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CHAPTER III
GREEN SYNTHESIS OF SILVER NANOPARTICLES USING Pteridium aquilinum AQUEOUS EXTRACT AND ITS CHARACTERIZATION
Pages from 60 to 85
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60
CHAPTER III
3.1. INTRODUCTION
Nanotechnology emerges from the physical, chemical, biological and engineering
sciences where novel techniques are being developed to probe and manipulate single
atoms and molecules. The term nano is adapted from the Greek word meaning “dwarf.”
When used as a prefix, it implies 10–9
. A nanometer (nm) is one billionth of a meter, or
roughly the length of three atoms side by side. A DNA molecule is 2.5 nm wide, a protein
approximately 50 nm, and a flu virus about 100 nm. A human hair is approximately
10,000 nm thick. A nanoparticle is a microscopic particle with at least one dimension less
than 100 nm. The science and engineering of nanosystems is one of the most challenging
and fastest growing sectors of nanotechnology.
Nanoscience is a relatively new branch of science dedicated to the improvement
and utilization of devices and structures ranging from 1 to 100 nm in size, in which new
chemical, physical, and biological properties, not seen in bulk materials, can be observed
(Roco, 1998). Nanomaterial science and technology have generated great enthusiasm in
recent years because these novel technologies are guaranteed to have an impact on the
energy, chemical, electronic, and aerospace industries (Rao and Cheetham, 2001).
The physicochemical properties of nanomaterials significantly depend on their
three dimensional morphologies - size, shapes and surface topography - the surrounding
media, and their arrangement in space. The correlation of these parameters with the
relevant physical and chemical properties is a fundamental requirement for the discovery
of novel properties and applications as well as for advancing the fundamental and
practical knowledge required for the design and fabrication of new materials.
Nanometre sized particles are also found in the atmosphere where they originate
from combustion sources (traffic, forest fires), volcanic activity, and from atmospheric
gas to particle conversion processes such as photochemically driven nucleation. In fact,
nanoparticles are the end product of a wide variety of physical, chemical and biological
processes, some of which are novel and radically different, others are quite common.
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Nanoparticles are the simplest form of structures with sizes in the nm range.
In principle any collection of atoms bonded together with a structural radius of < 100 nm
can be considered as a nanoparticle. These can include, e.g., fullerens, metal clusters
(agglomerates of metal atoms), large molecules, such as proteins, and even hydrogen-
bonded assemblies of water molecules, which exist in water at ambient temperatures.
The nanosize of these particles allows various communications with biomolecules on the
cell surfaces and within the cells in way that can be decoded and designated to various
biochemical and physiochemical properties of these cells (Mody et al., 2009).
Nanoparticles are classified into major two types viz. organic and inorganic
nanoparticles. Carbon nanoparticles are called the organic nanoparticles. Magnetic
nanoparticles, noble metal nanoparticles (platinum, gold and silver) and semiconductor
nanoparticles (titanium dioxide and zinc oxide) are grouped as inorganic nanoparticles.
Inorganic nanoparticles are increasingly used in drug delivery due to their distinctive
features such as ease of use, good functionality, biocompatibility, ability to target specific
cell and controlled release of drugs.
Metallic nanoparticles (NPs) have attracted the attention of the scientific
community and technologists due to their ever-emerging, numerous, and fascinating
applications in various fields, including biomedical sciences and engineering. Gold and
silver have a broad absorption band in the visible region of the electromagnetic spectrum
(Kreibig and Vollmer, 1995; Mulvaney, 1996). The properties of these metals changes,
which depends upon their shape, size, and the surrounding medium, and they have been
used in advanced technologies in medicine, opto-electronics, and chemical catalysis,
in sensors, for drug delivery, and for etching and cutting (Che and Bennett, 1989;
Elghanian et al., 1997; Haruta, 1997; Valden et al., 1998; Fujimoto, 2003; Kruusing, 2004;
El-Sayed et al., 2006; Aurel et al., 2007; Jain et al., 2009).
Silver is a naturally occuring precious metal, most often as a mineral ore in
association with other elements. It has been positioned as the 47th
element in the periodic
table, having an atomic weight of 107.8 and two natural isotopes 106.90 Ag and
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108.90 Ag with abundance 52 and 48%. It has been used in a wide variety of applications
as it has some special properties like high electrical and thermal conductivity (Nordberg
and Gerhardsson, 1988).
Silver is widely known as a catalyst for the oxidation of methanol to
formaldehyde and ethylene to ethylene oxide (Nagy and Mestl, 1999). Colloidal silver
has particular interest because of distinctive properties, such as good conductivity,
chemical stability, catalytic, antibacterial and antimicrobial activity (Frattini et al., 2005;
Bhainsa and D’Souza, 2006). For centuaries silver has been in use for the treatment of
burns and chronic wounds. As early as 1000 B.C. silver was used to make water potables
(Richard et al., 2002; Castellano et al., 2007). In 1700, silver nitrate was used for the
treatment of venereal diseases, fistulae from salivary glands, and bone and perianal
abscesses (Klasen, 2000; Landsdown, 2002).
The optical properties of spherical silver nanoparticles are highly dependent on
their diameter. As the size of the silver particles increases, its unique plasmonic signature
shifts towards the red region of the visible spectrum and both the dipole and quadrupole
peaks are clearly expressed. The total optical extinction is comprised of absorption and
scattering. At small particle size silver nanoparticles are primarily absorbing and have a
clear yellow color in solution. As the silver particles get larger, the scattering portion of
the extinction increases. This increase in the scattering component results in the solution
becoming grayer in color. Nanoparticles have found usage in many applications such as
catalysis, sensors, drug delivery, opto-electronics, and magnetic devices (Aurel et al., 2007;
Chan and Nie, 1998; Vaseashta and Dimova-Malinovska, 2005).
Nanoparticles of a wide range of materials can be prepared by a variety of methods that
include atomic manipulation with scanning probe methods, self-organized growth, and
the controlled deposition of nanoclusters from the gas phase (Bromann et al., 1996).
In this study, we describes a rapid and eco-friendly method for green synthesis of silver
nanoparticles using leaf extract of Pteridium aquilinum, as both the reducing and
stabilizing agent, and demonstrate its suitability for synthesis of silver nanoparticles.
Recently, the utilization of biological systems has emerged as a novel method for
the synthesis of nanoparticles. These approaches have many advantages over chemical,
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63
physical, and microbial synthesis (Keki et al., 2000; Kowshik et al., 2003; Yu, 2007;
He et al., 2007 Basavaraja et al., 2008; Jha and Prasad, 2010) because there is no need of
the elaborated process of culturing and maintaining the cell, hazardous chemicals,
high-energy requirements, and wasteful purifications. Recently, several plants have been
successfully used and reported for efficient and rapid extracellular synthesis of silver,
copper, and gold nanoparticles such as broth extracts of neem (Shankar et al., 2004),
Ocimum sanctum (Ahmad et al., 2010), Pongamia pinnata (Raut et al., 2010), Eclipta
prostrate (Rajkumar and Rahuman, 2011), Annona squamosa (Naresh Kumar et al., 2011),
Nerium oleander (Roni et al., 2013). This tends us to search for a new and easily
available green reductant.
