ISSN: 0970-020 X

ORIENTAL JOURNAL OF CHEMISTRY CODEN: OJCHEG

An International Open Free Access, Peer Reviewed Research Journal
2017, Vol. 33, No.(6):
Pg. 2959-2969
www.orientjchem.org

Synthesis of Copper-Chitosan Nanocomposites and their

Applications in Treatment of Local Pathogenic
Isolates Bacteria
Sohier M.Syame*1,2, W.S.Mohamed3, Rehab K. Mahmoud4and Shimaa T. Omara1

1
Department of Microbiology and Immunology, National Research Centre,
33 Bohouth St. Dokki, Postal Code 12311, Giza, Egypt.
2
Department of Polymer, National Research Centre, 33 Bohouth St. Dokki,
Postal Code 12311, Giza, Egypt.
3
Department of Chemistry, Faculty of Science, Beni-Suif University, Egypt.
4
Department of Chemistry, Faculty of Science, Beni-Suif University, Egypt.
*Corresponding author E-mail: sohiersyame@yahoo.com

http://dx.doi.org/10.13005/ojc/330632

(Received: May 23, 2017; Accepted: July 01, 2017)

ABSTRACT

Development of nanotechnology, nanoparticles based product and its application is generating

interest of many researchers due to its promising biological achievement. However, it is well known
that inorganic nanomaterials are good antimicrobial agents. Among the various nanoparticles, metal
nanoparticles as copper assume special importance due to its low cost and easy availability. In this study,
the green synthesis method as eco-friendly approach is used to produce biologically copper
oxide nanoparticles from Ficus carica leaf extract, stable CuO NPs were formed. The synthesized
nanoparticles is characterized through the UV-Vis Spectrophotometer as it found to be 437 nm,
Transmission Electron microscopy (TEM) investigated particle sizes in the range 51-62 nm and
typical XRD patterns of the formed CuO NPs with high phase purity were obtained. Chitosan
stabilizer as naturally occurring polymers was added to the prepared copper nanoparticles in
different amounts to obtain Chitosan-CuO with different CuO percentage for long-term stability,
for prevention the agglomeration of nanoparticles and enhancing their antibacterial efficacy.
FTIR spectroscopy analysis was performed to copperoxide nanoparticle, chitosan, copperoxide
chitosan composite to confirm that CuO nanoparticle was mixed with polymer. The antibacterial
efficacy of chitosan, copper nanoparticles alone was studied against 22 bacterial pathogen like
Coaggulase +ve S. aureus, methicillin-resistant Coaggulase -ve S. aureus, Klebsiella pneumonia as
gram positive bacteria, Escherichia coli, e.coli o157 Salmonella typhi, Pseudomonas aeruginosa
as gram negative bacteria that showed antibacterial activity against Gram positive as well as
Gram negative bacteria. Antimicrobial activities of polymer/metal composites (Co oxide-chitosan
nanoparticle) is studied against the same bacterial pathogen. The effect of the prepared Chitosan-
CuO composite on ultrastructure of bacterial cells were evaluated by scanning electron microscopy
(SEM), it was found that the antibacterial activity of Cu-chitosan nanoparticle composite is more
greater than antibacterial activity of copper nanoparticles and chitosan alone that indicate the
addition of chitosan stabilizer enhance at great extent the antimicrobial activity of CuO NPs.

Keywords: Copper oxide nanoparticle, Copper oxide –Chitosan nano particle composite,
Ficus Carica leaf extract, Anti-microbial activity.
2960 Syame et al., Orient. J. Chem., Vol. 33(5), 2959-2969 (2017)

