf oil and O. vulgare. The maximum zone of inhibition was >40 mm for O. vulgare oil at a volume of 25 μl when compared to piper betel leaf oil and fluconazole suspension, that is, 17 mm and >35 mm at 75 μl, respectively, as depicted in [Table 1] and [Figure 1].
Table 1: Zone of inhibition of Candida albicans by different agents and volumes

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Figure 1: In vitro antifungal activity of piper betel leaf oil and Origanum vulgare oil against Candida albicans. Exponentially growing C. albicans cells were cultured with varying concentrations of different agents. (A) Zone of inhibition with piper betel leaf oil. (B) Zone of inhibition with O. vulgare oil

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MIC of the tested agents was expressed in terms of whether the organism was sensitive or resistant at a particular concentration. In this study, the broth macro-dilution technique was employed to determine the susceptibility of C. albicans to O. vulgare and piper betel leaf oil and fluconazole as positive control as shown in [Figure 2]. It was found that MIC of O. vulgare oil was 0.4%, and that of fluconazole and piper betel leaf oil was 0.8% and 1.6%, respectively, as displayed in [Table 2].
Figure 2: Broth macro dilution technique employed for different agents to determine MIC. (A) Broth containing piper betel leaf oil. (B) Broth containing Origanum vulgare oil. (C) Broth containing fluconazole

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Table 2: Minimum inhibitory concentration of different agents against Candida albicans

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  Discussion Top


Nowadays, the indiscriminate use of commercial antimicrobial drugs has caused multiple drug resistance in human pathogenic microorganisms. The resistant strains of C. albicans have become a cause of major health concerns, and novel antifungal agents are required to tackle this problem. This situation forced the scientists to search for new and effective antimicrobial agents to replace the current regimens.

Since a long time, medicinal plants have played a vital role in promoting the health of mankind. Traditional medicine has remained as the most affordable and easily accessible source of treatment in the primary health care system of resource-poor communities and countries. About 80% of the population in the developing countries use traditional medicines, because they cannot afford the high cost of western pharmaceuticals and health care, and because traditional medicines are more acceptable from a cultural and spiritual perspective.[10]

In this study, the in vitro antifungal activities of piper betel leaf oil, O. vulgare essential oil, and fluconazole suspension against C. albicans were compared. This study has significance in the wake of growing trend in the usage of herbal products in medicine.

C. albicans is a dimorphic, yeast-like fungus, which resides as a commensal in the muco-cutaneous cavities of the skin, the vagina and the intestine of humans. It can cause infections under altered physiological and pathological conditions such as infancy, pregnancy, diabetes, prolonged broad-spectrum antibiotic administration, steroidal chemotherapy and acquired immune deficiency syndrome. With regard to oral candidiasis, C. albicans generally accounts for around 50% of the cases; a similar prevalence of C. albicans occurs in patients with systemic candidiasis.[11]

The betel leaf, commonly known as ‘Paan’ or ‘Nagvalli’ (family − Piperaceae), and Origanum oil or Ajwain are evergreen, perennial creepers cultivated in most parts of India. Betel leaves are associated with every sphere of human life including social, cultural and religious, and this association is very much relevant even in modern times. In Indian folk medicine, betel leaf is popular as an antiseptic and is commonly applied on wounds and lesions because of its healing effects.[12] Essential oil extracted from betel leaf and O. vulgare exhibited inhibitory effect on fungal species of clinical significance.[7],[8] Therefore, this study aimed to compare the antifungal activities of piper betel leaf oil, O. vulgare essential oil and fluconazole suspension on C. albicans.

Agar well diffusion method is considered to be a standard, simple, cost-effective and qualitative method for assessing the antimicrobial property of the drug.[13] The maximum zone of inhibition by well diffusion method for O. vulgare oil was >40 mm, which was more when compared with fluconazole, that is, >35 mm. Piper betel leaf oil produced a maximum zone of inhibition of 17 mm, which was very less when compared to fluconazole probably because of less diffusion through agar medium.

To substantiate the results obtained from the diffusion method, MIC of piper betel leaf oil and O. vulgare essential oil was compared with fluconazole in this study using the broth macro-dilution method. MIC is considered to be more meaningful in assessing the antimicrobial property of test agent, because quantitative results will be obtained.[13]

C. albicans was most sensitive for O. vulgare oil when compared to piper betel leaf oil and fluconazole concentrations. In a study conducted by Ali et al.[5] using hydroxychavicol isolated from piper betel leaf, MIC values were 12.5–50% against C. albicans, which were higher than this study because of the different strains of Candida being used. Furthermore, piper betel leaf oil was reported to inhibit the growth of C. albicans with an inhibition zone of 12 mm in the study conducted by Saxena et al.,[14] which used disk diffusion and broth dilution assays against various bacterial and fungal strains.MIC result of this study was double compared to the study conducted by Manohar et al.,[15] in which C. albicans was found to be sensitive to O. vulgare at a concentration of 0.2% using broth micro-dilution method. Another study reported by Adams et al.[16] concluded that antifungal effect of Origanum essential oil was relatively higher at 4% when compared to that at 2%.

