Green Synthesis of silver nanoparticles using leaf extracts of Clitoria ternatea and Solanum

nigrum and study of its antibacterial effect against common nosocomial pathogens
1

N. Krithiga A. Rajalakshmi and A. Jayachitra

1*

Department of plant biotechnology, school of biotechnology, madurai kamaraj

university, Madurai- 6250021, india
corresponding author*- jchitra21@gmail.com
ABSTRACT
Bionanotechnology has emerged up as integration between biotechnology and nanotechnology
for developing biosynthetic and environmental friendly technology for synthesis of nano
materials. Silver has been known to have effective bactericidal properties for centuries.
Nowadays, silver based topical dressings have been widely used as a treatment for infection in
burns, open wounds, and chronic ulcer. As the pathogenic organisms are getting evolved day by
day due to mutation and gaining antibiotic resistance, a n important industrial sector of
nanosciene deals with the preparation and study of nanoparticles in antibacterial clothings, burn
ointments and coating for medical device. The size of nanomaterials is much smaller than that of
most biological molecules and structures, therefore nanomaterials can be useful in both in vivo
and in vitro biomedical research application. The purpose of the study is to s ynthesis and
characterizes the plant mediated silver nanoparticles using Clitoria ternatea and Solanum nigrum
. Further investigation of the shape and size of nanoparticle was done by X-ray diffraction and
Scanning electron microscopic studies. A silver nanoparticle at different concentration was
assessed for its antibacterial effect, against various nosocomial pathogens.

KEY WORDS: silver nanoparticles, Clitoria ternatea, Solanum nigrum. Antibacterial activity

INTRODUCTION

Nanotechnology is the fastest growing area of manufacturing in the world today and there
is an increasingly frantic search for new nanomaterials and methods to make them. It has been
well known that living cells are the best examples of machines that operate at the nano level and

perform a number of jobs ranging from generation of energy to extraction of targeted materials
1

at very high efficiency .

One of the fields in which nanotechnology finds extensive applications is nanomedicine,

an emerging new field which is an outcome of fusion of nanotechnology and medicine. Medicine
is no more physician job exclusively, the materials and devices designed at the level of nanoscale
are for diagnosis, treatment, preventing diseases and traumatic injury, relieving pain and also in
2

the overall preservation and improvement of health .Nanotechnology can improve our
understanding of living cells and of molecular level interactions. A number of nanoparticles
based therapeutics have been approved clinically for infections, vaccines and renal
3

diseases .Oligodynamic silver having antimicrobial efficacy extends well beyond its virotoxicity
4

and it have lethal effects spanned across all microbial domains . The application of silver
nanoparticles in drug delivery, drug discovery and new drug therapies have declare war on many
dead full diseases and they use the body natural transport pathway and natural mechanism of
5

uptake of the drug by the diseased cells .

Nanoparticles can be synthesized using various approaches including chemical, physical,

and biological. Although chemical method of synthesis requires short period of time for synthesis
of large quantity of nanoparticles, this method requires capping agents for size stabilization of the
nanoparticles. Chemicals used for nanoparticles synthesis and stabilization are toxic and lead to
non-ecofriendly byproducts. The need for environmental non-toxic synthetic protocols for
nanoparticles synthesis leads to the developing interest in biological approaches which are free
from the use of toxic chemicals as byproducts. Thus, there is an increasing demand for Green
nanotechnology. Many biological approaches for both extracellular and intracellular
6

nanoparticles synthesis have been reported till date using microorganisms including bacteria ,
7-8

fungi

and plants

9-11

Plant provide a better platform for nanoparticle synthesis as they are free from toxic
chemicals as well as provide natural capping agents. Moreover, use of plant extracts also reduces
the cost of microorganisms isolation and culture media enhancing the cost competitive feasibility
over nanoparticles synthesis by microorganisms

12

Nanobiotechnology is the most active areas of research in modern material science.

