TITLE: Antimicrobial potential of Asphodelus tenuifolius (cav.)
Name of authors:
1. Dr. A.S. Dangi
MSc. PhD
Deptt. of Microbiology
PT.BDS PGIMS,Rohtak
2. Dr. Aparna*
MBBS,MD(Microbiology)
Professor, Deptt. of Microbiology
PT.BDS PGIMS, Rohtak
3 Dr. Madhu Sharma
MBBS,MD(Microbiology)
Professor, Deptt. of Microbiology
PT.BDS PGIMS,Rohtak
4 Dr. J.P.Yadav
MSc.PhD
Prof & Head,Deptt of Genetics
MDU,Rohtak
5 Dr. D.R. Arora
MBBS,MD(Microbiology)
SGT Medical college,Budhera,Gurgaon
6 Dr. Uma chaudhary
MBBS,MD(Microbiology)
Sr.Prof & Head, Deptt. of Microbiology
PT.BDS PGIMS,Rohtak
3. Corresponding author : Dr. Aparna*
MBBS,MD(Microbiology)
Professor,Deptt. of Microbiology
PT.BDS PGIMS,Rohtak
E-mail : aparnaparmar2008@rediffmail.com
Abstract
With the upcoming increase in drug resistant bacteria, there is a need to develop new
antimicrobial agents. The demand for more and more drugs from plant sources is continuously
increasing. Asphodelus tenuifolius (cav.) is a known medicinal plant used for rubbing on the bite
of bees and wasps for relief. The present study was carried out to investigate antimicrobial
activity against some pathogenic bacteria and fungi. Asphodelus tenuifolius (cav.) exhibited
antibacterial activity against Vibrio cholerae, Staphylococcus aureus, Pseudomonas aeruginosa,
Proteus mirabilis, Salmonella Typhi and Shigella flexneri. No antifungal activity was observed.
Therefore, the role of Asphodelus tenuifolius as an antibacterial agent is quite evident and it
should be evaluated further as a therapeutic agent for use in future.
Key Words: Asphodelus tenuifolius (cav.), antibacterial activity, antifungal activity
INTRODUCTION
India is a rich meadow of medicinal plants and its wealth of medicinal and aromatic
plants are known the world over. A large proportion of the Indian population even today depends
on the Indian system of medicine – Ayurveda, an ancient science of life. India enjoys the
privilege of having the time tested traditional system of medicines based on natural products.
Medicinal and aromatic plants are one of the major natural resources of our country occurring in
a diverse ecosystem. Most of the drugs used in primitive medicine were obtained from the plants.
India’s rich heritage of plants is because of its wide diversity in soil and climatic conditions.
Thus it has one of the oldest, richest and diverse folk traditions associated with the use of
medicinal plants in a healthcare system to promote, safeguard and relieve pain, heal wounds and
refresh mind, muscles and nerves1.
Since the introduction of antibiotics there has been tremendous increase in the resistance
of diverse bacterial pathogens2,3. This shift in susceptibility greatly affects our ability to
successfully treat patients empirically. The demand for more and more drugs from plant sources
is continuously increasing. It is therefore essential to evaluate plants of medicinal value
systematically for various ailments that are used in traditional medicine4. Hence, there is a need
to screen medicinal plants for their promising biological activity.
In the present study, we have evaluated the antimicrobial effects of extracts of
Asphodelus tenuifolius (cav.) It is an annual stemless herb which is variable in size. Leaves are
hollow with several scapes. It is simple or branched in the upper part and has white flowers.
Traditionally the paste of plant was used for rubbing on the bite of bees and wasps for relief. The
seeds are generally taken for cold and hemorrhoids, a febrifuge and also used for rheumatic pain.
Seeds are also used as diuretic agent, for healing wounds and they are applied externally to
ulcers and for inflammed parts5.We studied the efficacy of extracts ofshade dried seeds of A.
tenuifolius in different solvents against bacterial and fungal pathogens.
MATERIALS AND METHODS
The plant as a whole was collected from different parts of Haryana and it was confirmed
at Forest Research Institute, Dehradun and herbarium of the department of Genetics, M.D.
