Physica E 44 (2011) 665–671

Contents lists available at SciVerse ScienceDirect

Physica E
journal homepage: www.elsevier.com/locate/physe

Green synthesis and characterization of gold nanoparticles using extract of

anti-tumor potent Crocus sativus
R. Vijayakumar n, V. Devi, K. Adavallan, D. Saranya
Department of Physics, Annamalai University, Annamalainagar 608002, Tamil Nadu, India

a r t i c l e i n f o abstract

Article history: In the present study, we have explored anti-tumor potent Crocus sativus (saffron) as a reducing agent
Received 10 September 2011 for one pot size controlled green synthesis of gold nanoparticles (AuNps) at ambient conditions. The
Received in revised form nanoparticles were characterized using UV–vis, scanning electron microscope (SEM), high resolution
28 October 2011
transmission electron microscopy (HRTEM), X-ray diffraction (XRD) and FTIR analysis. The prepared
Accepted 1 November 2011
Available online 7 November 2011
AuNPs showed surface Plasmon resonance centered at 549 nm with average particle size of 15 7 5 nm.
Stable, spherical and triangular crystalline AuNPs with well-defined dimensions were synthesized
using anti-tumor potent Crocus sativus (saffron). Crystalline nature of the nanoparticles is confirmed
from the HR-TEM, SAED and SEM images, and XRD patterns. From the FTIR spectra it is found that the
biomolecules are responsible for capping in gold nanoparticles.
& 2011 Elsevier B.V. All rights reserved.

1. Introduction
Metal nanoparticles (NPs) have attracted considerable atten-
tion for their unusual chemical and physical properties such that
they show great potential applications in biotechnology, catalysis,
medical imaging, novel electronics and optics [1–6]. Gold nano-
particles have been considered as an important area of research
due to their unique and tunable surface Plasmon resonance (SPR)
and their applications in biomedical science including drug
delivery, tissue/tumor imaging, photothermal therapy and
immuno-chromatographic identification of pathogens in clinical
specimens [7]. Many methods have been developed for synthe-
sizing metal NPs, especially gold nanoparticles (AuNPs) [8–13].
Recently there is an ever-growing need to develop clean, non-
toxic and environmentally friendly (‘‘green chemistry’’) synthetic
procedures and researchers have been looking at biological
systems for inspiration [14–16]. From the green chemistry
approach, it has been concluded that the biosynthesized gold
nanoparticles are biocompatible, inert [17], bind readily to a large
range of biomolecules such as proteins/enzymes [18,19], DNA
[20], amino acids [21,22] and expose large surface area for the
immobilization ion of such biomolecules and environmentally
friendly chemicals.
There are several reports on biological methods of nano-
particles synthesis using microorganisms such as Fusarium oxy-
sporum [23], Fusarium semitactum [24,25], Cladosporium sp. [26],
Rhodopseudomonas sp. [27], Megatherium sp. [28], which have
n Coriander leaf for the synthesis of gold NPs. Shukla et al. [43] used
Corresponding author. Tel.:þ 91 4144 238282; fax: þ91 4144 238080.
E-mail address: vijay_phy@yahoo.com (R. Vijayakumar).
been exploited for toxic metals recovery via reduction of the
metal ions. There have been numerous reports on the synthesis of
gold NPs using plant materials as environment friendly substi-
tutes to chemical and physical methods. The work reported on the
microorganisms and plants in the synthesis of Au nanoparticles
has been reviewed by Mohanpuria et al. [29] and very recently by
Narayanan and Sakthivel [30]. Kasturi et al. [31] have reported the
synthesis of silver and gold nanoparticles using purified apiin
compound extracted from henna leaf. Shankar et al. [32] synthe-
sized gold NPs using Neem leaf broth and they have reported that
the formation of pure metallic NPs is possibly facilitated by
reducing sugars and terpenoids present in the Neem leaf broth.
They have also synthesized gold NPs using Geranium leaves and
Lemongrass extract [33,34]. Gardea-Torresdey et al. [35] synthe-
sized gold NPs within live Alfalfa plants and this method can be
very efficient in decontaminating soil polluted with heavy metal
ions. Ankamwar et al. [36] reported the synthesis of gold
nanotriangles using Tamarind leaf extract and studied their
potential applications in vapor sensing. The synthesis of gold
nanotriangles using Aloe vera extract was investigated by Chan-
dran et al. [37]. Huang and co-workers [84] demonstrated a
bioreduction approach to gold NPs using sundried biomass of
Cinnnamomum camphora leaf. Song et al. [38] demonstrated the
rapid synthesis of gold NPs using Magnolia kobus and Diopyros
kaki leaf extracts. Wang et al. [39] reported the extracellular
synthesis of gold NPs using Skull cup herb. Nune et al. [40]
synthesized gold NPs using Tea leaves and Nestor et al. [41]
studied the optical properties of gold NPs synthesized using
Camellia sinensis extract. Narayanan and Sakthivel [42] used
Soybeans as a phytochemical reservoir for the production and

