5

Chapter 2
REVIEW OF RELATED LITERATURE
2.1 Phyllanthus niruri Linn.
Phyllanthus niruri Linn. (P. niruri) is shown in Figure 1, locally known as sampasampalukan, surusampalok, talikod, or taltalikod, San Pedro, malakirum-kirum,
turutalikod and other tags depending on the local or cultural terms in the Philippines
(Juario et. al, 2014). It is commonly ignored because it was regarded as a roadside and
garden weed which is found throughout the Philippines. P. niruri is a branching herb with
small oblong leaves and fruits in its branches.
Phytochemical analysis of the leaf reported that it consists of multiple compounds
which include alkaloids, saponins, tannins, oxalate, flavonoids, glycosides, lignins,
terpenoids, polyphenols and coumarins. While mineral constituents include lead,
phosphorus, magnesium, copper, calcium, iron, nitrogen, zinc, selenium, sodium and
potassium. P. niruri have been reported to have hepatoprotective effect, inhibiting HIV
replication, lipid lowering activity, antidiabetic activity, anti-malarial activity, antispasmodic activity, analgesic activity, antioxidant activity and inhibiting chromosomal
aberrations (Montejo et. al, 2014), anti-inflammatory, anti-fungal, anti-viral, antibacterial, and inhibitory effect on renal stone formation (Narendra et al., 2012).

Figure 1 Phylanthus niruri

2.2 Nanotechnology
Nanotechnology is a new technology for the modern research dealing with
synthesis, manipulation of particles structure ranging from approximately 1 to 100 nm in
size. The properties within this size range changes chemical, physical and biological in
fundamental ways of both individual atoms or molecules and their corresponding bulk.
Novel applications of nanoparticles and nanomaterials are paramount and growing
rapidly on various fields like health care, biomedical, cosmetics, environment, material
science, surface chemistry, energy science, chemical industries, etc. due to the completely
new or enhanced properties based on size, distribution and morphology of these
nanoscale materials. (Ahmed S et al., 2015).
Nanoparticles are generally prepared by a variety of chemical and physical
methods such as ultraviolet irradiation, aerosol technologies, lithography, laser ablation,
ultrasonic fields and photochemical reduction which uses expensive and toxic that are

responsible for various biological risks chemicals (Ahamed M et al., 2011). The
development of biologically-inspired experimental processes for the syntheses of
nanoparticles is evolving into an important branch of nanotechnology. There are two
approaches which are involved in the synthesis of silver nanoparticles (AgNP); top to
bottom approach and bottom to top approach shown in Figure 2.

Figure 2 Different approaches of synthesis of silver nanoparticles. Adapted from

Ahmed S, Ahmad M, Lal Swami B, Ikram S, 2015
In bottom to top approach, nanoparticles can be synthesized using chemical and
biological methods by self-assemble atoms to new nuclei which grow into a particle of
nanoscale while in top to bottom approach, suitable bulk material break down into fine
particles by size reduction with various lithographic techniques e.g. grinding, milling,
sputtering and thermal or laser ablation see Figure 3.

Figure 3 Protocols employed for synthesis of nanoparticles (a) Top to Bottom Approach
and (b) Bottom to Top Approach. Adapted from Ahmed S, Ahmad M, Lal
Swami B, Ikram S, 2015
2.3 Silver Nanoparticles
Synthesis of silver nanoparticles from colloidal silver has drawn interest by
researchers in scientific community because of its unique properties like chemical
stability, catalytic activity (Logeswari P et al., 2015), size and shape depending optical,
electrical and magnetic properties which can be incorporated into antimicrobial
applications, biosensor materials, composite fibers, cryogenic superconducting materials,
cosmetic products, and electronic components. Several physical and chemical methods
have been used for synthesizing and stabilizing silver nanoparticles. The most popular
chemical approaches, including chemical reduction using a variety of organic and
inorganic reducing agents, electrochemical techniques, physicochemical reduction, and
radiolysis are widely used for the synthesis of silver nanoparticles. Recently, nanoparticle
synthesis is among the most interesting scientific areas of inquiry, and there is growing
attention to produce nanoparticles in green chemistry using environmentally friendly

