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CHAPTER 6

CYTOTOXICITY, ANTIBACTERIAL, ANTIFUNGAL,

ANTICANCER ACTIVITY OF SILVER NANOPARTICLES

SYNTHESIZED FROM WRIGHTIA TINCTORIA

6.1 INTRODUCTION

Silver nanoparticles are being used in numerous technologies and

incorporated into a wide array of consumer products that take advantage of

their desirable optical, conductive, and antibacterial properties.

Diagnostic Applications: Silver nanoparticles are used in biosensors and

numerous assays where the silver nanoparticle materials can be used as

biological tags for quantitative detection.

Antibacterial Applications: Silver nanoparticles are incorporated in apparel,

footwear, paints, wound dressings, appliances, cosmetics, and plastics for

their antibacterial properties.

Conductive Applications: Silver nanoparticles are used in conductive inks

and integrated into composites to enhance thermal and electrical conductivity.

Optical Applications: Silver nanoparticles are used to efficiently harvest

light and for enhanced optical spectroscopies including metal-enhanced

fluorescence (MEF) and surface-enhanced Raman scattering (SERS).

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Wrightia tinctoria (veppallai) with its rich source of polyphenolic

compounds was exploited for the reduction and capping of silver

nanoparticles (Ag NPs), making it a complete green chemical route. The

reduction of Ag+ to Ag0 was observed by the color change from pale yellow to

dark yellow.

6.2 MATERIALS AND METHODS

6.2.1 Synthesis of Silver Nanoparticles

The silver nitrate (AgNO3) was purchased from Sigma-Aldrich

chemicals. Wrightia tinctorialeaves were collected from chengalpet. The leaf

extract was used for the reduction of Ag+ ions to Ag°. 50g of finely cut leaves

were thoroughly washed, dried and immersed in 100 ml of distilled water

contained in a 500 ml Erlenmeyer flask. The mixture was boiled for 15 to 20

minutes in a hot plate. Then the extract was further filtered through Whatman

No. 1 filter paper and stored at 4°C for further experiments. 1mM aqueous

AgNO3 solution was prepared. 5 ml of the leaf extract was added to 45 ml of

1 mM AgNO3 (aqueous) solution and kept in dark for 48 hours for the

formation of silver nanoparticles.

6.2.2 Characterization of the Silver Nanoparticles

The nanoparticles were characterized by UV spectroscopy (Perkin

Elmer Lambda Spectrophotometer), FTIR analysis, XRD analysis, (X-ray

diffraction method) and SEM - EDAX analysis (Scanning Electron Microscopy).

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6.2.2.1 UV spectrophotometric analysis

Samples for UV/Vis spectrophotometry are most often liquids,

although the absorbance of gases and even of solids can also be measured.

Samples are typically placed in a transparent cell, known as a cuvette.

Cuvettes are typically rectangular in shape, commonly with an internal width

of 1 cm. (This width becomes the path length, L, in the Beer-Lambert

law.) Test tubes can also be used as cuvettes in some instruments. The type of

sample container used must allow radiation to pass over the spectral region of

interest. The most widely applicable cuvettes are made of high quality fused

silica or quartz glass because these are transparent throughout the UV, visible

and near infrared regions. Glass and plastic cuvettes are also common,

although glass and most plastics absorb in the UV, which limits their

usefulness to visible wavelengths.

Specialized instruments have also been made. These include

attaching spectrophotometers to telescopes to measure the spectra of

astronomical features. UV-visible micro spectrophotometers consist of a UV-

visible microscope integrated with a UV-visible spectrophotometer. These are

commonly used for measuring thin film thickness in semiconductor

manufacturing, materials science research, measuring the energy content of

coal and petroleum source rock, and in forensic laboratories for the analysis

of microscopic amounts of trace evidence as well as questioned documents.

A complete spectrum of the absorption at all wavelengths of

interest can often be produced directly by a more sophisticated

spectrophotometer. In simpler instruments the absorption is determined one

wavelength at a time and then compiled into a spectrum by the operator. By

determined as a function of wavelength.

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6.2.2.2 XRD analysis

Collidge tube is arranged and a power of 1.2kw is produced form

40 kv & 30 mA in gerator to produce the X-ray of wavelength 1.5406 Å. The

prepared sample is mounted on the focusing circle. The x-rays are allowed to

fall on the sample and the counter is initially set at an angle 0°. The program

is set to run such that the detector counter moves through the required angle at

specific counts & scans the sample.

