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CHAPTER 2
LITERATURE REVIEW
2.1 ANTIMICROBIAL ACTIVITY
2.1.1 Introduction
Antimicrobials are typically liquids. Antimicrobial liquids kill or
inhibit the growth of microorganisms such as bacteria, fungi and protozoans.
Antimicrobial drugs (e.g. penicillin) are selective and kill microbes
(microbiocidal) or prevent their growth (microbiostatic). Disinfectants are
non-selective antimicrobial substances (e.g. bleach) and are used on non-
living objects or the outside of the body.
With the emergence and increase of microbial organisms resistant
to multiple antibiotics and the continuing emphasis on health-care costs,
many researchers have tried to develop new effective antimicrobial reagents
free of resistance and cost. The most important problem caused by the
chemical antimicrobial agents is multidrug resistance. Generally, the
antimicrobial mechanism of chemical agents depends on the specific binding
with surface and metabolism of agents onto the microorganism.
Various microorganisms have evolved drug resistance over many
generations. So far, antimicrobial agents based on chemicals have been
effective for therapy; however, they have been limited to use for medical
devices and in prophylaxis in antimicrobial facilities. Therefore, an
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alternative way to overcome the drug resistance of various microorganisms is
required desperately, especially in medical devices, etc.
Nanotechnology is expected to open some new aspects to fight and
prevent diseases using atomic scale tailoring of materials. The ability to
uncover the structure and function of biosystems at the nanoscale stimulates
research leading to improvement in biology, biotechnology, medicine and
healthcare. The size of nanomaterials is similar to that of most biological
molecules and structures; therefore, nanomaterials can be useful for both in
vivo and in vitro biomedical research and applications. The integration of
nanomaterials with biology has led to the development of diagnostic devices,
contrast agents, analytical tools, physical therapy applications, and drug
delivery vehicles.
2.1.2 Silver Nanoparticles
The fight against infections is as old as civilization. Silver, for
instance had already been recognized in ancient Greece and Rome for its
infection-fighting properties and it has a long and intriguing history as an
antibiotic in human health care. Modern day pharmaceutical companies
developed powerful antibiotics which also happen to be much more profitable
than just plain old silver. This is an apparent high-tech solution to get
undesirable microbes such as harmful bacteria under control. However,
thanks to emerging nanotechnology applications, silver is making a comeback
in the form of antimicrobial nanoparticle coatings. As even the most powerful
antibiotics become less and less effective, researchers have begun to re-
evaluate old antimicrobial substances such as silver and as a result,
antimicrobial nano-silver applications have become a very popular early
commercial nanotechnology product.
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Silver et al (1996) outlined the antibacterial effects of Ag salts and
indiacated that Ag is currently used to control bacterial growth in a variety of
applications, including dental work, catheters, and burn wounds.
Zhao et al (1998) explained that Ag ions and Ag-based compounds
are highly toxic to microorganisms, showing strong biocidal effects on as
many as 12 species of bacteria including Escherichia coli.
Russel and Hugo (2000) reported on the antimicrobial properties of
AgNPs and stated that Ag ions and Ag salts have been used for decades as
antimicrobial agents in various fields because of their growth-inhibitory
capacity against microorganisms. The mechanism of the inhibitory effects of
Ag ions on microorganisms is partially known.
Mirkin and Taton (2000) stated that reducing the particle size of
materials is an efficient and reliable tool for improving their biocompatibility.
Aymonier et al (2002) have shown that hybrids of silver
nanoparticles with amphiphilic hyperbranched macromolecules exhibited
effective antimicrobial surface coating agents.
In contrast, Sondi and Salopek-Sondi (2004) reported that the
antimicrobial activity of silver nanoparticles on Gram-negative bacteria was
dependent on the concentration of Ag nanoparticle, and was closely
associated with the formation of pits in the cell wall of bacteria.
Furno et al (2004) identified usage of silver nanoparticles for
impregnation of polymeric medical devices to increase their antibacterial
activity. Silver impregnated medical devices like surgical masks and
implantable devices showed significant antimicrobial efficiency.
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Furno et al (2004) concluded that Ag ions and Ag-based
compounds have strong antimicrobial effects. These inorganic nanoparticles
have a distinct advantage over conventional chemical antimicrobial agents.
Morones et al (2005) pointed out that the silver nanoparticles were
found to be cytotoxic to E. coli. It was also shown that the antibacterial
activity of silver nanoparticles was size dependent. Silver nanoparticles
mainly in the range of 1 -10 nm attach to the surface of cell membrane and
drastically disturb its proper function like respiration and permeability.
Baker et al (2005) reported that silver nanoparticles were found to
be completely cytotoxic to E. coli for surface concentrations as low as 8 μg of
Ag/cm2.
Gogoi et al (2006) investigated the antibacterial effect of silver
nanoparticles against the fluorescent bacteria.The green fluorescent proteins
(GFPs) were adapted to these studies. The general understanding is that silver
nanoparticles get attached to sulfur containing proteins of bacteria cell
causing the death of the bacteria. The fluorescent measurements of the cell-
free supernatant reflected the effect of silver on recombination of bacteria.
Shahverdi et al (2007) have been studied the high synergistic
activity of silver nanoparticles.
Microbes cannot build up resistance against silver as they are doing
against conventional and narrow-target antibiotics, because the metal attacks
a broad range of targets in the organisms, which means that they would have
to develop a host of mutations simultaneously to protect themselves (Pal et al
2007).
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Kong and Jang (2008) were studied the antibacterial properties of
the biosynthesized silver nanoparticles when incorporated on to textile fabric
resulting in effective inhibition.
Lara et al (2009) proposed another mechanism of bactericidal
action based on the inhibition of cell wall synthesis, protein synthesis
mediated by the 30s ribosomal subunit and nucleic acid synthesis.
Nanoparticles bind with a viral envelope glycoprotein and inhibit the virus by
binding to the disulfide bond regions of the CD4 binding domain within the
HIV-1 viral envelope glycoprotein gp120.
According to Asha Rani (2009), the silver nanoparticles exhibited a
prominent metabolic arrest of fibroblast cells (IMR-90) at higher
concentrations and the toxicity depends on size of the nanoparticles. AgNPs
exhibited strong antibacterial activity against all human pathogens even at the
lowest concentrations used, except against K. pneumoniae.
Virender and Sharma (2009) reported the overview of silver
nanoparticles (AgNPs) preparation by green synthesis approaches that have
advantages over conventional methods. Silver is known for its antimicrobial
properties and has been used for antimicrobial applications and even has
shown to prevent HIV binding to host cells. AgNPs may attach to the surface
of the cell membrane disturbing permeability and respiration functions of the
cell. Smaller AgNPs having the large surface area available for interaction
would give more bactericidal effect than the larger AgNPs.
Enhanced antibacterial activities have been reported in AgNPs
modified by surfactants, as SDS and Tween 80, and polymers, as PVP 360.
The antibacterial effect of Tween80 modified AgNPs was not significant.
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Rai et al (2009) outlined that the silver nanoaparticles have
important applications in the field of biology such as antibacterial agents and
DNA sequencing. Antibacterial property of silver nanoparticles against
Staphyloccocus aureus, Pseudomonas aeruginosa and Escherichia coli has
been investigated.
But Amro et al (2010) suggested that metal depletion may cause the
formation of irregularly shaped pits in the outer membrane. It also change
membrane permeabilitydue to progressive release of lipopolysaccharide
molecules and membrane proteins. Ag+ ions and Ag salts have been used for
decades as antimicrobial agents in various fields since many decades because
of their growth-inhibitory capacity against microorganisms.
Shankar et al (2010) demonstrated the formation of silver
nanoparticles with the addition of silver nitrate to the leaf extract of neem
with antimicrobial activity.
Amro et al (2010) suggested that silver metal depletion may cause
the formation of irregularly shaped pits in the outer membrane and change
membrane permeability, which is caused by the progressive release of
lipopolysaccharide molecules and membrane proteins.
Lara et al (2011) examined AgNPs as antibacterial virucidal agents.
Ag+ ions and Ag-based compounds are toxic to microorganisms, possessing
strong biocidal effects on at least 12 species of bacteria including
multiresistant bacteria like Methicillin-resistant Staphylococcus aureus
(MRSA), as well as multidrug-resistant Pseudomonas aeruginosa, ampicillin-
resistant E. coli O157: H7 and erythromycin-resistant S. pyogenes. AgNPs
interact with a wide range of molecular processes within microorganisms
resulting in a range of effects from inhibition of growth, loss of infectivity to
cell death which depends on shape, size, and concentration of AgNPs and the
35
sensitivity of the microbial species to silver. Also, the positive charge on the
Ag+ ion is crucial for its antimicrobial activity through the electrostatic
attraction between the negatively charged cell membrane of the
microorganism and the positively charged nanoparticles. Gram-negative
bacteria may also depend on the concentration of AgNPs and is closely
associated with the formation of pits in the cell wall of bacteria.
Consequently, AgNPs accumulated in the bacterial membrane disturbing the
membrane permeability, resulting in cell death.
Dipankar and Murugan (2012) studied the synthesis,
characterization of silver nanoparticles using Iresine herbstii and evaluation
of their antibacterial avtivities. Silver ion and silver-based compounds are
highly toxic to microorganisms which show a strong biocidal effect against
the microbial species. Staphylococcus aureus, Enterococcus faecalis,
Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa are
the test bacterials pathogens used to determine the antibacterial activity of
silver nanoparticles by Mueller–Hinton agar plates method. The antimicrobial
effect was dose-dependent and increased linearly with the increased
concentration of the test sample.
