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IJPRD, 2015; Vol 6(11):January-2015 (070 – 075)
International Standard Serial Number 0974 – 9446
-------------------------------------------------------------------------------------------------------------------------------------------------EVALUATION OF ANTIMICROBIAL AND ANTIOXIDANT PROPERTIES OF THE BIOLOGICALLY SYNTHESIZED
GOLD NANOPARTICLES OF OCIMUM PILOSUM WILLD
Thomas Bennans1*,
D K Sathish2, Lulu Maria Thomas3
1
Department of Botany, FMN College, Kollam, TN, India.
Department of Botany, University College, Thiruvananthapuram, TN, India
3
Department of Pharmacology, Sree Ramakrishna Institute of Paramedical Sciences, Coimbatore, TN, India
2
ABSTRACT
Plant extracts mediated biosynthesis of nanoparticles has been
emerging as the current trend in the drug delivery mechanism of
pharmacology. The immense potential of nano biotechnology
makes it an intensively researched field in modern medicine. Gold
nano particles (AuNPs) offer promising therapeutic possibilities
due to its higher biocompatibility, high surface reactivity and
inhibitory activity against a variety of cellular disorders. The
present study involves the free synthesis of stable gold
nanoparticles (AuNPs) using the water extracts of Ocimum
pilosum and the evaluation of their anti microbial and anti oxidant
activity. Visual observation and ultraviolet–visible spectroscopy
and SEM were used to characterize the synthesized AuNPs. The
absorption maxima was obtained at 545 nm within a time frame
of 7200 seconds beyond which the encapsulation of particles
showed a stationary phase. These particles were screened for the
free radical scavenging activity and anti microbial activity and the
results were compared. The in vitro anti microbial activity was
carried out by Agar diffusion method and the free radical
scavenging activity was determined by DPPH method. The
inherent antimicrobial activity of gold metals along with that of
plant extracts has yielded a higher level of antimicrobial as well as
anti oxidant activity under experimentation. This new biomimetic
approach using traditional medicinal plants to synthesize
biocompatible antibacterial and free radical scavenging AuNPs
could easily be scaled up for additional biomedical applications.
Correspondence Author
Thomas Bennans
Department of Botany, FMN College,
Kollam, TN, India
Key words: Ocimum pilosum, Gold nano particles, AuNPs,
antimicrobial, antioxidant.
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International Journal of Pharmaceutical Research & Development
ISSN: 0974 – 9446
The herb Ocimum pilosum (Willd.), a
INTRODUCTION
popular garden and ornamental plant, is a member
Over the last few years, researchers
of the family Lamiaceae. The plant grows in the
have been aiming at identifying and validating
subtropical regions and is widely distributed
plant-derived substances for the treatment of
throughout India. All the aerial parts were used as
various diseases. Interestingly it is estimated that
a single effective prescription in folk medicine for
more than 25% of the modern medicines are
treatment of cold associated symptoms.
directly or indirectly derived from plants. It is worth
mentioning that Indian medicinal plants are
considered as a vast source of several
MATERIALS AND METHODS
pharmacologically principles and compounds that
Plant material
are commonly used as home remedies against
The study involves the green synthesis of gold nano
multiple ailments (1). Since early 1990s, the use of
particles with extracts of Ocimum pilosum leaves.
forest products for medicine has been emerging as
Ocimum pilosum was collected locally and
a vital income generating resource for the
confirmed to their identified taxonomic position.
development of various social groups; hence, there
The method was carried out in three parts viz. The
is an increased attention for their long-term
plant extract is allowed to mediate the synthesis of
sustainability (2).
AuNPs, followed by its confirmation and
Research is being carried out all over
characterisation. It was screened for antimicrobial
the world to develop environmentally safe, non
and anti oxidant activity
toxic and economically viable plant based products
for the remedy of various ailments. In this context,
Preparation of plant extract.
biosynthesis of nanoparticles mediated by plant
The plant was washed in running water and blotted
extracts has emerged as a promising area for
dry. The leaves were selected harvested and shade
developing environmentally safe and target specific
dried for four days. Dried leaves were then
drug delivery method in pharmacological research.
powdered using mortar and pestle. Two grams of
Depending on the type of particle, the active
fine filtered powder was boiled in 100ml distilled
substance can be encapsulated or attached to the
water for 5 minutes and cooled to room
temperature. The filtrate was decanted and filtered
surface. This means that even if they dissolve
first through whatmann filter paper (pore size
poorly in water, they can be transported in an
0.45µm) and then through 0.22µm filters. The
aqueous solution, such as blood, and are better
filtrate was stored in sterile vials and kept in
protected against degradation by enzymes. Besides
ambient temperature.
acting as a delivery system, in some cases
nanoparticles can act as an active substance.