Nanoparticles exhibit completely new or improved properties based on specific characteristics
such as size, distribution and morphology

13

Antibacterial activity of the silver containing materials used in medicine to reduce

infections in burn treatment
16

prostheses

human skin

14

and arthroplasty

15

, as well as to prevent bacteria colonization on

17

,catheters , vascular grafts, dental materials

18

, stainless steel materials

19

, and

20

Silver nanoparticles are widely used for its unique properties in catalysis, chemical
21

sensing, biosensing, photonics, electronic and pharmaceuticals . Silver nanoparticles have a

22

great potential for use in biological including antimicrobial activity . Antimicrobial capability
of silver nanoparticles allows them to be suitably employed in numerous household products
23

such as textiles, food storage containers, home app liances and in medical devices . Silver is an
effective antimicrobial agent exhibits low toxicity

24

. The most important application

of silver and silver nanoparticles is in medical industry such as tropical ointments to prevent
25

infection against burn and open wounds . Silver nanoparticles play a profound role in the field
of biology and medicine due to their attractive physiochemical properties. Silver products have
long been known to have strong inhibitory and bactericidal effects, as well as a broad spectrum
of antimicrobial activities, which has been used for centuries to prevent and treat various
diseases, most notably infections

26

.Silver nanoparticles are reported to possess anti- fungal, anti27

inflammatory, anti-viral, anti-angiogenesis and antiplatelet activity .

MATERIALS AND METHODS

Plant material
Clitoria

ternatea habit is a twining shrub with

alternate imparipinnate leaves. About their

flowers are solitary or in pairs .The classification of Clitoria ternatea is given below
Family
Subfamily
Genus
Species
Botanical name
Common name

Fabaceae
Faboideae
Clitoria
ternatea
Clitoria ternatea.
Sankupushpam

Solanum nigrum habit dicot weed in Solanaceae family it is a African paediatric plant used for
several aliments that are responsible for to infant mortality especially to convulsions. It is an
annual branched herb of up to 90 cm high dull dark green leaves, juicy, ovate or lanceoate, and
toothless to slightly toothed on the margins . The classification of Solanum nigrum is given
below
Family
Genus
Species
Botanical name
Common name

Solanaceae
Solanum
nigrum
Solanum nigrum
night shade

Collection and Preparation of plant mate rials:

Fresh leaves of two different plant i.e. Clitoria ternatea and Solanum nigrum free from
diseases were collected from Madurai then washed thoroughly 2-3 times with tap water and once
with sterile water. 20 g of fresh leaves were finely chopped and added to 100 mL of Distilled
0

water and stirred at 60 C for 1 h. After boiling, the mixture was cooled and filtered with
Whatman paper No. 1. Filtrate was collected.

Synthesis of silver nanoparticles

12

0.1M of aqueous solution of silver nitrate (AgNO3) was prepared and used for the
synthesis of silver nanoparticles. 5mL of leaf extract Clitoria ternatea and Solanum nigrum was
added to 45 mL of 0.1M AgNO3 solution for bioreduction process at room temperature.

UV-Visible absorbance spectroscopy

26

UV-Visible spectroscopy analysis was carried out on a Systronic UV-Visible absorption

spectrophotometer 117 with a resolution of 1nm between 200-1000nm processing a scanning
speed of 200nm/min. Equal amounts of the suspension (0.5ml) was taken a nd analysed at room
temperature. The progress of the reaction between metal ions and the leaf extract were monitored
by UV-Visible spectra of silver nanoparticles in aqueous solution with different wavele ngth in
nanometers from 340 to 800nm. The reduction of silver ions and formation of silver
nanoparticles occurred within an hour of reaction .Control was maintained by using AgNo 3.

Fourier transforms Infrared spectroscopy (FTIR)

For FTIR measurements, the synthesized Silver nanoparticles solution was

centrifuged at 10000 rpm for 30 minutes. The pellet was washed thrice with 5 ml of
deionised water to get rid of the free proteins or enzymes that are not capping the silver
nanoparticles. The pellet was dried by using vaccum drier. It was analysed by FTIR.