Start angle : 10°

Stop angle : 70°

Step input : 0.02

Time : 1sec

As the X-ray beam is diffracted by the sample and detected at

various angles, the output peak is generated on the computer screen. The

output is a graph with different peaks corresponding to different planes of the

crystal and the graph drawn was in betwee - axis and intensity in the

y-axis. The obtained peak is compared with the data in JCPDS tool which is a

standard.From this even the particle size of the crystal was calculated using

formula.

6.2.2.3 SEM analysis

All samples must also be of an appropriate size to fit in the

specimen chamber and are generally mounted rigidly on a specimen holder

called a specimen stub. Several models of SEM can examine any part of a

6-inch (15 cm) semiconductor water, and some can tilt an object of that size

to 45°.

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For conventional imaging in the SEM, specimens must be

electrically conductive, at least at the surface, and electrically grounded to

prevent the accumulation of electrostatic charge at the surface. Metal objects

require little special preparation for SEM except for cleaning and mounting

on a specimen stub. Nonconductive specimens tend to get charged when

scanned by the electron beam, and especially in secondary electron imaging

mode, this causes scanning faults and other image artifacts. They are

therefore usually coated with an ultra thin coating of electrically-conducting

material, commonly gold, deposited on the sample either by low vacuum

sputter coating or by high vacuum evaporation. Conductive materials in

current used for specimen coating include gold, gold/palladium alloy,

platinum, osmium, iridium, tungsten, chromium and graphite. Coating

prevents the accumulation of static electric charge on the specimen during

electron irradiation. Here the semi-conductive nano particles of silver were

coated using gold for SEM characterization

6.2.2.4 FTIR analysis

Although a homogeneous mixture will give the best

results, excessive grinding of the potassium bromide is not required. The

finely powdered potassium bromide will absorb more humidity (it is

hygroscopic) from the air and therefore lead to an increased background in

certain ranges. Some KBr was transferred out of the oven into a mortar.

About 1 to 2 % of the sample was added, mixed and ground to a fine powder.

For very hard samples, the sample was added first, ground, then the KBr was

added and then ground again.

The sample must be very finely ground to reduce scattering losses

and absorption band distortions. Two stainless steel disks was taken out of the

desiccator. A piece of the precut cardboard (in the tin can next to the

oven)was placed on top of one disk and the cutout hole was filled with the

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finely ground mixture. The second stainless steel disk was kept on top and the

sandwich was transferred onto the pistil in the hydraulic press. With a

pumping movement, the hydraulic pump was moved downward. The pistil

will start to move upward until it reaches the top of the pump chamber. Then,

the pump was moved until the pressure reaches 20,000 PRF. Leave for a few

seconds and with the small lever on the left side and the pressure was released

(hold until the sample and pistil are all the way down). The disks were

removed and pull apart. The film was removed, which should be homogenous

and transparent in appearance. It was inserted into the IR sample holder and

attached with a scotch tape. Then, running the spectrum was performed.

6.2.3 Biomedical Applications

6.2.3.1 Disk diffusion method and well diffusion method

Clinical isolates of Bacillus cereus ATCC 10987, Bacillus subtilis

MTCC 1133, Staphylococcus aureus MTCC 96, Micrococcus luteus ATCC

4698, Vibrio cholerae ATCC 14035, Escherichia coli MTCC 118, Salmonella

typhi MTCC 733 andKlebsiella pneumonia MTCC 109 were collected from

KMCH, Coimbatore (India). Each test strain was inoculated in Mueller

Hinton liquid medium (broth) and incubated in a temperature controlled

shaker (120 rpm) at 30 °C overnight.

Fungal cultures Candida albicans MTCC 183, Candida

parapsilosis MTCC 2509, and Candida tropicalis MTCC 184 used for the

experimental analysis were maintained on potato dextrose agar (PDA) at

28°C. Reference antibiotics such as Erythromycin, amoxicillin,

chloramphenicol, rifamycin and flucanazole used for the antibacterial and

antifungal analysis respectively, were purchased from Sigma Aldrich,

Bangalore, India and Merck Limited, Mumbai, India.

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6.2.3.2 Thin layer chromatography of AMOXYCILLIN (a reference

antibiotic)

It is used to separate the individual compounds formulated in the

antibiotics (crude). The separation of the compound also depends on the type

of the solvent used. The antibiotic representing maximum zone of inhibition

(amoxycillin) based on disk diffusion method was used for TLC analysis. A

sample of 10 mg/ml concentration of antibiotics in methanol was prepared.