Raju Vivek et al (2012) studied the biological method for the
synthesis of silver nanoparticles (AgNPs) using Annona squamosa leaf
extract and its cytotoxicity against human breast cancer cells (MCF-7) and
normal breast epithelial cells (HBL-100) in vitro. The mechanisms for AgNPs
induced toxicity may be related with mitochondrial damage, oxidative stress,
DNA damage and induction of apoptosis. In line with this, in the present
study it is stated that induction of apoptosis could be the possible mechanism
for anti-proliferative activity of biosynthesized AgNPs. The dose dependent
cytotoxicity was observed in AgNPs treated MCF-7 cells. The results
obtained can be considered as a proof cytotoxic effect of biosynthesized
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AgNPs against breast cancer MCF-7 cell line compared with HBL-100
normal breast cell line. Induction of apoptosis by biosynthesized AgNPs by
morphological changes of AgNPs treated cells MCF-7 cells when compared
with the untreated cells.
Montazer et al (2012) explained antibacterial activity of In situ
synthesis of nano silver on cotton using Tollen’s reagent. Two bacteria,
Staphylococcus aureus and Escherichea coli were used to test the
antibacterial activity of silver nanoparticles. Ag nano particles were assumed
to generate into the intra molecular and produced durable antibacterial
properties.
Friedman et al (2012) synthesized silver nanoparticle platform
evincing steady delivery. Their findings indicated inhibition of methicillin-
resistant Staphylococcus aureus (MRSA) proliferation by Silver nanoparticle
bacteriostatical functions. Mechanism of Ag-NP has a capacity to perturb the
cell wall architecture resulting in cellular edema and subsequent cellular lysis.
In addition, Ag-NPs also proved to be efficacious against another etiological
agentce, Acinetobacter baumannii (Ab). Their investigation includes resistant
and nosocomially relevant Gram-positive (Streptococcus pyogenes and
Enterococcus faecalis) and Gram-negative (Escherichia coli, Klebsiella
pneumoniae and Pseudomonas aeruginosa) bacteria.
Vijayakumar et al (2013) reported silver nanoparticles synthesized
Asteraceae have great susceptibility to different microbes. The antimicrobial
activities of inorganic metal oxide nanoparticles, such as ZnO, MgO, TiO2
and SiO2, and their selective toxicity to biological systems suggests a
potential application as therapeutics, diagnostics, surgical devices and
nanomedicine-based antimicrobial agents. Antibacterial activity of the
synthesised AgNPs was determined using the agar well diffusion assay
37
method. They suggested that the antimicrobial activity of silver nanoparticles
is due to the electrostatic attractions between the negatively charged cell
membrane of microorganisms and the positively charged nanoparticles.
The antimicrobial activity of AgNPs on Gram-negative bacteria
was dependent on the concentration of the silver nanoparticles used, and was
closely associated with the formation of pits in the cell wall of bacteria.
Following this, silver nanoparticles accumulate in the bacterial membrane and
cause permeability, resulting in cell death.
Priyadarshini et al (2013) described the synthesis of anisotropic
AgNPs using B. flexus S-27 bacterial strain showing effective antibacterial
property. The antibacterial activity of the AgNPs was examined by the
standard Kirby–Bauer disc diffusion method against multi-drug resistant
(MDR) strains such as E. coli, B. subtilis, S. pyogenes and P. aeruginosa. The
Gram negative bacterium E. coli showed maximum zone of inhibition which
may be due to the cell wall of Gram positive bacteria composed of a thick
peptidoglycan layer. The interaction with silver cations lead to the increased
membrane permeability causing the changes in cell structure. In other words
AgNPs are attached to the negatively charged bacterial cell wall followed by
rupture leading to denaturation of protein and finally cell death.
Lok et al (2013) elucidated that AgNPs exhibited destabilization of
the outer membrane and rupture of the plasma membrane, thereby causing
depletion of intracellular ATP. Silver has a greater affinity to react with
sulphur or phosphorus-containing biomolecules in the cell. Thus sulphur-
containing proteins in the membrane or inside the cells and phosphorus-
containing elements like DNA are likely to be the preferential sites for silver
nanoparticle binding.
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Moustafa and Fouda et al (2013) investigated the antimicrobial
activity of carboxymethyl chitosan/polyethylene oxide nanofibers embedded
silver nanoparticles. Zone of inhibition test was performed to evaluate the anti
microbial activity of silver nanoparticles. Results illustrated that S. aureus
was the most sensitive microbe against antimicrobial disk (AMC), CMCTS–
PEO–AgNPs nanofiber and AgNPs solution with inhibition zone 30, 22 and
15 millimeters (mm) respectively. It was observed that CMCTS–PEO–AgNPs
nanofibers are the most effective silver containing material against all tested
microbes. Compare to antibiotics these are less hazardous material.
Das et al (2013) demonstrated that ethanolic extracts of P.
decandra, G. sempervirens, H. Canadensis and T. occidentalis are used for
biosynthesis of silver nanoparticles. Antimicrobial activity was evaluated by
cell viability assessment and minimum inhibitory concentration of silver
nanoparticles. The cause of A375 cell death induced by silver nanoparticles
was apoptosis, was performed by flow cytometric analysis.
Roopan et al (2013) used Coconut-coir (C. nucifera) for the
reduction of silver nitrate into silver nanoparticles. A. stephensi and C.
quinquefasciatus larvae were collected from stagnant water area of
Melvisharam, Vellore, Tamilnadu. The larvicidal activity was assessed by the
procedure of world health organization (WHO) with some modification.
Mortality was assessed after 24 h to determine the acute toxicities on fourth
instar larvae of A. stephensi and C. quinquefasciatus. Synthesized AgNPs
were subjected to a dose–response bioassay for larvicidal activity against A.
stephensi and C. quinquefasciatus. Different concentrations ranging from 4,
2, 1, 0.5 and 0.25 mg/L for synthesized AgNPs were prepared for larvicidal
activity. The numbers of dead larvae were counted after 72 h of exposure, and
the percent mortality was reported from the average of five replicates.
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Dar et al (2013) studied extracellular synthesis of silver
nanoparticles from fungal Cryphonectria sp. isolated from chestnut trees.
Silver has inhibitory effect on microbes in both medical treatment and
industrial processes. The use of AgNPs in several pathogenic bacteria
developed resistance against various antibiotics. Antimicrobial activity was
evaluated against three human pathogenic bacteria S. aureus, S. typhi, and E.
coli. The antibacterial activities of AgNPs were determined by disk diffusion
method. AgNPs showed high activity against both S. aureus and E. coli and
less against S. typhi.
Jasmine Kaur and Kulbhushan Tikoo (2013) synthesize AgNPs to
control bacterial growth in a variety of applications including dental work,
catheters and burn wounds. AgNPs are also used in washing clothes due to
their anti-microbial property to inhibit the bacterial growth and thereby
making the fabric odor resistant. The anti-microbial activity of silver
nanoparticles was evaluated against E.coli and S.typhii by colony counting
method. Metabolic activity was determined by MTT assay. Silver
nanoparticles have been reported to alter the sulfur containing proteins of cell
membrane, thereby damaging the cell membrane of the bacteria.
Higher negative zeta potential of TSNPs depict lower aggregation
and uniform size distribution and hence more efficient in killing bacteria.
Action of AgNPs on respiratory chain has been proposed to be one of the
mechanisms of anti-microbial activity. E.coli contains two NADH
dehydrogenase, which have cysteine residues. These residues have high
affinity towards Ag. When silver binds to these potential enzymes, passage of
electrons to oxygen at the terminal oxidase is inhibited. This results in
generation of ROS and thus bacterial death. As BSNPs form aggregates at
higher concentration, it becomes difficult for them to enter the cells and
interfere with the molecular pathways. TSNPs easily penetrate the cells and
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hence can disturb the respiratory chain of the microbes resulting in the
bacterial cell death. Oxidative stress in the cells after treatment with TSNPs
and BSNPs causes’ surface oxidation of AgNPs upon contact with proteins in
the cytoplasm liberating Ag+ ions which can amplify the toxicity.
Bindhu and Umadevi (2013) carried out synthesis of silver
nanoparticles using leaf extract of Hibiscus cannabinus and showed good
antimicrobial activity against Escherichia coli, Proteus mirabilis and Shigella
flexneri. Silver nanoparticles are reported to possess anti-fungal, anti-
inflammatory, anti-viral, anti-angiogenesis, antiplatelet activity besides
effective antimicrobial agent against various pathogenic microorganisms.
Bacterial sensitivity to antibiotics is commonly tested using a disc diffusion
method. Bacterial growth inhibition around the well is due to the release of
diffusible inhibitory compounds from silver nanoparticles. Smaller particles
having the larger surface area available for interaction will give more
bactericidal effect than the larger particles.
