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journal of herbal medicine 4 (2014) 83–88
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Original Research Article
Inhibitory effect of Tridax procumbens against
human skin pathogens
R.S. Policegoudraa,∗, P. Chattopadhyaya, S.M. Aradhyab,
R. Shivaswamyc, L. Singh d,V.Veer
a
aDepartment of Pharmaceutical Technology, Defence Research Laboratory, Tezpur 784001, Assam, India
bDepartment of Fruit and Vegetable Technology, Central Food Technological Research Institute, Mysore 570020,
Karnataka, India
cDepartment of Central Instrumentation Facility Services, Central Food Technological Research Institute, Mysore
570020, Karnataka, India
dDirectorate of Life Sciences (DLS), DRDO HQ, New Delhi, India
article info
Article history:
Received 31 October 2011
Received in revised form
4 December 2013
Accepted 8 January 2014
Available online 26 January 2014
Keywords:
Tridax procumbens
Dermatophytes
Antifungal activity
GCMS
MIC
Fatty acids
abstract
Tridax procumbens is a common herb with significant medicinal properties traditionally used
in the treatment of many skin diseases. The methanol extract of T. procumbens exhibited high
antifungal activity against clinically important human skin pathogens such as Microsporum
fulvum,Microsporum gypseum,Trichophyton mentagrophytes,Trichophyton rubrum,Trichosporon
beigelii and Candida albicans with low MIC values. The fractionation of a methanol extract
with dichloromethane yielded an oily viscous fluid with antifungal activity which was sep-
arated and characterized. The GC–MS analysis revealed the presence of 26 compounds. The
major constituents were characterized as 9,12-octadecadienoic acid ethyl ester (18.04%),
5␣-cholestane (12.42%), hexadecanoic acid ethyl ester (4.86%) and 9-octadecenoic acid ethyl
ester (4.72%). This study demonstrated the efficacy of this herb against clinically important
dermatophytes and also justified its traditional use.
© 2014 Elsevier GmbH. All rights reserved.
1. Introduction
Plants are a good source of novel bioactive molecules with
therapeutic potential. There is a plethora of pharmaceutically
important molecules, but only a small percentage of plants
have been explored for their phytochemical constituents
(Hostettmann et al., 1998; Balandrin et al., 1985). Tridax procum-
bens is a common weed native to tropical America and
∗Corresponding author. Tel.: +91 3712 258836; fax: +91 3712 258534.
E-mail addresses: poligene@rediffmail.com,poligene@gmail.com (R.S. Policegoudra).
distributed in tropical Africa, Australia and Asia. It is exten-
sively used in the Indian Ayurvedic system of medicine for the
treatment of diarrhoea, as an insect repellent, hair tonic and
wound healer, i.e. the leaf juice is used to check haemorrhage
from cuts and bruises (Srivastava et al., 1984; Udupa et al.,
1991; Saraf et al., 1992; Bhat et al., 2007). It is a well known
remedy for liver disorders and has been shown to have antidi-
abetic activity (Vilwanathan et al., 2005; Bhagwat et al., 2008).
T.procumbens has demonstrated significant anti-inflammatory
2210-8033/$ – see front matter © 2014 Elsevier GmbH. All rights reserved.
http://dx.doi.org/10.1016/j.hermed.2014.01.004
Author's personal copy
84 journal of herbal medicine 4 (2014) 83–88
and antimicrobial activity (Nia et al., 2003; Mahato and
Chaudhary, 2005). Interestingly some animal studies have
shown that it may have potent immune-modulating property
(Tiwari et al., 2004; Oladunmoye, 2006). T. procumbens contains
alkaloids, carotenoids, saponins, flavonoids, flavones, glyco-
sides and tannins from the leaves of this plant (Raju and
Davidson, 1994; Yadawa and Saurabh, 1998; Ali et al., 2001;
Jude et al., 2009). It also contains lipid constituents (Verma
and Gupta, 1988) plus saturated and unsaturated fatty acids
(Gadre and Gabhe, 1988). The presence of -sitosterol-3-O--
d-xylopyranoside in the flowers of T. procumbens was reported
by Saxena and Albert (2005).
