Available Online at http://www.recentscientific.com
International Journal of Recent Scientific Research
Vol. 3, Issue, 3, pp.149 -154, March, 2012
International Journal
of Recent Scientific
Research
ISSN: 0976-3031
STUDIES ON THE ANTIFUNGAL ACTIVITY OF Turnera subulata AND Acacia nilotica
AGAINST SELECTED FUNGAL PATHOGENS
1*Prabhahar. C., 2Saleshrani, K., 3Saranraj, P and 1Tharmaraj, K
1
Department of Zoology, Annamalai University, Annamalai Nagar.608 002, Chidambaram, Tamil Nadu, India.
2
Department of Zoology,.Manonmaniam Suntaranar University, Thirunelveli, Tamil Nadu, India.
3
Department of Microbiology, Annamalai University, Annamalai Nagar - 608 002, Chidambaram, Tamil Nadu, India.
ARTICLE INFO
ABSTRACT
Article History:
Several hundreds of plant genera are used medicinally and plants are vital sources
for potential and powerful drugs. The present study was conducted to screen the
pharmacological activity of the ethanol and ethyl acetate extract of Turnera
subulata and Acacia nilotica for its antifungal activity against pathogenic fungi.
Six different fungal isolates viz., Candida albicans, Candida glabrata,
Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger and Penicillium
chrysogenum were tested for its antifungal activity. The collected leaf samples
were powdered and the bioactive compounds were extracted by using ethanol and
ethyl acetate in a Soxhelet extractor. The antifungal activity was determined by
using Well diffusion method. Ethanol and ethyl acetate extracts with different
concentrations (100mg/ml, 200mg/ml and 300mg/ml) were mixed with 1 ml of
Dimethyl sulfoxide (DMSO) and added into the well. The inhibitory effect of
ethanol extract was relatively high when compared to ethyl acetate extract. The
extract of Turnera subulata showed maximum zone of inhibition against fungal
pathogens when compared to Acacia nilotica. This study also encourages
cultivation of the highly valuable plant in large scale to increase the economic
status of the cultivators and provide a support to use of the plant in traditional
medicine.
Received 10th January, 2012
Received in revised form 12th February, 2012
Accepted 28th February, 2012
Published online 25th March, 2012
Key words:
Antifungal activity, Ethanol extract,
Ethyl acetate extraction , Zone of
inhibition, Turnera subulata, Acacia
nilotica, Fungi and Phytochemical
analysis.
© Copy Right, IJRSR, 2012, Academic Journals. All rights reserved.
INTRODUCTION
India has a rich heritage of knowledge on plant based
drugs both for use in preventive and curative medicine. A
country like India is very much suited for development of
drugs from medicinal plant. Because of its vast and wide
variations in soil and climate, the Indian sub – continent is
suitable for cultivation of large number of medicinal and
aromatic plant which can be used as raw materials for
pharmaceutical, perfumery, cosmetics, flavour and food
and agrochemical industries. A large number of these
plants grow wild and exploited especially for use in
indigenous pharmaceutical houses. Some of these plants
produce valuable drugs which have high export potential.
(Rathish et al., 2005).
Nature has been a source of medicinal agents for
thousands of years and an impressive number of modern
drugs have been isolated from natural sources, many
based on their use in traditional medicine. Various
medicinal plants have been used for years in daily life to
treat disease all over the world. They have been used as a
source of medicine. The widespread use of herbal
remedies and healthcare preparations, such as
thoseescribed in ancient texts like the Vedas and the
Bible, has been traced to the occurrence of natural
* Corresponding author: +919443185302
E-mail address: prabhaharc@yahoo.com
products with medicinal properties. In fact, plants
produce a diverse range of bioactive molecules, making
them a rich source of different types of medicines. Higher
plants, as sources of medicinal compounds, have
continued to play a dominant role in the maintenance of
human health since ancient times (Farombi, 2003). Over
50% of all modern clinical drugs are of natural product
origin and natural products play an important role in drug
development programs in the pharmaceutical industry
(Baker et al., 1995).
