Artificial Cells, Nanomedicine, and Biotechnology

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Biosynthesis of silver nanoparticles from

Catharanthus roseus leaf extract and assessing
their antioxidant, antimicrobial, and wound-
healing activities

Hanady S. A. Al-Shmgani, Wasnaa H. Mohammed, Ghassan M. Sulaiman & Ali

H. Saadoon

To cite this article: Hanady S. A. Al-Shmgani, Wasnaa H. Mohammed, Ghassan M. Sulaiman

& Ali H. Saadoon (2016): Biosynthesis of silver nanoparticles from Catharanthus roseus leaf
extract and assessing their antioxidant, antimicrobial, and wound-healing activities, Artificial
Cells, Nanomedicine, and Biotechnology, DOI: 10.1080/21691401.2016.1220950

To link to this article: http://dx.doi.org/10.1080/21691401.2016.1220950

Published online: 18 Aug 2016.

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ARTIFICIAL CELLS, NANOMEDICINE, AND BIOTECHNOLOGY, 2016
http://dx.doi.org/10.1080/21691401.2016.1220950

RESEARCH ARTICLE

Biosynthesis of silver nanoparticles from Catharanthus roseus leaf extract and

assessing their antioxidant, antimicrobial, and wound-healing activities
Hanady S. A. Al-Shmgania, Wasnaa H. Mohammedb, Ghassan M. Sulaimanb and Ali H. Saadoonb
a
Biology Department, College of Education for Pure Science/Ibn al-Haitham, University of Baghdad, Baghdad, Iraq; bBiotechnology Division,
Applied Science Department, University of Technology, Baghdad, Iraq

ABSTRACT ARTICLE HISTORY

Biosynthesis of silver nanoparticles (AgNPs) from Catharanthus roseus leaf extract was carried out, and Received 15 May 2016
their characterization, as well as antioxidant, antimicrobial, and wound-healing activities were evaluated. Revised 28 July 2016
Color change, UV-vis spectrum, XRD, FTIR, and AFM assessments supported the biosynthesis and char- Accepted 2 August 2016
acterization of AgNPs. The synthesized AgNPs showed strong in vitro antioxidant and antimicrobial Published online 17 August
2016
activities against various pathogens. The in vivo assessment of wound healing in AgNPs-treated mice
revealed their effectiveness in closuring and reducing size of wounds. Such potent bioactivity may jus- KEYWORDS
tify their biomedical use as antioxidant and antimicrobial agents for controlling various health-related Silver nanoparticles;
diseases, particularly in wound healing. antioxidant; antimicrobial;
wound healing

Introduction
In recent years, nanoparticles have been addressed to play a
vital role in medicine, science, and various biotechnological
fields. They have various sizes, shapes, and high surface-
to-volume ratios; therefore, nanoparticles are potentiated for
various biomedical applications, such as target drug delivery,
imaging and biosensor, diagnosis, and disease therapy (Wang
and Wang 2014). Metal and metal oxide nanoparticles have
been further explored, and their biological applications have
remarkably been demonstrated; for instance, anticancer, anti-
oxidant, anti-inflammatory, wound-healing, and antimicrobial
activities (Palaniselvam et al. 2014, Schrofel et al. 2014, Singh
et al. 2013).
Silver is used to treat infected wounds long before antibi-
otics were introduced in modern medicine (Singh et al. 2013).
However, the use of silver is limited because of its high tox-
icity and the availability of antibiotics. Recently, silver nano-
particles (AgNPs) have attracted the scientific attention of
researchers regarding their use as potential antimicrobial
agents (Narayanan and Sakthivel 2011). They possess a wide
range of applications in medicine; including pharmaceutical,
cosmetics, and medical devices because of their broad bac-
tericidal and fungicidal spectrum (Palaniselvam et al. 2014).
Green synthesis of nanoparticles is gaining interest world-
wide because of its advantages, such as being ecofriendly,
non-toxic, and economic, over chemical and traditional phys-
ical methods (Mohanpuria et al. 2008). Several biological meth-
ods have been used for nanoparticle production from
organisms involving bacteria, fungi, and plant extracts (Aruna
et al. 2014, Hemanth et al. 2010, Natarajan and Ramchandra
2010). AgNP synthesis from plant extracts has been developed
because it is easily obtained and safe and has large variety of
metabolites supporting silver ion reduction. A mechanism pro-
posed that phytochemicals are directly involved in the allevi-
ation of ions and realization of AgNPs (Jha et al. 2009).
Catharanthus roseus (Madagascar periwinkle or Sadabahar),
which belongs to the plant family Apocynaceae, has been
widely introduced as one of the important medicinal plants
that is used to treat different diseases in folk medicine
(Appidi et al. 2008, Singh et al. 2001). The leaves and roots of
C. roseus are rich in phytochemicals; for instance alkaloids,
which have been demonstrated to have anticancer and anti-
hypertensive effects (Ei-Sayed and Verpoorte 2005).
The present study aimed to determine the biological syn-
thesis of AgNPs produced from C. roseus leaf aqueous extract
and characterize these nanoparticles using X-ray diffraction
(XRD), Fourier transform infrared (FTIR), and atomic force
microscopy (AFM). Moreover, antioxidant effect, wound-heal-
ing property, and antimicrobial activity of synthesized AgNPs
against clinical isolates of bacteria (Escherichia coli,
Staphylococcus aureus, Klebsiella pneumonia, Citrobacter koseri,
Pseudomonas aeruginosa) and the fungus Candida albicans
were evaluated.
Materials and methods
Materials
Brain heart agar (BHA), Sabouraud dextrose agar (SDA), silver
nitrate (AgNO3), and 2,2-diphenyl-1-picrylhydrazyl (DPPH)
were purchased from Sigma-Aldrich company (Germany).