Biosynthesis of nanoparticles is a kind of bottom up approach where the main
reaction occurring is reduction/oxidation. The microbial enzymes or the plant
phytochemicals with anti-oxidant or reducing properties are usually responsible for
reduction of metal compounds into their respective nanoparticles.
Pteridium aquilinum L. Kuhn (Dennstaedtiaceae) also known as bracken fern, is a
cosmopolitan species with world-wide distribution (USDA, 2006). The medicinal use of
natural products has played a very important role in treatment of many diseases and
insecticidal activities. The aim was to demonstrate the reducing effect of Pteridium
aquilinum in the biosynthesis of silver nanoparticles. Many studies have highlighted the
fact that phytochemical constituents present in the plant extracts play a major role in the
reduction of silver ions into metallic silver and subsequent capping to prevent
agglomeration. One of the important groups of phytochemicals, that is flavonoids have
proven to be have pharmacological properties like anti-inflammatory, anti-allergic,
anti-bacterial, and anti-viral properties (Cook and Samman, 1996; Murray, 1998; Cushnie
and Lamb, 2005) and have also been found to have cytotoxic antitumor properties and to
be effective in neurodegenerative diseases (de Rijke et al., 2006; Chebil et al., 2006).
Flavonoids are free radical scavengers acting as antioxidants against free radicals
(Pal et al., 2009). Natural antioxidants that are present in herbs and spices are responsible
for inhibiting or preventing the deleterious consequences of oxidative stress. Spices and
herbs contain free radical scavengers like polyphenols, flavonoids and phenolic compounds.
Flavonoids prevent synthesis of PGs that suppress T-cells (Bitis et al., 2010); there are a
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64
huge number of research studies done, in which silver nanoparticles are synthesized using
plant extracts rich in flavonoids. Hence the (Chapter I) mainly concentrated on the
identification of phenolic compounds particularly flavonoids. In the present study,
Pteridium aquilinum has been used as a reducing agent in the synthesis of silver
nanoparticles.
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3.2. REVIEW OF LITERATURE
The uprising in material science has been hosted by previous few decades.
There has been a substantial research interest in the area of using particulate systems to
accomplish various approaches. The conception or synthesis of material with nanometer-
scale precision (nanoparticles), by means of material science, is nanotechnology.
Nanoscience and nanotechnology are the study and application of extremely small things
and can be used across all the other scientific fields, such as chemistry, biology, physics,
material science, and engineering conducted at the nanoscale, which is about 1 to 100
nanometers. Particles are further classified according to diameter. Coarse particles cover
a range between 10,000 and 2,500 nanometers. Fine particles are sized between 2,500
and 100 nanometers. Ultrafine particles or nanoparticles are sized between 1 and 100
nanometers.
The concept of nanotechnology though considered to be a modern science, has its
history dating to as back as the 9th
centuary. Nanoparticles of gold and silver were used
by the artisans of Mesopotamia to generate a glittering effect to pots. The first scientific
description of the properties of nanoparticles was provided in 1857 by Michael Faraday
in his famous paper “Experimental relations of gold (and other metals) to light”
(Faraday, 1857). In 1959, Richard Feynman gave a talk describing molecular machines
built with atomic precision. This was considered the first talk on nanotechnology.
This was entitled “There‟s plenty of space at the bottom”. The term "Nanotechnology"
was first defined by Norio Taniguchi of the Tokyo Science University in 1974.
Nanotechnology strategies are expected to involved in the creation and/or manipulation
of materials on the nanometer scale, either by scaling up from single groups of atoms or
by refining or reducing bulk materials into nanoparticles (NPs) (Jabir et al., 2012).
Nanoparticles are commonly synthesized by either top-down or bottom-up
approaches. Top-down approach is based on the mechanical method of size reduction by
breaking down the bulk materials gradually to nanoscale structures. Bottom up approach
is based on the assembly of atoms or molecules to molecular structures in nanoscale
range. Both chemical and biological synthesis of nanoparticles rely on the bottom-up
approach (Vijayaraghavan and Nalini, 2010; Narayanan and Sakthivel, 2010).
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Synthesis of noble metal nanoparticles for applications such as catalysis,
electronics, optics, environmental, and biotechnology is an area of constant interest in the
whole world. Gold and silver have been used mostly for the synthesis of stable
dispersions of nanoparticles, which are useful in areas such as photography, catalysis,
photonics, opto-electronics, biomedicine, etc. (Virender et al., 2009). Silver has known to
be a metal that came into use even before Neolithic revolution. The novel properties of
NPs have been exploited in a wide range of potential applications in medicines,
cosmetics, renewable energies, environmental remediation and biomedical devices
(Lu et al., 2007; De et al., 2008; Ghosh Chaudhuri and Paria, 2012). Among them, silver
nanoparticles (Ag-NPs or nanosilver) have attracted increasing interest due to their
unique physical, chemical and biological properties compared to their macro-scaled
counter parts (Sharma et al., 2009).
Silver nanoparticles (Ag-NPs) have distinctive physico-chemical properties,
including a high electrical and thermal conductivity, surface-enhanced Raman
scattering, chemical stability, catalytic activity and non-linear optical behavior
(Krutyakov et al., 2008). Besides, Ag-NPs exhibit broad spectrum of bactericidal and
fungicidal activity (Ahamed et al., 2010) that has made them extremely popular in a
diverse range of consumer products, including plastics, soaps, pastes, food and textiles,
increasing their market value (Garcıa-Barrasa et al., 2011; Fabrega et al., 2011;
Dallas et al., 2011). Owing to the excellent antimicrobial activity, incorporation of Ag
nanoparticles in medical field as well as air and water purification will continue to be a
growing trend. The increasing use of Ag nanoparticles in consumer products will provide
stimulus for the development of an eco-friendly production method which is not only
cost-effective but also able to retain its antimicrobial activity.
There are many ways depicted in various literatures to synthesize silver
nanoparticles. These include physical, chemical, and biological methods. The physical
and chemical methods are numerous in number, and many of these methods are
expensive or use toxic substances which are major factors that make them „not so
favored‟ methods of synthesis. An alternate, feasible method to synthesize silver
nanoparticles is to employ biological methods of using microbes and plants.
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The use of plants in the recovery of noble metals from ore mines and runoffs is
known as phytomining. In recent times, the plant extracts are widely used as a viable and
facile alternative strategy for the synthesis of metal nanoparticles rather than physical and
chemical methods. Here we present a simple green synthetic methodology of silver
nanoparticles using Pteridium aquilinum aqueous extract as a reducing and capping
agents.
Biosynthesis of nanoparticles is a type of bottom up approach which employs a
biological system or its components for the formation of nanoparticles, where the main
reaction is reduction of raw metal into nanoparticles. The process of biological route is
due to metal tolerance of biological entities (Li et al., 2007). Biological entities in
synthesis of nanoparticles may vary from simple prokaryotic bacteria to eukaryotes such
as fungi and plants. Compared to microorganisms, plants have better advantages wherein
plant mediated synthesis is a one-step protocol towards synthesis whereas microorganisms
during the course of time may lose their ability to synthesize nanoparticles due to
mutations. Further preservation of microorganisms and maintenance of cultures in active
form are very laborious and time consuming. While in plants it is easy and safe with one
step protocol towards synthesis; hence research on plants has expanded rapidly (Cui and
Gao, 2003).
