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International Journal of Herbal Medicine 2017; 5(5): 192-195
E-ISSN: 2321-2187
P-ISSN: 2394-0514
IJHM 2017; 5(5): 192-195
Received: 14-07-2017
Accepted: 15-08-2017
Prashith Kekuda TR
Department of Microbiology,
S.R.N.M.N College of Applied
Sciences, N.E.S Campus, Balraj
Urs Road, Shivamogga,
Karnataka, India
Vinayaka KS
Department of Botany,
Kumadvathi First Grade College,
Shimoga Road, Shikaripura,
Karnataka, India.
Praveen Kumar SK
Department of Studies in
Biochemistry, Karnatak
University, Dharwad,
Karnataka, India
Correspondence
Prashith Kekuda TR
Department of Microbiology,
S.R.N.M.N College of Applied
Sciences, N.E.S Campus, Balraj
Urs Road, Shivamogga,
Karnataka, India
Antimicrobial activity of Salix tetrasperma Roxb.
(Salicaceae)
Prashith Kekuda TR, Vinayaka KS and Praveen Kumar SK
Abstract
Salix tetrasperma Roxb. belongs to the family Salicaceae. The plant is traditionally used and is reported
to exhibit several bioactivities. The present study was carried out to investigate antimicrobial activity of
leaf extract of S. tetrasperma. Extraction of shade dried and powdered leaf material was carried out by
maceration process. Antibacterial activity was evaluated by agar well diffusion method against 4 Gram
positive and 4 Gram negative bacteria. Antifungal activity was determined against 4 seed-borne fungi by
poisoned food technique. The extract exhibited concentration dependent activity against test bacteria.
Among Gram positive and Gram negative bacteria, S. epidermidis and P. aeruginosa displayed highest
susceptibility to extract respectively. Inhibition of test fungi by extract was concentration dependent.
Among fungi, marked and least susceptibility to extract was shown by Rhizopus sp. and Cladosporium
sp. respectively. In suitable form, the leaf of S. tetrasperma can be used to treat bacterial infections and to
manage seed-borne fungal diseases of plants.
Keywords: Salix tetrasperma, Maceration, Agar well diffusion, Poisoned food technique
1. Introduction
Antibiotic resistance is a global problem and is a serious and widespread problem in
developing countries. Emergence of resistant pathogens is mainly due to indiscriminate use of
antibiotics and the resistant pathogens are of serious concern in both hospitals and the
community. Bacteria such as Staphylococcus aureus, Mycobacterium tuberculosis,
Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa are few among the
antibiotic resistant bacteria. Antibiotic resistant bacteria are known limit the efficacy of current
drugs and cause high mortality each year due to failure of treatment. This situation triggered
immense research on searching for alternatives for disease therapy. Natural products, in
particular botanicals, offer as an important alternative strategy for therapy against infectious
diseases. Extracts and purified compounds from higher plants have shown to exhibit potent
inhibitory activity against a wide range of pathogenic bacteria including antibiotic resistant
bacteria [1-7].
Seed is an important input in the production of several crops. Seeds are considered as passive
carriers of several pathogenic microorganisms. Fungi such as species of Aspergillus,
Helminthosporium, Cladosporium, Penicillium, Rhizopus, Fusarium, Curvularia, Alternaria
and Cercospora are associated with seeds. Many seed-borne fungi are known to cause
reduction in seed quality, seed abortion, seed rot, reduction of germination ability and seedling
damage. Besides, some seed-borne fungi such as Aspergillus and Penicillium are known to
elaborate certain toxins (mycotoxins) which cause severe health hazards on consumption.
Fungal diseases of plants are one of the major constraints in successful crop production and are
known to cause severe yield loss. The unrestrained use of synthetic fungicides for the control
of phytopathogenic fungi and diseases of crop plants resulted in serious threat to human health
as well as environment. Extensive use of fungicide is leading to disturbed biodiversity (due to
effects on non-target organisms) and development of resistance in the plant pathogens. Hence
there is immense scope for searching alternative strategies for diseases control. The use of
natural products such as plants and biological control agents appears to be the best alternatives
for chemical agents [8-11].
The genus Salix comprises about several species mainly distributed in the temperate regions of
the world and in higher altitudes of the tropics. Salix tetrasperma Roxb., belonging to the
family Salicaceae, is a small sized tree characterized by glabrous to silky pubescent twigs and
simple, shiny, elliptic-lanceolate leaves with acute apex and characteristic margin. Bark is steel
grey in color, farrowed and scaly in appearance. Flowers are unisexual, sessile and are in
catkin. Fruit is a 2-valved capsule, dry and dehiscent. The plant is commonly known as Indian
willow and often grows along water bodies. The plant is found distributed in various parts of
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International Journal of Herbal Medicine
Karnataka such as Bangalore, Belgaum, Chikamagalur,
Shimoga, Mysore, Kodagu, Hassan and Uttara Kannada [12, 13].