Elhusseiny and Hassan (2013) produced silver nanoparticles and
tested their antibacterial, antiviral, anti - inflammatory and antitumour
activity. Besides antimicrobial activity of the synthesized polymeric
nanoparticles (platinum and palladium complexes) against pathogenic
bacterial strains Staphylococcus aureus (Gram-positive bacteria), Escherichia
coli (Gram-negative bacteria), pergillums flavus (filamentous fungi) and
Candida albicans (yeast). The antimicrobial activity of the tested samples
was determined using a modified Kirby-Bauer disc diffusion method. The
antimicrobial activity may be attributed to the presence of the sulfonic active
group which may react easily with the bacteria’s cell wall forming a matched
ion-pair. Anti tumour activity was assessed by SulfoRhodamine-B (SRB)
assay for cytotoxic activity against the following tumor cell lines: Liver
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carcinoma cell line (HEPG2), Breast carcinoma cell line (MCF7), Colon
carcinoma cell line (HCT 116).
Ghassan Mohammad Sulaiman et al (2013) synthesized silver
nanoparticles from leaves extract of Eucalyptus chapmaniana and then tested
the antimicrobial effect of silver nanoparticles against different pathogenic
bacteria, yeast, and its toxicity against human acute promyelocytic leukaemia
(HL-60) cell lines. Test for antimicrobial activity of silver nanoparticles was
assessed by agar well diffusion method against different pathogenic
microorganisms Escherichia coli, Pseudomonas aeruginosa, Klebsiella.
pneumoniae, Proteus volgaris (Gram negative), Staphylococcus aureus
(Gram positive) and Candida albicans (Yeast). Cell viability was evaluated
by MTT colorimetric method. The antimicrobial effect was dose-dependent
and was more against gram-positive bacteria than gram-negative bacteria.
2.1.3 Gold Nanoparticles
Ascencio et al (2003) used dried powder of alfalfa was used in the
synthesis of novel nanomaterials based on bimetallic particles of rare earth
metals, for example i.e europium–gold (Eu–Au) nanoparticles which finds
wide applications in nuclearmedicine and nanophotonics.
Using geranium stem extract, Shankar et al (2004) biosynthesized
spherical nanoparticles in the size range of 8.3–23.8 nm with an average size
of ~14 nm. X-ray diffraction (XRD) pattern showed broad diffraction peaks
indicating crystalline and nanoscale dimensions of particles.
Shankar et al (2004) synthesized gold nanotriangles and spherical
nanotriangles from lemongrass extract with the size of 0.05–1.8 μm. Atomic
force microscopic (AFM) imaging of nanotriangles showed the thickness of
14 nm and edge length of 440 nm.
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Ankamwar et al (2005) used the fruit extract of Indian gooseberry
to extracellularly reduce gold ions to synthesize highly stable gold
nanoparticles. TEM analysis showed the particle size in the range of 15–25
nm. Using tamarind leaf extract, gold nanotriangles and hexagons were
synthesized. The edge-length of nanotriangles was 100–500 nm with
thickness in the range of 20–40 nm. FTIR analysis showed the characteristic
carbonyl stretch vibrations possibly from the acid groups of tartaric acid
present in the tamarind leaf extract.
Chandran et al (2006) demonstrated the formation of gold
nanoparticles from Aloe vera extract using UV–Vis–NIR spectroscopy,
showing a relatively increased intensity of transverse band in comparison
with longitudinal band. FTIR spectrum confirmed the presence of carbonyl
groups as stabilizing and capping agent of nanoparticles. TEM analysis
showed the average size of spherical and nanotriangles as 50–350 nm.
Ghule et al (2006) have showed the remnant water from soaked
chickpea seeds (Cicer arietinum) has also been used for the synthesis of
microscale sized triangular gold prism (~25 nm thick) at room temperature.
The exudates of chickpea seeds rich in proteins, amino acids and other
biomolecules mediated Au3+
ion reduction, assembly, growth, sintering and
stabilization of triangular gold prisms.
Similarly Huang et al (2007) used sun dried leaves of
Cinnamomum camphora was used for the first time in the synthesis of gold
nanotriangles with flat and plate-like morphologies in the size range of 55–80
nm and these particles showed absorbance in the NIR region. TEM analysis
showed the size of nanoparticles.AFM study showed the thickness of
nanotriangles as 7 nm and the investigation of surface functional groups as
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capping agents by FTIR spectrum revealed the presence of water-soluble
heterocyclic compounds like alkaloids, flavones and anthracenes.
Narayanan et al (2008) similarly used the leaf extract of coriander
and Coleus amboinicus to synthesis gold nanoparticles of size 20.65±7.09 nm
and 20.5±11.45 nm respectively. UV-Vis spectroscopic analysis showed SPR
band at 536 nm with color change to pinkish-ruby color indicating the
formation of nanoparticles. TEM analysis showed the formation of spherical,
triangular, truncated triangular, hexagonal and decahedral nanoparticles.
Ramezani et al (2008) reported TEM analysis of the gold
nanoparticles produced by the methanolic extract of Eucalyptus
camaldulensis leaves with size ranging between 1.25 and 17.5 nm with an
average size of 5.5 nm. Similarly, the methanolic extract of Pelargonium
roseum leaves reduced gold ions to gold nanoparticles and TEM analysis
showed the size between 2.5 and 27.5 nm with an average size of 7.5 nm.
Vilchis-Nestor et al (2008) demonstrated the presence of
polyphenols in green tea leaf extract when involved in the synthesis of gold
nanoparticles within 24 h of reaction and TEM analysis also showed
polydispersed gold particles with anisotropic nanotriangles and irregular
contours with an average particle size of ~40 nm.
Kasthuri et al (2009) also reported the biological synthesis of gold
nanoparticles using the biocompatible compound i.e apiin from Lawsonia
inermis. Extraction of apiin was done with methanol and subsequently with
ethyl acetate from the air-dried leaves of henna, which acts as the reducing
and stabilizing agent in the formation of gold nanoparticles.
Philip (2009) produced different sized and shaped gold
nanoparticles using the leaf extract of Hibiscus rosa-sinensis by varying the
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ratio of metal salt and extract. These nanoparticles were mainly spherical,
triangular, hexagonal and dodecahedral with the size of ~14 nm. FTIR spectra
showed that the gold nanoparticles were stabilized through amino (-NH2)
groups.
Raghunandan et al (2009) showed that guava leaf extract which has
anti-malignant activity against cancer cells was also capable of synthesizing
gold nanoparticles. Microwave-assisted aqueous leaf extract was made to
produce polyshaped AuNPs and UV–Vis spectroscopic analysis showed the
rapid reduction of gold ions up to 90% within 5 min to form metallic gold
nanoparticles. This is the fastest method so far reported in microorganisms
and plants. FTIR analysis showed absence of amide peaks, which was
characteristic for proteins.
Song et al (2009) used the leaf extracts of both Magnolia kobus and
Diopyros kaki to synthesize gold nanoparticles for antimicrobial applications.
M. kobus leaf broth took 3 min for the reduction of 90% of gold ions to gold
nanoparticles at 95°C. FTIR analysis showed the surface molecules of M.
kobus as proteins and metabolites such as terpenoids. In general, TEM
analysis showed the particle size ranging from 5 to 300 nm with a mixture of
triangles, pentagons, hexagons and spheres.
Furthermore, Wang et al (2009) demonstrated the extracellular
synthesis of gold nanoparticles using the extract of herbaceous plant,
Scutellaria barbata as the reducing agent. UV–Vis spectroscopic analysis
showed the presence of SPR band centered at 540 nm and the TEM analysis
showed the presence of well-dispersed nanoparticles in the size range of
5-30 nm.
Smitha et al (2009) synthesized gold nanoparticles using the leaf
broth of Cinnamomum zeylanicum as reducing agent. When the concentration
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of extract was increased, the morphology of nanoparticles was changed from
prism to spherical with an average size of 25 nm.
On same lines, Ghodake et al (2010) used a single step room
temperature biosynthetic route for gold nanoparticles with the pear fruit. The
alkaline pH 9.0 of the pear fruit extract induced the formation of gold
triangles with edge length of 200-500 nm and hexagonal nanoplates with
thickness of 12-20 nm.
Dubey et al (2010) reported that tansy fruit extract was used as a
reducing agent in the synthesis of gold nanoparticle from auric acid. TEM
images showed the formation of spherical and triangular nanoparticles with
an average size of 11 nm. FTIR analysis confirmed the involvement of
carbonyl group in the synthesis and presumed that (-COOH) ions cover the
surface, imparting the negative charge to the nanoparticles.
Singh et al (2010) reported the synthesis of gold nanoparticles
using the aqueous extract of clove (Syzygium aromaticum). TEM analysis
showed the formation of different morphologies of triangular and polygonal
in the size range of 100 to 300 nm with change in the concentration of extract.
Ankamwar et al (2010) have shown that the leaf extract of almond
(Terminalia catappa) reduced gold ions to highly stable spherical gold
nanoparticles in the size range from 10 to 35 nm with an average size of
21.9 nm.
Arulkumar and Sabesan (2010) have reported methanolic extract of
Mucuna pruriens plant seeds used in the synthesis of monodispersed spherical
gold nanoparticles in the size range of 6-17.7 nm.
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Bankar et al (2010) synthesized gold nanoparticles by using Banana
peel extract with 300 nm showed antimicrobial activity towards bacterial such
as Shigella sp., Citrobacter koseri, Escherichia coli, Proteus vulgaris and
Enterobacter aerogenes respectively.
Das et al (2010) used ethanolic leaf extract of Centella asiatica for
the synthesis of gold nanoparticles with an average size ranging from 9.3 to
10.9 nm. The phytochemicals present in the leaf extract were involved in the
reduction and stabilization of nanoparticles.
