Research Article Kokati Venkata Bhaskara Rao et al.

/ Journal of Pharmacy Research 2010, 3(3),539-542

ISSN: 0974-6943 Available online through
[Link]

In vitro anti-Candida activity of Calotropis gigantea.

Gaurav Kumar, Loganathan Karthik and Kokati Venkata Bhaskara Rao*.
*Environmental Biotechnology Division, School of Bioscience and Technology, VIT University, Vellore, TN - 632 014, India
Received on: 17-11-2009; Revised on: 19-12-2009; Accepted on:08-02-2010

ABSTRACT

The aim of this study is to screen leaves of Calotropis gigantea for the anti-Candida properties against four clinical isolates of Candida.
Aqueous, methanol, ethanol and petroleum ether extracts of the leaves of Calotropis gigantea were screened against clinical isolate of
Candida albicans, C. parapsilosis, C. tropicalis and C. krusei. The in vitro anti-Candida assay was performed by agar well diffusion
method in potato dextrose agar (PDA). Minimal inhibitory concentration (MIC) test was performed by modified agar well diffusion assay in
PDA. The inhibitory effect of aqueous extract on all four tested Candida species was significantly higher than the methanol, ethanol and
petroleum ether extracts. Aqueous extract showed low MIC values against C. tropicalis (125 µg/ml), C. krusei (250 µg/ml) and C. albicans
(500 µg/ml), followed by the methanol extract against C. tropicalis, C. krusei (500 µg/ml) and petroleum ether extract against C. tropicalis (500
µg/ml). The results prove Calotropis gigantea as a potent source of natural anti-Candida compounds.

Keywords: Calotropis gigantea; medicinal properties; minimal inhibitory concentration; well diffusion method.

INTRODUCTION

Candida is thin walled small yeast that associates with wound healing activity,[5] anti-diarrhoeal activity,[6, 7] anti-pyretic ac-
humans as commensal and/or as pathogen. Candida is usually an tivity,[8] CNS activity,[9] analgesic activity.[10] Calotropis gigantea also
opportunistic pathogen and can cause disease in humans, especially reported to cure toothache, earache, sprain, anxiety, pain, epilepsy,
in immunocompromised patient. Nearly 150 species of Candida have mental disorders and contain pregnancy interceptive properties.[11]
been identified, out of them C. albicans is one of the most pathogenic
species and it cause candidiasis. [1] C. krusei, C. tropicalis, C. In previous studies Calotropis gigantea has already been
dubliniensis, C. parapsilosis etc. are some other pathogenic species reported to possess antibacterial and antifungal properties. [4, 12] Cur-
from genus Candida. Most Candida infections can be treated topical rent study is mainly focused on the anti-Candida activity of Calotropis
administration of antifungal drugs such as clotrimazole, miconazole, gigantea against four clinical isolates of Candida in vitro.
nystatin, tioconazole or oral administration of drugs such as
fluconazole and amphotericin B. Widespread and overuse of these MATERIALAND METHODS
drugs, leading microbes to develop a resistance mechanism against
that particular drug especially in hospital environment.[2] To over- Plant material
come this problem scientists are looking for the isolation of new
potential therapeutic compounds from plants. Calotropis gigantea plant was collected from the waste-
land of Vellore district, TN, India, during December 2008. Plant was
Plants have been utilized for medication since thousands of identified in Herbal Garden of VIT University, TN, India.
years in traditional medicine systems and well accepted as one of the
main source of drug discovery and development.[3] Calotropis Preparation of plant extract
gigantea is a widely growing plant native to Asian continent and
known as milkweed. Calotropis gigantea has been reported to con- Fresh and mature leaves of Calotropis gigantea were col-
tain a number of medicinal properties such as antimicrobial activity,[4] lected and washed properly with tap water followed by distilled wa-
ter, then shade dried at room temperature. Leaves were powdered
*Corresponding author. using a mechanical grinder. The leaf powder was extracted in water,
Dr. Kokati Venkata Bhaskara Rao methanol, ethanol and petroleum ether. Ten grams of leaf powder
Assistant Professor (Senior), Environmental Biotechnology Division
School of Bioscience and Technology was soaked in 100 ml of distilled water, methanol, ethanol and petro-
VIT University, Vellore, TN - 632 014, India leum ether and loaded on a shaker at a speed of 120 rpm for 48 hour at
room temperature. Mixtures were filtered by using filter cloth followed
by whatman number 1 filter paper. The filtrate was concentrated by