In tropical and subtropical countries, the infectious dis-
eases that affect the skin and mucosal membranes are a
severe problem. A number of these infections are most fre-
quently caused by dermatophytes and yeasts (Hay, 2006). Due
to an increase in the number of immune-suppressed patients
in the last decade, there are more reports of systemic and
superficial mycoses such as aspergillosis, candidiasis, and
fungal infections (Gabardi et al., 2007; Nucci and Marr, 2005;
Pfaller et al., 2006). Several reports have shown that the ther-
apeutic potential of plant extracts against many diseases like
skin and respiratory infection is due to their high antimi-
crobial activity against bacteria, yeasts and dermatophytes
(Janssen et al., 1987; Rios et al., 1988; Griffin et al., 1999). The
increasing recognition and importance of fungal infections
in regard to resistance to antifungal drugs have stimulated
the search for safe, natural therapeutic alternatives (Pina-Vaz
et al., 2004). The use of indigenous folk medicines for the treat-
ment of fungal infections may offer new effective remedies
(Seneviratne et al., 2007; Li et al., 2008; Webster et al., 2008).
The present study explored the bioactive constituents of a
methanol extract of T. procumbens and its antifungal activity
against dermatophytes.
2. Materials and methods
2.1. Plant material and extraction
T. procumbens was collected from Tezpur, Assam, India dur-
ing September 2011. The plant was identified by Dr Jayshree
Das, Pharmaceutical Technology Division, Defence Research
Laboratory, Tezpur and a voucher specimen stored in their
herbarium. The flowers were separated and only the aerial
parts were dried in an oven at 50◦C for 72 h and powdered
in a grinder. The powder (100g) was extracted with 1000 ml of
methanol for 48 h. The extract was concentrated to dryness
using a vacuum rotary evaporator (Buchi Rotavapor) at 50 ◦C
to remove all traces of methanol. The dried extract was then
stored at 4 ◦C.
2.2. Fractionation of extract
The methanol extract was fractionated with dichloromethane.
The dichloromethane soluble fraction was separated and
yielded an oily, viscous fluid. This oily fraction was subjected
to GC–MS analysis.
2.3. GC–MS analysis
The GC–MS analyses were performed in EI mode on a GCMS,
Perkin Elmer, Turbomass gold, GC-Autosample xL (Perkin
Elmer International, Boesch, Huenenberg, Switzerland) sys-
tem with Elite-1 fused capillary column (composed of 100%
dimethylpolysiloxane), 30 m ×0.25 mm ×0.25 m, directly
coupled to mass detector. The mass spectrometer was oper-
ated at 70 eV. Injection conditions were as follows: Column
temperature 40–250 ◦C at a rate of 5 ◦C/1min; carrier gas was
He: 1 ml/min; sample injection volume 1 l. The constituents
of the essential oils were identified based on a comparison of
mass spectra with those of data in the National Institute of
Standards and Technology (NIST) libraries.
2.4. Antifungal activity
2.4.1. Microbial strains and culture conditions
The fungal strains used in this study included Microsporum
fulvum (MTCC 8478), Microsporum gypseum (MTCC 8469), Tri-
chophyton mentagrophytes (MTCC 8476), Trichophyton rubrum
(MTCC 8477) and Candida albicans (MTCC 854) obtained from
the School of Tropical Medicine, Kolkata. Trichosporon beigelii
was isolated from a clinical sample by standard NCCLS (2002)
method. All fungal strains were maintained on Sabouraud
dextrose agar (SDA) medium (Himedia, Mumbai) at 28–30◦C
for 10 days.
2.4.2. Preparation of spore suspension
The 10-day-old cultures were used for the preparation of
inoculums. The spores were scraped with a sterile loop and
macerated in sterile saline (0.85%) solution. The final spore
suspension was adjusted to 105CFU/ml.
2.4.3. Agar well diffusion method
An antifungal assay was carried out using the modified
method of Kariba et al. (2001). The methanol extract (5 mg/ml)
was reconstituted in dimethyl sulphoxide (DMSO) to assess
the antifungal activity. The SDA media was inoculated with
spore suspension (105CFU/ml) of the test fungi. The test sam-
ple was placed in the 6 mm agar well. The plates were then
incubated at 28 ±1◦C. Griseofulvin was used as a standard and
DMSO served as the control. The zone of inhibition around the
well was determined as antifungal activity. Values are given as
mean and SD of tests performed in triplicate.
