59

RESULTS AND DISCUSSION

Collection and screening of plant material for potent gold and silver nanoparticle

synthesis

Collection of Plants and Extract preparation

The healthy leaves of Cassia auriculata, Cassia tora, Cassia occidentalis, Cassia

sophera, Cassia fistula were collected from the botanical garden Gulbarga University,

Gulbarga. The leaves were gently washed with soap solution and bavistine to remove the

dust and any other contaminants, shade dried at room temperature. Dried leaves were

powdered and 5% of aqueous extracts were prepared by boiling the leaf powder in

distilled water for 5-10minutes, centrifuged and supernatants were used as reducing

agent.

Screening and Biosynthesis of AuNPs and AgNPs

Initially, the color of the reaction mixture confirmed the reduction AuNPs and AgNPs

obtained by adding the leaf extracts into 1mM chloroauric chloride and silver nitrate

aqueous solution (Plate-2, Fig.1a & b), various colors of the gold nanoparticles and

brown color of silver nanparticles were observed. Extracellular synthesis of AgNPs and

AuNPs was observed in three plants i.e, Cassia auriculata, Cassia fistula and Cassia tora

whereas intracellular synthesis was observed in Cassia occidentalis and Cassia sophera.

In extracellular gold nanoparticle synthesis the color varies depending on the size and

shape of the AuNPs, smaller particles appears pink while large particles appear purple.

Intracellular synthesis of AgNPs and AuNPs is not yet fully understood. Intracellular

60

synthesis always takes longer reaction times and also demands subsequent extraction and

recovery steps. Hence we have concentrated on extracellular synthesized nanoparticles

for further characterization and bioassay. Senapati et al. (2012) reported the intracellular

production of gold nanoparticles using an alga Tetraselmis kochinensis. Rai & Durn

(2011) stated the mechanism for intra and extracellular synthesis of nanoparticles differs

in various biological agents. Basavaraja et al. (2008) reported about the significant brown

color of extracellular synthesized silver nanoparticles. The increase in color intensity with

incubation time revealed enhanced nanoparticles synthesis. The size of the nanoparticles

depends on color of the colloidal solution (Jain et al. 2006). Vahabi et al. (2011)

explained the color intensity, indicates the reduction of ions and formation of

nanoparticles due to excitation of surface plasmon resonance of the metallic

nanoparticles. (Table.2) shows the UV-Visible spectra of the synthesized AuNPs and

AgNPs, time taken for reduction size and color of the nanoparticles.

Cassia Plant

Time for reduction

of Nanoparticles

Wavelength /

Particle Size (nm)

Gold Silver Gold Silver

1. Cassia auriculata (E)

2. Cassia fistula (E)

3. Cassia occidentalis(I)

4. Cassia sophera(I)

5. Cassia tora(E)

8 min

25 min

4 hours

24 hours

24 hours

3 min

15 min

1hours

24hours

24hours

535

556

545

-

-

14

29

23

-

-

427

441

432

-

-

17

35

21

-

-

Table.2. Time of reduction, wavelength and size of AuNPs and AgNPs of Cassia species.

(E- Extracellular, I- Intracellular synthesis).

61

Hence both extra and intracellular synthesis of AuNPs and AgNPs were observed in

screening and based on the results obtained we decided to carry work on Cassia

auriculata.

Phytochemical Analysis

The results of preliminary phytochemical analysis of aqueous leaf extracts revealed

relative distribution of the secondary metabolites responsible for the reduction of

AuNPs and AgNPs. Phytochemical analysis reveals presence of phenols, flavoniods,

terpenoids, tannins, cardiac glycosides, sterols and saponins (Table.3).

Medicinally valuable angiosperms have the greatest potential for the synthesis of metallic

nanoparticles with respect to quality and quantity (Song and Kim 2009). Medicinal plants

possess important phytochemicals and active compounds which are medicinally valuable

and exhibit significant properties in the treatment of various diseases. These

phytochemicals are not only important in medicines, but also responsible for the

reduction and stabilization of metal nanoparticles. Shukla et al. (2008) Li et al. (2007),

Jacob et al. (2008) reported that the phytochemicals such as water soluble proteins,

polyphenols, and sugars present in plant extracts plays a significant role in reducing and

stabilizing the NPs. Ankamwar et al. (2005b) reported the antibacterial and antioxidant

properties of biomolecules present in the plant extract have facilitated excellent stability

of the nanoparticles. Most of these phytochemicals are water soluble and can be easily

extracted in water without losing their properties. Such phytochemicals act as metal

reducing and capping agents, there by resulting in one step biosynthesis of metal

nanoparticles.

62

The main water soluble phytochemicals are flavones, organic acids, proteins and

quinones responsible for immediate reduction. According to Hemen Dave et al. (2012),

the genus Cassia have important source of naturally occurring bioactive quinones and

secondary metabolites which might have helped in rapid reduction of nanoparticles.

Therefore compared to bacteria and fungi, plants are better candidates for the rapid

synthesis of nanoparticles. Polarity based fractionation leads to the separation of only

Table.3. Phytochemicals screening of Cassia species. CA:Cassia auriculata, CF: Cassia

fistula, CT: Cassia tora, CS: Cassia sophera, CO: Cassia occidentalis

63

water soluble bioactive components. However Shankar et al. (2004), Parashar et al.

(2009), Elavazhagan et al. (2011) described various extraction procedures for plant

extract preparation from green and dry leaves (upon boiling). Philip et al. (2011) reported

that saponin present in aqueous extract was responsible for the mass production of silver

and gold nanoparticles. Jha et al. (2009) assumed the formation of pure metallic

nanoparticles and bimetallic nanoparticles by reduction of the metal ions was possibly

facilitated by reducing sugars or terpenoids. Jiang et al. (2006) reported that

phytochemicals were directly involved in the reduction of ions and formation of silver

nanoparticles. However Xie et al. (2007) assumed that these polyphenolic compounds,

quinones and other coordinating phytochemicals present in the leaf extract aided in

stabilization of AuNPs. Proteins, polysaccharide and phytochemicals play a role in the

synthesis of AgNPs and AuNPs (Park et al. 2011, Rai et al. 2009).

Characterization of Nanoparticles

UV-Vis Spectroscopy

The formation of various nanoparticles from their different salts gives characteristic

peaks at different absorptions due to surface plasmon resonance. As the plant extract was

mixed in the aqueous solution of ion complex, the color started changing from ruby red

and brown due to reduction of ions and excitation of surface plasmon vibrations, which

indicate the formation of nanoparticles.

64

Fourier transform infrared (FTIR)

Fourier transform infrared (FTIR) spectroscopy is a chemical analytical technique. It is

used to determine the nature of associated molecules of plants or their extracts with

nanoparticles. Based upon the wavenumber, infrared light can be categorized as far

infrared (4-400 cm−1

), mid infrared (400-4000cm-1

) and near infrared (4000-14000cm-1

).

Transmission electron microscopy

It is mainly used for size and morphological studies of nanoparticles.

