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Biological synthesis of copper nanoparticles using Magnolia kobus leaf extract

and their antibacterial activity

Article  in  Journal of Chemical Technology & Biotechnology · March 2013

DOI: 10.1002/jctb.4052

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Research Article
Received: 14 December 2012 Revised: 24 January 2013 Accepted article published: 21 February 2013 Published online in Wiley Online Library: 3 April 2013

(wileyonlinelibrary.com) DOI 10.1002/jctb.4052

Biological synthesis of copper nanoparticles

using Magnolia kobus leaf extract and their
antibacterial activity
Hyo-Jeoung Lee, Jae Yong Song and Beom Soo Kim∗

Abstract
BAKGROUND: Biological methods for metal nanoparticle synthesis using plant extracts have been suggested as possible
ecofriendly alternatives to chemical and physical methods. In the present study, copper nanoparticles were biologically
synthesized using Magnolia kobus leaf extract as reducing agent and their antibacterial activity was evaluated against
Escherichia coli.

RESULTS: On treatment of aqueous solution of CuSO4 ·5H2 O with Magnolia kobus leaf extract, stable copper nanoparticles
were formed. UV–vis spectroscopy was used to monitor the quantitative formation of copper nanoparticles. The synthesized
nanoparticles were characterized with inductively coupled plasma spectrometry (ICP), energy dispersive X-ray spectroscopy
(EDS), X-ray photoelectron spectroscopy (XPS), and high-resolution transmission electron microscopy (HR-TEM). Electron
microscopy analysis of copper nanoparticles indicated that they ranged in average size from 37 to 110 nm. Antibacterial
tests were carried out by counting viable E. coli cells after 24 h growth in shake flasks containing latex foams coated with
copper nanoparticles. As a result, foams coated with biologically synthesized copper nanoparticles showed higher antibacterial
activity compared with foams untreated and foams treated with chemically synthesized copper nanoparticles using sodium
borohydride and Tween 20. The antibacterial activities were inversely proportional to the average nanoparticle sizes.

CONCLUSION: The present results show that stable copper nanoparticles can be ecofriendly synthesized using Magnolia kobus
leaf extract, offering an inexpensive alternative to antibacterial silver nanoparticles.

c 2013 Society of Chemical Industry

Keywords: copper nanoparticles; Magnolia kobus; leaf extract; antibacterial activity

INTRODUCTION synthesis using microorganisms, enzymes, and plants or plant

Silver nanoparticles have been used extensively in many
antibacterial fields for several decades.1,2 Although only a few
studies have reported the antibacterial properties of copper
nanoparticles, they show copper nanoparticles hold significant
promise as a bactericidal agent.3,4 The antibacterial properties
of silver and copper nanoparticles were compared using various
microbial strains.4 Other nanoparticles, such as platinum, gold,
iron oxide, silica and its oxides, and nickel have not shown
bactericidal effects in studies with Escherichia coli.5 It was
also reported that copper nanoparticles demonstrated superior
antibacterial activity compared with silver nanoparticles.6 Silver
and copper nanoparticles were supported on various materials,
such as carbon, polyurethane foam, polymers and sepiolite for
bactericidal applications.2,7 – 9 The antibacterial activities of metal
nanoparticles were found to be related to their shapes and sizes.10
Production of metal nanoparticles can be achieved through
different methods. Chemical approaches are the most popular
methods for the production of nanoparticles. However, some
chemical methods cannot avoid the use of toxic chemicals in the
synthesis protocol. Since metal nanoparticles are widely applied
to human contacting areas, there is a growing need to develop
environmentally friendly processes of nanoparticle synthesis that
extracts have been suggested as possible ecofriendly alternatives
to chemical and physical methods.11 Using plants for nanoparticle
synthesis can have advantages over other biological processes
because it eliminates the elaborate process of maintaining cell
cultures and can also be suitably scaled up for large-scale
nanoparticle synthesis.11
Recently, we reported engineering approaches such as rapid
synthesis of silver, gold, platinum, and bimetal nanoparticles
using plant extracts and size/shape control of the synthesized
nanoparticles.12 – 16 Several plant leaf extracts were screened and
compared for their synthesis of nanoparticles. The synthesis rate
was highest with Magnolia kobus leaf broth as reducing agent.
The effects of reaction conditions such as temperature and
composition of the reaction mixture were also investigated to
control the size and shape of nanoparticles.
∗
Correspondence to: Beom Soo Kim, Department of Chemical Engineering,
Chungbuk National University, Cheongju, Chungbuk 361–763, Republic of
Korea. E-mail: bskim@chungbuk.ac.kr
Department of Chemical Engineering, Chungbuk National University,
1971

