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Karbala International Journal of Modern Science xx (2015) 1e11
http://www.journals.elsevier.com/karbala-international-journal-of-modern-science/

In vitro antibacterial and cytotoxicity studies of ZnO nanopowders

prepared by combustion assisted facile green synthesis
Prashanth G.K. a,b, Prashanth P.A. b,c,*, Utpal Bora d, Manoj Gadewar d,
Nagabhushana B.M. e, Ananda S. f, Krishnaiah G.M. a, Sathyananda H.M. a
a
Department of Chemistry, Sir M. Visvesvaraya Institute of Technology, Bengaluru, 562 157, India
b
Research and Development Centre, Bharathiar University, Coimbatore, 641 046, India
c
Department of Chemistry, Sai Vidya Institute of Technology, Bengaluru, 560 064, India
d
Department of Bioscience and Bioengineering, Indian Institute of Technology, Guwahati, 781 039, India
e
Department of Chemistry, M. S. Ramaiah Institute of Technology, Bengaluru, 560 054, India
f
Department of Chemistry, University of Mysore, Mysuru, 570 006, India
Received 17 July 2015; revised 5 September 2015; accepted 25 October 2015

Abstract

This research work presents the synthesis, characterization, evaluation of the antibacterial and anticancer activity of ZnO
nanopowders prepared by solution combustion method using the bio fuels Punica granatum L and Tamarindus indica L. The X-ray
diffractograms of all the samples revealed the hexagonal Wurtzite structure with the standard JCPDS pattern of zincite [36e1451].
Surface morphology of the samples was studied by SEM. Particle shapes and sizes were determined by TEM. Qualitative
phytochemical screening of the aqueous fruit extracts of Punica granatum L and T. indica L revealed the presence of many phyto-
components in them. Toxicity of the nanopowders was tested on Gram-negative Escherichia coli MTCC 7410 and Pseudomonas
aeruginosa MTCC 7903 by disk diffusion method. Minimum inhibitory concentration was determined by microbroth dilution
technique. Anticancer activity of ZnO powders was tested against breast cancer cell line MCF-7 by MTT assay. The cytotoxicity
was assessed by hemolytic activity.
© 2015 The Authors. Production and hosting by Elsevier B.V. on behalf of University of Kerbala. This is an open access article
under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Keywords: ZnO nanopowders; Green synthesis; Antibacterial; MTT assay; Hemolytic cytotoxicity

1. Introduction dependent properties. ZnO being a wide band gap

In recent years, ZnO has received more attention
due to its unique morphology and dimension
* Corresponding author. Department of Chemistry, Sai Vidya
Institute of Technology, Bengaluru, 560 064, India. Tel.: þ91 96633
13591.
E-mail address: prsnthmysore@gmail.com (P. P.A.).
Peer review under responsibility of University of Kerbala.
semiconductor (3.3 eV) has received more attention as
it possesses a wide range of useful properties including
electrical, chemical, optical and magnetic properties
[1e3]. ZnO has gained much importance as it can be
applied in many applications such as for gas sensing,
catalyst, for semiconductors, UV-shielding materials,
nano generators, an antibacterial agent, cosmetics as
well as medicinal applications [4e8].