X-ray diffraction (XRD)

This technique is used to establish the metallic nature of particles. X-rays are

electromagnetic radiation with typical photon energies in the range of 100eV-100keV,

short-wavelength X-rays (hard X-rays) in the range of a few angstroms to 0.1 Å (1-120

keV) are used. The formation of nanoparticles synthesized was further supported by X-

ray diffraction (XRD) measurements. The Bragg reflections corresponding to the (111),

(200), (220), (310) sets of lattice planes were observed that may be indexed on the basis

of the FCC structure of gold and silver NPs. All the peaks match well with the standard

JCPDS file 04-0783. The XRD pattern clearly shows nature of nanoparticles.

Thermogravimetric analysis (TGA)

Thermogravimetric analysis (TGA) is a thermodynamic technique measures the change

of mass as a function of temperature in a preselected environment. Data obtained from

this technique are usually expressed in percentage weight loss versus temperature, and

65

this is quite useful in evaluating the thermal stability and reaction rates at various

atmospheric conditions. It is also a good method for establishing purity of materials.

Effect of Leaf extracts concentration on formation of Gold Nanoparticles

Dried leaves were powdered and aqueous extract of Cassia auriculata was prepared with

different concentrations (1%, 5%, and 10%) by boiling the powder in double distilled

water, filtered and supernatant was used as reducing agent and 1mM tetracholoroauric

acid (HAuCl4) was kept constant. The procedures were performed at room temperature.

As the concentration of leaf extract increased there was an increase in particle size in

AuNPs. 1% leaf extract mediated synthesis resulted in ruby red color of the solution due

to surface plasmon resonance which confirms the formation of nanoparticles. UV-Vis

spectra reveals absorbance at 540nm, crystallite size of nanoparticle calculated by XRD

was 10nm and TEM image shows particles were spherical and polydispersed (Plate-3,

Figure.2). Reduction time for the synthesis was 1minute in all the three solution. 3% of

leaf extract mediated synthesis resulted in blue color of the solution which confirms the

formation of nanoparticles. UV-Vis spectra reveals absorbance at 534nm, crystallite size

of nanoparticle was 12nm and TEM image shows particles were spherical and

polydispersed (Plate-4, Figure.3). 10% of leaf extract mediated synthesis resulted in blue

color of the solution which confirms the formation of nanoparticles. UV-Vis spectra

reveals absorbance at 530nm, crystallite size of nanoparticle was 15nm and TEM image

shows particles were spherical and polydispersed (Plate-5, Figure.4). The concentrations

of leaf extract have affected the particles size but have no effect on shape of the

nanoparticles.

66

Effect of HAuCl4 concentration on Synthesis of Gold Nanoparticles

It has been reported that the nanoparticles size and shape mainly depends on different

factors like concentration of leaf extract, salt solution, PH and temperature (Armendariz

et al. 2004, Rai et al. 2006). Three concentrations (0.5mM, 1mM and 2mM) of HAuCl4

were used by keeping the leaf extract concentration 1% as constant. It was observed that

there was not much difference on particles size and morphology. 1% of leaf extract and

0.5mM Tetracholoroauric acid (HAuCl4) mediated synthesis resulted in blue color of the

solution which confirms the formation of nanoparticles. UV-Vis spectra reveals

absorbance at 530nm, crystallite size of nanoparticle by XRD calculation was 14nm and

TEM image shows particles were spherical and polydispersed (Plate-6, Figure.5). 1% of

leaf extract and 2mM tetracholoroauric acid (HAuCl4) mediated synthesis resulted in

ruby red color which confirms the formation of nanoparticles. UV-Vis spectra reveals

absorbance at 535nm, crystallite size of nanoparticle by XRD calculation was 20nm and

TEM image shows particles were spherical and polydispersed (Plate-7, Figure.6).

Synthesis and Characterization of Silver Nanoparticles

Effects of Leaf extract concentration on Synthesis of Silver Nanoparticles

For the synthesis of silver nanoparticles the three different concentrations (1%, 5%, and

10%) of leaf extracts of Cassia auriculata were prepared by boiling the powder in double

distilled water, filtered and supernatant was used as reducing agent. The concentration of

Silver nitrate (AgNO3) was kept constant (1mM). As the concentration of leaf extract

increased there was decrease in particle size of AgNPs. 1% leaf extract mediated

synthesis resulted in brown color of the solution due to surface plasmon resonance which

67

confirms the formation of silver nanoparticles. UV-Vis spectra reveals absorbance at

435nm, crystallite size of nanoparticle was 35nm and TEM images shows particles are

spherical and polydispersed (Plate-8, Figure.7). Reduction time for the synthesis was 4-6

minutes in all the three solution. 3% leaf extract mediated synthesis resulted in brown

color of the solution which confirms the formation of nanoparticles. UV-Vis spectra

reveals absorbance at 430nm, crystallite size of nanoparticle by XRD calculation was

25nm and TEM images shows particles were spherical and polydispersed (Plate-9,

Figure.8). 5% leaf extract mediated synthesis resulted in dark brown color of the solution

which confirms the formation of nanoparticles. UV-Vis spectra reveals absorbance at

420nm, crystallite size of nanoparticle was 11nm and TEM image shows particles were

spherical and polydispersed (Plate-10, Figure.9). The concentrations of leaf extract have

affected the particles size but no difference was observed in morphology of the silver

nanoparticles. There was no marked difference in the shape at various biomaterial

dosages as reported by Huang et al. (2007).

Effect of AgNO3 concentration on synthesis of Silver Nanoparticles

Three concentrations (0.5mM, 1mM and 2mM) of AgNO3 were used by keeping the leaf

extract concentration 1% as constant. Varying the concentrations of AgNO3 did not show

much difference on morphology of silver nanoparticles.

1% of leaf extract and 0.5mM AgNO3 mediated synthesis resulted in brown color of the

solution which confirms the formation of nanoparticles. UV-Vis spectra reveals

absorbance at 450nm, crystallite size of nanoparticle by XRD calculation was 20nm and

TEM image shows particles were spherical and polydispersed (Plate-11, Figure.10). 1%

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of leaf extract and 2mM AgNO3 mediated synthesis resulted in ruby red color of the

solution which confirms the formation of nanoparticles. UV-Vis spectra reveals

absorbance at 440nm, crystallite size of nanoparticle by XRD calculation was 30nm and

TEM image shows particles were spherical and polydispersed (Plate-12, Figure.11).

Engineered AgNPs are typically stabilized against aggregation through adsorption or

covalent attachment of organic compounds prior to evaluating their environmental fate

and toxic effects. These molecules on the surface of the nanoparticles provide

electrostatic, steric, or electrosteric repulsive forces between particles, allowing them to

resist aggregation (Hotze et al. 2010). These molecules, if applied during synthesis, are

also often referred to as “capping agents” and allow the control of size and shape (Wiley

et al. 2005).

