This article appeared in a journal published by Elsevier.

The attached
copy is furnished to the author for internal non-commercial research
and education use, including for instruction at the authors institution
and sharing with colleagues.
Other uses, including reproduction and distribution, or selling or
licensing copies, or posting to personal, institutional or third party
websites are prohibited.
In most cases authors are permitted to post their version of the
article (e.g. in Word or Tex form) to their personal website or
institutional repository. Authors requiring further information
regarding Elsevier’s archiving and manuscript policies are
encouraged to visit:
http://www.elsevier.com/copyright
 Author's personal copy

Process Biochemistry 44 (2009) 1133–1138

Contents lists available at ScienceDirect

Process Biochemistry
journal homepage: www.elsevier.com/locate/procbio

Biological synthesis of gold nanoparticles using Magnolia kobus

and Diopyros kaki leaf extracts
Jae Yong Song, Hyeon-Kyeong Jang, Beom Soo Kim *
Department of Chemical Engineering, Chungbuk National University, Cheongju, Chungbuk 361-763, Republic of Korea

A R T I C L E I N F O A B S T R A C T

Article history: Leaf extracts of two plants, Magnolia kobus and Diopyros kaki, were used for ecofriendly extracellular
Received 5 January 2009 synthesis of metallic gold nanoparticles. Stable gold nanoparticles were formed by treating an aqueous
Received in revised form 6 June 2009 HAuCl4 solution using the plant leaf extracts as reducing agents. UV–visible spectroscopy was used for
Accepted 10 June 2009
quantification of gold nanoparticle synthesis. Only a few minutes were required for >90% conversion to
gold nanoparticles at a reaction temperature of 95 8C, suggesting reaction rates higher or comparable to
Keywords: those of nanoparticle synthesis by chemical methods. The synthesized gold nanoparticles were
Biological synthesis
characterized with inductively coupled plasma spectrometry (ICP), energy-dispersive X-ray spectro-
Gold
Nanoparticles
scopy (EDS), scanning electron microscopy (SEM), transmission electron microscopy (TEM), atomic force
Plant extracts microscopy (AFM), X-ray photoelectron spectroscopy (XPS), Fourier-transform infrared spectroscopy
Magnolia kobus (FTIR), and particle analysis using a particle analyzer. SEM and TEM images showed that a mixture of
Diopyros kaki plate (triangles, pentagons, and hexagons) and spherical structures (size, 5–300 nm) were formed at
lower temperatures and leaf broth concentrations, while smaller spherical shapes were obtained at
higher temperatures and leaf broth concentrations.
ß 2009 Elsevier Ltd. All rights reserved.

1. Introduction
Nanoparticles, generally considered as particles with a size of
up to 100 nm, exhibit completely new or improved properties as
compared to the larger particles of the bulk material that they are
composed of based on specific characteristics such as size,
distribution, and morphology [1]. Nanoparticles of noble metals,
such as gold, silver, and platinum, are widely applied in products
that directly come in contact with the human body, such as
shampoos, soaps, detergent, shoes, cosmetic products, and tooth-
paste, besides medical and pharmaceutical applications. Gold has a
long history of use. Red colloidal gold has been used as medicine for
revitalization in China and India [2]. Gold nanoparticles have found
use in diagnostic and drug delivery applications [3]. Therefore,
there is a growing need to develop environmentally friendly
processes for nanoparticle synthesis without using toxic chemi-
cals. Biological methods for nanoparticle synthesis using micro-
organisms, enzymes, and plants or plant extracts have been
suggested as possible ecofriendly alternatives to chemical and
physical methods [4].
Using plants for nanoparticle synthesis can be advantageous
over other biological processes because it eliminates the elaborate
* Corresponding author. Tel.: +82 43 261 2372; fax: +82 43 269 2370.
E-mail address: bskim@chungbuk.ac.kr (B.S. Kim).
process of maintaining cell cultures and can also be suitably scaled
up for large-scale nanoparticle synthesis [5]. Gardea-Torresdey
et al. [6,7] demonstrated gold and silver nanoparticle synthesis
within live alfalfa plants from solid media. Extracellular nano-
particle synthesis using plant leaf extracts rather than whole plants
would be more economical owing to easier downstream proces-
sing. Pioneering works on nanoparticle synthesis using plant
extracts have been carried out by Sastry and others [5,8–13] who
reported that nanoparticles can be synthesized using plant extracts
at rates comparable to those of chemical methods. The shape of
nanoparticles plays a crucial role in the modulation of their optical
properties. Gold nanotriangles were formed when lemongrass
(Cymbopogon flexuosus) leaf extract was reacted with aqueous
AuCl4 ions [10]. Gold and silver nanotriangles, in particular, are
promising because they may find potential applications in the
treatment of cancer hyperthermia and in infrared radiation-
absorbent optical coatings [13].
There have been recent reports on phytosynthesis of silver and
gold nanoparticles by employing coriander leaves [14], sundried
Cinnamomum camphora leaves [15], phyllanthin extract [16], and
purified apiin compound extracted from henna leaves [17]. In case
of sundried C. camphora leaves, the polyol and water-soluble
heterocyclic components were mainly found to be responsible for
the reduction of silver or chloroaurate ions and stabilization of
nanoparticles, respectively [15]. In phyllanthin-assisted silver and
gold nanoparticle synthesis, the rate of reduction of HAuCl4 was