http://dx.doi.org/10.1016/j.kijoms.2015.10.007
2405-609X/© 2015 The Authors. Production and hosting by Elsevier B.V. on behalf of University of Kerbala. This is an open access article under
the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Please cite this article in press as: P. G.K. et al., In vitro antibacterial and cytotoxicity studies of ZnO nanopowders prepared by combustion
assisted facile green synthesis, Karbala International Journal of Modern Science (2015), http://dx.doi.org/10.1016/j.kijoms.2015.10.007
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2 P. G.K. et al. / Karbala International Journal of Modern Science xx (2015) 1e11
In recent days controllable synthesis of ZnO nano-
materials of desired size and shapes has been the
subject of investigation by researchers because it has
been found that the properties of ZnO nanoparticles are
size and morphology dependent. Several methods have
been developed to synthesize ZnO nanoparticles such
as precipitation, hydrothermal, combustion, solegel,
chemical vapor deposition, spray pyrolysis, sono-
chemical [9e15]. Among various methods developed,
combustion synthesis is a simple, convenient and
efficient method which involves the redox reaction
between an oxidizing reagent usually desired metal
salts and a reducing agent forming highly pure prod-
ucts within a very short time [11,16,17].
The attempts of biosynthesis of nanoparticles star-
ted as the physical and chemical processes were costly.
It was observed that many a times, chemical methods
lead to the presence of some of the toxic chemicals
absorbed on the surface of nanoparticles that may have
adverse effects in medical applications [18]. This
problem can be overcome by synthesizing nano-
materials by green methods [19]. Green synthesis
provides advancement over chemical and physical
methods as it is cost effective, environment friendly
and in this method there is no need of using high
pressure, toxic chemicals [20]. Biological entities have
been shown to serve as both reducing and stabilizing
agents for the synthesis of metallic nanoparticles [21].
Among the various categories of biological com-
pounds, phytochemicals are emerging as a useful nat-
ural resource for the synthesis of metallic nanoparticles
[22]. Plant materials such as Pomegranate Poly-
phenols, Murraya koenigii, Calotropis gigantean,
Ananas comosus and Acacia leucophloea have been
used as fuels to synthesize various nanomaterials
[23e27].
Pomegranate belongs to punicaceae family. It is one
of the important horticulture fruits to the Mediterra-
nean climate. Number of studies has been reported on
the antioxidant properties of pomegranate constituents
[28]. Literature shows pomegranate contains flavo-
noids, anthocyanidins and shows potent antioxidant
activity [29]. Tamarindus indica L (tamarind)
commonly known as tamarind tree is one of the most
important multipurpose tropical fruit tree species in the
Indian subcontinent. It has numerous chemical values
and is rich in phytochemicals. Hence the plant is re-
ported to possess antidiabetic, antimicrobial, anti-
venomic, antioxidant, antimalarial, hepatoprotective,
antiasthmatic, laxative and anti-hyperlipidemic activ-
ities. Every part of the plant, from root to leaf tips is
useful for human needs [30]. Almost all parts of the
Please cite this article in press as: P. G.K. et al., In vitro antibacterial and cytotoxicity studies of ZnO nanopowders prepared by combustion
assisted facile green synthesis, Karbala International Journal of Modern Science (2015), http://dx.doi.org/10.1016/j.kijoms.2015.10.007
tree find some use or the other in food, chemical,
pharmaceutical, textile industries, as fodder, timber,
and fuel. The pulp contains organic acids, such as
tartaric acid, acetic acid, citric acid, formic acid, malic
acid and succinic acid [31].
The interaction of nanoparticles with microorgan-
isms and biomolecules is an expanding area of
research, which is still largely unexplored yet. The
present study reports the green synthesis of ZnO
nanopowders using two different fuels. The fuels used
were the aqueous fruit extracts of P. granatum L and
T. indica L. The effect of as formed ZnO nanopowders
was investigated against Gram-negative bacteria
namely Escherichia coli MTCC 7410 and Pseudo-
monas aeruginosa MTCC 7903 by disk diffusion
method. Minimum inhibitory concentration (MIC)
was determined by micro broth dilution technique.
Anticancer activity of ZnO nanopowders was tested
against breast cancer cell line MCF-7 by MTT (3-(4,
5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bro-
mide) assay. Hemolysis assay was performed to
evaluate the biocompatibility of these ZnO nano-
powders in vitro. Biological activity of ZnO nano-
powders prepared by SCS using bio fuels was
compared with that of the commercially available
ZnO nanopowder.