1386-9477/$ - see front matter & 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.physe.2011.11.002
666 R. Vijayakumar et al. / Physica E 44 (2011) 665–671
stabilization of gold NPs. Katti et al. [44] synthesized gold NPs using
Cumin seeds and Sharma et al. [45] reported the synthesis of gold
NPs using the seeds of Sesbania drummondi. Recently the synthesis
of gold NPs have been reported using plants such as Coleus
ambonicus Lour [46], Terminalia catappa [47], Chenopodium album
[48], Sorbus aucuparia [49] and Syzygium aromaticum [50]. The
extracts of fruits like Gooseberry [51], Tansy [52], Papaya [53], Pear
[54] and Lemon [55] were also used for the synthesis of gold NPs.
Philip and coworkers reported biosynthesis of gold NPs using Honey
[56], Mushroom [57], C. zeylanicum [58], H. rosa-sinensis [59], O.
sanctum [60], M. indica [61], Murraya koenigil [62] and A. occidentale
[63]. Very recently, they have also synthesized gold NPs using
aqueous extract of Macrotyloma uniflorum [64]. Bankar et al. [65]
have shown that Banana peel extract is an efficient green material
for the rapid and consistent synthesis of gold NPs. Recently, the leaf
extract of antidiabetic potent Cassia auriculata has also been used in
the synthesis of Au nanoparticles [66]. Very recently Castro et al.
[67] have reported the synthesis of gold nanowires from Sugar
beet pulp.
In biosynthesis of nanoparticles, it is always beneficial to use
plant or part of plant rather than the organisms as it avoids
pathogenic, laborious, time consuming and hygiene maintenance
procedures of cell culture [68]. In continuation of the efforts for
synthesizing gold nanoparticles by green route, a facile, rapid and
single-pot aqueous biosynthesis of gold nanoparticles using the leaf
extract of anti-tumor potent Crocus sativus (saffron) has been
reported here. Saffron contains more than 150 volatile and aroma-
yielding compounds. It also has many nonvolatile active compo-
nents, many of which are carotenoids, including zeaxanthin, lyco-
pene and various a- and b-carotenes. Saffron has many medicinal
uses [69]. A 2010 double-blind, placebo-controlled study found that
saffron helped in mild to moderate Alzheimer’s disease [70].
Crocetin, an important carotenoid constituent of saffron, has shown
significant potential as an anti-tumor agent in animal models and
cell culture systems [71]. Saffron inhibits DMBA-induced skin
carcinoma in mice when treated early [72]. Both saffron stigma
and petals are said to be helpful for depression [73]. Saffron was
found to be effective in relieving symptoms of PMS [74]. Saffron,
crocins and crocetin inhibit breast cancer cell proliferation [75].
Crocus sativus (most saffron research refers to the stigmas but often
this is not made clear in research papers) inhibits histamine H1
receptors in animals, suggesting a potential use in allergic disorders
[76]. Histamine is a biological amine that plays an important role in
allergic responses. Saffron may have a protective effect on the heart
[77]. A recent (2011) double blind, human trial found use of 100 mg
of saffron daily to have temporary immunomodulatory activities
[78]. In the present investigation, the synthesis of gold nanoparticles
emphasizes the potentiality of Crocus sativus (saffron) in reducing
and stabilizing gold nanoparticles.
2. Materials and methods
2.1. Materials
Chloroauric acid (HAuCl4  3 H2O) was procured from Sigma-
Aldrich Chemicals, India and used as received. All other reagents
were of analytical grade with maximum purity. All glasswares
have been washed with distilled water and dried in oven before
use. Saffron has been collected from Baby brand Saffron, USMS
Saffron Co. Inc., New Delhi, India.
2.2. Synthesis of gold nanoparticles
In this study a simple and rapid green synthesis of AuNPs
using extract of Crocus sativus (saffron) has been reported. Crocus
sativus (saffron) extract was prepared from 50 mg of saffron
boiled in 50 ml of ultra pure deionized water in Borocil RB flask
for 15 min. Whatman filter paper (No. 41) filtered extract was
stored at room temperature for further use, being usable for
1 week. For the synthesis of gold nanoparticles the effect of
extract quantity and concentration of metal ion solution were
also evaluated to optimize the synthesis route producing the
metal nanoparticles. The effect of extract concentration in the
mixed solution on the biosynthesis of gold nanoparticles was first