Rapid Phytosynthesis and Characterization of Gold and Silver Nanoparticles under

Microwave Irradiation

The biosynthesis of nanoparticles was performed under microwave oven using 1%

Cassia auriculata leaf extract and 1mM salt solution. Leaf extract was taken in a 250 mL

conical flask and immediately the whole mixture was put in a domestic microwave oven

(Samsung Model GW73BD). The mixture was subjected to several short burst of

microwave irradiation as described in Materials and Methods. The reduction of aqueous

AuCl4 ions during reaction with the Cassia auriculata leaf extract was followed by UV-

Vis spectroscopy. It was found that the colorless solution turned ruby red within 30

seconds of microwave irradiation, the UV-Vis absorption spectra recorded shows a strong

resonance at 524 nm and peaks arises due to the excitation of surface plasmon vibrations

in the gold nanoparticles, crystallite size of nanoparticle by XRD calculation was 15nm

69

and TEM images shows particles are spherical and polydispersed (Plate-13, Figure.12).

The ability of microwave irradiation to bring about reduction of Au+ to Au

0 within 30sec

indicates the superiority of this method. However in case of silver nanoparticles synthesis

the reduction of aqueous Ag ions during reaction with the Cassia auriculata leaf extract

may be easily followed by UV-Vis spectroscopy. The solution started changing the color

from yellowish to brown in 55 seconds due to reduction of silver ions, which indicates

formation of silver nanoparticles (Lopez et al. 2013). The UV-Vis absorption spectra

recorded shows a strong resonance at 440 nm due to the excitation of surface plasmon

vibrations in the silver nanoparticles, crystallite size of nanoparticles calculated by XRD

was 15nm and TEM images shows particles were spherical and polydispersed (Plate-14,

Figure.13). We reported fastest synthesis as compared to others who got in 90secs

(Deshpande et al. 2011) and 5minutes (Renugadevi et al. 2012). The template method

provides good control over the shape and dimensions of nanoparticles and is faster under

the microwave irradiation method. The microwave irradiation effectively utilizes the

internal heat generated within the matrix and is able to bring about the complete

reduction within short duration of time. The solution containing ions moves through the

solution under the influence of an electric field, resulting in expenditure of energy due to

an increased collision rate, converting the kinetic energy to heat.

70

Rapid Phytosynthesis and Characterization of Gold and Silver Nanoparticles under

Sunlight Irradiation

Sunlight-induced rapid synthesis of gold and silver nanoparticles was investigated. Leaf

extract (1%) Cassia auriculata was treated with 1mM tetracholoroauric acid and silver

nitrate in a separate conical flask and were irradiated to sunlight during the summer,

afternoon of a sunny day 45º C temperature was recorded. It was found that the colorless

solution turned ruby red within 55 seconds and reddish brown within 1minute of sunlight

exposure. The UV-Vis absorption spectra of gold nanoparticles recorded at 540nm,

crystallite size calculated by XRD was 10nm and TEM images shows particles were

spherical and polydispersed (Plate-15, Figure.14). The UV-Vis absorption spectra of

silver nanoparticles was recorded at 440nm, crystallite size was 20nm and TEM images

shows particles were spherical and polydispersed (Plate-16, Figure.15). The visible light

radiation significantly prompted the synthesis of silver NPs using a culture supernatant of

the Klebsiella pneumonia bacterium (Mokhtari et al. 2009). Ravishankar et al. (2011)

reported the AgNPs synthesis within 1minute from Pleurotus florida extract. Karimi

Zarchi et al. (2012) synthesized smaller sized biogenic silver NPs were fabricated under

direct sunlight radiation. The concentrations used and particles size obtained of

biosynthesized gold and silver nanoparticle during the course of work given in (Table.3).

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FTIR analysis

The FTIR spectrum indicates various functional groups present at different positions. IR

spectroscopy study has confirmed that the carbonyl group of amino acid residues and

peptides of proteins has a stronger ability to bind metal, so that the proteins could most

possibly form a coat over the metal nanoparticles (i.e., capping of AuNP and AgNP) to

prevent the agglomeration of the particles, and thus stabilized in the medium. The peaks

in the region between 3412 cm-1

to 2849 cm-1

were assigned to O-H stretching of alcohol

Table. 3. Particles size of Gold and Silver nanoparticles synthesized from Cassia

auriculata leaf extract

72

and phenol compounds and aldehyde –C-H- stretching of alkanes. The peaks in the

region 1597 cm-1

to 1508 cm-1

and 1385 cm-1

to 827 cm-1

corresponds to N-H(bond) of

primary and secondary amides and –C-N- stretching vibration of amines or –C-O-

stretching of alcohols, ethers, carboxylic acids and anhydrides (Plate-17, Figure.16). The

presence of important phytochemicals (Usha & Bopaiah 2012) and essential amino acids

(Gaikwad et al. 2010) in C. auriculata might have facilitated the synthesis and

stabilization of nanoparticles. The FTIR analysis reveals the dual function of biological

molecules, which might contribute to the reduction and stabilization of gold and silver

nanoparticles in the aqueous medium.

Identification of Protein Capped on Gold and Silver Nanoparticles

Protein polar groups interact with polar solvent and get dissolved. The proteins identified

in aqueous dry leaves extract are polar proteins. The estimated amount of proteins found

associated with the AuNPs (120µg/mg), AgNPs (144µg/mg) and plant extract

(190µg/mg). The SDS analysis revealed presence of two proteins of 15kD and 42kD.

(Plate-18, Figure.17) shows the bands of proteins present in plant extract and associated

with AuNPs and AgNPs. The observation specifies the presence and binding of proteins

with nanoparticles which lead to their possible stabilization and functionalization of NPs.

The presence of proteins was also confirmed by FTIR analysis of Cassia auriculata

(Udayasoorian et al. 2011). The peaks were assigned to stretching of N-H, O-H and C=O

of primary and secondary amides. These proteins could be responsible for the synthesis

as well as stability of nanoparticles. Hence water soluble phytochemicals and water

soluble proteins facilitated the reduction of nanoparticles and immediately proteins may

adsorbed on the surface of nanoparticles making the solution stable. Previous studies

73

show that the proteins get bound to surface of nanoparticles immediately upon contact

(Elechiguerra et al. 2005). We believed sooner the proteins gets adsorbed to the

nanoparticles lesser will be the particle size leading to stabilization. Kaur et al. (2009)

suggested impact of proteins on the shape of SeNPs. The proteins can bind to the gold

and silver nanoparticles either through free amine groups or cysteine residues (Gole et al.

2001). Maliszewska et al. (2013) demonstrated the stabilization of silver nanoparticles

occurs by electrostatic repulsion due to the negative charge of protein molecules, and

conformational changes occur when proteins adsorb onto nanoparticles (Fei et al. 2009).

Gaikwad et al. (2010) detected ten amino acids in the leaves of Cassia auriculata.

Among that D-Threonine, L-Ornithrine hydrochloride, Aspartic acid, L-Cystine and

Hydroxyproline were polar. The reported proteins have significant role in plant and

human as well which makes the NPs biocompatible. There might be possibility that these

polar proteins have assisted in reduction and stabilization of nanoparticles. (Plate-19,

Figure.18) shows the hypothesized mechanism of reduction and stabilization of NPs.