INTRODUCTION particles at their reactivity and its rates of surface

reactions23,24, so the immobilization of colloidal metal
Resistance of microorganisms to antibiotics nano particles with naturally occurring polymers is
have been increased, so many attempts have very important for prevention the aggregation and
been demonstrated for synthesis of resistant create a great positional stability to the nano particles
antimicrobial compounds against many pathogenic to be used in a wide range of application, as well
microorganisms which cause dangerous threat to as such alternation in chemicophysical properties
health1. Nanotechnology is an important research field may enhance minimum bactericidal concentration
concerning with the manipulation and design of small (MBC)/minimum inhibitory concen-tration (MIC),
particles, in the range of 100 nm or less called nano antibacterial activity values25. Chitosan is the second
particles2-4 that showed a wide range of properties naturally occurring biopolymer, it is composed of
which differ from its bulk material as electrical, N-acetylglucosamine and glucosamine units, it is a
optical, magnetic, catalytic and biological activity as nontoxic bio-degradable and biocompatible polymer
they exhibit a wide range of anti-microbial activity have many applications in the biomedical and
against different species of Gram-negative, Gram- pharmaceutical fields26. It is recorded that chitosan
positive bacteria and fungi5-7. Metal nano particles of lower molecular weight has been showed strong
(NPs) as (NiO, CuO, Sb2O3 and ZnO,) have been bacterial and superior biological activities than high
subjected of research interest in the last few years molecular weight. It has been recorded that chitosan
especially CuO NPs which gained great attention of act as antibacterial and anti-fungal substance27. As
researchers due to its potential industrial uses as chitosan disrupting the cell membrane by binding
catalytic process, gas sensors, solar cells and super to the bacterial cell surface of the negative charge,
conductors1,8, also its application in wound dressings changing its permeability that leading to come out
and biocidal properties9-11. Its well-known that copper of material inside the bacterial cells causes its
is greatly toxic to microorganisms such as bacteria death28. Chitosan has antibacterial activity only in
(Pseudomonas aeruginosa, Staphylococcus aureus, an acidic medium as its not soluble above pH 6.5.
E.coli ,) to be considered an effective bactericidal can There are many factors affect the antibacterial activity
be used in water treatment and food packages12. The of chitosan as degree of polymerization, pH of the
synthesis of stable uniformly-shaped CuO NPs has solution and chitin type27. Chitosan exhibits greater
showed great difficulty due to presence of some toxic antibacterial activity against Gram-positive than
chemicals formed by Chemical synthesis methods Gram-negative bacteria, as cell wall in Gram-positive
absorbed on the surface to cause harmful effects bacterium, is composed of one layer of peptide
in medical applications. The development of polyglycogen with large numbers of pores that
eco-friendly methods for the preperation of these allow foreign materials to enter into the cell easily
nano materials as new inexpensive acceptable in contrast to gram-negative bacterium of peptide
methodology of low cost production and less time polyglycogen thin cell wall membrane and bilayer
required have been considered from the most outer membrane from of lipopolysaccharide,
important aspects today, scientists used plant phospholipids and lipoprotein , that act as a barrier
extracts for greens ynthesis of different nanoparticles against foreign materials29,30,31. In this paper, CuO
such as Euphorbia tirucalli13, neem14, Cinnamomum NPs were prepared from plant extract of Ficus carica.
camphora15, Emblica officinalis16, tamarind17, lemon The obtained product was charac-terized with the aid
grass18 and alfalfa19,20. Polymer nanocomposites of PXRD, UV–visible, and TEM. Further, chitosan was
which consists of metal nanostructures and prepared and added by different concentration to the
polymers are of special prac-tical interest today. The synthesized nanoparticles to produce copperoxide
communication of biology with nanotechnology chitosan nanocomposite finally CuO Nps, chitosan
gives the chance for the development of nanoscale and copperoxide chitosan nanocomposite was
materials can be used in several applications at evaluated for antibacterial activities by employing,
biological science and clinical medicine21,22. The post- Coaggulase +ve S. aureus, methicillin-resistant
synthesis stability of metal nanoparticles in relation to Coaggulase -ve S. aureus as Gram positive bacteria,
its shape and size is very crucial as nanoparticles Escherichia coli 0157, Pseudomonas aeruginosa,
of weakly aggregation or dispersed in suspension Salmonella typhi and Klebsiella pneumonia as Gram
different from those of strongly aggregated nano negative bacteria using agar disc diffusion method
 Syame et al., Orient. J. Chem., Vol. 33(5), 2959-2969 (2017) 2961

Material and methods The crystal structure was characterized by X-ray

diffractometer, (XRD-6000, Shimadzu) equipped
Preparation of plant leaf extract with CuKα radiation source (λ =0.154056 nm) using
The powder of Ficus carica was purchased, Ni as filter at a setting of 30 kV/30mA. All XRD data
weighed 5 g and dissolved in 100mL of distilled water were collected under the experimental conditions
in the angular range 3o ≤ 2θ ≤ 80o. FT-IR analysis
and boiled for 20 min. at 50 oC. The extract is filtered
is used to identify and get an approximate ides of
by Whatmann No1 filter Paper before centrifuging at
the possible biomolecules that are responsible for
1200 rpm for 2 min. to remove biomaterials .Then capping and stabilization of the CuO-NPs with the
the filtrate is stored in a tight seal pack under 4 oC Ficus carica leaf extract.
for further use.
Preparation of chitosan-CuO nanocomposite:
Synthesis of copper oxide (CuO) nanoparticles Different amounts of the prepared CuO
using Ficus carica extracts were sonicated in chitosan solution for 20 min. and
20 mL solution of Ficus carica extract then stirred at room temperature for 8 h and washed
was introduced drop wise into 80 mL of 1mM (1mM) with ultra pure water by ultracentrifugation to remove
solution of copper sulphate(CuSO4.5H2O) in a 250 unbound Chitosan, then collected and dried at room
ml Erlenmeyer flask under continuous stirring . After temperature to obtain Chitosan-CuO with different
the complete addition of extract, the flask was then CuO percentage.
kept stirring for overnight at room temperature. Within
a particular time ,the colour solution was changed Characterization of the formed nano composite
into straw yellow, which indicates the formation of by fourier transforms infra red spectroscopy
copper oxide nanoparticles as seen in Fig.(1). CuO FTIR spectra chitosan nanocomposites
nano particles solution was purified by repeated were recorded on a FTIR spectrophotometer
centrifugation at 12,000 rpm for 15 min. followed (Thermo Nicolet, NEXUS, TM) in the range of
by re-dispersion of the pellet in deionized water to 4000–400 cm-1 using KBr pellets.
remove any unwanted biological materials. Then
the CuO nano particles were dried in oven at 80°C. Evaluation of antibacterial activity In Vitro
The obtained products of CuO nano particles were materials
stored in air tight container for further analysis. Twenty two bacter ial strains from
microbiology and immunology department, National
Characterization of copper oxide nanoparticles Research Center, Cairo, Egypt were isolated. They
The synthesized copper Nanoparticles include Coaggulase +ve S. aureus, methicillin-
were characterized through UV-Vis spectro- resistant Coaggulase -ve S. aureus as Gram-positive
photometer HITACHI U2300. The reduction of
bacteria and Escherichia coli, E.coli 0157, Klebsiella
copper Nanoparticles was Monitored by UV-
pneumoniae, Salmonella typhi, Pseudomonas
spectrophotometer range of absorbance from 250-
aeruginosa, etc as Gram-negative bacteria. The
480nm. The Morphology and mean particle size most frequent and abundant bacteria in many
of Copper oxide nanoparticles synthesized by this
disease infection represent Gram positive and
green method were determined by transmission
Gram negative bacteria, respectively were selected.
electronic microscopy( SEM) and (TEM). The SEM
Fresh inoculants for antibacterial assessment were
analysis was established by using Supra Zeiss with
prepared on nutrient broth at 37°C for 24 hours.
1 nm resolution at 30 kV with 20 mm Oxford EDS
detector. The elemental composition in the reaction Test method
mixture was determined by EDX analysis ,where the The antibacterial spectrum of copper oxide
TEM images were obtained by (JEM-1230-electron
nanoparticles, chitosan, Cu-chitosan nanoparticle
microscopy operated at 60 KV). Before taking a TEM
composite samples were determined against the test
image the sample was diluted at least 10 times by
bacteria by disc well - diffusion method on an agar
water. A drop of well dispersed diluted sample was
plate28. Briefly incubated cultures of bacteria were
placed onto a copper grid (200 mesh and covered
with a carbon membrane) and dried at ambient swabbed uniformly on the individual plates using
temperature. A drop of phosphotungestic acid (0.4%) sterile cotton swabs on the Muller Hinton Agar, 50
as a stain was deposited over the dried sample. µl of copper oxide nanoparticles, chitosan and Cu-
2962 Syame et al., Orient. J. Chem., Vol. 33(5), 2959-2969 (2017)