methods. Green synthesis approaches include mixed-valence polyoxometalates,

polysaccharides, Tollens, biological, and irradiation method which have advantages over
conventional methods involving chemical agents associated with environmental toxicity
(Korbekandi H and Iravani S, 2012).
2.4 Green Synthesis
Green synthesis of Ag NPs involves three main steps, which must be evaluated
based on green chemistry perspectives, including (1) selection of solvent medium, (2)
selection of environmentally benign reducing agent, and (3) selection of nontoxic
substances for the Ag NPs stability (Bhosale RR et al., 2014). The synthesis of silver
nanoparticle via green route is environment friendly, cost effective and easily scaled up
for large scale syntheses of nanoparticles moreover the use of high temperature, pressure,
energy and toxic chemicals is not needed. Several literature has been reported in presentday on biological syntheses of silver nanoparticles using microorganisms including
bacteria, fungi and plants; because of their antioxidant or reducing properties typically
responsible for the reduction of metal compounds in their respective nanoparticles.
Although among the various biological methods of silver nanoparticle synthesis, microbe
mediated synthesis is not of industrial feasibility due to the requirements of highly aseptic
conditions and their maintenance. Therefore, the use of plant extracts for this purpose is
potentially advantageous over microorganisms due to the ease of improvement, the less
biohazard, elaborate process of maintaining cell cultures and its cost feasibility over
nanoparticles synthesized by microorganisms (Ahmed et al., 2015).

10

2.4.1 Green Synthesis of Silver Nanoparticles (AgNP) using Plant Extracts

One way to synthesize AgNP is the use of plant extracts that serves as reducing
agent and stabilizing agent in the reduction of Ag + ions into Ag0. The synthesis of AgNP
using plant extracts involves reduction-oxidation reactions. The reduction of Ag + ion to
Ag0 is due to the reducing agents that lose or donates electrons. The plant phytochemicals
with antioxidant or reducing properties that act on the respective compounds and give the
desired nanoparticles which involves the reduction and stabilization of silver ions by
combination of biomolecules such as proteins, amino acids, polysaccharides, alkaloids,
tannins, phenolics, saponins, steroids, terpenoids, vitamins, flavonoids (Ahmed S et al.,
2015), carboxylic acids, ketones, amides, organic acids, quinones (Jha AK et al., 2009)
and coenzymes (Kulkarni N. and Mudappur U, 2014) that are already established having
medicinal properties responsible for the immediate reduction of the ions. It was suggested
that the phytochemicals are involved directly in the reduction of the ions and formation of
silver nanoparticles. The three major components involved in the preparation of
nanoparticles using biological methods are the solvent medium for synthesis, the
environmentally friendly reducing agent, and a nontoxic stabilizing agent (Ahmed S et
al., 2015).
Some of the mechanism proposed in the synthesis of AgNP using hydrophytes.
The antioxidant ascorbate is oxidized in antioxidative reactions, and a system for the
regeneration of ascorbate is critical formaintaining the antioxidative system.
Dehydroascorbate (DHA) reductase (DHAR) catalyze the re-reduction of DHA to
ascorbate. Under alkaline conditions catechol (a dihydric Phenol) can get transformed
into protocatechaldehyde and finally to protocatecheuic acid. In both the cases reactive

11

hydrogen gets liberated which participates in the synthesis of silver nanoparticles, see
Figure 4.

Figure 4 Mechanism of biosynthesis of Ag nanoparticles using hydrophytes.

