ISSN 1517-8382
ABSTRACT
Antifungal properties of some essential oils have been well documented. Clove oil is reported to have strong
antifungal activity against many fungal species. In this study we have evaluated antifungal potential of
essential oil of Syzygium aromaticum (L.) against some common fungal pathogens of plants and animals
namely, Fusarium moniliforme NCIM 1100, Fusarium oxysporum MTCC 284, Aspergillus sp., Mucor sp.,
Trichophyton rubrum and Microsporum gypseum. All fungal species were found to be inhibited by the oil
when tested through agar well diffusion method. Minimum inhibitory concentration (MIC) was determined
for all the species. Column chromatography was performed to separate the eugenol rich fraction from clove
oil. Out of seven fractions maximum activity was obtained in column fraction II. TLC and HPLC data
confirmed presence of considerable Eugenol in fraction II and clove oil. Microscopic study on effect of
clove oil and column fraction II on spores of Mucor sp. and M. gypseum showed distortion and shrinkage
while it was absent in other column fractions. So it can be concluded that the antifungal action of clove oil is
due to its high eugenol content.
toxicity, efficacy and cost, and their frequent use has led to the
effectively
inhibit
the
growth
of
wide
range
of
*Corresponding Author. Mailing address: Research & Development Center, Kilpest India Ltd., Govindpura, Bhopal-462023, India.; Tel.: 91 9893573161.; Email: ranaindersingh@yahoo.com
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(16).
Clove oil (Syzygium aromaticum) is widely used as a
hours and the oil was drained off and dried over anhydrous sodium
sulphate (4).
oil (EO) was done through agar well diffusion assay (17). Spore
The major constituent of the clove oil (eugenol) was also purified
l of
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along with 45
l.
negative control.
column fractions 5
with Tween-20 alone was set as control. The plate with lowest
reached the top, the plate was air dried and exposed to iodine
Separation
of
clove
EO
fractions
through
column
chromatography
and poured into a glass column (60 x 3 cm, ASGI, India) with
milliliter of clove oil was loaded onto column bed and eluted
ether:chloroform
(5:5),
chloroform:ethyl
acetate
(3:2),
together as 100%.
l was
1271
DMSO.
Figure 1. Mycelial inhibition (mm) of various fungal strains with clove EO (10 l), Ketoconazole (1mg/ml)
and DMSO (10 l)
1272
1 l/ml concentration.
l/ml and F.
MIC to evaluate the antifungal activity of the clove oil and its
Table 1. MIC of clove oil against fungal strains after 5 days of incubation
Mycelial diameter (mm)
Fungal strain
Mucor sp.
Aspergillus sp.
F. moniliforme
F. oxysporum
Microsporum
gypseum.
Trichophyton
rubrum
Negative
Control
100
100
100
67.31.7
1
100
100
31.30.4
27.60.4
2
100
100
100.8
9.60.4
4
320.8
31.30.9
8.60.4
8.30.4
8
210.8
20.60.4
7.60.9
7.30.4
9
3.30.4
6.30.4
3.30.4
10
5.60.4
-
11
2.60.4
-
12
-
19.30.9
4.60.4
4.30.4
3.60.4
3.30.9
27.60.4
110.8
80.8
5.60.4
3.60.9
fractions were oily and light colored while polar fractions were
Table 2. Quantity, consistency and color of six column fractions and clove
Column Fractions eluted with following solvent
Ether I
Ether: Chloroform (5:5) II
Chloroform: Ethyl acetate (3:2) III
Chloroform: Ethyl acetate (5:5) IV
Chloroform: Ethyl acetate (2:8) V
Methanol VI
Weight of
samples (g)
5.49
2.22
0.58
0.03
0.08
0.05
Consistency/
color
Light colorless Oil
Viscous yellow oil
Semisolid yellow
Brown solid
Brown solid
Dark brown solid
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Inhibition zone
in eugenol 95% (5 l)
22.6
14
15
14.6
21.3
17
fraction II at lane 2 and for eugenol standard was 0.85 cm. The
proves that the antifungal activity of the clove oil is due to its high
eugenol content.
inhibition zones were also observed in this fraction. The non polar
constituent
of
clove
oil
against
the
dermatophytes
T.
Figure 2. TLC plate showing column fractions (I, II, III, IV, V, VI) respectively in lane 1, 2, 3, 4, 5, 6. Lane 2 (column fraction II)
showed presence of substantial amount of eugenol when compared with standard eugenol spot (5 l) at lane 7.
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be attributed to eugenol.
Figure 3. A: HPLC elution profile of eugenol (95% LR grade) with retention time of 3.056 min confirmed 90.55% eugenol. B:
HPLC elution profile of clove oil confirmed presence of 68.73% eugenol with retention time of 3.045 min
citratus.
and conidia with clove oil, lysis and distortion of spores was
observed in all the fungal isolates. The effect was profound and
5B). Similar effects were noted when spores were treated with
antifungal
standard eugenol.
tests
that
cinnamaldehyde,
-methyl
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Figure 4. A: Showing Mucor sp. spores of control. 4B Showing deformities/ distortion of spores of Mucor sp. upon treatment with
column fraction II. Fig. 5A: Showing M. gypseum spores of control. 5B Showing leakage of cellular components of spores of M.
gypseum at tips and near center upon treatment with column fraction II
ACKNOWLEDGEMENTS
active against all the tested fungal strains with low MIC values.
Kilpest India Limited for providing funds and lab facility for
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