2. Experimental details
2.1. Fruits collection
Fruits of P. granatum L (PG) were collected from
local market and fruits of T. indica L (TI) were
collected in and around Hunasamarnahalli, Bengaluru.
It was ensured that the fruits were uninfected. Fruits
were washed individually under running tap water to
remove any traces of soil particles and other dirt. Then
they were washed with distilled water and air dried at
room temperature. Fruits of T. indica L were cut into
pieces and dried in shade at room temperature for
15e20 days.
2.2. Preparation of aqueous extracts of P. granatum L and
T. indica L
30 g of the fresh seeds of P. granatum L and 10 g of
the dried fruits of T. indica L were subjected to soxhlet
extraction separately for 72 h using 60 mL of double
distilled water. The aqueous solutions obtained after
extraction were filtered using Whatman No.1 filter
paper. Filtrates were collected and stored for further
use.
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P. G.K. et al. / Karbala International Journal of Modern Science xx (2015) 1e11
2.3. Green synthesis of ZnO nanopowders
All the reagents used in the experiment were of
analytical grade without further purification. ZnO
nanopowder (>99% purity, SigmaeAldrich) of particle
size less than 100 nm was used as the commercial
nanopowder. 3 g of zinc nitrate hexahydrate
[Zn(NO3)2.6H2O] was dissolved in 40 mL of distilled
water. Then 10, 11 and 12 mL of the aqueous fruit
extract of P. granatum L were added separately to it.
The reaction mixture was mixed well using with the
help of a magnetic stirrer for 10 min and crystallizing
dish containing the reaction mixture was placed in a
preheated muffle furnace at 375 ± 10  C. Within a
short while the solution boiled to form a gel followed
by decomposition with the evolution of gases. Similar
procedure was repeated by taking 3, 5 and 7 mL of the
aqueous fruit extract of T. indica.
Samples prepared above were ground into fine
powders and labeled as ZnO (PG-1, PG-2, PG-3) and
ZnO (TI-1, TI-2, TI-3) for ZnO powders obtained
using fruit extract of P. granatum and T. indica
respectively.
2.4. Characterization techniques
The phase of as-synthesized ZnO nanopowders
was characterized between wide range of Bragg an-
gles by powder X-ray diffraction using PANalytical
X'pert diffractometer with Cu Ka radiation
(l ¼ 1.5418 Å) as the source. The formation of ZnO
and the absence of any other functional groups from
the precursors were confirmed using Fourier Trans-
form Infrared Spectroscopy (FTIR) which was carried
out on PerkineElmer spectrophotometer (Model:
Spectrum 1000) using KBr disc method within the
range of 350e3000 cm1. The weight change with
heat was measured using thermogravimetric analysis
(TGA) to check the amount of impurities which might
have derived from the fruit extracts in ZnO nano-
powders. TGA studies were carried out using a TG-
DTA/DSC apparatus (NETZSCH STA 409 PC/PG).
The surface morphology of the samples was studied
by scanning electron microscopy (SEM) performed
on HITACHI S3400N. The particle shape and size
were determined by Transmission Electron Micro-
scopy (TEM), carried out on Philips CM200. To un-
derstand which components are responsible for
combustion, qualitative phytochemical examinations
were carried out for the aqueous fruit extracts of
Punica granatum L and T. indica L as per the stan-
dard methods [32e34].
Please cite this article in press as: P. G.K. et al., In vitro antibacterial and cytotoxicity studies of ZnO nanopowders prepared by combustion
assisted facile green synthesis, Karbala International Journal of Modern Science (2015), http://dx.doi.org/10.1016/j.kijoms.2015.10.007
3
2.5. Bio studies
2.5.1. Screening of ZnO nanopowders for antibacterial
activity
The antibacterial activity of ZnO nanopowders was
carried out by disk diffusion method in Mueller Hinton
(MH) agar media. Homogeneous dispersion ZnO of
nanopowders of 100 mg/mL was prepared by ultra-
sonication. Cell suspensions prepared from E. coli and
P. aeruginosa bacterial cultures grown on Trypticose
soya broth (adjusted to 1e2 x 105 cells/mL) were used
as inoculums. 100 mL of test culture was inoculated on
MH agar plates (90 mm). Dispersion of ZnO nano-
powders (10 mL, 100 mg/mL) and ciprofloxacin (10 mL,
1 mg/mL) were impregnated on 6 mm sterile Whatman
No. 1 disks. Test compounds and standard disks were
placed on agar plates and they were incubated at 35  C
for 24e48 h and observed for zone of inhibition
around the disk. Ciprofloxacin was used as the stan-
dard. Sterile water served as the negative control.
2.5.2. Determination of MIC by micro broth dilution
technique
Cell suspensions from bacterial cultures grown on
trypticose soya broth (adjusted to 1e2  105 cells/mL)
were used as inoculumns. Ciprofloxacin of 1e64 mg/
mL (two fold dilutions) in MH broth and aqueous