Developing a medicinal plants sector, across the
various states of India has become an important issue.
Different stockholders in the medicinal plants sector have
projected Tamil Nadu, one of the southern states, as an
“Herbal State”. This nation has made medicinal plants as
a commodity of high value across the state. At the same
time, realizing the continuous depletion of this valuable
resource, attempts are being made for its large-scale
cultivation and multiplication in order to meet its
escalating demand as well as long-term sustainability.
There are many aspects of research associated with the
medicinal plants sector. The significant contribution to the
society, traditional medicine has experienced very little
attention in modern research and development and less
International Journal of Recent Scientific Research, Vol. 3, Issue, 3, pp. 149 -154, March, 2012
effort has been done to upgrade the practice (Giday et al.,
2003).
MATERIALS AND METHODS
Collection and Drying of Plant materials
In the modern world multiple drug resistance has
developed against many microbial infections due to the
indiscriminate use of commercial antimicrobial drugs
commonly used in the treatment of infectious disease. In
addition to this problem, antibiotics are sometimes
associated with adverse effects on the host including
hypersensitivity, immune-suppression and allergic
reactions. This situation forced scientists to search for
new antimicrobial substances. Given the alarming
incidence of antibiotic resistance in bacteria of medical
importance, there is a constant need for new and effective
therapeutic agents. Therefore, there is a need to develop
alternative antimicrobial drugs for the treatment of
infectious diseases from medicinal plants (Agarwal et al.,
1996).
Mature leaves of Acacia nilotica and Turnera subulata
were collected from the Herbal garden, Department of
Microbiology, Annamalai University, Chidambaram,
Tamil Nadu. The leaves were washed thoroughly three
times with water and once with distilled water. The plant
materials were air dried and powdered. The powdered
samples were hermetically sealed in separate polythene
bags until the time of extraction.
Preparation of plant extract
40 g of powdered leaves Acacia nilotica and Turnera
subulata were extracted successively with 200 ml of
ethanol at 56-60C and ethyl acetate at 40-50C in
Soxhelet extractor until the extract was clear. The extracts
were evaporated to dryness and the resulting pasty form
extracts were stored in a refrigerator at 4C for future use
(Chessbrough, 2000).
The use of plants and plant products as medicines
could be traced as far back as the beginning of human
civilization. The earliest mention of medicinal use of
plants in Hindu culture is founds in “Rig Veda”, which is
said to have been written between 4500-1600 B.C. and is
supposed to be the oldest repository of human knowledge.
It is Ayurveda, the foundation of medicinal science of
Hindu culture, in its eight division deals with specific
properties of drugs and various aspects of science of life
and the art of healing (Rastogi and Mehrotra, 2002).
Nowadays, there has been a revival of interest in herbal
medicines. This is due to increased awareness of the
limited ability of synthetic pharmaceutical products to
control major diseases and the need to discover new
molecular structures as lead compounds from the plant
kingdom. Plants are the basic source of knowledge of
modern medicine. The basic molecular and active
structures for synthetic fields are provided by rich natural
sources. This burgeoning worldwide interest in medicinal
plants reflects recognition of the validity of many
traditional claims regarding the value of natural products
in health care.
Test microorganisms
Six pathogenic fungi, viz., Candida albicans, Candida
glabrata, Aspergillus fumigatus, Aspergillus flavus,
Aspergillus niger and Penicillium chrysogenum were
collected from obtained from Rajah Muthaiya Medical
College Hospital, Chidambaram. The cultures were subcultured and maintained on Sabouraud’s dextrose agar
slants and stored in refrigerator at 4C.
Determination of antifungal activity
Inoculum preparation
Fungal inoculum was prepared by inoculating a loopful of
test organisms in 5 ml of Sabouraud’s dextrose and
incubated at room temperature for 3 days.
