GREEN SYNTHESIS OF SILVER NANOPARTICLE BY USING LEAF EXTRACT OF Murraya koenigii

1. INTRODUCTION

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1.1 INTRODUCTION
Nanoparticles, generally considered as particles with a size up to 100 nm,
exhibit completely new or improved properties as compared to the bulk
material that they are collected based on particular characteristics such as
size, distribution, and morphology . Nanoparticles of noble metals, such as
gold, silver and platinum are broadly applied in many fields and also directly
come in contact with the human body, such as shampoos, soaps, detergents,
shoes, cosmetic products, and tooth paste, besides medical and
pharmaceutical applications .

In present days, nanoparticles based on their electrical, optical, magnetic,

chemical and mechanical properties are used in various areas, such as the
medical sector for diagnosis, antimicrobial, drug delivery and also they are
also used in the electronic and optoelectronic industry in the chemical sector
for catalysis for environmental protection and energy conversion.

Nanoparticle synthesis is generally carried out by physical and chemical

methods, such as laser ablation, pyrolysis, chemical or physical vapour
deposition, lithography electro deposition, sol gel etc., which are costliest,
human hazardous, and not eco-friendly. Because of the use of toxic and
hazardous reagents emits toxic byproducts in environment. Compared to
physical and chemical methods, the green synthesis is low cost, ecofreindly,
competent, and fast method for producing nanoparticles. Currently, there is a
growing need to develop environmentally benevolent nanoparticles synthesis
processes that do not use toxic chemicals in the synthesis protocol [9]. So the
researchers in the field of nanoparticles synthesis and assembly have turned
to biological inspiration.

Nanoparticle synthesis is an essential area of research in nanotechnology

deals with the diverse chemical composition, dimension and controlled mono
disparity. Indeed, nanoparticles shape control is a recent addition to the list of
demands being made of newly emerging synthetics method. Nanoparticles are
the fundamental building blocks of nanotechnology. The most important and
distinct property of nanoparticles is that they exhibit a larger surface area to

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volume ratio. Nanoparticles are made from noble metals; in particular Ag, Pt.,
Au, and Pd. Metal nanoparticles have marvelous applications in the area of
catalysis, optoelectronics, diagnostic biological probes, and display devices.
Among the above four silver nanoparticles play a major role in the field of
biology and medicine 3 . Silver has been describing as an ‘oligodynamic.

Silver is a nontoxic, safe inorganic antibacterial agent and is capable of killing

about 650 microorganisms that cause diseases. Usually physical agents such
as UV radiation and chemical agents such as chlorine, silver nitrate, etc. are
commonly used 4 . Silver has been widely used for the development of many
biological and pharmaceutical processes, products, and appliances such as
coating materials for medical devices, orthopedic or dental graft materials,
topical aids for wound repair, water sanitization, textile products, and even
washing machines. The use of nano-sized silver particles as antimicrobial
agents has become more common as technological advances make their
production more economical.

1.2 Plant description

Classification
Common Name : Curry Leaves
Family : Rutaceae
Plant part taken : Leaves
Genus : Murraya
Species : koenigii
Botanical name : Murraya koenigii
Fig. 1 Murray koenigii leaf

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Description :

It is a tropical to sub-tropical tree which is native to India. The leaves are

highly valued as seasoning in southern and west-coast India cooking. The
leaves are used as a herb in Ayurvedic medicinne. It is valued as in anti-
diebetic, antioxidant, antimicrobial, anti-inflammatory, Anti-
hypercholesterolemic etc. It contains carbazolealkanoid that can induce
apoptosis in cancerous cells in liv

In biosynthesis, many prokaryotic and eukaryotic micro organisms such as

bacteria, fungi, yeast, and macro organisms like plants are using for
nanomaterial synthesis either intra or extracellulary. Compare to micro
organisms’ plants are the rich source of nature and are easily available in
nature and also their enzymatic activity is more. Grounding on these potential
properties we selected plant extract for synthesis of Silver nanoparticles. In
the present work, we used Murraya koenigii leaves extract for AgNPs
synthesis. Murraya koenigii, it is a tropical tree belongs to family Rutaceae.
Plant is widely grown; its leaves edible Because of its evergreen properties,
easy availability and more metabolic rate we selected Murraya koenigii for
silver nanoparticles synthesis.