investigated. The different concentrations of extract in the mixed
solution were obtained by changing the volume of the added
extract solution. Different volumes (0.1–5 ml) of the extract
solution were added to the HAuCl4 solution of different concen-
trations. The concentration of extract and metal ion solution to be
used were optimized to 4 ml and 0.5 mM, respectively. Gold
nanoparticles can be prepared and the stability of particles was
evaluated without any additional stabilizing chemicals/and or
physical steps. It was observed that the colloidal solution main-
tains its stability and uniformity for more than six weeks. The
evolution of Au nanoparticles from the reduction of different
ratios of HAuCl4 with optimized concentration of the extract has
been evaluated through monitoring of Surface Plasmon behavior
as a function of time. Further, 4 ml of the extract was added to
20 ml of 0.5 mM HAuCl4 and the solution was placed in orbital
shaker at room temperature, for reduction of Au3 þ to Au0. The
bio-reduction of AuCl4 ions solution was monitored by periodic
sampling of aliquots (2 ml) of aqueous component and measuring
UV–vis spectra of the solution. Fig. 1 shows the color change
involved in the formation of gold nanoparticles. The reaction was
rapid as the purple color appeared within 10 min and appearance
of purple color in the reaction confirmed the formation of gold
nanoparticles and there was no further color change. The light
absorption pattern of the biomass was kinetically monitored and
was in the range of 549 nm recorded using Shimadzu dual-beam
UV–vis spectrometer (model UV-1650PC) operated between
200 nm and 800 nm. The experiment was duplicated thrice to
confirm the reduction reaction.
For the crystallinity studies, films of AuNPs formed on glass
substrates by drop coating the colloidal nanoparticles were used
for X-ray diffraction (XRD) study. The data were obtained using
PAN ANALYTICAL X1 pert diffractometer by Cu-Ka1 radiation with
l of 1.5406 Å and nickel monochromatic filtering wave at tube
voltage of 40 kV and tube current of 30 mA. Size distributions and
Fig. 1. (A) Crocus sativus (Saffron) extract, (B) Auric chloride and (C) purple color
indicating the formation of gold nanoparticles. (For interpretation of the refer-
ences to color in this figure legend, the reader is referred to the web version of this
article.)
 R. Vijayakumar et al. / Physica E 44 (2011) 665–671
shape of the nanoparticles were obtained from High Resolution
TEM (HR-TEM) photographs. HR-TEM measurements were per-
formed on a TECNAI G2 FEI F12 model TEM instrument operated
at an accelerating voltage of 200 eV. Samples for HR-TEM were
prepared by placing a drop of the gold colloidal solution on a HR-
TEM copper grid (200 meshes, carbon-coated, colloid on-covered).
The films on the HR-TEM grids were allowed to dry for several
hours after the extra solution was removed using blotting paper.
For FTIR measurements, the bioreduced chlorauric solution
was centrifuged at 10,000 rpm for 15 min. The pellet was washed
three times with 20 ml of deionized water to get rid of the free
proteins/enzymes that are not capping the gold nanoparticles.
The samples were dried and grinded with KBr pellets and
analyzed on an Avatar 330 FTIR instrument in the diffuse
reflectance mode operating in the range of 400–4000 Cm  1 and
resolution of 4 Cm  1.
3. Results and discussion
In this paper, we report on the use of anti-tumor potent Crocus
sativus (saffron) extract for the synthesis of gold nanoparticles
(AuNPs) from aqueous HAuCl4 solution. The primary color of the
solution mixture of extract and aqueous HAuCl4 was colorless.
The intensity of the color of reaction mixture changes to dark
purple after 2 h of reaction. This shows that the reaction medium
behaves with time kinetics; the same is shown in Fig. 1. The color
change in the solution indicates the formation of AuNPs. This
observation shows that the reduction of Au3 þ ions takes place in
the solution due to the addition of extracts. Color of the AuNPs
found to be dark purple in water is due to excitation of the surface
Plasmon resonance (SPR) in metal nanoparticles [79]. It is
observed that the SPR band occurs at 549 nm and there was an
increase in intensity till 24 h as a function of time without any
shift in the peak wavelength as is shown in Fig. 2. The morphol-
ogy and size of the particles are determined by the HR-TEM