Peptides of proteins possesses strong ability to stick on metals due to adsorption and

forms coating over the nanoparticles (capping of AgNP/AuNP). This prevents

agglomeration of the particles in the medium. Adsorption of proteins at the nano-bio

interface is assisted by several forces such as hydrogen bonds, solvation forces, Van der

Waals interactions etc. The binding of amines on gold nanoparticles was reported in

Cinnamomum zeylanicum (Rai & Durn 2011). The proteins were active biomolecule

present in the plants responsible for the reduction of noble metal nanoparticles (Sheny et

al. 2011). Fayaz et al. (2011) reported involvement of tyrosine residue in the reduction of

gold ion from Maduca longifolia extract. Capping agents have become increasingly

74

prevalent in NP synthesis and are primarily used to stabilize the highly reactive NP

surfaces (Dylan et al. 2011). Organic molecules bound to the NP core impart stability

through electrostatic repulsion, steric stabilization or a combination of both mechanisms

(Lead et al. 2009). In addition to stabilizing properties, capping agents were also utilized

to alter surface chemistry of NPs for specific applications such as drug delivery, to

control size and shape during NP synthesis (Tolaymat et al. 2010, Murugadoss et al.

2010).

Immobilization study of AuNPs and AgNPs

In the present investigation we have developed a simple, inexpensive and well-suitable

network hydrogel system, effectively used for obtaining highly dispersed nanoparticles

by using Cassia auriculata leaf extract as reducing agent. The swollen hydrogel acts as

the nanoreactors that immobilize the nanoparticles, offering large free space between the

cross-linked networks for the nucleation and growth of the nanoparticles. Alginate has

potential to form edible films, exhibit poor water resistance due to hydrophilicity owing

to presence of carboxylic and hydroxyl groups. Alginate has been cross-linked with

divalent Ca2+

which can ionically interact to produce strong gels or insoluble polymers.

Preparation and Characterization of Au-BNC and Ag-BNC

The alginate hydrogel was prepared by gel casting technique. Pure alginate films were

transparent, however the gold containing film was pink, characteristic color of gold

nanoparticles (Peter 2011), and silver containing films were brown which is characteristic

of silver nanoparticles (Liz-Marzan 2004). The formation of the nanoparticles in the

hydrogel networks was indicated by the appearance of color (Plate- 20, Figure.19).

75

UV-Visible spectra of Bionanocomposite

Pure alginate did not show any absorbance in the visible region, whereas AuNPs and

AgNPs embedded film had an absorbance centered at 526nm and 424nm. The absorbance

was due to the reduction of ions and excitation of surface plasmon vibrations, which

indicate the formation nanoparticles (Kuila et al. 2007, Zhang et al. 2008) and confirms

the nanoparticle formation (Plate-20, Figure.20). The gel had diverse surface charges and

sizes of the AgNPs inside which caused expansion of the hydrogel networks (Murali

Mohan et al. 2006). The immobilization of nanoparticles has strong localization of

particles within the gel networks. Therefore, plant extract can stimulate the nanoparticles

stabilization to a greater extent when compared to conventional hydrogel systems where

chemicals methods are used for immobilization of metal nanoparticles on alginate films

(Anubha et al. 2009, Sandip et al. 2010, Vincent et al. 2010).

FT-IR analysis

FT-IR spectroscopy confirmed chemical structure and potential interactions between

alginate and nanoparticles. The peak at 3268cm-1

represents –OH stretching frequency

due to hydrogen bonding, while in the region 1590-1000cm-1

represents C=O stretching

of carboxylic acid, ethers, esters or N–H bending (amide II) present due to plant extract.

The band at 1026cm-1

related to O-glycosidic bonding between β-D-mannuronic and α-L-

guluronic acid residues and indicates the degree of stability of the linear chain in the

alginate (Torres et al. 2005). The peaks near 500cm-1

region represents the formation of

metallic bonding formed due to AuNPs and AgNPs (Plate-21, Figure.21).

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TEM images of Gold and Silver Bionanocomposites

TEM images of Au-BNC and Ag-BNC shows the particles were uniform, spherical and

discrete with particle size varies between 5-15nm (Plate-22, Figure.22). Most of the

nanoparticles were separated from each other due to protection by alginate which

indicates good stabilization.

Measurement of dry films width

The width of AuNPs and AgNPs embedded dry films were measured by using

micrometer and found 0.8µm of gold film and 1.3µm of silver film.

Fresh and Dry weight/ Moisture content

The fresh weight of AgNPs containing film was about 15.66mg and AuNPs film was

15.33mg, similarly dry weight Ag-BNC was 3.33mg and Au-BNC was 2.77mg. The

weight loss observed in Au-BNC was 79.66% and Ag-BNC was 88.57%.

Swelling and degrading studies

The swelling behavior of an antibacterial film is an important aspect of its successful

biomedical application. The invitro degradability of the composite was performed by

soaking the dry hydrogels in phosphate buffer (pH-7.0) commonly used in biological

research and distilled water. The dry films soaked in double distilled water have shown

slight swelling after 2days and no signs of degradation were observed even after a month.

The dry films started swelling in 2 minutes and completely degraded within 6 minutes in

phosphate buffer. The osmolarity and ion concentrations of the buffer matches those of

the human body, hence these composites can be exploited in drug delivery applications.

The films obtain a high swelling degree in a short period of time, causing their

dissolution due to ionic changes between the calcium ions present in the hydrogel

77

structure present in the medium. The ionic strength of the medium influences the swelling

capacity of ionic gels (Ravindra et al. 2012). Therefore, the use of biopolymer,

biodegradable and biocompatible cross-linked hydrogel templates are more convenient

for developing metal nanoparticles than conventional non-aqueous systems of non

biocompatible polymeric systems for biomedical applications (Brandon et al. 2009,

Armentano et al. 2010).

Thermal Studies

The thermal analysis (TGA) graphs (Plate-22, Figure.23) of Au-BNC and Ag-BNC film

shows the weight loss took place in three steps. In the first step 10.33%-15.89% weight

losses was observed between 100-180°C due to absorbed moisture of hydroxyl molecules

present in the biopolymer. The second major weight loss of 37.72% was observed from

200-450°C due to organic moieties present around the noble metal nanoparticles with the

presence of alginic polymer network. The last stage where 5-23% weight loss occurred

from 500-870°C due to decomposition of Ca+. The remaining 30-40 % residue is AuNPs

and AgNPs respectively. Hence the multistep gravimetric study indicates good thermal

stability of Au-BNC and Ag-BNC films as compared to reported earlier (Murthy et al.

2008, Yonghyun Kim et al. 2011, Ravindra et al. 2012, Madhusudana Rao et al. 2013).

Application Studies

Antimicrobial assay

From the onset of nanotechnology the antimicrobial effect of silver nanoparticles has

regained its lost status and thus provides a faster wound healing along with its efficacy

against microbial resistance to antibiotics.

78

Antimicrobial assay of biosynthesized silver nanoparticles was examined against gram

negative bacteria E. coli and gram positive B. subtilis, fungal strains like A. niger and A.

flavus. The AgNPs shows significant inhibitory activity against bacteria and fungi.