Dubey et al (2010) synthesized gold nanoparticles with spherical,
triangular, and hexagonal shapes with an average size of 18 nm using the leaf
extract of Sorbus aucuparia. Rosa rugosa is an ornamental plant commonly
known as Japanese rosa in Eastern Asia. The extract of this plant is used in
herbal medicines and vitamin products.
Dubey et al (2010) reported the synthesis of gold nanoparticles
using the leaf extract of R. rugosa within 10 min. TEM microscopic images
revealed the formation of triangular and hexagonal gold nanoparticles.
Gupta et al (2010) prepared gold nanoparticles of size 20 nm in
diameter by addition of chloroauric acid to green tea leaf extract at room
temperature. The synthesized gold nanoparticles were used as catalyst for the
reduction of methylene blue in the presence of Sn (II) in aqueous solution.
Khalil et al (2010) synthesized various shaped gold nanoparticles
such as triangle, hexagonal and spherical in the size range of 50–100 nm upon
incubation of hot water olive leaf extract with HAuCl4 for 20min.
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Mishra et al (2010) used leaf extract of zero-calorie sweetener herb,
Stevia rebaudiana for the synthesis of well-dispersed octahedral fcc
structured gold nanoparticles of size 8–20 nm.
Philip (2010) reported the facile synthesis of gold nanoparticles
using fresh and dry leaf extract of Mangifera indica. TEM analysis revealed
the formation of monodispersed gold nanoparticles of 17 nm size.
Raju et al (2011) utilized aqueous green extract (unboiled), boiled
extract and green biomass of Semecarpus anacardium leaf for the synthesis of
gold nanoparticles in ambient conditions. The particles were polydispersed in
the range of 13-55 nm.
Montes et al (2011) prepared gold nanoparticles by using the
aqueous and isopropanol extract of alfalfa biomass as reducing agents at pH
3.5 and 3.0 anisotrophic gold nanoparticles and gold nanoplates. When
isopropanol extract was used, decahedral and icosahedral nanoparticles of
about 30-60 nm were formed.
Noruzi et al (2011) have shown that aqueous petal extract of rose
flower as reducing and stabilizing agent in the synthesis and antibacterial
activity of gold nanoparticles. TEM analysis showed polydispersed
nanoparticles with spherical, triangular, and hexagonal shapes with an
average size of 10 nm.
Vineet kumar and Sudesh kumar (2011) reported green, rapid and
extracellular synthesis of polyshaped (i.e. triangular, pentagons, hexagonal,
and spherical) gold nanoparticles (GNPs) using Bauhinia variegate leaf
extract. Higher temperature (800C) and LE ratio at 1 mM basic metal ion
concentration leads to the synthesis of spherical shaped GNP.
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Ghoreishi et al (2011) successfully bio synthesized green synthesis
of gold nanoparticles and silver nanoparticles using the flower extract of Rosa
damascena as a reducing and stabilizing agent for electro chemistry
applications.
Elavazhagan and Arunachalam (2011) used an aqueous leaf extract
of Memecylon edule (Melastomataceae) to synthesize silver and gold
nanoparticles consisted of a mixture of triangles and truncated triangles for
antibacterial applications.
Mondal et al (2011) reported the formation of AuNPs using the leaf
extract of mahogany with SPR band centered at 537 nm. The polyols reduced
Au+3 by oxidizing to α,β-unsaturated carbonyl group or simple cyclic ketones.
Similarly, Sheny et al (2011) used the leaf extract and powder of
Anacardium occidentale for the synthesis of AuNPs. UV-vis spectra showed
SPR bands around 529 nm and 526 nm for leaf extract and dried leaf powder
respectively and these particles were mostly spherical with average sizes of
6.5 nm and 17 nm.
Castro et al (2011) demonstrated that the proteins present in the
sugar beet pulp were the principal biomolecules involved in the reduction of
gold ions to anisotrophic gold nanostructures such as rods, triangular and
hexagonal. Similarly, he demonstrated the synthesis of gold nanowires using
sugar beet pulp as reductor and capping agent.
Dwivedi and Gopal (2011) carried out the single-pot biosynthesis
of quasi-spherical gold nanoparticles in the size range of 10-30 nm with an
average size of 10 nm using an obnoxious weed, Chenopodium album.
49
Viminalis and Leonard et al (2011) reported that when Korean red
ginseng root was mixed with chloroauric acid and ultrasonically irradiated at
a frequency of 38 kHz and at a power of 100W, it produced biocompatible
gold nanoparticles in 1h. FTIR study showed the presence of ginsenosides,
polysaccharides, flavones, and other enormous phytochemicals on the surface
of nanoparticles, which served as excellent reducing and coating material in
the formation of nanoparticles and prevented it from aggregation. TEM
analysis showed the formation of 16.2±3 nm sized spherical nanoparticles.
Philip and Unni (2011) demonstrated that the aqueous extract of
Ocimum sanctum (Krishna tulsi) can also be used as a reducing agent for the
synthesis of hexagonal gold nanoparticles in the size range of 30 nm with two
SPR bands at room temperature.
Philip et al (2011) demonstrated a facile bottom-up method for the
synthesis of gold nanoparticles using the leaf extract of Murraya koenigii as
reducing and stabilizing agent at 373 K. TEM micrograph study revealed the
formation of nearly spherical nanoparticles with the size of 20nm.
Hongjie An and Bo Jin (2012) stated that gold nanoparticles-DNA
binding and its implication in medical biotechnology. Gold nanoparticle
(AuNP) and silver nanoparticle (AgNP) are usually functionalized with
thiolated oligonucleotides generating DNA–nanoparticle probes for specific
DNA hybridization and recognition of complementary sequences of interest.
The inhibition of the in vitro production of hepatitis B virus RNA and
extracellular virions were also studied.
Nagaraj et al (2012) synthesized gold nanoparticle by using
Caesalpinia pulcherrima (Peacock flower) flower extract as reducing agent.
The TEM analysis shows that products have spherical morphology with size
ranging between 10-50 nm. The study also indicates that gold nanoparticles
50
show good antimicrobial activities when compared to the standard antibiotics
against Aspergillus niger, Aspergillus flavus, E.coli and Streptobacillus
species.
Hongjie An and Bo Jin (2012) demonstrated the possible
interaction between nanoparticles and DNA and its application in medical
biotechnology. ss-DNA is flexible and favors the wrapping around Au-NP,
while ds-DNA is relatively rigid and not favorable for wrapping around the
Au-NP. The DNA structure may play an important role in DNA-gold
nanoparticle interactions.
Ramamurthy et al 2013 studied bio reduction of chloro auric acid
(HAuCl4) is achieved extracellularly by using the aqueous extract of Solanum
torvum (S. torvum) fruit. Gold nanoparticles serve as strong zone of against
Escherichia coli, Pseudomonas and Bacillus species.
2.2 ANTI OXIDANT ACTIVITY
2.2.1 Introduction
A free radical has one or more unpaired electrons. An electron
without a partner is highly unstable and very reactive. To gain stability, a free
radical attacks another stable but vulnerable compound and steals an electron.
After losing an electron, the previously stable molecule becomes a free
radical and then it attacks another molecule stealing an electron. This process
results in an electron-stealing chain reaction with one free radical producing
another free radical (Moses Gomberg et al 1900).
The main characteristic of an antioxidant is its ability to trap free
radicals. Many reactive oxygen species (ROS) including the hydroxyl radical,
hydrogen peroxide and the peroxide radical are known to cause oxidative
damage to living systems. ROS also play a significant role in human diseases
51
such as cancer, atherosclerosis, hypertension and arthritis (Halliwell and
Gutteridge 1984, Frenkel 1992). Given this negative impact, there is
increasing interest in discovering natural antioxidants. Further, these natural
antioxidants are likely to be more desirable than chemically produced analogs
because some of the latter are reportedly carcinogenic (Imaida et al 1983).
Free radicals especially damage polyunsaturated fatty acids in
lipoproteins and in cell membranes, affecting transport of compounds in and
out of cells. Free radicals also damage cell proteins (altering functions) and
DNA (creating mutations). If free radical damage, oxidative stress, becomes
extensive, health problems can develop. Oxidative stress has been identified
as a causative factor in cognitive performance, the aging process, and in the
development of diseases such as cancer, arthritis, cataracts, and heart disease
(Richard 1988).
An antioxidant is a molecule that inhibits the oxidation of other
molecules. Oxidation is a chemical reaction that transfers electrons or
hydrogen from a substance to an oxidizing agent. Oxidation reactions can
produce free radicals. In turn, these radicals can start chain reactions. When
the chain reaction occurs in a cell, it can cause damage or death to the cell.
Antioxidants terminate these chain reactions by removing free radical
intermediates, and inhibit other oxidation reactions. They do this by being
oxidized themselves, so antioxidants are often reducing agents such as thiols,
ascorbic acid, or polyphenols. Antioxidants can end the chain reaction of
forming new free radicals by donating one of their own electrons. When
antioxidants donate an electron they do not become a free radical because
they are stable in either form.
A rapid, simple and inexpensive method to measure antioxidant
capacity of food involves the use of the free radical, 2,2-Diphenyl-1-
52
picrylhydrazyl (DPPH). DPPH is widely used to test the ability of compounds
to act as free radical scavengers or hydrogen donors, and to evaluate
antioxidant activity of foods (Huang et al 2005). Because DPPH and peroxyl
radicals have similar electronic structures the reaction rate of DPPH and
antioxidants give a good approximation for scavenging activities with lipid
peroxyl radicals (Brandwilliams et al 1995, Valgimigli et al 1995). It has also
been used to quantify antioxidants in complex biological systems in recent
years. The DPPH method can be used for solid or liquid samples and is not
specific to any particular antioxidant component, but applies to the overall
antioxidant capacity of the sample. A measure of total antioxidant capacity
helps understand the functional properties of foods (Aruoma et al 2003).
