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Eco-friendly synthesis of zinc oxide nanoparticles using Cinnamomum Tamala leaf

extract and its promising effect towards the antibacterial activity

Happy Agarwal, Amatullah Nakara, Soumya Menon, VenkatKumar Shanmugam

PII: S1773-2247(19)30736-1
DOI: https://doi.org/10.1016/j.jddst.2019.101212
Reference: JDDST 101212

To appear in: Journal of Drug Delivery Science and Technology

Received Date: 23 May 2019

Revised Date: 7 August 2019
Accepted Date: 9 August 2019

Please cite this article as: H. Agarwal, A. Nakara, S. Menon, V. Shanmugam, Eco-friendly synthesis
of zinc oxide nanoparticles using Cinnamomum Tamala leaf extract and its promising effect towards
the antibacterial activity, Journal of Drug Delivery Science and Technology (2019), doi: https://
doi.org/10.1016/j.jddst.2019.101212.

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Eco-friendly synthesis of zinc oxide nanoparticles using Cinnamomum
Tamala leaf extract and its promising effect towards the antibacterial activity
Happy Agarwal, Amatullah Nakara, Soumya Menon and VenkatKumar Shanmugam*
School of Bio-Sciences and Technology, Vellore Institute of Technology, Vellore – 632014,
TN, India
*Corresponding Author Mail id: venkatkumars@vit.ac.in; drvenkatshanmugam@gmail.com

Abstract
Green synthesis of zinc oxide nanoparticles (ZnO NPs) using plant extracts provides an eco-
friendly and promising substitute to the conventional methods of chemical synthesis. The
present study focuses on the fabrication of nano-sized ZnO particles by using zinc oxide as a
precursor molecule and leaf extract of Cinnamomum Tamala as a reducing and capping agent.
The morphology and structural properties of these concocted ZnO NPs were characterized by
UV-Visible (UV-Vis) Spectrophotometry, Zeta Potential analysis, X-Ray Diffraction (XRD)
analysis, Scanning Electron Microscope (SEM), Energy Dispersive X-Ray (EDX) analysis and
Fourier-Transform Infrared (FT-IR) Spectroscopy. The nanoparticles have a particle size of
26.57 nm and were in the hexagonal wurtzite phase which was confirmed by XRD analysis.
Zeta potential analysis confirmed the moderate stability of the nanoparticles and SEM analysis
confirmed that the nanoparticles were spherical and hexagonal in shape. The antibacterial
properties of the nanoparticle against Staphylococcus aureus were studied by broth dilution
method, protein leakage analysis, membrane stability analysis, and growth curve analysis on
selected bacterial species. The results showed time and concentration-dependent reduction in
bacterial growth by leakage of intracellular proteins and cellular contents due to membrane
damage. This method of synthesis of ZnO NPs from Cinnamomum Tamala is simple, eco-
friendly, cost-effective and convenient and, therefore, is expected to have applications in bio-
remediation, drug deliveries, catalysis, and other medical fields.

Keywords: Zinc oxide nanoparticles; Cinnamomum Tamala; leaf extract; green synthesis;
antibacterial activity

