Journal of Science: Advanced Materials and Devices 3 (2018) 440e451

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Journal of Science: Advanced Materials and Devices

journal homepage: www.elsevier.com/locate/jsamd

Original Article

Cytotoxicity, antibacterial and antifungal activities of ZnO

nanoparticles prepared by the Artocarpus gomezianus fruit mediated
facile green combustion method
R. Anitha a, K.V. Ramesh b, T.N. Ravishankar c, K.H. Sudheer Kumar d,
T. Ramakrishnappa d, *
a
Department of Biochemistry, Bharathiar University, Coimbatore, 641 046, India
b
PG Department of Biochemistry, Dayananda Sagar College, Bangalore, 560 078, India
c
Department of Chemistry, Global Academy of Technology (GAT), Rajarajeshwarinagar, Off Mysore Road, Ideal Homes Township, Bangalore, 560098,
Karnataka, India
d
Department of Chemistry, BMS Institute of Technology and Management, Avalahalli, Doddaballapura Main Road, Yelahanka, Bangalore, 560064, India

a r t i c l e i n f o a b s t r a c t

Article history: Spherical nanoparticles of zinc oxide (ZnO NPs) were synthesized by an eco-friendly green combustion
Received 27 July 2018 method using citrate containing Artocarpus gomezianus fruit extract as a fuel. The morphology, com-
Received in revised form positions and structure of the product were characterized by Powder X-ray Diffraction (PXRD), Scanning
4 November 2018
Electron Microscopy (SEM), Transmission Electron Microscopy (TEM), Fourier Transform Infra-red (FTIR),
Accepted 6 November 2018
Available online 22 November 2018
UVeVisible (UVeVis) and Raman Spectroscopy. Highly uniform spherical zinc oxide NPs were subjected
to cytotoxicity, antifungal and antibacterial activities. PXRD patterns show that the product formed
belongs to a hexagonal wurtzite system. SEM micrographs reveal that the particles are agglomerated. The
Keywords:
Green synthesis
TEM images demonstrate that the particles are highly uniform spherical in shape and loosely agglom-
ZnO nanoparticles erated. Scherrer's method and WeH plots were used to calculate the average crystallite sizes, yielding 39,
Anticancer activity 35, 31 and 40, 37, 32 nm for ZnO NPs prepared with 5, 10 and 15 mL of 10% Artocarpus gomezianus fruit
MCF-7 extract, respectively. These results were confirmed by the TEM observations. Breast cancer cell lines
Antibacterial (MCF-7) were subjected to in vitro anticancer activity. MTT assay revealed a good anticancer activity of
Antifungal ZnO NPs against MCF-7. Zone of the inhibition method shows that the spherical ZnO NPs also exhibit
significant antibacterial activity against staphylococcus aureus and antifungal activity against Aspergillus
niger. The synthesized ZnO NPs can find plausible biological applications.
© 2018 The Authors. Publishing services by Elsevier B.V. on behalf of Vietnam National University, Hanoi.
This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

1. Introduction ZnO NPs gained also significant attention due to their implications
for cancer therapy [4]. It has been found from studies that ZnO NPs
Inorganic materials such as metals and metal oxides due to their cause cytotoxicity to many types of cells such as HepG2, MCF-7,
stability are more advantageous in many aspects than organic com- HT29, Caco-2, rat C6, HeLa, THP-1 [5e8]. In addition, ZnO NPs
pounds [1]. Among the metal oxides, zinc oxide nanoparticles (ZnO exhibit antibacterial and antifungal activity. They can decrease the
NPs) have received a special attention as an anticancer, antibacterial viability and attachment of microbes on biomedical surfaces [9].
and antifungal material. ZnO NPs exhibit improved properties ZnO NPs can be chemically synthesized by different methods such
compare to bulk materials and these novel properties are attributed as, spray pyrolysis, hydrothermal treatment, sol-gel process, co-
to the changes in specific characteristics such as morphology and size precipitation, combustionor sonochemical, etc. [10e12]. Generally
of the particles [2]. ZnO NPs have a wide range of applications in solar the chemicals used in the synthesis and stabilization are toxic and
cells, catalysts, gas sensors, luminescent devices etc. [3]. Nowadays, lead to by-products which are non eco-friendly and cause danger to
human beings and the environment [13]. The generations of toxic by-
products can be avoided using a green chemistry approach, for
* Corresponding author. instance, using plants for the synthesis of ZnO NPs. Hence, the green
E-mail address: [email protected] (T. Ramakrishnappa). combustion synthesis is an eco-friendly alternative wet-chemical
Peer review under responsibility of Vietnam National University, Hanoi.