X-Ray diffraction

28

A thin film of the silver nanoparticle was made by dipping a glass plate in a
solution and carried out for X-ray diffraction studies. The crystalline silver nanoparticle
was calculated from the width of the XRD peaks and the average size of the nanoparticles
29

can be estimated using the DebyeScherrer equation

D= k / cos
Where D = Thickness of the
nanocrystal, k = Constant,
= Wavelength of X-rays,

= Width at half maxima of (111) reflection at Braggs

angle 2, = Bragg angle.
The size of the silver nanoparticle was made from the line broadening of the (111)
reflection using the Debye-Scherrer formula. According to the formula,
Constant (K) = 0.94
Wave length () = 1.5406 x 10

-10

Scanning Electron Microscopy

30

The pellet was subjected for SEM analysis. Thin films of the sample were prepared on a
carbon coated copper grid by just dropping a very small amount of the sample on the
grid, extra solution was removed using a blotting paper and then the film on the SEM
grid were allowed to dry for analysis.
31

Anti bacterial Assays

The antibacterial assays were done pathogens like nosocomial pathogens
such as
Bacillus subtilis, Staphylococcus auerus, Streptococcus pyogenes, Escherichia coli,

Pseudomonas aeroginosa and Klebsiella aerogenes by standard disc diffusion method.

Briefly

Luria Bertani (LB) broth/agar medium was used to cultivate Bacteria. Fresh overnight
culture of inoculum (100l) of each culture was spread on to Muller Hinton Agar (MHA)
plates. Sterile paper disc of 5mm diameter containing 30 g/ml silver nanoparticles along
with standard antibiotic (30g/ml) containing discs were placed in each plate as control.
o

The plates were incubated at 37 c for overnight. Next day the inhibition zones around the
discs were measured.
RESULTS

Plate 1 (a): Synthesis of silver nanoparticles using Clitoria ternatea

A Fresh Leaf extract B Silve r nitrate before addition of leaf

extract C After addition of leaf extract

Plate 1 (b): Synthesis of silver nanoparticles using Solanum

nigrum

A Fresh Leaf extract B Silve r nitrate before addition of leaf

extract C After addition of leaf extract

Figure 1 a UV-VIS spectroscopy for silver nanoparticles sythesised using Clitoria ternatea
leaves extracts

absobance in O.D units

Clitoria ternatea leaf extract

1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
250 270 300 310 330 350 400 430 450 470 510 570 610 650 670 710 750 800
wavelength in nm

Fig 1 b UV-VIS spectroscopy for silver nanoparticles sythesised using Solanum nigrum leaves
extracts

absorbance in O.D units

Solanum nigrum leaf extract

1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
250 270 300 310 330 350 400 430 450 470 510 570 610 650 670 710 750 800
wavelength in nm

Fig 2(a): FTIR spectrum of Silver nanoparticles synthesized by using the

leaf extact of Clitoria ternatea

Fig 2(b): FTIR spectrum of Silver nanoparticles synthesized by using the

leaf extact of Solanum nigrum

Figure 3(a): XRD Pattern of silver nanoparticles synthesized using

Clitoria ternatea leaf extract
Counts
outside work2_893
300

200

100

0
20

30

40

50

60

70

Position[ 2 Theta]
Position [2Theta] (Copper (Cu))

Pos. [2Th.] Height [cts]

28.0784

21.71

FWHM
[2Th.]
0.5353

d-spacing
[]
3.17800

Rel. Int. [%]

Particle size

7.00

15.98

32.5020

64.99

0.3346

2.75486

20.94

25.84

38.3386

310.37

0.3011

2.34783

100.00

29.18

44.5440

78.60

0.5353

2.03411

25.32

16.75

46.5090

39.65

0.4015

1.95265

12.77

22.50

57.7165

14.33

0.5353

1.59732

4.62

17.70

64.7555

73.34

0.5353

1.43966

23.63

18.36

77.6973

76.20

0.6528

1.22803

24.55

Average particle size of the nano particles

16.32

20

3(b): XRD Pattern of silver nanoparticles synthesized using Solanum

nigrum

leaf extra

Counts
300

outside work2_894

200

100

20

30

40

50

60

70

o
Position [2Theta] (Copper (Cu))

Position[ 2 Theta]

Pos. [2Th.] Height [cts]

FWHM

d-spacing

[2Th.]