The use of plant systems has been considered a green route and a reliable method
for the biosynthesis of nanoparticles owing to its environmental friendly nature
(Bhattacharya and Gupta, 2005). It is evident from the earlier reports that plants have
been exploited successfully for rapid and extracellular biosynthesis of nobel metal
nanoparticles (Kim, and Song, 2010).
The first successfully report of synthesis of nanoparticles, assisted by living plants
appeared in 2002 when it was shown that gold nanoparticles, ranging in size from 2 to
20 nm, could form inside alfalfa seedlings (Gardea-Torresday et al., 2002). Subsequently
it was shown that alfalfa also could form silver nanoparticles when exposed to a silver
rich solid medium (Gardea-Torresdey et al., 2003).
The production of gold and silver nanoparticles using Geranium extract
(Shankar et al., 2004), Aloe vera plant extracts (Chandran et al., 2006), sundried
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Cinnamomum camphora and Azadiracta indica leaf extract has been explained
(Huang et al., 2007; Shankar et al., 2004; Patil et al., 2012). Leela and Vivekanandan (2008)
performed an interesting experiment using the leaf extracts of plants, namely, Helianthus
annus, Basella alba, Oryza sativa, Saccharum officinarum, Sorghum bicolar, and
Zea mays, and concluded that among all the tested plant extracts, H. annus exhibited the
strongest potential for rapid reduction of silver ions.
Raveendran et al. (2003) synthesized starch AgNPs using starch as a capping
agent and β-d-glucose as a reducing agent in a gently heated system. The starch in the
solution mixture avoids use of relatively toxic organic solvents. Additionally, the binding
interactions between starch and AgNPs are weak and can be reversible at higher
temperatures, allowing separation of the synthesized particles (Amanullah and Yu, 2005).
Emblica officinalis fruit extract was used for fabrication of gold and
silver nanoparticles of 10 nm, showed a maximum absorption of light at 430 nm
(Ankamwar et al., 2005). Mohan et al. (2011) reported that the AgNP-grafted carbon
nanotubes and Cu-grafted carbon nanotubes may be used as effective antimicrobial
materials that find applications in biomedical devices and antibacterial controlling
system. A low melting point soda-lime glass powder containing CuNPs with high
antibacterial (against Gram-positive and Gram-negative bacteria) and antifungal activity
have been reported by Esteban-Tejeda et al. (2009). Raghunandan et al. (2009)
studied the synthesis of stable polyshaped gold nanoparticles using leaf extract from
Psidium guajava.
Ghule et al. (2006) synthesized Au nanoparticles of triangular size using bean
extract of Cicer arietinum. The synthesis of circular, triangular, hexagonal shaped gold
nanoparticles of 10-45nm size using the leaf extract of Memecylon edule was reported by
Elavazhagan and Arunachalam (2011). The recent reports on phytosynthesis of nobel
metal nanoparticles have been summarized in Tables 3.1.
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Table 3.1. Phyto-synthesis of Silver Nanoparticles
Plants Part’s
used NPs
Particle
Size (nm) Particle’s shape References
Carica papaya L fruits Ag 15 nm Cubic, hexagonal
shape
Jain et al. (2009)
Eucalyptus
hybrida (Safeda)
leaves Ag 50 - 150 nm Cubic Dubey et al. (2009)
Scutellaria
barbata
Whole
plant
Ag 5-30 Spherical/triangular Wang et al. (2009)
Clerodendrum
Inerme
leaves Ag -- Spherical Arshad
Farooqui et al. (2010)
Trianthema
decandra
Roots Ag 15nm Cubic and
hexagonal
Geethalakshmi and
Sarada, 2010
Acalypha indica leaves Ag 20-30 Spherical Krishnaraj et al.
(2010)
Hibiscus Rosa
sinensis
leaves Ag,
Au
14 Spherical/prism Philip (2010)
Eclipta prostrata leaves Ag 45nm Spherical with a
small percentage of
elongated particles
Rajakumar and
Rahuman (2011)
Phyllanthus
amarus
leaves Ag 32-53 nm Spherical, Cubic Annamalai et al.
(2011)
Citrus sinensis Peel
extract
Ag 35±2 Spherical Kaviya et al. (2011)
Vitex Negundo L. leaves Ag 18.2 ±
8.9 nm
Spherical Zargar et al. (2011)
Mangifera indica leaves Ag 20 nm Triangular,
Hexagonal and
nearly spherical
Philip (2011)
Phyllanthus
niruri
leaves Ag 32-53nm -- Krishnamoorthy,
Jayalakshmi, 2012
Annona
squamosa
Peel
extract
Ag 35±5 Irregular Spherical Kumar et al. (2012)
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Plants Part’s
used NPs
Particle
Size (nm) Particle’s shape References
Boswellia serrata gum
olibanum
Ag 7.5 ± 3.8 Spherical Kora et al. (2012)
Paederia
foetida L.
leaves Ag 24nm Spherical Lavanya et al.
(2013)
Pedilanthus
tithymaloides
leaves Ag 15 and
30 nm
Spherical shape Sundaravadivelan
et al. (2013)
Cynodon
dactylon
leaves Ag 8–10 nm Spherical Sahu et al. (2013)
Coleus
aromaticus
leaves Ag 40–50 nm. Spherical Vanaja and
Annadurai, (2013)
Origanum
vulgare
leaves Ag 136±10.09 Spherical Sankar et al. (2013)
Nanoparticles are generally characterized by their size, shape, surface area, and
dispersity (Jiang et al., 2006). A homogeneity of these properties is important in many
applications. The common techniques of characterizing nanoparticles are as follows:
transmission and scanning electron microscopy (TEM, SEM), atomic force microscopy
(AFM), dynamic light scattering (DLS), X-ray photoelectron spectroscopy (XPS),
X-ray diffractometry (XRD), Fourier transform infrared spectroscopy (FTIR), and
UV–Vis spectroscopy (Lee et al., 2003; Zhang et al., 2004; Hutter and Fendler, 2004;
Choi et al., 2007; Vilchis-Nestor et al., 2008). Nevertheless, the use and knowledge of
these techniques for nanoparticle characterization still need to be further improvement
and become a standard tool in nanobiotechnology.
Li et al. (2007) synthesized silver nanoparticles using the Capsicum annum L.
extract. Capsicum annum L. extract is known to contain a number of biomolecules such
as proteins, enzymes, polysaccharides, amino acids and vitamins. These biomolecules
could be used as bioreductants to react with metal ions and they could also be used as
scaffolds to direct the formation of nanoparticles in solution. The mechanism responsible
for the reduction was postulated as follows: the silver ions were trapped on the surface of
proteins in the extract via electrostatic interactions. This stage was the recognition
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71
process. The silver ions were then reduced by the proteins leading to changes in their
secondary structure and the formation of silver nuclei. The silver nuclei subsequently
grew by the further reduction of silver ions and their accumulation of the nuclei.