https://doi.org/10.1016/j.jsamd.2018.11.001
2468-2179/© 2018 The Authors. Publishing services by Elsevier B.V. on behalf of Vietnam National University, Hanoi. This is an open access article under the CC BY license
(http://creativecommons.org/licenses/by/4.0/).
 R. Anitha et al. / Journal of Science: Advanced Materials and Devices 3 (2018) 440e451 441

method. This method has proved to be an excellent technique for (JOEL JSM 840 A) with gold as contrast enhancing material covered
preparing several grams due to its low processing temperature, short by the sputtering technique. TEM analysis was carried out using
processing time, cost effectiveness. It shows good ability to achieve the Hitachi H-8100 (accelerating voltage up to 200 KV, LaB6
high purity in making multiphase or single complex oxides [14,15]. Filament) equipped with EDS (Keney Sigma TM Quasar, USA). The
The main advantages of synthesis of ZnO NPs via the solution FTIR studies were performed by using the Perkin Elmer Spec-
combustion method towards biological activities are: (i) A larger trometer with KBr pellets. Raman spectrum was obtained at room
surface area with high porosity (as in the case of nanoparticles temperature in a back scattering geometry using a 632 nm HeNe
fabricate by solution combustion method) ensures an increased laser with a JobinYvonLabRam HR spectrometer (LABRM-HR). The
range of probable interaction with bio-organics present on the UVeVisible absorption spectrum was obtained on the SL 159
viable cell surface [16]. (ii) The considerable antimicrobial activities ELICO UVeVIS Spectrometer. Flow cytometry measurements were
of inorganic metal oxide nanoparticles such as ZnO NPs and their done by using the BD FACS Calibur Flow Cytometry.
selective toxicity to biological systems suggest their potential
application as antimicrobial agents in therapeutic, diagnostic, sur-
gical devices and in nano-medicine as well [17]. (iii) The advantages
2.4. Anticancer activity by MTT assays
of using ZnO NPs as antimicrobial agents are their greater effec-
tiveness on resistant strains of microbial pathogens, less toxicity
The anticancer activity was checked by the 3 e (4,5-
and good heat resistance. In addition, they provide mineral ele-
dimethylthiazol-2-yl) - 2,5 - diphenyltetrazolium bromide (MTT)
ments essential to human cells and even small amounts of them
assay. The monolayer cell (Mammalian breast cancer fibroblast
exhibit strong activity. (iv) The solution combustion method is a
cells) culture was trypsinized and the cell count was adjusted such
very simple, low-cost one, using which highly pure and highly
that 200 mL of suspension contained approximately 20,000 cells. To
crystalline size nanoparticles can be obtained.
each well of the 96 wells microtitre plate, 200 mL of the diluted cell
Many articles have reported on the acute toxicity of ZnO NPs.
suspension was added and incubated at 37
C and 5% CO2 atmo-
However, a citrate containing A. gomezianus fruit mediated spherical
sphere for 24 h. After 24 h 200 mL of different test concentrations of
ZnO NPs has not been discussed so far. In this study highly uniform
test drugs were added on to the partial monolayer. The plate was
spherical ZnO NPs were successfully prepared by an eco-friendly
then incubated at 37
C and 5% CO2 atmosphere for 24 h. Media
green combustion method using different volumes of citrate con-
containing 10% MTT reagent was then added to each well and the
taining Artocarpus gomezianus fruit source as a fuel. The as-prepared
plate was incubated at 37
C and 5% CO2 atmosphere for 3 h. Then
ZnO NPs were used to study in detail the anticancer, antibacterial
100 mL of solubilization solution DMSO (DIMETHYL SULFOXIDE)
and antifungal activities.
was added and the plate was gently shaken to solubilize the formed
formazan. The absorbance was measured by microplate reader at a
2. Experimental
wavelength of 570 nm. After subtracting the background and the
blank, the percentage growth inhibition was calculated and the
2.1. Chemicals
concentration of the test drug needed to inhibit the cell growth by
50% (IC50) was generated from the dose-response curve for the cell
The chemicals used for the synthesis were of analytical grade
line.
and were used without any further purification. Zinc nitrate was
The cell viability was expressed as follows:
procured from Merck. The glassware used in the laboratory were
cleaned with a fresh solution of HCl/HNO3 (1:3, v/v), washed Test
thoroughly with double distilled water and dried. Double distilled Cell vialbility ¼  100% (1)
Control
water was used for all the experiments.

