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Table of Contents   
ORIGINAL RESEARCH  
Year : 2013  |  Volume : 24  |  Issue : 4  |  Page : 401-405
Antimicrobial activity of plant extracts on Candida albicans: An in vitro study


1 Department of Oral Pathology and Microbiology, Teerthanker Mahaveer Institute of Dental Sciences and Research Centre, Teerthanker Mahaveer University, Bagarpur, Delhi Road, Moradabad, Uttar Pradesh, India
2 Department of Oral Pathology and Microbiology, MGM Dental College and Hospital, Kamothe Navi Mumbai, Maharashtra, India
3 Department of Oral Pathology and Microbiology, Pacific Dental College and Research Centre, Udaipur, Rajasthan, India

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Date of Submission27-Apr-2012
Date of Decision24-Sep-2012
Date of Acceptance28-Nov-2012
Date of Web Publication19-Sep-2013
 

   Abstract 

Background and Objectives: Plants as sources of medicinal compounds have continued to play a predominant role in the maintenance of human health since ancient times. Even though several effective antifungal agents are available for oral candida infections, the failure is not uncommon because isolates of Candida albicans may exhibits resistance to the drug during therapy. The present study was conducted to evaluate the antimicrobial effects of few plant extracts on Candida albicans. An additional objective was to identify an alternative, inexpensive, simple, and effective method of preventing and controlling Candida albicans.
Materials and Methods: Fine texture powder or paste form of leaves was soaked in sterile distilled water and 100% ethyl alcohol, which were kept in refrigerator at 4°C for 24 h. Then filtrates were prepared and kept in a hot air oven to get a black shining crystal powder/paste form. Stock solutions of plant extracts were inoculated on petri plates containing species of Candida albicans and incubated at 25 ± 2°C for 72 h.
Results: Alcoholic curry leaves showed the maximum zone of inhibition on Candida albicans followed by aqueous tea leaves. The other plant extracts like alcoholic onion leaves, alcoholic tea leaves, alcoholic onion bulb, alcoholic aloe vera, and alcoholic mint leaves also inhibited the growth of Candida albicans but lesser extent.
Conclusion: The present study renders few medicinal plants as an alternative medicines to the field of dentistry which can be used adjunct to conventional therapy of oral candidasis.

Keywords: Alternative medicine, antimicrobial activity, biological agents, Candida albicans, complementary therapies, phytotherapy, plant extracts

How to cite this article:
Doddanna SJ, Patel S, Sundarrao MA, Veerabhadrappa RS. Antimicrobial activity of plant extracts on Candida albicans: An in vitro study. Indian J Dent Res 2013;24:401-5

How to cite this URL:
Doddanna SJ, Patel S, Sundarrao MA, Veerabhadrappa RS. Antimicrobial activity of plant extracts on Candida albicans: An in vitro study. Indian J Dent Res [serial online] 2013 [cited 2014 Oct 31];24:401-5. Available from: http://www.ijdr.in/text.asp?2013/24/4/401/118358
Oral candidal infections are considered opportunistic and hence the epithet "the disease of the diseased" given to this condition. Its occurrence has been increased remarkably since the prevalent use of antibiotics, and also in denture wearers, diabetics, HIV infected individuals, patients under chemotherapy, and transplant recipients. [1],[2],[3] Even though several effective antifungal agents are available for oral candidal infections, [4] the failure is not uncommon because isolates of Candida albicans (C. albicans) may exhibits intrinsic (primary) resistance or secondary resistance to the drug during therapy. [3] To cope with the wide-spread problem of antimicrobial resistance, antimicrobial alternatives have been proposed. [5],[6],[7]

Use of natural products for the control of fungal diseases is considered as an interesting alternative to synthetic fungicides due to their lower negative impact, reduced cost, and adverse reactions to plant preparations compared to modern conventional pharmaceuticals. India is considered to be a rich emporium of drug plants, mainly used in preventive and curative medicine. [8] Screening of medicinal plants for antimicrobial agents has gained much importance. Hence, there is urgent need to promote the traditional preventive measures that are acceptable, easily available, and cost effective.


   Aims and Objective Top


The present study aimed to identify inexpensive, simple, and effective plants which can prevent and control the growth of C. albicans.

To evaluate the antimicrobial effects of plants like tea leaves, onion leaves, onion bulb, aloe vera, mint leaves, and curry leaves on C. albicans involved in causing oral candidiasis.