chitosan nanoparticle composite samples with different

concentration of 1,2,3,5 %were loaded in sterile discs,
Plates were incubated at 37°C for 24 hours.The inhibition
effect was verified by the presence of inhibition zones
around the discs where the solution was deposited and
sized for analysis and comparison.

Morphological analysis by scanning electron

microscope
Bacterial cultures of E. coli O157:H7, E.coli
0157:H7, methicillin-resistant Coaggulase -ve S.
aureus were reated with 2 mg/ml copper oxide Fig.2. UV-Vis absorption spectra of Copper oxide
nano particles for 8 hours. Aliquots of 1 mL samples nanoparticle materials synthesized with (1mM)
were centrifuged for 2 min. at 4000 rpm and the solution of copper sulphate(CuSO4.5H2O) and Ficus
cell pellets were resuspended in 0.1 mL MH broth. carica extracts
Subsequently, 20 µl of each concentrated sample TEM analysis of CuO nano particles
was deposited and spread onto a glass coverslip The shape and size of the synthesized
pre-washed with acetone and ethanol. After drying CuO-NPs were analysed by TEM analysis .Fig. 4
the slips for 15 min. at 37°C, the bacterial cells were shows the TEM image of biosynthesized CuONPs.
subjected to fixation, dehydration, and critical point The synthesized CuO-NPs have particle size in nano
drying for SEM analysis as described previously32. range with spherical shape.
results

Synthesis of copper oxide nanoparticles (visual

inspection)
After 28 h of reaction, The reaction mixture
colour change from Light to dark colour, that can be
given by below (Fig.1) the reduction of Cu+ ions it
show the dark brown colour as the Surface Plasmon
vibration was the excited in a metal nano particles.

Characterization of copper oxide nanoparticles

UV-spectrophotometer Fig.3. SEM image of the synthesized CuO
The reduction of Cu+ ions was monitored nanoparticles
by UV-Vis Spectrophotometer at range( 250-500) As seen in Fig. 5 shows the X-ray diffraction
for the metal ions stability. The peak was obtained (XRD) pattern of the CuO powder synthesized from
at 437nm as showed in Figure. 2. of copper sulphate(CuSO4.5H2O) in the presence
of Ficus carica extracts .The XRD pattern revealed
Scanning electron microscope (SEM) analysis the orientation and crystalline nature of copper
SEM images revealed that the synthesized oxide nano particles. The peak are indexed at
copper oxide nanoparticles are clustered and the (110), (-111), (111), (-202), (020), (202), (-113),
surfaces of the aggregates are rough. The SEM (-311), (220), (311), and (004) planes with position
images indicated that the size of crystalline CuO with 2θ values of 32.48o, 35.48o, 38.95o, 48.74o,
nanoparticles particles from the SEM scale are 53.44o, 58.33o, 61.53o, 65.78o, 66.24o, 72.42o and
ranged between 51 to 62 nm Figure.3. 75.04o. No another diffraction peaks of other phases
are detected, investigating the phase purity of
CuO-NPs. The average crystallite size of the
synthesized copper oxide nanoparticles was calculated
to be 14 nm using Debye-Scherrer equation (Klug
and Alexander, 1954): D=Kλ/βcosθ.Where D is
the crystallite size of copper oxide nanoparticles, λ
represents wavelength of x-ray source 0.1541 nm)
used in XRD, β is the full width at half maximum of the
diffraction peak, K is the Scherrer constant with value
Fig.1. Formation Copper oxide nanoparticle from 0.9 to 1 and θ is the Bragg angle.
 Syame et al., Orient. J. Chem., Vol. 33(5), 2959-2969 (2017) 2963

Coaggulase -ve Staphylococcu saureus. Table. (1)

represents the antibacterial activity of CuO NPs
for various bacteria by the disc diffusion assay.
Results showed that CuO NPs demonstrated
excellent antimicrobial activity against wide range of
bacteria also CuO nanoparticles showed significant
antibacterial activity on Gram negative bacterial
strains than Gram positive one. According to zone
of inhibition copper nano particles strongly inhibited
the growth of Gram negative bacteria as the zone
of inhibition recorded for E.coli 0157 (6.6 mm),
Pseudomonas aeruginosa (6.3 mm), Salmonella
typhimurium, Klebsiella pneumoniae subsp. Ozaenae
Fig.4. TEM image of the synthesized CuO
(6.1mm), Aeromonas (6 mm). On the other hand these
nanoparticles X-ray diffraction Analysis
nano particles showed a low inhibitory effect on the
growth of Coaggulase +ve Staphylococcu saureus
(3.1mm) and methicillin-resistant Coaggulase -ve
Staphylococcu saureus (3.7 mm).