Recent in vitro and in vivo studies have revealed
Synthesis of gold nanoparticles
Synthesis of gold nano particles was carried out by
that silver and gold nanoparticles are endowed
reacting plant extracts with appropriate aliquots of
with innate antiplatelet properties (3).
Auric chloride. 1mM Auric chloride was carefully
In the present investigation gold
weighed and added to 180 ml of deionised water in
nanoparticles (AuNPs) are used for the preparation
of plant extract coated nanoparticles. The use of
250ml Erlenmeyer flask in dark followed by the
addition of 20ml of plant extract (4). The flask was
plant extracts in the synthesis of nanoparticles has
then incubated in rotary shaker at 160rpm in the
several advantages compared to methods relying
dark at room temperature. The reaction was
on microorganisms as agents. Hence the study aims
allowed to cool at room temperature for the
at the evaluation of the antioxidant and
formation of gold ions. The filtrate of the plant
antimicrobial activities of the leaf extracts of
extract acts as reducing or capping agent for the
Ocimum pilosum in association with gold
AuNPs. Characterisation of the encapsulated gold
nanoparticles.
nano particles was carried out by UV Visible
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71
International Journal of Pharmaceutical Research & Development
ISSN: 0974 – 9446
spectroscopy
and
Transmission
electron
was 100 µg/ml. From stock solution 0.25µl, 0.5µl,
microscopic analysis(5).
0.75µl and 1µl were taken in four test tubes and
the final volume of each test tube was made up to
UV Visible Spectroscopy
3 µl with methanol. Freshly prepared DPPH
The AuNPs were characterised using UV-Visble
solution( 0.004% w/v) was added into the test
spectrophotometer. The scanning range of the
tubes containing extract. After 30 minutes the
absorbance was taken at 517nm using a
sample was 300-800nm. Base line correction of the
spectrophotometer. Ascorbic acid was used as the
spectrophotometer was carried out using a blank
reference standard and the scavenging activity and
reference. The UV- Visible absorption spectra of all
the samples (concentration) were recorded along
IC50 values were calculated.
with the resulting data recorded in graphical
format.
RESULTS AND DISCUSSION
UV Spectral analysis
Scanning Electron Microscopy
The formation of AuNPs using extracts of Ocimum
SEM was adopted to detect the formation of
pilosum Willd and could be visually observed due
AuNPs. Preparation for SEM was done by the
to the red brown coloration of the sample. It was
method of Salini and Mohankumar (6). It was done
confirmed by the spectroscopic study. The colour
by fixing the samples in 2.5% glutaraldehyde in
exhibited by the nanoparticles was due to the
0.1M cacodylate buffer with pH 7.4 for 6 hours at
coherent excitation of all the free electrons within
1-4˚C. The fixed sample was washed four times
the conduction band leading to an in- phase
thoroughly in cold 0.1 M cacodylate buffer (pH 7.4)
oscillation which is known as the Surface Plasmon
for 30 minutes. It was then subjected to osmication
resonance. Figure 1 demonstrates the absorption
by again fixing it in 2 % osmium tetroxide in 0.05M
spectrum of auric chloride and there is no
cacodylate buffer pH 7.4 overnight in a
characteristic absorption peak in between 500 and
refrigerator. The osmicated tissues were
550 nm. In order to show the formation of gold
dehydrated in a graded ethanolic series embedded
nano particles from auric chloride and Ocimum
in synthetic resin and used at 60˚C for 24 hours. A
pilosum extract it was essential to find their
small quantity was taken to load the carbon coated
absorbancy alone. Figure 2 shows the absorption
copper grids for analysis. Dark and bright filed
spectrum of the. Ocimum pilosum leaf extract
images of samples were recorded.
which clearly indicates the absence of absorption
peak in between 500 -550 nm. Figure 3 reveals the
Anti microbial activity
formation of extract encapsulated gold nano
It was detected by agar well diffusion method. The
particles. The absorption maxima was obtained at
log phase cultures were spread on nutrient agar
545 nm which indicates the encapsulation of
medium plates using a sterile cotton swab in order
AuNPs. A time scan of 0 to 24 hours was set to find
to get a uniform microbial growth on test plates.
out the formation of AuNPs. It was found that at
Four wells of uniform size were made with a cork
7200 seconds the formation was stand still which
resulted in a sigmoid curve.
borer in each plate and 25, 50, 75 and 100µl of the
encapsulated gold nano particles were pipetted
The scanning electron microscopic study
directly into the well. The plates were incubated in
The scanning electron microscopic study was done
upright positions at 37˚ C for 18 to 48 hours.
for detecting the formation of AuNPs. The
micrographs
obtained
demonstrate
the
Anti oxidant activity
appearance and distribution of Au NPs at different
It was done using DPPH Free radical scavenging
magnifications. Figure 4 depicts the clubbed
assay of Lee et al., 2003(7). DPPH solution was
electron microscopic view of gold nanaoparticles as
prepared in methanol. The concentration of the
dark bodies because its diameter was 0.5 mm
biosynthesized gold nano particles extract solution
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72
International Journal of Pharmaceutical Research & Development
whereas in figure 5, comparatively larger particles
are seen because the AuNPs shown are of the size
of 100 nm and 50nm respectively.
