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Enhanced antibacterial activity of Iron Oxide

Magnetic Nanoparticles treated with Argemone
Mexicana, L. leaf extract: an In vitro study
ARTICLE in MATERIALS RESEARCH BULLETIN SEPTEMBER 2013
Impact Factor: 1.97 DOI: 10.1016/j.materresbull.2013.05.059

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Materials Research Bulletin 48 (2013) 33233327

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Materials Research Bulletin

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Enhanced antibacterial activity of iron oxide magnetic nanoparticles treated with

Argemone mexicana L. leaf extract: An in vitro study
S. Arokiyaraj a, M. Saravanan b,*, N.K. Udaya Prakash a, M. Valan Arasu c, B. Vijayakumar a, S. Vincent d
a

R&D, Vel Tech High Tech Dr.Rangarajan Dr.Sakunthala Engineering College, Avadi, Chennai 600062, India
Department of Biotechnology, SRM University, Kattankulathur, Chennai 603203, India
c
Department of Bio-Environmental Chemistry, Chungnam National University, Daejeon 305 764, South Korea
d
Department of Advanced Zoology and Biotechnology, Loyola College, Chennai 600034, India
b

A R T I C L E I N F O

A B S T R A C T

Article history:
Received 8 November 2012
Received in revised form 30 April 2013
Accepted 7 May 2013
Available online 24 May 2013

The present study intended for the chemical synthesis of iron oxide nanoparticles (IO-NPs) followed by
characterization and evaluation of antibacterial activity after treating with Argemone mexicana L. leaf
extract. The formation of IO-NPs was conrmed by the colour change and further examined by UVvis
spectroscopy. The morphology was characterized by using SEM and TEM, which showed spherical
particles of uniform size ranged between 10 and 30 nm and the crystallites were determined through
XRD. The peaks in XRD pattern are in good agreement with that of face-centered cubic form of iron oxide
nanoparticles. FT-IR spectroscopy conrmed the attachment of bioactive molecules of plant on the IONPs surfaces. Furthermore, the antibacterial efcacy of IO-NPs, plant extract and IO-NPs treated with
plant extract was screened against Escherichia coli MTCC 443, Proteus mirabilis MTCC 425 and Bacillus
subtilis MTCC 441. The results showed a noteworthy inhibition on P. mirabilis and E. coli with IO-NPs
treated plant extract. This outcome may pave a way for using the magnetic nanoparticles as a drug
carrier system to cure bacterial diseases.
2013 Elsevier Ltd. All rights reserved.

Keywords:
A. Magnetic materials
B. Chemical synthesis
C. Electron microscopy
C. X-ray diffraction

1. Introduction
Antibiotic resistance, a well-known phenomenon in nature
assumes signicant public health importance when it gets
amplied many folds due to human misuse and neglect [1]. It
has become a serious public health concern with economic and
social implications throughout the world [2]. To overcome this, a
newer area of research, i.e. nanoparticles in controlling bacterial
growth is carried out by many authors. The antimicrobial activities
of aluminum oxide, silver nanoparticles, gold nanoparticles and
iron oxide nanoparticles have been previously reported [35]. Due
to their antibacterial activities, metallic nanoparticles represent an
effective solution for overcoming bacterial resistance [6].
Magnetic nanoparticles have applications in biomedicine due to
their controllable size of less than 100 nm which give them the
ability to attach with microbial cells. Magnetic biomaterials
provide the ability to be directed and concentrated within the
target tissue by means of external magnetic eld and be removed
once therapy was completed [7]. The drug loaded magnetic
nanoparticles helps in the controlled release of drug which reduce
side effects due to their lower dosage and minimize or prevent

* Corresponding author. Tel.: +91 9443077097; fax: +91 044 27453903.

E-mail address: bioinfosaran@gmail.com (M. Saravanan).
0025-5408/$ see front matter 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.materresbull.2013.05.059

drug degradation by using pathways other than gastrointestinal

and have several advantages such as, small particle size, large
surface area, magnetic response, bio-compatibility and nontoxicity [8].
The synergistic impact of nanoparticles as antimicrobials is
already studied and showed enhanced result. The synergistic
impact of silver nanoparticles with b-lactam antibiotics as an
antibacterial agent [9], silver nanoparticles and antibiotics [10] as
an effective antibacterial agent, antibacterial effect of iron oxide
and titania [11], magnetic nanoparticle and microwave exposure
[12], magnetic nanoparticle with glycol chitosan [13] and magnetic
nanoparticles and cephalosporins [14] were reported.
Similarly, the antiseptic qualities of medicinal plants have been
long recognized. A major part of total population in developing
countries still uses folklore medicines obtained from plant
resources [15]. Biologically active compounds which are present
in plants have always been of great interest. Recently there has
been a revival of interest in herbal medications due to a perception
that there are lower incidences of adverse reactions to plant
preparations compared to synthetic pharmaceuticals [16].
Argemone mexicana L. (Papaveraceae), commonly known as
Mexican prickly poppy, a native of Tropical America is introduced
and naturalized in India and generally occurs as wasteland weed in
almost every part of the country. It is a prickly, glabrous, branching
herb with yellow juice and showy yellow owers. The plant