Fig.5. XRD pattern of the synthesized copper

oxide nano particles Fourier transform infrared
spectroscopy analysis

Fourier Transform Infrared (FTIR) spectra (a)

of CuO nano particles, Chitosan and chitosan-CuO
composite samples are shown in Fig. 6 from it is clear
that. A sharp peak at 3388 cm-1 can be indicated
to hydrogen bonded O-H groups of alcohols and
phenols and also to the presence of amines N-H
of amide ,The major peak was observed to be 513
cm-1 should be a stretching of Cu-O, While the bands
at 1465 cm-1 and the peak at 1319 cm-1, 1346 cm-1
assigned to C=O stretching and N-H bending. While
FTIR of chitosan was showed that the intense and
wide band at 3220 cm-1 is attributed to the vibration (b)
of -OH group, the characteristic peaks at 1513 cm-1
and 1040 cm-1 are due to the vibration of C=O . In
chitosan-CuO composite spectra, the intense bands
at 3480 cm-1 and 1040 cm-1 are attributed to the
stretching vibration of N-H and Cu-O respectively.

Activity of copper oxide nanoparticles

Antimicrobial efficacy of CuO-NPs was
analyzed against 22 pathogenic bacterial isolates
represented grame negative, gram positive
bacteria as E.coli 0157, Pseudomonas aeruginosa, (c)
Salmonella typhimurium, Klebsiella pneumoniae
Fig. 6. FT-IR of synthesized (A) CuO-NPs, (B) Chitosan
subsp. Ozaenae, Aeromonas, coaggulase +ve and (C) chitosan-CuO composite samples. in Ficus,
Staphylococcu saureus and methicillin-resistant leaf extract
2964 Syame et al., Orient. J. Chem., Vol. 33(5), 2959-2969 (2017)

The result of antibacterial activity of chitosan strains using disc well diffusion method. (Fig.7),
Chitosan was showed antibacterial activity Table 3 shows the effect of CuO nanoparticles with
against gram negative, gram positive microor-ganisms chitosan at different concentration 1%, 2%,3%,5%,
as shown in table.2, the zone of inhibition of the chitosan chitosan with CuO nanoparticles showed significant
vary from 2 to 3.9 mm depending on the strain of the antibacterial activity on Gram negative bacteria than
bacteria as it showed the maximum values against Gram positive one. CuO nanoparticles with chitosan
Gram positive microorganism as Coaggulase +ve of concentration 3%, 5% strongly inhibited the growth
Staphylococcu saureus 3.6 mm and 3.9 mm for of bacteria as the zone of inhibition recorded from
methicillin-resistant Coaggulase -ve Staphylococcu 4.9 mm for Serratia fonticola to 8 mm for E.coli o157
saureus where diameter of zone of inhibition of the at concentration 3% for CuO nanoparticles with
gram negative microorganisms was ranged from 2 mm chitosan, while the zone of inhibition for the same
for Shigella sonnei to 2.9 mm for E.coli O157. concentration ranged from 3.9 mm to 4.1 for the
Coaggulase +ve and methicillin-resistant Coaggulase
Antibacterial activity of chitosan with CuO -ve S. aureus Gram positive bacteria. The zone of
nanoparticles (mm.) inhibition for CuO nanoparticles with chitosan of
The antibacterial activity of the CuO concentration 5% recorded from 5.2 mm for Serratia
nanoparticles with chitosan were evaluated against fonticola to 8.5 mm for E.coli o157 while the zone of
2 Gram positive and twenty Gram negative bacterial inhibition for the same concentration ranged from 4.2
Table. 1: Antibacterial activity of copper oxide mm to 4.3 for the Coaggulase +ve and methicillin-
nanoparticles resistant Coaggulase -ve S. aureus gram positive
bacteria. The zone of inhibition for CuO nanoparticles
No. Bacteria (mm)
CuO Table. 2: Antibacterial activity of chitosan
nanoparticles %
No. Bacteria (mm)
(chitosan)
1. E.coli o157:H7 6.6±1mm
2. Shigella sonnei 5.9±1mm
1. E.coli o157:H7 2.9±1mm
3. Shigella flexeneri 5±1mm
2. Shigella sonnei 2±1mm
4. Salmonella typhimurium 6.1±1mm 3. Shigella flexeneri 2.4±1mm
5. S.enteritidis 3±1mm 4. Salmonella typhimurium 2.7±1mm
6. S.montevideo 3±1mm 5. S.enteritidis 2.4±1mm
7. Pseudomonas aeruginosa 6.3±1mm 6. S.montevideo 2.2±1mm
8. Serratia liquefaciens 4±1mm 7. Pseudomonas aeruginosa 2.8±1mm
9. Citrobacter freundii 5±1mm 8. Serratia liquefaciens 2.5±1mm
10. Enterobacter intermedius 4.5±1mm 9. Citrobacter freundii 2.5±1mm
11. Proteus mirabilis 5±1mm 10. Enterobacter intermedius 2.3±1mm
12. Enterobacter cloaca 4.5±1mm 11. Proteus mirabilis 2.3±1mm
12. Enterobacter cloaca 2.1±1mm
13. Yersinia pseudotuberculosi 4.2±1mm
13. Yersinia pseudotuberculosis 2.8±1mm
14. Proteus penneri 4.1±1mm
14. Proteus penneri 2.6±1mm
15. Aeromonas 6±1mm
15. Aeromonas 2.7±1mm
16. providencia stuartii 5.4±1mm 16. providencia stuartii 2.3 ±1mm
17. Proteus vulgaris 5±1mm 17. Proteus vulgaris 2.2±1mm
18. Erwinia cacticida 4.7±1mm 18. Erwinia cacticida 2.3±1mm
19. Serratia fonticola 4.2±1mm 19. Serratia fonticola 2.2±1mm
20 Klebsiella pneumoniae subsp. 6.1±1mm 20 Klebsiella pneumoniae 2.5±1mm
Ozaenae subsp. Ozaenae
21 Coaggulase +ve S. aureus 3.1±1mm 21 Coaggulase +ve S. aureus 3.6±1mm
22 Methicillin-resistant Coaggulase 3.7±1mm 22 methicillin-resistant Coaggulase 3.9±1mm
-ve S.aureus -ve S. aureus
 Syame et al., Orient. J. Chem., Vol. 33(5), 2959-2969 (2017) 2965