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2. REVIEW OF
LITERATURE

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2.1 Nanoparticles:
Use of biological systems for the synthesis of nanometal is rapidly
gaining importance due to their anomalous optical (Iravani et al., 2011),
chemical (Krolikowska et al., 2003), photo electrochemical (Kumar et al.,
2003) and electronic (Chandrasekharan et al., 2000) properties. There is a
need for an eco-friendly method of nanoparticle synthesis.

Apart from physical and chemical methods, biological systems are found to be
efficient Nano-factories for nanoparticle synthesis since they possess reducing
agents such as enzymes, which can reduce metals. The nanoparticles
synthesized by various living systems have been shown to be coated with
peptides or proteins. This leads to a similar charge distribution all over the
surface of nanometal which results in repulsion between them. These inter
particle repulsive forces prevent aggregation and so, nano metal solutions
synthesized by microbes and algae have been shown to be extremely stable
even after a period of six months.

2.2 Types of Nanoparticle

Nanoparticles can be broadly grouped into two, namely, organic

nanoparticles which include carbon nanoparticles (fullerenes) while, some of
the inorganic nanoparticles include magnetic nanoparticles, noble metal
nanoparticles (like gold and silver) and semi-conductor nanoparticles (like
titanium oxide and zinc oxide). There is a growing interest in inorganic
nanoparticles i.e. of noble metal nanoparticles (Gold and silver) as they
provide superior material properties with functional versatility. Due to their
size features and advantages over available chemical imaging drug agents
and drugs, inorganic particles have been examined as potential tools for
medical imaging as well as for treating diseases. Inorganic nonmaterial have
been widely used for cellular delivery due to their versatile features like wide
availability, rich functionality, good compatibility, and capability of targeted
drug delivery and controlled release of drugs (Jiang et al., 2010).

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2.3 Synthesis of Nanoparticles

Silver nanoparticles are of interest because of the unique properties
(e.g., 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 (Xu et al.,
2006 and Klaus, et al., 1999).

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 using environmentally friendly methods (green
chemistry).

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. The development of green processes for the
synthesis of silver nanoparticles is evolving into an important branch of
nanotechnology (Majuru et al., 2009 and Jain et al., 2005).

Biologically synthesized AgNPs have drawn the attention of scientists (Buzea

et al., 2007), because of their extensive application in the development of new
technologies in the areas of electronics, material sciences and medicine at the
nanoscale (Fahlman et al., 2007, Liu et al., 2007, and Wang et al., 2008).
AgNPs are highly toxic against human pathogen showing a strong biocidal
effect against microbial species because these are highly reactive species 21
with a large surface area. AgNPs exhibit potent antimicrobial activity that
is produced by using microbes and plant extracts (Riehemann, et al., 2009).

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Nanoparticles play an important role in several aspects such as targeted drug

delivery, diagnosis, tissue engineering and antimicrobial activities (Sinha, et
al., 2006, Cho et al., 2008; Jiang, et al., 2010). The use of environmentally
benign materials like herbal extract, callus extract ( Klaus, et al., 1999),
bacteria, fungi and enzymes in green process for the synthesis of AgNPs
(Senapati et al., 2005), which offers many advantages over traditional,
chemical and physical methods in terms of cost effective, eco friendliness and
compatibility for pharmaceutical and biomedical applications as they do not
use toxic chemicals in the synthesis protocols, large scale production, easily
affordable and there is no need to use high pressure, energy, temperature and
toxic chemicals (Jiang et al., 2010 and Xu et al., 2006).