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S. Arokiyaraj et al. / Materials Research Bulletin 48 (2013) 33233327

contains alkaloids as berberine, protopine, sarguinarine, optisine,

chelerytherine and the seed oil contains myristic, palmitic, oleic,
and linoleic acids. In Ayurveda, the plant is used as a diuretic and
purgative. Roots, leaves, seeds and yellow juice are used to cure
leprosy, skin-diseases, inammations and bilious fevers [17].
The objective of this study is to synthesize iron oxide magnetic
nanoparticles (magnetite) by chemical co-precipitation method
and to characterize using UVvisible spectrophotometer, XRD, FTIR, SEM, TEM and evaluation of antibacterial activity after treating
with A. mexicana L. leaf extract. In the present study a new
approach has been taken to study the antibacterial property of A.
mexicana ethanolic extracts treated with IO-NPs by disc diffusion
method against Escherichia coli, Proteus mirabilis and Bacillus
subtilis. To the best of our knowledge, the synergistic impact of
magnetic nanoparticles of iron oxide treated with any of the plant
extract as an antibacterial agent is not yet reported so far in the
earlier literature and hence this study is conducted to nd
the antibacterial activity of magnetic nanoparticles.

particle size of IO-NPs was calculated using full width at half

maximum of face-centered cubic (3 1 1) using DebyeScherrer
equation (D = Kl/b cos u) from the highest intensity of XRD pattern
[18]. Fourier transform infrared (FT-IR) spectrometer was used to
test the physicochemical interaction between leaf extract of A.
mexicana and IO-NPS. FT-IR spectra were recorded in the range
4000400 cm 1 with Thermo Nicolet Avatar 370 FT-IR spectrophotometer, using KBr pellet method. The morphology of the
synthesized IO-NPs was determined by scanning electron microscope (JSM-6390LV, JEOL, Japan) resolution at 300 nm at an
accelerating voltage of 15 kV. A thin lm was prepared by drop
coating synthesized IO-NPs on to carbon coated copper SEM grids
and then the extra sample solution was removed using a blotting
paper. The thin lm on the SEM grid was allowed to stand for 5 min
to dry prior to measurement. The ne powder of IO-NPs has been
dispersed in ethanol on carbon coated copper grid and the size and
shapes of the IO-NPs were obtained with TEM (TEM-JEOL 1200EX,
Japan) at an accelerating voltage of 120 kV.

2. Materials and methods

2.4. Microorganisms

2.1. Plant material collection and preparation of the extract

Three bacterial strains chosen for studies of antibacterial

assay were two Gram positive bacteria such as E. coli MTCC 443,
P. mirabilis MTCC 425 and one Gram negative bacteria B. subtilis
MTCC 441 consigned from Institute of Microbial Technology
(IMTECH), Chandigarh, India. Bacterial cultures were prepared in
Mueller Hinton Broth (Hi-Media, Pvt. Ltd., Mumbai, India) and
maintained for 24 h at 37 8C. The cell suspensions were diluted
with sterile MHB to provide an initial cell count of about
106 CFU/ml.

