A.k.jha 2010

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International Journal of Green Nanotechnology: Physics
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Green Synthesis of Silver Nanoparticles Using Cycas
Leaf
Anal K. Jha
a
& K. Prasad
b
a
Department of Chemistry , T. M. Bhagalpur University , 812007, India
b
Department of Physics , T. M. Bhagalpur University , Bhagalpur, 812007, India
Published online: 26 Mar 2010.
To cite this article: Anal K. Jha & K. Prasad (2010) Green Synthesis of Silver Nanoparticles Using Cycas Leaf, International
Journal of Green Nanotechnology: Physics and Chemistry, 1:2, P110-P117, DOI: 10.1080/19430871003684572
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International Journal of Green Nanotechnology: Physics and Chemistry, 1:P110–P117, 2010
Copyright c Taylor & Francis Group, LLC
ISSN: 1943-0876 print / 1943-0884 online
DOI: 10.1080/19430871003684572
Green Synthesis of Silver Nanoparticles Using Cycas Leaf
Anal K. Jha
K. Prasad
ABSTRACT. A green, low-cost, and reproducible Cycas leaf–negotiated synthesis of silver nanoparti-
cles is reported. X-ray and transmission electron microscopy (TEM) analyses are performed to ascertain
the formation of Ag nanoparticles. Nanoparticles almost spherical in shape having the size of 2–6 nm
are found. Rietveld analysis to the X-ray data indicated that Ag nanoparticles have fcc unit cell structure.
Ultraviolet (UV)-visible study revealed the surface plasmon resonance at 449 nm. An effort has been
made to understand the possible involved mechanism for the biosynthesis of Ag nanoparticles. Present
procedure offers the benefit of eco-friendliness and amenability for large-scale production through
scaling up.
KEYWORDS. biosynthesis, green synthesis, nanobiotechnology, nanoparticle, nano silver
INTRODUCTION
Phytodiversity have not only been silent par-
ticipants of the exponential process of evolution,
rather have indeed survived the extremities of en-
vironmental rigors themselves by the prodigally
treasuring different valued metabolites. These
metabolites (both primary and secondary) have
played an indispensable role for the surviving
humanity. Right from cyanobacteria to giant tree
ferns and pines are unique due to their wide
yet well-organized treasures of metabolites, ca-
pable of interacting with each other, obeying
the principles of thermodynamics. Cycas, which
belongs to the family Cycadaceae, is a com-
mon gymnospermic plant and is the commer-
cial source of sago. Along with fatty acids (like
palmitic, stearic, oleic, and behenic acids), they
Received 1 January 2010; accepted 4 February 2010.
Anal K. Jha is affiliated with the Department of Chemistry, T. M. Bhagalpur University, 812007, India
K. Prasad is affiliated with the Department of Physics, T. M. Bhagalpur University, Bhagalpur 812007, India
The authors gratefully acknowledge Dr. Kamlesh Prasad, SLIET, Longowal, India, for arranging the TEM
micrograph and K. AmarNath, HEG Ltd., Bhopal, India, for kindly providing XRD data.
Address correspondence to K. Prasad,University Department of Physics, T. M. Bhagalpur University,
Bhagalpur 812007, India. E-mail: k.prasad65@gmail.com
are rich in flavonoids broadly belonging to the
class of phenolic compounds.[1]
The term phe-
nolic compound embraces a wide range of plant
substances that bear in common an aromatic
ring with one or more hydroxyl substituents.
Some 10 classes of flavonoids are recognized but
they are mainly hydrophilic compounds and are
present in all vascular plants. Whereas flavones
and flavonols are universal, isoflavones and espe-
cially biflavonyls are of limited distribution and
confined to the gymnosperms only. Cycas leaves
have been found to contain Amentiflavone and
Hinokiflavone as characteristic biflavonyls.[2–5]
Synthesis of metallic and/or oxide nanopar-
ticles taking assistance of plant extracts has at-
tained an upsurge in the immediate past.[6–12]
Although a lot of works have been done in bi-
ologically assisted synthesis of metal and oxide
P110
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A. K. Jha and K. Prasad P111
nanoparticles in the name of green synthesis in
the recent past, the actual issue has probably
not been addressed and that is the potential and
promises of plant systems. They are widely dis-
tributed along the ecological boundaries, are eas-
ily available and safe to handle, equipped with a
broad weaponry of metabolites, and, above all,
they are truly green while undertaking any chem-
ical protocol. Such studies could prove to have an
enormous impact in the immediate future if plant
tissue culture and downstream processing proce-
dures are applied in order to synthesize metallic
as well as oxide nanoparticles on industrial scale.