do not use toxic chemicals. Biological methods for nanoparticle Cheongju, Chungbuk 361-763, Republic of Korea

J Chem Technol Biotechnol 2013; 88: 1971–1977 www.soci.org

c 2013 Society of Chemical Industry
 www.soci.org
In this study, we report for the first time biological synthesis
of stable copper nanoparticles using plant leaf extract. Using
Magnolia kobus leaf broth as reducing agent, we investigated
the effects of reaction temperature and composition on
synthesis rate and particle size of copper nanoparticles. We also
evaluated antibacterial characteristics of the synthesized copper
nanoparticles by coating on latex foam products.
MATERIALS AND METHODS
Materials
Copper (II) sulfate pentahydrate (CuSO4 ·5H2 O; 99.5% purity) was
purchased from Samchun Chemical (Korea). Latex foam was
supplied from Latex Korea Co. (Cheongju, Korea).
Synthesis and characterization of copper nanoparticles
For biological synthesis of copper nanoparticles, Magnolia kobus
leaves were collected and dried for 2 days at room temperature.
The plant leaf broth solution was prepared by taking 25 g of
thoroughly washed and finely cut leaves in a 1 L beaker with
500 mL of sterile distilled water and then boiling the mixture for
5 min before finally decanting it. It was stored at 4 ◦ C and used
within a week.
Typically, 30 mL of leaf broth was added to 170 mL of 1 mmol L−1
aqueous CuSO4 ·5H2 O solution for the reduction of copper ions.
The effects of temperature on synthesis rate and particle size of
the prepared copper nanoparticles were studied by carrying out
the reaction in a water bath at 25–95 ◦ C with reflux. The leaf broth
concentrations were also varied between 5 and 20% by volume.
The copper 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.
The purified copper particles were freeze-dried, and
their structure and composition were analyzed by high-
resolution transmission electron microscopy (HR-TEM; JEOL-
2010), energy-dispersive X-ray spectroscopy (EDS; Sigma), and
X-ray photoelectron spectroscopy (XPS; ESCALAB 210). Copper
concentrations were determined using inductively coupled
plasma spectrometry (ICP; JY38Plus), and the average particle
size ascertained from TEM micrographs.
For chemical synthesis of copper nanoparticles, 4 mL of Tween
20 and 6 mL of sodium borohydride was added to 190 mL of
1 mmol L−1 aqueous CuSO4 ·5H2 O under 300 rpm stirring. The
reaction medium was allowed to react for 2 h at 25 ◦ C.
Coating of copper nanoparticles on latex foams
The synthesized copper nanoparticles were used to dip-coat latex
foams to evaluate antibacterial characteristics. Latex foams were
washed, sterilized and dried before use. Latex foam (2 × 2 ×
2 cm) was added to solution containing copper nanoparticles
synthesized with 1 mmol L−1 CuSO4 ·5H2 O and 15% Magnolia
kobus leaf broth. The weight of latex foam was 5 g and the
total volume of solution was 100 mL. The copper colloid solution
containing latex foam was placed in a shaking incubator for 1 h at
37 ◦ C. The resulting foams coated with copper nanoparticles were
dried in an oven for 24 h at 50 ◦ C.
Antibacterial acitivity
The antibacterial activity was tested against gram-negative
Esherichia coli strain (ATCC 25922). A 100 mL sample of LB broth
1972
(yeast extract 5 g L−1 , tryptone 10 g L−1 , and NaCl 5 g L−1 ) was
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c 2013 Society of Chemical Industry
H-J Lee, JY Song, BS Kim
added to 250 mL sterile flask and then inoculated with 1 mL of the
E. coli culture solution grown on LB broth for 12 h at 37 ◦ C.
Sterile latex foams coated with copper nanoparticles (5 g) were
placed in the flasks and shaken for 24 h at 37 ◦ C. Then, 100 µL of
the cultured solution was transferred to a nutrient agar medium
(DifcoTM LB Agar). After 24 h of incubation at 37 ◦ C, colonies of
viable cells were counted with suitable dilution and reported as
colony forming units (CFU) per mL. Uncoated foams were used
as control. Antibacterial activity was defined as the percentage of
microbe reduction17 and was calculated as follows.