   Materials and Methods Top


The following materials are used in this study

  1. Plant extracts of
    • Tea leaves (Camellia sinensis),
    • Onion leaves (Allium cepa),
    • Onion bulb (Allium cepa),
    • Mint leaves (Mentha piperita/Mentha Arvensis),
    • Aloe (Aloe vera)
    • Curry leaves (Murraya koenigii)
  2. Species of Candida albicans (MTCC 183)
Preparation of the plant extracts

Fresh leaves were thoroughly cleaned twice using distilled water. They were cut into pieces with the help of scissors/knife and grinded using a sterile electric grinding jar into fine texture powder/paste form. Aqueous and alcoholic plant extracts were prepared by dissolving the powdered or paste form of plant materials in sterile distilled water and 100% ethyl alcohol, respectively, in the ratio of 1:5 i.e., 20 gm of plant material in 100 ml of water/alcohol in a sterile 250 ml glass flask. Flasks were then plugged with cotton and kept in refrigerator at 4°C for 24 h. These were then filtered and kept in a hot air oven for 5-7 days at 30 ± 2°C to completely evaporate the solvent and to get a black shining crystal powder/paste form. Stock solutions of alcoholic extracts were prepared by dissolving 100 mg of the alcoholic extracts in 10 ml 50% of Dimethyl Sulfoxide (DMSO). Stock solutions of aqueous extracts were prepared by dissolving 100 mg of the aqueous extracts in 10 ml of sterile distilled water to get the 10 mg/ml. The stock solution of positive control (Flucanozole) was also prepared by dissolving 100 mg in 10 ml of sterile distilled water to get the 10 mg/ml. Stock solutions of each plant extracts were numbered [Table 1]. [9],[10]
Table 1: Antimicrobial activity of aqueous and alcoholic plant extracts against Candida albicans

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Procurement of microorganism

Freeze dried form of the microorganism C. albicans was obtained from Microbial Type Culture Collection (MTCC), Chandigarh.

Preparation of culture media for the study

According to the manufacturer's instruction, Ampoule containing freeze dried form of the microorganism was opened and the contents were added to the Yeast Extract Peptone Dextrose (YEPD) broth which was incubated at 25 ± 2°C for 72 h. The 30 ml of molten sterile agar was poured aseptically in each four sterile petri plates and were allowed to solidify at room temperature. Hundred microliter of inoculum was spread with a sterile steel spreader to prepare a lawn of microorganism. [1]

Ditch plate method

In three plates four wells were prepared. These wells were given numbers from 1 to 12. The wells were filled with fixed volume (250 μl) of respective stock solution of plant extract using micropipette. Then on the 4 th plate three wells were prepared in the similar fashion. Flucanozole was used as positive control, and 50% DMSO and sterile distilled water were maintained simultaneously as negative controls in the same plate. Then all the plates were incubated in an upright position at 25 ± 2°C for 24 h. The whole procedure was repeated twice. The inhibition zones were measured on the underside of the plates, using Hi-media zone scale after 24 and 48 h. [1]

Statistical analysis

The collected data were analyzed using statistical tests like mean value and analysis of variance (ANOVA) on an SPSS (Version 11.6) software database.


   Results Top


The means of the zones of inhibition of C. albicans by aqueous and alcoholic plant extracts at 24 and 48 h were measured. Alcoholic curry leaves showed strongest growth inhibition of C. albicans with 23.3 ± 0.14 and 24.05 ± 0.07, followed by aqueous tea leaves with 17.95 ± 0.07 and 18.10 ± 0.14 mm at 24 h and 48 h, respectively. Alcoholic onion leaves, alcoholic tea leaves, alcoholic onion bulb, alcoholic aloe vera, and alcoholic mint leaves extracts showed 17.95 ± 0.07 mm and 18 ± 0.0 mm, 16 ± 0.07 mm and 15.95 ± 0.0 mm, 13 ± 0.0 mm and 13.05 ± 0.07 mm, 13 ± 0.0 mm and 13 ± 0.0 mm and 12.95 ± 0.07 mm and 13 ± 0.0 mm at 24 and 48 h, respectively [Figure 1] and [Figure 2]. Other extracts did not demonstrate any antimicrobial activity. In general all the plant extracts showed antimicrobial activities extending up to 48 h at the critical difference of 5% (P ≤ 0.05) [Table 1].
Figure 1: Antimicrobial activity of plant extracts against C. Albicans after 24 h

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Figure 2: Antimicrobial activity of plant extracts against C. Albicans after 48 h

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   Discussion Top


C. albicans is the most common candida species residing in the oral cavity in both health and disease, and is the agent of most oral candida infections. Several effective antifungal agents are available for the management of candidiasis. But isolates may exhibit intrinsic or secondary resistance to the drug during therapy. [11] So the use of natural products as alternative agents for the control of fungal diseases is considered as an interesting alternative to synthetic fungicides. [12]