1359-5113/$ – see front matter ß 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.procbio.2009.06.005
 Author's personal copy

1134 J.Y. Song et al. / Process Biochemistry 44 (2009) 1133–1138

greater than that of AgNO3, at a given constant level of phyllanthin

extract [16]. The size and shape of the nanoparticles could be
controlled by varying the concentration of the phyllanthin extract
thereby to tune the optical properties of the nanoparticles.
Shankar et al. [5] reported pure metallic silver and gold
nanoparticle synthesis by the reduction of Ag+ and Au3+ ions using
Neem (Azadirachta indica) leaf broth. The time required for >90%
reduction of Ag+ and Au3+ ions using Neem leaf broth was
approximately 4 and 2 h, respectively. In order to enable the
biological methods of nanoparticle synthesis to compete with the
chemical methods, there is a need to achieve faster synthesis rates
with the former. We recently reported that silver nanoparticles
could be rapidly prepared using screened plant extracts [18],
requiring only 11 min for >90% conversion at a reaction
temperature of 95 8C using Magnolia leaf broth. The size of the
silver nanoparticles could also be controlled by changing the
reaction conditions. In this study, we carried out rapid synthesis of
gold nanoparticles using the leaf extracts of two screened plants,
i.e., Magnolia kobus and Diopyros kaki. We also investigated the Fig. 1. UV–vis spectra recorded as a function of the reaction time for the reaction of
effects of reaction conditions such as the reaction temperature and 1 mM HAuCl4 solution with 5% Magnolia kobus leaf broth.

