Colloids and Surfaces B: Biointerfaces 96 (2012) 6974

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Colloids and Surfaces B: Biointerfaces

journal homepage: www.elsevier.com/locate/colsurfb

Biosynthesis of silver nanoparticles from Tribulus terrestris and its

antimicrobial activity: A novel biological approach
V. Gopinath a , D. MubarakAli b , S. Priyadarshini a , N. Meera Priyadharsshini a ,
N. Thajuddin b , P. Velusamy a,
a
b

Department of Biotechnology, School of Bioengineering, SRM University, Kattankulathur, Chennai 603203, Tamil Nadu, India
Department of Microbiology, School of Lifesciences, Bharathidasan University, Tiruchirappalli 620024, India

a r t i c l e

i n f o

Article history:
Received 27 December 2011
Accepted 30 March 2012
Available online 6 April 2012
Keywords:
Biosynthesis
Silver nanoparticles
TEM
Tribulus terrestris L.
Fruit bodies
Antibacterial
Green chemistry

a b s t r a c t
In the recent decades, increased development of green synthesis of nanoparticles is inevitable because of
its incredible applications in all elds of science. There were numerous work have been produced based
on the plant and its extract mediated synthesis of nanoparticles, in this present study to explore that the
novel approaches for the biosynthesis of silver nanoparticles using plant fruit bodies. The plant, Tribulus
terrestris L. fruit bodies are used in this study, where the dried fruit body extract was mixed with silver
nitrate in order to synthesis of silver nanoparticles. The active phytochemicals present in the plant were
responsible for the quick reduction of silver ion (Ag+ ) to metallic silver nanoparticles (Ag0 ). The reduced
silver nanoparticles were characterized by Transmission Electron Microscope (TEM), Atomic Force Microscope (AFM), XRD, FTIR, UVvis spectroscopy. The spherical shaped silver nanoparticles were observed
and it was found to be 1628 nm range of sizes. The diffraction pattern also conrmed that the higher
percentage of silver with ne particles size. The antibacterial property of synthesized nanoparticles was
observed by KirbyBauer method with clinically isolated multi-drug resistant bacteria such as Streptococcus pyogens, Pseudomonas aeruginosa, Escherichia coli, Bacillus subtilis and Staphylococcus aureus. The
plant materials mediated synthesis of silver nanoparticles have comparatively rapid and less expensive
and wide application to antibacterial therapy in modern medicine.
2012 Elsevier B.V. All rights reserved.

1. Introduction
Nanotechnology provides the tools and technology platform for
the investigation of biological systems, and biology offers inspiration models for bio-assembled components to nanotechnology.
Nanostructured materials showed many aspects of interesting
characteristics, i.e., optical, catalytic, that greatly depends on the
size and shape of nanoparticles as an effect of quantum connement of electrons. Metal nanoparticles are extensively used in
many electrochemical, electro analytical and bio-electrochemical
applications owing to their extraordinary electro catalytic activity
[1].
Nanoparticle research is inevitable today not because of only
application and also by the way of synthesis. Route of synthesis of
nanoparticles by physical and chemical methods may have considerable environmental defect, technically laborious and economically expensive. Many researchers have explored the technological

Corresponding author. Tel.: +91 8939731191.

E-mail addresses: vels73@gmail.com,
velusamy.p@ktr.srmuniv.ac.in (P. Velusamy).
0927-7765/$ see front matter 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.colsurfb.2012.03.023