CONTACT Ghassan M. Sulaiman gmsbiotech@hotmail.com Biotechnology Division, Applied Science Department, University of Technology, Baghdad, Iraq
ß 2016 Informa UK Limited, trading as Taylor & Francis Group
2 H. S. A. AL-SHMGANI ET AL.
Microorganisms
E. coli, S. aureus, K. pneumonia, C. koseri, P. aeruginosa, and C.
albicans were used to investigate the antimicrobial potential
of prepared AgNPs. They were identified and kindly supplied
by Division of Biotechnology, Department of Applied Science,
University of Technology, Baghdad, Iraq.
Experimental mice
For wound-healing experiment, 6- to 8-week-old male albino
mice (20–22 g) were used. The animals were housed in sterile
polypropylene cages and maintained at standard laboratory
conditions (12 h of light–dark cycle, 25 ± 3
C and
35–60% humidity), and had free access to food and water (ad
libitum).
Preparation of leaves extract
Fresh leaves of C. roseus were collected from Al-Tarmyia
(a region located 60 km northeast the capital Baghdad) and
washed, air-dried, and ground to produce powder. Leaf
extract was prepared via Soxhlet extraction method according
to Sulaiman and co-workers (Sulaiman et al. 2013), where
methanol was used as a solvent. After filtration of extract
(Whatman filter paper No. 1), the solvent was evaporated in
rotary evaporator, and the obtained extract was reconstituted
to 10 mL with deionized distilled water and maintained at
4
C for further experiments.
Synthesis of silver nanoparticles
An aqueous solution of 2 mM AgNO3 was prepared according
to the method of Sulaiman and co-workers using distilled
water at room temperature (Sulaiman et al. 2013). Briefly, the
aqueous solution (90 mL) was mixed with 10 mL of leaf
extract at 70
C and stirred magnetically at 1000 rpm for
3 min. The bioreduced aqueous component was subjected for
characterization of AgNPs.
Characterization of silver nanoparticles
The characterization of synthesized AgNPs was first moni-
tored by measuring the UV-vis spectrum using Hitachi U-2910
Spectrophotometer (Tokyo, Japan). UV-vis spectroscopic ana-
lysis was performed via continuous scanning at the range of
280–760 nm. Then, XRD analysis was carried out, in which,
purified AgNP solution drop-coated on glass was observed
using XRD-6000 X-ray diffractometer (Shimadzu, Japan) oper-
ated at a voltage and current of 40 kV and 30 mA, respect-
ively, with Cu Ka radiation in 2h configurations. The size was
calculated by using the following Scherrer formula was used:
D ¼ 0.94 k/b Cos h. FTIR analysis was carried out by using
FTIR 8400S spectrometer in attenuated total reflection mode
and spectral range of 4000–400 cm1 with a resolution of
4 cm1. Finally, the morphology of the synthesized AgNPs
was determined via AFM analysis using AA 3000 Scanning
Probe Microscope.
DPPH-free radical scavenging assay
The antioxidant activity of synthesized AgNPs was manifested
through DPPH method. One milliliter of DPPH (0.1 mM; in
ethanol) was added to the AgNPs. The reaction mixture was
shaken and incubated in the dark place for 30 min. The
absorbance was measured at 517 nm against a blank
(ethanol). The reaction mixture with lower absorbance indi-
cated a higher percentage of scavenging activity. The equa-
tion used for calculating DPPH-free radical scavenging activity
is as follows.
DPPH scavenging activity ð%Þ ¼ Ac  As=Ac  100
where Ac and As are the peak intensity for control (DPPH)
and test sample solvent, respectively.
Antimicrobial assessment of silver nanoparticles
The synthesized AgNPs were tested for antimicrobial activity
via agar-well diffusion method. The pure cultures of patho-
genic bacteria were sub-cultured on BHA. Each strain was
swabbed uniformly onto individual plate using sterile cotton
swabs. The discs (8 mm, diameter) containing the prepared
nanoparticle solution were placed on agar medium and
incubated at 37
C for 24 h. For antifungal measurement,
wells with a diameter of 8 mm were made on SDA plates
by using the gel puncture method. Aliquot 50 lL of pre-
pared nanoparticle solution were poured onto each well of
all plates, and these plates were incubated at 30
C for
48 h. The diameter of inhibition zone was measured in milli-
meter and recorded as mean ± SD of a triplicate assessment
according to the standard protocol. The plant extract and
silver nitrate were also tested in a similar manner. In add-
ition, amoxicillin (30 lg mL1) and fluconazole (5 lg mL1)
were used as a control for tested bacteria and C. albicans,
respectively.
Excision wound model and AgNPs administration
The wound-healing experimental protocols were approved
by the Biotechnology Division Committee, Applied Science
Department, University of Technology, Baghdad, Iraq. Six
mice were distributed into three groups (each of two ani-
mals): control (Group I), treated with plant extract only
(Group II), and treated with AgNPs (Group III). For wound
excision, the dorsal side hair of mice was shaved using ster-
ile surgical blade, and (2  2 cm2) full-thickness excision
wound was done via removing a patch of skin under
chloroform anaesthesia. The wound of control mice received
2 mL of normal saline, while wounds of treated mice
received 2 mL of either plant extract or synthesized AgNP
(2 mM) that was put in the dressing (size: 2  2 cm2) fol-
lowed by topical application to the wound bed, once daily
for a period of 12 days. Wound tissue contraction was
monitored by tracking the wound healing at 0, 1, 4, 8, and
12 days. For a visual comparison, the wounds of control
and treated mice were photographed on scheduled days,
and images were inspected.
 ARTIFICIAL CELLS, NANOMEDICINE, AND BIOTECHNOLOGY 3