2.2. Preparation of ZnO NPs

2.5. Anticancer activity by apoptosis assay
The citrate containing Artocarpus gomezianus fruit source was
collected from Mangalore, Karnataka. The collected fresh, healthy The detection of the apoptosis and necrosis was done by
fruits were washed thoroughly using double distilled water and cut the flow cytometry. The principle of this method is that the
into small pieces. Then small pieces were dried at room tempera- enhancement in the intensity of the side scattered light with the
ture for 10 days under dust free conditions and subsequently intensity of the forward scattered light in the cells reveals that
grinded into a fine powder. 10 g of this fine powder were boiled in the enhanced granularity of cells is correlated to cellular uptake of
100 mL doubled distilled water to prepare a 10% crude solution, the NPs. Scattered light is defined as the laser light scattered at
then filtered and stored in refrigerator for further usage. In a typical about a 90
angle to the axis of the laser beam and forward
synthesis, 5 mL of the 10% crude solution was added to 3 g of scattered light is the laser light scattered at narrow angles to the
Zn(NO3)2.6H2O which is already dissolved in 10 mL of double axis of the laser beam. Cells were cultured in a 6-well plate and
distilled water. This reaction mixture was well mixed using a then incubated in a CO2 incubator at 37
C for 24 h. Then 180 mL of
magnetic stirrer for about 5e10 min and then placed in a preheated the trypsin-EDTA solution were added and the mixture was
muffle furnace maintained at about 400 ± 10
C. The reaction incubated at 37
C for 3e4 min. The tubes containing the mixture
mixture boils froths and thermally dehydrates to form foam. The were then centrifuged for 5 min to carefully decant the superna-
whole process was completed in a few min. Similar procedure was tant. The cells were resuspended in a 1X Binding Buffer. 100 mL of
repeated by taking 10 and 15 mL of the 10% crude sample. the solution (1  105 cells) was transferred to a 5 mL culture tube
and 5 mL of FITC (Fluorescein Isothiocyanate) Annexin V was
2.3. Characterization of ZnO NPs added, slowly stirred and the incubated for 15 min at room tem-
perature (25
C) in the darkness. Further, 5 mL of Propidium Iodide
PXRD data were recorded on the PANalyticalX'Pert Pro X-ray (PI) and 400 mL of 1X Binding buffer were added to each tube and
Diffractometer with the graphite monochromatised Cu-Ka circularly shaked gently. The analysis was performed by flow
(1.5418 Å) radiation. The surface morphology was observe by SEM cytometry after the addition of PI.
442 R. Anitha et al. / Journal of Science: Advanced Materials and Devices 3 (2018) 440e451

2.6. Anticancer activity by CAM method spread onto the four well bored media. 100 mL of different
working concentrations of the sample were loaded to four wells.
The anticancer activity has also been checked by the Chlorio- The plate was kept at 37
C for the incubation along with a
allantoic Membrane (CAM) assay. Whatmann filter paper bud control concerning DMSO and antibiotic (ampicillin) for a period
containing the compound ZnO NP concentration corresponding to of 17 h.. The zone of inhibition was recorded following the in-
its respective IC50 value was implanted on the chick embryo chorio- cubation period.
allantoic membrane through a hole cut in to the shell of the egg.
The incubation period may range from 1 to 3 days. Afterwards, time
angiogenesis can be quantified through an image analysis. 2.8. Evaluation of the antifungal activity