Determination of antifungal activity by Agar well
Diffusion Method
Sabouraud’s Dextrose agar plates were inoculated with
test organisms by spreading the fungal inoculum on the
surface of the media. Wells (5 mm in diameter) were
punched in the agar. Ethanol and ethyl acetate extracts
with different concentrations (100mg/ml, 200mg/ml and
300 mg/ml) were mixed with 1 ml of Dimethyl sulfoxide
(DMSO) and added into the well. Well containing DMSO
alone act as a negative control. The plates were incubated
at room temperature for 3 days. The antifungal activity
was assessed by measuring the diameter of the zone of
inhibition (in mm).
Antimicrobials of plant origin have enormous
therapeutic potential. They are effective in the treatment
of infectious diseases while simultaneously mitigating
many of the side effects that are often associated with
synthetic antimicrobials. The beneficial medicinal effects
of plant materials typically result from the combinations
of secondary products present in the plant. Turnera
subulata and Acacia nilotica plants are used in folk
therapy in the treatment of skin, GIT and respiratory
problems while seeds of later plant used in the treatment
of diabetic patients. The (aqueous and ethanolic) extracts
of all parts of Turnera subulata and Acacia nilotica plants
has been carried out on different microbes for
antibacterial study which contains other chemical
constituents of plants. The present study is focussed on
the pharmacological screening of Turnera subulata and
Acacia nilotica for its antifungal activity against fungal
pathogens.
RESULTS AND DISCUSSION
Medicinal plants are a source of great economic value all
over the world. Nature has been showed on us a very rich
botanical wealth and a large number of diverse types of
plants grow in different parts of the country. India is rich
in all the 3 levels of biodiversity, namely species
diversity, genetic diversity and habitat diversity. In India,
thousands of species are known to have medicinal value
and the use of different parts of several medicinal plants
to cure specific ailments has been in vogue since ancient
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International Journal of Recent Scientific Research, Vol. 3, Issue, 3, pp. 149 -154, March, 2012
results were showed in Table-1 and Table-2. The ethanol
extract of Turnera subulata (300mg/ml) recorded
maximum zone of inhibition against Candida glabrata
(22mm), followed by Aspergillus flavus (21mm),
Aspergillus niger (19mm), Aspergillus fumigatus (18mm),
Penicillium chrysogenum (13mm) and Candida albicans
(12mm). For ethyl acetate extract, maximum zone of
inhibition was seen against Candida glabrata (20mm)
followed by Candida albicans (13mm), Aspergillus niger
times. Herbal medicine is still the main stay of about 7580% of the whole population, and the major part of
traditional therapy involves the use of plant extract and
their active constituents (Akerele, 1993). Following the
advent of modern medicine, herbal medicine suffered a
setback, but during last two or three decades advances in
Phytochemistry and in identification of plant compounds
effective against certain diseases have renewed the
interest in herbal medicines (FAO, 1990).