Journal of Pharmacy Research [Link] [Link] 2010 539-542

 Kokati Venkata Bhaskara Rao et al. / Journal of Pharmacy Research 2010, 3(3),539-542

using rotary evaporator. The concentrated extracts were dried by lyo- Determination of minimum inhibitory concentration (MIC)
philizer. Dried extracts were collected in air tight containers and stored
at room temperature for further [Link] extracted powder was dis- The MIC of all four extracts was determined by modified
solved in dimethyl sulfoxide (DMSO) (10% w/v) as 100 mg/ml stock agar well diffusion method.[15, 16] All the extracts were dissolved in
solution and sterilized by using 0.45µ syringe filter. DMSO to obtain a concentration range of 125, 250, 500, and 1000 µg/
ml. The concentration of test cultures was adjusted to 0.5 McFarland
Test microorganism standards by using a spectrophotometer. Test organisms were lawn
cultured on the PDA plates. Agar surface was bored by using a ster-
The following four clinical isolates of Candida were used in ilize cork borer of 7 mm diameter. A 100 µl of each dilution was poured
the study: C. albicans, C. parapsilosis, C. tropicalis and C. krusei. in to wells. All test plates were incubated at 37°C for 24 hours. The
All isolates were isolated from clinical samples and maintained on minimum concentration of each extract showing a clear zone of inhibi-
PDA at 4°C. All test organisms were inoculated in potato dextrose tion was considered to be MIC. Experiment was performed in tripli-
broth (PDB) and incubated overnight at 37°C to make a homogenized cates.
suspension.
RESULTS AND DISCUSSION
Positive and negative control
Herbs and plants are being used as a source of medicine
Fluconazole disc (10µg/disc) was used as positive control from long time and it is quite popular in tribal and rural population. In
(PC) and DMSO was used as negative control (NC). the current scenario many plants and plant derivatives used to obtain
drugs.[17] In the current study four extracts of Calotropis gigantea
Determination of anti-Candida activity were analyzed for anti-Candida activity against four clinical isolates
of Candida. The results of the anti-Candida assay of the aqueous,
Anti-Candida testing methanolic, ethanolic, petroleum ether extracts of Calotropis gigantea,
standards DMSO and Fluconazole are presented in Tables 1 and Fig-
Anti-Candida activity of the crude extracts wre determined ure 1. All four leaf extracts showed anti-Candida activity against C.
by the agar well diffusion method with some modifications. The con- albicans, C. tropicalis and C. krusei, whereas only aqueous and
centrations of test suspensions were adjusted to 0.5 McFarland stan- methanol extract showed activity against C. parapsilosis.
dards by using a spectrophotometer. The Candida cultures were in-
oculated on PDA plates by using sterilised cotton swabs. In each of Results of relative percentage inhibition with respect to posi-
these plates, five wells were cut out using a standard cork borer (7 tive control are reported in Table 2 and Figure 2. Aqueous extract
mm). Using a micropipette, 100 µl of each extracts and negative con- showed high relative parentage inhibition against C. tropicalis
trol (DMSO) was added in to different wells. A positive control (108.1%) and low relative parentage inhibition against C. parapsilosis
(Fluconazole) disc was placed in the plate. Plates were incubated for (5.4%). Methanol, ethanol and petroleum ether extract showed maxi-
24 hours at 37°C. Anti-Candida activity was evaluated by measuring mum relative parentage inhibition against C. krusei, whereas no/less
the zone of inhibition. Experiment was performed in triplicates.[13] relative parentage inhibition against C. [Link] of MIC
are listed in Table 3 and Figure 3. Aqueous extract showed signifi-
Determination of relative percentage inhibition cantly low MIC values compare to methanol, ethanol and petroleum
ether extracts. The MIC values of the aqueous extracts were found
The relative percentage inhibition with respect to positive 125, 250, 500 and 1000 µg/ml for C. tropicalis, C. krusei, C. albicans
control was calculated by using the following formula.[14] and C. parapsilosis respectively. The MIC values of the methanol
extracts were found 500 µg/ml for C. tropicalis and C. krusei and 1000
µg/ml for C. albicans and C. parapsilosis. The MIC values of the
ethanol extracts were found 1000 µg/ml for C. albicans, C. tropicalis
1 0 0 × (a -b ) and C. krusei respectively. The MIC values of the petroleum ether
Relative percentage inhibition of the test extract =
(c -b ) extracts were found 500 µg/ml for C. tropicalis and 1000 µg/ml for C.
albicans and C. krusei respectively.
Where,
a: total area of inhibition of the test extract Earlier studies proved the strong antimicrobial effect of the
b: total area of inhibition of the solvent root extract of Calotropis gigantea against pathogenic bacteria. Some
c: total area of inhibition of the standard drug other studies reflect Calotropis gigantea as a potent source of vari-
ety of medicinal compounds. [4-11] We conclude that Calotropis
The total area of the inhibition was calculated by using area
= pr2; where, r = radius of zone of inhibition. gigantea represents an unexploded source of potentially useful anti-
Candida compounds and it worth for future clinical use. In addition,
urgent measures have to be taken to preserve the traditional knowl-
edge about medicinal plants.
Journal of Pharmacy Research [Link] [Link] 2010 539-542
 Kokati Venkata Bhaskara Rao et al. / Journal of Pharmacy Research 2010, 3(3),539-542
Table: 1 Anti-Candida assay of Calotropis gigantea against tested or- Figure2 : Relative percentage inhibition of Calotropis gigantea on
ganisms tested organisms
Test organisms Inhibition zone diameter (mm)
ME EE PE AE PC NC