Asha Rani et al. (2009) investigated the cytotoxicity and genotoxicity of starch
coated Ag-NPs of a normal human lung fibroblast cells (IMR-90) and human
glioblastoma cells (U251). The results indicated mitochondrial dysfunction, induction of
ROS by Ag-NPs which in turn set off DNA damage and chromosomal aberrations.
A possible mechanism of toxicity was proposed which involves disruption of the
mitochondrial respiratory chain by Ag-NPs leading to production of ROS and
interruption of ATP synthesis, which in turn causes DNA damage. It was anticipated that
DNA damage is augmented by deposition, followed by interactions of Ag-NPs to the
DNA leading to cell cycle arrest in the G2/M phase.
Silver in its metallic state is inert but it reacts with the moisture in the skin and the
fluid of the wound and gets ionized. The ionized silver is highly reactive, as it binds to
tissue proteins and brings structural changes in the bacterial cell wall and nuclear
membrane leading to cell distortion and death. Silver also binds to bacterial DNA
and RNA by denaturing and inhibits bacterial replication (Castellano et al., 2007).
Klaus et al. (1999) has described the significance of biosorption and bioreduction of
silver ions by dried Pseudomonas stutzeri AG259. Lin et al. (1998) explained that in
general, silver ions from silver nanoparticles are believed to become attached to the
negatively charged bacterial cell wall and rupture it, which leads to denaturation of
protein and finally cell death. Mukherjee et al. (2001a; 2001b) have opened the field to
the synthesis of metal nanoparticles by eukaryotic organisms like Verticillium sp.
They demonstrated that the shift from bacteria to fungi had the advantage that processing
and handling of the biomass would be much simpler.
Safaepour et al. (2009) directly used geraniol extract for the reduction of silver
ions and found that geraniol possesses the ability to synthesize silver nanoparticles by
reducing silver ions. The study reported the synthesis of uniformly dispersed silver
nanoparticles in the size range of 1–100 nm. Silver nanoparticles were successfully
synthesized using the latex of Jatropha curcas. The plant, J. curcas, is commercially important,
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72
as bio-diesel is extracted from its seeds on an industrial scale. Crude latex was obtained
by cutting the green stems of J. curcas plants (Bar et al., 2009). Saifuddin et al. (2009)
investigated the extracellular synthesis of silver nanoparticles (5–50 nm) in the presence
of silver ions and Bacillus subtilis supernatant solution using microwave (MW)
irradiation.
Dubey et al. (2010) reported the synthesis of gold nanoparticles with Spherical,
triangular and hexagonal shapes and about 18nm size using leaves of Sorbus aucuparia.
In addition, synthesis of Quasi-spherical and spherical gold nanoparticles have been
reported on the reduction of AuCl4− by the leaf extract and root extract of Chenopodium
album and Panax ginseng C.A. Meyer. Through this study they have synthesized
nanoparticles with a size of 10-30nm and 16.2-3.0 nm, respectively. Dwivedi and
Gopal (2010) reported the synthesis of silver and gold nanoparticles (10-30 nm) using leaf
extract of Chenopodium album.
Elumalai et al. (2010) have reported that the aqueous extract of shade dried leaves
of Euphorbia hirta was used for the synthesis of AgNPs and their antibacterial activities.
Forough and Farhadi (2010) reported the synthesis of stable silver nanoparticles using
aqueous extracts of the manna of hedysarum plant and the soap-root (Acanthe phylum
bracteatum) plant were used as reducing and stabilizing agents. The average diameter of
the prepared nanoparticles in solution was about 29-68 nm.
Raghunandan et al. (2011) reported the microwave-assisted rapid extracellular
synthesis of stable bio-functioned silver nanoparticles from guava (Psidium guajava) leaf
extract and the resulted nanoparticles were spherical in shape with 26±5 nm in size.
Gnanadesigan et al. (2012) demonstrated the antibacterial potential of biosynthesis of
silver nanoparticles with spherical shape of about 71-110nm using Avicennia marina
mangrove plant. Satyavani et al. (2011) highlighted the possibility of tissue culture-
derived callus extract from Citrullus colocynthis (L.) for the synthesis of silver
nanoparticles. The resulted nanoparticle has a spherical shape and size of 31 nm.
Similarly, other researchers also reported the formation of silver nanoparticles using
rhizome extract of Dioscorea batatas, which gave a circular and flower shaped
nanoparticles (Nagajyothi and Lee, 2011).
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73
Valodkar et al. (2011) studied the synthesis of silver nanoparticles using stem
latex of a medicinally important plant, Euphorbia nivulia. The synthesized latex capped
silver nanoparticle formulation is toxic to human lung carcinoma (A549) cells in a dose
dependent manner. Thus plant latex solubilizes the AgNPs in water and acts as a
biocompatible vehicle for transport of AgNPs to tumor/cancer cells. Banerjee and
Narendhirakannan (2011) used an extract of Syzygium cumini (jambul) seeds to produce
silver nanoparticles. The seed extract had antioxidant properties in in vitro. The nanoparticles
formed using the extracts were found to have higher antioxidant activity compared with
the seed extract. This may have been due to a preferential adsorption of the antioxidant
material from the extract onto the surface of the nanoparticles. Silver nanoparticles have
been synthesized (at room temperature and 60ºC) using Polyalthia longifolia leaf extract
as a reducing and capping agent along with D-sorbitol used to increase the stability of the
nanoparticles (Kaviya et al., 2011).
Yilmaz et al. (2011) reported the synthesis of silver nanoparticles using leaves of
Stevia Rebaudiana which was spherical and polydispersed nanoparticles with diameters
below 50 nm. Krishnamurthy et al. (2011) assayed the seed extracts of Cuminum
cyminum for the reduction of plant seed extract with AuNPs. The results indicated that all
the tested leaf extracts have the ability to produce gold nanoparticles with spherical
shaped, 1-10nm nanoparticles. Liu et al. synthesized gold nanoparticle using extracts of
Chrysanthemum and tea beverages; A nanoparticle based assay was developed for
quantifying the antioxidant properties of teas (Liu et al., 2012). The presences of various
secondary metabolites, enzymes, proteins and/or other reducing agents with electron-
shuttling compounds are usually involved in the synthesis of metal nanoparticles by plant
components. Inbakandan et al. (2012) reported the biosynthesis of silver nanoparticles
using the extract of marine sponge, Acanthella elongata with the size, ranging from
15 nm to 34 nm and spherical shaped polydispersed particles.
Song et al. (2012) studied and reported the antibacterial latex foams coated with
biologically synthesized silver nanoparticles using Magnolia kobus leaf extract, which
was 25nm in size. Daisy and Saipriya (2012) synthesized gold nanoparticles (55–98 nm)
using an aqueous extract of Cassia fistula. Extracts of C. fistula bark are known to be
hypoglycemic. Gold nanoparticles made using the extract were found to be superior to
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74
the extract as hypoglycemic agents in rats for the management of diabetes mellitus
(Liu et al., 2012). Clearly, the particles concentrated the hypoglycemic agent from the
extract on their surfaces. The biosynthesis of gold nanoparticles (AuNPs) and silver
nanoparticles (AgNPs) from saponin isolated aqueous extract of Trianthema decandra
was reported by Geethalakshmi and Sarada (2013).