1. Introduction
Nanoparticles exhibit better physical, chemical as well as biological properties than their bulk
counterparts because of which they have gained significant interest in the research field [1].
Some of these properties include better biocompatibility, UV-blocking properties, absorption of
solar radiations, lower melting points, ability to form suspensions, superparamagnetism in
1
magnetic materials, easy diffusion at elevated temperatures and better electrical conductivity
[2]. This has augmented research on the synthesis that allows desirable control of the size and
shapes at the nanoscale for various industrial, environmental, chemical, biological and medical
applications. Some of these applications include drug and gene delivery using nanoconjugates,
tissue engineering, bio-sensing, bio-labelling, use as antimicrobial agents, detection of
pathogens, hyperthermia tumor destruction, bone regeneration due to migration, proliferation
and adhesion capabilities, medical imaging, phagokinetic studies, synthesis of nanocomposites,
nanomedicines, nanoceramics and nanopolymers [3][4].
Ionic metal oxide nanoparticles, particularly zinc oxide nanoparticles have kindled much
interest due to their wide variety of physical [5][6] and chemical properties [7][8] and also
their antimicrobial properties [9][10]. According to recent studies, zinc oxide nanoparticles
induce cell death by photoactivation and oxidative bursting in eukaryotes and inhibit the
growth of prokaryotes by cytotoxicity [11]. Nanoparticles have been synthesized from metals
like silver(Ag), gold(Au), copper(Cu) and iron(Fe) [12] and metal oxides like zinc oxide(ZnO)
[13], cupric oxide(CuO) [14], magnesium oxide(MgO) [15], silver oxide(AgO) [16] and
titanium dioxide(TiO2) [17]. Metal oxide nanoparticles are used in optoelectronics and
piezoelectric devices since their high density and mechanical stability renders them eccentric
physical and chemical properties [18][19]. Among the metal oxide nanoparticles, ZnO
nanoparticles have allured researchers due to their broad spectrum of antibacterial activity and
easily tuneable chemical behaviors. ZnO NPs have potential implementations as bacteriostatic
agents, coating agents, ZnO-coated carbon nanotubes (CNTs), ZnO nanowires, bio-imaging
and also in catalysis, drug and gene delivery systems and cosmetics [20][21]. ZnO NPs exhibit
some special characteristic properties like high photocatalytic activity, excellent thermal and
chemical stability, and UV filtering, anti-corrosive and luminescence properties [22]. ZnO NPs
are also applied in manufacturing industries due to their waterproofing and anti-aging
properties and also due to their property of improving toughness and integrity of various
polymers. Most prominently, ZnO NPs find applications in biological and biomedical fields
due to their antiseptic, anti-inflammatory, anti-cancerous, wound healing and antibacterial
activity against a wide range of pathogenic bacteria [23] as well as some multi-drug resistant
(MDR) bacteria [24]. Nano ZnO is readily absorbed into the body due to its particle size. Zinc
acts as a co-factor of many essential enzymes and has also been regarded as safe by the Food
and Drug Administration (FDA). Therefore, ZnO NPs also find applications as food additives
and components of food packaging agents due to their biocompatibility and anti-microbial
activity [23].
2
Chemical synthesis of nanoparticles using methods like laser ablation, lithography, and
ultrasonic fields can generate toxic by-products as well as require high pressure and/or
temperature conditions. They may also lead to the absorbance of some toxic chemicals on the
surface of the nanoparticle which could have adverse effects in medical and research-based
applications [25]. Therefore, biosynthesis of nanoparticles using ‘green’ sources like plant
extracts is the need of the hour. Synthesis of nanoparticles from plant sources provides a
simplistic as well as a cost-effective method of nanoparticle synthesis [26].

Cinnamomum tamala belonging to family ‘Lauraceae’ is used as a spice in food industries

because of its aroma. Flavonoids, tannins, alkaloids, terpenoids, saponins, and sterols have
been detected in C. tamala leaves. C. tamala leaf extracts have been used in Ayurvedic
medicine in the treatment of diarrhea, rheumatism, colic, bladder disorders, anorexia and
coryza [27]. Studies have shown that C. tamala leaves are antihelminthic, antimicrobial, anti-
dermatophyte and diuretic and also have hypoglycemic and hypolipidemic properties. They
have been used in the treatment of inflammation and cardiac disorders [28]. The essential oil
obtained from the leaves has the quorum-sensing inhibitory potential [29]. It is also
characterized by a high content of eugenol and sesquiterpenoids and is used as a stimulant and
also has anti-flatulent, anti-biofilm, astringent and carminative properties and is therefore used
in pharmaceutical preparations [30].
In this study, plant leaf extracts of Cinnamomum tamala having remarkable therapeutic
properties are used as surface stabilizing agents for the ‘green synthesis’ of ZnO NPs having
antibacterial properties. The structural properties of the synthesized NPs have also been
analyzed using standard characterization experiments. The anti-bacterial property of the NPs
against Staphylococcus aureus was examined using broth dilution assay, protein leakage
assay, and membrane stability assay and growth curve analysis of S. aureus culture inoculated
with the NPs.