In the present in vitro study, few plant extracts like tea leaves, onion leaves and bulb, aloe vera, mint leaves, and curry leaves with medicinal values are screened for evaluating their antimicrobial activity against C. albicans. C. albicans was strongly inhibited by the alcoholic curry leaves followed by aqueous tea leaves. The alcoholic onion leaves, alcoholic tea leaves, alcoholic onion bulb, alcoholic aloe vera, and alcoholic mint leaves also inhibited the growth of C. albicans in a decreasing order, but not as strong as the above extracts. Remaining extracts did not show any inhibitory activity. This suggests that plant extracts can be used to inhibit the growth of C. albicans and thus they can be implicated in the prevention and treatment of oral candidal infections. In the present study, we confirm the inhibitory effect of aloe vera and Onion against C. albicans, which has been described before by Richerd wynn (2005) [13] and Cowan M.M. (1999). [14]

Antimicrobial activity of all plant extracts was observed at 24 h and 48 h, respectively, in order to know their efficacy at different time intervals. This assay showed that the inhibition zone increased at 48 h compared to 24 h. There was no statistical significant difference in the inhibitory zones at 24 and 48 h with maximum inhibition at 24 h.

The efficacy of plants and their extracts is due to the presence of several primary and/or secondary metabolites such as phenolics, polyphenols, tannins, quercetin, flavones, flavonols, alkloids, terpenoids, lectins, polypeptides, and complex mixtures. [14] Although phytochemicals (plant derived metabolites) are antimicrobial in nature but they also produce other biological activities in oral cavity like induction of immunity, which indirectly reduces the risk of oral diseases. [15]

Math, and Balasubramaniam (2004) [16] have reported curry leaves consists of carotene, beta carotene, folic acid, riboflavin, iron, calcium, phosphorus, and zinc and proposed that Chlorophyll of curry leaves is an anticariogenic agent. Nevin and Bibby (1951), [17] have reported that chlorophyll is bacteriostatic with respect to many oral microorganisms including streptococci and lactobacilli. This suggests that in present study also, chlorophyll of curry leaves may be responsible for the maximum antimicrobial activity with zone of inhibition 24.05 mm against C. albican. The similar study by Chandrabhan Seniya et al. (2011) [18] have also shown intermediate antimicrobial effect of curry leaves leaf extract against C. albicans.

It has been reported that the green tea leaves contains several catechin polyphenols units of flavonoid, in which epigallocatechin gallate and epicatechin can disrupt microbial membranes and these compounds have been found to possess antimicrobial action as well as anticariogenic. [14],[19] Hamilton-Miller, (2001) [20] have reported that rinsing mouth with 20 ml of a tea catechin solution results in antiplaque effect up to 90 min. Yassien (2010) [21] have reported inhibitory effect of green tea extracts on growth C. albicans which is consistent with the present study.

Mint leaves are mainly made up of menthol, menthone, methyl esters, and terpenoids, i.e. monoterpenes derivatives. The antimicrobial activity of terpenoids on microorganisms is mainly by membrane disruption. [14] Sunita Bansod and Mahendra Rai (2008) [22] have shown that the Mint oil to possess antimicrobial action on Aspergillus fumigatus and A. niger. The present study is consistent with data obtained from other studies which demonstrated positive results for mint leaves as well. Kumar et al. (2011) [23] found a significant antifungal activity by both aqueous, ethonolic, and methoholic Mint leave extracts activity on C. albicans. Höfling et al. (2010) [24] have been reported antifungal activity on C. albicans by methonolic mint leave extracts.

Chemical composition of the aloe vera gel is complex mixture. It contains latex compound which has been found to be bacteriostatic in nature. [25]

Plants extract creates an oral environment which is unfavorable for microbes. This indirectly reduces the microbial colonization and modifies oral environment. Although phytochemicals (plant derived metabolites) are antimicrobial in nature but they also produce other biological activities in oral cavity like induction of immunity, which indirectly reduces the risk of oral diseases. [15]

Even though many plants are used as home remedies for oral diseases, their antimicrobial effects on particular pathogenic microorganisms should be evaluated, and implied in the practice. Thus, the present study provides evidence for the presence of one or more soluble constituents in the plants used, which interfere the growth and metabolism of C. albicans which may prevent oral candidal infections. Hence, they can be used as alternative medicine in the patients who develop primary or secondary resistance.

Using of these plant extracts as home remedies or adding to dentifrices, mouthwashes, and varnishes may create an oral environment which is unfavorable for C. albicans. However using the curry leaves, tea leaves and onion particularly by the denture wearers, immune-compromised and diabetic patients may have great help in preventing the oral candidal infections without any side effects.


   Conclusion Top


The present study renders few easily available household medicinal plants like tea leaves, curry leaves, onion, mint leaves, and aloe vera to the field of dentistry which can be used as alternative medicines and adjunct to conventional therapy which will be a great help in developing countries with financial constraints and limited oral health care facilities for populations. This study was conducted in vitro. The duration of the contact of the extracts with C. albicans in the oral cavity in vivo is not clear. A future study for fractionation of potentially active components of the plants is indicated.