[]

Rel. Int. [%]

Particle size

28.0437

132.93

0.2007

3.18185

44.71

42.63

32.4911

297.33

0.1840

2.75576

100.00

46.99

38.3743

32.86

0.5353

2.34574

11.05

16.42

46.4844

188.60

0.2175

1.95363

63.43

41.53

55.1135

49.99

0.3680

1.66643

16.81

25.44

57.6944

46.58

0.3346

1.59788

15.66

28.32

67.7475

14.42

0.8029

1.38318

4.85

12.45

74.7433

5.61

0.9792

1.26906

1.89

10.66

Average particle size of the nano particles

28

Figure 4(a): SEM micrograph of Silver nanoparticles synthesized by using

the leaf extract of Clitoria ternatea

Figure 4 (b): SEM micrograph of Silver nanoparticles synthesized by

using the leaf extract of Solanum nigrum

The reduction of silver nitrate using the plant leaf extract was viewed by the colour change in the
reaction solutions (plate 1a and b). In UV-Vis spectra recorded for the reaction solution of
reduced silver nitrate by leaf extract of Clitoria terntea and Solanum nigrum (Fig 1 a and b). The
maximum absorbance peak was seen at 420 and 440 nm for Clitoria terntea and Solanum nigrum
respectively.

FTIR measurements carried out to identify the possible biomolecules responsible for the
capping and efficient stabilization of the silver nanoparticles synthesized by the plant extacts.
(Fig 2 a and b) show the leaf broth of Clitoria terntea and Solanum nigrum respectively
-1

Absorbance bands of Clitoria terntea were observed at 3317.34 Cm

-1

stretch, 3317.34 Cm

-1

stretch, 1394.44 Cm

-1

stretch, 752.19 Cm

-1

assigned to O-H (s) stretch, 1614.31 Cm

assigned to C=C aromatic

-1

assigned to C-H alkenes stretch, 1191.93 Cm

-1

and 655.75 Cm

assigned to O-H (s)

assigned to C-N amines

assigned to C-H alkenes stretch. Absorbance bands of

-1

Solanum nigrum were observed at 3317.34 Cm

-1

assigned to C-H (s) stretch, 1606.59 Cm

-1

assigned to C=C aromatic stretch, 1394.44 Cm

-1

assigned to C-H alkenes stretch, 1191.93 Cm

-1

-1

assigned to O-H (s) stretch, 2933.88 Cm

-1

assigned to C-N amines stretch,1122.49 Cm

-1

assigned to C-N amines stretch 752.19 Cm and 655.75 Cm assigned to C-H alkenes stretch.

The silver oxide molecules formed are necessarily subjected to XRD analysis for the
measurement of size of these particles. (Fig 3a and b) show the XRD pattern obtained for the
silver nanoparticles synthesized using the leaf extract of Clitoria terntea and Solanum nigrum
The intense peak of nanoparticles 28, 33, 38, 44, 46, 55, 58, 65 and 77 in Clitoria terntea 28, 32,
39, 45, 55, 57, 65, 69, 75 and 77 in Solanum nigrum were appeared which are indexed as
crystalline silver. The sharpening of the peaks clearly indicates that the particles are the spherical
nanoparticles. The Average size of the silver nanoparticles is estimated by using the Debye
Scherrers formula. The average of silver nanopartic les synthesized by Clitoria terntea is 20nm
and average of silver nanoparticles synthesized by Solanum nigrum is 28nm.