From this solution, 4µl of the sample prepared was taken and spotted on the

silica coated TLC plates. It was then kept in slanting position with the solvent

to run under capillary pressure. Here methanol and chloroform in the ratio of

3:7 was used as a solvent. The spots were then identified both in the UV

light, far light and in the iodine chamber. The Rf values were calculated.

6.2.3.3 Bioautography Agar over layer method

The developed TLC plates were kept in the sterile petriplates. Then

the nutrient agar prepared was poured over the thin layer which was further

spreaded over the entire petridish. 24 hours cultures of ( V.cholerae) were

swabbed on it. The plates were then incubated at 37 º C for 24 hours. Zone of

inhibition obtained at varying separation point were observed.

Similarly chromatogram was developed by loading 1µg

concentration of nanoparticle impregnated with rifamycin (antibiotic). The

characteristic zone of inhibition thus obtained at the separation point by agar

over layer method was investigated. The results were discussed.

6.2.3.4 MTT assay

A549 cell lines were obtained from National centre for cell

sciences Pune (NCCS). The cells were maintained in Minimal Essential

111

Media supplemented with 10% FBS, penicillin (100 U/ml), and streptomycin

2 at 37 °C.

MEM was purchased from Hi Media Laboratories Fetal bovine

serum (FBS) was purchased from Cistron laboratories Trypsin,

methylthiazolyl diphenyl- tetrazolium bromide (MTT), and Dimethyl

sulfoxide (DMSO) were purchased from (Sisco research laboratory ).

Cells (1 × 105/well) were plated in 24-well plates and incubated in

37°C with 5% CO2 condition. After the cell reaches the confluence, the

various concentrations of the samples were added and incubated for 24 hours.

After incubation, the sample was removed from the well and washed with

phosphate-buffered saline (pH 7.4) or MEM without serum. 100 µl/well (5

mg/ml) of 0.5% 3-(4, 5-dimethyl-2-thiazolyl)-2,5-diphenyl-tetrazolium

bromide (MTT) was added and incubated for 4 hours. After incubation, 1ml

of DMSO (Dimethyl sulphoxide) was added in all the wells. The absorbance

at 570 nm was measured with UV- Spectrophotometer using DMSO as the

blank.

6.2.3.5 DNA fragmentation assay

The DNA was isolated from the cancer cells. Then the DNA was

dissolved by adding to each tube 20-50 µl of TE (Tris EDTA) solution and

the tubes were placed at 4°C. The samples were mixed with loading buffer by

adding 10X loading buffer to a final concentration of 1X. The addition of

loading buffer to samples allows to load in wells more easily and to monitor

the run of samples. The voltage was set up to 100V and runing the gel

through electrophoresis in standard TE buffer was performed.. During

electrophoresis, it is possible to monitor the migration of samples by

following the migration of bromophenol blue dye contained in the loading

dye. The process was stopped by switching off the power supply, when the

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dye reaches about 3 cm from the end of the gel. The gel was then placed on a

UV Transilluminator to visualize the DNA.

6.2.3.6 DPPH assay

DPPH (1,1-diphenyl-2-picrylhydrazyl) is characterized as a stable

free radical by virtue of the delocalisation of the spare electron over the

molecule as a whole, so that the molecules do not dimerise, as would be the

case with most other free radicals. The delocalisation also gives rise to the

deep violet colour, characterized by an absorption band in ethanol solution

centered at about 520 nm. When a solution of DPPH is mixed with that of a

substance that can donate a hydrogen atom, then this gives rise to the reduced

form (Blois,1958) with the loss of this violet colour (although there would be

expected to be a residual pale yellow colour from the picryl group still

present).

AH, the primary reaction is

step. This latter radical will then undergo further reactions which control the

overall stoichiometry, that is, the number of molecules of DPPH reduced

(decolorized) by one molecule of the reductant.

Aliquots of 3.7 ml of absolute methanol were taken in all the test

tubes. 3.8 ml of absolute methanol was added to blank. 100µl of BHT was

added to the tube marked as standard and 100µl of respective samples to all

other tubes marked as tests. 200µl of DPPH reagent was added to all the test

tubes including blank. All the test tubes were incubated at room temperature

113

in dark condition for 30 minutes. The absorbance of all samples was read at

517nm.

6.2.3.7 FRAP assay

-

tripyridyltriazine complex to form. FRAP values are obtained by comparing

the absorbance change at 593 nm test reaction mixtures with those containing

ferrous ion in known concentrations.