A. mexicana was collected from Chengelpet district belonging to

the state of Tamil Nadu, India and it was acknowledged by one of
the authors (N.K. Udaya Prakash, Botanist) and deposited in the
herbarium collections, Department of Biotechnology, Veltech High
Tech Dr.RR Dr.SR Engineering College, Chennai, India. The leaves
that are collected were investigated for pathogenic infections and
healthy leaves were selected after the examination. Thus the
selected leaves were washed in running tap water for removing
the surface pollutants. The washed leaves were dried at room
temperature for 23 days under dark condition. After drying the
leaves were powdered using electric blender. The extract was
prepared using Soxhlet apparatus with ethanol as a solvent. 15 g of
powder was made into three bags containing 5 g each, were used in
Soxhlet apparatus to form extract. Approximately 150 mL of
ethanol was used for making extract. The plant extract (PE), thus
formed was ltered and concentrated using rotary evaporator. The
concentrated extract was stored in refrigerator for further use.
The stored product was reconstituted again by using the same
solvent for required concentration.
2.2. Synthesis of iron oxide nanoparticles
The iron oxide nanoparticles (magnetite) were synthesized by
using co-precipitation method in alkaline media. Solutions of Fe2+
and Fe3+ with the molar ratio of 0.0125 M and 0.025 M in deionized
water were stirred and 29.5% of ammonium hydroxide was added
at a rate of 0.2 mL/min to adjust the pH at 10 in a non-oxidizing
environment. The black precipitate, thus obtained was washed
thrice with deionized water and twice with ethanol. The black
colored nanoparticles were separated by magnetic decantation to
form magnetite. The magnetites formed were washed 3 times with
deionized water and 2 times with ethanol, and dried at room
temperature.
2.3. Characterization methods
The synthesized nanoparticles were analyzed using ultra
violetvisible spectroscopy (UVvis) (Perkin-Elmer Lambda 2
Spectrophotometer) in 300800 nm wave length range. The XRD
patterns of the synthesized magnetite nanoparticles were recorded
by X-ray diffractometer (Bruker D8 Advance, Bruker AXS,
Germany) equipped with Ni lter and CuKa (l = 1.54056 A)
radiation with scan range of 1070 A at 40 kV and 40 mA. The

2.5. Treatment of IO-NPs with ethanolic extract of A. mexicana

Plant extract of A. mexicana and synthesized particles (IO-NPs)
were taken in the ratio of 1:1 and dissolved in 1 mL of ethanol. The
treated mixture was incubated in BOD incubator shaker at 45 8C for
12 h. After incubation this mixture was used for antibacterial assay
to nd the bactericidal activity.
2.6. Antibacterial assay of IO-NPs with ethanolic extract of A.
mexicana
The antibacterial efcacy was assayed by the standard Kirby
Bauer disc diffusion method against the bacterial pathogen [19].
The bacterial suspension (106 CFU/ml) swabbed on the Muller
Hinton Agar (MHA) plates using sterile cotton swab. The sterile
Whatman No. 1 paper disc of 6 mm dimension was impregnated
with the three solution components such as IO-NPs alone,
ethanolic extract of A. mexicana and IO-NPs treated with A.
mexicana in three different concentrations i.e. 12.5 mg/disc, 25 mg/
disc and 50 mg/disc respectively. The discs with streptomycin
(Himedia, 30 mg/disc) was placed on the MHA plates maintained as
control. The disc were gently pressed and incubated in inverted
position for 24 h at 37 8C. After the incubation period, the
susceptibility of the test organisms was determined by measuring
the diameter of the zone of inhibition using Himedia zone scale and
the obtained results were tabulated for evaluation. These
experiments were performed in triplicates for achieving the
optimum results.
3. Results and discussion
3.1. Synthesis and characterization of IO-NPs
UVvis absorption spectrum measures the wavelength of the
light that the nanoparticles absorb. The absorption spectrum of

S. Arokiyaraj et al. / Materials Research Bulletin 48 (2013) 33233327

3325

Fig. 3. XRD pattern of iron oxide magnetic nanoparticles.

Fig. 1. UVvisible spectral analysis of synthesized IO-NPs and the peak noted
around 315 nm.

Fig. 2. (a) FT-IR spectroscopy analysis of IO-NPs and Argemone mexicana leaf extract
and (b) FT-IR spectroscopy analysis of IO-NPs treated with Argemone mexicana.

the synthesized IO-NPs recorded the peak at 315 nm (Fig. 1).

Parallel results were observed by Cornell and Schwertmann, 2003
[20]. The FT-IR spectrum of the magnetite showed three distinct
bands at 566 cm 1, 1635 cm 1 and 3426 cm 1 (Fig. 2a). The FeO
stretching was established by vibration at 566 cm 1, OH
stretching vibration at 3426 cm 1 and OH distorted vibration
at 1635 cm 1 are due to surface adsorbed water. Similar results
were reported by Yang et al. [21]. FT-IR spectrum of plant extract
shows HCH stretching and bending vibrations at 2917
2849 cm 1 and 14661400 cm 1 respectively (Fig. 2a) due to nalkane [22]. The FT-IR spectrum of Fe3O4 nanoparticles after
treatment with the plant extract also showed HCH stretching
vibrations at 2972 and 2882 cm 1 and HCH bending vibrations
at 1450 cm 1 (Fig. 2b). From the FT-IR data, it is clear that the
bioactive molecules present in the leaves extract of A. mexicana
interacted with the synthesized Fe3O4 nanoparticles.
The XRD pattern of the synthesized magnetite displayed six
characteristic peaks (2u = 30.308, 35.718, 43.318, 53.318, 57.618 and
62.818), marked by their indices [(2 2 0), (3 1 1), (4 0 0), (4 2 2),
(5 1 1), and (4 4 0)] are quite identical to the peaks of Fe3O4 crystal
with the cubic spinal structure (Fig. 3). The results are in
agreement with the standard XRD pattern of Fe3O4 (JCPDS 750033). From the Scherrers formula the average crystallite size of
the nanoparticles was found to be 11 nm. Similar results were
obtained by Gerko [23].
Fig. 4 shows the micrographs of scanning electron microscope
of the synthesized magnetite, the morphology of the particles was

Fig. 4. SEM image of synthesized IO-NPs.