Extracts from natural sources are suitably scaled up for large scale
biosynthesis of AgNPs in a controlled manner according to their shape, size
and sensitivity (Kruis et al., 2000, Magnusson et al., 1999 and Jung et al.,
2006).The aim of this chapter is, therefore, to reflect on the current state and
future prospects, especially the potentials and limitations of the above
mentioned techniques for industries.

Physical approaches: Most important physical approaches include

evaporation-condensation and laser ablation. Various metal nanoparticles
such as silver, gold, lead sulfide, cadmium sulfide, and fullerene have
previously been synthesized using the evaporation-condensation method. The
absence of solvent contamination in the prepared thin films and the
uniformity of nanoparticles distribution are the advantages of physical
approaches in comparison with chemical processes (Senapati et al., 2005 and
Kruis et al., 2000). It was demonstrated that silver nanoparticles could be
synthesized via a small ceramic heater with a local heating source (Magnusson
et al., 1999).

The evaporated vapor can cool at a suitable rapid rate, because the
temperature gradient in the vicinity of the heater surface is very steep in
comparison with that of a tube furnace. This makes possible the formation of
small nanoparticles in high concentration. This physical method can be useful

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as a nanoparticle generator for long-term experiments for inhalation toxicity

studies, and as a calibration device for nanoparticle measurement equipment
(Magnusson, et al., 1999). Silver nanoparticles could be synthesized by laser
ablation of metallic bulk materials in solution (Jung et al., 2006 and Dolgaev et
al., 2002). The ablation efficiency and the characteristics of produced
nanosilver particles depend upon many factors such as the wavelength of the
laser impinging the metallic target, the duration of the laser pulses (in the
femto-, pico- and nanosecond regime), the laser fluence, the ablation time
duration and the effective liquid medium, with or without the presence of
surfactants (Sylvestre et al., 2004 and Kawasaki, 2006).

One important advantage of laser ablation technique compared to other

methods for production of metal colloids is the absence of chemical reagents
in solutions. Therefore, pure and uncontaminated metal colloids for further
applications can be prepared by this technique (Tarasenko, 2006).

Chemical approaches: The most common approach for synthesis of silver

nanoparticles is chemical reduction by organic and inorganic reducing agents.
In general, different reducing agents such as sodium citrate, ascorbate, sodium
borohydride (NaBH4), elemental hydrogen, polyol process, Tollens reagent,
N, N-dimethylformamide (DMF), and poly (ethylene glycol)-block copolymers
are used for reduction of silver ions (Ag+) in aqueous or non-aqueous
solutions.

The aforementioned reducing agents reduce silver ions (Ag+) and lead to the
formation of metallic silver (AgO), which is followed by agglomeration into
oligomeric clusters. These clusters eventually lead to formation of metallic
colloidal silver particles (Tsuji et al., 2002 and Merga et al., 2007). It is
important to use protective agents to stabilize dispersive nanoparticles during
the course of metal nanoparticle preparation, and protect the nanoparticles
that can be absorbed on or bind onto nanoparticle surfaces, avoiding their
agglomeration (Evanoff et al., 2004). The presence of surfactants
comprising functionalities (e.g., thiols, amines, acids, and alcohols ) for

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interactions with particle surfaces can stabilize particle growth, and protect
particles from sedimentation, agglomeration, or losing their surface
properties. Recently, a simple one-step process, Tollens method, has been
used for the synthesis of silver nanoparticles with a controlled size. In the
modified Tollens procedure, silver ions are reduced by saccharides in the
presence of ammonia, yielding silver nanoparticle films (50-200 nm), silver
hydrosols (20-50 nm) and silver nanoparticles of different shapes Oliveira et
al., 2005).

Biological approaches: In recent years, the development of efficient green

chemistry methods employing natural reducing, capping, and stabilizing
agents to prepare silver nanoparticles with desired morphology and size have
become a major focus of researchers. Biological methods can be used to
synthesize silver nanoparticles without the use of any harsh, toxic and
expensive chemical substances (Yin et al., 2002 and Ahmad, et al., 2003).