approach for the synthesis. For example, silver ions are reduced
by chemical, electrochemical, radiation, photochemical methods;
LangmuirBlodgett and biological techniques were reviewed [2].
Biological route synthesis of nanoparticles have much attention
focused by researcher for the reason that to elucidate the mechanism of synthesis. Almost all type of biological entity has been used
for the synthesis of nanoparticles with size and shape controlled
manner. First and foremost, a microorganism, Pseudomonas stutzeri
AG259 have used for the synthesis of silver nanoparticles [3], later
on actinomycetes [4], fungi [5], cyanobacteria [6], biomolecules [7]
and different parts of plant materials. The plant material based
production of nanomaterials has wide range of application such
as antimicrobial property. Various plant materials have been studied so far for the synthesis of silver, gold, platinum and titanium
nanoparticles in different sizes and shapes were tabulated (Table 1).
In recent days, different parts of plant materials have been studied
for this direction more exclusively such as extracts [1], fruit [8],
bark [9], fruit peels [10], root [2] and callus [11].
In this present study to explore that the novel approaches for
the biosynthesis of silver nanoparticles using dried fruit bodies
from the plant, Tribulus terrestris L. is belongs to the family Zygophyllaceae and it has long been used in the traditional Chinese and

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V. Gopinath et al. / Colloids and Surfaces B: Biointerfaces 96 (2012) 6974

Table 1
Various plant materials used for the biosynthesis of nanoparticles and its size range.
S. No.

Plant materials

Metal nanoparticles

Particle size

Reference

1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.

Acalypha indica
Magnolia kobus and Diopyros kaki
Cinnamomum camphora
Citrus sinensis
Curcuma longa
Cassica stula
Sorbus aucuparia (leaf)
Geranium (leaf)
Sorghum (bran)
Aloe vera
Azadirachta indica
Carica papaya
Emblica ofcinalis
Eclipta prostrata
Henna (apiin)
Mangifera indica
Tanacetum vulgare
Parthenium hysterophorus
Medicago sativa
Citrus sinensis

Ag
Au
Au and Ag
Ag
Ag
Ag
Ag and Au
Ag
Fe and Ag
Ag and Au
Au, Ag and Au Ag
Ag
Ag and Au
Ag
Au and Ag
Ag
Ag and Au
Ag
Ag
Ag

2030 nm
5300 nm
5580 nm
312 nm
2130 nm
5060 nm
16 nm and18 nm
1640 nm
10 nm and 50 nm
15.2 4.2 nm
50100 nm
6080 nm
1020 nm, 1525 nm
3560 nm
21 nm and 39 nm
20 nm
16 nm and 11 nm
5280 nm
86108 nm
35 nm

[14]
[15]
[16]
[17]
[18]
[19]
[20]
[21]
[22]
[23]
[24]
[25]
[26]
[27]
[28]
[29]
[30]
[31]
[32]
[33]

Indian systems of medicine for the treatment of various ailments

and is popularly claimed to improve sexual functions in man [12]. It
is used in the folk medicine of India, China, Bulgaria and South Africa
against sexual impotence, oedemas, abdominal distention and cardiovascular diseases [13]. This present investigation deals with T.
terrestris L. plant fruit bodies mediated synthesis and characterization of silver nanoparticles and their biomedical application.
2. Materials and methods
2.1. Collection of plant materials
The dried fruit bodies of the T. terrestris L. was collected from
local market in Chennai, India. The fruit bodies were rinsed with
water thrice followed by Milli-Q water to remove the ne dust
materials and then, the fruit bodies were dried under direct sun
light for 1 week to completely remove the moisture.
2.2. Preparation of fruit extracts
The dried fruits were pulverized well with mortar and pestle
to make a powder. Five grams of powder sample was mixed into
100 ml of deionized water and the mixture was boiled for 10 min.
After cooling the fruit extract was ltered with Whatman No. 1
lter paper. The ltrate was stored at 4 C for further use.
2.3. Synthesis of silver nanoparticles
The 100 ml of aqueous ltrate extract of T. terrestris L. was taken
into 250 ml of Erlenmeyer ask. Then the extract was mixed into
Silver nitrate (AgNO3 ) to make the nal volume concentration of
1 mM solution. The reaction mixture was kept into dark room condition until the colour change was arisen. The reaction solution
colour changes have observed for the characterization of silver
nanoparticles.
2.4. Characterization of nanoparticles
The synthesized silver nanoparticles were characterized by
UVvis spectroscopy periodically for a week in order to observe
rapid reduction silver nanoparticles by the action of plant extracts.
The biologically reduced brown colour solution mixture was
scanned by Perkin Elmer preclsley, Lambda 25 instrument operated
at a resolution of 1 nm. In this analysis the fruit extract without