0.50
ISSN: 0974 – 9446
ABS
0.45
0.40
0.35
0.30
0.25
0.20
0.15
Figure 5
0.10
0.05
0.00
300
350
400
450
500
550
600
650
700
750
nm
800
700
750
nm
800
Legends
Figure 1: Absorption spectrum of auric chloride
between 500 and 550 nm.
Figure 2 : Absorption spectrum of Ocimum pilosum
between 500 and 550 nm.
Figure 3 : Absorption spectrum of Ocimum pilosum
extract coated AuNPs at 545 nm
Figure 4 : Clubbed electron microscopic view of
gold nanaoparticles as dark bodies under SEM.
Figure 5 : Enlarged view of gold nanaoparticles
under SEM.
Figure 1
2.0
ABS
1.9
1.8
1.7
1.6
1.5
1.4
1.3
1.2
1.1
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
300
350
400
450
500
550
600
650
Figure 2
ABS
1.2
1.1
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
s
0
1000
2000
3000
4000
Figure 3
5000
6000
7000
Anti microbial activity
Table 1 demonstrates the result of disc diffusion
assay technique and it shows that the leaf extract
coated gold nano particles have maximum activity
against Staphylococcus aureus followed by
Escherichia coli. The anti fungal activity was highest
against Candida albicans. All concentrations of
AuNPs under study showed anti microbial activity.
The antibacterial properties of drugs coated gold
nanoparticles were higher when compared with
the pure drugs. The small size of gold
nanoparticles, large surface area and high
penetrating power might be the reason for the
enhanced activity and hence such nanoparticles
could effectively bind to the substrates on the
outer membrane and cell membranes of organisms
(8, 9).
Figure 4
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International Journal of Pharmaceutical Research & Development
ISSN: 0974 – 9446
Table 1. Inhibitory effect Ocimum pilosum leaf extract mediated AuNPs against pathogenic organisms
Zone of inhibition for micro organisms
Sl No
1
2
3
4
Sl. No
1
2
3
4
Concentration of
AuNPs (µg/ml)
25
50
75
100
Escherichia
coli
6.2±1
8.5±0.59
14±2.08
20 ± 0.25
Staphylococcus aureus
7.30 ± 0.57
10.80 ± 0.85
16.25 ± 0.39
24.00 ± 1.70
Candida
albicans
3.9 ± 0.88
9.8 ± 0.65
15.9 ± 0.45
21.9 ± 1.65
Table 2: Antioxidant activity of AuNPs of Ocimum pilosum.
Concentration
of Optical density Optical density IC50
Ascorbate and AuNPs of ascorbate
of AuNPs
(µg/ml)
25
0.162
0.102
37%
50
0.650
0.312
52%
75
0.890
0.285
67.9%
100
1.050
0.312
70.28%
Antioxidant activity
Table 2 explains the nature of decrease of
concentration of DPPH radical at different
concentrations of the AuNPs. It was found that the
significant free radical scavenging activity was
noted at 100µl AuNPs. The IC50 inhibition
concentration at which there was 50% reduction of
free radical resulted at 50µl concentration. The
substantial decrease in the concentration of DPPH
radical was due to the scavenging ability of gold
nanoparticles formed by Ocimum pilosum leaf
extract. Similar results were obtained where Gold
nanoparticles embedded 3,6-dihydroxyflavone
individually showed maximum percent of inhibition
(72.04 %) at the 100 lg/ml concentration compared
to native 3,6-dihydroxyflavone (64.21 %).The
inclusion of gold nanoparticle embedded 3,6dihydroxyflavone with other dietary nutrients
lutein and selenium methyl selenocysteine further
increased maximum inhibition (87.13 %) at the
same concentration (10).
CONCLUSION
The present investigation demonstrates the
encapsulated gold nano particles of Ocimum
pilosum leaf extract showed typical features of
AuNPs lie in range of 540-550 nm. The absorption
maxima λ of the sample extract was obtained at
545nm. The fine structural analysis of the AuNPs
under SEM provided sufficient data for its
confirmation. Considering the practical relevance
of the AuNPs for the welfare of mankind, it could
be used as a potent antimicrobial and antioxidant
agent (11). The major observation regarding the
antimicrobial activity is that the consumption of
this safer compound could stimulate the efficiency
of our immune system whereas the anti oxidant
potential imparts the scavenging of reactive oxygen
species from living body. Applications of such
nanoparticles in medical and other applications
make this method potentially useful for their largescale synthesis and commercial utilization.
ACKNOWLEDGEMENTS
We express our sincere gratitude to Kerala State
Council for Science, Technology and Environment
for their financial support.
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