NewBioWorld A
Journal of Alumni Association of Biotechnology (2021) 3(1):11-17
RESEARCH
ARTICLE
Antimicrobial
activity of Azadirachta indica (Neem) leaf extract on gram positive and
gram-negative bacteria
Apurva
Singh1, Dristi Verma2, Shubhra Tiwari3*, S.K.
Jadhav4
1MATS
University, Raipur, Chhattisgarh,
India
2School of
Studies in Biotechnology, Pt. Ravishankar Shukla University, Raipur, Chhattisgarh, India
3School of
Studies in Biotechnology, Pt. Ravishankar Shukla University, Raipur, Chhattisgarh, India
4School of
Studies in Biotechnology, Pt. Ravishankar Shukla University, Raipur, Chhattisgarh, India
*Corresponding Author Email- shubhratiwari77@gmail.com
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ARTICLE INFORMATION
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ABSTRACT
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Article history:
Received
08 February 2021
Received in revised form
11 March 2021
Accepted
Keywords:
Antibacterial;
Azadirachta indica; Klebsiella oxytoca;
Nanoparticle; Staphylococcus
aureus
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Infectious diseases produced by bacteria are an urgent
healthcare concern worldwide. Several infections in humans are caused by
bacterial agents such as Bacillus
subtilis, Escherichia coli, Pseudomonas aeruginosa, Proteus vulgaris, and Staphylococcus aureus. The current
development of resistance to antibiotics and accompanying toxicity issues is
causing a rise in studies into the antibacterial role of plants against
resistant strains due to relative effectiveness and safety. The current work
was done to check the antibacterial activity of Azadirachta indica leaves (Neem) along with the green
synthesis of silver nanoparticles. Methanol and Acetone extracts of Neem
leaves were tested against Klebsiella
oxytoca and Staphylococcus
aureus, both of which are resistant to antibiotics. Silver
nanoparticle synthesized in methanol was very effective against Klebsiella oxytoca (13.5mm).
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Introduction
The World Health Organization has revealed that the
world is entering post-antibiotic time, and the present antibiotics are known
to become inefficient for future use. Also, it is causing toxic side effects, and
a hike in the cost of antibiotics has placed a burden on the public, making
them suffer badly (Maleki et al. 2017). According to various studies, medicinal
plants are a better and safer alternative to current antibiotics
(Theuretzbacher, 2013; Saleem, 2014). Azadirachta indica (Neem) is a
plant found widespread with lots of health benefits and is used to treating
various infectious diseases caused by harmful microorganisms like E. coli,
S. aureus, K. oxytoca, Pseudomonas aeruginosa etc. Neem leaves shows various
properties like antibacterial, antifungal, anti-diabetic contraception,
anti-helminthic and sedative. Neem constitutes of complex of various
constituents like nimbin, nimbidin, nimbolide and limonids and such types of
ingredients play an important role in disease management through the modulation
of various genetic pathways and other activities (Alzohairy, 2016).
DOI: 10.52228/NBW-JAAB.2021-3-1-4
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Nanobiotechnology is currently one
of the strongest and most rapidly evolving areas of study in current material
science, with plants and various products of plants playing an important role
in the production of nanoparticles (NPs). Particles having a size less than 100
nm are generally referred to as NPs (Banerjee et al. 2014). The essential
component of nanotechnology is nanoparticle formation because of the high
surface area to volume ratio (Lalitha et al. 2013). Nanoparticles are quickly
expanding in a variety of fields due to their completely new and improved
properties based on their size, distribution, and morphology (Shrivastava et
al. 2007). The formation of metal nanoparticles has attracted the interest of
researchers and nanotechnologists due to their microcidal characteristics.
Copper, silver, zinc, platinum, etc. are well-known metals that show
electromagnetic, catalytic, and antibacterial activity. Recently, silver and
salts of silver are well known for their antifungal properties and are emerging
rapidly in the field of medical sciences. Recently, silver, and silver salts
have been well known for their antifungal properties, and they are rapidly
gaining popularity in the world of medical sciences (Banerjee et al. 2014). The
nanoparticles encapsulated with neem extract display antibacterial activity
when an aqueous extract of neem leaves converts silver salt to silver nitrate
(Roy et al. 2017). Silver nanoparticles have antibacterial properties against a
variety of viruses, including hepatitis B, respiratory syncytial virus, herpes
simplex virus type, and monkey pox. Silver nanoparticles are employed in a
variety of applications including clothing, catheters, electric home
appliances, and biomedical implants. Silver nanoparticles have well-known
antibacterial properties, and various silver compounds were employed in the
treatment of burns.