Excess free radicals generated in the body play key roles in many
degenerative diseases of aging such as cancer, cardiovascular disease,
cataracts, a weak immune system and brain dysfunction. Inorganic
nanoparticles have been found to be effective at scavenging oxygen-based
free radicals (Dipankar and murugan 2012).
2.2.2 Silver Nanoparticles
Dipankar and Murugan (2012) studied the synthesis and
characterization of silver nanoparticles using Iresine herbstii and evaluation
of their antioxidant and cytotoxic activity. Superoxide anions are free radicals
generated by the transfer of one electron and play an important role in the
formation of other reactive oxygen species such as hydrogen peroxide,
hydroxyl radical, or singlet oxygen in living systems.
Szydłowska-Czerniak et al (2012) developed a novel silver
nanoparticle-based method for determination of antioxidant capacity of
rapeseed and its products against 2,2'-diphenyl-1-picrylhydrazyl (DPPH) free
radical.
53
Sharma et al (2012) studied silver nanoparticle-mediated
enhancement in growth and antioxidant status of Brassica juncea. Synthesized
silver nanoparticle treatment induced the activities of specific antioxidant
enzymes, resulting in reduced reactive oxygen species levels.
Bunghez et al (2012) studied antioxidant effects of silver
nanoparticles which were synthesized by using ornamental plants (Hyacinthus
orientalis L. and Dianthus caryophyllus L.) by using chemiluminiscent
method. The herbal silver nanoparticles exhibited high values of antioxidant
activity ranging between 88.30 and 97.38%, white carnation–AgNPs having
the strongest antioxidant properties (AA = 97.38%).
Niraimathi et al (2013) investigated on biosynthesis of silver
nanoparticles using Alternanthera sessilis (Linn.) extract and antioxidant
activities. Free radical scavenging activity of the AgNPs on DPPH radical
was found to increase with increase in concentration, showing a maximum of
62% at 500 µg/ml. The standard gallic acid, however, at this concentration
exhibited 80% inhibition. The IC50 value was found to be 300.6 µg/ml.
Inbathamizh et al (2013) studied in vitro evaluation of antioxidant
and anticancer potential of Morinda pubescens synthesized silver
nanoparticles. The decolorization from purple DPPH radical to yellow
DPPHH molecule by the sample in a dose-dependent manner with an IC50
value of 84±0.25 µg/ml indicated the sample’s high radical scavenging
activity, which was closer to that of the standard whose IC50 value was found
to be 80±0.69 µg/ml.
2.2.3 Gold Nanoparticles
Gold is a well-known biocompatible metal and colloidal gold was
used as a drinkable sol that exerted curative properties for several diseases in
54
ancient times (Daniel et al 2004).Because of its low cytotoxicity gold
nanoparticles have been widely used as the platform material in the fields of
biodiagnostics (Nam et al 2003), drug/DNA delivery (Paciotti et al 2004,
Prow et al 2006), cell imaging (Bielinska et al 2002), immunostaining (Roth
et al 1996), biosensing (Penn et al 2003) and electron microscopy markers
(Baschong et al 1998). The surface of gold nanoparticles can be facilely
modified with a variety of functional groups by ligand exchange reaction and
terminal group coupling reaction (Templeton et al 2000, Ingram et al 1997).
The designable, stepwise ligand exchange is expected to serve as an efficient
avenue to prepare a variety of multiantioxidant-functionalized
nanocomposites which would present a new model for the investigation of the
cooperative antioxidant interactions (Palozza et al 1992, Zhou et al 2005).
Accordingly, in the present workl it is hypothesized that the assembly of
antioxidant ligands on AuNPs could provide the possibility of improving
antioxidant activity.
However some precursors of nanoparticles and gold nanoparticles
without capped monolayer may be toxic (Pernodet et al 2006), the well-
capped gold nanoparticles are innocuous to cellular function by in vitro
human cell experiments (Connor et al 2005). Furthermore, Shukla et al (2005)
investigated a detailed morphological study of the metabolism of well-capped
gold nanoparticles in RAW 264.7 macrophages.
Pernodet et al (2006) studies showed the capping of gold
nanoparticles with biomolcule will exhibiting the high antioxidant activities
which vary by depending on the functional groups of biomolecules and its
orientation.
Krpetic et al (2009) reported that the Cape aloe components like
aloin A and aloesin were used as stabilizing agents to form gold nanoparticles
55
employing sodium borohydride, citric acid, and ascorbic acid as reducing
agents and NaAuCl4 as metal precursors.
Shah and Vohora (2009) reported that the immunomodulatory,
antioxidative and restorative activities of Swarna Bhasma in cerebral
ischaemic rats have revealed their perceptive applications in the treatment of
ischaemia and cerebral damages. The major drawback of ionic gold lies on
the fact that they are easily inactivated by complexation and precipitation thus
limiting their desired functions in human system.
BarathManiKanth et al (2010) analysed the effect of gold
nanoparticles on oxidative stree and its related diseases. Thioredoxin-
interacting protein (Txnip) is responsible for the antioxidative mechanism
through the regulation of cellular redox balance.
Ramamurthy et al 2013 studied bio reduction of chloro auric acid
(HAuCl4) is achieved extracellularly by using the aqueous extract of Solanum
torvum (S. torvum) fruit. Gold nanoparticles serve as strong hydroxyl,
superoxide, nitric oxide and DPPH radical scavengers in contrast to their
corresponding metal oxides. The radical quenching properties of gold
nanoparticles were found to correlate with in vitro DNA protective effect.
Mohanan et al 2013 study reports the biological synthesis of gold
nanoparticles by the reduction of HAuCl4 by using citrus fruits (Citrus limon,
Citrus reticulata and Citrus sinensis) juice extract as the reducing and
stabilizing agent.
56
2.3 ANTICANCER STUDIES
2.3.1 Introduction
One of the major applications of nanotechnology is in biomedicine.
Nanoparticles can be engineered as nanoplatforms for effective and targeted
delivery of drugs and imaging labels by overcoming the many biological,
biophysical, and biomedical barriers. For in vitro and ex vivo applications, the
advantages of state-of-the-art nanodevices (eg, nanochips and nanosensors)
over traditional assay methods are obvious (Grodzinski et al 2006, Sahoo et al
2007). However, several barriers exist for in vivo applications in preclinical
and potentially clinical use of nanotechnology i.e.biocompatibility, in vivo
kinetics, tumor targeting efficacy, acute and chronic toxicity, ability to escape
the reticuloendothelial system (RES), and cost-effectiveness (Cai and Chen
2007).
Nanotechnology, an interdisciplinary research field involving
chemistry, engineering, biology, and medicine, has great potential for early
detection, accurate diagnosis, and personalized treatment of cancer (Cai and
Chen 2007).
Cancer nanotechnology is an interdisciplinary area with broad
potential applications in fi ghting cancer, including molecular imaging,
molecular diagnosis, targeted therapy, and bioinformatics. The continued
development of cancer nanotechnology holds the promise for personalized
oncology in which genetic and protein biomarkers can be used to diagnose
and treat cancer based on the molecular profile of each individual patient.
Nanotechnology, an interdisciplinary research field involving chemistry,
engineering, biology, and medicine has great potential for early detection,
accurate diagnosis, and personalized treatment of cancer (Cai et al 2007).
With the size of about one hundred to ten thousand times smaller than human
57
cells, these nanoparticles can offer unprecedented interactions with
biomolecules both on the surface of and inside the cells, which may
revolutionize cancer diagnosis and treatment.
Nowadays, the use of existing chemotherapeutic drugs are limited
with poor specificity, high cost, high toxicity, side effects and the emergence
of drug esistance. Despite the progression of early diagnosis and treatment, it
is imperative to discover alternative therapies, tools and drugs to conquer the
situation. Nanodrug particles and development of nanoformulations for drug
delivery were intensively studied, which includes the production, stability,
characterization, formulation, delivery, and biological fate. In recent times,
biosynthesis of nanomaterials is exposed as a viable and facile alternative
strategy, mainly because of its green chemistry principles.
Lung cancer (both small cell and non-small cell) is the leading
cause of cancer death for both men and women. More people die of lung
cancer than of colon, breast, and prostate cancers combined. Lung cancer is
rare in people under the age of 45. The average lifetime chance that a man
will develop lung cancer is about 1 in 13. For a woman it is about 1 in 16.
These numbers include both smokers and non-smokers. For smokers the risk
is much higher, while for non-smokers the risk is lower (Thun et al 2008).
For the in vitro anticancer studies A549 cell line has been used
model cancer cell line. A549 cells are adenocarcinomic human alveolar basal
epithelial cells. The A549 cell line was first developed by Giard et al (1972)
through the removal and culturing of cancerous lung tissue in the explanted
tumor a of 58-year-old caucasian male.In nature, these cells are squamous and
responsible for the diffusion of some substances, such as water and
electrolytes across the alveoli of lungs. If A549 cells are cultured in vitro,
they grow as monolayer cells getting adherent to the culture flask. A549 cell
58
line are widely used as in vitro model for a type II pulmonary epithelial cell
model for drug metabolism and as a transfection host (Giard and Aaronson
et al 1973).