Adapted from Jha AK, Prasad K, Prasadc K, Kulkarni AR, 2009
However, each plant varies as the phytochemical involved varies, the major
mechanism involved is the reduction of the ions (Prabhu S and Poulose EK, 2012).
Moreover the color, shape, size and stability of silver nanoparticles depend on the
concentration or volume of the reducing agent (Mukherjee et. al, 2014)
2.5 Phytochemical Screening
Phytochemical screening is a process of identifying the presence of active
components in a plant. This process can give scientists the knowledge of the desirable
constituents in plants. It gives information on the presence of commercially important
compounds such as tannins, oils, gums, precursors for the synthesis of such complex

12

compounds. The major chemical substances in plants have been the alkaloids and
steroidal sapogenins, however, other diverse group naturally occurring phytochemicals
such as flavonoids, tannins, trepernoid (Adnan et al. 2009).
2.6 Antibacterial Activity of AgNP
Silver is a well-known antimicrobial agent against a wide range of over 650
microorganisms from different classes such as gram-negative and gram-positive bacteria,
fungi or viruses. However the exact mechanisms of antimicrobial or toxicity activities by
silver nanoparticles are still in investigation and a well debated topic. The antimicrobial
properties of silver nanoparticles from different plant sources depend on; (1) Size and
environmental conditions (pH, ionic strength) and (2) Capping agent (Ahmed S. et al.,
2015).
Several mechanisms of AgNP have been reported of its antimicrobial acitivity or
toxicity activity. It has the ability to anchor to the bacterial cell wall and subsequently
penetrate it, thereby causing structural changes in the cell membrane like the permeability
of the cell membrane and death of the cell. There is formation of pits on the cell surface,
and there is accumulation of the nanoparticles on the cell surface. The formation of free
radicals by the silver nanoparticles may be considered to be another mechanism by which
the cells die. There have been electron spin resonance spectroscopy studies that suggested
that there is formation of free radicals by the silver nanoparticles when in contact with the
bacteria, and these free radicals have the ability to damage the cell membrane and make it
porous which can ultimately lead to cell death. It has also been proposed that there can be
release of silver ions by the nanoparticles, and these ions can interact with the thiol
groups of many vital enzymes and inactivate them. The bacterial cells in contact with

13

silver take in silver ions, which inhibit several functions in the cell and damage the cells.
Then, there is the generation of reactive oxygen species, which are produced possibly
through the inhibition of a respiratory enzyme by silver ions and attack the cell itself.
Silver is a soft acid, and there is a natural tendency of an acid to react with a base, in this
case, a soft acid to react with a soft base. The cells are majorly made up of sulfur and
phosphorus which are soft bases. The action of these nanoparticles on the cell can cause
the reaction to take place and subsequently lead to cell death.
Another fact is that the DNA has sulfur and phosphorus as its major components;
the nanoparticles can act on these soft bases and destroy the DNA which would definitely
lead to cell death. The interaction of the silver nanoparticles with the sulfur and
phosphorus of the DNA can lead to problems in the DNA replication of the bacteria and
thus terminate the microbes. (Prabhu S and Poulose EK, 2012)
2.7 Disc Diffusion Method
This is a method to detect the antimicrobial potential of a sample. The inoculum is
prepared by getting a colony and dissolving in the normal saline solution. The turbidity of
the suspension is standardized to match that of a 0.5 McFarland standard (corresponds to
approximately 1.5 X 108 CFU/ml). The adjusted suspensions must be used within 15
minutes. The plate inoculum must be warm to room temperature so that any excess
moisture is absorbed into the medium. It can expedite this step by placing the plates in
the incubator with their lids ajar for 1015 minutes. All inoculum plate must be in the
standard depth depending on what type. In the inoculation of the plates, inoculate the
plate starting at the top surface with swab covering the entire plate by streaking back and
forth from edge to edge. Rotate the plate approximately 60 and repeat the swabbing