AgNPs exerted highest toxicity against A. niger and intermediary effects on E. coli,

B. subtilis and A. flavus (Plate-23, Figure. 24 & 25). AgNPs synthesized at 5%

concentration were found to be significantly toxic to the fungi whereas standard antibiotic

Clotrimazole did not show any zone of inhibition at 50µg/ml on A.niger (Plate-24,

Figure.26 & 27). AgNPs synthesized at 3 % concentration were found better on bacteria

tested followed by 1%. The maximum toxicity observed in AgNPs synthesized from 3%

and 5% of leaf extracts. The zone of inhibition in figures and graphical presentation given

in plates mentioned above. The reason could be that the smaller size of the particles leads

to increased membrane permeability and cell destruction. Our results are in agreement

with Boswellia ovalifoliata (Savithramma et al. 2011).

The toxic effects of silver on bacteria have been investigated for more than 60 years

(Franke et al. 2001). Several mechanisms have been proposed to explain the inhibitory

effect of the AgNPs on bacteria. The exact mechanism behind the activity of nanosilver

on bacteria not yet fully elucidated, the three most common mechanisms of toxicity

proposed up to now are: uptake of free silver ions followed by disruption of ATP

production and DNA replication (Lok et al. 2006), formation of reactive oxygen species

(ROS) (Park et al. 2009) and direct damage to cell membranes (Raffi et al. 2008). It was

also suggested that silver ions released by AgNPs can interact with phosphorus moieties

in DNA, resulting in inactivation of DNA replication or can react with s-containing

proteins leading to the inhibition of enzymatic functions. AgNPs are highly reactive

79

because they generate Ag+ ions, whereas metallic silver is relatively unreactive (Morones

et al. 2005). Moreover, silver ions are known to produce reactive oxygen species (ROS)

that are detrimental to cells, causing damage to proteins, lipids, and nucleic acids (Hwang

et al. 2008). Ag+

may displace native metal cations from their usual binding sites in

enzymes (Ghandour et al.1988). Furthermore, silver ions inhibit oxidation of glucose,

glycerol, fumarate, and succinate in E. coli (Ahearn et al. 1995). This indicates that

AgNPs may target the bacterial membrane, leading to a dissipation of the proton motive

force (Lok et al. 2006). Consequently, the nanoparticles preferably attack the respiratory

chain, cell division finally leading to cell death (Rai et al. 2009). Holt and Bard 2005

found that Ag+ inhibited respiration of E. coli by determining change of oxygen dissolved

in culture resolution (Holt and Bard 2005). Besides, Kim et al. 2008 found that Ag+

interacted with thiol (–SH) group of cysteine by replacing the hydrogen atom to form

–S-Ag, thus hindering the enzymatic function of affected protein to inhibit growth of

E. coli.

According to Ashkarran et al. (2012) species sensitivity also depends on the structure

of the cell wall composition in Gram-positive and Gram-negative bacteria. Several

additional factors influence the susceptibility or tolerance of bacteria to NPs. The

antibacterial effect of AgNPs found higher than CuNPs against E. coli (–) and S. aureus

(+) bacteria (Lu et al. 2009). The shape and size of nanoparticles have differential effect

on microorganisms. Tavares et al. (2009) speculated shape-dependent interaction of

AgNPs having different shapes with different effective surface areas in gram-negative

bacterium E. coli. Choi et al. (2008) observed that spherical and hexagonal AgNPs were

adsorbed onto the bacterial cell surface, causing cell surface depression. Shrivastava et al.

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(2011) studied the dose dependent effect of silver nanoparticles (in the size range of 10-

15 nm) on the Gram-negative and Gram-positive microorganisms. Bard et al. (2005) have

reported micromolar levels of Ag+ ions to uncouple respiratory electron transport from

oxidative phosphorylation, inhibit respiratory chain enzymes, or interfere with the

membrane permeability to protons and phosphate. In addition, higher concentrations of

Ag+ ions have shown to interact with cytoplasmic components and nucleic acids (Dibrov

et al. 2002).

Sotiriou and Pratsinis (2010) proposed that the antibacterial activity of AgNPs depends

on size. They provided evidence that smaller size of AgNPs releases many Ag+ ions and

these ions dominate antibacterial activity. Patil et al. (2012) have investigated

antibacterial property of AgNPs against S. aureus, P. aeruginosa and E. coli. The plant

extract reduced AgNPs shows good fungicidal effect on A. flavus, A. niger and A.

fumigates (Singh et al. 2010). The fungistatic or fungicidal effect of AgNPs were due to

the inhibitory action of plant metabolite reduced silver and the mechanism involved was

cytoplasm granulation, cytoplasmic membrane rupture and inactivation or inhibition of

intracellular and extracellular enzymes. These biological events could take place

separately or culminating with mycelium germination inhibition (Cowan 1999).

The bactericidal activity of nanoparticles depends on the stability in the cultured medium

too. Nowadays the main problem is bacteria have developed resistance towards many

antibacterial agents. Hence there is increase in use of AgNPs in various fields against

microorganisms, so there is need to prepare the AgNPs with cost effective methods and

to find out the mechanism of antimicrobial activity. There are alarming reports of

opportunistic fungal infections. The infections caused by opportunistic fungi were

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included under new spectrum of fungal pathogens. The results suggested that AgNPs

have exerted antifungal activity by disrupting the structure of cell membrane and

inhibiting the normal budding process due to the destruction of membrane integrity. The

present study indicates AgNPs have considerable antimicrobial activity.

Effect of Gold nanoparticles on seed germination

The impact of biologically synthesized AuNPs investigated on seed germination and

seedling growth in Pennisetum glaucum. AuNPs did not affect the seed living process but

have enhanced the seed germination and seedling growth compared to the control. Seed

germination enhancement have been speculated as dispersed nanoparticles can penetrate

through the seed coat and create “nano-holes” on seed coats, resulting in improved

germination conditions, slow and minimal release of Au ions could be one of the reason

for AuNPs to have no major effect on germination of Pennisetum glaucum seeds. The

highest percentage of seed germination was 86.66% and increased seedling length at

50µg/ml of gold nanoparticles followed by 20µg/ml. The seed germination and seedling

growth in this study was positively altered by nanoparticles. Thus, nano treated seeds can

be used to lower the environmental impacts of fungicides/pesticides and reduce the cost

of agricultural production. The nanoparticles after penetrating the seed coats, could

contact radicals directly. The root elongation of sensitive plant species would have a dose

dependent response. (Plate-25, Figure. 28 & 29) shows the effect of AuNPs on seed

germination, root length and shoot length of Pennisetum glaucum. Hence higher

concentration of AuNPs used in the study increased the germination percent with root,

shoot and total seedling length. This biological response of seed germination and

elongation could be highly dependent on both plant type and nanoparticle properties.

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These results could be important for both areas of plant biology and nanotechnology. The

strategy of using biologically synthesized AuNPs to agricultural crop plants reduces the

cost of production and the environmental impact. The accumulated gold nanoparticles

move through the vasculature of the plant (Sabo et al. 2011). Compared to plant cell

walls and membranes the penetration of nanoparticles into seeds is expected to be

difficult due to thick seed coat (Srinivasan and Saraswathi 2010). The bioaccumulation,

biomagnification and biotransformation of engineered nanoparticles in food crops are still

not well understood (Rico et al. 2010). It has been suggested that metal transport proteins

may be involved in transporting gold across membranes (Bali et al. 2010) and Gardea-

Torresdey et al. (2002) have hypothesized that the NPs transport was active and the

translocation through the plant occurs through the water transport pathway (Sharma et al.