The silver nanoparticles synthesized using Clitoria terntea and Solanum nigrum leaf
extract were optimized by various parameter such as Temperature, pH and Time (Fig 3a and b)

shows that temperature increased, the rate of silver nanoparticles formation also increased. The
size is reduced initially due to the reduction in aggregation of the growing nanoparticles. (Fig 4a
and b) shows that acidic condition suppresses the formation of silver nanoparticles but the basic
condition enhances the formation of silver nanoparticles. Large nanoparticles were formed at
lower pH (pH 4), where as small and highly dispersed nanoparticles were formed at high pH (pH
9). At neutral pH, the normal size of the nanoparticles are formed.(Fig 5a and b) shows that
duration of reaction increases, more silver nanoparticles are formed. Due to the instability of the
silver nanoparticles formed, an optimum duration is required, as silver nanoparticles
agglomeration after the optimum duration resulting in larger particle sizes. The optimum time
required for the completion of reaction from our study was 60 min.

The silver oxide molecules formed are necessarily subjected to XRD analysis for the
measurement of size of these particles. (Fig 3a and b) show the XRD pattern obtained for the
silver nanoparticles synthesized using the leaf extract of Clitoria terntea and Solanum nigrum
The intense peak of nanoparticles 28, 33, 38, 44, 46, 55, 58, 65 and 77 in Clitoria terntea 28, 32,
39, 45, 55, 57, 65, 69, 75 and 77 in Solanum nigrum were appeared which are indexed as
crystalline silver. The sharpening of the peaks clearly indicates that the particles are the spherical
nanoparticles. The Average size of the silver nanoparticles is estimated by using the Debye
Scherrers formula. The average of silver nanoparticles synthesized by Clitoria terntea is 20nm
and average of silver nanoparticles synthesized by Solanum nigrum is 28nm.

(Fig 4a and b ) shows the SEM image of the silver nanoparticles in Clitoria terntea and
Solanum nigrum. Uniformly distributed silver nanoparticles on the surface of the cells are
observed. However, it does not indicate that all the silver nanoparticles are bound to the surface
of the cells, because those dispersing in the solution may also deposit on to the surface of the
cells during the drying process, which is a necessary step b efore SEM.
Fig 5 antibiotic tetracyclin was used as positive control and water as negative control. The zone
of inhibition was observed for antibiotic and no zone was formed for negative control.(I attach a
alternate graph with controls

B.s bacillus subtilis, S.a Staphylococcus auerus, S.p Streptococcus pyogenes, E.c
Escherichia coli, P.a Pseudomonas aeroginosa, K.a Klebsiella aerogenes

Fig (5 a) shows the antibacterial activity of silver nanoparticles using Clitoria ternatea
leaf extract against nosocomial pathogens and compared with the standard drug.

Fig (5 b) shows the antibacterial activity of silver nanoparticles using Solanum nigrum
leaf extract against nosocomial pathogens and compared with the standard drug.
The formation of silver nanoparticles using plant leaf extract of Clitoria terntea and Solanum
nigrum was viewed by the colour change from the colourless to Yellowish brown. Similarly,
Saatry

32

reported that the silver nanoparticles exhibited striking colours, from light Yellow to

brown. Further, Shankar

33

reported that Silver nanoparticles exhibited Yellowish brown colour

in aqueous solution due to excitation of surface plasmon vibrations in s ilver nanoparticlesBy

using UV-Visible spectrum the maximum absorbance peak for Clitoria terntea and Solanum
nigrum was seen at 420 and 440 nm respectively.Similarly, Prasad and Elumalai,

34

reported that

Absorption spectra of silver nanoparticles formed in the reaction media has absorbance peak at
430-440 nm. Ravichandran et al

35

reported that Absorption spectra of silver nanoparticles

formed in the reaction media has absorbance peak at 438 nm.Different parameters were
optimized including temperature, pH and time which had been identified as factors affecting the
yields of silver nanoparticles. Gilaki