Niraimathi et al. (2013) investigated antimicrobial and antioxidant activity of
green synthesized AgNPs using Alternanthera sessilis. Abboud et al. (2013) investigated
the biosynthesis of silver nanoparticles (AgNPs) using onion (Allium cepa) under
microwave irradiation. Further, these synthesized silver nanoparticles were found to
exhibit high antibacterial activity against two different strains of bacteria Escherichia coli
(Gram negative) and Staphylococcus aureus (Gram positive). The extracellular synthesis
of silver nanoparticles by the brown seaweed Sargassum wightii and their antibacterial
effects against some selected human pathogens (Shanmugam et al., 2013).
Awwad et al. (2013) synthesized silver nanoparticles using carob leaf extract for
reduction of Ag+ ions to Ag
0 nanoparticles from silver nitrate solution within 2 min of
reaction time at ambient temperature. It was also shown that the average size of silver
nanoparticles can be controlled to 5 to 40 nm by varying the concentration of silver
nitrate and the volume of carob leaf extract. Further, biosynthesized silver nanoparticles
are found to be highly effective against Escherichia coli bacteria. Vanaja et al. (2013)
reported the synthesis of silver nanoparticles by the stem extract of Cissus
quadrangularis as a reducing agent and find the effective factors for its synthesis process
by varying the pH, temperature, metal ion concentration, and time duration. Furthermore,
synthesized silver nanoparticles show more antibacterial activity against Klebsiella
planticola and Bacillus subtilis, which was analyzed by disc diffusion method.
Subramanian et al. (2013) studied the antioxidant activity of the acetone and methanol
extracts of the stem bark of the plant, Shorea roxburghii and it can be used as a green
reducing agent for the synthesis of Ag nanoparticles.
Vijayakumar et al. (2013) reported that a simple and eco-friendly chemical
reaction for the synthesis of silver nanoparticles (AgNPs) from Artemisia nilagirica
(Asteraceae) has been developed. Silver nitrate was used as the metal precursor and
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75
hydrazine hydrate as a reducing agent. The morphology of the AgNPs was determined
by SEM and the average diameter of the particles was determined as 70–90 nm.
Raju et al. (2013) studied the green synthesis of silver nanoparticles (AgNPs) using
Semecarpus anacardium L. leaf extract and transmission electron microscopy (TEM)
analysis showed that the synthesized AgNPs varied from 10 to 25 nm and has spherical
shape. Gopinath et al. (2013) investigated the synthesis of spherical gold nanoparticles
(AuNPs) using aqueous leaf extract of Terminalia arjuna. T. arjuna contains arjunetin,
leucoanthocyanidinsand hydrolyzable tannins, which are found to be responsible for the
bio-reduction of AuNPs. Furthermore, the efficacy of the synthesized AuNPs induces the
mitotic cell division and pollen germination.
Leaf extract of Morinda citrifolia L. was assessed for the synthesis of silver
nanoscale particles at different temperatures and reaction times (Sathishkumar et al., 2012).
Emeka et al. (2014) reported to explore the potential of pineapple leaf towards reduction,
capping and stabilization of silver compounds.
Vidhu and Philip (2014) reported a green synthetic route for the production of
highly stable, bio-inspired silver nanoparticles using dried Saraca indica flower and they
found that the efficiency of synthesized nanoparticles as an excellent catalyst is proved
by the reduction of methylene blue which is confirmed by the decrease in the absorbance
with time and is attributed to electron relay effect. Similarly, the reducing and capping
potential of Phoenix dactylifera extract for the synthesis of gold nanoparticles exhibited
good catalytic activity for the degradation of 4-nitrophenol (Zayed and Eisa, 2014).
Guo et al. (2014) studied the synthesis of gold nanoparticles (AuNPs) using a
flavonol (Dihydromyricetin) without adding external surfactant, capping agent or
template. The use of single active substance of the plant extract provides an important
protocol for the exploration of the biosynthesis mechanism. The synthesis of silver
nanoparticles using the leaf extracts of Caesalpinia coriaria and the synthesized AgNPs
were found to show potential antimicrobial activity against multidrug resistant
Gram-positive (Escherichia coli and Pseudomonas aeruginosa) and Gram-negative
(Klebsiella pneumoniae, and Staphylococcus aureus) clinically isolated human pathogens
(Jeeva et al., 2014). The synthesis of silver nanoparticles (AgNPs) using aqueous seed
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76
extract of Manilkara zapota (L.) under ambient conditions and the DLS showed
distribution of AgNPs in colloidal solution in the range of 40–100 nm. The synthesized
AgNPs showed excellent antimicrobial activity against Candida species (Otari et al., 2014).
Bracken fern (Pteridium aquilinum) has many uses as food, medicine, and a
variety of other things. Several sources give recipes and medicinal formulas as well as
step-by-step processes for making dyes or baskets. Much scientific research has been
done and continues with regard to Pteridium and forestry, chemistry, agriculture,
horticulture, and pest control. The young shoot or fiddlehead can be eaten raw as in a
salad but cooking is recommended to reduce the enzyme thiaminase (Peterson and
Allen, 1977). Moura et al. (1988) observed chromosome aberrations in cattle raised on
bracken fern pasture.
Hassan et al. (2007) evaluated the antibacterial activity, phytochemical content
and risk assessments of the P.aquilinum leaf extract. The presence of tannins, volatile
oils, cardiac glycosides and anthraquinone glycosides, in the extracts of P.aquilinum has
earlier been associated with antimicrobial activity (Hostettman and Nakanishi, 1979;
Okwute and Hann, 1999). It is probable that the antibacterial agents in the extracts of
P. aquilinum act by inhibition of nucleic acid, protein and membrane phospholipid
biosynthesis (Franklin et al., 1987). Kardong et al. (2013) observed the phytochemical
constituent‟s such as alkaloids, tannins, saponin, terpenoids, flavonoids, phenols and
cardiac glycosides and the absence of anthraquinone and steroid were recorded in the
Pteridium aquilinum and also he studied the antioxidant and antimicrobial activity
against Bacillus subtilis, Streptococcus aureus, Proteus vulgaris and Escherichia coli.
With regards of previous study, in Chapter I & II we observed the various
phytochemicals and identified the active compounds in the ethanolic extracts of
P. aquilinum and also to assess the antioxidative activity. Based on the literature cited
above the selected plant, P. aquilinum is given importance and used to reduce silver
nanoparticles from silver nitrate.
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77
3.3. AIM AND OBJECTIVES
The principal objectives of this present chapter work are:
Green synthesis of silver nanoparticles using leaf extract of Pteridium aquilinum.
Characterization of these synthesized silver nanoparticles using by various
techniques, such as uv-visible spectroscopy, X-ray Diffraction (XRD), scanning
electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDX) and
Fourier transform infrared spectroscopy (FTIR).