leaf broth concentration on the particle size and morphology of the

synthesized gold nanoparticles.
2. Materials and methods
2.1. Synthesis of gold nanoparticles
Magnolia (M. kobus) and Persimmon (D. kaki) leaves were collected and dried for
2 d at room temperature. The plant leaf broth solution was prepared by taking 5 g of
thoroughly washed and finely cut leaves in a 300-mL Erlenmeyer flask along with
100 mL of sterile distilled water and then boiling the mixture for 5 min before
finally decanting it. The solutions were stored at 4 8C and used within a week.
Typically, 10 mL of leaf broth was added to 190 mL of 1 mM aqueous HAuCl4
solution for the reduction of Au3+ ions. The effects of temperature on the synthesis
rate and particle size/shape of the prepared gold nanoparticles were studied by
carrying out the reaction in a water bath at 25–95 8C with reflux. The leaf broth
concentrations were also varied between 5 and 50% by volume. The gold
nanoparticle solution thus obtained was purified by repeated centrifugation at
15,000 rpm for 20 min followed by redispersion of the pellet in deionized water.
2.2. Characterization of gold nanoparticles
UV–vis spectra were recorded as a function of the reaction time on a UV-1650CP
Shimadzu spectrophotometer operated at a resolution of 1 nm. The purified gold
particles were freeze-dried, and their structure and composition were analyzed by
high-resolution transmission electron microscopy (HR-TEM; JEOL-2010), field-
emission scanning electron microscopy (FE-SEM; LEO-1530), atomic force
microscopy (AFM; Dimension 3100), energy-dispersive X-ray spectroscopy (EDS;
Sigma), X-ray photoelectron spectroscopy (XPS; ESCALAB 210), and Fourier-
transform infrared spectroscopy (FTIR; Bomem MB100). Gold concentrations and
conversion were determined using inductively coupled plasma spectrometry (ICP;
JY38Plus). The average particle size and distribution were measured using a particle
analyzer (NICOMPTM 380 ZLS).
between the gold concentration determined by ICP and the
absorbance at 540 nm. It was reported that flat gold nanoparticles
absorb the wavelength in the NIR region of the electromagnetic
spectrum, which corresponds to the longitudinal surface plasmon
absorption [13]. From Fig. 1, it is obvious that absorption of longer
wavelengths in the NIR region increases with time, suggesting the
formation of flat gold nanoparticles using Magnolia leaf broth as
the reducing agent.
Fig. 2 shows the time courses of gold nanoparticle synthesis
with different reaction temperatures obtained using Persimmon
leaf broth. As the reaction temperature increased, the gold
nanoparticle synthesis rate increased. The conversion at 95 8C
was 79% after 1 min and reached almost 100% after 5 min. At room
temperature, there was an initial lag period for the formation of
gold nuclei, and the final conversion was only ca. 60%. The result
using Magnolia leaf broth as the reducing agent is shown in Fig. 3A.
It required only 3 min for >90% conversion to gold nanoparticles at
95 8C, which was faster than the time required for silver
nanoparticle synthesis using the same leaf broth, i.e., 11 min,
owing to the higher reduction potential of Au3+ ions than Ag+ ions
[18]. The final conversion to gold nanoparticles was almost 100% at
all reaction temperatures; however, the synthesis time was longer
at room temperature. The increase in the reaction rate with the
reaction temperature was also reported by Rai et al. in case of gold
nanotriangle synthesis using lemongrass extract [11] and by our
group in case of silver nanoparticle synthesis using Persimmon and