Table 1 Antifungal activity of ethanol extract of Turnera subulata
S.No
Fungi
1
2
3
4
5
6
Candida albicans
Candida glabrata
Aspergillus fumigatus
Aspergillus flavus
Aspergillus niger
Penicillium chrysogenum
Concentration of the extract (mg/ml) and zone of inhibition (mm)
DMSO
100 mg/ml
200 mg/ml
No zone
8mm
10mm
No zone
14mm
28mm
No zone
7mm
12mm
No zone
11mm
15mm
No zone
10mm
14mm
No zone
10mm
12mm
300 mg/ml
12mm
22mm
18mm
21mm
19mm
13mm
Table 2 Antifungal activity of ethyl acetate extract of Turnera subulata
S.No
1
2
3
4
5
6
Fungi
Candida albicans
Candida glabrata
Aspergillus fumigatus
Aspergillus flavus
Aspergillus niger
Penicillium chrysogenum
Concentration of the extract (mg/ml) and zone of inhibition (mm)
DMSO
100 mg/ml
200 mg/ml
300 mg/ml
No zone
No zone
7mm
13mm
No zone
9mm
14mm
20mm
No zone
No zone
6mm
8mm
No zone
No zone
No zone
No zone
No zone
No zone
7mm
9mm
No zone
No zone
No zone
6mm
Table 3 Antifungal activity of ethanol extract of Acacia nilotica
S.No
1
2
3
4
5
6
Fungi
Candida albicans
Candida glabrata
Aspergillus fumigatus
Aspergillus flavus
Aspergillus niger
Penicillium chrysogenum
Concentration of the extract (mg/ml) and zone of inhibition (mm)
DMSO
100 mg/ml
200 mg/ml
300 mg/ml
No zone
12mm
14mm
15mm
No zone
No zone
6mm
8mm
No zone
8mm
10mm
13mm
No zone
15mm
19mm
25mm
No zone
No zone
No zone
No zone
No zone
6mm
7mm
11mm
Table 4 Antifungal activity of ethyl acetate extract of Acacia nilotica
S.No
1
2
3
4
5
6
Fungi
Candida albicans
Candida glabrata
Aspergillus fumigatus
Aspergillus flavus
Aspergillus niger
Penicillium chrysogenum
Concentration of the extract (mg/ml) and zone of
inhibition(mm)
DMSO
100 mg/ml
200 mg/ml
30mg/ml
No zone
7 mm
9 mm
10 mm
No zone
No zone
No zone
No zone
No zone
6 mm
9 mm
12 mm
No zone
10 mm
12 mm
15 mm
No zone
No zone
No zone
No zone
No zone
No zone
No zone
7mm
The beneficial medicinal effects of plant materials
typically result from the secondary products present in the
plant although, it is usually not attributed to a single
compound but a combination of the metabolites. The
medicinal actions of plants are unique to a particular plant
species or group, consistent with the concept that the
combination of secondary products in a particular plant is
taxonomically distinct (Parekh et al., 2005). The
screening of plants usually involves several approaches,
Ethno botanical approach is one of the common methods
that are employed in choosing the plant for
pharmacological study.
(9mm), Aspergillus fumigatus (8mm) and Penicillium
chrysogenum (6mm). No zone of inhibition was recorded
against Aspergillus flavus and DMSO control. The
ethanol extract of Turnera subulata showed more fungal
inhibitory activity when compared to ethyl acetate extract.
Kawther Abeb (2007) evaluated the antibacterial
activity of dill and fennel against Mycobacterium sp,
Staphylococcus aureus, Bacillus subtilis, Escherichia coli,
Pseudomonas aeruginosa and Candida albicans. The
results showed that the volatile oils extracted by steam
distillation method from roots, stem and leaves of dill and
fennel plants did not show antibacterial or anticandidal
activities. Seed extracts from both dill and fennel
exhibited varying degrees of growth inhibition of
The antifungal activity of the ethanol and ethyl acetate
extract of Turnera subulata was investigated and the
151
International Journal of Recent Scientific Research, Vol. 3, Issue, 3, pp. 149 -154, March, 2012
Candida albicans, Candida tropicalis and Candida
glabrata. Extracts of dill and fennel seeds prepared by
simple solvent extraction method, using acetone,
petroleum ether, methanol and chloroform, did not show
any antimicrobial activity against common bacterial or
fungal pathogens. Growth of some Mycobacterium sp.
was inhibited by the seed extracts of both fennel and dill.
Their results suggested that the anticandidal and
antimycobacterial properties of these two herbs may be
further investigated to explore the possibility of using
them in the treatment of candidal or mycobacterial
infections. Sundaram Ravikumar et al. (2010) screened
the in vitro antibacterial and antifungal activity of the
chloroform extracts of the seventeen different coastal
medicinal plants against different gram positive and gram
negative and fungal ornamental fish pathogens. Of the
selected plants Datura metel showed wide range of
antimicrobial activity against many of the fish pathogens.