A standard solution of 1mM ferrous sulphate(control) was prepared

by dissolving 0.139g of FeSO4 .7H2O in 580 ml of distilled water. Serial

dilutions were made and the absorbance at 593nm measured by performing

the assay as described above with ferrous sulphate in place of test samples.

1 ml of distilled water and 80 µl of test sample was taken in the

standard 4ml plastic cuvette. 600 µl of incubated FRAP Reagent was added to

the cuvette, which was briefly inverted to mix the solutions. The reagent

blank was also prepared as described above but 80 µl of distilled water was

added instead of test sample. Change in absorbance at 593nm (as a result of

the reduction of the Fe3+ -TPTZ (Tri Pyridyl-S-Triazine) complex at low pH)

was recorded at exactly at 4 minutes using spectrophotometer. Each test

sample dilution was tested in triplicate to calculate the mean absorbance.

6.2.3.8 Scratch wound healing assay

The Vero cell lines were used for wound healing assay. The cells

were seeded into the plate and incubated for 24 hrs. After incubation, the cells

were observed for growth. The samples were weighed and dissolved in

DMSO. It was serially diluted at different concentrations and the

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concentration was chosen, based on IC50 values obtained from MTT Assay

(125 to 62.5 µg/ml).The medium was discarded and the plate was kept under

microscope. A sterile tip was used and wound was created. The desired

concentrations (125 to 62.5 µg/ml) were added to the respective wells and

incubated. After 4 hrs incubation, the plate was observed for the growth of

cells.

6.3 RESULTS AND DISCUSSION

6.3.1 Formation of Silver Nanoparticles

Appearance of the brownish red colour indicates the formation of

silver nanoparticles. The formed nanoparticles were found to be stable and are

uniformly distributed throughout the solution. FTIR wave number (s1)1385,

(s2)1375, (s3)1378, (s4)1377 characteistically corresponds to absorbance

between 400 to 440 nm, an indicative for the formation nanoparticle

Figure 6.1.

Figure 6.1 FTIR wave number characteriestic of absorbance indicating the formation of silver nanoparticle

115

6.3.2 Characterization of the Silver Nanoparticles

6.3.2.1 UV spectrophotometry analysis

The characteristic peaks were observed in between 410 nm and 440

nm, which confirmed the presence of silver nanoparticle formation. The

broadening of the peak indicates the uniform distribution and polydispersion

of silver nanoparticles.The size and shape of the nanoparticle was determined

by the frequency and width of the surface plasmon absorption produced.

6.3.2.2 X-RD analysis

The scintillation detector present in the instrument moves through

the required angle at specific counts and scans the sample between 100 and

700 - axis and

intensity on y-axis. The obtained graph containing different peaks

corresponding to different planes of the crystal was compared with the

standard data in JCPDS tool. From the result obtained, the average size of the

where,

is full width at half maximum of peak (FWHM), and D is the average particle

size. Depending upon the characteristic peaks obtained in Fig.6.2, the X-RD

pattern defines the distribution of the size of the particle as 66.5 nm, 50.15

nm, 50.94 nm, 51.96 nm, 34.9 nm, 35.93 nm, 54.41 nm, 36.91 nm, 56.62 nm,

39.55 nm and 39.95 nm, respectively. Based upon the characteristic peak

e crystal was

calculated and this ranges from 50nm to 400 nm in diameter. The particles are

characterized using XRD, UV {visible, photoluminescence and Raman

spectroscopy.The absorption feature (peak) at480 nm which is considerably

116

blue-shifted relative to the peak absorption of bulk CdS indicating quantum

size effect. the photoluminescence (PL) spectra of nanoparticles of CdS for

different excitation wavelengths of 300, 240 and 230 nm (energies 4.1, 5.1

and 5.4 eV).The Raman spectrum of nanoparticles of CdS is in the range 180-

400 cm-1. The spectrum exhibits a strong but broadpeak at 302 cm-1

corresponding to the LO phonon mode (KaruppasamyKandasamy et al 2009).

X-ray diffraction patterns of CdS nanoparticles are characteristic of

the hexagonal phase and t values were found to be 4.124 and

6.686Å. The XRD peaks are very broad indicating the presence of very fine

grains of particles. The XRD pattern exhibits broad peaks of 26.778, 43.928

and 51.6780. The size of the particle increases with the concentration of the

capping agent increased (Manickathai et al 2008).

