Materials Science and Engineering C 43 (2014) 5864

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Materials Science and Engineering C

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

Preparation of Au and Ag nanoparticles using Artemisia annua and their

in vitro antibacterial and tyrosinase inhibitory activities
Nagaraj Basavegowda 1, Akber Idhayadhulla 1, Yong Rok Lee 1
School of Chemical Engineering, Yeungnam University, Gyeongsan 712-749, Republic of Korea

a r t i c l e

i n f o

Article history:
Received 18 March 2014
Received in revised form 14 May 2014
Accepted 30 June 2014
Available online 9 July 2014
Keywords:
Metal nanoparticles
Artemisia annua
Reducing agent
Antibacterial
Antityrosinase
Leaf extract

a b s t r a c t
This work describes a plant-mediated approach to the preparation of metal nanoparticles using leaf extract of Artemisia annua (A. annua), an ethno-medicinal plant widely found in Asia, which was used as reducing and stabilizing agent. A. annua is used in traditional Chinese medicine to alleviate fever. Au and Ag nanoparticles were
prepared using a one-step aqueous method at room temperature without any toxic chemicals. The formation
of Au and Ag nanoparticles was monitored by UVvis spectroscopy. Synthesized nanoparticles were characterized by X-ray diffraction (XRD), transmission electron microscopy (TEM), energy dispersive X-ray analysis
(EDX), Fourier transform infrared (FT-IR) spectroscopy, and thermogravimetric analysis (TGA). TEM analysis of
Au nanoparticles showed that they had triangular and spherical shapes with sizes ranging from 15 to 40 nm.
The silver nanoparticles were predominantly spherical and uniformly sized (3050 nm). The Au and Ag nanoparticles produced showed signicant tyrosinase inhibitory and antibacterial effects. These results suggest that the
synthesized nanoparticles provide good alternatives in varied medical and industrial applications.
2014 Elsevier B.V. All rights reserved.

1. Introduction
In this era of modern material technology, noble nanoparticles like
gold and silver are attracting considerable attention because of their
high surface areas, small sizes, greater activities and their potential usages in elds like photonics [1], microelectronics [2], drug delivery [3],
food industries [4], agriculture [5], textile industries [6], and water
treatment [7]. In particular, biosynthesized gold nanoparticles have a
large number of applications for drug delivery [8], immunoassays [9],
cancer therapy [10], catalysis [11], imaging [12] opto-electronic devices
[13,14], immune staining, and biosensors [15]. On the other hand, silver
nanoparticles are considered important due to their applications in
antimicrobial ointments/creams [16], catalysis [17], label-free colorimetric assay [18], photocatalysts [19], medical diagnostics, therapeutics
[20,21], solar energy [22], and their anti-oxidant [23], anti-bacterial [24]
and cytotoxic [25] effects. Several techniques have been developed for
the synthesis of noble metal nanoparticles, including chemical, physical,
and biological techniques. Of these, plant-mediated synthetic methods
are preferred because they are cheap, solvent free, do not require a
stabilizing or reducing agent, and safe [26,27] whereas chemical and
physical methods require some reducing agents, solvents, surfactants,
capping agents, and thus, are potentially dangerous to biological
systems and raise environmental concerns. Since the beginning of this
century, researchers have been working on the extracellular and intracellular synthesis of metal nanoparticles using bacteria, fungi, yeast,

E-mail address: yrlee@yu.ac.kr (Y.R. Lee).

Tel.: +82 53 810 2529; fax: +82 53 810 4631.

http://dx.doi.org/10.1016/j.msec.2014.06.043
0928-4931/ 2014 Elsevier B.V. All rights reserved.