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78
3.4. MATERIALS AND METHODS
3.4.1. Collection of plant materials
Leaves of Pteridium aquilinum were collected from in and around Valparai,
Western Ghats, Pollachi, Tamil Nadu, India The leaves were washed well with distilled
water and dried for 2 days at room temperature.
3.4.2. Preparation of leaf broth
The plant leaf broth solution was prepared by taking 10g of thoroughly washed
and finely cut leaves in a 300 mL Erlenmeyer flask with 100 mL of sterile distilled water
and then boiled the mixture for 5 min before finally decanted it. They were stored at 4oC
and used within a week.
3.4.3. Synthesis of silver nanoparticles
10 mL of leaf broth was added to 190mL of 1mM aqueous AgNO3 solution for
reduction of Ag+ ions. The effects of reaction time on synthesis rate and particle size of
the prepared silver nanoparticles were studied by carrying out the reaction in water bath
for 10min to 4h at 95oC with reflux.The silver nanoparticle solution thus obtained was
purified by repeated centrifugation at 15,000 rpm for 20 min followed by redispersion of
the pellet in deionized water.
3.4.4. Characterization of silver nanoparticles
UV-vis spectra were recorded as a function of reaction time on a UV-3600
Shimadzu spectrophotometer operated at a resolution of 1nm. After freeze drying of the
purified silver particles, the structure and composition were analyzed by 10 kV Ultra
High Resolution Scanning Electron Microscope (FEI QUANTA- 200 SEM) and energy
dispersive X-ray spectroscopy. The surface groups of the nanoparticles were qualitatively
confirmed using FTIR spectroscopy. FTIR spectra were recorded on a perkin-Elmer
spectrum 2000 FTIR spectrophotometer. X-ray diffraction using Cukα radiation
(PAN anlytical X’pert Pro MPD diffractometer) was used to determine the crystalline
structure of silver nanoparticles. Powder X-ray analysis was carried out using a Philips
Model PW 1050/37 diffractometer, operating at 40 kV and 30 mA, with a step size of
0.02° (2θ).
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79
3.5. RESULTS AND DISCUSSION
Many researchers have widely used noble nanoparticles (NPs) in various
technological applications because of their unique properties. Metal nanoparticles are
generally obtained from noble metals like silver, gold, platinum, titanium, copper and tin.
Among the noble metals, silver nanoparticles exhibit tremendous applications in
spectrally selective coatings for solar energy absorption, optical receptors, bio-labeling,
intercalation materials for electrical batteries, filters, antimicrobial, anti-malarial
agents and sensors (Navarro et al., 1997; Kowshik et al., 2003; Duran et al., 2005;
Shahverdi et al., 2007; Smitha et al., 2008; Kalimuthu et al., 2008; Mukherjee et al., 2008;
Sangi and Verma, 2009). Silver nanoparticles are being extensively synthesized using
many different biological sources including fungi, bacteria and plants (Shaligram et al., 2009;
Shivaji et al., 2011). Among them the plant mediated nanoparticle synthesis is getting
more popular because of the high reactivity of plant extract and easy availability of plant
materials. This method of nanoparticle synthesis involves non-toxic chemicals and
termed as green chemistry procedure.The present experimental investigation reports the
green synthesis of silver nanoparticles using Pteridium aquilinum. This method utilizes a
non-toxic, renewable P. aquilinum which functions as both reducing and stabilizing agent
during synthesis.
3.5.1. UV-VISIBLE ABSORPTION SPECTROSCOPY STUDIES
The nanoparticles were primarily characterized by UV–visible spectroscopy, which
proved to be a very useful technique for the analysis of nanoparticles (Sastry et al., 1998).
When the leaves extract was mixed with silver nitrate solution its color started to change.
As the Pteridium aquilinum leaf extract was mixed with aqueous solution of the silver
nitrate, it started to change the color from yellowish to brown due to reduction of silver
ion; which indicated the formation of silver nanoparticles (Fig. 3.6a). It is generally
recognized that UV–Vis spectroscopy could be used to examine the size and shape-
controlled nanoparticles in aqueous suspensions (Shrivastava and Dash, 2010). The UV
absorption spectra of silver nanoparticles recorded from the reaction medium as a
function of reaction time (10min, 30min, 60min, 120min and 240min) using 10%
P.aquilinum leaf broth with 1mM AgNO3 at 95oC is shown in fig. 3.1-3.5.
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Fig 3.1. UV Visible Spectra recorded as the function of Concentration of Pteridium
aquilinum leaves in a reaction with an aqueous solution of 1mM AgNO3 OD
result at 10 min (95°C)
200 300 400 500 600 700 800
0.0
0.2
0.4
0.6
0.8
1.0
1.2
Ab
so
rban
ce (
a.u
)
Wavelength (nm)
10 min
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Fig 3.2. UV Visible Spectra recorded as the function of Concentration of Pteridium
aquilinum leaves in a reaction with an aqueous solution of 1mM AgNO3 OD
result at 30 min (95°C)
200 300 400 500 600 700 800
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
Ab
so
rban
ce (
a.u
)
Wavelength (nm)
30 min
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Fig 3.3. UV Visible Spectra recorded as the function of Concentration of Pteridium
aquilinum leaves in a reaction with an aqueous solution of 1mM AgNO3 OD
result at 60 min (95°C)
200 300 400 500 600 700 800
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
Ab
so
rban
ce (
a.u
)
Wavelength (nm)
60 min
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Fig 3.4. UV Visible Spectra recorded as the function of Concentration of Pteridium
aquilinum leaves in a reaction with an aqueous solution of 1mM AgNO3 OD
result at 120 min (95°C)
200 300 400 500 600 700 800
0.0
0.5
1.0
1.5
2.0
Ab
so
rban
ce (
a.u
)
Wavelength (nm)
120 min
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Fig 3.5. UV Visible Spectra recorded as the function of Concentration of Pteridium
aquilinum leaves in a reaction with an aqueous solution of 1mM AgNO3 OD
result at 240 min (95°C)
200 300 400 500 600 700 800
0.0
0.5
1.0
1.5
2.0
2.5
Ab
so
rban
ce (
a.u
)
Wavelength (nm)
240 min
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Fig 3.6. The extract of Pteridium aquilinum before and after synthesis of AgNPs. b UV–vis
spectra of aqueous silver nitrate with P. aquilinum aqueous leaf extract at
different time intervals
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80
The change of color among the different time periods used might be due to the
variation in concentration, size and shape of the particles. Consequently, absorbance
peaks can be used as a tool to predict particle size and stability. Smaller AgNPs will have
an absorbance maximum around 420 nm, which increases with size and disappears when
particle size falls outside nanodimensions. Earlier, studies on the biosynthesis of gold
nanoparticles using Stenotrophomoas maltophilia have suggested an absorption maximum at
530 nm (Nangia et al., 2009) whereas, we found a sharp shift in peak maxima with a
maximum absorption at 420 nm as a function of reaction time where 2.10 a.u in 240 min
(Fig 3.6b). It is seen that the surface plasmon peak of silver nanoparticles at 420 nm
increases steadily as the reaction time increases and the peak gets saturated after 120min
of reaction time indicating that silver nitrate is completely reduced. The absorption peak
varied as the function of reaction time and concentration of silver nitrate changed.