adding the silver nitrate was used as a control. The FTIR is performed to the extract which was exposed before and after addition
to the silver nitrate solution. The samples were mixed with KBr
to make a pellet and it was placed into the sample holder. The
spectrum was recorded at a resolution of 4 cm1 . X-ray diffraction
pattern was obtained from lyophilized silver nanoparticles by powder diffraction method, where it gives grain size and shape of the
particles by h, k and l index value.
The morphological analysis of the particle was done with transmission electron microscopy (TEM). The drop of aqueous silver
nanoparticle sample was loaded on carbon-coated copper grid and
it was allowed to dry for an hour. The TEM micrograph images were
recorded on JEOL instrument 1200 EX instrument on carbon coated
copper grids with an accelerating voltage of 80 kV. The clear microscopic views were observed and documented in different range
of magnications. The synthesized silver nanoparticles structure
morphology and size of the nanoparticle were further characterized by the atomic force microscopy (AFM) images. The microscopic
images were recorded with silicon cantilever with force constant
0.220.77 N/m, tip height 1012 nm in the contact mode.
2.5. Screening of antibacterial activity against MDRM
The antibacterial activity was assayed by the standard
KirbyBauer disc diffusion method. The synthesized silver
nanoparticles antibacterial activity was observed against multi
drug resistant microorganisms (MDRM) such as Streptococcus pyogens, Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis and
Staphylococcus aureus. The bacterial suspension was swabbed on
the Muller Hinton Agar (MHA) plates using sterile cotton swab.
The sterile 6 mm paper disc was impregnated into the MHA plates
for the inoculation of synthesized silver nanoparticles and control.
15 l of silver nanoparticles was inoculated into 4 discs of all the
plates. The centre disc of all the plate was loaded 15 l of T. terrestris L. fruit extract used as a control. The plates were incubated
at 35 C for 24 h. After the incubation period, the zone of inhibition
were observed and tabulated.
3. Results and discussion
3.1. Biosynthesis of silver nanoparticles
The nanoparticle synthesis reaction was started after the fruit
extract was introduced into 1 mM aqueous (AgNO3 ) solution. After

V. Gopinath et al. / Colloids and Surfaces B: Biointerfaces 96 (2012) 6974

71

Fig. 1. UVvis spectra of fruit extract alone showed SPR peak at 280300 nm (a), fruit extract reduced silver nanoparticles SPR peak at 435 nm (b). The gure inset shows
fruit extract alone (a) and synthesized silver nanoparticles (b).

36 h of incubation in dark room condition the colourless reaction

mixture was turned into dark brown colour solution corroborate
the silver nanoparticle synthesis (Fig. 1). The colour change was
occurring because the active molecules present in the fruit extract
reduce the silver metal ions into silver nanoparticles. The intensity
of the colour change was increased direct proportion to the incubation period of nanoparticle synthesis. It may be due to the excitation
of surface plasmon resonance (SPR) and reduction of AgNO3 [34].
3.2. Characterization of silver nanoparticles
UVvis spectra were recorded for the fruit extract of T. terrestris
was introduced with 1 mM AgNO3 solution at various time intervals. After 36 h of incubation in the dark room condition yellow
colour reaction mixture was turned into dark brown. It may due
to the excitation of surface plasmon resonance for the synthesised
silver nanoparticles. The silver nitrate fruit extract reaction mixture exhibits the strong peak at 435 nm (Fig. 1). Silver nanoparticles
exhibit unique and tunable optical properties on account of their
surface plasmon resonance, dependent on shape and size distribution of the nanoparticles. The fruit extract of T. terrestris without
adding the silver nitrate was used as a control showed the peak at
280300 nm. It may due to the reducing protein molecules present
in the fruit body extract of T. terrestris.
The major factor liable for the biological reduction of silver ions
(Ag+ ) into silver nanoparticles (Ag0 ) present in the fruit extract of
T. terrestris were identied using FTIR spectroscopy. The biologically synthesized SNPs and the powdered fruit body were mixed
with the Potassium bromide to make a pellet. The IR spectrum
of fruit extract alone showed the distinct peak in the range of
3488.76, 2811.25, 2727.52, 2164.11, 1591.36, 1388.31, 1348.56,
1124.13 and 766.16 cm1 (Fig. 2a). The spectrum of IR peak at
3488.76 was referred as the strong stretching vibrations of O H
functional group [35]. The corresponding peaks at 2811.25 and
2727.52 cm1 due to the C H vibrational mode [29]. The vibrational
mode at 2164.11 cm1 corresponds to C C variables present in the
plant protein. The medium of the intense peak at 1613.12 cm1
was mainly attributed due to the carboxyl group of C C stretching