Figure 6.2 XRD analysis indicating the formation of silver nanoparticle

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6.3.2.3 SEM EDAX analysis

The surface morphology of the formed silver nanoparticles was observed and was represented as nanoclusters. Besides, the elemental composition was checked by EDAX, which indicates unique peak for silver which constitutes major composition.Electron-diffraction patterns and high-resolution lattice images obtained with transmission electron microscopy (TEM) indicated that the CdS nanoparticles were crystalline with dspacings corresponding to the zinc blende structure ( dspacings, 0.336, 0.206, 0.176, 0.133, 0.118 nm) (Limin Qi et al 2001). Absorption, fluorescence spectroscopy, and transmission electron microscopy were employed for characterization, which revealed that the prepared silver nanoparticles had a well-resolved cubic structure and were monodisperse in size Figure 6.3. It was also found that the silver nanoparticles were dispersed in solution as single entities and showed a very good resistance against oxidation for months, according to their polymer shell. The particle size was controllable in the range between 2 and 4 nm by adjusting the polymer concentration and choice of the solvent (Rajeev Prabhu & Abdul Khadar 2005).

Figure 6.3 SEM image for the formation of silver nanoparticle

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6.3.2.4 FTIR analysis

The FTIR spectrum of silvernanoparticles was shown in

Figure 6.4. The higher energy region peaks at 3784 cm-1 and 2426cm-1 are

assigned to O-H stretching of silver nanoparticles. Also a stretch of N-H

amine group was observed at 3399 cm-1 and 1615 cm-1 respectively. A strong

stretch of N-O nitro group was observed at 1384 cm-1. An alkene =C-H

bending was observed at 805 cm-1. Also a strong alkyl halide C-I stretch was

observed at 463cm-1 .Upon verification with the present database, the

spectrum matches with that of silver and partially with the spectrum of Al2O3.

Figure 6.4FTIR analysis for the formation of silver nanoparticle

4000.0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 600 400.00.0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100.0

cm-1

%T

3916.0

3784.3

3399.1

2426.1

1615.2 1384.3

805.1

463.1

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6.3.3 Applications

6.3.3.1 Antibacterial and antifungal Activity of Silver Nanoparticles

Table 6.1 Zone of inhibition produced by silver nanoparticles, reference antibiotic Amoxycillin, and AgNps with amoxycillin

Pathogenic Bacterial strains

Zone of inhibition (mm)

Amoxycillin

50µg/ml

AgNps

25µg/ml

AgNps

50µg/ml

AgNps &

Amoxycillin 50µg/ml

B.cereus ATCC10987 12 11 13 13

B.subtilis MTCC1133 18 16 17 21

S. aureus MTCC 96 15 22 20 24

M. luteus ATCC 4698 18 21 21 21

V. cholerae ATCC 14035 31 18 17 18

E. coli MTCC 118 20 22 25 24

S. typhi MTCC 733 15 21 23 22

K. pneumoniae MTCC109 24 27 29 29

Pathogenic fungal strains

Zone of inhibition (mm)

Flucanazole

50µg/ml

AgNps

25µg/ml

AgNps

50µg/ml

AgNps &

Flucanazole 50µg/ml

C. albicans MTCC 124 28 16 17 15

C. parapsilosis MTCC 2509 - 15 16 15

C. tropicalis MTCC 184 - 19 20 20

120

DETERMINATION OF THE ANTIBACTERIAL AND ANTIFUNGAL ACTIVITY OF THE SILVER NANOPARTICLES SYNTHESIZED THROUGH

WRIGHTIA TINCTORIA PLANT EXTRACT WITH REFERENCE TO AMOXYCILLIN AND FLUCANAZOLE

Zone of inhibition against Vibrio cholerae ATCC 14035

Zone of inhibition against Candia albicans MTCC 124

AB

C

D E

AB

C

D E

Amoxycillin(50 g/ml)

Amoxycillin&AgNps(50 g/ml

AgNps(25 g/ml)AgNps (50 g/ml

Flucanazole(50 g/ml)

Flucnazole&AgNps(50 g/ml)

AgNps(25 g/ml

AgNps(50 g/ml

Figure 6.5(a) Zone of clearance producedby AgNps in comparison with amoxicillin

Figure6.5(b) Zone of clearance produced by AgNps in comparison with flucanazole

nearly 11mm, 13mm and 13mm diameter of zonal expansion respectively

against B.cereus ATCC 10987, on well diffusion assay. Similarly, with

respect to B.subtilis

and combination exhibited 16mm and 17mm diameter of inhibitory zone,

respectively.