CFU/ml (sample)
Antibacterial activity (%) = 1 − × 100
CFU/ml (control)
RESULTS AND DISCUSSION
Synthesis and characterization of copper nanoparticles
Reduction of the copper ion to copper nanoparticles during
exposure to the plant leaf extract could be followed by color
change and thus UV–vis spectroscopy. It is observed that the
maximum absorbance occurs at c. 560 nm and steadily increases
in intensity as a function of reaction time. We quantitatively
monitored the concentrations of copper nanoparticles and
conversion by measuring the absorbance at 560 nm. A linear
relationship was obtained between the copper concentration
determined by ICP and the absorbance at 560 nm (data not
shown).
Figure 1(a) and (b) show TEM images obtained with 15–20%
Magnolia leaf broth and 1 mmol L−1 CuSO4 ·5H2 O solution at 95 ◦ C.
It is shown that relatively spherical nanoparticles are formed
with diameter 50–250 nm. Figure 2 shows the time courses of
copper nanoparticle production at different reaction temperatures
obtained with 15% Magnolia leaf broth and 1 mmol L−1
CuSO4 ·5H2 O. As the reaction temperature was increased, both
synthesis rate and conversion to copper nanoparticles increased.
The conversion after 24 h was about 70% at 25 ◦ C and 80–100%
at 60 and 95 ◦ C. The average particle size decreased from 110 nm
at 25 ◦ C to 37 nm at 95 ◦ C as summarized in Table 1. The reason
for decrease in particle size with temperature can be discussed
as follows. As the reaction temperature increases, the reaction
rate increases and thus most copper ions are consumed in the
formation of nuclei, stopping the secondary reduction process on
the surface of the preformed nuclei. Similar trends were observed
with gold, silver, and platinum nanoparticles synthesized using
plant extracts.12 – 14,18
Effects of different Magnolia leaf broth concentrations at 5–20%
were investigated on copper nanoparticle formation at 1 mmol L−1
CuSO4 ·5H2 O (Fig. 3). The reaction rate was highest at 20% leaf
broth concentration. With increasing leaf broth concentration,
the average particle size decreased up to 15% leaf broth
concentration and then increased at 20% leaf broth concentration.
We previously found that large hexagonal or triangular gold
nanoparticles were produced by reacting HAuCl4 with a low
concentration of Magnolia leaf extract, but that increasing the
broth concentration led to smaller spherical nanoparticles being
formed.13 It is considered that too many capping materials
from the plant leaf broth caused some aggregation of copper
particles at high leaf broth concentration of 20% probably due
to the interaction between nanoparticles, which are surrounded
by proteins and metabolites such as terpenoids and reducing
sugars. Figure 4 shows the effects of CuSO4 ·5H2 O concentration on
conversion to copper nanoparticles obtained with 15% Magnolia
J Chem Technol Biotechnol 2013; 88: 1971–1977
Biological synthesis of copper nanoparticles using Magnolia kobus leaf extract
(a)
(b)
Figure 1. TEM images of copper nanoparticles formed with (a) 15% and
(b) 20% Magnolia kobus leaf broth in 1 mmol L –1 CuSO4 ·5H2 O solution at
95 ◦ C.
leaf broth concentration. The times required for more than 90%
conversion were 1600, 1400, and 200 min at 95 ◦ C, respectively,
when CuSO4 ·5H2 O concentrations were 0.5, 1, and 2 mmol L−1 ,
respectively. These are slower than the 150 min to achieve more
than 90% conversion to platinum nanoparticles with Persimmom
leaf broth at 95 ◦ C, and much slower than the 3 and 11 min
required for comparable levels of conversion when synthesizing
gold and silver nanoparticles, respectively, with Magnolia leaf
broth at the same temperature.12 – 14 The relatively low rate of
copper nanoparticle synthesis is possibly due to difficulty in initially
forming copper nuclei, indicating that achieving close to 100%
conversion to copper nanoparticles requires longer reaction times
than those required for gold, silver, or platinum nanoparticles.
EDS and XPS spectra recorded from the synthesized copper
nanoparticles are shown in Fig. 5. EDS profile showed copper
signals along with oxygen and carbon peak, which may originate
from the biomolecules that are bound to the surface of the copper
nanoparticles. The XPS spectrum showed characteristic copper
J Chem Technol Biotechnol 2013; 88: 1971–1977