vibration present in the plant fruit body [36]. The IR bands of 1348
and 1125.13 cm1 were due to the strong stretching vibrations of
C N aromatic and aliphatic amine. The FTIR spectrum of silver
nanoparticles showed the distinct peak in the range of 3419.18,
2811.83, 2726.33, 2171.34, 1613.12, 1348.66, 1125.13, 763.75 cm1
(Fig. 2b). The comparison of FTIR spectrum between the fruit extract
and silver nanoparticles were observed only minor changes in the
position as well as the absorption bands. Due to the silver nanoparticles the O H stretching vibration shifted from 3488.76 to 3419.18.
The X-ray diffraction studies were performed to conrm the
crystalline structure of synthesized silver nanoparticles. XRD spectrum of fruit extract reduced silver nanoparticle were showed the
four distinct diffraction peak at 38.1 , 44.3 , 64.4 and 77.4 the
lattice plane value was observed which may indexed at 1 1 1, 2 0 0,
2 1 1 and 2 2 0 of the cubic silver (Fig. 3). The lattice constant calculated from the diffraction spectrum was a = 4.0857 A and the
resultant data was matched with the database Joint Committee

Fig. 2. FTIR analysis of reducing protein responsible for the synthesis of silver
nanoparticles (b) and the fruit body alone (a).

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V. Gopinath et al. / Colloids and Surfaces B: Biointerfaces 96 (2012) 6974

Fig. 3. XRD spectrum of silver nanoparticles synthesized from fruit body of Tribulus
terrestris.

on Powder Diffraction Standards (JCPDS) le no. 01-087-0717. The

resultant XRD spectrum clearly suggests that the silver nanoparticles synthesized from the fruit extract of T. terrestris L. was
crystalline which strongly proves in AFM and TEM micrograph
images.
The synthesized silver nanoparticles structure morphology and
size of the nanoparticle were characterized by the AFM images. The
resultant silver nanoparticle images were observed as spherical in
shape shown in Fig. 4(a). It has been reported that the topographical image of irregular silver nanoparticles have documented [37],
where it can be clearly shown that apart from nanoisland formation
there is also an agglomeration of silver. The particle size of the silver nanoparticles ranges in size from 160 to 180 nm and cannot be
controlled by varying the synthesis condition. The fabricated silver
nanoparticles were imaged by AFM to understand the exact conguration of the fabricated silver nanoparticles and also used to verify
that the silver nanoparticles were more or less homogenous in size
and were spherical in shape [38] The particle size was measured
using line prole determination of individual particles in the range
of 24.631 nm. And the silver nanoparticle crystalline structure and
size were further characterized by TEM. The biologically synthesized silver nanoparticles using the dried fruit extract of T. terrestris

Fig. 5. TEM micrograph image of synthesized silver nanoparticles showed the average size of 22 nm spherical shaped particles.

structural morphology and crystallinity were further conrmed by

TEM micrograph images. The aliquot of silver nanoparticle solution was placed into a drop coated copper grid and the sample was
allowed to dry. The TEM images were recorded at different magnication to nd the individual particles. The synthesized silver

Fig. 4. AFM images of silver nanoparticles synthesized using fruit body extract of Tribulus terrestris showed the spherical shaped particle size of 22.473 nm, (a) particle size
determination prole of the AFM image (b).