Statistical analysis
The data analyses were evaluated using ANOVA test with
SPSS/14 computer software (SPSS Inc., Chicago, IL). Results
were presented as the mean ± SD and statistical significance
levels were set at P  .05.

Results and discussions

Characterization of silver nanoparticles
In present work, AgNPs were synthesized from C. roseus leaf
extract. In general, AgNPs produced brown solution in water
(Figure 1). The observed color change (brownish), when the
Figure 1. Photograph showing color changing (A) aqueous leaf extract of
plant extract was mixed with AgNO3 solution, suggested the Catharanthus roseus (B) changing color from yellowish to reddish brown after
reduction of Agþ to nanoparticles. The metal nanoparticles adding 2 mM AgNO3 and exposing to heat at 70
C for 3 min. (C) 2 mM AgNO3
only.
have free electrons, which provide the surface plasmon res-
onance (SPR) absorption band (Narayanan et al. 2012). This
specific color alteration was caused by the excitation of SPR
in the metal nanoparticles. The color change of the leaf
extract was observed when the extract was incubated with
the AgNO3 solution.
The absorption spectrum of the incubated solution at dif-
ferent wavelengths ranging within 200–600 nm revealed a
peak at 425 nm (Figure 2). AgNPs prepared from other plant
extracts showed SPR peak at 420 nm (Logeswari et al. 2013,
Maria et al. 2015, Mulvaney 1996). The frequency and width
of the SPR absorption band based on the shape and size of
the metal nanoparticles, dielectric constant of the metal itself
(the composition of the particles) and the dielectric constant
of surrounding medium. Also, a single SPR band reveals to
the spherical shape, while two or more SPR bands correspond
to the anisotropic particles (Udayasoorian et al. 2011). UV-vis
spectroscopy technique could be used to examine shape-
and size-controlled nanoparticles in aqueous suspensions
Figure 2. UV-vis absorption spectra of reduction of silver ions to silver
(Wiley et al. 2006). Thus, the UV-vis results indicated that C. nanoparticles.
roseus leaf extract can possibly reduce Ag to AgNPs, and
such finding was further inspected to confirm the synthesis
of AgNPs from C. roseus. AgNPs and provide information regarding the functional
The XRD pattern of AgNPs produced from the leaf extract groups. The representative spectra of AgNPs are shown in
was further illustrated and confirmed by the characteristic Figure 4. The FTIR signals of AgNPs were observed at 3434,
peaks observed in the XRD spectra (Figure 3). Four Bragg’s 2024, 1624, and 1213. The absorption peak at 1213 and
reflection patterns at 2h, namely, 32.00
, 38.50
, 44.40
, 1624 was attributed to the C–O and N–H stretching, which
64.50
, and in the entire spectrum of value ranging within was possibly caused by the presence of carboxylic acid and
10–90, were interpreted from XRD. These patterns corre- amide groups, respectively. The strong absorption peaks at
sponding to (122), (111), (200), and (220), respectively. The 3434 were caused by the N–H bond for amine groups,
set of lattice planes were observed and compared with the which were used for the stabilization of AgNPs. However,
reference values of Joint Committee on Powder Diffraction the presence of the amide group characteristic proteins/
Standards (JCPDS: 89-3722) and further on the basis that they enzymes is responsible for the reduction of AgNO3 to Ag
can be indexed as face-centered-cubic structure of silver with (Mohamed et al. 2014). AFM was used to observe the
a diameter around of 20 nm. Therefore, the XRD pattern sample’s surface morphology and roughness. Figure 5