2. Materials and methods

2.1 Preparation of plant extracts

Fresh leaves of Cinnamomum tamala were collected and washed thoroughly with running tap
water followed by Milli Q water. The leaves were air-dried and crushed using a sterile pestle
and mortar. These crushed leaves (10 g) were boiled with 100 mL of Milli Q water in a water
bath at 100˚C for 20 min. The extract was filtered in a separate conical flask.

3
2.2 Preparation of ZnO NPs
50 mL of 0.01 M zinc oxide solution was prepared to which 10 mL of the obtained plant
extract was added. This mixture was stirred for 3-4 hours at 700 rpm and 60˚C using a
magnetic stirrer until the color of the mixture gradually changed from white to light yellow.
After stirring, the solution was centrifuged at 5000 rpm for 5 min. The supernatant was
discarded and the pellet obtained was washed and dried in a hot air oven for 2 days at 80˚C.
The dried pellet was collected in an Eppendorf and used for characterization and further
experiments.

2.3 Characterization of NPs

The obtained ZnO NPs were measured for their maximum absorbance using UV-Visible
Spectrophotometry in the range of 300-700 nm. The stability of the NPs was determined by
Zeta Potential analysis. X-Ray Diffraction (XRD) analysis was used to determine the
crystallinity of the NPs and Scanning Electron Microscope (SEM) was used to characterize the
shape and external morphology of the NPs. Energy Dispersive X-ray (EDX) analysis was used
for the elemental analysis or chemical characterization of the NPs. Fourier-Transform Infrared
(FT-IR) spectroscopy was used to identify the functional groups present in the NPs.

2.4 Anti-bacterial activity screening of ZnO NPs

2.4.1 Broth Dilution Assay

The anti-bacterial effect of ZnO NPs was evaluated against Staphylococcus aureus in nutrient
broth medium. 20 µL of log-phase culture was inoculated to 5 mL of nutrient broth medium
containing different concentration (20, 40, 60, 80, 100 µg/mL) of ZnO nanoparticle.
Nanoparticle free broth medium was used as a control. Test tubes were incubated at 37°C for
24 h and the O.D. was recorded at 600 nm using UV-Visible Spectrophotometer. The IC50
value was determined based on the graph.
% Inhibition was calculated using the below-mentioned formula

2.4.2 Protein Leakage Analysis

4
The analysis was performed using a standard Bradford assay [31]. Fresh 12 h culture of
Staphylococcus aureus was used for the experiment. 100 µL from this fresh S. aureus culture
was mixed with well sonicated 2, 1, 0.5, 0.25 and 0.125 mg/mL ZnO NPs in 10 mL nutrient
broth. NP-free broth inoculated with culture was used as the control. These were incubated at
37˚C for 5 h and then centrifuged at 6500 rpm for 15 min. For each sample, 100 µL
supernatant was mixed with 1 mL of Bradford reagent. Optical density (OD) was measured at
595 nm after 10 min of incubation in the dark. Bovine Serum Albumin (BSA) was used as the
standard protein.

2.4.3 Membrane Stability of S. aureus

100 µL of a fresh culture of S. aureus was mixed with PBS buffer containing 2, 1, 0.5, 0.25 and
0.125 mg/mL ZnO NPs in 10 mL nutrient broth. NP-free broth containing culture was used as a
control. These were incubated at 37˚C for 30 min. The cells were centrifuged at 6500 rpm for
15 min. The pellet obtained in each was treated with 1 mL of 0.15% SDS solution. The OD at
595 nm was measured every 5 min.

2.4.4 Growth Curve

Two flasks containing 100 mL of nutrient broth were each inoculated with 100 µL of a fresh
12 h culture of Staphylococcus aureus. 1 mg ZnO NPs were dissolved in 1 mL of Milli Q
water and sonicated. One flask was NP-free and was used as the control to track the normal
growth of the bacteria while 1 mg/mL sonicated solution of ZnO NPs was inoculated in the
other. The flasks were kept in a shaker at 180 rpm. O.D. measurements from each flask were
taken every 1 h at 600 nm using a spectrophotometer. The comparative growth rate of bacteria
with and without the NPs was analyzed by plotting a graph of O.D. versus time.