dispersions of ZnO nanopowders of 16e1024 mg/mL
(two fold dilutions) in MH broth were tested against
the test cultures. RPMI media (MH broth) inoculated
with culture and without ZnO nanopowders was used
as the control. 90 mL of the dispersion of test com-
pound of different concentrations was mixed with
10 mL inoculum in 96 well plates in triplicate. 90 mL of
RPMI media (MH agar) without drug mixed with
10 mL inoculums was used as the control. Treated
bacterial cultures were incubated at 35  C. The test
plates were observed after 24e48 h and optical density
was measured at 600 nm in tecan plate reader. Percent
inhibition was calculated as follows [absorbance con-
trol (untreated) - absorbance (treated)]/absorbance
control. MIC was determined as minimum concentra-
tion of ZnO nanopowder giving a minimum of 50%
inhibition of OD as compared with the control.
2.5.3. Assessment of anticancer activity by MTT assay
Cells were cultured in Dulbecco's Modified Eagle's
Medium (DMEM), supplemented with 11% fetal
bovine serum at 37  C with 5% CO2. Cells were plated
in 96-well plates (105 cells/well) containing 100 mL of
medium. After 24 h, ZnO nanopowders at different
concentration dispersed in Dimethyl sulfoxide
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4 P. G.K. et al. / Karbala International Journal of Modern Science xx (2015) 1e11
Fig. 1. Powder XRD pattern of ZnO nanopowders.
(DMSO), were added to each well and incubated for
24 h. Control received the same amount of DMSO.
Growth of the cells was quantified by the ability of
living cells to reduce the yellow dye MTT to a blue
formazan product. At the end of 24 h of incubation the
medium in each well was replaced by fresh medium
(100 mL) containing 0.5 mg/mL of MTT. Four hours
later, the formazan product of MTT reduction was
dissolved in DMSO and absorbance was measured
using a microplate reader. Effect of ZnO nanopowders
was quantified as the percentage of control absorbance
of reduced dye at 570 nm.
2.5.4. Assessment of cytotoxicity by hemolysis
The hemolysis activity test was performed against
ZnO nanopowders by following the procedure as re-
ported in the literature [35]. The percentage of hemo-
lytic index was calculated by using the following
formula [36].
ðOD test sample  OD negative controlÞ
Percent hemolysis ¼
ðOD positive control  OD negative controlÞ
Please cite this article in press as: P. G.K. et al., In vitro antibacterial and cytotoxicity studies of ZnO nanopowders prepared by combustion
assisted facile green synthesis, Karbala International Journal of Modern Science (2015), http://dx.doi.org/10.1016/j.kijoms.2015.10.007
where, OD is the optical density value. Triton X-100
and phosphate buffer saline (PBS) of pH 7.4 served as
positive and negative controls respectively.
3. Results and discussion
3.1. Crystal structure
Crystal structures of the as-prepared ZnO nano-
powders synthesized using different volumes of the
bio fuels, P. granatum L and T. indica L are as shown
in Fig. 1. All the diffraction patterns agree with the
standard Joint Committee on Powder Diffraction
(JCPDS) pattern of zincite [36e1451]. The crystal
structure of the ZnO nanopowders was evaluated
using DIAMONDecrystal and molecular structure
visualization software with the help of PXRD data.
This confirms the hexagonal Wurtzite (P63mc)
structure of ZnO with lattice parameters
a ¼ 3.24982 Å and c ¼ 5.20661 Å. The crystal lattice
and structural parameters of ZnO nanopowders are
presented in Table 1. The packing diagram of ZnO
nanopowders is as shown in Fig. 2. The crystallite
size was calculated using Scherrer equation, D ¼ k l/
b Cosq, where D is the crystallite size, k is the shape
constant (~0.9), l is the X e ray wavelength, q is the
Bragg's angle and b is the line broadening at half the
maximum intensity (FWHM) in radians. The average
crystallite sizes of all ZnO nanopowders prepared are
given in Table 2. The decrease in crystallite size with
increase in fuel/oxidant molar ratio may be due to
number of moles of gaseous products liberated. As
more gases are liberated with increase in fuel to
oxidant molar ratio, the agglomerates disintegrate and
additional heat is carried away from the system
thereby hindering the particle growth, which in turn
produces nanoparticles of smaller size with high
specific area [37]. Also, an increase in the crystallite
size with the increase in fuel/oxidant ratio was
observed. This might be due to increase of flame
temperature which assists the crystal growth [38].
Similar observations have been reported by other
researchers [39]. As the samples ZnO (PG-2) and (TI-
2) exhibited better crystallinity, they were selected for
further characterization and bio studies.
 100
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P. G.K. et al. / Karbala International Journal of Modern Science xx (2015) 1e11 5