and many other plant sources [2831]. Furthermore, the synthesis of

nanoparticles using plant extracts is more advantageous than microbial
routes, which require elaborate processes for maintaining cell cultures
and reducing biohazards [32].
In this work, we devised simple and rapid syntheses of Au and Ag
nanoparticles using the leaf extract of Artemisia annua (sweet wormwood). The procedure developed is rapid, simple and viable. A. annua
is used in Chinese traditional medicine to relieve fever, and in combination with other herbs, to treat headache, jaundice, nosebleeds, and dizziness. The leaves of A. annua contain artemisinin, which is a potent
anti-malarial agent [33], and can kill Plasmodium falciparum the most
deadly malarial parasite. A. annua leaf extract contains antioxidant compounds [34] and possesses strong antibacterial [35], anti-inammatory
[36], plant growth regulatory [37], and antitumour [38] activities. Different plant parts of A. annua contain sesquiterpenoids, avonoids, coumarins, triterpenoids, steroids, phenolics, purines, lipids, and aliphatics
[39]. Recently, the green synthesis of AgNPs using A. annua and their antibacterial and antioxidant has been reported by Johnsona group [40],
but the synthesized AgNPs were never characterized by FTIR, TEM,
SAED, and XRD patterns to study the morphology and size of the
nanoparticles. The evaluation of antibacterial activity of AuNPs and
tyrosinase inhibitory activities of Au and Ag nanoparticles synthesized
by A. annua leaf extract has not been reported so far.
Here, we describe the synthesis and characterization of Au and Ag
nanoparticles using the aqueous extract of the shade dried leaves of
A. annua. In addition, as a biological application of this work, the evaluation for their antibacterial and tyrosinase inhibitory activities is also
reported.

N. Basavegowda et al. / Materials Science and Engineering C 43 (2014) 5864

2. Materials and methods

59

cooled energy-dispersive X-ray analysis (EDAX) detector using an

ultrathin window.

2.1. Chemicals and microorganisms

Silver nitrate, chloro auric acid, mushroom tyrosinase (EC 1.14.18.1),
and L-DOPA were obtained from Sigma-Aldrich. All glassware were
washed with distilled water and dried in an oven before use. Air dried
leaves of A. annua by keeping in shady area were obtained at a local
market in Yeongchon, South Korea. The standard bacterial strains
Escherichia coli (KCTC-1924), Pseudomonas aeruginosa (KCTC-2004),
Enterobacter aerogenes (KCTC-2190), and of the Gram negative bacteria
Bacillus cereus (KCTC-1012) and Staphylococcus aureus (KCTC-1916)
were obtained from the Korean Collection for Type Cultures (KCTC).
2.2. Preparation of A. annua extract
Dried A. annua leaves were rst ground to a ne powder and then
screened using a 20 mesh sieve. The aqueous extract was prepared by
adding 4 g of powdered leaves to 200 mL of deionized Milli-Q water
and boiled for 5 min. The solution obtained was then ltered through
a 0.2 m lter and stored at 4 C for further use, within a week.
2.3. The synthesis of Au and Ag nanoparticles
Aqueous solutions of 5 mM (200 mg/100 mL) chloroauric acid
trihydrate (HAuCl43H2O) with pH 2.61 at 19.5 C and 5 mM (85 mg/
100 mL) silver nitrate (AgNO3) with pH 4.74 at 19.5 C, were used to
synthesize Au and Ag nanoparticles. A. annua extract (5 mL) was
carefully added to 50 mL of previously prepared salt solutions (5 mM)
and stirred for 10 min at room temperature. Bioreduction occurred
within a short time as indicated by a color change to dark pink and
dark brown indicating the formations of Au and Ag nanoparticles,
respectively. The nanoparticles obtained were puried by centrifugation in a Beckman Coulter's Avanti J-E centrifuge (USA) at 10,000 rpm
for 20 min. After drying at 40 C for 2 h, AuNPs (21 mg) and AgNPs
(20 mg) bearing bioactive molecules were obtained.

