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Phyto-assisted green synthesis of zinc oxide nanoparticles and its

antibacterial and antifungal activity

Article · January 2019

DOI: 10.31830/2348-7542.2019.107

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Res. on Crops 20 (4) : 725-730 (2019) DOI : 10.31830/2348-7542.2019.107
With five figures
Printed in India
Phyto-assisted green synthesis of zinc oxide nanoparticles and its
antibacterial and antifungal activity

MARYAM BAYAT*, ELENA CHUDINOVA, MEISAM ZARGAR, MARINA LYASHKO,

KALISA LOUIS AND FREHIWOT KEBEDE ADENEW

Department of AgroBiotechnology
Institute of Agriculture, RUDN University, 117198, Moscow, Russia
*(e-mail : maryambayat1313@yahoo.com)

(Received : October 24, 2019/Accepted : December 12, 2019)

ABSTRACT

Zinc oxide (ZnO) has broad applications in different areas. Green synthesis is an
alternative to conventional physical and chemical methods. In our research, zinc oxide
nanoparticles (ZnO NPs) were synthesized by the use of leaf extract of strawberry plants
under different parameters. ZnO NPs biosynthesis was confirmed with UV-visible
spectrophotometer and then characterized by Scanning Electron Microscopy (SEM). The
significant antibacterial and antifungal efficacy of ZnO NPs was attained. The synthesized
ZnO NPs indicated antimicrobial activity against both Pseudomonas aeruginosa and Botrytis
cinerea pathogens. Different concentrations of ZnO NPs were tested against P. aeruginosa.
The results indicated that ZnO nanoparticles synthesized with strawberry leaves extract
showed antibacterial activity against tested bacteria. The most effective concentrations
were 26 and 42 mg/ml for non-calcinated and calcinated ZnO NPs, respectively. Different
ZnO NPs concentrations caused inhibition in the fungal growth of B. cinerea, and the
inhibition enhanced with the increase in the concentration of NPs. Overall, the results
showed a rapid, cost-effective, environmentally friendly and convenient method for ZnO
NPs synthesis, which could be used as a potential antimicrobial agent against bacterial
and fungal diseases.

Key words : Antibacterial, antifungal activity, spectrophotometer, strawberry, zinc oxide