1.500
1.000
Abs.
24 h
0.500 1h 12h
5min
2h
0.000
225.00 400.00 600.00
nm.
Fig. 2. UV–vis spectrum recorded as a function of time of reaction of aqueous
solution of chloroauric acid with Crocus sativus (Saffron) extract.
667
images and are shown in Fig. 3(A–C). The HR-TEM images showed
that biosynthesized gold nanoparticles presented variable shape,
most of them were spherical in nature with some others having
occasionally triangular shape. The fringe spaces were observed
from the image (Fig. 3C) for the colloid, which correspond to the
spacing between the (1 1 1) plane of fcc gold [64]. The gold
nanoparticles formed were predominantly monodisperse with
diameter ranging from 11 to 20 nm where maximum number of
gold nanoparticles having average size is around 15 nm. The
selected area electron diffraction (SAED) patterns (Fig. 3D) reveal
that the particles are crystalline in nature. The SAED pattern
shows the diffraction ring from inner to outer, which can be
indexed as (1 1 1), (2 0 0), (2 2 0), (3 1 1) and (2 2 2) reflections of
fcc gold. Fig. 4 shows the XRD pattern suggesting the crystalline
nature of the AuNPs synthesized from anti-tumor potent Crocus
sativus (Saffron) extract. The intense diffraction peaks due to
AuNPs are clearly observed at (1 1 1), (2 0 0) and (2 2 0) reflec-
tions of fcc structure of metallic gold. These agree well with the
reported standards in JCPDS file no. 04-0784. This study reveal
that the synthesized AuNPs are of pure crystalline gold. The mean
particle diameter of AuNP was calculated from the XRD pattern
according to the line width of the (1 1 1) refraction peak using the
Debye Scherer’s equation. The calculated average particle size of
the Au was found to be 15 nm, which was also in line with the
observation of the HR-TEM study. Fig. 5 shows the SEM image of
synthesized AuNPs on micrometric scale. The nanoparticles were
uniformly distributed and are found to have formed in an
aggregated state. On careful observation, AuNPs are found to be
surrounded by a thin layer of bio-moieties (mostly flavonoids),
which seem to be responsible for reducing Au3 þ ions to AuNPs.
Most of the particles are spherical in shape and monodispersed in
nature with diameters in the range of 11–20 nm. The biosynthe-
sized gold nanoparticles were studied using FTIR. Fig. 6 shows the
FTIR bands at 1636 cm  1 1384 cm  1, 1075 cm  1, 1047 cm  1,
3411 cm  1, 1245 cm  1, 1400 cm  1, 2343 cm  1 and 2926 cm  1.
This represents different functional groups of adsorbed biomole-
cules on the surface of the nanoparticles and also indicates the
influence of organic moieties on the formation of Au nanoparti-
cles and for stabilization in the aqueous medium. The peak at
1636 cm  1 indicates –CQC– aromatic stretching [80]. In addi-
tion, clearly more absorption occurs in broad spectral range of
1384 1075 cm  1 showing the interactions between gold nano-
particles. The peak at 1047 cm  1 indicates the presence of
amine groups [81]. The strong band observed at 1245 cm  1
corresponds to the amide I–III bands of polypeptide/proteins
[82]. The band at 3411 cm–1 is reported to occur due to amine
group stretching vibrations superimposed on the side of hydroxyl
group [83]. It is suggested that the 2343 cm(  1) band is due to
CO(2) dissolved in water, most likely produced at the final point
of fermentation of amino acid by the microorganism. A major
peak was observed at 2926 cm  1 that could be assigned to C–H
stretching vibrations of methyl, methoxy and methylene groups.
The peaks at 1636 cm  1, 1384 cm  1 and 1075 cm  1 clearly show
the presence of flavonoids, tannins and terpenoids. This clearly
shows that the freely water soluble flavonoids present in the
saffron solution could have been adsorbed on the surface and the
same may be induced in the catalytic reduction of Au3 þ ions to
Au0 nanoparticles with interaction of carbonyl groups or p-
electrons. The internal conversion mechanism of ketone group
to carboxylic acid in flavonoids may also be involved in the Au3 þ
ion reduction. Specially the conversion of CQO group of terpenes
to –C(O)QO group may be responsible for the reduction of gold
800.00
ions. In addition to that the peaks at 1245 cm  1, 3411 cm  1,
2343 cm  1 and 2926 cm  1 indicate that the protein molecules in
the saffron may also participate in the process of formation of
nanoparticles.
668 R. Vijayakumar et al. / Physica E 44 (2011) 665–671