The examination of the antifungal activity of 15 mL of 10%

2.7. Evaluation of the antibacterial activity Artocarpus gomezianus fruit extract mediated ZnO NPs was carried
out by the zone of inhibition method in potato dextrose agar (PDA)
The antimicrobial activity of 15 mL of 10% Artocarpus gomezianus media against Aspergillus niger. An autoclaved petriplate was filled
fruit extract mediated ZnO NPs was examined by the zone of in- with the steriled potato dextrose agar (PDA) media. 100 mL of spore
hibition method in Muller Hinton agar (MHA) media against Gram- suspension (Artocarpus niger) was spread onto the four wells.
Positive Staphylococcus spp. 100 mL of different working concentrations of the sample were
Agglomeration was prevented by the standard sonication loaded to the four wells. The plate was kept for incubation along
method, in which particles were dispersed in the media using the with a control concerning DMSO and antifungal (fluconazole) at
sonicator. The ZnO NPs obtained from 5 mL, 10 mL and 15 mL of 10% room temperature for a period of 96 h. The zone of inhibition was
crude samples were dissolved in Di Methyl Sulphoxide (DMSO) to recorded following the incubation period.
give a concentration of 10 mg/mL. They were marked as stock, to
carry out the minimum inhibitory concentration for the ZnO NPs.
Working concentrations of 5 mg/mL, 0.5 mg/mL, 0.05 mg/mL and 3. Result and discussion
0.005 mg/mL. 0.5 mL was prepared by the serial dilution of 0.5 mL
of the stock solution. 3.1. Crystal structure
An autoclaved petriplate was filled with Sterile Muller Hinton
agar. 100 mL of 24 h. The test culture (Staphylococcus aureus) was Fig. 1 shows the PXRD patterns of spherical ZnO NPs prepared by
different volume of citrate containing 10% Artocarpus gomezianus
fruit extract (5e15 mL) as fuel via a green solution combustion
method. The diffraction peaks are shown corresponding to the
hexagonal wurtzite structure of ZnO (JCPDS NO. 36-1451) and
average crystallite size (d) was estimated using the Scherrer's
equation [18].

kl
d¼ (2)
b cos q
The shift of peaks and the widening of lines in PXRD profile arise
due to the micro strain in the nanoparticles. The Williamson and
Hall (WeH) graphs (not shown here) were used to calculate the
micro strain in ZnO NPs using the relation [19].

kl
bhkl cosqhkl ¼ þ 4ε sinqhkl (3)
D

where ε is the strain associated with the NPs. Equation (3) repre-
sents a straight line between bcosq (Y-axis) and 4sinq (X-axis). The
slope of the line of the W-H graphsl gives the strain (ε) and inter-
cept (0.9l/D) of this line on the Y-axis gives the average crystallite
size (D) for the 5, 10 and 15 mL of 10% A. gomezianus fruit extract
mediated ZnO NPs. The obtained mean crystallite size from
Scherrer's method and WeH graphs are tabulated in Table 1. As the
volume of the citrate containing 10% Artocarpus gomezianus fruit
extract increases, the broadening of the lines also increases, indi-
Fig. 1. PXRD patterns of ZnO NPs prepared with (a) 5, (b) 10 and (c) 15 mL of 10% cating that the particle sizes decreases and it is in good agreement
A. gomezianus fruit extract. with TEM results.

Table 1
Average crystallite size and strain of ZnO NPs synthesized by 5, 10 and 15 mL of Artocarpus gomezianus fruit extract.

Fruit extract (mL) Average crystallite size (nm) Strain (104)

Scherrer's method (d) WeH plots method (D)

5 39 40 2.42
10 35 37 3.17
15 31 32 5.36
 R. Anitha et al. / Journal of Science: Advanced Materials and Devices 3 (2018) 440e451 443

3.2. Morphological studies between 1400 and 1649 cm1 is due to the stretching mode of C]
O. The peak at 2350 cm1 arises due to CO2 absorption from the
Fig. 2 shows the SEM micrographs and the EDS of the ZnO atmosphere on the metallic cations. The bands at 421 and 590 cm1
nanoparticles prepared using 5, 10 and 15 mL of citrate containing correspond to the bonding between ZneO [20].
Artocarpus gomezianus fruit extract as a fuel. The combustion
product is more influenced by the type of the fuel used. The nature 3.4. UVeVis analysis
of the combustion differs from flaming to non-flaming type.
Generally, flaming reaction is associated with the release of large Fig. 5 shows the UVeVis spectrum of the ZnO NPs prepared at
quantity of gases. The SEM micrographs (Fig. 2 (a), (c) and (e)) show room temperature using 15 mL of citrate containing A. gomezianus
the agglomeration, voids and pores. The pores and voids can be due fruit extract as a fuel. At the wavelength of 367 nm, the charac-
to the huge quantity of gases escaping out of the reaction mixture teristic absorption peak in the ZnO NPs spectrum is observed. Due
during the combustion (flaming). to the electron transitions from the valence band to the conduction
The energy dispersive spectrometry (EDS) analysis was used to band (O2p-Zn3d), the characteristic absorption peak of the ZnO NPs
determine the composition of ZnO NPs prepared using 5, 10 and can be assigned [21]. From this absorption spectrum, using Tauc
15 mL of citrate containing Artocarpus gomezianus fruit extract as a equation, the band-gap of the ZnO thin film was calculated [22]:
fuel and results are shown in Fig. 2 (b), (d) and (f), respectively. The