S.aureus MTCC

96, M.luteus ATCC 4698, V. cholerae ATCC 14035, E. coli MTCC 118, and

S. typhi MTCC 733, Klebsiella pnuemonea MTCC 1303 showed more or less

22±1 mm, 21±2 mm and 18±2 mm, 25±2 mm, 23±1 mm, 27±2 mm diameter

121

of zone of inhibition Table 6.1. Combined effects of the silver nanoparticle

and erythromycin pose equal effects as that of nanoparticle. All the results

obtained were compared with the control (reference antibiotics) in order to

check the efficiency of silver nanoparticles. Almost, the nanoparticles derived

from the four sources depicted nearly the similar types of results, and

exhibited it as a potential antimicrobial agent.

combinations (silver nanoparticle and flucanazole) exhibited 16 mm and 17

mm diameter of inhibition zone against C.albicans MTCC 183, which was

found to be half of the effect of the control. But, for C.parapsilosis MTCC

2509 and C tropicalis MTCC 184 species, control becomes ineffective. No

characteristic zone of inhibition was observed for the selected reference

pronounced effect of 16mm and 20mm diameter of increase in the fold area

of inhibition zone against C.parapsilosis MTCC 2509 and C tropicalis

MTCC 184, species respectively. This inferred the antifungal property of

silver nanoparticles over broad range of pathogens tested. All the results

obtained were comparatively analysed with the control (reference antibiotics)

Figure 6.5(a) & Figure 6.5(b) . Not only does the silver nanoparticle serve as

a good antibacterial agent, but also capable of using as a potential antifungal

agent against broad spectrum of bacterial and fungal pathogens taken under

clinical investigation.

6.3.3.2 Thin layer chromatography of amoxycillin (a reference

antibiotic)

It is used to separate the individual compounds formulated in the

antibiotics (crude). The separation of the compound also depends on the type

of the solvent used. The antibiotic representing maximum zone of inhibition

(amoxycillin) based on disk diffusion method was used for TLC analysis. A

122

sample of 10 mg/ml concentration of antibiotics in methanol was prepared.

From this solution, 4 µl of the sample prepared was taken and spotted on the

silica coated TLC plates. It was then kept in slanting position with the solvent

to run under capillary pressure. Here methanol and chloroform in the ratio of

3:7 was used as a solvent. The spots were then identified both in the UV

light, far light and in the iodine chamber. The Rf values were calculated

Figure 6.6 (a) and Figure 6.6 (b).

Figure 6.6(a) Detection by U-V light

Figure 6.6(b) Detection through iodine vapours

6.3.3.3 Bioautography analysis of antibiotics and antibiotics with

nanoparticles

A characteristic zone of inhibition (from the separation point on the

thin layer to the agar on the petridish) diffused radially was observed. Rf

value nearer to 0.659 indicates the antibacterial efficiency inferred

amoxycillin. However, in combination with nanoparticle, the zone of

inhibition was shared between nanoparticle and rifamycin at two different

points.

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In Figure 6.7 (a), amoxycillin (derivative) showed a single broader

characteristic zone (in red colour) against V.cholerea. But, in Figure 6.7 (b),

two characteriestic zones were observed, a black zone representing the

nanoparticle, and a red zone representing amoxycillin. A significant

observation of this technique based analysis was perfectly on the zone

produced at reference points. The zone will radially outwards from thin layer

to the petriplate in circular motion, thereby inferring the antibacterial

efficiency of the particles

ZONE OF CLEARANCE PRODUCED BY THE AMOXYCILLIN AND WRIGHTIA TINCTORIA DERIVED SILVER NANOPARTICLES SEPERATED THROUGH THIN LAYER CHROMATOGRAPHY

Seperated amoxycillin diffused over Vibrio cholerae (red zone )

Unseperated Ag Nps diffused over Vibrio cholerae (black zone).

Seperated amoxycillin diffused over Vibrio cholerae (red zone).

Figure6.7(a) Zone ofclearance

producedby the amoxicillin seperated through TLC

Figure6.7(b) Zone of clearance produced bythe separated amoxicillin and unseperated AgNps through TLC

6.3.3.4 Cytotoxic effects of silver nanoparticles on bovine bone marrow

cells CD45-/CD14-and chick embryonic cells CD45-/ CD14-

Chick embryonic cells and bovine bone marrow cells does not

124

viable cells started decreasing. This was proved by the MTT assay, wherein

proportional to the decrease in the cellular proliferation at 24 hrs and 48 hrs of

incubation respectively. With reference to the assay performed, it was

concluded that

concentration for the silver nanoparticles (AgNps) derived using Wrightia

tinctoria plant extracts offered the cell viability Figure 6.8(a) and

Figure 6.8(b).