Table 1
Crystal lattice and structural parameters of ZnO nanopowders.
Atom Oxidation state Wyckoff notation x y z Occupancy
Zn þ2 (2b) 0.3333 0.3333 0 1
O 2 (2b) 0.3333 0.3333 0.3821 1
Crystal system ¼ Hexagonal, Lattice parameters; a ¼ 3.24982 Å, c ¼ 5.20661 Å; Space group ¼ P63 mc (No. 186).

Fig. 2. Packing diagram of ZnO nanopowders.

Table 2
Average crystallite size of ZnO nanopowders.
Sample Volume of the Average crystallite
Fig. 3. FTIR spectra of ZnO nanopowders.
fuel used (mL) size (nm)
ZnO (PG-1) 10 20
ZnO (PG-2) 11 07 transmittance bands at 435 cm1 correspond to the
ZnO (PG-3) 12 04 ZneO bonding and confirm the presence of ZnO
ZnO (TI-1) 03 08 particles.
ZnO (TI-2) 05 10
ZnO (TI-3) 07 04
3.3. TG analysis

3.2. FTIR analysis
FTIR spectra of ZnO (PG-2) and ZnO (TI-2) are
shown in Fig. 3. The absorption band near 3400 cm1
was due to OeH mode (water absorbed from the
moisture), 1400-1515 cm1 were due to C]O
stretching mode and the band near 2345 cm1 was due
to absorption of atmospheric CO2 on the metallic
cations. Hence these peaks can be neglected. The
TG curves of ZnO (PG-2) and ZnO (TI-2) are shown
in Fig. 4 (a and b) respectively. The temperature scale
for the measurement was from 25  C 1480  C. In both
the samples the weight increased up to 100  C. The
probable reason for increase in weight is due to OH
bonding that might have come from the reaction with
moisture [40]. Both the samples show that the weight
decreased continuously up to 1480  C. The total weight
decrease for ZnO (PG-2) and ZnO (TI-2) were 18% and

Please cite this article in press as: P. G.K. et al., In vitro antibacterial and cytotoxicity studies of ZnO nanopowders prepared by combustion
assisted facile green synthesis, Karbala International Journal of Modern Science (2015), http://dx.doi.org/10.1016/j.kijoms.2015.10.007
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6 P. G.K. et al. / Karbala International Journal of Modern Science xx (2015) 1e11

Fig. 4. TG curves of (a) ZnO (PG-2) and (b) ZnO (TI-2).

12.5% respectively. The microvoids in the samples
could be the reason in allowing weight loss over a
period of 1480  C. ZnO (PG-2) shows a continuous
decrease in weight because the TG analysis was carried
out in N2 ambient. Oxygen out-diffusion from ZnO
matrix probably has resulted in the formation of oxygen
deficient ZnO compound (ZnO1-d). The weight loss was
also might be due to thermal desorption of water and
carbon dioxide from the surface of ZnO particles. TG
curve of ZnO (IT-2) shows a clear plateau formed at a
temperature between 200  C and 600  C which is the
indication of formation of nanocrystalline ZnO. Beyond
this temperature weight loss was observed due to oxy-
gen removal form ZnO matrix. Furthermore, no obvious
peaks were seen in the curves as shown in Fig. 4 (a and
b), which may indicate the formation of hexagonal
Wurtzite structure of ZnO due to temperature released
(~1400  C) during combustion process. In both the
curves no transition was seen anywhere. TGA results
also indicate that both the samples absorb a lot of N2
and slowly release them over a period of time. This is a
direct indication that the nanopowders are not only pure
but also very porous in nature. Thus TGA studies
confirm the absence of any reminder from the fruit
extracts after the combustion process.
3.4. Morphology
Surface morphology of the as-formed ZnO (PG-2)
and ZnO (TI-2) was studied using the SEM. The SEM
micrographs are shown in Fig. 5 (a and b) for ZnO
(PG-2) and ZnO (TI-2) respectively. The SEM analyses
revealed the presence of agglomerates of the nano-
particles. The micrographs showed that the
morphology is replete with voids and pores, the reason
for which can be traced to the large amounts of hot
gases that escape out of the reaction mixture during
combustion. The crystallites are interlinked to one
another to make large network systems with irregular
pore sizes and shapes. SEM images indicate that the
products are made of clusters, circular nanoparticles
and both the samples are highly agglomerated.