14

procedure. Rotate the plate 60 again and swab the entire plate a third time. This will
ensure that the inoculum is evenly distributed. Then the antimicrobial disks can be
applied the total number of disks that can be applied depends on the size of plate and
bacteria. Press each disk down firmly to ensure complete, level contact with the agar
(Coyle M, 2005).
The size of the zone inhibition is an area on an agar plate where growth of a
control organisms is prevented by an antibiotic usually placed on the agar sample.
Antibiotic then diffuses into agar away from the disk. If organisms are sensitive to the
antibiotic, they will not grow near the disk. The size of the zone is proportional how
sensitive the organism is. If the organism is resistant to the antibiotic, it will grow right
up to the disk. If the antibiotic works successfully a clear ring will appear around the disk
in 24 or 48 hours. The ring is called as the zone of inhibition; the larger the zone of the
inhibition, the more effective that the antibiotic is against the particular bacterium.
Usually related to the level of antimicrobial activity present in the sample, or the product
larger zone of inhibition usually means that the antimicrobial activity is more potent.
Typically, several million bacterial cells are spread on the agar-plate, and if their growth
is inhibited, a clear zone of inhibition is observed around the antibiotic impregnated
disc. If the bacteria are resistant to the antibiotic, a confluent lawn of growth
(opaqueness) is observed. (Carriaga et al., 2009).
2.7.1 Positive Control
It is important to run a medium control and both negative- and positive- controls
as the test is complex and the controls have known outcomes to indicate if the media and
reagents are reacting appropriately. Quality control tests are performed each time clinical

15

isolates are tested, using QC strains. The medium control and negative control should
each always yield a negative reaction; a positive control should always yield a positive
reaction. If these results do not occur, start the test over with a new suspension, new
media, and new reagents (Perilla MJ et al., 2003). The positive control used for
Staphylococcus aureus, Escherichia coli, Haemophilus influenzae and Streptococcus
pneumoniae were erythromycin, gentamycin ampicillin and vancomycin was the choice
of antibiotic described in Clinical and Laboratory Standards Institute antimicrobial
susceptibility testing standards.
2.8 Test Organism for Antimicrobial Screening
Antimicrobials agent are drugs that is used in the treatment of infectious disease:
antibiotics, which are natural substances produced by certain groups of microorganisms,
and chemotherapeutic agents, which are chemically synthesized. (Kenneth Todar). The
following are the test organisms that was used in the study:
2.8.1 Escherichia coli
Escherichia coli is a gram-negative, facultatively anaerobic, rod-shaped bacterium
of the genus Escherichia that is commonly found in the lower intestine of warm-blooded
organisms (Singleton P, 1999). It is not usually pathogenic, however, it can be a cause of
urinary tract infections and certain strains produce enterotoxins that cause travelers
diarrhea and occasionally cause very serious foodborne diseases (Tortora et al.,2007).
2.8.2 Staphylococcus aureus
Staphylococcus aureus is an aerobic gram-positive bacterium, grow in clusters
and are facultative aerobes, further they produce acids from glucose both aerobically and

16

anaerobically. S. aureus can cause serious infections in humans and is the most
predominant bacteria found in chronic wounds. Moreover, it is the most
problematic bacterium in burn and surgical wound infections. S. aureus can be
recognized by its yellow pigment and it can be found in pathological conditions as boils,
pimples, arthritis and meningitis .Studies have shown that S. aureus has been found
in wounds together with other bacteria, such as P. aeruginosa (Sandstrm S, 2011).

2.8.3 Fastidious Microorganisms

A fastidious organism is any organism that has a complex nutritional requirement.
In other words, a fastidious organism grow when specific nutrients are included in its
diet. The more restrictive term fastidious microorganism is often used in the field of
microbiology to describe microorganisms that grow only in special nutrients which are
present in their culture medium (Sridhar Rao PN, 2012).
2.8.3.1 Haemophilus influenzae
Haemophilus influenzae is a common etiologic agent of diseases such as
pneumonia, meningitis, otitis media, and conjunctivitis in human. Meningitis caused by
H. influenzae occurs almost exclusively in children less than five years of age, and most
invasive H. influenzae disease is caused by organisms with the type b polysaccharide
capsule (H. influenzae type b, commonly abbreviated as Hib). There are conjugate
vaccines to prevent H. influenzae infections caused by serotype b, though they are not
widely available in some parts of the world. No vaccines for the other serotypes or for
unencapsulated strains have been developed. Although meningitis is the most severe

17

presentation of disease, H. influenzae pneumonia causes more morbidity than H.