with chitosan at concentration 1% vary from 4.2 Scanning electron microscope for detection of
mm to 6.6 mm on Gram negative bacteria, 3.3 mm morphological changes in bacteria by copper
to 3.8 mm on Gram positive bacteria, at the same oxidnanoparticle
As seen in Fig.8 SEM analysis showed
time the zone of inhibition for CuO nanoparticles
distribution an great changes in the cell morphology
with chitosan at concentration 2% ranged from 4.5
of E.coli O157:H7 (A,B), methicillin-resistant
mm to7 mm on Gram negative bacteria, 3.6 mm to Coaggulase -ve S. aureus (C,D) bacteria treated
3.9 mm on Gram positive bacteria. with Copper oxide nanoparticles.
Table. 3: Antibacterial activity of chitosan with CuO nanoparticles (mm.)

No. Bacteria Chitosan with CuO nanoparticles (mm.)

1% 2% 3% 5%

1. E.coli o157:H7 6.6±1mm 7±1mm 8±1mm 8.5±1mm

2. Shigella sonnei 6.2±1mm 6.5±1mm 7±1mm 8±1mm
3. Shigella flexeneri 5.3±1mm 6±1mm 6.5±1mm 7±1mm
4. Salmonella typhimurium 6.4±1mm 7±1mm 7.5±1mm 8±1mm
5. s.enteritidis 4±1mm 4.3±1mm 4.8±1mm 5±1mm
6. s.montevideo 5±1mm 5.5±1mm 5.7±1mm 5.9±1mm
7. Pseudomonas aeruginosa 6.5±1mm 6.8±1mm 7±1mm 7.3±1mm
8. Serratia liquefaciens 4.2±1mm 4.5±1mm 4.9±1mm 5.1±1mm
9. Citrobacter freundii 5.1±1mm 5.6±1mm 5.9±1mm 6.2±1mm
10. Enterobacter intermedius 4.8±1mm 5±1mm 5.3±1mm 5.8±1mm
11. Proteus mirabilis 5.1±1mm 5.3±1mm 5.8±1mm 6±1mm
12. Enterobacter cloaca 4.7±1mm 4.9±1mm 5.3±1mm 5.8±1mm
13. Yersinia pseudotuberculosis 5±1mm 5.5±1mm 6±1mm 6.3±1mm
14. Proteus penneri 4.6±1mm 4.9±1mm 5±1mm 5.3±1mm
15. Aeromonas 6±1mm 6.4±1mm 6.7±1mm 6.9±1mm
16. providencia stuartii 5.8±1mm 6±1mm 6.4±1mm 6.8±1mm
17. Proteus vulgaris 6±1mm 6.3±1mm 6.5±1mm 6.9±1mm
18. Erwinia cacticida 4.9±1mm 5±1mm 5.4±1mm 5.8±1mm
19. Serratia fonticola 4.4±1mm 4.7±1mm 4.9±1mm 5.2±1mm
20 Klebsiella pneumoniae 6.3±1mm 6.6±1mm 6.9±1mm 7.1±1mm
subsp. Ozaenae
21 Coaggulase +ve S. aureus 3.3±1mm 3.6±1mm 3.9±1mm 4.2±1mm
22 Methicillin-resistant Coaggulase 3.8±1mm 3.9±1mm 4.1±1mm 4.3±1mm
-ve S. aureus

Discussion that the amino acids, enzymes and abundance of

carboxylate and hydroxyl groups present in plants
Now day the field of nanotechnology and might play an important role at the formation
its based product of nanoparticles applications of copper hydroxide which hydrolyzed later into
are increased due to its many great biological nanocrystalline CuO34. It is well known that copper
effectiveness. In the present investigation, the oxide nanoparticles optical properties are strongly
green synthesis method is used to synthesize dependent on their size, shape and the structure
the Copper oxide nanoparticles using aqueous of the nanoparticles that can be studied by using
leaf extract of Ficus carica which is eco-friendly the UV/Vis spectra. The typical UV/Vis spectra of
cost effective approach when compared to other the Copper oxide nanoparticles synthesized are
reported several method used for preparing copper shown in Fig.2. The nanoparticles exhibited one
oxide nanoparticles 33, as it has been reported characteristic absorbance peak at 437 nm ,however
2966 Syame et al., Orient. J. Chem., Vol. 33(5), 2959-2969 (2017)