The synthetic techniques that have evolved for the
synthesis of silver nanoparticles are chemical reduction of silver ions in
aqueous solution without or with stabilising agents and chemical photoreduction
in reverse micelles (Abou El-Nour et al. 2010). Most nanoparticle production
methods involved the use of dangerous chemicals or high energy needs, both of
which are challenging and inefficient (Rautela and Rani 2019). Green synthesis
of nanoparticles provides an advantage over other approaches due to its low
cost, environmental friendliness, simplicity, and reproducibility, which
typically result in more stable material (Mittal et al. 2014). It can be used
as an economical and viable alternative for the synthesis of large-scale metal
nanoparticles. Plants, bacteria, and fungi are the primary sources of silver
nanoparticles.
Present work emphasis on, the green synthesis of
silver nanoparticles with neem leaves, and its antibacterial effects at
different concentrations by the disc diffusion method.
Material and Method
1. Collection of plant material
Azadirachta
indica commonly known as Neem leaves were collected from
the premises of School of Studies in Biotechnology of Pt. Ravishankar Shukla
University Raipur, (C.G.) and were thoroughly washed 2-3 times with simple and
distilled water and were dried in shade for further use (Fig.1).
Figure 1:
Fresh neem leaves
2. Microbial
Culture
The antibacterial assay of plant extracts was tested
against gram-positive bacteria (Staphylococcus aureus) and gram-negative
bacteria (Klebsiella oxytoca). All bacterial cultures were procured from
the School of Studies in Biotechnology of Pt. Ravishankar Shukla University
Raipur. The bacterial culture was timely subculture in nutrient broth medium.
3. Preparation
of methanolic extract of Neem (Azadirachta indica)
10 g and 15 g of dried neem leaves were taken, 10 ml
of methanolic solvent were added in each and finely crushed in the motor and
pastel. The extract was filtered out and stored in refrigerator for further
use.
4. Preparation
of acetone extract of Neem (Azadirachta indica)
10 g and 15 g of dried neem leaves were taken, 10 ml
of acetone solvent were added in each and finely crushed in the motor and
pastel. The extract was filtered out and stored in refrigerator for further
use.
5.
5. Preparation of aqueous
extract of Neem (Azadirachta indica)
20 g of air-dried neem leaves were taken, finely
chopped, and added to 100 ml of distilled water and boiled for 10 minutes. The
extract was then cooled, filtered out using Whatman filter paper, and stored
for further use. This extract was used for the green synthesis of silver
nanoparticles.
6.
6. Preparation of nutrient
agar medium (NAM) for antibacterial assay
To test the antibacterial activity, nutrient agar
media was prepared by using following composition, for 1000ml- peptone (5g),
sodium chloride (5g), beef extract (3g), agar-agar (15g) followed by
autoclaving the media at 121℃ for 20minutes at 15 psi pressure
7.
7. Antibacterial screening
by well diffusion method
The antibacterial assay was performed using the agar
well diffusion method. A 100μl of the bacterial suspension was spread evenly on
the nutrient agar plates using a sterile spreader. A different concentration of
methanolic and acetone extract of neem (20µl -200 µl) was introduced in the
well. The plates were incubated at 37°C for 24 hours. The zone of inhibition
was measured with an antibiotic zone scale in mm.
8.
8. Green synthesis of
silver nanoparticle and study of antibacterial properties
a.
With methanolic extract:
A 1 mM silver nitrate solution was prepared in a
ratio of 1:10, where 90 ml of silver nitrate and 10 ml of methanolic solvent
were used, followed by continuous magnetic stirring for 1 hour. This experiment
was performed under dark conditions to minimize the photoactivation of silver
nitrate at room temperature.
b. With aqueous extract:
1mM silver nitrate solution was prepared and then to
5 ml silver nitrate solution was added separately to 1ml, 2ml, 3ml, 4ml, 5 ml
of neem extract. This experiment was performed and incubated in dark chamber to
minimize the photoactivation of silver nitrate at room temperature.
9.
9. Characterization of
silver nanoparticle
The green synthesis of silver nanoparticles was
confirmed by observing the colour change in the reaction mixture using
UV-visible spectroscopy. At 200–800 nm wavelengths, spectral analysis was
observed.
10. Antimicrobial activity
In order to study the antimicrobial activity of neem
extract and silver nanoparticles, gram-positive bacteria (S. aureus)
and gram-negative bacteria (K. oxytoca) were taken, and the diffusion
method was applied. A culture of S. aureus and K. oxytoca was
spread on a nutrient agar plate, and then a well was bore on this plate.