Our results are similar to the previous work where the colour of fresh suspension of
Vitex negundo and silver nitrate solution was also dark brown (Zargar et al., 2011).
In this study, the formation of silver nanoparticles reduced by active properties of
P. aquilinum was investigated. The appearance of a yellowish brown color in the reaction
vessels suggested the formation of silver nanoparticles (Ahmad et al., 2003). Metallic
nanoparticles display characteristic optical absorption spectra in the UV–visible region
called surface plasmon resonance (SPR).The silver nanoparticles exhibit yellowish brown
color in aqueous solution due to excitation of surface plasmon vibrations in silver
nanoparticles (Shankar et al., 2004). Noble metals are known to exhibit unique optical
properties due to the property of Surface Plasmon Resonance (SPR) which is the
collective oscillation of the conduction of electrons in resonance with the wavelength of
irradiated light. Previous reports clarify the presence of AgNPs exhibiting yellowish
brown color in solution due to excitation of surface plasmon vibrations (Rai et al., 2006).
When metal nanoparticles form in solution, they must be stabilized against the van der
Waals forces of attraction, which may otherwise cause coagulation. Physisorbed
surfactant and polymers may cause steric or electrostatic barriers or purely electrostatic
barriers around the particle surface and may thereby provide stabilization (Mulvaney, 1996).
UV-vis absorption spectrum shows peaks characteristic of the surface plasmon resonance
of nanosized particles (Armendariz et al., 2002; Gardea-Torresdey et al., 2002; 2003).
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81
The silver nanoparticle solution thus obtained was centrifuged at 15,000 rpm for
20 min, after which the pellet was redispersed in deionized water and filtered through
Millipore filter (0.45µm) to get rid of any uncoordinated biological molecules.
The purified pellets were then freeze-dried, powdered, and used for XRD, FTIR, SEM,
and EDX analyses.
3.5.2. XRD STUDIES
As a primary characterization tool for obtaining critical features such as crystal
structure, crystallite size, and strain, x-ray diffraction patterns have been widely used in
nanoparticle research. The diffractometer was operating at 40 kV and 30 mA, with a step
size of 0.02° (2θ). The scanning was done in the region of 35˚to 85˚ for 2θ. The XRD
pattern showed numbers of Bragg reflections that may be indexed on the basis of the
face-centered cubic structure of silver. A comparison of our XRD spectrum with the
standard, (JCPDS- 87-0598 and 41-1402) confirmed that the silver particles formed in
our experiments were in the form of nanocrystals, as evidenced by the peaks at 2θ values
of 46.44°, 55.05°, 57.75°, 68.05°, 75.01° and 77.75° assigned to the (100), (006), (103),
(112), (114) and (201) Bragg reflections, respectively, which may be indexed based on
the face-centered cubic structure of silver (Fig 3.7).The noise observed might be due to
the presence of various crystalline biological macromolecules in the aqueous extract of
P. aquilinum. The result shows that the Ag+ of silver nitrate had reduced to Ag
0 by
P. aquilinum. These sharp Bragg peaks might have resulted due to the capping agent
stabilizing of the nanoparticle. This result is in agreement with a previous result, where
AgNPs were synthesized using leaf extract of Acalypha indica and their antibacterial
activity against water-borne pathogens was investigated (Krishnaraj et al., 2010).
The XRD pattern of pure silver ions was known to display peaks at 2θ=7.9°, 11.4°, 17.8°,
30.38°, and 44° (Gong et al., 2007). The XRD patterns of Ag/extract indicated that the
structure of silver nanoparticles is face-centered cubic (fcc) (Shameli et al., 2010). Hence
from the XRD pattern it is clear that AgNPs formed using P. aquilinum leaf broth were
essentially crystalline. The XRD patterns displayed here are consistent with earlier
reports (Satishkumar et al., 2009; Bar et al., 2009). Dubey et al. (2009) reported the size
of silver nanocrystallites as estimated from the full width at half maximum of the (111)
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Fig 3.7. XRD pattern of bio-synthesized AgNPs using P. aquilinum aqueous leaf extract
40 50 60 70 80
500
1000
1500
2000
2500
3000
3500
(201)
(114) (112)
(103) (006)
(100)
Inte
nsit
y (
a.u
)
2 (degree)
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82
peak of silver using the Scherrer formula was 20–60 nm. Therefore XRD results also
suggest that crystallization of the bioorganic phase occurs on the surface of the silver
nanoparticles.
3.5.3. FOURIER TRANSFORM INFRARED SPECTROSCOPY (FTIR) STUDIES
FTIR spectroscopy analysis was carried out to identify the biomolecules
responsible for capping of the bioreduced AgNPs synthesized using plant extract.
For FTIR measurements, the Ag nanoparticles solution was centrifuged at 15,000 rpm for
20 min. The pellet was washed five times with 2ml of de-ionized water to get rid of the
free proteins/ enzymes that are not capping the silver nanoparticles. The samples were
freeze dried and analyzed on a perkin-Elmer spectrum 2000 FTIR spectrophotometer in
the diffuse reflectance mode operating at a resolution of 4 cm−1
. Figure 3.8 shows the
FTIR spectra of aqueous silver nanoparticles prepared from the Pteridium aquilinum leaf
extract peaks at 3443.28, 2923.56, 1440, 1091.65 and 612.28 cm−1
.The results of the
FTIR values of synthesized silver nanoparticles showed the presence of various
functional groups such as alkane groups, methylene groups, alkene groups, amine groups,
and carboxylic acids, and these functional groups are the major classes in many of the
chemical groups and these chemical groups are previously proved to have potential
reducing agents in the synthesis of silver nanoparticles (Cho et al., 2005).
The FTIR peak located at around 2,359 cm-1
was attributed to the N–H stretching
vibrations or the C=O stretching vibrations. A broad intense band at 3,402 cm-1
in the
spectra can be assigned to the N–H stretching frequency arising from the peptide linkages
present in the proteins of the extract (Mukherjee et al., 2008).The peaks at 1,027–1,092 cm−1
correspond to the C–N stretching vibration of aliphatic amines or to alcohols/phenols,
representing the presence of polyphenols (Songa et al., 2009). This suggests that the
biological molecules could possibly perform dual functions of reduction and stabilization
of silver nanoparticles in the aqueous medium, possibly by in situ oxidation of hydroxyl
groups and by the intrinsic carbonyl groups, as well as those produced by oxidation with
air. The proposed mechanism was also substantiated by the FTIR data. Huang et al. (2007)
verified by FTIR the synthesis of silver nanoparticles in the presence of reductive
biomolecules present in Cinnamomum camphora leaf extract. In the FTIR spectra, the
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Fig 3.8. FTIR spectra of vacuum-dried powder of synthesized AgNPs using Pteridium
aquilinum leaf extract
10
91
.65
14
40
34
43
.28
29
23
.56
61
2.2
8
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83
presence of functional groups like –C–O–C, –C–O–, –C=C–, and –C=O–, derived from
several heterocyclics, was observed. These bioactive compounds are presumed to act as
reducing and capping agents for the silver nanoparticles.