2007). The most logical hypothesized mechanism that metal transporters used for

essential metals are able to transport gold without having evolved to do so. The

nanoparticle effectively penetrates seed coat and influence the seed germination and plant

growth (Khodakovskaya et al. 2009). The reason could be that the NPs can penetrate

through seed coat and may be activate the embryo which leads to fastest germination. On

the contrary when nanoparticles were applied on leaf surfaces, entered through the

stomatal openings or through the bases of trichomes and then translocated to various

tissues (Uzu et al. 2010). Lin and Xing (2007) reported the positive effects of suspensions

of MWCNTs on seed germination and root growth of six different crop species radish

(Raphanus sativus), rape (Brassica napus), rye grass (Lolium perenne), lettuce (Lactuca

sativa), corn (Zea mays) and cucumber (Cucumis sativus). Nano-TiO2 (anatase) improved

plant growth by enhanced nitrogen metabolism (Yang et al. 2006) that promotes the

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absorption of nitrate in spinach. Carbon nanotubes (CNTs) were found to penetrate

tomato seeds and affect their germination, growth rates and water uptake inside seeds

(Khodakovskaya et al. 2009). Our results are in agreement that the biological synthesized

AuNPs enhanced the germination and seedling growth of biofuel producing crop. The

AgNPs extended the vase life of gerbera flowers by inhibiting microbial growth and

reduced vascular blockage which results the increased water uptake and maintained the

turgidity (Liu et al.2009). The biological synthesized AgNPs enhanced the germination in

Pearl millet but reduced the growth of seedlings (Asra and Srinath 2014). With concerns

of toxicity, major attention has been driven to their uptake, translocation and

biotransformation in the plant systems. Although too much of anything is toxic,

observations have been made on the beneficial aspects of various nanomaterials and

enhanced water and nutrient uptake, gaseous exchange, and delivery of crucial chemicals

to the plants. Chilopsis linearis root growth was inhibited in the presence of gold above

0.8 mM (Rodriguez et al. 2007). Starnes et al. (2010) reported gold ions inhibited alfalfa

growth in liquid culture above 250µM and inhibited growth of Arabidopsis thaliana on

solid medium above 100µM gold. The toxicity of gold NPs in plants is concentration

dependent. From this it can be hypothesized that the least concentrations found worthy to

the plants whereas higher doses may affect the growth of the plants. Guadalupe de la

Rosa et al. (2009) investigated that up to 160mg/L, AuNPs did not reduce corn seed

germination or plant growth. The interaction of plant cell with the nanoparticles results in

modification of plant gene expression and associated biological pathways which

ultimately affect plant growth and development. The effect of NPs investigated in various

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studies was dependent upon the type of plants used, nanoparticles size, shape and charge

also the particular environmental conditions.

Nano-encapsulated agrochemicals should be designed in such a way that they possess all

necessary properties such as effective concentration (with high solubility, stability and

effectiveness), time controlled release in response to certain stimuli, enhanced targeted

activity and less ecotoxicity with safe and easy mode of delivery thus avoiding repeated

application (Boehm et al. 2003). The study shows positive effect of green nanogold on

biofuel plant. Apart from the good growth in certain environmental conditions biofuel

crops get infected by various fungal, bacterial, viral diseases and nematodes, parasites.

The control of parasitic weeds with nanocapsulated herbicides could reduce the

phytotoxicity of herbicides on crops (Perea and Rubiales 2009). Surface modified

hydrophobic nanosilica has been successfully used to control a range of agricultural

insect pests (Barik et al. 2008). The use of such nanobiopesticide will be more acceptable

since they could be safe for plants and cause less environmental pollution in comparison

to conventional chemical pesticides. Hence nanoparticulate formulations can be used as

novel agrochemicals with high specificity and improved functions.

Effect of Silver nanoparticles on seed germination

The biosynthesized AgNPs were used to study its influence on seed germination, shoot

length, root length in Pennisetum glaucum. Silver nanoparticles did not adversely affect

the seed living process. Infact AgNPs have enhanced the seed germination compared to

the control. As mentioned above seed germination enhancement could be due to

dispersed nanoparticles can penetrate through the seed coat and create “nano-holes” on

seed coats, resulting in improved germination conditions, slow and minimal release of

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Ag+ ions could be one of the reasons for AgNPs to have no major effect on germination

of Pennisetum glaucum seeds. The highest percentage of seed germination was 93.33% at

50mg/L of silver nanoparticles. Since results revealed treating seeds with silver

nanoparticles did not reduce germination, so possible to use this treatment in agricultural

practices. This may explain that seed germination in this study was not greatly altered by

nanoparticles. Silver nanoparticles can protect seeds against fungi as well as the

conventional fungicide. Results of seed protection test indicate that silver nanoparticles

may be an alternative to conventional fungicides for protecting seeds against fungi. Thus,

nano treated seeds can be used to lower the environmental impacts of fungicides and

reduce the cost of agricultural production. The silver nanoparticles at 50µg/ml decreased

the root length and shoot length followed by 20µg/ml. Nanoparticles adhere to plant roots

and exert physical or chemical toxicity in plants. Radicals, after penetrating the seed

coats, could contact the nanoparticles directly. The root elongation of sensitive plant

species would have a dose-dependent response. Sresty and Rao (1999) predicted that

roots are the first target tissue to confront with excess concentrations of pollutants. Toxic

symptoms seem to appear more in roots rather than in shoots. The nanoparticles

presumably cross the epidermis and the root cortex by the apoplastic route and then

endodermis via protoplasts, to reach the central part of the roots and transported to

shoots. The impact of AgNPs can be evaluated in three levels; physiological

phytotoxicity, cellular accumulation and subcellular transport of AgNPs. AgNPs

accumulated progressively in the sequence: border cells, root cap, columella and

columella initials. AgNPs were apoplastically transported in the cell wall and found

aggregated at plasmodesmata (Jane et al. 2013). Breakage of cell wall and vacuoles in

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root cells of test species described the toxicity of particles as the pore size of the cell

walls smaller than the nanoparticles which can be taken up and translocated through

stems to leaves. Hence AgNPs could penetrate the plant system and may interfere with

intracellular components, causing damage to cell division. It was investigated that AgNPs

impaired the stages of cell division and caused cell disintegration (Kumari et al. 2009).

The effect of silver nanoparticles on seed germination, root length and shoot length of

Pennisetum glaucum is given in (Plate-26, Figure.30 & 31). Higher the concentration of

AgNPs decreased the root, shoot and total seedling length. It was observed that higher

concentration of nanoparticles had adverse effect on plant species. Inhibition of seed

germination and root elongation found to be highly dependent on both plant type and

nanoparticle properties.

Nanotoxicology is the study of the toxicity of nanomaterials. Because of the size effects

and large surface area, nanomaterials have unique properties compared with their larger

counterparts (Cristina et al. 2007). Seed germination and root elongation is a rapid and

widely used acute phytotoxicity test with several advantages: sensitivity, simplicity, low

cost and suitability for unstable chemicals or samples (Munzuroglu and Geckil 2002).