3. Results and discussion

3.1. Effect of reaction temperature

Fig. 1 shows the UV–vis spectra recorded from the aqueous

solution of 1 mM HAuCl4 as a function of the reaction time using
Magnolia leaf broth at 25 8C. The maximum absorbance was
observed to occur at ca. 540 nm, and the intensity steadily
increased to saturation as a function of the reaction time. Gold
nanoparticles are known to exhibit a ruby-red color in aqueous
solutions due to excitation of the surface plasmon vibrations in the
gold nanoparticles [5]. Reduction of the gold ion to gold
nanoparticles during exposure to the plant leaf extracts could
be detected by the color change and, thus, by UV–vis spectroscopy.
Since the peak wavelength did not shift during the reaction, we
could quantitatively monitor the concentrations of the gold
nanoparticles and, consequently, the conversion by measuring Fig. 2. Time courses of gold nanoparticles synthesized by the reaction of 1 mM
the absorbance at 540 nm. A linear relationship was obtained HAuCl4 and 5% Diopyros kaki leaf broth at different reaction temperatures.
 Author's personal copy
J.Y. Song et al. / Process Biochemistry 44 (2009) 1133–1138
Fig. 3. (A) Time courses of gold nanoparticles synthesized by the reaction of 1 mM
HAuCl4 and 5% Magnolia kobus leaf broth at different reaction temperatures. (B)
Effect of the reaction temperature on the average gold nanoparticle size.
Magnolia leaf broth [18]. The average particle sizes with the
reaction temperatures are shown in Fig. 3B. The particle size
decreased from 110 nm at 25 8C to 40 nm at 95 8C. We observed a
similar trend with silver nanoparticle synthesis using Persimmon
and Magnolia leaf broth and discussed the reason behind the
decrease in the particle size with temperature as follows. As the
reaction temperature increases, the reaction rate increases and,
thus, most gold ions are consumed in the formation of the nuclei,
thereby stopping the secondary reduction process on the surface of
the preformed nuclei [18].
Fig. 4 shows the TEM images obtained by the reaction of 5%
Magnolia leaf broth and 1 mM HAuCl4 solution at three different
temperatures (A: 25 8C, B: 60 8C, and C: 95 8C). A mixture of plate
(triangles, pentagons, and hexagons) and spheres was obtained at
25 and 60 8C, while mainly spherical shapes were obtained at
95 8C. A similar trend is also observed in the SEM images obtained
by the reaction of 5% Persimmon leaf broth and 1 mM HAuCl4
solution at various temperatures (Fig. 5). It is clear that the
triangles, pentagons, and hexagons are plate structures with sizes
of up to 300 nm, and most plates disappeared at 95 8C. The change
in morphology with the reaction temperature can be discussed in
Fig. 4. TEM images of the gold nanoparticles formed by the reaction of 1 mM HAuCl4 and 5% Magnolia kobus leaf broth at different reaction temperatures: (A) 25 8C, (B) 60 8C,
and (C) 95 8C.
1135
relation to the reaction rate. At higher temperatures, most gold
ions first form nuclei, and the secondary growth of the particles
stops because the reaction rate is very high. Fig. 6 is AFM image of
one gold nanoparticle synthesized with 5% Magnolia leaf broth at
25 8C. It is shown that the nanoplate structure was formed with a
width of 250–300 nm and a thickness of 5–7 nm. The EDS and XPS
spectra recorded from the gold nanoparticles are shown in Fig. 7A
and B, respectively. The EDS profile shows a strong gold signal
along with weak oxygen and carbon peaks, which may have
originated from the biomolecules bound to the surface of the gold
nanoparticles. It has been reported that nanoparticles synthesized
using plant extracts are surrounded by a thin layer of some capping
organic material from the plant leaf broth and are, thus, stable in
solution up to 4 weeks after synthesis [5,18]. This is another
advantage of nanoparticles synthesized using plant extracts over
those synthesized using chemical methods. The XPS spectrum
shows the characteristic gold peaks on the surface of the
nanoparticles, suggesting successful gold nanoparticle synthesis
using plant leaf broth.
3.2. Effect of leaf broth concentration
We further investigated the possibility of controlling the
particle size and shape by changing the composition of the
reaction mixture. Fig. 8 shows the TEM images of the gold
nanoparticles synthesized using different Magnolia leaf broth
concentrations (A: 10%, B: 20%, and C: 50%) with 1 mM HAuCl4 at
60 8C for 30 min. As compared to results of the 5% leaf broth
concentration shown in Fig. 4B, large plate structures could not be
detected and most particles were spherical at leaf broth
concentrations >10%. The particle size was observed to decrease
with an increase in the leaf broth concentration (Fig. 8D). Similar
changes in the shapes and size were observed on phyllanthin-
assisted gold nanoparticle biosynthesis [16]. The use of a low
concentration of the plant extract reacting with HAuCl4 led to
the formation of hexagonal or triangular gold nanoparticles, while
the shape of the nanoparticles changed to spherical on increasing
the concentration of the phyllanthin extract. Control of the shape
and size of metallic nanoparticles enables tuning of their optical,
electronic, magnetic, and catalytic properties. Gold nanoparticles
synthesized by chemical methods are generally spherical, and
nanostructures with a triangular morphology are rare. It has been
reported that the shape and size of nanoparticles synthesized using
plant extracts could be controlled by chemical and physical
methods [11,13,19].
3.3. FTIR analysis of gold nanoparticles
FTIR analysis was used for the characterization of the
extract and the synthesized nanoparticles (Fig. 9). The FTIR
spectra of the M. kobus leaf extract before and after bioreduction
did not show any significant changes. The FTIR spectrum of the
 Author's personal copy

1136 J.Y. Song et al. / Process Biochemistry 44 (2009) 1133–1138

Fig. 5. SEM images of the gold nanoparticles formed by the reaction of 1 mM HAuCl4 and 5% Diopyros kaki leaf broth at different reaction temperatures: (A) 25 8C, (B) and (C)
60 8C, and (D) 95 8C.