3. Results and Discussions

3.1 UV-Visible Spectral Analysis

The absorption spectra of ZnO NPs were recorded using double beam UV-Vis
Spectrophotometer (Fig 1). The absorption spectrum for the sample was recorded in the range
of 300-700 nm. The absorption maximum was obtained at 340 nm which confirms the
synthesis of ZnO nanoparticles [32] since the absorption maximum for bulk ZnO occurs at
around 380-385 nm [33]. This occurs due to a phenomenon called Blue Shift in which the

5
reduction in the size of ZnO causes a decrease in distance between two valence bands, thereby,
causing an increase in the frequency. This leads to a corresponding decrease in wavelength
towards the blue end of the spectrum which is why the absorption wavelength of nano ZnO is
lesser than bulk ZnO [34][35]. A similar absorption maximum in the range of 340-360 nm has
also been reported in previous literature wherein ZnO NPs have been synthesized using
Laurus nobilis L. [36], Solanum torvum L. [37] and Ixora cocccinea [11] leaf extracts. From
the significant sharp absorption peak of ZnO NPs and in accordance with previously reported
results, it can be inferred that the particle size distribution is narrow, corresponding to and
further confirming the nanosize of the particles [38]. The sharp absorption peak also infers a
monodispersed nature of distribution [39] and indicates the spherical shape of the NPs [37]. It
was observed that after 3 h, the curve started to decline indicating that synthesis of ZnO NPs
stopped after 3 h and therefore, 3 h readings were used for characterization henceforth.

(Fig 1) UV-Vis spectrum of synthesized ZnO NPs

3.2 Zeta Potential Analysis

Zeta Potential is the potential difference between the mobile solvent and the stationary layer of
the solvent attached to the dispersed NPs. The surface charge of nanoparticles is a fundamental
attribute which is important in determining the colloidal stability, self-assembly and structure
and function of nanoparticles [40]. The surface charge of the synthesized NPs was determined
by Zeta Potential analysis (Fig 2). The NPs showed a mean Zeta Potential of -20.9 mV
indicating that they are moderately stable. The stability of the NPs is directly proportional to the
magnitude of the charge on them. A large positive or negative value of Zeta Potential indicates
6
better physical colloidal stability due to the electrostatic repulsions between the individual
particles. On the other hand, particles with a lesser magnitude of Zeta Potential may lead to
aggregation due to the action of Van der Waals forces [41]. Previously reported literature,
where ZnO NPs have been synthesized using Cardiospermum halicacabum leaf extracts, reports
the Zeta Potential of ZnO NPs in the range of -32.06 to -17.89 mV and indicates moderate
stability of the NPs, similar to results obtained in this literature [42]. Some of the most
important factors affecting Zeta Potential are pH and composition of the solvent and the ionic
strength of the medium. The stationary layer of solvent surrounding the particle forms a solvent
film around the particle. The nature of the solvent determines the aggregation effect and
subsequently the Zeta Potential of the particles. A hydrophilic solvent film results in repulsion
between individual particles to maximize contact with water. On the other hand, a hydrophobic
solvent film around the particles enhances attraction and thereby, aggregation between
individual particles, decreasing the Zeta Potential of individual NPs [43]. The ionic strength of
the dispersion medium also affects the Zeta Potential. The Zeta Potential is inversely
proportional to the ionic strength of the aqueous phase. The ionic strength of the dispersion
medium determines the interaction with water molecules and thereby, the net Zeta Potential
[44]. The solvent used in this analysis is Milli Q water and due to its amphipathic nature, has no
effect on the Zeta Potential measurement of the NPs. Therefore, the effects of solvents and ionic
strength determine the aggregation effect as well as the stability of the NPs. Based on the above
theory, NPs dissolved in a hydrophilic solvent and with less ionic strength may be a plausible
candidate for drug delivery due to less aggregation and better stability.