clearly revealed that AgNPs are crystalline in nature (Shameli shows crystalline particles with grains sized 10–88 nm in
et al. 2011). The sharp peaks clearly indicate the cubic crystal- diameter with mean size of about 49 nm. A few aggrega-
line nature of the synthesized AgNPs which is in nanoscale tions of AgNPs are also observed in some places, indicating
and agreement with report of Bhakya and co-workers agglomeration after several weeks of AgNPs preparation.
(Bhakya et al. 2016). Hence, C. roseus leaf extract is promising Such finding confirmed the crystalline structure obtained by
target in the development of AgNPs. XRD assay. However, FESEM technique is required in a fur-
FTIR spectroscopic studies were conducted to investigate ther analysis to visualize the size and shape of the synthe-
the plausible mechanism behind the formation of these sized AgNPs.
4 H. S. A. AL-SHMGANI ET AL.

Figure 3. XRD pattern of silver nanoparticles formed after reaction with Catharanthus roseus leaf extract.

Figure 4. The FTIR spectra of silver nanoparticles.

Figure 5. FM image of silver nanoparticles film showing uniformly distributed nanoparticles and some agglomeration.
 ARTIFICIAL CELLS, NANOMEDICINE, AND BIOTECHNOLOGY 5

In vitro antioxidant activity of silver nanoparticles

Figure 6. DPPH-free radical scavenging activity of silver nanoparticles formed
after reaction with Catharanthus roseus leaf extract. Results are expressed as per-
centage decrement of absorbance at 517 nm with respect to control. Each value
represents the mean ± SD of three experiments.
The antioxidant activity of bioconjugated AgNPs was eval-
uated using DPPH scavenging assay. DPPH is a stable com-
pound and accepts hydrogen or electron from AgNPs. The
color is changed from purple to yellow after reduction, which
can be quantified by its decreased absorbance at wavelength
of 517 nm. Figure 6 shows the dose-dependent increase in
the inhibition percentage of synthesized AgNPs at 50, 150,
and 300 lg mL1. The 300 lg mL1 concentration exhibited a
higher inhibition (82%) compared to that of the other two
concentrations. The disappearance of purple color when syn-
thesized AgNPs were added might be caused by the pres-
ence of antioxidant in the medium. The antioxidant property

Figure 7. Antimicrobial activity assay of silver nanoparticles against different pathogens by the well diffusion method. (A) Amoxicillin/or fluconazole (B)
Catharanthus roseus leaf extract, (C) silver nitrate, (D) synthesized silver nanoparticles at 100 lg mL1, and (E) synthesized silver nanoparticles at 200 lg mL1.
Antibiotic amoxicillin at concentration 30 lg mL1 was used as a control for all tested bacteria while, fluconazole at concentration 5 lg mL1 was used as a control
for Candida albicans.
6 H. S. A. AL-SHMGANI ET AL.

of AgNPs could be explained to functional groups adhered to

them which were produced from the C. roseus leaf extract.
Superoxide anions are free radicals generated by the trans-
fer of one electron and play a vital role in the formation of
other reactive oxygen species (ROS), such as hydrogen perox-
ide, hydroxyl radical, or singlet oxygen within living systems
(Stief 2003). Antioxidants are beneficial for the management
of many deleterious diseases because of their scavenging
ability. Antioxidants can also react with nitric oxide to form
peroxynitrite, which can generate toxic radicals, such as the
hydroxyl radical (Halliwell 1997). Although the mechanism of
action in a more detailed level needs more advanced experi-
mental proofs, AgNPs are promising as a potential biological
label.