CYTOTOXIC EFFECTS OF SILVER NANOPARTICLES DERIVED FROM WRIGHTIA TINCTORIA ON THE CELLULAR

PROLIFERATION

0

0.5

1

1.5

2

2.5

0 100 200 300 400 500 600

Conentration ( M)

Wrightia tinctoria derived AgNps activity on chick

embryonic cell proliferation

24 hrs48 hrs

0

0.5

1

1.5

2

2.5

3

0 200 400 600

Concentration ( M)

Wrightia tinctoria derived AgNpsactivity on bovine bone marrow

cell proliferation

24 hrs

48 hrs

Figure6.8 (a) Cytotoxic effects of

silver nanoparticles derived from wrightia tinctoriaon the cellular proliferationof chick embryonic cd45-/cd14- stem cells

Figure6.8 (b) Cytotoxicity effects of silver nanoparticles derived from wrightiatinctoriaon thecellular proliferation of bovine bone marrow cells cd45-/cd14- stem cells

125

6.3.3.5 Anticancer activity of silver nanoparticles on A549 cell line

(Adenocarcinomic human alveolar basal epithelial cells)

With respect to biological and clinical applications, the ability to

control and manipulate the accumulation of nanoparticles for an extended

period of time inside a cell can provide sensitivity towards diagnosis and

therapeutic efficiency.. In general, silver nanoparticles should serve as one of

the best ways of treating diseases that involve cell proliferation and cell death.

Measurements were performed and the concentration required for a

50% inhibition (IC50) was determined graphically (Table 6.2). The % cell

viability was calculated using the following formula,

%cell viability = A570 of treated cells / A570 of control cells × 100

Graphs were plotted using the % of Cell Viability at Y-axis and

concentration of the sample in X-axis. Cell control and sample control was

included in each assay to compare the full cell viability in Cytotoxicity and

anti-cancer activity assessments.

For Wrightia tinctoria

concentration offered 50% cell viability (Figure 6.9 & Figure 6.10). This

assay confirmed the anti cancer potential of the silver nanoparticles.

126

Table 6.2 Anticancer effect of silver nanoparticles derived from Wrightia tinctoria on A549 cell line

S.No Concentration (µg/ml)

Dilutions Absorbance (O.D)

Cell viability (%)

1 1000 Neat 0.12 21.05

2 500 1:1 0.17 29.82

3 250 1:2 0.23 40.35

4 125 1:4 0.27 47.36

5 62.5 1:8 0.32 56.14

6 31.2 1:16 0.40 70.17

7 15.6 1:32 0.45 78.94

8 7.8 1:64 0.49 85.96

9 Cell control - 0.57 100

ANTICANCER ACTIVITY OF SILVER NANOPARTICLE ON A549 CELL LINE (ADENOCARCINOMIC HUMAN ALVEOLAR BASAL

EPITHELIAL CELLS)

IC50 values of AgNps derived from Wrightia tinctoria on A549 tumour cell line

Figure 6.9 IC50 values of AgNps derived from Wrightia tinctoriaon A549 tumour cell line

127

IN VITRO CYTOTOXICITY ASSAY (MTT ASSAY) FOR THE EVALUATION OF THE ANTICANCER ACTIVITY OF SILVER

NANOPARTICLE DERIVED FROM WRIGHTIA TINCTORIA ON A549 CELL LINE

Normal A549 cell line

Toxicity- 250µg/mlToxicity- 1000µg/ml

Toxicity- 125µg/ml Toxicity- 62.5µg/ml

Figure 6.10 In vitro cytotoxicity assay (MTT assay) for the evaluation of the anticancer activity of silver nanoparticle derived from wrightia tinctoria on A549 cell line

6.3.3.6 Evaluation of the anti apototic activity of the silver

nanoparticles by DNA fragmentation assay

It was observed that there was no cleavage in the control, where as

the DNA isolated from the cancer cell A549 was found to be cleaved into two

to three fragments on the well treated with various concentration of silver

nanoparticles .This investigation depicted the anti apoptotic behavior of the

silver nanoparticles derived from Wrightia tinctortia (Figure 6.11).

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Programmed cell death was also be resulted by the activation of

caspase 3, which leads to to the cleavage of caspase substrates, resulting in

the fragmentation of DNA. This induces the apoptotic signal of the cell by the

trafiicking of the nanoparticle.