7
(Fig 2) Zeta Potential Analysis of synthesized NPs

3.3 Fourier Transform Infrared (FT-IR) Spectroscopy

FT-IR Spectroscopy was used to identify the chemical bonds present in the synthesized NPs.
An FT-IR Spectrometer operating in the percent transmittance (%T) mode at a resolution of
4000-400 cm-1 was used to obtain the IR spectrum. The presence of ZnO NPs is indicated by the
absorption peak at 460.99 cm-1 due to the stretching vibrations of Zn-O [45]. The other
chemical bonds present in the nanoparticle are depicted in the graph (Fig 3) and are obtained
from existing literature [46][47]. The broad stretch of the absorption band at 3319.49 cm-1
corresponds to O-H stretching of alcohol or phenolics from the plant extract. The absorption at
2922.16 cm-1 attributes to C-H stretching from the aliphatic functional groups from the
phytochemicals. The other peaks depicted correspond to the other functional groups present in
the phytochemicals from the plant extract. Presence of prominent peaks from the
phytochemicals leads to the conclusion that the phytochemicals successfully act as capping
agents for the ZnO NPs. These phytochemicals interact with the surface of the NPs and aid in
its stabilization [37][48]. Similar results have also been previously reported where ZnO NPs
have been synthesized using Solanum torvum L. [37] and Bauhinia tomentosa [9] leaf extracts.

(Fig 3) FT-IR Spectra of synthesized ZnO NPs

8
3.4 X-Ray Diffraction (XRD) Analysis

XRD spectrum of the synthesized NPs was obtained for 2θ values ranging from 20-90˚ using
an X-Ray Diffractometer at λ=1.5406 A˚. The prominent peaks were observed at 2θ values
with lattice planes at 31.8165˚(100), 34.4724˚(002), 36.322˚(101), 47.6097˚(102),
56.6208˚(110), 62.9286˚(103), 68.0033˚(112), 69.1416˚(201) (Fig 4) which are in close
agreement with JCPDS Data Card No: 36-1451. Peaks with similar lattice planes have also
been obtained in previous literature of biosynthesis of ZnO NPs using Laurus nobilis leaf
extract [36] and Costus pictus leaf extract [49]. This confirms the hexagonal wurtzite structure
of the NPs. The sharp diffraction peaks obtained indicate a good degree of crystallinity of the
NPs. Absence of other prominent diffraction peaks other than those attributed to those of the
NPs indicates the good degree of purity of the synthesized ZnO NPs [50]. The crystallite size
of the NPs was calculated as 26.57 nm which was obtained using the Debye-Scherrer formula.

where k is a constant equal to 0.90, λ is the wavelength of the incident X-ray, β is the FWHM
in radian, D is the crystallite size and θ is the Bragg’s angle in radian.
This is similar to existing literature where ZnO NPs synthesized using Costus pictus leaf
extract having a crystallite size of 29.11 nm [49] and Solanum nigrum leaf extract mediated
synthesis of ZnO NPs reports a crystallite size between 20-30 nm [10]. Another literature
where ZnO NPs have been synthesized using Camellia sinensis leaf extract also showed
similar lattice planes in the XRD spectrum of the NPs and average crystallite size of 30-40 nm
[48]. According to previous literature, a crystallite size of less than 100 nm produces
broadened diffraction peaks in case of NPs since such particles have very less parallel
diffraction planes. Peak broadening is a common phenomenon in small crystallite sized NPs
and this crystallite size calculated is the measure of smallest precise regions or coherently
diffracting domains present in the individual crystal. Particle size is the total size of the
particle comprising all the crystals [51]. However, in the case of nanomaterials, particle size
may be the same as crystallite size if the NPs are well dispersed and defined by a single
boundary. Therefore, for single-crystal NPs particle size is equal to the crystallite size, but in
other cases, the crystallinity of the NPs depends on the preparation method used. Hence, XRD
analysis if often restricted to the calculation of crystallite size and high-resolution microscopy
techniques are employed if particle size has to be calculated [52].

9
(Fig 4) XRD Spectrum of synthesized ZnO NPs

3.5 Scanning Electron Microscope (SEM) analysis

The external morphology of the synthesized NPs was examined using SEM. The SEM image
(Fig 5) revealed that most of the synthesized NPs were spherical and hexagonal in shape. Due
to the moderate stability of the NPs inferred by the Zeta Potential result, the NPs are bound to
interact with each other. Studies of NP aggregation due to particle-particle interactions show
that ionic strength and pH affect the aggregation of NPs [53][54]. Since the NPs have a negative
Zeta potential, the NPs interact with each other by Van der Waals interactions which causes
them to come relatively close to each other, leading to aggregation. This aggregation of NPs
also has an effect on their stability. Similar aggregation effect has also been observed in
previous literature where ZnO NPs have been synthesized using Cardiospermum halicacabum
leaf extracts [42].