Antimicrobial assay of silver nanoparticles

The synthesized AgNPs manifested good antibacterial prop-
erty against gram-negative bacteria, such as E. coli, C. koseri,
K. pneumonia, P. aeruginosa, and S. aureus. They also showed
antifungal activity against C. albicans. AgNPs exhibited more
significant effect than the use of plant extract or silver nitrate
only (Figure 7). Researchers suggested different mechanism(s)
of the AgNPs action onto bacteria. Yliniemi and Vahvaselka
(Yliniemi and Vahvaselka 2008) demonstrated the AgNPs’ bac-
terial cell membrane permeability and respiration function,
which lead to cell death. The smaller size of AgNPs provides
a large surface area that is available for interaction, which
would provide more considerable effect than the larger
Agþ (Rai et al. 2009).
In addition, AgNPs do not only attach to the bacterial cell
surface but also enter inside the bacteria, which results in a
disruption of adenosine triphosphate (ATP) production and
DNA replication, generation of ROS and direct damage to cell
structures (Sahayaraj and Rajesh 2011). Furthermore, the bac-
tericidal effect of silver might be attributed to the inactivation
of phosphomannose isomerase, which catalyzes the conver-
sion of mannose-6-phosphate to fructose-6-phosphate. The
latter is an important intermediate of glycolysis, which is the
most common pathway in bacteria for sugar catabolism
(Beddy et al. 2004).
Figure 8. Photographs of wounds from animals elucidation on different days of
(A) negative control, (B) silver nanoparticles, and (C) Catharanthus roseus leaf
Wound-healing activity of silver nanoparticles extract-treated mice.

The wound-healing activity of synthesized AgNPs was proven

in male albino mice using an excision wound model. As
shown in Figure 8, AgNP-treated animals showed better
wound-healing activity compared with that of either nega-
tive- or positive-control groups. AgNP-treated wounds exhib-
ited no evidence of microbial contamination, bleeding or pus
formation during treatment, whereas control wounds
revealed notable inflammation. On the 4th day onwards,
AgNP-treated group recorded distinguished wound closure
and reduced wound size, which were enhanced during the
remaining days of treatment compared to controls. At the
end of the experiment, the AgNP-treated wound showed
approximately 98% of closure, whereas control wound
appeared approximately 85% of closure (Figure 8).
The reduced wound size and enhanced wound closure might
be due to the bactericidal effects of AgNPs against bacterial
contamination in the wound area, which can consequently
restore tissue integrity and often results in a satisfactory
repair of damaged sites (Chatterjee et al. 2014).
Tian et al. demonstrated the potential role of AgNPs on
wound healing in an animal model and executed that rapid
healing and better cosmetic appearance occur in a dose-
dependent manner (Beddy et al. 2004). Furthermore, AgNPs
showed positive effects through their antimicrobial potentials,
reduction in wound inflammation through decreasing
lymphocyte and mast cell infiltration, and amendment of
fibrogenic cytokines (Tian et al. 2007). Similarly, Lee et al.

investigated the effect of AgNPs in dermal contraction and
epidermal reepithelialization during wound healing and sug-
gested that AgNPs could increase the rate of wound closure.
This property was interpreted through the promotion of
reproduction and migration of keratinocytes (Lee et al. 2010).
Additionally, AgNPs could boost the differentiation of fibro-
blasts into myofibroblasts, thereby inducing wound contrac-
tion (Gunasekaran et al. 2012).
Conclusion
C. roseus leaf extract-synthesized AgNPs exhibited a strong
antimicrobial activity against several pathogens that were
tested in present study. The tailored AgNPs prompted
wound-healing potential in albino male mice using wound
closure assay. The AgNPs also enhanced the wound-healing
activity in mice by inhibiting the pathogenic bacterial growth
in the wound area. On the basis of our previous and current
findings, it is possible to conclude that the biosynthesized
AgNPs could be considered as cost-effective, antioxidant,
and effective therapeutic agent for controlling bacterial and
fungal growth.
Disclosure statement
The authors declare that there are no conflicts of interest. The authors
alone are responsible for the content and writing of the paper.
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