Hydroxychavicol and carvacrol are the major constituents of piper betel leaf oil and O. vulgare oil, respectively,[5],[15] and are responsible for the antifungal and antibacterial activities. They alter the cell membrane structure resulting in the disruption of the permeability barrier of microbial membrane structure.[15]

O. vulgare oil was found to be a more effective antifungal agent against C. albicans when compared to piper betel leaf oil and fluconazole. This study helps in promoting traditional medicine for modern dentistry, as piper betel leaf and O. vulgare essential oil are easily accessible, economically feasible, culturally acceptable and less toxic.

The results from this study not only encourage the researcher to evaluate the efficacy of piper betel leaf oil and O. vulgare oil in other forms of systemic and superficial fungal infections, but also to explore its broad-spectrum effect against other pathogenic manifestations including malignancies. Further research may be performed to study the detailed mechanism of the action of betel leaves and Origanum oil in various metabolic activities in human, which will be beneficial to mankind.


  Conclusion Top


The result of this study clearly demonstrates that the O. vulgare oil is more effective as an antifungal agent against C. albicans when compared to piper betel leaf oil and fluconazole.

The result of this study helps in promoting traditional medicine for modern dentistry, as piper betel leaf oil and O. vulgare essential oil are easily accessible, economically feasible, culturally acceptable and less toxic. Therefore, these essential oils can be used in the development of antimicrobial agents as an herbal remedy, as an alternative to synthetic antimicrobial agents and for patients allergic to synthetic counterparts.

Acknowledgements

Our heartfelt gratitude to Dr. Krishore G. Bhat, Professor and Head, Department of Microbiology, and the Director, Department of Molecular Biology and Immunology, Maratha Mandal’s NGH Institute of Dental Sciences & Research Centre for his help in conducting the microbial laboratory analysis.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Coco BJ, Bagg J, Cross LJ, Jose A, Cross J, Ramage G. Mixed Candida albicans and Candida glabrata populations associated with the pathogenesis of denture stomatitis. Oral Microbiol Immunol 2008;23:377-83.  Back to cited text no. 1
    
2.
Akpan A, Morgan R. Oral candidiasis. Postgrad Med J 2002;78:455-9.  Back to cited text no. 2
    
3.
Ramage G, Jose A, Coco B, Rajendran R, Rautemaa R, Murray C et al. Commercial mouthwashes are more effective than azole antifungals against Candida albicans biofilms in vitro. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2011;111:456-60.  Back to cited text no. 3
    
4.
Pappas PG, Rex JH, Sobel JD, Filler SG, Dismukes WE, Walsh TJ et al. Guidelines for treatment of candidiasis. Clin Infect Dis 2004;38:161-89.  Back to cited text no. 4
    
5.
Ali I, Khan FG, Suri KA, Gupta BD, Satti NK, Dutt P et al. In vitro antifungal activity of hydroxychavicol isolated from Piper betle L. Ann Clin Microbiol Antimicrob 2010;9:7.  Back to cited text no. 5
    
6.
WHO Traditional Medicine Strategy. 1st ed. Geneva: World Health Organization; 2002.  Back to cited text no. 6
    
7.
Patra B, Das MT, Dey SK. A review on Piper betle L. J Med Plants Stud 2016;4:185-92.  Back to cited text no. 7
    
8.
Cleff MB, Meinerz AR, Xavier M, Schuch LF, Meireles MC, Rodrigues MR et al. In vitro activity of Origanum vulgare essential oil against Candida species. Braz J Microbiol 2010;41:116-23.  Back to cited text no. 8
    
9.
Caburian AB, Osi MO. Characterization and evaluation of antimicrobial activity of the essential oil from the leafs of Piper betle L. E-Int Sci Res J 2010;2:2-13.  Back to cited text no. 9
    
10.
Hosseinzadeh S, Jafarikukhdan A, Hosseini A, Armand R. The application of medicinal plants in traditional and modern medicine: A review of Thymus vulgaris. Int J Clin Med 2015;6:635-42.  Back to cited text no. 10
    
11.
Williams D, Lewis M. Pathogenesis and treatment of oral candidosis. J Oral Microbiol 2011;3:5771.  Back to cited text no. 11
    
12.
Sripradha S. Betel leaf − The green gold. J Pharm Sci Res 2014;6:36-7.  Back to cited text no. 12
    
13.
Jorgensen JH, Ferraro MJ. Antimicrobial susceptibility testing: A review of general principles and contemporary practices. Clin Infect Dis 2009;49:1749-55.  Back to cited text no. 13
    
14.
Saxena M, Khare NK, Saxena P, Syamsundar KV, Srivastava SK. Antimicrobial activity and chemical composition of leaf oil in two varieties of Piper betle from northern plains of India. J Sci Ind Res 2014;73:95-9.  Back to cited text no. 14
    
15.
Manohar V, Ingram C, Gray J, Talpur NA, Echard BW, Bagchi D et al. Antifungal activities of Origanum oil against Candida albicans. Mol Cell Biochem 2001;228:111-7.  Back to cited text no. 15
    
16.
Adams A, Kumar S, Clauson M, Sahi S. Anti-yeast activities of Origanum oil against human pathogenic yeasts. Adv Biosci Biotechnol 2011;2:103-7.  Back to cited text no. 16
    


    Figures

  [Figure 1], [Figure 2]
 
 
    Tables

  [Table 1], [Table 2]



 

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