nanoparticles

INTRODUCTION nutrient. Nanotechnology can ameliorate the

Nanoscience deals with the production
and usage of material with nanoscale
dimension. Nanoscale zinc oxide nanoparticles
(ZnO NPs) had been in current researches
because of their potential applications like
antibacterial, antifungal, wound healing,
antioxidant and optic properties (Kumar et al.,
2013; Happy Agarwal et al., 2017;
Madhusudanan et al., 2019). Hence, several
potential benefits such as increase of quality
and safety of food, diminish in farming inputs,
enrichment of absorbing nanoscale nutrients
from the soil allow nanotechnology to be quite
significant (Krishnamoorthy et al., 2012;
Hashemi et al., 2016). The importance of
nanomaterials in cropping systems is to
decrease the amount of spread chemicals,
reduce nutrient losses in fertilization and
enhance grain yield through managing pest and
agricultural systems and foods with novel tools
for the managing of rapid disease diagnostic,
increasing the plant capacity to absorb
nutrients (Alavi and Karimi, 2018).
The importance of nanotechnology in
cropping systems involves specific applications
like nanofertilizers and nanopesticides to trail
products and nutrients levels for enhancing the
crop productivity. Synthesis of nanoparticles
is mediated through chemical and green
techniques. Thus, we need an environment-
friendly and cost-effective strategy for
nanoparticle synthesis (Happy Agarwal et al.,
2017; Zargar et al., 2017). Because of the large
dose of chemicals and extreme environment
employed in the chemical production of NPs,
green methods by using plants, fungus,
bacteria and algae have been introduced and
adapted (Wani and Shah, 2012). Currently,
several microorganisms and plant extracts were
726 Bayat, Chudinova, Zargar, Lyashko, Louis and Adenew
used to synthesize inorganic nanostructures.
Size, orientation and physical characteristics
of nanoparticles influence the performance and
reproducibility of a potential device, hence,
making the synthesis of shape and size
controlled nanocrystals is an essential
component for any practical application
(Prakash and Paul, 2012; Nagarajan and
Kuppusamy, 2013).
Antibacterial and antifungal efficacy of
ZnO-NPs has recently obtained favourable
interest globally especially by using
nanotechnology to synthesize particles in the
nanometer region. There are many
microorganisms in the range from hundreds
of nanometers to tens of micrometers. ZnO-
NPs have attractive antimicrobial
characteristics because of enhanced specific
surface area as the diminished particle size
leading to increased particle surface. ZnO is a
safe biomaterial that possesses photocatalysis
effects on chemical and biological species
(Sirelkhatim et al., 2015). The main aim of this
research was the phyto-assisted green
synthesis of zinc oxide nanoparticles using
extracts of strawberry leaves and investigation
of their antibacterial and antifungal efficiency
against Pseudomonas aeruginosa and Botrytis
cinerea.
MATERIALS AND METHODS
Materials
Strawberry leaves were collected from
a field near the Moscow, Russia. All the
chemicals were of analytical grade purity. Zinc
acetate dihydrate, Zn(CH2COO)2.2H2O was used
as the precursor for the synthesis of ZnO
nanoparticles.
Fig. 1. Schematic presentation of strawberry leaf extract preparation.
Plant Extract Preparation
Strawberry leaves were collected,
washed and rinsed with distilled water, shade-
dried at room temperature, grounded and kept
for further use. Twenty g of leaf powder was
mixed with 200 ml of distilled water, boiled and
stirred for 1 h. The mixture was cooled at room
temperature and then filtered with a fabric filter
and centrifuged for 20 min at 10000 rpm. The
supernatant was filtered through paper filter
and stored at 40C for further use. The extract
was used for ZnO nanoparticles biosynthesis.
Fig. 1 represents the schematic procedure of
the strawberry leaf extract preparation.
Green Synthesis of ZnO Nanoparticles
Zinc acetate dihydrate, Zn(CH2COO)2.
2H2O was used as precursor for the synthesis
of ZnO nanoparticles. Hundred ml extract was
diluted by adding 100 ml distilled water and
heated to 70oC. Then 20 g of zinc acetate salt
was dissolved in this solution and subjected to
continuous heating and magnetic stirring at
70 o C and 800 rpm and addition of 2M
potassium hydroxide solution drop by drop till
the pH becomes 11. A progressive change in
the colour of solution from sturdy brown to
yellowish white during the process confirmed
the formation of nanoparticles. The formation
of precipitations increased gradually and finally
dense precipitations were obtained.
Resultant solution was kept for three
days at room temperature. The supernatant
was discarded, and the pellet was washed with
distilled water and centrifuged at 4000 rpm for
25 min for two times. The as-obtained viscous
gel was loaded into the oven at 90oC to produce
a brown colour dried mass and then ground to
 Phyto-assisted green synthesis of zinc oxide nanoparticles
make fine powders of ZnO nanoparticles. The
particles were divided into two parts : One part
calcinated in furnace at 400oC for 2 h and the
other part remained without any further
manipulation. A schematic representation of
the experimental procedure is shown in Fig. 2.
UV-Visible Absorption Spectroscopy
A UV-Vis spectrophotometer model
Varian Cary 50 was employed to confirm the
synthesis of NPs in the range of 200 to 800
nm.
Scanning Electron Microscopic (SEM)
Analysis
The structural morphology of
synthesized ZnO nanoparticles was studied by
Scanning Electron Microscopy (SEM) using
JEOL - JSM-6490 instrument.
Antibacterial Activity
The antibacterial potential of both the
types of synthesized ZnO NPs was determined
against pathogenic bacterium Pseudomonas
aeruginosa by a broth dilution method.
LB medium containing 2 g triptone, 2 g
NaCl, 1 g yeast extract and 200 ml distilled
water was used as liquid medium. For
preparation of LB agar, 3 g agar was added to
200 ml LB solution. The bacteria were
inoculated in the LB medium. The overnight
grown cultures of tested bacteria were used for
Fig. 2. Schematic presentation of ZnO NPs green synthesis.
727
the assay. Bacterial culture without ZnO NPs
served as the positive control. Then amounts
of 10 µl of night cultured bacterial solution
poured in tubes and let them to be incubated.
Ten µl of each tube with no visible bacterial
growth were placed in LB agar plates and
incubated for 24 h.
Antifungal Activity
Pathogenic fungi, Botrytis cinerea was
used in this study. Oat meal agar was applied
for cultivation of fungi. Agar dilution method
was selected for antifungal tests. ZnO NPs
added to the autoclaved media with different
concentrations and also a NP free solution were
poured into the Petri dishes and allowed to
solidify. A sample (1 cm) of mycelial was
obtained from the edge of 7-day old fungal
cultures and inoculated in the center of each
Petri dish. Then the Petri dishes were incubated
at 25°C. The antifungal property of ZnO NP was
evaluated by measuring the diameter of fungal
colonies at the time intervals.
RESULTS AND DISCUSSION
In the present investigation, for the first
time, the synthesis of zinc oxide nanoparticles
was carried out by a novel, ecofriendly, cost
effective, biocompatible, safe approach using
strawberry leaf extract as a natural reducing
and capping agent. It is known that the green
synthesis of ZnO nanoparticles is much safer
and environmentally-friendly as compared to
728 Bayat, Chudinova, Zargar, Lyashko, Louis and Adenew