The plant is used as fuel wood, and in making furniture, roof,
fence and agricultural tools. The twigs are used to make
baskets [14-16]. Various parts of S. tetrasperma are used
traditionally for the treatment of ailments or diseases such as
pain, fever, rheumatism, epilepsy, venereal diseases,
swellings, piles and bladder stone [17-21]. Several bioactivities
such as analgesic [13], anti-inflammatory [13], antipyretic [22],
antioxidant [13, 23], antimicrobial [24, 25], hypoglycaemic [26],
laxative [27], diuretic [27], cytotoxic [28], insecticidal [24] and
CNS activity [29]. The present study was carried out to
investigate antibacterial and antifungal activity of solvent
extract obtained from leaves of S. tetrasperma.
2. Materials and Methods
2.1. Media and chemicals
Three culture media viz. Nutrient agar, Nutrient broth and
Potato dextrose agar and chemicals viz. methanol,
Streptomycin and Dimethyl sulfoxide (DMSO) were
purchased from HiMedia laboratories, Mumbai, India.
2.2. Plant material and extraction
The plant S. tetrasperma was collected at Siddarahalli, near
Matturu, Shivamogga, Karnataka during January 2017. The
plant was authenticated by Prof. D. Rudrappa, Department of
Botany, S.R.N.M.N College of Applied Sciences,
Shivamogga. The leaves were separated, washed, dried under
shade and powdered. Extraction of leaf powder (10g) was
carried out by maceration technique using methanol (100ml).
The leaf material was left in methanol for two days in
stoppered container and later filtered [30]. The leaf extract (LE)
obtained was used for antimicrobial studies.
2.3. Test bacteria and inoculum preparation
Four Gram positive bacteria (Staphylococcus aureus NCIM
5345, Staphylococcus epidermidis NCIM 2493, Bacillus
subtilis NCIM 2063 and Bacillus cereus NCIM 2016) and
four Gram negative bacteria (Klebsiella pneumoniae NCIM
2957, Escherichia coli NCIM 2065, Pseudomonas aeruginosa
NCIM 2200 and Salmonella typhimurium NCIM 2501) were
used. The test bacteria were maintained on in nutrient agar
slants under refrigeration condition. The test bacteria were
seeded into sterile nutrient broth tubes and incubated
overnight at 37 oC.
2.4. Antibacterial activity of LE of S. tetrasperma
Agar well diffusion method as employed by Raghavendra et
al.[31] was used to evaluate antibacterial activity of two
concentrations of LE (10mg/ml and 20mg/ml) of S.
tetrasperma. The method involved swabbing of overnight
growth broth cultures of test bacteria on the surface of sterile
nutrient agar plates aseptically followed by punching wells of
8mm diameter using a sterile cork-borer. The wells were
labeled and filled with 100µl of LE, reference antibiotic
(Streptomycin, 1mg/ml of sterile distilled water) and DMSO.
The plates were left undisturbed for 24 hours at 37oC
followed by measuring the diameter of inhibition zones
formed around the wells.
2.5. Test fungi
Four seed-borne fungi viz. Cladosporium sp.,
Helminthosporium sp., Rhizopus sp. and Penicillium sp.
isolated previously from paddy seeds were used. The test
fungi were maintained on potato dextrose agar slants in
refrigerator.
2.6. Antifungal activity of LE of S. tetrasperma
Poisoned food technique was employed to evaluate antifungal
potential of LE of S. tetrasperma. Potato dextrose agar was
prepared, autoclaved, poisoned with LE (0.5 and 1.0mg/ml of
medium) and poured into sterile plates. The control (without
LE) and poisoned potato dextrose agar plates were inoculated
aseptically with well sporulated (7 days old) cultures of test
fungi and the plates were incubated at room temperature for 5
days. Later, the diameter of fungal colonies was measured
using a ruler. Extent of inhibition of mycelial growth of fungi
(%) was calculated using the formula:
Mycelial growth inhibition (%) = (Dc – Dt / Dc) x 100, where
Dc refers to colony diameter of fungi in control plates and Dt
denotes diameter of fungal colonies in poisoned plates[11].
2.7. Statistical analysis
Antibacterial and antifungal experiments were carried out in
triplicate. The results were presented as Mean ± Standard
deviation (S.D).
3. Results and Discussion
3.1. Antibacterial activity of LE of S. tetrasperma
In the present study, the LE of S. tetrasperma was found to
exhibit concentration dependent inhibitory activity against test
bacteria as noticed by an increase in the diameter of zones of
inhibition around wells on increasing in the concentration of
extract. LE exhibited varied inhibitory activity against test
bacteria. Marked and least inhibitory activity was observed
against S. epidermidis and E. coli respectively. At 10mg/ml
concentration, LE was not effective in inhibiting E. coli.