influenzae meningitis.
H. influenzae are characterized as small, gram-negative bacilli or coccobacilli that
require X and V growth factors, grow on chocolate agar (but not on sheep blood agar),
and have a pungent indol smell. H. influenzae is a fastidious organism requiring media
containing haemin (X factor) and nicotinamide adenine dinucleotide (NAD, V factor) for
growth. The standard medium is chocolate agar, which is often prepared with horse
blood, a good source of both X and V factors. Heating the blood is necessary to make
both factors available to the organism. Supplemented chocolate agar is superior to
unsupplemented medium for growth of H. influenzae and is the medium of choice.
Although some strains of H. influenzae may grow on unsupplemented chocolate agar,
supplements must be added to reliably support the growth of most strains (Manual for the
Laboratory Identification and Antimicrobial susceptibility testing of Bacterial Pathogens
of Public Health Importance in Developing World, page 5-9)
2.8.3.2 Streptococcus pneumoniae
Streptococcus pneumoniae is a common agent of lower and upper respiratory
diseases, such as pneumonia, meningitis and acute otitis media (middle ear infections),
affecting children and adults worldwide. This bacterial pathogen is the cause of
approximately 40% of acute otitis media. Meningitis in infants, young children and the
elderly is often caused by S. pneumoniae. Persons who have sickle cell disease, anatomic
asplenia, or are immunocompromised also have increased susceptibility to S. pneumoniae
infection. Pneumococcal meningitis is the most severe presentation of disease, but most
illnesses and deaths result from pneumococcal pneumonia.

18

S. pneumoniae are gram-positive diplococci or chains of cocci. On blood agar and

chocolate agar plates, S. pneumoniae colonies appear small, greyish and mucoid (i.e.,
watery), and are surrounded by a greenish zone of alphahemolysis (-hemolysis).
Colonies of pneumococci and -hemolytic viridans streptococci each appear raised when
young; however, after 2448 hours, the center of pneumococcal colonies becomes
depressed, whereas viridans streptococcal colonies retain their raised appearance. A 3x
hand-lens or a microscope (30x50x) can therefore be a useful aid in differentiating
pneumococci from -hemolytic viridans streptococci. Laboratory differentiation between
S. pneumoniae and viridans streptococci is accomplished by optochin and bile solubility
testing: pneumococci are susceptible to optochin and bile-soluble, while viridans
streptococci are not. Commercially available slide agglutination tests can also be used for
identification of pneumococci. For optimal results plates for pneumococcal identification
assays should be incubated in a 5% CO2 atmosphere. (Manual for the Laboratory
Identification and Antimicrobial susceptibility Testing of Bacterial Pathogens of Public
Health Importance in Developing World, page 45-46)
2.9 Review of Related Studies
The following are the studies conducted on the synthesis of silver nanoparticle via
green route from various plant extracts. The results and parameters such as
characterization of AgNPs, concentration of the solvent and reagent used were presented
in this section.
The silver nanoparticles were green synthesized using leaf extract of Euphorbia
hirta weed. The solution containing plant extract and 1 mM AgNO 3 were heated at 50 to
95 0C then a change of color was observed in the heating process. Synthesized silver

19

nanoparticle revealed to possess an effective antifungal property against Candida