The present investigation is an effort in this di-
rection. In this work, Cycas-negotiated synthe-
sis of silver nanoparticles (abbreviated Ag NPs)
has been reported so that gymnosperms could
also be taken as potential candidate plant speci-
mens for the synthesis of metal as well as oxide
nanoparticles. An effort has been also been made
to understand the possible involved mechanism
for the biosynthesis of Ag NPs.
MATERIALS AND METHODS
Synthesis of Silver Nanoparticles Using
Cycas Leaf Broth
Known weight (5 g) of freshly collected, taxo-
nomically authenticated healthy leaves of Cycas
(inset Figure 1a) were taken and washed thor-
oughly in flush of tap water in the laboratory
for 10 min in order to remove the dust particles,
cut into small pieces, and rinsed briefly in sterile
distilled water. Then the sample was placed in a
250-mL beaker containing 200 mL 50% ethanol
(EtOH) and was placed on boiling steam bath
for 15 to 20 min until the color of the solvent
changes to light brown. This solution was not
treated with activated charcoal in order to avoid
adsorption of the probable candidate flavonoid
pigments along with chlorophylls and their con-
geners. The extract was cooled to room temper-
ature, pressed, and filtered firstly through sterile
serene cloth.
This solution was treated as source extract
and was utilized in subsequent procedures. Next,
40 mL of the source extract was doubled in
volume by adding 40 mL of sterile distilled
water. The extract solution was treated with 20
mL of 0.25 M AgNO3 solution and warmed
again on the steam bath for 10 min until the color
of the solution changes to reddish brown and
was allowed to cool and incubate in the labora-
tory ambience. Concurrently, ultraviolet-visible
(UV-vis) spectrophotometric study was pursued
in which 50% EtOH extract of Cycas was taken
as blank. The deposition gets distinctly visible
in the flask within 10 min, which was left for 4
h (inset, Figure 1a) and subsequently filtered.
Characterization
The formation of single-phase compound
was checked by X-ray diffraction (XRD) tech-
nique. The XRD data were collected using X-
ray diffractometer (XPERT-PRO, PW3050/60)
at room temperature, with CuKα radiation (λ =
1.5406 ˚A) between 20◦
and 90◦
. The XY (2θ ver-
sus intensity) data obtained from this experiment
were plotted with the WinPLOTR program and
the angular positions of the peaks were obtained
with the same program.[13]
The dimensions of
the unit cell, hkl values, and space group of
Ag NPs were obtained using the TREOR pro-
gram in the FullProf 2000 software package and
then refinement was carried out through the pro-
file matching routine of FullProf.[14]
The Bragg
peaks were modeled with pseudo-Voigt function
and the background was estimated by linear in-
terpolation between selected background points.
The lattice strain of Ag NPs was estimated by
analyzing the broadening of X-ray diffraction
peaks, using Williamson-Hall approach[15]
:
η cos θ = (Kλ/D) + 2( ξ/ξ) sin θ (1)
where η is diffraction full peak width at half
intensity (FWHM), ξ/ξ is the lattice strain,
and K is the Scherrer constant (0.89). The terms
Kλ/D and 2( ξ/ξ) sin θ, respectively, represent
the Scherrer particle size distribution and the
strain broadening. A Lorentzian model was ap-
plied to estimate the values of η. Transmission
electron microscopy (TEM) analysis of Ag NPs
was performed with Hitachi H-7500, operated at
80 kV. The specimen was suspended in distilled
water, dispersed ultrasonically to separate indi-
vidual particles, and two drops of the suspension

P112 INTERNATIONAL JOURNAL OF GREEN NANOTECHNOLOGY: PHYSICS AND CHEMISTRY
FIGURE 1. (a) TEM photograph, (b) SAED pattern, and (c) particle size distribution of Ag NPs.