2.4.4. Powder X-ray diffraction (XRD) analysis

Both Au and Ag nanoparticles were subjected to X-ray diffraction.
Nanoparticles were dried at 80 C and lightly hand-ground to ne powders. X-ray powder diffraction data was acquired using a PANalytical
X'Pert MRD model operated at 30 kV and 40 mA with Cu K radians
( = 1.5406 ) at 2 angle conguration. The scan range selected was
from 20o to 80.
2.4.5. Thermo gravimetric analysis (TGA)
TGA experiments were performed for both the AuNPs and AgNPs
using TG-DTA, SDT-Q600 V20.5 Build 15. The sample (5 mg) was placed
in a platinum sample pan and heated under N2 atmosphere at a rate of
10 C min1 to 1000 C.
2.5. Antibacterial activity
The antibacterial screenings of Au and Ag nanoparticles against
pathogenic bacteria, that is, Gram positive bacteria viz., E. coli (KCTC1924), P. aeruginosa (KCTC-2004), and E. aerogenes (KCTC-2190); and
the Gram negative bacteria viz., B. cereus (KCTC-1012) and S. aureus
(KCTC-1916) were performed using the agar well diffusion method
[41]. Bacteria were inoculated into 5 mL of sterile Mueller-Hinton
broth and incubated at 37 C for 8 h. Cultures were swabbed on the
surfaces of sterile nutrient agar plates using a sterile cotton swab. Sterile
lter paper discs (8 mm diameter) were moistened with the compound
solution of Au and Ag nanoparticles. Both Au and Ag nanoparticles were
suspended in DMSO, which did not affect microorganisms, at different
concentrations (1000, 100, 10 or 1 g/mL). Using a micropipette, 50 L
of the Au or Ag nanoparticle suspensions were added to paper discs in
agar plates. Agar plates were incubated at 37 C for 24 h. Antibacterial
activities were determined by measuring the widths of inhibitory
zones (mm) with ampicillin as positive reference.
2.6. Tyrosinase inhibitory activity

2.4. Characterization of the synthesized Au and Ag nanoparticles

2.4.1. UVvis spectral analysis
Bioreductions of Au and Ag salts were monitored by UVvis spectroscopy. Absorption spectra were obtained at different times using an
Optizen 3220 (double beam) UVvis spectrophotometer using a quartz
cuvette and distilled water as a reference. UVvis spectral readings were
recorded between 450 and 650 nm for Au nanoparticles and 350 and
550 nm for Ag nanoparticles.
2.4.2. Fourier transform infra-red (FT-IR) spectroscopy
FT-IR analysis was performed to identify the biomolecules in
A. annua extract possibly responsible for the reductions of the metals
and for the stabilizations of nanoparticles. After bioreductions, solutions
were centrifuged at 10,000 rpm for 20 min and pellets were washed
twice with deionized water to remove free proteins. The obtained
pellets were then thoroughly dried and ground with KBr (potassium
bromide) to make discs, which were examined in a JASCO FT-IR spectrophotometer in the wavelength range of 4004000 cm1.
2.4.3. Transmission electron microscopy (TEM) and EDAX
The morphology, size, and shape of Au and Ag nanoparticles were
determined by transmission electron microscope. Samples were prepared by placing a drop of the Au and Ag nanoparticle solutions on
carbon-coated copper grids and allowing the solvent to evaporate in
air at ambient temperature. TEM measurements were performed
using a FEI Tecnai G2 F20 ST FE-TEM, operated at 200 kV, a point resolution of 0.24 nm, and a Cs of 1.2 mm. The chemical composition of the Au
and Ag nanoparticles was analyzed using a Genesis liquid nitrogen

Tyrosinase inhibitory activity was measured using a modied spectrophotometric method [42,43]. Mushroom tyrosinase and L-DOPA as
substrates were used for the bioassay. The reaction mixture (nal
volume 3.0 mL) contained 1.5 mM L-DOPA, 0.1 mM sodium phosphate
buffer (pH 6.5), and 12.43 U of mushroom tyrosinase, and test sample
of 100 g/mL was incubated at 30 C for 2 min. The formation of
dopachrome was monitored spectrophotometrically at 475 nm using
an Optizen 3220 (double beam) UVvisible spectrophotometer. Kojic
acid was used as a positive control. Tyrosinase inhibitory activity (%)
was calculated using the following equation:
Tyrosinase inhibitory activity %