EVALUATION OF THE ANTI APOPTOTIC ACTIVITY OF THE SILVER NANOPARTICLES DERIVED FROM WRIGHTIA TINCTORIA

BY DNA FRAGMENTATION ASSAY

Lane M: Marker(100bp DNA Ladder) Lane 1: ControlLane 2: Con 125µg/mlLane 3: Con 250µg/ml Lane 4: Con 62.5µg/ml

M 1 2 3 4

Figure 6.11 Evaluation of the anti apoptotic activity of the silver

nanoparticles derived from wrightia tinctoria by DNA fragmentation assay

6.3.3.7 Evaluation of antioxidant activity of the silver nanoparticles by

DPPH (1, 1-Diphenyl-2-Picrylhydrazyl)

Based on the DPPH assay performed, it was found that, a good free

radical scavenging potential was observed for the silver nanoparticles derived

using the plants extracts of Wrightia tinctoria.

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The percentage antioxidant activity of the silver nanoparticle

derived using wrightia tinctoria leaf extract was found to be 30.55%

Figure 6.12

Figure 6.12 Evaluation of % antioxidant activity of the silver nanoparticles derived from Wrightia tinctoriaby DPPH assay

6.3.3.8 Evaluation of antioxidant activity of the silver nanoparticles by

frap (Ferric ion reduction potential) assay

The FRAP value of the silver nanoparticle was found to be

nanoparticles derived form Wrightia tinctoria was justified Figure 6.13.

0

20

40

60

80

100

% of antioxidant activity

130

Figure 6.13 Evaluation of % antioxidant activity of the silver nanoparticles derived from Wrightia tinctoriaby FRAP assay

6.3.3.9 Evaluation of wound healing activity of silver nanoparticles on

vero cell lines

Silver nanoparticles afford a good wound healing potential and this

was manipulated by scratch wound healing assay on Vero cell line. After

24hrs and after 48hrs of incubation with silver nanoparticles Figure 6.14,

migration of cells occurred which facilitate the wound healing activity of the

cells was justified. Silver nanoparticle exhibit to increase the

polymorphonuclear cell apoptosis, but he activity of matrix metalloproteinase

(MMP) remains low, conferred an anti-inflammatory potency.

0

50

100

150

200

250

AgNPS 50 g/ml AgNps

100 g/ml control

FRAP Value(mM/L)

FRAP Value(mM/L)

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ASSESSMENT OF WOUND HEALING ACTIVITY OF THE SILVER NANOPARTICLES DERIVEN FROM WRIGHTIA TINCTORIA ON

VERO CELL LINES

Wound created on vero Cell lineWound healed after 24 hrs of

incubation with silver nanoparticle

(a) (b)

Figure 6.14 Assessment of wound healing activity of the silver nanoparticles derived from wrightia tinctoria on vero cell lines(a) scratched cells (b) migrated cells

6.3.3.10 Evaluation of cell viability of silver nanoparticle /BSA scaffold

on mesenchymal stem cells by in vitro cytotoxicity assay

from Wrightia tinctoria was blended with BSA 10mM (Sigma Aldrich)

Bovine Serum Albumin . The contents were then lyophilized at -80 C for

the formation of scaffold. MTT assay was perfomed by adding the

nanoparticle solution on the mesenchymal stem cells seeded on th 96 well

microtitre plate.The absorbance were read at 570nm.It was observed that after

12 hr, 24 hrs and 48 hrs of incubation , the bio scaffold blended with silver

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nanoparticle and BSA showed celluar proliferation and supports cell viability

(Figure 6.15).

Figure 6.15 Effect of Wrightia tinctoria AgNps /BSA scaffold on mesenchymal stem cells by invitro cytotoxicity assay

6.4 CONCLUSION

From this summary, it was concluded that plant mediated synthesis of

silver nanoparticles possess potential antimicrobial applications. The

characterization analysis proved that the particle so produced in nanodimensions

would be equally effective as that of antibiotics and other drugs in pharmaceutical

applications. The use of Wrigthia tinctoria derived silver nanoparticles in drug

delivery systems might be the future thrust in the field of medicine. It was

concluded that the silver nanoparticles can serve as a potential drug with various

clinical and pharmacological properties, thereby demonstrating enhanced

characteristic anti cancer activity, anti apoptotic activity, anti oxidant activity,

wound healing activity and antimicrobial activity.

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

12 hrs 24 hrs 36 hrs 48 hrs 60 hrs 72 hrs

AgNps/BSA scaffold showing cell viability on mesenchymal stem cells

g/ml+40 g/ml

g/ml+30 g/ml

g/ml+20 g/ml

g/ml+10 g/ml

g/ml

control