Fig. 7. Characterization of the gold nanoparticles formed by the reaction of 1 mM

Fig. 6. AFM image of the gold nanoparticles formed by the reaction of 1 mM HAuCl4 HAuCl4 and 5% Magnolia kobus leaf broth at 25 8C. (A) Spot profile EDS spectrum and
and 5% Magnolia kobus leaf broth at 25 8C. (B) XPS spectrum.
 Author's personal copy
J.Y. Song et al. / Process Biochemistry 44 (2009) 1133–1138
Fig. 8. TEM images of the gold nanoparticles formed by the reaction of 1 mM HAuCl4
and various concentrations of the Magnolia kobus leaf broth at 60 8C for 30 min: (A)
10%, (B) 20%, and (C) 50%. (D) Effect of leaf broth concentration on the average gold
nanoparticle size.
Fig. 9. FTIR spectra of the Magnolia kobus extract (curve 1) and of the gold
nanoparticles synthesized by the reduction of AuCl4 with the Magnolia kobus leaf
extract (curve 2).
leaf extract (curve 1) showed bands at 3332 and 1637 cm 1. The
intense broad absorbance at 3332 cm 1 is the characteristic of the
hydroxyl functional group in alcohols and phenolic compounds.
The band at 1637 cm 1 can be assigned to the amide I band of the
proteins released by the Magnolia leaves or to C5 5C groups/
aromatic rings. The FTIR spectrum of the gold nanoparticles (curve
2) showed bands at 1024, 1227, 1629, 1736, and 2916 cm 1 along
with other small bands. The band at 1024 cm 1 corresponds to the
C–N stretching vibration of aliphatic amines or to alcohols/
phenols. The weaker bands at 1227 and 1629 cm 1 correspond to
the amide III and amide I bands of proteins, respectively. The
bands at 1736 and 2916 cm 1 can be assigned to the carbonyl
groups and secondary amines, respectively. This indicates that
gold nanoparticles synthesized using the M. kobus extract are
surrounded by some proteins and metabolites such as terpenoids
having functional groups of amines, alcohols, ketones, aldehydes,
and carboxylic acids.
1137
Regarding the mechanism of biological nanoparticle synthesis,
it has been reported that reduction occurs due to the NADH-
dependent reductase released into the solution in case of gold
nanoparticles synthesized extracellularly by the fungus Fusarium
oxysporum [20]. It has also been suggested that nitroreductase
enzymes may be involved in silver nanoparticle synthesis using the
culture supernatants of Enterobacteria [21]. In case of Neem leaf
broth, terpenoids are believed to be the surface-active molecules
stabilizing the nanoparticles, and the reaction of the metal ions is
possibly facilitated by the reducing sugars and/or terpenoids
present in the Neem leaf broth [5]. Terpenoids (isoprenoids) are a
large and diverse class of naturally occurring organic chemicals
derived from five-carbon isoprene units assembled and modified
in a variety of ways. These lipids can be found in all classes of living
things and are the largest group of natural products. Thus, many
plant extracts can be used to synthesize metallic nanoparticles
owing to the existence of terpenoids and reducing sugars in them.
In our process of screening of plant extracts with high nanoparticle
synthesis capabilities, all the plants tested could synthesize gold
and silver nanoparticles, albeit with different synthesis rates [18].
In conclusion, we proposed an ecofriendly method for gold
nanoparticle synthesis using plant extracts. The proposed method
requires only a few minutes for >90% conversion by using
Magnolia and Persimmon leaf broths and by increasing the
reaction temperature to 95 8C; the reaction rate thus obtained was
higher or comparable to the rate of gold nanoparticle synthesis by
chemical methods. The particle size ranging from 5 to 300 nm and
the shape of the plate and spherical structures could be controlled
by changing the reaction temperature and leaf broth concentra-
tion. This environmentally friendly method of biological gold
nanoparticle synthesis can potentially be applied in various