ABCD
 100
AB

where A = absorbance of the blank after incubation, B = absorbance of

the blank solution before incubation, C = absorbance of the sample
solution after incubation, and D = absorbance of the sample solution
before incubation.
3. Results and discussion
3.1. UVvis study of Au and Ag nanoparticles
UVvis measurements were performed to identify the formation of
Au and Ag nanoparticles and spectrum showed surface plasmon resonance (SPR) for both Au and Ag nanoparticles [44]. Due to the reaction
of Au salt and leaf extract, the color of the solution changed from yellow
to dark pink indicating the formation of Au nanoparticles. We can

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N. Basavegowda et al. / Materials Science and Engineering C 43 (2014) 5864

presume that the avonoid and phenolic acids present in the leaf extract
may act as reducing, and capping agents [45].
As biosynthesis proceeded, the color changed from light yellow to
dark pink in different time intervals. Aliquots of reaction mixture were
withdrawn at different times and scanned on a spectrometer. The gold
SPR band occurred at 540 nm and an increase in intensity was observed
up to 12 h without any shift in wavelength [46]. Fig. 1a shows the UV
Vis spectra of the Au nanoparticles obtained and the inset shows
photographs of the color change at different time intervals. The absorption band for Au nanoparticles was broad and remained at 540 nm
throughout the reaction, which indicated that the particles remained
well dispersed.
The silver nitrate and leaf extract solution turned from yellow to
dark brown (due to surface plasmon vibrations), indicating the formation of Ag nanoparticles. Fig. 1b shows characteristic absorption bands
of Ag nanoparticles at 435 nm and the inset shows photographs of
color changes at set times. The observed increase in intensity was due
to nanoparticle formation [47]. It is well known that the absorption
band between 400 and 440 nm is due to SPR [48]. The rate of formation
of AuNPs and AgNPs was found to be slower at lowest concentration
and hence causing weaker absorbance [49].
3.2. TEM and EDAX analyses of Au and Ag nanoparticles
A transmission electron microscope was used to analyze the morphology and size of the Au and Ag nanoparticles produced. Aliquots of
Au and Ag nanoparticle solutions were placed on a carbon coated
copper grid and allowed to dry under ambient conditions. TEM images
were recorded at different magnications (Figs. 2 and 3). Fig. 2a
shows that Au nanoparticles are almost all spherical with a few triangular and irregularly shaped particles. Triangular particles were bigger
than spherical particles. Au nanoparticles had sizes ranging from 15 to
40 nm, and most were ~ 25 nm. As can be seen from the micrograph,
smaller Au nanoparticles agglomerated to form non-spherical, slightly
larger particles. Particle size variations were probably due to different
growth times [50]. This result is in agreement with the shape of the
SPR band (Fig. 1(a)(F)). Insets of TEM images (Fig. 2a) show selected
area electron diffraction (SAED) patterns corresponding to the (111),
(200), (220), and (311) face centered cubic planes, which conrmed
the crystalline nature of Au nanoparticles. Energy dispersive X-ray
analysis (EDAX) (Fig. 2b) revealed a strong signal in the gold region
and conrmed the formation of Au nanoparticles, which showed a
typical optical absorption peak at ~ 2.22 keV due to surface plasmon
resonance. The presence of C and Cu was possibly due to carbon coated
copper grid used for sample preparation.