Synthetic silver nanoparticles (from methanolic and aqueous extracts) of
different concentrations, from 20 µl to 200 µl were poured into the well and
incubated overnight at 37 °C. As a control, silver-free agar plates cultured
under the same conditions were used.
Results
and Discussion
1. Antibacterial
activity of gram-positive bacteria (Staphylococcus aureus) with different solvent of neem extract
1.1 Methanolic extract
Staphylococcus aureus, a gram-positive bacterium was
spread on the nutrient agar plate and wells were bore on it. 10 g and 15 g in 10 ml were the concentration of methanolic
neem extract and volume of extract was 20-200µl. Different zone of inhibition
was observed (Table 1 and 2) (Fig.2 and Fig.3).
1.2 Acetone extract: Staphylococcus
aureus, a gram-positive bacterium was spread on the nutrient agar plate and
wells were bore on it. 10 g and 15 g in 10 ml were the concentration of acetone
neem extract and volume of extract was 20-200µl. Different zone of inhibition
was observed (Table 3 and 4) (Fig 4).
2. Antibacterial
activity of gram-negative (Klebsiella oxytoca) bacteria with different
solvent of neem extract
2.1. Methanolic extract: Klebsiella oxytoca, a
gram-negative bacterium was spread on the nutrient agar plate and wells were
bore on it. 10 g and 15 g in 10 ml were the concentration of methanolic neem
extract and volume of extract was 20-200µl. Different zone of inhibition was
observed (Table 5 and 6).
2.2. Acetone extract: Klebsiella oxytoca, a
gram-negative bacterium was spread on the nutrient agar plate and wells were
bore on it. 10 grams in 10 ml and 15 grams in 10 ml were the concentration of
methanolic neem extract and volume of extract was 20-200µl. Different zone of
inhibition was observed (Table 7 and 8).
3. Green
synthesis of silver nanoparticle with neem extract
After addition of aqueous neem extract to silver
nitrate solution the distinct colour change was observed in Sample A (10 ml
silver nitrate solution +1ml aqueous neem extract) the absorbance peak was
absorbed at 317 nm. In Sample B (10 ml
silver nitrate solution +2ml aqueous neem extract) the absorbance peak was
observed at 392 nm. In Sample C (10 ml silver nitrate solution +3 ml aqueous
neem extract) the absorbance peak was observed at 422 nm. In Sample D (10ml
silver nitrate solution +4ml aqueous neem extract) the absorbance peak was
observed at 408 nm. In Sample E (10 ml silver nitrate solution +5ml aqueous
neem extract) the absorbance peak was observed at 412 nm. In sample C, D, E the
absorbance peak was seen at 422, 408, 412. Since sample C was nearest to the
expected peak it was used for antibacterial assay (Fig.5) (Roy et al. 2017).
For antibacterial activity, 15g methanolic neem
extract and gram-negative bacteria was chosen as it showed best result (Table
9). The characterization of methanolic silver nanoparticle was confirmed by
UV-Vis peak at 394nm. The zone of inhibition was 13.5 mm with K.oxytoca
(Fig.6). On the other side, aqueous synthesized silver nanoparticle showed peak
at 287nm with low absorbance, so it was not considered for antibacterial
activity. Banerjee et al (2014) performed antibacterial test using banana, neem
and tulsi leaf with Bacillus and E. coli cultures. They found the
maximum zone of inhibition (16±0.016mm) with green synthesis of silver
nanoparticle using banana leaf extract against Bacillus cultures.
Phanjom and Ahmed (2017) synthesized silver nanopartciles using Aspergillus
oryzae (fungal extract) to check its antibacterial effect against
gram-positive (Bacillus subtilis, Staphylococcus aureus) and
gram-negative bacteria (Escherichia coli, Klebseilla pneumoniae). The
gram-negative bacteria (E. coli) showed better anti-bacterial effect.