c 2013 Society of Chemical Industry
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Cu Nanoparticle Concentration (mg/L)
70 100
60
80
Conversion (%)
50
40 60
30
40
95°C
20
60°C
25°C 20
10
0 0
0 200 400 600 800 1000 1200 1400
Time (min)
Figure 2. Time courses of copper nanoparticle formation obtained with
1 mmol L –1 CuSO4 ·5H2 O and 15% Magnolia kobus leaf broth at different
reaction temperatures.
peaks, suggesting that copper nanoparticles were successfully
synthesized using Magnolia leaf broth.
Copper nanoparticles were also synthesized using a well-known
chemical method for comparison. Sodium borohydride and Tween
20 were used as reducing and stabilizing agent, respectively. It was
observed that chemically synthesized copper nanoparticles were
oxidized, aggregated and settled down after 24 h (Fig. 6(a)), while
copper nanoparticles synthesized using Magnolia leaf extract
were stable for over 30 days because some capping materials
surrounded the surface of nanoparticles (Fig. 6(b)). This is another
advantage of using plant extract for nanoparticle synthesis over
using a chemical method.
The mechanism by which nanoparticles form in biosynthesis
procedures is not clear. It has been reported that reduction occurs
due to the NADH-dependent reductase released into the solution
in the case of gold nanoparticles synthesized extracellularly by
the fungus Fusarium oxysporum.19 It has also been suggested that
nitroreductase enzymes may be involved in silver nanoparticle
synthesis using the culture supernatants of Enterobacteria.20
Proteins in an extract of the unicellular green alga Chlorella vulgaris
were thought to be involved in the biological synthesis of silver
nanoplates using this organism.21 Xie et al.21 designed a simple
bifunctional tripeptide (Asp-Asp-Tyr-OMe) that had a Tyr residue
as a reduction source and 2 carboxyl groups in each of the Asp
residues as shape-directors, and found that this produced a good
yield of silver nanoplates with low polydispersity. In the 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.11 In our previous
study on gold nanoparticle synthesis using Magnolia kobus leaf
extract, the FTIR spectrum of the leaf extract showed characteristic
bands of the hydroxyl functional group in alcohols and phenolic
compounds and the amide I band of the proteins released by
the Magnolia leaves.13 It appears that the reduction of copper
ions and stabilization of synthesized copper nanoparticles is the
responsibility of some proteins and metabolites such as terpenoids
and reducing sugars having functional groups of amines, alcohols,
1973
ketones, aldehydes, and carboxylic acids.
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 www.soci.org H-J Lee, JY Song, BS Kim

Table 1. Summary of antibacterial activity and average size of copper nanoparticles synthesized with different synthesis methods, synthesis
temperatures, and leaf broth concentrations. The experiments were performed in triplicate and all data reported are average values.