University, Rohtak, and Haryana. Voucher specimen (MDU 6807) was deposited in the Deptt. of
Genetics, MDU,Rohtak. The plant was trimmed, cleaned and dried. A total of 320 gm of dried
seeds of A.tenuifolius was used for extraction of the organic constituents in Soxhlet apparatus.
The extraction of plant material was made in different solvents viz petroleum ether, ethanol,
methanol and water sequentially according to their increasing polarity. Dried extracts were
lyophilized and were kept in the refrigerator in screw capped vials. The extracts were also
subjected to preliminary phytochemical analysis. The extracts were tested for their antimicrobial
activities against nine bacterial and two fungal strains. The bacterial strains included,
Staphylococcus aureus (ATCC 259323), Escherichia coli (ATCC 25922), Klebsiella
pneumoniae (ATCC 700603), Pseudomonas aeruginosa (ATCC 27853), Proteus mirabilis
(ATCC 43071), Salmonella Typhi (ATCC 13311), Shigella flexnerii (ATCC 12022), Vibrio
cholerae(ATCC 39315), and Serratia marcescens(ATCC 27137). Two fungal strains were
Candida albicans(ATCC 10231) and Aspergillus niger (ATCC 16404).
The bacterial strains were grown on nutrient agar plates and fungal strains on Sabouraud
dextrose agar slants. The susceptibility testing was done on Mueller-Hinton agar as per modified
Kirby Bauer disc diffusion method. The extract discs were prepared by using sterilized What
man filter paper No. 1 discs of 6mm in diameter, soaked in 30 µl of extract dissolved in DMSO.
Antibiotic disc of amikacin (30µg/ml) was used as positive control for bacteria and Ketoconazole
(10µg/ml) for fungi and DMSO added discs were used as negative control to determine possible
inhibitory activity of the diluents of extracts6.
The MIC and MFC were detected by: 1) Macro broth dilution method as per NCCLSTwo fold serial dilution of all extracts (64-0.025mg/ml) were prepared in tubes containing
DMSO as diluents. Bacterial suspensions were adjusted to the 0.5 McFarland’s standard (108
CFU/ml) and 104 spores/ml for fungi. To the final inoculates of bacterial suspensions, different
dilutions of plant extract and Mueller-Hinton broths were added to all the tubes and they were
incubated at 37ºC for 24 hrs. for bacteria and 24-48 hrs. for fungi in BOD incubator. The MIC
was the lowest concentration of the extract at which there was no visible growth of the
organism6.
2) By micro broth dilution method- It was done in 96 wells micro titre plates by Sarker et
al7 with slight modification. The crude extracts were first diluted to the highest concentration
(40,000-625 µg/ml) to be tested and 50 µl of normal saline was distributed from 2nd to 9th well.
50 µl from each extract was pipette into the first test well of each micro titre line which acted as
sterility control, and then 50 µl of scalar dilution of the plant extract was transferred from the
second to the ninth well. To each well 10µl of resazurin indicator solution was added. Then 30µl
of Mueller-Hinton broth was added to each well. Finally, 10µl of bacterial suspension was added
to each well. A column with a broad-spectrum antibiotic (streptomycin) was used as the positive
control. The test was put up in triplicate. The plates were incubated at 37ºC for 24 hrs. The color
change was assessed visually. Any color change from purple to pink was taken as positive. The
lowest concentration at which the color change occurred was taken as MIC value. The average of
three values was calculated and it was taken as MIC for the test.
RESULTS AND DISCUSSION
The dried seeds of A. tenuifolius showed maximum yield and extractive values in
aqueous (8.2 gm, 2.56%) followed by methanol (3.6 gm, 1.12%), ethanol (2.4 gm, 0.75%) and
petroleum ether(2.2gm, 0.68%). The other physical properties are shown in Table 1.
Antimicrobial activity and MIC values of all extracts have been shown in Table 2 and 3.
The ethanol extracts showed maximum inhibition against Vibrio cholerae (16 mm, MIC 16
µg/ml) followed by Proteus mirabilis, Salmonella Typhi, Shigella flexneri, Staphylococcus
aureus, Pseudomonas aeruginosa and Serratia marcescens. The methanol extracts also exibited
strong antibacterial activity against V.cholerae while the aqueous extract showed maximum
antibacterial activity against P.mirabilis. The petroleum ether extract did not exhibit any
antibacterial activity against any test organism.