the electromagnetic radiation formed the surface well diffusion method. CuO nanoparticles showed
plasmon absorption in copper oxide nanoparticles significant antibacterial activity against all bacterial
through excitation of collective oscillation free strains as seen in Table 1 and agree with previous
electron conduction band, this type of resonance results of Hassan et al., and Vinod et al., who
occur when the wavelength of the incident light far demonstrated that Copper oxide (CuO) nanoparticles
exceeds the particle diameter35. Surface plasmon synthesized from leaf extracts acts as an effective
absorption band with a maximum at 437 nm reported antimicrobial agent against game positive infectious
the formation of copper oxide nanoparticles 36. organisms such as Staphylococcus aureus, Bacillus
Fig. 3 and 4 show the typical SEM and TEM images subtilis and grame negative organisms as E. coli,
of the CuO-NPs, respectively that showed the Vibrio cholerae and Pseudomonas aeruginosa34,41.
particles are nearly spherical in nature with rough The bactericidal effects observed in this study as
agglomeration of average diameter around 51-62 a result of Cu2+ ions released in solution by the
nm. The highly agglomeration of the nanoparticles NPs that binding to the bacterial cell surface of the
synthesis by this method may be explained by negative charge, changing its permeability that
the increase in the catalytic activity of the surface leading to come out of material inside the bacterial
nanoparticles 37, that can illustrate the higher cells causes its death . The mechanism investigated
antibacterial activity of the CuO-NPs. The XRD by Azam et al and Stochs et al., reported that the
pattern in Fig.5 revealed the orientation and copper ion causes disorders of the cellular proteins,
crystalline nature of copper oxide nanoparticles that lipids and helical structure of DNA molecules through
agreement with the International Center of Diffraction crosslinking between and within the nucleic acid
Data card (JCPDS-80-1916) to confirm the formation strands and destroy the biochemical process,
of a crystalline monoclinic structure38 also sharp also the reaction of copper with sulfhydryl (-S-H)
peaks structures in XRD patterns and crystallite groups and oxygen on the cell wall forms R-S-S-R
size less than 100 nm indicated the nanocrystalline bonds that blocks respiration centers of the cell and
nature of CuO-NPs as the highest peak observed cause its damage42,43. The diameter of inhibition
in XRD pattern represented crystallite size of 14 zone indicated susceptibility of microbes,the
nm using Debye-Scherrer equation as described. susceptible strains to CuO-NPs showed larger zone
FT-IR analysis in Fig.6 is used to determine an of inhibition, while resistant strains showed smaller
approximate ides possible biomolecules that are zone of inhibition. According to zone of inhibition the
responsible for stabilized and capping the formed CuO-NPs as seen in Table 1 exhibited more
CuO-NPs with Ficus carica leaf extract. The FTIR inhibitory activity towards gram negative than
analysis of CuONPs Fig. 6(A) investigated that they gram positive bacteria as seen in (Fig.7, Table
might surround by the any of these organic molecules 1 ) because there is a difference in structure of
such as alkaloids, terpenoids, fatty acids and the bacterial cell wall as gram negative cell wall
polyphenols which have reducing, capping capacity consists of single peptidoglycan layer while gram
and have a big role in the reduction of copper ions positive cell wall have several peptidoglycan
to copper nanoparticles that results agreement layers12, in contrast to J. Emima Jeronsia et al.,
with Kalainila et al., who reported the same type who observed that the Grampositive bacterial
of results39. Chitosan-CuO composite FTIR spectra strains have higher sensitivity to CuO-NPs than
as seen in Fig. (6)C displayed the characteristic Gramnegative44. In our research we demonstrated
bands of both Chitosan and CuO. The band at 400 the antibacterial activity of chitosan towards
cm-1 was ascribed to the stretching mode of Cu-O40. Gram positive and gram-negative bacteria as
When compared to the pure chitosan FTIR spectra in recorded in Table.2 and agreed with many
Fig. (6)B ,the bands represented the amide, amino investigation recorded that chitosan shows
and hydroxyl groups were shifted. The shift of FTIR antibacterial activity towards both gram-negative
bands confirmed that the CuO nanoparticle was and Gram positive bacteria due to the nature of
mixed with polymer. The antibacterial activity of the chitosan surface of as polycation of high charge
CuO nanoparticles, chitosan and CuO nanoparticles density which tightly attached and absorbed onto
with chitosan composite at different concentration the surface of bacterial membrane to disrupt the
were evaluated against two Gram positive and cell membrane, thus kill bacterial cells27,28,45,46. Also
twenty Gram negative bacterial strains using disc Chitosan can form various chemical bonds with
 Syame et al., Orient. J. Chem., Vol. 33(5), 2959-2969 (2017) 2967

metal particles and prevents agglomeration, thus tested bacteria strains coli O157:H7, and methicillin-
increasing the stability of the NPs26. resistant Coaggulase -ve S. aureus , as it find the way
into the bacterial cells causing great lipid bilayer and
membrane protein damage as reported in previous
studies 9,10,11,24, 52,53,54.

Fig.7. Antibacterial activity of Chitosan with CuO

nanoparticles at concentration 2, 3, 5%, negative
control © : Zone of inhibition against (a) Salmonella
typhimurium, (b) methicillin-resistant Coaggulase
-ve S. aureus and (c) E.coli O157 bacteria.