Fig. 5. SEM images of (a) ZnO (PG-2) and (b) ZnO (TI-2) respectively.

Please cite this article in press as: P. G.K. et al., In vitro antibacterial and cytotoxicity studies of ZnO nanopowders prepared by combustion
assisted facile green synthesis, Karbala International Journal of Modern Science (2015), http://dx.doi.org/10.1016/j.kijoms.2015.10.007
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P. G.K. et al. / Karbala International Journal of Modern Science xx (2015) 1e11 7

Fig. 6. TEM images of (a) ZnO (PG-2) and (b) ZnO (TI-2) respectively.

Table 3
Results of phytochemical screening of the aqueous fruit extracts of Punica granatum L and Tamarindus indica L granatum L and Tamarindus indica
L.
Constituent Test Result Constituent Test Result
PG TI PG TI
Alkaloids Mayer's reagent test e e Tannins and Phenolic compounds KillereKilliani test þ e
Wagner's reagent test e e Ferric chloride test þ e
Hager's reagent test e e Lead acetate test þ e
Carbohydrates Molish's test þ þ Saponins Froth test e e
Barfoed's test þ þ Proteins and Amino acids Biuret test e e
Reducing sugars Fehling's test þ þ Ninhydrin test e e
Benedicts's test þ þ Triterpenoids and Steroids Salwonski test e e
Flavanoids Alkaline reagent test e e Libermann and e e
Lead acetate test e e Burchard's test e e
Glycoside Legal's test e e Carboxylic acids Sodium bicarbonate test þ þ
Bomtrager's test e e Ester test þ e
þ is present.
e is absent.

Table 4
Results of antibacterial activity of ZnO nanopowders and standard antibiotic (Zone of inhibition in mm).
Test sample Concentration (10 mL, mg/mL) Test organisms
E. coli P. aeruginosa
ZnO (PG-2) 100 14.00 ± 1.0 14.00 ± 0.0
ZnO (TI-2) 100 13.50 ± 1.0 12.50 ± 1.0
ZnO (Commercial) 100 9.50 ± 0.00 12.00 ± 0.00
Ciprofloxacin 1.0 16.00 ± 0.0 18.00 ± 0.0

The TEM images of ZnO (PG-2) and ZnO (TI-2)
are shown in Fig. 6 (a and b) respectively. The TEM
study was carried out to understand the crystalline
characteristics and size of the nanoparticles. The im-
ages of ZnO (PG-2) and ZnO (TI-2) confirm that the
particles are almost spherical with non uniform thick-
ness. The average particle size by histogram was found
to be 10 nm and 14 nm for ZnO (PG-2) and ZnO (TI-2)
respectively.
3.5. Product formation mechanism
Zn(NO3)2 and the fuel (aqueous fruit extracts of
Punica granatum L and T. indica L) were mixed in

Please cite this article in press as: P. G.K. et al., In vitro antibacterial and cytotoxicity studies of ZnO nanopowders prepared by combustion
assisted facile green synthesis, Karbala International Journal of Modern Science (2015), http://dx.doi.org/10.1016/j.kijoms.2015.10.007
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distilled water. When this mixture was heated to
375 ± 10  C, initially the wet powder undergoes ther-
mal dehydration followed by decomposition of zinc
nitrate and fuel. Then it ruptures in to a flame and
yields porous, agglomerated powders. The reaction
was selfepropagating and the temperature produced
was sustained for a length of few seconds.
The probable reaction mechanism of formation of
ZnO is as follows
samples exhibited almost similar antibacterial efficacy
against both the bacteria selected in our studies. The
detailed mechanism of the bioactivity of ZnO is still
under discussion. Many mechanisms have been pro-
posed related to this: (a) One of the possible mecha-
nisms is based on the abrasive surface texture of ZnO
e binding of ZnO nanoparticles to the bacterial surface
is due to electrostatic forces that directly kill bacteria
[43], (b) mechanical destruction of the cell membrane