V. Gopinath et al. / Colloids and Surfaces B: Biointerfaces 96 (2012) 6974

nanoparticles were observed in spherical shape and average size of

the particles was 22 nm (Fig. 5). The variation in the particle sizes
such as 20, 40, 53 and 70 nm difference in size is possibly due to the
fact that the nanoparticles are being formed at different times [28].

73

3.3. Antimicrobial activity of silver nanoparticles

The biologically synthesized silver nanoparticles showed excellent antimicrobial activity against clinically isolated Multi drug

Fig. 6. Antimicrobial activity of silver nanoparticles against multidrug resistant pathogen Escherichia coli, Bacillus subtilis, Streptococcus pyogens, Pseudomonas aeruginosa and
Staphylococcus aureus.

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V. Gopinath et al. / Colloids and Surfaces B: Biointerfaces 96 (2012) 6974

Table 2
Mean zone of inhibition of synthesized AgNPs against various pathogenic bacteria.
Study no.

Pathogenic bacteria

Zone of diameter in mm
(mean of four replicates)

1
2
3
4
5

Staphylococcus aureus
Bacillus subtilis
Escherichia coli
Pseudomonas aeruginosa
Streptococcus pyogens

9.75
9.25
10.75
9.25
10

resistant human pathogens such as Gram positive bacteria S. pyogens, Staphylococcus aureus, B. subtilis and Gram negative bacteria
P. aeruginosa and E. coli. The mean inhibitory zone of the four replicates of diameter was measured and to be tabulated (Table 2). The
Gram negative bacterium E. coli showed maximum zone of inhibition at 10.75 mm which may due to the cell wall of Gram positive
bacteria composed of a thick peptidoglycan layer, which consisting of linear polysaccharide chains cross linked by short peptides
thus forming more rigid structure leading to difcult penetration
of the silver nanoparticle compared to the gram negative bacteria where the cell wall possesses thinner peptidoglycan layer [39].
The high bactericidal activity is certainly due to the silver cations
released from Ag nanoparticles that act as reservoirs for the Ag+
bactericidal agent. Big changes in the membrane structure of bacteria as a result of the interaction with silver cations lead to the
increased membrane permeability of the bacteria [40,41]. Lin et al.
explained that in general, silver ions from silver nanoparticles are
believed to become attached to the negatively charged bacterial
cell wall and rupture it, which leads to denaturation of protein
and nally cell death [42]. The attachment of either silver ions or
nanoparticles to the cell wall causes accumulation of envelope protein precursors, which results in dissipation of the proton motive
force. On the other hand, [43] elucidated that silver nanoparticles exhibited destabilization of the outer membrane and rupture
of the plasma membrane, thereby causing depletion of intracellular ATP. Sarkar et al. reported that for E. coli (ATCC 10536) and
Staphylococcus aureus (ML 422), silver nanoparticles demonstrated
greater bactericidal efciency compared to penicillin [44]. Silver
has a greater afnity to react with sulfur- or phosphorus-containing
biomolecules in the cell. Thus, sulfur-containing proteins in the
membrane or inside the cells and phosphorus-containing elements
like DNA are likely to be the preferential sites for silver nanoparticle
binding [45,46] (Fig. 6).
4. Conclusion
In this study we have demonstrated that use of plant fruit
powder extract of T. terrestris as a reducing agent can effectively
produce the spherical shape silver nanoparticles followed by the
green chemistry approach. The synthesized silver nanoparticles
using fruit extract of T. terrestris proved excellent antimicrobial activity against clinically isolated multidrug resistant human
pathogens. The antimicrobial activity of silver nanoparticle was
well demonstrated by the clear zone of inhibition against S. pyogens, Staphylococcus aureus, B. subtilis, P. aeruginosa and E. coli.
The powder diffraction study showed the face centred cubic silver nanoparticles were stable after 6 months in room temperature,
TEM and AFM studies revealed that spherical shaped nanoparticles in the range of 24 nm. Moreover biological synthesis of silver
nanoparticles using plant material was the most conventional and

eco-friendly method compare to the chemical and physical synthesis.

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