2.4.4. Minimum inhibitory concentration (MIC)
The MIC was assessed according to the agar dilution method of
Kariba et al. (2001) with modifications. The methanol extracts
and fractions were dissolved in DMSO and concentrations
ranging from 32 to 0.06 mg/ml were incorporated into SDA
growth medium. The resulting SDA medium was inoculated
with spore suspension (105CFU/ml) of the test fungi. The
plates were incubated at 28±1◦C for 10 days. The minimal
inhibitory concentration was recorded as the lowest con-
centration that produced no visible fungal growth. All the
experiments were carried out in triplicate.
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journal of herbal medicine 4 (2014) 83–88 85
3. Results and discussion
3.1. Extraction and fractionation
The extraction of T. procumbens powder with methanol yielded
28 g of extract. This extract showed antifungal activity against
dermatophytes hence the fractionation was carried out to
identify the bioactive components. The methanol extract was
fractionated with dichloromethane (DCM). This DCM solu-
ble fraction obtained was an oily, viscous fluid. This fraction
showed antifungal activity against dermatophytes hence the
bioactive components were characterized.
3.2. GCMS analysis of bioactive fraction
The GCMS analysis of the bioactive fraction from the methanol
extract of T. procumbens yielded 26 components (Table 1). The
bioactive fraction was mainly composed of alicyclic hydrocar-
bons, fatty acids and steroids. The major components were
identified as 9,12-octadecadienoic acid ethyl ester (18.04%),
5␣-cholestane (12.42%), hexadecanoic acid ethyl ester (4.86%)
and 9-octadecenoic acid ethyl ester (4.72%). The structure of
all the bioactive components characterized from the DCM
fraction of T. procumbens is represented in Fig. 1. This is
the first report of the identification of 5␣-cholestane from
plants. Cholestane glycosides and rhamnosides are reported
earlier from different plants and they are known for their
potent cytotoxicity activity against malignant tumour cells
(Liu et al., 2008; Kuroda et al., 2002). This is also the first
report of the presence of different siloxanes like heptamethyl
trisiloxane, octamethyl trisiloxane, 1H-indole-2,3-dione-5-
methyl-1-(trimethylsilyl) and decamethyl tetrasiloxane. The
role of these siloxanes in the plant’s metabolic pathways is
yet to be understood.
3.3. Antifungal activity
The agar well diffusion assay clearly showed the inhibition
of all dermatophytes by the methanol extract with zones
of inhibition ranging from 17 to 25 mm (Fig. 2). C. albicans
was highly susceptible whereas T. mentagrphytes was less
susceptible to the methanol extract. The oily, viscous DCM
fraction also showed high antifungal activity against all the
test organisms. This fraction was also most effective against
C. albicans with 32 mm zone of inhibition, M. fulvum and
T. rubrum however were less susceptible to this fraction.
The antifungal activity of T. procumbens may be due to the
presence of many bioactive compounds including phenols,
flavonoids, saponins, sterols and fatty acids as reported ear-
lier (Manjamalai et al., 2010). The bioactive compounds such
as 8,3-dihydroxy-3,7,4-trimethoxy-6-O-ˇ-d-glucopyranosyl
flavones, 6,8,3-trihydroxy-3,7,4-trimethoxyflavone, puerarin,
esculetin, oleanolic acid, betulinic acid, centaurein, bergenin
and centaureidin have previously been isolated and charac-
terized from this plant (Xu et al., 2010; Jachak et al., 2011).
These bioactive compounds may have some role in antifungal
activity.
The MIC for the methanol extract ranged from 1.6 to12.8 mg
for all the test organisms (Fig. 3). Among the test organisms,
C. albicans and T. beigelii were inhibited at a low MIC of 1.6mg
each. The DCM fraction was also most effective against C.
Table 1 – Chemical constituents of DCM fraction of methanol extract of T.procumbens.