Inset: Photograph showing Cycas leaf, deposition of Ag NPs and its bottom view.
was deposited onto holey-carbon–coated copper
grids and dried under infrared lamp. The absorp-
tion spectra of the sample were measured by
a computer-controlled UV-visible spectropho-
tometer (DR 5000; Hach, Germany).
RESULTS
Figure 1a shows the TEM image recorded
from drop-coated ﬁlm of Ag NPs synthesized by
treating AgNO3 solution with Cycas leaf broth
for 4 h. The micrograph clearly shows individ-
ual nanoparticles, almost spherical in shape with
diameters in the range of 2–6 nm. The measure-
ment of size was performed along the largest
diameter of the particles. Figure 1b shows the
selected area electron diffraction (SAED) pat-
tern obtained from Ag NPs. The Scherrer rings,
characteristic of fcc silver, are clearly seen, con-
ﬁrming nanocrystalline nature of Ag NPs as ob-
served in the TEM image (Figure 1a). The parti-
cle size histogram of Ag NPs (Figure 1c) shows
the distribution of particle sizes, which follows
a lognormal type of distribution:
D = Do + A/
√
2π.w.x
· exp − ln (x/xc)2
/ (/2.w)
2
(2)
where A, w, and xc are respectively the area,
full width at half height, and center of the curve.
The values of the parameters A, Do, w, and xc
are respectively estimated to be 116.37, −1.941,
0.40, and 3.29. The sizes of particles are ranged
from 2 to 6 nm and the average particle size
comes out to be 3.29 ± 0.22 nm. Also, it is

TABLE 1. The crystal data and refinement factors of Ag NPs obtained from X-ray powder
diffraction data
Parameters Results Description of parameters
Crystal system fcc Rp (profile factor) = 100[ |yi − yic|/ |yi|], where yi is the observed intensity and yic is
Space group Fm¯3m the calculated intensity at the ith step.
a ( ˚A) 4.0913 Rwp (weighted profile factor) = 100[ ωi|yi − yic|2/ ωi(yi)2]1/2, where ωi = 1/σ2
i and σ2
i
is variance of the observation.
V ( ˚A3) 68.4808 Rexp (expected weighted profile factor) = 100[(n − p)/ ωi(yi)2]1/2, where n and p are
the number of profile points and refined parameters, respectively.
Rp 45.3 RB (Bragg factor) = 100[ |Iobs − Icalc|/ |Iobs|], where Iobs is the observed integrated
intensity and Icalc is the calculated integrated intensity.
Rwp 41.5 RF (crystallographic RF factor) = 100[ |Fobs − Fcalc|/ |Fobs|], where F is the structure
factor, F =
√
(I/L), where L is Lorentz polarization factor.
Rexp 28.1 χ2 = ωi(yi − yic)2.
RB 0.0278 d(Durbin–Watson statistics) = {[ωi(yi − yic)-ωi−1(yi−1 − yic−1)]2}/ [ωi(yi − yic)]2.
RF 0.0533 QD = expected d.
χ2 2.177 S (goodness of fit) = (Rwp/Rexp).
d 1.0885
QD 1.8901
S 1.612
observed that the Ag NPs are scattered over the
surface and no aggregates are noticed under the
TEM. The difference in size is possibly due to
the fact that the nanoparticles are being formed
at different times.[7]
To ascertain the crystal structure of Ag NPs,
XRD study was carried out. Rietveld refinements
on the XRD data of Ag NPs were done, select-
ing the space group Fm¯3m. Figure 2 depicts the
observed, calculated, and difference XRD pro-
files for Ag NPs after final cycle of refinement.
It can be seen that the profiles for observed and
calculated ones are perfectly matching, which is
well supported by the value of χ2
(= 2.18). The
profile fitting procedure adopted was minimizing
the χ2
function.[16]
The XRD analyses indicated
that Ag NPs has a face-centered cubic (fcc) unit
cell having the sets of lattice planes (111), (200),
(220), (311), and (222). The crystal data and re-
finement factors of Ag NPs obtained from XRD
data are summarized in Table 1. The lattice pa-
rameter as obtained for Ag NPs is in good agree-
ment with the literature report (JCPDS file no.
04-0783). Also, it is important to note that the in-
tensity ratio I111/I200 comes out to be 9, which
is much higher than the conventional value ‘4’.