n
EDS measurements revealed the presence of Zn and O peaks for ahv ¼ A hv  Eg (4)
ZnO.
Fig. 3 shows the TEM image of the ZnO NPs prepared using 5, 10 where ahn is the photon energy, Eg is the band gap, n ¼ 1/2 for the
and 15 mL of citrate containing Artocarpus gomezianus fruit extract direct band gap transition and A is a constant which is different for
as a fuel. It clearly shows that the nanoparticles are of sizes in the different transitions. The progress information gives the best linear
range 10e30 nm and spherical in shape of rather uniform fit in the band edge location for n ¼ 1/2. The band gap was observed
dimension. as 3.39 eV which is somewhat more prominent than that of the
massive ZnO (~3.37 eV). This band gap upgrade emerges because of
3.3. FTIR analysis the size impact of the nanoparticles.

FTIR spectra of ZnO NPs prepared using 5, 10 and 15 mL of citrate 3.5. Raman analysis
containing Artocarpus gomezianus fruit extract are shown in Fig. 4
(a-c). The absorption band near 3450 cm1 is due to the hydroxyl Raman spectroscopy can give information on the vibrational
group of H2O adsorbed on the ZnO NPs. The transmittance band properties of the ZnO NPs. Fig. 6 shows the Raman spectrum of the

Fig. 2. SEM micrographs ((a), (c) and (e)) and EDS ((b), (d) and (f)) of ZnO NPs prepared with (a) 5, (b) 10 and (c) 15 mL of 10% A. gomezianus fruit extract.
444 R. Anitha et al. / Journal of Science: Advanced Materials and Devices 3 (2018) 440e451

Fig. 3. TEM images of ZnO NPs prepared by (a) 5, (b) 10 and (c) 15 mL of 10% A. gomezianus fruit extract.

Fig. 5. UVeVis spectrum of ZnO NPs prepared by 15 mL of 10% Artocarpus gomezianus

fruit extract.

Fig. 4. FTIR spectra of ZnO NPs prepared by (a) 5, (b) 10 and (c) 15 mL of 10% A.
gomezianus fruit extract.

sample in the range of wavelengths between 200 and 800 cm1.

The peak at 436 cm1 relates to E2 (high), which is moved by
3 cm1. The peak at 582 cm1 is set between E1 (LO) and A1 (LO),
which is a great concurrence with the literature data [23]. The peak
at 330 cm1 is because of the second-order Raman scattering. The
peak at 379 cm1 relates to A1 (TO) and that at 410 cm1 compared
to E1 (TO) vibrational modes of ZnO nanocrystals [24].

3.6. Product formation mechanism

Zn(NO3)2.6H2O and the aqueous 10% A. gomezianus fruit extract Fig. 6. Raman spectrum of ZnO NPs prepared by 15 mL of 10% Artocarpus gomezianus
as a fuel were mixed in distilled water. When this mixture was fruit extract.
 R. Anitha et al. / Journal of Science: Advanced Materials and Devices 3 (2018) 440e451 445

Fig. 7. (a) Morphologies of normal MCF-7 cells in the absence of the ZnO NPs, (b) MCF-7 cells treated with A. gomezianus fruit extract only, (c) MCF-7 Untreated, (d) MCF-7 against
10 mM ZnO NPs, (e) MCF-7 against 50 mM ZnO NPs (f) MCF-7 against 100 mM ZnO NPs (g) MCF-7 against 200 mM ZnO NPs (h) MCF-7 against 300 mM ZnO NPs (i) MCF-7 against
500 mM ZnO NPs.
446 R. Anitha et al. / Journal of Science: Advanced Materials and Devices 3 (2018) 440e451

heated to 400 ± 10
C, in the beginning the wet powder undergoes

the thermal dehydration. Then it undergoes the decomposition of
Zn(NO3)2.6H2O and of the fuel. Then it breaks down into a flame,
yielding porous, agglomerated powders. The reaction was self-
propagating and the heat released was sustained for a length of
few seconds. The probable formation mechanism of ZnO NPs is as
the following

Zn(NO3)2.6H2O þ fruit extract / ZnO nanoparticles þ Gaseous

products

Phytochemicals present in the aqueous fruit extracts react with

the Zn ions. Fruit extracts act as reducing and stabilizing agents.