Sl. no Retention time Mass Compound name % Amount of compounds
1 3.18 158 2-Propyl-1-heptanol 0.55
2 3.29 128 3-Octen-1-ol 0.51
3 3.42 136 ␣-Methyl benzeneethanol 0.47
4 3.49 114 2,3-Dimethylhexane 0.21
5 3.6 114 2-Methylheptane 0.19
6 3.74 114 2,4-Dimethylheptane 0.22
7 3.87 128 2-Propenyl butanoate 0.09
8 5.59 222 Heptamethyl trisiloxane 0.13
9 6.77 236 Octamethyl trisiloxane 0.08
10 9.2 233 1H-indole-2,3-dione-5-methyl-1-(trimethylsilyl)- 0.24
11 12.72 310 Decamethyl tetrasiloxane 0.39
12 26.74 204 1,3-Cyclohexadiene,5-(1,5-dimethyl-4-hexenyl)-2 methyl 0.15
13 38.39 278 Dibutyl phthalate 0.06
14 39.12 256 Hexadecanoic acid 0.08
15 39.89 284 Hexadecanoic acid, ethyl ester 4.86
16 43.59 308 9,12-Octadecadienoic acid, ethyl ester 18.04
17 43.78 310 9-Octadecenoic acid ethyl ester 4.72
18 44.29 322 Isopropyl linoleate 0.29
19 44.47 312 Octadecanoic acid, 2-methyl, methyl ester 0.33
20 49.22 282 2,6,11-Trimethyl dodecane 0.18
21 51.35 296 2,6,10,14-Tetramethyl heptadecane 0.71
22 53.46 366 7-Hexyl eicosane 0.81
23 55.72 282 Eicosane 0.88
24 58.41 372 5␣-Cholestane 12.42
25 58.86 416 14-Methyl cholest-8-ene-3,6-diol 0.09
26 58.95 400 3,4-Epoxy-2-methyl cholestane 0.06
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86 journal of herbal medicine 4 (2014) 83–88
Fig. 1 – Phytochemical constituents of Tridax procumbens.
Fig. 2 – Antifungal activity of methanol extract and DCM
fraction against dermatophytes and yeasts. (1) Microsporum
fulvum (Mf); (2) Microsporum gypseum (Mg); (3) Trichophyton
mentagrophytes (Tm); (4) Trichophyton rubrum (Tr); (5)
Trichosporon beigelii (Tb); (6) Candida albicans (Ca).
Fig. 3 – MIC of methanol extract and DCM fraction against
dermatophytes and yeasts. (1) Microsporum fulvum (Mf); (2)
Microsporum gypseum (Mg); (3) Trichophyton mentagrophytes
(Tm); (4) Trichophyton rubrum (Tr); (5) Trichosporon beigelii
(Tb); (6) Candida albicans (Ca).
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journal of herbal medicine 4 (2014) 83–88 87
albicans with an MIC of 0.2 mg. The DCM fraction was effec-
tive against all the dermatophytes with MIC values ranging
from 0.4 to 3.2 mg. This investigation clearly demonstrated
the potential antifungal activity of the methanol extract of T.
procumbens and DCM fraction. The MIC values for Griseofulvin
ranged from 0.25 to 4.0 g/ml against the dermatophytes.
The antifungal activity of the methanol extract may be
due to the presence of various bioactive compounds reported
earlier. Whereas the DCM fraction may be due to major bioac-
tive compounds like 9,12-octadecadienoic acid ethyl ester,
cholestane, hexadecanoic acid ethyl ester and 9-octadecenoic
acid ethyl ester. The cumulative effect of other compounds
may also play an important role in antifungal activity.
The antimicrobial activity of unsaturated fatty acids has
long been known (Knapp and Melly, 1986). The antidermato-
phytic activity of the DCM fraction may be attributed to the
presence of unsaturated fatty acids and other components.
This is the first report on the presence of 5␣-cholestane and
different siloxanes from plant sources. The antifungal activity
of the active fraction against dermatophytes may be attributed
to the cumulative effect of all the components reported in
Table 1. This investigation has clearly shown the efficacy of
this medicinal herb against some clinically important human
skin pathogens and validated its use in traditional medicine.
Further clinical trials are needed to study the in vivo effective-
ness of this herb.
4. Conclusion
The present investigation revealed the presence of alicyclic
hydrocarbons, fatty acids and steroids in the bioactive frac-
tion of methanol extract of T. procumbens. This is the first
report on the presence of 5␣-cholestane and siloxanes from
this plant. The antifungal activity of the active fraction may be
attributed to the presence of fatty acid derivatives and other
constituents. More work needs to be carried out to identify
the bioactivity of cholestane and siloxanes in regard to their
possible contribution to antifungal activity.
Appendix A. Supplementary data
Supplementary data associated with this article can be found,
in the online version, at doi:10.1016/j.hermed.2014.01.004.
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