He concluded that the Datura metel has been used as a
putative antimicrobial drug in the aquaculture
maintenance.
more fungal inhibitory activity when compared to ethyl
acetate extract.
Ali Rehman et al. (2002) tested the aqueous and
ethanol extracts of Acalypha indica against Microsporum
canis, Aspergillus fumigatus, Candida albicans,
Escherichia coli and Staphylococcus aureus by disc
diffusion method. There was no zone of inhibition of
Acalypha indica towards Aspergillus fumigatus and
Candida albicans. Anand et al. (2007) tested the
medicinal plant extracts of Curcuma longa, Acalypha
indica, and Anona sqamosa by Cold percolation method
against Dermatophytic isolates. Curcuma lounga showed
antifungal effect against Trichophyton rubrum and
Microsporum gypseum. These two organisms were found
to be resistant towards Acalypha indica and Anona
sqamosa. The other dermatophytes were resistant to all
medicinal plants tested.
Satish et al. (2007) tested the aqueous extract of 52
plants from different families for their antifungal potential
against eight important species of Aspergillus such as
Aspergillus candidus, Aspergillus columnaris, Aspergillus
flavipes, Aspergillus flavus, Aspergillus fumigatus,
Aspergillus niger, Aspergillus ochraceus, and Aspergillus
tamarii. Among fifty-two plants tested, aqueous extract of
Acacia nilotica, Achras zapota, Datura stramonium,
Emblica officinalis, Eucalyptus globules, Lawsonia
inermis, Mimusops elengi, Peltophorum pterocarpum,
Polyalthia longifolia, Prosopis juliflora, Punica granatum
and Sygigium cumini have recorded significant antifungal
activity against one or the other Aspergillus species
tested. Aspergillus flavus recorded high susceptibility and
hence solvent extracts viz., petroleum ether, benzene,
chloroform, methanol and ethanol extracts of all the
twelve plants were tested for their antifungal activity.
Among the solvent extracts tested, methanol gave more
effective than ethanol, chloroform, benzene and
petroleum ether.
Siva Sakthi et al. (2011) evaluated the antibacterial
potentiality of ethanol and ethyl acetate solvent extracts of
mature leaves of Datura metel against nine pathogenic
bacteria isolates viz., Staphylococcus aureus, Bacillus
subtilis, Bacillus cereus, Escherichia coli, Salmonella
typhi, Shigella flexneri, Klebsiella pneumoniae, Vibrio
cholerae and Pseudomonas aeruginosa. The ethanol
extract of Datura metel (100 mg/ml) showed maximum
zone of inhibition (26 mm) against Pseudomonas
aeruginosa, Escherichia coli and Bacillus subtilis.
Staphylococcus aureus showed less zone of inhibition (8
mm). The ethyl acetate extract of Datura metel (100
mg/ml) showed maximum zone of inhibition (19 mm)
against Escherichia coli. There was no zone of inhibition
against Pseudomonas aeruginosa. The findings of the
present study coincide with the results of Siva Sakthi et
al. (2011). In this study, ethanol was best solution for
extracting the effective antifungal substances from the
medicinal plant Acacia nilotica and Turnera subulata
than ethyl acetate. This could be related to the presence of
bioactive metabolites present in Datura metel which are
not soluble in ethyl acetate but they can be soluble in
ethanol.
Saranraj et al. (2010) investigated the antibacterial
potentiality of ethanol and ethyl acetate extract of
Acalypha indica leaves against human pathogenic bacteria
and concluded that and concluded that the ethanol extract
showed more inhibitory activity against human
pathogenic bacteria when compared to ethyl acetate
extract. The findings of the present study coincide with
the results of Saranraj et al. (2010). The findings of the
present study showed that the ethanol was best solution
for extracting the effective antifungal substances from the
medicinal plant Acacia nilotica than ethyl acetate. This
could be related to the presence of bioactive metabolites
present in Acacia nilotica which are not soluble in ethyl
acetate but they can be soluble in ethanol.