3.7. Anticancer activity

In vitro experiments can be easy and rapid to perform and can

provide a range from 10 to 500 mg/mL of the in vivo toxicity. The
cytotoxicity results of in vitro experiments were taken after 24 h Fig. 8. Cell viability of MCF-7 cells calculated by MTT assay. Cells were incubated for
of the incubation with different concentrations of ZnO NPs 24 h with the ZnO NPs prepared with 15 mL of 10% A. gomezianus fruit extract.
ranging from 10 to 500 mg/mL, prepared with 15 mL of 10%
A. gomezianus fruit extract and are shown in Fig. 7. Cells at different
concentrations of ZnO NPs show different stages of cell death/ne- 5, 10 and 15 mL fruit extract mediated ZnO NPs at 0.5 mg/100 mL
crosis [25]. The drug of nano ZnO shows necrosis of the MCF-7 cells exhibited rather similar antibacterial and antifungal efficacy
at 100 mM indicating its toxicity is approximately near to the against Gram-Positive Staphylococcus aureus and Aspergillus niger,
standard drug camptothecin whose toxicity level is 50 mM [26]. The respectively. Nanoparticles provide relatively larger active surface
cytotoxic effect of ZnO NPs in MCF-7 cell lines is presented in Fig. 8. area and thus, a higher amount of those Zn atoms that trigger a
The results obtained infers an inverse relation between the drug toxicity effect of ZnO towards the bacteria [27]. The detailed
concentration and the cell viability. The percentage growth inhi-
bition was found by subtracting the background and blank. The
concentration of the nano ZnO drug required to inhibit cell growth
by 50% (IC50) was got from the dose-response curve. So we have got
the inference of IC50 with the value of 9.3495 mg/mL.
Fig. 9 (a) and (b) show the flow cytometry data of SSC-H versus
FSC-H, untreated and treated with ZnO NPs prepared with 15 mL of
10% Artocarpus gomezianus fruit extract. It is clear that the SSC in-
tensity is an indicator for the uptake of the ZnO NPs. Fig. 10 shows a
graph of the flow cytometry analysis of MCF-7 cells with Annex in-
V Fluorescein isothiocyanate (FITC) of which graph, in the lower left
corner are the living cells. From the graph, it is clear that all the cells
undergo an apoptotic pathway as indicated by the presence of the
small amounts of cells in each plot. And in the FITC count graph of
log counts we can observe that there is a sharp peak obtained
indicating the path taken by cell during apoptotic pathway which is
expelled by the color of fluorochrome FITC linked to the cell. Fig. 11
shows the graphical representation of counts versus FITC Annex in-
V for ZnO NPs.
Fig. 12 shows the CAM assay involving the implantation of the
experimental drug on the blood vessels of a chick embryo. This
allows nanoparticles to observe the thinning/disappearance of
blood vessels which can be related to the destruction of tumors
(cancerous tumors) via the disruption of the blood vessel devel-
opment or inhibiting the formation of new blood vessels inside the
tumor, thereby inhibiting the further spread of cancer. This is
another proof of the anti-cancer properties of the ZnO
nanoparticles.

3.8. Antibacterial and antifungal assay

Figs. 13 and 14 show the photographs illustrating the antibac-

terial and antifungal activities of the ZnO NPs prepared with 15 mL
of 10% A. gomezianus fruit extract using the zone of inhibition
method against Staphylococcus aureus and Artocarpus niger,
respectively. The zone of inhibition was observed against the ZnO
NPs and results are summarized in Table 2. The results indicate that Fig. 9. Graph of SSC-H versus FSC-H (a) untreated and (b) treated with ZnO NPs.
 R. Anitha et al. / Journal of Science: Advanced Materials and Devices 3 (2018) 440e451 447


O2
2 , H2O2, OH which harm the DNA, cell films or cell proteins, and
may at long last prompt the hindrance of the bacterial development
and in the end leading to the bacterial death.