36

reported that pH, Strength of elements, Plant sources,

Time and Incubation temperature nanoparticles synthesis reaction mixture, the synthesis
methods, it is possible to create a wide range of different nanoparticles. FTIR analysis confirmed
+

that the bioreduction of Ag ions to silver nanoparticles are due to the reduction by capping
material of plant extract. Similarly, Gole et al.,

37

reported that Proteins present in the extract can

bind to silver nanoparticles through either free amino or carboxyl groups in the proteins. Prasad
et al.,

38

reported that the carboxyl (-C=O), hydroxyl (-OH) and Amine (-NH) groups of leaf

extracts are minely involved in fabrication of silver nanoparticles. The average of silver
nanoparticles synthesized by Clitoria terntea 20nm and average of silver nanoparticles
synthesized by Solanum nigrum. The observed result was in accordance to the results of Raut et
al., (2009) where the XRD studies reveals that silver nanoparticles which are polydisperesed and
ranged in size from 10-50nm with an average size of 27nm. Marimuthu vivek et al.,

39

reported

that the silver nanoparticles synthesized from Gelidiella acerosa extact of XRD pattern reveals
that the average size of the silver nanoparticles was 23nm.In these SEM micrograph, it was
observed that the spherical nanoparticles were in the size ranging from 10 -50nm with a variety

of morphology. Savithramma et al.,

30

reported that relatively spherical shape silver nanoparticles

formed with diameter ranging from 30-40 nm in Boswellia ovalifoliolata and 40 nm in Shorea
tumbuggaia.
Silver nanoparticles obtained from the Clitoria terntea and Solanum nigrum have very
strong inhibitory action against Pseudomonas aeruginosa followed by Staphylococcus aureus,
Escherichia coli and Streptococcus viridans. Similarly, Jain et al.,

40

reported that antibacterial

assay was done using Papaya fruit extract mediated silver nanoparticles on human pathogen,
showed highly toxic against multidrug resistance bacteria. Kumar Prasad et al.,

41

reported that

silver nanoparticles were fairly toxic to Pseudomonas aeruginosa while they showed a moderate
toxicity against P. vulgaris, E. coli, B. subtilis and P. putida. However, nanoparticles exhibited
low toxicity against S. typhi. The silver nanoparticles of Clitoria terntea and Solanum nigrum
were against the pathogens growth was inhibited.
CONCLUSION
A critical need in the field of nanotechnology is the development of a reliable and ecofriendly process for synthesis of metallic nanoparticles. Nanoparticles are being viewed as
fundamental building blocks of nanotechnology. Silver nanoparticles play a profound role in the
field of biology and medicine due to their attractive physiochemical properties. In the present
study, we have demonstrated that use of a natural, low cost biological reducing agent, Clitoria
ternatea and Solanum nigrum leaves extracts can produce metal nanostruc tures, through
efficient green nanochemistry methodology, avoiding the presence of toxic solvents and waste.
The biosynthesized silver nanoparticles using Clitoria ternatea and Solanum nigrum leaves
extract proved to be excellent against nosocomial pathogens and also compared to that the silver
nanoparticles of Clitoria ternatea showed high activity than the silver nanoparticles of Solanum
nigrum against nosocomial pathogens . The antimicrobial activity is well demonstrated by disc
diffusion method. The present study showed a simple, rapid and economical route to synthesize
silver nanoparticles. The use of Clitoria ternatea and Solanum nigrum has the added advantage
that this plant can be used by nanotechnology processing industries. Prepared nanoparticles can
be used as bactericidal and in wound healing, water purification and also in the field of medicine
due to these applications, this method is potentially exciting for the large-scale synthesis of
nanoparticles.

ACKNOWLEDGEMENT:
We would like to thank Central Electrochemical Research Institute (CECRI), Karaikudi,
Tamilnadu for their technical assistance in Electron Microscopy and XRD Spectral analysis
during our research.
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