C. albicans 9 8 10 12 22 0
C. tropicalis 13 10 11 26 25 0
C. krusei 20 16 13 20 22 0
C. parapsilosis 6 0 0 7 30 0

MA: methanol extract; EE: ethanol extract; PE: Petroleum ether extract; AE:
aqueous extract
PC: positive control; NC: negative control
Each value is a mean of three replicates values
Zone of inhibition not include the diameter of the well
Table: 2 Relative percentage inhibition of Calotropis gigantea on
tested organisms

Test organisms Relative percentage inhibition (In %)

ME EE PE AE

C. albicans 16.7 13.2 20.6 29.7 Figure3 : Minimum inhibitory concentration of Calotropis
C. tropicalis 27.0 16.0 19.3 108.1
C. krusei 82.6 52.8 34.9 82.6 gigantea on tested organisms
C. parapsilosis 4.0 0 0 5.4

MA: methanol extract; EE: ethanol extract; PE: Petroleum ether extract; AE:
aqueous extract
Table: 3 Minimum inhibitory concentration of Calotropis gigantea

Test organisms MIC (µg/mL)

ME EE PE AE

C. albicans 1000 1000 1000 500

C. tropicalis 500 1000 500 125
C. krusei 500 1000 1000 250
C. parapsilosis 1000 - - 1000

MA: methanol extract; EE: ethanol extract; PE: Petroleum ether extract; AE:
aqueous extract
Each value is a mean of three replicates values

Figure 1 Anti-Candida activity of Calotropis gigantea ACKNOWLEDGEMENTS:

The authors wish to thank the Management and Staff of VIT
University, Vellore, TN, India for providing necessary facilities to carry
out this study.

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Source of support: Nil, Conflict of interest: None Declared

Journal of Pharmacy Research [Link] [Link] 2010 539-542