Synthesis method Synthesis Leaf broth Antibacterial Average

of copper nanoparticles temperature (◦ C) concentration (%) activity (%) particle size (nm)

Chemical method 25 — 17 150

Biological method 25 15 40 110
Biological method 60 15 95 90
Biological method 95 5 70 91
Biological method 95 10 89 58
Biological method 95 15 99 37
Biological method 95 20 75 82

70 100 (a)
Cu Nanoparticle Concentration (mg/L)

60
80
50 leaf broth 5%

Conversion (%)
leaf broth 10%
40 60
leaf broth 15%
leaf broth 20%
30 40
20
20
10

0 0
0 200 400 600 800 1000 1200 1400
Time (min)

Figure 3. Time courses of copper nanoparticle formation obtained with

1 mmol L –1 CuSO4 ·5H2 O and various concentrations of Magnolia kobus
(b)
leaf broth at 95 ◦ C.

Cu2p
100
90
Intensity (a.u.)

80
70
Conversion (%)

60
50
40
30 2 mM CuSO4 5H2O
20 1 mM CuSO4 5H2O
10 0.5 mM CuSO4 5H2O 970 960 950 940 930 920
0
Binding Energy (eV)
0 200 400 600 800 1000 1200 1400 1600 1800
Figure 5. Characterization of copper nanoparticles formed with 1 mmol L –1
Time (min) CuSO4 ·5H2 O and 15% Magnolia kobus leaf broth at 95 ◦ C. (a) Spot profile
EDS spectrum. (b) XPS spectrum.
Figure 4. Time courses of copper nanoparticle formation obtained with
5% Magnolia kobus leaf broth and various concentrations of CuSO4 ·5H2 O
at 95 ◦ C. coated (Fig. 7(b)) due to the copper nanoparticles incorporated.
Figure 8(a) and (b) show photographs of E. coli cultures grown
Antibacterial property of foams containing copper with untreated foams and foams treated with copper nanoparti-
nanoparticles cles, respectively. The colony count of untreated latex foam was
These nanoparticles were coated on the surface of latex foam 22 400 CFU mL−1 (Fig. 8(a)), which decreased to 190 CFU mL−1
products using dip coating (exposure to nanoparticle solution) (Fig. 8(b)) by copper nanoparticle treatment. Table 1 summa-
and their antibacterial properties were tested against E. coli. The rizes antibacterial activities of copper nanoparticles synthesized
incorporation of copper nanoparticles in latex foam was observed using Magnolia leaf extract at various temperatures and leaf broth
by color change of the coated foams. The apparent colors of foams concentrations compared with chemically synthesized copper
1974

changed from white for the uncoated (Fig. 7(a)) to brown for the nanoparticles. The antibacterial activities were 40, 95, and 99%,