The bio reduction of metal ions by combinations of biomolecules found in the

extracts of certain organisms (e.g., enzymes/proteins, amino acids,
polysaccharides, and vitamins) is environmentally benign, yet chemically
complex. Many studies have reported successful synthesis of silver
nanoparticle using organisms (microorganisms and biological systems)
(Ankamwar et al., 2005 and Korbekandi et al., 2009).

The study of biosynthesis of nanomaterials offers a valuable contribution into

materials chemistry. The ability of some microorganisms such as bacteria and
fungi to control the synthesis of metallic nanoparticles should be employed in
the search for new materials (Sandhiya et al., 2009).

Biosynthetic methods have been investigated as an alternative to chemical

and physical ones. These methods can be divided into two categories
depending on the place where the nanoparticles or nanostructures are
created as many microorganisms can provide inorganic materials either
intracellularly or extracellularly (Mandal et al., 2006). For example, bacteria
Pseudomonas strutzeri isolated from silver mine materials is able to reduce

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Ag+ ions and accumulates silver nanoparticles, the size of such nanoparticles
being in the range 16–40 nm, with the average diameter of 27 nm (Mann,
1996).).

The examples also include magneto tactic bacteria which produce magnetite
(Fe3O4) or greigite (Fe3S4) and diatoms which produce siliceous material
(Klaus-Joerger T.et al., 2001).

The intracellular methods need special ion transportation system into the
microbial cell. Formation of magnetite particles proceeds through a sequence
of events: reduction of Fe (III) to Fe (II), precipitation of amorphous oxide and
subsequent transformation to magnetite (Klaus et al., 2001),). Gold
nanoparticles have also been synthesized in human cells, both in cancer and
non-cancer ones (Mann, 2001); the scanning microscopic images confirmed
that their morphologies differed significantly.

This behavior can have an implication to cancer diagnostics. In contrast,

extracellular synthesis of nanoparticles occurs in alkalo thermophilic
actinomycete, Thermomonospora sp., which reduces gold ions. The metabolic
activity of microorganisms can lead to precipitation of nanoparticles in
external environment of a cell, the fungi being extremely good candidates for
such processes.

The extracellular synthesis of silver and gold nanoparticles by the fungus

Colletotrichum sp. (Sandhiya, et al., 2009) or Aspergillus fumigatus has been
reported (Vankataraman et al., 2005).

A novel biological method for synthesis of silver nanoparticles using

Vericillum was proposed by (Bhanska et al., 2006 and Mukherjee, 2001), a
two-step mechanism was suggested. The first step involves trapping of Ag+
ions at the surface of the fungal cells. In the second step, enzymes present in
the cell reduce silver ions. The extracellular production of metal nanoparticles
by several strains of the fungus Fusarium oxysporum has been described by
Duran et al., 2005.

The presence of hydrogenasein in the F. oxysporium broth was demonstrated.

This extracellular enzyme shows excellent redox properties and it can act as

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an electron shuttle in metal reduction. It was evident that electron shuttles or

other reducing agents (e.g., hydroquinones) released by microorganisms are
capable of reducing ions to nanoparticles.

The Neem (Azadirachta indica) leaf broth and aqueous solution of silver
nitrate or chloroauric acid were used for the extracellular synthesis of pure
metallic silver and gold particles (Shankar et al., 2004). The time required for
Ag+ and Au3+ ions to reduce was 4 h and 2 h, respectively, being extremely
short compared to both bacteria and fungi (24 h and 120 h). Surface active
constituents of the leaf broth stabilize nanoparticle suspensions – an aqueous
suspension showed stability even after 4 weeks.