albicans, C.kefyr, A.niger. The silver nanoparticles were confirmed using UV-vis spectra
analysis and SEM analysis which revealed that the particles sizes were 40-50nm.The
study emphasizes the use of plant medicinal for the synthesis of silver nanoparticle with
antifungal effect (Elumalai EK et al., 2010).
A study conducted by Renugadevi K et. al (2012), silver nanoparticle was
synthesized via biological method using the leaf extract of Baliospermum montanum as a
reducing agent under microwave irradiation condition. Then the silver nanoparticle was
characterized by UV analysis, SEM-EDAX. The particle size was studied by SEM
analysis and were found to be in the range of 10-60nm. The antibacterial and anticancer
potentials of the silver nanoparticle were studied. The silver nanoparticle has shown
maximum activity against E.coli among the tested bacteria. The silver nanoparticle shown
cytotoxicity effect by MTT assay against Vero cell line and Hep2 cell line.
Abboud Y et al. (2013) used onion (Allium cepa) in the biosynthesis of silver
nanoparticles (AgNPs) under microwave irradiation. Influence of various reaction
parameters such as microwave irradiation power and microwave irradiation time was
analyzed. The synthesized nanomaterial was characterized by UV-visible absorption
spectroscopy at 300 Watts for 30 seconds a distinct peak showed at 450 nm confirming
the formation of silver nanoparticles and Fourier transform infrared spectrum analysis for
the phytochemicals involved in the reduction and stabilization of silver nanoparticles. Xray diffraction (XRD), transmission electron microscopy (TEM) confirm the formation
and the crystalline nature of the synthesized nanomaterial and the average size of the
silver nanoparticle was 38.7 based on the size distribution. Further, these nanoparticles

20

were found to exhibit high antibacterial activity against two different strains of bacteria
Escherichia coli (Gram negative) and Staphylococcus aureus (Gram positive).
In the study of Pal J et al. (2013) silver nanoparticles was synthesized using
Benzo-18-crown-6 which acts as both reducing and stabilizing reagent in the reaction by
microwave irradiation. Different irradiation time and energy were studied ranging from 3
to 12 minutes and 180W to 450W respectively. Silver nanoparticles are analyzed using
transmission electron microscope and UVvisible spectroscopic technique. The silver
nanoparticles prepared in this way are uniform and stable, which can be stored at
refrigerator for 5 months. Appearance of surface plasmon band at 420 nm indicated the
formation of silver nanoparticles. Highly monodispersed stable silver nanoparticles were
obtained within 3 min of microwave irradiation. Through transmission electron
microscopy, silver nanoparticles were observed to be spherical having a particle size
range of 6 to11 nm.
Kahrilas GA et al. (2013) presented a one-step microwave synthesis using the
BoxBenhken design for three factors (time, temperature, and pressure). Aqueous
extracts from the peels of citrus fruits (orange, grapefruit, tangelo, lemon, and lime) were
used for the synthesis of AgNPs using microwave technology, though the synthesis of
AgNPs was only successful using the orange peel extract. Nanospheres of TEM mean
diameter (with standard deviation) of 7.36 8.06 nm were successfully synthesized in 15
min by reducing Ag+ ions (from AgNO3) with orange peel extract, which also served as a
capping agent. Creation of AgNPs was confirmed using UVvisible spectroscopy,
fluorescence emission spectroscopy, powder X-ray diffraction, and transmission electron
microscopy, while size analysis was gathered from both transmission electron

21

microscopy as well as dynamic light scattering. Analysis of all citrus peel extracts by gas
chromatographymass spectrometry indicated that the putative compounds responsible
for successful AgNP synthesis with orange extract were aldehydes. The creation of
AgNPs using environmentally benign reagents in minimal time paves the way for future
studies on AgNP toxicity without risking interference from potentially toxic reagents and
capping agents.
Geetha N et al., (2014) synthesized silver nanoparticles using Cymbopogan
Citratus (Dc) Stapf. also known as lemon grass. The silver nanoparticles were formed
after 3 hours of incubation at 37C using aqueous solution of 5 mM silver nitrate
(AgNO3) and synthesized silver nanoparticles were characterized by UV-vis, XRD,
SEM,EDS and FTIR. The antibacterial activity of synthesized silver nanoparticles was
investigated by disc diffusion method.
Siby J and Beena M, 2014 synthesized silver nanoparticles (AgNPs) in aqueous
medium by a simple, efficient and economic microwave assisted synthetic route using
pectin as the reducing agent and the biopolymer pectin as stabilizer. The synthesized
AgNPs were characterized by UV-vis. spectroscopy, Energy dispersive X-ray (EDX), Xray diffraction (XRD) and Transmission electron microscopy (TEM) techniques. TEM
images suggest that the nanoparticles are of spherical shape with an average diameter of
18.84 nm. The reduction of 4-nitrophenol to 4-aminophenol by NaBH4 in aqueous
medium was selected as a model reaction to investigate the catalytic activity of AgNPs.
The pectin stabilized silver nanoparticles (AgNPpectin) were found to exhibit very good
catalytic activity and the reaction followed pseudo-first order kinetics. The rate of