The chitosan medium increase the efficiency

of the antimicrobial activity of copper oxide nanoparticles
against Gram negative, Gram positive organisms
that indicated by the zones of inhibition as shown in Fig. 8. Scanning electron micrographs of E.coli O157:H7
Table.3, it was observed that zones of inhibition against (A) and methicillin-resistant Coaggulase –ve S. aureus
(C) . SEM images were taken from the bacterial cells coli
Gram negative and gram positive bacteria for CuO
O157:H7 (B), and methicillin-resistant Coaggulase -ve
nanoparticles with Chitosan at concentration 1%, S. aureus (D) treated with 2 mg/ml CuO nanoparticles
2%, 3%, and 5% respectively was higher than zones for 8 h. The control cells were incubated under the
of inhibition of CuO nanoparticles or chitosan alone, same conditions without nanoparticles
that agreement with study demonstrated that copper
Conclusion
oxide nanoparticles embedded within polypropylene
exhibiting stronger antimicrobial activity than metal we have demonstrated a simple biological
copper nanoparticles47 as coatings based on copper approach to fabricate highly stable copperoxide
/ polymer which called ex situ, the matrix is act as nanoparticles embedded in a natural polymer by
green method from Ficus carica plant extract .The
dispersion medium and the polymer incorporated
antibacterial properties of copper oxide nanoparticle,
into the synthesized cupper nanoparticles to chitosan and Copper oxide chitosan nanoparticle
develop antimicrobial activity48,49. The mechanisms composite were investigated against two Gram-
for the antimicrobial behavior of metal/polymer positive and twenty Gram-negative bacteria. The
nanocomposites based on thermoplastic matrices results investigated that Addition of polymers to
explained by the adsorption of bacteria on the metal-based particles enhanced to great extent
antimicrobial properties. Although there are many
polymer surface that cause water to be diffused
research investigated copper oxide nanoparticle
through the polymer matrix leading to corrosion embedded into different polymer matrices forming
processes and release of metal ions and water composite, further research is very important to
that dissolved in oxygen to the metal/polymer support the development of bioactive polymeric
nanocomposite surface causing destroy bacterial materials can be easily fabricated into fiber and film
for producing promise biocide materials for further
membrane25,50,47,51,30. The morphological changes
extension of applications can be used as in bed
and disruption of the bacterial cell membrane of lining, cotton ban-dages, wound dressing as well as
copper oxide nanoparticlen treated bacteria was for medical in hospital equipment or in prostheses
supported by SEM analysis as seen in Fig.8, the and food applications.

References
1. Ren,G ; Hu ,D; Cheng,C ; .Vargas-Reus, M; 2. Bai, C. Liu, M. Nano Today. 2012, 7, 258–281.
Reip,P ; Allaker ,RP, International Journal of 3. Thanh, N. T. K. ; Green, L. A. W. Nano Today.
Antimicrobial Agents. 2009, 33, 587–590. 2010, 5, 213–230
2968 Syame et al., Orient. J. Chem., Vol. 33(5), 2959-2969 (2017)

4. Brege, J. J.; Hamilton, C. E; Crouse, C. A 22. Pa u l , D. R . ; R o b e s o n , L . M . Po l y m e r

; Barron, A. R. Nano Lett. 2009, 9, 2239– nanotechnology: Nanocomposites. Polymer.
2242. 2008, 49, 3187–3204.
5. Comfort, K. K ; Maurer, E. I ; Braydich-Stolle, 23. Lemire, J.A.; Harrison, J.J.; Turner, R.J. Nat.
L. K ; Hussain, S. M. ACS Nano. 2011, 5, Rev. Microbiol. 2013, 11, 371–384.
10000–10008 . 24. Gunawan, C.; Teoh, W.Y.; Marquis, C.P.; Amal,
6. Yang,S ; Wang, C; Chen,L; Chen,S ;. Chem. R.. ACS Nano. 2011, 5, 7214–7225.
Phys., 2010, 120, 296–301. 25. Muñoz-Bonilla, A; Fernández-García, M..
7. Debbichi,L ; Marco de Lucas,M.C ; Pierson, Prog. Polym. Sci. 2012, 37, 281–339.
J.F; Krüger, P ; Phys, J .Chem. C . 2012, 116, 26. Muzzarelli, R.A. . Carbohydr Polym. 2011, 84,
10232–10237. 54-63.
8. Baek , Y.-W ; An, Y.-J “Science of the Total 27. Avadi ,M ;Sadeghi, A ;Tahzibi ,A; Bayati, K;
Environment. 2011 , 409 (8), 1603–1608. Pouladzadeh, M ;Zohuriaan-Mehr, M; Rafiee-
9. Bondarenko, O; Ivask,A; Käkinen,A; Tehrani, M. 2004, 40 , 1355-1361.
Kahru,A. Environmental Pollution .2012 , 169
28. Abou-Zeid, N; Waly, A; Kandile, N; Rushdy,
,81–89.
10. Jadhav,S; Gaikwad,S; Nimse, M; Rajbhoj, A; El-Sheikh, M; Ibrahim, H.. Carbohydrate
A. Journal of Cluster Science.2011, 22 , Polymers, 2011, 84 (1), 223-230.
121–129. 29. Lu, Y; Cheng, D; Lu, S; Huang, F; Li, G.. Textile
11. Studer,A.M ; Limbach,L.K; Van Duc, Research Journal, 2014, 84 , 2115-2124.
L.Toxicology Letters . 2010 , 197(3) 169– 30. Cioffi, N.; Torsi, L.; Ditarantano, N.; Tantalillo,
174 G.; Ghibelli, L.; Sabbatini, L.; Bleve-Zacheo,
12. Li Q; Mahendra S; Lyon D.Y; Brunet ,L; Liga, T.; D’Alessio, M.; Zambonin, P.G.; Traversa, E.
M. Water Research . 2008 , 42: 4591-4602. Chem. Mater. 2005, 17, 5255–5262.
13. R a v i k u m a r, B . S ; N a g a b h u s h a n a , H ; 31. Pinto, R.J.; Daina, S.; Sadocco, P.; Pascoal
Sunitha,D.V ; Sharma,S.C ; Nagabhushana, Neto, C.; Trindade, T. Biomed. Res. Int. 2013,
B.M ; Shivakumara,C ; Alloys. J . Compd. doi: 10. 1155/2013/280512.
2014, 585 , 561–571. 32. Xie, Y; He, Y; Irwin, P.L; Jin, T; Shi, X. Appl
14. Sastry,M; Shankar,S.S; Rai,A; Ahmad ,A. J. Environ Microbiol. 2011, 77, 2325–2331. doi:
Colloid Inter face Sci . 2004, 275 , 496–502. 10.1128/AEM.02149-10.
15. Huang,J ; Li, Q; D; Sun, Y;Lu, Y; Su, X. 33. Sankar, R; Manikandan, P; Malarizhi, V;
Yang, H. Wang, Y.Wang, W. Shao, N. He, J. Fathima, T; Shivashangari, K.S; Ravikumar,
Hong, C. Chen, Nanotechnology. 2007,18, V. Spectrochimica Acta A: Molecular and
105104–105115 Biomolecular Spectroscopy. 2014, 121, 746-
16. Ankamwar,B; Chinmay,D; Absar,A; Murali, 750.
S. Nanosci.J. Nanotechnol. 2005,10, 1665– 34. Vinod, Vellora.; Thekkae, Padil ; Miroslav, Cernik..
1671. Int. J. Nanomedicine. 2013, 8, 889 – 898
17. Ankamwar,B ; Chaudhary,M ; Murali,S. Nano- 35. Raja ,Naikaa ; Lingaraju,K ; Manjunath,K;
Metal Chem. 2005, 135 ,9–26. Danith Kumar, G; Nagarajuc, G; Suresh,
H; Nagabhushanaea,C .Journal of Taibah
18. Shankar,S.S ; Rai, A; Ankamwar,B; Ahmad,A;
University for Science 2015, 9, 7–12.
M. Sastry,M. Nat.Mater. 2004 , 3, 482–488. 36. Dhaneswar, D ; Nathb,B.C ; P. Phukonc, P;
19. Gardea-Torresdey, J.L ; Parsons,J.G; Doluia,S.K; ColloidsSurf.J ; B: Biointerfaces.
Gornez,E; J. Videa,J; Troiani,H.E ; Santiagol, 2013, 101, 430–433.
P. Nano Lett. 2002, 2 ,397–401. 37. Awwad, A.M; Albiss, B.A;Salem, N.M. SMU
Medical Journal 2015, 2, 1.
20. Gardea-Torresdey, J.L ; Parsons,J.G;
38. The XRD patterns were indexed with reference
Gornez,E; J. Videa,J; Troiani,H.E ; Yacaman, to the crystal structures from the ASTM charts:
M Langmuir .2003 , 19,1357–1361. Cu (chart card number 03-1005), CuO (chart
21. De Azeredo, H.M.C. Nanocomposites for food card number 44-0706), Cu 2O (chart card
packaging applications. Food Res. Int. 2009, number 05-0667 and 03-0898) and Cu4O3
42, 1240–1253. (chart card number 33-0480).
 Syame et al., Orient. J. Chem., Vol. 33(5), 2959-2969 (2017) 2969