 
   References Top

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2.Akpan A, Morgan R. Oral candidiasis. Postgrad Med J 2002;78:455-9.  Back to cited text no. 2
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3.Al-Bagieh NH, Idowu A, Salako NO. Effect of aqueous extract of miswak on the in vitro growth of Candida albicans. Microbios 1994;80:107-13.  Back to cited text no. 3
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4.Al-hebshi N, Al-haroni M, Skaug N. In vitro antimicrobial and resistance-modifying activities of aqueous crude khat extracts against oral microorganisms. Arch Oral Biol 2006;51:183-8.  Back to cited text no. 4
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6.Almas K. The antimicrobial effects of extracts of Azadirachta indica (Neem) and Salvadora persica (Arak) chewing sticks. Indian J Dent Res 1999;10:23-6.  Back to cited text no. 6
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8.Bagg J, MacFarlane TW, Poxton IR, Millar CH, Smith AJ. Essentials of microbiology for dental students. 2 nd ed., London: Oxford University Press; 1999.  Back to cited text no. 8
    
9.Duraipandiyan V, Ayyanar M, Ignacimuttu S. Antimicrobial activity of some ethanomedicinal plants used by Paliyar tribe from Tamilnadu, India. BMC Complement Altern Med 2006;6:35.  Back to cited text no. 9
    
10.Mbata TI, Debiao L, Saikia A. Antimicrobial activity of the crude extract of Chinese green tea (camellia sinensis) on Listeria monocytogenes. Afr J Biotechnol 2008;7:1571-3.  Back to cited text no. 10
    
11.Ellepola AN, Samaranayake LP. Oral candidial infections and antimycotics. Crit Rev Oral Biol Med 2000;11:172-98.  Back to cited text no. 11
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12.Bianchi A, Zambonelli A, D′Aulerio AZ. Ultra structural studies of effects of allium sativum on phytopathogenic fungi in vitro. Am Phytopathol Soc 1997;81:1241-6.  Back to cited text no. 12
    
13.Wynn LR. General dentistry. Available from: http://www.agd.org. [Last cited in 2005].  Back to cited text no. 13
    
14.Cowan MM. Plant products as antimicrobial agents. Clin Microbiol Rev 1999;12:564-82.  Back to cited text no. 14
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15.Haslam E. Natural polyphenols (vegetable tannins) as drugs: Possible modes of action. J Nat Prod 1996;59:205-15.  Back to cited text no. 15
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16.Math MV, Balasubramaniam P. Curry leaves. Br Dent J 2004;197:519.  Back to cited text no. 16
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17.Nevin TA, Bibby GB. The effect of water soluble chlorophyll on pure cultures of microorganisms commonly found in the oral cavity. J Dent Res 1951;30:469.  Back to cited text no. 17
    
18.Argal MS, Kumar S, Chaudhary HS, Thakkar RM, Verma SK, Seniya C. The efficacy of Muirraya koeniggi leaf extract on some bacterial and fungal strain by disk diffusion method. J Chem Pharm Res 2011;3:697-704.  Back to cited text no. 18
    
19.Ooshima T, Minami T, Aono W, Izumitani A, Sobue S, Fujiwara T, et al. Oolong tea polyphenols inhibit experimental dental caries in SPF rats infected with mutans streptococci. Caries Res 1993;27:124-9.  Back to cited text no. 19
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20.Hamilton-Miller JM. Anti-cariogenic properties of tea (Camellia sinensis). J Med Microbiol 2001;50:299-302.  Back to cited text no. 20
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21.Yassien SN. Study the Inhibitory Effect of Green Tea Extracts on Growth of Some Dermatophytes. Al- Anbar J Vet Sci 2010;3:103-9.  Back to cited text no. 21
    
22.Bansod S, Rai M. Antifungal Activity of Essential Oils from Indian Medicinal Plants Against Human Pathogenic Aspergillus fumigatus and A. niger. World J Med Sci 2008;3:81-8.  Back to cited text no. 22
    
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24.Höfling JF, Anibal PC, Obando-Pereda GA, Peixoto IA, Furletti VF, Foglio MA, et al. Antimicrobial potential of some plant extracts against Candida species. Braz J Biol 2010;70:1065-8.  Back to cited text no. 24
    
25.Osato JA, Santiago LA, Remo GM, Cuadra MS, Mori A. Antimicrobial and antioxidant activities of unripe papaya. Life Sci 1993;53:1383-9.  Back to cited text no. 25
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Correspondence Address:
Sunitha Jagalur Doddanna
Department of Oral Pathology and Microbiology, Teerthanker Mahaveer Institute of Dental Sciences and Research Centre, Teerthanker Mahaveer University, Bagarpur, Delhi Road, Moradabad, Uttar Pradesh
India
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DOI: 10.4103/0970-9290.118358

PMID: 24047829

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