chemical synthesis (Ogunyemi et al., 2019). The
reaction mechanism of ZnO NPs formation
could be illustrated as bioreduction of zinc ions
by various plant metabolites or phytochemical
components of the plant extract, including
terpenoids, polyphenols, sugars, alkaloids,
phenolic acids, tannins, glycosides, flavonoids,
proteins, etc., which resulted in complexation
and then hydrolysis reaction of zinc hydroxide
as there were the hydroxyl groups in the
polyphenol. These phytochemicals also acted
as capping agent or stabilizer for nanoparticles
to control the growth of them and preventing
them from aggregation. The synthesis of zinc
oxide by strawberry leaves resulted in end
product of light brown precipitate. The final step
was calcination and decomposition reaction to
yield ZnO NPs, a pale white powder (Sintubin
et al., 2012; Makarov et al., 2014).
Synthesized ZnO NPs were subjected to
UV-visible spectrophotometer to confirm the
formation of NPs at the first step. The UV-Vis
spectra of calcinated ZnO NPs showed an
absorption band at 393 nm and non-calcinated
ZnO NPs showed different absorbance peaks
at the range of 210 to 360 nm, which is the
characteristic wavelength range of zinc oxide
nanoparticles and is consistent with previous
studies (Fig. 3).
Scanning electron microscopy captured
the surface morphology and size of the ZnO
NPs (Fig. 4), which clearly demonstrated the
presence of quasi-spherical agglomerated nano-
scaled particles with sizes up to 225 nm and
spherical shaped ZnO NPs with size of about
30 to 40 n m. The syn th esized ZnO
nanoparticles were agglomerated. It was found
that usage of natural materials as reducing
agents sometimes led to particles agglomeration
(Chikkanna et al., 2019).

Fig. 3. UV-Vis spectra of (a) Calcinated ZnO NPs and (b) non-calcinated ZnO NPs.

Fig. 4. SEM images of synthesized ZnO NPs.

 Phyto-assisted green synthesis of zinc oxide nanoparticles
The antimicrobial activity of ZnO NPs
was demonstrated using broth dilution method.
Different concentrations of ZnO NPs were tested
against P. aeruginosa. The results indicated that
ZnO nanoparticles synthesized with strawberry
leaves extract showed antibacterial activity
against tested bacteria. According to the results,
the inhibitory effect of ZnO increased with the
increase in concentration (Fig. 5).
According to the results of this study,
the effective concentrations (EC) were 26 and
42 mg/ml for non-calcinated and calcinated
ZnO NPs, respectively. These results showed
that ZnO NPs had undergone an interaction
with the bacterial cell and it could be concluded
that these ZnO NPs had shown antibacterial
property on this class of bacteria. At very low
concentration of ZnO we didn’t observe any
antibacterial activity. It may be because of the
presence of lower amount of ZnO NPs which
sometimes acted as a nutrient for organisms.
Antimicrobial activity was always higher above
5 µg/ml concentration.
Many different bactericidal and
bacteriostatic mechanisms could be
represented (Zheng et al., 2015). For example,
there was presence of amines and carboxyl
groups on their cell surface and greater affinity
of ZnO ions towards these groups. Also,
extremely large surface area of ZnO NPs could
result in better contact with microorganisms.
Zinc ions released subsequently may be bound
to DNA molecules and led to disorder of the
helical structure by cross-linking within and
between the nucleic acid strands. Zinc ions
inside bacterial cells were also involved in the
disruption of biochemical processes
Fig. 5. CFU vs. concentration of ZnO NPs for (A) calcinated and (B) non-calcinated.
729
(Chikkanna et al., 2019).
Another mechanism is the formation of
reactive oxygen species (ROS) such as hydrogen
peroxide (H2O2), hydroxyl radicals (OH-) and
peroxide (O2-2) which contribute in different
mechanisms such as cell wall damage as a
result of ZnO-localized interaction, increased
membrane permeability, internalization of
nanoparticles because of loss of proton motive
force and uptake of toxic soluble zinc ions
(Sirelkhatim et al., 2015). Paper disk diffusion
bioassays did not show good results because
of the difficulties of diffusion of solid
nanoparticles through agar medium (Bhuyan
et al., 2015).
The results of the study indicated that
various concentrations of ZnO NPs caused
inhibition in the fungal growth of B. cinerea,
and the inhibition increased with the increase
in the concentration of NPs. The highest
concentration resulted in the maximum
inhibition. B. cinerea biomass was mainly
composed of hyphae. It was revealed that ZnO
NPs inhibited the growth of B. cinerea by
affecting cellular functions, which caused
deformation in fungal hyphae (Hel et al., 2011).
Agro-waste based green synthesized ZnO NPs
possess small sized particles, enhance the good
antimicrobial effect. This research leads to
develop a new strategy of cost-effective
synthesis and reducing usage of chemicals in
further studies. Green synthesis of
nanoparticles enhances importance because of
its cost-effectiveness, reduction of toxicity and
antimicrobial activity. Here, we have illustrated
synthesis of zinc oxide nanoparticles by the use
of strawberry leaves as a reducing agent.
 730 Bayat, Chudinova, Zargar, Lyashko, Louis and Adenew
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