Vaghasiya and Chanda 8 evaluated the antibacterial activity of methanol and acetone
extract of leaves and stem of A.tenuifolius and found that the methanol extract exhibited activity
against S.aureus (9 mm), Bacillus cereus (13 mm), Citrobacter freundii (10 mm) and acetone
extract was active against Klebsiella pneumoniae (17 mm).The methanol extract also showed
antifungal activity against Candida tropicalis with an inhibition zoneof 11 mm. However we
could not detect any antifungal activity of A.tunifolius.
The preliminary phytochemical evaluation study revealed the presence of anthraquinones,
anthrocyanins and cardiac glycosides from A.tenuifolius.
Antibiotics provide the main basis for the therapy of microbial infections. Since the
discovery of these antibiotics and their uses as chemotherapeutic agents there was a belief in the
medical fraternity that this would lead to the eventual eradication of infectious diseases.
However, overuse of antibiotics has become the major factor for the emergence and
dissemination of multidrug resistant (MDR) strains of several groups of microorganisms. Thus,
in light of the evidence of rapid global spread of resistant clinical isolates, the need to find new
antimicrobial agents is of paramount importance. However, past records reveal that even new
families of antimicrobial agents become ineffective after awhile. Thus the researchers are
increasingly turning their attention to herbal products, looking for new leads to develop better
drugs against MDR strains. Advances in the area of cell cultures for the production of medicinal
compounds has made possible the production of a wide variety of pharmaceutical agents.
Table-1 Physical properties of extract of Asphodelus tenuifolius(cav.) obtained from 320 gm dry
weight of plant material
S.No.
Solvent
Yeild(gm)
used for
Extractive
Color
Odor
value %
Physical
state
extraction
1.
Petroleum
2.2
0.68
Brown
Light smell Viscous
2.4
0.75
Dark
Light smell Viscous
ether (6080º C)
2.
Ethanol
brown
3.
4.
Methanol
Aqueous
3.6
8.2
1.12
2.56
Dark
Tobacco
brown
like
Black
Pungent
smell
Viscous
Solid
Table 2 Antimicrobial activity of plant extract of Asphodelus tenuifolius in different solvents
measured by zones of inhibition(mm)
Pathogens
Positive
Petroleum
Ethanol
Methanol
Aqueous
Control
ether
S.aureus
30±0.17
0
13±0.222
14±0.32
12±0.18
E.coli
32±0.18
0
0
0
0
K.pneumoniae 24±0.24
0
0
0
0
P.aeruginosa
27±0.12
0
13±0.23
15±0.22
14±0.16
Proteus
23±0.24
0
14±0.22
16±0.11
16±0.32
S.Typhi
25±0.34
0
14±0.31
15±0.34
15±0.16
Shigella
28±0.26
0
14±0.25
13±0.14
12±0.40
V.cholerae
22±0.18
0
16±0.19
18±0.32
14±0.22
Serratia
28±0.43
0
11±0.16
12±0.32
0
28±0.36
0
0
0
0
25±0.28
0
0
0
0
mirabilis
flexneri
marcescens
Candida
albicans
Aspergillus
niger
DMSO 30µl/disc taken as negative control (zero)
Amikacin 30mcg/disc taken as positive control for bacteria.
Fluconazole 10mcg/disc taken as positive control for fungus (Candida albicans)
Ketoconazole 10mcg/disc taken as positive control for Aspergillus niger
All values are representation as mean ± S.E.(standard error)
Table 3 Minimum inhibitory concentration of Asphodelus tenuifolius in µg/ml
Pathogen
Petroleum ether
Ethanol
Methanol
Aqueous
S.aureus
0
16
8
32
E.coli
0
0
0
0
K.pneumoniae
0
0
0
0
P.aeruginosa
0
16
16
16
Proteus
0
32
16
16
S.Typhi
0
32
16
32
Shigella flexneri
0
32
16
32
Vibrio cholera
0
16
16
32
Serratia
0
32
16
0
0
0
0
0
0
0
0
0
mirabilis
marcescens
Candida
albicans
Aspergillus
niger
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