Among Gram positive and Gram negative bacteria, S.
epidermidis and P. aeruginosa displayed highest
susceptibility to extract respectively. Reference antibiotic
displayed higher inhibition of test bacteria when compared to
LE. Overall, LE as well as reference antibiotic were more
active against Gram positive bacteria when compared to Gram
negative bacteria. In an earlier study, Islam et al.[24] also
observed the inhibition of leaf extract of S. tetrasperma
against Gram positive bacteria while most of Gram negative
bacteria remain unaffected. In another study, a herbal
formulation containing S. tetrasperma was shown to inhibit
acne causing Propionibacterium acnes and Staphylococcus
epidermidis[32]. Abdel-Hameed et al.[33] evaluated
antibacterial activity of leaves of S. tetrasperma and observed
that the plant was not effective in exhibiting antibacterial
activity at 1mg/ml concentration tested. The lower
susceptibility of Gram negative bacteria to LE of S.
tetrasperma and antibiotic could be attributed to the presence
of an outer membrane which might have acted as an
additional barrier for the entry of LE and antibiotic.
Table 1: Antibacterial activity of leaf extract of S. tetrasperma
Test bacteria Zone of inhibition in cm (Mean± S.D)
LE
10mg/ml LE
20mg/ml Antibiotic DMSO
S. aureus 1.03±0.00 1.30±0.00 2.86±0.05 0.00±0.00
S. epidermidis 2.10±0.10 2.66±0.05 3.30±0.00 0.00±0.00
B. cereus 1.73±0.05 2.10±0.00 3.10±0.10 0.00±0.00
B. subtilis 1.60±0.00 1.80±0.10 3.20±0.00 0.00±0.00
K. pneumoniae 1.33±0.05 1.60±0.00 2.53±0.05 0.00±0.00
P. aeruginosa 1.50±0.10 1.70±0.00 2.60±0.00 0.00±0.00
S. typhimurium 1.30±0.00 1.53±0.05 2.50±0.00 0.00±0.00
E. coli 0.00±0.00 1.10±0.00 2.20±0.10 0.00±0.00
3.2. Antifungal activity of LE of S. tetrasperma
A considerable reduction in mycelial growth of test fungi was
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International Journal of Herbal Medicine
observed in plates poisoned with LE of S. tetrasperma. The
LE displayed concentration dependent inhibition of test fungi
(Table 2). Among fungi, marked and least susceptibility to
extract was shown by Rhizopus sp. and Cladosporium sp.
respectively. At extract concentration of 1mg/ml, an
inhibition of >60% of all fungi was observed. At 0.5mg/ml
concentration, Cladosporium sp., Rhizopus sp.,
Helminthosporium sp. and Penicillium sp. were inhibited to
25.79%, 39.70%, 27.20% and 33.33% respectively. At
1.0mg/ml concentration, Cladosporium sp., Rhizopus sp.,
Helminthosporium sp. and Penicillium sp. were inhibited to
61.13%, 70.14%, 62.79% and 63.63% respectively. In a
similar study, Pushpavathi et al.[25] observed marked
inhibitory activity of leaf extract of S. tetrasperma against
seed-borne fungi isolated from sorghum seeds. The study of
Abdel-Hameed et al.[33] showed no inhibitory activity of leaf
extract of S. tetrasperma against Candida albicans and
Aspergillus niger. In a study by Deepak et al.[34], S.
tetrasperma failed to show inhibition of zoosporangium
formation of Sclerospora graminicola, causal agent of downy
mildew disease of pearl millet.
Table 2: Colony diameter of test fungi in control and poisoned
plates
Test fungi Colony diameter in cm
Control LE 0.5mg/ml LE 1.0mg/ml
Cladosporium sp. 2.83±0.05 2.10±0.10 1.10±0.10
Rhizopus sp. 6.80±0.00 4.10±0.00 2.03±0.05
Penicillium sp. 3.30±0.00 2.20±0.10 1.20±0.00
Helminthosporium sp. 4.30±0.10 3.13±0.05 1.60±0.00
4. Conclusion
In the present study, the LE of S. tetrasperma exhibited
concentration dependent inhibitory activity against bacteria
and fungi. The plant appears to be a suitable candidate for
developing herbal formulation and lead drugs for the
treatment of infectious bacterial diseases and management of
plant diseases caused by seed mycoflora.
5. Acknowledgements
Author is thankful to head of the department of Microbiology
and Principal, S.RN.M.N College of Applied Sciences and
N.E.S, Shivamogga for support. Author also thanks Prof. D.
Rudrappa for assisting in the collection and identification of
plant material.
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