S. Arokiyaraj et al. / Materials Research Bulletin 48 (2013) 33233327

3326

Table 1
Antibacterial activity of IO-NPS, plant extract and IO-NPs treated with Argemone mexicana.
Bacteria

E. coli
P. mirabilis
B. subtilis

IO-NPs (mg/disc)

Plant extract (mg/disc)

IO-NPs + plant extract (mg/disc)

Streptomycin (mg/disc)

12.5

25

50

12.5

25

50

12.5

25

50

30

7
7
7

7
7
7

8
8
8

8
10
8

10
12
9

11
16
7

12
17
8

13
18
10

14
14
9

Values are expressed in mean of three independent experiments, () no zone of inhibition.

observed to be spherical. The size and morphology of the

synthesized magnetite were also characterized by transmission
electron microscopy (Fig. 5). The magnetites were found to have
smooth surface with well dispersed particles and the size ranged
between 10 and 30 nm. The results are in agreement with
observation from SEM images [24].
3.2. Antibacterial activity
The antibacterial assay of IO-NPs, ethanolic extract of A.
mexicana and combination effect of IO-NPs with A. mexicana
against the bacterial pathogens was identied (Table 1 and Fig. 6).

Fig. 5. TEM image of iron oxide nanoparticles.

The mean zone diameter of different concentration level of IO-NPs

was also determined. Based on the observations, it can be
perceived that very lesser amount of activity against the tested
bacterial strains was reported. Similar antibacterial effect of iron
oxide nanoparticles was reported by Tran et al. [25]. The
bactericidal effect of IO-NPs may be due to their smaller size.
Lee et al. [26] reported that the inactivation of E. coli by zero-valent
iron nanoparticles could be because of the penetration of the small
particles (sizes ranging from 10 to 80 nm) into E. coli membranes,
leading to oxidative stress and causes disruption of the cell
membrane.
The ethanolic extract of A. mexicana showed moderate activity,
and this inhibitory activity is may be due to the presence of
phytochemical alkaloids. This phytochemical is known to be a
DNA intercalator and an inhibitor of DNA synthesis through
topoisomerase inhibition [27,28]. Whereas the IO-NPs treated
with ethnaolic extract of A. mexicana showed higher activity
against the E. coli and P. mirablis which is reected in the mean
zone values of 13 mm and 18 mm respectively. The recorded
activity is higher than that of the standard streptomycin (14 mm).
In the case of B. subtilis the activity was found to be less and
maximum inhibition was observed at 50 mg/disc. Taylor et al. [29]
have also reported similar concentration dependent bacteria
inhibition. Mechanism: the antibacterial drugs and antibiotics
develop oxidative stress by generating reactive oxygen species
[30]. Reactive oxygen species (ROS) can cause damage to proteins
and DNA in bacteria [31]. This bactericidal property has been
evidenced by Park et al. [32] from silver metals. So, the inhibitory
property of IO-NPs treated with ethanolic extract of A. mexicana
could be due to the synergistic effect of the phytochemicals and
generation of ROS by IO-NPs.
4. Conclusion
The synthesized IO-NPs treated with leaf extract of A. mexicana
L. proved to have outstanding antimicrobial efcacy against the
bacterial pathogens. The biological approach on IO-NPs treated
with A. mexicana L. leaf extract material is the most rapid and ecofriendly method and it has a wide scope in opting as an excellent
drug delivery system. The present study proved that the
immobilized nanomaterials of magnetite can effectively improve
the drug loading and the antibacterial efciency against the
microbial pathogens. It is conclude that further studies on this eld
are of enormous importance in developing eco-friendly bionanomaterial and is highly recommended for biomedicines.
Conict of interest statement
The authors have declared no conict of interest.
Acknowledgement

Fig. 6. The antibacterial assay of (a) IO-NPs alone, (b) ethanolic extract of Argemone
mexicana and (c) combination effect of IO-NPs with Argemone mexicana against the
bacterial pathogens.

The authors express their gratitude to Sophisticated Analytical

Instrumentation facility, Indian Institute of Technology, Chennai
600 036, India for the characterization study.

S. Arokiyaraj et al. / Materials Research Bulletin 48 (2013) 33233327

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