There are numerous methods available using various approaches

including chemical, physical, and biological protocols for the synthesis of
nanoparticles. Chemical method of synthesis is advantageous as it takes short
period of time for synthesis of large quantity of nanoparticles. However, in
this method capping agents are required for size stabilization of the
nanoparticles. Moreover, chemicals reagents used generally for nanoparticles
synthesis and stabilization are toxic and lead to by products that are not
environment benign.

The need for eco-friendly non-toxic methods for nanoparticles synthesis is

developing interest in biological approaches which are free from the use of
toxic chemicals as byproducts. A variety of natural sources are there for metal
nanoparticle synthesis including plants, fungi, yeast, actinomycetes, bacteria
etc. The unicellular and multicultural organisms can produce intracellular and
extra cellular inorganic nanoparticles.

Plant in Nanoparticles Synthesis: Plants offer a better option for synthesis of

nanoparticle as the protocols involving plant sources are free from toxic
chemicals; moreover, natural capping agents are readily supplied by the
plants. Sastry et al., reported the synthesis of gold and silver nanoparticles
using geranium extracts (Shankar et al., 2003). Further, gold nano triangles
and silver nanoparticles were synthesized using Aloe Vera plant extracts
(Shahverdi et al., 2007). Most reports available on the synthesis of silver or
gold nanoparticles use broths resulting from boiling fresh plant leaves.

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However, Huang et al., 2007; synthesized silver and gold nanoparticles using
the sundried Cinnamomum camphora leaf extract (Singaravelu et al., 2007). A
simple green synthesis method for production of well-defined silver
nanowires was reported recently by Lin et al, 2008. The method involves
reduction of silver nitrate with the broth of sundried Cassia fistula leaf at
room temperature without using any additive.

Yeast in Nanoparticle Synthesis: Kowshik et al., have demonstrated that

MKY3, a silver-tolerant yeast species, when challenged with soluble silver in
the log phase of growth, majority of silver precipitate extracellularly as
elemental nanoparticles. When challenged with soluble silver in the log phase
of growth, based on differential thawing of the sample, for separation of the
Metallic nanoparticles from the medium (Pedit et al., 1999).

Fungi in Nanoparticle Synthesis: The fungi taking the center stage of studies
on biological generation of nanoparticles because of the tolerance and
bioaccumulation (Loveley et al., 1987). The advantages of using fungi in their
scale up process (e.g., using a thin solid substrate fermentation method) is
that they are efficient secretor of extra cellular enzymes; it can easily obtain
large scale production of enzymes. Further advantages of using fungal
mediated green approach for synthesis of metallic nanoparticles include
economic viability and ease in handling biomass. The main drawback of
biosynthesis of nanoparticles synthesis in eukaryotic organisms lies in the
problem of genetic manipulation of the organism as a mean to over express
the enzymes which is relatively much more difficult in eukaryotes than that in
prokaryotes. Mukherjee et al. demonstrated “green synthesis” of highly
stabilized nanocrystalline silver particles by a nonpathogenic and
agriculturally important fungus, Trichoderma asperellum (Slawson et al.,
1992).

Algae in Nanoparticles Synthesis: Algae are similar to yeast for biosynthesis of

nanoparticle, still very few reports used algae as a “Biofactory” for the
nanoparticles synthesis. The marine algae used for the biosynthesizing highly
stable extracellular gold nanoparticles in a relatively short time period
compare to other biosynthesizing process. Palladium and platinum

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nanoparticles starting with their corresponding metallic chloride- containing

salts have been investigated (Konishi et al., 2004).

Actinomycetes in Nanoparticles Synthesis: The monodispersity of the silver

and gold nanoparticles produced either intracellularly or extracellularly is of
interest to scientists. But it was not very high and was far inferior to that
obtained by conventional methods. Most of the actinomycetes especially the
thermophilic actinomycetes, Thermomonospora species was exposed to gold
and silver irons and the metals got reduced extracellularly (Liangwei et al.,
2007).