However mechanism of nanotoxicity remains unknown it would depend on the chemical

composition, chemical structure, particle size and surface area of the nanoparticles.

Toxicity of nanoparticles may be attributed to two different actions (1) a chemical

toxicity based on the chemical composition e.g. release of toxic ions and (2) stress or

stimuli caused by the surface, size and shape of the particles (Brunner et al. 2006).

Smaller NPs would have higher surface energy and thus proved more toxic to the cell

(Krug and Wick 2011). In terms of metallic nanoparticles (MNPs), copper nanoparticles

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have shown to be toxic on two crop species, mung bean (Phaseolus radiatus) and wheat

(Triticum aestivum) as demonstrated by the reduced seedling growth rate (Lee et al.

2008). Kumari et al. (2009) studied AgNPs could disrupt cell division process causing

chromatin Bridge, stickiness and cell disintegration in Allium cepa. Another study on root

cells of Allium cepa reported that ZnO NPs exert cytotoxic and genotoxic effects,

including lipid peroxidation, decreasing of the mitotic index and increasing the

micronuclei and chromosomal aberration indexes (Kumari et al. 2011). According to

Raskar and Laware (2014) the lower concentration of ZnO NPs was not harmful to the

cell division and early seedlings growth in onion. Up to this point, most evidence on

phytotoxicity appeared to support the hypothesis that surface area as most important

factor on the phytotoxicity of nanoparticles to plants at both seedling and cellular levels.

Seed germination in zucchini was unaffected by bulk or nano Ag, Cu, Si, MWCNTs or

ZnO even at 1000 mg/L (Stampoulis et al. 2009). The silver nanosolution coated seeds

recorded better germination than the seeds treated with fungicide and concluded to use

silver nano coating instead of using fungicides (Karimi et al. 2012). Silver NPs even at

the highest dose employed (4000 ppm) did not disturb the germination of the seedlings or

the growth of Ricinus communis (Jyothsna and Pathipati 2013). Yang and Watts (2005)

reported Nano-TiO2 promoted photosynthesis and nitrogen metabolism and improved

growth of spinach. TiO2 NPs via the electrospray promoted the germination of lettuce

(Lactuca sativa) seeds (Stephen et al. 2014) and wheat (Mahmoodzadeh and Aghili

2014). Similarly nano SiO2 significantly enhanced the seed germination in tomato

(Siddiqui and Al-Whaibi 2014). The multiwall carbon nanotubes penetrated tomato seeds

and increased the germination rate up to 90% (compared to 71% in control) by rising

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water uptake, also increased plant biomass (Khodakovskaya et al. 2009). Our results are

in agreement that biologically synthesized silver NPs enhanced the percentage of seed

germination in Pennisetum glaucum. Majumdar and Ahmed (2011) reported significant

inhibition in rate of seed germination, root and shoot growth by silver nanoparticles when

compared with silver ions in Oryza sativa, Brassica campestris and Vigna radiata. The

silver ions inhibited barley seed germination (El-Temsah and Joner 2012). Reports on

various size of AgNPs showed that Ag colloid (0.6±2nm) had greater toxicity in flax,

barley and rye grass than AgNPs of 5 and 20nm (Locke et al. 2000). Engineered

nanoparticles adhere to plant roots and exert physical or chemical toxicity in plants.

Lin et al. (2009) reported the root growth inhibition by using different nanoparticles on

different plant species. AgNPs also inhibited the growth of Lemna minor (Gubbins et al.

2011). AgNPs significantly decreased plant biomass, plant tissue nitrate-nitrogen content,

chlorophyll a/b and chlorophyll fluorescence (Fv/Fm) in an aquatic macrophyte Spirodela

polyrhiza (Jiang et al. 2012). Nanoparticles phytotoxicity on various edible plants and the

potential impact of NPs on agricultural processes was examined (Mondal et al. 2011,

Rico et al. 2011). TiO2 NPs were efficiently taken up by plant roots and localized in the

parenchymal region and vascular cylinder in Triticum aestivum, Brassica napus and

Arabidopsis thaliana (Larue et al. 2011). AgNPs caused changes in proteins involved in

the redox regulation and in the sulfur metabolism. AgNPs altered some proteins related to

the endoplasmic reticulum and vacuole (Vannini et al. 2013). The nanoparticles may also

bind with different cytoplasmic organelles and interfere with the metabolic processes at

that site (Jia et al. 2005). Yin et al. (2011) has been suggested that phytotoxicity depends

on plant selection, concentrations tested, size, and exposure conditions. Silver

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nanoparticles during penetrations inside the root cell, damaged the cell wall as well as

vacuoles to enter and concluded that it may be due to the penetration of larger particles

entering through small pores of cell walls. Ruffini and Cremonini (2009) reported AgNPs

mostly affected the shoot and root growth than seed germination. A comparative study of

nano TiO2 and AgNPs on tomato (Lycopersicon esculentum) mature plants, AgNPs

exhibited low chlorophyll contents, higher SOD, and less fruit production, but nano-TiO2

treated plants showed only higher SOD values at the highest (5000 mg/kg) levels of

treatment (Uhram et al. 2013). However Krishnaraj et al. (2012) reported AgNPs treated

plant (Bacopa monnieri) had higher levels of protein and carbohydrate while

comparatively lower level of total phenol content (TPC), CAT and POX activities,

reflecting the better plant growth with reduced toxicity. Nanoparticles may increase

lipid membrane peroxidation upon contact to cells due to the reactive oxygen species

(ROS) (Nel et al. 2006). The entry of nanosilver into the cell has damage DNA (ATSDR

1990). Copper oxide NPs damaged DNA in some agricultural and grassland plants

(Raphanus sativus, Lolium perenne, and Lolium rigidum) (Atha et al. 2012). MWCNTs

induced increased oxidative stress and decreased cell proliferation, which eventually led

to complete cell death in Rice (Tan et al. 2009). While examining the acute toxic effects

of AgNPs in different organisms, Liu and Hurt (2010) suggested that the toxic effects of

silver nanoparticles was due to the presence of nanoparticles and secondly by the release

of Ag+ ions from nanoparticles and the free radicals generated during the AgNPs

suspension.

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Cytotoxicity and Genotoxicity of Biosynthesized Gold and Silver Nanoparticles on

Human Cancer Cell Lines

The study describes dose dependent cytotoxicity and genotoxicity of biologically

synthesized AuNPs and AgNPs on A549, LNCap-FGC, MDA-MB. The half maximal

inhibitory concentration (IC50) was calculated as the concentration required for

inhibiting the growth of cancer cells in culture by 50 % compared to the untreated cells.

