CHAPTER III GREEN SYNTHESIS OF SILVER ...shodhganga.inflibnet.ac.in/bitstream/10603/40511/7/07...CHAPTER III GREEN SYNTHESIS OF SILVER NANOPARTICLES USING Pteridium aquilinum AQUEOUS

CHAPTER III

GREEN SYNTHESIS OF SILVER NANOPARTICLES USING Pteridium aquilinum AQUEOUS EXTRACT AND ITS CHARACTERIZATION

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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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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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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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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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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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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


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


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.


http://www.scialert.net/asci/result.php?searchin=Keywords&cat=&ascicat=ALL&Submit=Search&keyword=antibacterial+activity

http://www.scialert.net/asci/result.php?searchin=Keywords&cat=&ascicat=ALL&Submit=Search&keyword=antimicrobial+activity

http://scialert.net/fulltext/?doi=jpt.2007.168.175&org=10#179586_ja

http://scialert.net/fulltext/?doi=jpt.2007.168.175&org=10#179624_ja

http://scialert.net/fulltext/?doi=jpt.2007.168.175&org=10#16884_b

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).


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).


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.


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





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





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





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





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


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


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).


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)


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)


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


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


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


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


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


Fig 3.10. EDX spectrum of biosynthesized silver nanoparticles using leaf broth of

Pteridium aquilinum


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).


http://www.ncbi.nlm.nih.gov/pubmed?term=Khan%20M%5BAuthor%5D&cauthor=true&cauthor_uid=23620666

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