Fig. 3. HR-TEM images indicating the presence of spherical and triangular nanoparticles recorded at various magnifications (A–C). (D) The selected area electron
diffraction (SAED) pattern.

Fig. 5. (A) SEM images of gold nanoparticles obtained using Crocus sativus
(Saffron) extract.

Fig. 4. XRD pattern of gold nanoparticles obtained using Crocus sativus (Saffron)
extract.
In this present study the synthesis of nanoparticles using
biological components and the potential applications of bio-
synthesized nanoparticles as new advanced nanomaterials are
currently discussed. For this purpose, the plant with highest anti-
tumor potent Crocus sativus has been selected to synthesize
AuNPs by a simple and eco-friendly method. Several concentra-
tions of gold ions and extract solution for the experiment have
been investigated, compared with other concentrations of gold
ions and extract solution; we found that gold nanoparticles with
uniform and appropriate size can be biosynthesized only in the
concentration of 1 mM HAuCl4 solution and 4 ml extract solution.
Therefore, 0.5 mM HAuCl4 solution and 4 ml extract solution were
selected to do the above experiments. It is well known that gold
nanoparticles exhibit purple color in aqueous solution due to the
excitation of surface Plasmon vibrations in gold nanoparticles.
Therefore, reduction of the gold ions to gold nanoparticles during
exposure to the extract could be followed by UV–vis spectroscopy
 R. Vijayakumar et al. / Physica E 44 (2011) 665–671 669

Fig. 6. FT-IR spectra of (A) Crocus sativus (Saffron) extract, (B) gold nanoparticles synthesized by Crocus sativus (Saffron) extract.

and color change. The appearance of the purple color and the
maximum absorbance were indications of the formation of
colloidal gold particles in the medium. The increase in intensity
could be due to increasing number of nanoparticles formed as a
result of reduction of gold ions by extract. With increasing
incubation time till 24 h, the absorbance at around 549 nm did
not increase anymore, which suggested that almost all of the gold
ions convert to gold nanoparticles. Since the peak wavelength
did not shift during the reaction, we could quantitatively monitor
the concentrations of gold nanoparticles by measuring the
670 R. Vijayakumar et al. / Physica E 44 (2011) 665–671
absorbance at 549 nm. Proteins could most possibly form a coat
covering the AuNPs to prevent agglomeration of the particles
stabilizing them in the medium. This evidence suggests that the
biological molecules could possibly perform the function for
the formation and stabilization of the AuNPs in aqueous medium.
The stabilization of nanoparticles and the functional groups of the
compounds responsible were identified using FT-IR studies. The
results obtained from the HR-TEM and SEM study gives a clear
indication regarding the shape and size of the nanoparticles. The
AuNPs formed were predominantly monodisperse with diameter
of 11–20 nm. On careful observation of various magnifications of
HR-TEM images of gold nanoparticles it is noted that the particles
are of uniform size. The crystalline nature of the AuNPs can be
studied by measuring XRD pattern. The XRD pattern measured in
this case resulted in three intense peaks observed in the spec-
trum, which agrees with the Bragg’s reflection of AuNPs. The
average size of the AuNPs was calculated using Debye–Scherer’s
equation. Crystalline nature of NPs is evident from bright circular
spots in the SAED pattern, clear lattice fringes in the HR-TEM
images and peaks in the XRD pattern. On storage of the bio-
functionalized AuNPs synthesized using anti-tumor potent Crocus
sativus (Saffron), it was observed that the colloidal solution
maintains its stability and uniformity for more than 6 weeks.
4. Conclusion
The biosynthesis of gold nanoparticles using anti-tumor potent
Crocus sativus (Saffron) has been reported in this study. It has
been demonstrated that the Crocus sativus (Saffron) extract is
capable of producing fairly mono-dispersed spherical and trian-
gular gold nanoparticles with well defined dimensions with the
size of 11–20 nm. Only 24 h of reaction time is required for
conversion of almost all gold ions to gold nanoparticles at the
room temperature. Using the clean-green chemistry involved in
this way, we can think of the industrial scaling of nanoparticles.
This field of gold nanoparticles biosynthesis is leading to new
avenues in material science, chemistry, nanobiotechnology and
biotechnology. This simple procedure for the biosynthesis of gold
nanoparticles has several advantages such as cost-effectiveness,
compatibility for biomedical and pharmaceutical applications as
well as for large-scale commercial production. In future, it would
be significant to select such plants, to understand the clear
mechanism of biosynthesis and to technologically engineer
the nanoparticles in order to achieve better control over size,
shape and absolute monodispersivity to utilize the potential of
herbal medicine in nanoscience for anti-tumor or biomedical
applications.
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