This indicates that the nanoparticles are abun-
dant in (111) plane. Thus, diffraction intensity
of (111) plane should be greatly enhanced in
comparison to that of other planes. This result
is in consistence with the earlier reports.[7,12,17]
The broadening of X-ray peaks observed is pri-
marily due to the small particle size. Inset of
Figure 2 is the Williamson-Hall plot for Ag NPs.
The lattice strain is estimated to be 0.0044 us-
ing the linear least-square fitting of ηcosθ− sinθ
data. The low value of lattice strain might be due
the fact that the procedure adopted in the syn-
thesis of nanoparticles is natural (biosynthetic)
one. Also, the structure of Ag NPs as observed
in SAED pattern resembles the XRD result.
Figure 3 shows the UV-vis spectra recorded
on 50% EtOH Cycas extract AgNO3 (0.25 M)
solution as a function of time of reaction. Im-
mediately after addition of AgNO3 solution to
the extract, the color of extract changes to yel-
lowish brown. At this stage, formation of metal
nanoparticles due to reduction was followed by
UV-vis spectroscopy. The generation of color
is due to excitation of surface plasmons in
metal nanoparticles.[18]
The silver surface plas-
mon resonance was observed at 449 nm, which
steadily increased in intensity as a function of
time of reaction (ranging from 30 to 240 min)
(inset Figure 3) without showing any shift of
the wavelength maximum. This simply indicates

FIGURE 2. Rietveld refined pattern of Ag NPs in the space group Fm¯3m. Symbols represent the
observed data points and the solid lines their Rietveld fit. Inset: Williamson-Hall plot for Ag NPs.
longitudinal plasmon vibrations. Also, the plas-
mon bands are broadened with an absorption
tail in the longer wavelengths, which may be
due to the size distribution of the particles.[19]
The inset of Figure 3 depicts the reduction of
the silver ions taking place at a faster rate and
that saturation of data occurs at 240 min, which
FIGURE 3. UV-vis spectra for Ag NPs recorded
as function of time of reaction of 0.25 M aqueous
solution of AgNO3 with Cycas. Inset: Absorbance
at λmax (ξ) as a function of time with nonlinear fit.
clearly indicates the completion of reaction. The
absorbance at λmax(ξ) – time data were modeled
with the function:
ξ(t) = ξs − κ.rt
(3)
where the parameters ξs and r (0 < r ≤ 1) are
respectively the saturation value of absorbance
and the rate of reaction. κ is the material de-
pendent constant, which may be taken as the
response range of the reaction. Here, ξ|t=0 is the
initial value of absorbance, representing the con-
trol value. For the present study, the estimated
value of ξ|t=0 comes out to be 0.13, which is
very close to the experimental value 0.11 (Fig-
ure 3). It is observed that the experimental data
fits excellently well (R2
= .99665) with the pro-
posed theoretical model (inset Figure 3). The
computer fitting of ξ(t) data with Eqn. 3 pro-
vided the model parameters: ξs = 2.91443, κ =
2.77805, and r = 0.96912. Further, it seems that
the present procedure of biosynthesis of Ag NPs
proceeds with a much faster pace and bioreduc-
tion of silver ions is complete within 4–6 h. In
earlier studies on synthesis of silver nanoparti-
cles employing bacteria,[20–23]
fungi,[19,24–26]
or
plant extracts,[6–10,27–29]
the time required for

FIGURE 4. Mechanism for the biosynthesis of Ag NPs using Cycas leaf broth.
completion (i.e., complete reduction of Ag ions)
ranged from 12 to 120, and are rather slow.
DISCUSSION
Salt/metal ion, as well as drought, chill-
ing and extreme temperatures, increase the lev-
els of reactive oxygen species (ROS). Exces-
sive levels of ROS lead to oxidative damage
to cellular molecules, aging, and cell death.
