Antifungal efficacy of <i>Punica granatum</i>, <i>Acacia nilotica</i>, ... http://www.ijdr.in/printarticle.asp?issn=0970-9290;year=2010;volume=21...

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ORIGINAL RESEARCH
Year : 2010 | Volume : 21 | Issue : 3 | Page : 334--336

Antifungal efficacy of Punica granatum, Acacia nilotica, Cuminum cyminum and Foeniculum vulgare on Candida
albicans: An in vitro study
Mithun B.H Pai1, GM Prashant1, KS Murlikrishna2 , KM Shivakumar3, GN Chandu1 ,
1 Department of Community Dentistry, College of Dental Sciences, Davangere, Karnataka - 577 004, India
2 Department of Pharmacognacy, Bapuji College of Pharmacy, Davangere, Karnataka - 577 004, India
3 Department of Public Health Dentistry, Sharad Pawar Dental College, DMIMS University, Sawangi (M) - 442 004, India
Correspondence Address:
G N Chandu
Department of Community Dentistry, College of Dental Sciences, Davangere, Karnataka - 577 004
India

Abstract
Background: The establishment and maintenance of oral microbiota is related not only to interbacterial coaggregations but also to interactions of these bacteria with yeasts. Hence, it is
important for agents used in the treatment of oral diseases to have antifungal properties for effective therapy. Objective: The main purpose of this study was to evaluate the in vitro
antifungal efficacy of Punica granatum, Acacia nilotica, Cuminum cyminum and Foeniculum vulgare on Candida albicans. Materials and Methods: The pomegranate peel is separated,
dried and powdered. Fennel, cumin and acacia bark obtained from the tree are powdered. Candida is inoculated at 37C and seeded on Sabourauds agar medium. Sterilized filter papers
saturated with 30 l of the extracts are placed on the seeded plates and inoculated at 24 and 48 h. Zones of inhibition on all four sides are measured around the filter paper with a vernier
caliper. The experiments were repeated on four plates, with four samples of each extract on one plate for all of the extracts. Results: All the above-mentioned ingredients showed
antifungal property, with Punica granatum showing the highest inhibition of Candida albicans with a mean zone of inhibition of 22 mm. P-values <0.05 were obtained for Punica granatum
when compared with the other extracts. Conclusion: The results showed the potential use of these products as cheap and convenient adjuvants to pharmaceutical antifungal products.

How to cite this article:

Pai MB, Prashant G M, Murlikrishna K S, Shivakumar K M, Chandu G N. Antifungal efficacy of Punica granatum, Acacia nilotica, Cuminum cyminum and Foeniculum vulgare on
Candida albicans: An in vitro study.Indian J Dent Res 2010;21:334-336

How to cite this URL:

Pai MB, Prashant G M, Murlikrishna K S, Shivakumar K M, Chandu G N. Antifungal efficacy of Punica granatum, Acacia nilotica, Cuminum cyminum and Foeniculum vulgare on
Candida albicans: An in vitro study. Indian J Dent Res [serial online] 2010 [cited 2015 Aug 31 ];21:334-336
Available from: http://www.ijdr.in/text.asp?2010/21/3/334/70792