10
(Fig 5) SEM image of the synthesized ZnO NPs

3.6 Energy Dispersive X-Ray Spectroscopy (EDX) analysis

EDX was carried out to determine the elemental composition of the NPs. In (Fig 6) presence of
zinc and oxygen signals from the NPs indicate that the NPs are pure in nature. The elemental
analysis of the NPs yielded 31.44% of zinc and 68.56% of oxygen (atomic %) which correlates
with the already reported results in which similar elemental composition has been observed
[55]. Log phase culture of Staphylococcus aureus inoculated with the NPs was also examined
for its elemental composition using EDX. The nanoparticle free culture was used as the control.
Elemental analysis of these showed that zinc was present only in the culture inoculated with the
NPs (Fig 7) and not in the control (Fig 8) which confirms the entry of the ZnO NPs into the
bacterial cells.

11
(Fig 6) EDX graph and elemental analysis of the NPs

(Fig 7) EDX graph and elemental analysis of bacterial culture treated with ZnO NPs

(Fig 8) EDX graph and elemental analysis of bacterial culture without ZnO NPs (Control)

3.7 Broth Dilution Assay

From the graph (Fig 9), it is seen that as the concentration of the NP increases, the percentage
of growth inhibition also increases. This can be due to the attachment of ZnO NPs to bacterial
cell walls and the subsequent release of Zn2+ ions into the bacterial cytoplasm. This indicates
that the synthesized ZnO NPs have antibacterial activity against Staphylococcus aureus. This
leads to the conclusion that the cell viability of S. aureus cells is dependent on the exposure
time as well as the concentration of ZnO NPs. Similar results have also been obtained in
previous studies where chemically synthesized ZnO NPs had a similar trend in growth
12
inhibition of the cyanobacterium Spirulina platensis [56] and also in bio-synthesized ZnO NPs
using Pandanus odorifer leaf extract where the NPs exhibited an inhibition in growth of
Bacillus subtilis and Escherichia coli with an increasing concentration of ZnO NPs from 50 to
100 μg/mL [57].

(Fig 9) Broth Dilution graph of bacterial culture treated with different concentrations of NPs

3.8 Protein Leakage analysis

From the graph (Fig 10), it is seen that as the concentration of NP increases, there is an increase
in degradation of the LPS layer of the outer cell membrane of bacteria. This is confirmed by the
increase in the OD with an increase in NPs concentration. The amount of cellular protein
released from bacterial cells is directly proportional to the concentration of the NPs which
confirms the anti-bacterial nature of the NPs [58]. These results indicate that most of the NPs
treated cells released their intracellular material into the extracellular matrix (cell suspension).
Upon internalization of ZnO NPs, local dissolution of ZnO occurs, releasing Zn2+ in the
bacterial cytoplasm. This leads to loss of proton motive force and subsequently, creates pores in
the cell membrane. This is one of the major reported mechanism and is used widely by ZnO NPs
as an anti-bacterial technique by internalization into the cells and loss of cell membrane
integrity, leading to leakage of intracellular components leading to cell death [59]. Similar
results have also been reported in previous literature where biosynthesized ZnO NPs using leaf
extracts of Aristolochia indica showed prominent protein leakage upon treatment of the NPs
with Escherichia coli, Staphylococcus aureus and multi-drug resistant strain of Acinetobacter
13
baumannii. The literature also concludes the mechanism of the bactericidal effect is the damage
to the cell membrane by the NPs causing an effluence of protoplasmic inclusions into the cell
suspension [60]. Another literature also depicts a similar trend wherein chemically synthesized
ZnO NPs show a concentration as well as time-dependent protein leakage in Klebsiella
pneumoniae [61].