products that directly come in contact with the human body,
such as cosmetics, foods, and consumer goods, besides medical
applications.
Acknowledgments
This research was financially supported by the Ministry of
Knowledge Economy (MKE) and Korea Industrial Technology
Foundation (KOTEF) through the Human Resource Training Project
for Strategic Technology.
References
[1] Willems, van den Wildenberg. Roadmap report on nanoparticles. Barcelona,
Spain: W&W Espana sl; 2005.
[2] Bhattacharya R, Murkherjee P. Biological properties of ‘‘naked’’ metal nano-
particles. Adv Drug Deliv Rev 2008;60:1289–306.
[3] Bhumkar DR, Joshi HM, Sastry M, Pokharkar VB. Chitosan reduced gold
nanoparticles as novel carriers for transmucosal delivery of insulin. Pharm
Res 2007;24:1415–26.
[4] Mohanpuria P, Rana NK, Yadav SK. Biosynthesis of nanoparticles: technolo-
gical concepts and future applications. J Nanopart Res 2008;10:507–17.
[5] Shankar SS, Rai A, Ahmad A, Sastry M. Rapid synthesis of Au, Ag, and bimetallic
Au core Ag shell nanoparticles using Neem (Azadirachta indica) leaf broth. J
Colloid Interface Sci 2004;275:496–502.
[6] Gardea-Torresdey JL, Parsons JG, Gomez E, Peralta-Videa J, Troiani HE, Santiago
P, et al. Formation and growth of Au nanoparticles inside live alfalfa plants.
Nano Lett 2002;2:397–401.
[7] Gardea-Torresdey JL, Gomez E, Peralta-Videa J, Parsons JG, Troiani HE, Santiago
P, et al. Alfalfa sprouts: a natural source for the synthesis of silver nanopar-
ticles. Langmuir 2003;19:1357–61.
[8] Shankar SS, Ahmad A, Pasricha R, Sastry M. Bioreduction of chloroaurate ions
by geranium leaves and its endophytic fungus yields gold nanoparticles of
different shapes. J Mater Chem 2003;13:1822–6.
[9] Shankar SS, Ahmad A, Sastry M. Geranium leaf assisted biosynthesis of silver
nanoparticles. Biotechnol Prog 2003;19:1627–31.
[10] Shankar SS, Rai A, Ankamwar B, Singh A, Ahmad A, Sastry M. Biological
synthesis of triangular gold nanoprisms. Nat Mater 2004;3:482–8.
[11] Rai A, Singh A, Ahmad A, Sastry M. Role of halide ions and temperature on the
morphology of biologically synthesized gold nanotriangles. Langmuir
2006;22:736–41.
 Author's personal copy
1138 J.Y. Song et al. / Process Biochemistry 44 (2009) 1133–1138
[12] Rai A, Chaudhary M, Ahmad A, Bhargava S, Sastry M. Synthesis of
triangular Au core–Ag shell nanoparticles. Mater Res Bull 2007;42:
1212–20.
[13] Chandran SP, Chaudhary M, Pasricha R, Ahmad A, Sastry M. Synthesis of gold
nanotriangles and silver nanoparticles using Aloe vera plant extract. Biotech-
nol Prog 2006;22:577–83.
[14] Narayanan KB, Sakthivel N. Coriander leaf mediated biosynthesis of gold
nanoparticles. Mater Lett 2008;62:4588–90.
[15] Huang J, Li Q, Sun D, Lu Y, Su Y, Yang X, et al. Biosynthesis of silver and gold
nanoparticles by novel sundried Cinnamomum camphora leaf. Nanotechnology
2007;18:105104.
[16] Kasthuri J, Kathiravan K, Rajendiran N. Phyllanthin-assisted biosynthesis of
silver and gold nanoparticles: a novel biological approach. J Nanopart Res
2009;11:1075–85.
[17] Kasthuri J, Veerapandian S, Rajendiran N. Biological synthesis of silver and gold
nanoparticles using apiin as reducing agent. Colloids Surf B Biointerf
2009;68:55–60.
[18] Song JY, Kim BS. Rapid biological synthesis of silver nanoparticles using plant
leaf extract. Bioprocess Biosyst Eng 2009;32:79–84.
[19] Ogale SB, Ahmad A, Pasricha R, Dhas VV, Syed A. Physical manipulation of
biological and chemical syntheses for nanoparticle shape and size control.
Appl Phys Lett 2006;89:263105.
[20] Mukherjee P, Senapati S, Mandal D, Ahmad A, Khan MI, Kumar R, et al.
Extracellular synthesis of gold nanoparticles by the fungus Fusarium oxy-
sporum. ChemBioChem 2002;5:461–3.
[21] Shahverdi A, Minaeian S, Shahverdi HR, Jamalifar H, Nohi A-A. Rapid synthesis
of silver nanoparticles using culture supernatants of Enterobacteria: a novel
biological approach. Process Biochem 2007;42:919–23.