Figure
2: Zone of inhibition shown by 10g/10ml
methanolic neem extract in S. aureus
Figure 3: Zone of inhibition shown by 15g/10ml
methanolic neem extract in S. aureus
Figure 4: Zone of inhibition shown by 15g/10ml
acetone neem extract in S. aureus
Table 1: Antibacterial activity of methanolic
extract (10g/10ml) of Azadirachta indica
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Bacteria
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Methanolic extract of Azadirachta
indica
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Staphylococcus
aureus
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10g/10ml
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Volume of leaf extract taken (in µL)
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20
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40
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60
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80
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100
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120
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140
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160
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180
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200
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Zone of inhibition (in mm)
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8
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10
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11
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14
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14
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NZ
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NZ
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NZ
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NZ
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NZ
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Table 2: Antibacterial activity of methanolic
extract (15g/10ml) of Azadirachta indica
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Bacteria
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Methanolic extract of Azadirachta
indica
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Staphylococcus
aureus
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15g/10ml
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Volume of leaf extract taken (in µL)
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20
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40
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60
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80
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100
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120
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140
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160
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180
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200
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Zone of inhibition (in mm)
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8
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10
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12
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14
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15
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23
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21
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21
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21
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21
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Table 3: Antibacterial activity of Acetone
extract (10g/10ml) of Azadirachta indica
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Bacteria
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Acetone extract of Azadirachta
indica
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Staphylococcus
aureus
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10g/10ml
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Volume of leaf extract taken (in µL)
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20
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40
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60
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80
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100
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120
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140
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160
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180
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200
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Zone of inhibition (in mm)
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10.6
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12.5
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14.3
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15.4
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18.2
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18.4
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19.4
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19.1
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19.1
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19
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Table 4: Antibacterial activity of Acetone
extract (15g/10ml) of Azadirachta indica
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Bacteria
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Acetone extract of Azadirachta
indica
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Staphylococcus
aureus
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15g/10ml
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Volume of leaf extract taken (in µL)
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20
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40
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60
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80
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100
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120
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140
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160
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180
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200
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Zone of inhibition (in mm)
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14.2
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16.2
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16.5
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19.1
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20.1
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20.2
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24.1
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24.2
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24
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24
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Table
5: Antibacterial activity of methanolic
extract (10g/10ml) of Azadirachta indica
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Bacteria
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Methanolic extract of Azadirachta
indica
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Klebsiella
oxytoca
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10g/10ml
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Volume of leaf extract taken (in µL)
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20
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40
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60
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80
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100
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120
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140
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160
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180
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200
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Zone of inhibition (in mm)
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14
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15
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16
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17
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18
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21
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21
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21
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21
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21
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Table
6: Antibacterial activity of methanolic
extract (15g/10ml) of Azadirachta indica
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Bacteria
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Methanolic extract of Azadirachta
indica
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Klebsiella
oxytoca
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15g/10ml
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Volume of leaf extract taken (in µL)
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20
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40
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60
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80
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100
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120
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140
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160
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180
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200
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Zone of inhibition (in mm)
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10
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13
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15
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15
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17
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18
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19
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18
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18
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18
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Table
7: Antibacterial activity of acetone
extract (10g/10ml) of Azadirachta indica
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Bacteria
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Acetone extract of Azadirachta
indica
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Klebsiella
oxytoca
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10g/10ml
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Volume of leaf extract taken (in µL)
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20
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40
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60
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80
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100
|
120
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140
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160
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180
|
200
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Zone of inhibition (in mm)
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14
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17
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18
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17
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21
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20
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26
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28
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28
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28
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Table
8: Antibacterial activity of acetone
extract (15g/10ml) of Azadirachta indica
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Bacteria
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Acetone extract of Azadirachta
indica
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Klebsiella
oxytoca
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15g/10ml
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Volume of leaf extract taken (in µL)
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20
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40
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60
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80
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100
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120
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140
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160
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180
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200
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Zone of inhibition (in mm)
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14
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15
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16
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17
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20
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22
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24
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27
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28
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28
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Table 9: Antibacterial activity of green silver nanoparticle of Azadirachta
indica
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Nanoparticle
Extract
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Bacteria Staphylococcus aureus
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Klebsiella oxytoca
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Conc. (g/mL)
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Solvents
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Solvents
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Aqueous
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Methanol
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Aqueous
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Methanol
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Zone
(mm)
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10g/10mL
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5.5
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10
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No Zone
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11.5
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15g/10mL
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7.5
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12.5
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10.5
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13.5
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Figure 5:
Colour change of neem extract after addition of silver nitrate
Figure 6:
Zone of inhibition shown by green synthesized silver nanoparticle against K.
oxytoca.
Conclusion
Neem is known for its
medicinal values and is one of the most useful plants for the prevention of
various diseases. In this study, the antimicrobial activity of neem leaf was
examined for different solvents, and its antibacterial activity was tested
against S. aureus and K. oxytoca using the agar well diffusion
method. The reduction of silver nitrate solutions using neem leaves was
performed for the biosynthesis of silver nanoparticles. The antibacterial
activity of green synthesized nanoparticles and their stability have important
applications for industrial purposes and pharmaceutical research.
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