CHAPTER 2 LITERATURE REVIEW - Shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/25097/7/07... ·...

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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

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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

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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).


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.


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).


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


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


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)


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


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



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



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



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


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



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



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



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


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


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


graveolens Root Gold(Au

0)

Spherical and

triangle

11.4–34

nm


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)




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




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




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




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




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

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