3.9. Mechanism of antibacterial and antifungal activities

Although the exact mechanism of antibacterial and antifungal

activities of ZnO nanoparticles is still unknown, the antimicrobial
activity of these nanoparticles was attributed to several mecha-
nisms, including the release of antimicrobial ions [30], the
interaction of nanoparticles with microorganisms followed subse-
quently by damaging the integrity of the bacterial cells [31] and the
formation of reactive oxygen species (ROS) by the effect of the light
radiation [32]. The release of the Zn2þ antimicrobial ions has been
suggested as a reasonable hypothesis about the toxicity of ZnO
Fig. 10. Graph of Flow cytometry analysis of MCF-7 cells with Annex in-V Fluorescein against S. cerevisiae [33]. According to this author, the toxicity of
isothiocyanate (FITC) of which graph in the lower left area are the living cells. ZnO nanoparticles could result from the solubility of the Zn2þ ions
in the medium containing the microorganisms. However, the sol-
ubility of the metal oxides, such as ZnO is a function of their con-
centration and the time [33]. Thus, low concentrations of
solubilized Zn2þ ions can trigger a relatively high tolerance by the
microorganism. In the case of yeast, labile Zn2þ ions rapidly accu-
mulates in dynamic vesicular compartments (vacuoles and zinco-
somes), which are an important cellular defense system to buffer
both the zinc excess and deficiency [34].
In addition, there are differences in the metabolic processes of
the Zn2þ ions, which depend on characteristics intrinsic to each
microorganism. This could be one of the possible reasons for the
observed differences in toxicity thresholds of ZnO nanoparticles
in various microorganisms. In this way, Reddy et al. [35] studied
the toxicity of ZnO nanoparticles on E. coli and S. aureus. The
Fig. 11. Graphical representation of counts vs FITC Annex in-V for ZnO nanoparticles results showed complete inhibition of E. coli growth at concen-
prepared with 15 mL of 10% A. gomezianus fruit extract. trations 3.4 mM, while the growth of S. aureus was completely
inhibited at concentrations 1 mM. Moreover, Reddy et al.
observed that cells of E. coli treated with 1 mM of ZnO showed a
reaction system of the bioactivity of ZnO is still under debate. consistent increase in the number of colony forming units
Numerous systems have been proposed and are identified with the (CFU) compared to control, due to the preference of this micro-
features, such as: (i) One of the conceivable mechanism depending organism for low concentrations of Zn2þ in the growth me-
on the grating surface of ZnO, actually the ZnO NPs to the bacterial dium. Conversely, S. aureus showed an efflux mechanism of Zn2þ
surface is due to the electrostatic powers that straightforwardly during the exposure to ZnO nanoparticles in the millimolar
eliminate microorganisms [28], (ii) The entering ZnO NPs can range, indicating that the sufficient ion concentration results in
connect with the layer lipids and casues the pulverization of the cell undesirable and potentially toxic conditions to this microor-
membrane, which prompts the lost of the membrane uprightness ganism. Thus, concerning the effect of ZnO against E. coli at low
and breakdown, and lastly leads to the bacterial demise [29], (iii) concentrations, rather than exercising antimicrobial activities,
The arrival of the Zn2þ particles from the ZnO nanoparticles and (iv) the ZnO nanoparticles may actually increase the bacterial growth.
The generation of the very responsive species of, for example, Zhang et al. [31] studied the effect of ZnO NPs on E. coli cells, and

Fig. 12. (a) Implantation of the drug ZnO 100 mM (IC50 value) and (b) Thinning of blood vessels seen preceding the site of the drug implant.
448 R. Anitha et al. / Journal of Science: Advanced Materials and Devices 3 (2018) 440e451

Fig. 13. Photographs showing the antibacterial activity of the ZnO NPs prepared by 15 mL of 10% Artocarpus gomezianus fruit extract in the zone of inhibition method with
Staphylococcus aureus 0.0005 to 0.5 (mg/100 mL) of samples 1, 2, 3, respectively.
 R. Anitha et al. / Journal of Science: Advanced Materials and Devices 3 (2018) 440e451 449

Fig. 14. Photographs showing the antifungal activity of the ZnO NPs prepared with 15 mL of 10% Artocarpus gomezianus fruit extract in the zone of inhibition method with
Aspergillus niger 0.0005 to 0.5 (mg/100 mL) of samples 1,2,3, respectively.
450 R. Anitha et al. / Journal of Science: Advanced Materials and Devices 3 (2018) 440e451

Table 2
Zone of inhibition of the ZnO NPs prepared by 10% Artocarpus gomezianus fruit extract against Staphylococcus aureus and Aspergillus niger.