Phytochemicals present in aqueous fruit extracts /Reaction with Zn ions

Plant extracts act as reducing and stabilizing agents ZnO nanopowders

þGaseous products

3.6. Qualitative phytochemical analysis of the caused by penetration of the nanoparticles - ZnO
aqueous fruit extracts of P. granatum L and T. causes damage to the membrane [44], (c) release of
indica L Zn2þ ions from the nanoparticles [45e47] and (d) the
generation of highly reactive species such as OH.,
To understand which components of the bio fuels H2O2, O2 2 [48e52].
are responsible for combustion a detailed qualitative Results of MIC studies of ZnO nanopowders and the
phytochemical examination was carried out for the standard antibiotic against E. coli MTCC 7410 and P.
aqueous fruit extracts of P. granatum L (PG) and T. aeruginosa MTCC 7903 are presented in Table 5. The
indica L (TI). The results are presents in Table 3. These effect of ZnO nanopowders and the standard antibiotic
studies confirm the presence of many phytochemicals on the growth of the E. coli MTCC 7410 and P. aeru-
in the aqueous extracts of both the fruits. The results ginosa MTCC 7903 is represented in Fig. 7 (a and b)
are in almost agreement with the literature [41,42]. respectively. Comparison of the results of antibacterial
Phytochemicals such as carbohydrates, reducing activity of ZnO (PG-2) and ZnO (TI-2) revealed that
sugars, carboxylic acids which are present in aqueous both the samples exhibited almost similar activity
fruit extracts might be responsible for the combustion against E. coli and P. aeruginosa. It was observed that
process in SCS. the sample ZnO (PG-2) had a slight better bactericidal
activity than ZnO (TI-2) and ZnO (commercial). This
3.7. Evaluation of antibacterial studies finding might be due to different particle sizes of the
two samples as observed from the TEM images Fig. 6 (a
The zone of inhibition observed against ZnO and b). Smaller particles size provides relatively larger
nanopowders and the standard antibiotic is summa- surface area and higher amount of Zn atoms that trigger
rized in Table 4. The results indicate that both the toxicity effect of ZnO towards the bacteria [46,7].
Enhanced bio activity of the sample ZnO (PG-2) with
smaller particle size might be due to its large surface
Table 5
Results of MIC studies of ZnO nanopowders and standard antibiotic area to volume ratio [52]. According to the MIC values,
against test organisms. P. aeruginosa was more resistant to ZnO nanoparticles
Organism Sample MIC value (mg/mL) than E. coli. This resistance of P. aeruginosa compared
to E. coli might be due to the presence of a cytochrome
E. coli ZnO (PG-2) 512
ZnO (TI-2) 512 oxidase in P. aeruginosa [49].
ZnO (Commercial) 1024
Ciprofloxacin 1.0 3.8. Anticancer activity by MTT assay
P. aeruginosa ZnO (PG-2) 1024
ZnO (TI-2) 1024
Cytotoxic effect of ZnO nano powders in cell lines
ZnO (Commercial) >1024
Ciprofloxacin 1.0 is presented Fig. 8. It was observed from the results

Please cite this article in press as: P. G.K. et al., In vitro antibacterial and cytotoxicity studies of ZnO nanopowders prepared by combustion
assisted facile green synthesis, Karbala International Journal of Modern Science (2015), http://dx.doi.org/10.1016/j.kijoms.2015.10.007
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P. G.K. et al. / Karbala International Journal of Modern Science xx (2015) 1e11 9

Fig. 7. Effect of ZnO nanopowders and standard antibiotic on the growth of (a) Escherichia coli MTCC 7410 and (b) Pseudomonas aeruginosa
MTCC 7903 respectively.