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c 2013 Society of Chemical Industry J Chem Technol Biotechnol 2013; 88: 1971–1977
Biological synthesis of copper nanoparticles using Magnolia kobus leaf extract
(a)
24h
(b)
30 day
Figure 6. Photographs of copper nanoparticle solution obtained by (a)
chemical synthesis using sodium borohydride and Tween 20, and (b)
biological synthesis using Magnolia kobus leaf broth.
respectively, when the synthesis temperatures were 25, 60, and
95 ◦ C, respectively. The antibacterial activities were inversely pro-
portional to the average nanoparticle sizes, suggesting that smaller
nanoparticles synthesized at higher temperature showed higher
antibacterial activity due to the larger specific surface area and
higher content of copper nanoparticles. Chemically synthesized
copper nanoparticles at 25 ◦ C showed 17% of antibacterial activity.
When the leaf broth concentrations were 5, 10, 15, and 20%, the
antibacterial activities were 70, 89, 99, and 75%, respectively, which
was also inversely proportional to the average nanoparticle sizes.
The antimicrobial properties of silver nanoparticles are well-
established and several factors have been reported to influence
silver nanoparticle toxicity like particle size, shape, crystallinity, and
capping agents, as well as solution pH, ionic strength, specific ions
and ligands, and macromolecules.22 Smaller particle sizes tend to
enhance antibacterial properties because there is a large number
of atoms on the surface available to interact with bacteria or to
release a higher amount of silver ions with decreasing size,23,24
which is consistent with our data on copper nanoparticles. Higher
stability produced higher antibacterial properties because non-
stable nanoparticles tend to form aggregates and thus surface
area is reduced.25 It was also reported that biologically synthesized
silver nanoparticles had greater bactericidal activity than did
chemically synthesized silver nanoparticles,26,27 which is also
consistent with our data on copper nanoparticles. These results
show that stable nanoparticles synthesized using plant extracts
may have advantages in antibacterial applications compared with
chemically synthesized nanoparticles. Currently, the mechanism
of antimicrobial action of silver or copper nanoparticles is only
partially understood. Several studies propose that nanoparticles
J Chem Technol Biotechnol 2013; 88: 1971–1977

c 2013 Society of Chemical Industry
www.soci.org
(a)
(b)
Figure 7. Photographs of foams (a) untreated and (b) dip-coated with
copper nanoparticles formed with 1 mmol L –1 CuSO4 ·5H2 O and 5%
Magnolia kobus leaf broth at 95 ◦ C.
attach to the surface of cell membrane disturbing the permeability
and respiration function of the cell.28,29 The damage to the cell
may be caused by the interaction of nanoparticles with phosphate
or sulfur-containing compounds such as DNA. Recently, there is
concern that nanoparticles may be harmful to human health.
However, sufficient information is not available on the adverse
effects of nanoparticles on human health. Further research is
required to fully understand the mechanisms of nanoparticle
toxicity and to safely exploit the tremendous antimicrobial
properties of copper without negatively impacting human health
and the environment.
CONCLUSION
We proposed an ecofriendly method for copper nanoparticle syn-
thesis using plant extract. The conversion to copper nanoparticles
was more than 90% using Magnolia leaf broth when the reaction
temperature was increased to 95 ◦ C. The average particle size
ranged from 37 to 110 nm and could be controlled by changing
1975
the reaction temperature and leaf broth concentration. The
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 www.soci.org
(a)
(b)
Figure 8. Photographs of E. coli cultures grown with foams (a) untreated
and (b) dip-coated with copper nanoparticles formed with 1 mmol L –1
CuSO4 ·5H2 O and 15% Magnolia kobus leaf broth at 95 ◦ C. E. coli cells were
grown in LB media containing latex foams for 24 h at 37 ◦ C and then 100 µL
of cultured solution was transferred to agar medium with suitable dilution
and incubated for another 24 h.
growth of E. coli in shake flask culture was significantly suppressed
by treatment with copper nanoparticles on latex foams. The
antibacterial activities were inversely proportional to the average
nanoparticle sizes. The biologically synthesized copper nanopar-
ticles using plant extract showed higher antibacterial activity
than copper nanoparticles chemically synthesized using sodium
borohydride and Tween 20. Copper nanoparticles synthesized
using Magnolia leaf extract were stable for over 30 days due to
capping materials surrounding the surface of the nanoparticles,
which is another advantage of using plant extract for nanoparticle
synthesis over using a chemical method. These environmentally
friendly synthesized copper nanoparticles can be used as an
inexpensive alternative to antibacterial silver nanoparticles.
ACKNOWLEDGEMENTS
This research was supported by Basic Science Research Program
1976
through the National Research Foundation of Korea (NRF)
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c 2013 Society of Chemical Industry
H-J Lee, JY Song, BS Kim
funded by the Ministry of Education, Science and Technology
(2012R1A1A2006375).
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