Fruit extract (mL) Sl. No. Concentration (mg/100 mL) Zone of inhibition (mm)

Staphylococcus aureus Aspergillus niger

05 1 0.5 16.0 ± 0.66 23.0 ± 0.57

2 0.05 11.0 ± 0.33 16.0 ± 0.88
3 0.005 10.5 ± 0.51 14.0 ± 0.66
4 0.0005 ne 13.5 ± 0.33
10 1 0.5 10.5 ± 0.79 22.0 ± 0.33
2 0.05 ne 18.0 ± 1.20
3 0.005 ne 14.5 ± 0.51
4 0.0005 ne 13.0 ± 0.33
15 1 0.5 12.25 ± 0.57 25.0 ± 0.79
2 0.05 11.0 ± 0.51 20.0 ± 1.15
3 0.005 ne 15.0 ± 0.51
4 0.0005 ne ne

Values are mean inhibition zone (mm) ± S.D of three replicates.

Note: ‘ne’ indicates no effect.

that, 15 mL, 10% citrate containing A. gomezianus fruit extract

mediated ZnO NPs are desirable in shape and size. FTIR and Raman
spectra affirmed the formation of ZnO. The optical band gap of the
ZnO nanoparticles was acquired to be 3.39 eV. The as-synthesized
ZnO NPs are found to be potentially usable as an alternative anti-
cancer drug other than the standard camptothecin one. The
cytotoxicity results of the in vitro experiments were obtained after
24 h of the incubation with different concentrations of the ZnO
NPs prepared with 15 mL of 10% Artocarpus gomezianus fruit
extract, ranging from 10 to 500 mg/mL showing that different
concentrations of ZnO NPs caused different stages of the cell
death/necrosis. The nano sized ZnO drug showed necrosis of the
MCF-7 cells at 100 mM indicating its toxicity to be approximately
near to the standard drug camptothecin whose toxicity level is
50 mM. The percentage growth inhibition was found by subtract-
Fig. 15. Possible mechanism of antibacterial and antifungal activities by using ZnO NPs ing the background and the blank data. The concentration of the
prepared with 15 mL of 10% Artocarpus gomezianus fruit extract. nanosized ZnO drug required to inhibit cell growth was found
from the dose-response curve to be at 50% (IC50) and the infer-
ence of IC50 value ¼ 9.3495 mg/mL. Furthermore, our study has
as a result they pointed out that the interaction between the ZnO shown that the ZnO NPs exhibit significant antibacterial and
nanoparticles and the E. coli cells is caused by electrostatic forces. antifungal activities and, thus, can be useful for biological appli-
According to Stoimenov et al. [28], the global charge of bacterial cations. The antibacterial and antifungal activities of the ZnO NPs
cells at biological pH values is negative, due to the excess of prepared with 15 mL of 10% A. gomezianus fruit extract were
carboxylic groups, which are dissociated and provide a negative evaluated by the zone of inhibition method against Staphylococcus
charge to the cell surface Conversely, ZnO nanoparticles have a aureus and A. niger, respectively. The zone of inhibition was
positive charge, with a zeta potential of þ24 mV, [31]). As a observed against the ZnO NPs and is summarized. These results
result, opposite charges between the bacteria and the ZnO ano- indicate that the 5, 10 and 15 mL fruit extract mediated ZnO NPs at
particles generate electrostatic forces, leading to a strong binding 0.5 mg/100 mL exhibited rather similar antibacterial and anti-
between the nanoparticles and the bacteria surface and, conse- fungal efficacy against the Gram-Positive Staphylococcus aureus
quently, producing the cell membrane damage. The possible and Aspergillus niger, respectively.
mechanism of the antimicrobial activity of the ZnO nanoparticles
is still unknown. However, is it could be possibly suggested in a
schema, which is shown in Fig. 15. References

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