(Fig 10) Protein Leakage analysis graph of bacterial culture treated with ZnO NPs

3.9 Membrane stability analysis

From the graph (Fig 11), it is seen that as the concentration of the NP increases, the O.D.
decreases which is a direct indication of a reduction instability of the outer membrane of the
bacterial cells. Also, it is observed that the time the NP is allowed to act on the bacterial
culture is directly proportional to the destabilization of the membrane by the NPs. The OD of
ZnO NPs treated cells showed a concentration-dependent decrease within 30 min of incubation
with the NPs in the presence of SDS. NPs uptake by cells involves electrostatic interactions
with the cell membrane or Van der Waals interactions. Interaction of positively charged Zn2+
with the negatively charged cell membrane is the most prominent mechanism of uptake. NPs
can be internalized through endocytosis or membrane diffusion through pores in the cell
membrane. Mechanism of binding and uptake depends on the shape, size and charge on the
NPs. Considerable binding of ZnO NPs to the cell membrane followed subsequently by
endocytosis disrupts the integrity and morphology of the phospholipid bilayer in the cell
membrane [62]. Some previous reports also depict similar results were chemically synthesized
ZnO NPs showed time as well as concentration-dependent destabilization in the cell
14
membrane of Klebsiella pneumoniae [61].

(Fig 11) Membrane stability analysis graph of bacterial culture treated with different
concentrations of ZnO NPs

3.10 Growth Curve

The OD at 595 nm is due to the scattering of light by bacterial cells and is a function of
bacterial cell density. It, therefore, correlates with the growth of the bacterial colonies. As
observed in (Fig 12), time-dependent changes in bacterial growth were observed in the culture
treated with ZnO NPs. A reduction in growth was observed in NP-treated culture which proved
the anti-bacterial property of the ZnO NPs. Some previous literature also reports similar
findings where chemically synthesized ZnO NPs suppresses cell viability and growth of
Bacillus subtilis in a concentration-dependent manner [63] and on a similar line, another study
showing that chemically synthesized ZnO NPs inhibit the growth of Pseudomonas putida with
increasing concentrations and decreasing the agglomerate size of the NPs [64]. This
antibacterial activity of the NPs is probably due to electrostatic interaction with the cell
membrane of the bacteria and internalization of the ZnO NPs in the bacterial cell which leads to
the production of reactive oxygen species (ROS) and membrane damage [65]. ROS are strong
oxidizing agents which oxidize lipids and proteins present in the cell and consequently cause
DNA damage. This causes oxidative stress and leads to a disruption in normal cellular functions
due to the inactivity of essential proteins and also disarray in replication and protein synthesis
due to DNA damage. It also alters or inhibits metabolism or respiratory cycles of the bacteria.

15
All these mechanisms finally lead to cell death and therefore, suppression of bacterial growth
causing a decline in OD [62].

(Fig 12) Growth curve of bacterial culture with and without NPs

4. Conclusion

The biosynthesis of zinc oxide nanoparticles using leaf extract of Cinnamomum Tamala
proves to be a cost-effective and eco-friendly method for synthesis of nanoparticles. The
synthesized zinc oxide nanoparticles were characterized using UV-Vis Spectrophotometer,
FT-IR, XRD, SEM, EDX and Zeta Potential analysis. SEM analysis showed that the
nanoparticles are spherical in shape. FT-IR analysis shows that the peak at 460.99 cm-1 is the
characteristic absorption of the zinc oxide (Zn-O) bond which confirms the formation of zinc
oxide nanoparticles. XRD analysis confirms the formation of nanoparticles with a particle size
of 26.57 nm and in the hexagonal wurtzite phase which is the form with the highest stability of
zinc oxide at ambient conditions. The Zeta potential of the nanoparticles was -20.9 mV which
indicates that the nanoparticles have moderate stability. The anti-bacterial property of the
synthesized nanoparticles against Staphylococcus aureus has also been proved using broth
dilution analysis, protein leakage analysis, membrane stability analysis and growth curve
analysis. The nanoparticles synthesized using this method are expected to have more extensive
applications in bioremediation, catalysis, drug delivery systems, and other medical fields.

16
Acknowledgments

The author would like to thank the Vellore Institute of Technology (VIT) for providing the seed
money to carry out the research work.

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Highlights
• Biosynthesis of Zinc oxide nanoparticles using Cinnamomum tamala leaf extract
• Anti-bacterial property of the NPs against Staphylococcus aureus
• Growth curve analysis of Staphylococcus aureus culture inoculated with the NPs
 Conflict of Interests
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