The resultant Ag nanoparticle images were spherical, although a few

were irregular (Fig. 3a). The particle sizes of the Ag nanoparticles
produced ranged from 30 to 50 nm (average 30 nm). This result is in
agreement with the shape of the SPR band (Fig. 1(b)(F)). Insets of
TEM images show that the diffraction rings correspond to the (111),
(200), (220), and (311) face centered cubic planes and it conrmed a
crystalline nature of silver (Fig. 3a). The EDAX prole of Ag nanoparticles showed strong signals for silver atoms (Fig. 3b). Metallic silver
nanocrystals generally show an absorption peak at ~3 keV due to SPR
[51]. Signals from carbon and copper were also observed.
3.3. Powder X-ray diffraction studies of Au and Ag nanoparticles
The XRD patterns of synthesized Au nanoparticles are shown in
Fig. 4a. The scanning range (2) was selected between 20 and 80.
Diffraction peaks were observed at 2 values of 38.17, 44.37,
64.56, and 77.54, which corresponded to the (111), (200), (220),
and (311) reections of face-centered gold [52]. The calculated
lattice values (a = 4.0796 ) were consistent with the Joint Committee on Powder Diffraction Standard (JCPDS) le (no. 04-0784). A few
additional sharp unassigned peaks at 27.87 and 32.30 were also
observed. These peaks were due to the crystallizations of proteins
and other bioorganic compounds on the surfaces of Au nanoparticles,
which concurred with previous reports [53].
Fig. 4b shows the XRD patterns of Ag nanoparticles synthesized
using the leaf extract of A. annua. A number of Bragg reections with
2 values of 38.10, 44.37, 64.17 and 77.54, corresponded to the
(111), (200), (220) and (311) face centered cubic structures of silver
[54]. The calculated lattice constant (a = 4.0800 ) was in good agreement with the reported value (JCPDS 4-783; a = 4.0862 ). Similar results have been reported for Ag nanoparticles, previously [55].
3.4. FT-IR analysis of Au and Ag nanoparticles
FT-IR measurements were carried out to identify possible biomolecules in A. annua leaf extract responsible for the reductions and stabilizations of Au and Ag nanoparticles. The spectra of leaf powder of A. annua
and of Au nanoparticles (Fig. 5a) revealed strong bands at 3366 and
3396, 2925 and 2925, 1643 and 1629, 1399 and 1383 and 1042 and
1070 cm1, respectively. The band at 3366 cm1 in A. annua leaf powder
was attributed to O\H stretching [56] and this band was shifted to
3396 cm1 in Au nanoparticles presumably because of protein binding.
The small peaks (2925 and 2925) cm1 were attributed to C\H
stretching [57]. The absorption peaks located at (1643 and 1629) cm1
was attributed to carbonyl stretching vibrations of protein amides [58].

Fig. 1. (a) UVvis spectra and inset diagram showing color changes at different time intervals: (A) gold chloride solution (B) leaf extracts (C) reduction after 30 min, (D) reduction after 1 h,
(E) reduction after 6 h, and (F) reduction after 12 h, (b) UVvis spectra and inset diagram showing color changes at different time intervals: (A) silver nitrate solution (B) leaf extracts
(C) reduction after 30 min, (D) reduction after 1 h, (E) reduction after 6 h, and (F) reduction after 12 h.

N. Basavegowda et al. / Materials Science and Engineering C 43 (2014) 5864

61

Fig. 2. (a) TEM micrograph of Au nanoparticles (inset shows the SAED pattern of nanocrystalline gold), and (b) EDAX result showing the formation of Au nanoparticles.

Fig. 3. (a) TEM micrograph of Ag nanoparticles (inset shows the SAED pattern of nanocrystalline silver), and (b) EDAX result showing the formation of Ag nanoparticles.

The medium sized peaks at 1399 and 1383 cm1 were attributed to
C\N stretching vibrations of aromatic amines [59], and the bands at
1042 and 1070 cm1 to C\O\C stretching [60]. Almost similar results
were obtained for Ag nanoparticles, but the peak at 1070 cm1 shifted
to 1242 cm1 as shown in Fig. 5b.

3.5. TGA results of Au and Ag nanoparticles

TGA spectra of both AuNPs (Fig. 6a) and AgNPs (Fig. 6b) showed
signicant weight loss when heated from 10 C min 1 to 1000 C.

This result indicated that bioactive molecules capped on both AuNPs

and AgNPs were completely degraded on high temperature.
3.6. Antibacterial activities of Au and Ag nanoparticles
In the present study, the antibacterial activities of synthesized Au
and Ag nanoparticles and of crude leaf extract were tested against
human pathogens, that is, Gram-negative E. coli, P. aeruginosa, and
E. aerogenes, and Gram-positive S. aureus, and B. cereus at (1, 10,
100, 1000 g/mL) different concentrations using the disc diffusion
method. Ag nanoparticles had zones of inhibition of 22 mm and 23

Fig. 4. (a) XRD patterns of Au nanoparticles and (b) XRD patterns of Ag nanoparticles synthesized using A. annua leaf extract.