Full Text
Punica granatum, (pomegranate), Acacia nilotica (jalli/babul), Cuminum cyminum (cumin, jeera) and Foeniculum vulgare (fennel, saunf)) have been widely approved for their
antimicrobial properties. [1],[2] The antimicrobial property of any material not only refers to its antibacterial properties but also includes its antifungal, antiprotozoal and antiviral properties,
contrary to which all the research is concentrated only toward finding the antibacterial property of the material. As oral microbiota not only consist of bacteria but also contain other
microorganisms that cause mixed infections in the oral cavity, a broader spectrum of activity of oral care products is needed. The establishment and maintenance of oral microbiota is
related not only to interbacterial coaggregations but also to interactions of these bacteria with yeasts such as Candida albicans. Fungi are frequently isolated in several oral sites,
including the tongue, buccal mucosa, palate, dental biofilm, subgingival microbiota, carious lesions and prosthetic appliances. Studies have suggested a possible relation between
Candida albicans and periodontal disease, dentin and/or root caries. These studies showed that Candida albicans has a similar capacity of colonizing hydroxyapatite as Streptococcus
mutans; however, using different mechanisms. Enamel and dentin demineralization produced by fungal organic acids as well as the presence of cells with Candida albicans hyphe
invading dentinal tubules prove this ability of the fungi to invade and destroy organic and inorganic dental tissues. This microorganism adheres to hydroxyapatite, especially through
electrostatic interactions, and, at smaller numbers, Candida albicans also has the ability to dissolve hydroxyapatite at a larger rate compared to Streptococcus mutans hence potentiating
it as a pioneer microbe in the causation of caries, which is a common oral disease. [3] Hence, it is desirable for all antibacterial products to have antifungal properties to prevent infections
caused by both bacteria and fungi.
Objective
The aim of this study was to evaluate the antifungal efficacy of Punica granatum (pomegranate), Acacia nilotica (jalli/babul), Cuminum cyminum (jeera) and Foeniculum vulgare (saunf)
on Candida albicans in vitro.

Materials and Methods

Collection of materials
The Punica (pomegranate) fruits for the study were obtained from the local market in Davangere in the month of October. After washing, the peel was separated from the mesocarp and
the inner and the outer rinds were obtained. The acacia (jalli/babul) bark wass obtained from a tree in Davangere city. Both the specimens were sun dried for 3 days. The dried seeds of
cumin (jeera) and fennel (saunf), which were obtained from a grocery shop, and all other materials were ground in an electric grinder to produce a powder. All specimens were identified
by a botanist and a pharmacognocist for their authenticity at the Department of Pharmacognocy, Bapuji College of Pharmacy, Davangere. Pure cultures of Candida albicans were
obtained from the Department of Microbiology, Jagadguru Jaydeva Murugarajendra Medical College, Davangere.
Preparation of extracts
Preparation of aqueous decoction

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Punica and acacia contain tannins as active ingredients [4] that are dissolved in water easily and hence an aqueous decoction method was devised to produce the extract. Aqueous
decoctions were prepared by boiling 10 g punica and acacia powdered in 100 ml sterile distilled water over a low flame for 15 min. The flasks were then plugged and removed from heat
and allowed to cool for 45 min. After cooling, the contents of the flasks were filtered with double filter paper and sterile filters to remove any impurities. [5],[6]
Preparation of essential oil extracts
Cumin (jeera) and fennel (saunf) contain essential oils as active ingredients. The sample is prepared by the method reported by Hanafy and Hatem, [7] where 500 ml of diethyl ether is
added to 200 g of chopped spices and the mixture is left for 6 h. The mixture was periodically agitated during this period once every 15 min. Afterwards, it was filtered and the ether was
vaporized in an evaporator at 60C. The dark-colored oily extract obtained at the end of these processes was used in a non-diluted form for the analyses.
The sample extracts were kept in the refrigerator (4C) until the analyses were accomplished.
Antimicrobial tests
The disc diffusion method was used to determine the antimicrobial activity in vitro. In the present study, with the help of sterile cotton swabs, individual microorganisms grown in
Sabouraud broth was swabbed over the surface of Sabouraud agar plates. Filter discs uniformly loaded with 30 l of the extract were placed with sterile tweezers onto the prepared
plates. The zones of microbial inhibition were measured after the time intervals of 18 and 24 h on the underside of the Petri dishes using vernier callipers. The zones of inhibition on the
growth of test strains were defined by the area where visible growth had been inhibited completely. The experiments were repeated on four plates with four samples of each extract on
one plate for all the extracts. All the measurements of zone of inhibition were carried out by a single examiner. Calibration of examiner was done prior to and during the study by
re-examining 5% of the samples to minimize intra-examiner variability. Intra-examiner agreement was determined using kappa statistics (k). Intra-examiner agreement score (k=0.94) was
almost perfect thus meeting the scientific requirement for validity and reliability.
Statistical analysis

Mean value and standard deviation

First, the whole of the diameter of the zone of inhibition was measured and later the diameter of the filter disc was deducted from it. This gave the actual zone of inhibition around the filter
disc. The means of all the individual filter discs and all the four plates were taken for means.One-way analysis of variance.Tukey post hoc test.P-values <0.05 were considered
statistically significant.