Viruses in Nanoparticle Synthesis: Biological synthesis of nanoparticles has

been extended to biological particles like viruses, proteins, peptides and
enzymes. Cowpea chlorotic mottle virus and cowpea mosaic virus have been
used for the mineralization of inorganic materials (Sastry et al., 2003).
Tobacco mosaic virus has shown to direct successfully the mineralization of
sulphide and crystalline nanowires. One step further, peptides capable of
nucleating nanocrystal growth have been identified from combinatorial
screens and displayed on the surface of M13 bacteriophage (Mukherjee et al.,
2008).

Bacteria in Nanoparticles Synthesis: Bacteria have been most extensively

researched for synthesis of nanoparticles because of their fast growth and
relative ease of genetic manipulation. Slawson et al., found the Silver
producing bacteria isolated from the silver mines Pseudomonas stutzeri
AG259 where the silver nanoparticles were accumulated in the periplasmic
space but the particles size ranges from 35 to 46 nm. The Lactobacillus strains
present in the milk were exposed to larger concentration of nanoparticles to
produced silver, gold, and alloy crystals of defined morphology (Singaravelu
et al., 2007). Bacteria have also been used to synthesize gold nanoparticles
(Shankar et al., 2003).

The pH was an important factor in controlling the morphology of

bacteriogenic nanoparticles and location of the deposition. These
nanoparticles were used in many application e.g. direct electrochemistry of

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proteins (Chandran et al., 2006). The most important application of bacterium

would be in industrial silver recovery.

The left side of the figure shows the possible mechanism of silver
nanoparticle synthesis at lower concentration which may involve nitrate
reductase enzyme. B- The right side of the figure shows the possible
mechanism for the induction of apopt Possible mechanisms of the duality in
functions of silver nitrate in bacteria.

A-osis by silver nitrate which may involve inactivation of thiol group

containing proteins (e.g. NADH dehydrogenase II) and direct binding of silver
to DNA thus stopping replication that certainly leads to apoptosis ( Source:
Sureshbabu et al, 2010).

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03 OBJECTIVES
 Green synthesis of silver nanoparticle
 Characterization of silver nanoparticle
 Atimicrobial activity of synthesized silver nanoparticle

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04 MATERIALS AND
METHOD

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MATERIALS AND METHOD

4.1 Materials
Silver nitrate was purchased from Venkatesh chemicals Gulbarga. Double
distilled water was used throughout the process. Whatman no.1 filter papers
were used for filtration. Glass wares used for the complete reactions were
washed well and rinsed with double distilled water and dried in hot air oven
before use.

4.2 METHODOLOGY :

 Sample collection fresh leaves of Murraya koenigii were collected in

sterilized polythene bag and brought to botany department
laboratory and stored in laboratory conditions for further studies.
 Preparation of leaf extract.
 Green synthesis of silver nanoparticles.
 UV-Visible absorption spectroscopy.
 Transmission Electron Microscopy (TEM).
 X-ray diffraction analysis (XRD).

4.3 sample collection

Murraya koenigii fresh leaves were collected from Gurmitkal region distric
Yadgir. Were collected in sterilized polythene bag brought to botany
department and stored in laboratory conditions for further studies.

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Fig. 2 Murraya koenigii plant.

4.4 Preparation of leaf extract

The fresh leaves were washed several times using normal and distilled water
to remove impurities present on the leaves. The cleared leaves were dried
under shade with closed pack to remove moisture completely. Dried leaves
were cut into small pieces and grinded to powder By using mechanical grinder
the dried leaves were powdered.

The 25g of plant powder was taken along with 500 ml of distilled water in a
round bottom flask and then allowed to boil at 60°C for 30 min under reflux
condition, then it was cooled down to room temperature. Thus the prepared
solution was initially filtered through normal filter paper there by powdered
leafy. Materials will be filtered out, the filtrate was again filtered through
Whatman No.1 filter paper to get clear solution. The filtrate was then stored at
4°C which is to be used for further characterization and future works.