The antioxidative system is important for the
maintenance of intracellular ROS at appropriate
levels.[30]
Mechanisms of metal detoxiﬁcation
by biomolecules proceeds as cascade of events,
such as induction of proteins such as metal-
lothionein, heat-shock protein, phytochelatins,
and ferritin, transferring; or by triggering an-
tioxidant enzymes such as superoxide dismu-
tase, catalase, glutathione, and peroxidase; or
through high turnover of organic acids such as
malate, citrate, oxalate, succinate, aconitate, α-
ketoglutarate, etc. Various types of heavy metal
adaptation strategies in plants have exhaustively
been studied.[31]
Primarily two types of mech-
anisms may explain the resistance to the tox-
icity of metal ions in plants. The prominent
metal complexation processes are the synthesis
of phytochelatins and of other metal-chelating
peptides.[31,32]
Phytochelatins were found to be
induced by Ag, Bi, Cd, Cu, Hg, Ni, Sn, W, and
Zn, whereas no induction was noticed in the
case of Na, Mg, Al, Ca, V, Cr, Sb, Te, Mn, Fe,
Co, and Cs. Glutathione functions as a precursor
of phytochelatin synthesis. Metal-induced phy-
tochelatin production decreases cellular levels
of glutathione. Glutathione and its homologues,
viz. homoglutathione and hydroxymethylglu-
tathione, are the abundant low-molecular-weight
thiols in plants. Glutathione was implicated to
play a major role in plants exposed to metal
stress.[31]
Metallothionein (MT) is a family of cysteine-
rich, low-molecular-weight (MW ranging from
3500 to 14000 Da) proteins. MTs have the ca-
pacity to bind both physiological (such as zinc,

copper, selenium) and xenobiotic (such as cad-
mium, mercury, silver, arsenic) heavy metals
through the thiol group of its cysteine residues,
which represents nearly the 30% of its amino
acidic residues. The diversity of the plant MT
gene family suggests that these may differ not
only in sequence but also in function. There
is little information about MT genes in non-
flowering plant species. However, genes encod-
ing type 3 MTs have been cloned from several
gymnosperms.[33]
Antioxidant action of phenolic compounds is
due to their high tendency to chelate metals. Phe-
nolics possess hydroxyl and carboxyl groups,
able to bind particularly iron and copper. They
may inactivate iron ions by chelating and addi-
tionally suppressing the superoxide-driven Fen-
ton reaction, which is believed to be the most im-
portant source of ROS.[34,35]
Tannin-rich plants
such as tea, which are tolerant to Mn excess,
are protected by the direct chelation of Mn.
Plants contain two major types of peroxidases,
which can be divided into two groups: perox-
idases that use ascorbic acid as the preferen-
tial electron donor, and those that use phenolics.
Phenolics, especially flavonoids and phenylo-
propanoids, are oxidized by peroxidase, and act
in H2O2-scavenging system. Their antioxidant
action resides mainly in their chemical structure.
There is some evidence of induction of phenolic
metabolism in plants as a response to multiple
stresses (including heavy metal stress).[34]
Reduction is accomplished due to phy-
tochemicals (flavonoids or other any other
polyphenols) or phyotchelatins/glutathiones/
metallothioneins present in Cycas leaves paren-
chyma. Reduction of particle size by the
leaf broth in a tick compared to bacteria
or fungi may be perceived as an apprecia-
ble advancement. It is well established that
the biflavanone, tetrahydrohinokiflavone, to-
gether with amentoflavone, has been found in
the leaves of Cycas beddomei.[36]
In Cycas
leaf broth having well-defined metabolite trea-
sure, the process of nano-transformation might
have resulted due to redox activities of ascor-
bic/dehydroascorbic acid and amenti/hinoki
flavones and involvement of ascorbates/glutathi-
ones/metallothioneins (Figure 4), leading to the
reduction of silver ions present in the solution.
Therefore, compared to bacteria or fungi, plant
cells are much suitable candidates for the syn-
thesis of metallic nanoparticles. The significant
reduction in reaction time with Cycas leaf is
an important result and will enable nanoparti-
cle biosynthesis methods to compete with other
plant-assisted biosynthesis routes for the forma-
tion of silver nanoparticles that are currently
much more rapid and reproducible.
CONCLUSION
In conclusion, the present biotechnological
method is capable of producing Ag nanoparti-
cles. Also, it is a green, high-yield, fast, and low-
cost approach. Reduction is accomplished prob-
ably due to phytochemicals such as polyphenols,
glutathiones, metallothioneins, and ascorbates,
which may help in the production of Ag NPs as
a result of the detoxification procedure.
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