FT-IR study reveals the multi-functionality of the aqueous extract of P.aquilinum,
where reduction and stabilisation occurs simultaneously. Fig. 3.8 shows the FTIR spectra
of aqueous silver nanoparticles prepared from the P.aquilinum leaf extract shows
transmittance peaks at 612.28 (C–H bend alkenes), 1091.65 (C–N stretching vibration of
aliphatic amines), 1440 (O-H bend carboxylic acids), 2923.56 (C-H stretch alkenes) and
3443.28 (O–H stretching alcohols group).These compounds may be responsible for
production of AgNPs from leaves of P.aquilinum.These peaks indicate that the carbonyl
group formed amino acid residues and that these residues ‘‘capped’’ the silver nanoparticles
to prevent agglomeration, thereby stabilizing the medium (Sathyavathi et al., 2010).
FTIR peaks that were corresponding to aromatic rings, geminal methyls, and ether
linkages indicate the presence of flavones and terpenoids responsible for the stabilization of
the AgNPs synthesized by the Sesuvium portulacastrum leaf extract (Nabikhan et al., 2010).
The absorbance peaks at 1,620–1,636 cm−1
represent carbonyl groups from
polyphenols such as catechin gallate, epicatechin gallate, epigallocatechin, epigallocatechin
gallate, gallocatechin gallate and theaflavin; the results suggest that molecules attached
with AgNPs have free and bound amide groups. These amide groups may also be in the
aromatic rings. This concludes that the compounds attached with the AgNPs could be
polyphenols with an aromatic ring and bound amide region (Kumar et al., 2010). In the
present study the leaf extract of P. aquilinum is rich in different types of plant secondary
metabolites such as alkaloid, flavonoid, phenolic, protein, carbohydrate, saponin, tannin
and glycosides. Evidence for the presence of polyphenolic compounds (i.e. kaempferol)
was obtained from the High Performance liquid Chromatography analysis (Chapter I).
The AgNPs can be stabilized by the polyphenolic compounds, kaempferol, as well as the
other coordinating phytochemicals present in the leaf extract. An immediate reduction of
silver ions in the present investigation might have resulted due to water soluble
phytochemicals like flavonoids present in the P. aquilinum leaf, silver reduction and
fabrication accomplished due to phytochemicals (flavonoids or other polyphenols), some
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84
proteins and metabolites such as terpenoids having functional groups of alcohols, alkenes
present in P. aquilinum leaves may be considered as a significant advance in this
direction.
3.5.4. SCANNING ELECTRON MICROSCOPE (SEM) AND EDX STUDIES
The scanning electron microscope uses a beam of high-energy electrons to
produce a variety of signals at the surface of specimens used. The signals show
information about the sample including chemical composition, and crystalline structure,
external morphology (texture) and orientation of materials which make up the sample.
SEM analysis is normally considered to be non-destructive because the x-rays generated
do not lead to loss of volume of the sample, so it becomes possible to repeatedly analyze
the same materials. A scanning electron microscope is a kind of electron microscope
which images a sample by scanning it using a high-energy electron beam. The electrons
then interact with the atoms making up the sample, thus producing signals which reveal
information about the sample's composition, surface topography and other properties
such as electrical conductivity.
For the SEM studies, reaction mixtures were air-dried on silicon wafers.
As a result, a coffee ring phenomenon was observed. It is well-known that when liquids
that contain fine particles were evaporated on a flat surface, the particles accumulate
along the outer edge and form typical structures (Chen and Evans, 2009). Figure 3.9a and
3.9b are SEM images, obtained with 10% P. aquilinum leaf broth at 95oC. The 10 kV Ultra
High Resolution Scanning Electron Microscope (FEI QUANTA- 200 SEM) has been
used. The SEM images shows that the silver nanoparticles synthesized using leaf broth of
P. aquilinum was spherical in shape with the size measured at 35–65 nm. In addition, the
SEM image shows that the ‘‘capped’’ silver particles were stable in solution for at least
8 weeks. Similarly, Ankanna et al., reported the SEM micrograph analysis of the silver
nanoparticles indicated that they were well-dispersed and ranged in size 30-40nm
(Ankanna et al., 2010). The silver nanoparticles formed were predominantly cubical with
uniform shape. It is known that the shape of metal nanoparticles considerably change
their optical and electronic properties (Xu and Käll, 2002). The particle shape of
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Fig 3.9. SEM micrograph showing the morphological characteristics of silver
nanoparticles synthesized using leaves of Pteridium aquilinum leaf extract.
a Higher magnification, b Lower magnification
a
b
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Fig 3.10. EDX spectrum of biosynthesized silver nanoparticles using leaf broth of
Pteridium aquilinum
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85
plant-mediated AgNPs were mostly spherical with exception of neem (Azadirachta
indica) which yielded polydisperse particles both with spherical and flat plate-like
morphology with 5–35 nm in size (Shankar et al., 2004).
Earlier authors reported that the SEM analysis of the silver nanoparticles ranging
from 55 to 80 nm in side and triangular or spherical gold nanoparticles were fabricated
using the novel sundried biomass of Cinnammum canphora leaf (Huang et al., 2007).
EDX attachment present with the SEM is known to provide information on the chemical
analysis of the fields that are being investigated or the composition at specific locations
(spot EDX). Fig. 6 is a representative profile of the spot EDX analysis reveals strong
signal in the silver region and confirms the formation of silver nanoparticles. A distinct
signal and high atomic percent values for silver were obtained. These results are
consistent with an earlier report on silver nanoparticle synthesis by the fungus
Trichoderma viride (Fayaz et al., 2010). Metallic silver nanocrystals generally show
typical optical absorption peak approximately at 3 keV due to surface plasmon resonance
(Magudapatty et al., 2001). A weak signal from ‘O’ is recorded it may due to the
presence of organic moieties from the enzymes or proteins in the leaf extract. It has been
reported that nanoparticles synthesized using plant extract are surrounded by a thin layer
of some capping organic material from the plant leaf broth that remains stable in the
solution even after synthesis.
The results of the study are favorably supported by many studies such as, the bark
powder and water extract from Cynnamn zeylanicum tree were used for silver synthesis
(Sathishkumar et al., 2009); Leaf extracts of two plants Magnolia kobus and Diopyros
kaki were used for extracellular synthesis of gold nanoparticles (Song et al., 2009);
The extract from Black Tea has been employed as a reducing agent for the synthesis of
Au and Ag nanoparticles (Begum et al., 2009); Synthesis of silver nanoparticles by
different plant part extracts of Portulaca oleracea L. (Asghari et al., 2014); Green
synthesis of silver nanoparticles mediated by Pulicaria glutinosa extracts (Khan et al., 2013);
Synthesis of silver nanoparticles using Lantana camara fruit extract and its effect on
pathogens (Sivakumar et al., 2012); Green synthesis of silver nanoparticles using extracts
of Ananas comosus (Ahmad and Sharma, 2012).
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