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Murraya koenigii leaves powder 25g plant powder+500ml of D.W.in

Fig. 3 in round bottom flask

Fig.4

4.5 Green synthesis of silver nanoparticles

50 mL of 1 mM aqueous solution of silver nitrate (AgNO3) was taken in 100 mL
conical flask.
Then the prepared leaf extract solution with various concentrations from 5, 10, and
15 mL was added separately and under continuous stirring for 20 min. After the
complete addition of leaf extract, the colourless solution changed into pale
yellow colour,

After 24, 48 and 72 hours of time interval colour changed from pale yellow to
reddish brown colour due to reduction of Silver nitrate to Silver ions which
indicates formation of silver nanoparticles.

Then the solution was centrifuged at10,000 rpm for 15 min , consequently
dispersed in double distilled water to remove any heavy-handed biological
materials .

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Fig.5 Aqueous plant extract of Murraya koenigii

Fig.6 (A)50ml of AgNO3+15ml of plant extract ( B)50ml of AgNO3+10ml of plant extract

Fig. 6 Fig. 7

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( C) 50ml of AgNO3+5ml of plant extract ( D ) 50 ml DW+15ml plantextract

Fig. 8 Fig. 9

Biosynthesis of silver nanoparticles-color change reaction: conical flask containing the

aqueous plant extract of Murraya koenigii

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4.6 Characterization of synthesized silver nanoparticles

 UV- Visible spectroscopy

UV-Visible spectroscopy is simplest way to confirm the formation of

nanoparticles.
The reduction of Silver ions was confirmed by qualitative testing of supernatant by
UV- Visible spectrophotometer. The UV –Visible spectroscopy measurements
were performed on Elico spectral photometer as a resolution of 1nm from 200 to
800 nm with distilled water as blank reference.

 XRD study
Powdered sample was used for X-ray diffraction; analysis for silver nanoparticles
was performed by using monochromatic Cu kα radiation (λ=1.5406 A°) operated
at 40 kV and 30 mA at 2θ angle pattern.The Coherently diffracting
Crystallography domain size of the Silver nano particle was calculated from the
width of the XRD peaks using scherrer formula.

 TEM (Transmission Electron Microscopy) analysis

Samples were prepared for Transmission electron microscopic Analysis (IIT

Mumbai) TEM Technique was employed to see the size and shape of the
synthesized silver nanoparticles, the dilute drops of suspension were allowed to dry
slowly on carbon-coated grids for TEM measurement.

 Antimicrobial activity
The bacterial sensitivity of pathogens were tested using Well diffusion method
[28].The antimicrobial study of AgNPs was estimated against pathogenic bacteria
such as E-coli, Pseudomonas aeruginosa and Staphylococcus aureus. Nutrient agar
was used to cultivate the bacteria. Medium was sterilized by using autoclave then
poured into petriplates. After solidification of medium the holes were punched on
the solidified medium by using stainless steel cylinder.

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The prepared Silver nanoparticles were added into the holes with different
concentrations like 25 µl, 50µl and 100 µl. Plates were incubated for 24 hr. at
37°C. After incubation period zone of inhibition was measured around the well.

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5. RESULT AND
DISCUSSION

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 GREEN SYNTHESIS OF SILVER NANOPARTICLE BY USING LEAF EXTRACT OF Murraya koenigii

RESULT AND DISCUSSION

5.1 UV-Vis spectroscopy analysis
The reduction of silver ions to silver nanoparticles was confirmed by UV-
Visible spectroscopy analysis and shown in table. It is well known that the
silver nanoparticles shows yellowish brown colour in water. These colours
occur due to the observable fact of surface Plasmon excitations in the metal
nanoparticles [29]. When the plant extract was added into the AgNO3 solution
the pale yellow colour solution was obtained. After 20 minutes,

Three different concentrations that are 5 ml, 10 ml, and 15 ml of Murraya

koenigii plant extracts screened for Biological synthesis of Silver
nanoparticles. Plant extract was treated with 1 Mm Silver nitrate in 100 ml
conical flask the reduction of silver ion into silver nanoparticles during
exposure to plant extract was followed by changing color, colorless to dark
brown.
Interestingly, 10 ml and 15 ml concentration plant extracts were changed the
color within 24 hours from colorless to brown whereas 5 ml concentration
plant extract changed the color within 72 hours.
the UV VIS-Spectroscopy of the synthesized silver nanoparticles were in the
range of 420,425, and 430 respectively.