Result

Punica granulatum (pomegranate) showed the highest antifungal activity against Candida albicans (P=0.00, which was highly significant), followed by Acacia nilotica (jalli/babul),
Cuminum cyminum (jeera) and Foeniculum vulgare (saunf), which had least antifungal activity at the end of 48 h. The difference between the plant extracts was significant at the end of
24 h (P=0.00) whereas the difference between Acacia nilotica (jalli/babul) andCuminum cyminum was not statistically significant at 48 h, with P-values of 0.538 (> 0.05), of which Acacia
nilotica showed the higher zones of inhibition than Cuminum cyminum .
[Table 1] and [Table 2] show the antifungal activity extracts of punica, acacia, cumin, fennel at 24 and 48 h, respectively. Graph 1[SUPPORTING:1] shows the difference between the
plant extract at 24 and 48 h, respectively.{Table 1}{Table 2}

Discussion

Punica granulatum (pomegranate), Acacia nilotica (jalli/babul) and Cuminum cyminum (jeera) showed antifungal property at both 24 and 48 h, whereas Foeniculum vulgare (saunf)
inhibited Candida albicans at only 48 h and no activity was observed at 24 h even after using the extract at 100% concentration.
Punica granulatum (pomegranate) and Acacia nilotica (jalli/babul) contain tannins. Tannins are known to precipitate proteins and might be involved in the inhibitory mechanism of the
extracts. However, the exact inhibitory mechanism is not known. [8] Acacia nilotica (jalli/babul) contains an additional product known as methyl gallate, which possesses antimicrobial
activity by accelerating DNA damage by a ferric-bleomycin system, [9] and the result of the present study is similar to studies conducted by Vasconceles et al. and Jennifer Beckman,
where the pomegranate extract showed inhibition of Candida albicans, which is similar to this study, whereas Nascimento et al. could not elicit any antifungal activity from the
pomegranate extract, which may be because the authors used the seeds of the fruit, which a contain lower concentration of tannins compared to the outer carp.
Acacia nilotica, commonly called babul, has been used as an oral hygiene adjuvant from ages, but still, studies are lacking on the antifungal property of babul on the most common oral
fungus. Of the few studies conducted in the past, antifungal efficacy was demonstrated on animals with direct use of leaves. But, in the present study, the bark of the tree is used, which
contains a higher amount of antimicrobial agents and thus so there is demonstration of significant antifungal activity, which is consistent with the studies of Runoro, where zones of
inhibition were <10 mm. However, in the present study, the zone of inhibition of Acacia nilotica was 7.62 mm at the end of 48 h.
The extracts of Cuminum cyminum (jeera) and Foeniculum vulgare (saunf) have evoked interest as sources of natural products for their potential uses as alternative remedies to heal
many infectious diseases as they contain essential oils. [7] In the present study, the essential oil extracts demonstrated antifungal activity but to a lesser extent than the aforementioned
plants. However, most of the studies concerning Cuminum showed that they have a mild antifungal activity, which is consistent with the data of the present study, and fennel showed no
activity at the end of 24 h, with results similar to those reported in the study of Agaoglu et al.
The present study is just a venture from the usual clinical approach that is so commonly followed. The use of medicinal plants against Candida can be a viable alternative to other
antifungal agents as these offer a cheap and effective module used in the control of both bacteria and yeasts responsible for mixed oral infections such as caries, periodontal disease and
stomatitis. The present study concentrates on Candida albicans only because there are innumerable studies on the antibacterial properties of the above-titled plants and hence more
studies in vivo regarding the antimicrobial properties of the above-mentioned plant extracts are necessary. Further, it is recommended that as these plants are commonly available, they
can be amalgamated with present materials used as preventive measures to improve their efficacy. The active ingredients from these plants can be isolated and studied further for their
use in dentistry with respect to their functions as antimicrobial, anti-inflamatory or immunomodulators.

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