22

reaction was found to increase with increasing temperature and the activation energy was
found to be 47.3 kJ mol-1.
Vijayashree IS et al. (2014) conducted a study on the microwave irradiated
synthesis of silver nanoparticles (AgNP) using apple which acts as both reducing and
capping media. AgNPs were characterized by UVvisible spectroscopy, XRD, FT-IR and
TEM. The kinetics of reduction of aqueous silver ions during reaction with the apple fruit
extract were monitored with the help of UV-visible spectroscopy. The XRD pattern of
AgNPs was found agreeing with the fcc structure of Ag metal. Further, where TEM
analysis exhibited formation of spherical shaped nanoparticles in the range of 1045nm;
FTIR analysis was carried out to identify the functional groups which were responsible
for reduction/capping of AgNPs and conclude that the characterized AgNPs carry the
potential for adoption in various medical and industrial applications. The antimicrobial
activity of the AgNP was found to be effective against E. coli and exhibits very good
antioxidant property.
Augustin R and Kalarikkal N (2014) reported that the green synthesis of
nanoparticles is widely accepted due to the less toxicity in comparison with chemical
methods and a novel cost-effective and eco-friendly method for the rapid green synthesis
of silver nanoparticles using aqueous leaf extracts of Piper nigrum leaves by microwave
irradiation was conducted. Their findings suggested that silver nanoparticles obtained
using this method produced with controllable size within a few minutes. The fabricated
nanoparticles possessed excellent antibacterial property against both Gram-positive and
Gram-negative bacteria.

23

Saware K et. al (2014) used Ficus benghalensis (F.B.) leaf extract to synthesize
ecofriendly silver nanoparticles. It was reported that the stable and spherical SNPs of
variable size ranging from 10-50 nm. The formation of SNP was monitored using
ultraviolet-visible spectroscopy (UV-Vis) technique for surface Plasmon resonance and it
was found at 435 nm and absorbance peak at 280 and 240 nm are also found and
indicates the protein and hydroxyl anthraquinones capping. The kinetics of effect of time,
pH, ionic strength, Concentration of leaf extract and temperature on the formation of SNP
was studied using UV-Vis spectroscopy. Fluorescence spectroscopy and Fouriertransform infrared spectroscopy (FTIR) studies indicate the involvement of protein as a
capping agent and Quinones as reducing agents. The morphology of the SNPs was
determined AFM (Atomic Force Microscopy) and FESEM (Field emission Scanning
electron microscopy) along with EDAX. X-ray diffraction (XRD) study was carried and
found to be face centred cube (fcc).Thermal Gravimetric analysis (TGA) was performed
to understand the thermal behaviour of biosynthesized silver nanoparticles. The results
indicated that the proteins, which have amine groups, have played a significant role in
stabilizing SNPs in the solutions.
In study of Akele ML et al. (2015) the synthesized silver nanoparticle by green
microwave-assisted which is a simple, low cost and eco-friendly technique using gum
acacia prepared in 0.5% (w/v) in Milli-Q water that serves as both reducing and capping
agent was conduted. The catalytic activity of the synthesized silver nanoparticles was
studied. The formation of the AgNPs was identified through the change in color of the
solution from colorless to yellow. The effects of reaction conditions such as concentration
of GA, AgNO3and irradiation time on the synthesis of AgNPs were studied. The