The effect of synthesized AuNPs and AgNPs on human carcinoma cell lines was

investigated by knowing IC50 values using MTT assay (Plate-27, Figure.32). The IC50

of AuNPs obtained at 10µg/ml on all the cancer cell lines and increased concentration of

AuNPs 30µg/ml resulted in 100 % cell lysis. The IC50 for AgNPs could be less than

10µg/ml, because at this concentration AgNPs has resulted in 100 % cell lysis of cancer

cell lines (Plate-28, Figure.33). Longer exposures could result in additional toxicity to the

cells. These results demonstrate that AuNPs and AgNPs facilitated concentration

dependent increase in toxicity. However, the actions of AuNPs and AgNPs depend on

size, shape, conditions of media, type of cells, dose and exposure time. Since most Nano

toxicological screening studies found simpler to perform in in vitro on cell cultures,

though these results may not accurately predict the in vivo toxicity. Griffith and Swartz

(2006) provided a basis for understanding the mechanism of toxicity and nanoparticle

uptake at the cellular level. Goodman et al. (2004) proved AuNPs as biologically inert

and non-toxic. The cytotoxicity induced by gold nanoparticles depends on size, shape,

functional group, charge as well as on the method of cellular uptake (Chen et al. 2009). It

was found that 1.4nm gold nanospheres triggered necrosis, mitochondrial damage and

oxidative stress on all examined cell lines, and found no evidence of cellular damage for

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15nm gold nanospheres bearing the same surface group (Pan et al. 2009). The result

highlights the possible size dependent toxicity of gold nanoparticles, while many studies

focused on determining the lethal dosage of nanoparticles (LD50, dose required to kill

half of the population). In addition, the potential cytotoxicity of AgNPs against cancer

was demonstrated (Sriram et al. 2010). Franco-Molina et al. (2010) reported the

cytotoxic effect of colloidal silver on MCF7 human breast cancer cells through induction

of apoptosis. AgNPs disrupts normal cellular function, affects the membrane integrity

and induces various apoptotic signaling genes of mammalian cells leading to

programmed cell death (Sanpui et al. 2011). Gopinath et al. (2008) observed the IC50

value and molecular mechanism of 10-15nm size of AgNPs mediated cytotoxicity in

BHK21 (noncancer) and HT29 (cancer) cells at 27µg/ml, whereas in our study IC50

value was lesser than the concentration reported. The present IC50 results revealed that

Cassia auriculata mediated synthesized AgNPs shows more efficacy than the previously

report. In a study on the effects of AgNPs on skin using the human derived keratinocyte

HaCaT cell line model, AgNPs caused concentration and time dependent decrease of cell

viability, with IC50 values of 6.8±1.lM (MTT assay) and 12±1.2lM (SRB assay) after 7

days of contact (Zanette et al. 2011). However in our experiment the incubation time of

cell with nanoparticles was only 4hrs which has revealed 100 % cell lysis in short period

of time and made these nanoparticles more efficient. Using an MTT assay, comparison of

effective concentration (EC50) values of AgNPs of different sizes (5nm, 20nm and 50

nm) and surface areas on different cell types (A549, HepG2, MCF-7 and SGC-7901

cells) were also evaluated (Liu et al. 2010). The silver nanoparticles on Human

epidermoid larynx carcinoma cell line exhibited a dose dependent toxicity and the

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viability of Hep-2 cells was decreased to 50% (IC50) at the concentration of 500nM

(Kaliyamurthi et al. 2012). In the present study silver nanoparticles were found to be

more toxic than gold nanoparticles as reported earlier (Foldbjerg et al. 2011). The

flavonoid conjugated AgNPs were devoid of anticancer effect and found synergic effect

of AuNPs on cell lines (Raghunandan et al. 2011), lower cytotoxicity of AgNPs

compared to AuNPs may attributed to the difference in surface charges between NPs

(Yen et al. 2009). The cytotoxic effects of silver nanoparticles were due to active

physicochemical interaction of silver atoms with the functional groups of intracellular

proteins as well as with the nitrogen bases and phosphate groups in DNA (Moaddab et al.

2011).

DNA Fragmentation

The impact of AuNPs and AgNPs in DNA fragmentation was investigated. The assay

involves extraction of DNA from a lysed cell homogenate followed by agarose gel

electrophoresis. The gel after electrophoresis clearly revealed the intensity of all treated

DNA samples has diminished, possibly because of the cleavage of the DNA (Plate-29,

Figure.34). The metals produced ROS such as hydroxyl radical (OH), superoxide radical

(O2-

) or hydrogen peroxide (H2O2) which cleaves the DNA. These nanoparticles produce

oxidative stress which causes direct damage to the DNA in which a single electron may

be accepted or donated by the metal. Excessive production of ROS in the cell known to

induce apoptosis (Martindale and Holbrook 2002) and plays an important role in

apoptosis induced by AgNPs (Carlson et al. 2008). AgNPs were also reported to induce

severe structural damage and accumulate in mitochondria, which eventually contributed

to oxidative stress (Asharani et al. 2009b). Generation of excessive intracellular ROS

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leads to apoptosis and necrosis (Hackenberg et al. 2011) because increased ROS levels

correlate with massive DNA breakage and high levels of apoptosis and necrosis (Singh

and Ramarao 2012). However a number of mechanisms affect the ability of nanoparticles

to cause DNA damage. ROS may cause DNA–protein crosslinks, damage to the

deoxyribose phosphate backbone, and specific chemical modifications of purine and

pyrimidine bases (Dizdaroglu 1991). ROS can also modify the DNA bases and cause

strand scission by degrading the ribose ring (Yang and Gao 2002). The DNA damage by

gold nanoparticles further supports the fact that gold nanoparticles induced apoptosis in

HL-60 cells (RaviGeetha et al. 2013). Highly reactive ROS caused oxidative harm to

DNA and cell enzymes (Boonstra and Post 2004). Panda et al. (2011) suggested that

AgNPs induced DNA damage was apparently mediated through ROS. Cadet et al. (2010)

reported that H2O2 being highly reactive with Ag, yielded OH radicals which in turn

damaged DNA.

Antibacterial Activity of Au-BNC and Ag-BNC Bio-nanocomposites

The Au-BNC and Ag-BNC nanocomposites evaluated to determine the antibacterial

activity. The bionanocomposites were found antibacterial against E. coli (Gram negative)

and B. subtilis (Gram positive) strains as indicated by zones of inhibition around the films

in bacterial culture plates and there was no zone of inhibition found around the control

alginate film indicating no antibacterial activity of alginate (Plate-30, Figure.35 & 36).

The nanoparticles embedded throughout the networks releases ions within aqueous media

and interact with the bacteria. It has been reported that when metallic silver reacts with

moisture or human fluids, releases silver ions, damaging bacteria or inhibiting bacterial

replication (Blaker et al. 2004). Ag-BNC was found better than Au-BNC in antibacterial

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activity which confirms that AgNPs have strong effects on microbes as reported in the

literature. Clinical guidelines recommended the use of silver dressings for wounds

healing by preventing microorganisms (Best Practice Statement 2011) and have

beneficial effects on cost of wound management and on quality of life parameters

(Opasanon et al. 2010). From the results, the bionanocomposite hydrogels showed better

activity towards the gram-negative bacteria than gram-positive bacteria, due to the

presence of high lipid content in the gram negative bacterial cell wall (Madhusudana et

al. 2013). The possible mode of increased toxicity of bionanocomposite may be due to

nanoparticles releases ions easily and interact with lipid layer of cell membrane (Murali

Mohan et al. 2007) thus extending their application for antibacterial purposes. The

composites of silver nanoparticles within polymers results in the improvement of

antimicrobial activities at lower concentrations (Madhusudana et al. 2013).