39. Kalainila, P; Subha, V; Ernest, S; ravindran, 47. Delgado, K.; Quijada, R.; Palma, R.; Palza,
R.S. Asian J Pharm Clin Res. 2014, 7, 39- H. Lett. Appl. Microbiol. 2011, 53, 50–54.
43. 48. Zhang, W.; Zhang, Y.H.; Ji, J.H.; Zhao, J.;
40. Rahnama, A; Gharagozlou, M. .Optical and Yan, Q.; Chu, P.K. Polymer 2006, 47, 7441–
Quantum Electronics. 2012 , 44, 313–322 7445.
41. Hassan, M.S; Amna, T; Yang, O.B; Newehy, 49. Cometa, S.; Iatta, R.; Ricci, M.A.; Ferretti, C.;
M.H. E; Al-Deyab, S.S; Khil, M.S. Colloids Surf de Giglio, E. J. Bioact. Compat. Polym. 2013,
B:Biointerfaces. 2012, 97, 201 – 206. 28, 508–522.
42. Azam, A; Ahmed, A.S; Oves, M; Khan, M.S; 50. Palza, H.; Gutiérrez, S.; Delgado, K.; Salazar,
Habib, S.S; Memic, A. Int J Nanomedicine. O.; Fuenzalida, V.; Avila, J.; Figueroa, G.;
2012, 7, 60039. Quijada, R .Macromol. Rapid Commun. 2010,
43. Stohs, S.J; Bagchi ,D. Free Radic Biol Med . 31, 563–569.
1995, 18, 32136. 51. Vimala, K.; Sivudu, K.S.; Mohan, Y.M.;
44. Emima Jeronsia, J ; Vidhya Raj, D.J ; Allwin Sreedhar, B.; Raju, K.M. Carbohydr. Polym.
, L; Joseph, K; Rubini,1 ; Jerome Das,S . J 2009, 75, 463–471.
Med Sci .2016, 36,145151 52. Matai, I. Colloid. Surface B. 2014, 115,
45. Lahmer. R.A; Williams, A.P; Townsend, S; 359–367.
Baker, S; Jones, D.L. Food Control. 2012, 53. Newcomb, C. J. Nat Commun. 2014, 5,
26, 206-211 1024–1031
46. Lahmer. R.A; Williams, A.P; Townsend, S; 54. Lu, Z.X; Zhou, L; Zhang, Z.L; Shi, W.L; Xie,
Baker, S; Jones, D.L. American Journal of Z.X; Xie, H.Y; Pang, D.W; Shen, P. Langmuir.
Food, Nutrition and Health. 2016, 1, 7-12 2003, 19, 8765–8768.