The antifungal activity of the ethanol and ethyl acetate
extract of Acacia nilotica was evaluated and the results
were showed in Table-3 and Table-4. The ethanol extract
of Acacia nilotica (300mg/ml) recorded maximum zone
of inhibition against Aspergillus flavus (25mm) followed
by Candida albicans (15mm), Aspergillus fumigatus
(13mm), Penicillium chrysogenum (11mm) and Candida
glabrata (8mm). No zone of inhibition was recorded
against Aspergillus niger. For ethyl acetate extract,
maximum zone of inhibition was recorded against
Aspergillus flavus (15mm), Aspergillus fumigatus
(12mm), Candida albicans (10mm) and Penicillium
chrysogenum (7mm). No zone of inhibition was seen
against Candida glabrata, Aspergillus niger and DMSO
control. The ethanol extract of Acacia nilotica showed
Recently, Saranraj et al. (2011) screened the
pharmacological activity of the ethanol and ethyl acetate
extract of Datura metel and Acalypha indica for its
antifungal activity against pathogenic fungi. Six different
fungal isolates viz., Candida albicans, Candida glabrata,
Aspergillus fumigatus, Aspergillus flavus, Aspergillus
niger and Penicillium chrysogenum were tested for its
antifungal activity. The collected leaf samples were
152
International Journal of Recent Scientific Research, Vol. 3, Issue, 3, pp. 149 -154, March, 2012
powdered and the bioactive compounds were extracted by
using ethanol and ethyl acetate in a Soxhelet extractor.
The antifungal activity was determined by using Well
diffusion method. Ethanol and ethyl acetate extracts with
different concentrations were mixed with 1 ml of
Dimethyl sulfoxide and added into the well. The
inhibitory effect of ethanol extract was relatively high
when compared to ethyl acetate extract. The extract of
Datura metel showed maximum zone of inhibition against
fungal pathogens when compared to Acalypha indica.
The findings of the present investigation suggests that the
organic solvent extraction was suitable to verify the
antimicrobial properties of medicinal plants and they
supported by many investigation. The present study
justifies the claimed uses of leaves in the traditional
system of medicine to treat various infectious diseases
caused by the microbes. This study also encourages
cultivation of the highly valuable plant in large scale to
increase the economic status of the cultivators in the
country. The obtained results may provide a support to
use of the plant in traditional medicine. Based on this
further chemical and pharmacological investigations can
be done to isolate and identify minor chemical
constituents in the seeds and to screen other potential
bioactivities may be recommended.
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CONCLUSION
The present research concluded that the organic solvent
extraction was suitable to verify the antimicrobial
properties of medicinal plants and they supported by
many investigation. The investigation on antifungal
activity of herbal plant extracts of Turnera subulata and
Acacia nilotica showed that the ethanol extract shows
promising antifungal activity against fungal pathogens
when compared to ethyl acetate extract. Phytochemical
analysis showed that the antibacterial activity of Turnera
subulata and Acacia nilotica was due to the presence of
phytochemical compounds like alkaloids, tripenoid,
steroids, flavonoid, triterpenes, phenolic compounds and
tannins. The extract of Turnera subulata showed
maximum zone of inhibition against fungal pathogens
when compared to Acacia nilotica The results also
indicated that scientific studies carried out on medicinal
plants having traditional claims of effectiveness might
warrant fruitful results. These plants could serve as useful
source of new antimicrobial agents. The present study
justifies the claimed uses of leaves in the traditional
system of medicine to treat various infectious diseases
caused by the microbes. This study also encourages
cultivation of the highly valuable plant in large scale to
increase the economic status of the cultivators in the
country. The obtained results may provide a support to
use of the plant in traditional medicine.
Acknowledgement
The authors are deeply indebted to Professor and Head,
Department of Zoology, Annamalai University,
Annamalai Nagar, Tamil Nadu, India for their inspiring
help, constant support and for providing adequate
laboratory facilities in the department to carry out the
research work.
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