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Original Research www.ijdr.in

Antifungal efficacy of Punica granatum, Acacia nilotica,

Cuminum cyminum and Foeniculum vulgare on Candida
albicans: An in vitro study

Mithun BH Pai, Prashant GM, Murlikrishna KS1, Shivakumar KM2, Chandu GN

Department of Community
Dentistry, College of Dental
ABSTRACT
Sciences, 1Department of
Pharmacognacy, Bapuji College
of Pharmacy, Davangere,
Karnataka - 577 004,
2
Department of Public Health
Dentistry, Sharad Pawar Dental
College, DMIMS University,
Sawangi (M) - 442 004, India
Received : 01-07-09
Review completed : 08-09-09
Accepted : 21-05-10
PubMed ID : ***
DOI: 10.4103/0970-9290.70792
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 37˚C and seeded
on Sabourauds agar medium. Sterilized filter papers saturated with 30 µl 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.
Key words: Antifungal proper, babul, candida, cumin, fennel, pomegranate

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
Address for correspondence:
Dr. Chandu GN
E-mail: codspcd@gmail.com
Indian J Dent Res, 21(3), 2010
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
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Antifungal efficacy of plant extracts Pai, et al.
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
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 60°C. 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 (4°C) 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 µl 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
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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) and Cuminum 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.
Tables 1 and 2 show the antifungal activity extracts of punica,
acacia, cumin, fennel at 24 and 48 h, respectively. Graph 1
shows the difference between the plant extract at 24 and 48
h, respectively.
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.
25
20 Mean
15 24h
Mean
10
48h
5
0
Punica Acacia Cumin Fennel
Graph 1: Difference between the punica, acacia, cumin, fennel extracts
of at 24 h and 48 h, respectively
Indian J Dent Res, 21(3), 2010
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Antifungal efficacy of plant extracts Pai, et al.
Table 1: Antifungal activity of the punica, acacia, cumin,
fennel extracts at 24 h
Mean SD F-value P-value Tukey post
hoc test
Punica 18.8 1.93 554.5 0.000 1>2
(pomegranate) (P<0.05) >3>4
Acacia (jalli/babul) 4.62 0.88
Cumin (jeera) 1.81 0.9
Fennel (saunf) 0 0
Means and standard deviations in millimeter
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
Indian J Dent Res, 21(3), 2010
Table 2: Antifungal activity of punica, acacia, cumin, fennel
extracts after 48 h
Mean SD F-value P-value Tukey post
hoc test
Punica 22 2.9 192.4 0.000 1>2
(pomegranate) (P<0.05) 3>4
Acacia (jalli/babul) 7.62 1.6
Cumin (jeera) 6.5 1.3
Fennel (saunf) 2.5 1.3
Means and standard deviations in millimeter
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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How to cite this article: Pai MBH, Prashant GM, Murlikrishna KS,
Shivakumar KM, Chandu GN. Antifungal efficacy of Punica granatum, Acacia
nilotica, Cuminum cyminum and Foeniculum vulgare on Candida albicans: An
in vitro study. Indian J Dent Res 2010;21:334-6
Source of Support: Nil, Conflict of Interest: None declared.
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