24

synthesized AgNPs were characterized by UV-Vis spectroscopy, Fourier transform

infrared spectroscopy (FTIR), Transmission electron microscopy (TEM), and X-ray
diffraction (XRD).The reaction parameters significantly affected the rate of formation,
size and distribution of the AgNPs. XRD analysis revealed that the particles were facecentered cubic (FCC). The stabilized AgNPs, in the presence of NaBH4 as a reducing
agent, demonstrated excellent catalytic activity in reducing 4-nitrophenol (4-NP) to 4aminophenol (4-AP). The kinetics of the reaction was found to be of pseudo-first-order
with respect to the 4-NP.
A bioinspired method of silver nanoparticle (AgNP) synthesis utilizing the highly
invasive terrestrial weed mimosa (Mimosa pudica), is presented in the study of Ganaie
SU et al. (2015). Aqueous extract of the leaves of the weed served as reducing as well as
stabilising agent which was employed in various proportions with Ag (I) solution, for the
synthesis at ambient temperature of (293 0C). The effect of several key variables that
influence the shape/size of the AgNPs concentrations of the extract relative to Ag (I),
temperature, interaction time, stirring and pHwas studied employing UV-visible
spectrophotometry, scanning electron microscopy (SEM), transmission electron
microscopy (TEM), dynamic light scattering (DLS) and X-ray diffraction (XRD). The
study provides the basis with which processes for synthesizing AgNPs of desired shapes
and sizes can be developed using mimosa as the bioagent. All routes being simple, nonpolluting, inexpensive, and non-hazardous, raise the possibility of large scale utilization
of mimosa, thereby offering a means to reduce the ecological degradation that is causes.
Parveen M et al. (2016) also presented an environmental friendly and cost
effective biosynthesis of the silver nanoparticles. In this study, microwave assisted

25

synthesis of silver nanoparticles (AgNPs) has been demonstrated using aqueous leaf
extract and ethanolic leaf extract of Fraxinus excelsior reducing aqueous AgNO3
solution. The synthesized nanoparticles have been characterized on the basis of fourier
transform infrared spectroscopy (FT-IR), UVVis spectroscopy, scanning electron
microscopy (SEM), transmission electron microscopy (TEM) and energy dispersive Xray (EDX) analysis. The presence of a characteristic surface plasmon resonance (SPR)
absorption band at 425 nm in UVVis reveals the reduction of silver metal ions into
silver nanoparticles. TEM analysis showed that the synthesized silver nanoparticles
formed were predominantly spherical and polydisperse with diameters in the range 2540
nm. FTIR analysis was carried out to probe the possible functional group involved in the
synthesis of AgNPs. Further leaf extracts and AgNPs were evaluated for antiradical
scavenging activity by 1,1-diphenyl-2-picryl-hydrazyl (DPPH) assay.
2.10 Synthesis
The eleven reviewed related studies used different aqueous and ethanolic plant
extracts to synthesize silver nanoparticles (AgNPs) via green route. Two of the reviewed
studies used pectin in the study of Siby J and Beena M (2014) while benzo-18-crown-6
was used by Pal J et al. (2013) to produce silver nanoparticles. The reviewed studies dealt
about the synthesis of AgNP using leaves, fruit, root crops, sap and derived compounds
from plants of various plant and chemicals moreover three of the reviewed studies used
weeds and grass that serves as the reducing and stabilizing agent. Eleven studies used
water as solvent in the extraction of plant extracts while Parveen M et al. (2015) used
ethanol as a solvent in the extraction of Fraxinus excelsior leaves. Elumalai EK et al.,
(2010), Geetha N et al., (2014) and Ganaie SU et al., (2015) used heating and ambient

26

temperature respectively to synthesize silver nanoparticles. These studies are similar to

the present study since the method and approach used were from green route and bottom
to top approach but differs from plant sample and test organisms to be used for the
antimicrobial activity of the synthesized AgNPs. The methodology that will be used in
this study was adapted with modifications in the study of Pal J et al. (2013).
The current study synthesized AgNP using ethanolic Phyllanthus niruri leaf
extract as a reducing and stabilizing agent. The synthesized AgNP was characterized
using UV-vis, SEM-EDX and FT-IR. The comparison of antimicrobial activity of the
synthesized AgNP from aqueous Phyllanthus niruri leaf extract was conducted using the
following test organisms E. coli, S. aureus, H. influenzae and S. pneumoniae.