2.3.1 Silver Nanoparticles
The emergence of multiple drug resistance and development of
severe side effects to various chemotherapies now a day’s cancer becomes the
most distressing and life-threatening disease that enforces severe mortality
worldwide. To conquer this problem there is an urgent need to develop
therapeutic modalities for the early diagnosis and treatment of cancer with
minimal side effects. Recent research in the nano-oncology has led to the
development varied nanoscale materials, devices and therapeutic agents for
the early diagnosis and treatment of cancer.
Now-a-days synthesis and characterization of silver nanoparticles
(AgNPs) through biological entity is quite interesting to employ AgNPs for
various biomedical applications in general and treatment of cancer in
particular.Among different nanoparticles exploited, silver nanoparticles
(AgNPs) are one of the promising nanoproduct widely used in the field of
nanomedicine because of their unique properties. Before implementing the
various applications of silver nanoparticles, it is necessary to investigate the
potential toxicological impacts of silver nanomaterials. The literature
available in this regard is limited.
Liau et al (1997) stated that the silver nanoparticles have received
considerable attention due to their attractive physicochemical properties when
compared to different novel metal nanomaterials. The strong toxicity that
silver exhibits in various chemical forms to a wide range of microorganisms
is very well known.
59
Lam et al (2004) reported that nanotubes induced lung tissue
damage in mice resulting in granulomas.
Shankar et al (2004) reported AgNPs having pinnacle antimicrobial
activity against Gram-positive and Gram-negative bacteria, fungi, protozoa
and certain viruses. Apart from this, recently the antitumor effect of AgNPs
has been reported against different cancerous cell lines. In biomedical
applications, silver is currently used for the treatment of burn and chronic
wounds with new products containing nano-size silver currently being
developed and introduced commercially. Other potential applications include
antimicrobial/antiviral coatings, paints, creams, lotions, fabrics, etc, for
medical, industrial and consumer markets.
Oberdorster (2004) indicated that nanomaterials (Fullerenes C60)
induced oxidative stress in a fish model, as demonstrated by a significant
elevation of lipid peroxidation and marginal GSH depletion.
Another report by Warheit et al (2004) investigated acute lung
toxicity and observed that intra-tracheally instilled single-wall carbon
nanotubes produced granulomas in rats at very high doses. Although, in vitro
data is not a substitute for whole animal studies, use of simple in vitro models
with end points that reveal a general mechanism of toxicity can be a basis for
further assessing the potential risk of chemical/material exposure. Also, based
on general observations on dosing solutions, turbidity tend to increase as
concentration increases. A single MTT assay was also conducted to determine
an appropriate dose range by testing varying concentrations in the range of 0-
250 μg/ml. It was found that Ag-NP had extensive (> 98%) toxicity beyond
100 μg/ml when exposed to the smaller sizes of Ag-NP. Based on the success
of these initial tests, an appropriate preparation method and dose range was
developed, serving as a base for the experiments.
60
Elechiguerra et al (2005) illustrated nanosilver’s potentially huge
impact on the fight of AIDS; demonstrating the ability of nanosilver (1-10 nm
range) to attack HIV-1 preventing interaction of the virus with host cells.
Piao et al (2011) reported that highly reactive hydroxyl radicals
released by AgNPs attack cellular components including DNA, lipids, and
proteins to cause various kinds of oxidative damages. Furthermore, the results
showed that AgNPs were found to be increase the DNA tail length in a comet
assay, which measures DNA strand breaks as well as alkali labile sites.
Jacob et al (2012) explained the factors that affect the toxicity of
silver nanoparticles.Several factors influence toxicity of AgNPs such as dose,
time and size of the particles and it was found that biogenic AgNPs show
doze dependent toxicity against MCF-7 cells. AgNPs treated MCF-7 cells
showed most readily noticeable effect is the alteration in cell shape apparent
morphological variations such as coiling and cell shrinkage compared to
control cells. Biologically synthesized AgNPs because cellular damage in
Hep-2 cell line through the formation of ROS were reported elsewhere.
Dipankar and Murugan (2012) reported the cytotoxicity of AgNPs
performed using the HeLa cell line with the trypan blue assay. Cytotoxic
activity was extremely sensitive to the size of the nanoparticles and the
viability measurements considerably decreased with increasing doses.
Jeyaraj and Rajesh et al (2013) observed cytotoxicity and apoptotic
effect of biogenic AgNPs using P. hexandrum leaf extract. It was also noticed
that AgNPs initiates the cancer cell death by decreasing cell proliferation,
increasing intracellular ROS, DNA damage and apoptosis.
61
2.3.2 Gold Nanoparticles
Gold nanoparticle is unique in a sense because of its intriguing
optical properties which can be exploited for both imaging and therapeutic
applications. The future of nanomedicine lies in multifunctional
nanoplatforms which combine both therapeutic components and
multimodality imaging. The ultimate goal is that nanoparticle-based agents
can allow efficient, specific in vivo delivery of drugs without systemic
toxicity. The dose delivered as well as the therapeutic effi cacy can also be
accurately measured noninvasively over time. AuNPs have long been used in
cancer diagnosis, with many advantages over quantum dots and organic dyes
of low toxicity, much better contrast than organic dyes and surface enhanced
optical properties.
Gold nanoparticles have been investigated in diverse areas such as
in vitro assays, in vitro and in vivo imaging, cancer therapy, and drug
delivery. In order to be useful for cancer treatment, the AuNPs must be non-
cytotoxic (i.e. non toxic for cells) for normal cells. This biocompatibility of
gold nanoparticles helps to high utilization in biomedical fied.
Sipkins et al (1998) has been reported in vivo imaging using gold
nanoparticles as contrast agents for biomedical imagng and electrochemical
applications.
Mirkin et al (2002) group studied the expression of some antigens
that are important in cancers, and AuNPs functionalized with antibodies can
then allow the diagnosis of cancer. Experiments using Prostate-cancer-
Specific Antigens (PSA) also have been carried out by this research group.
The AuNPs that are functionalized with antibodies for PSA are incubated
with PSA antigens.
62
Bardhan et al (2002) have carried out experiments under
physiological conditions upon functionalizing AuNPs only with PEG.
Phototherapy uses the optical heating to destroy cancer cells. The irradiation
of AuNPs with visible light in the SPB leads to light energy absorption that is
relaxed thermally within one picosecond.
Gao et al (2004) postulates in vivo targeted cancer imaging using
nanoparticles.
Michalet et al (2005) reports show that Cell and phantom imaging
using gold nanoparticle serves as a proof-of-principle for their potential
applications in live animals or cancer patients.
Zharov et al (2005) showed that the absorbance wavelength (in the
visible range) of small gold nanospheres is not optimal for in vivo
applications, besides investigating the assembly of gold nanoclusters on the
cell membrane.
Chan et al (2006) research shows the effect of AuNPs size on Hela
cells due to internalization time. The internalization time of AuNPs
measuring between 14 and 74 nm is independent of their size. However, this
difference modifies the number of internalized particles.
Rotello et al (2008) has reported the synthesis and use of AuNPs
having a core size of 2.5 nm encapsulating tamoxifen (TAF) and b-lapachone
(LAP), two anti-cancer drugs. They also reveals that the accumulation of
AuNPs near cancer cells is because of the Enhanced Permeability and
Retention (EPR) effect and the vectorization is called a ‘‘passive’’ one.
Sheetal Dhar et al (2010) has reported the cellular uptake studies
and cytotoxic effect of biosynthesized gold nanoparticles human glioma cell
line LN-229 and human glioma stem cell line HNGC-2. The gold
63
nanoparticles showed greater cytoxicity by killing the glioma cell lines and
the glioma stem cell lines also.
Audrey and dider (2012) results indicated that some AuNPs are
toxic at a concentration of 100 mM for cancer cells, but not for immune cells.
The positively charged AuNP ligands are usually toxic at concentrations
weaker than those at which negatively charged ligands would be cytotoxic.
Lokina and narayanan (2013) studies shows that cytotoxicity on
hela cancer cell of gold nanoparticles synthesized from grape fruit extract was
very inevitable results with addition to antimicrobial activity.