Plant extracts Time taken UV – peaks Colour

& for in
concentration reduction nm
Murayya koenigii 5 ml 72 hours 430-490 Colorless- Brown
Murayya koenigii 10 ml 24 hours 420-470 Colorless- Brown
Murayya koenigii 15 ml 24 hours 420-470 Colorless- Brown

Table 1: UV-VIS Spectrum analysis shows time interval for changing color of plant
extracts.

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 GREEN SYNTHESIS OF SILVER NANOPARTICLE BY USING LEAF EXTRACT OF Murraya koenigii

Fig. 10

5.2 XRD analysis

Obtained Silver naonoparticles were purified by repeated centrifugation at 3000
rpm for 40 minutes by redispersing silver nanoparticles pellet into 10 ml double
distilled water.

After drying silver nanoparticles in room temperature structure and composition

analysis was carried out by XRD .The crystallite domain size was calculated by the
width of the XRD peaks using Scherer formula D=0.96 ƛ/β cos θ, where D is
crystalline domain size perpendicular to reflecting planes, ƛ is the x-ray
wavelength, β is the full width at half maximum and θ is the diffraction angle.

The average particle size was (example 30-35 nm.) XRD analysis, peaks assigned
to the corresponding diffraction signals ( examples (111), (200), (220), and (311)
)facets of Silver. The mean particle diameter of silver nanoparticles was calculated
from the XRD pattern according to the line width.

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 GREEN SYNTHESIS OF SILVER NANOPARTICLE BY USING LEAF EXTRACT OF Murraya koenigii

5.3 TEM (Transmission Electron Microscopy) Analysis

Fig. 11
Sample was prepared for Transmission electron microscopic Analysis (IIT
Mumbai) TEM Technique was employed to see the size and shape of the
synthesized silver nanoparticles; the particle sizes around example (30% of
particles 25%and 20% ranges. )
The TEM image suggests that the particles are polydispersed and are
rounding spherical in shape.

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 GREEN SYNTHESIS OF SILVER NANOPARTICLE BY USING LEAF EXTRACT OF Murraya koenigii

image of TEM
Fig. 12

5.4 Antimicrobial study on AgNPs

The antimicrobial activity of synthesized AgNPs by using aqueous AgNO3 and
plant extract, against both gram positive and gram negative pathogenic bacteria.

The well diffusion method was used to analyze the antimicrobial study of
pathogens such as E-coli, Pseudomonas aeruginosa (gram negative) and
Staphylococcus aureus (gram negative). The zone of inhibition (ZOI) was
increased when increasing the concentration of Silver nanoparticles. However the
zone of inhibition. Maximum zone of inhibition and Minimum ZOI was observed
from bacteria, when compared to other pathogens.

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 GREEN SYNTHESIS OF SILVER NANOPARTICLE BY USING LEAF EXTRACT OF Murraya koenigii

6 CONCLUSION

In the present study Silver nanoparticles were Green synthesized using

Murraya koenigii plant leaf extract.
The plant extract in different concentration i.e. 5 ml 10 ml and 15 ml are
challenged with 1mM Silver nitrate; change of mixture from color less to
brown indicates the synthesis of Silver nanoparticles in the reaction mixture.
And the crystallite domain size of synthesized silver nanoparticles was
measured by XRD analysis, shape and size of the silver nanoparicles was
studied by TEM analysis. Results conclude that Murraya koenigii plant leaf
extract is potential producer of Silver nanoparticles .

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7. REFERENCES

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