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N. Basavegowda et al. / Materials Science and Engineering C 43 (2014) 5864

Fig. 5. (a) FT-IR spectra of Au nanoparticles and (b) FT-IR spectra of Ag nanoparticles synthesized from A. annua leaf extract.

Fig. 6. (a) TGA spectra of AuNPs and (b) AgNPs.

mm against E. aerogenes and S. aureus, respectively, whereas plant

extract and standard ampicillin had zone of inhibitions of 20 mm
and 23 mm and 21 mm and 23 mm, respectively, at a concentration
of 100 g/mL.
The Au nanoparticles exhibited zones of inhibition of 18 and 23 mm
against E. coli and B. cereus, respectively as compared with 18 and
14 mm for plant extract and 18 and 23 mm for standard, respectively.
The as synthesized Au and Ag nanoparticles had higher activity than the
plant extract but lower activity than the standard ampicillin at 100 g/
mL. Values are summarized in Table 1.

most skin-whitening agents, although some, such as, arbutin have been
suggested to be toxic [61]. In particular, hydroquinone has been used
extensively in cosmetic formulations, but has been dropped because of
its undesirable side effects [62].
The identication of natural tyrosinase inhibitors is of enormous importance to the cosmetic industry, because they are likely to have fewer
side effects. Although the tyrosinase inhibitory activities of Au and Ag
nanoparticles are lower than that of standard Kojic acid (Fig. 7), Au
nanoparticles signicantly inhibited tyrosinase activity by as much as
65.49% at 100 g/mL. Curves showing the Au nanoparticle inhibition
of oxidation reaction of L-DOPA by tyrosinase are shown in Fig. 8.

3.7. Tyrosinase inhibitory activities of Au and Ag nanoparticles

4. Conclusions
During preliminary screening using mushroom tyrosinase as a
representative enzyme, we observed that Au nanoparticles signicantly
inhibited tyrosinase L-DOPA oxidation. Tyrosinase is a cellular target for

The medicinally important aqueous extract of A. annua was found to

act as reducing and capping agent for Au and Ag nanoparticle production.

Table 1
Antibacterial activity of Au and Ag nanoparticles against different bacterial species. Antibacterial activities were quantied by measuring the width of zone of inhibition (mm).
Compound

Concentration g/mL

Diameter of growth inhibition zone (mm)

Gram-negative

AgNPs

AuNPs

Plant extract

Ampicillin
() not active.

1000
100
10
1
1000
100
10
1
1000
100
10
1
100

Gram-positive

E. coli

P. aeruginosa

E. aerogenes

S. aureus

B. cereus

13
12
12
10
18
15
10
10
18
15
12

17

15
14
12
10
10
8
8

10

22

22
20
18
13
16
14
14
13
20
16
14
10
21

25
20
10
8
18
16
14
11
23
21
10
10
23

16
14
12
12
23
18
14
11
14
12
12
12
19

N. Basavegowda et al. / Materials Science and Engineering C 43 (2014) 5864

Fig. 7. Tyrosinase inhibitory activities.

Fig. 8. Curve showing the oxidation of L-DOPA by mushroom tyrosinase in the presence of
Au and Ag nanoparticles.

The Au and Ag nanoparticles produced had triangular and spherical

shapes respectively, and average particle sizes of 25 and 30 nm, respectively. FT-IR analysis conrmed that biomolecules present in A. annua
were responsible in reducing and capping the Au and Ag nanoparticles
produced. UVvisible and TEM results indicated that the produced
nanoparticles were stable and XRD and EDAX conrmed their elemental
make-ups. The study also shows that the Au and Ag nanoparticles
synthesized had signicant tyrosinase inhibitory and antibacterial activities. The proposed synthetic method is simple and eco-friendly because
it does not require any extra surfactant or reductant. Our ndings suggest
that these nanoparticles synthesized by an environmental-friendly
method could be used in important materials, such as, cosmetics,
nutraceuticals, and medicines because of bearing bioactive molecules.
Acknowledgment
This research was supported by the Nano Material Technology
Development Program of the Korean National Research Foundation
(NRF) funded by the Korean Ministry of Education, Science and
Technology (2012M3A7B4049675).
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