64
Table 2.1 List of previous works done on green synthesis of silver nanoparticles by using plants
S.No Author(s) Year Common Name Botanical name Part Particle Morphology Size
1 Shankar et al 2004 Geranium Pelargonium
graveolens Leaf Silver (Ag
0) Spherical 16–40 nm
2 Shankar et al 2004 Neem Azadirachta
indica Leaf Silver (Ag
0) Spherical 5–35 nm
3 Chandran et al 2006 Aloe vera Aloe barbadensis Leaf Silver (Ag0) Spherical 15.2±4.2 nm
4 Huang et al 2007 Camphor tree Cinnamomum
camphora Leaf Silver (Ag0)
Flat, spherical,
rods and wires 5–40 nm
5 Li et al 2007 Bell pepper Capsicum
annuum Fruit Silver (Ag0) Spherical 10±2 nm
6 Narayanan and
sakthivel 2008 Indian Borage
Coleus
amboinicus Leaf Silver (Ag0)
Spherical,
triangle
decahedral
and hexagonal
4.3–55 nm
7 Leela and
Vivekanandan 2008 Maize Zea mays Leaf Silver (Ag
0) Spherical 15 nm
8 Leela and
Vivekanandan 2008 Sorghum
Sorghum
bicolour Leaf Silver (Ag
0) Spherical 40-70 nm
9 Leela and
Vivekanandan 2008 Rice Oryza sativa Leaf Silver (Ag
0) Spherical 40 nm
65
Table 2.1(Continued)
S.No Author(s) Year Common Name Botanical name Part Particle Morphology Size
10 Leela and
Vivekanandan 2008 Sugarcane
Saccharum
officinarum Leaf Silver (Ag
0)
Spherical and
rods 10-25 nm
11 Leela and
Vivekanandan 2008 Spinach Basella alba Leaf Silver (Ag
0) Spherical 30 nm
12 Leela and
Vivekanandan 2008 Sunflower
Helianthus
annuus Leaf Silver (Ag0) Spherical 20-4 0nm
13 Song and Kim 2008 Ginkgo Ginko biloba Leaf Silver (Ag0) Spherical 35 nm
14 Song and Kim 2008 Pine Pinus desiflora Leaf Silver (Ag0) Spherical 20 nm
15 Vilchis-Nestor et
al 2008 Tea plant Camellia sinensis Leaf Silver (Ag
0) Nanotriangles ~40 nm
16 Huang et al 2008 Camphor tree Cinnamomum
camphora Leaf Silver (Ag
0)
spherical and
nanotriangle 55–80 nm
17
Singaravelu
Vivekanandhan et
al
2009 Soybean Glycine max Leaf Silver (Ag0) Spherical 25–100 nm
18 Jha et al 2009 Daisy plant Eclipta Leaf Silver (Ag0) Spherical 2–6 nm
19 Dubey et al 2009 Safeda Eucalyptus
hybrida Leaf Silver (Ag0) Spherical 50–150 nm
20 Safaepour et al 2009 Geranium Pelargonium
graveolens Leaf Silver (Ag0) ellipsoidal 1–10 nm
66
Table 2.1(Continued)
S.No Author(s) Year Common Name Botanical name Part Particle Morphology Size
21 Raut et al 2009 Gliricidia Gliricidia sepium Leaf Silver (Ag0) Spherical 10–50 nm
22 Parashar et al 2009 Parthenium Parthenium
hysterophorus Leaf Silver (Ag0) Irregular ~50 nm
23 Parashar et al 200 Peppermint Mentha piperita Leaf Silver (Ag0)
Triangular,
spherical and
ellipsoidal
5–30 nm
24 Namrata et al 2009 Papaya Carica papaya Callus Silver (Ag0) Spherical 60–80 nm
25 Bar et al 2009 Jatropha Jatropha curcas Seed Silver (Ag0) Spherical 15–50 nm
26 Bar et al 2009 Jatropha Jatropha curcas Latex Silver (Ag0) Spherical 20–40 nm
27 Song and Kim 2009 Persimmon Diospyros kaki Leaf Silver (Ag0) Spherical 32 nm
28 Song and Kim 2009 Magnolia Magnolia kobus Leaf Silver (Ag0) Spherical 25 nm
29 Song and Kim 2009 Platanus Platanus
orientalis Leaf Silver (Ag
0) Spherical 15 nm
30 Krpetic et al 2009 Cape aloe Aloe ferox Leaf Silver (Ag0) Spherical 5 nm
31 Kasthuri et al 2009 Phyllanthus Phyllanthus
amarus Leaf Silver (Ag
0)
Quasi-
spherical and
ellipsoidal
30 nm
32 Kasthuri et al 2009 Henna Lawsonia
inermis Leaf Silver (Ag
0)
Quasi-
spherical 21–39 nm
67
Table 2.1(Continued)
S.No Author(s) Year Common Name Botanical name Part Particle Morphology Size
33 Govindaraju et al 2010 Turkey berry Solanum torvum Fruit Silver (Ag0) Spherical 14 nm
34 Saxena et al 2010 Onion Allium cepa Stem Silver (Ag0) Spherical 33.67 nm
35 Prabhu et al 2010 Chaste tree Vitex negundo Leaf Silver (Ag0) Spherical 35 nm
36 Sathyavathi et al 2010 Coriander Coriandrum
sativum Leaf Silver (Ag
0)
Spherical 26 nm
37 Sathishkumar et al 2010 Alfalfa Medicago sativa Seed Silver (Ag0)
Spherical,
flower-like
and triangular
5–108 nm
38 Cruz et al 2010 Lemon Verbena Lippia citriodora Leaf Silver (Ag0) Spherical 15–30 nm
39 Farooqui et al 2010 Glory bower Clerodendrum
inerme Leaf Silver (Ag0) Spherical 40-70 nm
40 Kumar et al 2010 Jambul Syzygium cumini Seed Silver (Ag0) Spherical 92, 73 nm
41 Sathishkumar et al 2010 Turmeric Curcuma longa Tuber Silver (Ag0)
Quasi-
spherical,
triangular and
rod
10-20 nm
42 Roy and barik 2010 Water primrose Ludwigia
adscendens Leaf Silver (Ag0)
Spherical
andcubic 100–400 nm
43 Nabikhan et al 2010 Saltmarsh plant Sesuvium
portulacastrum
Leaf
callus Silver (Ag0) Spherical 5–20 nm
68
Table 2.1(Continued)
S.No Author(s) Year Common Name Botanical name Part Particle Morphology Size
44 Kora et al 2010 Buttercup tree Cochlospermum
gossypium Exudate Silver (Ag
0)
Hexagonal
Polygonal,
spherical
30-40 nm
45
Jha and prasad 2010 sago palm Cycas revoluta Leaf Silver (Ag
0) Spherical 2–6 nm
46 Geethalakshmi and
Sarada 2010 Sangamner
Trianthema
decandra Root Silver (Ag
0) Spherical 50 nm
47 Elumalai et al 2010 Asthma weed Euphorbia hirta Leaf Silver (Ag0) Spherical 40–50 nm
48 Bankar et al 2010 Banana Musa
paradisiaca Peel Silver (Ag0) Spherical 20-30 nm
49 Ankanna et al 2010 Indian Olibanum Boswellia
ovalifoliolata Stem Silver (Ag0) Polydispersed 30–40 nm
50 Ahmad et al 2010 Basil Ocimum
basilicum
Root,
Stem Silver (Ag0) Spherical
10±2 nm,
5±1.5 nm
51 Krishnaraj et al 2010 Indian Nettle Acalypha indica Leaf Silver (Ag0) Spherical 20–30 nm
52 Dubey et al 2010 Rosa rugosa Japanese Rosa Leaf Silver (Ag0) Spherical 12 nm
53 Dubey et al 2010 European
mountain ash
Sorbus
aucuparia Leaf Silver (Ag0)
Spherical,
triangular and
hexagonal
16 nm
69
Table 2.1(Continued)
S.No Author(s) Year Common Name Botanical name Part Particle Morphology Size
54 Dubey et al 2010 Common Tansy Tanacetum
vulgare Fruit Silver (Ag
0)
Spherical and
triangular 16 nm,
55 Philip 2010 Hibiscus Hibiscus rosa
sinensis Leaf Silver (Ag
0) Spherical 13 nm
56 Kaviya et al 2011 Orange Citrus sinensis Peel Silver (Ag0) Spherical 35 nm and
10 nm
57 Sathyavathi et al 2011 Drumstick Tree Moringa oleifera Leaf Silver (Ag0) Spherical 5–80 nm
58 Linga Rao and
Savithramma 2011 Svensonia
Svensonia
hyderabadensis Leaf Silver (Ag0) Spherical 45 nm
59 Mahitha et al 2011 Water hyssop Bacopa moniera Plant Silver (Ag0) Spherical 10–30 nm
60 Prathna et al 2011 Lemon Citrus limon Fruit Silver (Ag0) Spherical 50 nm
61 Velmurugan et al 2011 Oil palm Elaeis guineensis Biosolid Silver (Ag0) Spherical 5–50 nm
62 Santhoshkumar et
al 2011 Indian Lotus
Nelumbo
nucifera Leaf Silver (Ag0) Spherical 20–80 nm
63 Veerasamy et al 2011 Mangosteen Garcinia
mangostana Leaf Silver (Ag
0) Spherical 35 nm
64 Sheny et al 2011 Cashew Anacardium
occidentale Leaf Silver (Ag
0) Spherical 15.5 nm
65 Samiran Mondal et
al 2011 Mahogany
Swietenia
mahogany Leaf Silver (Ag
0) Spheroidal 25 nm
70
Table 2.1(Continued)
S.No Author(s) Year Common Name Botanical name Part Particle Morphology Size
66 Philip et al 2011 Curry tree Murraya koenigii Leaf Silver (Ag0) Spherical 10 nm
67 Philip et al 2011 Krishna tulsi Ocimum sanctum Leaf Silver (Ag0) Spherical 10–20 nm
68 Philipand Unni 2011 Mango tree Magnifera indica Leaf Silver (Ag0)
Triangular,
hexagonal and
spherical
~20 nm
69 Babu and prabu 2011 Rooster tree Calotropis
procera Flower Silver (Ag0) Cubical 35 nm
70 Ahmad et al 2011 Tick clover Desmodium
triflorum Plant Silver (Ag0) Spherical 5–20 nm
71 Guidelli et al 2011 Natural rubber Hevea
brasiliensis Latex Silver (Ag
0)
Spherical and
oval phase 90–400 nm
72 Dwivedi and
Gopal 2011 Pig weed
Chenopodium
album Plant Leaf Silver (Ag
0)
Quasi-
spherical 12 nm
73 Singh et al 2011 Clove Syzygium
aromaticum
Flower
bud Silver (Ag
0)
Spherical and
triangular 30 nm
74 Venkata Subbaiah
et al 2013 Vinca rosea
Catharanthus
roseus Linn. G.