120

100
% of cell viability

80

ZnO (PG-2)
60 ZnO (TI-2)
ZnO (Commercial)

40

20

0
10

20

30

40

50

60

70

80

90
l

0
ro

10
nt
Co

Concentration of ZnO nanopowders (μg/ml)

Fig. 8. Cytotoxic effect of ZnO nano powders in MCF-7 cell lines. Cells were treated with various concentrations (0, 10, 20, 30, 40, 50, 60, 70, 80,
90, 100 mg/mL) of ZnO nanopowders for 24 h grown in a serum free media. The percentage of cell death induced was determined using the MTT
assay.

Table 6 specific and proliferation dependent manner by rapidly

Results of blood hemolysis by ZnO nanopowders. dividing cancer cells being the most susceptible and
Sample concentration Percent hemolysis quiescent cells being the least sensitive [53,54].
(mg/mL) ZnO (PG-2) ZnO (TI-2) ZnO (commercial) However, the anticancer activity of ZnO nanoparticles,
0.25 0.07 0.07 1.22 in particular the mechanism of apoptosis in cancer
0.5 0.52 0.74 1.78 cells due to ZnO nanoparticles is still not clear.
1.0 1.19 1.56 2.43
2.5 3.20 3.57 3.41 3.9. Hemolysis
5.0 5.73 5.44 7.30

The results of cytotoxicity studies by hemolysis

that the sample ZnO (PG-2) had a slight higher cyto-
toxic effect on MCF-7 than ZnO (TI-2). The viability
values indicate ZnO nanopowders prepared by green
synthesis at the highest concentrations used in our
studies (100 mg/mL) exhibited around 58% cell
viability where as commercial ZnO nanopowder
exhibited around 66% cell viability. Literature shows
that ZnO nanoparticles induce cytotoxicity in a cell
assay are presented in Table 6. The interaction of ZnO
nanopowders with RBC revealed a hemolysis per-
centage of greater than 5 at a concentration of 5 mg/
mL of ZnO nanopowders. Since 5% hemolysis is
considered as permissible limit for biomaterials, till a
concentration of 2.5 mg/mL of ZnO nanoparticles can
be taken for hemolysis activity [55]. These results are
in agreement with the literature [35]. Therefore, within

Please cite this article in press as: P. G.K. et al., In vitro antibacterial and cytotoxicity studies of ZnO nanopowders prepared by combustion
assisted facile green synthesis, Karbala International Journal of Modern Science (2015), http://dx.doi.org/10.1016/j.kijoms.2015.10.007
 + MODEL
10 P. G.K. et al. / Karbala International Journal of Modern Science xx (2015) 1e11
the limitation of this study it can be concluded that the
concentrations of ZnO nanopowders employed in the
present antibacterial and anticancer studies are
biocompatible.
4. Conclusions
ZnO nanopowders were prepared by low cost SCS
using aqueous fruit extracts of Punica granatum L and
T. indica L as fuels. The samples were characterized by
PXRD, FTIR, SEM and TEM. TG analysis confirmed
the absence of any reminder from the fruit extracts in
both ZnO nanopowders. The samples exhibited
considerable antibacterial activity against E. coli and P.
aeruginosa. Within the limitations of this study, the
MTT assay results suggest that when the concentration
of green synthesized ZnO nanopowders was 100 mg/
mL, the viability of MCF-7 cells dropped to around
58%. All the samples exhibited permissible hemolysis
activity till a concentration of 2.5 mg/mL. The results
indicate the enhanced bio activity of ZnO powders
prepared by SCS using bio fuels as reducing agents
than the commercial ZnO nanopowder which was
employed in our present studies. Therefore this study
successfully demonstrates the convenient utilization of
bio fuels to synthesize ZnO nanopowders and their
plausible biological applications.
Acknowledgments
The authors G.K. Prashanth, Dr. G.M. Krishnaiah
and H.M. Sathyananda thank the management of Sri
KET and Dr. M.S. Indira, Principal of Sir MVIT for the
support and encouragement extended towards this
project. The authors thank Mr. Tejabhiraman, Research
Scholar, SRM University for his valued suggestions.
The authors acknowledge SAIF, IITB for TEM, CIF,
Pondicherry University for SEM and IISc, Bengaluru
for TG analyses.
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