Donn
Dried
leaves Silver (Ag0) Spherical 27-30 nm
75 Sumana et al 2013 Indian Mulberry Morinda
citrifolia Root Silver (Ag0) Spherical 30-55 nm
71
Table 2.1(Continued)
S.No Author(s) Year Common Name Botanical name Part Particle Morphology Size
76 Jeyaraj et al 2013 Agati
Sesbania
grandiflora
(Linn.)
Leaves Silver (Ag0) Spherical 22 nm
77 2013 Bogong gum Eucalyptus
chapmaniana Leaves Silver (Ag
0) Spherical 40 nm
78 Sreemanti Das et
al 2013 Emerald Green
Thuja
occidentalis
Whole
plant Silver (Ag
0) Spherical 122 nm
79 Sreemanti Das et
al 2013 Yellow puccoon
Hydrastis
Canadensis
Whole
plant Silver (Ag0) Spherical 111 nm
80 Sreemanti Das et
al 2013 Yellow jasmine
Gelsemium
Sempervirens
Whole
plant Silver (Ag0) Spherical 112 nm
81 Sreemanti Das et
al 2013 Poke weed
Phytolacca
decandra
Whole
plant Silver (Ag0) Spherical 90.87 nm
82 Selvaraj et al 2013 Coconut tree Cocos nucifera Coir Silver (Ag0) Spherical 23 nm
83 Umesh and
Vishwas 2013 Jackfruit
Artocarpus
heterophyllus
Lam.
Seed Silver (Ag0)
Spherical and
irregular 10.78 nm
84 Ning and Wei-
hong 2013 Mango
Mangifera indica
Linn Peel Silver (Ag
0) Spherical 7–27 nm
85 Yongqiang et al 2013 Aloe Aloe vera Leaf Silver (Ag0) Spherical 20 nm
72
Table 2.2 List of previous works done on green synthesis of gold nanoparticles by using plants
S.No Author(s) Year Common
Name Botanical name Part Particle Morphology Size
1 Shankar et al 2003 Geranium Pelargonium
graveolens Leaf Gold(Au
0)
Decahedral and
icosahedral 20–40 nm
2 Shankar et al 2004 Neem Azadirachta indica Leaf Gold(Au0)
Spherical,
triangle and
hexagonal
50–35 nm
3 Shankar et al 2004 Geranium Pelargonium
graveolens Root Gold(Au
0)
Spherical and
triangle
11.4–34
nm
4 Shankar et al 2004 Geranium Pelargonium
graveolens Stem Gold(Au
0) Spherical
8.3–23.8
nm
5 Ankamwar et al 2005 Tamarind Tamarindus indica Leaf Gold(Au0) Flat-triangle and
hexagonal 20–40 nm
6 Ankamwar et al 2005 Indian
gooseberry Emblica officinalis Fruit Gold(Au0) Triangle 15–25 nm
7 Shankar et al 2005 Lemongrass Cymbopogon
flexuosus Leaf Gold(Au0)
Triangle and
spherical
0.05–1.8
μm
8 Ghule et al 2006 Chickpea Cicer arietinum Beans Gold(Au0) triangular prism
~ 25 nm
thick
9 Chandran et al 2006 Aloe vera Aloe barbadensis Leaf Gold(Au0)
Spherical and
triangle
50–350
nm
73
Table 2.2 (Continued)
S.No Author(s) Year Common
Name Botanical name Part Particle Morphology Size
10 Huang et al 2007 Camphor tree Cinnamomum
camphora Leaf Gold(Au
0)
Flat and plate-
like triangle 55–80 nm
11 Vilchis-Nestor et
al 2008 Tea plant Camellia sinensis Leaf Gold(Au0) Nanotriangles ~40 nm
12 Ramezani et al 2008 Rose
geranium
Pelargonium
roseum Leaf Gold(Au0) Hexagonal
2.5–27.5
nm
13 Ramezani et al 2008 Red river
gum
Eucalyptus
camaldulensis Leaf Gold(Au0)
Traiagle and
hexagonal
1.25–17.5
nm
14 Narayanan and
Sakthivel 2008 Coriander
Coriandrum
sativum Leaf Gold(Au0)
Spherical,
triangle and
truncated
triangle
20.6±7.09
nm
15 Smitha et al 2009 Cinnamon Cinnamomum
zeylanicum Leaf Gold(Au
0)
Nanoprism and
spherical 25 nm
16 Wang et al 2009 Barbated
Skullcup Scutellaria barbata Plant Gold(Au0)
Spherical and
triangles 5–30 nm
17 Song and kim 2009 Persimmon Diopyros kaki Leaf Gold(Au0)
Triangles,
pentagons,
hexagons and
spherical
~300 nm
74
Table 2.2 (Continued)
S.No Author(s) Year Common
Name Botanical name Part Particle Morphology Size
18 Song et al 2009 Magnolia Magnolia kobus Leaf Gold(Au0)
Triangles,
pentagons,
hexagons and
spherical
5–300 nm
19 Raghunandan et
al 2009 Guava Psidium guajava Leaf Gold(Au0)
Spherical,
triangular and
hexagonal
27±3 nm
20 Krpetic et al 2009 Cape aloe Aloe ferox Leaf Gold(Au0)
Spherical and
triangular
6–35 nm,
4–45 & 50
nm
21 Kasthuri et al 2009 Phyllanthus Phyllanthus amarus Leaf Gold(Au0)
Hexagonal,
triangular, rod-
shaped and
spherical
18–38 nm
22 Kasthuri et al 2009 Henna Lawsonia inermis Leaf Gold(Au0)
Spherical and
triangular 7.5–65 nm
23 Ghodake et al 2010 Pear Pyrus species Fruit Gold(Au0)
Triangular and
hexagonal
3plates
200–500,
12–20 nm
24 Philip 2010 Mango tree Magnifera indica Leaf Gold(Au0) Spherical 17 nm
75
Table 2.2 (Continued)
S.No Author(s) Year Common
Name Botanical name Part Particle Morphology Size
25 Anuj et al 2010 Sweet leaf Stevia rebaudiana Leaf Gold(Au0) Octahedral 8–20 nm
26 Castro et al 2010 Sugar beet Beta vulgaris Pulp Gold(Au0)
Nanorods,
triangular and
Spherical
25 nm, 20
nm, 165
nm
27 Subramanian and
Muthukumaran 2010 Velvet bean Mucuna pruriens Seed Gold(Au
0) Spherical 6–17.7 nm
28 Ankamwar 2010 Almond Terminalia catappa Leaf Gold(Au0) Spherical 21.9 nm
29 Singh et al 2010 Clove Szyygium
aromaticum
Flower &
Bud Gold(Au0)
Triangular and
polygonal
100–300
nm
30 Dubey et al 2010 Common
Tansy Tanacetum vulgare Fruit Gold(Au0)
Spherical and
triangular 11 nm
31 Philip 2010 Hibiscus Hibiscus rosa
sinensis Leaf Gold(Au
0)
Spherical,
triangular and
hexagonal
14 nm
32 Narayanan and
Sakthivel 2010
Indian
Borage Coleus amboinicus Leaf Gold(Au
0)
Spherical,
truncated
triangle and
hexagonal
4.6–55.1
nm
76
Table 2.2 (Continued)
S.No Author(s) Year Common
Name Botanical name Part Particle Morphology Size
33 Noruzi et al 2011 Rose Rosa hybrida Petals Gold(Au0)
Spherical,
triangular and
hexagonal
10 nm
34 Montes et al 2011 Alfalfa Medicago sativa Biomass Gold(Au0)
Decahedral,
icosahedral and
Nanoplates
30–60 nm
35 Raju et al 2011 Markingnut
Tree
Semecarpus
anacardium Leaf Gold(Au
0)
Triangle and
hexagonal 13–55 nm
36 Sheny et al 2011 Cashew Anacardium
occidentale Leaf Gold(Au
0) Spherical 6.5, 17 nm
37 Samiran Mondal
et al 2011 Mahogany
Swietenia
mahogany Leaf Gold(Au
0)
Spheroidal,
triangles and
hexagonals
50 nm
38 Philip et al 2011 Curry tree Murraya koenigii Leaf Gold(Au0) Spherical 20 nm
39 Philip and Unni 2011 Krishna tulsi Ocimum sanctum Leaf Gold(Au0) Hexagonal 30 nm
40 Mohanan and
Soundarapandian 2013 Citrus Citrus sinensis Fruit Gold(Au
0)
Spherical,
triangular and
hexagonal
56.7 nm
77
Table 2.2 (Continued)
S.No Author(s) Year Common
Name Botanical name Part Particle Morphology Size
41 Mohanan and
Soundarapandian 2013 Citrus
Citrus reticulate
Fruit Gold(Au
0)
Spherical,
triangular and
hexagonal
43.4 nm
42 Mohanan and
Soundarapandian 2013 Citrus Citrus limon Fruit Gold(Au
0)
Spherical,
triangular and
hexagonal
32.2 nm
43 Ramamurthy et al 2013 Hativekuri Solanum torvum Fruit Gold(Au0)
Spherical,
triangular and
hexagonal
5-50 nm